Synthesis and Biological Activity of Biuret And

Home , Biuret

SYNTHESIS AND BIOLOGICAL ACTIVITY OF BIURET AND

DITHIOBIURET METAL COMPLEXES

SAIRA SHERZAMAN

Regd. No. 99-GMDG-3040

(Session 2007- 2010)

Department of Chemistry

Faculty of Sciences

University of Azad Jammu and Kashmir, Muzaffarabad, Pakistan SYNTHESIS AND BIOLOGICAL ACTIVITY OF BIURET AND

DITHIOBIURET METAL COMPLEXES

SAIRA SHERZAMAN

Regd. No. 99-GMDG-3040

A Thesis

Submitted in partial fulfillment of the requirement for the degree of

Doctor of Philosophy

Inorganic Chemistry

Department of Chemistry Faculty of Sciences The University of Azad Jammu and Kashmir, Muzaffarabad, Pakistan

Certificate of Approval

This is to certify that the research work presented in this thesis, entitled "Synthesis and Biological Activity of Biuret and Dithiobiuret Metal Complexes” was conducted by Saira Sherzaman under the supervision of Prof. Dr. Sadiq-ur-Rehman. No part of this thesis has been submitted anywhere else for any other degree. This thesis is submitted to the Department of Chemistry in partial fulfillment of the requirements for the degree of Doctor of Philosophy in field of Chemistry, Department of Chemistry, University of Azad Jammu and Kashmir, Muzaffarabad.

Student Name: Saira Sherzaman Signature: ______

Examination Committee: a) External Examiner: Prof. Dr. Rashid Ahmad Signature: ______Department of Chemistry University of Malakand, Pakistan.

b) Internal Examiner: Prof. Dr. Sadiq-ur-Rehman Signature: ______Department of Chemistry University of Azad Jammu & Kashmir, Muzaffarabad

Prof. Dr. Sadiq-ur-Rehman (Supervisor) Signature: ______

Prof. Dr. Khawaja Ansar Yasin (Chairman) Signature: ______

Prof. Dr. Muhammad Qayyum Khan (Dean) Signature: ______

Prof. Dr. Azhar Saleem (Director, ASR) Signature: ______

CERTIFICATION

Certified that the contents and form of thesis entitled “Synthesis and Biological Activity of Biuret and Dithiobiuret Metal Complexes” submitted by Saira Sherzaman have been satisfactory for the requirement of PhD in Chemistry.

SUPERVISORY COMMITTEE

Supervisor: Prof. Dr. Sadiq-ur-Rehman ______Department of Chemistry University of Azad Jammu & Kashmir, Muzaffarabad

Member 1: Prof. Dr. Khawaja Ansar Yasin ______Department of Chemistry University of Azad Jammu & Kashmir, Muzaffarabad.

Member 2: Dr. Muhammad Naeem Ahmed ______Assistant Professor Department of Chemistry University of Azad Jammu & Kashmir, Muzaffarabad.

External Examiner: Prof. Dr. Rashid Ahmad ______Department of Chemistry University of Malakand, Pakistan.

______Chairman Department of Chemistry

______Dean Director Faculty of Science Advanced Studies & Research

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DEDICATION

I would like to dedicate my thesis to my beloved husband Dr. Abdul Rehman for supporting me all the way

and my kids.

CONTENTS LISTS OF SCHEMES ...... xi LIST OF FIGURES ...... xii LIST OF TABLES ...... xiv ABBREVIATION...... xvi ACKNOWLEDGEMENT ...... xvi ABSTRACT ...... xx Chapter 1 ...... 1 INTRODUCTION ...... 1 1.1 BIURET, THIOBIURET AND DITHIOBIURET METAL COMPLEXES 1 1.2 BRIEF INTRODUCTION OF TRANSITION METALS ...... 4 1.3 REVIEW OF LITERATURE...... 6 1.3.1 Background Synthesis of Biurets, Thiobiurets and Dithiobiurets ...... 6 1.3.2 Synthesis of Thiobiuret ...... 9 1.3.3 Transition Metal Complexes of Thiobiuret ...... 12 1.3.4 Synthesis of Dithiobiurets ...... 13 1.3.5 Transition Metal Complexes of Dithiobiurets ...... 15 1.4 APPLICATIONS OF BIURET THIOBIURET AND DITHIOBIURET .... 16 METAL COMPLEXES ...... 16 1.4.1 Biological Applications…………………………………………………..16 1.4.2 Non-Biological Applications...... 21 1.4.2.1 Precursors for preparation of nanoparticles ...... 21 1.4.2.2 As a precursor of heterocyclic compounds ...... 22 1.4.2.3 Potential as extreme pressure lubricant additives ...... 23 1.4.2.4 Use as a corrosion inhibitor ...... 23 1.4.2.5 As floating aid ...... 24 1.4.2.6 As liquefying agents ...... 24 1.4.2.7 Use in textile industry ...... 24 1.4.3 Agricultural Applications ...... 24 1.4.3.1 Insect growth regulator ...... 25 1.4.3.2 Effect on seed germination and plant growth...... 25 1.4.3.3 Antifungal activity...... 26 1.4.3.4 Herbicidal activity ...... 26 1.4.4 Miscellaneous Uses ...... 27 1.5 STRUCTURAL ELUCIDATION TECHNIQUES ...... 28 1.5.1 Infrared Spectroscopy ...... 28 1.5.2 NMR Spectroscopy...... 29 1.5.3 X-ray Crystallography ...... 30 1.5.3.1 Crystal structure determination ...... 30 1.6 BIOLOGICAL STUDIES ...... 30 1.7 SCOPE OF STUDY ...... 31 1.8 PLAN OF WORK ...... 31

Chapter 2………………………………………………………………………… 33 2.1 CHEMICALS ...... 33 2.2 INSTRUMENTATION ...... 35 2.3 GENERAL PROCEDURE (A) FOR SYNTHESIS OF BIURET, THIOBIURET AND DITHIOBIURET METAL COMPLEXES ...... 36 2.4 SYNTHESIS OF THIOBIURET AND DITHIOBIURET NICKEL(II) COMPLEXES USING DIFFERENT AMINES ...... 38 2.4.1 Bis[1,1,5,5-tetra ethyl-4-thiobiureto]Nickel(II) 1 ...... 38 2.4.2 Bis[1,1-diethyl-5-( benzylmethyl)-4-thiobiureto]Nickel(II) 2 ...... 38 2.4.3 Bis[1,1-diethyl-5-(3-hydroxypyridine)-4-thiobiureto]Nickel(II) 3 ...... 39 2.4.4 Bis[1,1-diethyl-5-(1-(2-furoyl)-pipraziyl)-4-thiobiureto]Nickel(II) 4 .. 40 2.4.5 Bis[1,1-diethyl-5-(4-nitrophenyl)-4-thiobiureto]Nickel(II) 5 ...... 40 2.4.6 Bis[1,1-diethyl-5-(aminobenzyl)-4-thiobiureto]Nickel(II) 6 ...... 41 2.4.7 Bis[1,1- diethyl-5-(2-hydroxy benzoamide)-4-thiobiureto]Nickel(II) 7 41 2.4.8 Bis[1,1-diethyl-5-(methyl benzyl)- 4-thiobiureto]Nickel(II) 8 ...... 42 2.4.9 Bis[1,1-diethyl-5-(dodecylamine)-4-thiobiureto]Nickel(II) 9 ...... 42 2.4.10 Bis[1,1-diethyl-5-(2,4-dinitrophenylhydrazine)-4-thiobiureto]Nickel(II) 10 ...... 43 2.4.11 Bis[1,1-diethyl-5,5-dipropyl-4-thiobiureto]Nickel(II) 11 ...... 43 2.4.12 Bis[1,1,5,5-tetramethyl-2,4-dithiobiureto]Nickel(II) 12 ...... 44 2.5 SYNTHESIS OF BIURET, THIOBIURET AND DITHIOBIURET COPPER(II) COMPLEXES USING DIFFERENT AMINES...... 44 2.5.1 Bis[1,1,5,5-tetra ethyl-4-thiobiureto]Copper(II) 13 ...... 45 2.5.2 Bis[1,1-diethyl-5-( benzylmethyl)-4-thiobiureto]Copper(II) 14 ...... 45 2.5.3 Bis[1,1-diethyl-5-(3-hydroxypyridine)-4-thiobiureto)]Copper(II) 15 .... 46 2.5.4 Bis[1,1-diethyl-5-(1-(2-furoyl)-pipraziyl)-4-thiobiureto]Copper(II) 16 46 2.5.5 Bis[1,1-diethyl-5-(4-nitrophenyl)-4-thiobiureto]Copper(II) 17 ...... 47 2.5.6 Bis[1,1-diethyl-5-(aminobenzyl)-4-thiobiureto]Copper(II) 18 ...... 48 2.5.7 Bis[1,1- diethyl-5-(2-hydroxy benzoamide)-4-thiobiureto]Copper(II) 19 ...... 48 2.5.8 Bis[1,1-diethyl-5-(methyl benzyl)- 4-thiobiureto]Copper(II) 20 ...... 49 2.5.9 Bis[1,1-diethyl-5-(dodecylamine)-4-thiobiureto]Copper(II) 21 ...... 49 2.5.10 Bis[1,1-diethyl-5-(2,4-dinitrophenylhydrazine)-4-thiobiureto]Copper(II) 22...... 50 2.5.11 Bis[1,1-diethyl-5,5-dipropyl-4-thiobiureto]Copper(II) 23 ...... 50 2.5.12 Bis[1,1,5,5-tetramethyl-2,4-dithiobiureto]Copper(II) 24 ...... 51 2.6 SYNTHESIS OF BIURET, THIOBIURET AND DITHIOBIURET COBALT(II) COMPLEXES USING DIFFERENT AMINES ...... 51 2.6.1 Tris[1,1,5,5-tetra ethyl-4-thiobiureto]Cobalt(II) 25 ...... 52 2.6.2 Tris[1,1-diethyl-5-(benzylmethyl)-4-thiobiureto]Cobalt(II) 26 ...... 52 2.6.3 Tris[1,1-diethyl-5-(3-hydroxypyridine)-4-thiobiureto)]Cobalt(II) 27 .... 53

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2.6.4 Tris[1,1-diethyl-5-(1-(2-furoyl)-pipraziyl)-4-thiobiureto]Cobalt (II) 28 ...... 54 2.6.5 Tris[1,1-diethyl-5-(4-nitrophenyl)-4-thiobiureto]Cobalt(II) 29 ...... 54 2.6.6 Tris[1,1-diethyl-5-(aminobenzyl)-4-thiobiureto]Cobalt(II) 30 ...... 55 2.6.7 Tris[1,1-diethyl-5-(2-hydroxy benzoamide)-4-thiobiureto]Cobalt(II) 31 ...... 55 2.6.8 Tris[1,1-diethyl-5-(methyl benzyl)-4-thiobiureto]Cobalt(II) 32 ...... 56 2.6.9 Tris[1,1-diethyl-5-(dodecylamine)-4-thiobiureto]Cobalt(II) 33 ...... 56 2.6.10Tris[1,1-diethyl-5-(2,4-dinitrophenylhydrazine)-4-thiobiureto]Cobalt(II) 34 ...... 57 2.6.11Tris[1,1-diethyl-5,5-dipropyl-4-thiobiureto]Copper(II) 35 ...... 57 2.7 SYNTHESIS OF COMPLEXES OF BIURET, THIOBIURET AND DITHIOBIURET CADMIUM(II) COMPLEXES USING DIFFERENT AMINES...... 58 2.7.1 Bis[1,1,5,5-tetramethyl-2,4-dithiobiureto]Cadmium(II) 36 ...... 59 2.8 SYNTHESIS OF BIURET, THIOBIURET AND DITHIOBIURET ZINC(II) COMPLEXES USING DIFFERENT AMINES ...... 59 2.8.1 Bis[1,1,5,5-tetra ethyl-4--thiobiureto] Zinc(II) 37 ...... 60 2.8.2 Bis[1,1-diethyl-5-(benzylmethyl)-4-thiobiureto]Zinc(II) 38 ...... 60 2.8.3 Bis[1,1-diethyl-5-(3-hydroxypyridine)-4-thiobiureto)]Zinc(II) 39 ...... 61 2.8.4 Bis[1,1-diethyl-5-(1-(2-furoyl)-pipraziyl)-4-thiobiureto]Zinc(II) 40 ... 61 2.8.5 Bis[1,1-diethyl-5-(4-nitrophenyl)-4-thiobiureto]Zinc(II) 41 ...... 62 2.8.6 Bis[1,1-diethyl-5-(aminobenzyl)-4-thiobiureto] Zinc(II) 42 ...... 62 2.8.7 Bis[1,1- diethyl-5-(2-hydroxy benzoamide)-4-thiobiureto]Zinc(II) 43 63 2.8.8 Bis [1,1-diethyl-5-(methyl benzyl)-4-thiobiureto]Zinc(II) 44 ...... 63 2.8.9 Bis[1,1-diethyl-5-(dodecylamine)-4-thiobiureto]Zinc(II) 45 ...... 64 2.8.10 Bis[1,1-diethyl-5-(2,4-dinitrophenylhydrazine)-4-thiobiureto]Zinc(II) 646 ...... 64 2.8.11 Bis[1,1-diethyl-5,5-dipropyl-4-thiobiureto]Zinc(II) 47 ...... 65 2.9 PROCEDURE (B) ADAPTED FOR SYNTHESIS OF BIURET, THIOBIURET AND DITHIOBIURET METAL COMPLEXES ...... 65 2.10 SYNTHESIS OF THIOBIURET METAL COMPLEXES USING DIFFERENT AMINES ...... 67 2.10.1 Bis[(Z)-3-(3,3-dimethylbutanoyl)-1,1-diethyl-2-thiobiureto]Nickel(II) 48 ...... 67 2.10.2 Bis[(Z)-3-(3,3-dimethylbutanyol)-1-(2-hydroxyphenyl)-2-thiobiurato] Nickel(II) 49 ...... 68 2.10.3 Bis[(Z)-3-(3-dimethylbutanoyl)-1-(3-morpholinopropyl)-2-thiobiureto] Nickel(II) 50 ...... 68 2.10.4 Bis[(Z)-3-(3,3-dimethylbutanoyl)-1,1-diethyl-2-thiobiureto]Copper(II) 51 ...... 69

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2.10.5 Bis[(Z)-3-(3,3-dimethylbutanoyl)-1-(2-hydroxyphenyl)-2-Copper(II) 52 ...... 70 2.10.6 Bis[(Z)-3-(3,3-dimethylbutanoyl)-1-(3-morpholinopropyl)-2- thiobiureto]Copper(II) 53 ...... 70 2.10.7 Tris[(Z)-3-(3,3-dimethylbutanoyl)-1,1-diethyl-2-thiobiureto]Cobalt(II) 54 ...... 71 2.10.8 Tris[(Z)-3-(3,3-dimethylbutanoyl)-1-(2-hydroxyphenyl)-2-thiobiureto] Cobalt(II) 55 ...... 72 2.10.9 Tris[(Z)-3-(3,3-dimethylbutanoyl)-1-(3-morpholinopropyl)-2- thiobiureto]Cobalt(II) 56 ...... 72 2.10.10 Bis [(Z)-3-(3,3-dimethylbutanoyl)-1,1-diethyl-2-thiobiureto]Zinc(II) 57 ...... 73 2.10.11 Bis[(Z)-3-(3,3-dimethylbutanoyl)-1-(2-hydroxyphenyl)-2-thiobiureto] Zinc(II) 58 ...... 74 2.10.12 Bis[(Z)-3-(3,3-dimethylbutanoyl)-1,1-diethyl-2-thiobiureto] Cadmium(II) 59 ...... 74 2.10.13 Bis[(Z)-3-(3,3-dimethylbutanoyl)-1-(2-hydroxyphenyl)-2-thiobiureto] Cadmium(II) 60 ...... 75 2.11 BIOLOGICAL ACTIVITIES ...... 75 2.11.1 Antimicrobial Assay ...... 76 2.11.1.1 Assay procedure ...... 77 2.11.2 Antioxidant Assay ...... 79 2.11.2.1 DPPH free radical scavenging assay...... 79 2.11.2.2 Assay procedure ...... 80 2.11.3 Total Antioxidant Capacity ...... 80 2.11.3.1 Assay procedure ...... 80 2.11.4 Reducing Power Assay ...... 81 2.11.4.1 Assay procedure ...... 81 2.11.5 Protein Kinase Inhibition Assay ...... 83 2.11.5.1 Assay procedure ...... 83 2.11.6 ABTS Radical Cation Decolourization Bioassay ...... 85 2.11.7 Anticancer Assay ...... 87 2.11.7.1 Assay Procedure ...... 88 2.12 ELECTROLYTIC BEHAVIOUR...... 91 chapter3………………………………………………………………………….. 92 RESULTS AND DISCUSSIONS ...... 92 3.1 SYNTHESIS OF THIOBIURET AND DITHIOBIURET NICKEL(II) COMPLEXES (1-12) ...... 93 3.1.1 UV-Visible Analysis of Thiobiuret and Dithiobiuret Nickel(II) Complexes (1- 12) ...... 95 3.1.2 Infrared Spectroscopic Characterizationof Thiobiuret and Dithiobiuret Nickel(II) Complexes (1-12) ...... 97

3.2 SYNTHESIS OF THIOBIURET AND DITHIOBIURET COPPER(II) COMPLEXES (13-24) ...... 99 3.2.1 UV-Visible Analysis of Thiobiuret and Dithiobiuret Copper(II) Complexes (13-24) ...... 100 3.2.2 Infrared Spectroscopic Characterization of Thiobiuret and Dithiobiuret Copper(II) Complexes ...... 101 3.3 SYNTHESIS OF THIOBIURET AND DITHIOBIURET COBALT(II) COMPLEXES (25-35) AND CADMIUM(II) COMPLEX (36) ...... 102 3.3.1 UV-Visible Analysis of Thiobiuret and Dithiobiuret Cobalt(II) and Cadmium(II) Complexes ...... 104 3.3.2 Infrared Spectral Characterization of Thiobiuret and Dithiobiuret Cobalt(II) and Cadmium(II) Complexes ...... 105 3.4 SYNTHESIS AND CHARACTERIZATION OF THIOBIURET AND DITHIOBIURET ZINC(II) COMPLEXES (37-47) ...... 106 3.4.1 UV-Visible Analysis of Zinc(II) Complexes (37-47) ...... 108 3.4.2 Infrared Spectroscopic Characterization of Thiobiuret and Dithiobiuret Zinc(II) Complexes (37-47) ...... 109 3.5 SYNTHESIS OF BIURET AND DITHIOBIURET METAL COMPLEXES USING NEW METHOD B ...... 110 3.5.1 UV-Visible Analysis of Thiobiuret and Dithiobiuret Metal Complexes (48-60) ...... 111 3.5.2 Infrared Spectroscopic Characterization of Thiobiuret and Dithiobiuret Metal Complexes (48-60) ...... 113 3.6 NMR SPECTROSCOPIC STUDIES ...... 115 3.6.1 1H NMR Spectroscopy ...... 115 3.6.2 13C NMR Spectroscopy ...... 119 3.7 CONDUCTANCE ...... 120 3.8 X-RAY CRYSTALLOGRAPHY ...... 121 3.9 BIOLOGICAL ACTIVITIES ...... 132 3.9.1 Antibacterial Activity ...... 132 3.9.2 Antifungal Activity ...... 133 3.9.3 Total Antioxidant Capacity ...... 134 3.9.4 Reducing Power Assay ...... 134 3.9.5 DPPH Free Radical Scavenging Assay ...... 135 3.9.6 Protein Kinase Inhibition Assay ...... 135 3.9.7 ABTS Radical+ Decolourization Bioassay ...... 136 3.9.8 Anticancer Activity ...... 137 SUMMARY ...... 138 LITERATURE CITED ...... 141 APPENDICES ...... 156 LIST OF PUBLICATIONS ...... 162

LISTS OF SCHEMES Scheme 1.1: Synthesis of biuret by thermal decomposition of urea nitrate. 6

Scheme 1.2: Synthesis of dithiobiuret by reaction of dicynamides and hydrogen

sulphide. 7

Scheme 1.3: Synthesis of thiobiuret by reaction of N-cyanoureas with hydrogen

sulphide. 7

Scheme 1.4: Synthesis of thiobiuret ligand by cyclohexanalcarbonyl with KSCN

and amine. 10

Scheme 1.5: Chemical reaction of formation of N-(6-methylpyridin-2-yl

carbamothioyl)biphenyl-4-carboxamide. 11

Scheme 1.6: Schematic view of synthesis of disubstituted thiourea. 11

Scheme 1.7: Preparation of ligand N-(biphenyl-2-thiocarbomoyl)-4-

phenylcarboxamide. 12

Scheme 1.8: Synthesis of complex by reaction of ligands with metallic salts. 13

Scheme 1.9: Preparation of disubstituted thiourea copper (I) complex. 13

Scheme 1.10: Synthesis of phenylated dithiobiuret by reaction of

isoperthiocyanic acids with aniline. 14

Scheme 1.11: Formation of dithiobiuret by Hetch and Wunderlich’s cyanourea

synthesis. 14

Scheme 1.12: Synthesis of trisubsituted dithiobiuret. 15

Scheme 1.13: Synthesis of heterocyclic precursor. 23

Scheme 2.1: General scheme for synthesis of biuret, thiobiuret and dithiobiuret

metal complexes by method A. 37

Scheme 2.2: General scheme for synthesis of thiobiuret metal complexes by method B. 66

LIST OF FIGURES

Figure 1.1: General structures of thiobiuret and dithiobiuret metal 01

complexes.

Figure 1.2: Some biological active thiobiuret and dithiobiuret compounds. 04

Figure 1.3: 1-Aroyl-3-aryl thioureas have potential antibectarial substance. 17

Figure 1.4: 1-(5-Bromopyridin-2-yl)-3-(2-(pyridin-2-yl)ethyl)thiourea 17

active against HIV.

Figure 1.5: (S)-(N)-(3-chlorophenyl)-3-methyl-4-(3-methylbut-2-enyl)-1,4- 17

diazepane-1-carbothioamide against HIV.

Figure 1.6: 1,3-bis(4-(Isopentyloxy)phenyl)thiourea used for TB treatment. 18

Figure 1.7: 1-(Naphthalen-4-yl)thiourea and 1-phenylthiourea used as 18

rodenticide.

Figure 1.8: 1- (2,4,6-Trichlorophenyl)thiourea potential as antithyroid 19

drugs.

Figure 1.9: 1- (2,6-Dimethylphenyl) thiourea show hypertensive activity. 19

Figure 1.10: Anticancer active drugs used for cancer treatment. 20

Figure 1.11: 2-(tert-butylimino)-3-isopropyl-5-phenyl-1,3,5- thiadiazinan-4- 25

one control the growth of insects.

Figure 1.12: N-benzyol-N-(3-methylpyrid-2-yl)thiourea used as Plant growth 26

regulator.

Figure 1.13: Thiourea derivatives showing antifungal activity. 26

Figure 1.14: 1-phenyl-3-(thiazol-2-yl)thiourea herbicidal active compound. 27

Figure 3.1: ORTEP structure of complex 12 bis(1,1,5,5-tetramethyl-2,4- 121

dithiobiureto)nickel(II).

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Figure 3.2: ORTEP structure of 36 bis(1,1,5,5-tetramethyl-2,4- 125

dithiobiureto) cadmium(II).

Figure 3.3: ORTEP structure of 24 bis(1,1,5,5-tetramethyl-2- 127

thiobiureto) copper(II).

Figure 3.4: ORTEP structure of 48 bis[Z-3-(3,3-dimethylbutanoyl)-1,1- 128

diethyl-2-thiobiureto]nickel(II) complex.

Figure 3.5: Packing pattern of complex 48 showing van der waal 128

attractions.

Figure 3.6: ORTEP structure of 54 tris [Z-3-(3,3-dimethylbutanoyl)-1,1- 129

diethyl-2-thiobiureto]cobalt(II)

Figure 3.7: Packing pattern of complex 54 showing van der waal attraction. 130

Figure 3.8: Antibacterial activity of tb and dtb metal complexes. 133

Figure 3.9: Antifungal activity of thiobiuret and dithiobiuret metal 134

complexes.

Figure 3.10: Total antioxidant capacity, reducing capacity and DPPH % 135

scavenging of thiobiuret and dithiobiuret metal complexes.

Figure 3.11: Protein Kinase 1nhibition activity of biuret, thiobiuret and 136

dithiobiuret metal complexes.

Figure 3.12: ABTS antioxidant potential of thiobiuret and dithiobiuret metal 137

complexes.

Figure 3.13: Anticancer activity of tb and dtb metal complexes 138

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LIST OF TABLES

Table 2.1: List of chemicals acquired for synthesis of thiobiuret and 33

dithiobiuret metal complexes.

Table 2.2: Instruments used in the preparation and characterization of 36

thiobiuret and dithiobiuret metal complexes.

Table 2.3: Antibacterial activity of tb and dtb metal complexes. 78

Table 2.4: Antifungal activity of biuret and dithiobiuret metal 79

complexes.

Table 2.5: Antioxidant activities of tb and dtb metal complexes. 82

Table 2.6: Protein Kinase inhibition activity of thiobiurets and 84

dithiobiuret metal complexes.

Table 2.7: ABTS antioxidant activities of thiobiuret and dithiobiuret 86

metal complexes.

Table 2.8: Anticancer activity of thiobiuret and dithiobiuret metal 90

complexes.

Table 3.1: Physical properties of synthesized thiobiuret and 94

dithiobiuret nickel(II) complexes.

Table 3.2: UV-visible absorption data of nickel(II) complexes 1-12. 96 96

Table 3.3: FTIR data of complexes of nickel(II) 1-12. 98

Table 3.4: Physical properties of tb and dtb copper(II) complexes. 99

Table 3.5: UV-visible absorption data of copper(II) complexes. 13-24. 100

Table 3.6: FTIR data of complexes of copper(II)13-24. 102

Table 3.7: Physical properties of synthesized tb and dtb cobalt(II) 103

complexes.

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Table 3.8: UV- visible absorption data of cobalt(II) complexes 25-35. 104

Table 3.9: FTIR data of cobalt(II) and cadmium(II) complexes 25-36. 106

Table 3.10: Physical properties of synthesized thiobiuret and 107

dithiobiuret zinc(II) complexes.

Table 3.11: UV-Visible absorption data of zinc(II) complexes 37-47. 108

Table 3.12: FTIR data of zinc(II) complexes 37- 47. 109

Table 3.13: Physical properties of tb and dtb of metal complexes. 111

Table 3.14: UV-visible absorption data of complexes 48-60. 112

Table 3.15: FTIR data of complexes 48-60. 114

Table 3.16: 1H NMR data of representative complexes. 116

Table 3.17: 13C NMR data of some representative metal complexes of 120

thiobiuret and dithiobiuret.

Table 3.18: X-ray diffraction data and structure refinement parameterss 122

for complexes 12, 24, 36, 48, 54.

Table 3.19: Selected bond lengths [Å] and angles [°] for complex 12. 124

Table 3.20: Bond lengths [Å] and angles [°] for complex 36. 126

Table 3.21: Bond lengths [Å] and angles [°] for complex 24. 127

Table 3.22: Bond lengths [Å] and angles [°] for complex for complex 48 129

Table 3.23: Bond lengths [Å] and angles [°] for complex for complex 54 130

ABBREVIATION

ABTS 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) A. flavus Aspergilus flavus AIDS Acquired immune Deficiency Syndrome A.niger Aspergilus niger APDTB 1-anisidyl-5-phenyl-2,4-dithiobiuret B. subtilis Bacillus subtilis CPDTB 1-chorophenyl-5-diphenyl-2,4-dithiobiuret d Doublet dd Double doublet DMSO Dimethylsulfoxide DNA Deoxyribonucleic Acid DPDTB 1,5-diphenyl-2,4-dithiobiuret DPPH 2,2-diphenyl-1-picrylhydrazyl Dtb Dithiobiuret E. coli Escherichia coli FTIR Fourier transform infrared HDL High density lipoprotein HIV Human Immune Deficiency Virus

IC50 Half Maximal Inhibitory concentration IEs Inhibition efficiencies IGRs Insect Growth Regulators J Coupling constant m Multiplet MeCN Acetonitrile mg Milligram Ml Milli liter Mm Milli meter Mm Milli molar m.p Melting point NA Nutrient agar

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NMR Nuclear magnetic resonance OD Optical density PBS Phosphate buffered saline PMA Phorbol myristate acetate ppm Parts per million q Quartet R Resistance s Singlet S. aureus Staphylococcus aureus t Triplet tb Thiobiuret TB Tuberculosis THF Tetrahydrofuran TIBO Tetrahydroimidazobenzodiazepinthiones TPDTB 1-tolyl-5-phenyl-2,4-dithiobiuret UV Ultraviolet XRD X-ray Diffraction µg Microgram

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ACKNOWLEDGEMENT

All praise and thanks are due to the ALMIGHTY ALLAH WHO always guides me to the right path and HAS helped me to complete this thesis. I offer my humblest and sincere thanks to the HOLY PROPHET HAZRAT MUHAMMAD

(Peace be upon Him) WHO exhorts his followers to seek knowledge from cradle to grave.

First and foremost I would like to record my gratitude to my supervisor affectionate Prof. Dr. Sadiq-ur-Rehman Chairman Department of Chemistry, The

University of Azad Jammu and Kashmir Muzaffarabad for his supervision, advice, patience and professional guidance from the early stage of this research, as well as giving me extraordinary experiences throughout the work. Above all and the most needed, he provided me unflinching encouragement and support in various ways. I am really indebted to him more than he knows.

I am thankful to Dr. M. Naeem Ahmad Assistant Professor Department of

Chemistry the University of Azad Jammu and Kashmir Muzaffarabad for his generous cooperation for providing me links for different bioassays and helping me in XRD analysis.

My special thanks goes to Dr. Muhammad Nawaz Tahir Department of

Physics, University of Sargodha for his help in studies of XRD. It is injustice if I don’t mention Dr. Bilal Ahmed Khan Assistant Professor Department of

Chemistry the University of Azad Jammu and Kashmir Muzaffarabad for his immense help and assistance in NMR analysis. Thank you very much.

My deepest gratitude is reserved for my torch bearer brother Prof. Dr. M.

Siddique Awan for his guidance, valuable suggestion, encouragement and support.

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I would like to pay tribute to my mother for her unconditional love, prayers and encouragement. I am also thankful to Dr. Saiqa Andaleeb Department of

Zoology and Dr. Abdul Rehman Assistant Professor of Biochemistry for their help and guidance. I am also thankful to all the faculty members and technical staff of Department of Chemistry, University of Azad Jammu and Kashmir. I am highly indebted to the Department of Higher Education/Colleges for granting me study leave. My special thanks goes to my junior M. Phil Scholar Rehana Khadim for her help in composing thesis.

At the end my special, profound and affectionate thanks goes to my loving husband Dr. Abdul Rehman who has been struggling with me, hand by hand to secure and shape brighter future. I am indebted for his suffering and sacrifices during my studies. I am unable to express my feelings for my kids Muazzama,

Samiullah, Tayyaba and infant Ayesha. They are real source of inspiration and happiness for me.

SAIRA SHERZAMAN

xix

ABSTRACT

A variety of bis and tris thiobiureto and dithiobiureto Co(II), Ni(II), Cd(II),

Cu(II), Zn(II) metal complexes were synthesized by reacting N,N-dialkylcarbamoyl or N,N-dialkylthiocarbamoyl with potassium thiocyanide and twelve different commercially available amines. These complexes were characterized by spectroanalytical (UV-Vis, FT-IR, 1H-NMR and 13C-NMR) and single crystal X- ray diffraction techniques. To find percentage composition of atoms i.e. C, H and N present in synthesized complexes were also determined by CHN analysis. The obtained data from UV-Visible, FT-IR, 1H NMR, 13C NMR and elemental analysis were compared with the reported values which satisfactorily justified the synthesis of thiobiuret and dithiobiuret metal complexes. The structure of five complexes such as bis(1,1,5,5-tetramethyl-2,4-dithiobiureto)nickel(II) 12, bis(1,1,5,5- tetramethyl-2-thiobiureto)copper (II) 24, bis (1,1,5,5-tetramethyl-2,4-dithiobiureto) cadmium (II) 36, bis[(Z)-3-(3,3-dimethylbutanyol)-1,1-diethyl-2-thiobiureto]nickel

(II) 48 and tris[(Z)-3-(3,3-dimethylbutanyol)-1,1-diethyl-2-thiobiureto] cobalt(II)

54 was unambiguously confirmed with single crystal X-ray analysis, establishing that the geometry of Ni(II), Cd(II) complexes (12, 36) is tetrahedral, square planar for Cu(II) and Ni(II) (24, 48) and octahedral geometry for (54) Co(II) complexes.

Synthesized complexes were considered to evaluate their conductance and results indicate that they possess differential levels of electrolytic characteristics, which indicates that complexes had positive electrolytic behavior, whereas few complexes showed suppressed electrolytic properties.

Regarding the assessment of antibacterial, antifungal, protein kinase inhibition, total antioxidant capacity, reducing power, DPPH radical scavenging,

ABTS antioxidant potential and anticancer assay; fourteen synthesized complexes were used for antibacterial and antifungal activity, thirty two complexes were considered for rest of the abovementioned assays.

Out of fourteen complexes 8, 12, 13, 14, 27, 32, 38 had significant activities against three bacterial strains i.e. Echerishia coli, Staphylococcus aureus and

Pseudomonas aeruginosa due to NCS containing moieties and 2, 3, 4, 12, 14, 27,

38, 47 possess differential levels of inhibition against Aspergilus niger, Flavus solani, Mucor species and Aspergilus flavus microbes.

Out of thirty two said complexes on the other hand, 3, 5, 7, 10, 13, 15, 17,

19, 28, 46 and 1-8, 10, 12, 13, 14, 16-19, 22, 28-31, 43, 46 complexes were found as potential agents for protein kinase inhibition and antioxidant activity, respectively. Furthermore, all said synthesized transition metal complexes possessed significant inhibition activity against the cancer cell line.

In all, biological findings obtained through the present study indicate that almost all synthesized complexes are promising candidates to combat cancer effect by suppressing oxidative stresses, and as well as anti-microbial activity to control infections. However, further comprehensive animal based studies are warranted to confirm our results to be translated them as drugs/medicines in future.

xxi

Chapter 1

INTRODUCTION

1.1 BIURET, THIOBIURET AND DITHIOBIURET METAL COMPLEXES

Biurets and their derivatives such as thiobiurets, dithiobiurets are already established and well characterized stable and neutral coordination compounds of wide variety of transition metal ions. The urea and its derived thiourea compounds have become the attention of researchers due to their vital use for the isolation and separation of metallic ions. Due to three active coordination sites, which are containing sulfur, nitrogen and oxygen atoms of CS, amino (NH) and (CO) of carbonyl groups respectively, these ligands have a wide range of applications in daily life. These coordinating sites as shown in Figure 1.1 give paramount status to these complexes to be used in every discipline of life.

M M O S S S

R R R R N N N N N N

R R R R

M = Co, Ni, Cd, Cu, Zn

Figure 1.1: General structures of thiobiuret and dithiobiuret metal

complexes.

In twentieth century, development of antimicrobial drugs, especially antibiotics is regarded as a great milestone to cure communicable maladies, which are major causes of motalities in developing world. However, various kinds of resistance development make available drugs as less in most of the cases not efficacious to combat disease causing microbial agents to overcome the widespread of contagious diseases at global scale Recently, it is manifested that by the

copulation of organic compounds with metal containing ligands could lead to improve drug efficacy. As in few past studies, it has already been demonstrated that thiobiuret and dithiobiuret metal complexes have antimicrobial potential to overwhelm various transmittable sicknesses (Ajibade and Zulu, 2011).

Due to delocalization of an adequate number of lone pair of electrons, the oxidation state of biuret, thiobiuret and dithiobiuret becomes unique to form metallic complexes. Furthermore, its chelation is also possible through one/two sulphur atoms or through one/two nitrogen atoms and through oxygen atom either in form of a neutral/uninegative or dinegative ion donor, whatever is the state

(Khan et al., 2009).

Among several metal complexes, thioureas show a wide range of antimicrobial activity by binding to metals in microbes. Because of this potential researchers are paying profound attention to explore and develop more vital complexes under the domain of coordination chemistry. They are focusing to understand chemical composition, structure and physicochemical properties of thio compounds and their metallic complexes which might become potential antineoplastic drugs. Besides binding to metal ions in micro-organisms, thio compounds can be used as antidotes in case of heavy metals poisoning. Thus, it was supposed to be valuable that by investigating appendages of versatile ligands such as 5-isopropylidene-l-methyl-2,4-dithiobiuret hydrochloride and 1,5- disubsituted-2,4-dithiobiurets with various metals including mercury (Hg), copper

(Cu), zinc (Zn), cadmium (Cd), nickel (Ni) and cobalt (Co). In a previous study,

Cu(I) complexes of 2,4-dithiobiuret and Ni complexes of 2-(2,4-dithiobiuret) in

ethanol have been reported to regulate a balance of metal in microbes (Naik et al., 1999).

Among numerous organic reagents, compounds containing carbonyl and thiocarbonyl moieties play key role as ligands to donate pair of electrons to the transition metal ions. Out of all, urea and thiourea derivatives are found to coordinate to a wide range of metal centers as neutral ligands such as monoanions or dianions. In thiourea derivatives oxygen, nitrogen and sulfur atoms provide a conflux of linking potential. Both types of said ligands of metal complexes exhibit a wide range of antimicrobial activities including antitubercular, antibacterial, antifungal, antithyroid, antihelmintic, rodenticidal, insecticidal, herbicidal, and plant growth regulator properties (Naik et al., 1999).

In these complexes compounds having carbonyl and thiocarbonyl moieties play an important role as prospective donor ligands for ions of transition metals in organic reagents. Derivatives of urea and thiourea are potent and multidimensional ligands, these act like a neutral ligand when coordination shows with metallic centers. These ligands provide a multitude of bonding possibilities due to donor atoms (oxygen, nitrogen, sulfur) of urea and thio urea derivatives (Arslan et al.,

2009). These metal complexes having such ligands exhibit a wide span of antimicrobial activities such as anti (bacterial, fungal, tubercular, thyroid, helmintic) and rodenticidal, insecticidal, herbicidal and also play vital role in regulating mechanism of plant growth. Some of the biological active compounds are shown in Figure 1.2.

O S R N N H H N N R

O N O S 2

Antifungal Antibectarial S HN N

Cl N

Antituberculars

Figure 1.2: Some biological active thiobiuret and dithiobiuret compounds.

1.2 BRIEF INTRODUCTION OF TRANSITION METALS

Metals e.g. copper (Cu), zinc (Zn), magnesium (Mg), iron (Fe) and manganese (Mn) etc. are found in the body in very small amounts, therefore, these are termed as trace elements or micro-nutrients. Cobalt, copper, nickel and zinc are recognized as essential metals. Each essential elements do work in three stages while involve in biological activity, trace levels necessary for best growth and development, homeostatic levels (storing levels) and lethal levels (Mahbubul

Hasan, 2010). Vitally important elements carried out their biological function appropriately and can be considered like vitamins, as normal nutritional content without which healthy life and growth are not possible. Metal based nanoparticles i.e. Ni-nanoparticles have been proved vital bactericidal agents even in very low quantities (Harish et al., 2011).

Besides this, previously published studies indicate that Co complexes possess anti cancerous, antimicrobial and antitumor effects (Bruijnincx and Sadler,

2008; Jungwirth et al., 2011; Ahmad et al., 2014), antimicrobial (Kamalakannan and Venkappayya, 2002; Temitayo Olufunmilayo et al., 2012; Ayodele, 2016) and antitumor agents (Osinsky et al., 2003). Recently, Co nanoparticles have been proved as viable means to deliver drugs by targeting a specific organ.

(Chattopadhyay et al., 2013; Javed et al., 2015).

These crucial metals show various oxidation states and their complexes give intense color. Metals can show paramagnetism hooked on metal oxidation state and ligand domain. Coordination compounds are formed when transition metals having more than one oxidation state (+1, +2, +3, +4, +5, +6, +7) combine with ligands (Gray et al., 1983). Structural geometries of complexes and specific coordination numbers depend on metal and its d-electrons.

Transition metal ions play a vital role in natural processes that occur in the human body (Jäger, 1995; Chen et al., 1996). Ions of these metals Zn, Cu and Ni are the most lavish transition metals in human’s body. They have active sites and behave as structural components of the enzymes (Butter, 1985; Cotton and

Wilkinson, 1988). The coordination chemistry of biologically important metal ions with mixed ligands has been broadly studied the recent developments. Metal complexes containing nitrogen and sulfur donor moieties are shown as potential antibacterial and antifungal agents (Crim and Petering, 1967) and also a component of several vitamins and drugs (Kato and Muto, 1988; Nagar, 1990).

In industrial zone production process run through these metals Co, Cu, Ni,

Zn and Cd, they are widely used. In biological systems these crucial metals present

in very minute amounts as essential elements display vital role in pathways of many bioinorganic reaction mechanisms. In order to study their role as metallic ions in biological organisms and their systems, structural characterization of these bioinorganic molecules and their metal complexes show broad band spectrum of applications.

1.3 REVIEW OF LITERATURE

1.3.1 Background Synthesis of Biurets, Thiobiurets and Dithiobiurets

Biuret, is the ancestor compound of a large and interesting class of organic substances. It was first identified by Wiedemann who, working in the Magnus laboratory in Berlin, isolated the new compound from the products of the thermal decomposition of urea or urea nitrate. On the basis of its utmost composition,

Wiedemann correctly interpreted its formation by the loss of ammonia from two molecules of urea, and therefore decided the name which is still in use today

(Kurzer, 1956). The reaction was represented by the following scheme 1.1.

2C2H4 N2O4 C4H5N3O4 + NH3

Scheme 1.1: Synthesis of biuret by thermal decomposition of urea nitrate.

This first account on this subject, published only twenty years after

Wohler’s classical synthesis of urea, thus contains no less than the discovery of biuret. In 1945 dithiobiuret was first time prepared in the laboratories of the

American cyanamid company by the mixing of dicyandiamide and pressurized H2S in dry inert solvents (Kurzer, 1956). The reaction occurs probably in two steps: dicyandiamide simply adds the elements of hydrogen sulfide when treated with this gas at atmospheric pressure, to yield guanylthiourea while small quantities of

dithiobiuret are formed as a by-product; quantity of dithiobiuret rise slowly as the time of reaction is increased. Following synthesis is given below in scheme 1.2.

NH2C(=NH)NHCN + H2S NH2C(=NH)NHCSNH2

+ H S NH2C(=NH)NHCSNH2 2 NH2CSNHCSNH2

Scheme 1.2: Synthesis of dithiobiuret by reaction of dicynamides and

hydrogen sulphide.

Dithiobiuret is also synthesized from the interaction of metallic dicyanamides and hydrogen sulfide. Annual developments of biuret and its related compounds was reviewed by (Kurzer, 1956).

Thiobiuret was first synthesized by Wunderlich in 1886 (Lotsch and

Schnick, 2004) from N-cyanoureas (Kurzer and Taylor, 1962) and hydrogen sulfide as shown in scheme 1.3.

NH2CONHCN + H2S NH2CONHCSNH2

Scheme 1.3: Synthesis of thiobiuret by reaction of N-cyanoureas with

hydrogen sulphide.

Thiobiuret (mono, di) present as ambidextrous species and have been frequently used as potent anti (malarials, tuberculars, hypoglycemic, inflammatory) reagents identified by (Curd et al., 1949; Foye and Hefferren, 1953; Rastogi et al.,

2001; Rastogi et al., 2012). Another bright aspect of these compounds is that they have earned an eminent name in agricultural sectors their herbicidal, insecticidal, fungicidal and pesticidal action is identified by (Bellina, 1977; Vassilev and

Strashimirov, 1979). The biological activity of these compounds mainly ascribed by existence of this moiety N-C-S which is responsible for intensify chelation

with transition metal ion is narrated by (Joshi and Giri, 1963; Matolcsy, 1971).

Many compounds were reported as potential chemotherapeutic agents (Borkovec et al., 1971).

It is very fascinating that these complexes not only exhibit potential in biological fields but lots of researchers have proved them beneficial in an industrial sector. Physiological and potential chemotherapeutic properties of numerous derivatives have been studied, and possible technical application, particularly in the field of plastics and resins, are embodied in an extensive patent literature

(Ramasamy et al., 2010a) and these urea and thiobiuret derivatives have potential as extreme pressure lubricant additives and corrosion inhibitor (Quraishi et al.,

2000) and (Rastogi et al., 2005; Rastogi et al., 2011; Rastogi et al., 2012). This class of compounds was used as ancestor for CoS, ZnS thin films or nanoparticles.

During recent years the chemistry of biuret and related compounds has attracted attention according to different protocols. These compounds are used as ligands for synthesis of metal complexes, easy characterization and their large scope of microbial studies. Khan et al., (2009) worked on synthesis 1,5- disubstituted 2, 4-dithiobiurets and studied their complexation behavior. Synthesis, spectroanalytical and biological studies of transition metal complexes of 5- isopropylidene-1-Methyl-2, 4-dithiobiuret is studied by (Naik et al., 1999). Work on experimental preparation and structural characterization of molybdenum and tungsten complexes of 1-Aryl- 2,4- dithiobiurets has been carried out by (Rastogi et al., 2001). Ajibade and Zulu, (2011) made a proposal on the synthesis of complexes of diisopropylthiourea its analysis and antibacterial studies. To investigate their importance in agricultural sector they synthesized some novel

benzoylated N-glucosyl thiobiurets. Jain and Deshmukh, (2011) has worked on such complexes. Most of these complexes showed potential in antimicrobial and anticancer activities therefore antimicrobial and anticancer valuation of 1-aryl-5-(o

-thoxyphenyl)-2-S-benzyl Isothiobiurets was synthesized and studied by (Ansari et al., 2014).

1.3.2 Synthesis of Thiobiuret

Mushtari and Yuosf, (2009) reported that there is a lot of technological developments of thiobiurets. They were applied as catalysts in the extraction of toxic metals. They have properties like antihelmintic, antitubercular, antithyroid, rodenticidal and insecticidal which are helpful in the synthesis of many drugs. Both thiourea derivatives ligands and their complexes enhanced plant growth regulating mechanisms identified by Arslan et al.,(2009). The thiobiurets have been applied in pre-concentration, solvent (liquid-liquid) extraction and chromatographic separations, because of their ability to form multi-bonding systems due to electron pair donor ligands (Yesilkaynak et al., 2010).

(Arslan et al., 2009) was reported that the thiobiuret coordinate to nickel

(II) and copper (II) metals. They have synthesized four thiobiuret ligands as shown in below schemes. These complexes were tested against different microbes for checking their antimicrobial activity. For the synthesis of thiobiurets reaction is carried between the cyclohexane carbonyl chloride and potassium thiocyanide in dry acetone after this secondary amine is added. After this condensation purification was carried out with ethanol-dichloromethane. It is concluded that the presence of cyclohexyl group lower antimicrobial of these molecules than other thiourea derivatives. The ligand prepared by this method is shown in scheme

1.4.

O O O S

Cl KSCN NCS HNR N NR 2 H 2

Scheme 1.4: Synthesis of thiobiuret ligand by cyclohexanalcarbonyl with KSCN

and amine.

By studying the experimental work conducted by (Yesilkaynak et al., 2010) is totally different, than the reagents used by (Arslan et al., 2009). They used biphenyl-4-carbonyl chloride aromatic compound as the starting reagent. The method adopted is the same as used by (Arslan et al., 2009). The reaction is started by mixing biphenyl-4-carbonyl chloride with potassium thiocyanate. The resultant mixture was heated and refluxed for 2 hours then chilled at 25 oC. After this adding

6-methylpyridin-2-amine in acidic medium and stirred the mixture for half an hour.

For acidification hydrochloric acid is mixed into the reaction mixture. Crude solid product is obtained after filtration and then recrystallization is carried out by using mixture of ethanol and dichloromethane. They found using X-ray diffraction technique in their study the compound has intermolecular hydrogen bonds between its molecules (Yesilkaynak et al., 2010).

The chemical reaction for the formation of N-(6-methylpyridin-2-yl- carbamothioyl) biphenyl-4-carboxamide is shown in scheme 1.5.

O S O N NH O 2 N N N NCS H H Cl KSCN

Scheme 1.5: Chemical reaction of formation of N-(6-methylpyridin-2-yl

carbamothioyl) biphenyl-4-carboxamide.

For the preparation of disubstituted thiourea ligand, the reagent benzoyl isothiocyanate was used as starting material and was reacted with substituted amine added in dry acetone. Reacting mixture was refluxed on heating with continued stirring. Then cold water was added with this material at cooling. The final product was obtained and washed with distilled water. After this, crude product was dried at slow rate. To investigate cytotoxic activity, thiobiuret derivatives were screened against the human cells. These thiobiuret derivatives have the capability to behave as cytotoxic agents (Rauf et al., 2009) (scheme 1.6).

O Cl O NCS O OH NH2 R R O H DMF KSCN N SOCl2 heat Acetone HN Acetone S Scheme1.6: Schematic view of synthesis of disubstituted thiourea.

A new synthetic approach was studied by (Musad et al., 2011) in his research work. He used acetonitrile, instead of acetone. In their new schematic approach for formation of N-(biphenyl-2-thiocarbomoyl)-4-phenylcarboxamide they had utilized 2-aminobiphenyl mixed with benzoyl isothiocyanate, in dry solvent acetonitrile. The resultant product was heated and refluxed for 2hrs. For

acidification HCl is added to the mixture. For lowering temperature chilled cold water was added and solid product is formed as shown scheme 1.7.

O S

SCN N N dry acetonitrils H H O NH2

Scheme 1.7: Preparation of ligand N-(biphenyl-2-thiocarbomoyl)-4-

phenylcarboxamide.

1.3.3 Transition Metal Complexes of Thiobiuret

Thiobiurets are much multifaced and versatile ligands having three multi- bonding cities. It has electron donor atoms oxygen, nitrogen and sulfur in its structural geometry (Yesilkaynak et al., 2010). Saeed et al., (2009) have reported in his studies that, thiobiuret possess both carbonyl and thiocarbonyl moieties that make it powerful reagent to form complexes with transition metal ions. In many protocols it will act as an ambidentate donor ligand by implementation of this potential of thiourea it is used for metal complexation.

This work was done by Arslan et al., (2009), concluded that similar metallic complexes were formed by mixing of the this ligands with ionic salts at 25 oC. The copper (II) complexes of thiobiuret were also subjected for antifungal assay against the fungal strains and comparative studies of nickel (II) complexes is done.

O S NR2 M O S N M N N NR2 H S O R2N

Scheme 1.8: Synthesis of complex by reaction of ligands with metallic salts.

Metals used in this scheme were Ni and Cu Rauf et al., (2009) have described that, preparation of metal complexes, CuCl powder was added in acidified methanol having thiourea derivative. Stirring was continued for a few hrs.

The final product formed was separated out. Washing was done by methanol, for recrystallization dissolved this solid in dichloromethane. Synthesis of the resultant products is displayed in scheme 1. 9.

O H R HN N R S R O R H Metallic salt N HN S M Cl N Methanol H H S N S NH O O NH

Scheme 1.9: Preparation of disubstituted thiourea copper (I) complex.

1.3.4 Synthesis of Dithiobiurets

1-Subsituted dithiobiuret compound was synthesized by the

isoperthiocyanic acids with aniline give phenylated dithiobiuret as shown in

scheme 1.10.

C H NHCSNHCSNH C2H2N2S3 + C6H5NH2 6 5 2

Scheme 1.10: Synthesis of phenylated dithiobiuret by reaction of

isoperthiocyanic acids with aniline.

Hetch and Wunderlich’s cyanourea synthesis is used in the preparation of l-substituted dithiobiurets, the interaction of isothiocyanates and sodium cyanamide affords the required intermediate 1-substituted cyanothioureas. Their subsequent treatment, in ammoniacal solutions containing ammonium chloride with hydrogen sulfide produces dithiobiurets in good yields as shown in scheme 1.11.

H2S RNCS + NHNaCN RN=C(SNa)NHCN RNHCSNNaCSNH2

Scheme 1.11: Formation of dithiobiuret by Hetch and Wunderlich’s cyanourea

synthesis.

The reaction has been generally employed for the preparation of numerous alkyl, aryl and heterocyclic derivatives. From aryldicyandiamides low yields of l- substituted dithiobiuret have been isolated as a byproduct in the production of p- chlorophenylguanylthiourea from the dicyandiam (Modest, 1956). The isoperthiocyanic acid synthesis is readily used for the preparation of 1,1- disubstituted dithiobiurets. Trisubstituted dithiobiurets are synthesized from thiocarbamyl thiocyanates. Thiocarbamyl thiocyanates are obtained from the reaction of thiocarbamyl chlorides and potassium thiocyanate in absolute ethanol or from lead dithiocarbamates and cyanogen bromide in dry benzene. The reaction proceeds with addition of aniline at room temperature, and loss of thiocyanic acid, to form trisubstituted thioureas. Near their melting points, however, thiocarbamyl thiocyanates isomerizes to the isothiocyanates, which now condense with one

molecule of aniline to produce trisubstituted dithiobiuret ; 1-methyl-1,5-diphenyl-

2,4-dithiobiuret has been prepared by (Koch and Wentrup, 2012) this route is shown in scheme 1.12.

R2NCSCl KSCN R2NCSSCN KCl

(R2NCSSH)2Pb 2CNBr 2R2NCSSCN PbBr2

R NCSHNHC H C H NH HSCN R2NCSSCN 2C6H5NH2 2 6 5 6 5 2.

C6H5N(CH)3CSSNCN C6H5N(CH)3CSNCS

C6H5N(CH)3CSNCS C6H5NH2 C6H5N(CH)3CSNHCSNHC6H5

Scheme 1.12: Synthesis of trisubstituted dithiobiuret.

1.3.5 Transition Metal Complexes of Dithiobiurets

Dithiobiuret (dtb) is a good donor ligand for d-block metals (Burman et al.,

1980). It has plenty of non-bonding π electrons that show important structural influence upon complexation. It is derived from the ligand biuret, (Girling and

Amma, 1976) which binds through oxygen. The 2,4-dithiobiuret (dtb), 2,4- thiobiuret (tb) and their derivatives are well known neutral and monoanionic (S),

(S)- and (O), (S)- bidentate ligands (Girling and Amma, 1976; Armstrong et al.,

2004). In a comparative study of the biuret reaction, this ligand has the tendency to rapidly lose a hydrogen from the central nitrogen atom (Armstrong et al., 2004;

Crane and Whittingham, 2004b). A variety of simple (N)1,(N)1,(N)5,(N)5- tetrasubstituted-2,4-dithiobiurets and 2,4-dithiobiurets have been prepared and their coordination chemistry has been investigated in detail (Shibuya and

Nakanishi, 1987). By using the single-crystal X-ray technique structure is established of these (N)1,(N)1,(N)5,(N)5-tetramethyl and (N)1,(N)1,(N)5,(N)5-

tetraethyl-2,4-dithiobiuret cobalt(II) and (N)1,(N)1,(N)5,(N)5-tetra-iso-propyl-2- thiobiuretcobalt (II) complexes have been identified by (Ramasamy et al., 2010b).

1.4 APPLICATIONS OF BIURET THIOBIURET AND DITHIOBIURET METAL COMPLEXES Biuret, thiobiuret and dithiobiuret metal complexes present a wide range of applications in the biocidal, industrial and agricultural sectors. All significances of these complexes are divided into following groups:

 Biological Applications

 Non-Biological Applications

 Agricultural Applications

1.4.1 Biological Applications

Biuret and its derivatives such as thiobiurets, dithiobiurets possess interesting biological properties such as antibacterial, herbicidal, and fungicidal.

Thiobiurets (mono and di) are important derivatives of (thio) urea which can increase the biological activity of (thio) ureas. Mono and dithiobiuret derivatives are effective against bacteria, fungi, herbs, and matricides (Bellina, 1977). These derivatives have great potential to act as antibacterial substances such as 1-aroyl-3- aryl thioureas as shown Figure 1.3 have potential against B. Subtilis, S. Aurous and

E. Coli (Saeed et al., 2009).

R O R H N HN S

Figure 1.3: 1-Aroyl-3-aryl thioureas have potential antibacterial substance.

Many thiourea and its derivatives show potential against HIV (Tsogoeva et al., 2005). Patients of HIV are a bearer of great danger of TB and other

infections. So there was a need to develop a single class of drug that can be used for the treatment of both diseases simultaneously (de Souza et al.) like compounds shown in Figure 1.4 and 1.5. In this regard thiourea derivatives act as an optimistic class. Due to this development patients can stay away from a pill burden as well expected toxicity developed by treatment of HIV and TB.

Br S

N N N N H H

Figure 1.4: 1-(5-Bromopyridin-2-yl)-3-(2-(pyridin-2-yl)ethyl)thiourea active

against HIV.

S HN N

N Cl

Figure 1.5: (S)-(N)-(3-chlorophenyl)-3-methyl-4-(3-methylbut-2-enyl)-1,4-

diazepane-1-carbothioamide against HIV.

H H N N S O O

Figure 1.6: 1,3-bis(4-(Isopentyloxy)phenyl) thiourea used for TB treatment.

Tetrahydroimidazobenzodiazepinthiones (TIBO) derivative (9-chloro

TIBO), (Trovirdine) is shown in Figure 1.4 are used for HIV treatment. An impressive number of currently used drugs can be regarded as thiourea derivatives for example, thyreostatic: carbimazole, propylthiouracil, methylthiouracil, and ultrashortnarcotic: thiamylal. Thiourea shows considerable toxicity towards higher

organisms and is used` as an insecticide (Ruder and Kayser, 1994) and rodenticide

(Kawalek et al., 1979). Alpha naphthyl thiourea and phenyl thiourea are frequently treated as rodenticide. Especially alpha naphthyl thiourea is functional in brown rat

(Richter, 1945) as represented in Figure 1.7.

NH HN 2 S

H2N N H

Figure 1.7: 1-(Naphthalen-4-yl)thiourea and 1-phenylthiourea used as

rodenticide.

Dithiobiuret is the analogous compound to urea with a change of oxygen atom by sulfur atom, also thiobiurets have a noticeably a broad band of applications. The reactivity behavior of urea and thiourea is significantly different due to electronegativity difference between sulfur atom and oxygen atom. Thiourea compounds are considering basic functional units in the preparation of heterocyclic compounds. Substituted thioureas have gained gigantic importance in the synthesis of biological active compounds. These compounds are screened against verities of biological activities such as antiviral, anticancer, anti-HIV, HDL-elevating, and analgesic. All complexes showed positive response. Acyl thiourea derivatives are familiar for showing a broad range of biological activities like anti (bacterial, fungal), herbicidal, insecticidal action and help in regulating plant growth mechanism. Thiobiuret and dithiobiuret are considered important (O) and (S) donor family of organic chemistry due to the manifold application in following

disciplines such as drugs for treatment of co-infections. In antithyroid drugs antiepileptic drugs these compounds play very active role shown in Figure 1.8.

Cl H N NH2

S Cl Cl

Figure 1.8:1- (2,4,6-Trichlorophenyl)thiourea potential as antithyroid drugs.

These mono and disubstituted phenylthioureas act as anti-hypertensive compounds (Loev et al., 1972) as represented in Figure 1.9. These show antihypertensive activity but the greatest activity was observed for this compound.

NH2 NH

Figure 1.9: 1- (2,6-Dimethylphenyl) thiourea show hypertensive activity.

One of the most important uses of thiourea derivatives is their cytotoxic activity against human cell lines. Thioureas are frequently applied as anti-cancer therapeutics in many clinical trials. In medical science research is continued for the discovery of most adaptable and safer anticancer agent. Ureas, thiourea and benzothiazoles are the considered most influential anticancer drugs. DNA topoisomerase or HIV reverse transcriptase inhibitors is produced by reaction of benzothiazoles with ureas and thioureas. Novel thioureas having general formula were formed and subjected to test for their anti-cancer activity as shown in Figure

1.10.

O OH OH S R H H R H H H N N N N N N H NH S O O S S

Figure 1.10: Anticancer active drugs used for cancer treatment.

Most thioureas were efficient in cytotoxicity (Saeed et al., 2010). On the other hand, some thiourea derivatives are present in market as commercial fungicides. In complex interfering materials they are applied as selective analytical reagents, particularly for the determination of metals. Benzoyl thiourea compounds are one of vital thiourea derivatives which have many biological activities reported including these antimicrobial, antitubercular, herbicidal, insecticidal and pharmaceutical properties and behave as chelating agents. Including this benzoyl thiourea derivatives were used in bio-analytical applications.

The ligands N-benzoyl-N-alkylthiourea and N-benzoyl-N,N-dialkylthiourea have freshly attracted attention of researchers in the approach of their function as strongly selective agents for isolation and separation of metallic cations and their concentrations, one particular advantage for these species is the coordination of obnoxious compounds, which can be obtained in the organism by one or several other ligands of appropriate structure.

Mostly thiourea and its derivatives form stable complexes by coordinating with diversity of transition metal ions (Ajibade and Zulu, 2011). Many derivatives of thiourea metal complexes which have potential for biological activity has been successfully tested for various biological actions: anti (depressant, anesthetic, convulsant, thelmintic, histaminic, tussive, analgesic etc) (Alkherraz et al., 2014).

Dithiobiuret derivatives are used for repelling birds, rodents, leporine animals, and ruminants as reported by (Hermann et al., 1976). Mono and dithiobiuret showed effective growth regulating activity. 1-Allyl-2-thiobiuret regulates the growth of germinating wheat and cucumber seeds. (Oliver et al.,

1973; Pandeya et al., 1987) have reported insect chemosterilising action of dithiobiuret derivatives in male house flies. Biuret and its derivatives also showed analgesic, anticonvulsant (Ansari et al., 2014) and hypnotic activity (Asif, 2001).

Glycosyl urea and their biuret derivatives are reported as potential glycol enzyme inhibitor (Felföldi et al., 2010).

Some substituted thiobiurets act as analgesic agents an analogue of dialkylaminoalkylester of N-phenylaminocarboximidic acids displayed, hypoglycemic, local anesthetics and analgesic activity. Pharmacological screening of certain 1,3-diketone derivatives and urea derivatives of 2-amino-N- pyridylbenzene-propanamides were considered to have analgesic potential and better tolerance profile (Asif, 2001).

1.4.2 Non-Biological Applications

In the last two decades these complexing agents are widely used as efficient ligand in coordination chemistry and have strong potential applications in industry.

1.4.2.1 Precursors for preparation of nanoparticles

Cobalt complexes of (N)1,(N)1,(N)5,(N)5-tetramethyl-2,4-dithiobiuret, and

(N)1,(N)1,(N)5,(N)5-tetraisopropyl-2-thiobiuret were applied as one-molecule precursors for the synthesis of CoS nano materials by using process thermolysis in oleylamine, hexadecylamine and octadecylamine. Cobalt sulphides form semiconductor materials which have appreciable potential for electronic

instruments and appliances. They can be utilized in solar energy as absorber magnetic ultra-high-density recordings, electrodes for Li-ion cells and for hydrodesulphurization and dehydrodearomatization function as catalysts

(Ramasamy et al., 2010b).

The complexes of (N)1,(N)1,(N)5,(N)5-tetraalkyl-2-4-dithiobiurets Zn(II) and

(N)1,(N)1,(N)5,(N)5-tetraalkyl-2-thiobiurets Cd(II) were used as building blocks of zinc sulphide or cadmium sulphide nanoparticles have been functional for a large variety of technological uses participating in photo luminescent instruments, such as electro-opticmodulators, detectors and sensors, photovoltaic cells/batteries, electroluminescent and antireflection coatings. Especially they are frequently used in displaying techniques like a host lattices used in doped phosphor materials, whose emission wavelength is used as a doping metal (Ramasamy et al., 2011).

1.4.2.2 As a precursor of heterocyclic compounds

The heterocyclic compounds such as furoquinolines, indoloquinolines, cyclopentaquinolines, isofuroquinolines, and related ring systems are synthesized by reaction of thioureas, N-arylsubstituted thiocarbamates and thioamides and presences of tris(trimethylsilyl) silane and exposure of U.V light (Du and Curran,

2003). Another schematic approach that is reported is the utilization of TsCl/

NaOH as starting material 2-aminoethyl-thioureas, 2-hydroxyethyl-thioureas, and

2-marceptoethyl-thioureas in a large variety of 2-amino-substituted heterocyclic of various ring size and substitution is synthesized respectively as shown in scheme

1.13.

S H Rn N TsCl N R N NH2 XH R Rn H NaOH X

Where X=C, O, NH

Scheme 1.13: Synthesis of heterocyclic precursor.

1.4.2.3 Potential as extreme pressure lubricant additives

Molybdenum and tungsten complexes of dithiobiuret have economic potential as an extreme pressure lubricant additives.

1.4.2.4 Use as a corrosion inhibitor

Molybdenum and tungsten complexes of substituted dithiobiurets are reported as corrosion inhibitors for structural material steel in the presence of

H2SO4. Mild steel is commonly applied as a low price building stuff for modeling of storage tanks, shipping drums, pipe aliments, etc. These apparatus may face critical attack of H2SO4 during rinsing, food preservation process, acidizing in mines and oil reservoirs and also transportation. The inhibitors used for protection of these systems are pure organic in nature containing multiple donor atoms (S),

(O) and (N) and these complexes also act as inhibitors of amidic corrosion to prevent corrosion of structural material and get rid of it by adsorbing on surface.

Rastogi et al., (2004) have reported that substituted dithiobiurets, namely

1,5-diphenyl-2,4-dithiobiuret (DPDTB) and 1-tolyl-5-phenyl-2,4-dithiobiuret

(TPDTB) are inhibitors according to his protocol. The inhibition efficiencies (IEs) of these 1-anisidyl-5-phenyl-2,4-dithiobiuret(APDTB) and 1-chorophenyl-5- diphenyl-2,4-dithiobiuret (CPDTB) ligands are lower than their complexes. Such corrosion inhibition behavior was also studied by (Quraishi et al., 2000). Thiourea

and thiourea derivatives can act as corrosion inhibitors due to the presence of thio atom. The metallic corrosion was inhibited by compounds having multiple donor atoms like oxygen, nitrogen and sulphur present in biurets and thiobiurets (Edrah and Hasan, 2010).

1.4.2.5 As floating aid

Thiourea derivatives find widespread uses in the mining industry such as used as floating aid for sulfidic ore extractions (El Aamrani et al., 1999).

1.4.2.6 As liquefying agents

In the USA, biuret and dithiobiuret compounds are used as a liquefying agent in animal hide glue, where thiourea is present in concentration of 10–20%

(Kubota and Asami, 1985).

1.4.2.7 Use in textile industry

In textiles complexes of dithiobiuret are used in many procedures like dyeing, bleaching and finishing process. Dithiobiuret which will contain <0.02% thiourea for this stage is used as a fire proof reagent to the cloth in finishing process. Many investigation is made by researchers on a textile industry on the prevalence of hypothyroidism gave typical proportion of five μg thiourea/m3 at an inlet of the domestic exhaust ventilation of the finishing devices (Roberts et al.,

1999).

1.4.3 Agricultural Applications

Biurets and thiobiurets have multi-dimensional uses in agriculture fields.

They are applied as to overcome the growth rate of insects, seed germination and help in development and growth of plants as fungicide and herbicide (Yonova and

Stoilkova, 2004).

1.4.3.1 Insect growth regulator

IGRs used to control the growth of insects by ruining their life cycle. There are two types of IGRs; hormonal IGRs and chitin synthesis inhibitors. The plant hooper is a brown insect of rice fields. This worm demolished the rice crop by gulping cell sap and source of transmitting viral infections. Derivatives of these compounds shown in Figure 1.11 monitor the breathing of insects by finishing nymph at a conc. less than 1 ppm. This is friend of environment not inhibit beneficial insects (Tunaz and Uygun, 2004).

S NC(CH3)3

N N

CH(CH3)2 O

Figure 1.11: 2-(tert-butylimino)-3-isopropyl-5-phenyl-1,3,5-thiadiazinan-4-one

control the growth of insects.

1.4.3.2 Effect on seed germination and plant growth

Many organic compounds control the germination and growth rate of seed.

These compounds affect the growth rate of a root system of linseed plant.

Development of root is decreased up to fifty percent at conc. 0.18 μ. At higher concentrations, tomatoes and green leafy vegetables have same effects were observed. Trifluralin compound has same effect. A research of analogues told that this structural influence is extremely specific for this activity. Most of the compounds belonging to this class showed plant growth regulating properties

(Brown and Harris, 1973) as shown in Figure 1.12.

O S

N NH H N

Figure 1.12: N-benzyol-N-(3-methylpyrid-2-yl)thiourea used as

plant growth regulator.

1.4.3.3 Antifungal activity

Fungicides are called the chemicals or organisms that are applied to fungus and fungal spores. Fungicides play vital role in agriculture industry because funguses are responsible for serious damage to cultivated crops and decreased their yield (Rodriguez-Fernandez et al., 2005). Following compounds and their complexes in Figure 1.13 show positive antifungal activity against these yeast

Saccharomyces cerevisiae and Penicillium digitatum.

S S

N H2N N H N N H 2 H Phenyl thiourea pyridyl thiourea

H H N N O N O N S S 3-benzyol-1-butyl-1-methyl thiourea 3-benzyol-ethylisopropylthiourea

Figure 1.13: Thiourea derivatives showing antifungal activity.

1.4.3.4 Herbicidal activity

Following compounds shown in Figure 1.14 showed herbicidal activity against seedlings of cucumber and wheat (Yonova and Stoilkova, 2004). These

showed significant activity against root and stalk of Amaranthus retroflexus

(Yonova and Stoilkova, 2004; Ke and Xue, 2006).

S N

S N N H H

Figure 1.14: 1-phenyl-3-(thiazol-2-yl)thiourea herbicidal active compound.

1.4.4 Miscellaneous Uses

For manufacturing of flame proof resins and a vulcanization accelerator compounds of thiourea are used. It is noted that in European countries these molecules is not only applied in the leaching of ore mines and not processed to thiourea dioxide but following use pattern is recorded, precipitation of heavy metals, corrosion inhibitor, auxiliary agent in diazo paper (light-sensitive photocopy paper) and other types of copy paper, metal cleaning, including silver polish, electroplating/electroforming, additive in slurry explosives, vulcanization accelerators, processing to organic intermediates, mercaptosilanes, resin modification, and chemicals industry and miscellaneous. Fertilizers of thiourea inhibit the nitrification process in Japan (Kubota and Asami, 1985).

Thiobiuret is emitted by factories of electronic components, accessories and builder of aircraft and aircraft parts. Organic derivatives of urea and thiourea is used in synthesis of pharmaceuticals (hydrotherapeutic, antiseptic, narcotic, and tuberculostatic agents) and formation of vulcanization accelerators (Mertschenk et al., 1995).

1.5 STRUCTURAL ELUCIDATION TECHNIQUES

Metal complexes of thiobiuret and dithiobiuret ligands can be studied by variety of techniques. In this era the use of UV-Vis, infrared spectroscopy, (1H,

13C) nuclear magnetic resonance spectroscopy and single crystal X-ray analysis are excellent tools for the structural characterization of metal complexes.These paramount states techniques are precisely reviewed over here.

1.5.1 Infrared Spectroscopy

The infrared spectral studies are a quite fruitful in detecting the way of coordination of substances like ligands. By understanding, examining the position and direction of the bonding atoms, their moving shifts of the ligands after complexation, and made comparisons to their positions in the free state. The manner of coordination can be purposed for all the synthesized complexes in solid state and were recorded in infrared spectral region 4000–400 cm−1 and assigned stretching frequencies on careful comparison. In spectroscopic studies the characteristic stretching frequencies of thiobiuret ligands and dithiobiuret are

υ(C=O) 1654-1668 cm-1, υ(N-H) 3286- 3322 cm-1, υ(C-N) 1249-1273 cm-1 and

υ(C=S) 843-854 cm-1 (Halim et al., 2012).

According to literature structural investigation of copper complexes

(thiourea derivatives) infrared spectra of the ligands showed strong band of high intensity at 1715-1730 cm-1, which give characteristic peak of carbonyl moiety

(C=O). After formation of the complex, this band shifted to the lower frequency at

1651-1670 cm-1 indicating expected bonding possibility of carbonyl oxygen atom of ligand with metal ion. At a time, appearance of a strong band was assigned at

820-840 cm-1 of ligand at free state, which was attributed to the C=S group,

absences of this band in the complexes, indicated bonding through sulphur atom contributed to the formation of the complexes.

Infrared spectra of diisopropylthiourea metal complexes bands in the region 3450–3100 cm−1 recognizes the N–H stretching vibrations that appear as a broad band at 3450 cm−1 in the spectra of the ligand shifted to higher region of frequency in the complexes. The band appeared at 1480 cm−1 in the ligand and moved to higher region about 1486–1524 cm−1 in the spectra of the complexes.

These bands are assigned which are responsible to increase in the double bond character on formation of complex (Ajibade and Zulu, 2011).

1.5.2 NMR Spectroscopy

Nuclear magnetic resonance spectroscopy is spectral technique to acquire data about the structure, to study chemical changes in reaction mechanism and intra and inter molecular forces etc. 1H, 13C analyses comprehensively impart highly valuable statistics and hence are used for the structural elucidation of biuret, thiobiuret and dithiobiuret metal complexes.

According to literature NMR study of this complex bis(1N,1N,5N,5N- tetraethyl-2,4-dithiobiureto) cadmium(II) showed shifts are 1H NMR (300 MHz,

CDCl3, Me4Si): 3.25 (q, 8H), 3.8 (q, 8H) 1.2 (t,12H), 1.4 (t,12H) these signals appeared respectively as shifts observed in spectra of metal and ligand. Shifts of proton signals probably change in spectra of complex due to electronic environment of different proton as consequence of coordination. Signal present due to presence of amino group –NH proton appearing at δ 6.52 ppm in ligand completely disappears in complexes due to thioenolisation. The structural investigation of copper complexes (thiourea derivaties) in spectral studies of ligand

attributed only the band at δ11.77 ppm for NH group between C=S, C=O functional group is absent, indicating the deprotonation of amide proton and formation of C=N bond in spectra of complexes.

1.5.3 X-ray Crystallography

Single crystal X-ray diffraction imparts a direct way of ascertaining structures of the crystalline solids. This tool determines accurate structural information, atom orientations and data about thermal displacement parameters.

1.5.3.1 Crystal structure determination

A cell unit of crystal of mm dimension is required for the crystal structure determination. A suitable and proper dimensional crystal entity is applying on a glass fiber on a goniometer and mechanical devices for rotation, tilting the crystal in each direction is precisely applied to measured angular displacements. The crystal is then hit with X-rays and information is collected. From this valuable data, maps of electron density through various cross-sections of the crystal are produced.

The details of procedure is reported in the literature (Harrison, 1979) .

1.6 BIOLOGICAL STUDIES

Biological studies play an important role in exploration of drugs. Presently there is demand for invention is having less side effects drugs is increased.

Information is collected to acquire data of active substances and its utilization in potential drug therapy against different diseases the necessary screens can be done by the combination of many bioassays. Bioassays are the basic tool to acknowledge the quality of bioactive compounds (Bohlin and Bruhn, 1999). In present work primary bioassays are carried out to predict the nature of synthesized complexes

because these procedures run fastly, tolerant of impurities, reproducible and compatible with dimethylsulphoxide used as a negative control (Ghosh, 2007).

1.7 SCOPE OF STUDY

Transition metal complexes with biuret, thiobiuret and dithiobiuret ligands are well known since biurea, thiourea and dithiourea compounds can play an important role in various applications in many disciplines of life. Due to the monodentate or bidentate nature of ligand, it can be utilized for the synthesis of thiobiureto and dithiobiureto metal complexes. By using this multitude of combining capacities of these ligands we can utilize different approaches to synthesize metal complexes and structurally characterize them. Besides that, the microbial assays and other biological assays of the complexes can be analyzed which can give us a good results.

1.8 PLAN OF WORK

As an important family of (S-) and (O-) donor ligands such as dtb and tb can be used for the synthesis of new metal complexes. Therefore, we investigated the chemistry of dtb and tb ligands with the following divalent metal ions to form some novel metal complexes.

 Co(II), d7-metal ion

 Ni(II), d8-metal ion

 Cd(II), d8-metal ion

 Cu(II), d9-metal ion

 Zn(II), d10-metal ion

The synthesized complexes will then be characterized by spectroanalytical methods such as elemental analysis, UV-Vis, FTIR, NMR, and X-ray

crystallography. Fully characterized complexes will then be screened for microbial studies and other biological assays to investigate their biological active nature.

Chapter 2

MATERIALS AND METHODS

This chapter presents all experimental and bio analytical procedures used in this study to gain the objectives of the research.

2.1 CHEMICALS

Main chemicals used in this synthesis are diethyl and dimethyl-carbamoyl chloride or dimethyl thiocarbamoyl chloride and 3,3-dimethylbutyryl chloride were purchased from sigma Aldrich. Organic solvents acetonitrile, methanol, chloroform dimethylsulfoxide and THF were also purchased from Sigma Aldrich, Riedel-De

Haen. Solvents were dried from moistures and other impurities at standard procedures then freshly collected and used accordingly (Chai and Armarego,

2003). List of chemical and solvents used for preparation of thiobiuret and dithiobiuret metal complexes are given below in table 2.1.

Table 2.1: List of chemicals acquired for synthesis of thiobiuret and dithiobiuret

metal complexes.

Sr. No Chemicals/Solvents Manufacturer

1 N,N-diethylcarbamoyl chloried (99%) Sigma Aldrich

2 N,N-dimethylthiocarbamoyl chloride (99)% Sigma Aldrich

3 3,3-dimethylbutyryl chloride (99%) Sigma-Aldrich

4 Potassiumthiocynate (99%) Merck

5 Diethyl amine (99%) Sigma-Aldrich

6 Dimethylamine Sigma-Aldrich

7 Dipropyl amine (99%) Sigma-Aldrich

Sr. No Chemicals/Solvents Manufacturer

8 N-benzylmethylamine (99%) Sigma-Aldrich

9 1-(2-furoyl)-piprazine (97%) Sigma-Aldrich

10 Dodecylamine (98%) Alfa Aesar

11 Benzylamine (98%) Alfa Aesar

12 4-nitroaniline (98%) Merck

13 Phenylhydrazine (98%) Merck

14 2-hydrazinylphenol (97%) Merck

15 2,4-dinitrophenyl hydrazine (98%) Merck

16 Nickel(II) acetate tetrahydrate (98%) Sigma-Aldrich

17 Dimethylcarbamyl chloride Sigma-Aldrich

18 Cobalt(II) acetate tetrahydrate (99%) Sigma-Aldrich

19 Zinc(II) acetate dihydrate (98%) Sigma-Aldrich

20 Cupric(II) acetate monohydrate (98%) BDH

21 2-amino-3-hydroxy pyridine (98%) Merck

22 Acetonitrile (98%) Sigma-Aldrich

23 2-amino phenol (99%) Sigma-Aldrich

24 Methanol (98%) Sigma-Aldrich

25 Dichloromethane (99%) Sigma-Aldrich

26 Dimethylsulfoxide (99%) Riedel-De Haen

27 Tetrahydrofuran (99%) Riedel-De Haen

28 Acetone (99%) Merck

29 n-Hexane(99%) Fisher Scientific

30 Chloroform (99%) Sigma-Aldrich

2.2 INSTRUMENTATION

The melting points of synthesized complexes were noted on Griffen (Japan) digital electrothermal melting point instrument. The Fourier transform infrared spectral data collected for solid samples was in KBr pellets and liquid samples on neat KBr cell FTIR-8400 SHIMADZU spectrophotometer within spectral range of

4000-400 cm-1.

Multinuclear magnetic resonance (1H,13C) spectral data was determined on a Bruker AMX 400 MHz-FTNMR and a Bruker Advance 600 MHz-FTNMR taking CDCl3/DMSO as internal Standard (Wrackmeyer, 1999). Chemical shifts (δ) were read out in ppm and coupling constants (J) were assigned in Hz. The multiplicities of absorption bands in the 1H NMR spectrum are put in writing as (s for singlet, d for doublet, t for triplet, q for quartet, m for multiplet). For electronic analysis of synthesized compounds UV spectrophotometer Shimadzu 1601 was used. CHNS Analyzer elementar Analysensysteme GmbH-vario EL III was used for determination of percentage of carbon, hydrogen and nitrogen elements in synthesized complexes. The X-ray parameters were measured on Bruker Kappa

Apex-IICCD diffractometer equipped with graphite monochromator and Mo-Kα radiation source, the geometry was elaborated by direct method and solved by applying SHELXL 2013 (Sheldrick, 2013) the figure was design with add of

ORTEP II.

Table 2.2: Instruments used in the preparation and characterization of thiobiuret

and dithiobiuret metal complexes.

S. No. Instrument Manufacturer 1 IR spectrophotometer FTIR-8400s SHIMADZU Japan 2 NMR spectrophotometer Bruker AMX 400 MHz FTNMR 3 Melting point apparatus Griffin 4 Electric hot plate MSH-20A, Germany 5 Condensers Pyrex 6 UV-Vis spectrophotometer Shimadzu 1601 7 Microwave Oven Haier 8 Weighing balance Sartorius GMBH, Germany 9 Conductivity meter BANTE DDS-12DW USA

2.3 GENERAL PROCEDURE (A) FOR SYNTHESIS OF BIURET,

THIOBIURET AND DITHIOBIURET METAL COMPLEXES

A solution of substituted carbamoyl chlorides or thiocarbamyl chlorides and potassium thiocyanate with molar ratio 1:1 was heated at reflux for two hours in dry acetonitrile (30ml) with continued stirring. After reflux, the solution was chilled at room temperature 25 oC. A 60% aqueous solution of substituted amine was added and started stirred for 30 minutes. Stirring was continued for 30 min by addition of metal acetate hydrated (1/2 mol). The reaction was proceeding in a 250 ml two-necked round bottom flask, fitted with a water condenser for refluxing, and a magnet bar. The product in each case was obtained after a few min as a precipitate by adding distilled water (150 ml). The crude product was always subjecte for filtration, washed with methanol, air dried and recrystallized by slow

evaporation at room temperature from chloroform or dichloromethane to get crystals of synthesized complexes suitable for a single crystal X-ray studies.

R R

R N S N Cl KSCN R C N C C CH3CN Z Z Reflux 2hr

stirr at room temp R HN R R

N R R Z C R M(OAc)2 H N M N N N CH3OH R C C S C H2O R N R Z S

R n Z = O, S

R = Me, Et,

M = Co, Ni, Cu,

Cd, Zn

n = 2, 3

Scheme 2.1: General scheme for synthesis of biuret, thiobiuret and dithiobiuret

metal complexes by method A.

2.4 SYNTHESIS OF THIOBIURET AND DITHIOBIURET NICKEL (II)

COMPLEXES USING DIFFERENT AMINES

2.4.1 Bis [1,1,5,5-tetra ethyl-4-thiobiureto]Nickel(II) 1

C2H5 C H N 2 5 N C C N C H C2H5 O S 2 5 Ni S O C2H5 C2H5 N C C N N C H C2H5 2 5

Adopted procedure A for synthesis, diethylamine is used. Purple crystalline

o -1 solid, m.p 138 C, yield 60% selected IR (KBr cm ); vmax Aliphatic C-C 2929,

C=N 1541, C=S 1076, 1006 C=O 1652, N-C 1174, M-O 649, M-S 526, UV-vis λ

1 (nm); 274, 305, 503, H NMR (CDCl3): δ 1.07 (br. s, 12H, 4CH3), 3.20 (d, 4H,

4CH2) 3.33 (d, 4H, 4CH2). Anal. Calc. For (C10H20N3OS)2Ni (MW = 518): C =

46.3; H = 7.7; N = 16.2. Found values: C = 46.0; H = 7.1; N = 16.0.

2.4.2 Bis [1,1-diethyl-5-( benzylmethyl)-4-thiobiureto]Nickel(II) 2

C H CH 2 5 N 3 N C C N C H 2 5 O S Ni

S O C H H3C 2 5 N C C N N C2H5

Adopted procedure A for synthesis, N-benzylmethylamine is used. Purple

o -1 solid product, m.p 110 C, yield 52% selected IR (KBr cm ); vmax Aliphatic C-C

2925, C=N 1541, C=S 1076, 1000, C=O 1710, N-C 1178, Ar C=C 1473, M-O 543,

1 763, M-S 489. UV-vis λ (nm); 270, 302, 506, H NMR (CDCl3): δ 0.81 (br. s, 6H,

2CH3), 3.15-3.25 (m, 4H), 2.81 (s, 3H), 7.03-7.24 (m, 5H). Anal. Calc. For

(C13H18N3OS)2Ni (MW = 586): C = 53.2; H = 6.1; N = 14.3. Found values: C =

53.0; H = 5.4; N = 14.0.

2.4.3 Bis [1,1-diethyl-5-(3-hydroxypyridine)-4-thiobiureto]Nickel(II) 3

OH C H H 2 5 N N C C N C H N 2 5 O S

HO Ni S O C2H5 N C C N N H N C2H5

Procedure adapted A for synthesis, amine is 2-amino-3-hydroxy pyridine.

o -1 Purple solid product, m.p 150 C, yield 55% selected IR (KB cm ); vmax Aliphatic

C-C 2931, C=N 1515, C=S 1076, 999, C=O 1610, N-C 1166, M-O 532, M-S 490,

O-H 3403, UV-vis λ (nm); 275, 322, 516, Anal. Calc. For (C11H15N4O2S)2Ni (MW

= 592): C = 44.5; H = 5.0; N = 18.9. Found value: C = 44.0; H = 4.5; N = 18.3.

2.4.4 Bis[1,1-diethyl-5-(1-(2-furoyl)-pipraziyl)-4-thiobiureto]Nickel(II) 4

C H 2 5 N N C C N C H 2 5 O S N O Ni O

S O C2H5 N C C N N N C2H5 O O

Procedure adopted A for synthesis, amine used is 1-(2-furoyl peprazine).

o -1 Purple solid product, m.p 190 C, yield 65% selected IR (KBr cm ); vmax Aliphatic

C-C 2925, Ar C=C 1427, C=N 1571, C=S 1081, 1004, C=O 1612, N-C 1176, M-O

1 595, M-S 475, UV-vis λ (nm); 277, 322, 506, H NMR (CDCl3): δ 1.41 (br. s, 12H,

13 4CH3), 3.54-3.66 (m, 8H, 4CH2) 4.13-4.28 (d, 8H) 6.83 (s, 3H), C NMR (CDCl3):

δ 31, 13, 170, 43, 111. Anal. Calc. For (C15H20N4O3S)2Ni (MW = 732): C = 49.1;

H = 6.7; N = 15.3. Found values: C=48.5; H = 6.0; N = 15.0.

2.4.5 Bis [1,1-diethyl-5-(4-nitrophenyl)-4-thiobiureto]Nickel(II) 5

C2H5 C2H5 N HN NO N 2 C C

O S Ni S O C2H5 C O2N N C N H N C2H5

Procedure adapted A for synthesis, amine used is 4-nitroanniline. Purple

o -1 solid product, m.p 160 C, yield 56%, selected IR (KBr cm ); vmax Aliphatic C-C

2929, Ar C=C 1423, C=N 1598, C=S 1058, C=O 1685, N-C 1139, 1004, M-O 526,

1 M-S 493, UV-vis λ (nm); 274, 341, 506. H NMR (CDCl3): δ 0.41 (br. s, 12H,

4CH3), 3.19 (m, 8H 4CH2), 7.03-7.20 (m, 8H), 2.58 (s, 2H). Anal. Calc. For

(C12H15N4O3S)2Ni (MW = 648): C = 44.4; H = 4.6; N = 17.2. Found values: C =

44.0; H = 4.0; N = 16.5.

2.4.6 Bis[1,1-diethyl-5-(aminobenzyl)-4-thiobiureto]Nickel(II) 6

C2H5 N N C C N NH2 C2H5 O S

Ni S O C2H5 H2N N C C N N C2H5

Procedure adapted for synthesis A, amine used is phenyl hydrazine. Purple

-1 viscous liquid product, yield 62% selected IR (neat cm ); vmax Aliphatic C-C 2929,

Ar C=C 1404, C=N 1593, C=S 1076, 1010, C=O 1666, N-C 1153, N-H 3191, M-O

543, M-S 489, UV-vis λ (nm); 225, 320, 431, Anal. Calc. For (C12H16N4OS)2Ni

(MW = 586): C = 49.1; H = 4.9; N = 19.1. Found values: C = 48.5; H = 4.5; N =

18.5.

2.4.7 Bis[1,1- diethyl-5-(2-hydroxy benzoamide)-4-thiobiureto]Nickel(II) 7

H O C2H5 N N C C N NH C C H OH 2 5 O S

S O O H C2H5 C HN N C C N HO N C2H5

Procedure adopted A for synthesis, amine used is 2-hydrazinylphenol.

o -1 Purple solid product, m.p 80 C, yield 56% selected IR (KBr cm ); vmax Aliphatic

C-C 2916, Ar C=C 1404, C=N 1544, C=S 1081, 1010, C=O 1629, N-C 1172, O-H

3411, M-O 621, M-S 475, UV-vis λ (nm); 236, 321, 469. Anal. Calc. For

(C13H17N4O3S)2Ni (MW = 676): C = 57.6; H = 5.0; N = 16.5. Found values: C =

57.1; H = 4.5; N =16.0.

2.4.8 Bis[1,1-diethyl-5-(methyl benzyl)- 4-thiobiureto]Nickel(II) 8

C2H5 N N C C NH

C2H5 O S Ni

H S C H O 2 5 N C C N N C H 2 5

Procedure adopted A for synthesis, amine used benzyl amine. Purple

-1 viscous liquid, yield 55% selected IR (neat cm ); vmax Aliphatic C-C 2929, Ar

C=C 1404, C=N 1645, C=S 1076, 1027, C=O 1699, N-C 1180, M-O 524, M-S

1 495, UV-vis λ (nm); 271, 334, 466, 547. H NMR (CDCl3): δ 0.72 (br. s, 12H,

4CH3), 3.97 (d 8H, 4CH2), 6.88-6.93 (m, 10H), 4.44-4.43 (d, 4H), 2.67 (s, 2H).

Anal. Calc. For (C13H1 8N3OS)2Ni (MW = 586): C = 53.2; H = 4.4; N = 14.3.

Found values: C = 53.0; H = 4.0; N = 14.0.

2.4.9 Bis[1,1-diethyl-5-(dodecylamine)-4-thiobiureto]Nickel (II) 9

C2H5 H N CH N C C N 3 O C2H5 S Ni

S O C2H5

H3C N C C N N H C2H5

Procedure adopted A for synthesis, amine used dodecylamine. Purple

-1 viscous liquid, yield 53% selected IR (neat cm ); vmax Aliphatic C-C 2923, C=N

1550, C=S 1080, C=O 1618, N-C 1180, M-O 628, M-S 426, UV-vis λ (nm); 321,

466, 531. Anal. Calc. for (C18H33N3O)2Ni (MW = 736): C = 58.6; H = 8.9; N =

11.4. Found values: C = 58.1; H = 8.3; N = 11.0.

2.4.10 Bis[1,1-diethyl-5-(2,4-dinitrophenylhydrazine)-4-thiobiureto]Nickel(II) 10 N C2H5 N C C N NH2 C2H5 O S N2O

Ni N2O

S O C2H5 H2N N C C N N C2H5 ON2

N2O

Procedure adopted A for synthesis, amine used 2,4-dinitrophenyl hydrazine.

o -1 Purple solid, m.p 143 C yield 61% selected IR (KBr cm ); vmax Aliphatic C-C

2931, C=N 1544, C=S 1058, C=O 1618, N-C 1134, M-O 511, M-S 428, UV-vis λ

(nm); 342, 556. Anal. Calc. for (C12H15N6O5S)2Ni (MW = 768): C = 37.5; H = 3.9;

N = 21.8. Found values: C = 37.0; H = 3.5; N = 21.0.

2.4.11 Bis[1,1-diethyl-5,5-dipropyl-4-thiobiureto]Nickel(II) 11

H3C C H N 2 5 CH3 N C C N C H CH 2 5 O S 3 CH3 Ni

CH3 S O C2H5 H3C N C C N C2H5 H3C N

H3C Procedure adopted A for synthesis, amine used dipropyl amine. Purple

-1 viscous liquid, yield 64% selected IR (KBr cm ); vmax Aliphatic C-C 2968, C=N

1552, C=S 1062, 1035, C=O 1612, N-C 1172, M-O 570, M-S 432, UV-vis λ (nm)

344, 508. Anal. Calc. for (C12H24N3OS)2Ni (MW = 574): C = 50.1; H = 8.3; N =

14.6. Found values: C = 49.5; H = 7.5; N =14.0.

2.4.12 Bis [1,1,5,5-tetramethyl-2,4-dithiobiureto]Nickel(II) 12

H3C N CH3 N C C N H3C CH3 S S

S S H3C CH3 N C C N H C 3 N CH3

Procedure adopted A for synthesis, amine used dimethyl amine. Purple

-1 colored solid, yield 73% selected IR (KBr, cm ); vmax C-C 2921, C=N 1506, 1495,

C-N 1133, 1114, C=S 1049, 911, M-S 463, UV-vis λ (nm); 265, 370, 520. 1H NMR

13 (CDCl3): δ 3.10 (br. s, 12H, 4CH3), 3.07 (br. s, 12H, 4CH3), CNMR (CDCl3): δ

173, 41, 39. Anal. Calc. for (C6H12N3S2)2Ni (MW = 439): C = 41.0; H = 5.4; N =

19.1. Found values: C = 40. 5; H = 5.0; N = 18.5

2.5 SYNTHESIS OF BIURET, THIOBIURET AND DITHIOBIURET

COPPER(II) COMPLEXES USING DIFFERENT AMINES

The copper(II) complexes (13-24) were synthesized by the reaction of commercially available secondary amine and in situ generated 3,3- diethyllthiocarbamoylthiocynate or N,N-dimethyldithiocarbamoylcynate in acetonitrile with addition of cupric(II) acetate monohydrated according to scheme

2.1. Crude product of muddy grey color obtained was recrystallized from chloroform. Complex 24 give grey fine crystals were obtained for single crystal X- ray studies on evaporating solvent at slow rate for several days.

2.5.1 Bis [1,1,5,5-tetra ethyl-4-thiobiureto]Copper(II) 13

C2H5 C2H5 N N C C N C H C2H5 O S 2 5 Cu

S O C2H5 C2H5 N C C N N C2H5 C2H5

Adopted procedure A for synthesis, diethylamine is used. Muddy gray

o -1 crystalline solid, m.p 120 C, yield 68% selected IR (KBr cm ); vmax Aliphatic C-

C 2929, C=N 1473, C=S 1075, C=O 1616, N-C 1172, M-O 550, M-S 655, UV-vis

1 λ (nm); 266, 346, 508. H NMR (CDCl3): δ 1.03 (br. s, 12H, 4CH3), 3.19 (d, 4H,

4CH2) 3.30 (d, 4H, 4CH2). Anal. Calc. for (C10H20N3OS)2Cu (MW = 523): C =

45.8; H = 7.6; N = 16.0. Found values: C = 45.2; H = 7.0; N = 15.5.

2.5.2 Bis [1,1-diethyl-5-( benzylmethyl)-4-thiobiureto]Copper(II) 14

C2H5 N CH3 N C C N C H 2 5 O S Cu

S O C H H3C 2 5 N C C N N C2H5

Adopted procedure A for synthesis, N-benzylmethylamine is used. Muddy

o -1 gray solid product, m.p 150 C, yield 53% selected IR (KBr, cm ); vmax Aliphatic

C-C 2925, C=N 1475, C=S 1080, C=O 1616, N-C 1153, Ar C=C 1473, M-O 510,

1 M-S 648. UV-vis λ (nm); 270, 347, H NMR (CDCl3): δ 1.08 (br. s, 12H, 4CH3),

3.26-3.37 (m, 8H), 2.82 (s, 6H), 7.05-7.23 (m, 10H). Anal. Calc. for

(C13H18N3OS)2Cu (MW = 591): C = 52.7; H = 5.0; N = 14.2. Found values: C =

52.2 ; H = 4.4; N = 13.6.

2.5.3 Bis [1,1-diethyl-5-(3-hydroxypyridine)-4-thiobiureto)]Copper(II) 15

OH C H H 2 5 N N C C N N C2H5 O S

HO Cu S O C2H5 N C C N N N H C2H5

Procedure adapted A for synthesis, amine is 2-amino-3-hydroxy pyridine.

o -1 Muddy gray solid product, m.p 135 C, yield 54% selected IR (KBr, cm ); vmax

Aliphatic C-C 2931, C=N 1554, C=S 1083, C=O 1612, N-C 1161, M-O 582, M-S

513, O-H 3403, UV-vis λ (nm); 270, 353, 595. Anal. Calc. for (C11H15N4O2S)2Cu

(MW = 597): C = 44.2; H = 5.0; N = 18.7. Found value: C = 44.0; H = 4.9; N =

18.2.

2.5.4 Bis [1,1-diethyl-5-(1-(2-furoyl)-pipraziyl)-4-thiobiureto]Copper(II) 16

C H 2 5 N N C C N C H 2 5 O S N O Cu O

S O C2H5 N C C N N N C2H5 O O

Procedure adopted A for synthesis, amine used is 1-(2-furoyl peprazine).

o -1 Muddy gray solid product, m.p 162 C, yield 64 %, selected IR (KBr, cm ); vmax

Aliphatic C-C 2937, Ar C=C 1427, C=N 1527, C=S 1076, C=O 1604, N-C 1132,

1 M-O 595, M-S 622, UV- vis λ (nm); 266, 345. H NMR (CDCl3): δ 1.25 (br. s, 6H,

13 2CH3), 1.41 (br. s, 6H, 2CH3), 4.13-4.28 (d, 8H) 6.83 (s, 3H), C (CDCl3): δ 36,

15, 169, 45, 117. Anal. Calc. for (C15H20N4O3S)2Cu (MW = 737): C = 48.8; H =

5.6; N = 15.1. Found values: C=48.5; H = 5.0; N = 14.5.

2.5.5 Bis[ 1,1-diethyl-5-(4-nitrophenyl)-4-thiobiureto]Copper(II) 17

C2H5 C2H5 N HN NO N 2 C C

O S Cu S O C2H5 C O2N N C N H N C2H5

Procedure adapted A for synthesis, amine used is 4-nitroanniline. Muddy

o -1 gray solid product, m.p 140 C, yield 55% selected IR (KBr, cm ); vmax Aliphatic

C-C 2933, Ar C=C 1423, C=N 1537, C=S 1083, C=O 1614, N-C 1211, M-O 594,

M-S 663, UV-vis λ (nm); 347. Anal. Calc. for (C12H15N4O3S)2Cu (MW = 653): C =

44.1; H = 4.5; N = 17.1. Found values: C = 43.5; H = 4.0; N = 16.5.

2.5.6 Bis[1,1-diethyl-5-(aminobenzyl)-4-thiobiureto]Copper(II) 18

C2H5 N N C C N NH2 C2H5 O S

S O C2H5 H2N N C C N N C2H5

Procedure adapted A for synthesis, amine used is phenyl hydrazine. Muddy

o -1 gray solid product, m.p 130 C, yield 57% selected IR (KBr, cm ); vmax Aliphatic

C-C 2929, Ar C=C 1404, C=N 1556, C=S 1081, C=O 1616, N-C 1153, N-H 3191,

M-O 528, M-S 617, UV-vis λ (nm); 333, Anal. Calc. for (C12H16N4OS)2Cu (MW =

593): C = 48.7; H = 5.4; N = 18.9. Found values: C = 48.2; H = 5.0; N = 18.2.

2.5.7 Bis[1,1- diethyl-5-(2-hydroxy benzoamide)-4-thiobiureto]Copper(II) 19

H O C2H5 N N C C N NH C C H OH 2 5 O S Cu

S O O H C2H5 C HN N C C N HO N C2H5

Procedure adopted A for synthesis, amine used is 2-hydrazinylphenol.

o -1 Muddy gray solid product, m.p 100 C, yield 55% selected IR (KBr, cm ); vmax

Aliphatic C-C 2952, Ar C=C 1404, C=N 1541, C=S 1081,C=O 1616, N-C 1170,

O-H 3411, M-O 553, M-S 663, UV-vis λ (nm) 233, 364. Anal. Calc. for

(C13H17N4O3S)2Cu (MW = 681): C = 57.6; H = 5.0; N = 16.5. Found values: C =

57.1; H = 4.5; N =16.0.

2.5.8 Bis[1,1-diethyl-5-(methyl benzyl)- 4-thiobiureto]Copper(II) 20

C2H5 N N C C NH

C2H5 O S

S H O C2H5 N C C N N C2H5

Procedure adopted A for synthesis, amine used benzyl amine. Muddy gray

-1 viscous liquid, yield 60% selected IR (neat, cm ); vmax Aliphatic C-C 2929, Ar

C=C 1404, C=N 1537, C=S 1095, C=O 1645, N-C 1134, M-O 551, M-S 451, UV- vis λ (nm); 226, 309, 420. Anal. Calc. for (C13H1 8N3OS)2Cu (MW = 591): C =

52.7; H = 6.0; N = 14.2. Found values: C = 52.5; H = 5.5; N = 13.7.

2.5.9 Bis[1,1-diethyl-5-(dodecylamine)-4-thiobiureto]Copper(II) 21

C2H5 N H CH N C C N 3

C2H5 O S Cu

S O C2H5

H3C N C C N N C2H5 H

Procedure adopted A for synthesis, amine used dodecylamine. Muddy gray

o -1 solid, m.p 83 C, yield 61% selected IR (KBr, cm ); vmax Aliphatic C-C 2921,

C=N 1556, C=S 1098, C=O 1645, N-C 1218, M-O 625, M-S 617, UV-vis λ (nm);

548. Anal. Calc. for (C18H33N3OS)2Cu (MW = 741): C = 58.2; H = 8.9; N = 11.3.

Found values: C = 57.5; H = 8.5; N = 10.5.

2.5.10 Bis[1,1-diethyl-5-(2,4-dinitrophenylhydrazine)-4thiobiureto]Copper(II)

N C2H5 N C C N NH2 C2H5 O S N2O

Cu N2O

S O C2H5 H2N N C C N N C2H5 ON2

N2O

Procedure adopted A for synthesis, amine used 2,4-dinitrophenyl hydrazine.

o -1 Muddy gray solid, m.p 121 C, yield 60% selected IR (KBr, cm ); vmax Aliphatic

C-C 2931, C=N 1550, C=S 1105, C=O 1618, N-C 1126, M-O 418, M-S 626 UV- vis λ (nm); 367, 415, Anal. Calc. for (C12H15N6O5S)2Cu (MW = 773): C = 37.2; H

= 3.8; N = 21.7. Found values: C = 36.5; H = 3.5; N = 21.2.

2.5.11 Bis[1,1-diethyl-5,5-dipropyl-4-thiobiureto]Copper(II) 23

H3C C H 2 5 N CH3 N C C N C H CH 2 5 O S 3 CH3 Cu

CH3 S O C2H5 H3C N C C N C H H3C N 2 5 H3C

Procedure adopted A for synthesis, amine used dipropyl amine. Muddy

-1 gray viscous liquid, yield 62% selected IR (KBr, cm ); vmax Aliphatic C-C 2933,

C=N 1545, C=S 1060, C=O 1600, N-C 1126, M-O 435, M-S 617, UV-vis λ (nm);

1 521. H NMR (CDCl3): δ 0.85 (br. s, 6H, 2CH3), 0.92 (br. s, 6H, 2CH3), 3.26-3.30

13 (m, 4H), 3.51-3.61 (br. s, 12H, 4CH3), 1.24-1.60 (br. s, 4H), C NMR (CDCl3): 19,

29, 40, 52, 160, Anal. Calc. for (C12H24N3OS)2Cu (MW = 579): C = 49.7; H = 8.2;

N = 14.5. Found values: C = 49.5; H = 7.5; N = 14.0.

2.5.12 Bis[1,1,5,5-tetramethyl-2,4-dithiobiureto]Copper(II) 24

H3C N CH3 N C C N H3C CH3 S S

S S H3C CH3 N C C N H3C N CH3

Procedure adopted A for synthesis, amine used dimethyl amine. Muddy

-1 grey solid, yield 55% selected IR (KBr, cm ); vmax Aliphatic C-C 2926, 2847,

C=N 1540, C-N 1197, C=S 1032, M-S 469, UV-vis λ (nm); 260, 355. 1H NMR

(CDCl3): δ 3.37 (s, 6H, 2CH3), 3.31 (br. s, 6H, 2CH3), 3.25 (s, 6H, 2CH3), 3.07 (br.

13 13 S, 6H, 2CH3). C NMR (CDCl3): Due to paramagnetic nature of the compound C

NMR is silent. Anal. Calc. for (C6H12N3OS)2Cu (MW = 412): C = 34.9; H = 4.1; N

= 20.3. Found values: C = 34.2; H = 3.9; N = 20.0.

2.6 SYNTHESIS OF BIURET, THIOBIURET AND DITHIOBIURE

COBALT(II) COMPLEXES USING DIFFERENT AMINES

Cobalt(II) complexes (25-35) with thio and dithiobiuret ligand is synthesize by reacting 3,3-diethylcarbamoyl or 3,3-dimethylcarbamyl chloride with potassium thiocyanide and secondary amines using solvent acetonitrile with addition of cobalt acetate tetrahydrate according to scheme 2.1. Crude product of green color

obtained was recrystallized from chloroform. Green fine crystals obtained and subjected for single crystal X-ray studies on slow evaporation after placing for several days.

2.6.1 Tris [1,1,5,5-tetra ethyl-4-thiobiureto]Cobalt(II) 25

C H C2H5 N 2 5 N C C N C H C2H5 2 5 O S C2H5 N S C H Co O 2 5 C H C N 2 5 C C2H5 N O S C C N C2H5 N N C2H5 C2H5 C2H5

Adopted procedure A for synthesis, diethylamine is used. Dark green

-1 viscous liquid, yield 62% selected IR (KBr, cm ); vmax Aliphatic C-C 2918, C=N

1556, C=S 1072, C=O 1693, N-C 1172, M-O 655, M-S 478, UV-vis λ (nm); 266,

313, 623. Anal. Calc. for (C10H20N3OS)3Co (MW = 749): C = 52.8; H = 8.0; N =

16.8. Found vales: C = 52.5; H = 7.5; N = 16.5.

2.6.2 Tris [1,1-diethyl-5-(benzylmethyl)-4-thiobiureto]Cobalt(II) 26

CH C2H5 N 3 N C C N C H 2 5 O S

N S C H Co O 2 5 H C C N 3 C C2H5 N O S C C N C2H5 N N H3C C2H5

Adopted procedure A for synthesis, N-benzylmethylamine is used. Dark

o -1 green solid product, m.p 111 C, yield 54% selected IR (KBr, cm ); vmax Aliphatic

C-C 2925, C=N 1514, C=S 1076, C=O 1635, N-C 1122, Ar C=C 1473, M-O 520,

M-S 472, UV-vis λ (nm); 266, 318, 621. Anal. Calc. for (C13H18N3OS)2Co (MW =

851): C = 54.9; H = 6.3; N = 14.8. Found values: C = 54.2 ; H = 6.0; N = 14.5.

2.6.3 Tris[1,1-diethyl-5-(3-hydroxypyridine)-4-thiobiureto)]Cobalt(II) 27

HO H N C2H5 N N C C N N C2H5 O S

N C H S Co O 2 5 H C N C C2H5 N O S C C N C H N N N 2 5 H

C2H5

Procedure adapted A for synthesis, amine is 2-amino-3-hydroxy pyridine.

o -1 Dark green solid product, m.p 140 C, yield 55% selected IR (KBr, cm ); vmax

Aliphatic C-C 2929, C=N 1544, C=S 1078, C=O 1627, N-C 1153, M-O 574, M-S

458, O-H 3403, UV-vis λ (nm); 257, 342, 546, 619, Anal. Calc. for

(C11H15N4O2S)2Co (MW = 869): C = 45.5; H = 5.2; N = 19.3. Found value: C

=45.0; H = 4.5; N = 19.2.

2.6.4 Tris[1,1-diethyl-5-(1-(2-furoyl)-pipraziyl)-4-thiobiureto]Cobalt (II) 28

O C2H5 N N C C N N O C2H5 O O O S N S Co C2H5 N C O N C C H N O S 2 5 C C N C2H5 N N C H N 2 5 O O

Procedure adopted A for synthesis, amine used is 1-(2-furoyl peprazine).

o -1 Dark green solid product, m.p 115 C, yield 63%, selected IR (KBr, cm ); vmax

Aliphatic C-C 2925, Ar C=C 1427, C=N 1542, C=S 1078, C=O 1620, N-C 1153,

M-O 595, M-S 644, UV-vis λ (nm); 267, 313, 626. Anal. Calc. for

(C15H20N4O3S)2Co (MW = 974): C = 55.4; H = 6.4; N = 17.2. Found values:

C=55.1; H = 6.0; N = 16.5.

2.6.5 Tris [1,1-diethyl-5-(4-nitrophenyl)-4-thiobiureto]Cobalt(II) 29

H C2H5 N N N C C C H O2N 2 5 O S NO2 S Co C2H5 N C O N H C C H N O S 2 5 C C N C H N 2 5 HN C2H5

NO2

Procedure adapted A for synthesis, amine used is 4-nitroanniline. Dark

o -1 green solid product, m.p 145 C, yield 66%, selected IR (KBr, cm ); vmax Aliphatic

C-C 2931, Ar C=C 1423, C=N 1598, C=S 1078, C=O 1652, N-C 1174, M-O 555,

M-S 493, UV- vis λ (nm); 334 Anal. Calc. for (C12H15N4O3S)2Co (MW = 944): C

=45.7; H = 4.7; N = 17.7. Found values: C = 45.2; H = 4.2; N = 17.2.

2.6.6 Tris[1,1-diethyl-5-(aminobenzyl)-4-thiobiureto]Cobalt(II) 30

NH2 C2H5 N N N C C C2H5 O S

S Co C2H5 N C O N C C2H5 H2N N O S C C N C H N 2 5 N

C2H5 H2N

Procedure adapted A for synthesis, amine used is phenyl hydrazine. Dark

o -1 green solid product, m.p 150 C, yield 54% selected IR (KBr, cm ); vmax Aliphatic

C-C 2929, Ar C=C 1404, C=N 1556, C=S 1081, C=O 1616, N-C 1178, N-H 3191,

M-O 525, M-S 617, UV-vis λ (nm); 309, 614, Anal. Calc. for (C12H16N4OS)2Cu

(MW = 851): C = 50.7; H = 5.6; N = 18.9. Found values: C= 50.0; H = 5.0; N =

19.2.

2.6.7 Tris [1,1-diethyl-5-(2-hydroxy benzoamide)-4-thiobiureto]Cobalt(II) 31

H O N NH C C2H5 N N C C OH C2H5 O S O H S Co C2H5 C HN N C O N C HO C2H5 N O S C C N C2H5 N O HN C2H5 N C OH H

Procedure adopted A for synthesis, amine used is 2-hydrazinylphenol. Dark

o -1 green solid product, m.p 80 C, yield 55% selected IR (KBr, cm ); vmax Aliphatic

C-C 2929, Ar C=C 1404, C=N 1544, C=S 1083, C=O 1614, N-C 1151, O-H 3411,

M-O 547, M-S659, UV-vis λ (nm); 315, 543, Anal. Calc. for (C13H17N4O3S)2Co

(MW = 986): C = 47.4; H = 5.1; N = 17.0. Found values: C = 47.1; H = 4.4; N =

16.5.

2.6.8 Tris [1,1-diethyl-5-(methyl benzyl)-4-thiobiureto]Cobalt(II) 32

C2H5 N N C C N H C2H5 O S H S Co O C2H5 N C N C C H N O S 2 5 C C N C2H5 N HN C2H5

Procedure adopted A for synthesis, amine used benzyl amine. Dark green

-1 viscous liquid, yield 55% selected IR (neat, cm ); vmax Aliphatic C-C 2923, Ar

C=C 1404, C=N 1552, C=S 1081, C=O 1633, N-C 1168, M-O 551, M-S 420, UV- vis λ (nm); 531, 623. Anal. Calc. for (C13H1 8N3OS)2Co (MW = 851) : C = 54.9; H

= 6.3; N = 14.8. Found values: C = 54.2; H = 6.0; N = 14.3.

2.6.9 Tris [1,1-diethyl-5-(dodecylamine)-4-thiobiureto]Cobalt(II) 33

H C2H5 N N C C N CH3 C2H5 O S

C H S Co O N 2 5 N C H3C C C2H5 H N O S C C N C2H5 N HN CH C2H5 3

Procedure adopted A for synthesis, amine used dodecyl amine. Dark green

-1 solid, yield 56% selected IR (KBr, cm ); vmax Aliphatic C-C 2921, C=N 1556,

C=S 1004, C=O 1645, N-C 1103, M-O 520, M-S 617, UV-vis λ (nm); 621, Anal.

Calc. for (C18H33N3OS)2Co (MW = 1076): C = 60.2; H = 9.2; N = 11.7. Found values: C = 59.7; H = 8.5; N = 11.5.

2.6.10 Tris[1,1-diethyl-5-(2,4-dinitrophenylhydrazine)-4-thiobiureto]Cobalt(II)

N2O H2N C2H5 N N C C N N2O C2H5 O S C H NH2 2 5 S Co O N ON N C 2 C C H N O S 2 5 C N N2O C C2H5 N N NH2 C2H5 NO2

N2O

Procedure adopted A for synthesis, amine used 2,4-dinitrophenyl hydrazine.

o -1 Dark green solid, m.p 120 C, yield 65% selected IR (KBr, cm ); vmax Aliphatic C-

C 2931, C=N 1550, C=S 1105, C=O 1618, N-C 1126, M-O 418, M-S 626, UV-vis

λ (nm); 285, 344, Anal. Calc. for (C12H15N6O5S)2Co (MW = 844): C = 48.8 ; H =

5.0; N = 28.5. Found values: C = 48.2; H = 4.5; N = 28.0.

2.6.11 Tris[1,1-diethyl-5,5-dipropyl-4-thiobiureto]Cobalt(II) 35

H3C C2H5 CH3 N N CH3 C2H5 N C C O S CH3 CH3 C2H5 H3C S Co O N N C C C2H5 H3C N O S C C N H C 3 C2H5 N CH N 3 C H 2 5 CH3 H3C CH3

Procedure adopted A for synthesis, amine used dipropyl amine. Dark green

-1 viscous liquid, yield 60% selected IR (neat, cm ); vmax Aliphatic C-C 2925, C=N

1556, C=S 1076, C=O 1600, N-C 1172, M-O 520, M-S 474, UV-vis λ (nm) 650,

1 H NMR (CDCl3): δ 0.86 (br. s, 6H, 2CH3), 0.88 (br. s, 6H, 2CH3), 3.26-3.30 (m,

13 4H) 3.51-3.58 (br. s, 12H, 4CH3), 1.51-1.58 (br. s, 4H), CNMR (CDCl3): 11, 21,

44, 58, 164. Anal. Calc. for (C12H24N3OS)2Co (MW = 833): C = 51.8; H = 8.6; N =

14.5. Found values: C = 51.2; H = 8.2; N =14.8.

2.7 SYNTHESIS OF COMPLEXES OF BIURET, THIOBIURET AND

DITHIOBIURET CADMIUM(II) COMPLEXES USING DIFFERENT

AMINES

Complex (36) was synthesized by the similar method A, described above with use of cadmium acetate tetrahydrate and followed scheme 2.1. Crude product of yellow color obtained was recrystallized from chloroform. Yellow fine crystals obtained on slow evaporation after placing for several days. The structural investigation of complex 36 was carried out by X-ray diffraction studies.

2.7.1 Bis [1,1,5,5-tetramethyl-2,4-dithiobiureto]Cadmium(II) 36

H3C N CH3 N C C N H3C CH3 S S

S S H3C CH3 N C C N H C 3 N CH3

Procedure adopted for this synthesis is A, amine used dimethyl amine.

o -1 Yellow crystalline solid, m.p 170 C, yield 70% selected IR (KBr, cm ): vmax C=C

1 2921, 2857, C=N 1568, C-N 1108, C=S 1053, M-S 445, H NMR (CDCl3): δ 3.39

13 (br. s, 12H, 4CH3), 3.17 (br. s, 12H, 4CH3), C NMR (CDCl3): δ 173, 42, 39.

Anal. Calc. for (C6H12N3S2)2Cd (MW = 493): C = 29.2; H = 4.8; N = 17.0. Found values: C = 29.0; H = 4.5; N = 16.5.

2.8 SYNTHESIS OF BIURET, THIOBIURET DITHIOBIURET ZINC(II)

COMPLEXES USING DIFFERENT AMINES

The zinc(II) complexes (37-47) were synthesized by the reaction of commercially available secondary amine and in situ generated 3,3- diethyllthiocarbamoylthiocyanate or N,N-dimethyldithiocarbamoylcyanate in acetonitrile with addition of zinc(II) acetate dihydrated according to scheme 2.1.

Crude product of off-white color obtained was recrystallized from chloroform.

2.8.1 Bis [1,1,5,5-tetra ethyl-4--thiobiureto] Zinc(II) 37

C H C2H5 N 2 5 N C C N C H C2H5 2 5 O S Zn

S O C H C2H5 2 5 N C C N C H C2H5 2 5 N Procedure adopted A for synthesis, diethylamine is used. Off-white solid,

o -1 m.p 102 C, yield 54% selected IR(KBr, cm ): vmax Aliphatic C-C 2929, C=N

1521, C=S 1074,C=O 1612, N-C 1122, M-O 619, M-S 500, UV-vis λ (nm);548,

1 554, H NMR (CDCl3): δ 1.08(br. s, 12H, 4CH3), 3.17(br. s, 8H, 4CH2).Anal. Calc. for (C10H20N3OS)2Zn (MW = 525): C = 45.7; H = 7.6; N = 16.0. Found value: C =

45.0; H = 7.1; N = 15.5.

2.8.2 Bis [1,1-diethyl-5-(benzylmethyl)-4-thiobiureto]Zinc(II) 38

C H CH 2 5 N 3 N C C N C H 2 5 O S Zn

S O C H H3C 2 5 N C C N N C2H5

Adopted procedure A for synthesis, N-benzylmethylamine is used. Off- white solid product, m.p decomposed at heat, yield 52% selected IR (KBr cm-1); vmax Aliphatic C-C 2933, C=N 1560, C=S 1085, 1000, C=O 1610, N-C 1128, Ar

C=C 1473, M-O 622, M-S 459, UV-vis λ (nm); 412, Anal. Calc. for

(C13H18N3OS)2Zn (MW = 593): C = 52.6 ; H = 4.3 ; N = 14.1. Found values: C =

52.1; H = 4.0; N = 14.0.

2.8.3 Bis [1,1-diethyl-5-(3-hydroxypyridine)-4-thiobiureto)]Zinc(II) 39

OH C H H 2 5 N N C C N C H N 2 5 O S Zn HO S O C2H5 N C C N N H N C2H5

Procedure adapted A for synthesis, amine is 2-amino-3-hydroxy pyridine.

o -1 Off white solid product, m.p 145 C, yield 59% selected IR (KBr cm ); vmax

Aliphatic C-C 2931, C=N 1548, C=S 1076, 999, C=O 1616, N-C 1126, M-O 525,

M-S 609, O-H 3403, UV-vis λ (nm) 368, Anal. Calc. for (C11H15N4O2S)2Zn (MW

= 599): C = 44.0; H = 5.0; N = 18.6. Found value: C = 43.7; H = 4.5; N = 18.1.

2.8.4 Bis [1,1-diethyl-5-(1-(2-furoyl)-pipraziyl)-4-thiobiureto]Zinc(II) 40

C H 2 5 N N C C N C H 2 5 O S N O Zn O

S O C2H5 N C C N N N C2H5 O O

Procedure adopted A for synthesis, amine used is 1-(2-furoyl peprazine).

-1 Off-white viscous product, yield 54% selected IR (KBr cm ); vmax Aliphatic C-C

2921, Ar C=C 1427, C=N 1573, C=S 1091,1004, C=O 1645, N-C 1122, M-O 543,

M-S 435, UV- vis λ (nm); 277, Anal. Calc. for (C15H20N4O3S)2Zn (MW = 759): C

= 47.4; H = 5.5; N = 14.7. Found values: C = 47.0; H = 5.1; N = 14.2.

2.8.5 Bis [1,1-diethyl-5-(4-nitrophenyl)-4-thiobiureto]Zinc(II) 41

C2H5 C2H5 N HN NO N 2 C C

O S Zn S O C2H5 C O2N N C N H N C2H5

Procedure adapted A for synthesis, amine used is 4-nitroanniline. Off-white

-1 viscous liquid product, yield 55%, selected IR(neat cm );vmax Aliphatic C-C 2929,

Ar C=C 1423, C=N 1598, C=S 1058, C=O 1685, N-C 1139, 1004, M-O 526, M-S

493, UV- vis λ (nm); 274, 341. Anal. Calc. for (C12H15N4O3S)2Ni (MW = 648): C

=43.9; H = 4.5; N = 17.0. Found values: C = 43.5; H = 4.0; N = 16.5.

2.8.6 Bis[1,1-diethyl-5-(aminobenzyl)-4-thiobiureto] Zinc(II) 42

C2H5 N N C C N NH2 C2H5 O S Zn

S O C2H5 H2N N C C N N C2H5

Procedure adapted A for synthesis, amine used is phenyl

-1 hydrazine. Off-white liquid viscous product, yield 56% selected IR(neat cm ); vmax

Aliphatic C-C 2929, Ar C=C 1404, C=N 1593, C=S 1076,1010, C=O 1666, N-C

1153, N-H 3191, M-O 543, M-S 489, UV-vis λ (nm); 320, 431, Anal. Calc. for

(C12H16N4OS)2Zn (MW = 593): C = 48.5; H = 5.3; N = 18.0. Found values: C =

48.1; H = 5.0; N = 17.5.

2.8.7 Bis[1,1- diethyl-5-(2-hydroxybenzoamide)-4-thiobiureto]Zinc(II) 43

H O C2H5 N N C C N NH C C H OH 2 5 O S Zn

S O O H C2H5 C HN N C C N HO N C2H5

Procedure adopted A for synthesis, amine used is 2-hydrazinylphenol. Off-

o -1 white solid product, m.p 50 C, yield 58% selected IR(KBr cm ); vmax Aliphatic C-

C 2916, Ar C=C 1404, C=N 1544, C=S 1081, 1010 C=O 1629, N-C 1172, O-H

3411, M-O 621, M-S 475, UV-vis λ (nm); 278, 335. Anal. Calc. for

(C13H17N4O3S)2Zn (MW = 683): C = 45.6; H = 4.9; N = 16.3. Found values: C =

45.2; H = 4.2; N = 16.0.

2.8.8 Bis [1,1-diethyl-5-(methyl benzyl)-4-thiobiureto]Zinc(II) 44

C H 2 5 N N C C NH C2H5 O S

S H O C2H5

N C C N N C2H5

Procedure adopted A for synthesis, amine used benzyl amine. Off-white

-1 viscous liquid, yield 55% selected IR (neat cm ); vmax Aliphatic C-C 2929, Ar

C=C 1404, C=N 1645, C=S 1076, 1027, C=O 1699, N-C 1180, M-O 524, M-S

495, UV-vis λ (nm);321, 531.Anal. Calc. for (C13H1 8N3OS)2Zn (MW = 593): C =

52.6; H = 6.0; N = 14.0. Found values: C = 52.1; H = 5.5; N = 13.5.

2.8.9 Bis[1,1-diethyl-5-(dodecylamine)-4-thiobiureto]Zinc(II) 45

C2H5 N H CH N C C N 3 O C2H5 S Zn

S O C2H5

H3C N C C N H N C2H5

Procedure adopted A for synthesis, amine used dodecyl amine. Off-white

-1 viscous liquid, yield 52% selected IR (neat cm ); vmax Aliphatic C-C 2923, C=N

1550, C=S 1080, C=O 1618, N-C 1180, M-O 628, M-S 426, UV-vis λ (nm); 321,

628. Anal. Calc. for (C18H33N3O)2Zn (MW = 743): C = 58.1; H = 8.8; N = 11.3.

Found values: C = 57.7; H = 8.2; N = 11.0.

2.8.10 Bis[1,1-diethyl-5-(2,4-dinitrophenylhydrazine)-4-thiobiureto]Zinc(II) 46

C2H5 N N C C N NH2 C2H5 O S N2O

Zn N2O

S O C2H5 H2N N C C N N C2H5 ON2

N2O Procedure adopted A for synthesis, amine used 2,4-dinitrophenyl hydrazine.

o -1 Off-white solid, m.p 143 C yield 61% selected IR(KBr cm ); vmax Aliphatic C-C

2931, C=N 1544, C=S 1058, C=O 1618, N-C 1134, M-O 511, M-S 428, UV-vis λ

1 (nm); 342, 415, 556. H NMR (CDCl3): δ 1.29 (br. s, 12H, 4CH3), 3.43 (br. s, 8H,

13 4CH3). 7.95-7.97 (m, 6H), 4.04 (m, 4H) C NMR (CDCl3): δ 17, 25, 48, 116, 130,

155. Anal. Calc. for (C12H15N6O5S)2Zn (MW = 775): C = 37.1; H = 3.8; N = 21.0.

Found values: C = 36.4; H = 3.5; N = 20.5.

2.8.11 Bis[1,1-diethyl-5,5-dipropyl-4-thiobiureto]Zinc(II) 47

H3C C H N 2 5 CH3 N C C N C H CH 2 5 O S 3 CH3 Zn

CH3 S O C2H5 H3C N C C N C H H3C N 2 5 H3C Procedure adopted A for synthesis, amine used dipropyl amine. Off-white

-1 viscous liquid, yield 54% selected IR (neat cm ); vmax Aliphatic C-C 2925, C=N

1558, C=S 1080, C=O 1614, N-C 1180, M-O 510, M-S 621, UV-vis λ (nm); 215,

365, Anal. Calc. for (C12H24N3OS)2Ni (MW = 581): C = 49.5; H = 8.2; N = 14.4.

Found values: C = 49.0; H = 7.5; N =14.0.

2.9 PROCEDURE (B) ADAPTED FOR SYNTHESIS OF BIURET

THIOBIURET AND DITHIOBIURET METAL COMPLEXES

A solution of 3,3-dimethylbutyryl chloride and potassium thiocynate were added into dry acetonitrile (40ml) and stirred for two hours continuously at room temperature. After this was added substituted amines and stirred for more 30 min.

Metal acetate (hydrated) was then added and stirring was continued for 1 hour.

During stirring, excess of distilled water was added and a colored crude product obtained. It was separated by after filtration. Recrystallization was performed by

using chloroform tetrahydrofurain mixture. Crystalline solid were characterized by

XRD.

O R O R KSCN R R Cl CH3CN NCS R R

HNR R = Me, Et 2

M = Co, Ni, Cu, Cd, Zn

O R S R R N N R H R

M(OAc) 2 H2O R R N N R

R R O S M

O S R R R N N R R

Scheme 2.2: General scheme for synthesis of thiobiuret metal by method B.

2.10 SYNTHESIS OF THIOBIURET METAL COMPLEXES USING

DIFFERENT AMINES

2.10.1 Bis[(Z)-3-(3,3-dimethylbutanoyl)-1,1-diethyl-2-thiobiureto]Nickel(II) 48

C2H5 N N C C2H5 S O Ni

O S

C C2H5 N N C2H5 Procedure adopted is B for synthesis, diethylamine is used. Purpled colored

o -1 solid, m.p 80 C, yield 67% selected IR ( KBr cm ); vmax Aliphatic C-C 2929,

C=N 1558, C=S 1076, C=O 1616, N-C 1130, M-O 524, M-S 428, UV-vis λ (nm);

1 275, 536, H NMR (CDCl3): δ 1.11 (br. s, 6H, 2CH3), 1.21 (br. s, 6H, 2CH3), 3.57-

3.77 (m, 8H, 4CH2), 0.98 (br. s, 18H, 6CH3), 2.13 (br. s, 4H, 2CH2). Anal. Calc. for

(C11H21N2OS)2Ni (MW = 517): C =51.1; H = 8.1; N = 10.8. Found values: C =

50.5; H = 7.5; N = 10.2.

2.10.2 Bis[(Z)-3-(3,3-dimethylbutanyol)-1-(2-hydroxyphenyl)-2-

thiobiurato]Nickel(II) 49

NH C O S N Ni N C S O C HN

Procedure adopted B for synthesis, 2-amino phenol is used. Purple colored

o -1 solid, m.p 160 C, yield 71% selected IR ( KBr cm ); vmax Aliphatic C-C 2958,

C=N 1544, C=S 1037, C=O 1608, N-C 1141, M-O 597, M-S 536, N-H, 3218, UV- vis λ (nm); 262, 504. Anal. Calc. for (C13H18N2O2S)2 Ni(MW = 588): C =53.0; H =

6.1; N = 9.5. Found values: C = 52.5; H = 5.5; N = 9.1.

2.10.3Bis[(Z)-3-(3-dimethylbutanoyl)-1-(3-morpholinopropyl)-2-

thiobiureto]Nickel(II) 50

O N

N O S NH Ni HN S O N

Procedure used B for synthesis, 3-morpholinopropylamine is used. Purpled

o -1 colored solid, m.p 116 C, yield 61% selected IR (KBr cm ); vmax Aliphatic C-C

2948, C=N 1564, C=S 1009, C=O 1610, N-C1141, N-H 3228, M-O 621, M-S 501,

UV-vis λ (nm); 262, 517. Anal. Calc. for (C14H27ON3S)2Ni (MW = 657): C = 51.1;

H = 8.2; N = 12.7. Found values: C = 50.5; H = 8.0; N =12.2.

2.10.4 Bis[(Z)-3-(3,3-dimethylbutanoyl)-1,1-diethyl-2-thiobiureto]Copper(II) 51

C2H5 N N

C2H5 C S O Cu

O S

C C2H5 N N C2H5

Procedure adopted B for synthesis, amine used diethyl amine. Muddy grey

o -1 solid, m.p 110 C, yield 62% selected IR (KBr cm );vmax Aliphatic C-C 2935,

C=N 1558, C=S 1043, C=O 1620, N-C 1193, M-S 617, M-S 518, UV-vis λ (nm);

1 557, 688. H NMR (CDCl3): δ 1.10 (br. s, 6H, 2CH3), 1.20 (br. s, 6H, 2CH3), 3.56 -

3.78 (m, 8H, 4CH2), 0.98 (br. s, 18H, 6CH3), 2.15 (br. s, 4H, 2CH2). Anal. Calc. for

(C11H21N2OS)2Cu (MW = 521): C = 50.6; H = 8.0; N = 10.0. Found values: C = 5

0.1; H = 7.6; N = 9.5.

2.10.5 Bis[(Z)-3-(3,3-dimethylbutanoyl)-1-(2-hydroxyphenyl)-2-Copper(II) 52

NH C O S N Cu N C S O C HN

Procedure adopted B for synthesis, 2-amino phenol is used. Muddy grey

o -1 solid, m.p 142 C, yield 79%, selected IR (KBr cm ); vmax Aliphatic C-C 2931,

C=N 1558, C=S 1018, C=O 1602, N-C 1147, N-H 3251, M-O 530, M-S 434, UV- vis λ (nm); 241, 557,688, Anal. Calc. for (C13H18N2O2S)2Cu (MW = 593): C =

52.6; H = 6.0; N = 9.4. Found values: C = 52.0; H = 5.5; N = 9.0.

2.10.6 Bis[(Z)-3-(3,3-dimethylbutanoyl)-1-(3-morpholinopropyl)-2-

thiobiureto]Copper(II) 53

O N

N O S NH Cu HN S O N

Procedure adopted B for synthesis. 3-morpholinopropylamine is used.

-1 Muddy grey solid, yield 57%, selected IR (KBr cm ); vmax Aliphatic C-C 2945,

C=N 1585, C=S 1110, C=O 1652, N-C 1235, N-H, 3438, M-O 514, M-S 464, UV-

vis λ (nm); 248. Anal. Calc. for (C14H27ON3S)2Cu (MW = 663): C =50.6; H = 8.1;

N = 12.0. Found values: C = 50.1; H = 7.5; N = 11.5.

2.10.7 Tris[(Z)-3-(3,3-dimethylbutanoyl)-1,1-diethyl-2-thiobiureto]Cobalt(II)

C2H5 N C H 2 5 N C

O S O Co N S C S C2H5 O C N N C2H5 N C2H5 C2H5

Procedure used B for synthesis, diethyl amine is used. Dark green solid

o -1 yield, m.p 120 C, 52% selected IR (KBr cm ); vmax Aliphatic C-C 2933, C=N

1525, C=S 1076, C=O 1645, N-C 1174, M-O 516, M-S 447, UV-vis λ (nm);265,

1 536, 605, H NMR (CDCl3): δ 1.12 (br. s, 6H, 2CH3), 1.25 (br. s, 6H, 2CH3), 3.65-

3.99 (m, 8H, 4CH2), 0.98 (br. s, 18H, 6CH3), 2.16 (br.s, 4H, 2CH2). Anal. Calc. for

(C11H21N2OS)3Co (MW = 746): C = 52.1; H = 8.3; N = 11.0. Found values: C =

51.7; H = 8.0; N = 10.5. XRD data of this complex is presented and discussed in next chapter.

2.10.8 Tris[(Z)-3-(3,3-dimethylbutanoyl)-1-(2-hydroxyphenyl)-2-

thiobiureto]Cobalt(II) 55

NH N C O S C N Co O C S S HN O NH OH C N HO

Procedure adopted B for synthesis, 2-aminophenol is used. Dirty green

o -1 solid, m.p 150 C, yield 87% selected IR (KBr cm ); vmax Aliphatic C-C 2952,

C=N 1560, C=S 1039, C=O 1645, N-C 1145, N-H 3230, M-O 592, M-S 532, UV- vis λ (nm); 278, 320. Anal. Calc. for (C13H18N2O2S)3Co (MW = 857): C = 54.6; H

= 6.3; N = 9.8. Found values: C = 54.0; H = 6.0; N = 9.5.

2.10.9 Tris[(Z)-3-(3,3-dimethylbutanoyl)-1-(3-morpholinopropyl)-2-

thiobiureto]Cobalt(II) 56

N H N N

S O

O Co N S S O N N N HN H O

N O

Procedure adopted B for synthesis, 3-morpholinopropylamine is used.

o Muddy greenish solid, m.p 220 C, yield 49% selected IR (cm-1); vmax Aliphatic C-

C 2952, C=N 1550, C=S 943, C=O 1652, N-C 1116, M-O 621, M-S 501, N-H

3438, UV-vis λ (nm); 259, 547, Anal. Calc. for (C14H27ON3S)3Co (MW = 914): C

= 55.1; H = 8.8; N = 13.7. Found values: C = 54.7; H = 8.2; N = 13.1.

2.10.10 Bis [(Z)-3-(3,3-dimethylbutanoyl)-1,1-diethyl-2-thiobiureto]Zinc(II) 57

C2H5 N N

C2H5 C S O Zn

O S

C C2H5 N N C2H5

Procedure adopted B for synthesis, diethylamine is used. Off-white solid,

o -1 m.p 234 C, yield 56% selected IR (KBr cm ); vmax Aliphatic C-C 2946, C=N

1538, C=S 1070, C=O 1631, N-C 1180, M-O 546, M-S 490, UV-vis λ (nm); 548,

1 13 554, H NMR (CDCl3): δ 3.10 (br. s, 12H, 4CH3), 3.07 (br. s, 12H, 4CH3), C

(CDCl3): δ 173, 41, 39. Anal. Calc. for (C11H21N2OS)2Zn (MW = 523): C = 50.4; H

= 8.0; N = 10.7. Found values: C = 50.0; H =7.7; N =10.5.

2.10.11Bis[(Z)-3-(3,3-dimethylbutanoyl)-1-(2-hydroxyphenyl)-2-

thiobiureto]Zinc(II) 58

NH C O S N Zn N C S O C HN

Procedure adopted B for synthesis, 2-aminophenol is used. Off-white solid,

o -1 m.p 200 C, yield 73% selected IR (KBr cm ); vmax Aliphatic C-C 2960, C=N

1568, C=S 1010, C=O 1606, N-C 1149, N-H 3226, M-O 624, M-O 529, UV-vis λ

(nm); 280. Anal. Calc. for (C13H18N2O2S)2Zn (MW = 595): C =52.4; H = 6.0; N =

9.4. Found values: C = 50.0; H = 5.5; N = 9.1.

2.10.12Bis[(Z)-3-(3,3-dimethylbutanoyl)-1,1-diethyl-2-

thiobiureto]Cadmium(II) 59

C2H5 N N C C2H5 S O Cd

O S

C C2H5 N N C2H5 Procedure adopted B for synthesis, diethylamine is used. Yellow solid, m.p

o -1 175 C, yield 59% selected IR (KBr cm ); vmax Aliphatic C-C 2959, C=N 1558,

C=S 1070, C=O 1612, N-C 1193, M-O 684, M-S 518, UV-vis λ (nm); 298, 552,

605. Anal. Calc. for (C11H21N2OS)2Cd (MW = 570): C = 46.6; H = 7.3; N = 9.8.

Found values: C = 46.0; H = 7.0; N =9.3.

2.10.13Bis[(Z)-3-(3,3-dimethylbutanoyl)-1-(2-hydroxyphenyl)-2-

thiobiureto]Cadmium(II) 60

NH C O S N Cd N C S O C HN

Procedure adopted B for synthesis, 2-aminophenol is used. Yellow solid

o -1 product, m.p 198 C, yield 53% selected IR (KBr cm ); vmax Aliphatic C-C 2923,

C=N 1548, C=S 1020, C=O 1635, N-C 1140, N-H 3238, M-O 597, M-S 520, UV– vis λ (nm); 279, Anal. Calc. for (C13H18N2O2S)2Cd (MW = 642): C = 48.5; H =

5.6; N = 8.7. Found values: C = 48.0; H = 5.0; N = 8.2.

2.11 BIOLOGICAL ACTIVITIES

Generally, microorganisms have an inherent character and acquire resistance to drugs; these are active as therapeutic agents. In the last three decades, the pharmaceutical industry has produced a large variety of new antibiotics and the resistance of microbes to these drugs has been increased (Faúndez et al., 2004).

Metal ion complexes are performing a vital role in the development of antibiotics. Copper and cobalt ions are well known as inhibitor to retard the growth of harmful bacteria and fungi since centuries (Stanila et al., 2011). Contagious diseases separated by bacteria and fungi have been successfully treated by

antimicrobial drugs (antibacterial and antifungal) since ancient time (Alfallous and

Aburzeza). By decreasing the death rate due to infectious diseases, antimicrobial therapy has effectively prolonged the average life expectancy. Due to the constant use and mishandling of antimicrobial drugs they are the big source of producing more resistant pathogens which are frequently use as antimicrobials. The infectious diseases treatment have been made more complex by the presence of multidrug resistant strains as compared to the ﬁrst half of last century (Grare et al., 2007).

Fungal infections are becoming common in patients undergoing chemotherapy, organ transplants and infected with AIDS with suppressed immune systems (Ghannoum and Rice, 1999). Moreover, the development of fungal resistance, has made antifungal drugs less effective (Eliopoulos et al., 2002). As a result, recently the cases of microbial infections in many countries of the world have been increased due to antimicrobial resistance. There is the need for the synthesis of new antimicrobial compounds to face this alarming situation (Kabbani et al., 2007).

Bioassays are a basic key in developing the quality and standard of bioactive compounds (Bohlin and Bruhn, 1999). In the present study, primary bioassays are studied because they are common in nature, easy to handle, predictive fast, tolerant against impurities, reproducible and compatible with dimethylsulfoxide (Ghosh, 2007) and help in characterization of bioinorganic nature of synthesized complexes.

2.11.1 Antimicrobial Assay

In the present era due to inappropriate use of antibiotics resistant bacteria and fungi are becoming an alarming problem worldwide. Antibiotics were

considered as life saving drugs but in the last few decades many microbes become multi drug resistance. To solve this challenge many inventions come in drug history and derivatives of exctant drugs are introduced (Hawkey, 2008). All the newly synthesized compounds were screened for antimicrobial activity against pathogenic bacteria measured by disc diffusion method (antibacterial activity)

(Kavitha et al., 2016) and fungi zone of inhibition measured by sabouraud dextrose agar (antifungal activity). Antibacterial activity analysis proceeds against

24 hours old cultures of bacterial pathogens and antifungal activity in 72 hours old fungal pathogens and left to grow.

Echerishia coli, Staphylococcus aureus and Pseudomonas aeruginosa were used in the present work to investigate antibacterial activity. Asperjilus niger,

Flavus solani, Mucor species and Aspirjilus flavus were used to determined antifungal activities of synthesized complexes.

2.11.1.1 Assay procedure

Newly synthesized compounds were screened against microbial pathogens by agar diffusion methods. To start, filter paper discs of 6 mm were swaged with test compounds. Stock solutions containing 50 µg/discs in dimethylsulfoxide were carefully impregnated on agar culture plates, which had already been inoculated separately with pathogens. Subsequently, impregnated discs were incubated at

37 oC for 24 and 48 hours for antibacterial and antifungal activity, respectively.

The zone of inhibition was measured after 24 hours in ppm and compared with positive control. Cefexime and clotrimazole were used as references to evaluate antibacterial and antifungal activity respectively. DMSO alone was used as negative control and two said drugs i.e. cefexime and clotrimazole were used as

positive controls. Former was used to ascertain bacterial inhibition, while the later was used for fungal inhibition. Fourteen compounds were subjected for this assay, separately. Results are shown in tables 2.3 and 2.4.

Table 2.3: Antibacterial activity of tb and dtb metal complexes.

Complex Zone of inhibition 50µg/disc (mm) No E. coli S. aureus P. aeruginosa 1 5 2 7 2 - - 7 4 5 4 5 8 14 10 7 11 5 - - 12 10 6 13 13 13 5 11 14 8 11 10 27 11 10 9 31 5 1 8 32 13 14 10 38 15 11 8 43 - 9 11 46 - 4 7 Cefexime Pc = 20 Pc = 20 Pc = 20

- = Means no activity pc = means positive control which is cefexime.

Table 2.4: Antifungal activity of biuret and dithiobiuret metal complexes.

Complex No Zone of inhibition 50 µg/disc (mm) A. niger F. solani Mucor. sp A. Flavus 2 7 _ - 18 3 - - - 22 4 - 15 - - 8 6 - - - 12 - 8 - 17 14 13 - 20 19 - - 7 - 22 - - 7 - 27 20 - - - 31 - - 7 - 34 - 7 - 38 - - - 16 43 12 - - - 47 22 Clotrimazole Pc = 25 Pc = 25 Pc = 25 Pc = 30

- = Means no activity pc = means positive control which is clotrimazole.

2.11.2 Antioxidant Assay

Antioxidant potential of synthesized complexes was determined using different assays which were discussed below:

2.11.2.1 DPPH Free Radical Scavenging Assay

Antioxidant potential of metal complexes against DPPH was determine by method as described by (Clarke et al., 2013).

2.11.2.2 Assay procedure

The 2,2-diphenyl-1-picrylhydrazyl free radical scavenging assay was carried out by using 96 well plates. Initially, the stock solution was prepared in respective well plates by taking 20 µL of test sample and then added 180 µL of

DPPH solution to make 200 µL final volume. Ascorbic acid was used as standard and DMSO as negative control. The mixture was incubated at room temperature and after half hour color change was observed from violet to yellow which is regarded as an indicator of oxidation potential of tested complexes. Absorbance of tested compounds was recorded at 517 nm on microplate reader. Scavenging activity of sample was calculated as below;

DPPH % scavenging = (OD of control - OD of sample under observation) /

(Absorbance of control × 100)

OD = optical density (absorbance)

2.11.3 Total Antioxidant Capacity

Phosphomolybdenum method was applied to determine total antioxidant capacity of synthesized complexes, as reported by (Dillard and German, 2000).

2.11.3.1 Assay procedure

Assay was proceeded by preparing one mL of reaction mixture by adding

100 µL of reagent solution into sample. Reagent solution was made of ammonium molybdate (4 mM), sodium phosphate (28 mM) and sulfuric acid (0.6M). At 95 oC mixture was incubated for 90 minutes, which was successively cooled and absorbance was measured at 695 nm. Dimethylsulfoxide was used blank. An antioxidant potential of the synthesized complexes was expressed as ascorbic acid equivalent.

2.11.4 Reducing Power Assay

Reducing power assay for synthesized compounds was performed as described by (Shah et al., 2013).

2.11.4.1 Assay procedure

Reducing potential of samples was calculated by taking 100 µL of test sample, 250 µL phosphate buffer and 250 µL potassium ferricyanide in total volume of 600 mL was incubated for 30 minutes. Then after adding of 250 µL of trichloroacetic acid (10%), solution was placed on incubation, which was centrifuged at 3000 rpm for few minutes. After this 250 µL of complex supernatant was taken and poured in their respective well with addition of 20 µL of 0.1% solution ferric cyanide and 30 µL of distilled water. Absorbance of solution was calculated at 700 nm. DMSO was used for negative control. Ascorbic acid equivalent was used for expressing of reducing power of metal complexes. Results are shown in table 2.5.

Table 2.5: Antioxidant activities of tb and dtb metal complexes.

Complex Total Reducing DPPH no antioxidant power Assay Capacity µg AAE/mg µg AAE/mg % scavenging at 200 µg/ml

1 23.8 47.8 66.2

2 42.1 65.8 63.9

3 39.4 65.8 41.5

4 39.9 69.9 80.0

5 23.4 72.0 78.6

6 56.7 54.7 79.8 7 141.4 63.7 60.8 8 43.6 49.9 39.9 10 53.8 52.6 47.2 12 44.4 53.3 50.8 13 33.1 103.9 50.7 14 45.1 69.9 62.9 15 49.0 50.6 6.2 16 40.7 59.6 49.9 17 44.2 52.6 73.1 18 81.6 80.1 66.6 19 81.1 113.6 50.8 21 37.8 99.7 10.7 22 104.3 52.6 42.6 25 66.8 68.6 51.5 26 12.3 58.2 0.2 27 10.0 79.6 28.3

Complex Total Reducing DPPH No antioxidant power Assay Capacity µg AAE/mg µg AAE/mg % scavenging at 200 µg/ml

28 23.7 57.5 18.2 29 17.7 58.9 72.9 30 20.5 64.4 38.5 31 25.1 52.6 46.6 34 15.2 62.3 15.0 37 17.4 43.6 7.7 38 9.5 56.8 50.3 43 29.0 57.7 20.3 46 32.7 57.5 73.4

Ascorbic acid was used as positive control (DPPH IC50 = 0.032 µg/ml)

2.11.5 Protein Kinase Inhibition Assay

Protein kinase inhibition activity of synthesized complexes was checked by observing hyphae formation in purified isolates of streptomyces 85E strain (Fatima et al., 2015).

2.11.5.1 Assay procedure

Bacterial garden was permitted to develop by spreading spores (mycelia fragments) of fresh culture of Streptomyces drawn on sterile plate’s having ISP4 medium. Stock solution was prepared on dissolving 25 µg of sample in solvent

DMSO was planted on to sterile 6mm filter paper discs. The loaded paper discs with final concentration of 100 µg/discs were applied on the plates seeded with streptomyces 85E microbes. DMSO and surfactant was used as negative and

positive control respectively. Incubation was carried out at room temperature for 72 hours and results were interpreted as bald zone of inhibition around sample and infused discs, as shown table 2.6.

Table 2.6: Protein kinase inhibition activity of thiobiurets and dithiobiurets.

Complex No Zone of inhibition at 25 µg/disc

Blade zone Clear Zone

1 - 6

2 - 7.0

3 30 16.0

4 30 8

5 19 15

6 25 12.0

7 20 -

8 - 10.0

10 24 7.0

12 20 16

13 24 9

14 36 14.0

15 46 10

16 32 8.0

17 36 10

18 34 7.0

19 30 6.0

21 - 7.0

Complex No Zone of inhibition at 25 µg/disc

Blade zone Clear Zone

22 - 10

25 - -

26 - -

27 30 13.0

28 24 -

29 14 -

30 - 7.0

31 - 5.0

34 - 5.0

37 - 5.0

38 - 5.0

43 34 12.0

46 - 5.0

Standard 10 µg/discs

Surfactant 18 mm

2.11.6 ABTS Radical Cation Decolourization Bioassay

The antioxidant potential of the complexes was determined by ABTS radical cation decolourization bioassay. The stock solution was prepared by 7 mM ABTS solution and 2.5 mM solution of potassium persulfate. Then testing solution was prepared by combing the two stock solutions in equal quantities and kept them to react overnight at room temperature in dark. The dilution of ABTS solution was carried out by addition of distilled water to obtain an absorbance 2.00 at 405 nm

using a spectrophotometer. For each reading fresh ABTS solution was prepared.

The sixteen complexes were assessed under this procedure and results are shown in table 2.7.

Table 2.7: ABTS antioxidant activities of thiobiuret and dithiobiuret metal

complexes.

Complex No Conc. PSP (%) IC50 μg/ml Antioxidant potential μg/ml 9 20 51.8 60.9 Active 40 65.1 60 72.5 11 20 30.9 124.2 Active 40 43.4 60 72.7 19 20 43.6 41.9 Active 40 51.4 60 57.7 23 20 41.7 124.2 Active 40 55.6 60 76.9 30 20 62.2 29.4 Active 40 70.3 60 72.12 20 79.3 46.3 Active 33 40 89.2 60 94.9 35 20 76.6 102.5 Active 40 82.4 60 62.2

Complex No Conc. PSP (%) IC50 μg/ml Antioxidant potential μg/ml 48 20 36.2 62.7 Active 40 43.4 60 57.3 49 20 18 78.3 Active

40 75.5 60 84.5 51 20 93.4 8.0 Active 40 95.1 60 96.1 20 57.5 19.9 Active 52 40 72.3 60 73.6 20 41.9 34.5 Active 53 40 55.2 60 75.9 20 58.6 39.9 Active 54 40 66.4 60 72.05 55 20 60.7 0.8753 Active 40 84.1 60 89 56 20 49.9 26.8 Active 40 55.6 60 74.4 58 20 22.2 32.9 Active 40 41.5 60 54.8 60 20 32.4 35.5 Active 40 49.9 60 67.9

2.11.7 Anticancer Assay

Cancer is still continuing to be a vital health issue worldwide. The discovery of new anticancer therapeutic agents is one of the fundamental objectives in medicinal chemistry as cancer causes about 13% of all the death. International research is going on to developed new, effective protective and active anticancer agents to target the prevention or cure of this acute illness (Ahsan et al.,

2013). Anticancer drug discovery and development is one of the most essential and rapidly changing avenues for medicinal chemist. The requirement for new chemotherapeutics in cancer is evident due to the limited capacity of drugs to cure or significantly prolong the survival of patients with disseminated tumors or certain leukemias (Rana et al., 2014).

Literature survey revealed that thiourea and its derivatives showed noteworthy anti-cancer activity. Drugs having thiobiuret and dithiobiuret entities are being used as anti-cancer remedial in many clinical trials. Ureas, thioureas and benzothiazoles are considered as powerful anticancer drugs. DNA topoisomerase or HIV reverse transcriptase inhibitors are produced by combination of both ureas and thioureas with benzothiazoles (Saeed et al., 2010; Ahsan et al., 2013).

2.11.7.1 Assay Procedure

The human monocytic leukaemia cell line THP-1 was used for investigation of anticancer activity. The cells were cultivated in the RPMI 1640 medium assisted with 10 percent FBS, 2 mM glutamine, 100 U/mL of penicillin and 100 µg/mL of streptomycin in a steamy atmosphere having 5% carbon dioxide at 37 °C. For getting a concentration of 500000 cells/mL stabilized cells were split into micro titration plates and the differentiation to macrophages was induced by phorbol

myristate acetate dissolved in dimethylsulfoxide (DMSO) at the final concentration of 20 µg/ml. These cells were incubated for 24 hours. In comparison with monocytes, differentiated macrophages tend to clink at the bottoms of the cultivated plates.

Cells were incubated with a fresh complete RPMI medium, i.e. containing antibiotics and FBS, without PMA for next twenty four hours. The medium was then aspirated, and the cells were cleaned with PBS and cultivated for next 24 hours in serum-free RPMI 1640 medium. For the detection of inflammatory response that’s showing inhibition these prepared macrophages were used. As results are given in table 2.8.

Table 2.8: Anticancer activity of thiobiuret and dithiobiuret metal complexes.

Complex no THP1 cancer cell line % inhibition at 20 µg/ml 1 98 2 85 3 87 4 58 5 88 6 92 7 33 8 50 10 73 12 90 13 95 14 98 15 62 16 48 17 83 18 92 19 75 21 60 22 52

Complex no THP1 cancer cell line % inhibition at 20 µg/ml 25 43 26 65 27 55 28 95 29 83 30 96 31 55 34 73 37 95 38 99 39 98 43 46 46 76

2.12 ELECTROLYTIC BEHAVIOUR

Conductance of the synthesized complexes was investigated by using conductometer BANTE DDS-12DW USA with equimolar solutions of tested complexes using dimethylsulfoxide as solvent at a room temperature.

Chapter 3

RESULTS AND DISCUSSIONS

Dithiobiuret and thiobiurets has been investigated as multifaceted compounds. They have been recruited as antimalarial (Curd et al., 1949), hypoglycemic (Rastogi et al., 2001), anti-inflammatory (Asif, 2001) and anti tuberculars (Rastogi et al., 2001) agents. In addition to this they are also reported for their striking insecticidal (Atchison, 1989) herbicidal, pesticidal (Johnson,

1968) and fungicidal action. The biological activity of these compounds mainly ascribed (N-C-S) multiple bond donor moiety which enhanced the chelation power with metal ion. It is very fascinating to note that zone of these compounds is not only spread out to biological field but they are equally recognized in the industrial/material chemistry (Thimmaiah et al., 1985).

Thiobiurets and dithiobiurets compounds are becoming building blocks for the synthesis of nanoparticles (Ramasamy et al., 2010a). It has an abundance of delocalized lone pair of electrons that make it suitable for the stabilization of unusual oxidation states. Moreover, it could possibly chelate through the two/one sulfur atoms or through two/one nitrogen atoms (Khan et al., 2009).

Looking at the broad band spectrum of this class of compounds here in synthesized sixty thiobiuret and dithiobiurt metal complexes with new and reported methods. In the present study, synthesis of complexes is a one-pot synthesis. It consists of (1-12) dithiobiuret and thiobiuret complexes of nickel(II), (13-24) complexes of copper(II) (25-35) cobalt (II) (36) of cadmium(II) and (37-47) complexes of zinc(II) were derived from reaction of N,N-dialkylthiocarbamoyl or

N,N-dialkylcarbamoyl with potassium thiocyanide and twelve different secondary

amines. The (48-60) complexes of dithiobiuret and thiobiuret of these (Ni, Cu, Co,

Zn, Cd) metals were obtained by reaction of 3,3-dimethylbutrylchloried with potassium thiocynaide using a new method as shown scheme 2.2 in previous chapter.

Mostly the prepared solids are crystalline in nature with few exceptions which are viscous liquids. The synthesized complexes 12, 24, 36, 48 and 54 give a fine crystal after recrystallization. These complexes were subjected to XRD analysis to determine their exact geometry. The important discussion, findings and description of results of synthesized complexes are described below.

3.1 SYNTHESIS OF THIOBIURET AND DITHIOBIURET NICKEL(II)

COMPLEXES (1-12)

Nickel(II) complexes with thio and dithiobiuret ligand is synthesized from

3,3-diethylcarbamoyl and 3,3-dimethylcarbamyl chloride with potassium thiocynaide and secondary amines using solvent acetonitrile according to scheme

2.1 as shown in previous chapter.

Twelve complexes of nickel thiobiurets and dithiobiurets were synthesized by using different secondary amines in presence of nickel(II) acetate tetrahydrated.

All the synthesized complexes were obtained in good yield. Mostly the complexes were obtained in purple crystalline solid with good purity and few were also in viscous liquid. The synthesized complexes were characterized by UV-visible, IR,

1H NMR, 13C NMR and elemental analysis. The structure of complex 12 was confirmed through single crystal XRD. Physical properties of thiobiuret and dithiobiuret of nickel complexes is given below in table 3.1.

Table 3.1: Physical properties of synthesized thiobiuret and dithiobiuret nickel(II) complexes.

Elemental Analysis Complex Yield Melting point Molecular formula o Mol.Mass No % C Calculated Found C H N C H N 1 (C10H20N3OS)2Ni 60 138 518 46.3 7.7 16.2 46.0 7.1 16.0

2 (C13H18N3OS)2Ni 52 110 586 53.2 6.1 14.3 53.0 5.4 14.0

3 (C11H15N4O2S)2Ni 55 150 592 44.5 5.0 18.9 44.0 4.5 18.3

4 (C15H20N4O3S)2Ni 65 190 732 49.1 6.7 15.3 48.5 6.0 15.0

5 (C12H15N4O3S)2Ni 56 160 648 44.4 4.6 17.2 44.0 4.0 16.5

6 (C12H16N4OS)2Ni 62 Viscous 586 49.1 4.9 19.1 48.5 4.5 18.5

7 (C13H17N4O3S)2Ni 56 80 676 57.6 5.0 16.5 57.1 4.5 16.0

8 (C13H1 8N3OS)2Ni 55 Viscous 586 53.2 4.4 14.3 53.0 4.0 14.0

9 (C18H33N3OS)2Ni 53 Viscous 736 58.6 8.9 11.4 58.1 8.3 11.0

10 (C12H15N6O5S)2Ni 61 143 768 37.5 3.9 21.8 37.0 3.5 21.0

11 (C12H24N3OS)2Ni 64 Viscous 574 50.1 8.3 14.6 49.5 7.5 14.0

12 (C6H12N3S2)2Ni 73 131 439 41.0 5.4 19.1 40.5 5.0 18.5

3.1.1 UV-Visible Analysis of Thiobiuret and Dithiobiuret Nickel(II)

Complexes (1- 12)

Absorption studies of the synthesized complexes were performed on UV- visible spectrophotometer SHIMADZU 1601. Complexes were dissolved in

DMSO, meanwhile moved to a quartz cell to measure absorbance. Ultraviolet- visible spectroscopy is an important analytical technique which informed us about the geometries and conjugated systems of compounds. The electronic spectra are recorded at 200-400 nm is the range of UV region while 400-800 nm is visible region.

The common possible transitions found in UV-visible spectroscopic spectra of synthesized metal complexes are between the π orbitals to π* orbitals, charge transfer transition between metal and ligand and d-d transition. Some complexes also show n-π* transitions.

The electronic absorption data for nickel(II) complexes is displayed in

Table 3.2. These bands are categories into three groups: The transitions noted in the absorption spectra are π -π* are bands observed at 265-277 nm and charge transfer transitions are assigned at 302-370 nm while d-d transitions are seen in the visible region at 466-556 nm. A few complexes also shows n-π* at 225 nm and 236 nm.

These transitions give hints in relevance to the possible structures of complexes.

The observed absorption bands suggest a possible tetrahedral geometry of Ni(II),

Zn(II), Cd(II), square planar of Cu(II), Ni(II) and octahedral geometry of Co(II) complexes. On comparison with the literature values it has been resulted that the possible geometries of the metal centers are compatible (Ramasamy et al., 2010a).

The absorption spectra of complexes 1, 4 is shown in appendices.

Table 3.2: UV-visible absorption data of nickel(II) complexes (1-12).

Wavelength λ Comp No Transitions (nm) 1 274, 305, 503 π -π*, charge transfer, d-d

2 270, 302, 506 π -π*, charge transfer, d-d

3 275, 322, 516 π -π*, charge transfer, d-d

4 277, 322, 506 π -π*, charge transfer, d-d

5 274, 341, 506 π -π*, charge transfer, d-d

6 225, 320, 431 n-π*, charge transfer, d-d

7 236, 321, 469 n-π*, charge transfer, d-d

8 271, 334, 547 π -π*, charge transfer, d-d

9 321, 466, 531 charge transfer, d-d

10 342, 556 charge transfer, d-d

11 344, 508 charge transfer, d-d

12 265, 370, 520 π -π*, charge transfer, d-d

3.1.2 Infrared Spectroscopic Characterization of Thiobiuret and

Dithiobiuret Nickel(II) Complexes (1-12)

Infrared spectra of the synthesized nickel(II) complexes (1−12) have been collected by preparing solid samples as KBr pellets and liquids samples using KBr cell in the spectral range of 4000-400 cm−1. In spectroscopic studies the important

IR bands of all thiobiuret and dithiobiuret ligands showed the following stretching frequencies υ(C=O), υ(N-H), υ(C-N) and υ(C=S) at 1654–1668, 3286-3322, 1249-

1273 and 843-854 in cm-1 according to literature (Mohanan and Murukan, 2005).

When complexation occurred this υ(C=O) band is moved to a lower region suggesting coordination to metallic ion. The υ(C=S) band is shifted to higher frequency, but this vibration could be assigned clearly due to appearance of broad peaks (Halim et al., 2012).

According to literature structural investigation of infrared spectral data, the ligands give an intense band at 1725-1730 cm-1, which is the characteristic stretch of a carbonyl group (C=O) while in synthesized complexes this band shifted to the lower side, suggesting involvement of carbonyl oxygen atom in coordination with metal ion.

Synthesized complexes (1-12) were shown this peak at 1610-1699 cm-1 which proved formation of thiobiurets metal complexes. At a time a strong band was seen at 820-840 cm-1 which was assigned to the C=S stretching in free ligand, significantly shifted to higher frequency 1050-1081 cm-1 as shown nickel complexes showing that sulfur, atom attributed to the formation of the complexes.

Infrared spectra of metal complexes the N–H vibrations that disappeared in complexes as due to formation of N=C bond a strong peak was observed at 1500-

1598 cm-1. The alkyl moieties present in complexes are showed their frequencies in range of 2916-2968 cm-1. The peaks at 511-649 cm-1 showed the formation M-O bond and 426-526 cm-1 showed formation of M-S bond which proved the synthesis of nickel thiobiuret and dithiobiuret complexes. The IR data is shown in table 3.3 is given below. The FTIR spectra of complex 1, 2, 4 is shown in appendices.

Table 3.3: FTIR data of nickel(II)complexes (1-12).

Comp υ cm-1

No C=N C=O C=S C-N C-C M-O M-S

1 1541 1616 1076 1174 2929 649 526

2 1541 1710 1076 1178 2925 543 489

3 1515 1614 1076 1166 2931 532 490

4 1571 1612 1071 1176 2925 595 475

5 1598 1685 1058 1139 2929 526 493

6 1593 1666 1076 1153 2922 543 489

7 1544 1629 1081 1172 2916 621 475

8 1645 1699 1076 1180 2929 524 495

9 1550 1618 1080 1180 2923 628 426

10 1544 1618 1058 1134 2931 511 428

11 1552 1612 1062 1172 2968 570 432

12 1506 - 1049 1133 2921 - 463

3.2 SYNTHESIS OF THIOBIURET AND DITHIOBIURET COPPER(II)

COMPLEXES (13-24)

The copper(II) complexes (13-24) were synthesized by the reaction of commercially available secondary amine and in situ generated 3,3- dialkylthiocarbamoylthiocynate or N,N-dialkyldithiocarbamoylcynate in acetonitrile with addition of cupric(II) acetate monohydrated. Crude muddy grey color solid obtained was recrystallized from chloroform. Gray fine crystals of complex 24 were obtained for XRD. All the synthesized complexes were obtained in good yield. Mostly the complexes were obtained in muddy grey crystalline solid with good purity and few were also in liquid state. The synthesized complexes were characterized by UV-visible, IR, 1H NMR, 13C NMR and elemental analysis.

Physical parameters of thiobiuret and dithiobiuret of copper complexes are displayed by table 3.4 as shown below.

Table 3.4: Physical properties of tb and dtb copper(II) complexes.

Comp Molecular Yield Melting Mol. Elemental Analysis No formula % point oC Mass Calculated Found C H N C H N 13 (C10H20N3OS)2Cu 68 120 523 45.8 7.6 16.0 45.2 7.0 15.5

14 (C13H18N3OS)2Cu 53 150 591 52.7 5.0 14.2 52.2 4.4 13.6

15 (C11H15N4O2S)2Cu 54 135 597 44.2 5.0 18.7 44.0 4.9 18.2

16 (C15H21N4O3S)2Cu 64 162 737 48.8 5.6 15.1 48.5 5.0 14.5

17 (C12H15N4O3S)2Cu 55 140 653 44.1 4.5 17.1 43.5 4.0 16.5

18 (C12H16N4OS)2Cu 57 130 591 48.7 5.4 18.9 48.2 5.0 18.2

19 (C13H17N4O3S)2Cu 55 100 681 45.8 4.9 16.4 45.2 4.5 16.0

100

Comp Molecular Yield Melting Mol. Elemental Analysis No formula % point oC Mass Calculated Found C H N C H N 20 (C13H1 8N3OS)2Cu 60 Viscous 591 52.7 6.0 14.2 52.5 5.5 13.7

21 (C18H33N3OS)2Cu 61 83 741 58.2 8.9 11.3 57.5 8.5 10.5

22 (C12H15N6O5S)2Cu 60 121 773 37.2 3.8 21.7 36.5 3.5 21.2

23 (C12H24N3OS)2Cu 62 Viscous 579 49.7 8.2 14.5 49.5 7.5 14.0

24 (C6H12N3OS)2Cu 55 158 412 34.9 4.1 20.3 34.2 3.9 20.0

3.2.1 UV-Visible Analysis of Thiobiuret and Dithiobiuret Copper(II)

Complexes (13-24)

The electronic spectra of copper(II) complexes were given π-π*, charge transfer and d-d transitions. Absorption bands mostly in range of 260-270 nm were assigned π -π* transition. In synthesized complexes absorption at 333-367 nm was due to charge transfer transition. The electronic spectrum shown strong absorption in range of 420-595 nm which assigned d-d transition were appeared in visible region. Result of electronic spectra is shown in table 3.5.

Table 3.5: UV-visible absorption data of copper(II) complexes.

Complex No Wavelength λ (nm) Transitions 13 266, 346, 508 π -π*, charge transfer, d-d 14 270, 347 π -π*, charge transfer 15 270, 353, 595 π -π*, charge transfer, d-d 16 266, 345 π -π*, charge transfer 17 347 charge transfer 18 333 charge transfer 19 233, 364 n-π*, charge transfer 20 226, 309, 420 n-π*, charge transfer, d-d

101

Complex No Wavelength λ (nm) Transitions

21 548 d-d 22 367, 415 charge transfer, d-d

23 521 d-d

24 260, 355 π -π*, charge transfer

3.2.2 Infrared Spectroscopic Characterization of Thiobiuret and

Dithiobiuret Copper(II) Complexes

Infrared spectra of the synthesized thiobiuret and dithiobiuret copper(II) complexes (13−24) have been measured as KBr pellets for solid samples neat liquids as KBr cell for viscous liquids in the range of 4000-400 cm−1. In spectroscopic studies the important functionality peaks of all thiobiuret and dithiobiuret ligands are υ(C=O), υ(N-H), υ(C-N) and υ(C=S) showed the vibrational frequencies at 1654-1668 cm-1, 3286-3322 cm-1 , 1249-1273 cm-1 and

843- 854 cm-1. According to literature (Halim et al., 2012) structural investigation of infrared spectra of the ligands gives a strong band at 1715-1730 cm-1, which is the characteristic peak of carbonyl group (C=O) while in complexes this band shifted to the region 1650-1670 cm-1, indicating the co-ordination of carbonyl oxygen atom of ligand with metal ion.

Synthesized complexes (13-24) showed this peak at 1600-1645 cm-1 to represent coordination. The C=S bond gives a characteristic peak at 1032-1100cm-1 region of higher frequency. Position of C=N was located at 1473-1556 cm-1 showed formation of complexes due to disappearance of N-H band. M-O bands was found at 510-595 cm-1 and M-S bond at 451-663 cm-1 were proved for formation of synthesized complexes. The FTIR data of copper(II) complexes represented in table 3.6 as given below.

102

Table 3.6: FTIR data of complexes of copper (13-24).

Comp υ cm-1 No C=N C=O C=S C-N C-C M-O M-S 13 1473 1616 1075 1172 2929 550 655 14 1475 1616 1080 1153 2925 510 648 15 1554 1612 1083 1161 2931 582 513 16 1527 1604 1076 1132 2937 595 622 17 1537 1614 1083 1211 2933 594 663 18 1556 1616 1081 1153 2929 528 617 19 1541 1616 1081 1170 2952 553 663 20 1537 1645 1095 1134 2929 551 451 21 1556 1645 1098 1218 2921 525 617 22 1550 1618 1105 1126 2931 418 626 23 1545 1600 1060 1126 2933 435 617 24 1540 - 1032 1193 2926 - 469

3.3 SYNTHESIS OF THIOBIURET AND DITHIOBIURET COBALT(II)

COMPLEXES (25-35) AND CADMIUM(II) COMPLEX (36)

Complexes (25-35) and 36 was synthesized by the reaction of commercially secondary amines and in situ generated 3,3-dialkylthiocarbamoylthiocyanate or

N,N-dialkyldithiocarbamoylcynate in acetonitrile with addition of cobalt(II) acetate tetrahydrate/cadmium acetate tetrahydrate. All the synthesized complexes were obtained in good yield. Mostly the complexes of cobalt were obtained in green crystalline solid and cadmium 36 was yellow in color with good purity and few were also in liquid state. Complex 36 was subjected for XRD analysis to determine its geometry. The synthesized complexes were characterized by UV-vis, IR, 1H

NMR, 13C NMR and elemental analysis. Physical properties of thiobiuret and dithiobiuret of cobalt and cadmium complexes is given in table 3.7 as shown below.

103

Table 3.7: Physical properties of synthesized tb and dtb cobalt(II) complexes

Elemental Analysis Comp Molecular Yield Melting Mol. Calculated Found No formula % point oC mass C H N C H N

25 (C10H20N3OS)3Co 62 Viscous 749 52.8 8.0 16.8 52.5 7.5 16.5

26 (C13H18N3OS)3Co 54 111 851 54.9 6.3 14.8 54.2 6.0 14.5

27 (C11H15N4O2S)3Co 55 I40 869 45.5 5.2 19.3 45.0 4.5 19.0

28 (C15H21N4O3S)3Co 63 115 974 55.4 6.4 17.2 55.1 6.0 16.5

29 (C12H15N4O3S)3Co 66 145 944 45.7 4.7 17.7 45.2 4.2 17.2

30 (C12H16N4OS)3Co 54 150 851 50.7 5.6 19.7 50.0 5.0 19.2

31 (C13H17N4O3S)3Co 55 80 986 47.4 5.1 17.0 47.1 4.4 16.5

32 (C13H1 8N3OS)3Co 55 Viscous 851 54.9 6.3 14.8 54.2 6.0 14.3

33 (C18H33N3OS)3Co 56 Viscous 1076 60.2 9.2 11.7 59.7 8.5 11.5

34 (C12H15N6O5S)3Co 65 120 884 48.8 5.0 28.5 48.2 4.5 28.0

35 (C12H24N3OS)3Co 60 Viscous 833 51.8 8.6 15.1 51.2 8.2 14.8

36 (C6H12N3S2)2Cd 70 170 493 29.2 4.8 17.0 29.0 4.5 16.5

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3.3.1 UV-Visible Analysis of Thiobiuret and Dithiobiuret Cobalt(II) and

Cadmium(II) Complexes

The electronic spectra of cobalt(II) complexes were given π-π*, charge transfer and d-d transitions. Absorption bands mostly in the range of 257-285 nm were assigned π-π* transition in synthesized complexes. Absorption at 309-344 nm was due to charge transfer transition. The electronic spectrum showed strong absorption in range of 531-650 nm which assigned d-d transition was appeared in visible region. Result of electronic spectra is represented by table 3.8 as given below.

Table 3.8: UV- visible absorption data of cobalt(II) complexes 25-35.

Comp. No Wavelength λ (nm) Transition

25 266, 313, 623 π-π*, charge transfer, d-d

26 266, 318, 621 π-π*, charge transfer, d-d

27 257, 342, 546, 619 π-π*, charge transfer, d-d

28 267, 313, 626 π-π*, charge transfer, d-d

29 334 charge transfer

30 309, 614 charge transfer, d-d

31 315, 543 charge transfer, d-d

32 531, 623 d-d

33 621 d-d

34 285, 344 π-π*, charge transfer

35 650 d-d

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3.3.2 Infrared Spectral Characterization of Thiobiuret and Dithiobiuret

Cobalt(II) and Cadmium(II) Complexes

The solid state and liquid state IR spectra of synthesized thiobiuret and dithiobiuret cobalt and cadmium complexes are notified in the spectral region

4000–400 cm−1. The υ(C=O) band are shifted to lower frequencies suggesting coordination of the metal with ligand. The vibrational frequency of υ(C=S) bands are shifted to higher frequency, but this vibration could be assigned clearly due to appearance of broad peaks (Halim et al., 2012). According to literature, structural investigation of infrared spectra of the ligands of thiobiuret and give a stretch at

1715-1730 cm-1, which is due to presence of a carbonyl group (C=O) while in complexes this band moves to lower shift 1600-1670 cm-1, indicating the involvement of oxygen atom of carbonyl group ligand with metal ion.

Synthesized complexes (25-35) showed this peak at 1600-1693 cm-1 and

C=S give a characteristic band shifted to higher frequency at 1004-1080 cm-1 indicating that formation of dithiobiuret and thiobiuret cobalt complexes. Position of C=N absorption band at 1514-1598 cm-1 represents formation of double bond by removal of NH proton. M-O bands found at 478-595 cm-1 and M-S bond at 420-

459 cm-1 were proved for formation of synthesized complexes. The FTIR data of cobalt and cadmium complexes is represented in table 3.9 given below.

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Table 3.9: FTIR data of cobalt(II) and cadmium(II) complexes (25-36).

Complex υ cm-1 No C=N C=O C=S C-N C-C M-O M-S

25 1556 1693 1072 1172 2918 478 655

26 1514 1635 1076 1122 2925 520 472

27 1544 1627 1078 1153 2929 574 458

28 1542 1620 1078 1153 2925 595 644

29 1598 1652 1078 1174 2931 555 493

30 1556 1616 1081 1176 2929 525 617

31 1544 1614 1083 1151 2929 547 659

32 1552 1633 1081 1168 2923 551 420

33 1556 1645 1004 1103 2921 520 617

34 1548 1620 1060 1128 2933 532 624

35 1556 1600 1076 1172 2925 520 474

36 1568 - 1053 1108 2921 - 445

3.4 SYNTHESIS AND CHARACTERIZATION OF THIOBIURET AND

DITHIOBIURET ZINC(II) COMPLEXES (37-47)

Complexes (37-47) were synthesized by the reaction of commercially found secondary amine and in situ generated 3,3-dialkylthiocarbamoylthiocyanate or

N,N-dialkyldithiocarbamoylcyanate in acetonitrile with addition of zinc(II) acetate dihydrated described in previous chapter followed scheme 2.1. All the synthesized complexes were obtained in good yield. Mostly the complexes obtained in off

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white crystalline solid state with good purity and few were also in liquid state. The synthesized complexes were characterized by elemental analysis UV-vis, IR, 1H

NMR and 13C NMR spectral data. Physical properties of thiobiuret and dithiobiuret of cobalt(II) and cadmium(II) complexes is given in table 3.10 as shown below.

Table 3.10: Physical properties of synthesized thiobiuret and dithiobiuret zinc(II)

complexes 37-47.

Elemental Analysis Comp Molecular Yield Melting Mol. No formula % point oC Mass Calculated Found C H N C H N

37 (C10H20N3OS)2Zn 54 102 525 45.7 7.6 16.0 45.0 7.1 15.5

38 (C13H18N3OS)2Zn 52 Decompo 593 52.6 4.3 14.1 52.1 4.0 15.4 sed

39 (C11H15N4O2S)2Zn 59 145 599 44.0 5.0 18.6 43.7 4.5 18.1

40 (C15H21N4O3S)2Zn 54 Viscous 759 47.4 5.5 14.7 47.0 5.1 14.2

41 (C12H15N4O3S)2Zn 55 Viscous 655 43.9 4.5 17.0 43.5 4.0 16.5

42 (C12H16N4OS)2Zn 56 Viscous 593 48.5 5.3 18.0 48.1 5.0 17.5

o 43 (C13H17N4O3S)2Zn 58 50 C 683 45.6 4.9 16.3 45.2 4.2 16.0

44 (C13H1 8N3OS)2Zn 55 Viscous 593 52.6 6.0 14.0 52.1 5.5 13.5

45 (C18H33N3OS)2Zn 52 Viscous 743 58.1 8.8 11.3 57.7 8.2 11.0

46 (C12H15N6O5S)2Zn 55 Viscous 775 37.1 3.8 21.0 36.4 3.5 20.5

47 (C12H24N3OS)2Zn 54 Viscous 581 49.5 8.2 14.4 49.0 7.5 14.0

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3.4.1 UV-Visible Analysis of Zinc(II) Complexes (37-47)

The electronic spectra of zinc(II) complexes were given π-π*, charge transfer and d-d transitions. Bands mostly in the range of 274-278 nm were assigned π-π* transition in synthesized complexes. Absorption at 320-368 nm was due to charge transfer transition. The electronic spectrum showed strong absorption in range of 412-628 nm which was assigned to a d-d transition which appeared in visible region. The results of electronic spectra is shown in table 3.11.

Table 3.11: UV-visible absorption data of zinc(II) complexes.

Complex No Wavelength λ (nm) Transitions

37 548, 554 d-d

38 412 d-d

39 368 charge transfer

40 277 π-π*

41 274, 341 π-π*, charge transfer

42 320, 431 charge transfer, d-d

43 278, 335 π-π*, charge transfer

44 321, 531 charge transfer, d-d

45 321, 628 charge transfer, d-d

46 278, 350 π-π*, charge transfer

47 215, 365 n-π*, charge transfer

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3.4.2 Infrared Spectroscopic Characterization of Thiobiuret and

Dithiobiuret Zinc(II) Complexes (37-47)

The liquid state and few solid IR spectra of synthesized thiobiuret and dithiobiuret zinc complexes were read out in the spectral region of 4000–400 cm−1.

According to literature, structural investigation of infrared spectra of the ligands give intense band was observed at 1715-1730 cm-1, which is the important peak of carbonyl group (C=O) while in complexes this band appeared at 1600-1670 cm-1, indicating the co-ordination of oxygen atom of ligand with metal ion. Synthesized complexes (37-47) showed this peak at 1600-1699 cm-1. The C=S gives a characteristic band shifted to higher frequency at 1004-1091 cm-1. The vibrational stretch at 1521-1598 cm-1 is due to formation of C=N bond. M-O bands found at

510-628 cm-1 and M-S bond at 426-495 cm-1 were proved for formation of synthesized complexes. The FT-IR reading of zinc complexes is displayed in table

3.12 given below.

Table 3.12: FTIR data of zinc complexes (37- 47).

Complex υ cm-1 No C=N C=O C=S C-N C-C M-O M-S 37 1521 1600 1074 1122 2929 619 465 38 1560 1610 1085 1128 2933 622 459 39 1548 1616 1076 1126 2931 525 609 40 1573 1645 1091 1122 2921 543 435 41 1598 1685 1004 1139 2929 526 493 42 1593 1666 1010 1153 2929 543 489 43 1612 1635 1020 1168 2952 550 655 44 1645 1699 1027 1180 2929 524 495 45 1550 1618 1080 1180 2923 628 426 46 1544 1618 1058 1134 2931 514 428 47 1558 1614 1080 1180 2925 510 621

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3.5 SYNTHESIS OF BIURET, THIOBIURET AND DITHIOBIURET

METAL COMPLEXES USING NEW METHOD B

Metal complexes (48-60) was synthesized by ligand derived from the reaction of commercially available secondary amine and in situ generated 3,3- dimethylbutanoylthiocyanate in acetonitrile with addition of metal acetate hydrated of Co, Ni, Cu, Cd and Zn. All the synthesized complexes were obtained in good yield. Mostly the complexes were obtained as colored crystalline solid states with good purity.

The synthesized complexes were characterized by UV-vis, IR, 1H NMR,

13C NMR and elemental analysis. Complex 48 and 54 give fine colored crystals which were subjected to XRD crystallography. Physical properties of thiobiuret and dithiobiuret of metal complexes are shown in table 3.13.

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Table 3.13: Physical properties of tb and dtb of metal complexes.

Meltig Elemental analysis Comp Molecular Yield Mol. point Calculated Found No Formula % o Mass C C H N C H N

48 (C11H21N2OS)2Ni 67 80 517 51.1 8.1 10.8 50.5 7.5 10.2

49 (C13H18N2O2S)2 Ni 71 160 588 53.0 6.1 9.5 52.5 5.5 9.1

50 (C14H27ON3S)2Ni 61 116 657 51.1 8.2 12.7 50.5 8.0 12.2

51 (C11H21N2OS)2Cu 62 110 521 50.6 8.0 10.0 50.1 7.6 9.5

52 (C13H18N2O2S)2Cu 79 142 593 52.6 6.0 9.4 52.0 5.5 9.0

53 (C14H27ON3S)2 Cu 57 180 663 50.6 8.1 12.0 50.1 7.5 11.5

54 (C11H21N2OS)3Co 52 120 746 52.1 8.3 11.0 51.7 8.0 10.5

55 (C13H18N2O2S)3Co 87 150 857 54.6 6.3 9.8 54.0 6.0 9.5

56 (C14H27ON3S)3Co 50 220 914 55.1 8.8 13.7 54.7 8.2 13.1

57 (C11H21N2OS)2Zn 56 234 523 50.4 8.0 10.7 50.0 7.7 10.5

58 (C13H18N2O2S)2Zn 73 200 595 52.4 6.0 9.4 52.0 5.5 9.1

59 (C11H21N2OS)2Cd 59 175 570 46.6 7.3 9.8 46.0 7.0 9.3

60 (C13H18N2O2S)2Cd 53 198 642 48.5 5.6 8.7 48.0 5.0 8.2

3.5.1 UV-Visible Analysis of Thiobiuret and Dithiobiuret Metal Complexes

(48-60)

The electronic spectra of nickel complexes were given π-π*, d-d transitions.

The absorption peaks in range of 262-275 nm and 504-536 were assigned π-π* and

d-d transition respectively in synthesized complexes. Absorption at 320 nm was

due to charge transfer transition of cobalt complexes. The electronic spectrum

showed a strong absorption in range of 504-536 nm which was assigned to d-d

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transition and which appeared in visible region. The complexes of copper were given π-π*, and d-d transitions absorption bands appeared at 241- 248 nm, 557-688 nm respectively. The complexes of cobalt showed three type of transition π-π*, charge transfer and d-d transitions at 259-278, 320, 547-605 nm respectively. Zinc and cadmium complexes were given π-π* and d-d transitions at 280 nm, 298 nm and 548-605 nm. Results of electronic transition is shown in table 3.14.

Table 3.14: UV-visible absorption data of complexes 48-60.

Complex Wavelength λ (nm) Transitions No

48 275, 536 π-π*, d-d

49 262, 504 π-π*, d-d

50 262, 517 π-π*, d-d

51 557, 688 d-d

52 241, 557, 688 π-π*

53 248 π-π*

54 265, 536, 605 π-π*, d-d

55 278, 320 π-π*, charge transfer

56 259, 547 π-π*, d-d

57 548, 554 d-d

58 280 π-π*

59 298, 552, 605 π-π*, d-d

60 279 π-π*

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3.5.2 Infrared Spectroscopic Characterization of Thiobiuret and

Dithiobiuret Metal Complexes (48-60)

Infrared (IR) spectroscopy is an important characterization technique which is used for the analysis of 2,2-diethyl amine, 2-aminophenol and 3- morpholinopropylamine based thiobiuret metal complexes. The IR spectra of 2,2- diethyl amine, 2-aminophenol and 3-morpholinopropylamine based thiobiuret complexes of nickel, copper, cobalt, zinc and cadmium were recorded in range of

4000-400 cm-1 by KBr disc method.

Stretching bands of synthesized complexes were compared and assigned on careful comparison. According to literature carbonyl group stretch at 1715-1730 cm-1 in the thiobiurets ligand. This frequency will be shifted to lower side after complexation as shown in synthesized complexes have given an absorption bands at 1600-1652 cm-1 which specifies the C=O stretch. The appearing peaks in the region of 1025-1276 cm-1 specify the nitrogen to carbon bond.

In these complexes v(N-H) peak disappears in the region of 3218-3438 cm-1 which specifying the non-participation of the v(N-H) band of the ligand in complex formation. It is in agreement with the literature report (Naik et al., 1999). The appearing peak in region 1525-1585 cm-1 give the formation of C=N bond. They move towards red shift and absorb at lower region which directs the delocalization electrons on the metal and the C=N double bond character decreases (Sączewski et al., 2006).

The C=S stretching band was observed at 1010-1193 cm-1. The thiobiuret behaves as a bidentate ligand and is chelated to form a desired complex. Zinc and cadmium have a distorted tetrahedral geometry. The distortion reflects the variation

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of bond distances of metal to sulfur. The comparison of peaks data suggest an octahedral geometry of cobalt along with two water molecules (Ramasamy et al.,

2011). The IR peaks appeared at 518-548 cm-1 openly showed the formation of

M-O bond formation (Sovilj et al., 2000). Characteristics IR bands of the investigated metal complexes (48-60) are presented in the Table 3.15 FTIR spectra of complex 48, 54 is shown in appendices.

Table 3.15: FTIR data of complexes (48-60).

Comp υ cm-1

No C=N C=O C=S C-N C-C M-O M-S

48 1558 1616 1076 1130 2929 524 428

49 1544 1608 1037 1141 2958 597 536

50 1564 1610 1009 1141 2948 621 501

51 1558 1620 1043 1193 2935 617 518

52 1558 1602 1018 1147 2931 530 439

53 1585 1652 1110 1235 2935 514 664

54 1525 1645 1076 1126 2933 516 447

55 1560 1645 1039 1145 2952 592 532

56 1550 1652 945 1116 2952 621 501

57 1538 1631 1070 1180 2946 546 490

58 1568 1606 1010 1149 2960 624 529

59 1558 1612 1193 1070 2959 684 518

60 1548 1635 1020 1140 2923 597 520

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3.6 NMR SPECTROSCOPIC STUDIES

Nuclear magnetic resonance (NMR) spectroscopy is a very good technique to obtain structural information, to study chemical changes in the mechanism of reactions, inter and intra interactions of molecules etc. 1H, 13C NMR analyses provides highly valuable data and hence are frequently used for the characterization of coordination complexes like thiobiuret and dithiobiuret metal complexes. It is a latest tool for doing structural analysis of metal complexes and is equally important for elucidating the coordination geometry of ligands around metal.

3.6.1 1H NMR Spectroscopy

Multinuclear NMR (1H) spectra were scanned by using Bruker ARX 300

MHz-FT-NMR and a Bruker 400 MHz-FT-NMR in deutrated chloroform as solvent, and tetramethylsilane as an internal reference. Stretching shifts (δ) and coupling constants (J) are assigned in ppm and Hz respectively. The multiplicities of absorption bands in the 1H NMR spectrum are put in writing as s for singlet, d for doublet, t for triplet, q for quartet, m for multiplet).

1H NMR supported the successful synthesis of target metal complexes. In representative complexes, ligands are same bearing similar types of protons i. e.

CCH3, CCH2 and NCH2 are present. The shift observed in proton spectra of metal complexes are CCH3 proton of ethyl group was shown singlet broad peaks at 0.86 -

1.41 ppm while chemical shifts of CCH2 proton of ethyl group was give doublet and multiplet peaks at 3.15 to 3.97 ppm. NCH2 aliphatic proton of methyl group was resonated in range of 2.81-3.78 ppm as broad singlet. Aromatic protons resonated in range of 6.98-7.98 ppm in multiplet peaks. According to literature the

N–H signals are seen at 8.02 ppm in the 1H NMR spectra of ligand and disappeared

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in the corresponding spectra of complexes as a consequence of coordination, in agreement with IR results.

In addition, all the other chemical shift values of 1H NMR data agree with the literature (Ugur et al., 2006) and expected structure of the complexes. The spectra of complexes showed peak at δ 8.02 ppm for proton of NH between carbonyl group and thio group disappeared, showing the removal of this proton and forming C=N bond this gives a confirmation of desired synthesis. The proton data of some representative nickel(II), cobalt(II), copper(II), zinc(II) and cadmium(II) thiobiuret and dithiobiuret complexes are shown in table 3.16. Representative 1H-

NMR spectra of complexes 1, 48 and 54 are given in appendices.

Table 3.16: 1HNMR data of representative complexes.

R R R R N N R R N N R R R C S Z C S O N M N M C Z S C O S R N N R R R R R R N N R R

H2N N R= CH3, C2H5, NH N , CH , N , No2 3 H C O O 3 No2

Comp δ (ppm) multiplicity, Integration and coupling constant J lex No

CH3 CH2 CH3 H CH2 -C=CH NH CH Ethyl Ethyl Aliphatic Phenyl Aliphatic

1 1.07 (12H, br, s) 3.20 (d, J = 4Hz, 4H) ------3.33 (d, J = 4Hz, 4H) 2 0.81 (12H, br, s) 3.15-3.25 (8H, m) 2.81 (6H, s) 7.03-7.24 - - - - (10H, m)

4 1.4 (12H, br, s) 3.54-3.66 (8H, m) - - 4.13-4.28 6.83 (dd, J1 =3.39, - - (8H, br, s) J2 =1.88 Hz, 2H) 5 0.41 (12H, br, s) 3.19 (8H, m) - 7.03-7.20 - - 2.58 - (8H, m) (2H, s) 8 0.72 (12H, br ,s) 3.97 (d, J = 8H) - 6.88-6.93 4.44-4.43 - 2.67 - (10H, m) (d , J =2Hz, (2H, s) 4H) 12 - - 3.07-3.10 - - - - - (12H,br,s) 13 1.03 (12H, br, s) 3.19 (d, J = 4Hz, 4H) ------3.30 (d, J = 4Hz, 4H) 14 1.08 (12H, br, s) 3.26-3.37 (8H, m) 2.82 (6H, s) 7.05-7.23 - - - - (10H, m) 16 1.25 (6H, br, s) 3.57 (d, J =7.3, 4H) - - 4.13(4H, br, s) 6.82 (dd, J1 =3.39, J2 - - 1.41 (6H, br, s) 3.67 (d, J =7.03, 4 H) 4.28(4H, br, s) =1.86, 3H)

23 0.85 ( 6H, br, s) 3.26-3.30 (4H, m) 3.51-3.61 (12H, br, s) - -- - 1.24-1.60 0.92 (6H, br, s) (4H, br, s)

24 - - 3.25 (6H, br,s) - - - - - 3.37 (6H, br, s) 35 0.86 (6H, s) 3.20-3.30 (4H, m) 3.51-3.58 (12H, br ,s) - - - - 1.51-1.58 0.88 (6H, s) (4H, br, s) 36 - - 3.17(12H, br, s) - - - - - 3.39 (12H, br, s)

117

118

37 1.08 (br, s, 12H) 3.17 (br, s, 8H) ------

46 1.29 (12H, br, s) 3.43 (d, J =4Hz, 8H) - 7.95-7.97 - - 4.04 - (6H, m) (4H, s)

48 1.11(6H, br, s) 3.57-3.77 (8H, m) 0.98 (18H, br, s) - 2.13 - - - 1.2 (6H, br, s) (4H, br, s)

51 1.10 (6H, br, s) 3.56-3.78 (8H, m) 0.98 (18H, br, s) - 2.15 - - - 1.20 (6H, br, s) (4H, br, s)

54 1.12 (6H, br, s) 3.65-3.99 (8H, m) 0.98 (18H, br, s) - 16 (4H, br, s) - - - 1.25 (6H, br, s) 2.41(4H, br, s)

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3.6.2 13C-NMR Spectroscopy

13C-NMR spectra was scanned by using Bruker ARX 300 MHz-FT-NMR and a Bruker 400 MHz-FT-NMR in deturated chloroform as solvent, and tetramethylsilane as an internal reference. Mostly the synthesized complexes are paramagnetic in nature so 13C-NMR is silent .13C-NMR data of some representative metal complexes supported this synthesis. The most deshielded signal in range of

150-173 ppm because of carbonyl carbon. The methyl carbon of ethyl group resonates in range of 21-39 ppm. CH2 carbon of ethyl group show peak at 11-19 ppm. The thionoyl carbon resonate at the range of 40-48 ppm of selected metal complexes of thiobiuret metal complexes. The 13C-NMR data of some representative nickel(II), cobalt(II), copper(II), zinc(II) and cadmium(II) thiobiuret and dithiobiuret complexes are shown in table 3.17. Representative 13C-NMR spectra of complex 4 are given in appendices.

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Table 3.17: 13C NMR data of some representative metal complexes of thiobiuret

and dithiobiuret.

2 1 H C 3 4 R H3C 2 N C N C N H C C R 3 O S H2 Ni

S O H2C CH3 R N C N C N R C CH3 H2

H2N N N R= CH ,C H , CH3 3 2 5 , N , No2 H3C O O

No2

Complex No δ (ppm) Chemical shifts

C-1 C-2 C-3 C-4 Ar-C C=C C(CH3)2 4 31 13 170 43 - 111 - 12 39 - 170 41 - - - 16 36 15 169 45 - 117 - 23 29 19 160 40 - - 52 35 21 11 164 44 - - 58 36 39 - 173 42 - - - 46 25 17 155 48 130 116 -

3.7 CONDUCTANCE

It has been observed that mostly all of the complexes showed electrolytic behavior. The conductance values range from 1.95-21.2 μS. Synthesized complexes were considered to evaluate their conductance and results indicate that they possess differential levels of electrolytic characteristics, which indicates that

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complexes had positive electrolytic behavior, whereas few complexes showed suppressed electrolytic properties.

3.8 X-RAY CRYSTALLOGRAPHY

The X-ray structure of complexes 12, 24, 36, 48, 54 was unambiguously illustrated by single crystal X-ray diffractional analysis. The X-ray parameters were measured on Bruker Kappa Apex-IICCD diffractometer equipped with graphite monochromator and Mo-Kα radiation source. The ORTEP plots and unit cell diagrams along with selected bond lengths, bond angles and dihedral angles of complex 12 are given in the caption to Figure 3.1 and table 3.18 and 3.19.

Figure 3.1: ORTEP structure of complex 12 bis (1,1,5,5-tetramethyl-2,4- dithiobiureto) nickel(II).

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Table 3.18: X-ray diffraction data and structure refinement parameters for complexes 12, 24, 36, 48, 54.

Crystal Complex Complex Complex Complex Complex parameters 12 24 36 48 54

Chemical formula C12H24N6NiS4 C12H24CuN6O2 S2 C12H24CdN6S4 C22H42N4NiO2S2 C34H63CoN6O3S3

Mr 439 412 493 517 746 Crystal system, Triclinic, Monoclinic, Monoclinic, Monoclinic, Trigonal, Space group P-1 P2(1)/c P2(1)/c P2(1)/c P3 Temperature (K) 150 150 100 296 296 a 7.5520(3) 8.9010(3) 12.3267(4) 20.1555(4) 13.5545(5)

b 8.2730(3) 14.0820(5) 12.0136(3) 12.5641(3) - c (Å) 8.320(3) 14.3420(7) 13.5508(4) 11.158(2) 14.4071(6)

β (°) 85.253(2) 98.932(2) 105.594(4) 95.046 (1) -

V (Å3) 457.16(3) 1775.88(12) 1932.85(10) 2814.85 (10) 2292.31 (19)

Z 1 4 4 4 2

Radiation type Mo Kα Mo Kα Mo Kα Mo Kα Mo Kα μ (mm−1) 1.524 1.482 1.568 0.86 0.72

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Crystal size (mm) 0.30 x 0.30 x 0.50 x 0.30 x 0.05 0.26 x 0.14 x 0.08 0.45 × 0.38 × 0.30 0.42 × 0.36 × 0.26 0.20 No. of reflections 3891 15554 37275 6416 3353

No. of parameters 110 216 216 290 156

R (int) 0.0584 0.084 0.0342 0.025 0.033

Δρmax, Δρmin (e 0.681, -1.045 1.044, -1.435 0.532, -0.352 0.38, −0.24 0.31, −0.38 Å−3)

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The X-ray structures of isolates the complexes 12 and 36 consist of

2+ molecular entities of (C6H12N3S2)2M, where M is Ni(II) or Cd(II), with only van der Waals interaction between these units. There was found no proof interatomic interactions of hydrogen bonding distances, of M-M interaction or any metal- adjacent ligand bonding. The metal present on a crystallographic center of symmetry and is connected to four sulfur atoms from two different ligands moieties in a planner and tetrahedral MS4 array, respectively. In complex 12 the S-Ni-S angles are not 90˚ but expended to 97.29 (2)˚ with chelate ring, table 3.19. The M-S bond distance is little bit shorter than that found in Pd, Pt complexes with ligand thiourea (Berta et al., 1970; Spofford and Amma, 1972). It is interesting point to be noted that the ligand symmetry is not planar in this complex, but the individual

SC(N,N) units are quite planar.

Table 3.19: Representative bond lengths [Å] and angles [°] for complex 12.

`Ni(1)-S(2) 2.1672(6) S(2)-Ni(1)-S(2) 180.00(4) Ni(1)-S(2) 2.1672(6) S(2)-Ni(1)-S(3) 82.71(2) Ni(1)-S(3) 2.1739(6) S(2)-Ni(1)-S(3) 97.29(2) Ni(1)-S(3) 2.1739(6) S(2)-Ni(1)-S(3) 97.29(2) S(2)-C(7) 1.733(2) S(2)-Ni(1)-S(3) 82.71(2) S(3)-C(8) 1.739(2) S(3)-Ni(1)-S(3) 180.00(4) N(4)-C(7)-S(2) 130.30(18) C(7)-S(2)-Ni(1) 116.60(8) N(6)-C(7)-S(2) 115.34(18) C(8)-S(3)-Ni(1) 116.00(8) N(4)-C(8)-N(5) 114.5(2)

The complex 36 was also established it geometry by X-ray crystallography and resolved by ORTEP plots along with bond length, bond angles and dihedral angles are shown in table 3.20 and Figure 3.2.

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Figure 3.2: ORTEP structure of 36 bis (1,1,5,5-tetramethyl-2,4-dithiobiureto)

cadmium(II).

The structure of complex 36 tells us that the Cd(II) has a distorted tetrahedral geometry, shown in fig.3.2. The bite angles of the ligand is 99.01 (3)˚ and 96.44

(3)˚ as shown in table 3.20 are slightly smaller than the perfect tetrahedral angle, but the bidentate ligand planes Cd-S(1)C(1) and Cd-S(2)C(2) are almost both perpendicular with each other. The overall ligands however, are not planar due to torsional twists about the central N(1) and N(4) atoms respectively, and the corresponding bond angles on these nitrogen atoms are 131.70(12)˚ and

133.13(12)˚, table 3.20.

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Table 3.20: Bond lengths [Å] and angles [°] for complex 36.

Cd-S(2) 2.5003(4) Cd-S(2) 2.5003(4) Cd-S(4) 2.5107(4) Cd-S(4) 2.5107(4) Cd-S(3) 2.5173(4) Cd-S(3) 2.5173(4) Cd-S(1) 2.5787(4) Cd-S(1) 2.5787(4) S(1)-C(1) 1.7482(13) S(1)-C(1) 1.7482(13) S(2)-C(2) 1.7459(14) S(2)-C(2) 1.7459(14) S(3)-C(7) 1.7448(13) S(3)-C(7) 1.7448(13) S(4)-C(8) 1.7496(14) S(4)-C(8) 1.7496(14) N(1)-C(1) 1.3190(18) C(1)-N(1)-C(2) 131.70(12) N(1)-C(2) 1.3199(18) C(8)-N(4)-C(7) 133.13(12)

According to crystal data structure of complex 24 shows that Cu(II) has established a square planar geometry similar to 12 as shown fig.3.3 and the planes of bond distances observed is compatible to the corresponding homoleptic Ni(II) complex is studied by Crane and Whittingham (2004). It shows that the formal negative charge is predominately localized on the sulfur atom. The relatively long

C-S and short C-O average bond lengths 1.754(5) and 1.269(3) Å is displayed in table 3.21 are consistent with mostly single- and double-bond characters, respectively and this bond localization is also reflected in the average C-N bond distances to the central N atom, viz. 1.327(6) Å in the (iso)thiourea group and 1.348

(7) Å in the urea group (Crane and Whittingham, 2004a).

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Figure 3.3: ORTEP structure of 24 bis(1,1,5,5-tetramethyl-2- thiobiureto)

copper(II).

Table 3.21: Bond lengths [Å] and angles [°] for complex 24.

C(1)-N(1) 1.327(6) C(2)-O(1)-Cu(1) 133.7(3) C(1)-N(2) 1.338(5) C(8)-O(2)-Cu(1) 133.4(3) C(1)-S(1) 1.754(5) C(1)-S(1)-Cu(1) 108.30(15) C(2)-O(1) 1.269(5) C(7)-S(2)-Cu(1) 108.35(18) C(2)-N(1) 1.342(5) O(2)-Cu(1)-O(1) 84.37(13) C(2)-N(3) 1.347(6) O(2)-Cu(1)-S(2) 94.19(10) C(3)-N(2) 1.453(6) O(1)-Cu(1)-S(2) 172.13(12) O(1)-Cu(1) 1.913(3) O(2)-Cu(1)-S(1) 173.81(11) O(2)-Cu(1) 1.909(3) O(1)-Cu(1)-S(1) 93.35(9) S(1)-Cu(1) 2.2307(12) S(2)-Cu(1)-S(1) 88.83(5) S(2)-Cu(1) 2.2297(14) N(1)-C(1)-N(2) 115.3(4)

128

The structure of complexes 48 and 54 was confirmed by single crystal X- ray analysis. The ORTEP plot along with selected bond length bond angles and dihedral angle is shown in table 3.18, 3.22 and 3.2 3 and Figures 3.4 and 3.5.

Figure 3.4: ORTEP structure of 48 bis[(Z)-3-(3,3-dimethylbutanoyl)-1,1-diethyl-

2-thiobiureto] nickel(II) complex.

Figure 3.5: Packing pattern of complex 48 showing van der waals attractions.

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Table 3.22: Bond lengths [Å] and angles [°] for complex 48

Ni-S1 2.14 (6) S1-Ni-O1 94.9 (15) Ni-O1 1.85 (15) S1-C1-N2 126.5 (18) S1-C1 1.73 (2) S1-C1-N1 117.6 (17) C1-N1 1.33 (3) C2-N1-C4 116.2 (2) C1-N2 1.34 (3) O1-C6-N2 128.4 (2) N2-C6 1.32 (3) N2-C6-C7 116.0 (2) O1-C6 1.26 (3) C6-C7-C8 116.0 (2) C6-C7 1.51 (3) N2-C1-N1 115.6 (19) N1-C4 1.47 (3) N1-C2 1.48 (3)

Figure 3.6: ORTEP structure of 54 tris[(Z)-3-(3,3-dimethylbutanoyl)-1,1-diethyl-2-

thiobiureto] cobalt(II).

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Figure 3.7: Packing pattern of complex 54 showing van der waals attraction

Table 3.23: Bond lengths [Å] and angles [°] for complex 54.

Co-S1 2.21 (5) S1-Co-O1 95.6 (4) Co-O1 1.92 (12) S1-C7-N1 128.3 (14) O1-C1 1.26 (2) S1-C7-N2 117.4 (14) C1-N1 1.32 (2) O1-C1-N1 130.0 (17) S1-C7 1.73 (18) O1-C1-C2 115.5 (16) C7-N1 1.34 (2) C1-C2-C3 115.8 (16) C7-N2 1.34 (2) N1-C7-N2 114.2 (16) N2-C10 1.48 (3) C8-N2-C10 116.1 (16) N2-C8 1.46 (3) C1-C2 1.51 (3)

The molecular formulas of complexes 48 and 54 consist of

[C22H42N4NiO2S2] and [C33H63CoN6O3S3] entities, respectively. The Ni complex

(48) crystallized in the monoclinic crystal system having space group P21/c, whereas Co complex (54) crystalized in trigonal crystal system having P3 space group. The crystal structure data of both complexes is given in table 3.18 and

ORTEP plots drawn at 50% probability level are shown in the Figure 3.4 and 3.6.

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Analysis of ORTEP plots shows that the metal is localized on a crystallographic center of symmetry and is bound with two thiobiuret ligands (four

S and O atoms) in Ni (II) complex 48, where as in Co(II) complex 54, the central metal atom is bound with three molecules of ligand. The Ni(II) complex is neutral homoleptic having square planar geometry and all non-hydrogen atoms are located on a crystallographic mirror plane Figure .3.4. The Ni(II) ion is square planar with

S and O donor set, which are mutually fac to each other and respective planes are parallel to each other. The bond distances from central metal atoms (Ni-S1 = 2.14

Å and Ni-O1 = 1.85 Å) shown in table 3.22 are indicative of localization of -ive charge on the S atom. The relatively longer C-S (1.73 Å) and shorter C-O (1.26 Å) bond lengths are indicative of single and double bond character, respectively

(Crane and Whittingham, 2004a).

The Co(II) complex is also neutral homoleptic and has octahedral geometry.

All three thiobiuret ligands in 54 are twisted and show significant deviation from planarity. The pattern of bond lengths is similar to that observed in the corresponding homoleptic nickel(II) complex 48 (Crane and Whittingham, 2004b)

.The single crystal X-ray diffraction studies depicted that both Ni(II) and Co(II) complexes exist as isolated molecular units of (C11H21N2SO)2Ni and

(C11H21N2SO)3Co, respectively. No evidence of hydrogen bonding, any metal- adjacent ligand and metal-metal interactions is observed in crystal packing unit cell diagrams of both complexes 48 and 54 (Van der Waals interactions are observed among different proton and carbon atoms of both complexes) (Figure. 3.5 and 3.7).

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3.9 BIOLOGICAL ACTIVITIES

Selected complexes were tested for eight different type of bioassays antibacterial, antifungal, protein kinase inhibition assay, total antioxidant capacity, reducing power assay, DPPH radical scavenging assay, ABTS+ decolourization antioxidant bioassay and anticancer assay.

3.9.1 Antibacterial Activity

Thiobiuret and dithiobiuret metal complexes were examined for antibacterial activities against three types of bacteria through agar well diffusion method (Atta-ur-Rahman and Thomsen, 2001). Fourteen selected complexes were screened for testing their antibacterial potential against three bacterial microbes

Echerishia coli, Staphylococcus aureus and Pseudomona aeruginosa.

The selected synthesized complexes showed good result. Complexes no 8,

12, 13, 14, 27, 32 and 38 showed significant antibacterial activities against all bacterial strains. Complexes 1, 4, 31 had given moderate to minimum inhibition values. Complex 2 was silent against Echerishia coli, Staphylococcus aureus and showed moderate activity against Pseudomonas aeruginosa. The 43 and 46 were silent against Echerishia coli, but showed moderate activity against Staphylococcus aureus and Pseudomona aeruginosa. Complex 11 showed minimum value against

Echerishia coli and showed no activity against Staphylococcus aureus and

Pseudomonas aeruginosa. The results are displayed in Figure 3.8

133

Figure 3.8: Antibacterial activity of tb and dtb metal complexes.

3.9.2 Antifungal Activity

Thirty two selected complexes were scanned for their antifungal potential against A. niger, F. Solani, M. species and A. flavus. Fourteen complexes were shown to give positive response while all other compounds are silent against these four fungal strains. Compound 2, 4, 14, 27 and 43 showed significant activity against Aspergilus niger. Complexes 4, 12 and 47 showed good activities against F.

Solani 47 have excellent antifungal potential. Complexes 19, 22, 31 and 34 shows moderate activity against Mucor species and 2, 3, 12, 14, 38 showed maximum zone of inhibition against Aspergilus flavus. The results are represented in Figure

3.9 as given below.

134

Figure 3.9: Antifungal activity of thiobiuret and dithiobiuret metal complexes

3.9.3 Total Antioxidant Capacity

Thirty two assorted compounds were examined for their total antioxidant behavior. All the complexes exhibited good result. Complex number 7, 18, 19, 22 were showed excellent result. The 2-4, 6, 8, 10, 12, 14-17 complexes showed good result .Complexes 1, 5, 13, 21, 28, 30, 31, 43, 46 showed significant antioxidant behavior. 26-30, 34-38 exhibited adequate antioxidant behavior. Results are shown in Figure 3.10.

3.9.4 Reducing Power Assay

High values of ascorbic acid equivalent number show the more reducing power. Thirty one complexes were scanned for reducing potential and indicate higher values. All the complexes are very active. Complexes 13, 19, 21 showed maximum values. Complexes 1-12, 22-46 were good reducing potential. Results are shown in Figure 3.10.

135

3.9.5 DPPH free Radical Scavenging Assay

Thirty two complexes were subjected for DPPH free radical scavenging activity. As standard reference Gallic acid was used. All the tested complexes showed excellent antioxidant activity due to multiple binding cities of free radicals.

Only 15, 21, 26 and 37 showed minimum scavenging. The most active complexes were found 4, 5, 6 and 46 which indicates maximum scavenging at 200 µg/ml.

Results are presented in Figure 3.10 as given below.

Figure 3.10: Total antioxidant capacity, reducing capacity and DPPH %

Scavenging of thiobiuret and dithiobiuret metal complexes.

3.9.6 Protein Kinase Inhibition Assay

Thirty two complexes were assested for their potential by measuring zone of protein kinase inhibition. The result revealed that complexes 3-7, 10-19, 27-29

136

and 43 had maximum inhibition at 25µg/disc. Complexes 1, 2, 8, 21-26, 30-38 and

46 have no inhibition in blade zone. Results are shown in Figure 3.11.

Figure 3.11: Protein Kinase inhibition activity of biuret thiobiuret and dithiobiuret

metal complexes

3.9.7 ABTS Radical+ Decolourization Bioassay

Seventeen complexes were screened ABTS radical+ decolourization bioassay to investigate antioxidant potential. According to the results all the complexes had excellent antioxidant potential except 51 and 55 as shown in Figure

3.12.

137

Figure 3.12: ABTS antioxidant potential of thiobiuret and dithiobiuret metal

complexes.

3.9.8 Anticancer Activity

Thiobiuret and dithiobiuret of Ni(II), Cu(II), Co(II) and Zn(II) complexes were tested against THP-1 cancer cell line for anticancer activity. To evaluate the biological activity of the drugs on cancer cells, we examined their growth inhibiting effects on human cell lines in vitro and compared the results against negative controls. All the 32 metal complexes were under investigation showed promising efficiency. Complexes 1-3, 5, 6, 12-14, 17, 18, 28-30, 37-39 showed maximum inhibition at 20 µg/ml. They showed strong activity as anticancer agents

Thus, the ultimate purpose of the current work seems fulfilled to synthesized complexes with high efficacy against cancer cells. The complexes 4, 7, 8, 15, 16,

19, 21-27, 31, 34, 43, 46 showed good activities against cancer cell. The results are shown in Figure 3.13.

138

Figure 3.13: Anticancer activity of tb and dtb metal complexes.

SUMMARY

Two series of bis and tris thiobiureto, dithiobiureto Co(II), Ni(II), Cd(II),

Cu(II) and Zn(II) metal complexes are synthesized to achieve the aim of present work. First series of these metal complexes (1-47) were prepared by the reaction of commercially available secondary amine and in situ generated 3,3- dialkylthiocarbamoylthiocyanate or N,N-dialkyldithiocarbamoylcyanate in acetonitrile. Second series of metal complexes (48-60) by the reaction of commercially available secondary amine and in situ generated 3,3- dimethylbutanoylthiocyanate in acetonitrile.

First and second series of complexes were identified by spectral analytical tools such as elemental analysis UV-visible, FTIR, 1H-NMR, 13C-NMR spectroscopy and single crystal X-ray diffraction. To find the percentage composition of atoms i.e. C, H and N present in said synthesized complexes was

139

also calculated. The desired products were also obtained in good to excellent yield.

Presence of NCS functionality in these compounds gave them special properties.

Mostly complexes showed electrolytic behavior, few were silent due to diamagnetic properties. The obtained data from UV-visible, FT-IR, 1H-NMR, 13C-

NMR and elemental analysis was compared with the literature values which satisfactorily justified the synthesis of thiobiuret and dithiobiuret metal complexes.

The geometry of five complexes 12, 24, 36, 48, 54 was established through

X-ray crystallography, and concluded that the geometry of Ni(II), Cd(II) complexes is tetrahedral, square planer for Cu(II) and Ni(II) and octahedral geometry for Co(II) complexes.

Fourteen selected complexes were tested for investigating their antibacterial potential. Complex no 8, 12, 13, 14, 27, 32, 38 showed significant antibacterial activity against all bacterial strains. Thirty two complexes were screened for different biological activities including antifungal, total antioxidant activities, protein inhibition assay and anticancer activities. Synthesized compounds showed good to significant activities. Excellent cytotoxicity result was observed of synthesized complexes. These may be promising drug candidates for the future.

Furthermore, all the said synthesized transition metal complexes possessed significant inhibition activity against the cancer cell lines.

In all, findings obtained through the present study indicate that almost all synthesized complexes are promising candidates to combat cancer effects by suppressing oxidative stresses, and as well as anti-microbial activity to control infections.

140

However, further comprehensive animal based studies are warranted to confirm our results to be translated them as drugs/medicines in future.

141

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APPENDICES

Appendix I: Representative UV-Visible Spectrum of complex 1.

Appendix II: Representative UV-Visible Spectrum of complex 2.

157

Appendix III: Representative UV-Visible Spectrum of complex 4.

Appendix IV: Representative UV Spectrum of complex 48 and 54.

158

52.5

424.31

526.53

486.03

977.84

669.25

941.20 37.5 1006.77

649.97

877.55

813.90

30 1174.57

1616.24

3126.40

22.5 711.68

2867.95

765.69

15 3903.65

1222.79

1076.21

1298.00

3384.84

3566.14 1319.22

1128.28

3421.48

2929.67 7.5

2972.10

1373.22

1652.88

1255.57

-0 1404.08

1423.37

1541.02 1352.01

1436.87

1517.87

1477.37 4000 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 600 400 SA 1 1/cm

Appendix V: Representative FTIR Spectrum of Complex 1.

22.5

17.5

2088.76

810.05

12.5

2281.63

513.03

478.31 7.5

2528.50

881.41

5 453.24

595.96

640.32

665.40

2792.73

850.55 2.5

715.54

933.48

779.19

1081.99 -0 2867.95

3211.26

761.83

3103.25

1244.00

2925.81

2979.82

1571.88 1315.36 1157.21

1176.50 1004.84

4000 3600 3200 2800 2400 2000 1800 16001612.38 1400 1350.08 1200 1000 800 600 400

1539.09 1400.22 SA4 1/cm Appendix V1: Representative FTIR Spectrum of Complex 4.

159

Appendix VII: Representative FT-IR Spectrum of Complex 48.

Appendix VIII: Representative FT-IR spectrum of Complex 54.

160

Appendix IX: Representative 1H NMR Spectrum of Complex 1.

Appendix X: Representative 13C NMR Spectrum of Complex 4.

161

Appendix XI: Representative 1H NMR Spectrum of Complex 48.

Appendix XII: Representative 1H NMR Spectrum of Complex 54.

162

LIST OF PUBLICATIONS

1. Saira Sherzaman, Sadiq-ur-Rehman, Saqib Ali, Saira Shazadi and

Madeleine Helliwell, Poly[l2-chlorido-nonamethyl-l3-nitratotritin(IV)].

Corrigendum, Acta Crystallographica, E64, 26, 2008.

2. Saira Sherzaman, Sadiq-ur-Rehman, Muhammad Aziz Choudhary,

Muhammad Aslam Mirza and Khawaja Ansar Yasin, Synthesis, Structure

Elucidation and Study of Antimicrobial Activity of Stannic(Iv) Derivatives.

Asian Journal of Chemistry; 25, 17 (2013), 9688-9692.

3. Saira Sherzaman, Sadiq-ur-Rehman, Muhammad Naeem Ahmed, Bilal

Ahmad Khan, Tariq Mahmood, Khurshid Ayub and Muhammad Nawaz

Tahir, Thiobiuret based nickel(II) and cobalt(II) complexes: Synthesis,

molecular Structures and DFT Studies. Journal of Molecular Structure 1148

(2017) 388-396.