Chapter-2

Materials and Methods

Materials and experimental techniques employed at various stages of

investigation for the preparation of the newly synthesized ligand metal

complexes are described in this chapter. .

Synthesis of ligand(L):2-amino-4-phenyl-thiazole

Chemicals used:

5-nitro salicyaldehyde, 4-chloro phenacyl bromide, potassium tert-butoxide,

dichloromethane, hydrazine hydrate, benzaldehyde were of analytical reagent

grade procured from avra synthesis.

Synthesis of (4-chlorophenyl)(5-nitrobenzofuran-2-yl)methanone (III)

A mixture of 5-nitro salicyaldehyde (I) (2 mmol), 4-chloro phenacyl bromide (II) (2

mmol) and potassium tert-butoxide (2 mmol) in 10 mL of dichloromethane (DCM)
containing molecular sieves was refluxed at 30 0C for 3 h. Progress of the reaction
was monitored by TLC using hexane:ethyl acetate (8:2) mixture as mobile phase.
After the completion of the reaction, the reaction mixture was washed with 10% HCl
solution followed by water. The organics were dried over anhydrous sodium sulfate.
The yellow solid was obtained by desolventizing in a rotary evaporator at room
temperature affords (4-chlorophenyl)(5-nitrobenzofuran-2-yl)methanone (III).
Synthesis of ((4-chlorophenyl)(5-nitrobenzofuran-2-

yl)methylene)hydrazine (IV)

(4-chlorophenyl)(5-nitrobenzofuran-2-yl)methanone (III) (0.1mol) in 15 ml ethanol

was added to a solution of hydrazine hydrate (0.14 mol) and sodium acetate (0.14
mol) the mixture was heated at 80–90 oC for 1-2 hrs and then left to cool to room
temperature. The precipitate was collected and purified by crystallization from
ethanol to obtained compound (IV).

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 Chapter-2

Scheme 1: Synthesis of benzofuran based ligand (IV)

2.1.2. Metals Used In Complexes

Copper: Copper is one of the important transition element. It occurs in the range

of oxidation states +1, +2, +3. In cupric compounds and its ions undergo

immediate coplexation yielding variety of coordination complexes. They occupy

very top position in inorganic chemistry due to their important role in oxidation,

coupling reactins and halogenations. Oxidantion of phenol by copper oxine

complexes provide model for phenol oxidizing enzymes. Copper also involved in

many biological processes. For example Cu(DMG)2 shows some activity against

cancer and shows increasing life span, but only DMG is not active againt cancer.

This shows necessary of complexes in biological process.

Cobalt: Cobalt is an important metal with its oxidantion state +2 and +3 in its

coordination complexes. It exhibit substitution variety in nature. These facts can

be observed through their oxidation states, coordination number, geometric

structure, liability, stability as well many other aspects of their chemistry. Cobalt

compounds have some synergic effects on certain antibiotics notably Penicillin.

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 Chapter-2

Being a vital element in animal nutrition, cobalt and its biological importance has

been reviewed by Nicholls.

Nickel:Nickel is a chemical element with symbol Ni and atomic number 28. It is a

silvery-whitelustrous metal with a slight golden tinge. Nickel belongs to the

transition metals and is hardand ductile. Pure nickel, powdered to maximize the

reactive surface area, shows a significantchemical activity, but larger pieces are

slow to react with air under standard conditions because anoxide layer forms on

the surface and prevents further corrosion (passivation).

Even so,pure native nickel is found in Earth's crust only in tiny amounts,

usually in ultramafic rocks. [4][5] andinthe interiors of larger nickel–iron

meteorites that were not exposed to oxygen when outside

Earth'satmosphere.Meteoric nickel is found in combination with iron, a

reflection of the origin of those elements as majorend products of supernova

nucleosynthesis. An iron–nickel mixture is thought to

composeEarth's inner core. Nickel is a silvery-white metal with a slight

golden tinge that takes a high polish. It is one of only fourelements that are

magnetic at or near room temperature, the others

beingiron, cobalt and gadolinium. Its Curie temperature is 355 °C (671 °F),

meaning that bulk nickel isnon-magnetic above this temperature. [9] The unit

cell of nickel is a face-centered cube with the latticeparameter of 0.352 nm,

giving an atomic radius of 0.124 nm. This crystal structure is stable topressures

of at least 70 GPa. Nickel belongs to the transition metals and is hard and ductile.

Manganese: Manganese is the member of the first transition series having the

atomic number 25, having variable oxidation states from +2 to +7; most stable

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 Chapter-2

among them is +2 since 3d subshell is half filled. The electronic configuration is

[Ar] 3d5 4s2. It is an important nutrient in all the forms of life. Manganese plays

very important role in photosynthesis in oxygen evolution in chloroplast. It is

also found in various enzymes tranferases, hydrolases, isomerases, superoxides ,

superoxide dismutase etc..,

2.3.1. Preparation of benzofuran fused metal complexes: (BM1-BM4)

Ethanolic solution of ligand (3) (2 mmol) was slowly added to ethanolic solution
of MCl2.nH2O (1 mmol, where n = 0, 1, 2, 3…) [Metal= CuCl2, NiCl2, CoCl2, MnCl2]
and the mixture was kept on a water bath at 70 oC with continuous stirring for
one hour. The solid complex formed was filtered and washed with ethanol then
with ether.

Metal= CuCl2, NiCl2, CoCl2, MnCl2 X=Cl

Figure2.1. General structure synthesized benzofuran fused metal complexes

(BM1-BM4).

Physico-chemical Techniques

Infrared spectral measurements (IR)

Infrared spectral measurements were made for the prepared compounds

using a Jasco FT-IR-4100 type double beam spectrophotometer. Infrared spectra

of the compounds were recorded by Nujol mull method. Generally a composition

of the infrared spectrum of the organic compounds is useful to find out the

functional groups attached to the central organic nucleus ring. Complete analysis

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 Chapter-2

of the spectra has not been attempted. However, attention has been focused on a

limited number of bands, which provide considerable structural information in

order to suggest the most probable manner of bond formation of the functional

groups.

Electronic spectra measurements (ESR)

The electronic spectra of the complex in the visible region were recorded

in ELICO SL 117 scanning mini spectrophotometer provide with the quartz cell.

The working range of the spectrophotometer is 900-400 nm. The spectra of the

complexes were recorded using DMF/DMSO as the solvent. The electronic

spectral measurements were used for assigning the stereochemistries of metal

ions in the complexes based on the positions and number of d-d transition peaks.

Electrical conductance measurements

The electrical conductance measurements of the synthesized complexes

were done to know whether the anions of the metal salts remain inside or

outside the coordination sphere of the central metal ion. The conductance data

were recorded in 10-3 M DMF solutions at room temperature using an Elico Cm-

180 conductometer. The cell constant of the conductivity cell used was 0.5 cm-1.

Magnetic susceptibility measurements

The magnetic susceptibility measurements of the complexes were

carried out in order to find out the effective magnetic moment per each metal in

the complexes. The number of unpaired electrons possessed by the metal ion can

be determined from the effective magnetic moment of the metal ion. On the basis

of unpaired electrons, it is possible to infer the valence state of the metal ion in

the complex and further if there are more than three d-electrons and the

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 Chapter-2

complex is of octahedral stereochemistry whether the bonding is of spin-free or

spin-paired type. µeff = [n(n+1)]1/2 B.M.

The magnetic susceptibilities of the solid complexes were determined by

the Gouy method at room temperature. The effective magnetic moment per

metal atom was calculated from the expression.

µeff = 2.84[χmT]1/2 B.M.

whereχmis the molar susceptibility of the complex obtained after applying the

diamagnetic corrections [1]by the use of pascals constants for other atoms and

groups in the complex. Hg[Co(SCN)4] was used as a calibrant.

2.2.5. 1H NMR

The 1H NMR spectra of the molecules were recorded using TMS as a

internal reference in CDCl3 solvent. The proton NMR spectra were determined on

Bruker AC-400 MHz High resolution Multinuclear FT-NMR Spectrometer at

University of Mysore, Mysuru, Karnataka, India.

2.2.6. Mass Spectra

Mass spectra were recorded on a Water-Q-TOF ultima spectrometer.

2.2.7. Elemental analysis (C, H, N and S)

Carbon, nitrogen and hydrogen contents of the molecule were performed

on a Thermo Finnigan Flash EA 1112 elemental analyzer.

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 Chapter-2

RADICAL SCAVENGING ACTIVITY:

Free radicals are types of Reactive oxygen species (ROS),which include all
highly reactive, oxygen-containing molecules. Free radicals may be defined as
chemical species associated with an odd or unpaired electron. They are neutral,
short lived, unstable and highly reactive to pair up the odd electron and finally
achieve stable configuration. They are capable of attacking the healthy cells of
the body, causing them to lose their structure and function. Cell damage caused
by the free radicals appears to be a major contributor to aging and degenerative
diseases of aging such as cancer, cardiovascular disease, cataracts, immune
system decline, liver diseases, diabetes mellitus and stress among others.

Radical scavengers are antioxidants capable of trapping radicals.

Antioxidants are the substances that when present in low concentrations
compared to those of an oxidizable substrate significantly delays or prevents
oxidation of that substance. Apart from their role of health benefactors,
antioxidants are added in foods to prevent or delay oxidation of food initiated by
free radicals formed during their exposure to environmental factors such as air,
light and temperature.

Two models that are employed for evaluating antioxidant activity are,

 In vitro model.
 In vivo model.

In-vitro models for evaluating antioxidant activity:

 Conjugated diene assay.

 DPPH Method (2,2-diphenyl-1-picryl hydrazyl).
 Super oxide radical scavenging activity.
 Hydroxyl radical scavenging activity.
 Nitric oxide radical inhibition activity.
 Reducing Power Method.
 Phospho molybdenum Method.
 Peroxynitrite radical scavenging activity.

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 Chapter-2

 ABTS (2,2-azinobis(3-ethyl benzothiazoline-6-sulfonicacid)diamonium

salt)Method.
 DMPD(N,N-dimethyl-p-phenylene diamine dihydrochloride)Method.
 Oxygen Radical Absorbance Capacity(ORAC).
 Xanthine oxidase Method.
 FRAP Method.
 TRAP Method.
 Cytochrome C test.
 Erythrocyte ghost system.
 Microsomal lipid peroxidation or Thiobarbutaric acid(TBA)assay.

In-vivo model for evaluating antioxidant activity:

 Immunological studies.
 Phagocytosis.
 Lipid peroxidation.
 Estimation of superoxide dismutase.
 Determination of catalase activity.
 Determination of the reduction in the amount of glutathione.

Even though a number of methods are available for the

determination of antioxidant activity, the assay employing the stable 2,2-
diphenyl-1-picryl-hydrazyl radical(DPPH) has received more attention owing to
its ease of use and convince. This assay is the most widely used ‘in vitro’ test to
assess free radical scavenger capacities.

DPPH radical scavenging assay:

A rapid, simple and inexpensive method to measure antioxidant capacity

involves the use of the free radical,2,2-Diphenyl-1-picrylhydrazyl (DPPH).DPPH
is widely used to test the ability of compounds to act as free radical scavengers
or hydrogen donors, and to evaluate antioxidant activity of foods. The DPPH
method can be used for solid or liquid samples and is not specific to any
particular antioxidant component, but applies to the overall antioxidant capacity
of the sample.
Principle:

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 Chapter-2

A simple method that has been developed to determine the antioxidant

activity utilizes the stable 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical. The
molecule of DPPH is characterized as a stable free radical by virtue of the
delocalisation of the spare electron over the molecule as a whole, so that the
molecules do not dimerise, as would be the case with most other free radicals.
The delocalisation is responsible to the purple colour of the compound.

Fig; 2,2-Diphenyl-1-picrylhydrazyl(DPPH)

(Free radical)(Purple colour)

When a solution of DPPH is mixed with that of a substance that can donate
a hydrogen atom, then this give rise to the reduced form DPPH-H (2,2-Diphenyl-
1-picrylhydrazin) that will be yellow colour due to the picryl group still present.

Fig; 2,2-Diphenyl-1-picrylhydrazyl(DPPH)

(non radical)(Yellow colour)

DPPH RADICAL SCAVENGING ACTIVITY

Materials required:
 DPPH.
 Distilled ethyl alcohol.
 Ascorbic acid(AA).
 Micro pipette(10ml).
 Cuvet.

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 Chapter-2

 Standard flask.
 Test tubes.
 Spectrophotometer.

The compound under study was dissolved in distilled ethanol (50mL) to

prepare 1000μ M solution. Solutions of different concentrations (10μm, 25μm,
50μm, 100μm 200μm, 500μm) were prepared by serial dilution and the free
radical scavenging activity was studied. The DPPH scavenging effect was carried
out according to the method first employed by Blois. Each compound solution
having different concentrations were taken in different test tubes. 4mL of 1mM
ethanol solution of DPPH was added and shaken vigorously. The tubes were then
incubated in the dark room at room temperature for 20 minutes.

A DPPH blank was prepared without compound and ethanol was used for
the base line correction. Changes (decrease) in the absorbance at 517nm were
measured using a UV-Visible spectrophotometer and the remaining DPPH was
calculated. The percent decrease in the absorbance was recorded for each
concentration, and percentage quenching of DPPH was calculated on the basis of
the observed decrease in the absorbance of the radical.

The radical scavenging activity was expressed as the inhibition

percentage and calculated using the formula:

Radical Scavenging Activity (%) = [(AO-A1) / A0 × 100]

Where, A0 is the absorbance of control (blank, without compound)

A1 is the absorbance of the compound

The radical scavenging activity of ascorbic acid was measured and

compound with the newly synthesized compounds. IC50 (50% inhibitory
concentration) was calculated by plotting graph (concentration v/s RSA).

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