CHAPTER III

REAGENTS AND EXPERIMENTAL METHODS

The details regarding various reagents used, the procedures for the

preparation of the ligands and the methods adopted for the analysis of the

complexes are presented in this chapter. A brief description of the instruments

used for the physico-chemical studies, their operational characteristics and the

procedure for antimicrobial tests employed are also given.

REAGENTS

Metal Salts

The following metal salts were used as the starting materials for the

preparation of the complexes. MoCl5 (Alfa Aesar, Lancaster), MoO3 (Loba

Chemie, Mumbai), RuCl3.3H2O (Loba Chemie, Mumbai), (UO2NO3)2.6H2O

(BDH, England), NbCl5.5H2O (E-Merck, Germany) were used for the

preparation of complexes.

Dioxouranium(VI) carbonate was precipitated by adding AR Na2CO3 to

a saturated solution of uranyl nitrate. Dioxouranium carbonate was dissolved in

minimum quantity of appropriate dilute acids to get solutions of chloride and

bromide. These solutions were evaporated on a water bath to get crystals of the

salts.

Solvents

Commercially available methanol, ethanol, DMF, DMSO and benzene

were purified by standard procedures (Weissberger, 1956). BDH spectroscopic

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55

grade methanol was used as such for conductivity measurements. Nitrobenzene

used for conductivity measurements was purified by repeated distillation of

BDH samples over phosphorous(V) oxide.

Other reagents

Salicylaldehyde, 4-chloro-m-cresol, 2-naphthol (E-Merck, Germany) 2-

methoxyphenol, isoeugenol (Sisco Research Lab, Mumbai), 3-

ethoxysalicylaldehyde (Alfa Aesar, Lancaster) and 4-aminoantipyrine (Fluka,

Switzerland) were used as such for the preparation of ligands.

All other reagents used in the present investigation were of BDH AR or

E. Merck GR or other analytical reagent grade chemicals.

EXPERIMENTAL

Preparation of Ligands

1. 2,3-dimethyl-1-phenyl-4-(2-hydroxy-3-ethoxybenzylidene

amino)pyrazol-5-one (APES)

The Schiff base, APES (C20H21N3O3) was prepared by mixing

methanolic solutions of 3-ethoxysalicylaldehyde (0.05mol, 50 mL) and 4-

aminoantipyrine (0.05mol, 50 mL) and stirred the mixture for ~30 minutes. The

pale yellow solid, separated on concentration, was filtered, washed with

methanol and dried. It was characterized by elemental analysis, IR, UV-vis.,

1H NMR, 13C NMR and single crystal X-ray diffraction data.

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56

N

N

NO

H

O

O

CH3

H3C

Fig. III (1)

Structure of APES

2. 2,3-dimethyl-1-phenyl-4-(2-hydroxybenzylideneamino)pyrazol-5-one

(AAPS)

The Schiff base, AAPS (C18H17N3O2) was prepared by mixing

methanolic solutions of salicylaldehyde (0.05 mol, 50 mL) and 4-

aminoantipyrine (0.05 mol, 50 mL) and refluxed the mixture for 2 hours. The

pale yellow solid, separated on concentration, was filtered, washed with

methanol and dried. It was characterized by elemental analysis, IR, UV-vis.,

1H NMR and single crystal X-ray diffraction data.

Fig. III (2)

Structure of AAPS

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3. 2,3-dimethyl-1-phenyl-4-(2-hydroxy-3-methoxyphenylazo)pyrazol-5-

one (MPAP)

The ligand, MPAP (C18H18N4O3) was synthesized from 4-

aminoantipyrine and 2-methoxyphenol by diazotisation and coupling as given in

the literature (Vogel, 1994).

4-aminoantipyrine (10 g, 50 mmol) was converted to the

hydrochloride using 1:1 hydrochloric acid (28 ml) and the solution was cooled

below 0 °C in an ice-salt bath. A solution of sodium nitrite (3.8 g, 55 mmol) in

water (20 ml) was chilled using the ice-salt bath.

The pre-cooled nitrite solution was then added in small volumes to the

cold amine hydrochloride solution with good stirring. The temperature was

always kept below 10 °C and small amounts of crushed ice were used when

required. The last part of the nitrite solution was added slowly and drop wise

till a slight excess of nitrous acid was present which was indicated by an

immediate blue colour, imparted to a starch potassium iodide paper. After

keeping the diazonium chloride solution in ice bath for a few min, the

temperature was allowed to rise to about 5 °C.

2-Methoxyphenol (7 ml, 50 mmol) was dissolved in 45 ml of 10%

sodium hydroxide solution. The solution was then cooled below 5 °C in an ice

bath followed by the direct addition of ~25g of crushed ice. The cold diazonium

chloride solution was added very slowly to the solution of 2-methoxyphenol

with vigorous stirring. The colour of the solution became reddish yellow and a

solid product separated slowly. After the addition of the entire amount of diazo

compound, the mixture was allowed to stand in the bath for about 30 min. with

occasional stirring. The solid product obtained was then filtered under gentle

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58

suction, washed well with cold water and recrystallized from alcohol. The

ligand was characterized by elemental analysis, IR, FAB mass and NMR spectra.

Fig. III (3)

Structure of MPAP

4. 2,3-dimethyl-1-phenyl-4-(2-hydroxy-3-methoxy-5-prop-1-

enylphenylazo)pyrazol-5-one (IEAP)

The above synthetic procedure was repeated for the ligand IEAP,

C21H21N4O3 appropriately adapting isoeuginol (8.2 g, 0.05 mol) in place of 2-

methoxyphenol. It was characterized by elemental analysis, IR, UV and NMR

spectra.

Fig. III (4)

Structure of IEAP

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5. 2,3-dimethyl-1-phenyl-4-(5-chloro-2-hydroxy-4-methylphenylazo)

pyrazol-5-one (CCAP)

The above synthetic procedure was repeated for the ligand, CCAP,

C18H17ClN4O2 appropriately adapting 4-chloro-3-cresol (7.1g, 0.05 mol) in

place of 2-methoxyphenol. It was characterized by elemental analysis, IR, UV and

NMR spectra.

Fig. III (5)

Structure of CCAP

6. 2,3-dimethyl-1-phenyl-4-(2-hydroxynaphthylazo)pyrazol-5-one

(NAAP)

The above synthetic procedure was repeated for the ligand NAAP,

C21H18N4O2 appropriately adapting β-Naphthol (6.2g, 0.05 mol) in place of 2-

methoxyphenol. It was characterized by elemental analysis, IR, UV,1H NMR and

single crystal X-ray diffraction studies.

N

N

N

N

OH

O

Cl

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N

N

N

N

O

OH

Fig. III (6)

Structure of NAAP

Analysis of the complexes

1. Estimation of metals

a. Estimation of molybdenum

Molybdenum in the complexes was estimated (Vogel, 1996) gravimetrically

as oxinate, MoO2(C9H6ON)2.

About 0.2 g of the complex was weighed accurately and digested with

10 mL of conc. sulphuric acid and a few drops of perchloric acid. The resulting

clear solution was quantitatively transferred into a 500 mL beaker. A drop of

methyl red indicator was added and neutralized with conc. ammonia solution.

The solution was then acidified with a few drops of 1 M sulphuric acid and then

5 mL of 2 M ammonium acetate were added and diluted to 100 mL. After

adjusting the pH between 3.3 and 7.5, the solution was heated to boiling and

3% solution of oxine in dil. acetic acid was added drop wise with stirring till the

supernatant liquid became perceptibly yellow. The boiling and stirring were

continued for 3 min. The precipitated molybdenum oxinate was filtered through

a sintered glass crucible (G4), washed with hot water until free from the reagent,

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61

dried to constant weight at 130-140°C and weighed as MoO2(C9H6ON)2.

b. Estimation of Niobium

Niobium was estimated gravimetrically as Nb2O5

c. Estimation of Ruthenium

The metal percentage in the ruthenium(III) complexes were determined by

ICP-AES spectrometer in STIC, Kochi.

d. Estimation of Uranium

Uranium in the complexes was estimated (Vogel, 1996) gravimetrically

by oxalate-oxide method. About 0.1 to 0.2g of the complex was digested with

con. HNO3 (20ml) and a few drops of perchloric acid and the resulting solution

was evaporated to dryness. The residue was extracted with water (50ml) and tha

PH of the solution was adjusted to 7 by adding ammonia or hydrochloric acid as

the case may be. The resulting solution was heated to boiling and a saturated

solution of oxalic acid(10ml) was added with stirring. The metal oxalate

precipitated was filtered through whatmann No.42 filter paper and washed

several times with 2% solution of oxalic acid. The precipitate was ignited in a

silica crucible and weighed as U3O8. From the weight of the oxide the amount

of uranium in the respective complexes were calculated.

2. Estimation of halides

A known weight of the complex was fused with excess of AR sodium

carbonate at 450 20 °C. The residue was extracted with water, transferred into

a beaker and AR 1:1 nitric acid was added till the decomposition of carbonate

was completed. The solution was filtered, free of any carbon particles. The

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62

chloride and bromide were estimated by Volhard’s method (Vogel, 1996).

3. Estimation of perchlorate

Perchlorate in the complexes was estimated by Kurz’s (1958) method.

The weighed complex was heated with excess sodium nitrite in a muffle furnace at

500 20 °C to reduce it to the chloride. The residue was extracted with water,

and the chloride in it was estimated by Volhard’s method (Vogel, 1996).

4. Estimation of carbon, hydrogen , nitrogen and sulphur

Carbon, hydrogen, nitrogen and sulphur of the complexes were estimated

by microanalytical methods in STIC, Kochi.

PHYSICO-CHEMICAL STUDIES

Electrical conductance

Molar conductances of the complexes in nitrobenzene and methanol

were measured at room temperature (28 2 °C) using an Elico direct reading

conductivity meter, with cell constant 0.96 cm-1. The solution used had a

concentration of ~10-3 M.

Magnetic measurements

Magnetic susceptibilities of the complexes were determined on a Gouy

balance at room temperature using Hg[Co(NCS)4] as calibrant. Diamagnetic

corrections for various atoms and structural units were computed using Pascal’s

constant (Dutta and Syamal, 1992). Effective magnetic moments were

calculated from the corrected magnetic susceptibilities using the relation.

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'eff Mμ =2.84 χ xT

where 'Mχ is the corrected molar susceptibility and T is the absolute

temperature.

Infrared spectra

The infrared spectra of the ligands and the complexes were recorded on

a Perkin-Elmer 397 IR spectrophotometer in the range 400-4000 cm-1 using

KBr pellets at NIIST, Thiruvananthapuram and also on a Thermo Nicolet,

Avatar 370 FTIR spectrometer at STIC, Kochi.

Electronic spectra

The electronic spectra of the ligands and the complexes in methanol

were recorded on JASCO V-550 UV-Vis. spectrophotometer in the range 200-

900 nm at NIIST, Thiruvananthapuram.

Nuclear magnetic resonance (NMR) spectra

1H NMR spectra of the ligands and the complexes were recorded on a

300 MHz (Bruker Advance dpx-300) and on a Bruker Avance III, 400MHz

FT-NMR spectrometer using TMS as reference material at NIIST,

Thiruvananthapuram and STIC, Kochi respectively.

Electron paramagnetic resonance (EPR) spectra

The EPR spectra of oxomolybdenum(V) and ruthenium(III) complexes

were recorded in the liquid nitrogen temperature using a JEOL JES - FA200

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64

ESR Spectrometer with X and Q band with TCNE (Tetracyanoethylene) as

marker at the SAIF, IIT, Mumbai, India.

Mass spectra

The FAB mass spectra of the ligand and the complexes were recorded in

a JEOL JMS 600H mass spectrometer at NIIST, Thiruvananthapuram.

X-ray powder diffraction

X-ray powder diffraction patterns of the complexes were recorded on a

Philips X-ray diffractometer (PW 1710) at NIIST, Thiruvananthapuram and on

a Bruker AXS D8 Advance X-ray powder diffractometer at STIC, Kochi.

Copper X-ray tubes, for which the wavelength of the strongest radiation (K) is

approximately 1.54 Å, was used for the diffraction studies.

Single crystal X-ray diffraction

The crystallographic data were collected using Bruker Kappa Apex2

CCD diffractometer with graphite monochromated Mo K ( = 0.71073 Å)

radiation at STIC, Cusat, Kochi. The program SAINT/XPREP was used for

data reduction and APEX 2/SAINT for cell refinement (Bruker, 2004). The

structure was solved using SIR92 (Altornare et al., 1993) and refinement was

carried out by full-matrix least squares on F2 using SHELXL-97 (Sheldrick,

1997). All non-hydrogen atoms were refined with anisotropic thermal

parameters. All hydrogen atoms with the exception of those on nitrogen atoms

were geometrically fixed and refined using a riding model. Molecular graphics

employed were ORTEP 3 (Farrugia, 1997) and MERCURY (Bruno et al.,

2002).

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Scanning electron microscopy

Scanning electron microscopy (SEM) is a method for high resolution

surface imaging. The SEM uses an electron beam for surface imaging. The

advantages of SEM over light microscopy are greater magnification and much

larger depth of field. The SEM images were recorded on a JEOL Model JSM-

6390LV at STIC Kochi.

Thermogravimetry

The TG and DTG curves of the complexes were recorded on a Mettler

Toledo STARe system at the Diamond thermal analysis system at STIC, Kochi.

Irradiation studies

The irradiation studies of the samples were done at the Rubber Research

Institute of India, Kottayam. The dried samples sieved to uniform mesh size of

100-120 were sealed in vacuum in glass ampoules and were exposed to gamma

irradiation to a dose of 800 kGyh-1 using 60Co γ-ray in Gamma chamber 5000

cm3, self shielded at constant intensity under room temperature at a dose rate of

1.85 kGyh-1. After irradiation, the samples were mixed uniformly and stored

over P4O10 in vacuum desiccators. The thermal, XRD and SEM studies were

done within one week of irradiation.

Antimicrobial studies

Antitubercular activity of some of the ligands and a few of the metal

complexes against Mycobacterium tuberculosis H37 Rv and Lactobacillus

leichmannii were done by Resazurin assay method, at the Rajeev Gandhi centre

for Biotechnology, Thiruvananthapuram.

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66

The effect of the extracts was checked for their activity against M.

tuberculosis H37Rv/ Lactobacillus leichmannii . The medium used was

Middlebrook 7H9 broth (Difco BBL) (containing OADC supplement and 0.5%

glycerol). The compounds were added to 450 L of the medium taken in 2 ml

tubes to make final concentrations of 1, 10 and 100 g/mL. To these tubes

50 L of the M. tuberculosis/ L. leichmannii culture diluted to Mc Farland

standard 0.5 was added. Control tubes consisted of the medium with the

bacterial culture to which the same volume of DMF was added. The tubes were

incubated at 37 ºC for 7 days. At the end of incubation 25 L of 0.01% of

Resazurin (Sigma) dye was added to the tubes and kept for incubation until the

dye turned pink in the control tubes. Change of colour from blue to pink would

indicate growth while lack of colour change would suggest inhibition of

growth.

Some of the ligands and their complexes were screened in vitro for their

possible antibacterial activities against Escherichia coli 585, Vibrio cholera,

Bacillus cereus 2248 and Streptococcus aureus 1938 using the disc diffusion

method (Anantharaman, 1999) (Kirby Bauer Method) and antifungal activity

against Aspergilus flavus and Penicillium crysogenum.

In vitro antibacterial activity was screened by using Mueller Hinton

Agar (MHA), obtained from Himedia (Mumbai). The MHA plates were

prepared by pouring 15 mL of molten media into sterile petriplates (100 mm).

The plates were allowed to solidify for 5 min. and 0.1% inoculum suspension

was swabbed uniformly. All the plates were allowed to air dry in sterile

conditions and swabbed with the pure culture of bacteria on the MHA plates.

Already prepared sterile discs (6 mm) impregnated with the compounds of the

study were placed above the seeded plates aseptically with a sterile forceps. A

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67

disc was also used in pure chloroform to provide a control. The plates were

incubated for 24 h at 37 C and the zone of inhibition of the antibiotic

compound against the bacteria can be measured by measuring the zones of

inhibition around the bacterial cultures and was measured by the diameter of

the zone in millimeters.

Molecular modeling studies

The molecular modeling was constructed using modeling and analysis

software (Chem Bio Office Ultra, 2008). The possible 3D structures of ligands and

the complexes, were optimized by molecular mechanics calculations, MM2 giving

the lowest energy CHEM 3D models.