CHAPTER 2

24

Chapter 2

Synthesis, Characterization and Bioactivity evaluation of Metal Complexes.

Section I Synthesis, Thermal and Electrochemical study of Metal Complexes.

Section II Bioactivity evaluation of Metal Complexes.

Publications

Synthesis, Characterization and electrochemical study of bioactive

metal complexes, communicated to International Journal of Chemical

and Analytical Research

Thermal evaluation of bioactive para - Hydroxy - meta - nitro

acetophenone and it’s Metal Complexes, communicated to Der

Pharma Chemica Acta

25

Introduction Inorganic chemistry is of fundamental importance not only as a basic science

but also as one of the most useful sources for modern technologies.

Elementary substances and solid-state inorganic compounds are widely used

in the core of information, communication, automotive, aviation and space

industries as well as in traditional ones. Inorganic compounds are also

indispensable in the frontier chemistry of organic synthesis using metal

complexes, homogeneous catalysis, bioinorganic functions, etc.

One of the reasons for the rapid progress of inorganic chemistry is the

development of the structural determination of compounds by X-ray diffraction

and other analytical instruments. It has now become possible to account for

the structure-function relationships to a considerable extent by the

accumulation of structural data on inorganic compounds. More than ten

million organic compounds are known at present and infinite numbers of

inorganic compounds are likely to be synthesized by the combination of all the

elements. Recently, really epoch making compounds such as complex copper

oxides with high-temperature superconductivity and a new carbon allotrope

C60 have been discovered and it is widely recognized that very active

research efforts are being devoted to the study of these compounds. By the

discoveries of new compounds, new empirical laws are proposed and new

theories are established to explain the bondings, structures, reactions and

physical properties. However, classical chemical knowledge is essential

before studying new chemistry1. Co-ordination compounds

Coordination compounds, or complexes, are integral molecular or ionic units

consisting of a central metal ion (or atom), bonded to a defined number of

ligands in a defined geometrical arrangement. The ligands can be ions or

(induced) dipolar molecules. Each ligand provides a free electron pair, i.e. the

ligands are Lewis bases, while the metal in the coordination centre is the

Lewis acid. The bonding can thus be described in terms of Lewis acid/Lewis

base interaction. Other descriptions of the bonding situation are:

(i) donor bond; (ii) coordinative covalent bond, often denoted by L→M, where

L = ligand and M = metal.

26

Complexes tend to be stable when the overall electron configuration at the

metal centre (the sum of metal valence electrons plus electron pairs provided

by the ligands) is 18 (or 16 for the late transition metals).

M + nL ' [MLn] q (n = number of ligands, q = charge of the complex)

c (MLn)/c(M) cn(L) = K

K is the stability constant or complex formation constant (pK = -logK); its

inverse, K-1, is termed dissociation constant2 .

The rich diversity of coordination chemistry provides exciting prospects for the

design of novel therapeutic agents with unique mechanisms of action. Central

to such discovery is the understanding of both the kinetics and

thermodynamics of reactions of metal complexes under conditions of

biological relevance and consideration of the roles of both the metal and their

ligands in recognition processes3. There is a wide range of potential

applications of inorganic compounds and metal coordination complexes. Formation of complexes

The chemistry of the transition metal complexes exhibited due to small, highly

charged ions which have vacant orbitals of suitable energy to accept lone

pairs of electrons donated by other groups or ligands. In case of transition

metals in high oxidation states, highly charged ions can strongly bind electro

statically a wide variety of negative or polar ligands. In the case of transition

metals in low oxidation states, the electrons in the d orbital become involved

in π bonding with ligands. The majority of complexes are octahedral or

tetrahedral besides these trigonal bipyramidal, pentagonal bipyramid etc.

have been established. The bonding can either be electrostatic or covalent or

in many cases intermediate between the two extremes4.

Some transition metals are essential for the normal function of living

organisms. Metallo-drugs are becoming an interesting research area after the

discovery of cisplatin5.Copper complexes are known to have a broad

spectrum of biological action in certain concentration6-7.Bioactive

Copper+2complexes with phenoxyalkanoic acids and nitrogen donor

heterocyclic ligands have been reported as dinuclear or uninuclear neutral

complexes8. Complexes of La, Eu, Gd, Tb, Dy, Ho and Er) with Iso nicotinoyl

hydrazone of 1-Phenyl-3-methyl-5-hydroxy-4-pyrazolyl phenyl ketone have

been acknowledged .The La and Eu complexes possess antitumor activity

27

and the inhibitory rates for leukemia cells9. Bis (pyridine-2,6-diimine) (I) metal

complexes of Mn, Fe, Co, Ni, Cu, Zn have been cited10.Complexes of life

essential metal ions like VO+2,Co+2,Ni+2and Cu+2 with the Schiff bases derived

from 2-hydroxyacetophenone/2-chlorobenzaldehyde with 2-amino- 4-

chlorophenol (II) answer the problem of multi-drug resistance (MDR)11. Metal

complexes of Mn+2, Co+2, Ni+2, Cu+2 and Cd+2with bis (acetophenone)

ethylene di-amine and 5-chloro-salicylidene-aniline or its 5-bromo

salicylidene-aniline derivative have been reported12.

Inorganic and bio-chemistry describes the mutual relationship between these

two sub-disciplines, with focus upon the function of inorganic “substances“ in

living systems, including the transport, speciation and eventually,

mineralization of inorganic materials and including the use of in organics in

medicinal therapy and diagnosis. These “substances” can be metal ions (such

as K+, ferrous and ferric), composite ions (e.g. molybdate), coordination

compounds (like cisplatin and carbonyl technetium), or inorganic molecules

such as CO, NO, O3.

Metallome

Medicinal inorganic chemistry on the one hand and bio-mineralization on the

other hand, are important integral parts. Inorganic reactions have possibly

played an important role in the formation and development of organic “life

molecules” in the pre-biotic area and from the very beginning of life on

Earth13. Chemical and elemental analysis of humans in vivo using improved

body composition models showed that about 99% of mammals' mass are the

elements carbon, nitrogen, calcium, sodium, chlorine, potassium, hydrogen,

phosphorus, oxygen and sulfur. The organic compounds (proteins, lipids and

carbohydrates) contain the majority of the carbon and nitrogen and most of

the oxygen and hydrogen is present as water. The entire collection of metal

containing bio-molecules in a cell is called the metallome14. Clinical uses

In the process, mystical/magical use of metals in medicine has given way to

rational design and testing of new metallo-pharmaceutical compounds.

Presently the application of metal ions includes bismuth compounds, lithium

carbonate and cis-platin in clinical use.

28

“Metals in medicine” also covers a wealth of newer diagnostic and therapeutic

compounds, such as gadolinium-based contrast agents for magnetic

resonance imaging (MRI), samarium-based compounds for relief of bone pain

in intractable cancer and lanthanum carbonate for the treatment of

hyperphosphatemia. Advances in our understanding of bioinorganic chemistry

and increasingly sensitive analytical instrumentation enable an accelerated

pace of development in medicinal inorganic chemistry15. The asserted

biological relevance of metal ions and complexes led to their wide applications

in the branch of medicinal inorganic chemistry.

Novel bioactive materials − Bone substitute

Some ceramics, such as Bio-glass, sintered hydroxy apatite and glass-

ceramic A-W, spontaneously bond to living bone. They are called bioactive

materials and are already clinically used as important bone substitutes,

compared with human cortical bone development of bioactive materials with

improved mechanical properties has been developed.

It is induced by functional groups, such as Si–OH, Ti–OH, Zr–OH, Nb–OH,

Ta–OH, –COOH, and PO4H2. These fundamental findings provide methods

for preparing new bioactive materials with different mechanical properties.

Tough bioactive materials can be prepared by the chemical treatment of

metals and ceramics by NaOH and heat treatments of titanium metal, titanium

alloys and tantalum metal and by H3PO4 treatment of tetragonal zirconia. Soft

bioactive materials can be synthesized by bioactive silica or

polydimethylsiloxane or polytetramethyloxide, at the molecular level to form

an inorganic–organic nano-hybrid. Metallic materials have been widely used

for these applications because of their good mechanical properties and

biocompatibility. However, none of these metallic materials can directly bond

to living bone16.

Industrial Applications − Biomolecules The chemical industry is now turning more and more to enzymatic and

fermentation processes in order to obtain enantiomerically pure amino acids,

amino alcohols, amines, alcohols and epoxides as intermediates for the

pharmaceutical industry and agro chemistry, where both a high degree of

purity and large quantities of compounds are required17.

29

The major advantage of whole living cells over isolated NAD(P)H-dependent

carbonyl reductases for use in reduction processes is that the cells regenerate

their own cofactors. Further, they are easy to produce and handle and are of

relatively low cost. This review focuses on the potential of whole cells of living

organisms, mainly yeast cells, to reduce acetophenones and α,β-unsaturated

carbonyl compounds (aldehydes and ketones) furnishing relevant chiral

building blocks for fine chemicals and the pharmaceutical industries18.

Dinuclear Schiff base complexes of copper+2ions were used successfully in

hydroxylation of phenol19. Co (salen) and its analogues, (I, II, III, IV and V)

have been used for catalysing the oxidation of phenols and alcohols with

dioxygen as oxidant (VI)20. The pronounced biological effect of

organometallics led to their wide applications in fields like medicine and

agriculture .The synthesized complexes may have great value of medicines.

Therefore, it was planned to study the bioactivities of the organic ligand and

some of its metal complexes in this work.

(I) (II) (III)

(IV) (V) (VI)

30

Section I Synthesis, Thermal and Electrochemical study of Metal

Complexes. 2. 1. 1 Introduction Investigations of the bioactive metal complexes are interesting in various

industries like medicine, pharmaceutical etc. It is due to the aspects on the

widely used therapy of different states of anemia as well as in metabolism of

disorder. A new era of investigation is on the catalytic activity of various types

of organo metallic complexes.

In the field of bio-coordination chemistry, investigations are based on the

synthesis and characterizations of different metal complexes of ligands that

are present in biological systems, or synthetic ligands, which will serve like the

model-molecules for complex bio-molecular structures.

The complex compounds of bio-metals with different types of drugs are the

most promising tool for introducing the required metal into the body. The

application of anti-inflammatory agents as complexes with some bio-metals

decreases their toxicity and increases their therapeutic effect anti-ulcerogenic,

cytotoxic and other helpful properties, unusual to non-complexed agents,

appear to understand the roles of these metal ions in biological systems. To

know the coordination chemistry along with structure and bonding of metal

ions in their active sites by applying new analytical approaches21.Metal ions

bind to ligands via interactions that are strong and selective. Some stereo

selective and stereo specific complexes are of importance. The ligands impart

their own functionality and can tune properties of the overall complexes that

are unique from those of the individual ligand or metal. Metal ions change

their oxidation states are investigated by cyclic voltametry. The

thermodynamic and kinetic properties of metal-ligand interactions influence

ligand exchange reactions22.With this rationale the present work on the metal

complexes of the synthesized bio- active ligand has been proposed.

2. 1. 2 Review of Literature Heterocyclic base adducts of copper+2 complexes have been synthesized by

the reaction of copper+2 chloride with 5-chloro-2-hydroxy acetophenone N(4)

31

methyl thiosemicarbazone in presence of heterocyclic base like pyridine (py),

2,2’-bipyridine (bipy), 1,10-phenanthroline (Phen), α/β-picoline23. A series of

homo- and heteropolynuclear copper+2 complexes of N,N″-bis[1-biphenyl-2-

hydroxyimino-2-(4-acetylanilino)-1-ethylidene]-diamines had been prepared

and characterized by different physical techniques24.

The coordination chemistry of nickel metal complexes with salen-type ligands

has achieved a special status25-28. Different substituted hydroxy

acetophenone and quinoline carbaldehyde by Claisen Schmidt condensation

give 1-[substituted aryl] -3‐substituted hetero aryl] ‐2‐propene‐1‐ones,various

types of chalcones, which with Cu+2, Ni+2 , Co+2, Mn+2 and Zn+2 produce

complexes ,exhibited improved bioactivity than respective chalcones29. 2-

Hydroxy-4-methacryloxyacetophenone-semicarbazone with the polymer metal

chelates of Cu+2 and Ni+2 of poly (HMAPS) had been synthesized30. 2-

Hydroxy acetophenone, with 4-chloro-2, 6-diaminopyrimidine in the presence

of Cu+2 , Ni+2 , Mn+2 , Co+2 and Vo+4 ions result with Schiff base condensation

complexes. These mononuclear complexes were characterized by physico-

chemical techniques having biological applications31.

The conventional and microwave synthesis, physico-chemical

characterization and bio-inorganic studies of Schiff bases involving methyl

isobutyl ketone with 2-amino-4-chlorophenol and 2-hydroxy acetophenone

with isonicotinic acid hydrazide and their metal chelates with Ni+2 and Cu+2

had been reported32.A series of Cu+2, Co+2 and Ni+2 complexes with the 1-(2- -

hydroxyphenyl)-3-phenyl-2-propen-1-one, N2-[(3,5-dimethyl-1H-pyrazol-1--

yl)methyl] hydrazone ligand, C21H22N4O (LH), were synthesized by the

reaction of 1-(2-hydroxyphenyl)-3-phenyl-2-propen-1-one, hydrazone with

(3,5-dimethyl-1H-pyrazol-1-yl)methanol33.

2. 1. 3 Present Work Three novel complexes (compounds 2 − 4) are synthesized in the present

study by carrying out the reaction of Cu+2, Ni+2 and Co+2 chlorides with the

synthesized bio-active molecule (compound1).Structures have been

determined by the appraisal of UV, IR, LC-MS, AAS, magnetic susceptibility

and CV. The study of thermal properties such as order of reaction, energy of

32

activation and kinetics is furnished with the help of Thermo gravimetric

Analysis – Differential Thermal Analysis (TGA – DTA) .The data obtained

from thermal analysis is provided using Coats and Redfern Equation and

computer programme.Irreversible and reversible electrochemical / redox

behavior and electron transfer along with rate constant have been evaluated

for the first time with help of cyclic voltammetry technique. Antioxidant

potential of synthesized complexes has been reported (Section II).

2. 1. 4 Results and Discussion

Compound 2 The reaction of compound 1 with an ethanoic solution of copper (II) chloride

dihydrate (CuCl2.2H2O) in 1:2 mole ratio resulted in light green crystalline

crude solid. By the treatment of selective solvents and washings as by usual

protocol method by purification it yielded compound 2, a copper complex of

the ligand, hydroxy acetophenone derivative, compound 1.

Chemical ionization MS (LC-MS) spectrum of compound 2 (Fig 1) shows

molecular ion peak at m/z 449.The mass data indicates the molecular formula

to be C8H6NO4 Cu2Cl4. In order to study the binding mode of ligand in the

complexes IR has been studied.

IR spectrum of the free ligand is compared with the IR spectrum (Fig 2) of

complex .Peaks detected at 3612-3100 cm-1 is due to hygroscopic nature of

the complex. A sharp band of nitro group at 1543 cm-1 allocated to ligand is

broadened and merged in aromatic region which is noticed as a broad peak at

1566 cm-1 due to aromatic stretching frequency. The strong bands at 1320

cm-1 and 1312 cm-1 are seen in ligand for nitro group in its symmetric

stretching is found to shift to 1333 cm-1 suggesting coordination must be

through nitro group. The finger print stretching pattern at 763 cm-1 to 532 cm-1

has been completely changed in complex with excess frequencies due to

presence of co-ordinated chlorine atoms of metal salts. .Additional

frequencies are seen for Metal- Ligand bonding at 494 cm-1 & 455 cm-1.

33

UV- visible spectrum depicts a band with λmax = 796 nm (Fig 3) The stronger

evidence for John –Teller effects in transition metal compounds brings to light

on structural studies of solids containing d9Cu2+ ion complexes. Crystal field

parameters cannot be evaluated from UV-Vis spectra. Due to these

observations Tanable Sugano Diagrams cannot be reported. This indicates

that there must be some type of distortion by either elongation or compression

which leads to destabilization of Cu (II) complex. Experimental measurements

displayed that the distortion by elongation along Z axis.

In measurement of magnetic susceptibility of copper complex it gets

repelled by magnetic field indicating diamagnetic properties. There may be

pairing of unpaired electrons of two Cu ions. Copper complex is green in

colour because of a single absorption band in the region of 1100- 1200 cm-1.

M

Cl

Cl

Cl

Cl

L M

Compound 2 (M2LCl4), [M = Cu]

4-hydroxy-3-Nitro-acetopnenone Cu (II)

di µ Chloro dichloro copper (II)

Compound 3

The reaction of compound 1 with an ethanoic solution of nickel (II) chloride

hexahydrate (NiCl2.6H2O) in 1:2 mole ratio resulted in fluorescent light green

crystalline crude solid. By the treatment of selective solvents and washings as

by usual protocol method by purification it yielded compound 3, a Nickel

complex of the ligand, hydroxy acetophenone derivative, compound 1.

CI-MS (LC-MS) spectrum of compound 3 (Fig 4) depicted molecular ion peak

at m/z 493.The mass data indicates the molecular formula to be C8H12NO7

Ni2Cl4. In order to study the binding mode of ligand in the complexes. IR

spectrum of the free ligand is compared with the IR spectrum (Fig 5) of

34

complex .Peak detected at 1543 cm-1 of nitro group has been changed as it

shows significant decreasing frequency with its changed stretching pattern at

1520 cm-1 & 1500 cm-1.A very strong peaks at 1320 cm-1 and 1312 cm-1 have

been converted into 1344 cm-1for nitro group in its symmetric stretching. An

intense peak at 876 cm-1, 841 cm-1 & 822 cm-1are the characteristic

frequencies of coordinated water molecules.

Presence of coordinated water molecules is also confirmed from TGA

examinations. The finger print stretching pattern at 768 cm-1 to 552 cm-1

has been completely changed in complex with excess frequencies due to

presence of co-ordinated chlorine atoms of metal salts .Some more additional

frequencies are analyzed which are indicative for Metal- Ligand bonding at

503 cm-1 and 438 cm-1.

UV- visible spectrum illustrates a band with λmax = 430 nm (Fig 6) .Crystal

field parameters are evaluated from UV-Vis Spectra and Tanable Sugano

Diagrams are denoted (Table 2).

In measurement of magnetic susceptibility of Nickel complex Ni = d8 system

has T type ground term. It proves difference between effective magnetic field

(µ) calculated and observed indicates orbital contribution to magnetic

moment. µ observed = 3.89 BM and µspin only = 2.83 BM has two unpaired

electrons in complex with tetrahedral geometry.

M

Cl

Cl

Cl

Cl

L M3 H2O

Compound 3 [M2LCl4] 3H2O, [M = Ni]

4-hydroxy-3-Nitro-acetopnenone Ni (II),

di µ Chloro Nickel (II) chloride trihydrate

35

Compound 4 The reaction of compound 1 with an ethanoic solution of cobalt (II) chloride

hexahydrate (CoCl2.6H2O) in 1:2 mole ratio resulted in blue crystalline crude

solid. By the treatment of selective solvents and washings as by usual

protocol method by purification it yielded compound 4, a cobalt complex of

the ligand, hydroxy acetophenone derivative, compound 1. Chemical ionization MS (LC-MS) spectrum of compound 4 (Fig 7) shows

molecular ion peak at m/z 547.The mass data indicates the molecular formula

to be C8H10NO6Co2Cl6. In order to study the binding mode of ligand in the

complexes IR has been studied.

IR spectrum of compound 4 (Fig 8) shows peaks at 3488 cm-1 and 3195 cm-1

indicating the presence of water molecules which is confirmed by TG analysis.

Frequency detected at 1543 cm-1 of nitro group has been changed as it shows

significant decreasing frequency with its changed stretching pattern at 1520

cm-1 & 1500 cm-1. Very strong peaks at 1320 cm-1 &1312 cm-1 have been

converted into 1329 cm-1for nitro group in its symmetric stretching. The finger

print stretching pattern at 763 cm-1 to 532 cm-1 has been completely changed

in complex with excess frequencies due to presence of co-ordinated chlorine

atoms of metal salts. Some more additional frequencies are analyzed which

indicative for Metal- Ligand are bonding at 494 cm-1 and 455 cm-1.

UV- visible spectrum illustrates a band with λmax = 430 nm (Fig 9) .Crystal

field parameters are evaluated from UV-Vis Spectra and Tanable Sugano

Diagrams are denoted (Table 2).

Magnetic susceptibility of cobalt complex (compound 4) system has T type

ground term. It shows difference between effective magnetic field (µ)

calculated and observed indicates orbital contribution to magnetic moment.

µ observed = 5.012 BM and µspin only = 4.9 BM has four unpaired electrons in

complex with octahedral geometry. Paramagnetic nature of the compound 4

was examined by Gouy method. The structure is reported.

36

M

Cl

Cl

Cl

Cl

L M

Cl

ClH2O

H2O

Compound 4 [M2LCl6 2H2O], [M = CO]

Aqua-Chloro-4-hydroxy-3-Nitro-acetopnenone Co (III)

di µ Chloro aqua tri Chloro Cobalt (III)

2. 1. 5 Physical properties and Analytical data All the metal complexes synthesized are coloured solids, stable at room

temperature and can be stored for long periods. They are soluble in common

organic solvents such as dimethyl formamide, dimethyl sulphoxide,

acetonitrile and methanol. The ligand was prepared as described in the

experimental part, crystallized and dried in air and focused to physical and

spectral analysis. Analytical and physical data of compound 1, 4 - hydroxy –

3 - nitro-acetophenone with its metal complexes are presented (Table 1).

The complexes are formed in 1:2 ratios of the ligand and metal. The metal

content of the complexes was determined by subjecting the samples to

Atomic Absorption Spectroscopy. These results were compared with the

residue from thermo gravimetric analyses. The analytical data of the

complexes are consistent with the proposed molecular structures assuming

molar metal to ligand ratios of 1:2.The magnetic moment of the complexes

was measured at room temperature. Both nickel and cobalt complexes have

paramagnetic character while copper have diamagnetic character.

2.1.6 Evaluation of Crystal Field parameters from UV-Vis Spectra and Tanabe Sugano Diagrams The six coordinated high spin Cobalt (III) complex compound 4 shows only

one transition 5T2g → 5Eg and it is sensitive to John Teller distortion. It exhibits

a spectrum with split bands at 613 nm (16313 cm-1) and 658 nm (15198cm-1).

Here 10Dq taken as 15755.5cm-1 mean position of split bands.

37

Consider λ for CO (III) 145 cm-1 in weak octahedral field and 10Dq value taken

from UV-Vis spectrum of cobalt complex. µeff = 5.08BM and mean frequency

at 635.5nm gives brown colour to the complex.

Nickel complex has absoption bands at 9328 cm-1,9990cm-1 and 11820cm-1

etc. The energy level diagram of tetrahedral nickel (II) (d8) should be like that

of octahedral d2 configuration. The three transitions are expected as,

3T1 → 3T2 (1ע) E = 8Dq …………………………..Eq.1 3T1 → 3A2 (2ע) E = 18Dq …………………………Eq.2 3T1 → 3T1 (P) (3ע) E = 6Dq + 15B …………………...Eq.3

From the following equation,

9328 cm-1 + 9990 cm-1

29659 cm-1

The value 9659 cm-1 is obtained .It is too high for tetrahedral complex

because 1ע transition for [Ni (H2O) 6]2+ appears at 8500/ cm34. As Δt = 4/9 Δ0

Where, Δt is crystal field stabilization energy for tetrahedral complex and Δ0

is crystal field stabilization energy for octahedral complex.

Therefore, 9659 cm-1 taken as 2ע,

cm-1 = 18Dq 9659 = 2ע

The value of Dq = 536.6 cm-1.

Substituting the value of Dq in equation no. 3 gives the value of B .11820 = 3ע

i.e. 573 cm-1. β = B/B0 , Substituting the value of B and B0

B 573 β' = -------- = ----------- B0 1041 β' = 0.55 Nephelauxetic Ratio

B = Interelectronic Repulsion parameter in complex

B0 = Interelectronic Repulsion parameter in free Ni2+ion.

β‘ the Nephelauxetic Ratio is always less than one and it decreases with

increasing delocalization. Therefore the complex has more covalent

character. The Crystal Field parameters of the complexes calculated using

Tanabe Sugano Diagram and energy ratio are reported (Table 2).

38

2.1.7 Non isothermal Thermogravimertic Analysis The non isothermal Thermo gravimetric curves for solid phase thermal

decompositions of Cu (II), Ni (II) and CO (II) complexes of compound 1 are

presented. Cu and Ni complexes decompose in two steps while Co complex

decomposes in three steps.

The thermal kinetic parameters such as order of reaction η’ and energy of

activation ‘Ea’ were determined from computer programme of rising

temperature expression of Coats and Redfern35-35a. The order of reaction in

the expression was varied until a straight line was obtained from the plot of,

{1-(1-α) 1-n} 1 In -------------- Vs --- {(1-n) T2} T

The criterion for straight line is closeness of correlation coefficient ‘r’ to

positive or negative 0.999. Slope of the line gives energy of activation as,

Ea = Slope x R, where R is Gas constant (8.314 J/deg/mol).The details of this

analysis are discussed.

Compound 2 The TG of compound 2 (Fig 10) demonstrates the mass loss as a function of

temperature which is seen in two steps. In the first step the decomposition of

the molecule up to temperature 110 0C occurs with % weight loss 23.83

(observed) and 23.65(calculated) with removal of three chlorine atoms.

The second step of decomposition shows % weight loss 64.80(observed) and

64.80(calculated) in the temperature range 110-6500C corresponding to the

removal of one chlorine atom and the organic ligand C8H6NO2.

Residue at 6500C is 35.85% (observed) and 35.43 %(calculated) as 2CuO

gives composition of metal complex M2LCl4 was observed. The complete

decomposition of the complex is confirmed by constant platue after 6500C.

The kinetic data for step I & step II given in (Table 3 & 4) and their kinetic

plots are presented in (Fig 11 & 12).The kinetic parameters are reported in

(Table 5).

39

Compound 3 Similarly TG of compound 3 displays in (Fig 13) the mass loss as a function

of temperature which is seen in two steps. In the first step the decomposition

of the molecule up to temperature 217 0C occurs with % weight loss 39.7

(observed) & 39.69(calculated) with removal of four chlorine atoms and loss

of three water molecules. The second step of decomposition confirms %

weight loss 72.75 (observed) and 72.96(calculated) in the temperature range

217-5300C corresponding to the loss of one organic ligand C8H6NO2.Residue

at 5300C is 30.41% (observed) and 30.28 %(calculated) as 2NiO gives

composition of metal complex [M2LCl4] 3 H2O was observed that complete

decomposition of the complex is confirmed by constant platue after 5300C. The kinetic data for step I & step II given in (Table 6 & 7) and their kinetic

plots are presented in (Fig 14 & 15) respectively. The kinetic parameters are

reported in (Table 8) .

compound 4 Similarly TG of compound 4,showin in (Fig 16) exhibits the mass loss as a

function of temperature which is seen in three steps. In the first step the

decomposition of the molecule up to temperature 100 0C occurs with % weight

loss 19.2 (observed) & 19.45(calculated) with removal of three chlorine

atoms. The second step of decomposition confirms % weight loss 36.13

(observed) and 35.72 (calculated) in the temperature range 100-2050C

corresponding to the loss of two chlorine atoms and one water molecule.

The third step of decomposition confirms % weight loss 73.07 (observed) and

72.58 (calculated) in the temperature range 205-5250C corresponding to the

loss of one chlorine atom , one water molecule and a part of organic ligand as

C8H6NO2. Residue after 5300C is 26.91% (observed) and 27.59 %

(calculated) as 2CoO gives composition of metal complex [M2LCl62H2O]. The

kinetic data for step I ,II & III are given in (Table 9,10 & 11) and their kinetic

plots of steps I ,II & III are presented in (Fig 17,18 & 19).The kinetic

parameters are reported in (Table 12) .

All the observed values for decomposition of each step are in superior

conformity with the calculated values. The final decomposition products of all

the complexes (compound 2 - 4) are in accordance with calculated

40

percentage reside. First time the kinetic parameters such as the reaction

order and activation energies of metal complexes are determined.

Decomposition of metal complexes starts at approximately 450C where as that

of ligand starts at 100 0C.This data suggests that metal complexes are less

stable than ligand. Copper complex, compound 2 have less activation energy

than ligand and Nickel and Cobalt complexes. Therefore copper complex is

more active than Nickel and Cobalt complex, which is in accordance with the

bioactivity results (Section II of this chapter). Activation energy of water

molecule in Nickel complex, compound 3 is less as compared to cobalt

complex and evolved at lower temperature than that in cobalt complex.

Therefore, water molecules in Nickel complex are adsorbed water molecules

and they are outside the coordinated sphere while water molecules in cobalt

complex compound 4 are coordinated to cobalt.

2.1.8 Electrochemical Studies Cyclic Voltametry

The electrochemical behavior of complexes is studied in aprotic solvent. The

cyclic voltammograms of solvent acetonitrile are presented at various scan

rates are presented (Fig 20).To get accurate measurements of ip current CVs

of solvents are tested for residual current prior to use, to avoid errors due to

dissolve oxygen or other redox impurities. The CV waves are corrected for

solvent background using Gutmann’s correction36.The redox peak potentials

for complexes, compound 2-4, are presented (Table 13, Fig 21 - 23).

Mechanism of reaction

In CV the reversible heterogeneous charge transfer reaction showing ideal

Nernstian reversible mechanism should posses following characteristics 37-38.

I. For reversible wave in SEV (Stationary electrode voltammogram) EP is

independent of scan rate (v) and peak current , ip is proportional to v1/2 where

the peak current is defined by Randles39 and Sevcik40.

II. The diffusion coefficient of oxidized (Do) and reduced (Dr) forms must be

same.

III. For chemically reversible system, ΔEP = Epa - Epc = 0.059/n

41

Where n = no. of electrons transferred and Epa and Epc are the anodic and

cathodic peak potentials in volts respectively.

In this reaction the potential midway between the two peak potentials is formal

electrode potential (corrected for reference electrode being used) of the redox

couple. Epa + Epc E0’ = -------------- 2 IV. Plot of ipa and ipc Vs v1/2 should be linear with intercepts at the origin for

the reversible couple.

V. ipa and ipc should be similar in magnitude, with no kinetic complications so

that ipa/ipc ~ 1.

VI. The current function Ψp ~ 0.466 which is dimensionless and is

proportional to concentration C0 and scan rate v.

Ψp = ip/n F A D ½ C0 (nFv/RT)1/2

Where , ip = Peak current in (amperes)

F = Faraday constant = 96500 Coulombs

A = Electrode area cm2

D = Diffusion coefficient (cm2/s)

C0 = Concentration of redox species (mol/cm3)

n = no. of electron in step

v = scan rate per sec

R = Gas constant (1.9872 Cal K-1 mol -1 )

T = Absolute temperature (Kelvin)

The quasi –reversible systems were first exampled by Matsuda and Ayabe41

in which ip may not be proportional to v1/2 and peak parameters ip,Ep,Ep/2 are

functions of α , the charge transfer coefficient. The expression of current

function of quasireversible reaction should be same as that of reversible

reaction with modification of number of electrons ‘ n’ by apparent number of

electrons with transfer coefficient Viz, α n a. Matsuda etal41had used Ψ(E)

notation for current functions. Ψ and Ψp according to Bard etal42 relates to

heterogeneous rate constant of charge transfer K0 as follows :

Ψ = (RT/π n F v D )1/2 K0

42

In Quasireversible reaction for rate constant Kf and Kb notation may be used

in case of forward and backward reactions. Both of them must be of same

order of magnitude over most of the potential range.

Electrochemical irreversibility is caused by slow electron exchange of the

redox species with the working electrode. If Kf >> Kb for the cathode peak and

Kb >> Kf for irreversible character.

Irreversibility of charge transfer reaction is specified by a separation of peak

potentials (ΔEP ) that is greater than 0.059/n volts and that is increasing with

increasing v , here ΔEP corresponds to Epa- Epc .

CV used to detect and characterise coupled chemical reaction, Nicolson and

Shain43,43a had presented types of electrode processes coupled with chemical

reactions which are depending on ratios of ipa/ipc.

CV of copper complex (Compound 2) Copper complex of the compound 1 shows two reduction peaks Epc1 and Epc2

seperated sufficiently from each other indicates a chemical reaction coupled

between two charge transfers ErCrEr mechanism43.Results are reported (Table 14 & 15 ).

Epc1 = o.2 v,Epc2 = -1.2 v ,Ipc1 = 0.93 x 10-5 Å and Ipc2 = 1.07 x 10 -5 Å

Epa1 = 0.8 v,Epa2 = 0 v, Ipa1 = 0.4310 -5 Å and Ipa2 = 1.78 10 -5 Å

Ipa/Ipc Vs scan rate variation plot (Fig 24) shows b type of mechanism for

both electron transfers i.e. ErCrEr. Ko values for first and second redox peak

is 5.2 x 10 -7 cm/s and 8.25 cm/s respectively .It explains slow quasi reversible

electron transfer between which there is a reversible chemical reaction

ErCrEr. Results are reported (Table 16 & 17 ).

CV of Nickel complex (compound 3)

It confirms two Epc1 and Epc2 values 0.9 v and -0.8 v respectively ,separated

from each other sufficiently with large voltage shows EirCEr mechanism

where the irreversible electron transfer rate is constant is 4.2 x10-1 cm/s.

Therefore fast electron transfer occurs and corresponding Epa1 value is not

seen at all scan rates. Irreversible electron transfer but Epa2 is at -0.5v and

EPC2 is -0.8v.ΔEp2 is -0.3v. Hence, it is a Quasireversible electron transfer. Results are reported (Table 18 ).

43

CV of Cobalt complex (compound 4) Epc and Epa values 0.95 v and -0.8 v .It shows b type (Fig 25) of electron

transfer CrEr. A reversible chemical reaction followed by a reversible electon

transfer. Rate constant 1.81 x 10-7 cm/s shows sluggish electron transfer. Results are reported (Table 19 & 20).

2.1.9 Conclusion In this study, Cu (II), Ni (II) and Co (II) complexes have been synthesized and

characterized by using various spectroscopic methods. Their thermal and

electrochemical studies are performed to get important parameters. The

values of different energies connected with the complexes and their redox

potentials are the facts to the stability and active nature of the compounds.

2.1.10 Experimental Compound 2-4

The ethanolic solution of compound 1 (0.001 mol) was added slowly with

constant stirring to the ethanolic solution of Copper (II) chloride

dihydrate,Nickel (II) chloride hexahydrate and cobalt (II) chloride hexahydrate

(0.002 mol) separately. The colour of the resulting solution changes from

bottle green to light green,fluroscent green and blue respectively for Cu (II),Ni

(II) and Co (II) . The resulting mixture was refluxed for one and the half hour.

The solvent was removed under reduced pressure.The residue was triturated

with chloroform.Chloroform soluble part was separated. Residue after

washing with diethyl ether,ethanol yielded compounds 2 ,3 and 4.

Melting point/s decomposition temperatures of these compounds were

recorded/ and TLC was recorded. The IR and UV absorption spectra of

compounds 2-4 were obtained by using ‘Schimadzu FTIR 3600

spectrophotometer’ and ‘UV-Vis 1700 spectrophotometer’. LC-MS spectra

were recorded on LC-MS-MS Perkin Elmer Applied Bio-systems SCIEX-2000

at room temperature. Cyclic voltammograms of the compounds 2-4 were

recorded in acetonitrile solution used as solvent at 300K on ‘CHH Instrument

44

Element Analyzer’ which is composed of three – electrode cell. Here internal

standard comprises as a reference electrode Ag/AgCl and auxillary Pt with

working Pt electrodes. Electrochemical behavior of compound 1 was studied

in aprotic solvent Acetonitrile at room temperature. Voltammograms of

acetonitrile were recorded to eliminate the effect of solvent.

Thermogravimertic experiments were carried out from room temperature to

7000C in air at heating rate of 30C/min. following the same procedure as

mentioned previously in Chapter 1, Section II.

Magnetic susceptibilities were measured at room temperature by Guoy

method. In the Gouy method, the balance measures the apparent change in

the weight of the sample created by the sum of the diamagnetic repulsion and

paramagnetic attraction for the applied field using Hg [Co(NSC)4] as the

caliberant, which have Xg values of 1.644 x 10-5 erg . G-2 . cm-3, Diamagnetic

corrections were applied using Pascal’s constants 44.

Table 1 Analytical and Physical data of Complexes

Sr.No.

Metal

Complex Mol.Wt. Decom.Temp.(0C)

Metal Content

(%)

TGA AAS

1 Compound 2 449 250 35.85 35.40

2 Compound 3 493 250 30.28 30.39

3 Compound 4 547 250 26.91 26.20

Table 2 Crystal Field parameters of Metal complexes 34

Compound B (cm-1) B0 (cm-1) β' (cm-1) 10 Dq (cm-1)

3 573 1041 0.55 5366

45

Table 3 Kinetic data from TG of compound 2 in air atmosphere (Step I) Initial % Wt loss = 0 Final % Wt loss = 23.83

T ( Kelvin) Wt 1/T F (α)

313 4.69 0.0032 -12.99

323 7.34 0.0030 -12.60

334 9.98 0.0029 -12.31

356 16.60 0.0028 -11.74

368 19.25 0.0027 -11.55

379 20.57 0.0026 -11.47

380 23.20 0.0025 -11.13

Table 4 Kinetic data from TG of compound 2 in air atmosphere (Step II) Initial % Wt loss = 23.83 Final % Wt loss = 64.80

T ( Kelvin) Wt 1/T F (α)

389 2.04 0.0025 -14.85

522 4.69 0.0019 -14.50

562 6.01 0.0017 -14.34

662 8.66 0.0015 -14.19

761 11.31 0.0013 -14.07

864 13.96 0.0011 -13.97

920 15.28 0.0010 -13.91

Table 5 Data from Dynamic TGA for compound 2 (Air atmosphere)

Step Temp.range

(0C)

% wt.

Loss

Observed

% Wt.

Loss

Calculated

Loss of

probable

moiety

Order

of

reaction

(n)

Ea

(KJ/mol)

I 40-110 23.83 23.65 3 Cl 0.7 21.73

II 110-650 64.80 64.58 1Cl+

C8H6NO2

2.9 5.30

46

Table 6 Kinetic data from TG of compound 3 in air atmosphere (Step I) Initial % Wt loss = 0 Final % Wt loss = 39.70

T ( Kelvin) Wt 1/T F (α)

320 4.81 0.0030 -13.62

345 7.45 0.0029 -13.23

362 11.42 0.0028 -12.81

390 18.03 0.0026 -12.34

442 21.99 0.0024 -12.13

465 29.93 0.0022 -11.64

489 33.90 0.0022 -11.31

Table 7 Kinetic data from TG of compound 3 in air atmosphere (Step II) Initial % Wt loss = 39.70 Final % Wt loss = 72.75

T ( Kelvin) Wt 1/T F (α)

490 1.60 0.0019 -15.51

566 2.70 0.0018 -15.06

588 4.20 0.0017 -14.62

612 6.60 0.0016 -14.13

655 9.90 0.0015 -13.66

742 15.50 0.0014 -12.94

789 19.50 0.0013 -12.43

Table 8 Data from Dynamic TGA for compound 3 (Air atmosphere)

Step Temp.range

(0C)

% wt.

Loss

Observed

% Wt.

Loss

Calculated

Loss of

probable

moiety

Order of

reaction

(n)

Ea

(KJ/mol)

I 45 - 217 39.70 39.69 4 Cl + 3 H2O

1.3 20.92

II 217 - 530 72.75 72.96 C8H6NO2 2.8 48.78

47

Table 9 Kinetic data from TG of compound 4 in air atmosphere (Step I)

Initial % Wt loss = 0.0 Final % Wt loss = 19.20

T ( Kelvin) Wt 1/T F (α) 320 1.48 0.0031 -14.05 334 3.72 0.0030 -13.08 345 5.96 0.0029 -12.54 351 7.64 0.0028 -12.22 356 9.88 0.0028 -11.81 362 11.56 0.0028 -11.53

Table 10 Kinetic data from TG of compound 4 in air atmosphere (Step II)

Initial % Wt loss = 19.20 Final % Wt loss = 36.13

T ( Kelvin) Wt 1/T F (α) 390 21.07 0.002564 -13.9582 401 22.19 0.002494 -13.4255 412 24.99 0.002427 -12.4654 423 26.67 0.002364 -11.9982 440 28.91 0.002273 -11.363 445 30.03 0.002247 -10.9868 468 32.27 0.002137 -10.0679

Table 11 Kinetic data from TG of compound 4 in air atmosphere (Step III)

Initial % Wt loss = 36.13 Final % Wt loss = 73.07

T ( Kelvin) Wt 1/T F (α) 485 37.32 0.002062 -15.756 540 38.44 0.001852 -15.2611 589 40.12 0.001698 -14.8152 622 41.24 0.001608 -14.6257 679 42.92 0.001473 -14.4361 742 45.16 0.001385 -14.1578 770 46.84 0.001316 -13.9956

Table 12 Data from Dynamic TGA for compound 4 (Air atmosphere) Step Temp.range

(0C) % wt. Loss Observed

% Wt. Loss Calculated

Loss of probable moiety

Order of reaction (n)

Ea (KJ/mol)

I 45 - 100 19.20 19.45 3 Cl 1.7 62.37 II 110 - 205 36.13 35.72 2 Cl + H2O 3.0 75.76 III 205 - 525 73.07 72.58 Cl + H2O+

C8H6NO2 2.9 19.32

48

Table 13 Electro Chemical data for compound 2 - 4 in aprotic solvent at 100 mv scan rate

Compound Epc1 (v) Epc2 (v) Epc3(v) Epa1 (v) Epa2 (v) Epc3 (v)

2 0.2 -1.2 - 0.8 0 -

3 0.9 - - - - -

4 0.95 - - -0.8 - -

Table 14 Electro Chemical data for quasi reversible charge transfer in

aprotic solvent with scan rate variations for 1st redox couple (compound 2)

ν ν1/2 Ipa I pc Ipa/Ipc Ipc/ν1/2 λ Epc Epa ΔEp E1/2 τ (v/sec) (A) (A) (V) (V) (V) (V) (V) (Sec) 0.05 0.22 -0.31 0.96 -0.48 4.70 -1.40 0.25 0.75 0.50 0.5 38.00 0.10 0.32 0.43 1.40 0.93 4.43 -1.40 0.20 0.80 0.60 0.5 19.00 0.15 0.39 0.55 0.9 6.25 -0.52 -1.40 0.85 0.10 0.75 0.47 12.50 0.25 0.50 0.45 0.82 7.00 -0.50 -1.40 0.1 0.92 0.82 0.51 7.64

Table 15 Electro Chemical data for quasi reversible charge transfer in

aprotic solvent with scan rate variations for 2nd redox couple (Compound 2)

ν ν1/2 Ipa I pc Ipa/Ipc Ipc/ν1/2 λ Epc Epa ΔEp E1/2 τ (v/sec) (A) (A) (V) (V) (V) (V) (V) (Sec)

0.05 0.22 1.56 0.94 -0.48 4.70 -1.40 -1.05 -0.08 -0.97 0.56 16.70 0.10 0.32 1.78 1.07 0.93 4.43 -1.40 -1.2 0 -1.2 -0.60 8.00 0.15 0.39 2.1 1.8 6.25 1.3 -1.40 1.3 0.02 1.22 -0.68 4.80 0.25 0.9 1.45 0.82 7.00 -0.50 -1.40 1.4 0.05 1.45 0.675 2.90

49

Table 16 Ko values for various Scan rates 1st redox couple (Compound 2)

Scan rate v/s

ipc x10-5 (A)

Epc (v)

Epa (v)

ΔEp (v) αna

Dx10-10

(cm2/s) Kox10-7cm/s D 1/2 0.05 1.05 -0.25 0.75 0.50 0.11 1.64 4.32 4.05 0.10 0.93 0.20 0.80 0.60 0.09 1.49 5.83 3.86 0.15 0.55 0.85 0.10 -0.75 0.07 9.30 5.64 3.05 0.20 0.82 0.1 0.92 -0.70 0.08 4.63 5.14 2.15 Average rate constant ( Ko) = 5.23x10-7 cm/s Co = 0.001M (mol/cm-3

) A = 0.2826 cm2

Quasi reversible charge transfer Table 17 Ko values for various Scan rates 2nd redox couple

(compound 2)

Scan rate v/s

ipc x10-5 (A)

Epc (v)

Epa (v)

ΔEp (v) αna

Dx10-10

(cm2/s) Kox10-7cm/s D1/2

0.05 0.94 -1.05 -0.08 -1.1 0.05 3.04 5.89 5.51

0.10 1.07 1.2 0 -1.15 0.05 1.97 6.71 4.44 0.15 1.8 1.3 0.02 -0.5 0.11 3.72 11.28 6.10 0.20 1.45 1.4 0.55 0.55 0.10 1.44 9.09 3.80 Average rate constant ( Ko) = 8.24x10-7 cm/s Co = 0.001M (mol/cm-3

) A = 0.2826 cm2

Quasi reversible charge transfer Table 18 Ks values for various Scan rates (compound 3)

Scan rate ע(V/ s) αna Ks x10-2 (cm/s) 0.05 0.0428 5.3 0.1 0.1252 31.53 0.15 0.08887 20.85 0.2 0.01379 110.17 0.25 0.01407 86.44 Average rate const (Ks) = 4.1962x10-1 cm/s Co =0.00M (mol/cm-3), A= 0.2826 cm2 Irreversible Charge Transfer, [Eo = 0.222 v (Ag/AgCl)]

50

Table 19 Electro Chemical data for quasi reversible charge transfer in aprotic solvent with scan rate variations (compound 4)

ν ν1/2 Ipa I pc Ipa/Ipc Ipc/ν1/2 λ Epc Epa ΔEp E1/2 τ

(v/sec) (A) (A) (V) (V) (V) (V) (V) (Sec)

0.05 0.22 0.05 -1.54 -0.03 6.89 1.35 0.95 -0.70 -1.65 0.13 29.50

0.10 0.32 0.05 -0.05 -1.00 0.16 1.35 0.95 -0.80 -1.75 0.08 14.25

0.15 0.39 0.04 -0.02 -2.00 0.05 1.40 0.85 -0.75 -1.60 0.05 9.66

0.20 0.45 0.03 -0.03 -1.00 0.07 1.35 0.90 -0.75 -1.65 0.08 7.13

0.25 0.50 0.02 -0.06 -0.33 0.12 1.35 1.05 -0.75 -1.80 0.15 6.00

0.30 0.55 0.04 -0.06 -0.67 0.11 1.40 1.00 -0.75 -1.75 0.13 5.08

Table 20 Ko values for various Scan rates (compound 4)

Scan

rate v/s

ipc x10-5

(A)

Epc

(v)

Epa

(v)

ΔEp

(v) αna

Dx10-10

(cm2/s) Kox10-7cm/s D1/2

0.05 -1.54 0.95 -0.70 -1.65 0.04 0.08 9.65 0.90

0.10 -0.05 0.95 -0.80 -1.75 0.03 0.04 0.31 0.21

0.15 -0.20 0.85 -0.75 -1.60 0.04 0.05 0.12 0.68

0.20 -0.30 0.90 -0.78 -1.68 0.04 0.08 0.18 0.88

0.25 -0.60 1.02 -0.72 -1.74 0.03 0.02 0.37 0.16

0.30 -0.60 1.02 -0.75 -1.80 0.03 0.02 0.37 0.14

Average rate constant ( Ko) = 1.81x10-7 cm/s Co = 0.001M (mol/cm-3)

A = 0.2826 cm2

Quasi reversible charge transfer

Table 21 Type of reaction mechanism and redox potentials

compound Type of Mechanism Rate constant (cm/sec )

2 (1st redox potential ) ErCrEr 5.20 x 10-7

2 (2nd redox potential ) ErCrEr 8.25 x 10-7

3 EirCEr 4.20 x 10-1

4 CrEr 1.81 x 10-7

51

Spectral data of compound 2

Fig 1 LC-MS

Fig 2 IR

Fig 3 UV-Vis

52

Spectral data of compound 3

Fig 4 LC-MS

Fig 5 IR

Fig 6 UV-Vis

53

Spectral data of compound 4

Fig 7 LC-MS

Fig 8 IR

Fig 9 UV-Vis

54

Fig 10 Dynamic TGA in Air atmosphere compound 2

Fig 11 Kinetic plot of 1/T Vs f (α) from TG of compound 2 (Step I)

Fig 12 Kinetic plot of 1/T Vs f (α) from TG of compound 2 (Step II)

y = -2614.x - 4.472R² = 0.993

-13.5-13

-12.5-12

-11.5-11

-10.5-10

0.0015 0.002 0.0025 0.003 0.0035

ln[1

-(1-a

)1-n /(1

-n)T

2 ]

1/T K-1

Coats-Redfern plot

y = -637.6x - 13.23R² = 0.994

-16

-15

-14

-13

-12

-11

-10

0.0015 0.002 0.0025 0.003

ln[1

-(1-a

)1-n /(1

-n)T

2 ]

1/T K-1

Coats-Redfern plot

55

Fig 13 Dynamic TGA in Air atmosphere compound 3

Fig 14 Kinetic plot of 1/T Vs f (α) from TG of compound 3 (Step I)

Fig 15 Kinetic plot of 1/T Vs f (α) from TG of compound 3 (Step II)

y = -2517.x - 5.931R² = 0.994

-14-13.5

-13-12.5

-12-11.5

-11-10.5

-10

0.0015 0.002 0.0025 0.003 0.0035

ln[1

-(1-a

)1-n /(1

-n)T

2 ]

1/T K-1

Coats-Redfern plot

y = -5868.x - 4.594R² = 0.994

-16

-15

-14

-13

-12

-11

-10

0.0015 0.0016 0.0017 0.0018 0.0019

ln[1

-(1-a

)1-n /(1

-n)T

2 ]

1/T K-1

Coats-Redfern plot

56

Fig 16 Dynamic TGA in Air atmosphere compound 4

Fig 17 Kinetic plot of 1/T Vs f (α) from TG of compound 4 (Step I)

Fig 18 Kinetic plot of 1/T Vs f (α) from TG of compound 4 (Step II)

Fig 19 Kinetic plot of 1/T Vs f (α) from TG of compound 4 (Step III)

y = -7502.x + 9.237R² = 0.994

-15

-14

-13

-12

-11

-10

0.0015 0.002 0.0025 0.003 0.0035

ln[1

-(1-a

)1-n /(1

-n)T

2 ]

1/T K-1

Coats-Redfern plot

y = -9113.x + 9.452R² = 0.992

-15

-14

-13

-12

-11

-10

0.0015 0.002 0.0025 0.003

ln[1

-(1-a

)1-n /(1

-n)T

2 ]

1/T K-1

Coats-Redfern plot

y = -2324.x - 10.93R² = 0.994

-18

-16

-14

-12

-10

0.0015 0.0017 0.0019 0.0021

ln[1

-(1-a

)1-n /(1

-n)T

2 ]

1/T K-1

Coats-Redfern plot

57

Fig 20 Cyclic voltammograms of CH3CN at various scan rates

50mv/sec 100 mv/sec

150 mv/sec 200mv/sec

250mv/sec 300mv/sec

58

Fig 21 Cyclic voltammograms in CH3CN at various scan rates

(Compound 2 )

50mv/sec 100 mv/sec

150 mv/sec 250mv/sec

59

Fig 22 Cyclic voltammograms in CH3CN at various scan rates

(Compound 3)

50mv/sec 100 mv/sec

150 mv/sec 200mv/sec

250mv/sec 300mv/sec

60

Fig 23 Cyclic voltammograms in CH3CN at various scan rates (Compound 4)

50mv/sec 100mv/sec

150mv/sec 200 mv/sec

250 mv/sec 300mv/sec Fig 24 Plot of Reaction Mechanism for compound 2 & 4

0

1

2

3

0 0.2 0.4

ipa

/ipa

c x

1o-5

v (v/s)

1st redox potential

2nd redox potential -2

0

2

4

0 0.2 0.4

ipa

/ipc

x 1

o-5

v (v/s)

compound 4

compound 4

61

Section II Bioactivity evaluation of Metal Complexes 2. 2. 1 Introduction A methodological consideration for characterizing potential antioxidant actions

of bioactive components in plant parts has become challengeable scenario.

The study of free radicals and antioxidants in biology is producing medical

revolution that promises a new age of health and disease management. From

prevention of the oxidative reactions in foods, pharmaceuticals and cosmetics

to the role of reactive oxygen species (ROS) in chronic degenerative diseases

including cancer, autoimmune, inflammatory, cardiovascular and

neurodegenerative (e.g. Alzheimer’s disease, Parkinson’s disease, multiple

sclerosis, Downs syndrome) and aging challenges continue to emerge from

difficulties associated with methods used in evaluating antioxidant actions in

vivo45. Antioxidants are substances that delay or prevent the oxidation of

cellular oxidizable substrates.

The various antioxidants exert their effect by scavenging superoxide or by

activating a battery of de-toxifying / defensive proteins. The prevention of

oxidation is an essential process in all the aerobic organisms. The decreased

antioxidant protection may lead to cytotoxicity, mutagenicity .and/or

carcinogenicity46. 2,2’’-diphenyl-1-picrylhydrazyl (DPPH) is a stable free

radical containing an odd electron in its structure and usually used for

detection of the radical scavenging activity in chemical analysis47. The

reduction capability of DPPH radicals was determined by decrease in its

absorbance at 517 nm induced by antioxidants48.

This assay provides an easy and rapid way to evaluate the antiradical

activities of antioxidants. DPPH was used as stable free radical electron

acceptor or hydrogen radical to become a stable diamagnetic molecule 49

.Many activities of metal ions in biology have stimulated the development of

metal-based therapeutics. Metal-based chemotherapeutic compounds have

been investigated for potential medicinal applications, including superoxide

dismutase mimics and metal-based NO donors/scavengers. These

compounds have the potential to modulate the biological properties of

62

superoxide anion and nitric oxide. Thus metal complexes become as potential

therapeutics50.

2 . 2 . 2 Review of Literature Antioxidant potential of rare earth complexes, Yt+3complex and Eu+3 complex

, with naringenin-2-hydroxy benzoyl hydrazone ligand were determined by

hydroxyl radical scavenging method51.Certain flavonoid complexes inhibit the

oxidation of ascorbic acid; this behaviour has been attributed to the ability of

flavonoids to act as free radical acceptors and also to remove catalytic metal

ions by complexation52.Quercetin (3,3′,4′,5,7-pentahydroxyflavone) one of the

most abundant dietary flavonoids, has been investigated in the presence of

Cu(II) in methanol were reported antioxidant activity by using the 1,1-

diphenyl-2-picrylhydrazyl (DPPH) radical scavenging method53. Antioxidant

potential of Cr+3 , Ni+2 & Cu+2complexes of 3-aminocoumarin were reported 54.

Biological significance

Copper+2 is a biologically active, essential ion, creating ability and positive

redox potential allow participation in biological transport reactions. Cu+2

complexes possess a wide range of biological activity and are among the

most potent antiviral, antitumor and anti-inflammatory agents55. Copper atom

is an essential nutrient involved in the catalytic function of many enzymes

such as copper-zinc superoxide dismutase56-57.

Copper has an important role in the metabolism and transition of iron in the

body. Copper deficiency has been reported to cause hematologic disorders,

hypo-pigmentation, defective connective tissue cross-linking and ataxia 58-59.

Microcytic hypo chromic anemia is one of the outcomes of copper

deficiency60. Copper is an essential component of several endogenous

antioxidant enzymes and that free radicals have been proposed to play a role

in the process of carcinogenesis61. Nickel

Very small amounts of nickel have been shown to be essential for normal

growth and reproduction in some species of animals; therefore, small

amounts of nickel may also be essential to humans62.

63

Cobalt

However, trace amounts of cobalt are needed in the diet because cobalt is an

integral metal of vitamin B12 63.

Medicinal exploitation Cobalt complexes possess in vivo insulin-like properties64,antifungal 65 and

antioxidant activity66 .Binuclear Cu+2, Co+2 and Ni+2 complexes derived from

N1-ethyl-N2-(pyridin-2-yl) hydrazine-1,2-bis(carbothioamide) had been

testimonied the anti-oxidant, anti-hemolytic and cytotoxic activities of the

compounds67. The chelate complexes of bio-chanin A-(4'-methoxy-5,7-

dihydroxy-isoflavone) with copper+2and nickel+2 ions demonstrate the antiviral

,anti-cancer and antioxidant activity68.Bis(N-allylbenzimidazol-2-

ylmethyl)benzylamine (babb) and two of its complexes, [Cu(babb)(pic)2]·H2O

and [Co(babb)2](pic)2 (pic=picrate) exhibited potential antioxidant properties69.

4,4’-Bis-({2-[(2-hydroxy-phenylimino)-methyl]-benzylidene}-amino)-biphenyl-3,

3’-diol and its metal complexes (Cu, Ni and VO) were enhanced significant

DPPH activity70.

2. 2. 3 Present Work The model of the scavenging of the stable DPPH radical is extensively applied

to evaluate antioxidant activities in less time than that is required by other

methods. DPPH is a stable free radical that can accept an electron or

hydrogen radical and get converted to a stable, diamagnetic molecule. DPPH

has an odd electron and so has a strong absorption band at 517 nm, when

this electron becomes paired off, the absorption decreases stoichiometrically

with respect to the number of electrons or hydrogen atoms taken up71 .

The antioxidant potential of the complexes were measured

spectrophotometrically using a standard protocol using DPPH and nitric oxide

(NO).Taking into consideration the investigation of these chemical moieties

have been related to the antioxidant system.

.

2. 2. 4 Results and Discussion Hydrogen atom or electron-donation ability of the corresponding compounds

were measured spectrophotometrically from the bleaching of the purple-

64

colored methanol solution of 2,2-diphenyl-1-picrylhydrazyl (DPPH)72.All the

compounds [2,3 & 4] have shown encouraging antioxidant activities. The

compounds having p-hydroxy and m-nitro groups on their phenyl rings,

exhibited the remarkable scavenging capacity of active radical species.

Results are reported (Table 21 & 22).

2. 2. 5 Conclusion In conclusion it can be pointed out that Cu (II) complex is found to be more

active in both the assays. The Ni (II) and Co (II) complexes as compared to

standard are less active. Further studies are required to explore these

complexes as drugs.

Table 22 Ic50 values by DPPH assay

Ic 50 values µg/ml

Standard 3.02 Cu- complex 6.83 Ni-complex 13.03 Co-complex 20.68

Table 23 Ic50 values by NO

Extracts/

Standard

IC50 (µg/ml)

Compound 2 20.68

Ascorbic acid 13.11

2. 2. 6 Experimental The results are obtained by the standard protocol.5 mL of various dilutions of

the compounds [ 2,3 & 4] in dimethyl sulfoxide (DMSO; final concentration 0-

80 µM) were mixed with 0.5 mL of 0.004% methanol solution of DPPH. After

an incubation period of 30 min at 370C, the absorbance of the samples was

measured at 517 nm. Ascorbic acid was used as reference compound.

Inhibition of DPPH free radical in percent (I%) was calculated in following way:

65

I % = (Ablank – Asample/Ablank) x 100; where Ablank is the absorbance of the

control reaction (containing all reagents except the test compound), and

Asample is the absorbance of the test compound. Compound concentration

providing 50% scavenging (IC50) was calculated from the graph plotting

inhibition percentage against compound concentration.

Scavenging Capacity of Nitric Oxide

Nitric oxide (NO) is an abundant reactive radical that acts as an important

oxidative biological signaling molecule in a large variety of diverse

physiological processes, including neurotransmission, blood pressure

regulation, defence mechanisms, smooth muscle relaxation and immune

regulation73-76. sodium nitroprusside in aqueous solution at physiological pH

spontaneously generates nitric oxide, which interacts with oxygen to produce

nitrite ions that can be estimated using Greiss reagent77. Sodium nitroprusside

in phosphate buffer pH 7.4 was incubated with the novel compounds

dissolved in DMSO (final concentration 0-100 µM) and the mixtures were

incubated at 25°C for 120 min. Control experiment was conducted with equal

amount of solvent in an identical manner. At intervals, 0.5ml of incubated

solution was taken and diluted with 0.5 ml of griess reagent .The absorbance

of the chromophore formed during diazotization of nitrite with sulfanilamide

and subsequent N-naphthylethylenediamine dihydrochloride was measured at

546 nm. Dose dependent nitric oxide scavenging effects of the synthesized

compounds were expressed as IC50 values. Ascorbic acid was used as the

reference compound. (Fig 25 & 26).

Fig 26 DPPH assay Fig 27 Nitric Oxide assay

0

20

40

60

80

100

1 2 3 4 5

%an

tira

dica

l act

ivit

y

conc.µg/ml

DPPH Assayconc.µg/ml

Compound 4

Compound 3

Compound 2

standard

0

20

40

60

80

100

%an

tira

dica

l act

ivit

y

conc.µg/ml

NO assay

Standard

Compound 2

66

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