synthesis, characterisation, thermoacoustical

SYNTHESIS, CHARACTERISATION, THERMOACOUSTICAL, ANTIMICROBIAL AND MOLECULAR DOCKING STUDIES OF

METAL COMPLEXES OF MANNICH BASES

A Thesis submitted to the Bharathidasan University, Tiruchirappalli – 620 024 for the award of the Degree of

DOCTOR OF PHILOSOPHY

in

CHEMISTRY

by

Mr. S. FAROOK BASHA

Under the guidance of Dr. M. SYED ALI PADUSHA M.Sc., Ph.D.,

Since 1951

PG AND RESEARCH DEPARTMENT OF CHEMISTRY JAMAL MOHAMED COLLEGE (Autonomous)

College with Potential for Excellence Accredited at „A‟ Grade by NAAC – CGPA 3.6 out of 4.0

(Affiliated to Bharathidasan University) TIRUCHIRAPPALLI – 620 020

DECEMBER 2015

PG & RESEARCH DEPARTMENT OF CHEMISTRY JAMAL MOHAMED COLLEGE (Autonomous)

College with Potential for Excellence Accredited with “A” Grade by NAAC - CGPA 3.6 out of 4.0

(Affiliated Bharathidasan University) Tiruchirappalli-620 020, Tamil Nadu, India

Since 1951

Dr. M. Syed Ali Padusha, M.Sc., Ph.D., Email: [email protected] Associate Professor Mobile: +91 98654 47289

Date:

CERTIFICATE

This is to certify that the thesis entitled “Synthesis, Characterisation,

Thermoacoustical, Antimicrobial and Molecular Docking Studies of Metal

Complexes of Mannich Bases” submitted by S. FAROOK BASHA (Ref. No:

27785/Ph.D.1/Chemistry/Part-time/October 2011) is a bonafide record of

research work done by him under my guidance in the PG & Research

Department of Chemistry, Jamal Mohamed College, Tiruchirappalli and that

thesis has not previously formed the basis for the award of any degree or any

other similar title. The thesis is the outcome of original research work done by

the candidate under my overall supervision.

Date: (M. SYED ALI PADUSHA)

Station: Tiruchirappalli

S. FAROOK BASHA Research Scholar, PG and Research Department of Chemistry, Jamal Mohamed College (Autonomous), Tiruchirappalli – 620 020.

DECLARATION

I hereby declare that the thesis entitled “Synthesis, Characterisation,

Thermoacoustical, Antimicrobial and Molecular Docking Studies of Metal

Complexes of Mannich Bases” which submit for the award of the degree of

Doctor of Philosophy in the Bharathidasan University is the original work

carried out by me under the guidance and supervision of Dr. M. Syed Ali

Padusha, Associate Professor, Department of Chemistry, Jamal Mohamed

College (Autonomous), Tiruchirappalli – 620 020.

I further declare that this work has not been submitted earlier in full or in

parts to any other university for the award of any other degree or diploma.

Place: Tiruchirappalli-20 (S. FAROOK BASHA)

Date: 2015

ACKNOWLEDGEMENT

First and foremost my head bows with rapturous dedication

within my heart to the Almighty God. I wish to make devote

supplication to the Almighty God, “the great scientist of this lovely

world” without whose blessings and benevolence my endeavours would

not have reached to the zenith of success.

I deem it to be my proud privilege to express my whole heartedly

sense of gratitude and thanks to the President, Secretary and

Correspondent, Treasurer, Assistant Secretary and Members of the

noble management committee of this great institution, Jamal Mohamed

College (Autonomous), Tiruchirappalli, for giving me an opportunity to

work and to do the research work in part-time.

I express my sincere thanks to Dr. M. Mohamed Salique,

Principal, Jamal Mohamed College (Autonomous), Tiruchirappalli, for

his encouragement and support in the execution of the research work.

I am bound to extend my special thanks to Dr. M. Mohamed

Sihabudeen, Associate Professor and Head, Post Graduate and

Research Department of Chemistry, Jamal Mohamed College

(Autonomous), Tiruchirappalli, for his moral care, motivation and

evergreen support in the Department to do the research work

successfully.

I would like to express my deep sense of gratitude and

acknowledge my sincere indebtedness to my research advisor,

Dr. M. Syed Ali Padusha, Associate Professor, Post Graduate and

Research Department of Chemistry, Jamal Mohamed College

(Autonomous), Tiruchirappalli, for his unceasing interest, incessant

encouragement, constructive suggestions and gifted guidance

throughout the progress of this research work. I consider myself

fortunate in having a guide like him and my gratefulness to him cannot

be expressed in words. I pray to the Almighty, that I may come to his

expectations in present as well as in future.

I wish to record my thanks and gratitude to the Doctoral

Committee Members Dr. A. Jafar Ahamed, Associate Professor, Post

Graduate and Research Department of Chemistry, Jamal Mohamed

College (Autonomous), Tiruchirappalli and Dr. Shameela Rajam,

Associate Professor, Post Graduate and Research Department of

Chemistry, Bishop Heber College (Autonomous), Tiruchirappalli, for

their valuable suggestions and support throughout the research

programme.

I owe my respectful thanks to Dr. M. Sheik Mohamed, Dr. R.

Khader Mohideen and Dr. A.M. Mohamed Sindhasha, Former

Principals, Jamal Mohamed College (Autonomous), Tiruchirappalli, for

their continuous support to me in this great institution, Jamal

Mohamed College (Autonomous), Tiruchirappalli.

I express my sincere thanks whole heartedly to Dr. T. Janakiram,

Dr. K. Sithick Ali, Dr. A. Abdul Jameel, and Dr. M.I. Fazal Mohamed,

Former Heads of the Department of Chemistry, Jamal Mohamed

College (Autonomous), Tiruchirappalli, and to Dr. S.M. Mazhar Nazeeb

Khan, Controller of Examinations, Jamal Mohamed College

(Autonomous), Tiruchirappalli, for their support and constant

encouragement.

I express my sincere thanks to Dr. M. Seeni Mubarak, Associate

Professor, Post Graduate and Research Department of Chemistry,

Jamal Mohamed College (Autonomous), Tiruchirappalli, for his valuable

suggestions during the course of the research work.

I express my thanks to all of the faculty members of the Post

Graduate and Research Department of Chemistry, Jamal Mohamed

College (Autonomous), Tiruchirappalli, for their parental support and

co-operation in the Department.

I wish to record a sincere thanks to the non-teaching staff

members of the College, for their support and encouragement to me for

completing the research work successfully.

I convey my sincere thanks whole heartedly to

Prof. A. Balasundaram and Prof. V. Jeevanandham, Assistant

Professors, Jamal Mohamed College of Teacher Education,

Tiruchirappalli, for their suggestions, co-operation and their great

efforts for doing the research work successfully.

I express my wholeheartedly thanks to Prof. Y. Moydheen Sha,

Assistant Professor of Commerce, Dr. A. Raja, Assistant Professor of

Microbiology, Prof. M. Mohamed Rafi, Assistant Professor of Chemistry

and to my research mates Prof. Mashood Ahamed and

Mr. T. Chandrasekaran and to the College Administrative Staff

members Mr. Kajamideen, Mr. M. Mohamed Ali,

Mr. M. Mohamed Rafi, Mr. M. Mohamed Azarudeen and to all of the

research scholars, for their kind help and support to carry out my

research work successfully.

I owe my sincere thanks whole heartedly to my brotherhood

friends Mr. S. Alaudeen and Mr. Y. Mohamed Zameer, by whom I get

this achievement with tremendous support and marvellous

cooperation. They stood beside me with their helping hands and moral

support at every stage of my life. I gratefully thank them by the grace of

the Almighty.

I feel great pleasure to acknowledge my deepest sense of

indebtedness to my family. My words fail to express my feeling and

acknowledging the tremendous debt to my father Mr. B. Shebbeer

Basha and to my mother Mrs. S. Aziza Bi, because they are the basic

stone of my life. Nobody is able to give justice in giving entirely and

adequately thanks to parents for their keen care.

I owe my special thanks to my brother-in-law, Mr. S. Hakeem Ali,

my sister Mrs. S. Peerani, Assistant Professor of English and to my

brother Mr. S. PeerBasha, Assistant Professor of Information

Technology and Mrs. Dilrash Roshini, for their endless and continuous

support in my life. They helped me a lot with continuous source of

inspiration, motivation and devotion to reach the goal successfully.

I cherish my thanks to my better half Mrs. F. Thabassum

Thahira and my kids F. Munawwara Siddiqua, F. Mohamed Abubacker

Siddiq, H. Shifana and H. Jamal for their patience, encouragement and

tremendous help in my life and also to complete the research

programme successfully.

Once again, I thank the Almighty, for giving me enough strength,

good health and knowledge for completing the task with utmost

satisfaction.

S. Farook Basha

TABLE OF CONTENTS

Chapter No. Title Page No.

I Introduction 1 - 41

II

Synthesis and Characterisation of 2-[(morpholin-4-yl) (pyridin-3-yl) methyl] hydrazinecarboxamide (MPH) and their Mn(II), Co(II), Ni(II), Cu(II) and Zn(II) metal complexes.

42-58

III

Synthesis and characterisation of Nꞌ-(morpholino (thiophen-2-yl) methyl) pyridine-3-carbohydrazide (MTN) and their Co(II), Ni(II), Cu(II) and Zn(II) metal complexes

59 - 71

IV

Synthesis and characterisation of 1-(furan-2-yl) (morpholino) (methyl)-3-phenyl urea (MFP) and their Co(II), Ni(II), Cu(II) and Zn(II) metal complexes

72 - 85

V Thermoacoustical Studies 86 - 107

VI Biological Studies 108 - 215

Conclusion 216 - 220

LIST OF TABLES

Table No. Table Page No.

2.1. Elemental analysis and molar conductance of MPH and its metal complexes

45

2.2. Characteristic Infrared bands of MPH and its metal (II) complexes (ν cm-1)

46

2.3. Electronic Spectral bands of MPH and its metal complexes

48

3.1. Elemental analysis and molar conductance of MTN and its complexes

61

3.2. Characteristic Infrared bands of MTN and its metal complexes

63

3.3. UV-Vis spectral data and Magnetic Moment of MTN and its Complexes

5

4.1. Elemental analysis and molar conductance of MFP and its complexes

75

4.2. Characteristic IR bands of the MFP and its metal complexes (νcm-1)

75

5.1. Measured value of Ultrasonic Velocity (u), Density (ρ) and coefficient Viscosity (η) of the two binary systems of aqueous MPH and MTN in DMSO at different temperatures

94

5.2 Computed values of adiabatic compressibility (κ), intermolecular free length (Lf), molar volume (Vm) of two binay systems of MPH and MTN at different temperatures

94

5.3 Computed values of Relaxation time (τ), Specific acoustic impedance (Z) and LJP of two binary systems of MPH and MTN at different temperatures

95

5.4. Computed values of internal pressure (πi), free volume (Vf) and molecular cohesive energy (MCE) of two binary systems of MPH and MTN at different temperatures

95

5.5 Computed values of Available volume (Va), Gibbs free energy(∆G) and Absorption coefficient (α/f2of two binary systems of aqueous sample1 and sample 2 at different temperatures.

95

6.1. Haemolytic variables in literature 157

6.2 Biochemical characterizations values of isolated strains from infected patient samples in Government Hospital, Tiruchirappalli

175

6.3 Antimicrobial activity of individual metal ions 176

6.4 Antimicrobial activity of individual ligands 177

6.5 Antimicrobial activity of metal and ligand combined compound (complex)

178

6.6 Descriptive statistics for antimicrobial activity of metal ions

183

6.7 Descriptive statistics for antimicrobial activity of ligands

184

6.8 Descriptive statistics for antimicrobial activity of combination of metal ion and ligand (1:1 ratio)

185

6.9 Larvicidal activity of ligands 191

6.10 Parameters of the docked models for MPH 191

6.11 The molecular docking studies of metal complexes of MPH with Extended-spectrum β-lactamase

196

LIST OF FIGURES

Figure No. Figure Page

No.

1 Proposed structure of metal complexes of 2-[(morpholin-4-yl) (pyridin-3-yl) methyl] Hydrazinecarboxamide (MPH) (M = MnII, CoII, NiII, CuII and ZnII)

50

2 IR spectrum of 2-[(morpholin-4-yl) (pyridin-3-yl) methyl] Hydrazinecarboxamide (MPH)

51

3 IR spectrum of CoII complex of 2-[(morpholin-4-yl) (pyridin-3-yl) methyl] Hydrazinecarboxamide (MPH)

51

4 IR spectrum of CuII complex of 2-[(morpholin-4-yl) (pyridin-3-yl) methyl] Hydrazinecarboxamide (MPH)

52

5 IR spectrum of NiII complex of 2-[(morpholin-4-yl) (pyridin-3-yl) methyl] Hydrazinecarboxamide (MPH)

52

6 IR spectrum of MnIIcomplex of 2-[(morpholin-4-yl) (pyridin-3-yl) methyl] Hydrazinecarboxamide (MPH)

53

7 UV spectrum of CoII complex of 2-[(morpholin-4-yl) (pyridin-3-yl) methyl] Hydrazinecarboxamide (MPH)

53

8 UV spectrum of Mn(II) complex of 2-[(morpholin-4-yl) (pyridin-3-yl) methyl] Hydrazinecarboxamide (MPH)

54

9 1H NMR of 2-[(morpholin-4-yl) (pyridin-3-yl) methyl] Hydrazinecarboxamide (MPH)

54

10 13C NMR of 2-[(morpholin-4-yl) (pyridin-3-yl) methyl] Hydrazinecarboxamide (MPH)

55

11 Cyclic voltammogram of NiIIcomplex of 2-[(morpholin-4-yl) (pyridin-3-yl) methyl]hydrazinecarboxamide (MPH)

55

12 TGA curve of Cu (II) complex of 2-[(morpholin-4-yl) (pyridin-3-yl) methyl] Hydrazinecarboxamide (MPH)

56

13 EPR spectrum of Cu (II) complex of 2- [(morpholin-4-yl) (pyridin-3-yl) methyl] Hydrazinecarboxamide (MPH)

56

14 Mass spectrum of 2-[(morpholin-4-yl) (pyridin-3-yl) methyl] Hydrazinecarboxamide (MPH)

57

15 IR SPECTRUM OF N(Morpholino (thiophen-2-Yl) methyl) nicotinohydrazide)(MTN)

67

16 IR SPECTRUM OF Cu (II) of N(Morpholino (thiophen-2-Yl) methyl) nicotinohydrazide)(MTN)

67

17 IR spectrum of Ni (II) of N(Morpholino (thiophen-2-Yl) methyl) nicotinohydrazide)(MTN)

68

18 IR spectrum of Zn (II) of N(Morpholino (thiophen-2-Yl) methyl) nicotinohydrazide)(MTN)

68

19 1H NMR of N(Morpholino (thiophen-2-Yl) methyl) nicotinohydrazide) (MTN)

69

20 13C NMR of N(Morpholino (thiophen-2-Yl) methyl) nicotinohydrazide) (MTN)

69

21 EPR spectrum of Cu (II) complex of N(Morpholino (thiophen-2-Yl) methyl) nicotinohydrazide) (MTN)

70

22 TGA curve of N(Morpholino (thiophen-2-Yl) methyl) nicotinohydrazide) (MTN)

70

23 Proposed structure for metal complexes of 1-(furan-2-yl) (morpholino) (methyl)-3-phenyl urea

79

24 IR spectrum of 1-(furan-2-yl) (morpholino) (methyl)-3-phenyl urea (MFP)

81

25 IR spectrum of CuII complex of 1-(furan-2-yl) (morpholino) (methyl)-3-phenyl urea (MFP)

81

26 IR spectrum of NiII complex of 1-(furan-2-yl) (morpholino) (methyl)-3-phenyl urea (MFP)

82

27 IR spectrum of ZnII complex of 1-(furan-2-yl) (morpholino) (methyl)-3-phenyl urea (MFP)

82

28 UV spectrum of CuII complex of 1-(furan-2-yl) (morpholino) (methyl)-3-phenyl urea (MFP)

83

29 UV spectrum of NiII complex of 1-(furan-2-yl) (morpholino) (methyl)-3-phenyl urea (MFP)

83

30 1H NMR of 1-(furan-2-yl) (morpholino) (methyl)-3-phenyl urea (MFP)

84

31 13C NMR of 1-(furan-2-yl) (morpholino) (methyl)-3-phenyl urea (MFP)

84

32 TGA curve of Cu(II) Chloro complex of 1-(furan-2-yl) (morpholino) (methyl)-3- phenyl urea (MFP)

85

33 Mass spectrum of 1-(furan-2-yl) (morpholino) (methyl)-3-phenyl urea (MFP)

85

34 Plots for ultrasonic velocity versus concentration for aqueous a) MPH and b) MTN at different temperatures () T = 303 K, (•) 308 K and () 313 K

97

35 Plots for adiabatic compressibility versus concentration for aqueous a) MPH and b) MTN at different temperatures () T = 303 K, (•) 308 K and () 313 K

98

36 Plots for internal pressure versus concentration for aqueous a) MPH and b) MTN at different temperatures () T = 303 K, (•) 308 K and () 313 K

99

37 Plots for Enthalpy versus concentration for aqueous a) MPH and b) MTN at different temperatures () T = 303 K, (•) 308 K and () 313 K

100

38 Plots for Gibbs free energy versus concentration for aqueous a) MPH and b) MTN at different temperatures () T = 303 K, (•) 308 K and () 313 K

101

39 Antibacterial activity of metal ions of MPH by agar Disc diffusion method

179

40 Antibacterial activity of metal ions and ligand MPH alone by agar Disc diffusion method

179

41 Antibacterial activity of metal ions and ligand MTN alone by agar Disc diffusion method

180

42 Antibacterial activity of metal ions and ligand MFP alone by agar Disc diffusion method

181

43 Combined Antibacterial activity of ligand MPH and metal ions (1:1) by agar well diffusion method

181

44 Antimicrobial activity of B1 and B2 metal ions (alone)

186

45 Antimicrobial activity of B3, B4 metal ions (alone) and positive control

186

46 Antimicrobial activity of ligands L1, L2 and L3 (alone) and positive control

187

47 Antimicrobial activity of combination of metal ion (B1 and B2) and ligand (L1) (Metal ion + Ligand = 1:1 ratio)

187

48 Antimicrobial activity of combination of metal ion (B3 and B4) and ligand (L1) (Metal ion + Ligand = 1:1 ratio), and positive control

188

49 Heamolytic assay of ligand samples 188

50 Titer plate method for analysis of heamolytic (using human RBC) assay against ligand samples

189

51 Triplicate of Control flask containing 0.5% of DMSO with (n=20 )3rd instar larvae

189

52 Testing of Ligand at 250,500 and 1000µg/ml with (n= 20) 3rd instar larvae

190

53 Formation of pupa at 1000µg/ml 190

54 Structure for Ebola virus 192

55 Ebola virus in Rasmol Visualization 192

56 Structure for the (morpholin-4-yl) (pyridin-3-yl) methyl] hydrazinecarboxamide

193

57 Receptor (Ebola) ready to dock with the Ligand L1

193

58 Receptor (Ebola) docked with the Ligand L1 194

59 Docked structure with the binding visualizations

194

60 Structure for the Ligand L1(morpholin-4-yl) (pyridin-3-yl) methyl] hydrazinecarboxamide with Phymol

195

61 Structure for the target receptor for cancer 195

62 Docking of receptor (cancer) and MPH 196

63 Docking study of CoII complex of MPH with extended-spectrum β-lactamase

197

64 Docking study of CuII complex of MPH with extended-spectrum β-lactamase

199

65 Extended-spectrum β-lactamase with compound Ciprofloxacin

199

Chapter I

INTRODUCTION

Chemistry has accomplished rapid progress in understanding the

properties of all of the elements. Among the various branches of

chemistry, inorganic chemistry is of fundamental importance not only

as a basic science but also as one of the most useful sources for

modern technologies. The main purpose of inorganic chemistry in near

future will be the synthesis of the compounds with unexpected bonding

modes and structures and with the discoveries of novel reactions and

physical properties of new compounds. Inorganic compounds are also

indispensable in the frontier chemistry of organic synthesis using metal

complexes, homogeneous catalysis, bioinorganic functions etc.

Coordination chemistry plays a vital role and it is one of the most

active research fields in inorganic chemistry. Coordination chemistry

assumed a vital significance with the development of bioinorganic

chemistry, which is mainly the chemistry of coordination compounds

[1]. Coordination chemistry, emerged from the work of Alfred Werner, a

Swiss chemist who examined different compounds composed of cobalt

(III) chloride and ammonia. The coordination chemists have showed

their major interest in the stereochemistry of the coordination

compounds. Nowadays, the research on coordination complexes has

been increased due to their magnetic, optical, electronic properties and

also due to their complex structures.

Due to the catalytic and bioinorganic relevance, the chemistry of

transition metal complexes has received a considerable interest in the

research field [2-7]. The literature survey clearly reveals that transition

1

metal ions have been subjected to detailed investigations. Nowadays,

very large number of ligands are widely used with many number of

transition metal ions. Research on higher dimensional compounds

such as multinuclear complexes, cluster compounds and solid-state

inorganic compounds in which metal atoms and ligands are bonded in

a complex manner is becoming much easier. Most of the molecular

compounds of transition metals are metal complexes and

organometallic compounds in which ligands are coordinated to metals.

These molecular compounds include not only mononuclear complexes

with a metal centre but also multinuclear complexes containing several

metals, or cluster complexes having metal-metal bonds. Among the

various types of ligands, the heterocyclic bases containing oxygen and

nitrogen donors are considered to be the potential ligand centres for the

coordination of the metal atom. In the recent years, a variety of ligands

have been studied are the ligands containing oxygen and nitrogen as

donor [8-14]. Due to this, the coordination chemistry of nitrogen-

oxygen donor ligands has made a considerable interest in the field of

research. The Mannich and Schiff base compounds generally contain

the nitrogen, oxygen and sulphur donor atoms as donor and these

types of compounds exhibits a wide range of biological properties such

as antibacterial, antifungal [15-28], anti HIV [19-23 & 29-30], antiviral

[31-32], anticonvulsant [33-36], antitubercular [37-39] and anticancer

[40-42].

Mannich Reaction

The Mannich reaction is a classical method for the preparation of

β-amino ketones and aldehydes. Mannich reaction is one of the most

2

important basic reaction types in organic chemistry. It is the key step

in the synthesis of numerous pharmaceuticals and natural products.

The amino alkylation of CH-acidic compounds was described by

several authors as early as the 19th century. However, it was Carl

Mannich, who was the first to recognize the enormous significance of

this reaction type, and it was he who extended the chemistry into a

broad based synthetic methodology through systematic research. Since

then this reaction that now carries his name has developed into one of

the most important C C bond forming reactions in organic chemistry

[43-44].

Thus, the Mannich reaction is a three-component reaction, which

involves the condensation of a compound capable of supplying one or

more active hydrogen atoms with an aldehyde (usually formaldehyde)

and an N-H derivative (ammonia, any primary or secondary amine or

amide) in the presence of an acid to give β-amino carbonyl compounds.

In the Mannich reaction, the Mannich base is the end product, which is

a nucleophilic addition of an amine to a carbonyl group followed by

dehydration to the Schiff base.

The activation of aldehyde, primary or secondary amines or

ammonia are employed in the above reaction. For forming the

intermediate enamine, the tertiary amines are not used due to the lack

of an N–H proton. The nucleophiles (α-CH-acidic compounds) employed

for this reaction include carbonyl compounds, nitriles, acetylenes,

3

aliphatic nitro compounds, α-alkyl-pyridines or imines. It is also

possible to use activated phenyl groups and electron-rich heterocycles

such as furan, pyrrole, thiophene and indole particularly as active

substrate.

The most commonly used solvents for the Mannich reaction are

alcohols (ethanol, methanol and isopropanol), water and acetic acid.

The reaction is usually carried out by mixing the reactants in equimolar

amounts. However, an amine and aldehyde are allowed to react first

followed by the elimination with the substrate. There is no general rule

in selecting the reagents and reaction condition.

Importance of Mannich Bases

In organic synthesis, Mannich bases and their derivatives

exhibits a wide range of applications. Mannich bases are very reactive.

They can easily be transformed into numerous other compounds.

Several pharmacological properties can be obtained on account of

the reactivity of the Mannich bases. Many amino alkyl derivatives have

been prepared in order to correlate their structure and reactivity with

their pharmacological activities [45]. The Mannich synthesis introduces

a basic function which can render the molecules soluble in aqueous

solvents when it is transformed into the iminium salt.

Mannich bases also act as important pharmacophores or

bioactive leads which are further used for synthesis of various potential

agents of high medicinal value which possess amino alkyl chain. The

examples of clinically useful Mannich bases which consist of amino

alkyl chain are cocaine, fluoxetine, atropine, ethacrynic acid,

4

trihexyphenidyl, procyclidine, ranitidine, biperiden [46-48], and so

forth.

Mannich bases are known to play a vital role in the development

of synthetic pharmaceutical chemistry.

Structures of some pharmacologically important Mannich bases

are given in the figures a, b, c, d, and e.

OMe

OH

NMe2

Tramadol (Analgesic)

(a)

N

Osnervan (Antiparkinsonic)

OH

(b)

OnPr

O

N

Falicain (Anesthetic)

(c)

NH

Et

MeN

O

O

Moban (Neuroleptic)(d)

NH

OHO

Be - 2254 (Antihpertensive)(e)

Besides the biological activities, Mannich bases are also known

for their uses: detergent additives, resins, polymers, surface active

5

agents, and so forth. Pro drugs of Mannich bases of various active

compounds have been prepared to overcome the limitations [49].

Mannich bases (optically pure chiral) of 2-naphthol are employed

for catalysis (ligand accelerated and metal mediated) of the enantio

selective carbon-carbon bond formation. Mannich bases and their

derivatives are intermediates for the synthesis of bioactive molecules

[50, 51].

Mannich reaction is widely used for the construction of nitrogen

containing compounds [52]. Mannich bases are used in agrochemicals

as plant growth regulators.

Denton and his co-workers [53] indicated that the reduction of

the piperidino Mannich bases to the corresponding amino alcohols led

to the increase in antispasmodic activity.

Mannich bases derived from bis-[2-chloroethyl]-amine, melamine

or ethyldiamines are found to possess antimicrobial and cardio tonic

activities [54-56].

It has been reported that the aminobenzylated Mannich bases of

3-bromobenzaldehyde with suitable secondary amines and the

compounds containing amide moiety were found to possess many

interesting biological properties [57].

GENERAL SURVEY OF THE LIGAND CONSTITUENTS

Aldehydes

Survey of literature reveals that a large number of reports are

available for the synthesis of Mannich reaction by using aldehydes

other than Formaldehyde. Aromatic aldehydes, aliphatic aldehydes and

6

heteroaldehydes are some of the types of aldehydes which are used in

the Mannich reaction. A large number of reports are available in the

literature, benzaldehyde as a reactant in the synthesis of Mannich

Base. Much work has been carried out by using chloro, bromo, nitro

and amino substituted benzaldehydes.

A survey on the literature [58] shows that in the orientation of

the Mannich base reaction, the unsymmetrical dialkylketone,

secondary aliphatic amine and formaldehyde condensation takes place

mainly on the C – atom having less number of carbon unless this C is

sterically hindered. But in the case of aromatic aldehydes, due to steric

hindrance, the reaction seems to take place on the C - atom which are

having more number of hydrogen atoms. The second bulk aldehydic

group cannot remove other hydrogen atoms to incorporate itself and

hence the reaction does not proceed further.

Amines

Amines and their derivatives are important functional groups in a

variety of natural products. Drugs based on amine moieties have been

extensively used in the pharmaceutical industries [59]. Amines and

their derivatives act as precursors to a variety of biologically active

compounds like pharmaceuticals and agrochemicals [60]. The direct

reductive amination of aldehydes and ketones is one of the most useful

methods for the synthesis of secondary and tertiary amines [61].

Among the various types of organic compounds, amines constitute an

important type by replacing one or more hydrogen atoms of ammonia

molecule by alkyl or aryl group. The Nitrogen atom of amines carries an

unshared pair of electrons and it is trivalent. The lower aliphatic

amines can form hydrogen bonds with water molecules and hence they

7

are soluble in water. However, solubility decreases with the increase in

molar mass of amines due to the increase in size of the hydrophobic

alkyl part and will cause the intermolecular association in the primary

and secondary amines. This is due to the hydrogen bonding between

nitrogen of the one and nitrogen of the other molecule.

This intermolecular association is more in primary amines than in

secondary amines as there are two hydrogen atoms available for

hydrogen bond formation in it. But in the case of tertiary amines, the

intermolecular association does not happens, due to the absence of

hydrogen atom available for hydrogen bond formation. The reaction of

the amines can be decided by the number of hydrogen atoms attached

to the nitrogen atoms and in this way only, the primary, secondary and

tertiary amines differ with each other in the reactions. Due to the

presence of unshared electron pair in the amines, they act as

nucleophiles. In the case of aniline or other aryl amines, the –NH2

group is attached directly to the benzene ring and it makes less for

protonation. In most of the Mannich reactions, the amines used are

either primary or secondary amine. But secondary amines are preferred

in Mannich base synthesis since it possess only one replaceable

hydrogen atom (R2NH). The product with primary amine is secondary

amine, which further reacts to give secondary amine [62].

The Mannich donors like ketones or aldehydes, will have the

chiral amines. The resulting enamine can attack a Mannich acceptor,

usually a pro chiral aldimine, by introducing one or two chiral centres

in the Mannich base. Several acyclic chiral amines, amino acids and

amides are served as amines in the synthesis of Mannich bases. In the

8

present study Morpholine has been employed as an amine for the

synthesis of Mannich Bases.

Active Hydrogen atom

A large number of reports are available by employing active

hydrogen atom as a substrate in the synthesis of Mannich base. The

amide moieties of the organic compounds has placed a significant role

in biology, because they are the repeating unit of polypeptide

macromolecules. The coordination chemists has made a considerable

interest on these polypeptide macromolecules, because they serve as

models for peptide interaction and to metalloenzymes [63, 64]. The

N – amino methylation of acyclic carboxamides has been carried out

with primary and secondary amines [65, 66].

A number of reports available in the literature using amide

moieties as substrates for the synthesis of Mannich base. A study

concerning amino methylation reaction in several benzamides and

chloroacetamides established that the reaction only takes place with

sufficiently basic amines or with amides derived from sufficiently strong

carboxylic acids [67] have synthesized Mannich bases employing

thiourea and thiocarboxamides as substrates.

Many researchers have employed acetamide, urea, thiourea and

their derivatives as a compound containing active hydrogen for the

synthesis of Mannich bases [68-78]. Besides, semicarbazide, nicotinic

acid hydrazide, benzohydrazide and piperazine carboxamide are also

employed as substrate in the Mannich base synthesis in recent years.

Considerable interest has been paid on the synthesis of Mannich

bases using heterocyclic derivatives, many new drugs has been

9

developed. A large number of pharmacological activities are present in

the heterocyclic compounds such as Pyrrole, furan and thiophene and

their derivatives. The potential entity of the heterocyclic compounds

which possess the pharmacological characteristics has been

established by the thiophene nucleus. The medicinal chemists has paid

a considerable interest on the physicochemical parameters of the

heterocyclic compounds in order to produce combinatorial library and

carry out exhaustive efforts in the search of lead molecules.

A probe into the literature clearly reveals that aldehydes like

formaldehyde, benzaldehyde and substituted benzaldehyde are used

along with secondary amines such as piperidine and piperazine etc.,

and a compound containing an active hydrogen atom like alkyl ketones,

phenols, carboxylic acid derivatives, heterocyclic compounds, alkynes

and amides are used for the synthesis of the Mannich bases. It has

been found that, a few reports are available for the synthesis of

Mannich bases using thiophene-2-carboxaldehyde and pyridine-3-

carboxaldehyde as the component.

Based on the above considerable facts, the present study is

aimed at the synthesis of Mannich bases by reacting Furfuraldehyde,

Thiophene-2-Carboxaldehyde and Pyridine-3-Carboxaldehyde,

Morpholine as an amine and the compounds such as semicarbazide,

nicotinic acid hydrazide and phenyl urea as the compound containing

active hydrogen atom. Employing the synthesized product of the

reactants as ligand, metal complexes of Mn (II), Co (II), Ni (II), Cu (II),

and Zn (II) have been prepared.

10

Major applications of the ligand constituents

Semicarbazide

Semicarbazide is prepared by treating urea with hydrazine [79].

OC (NH2)2 + N2H4→ OC (NH2) (N2H3) + NH3

Many derivatives of Semicarbazide and their metal complexes are

found as efficient drugs against influenza, tuberculosis and some kinds

of tumours. Semicarbazide is used in preparing pharmaceuticals

including nitro furan anti bacterials (furazolidone, nitrofurazone,

nitrofurantoin) and related compounds. Semicarbazide is used as a

detection reagent in thin layer chromatography (TLC). Semicarbazide

stains α-keto acids on the TLC plate, which must then be viewed under

ultraviolet light to see the results.

Clinical use of Semicarbazide (a SSAO-inhibitor) gives the

evidence that an inflammatory reaction can be reduced by blocking the

enzymatic activity of the enzyme Semicarbazide-sensitive amine oxidase

(SSAO). SSAO activity was found significantly increased in blood and

tissues in some pathological conditions. The enzyme activity has been

reported to be elevated in diabetes and cancer. The mean specific

activity of SSAO was significantly elevated in the group of patients

having prostate cancer with skeletal metastases. Semicarbazide (and

SSAO blockers) can reduce inflammatory response and can protect

against the progressive vascular complications caused by oxidative

stress. It also can reduce pain. The SSAO inhibitors also appears able

to protect endothelial cells against toxic effects of free radicals [80].

Semicarbazide itself is a standard enzyme inhibitor and new SSAO

inhibitors are in development. SSAO inhibitors significantly blocked the

11

catalytic activity of VAP-1 in tumour, attenuated tumour progression,

and reduced neo-angiogenesis [81]. Semicarbazide is a known inhibitor

of glutamic acid decarboxylase (GAD), the enzyme responsible for GABA

synthesis. GABA has emerged as a tumour signalling molecule in the

periphery that controls the proliferation of tumour cells and perhaps

tumour stem cells [82].

Morpholine

Morpholine is a compound having the chemical formula

O(CH2CH2)2NH. Morpholine may be produced by the dehydration of di

ethanolamine with sulphuric acid [83].

Morpholine is a common additive, in parts per

million concentrations, for pH adjustment in both fossil

fuel and nuclear power plant steam systems. Morpholine is used

because its volatility is about the same as water, so once it is added to

the water, its concentration becomes distributed rather evenly in both

the water and steam phases. Its pH adjusting qualities then become

distributed throughout the steam plant to provide corrosion protection.

Morpholine is often used in conjunction with low concentrations

of hydrazine or ammonia to provide comprehensive all-volatile

treatment chemistry for corrosion protection for the steam systems of

such plants. Morpholine decomposes reasonably slowly in the absence

of oxygen at the high temperatures and pressures in these steam

systems.

12

Morpholine undergoes most chemical reactions typical for other

secondary amines, though the presence of the ether oxygen withdraws

electron density from the nitrogen, rendering it less nucleophilic (and

less basic) than structurally similar secondary amines such

as piperidine. For this reason, it forms a stable chloramine [84].

It is commonly used to generate enamine [85]. Morpholine is

widely used in organic synthesis. For example, it is a building block in

the preparation of the antibiotic linezolid, the anticancer agent gefitinib

(Iressa) and the analgesic dextromoramide. In research and in industry,

the low cost and polarity of Morpholine lead to its common use as a

solvent for chemical reactions.

Morpholine is used as a chemical emulsifier in the process of

waxing fruit. Naturally, fruits make waxes to protect against insects

and fungal contamination [86].

Furfuraldehyde

Furfural is an organic compound derived from a variety of

agricultural by products, including corn, cobs, oat, wheat bran,

and sawdust. Furfural is a heterocyclic aldehyde, with the ring

structure. Its chemical formula is OC4H3CHO.

Furfural is an important renewable, non-petroleum based,

chemical feedstock. Hydrogenation of furfural provides furfuryl

alcohol (FA), which is a useful chemical intermediate and which may be

further hydrogenated to tetra hydro furfuryl alcohol (THFA). THFA is

used as a non-hazardous solvent in agricultural formulations and as an

adjuvant to help herbicides penetrate the leaf structure. It is used to

13

make other furan chemicals, such as furoic acid, via oxidation [87] and

furan itself via palladium catalyzed vapour phase decarbonylation [88].

Biological importance and Coordination Chemistry of Mn (II),

Co (II), Ni (II), Cu (II) and Zn (II) Metal Ions

Transition metal ions play an important role in the living systems

as metal binding systems. The active research of metal complexes has

made a considerable interest in the field of biochemical reactions. Many

researchers has made an interest on the role of metal ions in drug

metabolism, biology and cross linking of biomolecules from the various

disciplines such as chemistry, biology, medicine and agriculture.

Copper

Copper proteins have diverse roles in biological electron transport

and oxygen transportation, processes that exploit the easy inter

conversion of Cu (II) and Co (II) [89-92] the biological role for copper

commenced with the appearance of oxygen in earth's atmosphere [93].

The protein hemocyanin is the oxygen carrier in most mollusks and

some arthropods such as the horseshoe crab (Limulus polyphemus)

[94], because, hemocyanin is blue, these organisms have blue blood,

not the red blood found in organisms that rely on hemoglobin for this

purpose. Structurally related to hemocyanin are the laccases and

tyrosinases. Instead of reversibly binding oxygen, these proteins

hydroxylate substrates, illustrated by their role in the formation of

lacquers [95].

Copper is also a component of other proteins associated with the

processing of oxygen. In cytochrome c oxidase, which is required for

aerobic respiration, copper and iron cooperate in the reduction of

14

oxygen. Copper is also found in many superoxide dismutases, proteins

that catalyze the decomposition of superoxides, by converting it (by

disproportionation) to oxygen and hydrogen peroxide.

2HO2→ H2O2 + O2

Several copper proteins, such as the "blue copper proteins", do

not interact directly with substrates, hence they are not enzymes.

These proteins relay electrons by the process called electron transfer.

Photosynthesis functions by an elaborate electron transport

chain within the thylakoid membrane. A central "link" in this chain is

plastocyanin, a blue copper protein.

Copper, like all metals, forms coordination complexes with

ligands. In aqueous solution, copper (II) exists as [Cu (H2O) 6]2+. This

complex exhibits the fastest water exchange rate (speed of water

ligands attaching and detaching) for any transition metal aquo

complex. Adding aqueous sodium hydroxide causes the precipitation of

light blue solid copper (II) hydroxide. A simplified equation is:

Cu2+ + 2 OH−→Cu (OH) 2

Aqueous ammonia results in the same precipitate. Upon

adding excess ammonia, the precipitate dissolves, forming

tetraamminecopper (II):

Cu (H2O) 4(OH) 2 + 4 NH3→ [Cu (H2O) 2(NH3)4]2+ + 2 H2O + 2 OH−

Many other oxyanions form complexes; these include copper (II)

acetate, copper (II) nitrate, and copper (II) carbonate.Copper (II) sulfate

15

forms a blue crystalline pentahydrate, which is the most familiar

copper compound in the laboratory.

Cobalt

Cobalt is essential to all animals. It is a key constituent of

cobalamin, also known as vitamin B12, which is the primary biological

reservoir of cobalt as an "ultra trace" element [96, 97]. Bacteria in the

guts of ruminant animals convert cobalt salts into vitamin B12, a

compound which can only be produced by bacteria or archaea. The

minimum presence of cobalt in soils therefore markedly improves the

health of grazing animals, and an uptake of 0.20 mg/kg a day is

recommended for them, as they can obtain vitamin B12 in no other way

[98]. The cobalt deficiency was overcome by the development of "cobalt

bullets", dense pellets of cobalt oxide mixed with clay, which are orally

inserted to lodge in the animal's rumen.

Manganese

Manganese is a metal with important industrial metal alloy uses,

particularly in stainless steels.

In biology, manganese (II) ions function as cofactors for a large

variety of enzymes with many functions [99]. Manganese enzymes are

particularly essential in detoxification of superoxide free radicals in

organisms that must deal with elemental oxygen. Manganese also

functions in the oxygen-evolving complex of photosynthetic plants. The

element is a required trace mineral for all known living organisms but

is a neurotoxin. In larger amounts, and apparently with far greater

effectiveness through inhalation, it can cause a poisoning syndrome in

mammals, with neurological damage which is sometimes irreversible.

16

Nickel

Nickel (II) forms a very large number of complexes with a variety

of structures. Six-coordinated octahedral, five coordinated trigonal,

bipyramid and square pyramid and four coordinated tetrahedral and

square planar structures are some of them.

In aqueous solution, Ni2+ is present as [Ni (H2O)] 2+. The ligands

will easily replace the water molecules. In biological chemistry, Nickel is

a versatile metal and it is necessary of certain metallo-proteins. Nickel

acts as a tool for the study of the nucleic acid structure which is in the

form of tetraza macrocyclic complexes. Nickel plays a significant role in

pigmentation of metals which are found in ribo-nucleic acids.

Zinc

Zinc is the only transition metal, which requires by at least one

enzyme in each of the major classes of enzymatic activities. Zinc is a

versatile metal. It predominately forms four-coordinated tetrahedral

complexes. Complexes like [Zn (NH3)4]2+, [Zn (NH3)2Cl2], [Zn (Py)2 Cl2],

[Zn (CN) 4]2- are all tetrahedral. Zinc is a co-factor in a number of

enzyme systems. Zinc acts predominately as a Lewis acid and is found

in many metalloenzymes such as carboxypeptidase and carbonic

anhydrase for the following reasons:

1. Zinc (II) is active in many model systems.

2. Zinc (II) has no redox behaviour associated with it under

biological conditions.

3. The ligand exchange processes on Zinc (II) are rapid so that

substrates produced can be easily introduced and can be

removed.

17

Carbonic anhydrase, a Zinc metalloenzyme present in the

animals and plants, catalyses the interconversion of carbondioxide and

carbonates. Large number of diseases are associated with lowered zinc

level in blood. Various neurological diseases such as Alzheimer’s

disease, Parkinson’s disease, hypoxia-ischemia and epilepsy are closely

related to a disorder of zinc metabolism.

LITERATURE SURVEY OF MANNICH BASE AND ITS METAL

COMPLEXES

Metal complexes of Mannich base are formed by the strong ability

of the organic chelating ligands. These ligands contains amide moiety

which acts as a functional group. And also they exhibit a wide range of

biological properties such as bacteriocidal, fungicidal, herbicidal and

insecticidal activities [100-101].The amide group has two potential

binding sites viz., oxygen and nitrogen for complexation with metal ion.

Generally amide acts as a unidentate neutral ligand with oxygen

coordination to Lewis acid; however in the form of amidate anion

coordination takes place through nitrogen. The structure of amide,

probably a resonance hybrid of the canonical structures me and II, is

shown below.

CONH2

RCO

NH2

R

Vartale et al. have synthesized three Mannich bases, viz., 3,4-

Dihydro-2-N-methylmorpholino/pyrrolinido/piperidino-8-methoxy-2H-

1,2,4-triazino[3,4-]benzothiazole-3,4-dione by refluxing 3,4-Dihydro-8-

methoxy-2H-1,2,4-triazino[3,4-b]benzothiazole-3,4-dione, formaldehyde

18

and morpholine/ pyrolidene / piperidine with catalytic amount of

dioxane the compounds have been characterized by spectral studies.

Further, the compounds were screened for their antibacterial activities

against gram-positive and gram-negative bacteria [102].

Transition Metal Complexes of N-(1-Piperidinosalicylyl) acetamide

(PSA) and their Biological activities were studied by D. Sathya et al. IR

study of the PSA and its complexes show involvement of the nitrogen

atom from the piperidine ring and oxygen atom of amide are in

coordination with the metal ion. The electronic spectra of the metal

complexes suggest distorted octahedral structure for Cu (II) complex,

tetrahedral geometry for Co (II) complex, square planar geometry for Ni

(II) and Zn (II) complexes.

Mannich reaction of 4-Chlorobenzaldehyde, 4-aminopyridine, and

urea reported by G. Vishnuvardhanaraj et al., Der Chemica Sinica,

2013, 4(3).

19

Idhayadhulla et al. have synthesized six Mannich bases by fixing

Morpholine as an amine and changing the aldehyde and active

hydrogen compound. Elemental analysis and spectral studies have

been carried out to establish the structures of the compounds. The

synthesized compounds were screened for antimicrobial studies.

Structure activity relationship (SAR) has also been studied.

Bui Trung Hieu et al. prepared and characterized a series of

novel Mannich bases including (E)-1-(4-Chlorophenyl)-3-(4-

hydroxy-3-morpholine-4-yl-methyl-phenyl)-propenone. The synthesized

compounds were screened for their In vitro cytotoxic activity against the

human hepta cellular carcinoma, human lung carcinoma and human

breast cancer.

N-(morpholinobenzyl)benzamide, a Mannich base has been

prepared by the condensation of morpholine, benzamide and

benzaldehyde, and its Cu(II), Co(II), Ni(II) and Zn(II) complexes have

been synthesized by Raman et al. All the products have characterized

by spectral studies, magnetic moment, cyclic voltammetry and

conductance methods. All the complexes exhibit square planar

geometry. The synthesized Mannich base and its complexes have been

screened for their antimicrobial activities. The complexes have higher

antimicrobial activities than that of the free Mannich base and the

control.

Kadry et al. have synthesized two series of novel Mannich bases

such as1-(4-hydroxy-3-(morpholinomethyl) phenyl)-3-(1H-indole-3-yl)

prop-2-en-1- one and 1-(4-bromophenyl)-3-(4-hydroxy-3-methoxy-

5(morpholinomethyl) phenyl) prop-2-en-1-one. These compounds have

20

been characterized by elemental analysis, spectroscopic method and

chromatographic techniques and evaluated for their cytotoxic activity.

All the tested compounds exhibited a broad spectrum of antitumor

activity against renal cancer UO-31.

Mannich reaction of benzaldehyde, piperidine and semicarbazide

reported by * Dr. S. Ravichandran*, et al., Asian Journal of Biochemical

and Pharmaceutical Research Issue 3 (Vol. 1) 2011.

A Mannich base, N-[1-morpholino( 4-nitrobenzyl)] acetamide,

derived by the condensation of morpholine, 4-nitrobenzaldehyde and

acetamide, and its Cu(II), Co(II), Ni(II) and Zn(II) complexes have been

synthesized by Ravichandranet al. Both the ligand and the complexes

were characterized by molar conductance, magnetic susceptibility,

cyclic voltammetry and spectral studies. The antimicrobial activities of

the ligand and the complexes have also been studied and found that all

the complexes had higher activity than the free ligand and their

standard.

Kasim et al. have synthesized two Mannich bases viz. N, Nʹ-bis

(1- piperidino-4-methylbenzyl) urea by the condensation of urea,

piperidine and 4- methylbenzaldehyde and N, Nʹ-bis (1-piperidino-4-

chlorobenzyl) urea by the condensation of urea, piperidine and p-

chlorobenzaldehyde. These synthesized compounds have been

21

characterized on the basis of micro analytical and spectral studies.

Further these compounds were screened for their antimicrobial

activities against gram-positive and gram-negative bacteria.

Chakkaravarthy et al. have synthesized Mannich bases, such as

1- ((1H-benzo[d]imidazole-1-yl) methyl) urea (4) by the condensation

urea, benzimidazole and formaldehyde and 1-((3-hydroxynaphthalne-2-

yl) methyl) thiourea (5) by the condensation of thiourea, β-naphthol and

formaldehyde and characterized them by elemental analysis and

spectral studies.

4 5

The synthesized Mannich bases were screened for their

antimicrobial and anti-oxidant activity. The structure and biological

activity relationship denotes the higher activity in compound 4 may be

due to the presence of two N atoms in the benzimidazole adjoin with

amide group. The antioxidant activities of the synthesized Mannich

bases are due to the presence of electron releasing amide group in it. In

the above studies compound 4 shows better activities compared to the

compound 5.

Vasoya et al. have reported the synthesis of Mannich base by the

condensation of urea, piperidine/morpholine/indole/N-

methylpiperazine and 3-phenoxybenzaldehyde. The structure was

22

elucidated from elemental analysis and spectral data. It was screened

for its in-vitro antitubercular and antimicrobial activities.

R = piperidine/morpholine/indole/N-methylpiperazine

Aim and scope of the work

The organic synthetic researchers are being undertaken with an

aim of designing new routes based on the available literatures. A novel

series of compounds are prepared and their biological activities and

their pharmacological activities have been done for the newly

synthesised compounds. In the present work, the researcher, has made

an attempt to synthesise and characterize some Mannich bases, by

keeping Morpholine as a fixed reactant and by reacting this with

aldehydes namely Thiophene-2-Carboxaldehyde, Furfuraldehyde and

Pyridine-3-Carboxaldehyde by using substrates such as Nicotinic acid

hydrazide, Phenyl Urea and Semicarbazide. Various organic solvents

were used for this study. The present work is described in the following

chapters:

Chapter II

Synthesis and Characterization of Mn (II), Co (II), Ni (II), Cu (II)

and Zn (II) metal complexes of 2-[(morpholin-4-yl) (pyridin-3-yl)

methyl]hydrazinecarboxamide (MPH).

23

Chapter – III

Synthesis and Characterization of Co (II), Ni (II), Cu (II) and Zn (II)

metal complexes of (Morpholino (thiophen-2-Yl) methyl) nicotino

hydrazide (MTN).

Chapter - IV

Synthesis and Characterization of Co (II), Ni (II), Cu (II) and Zn (II)

metal complexes of 1-(furan-2-yl) (morpholino) (methyl)-3-phenyl urea

(MFP).

Chemicals used

All the chemicals used were of Merck and Sigma Aldrich

products, which are available commercially. The purchased chemicals

were used without any further purification.

Characterization techniques used

The synthesised ligands and their metal complexes have been

characterised by the following techniques.

1. Elemental Analysis.

2. Thin Layer Chromatography.

3. Infrared Spectroscopy.

4. Ultraviolet-visible Spectroscopy.

5. Nuclear Magnetic Resonance Spectroscopy.

6. Electron Paramagnetic Resonance Spectroscopy.

7. Mass spectroscopy.

8. Cyclic Voltammetry.

9. Magnetic Susceptibility Measurements.

10. Molar Conductance Measurements.

11. Thermal Analysis (TGA-DTA).

24

12. Thermo acoustical studies.

13. Antimicrobial and Molecular Docking Studies.

Elemental Analysis

The chemical analysis is quite helpful in fixing the stoichiometric

composition of the ligand as well as its metal complexes. The elemental

analysis is carried out to find out the molecular formula. The sample

was encapsulated in a thin tin foil and it is dropped into the furnace at

a temperature about 900˚ C. On ignition, the C, H, N and S present in

the sample converts to their respective combustion products. The

percentage of elements C, H, N and S is worked out by using Vario EL

III analyser available at the Sophisticated Test and Instrumentation

Centre, Cochin University, Kerala. By using the ACD/ Chemsketch /

Chemdraw / Freeware software, the elemental structures were drawn

based upon the elemental composition. By using the volumetric,

gravimetric and spectrophotometric methods, the metal content of the

complex were estimated.

Thin Layer Chromatography

Due to the extent separation and application of the compounds

in organic chemistry, the Thin Layer Chromatography is used as an

analytical tool to check the purity of the synthesised compounds by

using appropriate solvent as eluent.

Infrared Spectroscopy

In general, the spectra give sufficient information about the

structure of the compound. When Infrared light is passed through a

sample, some of the frequencies of the sample are absorbed while other

frequencies of the sample are transmitted through the sample without

25

being absorbed. The resulting plot of percent transmittance or percent

absorbance against frequency is an infrared spectrum. The IR

spectroscopy is widely used as a characterization technique for metal

complexes. The basic theory involved is that the stretching modes of

the ligands changes upon complexation due to weakening or

strengthening of the bonds involved in the bond formation resulting in

subsequent change in the position of the bands appearing in the IR

Spectrum. The changes in the structural features of the ligands are

observed as changes in bands observed, mainly in the fingerprint

region (4000-400 cm-1). The bands due to the metal ligand bonds are

mainly observed in the far IR region (600-100 cm-1).

For amines, the N-H stretching vibrations occur in the region

3300-3500 cm-1 in the dilute solution. Many workers have reported

medium intensity bands for (C-O-C) of furan ring vibrations in the

region 1020-1250 cm-1.The M-N stretching frequency is of particular

interest since it provides direct information regarding metal-nitrogen

coordinate bond. Different amines complexes exhibited the metal-

nitrogen frequencies in the 428-530 cm-1 region. Ketones, aldehydes,

carboxylic acids, carboxylic esters, lactones, acid halides, anhydrides,

amides and lactams show a strong stretching absorption band in the

region of 1870-1540 cm-1. Its relatively constant position and high

intensity and relative freedom from interfering bands make it as one of

the easiest band to recognize in infrared spectra.

Infrared spectra of the complexes were recorded on a Thermo

Nicolet Avatar 370 DTGS model FT-IR Spectrophotometer with KBR

pellets at St. Joseph’s College (Autonomous), Tiruchirappalli,

Tamil Nadu, India. For some of the complexes, the IR spectra were

26

recorded on a FT-IR Shimadzu IR Affinity-1 Spectrophotometer in the

range of 4000-400 cm-1 by using KBR pellets at Jamal Mohamed

College (Autonomous), Tiruchirappalli, Tamil Nadu, India.

Ultraviolet Spectroscopy

Most of the chemists use Ultraviolet spectroscopy as a valuable

tool to know about the important structural aspects of the complex.

The ligands which are considered as the organic compounds, shows

their absorption bands in the ultraviolet region of 200 – 300 nm of the

electromagnetic spectrum. Due to conjugation in some cases, these

bands extend over to higher wavelength region. In the transition metal

complex ions, there will be an interesting change in the electronic

properties of the system, due to the interaction with the metal ion. Due

to d-d absorption and charge transfer spectra from metal to ligand or

ligand to metal, new features or bands in the visible region can be

observed and this data can be processed to obtain information

regarding the structure and geometry of the compounds (38/EXPTL.

TECH).We can get the information about the possible distortions of

symmetry environment, magnitude of the ligand-field splitting (10 Dq),

certain bonding characteristics (charge-transfer bands) etc., from the

analysis of the spectrum of the complex.

The UV Spectrum of the compounds and complexes of the study

were recorded on Perkin Elmer, Lambda 35 model in the range

190-1100 nm at ACIC, St. Joseph’s College (Autonomous),

Tiruchirappalli.

27

Nuclear Magnetic Resonance Spectroscopy

1H NMR

The transitions between the magnetically inducted spin states are

known by the use of the NMR study. This study is concerned with the

magnetic properties of atomic nuclei with an integral value I. The

protons in an organic compound exposed to a powerful field, by the use

of this technique. The protons will process at different frequencies.

These processing protons are irradiating with steady changing

frequencies and observe the frequencies at which absorptions occur

and the signals obtained corresponding to the absorption is known as

NMR Spectrum. NMR spectroscopy is widely used to study the molecule

and it enables us to record the differences in the magnetic properties of

various magnetic nuclei present and to deduce the positions of this

nucleus within the molecule. By the use of the NMR spectroscopy, we

can deduce how many different kinds of environments there are in the

molecule and also which atoms are present in neighbouring groups. In

general, the number of atoms present in each of these environments is

measured. Therefore, the diagnostic features of the NMR Spectra are

the number of signals, position of signals, splitting pattern of signals

and area of signals. 1H NMR of the ligands were recorded using Bruker

300 MHz Avance–II FT-NMR Spectrometer with DMSO-d6 as the solvent

and TMS as internal standard at CARISM, SASTRA University,

Thanjavur, Tamil Nadu.

13C NMR

1H NMR and 13C NMR spectra differs both in the mode of

recording as well as in the appearance. The spin quantum number,

I for 12C is equal to zero since 12C isotope has an even number of

28

protons and even number of neutrons and hence no magnetic spin. It

is, therefore, non-magnetic and does not give any NMR signal. The

natural abundance of 13C is only about 1.1% and has an odd number

of neutrons. So, 13C has a spin quantum number equal to ½ and its

nuclear magnetic resonance can be observed in a magnetic field of

23,500 gauss at 25.2 mega cycles per second. 1H spectrum is normally

obtained by sweeping either the excitation frequency or the through the

region of precession frequencies. The inefficiency of this method is clear

from the fact that only one line can be observed at a given point in

time. The problem arises when 13C with intrinsically narrow lines

covering a wide absorption range is studied. It is, therefore,

advantageous to excite the whole band of frequencies simultaneously. It

is done by strong pulse of radio-frequency covering a large band of

frequencies which is capable of exciting all resonance of interest at

once. At the end of the pulse period, the nuclei will process freely with

their characteristic frequencies reflecting with the chemical

environment and exhibit chemical shifts. 13C NMR of the synthesized

compounds were recorded on 400 MHz Bruker Spectrometer at 298.6 K

using DMSO as solvent at CARISM, SASTRA University, Thanjavur, and

Tamil Nadu.

Electron Paramagnetic Resonance Spectroscopy

Electron Paramagnetic Resonance Spectroscopy is otherwise

known as Electron Spin Resonance (ESR) and Electron Magnetic

Resonance (EMR) spectroscopy. This technique is used for the study of

systems having uncompensated electron spins and deals with the

transition between Zeeman levels. An unpaired electron that is not

subject to interactions with other unpaired electrons or with magnetic

29

nuclei will show a single sharp absorption for its transition which in

turn corresponds to the position of magnetic field at which it comes

into resonance . Electron Paramagnetic Resonance Spectroscopy is a

powerful technique and it can be used to locate the distribution of

unpaired electron in a molecule and to some extent, decide the extent

to which electrons are delocalized over the ligands. The study gives the

numerical values for the parameters in the spin Hamiltonian

components of ʽgʼ tensor, detectable hyperfine splitting and principle

components of the hyperfine tensor.

The resonant position of EPR is referred to as the ʽgʼ value and it

is directly determined by the separation of the energy levels of the

system under investigation. The variation of the ʽgʼ value is interpreted

in terms of the first and the second order perturbation by the spin orbit

interactions. Investigation of the correlation of the line width with

chemical nature of bonding is of special importance.

The EPR spectrum of Cu (II) complexes of the synthesized ligands

were recorded on Jeol Model JES FA 200 spectroscope in X- band

frequencies at Room Temperature at SAIF, IIT, Madras.

Mass spectroscopy

For determining the molecular mass of the compound, the Mass

spectrometry is used and it is considered as one of the most accurate

method. In this technique, molecules are bombarded with a beam of

energetic electrons.

The molecules are ionized and broken up into many fragments,

some of which are positive ions. Each kind of ion has a particular ratio

30

of mass to charge, i.e. m/z ratio. For most ions, the charge is one and

thus, m/z ratio is simply the molecular mass of the ion.

The set of ions are analyzed in such a way that a signal is

obtained for each value of m/z that is represented. The relative

abundance of the ion is represented by the intensity of each signal. The

largest peak in the structure is called the base peak and its intensity is

taken as 100. Mass spectrum of a compound is a plot which represents

the intensities of the signals at various m/e values.

The mass spectrums of the synthesized compounds were

recorded on Jeol GC mate II Mass spectrometer at SAIF, IIT, Chennai,

and Tamil Nadu.

Cyclic voltammetry

The redox behaviour of the coordinated complexes can be studied

by the use of the Cyclic Voltammetry. The Cyclic Voltammetry studies

gives an insight into the stability of the compound under investigation

against electrolytic oxidation and reduction in the solution. In this

technique, the potential of a small stationary working electrode is

changed linearly with the time, starting from a potential where no

electrode reaction occurs and moving to potentials where reduction or

oxidation of a sample occurs. After traversing the potential region in

which one or more electrode reaction takes place, the direction of the

linear sweep is reversed and the electrode reactions of the

intermediates and products formed during the forward scan often can

be detected. The CV technique can be carried out using a suitable

reference, working and counter electrode, the selection of which can be

made depending on the nature of the compound and solvent used in

31

the presence of a supporting electrolyte. By selecting a range of voltages

and the variation involtammogram can be recorded at different sweep

rates. The peaks in the forward and the reverse sweeps can be

interpreted to assess the stability of the species. Depending upon the

nature of the voltammogram obtained, they may be termed as

reversible (IPA=Ipc), quasi - reversible (IPA>Ipc) and irreversible

process.

Cyclic Volta metric of all of the synthesized complexes were

carried out on Princeton voltammetry Applied instrumentation in the

frequency range -1 Hz to +1 MHz at ACIC, St. Joseph's College

(Autonomous), Tiruchirappalli, Tamil Nadu.

Magnetic susceptibility

Magnetic susceptibility studies can be used in conjunction with

electronic spectra to establish the geometry of the complexes.

Substances which contain one or more unpaired electrons have a

permanent magnetic moment which exists in the absence of a magnetic

field and also arises from the net spin and orbital momentum of the

electrons. Two properties of an unpaired electron, the spin and orbital

moments contribute to the magnitude of the paramagnetic moment.

The magnetic susceptibility measurements of the complexes are

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

atom 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. From the knowledge of unpaired electrons it is possible

to infer the valence state of the metal ion.

32

The magnetic susceptibility measurements of the powdered

sample were done at room temperature using a Sherwood Scientific

Magnetic Susceptibility Balance at

Conductivity Measurements

The molar conductance value of an electrolyte in a particular

solvent depends upon the various properties of the solvent such as

dielectric constant, viscosity, specific conductivity, purity, ligating

tendency towards metal ions and the temperature of measurement. The

electrical conductance measurements were done to see whether the

anions of the metal salts remain inside or outside the coordination

sphere of the central metal ion.

Molar conductivities of the complexes in Dimethyl formamide

solution (10-3μ) were measured at room temperature using a systronic

model 303 direct reading conductivity bridge at the PG and Research

Dept. of Chemistry, Jamal Mohamed College, Tiruchirappalli.

Thermo Gravimetric Analysis

Thermal methods of analysis are techniques in which changes in

physical and or chemical properties of a substance are measured as a

function of temperature. From the simultaneous recordings of

temperature, weight and heat capacities, the TG and DTA curves are

drawn. From the analysis of the complexes one can understand the

modes of decomposition of the complexes and the nature of the phase

changes taking place during the programmed heating. The thermal

stabilities and the decomposition temperatures can also be ascertained.

In addition, the purity of the complexes can also be checked. This

technique examines the various changes like thermal decomposition,

33

oxidation, solvent and water desorption, evaporation, sublimation etc.

which may take place with a consequent change in weight of the

sample when heated at a desired heating rate with proper furnace

atmosphere.

TG-DTG analysis of the complexes were carried out in a Perkin

Elmer Pyris 6 TG/DTA analyser at the temperature rate of 10.0 °C/min

in an atmosphere of nitrogen at the Department of Chemistry, SAIF,

Chennai, Tamil Nadu.

Thermo acoustical studies

Ultrasonic studies finds application in several industrial and

technological processes. The Ultrasonic investigations of liquid

mixtures consisting of polar and non-polar components are of highly

importance in understanding the physical nature and strength of

molecular interaction in the liquid mixtures.

In the present study, the Ultrasonic velocity, density, adiabatic

compressibility and viscosity measurements have been carried out for

tertiary mixture for various concentrations at different temperatures in

the Department of Physics, Jamal Mohamed College (Autonomous),

Tiruchirappalli, Tamil Nadu.

Antimicrobial and Molecular Docking studies

A preliminary biological study has been made on the synthesized

compounds and their metal complexes. The biological study includes

antibacterial and molecular docking studies, toxicity assay, larvicidal

studies and docking studies.

34

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41

CHAPTER II

Synthesis and Characterization of Mn (II), Co (II), Ni (II), Cu (II) and Zn (II) metal complexes of 2-[(morpholin-4-yl)

(pyridin-3-yl) methyl] Hydrazinecarboxamide (MPH)

This chapter deals with the Synthesis and Characterization of

Mn (II), Co (II), Ni (II), Cu (II) and Zn (II) metal complexes of

2-[(morpholin-4-yl) (pyridin-3-yl) methyl] Hydrazinecarboxamide (MPH).

Materials and Methods

All the chemicals and solvents used, were of Merck and Sigma

Alrich products (Analytical Reagent grade) and were used without

further purification. The synthesized ligand and their metal complexes

were characterized by the respective techniques, as discussed in

Chapter I.

Synthesis and Characterisation of 2-[(morpholin-4-yl) (pyridin-3-yl)

methyl] Hydrazinecarboxamide (MPH)

Pyridine-3-carboxaldehyde, Morpholine and Semicarbazide were

taken in the molar ratio of 1:1:1 and were reacted as shown in the

Scheme 1. To the ethanolic solution of semicarbazide (1.5g, 25 mmol)

taken in a round bottom flask, morpholine (2.1 mL, 25 mmol) and

pyridine-3-carboxaldehyde (2.6 mL, 25 mmol) were added. The reaction

mixture was kept on a magnetic stirrer and stirred under ice cold

condition for 3 h, the yellow coloured solid formed was filtered and

washed several times with petroleum ether (40-60%) and then dried.

The crude solid thus obtained was dried and recrystallized from

ethanol. The recrystallized product was dried over vacuum.

42

HN

O

Morpholine +

N

OPyridine-3-carboxaldehyde +

H2NNH

O

NH2

Semicarbazide

NH

HN

O

H2N

N

N

O

2-[(morpholin-4-yl) (pyridin-3-yl) methyl] hydrazinecarboxamide

Scheme 1: 2-[(morpholin-4-yl) (pyridin-3-yl) methyl] hydrazinecarboxamide (MPH)

Synthesis of MnII, CoII, NiII, CuII and ZnII metal complexes of MPH

To the methanolic solution of MPH (2.36 g, 10 mmole), copper (II)

chloride (0.85g, 5 mmole) was added in the molar ratio of 2:1. The

reaction mixture was taken in a round bottom flask and kept on a

magnetic stirrer and refluxed at 90 °C with continuous constant

stirring. After 2 h, the product separated as a green solid was filtered

and washed several times with methanol and then dried. The same

procedure was employed to prepare MnII, CoII, NiII and ZnII complexes

with slight modification in the reaction conditions.

H2O

43

Characterization of MPH and its MnII, CoII, NiII, CuII and ZnII

complexes

Elemental analysis

After ascertaining the elements that are present in the

synthesised compound, the proportionate percentage of various

elements was found out. The observed and calculated percentage

composition of C, H, N and O elements of the ligand MPH and its MnII,

CoII, NiII, CuII and ZnII complexes and the values of the molar

conductance of the complexes are presented in Table 2.1.

From the observed C, H, N and O values, the molecular formula

of the compound 2-[(morpholin-4-yl) (pyridin-3-yl) methyl]

Hydrazinecarboxamide (MPH)was assigned as C11H17N5O2 which was in

agreement with the calculated value. The molar conductance of the

complexes are in the range of μc is 23-31 Ω-1 mol-1cm2, in 10-3 M

DMSO. From the molar conductance values observed, it has been

revealed that, all the chloro complexes are non-electrolyte and the

anion was coordinated to the metal(II) ion [1, 2].

44

Table 2.1: Elemental analysis and molar conductance of MPH and its metal complexes

Compound and

Mol. Formula

Colour (

Yield %) M.P. ˚C

Elemental analysis found %

(Calculated %) Ω-1 mol-1cm2

C H N O A Cl

M P H = L

C 1 1 H 1 7 N 5 O 2

Yellow

(80) 145

52.58

(52.26)

6.82

(6.50)

27.87

(27.15)

12.73

(12.35) -- -- --

[ M n L 2 C l 2 ] C 2 2 H 3 4 C l 2 N 1 0 MnO 4

Pinkish

Brown

(72%)

196 43.58

(43.38)

6.47

(6.35)

23.46

(23.35)

8.93

(8.78)

7.67

(7.84) 9.90

(9.78) 28

[ C o L 2 C l 2 ] C 2 2 H 3 4 C l 2 N 1 0 C oO 4

Dark

Brown

(82%

284 43.34

(42.95)

6.43

(6.35)

23.33

(23.28)

8.88

(8.74)

8.18

(8.36) 9.84

(9.74) 22

[ N i L 2 C l 2 ] C 2 2 H 3 4 C l 2 N 1 0 N iO 4

Green

(79%) 271

43.45

(43.32)

6.44

(6.28)

23.33

(23.28)

8.88

(8.78)

8.15

(8.28)

9.84

(9.76) 26

[ C u L 2 C l 2 ] C 2 2 H 3 4 C l 2 N 1 0 C uO 4

Green

(86%) 250

43.06

(42.94)

6.39

(6.24)

23.18

(22.94)

8.83

(8.74)

8.76

(8.94)

9.78

(9.63) 24

[ Z n L 2 C l 2 ] C 2 2 H 3 4 Cl 2 N 1 0 ZnO 4

Colourles

s (84%) 182

43.24

(42.40)

5.13

(5.28)

18.49

(18.39)

10.80

(10.50)

8.52

(8.63)

11.51

(11.63) 30

Infrared Spectra

In general, a comparison of the infrared spectrum of the ligand

and that of its complexes will be of much help to find out the atom or

atoms, through which the atoms are attached to the metal ion. Infrared

spectrum of the compound and its metal complexes were recorded as

KBr disc. The infrared spectra of the ligand MPH and its MnII, CoII, NiII

and CuII complexes are shown in the Figures 2, 3, 4, 5 and 6

respectively. The infrared characterisation frequencies of the ligand

MPH and its MnII, CoII, NiII, CuII and ZnII complexes are presented in the

Table 2.2. A sharp band appeared at 3407 cm-1 is due to free ν(NH) of

semicarbazide. Being a single band is observed, the νN-H might be

involved in hydrogen-bonding [3]. A band at 2990 cm-1 is assigned to

45

ν(CH) of aromatic. A sharp band appears at 1669 cm-1 is assigned to

ν(C=O). A band appeared at 1142 cm-1 is assigned to ν(CNC) of

morpholine.

Table 2.2: Characteristic Infrared bands of MPH and its metal (II) complexes (ν cm-1)

Compounds ν(N-H) νC=O) ν(CNC) ν (C-O) ν (C-H) ν(M-N) ν(M-O)

MPH = L 3407 1669 1142 -- 3048 -- --

[ M n L 2 C l 2 ] 3405 1627 1122 -- 3045 605 570

[ C o L 2 C l 2 ] 3405 1632 1115 4.77 3040 615 575

[ N i L 2 C l 2 ] 3405 1645 1120 3.35 3024 602 552

[ C u L 2 C l 2 ] 3400 1630 1157 1.90 3042 594 563

[ Z n L 2 C l 2 ] 3402 1630 1125 -- 3038 608 567

Electronic spectra

The electronic spectra of the synthesised ligand MPH and its

MnII, CoII, NiII, CuII and ZnII metal complexes was recorded in 10-3 M

solution of DMSO at the wavelength range of 250-1100 nm and their

corresponding data and spectra are given in Table 2.3. and

Figures 7 and 8 respectively.

For CoII complex three transitions appearing at 10540, 15231

and 21683 cm-1 are assignable to 4T1g→4T2g(F), 4T1g(F)→4A2g(F) and

4T1g(F)→4T2g(P) transitions respectively. The effective magnetic moment

observed for the complex is spin octahedral complex with the ground

state as 4T1g, which is in agreement with the reported literature [4-7].

The electronic spectrum of NiII complex displays two bands, one

at 26734 cm-1 and other at 33472 cm-1. These bands are attributed to

46

(L→M) charge transfer transitions. The measured magnetic moment

value 3.35 BM favours octahedral geometry for the complex. The

possibility of tetrahedral environment around the metal ion is ruled out

because there is no band appeared below 10000 cm-1.

For CuII complex, two bands appearing at 14372 cm-1 and 19542

cm-1 are corresponding to 2Eg→2T2g and 2B1g→2B2g transitions

respectively which is in good agreement with the octahedral geometry.

The magnetic moments of CuII complex is found to be 1.80 BM, suggest

that the complex is magnetically diluted and have distorted octahedral

geometry [8, 9].

The electronic spectrum of MnII complex shows three bands in

the region 16000-21500, 22000-25500 and 26000-28000 cm-1

corresponding to the transitions 4A1g→6A1g, 4Eg(D) →6A1g and 4Eg(D)

→6A1g respectively which suggest the octahedral geometry for this

complex [10, 11].

The spectrum of ZnII complex exhibited bands at 37230-36540

cm-1 may be attributed to L→M charge transfer and it is diamagnetic as

expected.

47

Table 2.3: Electronic Spectral bands of MPH and its metal complexes

Compounds

Magnetic

moment

BM

Absorption

maxima

ν (cm-1)

Transition

assignment Geometry

MPH = L -- 38608 32570 11020

π→π* n→ π* --

[MnL2Cl2] 5.89 21500 25500 28000

4A1g→6A1g, 4Eg→6A1g

4Eg→6A1g (P)

Octahedral

[CoL2Cl2] 4.78 10540 15231 21683

4T1g→4T2g 4T1g→4A2g

4T1g→4T2g(F) Octahedral

[NiL2Cl2] 3.35 26734 33472 LMCT Octahedral

[CuL2Cl2] 1.80 14372 19542

2Eg→2T2g 2B1g→2B2g

Octahedral

[ZnL2Cl2] 0 37230 36540 LMCT Octahedral

Nuclear Magnetic Resonance spectra

1H NMR spectrum of the free ligand MPH was recorded in DMSO-

d6 medium using TMS as an internal standard. The spectrum of the

synthesised compound is shown in Figure 9. This spectrum has been

compared with the spectrum of Chemdraw Ultra 12.0 software.

A singlet at δ 10.4 is assigned to NH2 protons of semicarbazide.

A multiplet appears at δ 7.37 to 7.41 is assigned to protons of pyridine.

A singlet at δ 2.50 is assigned to NH adjacent to CH. A doublet at

δ 3.95 is assigned to CH adjacent to NH. A triplet at δ 2.58 may be

attributed to O-CH2 protons of morpholine and another at δ 3.95 may

be attributed to N-CH2 protons of Morpholine [11].

48

1H NMR spectrum of MPH was compared with 1H NMR spectrum

of Zn(II) complex. It has been observed that the signal due to NH-

proton adjacent to CH appeared at δ 2.50 in the spectrum of the ligand

has been found shifted slightly down field to δ 2.90 in the spectrum of

the complex. A signal due to N-CH2 protons of Morpholine is also

shifted to downfield and appeared at δ 2.8 in the complex. These

changes indicate the coordination of oxygen atom carbonyl adjacent to

NH and nitrogen atom of Morpholine.

13C NMR Spectrum of the ligand MPH is shown in Figure 10 and

was recorded in DMSO-d6 medium using TMS as an internal solvent. A

peak at δ 163.4 is assigned to carbonyl carbon. The peaks at δ 145,

142, 138, 131 and 129 are attributed to Pyridine ring. The peaks at δ

45.4 and 52 ppm are due to C-N-C and C-O-C of Morpholine. It has

been compared with the 13C NMR of spectrum of ZnII complex of MPH.

The peak due to carbonyl carbon appeared at δ 163 in the spectrum of

the ligand has been shifted to δ 168 in the spectrum of the complex.

Further, a peak δ 52 has been shifted to downfield in the spectrum of

the complex. These two changes are evidence for the involvement of

oxygen atom of carbonyl and nitrogen atom of Morpholine. The residue

of metal atom remains behind because under nitrogen atmosphere, no

metal oxide will be formed.

Mass Spectrum

Mass Spectrum of the ligand MPH was recorded by Electron

Ionization method and is shown in Figure 14. It shows a peak at m/z =

255.64 and it corresponds to molecular ion peak and a peak at m/z =

67.59 is due to the fragment CONH2. A peak at m/z 96.43 corresponds

to Morpholine group. A peak at m/z 222.59 corresponds to

49

semicarbazide group. The fragmentation pattern suggests the molecular

mass of MPH as 255.64. This value is also supported by nitrogen rule.

Thermal analysis

The complex [Cu(L)2Cl2] was heated in oxygen atmosphere with a

heating rate of 25˚C min-1 and the thermogram is shown in Figure 12.

It shows four different stages. The first step in the curve of thermogram

at 140 ˚C indicates the loss of solvent molecules [12]. The complex is

thermally stable upto 298 ˚C. A sharp inflation at 298 ˚C is attributed

to loss of chloride. The next stage between 310 ˚C-390 ˚C is because the

changes occur of decomposition of ligand moieties from the complex.

Based on the above data, the structure has been proposed for the

metal complexes of 2-[(morpholin-4-yl) (pyridin-3-yl) methyl]

hydrazinecarboxamide (MPH)

N

H2N

O

NH

N

O

NH

N

M

Cl

Cl

N

NH2

O

HN

N

O

HN

N

Figure 1: Proposed structure of metal complexes of 2-[(morpholin-4-yl) (pyridin-3-yl) methyl] Hydrazinecarboxamide (MPH)

(M = MnII, CoII, NiII, CuII and ZnII)

50

Figure 2: IR spectrum of 2-[(morpholin-4-yl) (pyridin-3-yl) methyl] Hydrazinecarboxamide (MPH)

Figure 3: IR spectrum of CoII complex of 2-[(morpholin-4-yl) (pyridin-3-yl) methyl] Hydrazinecarboxamide (MPH)

51

Figure 4: IR spectrum of CuII complex of 2-[(morpholin-4-yl) (pyridin-3-yl) methyl] Hydrazinecarboxamide (MPH)

Figure 5: IR spectrum of NiII complex of 2-[(morpholin-4-yl) (pyridin-3-yl) methyl] Hydrazinecarboxamide (MPH)

52

Figure 6: IR spectrum of MnIIcomplex of 2-[(morpholin-4-yl) (pyridin-3-yl) methyl] Hydrazinecarboxamide (MPH)

Figure 7: UV spectrum of CoII complex of 2-[(morpholin-4-yl) (pyridin-3-yl) methyl] Hydrazinecarboxamide (MPH)

53

Figure 8: UV spectrum of Mn(II) complex of 2-[(morpholin-4-yl) (pyridin-3-yl) methyl] Hydrazinecarboxamide (MPH)

Figure 9: 1H NMR of 2-[(morpholin-4-yl) (pyridin-3-yl) methyl] Hydrazinecarboxamide (MPH)

54

Figure 10: 13C NMR of 2-[(morpholin-4-yl) (pyridin-3-yl) methyl] Hydrazinecarboxamide (MPH)

Figure 11: Cyclic voltammogram of NiIIcomplex of 2-[(morpholin-4-yl) (pyridin-3-yl) methyl]hydrazinecarboxamide (MPH)

55

Figure 12: TGA curve of Cu (II) complex of 2-[(morpholin-4-yl) (pyridin-3-yl) methyl] Hydrazinecarboxamide (MPH)

Figure 13: EPR spectrum of Cu (II) complex of 2-[(morpholin-4-yl) (pyridin-3-yl) methyl] Hydrazinecarboxamide (MPH)

56

Figure 14: Mass spectrum of 2-[(morpholin-4-yl) (pyridin-3-yl) methyl] Hydrazinecarboxamide (MPH)

57

REFERENCES

1. Syed Ali Padusha M, Abdul Jameel A, Ind. J. Het. Cyclic. Chem, 2006, 16, 197.

2. Viswanathan M, Krishnan. G, Asian J. Chem, 2008, 16, 156.

3. Sharma Y R, Elementary Organic Spectroscopy, 2008, 4, 149.

4. Carlin R L, Stereochemistry of cobalt(II) complexes in Transition metal chemistry, Marcel Deckker, New York, 1972 88.

5. Cotton F A, Wilkinson, Advanced Inorganic Chemistry, vol. 1, 3rd edi., Wiley interscience, New York, 1972.

6. Chiswell B, Livingstone S E, J.Chem. Soc, 1960, 97.

7. Patel K S, Open Journal of Metal, 2012, 2, 49.

8. Alhadi A A, Bull.Chem.Soc.Ethiop, 2012, 26, 95.

9. Raman N, Esther S, Thangaraja C, J. Chem. Sci, 2004,116, 209.

10. Shelke V A, Jadhav S M, Shankarwar S G, Munde A S, Chondhekar T K, Soc. Ethiop Bull. Chem, 2011, 25, 381.

11. Vural Ufuk S, Hasan Mart H, Okkes D, Ozlem S, Vefa M, Koca M C, Bull. Chem. Soc. Ethiop, 2006, 20, 219.

12. Patel K S, Patel J C, Dholariya H R, Patel V K, Patel K D, Open Journal of Metal, 2012, 2, 49.

58

CHAPTER III

Synthesis and Characterization of Co (II), Ni (II), Cu (II) and Zn (II) metal complexes of N(morpholino (thiophen-2-

Yl) methyl) nicotino hydrazide (MTN)

This chapter deals with the Synthesis and Characterization of

Co (II), Ni (II), Cu (II) and Zn (II) metal complexes of N(morpholino

(thiophen-2-Yl) methyl) nicotino hydrazide (MTN).

Materials and Methods

All the chemicals and solvents used were of Merck and Sigma

Alrich products (Analytical Reagent grade) and were used without

further purification. The synthesized ligand and its metal complexes

were characterized by the respective techniques as discussed in

chapter I.

Synthesis and Characterization of N(morpholino (thiophen-2-Yl)

methyl) nicotinohydrazide) (MTN)

Morpholine, thiophene-2-carboxaldehyde and nicotinic acid

hydrazide were taken in the mole ratio of 1:1:1 and were reacted as

shown in the Scheme 2. To the methanolic solution of nicotinic acid

hydrazide (1.5g, 25 mmol) taken in a round bottom flask, morpholine

(2.1 mL, 25 mmol) and thiophene-2-carboxaldehyde (2.6 mL, 25 mmol)

were added. The reaction mixture was kept over a magnetic stirrer and

stirred well under ice cold condition for 2 h. The colourless solid formed

was filtered and washed several times with petroleum ether (40-60%),

the completion of the reaction was monitored by TLC technique. The

crude solid thus obtained was dried and recrystallized using ethanol.

The recrystallized product was dried over vacuum.

59

HN

O

Morpholine +

N

O

NH

H2N

Nicotinic acid hydrazide +

SO

Thiophene-2-carboxaldehyde

N O

N

H2N S

O

N

N(Morpholino (thiophen-2-Yl)methyl) nicotino hydrazide

Scheme 2

Synthesis of CoII, NiII, CuII and ZnII chloro complexes of MTN

The complexes are prepared by mixing hot methanolic solution to

the ligand MTN dissolved in methanol in 1:2 (metal:ligand) mole ratio.

The reaction mixture was kept over a magnetic stirrer with hot plate

and refluxed at 70 °C with continuous stirring. After 3 h, the precipitate

formed was filtered, washed with methanol and then dried over fused

calcium chloride.

Characterization of MTN and its CoII, NiII, CuII and ZnII

metal complexes

Elemental Analysis

After ascertaining the elements that are present in the

synthesised compound, the proportionate percentage of various

elements was found out. The observed and calculated percentage

-H2O

60

composition of elements of C, H, N, O and S elements of the

synthesized compound MTN and its CoII, NiII CuII and ZnII metal

complexes are presented in the Table 3.1. The results of the magnetic

susceptibility and conductivity measurements of all the complexes are

also presented in the same Table. From the observed C, H, N and S

values, the molecular formula for the compound (Morpholino (thiophen-

2-Yl) methyl) nicotino Hydrazide has been assigned as C15H18N4O2S

which is in agreement with the calculated value.

Table 3.1. Elemental analysis and molar conductance of MTN and

its complexes

Compound and Molecular Formula

Colour &

Yield

Molecular Weight

Melting Point

Elemental analysis % Found and (% Calculated) Ω-1 mol-1 cm2 C H N O S Cl

M T N C1 5H1 8N4O2S

White 70% 318.4 198 55.68

(55.05) 8.07 (8.12)

29.51 (29.41)

6.74 (6.53)

8.02 (8.14) -- --

C u ( M T N ) 2 C l 2 Blue 74% 645.1 226 54.53

(54.02) 7.63 (7.53)

31.79 (31.87)

6.06 (6.34)

7.88 (7.92)

16.42 (16.85) 98

N i ( M T N ) 2 C l 2 Green 78% 640.2 274 63.77

(63.04) 6.36 (6.44)

13.94 (13.23)

15.93 (15.88)

11.52 (11.50)

18.02 (18.68) 110

Z n ( M T N ) 2 C l 2 Creamy white

80% 646.9 256 66.88

(66.32) 7.37 (7.21)

16.62 (16.41)

11.44 (11.12)

11.53 (11.55)

17.98 (18.34) 110

C o ( M T N ) 2 C l 2 Brown 83% 635.2 234 62.86

(61.89) 7.25 (7.20)

16.52 (16.48)

11.31 (11.28)

11.56 (11.49)

17.86 (17.58) 96

Conductivity measurements

The molar conductivity values of copper complex is found to be of

65 – 118 ohm-1 cm2mol-2 suggesting the non-electrolytic nature of the

copper complex of MTN. For Co (II), Ni (II) and Zn (II) complexes, the

values lie in the range of 95-118 ohm-1 cm2mol-2. This suggests the

electrolytic behaviour of the metal complexes [1, 2].

Infra-Red Spectra (IR)

IR spectrum of the compound MTN is recorded as KBr disc and is

shown in Figure 16. A broad band at 3421 cm-1 is assigned to terminal

61

ν(NH2) and a sharp band at 3298 cm-1 is assigned to ν(N2H). A broad

band appearing at 1647 cm-1 is assigned to ν(C=O). The bands at 1396

cm-1 and 1244 cm-1 are assigned to ν(CNC) and ν(COC) of Morpholine.

The spectrum of MTN has been compared with the spectrum of

its complexes. By the comparison, it has been observed that, ν(C=O)

appeared at 1647 cm-1 in the spectrum of the ligand has been shifted to

downfield in the spectrum of the complexes. Ν(CNC) of the ligand

appeared at 1396 cm-1 in the spectrum of the ligand has also been

shifted to lower frequency by 15-25 cm-1 in the spectrum of the

complexes. No change has been observed in the vibrational frequencies

of other groups. Hence, it is concluded that the ligand acts as a neutral

bidentate ligand. The IR spectrum of both the ligand and the complexes

show intense bands at 3421 cm-1 which is the characteristic feature of

ν(N-H), indicating the existence of an –NH group [3-5]. This value is in

agreement with the literature values and are presented in Table 3.2.

A sharp band appeared at 1647 cm-1 can be attributed to the

ν(C=O) of the ligand, which in agreement with the literature value(1680

cm-1 to 1630 cm-1) of ν(C=O) of amide [6]. In the far IR region, all the

complexes exhibit bands around 572-505 cm-1 and 440-418 cm-1 which

are assignable to ν(M-O) [7] and ν(M-N) [8-10] respectively. Due to the

larger dipole moment change in the vibration of (M-O) band in

comparison to that of νM-N band, the band due to M-O usually appear

at higher frequency region which are sharp and stronger than (M-N)

band. These two ranges of bands collaborate the coordination of the

ligand with the metal ion.

62

Table 3.2: Characteristic Infrared bands of MTN and its metal

complexes

Compounds ν(NH) ν(CH) ν(C=O) ν(C=N) ν(M-O ν(M-N) M T N C15H18N4O2S

3421 2940 1647 1597 -- --

Co(MTN)2Cl2 3298 2844 1651 1585 505 431 Ni(MTN)2Cl2 3408 2912 1456 1570 568 438 Cu(MTN)2Cl2 3412 2900 1532 1500 572 440 Zn(MTN)2Cl2 3405 2808 1650 1500 560 436

UV-Vis spectra and magnetic measurements

The UV-Vis spectrum of MTN and its CoII, NiII, CuII and ZnII metal

complexes were recorded at room temperature in DMSO solvent. The

UV-Vis spectral data of MTN and its metal complexes are shown in

Table 3.3. The electronic absorption spectrum of the ligand MTN shows

an intense absorption peak at 33333 cm-1 is assigned to n→π*

transitions [11].

The Cu (II) ion with d9 configuration in a complex can be either

octahedral or tetrahedral or rarely square planar. The octahedral

coordinated Cu (II) ion has the ground state 2Eg (t2g)6 (eg)3. The only

excited state should then be 2tg (t2g)5 (eg)4. The Cu (II) complex under

present study shows a broad band in the region 12730 cm-1. This is

due to 2f2g →2f2g. The broadness of the band may be due to John-Teller

distortion [12].

Co (II) complex displays a band at 15635 cm-1 which corresponds

to 4A2→4T1 transitions. This favours octahedral geometry. The Ni (II)

complex is a diamagnetic suggesting octahedral geometry. It should

have a broad band at 14600 cm-1 assigned to 1A1g →1B1g transitions.

The spectra of this complex consistent with this arrangement.

63

Zn (II) complex exhibited a band assigned to L→M charge transfer, it is

a diamagnetic as expected.

Table 3.3: UV-Vis spectral data and Magnetic Moment of MTN and

its Complexes

Compounds Absorption maxima Assignments

Magnetic moment

(B.M.) Geometry

MTN = L 33333 n→π* -- --

[CoL2(H2O)2]Cl2 15635 4A2→4T1 4.75 Octahedral

[CuL2(H2O)2]Cl2 12730 2f2g →2f2g 1.82 Octahedral

[NiL2(H2O)2]Cl2 14600 1A1g →1B1g 3.60 Octahedral

[ZnL2(H2O)2]Cl2 0 L→M -- Distorted Octahedral

Nuclear Magnetic Resonance Spectra (NMR)

1H NMR spectrum of the ligand MTN was recorded in DMSO-d6

using TMS as internal standard at 300 MHz. The 1H NMR spectrum of

the compound is shown in Figure. The spectrum accounts for the

number of protons in the molecular formula. The ligand shows a

multiplet at δ 7.1 and δ 8.6-8.9 is assigned to nicotinic acid ring

protons and thiophene protons. A signal at δ 9.2 is assigned to NH

adjacent to C=O. The multiplet at δ 8.2 and δ8.4 is NH adjacent to CH

and CH is adjacent to NH.

13C NMR spectrum of the ligand MTN was recorded in DMSO-d6

at 100 MHz. The spectrum is shown in Figure. The peak at δ 159 is

assigned to carbonyl carbon. The peaks at δ 145, 142, 138, 131 and

129 are attributed to pyridine ring. Thiophene ring exhibits peaks at δ

144, 131, 129, 128.

64

Mass spectrum

Mass spectrum of MTN was recorded by electron ionization mode.

The spectrum shows a molecular ion peak at m/z 323 which confirms

the assigned molecular mass to the Mannich base MTN and also obeys

Nitrogen rule. There upon on fragmentation, it records intense signals

at m/z 148, 121 and 106 are due to the removal of thiophene ring,

NCH and NH groups respectively. Finally the signal at m/z 78 is due to

the presence of C5H4N+ cation after the removal of CO.

Electron Spin Paramagnetic Resonance Spectrum (EPR)

The EPR spectrum of CuII complex was recorded at room

temperature in DMF solution on the X-band at 9.1 GHz under the

magnetic field strength 3400 G and is shown in Figure 22. The analysis

of spectrum gives g = 2.21 and g = 2.02. The trend g>2.0023,

observed for the complex, under study, indicates that the unpaired

electron is localized in dx2-y2 orbital of the CuII ion and the spectral

features are characteristic for axial symmetry. Thus tetrahedral

elongated octahedral geometry is confirmed for the above complex

[13-15].

Cyclic Voltammetry

The electrochemical properties of Cu(II) complex of MTN were

investigated in DMSO solution containing 0.1 M ammonium chloride as

a supporting electrolyte by cyclic voltammetry. All the measurements

were carried out in 1 MM DMSO solution of the complex at room

temperature, in the potential range from +1.0 to -1, at the scan rate of

100 mVs-1. The Voltammogram shows a well-defined redox process

corresponding to the formation of MnII/Mn(0) couple at Epa = -0.985V

and the cathodic peak Epc = +0.506V. This couple is found to be

reversible with Ep = -1.49V and the ratio of cathodic to anodic currents

65

(ipc/ipa≈1) almost equal to one, corresponding to a simple one electron

process.

Thermo gravimetric Analysis

Thermo gravimetric analysis of copper complex of MTN was

carried out with 1.052 mg of the sample. It is scanned from 30 °C to

900 °C and the heating rate was suitably controlled at 15 °C/min.

under oxygen atmosphere. The complex decomposed in major three

steps and it is shown in Figure 22. The TG curve of the complex shows

an inflection at 100 °C, which may be due to the elimination of two

water molecules. Another inflection starts at 320 °C and ends at 350 °C

which indicates the decomposition of ligand moiety. Thereafter no

variation is found. This indicates the formation of stable metal oxide.

Based on the above data, a six coordinated octahedral geometry

has been proposed and four coordinated tetrahedral geometry has been

proposed for CuII, CoII, NiII and ZnII complexes of N(Morpholino

(thiophen-2-Yl) methyl) nicotinohydrazide).

Proposed Structure of the metal complex of MTN - M=CoII, CuII, NiII and ZnII

S

S

C

O

N

H

NH

NH C

N

O

Cl

Cl

C

O

N

H

N N

H H

C

ON

Me.H2O

66

Figure 16: IR SPECTRUM OF N(Morpholino (thiophen-2-Yl) methyl) nicotinohydrazide)(MTN)

Figure 17: IR SPECTRUM OF Cu (II) of N(Morpholino (thiophen-2-Yl) methyl) nicotinohydrazide)(MTN)

67

Figure 18: IR spectrum of Ni (II) of N(Morpholino (thiophen-2-Yl) methyl) nicotinohydrazide)(MTN)

Figure 19. IR spectrum of Zn (II) of N(Morpholino (thiophen-2-Yl) methyl) nicotinohydrazide)(MTN)

68

Figure 20: 1H NMR of N(Morpholino (thiophen-2-Yl) methyl) nicotinohydrazide) (MTN)

Figure 21. 13C NMR of N(Morpholino (thiophen-2-Yl) methyl) nicotinohydrazide) (MTN)

69

Figure 22: EPR spectrum of Cu (II) complex of N(Morpholino (thiophen-2-Yl) methyl) nicotinohydrazide) (MTN)

Figure 23: TGA curve of N(Morpholino (thiophen-2-Yl) methyl) nicotinohydrazide) (MTN)

70

REFERENCES

1. Rekha S, Nagasundara K R, Ind. J. Chem, 2006, 45, 2421.

2. Douglas X.W, Heloisa B, Amal A N, Fathy A E, Mohammed I A, Trans Met. Chem, 1999, 24421.

3. Raman N, Pitchikani Raja Y, Kulandaisamy, Indian Acad. Sci, 2001,113, 183.

4. Chandra S, Sharma A K, J. Indian Chem. Soc, 2009, 86, 690.

5. Yaul S R, Yaul A R, Pathe G B, Anand S, Amer-Eura. J. Sci. Res, 2009, 4, 229.

6. SharmaY R, Elementary Organic Chemistry, 4th edition, 2009, 148.

6. Sivakami M et al., Int. J. Pharm. Sci. Rev. Res., 2014, 27(1), 336-342.

7. Singh U K, Pandeya S N, Sethia S K, Pandey M, Singh A, Garg A, Kumar P, Int. J. Pharm. Soc. Drug. Res, 2010, 2 151.

8. Abdul Jameel A, Syed Ali Padusha M, Der Chemica Sinica, 2012, 3, 1098.

9. Shah T B, Gupta A, Indan J. Chem, 2010, 49, 578.

10. Ramesh M, Sabastiyan A, Der Chemica Sinica, 2012, 3, 534.

11. Kriza A, J. Serb.Chem. Soc, 2010, 75, 229.

12. N. Raman, C. Thangaraja and S.J. Raja, Ind. J. Chem., 44A 2005 693; K.R. Reddy, K.M. Reddy and K.N. Mahendra, Ind. J. Chem., 45A 2006 378

13. Sathya D ,Senthil Kumaran J, Priya S, Jayachandramani N, Mahlakshmi S Emelda A R, Int. J. Chem. Tech. Res, 2011, 3(1), 248.

14. Veeraraj A, Sami P and Raman N, Proc. Ind. Acad. Sci, 2000, 112, 515.

15. Raman N, Esther S, Thangarja C, J. Chem. Sci, 2004, 116, 209.

71

Chapter IV

Synthesis and Characterization of Co (II), Ni (II), Cu (II) and Zn (II) metal complexes of 1-(furan-2-yl) (morpholino)

(methyl)-3-phenyl urea (MFP)

This chapter deals with the Synthesis and Characterization of

Co (II), Ni (II), Cu (II) and Zn (II) metal complexes of 1-(furan-2-yl)

(morpholino) (methyl)-3-phenyl urea (MFP).

Materials and Methods

All chemicals and solvents used, were of Merck and Sigma

Aldrich products (Analytical reagent grade) and were used without

further purification. The synthesized ligand and its metal complexes

were characterized by the respective techniques discussed in Chapter I.

Synthesis of 1-(furan-2-yl) (morpholino) (methyl)-3-phenyl urea

(MFP)

Morpholine, Furfuraldehyde and 1-phenyl urea were taken in the

mole ratio of 1:1:1 and were reacted as shown in the Scheme 3. To the

ethanolic solution of phenyl urea (2.4 g, 25 mmol), taken in a round

bottom flask, furan-2-carbaldehyde (2.5 mL, 25 mmol) and morpholine

(2.1 mL, 25 mmol) were added. The reaction mixture was kept on a

magnetic stirrer and stirred well under ice-cold condition for 3 h.

After 3 h, the pale yellow coloured solid formed was filtered and washed

several times with petroleum ether (50-60 %). The crude solid thus

obtained was dried and recrystallized from ethanol. The recrystallized

product was dried in a vacuum desiccator over fused calcium chloride.

72

HN

O

Morpholine +

OO

Furfuraldehyde +

NH

O

H2N

1-Phenyl Urea

Scheme 3: Synthesis of 1-(furan-2-yl) (morpholino) (methyl)-3-phenyl urea (MFP)

Synthesis of CoII, NiII, CuII and ZnII metal complexes of MFP

To the methanolic solution of MFP (2.1 g, 10 mmol), copper (II)

chloride (0.83g, 5 mmol) dissolved in CH3OH:CHCl3 (1:1) v/v (10 mL)

was added. The reaction mixture was taken in a round bottom flask

and refluxed on a magnetic stirrer and refluxed at 60°C for 2 h with

continuous stirring. After 2 h the precipitate formed was filtered,

washed with methanol and then dried over fused calcium chloride.

The same procedure was employed for the preparation of CoII, NiII, and

ZnII chloro complexes. All the complexes prepared are of solid in nature

and are stable in air.

O

N

HNCO

HN

O

1-(furan-2-yl(morpholino)methyl)-3-phenylurea

73

Characterization of MFP and its CoII, NiII, CuII and ZnII complexes

Elemental analysis

After ascertaining the elements that are present in the

synthesised compound, the proportionate percentage of various

elements was found out. The observed and calculated percentage

composition of C, H, N and O elements of the MFP and its CoII, NiII, CuII

and ZnII metal complexes and the values of molar conductance of the

complexes are presented in the Table 4.1. From the observed C, H, N

and O values, the molecular formula for the compound 1-(furan-2-yl)

(morpholino) (methyl)-3-phenyl urea (MFP) was assigned as C16H19N3O3

which is in agreement with the calculated value. The

molar conductance values of the complexes are in the range of μc

10-35 Ω -1mol-1cm210-3M in the solution of DMSO. This indicates that

the metal complexes are non-electrolytic in nature [1].

Infra-Red spectra

The IR spectra of the synthesised compound MFP and its metal

complexes are recorded. These are shown in Figures 24, 25, 26 and 27.

Comparison of IR spectrum of the ligand and its complexes reveals that

the carbonyl stretching vibration of free MFP appeared at 1645cm-1 has

been shifted to lower frequency by 20 – 70 cm-1 in the spectrum of its

complexes. The C-N-C stretching vibration of free MFP appeared at

1103 cm-1 has been found shifted to lower frequency in the spectra of

its complexes. These changes indicate that oxygen atom of carbonyl

and nitrogen atom of morpholine are involved in coordination with the

metal ion. The presence of additional bands at 34 – 46 cm-1 in spectra

of the complexes indicates the presence of coordinated water molecules

[2].

74

Far IR spectrum of the complex show significant absorptions at

530, 461, and 405 cm-1 due to νM-O, 465, 392 and 313 cm-1 are due to

νM-N and at 352, 329 and 300 cm-1 are due to νM-Cl [3, 4].

Table 4.1. Elemental analysis and molar conductance of MFP and its complexes

Compound and

Molecular

Formula

Colour

& Yield

Melting

Point

Elemental analysis % Found and (% Calculated)

Ω-1

cm2

mol-1 C H N O Cl M

M F P = L

C 1 6 H 1 9 N 3 O 3

Pale

Yellow

80

150 63.77

(63.82)

6.36

(6.38)

13.94

(13.96)

15.93

(15.95) -- -- --

[ C u L 2 C l 2 ] Green

82 320

53.8

(54.20)

5.78

(5.85)

12.75

(13.28)

14.26

(14.78)

10.35

(10.97)

9.65

(9.80) 33.80

[ C o L 2 C l 2 ]

Dark

Green 335

58.8

(59.20)

6.36

(6.56)

13.75

(14.02)

13.26

(13.45)

11.45

(11.85)

8.59

(9.10) 26.95

[ N i L 2 C l 2 ]

Green 310

60.8

(62.85)

7.25

(7.32)

13.95

(14.23)

14.52

(14.78)

13.65

(14.60)

9.20

(9.53) 32.85

[ Z n L 2 C l 2 ]

Creamy

white 352

59.24

(60.02)

7.85

(7.96)

14.52

(14.68)

13.85

(14.02)

12.85

(13.50)

8.64

(8.84) 34.52

Table 4.2: Characteristic IR bands of the MFP and its metal complexes (νcm-1)

Compounds νN-H νC-H νC=O νM-N νM-O νM-Cl

M F P = L

C 1 6 H 1 9 N 3 O 3 3363 3217 1645 -- -- --

[CuCl2(MFP)(H2O)2 3313 3217 1635 465 530 352

[CoCl2(MFP)(H2O)2 3310 3212 1625 392 461 329

[MnCl2(MFP)(H2O)2 3312 3214 1614 313 445 327

[ZnCl2(MFP)(H2O)2 3310 3212 1597 315 405 301

75

1H NMR Spectra

1H NMR spectrum of the ligand and complexes is recorded using

TMS as an internal reference and DMSO-d6 solvent at ambient

temperature. The 1H NMR spectrum of MFP shows six due to six

different groups at various regions. A broad singlet at δ 8.73 is assigned

to the proton of NH which is located in between CO and NH. Multiplet

in the region δ 6.39 to δ 7.41 is assigned to the protons of the aromatic

ring. A broad singlet at δ 5.84 is assigned to proton of NH which is

adjacent to CH. A doublet at δ 5.56 to δ 5.59 is assigned to CH adjacent

to NH. The chemical shift of protons of N (CH2)2 and O (CH2)2 groups of

Morpholine occurs at δ 2.42 and δ 3.56 respectively.

1H NMR spectrum of MFP was compared with the 1H NMR

spectrum of its Zn (II) complex. The signals due to N (CH2)2 group of

Morpholine ring have experienced a downfield shift from δ 2.42 to

δ 2.89 in the spectrum of the complex. This change indicates that

involvement of nitrogen atom of N (CH2)2 of Morpholine in bonding with

the metal atoms [5, 6]. The NH protons on either side of carbonyl group

of amide have been found shifted to downfield in the spectrum of the

complex reveals, the coordination of oxygen atom of carbonyl to the

metal centre. The shift of electron density from the carbonyl oxygen to

the metal centre may cause depletion of electron density in the

aromatic ring. The spectrum of Zinc exhibited a signal at δ 59.20 which

is not seen in the spectrum of the free ligand (MFP). All the above

discussions are in agreement with the modes of coordination predicted

by IR data.

76

13C NMR Spectra

13C NMR spectrum of the ligand was recorded on Bruker-DRX-

400 MHZ using DMSO-d6 as solvent and TMS as internal standard. The

presence of carbonyl carbon is confirmed by the appearance of a signal

at δ 156.01. The signals exhibited at δ 154.68, δ 154.24, δ 152.20,

δ 152.12, δ 142.57, δ 140.51, δ 140.07, δ 128.68, δ 121.36, δ 117.92

are assigned to carbon atoms of benzene ring of Furan ring. The signals

due to O (CH2)2 and N (CH2)2 of Morpholine are appeared at δ 66.28 and

δ 56.01 respectively.

This spectrum was compared with the 13C NMR spectrum of Zn

(II) complex. The signals appeared at δ 56.01 in the spectrum of the free

ligand has been found shifted to δ 58.41 in the spectrum of the

complex. Further the signals due to aromatic carbon also shifted to

downfield. These changes clearly give further evidence for the

participation of oxygen atom of carbonyl and nitrogen atom of

Morpholine in coordination with the metal atom.

Conductivity Measurement

Using 10-3 solution of the complexes in DMSO, the molar

conductivity was measured on a systronic conductivity bridge with a

dip - type cell. The conductivity values are found to be 10-35 Ohm-1

cm2mol-1 for all chloro Cu (II), Co(II), Ni(II) and Zn(II). This supports the

non-electrolytic nature of metal complexes.

Mass Spectrum

The mass spectrum of MFP was obtained in an electron

ionisation method. It shows a molecular ion peak at m/z 311, which is

well fit the assigned structure of the compound. There upon further

77

fragmentation, it records intense signals at m/z = 267 and 225 which

are due to the removal of O (CH2)2 of morpholine ring respectively.

HN

C

HN

CH

O

NO

O

m/z=311

-44

HN

C

O

NHCH

N

O

CH2

CH2+ O(CH2)2

m/z=267

-42

HNC

O

HNCH

O

+ N(CH2)2

Based on the results of analytical, conductance, UV, IR, 1H NMR,

13C NMR and mass spectral data, hexa coordinated octahedral

geometry has been proposed for all the complexes.

78

2

31

4

5O

CH

O N

NH

C

O

NH

ClH2O

Cl

H2O

M

Figure 24: Proposed structure for metal complexes of 1-(furan-2-yl) (morpholino) (methyl)-3-phenyl urea

M = Co(II), Cu(II), Ni(II) and Zn(II)

Electronic spectra

The electronic spectrum of Co(II) complex displays three bands in

the region 12400-15800, 16200-19500 and 23800-27400 cm-1

corresponding to 3A2g→3T1g, 3A2g→3T1g and 3A2g→3T1g (P) respectively

which suggests an Octahedral geometry [7].

The electronic spectrum of the Zn(II) complex exhibits three

absorption bands at 10829, 16494 and 26743 cm-1 which are assigned

to 6A1g→4T1g (4G), 6A1g→4T2g (4G) and 6A1g→4T2g (4G) and to 6A1g→4Eg, 4A1g

(4G) transitions [respectively, which indicates the octahedral geometry

[9].

For Cu(II) complex of MFP three bands exhibited in the region

11500-15500, 16000-18200 and 22500-25500 cm-1 in the electronic

spectrum. These bands are corresponding to 2B1g→2B2g, 2B1g→2E1g, and

2B2g→2A2g respectively which are in good agreement with the distorted

octahedral geometry [10, 11]

79

Thermal analysis:

Thermal stability of a complex can be studied by the use of

Thermogravimetric analysis. This study will also give the information to

decide whether the water molecules are in the inner or outer

coordination of the central metal ion. By taking 2.058 mg of the

sample, this study was carried out at ambient temperature under

nitrogen atmosphere. This was controlled at 10˚c/min. The

Thermogravimetric curve of Cu(II) chloro complex shows an inflection at

170˚C, which may be due to the elimination of two water molecules.

Another inflection starts at 280˚C and ends at 350˚C which indicates

the decomposition of ligand moiety. Thereafter no variation is found.

This indicates the formation of stable metal oxide.

80

Figure 25: IR spectrum of 1-(furan-2-yl) (morpholino) (methyl)-3-phenyl urea (MFP)

Figure 26: IR spectrum of CuII complex of 1-(furan-2-yl)

(morpholino) (methyl)-3-phenyl urea (MFP)

81

Figure 27: IR spectrum of NiII complex of 1-(furan-2-yl)

(morpholino) (methyl)-3-phenyl urea (MFP)

Figure 28: IR spectrum of ZnII complex of 1-(furan-2-yl) (morpholino) (methyl)-3-phenyl urea (MFP)

82

Figure 29: UV spectrum of CuII complex of 1-(furan-2-yl) (morpholino) (methyl)-3-phenyl urea (MFP)

Figure 30: UV spectrum of NiII complex of 1-(furan-2-yl) (morpholino) (methyl)-3-phenyl urea (MFP)

UV-Vis Spectrum

ACICSt.Joseph's College, Trichy-2

Data Interval: 1.0000 nm

Spectrum Name: FMP-NI.SP

Instrument Model: Lambda 35 Scan Speed: 960.00 nm/min

Date: 5/29/2013

220.0 300 400 500 600 700 800 900 1000 1100.0

0.00

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

1.1

1.2

1.3

1.4

1.5

1.60

nm

A

373.94,0.25612

246.44,1.2152

83

Figure 31: 1H NMR of 1-(furan-2-yl) (morpholino) (methyl)-3-phenyl urea (MFP)

Figure 32: 13C NMR of 1-(furan-2-yl) (morpholino) (methyl)-3-phenyl urea (MFP)

84

Figure 33: TGA curve of Cu(II) Chloro complex of 1-(furan-2-yl) (morpholino) (methyl)-3- phenyl urea (MFP)

Figure 34: Mass spectrum of 1-(furan-2-yl) (morpholino) (methyl)-3-phenyl urea (MFP)

85

References

1. Paul R C, Kapila P A, Bedi S and Vasisht K K, J. Indian Chem. Soc., 1976, 53, 768-773.

2. K Hussain Reddy, M Suredra Babu, P Suresh Babu and S Dhayanantha, Ind. J. Chem., 43A 2004 1233.

3. B M Gate House, S E Livingstone and R S Nyholm, J. Inorg Nucl. Chem., 8 1958 75

4. Mohan et al., J. Ind. Chem. Soc., 83 2006 331.

5. Maurya R C, Patel P and Rajput S, Synth. React. Inorg. Met-org. Chem.2003, 23, 817-827.

6. Nakamoto K, Infrared and Raman spectra of Inorganic and Coordination compounds, Wiley Interscience, New York, 1971.

7. Ravichandran S, Raman N and Thangaraja C, Journal of Chem. Science., 2004, (116(4), 215-219.

8. Sabastiyan A and Yosuva Suvaikin, Advances in Applied Science Research., 2012, 3(1), 45-50.

9. Murali Krishna P, Shankara B S, Shashidhar Reddy N, Mahesh B and Basavaraj C, International Journal of Inorganic Chemistry., 2013, 10, 1.

10. Muhannd A Mahmoud, Abbas A Salih Al-Hamdani and Shaimaa R Bakir, Baghdad Science Journal, 2013, 9(2), 82-95.

11. Lever A B P, Inorganic Electronic spectroscopy, Elsevier Amstardam,1968, 32,420.

86

CHAPTER V

THERMOACOUSTICAL STUDIES

Introduction

This chapter deals with the thermo acoustical studies of the

Mannich bases 2-[(morpholin-4-yl) (pyridin-3-yl) methyl]

Hydrazinecarboxamide (MPH) and (Morpholino (Thiophen-2-Yl) Methyl)

Nicotino Hydrazide (MTN) by the measurements of parameters such as

ultrasonic velocity(U), density(ρ), viscosity(η), adiabatic compressibility

(κ), intermolecular free length (Lf), molar volume (Vm), Relaxation time

(τ), Specific acoustic impedance (Z), Lenard Jones Potential (LJP),

internal pressure (πi), free volume (Vf) and molecular cohesive energy

(MCE), Available volume (Va), Gibbs free energy(∆G) and Absorption

coefficient (α/f2).

Ultrasonic velocity measurements have been successfully

employed to detect and assess weak and strong molecular interactions,

present in binary [1, 2] and ternary liquid mixtures [3, 4]. The

ultrasonic studies find extensive applications in characterizing aspects

of physico-chemical behaviour of several liquid mixtures [5, 6]. A large

number of studies have been made on the molecular interaction in

liquid systems by various physical methods like Infrared [7, 8], Raman

Effect [9, 10], Nuclear magnetic resonance, Dielectric constant [11],

ultra violet [12] and ultrasonic method [13, 14]. In the recent years,

ultrasonic technique has become a powerful tool in providing

information regarding the molecular behaviour of liquids and solids

owing to its ability of characterising physiochemical behaviour of the

medium. The thermodynamic, acoustic and transport properties of

87

non-electrolyte liquid-liquid mixtures provide information about type

and extent of molecular interactions, and can be used for the

development of molecular models for describing the behaviour of

solutions [15-19].

Besides the solvent-solvent interactions, this study is helpful to

determine ion-solvent interactions ion solid interaction in the case of

solution containing electrolyte and molecular interactions between the

molecules of non-electrolyte and also interaction between non-

electrolytes and solvent. Many research work have been carried out to

study the molecular interaction between the liquid mixtures and the

solution of electrolyte.

The compounds 2-[(morpholin-4-yl) (pyridin-3-yl) methyl]

hydrazinecarboxamide (MPH) and (Morpholino (Thiophen-2-Yl) Methyl)

Nicotinohydrazide (MTN) are found to possess N-H-CO groups in its

structure. This has been characterised by spectral studies. The

compounds containing amide groups may involve inter and intra

molecular hydrogen bonding, because of the presence of CO and NH

groups. This present study is aimed at to validate the existence of

clusture of amide moieties in the synthesised compounds.

Experimental Procedure:

Accurately weighed amount of the synthesised compounds were

dissolved in the solvent DMSO to obtain solution in the concentration

range 1x10-3- 1x10-2 M. The ultrasonic velocities (U) have been

measured in ultrasonic interferometer operating at a frequency of

2 MHz with an accuracy of ±0.1%. The viscosities of the pure

compounds and their mixtures were determined using Ostwald’s

88

viscometer calibrated with double distilled water. The densities of the

solutions were measured accurately using 10 ml specific gravity bottles

in an electronic balance precisely and the accuracy in weighing

±0.1 mg. The temperature of the solutions and their mixtures was

maintained at 303 K, 308 K and 313 K using a thermostat. The

acoustical parameters such as adiabatic compressibility (κ),

intermolecular free length (Lf), molar volume (Vm), Relaxation time (τ),

Specific acoustic impedance (Z), LJP, internal pressure (πi), free volume

(Vf) and molecular cohesive energy (MCE), Available volume (Va), Gibbs

free energy (∆G) and Absorption coefficient (α/f2) were measured [20-24]

The study of various acoustical parameters and the factors

controlling the parameters helps to understand the structure of liquid

systems. In this part, the important acoustical parameters are defined.

Ultrasonic Velocity (U)

The relation used to determine the ultrasonic velocity is given by,

U = fλ ms-1

Where, f – frequency of ultrasonic waves, λ – wave length.

Adiabatic compressibility (β)

The acoustical parameters are related to compressibility and

hence the study of adiabatic compressibility provides better

understanding of the medium. The electrostatic field produced by the

structural arrangement of molecules which in turn pronounces the

effect of adiabatic compressibility.

89

From the measured values of the ultrasonic velocity (U) and the

density (ρ), the adiabatic compressibility (β) is calculated by using the

relation,

𝛽 = (1 𝑈2⁄ 𝜌) Kg-1 ms2

Free Length (Lf)

Free length is the distance between the surfaces of the

neighbouring molecules. Generally, when the ultrasonic velocity

increases, the values of the intermolecular free length decreases. The

decrease in intermolecular free length indicates the interaction between

the solute and solvent molecules due to which the structural

arrangement in the neighbourhood of constituent ions or molecules

gets affected considerably. The free length has been calculated using

the following formula given by Jacobson.

Lf = K / (Up) 1/2A°

Where k is the Jacobson’s constant. This constant is a temperature

dependent parameter. At any temperature (T) in Kelvin scale the value

of K is given as (93.875 + 0.345T) x 10-8.

Lenard Jones potential (LJP)

The Lenard Jones potential is given by

LJP = [6Vm/ Va]-13

Where Vm represents the molar volume and Va the available volume.

90

Internal Pressure (πi)

Internal pressure is a significant parameter in the study of

thermodynamic properties of liquids and liquid mixtures. It is a

measure of the resultant attractive and repulsive forces between the

interacting components in the mixture. A simple equation relation

using measurable parameter were,

𝜋𝑖 = 𝑏𝑅𝑇𝐾𝜂𝑈 (1

2)× 𝜌

32

𝑀𝑒𝑓𝑓76 𝑎𝑡𝑚

Where b stands for the cubic packing factor, which is assumed to be

two for liquid state including solutions, K, the temperature independent

constant, R, the molar gas constant, T, is the absolute temperature, η

is viscosity, U is the ultrasonic velocity, ρ is the density in Kgm-3 of the

liquid or liquid mixture. The reduction in internal pressure at lower

concentration may be attributed to breaking of inter-molecular forces.

The maximum value of internal pressure may be due to formation of

inter-molecular attraction between solvent ad solute molecules. In

other words it may be due to salvation.

Free Volume (Vf)

Free volume (Vf) is defined as the average volume in which the

centre of the molecules can move inside the hypothetical cell due to the

repulsion of surrounding molecules. Free volume can be calculated by

different methods. On the basis of dimensional analysis, obtained an

expression for free volume in terms of experimentally measurable

parameters like ultrasonic velocity, viscosity ad it is given by

𝑉𝑓 = (𝑀𝑒𝑓𝑓 𝑈 𝐾𝜂⁄ ) 𝑚3.

1/2

Where Meff is the effective molecular weight, which is expressed as

Meff = X1M1 + X2M2 +X3M3

91

Where X1 and M1 are the mole – fraction and molecular weight of the

component in the mixture respectively. K is the temperature

independent constant and the value of K is 4.28 x 109 and η is the

viscosity coefficient of the solution.

Cohesive energy (CE)

Cohesive energy is usually given as a product of internal pressure

and molar volume (Vm).

Available Volume (Va)

Another parameter, which can be calculated from the

experimental ultrasonic velocity do several investigators for study the

available volume pure as well as for ternary liquid systems. The

following relation is used for available volume.

Va = Vm 31 U mU∞

−

Where ρ the density of the liquid, Vm represents the molar volume, U

the ultrasonic velocity and U∞ is limiting value taken as 1600 ms-1 for

liquids.

Results and Discussion

Dimethyl Sulphoxide (DMSO) is a very good solvent and it is

widely used for the synthesis of organic compounds. Compounds

contain amide moieties (Urea, acetamide, semi and thio-semicarbazide,

phenyl urea) are easily soluble in water. But, in some synthesis water

may not be used as a solvent because of several reasons. In such cases,

DMSO is used instead of water, it may not cause the difficulty in

getting pure product.

92

DMSO is an organo sulphur compound with the formula

(CH3)2SO. This colourless liquid is an important polar aprotic solvent

that dissolves both polar and non-polar compounds and is miscible in a

wide range of organic solvents as well as water. Through oxygen it may

have the chances to form hydrogen bonding with other molecules.

Keeping in this view, binary liquid mixture containing DMSO and

MPH/MTN at five different concentrations were prepared and the

parameters studied at three different temperatures.

The experimental results of ultrasonic velocity(U), density(ρ),

viscosity(η), adiabatic compressibility (κ), intermolecular free length (Lf),

molar volume (Vm), Relaxation time (τ), Specific acoustic impedance (Z),

Lenard Jones Potential (LJP), internal pressure (πi), free volume (Vf) and

molecular cohesive energy (MCE), Available volume (Va), Gibbs free

energy(∆G) and Absorption coefficient (α/f2) are presented in Tables

5.1 – 5.5. The effect of temperature on these parameters are

represented graphically in Figures 35 - 39.

93

Table 5.1: Measured value of Ultrasonic Velocity (u), Density (ρ) and coefficient Viscosity (η) of the two binary systems of aqueous MPH and MTN in DMSO at different temperatures

Conc. mol dm-3

Velocity U /ms-1

Density ρ /kgm-3

Viscosity η /x10-3Nsm-2

303 K 308 K 313 K 303 K 308 K 313 K 303 K 308 K 313 K

2-[(morpholin-4-yl) (pyridin-3-yl) methyl] Hydrazinecarboxamide (MPH) 0.001 1486.9 1458.3 1437.4 1087.8 1083.3 1079.5 1.5531 1.519 1.4992

0.002 1483.8 1453.0 1429.6 1086.1 1082.3 1078.1 1.5442 1.5086 1.4821

0.003 1476.7 1448.8 1423.1 1085.2 1081.1 1077.4 1.5388 1.4931 1.476

0.004 1472.4 1444.6 1419.2 1084.3 1080.3 1076.1 1.5231 1.4857 1.4635

0.005 1470.4 1440.1 1412.8 1083.8 1079.3 1075.2 1.5173 1.4744 1.4582

N(morpholino (thiophen-2-Yl) methyl) nicotine hydrazide) (MTN) 0.001 1517.3 1501.4 1485.5 1089.7 1084.2 1080.4 1.7708 1.7209 1.6854

0.002 1508.5 1491 1480 1087.8 1083.2 1079.5 1.7513 1.6977 1.6623

0.003 1497.2 1482.3 1470.3 1086.6 1082.3 1078.5 1.7276 1.6708 1.6381

0.004 1488.6 1473.1 1457.4 1085 1081.1 1077.1 1.7104 1.6455 1.6122

0.005 1478.8 1460.1 1450 1084.8 1080.3 1076 1.6801 1.6227 1.5941

Table 5.2: Computed values of adiabatic compressibility (κ), intermolecular free length (Lf), molar volume (Vm) of two binay systems of MPH and MTN at different temperatures

Conc. mol dm-3

Adiabatic compressibility κ

/x10-9m2N-1

Free length Lf /A0

Molar volume Vm /x10-5m3mol-1

303 K 308 K 313 K 303 K 308 K 313 K 303 K 308 K 313 K

2-[(morpholin-4-yl) (pyridin-3-yl) methyl] Hydrazinecarboxamide (MPH) 0.001 0.415 0.434 0.448 0.4231 0.4362 0.4473 1.6638 1.6707 1.6765

0.002 0.418 0.437 0.453 0.4243 0.4380 0.4500 1.6741 1.6799 1.6865

0.003 0.422 0.440 0.458 0.4265 0.4395 0.4522 1.6833 1.6897 1.6955

0.004 0.425 0.443 0.461 0.4279 0.4409 0.4537 1.6924 1.6987 1.7053

0.005 0.426 0.446 0.465 0.4286 0.4425 0.4560 1.7009 1.7080 1.7145

N(morpholino (thiophen-2-Yl) methyl) nicotine hydrazide) (MTN) 0.001 0.3986 0.4092 0.4194 0.4143 0.4235 0.4326 1.6609 1.6694 1.6753

0.002 0.4039 0.4153 0.4229 0.4171 0.4266 0.4344 1.6716 1.6787 1.6845

0.003 0.4105 0.4205 0.4289 0.4204 0.4293 0.4375 1.6814 1.6881 1.6940

0.004 0.4159 0.4263 0.4371 0.4232 0.4322 0.4416 1.6916 1.6977 1.7041

0.005 0.4215 0.4342 0.4420 0.4260 0.4362 0.4441 1.6997 1.7068 1.7136

94

Table 5.3: Computed values of Relaxation time (τ), Specific acoustic impedance (Z) and LJP of two binary systems of MPH and MTN at different temperatures

Conc. mol dm-3

Relaxation time τ / x10-13 s

Specific acoustic impedance Z

/ X 106 Kg m-2 s-1

LJP / J mol-1

303 K 308 K 313 K 303 K 308 K 313 K 303 K

308 K

313 K

2-[(morpholin-4-yl) (pyridin-3-yl) methyl] Hydrazinecarboxamide (MPH) 0.001 8.6104 8.7913 8.9623 1.6174 1.5797 1.5516 71.88 54.74 46.04

0.002 8.6104 8.8031 8.9686 1.6115 1.5725 1.5412 69.61 52.30 43.33

0.003 8.6701 8.7729 9.0193 1.6025 1.5662 1.5332 64.85 50.49 41.26

0.004 8.6391 8.7868 9.0030 1.5965 1.5606 1.5272 62.23 48.77 40.09

0.005 8.6336 8.7827 9.0595 1.5936 1.5543 1.5190 61.07 47.03 38.28

N(morpholino (thiophen-2-Yl) methyl) nicotine hydrazide) (MTN) 0.001 9.4115 9.3884 9.4257 1.6534 1.6278 1.6049 103.0 84.36 70.84

0.002 9.4332 9.4002 9.3735 1.6409 1.6150 1.5976 91.91 75.07 67.00

X0.003 9.4570 9.3679 9.3680 1.6268 1.6042 1.5857 80.38 68.56 61.01

0.004 9.4853 9.3520 9.3960 1.6151 1.5925 1.5697 73.17 62.65 54.32

0.005 9.4429 9.3944 9.3950 1.6042 1.5773 1.5600 66.20 55.62 51.00

Table 5.4. Computed values of internal pressure (πi), free volume (Vf) and molecular cohesive energy (MCE) of two binary systems of MPH and MTN at different temperatures

Conc.

mol dm-

3

internal pressure πi / atm

Free volume Vf /x10-9m3mol-

Enthalpy H /k j

303 K 308 K 313 K 303 K 308 K 313 K 303 K

308 K

313 K

2-[(morpholin-4-yl) (pyridin-3-yl) methyl] Hydrazinecarboxamide (MPH) 0.001 38421 38893 39458 8.1457 8.1798 8.1637 63.92 64.97 66.15

0.002 38105 38598 39094 8.2475 8.2766 8.2951 63.79 64.84 65.93 0.003 37901 38217 38872 8.2895 8.4284 8.3481 63.80 64.57 65.91 0.004 37541 37956 38524 8.4388 8.5126 8.4784 63.53 64.47 65.69

0.005 37285 37647 38316 8.5278 8.6290 8.5249 63.42 64.30 65.69 N(morpholino (thiophen-2-Yl) methyl) nicotine hydrazide) (MTN)

0.001 40658 40819 41174 6.8974 7.0867 7.1959 67.53 68.14 68.97

0.002 40284 40439 40723 7.0008 7.2076 7.3569 67.34 67.88 68.59 0.003 39911 39992 40311 7.1154 7.3698 7.4996 67.10 67.50 68.28 0.004 39575 39569 39918 7.2104 7.5221 7.6327 66.94 67.17 68.02

0.005 39138 39239 39555 7.3839 7.6320 7.7571 66.52 66.97 67.78

95

Table 5.5: Computed values of Available volume (Va), Gibbs free energy(∆G) and Absorption coefficient (α/f2of two binary systems of aqueous sample1 and sample 2 at different temperatures.

Conc. mol dm-3

Available volume Va / x10-6m3mol-1

Gibbs free energy ∆G / x 10-20 k J mol-1

Absorption coefficient α/f2

/ X 10-14 NP m-1 S2

303 K 308 K 313 K 303 K 308 K 313 K 303 K 308 K 313 K

2-[(morpholin-4-yl) (pyridin-3-yl) methyl] Hydrazinecarboxamide (MPH)

0.001 1.1761 1.4796 1.7038 0.7077 0.7352 0.7624 1.1419 1.1888 1.2295

0.002 1.2158 1.5434 1.7961 0.7077 0.7357 0.7627 1.1442 1.1947 1.2371

0.003 1.2972 1.5968 1.8746 0.7106 0.7343 0.7651 1.1577 1.1941 1.2498

0.004 1.3497 1.6498 1.9270 0.7091 0.7350 0.7643 1.1569 1.1994 1.2509

0.005 1.3777 1.7069 2.0060 0.7088 0.7348 0.7670 1.1578 1.2026 1.2645

N(morpholino (thiophen-2-Yl) methyl) nicotine hydrazide) (MTN)

0.001 0.8585 1.0287 1.1988 0.7449 0.7631 0.7842 1.2231 1.2331 1.2512

0.002 0.9559 1.1436 1.2633 0.7459 0.7636 0.7818 1.2331 1.2432 1.2489

0.003 1.0803 1.2417 1.3732 0.7469 0.7622 0.7815 1.2456 1.2462 1.2564

0.004 1.1778 1.3465 1.5187 0.7482 0.7615 0.7828 1.2565 1.2519 1.2713

0.005 1.2875 1.4923 1.6065 0.7463 0.7634 0.7828 1.2592 1.2687 1.2777

96

Figure 35: Plots for ultrasonic velocity versus concentration for aqueous a) MPH and b) MTN at different temperatures () T = 303 K, (•) 308 K and () 313 K

0.001 0.002 0.003 0.004 0.0051410

1420

1430

1440

1450

1460

1470

1480

1490

Conc.

u / m

s-1

(a)

0.001 0.002 0.003 0.004 0.005

1450

1460

1470

1480

1490

1500

1510

1520

Conc.

u / m

s-1

(b)

97

Figure 36 Plots for adiabatic compressibility versus concentration for aqueous a) MPH and b) MTN at different temperatures () T = 303 K, (•) 308 K and () 313 K

0.001 0.002 0.003 0.004 0.0050.41

0.42

0.43

0.44

0.45

0.46

0.47

Conc.(a)

κ / x

10-9

m2 N

-1

0.001 0.002 0.003 0.004 0.005

0.40

0.41

0.42

0.43

0.44

Conc.

κ / 1

0-9 m

2 N-1

(b)

98

Figure 37 Plots for internal pressure versus concentration for aqueous a) MPH and b) MTN at different temperatures () T = 303 K, (•) 308 K and () 313 K

0.001 0.002 0.003 0.004 0.005

37500

38000

38500

39000

39500

Conc.(a)

π i / at

m

0.001 0.002 0.003 0.004 0.00539000

39500

40000

40500

41000

Conc.(b)

π i / at

m

99

Figure 38 Plots for Enthalpy versus concentration for aqueous a) MPH and b) MTN at different temperatures () T = 303 K, (•) 308 K and () 313 K

0.001 0.002 0.003 0.004 0.005

63.5

64.0

64.5

65.0

65.5

66.0

Conc.(a)

H / k

j

0.001 0.002 0.003 0.004 0.00566.4

66.8

67.2

67.6

68.0

68.4

68.8

Conc.(b)

H /

k j

100

Figure 39 Plots for Gibbs free energy versus concentration for aqueous a) MPH and b) MTN at different temperatures () T = 303 K, (•) 308 K and () 313 K

0.001 0.002 0.003 0.004 0.005

0.71

0.72

0.73

0.74

0.75

0.76

Conc.(a)

G /

x 10

-20 k

j m

ol-1

0.001 0.002 0.003 0.004 0.005

0.75

0.76

0.77

0.78

Conc.(b)

G /

x 10

-20 k

j m

ol

101

The excess properties derived from these physical property data

reflect the physico-chemical behaviour of the liquid mixtures with

respect to the solution, structure and intermolecular interactions

between the component molecules of the mixture. The trends of

changes have been interpreted by the earlier works reported by various

researchers [15-17]. It has revealed from the literature, that the trends

of changes are mainly depends on the differences in the size of the

molecule and strength of interactions taking place between the

component of the mixtures. The velocity of sound through liquid

mixtures could be helpful in assessing the degree of association

between the molecules. The molar sound velocity on non-associated

liquids has been found to be independent, while that for associated

liquids is dependent on temperature.

Velocity

From the Table 1, for both the samples it has been observed that,

as the concentration increases, velocity decreases. This decrease in

velocity is due to increased association between the solute and the

solvent molecules. The compounds MPH and MTN has NH-CO-NH

arrangement, this may interacts with solvent molecules (DMSO). This

shows that, the existence of hydrogen bonding present in the liquid

mixture. Further, it has been observed that when temperature

increases velocity decreases, this may be attributed to the increased

vibration or collision between the solvent and solute molecules.

Adiabatic Compressibility

It has been observed from the Table 2, the adiabatic

compressibility values computed for the samples at different

concentrations, has been found to increase as the concentration

102

increases. This increase in concentration is as a result of molecular

aggregation. Molecular aggregation takes place through hydrogen

bonding between the solute and solvent. This result clearly reveals the

existence of hydrogen bonding in the liquid mixture.

Free Length (Lf)

The free length (Lf) values measured at different concentrations

are listed in Table 2. It has been found that, Lf values are found to

increase as the concentration increases. In general, dilute solutions the

distance between the molecules will be large and hence, the length of

the non-covalent interaction will be less. While in concentrated

solutions, due to hydrogen bonding the distance between the molecules

will be less. Therefore, the intermolecular free length (Lf) is found to

increase in both the samples.

Free Volume

Free volume is one of the significant factors in explaining the

variations in the physico-chemical properties of liquid mixture. The free

space and its dependent properties has a close connection with

molecular structure and it may show interesting features about

interactions. This molecular interactions between like and unlike

molecules are influenced by structural arrangements along with the

shape and size of the molecules.

From the results of computed values of free volume of the binary

liquid mixture at different concentrations, it has been observed that

there is a increase in free volume as concentration increases. This

increase in free volume is attributed to the strong interaction between

the molecules when the solute concentration increases.

103

Acoustic Impedance

Acoustic impedance (Z) values measured for different

concentrations at three different temperatures are tabulated in Table 3.

The result show that for both the samples, the Z value decreases with

increase in concentration of the system. Similar trend has been

observed for both the samples when the temperature increases.

Relaxation time (τ)

The relaxation time (τ) computed for different concentrations

under different temperature for the binary liquid systems are presented

in Table 3. It has been noted that, the (τ) value increases with increase

in concentration of the systems. The dispersion of the ultrasonic

velocity in the system should contain information about characteristic

time (τ) of the relaxation process that causes dispersion. The relaxation

time which is in the order of 10-13 sec is due to structural relaxation

process and in situation. It is suggested that, the molecules get

rearranged due to cooperative process.

Gibb’s Free Energy

The computed values of Gibb’s Energy for the binary liquid

mixture of two samples are tabulated in Table 5. From the results, it

has been understood that the ( ) decreases with increase in

concentration of the system. This decrease in free energy confirms the

formation of hydrogen bonding in binary mixtures.

Enthalpy

The measured values of enthalpy for the binary liquid mixtures of

two samples are presented in Table 4. As the concentration of the

104

solute increases the enthalpy is found to increases. The same trend has

been observed when the temperature of the system increases.

Conclusion

The ultrasonic velocity, adiabatic compressibility, free length,

viscosity, free volume, free length, molar volume, density, internal

pressure, free energy and enthalpy have been measured. The measured

values of ultrasonic velocity, internal pressure, enthalpy and free

energy have been found to increase as the concentration increases. The

calculated values of adiabatic compressibility are found to decrease as

the concentration increases. These results are inferred that the strong

interaction is exist between the solvent (DMSO) and the solute (MPH

and MTN). Hence it is concluded that the association of solute

molecules occurs through hydrogen bonding.

105

References

1. Kannappan V & Jaya Shanthi R, Indian J Pure & Appl Phys, 43 (2005) 750.

2. Kannappan V, Xavier Jesu Raja S & Jaya Santhi R, Indian J Pure & Appl Phys, 41 (2003) 690.

3. Jayakumar S, Karunanithi N, Kannappan V & Gunasekaran S, Asian Chem Lett, 3 (1999), 224.

4. Neuman M S & Blum, J Am Soc, 86 (1964) 5600.

5. Dash S K & Swain B B, Chem. Papers, 48 (1994) 146.

6. Dash S K Chakravorthy V & Swain B B, Acoustic Lett, 19 (1996) 142.

7. Eyring H and Incaid K, Chem. Phys, 6.620 (1938)

8. Singh S, Singh R, Prasad N and Prakash S, Ind. J. Pure and Appl. Phy., 3, 156 (1977)

9. Ramamurthy M and Sastry O S, Indian J. Pure and Appl. Phys, 21, 579, (1983)

10. Hobbs M E and Bates W W, J. Am. Chem Soc., 74, 746, (1952)

11. Negakuva, J Am Chem Soc, 76, 3070, (1954).

12. Freedman E, J Chem Phys, 21, 1784, (1955).

13. Kannappan A N and Rajendran V, Indian J. Pure and Appl. Phys., 30(176, (1992)

14. Kinsler L E and Rray A R., Fundamentals of Acoustics, Wiley Edition, 1989.

15. J.M. Prausnitz, R.N. Lichtenthaler, E.G. Azevedo, Molecular Thermodynamic of Fluid-Phase Equilibria, 2nd Ed., Prentice Hall, Inc., 1986.

16. S.L. Oswal, N.B. Patel, J.Chem. Eng Data 40 (1995) 840-844.

17. S.L. Oswal, P. Oswal, J.P. Dave, J.Mol.Liq. 94 (2000) 203-219.

18. S.L. Oswal, K.D. Prajapati, N.Y. Ghael, S.P. Ijardar, Fluid Phase Equilib, 218 (2004) 131-140.

106

19. S.L. Oswal, R.L. Gardas, R.P. Phalak, J.Mol. Liq. 116 (2005) 109-118.

20. Kannappan, V, Jaya Santhi R & Malar E, J P, Phys Chem Liq, 40 (2002) 507.

21. Tabhane V A, Sangeeta Agarwal & Revetkar K G, J. Acous Soc Ind, 8 (2000) 369.

22. Anwar Ali & Anil Kumar Nain, Acoustics Lett, 19 (1996) 181.

23. Bhatt S C, Harikrishnan Semwal & Vijendra Lingwal, J Acous Soc Ind, 28 (2000) 275.

24. Nikam P S & Hiray, Indian J Pure & Appl. Phys, 29 (1991) 601.

25. Moelwyn Hughes E. A., Thorpe P.L., Proc. Roy. Soc. London 268 (1964) 574.

26. Prakash S., Srivastava S.B., Chem, Thermodynamics 7 (1975) 997.

27. Vileu R., Simion A., Rev. Roum, Chim 21 (1976) 117.

107

Chapter VI

Biological Studies

Introduction

Bacterial infection often produces pain and inflammation.

Inflammation remains a common with poorly controlled clinical

problem which can be life threatening in extreme form of allergy,

autoimmune diseases and rejection of transplanted organs. The

treatment options which can be used for inflammatory diseases are

unsatisfactory and complicated due to their lack of efficacy and adverse

effect profile. It seemed worthwhile to look for persons acting on more

than one pathway involved in inflammatory conditions [1].

Antimicrobial résistance (AMR) is a major public health threat. Despite

the need for new antimicrobials, very few effective molecules have been

brought to the market these last decades. The urgency for novel drug

candidates or for novel strategies to fight AMR is especially true when

considering the increasing resistance of Gram negative bacteria to all

known antibiotics. Our strategy for combatting this bacterial resistance

was first to focus on targets, not yet explored with antibiotics available

on the market.

Microorganisms Enterococcus faecalis Taxonomy

Domain : Bacteria Kingdom : Eubacteria Phylum : Firmicutes Class : Cocci Order : Lactobacillales Family : Enterococcaceae Genus : Enterococcus Species : faecalis

108

Enterococci are associated with both community and hospital

acquired infections. Enterococci can grow at a temperature range of 10˚

to 42°C and in environments with broad pH values. Some are known to

be motile. While there are over 15 species of the Enterococcus genus,

80-90% of clinical isolates are E. faecalis [2]. Enterococci are Gram-

positive cocci that typically form short chains or are arranged in pairs.

Under certain growth conditions they can elongate and appear

coccobacillary. 40% of the cell wall is made up of peptidoglycan, while

the rest of the cell wall is made up of a “rhamnose-containing

polysaccharide and a ribitol-containing teichoic acid” [3]. In general,

Enterococci are alpha-hemolytic. Some possess the group D Lancefield

antigen and can be detected using monoclonal antibody-based

agglutination tests. Enterococci are typically catalase negative, and are

anaerobic. They are able to grow in 6.5% NaCl, can hydrolyze esculin in

the presence of 40% bile salts and are pyrrolidonyl arylamidase and

leucine arylamidase positive.

Proteus mirabilis

Taxonomy

Kingdom : Bacteria

Phylum : Proteobacteria

Class : Gamma proteobacteria

Order : Enterobacteriales

Family : Enterobacteria

Genus : Proteus

Species : Proteus mirabilis

Proteus mirabilis was first discovered by a German pathologist

named Gustav Hauser. Hauser named this genus Proteus, after the

character in Homer’s The Odyssey that was good at changing shape

109

and evading being questioned [4], a name that seems apt given this

organism’s uncanny ability to avoid the host’s immune

system. P.mirabilis is a gram-negative, rod-shaped bacterium that can

be found as part of the micro flora in the human intestine. This

organism is not usually a pathogen, but does become a problem when

it comes into contact with urea in the urinary tract. From there,

infection can spread to other parts of the body. P.mirabilis accounts for

most of the urinary tract infections that occur in hospital settings and

for ninety percent of Proteus infections [5]. Proteus species are among

the commonly implicated pathogens in hospital as well as community

acquired infections [6]. This pathogen has a diverse mode of

transmission, and hence can cause infection in different anatomical

sites of the body. Some of the incriminating sources of transmission are

soil, contaminated water, food, equipments, intravenous solutions, the

hands of patients and healthcare personnel [7]. There are reports of 9.8

to 14.6% prevalence rates of Proteus infections in MGM [8].

Staphylococcus aureus

Taxonomy

Domain : Bacteria

Kingdom : Bacteria

Phylum : Firmicutes

Class : Cocci

Order : Bacillales

Family : Staphylococcaceae

Genus : Staphylococcus

Species : Staphylococcus aureus

Staphylococci sp. are spherical gram-positive bacteria, which are

immobile and form grape-like clusters. They form bunches because

110

they divide in two planes as opposed to their close relatives streptococci

which form chains because they divide only in one plane [9]. Colonies

formed by S.aureus are yellow (thus the name aureus, Latin for gold)

and grow large on a rich medium. Staphylococcus aureus and their

genus Staphylococci are facultative anaerobes which means they grow

by aerobic respiration or fermentation that produces lactic acid. As a

pathogen, it is important to understand the virulence mechanisms of

S. aureus especially the Methicillin-resistant Staphylococcus aureus

(MRSA) in order to successfully combat the pathogen

S. aureus is a versatile human pathogen with the ability to cause

a large spectrum of human diseases, ranging from skin lesions

(abscesses, impetigo) to invasive and more serious infections

(osteomyelitis, septic arthritis, pneumonia, endocarditis). The ability

of S. aureus to cause disease has been attributed to an impressive

spectrum of cell-wall-associated (protein A, clumping factors,

fibronectin binding proteins, and other adhesive matrix molecules)

factors, and extracellular toxins (coagulase, hemolysins, enterotoxins,

toxic-shock syndrome toxin 1, exfoliative toxins, and Panton-Valentine

leukocidin) as virulence determinants [10].

Pseudomonas aeruginosa Taxonomy

Domain : Bacteria

Kingdom : Eubacteria Phylum : Proteobacteria Class : Gamma Proteobacteria Order : Pseudomonadales Family : Pseudomonadaceae Genus : Pseudomonas

111

Species : aeruginosa

Pseudomonas aeruginosa is a gram-negative, rod-shaped,

asporogenous, and mono-flagellated bacterium that has an incredible

nutritional versatility. It is a rod about 1-5 µm long and 0.5-1.0 µm

wide. The opportunistic bacterial pathogen currently known as

P. aeruginosa has received several names throughout its history based

on the characteristic blue-green coloration produced during culture.

Lucke was the first to associate this pigment with rod-shaped

organisms. P. aeruginosa was not successfully isolated in pure culture

until 1882, when Carle Gessard reported in a publication entitled “On

the Blue and Green Coloration of Bandages” the growth of the organism

from cutaneous wounds of two patients with bluish-green pus [11].

The ability of P. aeruginosa to survive on minimal nutritional

requirements and to tolerate a variety of physical conditions has

allowed this organism to persist in both community and hospital

settings. In the hospital, P. aeruginosa can be isolated from a variety of

sources, including respiratory therapy equipment, antiseptics, soap,

sinks, mops, medicines, and physiotherapy and hydrotherapy pools

[12]. Community reservoirs of this organism include swimming pools,

whirlpools, hot tubs, contact lens solution, home humidifiers, soil and

rhizosphere, and vegetables.

Escherichia coli Taxonomy

Domain : Bacteria Kingdom : Eubacteria Phylum : Proteobacteria Class : Gamma Proteobacteria Order : Enterobacteriales

112

Family : Enterobacteriaceae Genus : Escherichia Species : coli

E.coli was first discovered in 1885 by Theodor Escherich, a

German bacteriologist. E.coli has since been commonly used for

biological lab experiment and research. E.coli is a facultative (aerobic

and anaerobic growth) gram-negative, rod shaped bacteria that can be

commonly found in animal feces, lower intestines of mammals, and

even on the edge of hot springs. They grow best at 37° C. E.coli is a

Gram-negative organism that cannot sporulate. E.coli can also be

classified into hundreds of strains on the basis of different

serotypes. E.coli O157:H7, for example, is a well-studied strain of the

bacterium E.coli, which produces Shiga-like toxins, causing severe

illness by eating cheese and contaminated meat [13]. Escherichia coli

represent a large array of genetic subtypes defined by the somatic (O)

and flagellar antigen (H). Most subtypes are harmless whilst some can

cause severe diarrhea. E. coli O157:H7 is an important subtype that

causes many food borne outbreaks worldwide in the past decades (2).

Other serotypes such as E. coli O26:H11, O111:H8, O103:H2,

O113:H21 and O104:H21 have also been implicated in causing

foodborne outbreaks [14].

Klebsiella pneumoniae Taxonomy

Domain : Bacteria Kingdom : Eubacteria Phylum : Proteobacteria Class : Gamma Proteobacteria Order : Enterobacteriales Family : Enterobacteriaceae

113

Genus : Klebsiella Species : pneumoniae

Bacteria belonging to the genus Klebsiella frequently cause

human nosocomial infections. Klebsiella is well known to most

clinicians as a cause of community-acquired bacterial pneumonia,

occurring particularly in chronic alcoholics and showing characteristic

radiographic abnormalities due to a severe pyogenic infection which

has a high fatality rate if untreated [15]. Klebsiella spp. are ubiquitous

in nature. Klebsiella probably have two common habitats, one being the

environment, where they are found in surface water, sewage, and soil

and on plants [16] and the other being the mucosal surfaces of

mammals such as humans, horses, or swine, which they colonize. In

this respect, the genus Klebsiella is like Enterobacter and Citrobacter

but unlike Shigella spp. or E. coli, which are common in humans but

not in the environment.

Antibiotic resistance

At present, serious infection caused by microorganism has

become difficult to treat because of their resistant to vast array of

antibiotics [17]. Antibiotic resistance is the reduction of effectiveness of

a drug when it is not intended to kill or inhibit a pathogen due to

altered enzyme targets [18]. Escherichia coli, Klebsiella pneumoniae,

Acinetobacter baumannii, Staphylococcus aureus, Streptococcus

pneumoniae, Enterococcus, and Mycobacterium tuberculosis are most

frequently reported as multi drug resistance producers among the

hospital acquired and community infection. Volume of antibiotic used

in hospitals and healthcare created a selective pressure on spread of

resistance, has become major threat to treat disease.

114

Spread of antibiotic resistance is contributed by indiscriminate

and widespread usage of antibiotics. Resistance is a pandemic and

complex have a wide range of physiological and biochemical mechanism

[19]. The molecular mechanism of drug resistance is a complex and

diverse among the microorganisms followed by nature and

anthropogenic activities. Furthermore, research on antibiotic resistance

reveals that the genetic mutation is a major global factor for the

evolution of antibiotic resistance by enzyme inactivation, efflux pump

inhibition, altering active site etc., [20]. Among the antibiotic

resistance, Beta-lactam antimicrobial agents exhibit the most common

bacterial infections and continue to be the prominent cause of

resistance to β-lactam antibiotics among Gram negative bacteria

worldwide. Mutation of β-lactamases results bacteria expanding their

activity even against the newly developed β-lactam antibiotics. These

enzymes are known as extended-spectrum β-lactamases (ESBLs) [21].

Molecular docking

Cancer can be defined as the uncontrolled growth of abnormal

cells. The epidermal growth factor receptor is responsible for the breast

cancer and the receptor – ligand interaction plays a significant role in

structural based drug designing. Similar to cancer study, the specific

receptors were found in the surface (antigen) of the Ebola virus. That

particular receptors were docked to the ligand morpholine and the

energy values are obtained separately. To detect the binding efficiency

and steric compatibility using docking studies. Docking is used to

predict the binding orientation of small molecule drug candidates to

their targets in order to predict the affinity and activity of the small

molecule. Hence docking plays an important role in the rational design

115

of drugs. The orientation of the ligand to the receptor as well as the

conformation of the ligand and receptor when bound to each other.

Docking is most recent technique which has been widely used for

virtual screening. Its default search function is based on Lamarckian

Genetic Algorithm (LGA), a hybrid genetic algorithm with local

optimization that uses a parameterized free-energy scoring function to

estimate the binding energy [22]. Each docking is comprised of multiple

independent executions of LGA and a potential way to increase its

performance is to parallelize the aspects for execution. Docking of small

molecules in the receptor binding site and estimation of binding affinity

of the complex is a vital part of structure based drug design.

Description and clinical significance of Enterococcus infection

Enterococci have proven to present a therapeutic challenge

because of their resistance to many antimicrobial drugs, “including

cell-wall active agents; aminoglycosides, penicillin and ampicillin, and

vancomycin” [23]. The Enterococci have the capacity to acquire a wide

variety of antimicrobial resistance factors, which present serious

problems in the management of patients with Enterococcal infections.

In general, Enterococcal isolates with lowered susceptibility to

vancomycin can be categorized as vanA, vanB, and vanC. vanA and

vanB pose the greatest threat because they are the most resistant and

the resistance genes are carried on a plasmid. Since the resistance

genes are carried on a plasmid they are readily transferable, E. faecalis

can transfer these plasmids by conjugation [24]. E. faecalis are also

resistant to teicoplanin. Enterococcal strains that are vancomycin-

dependent have been found, but are rare and less common than

116

vancomycin-resistant strains (referred to as “vancomycin-resistant

Enterococci” or “VRE”).

E. faecalis is generally considered a non-encapsulated organism,

shown by the “lack of a detectable mucoid phenotype”. However,

subsets of E. faecalis isolates possess a capsular polysaccharide. E.

faecalis can exchange genetic material (plasmids) by conjugation

processes induced by small peptide pheromones. Surface protein

“aggregation substances that recognize a specific ligand on recipient

cells” ensure successful connections for conjugation. E. faecalis also

have the capability to make surface pili which can lead to the formation

of a biofilm. The E. faecalis strains that cause endocarditis contain

large amounts of these pili. The pili allow for attachment to host

surfaces (e.g. the heart tissue). The strains of E. faecalis that cause

endocarditis produce the “biofilm significantly more often and also to a

greater degree than non-endocarditis isolates” [25].

Due to many public health dangers, the genome sequence data

from a strain of Enterococcus was necessary. The strain chosen for

genome DNA sequencing was E. faecalis V583, the first vancomycin-

resistant isolate in the United States. The genome of strain V583 was

sequenced by The Institute for Genome Research (TIGR). The

enterococcal genome shows E. faecalis is metabolically diverse and

contains a wide range of regulatory systems.. These differences

associated with antibiotic resistance or virulence suggested the

acquisition of genetic material from a foreign species through horizontal

transfer. It is still unknown whether the transfers are responsible for

the variations in DNA makeup.

117

The information contained in the genome of E. faecalis V583 will

greatly aid the understanding of how the organism has adapted to be a

versatile human pathogen. More studies like these will suggest new

medicines to bacterial infections caused by the Enterococci. The genome

also contains 3 Ebp (encoding for the endocarditis biofilm-associated

pili) operons which are important for biofilm production of E. faecalis

strain OG1RF. This strain uses these operons to produce surface pili. .

The surface pili are used for “attachment to the host surfaces and are

antigenic in humans during endocarditis”.

E. faecalis is a very diverse species of Enterococci. It interacts

with many other organisms and has effects on the environment. They

can be found in soil, water, and plants. Some strains are used in the

manufacture of foods whereas others are the cause of serious human

and animal infections (e.g. they are known to colonize the

gastrointestinal and genital tracts of humans). The Enterococci are

members of the bacterial community inhabiting the large bowel in

humans. They also are a natural part of the intestinal flora in most

other mammals and birds.

The Enterococci are also found in soil, plants, and water. When

they are found in water it is typically because the water had been

contaminated with fecal matter. The ecology of antibiotic resistance and

virulence gene transfer in the environment is still not well understood.

Insects, such as houseflies (HF), that develop in decaying organic

material can transmit antibiotic-resistant bacteria from the manure of

animals and other decaying organic substrates to residential settings

[26]. HF are perfect transmitters because of the live microbial

communities present in the habitats where they develop (e.g. feces).

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Adding to the good transfer qualities are the way in which HF

feed their young (regurgitation) and their attraction to human food. A

recent study screened for antibiotic resistance and virulence genes

in Enterococci from HF in fast-food restaurants in Kanasas. This study

showed that “houseflies in food-handling and serving facilities can

carry antibiotic-resistant and potentially virulent Enterococci that have

the capacity for horizontal transfer of antibiotic resistance genes to

other bacteria”. The effects that E. faecalis has on the environment tend

to be more negative. They typically contaminate water supplies that can

lead to infected plants as well as infections in people. The antibiotic

factors can also be transported by various insects (e.g. house flies) and

animals, leading to increasing numbers of virulent E. faecalis.

Enterococci have emerged as a major cause of nosocomial

infections, and within this group Enterococcus faecalis causes the

majority of human enterococcus infections. Since E. faecalis are

capable of surviving numerous environmental challenges (such as

temperature extremes and the presence of bile salts) and because they

can acquire resistance to multiple antibiotics, these bacteria have

become a major health problem. The National Nosocomial Infection

Surveillance (NNIS) system has reported increases in the incidence of

infections due to vancomycin-resistant Enterococci (VRE) since 1989.

This can mean serious health problems, which include the lack

of available antibiotic therapy for VRE infections, because most VRE

strains harbor resistance to multiple antibiotics besides vancomycin

(e.g. aminoglycosides and ampicillin). The transfer of vancomycin

resistant genes from VRE to other Gram-positive pathogens is a serious

public health concern. Enterococci can be carried on the hands of

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health care workers and be carried (transferred) from one patient to

another. It has been shown that VRE on the hands can persist for up to

60 minutes. The transmission from a health care worker’s hands to the

patient could take place upon contact with the patient’s intravenous or

urinary catheters. Rectal thermometers, not properly cleaned after use,

can transmit the VRE from patient to patient as well. Sometimes the

transmission can result in colonization of the patient’s GI tract with the

acquired strain. The new strain then becomes part of the patient’s

endogenous flora. The acquired strain, carrying antibiotic resistance

genes, is able to live in the GI tract. Infections then arise from these

newly acquired E. faecalis strains.

E. faecalis can cause many infections within the human body.

The most common infection caused by Enterococci is infection of the

urinary tract. E. faecalis can cause lower urinary tract infections (UTI),

such as cystitis, prostatitis, and epididymitis. E. faecalis are also found

in intra-abdominal, pelvic, and soft tissue infections. The E. faecalis

can cause nosocomial bacteremia. The source of bacteremia is most

often the urinary tract, occurring from an infected intravenous

catheter. Endocarditis is the most serious Enterococcai infection, as it

causes inflammation of the heart valves. In many cases of endocarditis,

antibiotic treatment fails and surgery to remove the infected valve is

necessary. Less common infections caused by E. faecalis include

meningitis, hematogenous, osteomyelitis, septic arthritis, and (very

rarely) pneumonia. Due to the resistance of Enterococci to many

antibiotics, treatment of these infections is difficult.

The Enterococci inhabit harsh environments, like the intestinal

tracts of humans and animals. Growth under these hostile conditions

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requires that E. faecalis have a metabolism that is flexible. E. faecalis

are capable of not only fermentation to produce lactic acid but also can

“catabolize a spectrum of energy sources from carbohydrates, glycerol,

lactate, malate, citrate, diamino acids and many α-keto acids”. It has

been shown that under selected growth conditions E. faecaliscan

enhance growth through oxidative phosphorylation using a proton

motive force established by electron transport. The tolerance of this

stress, combined with other severe growth conditions, allows the E.

faecalis to grow at 10 to 45°C, in bile salts, and at extremely low and

high pHs. In addition, E. faecalis can resist azide, detergents, heavy

metals, and ethanol.

In the intestine, E. faecalis derive most of their energy from the

fermentation of non-absorbed sugars. E. faecalis can also get energy by

degrading mucins, a carbohydrate that is heavily glycosylated and

produced by intestinal goblet cells. The E. faecalis uses a

“phosphoenolpyruvate phosphotransferase system (PTS) to sense

sugars outside the cell and couples uptake of sugars with

phosphorylation”. In doing so, less energy (ATP) is wasted compared to

how sugar is accumulated by non-PTS systems. Sugars metabolized

by E. faecalis are include: D-glucose, D-fructose, lactose, maltose (all

PTS substrates). E. faecalis is one of a few low-G+C content Gram-

positive bacteria that expel sugar during growth on glucose, a

phenomenon known as inducer expulsion. E. faecalis can even ferment

glycerol under aerobic and microaerophilic conditions.

Glycerol can cross the cell membrane using a protein called the

glycerol diffusion facilitator (GlpF). The GlpF makes the concentration

of glycerol inside and outside the cell equal, the protein is inhibited by

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glycolysis. E. faecalis are able to live in extreme alkaline pH and high

salt concentration. These traits require cation transport to maintain the

constant cytosolic ion composition essential for homeostasis. All cells

must expel excess sodium to maintain cytosolic concentrations in range

that favors homeostasis. Potassium is a major intracellular cation. The

potassium concentration within E. faecalis of 0.4 to 0.6 M is essential

for normal cellular metabolism, it “neutralizes intracellular anions,

activates diverse enzymes, and regulates cytosolic pH”. Although it is

known that KtrI and KtrII are K+ uptake systems (they are K+/H+

symporters), little more is known the proteins.

Enterococci have been studied for possible use as a probiotic (a

dietary supplement that contains living non-virulent microbial cells

that when ingested are thought to beneficially affect the composition of

the intestinal microflora). Administration of the E. faecalis strain has

been shown to reduce diarrhea. Due to the high disease causing

properties of E. faecalis, much more research has been conducted on

how to stop the virulence of E. faecalis than the beneficial use of E.

faecalis.

Description and clinical significance of Proteus infection

Urinary tract infections (UTIs) are very painful and can become

lethal if the infection spreads to other systems in the body. After

pneumonia, urinary tract infections are the most common problem in

long-term hospital patients. These infections are becoming more

difficult to treat because forty-eight percent of P.mirabilis strains are

resistant to amoxicillin, penicillin, fluoroquinolones and other broad-

range activity antibiotics. The pH of urine is usually neutral or slightly

acidic, but when a patient wears a catheter for extended periods of time

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crystalline deposits from the urine form a crust around the catheter

and obstruct urine from moving through the urethra. The encrusted

crystals on the catheter give P.mirabilis the opportunity to colonize in

large numbers and to hydrolyze the urea, thus increasing the

environmental pH through the production or ammonia [28].

The genome sequence of P.mirabilis was completed in March 28,

2008 by Melanie M. Pearson identifying more than 3,658 coding

sequences with 7 rRNA loci [29]. The genome’s total length is 4.063 Mb

with a 28.8% GC content. P.mirabilis also carries a single plasmid with

36, 298 nucleotides. The plasmid itself does not contain any virulence

genes but it may contain a bacteriocin and its immunity system. Within

the genome is a genomic island involved in pathogenicity that codes for

a type III secretion system comprising 24 genes used to inject bacterial

proteins into a host genome. This type III system appears to be

incorporated through horizontal gene transfer and is noted for its

relatively smaller G+C content compared with the rest of the genome

[29]. The genome sequence encodes 17 different types of fimbriae as

well as a 54 kb flagellar regulon. The flagella made by the strain all

come from a single locus. This information is characterized for a

specific uropathogenic strain of P.mirabilis, HI4320. It is the first

completed sequence of the bacterium out of more than 75 known

strains that were identified using one dimensional SDS PAGE of

cellular proteins mostly from human origin [30].

P.mirabilis has a bacillus morphology and is a gram-negative

bacterium. It is motile, alternating between vegetative swimmers and

hyper-flagellated swarmer cells. It also makes a variety of fimbriae.

P.mirabilis produces urease, an enzyme that converts urea into

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ammonia. Infection by P.mirabilis can therefore be detected by an

alkaline urine sample (pH 8 and up) with large amounts of ammonia.

P.mirabilis can be found as a free-living microbe in soil and water.

The organism is also normally found in the gastrointestinal tract of

humans. Some believe that P.mirabilis has access to the bladder by

infecting the periurethral area. P.mirabilis causes urinary tract

infections primarily through indwelling catheters. Usually the urinary

tract can wash out the microbe before it accumulates, but the catheter

prevents this from happening. P.mirabilis can then adhere to the

insides and outsides of the catheter, forming biofilm communities.

Once established, these microbes pass through the urethra via

swarming motility to the bladder. P.mirabilis binds to bladder epithelial

cells where it eventually colonizes [31]. P.mirabilis infection can also

lead to the production of kidney and bladder stones. The bacteria

colonize the stones as they form, making them less accessible to

antibiotic attack.

Pathogenicity of P.mirabilis is accomplished in the following two

steps. First the microorganism needs to colonize the urinary tract and

second, the microorganism needs to successfully evade host defenses.

Colonization of the urinary tract is done by using two of the four types

of fimbriae called Mannose-resistant fimbriae (MRF) and P.mirabilis

fimbriae (PMF). The importance of the MRF was determined in a study

done at the Mobley lab at the University of Michigan Medical School. In

their study they were able to successfully produce a nasal vaccine

against MRF that worked in mice. There are four possible mechanisms

by which P.mirabilis can use to evade the host defenses. The first is

production of an IgA-degrading protease which functions to cleave the

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secretory IgA. IgA is released by the host in an initial response to

infection [32].

The second immune system evasion mechanism is through three

unique flagellin genes, which have been shown to recombine and form

novel flagella capable of tricking the host’s defenses [33]. The third is

through expression of the MR/P fimbriae (mentioned above). The fourth

mechanism is the urease-mediated stone formation. Production of

ammonia by the action of urease results in stone formation, and these

stones in turn, help protect the bacteria. Urease and hemolysin are

known to cause damage to host epithelial cells. As mentioned above,

urease can damage host epithelial cells through the formation of

stones. Hemolysin damages cells because of its property as a potent

cytotoxin (Janson, 2003). P.mirabilis has seven virulence factors known

to aid pathogenicity.

The antigens found on the outer membrane of P.mirabilis can

potentially serve as targets for vaccines. So far, of the 37 identified

immuno-reactive antigens, 23 are surface-bound proteins. Studies have

shown that 2 iron acquisition proteins (PMI0842 and PMI2596)

increase the virulence of P.mirabilis in the urinary tract. Since both of

these proteins contribute to pathogenesis, they are good candidates for

vaccines. Once an effective vaccine is made for these antigens, further

research will determine whether or not these vaccines may be used

against other bacteria that cause complicated urinary tract infections,

such as Providencia and Morganella [33].

P.mirabilis can be commonly present in healthy individuals as

part of the normal mucosa. The bacterium becomes a significant

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problem mostly in individuals that have vulnerable immune systems

and are in danger of nosocomial transmission, such as hospital

patients [34]. Current studies show that there are a number of

antibiotics that were once effective against P.mirabilis that are now

useless due to extended spectrum beta lactamases (ESBLs).

Description and clinical significance of Staphylococcus infection

The Staphylococcus aureus genome, which is the most common

species among the Staphylococcus genome projects, is the most

completed genome sequence compared to any other microbial species.

The original genome map of Staphylococcus aureus was based on the

strain NCTC 8325, initiated by Peter A. Pattee and colleagues. By 2000,

the entire genome of strain 8325 had been sequenced and annotated.

Since then, at least six other ‘‘S.aureus’’ strains have been completed

(COL, N315, Mu50, MW2, MRSA252, MSSA476). The Staphylococcus

aureus strain NCTC 8325 complete circular genome map shows ~2,900

open reading frames, 61 tRNA genes, 3 structural RNAs, and 5

complete ribosomal RNA operons. This strain has about 33% G+C

content and an average gene length of 824 nucleotides with 85% coding

sequence, similar to other S.aureus strains. Half the coding sequence is

located predominantly on one replichore and the second half is located

predominantly on the other replichore.

Virulence factors are encoded by phages, plasmids, pathogenicity

islands and staphylococcus cassette chromosome [35]. Increased

resistance for antibiotics is encoded by a transposon (Tn 1546) that

was inserted into a conjugated plasmid that also encoded resistance to

other things including disinfectants. MRSA (Methicillin-

Resistant Staphylococcus aureus), which is resistant to the antibiotic

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methicillin, expresses a modified penicillin-binding protein encoded by

mecA gene. This was brought about by many evolutions thought

horizontal gene transfer of mecA to a wide variety of methicillin

susceptible S.aureus strains. The genes for antibiotic resistance

in Staphylococcus aureus are located on plasmids or other similar

structures. Evolution of this bacterium can occur through

asymptomatic colonization and/or during the course of the caused

disease. The increasing population of "super germs" and antibiotic

resistant pathogens have increased pressure on researchers to find

alternative, more effective ways of fighting these "super germs." DNA

sequencing of this microbe has already isolated the source code of its'

resistance to antibiotics, and further research will more than likely lead

us to the path of our next artillery against this and many other

pathogens.

Staphylococcus aureus is a gram-positive bacteria, which means

that the cell wall of this bacteria consists of a very thick peptidoglycan

layer. They form spherical colonies in clusters in 2 planes and have no

flagella [36]. Secretions are numerous, but include surface associated

adhesins, endotoxins, exoenzymes, and capsular polysaccharides. The

capsule is responsible for enhanced virulence of a mucoid strain.

Lactate is the end product of anaerobic glucose metabolism and acetate

and CO2 are the products of aerobic growth conditions. S.aureus can

uptake a variety of nutrients including glucose, mannose, mannitol,

glucosamine, N-acetylglucosamine, sucrose, lactose, galactose and

beta-glucosides.

Staphylococcus aureus is among the most common hospital

acquired pathogens. It is a normal inhabitant of the skin and mucous

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membranes in the nose of a healthy human. S.aureus is infectious to

both animals and humans and may only survive on dry skin. It can be

spread through contaminated surfaces, through the air and through

people. Though some host colonization can be benign, a puncture or

break in the skin can prompt this bacterium to enter a wound and

cause infections. The best preventive measure is simply regular hand

washing (preferably without antibacterial soaps or hand sanitizers, but

that’s another story) and daily bathing.

The MRSA, resistant to the antibiotic methicillin, was eventually

isolated. Consequently, vancomycin (the most powerful antibiotic in our

arsenal) became the primary antibiotic used to combat staphylococcus

infection. In 1997 a strain of S.aureus resistant to vancomycin was

isolated, and people are once again exposed to the threat of untreatable

staphylococcus infection. MRSA strains are currently a very significant

health care problem. The sequencing of the S.aureusgenome will

hopefully provide insight into how the organism generates such a

variety of toxins, and aid researchers in developing ways of combating

the versatile bacterium [37].

Description and clinical significance of Pseudomonas infections

A review of surveillance data collected by the CDC National

Nosocomial Infections Surveillance System from 1986 to 1998 shows

that P. aeruginosa was identified as the fifth most frequently isolated

nosocomial pathogen, accounting for 9% of all hospital-acquired

infections in the United States [38]. P. aeruginosa was also the second

leading cause of nosocomial pneumonia (14 to 16%), third most

common cause of urinary tract infections (7 to 11%), fourth most

frequently isolated pathogen in surgical site infections (8%), and

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seventh leading contributor to bloodstream infections (2 to 6%). Data

from more recent studies continue to show P. aeruginosa as the second

most common cause of nosocomial pneumonia, health care-associated

pneumonia, and ventilator-associated pneumonia and the leading

cause of pneumonia among pediatric patients in the intensive care unit

(ICU).

P. aeruginosa presents a serious therapeutic challenge for

treatment of both community-acquired and nosocomial infections, and

selection of the appropriate antibiotic to initiate therapy is essential to

optimizing the clinical outcome. Unfortunately, selection of the most

appropriate antibiotic is complicated by the ability of P. aeruginosa to

develop resistance to multiple classes of antibacterial agents, even

during the course of treating an infection. Epidemiological outcome

studies have shown that infections caused by drug-resistant P.

aeruginosa are associated with significant increases in morbidity,

mortality, need for surgical intervention, length of hospital stay and

chronic care, and overall cost of treating the infection [39].

P.aeruginosa a very ubiquitous microorganism, for it has been

found in environments such as soil, water, humans, animals, plants,

sewage, and hospitals (Lederberg and Joshua, 2000). In all oligotropic

aquatic ecosystems, which contain high-dissolved oxygen content but

low plant nutrients throughout, P.aeruginosa is the predominant

inhabitant and this clearly makes it the most abundant organism on

earth [40].

P.aeruginosa is an opportunistic human pathogen. It is

“opportunistic” because it seldom infects healthy individuals. Instead, it

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often colonizes immune compromised patients, like those with cystic

fibrosis, cancer, or AIDS [41]. It is such a potent pathogen that firstly, it

attacks up two thirds of the critically-ill hospitalized patients, and this

usually portends more invasive diseases. Secondly, P.aeruginosa is a

leading Gram-negative opportunistic pathogen at most medical centers,

carrying a 40-60% mortality rate. Thirdly, it complicates 90% of cystic

fibrosis deaths; and lastly, it is always listed as one of the top three

most frequent Gram-negative pathogens and is linked to the worst

visual diseases [42]. Furthermore, P.aeruginosa is a very important soil

bacterium that is capable of breaking down polycyclic aromatic

hydrocarbons and making rhamnolipids, quinolones, hydrogen cyanide,

phenazines, and lectins (NCBI). It also exhibits intrinsic resistance to a

lot of different types of chemotherapeutic agents and antibiotics,

making it a very hard pathogen to eliminate [43].

P.aeruginosa was first described as a distinct bacterial species at

the end of the nineteenth century, after the development of sterile

culture media by Pasteur. In 1882, the first scientific study on P.

aeruginosa, entitled “On the blue and green coloration of bandages,”

was published by a pharmacist named Carle Gessard. This study

showed P. aeruginosa’s characteristic pigmentation: P.aeruginosa

produced water-soluble pigments, which, on exposure to ultraviolet

light, fluoresced blue-green light. This was later attributed to

pyocyanine, a derivative of phenazine, and it also reflected the

organism’s old names: Bacillus pyocyaneus, Bakterium aeruginosa,

Pseudomonas polycolor, and Pseudomonas pyocyaneus (Botzenhardt et

al., 1993). P.aeruginosa has many strains, including Pseudomonas

aeruginosa strain PA01, Pseudomonas aeruginosa PA7, Pseudomonas

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aeruginosa strain UCBPP-PA14, and Pseudomonas aeruginosa strain

2192 (NCBI). Most of these were isolated based on their distinctive

grapelike odor of aminoacetophenone, pyocyanin production, and the

colonies’ structure on agar media [44].

P.aeruginosa is a Gram-negative microbe, it has an outer

membrane which contains Protein F (OprF). OprF functions as a porin,

allowing certain molecules and ions to come into the cells, and as a

structural protein, maintaining the bacterial cell shape. Because OprF

provides P.aeruginosa outer membrane with an exclusion limit of 500

Da, it lowers the permeability of the outer membrane, a property that is

desired because it would decrease the intake of harmful substances

into the cell and give P.aeruginosa a high resistance to antibiotics [45].

P.aeruginosa uses its single and polar flagellum to move around and to

display chemotaxis to useful molecules, like sugars. Its strains either

have a-type or b-type of flagella, a classification that is based primarily

on the size and antigenicity of the flagellin subunit. The flagellum is

very important during the early stages of infection, for it can attach to

and invade tissues of the hosts [46]. Similarly to its

flagellum, P.aeruginosa pili contribute greatly to its ability to adhere to

mucosal surfaces and epithelial cells. Specifically, it is the pili’s tip that

is responsible for the adherence to the host cell surface.

Overall, P.aeruginosa flagellum and pili have similar functionality

(for attachment) and structure (both are filamentous structures on the

surface of the cell), and their motility is controlled by RpoN, especially

during initial attachment to the human host and under low nutrient

conditions. When infecting its host, P.aeruginosa is starved for iron

because iron deprivation of an infecting pathogen is the key part in the

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humans’ innate defense mechanism. To overcome this

challenge, P.aeruginosa synthesizes two siderophores: pyochelin and

pyoverdin. P.aeruginosa then secrets these sideophores to the exterior

of the cell, where they bind tightly to iron and bring the iron back into

the cell. Additionally, P.aeruginosa can also use iron from enterobactin,

a special siderophore produced by E.coli for iron transport, to satisfy its

iron need [47].

P.aeruginosa is a facultative anaerobe; its preferred metabolism is

respiration. It gains energy by transferring electrons from glucose, a

reduced substrate, to oxygen, the final electron acceptor [48]. The

breakdown of glucose requires it to oxidize to gluconate in the

periplasm, then it will be brought inside the inner membrane by a

specific energy-dependent gluconate uptake system. Once inside,

gluconate is phosphorylated to 6-P-gluconate, which will enter the

central metabolism to produce energy for the cell [49].

When P.aeruginosa is in anaerobic conditions, however, P.aeruginosa

uses nitrate as a terminal electron acceptor [50]. Under oxidative-stress

conditions, P.aeruginosa synthesizes Fe- or Mn- containing superoxide

dismutase (SOD) enzymes, which catalyze the very reactive O- to H2O2

and O2.

Since P.aeruginosa can live in both inanimate and human

environments, it has been characterized as a “ubiquitous”

microorganism. This versatility is made possible by a large number of

enzymes that allow P.aeruginosa to use a diversity of substances as

nutrients. Most impressively, P.aeruginosa can switch from growing on

nonmucoid to mucoid environments, which comes with a large

synthesis of alginate. In inanimate environment, P.aeruginosa is

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usually detected in water-reservoirs polluted by animals and humans,

such as sewage and sinks inside and outside of hospitals.

P.aeruginosa groups tend to form biofilms, which are complex

bacterial communities that adhere to a variety of surfaces, including

metals, plastics, medical implant materials, and tissue. Biofilms are

characterized by “attached for survival” because once they are formed,

they are very difficult to destroy. Depending on their locations, biofilms

can either be beneficial and detrimental to the environment. For

instance, the biofilms found on rocks and pebbles underwater of lakes

and ponds are an important food source for many aquatic organisms.

On the contrary, those that developed on the interiors of water pipes

might cause clogging and corrosions [51].

P.aeruginosa rarely causes disease in healthy humans. It is

usually linked with patients whose immune system is compromised by

diseases or trauma. It gains access to these patients’ tissues through

the burns, for the burn victims, or through an underlying disease, like

cystic fibrosis. First, P.aeruginosa adheres to tissue surfaces using its

flagellum, pili, and exo-S; then, it replicates to create infectious critical

mass; and lastly, it makes tissue damage using its virulence factors

[52]. The pathogenesis of Ps. aeruginosa infections is multifactorial, as

suggested by the large number of cell-associated and extracellular

virulence determinants of these bacteria. The first step in Ps.

aeruginosa infections is the colonization of the altered epithelium.

Adherence of Ps. aeruginosa to the epithelium is mediated by fimbriae,

type 4 pili and flagella [53]. After colonization, Ps. aeruginosa produces

several extracellular virulence factors (alkaline protease and

staphylolytic protease, elastase, protease IV, heat-labile and heat-stable

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hemolysins, phospholipases C and exotoxins A, S, T, U, Y), responsible

for extensive tissue damage, bloodstream invasion, and dissemination

[54].

This disruption in the salt and water balance in the cell results in

the production of a thick mucus, which becomes the ideal home for

potential pathogens. P.aeruginosa attacks CF patients via airway and

once it is in, it uses its flagellum to go to the hypoxic zone, an oxygen-

depleted environment. At this location, P.aeruginosa undergoes a

transition from an aerobic to an anaerobic microbe and starts forming

biofilms anaerobically. Once this is formed, the P.aeruginosa in this

community can sense their population via quorum sensing, where they

secret low molecular weight pheromones that enable them to

communicate with each other [55]. This gives them the ability to resist

many defenses, including anti-Pseudomonas antibiotics such as

ticarcillin, ceftazidime, tobramycin, and ciprofloxacin, because once the

bacteria sense that their outer layer of biofilm is being destroyed, the

inner layers will grow stronger to reestablish the community [56].

P.aeruginosa is also resistant to many antibiotics and

chemotherapeutic agents due to their intrinsic resistance.

This is caused by the low permeability to antibiotics of the outer

membrane and by the production of β-lactamases against multidrug

efflux pumps and β-lactam antibiotics [57]. P.aeruginosa communicates

with other cells through quorum-sensing. This form of communication

allows the cells to regulate gene production which results in control of

certain cell functions. One of the enzymes responsible for quorum

sensing is tyrosine phosphatase (TpbA). This enzyme relays

extracellular quorum sensing signals to polysaccharide production and

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biofilm formation outside the cells [58]. P.aeruginosa attaches to

surfaces by way of biofilm production. Quorum-sensing can be a drug

target to cure infections caused by P. aeruginosa. Quorum-quenching is

used to blocks the signaling mechanism of quorum-sensing and

prevents biofilm formation in P. aeruginosa. Yi-Hu Dong and his

colleagues were able to prevent biofilm formation in mice under

laboratory conditions [59].

P.aeruginosa secrets many virulent factors to colonize the cells of

its host. For example, exotoxin A, the most toxic protein produced by P.

aeruginosa, catalyzes the ADP-ribosylation to form ADP-ribosyl-EF-2,

which inhibits the protein synthesis of the host’s cells. Moreover,

elastase, an extracellular zinc protease, attacks eukaryotic proteins

such as collagen and elastin and destroys the structural proteins of the

cell.

Pseudomonas aeruginosa is an environmentally ubiquitous

opportunistic pathogen. Epidermal infections often result from

P.aeruginosa infiltrating through a human host’s first line of defenses,

entering the body through the skin at the site of an open wound.

P.aeruginosa is a common member of hospital bacterial communities

where it can infect immunocompromised individuals including burn

victims. P.aeruginosa is a source of bacteremia in burn victims.

Following severe skin damage, the prevalence of P.aeruginosa in the

environment increases the probability of the organism accessing the

bloodstream through the burn victim’s exposed deep epidermal tissue.

Previous research of antibody-mediated host defenses indicates that on

the fifth day after the initial burn, Fc receptor expression is reduced in

polymorphonuclear leukocytes (PMNs). Without the Fc receptor, PMN

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chemotaxis is greatly reduced and the PMNs become less effective at

preventing infection.

P.aeruginosa can be transmitted to a host via fomites, vectors,

and hospital workers who are potential carriers for multiply-antibiotic-

resistant strains of the pathogen. Furthermore, any P.aeruginosa

already present on a burn victim’s skin before the injury can transform

from an innocuous organism on the surface of the skin to a source of

infection in the bloodstream and body tissues of the same individual.

The pili and flagella of P.aeruginosa play a vital role in the infection of

burns and wounds. Controlled infection of burn wounds on animal and

plant models with P.aeruginosa strains devoid of pili and flagella

demonstrate a trend of decreased virulence. Without these

morphological virulence factors, the bacteria exhibit a substantially

decreased survival rate at the wound site and a decreased ability to

disseminate within the host organism. The spread of P.aeruginosa

within host organisms is also dependent on the microorganism’s

elastase production and other protease mechanisms. Bacterial elastase

and other bacterial proteases degrade the host’s proteins, including the

structural proteins within membranes, disrupting the host’s physical

barriers against the spread of infection. Elastase also assists

P.aeruginosa in avoiding phagocytotic antibody-mediated cytotoxicity at

the site of the wound by inhibiting monocyte chemotaxis [60].

The two strains that have the complete genome sequence

are Pseudomonas aeruginosa PA01 and Pseudomonas aeruginosa PA14

(PGD). In 2000, a group of volunteer "Pseudomonas scientists",

including those from the Washington PathoGenesis Corportaion and

the Department of Biology of the University of California, San Diego,

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worked under the Pseudomonas aeruginosa Community Annotation

Project (PseudoCAP) to publish the complete genome sequence of

Pseudomonas aeruginosa PA01. This was done because knowing the

genomic sequence would provide new information about this bacterium

as a pathogen and about its ecological versatility and genetic

complexity. At 6,264,403 base pairs, its bacterial genome is the largest

to ever be sequenced. It also contains 5,570 predicted open reading

frames (ORFs), and thus it almost has the genetic complexity of simple

eukaryotes, such as Saccharomyces cerevisiae. Using whole-genome-

shotgun sampling, the complete 6.3 Mbp genome of Pseudomonas

aeruginosa PA01 is very much similar to the P. aeruginosa’s physical

map, with only one major exception, which is the inversion of about a

quarter of the Pseudomonas aeruginosa PA01 genome.

This inversion comes from the homologous recombination of the

rrnA and rrnB loci, and earlier studies on genomic sequence inversions

of ribosomal DNA loci in S. typhimurium and E.coli suggest that this

inversion might have adaptive significance [61]. The complete genome

sequence of Pseudomonas aeruginosa PA14 is currently being done by

Harvard Medical School scientists. The goal of this study is achieve a

public data of Pseudomonas aeruginosa PA14 genome. The shotgun-

sequencing phase of the project was finished in 2005, yielding 6.54

Mbp of PA14 sequence. It is currently being compared to the genome

of Pseudomonas aeruginosa PA01 and preliminary results have shown

that they are very similar but have several regions of marked

differences, such as the insertion of the 107911bp in PA14, which is

absent in PA01. Approximately, there is 96.3% of the DNA sequence of

PAO1 is in PA14, and 92.4% of PA14 DNA sequence is in PA01 .

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P. aeruginosa, as well as many other Pseudomonas, can degrade

aromatic hydrocarbons such as methylbenzenes, which are the by-

products of petroleum industries and are commonly used as solvents

for enamels and paints as well as in the production of drugs and

chemicals. Methylbenzenes are considered as environmental

contaminants that are present in the atmosphere, underground and

soils, and in surface water [62]. P.aeruginosa can break down toluene,

the simplest form of methylbenzene. P.aeruginosa degrades toluene

through the oxidation of the methyl group to aldehyde, alcohol, and an

acid, which is then converted to catechol. Hence, P.aeruginosa can be

used in pollution control [63].

Description and clinical significance of E. coli infections

E. coli comprises of non-pathogenic commensal isolates that

forms part of the normal flora of humans and various animals. In

humans, they are the major aerobic organism residing in the intestine,

typically with around 106 to 109 colony forming units per gram of

stool. The organism is also found in soil and water, usually as a result

of fecal contamination. Several variants or pathotypes of E. coli have

been described that cause infections of the gastrointestinal system (i.e.

intestinal pathogenic E. coli) while other pathotypes cause infections

outside the gastrointestinal system (i.e. extraintestinal pathogenic E.

coli) [64]. E. coli is the most common cause of urinary tract infections

(UTIs) in humans, and is a leading cause of enteric infections and

systemic infections [65]. The systemic infections include bacteremia,

nosocomial pneumonia, cholecystitis, cholangitis, peritonitis, cellulitis,

osteomyelitis, and infectious arthritis. E. coli is also leading cause of

neonatal meningitis

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Furthermore, enteric E.coli can be classified into six categories

based on its virulence properties [66]. These enteric E.coli can cause

several intestinal and extra-intestinal infections such as urinary tract

infection and mastitis. However, E.coli are not always harmful to

human bodies or other animals. Most E.coli live in our intestines, where

they help our body breakdown the food we eat as well as assist with

waste processing, vitamin K production, and food absorption. Extra

intestinal pathogenic E. coli (ExPEC), especially the uro-pathogenic E.

coli (UPEC) pathotype, is most commonly associated with human

infections due to E. coli outside the intestinal tract. UPEC are important

causes of lower urinary tract infections (UTIs) and systemic infections

in humans. The systemic infections include upper UTIs, bacteremia,

nosocomial pneumonia, cholecystitis, cholangitis, peritonitis, cellulitis,

osteomyelitis, infectious arthritis. ExPEC is also leading cause of

neonatal meningitis (referred to as NMEC) [67].

E.coli has only one circular chromosome, some along with a

circular plasmid. Its chromosomal DNA has been completely sequenced

by lab researchers. E.coli has a single chromosome with about 4,600

kb, about 4,300 potential coding sequences, and only about 1,800

known E.coli proteins. 70% of the chromosome is composed of single

genes (monocistronic), and 6% is polycistronic. Moreover, there are

many different strains of E.coli; each of these strains differs in its

genotype from wild-type E.coli. The genotype will then affect the

phenotype that is expressed, and further influences the physiology and

life cycle of each strain. Therefore, different strains of E.coli can live in

different kinds of animals. The natural biological process of mutation in

genomes is the major cause to produce so many different strains

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of E.coli. In addition, similar to most bacteria, E.coli can transfer its

DNA materials through bacterial conjugation with other related bacteria

to produce more mutation and add more strains into the existing

population.

Escherichia coli can be commonly found in lower intestines of

human and mammals. When E.coli locates in human large intestines, it

can help digestion processes, food breakdown and absorption, and

vitamin K production. Different strains of E.coli can be found in

different type of animals, so we can determine the source (from human

or from other animals) of the stools by examining which strain

of E.coli is present in the stools. E.coli can also be found in

environments at higher temperature, such as on the edge of hot

springs. E.coli is commonly used as an indicator in the field of water

purification. The E.coli-index can indicate how much human feces is in

the water. The reason why E.coli is used as an indicator is due to a

significant larger amount of E.coli in human feces than other bacterial

organisms. Most strains of "E.coli" are not harmful to their hosts;

however, more and more newly discovered strains are contributing into

existing population through mutation and evolution. Some can cause

severe disease, such as E.coli O157:H7.

Although E.coli in human large intestine can assist with waste

processing and food absorption, some strains of E.coli can cause severe

infections in many animals, such as humans, sheep, horses, dogs, etc.

The one that only found in humans is called enter aggregative E.coli.

Urinary tract infection, for example, can be caused by ascending

infections of urethra. Such infections can be found in both adult male

and female, and some infants can be infected as well. E.coli O157:H7 is

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one of the most infective strains that can cause food poisoning. It

belongs to enter hemorrhagic strain of the E.coli and can lead to bloody

diarrhea and kidney failure when one gets infected by contaminated

ground beef, unpasteurized milk or contaminated water. The toxin

that E.coli O157:H7 produces is a Shiga-like toxin which is a regulated

toxin that catalytically inactivate 60S ribosomal subunits of most

eukaryotic cells, blocking mRNA translation and thus causing cell

death [68]. Some important symptoms are diarrhea that is acute and

severe, either bloody or not bloody, stomach cramping, vomiting, and

loss of appetite, abdominal pain, and fever. The causes can usually

clear up on their own in 1-3 days with no treatment required. However,

patients should avoid dairy products because those products may

induce temporary lactose intolerance, and therefore make the diarrhea

worse.

E.coli plays an important role in current biological engineering

because of its manipulation and long laboratory history. It has been

widely used to synthesize DNA and proteins. Most results from E.coli

research can be applied to animals and humans. The most useful

contribution of recombinant DNA from E.coli is to use the manipulation

of E.coli to produce human insulin for diabetes patients.

Since E.coli can produce human enzymes through recombinant DNA

techniques, it is widely used as a very good tool to produce useful

compounds or enzymes for medication. In a recent study (Apr. 2007),

scientists has explored a new method to cure Alzheimer's disease (AD),

which is a neurodegenerative disorder characterized by a progressive

loss of cognitive function due to extra deposition of the longer form of

the amyloid peptide Abeta. In this study, scientists use E.coli to enable

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rapid production of the antigen and its purification [69]. In other

words, E.coli plays an important role to enable the rapid, continuous

production and purification in large amount of human Abeta sequence

by its unique expression system.

One recent study is working on the adherent-

invasive E.coli (AIEC) which can abnormally colonize the ileal mucosa of

Crohn disease (CD) patients and adhere to and invade intestinal

epithelial cells of CD patients. The study shows that this kind of CD-

associated AIEC strain can adhere to the brush border of primary ileal

enterocytes isolated from CD patients. The adhesion of AIEC is

dependent on type 1 pili expression on bacterial surface and on

carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6)

expression on the apical surface of ileal epithelial cells. CEACAM6 is an

essential receptor for AIEC strain to adhere the ileal eithelial cells in CD

patients. Moreover, this study also performs in vitro experiments

indicating that AIEC can boost its own colonization in CD.

Another recent study is focusing on exploring the antigens on the

outer membrane of the uropathogenic E.coli (UPEC) which can cause

uncomplicated urinary tract infection (UTI), and furthermore to design

a UTI vaccine to promote protective immunity against UPEC infection.

In this study, they apply an immune proteomics approach to vaccine

development that has been used successfully to identify vaccine targets

in other pathogenic bacteria. The outer membrane proteins of UPEC

from infected mice are separated by two-dimensional gel

electrophoresis and are identified by mass spectrometry. A total of 23

antigens have known roles in UPEC pathogenesis, such as ChuA, IroN,

IreA, Iha, IutA, and FliC. After identifying the antigens on outer

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membrane of UPEC, they demonstrate an antibody targeting directly on

these antigens during UTI. This study shows that these conserved outer

membrane antigens can be used as rational candidates for a UTI

vaccine.

Another recent study is focusing on genomic evolution

of E.coli O157:H7 strains which diverge into two distinct lineages,

lineages I and lineages II and appear to present different ecological

characteristics. Lineage I strains are more commonly associated with

human disease than lineage II. This experiment is carried out by

microarray-based comparative genomic hybridization (CGH) to identify

genomic differences among 31 E.coli O157:H7 strains which have

different lineage-specific polymorphism assay types. Among

31 E.coli O157:H7 strains, there are 15 lineage I, 4 lineageI/II, and 12

lineage Ii strains, respectively. From the CGH data, they conclude that

the presence of two dominant lineages subgroup E.coli O157:H7. The

genomic composition of these subgroups suggests that the genomic

divergence and lateral gene transfer have contributed to the evolution

of E.coli.

A long-term study has been done on genome evolution and

adaptation of Escherichia coli. Comparative genome sequencing done on

an experimental population of E.coli has allowed for further

investigation between the relationship between genome evolution and

organismal adaptation. E.coli was grown and sampled for almost 20

years with genome sequencing at generations 2,000, 5,000, 10,000,

15,000, 20,000 and 40,000 from an asexual population that evolved

with glucose as a limiting nutrient. Comparative genome sequencing

showed mutational differences between the ancestral and evolved

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genomes accumulating in a near-linear fashion, whereas the fitness

trajectory was strongly nonlinear. In particular, the rate of fitness

improvement decelerated over time. The discordance in rates of

genomic and adaptive evolution cannot be explained by the drift

hypothesis, but rather the discrepancy may be due to clonal

interference, compensatory adaptation, or changing mutation rates. In

clonal interference, sub-lineages with the most beneficial mutations

outcompete sub-lineages bearing mutations that are not as beneficial.

Most beneficial mutations dominate the early phase of evolution for

large populations in a new environment, but there are more potential

mutations that confer small advantages than large (adaptative) ones.

The population in the study retained a low ancestral mutation rate to at

least 20,000 generations but in later generations, however, this

population exhibited a greatly elevated rate of genomic evolution when

a mutator lineage became established later. Furthermore, there were no

synonymous mutations fixed in the first 20,000 generations and this is

consistent with the low point-mutation rate in E.coli and population-

genetic theory. These results indicate that it is important to explore

long-term dynamic coupling between genome evolution and adaptation

[70].

Description and Clinical Significance of Klebsiella pneumoniae

Klebsiella pneumoniae is a gram-negative that is associated with

nosocomial and community-acquired infections. Strains of

carbapenemase-producing Klebsiella pneumoniae (CPKP) have been

identified since the 1990s and have rapidly spread in many countries.

The most widespread carbapenemase enzymes include class A

carbapenemases (KPC types), class B or metallo-β-lactamases (Verona

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integron-encoded metallo-β-lactamase [VIM] and New Delhi metallo-β-

lactamase types [NDM]) and class D oxacillinases (OXA-48-like enzymes

[71]. The search for the pathogenic mechanisms of Klebsiella infections

has identified a number of bacterial factors that contribute to the

pathogenesis of these bacteria. Both in vitro and in vivo models have

been established to investigate the interaction of bacterial cells and the

host. As mentioned above, Klebsiellae usually develop prominent

capsules composed of complex acidic polysaccharides. The capsular

repeating subunits, consisting of four to six sugars and, very often,

uronic acids (as negatively charged components), can be classified into

77 serological types [72]. Capsules are essential to the virulence

of Klebsiella spp.

Due to their endotoxic properties, LPS are considered important

in the pathology of septicemia. Until recently, Klebsiella LPS O antigens

were generally considered to be masked by the capsule polysaccharides

and thus not to be exposed on surface, leaving them inappropriate as

vaccine candidates. Recent studies, however, demonstrated surface

exposure of O-antigens in strains expressing particular capsular

serotypes [73]. Most Klebsiella infections are acquired during hospital

stays and account for 5 to 7.5% of all nosocomial infections. The

morbidity and mortality of severe systemic infections, such as

bacteremia and pneumonia, remain high despite the use of appropriate

antibiotic therapy. Fatality rates of 20 to 50% in Klebsiella bacteremia

and of more than 50% in Klebsiella pneumoniae have been reported

[74]. Moreover, Klebsiella infections in pediatric wards have become a

major concern. In neonatal intensive care units, Klebsiella is one of the

three or four most common pathogen.

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Prevalence of ESBL pathogens

The most well-known of the “newer” β-lactamases was first

described in 1983 and have been named the extended-spectrum β-

lactamases or ESBLs. These enzymes have the ability to hydrolyse the

penicillins, cephalosporins and monobactams, but not the cephamycins

and carbapenems. ESBLs are inhibited by “classical” b-lactamase

inhibitors such as clavulanic acid, sulbactam and

tazobactam. Although ESBLs have been identified in a range of

Enterobacteriaceae, they are most often present in E. coli and K.

pneumoniae. The decreased susceptibility of Enterobacteriaceae to

carbapenems may be caused by either extended-spectrum beta-

lactamases (ESBLs) or AmpC enzymes as well as decreased drug

permeability caused by porin loss. Imported resistance to the β-lactams

involves the production of inactivating β-lactamases, for which several

families have been identified among clinical isolates of P. aeruginosa.

The variety, prevalence, and clinical significance of imported β-

lactamases in P. aeruginosa have been addressed in several reviews

over the last decade [75]. The most common imported β-lactamases

found among P. aeruginosa isolates are penicillinases belonging to the

molecular class A serine β-lactamases (PSE, CARB, and TEM families).

Within this group, enzymes belonging to the PSE family appear to be

the most prevalent. β-Lactam antimicrobial agents represent the most

common treatment for bacterial infections and continue to be the

leading cause of resistance to β-lactam antibiotics among Gram-

negative bacteria worldwide. The persistent exposure of bacterial

strains to a multitude of β-lactams has induced dynamic and

continuous production and mutation of β-lactamases in these bacteria,

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expanding their activity even against the newly developed β-lactam

antibiotics. These enzymes are known as extended-spectrum β-

lactamases (ESBLs) [76].

Treatment of these multiple drug-resistant organisms is a

therapeutic challenge. At the level of a wider geographic scale, the

incidence of ESBL-producing organisms is difficult to determine due to

various reasons, difficulty in detecting ESBL production and

inconsistencies in reporting. In recent surveys, a significant increase in

the ESBL rate was reported from all parts of the world [77]. Klebsiella

pneumoniae and Escherichia coli remain the major ESBL-producing

organisms isolated worldwide which are recommended to be routinely

tested for and reported by the Clinical and Laboratory Standards

Institute. Prevalence of ESBLs varies from an institute to another.

Previous studies from India and abroad have reported ESBL production

varying from 8 to 80%. However, there is paucity scientific information

available on antibiotic profile with rate of ESBL production in Klebsiella

pneumoniae isolates.

Keeping in view the above facts, the present study was

undertaken to find the prevalence of ESBL producers among Klebsiella

pneumoniae isolates at our institute [78]. Resistance to aminopenicillins

(e.g. ampicillin) and early-generation cephalosporins (e.g. cefazolin)

among E. coli is often mediated by the production of narrow-

spectrum b-lactamases such as TEM-1, TEM-2 and to a lesser extent

SHV-1 enzyme [79]. Most importantly among E. coli, is the increasing

recognition of isolates producing the so-called “newer b-lactamases”

that causes resistance to the expanded-spectrum cephalosporins

and/or the carbapenems. These enzymes consist of the plasmid-

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mediated AmpC b-lactamases (e.g. CMY types), extended-spectrum b-

lactamases (e.g. TEM, SHV, CTX-M types), and carbapenemases (KPC

types, metallo-b-lactamases (MBLs) and OXA-types).

Surveys from several countries worldwide have illustrated an

alarming trend of associated resistance to other classes of antimicrobial

agents among CTX-M-producing E. coli that included trimethoprim-

sulfamethoxazole, tetracycline, gentamicin, tobramycin and

ciprofloxacin. Studies consistently show that infections due to ESBL-

producing Enterobacteriaceae are associated with a delay in initiation

of appropriate antibiotic therapy, which consequently prolongs hospital

stays and increases hospital costs [80]. More importantly, failure to

initiate appropriate antibiotic therapy from the start appears to be

responsible for higher patient mortality.

Toxicity assay

The antibiotic resistant pattern and resistant of mosquito larvae

have been changing frequently. There is a need for establishing a

particular type of drug which is harmful to larvae without affecting

human system. The medicinal value of some chemical substances lies

in definite physiological action on the human body. So, there is a need

for exploring the Hemolytic activity of Red Blood Cells (RBC) resembles

the activities of other cells, the study was designed to check the effect

of ligand for larvicidal and hemolysis. Though a drug is very potential

against some diseases, it is of no value if toxic metals are present in

higher concentration

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Larvicidal assay

The mosquito is the principal vector of many of the vector-borne

diseases affecting human beings and other animals. Mosquitoes

constitute a major public health problem as vectors of serious human

diseases. In India, malaria is one of the most important causes of direct

or indirect infant, child, and adult mortality. About 2 million confirmed

malaria cases and 1,000 deaths are reported annually, although 15

million cases and 20,000 deaths are estimated by WHO South East

Asia Regional Office. India contributes 77% of the total malaria in

South East Asia. Mosquitoes are the primary source for the spread of

diseases among the arthropods. Organophosphates and insect growth

regulators are generally used for the control of mosquito larvae,

however, their continuous use has demonstrated undesirable effects on

non-target organisms, and their influence on the environment and

human health has been the main concern.

India is struggling to cope with outbreaks of mosquito borne

diseases. Overburden hospitals and clinics in several cities in the North

of India. Half of the entire human population, an estimated 3.3 billion

people, lives in malaria-risk areas around the world with about 250

million people infected annually. Malaria is believed to be responsible

for approximately one million deaths per year, particularly among

children under 5 year’s old and pregnant women. The other continents

contribute the remainder of the 35 million cases according to the report

of WHO in 2008. Anopheles subpictus is a complex isomorphic sibling

species and is recognised as a vector of malaria, a disease of great

socio-economic importance, and also a vector of some helminth and

arboviruses. Malaria and lymphatic filariasis (LF) are two of the most

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common and identifiable mosquito-borne parasitic diseases worldwide.

The highest malaria burden is found in Africa with an estimation of 212

million cases (86% globally) distributed in 45 countries.

Repeated use of synthetic insecticides for mosquito control has

disrupted natural biological control systems and led to resurgences in

mosquito populations. It has also resulted in the development of

resistance, undesirable effects on non-target organisms and fostered

environmental and human health concern, which initiated a search for

alternative control measures. The control of mosquito larvae is

dependent on regular applications of organophosphates and different

insecticides. The major drawback with the use of chemical insecticides

is that they are non-selective and could be more harmful to other non-

target organisms. Moreover, after few years the mosquitoes develop

resistance against the insecticides due to frequent use of them.

Pesticide exposure among humans has been linked to immune

dysfunction, various forms of cancer and birth defects. It is, therefore,

necessary to identify a safe, eco-friendly alternate source of larvicide in

order to reduce mosquito menace. Studies have been concentrated in

Aedes aegypti species because of its role in the arboviruses

transmission, responsible for yellow fever and dengue fever, diseases

which are endemic in South and Central America, Asia and Africa.

Dengue is a mosquito-borne viral disease found in three clinical forms:

dengue fever (DF), dengue haemorrhagic fever (DHF) and dengue shock

syndrome. It is endemic in more than 100 countries in Africa, the

Americas, the West Pacific and Asia (including Sri Lanka), and has

become the most important and the fastest-growing mosquito-borne

disease in these countries. A total of 40% of the world’s population

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(over 2.5 billion) has been reported to be at risk of dengue, and an

average of about 50 million cases of dengue infections are reported

each year.

Both vectors Aedes aegypti and Aedes albopictus lay eggs stuck

onto a damp surface just above the water level, in containers filled with

clean and clear water, preferably with a small amount of organic

matter. A single female oviposits about 100 eggs at a time, about three

times in its lifespan. These eggs hatch within 7 days or remain dormant

for about 1 year, resisting desiccation until the conditions become

favourable for hatching. Egg surveillance (ovitrap surveillance) and

larval surveillance have been recommended to determine the prevalence

of dengue vectors. In South and Southeast Asia, Aedes aegypti

Linnaeus (Diptera: Culicidae) is considered the primary vector of

dengue and is found mainly in urban areas. Aedes albopictus Skuse

(Diptera: Culicidae) is considered the secondary vector and is found

mainly in semi-urban and rural areas. Climatic, geographical,

environmental and social conditions in Sri Lanka provide favourable

conditions for the survival of both the vector species and the dengue

virus. Aedes aegypti is commonly known as the ‘yellow fever mosquito’,

since it transmits the yellow fever virus in Africa.

Aedes albopictus is known as the ‘Asian-tiger mosquito’ or ‘forest

day mosquito’ and was the native vector of the dengue virus in the

Oriental region among the arbovirus in India, distribution of all the

dengue virus types (DEN 1, 2, 3 & 4) is continuously expanding.

Remarkably the re-emergence of Chikungunya virus (CHIK) since 2005

is posing an additional and concurrent disease burden in the country

including the Maharashtra State. Both these viruses are borne by the

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mosquito, Aedes aegypti (L.). A. aegypti populations emerge either from

the rain filled container habitats and/or the manmade water storage

containers; the latter are apparently unaffected by rains. In India, both

these habitat types intersperse. As a consequence, A. aegypti

population prevalence indices vary sharply through seasons where the

rain filled habitats abound. On the other hand, its residential

populations varied slightly and were seasonally stable.

Vector control is facing a threat due to the emergence of

resistance to synthetic insecticides. Nowadays researchers are focusing

their research on a synthetic compound that kills the larvae at initial

stage itself. According to World Health Organisation (WHO), the one of

strategies is to destroy their vectors or intermediate hosts. The best

method is control of mosquito larvae using insecticides such as organo-

phosphates, natural products and heterocycles types. It is an urgent

need to develop new insecticides which are more environmentally safe

and also biodegradable and target specific against mosquitoes.

Larvicides play significant role in controlling mosquitoes at their

breeding and immature stages. Hydrazone derivatives possessed good

larvicidal activity in our previous study. In recent years, the chemistry

of carbon-nitrogen double bond of hydrazone is fast becoming the

backbone of condensation reaction in benzo-fused N-heterocycles.

Hydrazone containing azomethine –NHN=CH protons constitute an

important class of compounds for new drug development.

Hemolytic assay

In addition to synthesize biotechnologically valuable products, it’s

essential to evaluate pre clinical safety evaluation like local tolerance

test, in vitro haemolysis and biocidal effects are required to complete

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pharmacological end point. Toxicity of the active molecule is a key

factor during drug designing, and haemolytic activity represents a

useful starting point in this regard, it provides the primary

information on the interaction between molecules and biological

entities at cellular level. Haemolytic activity of any compounds is

an indicator of general cytotoxicity towards normal healthy cells.

The in vitro hemolysis assay evaluates hemoglobin release in the

plasma (as an indicator of red blood cell lysis) following test agent

exposure. The test is based on erythrocyte lysis induced by contact,

leachables, toxins, metal ions, surface charge or any other cause of

erythrocyte lysis. The method is based on release of hemoglobin, which

can be measured spectrophotometrically. This method is suited to

evaluate the haemocompatibility of biomaterials and medical devices

according to the international standard ISO 10993-4:2002. An

important haemolysis (destruction of red blood cells (RBC)) may result

in pathological conditions such as anaemia and renal failure

Hemolytic agents affect blood and blood components. Examples

of hemolytic agents include arsine, benzene, lead, antibiotics, bacteria’s

and viral infections. Available evidence suggests that the

administration of a hemolytic drug causes oxidative damage to either

the hemoglobin and/or the stroma of the sensitive cell. Heinz bodies

are visible manifestation of such damage.. The cell breakdown is often

evident to the naked eye in a pink to red tinge in the serum or plasma.

Hemolysis occurs when there is a break in a red blood cell’s membrane,

which causes the release of hemoglobin and other internal components

to leak into the surrounding fluid. The upper reference limit upper

reference limit for free hemoglobin free hemoglobin is 2 mg/dL for

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plasma and 5 mg/dL for serum. - Visually, hemolysis is defined as free

hemoglobin concentration > 30 - 50 mg/dL, conferring detectable

pink/red hue to serum or plasma. It becomes clearly visible clearly

visible in specimens containing as low as 0.5% lysed erythrocytes [81].

Haemolytic assays have long been used to measure free radical

damage and counteraction by antioxidants. It is useful for screening for

oxidising or antioxidising molecules. Many primary or secondary plant

metabolites have been found to protect cells from oxidative damage.

These compounds have been evidenced to stabilize RBC membrane by

scavenging free radicals and reducing lipid peroxidation. Drug-induced

immune hemolytic anemia (DIIHA) is rare and regard to the drug-

dependent mechanisms, which is universally accepted. Some drugs

bind covalently to proteins on the RBC membrane; thus, if conditions

are optimal (eg, high enough drug concentration), circulating RBCs will

be coated with drug; this does no harm to the RBCs, but if the patient

makes an IgG antibody to the drug the antibody will bind to the drug

on the RBC and the macrophages can interact, leading to Fc-mediated

extravascular RBC destruction. The prototype drug is penicillin;

cefotetan, but not ceftriaxone, can react by this mechanism [82].

Penicillin-type antibodies bind strongly to protein and can evoke

antibodies that appear to be directed mainly to the drug as one can

inhibit the antibodies with penicillin alone, in vitro, using hapten-

inhibition tests. Many other drugs appear to evoke antibodies that

mainly appear to be directed against a combination of drug plus RBC

membrane protein. Such antibodies are not inhibited by the drugs in

vitro and the drugs do not bind well to RBCs. Such antibodies are those

that were originally thought to be reacting by the immune complex

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mechanism [83]. Piperacillin can cause DIIHA and/or positive DATs; it

is the third most common drug to cause DIIHA. Although a semi-

synthetic penicillin, unlike other semi-synthetic penicillins (eg,

ampicillin), it reacts differently than penicillin G. In contrast to

penicillin, the in vivo RBC destruction can be complement-mediated;

most of the DATs are positive due to RBC-bound complement.

The denaturation of cell membrane proteins by proton-donating

groups is the major underlying mechanism for membrane damage. The

haemolytic activity is highly specific and seems to depend only on the

concentration of negatively charged groups that are accessible by the

cell membranes of erythrocytes. For example strong distortion of the

membrane after interaction with silica particles can lead to loss of

membrane flexibility and resiliency as well as the release of

haemoglobin (haemolysis). The toxic potency of the majority of

proteinaceous toxins is based on membrane interaction, pore formation

and, finally lysis and damage of cells. Such cytolysins are produced by

a variety of living organisms, particularly bacteria, certain insects,

poisonous reptiles and stinging marine invertebrates. These toxins need

to be secreted as water-soluble proteins but have to be transformed

into membrane proteins before penetrating the membrane. It can be

assumed that the toxin is concentrated on the membrane by means of

special cell surface features such as a protein receptor molecules, lipid

clusters or carbohydrate side chains [84].

The toxic activity of cell lysis is often measured by the lysis of red

blood cells (erythrocytes) and the detection of released haemoglobin

after cell disruption. Erythrocytes from diverse species have been

applied for haemolytic assays, and they express different

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susceptibilities for the same lysine. This phenomenon indicates that the

composition of erythrocyte cell membranes influences the haemolytic

effect of cytolytic toxins [85]. Cell lysis by surfactants is a process of

great fundamental and practical importance. Much research has been

done in order to understand the mechanism underlying this process,

mostly using erythrocytes as a convenient model system. Because the

human erythrocyte has no internal organelles and since it is the

simplest cellular model obtainable, it is the most popular cell

membrane system to study the surfactant membrane interaction [86].

As most known cyclotides are to some extent haemolytic, a

haemolytic activity assay was considered an appropriate method for an

initial biological activity screen and to assess the biological effects of

structural permutations in cyclotides. While a haemolytic activity assay

is a relatively simple procedure, it is a biological assay and therefore

subject to a range of variable factors. The previously reported HD50

values for kalata B1 are indeed widely divergent, with values of 50 µM

and 1510 µM [87]. Some of the haemolytic assays reported in the

literature used rat, sheep or bovine blood. Blood cells from different

mammalian species can have varying susceptibility to haemolysis [88].

Rat and sheep blood had drastically reduced susceptibility to

haemolysis by the peptides compared to human blood. In the context of

exploring drug for intended use in humans, assaying against blood

from other animals would therefore be misleading, as the peptides

would appear almost non-haemolytic in the case of rat and sheep

blood.

Some examples of variables in reported haemolytic assays of

amphipathic antimicrobial and haemolytic peptides are listed in Table

156

6.1, showing that haemolytic activity assays are not standardised

between laboratories. This chapter identifies a range of variables

involved in a haemolytic assay and examines how they can be

minimised for the highest possible assay reliability. The haemolytic

activities of seven native cyclotides are also reported.

Table 6.1: Haemolytic variables in literature

Blood source

RBC concentrations

100% lysis standard

Incubation temp./h Reference

Human 2.5% (v/v) Triton 1% 37°C 37°C 1hr Blondelle and

Houghten, 1991 Rat or sheep 107 cells/mL Triton 37°C /45

min Juvvadi et al.,1996

Bovine 0.5% Triton 1% 1% 37°C 1hr Bulet et al., 1996

Sheep 0.5% NS 39°C 1hr Otvos Jr. et al., 2000

Rat X Triton 1% 37°C 30 min Subbalakshmi et al., 2001

Docking studies

Since the early 1980s, a huge amount of molecular biology data

has been accumulated in the public databases, notably GenBank and

EMBL for DNA sequences, SWISS-PROT and PIR for protein sequences,

and PDB for three-dimensional biomolecular structures. These

databases, which we call factual databases, contain detailed

information on the biological macromolecules, mostly structural

information, as well as citations and links to other molecular biology

databases. Recent progress in genome sequencing has made it possible

to uncover complete structural information, albeit one dimensional, of

the biological macromolecules in a number of organisms from bacteria

to eukaryotes. This is driving new activities of computational functional

genomics that include the identification of biological functions of

157

unknown gene products, reconstruction of functional modules or

pathways from a set of genes in the genome, comparative analysis of

genes and genomes in different species, and analysis and simulation of

gene expressions in different cells or in different developmental stages

[89]. In order to facilitate such post-genome sequencing analyses, it has

become a high priority to construct a new breed of database that

defines functional aspects of genes, cells and organisms. But later the

bioinformatics analysis focused to the medical chemistry, in which the

ligands and metal complexes were graphically represented and find

their affinity/ binding level with the respective chemistry compounds

through different bioinformatics tools.

The ligand chemical database for enzyme reactions, which was

originally constructed by Nishioka and co-workers [90]. Ligand now

consists of two sections: the expanded ENZYME section and the new

compound section. The enzyme section is a collection of all known

enzymatic reactions classified according to the nomenclature of the

International Union of Biochemistry and Molecular Biology [91]. The

compound section is a collection of metabolic compounds, including

substrates, products, inhibitors, cofactors and effectors, and other

chemical compounds that play important functional roles in living cells.

Both sections are tightly coupled with the KEGG metabolic pathway

database: the enzyme section for the network of genes and the

compound section for the network of chemical compounds. The Ligand

database thus provides fundamental data on both biological and

chemical aspects of life.

The emergence of antibiotic-resistant strains of pathogenic

bacteria is an increasing threat to global health that underscores an

158

urgent need for an expanded antibacterial armamentarium. Gram-

negative bacteria, such as Escherichia coli, have become increasingly

important clinical pathogens with limited treatment. The increased

prevalence of pathogenic bacteria with resistance to clinically useful

antibiotics is a growing threat to public health. Despite this urgent,

unmet medical need, few novel classes of antibiotics and antibiotic

leads, e.g. linezolid, daptomycin, and platensimycin, have been

discovered in the last 30 years. Antibiotic-resistant Gram-negative

bacteria are of growing medical significance and consist of pathogens

such as Acinetobacter baumannii, Escherichia coli, Haemophilus

influenzae, Klebsiella pneumoniae, Neisseria meningitidis, and

Pseudomonas aeruginosa. E. coli and P. aeruginosa are commonly found

in hospital-acquired infections such as pneumonia and septicemia [92].

Therefore, the chemical compounds were analyzed whether the

compound is bind with respective cancer cell/ pathogens. It is less cost

and is not required for the wet chemistry analysis. So, now a day,

people are focusing the bioinformatics studies for predicting their

compounds may be act as an alternative drug or not.

MATERIALS AND METHODS

Sampling

The test cultures were isolated from the patient’s sample (Urine)

in Mahatma Gandhi Government Hospital at Tiruchirappalli, Tamil

Nadu, India, during May 2015. The samples were collected from the

patients with proper procedure during the morning time. All the

collected samples were separately stored in sterile containers and

stored in ice boxes at 4° C. The sterile storage containers contain the

transport medium for culture maintaining purposes. All the samples

159

were immediately transported in to the laboratory and were processed

within 6 h.

Isolation of the bacterial cultures

The patient samples were subjected to the pure culture

techniques for isolation of the individual bacteria among the bacterial

groups. The CLED and MacConkey agar medium was commonly used

for isolation of the bacterial culture and the spread/ streak plate

method was used for separation of pure (individual) culture.

Culture maintenance

All the test bacteria were grown in nutrient agar medium and

incubated at 37 ±1 °C for 24 - 48 h followed by frequent subculture to

fresh (Nutrient broth) medium and were used as test bacteria. The pure

cultures were separately maintained in the sterile test tubes and were

subjected for the identification.

Identification of the cultures

Gram Staining

The cultures from both medical samples were smeared on the

slide and heat fixed. The crystal violet dye was added and allowed to

cover the whole smear to act for 1 minute and rinsed with tap water.

Few drops of Grams-iodine was added and allowed to react for 30

seconds to 1 minute. It was decolorized with 95% ethanol and

immediately washed under running tap water. Then, safranine was

added and allowed to act for 1 minute, slide was rinsed with tap water

and blot dried and examined under oil immersion objective. Gram

positive cells were appeared in purple color and gram negative cells

were appeared in red color [1].

160

Motility determination

Hanging drop method

A small amount of Vaseline was placed at each corner of clean

cover glass. Two loopful of the 24 hour culture of the organism was

placed at the center of the cover glass. A depression slide was pressed

over the cover glass, such that the depressions cover the culture drop,

and quickly inverted. The completed preparation was observed

microscopically for motility [2].

Biochemical Characterization

Indole production test

Peptone broth was prepared and sterilized at 121ºC for 15

minutes at 15 1bs pressure. The culture was inoculated into the

peptone broth under aseptic condition and incubated at 37ºC for 24

hours. Few drops of Kovac’s reagent were added to the tube after

incubation. The formation of cherry red colored ring in the upper

surface of the medium indicates positive result for the identification of

bacterial isolates.

Methyl red test

MR – VP broth was prepared and sterilized at 121ºC for 15

minutes at 15 1bs pressure. The culture was inoculated into the broth

under aseptic condition and incubated at 37ºC for 24 hours. After

incubation, the indicator methyl red was added to the tubes. The

development of red colour indicates acid production and positive result.

Voges proskauer test

MR – VP broth was prepared and sterilized at 121ºC for 15

minutes at 15 1bs pressure. The culture was inoculated in to the broth

161

under aseptic condition and incubated at 37ºC for 24 hours. After

incubation the indicator Barritt’s reagent 1 and 2 were added. The

development of pink colour indicates the positive result.

Citrate utilization test

Simmon’s citrate agar was prepared and sterilized at 121ºC for

15 minutes at 15 1bs pressure. The medium was poured into the tube

and slant was prepared. The culture was streaked in the slant and

incubated at 37ºC for 24 hours. Development of blue colored slant after

incubation indicates the positive result.

Oxidase test

The deionized water wet disc was filled with few drops of 1%

tetramethyl-t-phenyline diamine dihydrochloride and inoculated a

loopful colony of pure culture of bacteria on the surface of the disc and

incubated for 1 min. If the area of inoculation turn dark blue to maroon

and finally to black colour indicates positive results.

Catalase test

Isolates were grown in Nutrient Agar Medium for 48-72 h at

50ºC. 3 % hydrogen peroxide was poured onto the colonies. Formation

of air bubbles indicates the presence of catalase enzyme.

Triple Sugar Iron test (TSI)

The growth of the organisms on the TSI slant indicates the type

of sugar fermented and in addition, identifying the hydrogen sulfide

products with an acid production and the color of the phenol red

indicator turns yellow. The alkaline reaction of the medium was

indicated by purple color production and hydrogen sulphide production

was indicated by formation of black color. The TSI agar medium was

162

prepared and dispensed 5.0 ml portion into the test tubes and,

sterilizes it and allowed to solidify in the slant position. A loopful

culture was streaked on to the slants and incubates at 37°C for 24

hours and results were observed.

Other biochemical tests

The starch hydrolysis, nitrate, casein hydrolysis, coagulase,

gelatin, ONPG, esculin, L-phenyl alanine, sporulation test were properly

performed by standard procedure.

Test microorganisms

The test strains were: E. coli (ESBL) (S1), Enterococcus fecalis

(S2), Proteus mirabilis (S3), Pseudomonas aeruginosa (S4) and

Staphylococcus aureus (S5).

Analysis of ESBL nature

The ESBL E.coli was isolated from the urine sample of the

patients and was identified by the proper biochemical test. The ESBL

E.coli was challenged against β-lactam group of antibiotics like

methicillin, amoxicillin, ampicillin and penicillin for identification of the

extended-spectrum beta-lactamases (ESBLs) nature of the E.coli.

Testing of antimicrobial activity

Microbial strains were tested for antimicrobial sensitivity using

the disc diffusion and well diffusion method. The test solutions were

prepared in 100 % DMSO solvent. This method was used to evaluate in

vitro antibacterial activity of test sample against certain human

pathogenic microorganisms on Muller Hinton Agar (MHA) plates. A

sterile cotton swab was used to inoculate the standardized bacterial

suspension (18 h) on surface of agar plate rotating the plate every 60°

163

to ensure homogeneous growth. For antimicrobial activity of metal ion

and ligand samples analysis, the 50 μL of test solution of 100 µg

concentration was poured in each disks, separately. In this study, four

metal ion (MnII(B1), CoII(B2), CuII(B3) and NiII(B4) and three ligand

(MPH(L1), MTN(L2) and MFP(L3) samples were analyzed separately. The

test samples coated disks (6-mm diameter) were placed in each dish by

a sterilized forceps, separately. But in the combination of metal ion and

ligand sample analysis, well diffusion method was used. The well (1 cm

in diameter and 4 mm in depth) was made by the sterile steel puncture

machine in the MHA plates. The 100 μL of test solution (Metal ion +

Ligand = 1:1 ratio = 50 µg concentration/ well) was added into each

well. Both type of plates were incubated at 37±1°C for 24–48 h (for

bacteria) and 25 ±1°C for 48-72 h (for fungus). After incubation, the

zone of inhibition was measured with ruler/HiAntibiotic ZoneScale-C.

The assays were performed in triplicate and the average values are

presented. Methicillin – 10mcg (for bacteria) and Itraconazole – 10mcg

(for fungus) was used as positive control.

Larvicidal assay

For the larvicide evaluation, World Health Organization protocols

were used with modifications. Eggs of A. aegypti from Entomological

research institute, Madurai were hatched by submerging them in

chlorine-free water at 27 ± 2°C, with 80 ± 5% of relative humidity. The

larvae were fed with fish food until L3 larval instar. Stock solutions of

synthesized compounds were prepared at 250, 500 and 1000 µg. mL-1

concentrations with 0.5% dimethyl sulfoxide (DMSO) in chlorine free

water. The stock solutions were placed in containers with 20 L3 larvae.

Each concentration had 60 larvae in triplicate. Aqueous solution of

164

0.5% DMSO was used as negative control. The LC50 were calculated

using probit analysis with a reliability interval of 95%. Larval mortality

percentage was estimated by using the following equation.

Larval mortality % = A – B / A × 100

Where, A = number of tested larvae and B = number of tested pupa

Heamolytic assay

Freshly collected human red blood cells from healthy individuals

were employed in this assay. Aliquots of 7 ml of blood were washed

three times with sterile saline solution (0.89%, w/v, NaCl, pyrogen free)

by centrifugation at 5000 rpm for 5 min. The cell suspension was

prepared by finally diluting the pellet to tyrode solution. A volume of

0.5 ml of the cell suspension was mixed with 0.5 ml diluent containing

10, 5 2.5 and 1.25 µg/ ml concentrations of individual ligand extracts

in saline solution. The mixtures were incubated for 12 h at 37° C and

centrifuged at 5000 rpm for 10 min. The free haemoglobin in the

supernatants was measured spectrophotometrically at 412 nm. Tyrode

and triton X were included as minimal and maximal haemolytic

controls. The haemolytic percent developed by the saline control was

subtracted from all groups. The experiment was done in triplicate and

mean ± S.D. was calculated [6]. The level of percentage hemolysis by

the compound was calculated according to the following formula:

As – At

Percent of hemolysis = Ac – At × 100

Here, As is the Absorbance of sample; At is the Absorbance of tyrode;

Ac is the Absorbance of triton x

165

Statistical analysis

Statistical analysis was performed with ORIGIN 8 software. The

descriptive data was given as a mean, standard deviation, standard

error and etc.

Docking analysis

The present study, two different bioinformatics tools were used

for analysis the mode of action of test chemical compounds against

cancer, virus and bacterial cells.

Docking Studies

Protein preparation

AutoDock is a suite of automated docking tool. It is designed to

predict how small molecules, such as substrates or drug candidates,

bind to a receptor of known 3D structure [8]. The protein retrieved from

PDB database. The protein structure of Extended-spectrum β-

lactamase (PDB ID: 4LEBN). All water molecules removed from all

protein structure and add with Kollmann charges was assigned.

Through which hydrogens were added, side chains were optimized for

hydrogen bonding. The energy minimized protein was then saved in

PDB format. Using MGLTools-1.4.6 nonpolar hydrogens were merged,

AutoDock atom type AD4 and Gasteiger charges were assigned and

finally saved in protein.pdbqt format.

166

Ligand preparation

Structure of ligands were drawn using ChemSketch, optimized

with 3D-geometry and the two-dimensional structures of synthetic were

converted into 3-D structure using the open Babel format molecule

converter and saved in PDB format for AutoDock compatibility.

MGLTools-1.4.6 (The Sripps Research Institute) was used to convert

ligand.pdb files to ligand.pdbqt files.

Active site prediction

The active site of the protein is the binding site or usually a

pocket at the surface of the protein that contains residues responsible

for substrate specificity which often act as proton donors or acceptors.

Identification and characterization of binding site is the key step in

structure based drug design. The binding site has been identified by

computational and literature reports. The active site region of the

protein is identified by castP. These servers analytically furnish the

area and the volume at the probable active site of each pocket to

envisage the binding site.

Docking protocol-MGL tools

Grid parameter files (protein.gpf) and docking parameter files

(ligand.dpf) have written using MGLTools-1.4.6. Receptor grids were

generated using 80x80x60 grid points in xyz with grid spacing of 0.375

Å. Grid box was centered co crystallized ligand map types were

generated using autogrid4. Docking of macromolecule was performed

using an empirical free energy function and Lamarckian Genetic

Algorithm, with an initial population of 250 randomly placed

individuals, a maximum number of 106 energy evaluations, a mutation

rate of 0.02, and a crossover rate of 0.80. One hundred independent

167

docking runs were performed for each ligand. Results differing by 2.0 Å

in positional root-mean square deviation (RMSD) were clustered

together and represented by the result with the most favourable free

energy of binding.

Docking protocol-Hex tools

The molecular docking between the target receptor and ligand

was performed by Hex tool. This tool is for interactive docking and can

able to run in any operating system. There are advanced versions in

this tool and it provides the energy values for all the models. The model

can visualize in any forms with docked parameters. The structure for

the ligand MPH can be visualized in the phymol viewer and docked with

the structure of ebola virus and the target receptor for cancer. The

ligand structure was drawn by chemsketch and subjected to docking.

Both the structures were docked and show energy values such as E-

max, E-min, E-shape, and E-total were calculated. The Net charges

with number of orientations were also calculated. Finally, the results

were compared based on the docking parameters.

Test microorganisms

The test microorganisms were isolated from the patient’s medical

samples and were identified by different biochemical test analysis Table

6.2. The identified test bacterial cultures were E. coli, E. faecalis, P.

aeruginosa, P. mirabilis and S. aureus. The identification of test cultures

were confirmed by the Bergy’s manual of determinative bacteriology.

Antimicrobial activity

The antimicrobial activity of test sample was examined with

various pathogenic microorganisms using the (measure the inhibition

168

zone) disk / well diffusion methods. The descriptive statistic of the

antimicrobial activity for three different samples were presented in

Table 6.3. The tested concentration produce zone of inhibition on MHA

plates for bacteria. In this study, four different samples such as metal

ions (B1, B2, B3 and B4), ligand (L1, L2 and L3) and combined metal

ion + ligand samples (B1+L1, B2+L1, B3+L1 and B4+LA) were

challenged against certain pathogenic microorganisms. In metal ion

(alone) samples, nil effect was recorded in the B1 samples against all

the microorganisms, except E. coli (ESBL) (8 mm). The nil was also

observed in B3 (E. faecalis) and B4 (E. coli - ESBL). The test sample B2

was most effective against E. faecalis while less significant activity was

observed against E. coli (ESBL). But in B3 sample, it was found to be

more effective against S. aureus while smaller effect was noticed in

E. faecalis. The B4 sample was effective against E. faecalis whereas nil

effect was observed in E. coli – ESBL and smaller effect against other

strains. The decreasing antimicrobial activity trends in metal ion

complex were: B2 > B3 > B4 > B1 Table 6.3. and Figure 39.

In ligand (alone) samples, nil effect was recorded in the L3

samples against E. coli (ESBL) and P. aeruginosa. In test bacterial

strains, the test sample L1 was most effective against E. faecalis and

P. mirabilis while smaller effect was noticed from E. coli (ESBL). But in

L2 sample was most effective against P. mirabilis while smaller effect

was noticed from E. coli (ESBL). The L3 sample was effective against S.

aureus whereas smaller effect was observed from E. coli (ESBL) and P.

aeruginosa. The decreasing antimicrobial activity trends in ligand

complex were: L1 > L2 > L3 Table 6.4.

169

In combination (metal ion + ligand = 1:1 ratio) samples, nil effect

was not recorded in any samples against all the microorganisms. The

test sample B1+L1 was most effective against E. faecalis while smaller

effect was noticed from P. mirabilis. But in B2+L1 sample was most

effective against E. faecalis and S. aureus while smaller effect was

noticed from E. coli (ESBL) and P. aeruginosa. The B3+L1 sample was

effective against S. aureus whereas smaller effect was observed from

P. mirabilis. The B4+L1 sample was effective against P. aeruginosa and

S. aureus whereas smaller effect was observed from E. coli (ESBL). The

decreasing antimicrobial activity trends in combined samples were:

B2+L1 > B3+L1 > B1+L1 > B4+L1 Table 6.5 and Figure.

All the microbial strains depict higher sensitivity to the test

samples (except few) when compared to the positive control. There is no

antimicrobial activity in solution devoid of sample used as a vehicle

control (concentrated DMSO), reflecting that antimicrobial activity was

directly related to the sample. This study shows that the test samples

have great promise as antimicrobial agent against microorganisms. It

may be concluded that the test complexes inhibits growth of microbes

to a good extent.

Descriptive statistics

In B1 sample, the mean, standard deviation, standard error and

variance level of S1was 8.3, 0.57, 0.33 and 0.33, respectively. But in

S2, S3, S4 and S5, the mean, standard deviation, standard error and

variance level were nil all the studies. In B4 sample, the mean,

standard deviation, standard error and variance level of S1, S2, S3, S4

and S5 were 0, 0, 0 and 0; 17.33, 1.52, 0.88 and 2.33; 8.33, 0.57, 0.33

170

and 0.33; 13.33, 1.52, 0.88 and 2.33; and 10, 1.0, 0.57 and 1.0,

respectively Table 6.6.

In L1 sample, the mean, standard deviation, standard error and

variance level of S1, S2, S3, S4 and S5 were 16.33, 1.52, 0.88 and

2.33; 19.66, 1.52, 0.88 and 2.33; 17.66, 1.52, 0.88 and 2.33; 20.33,

1.52, 0.88 and 2.33; and 18.33, 1.52, 0.88 and 2.33 respectively. In L2

sample, the mean, standard deviation, standard error and variance

level of S1, S2, S3, S4 and S5 were 13.0, 1.0, 0.57 and 1.0; 16.33,

1.52, 0.88 and 2.33; 15.0, 1.0, 0.57 and 1.0; 17.0, 1.0, 0.57 and 1.0;

14.0, 1.0, 0.57 and 1.0 respectively Table 67.

But in B1+L1 sample, the mean, standard deviation, standard

error and variance level of S1, S2, S3, S4 and S5 were 14.0, 1.0, 0.57

and 1.0; 26.33, 2.51, 1.45 and 6.33; 13.0, 1.0, 0.57 and 1.0; 10.0, 1.0,

0.57 and 1.0; 27.33, 2.51, 1.45 and 6.33, respectively. In B3+L1

sample, the mean, standard deviation, standard error and variance

level of S1, S2, S3, S4 and S5 were 17.0, 1.0, 0.57 and 1.0; 24.0, 2.0,

1.15 and 4.0; 14.0, 1.0, 0.57 and 1.0; 12.0, 1.0, 0.57 and 1.0; 27.0,

2.0, 1.15 and 4.0 respectively Table 6.8.

Larvicidal assay

Toxicity assays indicated that these derivatives could induce a

premature, abnormal, and lethal larval moult.

Compounds were screened in larval bioassays at concentrations

of 1000, 500 and 250µg/ml in a dose dependent manner and data for

their mortality was recorded in Table . Larvicidal activity was screened

for ligand 1 -3 at 72h suggested that third instar larvae at room

temperature. The compounds 1, and 3 are produced 70 and 63 %

mortality at 1000µg/mL and the compound 2 was found 16 % mortality

171

at concentration at 1000µg/mL. The larvicidal activities of 2-

[morpholin-4-yl) (pyridine-3-yl) methyl] hydrazine carboxamide( was

lower than those of ligands 1-(Furan-2-yl (morpholino) methyl)-3-

phenyl Urea and N-(morpholino (thiophen-2-yl) methyl) pyridine-3-

carbohydrazide. Compounds with carbonyl group commonly have high

larvicidal activities against testes A. aegypti. The number and position

of amine groups in the chemical moiety was also an important element

for the insecticidal activity and two amine was the most favorable

number for high activity. For example, Ligand 2 and 3 had high

insecticidal activities while ligand one showed lower activities.

Haemolytic assay

The determination of haemolysis is based on haemoglobin

absorbance at 550 nm, with subtraction of the interference of Ligand. A

significant difference was observed in washed RBC among L1, L2 and

L3 after 24 h of incubation in comparison with controls. A lower

haemolytic effect was observed at 1.25 μg/mL by L1. 2-[morpholin-4-

yl)(pyridine-3-yl)methyl]hydrazine carboxamide induced 14% of

haemolytic activity at 1.25 μg/mL while other Ligands 2&3 induced

high level of haemolytic activity 1.25 μg/mL indicates that the novel

compounds L2 and L3 showed significantly high haemolytic activity 

~ -68 and -52 % lysis of blood cells respectively at 1.25 μg/mL, which

can be attributed to the presence of electron donating methyl groups.

The ligand interference was avoided by the subtraction of the OD 550

nm of test sample suspended in the vehicle from the measured OD 550

nm at the same concentration. Negative control (tyrode) and positive

control (triton X-100 1%) induced 2% and 80% respectively of

haemolysis. Similar result was observed in L2 and L3 at 10 μg/mL

concentration. Anticancer activity is often enhanced by the presence of

172

electron releasing groups (Mologni et al., 2010). In contrast, the

compound L1 exhibited haemolytic activity below 10%. In view of the

observed differences in the % lysis of RBC values, it is inferred that the

electron withdrawing and electron donating nature of the substituent

groups have an influence on the haemolytic activity of the compounds

(Ding et al., 2012). Moreover, the cytotoxicity of the compounds viz. L2

and L3 can be optimized by making appropriate changes in the

molecular structures for the purpose of their use as toxic compounds to

control the uncontrolled proliferation of cells.

Docking studies

ESBL enzyme studies

Extended-spectrum β-lactamases (ESBLs) are a rapidly evolving

group of β-lactamases which share the ability to hydrolyze third-

generation cephalosporins and aztreonam but are inhibited by

clavulanic acid. They represent the first example in which β-lactamase–

mediated resistance to β-lactam antibiotics resulted from fundamental

changes in the substrate spectra of the enzymes. The total number of

ESBLs now characterized exceeds 200. These are detailed on the

authoritative website on the nomenclature of ESBLs hosted by George

Jacoby and Karen Bush (http://www.lahey.org/studies/webt.htm).

Published research on ESBLs has now originated from more than 30

different countries, reflecting the truly worldwide distribution of ESBL-

producing organisms. So Extended-spectrum β-lactamase is attractive

drug target for antibiotic resistance. Binding energies of the protein-

ligand (drug) interactions are important to describe how fit the drug

binds to the target macromolecule. We applied the docking procedures,

to dock copper compounds to dock with ESBL into the binding pocket

of this enzyme. Our docking simulation resulted in a very close target

173

protein structure, which supports our findings. The compounds Co(II)

and compound Cu(II) were highly interacted with ESBL and produced

halogen bond through amino acid interactions such as SER237

(Distance - 2.125) and GLN192 (Distance - 2.111)/ HIS197 (Distance -

2.146), respectively Figure and Table. Due to this we propose that

there is a high-potent inhibitory activity of copper compound.

MPH Result

The structure for Ebola virus and the structure of the ligand MPH

was displayed in the Figure 54. The target receptor was ready to dock

with the ligand in the Figure 55. The target receptor binds with the

ligand and it shows the E total value of 150.63 Figure 56. The E max

and E min value falls between -26.80.07 and -112.04. The model 1 has

201 residues in the ligand and shows the Net formal charge as 2. The

docked structure was displayed with binding visualizations in the

Figure 59. The structure of the MPH with phymol viewer was visualized

in the Figure 60 . The model of the target receptor was displayed in the

Figure 61 represents that the docked structure of the receptor and the

ligand. The model 2 has the E total value of -223.03 and E max is -

197.88. The model 2 has 201 residues in the ligand and shows the Net

formal charge as -3. The docked parameters are represented in the

Table 6.11. When the receptor (model 1) was docked with the ligand

MPH the Energy value obtained was 150.63 and the energy value for

model 2 was derived 223.03. From these results, we observed that the

ligand was docked with the target receptors effectively and we can able

to detect the binding affinity of the models. Hence, this compound

could be act as an alternative drug in near future with carried lot of

experiments done.

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Table 6.2: Biochemical characterizations values of isolated strains from infected patient samples in Government Hospital, Tiruchirappalli

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ultu

re

Slan

t

But

H2S

Gas

S1 + + - - Ac* Ac - + + - - - + + - - - + - + - - - Rods E. coli

S2 - - + + Ak* Ak - - - - - +/- + - - - - - + - - - + Cocci E. faecalis

S3 - - - + Ak Ak - - - + + - - + - - - - - + - - - Rods P. aeruginosa

S4 - + + + Ak Ac + + + - + - + + + - - - - + - + - Rods P. mirabilis

S5 - - + + Ac Ak - - - - + - + + - - + - - - + Cocci S. aureus

Ac* - Acid but; Ak* - Alkaline slant

175

Table 6.3: Antimicrobial activity of individual metal ions

S.No Test bacteria

Zone of inhibition (mm)

Test sample = 100 µg / disc Positive control

(Cefotaxime 30 mcg)

Negative control

(100 % DMSO) Remarks

B1 B2 B3 B4

1. E. coli (ESBL) 8 12 12 0 14 0

2. E. faecalis 0 22 0 17 18 0

3. P. aeruginosa 0 16 15 8 18 0

4. P. mirabilis 0 20 19 13 20 0

5. S. aureus 0 18 22 10 20 0

176

Table 6.4: Antimicrobial activity of individual ligands

S.No Test bacteria

Zone of inhibition (mm)

Test sample = 100 µg / disc Positive control

(Cefotaxime 30 mcg)

Negative control

(100 % DMSO) Remarks

L1 L2 L3

1. E. coli (ESBL) 16 13 0 14 0

2. E. faecalis 20 16 10 18 0

3. P. aeruginosa 18 15 0 18 0

4. P. mirabilis 20 17 10 20 0

5. S. aureus 18 14 14 20 0

177

Table 6.5. Antimicrobial activity of metal and ligand combined compound (complex)

S.No Test bacteria

Zone of inhibition (mm)

Test sample = 50 µg / well (100 µl sample)

Positive control

(Cefotaxime 30 mcg)

Negative control

(100 % DMSO) Remarks

B1+L1 B2+L1 B3+L1 B4+L1

1. E. coli (ESBL) 15 12 18 10 14 0

2. E. faecalis 26 32 24 17 18 0

3. P. aeruginosa 13 12 14 19 18 0

4. P. mirabilis 10 20 12 13 20 0

5. S. aureus 27 32 27 19 20 0

178

Figure 39: Antibacterial activity of metal ions of MPH by agar Disc diffusion method

Figure 40: Antibacterial activity of metal ions and ligand MPH alone by agar Disc diffusion method

179

Figure 41: Antibacterial activity of metal ions and ligand MTN alone by agar Disc diffusion method

180

Figure 42: Antibacterial activity of metal ions and ligand MFP alone by agar Disc diffusion method

181

Figure 43: Combined Antibacterial activity of ligand MPH and

metal ions (1:1) by agar well diffusion method

182

Table 6.6: Descriptive statistics for antimicrobial activity of metal

ions

S.No Sample

code Culture

code Mean SD* SE* Variance

1 B1 S1 8.33333 0.57735 0.33333 0.33333

2 B1 S2 0 0 0 0

3 B1 S3 0 0 0 0

4 B1 S4 0 0 0 0

5 B1 S5 0 0 0 0

6 B2 S1 12.33333 0.57735 0.33333 0.33333

7 B2 S2 22.33333 1.52753 0.88192 2.33333

8 B2 S3 16.33333 1.52753 0.88192 2.33333

9 B2 S4 20.33333 1.52753 0.88192 2.33333

10 B2 S5 18.33333 1.52753 0.88192 2.33333

11 B3 S1 12.33333 1.52753 0.88192 2.33333

12 B3 S2 0 0 0 0

13 B3 S3 14.66667 1.52753 0.88192 2.33333

14 B3 S4 18.66667 1.52753 0.88192 2.33333

15 B3 S5 22 2 1.1547 4

16 B4 S1 0 0 0 0

17 B4 S2 17.33333 1.52753 0.88192 2.33333

18 B4 S3 8.33333 0.57735 0.33333 0.33333

19 B4 S4 13.33333 1.52753 0.88192 2.33333

20 B4 S5 10 1 0.57735 1

21 PC* S1 14.33333 0.57735 0.33333 0.33333

22 PC S2 18.66667 1.1547 0.66667 1.33333

23 PC S3 17.66667 1.52753 0.88192 2.33333

24 PC S4 19.66667 1.52753 0.88192 2.33333

25 PC S5 20.33333 0.57735 0.33333 0.33333

SD* - Standard deviation; SE* - Standard Error; PC* - Positive control

183

Table 6.7. Descriptive statistics for antimicrobial activity of ligands

S.No Sample

code Culture

code Mean SD* SE* Variance

1 L1 S1 16.33333 1.52753 0.88192 2.33333

2 L1 S2 19.66667 1.52753 0.88192 2.33333

3 L1 S3 17.66667 1.52753 0.88192 2.33333

4 L1 S4 20.33333 1.52753 0.88192 2.33333

5 L1 S5 18.33333 1.52753 0.88192 2.33333

6 L2 S1 13 1 0.57735 1

7 L2 S2 16.33333 1.52753 0.88192 2.33333

8 L2 S3 15 1 0.57735 1

9 L2 S4 17 1 0.57735 1

10 L2 S5 14 1 0.57735 1

11 L3 S1 0 0 0 0

12 L3 S2 10 1 0.57735 1

13 L3 S3 0 0 0 0

14 L3 S4 10.66667 1.1547 0.66667 1.33333

15 L3 S5 14 1 0.57735 1

16 PC* S1 14.33333 0.57735 0.33333 0.33333

17 PC S2 18.66667 1.1547 0.66667 1.33333

18 PC S3 17.66667 1.52753 0.88192 2.33333

19 PC S4 19.66667 1.52753 0.88192 2.33333

20 PC S5 20.33333 0.57735 0.33333 0.33333

SD* - Standard deviation; SE* - Standard Error; PC* - Positive control

184

Table 6.8: Descriptive statistics for antimicrobial activity of

combination of metal ion and ligand (1:1 ratio)

S.No Sample

code Culture

code Mean SD* SE* Variance

1 B1+L1 S1 14 1 0.57735 1

2 B1+L1 S2 26.33333 2.51661 1.45297 6.33333

3 B1+L1 S3 13 1 0.57735 1

4 B1+L1 S4 10 1 0.57735 1

5 B1+L1 S5 27.33333 2.51661 1.45297 6.33333

6 B2+L1 S1 12 1 0.57735 1

7 B2+L1 S2 32 3 1.73205 9

8 B2+L1 S3 12 1 0.57735 1

9 B2+L1 S4 20 2 1.1547 4

10 B2+L1 S5 31.66667 2.51661 1.45297 6.33333

11 B3+L1 S1 17 1 0.57735 1

12 B3+L1 S2 24 2 1.1547 4

13 B3+L1 S3 14 1 0.57735 1

14 B3+L1 S4 12 1 0.57735 1

15 B3+L1 S5 27 2 1.1547 4

16 B4+L1 S1 10 1 0.57735 1

17 B4+L1 S2 17.33333 1.52753 0.88192 2.33333

18 B4+L1 S3 18.66667 1.52753 0.88192 2.33333

19 B4+L1 S4 13 1 0.57735 1

20 B4+L1 S5 20 1 0.57735 1

21 PC* S1 14.33333 0.57735 0.33333 0.33333

22 PC S2 18.66667 1.1547 0.66667 1.33333

23 PC S3 17.66667 1.52753 0.88192 2.33333

24 PC S4 19.66667 1.52753 0.88192 2.33333

25 PC S5 20.33333 0.57735 0.33333 0.33333

SD* - Standard deviation; SE* - Standard Error; PC* - Positive control

185

Figure 44: Antimicrobial activity of B1 and B2 metal ions (alone)

Figure 45: Antimicrobial activity of B3, B4 metal ions (alone) and positive control

186

Figure 46: Antimicrobial activity of ligands L1, L2 and L3 (alone)

and positive control

Figure 47. Antimicrobial activity of combination of metal ion (B1

and B2) and ligand (L1) (Metal ion + Ligand = 1:1 ratio)

187

Figure 48 Antimicrobial activity of combination of metal ion (B3 and B4) and ligand (L1) (Metal ion + Ligand = 1:1 ratio), and

positive control

Figure 49. Heamolytic assay of ligand samples

48

38 34

14

80

2

78 76 74 68

80

2

70 70 68

52

80

2 0

10

20

30

40

50

60

70

80

90

10µg 5µg 2.5µg 1.25µg Triton X Tyrode

Ligand1

Ligand 2

Ligand 3

188

Figure 50: Titer plate method for analysis of heamolytic (using human RBC) assay against ligand samples

Figure 51: Triplicate of Control flask containing 0.5% of DMSO with (n=20 )3rd instar larvae

189

Figure 52: Testing of Ligand at 250,500 and 1000µg/ml with (n= 20) 3rd instar larvae

Figure 53: Formation of pupa at 1000µg/ml

190

Table 6.9: Larvicidal activity of ligands

A. aegypti Ligand 1(µg/ml) Ligand 2(µg/ml) Ligand 3(µg/ml) Control

(DMSO) 1000 500 250 1000 500 250 1000 500 250

No of Larvae 60±0.02 60±0.002 60±0.06 60±0.02 60±0.12 60±0.02 60±0.22 60±0.02 60±0.002 60±0.002

No of Pupa 50±0.12 52±0.004 56±0.05 18±0.04 20±0.14 24±0.03 22±0.14 24±0.01 27±0.12 54±0.02

% of Larvicidal 16±0.22 13±0.004 6.6±0. 1 70±0.06 66±0.24 60±0.02 63±0.12 60±0.01 44±0.14 10±0.06

Table 6.10: Parameters of the docked models for MPH

Receptor1-Ligand Interaction Etotal Eshape Emin/K

j/mol Emax/Kj

/mol Estart/Kj/mol

/Rank Residues in the ligand

Net formal charge

Rank Orientations

Model 1 150.63 150.63 -112.04 -26.80 -0.18/306681 201 2 108143024 Model 2 -223.03 -223.03 -223.03 -197.88 -26.34/257654 201 -3 11476538

Operating System – Windows XP - Hex 4.5 Server control 2.1 – docking – default - receptor and ligand molecular interaction

191

Figure 54. Structure for Ebola virus

Figure 55: Ebola virus in Rasmol Visualization

192

Figure 56: Structure for the (morpholin-4-yl) (pyridin-3-yl) methyl] hydrazinecarboxamide

Figure 57. Receptor (Ebola) ready to dock with the Ligand L1

193

Figure 58. Receptor (Ebola) docked with the Ligand L1

Figure 59: Docked structure with the binding visualizations

194

Figure 60: Structure for the Ligand L1(morpholin-4-yl) (pyridin-3-yl) methyl] hydrazinecarboxamide with Phymol

Figure 61: Structure for the target receptor for cancer

195

Figure 62: Docking of receptor (cancer) and MPH

Table 6.11: The molecular docking studies of metal complexes of MPH with Extended-spectrum β-lactamase

S.No Compound name

Docking score

(Kcol/mol)

Inhibitory Concentrati

on (µm)

H-Bond Interaction

Distance

1 Compound Co - 5.11 4.2 SER237 N-H…O 2.125

2 Compound Cu - 5.2 2.3 GLN192 N-

H…O HIS197 N-H…O

2.111 2.146

3 Ciprofloxacin -4 389

SER235 N-H…O

SER130 N-H…O

1.793 1.958

196

Figure 63: Docking study of CoII complex of MPH with extended-spectrum β-lactamase

The amino acid residues SER237 was involved in interactions

with compound Co (II) the active site of ESBL. The length of hydrogen

bond formed 2.125Å. The IC50 values of this compound have 4.2 µm)

and low docking score (-5.11).

197

Figure 64: Docking study of CuII complex of MPH with extended-spectrum β-lactamase

The amino acid residues GLN192 and HIS192 were involved in

interactions with compound Cu (II) the active site of ESBL. The length

of hydrogen bond formed 2.111Å and 2.146Å. The IC50 values of this

compound have 2.3(µm) and low docking score (-5.2).

Figure 65: Extended-spectrum β-lactamase with compound

Ciprofloxacin

198

DISCUSSIONS

Antimicrobial activity

The emerging bacterial resistance causes a widespread problem

for the treatment of various infections. Therefore, the search for

antimicrobials is a never-ending task. Now-a-days a number of

hydrazone derivatives have been developed and evaluated for their

antibacterial activity. Aslan et al., (2012) investigated the antibacterial

activity of sulfonyl derivatives. Certain steroidal hydrazines have been

synthesized by Khan (2008) which possess in-vitro antibacterial activity.

Hydrazones bearing imidazoles have been synthesized and screened for

antibacterial activity against numerous bacterial strains by Abdel-

Wahab et al. (2011). Palekar et al., (2009) synthesized different

thiazolidinone derivatives using hydrazine hydrate and evaluated them

for their in-vitro antibacterial activity. In this study, three different

samples [metal ions (B1, B2, B3 and B4), ligand (L1, L2 and L3) and

combined metal ion + ligand samples (B1+L1, B2+L1, B3+L1 AND

B4+LA)] were challenged against certain pathogenic microorganisms for

antimicrobial studies. The nil effect was observed in metal ions and

ligand (alone) samples whereas no nil effect was noticed in the

combination samples. Wang et al., (2013) synthesized hydrazone

derivatives with significant antibacterial activity. Hydrazone derivatives

containing transition metal complex were synthesized and evaluated for

antimicrobial activity by Babahan et al. (2013) Ozkay et al., (2010)

synthesized novel benzimidazole derivatives bearing hydrazone moiety

with antibacterial activity against different bacterial strains.

Khalil et al., (2009) synthesized hydrazone derivatives and

reported them as potential antibacterial agent. Hydrazone derivatives

199

synthesized by Abdel-Aziz and Mekawey (2009) exhibited antibacterial

activity with minimum inhibitory concentration (MIC) of 75 μg/mL.

Good antibacterial activity of hydrazone derivatives was reported by

Bawa et al. (2009). In this present study, the decreasing antimicrobial

activity trends of metal ion complex were: B2 > B3 > B4 > B1. But in

ligand samples were: L1 > L2 > L3. The decreasing antimicrobial

activity trends in combined sample (metal ion + ligand) were: B2+L1 >

B3+L1 > B1+L1 > B4+L1. Among the ligand samples, ligand 1 gave a

better antimicrobial effect against most of the microorganisms.

Therefore, ligand 1 was combined with all the metal ions separately.

The antimicrobial activity of metal ion (alone) samples were compared

to the combination samples. Both the results were not varied, except

the position of B1 and B4 samples due to the effect of combination with

ligand. Most of the combination samples showed good antimicrobial

activity than the metal ion and ligand samples alone. It indicated that

the combination samples could be used for the alternative drug.

Hydrazone derivatives, synthesized by Sharma et al., (2011)

exhibited antibacterial activity against various bacterial strains.

Antibacterial activity of certain hydrazone derivatives was reported by

Kendall et al. (2007) Jubie et al. (2010) synthesized hydrazone

derivatives and reported them as promising antibacterial agents.

Govindasami et al. (2011) synthesized and evaluated vanillin related

hydrazone derivatives for their antibacterial activity. The activity of the

newer agents is mostly tested against virulent H37Rv strain. Kamal et

al., (2007) synthesized nitro heterocyclic based 1, 2, 4-

benzothiadizines, which exhibited MIC of 1 μg/mL. But in the present

study, the 100 µg concentration of both test samples (metal ion and

200

ligand) showed considerable activity against certain pathogenic

microorganism. But in the combination samples showed greater

antimicrobial effect against test microorganisms with smaller test

concentrations (50 µg). Raja et al., (2010) synthesized hydrazone

derivatives and reported to have MIC of 6.25 μg/mL. Telvekar et al.,

(2012) developed benzofuran-3-carbohydrazide derivatives with good

anti-tubercular activity. Hydrazone derivatives synthesized by Gemma

et al., (2009) exhibited MIC of 6.25 μg/mL.

Mahajan et al., (2011) synthesized ferrocene-based hydrazone

derivatives with significant antitubercular activity. 1H-indole-2,3-dione

based hydrazones, synthesized by Karali et al., (2007) exhibited half

maximal inhibitory concentration (IC50) of 7.6 μg/mL. Hydrazones,

synthesized by Eswaran et al., (2010) exhibited a MIC of 6.25 μg/mL.

Hydrazones synthesized by Imramovský et al., (2007) based on

isonicotinoylhydrazide, pyrazinamide, p-aminosalicylic acid,

ethambutol, and ciprofloxacin exhibited MIC of 0.78 μg/mL and 3.13

μg/mL respectively. Hearn et al., (2009) synthesized anti-tubercular

agents which showed MIC of 0.06 μg/mL and 0.20 μg/mL respectively.

But in the present study, common water and food borne pathogens

were used and the three different test samples were active against

certain pathogens. The results of antimicrobial activity of metal ions

(alone) showed considerable fluctuation between the four samples

depend on their nature. Similar pattern was also observed in ligand

(alone) samples. Nayyar and Jain (2008) synthesized disubstituted

quinolone based hydrazides with good activity profiles. Turan-Zitouni et

al., (2008) synthesized thiazolyl hydrazones having anti-tubercular

activity with MIC of 2.5 μg/mL. 4-(adamantan-1-yl)-2-substituted

201

quinoline based hydrazones synthesized by Nayyar et al., (2007)

showed MIC of 1.00 μg/mL.

Secci et al., (2012) developed novel Hydrazine derivative and

evaluated for in-vitro anti-Candidal activity which exhibited MIC of 0.25

μg/mL. Novel hydrazine thiazole derivatives have been synthesized by

Maillard et al., (2013) and reported to exhibit anti-Candidal activity

with MIC of 0.25 μg/mL. Altyntop et al., (2012) developed, evaluated

novel hydrazone derivatives for in-vitro anti-Candidal activity, and

reported to have MIC of 0.05 μg/mL. Hydrazide derivatives synthesized

and exhibited MIC of <15.62 μg/mL. Chimenti et al. (2007) synthesized

2-thiazolylhydrazones and reported to have potential activity against

various strains of Candida species. This present study, in metal ion

groups, the test samples B2 were most effective against E. faecalis and

S. aureus in B3 while smaller (nil) effect was noticed from all the metal

ion division, except B2.

In ligand samples, the test sample L1(MPH) was most effective

against E. faecalis whereas the test sample L3(MFP) showed smaller

effect was observed from E. coli (ESBL) and P. aeruginosa. But in

combination samples (metal ion + ligand), The test sample B1+L1 and

B3+L3 were most effective against E. faecalis while test samples B1+L1

and B4+L1 showed smaller effect against P. mirabilis and E. coli (ESBL),

respectively. Kocyigit-Kaymakcioglu et al. synthesized and evaluated

the antifungal activity of various 3-acetyl-2,5-disubstituted-2,3-

dihydro-1,3,4-oxadiazoles. Out of these, 4-Fluorobenzoic acid ([5-

bromothiophen-2-yl] methylene) hydrazide exhibited highest inhibitory

activity against Candida albicans, with MIC value of 125 μg/mL.

(2012). El-Sabbagh and Rady (2009) evaluated the antiviral activity of

202

hydrazone derivatives against hepatitis A virus. Tian et al., (2009)

synthesized hydrazone derivatives as potential targets of human

immunodeficiency virus-1 capsid protein. The half maximal effective

concentration (EC50) value of the agents was reported to be 0.21 and

0.17 μM respectively.

Heamolytic assay

Larvicidal activity

Larvicides play significant role in controlling mosquitoes at their

breeding and immature stages. Larvicidal activity of hydrazone

derivative also reported in previous studies (Tabanca et al., 2013;

Kocyigit-Kaymakcioglu et al., 2013; Narasimhan et al., 2010).

Hydrazones are present in many of the bioactive heterocyclic

compounds because of their various biological and clinical applications

such as antimicrobial, antiplatelet, anticancer, antifungal, antiviral,

antitumoral, antibacterial and antimalarial activities (Abdel-Aal et al.,

2010; Koçyiğit-Kaymakçıoğlu et al., 2009). Recently, our group has

been investigating the possible pharmacological potential of new

molecules that contain a amide-hydrazine scaffold. Amide Hydrazine

derivatives (L1-L3) synthesized via the nucleophilic addition–elimination

reaction of Morpholine and other compounds were tested for their

larvicidal activity. All synthetic amide hydrazine derivatives were also

screened against A. Aegypti for their larvicidal activity. The compounds

were first screened in larval bioassays at concentrations of 1000, 500,

and 250 ppm in a dose-dependent manner and percent mortality was

observed. Most of the researchers in previous work preserved the amide

moiety, which suggested that this structure was an important

pharmacophore in those compounds.

203

Ligand 2 carrying methyl substituent on phenyl ring showed the

highest deterrent effect on larvicidal activity. This result confirms that

phenyl and methyl substituted phenyl groups may have a role in this

larvicidal activity. Personal protection and reduction in mosquito

populations through chemical control are the effective ways to eliminate

mosquito borne diseases (Faradin and Day, 2002). The common

approach for the control of mosquito vectors and reducing arthropod

transmitted diseases is based on the use of chemical insecticides from

different chemical classes. However, frequent use of insecticides has

failed to achieve these objectives due to the development of insecticide

resistance among mosquito populations. Pesticides also pose concerns

on their toxic effects on human and animals and deterioration of

nontarget species in the ecosystem. In previous studies hydrazone

derivatives have been reported to exhibit a wide spectrum of biological

effects including antifungal and insecticidal activity in the literatures.

Our larvicidal results correlate with Legocki et al. (2003) reported that

2,4-dihydroxythiobenzoyl derivatives substituted with amide,

hydrazine, hydrazide, hydrazone, and semicarbazide groups showed

different levels of biological activity.

Docking studies

In the field of molecular modeling, docking is a method which

predicts the preferred orientation of one molecule to a second when

bound to each other to form a stable complex. Knowledge of the

preferred orientation in turn may be used to predict the strength of

association or binding affinity between two molecules using, for

example, scoring functions. The associations between biologically

relevant molecules such as proteins, nucleic acids, carbohydrates, and

204

lipids play a central role in signal transduction. Furthermore, the

relative orientation of the two interacting partners may affect the type of

signal produced (e.g., agonism vs antagonism) (Andrew Binkowski et

al., 2003). Therefore docking is useful for predicting both the strength

and type of signal produced.

Docking is frequently used to predict the binding orientation of

small molecule drug candidates to their protein targets in order to in

turn predict the affinity and activity of the small molecule. Hence

docking plays an important role in the rational design of drugs. We

wish to computerize all the molecular components and the network of

molecular interactions to describe, utilize and predict functional

aspects of living systems. The molecular components include not only

genes and gene products, namely DNAs, RNAs and proteins, but also

other chemical substances in living cells (Morris et al., 1998) [40].

Although the interests of most biologists are biased towards DNAs,

RNAs and proteins, we believe that small chemical substances and

metal ions must have played important roles, together with the

biological macromolecules, for small chemical substances and

biological macromolecules are nothing different when they interact with

each other to form a molecular network or assembly. Thus, the

complete catalogue of chemical substances, together with the complete

catalogues of all molecules.

In this study at MPH sample analysis, the E max and E min

value falls between -26.80.07 and -112.04. The model 1 has 201

residues in the ligand and shows the Net formal charge as 2. But, the

model 2 has the E total value of -223.03 and E max is -197.88. The

binding affinity and energy charge of this ligand against cancer cell and

205

Ebola virus were very high. This study was witnessed that this

compound may have good anticancer and antiviral activity. In ESBL

enzyme docking the compounds Co(II) and compound Cu(II) were highly

interacting with ESBL and it indicated that the high-potent inhibitory

activity of cobalt compound was observed from the affinity and energy

charge of the ESBL enzyme. This results showed that these compounds

may act as a good antimicrobial and anticancer agent to large extent in

near future.

206

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CONCLUSION

The chapter I of the thesis describes the introduction about

Mannich reaction and its applications. The detailed literature

survey of the Mannich base has been discussed. The results of

the metal complexes of the Mannich bases reported in the

literature have also been discussed. The aim and objective of the

present work have been discussed in this chapter.

The second chapter of the thesis describes the synthesis of

MPH and its characterisation by analytical and spectral methods

have been discussed. From the results of the analytical and

spectral methods, the structure of the compound has been

established. Employing MPH as ligand, metal complexes have

been prepared and characterised through analytical and spectral

methods. By comparing the results of characteristic frequencies of

ligand and the metal complexes prepared from it, the ligating

atoms are identified. In MPH, the nitrogen atom of Morpholine

and oxygen atom of carbonyl are act as donors. Thus, the ligand

MPH acts as a neutral bidentate ligand. From the electronic

spectral data, the geometries of the complexes have been

established. The presence of other ligating groups are identified

through thermal studies.

Synthesis of MTN has been discussed and the compound

MTN has been characterised by analytical and spectral methods.

The results of analytical and spectral data helped us to establish

216

the structure of the molecule. MTN has been used as the ligand

and the metal complexes have been prepared. The prepared metal

complexes have been characterised through analytical and

spectral methods. The results of molar conductance values reveal

that the complexes behave as non-electrolyte. The IR spectrum of

the ligand has been compared with the IR spectra of metal

complexes that have been prepared from the ligand (MTN). This

result shows that the ligand coordinated to the metal through the

nitrogen atom of the Morpholine and oxygen atom of carbonyl.

From the electronic spectral studies, the structures of the

complexes have been established. Further thermal studies has

been carried out to confirm the coordinated moieties. These are

discussed in chapter III of the thesis.

Chapter IV of the thesis describes the synthesis of MFP and

its characterisation. The compound MFP served as a ligand for the

synthesis of metal complexes. The synthesised complexes have

been characterised by analytical and spectral methods. The

results of the analytical and spectral studies clearly indicates that

the MFP acts as a neutral bidentate ligand. The coordination to

the metal from the ligand is occurred through oxygen and

nitrogen atoms has been established by the IR spectral studies.

The geometry of the metal complexes has been established from

the electronic spectral data.

Chapter V describes the thermoacoustical studies of MPH

and MTN. The binary liquid mixtures have been prepared for MPH

217

and MTN using DMSO as a solvent. In fact, water is a best solvent

for the dissolution of amide moieties, DMSO is generally employed

as a solvent for the dissolution of compound containing amide

groups by synthetic chemists. This is because the removal of

water molecules from the synthesised products is very difficult.

Hence, in the present investigation, DMSO is used as a solvent to

prepare liquid mixtures. The measured values of ultrasonic

velocity, internal pressure, enthalpy and free energy have been

found to increase as the concentration increases. The calculated

values of adiabatic compressibility are found to decrease as the

concentration increases. These results are inferred that the strong

interaction is exist between the solvent (DMSO) and the solute

(MPH and MTN). Hence it is concluded that the association of

solute molecules occurs through hydrogen bonding.

Chapter VI describes the antimicrobial activity of the ligands

and the metal complexes. In this study, three different samples

[metal ions (B1, B2, B3 and B4), ligand (L1, L2 and L3) and

combined metal ion + ligand samples (B1+L1, B2+L1, B3+L1 AND

B4+LA)] were challenged against certain pathogenic

microorganisms for antimicrobial studies. The nil effect was

observed in metal ions and ligand (alone) samples whereas no nil

effect was noticed in the combination samples. In this present

study, the decreasing antimicrobial activity trends of metal ion

complex were: B2 > B3 > B4 > B1. But in ligand samples were: L1

> L2 > L3. The decreasing antimicrobial activity trends in

218

combined sample (metal ion + ligand) were: B2+L1 > B3+L1 >

B1+L1 > B4+L1. Among the ligand samples, ligand 1 gave a better

antimicrobial effect against most of the microorganisms.

Therefore, ligand 1 was combined with all the metal ions

separately. The antimicrobial activity of metal ion (alone) samples

were compared to the combination samples. Both the results were

not varied, except the position of B1 and B4 samples due to the

effect of combination with ligand. Most of the combination

samples showed good antimicrobial activity than the metal ion

and ligand samples alone. It indicated that the combination

samples could be used for the alternative drug.

All synthetic amide hydrazine derivatives were also screened

against A. Aegypti for their larvicidal activity. The compounds

were first screened in larval bioassays at concentrations of 1000,

500, and 250 ppm in a dose-dependent manner and percent

mortality was observed. Ligand 2 carrying methyl substituent on

phenyl ring showed the highest deterrent effect on larvicidal

activity

Molecular docking studies has been performed for the metal

complexes of Cu (II) and Co(II). The study reveals ligand 1 sample

analysis, the E max and E min value falls between -26.80.07 and

-112.04. The model 1 has 201 residues in the ligand and shows

the Net formal charge as 2. But, the model 2 has the E total value

of -223.03 and E max is -197.88. The binding affinity and energy

charge of this ligand against cancer cell and Ebola virus were very

219

high. This study was witnessed that this compound may have

good anticancer and antiviral activity. In ESBL enzyme docking

the compounds Co and compound CU (II) were highly interacting

with ESBL and it indicated that the high-potent inhibitory activity

of copper compound was observed from the affinity and energy

charge of the ESBL enzyme. This results showed that these

compounds may act as a good antimicrobial and anticancer agent

to large extent in near future.

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IJSR - INTERNATIONAL JOURNAL OF SCIENTIFIC RESEARCH 59

Volume : 3 | Issue : 7 | July 2014 • ISSN No 2277 - 8179Research Paper

Chemistry

M. Syed Ali Padusha Post Graduate and Research Department of Chemistry Jamal Mohamed College(Autonomous), Tiruchirappalli – 620 020, Tamil Nadu South India

S. Farook Basha Post Graduate and Research Department of Chemistry Jamal Mohamed College(Autonomous), Tiruchirappalli – 620 020, Tamil Nadu South India

ABSTRACT A new Mannich base, namely N-(Morpholino(thiophen-2-yl)methyl)nicotino hydrazide(MTN) was syn-thesized through Mannich condensation by reacting thiophene-2-carboxaldehyde, morpholine and benzo-

hydrazide as substrate. The structure of the formed compound was characterised by IR, 1HNMR and mass spectroscopy and CHN analyses. Using the above compound as ligand, metal complexes were prepared and their structures were established by elemental analyses, IR , UV-visible spectra, molar conductivity and magnetic moment studies. The results of these study indicate the square pla-nar geometry for all the complexes. Further, the ligand and the metal complexes were tested for antimicrobial activity. Antimicrobial studies revealed that metal complexes possess higher activity than those of the metal salts and ligands.

Synthesis, Characterisation and Antimicrobial Activity Study of (Morpholino (Thiophen-2-Yl)Methyl) Nicotino Hydrazide and its Metal(II)

Complexes

KEYWORDS : N-(morpholino (thiophen-2-yl)methyl)

nicotinohydrazide(MTN),Mixed-ligand complexes, Antimicrobial activities,

Cu(II), Ni(II) and Zn(II).

INTRODUCTIONMannich reaction is a three component condensation reaction consisting of an aldehyde, an amine and a compound contain-ing an active hydrogen atom. Many researchers have studied the numerous applications of Mannich reactions [1, 2]. In the development of coordination chemistry, the metal complexes of Mannich bases play a major role. Mannich bases are of interest in various areas of application [3-7]. Recently much interest has been paid on the synthesis and characterisation of transition metal complexes containing a Mannich base due to their wide pharmaceutical properties [8-12]. Many metal ions are known to play very important roles in biological processes in the hu-man body [13, 14]. Metal ions like zinc(II) and copper(II) ions are the most abundant transition metals in human body, found either at the active sites or as structural components of a num-ber of enzymes [15, 16]. These metals and some of their com-plexes have been found to exhibit antimicrobial activities [17-19]. Metal complexes depends on the metal ions and the ligand. In some metal complexes, the drug action has been noticed very high, when compared with the ligand[20, 21]. Hydrazone derivatives are found to possess antimicrobial, antitubercular and anti-inflammatory activities. Particularly, the antibacterial, antifungal and anticancer activities of hydrazones and their complexes with some transition metal ions were studied and reported by R.N.Patel et al.

Following all these observations and as a part of our research on the coordination chemistry of multidendate ligands, We report here, the synthesis, characterization and antimicrobial activities the new copper(II), nickel(II) and zinc(II) mixed-ligand complexes of N-(morpholino(thiophen-2-yl)methyl)nicotinohydrazide(MTN).

EXPERIMENTALMaterialsAll reagents were commercially available and used without further purification. Solvents were distilled using appropriate drying agents subsequently prior to use. The bacterial cultures such as Staphylococcus aureus, Bacilus subtilis, Escherchia coli, Pseudomonas aeruginosa, Aspergillus niger, Rhizoctonia batai-cola obtained from Eumic Analytical Laboratory and Research Institute, Tiruchirappalli.

Physical measurementsMelting point was determined using open capillary tube and are uncorrected. The purity of the compound was checked by thin layer chromatography on glass plates using silica gel G as absor-bent and solvent system. 1HNMR spectrum was recorded on a Bruker Ultra Shield(300 MHZ) spectrometer using DMSO as a solvent and TMS as internal standard. Molar conductivity was determined using systronic conductivity bridge with a dip type cell using 10-3 M solution of complexes in DMSO using Perkin Elmer spectrophotometer, UV-visible spectra of complexes were

recorded using 10-3 M solution of complexes in DMSO for the range 4000-400 cm-1.

Synthesis of N-(morpholino(thiophen-2-yl)methyl)nicotinohydrazide(MTN)Thiophene-2-carboxaldehyde, nicotinic acid hydrazide and morpholine were taken in 1:1:1 ratio and were reacted as shown in the scheme I. Nicotinic acid hydrazide (13.7 g, 0.1 mol) was taken in a round bottom flask and 5 ml of water was added. To this solution, morpholine(8.7 mL, 0.1 mol) was added and stirred well for 15 min, by keeping the reaction mixture on a magnetic stirrer in an ice cold condition. After 2 h, the solid formed was filtered and washed with ethanol. The crude solid thus obtained was dried and recrystallised using ethanol and dried over vacuum.

Scheme I

Synthesis of Metal(II) complexesTo the 10 mL methanolic solution of MTN(0.85g, 0.1 mol), each of metal Cu(II), Ni(II) and Zn(II) (0.1) chloride dissolved in a mixture of methanol and Chloroform 1:1(v/v) was added slow-ly. This mixture was kept on a magnetic stirrer and stirring was continued for an hour. The solid separated out was washed, fil-tered and dried over vacuum.

Figure 1 - Structure of Metal Complexes

M = Cu(II), Ni(II) and Zn(II)

NH2 NH

O N

pyridine-3-carbohydrazide

+NH

O

+S

Othiophene-2-carbaldehyde morpholine

OH2

SN

O

NH NH

O

N

N'-[morpholin-4-yl(thiophen-2-yl)methyl]pyridine-3-carbohydrazide

N

NH

N

N

O

S

O

N

HN

N

N

O

S

O

M

60 IJSR - INTERNATIONAL JOURNAL OF SCIENTIFIC RESEARCH

Volume : 3 | Issue : 7 | July 2014 • ISSN No 2277 - 8179 Research Paper

Antimicrobial testsInvitro antimicrobial activities of the ligand, complexes and free metal ions were evaluated by the disc diffusion method against the microorganisms such as Staphylococcus aureus, Bacilus subtilis, Escherchia coli, Pseudomonas aeruginosa, Aspergil-lus niger, Rhizoctonia bataicola. Ampicillin and Amphotericin B were used as standard for bacteria and fungi. The microbial isolates were maintained on agar slant at 4˚C. The strains were sub cultured on fresh appropriate agar plate in an incubator for 18 h prior to any microbial test.

The nutrient agar medium was prepared and sterilized by auto-claving at 121˚C, 15 lbs pressure for 15 min and then aseptically poured the medium into the sterile petri plates and allowed to so-lidify the bacterial broth culture and these are swabbed on each petri plates by sterile buds. Then wells were made by well cutter.

The Kirby Bauer Agar(KBA) medium was used for the diffusion assays determination and Nutrient broth was used as microbial

growth medium. This procedure was repeated for each petri plate, then the petri plates were incubated at 37˚C for about 24h. After incubation, the plates were observed for the zone of inhi-bition. The effect produced by the sample was compared with the effect produced by the positive control. Nutrient agar(NA) was used for the activation of Bacillus species, while NA alone was used for the other bacteria.

Result and discussionThe results of the elemental analyses present in the Table in-dicate the stoichiometry of the metal complex is 1:2(M:L), Cu(MTN)2Cl2.H2O whose complex ion is similar to Ni(MTN)2Cl2.H2O and Zn(MTN)2Cl2.H2O. The complexes are very stable in air whereas the starting metal salts are hygroscopic in nature. The melting point of MTN was found to be 198˚C. The complexes are different in colour from the starting metal salts from which they are derived. The colour of the complexes are presented in table 1. The low conductance of the chelates supports the non-electrolytic nature of the metal complexes.

Table 1. Physical characterization, analytical and molar conductance data of the ligand(MTN) and its metal(II) complexes

No Molecular Formula Colour Mol.

Wt.Melting point(˚C)

Yield%

Found%(Calcd %) Molar Conductance(Ω-1mol-1cm2)

C H N O S Cl

01MTNC15H18N4O2S White 318.4 198 70 55.68

(55.05)8.07 (8.12)

29.51 (29.41)

6.74 (6.53)

8.02 (8.14) -- --

02 Cu(MTN)2Cl2 Blue 645.1 226 74 54.53 (54.02)

7.63 (7.53)

31.79 (31.87)

6.05 (6.34)

7.88 (7.92)

16.42 (16.85) 29

03 Ni(MTN)2Cl2 Green 640.2 274 78 63.77 (63.04)

6.36 (6.44)

13.94 (13.23)

15.93 (15.88)

11.52 (11.50)

18.02 (18.68) 21

04 Zn(MTN)2Cl2Creamy White 646.9 256 80 66.88

(66.32)7.37 (7.21)

16.62 (16.41)

11.44 (11.12)

11.53 (11.55)

17.98 (18.34) 34

Table 2: IR spectral data(cm-1) of MTN and its metal(II) complexes

Compounds ν (NH) ν(NH) ν(C=N) ν(CNC) ν(CH) ν(M-N) ν(M-O)MTN 3421 1625 1670 1290 2926 -- --Cu(MTN)2Cl2 3398 1610 1654 1288 2925 480 505

Ni(MTN)2Cl2 3200 1585 1625 1290 2930 440 512

Zn(MTN)2Cl2 3275 1598 1625 1290 2925 470 530

Infra Red spectraThe infrared spectral data of the ligand and its complexes are given in Table 2. In order to study the binding mode of the li-gand in the metal complexes, the IR spectrum of the ligand was compared with those of the corresponding metal complexes. In the infrared spectra of the complex, the band due to NH at 3421 cm-1 in the spectrum of the ligand has been found shifted to 20-30 cm-1 in the spectrum of the complexes indicating the co-ordination of N atom of NH with metal ion. The participation of the nitrogen atom in coordination with the metal ion is further supported by the appearance of new band which is attributed to ν(M-N) [22, 23].

For the ligand, the bands due to νC-O and νC=N appeared in the regions 1647 and 1155 cm-1 respectively. In the spectra of the complexes, the νC=O of the free ligand is not observed in-dicating the enolisation of C=O followed by deprotonation and complexation with metal ions. The ν(C=N) mode of the ligand has been found shifted to higher frequency in the spectra of the complexes supporting the coordination of oxygen atom of the carbonyl in binding with metal ions.

1HNMR spectra1HNMR spectrum of the ligand showed a multiplet between δ 6.9 to 7.2 is assigned to aromatic protons. A triplet at δ 2.5 and δ 3.4 are attributed N-CH2 and O-CH2 of morpholine. A broad singlet appeared at δ 3.8 is assigned to NH proton adjacent to CH and a singlet at δ 6.2 is due to the methine proton adjacent to NH. These results indicate that there is no interaction between NH and CH

protons. This might be due to nuclear quadrapole effect.

This spectrum is compared with the 1HNMR spectrum of the Zn(II) Chloro complex of MTN. It has been observed that a peak appeared at δ 9.8 in the spectrum of the ligand was found absent in the spectrum of the complex, suggesting the participation of oxygen atom after deprotoration. This arises due to –NH proton nearer to C=O undergoes tautomerisation as shown below.

UV-Vis SpectraThe UV-visible spectrum of copper complex in DMSO solution displayed a broad band at 11232 cm-1 and an another band at 23735 cm-1 are attributed to 2B1g 2A1g and 2B1g 2B2g transitions. These transitions are favour to square planar ge-ometry around the central metal ion. Distortion from perfect planar symmetry is supported by the existence of broad band which is further supported by the magnetic moment value(1.85 BM).

The electronic spectrum of nickel complex exhibited a band at 24547 cm-1 is assigned to 1A1g 1B1g transition which cor-roborates the Square planar geometry. The possibility of tetra-hedral is ruled out from the absence of any band below 10,000 cm-1 for nickel complexes.

NH

CO

N COH

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Volume : 3 | Issue : 7 | July 2014 • ISSN No 2277 - 8179Research Paper

REFERENCE1. A.N.M. Kasim and G.V. Venkatesa Prabhu, Asian J. Chem. 2000, 12, 379. | 2. R.S. Varma, N. Rastogi and A.P. Singh, Indian J. Heterocyclic Chem., 2002, 12, 159. | 3. P.S. Desai and K.R. Desai, J. Indian Chem., Soc., 1993, 70, 177. | 4. A. Abdul Jameel and M. Syed Ali Padusha, Indian. J. Heterocyclic

Chem., 2006, 16, 197. | 5. R.C. Paul, P.A. Kapila, S. Bedi and K.K. Vasisht, J. Indian Chem., Soc., 1976, 53, 768. | 6. A.N.M. Kasim, D. Venkappaya and G.V. Venkatesa Prabhu, Asian J. Ind. Chem. Soci., 1999, 76, 67. | 7. N. Raman and S. Ravichandran, S. Polish, J. Chem, 2004, 78, 2005. | 8. Ali Mohammed Ashraf and Shaharyar Mohammad, Bioorg.Med.Chem. Lett., 2009, 17, 3317. | 9. Reddy M. Vijaya Bhasker, Chung-Rensu, Chiou WenFei, Nan-Li Yi, Chen Rosemary Yin-Hwa, Kenneth F.B., Lee Kuo-Hsiung, Wu Tian-Shung, Bioorg.Med.Chem., 2008, 16, 7358. | 10. B.Singh, R.N. Singh and R.C. Aggarwal, Polyhedron, 1985, 4, 401. | 11. A.P. Mishra and S.K. Srivastavan, J. Ind. Coun. Chem. 1994, 10, 2. | 12. N. Raman, S. Esthar and C. Thangaraja, J. Chem. Sci., 2004 , 116, 209. | 13. Kaim. W. Schwederski, B. Bioinorganic Chemistry: Inorganic Elements of Life, John Wiley and Sons: London; 1996; pp 39-262. | 14. Xiao-Ming, C. Bao-Hui, Y.Xiao, C.H. Zhi-Tao, X. J. Chem. Soc., Dalton Trans, 1996, 3465. | 15. Cotton. F.A. Wilkinson G. Advanced Inorganic Chemistry, 5th ed., John Wiley and Sons: New York; 1988; pp 1358-1371. | 16. Greenwood, N.N.; Earnshaw, A. Chemistry of the Elements, Pergamon Press: Oxford 1984; pp 1392-1420. | 17. Faundez, G.; Troncoso, M.; Navarette, P.; Figueroa, G. BMC Microbiol, 2004, 4, 1471. | 18. Khan, F.; Patoare, Y.; Karim, P.: Rayhan, I.; Quadir, M.A.: Hasna, A. Pak. J. Pharm. 2005, 18, 57. | 19. Baena, M.I.; Marquez, M.C.; Matres, V.; Botella, J.; Ventosa, A. Curr. Microbiol. 2006, 53, 491. | 20. A. Abdul Jameel and M. Syed Ali Padusha, Research Journal of Pharmaceutical and Chemical Sciences. ISSN : 0975-8585. | 21. Prachi Arya et al., J. Chem. Pharm. Res, 2010, 626-630. | 22. Shayma, E-Journal of Chemistry. 7(4), 2010, 1598-1604. | 23. Vidyavati reddy, Nirdosh patil, Tukaram reddy and S.D. Angadi, E-Journal of Chemistry, 5, 2008, 529-538. |

Antimicrobial StudiesThe results of the antimicrobial activity of the MTN and its com-plexes are presented in Table 3. From the table, it is observed that the ligand and the metal complexes are more active than the free ligand and their standards. The increase in antimicro-bial activity is due to faster diffusion of metal complexes as a whole through the combined activity of the metal and the ligand.

Table 3 Antibacterial Activities of Metal(II) complexes

Complex

Inhibition zone(mm)Staphylococcus aureus Escherichia Coli Pseudomonas

aeruginosaBacilus subtilis

MTN 11 12 12 10Cu(II) 15 16 14 15Ni(II) 14 16 15 13Zn(II) 19 22 18 20CuCl2 12 12 10 11NiCl2 10 11 11 10ZnCl2 13 13 12 12Ampicillin 11 11 11 10DMSO -- -- -- --Metal Salt 13 15 14 11

Table 4 Antifungal Activities of Metal(II) complexes

ComplexInhibition zone(mm)Aspergillus niger Rhizoctonia bataicola

MTN 12 14Cu(II) 15 16Ni(II) 16 16Zn(II) 21 23Nutrient Agar 10 11

ConclusionThe ligand, MTN and its metal complexes have been synthe-sized and characterized by elemental analysis, IR, 1HNMR, UV and magnetic measurements. The results of UV spectral stud-ies and magnetic susceptibility studies confirms square planar geometry of the metal complexes. Antimicrobial screening of ligand and the metal complexes showed their excellent activity. The zone of inhibition of metal complexes are comparably high than the free ligand. The therapeutic promise of the investigated metal(II) complexes were found to exhibit higher antimicrobial activity than the ligand.

AcknowledgementThe authors would like to express their thanks and gratitude to the Management Committee and Principal, Jamal Mohamed College and the Head, Department of Chemistry, for providing necessary facilities.

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Research Paper Commerce Chemistry

Synthesis, Characterisation and Antimicrobial Studies of 2-[Morpholin-4-yl(Pyridine-3-yl)Methyl]

Hydrazinecarboxamide and its Transition Metal Complexes

S. Farook Basha PG & Research Department of Chemistry Jamal Mohamed College (Autonomous), Tiruchirappalli Tamil Nadu – 620 020

M. Syed Ali Padusha

PG & Research Department of Chemistry Jamal Mohamed College (Autonomous), Tiruchirappalli Tamil Nadu – 620 020

CoII, NiII, CuII and MnII complexes of Mannich base, as ligand, was prepared by condensation of aqueous semicarbazide, morpholine and pyridine-3-carboxaldehyde. The structure of the newly synthesized Mannich base was investigated by UV-Vis, IR, 1H-NMR, 13C-NMR, molar conductance and magnetic susceptibility studies. The antimicrobial activities of the

ligand and metals complexes have been screened in vitro against the organisms E.faecalis, Proteus mirabilis, Bacillus cereus, E.aerogens, ESBL E.coli, ESBL K.pneumoniae, by disc diffusion and well diffusion techniques. It is observed that the coordination of metal ions has pronounced effect on the microbial activities of the ligand. The metal complexes have higher antimicrobial effect than the free ligand.

ABSTRACT

KEYWORDS : Mannich base, Metal complexes, Disc and Well diffusion technique, Antimicrobial effect.

IntroductionThe Mannich reaction is a powerful C-C bond formation process and has wide applications for the preparation of diverse amino alkyl de-rivatives. The Mannich reaction involves the condensation of a com-pound consisting of an active hydrogen atom with aldehyde and an amine (10 or 20). Literature survey shows that the compounds con-taining amide moiety have a strong ability to form metal complexes and show a wide range of biological activities. Metal ions are known to play very important roles in biological processes in the human body1,2. For example, copper(II) ion was the most abundant tran-sition metal in humans. It was found either at the active sites or as structural components of a good number of enzymes3,4.. Mannich bases5 of heterocyclic molecules have been attracting the attention of the synthetic chemists for their wide range of antimicrobial proper-ties6,7. Semicarbazides and thiosemicarbazides are found to be asso-ciated with antibacterial and antifungal activities8. The present study reports the synthesis and characterization of Mannich base, [(mor-pholin-4-yl) (pyridin-3-yl)methyl]hydrazinecarboxamide(MPH) and its metal Cobalt(II), Nickel(II), Copper(II) and Manganese(II) complexes , which contains an amide moiety. The antimicrobial activities of the ligand and metal complexes have been screened in vitro against the following microorganisms: E.faecalis, Proteus mirabilis, Bacillus cereus, E.aerogens, ESBL E.coli, ESBL K.pneumoniae by disc diffusion and well diffusion method9,10.

ExperimentalMaterialsAll the reagents used, were of A.R. grade and the solvents used were highly purified compounds. The solvents were distilled according to the standard methods.

Physical measurementsBy the use of elemental analyzer, the elements C, H and N were ana-lysed. By previous literature procedures, the metal and anion contents of the complexes were estimated. Melting points were taken in an open capillaries and were uncorrected. IR spectra were recorded on a Shimadzu 8201 PC FTIR spectrophotometer and 1H-NMR spectra on a Bruker DRX-300 spectrometer(300MHz) using DMSO-d6 as sol-vent and TMS as an internal standard. Purity of the compounds was checked by TLC on Silica gel plates and was satisfactory. The solvent system employed was chloroform and the spots were identified by placing the plate in UV chamber(λmax 254 nm). Molar conductivity of the complexes was measured on a Systronic conductivity bridge with a tip type cell, using 10-3 M solution of the complexes in DMSO at room temperature. Magnetic susceptibility measurements of the complexes were done using a Gouy balance. Copper Sulphate was used as the calibrant. Antibacterial screening of newly synthesized

compound was carried out against E.Faecalis, P.mirabilis, B.cereus and E.aerogens, ESBL E.coli and ESBL K. pneumonia. Muller-Hinton agar was used as the medium for the study of antimicrobial activity of the ligand and the complexes by employing well-diffusion and disc diffu-sion techniques. Rifampicin and Cefatoxime were used as standard for the antimicrobial studies.

SynthesisSynthesis of [(morpholin-4-yl)(pyridin-3-yl)methyl]hy-drazinecarboxamide(MPH):Semicarbazide(2.6 g, 0.025 mol) was dissolved in water. To this solu-tion, morpholine(2.2 mL, 0.025 mol) was added dropwise with con-stant stirring by keeping the reaction mixture on a magnetic stirrer. After 15 minutes, pyridine-3-carboxaldehyde(2.8 mL, 0.025 mol) was added in drops and the reaction mixture was kept in ice cold condi-tion in an water bath over a magnetic stirrer and stirring was contin-ued for an hour. The yellow coloured solid formed was filtered and then recrystallised from ethanol. The purity of the compound was checked by TLC using silica gel.

HN

O

Morpholine

N

OPyridine-3-carbaldehyde

H2NNH

O

NH2

Semicarbazide

+ +

NH

NH2

O

HN

N

N

O

[(morpholin-4-yl)(pyridin-3-yl)methyl]hydrazinecarboxamide (MPH)

Synthesis of Co(II), Ni(II), Cu(II) and Mn(II) complexes of MPHCobalt(II) chloro complex was prepared by mixing ethanolic solu-tion of cobalt(II) chloride with ligand dissolved in chloroform in 1:2 (metal:ligand) molecular ratio. The reaction mixture was stirred un-der ice bath maintained at 5-10°C for 2 h. The bluish green coloured precipitate obtained was filtered, washed with 1:1 ethanolic-acetone mixture and then dried in vacuo.

Nickel(II) chloro complex was prepared by mixing metal salt with MPH in 1:1 mol ratio. To the ligand in chloroform-ethanol(1:1), the metal salt in ethanol was added and stirred for 1 h, under ice cold condi-tion on a water bath. The green coloured solid obtained was filtered,

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washed with chloroform-ethanol mixture and dried in vacuo.

Copper(II) chloro complex was prepared by mixing the ligand and the metal salts in 1:1 mol ratio in ethanolic medium. The reaction mixture was stirred well in an ice bath using a magnetic stirrer. The brown coloured solid formed was filtered, washed with ethanol and dried in vacuo.

Manganese(II) chloro complex was obtained by adding ethanolic solution of MPH to the metal solution in 1:1 molecular ratio. The re-action mixture was stirred well and gets warm on a hot water bath. The resulting mild pink coloured solid was washed with ethanol and dried in vacuo.

N

H2N

O

NH

N

O

NH

N

M

Cl

Cl

N

NH2

O

HN

N

O

HN

N

M = Cu(II), Co(II), Ni(II), Mn(II)

Results and discussionCharacterisation of MPH:The analytical and physical data of the ligand and the metal com-plexes are listed in Table 1. The analytical data are in good agreement with the general molecular formula proposed for all the complexes. The molar conductivities of the complexes are very low indicating the non-electrolyte nature. The complexes are very stable at room tem-perature in air.

The solubility of MPH was tested. It is soluble in methanol, ethanol, DMSO, chloroform and benzene. Melting point was determined us-ing melting point apparatus and is about 202°C. The molecular mass of the ligand was determined by Rast method using biphenyl as the solvent.

Table 1: Physical characterization, Analytical, Molar Con-ductance Data

Infrared spectraThe IR spectrum of the free ligand was compared with those of the metal complexes. This is used to determine the coordination sites in-volved in the coordinates. The IR spectrum gives the details regarding the nature of the functional group attached to the metal ion. The IR spectrum of the compound showed bands in the region of 3407 cm-1 assigned to (O-H) and (N-H). The bands located in the regions of 2231 and 1924 cm-1 were attributed to the aromatic and aliphatic C-H stretching vibration. The absorption band in the region of 1669 cm-1 was assigned to (C=O). The split bands from 1426 to 1407 cm-1 were due to the mixed (N-H) and (C-N) vibration. The bands in the region of 1142 cm-1 was due to out of plane bending vibrations of aromatic C-H.

1H-NMR spectraThe proton NMR spectrum of MPH was recorded using 300 MHz NMR spectrometer(BRUKER) by using DMSO as solvent. The spectrum showed the multiple peaks in the regions of 6.5 and 9.0 ppm were due to aromatic protons. A single peak appeared at 2.6 ppm was as-signed to methyl proton. Splitting of signal appeared at 2.5 and 3.5 ppm was assigned to C-H and N-H protons.

Based on the above physical and spectral data, the structure of the synthesized compound was confirmed as [(morpholin-4-yl)(pyridine-3-yl)methyl]hydrazinecarboxamide.

The molar conductance of 10-3 solution of the complex measured. The molar conductance was showed to 32 ohm-1 cm mol-1, which indicates the non electrolytic behavior of the complex. That is the ani-ons are present inside the coordination sphere.

The magnetic susceptibility of the complexes of [(morpholin-4-yl(pyri-dine-3-yl)methyl] hydrazinecarboxamide was determined using Gouy’s balance. The magnetic susceptibility value was 3.52 B.M.

Antimicrobial TestsThe ligand and metal mixed-ligand complexes were tested for antimi-crobial activities against six pathogens. The antimicrobial activities of the ligand and complexes were evaluated by the well-diffusion and disc diffusion techniques at the concenteration of 10 mg/mL and 50 mg/mL. Muller-Hinton agar was used as microbial growth medium. Rifampicin and Cefatoxime were used as reference antibiotic. The plates were inoculated at 37º±2º C for 24 h. Antimicrobial activity was evaluated by measuring the diameter of the inhibition zone(IZ) around the hole. Compounds were considered as active when the IZ was greater than 15 mm. The values are presented in Table 2.

Table 2. Antimicrobial activities of the ligand and metal mixed-ligand complexes

IZ = Inhibition Zone, A1 = [(morpholin-4-yl)(pyridin-3-yl)me-thyl]hydrazinecarboxamide, B1 = Cu(MPH)2Cl2,B2 = Co(MPH)-2Cl2, B3 = Ni(MPH)2Cl2, B4 = Mn(MPH)2Cl2, RIF = Rifampicin, CTX = Cefatoxime, NI = No Inhibitory Effect

CONCLUSIONThis paper describes the summary of Mannich reaction, mechanism, important properties and also describes about the metal coordination and importance of coordination compounds. The literature survey states that the coordination occurs through oxygen and nitrogen. The IR spectrum of the complex shows a negative shift in absorption band frequencies of C=O and C-N of pyridine which are suggesting the car-bonyl oxygen and nitrogen of pyridine involved in the coordination. Experimental techniques employed in the synthesis and characteriza-tion of [(morpholin-4-yl)(pyridin-3-yl)methyl]hydrazinecarboxamide and its complexes were also discussed in detail. Based on the analyt-ical and spectral studies, the structure of the ligand [(morpholin-4-yl)(pyridin-3-yl)methyl]hydrazinecarboxamide and its complexes were established. The electrolytic conductivity data of the complex indi-cates its non-electrolytic nature. The magnetic susceptibility value indicates the magnetic property of the complexes. Antimicrobial studies of these complexes against six pathogens shows that there is increased activity of the metal ions upon coordination to these ligand. The activity order is Co(MPH)2Cl2(B2)>Cu(MPH)2Cl2(B1)> Ni(MPH)2Cl2(B3) > Mn(MPH)2Cl2(B4). The metal complexes has been found to possess more activity than the free ligand.

AcknowledgementThe authors are thankful to the Management Committee, Principal, and to the Head, PG & Research Department of Chemistry, Jamal Mo-hamed College (Autonomous), Tiruchirappalli, Tamilnadu.

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REFERENCES 1. Kaim. W. Schwederski, B. Bioinorganic Chemistry: Inorganic Elements of Life. John Wiley and Sons: London, 1996; pp 39-262. | 2. Xiao-Ming, C.: Bao-Hui, Y.: Xiao, C.H.: Zhi-Tao, X.J. Chem Soc, Dalton Trans 1996, 3465. | 3. Cotton. F.A.: Wilkinson G. Advanced Inorganic Chemistry, 5th ed. John Wiley and Sons, New York, 1988, pp 1358-1371. | 4. Greenword, N.N.: Earnshaw, A.: Chemistry of the Elements, Pergamon Press, Oxford. 1984, pp

1392-1420. | 5. F. Aydogan, Z. Turgut, N.Ocal, Turk. J. Chem, 2002, 26, 159. | 6. C.R. Katica, D. Vesna, K. Vlado, G.M. Dora, B. Aleksandra, Molecules, 2001,6, 815-824. | 7. R. Valarmathi, S. Akilandeswari, V.N.I. Latha, G. Umadevi, Der ChemicaSinica, 2011, 2(5), 64-68. | 8. R.M. Silverstein, G.C. Bassier, T.C. Morrill, Spectrometric Identification of organic compounds 4th Ed. John Wiley and Sons, 1981. | 9. NCCLS, Performance Standards for Antimicrobial Disk Suspectibility Tests. Approved Standard NCCLS Publication M2-A5, Villanova, PA, U.S.A., 1993. | 10. C. H. Collins, P.M. Lyre and J. M. Grange, Microbiological Methods. 6th Ed. Butterworths, London, 1989. |