Institute of Chemistry University of the Punjab, Lahore

SYNTHESIS OF ECO-FRIENDLY DYES

A THESIS SUBMITTED TO THE UNIVERSITY OF THE PUNJAB

FOR THE AWARD OF DEGREE OF

DOCTOR OF PHILOSOPHY IN CHEMISTRY Session 2010

SUBMITTED BY: RANA AMJAD AYUB BHATTI

RESEARCH SUPERVISOR PROF. DR. MUNAWAR ALI MUNAWAR

INSTITUTE OF CHEMISTRY UNIVERSITY OF THE PUNJAB, LAHORE.

DEDICATDEDICATEDEDEDED

My Loving Family

Prof. Munawar Ali Munawar

Prof. Robert M. Christie

Mr. Shehzad ...H.HHHassanassanassan.. Pervaiz,

The prayers and guidance of all helped and enabled me to

accomplish this project.

Declaration

I hereby declare that the work described in this thesis was carried out by me under the supervision of Prof. Dr. Munawar Ali Munawar at the Institute of Chemistry, University of the Punjab, Lahore. I also hereby declare that the substance of this thesis has neither been submitted elsewhere for any other degree. I further declare that the thesis embodies the results of my own research work or advanced studies and that it has been composed by my self. Where appropriate, I have made acknowledgement of the work of others.

Rana Amjad Ayub Bhatti

APPROVAL CERTIFICATE

I hereby certify that Mr Rana Amjad Ayub Bhatti s/o M. Ayub Bhatti has conducted research work under my supervision, has fulfilled the condition and is qualified to submit the thesis entitled “Synthesis of Eco-friendly Dyes” in application for the degree of Doctor of Philosophy.

Dr. Munawar Ali Munawar (Supervisor) Professor of Organic Chemistry. Institute of Chemistry, University of The Punjab. Lahore, Pakistan.

ACKNOWLEDGEMENT

All praises to the Al-Mighty “Allah ” who provided us with knowledge, sight to observe and brain to think. Peace and blessings of Allah be upon the Holy Prophet “Hazrat Muhammad” (SAW) who exhorted his followers to seek for knowledge from cradle to grave. I wish to express my heartfelt appreciation and gratitude to my research supervisor Prof. Dr. Munawar Ali Munawar (Professor of Organic Chemistry, Institute of Chemistry, University of The Punjab), for his valuable guidance and continuous encouragement throughout the course of this work. It was indeed an honour and pleasure working with him. I have no words to thank Prof. Dr. Robert M. Christie , under whose guidance I had the opportunity to do part of my research work at the School of Textiles and Designs, Heriot Watt University, Edinburgh, U.K. which eventually enabled me to accomplish this research project successfully. It was indeed an honour to work with a leading Colour Chemist like him. His guidance and constant support during the period is highly appreciable. I also acknowledge Prof. Dr. Saeed Ahmed Nagra , Director, Institute of Chemistry, University of the Punjab and Prof. Dr. Ch. Jamil Anwar , Dean, Faculty of Science, University of the Punjab, for providing outstanding working environment. I would like to express my deep appreciation to Prof. Dr. Misbah-Ul-Ain and Prof. Dr. Makshoof Athar for their sincere and valuable advices and outstanding support through out the time period. I owe my sincere gratitude to Dr. Roger Spark, Dr. Alan Boyd, Dr. Redhuwan, Maggie Robertson and Ann Hardie for their helping hand in my research work at Heriot Watt University Edinburgh. I would also like to thank Higher Education Commission , Pakistan, for providing financial support to carry out this work and my parent organization Pakistan Council for Scientific & Industrial Research (P.C.S.I.R) for allowing and facilitating me to carry out this research project. I cannot forget to thank Wasim Ibrahim, Abdul Waqar Rajput, Wasim Kaimouz (University of Allipo, Syria), and friends of Shish group for making my stay at Galashiels a memorable one. I am also thankful to my research fellows at University of The Punjab for their cooperation and support during the whole period. Special thanks to my parents for their help during my studies and providing me inspiration to accomplish this goal. The acknowledgement would be incomplete if I don’t mention to thank my brother and sisters for their love and prayers for my success.

Rana Amjad Ayub Bhatti

LIST OF ABREVIATIONS DCM Dicholormethane DMSO Dimethyl sulpoxide

CHCl 3 Chloroform NMR Nuclear Magnetic Resonance spectrum s Singlet bs Broad singlet d Dublet t Triplet q Quartet Ar Aromatic ppm Parts per million ETAD Ecological & Technological association of dyestuff Manufacturing Industry IARC International Association for Research on Cancer

TABLE OF CONTENTS CHAPTER-1 INTRODUCTION ...... 1 1. Colour ...... 1 1.1. Constitution of colour...... 1 1.2 Theories of Colour and Constitution ...... 2 1.2.1. Classical theories ...... 2 1.2.2 Modern Theories ...... 4 (a) Valence Bond Theory (V.B.T) ...... 4 (b) Molecular Orbital Theory (M.O.T) ...... 5 1.3 Colorants ...... 6 1.4 Classification of Dyes ...... 9 1.5 Types of Dyes ...... 9 1.5.1. Acid dyes: ...... 9 1.5.2. Reactive dyes: ...... 10 Advantages of the Reactive Dyes ...... 10 1.5.3. Metal complex dyes ...... 11 1.5.4. Direct dyes: ...... 12 Disadvantages of Direct Dyes ...... 13 1.5.5. Basic dyes ...... 13 1.5.6. Mordant dyes ...... 14 1.5.7. Disperse dyes ...... 14 1.5.8. Pigment dyes: ...... 15 1.5.9. Vat dyes ...... 15 1.5.10. Anionic and Ingrain dyes ...... 16 1.5.11. Sulphur dyes ...... 16 1.5.12. Solvent dyes ...... 17 1.5.13. Fluorescent dyes ...... 17 1.5.14. Food dyes ...... 18 1.5.15. Natural dyes ...... 18 1.6 Azo Dyes ...... 19 1.6.1 Chemistry of Azo dyes ...... 20 1.6.2 Absorption spectra of the Azo compounds ...... 21

1.6.3 Effect of humidity on Azo dyes ...... 21 1.6.4 Effect of temperature on Azo Dyes ...... 21

1.6.5 Effect of Solvents on the λmax ...... 21 1.7. Synthesis of Azo Dyes...... 22 1.7.1. Diazotization ...... 22 1.7.2. Azo Coupling ...... 24 1.8. Structure of Azo Dyes...... 26 1.8.1 Monoazo dyes ...... 27 1.8.2 Disazo dyes ...... 28 1.8.3 Trisazo and Tetraazo dyes ...... 29 1.9 Coumarin Based Dyes...... 30 1.10. Flavone based Dyes ...... 37 1.11. Dye’s Toxicity...... 39 1.12. Testing of Mutagenicity ...... 44 Project Aims...... 46 CHAPTER-2 EXPERIMENTAL ...... 47 2. Materials and Methods ...... 47 2.1 Synthesis of Dye Intermediates...... 49 2.1.1. 4 ΄-Aminoflavone (67) ...... 49 2.1.1.1. 2-Acetylphenyl-4-nitrobenzoate (64): ...... 49 2.1.1.2 1-(2-Hydroxyphenyl)-3-(4-nitrophenyl)propane-1,3-dione (65): ...... 50 2.1.1.3 4΄-Nitroflavone (66): ...... 50 2.1.1.4. 4΄-Aminoflavone (67) ...... 51 2.1.2 Synthesis of 3 ΄-Aminoflavone (71) ...... 52 2.1.2.1 2-Acetylphenyl-3-nitrobenzoate (68) ...... 52 2.1.2.2 1-(2-Hydroxyphenyl)-3-(3-nitrophenyl)propane-1,3-dione (69): ...... 53 2.1.2.3. 3΄-Nitroflavone (70): ...... 53 2.1.2.4 3΄-Aminoflavone (71) ...... 54 2.1.3. 6-Aminoflavone (75) ...... 55 2.1.3.1 2-Acetyl-5-nitrophenyl benzoate (72) ...... 55 2.1.3.2 1-(2-Hydroxy-5-nitrophenyl)-3-phenylpropane-1,3-dione (73): ...... 56 2.1.3.3 6-Nitro flavone (74) ...... 56 2.1.3.4 6-Aminoflavone (75) ...... 57 2.2 General method of preparation of Dyes from the Intermediates (67, 71, 75)....58

2.2.1 Diazotization ...... 58 2.2.2 Coupling ...... 58 2.3 6-Amino-4-methylcoumarin (77) ...... 58 2.3.1 N-(4-Methyl-2-oxo-2H-chromen-6-yl)acetamide (76) ...... 58 2.3.2. 6-Amino-4-methylcoumarin (77) ...... 59 2.3.3 Dyes (61-75) from 6-Amino-4-methylcoumarin (77) ...... 59 2.3.3.1 Diazotization of 6-Amino-4-methylcoumarin (77) ...... 59 2.3.3.2 Coupling with the couplers (1-15) ...... 60 2.4. 4-Methyl-7-hydroxycoumarin (78)...... 60 2.4.1 Dyes from 4-Methyl-7-hydroxy-coumarin ...... 61 2.4.1.1 Diazotization ...... 61 2.4.1.2 Coupling ...... 61 2.5 Spectral and analytical data of the synthesised dyes ...... 62 Spectral and analytical data (Dyes 16-30) ...... 62 Spectral and analytical data (Dyes 31-45) ...... 69 Spectral and analytical data (Dyes 46-60) ...... 77 Spectral and analytical data (Dyes 61-75) ...... 84 Spectral and analytical data (Dyes 76-85) ...... 92 2.6 Dyeing of Polyester ...... 97 2.6.1 Stock dispersion solution preparation ...... 97 2.6.2 Dyeing Procedure ...... 97 2.7 Fastness Testing ...... 98 2.7.1 Colour fastness to sublimation ...... 98 2.7.2 Colour fastness to Perspiration (alkaline and acidic) ...... 99 Alkaline solution ...... 99 Acidic solution ...... 99 2.7.3 Colour fastness to washing ...... 100 2.7.4 Colour fastness to light ...... 100 CHAPTER-3 RESULTS & DISCUSSION ...... 102 3.1 Synthesis of Intermediates. (67, 71, 75)...... 102 3.1.1 4΄-Aminoflavone (67) ...... 103 3.1.2 3΄-Aminoflavone (71) ...... 103 3.1.3 6-Aminoflavone (75) ...... 103

3.2 Synthesis of Dyes (16-75) from the Intermediates (67, 71, 75) ...... 104 3.2.1 Dyes (16-30) from 4 ΄-Aminoflavone (67) ...... 106 3.2.1.1 Dyeing on Nylon Lycra ...... 107 3.2.2 Dyes (31-45) from 3 ΄-Aminoflavone (71) ...... 108 3.2.3 Dyes (46-60) from 6-Aminoflavone (75) ...... 109 3.3 Fastness studies...... 112 3.3.2 Fastness studies of dyes (16-30) ...... 112 3.3.1.1 Fastness studies of dyes dyed on Nylon-lycra ...... 117 3.3.2 Fastness studies of Dyes (31-45) ...... 119 3.3.3 Fastness studies of Dyes (46-60) ...... 123 3.4 6-Amino-4-methylcoumarin (77) ...... 127 3.4.1 Dyes (61-75) from 6-Amino-4-methylcoumarin (77) ...... 128 3.4.2 Fastness studies of dyes (61-75) ...... 131 3.5 4-Methyl-7-hydroxycoumarin (78)...... 135 3.5.1 Dyes (76-85) from 4-Methyl-7-hydroxycoumarin (77) ...... 136 3.5.2 Fastness studies of Dyes (76-85) ...... 138 3.6 Mutagenicity Assessment Data ...... 141 Conclusions...... 172 Bibliography...... 173

List of Schemes

Scheme 1 ...... 37 Scheme-2 ...... 102 Scheme-3 ...... 104 Scheme-4 ...... 110 Scheme-4 ...... 127 Scheme-5 ...... 129 Scheme-6 ...... 135 Scheme-7 ...... 136

List of Tables

Table 1. Table of complimentary colours and their wavelength ...... 6 Table-2 List of the carcinogenic amines according to the German Technical Rules for Dangerous Substances...... 43 Table-3 Calculations for the different depth of shades ...... 97 Table-4 Results from Light fastness testing of Dyes (16-30)...... 112 Table-5 Results from wash fastness testing of Dyes (16-30)...... 113 Table-6 Results from Sublimation fastness testing of Dyes (16-30)...... 114 Table-7 Results from Perspiration fastness (acidic / alkaline) of polyester dyed with dyes (16-30)...... 115 Table-8 Absorption spectra data for the dyes (16-30) ...... 116 Table- 8a Fastness studies of samples dyed on Nylon-lycra ...... 117 Table-9 Results from Light fastness testing of Dyes (31-45)...... 119 Table-10 Results from wash fastness testing of Dyes (31-45)...... 120 Table-11 Results from Sublimation fastness testing of Dyes (31-45)...... 121 Table-12. Results from Perspiration fastness (acidic/alkaline) of polyester dyed with dyes (31-45)...... 122 Table-13 Absorption spectra data for the dyes (31-45) ...... 122 Table-14 Results from Light fastness testing of Dyes (46-109)...... 123 Table-14(a) Results from wash fastness testing of Dyes (46-60) ...... 124 Table-15 Results from Sublimation fastness testing of Dyes (46-60)...... 125 Table-16 Results from Perspiration fastness (acidic / alkaline) of polyester dyed with dyes (46-60)...... 126 Table-17 Absorption spectra data for the dyes (46-60) ...... 126 Table-18 Results from Light fastness testing of Dyes (61-75)...... 132 Table-19 Results from wash fastness testing of Dyes (61-75)...... 132 Table-20 Results from Sublimation fastness testing of Dyes (61-75)...... 133 Table-21 Results from Perspiration fastness (acidic / alkaline) of polyester dyed with dyes (61-75)...... 134 Table-22 Absorption spectra data for the dyes (61-75)...... 135 Table-23 Results from Light fastness testing of Dyes (76-85)...... 138 Table-24 Results from wash fastness testing of Dyes (76-85)...... 139 Table-25 Results from Sublimation fastness testing of Dyes (76-85)...... 139

Table-26 Results from Perspiration fastness (acidic / alkaline) of polyester dyed with dyes (76-85)...... 140 Table-27 Absorption spectra data for the dyes (76-85)...... 141

List of Figures

Figure-1. Electromagnetic spectrum...... 2 Figure-2. Terms associated with the wavelength and the extinction coefficient changes ...... 22 Figure-3. Dyeing procedure of polyester fabric with Dyes (16-75)...... 98 Figure-4. Polyester fabric Dyed with dyes (16, 17, 18, 19, 20, 23, 24, 28 ) .. 118 Figure-5. Nylon-lycra dyed with the dyes (16, 17, 20, 22, 23, 24, and 28). ... 118 Figure 6 Dose response curve for Dye (16-20) using the standard mutagenicity assay and TA 98 without S9 activation...... 143 Figure 7 Dose response curve for Dye (21-25) using the standard mutagenicity assay and TA 98 without S9 activation...... 143 Figure 8 Dose response curve for Dye (26-30) using the standard mutagenicity assay and TA 98 without S9 activation...... 144 Figure 9 Dose response curve for Dye (31-35) using the standard mutagenicity assay and TA 98 without S9 activation...... 144 Figure 10 Dose response curve for Dye (36-40) using the standard mutagenicity assay and TA 98 without S9 activation...... 145 Figure 11 Dose response curve for Dye (41-45) using the standard mutagenicity assay and TA 98 without S9 activation...... 145 Figure 12 Dose response curve for Dye (46-50) using the standard mutagenicity assay and TA 98 without S9 activation...... 146 Figure 13 Dose response curve for Dye (51-55) using the standard mutagenicity assay and TA 98 without S9 activation...... 146 Figure 14 Dose response curve for Dye (56-60) using the standard mutagenicity assay and TA 98 without S9 activation...... 147 Figure 15 Dose response curve for Dye (61-65) using the standard mutagenicity assay and TA98 without S9 activation...... 147 Figure 16 Dose response curve for Dye (66-70) using the standard mutagenicity assay and TA98 without S9 activation...... 148 Figure 17 Dose response curve for Dye (71-75) using the standard mutagenicity assay and TA98 without S9 activation...... 148 Figure 18 Dose response curve for Dye (76-80) using the standard mutagenicity assay and TA98 without S9 activation...... 149

Figure 19 Dose response curve for Dye (81-85) using the standard mutagenicity assay and TA98 without S9 activation...... 149 Figure 20 Dose response curve for Dye (16-20) using the standard mutagenicity assay and TA 98 with S9 activation...... 150 Figure 21 Dose response curve for Dye (21-25) using the standard mutagenicity assay and TA 98 with S9 activation...... 150 Figure 22 Dose response curve for Dye (26-30) using the standard mutagenicity assay and TA 98 with S9 activation...... 151 Figure 23 Dose response curve for Dye (31-35) using the standard mutagenicity assay and TA 98 with S9 activation...... 151 Figure 24 Dose response curve for Dye (36-40) using the standard mutagenicity assay and TA 98 with S9 activation...... 152 Figure 25 Dose response curve for Dye (41-45) using the standard mutagenicity assay and TA 98 with S9 activation...... 152 Figure 26 Dose response curve for Dye (46-50) using the standard mutagenicity assay and TA 98 with S9 activation...... 153 Figure 27 Dose response curve for Dye (51-55) using the standard mutagenicity assay and TA 98 with S9 activation...... 153 Figure 28 Dose response curve for Dye (56-60) using the standard mutagenicity assay and TA 98 with S9 activation...... 154 Figure 29 Dose response curve for Dye (61-65) using the standard mutagenicity assay and TA98 with S9 activation...... 154 Figure 30 Dose response curve for Dye (66-70) using the standard mutagenicity assay and TA98 with S9 activation...... 155 Figure 31 Dose response curve for Dye (71-75) using the standard mutagenicity assay and TA98 with S9 activation...... 155 Figure 32 Dose response curve for Dye (76-80) using the standard mutagenicity assay and TA98 with S9 activation...... 156 Figure 33 Dose response curve for Dye (81-85) using the standard mutagenicity assay and TA98 with S9 activation...... 156 Figure 34 Dose response curve for Dye (16-20) using the standard mutagenicity assay and TA 100 without S9 activation...... 157 Figure 35 Dose response curve for Dye (21-25) using the standard mutagenicity assay and TA 100 without S9 activation...... 157

Figure 36 Dose response curve for Dye (26-30) using the standard mutagenicity assay and TA100 without S9 activation...... 158 Figure 37 Dose response curve for Dye (31-35) using the standard mutagenicity assay and TA100 without S9 activation...... 158 Figure 38 Dose response curve for Dye (36-40) using the standard mutagenicity assay and TA100 without S9 activation...... 159 Figure 39 Dose response curve for Dye (41-45) using the standard mutagenicity assay and TA100 without S9 activation...... 159 Figure 40 Dose response curve for Dye (46-50) using the standard mutagenicity assay and TA100 without S9 activation...... 160 Figure 41 Dose response curve for Dye (51-55) using the standard mutagenicity assay and TA100 without S9 activation...... 160 Figure 42 Dose response curve for Dye (56-60) using the standard mutagenicity assay and TA100 without S9 activation...... 161 Figure 43 Dose response curve for Dye (61-65) using the standard mutagenicity assay and TA100 without S9 activation...... 161 Figure 44 Dose response curve for Dye (66-70) using the standard mutagenicity assay and TA100 without S9 activation...... 162 Figure 45 Dose response curve for Dye (71-75) using the standard mutagenicity assay and TA100 without S9 activation...... 162 Figure 46 Dose response curve for Dye (76-80) using the standard mutagenicity assay and TA100 without S9 activation...... 163 Figure 47 Dose response curve for Dye (81-85) using the standard mutagenicity assay and TA100 without S9 activation...... 163 Figure 48 Dose response curve for Dye (16-20) using the standard mutagenicity assay and TA100 with S9 activation...... 164 Figure 49 Dose response curve for Dye (21-25) using the standard mutagenicity assay and TA100 with S9 activation...... 164 Figure 50 Dose response curve for Dye (26-30) using the standard mutagenicity assay and TA100 with S9 activation...... 165 Figure 51 Dose response curve for Dye (31-35) using the standard mutagenicity assay and TA100 with S9 activation...... 165 Figure 52 Dose response curve for Dye (36-40) using the standard mutagenicity assay and TA100 with S9 activation...... 166

Figure 53 Dose response curve for Dye (41-45) using the standard mutagenicity assay and TA100 with S9 activation...... 166 Figure 54 Dose response curve for Dye (46-50) using the standard mutagenicity assay and TA100 with S9 activation...... 167 Figure 55 Dose response curve for Dye (51-55) using the standard mutagenicity assay and TA100 with S9 activation...... 167 Figure 56 Dose response curve for Dye (56-60) using the standard mutagenicity assay and TA100 with S9 activation...... 168 Figure 57 Dose response curve for Dye (61-65) using the standard mutagenicity assay and TA100 with S9 activation...... 168 Figure 58 Dose response curve for Dye (66-70) using the standard mutagenicity assay and TA100 with S9 activation...... 169 Figure 59 Dose response curve for Dye (71-75) using the standard mutagenicity assay and TA100 with S9 activation...... 169 Figure 60 Dose response curve for Dye (76-80) using the standard mutagenicity assay and TA100 with S9 activation...... 170 Figure 61 Dose response curve for Dye (81-85) using the standard mutagenicity assay and TA100 with S9 activation...... 170 Figure 62 Dose Response Curve for Dyes (30,45, 60, 75 and 80) using the standard mutagenicity assay and TA98 with S9 activation...... 171 Figure 63 Dose Response Curve for Dyes (30,45, 60, 75 and 80) using the standard mutagenicity assay and TA98 with S9 activation...... 171

Abstract

This research project had three aspects, 1. Synthesis of novel dyes, 2. Assessing their applications i.e. fastness properties etc. 3. Testing the synthesised dyes for their mutagenicity.

Synthesis and evaluation of azo disperse dyes was done while using different aminoflavones (4 ΄-Aminoflavone, 3 ΄-Aminoflavone and 6- Aminoflavone), and coumarins (like 6-Amino-4-methylcoumarin and 4-Methyl- 7-hydroxy-coumarin). Dyes (16-75) have been synthesized by coupling the synthesized intermediates with the substituted aniline (1-10) as well as the naphthalene based couplers (11-15). The characterization of the dyes was done by Infrared, 1H-NMR, Mass Spectrometry, 13 C-NMR and Elemental analysis. Diazotization of the different heterocyclic amines (1a-10a). was carried out and the 4-Methyl-7-hydroxycoumarin was used as coupler to yield the dyes (76- 85). The synthesised dyes were applied on the polyester fabric and it proved to be effective as they gave good fastness properties. The dyes obtained had good fast colours ranging from orange to purple. Some of the synthesised dyes (16, 17, 20, 23, 24, 28) were also applied on the Nylon-lycra fabric and it gave good dyeing as well as good to moderate fastness properties. The yield of the dyes obtained was from 65-83 %. All the synthesised dyes were evaluated for their mutagenicity by undergoing standard Ames test. This was performed by using the Salmonella strains (TA 98 and TA 100) with and without S9 activation. The results of these mutagenicity results showed that almost all the dyes proved to be negative in the Ames test except the dyes having free amino group which can be attributed to be cause of their mutagenicity. The presence of the free amino group made these dyes more prone to be mutagenic as compared to the other dyes. 1. COLOUR Colour plays an important role in our every day life. It has a great impact and influence on our moods and emotions and helps us to enjoy our surroundings. The usage of colour is not a new one even in the prehistoric times it has been seen to be used in the body paints, colouring of the furs and skins and also on the cave wall paintings.

1.1. Constitution of colour Colour is basically the visual perceptual property by which the brain recognises the different qualities of the light which falls on the retina. The human eye recognises the light which falls between the wavelength range of 380-780 nm. Maxwell suggested that light has electromagnetic character and in 1905 Plank and Einstein gave the particle theory of electromagnetic radiation. Maxwell’s wave theory proposed that light is a stream of discrete energy particles or photons. According to the wave theory light can be characterized by its wavelength or frequency.

c= νλ Here c is the velocity of light, ν is the frequency and is λ the wave length of light. The particle theory of light deals with the characterisation of monochromatic radiation by the energy of the each photon. The Plank’s equation given below relates the energy of the photon to the frequency of the wave,

E= h ν h = Plank’s constant and its value is 6.63 x 10 -34 Js The electromagnetic spectrum comprises of the complete range of wavelengths of electromagnetic radiation and it starts from the short wavelengths such as the X-rays and γ rays to the radio waves having the long wavelengths. The visible region in the spectrum is from 380- 780 nm. The electromagnetic spectrum is shown in the Figure 1.

Figure-1. Electromagnetic spectrum. The colouring compounds commonly termed as colorants (dyes and pigments) have the ability to absorb selective radiations from the visible region. The absorption characteristics of the dyes can be measured in a solution by the UV-visible absorption spectroscopy. The Beer-Lambert law gives the relationship between the absorbance A of a dye solution and the concentration of an absorbing species and it is written as given below,

A= ε c l Where ε = the molar extinction coefficient having the units of mol -1 cm -1 c = concentration of solute expressed in mol l -1 l = path length of the sample expressed in cm.

1.2 Theories of Colour and Constitution 1.2.1. Classical theories Chemists have long been studying the relationship between the colour and the chemical constituent of the dyes. Grabe and Liberman in 1867 1 first proposed the relationship between colour and the constitution. It was proposed in 1876 by Witt 2 that chemically dyes consist of two groups, one is

called chromophores and the second auxochromes. Chromophores are principally responsible for the colour of the dye and the auxochromopores for the enhancement of the colour of the dye. Gomberg 3 in 1900 discovered triphenylmethyl as first coloured radical, and while working on this radical Baeyer 4 proposed the theory of halochromism according to which it was proposed that a colourless compound may become coloured on salt formation. In 1907 Baeyer 4 proposed a rapid oscillation between the two tautomeric forms of a compound like that of Doebner’s violet (1a-1b) , and a rapid flipping of the chloride ion from one amino group to another. Watson proposed Tautomeric Theory in 1914 5 and this helped further in the understanding of colour and the constitution relationship. This theory also further proposed that tautomerism is the basic requirement to achieve a coloured molecule.

- + - Cl H2N+ NH H2N NH 2 Cl 2

(1a) (1b)

The link between colour and the oscillation of electrons was first proposed by Adams and Rosenstein 6. The old concept of oscillation of atoms which gives rise to the adsorption of infrared radiation was rejected by them. In 1935 Bury highlighted the relationship between the colour of a dye and resonance 7. Bury also proposed that the colour of the Doebner’s violet was because of the electron that moved and not the movement of the atoms. This movement of electrons was explained by the concept of resonance (2a, 2b).

+ + H2N NH H2N NH2 2

(2a) (2b)

Bury also proposed that colour was due to the involvement of a chromagen in resonance in the molecule. In 1916 it was proposed by Lewis that colour is the result of selective absorption of light by valence electrons. The frequency of oscillation was suggested to be synchronised with light of a definite frequency.

1.2.2 Modern Theories The modern theories of colour i.e . Valence Bond Theory (V.B.T) and Molecular Orbital Theory (M.O.T) have evolved from the Einstein Plank Quantum Theory according to which energy adopts quantised values and is not continuous. Valence Bond Theory is based on the valence electron pair which are being localised between specific atoms while in the Molecular Orbital Theory electrons are considered as being distributed among a set of molecular orbitals of specific energies.

(a) Valence Bond Theory (V.B.T) Valence bond theory is also called as The Resonance Theory. The quantitative approach to the Valence Bond Theory was a failure in the case of the large molecules. After the publication of the Schrodinger’s equation qualitative approach to the Valence Bond Theory was put forward by London, Pauling and Slater. According to the Valence Bond Theory the electrons of a compound are localised in bonds between the atoms. The whole structure is considered to be the hybrid of the contributing forms. In order to apply the valence bond approach to the colour and constitution two assumptions were made which are,

(i) The ground state electronic state of the dye resembles the most stable resonance form. (ii) The first excited state of the dye resembles the less stable charge separated form.

(b) Molecular Orbital Theory (M.O.T) According to the Molecular Orbital Theory the electrons are considered to be a form of electromagnetic radiations. Because of the electronic distribution within a molecule electronically excited states are formed. This results in the absorption of the ultraviolet and visible light. According to the quantum theory molecule exists in a series of discrete energy states. The molecules are promoted to a higher energy state when the quanta of visible ultraviolet light are absorbed. The energy of the radiation absorbed and the absorption frequency is related by the following equation,

Eexcited state – E ground state = h ν Schrodinger’s wave equation in its simplest form is written as

E Ψ = H Ψ Here,

Ψ = molecular orbital wave function, a function of the momenta and coordinates of all the electrons in the molecule. E = energy associated with the molecular orbital. H = Hamiltonian operator (a complex differential operator which

operates on the molecular wave function Ψ). The overlap of atomic orbitals generates molecular orbitals obtained as a result of direct overlap is low energy. The sideways overlap gives rise to π molecular orbitals. The extended conjugation of dyes contains a frame work of σ bonds with an associated π system. The promotion of electrons from a π to an unoccupied π* orbital gives rise to the lowest energy transition. The absorption of organic dyes and pigments in the UV and visible regions of the spectrum are mainly because of the π- π* transitions.

1.3 Colorants Colorants are molecules that absorb light in the visible spectrum i.e . 380-780 nm and they are commonly classified into two main groups, organic and inorganic. The colorant can be either dye or pigment. The difference between pigment and dye is that pigments are insoluble in the medium in which they are applied whereas the dyes are soluble in the medium in which they are applied. Moreover pigments are mostly crystalline aggregates having particle size 1-10 µm and dyes must exist in monomolecular form. Besides that dyes usually exhibit special affinity for the substrate to which they are applied. A chemical compound appears coloured when it absorbs radiant energy with a wavelength that corresponds to light in the visible part of the spectrum. The light transmitted or reflected by the given compound produce the sensation of colour. So the light, that is being perceived is complimentary to that is absorbed. White light is basically the combination of seven colours. These seven colours are violet, blue, green, yellow, orange and red and they have wavelength range between 380-780 nm. When white light falls on any substrate it may be totally or partially absorbed, or reflected back.

Table 1. Table of complimentary colours and their wavelength8 Wavelength of light Colour Absorbed Visible Colour Absorbed (nm) 400-435 Violet Yellow Green 435-480 Blue Yellow 480-490 Green Blue Orange 490-500 Blue Green Red 500-560 Green Purple 560-580 Yellow Green Violet 580-595 Yellow Blue 595-650 Orange Green Blue 650-800 Red Blue Green

The substance appears to be white when the light is totally reflected and it appears black when it is totally absorbed. When a part of the incident light is absorbed and rest of it is reflected, the colour of substance is actually the colour of the reflected light. A Dye can be defined as an intensely coloured substance which when applied to a substrate, imparts colour to it. Dyes are retained in the substrate by absorption into the fiber or by ionic or covalent bonds. The usage of colour is quite old one and since the beginning of human kind the people have been using colorants for painting and dyeing of their surroundings, their skins and their clothes. Until the middle of the 19 th century almost all the colorants applied were from natural origin. Inorganic pigments such as soot, hematite, manganese oxide and ochre have been utilized within living memory. Recently in the Chauvet caves in France, Paleolithic rock paintings such as the 30,000 years old drawings provide ancient testimony of their application 9. Organic natural colorants have also been used as textile dyes. Most of these natural dyes are aromatic compounds, originating usually from plants ( e.g . alizarin and indigo) and from insects, fungi and lichens. The synthetic dye manufacturing was done accidentally in 1856 when the English Chemist Perkin 10 while attempting to synthesis quinine precursor obtained instead a bluish substance with excellent dyeing properties that latter became known as aniline purple. Perkin was not successful but he became interested in the reaction of other coal tar bases including the mixture of aniline and toluidines. He observed that the crude bases produced an intense bluish purple solution. This bluish purple solution dyed silk and this discovery led to the manufacture of a new colorant. The new dye was called Mauve or Mauveine (3) and was patented by 18 years old Perkin on 26 th August 1856. He was successful in establishing a dye industry by the name of Perkin and Sons.

CH3

N NH2

+ H3C N

H3C NH

CH3 (3)

In 1857 a factory was created for the production of this new dye and at that time no one dreamed of the far-reaching influence of these events on world history during the next century. The dye industry catalyzed the development of the general synthetic organic chemical industry in England, France, Germany and USA. This concept of research and development soon triggered others and new dyes began to appear in the market. In the beginning of the 20 th century synthetic dyestuff had almost completely supplanted natural dyes 11 . Madder, a wood extract is one of the most important the natural dyes. Its main constituent is alizarin (1, 2-dihydroxyanthraquinone) (4).

OH O HO

O (4) Picric acid (5) also has been used as a synthetic dye, was first prepared in the laboratory by treating indigo with nitric acid in 1877. Silk gave a bright greenish yellow colour when dyed with picric acid but the light fastness was poor, so it didn’t get any real significance.

O2N NO 2

NO 2 (5)

1.4 Classification of Dyes The dyes can be classified on the basis of two things, (1) Chemical structure (2) Usage or application method. The first type of classification is of more interest to chemists, who desire to know structurally the molecule of the dye. The second is of interest to dyers, who need to know which dye is appropriate to the material i.e. fabric etc they need to dye, and what will be the resultant colour. In the Colour Index which is edited since 1924 (and revised after every three months) by the Society of Dyers and Colourists and The American Association of Textile Chemists and Colourists the dyes have been classified into more than 15 types 12 .

1.5 Types of Dyes Generally dyes are classified into the following types;

1.5.1. Acid dyes: Acid dyes (6, 7) are the anionic compounds and are mainly used for dyeing nitrogen containing fabrics like silk, wool, modified acryl and polyamide. + They bind to the cationic NH 4 ions of those fabrics. Acid dyes are dyed in pH range from acid to neutral. Acid dyes are further classified into three subclasses: a) Simple acid dye- without any assistant (helper). b) Mordant acid dye- which needs a helper to fix to fiber. c) Premetalized acid dyes

In Premetalized acid dyes some metal ion is incorporated in the dye which forms a chelate which improves the fastness properties of the dye.

HO HO

N N

HO 3SN HO 3SN

Acid Red-88 (6) Acid Orange-7 (7)

1.5.2. Reactive dyes: The Reactive dyes form covalent bonds with NH-, -OH, or SH- groups in fibers (cotton, wool, silk, nylon). Reactive dyes (8, 9) are water soluble and contain a group capable of reacting with hydroxyl or amino groups in the cellulose, wool and other substrate.

Advantages of the Reactive Dyes (a) Permanency of the colour- Fibre Reactive Dyes can be easily said to be the most permanent of all dye types. This is because of a unique quality, of forming a covalent bond with the substrate (cellulose or protein molecule). The chemical bonds significantly improve the product's colour stability and wash ability. Thus, no doubt reactive dying of cotton is presently the most popular textile dying process in the world. (b) Easy washing- The fibres that are dyed with reactive dyes can be safely dyed even with white garments without the danger of colouring it. Moreover Reactive dyes offer, a) Improved wash fastness over direct dyes. b) Improved brightness over vat dyes. c) Improved shade range over azoic dyes. BASF revolutionized the reactive dyes by introducing reactive dyes containing a halobenzene nucleus linked to the chromophoric group via an amino linkage and additionally containing a second reactive group 13 .

SO3H Cl H N Cl N N CH 3 SO H N N N H 3 N N N N O N N H N N NH2 Cl Cl HN CH3 Cl SO3H O SO3H

C.I.Reactive Yellow-3 (8) C.I. Reactive Yellow-4 (9)

1.5.3. Metal complex dyes These dyes are strong complexes of metal atom (usually copper, chromium, nickel or cobalt) and one or more dye molecules. These dyes also find wide application in the field of textiles and leather. The examples of metal dyes are (10) and (11) .

O Et O C HN

N MeO S 2 N O

O Cr O O N SO Me N 2

NH C O Et O

C.I.Black-58 (10)

- - SO3 O3S

N N

O O Cr O O

N N

- O3S - SO3

C.I.Acid Blue-193 (11).

1.5.4. Direct dyes: Direct dyes are relatively large molecules with high affinity for cellulose fibers. These dyes are mostly azo dyes (with more than one azo bond) or phthalocyanine, oxazine or stilbene compounds. These dyes give full range of colours from full rich black to bright shades of all hues. Direct dyes differ from acid dyes due to their affinity towards cellulosic fiber like cotton.

SO 3Na NaO 3S NN N N

NaO 3S SO 3Na OH HO

NH2 H2N

Direct Blue 2-B (12)

Disadvantages of Direct Dyes

(a) Few Direct Dyes have low light fastness.

(b) Many Direct Dyes are dull in colour.

1.5.5. Basic dyes Basic dyes are cationic compounds and are used for dyeing acid-group containing fibers. They bind to the acid groups of the fibers. These dyes are water soluble and their dye part is present in cation. Their exceptional brightness is due to fluorescent nature. These dyes have affinity for wool, silk, leather, acrylic fiber and paper. These dyes can be applied on cotton if mordanted. The mordanting process usually requires buffering system of pH 5-6. Today urea is used for this purpose. The first synthetic dye Mauveine (5) belongs to this class of dye.

(H 3C) 2N N(CH 3)2

+ - N H2Cl

Auramine O, Basic Yellow-2 (13)

+ - (H 3C) 2N N (CH 3)2 Cl

N(CH 3)2

Crystal Violet, Basic Violet-3 (14)

1.5.6. Mordant dyes Mordant dyes (e.g. 15, 16) are fixed to fabrics by the addition of a mordant, a chemical that combines with the dye and fiber. These dyes have a little or no affinity for certain substrates. The process of fixing of these dyes is called mordanting. The mordants not only change the shade of original dyeing but also improve fastness of dyeing to both water and light. Alizarine red is an example of mordant dye. It is used for dying cotton and to some extent wool as well.

OH HO O HO OH NaO 3S N N

Mordant Red-14 (15) Mordant Black-17 (16)

1.5.7. Disperse dyes Disperse dyes have low solubility in water but they can interact with the synthetic fibre by forming dispersed particles. These dyes are non-ionic, water insoluble which can be dispersed in water in a very fine particle size. The general structure of the disperse dyes is small, non-ionic and planar with attached polar functional groups like –CN and –NO 2. These dyes can be utilized for the cellulose acetate, nylon, polyester and acrylic fibers. These dyes form the third largest group of dyes. The fastness properties of this range of dyes vary and this is dependent upon the fiber to which they are applied. Wash fastness is better when they are applied to hydrophobic fibers e.g . polyester or triacetate as compared to when applied on the secondary acetate or nylon. Examples of Disperse dyes are (17, 18).

NO 2 OH NH

N O2N NH N

NO 2 Disperse Yellow-1 (17) Disperse Orange (18)

1.5.8. Pigment dyes: These are the insoluble non-ionic compounds or insoluble salts that retain their crystalline or particulate structure throughout their application.

HO HO

NO 2 N N

H3C N O2NN

C.I.Pigment Red-3 (19) C.I.Pigment Red-1 (20)

1.5.9. Vat dyes Vat dyes are water insoluble dyes that are particularly and widely used for dyeing cellulose fibers. This class of dyes (21) is distinguished by the special method of application called vating operation. In this operation water insoluble dye is made soluble by chemical reduction of the chromophore, the ketonic group to alcoholic leuco compound, which forms water soluble, salt i.e. the leuco salt in the presence of alkali. After dyeing with vat solution the substance is reoxidised with air or oxidizing agent which reverses to form water insoluble colour leaving it on the fiber. The dye is thus mechanically trapped by the fiber.

NH O O HN

O Vat Blue-4 (21)

1.5.10. Anionic and Ingrain dyes These are the insoluble products of the reaction between a coupling component (mostly N, N-disubstituted anilines or phenols) and diazonium salt. These types of dyes (e.g . 22) are water insoluble and are directly applied on the substrate by some type of chemical action. These azoic dyes are mechanically trapped by the fiber. These colours are also called ‘Ice Colour’ because disazo component is kept at very low temperature to avoid its decomposition.

O2N N N

Red Ingrain Dye (22)

1.5.11. Sulphur dyes Sulphur dyes are water insoluble polymeric compounds containing disulfide (S-S) or oligosulphide (S-S) n linkage between the aromatics. The aromatic compounds from which these macromolecules are produced are phenols, aromatic amines, aminophenols etc. They are mainly used for dyeing cotton, silk, rayon and rubber goods. These dyes are water insoluble and are brought into soluble form by treatment with a hot solution containing sodium

sulphide or sodium hydrosulfite. About 20% of the synthetic dyes are Sulphur dyes and 38% of the cellulose fabrics are dyed with sulphur dyes 14 .

S H N N N NH 2 NH2 H2N 2

HO S O O S OH S 5-7 (23) 1.5.12. Solvent dyes Solvent dyes are non-ionic dyes and are used for dyeing substrates in which they can dissolve e.g . plastic, varnish, waxes, ink, and fats. They contain no sulphur or any other water insoluble group. These are soluble in organic solvents. Solvent Red-23 (24) is an example of Solvent dye.

N N N N

Solvent Red-23 (24) Solvent dyes have wide usage. They are applied to colour organic solvents, waxes, inks, hydrocarbon fuels, lubricants, and a variety of other hydrocarbon-based non-polar materials. Solvent-based dyes are used to add colours to a large variety of polymeric materials like PVC, nylon, polyester, acrylics, PMMA, polystyrene, and PETP.

1.5.13. Fluorescent dyes Fluorescent dyes are infact not dyes in the usual sense because they lack intense colour. They mask the yellowish tint of the natural fibers by absorbing ultraviolet light and weakly emitting visible blue. The examples of the florescent dyes are (25) and (26) .

NH2 NH2

N N

O N H H5C2 H5C2 N O O N O O

C2H5 C2H5

(25) (26)

1.5.14. Food dyes Food dyes are used for colouring of edible items. These dyes should qualify the international standards for being fit for edible usage. As these dyes are classed as food additives, they are manufactured to a higher standard than some industrial dyes. Food dyes can be direct, mordant and vat dyes, and their use is strictly controlled by legislation. Many are azo dyes, although anthraquinone and triphenylmethane compounds are used for colours such as green and blue. Some naturally-occurring dyes are also used.

1.5.15. Natural dyes The natural dyes can be used as mordant, vat, and direct, acid or solvent dyes in textile processing. Considering the toxic effects of the synthetic dyes there have been renewed efforts by the dyers and colourists to find the substitute of the synthetic dyes and for such cases the natural dyes are the best substitute. There are three categories of natural dyes, first ones are the dyes derived from the plant source like indigo (26) , second ones are derived from the animal sources called Cochineal (27) , and the third ones that are got from minerals (Ocher) . Natural dyes can provide the much needed alternative to the complex world of chemical dyes as they are environmentally friendly.

H O HO HO N O O HO

N HO OH OH HO O O H H3C O HO

Indigo (27) Carmine Dye (Cochineal) (28)

1.6 Azo Dyes These form the largest class accounting for more than half the total of disperse dyes. They now cover virtually the whole of the spectrum from greenish yellow, orange, brown, red, bright pink, violet, royal blue, navy blue, green to black. They contain an enormous variety of structural types 15 . In the beginning the azo dyes had certain drawbacks such as; 1. The range was rather limited particularly with regard to variety of shades and brightness. 2. Some dyes had relatively low light fastness. 3. Sublimation fastness was inadequate in many cases. The extension of the colour range (especially brighter colours) and improvements in fastness properties have enabled these dyes to challenge the unique position traditionally held by anthraquinone dyes. Their main advantage is an economic one because they are cheaper then anthraquinone disperses dyes and in addition they can be discharged. Many dyes are visible at concentration as low as 1 mg/L in water. Textile processing wastewater typically with a dye content in the range 10-200 mg/L 16 is therefore usually highly coloured and discharge in an open water presents an aesthetic problem. As dyes are designed to be chemically and protolytically stable they are highly persistent in natural environments. The release of dyes may therefore present an eco-toxic hazard and it can introduce the potential danger of bioaccumulation that may eventually affect man by transport through the food chain.

1.6.1 Chemistry of Azo dyes The history of the Azo dyes ages back to 1858 but now they account for more then 50 % of the total number of dyes which are being manufactured all over the world 17 . This important class of dyes contains the characteristic – N=N- chromophore. Such sort of dyes may have one or more than one azo groups and thus they are described as mono azo, bis azo, tris azo etc. A number of possible structural variations have given the advantage to the azo dyes to cover a huge range of hues 17 . The absorption and the fastness properties of the azo dyes are influenced by the presence of the substituent in the aryl ring which are known as the auxochromes. Another important factor with the azo dyes is the tautomeric equilibrium between the azo (29a) and the hydrazone (29b) forms. This position of the tautomer is significantly influenced by the polarity of the environment and the interaction with the substrate as well. It has been described that the position of the –OH group affects the light fastness properties of the dyes 17 .

R R

NH N NO N OH

Azo form (29a) Hydrazo form(29b)

The metal complexes of the azo dyes are also being widely used for the dyeing of the polyamide and protein fibers 17 . The process of metallization of the azo dyes can be done either during the dye synthesis or after the dyeing. The colour produced depends upon the metal selected and the dye ligands. This complex formation can be reversible also in some of the cases depending upon the changes in the pH and the stability of the metal ligand bond. A large number of the structural possibilities exist which can produce a pattern of the excited electronic states with associated transitions originating in the atomic orbital of the metal ion. Hence an unmetallized dye can produce a number of hues of the different tinctorial strengths by complexing it with

different metal ions. It is often difficult to quantify the transitions that lead to the absorption spectrum of a given complex.

1.6.2 Absorption spectra of the Azo compounds Aliphatic compounds absorb at short wavelength as compared to the aromatic azo compounds because of the presence of the conjugation in the azo group with the aromatic rings 18 . In such cases mostly two bands are seen one in the visible region and the other one in the UV region. The one in the UV region is stronger.

1.6.3 Effect of humidity on Azo dyes It has been seen that atmospheric moisture can promote the photophading of the azo dyes when they are used to dye the polymers. A number of studies show that there exists a quantitative relationship between the moisture contents and the fading of some dyes 19-23.

1.6.4 Effect of temperature on Azo Dyes It has been seen in a number of experimental studies that there is a correlation between the temperature and the photophading of the dyes. With the increase in the temperature photophading of the azo dye increases.

1.6.5 Effect of Solvents on the λmax The phenomenon which deals with the shift in the solvent polarity with shifts in the λmax of the dye either to the longer or the shorter wavelength is called as solvatochromism. This depends on the type of the interaction between the solvent and the ground and the excited states of the dye. For example with the increasing solvent polarity the excited state of the polar dyes molecules gets stabilized hence producing the bathochromic shift of the absorption band because of the decreased HOMO-LUMO gap. In the other case if the ground state of the dye molecule is more polar than the excited one then the interaction of the polar solvents with the ground state will be stronger than that of the excited state hence resulting in the hypsochromic shift due to an increased HOMO-LUMO gap. So the solvent polarity plays a 24 key role in determining the excitation energy . These shifts in the λmax can

also be accompanied by the changes in the magnitude of the extinction coefficient. Increase in the extinction coefficient is known as the hyperchromic and the decrease as the hypochromic effect. 24 . The summary of the possible effects on the solvent polarities on the absorption spectra is shown in the Figure-2.

Figure-2. Terms associated with the wavelength and the extinction coefficient changes

1.7. Synthesis of Azo Dyes Peter 25 was the first person who discovered aromatic diazo compounds in 1858. Preparation of azo compounds by the classical diazotization and coupling method has proven to be easy and also economically feasible hence numerous dyes and pigments have been synthesized. The synthesis of the azo dyes involve two steps i.e . Diazotization and coupling.

1.7.1. Diazotization Various methods of diazotization are reported in the literature 26 . But the main fundamental aspect of diazotization step is reaction of an aromatic primary amine with sodium nitrate in the presence of a mineral acid, in an aqueous medium.

OH O N O2N NH2 O2N N + + HNO2 2H2O

NO2 NO2

Diazotization process. (Equation-1)

In the diazotization step, the primary aromatic amine is reacted with a nitrosating species, e.g . HNO 2 generated in situ by reacting sodium nitrate with aqueous solution of a mineral acid e.g. HCl, H2SO 4 etc. The optimum acidity for the diazotization depends upon basicity of the amine being used. The reaction temperature is critical as the diazonium salts are instable and the reaction itself is exothermic. They are mostly synthesized at temperature below 5 0C. The diazotization reaction can be monitored by using the starch iodide paper. Nitrous acid concentration is generally required in a slight excess and the starch iodide paper turns blue once this excess has been achieved.

ArNH 2HX 2 + + NaNO2 ArN2X + NaX + 2H2O

- (X= Cl, Br, HSO 4, etc.)

Basic reaction scheme for diazotization.(Equation-2) A large excess of nitrous acid is not favorable for the stability of the diazonium salt as it causes the decomposition of the diazonium salt. Hence to avoid that, the excess nitrous acid must be destroyed. This is accomplished by the addition of urea or sulphamic acid.

NH 2SO 3H + HNO 2 N 2 + H 2SO 4 + H 2O. (Equation-3)

1.7.2. Azo Coupling Azo coupling reaction is the electrophilic substitution by the diazo group resulting in the loss of the hydrogen atom on the coupling component to form an azo compound. 1 Ar N

1 + 2 2 Ar N NX + Ar H N Ar + HX

Reaction of Diazonium salt with coupling component. (Equation-4)

The diazonium ions are relatively weak electrophiles and the aromatic compounds that carry strong donor substituents like OH, NH 2, NHR, NR 2 can undergo azo coupling. The rate of the azo coupling reaction which is usually carried out in an aqeous medium, at a given ambient temperature is dependent upon the nature of the diazo compound, the nature of the coupling component and the pH of the medium. The optimum coupling pH range for the aromatic amines is 4-9. They are converted into the more water soluble protonated form at this pH range.

+ + H + Ar--NH 2 Ar-NH 3 (Equation-5)

While for the phenols or napthols the optimum pH range is 7-9.

- - Ar—OH + OH Ar-O + H2O (Equation-6)

The generated anionic Ar-O- is more water soluble than the phenol and the anion is more powerfully electron releasing then OH hence it undergoes the electrophilic substitution more easily. Compounds containing active methylene group like 3-methyl-1-phenyl- 5-pyrazolone (30a) undergo the coupling reaction while forming enolate ion (30c) at the pH 5-9. Although at high pH level the concentration of the enolate ion increases but this can also lead to the side reactions because of the diazonium salt decomposition. Thus to avoid the formation of side products reaction is carried out at appropriate pH at which the coupling takes place at a

good rate. In such reactions only the conjugate base (30b) participates in the actual substitution step.

CH 3 CH3 CH 3 CH3 + + +H N -H N N O N O O N N N HO N

(30a) (30b) (30c) Ketonic form Conjugate Base Enolic form

The rate of the addition of the diazonium salt during the coupling has to be carefully controlled so that the excess of it is not present in the coupling component suspension, because it is difficult to remove at the end of the reaction and remains with the dye in the form of impurities. The presence of the excess diazo component can be confirmed by utilizing the H-Acid test. In coupling components carrying both the amino,as well as the hydroxyl groups, the site of the reaction is determined by controlling the pH of the reaction medium. In the case of the amino napthols e.g . 2-amino-8- hydroxy-6-sulphonic acid (Gama acid) (31) in the acidic media the coupling takes place at the 1- position i.e . on the aromatic ring which carries the amino group. But in the strongly alkaline media coupling takes place almost exclusively at 5 and the 7 position. 27-28 , the ring which carries the hydroxyl group. acidic OH alkaline NH2

HO3S

alkaline (31)

In the acidic media the diazo species involved in the azo coupling exists in the form of diazonium salt (32) . As the pH increases it is converted to the diazo hydroxide (33) and latter on into the non coupling diazotate (34). -OH -OH ArN 2+ X- Ar-N=N-OH Ar-N=N-O- +H + H (32) (33) (34)

Raising the temperature in most of the cases doesn’t exert a favorable influence in azo coupling, because the diazo decomposition reactions have a larger activation energies and therefore a larger temperature gradient then the coupling reactions. Whereas the reaction rate of the coupling increases by a factor of 2.0 to 2.4 for every 10 0C increase and that of decomposition reaction increases by that of 3.1 to 5.3 29 .

1.8. Structure of Azo Dyes Azo dyes can possess one, two or more azo groups. All of the azo dyes exist in the trans form which is more stable than the cis isomer 30 0 energetically . The bond angle is approximately 120 as all the nitrogens are sp 2 hybridised. Exception being when the hydrazone form is dominant. Usually the azo group is bonded to two groups with at least one of them being aromatic. The azo dyes can be conventionally categorised into sub-groups to indicate the appropriate method of synthesis using the letter A, B, E, Z and M31 . A carbocylic azo dye results when both A and E are aromatic and in the case of A and /or B is heterocyclic a heterocyclic azo dye results.

A: Primary aromatic Amine (normal diazo component). D: Primary aromatic Diamine (tetra-azo component). E: Coupling component capable of reacting with one diazonium salt ion to give the End component. Z: Coupling component capable of reaction with more than one diazonium ion. M: Coupling component containing a primary aromatic amino group

which after azo coupling reaction is susceptible to a second diazotization to furnish a second azo coupling (a Middle component). Some of the examples of the azo dyes are:

1.8.1 Monoazo dyes Examples of the monoazo dyes are (35, 36 and 37) .

HO NH N

N CH3 O

C.I.Disperse Yellow-16 (35)

HO N N

C.I.Solvent Yellow-14 (36)

O2NN C2H5 NN

CH 2CH 2OH

C.I.Disperse Red-1 (37) (A E method)

1.8.2 Disazo dyes Examples of disazo dyes are (38, 39) . these dyes are represented by A M E system which has an endless number of combinations and can cover the colour range from red to green.

L O

Cu C N O Cl H2N NH

N N N N N

SO 3H HO 3S NH N NH

HO 3S

SO 3H Reactive Brown (38)

HO 3S

N SO 3H N

N Cl

N N N

NH N NH

SO 3H

C.I.Reactive Brown-1 (39)

1.8.3 Trisazo and Tetraazo dyes The synthesis of these dyes requires at least three couplers and these dyes have dull colours like olive green, navy blue and black colours.

H2N NH2

N OMe

N MeN OH NH2 N

N N NH2 N

NaO 3S SO3Na

NH2

C.I.Direct Black-19 (40)

SO 3H

N HN N COOH

N OMe N HO 3S OH N N

Me N HN N NH

HO 3S SO 3H

C.I.Direct Green-26 (41)

N N

H2N

N N

NH2

N NH2 OH

N N N

HO 3S SO 3H

C.I.Direct Black-38 (42)

1.9 Coumarin Based Dyes Coumarin (43) 2H-1-Benzopyran-2-one or 1,2-Benzopyrone is a naturally occurring compound found in a number of plants like lavender, sweet clover grass, horse chestnut seeds and also in food plants like strawberries, apricots plums and cinnamon. In 1820 it was isolated and extracted for the first time by Vogel 32, 33 from tonka beans. It finds a number of uses like in perfumes and detergents, as a fixative and enhancement agent 34-36. Earlier it was used as food additives but nowadays it is discontinued because of its potential hyper toxicity. Coumarins play an important role in number of biological systems 37 . Hydroxyl coumarins have shown to provide good anti- bacterial, anti-tumoral, anticoagulant and anti-microbial activities 38-43 .Recent studies have shown that coumarin derivatives may act as potential anti-HIV agents 44 and also in the treatment of number of diseases like cancer, lymphoedema, rheumatic diseases and burns 39, 45-47.

O O

(43) Florescent coumarin derivatives have also been used as florescent whitening agents 48-50 and also as commercially important florescent dyes51-53 . Coumarin based compounds are also being used for dyeing the synthetic fibers 54-59 , plastics 60 and also as florescent pigments printing ink applications 61 ,

in detecting the destructive flaws, as tunable dye lasers 62-67 and also as solar energy collectors 68-69 . Most of the coumarin dyes can be represented by a common structure (44) if the oxygen atom is replaced by nitrogen the resulting dyes are called as carbostyryl dye. But these dyes are relatively hypsochromic and hence they have received less commercial importance.

H3C N X O

CH3 (44) [X=O or NH] The π electrons conjugation from an electron donor present at the position 7 to the acceptor at the position 2 (usually a carbonyl group) results in longer wavelength absorption as shown in (45a, 45b) . The donor is mostly dialkylamino but it can be hydroxyl or methoxy as well 70-71 .

H3C N O O H3C + - N O O CH3 CH3 (45a) (45b)

Migration of π electron in Coumarin nucleus

Some of the coumarin structures are shown (46) and it has been found that if π electron conjugation is extended and electron acceptor is incorporated at the positions 3 and 4, the absorption and the emission band is further shifted to the longer wavelength e.g . compound (46b) is colourless and (46c) is yellow.

2 R 3 R

1 R O O

(46)

Dye R1 R2 R3 46a H H H

46b OCH 3 H H

46c OCH 3 CN CN

Similarly the other examples of Coumarin dyes containing a benzimidazolyl (47a) , benzoxazolyl (47b) or benzothiazolyl (47c) group as an acceptor at the postion 3 are widely used in the dyeing of the synthetic fibers in greenish yellow shades 55,63,72-84 . Such sorts of dyes are also used in daylight florescent pigments and in dye lasers 55 .

NH2

H5C2 N O O

C2H5 (47)

Dye X R 47a NH H 47b O H 47c S H 47d O Cl

C.I Basic Yellow 40 (48) is also an important commercial product which imparts greenish yellow hues in acrylics and in daylight florescent pigments 85 .

H3C + N

N - Cl CH3 H5C2 N O O

C2H5 (48) Coumarin derivatives can also be modified at the position 2 to give rise to bathochromic shifts e.g . compound (49) shows violet red shades on polyacrylonitrile.

H5C2 + N

C2H5 H5C2 N O CN NC C2H5 (49) Coumarin derivatives are being used in the E.L devices 64, 86-92 and also as the electroluminescent materials in the flat panel displays 51,86 . The Coumarin derivatives (50) and (51) have been patented claiming there enhanced fluorescent efficiency, photo-oxidative stability and also being insensitive to pH 87. O N O X N O O N O O

R = H, CH 3 X = O, S, NH. (50) (51)

3-(2 ΄-Benzimidazolyl) coumarins of general structure (52) have been synthesized 93 and proven to be good florescent dyes. In such dyes the presence of an electron releasing group in the 7 position is important to ensure intense absorption and emission properties. Introduction of the additional amino group in either the 6 or the 8 position leads to a hypsochromic shift of the absorption band and loss of florescence.

1 R N

N H 2 R O O 3 R

(52)

Compound R1 R2 R3 52a H H H

52b H NEt 2 H

52c NO 2 H H

52d H H NO 2

52e NO 2 H NO 2

52f NH 2 H H

52g H H NH 2

52h NO 2 NEt 2 H

52i H NEt 2 NO 2

52j NH 2 NEt 2 H

52k H NEt 2 NH 2

Coumarin dyes of the type C-460 (53) and C-450 (54) have been used as laser grade dyes 94 .

CH3 CH3

H3C

H5C2 H5C2HNO O NOO C2H5 7-diethyl Amino 4-methyl Coumarin 7-Ethyl amino 4,6-dimethyl Coumarin C-460 (53) C-450 (54)

A series of new coumarin florescent dyes of the general structure (55), derived from the arylsulfonation of the parent benzothiazole, benzimidazole and benzoxazole dyes, have been synthesised 95 and found to give highly florescent greenish yellow shades when applied on polyester.

N SO 2NH

H5C2 N O O

C2H5 (55)

Where, Dye X R

55a S CH 3

55b NH CH 3

55c S OCH 3

55d NH OCH 3

55e O CH 3

55f O OCH 3

It has been seen by Xianjin et al. 96 that the introduction of a heterocylic substituent in the 3 rd position and then introduction of sulfonyl chloride into the heterocyclic substituent increases the electron attraction and results in the bathochromic effect and enhanced fastness to light and sublimation. Rogerro et al. 97 have synthesised new azoic dyes having the general formula (56). The colour of the synthesised dyes ranged from yellow to orange when polyester fabric was dyed with them. They had high dyeing strength and good overall characteristics particularly in the light fastness, fastness to wet and thermal treatments.

N R N R1

2 R NH

O 3 R XNO 4 R

(56) Where, X = NH, O. R = one or more alkyl, alkoxy, dialkylamino, di(alkoxy(hydroxyl)alkyl)amino, halo, cyano and nitro groups

R1 = H, CH 3, OCH 3, Cl.

R2 = H, CH 3, OCH 3, Cl.

R3 = CH 3, C2H5.

R4 = CH 3, C2H5 Yazdanbaksh et al . 98 have synthesised the hydroxyl coumarin dyes by coupling 4-hydroxy coumarin (57) with the diazonium salts prepared by the diazotization of the aniline and aniline derivatives (58). Their dissociation

constants were also studied and the dyes resulting from the coupling gave good colours ranging from red to yellow.

OH NH2

R O O (58) (57)

NH2 + - N2 Cl

0 conc HCl / 0 C + NaNO 2 R

+ - OH N2 Cl OH

0 (1) NaOH, 0 C / 30 min N N + R OO (2) HCl OO R

(Scheme 1): Synthesis of hydroxyl-azo dyes.

Here R= p-OCH 3, o-CF 3, m-CH 3, p-Cl, o-CH 3, m -CF 3, m-CH 3,

p-CF 3, p-CH 3, p-CN, H, p-NO 2

1.10. Flavone based Dyes Flavones, an important class of the natural compounds of flavoniod group are freely present in the nature. They have been used as antiproliferative 99 , anti-viral 100 , anti oxidant 101 or anti-fungal activities 102 . Besides this the synthesis of flavones and their derivatives have attracted considerable attraction due to their good anti-inflamatory 103 , anti-microbial 104 and herbicidal activities 105 . Flavones are also known to have good synthetic applications like photo-oxidation and reagent catalysed oxygenation reactions 106,107 . Because of these properties of flavones, many articles presenting their synthesis and structural modification are described in the

scientific literature 108,109 . Flavones (mostly hydroxyl flavones) are present in the plants are yellow compounds and are the main component of number of natural dyes used in textile dyeing. The main flavone yellow dye sources mentioned in traditional recipes are weld (Reseda luteola L.), young fustic (Cotinus coggygria Scop.), dyer’s greenweed (Genista tinctoria L.), sawwort (Serratula tinctoria L. Gaud.) and dyer’s camomile (Anthemis tinctoria L.) 110 . This yellow dye when used with alum as a mordant it produces a fast and bright yellow colours because of the presence of flavones luteolin (60) and apigenin (61) as the major constituent 111 . OH OH

HO O HO O OH

OH O OH O (60) (61)

In the past the mixtures of the hydroxyl flavones have been found to impart yellow coloured pigments in the members of the plants of the Chichriae subfamily 112 . Because of its dual florescence 113, 114 this compound has attracted the attention of the researchers. Besides that 3-hydroxyflavones dyes have been synthesised and used in the development of new florescent probes to study the solvent polarity 115 . Flavone dyes like 3-hydroxy-4΄-N,N-dimethylaminoflavone (62) and 4 ΄-N,N- dimethylamino-flavone-3-yl-methacrylate have been used as solvatochromegenic flourophores for the determination of water in acetone 116 .

H3C CH3 N

OR O

(62a) R = H (62b) R = COC(CH 3)CH=CH 2

4΄-(Dialkylamino)-3-hydroxyflavone dyes (63) have found to have their application in the bio-membrane studies. By substituting the 2-phenyl group with 2-thienyl group new dyes were synthesised which exhibit red shifted absorption and dual florescence. These dyes proved to be new flourophores for the synthesis of new florescent probes for bio membranes 117 .

2 1 R R N

O (63) The yellow coloration of the hydroxyflavones attracted the attention of researchers and Joseph 118 synthesized a series of 4 ΄-Aminoflavone and coupled it with β-napthol to give flavone-(4 ΄)-azo-beta-napthol dyes. These dyes were used to dye silk and wool to give fast yellow to orange colour. These dyes gave deeper colours then the most hydroxylated flavones occurring in nature.

1.11. Dye’s Toxicity In the recent decade society has become increasingly cautious for the protection of the Earth. Destruction of the forests, global warming and depletion of the ozone layer and the impact of various industries on the environment has become major issues. The textile industry also owes its responsibility towards a wide range of health, safety and environmental issues. In the earlier days the dyestuff selection, its application etc were not much looked from the angle of their environmental impact. But currently the dye manufacturers have to offer such a dye to the consumers which not only provides the water and the energy savings rather it also reduces pollution in the sense that they are environment friendly, having no or minimum impact on the environment. Until the late 19 th century most of the colorants used were from the natural sources. The most important and the major source was the plants and

to some extent insects and molluscs were also used. Thus the workers working in the dye industry at that time were less exposed to the chemicals and dyes as compared to those working in today’s dye industry. Dyestuff toxicity has been investigated in numerous studies. These toxicity studies (including mortality, genotoxicity, mutagenicity and carcinogenecity) range from tests with aquatic organisms to test with mammals. Chronic effects of azo dyes have been studied for several decades. The research was traditionally more focused on the effects of food colorants which were mostly azo compounds. The effects of occupational exposure to dyestuff of human workers in dyes manufacturing and dye utilizing industries have received much attention. Azo dyes in purified form are seldom directly mutagenic or carcinogenic except for some azo dyes with free amino groups 119 . The reduction of azo dyes leads to the cleavage of the dyes azo linkage(s) and formation of aromatic amines. Several aromatic amines are known carcinogens and mutagens. In mammals the reduction of azo dyes is mainly due to bacterial activity in the anaerobic parts of the lower gastrointestinal tract. Azo dyes can also be reduced in the various other organs especially the liver and the kidneys. After azo dyes reduction in the intestinal tract the released aromatic amines are absorbed in the urine .The acute toxic hazards of aromatic amines is carcinogenesis especially bladder cancer. The carcinogenicity mechanism probably includes the formation of acyloxy amines through N-hydroxylation and N-acetylation of the aromatic amines followed by O-acylation. These acyloxy amines can be converted to nitrenium and carbonium ions that bind to DNA and RNA, which include mutations and tumor formation 119 . The dye intermediate 2-Naphthylamine is a known human carcinogen 120 . Another well-known human carcinogen is benzidine 121 once widely used in dye synthesis. Benzidine and its salts have also proven to be potent human bladder carcinogens and it has been proven in a number of studies 122-129 . After such findings a number of countries have banned usage of such compounds. German Government has banned the use of these and other harmful amines as intermediate for the manufacture of dyestuffs.

In 1975 and then in 1982 the International Agency for Research on Cancer (IARC) summarized the literature on suspected azo dyes, mainly amino-substituted azo dyes and benzidine azo dyes 130 . Most of the dyes on the IARC list were taken out of production 131 . Exposure to aromatic amines may cause methemoglobinemia. Because of these amines the haeme i.e. iron of haemoglobin from Fe (II) to Fe (III) hence blocking the oxygen carrying capacity of haemoglobin. This results in characteristic symptoms like cyanosis of nose and lips, dizziness and weakness 132 .The extent of which various aromatic amines can cause methemogolobinemia varies however widely. The electron withdrawing nature of the azo linkages obstructs the susceptibility of azo dye molecules to oxidative reactions Therefore, azo dyes resist aerobic bacterial biodegradation. In contrast, breakdown of azo linkages by reduction under anaerobic conditions is much less specific. This anaerobic reduction implies decolourisation as the azo dyes are converted to usually colourless but potentially harmful aromatic amines 133 . Azo dyes under go a major metabolic pathway in mammals that results in the reduction and cleavage of the azo linkage. After cleavage of the azo linkage, the component aromatic amines are absorbed in the intestine and excreted in the urine. However, the polarity of azo dyes influences the metabolism and consequently the excretion. Sulfonation of azo dyes appears to decrease toxicity by enhancing urinary excretion of the dye and its metabolites. Sulfonated dyes are therefore used worldwide in foods, drugs, as drugs for oral application cosmetics 134 . High quality dyes are stable chemical compounds, which imply that they are not biodegradable. Dyes have to be very stable if they are expected to retain their colour when they are exposed to light, heat, detergents and perspirants etc over the lifetime of the goods that have been dyed with them. Environment friendly dyes are distinguished by their high exhaustion and high fastness. They are present at very low levels in the wastewater and they can be removed from the wastewater very effectively in the biological effluents treatment plants. In Germany concern about the hazards of dyes resulted in restriction of usage of such dyes and products made from them in 1995 and in 1998 the same restrictions became valid in Netherlands and Australia 135 . The carcinogenicity and mutagenic activity of certain aromatic amines

has been known for sometime 136-138 . Rehen et al .139 have reported that the workers who were working in the plant manufacturing unit of the dye fuchsin (magenta) from crude aniline appeared at his clinic with the symptoms of the bladder cancer and upon investigation it was found that this bladder cancer was associated with the workers occupation. When the bladder carcinogens were identified in the persons who were occupationally exposed two things have come forward, firstly the manufacture and the use of the 4- aminobiphenyl and 2-napthylamine has been banned totally, secondly it has been extensively studied in laboratory as to which aromatic amines are carcinogenic. Okajima et al .140 have shown that rats fed on the high doses of a benzidine dye developed an excess of tumors. Similarly Yoshida et al. 141 also showed that the benzidine dyes were reduced by E.Coli and the soil bacteria. When a number of studies showed the potential health and the environmental problems associated with the benzidine and dyes made with it, the Industrial consumers restricted their use and started using there substitutes. America which was the largest manufacturer of the benzidine based dyes stopped its production in August 1976. The British, German and Japanese Industries also stopped the production of the benzidine dyes. In 1974 Ecological and Toxicological Association of the Dyestuff Manufacturing Industry (ETAD) was founded to increase the awareness related to the dye toxicology and to minimize the possible damage to the environment. The purpose of ETAD is to coordinate the toxicological and ecological efforts of the dye manufacturers. It also identifies and assesses the risks caused by the dyes and their intermediates to the human health. ETAD has developed an online database containing the sources of toxicological, environmental and legal publications on colorants worth almost 12,900 documents concerning more then 2000 different dyes and pigments. Some other international organizations like International Assosiation for Research and Testing in the field of Textile Ecology refuses the eco-labels for the dyes which are carcinogenic 142 .

Table-2 List of the carcinogenic amines according to the German Technical Rules for Dangerous Substances.

Serial No. Name 1 4-Amino biphenyl 2 o-Aminoazo-toluene 3 o-Anisidine 4 4-Amino azo benzene 5 4-Amino-3-fluorophenol 6 6-Amino-2-ethoxynapthalene 7 Benzidine 8 p-Chloro aniline 9 4-Choloro-o-toluidine 10 4,4 ΄-Diamino diphenyl methane 11 3,3 ΄-Dichloro benzidine 12 3,3 ΄-Dimethoxybenzidine 13 3,3 ΄-Dimethyl benzidine 14 4-Methoxy-m-phenylene diamine 15 4-4΄-Methylenedi-o-toluidine 16 6-Methoxy-m-toluidine 17 4-4΄-Methylene bis(-2-choloraniline) 18 4-Methyl-m-phenylendiamine 19 2-Napthylamine 20 5-Nitro-o-toluidine 21 4,4 ΄-Oxydianiline 22 2,4,5-Trimethyl aniline 23 4.4’-Thiodianiline 24 o-Toluidine

Toxicology studies are concerned with a variety of aspects, primarily with (1) Acute toxicity (2) Irritation of skin and eyes. (3) Toxicity after repeated application (4) Sensitization (5) Mutagenicity (6) Carcinogenicity European Union EU directive 67/548/EEC defines the first step to determine whether a dye is hazardous or not by testing its acute toxicity. A list of azo dyes has been compiled by ETAD which upon reduction of the azo bond forms the aromatic amines which are listed in the above mentioned table. There are more then 500 azo dyes and out of them at least 142 are still available and being used in the International market 143 .

1.12. Testing of Mutagenicity In 1975 Salmonella mutagenicity assay was introduced 144 to screen for the existence of the mutagenic potential of the substances and was latter revised by Maron and Ames 145 in 1983. The Salmonella mutagenicity assay now often referred to as the Ames test, is used widely as an initial screening test procedure for the new compounds. This test involves the two or more strains of the micro organism Salmonella typhimurium and this is an in vitro method. Different types of bacterial strains are used and each is designed to detect a specific type of mutation e.g . TA 98 and TA 1538 for the frame shift mutations, TA 100 and TA 1535 are used for base pair substitution mutations. All these strains are developed in the absence of the amino acid histidine which is an essential component for growth. As a result the bacteria are unable to multiply unless they are incubated with an appropriate mutagen. Mutagenic activity can be quantitatively measured by counting the number of colonies present after incubating the bacteria with the test compound under standard conditions. The change in the bacteria is referred to as a reverse mutation and the colonies formed are termed as the reverent colonies. In addition to the histidine mutation the salmonella strains have been developed

with additional features that enhance their sensitivity to mutagenic compounds 146 . Prival modification method named after Prival and Mitchell in 1982 147 emerged from a search for a procedure suitable for testing benzidine based dyes. The original Ames test was modified to ensure the liberation of the parent diamines and the maximum possible mutagenic activity of the dyes. Five modifications were made to the standard mutagenicity test: 1- Instead of induced rat liver S9 uninduced hamster liver S9 is used. 2- The quantity of S9 used is three times that of the standard assay. 3- To facilitate the reductive cleavage of the azo bonds a reducing agent flavin mononucleotide (FMN) is used. 4- So as to facilitate the reductive cleavage exogenous glucose-6- phosphate is added. 5- A thirty minute pre-incubation step is employed before the addition of top agar. All the five changes are necessary for the optimal observation of the mutagenic activity. Like the introduction of the reducing agent flavin is necessary as the reduction of azo compounds can occur in mammals 148-152 . This test is now being actively used for the mutagenicity of the dyes and pigments. The Rat bacterial reduction system was used by Reid et al. 153-154 to determine the mutagenicity of a group of benzidine based disazo dyes. In this method, the dyes are first reduced using either rat cecal flora (bacteria with reducing capabilities), or hamster S9 mixture containing flavin mononucleotide (FMN). The reduction products are extracted from the crude mixture and subjected to oxidative metabolism using either induced rat liver S9 or uninduced hamster liver S9. The mutagenicity of the compounds is then measured using Salmonella typhimurium strain TA 1538.

PROJECT AIMS This study pertains to the synthesis of Environment friendly dyes. For the synthesis of environment friendly dyes flavones and coumarins were selected to be synthesised as dye intermediates because of their environment friendly nature. They are already present in nature hence the dyes made from them are more likely to be less toxic. Various Amino-flavones (like 4 ΄- Aminoflavone, 3 ΄-Aminoflavone and 6-Aminoflavone) and Amino-coumarins like 6-Amino-4-methyl-coumarin are to be used as the dye intermediates and the dyes prepared from them during the work of this study could serve as a good replacement of the currently used azo disperse dyes that have been proven to be mutagenic. The mutagenic studies i.e . Ames test will be carried out to ascertain their level of mutagenicity.

2. MATERIALS AND METHODS Equipment and Instrumentation Chemicals All the chemicals used were purchased from Sigma-Aldrich Chemical Co. and Acros Organics and were used without further purification.

Melting Points Melting points were determined using a Gallenkamp melting point apparatus and are uncorrected.

Infra-Red Spectroscopy IR spectra were taken on Perkin Elmer FTIR Spectrometer Spectrum RX 1 using KBr pellets.

Nuclear Magnetic Resonance 1 13 H NMR spectra were taken in DMSO-d6 and recorded at 300 M Hz. C NMR spectra were taken at 75 M Hz on a Brucker/XWIN-NMR instrument.

Chemical shifts are given in ppm relative to (Me) 4Si as internal standard (abbreviations: s, singlet;bs, broadsinglet; d, doublet; t, triplet; m, multiplet.).

Mass Spectrometery Mass spectra were recorded on Jeol JMS-HX110 and Agilent 6890 spectrometerElemental analyses were carried out on Perkin Elmer 2400–CHN Analyser.

Chromatography . Thin layer chromatography was carried out on Merck silica gel 60F 254 Flash chromatography as well as column chromatography was carried out by using Neutral Alumina Silica gel.

Dyeing Machine Dyeing was carried out in Zeltec- Poly-colour machine.

Light-fastness Light fastness was carried out using Heracus-Xenotest 1505 machine.

Wash-fastness Wash fastness studies were carried in Roaches Wastec-P machine.

2.1 Synthesis of Dye Intermediates 2.1.1. 4 ΄-Aminoflavone (67) 4΄-Aminoflavone was synthesised by three step process using method reported in the literature 155.

2.1.1.1. 2-Acetylphenyl-4-nitrobenzoate (64):

CH3

NO 2 O (64)

4΄-Nitrobenzoylchloride (4.9 gm, 0.25 mol) was added to 2- hydroxyacetophenone (3.4 gm, 0.25 mol) in pyridine (5 mL). The reaction mixture was stirred for 20 minutes and then poured into 1 M HCl (120 mL) containing crushed ice (50 gm). The precipitates were then filtered and washed with ice cold methanol (5 mL) followed by water (5 mL). The crude product was recrystalized from methanol to obtain 2-Acetylphenyl-4- nitrobenzoate (64) . (4.6 gm, 76 %). m.p 92 0C. -1 IR νmax (KBr/ cm ), 1679 (C=O) and 1611 (C=C). 1 H-NMR δΗ (DMSO-d6, 300 MHz) 7.56 (d, 2H, J = 7.06 Hz, Ar H), 7.31 (d,

2H, J = 7.01 Hz, Ar H), 7.26-7.01 (m, 3H, Ar H), 3.13 (s, 3H, C H3). MS EI (m/z), 281 (M +, 100%).

CHN Anal. Calcd for C 16 H13 N O4 : C, 67.83; H, 4.67; N, 4.14; O, 22.53 %. Found: C, 67.17; H, 4.86; N, 4.01; O, 22.30 %.

2.1.1.2 1-(2-Hydroxyphenyl)-3-(4-nitrophenyl)propane-1,3-dione (65):

OH NO 2

OO (65)

2-Acetylphenyl-4-nitrobenzoate (64) (2.85 gm, 0.2 mol) was dissolved in pyridine (9 mL) and heated up to 50 0C. Powdered potassium hydroxide (0.85 gm 0.3 mol) preheated in an oven at 100 0C was then added into the reaction mixture. After stirring for 15 minutes the reaction mixture was cooled to room temperature and acidified with 10 % acetic acid with continuous stirring during the addition. The precipitates were collected by filtration and dried in an oven at 50 0C and then recrystalized from methanol to get 1-(2- Hydroxyphenyl)-3-(4-nitrophenyl)propane-1,3-dione (65) .Yield (2.61 gm, 81%). m.p 117 0C. -1 IR νmax (KBr/ cm ), 1655 (C=O) and 3600-3546 (OH). 1 H-NMR δΗ (DMSO-d6, 300 MHz) 9.63(s, 1H, OH), 7.78 (d, 2H, J = 7.03 Hz, Ar H), 7.41 (d, 2H, J = 7.01 Hz, Ar H), 7.33-6.01 (m, 4H, Ar H),

3.69 (s, 2H, C H2CO). MS EI (m/z), 284 (M +, 100%).

CHN Anal. Calcd for C 15 H11 N O5: C, 63.13; H, 3.69; N, 4.64; O, 28.03 %. Found: C, 63.10; H, 3.84; N, 4.21; O, 28.10 %.

2.1.1.3 4΄-Nitroflavone (66):

NO 2

O (66)

1-(2-Hydroxyphenyl)-3-(4-nitrophenyl)propane-1,3-dione (65) (2.1 gm, 0.15 mol) was dissolved in glacial acetic acid (10mL) and concentrated sulphuric acid (0.4 mL) was added with continuous stirring. The reaction mixture was heated in a water bath for one hour with intermittent stirring. After one hour the reaction mixture was poured with stirring into crushed ice (50 gm). The ice was allowed to melt and the precipitates were filtered and washed with water until the washings were no longer acidic. The precipitates were dried in oven at 50 0C and then recrystalized in ethanol to obtain the white crystals of 4'-Nitroflavone (66). Yield (1.82 gm, 89%). m.p 139 0C. (lit. 156 140-142 0C). -1 IR νmax (KBr/ cm ), 1647 (C=O). 1 H-NMR δΗ (DMSO-d6, 300 MHz) . 7.78-7.36 (m, 4H, Ar H) , 7.21 (d, 2H, J = 7.03 Hz, Ar H), 7.13 (d, 2H, J = 6.91 Hz, Ar H), 6.69 (s, 1H, Ar H). MS EI (m/z), 266 (M +, 100%).

CHN Anal. Calcd for C 15 H9 N O4 : C, 67.43; H, 3.79; N, 5.26; O, 23.93 %. Found: C, 67.10; H, 3.84; N, 5.21; O, 24.90 %.

2.1.1.4. 4΄-Aminoflavone (67)

NH2

O (67)

4΄-Nitroflavone (66) (0.66 gm, 0.02 mol) was dissolved in ethanol (20 mL) and Pd/C (0.03 gm) was added. The reaction mixture was stirred under hydrogen atmosphere at room temperature (4 hours) and this reaction was monitored by TLC. The precipitates of 4'-Aminoflavone (67) were filtered, dried and recrystalized in methanol. Yield (0.58 gm, 79%). m.p 234 0C. (lit. 157 234-236 0C).

-1 IR νmax (KBr/ cm ), 3455 (NH 2)1655 (C=O). 1 H-NMR δΗ (DMSO-d6, 300 MHz) . 7.76-7.46 (m, 4H, Ar H), 7.33 (d, 2H, J =

7.13 Hz, Ar H), 7.25 (d, 2H, J = 6.94 Hz, Ar H), 6.69 (bs, 2H, N H2), 6.51 (s, 1H, Ar H). MS EI (m/z), 236 (M +, 100%).

CHN Anal. Calcd for C 15 H911 N O2 : C, 75.63; H, 4.73; N, 5.96; O, 13.97 %. Found: C, 75.10; H, 4.84; N, 5.45; O, 13.90 %.

2.1.2 Synthesis of 3 ΄-Aminoflavone (71) 3΄-Aminoflavone was synthesised by three step process using method reported in the literature 155.

2.1.2.1 2-Acetylphenyl-3-nitrobenzoate (68)

CH3

NO 2 (68)

3'-Nitrobenzoylchloride (4.9 gm, 0.35 mol) was added to 2- hydroxyacetophenone (3.4 gm, 0.25 mol) in pyridine (5 mL). The reaction mixture was stirred for twenty minutes and then poured into 1 M hydrochloric acid (120 mL) containing crushed ice (50 gm). The precipitates were filtered and washed with ice cold methanol (5 mL) followed by water (5 mL). The crude product was recrystalized in methanol to obtain 2-Acetylphenyl-3- nitrobenzoate (68). Yield (4.4 gm, 74 %). m.p 97 0C. -1 IR νmax (KBr/ cm ), 1671 (C=O) and 1613 (C=C). 1 H-NMR δΗ (DMSO-d6, 300 MHz) 7.26-7.01 (m, 8H, Ar H), 3.39 (s, 3H,

CH3). MS EI (m/z), 281 (M +, 100%).

CHN Anal. Calcd for C 16 H13 N O4 : C, 67.81; H, 4.63; N, 4.02; O, 23.47 %. Found: C, 67.77; H, 4.86; N, 3.81; O, 24.30 %.

2.1.2.2 1-(2-Hydroxyphenyl)-3-(3-nitrophenyl)propane-1,3-dione (69):

NO 2 OO (69)

2-Acetylphenyl-3-nitrobenzoate (68) (2.85 gm, 0.2 mol) was dissolved in pyridine (9 mL) and heated up to 50 0C. Powdered potassium hydroxide (0.85 gm 0.3 mol) preheated in an oven at 100 0C was then added into the reaction mixture. After stirring for 15 minutes the reaction mixture was cooled to room temperature and acidified with 10 % acetic acid with continuous stirring during the addition. The precipitates were collected by filtration and dried in an oven at 50 0C and recrystalized from methanol to get 1-(2- Hydroxyphenyl)-3-(3-nitrophenyl)propane-1,3-dione (69) (2.61 gm 81%), m.p 123 0C. -1 IR νmax (KBr/ cm ), 1659 (C=O) and 3549 (OH). 1 H-NMR δΗ (DMSO-d6, 300 MHz) 9.69 (s, 1H, O H), 7.83-7.13 (m, 8H, Ar H),

3.71 (s, 2H, C H2CO). MS EI (m/z), 284 (M +, 100%).

CHN Anal. Calcd for C 15 H11 N O5: C, 63.13; H, 3.69; N, 4.64; O, 28.03 %. Found: C, 63.10; H, 3.84; N, 4.21; O, 28.10 %.

2.1.2.3. 3΄-Nitroflavone (70):

NO 2

O (70)

1-(2-Hydroxyphenyl)-3-(3-nitrophenyl)propane-1,3-dione (69) (2.1 gm, 0.007 mol) was dissolved in glacial acetic acid (10 mL) and concentrated sulphuric acid (0.4 mL) was added with continuous stirring. The reaction mixture was heated in a water bath for one hour with intermittent stirring. After one hour the reaction mixture was poured into crushed ice (50 gm). The ice was allowed to melt and the precipitates were filtered, washed with water until the washings were no longer acidic. The precipitates were dried in oven at 50 0C and then recrystalized from ethanol to obtain white crystals of 3 ΄-Nitro flavone (70). (1.79 gm, 86 %), m.p 202-203 0C. (lit. 158 203 0C). -1 IR νmax (KBr/ cm ), 3415 (NH 2), 1649 (C=O). 1 H-NMR δΗ (DMSO-d6, 300 MHz) . 7.81-7.26 (m, 8H, Ar H), 6.71 (s, 1H, Ar H). MS EI (m/z), 266 (M +, 100%).

CHN Anal. Calcd for C 15 H9 N O4: C, 68.03; H, 3.66; N, 5.31; O, 24.03 %. Found: C, 67.89; H, 3.04; N, 5.41; O, 24.11 %.

2.1.2.4 3΄-Aminoflavone (71)

O NH2

O (71)

3΄-Nitroflavone (70) (0.66 gm, 0.02 mol) was dissolved in ethanol (20 mL) and Pd/C (0.03 gm) was added. The reaction mixture was stirred under hydrogen atmosphere at atmospheric pressure and room temperature (4 hours) and the reaction was monitored by TLC. The precipitates of 3΄- Aminoflavone (71) were filtered, dried and recrystalized from methanol. (0.55 gm, 80%). m.p 157 0C. (lit. 159 m.p 156-157 0C). -1 IR νmax (KBr/ cm ), 3449 (NH 2)1653 (C=O). 1 H-NMR δΗ (DMSO-d6, 300 MHz) . 7.83-7.36 (m, 8H, Ar H), 6.79 (bs, 2H,

NH2), 6.69 (s, 1H, Ar H). MS EI (m/z), 236 (M +, 100%).

CHN Anal. Calcd for C 15 H911 N O2: C, 75.53; H, 4.81; N, 5.81; O, 13.87 %. Found: C, 75.60; H, 4.85; N, 5.85; O, 13.88 %.

2.1.3. 6-Aminoflavone (75) 6-Aminoflavone was synthesised by three step process using method reported in the literature 155.

2.1.3.1 2-Acetyl-5-nitrophenyl benzoate (72)

CH3

O2N O (72)

Benzoylchloride (2.81 gm, 0.35 mol) was added to 2-hydroxy-5- nitroacetophenone (3.4 gm, 0.25 mol) in pyridine (5 mL). The reaction mixture was stirred for 20 minutes and then poured into 1 M hydrochloric acid (120 mL) containing crushed ice (50 gm). The precipitates were filtered and washed with ice cold methanol (5mL) followed by water (5mL). 2-Acetyl-5- nitrophenyl benzoate (72) was recrystalized in methanol. Yield (2.61 gm, 74 %). m.p 127 0C. -1 IR νmax (KBr/ cm ), 1668 (C=O) and 1613 (C=C). 1 H-NMR δΗ (DMSO-d6, 300 MHz) ) 7.66 (d, 2H, J = 7.03 Hz, Ar H), 7.41 (d,

2H, J = 6.97 Hz, Ar H), 7.26-7.01 (m, 4H, Ar H), 3.63 (s, 3H, C H3). MS EI (m/z), 281 (M +, 100%).

CHN Anal. Calcd for C 16 H13 N O4: C, 66.83; H, 4.77; N, 4.34; O, 22.43 %. Found: C, 67.07; H, 4.86; N, 4.21; O, 22.32 %

2.1.3.2 1-(2-Hydroxy-5-nitrophenyl)-3-phenylpropane-1,3-dione (73):

O2N OO (73)

2-Acetyl-5-nitrophenyl benzoate (72) (2.85 gm, 0.2 mol) was dissolved in pyridine (9 mL) and heated up to 50 0C. Powdered potassium hydroxide (0.85 gm 0.3 mol) preheated in an oven at 100 0C was added into the reaction mixture. After stirring for 15 minutes the reaction mixture was cooled to room temperature and acidified with 10 % acetic acid with continuous stirring during the addition. The precipitates were collected by filtration, washed and dried in an oven at 50 0C and then recrystalized from methanol to get 1-(2-Hydroxy-5- nitrophenyl)-3-phenylpropane-1,3-dione (73) (1.89 gm, 89%), m.p 135 0C -1 IR νmax (KBr/ cm ), 1649 (C=O) and 3603 (-OH). 1 H-NMR δΗ (DMSO-d6, 300 MHz) 9.69 (s, 1H, O H), 7.81 (d, 2H, J = 7.05 Hz, Ar H), 7.61 (d, 2H, J = 6.91 Hz, Ar H), 7.31-6.17 (m, 4H, Ar H),

3.89 (s, 2H, C H2CO). MS EI (m/z), 284 (M +, 100%).

CHN Anal. Calcd for C 15 H11 N O5: C, 62.73; H, 3.71; N, 4.61; O, 28.13 %. Found: C, 63.05; H, 3.74; N, 4.16; O, 28.14 %.

2.1.3.3 6-Nitro flavone (74)

O2N O (74) 1-(2-Hydroxy-5-nitrophenyl)-3-phenylpropane-1,3-dione (73) (2.1 gm, 0.15 mol) was dissolved in glacial acetic acid (10 mL) and concentrated sulphuric acid (0.4 mL) was added with continuous stirring. The reaction

mixture was heated in a water bath for one hour with intermittent stirring. After one hour the reaction mixture was poured into crushed ice (50 g). The ice was allowed to melt and the precipitates were filtered and washed with water until the washings were no longer acidic. The precipitates were dried in oven at 50 0C and then recrystalized from ethanol to obtain 6-nitroflavone (74) . (1.82 gm, 89 %), m.p 139 0C. (lit. 160 m.p 142 ). -1 IR νmax (KBr/ cm ), 1653 (C=O). 1 H-NMR δΗ (DMSO-d6, 300 MHz) . 7.68-7.37 (m, 4H, Ar H), 7.28 (d, 2H, J = 6.97 Hz, Ar H), 7.11 (d, 2H, J = 6.65 Hz, Ar H), 6.71 (s, 1H, Ar H). MS EI (m/z), 266 (M +, 100%).

CHN Anal. Calcd for C 15 H9 N O4: C, 66.83; H, 3.89; N, 5.23; O, 24.03 %. Found: C, 67.03; H, 3.74; N, 5.29; O, 24.78 %.

2.1.3.4 6-Aminoflavone (75)

H2N O (75)

6-Nitroflavone (74) (0.66 gm, 0.002 mol) was dissolved in ethanol (20 mL) and Pd/C (0.03 gm) was added. The reaction mixture was stirred under hydrogen atmosphere at atmospheric pressure and room temperature (4 hours) and the reaction was monitored by TLC. The precipitates of 6- Aminoflavone (75) were filtered, dried and recrystalized from methanol. (0.57 gm, 78%). m.p 201 0C. (lit. 161 201-203). -1 IR νmax (KBr/ cm ), 3465 (NH 2), 1651 (C=O). 1 H-NMR δΗ (DMSO-d6, 300 MHz) . 7.81-7.56 (m, 4H, Ar H), 7.30 (d, 2H, J =

7.03 Hz, Ar H), 7.27 (d, 2H, J = 6.97 Hz, Ar H), 6.97 (bs, 2H, N H2), 6.56 (s, 1H, Ar H). MS EI (m/z), 236 (M +, 100%).

CHN Anal. Calcd for C 15 H91 N O2: C, 75.61; H, 4.77; N, 5.86; O, 13.95 %. Found: C, 76.10; H, 4.84; N, 5.95; O, 13.80 %.

2.2 General method of preparation of Dyes from the Intermediates (67, 71, 75) 2.2.1 Diazotization Diazotization of Aminoflavones (67, 71, and 75) was carried out by dissolving (2mmol) of Aminoflavone in concentrated hydrochloric acid (8 mL). It was cooled to -5 0C. Sodium nitrite (0.15 gm in 2 mL of water) was added slowly in the reaction mixture and stirred for one hour below 0 0C and just before the coupling step sulphamic acid (0.05 gm) was added.

2.2.2 Coupling The coupler (1-15) (2mmol) was dissolved in water (20 mL) containing hydrochloric acid (1 mL) cooled to 5 0C. The diazonium salt solution was added drop wise in a 10 minutes time keeping the temperature below 5 0C. After the addition of the diazonium salt, solution of anhydrous sodium acetate (10 gm in 20 mL water) was added over 10 minutes time. After addition the stirring was continued for further one hour at room temperature. The dye obtained was filtered, dried and purified using the neutral alumina oxide column (Dichloromethane: methanol, 1: 1).

2.3 6-Amino-4-methylcoumarin (77) 2.3.1 N-(4-Methyl-2-oxo-2H-chromen-6-yl)acetamide (76)

CH3 O NH

CH3 OO (76) p-Acetylaminophenol (3.77gm, 0.02 mol) was dissolved in ethylacetoacetate (3.42 mL, 0.02 mol) and while stirring concentrated. sulphuric acid (35 mL) was added into it. The temperature of the reaction mixture was raised up to 150 0C and stirred at this temperature for half and hour and after that the reaction mixture was poured into ice cold water. The crude product was filtered, washed and recrystalized in ethanol to give N-(4- Methyl-2-oxo-2H-chromen-6-yl)acetamide (76). (2.81g, 78%). m.p. 129 0C.

-1 IR νmax (KBr/ cm ), 1653 (C=O). 3260 (NH) 1 H-NMR δΗ (DMSO-d6, 300 MHz) . 10.03 (s, 1H, N H), 7.81-7.17 (m, 3H,

Ar H), 6.81 (s, 1H, Ar H), 3.61 (s, 3H, C H3), 3.36 (s, 3H, C H3). MS EI (m/z), 216 (M +, 100%).

CHN Anal. Calcd for C 12 H11 N O3: C, 66.53; H, 5.89; N, 6.27; O, 22.10 %. Found: C, 67.03; H, 5.84; N, 6.31; O, 21.88 %.

2.3.2. 6-Amino-4-methylcoumarin (77)

CH3

H2N

OO (77)

N-(4-Methyl-2-oxo-2H-chromen-6-yl)acetamide (76) (2.2 g. 0.02 mol) were added in 0.5 M NaOH (15 mL) and stirred for two hours and after that poured into a beaker and acidified with concentrated hydrochloric acid to give the precipitates of the 6-Amino-4-methylcoumarin (77) which were recrystalized from ethanol (1.89g, 76%). m.p 179 0C. (lit. 162 180 0C) -1 IR νmax (KBr/ cm ), 1657 (C=O), 3643 (NH 2) 1 H-NMR δΗ (DMSO-d6, 300 MHz) .8.66 (bs, 2H, N H2), 7.71-7.27 (m, 3H,

Ar H), 6.29 (s, 1H, Ar H), 3.69 (s, 3H, C H3). MS EI (m/z), 176 (M +, 100%).

CHN Anal. Calcd for C 10 H9 N O2: C, 68.53; H, 5.19; N, 8.23; O, 14.09 %. Found: C, 67.13; H, 5.79; N, 8.29; O, 14.78 %.

2.3.3 Dyes (61-75) from 6-Amino-4-methylcoumarin (77) The dyes (61-75) were synthesised from 6-Amino-4-methylcoumarin by the process of diazotization and coupling. 2.3.3.1 Diazotization of 6-Amino-4-methylcoumarin (77) The diazotization was carried out by dissolving 6-Amino-4- methylcoumarin (2.2gm, 0.02mol) in concentrated hydrochloric acid (8mL). This reaction mixture was stirred and the temperature kept below 0 0C. Sodium nitrite (0.15gm in 2mL of water) was added slowly in the reaction

mixture. The stirring was continued for one hour at this temperature and sulphamic acid (0.05g) was added.

2.3.3.2 Coupling with the couplers (1-15) The coupler (0.02 mol) (1-15) was dissolved in hydrochloric acid solution (1 mL in 20 mL water). The mixture temperature was kept below 0 0C and the diazonium salt was added drop wise in 10 minutes time. After the addition of the diazonium salt solution the solution of anhydrous sodium acetate (10 gm in 20 mL water) was added in 10 minutes time. After the addition of the sodium acetate solution the reaction mixture was allowed to stir for one hour at room temperature to obtain the dyes. The dye obtained was filtered, dried and recrystalized.

2.4. 4-Methyl-7-hydroxycoumarin (78)

CH3

HO OO (78) 4-Methyl-7hydroxycoumarin was synthesized using the literature method 163 . Polyphosphoric acid (16gm) was added to a solution of resorcinol (1.1gm, 0.1mol) in (13gm, 0.1mol) of ethylacetoacetate. The mixture was stirred and heated at 75-80 0C for about 20 minutes, and then poured into ice- water. The pale yellow solid was collected by suction filtration, washed with cold water and dried at 60 0C. 4-Methyl-7-hydroxycoumarin was recrystalized from ethanol. Yield (1.8gm, 89 %) m.p.185 0C. (lit 163 m.p. 186 0C). -1 IR νmax (KBr/ cm ), 1655 (C=O), 1 H-NMR δΗ (DMSO-d6, 300 MHz) .9.33 (s, 1H, O H), 7.78-6.37 (m, 3H, Ar H),

6.19 (s, 1H, Ar H), 3.49 (s, 3H, C H3). MS EI (m/z), 175 (M +, 100%).

CHN Anal. Calcd for C 10 H8 O3: C, 68.13; H, 4.19; O, 27.09 %. Found: C, 68.03; H, 4.39; O, 26.78 %.

2.4.1 Dyes from 4-Methyl-7-hydroxy-coumarin 2.4.1.1 Diazotization Diazotization of heterocyclic amine (1a-10a) was carried out with nitrosyl sulphuric acid. The nitrosyl sulphuric acid was prepared by mixing sodium nitrite (1g) and concentrated sulphuric acid (7mL). The mixing was carried out at 70 0C for a time of 6-7 hours; it was further stirred for an additional 1 hour at 0 0C. The heterocylic amine (1a-10a) (0.02mol) was dissolved in hot glacial acetic acid (2.5mL) and then shifted to an ice bath maintaining temperature of 0 to -50C. The liquor was then added in portions in the total 30 minutes to a cold solution of nitrosyl sulphuric acid.

2.4.1.2 Coupling After the diazotization was complete the azo liquor was slowly added to the vigorously stirred solution of 7-hydroxy-4-methylcoumarin (3.52g, 0.002mol) in sodium carbonate solution (0.002mol in 2mL water). Sodium carbonate was added in portions periodically to maintain the pH of the reaction mixture at 7-8. Stirring was continued for about 1 hour maintaining the temperature at 0 to -50C and pH 7-8. The reaction was monitored by using the TLC technique using hexane: ethyl acetate (1:1) as the solvent mixture. After the reaction had completed the resulting dye was filtered, washed with cold water and dried in a hot air oven. The dyes obtained were recrystalized from hexane: ethyl acetate (1:1) mixture.

2.5 Spectral and analytical data of the synthesised dyes 2.5.1 (Dye 16) Yield 89.2%. MP 279-281 ºC. -1 IR νmax (KBr/ cm ), 3357 (N H), 1695 (C=O), 1605 (C=C) and 1551 (N=N) 1 H-NMR δΗ (DMSO-d6, 300 MHz) 8.02 ( s, 1H, N H) 7.78 (d, J = 7.2 Hz, 2H, H), 7.67 (d, J = 7.03 Hz, 2H, Ar H), 7.51 (s, 1H, Ar H), 7.30-6.48

(m, 6H, Ar H), 3.59 (q, J = 5.9 Hz, 4H, 2C H2 ), 4.03 (s, 3H, Ar-

OC H3), 3.77 (s, 3H, C H3 ), 3.39 (t, J = 5.2 Hz, 6H, 2C H3). 13 C-NMR δC (DMSO-d6 75 MHz) 69.1 (C=O), 163.1, 158.1, 156.8, 151.6, 142.3, 137.1, 134.6, 133.3, 131.1, 130.4, 128.0, 126.3,125.1, 123.3, 122.3, 117.1, 108.3 , 106.1, 94.7,62.5, 55.6, 49.0, 46.3. MS EI (m/z), 483 (100), 236 (48), 247 (26).

CHN Anal. Calcd for C 28 H28 N4 O4 (484.2): C, 69.10; H, 5.77; N, 10.23; O, 12.97 %. Found: C, 68.17; H, 5.86; N, 10.31; O, 12.95 %.

2.5.2 (Dye 17) Yield 83.0%. MP 238-240 ºC. -1 IR νmax (KBr/ cm ), 1675 (C=O), 1611 (C=C) and1556 (N=N) 1 H-NMR δΗ (DMSO-d6, 300 MHz) 7.76 (d, J = 7.3 Hz, 2H, Ar H), 7.67 (d, J = 7.1 Hz, 2H, Ar H), 7.61 (d, J = 6.9 Hz, 2H, Ar H), 7.52 (d, J = 6.3 Hz, 2H, Ar H), 7.41 (s, 1H, Ar H), 7.37-6.78 (m, 4H, Ar H), 3.88 (q,

J = 5.3 Hz, 4H, 2C H2 ), 3.62 (t, J = 5.1 Hz, 4H, C H2 ), 3.51 (t, J = 5.9 Hz, 2H, 2O H). 13 C-NMR δC (DMSO-d6 75 MHz) 169.1 (C=O), 158.1, 156.8, 151.6, 146.3, 141.6, 136.1, 134.3, 130.0, 126.3, 125.1, 123.5, 122.3, 121.3, 117.3, 113.1, 94.7, 62.1, 58.5. MS EI (m/z), 428 (100), 253 (55), 173 (31).

CHN Anal. Calcd for C 25 H23 N3 O4 (429.3): C, 69.72; H, 5.47; N, 9.23; O, 14.79 %. Found: C, 68.17; H, 5.33; N, 9.21; O, 14.66 %.

2.5.3 (Dye 18) Yield 73.2%. MP 268-270 ºC. -1 IR νmax (KBr/ cm ), 1679 (C=O) 1611 (C=C).and 1547 (N=N), 1 H-NMR δΗ (DMSO-d6, 300 MHz) 7.81 (d, J = 7.3 Hz, 2H, Ar H), 7.77 (d, J = 7.1 Hz, 2H, Ar H), 7.61 (d, J = 6.7 Hz, 2H, Ar H), 7.56 (d, J = 6.3 Hz, 2H, Ar H),7.51-6.78 (m, 4H, Ar H), 7.41 (s, 1H, Ar H), 4.51 (q,

J = 5.4 Hz, 2H, C H2), 3.81 (t, J = 5.0 Hz, 2H, C H2 ), 3.42 (q, J =

6.9 Hz, 2H, C H2), 3.21 (d, J = 6.5 Hz, 3H, C H3 ), 2.89 (t, J = 5.3 Hz, 1H, O H). 13 C-NMR δC (DMSO-d6 75 MHz) 169.0 (C=O), 157.3, 156.6, 152.6, 146.3, 141.6, 136.1, 134.3, 130.0, 126.3, 125.1, 123.5, 122.3, 121.3, 117.3, 113.1, 94.7, 62.1, 58.7, 49.3. MS EI (m/z), 412 (100). 256 (44), 156 (27).

CHN Anal. Calcd for C 25 H23 N3 O3 (413.2): C, 72.42; H, 5.77; N, 10.03; O, 11.57 %. Found: C, 68.17; H, 5.86; N, 10.21; O, 10.96 %.

2.5.4 (Dye 19) Yield 71.6%. MP 218-220 ºC. -1 IR νmax (KBr/ cm ), 1677 (C=O), 1610 (C=C).and 1551 (N=N) 1 H-NMR δΗ (DMSO-d6, 300 MHz) 7.75 (d, J = 6.3 Hz, 2H, Ar H), 7.67 (d, J = 7.3 Hz, 2H, Ar H), 7.57 (d, J = 6.7 Hz, 2H, Ar H), 7.49 (d, J = 6.8 Hz, 2H, Ar H),7.31-6.65 (m, 4H, Ar H), 6.81 (s, 1H, Ar H), 4.91 (q,

J = 5.8 Hz, 2H, C H2), 4.35 (d, J = 5.3 Hz, 2H, C H2 ), 4.14 (t, J =

5.0 Hz, 2H, C H2 ), 3.96 (t, J = 6.7 Hz, 2H, C H2 ), 3.75 (t, J = 5.2 Hz , 1H, O H). 13 C-NMR δC (DMSO-d6 75 MHz) 168.0 (C=O), 157.3, 156.6, 152.6, 146.3, 141.6, 136.1, 134.3, 130.0, 126.3, 125.1, 123.5, 122.3, 121.3, 117.3, 116.6, 94.7, 62.0, 58.1, 51.3, 47.5. MS EI (m/z), 438 (100), 247 (51), 186 (34).

CHN Anal. Calcd for C 26 H22 N4 O3 (438.31): C, 71.17; H, 5.17; N, 12.68; O, 10.88 %. Found: C, 70.17; H, 4.96; N, 12.21; O, 10.91 %.

2.5.5 (Dye 20) Yield 73.8%. MP 211-212 ºC. -1 IR νmax (KBr/ cm ), 1679 (C=O) 1614 (C=C).and 1555 (N=N) 1 H-NMR δΗ (DMSO-d6, 300 MHz) 7.81 (d, J = 6.23 Hz, 2H, Ar H), 7.63 (d, J = 7.2 Hz, 2H, Ar H), 7.59 (d, J = 6.7 Hz, 2H, Ar H), 7.41 (d, J = 5.4 Hz, 2H, Ar H), 7.31-6.89 (m, 4H, Ar H), 6.61 (s, 1H, Ar H), 3.91 (s,

6H, 2C H3). 13 C-NMR δC (DMSO-d6 75 MHz) 169.3 (C=O), 158.1, 156.3, 151.6, 145.3, 141.0, 136.3, 134.0, 130.3, 127.3, 125.1, 123.5, 122.3, 121.3, 117.3, 116.6, 94.7, 43.3. MS EI (m/z), 368 (100), 261 (51), 107 (28).

CHN Anal. Calcd for C 23 H19 N3 O2 (369.3): C, 74.10; H, 5.03; N, 11.23; O, 8.59 %. Found: C, 74.17; H, 4.99; N, 11.26; O, 8.59 %.

2.5.6 (Dye 21) Yield 81.2%. MP 217-219 ºC. -1 IR νmax (KBr/ cm ), 3357 ( NH ), 1679 (C=O) 1610 (C=C).and 1551 (N=N). 1 H-NMR δΗ (DMSO-d6, 300 MHz) 8.31 (s, 1H, N H), 7.75 (d, J = 7.6 Hz, 2H, Ar H), 7.61 (d, J = 7.1 Hz, 2H, Ar H), 7.67 (d, J = 7.2 Hz, 2H, Ar H), 7.54 (d, J = 6.8 Hz, 2H, Ar H),7.48-6.65 (m, 4H, Ar H), 4.75

(q, J = 5.1 Hz, 2H, C H2), 4.95 (q, J = 5.2 Hz, 1H, O H),.4.34 (t, J

= 4.4 Hz, 2H, C H2 ). 13 C-NMR δC (DMSO-d6 75 MHz) 169.1 (C=O), 158.3, 156.3, 152.6, 145.3, 142.0, 136.3, 134.3, 130.3, 127.3, 125.1, 123.5, 122.3, 121.3, 117.3, 116.6, 94.7, 64.3, 54.3. MS EI (m/z), 384 (100), 262 (42), 124 (27).

CHN Anal. Calcd for C 23 H19 N3 O3 (385.3): C, 71.65; H, 4.89; N, 10.23; O, 12.39 %. Found: C, 71.67; H, 4.99; N, 10.26; O, 12.59 %.

2.5.7 (Dye 22) Yield 76.2%. MP 220-222 ºC. -1 IR νmax (KBr/ cm ), 1672 (C=O) 1613 (C=C).and 1545 (N=N). 1 H-NMR δΗ (DMSO-d6, 300 MHz) 7.83 (d, J = 7.5 Hz, 2H, Ar H), 7.69-6.93 (m, 4H, Ar H), 7.63 (d, J = 7.1 Hz, 2H, Ar H), 7.51-6.53 (m, 7H,

Ar H), 6.38 (s, 1H, Ar H), 5.19 (q, J = 5.2 Hz, 2H, C H2 ), 4.81 (t, J

= 6.2 Hz, 2H, C H2), 3.88 (t, J = 7.2 Hz, 2H, C H2), 3.63 (s, 3H,

CH3), 3.33 (t, J = 6.2 Hz, 3H, C H3), 2.75 (t, J = 7.3 Hz, 1H, O H). 13 C-NMR δC (DMSO-d6 75 MHz) 169.0 (C=O), 156.6, 151.3, 142.3, 146.6, 137.3, 134.3, 132.0, 130.3, 126.1, 125.6, 124.3, 123.6, 122.3, 117.3, 114.6, 110.6, 94.1, 61.3, 59.3, 49.3, 43.6, 39.3. MS EI (m/z), 426 (100), 220 (32), 207 (26).

CHN Anal. Calcd for C 26 H25 N3 O3 (427.4): C, 73.10; H, 5.87; N, 9.23; O, 11.59 %. Found: C, 72.17; H, 4.99; N, 9.06; O, 11.60 %.

2.5.8 (Dye 23) Yield 73.5 %. MP 268-270 ºC. -1 IR νmax (KBr/ cm ), 3354 (NH), 1675 (C=O) 1610 (C=C).and 1557 (N=N). 1 H-NMR δΗ (DMSO-d6, 300 MHz) 8.13 (s, 1H, N H), 7.79 (d, J = 7.3 Hz, 2H, Ar H), 7.71 (d, J = 7.1 Hz, 2H, Ar H), 7.61-6.55 (m, 7H, Ar H), 6.38

(s, 1H, Ar H), 4.67 (q, J = 7.3 Hz, 4H, 2C H2), 4.33 (t, J = 6.1 Hz,

6H, 2C H3). 13 C-NMR δC (DMSO-d6 75 MHz) 169.1 (C=O), 168.1, 166.1, 156.8, 151.6, 142.3, 137.1, 134.6, 133.3, 131.1, 130.4, 128.0, 126.3, 125.1, 123.3, 122.3, 117.1, 108.3, 106.1, 94.7, 49.0, 44.2, 39.6. MS EI (m/z), 453 (100), 247 (49), 206 (24).

CHN Anal. Calcd for C 27 H26 N4 O3 (454.3): C, 71.33; H, 5.63; N, 12.23; O, 10.53 %. Found: C, 71.41; H, 5.63; N, 12.19; O, 10.47 %.

2.5.9 (Dye 24) Yield 69.7%. MP 228-230 ºC. -1 IR νmax (KBr/ cm ), 1671 (C=O) 1613 (C=C).and 1557 (N=N). 1 H-NMR δΗ (DMSO-d6, 300 MHz) 7.86 (d, J = 7.3 Hz, 2H, Ar H), 7.71 (d, J = 7.1 Hz, 2H, Ar H), 7.61-7.37 (m, 4H, Ar H), 7.28 (d, J = 6.2 Hz, 2H, Ar H), 7.14 (d, J = 6.1 Hz, 2H, Ar H), 6.71 (s, 1H, Ar H), 4.87

(q, J = 6.8 Hz, 4H, 2C H2), 4.32 (t, J = 6.3 Hz, 6H, 2C H3). 13 C-NMR δC (DMSO-d6 75 MHz) 169.3 (C=O), 158.1, 156.3, 151.6, 145.3, 141.0, 136.3, 134.0, 130.3, 127.3, 125.1, 123.5, 122.3, 121.3, 117.3, 116.6, 94.7, 47.3, 43.6. MS EI (m/z), 396 (100%), 246 (49), 148 (24).

CHN Anal. Calcd for C 25 H23 N3 O2 (397.3): C, 73.10; H, 5.03; N, 13.23; O, 7.59 %. Found: C, 73.17; H, 5.09; N, 13.26; O, 7.55 %.

2.5.10 (Dye 25) Yield 71.7%. MP 278-279 ºC. -1 IR νmax (KBr/ cm ), 1673 (C=O) 1613 (C=C).and 1557 (N=N). 1 H-NMR δΗ (DMSO-d6, 300 MHz) 7.77 (d, J = 7.2 Hz, 2H, Ar H), 7.66 (d, J = 6.8 Hz, 2H, Ar H), 7.59 (d, J = 7.2 Hz, 2H, Ar H), 7.43 (d, J = 6.3 Hz, 2H, Ar H), 7.39-6.78 (m, 4H, Ar H), 6.62 (s, 1H, Ar H), 4.38 (q,

J = 7.1 Hz, 2H, C H2), 4.16 (t, J = 6.2 Hz, 2H, C H2), 3.71 (t, J =

7.5 Hz, 2H, C H2), 3.41 (t, J = 6.7 Hz, 3H, C H3). 13 C-NMR δC (DMSO-d6 75 MHz) 169.3 (C=O), 158.0, 156.6, 152.6, 146.3, 141.6, 136.1, 134.3, 130.0, 126.3, 125.1, 123.5, 122.3, 121.3, 117.3, 116.0, 114.6, 94.7, 51.0, 44.6, 38.5, 36.6. MS EI (m/z), 422 (100%), 210 (37), 202 (26).

CHN Anal. Calcd for C 26 H22 N4 O2 (425.3): C, 73.10; H, 5.03; N, 13.23; O, 7.59 %. Found: C, 73.17; H, 5.09; N, 13.26; O, 7.55 %.

2.5.11 (Dye 26) Yield 86.4%. MP 211-213 ºC. -1 IR νmax (KBr/ cm ), 1676 (C=O) 1614 (C=C).and 1547 (N=N) 1 H-NMR δΗ (DMSO-d6, 300 MHz) 7.97-7.72 (m, 4H, Ar H), 7.69 (d, J = 6.3 Hz, 2H, Ar H), 7.51 (d, J = 7.3 Hz, 2H, Ar H), 7.47-6.73 (m, 5H, Ar H), 6.69 (s, 1H, Ar H), 6.63 (s, 2H, 2O H). 13 C-NMR δC (DMSO-d6 75 MHz) 168.3 (C=O), 158.3, 157.1, 151.0, 148.6, 144.3, 137.3, 132.0, 130.3, 126.3, 125.1, 122.6, 120.2, 118.3, 117.3, 111.1, 94.7. MS EI (m/z), 407 (100%), 210 (40), 202 (34).

CHN Anal. Calcd for C 25 H16 N2 O4 (408.3): C, 73.43; H, 3.63; N, 6.23; O, 15.59 %. Found: C, 73.17; H, 3.69; N, 6.23; O, 15.36 %.

2.5.12 (Dye 27) Yield 75.5%. MP 227-229 ºC. -1 IR νmax (KBr/ cm ), 1671 (C=O) 1614 (C=C).and 1566 (N=N). 1 H-NMR δΗ (DMSO-d6, 300 MHz) 7.73 (d, J = 6.4 Hz, 2H, Ar H), 7.67-6.91 (m, 4H, Ar H), 6.59 (d, J = 7.3 Hz, 2H, Ar H), 7.67-6.91 (m, 4H, Ar H), 6.31 (s, 1H, Ar H), 5.63 (s, 1H, O H). 13 C-NMR δC (DMSO-d6 75 MHz) 167.5 (C=O), 158.3, 157.1, 151.0, 146.6, 137.3, 135.0, 134.3, 130.1, 126.3, 125.1, 124.6, 122.1, 121.0, 118.3, 117.3, 109.1, 94.7. MS EI (m/z), 494 (100%), 260 (34), 235 (23).

CHN Anal. Calcd for C 25 H15 N2 O6 S (493.3): C, 60.60; H, 3.03; N, 5.23; O, 19.59; S 6.44%. Found: C, 60.17; H, 3.09; N, 5.26; O, 19.59; S, 6.37 %.

2.5.13 (Dye 28) Yield 67.5%. MP 269-270 ºC. -1 IR νmax (KBr/ cm ), 3426 (NH 2), 1676 (C=O), 1610 (C=C).and 1559 (N=N). 1 H-NMR δΗ (DMSO-d6, 300 MHz) 10.01 (s, 1H, O H), 7.96-7.21 (m, 4H, Ar H), 7.07 (d, J = 7.3 Hz, 2H, Ar H), 7.13 (d, J = 6.9 Hz, 2H, Ar H), 7.96-7.21 (m, 3H, Ar H), 6.69 (s, 1H, Ar H), 5.13 (bs, 2H,

NH2).

C-NMR δC (DMSO-d6 75 MHz) 169.2 (C=O), 158.1, 157.3, 151.6, 147.6, 137.3, 136.1, 135.0, 134.6, 130.1, 129.3, 126.3, 125.3, 124.3, 123.6, 122.1, 118.1, 117.0, 113.1, 94.7. MS EI (m/z), 596 (100%), 361 (42), 236 (32).

CHN Anal. Calcd for C 25 H15 N3 O6 S2 (597.3): C, 49.43; H, 2.33; N, 6.23; O, 23.49 %. Found: C, 49.17; H, 2.29; N, 6.17; O, 23.51 %.

2.5.14 (Dye 29) Yield 87.2%. MP 253-255 ºC. -1 IR νmax (KBr/ cm ), 3351 ( NH ), 1674 (C=O) 1611 (C=C).and 1535 (N=N). 1 H-NMR δΗ (DMSO-d6, 300 MHz) , 10.03 (s, 2H, 2O H), 7.86-7.23 (m, 7H, Ar H), 7.17 (d, J = 7.2 Hz, 2H, Ar H), 7.03 (d, J = 6.8 Hz, 2H, Ar H), 6.71 (s, 1H, Ar H). 13 C-NMR δC (DMSO-d6 75 MHz) 168.2 (C=O), 158.1 157.3, 151.6, 148.6, 137.3, 136.1, 135.0, 134.6, 130.1, 129.3, 126.3, 125.3, 124.3, 123.6, 122.1, 118.6, 117.0, 114.1, 94.7. MS EI (m/z), 612 (100%), 316 (35), 211 (42).

CHN Anal. Calcd for C 25 H16 N2 O4 (613.3): C, 73.43; H, 3.63; N, 6.23; O, 15.59 %. Found: C, 73.17; H, 3.69; N, 6.23; O, 15.36 %.

2.5.15 (Dye 30) Yield 79.5%. MP 217-219 ºC. -1 IR νmax (KBr/ cm ), 3640 (NH 2), 3359 (NH), 1673 (C=O) 1611 (C=C).and 1525 (N=N). 1 H-NMR δΗ (DMSO-d6, 300 MHz) 10.3 (s, 1H, O H), 7.89-7.33 (m, 8H, Ar H), 7.11 (d, J = 7.1 Hz, 2H, Ar H), 7.03 (d, J = 6.8 Hz, 2H,

Ar H), 6.73 (s, 1H, Ar H), 5.32 (bs, 2H, N H2 ). 13 C-NMR δC (DMSO-d6 75 MHz) 169.2 (C=O), 168.5, 157.3, 151.6, 150.6, 137.3, 135.1, 133.6, 134.1, 130.1, 126.3, 124.3, 123.0, 122.1, 118.1, 117.0, 113.1, 94.7. MS EI (m/z), 610 (100%), 302 (45), 308 (36).

CHN Anal. Calcd for C 25 H16 N3 O6 S (611.4): C, 58.34; H, 3.13; N, 8.21; O, 18.59; S, 6.13 %. Found: C, 58.67; H, 3.19; N, 8.23; O, 18.36 %.

2.5.16 (Dye 31) Yield 87.2%. MP 269-271 ºC. -1 IR νmax (KBr/ cm ), 3345 (NH), 1692 (C=O) and 1617 (C=C), and 1535 (N=N). 1 H-NMR δΗ (DMSO-d6, 300 MHz) 8.61 ( s, 1H, NH) 7.97-7.44 (m, 8H, Ar H), 7.31 (s, 1H, Ar H), 7.01 (s, 1H, Ar H), 6.74 (s, 1H, Ar H),

4.07 (s, 3H, Ar-OC H3), 3.41 (q, J = 7.2 Hz, 4H, 2C H2 ), 2.47 (s,

3H, C H3 ), 1.89 (t, J = 6.3 Hz, 6H, 2C H3). 13 C-NMR δC (DMSO-d6 75 MHz) .169.5 (C=O), 158.1, 157.3, 152.8, 146.1, 141.3, 135.6, 130.3, 129.6, 128.1, 126.3, 123.3, 122.1, 119.4, 117.0, 110.3, 107.1, 104.2, 125.1, 123.3, 122.3, 117.1, 108.3 , 106.1, 94.7, 55.6, 49.0, 38.2. MS EI (m/z), 483 (100%), 236.3 (48), 247 (26).

CHN Anal. Calcd for C 28 H28 N4 O4 (484.2): C, 69.11; H, 5.67; N, 10.23; O, 12.91 %. Found: C, 68.87; H, 5.83; N, 10.31; O, 12.85 %.

2.5.17 (Dye 32) Yield 84.3%. MP 237-239 ºC. -1 IR νmax (KBr/ cm ), 1675 (C=O), 1621 (C=C). and 1545 (N=N). 1 H-NMR δΗ (DMSO-d6, 300 MHz) 7.87-7.34 (m, 8H, Ar H), 7.22 (d, J = 7.8 Hz, 2H, Ar H), 7.01 (d, J = 7.19 Hz, 2H, Ar H), 6.69 (s, 1H, Ar H),

4.59 (t, J = 6.8 Hz, 2H, 2O H), 4.81 (q, J = 6.1 Hz, 4H, 2C H2),

3.51 (t, J = 5.7 Hz, 4H, 2C H2). 13 C-NMR δC (DMSO-d6 75 MHz) 169.6 (C=O), 158.1, 157.2, 152.6, 142.3, 135.3, 130.6, 129.1, 128.3, 123.0, 122.3, 121.3, 119.1, 117.5, 104.3, 121.3, 117.3, 114.3, 113.1, 94.7, 59.3, 62.1, 58.5. MS EI (m/z), 428.1(100%), 253 (53), 173 (42).

CHN Anal. Calcd for C 25 H23 N3 O4 (429.4): C, 69.72; H, 5.37; N, 9.13; O, 14.66 %. Found: C, 69.17; H, 5.22; N, 9.21; O, 14.03 %.

2.5.18 (Dye 33) Yield 77.3%. MP 285-287 ºC. -1 IR νmax (KBr/ cm ), 1679 (C=O) and 1611 (C=C), and 1543 (N=N). 1 H-NMR δΗ (DMSO-d6, 300 MHz) 7.73-7.33 (m, 8H, Ar H), 7.21 (d, J = 7.2 Hz, 2H, Ar H), 7.03 (d, J = 6.8 Hz, 2H, Ar H), 6.73 (s, 1H, Ar H),

4.66 (t, J = 5.3 Hz, 1H, O H), 3.73 (q, J = 5.1 Hz, 2H, C H2 ), 3.61

(t, J = 5.9 Hz, 2H, C H2), 3.48 (q, J = 6.89 Hz, 2H, C H2), 2.83 (s,

J = 6.89 Hz, 3H, C H3). 3 C-NMR δC (DMSO-d6 75 MHz) 169.3 (C=O), 138.1, 157.2, 152.6, 142.6, 135.3, 130.6, 129.1, 128.3, 123.6, 123.0, 122.3, 119.1, 117.5, 114.0, 104.3, 58.6, 44.3, 32.6. MS EI (m/z), 414 (100%), 256 (49), 155 (33).

CHN Anal. Calcd for C 25 H23 N3 O3 (413.4): C, 73.42; H, 5.62; N, 10.11; O, 11.33 %. Found: C, 69.17; H, 5.76; N, 10.31; O, 11.46 %.

2.5.19 (Dye 34) Yield 66.6%. MP 219-221 ºC. -1 IR νmax (KBr/ cm ), 1677 (C=O) and 1610 (C=C), and 1537 (N=N). 1 H-NMR δΗ (DMSO-d6, 300 MHz) 7.83-7.33 (m, 8H, Ar H), 7.13 (d, J = 5.3 Hz, 2H, Ar H), 6.83 (d, J = 5.3 Hz, 2H, Ar H), 6.63 (s, 1H, Ar H),

5.79 (t, J = 5.3 Hz, 1H, O H), 3.69 (q, J = 5.3 Hz, 2H, C H2), 3.56

(t, J = 5.3 Hz, 2H, C H2), 3.13 (t, J = 5.3 Hz, 2H, C H2), 3.03 (t, J =

5.3 Hz, 2H, C H2). 13 C-NMR δC (DMSO-d6 75 MHz) 169.0 (C=O), 163.1, 157.2, 152.6, 151.6, 142.2, 135.3, 130.6, 129.1, 128.3, 123.6, 122.3, 119.1, 117.5, 114.3, 58.6, 51.0, 44.3, 38.6. MS EI (m/z), 437 (100%), 247 (51), 188 (48).

CHN Anal. Calcd for C 26 H22 N4 O3 (438.3): C, 71.37; H, 5.19; N, 11.68; O, 10.65 %. Found: C, 71.47; H, 5.96; N, 11.69; O, 11.01 %.

2.5.20 (Dye 35) Yield 83.4%. MP 282-284 ºC. -1 IR νmax (KBr/ cm ), 1599 (C=O), 1614 (C=C), and 1535 (N=N). 1 H-NMR δΗ (DMSO-d6, 300 MHz) 7.89-7.37 (m, 8H, Ar H), 7.07 (d, J = 7.3 Hz, 2H, Ar H), 6.91 (d, J = 7.1 Hz, 2H, Ar H), 6.53 (s, 1H, Ar H),

3.46 (s, 6H, 2C H3. ). 13 C-NMR δC (DMSO-d6 75 MHz) 169.1 (C=O), 163.1, 157.2, 152.6, 151.3, 142.3, 135.3, 130.6, 129.1, 128.3, 123.6, 123.0, 122.3, 119.1, 117.5, 114.0, 40.3. MS EI (m/z), 369 (100%), 261 (42), 107 (33).

CHN Anal. Calcd for C 23 H19 N3 O2 (368.1): C, 74.22; H, 5.08; N, 10.83; O, 8.66 %. Found: C, 74.27; H, 5.19; N, 11.22; O, 8.66 %.

2.5.21 (Dye 36) Yield 75.2%. MP 277-279 ºC. -1 IR νmax (KBr/ cm ), 3354 ( NH ), 1671 (C=O), 1610 (C=C).and 1527 (N=N). 1 H-NMR δΗ (DMSO-d6, 300 MHz) 8.03 (s, 1H, NH ), 7.89-7.37 (m, 8H, Ar H), 7.17 (d, J = 7.5 Hz, 2H, Ar H), 6.97 (d, J = 7.3 Hz, 2H, Ar H), 6.43 (s, 1H, Ar H), 4.56 (t, J = 5.7 Hz, 1H, O H), 3.76 (q, J =

6.3 Hz, 2H, C H2), 3.23 (t, J = 5.1 Hz, 2H, C H2). 13 C-NMR δC (DMSO-d6 75 MHz) 169.1 (C=O), 158.1, 157.2, 152.6, 148.3, 141.3, 135.3, 130.6, 129.1, 128.3, 123.6, 123.0, 122.3, 119.1, 117.5, 113.6, 61.1, 46.1. MS EI (m/z), 384 (100%), 262 (36), 124 (26).

CHN Anal. Calcd for C 23 H18 N3 O3 (385.4): C, 70.85; H, 4.98; N, 10.73; O, 12.34 %. Found: C, 71.17; H, 4.69; N, 9.26; O, 12.66 %.

2.5.22 (Dye 37) Yield 76.4%. MP 242-244 ºC. -1 IR νmax (KBr/ cm ), 1662 (C=O) 1621 (C=C). and 1531 (N=N). 1 H-NMR δΗ (DMSO-d6, 300 MHz) 7.89-7.37 (m, 8H, Ar H), 7.13 (d, J = 7.3 Hz, 2H, Ar H), 6.72 (d, J = 7.1 Hz, 2H, Ar H), 6.53 (s, 1H, Ar H),

4.76 (t, J = 5.5 Hz, 1H, O H), 3.81 (q, J = 6.3 Hz, 2H, C H2), 3.63

(t, J = 5.3 Hz, 2H, C H2), 3.46 (q, J = 6.1 Hz, 2H, C H2), 3.25 (t, J

= 5.0 Hz, 2H, C H2), 2.19 (s, 3H, C H3) 13 C-NMR δC (DMSO-d6 75 MHz) 169.0 (C=O), 163.1, 157.2, 152.6, 141.3, 149.3, 135.3, 130.6, 129.1, 128.3, 125.3, 123.6, 122.3, 119.1, 117.5, 113.3, 61.1, 46.1. MS EI (m/z), 426 (100%), 220 (45), 207 (32).

CHN Anal. Calcd for C 26 H25 N3 O3 (427.6): C, 72.71; H, 5.71; N, 10.13; O, 11.19 %. Found: C, 72.11; H, 5.89; N, 9.36; O, 11.19 %.

2.5.23 (Dye 38) Yield 69.4 %. MP 275-277 ºC. -1 IR νmax (KBr/ cm ), 3354 ( NH ), 1675 (C=O), 1610 (C=C). and 1529 (N=N). 1 H-NMR δΗ (DMSO-d6, 300 MHz) 8.51 (s, 1H, NH ), 7.97-7.44 (m, 7H, Ar H), 7.31 (s, 1H, Ar H), 7.02 (d, J = 7.3 Hz, 2H, Ar H), 6.87 (d, J

= 6.4 Hz, 2H, Ar H), 3.43 (q, J = 5.3 Hz, 4H, 2C H2), 3.26 (t, J =

6.1 Hz, 6H, 2C H3), 3.19 (s, 3H, OC H3). 13 C-NMR δC (DMSO-d6 75 MHz) 168.3 (C=O), 158.1, 157.2, 152.6, 135.3, 133.3, 130.6, 129.1, 128.3, 124.3, 123.6, 122.3, 119.1, 117.5, 110.3, 106.6, 44.6, 22.3, 12.6. MS EI (m/z), 454 (100%), 247 (60), 206 (51).

CHN Anal. Calcd for C 27 H26 N4 O3 (453.1): C, 70.96; H, 5.66; N, 11.73; O, 10.31 %. Found: C, 71.11; H, 5.60; N, 12.10; O, 10.17 %.

2.5.24 (Dye 39) Yield 76.7%. MP 241-243 ºC. -1 IR νmax (KBr/ cm ), 1673 (C=O) 1623 (C=C). and 1548 (N=N). 1 H-NMR δΗ (DMSO-d6, 300 MHz) 7.77-7.31 (m, 8H, Ar H), 7.11 (d, J = 7.1 Hz, 2H, Ar H), 7.03 (d, J = 6.3 Hz, 2H, Ar H), 6.87 (s, 1H, Ar H),

3.43 (q, J = 5.3 Hz, 4H, 2C H2), 3.31 (t, J = 6.1 Hz, 6H, 2C H3). 13 C-NMR δC (DMSO-d6 75 MHz) 169.0 (C=O), 163.1, 157.2, 152.6, 151.3, 142.1, 135.3, 130.6, 129.1, 128.3, 124.0, 123.6, 122.3, 119.1, 117.5, 114.3, 44.6, 12.0. MS EI (m/z), 396 (100%), 246 (43). 149 (35).

CHN Anal. Calcd for C 25 H23 N3 O2 (397.4): C, 74.60; H, 5.73; N, 10.23; O, 8.19 %. Found: C, 73.77; H, 5.59; N, 11.16; O, 7.85 %.

2.5.25 (Dye 40) Yield 76.7%. MP 265-267 ºC. -1 IR νmax (KBr/ cm ), 1653 (C=O), 1611 (C=C). and 1544 (N=N). 1 H-NMR δΗ (DMSO-d6, 300 MHz) 7.89-7.21 (m, 8H, Ar H), 7.11 (d, J = 7.3 Hz, 2H, Ar H), 6.77 (d, J = 5.3 Hz, 2H, Ar H), 6.59 (s, 1H, Ar H),

3.66 (t, J = 7.0 Hz, 2H, C H2), 3.36 (q, J = 7.1 Hz, 2H, C H2), 3.36

(q, J = 7.3 Hz, 2H, C H2), 2.46 (t, J = 6.5 Hz, 3H, C H3). 13 C-NMR δC (DMSO-d6 75 MHz) 167.3 (C=O), 159.1, 157.2, 152.6, 151.3, 140.3, 135.3, 130.6, 129.1, 128.3, 124.1, 123.6, 122.3, 119.1, 117.5, 114.3, 51.2, 44.1, 16.6. MS EI (m/z), 422 (100%), 218 (44), 202 (22).

CHN Anal. Calcd for C 26 H22 N4 O2 (423.3): C, 73.66; H, 5.23; N, 13.03; O, 7.51 %. Found: C, 73.67; H, 5.07; N, 13.36; O, 7.60 %.

2.5.26 (Dye 41) Yield 83.0%. MP 261-263 ºC. -1 IR νmax (KBr/ cm ), 1656 (C=O), 1619 (C=C). and 1540 (N=N). 1 H-NMR δΗ (DMSO-d6, 300 MHz) 8.17 (s, 2H, 2OH), 7.89-7.33 (m, 4H, Ar H), 7.21-6.89 (m, 3H, Ar H), 6.71 (s, 1H, Ar H), 6.46 (d, J = 5.1 Hz, 2H, Ar H), 6.31 (d, J = 6.2 Hz, 2H, Ar H), 6.13 (d, J = 5.1 Hz, 2H, Ar H). 13 C-NMR δC (DMSO-d6 75 MHz) 168.5 (C=O), 158.1, 157.2, 152.6, 148.3, 144.0, 135.3, 132.3, 130.6, 129.1, 128.3, 126.3, 123.6, 122.3, 119.1, 117.5, 116.0, 111.3. MS EI (m/z), 408 (100%), 251 (52), 160 (32).

CHN Anal. Calcd for C 25 H16 N2 O4 (407.5): C, 73.47; H, 3.69; N, 6.79; O, 15.39 %. Found: C, 73.17; H, 3.61; N, 6.33; O, 15.13 %.

2.5.27 (Dye 42) Yield 81.2%. MP 211-213 ºC. -1 IR νmax (KBr/ cm ), 1669 (C=O), 1634 (C=C). and 1517 (N=N). 1 H-NMR δΗ (DMSO-d6, 300 MHz) 10.1 (s, 1H, OH), 7.89-7.33 (m, 9H, Ar H), 7.13 (d, J = 7.5 Hz, 2H, Ar H), 6.87 (d, J = 7.3 Hz, 2H, Ar H), 6.79 (s, 1H, Ar H), 13 C-NMR δC (DMSO-d6 75 MHz) 168.0 (C=O), 159.1, 157.2, 153.0, 152.6, 144.3, 137.0, 135.3, 130.6, 129.1, 128.3, 123.6, 122.3, 121.3, 119.1, 117.5, 109.3. MS EI (m/z), 508 (100%), 260 (35), 233 (27).

CHN Anal. Calcd for C 25 H15 N2 O6 S (509.1): C, 60.73; H, 3.13; N, 5.53; O, 19.61; S 6.24%. Found: C, 60.77; H, 3.03; N, 5.17; O, 19.49;S, 6.17 %.

2.5.28 (Dye 43) Yield 77.3%. MP 213-215 ºC. -1 IR νmax (KBr/ cm ), 1673 (C=O), 1613 (C=C). and 1527 (N=N). 1 H-NMR δΗ (DMSO-d6, 300 MHz) 10.1 (s, 1H, OH), 7.89-7.33 (m, 7H, Ar H), 7.21 (d, J = 7.3 Hz, 2H, Ar H), 6.88 (d, J = 7.0 Hz, 2H,

Ar H), 6.71 (s, 1H, Ar H), 5.43 (bs, 2H, N H2) 13 C-NMR δC (DMSO-d6 75 MHz) 169.0 (C=O), 163.1, 157.2, 152.6, 153.3, 148.0, 135.3, 130.6, 129.1, 128.3, 126.0, 123.6, 122.3, 119.1, 117.5, 109.3. MS EI (m/z), 596 (100%), 361 (42), 236 (37).

CHN Anal. Calcd for C 25 H14 N3 O9 S2 (595.3): C, 49.73; H, 2.39; N, 4.23; O, 24.49; S, 10.67 %. Found: C, 49.17; H, 2.41; N, 4.17; O, 23.51; S, 10.77 %.

2.5.29 (Dye 44) Yield 81.2%. MP 233-235 ºC. -1 IR νmax (KBr/ cm ), 1674 (C=O), 1611 (C=C), and 1529 (N=N). 1 H-NMR δΗ (DMSO-d6, 300 MHz) 10.4 (s, 2H, 2OH), 7.89-7.33 (m, 7H, Ar H), 7.23 (d, J = 7.1 Hz, 2H, Ar H), 7.08 (d, J = 5.0 Hz, 2H, Ar H), 6.61 (s, 1H, Ar H). 13 C-NMR δC (DMSO-d6 75 MHz) 167.0 (C=O), 158.6, 157.2, 152.3, 153.6, 148.1, 135.6, 130.6, 129.1, 128.3, 126.0, 123.6, 122.3, 119.1, 117.5, 109.6. MS EI (m/z), 612 (100%), 316 (35), 211 (41).

CHN Anal. Calcd for C 25 H14 N2 O10 S2 (613.3): C, 49.43; H, 2.63; N, 4.23; O, 26.59; S, 10.33%. Found: C, 49.17; H, 2.69; N, 4.02; O, 26.36; S, 9.17 %.

2.5.30 (Dye 45) Yield 72.6%. MP 261-263 ºC. -1 IR νmax (KBr/ cm ), 3466 ( NH 2), 1663 (C=O), 1614 (C=C), and 1544 (N=N). 1 H-NMR δΗ (DMSO-d6, 300 MHz) 10.1 (s, 1H, OH), 7.97-7.23 (m, 8H,

Ar H), 6.61 (s, 1H, Ar H), 5.96 (bs, 2H, N H2). 13 C-NMR δC (DMSO-d6 75 MHz) 167.3 (C=O), 157.6, 157.2, 152.6, 149.3, 135.3, 133.3, 130.6, 129.1, 128.3, 127.3, 126.0, 123.6, 122.3, 121.6, 119.1, 117.5, 113.3, MS EI (m/z), 610 (100%), 302 (45), 308 (36).

CHN Anal. Calcd for C 25 H15 N3 O9 S2 (611.3): C, 49.34; H, 2.13; N, 6.21; O, 23.59; S, 10.13 %. Found: C, 48.67; H, 2.19; N, 6.53; O, 23.36; S, 10.66 %.

2.5.31 (Dye 46) Yield 73.3%. MP 288-290 ºC. -1 IR νmax (KBr/ cm ), 3357 ( NH ), 1675 (C=O), 1618 (C=C). and 1540 (N=N). 1 H-NMR δΗ (DMSO-d6, 300 MHz) 10.09 (s, 1H, NH ), 7.78-7.41 (m, 6H, Ar H), 7.23 (d, J = 7.3 Hz, 2H, Ar H), 7.03 (d, J = 5.7 Hz, 2H,

Ar H), 6.97 (s, 1H, Ar H), 4.87 (s, 3H,Ar-OC H3), 3.89 (q, J = 5.4

Hz, 4H, 2C H2), 3.49 (t, J = 5.1 Hz, 4H, 2C H3), 3.49 (s, 3H, C H3). 13 C-NMR δC (DMSO-d6 75 MHz) 166.2 (C=O), 161.1, 158.3, 149.3, 147.6, 142.0, 130.6, 129.3, 127.0, 126.3, 124.0, 123.3, 118.0, 115.3, 110.3, 113.0, 97.6, 55.6, 32.6. MS EI (m/z), 484 (100%), 276 (44), 271 (36).

CHN Anal. Calcd for C 28 H28 N4 O4 (483.4): C, 69.34; H, 5.13; N, 11.21; O, 13.59 %. Found: C, 68.67; H, 5.19; N, 10.23; O, 13.36 %.

2.5.32 (Dye 47) Yield 77.2 %. MP 268-270 ºC. -1 IR νmax (KBr/ cm ), 1687 (C=O), 1611 (C=C). and 1517 (N=N). 1 H-NMR δΗ (DMSO-d6, 300 MHz) 7.92-7.42 (m, 4H, Ar H), 7.27 (d, J = 7.3 Hz, 2H, Ar H), 7.13 (d, J = 7.1 Hz, 2H, Ar H), 6.93 (d, J = 5.3 Hz, 2H, Ar H), 6.73 (d, J = 5.1 Hz, 2H, Ar H), 6.53 (s, 1H, Ar H), 4.87

(t, 4H, 2O H ), 3.91 (q, , J = 5.4 Hz, 4H, 2C H2 ), 3.47 (t, , J = 5.1

Hz, 4H, 2C H2). 13 C-NMR δC (DMSO-d6 75 MHz) 166.2 (C=O), 161.1, 158.3, 151.0, 147.6, 142.2, 130.6, 123.0, 129.3, 127.0, 126.3, 124.0, 123.3, 118.0, 113.6, 58.3, 44.3, 33.6. MS EI (m/z), 430 (100%), 218 (47), 216 (34).

CHN Anal. Calcd for C 25 H23 N4 O4 (429.2): C, 69.10; H, 5.17; N, 9.21; O, 14.87 %. Found: C, 68.87; H, 4.89; N, 9.31; O, 14.55 %.

2.5.33 (Dye 48) Yield 69.6 %. MP 308 ºC. -1 IR νmax (KBr/ cm ), 1669 (C=O), 1621 (C=C). and 1529 (N=N). 1 H-NMR δΗ (DMSO-d6, 300 MHz) 7.72-7.42 (m, 4H, Ar H), 7.38 (d, J = 7.3 Hz, 2H, Ar H), 7.30 (d, J = 7.0 Hz, 2H, Ar H), 7.11 (d, J = 6.5 Hz, 2H, Ar H), 7.03 (d, J = 6.1 Hz, 2H, Ar H), 6.23 (s, 1H, Ar H), 5.39

(t, J = 7.3 Hz, 1H, O H ), 4.29 (q, J = 7.2 Hz, 2H, C H2 ), 3.87 (t, J

= 7.0 Hz, 2H, C H2 ), 3.69 (q, J = 5.2 Hz, 2H, C H2 ), 3.17 (t, J =

5.1 Hz, 3H, C H3 ). 13 C-NMR δC (DMSO-d6 75 MHz) 166.2 (C=O), 161.1, 158.3, 152.3, 147.6, 142.0, 130.6, 129.3, 127.0, 126.3, 124.0, 123.3, 122.0, 118.0, 116.0, 114.5, 58.6, 49.0, 38.6. MS EI (m/z), 414 (100%), 221 (53), 194 (41).

CHN Anal. Calcd for C 25 H23 N3 O3 (413.3): C, 72.72; H, 5.27; N, 10.23; O, 11.49 %. Found: C, 72.17; H, 5.13; N, 9.79; O, 11.66 %.

2.5.34 (Dye 49) Yield 84.2%. MP 305-307 ºC. -1 IR νmax (KBr/ cm ), 1681 (C=O), 1613 (C=C). and 1557 (N=N). 1 H-NMR δΗ (DMSO-d6, 300 MHz) 7.82-7.12 (m, 4H, Ar H), 7.38 (d, J = 7.3 Hz, Ar H), 7.30 (d, J = 7.1 Hz, 2H, Ar H), 7.23 (s, 1H, Ar H), 7.11 (d, J = 5.7 Hz, 2H, Ar H), 7.23 (d, J = 5.1 Hz, 2H, Ar H), 4.79 (t, J

= 5.2 Hz, 1H, O H ), 3.89 (q, J = 5.3 Hz, 2H, C H2 ), 3.67 (t, J = 7.2

Hz, 2H, C H2 ), 3.43 (t, J = 5.7 Hz, 2H, C H2 ), 3.37 (t, J = 5.3 Hz,

2H, C H2 ). 13 C-NMR δC (DMSO-d6 75 MHz) 167.2 (C=O), 159.1, 158.3, 151.3, 147.6, 142.6, 130.6, 129.3, 127.0, 126.3, 124.0, 123.3, 120.6, 118.0, 114.3, 56.6, 46.3, 38.6. MS EI (m/z), 439 (100%) 217 (48), 221 (36).

CHN Anal. Calcd for C 20 H21 N3 O3 (438.2): C, 71.42; H, 5.17; N, 12.73; O, 10.57 %. Found: C, 70.17; H, 5.26; N, 11.21; O, 10.16 %.

2.5.35 (Dye 50). Yield 81.0%. MP 278-280 ºC. -1 IR νmax (KBr/ cm ), 1679 (C=O), 1623 (C=C). and 1520 (N=N). 1 H-NMR δΗ (DMSO-d6, 300 MHz) 7.92-7.32 (m, 4H, Ar H), 7.28 (d, J = 7.3 Hz, 2H, Ar H), 7.13 (d, J = 7.1 Hz, 2H, Ar H), 6.81 (d, J = 5.4 Hz, 2H, Ar H), 6.23 (, J = 5.1 Hz, 2H, Ar H), 6.13 (s, 1H, Ar H), 3.39 (s,

6H, 2C H3). 13 C-NMR δC (DMSO-d6 75 MHz) 166.2 (C=O), 159.1, 158.3, 152.3, 147.6, 143.0, 130.6, 129.3, 127.0, 126.3, 124.0, 123.3, 120.6, 118.0, 114.6, 42.3. MS EI (m/z), 370 (100%), 219 (52), 148 (28).

CHN Anal. Calcd for C 23 H19 N3 O3 (369.3): C, 74.17; H, 5.17; N, 11.68; O, 8.68 %. Found: C, 74.17; H, 4.96; N, 11.21; O, 8.91 %.

2.5.36 (Dye 51) Yield 83.0%. MP 309-311 ºC. -1 IR νmax (KBr/ cm ), 1669 (C=O) and 1619 (C=C), and 1525 (N=N). 1 H-NMR δΗ (DMSO-d6, 300 MHz) 8.07 (s, 1H, NH ), 7.92-7.42 (m, 4H, Ar H), 7.38 (d, J = 7.3 Hz, 2H, Ar H), 7.30 (d, J = 7.1 Hz, 2H, Ar H), 7.11 (d, J = 5.3 Hz, 2H, Ar H), 7.03 (d, J = 5.1 Hz, 2H, Ar H), 6.23 (s, 1H, Ar H), 4.69 (t, J = 7.3 Hz, 1H, O H), 3.89 (q, J =

7.0 Hz, 2H, C H2 ), 3.69 (t, J = 6.3 Hz, 2H, C H2 ). 13 C-NMR δC (DMSO-d6 75 MHz) 169.2 (C=O), 159.1, 158.3, 149.0, 147.6, 141.6, 130.6, 129.3, 127.0, 126.3, 124.0, 123.3, 122.6, 118.0, 113.3, 63.0, 48.3. MS EI (m/z), 386 (100%), 219 (47), 164 (37).

CHN Anal. Calcd for C 23 H19 N3 O3 (385.3): C, 70.89; H, 5.13; N, 10.23; O, 12.59 %. Found: C, 70.17; H, 4.98; N, 11.26; O, 12.59 %.

2.5.37 (Dye 52) Yield 86%. MP 278-280 ºC. -1 IR νmax (KBr/ cm ), 1689 (C=O), 1621 (C=C). and 1537 (N=N). 1 H-NMR δΗ (DMSO-d6, 300 MHz) 7.81-7.41 (m, 7H, Ar H), 7.37 (d, J = 7.3 Hz, 2H, Ar H), 7.29 (d, J = 7.1 Hz, 2H, Ar H), 6.23 (s, 1H, Ar H),

4.79 (t, J = 5.3 Hz, O H ), 3.88 (q, J = 5.1 Hz, C H2 ), 3.57 (t, J =

7.2 Hz, 2H, C H2 ), 3.39 (t, J = 7.1 Hz, 2H, C H2 ), 2.47 (t, J = 5.1

Hz, 3H, C H3 ). 13 C-NMR δC (DMSO-d6 75 MHz) 169.2 (C=O), 158.1, 157.3, 151.3, 147.6, 143.0, 133.0, 131.6, 127.3, 126.0, 124.0, 123.5, 118.0, 114.5, 111.6, 58.6, 44.6, 31.6, 23.0. MS EI (m/z), 428 (100%), 219 (48), 207 (38).

CHN Anal. Calcd for C 26 H25 N3 O3 (427.3): C, 73.65; H, 5.99; N, 9.23; O, 11.39 %. Found: C, 73.67; H, 5.89; N, 9.43; O, 11.59 %.

2.5.38 (Dye 53) Yield 86.5%. MP 306 ºC. -1 IR νmax (KBr/ cm ), 3354 ( NH ), 1677 (C=O), 1623 (C=C), and 1528 (N=N). 1 H-NMR δΗ (DMSO-d6, 300 MHz) 8.23 (s, 1H, N H), 7.78-7.41 (m, 7H, Ar H), 7.33 (d, J = 7.3 Hz, 2H, Ar H), 7.27 (d, J = 7.1 Hz, 2H,

Ar H), 6.97 (s, 1H, Ar H), 3.87 (s, 3H, C H3), 3.39 (q, J = 5.3 Hz,

4H, 2C H2), 2.89 (t, J = 5.1 Hz, 6H, 2C H3 ). 13 C-NMR δC (DMSO-d6 75 MHz) 169.2 (C=O), 157.1, 156.3, 149.6, 131.6, 129.0, 127.3, 126.3, 124.0, 123.3, 118.0, 110.2, 107.1, 46.3, 32.7, 24.7. MS EI (m/z), 454 (100%), 234 (51), 219 (36).

CHN Anal. Calcd for C 27 H26 N4 O3 (453.4): C, 71.10; H, 5.87; N, 12.23; O, 10.19 %. Found: C, 71.27; H, 5.88; N, 11.86; O, 10.60 %.

2.5.39 (Dye 54) Yield 66.6 %. MP 209-211 ºC. -1 IR νmax (KBr/ cm ), 1679 (C=O), 1610 (C=C), and 1549 (N=N). 1 H-NMR δΗ (DMSO-d6, 300 MHz) 7.73-7.43 (m, 4H, Ar H), 7.31 (d, J = 7.3 Hz, 2H, Ar H), 7.23 (d, J = 7.1 Hz, 2H, Ar H), 7.31 (d, J = 7.3 Hz, 2H, Ar H), 7.23 (d, J = 7.0 Hz, 2H, Ar H), 6.97 (s, 1H, Ar H), 3.49

(q, J = 5.3 Hz, 4H, 2C H2), 3.39 (t, J = 5.1 Hz, 6H, 2C H3 ). 13 C-NMR δC (DMSO-d6 75 MHz) 166.2 (C=O), 159.1, 158.3, 147.6, 143.6, 130.6, 129.3, 127.0, 126.3, 124.0, 123.3, 120.1, 118.0, 114.5, 46.7, 23.9. MS EI (m/z), 398 (100%), 220 (53), 177 (46).

CHN Anal. Calcd for C 25 H23 N3 O2 (397.3): C, 75.33; H, 5.89; N, 10.23; O, 8.53 %. Found: C, 76.11; H, 5.63; N, 10.59; O, 8.57 %.

2.5.40 (Dye 55) Yield 89.7%. MP 217-219 ºC. -1 IR νmax (KBr/ cm ), 1671 (C=O), 1613 (C=C), and 1528 (N=N). 1 H-NMR δΗ (DMSO-d6, 300 MHz) 7.80-7.43 (m, 4H, Ar H), 7.38 (d, J = 7.3 Hz, 2H, Ar H), 7.30 (d, J = 7.1 Hz, 2H, Ar H), 7.11 (d, J = 5.3 Hz, 2H, Ar H), 7.23 (d, J = 5.1 Hz, 2H, Ar H), 6.23 (s, 1H, Ar H), 3.79

(t, J = 7.3 Hz, 2H, C H2 ), 3.57 (q, J = 7.1 Hz, 2H, C H2 ), 3.29 (t, J

= 5.4 Hz, 2H, C H2 ),3.19 (t, J = 5.1 Hz, 3H, C H3 ). 13 C-NMR δC (DMSO-d6 75 MHz) 167.3 (C=O), 159.6, 157.3, 151.5, 147.0, 142.0, 130.3, 129.3, 126.5, 124.3, 123.0, 120.3, 118.6, 114.6, 46.7, 33.6. MS EI (m/z), 423 (100%), 218 (55), 201 (43).

CHN Anal. Calcd for C 26 H22 N4 O2 (422.3): C, 73.10; H, 5.33; N, 13.23; O, 7.59 %. Found: C, 72.17; H, 5.09; N, 12.86; O, 7.45 %.

2.5.41 (Dye 56) Yield 77.7%. MP 211 ºC. -1 IR νmax (KBr/ cm ), 1679 (C=O), 1617 (C=C), and 1523 (N=N). 1 H-NMR δΗ (DMSO-d6, 300 MHz) 8.03 (s, 2H, 2OH), 7.93-7.35 (m, 7H, Ar H), 7.29 (d, J = 7.4 Hz, 2H, Ar H), 7.16 (s, 1H, Ar H), 7.07 (d, J = 7.1 Hz, 2H, Ar H), 7.29 (d, J = 5.3 Hz, 2H, Ar H). 13 C-NMR δC (DMSO-d6 75 MHz) 163.3 (C=O), 155.6, 153.3, 151.0, 149.3, 148.6, 146.3, 142.3, 140.3, 128.6, 123.6, 122.3, 121.0, 120.6, 117.6, 113.3, 53.3, 49.3, 34.6, 24.6. MS EI (m/z), 408 (100%),

CHN Anal. Calcd for C 25 H16 N2 O4 (360.4): C, 73.10; H, 3.43; N, 6.23; O, 15.59 %. Found: C, 73.17; H, 3.39; N, 6.36; O, 15.55 %.

2.5.42 (Dye 57) Yield 84 %. MP 246-248 ºC. -1 IR νmax (KBr/ cm ), 1677 (C=O), 1624 (C=C), and 1521 (N=N). 1 H-NMR δΗ (DMSO-d6, 300 MHz) 10.03 (s, 1H, O H), 7.97-7.31 (m, 9H, Ar H), 7.27 (d, J = 7.1 Hz, 2H, Ar H), 7.19 (s, 1H, Ar H), 7.08 (d, J = 5.3 Hz, 2H, Ar H), 6.77 (s, 1H, Ar H). 13 C-NMR δC (DMSO-d6 75 MHz) 168.3 (C=O), 159.6, 157.3, 151.6, 147.0, 135.3, 130.3, 129.3, 128.3, 126.5, 124.3, 120.3, 118.6, 116.5, 106.5, MS EI (m/z), 492 (100%), 261 (58), 252 (34).

CHN Anal. Calcd for C 25 H15 N2 O7 S (493.3): C, 58.43; H, 3.53; N, 5.23; O, 21.59; S, 6.23 %. Found: C, 58.17; H, 3.69; N, 5.23; O, 21.36; S, 6.24 %.

2.5.43 (Dye 58) Yield 87.5%. MP 267-269 ºC. -1 IR νmax (KBr/ cm ), 1669 (C=O), 1624 (C=C), and 1526 (N=N). 1 H-NMR δΗ (DMSO-d6, 300 MHz) 10.01 (s, 1H, O H), 7.91-7.36 (m, 7H, Ar H), 7.28 (d, J = 7.1 Hz, 2H, Ar H), 7.17 (s, 1H, Ar H), 7.07 (d, J

= 5.3 Hz, 2H, Ar H), 6.76 (bs, 2H, NH 2). 13 C-NMR δC (DMSO-d6 75 MHz) 169.3 (C=O), 155.1, 153.3, 151.6, 150.6, 149.3, 135.1, 134.0, 130.6, 128.1, 127.3, 126.3, 122.3, 121.6, 120.6, 118.3, 109.6, 24.0. MS EI (m/z), 627 (100%), 361 (56), 236 (33).

CHN Anal. Calcd for C 25 H14 N2 Na 2 O 11 S2 (626.3): C, 47.60; H, 2.13; N, 4.23; O, 28.59; S 10.44%. Found: C, 47.17; H, 3.09; N, 4.26; O, 28.09; S, 10.37 %.

2.5.44 (Dye 59) Yield 71.5%. MP 245-247 ºC. -1 IR νmax (KBr/ cm ), 1676 (C=O), 1610 (C=C). and 1545 (N=N). 1 H-NMR δΗ (DMSO-d6, 300 MHz) 10.01 (s, 1H, 2O H), 7.93-7.34 (m, 7H, Ar H), 7.27 (d, J = 7.1 Hz, 2H, Ar H), 7.11 (s, 1H, Ar H), 7.03 (d, J = 5.3 Hz, 2H, Ar H). 13 C-NMR δC (DMSO-d6 75 MHz) 167.0 (C=O), 158.6, 157.2, 152.3, 153.6, 148.1, 135.6, 130.6, 129.1, 128.3, 126.0, 123.6, 122.3, 119.1, 117.5, 109.6. MS EI (m/z), 612 (100%), 316 (35), 211 (28).

CHN Anal. Calcd for C 20 H12 N2 O9 S2 (613.4): C, 49.43; H, 2.33; N, 6.23; O, 23.49 S 12.01 %. Found: C, 49.17; H, 2.29; N, 6.17; O, 23.51 S 12.06 %.

2.5.45 (Dye 60) Yield 84.2%. MP 305 ºC. -1 IR νmax (KBr/ cm ), 1679 (C=O), 1619 (C=C), and 1529 (N=N). 1 H-NMR δΗ (DMSO-d6, 300 MHz) 8.03 (s, 1H, O H), 7.83-7.35 (m, 8H, Ar H), 7.29 (d, J = 7.3 Hz, 2H, Ar H), 7.07 (d, J = 7.1 Hz, 2H,

Ar H), 7.16 (s, 1H, Ar H), 5.88(bs, 2H, NH 2). 13 C-NMR δC (DMSO-d6 75 MHz) 167.3 (C=O), 157.6, 157.2, 152.6, 149.3, 135.3, 133.3, 130.6, 129.1, 128.3, 127.3, 126.0, 123.6, 122.3, 121.6, 119.1, 117.5, 113.3, MS EI (m/z), 610 (100%), 308 (47), 302 (34).

CHN Anal. Calcd for C 20 H12 N2 Na 2 O 10 (611.3): C, 61.63; H, 3.63; N, 8.23; O, 19.59 S 6.77 %. Found: C, 61.17; H, 3.69; N, 8.33; O, 19.36; S, 6.89 %.

2.5.46 (Dye 61) Yield 72.2 %. MP 281-283 ºC. -1 IR νmax (KBr/ cm ), 3359 ( NH ), 1697 (C=O) and 1605 (C=C). 1 H-NMR δΗ (DMSO-d6, 300 MHz) 7.92 (s, 1H, NH ) 7.74-7.37 (m, 5H, Ar H),

6.21 (s, 1H, Ar H), 3.73 (s, 3H, -OCH 3), 3.59 (q, J = 7.1 Hz. 4H,

2C H2), 3.21 (s, 3H, C H3), 3.07 (t, J = 5.3 Hz, 6H, 2C H3), 2.23 (s,

3H, C H3). 13 C-NMR δC (DMSO-d6 75 MHz) 162.3 (C=O), 155.6, 153.0, 148.6, 128.6, 122.3, 121.2, 120.6, 24.6, 121.3, 120.3, 109.6, 106.6, 56.0, 48.6, 24.3, 17.6, 13.3. MS EI (m/z), 422 (100%), 280 (46), 143 (22).

CHN Anal. Calcd for C 23 H20 N4 O4 (421.2): C, 65.10; H, 6.17; N, 13.21; O, 15.17 %. Found: C, 64.87; H, 5.89; N, 6.31; O, 14.95 %.

2.5.47 (Dye 62) Yield 63.6 %. MP 238-240 ºC. -1 IR νmax (KBr/ cm ), 1675 (C=O) and 1611 (C=C). 1 H-NMR δΗ (DMSO-d6, 300 MHz) 7.73 (d, J = 5.3 Hz, 2H, Ar H), 7.33 (d, J = 7.3 Hz, 2H, Ar H), 7.24-6.37 (m, 3H, Ar H), 6.21 (s, 1H, Ar H),

3.56 (q, J = 5.3 Hz, 4H, 2C H2), 3.33 (t, J = 7.1 Hz, 4H, 2C H2),

2.37 (t, J = 5.1 Hz, 2H, 2O H), 2.03 (s, 3H, C H3). 3 C-NMR δC (DMSO-d6 75 MHz) 162.3 (C=O), 155.6, 153.0, 148.6, 146.0, 142.3, 128.6, 122.3, 123.0, 121.2, 120.6, 113.3, 64.6, 53.3, 24.6 MS EI (m/z), 366 (100%), 223 (47), 144 (26).

CHN Anal. Calcd for C 20 H21 N3 O4 (367.3): C, 65.72; H, 5.47; N, 11.23; O, 17.49 %. Found: C, 65.17; H, 5.43; N, 11.21; O, 17.66 %.

2.5.48 (Dye 63) Yield 78.2%. MP 265-267 ºC. -1 IR νmax (KBr/ cm ), 1679 (C=O) and 1611 (C=C). 1 H-NMR δΗ (DMSO-d6, 300 MHz) 7.71 (d, J = 7.3 Hz, 2H, Ar H), 7.35 (d, J = 7.1 Hz, 2H, Ar H), 7.29-6.57 (m, 3H, Ar H), 6.23 (s, 1H, Ar H),

3.55 (d, J = 5.3 Hz, 2H, C H2), 3.31 (d, J = 5.1 Hz, 2H, C H2 ),

2.69 (s, 3H, C H3). 2.37 (t, J = 7.3 Hz, 1H, O H), 1.69 (t, J = 5.3

Hz, 3H, C H3). 3 C-NMR δC (DMSO-d6 75 MHz) 162.3 (C=O), 155.6, 153.0, 148.6, 146.0, 142.3, 128.6, 122.3, 123.0, 121.2, 120.6, 113.3, 64.3, 58.3, 48.6, 34.6, 31.3. MS EI (m/z), 350 (100%), 206 (52), 144 (44).

CHN Anal. Calcd for C 20 H21 N3 O3 (351.2): C, 68.42; H, 6.17; N, 11.73; O, 13.57 %. Found: C, 68.17; H, 6.26; N, 11.21; O, 13.16 %.

2.5.49 (Dye 64) Yield 81.6%. MP 218-220 ºC. -1 IR νmax (KBr/ cm ), 1677 (C=O) and 1610 (C=C). 1 H-NMR δΗ (DMSO-d6, 300 MHz) 7.61 (d, J = 7.8 Hz, 2H, Ar H), 7.29 (d, J = 7.1 Hz, 2H, Ar H), 7.21-6.57 (m, 3H, Ar H), 6.27 (s, 1H, Ar H),

3.75 (q, J = 5.3 Hz, 2H, C H2), 3.69 (t, J = 5.1 Hz, 2H, C H2 ), 2.69

(t, J = 7.3 Hz, 2H, C H2 ), 2.37 (t, J = 7.2 Hz, 1H, O H ),1.79 (s,

3H, C H3). 13 C-NMR δC (DMSO-d6 75 MHz) 162.3 (C=O), 155.6, 153.0, 148.6, 146.0, 142.3, 128.6, 122.3, 123.0, 121.2, 120.6, 117.3, 113.3, 66.3, 61.0, 53.4, 24.6, 17.6. MS EI (m/z), 374 (100%), 230 (50), 143 (36).

CHN Anal. Calcd for C 21 H20 N4 O3 (376.3): C, 67.17; H, 5.17; N, 14.68; O, 12.68 %. Found: C, 67.17; H, 4.96; N, 14.21; O, 12.91 %.

2.5.50 (Dye 65) Yield 73.8%. MP 218-220 ºC. -1 IR νmax (KBr/ cm ), 3359 ( NH ), 1679 (C=O) and 1614 (C=C). 1 H-NMR δΗ (DMSO-d6, 300 MHz) 7.71 (d, J = 7.1 Hz, 2H, Ar H), 7.27 (d, J = 7.1 Hz, 2H, Ar H), 7.21-6.53 (m, 3H, Ar H), 6.37 (s, 1H, Ar H),

3.71 (s, 6H, 2C H3), 1.69 (s, 3H, C H3). 13 C-NMR δC (DMSO-d6 75 MHz) 162.3 (C=O), 155.6, 153.0, 148.6, 146.0, 142.3, 128.6, 122.3, 123.0, 121.2, 120.6, 113.3, 44.6, 24.6. MS EI (m/z), 306 (100%), 164 (35), 143 (23).

CHN Anal. Calcd for C 18 H17 N3 O2 (307.3): C, 70.10; H, 5.43; N, 13.23; O, 10.59 %. Found: C, 70.17; H, 4.98; N, 13.26; O, 10.59 %.

2.5.51 (Dye 66) Yield 86.2%. MP 218-220 ºC. -1 IR νmax (KBr/ cm ), 3357 ( NH ), 1679 (C=O) and 1610 (C=C). 1 H-NMR δΗ (DMSO-d6, 300 MHz) 8.17 (s, 1H, N H), 7.61 (d, J = 7.4 Hz, 1H, Ar H), 7.29 (d, J = 7.0 Hz, 2H, Ar H), 7.21-6.57 (m, 3H, Ar H), 6.27

(s, 1H, Ar H), , 3.69 (q, J = 5.4 Hz, 2H, C H2 ), 2.69 (d, J = 5.1 Hz

2H, C H2 ), 2.37 (s, 1H, O H ), 1.69 (s, 3H, C H3). 13 C-NMR δC (DMSO-d6 75 MHz) 162.3 (C=O), 155.6, 153.0, 148.6, 146.0, 142.3, 128.6, 122.3, 123.0, 121.2, 120.6, 113.3, 54.6, 65.3, 24.6 MS EI (m/z), 322 (100%), 180 (61), 144 (26).

CHN Anal. Calcd for C 18 H17 N3 O3 (323.3): C, 66.65; H, 4.99; N, 13.23; O, 14.39 %. Found: C, 66.67; H, 5.09; N, 13.23; O, 14.59 %.

2.5.52 (Dye 67) Yield 76.2%. MP 266-268 ºC. -1 IR νmax (KBr/ cm ), 1672 (C=O) and 1613 (C=C). 1 H-NMR δΗ (DMSO-d6, 300 MHz) 7.89-7.43 (m, 7H, Ar H), 6.30 (s, 1H, Ar H

), 3.79 (q, J = 7.3 Hz, 2H, C H2), 3.61 (s, 3H, C H3) 3.51 (t, J = 7.1

Hz, 2H, C H2), 3.33 (q, J = 5.3 Hz, 2H, C H2), 3.13 (s, 3H, C H3)

2.66 (t, J = 5.4 Hz, 1H, O H), 2.33 (s, 3H, C H3). 13 C-NMR δC (DMSO-d6 75 MHz) 162.3 (C=O), 155.3, 150.3, 149.3, 146.3, 142.3, 132.3, 128.3, 122.3, 123.6, 121.3, 120.0, 114.6, 110.3, 64.3, 58.6, 49.6, 36.0, 24.6, 18.3. MS EI (m/z), 364 (100%), 220 (42), 143 (23).

CHN Anal. Calcd for C 21 H23 N3 O3 (365.4): C, 69.10; H, 6.87; N, 11.23; O, 10.19 %. Found: C, 68.17; H, 6.88; N, 11.06; O, 10.60 %.

2.5.53 (Dye 68) Yield 86.6 %. MP 219-220 ºC. -1 IR νmax (KBr/ cm ), 3354 ( NH ), 1675 (C=O) and 1610 (C=C). 1 H-NMR δΗ (DMSO-d6, 300 MHz) 8.03 (s, 1H, NH ), 7.72-7.35 (m, 6H,

Ar H), 6.27 (s, 1H, Ar H), 3.69 (q, J = 5.3 Hz, 4H, 2C H2), 3.69 (t, J

= 5.1 Hz, 6H, 2C H3), 3.27 (s, 3H, C H3), 2.71 (s, 3H, C H3), 1.98

(s, 3H, C H3). 13 C-NMR δC (DMSO-d6 75 MHz) 169.3 (C=O), 163.0, 155.6, 153.3, 151.0, 149.3, 148.6, 146.3, 142.3, 140.3, 138.0, 128.6, 125.0, 123.6, 122.3, 121.0, 120.6, 119.2, 115.6, 113.3, 24.6, 17.6. MS EI (m/z), 392 (100%), 249 (44), 144 (26).

CHN Anal. Calcd for C 22 H23 N4 O3 (391.3): C, 67.33; H, 6.63; N, 14.23; O, 12.53 %. Found: C, 66.41; H, 6.63; N, 14.19; O, 12.57 %.

2.5.54 (Dye 69) Yield 79.7%. MP 217-219 ºC. -1 IR νmax (KBr/ cm ), 1671 (C=O) and 1613 (C=C). 1 H-NMR δΗ (DMSO-d6, 300 MHz) 7.68 (d, J = 5.3 Hz, 2H, Ar H), 7.37 (d, J = 7.3 Hz, 1H, Ar H), 7.23-6.53 (m, 3H, Ar H), 6.47 (s, 1H, Ar H),

3.71 (q, J = 5.3 Hz, 4H, 2C H2), 3.17 (t, J = 5.1 Hz, 6H, 2C H3),

1.77 (s, 3H, C H3). 13 C-NMR δC (DMSO-d6 75 MHz) 163.3 (C=O), 155.6, 153.3, 151.0, 149.3, 148.6, 146.3, 142.3, 140.3, 128.6, 123.6, 122.3, 121.0, 120.6, 113.3, 49.3, 24.6, 18.6. MS EI (m/z), 334 (100%), 192 (34), 143 (25).

CHN Anal. Calcd for C 20 H21 N3 O2 (335.3): C, 71.10; H, 6.03; N, 12.23; O, 9.59 %. Found: C, 71.17; H, 6.09; N, 12.06; O, 9.45 %.

2.5.55 (Dye 70) Yield 61.7%. MP 219-220 ºC. -1 IR νmax (KBr/ cm ), 1673 (C=O) and 1613 (C=C). 1 H-NMR δΗ (DMSO-d6, 300 MHz) 7.78 (d, J = 5.4 Hz, 1H, Ar H), 7.47 (d, J = 7.1 Hz, 1H, Ar H), 7.33-6.53 (m, 3H, Ar H), 6.54 (s, 1H, Ar H),

3.71 (t, J = 5.6 Hz, 2H, C H2), 3.27 (s, 3H, C H3) 3.17 (q, J = 5.1

Hz, 2H, C H2), 3.17 (t, J = 5.2 Hz, 2H, C H2), 1.77 (s, 3H, C H3). 13 C-NMR δC (DMSO-d6 75 MHz) 163.3 (C=O), 155.6, 153.3, 151.0, 149.3, 148.6, 146.3, 142.3, 140.3, 128.6, 123.6, 122.3, 121.0, 120.6, 117. 113.3, 53.3, 49.3, 24.6, 14.6. MS EI (m/z), 360 (100%), 218 (42), 143 (26).

CHN Anal. Calcd for C 21 H20 N4 O2 (360.4): C, 69.10; H, 5.43; N, 15.23; O, 8.59 %. Found: C, 70.17; H, 5.39; N, 15.36; O, 8.55 %.

2.5.56 (Dye 71) Yield 84.4%. MP 248-250 ºC. -1 IR νmax (KBr/ cm ), 1676 (C=O) and 1614 (C=C). 1 H-NMR δΗ (DMSO-d6, 300 MHz) 8.03 (s, 2H, 2O H), 787-7.32 (m, 6H, Ar H), 7.47 (d, J = 7.1 Hz, 2H, Ar H), 6.33 (s, 1H, Ar H), 2.71 (s,

3H, C H 3 ). 13 C-NMR δC (DMSO-d6 75 MHz) 166.3 (C=O), 152.1, 151.3, 149.6, 140.6, 144.3, 132.2, 130.0, 126.6, 122.3, 121.3, 120.6, 119.6, 118.6, 117.0, 113.6, 112.0, 24.6. MS EI (m/z), 407 (100%), 263 (51), 143 (26).

CHN Anal. Calcd for C 25 H16 N2 O4 (408.3): C, 73.43; H, 3.63; N, 6.23; O, 15.59 %. Found: C, 73.17; H, 3.69; N, 6.23; O, 15.36 %.

2.5.57 (Dye 72) Yield 79.5%. MP 265-267 ºC. -1 IR νmax (KBr/ cm ), 1671 (C=O) and 1614 (C=C). 1 H-NMR δΗ (DMSO-d6, 300 MHz) 8.03 (s, 1H, O H), 787-7.32 (m, 8H, Ar H),

6.33 (s, 1H, Ar H), 2.71 (s, 3H, C H 3 ). 13 C-NMR δC (DMSO-d6 75 MHz) 161.3 (C=O), 155.1, 153.3, 151.6, 150.6, 149.3, 135.1, 134.0, 130.6, 128.1, 127.3, 126.3, 122.3, 121.6, 120.6, 118.3, 109.6, 24.0. MS EI (m/z), 432 (100%), 289 (37), 143 (21).

CHN Anal. Calcd for C 20 H13 N2 Na O 6 S (432.3): C, 55.60; H, 3.13; N, 6.23; O, 20.59; S 7.44%. Found: C, 55.17; H, 3.09; N, 6.26; O, 20.59; S, 6.37 %.

2.5.58 (Dye 73) Yield 77.5%. MP 275-277 ºC. -1 IR νmax (KBr/ cm ), 1676 (C=O) and 1610 (C=C). 1 H-NMR δΗ (DMSO-d6, 300 MHz) 8.53 (s, 2H, O H), 7.87-7.32 (m, 8H,

Ar H), 5 .46 (bs, 2H, NH 2), 6.31 (s, 1H, Ar H), 2.87 (s, 3H, C H 3 ). 13 C-NMR δC (DMSO-d6 75 MHz) 160.3 (C=O), 153.1, 152.3, 149.4, 148.6, 136.1, 135.6, 129.0, 128.6, 126.1, 125.3, 122.3, 121.3, 120.6, 119.3, 117.1, 109.6, 21.3. MS EI (m/z), 532 (53), 390 (54), 142 (38).

CHN Anal. Calcd for C 20 H12 N2 O9 S2 (534.4): C, 49.43; H, 2.33; N, 6.23; O, 23.49 S 12.01 %. Found: C, 49.17; H, 2.29; N, 6.17; O, 23.51 S 12.06 %.

2.5.59 (Dye 74) Yield 74.2%. MP 250-252 ºC. -1 IR νmax (KBr/ cm ), 1674 (C=O) and 1611 (C=C). 1 H-NMR δΗ (DMSO-d6, 300 MHz) 8.53 (s, 2H, 2O H), 7.87-7.32 (m, 6H,

Ar H), 6.31 (s, 1H, Ar H), 2.87 (s, 3H, C H 3 ). 13 C-NMR δC (DMSO-d6 75 MHz) (C=O), 152.1, 151.3, 149.6, 144.6, 135.3, 134.1, 131.0, 130.6, 128.1, 127.3, 122.3, 121.3, 120.3, 119.6, 112.1, 103.6, 21.0. MS EI (m/z), 554 (100%), 413 (46), 144 (24).

CHN Anal. Calcd for C 20 H12 N2 Na 2 O 10 (556.3): C, 43.63; H, 2.63; N, 5.23; O, 29.59 S 11.77 %. Found: C, 73.17; H, 3.69; N, 6.23; O, 15.36 S 10.89 %.

2.5.60 (Dye 75) Yield 79.3%. MP 263-265 ºC. -1 IR νmax (KBr/ cm ) 3351 ( NH ), 1673 (C=O) and 1611 (C=C). 1 H-NMR δΗ (DMSO-d6, 300 MHz) 8.33 (s, 2H, O H), 7.97-7.33 (m, 6H,

Ar H), 6.33 (s, 1H, Ar H), 5.87 (bs, 2H, NH 2), 2.77 (s, 3H, C H 3 ). 13 C-NMR δC (DMSO-d6 75 MHz) 169.3 (C=O), 152.1, 150.5, 144.8, 134.6, 133.3, 130.1, 129.6, 123.3, 122.1, 121.4, 119.0, 118.3, 114.1, 21.6. MS EI (m/z), 446 (100%), 304 (51), 143 (27).

CHN Anal. Calcd for C 20 H14 N3 Na O 6 S (447.3): C, 53.64; H, 3.15; N, 9.21; O, 21.59; S, 7.13 %. Found: C, 53.67; H, 3.15; N, 9.23; O, 20.36; S, 6.99 %.

2.5.61 (Dye 76) Yield 72.0%. MP 142-143 ºC. -1 IR νmax (KBr/ cm ), 1698 (C=O) and 1591 (C=C), 1542 (N=N). 1 H-NMR δΗ (DMSO-d6, 300 MHz) 9.71 (s, 1H, O H), 7.43-6.31 (m, 5H,

Ar H), 3.77 (s, 3H, C H3). 13 C-NMR δC (DMSO-d6 75 MHz) 153.3 (C=O), 151.6, 143.3, 141.0, 127.3, 121.6, 119.0, 106.3, 97.3, 68.3, 21.3. MS EI (m/z), 287 (100%), 174 (52), 113 (28).

CHN Anal. Calcd for C 13 H11 N3 O3 S (289): C, 53.98; H, 3.83; N, 14.48; O, 16.58%. Found: C, 53.86; H, 3.81; N, 14.52; O, 16.51%.

2.5.62 (Dye 77) Yield 71.3%. MP 125-127 ºC. -1 IR νmax (KBr/ cm ), 1688 (C=O) and 1665 (C=C), 1552 (N=N). 1 H-NMR δΗ (DMSO-d6, 300 MHz) 8.73 (s, 1H, O H), 783-7.31 (m, 7H, Ar H),

2.91 (s, 3H, C H 3 ). 13 C-NMR δC (DMSO-d6 75 MHz) 161.3 (C=O), 153.1, 151.0, 148.6, 130.6, 123.3, 120.6, 112.3, 92.0, 24.6. MS EI (m/z), 338 (100%), 177 (57), 163 (36).

CHN Anal. Calcd for C 17 H13 N3 O3 S (339.3): C, 60.16; H, 3.86; N, 12.39; O, 14.15%. Found: C, 60.09; H, 3.78; N, 12.32; O, 14.09%.

2.5.63 (Dye 78) Yield 67.3%. MP 234-235 ºC. -1 IR νmax (KBr/ cm ), 1691 (C=O) and 1599 (C=C), 1532 (N=N). 1 H-NMR δΗ (DMSO-d6, 300 MHz) 8.73 (s, 1H, O H), 747-7.21 (m, 4H, Ar H),

2.82 (s, 3H, C H 3 ). 13 C-NMR δC (DMSO-d6 75 MHz) 163.3 (C=O), 152.1, 147.3, 133.6, 122.6, 119.3, 95.1, 84.0, 60.6, 58.1, 47.3, 44.6, 24.0. MS EI (m/z), 301 (100%), 173 (35), 123 (23).

CHN Anal. Calcd for C 14 H13 N3 O3 (303.3): C, 55.42; H, 4.34; N, 13.84; O, 15.86; S 10.51%. Found: C, 55.41; H, 4.31; N, 13.82; O, 15.78; S, 10.47 %.

2.5.64 (Dye 79) Yield 73.5%. MP 138 ºC. -1 IR νmax (KBr/ cm ), 1698 (C=O) and 1601 (C=C), 1532 (N=N). 1 H-NMR δΗ (DMSO-d6, 300 MHz) 8.51 (s, 2H, O H), 7.67-7.22 (m, 3H, Ar H), 7 06 (d, J = 7.3 Hz, 2H, Ar H), 6 86 (d, J = 7.1 Hz, 2H,

Ar H), 2.77 (s, 3H, C H 3 ). 13 C-NMR δC (DMSO-d6 75 MHz) 161.3 (C=O), 153.3, 142.3, 138.4, 121.6, 116.1, 103.3, 95.6, 69.0, 48.6, 21.7. MS EI (m/z), 368 (100%), 190 (58), 167 (39).

CHN Anal. Calcd for C 18 H15 N3 O4 S (369.3): C, 58.52; H, 4.09; N, 11.38; O, 17.33 S 8.68 %. Found: C, 58.48; H, 4.06; N, 11.29; O, 17.28 S 8.61 %.

2.5.65 (Dye 80) Yield 714.2%. MP 153-154 ºC. -1 IR νmax (KBr/ cm ), 1698 (C=O) and 1593 (C=C), 1572 (N=N). 1 H-NMR δΗ (DMSO-d6, 300 MHz) 8.63 (s, 1H, O H), 7.77-7.31 (m, 6H,

Ar H), 2.85 (s, 3H, C H 3 ). 13 C-NMR δC (DMSO-d6 75 MHz) 161.0 (C=O), 142.1, 131.3, 124.6, 114.6, 102.3, 84.1, 61.0, 50.3, 48.1, 37.3, 21.0. MS EI (m/z), 383 (100%), 173 (66), 107 (23).

CHN Anal. Calcd for C 17 H12 N4 O5 S (384.3): C, 53.12; H, 3.15; N, 14.57; O, 20.80; S, 8.34 %. Found: C, 53.09; H, 3.11; N, 14.52; O, 20.79 S 8.28 %.

2.5.66 (Dye 81) Yield 76.3%. MP 211-213 ºC. -1 IR νmax (KBr/ cm ) 1673 (C=O) and 1613 (C=C), 1552 (N=N). 1 H-NMR δΗ (DMSO-d6, 300 MHz) 8.43 (s, 1H, O H), 7.57-7.23 (m, 6H,

Ar H), 3.47 (s, 3H, C H3). 13 C-NMR δC (DMSO-d6 75 MHz) 163.3 (C=O), 150.1, 147.5, 134.5, 123.6, 113.3, 100.1, 89.6, 63.3, 52.1, 31.4, 29.0. MS EI (m/z), 372 (100%), 191 (35), 97 (17).

CHN Anal. Calcd for C 17 H12 N3 O3 S (373.8): C, 54.62; H, 3.24; N, 11.24; O, 12.84; S, 8.58 %. Found: C, 54.55; H, 3.18; N, 11.18; O, 12.77; S, 8.49 %.

2.5.67 (Dye 82) Yield 74.2%. MP 167-169 ºC. -1 IR νmax (KBr/ cm ), 1691 (C=O) and 1593 (C=C), 1533 (N=N). 1 H-NMR δΗ (DMSO-d6, 300 MHz) 8.37 (s, 1H, O H), 7.23-6.67 (m, 3H,

Ar H), 3.69 (s, 3H, C H3) 13 C-NMR δC (DMSO-d6 75 MHz) 161.1 (C=O), 145.6, 133.0, 128.3, 116.3, 102.3, 88.6, 67.3, 53.3, 44.6, 35.3, 24.6 MS EI (m/z), 286 (100%), 175 (65), 111 (16).

CHN Anal. Calcd for C 14 H13 N3 O4 (287.2): C, 58.52; H, 4.55; N, 14.62; O, 22.28 %. Found: C, 58.50; H, 4.47; N, 14.59; O, 22.21 %.

2.5.68 (Dye 83) Yield 69.2%. MP 171 ºC. -1 IR νmax (KBr/ cm ), 1697 (C=O) and 1585 (C=C), 1532 (N=N). 1 H-NMR δΗ (DMSO-d6, 300 MHz) 9.56 (s, 1H, O H), 7.89-7.31 (m, 3H,

Ar H), 3.73 (s, 3H, C H3). 13 C-NMR δC (DMSO-d6 75 MHz) 163.3 (C=O), 151.0, 147.3, 138.6, 120.6, 119.2, 115.6, 113.3, 24.6, 17.6. MS EI (m/z), 289 (100%), 175 (52), 113 (33).

CHN Anal. Calcd for C 12 H10 N4 O3 S (290.2): C, 49.65; H, 3.47; N, 19.31; O, 16.52; S, 11.05%. Found: C, 49.61; H, 3.44; N, 19.28; O, 16.48; S, 10.98%.

2.5.69 (Dye 84) Yield 77.0 %. MP 232-233 ºC. -1 IR νmax (KBr/ cm ), 1698 (C=O) and 1583 (C=C), 1533 (N=N). 1 H-NMR δΗ (DMSO-d6, 300 MHz) 8.66 (s, 1H, O H), 7.87 (s, 1H, NH ), 7.49-

6.43 (m, 3H, Ar H), 3.33 (s, 3H, C H3). 13 C-NMR δC (DMSO-d6 75 MHz) 160.3 (C=O), 148.3, 130.3, 129.0, 100.3, 74.3, 57.6, 46.6, 34.0, 28.6, 17.3. MS EI (m/z), 272 (100%), 249 (44), 144 (26).

CHN Anal. Calcd for C 12 H11 N5 O3 (273.3): C, 52.75; H, 4.06; N, 25.63; O, 17.57 %. Found: C, 52.66; H, 4.01; N, 25.60; O, 17.57 %.

2.5.70 (Dye 85) Yield 74.7%. MP 128 ºC. -1 IR νmax (KBr/ cm ), 1695 (C=O) and 1585 (C=C), 1531 (N=N). 1 H-NMR δΗ (DMSO-d6, 300 MHz) 8.91 (s, 1H, O H), 7.71 (d, J = 5.3 Hz, 2H, Ar H), 7.31 (d, J = 7.3 Hz, 2H, Ar H), 7.21-6.83 (m, 3H, Ar H),

3.77 (s, 3H, C H3). 13 C-NMR δC (DMSO-d6 75 MHz) 162.3 (C=O), 153.6, 151.0, 147.3, 141.3, 139.3, 130.3, 123.6, 122.3, 121.0, 113.3, 49.3, 24.6, 18.6. MS EI (m/z), 321 (100%), 175 (56), 147 (19).

CHN Anal. Calcd for C 17 H14 N4 O3 (322.3): C, 63.35; H, 4.38; N, 17.38; O, 14.89 %. Found: C, 63.22; H, 4.33; N, 17.29; O, 14.77 %.

2.6 Dyeing of Polyester 2.6.1 Stock dispersion solution preparation The dye is first ground into a fine powder using mortar and pestle. The mixture of dye (1.0 g), dispersing agent like Setamol WS (1.0 g), water (100 mL) and ceramic balls (250 g) were ball milled overnight to prepare the stock dispersion solution of the dyes. For calculating the amount of stock dispersion required for different percentages of shades (Table-3) the following formula was used,

Amount of stock solution required = % of shade x fabric weight conc. of stock Solution Table-3 Calculations for the different depth of shades % of shade Amount of stock Amount of Amount of water dispersion dispersing agent required in mL (1% stock (mL) dispersion) 0.1 5 20 75 0.5 25 20 55 1.0 50 20 30 2.0 100 20 ---

Weight of fabric = 5.0 Liquor ratio = 20: 1

2.6.2 Dyeing Procedure The dyeing was carried on the laser heated dyeing machine and the dyeing machine was initially heated up to 60 0C. The dyeing was started after the addition of the mixture of following: Stock dispersion solution = 25 mL Metaxil solution (1g/ 100 mL water) = 20 mL Deionised water = 55 mL The pH of the mixture is adjusted from 4.5 – 5.5 by the addition of acetic acid. The temperature is raised to 130 0C after keeping the temperature

of the machine for 20 minutes. The heating rate is kept at 2 /min. and it is kept at 130 0C for 60 minutes. It is then cooled down to 50 0C afterwards and the bath is then drained off. To remove any unfixed dye on the surface of the fabric it is reduction cleared at 70 0C for 20 minutes. For reduction clearing the solution of Sodium hydroxide (2.0 g/L) and Sodium dithionite solution (2.0 g/L) is used. The dyed samples are then dried in vacuum oven after calendaring they are ready for the fastness tests.

0 130 C 1 hour

0 2 C/ min 0 2 C/ min

0 20 minutes 60 C

0 50 C

Figure-3. Dyeing procedure of polyester fabric with Dyes (16-75).

2.7 Fastness Testing 2.7.1 Colour fastness to sublimation 165 The purpose of the colour fastness to sublimation is to determine the resistance of colour of the textile products to the action of dry heat. In this test the specimen of the textile is heated to a required temperature keeping it in close contact with a specified adjacent fabric. The electrically heated plates allow the fabric to be set in a flat position under a pressure of 4 ±1 kPa at a uniform temperature. The fabric to be tested along with the adjacent fabrics (cotton and polyester) are placed in the heating device and left for 30 seconds at 180 ± 2 0C. The fabric to be tested is taken out and left to dry for 4 hours in air before assessing the change in the colour by comparing it with the two sets of gray scales. The two sets of gray scales are for assessing the change in colour and the other one is for assessing the staining.

2.7.2 Colour fastness to Perspiration (alkaline and acidic) 166 The purpose of the colour fastness to perspiration is to determine the resistance of the colour of the textiles to the action of human perspiration. The textile specimen is kept in contact with the multi fiber strip and treated with two different solutions (acidic and alkaline) containing histidine. Both the textile specimen to be tested and the multi fiber strip is dipped in the solution and then placed between the two placed under a specified pressure in a testing device called as the perspirometer or the perspiration tester. The perspirometer is made up of a frame of stainless steel and the base of dimensions 60 mm x 115 mm is closely fitted so that the specimen which is kept inside faces the pressure of 12.5 kPa. The textile specimen size is 40 mm x 100 mm. The construction of the perspirometer is such that when the weight piece is removed during the test the pressure of 12.5 kPa remains unchanged. The specimen to be tested along with the multi fiber strip is kept in an oven maintained at the temperature of 37 0C+ 2 0C for 4 hours The alkaline and the acidic solutions contain the following,

Alkaline solution L-histidine monohydrochloride monohydrate = 0.5 g/L Sodium chloride = 5 g Disodium hydrogen orthophosphate dihydrate = 2.5 g The pH of the solution is adjusted at 8 with 0.1 mol/L sodium hydroxide solution

Acidic solution L-histidine monohydrochloride monohydrate = 0.5 g/L Sodium chloride = 5 g Sodium dihydrogen orthophosphate dehydrate = 2.5 g The pH of the solution is adjusted at 5.5 with 0.1 mol/L sodium hydroxide solution Both the specimen to be tested and the multi fiber is removed from the oven after 4 hours and dried again in an oven where the temperature doesn’t exceed 60 0C. Assessment is done by using the two sets of gray scale.

2.7.3 Colour fastness to washing 167 This test is done to assess the fastness to washing of the coloured textile specimen which is kept in contact with the multi fiber strip. The textile specimen is mechanically agitated under the specified conditions of time and temperature in a standard soap solution and then it is rinsed and dried. 5 g of soap and 2 g of anhydrous sodium carbonate / litre of water were used to prepare the soap solution. The solution is heated so as to enable the soap to be soluble in it and cooled down before sodium carbonate was added. The standard soap solution contains the following components: a) Free alkali calculated as Na 2CO 3 = 0.3 % maximum. b) Free alkali calculated as NaOH = 0.1 % maximum. c) Moisture content = less than 5%. d) Totally fatty matter = 850 g/kg minimum. e) Iodine value = 50 maximum. f) Florescent brightening agent = 0 %. The standard size of the specimen is 40 mm x 100 mm and the size of the adjacent multi fiber size is 40 mm x 100 mm. The specimen is placed in the container and the soap solution is poured into it in the ratio of 50: 1. The specimen is treated at 60 ±20C for 30 minutes. The wash fastness machine is preheated at 60 0C. At the end of the test the test specimen is removed and the test specimen is removed from the multi fiber and it is rinsed thoroughly to the remove the residual soap solution and it is then dried in the vacuum oven. The change of the colour and the staining extent is then assessed with the help of the gray scale.

2.7.4 Colour fastness to light 168 The colour fastness is assessed by testing the textile specimen by exposing it to the artificial Xenon light. Blue wool reference standard is set up along with the specimen. The standard blue wool reference is identified by the numerical numbers from 1 to 8, where 1 shows poor light fastness and 8 indicates very high fastness. There are five methods described in the British Standard 190 ranging from 1 to 5. Among these five methods method No 1 is considered to be most exact one and it is mostly preferred where the dispute

arises over the numerical rating. So in order to assess the light fastness the same method was followed. One third of the he specimen and the reference were covered. The samples were placed in the Xenon light testing machine and exposed to the xenon arc light. It was inspected periodically by removing the cover and test was continued until the exposed and the unexposed portion of the specimen was equal to the gray scale grade 3. During the testing if the reference 7 fades equal to the gray scale grade 4 before the test specimen does the exposure is terminated at this stage. The assessment is done comparing the exposed and the unexposed area of the specimen to that of the set of the blue wool reference.

Coumarins and flavones are widely present in nature and have an enormous potential of being used as the dye intermediates for the synthesis of the dyes. They have been previously used in number of uses ranging from the pharmaceutical to the dye industry. In the present study Aminoflavones and Aminocoumarins and hydroxycoumarins were synthesised. Aminoflavones (4΄-Aminoflavone, 3 ΄- Aminoflavone and 6-Aminoflavone) were diazotised coupled with the couplers (1-15) to get the dyes (16-75) . 4-Methyl-7-hydroxycoumarin was used as a coupler and it was coupled with the heterocyclic amines (1a-10a) to get the dyes (76-85) . In the second part the application aspect of the synthesised dyes was looked into by applying them to the polyester fabric and assessing their fastness properties. In the third part the synthesised dyes were subjected to the mutagenicity testing i.e . Ames test was performed to look for their mutagenicity

3.1 Synthesis of Intermediates. (67, 71, 75)

OH O R' + Cl CH3 O R O O R

R' NH2 R R' O O Pd/C CH3COOH O H2 H2SO4 R R O O O O

(Scheme-2) Synthesis of Amino-flavones. Intermediate R’ R

4΄-Aminoflavone 4-NO 2 H

3΄-Aminoflavone 3-NO 2 H

6-Aminoflavone H NO 2

3.1.1 4΄-Aminoflavone (67) 4΄-Nitroflavone (66) was synthesised using 2-hydroxyacetophenone and 4-nitrobenzoyl chloride using literature method (Scheme-2) 155 . 4 ΄- Nitroflavone was then reduced using Pd/C in hydrogen atmosphere under atmospheric pressure and at room temperature. The structure of the synthesised 4 ΄-aminoflavone was confirmed by 1H-NMR and EI Mass spectrometry and also by comparing its melting point by literature value.

3.1.2 3΄-Aminoflavone (71) For the synthesis of 3 ΄-Aminoflavone (71) the starting materials were 2- Hydroxyacetophenone and 3-Nitrobenzoylchloride and the method of synthesis which was followed was of the literature (Scheme-2) 155 . The yield of the product was 80% which was a fair yield for taking the product ahead as a dye intermediate. The 3 ΄-Nitroflavone formed was reduced by using the H 2 Pd/C under atmospheric pressure at room temperature which yielded 80% of 3΄-Aminoflavone. The synthesised 3 ΄-Aminoflavone was confirmed by m.p which was also at par with the m.p given in the literature. Moreover the spectroscopic studies i.e. 1H-NMR, Mass spectrometry also helped to confirm the same.

3.1.3 6-Aminoflavone (75) 6-Aminoflavone (75) was synthesised using 2-hydroxy-5- nitroacetophenone and benzoylchloride according to the literature method (Scheme-2) 155 . The yield of the product was 78 %. The reduction of the nitroflavone was carried out by using Pd/C under hydrogen atmosphere at atmospheric pressure and room temperature. The structure elucidation of the synthesised 6-aminoflavone was confirmed by 1H-NMR and Mass spectrometric analysis. Further the m.p of the synthesised 6-Aminoflavone given in the literature also confirmed the formation of the product.

3.2 Synthesis of Dyes (16-75) from the Intermediates (67, 71, 75)

O O

NH2 N+ N O O Diazotization

(61 or 71)

R4 R1 O R4 R1 N R N 2 R2 R3 N O N R O 3

(16-25) (31-45) N+ N O O R8 R1 Coupling R7 R2 R8 R1 N R7 N R2 O R6 R3 R5 R4 R6 R3 R5 R4 OR (26-30) (41-45) O

R1 R8 N R2 N R7 O

R3 R6 R4 R5

(Scheme-3) General structure of coupler (1-10)

4 R 1 R N 2 R 3 R

Where:

Coupler R1 R2 R3 R4

1 C2H5 C2H5 HNCOCH 3 OCH 3

2 C2H4OH C2H4OH H H

3 C2H5 C2H4OH H H

4 C2H4OH C2H4CN H H

5 CH 3 CH 3 H H

6 H C2H4OH H H

7 C2H5 C2H4OH CH 3 H

8 C2H5 C2H5 HNCOCH 3 H

9 C2H5 C2H5 H H

10 C2H5 C2H4CN H H

General structure of the couplers (11-15) ,

1 R8 R 7 2 R R

6 3 R R 5 4 R R Where:

Coupler R1 R2 R3 R4 R5 R6 R7 R8 11 H OH OH H H H H H

12 H OH H H H SO 3Na H H

13 NH 2 OH SO 3Na H H SO 3Na H H

14 H OH SO 3Na H H SO 3Na OH H

15 NH 2 OH H SO 3H H H H H

3.2.1 Dyes (16-30) from 4 ΄-Aminoflavone (67) A series of dyes from 4 ΄-Aminoflavone were synthesised using the couplers (1-15) . (Scheme-3). The substituted anilines proved to be good couplers as the dyes produced from them had good fastness properties. For the formation of the dyes diazotization was performed in concentrated hydrochloric acid and latter on coupled with couplers (1-15) . Polyester fabrics were dyed by the dyes (16-30) and it gave good fast colours as shown in (Table-4-8). Polyester fabric was dyed by using 2% stock dispersion solution of the dyes. The stock dispersion solutions of the dyes were prepared by first grounding the dye into a fine powder using mortar and pestle. The dye (1.0 g), dispersing agent Setamol WS (1.0 g), water (100 mL) and ceramic balls (250 g) were ball milled overnight to prepare the stock dispersion solution of the dyes. The pH of the stock solution was adjusted at 4.5 – 5.5 by the addition of acetic acid. The dyeing was carried on the laser heated dyeing machine preheated at 60 0C. The temperature of the dyeing machine was then raised to 130 0C. The heating rate was kept at 2 0C /min. and the temperature was kept at 130 0C for one hour. It was then cooled down to 50 0C afterwards and the dyed samples were taken out. To remove any unfixed dye on the surface of the fabric it was treated with alkaline solution of sodium hydroxide (2.0 g/L) and sodium dithionite (2.0 g/L) at 70 0C for 20 minutes. The dyed samples were then dried in vacuum oven and the fastness tests were carried. These dyes provided colours in the range of orange to purple with good levelness, brightness and depth on the fabric. The dyes synthesised were characterised by I.R, 1H-NMR, 13 C-NMR and Mass spectrometric results as shown in the experimental section (2.5.1- 2.5.15). The IR spectra of the dyes showed C=O stretching around 1650 cm -1. The azo group stretching vibration band appeared at 1500-1545 cm -1. 1H-NMR spectra of the dyes (16, 21, and 23) showed singlet attributing to the δ 8.02−8.13 corresponding to the single proton of the NH. The signals corresponding to the protons attached to the aromatic ring appeared around δ 6.67-7.78. For dyes (22) methyl protons attached to the phenyl group appeared as singlet in the region of δ 3.38. In case of the dye (16) the methoxy group exhibited a singlet in the region of 4.05. In dyes (26-30)

downfield signal at 10.2 integrating to the single proton might be attributed to the OH bonded to the aromatic ring. The pattern of peaks observed in the 13 C-NMR spectra also supported the formation of dyes. Almost all the dyes showed peaks in the range of 170- 167 ppm corresponding to the C=O of the flavone while the methylene signals were also from 106-48 ppm. All the other C atoms resonated in the expected regions. For the dyes (16-30) the base peak in the spectrum corresponding to the ion radical [M +] was observed. Moreover the peak corresponding to the cleavage at C-N bonds adjacent to the azo linkage were also observed as shown in the experimental section. (2.1.16- 2.1.30). Further the CHN analysis data also supported the proposed molecular formula and it was within the expected range. All the dyes were produced in a reasonable range varying from 65-89 %.

3.2.1.1 Dyeing on Nylon Lycra Apart from dyeing on the polyester some of the selected dyes (16, 17, 20, 23, 24 and 28) were used to dye the Nylon-lycra fabric. It gave good results in the sense of the fastness studies. The results as seen in the Table- 8a show that these dyes had good to moderate fastness properties. The stock dispersion solution was prepared in the same way as for the polyester dyeing i.e. ball milling the mixture of the dyes (1.0 g) along with the Dispersing agent Setamol-WS (1.0 g) water (100 mL) and ceramic balls (250 g). The pH was adjusted to 4.5-5.5 before adding the solution and the fabric in the dyeing machine. The dyeing was carried out by initially keeping the temperature of the machine at 60 0C and then raising it up to 130 0C at the heating rate of 2 0C/ minute. This temperature was maintained for 1 hour and then cooled to 50 0C. After achieving this temperature the machine was shut down and the dyeing vessels removed from the dyeing machine. The dyed fabrics were removed and reduction cleared using the solution of sodium hydroxide (2 g/L) and sodium dithionite (2g/L). The dyed samples were then dried and the fastness studies carried out. There shades are shown in Figure-5.

3.2.2 Dyes (31-45) from 3 ΄-Aminoflavone (71) The synthesised 3 ΄-Aminoflavone was used as the dye intermediate and the couplers (1-15) were used to synthesise the dyes (31-45). The (Scheme-3) was followed to synthesise the dyes. The synthesised dyes were confirmed by the spectroscopic studies i.e. 1H-NMR, Mass and elemental analysis. 3 ΄-Aminoflavone was diazotized using concentrated hydrochloric acid and coupled with the couplers (1-15) to yield the dyes (31-45). The synthesised dyes were purified using the column chromatography. The synthesised dyes were dyed on polyester fabric. They gave some good results of fastness of the dyes as shown in the fastness studies of these dyes (Table-9-13). The dyeing of polyester was done in 2% stock dispersion solution and it was prepared by overnight ball milling of the dyes (1.0 g) (31- 45) along with the Dispersing agent Setamol-WS (1.0 g) water (100 mL) and ceramic balls (250 g). All this was done in a closed glass container and after the overnight ball milling the stock dispersion solution was prepared using this solution. The pH of the stock dispersion solution was adjusted to 4.5-5.5 by using acetic acid. In this method the stock dispersion solution (25 mL) and polyester fabric (5g) were added to the vessels of the dyeing machine. The temperature of the dyeing machine was initially kept at 60 0C and then raised up to 130 0C at the heating rate of 2 0C/ minute. This temperature was maintained for 1 hour and then cooled to 50 0C. After achieving this temperature the machine was shut down and the dyeing vessels removed from the dyeing machine. The dyed fabrics were removed and reduction cleared using the solution of sodium hydroxide (2 g/L) and sodium dithionite (2g/L). The purpose of reduction clearing was to remove any of the unfixed dye from the surface of the fabric. The samples were then dried in a vacuum oven and fastness tests were carried. The synthesised dyes (31-45) were of good brightness and depth on the fabric. The characterisation of the dyes was done by I.R, 1H-NMR, Mass and Elemental Analysis the results of which are given in the experimental section (2.5.16-2.5.30). The I.R spectra of the dyes showed characteristic absorption bands around 1645 cm -1 due to the C=O group and the azo group stretching at 1525- 1550 cm -1.

The 1H-NMR spectra of the dyes (31, 36 and 38) showed the peak of NH in the range of δ 8.07−8.01. Peaks due to the aromatic protons clustered in the region of 6.66-7.79 and appeared as the multiplets. Peaks integrating to the three protons resonating around 2.19 were attributed to the Ar-CH 3 for the dye (37). The methoxy group showed the peak at around 4.07 for the dye (31). Further confirmation of the synthesised dyes was established from 13 C- NMR which showed characteristic peaks of the carbonyl compounds in the range of 169-167 while the rest of the C-atoms produced peaks according to the environment. The mass fragmentation pattern of all the synthesised dyes showed M + peak corresponding to the molecular mass of the product. The other common peaks obtained were because of the cleavage at the C-N bonds adjacent to the azo linkage. Further confirmation was established by the elemental analysis data which were well between the expected range.

3.2.3 Dyes (46-60) from 6-Aminoflavone (75)

O O

H N 2 Diazotization N+ O N O (75) (75a)

R4 R1 N R O 2 R3 R3 N N R O 2 N (46-55) O R1 R4

R R N+ 8 1 R R N O Coupling 7 2

(75a) R6 R3 R1 R8 O R5 R4 R2 R7 N N (56-60) R3 R6 O R4 R5 OR

R8 R1 O R7 R2 N N R6 R3 O R5 R4

(Scheme-4)

General structure of the coupler (1-10)

4 R 1 R N 2 R 3 R Where: Coupler R1 R2 R3 R4

1 C2H5 C2H5 HNCOCH 3 OCH 3

2 C2H4OH C2H4OH H H

3 C2H5 C2H4OH H H

4 C2H4OH C2H4CN H H

5 CH 3 CH 3 H H

6 H C2H4OH H H

7 C2H5 C2H4OH CH 3 H

8 C2H5 C2H5 HNCOCH 3 H

9 C2H5 C2H5 H H

10 C2H5 C2H4CN H H

General structure of the couplers (11-15) ,

1 R8 R 7 2 R R

6 3 R R 5 4 R R Where: Coupler R1 R2 R3 R4 R5 R6 R7 R8 11 H OH OH H H H H H

12 H OH H H H SO 3Na H H

13 NH 2 OH SO 3Na H H SO 3Na H H

14 H OH SO 3Na H H SO 3Na OH H

15 NH 2 OH H SO 3H H H H H

A series of dyes (46-60) was synthesised after diazotization of 6- aminoflavone in concentrated hydrochloric acid and coupling the diazonium salt formed with the couplers (1-15). (Scheme-4). The synthesised dyes were purified and applied on polyester fabric which yielded good fast colours of the range yellow to violet. The polyester fabrics were dyed by the synthesized dyes (46-60) using the 2% stock dispersion solution of the dyes. The dyes were powdered using the mortal and pestle and this powdered dye (1.0g), ceramic balls (250g) and a dispersing agent Setamol-WS (1.0g) were overnight ball milled. After the overnight ball milling the stock dispersion solution was added into the dyeing vessels of the dyeing machine. The pH of the stock solution was adjusted to 4.5-5.5 by adding few drops of acetic acid before addition to the dyeing machine which was preheated at 60 0C. The temperature was then raised up to 130 0C keeping the heating rate of 2 0C/minute. This temperature was kept at 130 0C for 60 minutes. After the completion of the time it was cooled to 50 0C and the dyeing vessels were taken off. The dyed fabrics were treated by the solution of sodium hydroxide (2g/L) and sodium dithionite (2g/L) so as to remove any unfixed dyes.

Fastness studies were carried out after drying the in a vacuum oven. The dyed fabrics showed good shade, level and brightness of the colours. The structure elucidation of the dyes was carried out using I.R, 1H- NMR, 13 C-NMR and Mass spectrometric and CHN results. The infra red spectra showed the characteristic absorption bands around 1530-1550cm -1 due to the presence of the azo group and also a absorption band around 1650cm -1 due to the presence of the C=O. The 1H- NMR spectra of the dyes (46, 53) showed the peak in the range of 10.6 which was attributed to the NH. The multiplets appearing in the range of 6.90-7.78 were because of the aromatic protons. The peak at around 2.40 was due to 13 the three protons of Ar-CH 3 of the dye (52). The C-NMR further helped to confirm the synthesised dyes as it gave the characteristic peaks of carbonyl in the range of 169-165 ppm. The rest of the C-atoms produced the peaks according to their respective environment. The elemental analysis data also helped to confirm the data of the synthesised dyes as it supported the proposed molecular formula and the results were in the expected range.

3.3 Fastness studies 3.3.2 Fastness studies of dyes (16-30) The light fastness was carried out using the Heracus-Xenotest 1505 fastness tester having the Xenon lamp. The dyed fabrics were exposed to the light along with the blue wool standard and rated according to the standard method 168 . The results are given in the Table-4.

Table-4 Results from Light fastness testing of Dyes (16-30). Dye Light fastness Dye Light fastness 16 4 24 4 17 4-5 25 3-4 18 4 26 3-4 19 4 27 4 20 3-4 28 4-5 21 3-4 29 3-4 22 3-4 30 3-4 23 4

As seen from the Table-5. the dyes (16-30) formed by the coupling of substituted anilines (1-10) showed better light fastness as compared to those formed by the naphthalene based couplers (11-15). Dyes (16-30) showed moderate to good wash fastness. Nylon was consistently the most stained component of the of the multi fiber. The wash fastness test was done to assess the fastness to washing of the coloured textile specimen which was kept in contact with the multi-fiber strip. The specimen was placed in the container and the standard soap solution was poured into it (soap: fabric. 50: 1) and heated at 60 ± 2 0C for 30 minutes. The textile specimen was mechanically agitated under the specified conditions of time and temperature in the standard soap solution. At the end of the test the samples were removed and the test specimens were removed from the multi fiber. It was rinsed thoroughly to the remove the residual soap solution and dried in the vacuum oven. The change of the colour and the staining extent was then assessed with the help of the gray scale and the results are given in the Table-5. In general the wash fastness of the disperse dyes tend to increase with the increasing molecular size of the dyes as depicted in Table-5.

Table-5 Results from wash fastness testing of Dyes (16-30).

Staining on Dye Change in colour Dicell Cotton Nylon PET Acrylic Wool 16 4-5 4 4-5 3-4 5 4-5 5 17 5 4 4-5 4 5 4 5 18 5 4-5 4-5 4 4-5 4-5 5 19 5 3 5 4-5 5 4-5 4-5 20 5 5 5 4-5 5 4-5 5 21 4-5 5 5 4-5 5 4-5 5 22 5 5 5 5 4-5 5 5

23 5 4 5 4 4-5 5 4-5 24 4-5 4 4-5 4 5 5 4-5 25 4-5 4 5 4 4-5 5 4-5 26 5 4 4-5 4-5 4-5 4 4-5 27 5 4 4 5 5 5 5 28 4-5 4 5 5 4 4-5 5 29 4-5 3-4 4-5 3-4 5 4 4 30 5 4-5 4-5 5 4 5 4-5

The sublimation fastness studies also showed good to moderate results for the synthesised dyes (16-30). It was assessed by keeping a composite specimen of the dyed polyester between the two un-dyed pieces of polyester and cotton in a precision press at 200 0C for 30 seconds. The electrically heated plates allowed the fabric to be set in a flat position under a pressure of 4 ± 1 kPa at a uniform temperature. The fabric to be tested along with the adjacent fabrics (cotton and polyester) were placed in the heating device and left for 30 seconds at 180 ± 2 0C. After that the fabric and the adjacent fabrics were taken out and left to dry for 4 hours in air. The change in the colour was assessed by comparing it with the two sets of gray scales. One set of gray scales was for assessing the change in colour and the other one for assessing the staining. The results are given in Table-6. It was seen that the dyes with high relative molecular mass showed best sublimation fastness. Dyes formed by coupling with the naphthalene based couplers generally showed excellent sublimation fastness.

Table-6 Results from Sublimation fastness testing of Dyes (16-30). Dye Colour Change Staining on PET Cotton 16 4-5 3 5 17 4 3-4 5 18 4-5 4 5 19 5 3-4 4 20 4-5 4 3

21 5 3-4 3-4 22 4-5 4 3 23 4 4 4 24 4-5 4 3 25 4 5 4 26 5 5 3 27 5 5 5 28 5 5 5 29 5 5 5 30 5 5 5

These dyes also gave good to moderate results for the perspiration fastness studies (Table-7) . Both acidic and alkaline perspiration fastness testing were done according to the British standard method 166 . In the case of perspiration fastness the dicell, cotton, nylon and polyester component of the multifiber adjacent fabric were particularly prone to staining during the test but the loss of colour did not occur perceptibly in the samples.

Table-7 Results from Perspiration fastness (acidic / alkaline) of polyester dyed with dyes (16-30).

Dye Colour Staining of adjacent fabric change Dicell Cotton Nylon Polyester Acrylic Wool 16 5/5 4/4 4-5/4-5 4/4 5/5 5/5 4/4 17 5/5 4-5/4-5 4/4 4-5/4-5 5/5 4/4 3/3 18 5/5 3/3 3-4/3-4 3/3 5/5 4-5/4-5 4-5/4-5 19 5/5 4/4 4-5/4-5 4/4 4-5/4-5 4/4 4/4 20 5/5 4-5/4-5 5/5 4-5/4-5 4-5/4-5 4-5/4-5 5/5 21 4-5/4-5 3/3 3/3 4-5/4-5 5/5 5/5 4/4 22 4-5/4-5 5/5 4-5/4-5 4-5/4-5 5/5 4/4 4-5/4-5 23 5/5 4/4 5/5 5/5 5/5 4-5/4-5 5/5 24 5/5 3/3 3-4/3-4 4/4 4-5/4-5 5/5 4/4 25 4/4 4-5/4-5 5/5 4-5/4-5 4/4 4-5/4-5 4/4

26 4/5 4/4 5/5 4-5/4-5 4/4 4-5/4-5 4-5/4-5 27 5/5 5/5 4-5/4-5 4-5/4-5 5/5 5/5 4-5/4-5 28 4-5/4-5 4/4 3-4/3-4 4/4 5/5 4/4 3/3 29 4-5/4-5 5/5 5/5 3-4/3-4 4/4 4/4 3-4/4 30 4/4 5/5 4/4 4/4 4/4 4-5/4-5 4-5/4-5

The visible absorption spectra of the dyes (16-30) were recorded in acetonitrile the results of which are summarised in the Table-8. The synthesised dyes (79-93) gave λmax from the range of 456-542 giving good fast orange to purple colour. The Table-8 shows the effects of the couplers on the colours and the λmax . It was observed that the colour of the dye is affected by the substituents in the coupler constituent. The coupler (1) gave the highest

λmax value.

Table-8 Absorption spectra data for the dyes (16-30)

Dye Colour λmax 16 Redish-Orange 542 17 Yellow 466 18 Orange-yellow 460 19 Orange 453 20 Orange 465 21 Orange 462 22 Orange 469 23 Redish-orange 505 24 Reddish-orange 456 25 Yellow 459 26 Yellow 451 27 Violet 472 28 Purple 493 29 Violet 481 30 Purple 497

3.3.1.1 Fastness studies of dyes dyed on Nylon-lycra The selected Dyes (7, 16, 17, 20, 23, 24 and 28) which were dyed on the Nylon-lycra fabric gave some good results as far as there fastness properties are concerned except dye (23 and 24) which showed poor properties in comparison with the one dyed on polyester. The results showed that these dyes could be successfully used for dyeing Nylon-lycra blend and it proved to be at par with the samples which were dyed on polyester fabric.

Table- 8a Fastness studies of samples dyed on Nylon-lycra

Dye Light Wash Sublimation fastness Perspiration fastness fastness fastness Colour Staining on Acidic Alkaline Change PET Cotton 7 4 3-4 4 4 4 4-5 4 16 4 4-5 4 3-4 4 4-5 4-5 17 4-5 4 4-5 4 3-4 4 4 20 4 4-5 4 4-5 4 4 4 23 3 3-4 4 4 3-4 4 3-4 24 3-4 4 3-4 4 4 3-4 4 28 4 4 4-5 5 5 4-5 4

Some of the dyed samples of the polyester and Nylon-lycra are shown in the Figure-4-5.

Dye 16 Dye-17 Dye 18 Dye 19

Dye 20 Dye 23 Dye 24 Dye 28

Figure-4. Polyester fabric Dyed with dyes (16, 17, 18, 19, 20, 23, 24, 28 )

Dye 16 Dye 17 Dye 20 Dye 22

Dye 23 Dye 24 Dye 28

Figure-5. Nylon-lycra dyed with the dyes (16, 17, 20, 22, 23, 24, and 28).

3.3.2 Fastness studies of Dyes (31-45) The dyed fabrics were assessed for their light fastness according to the standard test method 168 and the results shown in the Table-9 show that they showed good to moderate light fastness. It was carried out in the Xenon Light fastness tester.

Table-9 Results from Light fastness testing of Dyes (31-45).

Dye Light fastness Dye Light fastness 31 3-4 39 4 32 4 40 3-4 33 3-4 41 4 34 4 42 3 35 3-4 43 4 36 3-4 44 3-4 37 3-4 45 4 38 3-4

The wash fastness of the dyed samples was also carried out to assess the fastness of washing of the dyes when polyester was dyed with them. It was done using the standard test method 167 . The dyed fabrics along with the standard soap solution (50:1) were put in the wash fastness machine. A multi fiber strip was also attached along with. The machine was mechanically agitated and the temperature was maintained at 60 0C for 30 minutes. After this the sample was removed and rinsed with water to remove the residual soap solution and dried in vacuum oven. It was assessed using the gray scale and the results are shown in the Table-10.

Table-10 Results from wash fastness testing of Dyes (31-45).

Staining on Dye Change in colour Dicell Cotton Nylon PET Acrylic Wool 31 4 5 3-4 5 4-5 4-5 5 32 4 5 3-4 5 4-5 5 4-5 33 4-5 5 4 5 4 4 5 34 4-5 5 4 5 5 5 4-5 35 5 4-5 4 4-5 4-5 4-5 5 36 4 4-5 4 5 5 4-5 5 37 4-5 5 4 5 4 4-5 5 38 5 5 4 4-5 5 4 4 39 4-5 4-5 3-4 4-5 5 4 4-5 40 5 5 4-5 5 4 5 4 41 4 4-5 4-5 5 4-5 5 4-5 42 4-5 4-5 4 5 4-5 4 4 43 4 5 4 4-5 5 4-5 4-5 44 4 5 3-4 4-5 5 5 4 45 4-5 5 4 5 5 5 4-5

The sublimation fastness was carried out using the standard test method by keeping the dyed samples along with the adjacent fabrics (cotton and polyester) in a precision press for 30 seconds at the temperature of 200 0C. The electrically heated plates also exerted the pressure of 4 kPa. After the given time the samples were taken out and dried for 4 hours. The samples were then assessed against the gray scale and the results are shown in the Table-11 Again the dyes having the high molecular mass showed better sublimation fastness. Overall the synthesised dyes showed good to moderate sublimation fastness.

Table-11 Results from Sublimation fastness testing of Dyes (31-45).

Dye Colour Change Staining on PET Cotton 31 4 4 4 32 4 3 5 33 4-5 3-4 3-4 34 4-5 3-4 3-4 35 5 4 3 36 4-5 3-4 3-4 37 4-5 3-4 3 38 4 4 4 39 4 4 5 40 4 5 4 41 4-5 5 5 42 5 5 5 43 5 5 5 44 5 5 5 45 5 5 5

Perspiration fastness was carried out to look at the impact of the perspiration on the dyed fabrics. Both the alkaline as well as the acidic tests were carried out and the results are given in the Table-12 . Less loss of colour was seen in all of the samples and overall the dyes proved to have good to moderate Perspiration fastness.

Table-12. Results from Perspiration fastness (acidic/alkaline) of polyester dyed with dyes (31-45).

Dye Colour Staining of adjacent fabric change Dicell Cotton Nylon Polyester Acrylic Wool 31 4-5/5 4/4 5/4-5 3-4/3-4 5/5 4/5 5/4 32 4-5/4-5 4-5/4-5 3-4/3-4 4-5/4-5 5/5 3-4/3-4 3-4/3 33 5/5 3-4/3-4 3/3 3/3 5/5 4-5/4-5 4/4-5 34 5/5 4/4 4-5/4-5 3-4/3-4 4-5/4-5 4-5/4-5 3-4/4 35 5/5 4/4 4-5/4-5 4/4-5 4-5/4-5 4-5/4-5 5/5 36 4-5/4-5 3-4/3-4 3-4/3-4 4-5/4-5 3-4/4-5 5/5 4/4 37 4-5/4-5 4-5/4-5 4/4 4/4-5 4-5/4-5 4-5/4-5 4-5/4-5 38 5/4-5 4-5/4-5 4-5/4-5 5/5 5/5 4/4 5/5 39 5/5 3/3 3-4/4 4/4 4-5/4-5 4-5/4-5 5/5 40 4/4-5 4/4 5/5 4/4-5 4/4 4-5/4 4/4 41 4/5 3-4/4 5/5 4-5/4-5 4/4 4/4 4/4-5 42 5/5 4-5/5 4-5/4-5 4/4-5 5/5 4-5/4-5 4/4 43 4-5/4-5 3-4/3-4 3-4/3-4 3-4/3-4 5/5 4-5/4-5 3/3 44 4-5/4-5 5/4 5/4-5 3-4/4 4/4 5/5 4/4 45 4/4 4-5/4-5 4-5/4-5 3-4/3-4 4/4 4-5/4-5 4-5/4-5

Acetonitrile was used to make the solution of the dye and then the absorption maximum was recorded on Perkin Elmer UV spectrophotometer.

The effect of the different couplers on the colours and the λmax values can be seen from the results of Table-13.

Table-13 Absorption spectra data for the dyes (31-45)

Dye Colour λmax 31 Reddish orange 531 32 Dull yellow 489 33 Yellow 482 34 Orange 463 35 Orange-yellow 461

36 Orange-yellow 452 37 Yellow 470 38 Reddish-orange 507 39 Reddish-yellow 467 40 Reddish-yellow 461 41 Violet 453 42 Purple 503 43 Reddish-violet 496 44 Yellow 487 45 Reddish-violet 496

3.3.3 Fastness studies of Dyes (46-60) Light fastness was carried out using the standard method 168 using the Light fastness tester having the Xenon lamp. Blue wool standard was used as a reference along with the samples in the test. The samples along with the blue wool standards were exposed to the Xenon lamp and the results obtained are given in the Table-14 The dyed polyester fabrics had good to moderate light fastness properties.

Table-14 Results from Light fastness testing of Dyes (46-109).

Dye Light fastness Dye Light fastness 46 3-4 54 4 47 4 55 3-4 48 4 56 4 49 3-4 57 4 50 3-4 58 4 51 3 59 4 52 4 60 3 53 3-4

The sublimation fastness was performed to assess the fastness of washing of the dyed specimens. During the test the fabric samples were kept in contact with the multi-fiber strip. The dyed samples were dipped in the standard soap solution in the ratio (soap: fabric. 50:1). They were then mechanically agitated at 60 0C for 30 minutes and at the end the test samples were rinsed thoroughly with water to remove any remaining soap solution. The change in the colour and the staining extent was assessed by comparing with the gray scale and the results are given in the Table-14(a) .Two sets of gray scales were used to assess the change in colour and the extent of staining on the multi-fiber strip.

Table-14(a) Results from wash fastness testing of Dyes (46-60)

Staining on Dye Change in colour Dicell Cotton Nylon PET Acrylic Wool 46 5 4 4 4 5 4-5 5 47 4-5 4 4 5 5 4 5 48 5-4 4-5 4-5 4 4 4 5 49 5 3 4-5 5 4 4 4-5 50 5 5 4-5 4 5 4-5 5 51 4 5 4 4-5 5 4-5 5 52 5 5 5 5 4 4-5 5 53 4-5 3-4 4-5 4 5 4 4 54 4-5 4 4-5 4 5 4-5 4-5 55 4 4 4-5 4 4 5 4-5 56 5 4 4 5 4-5 5 4-5 57 4 5 5 5 4-5 5 5 58 4-5 4 4-5 5 4-5 4-5 5 59 4 5 5 4 5 4-5 5 60 5 4-5 4-5 5 5 5 4-5

The sublimation fastness studies were done by keeping the dyed fabrics along with the two un-dyed pieces of polyester and cotton in a press at 200 0C for 30 seconds. After that the fabric and the adjacent fabrics were taken out and dried for 4 hours in air. The change in the colour and staining on the adjacent fabrics was assessed using the gray scale. The dyes having high molecular mass showed good sublimation fastness results. The dyes (56-60) formed by the naphthalene based couplers had good sublimation fastness properties as compared to the dyes formed by the other couplers (1- 10).

Table-15 Results from Sublimation fastness testing of Dyes (46-60).

Dye Colour Change Staining on PET Cotton 46 5 4 4-5 47 5 4 4-5 48 4-5 4 5 49 5 3-4 4 50 4-5 4 3 51 5 3-4 3-4 52 4-5 4 3 53 5 4 4 54 4-5 4 4-5 55 4 5 4 56 5 5 4 57 5 5 5 58 5 5 5 59 5 5 5 60 5 5 5

The results ( Table-16 ) of perspiration fastness (both acidic and alkaline) show that the loss of the colour was not much and all the dyes had good to moderate fastness properties.

Table-16 Results from Perspiration fastness (acidic / alkaline) of polyester dyed with dyes (46-60).

Dye Colour Staining of adjacent fabric change Dicell Cotton Nylon Polyester Acrylic Wool 46 4-5/5 3/3 4-5/4-5 4/4 4-5/4-5 4-5/4-5 3/3 47 4-5/5 5/4 4/4 4-5/4-5 4/4 5/5 3-4/3 48 5/5 3/3 3-4/3-4 3/3 4-5/4-5 4-5/4-5 4-5/4-5 49 5/5 5/5 4-5/4-5 4/4 4-5/4-5 4/4 4/4 50 5/5 4-5/4-5 5/5 4-5/4-5 5/5 4-5/4-5 5/5 51 5/4-5 3/3 3/3 4-5/4-5 4-5/4-5 5/5 4/4 52 5/4-5 5/5 4-5/4-5 4-5/4-5 4-5/4-5 4/4 4-5/4-5 53 4-5/5 3-4/3-4 5/5 5/5 4/4 4-5/4-5 5/5 54 5/5 3/3 3-4/3-4 4/4 4-5/4-5 5/5 4/4 55 3-4/3-4 5/4-5 5/5 4-5/4-5 4/4 4-5/4-5 4/4 56 4/5 4/4 5/5 4-5/4-5 4/4 5/5 4-5/4-5 57 5/5 4-5/4-5 4-5/4-5 4-5/4-5 4/4 5/5 4-5/4-5 58 4-5/4-5 4/4 3-4/3-4 4/4 5/5 3-4/3-4 3/3 59 5/5 4-5/4-5 4-5/4-5 3-4/3-4 4/4 3-4/4 3-4/4 60 4/4 5/5 4/5 4/4 4/4 4/4-5 4-5/4-5

The absorption spectra of the dyes were recorded in acetonitrile and the results are shown in the Table-17 .and. The synthesised dyes (46-60) gave the λmax from the range of 452-531 giving fast yellow to violet colour. The coupler (1) gave the highest λmax value.

Table-17 Absorption spectra data for the dyes (46-60)

Dye Colour λmax 46 Reddish orange 531 47 Dull yellow 489 48 Yellow 482 49 Orange 463

50 Orange-yellow 461 51 Orange-yellow 452 52 Yellow 470 53 Reddish-orange 507 54 Reddish-yellow 467 55 Reddish-yellow 461 56 Violet 453 57 Purple 503 58 Reddish-violet 496 59 Yellow 487 60 Reddish-violet 496

3.4 6-Amino-4-methylcoumarin (77)

O CH + 3 conc. H 2SO 4 O NH H3C O

H5C2O HN CH3 CH3 OO O

NaOH Conc. HCl

CH3

H2N

(77)

(Scheme-4)

6-Amino-4-methylcoumarin was synthesised by using ρ- acetylaminophenol (3.02 gm, 0.02 mol) which was dissolved in ethylacetoacetate (2.06 mL, 0.02 mol) and while stirring concentrated sulphuric acid (35 mL) was added into it (Scheme-4) The reaction mixture

was heated up to 150 0C on a hot plate and stirred at this temperature for 30 minutes. The reaction mixture was then poured into ice cold water. The precipitates obtained were this of N-(2-Methyl-4-oxo-1,4-dihydroquinolin-6- yl)acetamide (76). It was further hydrolysed. The hydrolysis was done by adding N-(2-Methyl-4-oxo-1,4-dihydroquinolin-6-yl)acetamide (4.34 g, 0.02mol) in 0.5 M NaOH (15 mL). The reaction mixture was stirred for two hours and after that poured into a beaker and acidified by addition of concentrated hydrochloric acid (10mL) to give the precipitates of 6-Amino-4- methylcoumarin (77). The synthesised 6-Amino-4-methyl coumarin was characterised by 1H-NMR and Mass spectrometry.

3.4.1 Dyes (61-75) from 6-Amino-4-methylcoumarin (77) 6-Amino-4-methylcoumarin was used as an intermediate and dyes were synthesised from it by diazotizing 6-Amino-4-methylcoumarin and coupling it with the couplers (1-15) to yield the dyes (61-75). The dyes were synthesised using the Scheme-5.

CH3 CH3 NH N N 2 NaNO2 HCl, 0-5oC O O O O

(77) (77a)

Diazotization Step

R4 R 1 R R N 4 1 R N 2 R CH3 2 R3 N N

R3 O O (61-70)

CH3 N N R8 R1 R7 R2 O O Coupling R8 R1 (77a) CH R6 R3 3 R7 R2 N R5 R4 N R6 R3 O O R5 R4 (71-75) OR

R1 R8 CH 3 R2 R7 N N

R3 R6 O O R4 R5 Coupling step (Scheme-5) Where:

Coupler R1 R2 R3 R4

1 C2H5 C2H5 HNCOCH 3 OCH 3

2 C2H4OH C2H4OH H H

3 C2H5 C2H4OH H H

4 C2H4OH C2H4CN H H

5 CH 3 CH 3 H H

6 H C2H4OH H H

7 C2H5 C2H4OH CH 3 H

8 C2H5 C2H5 HNCOCH 3 H

9 C2H5 C2H5 H H

10 C2H5 C2H4CN H H

General structure of the couplers (11-15) ,

1 R 6 2 R R

5 3 R R 4 R Where:

Coupler R1 R2 R3 R4 R5 R6 11 H OH OH H H H

12 H OH H H SO 3Na H

13 H OH SO 3Na H SO 3Na H

14 H OH SO 3Na H SO 3Na OH

15 NH 2 OH H SO 3H H H

The diazotization of 6-Methyl-4-aminocoumarin was carried out in concentrated hydrochloric acid and keeping the reaction temperature below 00C. The diazonium salt formed was then coupled it with the couplers (1-15) . The diazotization was carried out at temperature below 0 0C so as to avoid the decomposition of the diazonium salt formed. The synthesised dyes (61-75) were dyed on polyester fabric and for dyeing 2% stock dispersion solution of the dye was used. This stock dispersion solution was prepared by ball milling of mixture of dye (1g), dispersing agent Setamol-WS (1g), water (100mL) and ceramic balls (250g). The dye was ground into a fine powder using mortal and pestle before adding it into the mixture. The pH of the solution was adjusted to 4.5-5.5 by using acetic acid before pouring it into the dyeing vessels. The dyeing was carried out in laser dyeing machine which was preheated at 60 0C. The temperature was raised up to 130 0C at the heating rate of 2 0C/minute. It was kept at this temperature for 1 hour. and then cooled to 50 0C. The dyed samples were taken out of the dyeing vessels. The dyed samples were then dipped into the solution of sodium hydroxide (2g/L) and sodium dithionite (2g/L). The purpose was to remove any un-fixed dye from the dyed samples. The samples were dried in a vacuum oven and afterwards the fastness

studies were carried out. These dyes provided colours in the range of 380 to 482 and the dyed fabrics showed good levels of shades. I.R, 1H-NMR, 13 C-NMR and Mass spectrometric results helped to elucidate the synthesised dyes as shown in the (2.5.45-2.5.60). The IR spectra of the dyes showed characteristic absorption band around 1646 cm -1 which was attributed to C=O stretching. Another vibration band appeared at 1510- 1546 cm -1 which may be due to the azo group stretching. 1H-NMR spectra of the dyes (61, 66 and 68) showed singlet attributing to the δ 8.03 corresponding to the single proton of the NH. The signals corresponding to the protons attached to the aromatic ring appeared around δ 6.67-7.78. The appearance of singlet at 3.73 integrating for three protons can because of the methoxy group of the dye (61). In compounds (71-75) downfield signal at around 8.10 integrating to the single proton might be attributed to the OH bonded to the aromatic ring. The pattern of peaks observed in the 13 C-NMR spectra also supported the formation of target compounds. The appearance of signals in the range of 170-167 ppm may be assigned to the C=O which also supports the IR indications. While the methylene signals were also from 68-48 ppm. All the other C atoms resonated in the expected regions. For the dyes (61-75) the base peak in the spectrum corresponding to the ion radical [M +] was observed. Moreover the peak corresponding to the cleavage at C-N bonds adjacent to the azo linkage was also observed as shown in the experimental section. (2.5.61-2.5.75). Further the CHN analysis data are also found in agreement with the calculated values. All the dyes were produced in a reasonable range varying from 67-89 %.

3.4.2 Fastness studies of dyes (61-75) The light fastness was carried out by exposing the samples of the dyed fabrics and the standard blue wool in the Xenon light fastness tester and rated according to the standard method 168 . The results are given in the Table-18.

Table-18 Results from Light fastness testing of Dyes (61-75).

Dye Light fastness Dye Light fastness 61 3-4 69 4 62 3 70 4 63 4 71 3-4 64 4 72 4 65 3-4 73 3-4 66 3-4 74 4 67 3-4 75 3-4 68 4

The synthesised dyes exhibited good to moderate wash fastness as shown in the Table-19. The wash fastness was done by mechanically agitating the dyed samples along with the multi-fiber strip in the standard soap solution. The temperature was kept at 60 0C for 30 minutes. At the end of the test the samples along with the multi-fiber were removed and rinsed thoroughly in water to remove any remaining soap solution. Gray scale was used to assess the change in colour and the staining extent and the results are depicted in Table-19.

Table-19 Results from wash fastness testing of Dyes (61-75).

Staining on Dye Change in colour Dicell Cotton Nylon PET Acrylic Wool 61 4-5 4-5 4 5 4 4 4-5 62 4 4-5 4 4-5 4-5 4-5 5 63 5 4 4-5 4 5 4 5 64 4-5 3-4 4-5 4-5 4 4 4 65 5 5 4-5 4 4-5 5 5

66 4 4-5 4 4 5 4-5 4-5 67 5 5 5 4-5 4-5 4-5 5 68 4 3-4 4-5 4 5 4-5 4 69 4-5 4 4-5 4-5 5 4-5 4-5 70 4 4 4-5 4-5 4 5 4-5 71 4-5 4 4 4-5 4-5 5 4-5 72 4 5 5 5 4-5 5 4-5 73 4-5 4 4-5 5 4-5 4-5 5 74 4 5 5 4 4-5 4-5 5 75 5 4-5 4-5 5 5 5 4-5

Sublimation fastness of the dyed fabrics was carried out by keeping the sample along with the two pieces of un-dyed pieces of polyester and cotton and the sample was pressed in a precision press at the temperature of 200 0C for 30 seconds. After that the samples and the adjacent fabrics were taken out of the press and allowed to dry for 4 hours. Two gray scales were used, one to assess the change in the colour and the other to assess the extent of staining on the adjacent fabric. The results are shown in the Table-20 .

Table-20 Results from Sublimation fastness testing of Dyes (61-75).

Dye Colour Change Staining on PET Cotton 61 4 3 4 62 4 3-4 4 63 4 3-4 4 64 5 3-4 4 65 4-5 4 3 66 5 3-4 3-4 67 4-5 3-4 3 68 4 4 4 69 4-5 3-4 3 70 4 5 4

71 5 5 3 72 5 5 5 73 5 5 5 74 5 5 5 75 5 5 5

The standard method 166 was followed to assess the perspiration fastness and the dyes (61-75) showed good to moderate fastness in this case as depicted in the Table-21 .

Table-21 Results from Perspiration fastness (acidic / alkaline) of polyester dyed with dyes (61-75).

Dye Colour Staining of adjacent fabric change Dicell Cotton Nylon Polyester Acrylic Wool 61 5/4 3-4/3-4 4-5/4 5/5 4/4 4/4 4/4 62 4/4 5/4 3-4/3-4 4-5/4-5 5/4 4/4 3/3 63 5/5 3/3 3-4/3-4 3/3 4/4 4-5/4-5 4-5/4-5 64 5/5 5/5 4-5/4-5 4/4 4-5/4-5 4/4 4/4 65 5/5 4-5/4-5 5/5 3/3 4-5/4-5 4-5/4-5 5/5 66 4-5/4-5 4/4 3/3 4-5/4-5 5/5 5/5 4/4 67 4-5/4-5 5/5 4-5/4-5 4-5/4-5 5/5 4/4 4-5/4-5 68 5/5 3/3 5/5 4/5 5/5 4-5/4-5 5/5 69 5/5 3/3 3-4/3-4 4/4 4-5/4-5 5/5 4/4 70 4/4 5/5 4-5/4-5 4-5/4-5 4/4 4-5/4-5 3-4/4 71 4/5 3-4/3-4 5/4-5 4/4 4/4 4-5/4-5 4-5/4-5 72 5/5 5/5 4-5/4 5/5 5/5 5/5 5/5 73 4-5/4-5 4/5 3-4/3 3-4/3-4 5/5 4/4 4/4 74 3/3 3/4 3-4/4 3-4/4 4/4 4/4 4/4 75 3/4 4/4 3/4 3-4/4 4/4 3-4/4 3-4/4

The absorption spectra was taken by making the solution of the dyes in acetonitrile and recording the on the spectrophotometer. The results are

shown in the Table-22

Table-22 Absorption spectra data for the dyes (61-75).

Dye Colour λmax 61 Red 460 62 Yellow 426 63 Yellow 431 64 Yellow 445 65 Yellow 421 66 Clear Yellow 389 67 Pale Yellow 406 68 Pale Yellow 403 69 Orange 409 70 Orange 414 71 Clear Yellow 380 72 Purple 482 73 Violet 481 74 Purple 479 75 Pale-Yellow 404 61 Reddish-Yellow 434

3.5 4-Methyl-7-hydroxycoumarin (78)

OH O CH3

0 75-80 C + H3C O

H5C2O Polyphosphoric acid OH HO OO

(78) (Scheme-6) 4-Methyl-7-hydroxycoumarin was synthesized by the literature method 143 by dissolving polyphosphoric acid (16gm,), resorcinol (1.1gm, 0.1mol) in (13mL, 0.1mol) of ethylacetoacetate. (Scheme-6) The mixture was

stirred and heated at 75-80 0C for about 20 minutes, and then poured into ice- water. The pale yellow crystals of 4-Methyl-7-hydroxycoumarin was collected by filtration, washed with cold water and dried at 60 0C. The compound was recrystalized in ethanol. The structure elucidation of the synthesized 4-Methyl- 7hydroxycoumarin was done by 1H-NMR and Mass spectrometry .

3.5.1 Dyes (76-85) from 4-Methyl-7-hydroxycoumarin (77)

Diazotizatiion Az N Az NH + 2 N

CH3

Az N + Coupling + N HO OO HO OO N N

Az Where Az;

N (1a) N (2a)

S S

N (3a) N (4a)

CH S S 3 H 3 CO

(5a) N (6a) N

O N 2 S C l S

(7a) CH 3 (8a) N N O N S

(9a) N (10a) N

NH N N

(Scheme-7)

The dyes (76-85) were obtained by the reaction (Scheme-7) . The diazotization and coupling was carried out as follows, The diazotization was carried out using nitrosyl sulphuric acid which was prepared by mixing sodium nitrite and concentrated sulphuric acid. The mixing was carried out at 70 0C for six hours and then stirred for one hour at 0 0C. The heterocylic amine (1a-10a) was dissolved in glacial acetic acid and the temperature was maintained at 0 0C. 7-Hydroxy-4methyl-coumarin was added in the solution of sodium carbonate and the pH was maintained at 7-8. The diazonium salt was added drop wise keeping the temperature at 0 0C.The dye obtained was filtered, washed and recrystalized from hexane:ethylacetate mixture (1:1) The structure elucidated of the synthesized dyes (76-85) was done with the help of I.R, spectroscopic data and the Elemental analysis. The appearance of the bands at 1648, 1531 and 1545 may be assigned to C=O, C=C and the –N=N- groups respectively. The edifying feature of 1HNMR spectra of dyes (84) is the presence of peak at 7.87 which was attributed to the NH and the multiplets in the range of 6.70-7.79 were because of the aromatic protons. In compounds (76-85) downfield signal at around 8.63 integrating to the single proton might be attributed to the OH bonded to the aromatic ring. The signal at 169 ppm can be assigned to the carbonyl carbon and the other carbons gave there peaks according to their environment. The data from the 13 C-NMR further helped to confirm the structures. For the dyes (76-85) the base peak which was that of the molecular radical ion was found moreover the characteristic peaks corresponding to the cleavage at C-N bond adjacent to the azo groups were also observed. Elemental analysis also helped to confirm the data of the synthesized dyes as it supported the proposed molecular formula. Moreover the elemental analysis results were in the expected range.

3.5.2 Fastness studies of Dyes (76-85) The synthesised dyes (76-85) were tested for their light-fastness using the British Standard method 168 and the results are depicted in Table-23 which show that the dyes showed moderate to good light fastness. Blue wool was used as the standard and the dyed samples were ranked by comparing them with the blue wool.

Table-23 Results from Light fastness testing of Dyes (76-85).

Dye Light fastness 76 4-5 77 4-5 78 3-4 79 4-5 80 3 81 4 82 4-5 83 4 84 3-4 85 3

Wash-fastness of the dyes (76-85) was carried out to assess the fastness of washing of the dyed fabrics. The dyed fabrics were kept in contact with the multi-fiber strip and dipped in the standard soap solution (soap: fabric, 50:1) and mechanically agitated in the machine while keeping the temperature at 60 0C for 30 minutes. The samples were removed at the end and washed thoroughly with water to remove any remaining soap solution. They were then compared with two sets of gray scale, one for assessing the change in colour and the other for assessing the extent of staining on the multi-fiber strip. The results are shown in Table-24.

Table-24 Results from wash fastness testing of Dyes (76-85).

Staining on Dye Change in colour Dicell Cotton Nylon PET Acrylic Wool 76 4 4-5 4 4-5 4-5 4 4-5 77 4 5 5 5 5 4 5 78 4 4-5 4-5 4-5 4-5 4 5 79 4 3-4 4-5 4-5 4 4 4 80 4 5 4-5 4 5 4-5 5 81 4 4- 5 4-5 5 4-5 5 82 4 5 5 5 4-5 4-5 5 83 4 3-4 4-5 4 5 4 4 84 3 3-4 4-5 4 5 4-5 4-5 85 4 4 4-5 4 4 5 4-5

For carrying out the sublimation fastness the dyed fabrics along with the two un-dyed pieces of polyester and cotton were kept in the precision press at 200 0C for 30 seconds and after the said time taken out dried and assessment done by comparing the dyed samples with the gray scale. The results showed that the dyes had good to moderate wash-fastness.

Table-25 Results from Sublimation fastness testing of Dyes (76-85).

Dye Colour Change Staining on PET Cotton 76 3-4 3-4 5 77 4-5 4 4 78 4 5 5 79 3 3-4 3-4 80 4-5 3-4 3

81 3-4 3-4 3-4 82 3 4 3 83 4-5 3-4 3 84 3-4 3-4 4 85 4 5 4

The loss of colour was not significant in the case of perspiration fastness (both acidic as well as alkaline) and all the dyes (76-85) showed good to moderate perspiration fastness.

Table-26 Results from Perspiration fastness (acidic / alkaline) of polyester dyed with dyes (76-85).

Dye Colour Staining of adjacent fabric change Dicell Cotton Nylon Polyester Acrylic Wool 76 4/4 3-4/3-4 4-5/4 4-5/5 4-5/4 4/4 4/4 77 5/4 5/4 4/3-4 4/4-5 4-5/4 4-5/4 3/3 78 4/5 3-4/3 4/3-4 3-4/3 4-5/4 4/4-5 4-5/4-5 79 4-5/5 4-5/5 4-5/4-5 4/4 4/4-5 4-5/4 4/4 80 5/4 4-5/4-5 5/5 3-4/3 4-5/4-5 4-5/4-5 5/5 81 4-5/4-5 4-5/4 3/3 4/4-5 5/5 4-5/5 3-4/4 82 4-5/4-5 5/5 5/4-5 4/4-5 5/5 4-5/4 4-5/4-5 83 5/4-5 3/3 4-5/5 4/4-5 4-5/5 4-5/4 5/5 84 5/4 3-4/3 4/3-4 4/4-5 4/4-5 5/4-5 4/4 85 4/4 5/5 4/4-5 4-5/5 4/4 4-5/5 4/4

The solution of the dyes were made in acetonitrile and their λmax were recorded on the Perkin Elmer UV spectrophotometer and the results in the

Table-27 show the change in the colour with the different couplers. The λmax was in the range of 351-481 giving yellow to reddish brown colours.

Table-27 Absorption spectra data for the dyes (76-85).

Dye Colour λmax 76 Orange 421 77 Orange 426 78 Bright Yellow 359 79 Reddish Brown 470 80 Reddish Brown 481 81 Bright yellow 462 82 Yellowish orange 413 83 Yellowish orange 419 84 Yellow 353 85 Yellow 355 76 Orange 421

3.6 Mutagenicity Assessment Data The mutagenicty testing of the dyes (16-85) was carried out using the Standard Ames test 169 Selected Salmonella typhimurium strains that revert from histidine dependence to histidine independence at an increased frequency in the presence of a mutagen 170 are used. In this test, Salmonella strains TA98 and TA100 were used. The test procedure was performed in the following way: The strains of bacteria from a fresh culture were added to agar (2-3 mL) (this was maintained at about 47-50°C), metabolic activation mixture (0.5 mL), a solution of test compound or solvent (0.01 mL and 0.1 mL). All this mixture was mixed gently and poured on plates that contained minimal- glucose agar (25 mL). After allowing the top agar to solidify (it takes about 20 minutes), the plates were incubated at 37°C for 72 hours. After the 72 hours the plates were taken out and the numbers of reverant colonies were counted. For every dose level used three replicates were used. The resulting plate counts were averaged and the average number of reverants was compared to the dose range. For each strain the dye undergoing evaluation was then judged mutagenic or nonmutagenic. The ones that were judged mutagenic were further characterized as weakly, moderately or strongly

mutagenic. Dyes producing the reverant counts that were two to four times the background count were judged weakly mutagenic while the ones producing reverants four to six times the background count were judged moderately mutagenic and those producing reverants more than six times were judged as strongly mutagenic. Figures-(6-62) gives the dose response curves for the dyes tested. The background count was established by the number of reverant colonies counted for a control test in which no dye was present. A mutagenic response was recorded if the number of reverant colonies counted were at least twice the background count. The test was performed using the standard mutagenicity assay and; (a) TA98 without S9 activation. (b) TA98 with S9 activation. (c) TA100 without S9 activation. (d) TA100 with S9 activation. Figures -(6-62) show the results of dose response for the dyes (16-85) using the standard mutagenicity assay and TA 98 without S9 activation, TA 98 with S9 activation, TA 100 without S9 activation and A 100 with S9 activation. As seen from the results of Figure (6-62) all the dyes (16-85) were negative in TA100 and TA98 without metabolic activation except the dyes (30, 45, 60 75 and 80) which showed mutagenicity in TA98 and slight mutagenicity in TA 100 as well. The dyes (30, 45, 60 75 and 80) showed the number of reverant colonies almost twice that of the control test. The mutagenicity of these dyes (30, 45, 60 and 75) can be anticipated because of the presence of the free amino group in the coupler which may be causing its increased mutagenicity. It was also observed that the dyes (30, 45, 60 and 75) showed a good dose response at the range of 50- 150 g but when the dose increased than 150 g the toxicity to the bacteria was observed. Thus a lower dose range curve was also drawn for dye (30, 45, 60, 75 and 80) to look at the effect of the lower dose and the results are shown in the Figure (61-62). These test results were similar to that of the high dose tests. It has been seen that these dyes were giving negative response at the dose level of 5 g but when the dose was increased from 65 g that it showed a significant increase in the colony growth. These test results showed that the dye (30, 45, 60, 75

and 80) was mutagenic in the higher dose range. Although dyes (28, 43, 56 and 73) had amino group in them but they were not so much mutagenic as compared to the dyes (30, 45, 60, 75, and 80) which may be attributed to the presence of two SO 3Na groups. Dye (80) also showed mutagenicity which might be attributed due to the presence of the nitro group in it. The nitro group can be reduced by enzymes to groups that cause adverse interactions with DNA. It has seen in the previous studies 171-173 as well that the azo dyes containing nitro group are often mutagenic and our results also support these findings. The presence of two sulphonic groups (sodium salt) in dyes (28,46,58 and 73) has reduced their mutagenecity as compared to the (30,45,60 and 75) which have one amino and one sulphonic group.

Dose Response Curve

100

80 Dye 16

60 Dye 17 Dye 18 40 Dye 19 Reverants 20 Dye 20

0 0 200 400 600 800 1000 1200 Dose

Figure 6 Dose response curve for Dye (16-20) using the standard mutagenicity assay and TA 98 without S9 activation.

Dose Response Curve

70 60 Dye 21 50 Dye 22 40 Dye 23 30 Dye 24 Reverants 20 Dye 25 10 0 0 200 400 600 800 1000 1200 Dose

Figure 7 Dose response curve for Dye (21-25) using the standard mutagenicity assay and TA 98 without S9 activation.

Dose Response Curve

100

80 Dye 26

60 Dye 27 Dye 28 40 Dye 29 Reverants 20 Dye 30

0 0 200 400 600 800 1000 1200 Dose

Figure 8 Dose response curve for Dye (26-30) using the standard mutagenicity assay and TA 98 without S9 activation.

Dye 31 40 Dye 32 30 Dye 33 Dye 34 20 Dye 35

0 0 200 400 600 800 1000 1200

Figure 9 Dose response curve for Dye (31-35) using the standard mutagenicity assay and TA 98 without S9 activation.

Dose Response Curve

50 Dye 36 40 Dye 37 30 Dye 38 20 Dye 39 Reverants 10 Dye 40 0 0 200 400 600 800 1000 1200 Dose

Figure 10 Dose response curve for Dye (36-40) using the standard mutagenicity assay and TA 98 without S9 activation.

Dose Response Curve

100

80 Dye 41

60 Dye 42 Dye 43 40 Dye 44 Reverants 20 Dye 45

0 0 200 400 600 800 1000 1200 Dose

Figure 11 Dose response curve for Dye (41-45) using the standard mutagenicity assay and TA 98 without S9 activation.

Dose Response Curve

70 60 Dye 46 50 Dye 47 40 Dye 48 30 Dye 49 Reverants 20 Dye 50 10 0 0 200 400 600 800 1000 1200 Dose

Figure 12 Dose response curve for Dye (46-50) using the standard mutagenicity assay and TA 98 without S9 activation.

Dose Response Curve

70 Dye 51 60 Dye 52 50 Dye 53 40 Dye 54 30 Dye 55 Reverants 20 Series6 10 Series7 0 0.2 400 600 800 1000 1200 Series8 Series9 Dose

Figure 13 Dose response curve for Dye (51-55) using the standard mutagenicity assay and TA 98 without S9 activation.

Dose Response Curve

100

80 Dye 56

60 Dye 57 Dye 58 40 Dye 59 Reverants 20 Dye 60

0 0 200 400 600 800 1000 1200 Dose

Figure 14 Dose response curve for Dye (56-60) using the standard mutagenicity assay and TA 98 without S9 activation.

Dose Response Curve

50 Dye 61 40 Dye 62 30 Dye 63 20 Dye 64 Reverants 10 Dye 65 0 0 200 400 600 800 1000 1200 Dose

Figure 15 Dose response curve for Dye (61-65) using the standard mutagenicity assay and TA98 without S9 activation.

Dose Response Curve

50 Dye 66 40 Dye 67 30 Dye 68 20 Dye 69 Reverants 10 Dye 70 0 0 200 400 600 800 1000 1200 Dose

Figure 16 Dose response curve for Dye (66-70) using the standard mutagenicity assay and TA98 without S9 activation.

Dose Response Curve

80 70 60 Dye 71 50 Dye 72 40 Dye 73 30 Dye 74 Reverants 20 Dye 75 10 0 0 200 400 600 800 1000 1200 Dose

Figure 17 Dose response curve for Dye (71-75) using the standard mutagenicity assay and TA98 without S9 activation.

Dose Response Curve

80 70 60 Dye 71 50 Dye 72 40 Dye 73 30 Dye 74 Reverants 20 Dye 75 10 0 0 200 400 600 800 1000 1200 Dose

Figure 18 Dose response curve for Dye (76-80) using the standard mutagenicity assay and TA98 without S9 activation.

Dose Response Curve

100 Dye 81 80 Dye 82 60 Dye 83 40 Dye 84

Reverants Dye 85 20 Series6 0 0 200 400 600 800 1000 1200 Dose

Figure 19 Dose response curve for Dye (81-85) using the standard mutagenicity assay and TA98 without S9 activation.

Dose Response Curve

90 80 70 Dye 16 60 Dye 17 50 Dye 18 40 30 Dye 19 Reverants 20 Dye 20 10 0 0 200 400 600 800 1000 1200 Dose

Figure 20 Dose response curve for Dye (16-20) using the standard mutagenicity assay and TA 98 with S9 activation.

Dose Response Curve

70 60 Dye 21 50 Dye 22 40 Dye 23 30 Dye 24 Reverants 20 Dye 25 10 0 0 200 400 600 800 1000 1200 Dose

Figure 21 Dose response curve for Dye (21-25) using the standard mutagenicity assay and TA 98 with S9 activation.

Dose Response Curve

100

80 Dye 26

60 Dye 27 Dye 28 40 Dye 29 Reverants 20 Dye 30

0 0 200 400 600 800 1000 1200 Dose

Figure 22 Dose response curve for Dye (26-30) using the standard mutagenicity assay and TA 98 with S9 activation.

Dose Response Curve

50 Dye 31 40 Dye 32 30 Dye 33 20 Dye 34 Reverants 10 Dye 35 0 0 200 400 600 800 1000 1200 Dose

Figure 23 Dose response curve for Dye (31-35) using the standard mutagenicity assay and TA 98 with S9 activation.

Dose Response Curve

50 Dye 36 40 Dye 37 30 Dye 38 20 Dye 39 Reverants 10 Dye 40 0 0 200 400 600 800 1000 1200 Dose

Figure 24 Dose response curve for Dye (36-40) using the standard mutagenicity assay and TA 98 with S9 activation.

Dose Response Curve

100

80 Dye 41

60 Dye 42 Dye 43 40 Dye 44 Reverants 20 Dye 45

0 0 200 400 600 800 1000 1200 Dose

Figure 25 Dose response curve for Dye (41-45) using the standard mutagenicity assay and TA 98 with S9 activation.

Dose Response Curve

80 70 60 Dye 46 50 Dye 47 40 Dye 48 30 Dye 49 Response 20 Dye 50 10 0 0 200 400 600 800 1000 1200 Dose

Figure 26 Dose response curve for Dye (46-50) using the standard mutagenicity assay and TA 98 with S9 activation.

Dose Response Curve

80 70 60 Dye 51 50 Dye 52 40 Dye 53 30 Dye 54 Reverants 20 Dye 55 10 0 0 200 400 600 800 1000 1200 Dose

Figure 27 Dose response curve for Dye (51-55) using the standard mutagenicity assay and TA 98 with S9 activation.

Dose Response Curve

120

100 Dye 56 80 Dye 57 60 Dye 58 40 Dye 59 Reverants 20 Dye 60 0 0 200 400 600 800 1000 1200 Dose

Figure 28 Dose response curve for Dye (56-60) using the standard mutagenicity assay and TA 98 with S9 activation.

Dose Response Curve

50 Dye 61 40 Dye 62 30 Dye 63 20 Dye 64 Reverants 10 Dye 65 0 0 200 400 600 800 1000 1200 Dose

Figure 29 Dose response curve for Dye (61-65) using the standard mutagenicity assay and TA98 with S9 activation.

Dose Reverants

50 Dye 66 40 Dye 67 30 Dye 68 20 Dye 69 Reverants 10 Dye 70 0 0 200 400 600 800 1000 1200 Dose

Figure 30 Dose response curve for Dye (66-70) using the standard mutagenicity assay and TA98 with S9 activation.

Dose Response Curve

80 70 60 Dye 71 50 Dye 72 40 Dye 73 30 Dye 74 Reverants 20 Dye 75 10 0 0 200 400 600 800 1000 1200 Dose

Figure 31 Dose response curve for Dye (71-75) using the standard mutagenicity assay and TA98 with S9 activation.

Dose Response Curve

120

100 Dye 76 80 Dye 77 60 Dye 78 40 Dye 79 Reverants 20 Dye 80 0 0 200 400 600 800 1000 1200 Dose

Figure 32 Dose response curve for Dye (76-80) using the standard mutagenicity assay and TA98 with S9 activation.

Dose Response Curve

100 Dye 81 80 Dye 82 60 Dye 83 40 Dye 84

Reverants Dye 85 20 Series6 0 0 200 400 600 800 1000 1200 Dose

Figure 33 Dose response curve for Dye (81-85) using the standard mutagenicity assay and TA98 with S9 activation.

Dose Response Curve

100

80 Dye 16

60 Dye 17 Dye 18 40 Dye 19 Reverants 20 Dye 20

0 0 200 400 600 800 1000 1200 Dose

Figure 34 Dose response curve for Dye (16-20) using the standard mutagenicity assay and TA 100 without S9 activation.

Dose Response Curve

100

80 Dye 21

60 Dye 22 Dye 23 40 Dye 24 Reverants 20 Dye 25

0 0 200 400 600 800 1000 1200 Dose

Figure 35 Dose response curve for Dye (21-25) using the standard mutagenicity assay and TA 100 without S9 activation.

Dose Response Curve

120

100 Dye 26 80 Dye 27 60 Dye 28 40 Dye 29 Reverants 20 Dye 30 0 0 200 400 600 800 1000 1200 Dose

Figure 36 Dose response curve for Dye (26-30) using the standard mutagenicity assay and TA100 without S9 activation.

Dose Response Curve

100 Dye 31 80 Dye 32 60 Dye 33 40 Dye 34

Reverants Dye 35 20 Series6 0 0 200 400 600 800 1000 1200 Dose

Figure 37 Dose response curve for Dye (31-35) using the standard mutagenicity assay and TA100 without S9 activation.

Dose Response Curve

100 Dye 36 80 Dye 37 60 Dye 38 40 Dye 39

Reverants Dye 40 20 Series6 0 0 200 400 600 800 1000 1200 Dose

Figure 38 Dose response curve for Dye (36-40) using the standard mutagenicity assay and TA100 without S9 activation.

Dose Response Curve

160 140 120 Dye 41 100 Dye 42 80 Dye 43 60 Dye 44 Reverants 40 Dye 45 20 0 0 200 400 600 800 1000 1200 Dose

Figure 39 Dose response curve for Dye (41-45) using the standard mutagenicity assay and TA100 without S9 activation.

Dose Response Curve

100

80 Dye 46

60 Dye 47 Dye 48 40 Dye 49 Reverants 20 Dye 50

0 0 200 400 600 800 1000 1200 Dose

Figure 40 Dose response curve for Dye (46-50) using the standard mutagenicity assay and TA100 without S9 activation.

Dose Response Curve

100

80 Dye 51

60 Dye 52 Dye 53 40 Dye 54 Reverants 20 Dye 55

0 0 200 400 600 800 1000 1200 Dose

Figure 41 Dose response curve for Dye (51-55) using the standard mutagenicity assay and TA100 without S9 activation.

Dose Response Curve

120

100 Dye 56 80 Dye 57 60 Dye 58 40 Dye 59 Reverants 20 Dye 60 0 0 200 400 600 800 1000 1200 Dose

Figure 42 Dose response curve for Dye (56-60) using the standard mutagenicity assay and TA100 without S9 activation.

Dose Response Curve

80 70 Dye 61 60 Dye 62 50 Dye 63 40 Dye 64 30 Reverants 20 Dye 65 10 Series6 0 0 200 400 600 800 1000 1200 Dose

Figure 43 Dose response curve for Dye (61-65) using the standard mutagenicity assay and TA100 without S9 activation.

Dose Response Curve

100

80 Dye 66

60 Dye 67 Dye 68 40 Dye 69 Reverants 20 Dye 70

0 0 200 400 600 800 1000 1200 Dose

Figure 44 Dose response curve for Dye (66-70) using the standard mutagenicity assay and TA100 without S9 activation.

Dose Response CUrve

100

80 Dye 71

60 Dye 72 Dye 73 40 Dye 74 Reverants 20 Dye 75

0 0 200 400 600 800 1000 1200 Dose

Figure 45 Dose response curve for Dye (71-75) using the standard mutagenicity assay and TA100 without S9 activation.

Dose Response Curve

120

100 Dye 76 80 Dye 77 60 Dye 78 40 Dye 79 Reverants 20 Dye 80 0 0 200 400 600 800 1000 1200 Dose

Figure 46 Dose response curve for Dye (76-80) using the standard mutagenicity assay and TA100 without S9 activation.

Dose Response Curve

100 Dye 81 80 Dye 82 60 Dye 83 40 Dye 84

Reverants Dye 85 20 Series6 0 0 200 400 600 800 1000 1200 Dose

Figure 47 Dose response curve for Dye (81-85) using the standard mutagenicity assay and TA100 without S9 activation.

Dose Response Curve

100

80 Dye 16

60 Dye 17 Dye 18 40 Dye 19 Reverants 20 Dye 20

0 0 200 400 600 800 1000 1200 Dose

Figure 48 Dose response curve for Dye (16-20) using the standard mutagenicity assay and TA100 with S9 activation.

Dose Response Curve

100

80 Dye 21

60 Dye 22 Dye 23 40 Dye 24 Reverants 20 Dye 25

0 0 200 400 600 800 1000 1200 Dose

Figure 49 Dose response curve for Dye (21-25) using the standard mutagenicity assay and TA100 with S9 activation.

Dose Response Curve

120

100 Dye 26 80 Dye 27 60 Dye 28 40 Dye 29 Reverants 20 Dye 30 0 0 200 400 600 800 1000 1200 Dose

Figure 50 Dose response curve for Dye (26-30) using the standard mutagenicity assay and TA100 with S9 activation.

Dose Response Curve

100

80 Dye 31

60 Dye 32 Dye 33 40 Dye 34 Reverants 20 Dye 35

0 0 200 400 600 800 1000 1200 Dose

Figure 51 Dose response curve for Dye (31-35) using the standard mutagenicity assay and TA100 with S9 activation.

Dose Response Curve

100

80 Dye 36

60 Dye 37 Dye 38 40 Dye 39 Reverants 20 Dye 40

0 0 200 400 600 800 1000 1200 Dose

Figure 52 Dose response curve for Dye (36-40) using the standard mutagenicity assay and TA100 with S9 activation.

Dose Response Curve

140 120 Dye 41 100 Dye 42 80 Dye 43 60 Dye 44 Reverants 40 Dye 45 20 0 0 200 400 600 800 1000 1200 Dose

Figure 53 Dose response curve for Dye (41-45) using the standard mutagenicity assay and TA100 with S9 activation.

Dose Response Curve

100

80 Dye 46

60 Dye 47 Dye 48 40 Dye 49 Reverants 20 Dye 50

0 0 200 400 600 800 1000 1200 Dose

Figure 54 Dose response curve for Dye (46-50) using the standard mutagenicity assay and TA100 with S9 activation.

Dose Response Curve

100

80 Dye 51

60 Dye 52 Dye 53 40 Dye 54 Reverants 20 Dye 55

0 0 200 400 600 800 1000 1200 Dose

Figure 55 Dose response curve for Dye (51-55) using the standard mutagenicity assay and TA100 with S9 activation.

Dose Repsonse Curve

100

80 Dye 56

60 Dye 57 Dye 58 40 Dye 59 Reverants 20 Dye 60

0 0 200 400 600 800 1000 1200 Dose

Figure 56 Dose response curve for Dye (56-60) using the standard mutagenicity assay and TA100 with S9 activation.

Dose Response Curve

80 70 60 Dye 61 50 Dye 62 40 Dye 63 30 Dye 64 Reverants 20 Dye 65 10 Series6 0 0 200 400 600 800 1000 1200 Series7 Dose

Figure 57 Dose response curve for Dye (61-65) using the standard mutagenicity assay and TA100 with S9 activation.

Dose Response Curve

80 70 Dye 66 60 Dye 67 50 Dye 68 40 Dye 69 30 Reverants 20 Dye 70 10 Series6 0 0 200 400 600 800 1000 1200 Dose

Figure 58 Dose response curve for Dye (66-70) using the standard mutagenicity assay and TA100 with S9 activation.

Dose Response Curve

80 70 Dye 71 60 Dye 72 50 Dye 73 40 Dye 74 30 Reverants 20 Dye 75 10 Series6 0 0 200 400 600 800 1000 1200 Dose

Figure 59 Dose response curve for Dye (71-75) using the standard mutagenicity assay and TA100 with S9 activation.

Dose Response Curve

160 140 120 Dye 76 100 Dye 77 80 Dye 78 60 Dye 79 Reverants 40 Dye 80 20 0 0 200 400 600 800 1000 1200 Dose

Figure 60 Dose response curve for Dye (76-80) using the standard mutagenicity assay and TA100 with S9 activation.

Dose Response Curve

120 100 Dye 81 Dye 82 80 Dye 83 60 Dye 84 40 Reverants Dye 85 20 Series6 0 0 200 400 600 800 1000 1200 Dose

Figure 61 Dose response curve for Dye (81-85) using the standard mutagenicity assay and TA100 with S9 activation.

Dose Response Curve

140 120 Dye 30 100 Dye 45 80 Dye 60 60 Dye 75 40 Dye 80 20 0 0 200 400 600 800 1000 1200 Dose Reverants

Figure 62 Dose Response Curve for Dyes (30,45, 60, 75 and 80) using the standard mutagenicity assay and TA98 with S9 activation.

Dose Reponse

120

100 Dye 30 80 Dye 45 60 Dye 60 40 Dye 75 Reverants 20 Dye 80 0 0 30 60 90 120 150 180 Dose

Figure 63 Dose Response Curve for Dyes (30,45, 60, 75 and 80) using the standard mutagenicity assay and TA98 with S9 activation.

CONCLUSIONS The results of this study indicates that the synthesis of the target environment friendly dyes was successful. Different dyes (16-75) which were synthesised using the different Aminoflavones (4 ΄-Aminoflavone, 3 ΄-Aminoflavone and 6-Aminoflavone) and Aminocoumarin (6-Amino-4-methylcoumarin) intermediates had good to moderate fastness properties. The use of the substituted anilines as well as the naphthalene based couplers proved to be successful as the dyes obtained were not only in good yield but also had good fastness properties. The toxicological assessment also proves that most of the dyes were non-mutagenic except for the one which had free amino groups. Their toxicity can be attributed to the presence of that free amino group in the coupler. Previous studies also support our work i.e the presence of the free amino group was the cause of the mutagenicity of dyes. On the other hand the dyes having Sodium salt of the sulphonic group along with the amino group were less mutagenic as compared to the ones having amino group. The presence of this group reduced the mutagenicity of the dyes. The dyes gave good results on the polyester fabric and some of the dyes were used for dyeing Nylon-lycra which also gave good results. So it can also be concluded that the other synthesised dyes can also be tested on Nylon-lycra fabric.

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