prr.hec.gov.pkprr.hec.gov.pk/jspui/bitstream/123456789/1134/1/2339S.pdf · i TABLE OF CONTENTS...

SYNTHESIS OF γ-KETOESTERS VIA ACID CATALYSED RING

OPENING OF SUCCINIC ANHYDRIDE WITH SUBSTITUTED

BENZYL ALCOHOLS

&

PHYTOCHEMICAL STUDIES OF BIOACTIVE COMPOUNDS FROM

CORIANDRUM SATIVUM Linn (UMBELLIFERAE)

BY

MUHAMMAD IQBAL

DEPARTMENT OF CHEMISTRY

GOMAL UNIVERSITY

DERA ISMAIL KHAN, KPK, (PAKISTAN)

2013

SYNTHESIS OF γ-KETOESTERS VIA ACID CATALYSED RING

OPENING OF SUCCINIC ANHYDRIDE WITH SUBSTITUTED

BENZYL ALCOHOLS

&



BY

MUHAMMAD IQBAL

Ph. D


GOMAL UNIVERSITY


2013

SYNTHESIS OF γ-KETOESTERS VIA ACID CATALYSED RING OPENING OF

SUCCINIC ANHYDRIDE WITH SUBSTITUTED BENZYL ALCOHOLS

&



A Thesis Submitted

For the fulfilment of the degree

Of

Doctor of Philosophy

In

CHEMISTRY

(Organic Chemistry)

BY

MUHAMMAD IQBAL


GOMAL UNIVERSITY


2013

IN THE NAME OF ALLAH

THE MOST MERCIFUL

&

THE MOST GRACIOUS

DEDICATED TO

MY

BELOVED WIFE

AND

CHILDREN

i

TABLE OF CONTENTS

SECTION CONTENTS Page #

TABLE OF CONTENTS i

LIST OF TABLES xiii

LIST OF FIGURES ix

LIST OF HISTOGRAMS & SCHEMES ix

ACKNOWLEDGEMENTS x

ABBREVIATIONS xii

TECHNIQUES xii

ABSTRACT 1

PART-I 4

Chapter 1: INTROUCTION 5

1 INTRODUCTION 6

1.1 General Overview 6

1.2 Benzyl Hydrogen Succinates (Monoesters) 6

1.3 Substituted Benzyl 4-Chloro-4-Ketohexanoates (Acid Chlorides) 8

1.4 γ-Ketoesters (Substituted Benzyl 4-Ketohexanoates) 9

1.5 Estimation of Biological Activity 13

1.5.1 Inhibition zones 14

1.5.2 Minimum inhibitory concentrations (MICs) 14

1.6 Aims and Objectives 16

Chapter 2: RESULTS AND DISCUSSION 17

2. RESULTS AND DISCUSSION 18

2.1 Characterization Monoester 1-56 18

2.2 Characterization of Monoester 30 19

2.3 Characterization of Acid Chlorides (1a-56a) 21

2.3.1 Characterization of 1a 21

2.3.2 Characterization of 30a 22

2.3.3 Characterization of γ-Ketoesters (1b-56b) 23

2.3.4 Characterization of 30b 24

2.4 Characterization of 57-60, 57a-60a and 57b-60b 24

2.5 Biological Activity and Structure Activity Relationship 1-29 25

Chapter 3: EXPERIMENTAL 27

3 EXPERIMENTAL 28

3.1 General Experimental 28

3.1.1 Fungal strains 28

3.1.2 Bacterial strains 29

ii

3.2 General Procedure for the Preparation of Monoesters 1-29 29

3.3 General Procedure for the Preparation of Monoesters 30-56 29

3.4 General Procedure for the Preparation of Monoesters (57-60) 30

3.5 General Procedure for Preparation of Mono-/di-/tri-benzyl) 4-Chloroketobutanoates

(1a-56a)

30

3.6 General Procedure for Alkyl 4-Chloro-4-Ketobutanoates (57a-60a) 30

3.7 Preparation of Diethyl Cadmium Reagent 31

3.8 General Procedure for Preparation of Benzyl-γ-Ketohexanoates (1b-56b) 31

3.9 General Procedure for Alkyl γ-Ketohexanoates (57b-60b) 31

3.10 Biological Activity 32

3.10.1 Measurement of zones of inhibition 32

3.10.2 Measurement of MICs 33

3.10.2.1 Preparation of stock solutions 33

3.10.3 Solutions of working concentrarions 33

3.10.4 Controls 34

3.12 Physical and Spectroscopic Data of Monoesters 1-60 35

3.12.1 2-Methoxybenzyl hydrogen succinate (1) 35



3.12.4 2,3-Dimethoxybenzyl hydrogen succinate (4) 36

3.12.5 2, 4-Dimethoxybenzyl hydrogen succinate (5) 36




3.12.9 2-Nirobenzyl hydrogen succinate (9) 37



3.12.12 2-Chlorobenzyl hydrogen succinate (12) 38



3.12.15 2-Bromobenzyl hydrogen succinate (15) 39



3.12.18 2-Iodobenzyl hydrogen succinate (18) 40



3.12.21 2-Methylbenzyl hydrogen succinate (21) 41



3.12.24 2-Hydroxybenzyl hydrogen succinate (24) 42



3.12.27 2-Aminobenzyl hydrogen succinate (27) 43



3.12.30 2,4-Dichlorobenzyl hydrogen succinate (30) 44


iii




3.12.35 2,3-Difluorobenzyl hydrogen succinate (35) 46




3.12.39 2,4-Dimethylbenzyl hydrogen succinate (39) 47




3.12.43 3,4-Dinitrobenzyl hydrogen succinate (43) 49

3.12.44 3,5-Dinitrobenzyl hydrogen succinate (44) 49

3.12.45 2,6-Diethoxybenzyl hydrogen succinate (45) 49

3.12.46 3,4-Diethoxybenzyl hydrogen succinate (46) 50

3.12.47 3,5-Dihydroxybenzyl hydrogen succinate (47) 50

3.12.48 2,3,4-Trimetxoxybenzyl hydrogen succinate (48) 50

3.12.49 2,4,5-Trimethoxbenzyl hydrogen succinate (49) 51

3.12.50 3,4,5-Trimethoxybenzyl hydrogen succinate (50) 51

3.12.51 3-Methoxy-4-nitrobenzyl hydrogen succinate (51) 51



3.12.54 2,4,6-Trichlorobenzyl hydrogen succinate (54) 52

3.12.55 2,4,5-Tribromobenzyl hydrogen succinate (55) 53

3.12.56 2,4,6-Trimethylbenzyl hydrogen succinate (56) 53

3.12.57 Isopropyl hydrogen succinate (57) 53

3.12.58 Isobutyl hydrogen succinate (58) 53

3.12.59 Isopentyl hydrogen succinate (59) 53

3.12.60 Benzyl hydrogen succinate (60) 54

3.13 Physical Characteristics of Acid Halides 1a-60a 54

3.13.1 2-Methoxybenzyl 4-chloro-4-ketobutanoate (1a) 54



3.13.4 2,3-Dimethoxybenzyl 4-chloro-4-ketobutanoate (4a) 55





3.13.9 2-Nitrobenzyl 4-chloro-4-ketobutanoate (9a) 56



3.13.12 2-Chlorobenzyl 4-chloro-4-ketobutanoate (12a) 57



3.13.15 2-Bromobenzyl 4-chloro-4-ketobutanoate (15a) 58



iv

3.13.18 2-Iodobenzyl 4-chloro-4-ketobutanoate (18a) 58



3.13.21 2-Methylbenzyl 4-chloro-4-ketobutanoate (21a) 59



3.13.24 2-Hydroxybenzyl 4-chloro-4-ketobutanoate (24a) 60



3.13.27 2-Aminobenzyl 4-chloro-4-ketobutanoate (27a) 61



3.13.30 2,4-Dichlorobenzyl 4-chloro-4-ketobutanoate (30a) 62





3.13.35 2,3-Difluorobenzyl 4-chloro-4-ketobutanoate (35a) 64




3.13.39 2,4-Dimethylbenzyl 4-chloro-4-ketobutanoate (39a) 65




3.13.43 3,4-Dinitrobenzyl 4-chloro-4-ketobutanoate (43a) 66

3.13.44 3,5-Dinitrobenzyl 4-chloro-4-ketobutanoate (44a) 67

3.13.45 2,6-Diethoxybenzyl 4-chloro-4-ketobutanoate (45b) 67

3.13.46 3,4-Diethoxybenzyl 4-chloro-4-ketobutanoate (46a) 67

3.13.47 3,5-Dihydroxybenzyl 4-chloro-4-ketobutanoate (47a) 68

3.13.48 2,3,4-Trimethoxybenzyl 4-chloro-4-ketobutanoate (48a) 68

3.13.49 2,4,5-Trimethoxbenzyl 4-chloro-4-ketobutanoate (49a) 68

3.13.50 3,4,5-Trimethoxybenzyl 4-chloro-4-ketobutanoate (50a) 69

3.13.51 3-Methoxy-4-nitrobenzyl 4-chloro-4-ketobutanoate (51a) 69



3.13.54 2,4,6-Trichlorobenzyl 4-chloro-4-ketobutanoate (54a) 70

3.13.55 2,4,5-Tribromobenzyl 4-chloro-4-ketobutanoate (55a) 70

3.13.56 2,4,6-Trimethylbenzyl 4-chloro-4-ketobutanoate (56a) 71

3.13.57 Isopropyl 4-chloro-4-ketobutanoate (57a) 71

3.13.58 Isopropyl 4-chloro-4-ketobutanoate (58a) 71

3.13.59 Isobutyl 4-chloro-4-ketobutanoate (59a) 71

3.13.60 Benzyl 4-chloro-4-ketobutanoate (60a) 71

3.14 Physical Characteristics of γ-Ketoesters 1b-56b 71

3.14.1 2-Methoxybenzyl γ-ketohexanoate (1b) 72



v

3.14.4 2,3-Dimethoxybenzyl γ-ketohexanoate (4b) 72





3.14.9 2-Nitrobenzyl γ-ketohexanoate (9b) 74



3.14.12 2-Chlorobenzyl γ-ketohexanoate (12b) 74



3.14.15 2-Bromrobenzyl γ-ketohexanoate (15b) 75



3.14.18 2-Iodobenzyl γ-ketohexanoate (18b) 76



3.14.21 2-Methylbenzyl γ-ketohexanoate (21b) 77



3.14.24 2-Hydroxybenzyl γ-ketohexanoate (24b) 78



3.14.27 2-Aminobenzyl γ-ketohexanoate (27b) 79



3.14.30 2,4-Dichlorobenzyl γ-ketohexanoate (30b) 80





3.14.35 2,3-Difluorobenzyl γ-ketohexanoate (35b) 81




3.14.39 2,4-Dimethylbenzyl γ-ketohexanoate (39b) 83




3.14.43 3,4-Dinitrobenzyl γ-ketohexanoate (43b) 84

3.14.44 3,5-Dinitrobenzyl γ-ketohexanoate (44b) 84

3.14.45 2,6-Diethoxybenzyl γ-ketohexanoate (45b) 85

3.14.46 3,4-Diethoxybenzyl γ-ketohexanoate (46b) 85

3.14.47 3,5-Dihydroxybenzyl γ-ketohexanoate (47b) 85

3.14.48 2,3,4-Trimetxoxybenzyl γ-ketohexanoate (48b) 86

3.14.49 2,4,5-Trimethoxbenzyl γ-ketohexanoate (49b) 86

3.14.50 3,4,5-Trimethoxybenzyl γ-ketohexanoate (50b) 86

vi

3.14.51 3-Methoxy-4-nitrobenzyl γ-ketohexanoate (51b) 86



3.14.54 2,4,6-Trichlorobenzyl γ-ketohexanoate (54b) 87

3.14.55 2,4,5-Tribromobenzyl γ-ketohexanoate (55b) 88

3.14.56 2,4,6-Trimethylbenzyl γ-ketohexanoate (56b) 88

3.14.57 Isopropyl γ-ketohexanoate (57b) 88

3.14.58 Isobutyl γ-ketohexanoate (58b) 89

3.14.59 Isopentyl γ-ketohexanoate (59b) 89

3.14.60 Benzyl γ-ketohexanoate (60) 89

BIBLIOGRAPHY 129

PART-II 141

Chapter 1: INTRODUCTION 142

1 Introduction 143

1.1 General Introduction 143

1.2 The Umbelliferae 145

Chapter 2: RESULTS AND DISCUSSION 147

2 RESULTS AND DISCUSSION 148

2.1 Essential Oil 148

2.2 Isolates 149

2.2.1 1-Oxo-11β,21β-dihydroxyoleane (1) 149

2.2.2 1-Oxo-11β-hydroxy-21β-O-acetyloleane (2) 152

2.2.3 1-Oxo-11β-hydroxy-21β-O-angeloyloleane (3) 153

2.2.4 1-Oxo-11β-O-angeloyl-21β-O-acetyloleane (4) 154

2.2.5 1-Oxo-11β, 21β-O-dibenzoyloleane (5) 155

2.2.6 28-β-D-glucopyranosyl 2α,3α,19α,24-tetrahydroxy-12-ene-oleaenoate (6) 157

2.2.7 28-β-D-glucopyranosyl 2β,3α,19α,24-tetrahydroxy-12-ene-oleaenoate (7) 159

2.2.8 28-β-D-glucopyranosyl 2α,3β,19α,24-tetrahydroxy-12-ene-oleaenoate (8) 160

2.2.9 28-β-D-glucopyranosyl 2β,3β,19α,24-tetrahydroxy-12-ene-oleaenoate (9) 161

2.2.10 28-β-D-glucopyranosyl 2β,3β,19β,24-tetrahydroxy-12-ene-oleaenoate (10) 162

2.2.11 28-β-D-glucopyranosyl 2α-O-acetyl,3α,19α,24-trihydroxy-12-eneoleaenoate (11) 163

2.2.12 28-β-D-glucopyranosyl 3α-O-acetyl,2α,19α,24-triahydroxy-12-eneoleaenoate (12) 165

2.2.13 28-β-D-glucopyranosyl 19α-O-acetyl,2α,3α,24-trihydroxy-12-eneoleaenoate (13) 166

2.3 Biological Activity 167

2.3.1 Antibacterial and antifungal activity 167

2.3.2 Anticancer activity 167

Chapter 3: EXPERIMENTAL 169

3 EXPERIMENTAL 170

3.1 General Experimental Procedure 170

3.2 Microorganisms 170

vii

3.2.1 Fungi and bacteria 170

3.2.2 Cancer cells 171

3.3 Collection of Plant Material 171

3.4 Hydrodistillation of Essential Oil from Seeds 172

3.5 GC Analysis of Essential Oil of C. sativum 172

3.6 Extraction 172

3.6.1 Isolation of compounds 172

3.6.2 Crude seed extract, solvent fractionation and isolation of 6-13 177

3.7 Physical and Spectroscopic Data of Isolates 178

3.7.1 1-Oxo-11β,21β-dihydroxyoleane (1) 178

3.7.2 1-Oxo-11β-hydroxy-21β-O-acetyloleane (2) 178

3.7.3 1-Oxo-11β-hydroxy-21β-O-angeloyloleane (3) 179

3.7.4 1-Oxo-11β-O-angeloyl-21β-O-acetyloleane (4) 179

3.7.5 1-Oxo-11β, 21β-O-dibenzoyloleane (5) 179

3.7.6 28-β-D-Glucopyranosyl, 2α,3α,19α,24-tetrahydroxy-12-ene-oleaenoate (6) 180

3.7.7 28-β-D-Glucopyranosyl, 2β,3α,19α,24-tetrahydroxy-12-ene-oleaenoate (7) 180

3.7.8 28-β-D-Glucopyranosyl, 2α,3β,19α,24-tetrahydroxy-12-ene-oleaenoate (8) 180

3.7.9 28-β-D-Glucopyranosyl, 2β,3β,19α,24-tetrahydroxy-12-ene-oleaenoate (9) 181

3.7.10 28-β-D-Glucopyranosyl, 2β,3β,19α,24-tetrahydroxy-12-ene-oleaenoate (10) 181

3.7.11 28-β-D-Glucopyranosyl, 2α-O-acetyl,3α,19α,24-tetrahydroxy-12-ene-oleaenoate (11) 181

3.7.12 28-β-D-Glucopyranosyl, 3α-O-acetyl,2α,19α,24-tetrahydroxy-12-ene-oleaenoate (12) 181

3.7.13 28-β-D-Glucopyranosyl, 19α O-acetyl,2α,3α,24-tetrahydroxy-12-ene-oleaenoate (13) 182

3.8 Biological Activities 182

3.8.1 Antifungal and antibacterial activity 182

3.8.2 Cytotoxicity 184

3.9 Statistical Analysis 184

BIBLIOGRAPHY 210

PUBLISHED WORK 218

viii

LIST of TABLES

PART-1

Table Title Page #

1 Physical characteristics of 1a-20a 91

2 Physical characteristics of 1b-20b 93

3 1H-NMR (300 MHz, CDCl3, δ, multiplicity, J in Hz) of 1-20 95



6 13

C-NMR (75 MHz, CDCl3, δ, DEPT, multiplicity) of 1-14 98

7 13


8 13


9 13


10 1H-NMR (300 MHz, CDCl3, δ, multiplicity, J in Hz) of 1a-28a 102

11 1H-NMR (300 MHz, CDCl3, δ, multiplicity, J in Hz) of 29a-56a 103

12 13

C-NMR (75 MHz, CDCl3, δ) of 1a-28a 104

13 13

C-NMR (75 MHz, CDCl3, δ) of 29a-56a 105

14 1H-NMR (300 MHz, CDCl3, δ, multiplicity, J in Hz) of 1b-27b 106

15 1H-NMR (300 MHz, CDCl3, δ, multiplicity, J in Hz) of 28b-56b 107

16 13

C-NMR (75 MHz, CDCl3, δ) of 1b-19b 108

17 13


18 13


19 Yield, mps/bps and Elemental Analysis of 57-60 and 57a-60a 111

20 Yield and bps of 57b-60b for comparison 112

21 Spectroscopic data of 57-60, 57a-60a and 57b-60b 113

22 Spectroscopic data of 59b-60b 114

23 Inhibition zones of monoesters 1-29 against fungi and bacteria 115

24 MICs of monoesters 1-29 against fungi and bacteria 116

PART-II

1 Natural product as anticancer drugs 186

2 Quantitative analysis of EO of C. sativum Linn 191

3 1H-NMR (C5D5N, 300 MHz) of compounds 1-5 isolated from C. sativum L. Seeds 192

4 13

C-NMR (DEPT, C5D5N, 75 MHz) of compounds 1-5 isolated from C. sativum L.

Seeds

193

5 Inhibition zones of 1-5 isolated from C. sativum against bacteria 194

6 Inhibition zones of 1-5 isolated from C. sativum against fungi 195

7 MICs of 1-5 isolated from C. sativum against bacterial strains 196

8 MICs of 1-5 from C. sativum against fungal strains 197

9 MBCs of 1-5 from C. sativum against bacterial strains 198

10 MFCs of 1-5 from C. sativum Seeds against fungal strains 199

11 1H-NMR (300 MHz, C5D5N) data of 6-9 isolated from C. sativum 200

12 1H-NMR (300 MHz, C5D5N) data of 10-13 isolated from C. sativum 201

13 13

C-NMR (75 MHz, C5D5N) data of 6-13 isolated from C. sativum 202

14 IC50 values (µM) of Compounds 1-13 isolated from C. sativum L. for various cell

lines

203

ix

LIST OF FIGURES

Figure Title Page#

1 General structures for various krtoesters 118

2 Preparation of monoesters (1-60), acid chlorides (1a-60a) and γ-ketoesters 119

3 Atom numbering in compounds for assignment of spectral data 121

4 Tetrahedral intermediate from reaction of RCdCl with CH3COCl 122

5 Proposed mechanism for ketone formation from acid chlorides and diethyl

cadmium 123

6 1H (

13C) NMR and 2DNMR: (i) COSY-45

0 and HOHAHA Bold-bond (ii)

HMBC (iii) NOESY (iv) HMQC for compound 1.

124

7 Presentation of 2D NMR interactions in 30 125

8 Presentation of 2D NMR interactions in 1b 126

9 Presentation of 2D NMR in 30b 127

10 Fragmentation pattern of compound 1 127

PART-II

1 FTIR of essential oils obtained from C. sativum 205

2 COSY and HOHAHA; HMBC Interaction in 1-5 206

3 NOESY Interaction in 1-5 207

4 COSY-45°, HOHAHA and HMBC in 6-13 208

5 ROESY Interaction 6-13 209

LIST OF HISTOGRAMS

1 Zone of inhibition (mm) of essential oil and other subfractions against

fungi

187

2 Zone of inhibition of (mm) essential oil and other subfractions against

bacteria

188

3 MIC (μg) of essential oil and other subfractions against fungi 189

4 MIC (μg) of essential oil and other subfractions against bacteria 190

SCHEMES

1 Extraction of compounds 1-5 175

2 Extraction of compounds 6-13 176

x

ACKNOWLEDGEMENTS

All admiration and prayer is purely for the Omnipotent, the most Merciful, the

Compassionate, and the Omniscient Al-Mighty ALLAH, whose clemency resulted into my

success. The ALLAH, Who created me, bestowed me with sound health and faultless body

and enabled me to perform and compose my research work in the form of thesis. I wish to pay

homage to the most perfect personality of the world Hazrat MUHAMMAD (PBUH), who

enlightened our minds to recognize our Creator.

I am highly indebted to my learned and able supervisor Professor Dr. Imam Bakhsh

Baloch for his valuable guidance, untiring working habit, brotherly behaviour and methodical

understanding of the subject of organic chemistry. I am extremely thankful to Professor Dr

Musa Kaleem Baloch, for his valuable discussions suggestions and instructions throughout

my career in general and during my Ph. D research in particular. My special thanks are for the

Chairman Department of Chemistry, Professor Dr Azim Khan Khattak for his extended

support and facilities during my research work. My special thanks go to Professor Dr Sher

khan Sadozai for his moral support during my career. I am extremely grateful to all my

talented teachers and colleagues for technical and moral support. I am also thankful to all my

friends, laboratory and office staff for their support and encouragement during my Ph. D

work.

I am highly obliged to my parent Organization “Gomal University Dera Ismail Khan”

the only existence of it in Dera Ismail Khan city provided me not only with the basic and

advanced knowledge of organic chemistry but also honour, respect, and job.

My special thanks are for those who extended moral support during completion of Ph. D

thesis.

xi

My prayers and thanks are due for my parents (Late), brothers, sisters, wife and

children whose moral, financial and inspiring support resulted in achieving this goal.

In the last I would like to acknowledge the cooperation and moral support of my eldest

brother, “A. W. Malik Pricipal (R) Govt College of Technology” throughout my life.

Muhammad Iqbal

xii

ABBREVIATIONS

Ac Acetyl

Aq Aqueous

Ar Aryl

n-Bu n-Butyl

C Concentration

CHCl3 Chloroform

CH2Cl2 Dichloromethane

DMSO Dimethyl sulphoxide

EtOAc Ethyl acetate

EIMS Electron impact mass spectrum

ESIMS Electrospray ionization mass spectrum

EtOH Ethanol

t-BuOH tert-Butanol

Et Ethyl

FABMS Fast atom bombardment spectrum

G Gram(s)

M+ Molecular ion

Me Methyl

MHz Megahertz

Min Minute(s)

mL milliliter(s)

Mm millimole(s)

m. p. melting point

m/z mass to charge ratio (in mass spectrometry)

Ph Phenyl

i-Pr Isopropyl

Rf Retention factor (in Chromatography)

Rt Room temperature

S Second (s)

THF Tetrahydrofuran

TLC Thin layer chromatography

xiii

TECHNIQUES

BB Broad (decoupled) band

CC Column chromatography

COSY Correlated spectroscopy

DEPT Distortionless enhancement by polarization

transfer

EI-MS Electron impact mass spectrum

FAB-MS Fast atom bombardment mass spectrometry

GC/MS Gas chromatography mass spectrometry

HMBC Heteronuclear multiple bond connectivity

HMQC Heteronuclear multiple quantum coherence

HOHAHA Homonuclear Hartmann-Hahn Spectroscopy

HR-EIMS High resolution electron impact mass spectrum

IR Infrared spectrophotometry

NMR Nuclear magnetic resonance

NOESY Nuclear overhauser effect spectroscopy

ROESY Rotating Frame Overhouser Enhancement

Spectroscopy

RP-HPLC Reverse Phase High Performance

Chromatography

TLC Thin layer chromatography

UV Ultraviolet

1

ABSTRACT

The thesis consists of two parts. The part-I deals with the synthesis of γ-ketoesters using succinic

anhydride as a starting material. The part-II is about extraction, isolation, purification and

characterization of the chemical constituents from Coriandrum sativum Linn (Umbelliferae).

It is an established fact that γ-ketoesters and their precursors are valuable synthons in synthetic

organic chemistry. These intermediates have potential applications in the preparation of

compounds of significant importance. In view of the important applications of γ-ketoesters, a

three-step reaction approach has been employed for their synthesis via acid catalyzed ring

opening of succinic anhydride with substituted benzyl alcohols.

Step-1: Sixty (1-60) new monoesters of succinic acid were prepared using succinic anhydride

and alcohols and toluene as a solvent. The catalyst employed was p-toluene sulfonic acid.

Step-2: In this step the prepared monoesters on treatment with thionyl chloride were converted to

corresponding acid chlorides (1a-60a).

Step-3: Finally, using diethyl cadmium reagent, acid chlorides obtained in the step-2 were

converted into corresponding novel γ-ketohexanoates (1b-60b). Diethyl cadmium was prepared

by employing ethyl magnesium bromide and cadmium bromide.

The final products were characterized by elemental analysis and spectroscopic techniques such as

UV, IR, NMR (1H and

13C) 1D and 2D, COSY-45°, DEPT, HMQC, HMBC, NOE, ROESY,

NOESY, mass spectrometry etc.

Monoesters (1-29) were screened for biological activity against three fungi and three bacteria

following disk diffusion protocol for inhibition zones and agar dilution method for minimum

inhibitory concentrations. Their structure activity relationship is also discussed.

2

All of the monoesters except nitro substituted were found to be active against fungi and bacteria.

In Part-II seeds of Coriandrum sativum Linn (Umbelliferae) were hydrodistilled for essential oil

and the obtained oil was analyzed by gas chromatograogy for its components. β-Linalool with

retention time 16.575 min and %age 79.86 was identified as major component by GC-MS. The

obtained essential oil was subjected to antifungal and antibacterial activity. Inhibition zones,

minimum inhibitory concentrations (MICs) against fungi and bacteria were assessed. Results of

these experiments showed that the essential oil was active against investigated microbes.

The whole plant (seeds, leaves, stems, flowers) was extracted in 95% aqueous MeOH. The

methanolic extract on solvent fractionation and repeated column chromatography on silica gel

afforded a bio-active fraction. The fraction on repeated chromatotrax preparative TLC yielded

most active fraction which under HPLC RP-18 fractionation afforded thirteen (1-13) compounds.

According to our knowledge, since compounds 1-5 have not been reported earlier from plant

kingdom and hence can be declared as new entities. The obtained compounds 1-13 were

subjected to antifungal and antibacterial activity. Inhibition zones, minimum inhibitory

concentrations (MICs) against fungi and bacteria were assessed. For isolates (6-13), cytotoxicity

against cancer cell lines (HL-60, SMMC-7721, A-549, MCF-7, and SW-480) was probed. IC50 of

compounds 6-13 against human cancer cell lines HL-60 were also assessed. Compounds (1-3, 6-

13) displayed antimicrobial and cytotoxicity against used human cell lines.

Among the tetra-hydroxysaponins (6-9) displayed IC50 =1.37± 0.02, 1.29 ± 0.02, 1.28 ± 0.02, and

1.07 ± 0.02, respectively, and compounds 10-13 displayed significant activity with IC50=1.27±

0.02, 1.02 ± 0.02, 1.02 ± 0.02, and 0.87 ± 0.02, respectively, standard (acarbose) showed IC50

0.82 µM.

3

Structure and relative configuration of the compounds 1-13 was established after recording and

analysis of spectroscopic data such as IR, 1D-NMR and 2D NMR (COSY-45, HOHAHA,

HMQC, HMBC and ROESY) and mass measurements. The data revealed that isolated

compounds are oleane triterpenoids and named as: 1-Oxo-11,21-dihydroxyoleane (1), 1-Oxo-11-

hydroxy-21-O-acetyloleane (2), 1-Oxo-11-hydroxy-21-O-angeloyloleane (3), 1-Oxo-11-O-angelo

yl-21-O-acetyloleane (4), 1-Oxo-11,21-O-dibenzoyloleane (5), 28-β-D-Glucopyranosyl 2α,3α,

19α,24-tetrahydroxy-12-ene-oleaenoate (6), 28-β-D-Glucopyranosyl 2β,3α,19α,24-tetrahydroxy-

12-ene-oleaenoate (7), 28-β-D-Glucopyranosyl 2α,3β,19α,24-tetrahydroxy-12-ene-oleaenoate (8),

28-β-D-Glucopyranosyl 2β,3β,19α,24-tetrahydroxy-12-ene-oleaenoate (9), 28-β-D-Glucopyrano-

syl 2β,3β,19α,24-tetrahydroxy-12-ene-oleaenoate (10), 28-β-D-Glucopyranosyl 2α-O-acetyl

,3α,19α,24-tetrahydroxy-12-ene-oleaenoate (11), 28-β-D-Glucopyranosyl 3α-O-acetyl,2α,19α,24-

tetrahydroxy-12-ene-oleaenoate (12) and 28-β-D-Glucopyranosyl 19α-O-acetyl,2α,3α,24-tetrahy-

droxy-12-ene-oleaenoate (13).

4

PART-I

SYNTHESIS OF γ-KETOESTERS

VIA

ACID CATALYSED RING OPENING

OF

SUCCINIC ANHYDRIDE

WITH

SUBSTITUTED BENZYL ALCOHOLS

5

CHAPTER-1

INTRODUCTION

6

1. INTROUCTION

1.1. General Overview

It was believed that a vital force, which could only be present in living organisms, was essential

for the synthesis of organic compounds (Kinne-Saffran and Kinne, 1999). Consequently, the

scientists believed that organic compounds could only be synthesized in living organisms like

plants and animals (Kinne-Saffran and Kinne, 1999). However, during early nineteenth century,

Wohler synthesized urea by heating ammonium cyanate and resulted in a revolution in the

synthetic organic chemistry (Kinne-Saffran and Kinne, 1999). The synthesis of urea not only led

to the termination of the so called “Vital Force Theory” but also delivered a new foundation to

synthetic organic chemistry and opened a new gateway for multitude of organic compounds.

After that, the scientists focused all their efforts to synthesize new organic compounds and

resulted the synthesis of astonishing and marvelous compounds and drugs.

In fact, the syntheses of valuable compounds from simple and easily available starting materials

have been the goal of the scientists. Dicarboxylic acids on esterification with alcohols could give

monoesters as well as diesters. Succinic anhydride is another compound that has enjoyed many

applications in the synthesis of monoesters. It is an excellent starting material and is readily

available in pure form and has been used in the synthesis of mono and di acid derivatives

(Izquierdo et al., 2011, Ronsheim et al., 2002). These are key intermediates in the synthesis of

valuable compounds of industrial, agricultural, medicinal and economic importance (Hilgenkamp

and Zercher, 2001)

1.2. Benzyl Hydrogen Succinates (Monoesters)

Monoesters can be derived from acid anhydrides, esterification of dicarboxylic acids and

hydrolysis of diesters (Huang et al., 2005, Williams et al., 2001, 2002).

7

Succinic anhydride is one of the several useful starting materials that provide monoesters when

treated with alcohols (Huang et al., 2005). The chemistry of succinic anhydride and succinic acid

has numerous applications in the preparation of daily life products (Williams et al., 2001, 2002).

Polymers of butylene with succinic acid and copolymer with succinic and adipic acid were

biodegradable (Maeda et al., 2005). Succinic anhydride on reaction with magnesium diethoxide

and ethylene oxide had been converted to a succinic acid-ethylene oxide polymer (Maeda et al.,

1997). These are also used in magnetic resonance imaging, gene transfection, tissue sealing and

drug delivery system (Luman et al., 2004). Monoesters are associated with amphiphilic and

lyotropic liquid crystal properties (Marchal et al., 2008; Nardello et al., 2006).

Monoesters are used in the preparation of isoprenoid building blocks (Ostermeier et al., 2003).

Using p-toluenesulphonic acid (TsOH) and D-glucose monoesters of succinc acid and ethanol

were prepared (Zhang et al., 2010). Other significant work in this respect is conversion of

succinic anhydride to monoesters on treatment with alcohols (van Rudloff, 1956).

Monoesters of succinates have been used as synthon in the preparation of flavonoids, terpenoids,

alkaloids, glycosides, vitamin K, vitamins, α-tocopherol, mammalian dolichols, archaic

membrane lipids, (S)-26-hydroxycholesterol, (-)-invictolide (Hekking et al., 2008; Jiang et al.,

2007). Monoesters from succinic anhydride are used as resolving agents ( Maeda et al., 1997).

Natural derivatives of succinic acid like dehydroandrographolide has been employed in in vitro

anti-HIV activities. Esters with diterpene alcohols displayed significant activity against HIV,

tumour and enzyme inhibition (Fujimaki, 1998). The derivatives of succinic acid with diterpenols

are even more biologically active against HIV, antitumor and in enzyme inhibition (Fujimaki,

1998; Vraka et al., 2006; Basak et al, 1999; Matsumura et al., 2000). Polymers formed with

glycerol are used as antiseptic (Vraka et al., 2006; Basak et al, 1999). The mnoesters also have

8

an effect on the chlorophyll contents and consequently on growth of plants (Todorov et al.,

1992). Some of them display phosphodiesterase inhibitory activity.

1.3. Substituted Benzyl 4-Chloro-4-Ketohexanoates (Acid Chlorides)

Acid chlorides are very reactive and synthetically useful compounds (Inoue et al., 2001). They

have been described as key intermediates in the homologation of carboxylic acids via Arndt

Eistert synthesis. Their use in Friedel-Crafts acylation of aromatic compounds to give ketones is

well recognized (Kobayashi et al., 1998; Yeung et al., 2002; Matsunami et al., 2007).

Acid chlorides have been frequently used in the synthesis of ketones on treatment with

organometallic reagents (von Rudloff, 1958). Numerous compounds of synthetic importance like

alkyl, aryl substituted heterocyclics, carbohydrates, amino acids and amides have been prepared

from acid chloride and zinc. Acid halides have been used as an acylating agent for several

triphenylphosphonium ylides (Meshram et al., 1998).

In another study 1-alkyl-2-methyleneaziridines and acid halides in non-polar solvents afforded

good yield of ring-opened en-amides as final products (Ennis et al., 2000). The selenoesters,

useful intermediates in the synthesis of natural products, were prepared by acid chlorides (Chen

and Zhang, 2000). The reaction of acid chlorides and diarylselenides catalyzed by Sm/CrCl3

afforded selenoesters (Liu and Zhang, 1999). Excellent yield of N, N-dimethyl amides were

obtained on reaction of acid chlorides with dimethylformamide (Lee et al., 2000; Sirinivasan and

Manisankar, 2010). Aliphatic, alicyclic, aromatic and heterocyclic acid chlorides have been

transformed into corresponding amides (Lee et al., 2000; Sirinivasan and Manisankar, 2010). An

excellent synthetic application of acid chlorides or acid anhydrides has been made in SmI2

catalyzed regioselective ring opening of three, four and five membered cyclic ethers (Kwon et al.,

2002).

9

Some alkoxy substituted acid chlorides readily under went acid catalyzed decarbonylation and

resulted in the formation of alkyl chloromethyl ethers.

Baruah and coworkers have successfully prepared β,γ-unsaturated ketones by treating acid

chlorides with allyl and crotyl bromide using finely divided cadmium as a catalyst and

tetrahydrofuran as a solvent (Baruah et all, 1996). Using organozinc reagents, ketones have been

synthesized from acid chlorides derived from aliphatic, cyclic and aromatic carboxylic acids in

the presence of palladium complexes as catalyst (Grey, 1983). Acid chlorides with organotin

compounds in the presence of Pd complex gave cross coupling reaction and yielded ketones.

Acid chlorides and alkyl boronic acids using palladium complex of triphenyphosphene and

CsCO3 as catalyst yielded ketones (Haddach and McCarthy, 1999). Acid chlorides are a good

source of acylsilanes (Geng et al., 1999). The above mentioned facts about synthetic utility of the

acid chlorides concluded beyond any doubt that these are intermediates of high synthetic utility in

the synthetic organic chemistry.

Therefore, a number of techniques have been proposed to synthesise such acid chlorides. Ruggli

and Maeder synthesized acid chlorides by treating carboxylic acid with oxaloyl chloride and

phosphorous oxychloride. Cason used thionyl chloride to transform monoesters into acid

chlorides. Carboxylic acids on treatment with cyanuric chloride (C3N3Cl3) in triethyl amine give

good yield of corresponding acid chlorides.

1.4. γ-Ketoesters (Substituted Benzyl 4-Ketohexanoates)

The name “ketoester” has been specified to bifunctional open and closed chain compounds

having ketone and ester functionality. The relative position of these two functions in the structure

of esters has been exploited to classify them into various categories. In α-ketoesters, the ketone

and ester functions are at adjacent positions (Agami et al., 2003) (Fig. 1a) and in β-ketoesters, the

10

ketone function is at 3 and ester at 1 position (Agami et al., 2003) (Fig. 1b). Bi-functional

compounds with a keto group at 4 and ester at 1 position are called 4-ketoester or gamma-

ketoester (γ- ketoesters, Fig. 1c). Both open chain and closed chain γ-ketoesters with varying size

of chain and ring have been synthesized. In the last decades, the agricultural, pharmaceutical and

medicinal importance of these esters has attracted the scientists (Ballini et al., 1998; Chaudhuri et

al., 2010). The ketoesters having ketone functionality at positions 5 and 6 are called δ and ω-

ketoesters, respectively (Ballini et al., 1998; Enders et al., 1997; King et al., 1996) (Fig. 1d).

γ-Ketoesters are used in the preparation of furans (Huang et al., 2005), benzotropolones (Kamal

et al., 2003), alkoxythiophenes (Sonpatki et al., 2001) and other heterocyles (Csende and Stajer,

2000). Their applications as intermediates in the preparation of cyclopentenone, natural flavor

(Starodubtseva, et al., 2008) and useful building blocks of pharmaceutical interests. Moreover,

ketoesters can be converted to various important compounds like butyrolactone, its dimer

(Williams et al., 2002) chiral γ-hydroxyesters (Benincori et al., 2004; Williams et al., 2001), γ-

hydroxycarboxylic acids (Csende and Stajer, 2000) and chiral auxiliaries (Romo and Meyers,

1991). The γ-ketoesters could be reduced to γ-hydroxyesters, which on subsequent hydrolysis

lead to γ-hydroxyl butyric acid (GHBA), a metabolite of γ-amino butyric acid present in

mammalian brain and used in the treatment of daytime sleepiness and cataplexy associated with

narcolepsy (Hog et al., 2008). Ketoesters can be reduced to corresponding secondary alcohols

using Cp2TiCl2 or Zn dust and water (Barrero et al., 2003). Presence of two functional groups in

ketoesters renders these compounds to own multifarious applications and their derivatives are

used in pharmaceutical and agrochemical industries (Nakamura et al., 2003; Pohmakotr et al.,

2007; Chaudhuri et al., 2010; Osawa et al., 2003). For example, the γ- ketoesters have been

catalytically reduced to chiral γ-hydroxyester and lactone derivatives by using RuCl3-BINAP-

11

HCl (Starodubtseva et al., 2008) and (COD) Ru(2-methylallyl)2-BINAP-HCl (Turova et al.,

2009). The γ- and δ-ketoesters and acids have also been reduced using yeasts to chiral hydroxyl

esters/acids which were further transformed to chiral lactones (Forzato et al., 2001; Benincori et

al., 2004). Their reduction to chiral hydroxyl esters has also been achieved by double

nucleophilic trifluoromethylation reactions (Singh et al., 2001). Chiral reduction of γ-ketoesters,

δ-ketoesters and lactones to corresponding optically active 1,4- and 1,5-diols by employing

Lipase from Pseudomonas cepacia have been reported (Kamal et al., 2003). These derivatives are

important synthons of some biologically active compounds like sex hormones and pheromones,

anti-asthma drugs, isoquinolines, lactam antibiotics, enzyme inhibitors, additives in food, and

perfumes (Huang et al., 2005; Starodubtseva et al., 2008; Heiss et al., 2001; Itoh et al., 2002;

Kizaki et al., 2001; Arends and Sheldon, 2004; Tojo and Fernández, 2006; Yamamoto et al.,

2002a, 2002b; Nanduri et al., 2001).

Keeping in view the importance of ketoesters and their derivatives numerous methods have been

put forward for their synthesis (Cason, 1946). Their synthesis by the reaction of aldehydes and

dimethyl maleate has also been reported (Starodubtseva et al., 2008). Reaction of methyl (or

ethyl) ester of 2,2,2-trifluoroacetic acid, dimethyl succinate and sodium in dry ether led to the

synthesis of γ-ketoesters which were further transformed to heterocyclic compounds (Wan et al.,

2009). Chiral 2-substituted γ-ketoesters have been prepared by the reaction of aryldiazoacetates

and enamines in the presence of copper and rhodium complex as catalysts in good yield (Zhao et

al., 2005). Synthesis of a number of γ-ketoesters has also been described by the reaction of

conjugated unsaturated ketones and α-nitrocycloalkanones (Ballini et al., 1998). Reaction of

aryldiazoacetates and enamines afforded γ-ketoesters via substituted enamines as intermediates

(Yan et al., 2004). These have also been prepared by a method involving cross coupling between

12

the monoester monochloride of succinic and glutaric acid and suitable Grignard compounds in

the presence of CuI (Benincori et al., 2004). Some other methods of practical worth for the

synthesis of γ-ketoesters include rearrangement of γ-hydroxy-α,β-unsaturated ketones in xylene

(Giardina et al., 2001). Kashima and coworkers prepared γ-ketoesters by the reaction of the

Grignard reagent and N-acylpyrazoles (Kashima et al., 2001). In aprotic solvents cyanide ion

catalyzed condensation of aromatic and heterocyclic aldehydes to α, β-unsaturated ketones,

esters, and nitriles resulted in the formation of corresponding γ-diketones, 4-oxo carboxylic

esters, and 4-oxo nitriles. (Ballini and Petrini, 2004). 4-Oxoesters were successfully prepared by

applying Nef reaction in which nitro group was converted to carbonyl yielding required esters

(Ballini and Petrini, 2004). Cowling and Mann prepared γ-ketoesters from oxyallyls and 2π-

systems (1, 1-dimethoxyethene). A good yield of γ-ketoesters was obtained by homologation of

aldehydes with methyl 5-triphenylphosphoranylidenelevulinate. γ-ketoesters were also obtained

by the reaction of bisdithiane, n-butyl lithium and alkyl or aryl halide in hexane. These have been

prepared from diethyl succinate via acetylation, hydrolysis, decarboxylation and esterification.

Formation of γ-diketones or γ-ketoesters from primary nitro alkanes with α, β-unsaturated

ketones or α, β-unsaturated esters, in the presence of two equivalents of 1, 8-Diazabicyclo [5.4.0]

undec-7-ene (DBU) have been reported (Ballini et al., 2002). Such compounds have been

synthesized from 3, 4-epoxyesters by treating with alcohols to get open chain 4-alkoxy-3-

hydroxyesters on treatment with BF3 (Izquierdo et al., 2011). The preparation of γ-ketoesters has

also been reported from the reaction of β-ketoesters and EtZnCH2I (Ronsheim et al., 2002). The

reaction of β-diketone with EtZnCH2I has been used to prepare γ-ketoesters (Hilgenkamp and

Zercher, 2001). Enamines and ethyldiazoacetate (EDA) in the presence of rhodium and copper

complex afforded γ-ketoesters (Huang et al., 2005). The γ-ketoesters have been prepared from the

13

esters having triple bond in acid part by addition of water in the presence of NaAuCl4 (Wang et

al., 2009). The γ-ketoesters were prepared from substituted γ -lactones and γ-lactone dimers using

Smarium iodide (SmI2) (Williams et al., 2001, 2002).

The revealed literature concluded that most of the techniques involved in synthesis are complex

in nature, require very expensive transition metal complexes, their ions or oxides as catalyst and

toxic solvents which may not be environment friendly (Starodubtseva et al., 2008; Forzato et al.,

2001; Benincori et al., 2004; Poliakoff et al., 2002; Arends and Sheldon, 2004; Tojo and

Fernández, 2006; Bandgar et al., 2005; Csende, 2002; Hilgenkamp and Zercher, 2001; Kashima

et al., 2001; Wang et al., 2009; Williams et al., 2001, 2002; Huang et al., 2005). Keeping in view

these facts some of the scientists came with anaerobic oxidation of hydroxyl esters to get

ketoesters. However, this technique faced criticism on the grounds that it is difficult to control

and to obtain a well-defined product ( Poliakoff et al., 2002).

The above aspects prompted us to synthesise several useful compounds (1-60, 1a-60a and 1b-

60b) by following a three-step reaction using diethyl ether as solvent and diethyl cadmium as

ethylating agent.

1.5. Estimation of Biological Activity

The prime objective of the scientists is to synthesize compounds having biological or chemical

applications. A compound is said to be biologically active if it possesses the ability to affect

(positive or negative) microorganisms, plants, animals or human beings on their exposure to it

(Andrew, 2001; Singh et al., 2011).

Though, all of the synthesized compounds do not display bio-activity but some of the synthesized

compounds have excellent biological activity and are used in many areas of health related

problems including various types of cancer (Ragavan et al., 2012). Therefore, it is necessary to

14

estimate the biological activity of the synthesized compounds to assess their applications or

utility. A technique commonly employed for the estimation of bioactivity of a compound is

treating certain pathogenic bacteria and fungi with it and comparing its activity to a standard drug

(EUCAST Documents, 2000, 2012; Doern, 2000). In estimating bioactivity the following

important parameters are measured.

1.5.1. Inhibition zones

Inhibition zones are qualitative measures of the biological activity of compounds against

microorganisms. The inhibition zone of a bioactive compound is defined as the diameter (mm) of

a clear circular zone (without any symptoms of growth of microorganism) around the filter paper

disk that was impregnated with the solution of the compound, placed on an appropriate

supportive media inoculated with the microorganism and incubated overnight. The inhibition

zones are directly related to the activity of the compound under consideration. The larger the

value of inhibition zone, the greater will be activity of the compound. Inhibition zone against

fungi and bacteria can be measured by employing either disk diffusion (EUCAST Definitive

Document, 2000) or agar well diffusion techniques (Khan et al., 2006).

1.5.2. Minimum inhibitory concentrations (MICs)

The second parameter which is of prime interest in estimating the bioactivity of the compounds is

minimum inhibitory concentration. It is a quantitative measurement of bioactivity which is

defined as the least or minimum quantity in mg/L (μg/mL) of a compound when added to a media

inhibits the growth of overnight culture of bacteria or fungi. The MICs values give information

about the activity of the compounds; low values correspond to high activities and vice versa. The

reported methods for the estimation of MICs of the compounds include agar dilution, agar well

dilution, broth dilution techniques and E-tests (Sen and Batra, 2012; Wanger et al., 1995; Clancy

15

and Ngyugen, 1999; Lozano-Chiu et al., 1998; Pfaller et al., 1998; (EUCAST Definitive

Document, 2000, 2012; Clinical Laboratory Standards Institute by Wayne 2006).

16

1.6. Aim and Objectives

Keeping in view the importance of γ-ketoesters and shortcomings pointed out in the techniques

involved in the synthesis of such compounds our aim was to synthesise these important

compounds by establishing a simple technique and investigate biological activity of the

synthesized compounds. For this purpose the following steps were taken;

a. Preparation of biologically active monoesters from succinic anhydride

b. Conversion of monoesters to acid chlorides

c. Preparation of γ-ketoesters from acid chlorides

d. Structural characterization of prepared compounds by various spectroscopic methods.

e. Biological activity of the synthesized monoesters

17

CHAPTER-2

RESULTS AND DISCUSSION

18

2. RESULTS AND DISCUSSION

The esterification of the alkyl, mono-, di-, and tri-substituted benzyl alcohols with succinic

anhydride afforded novel alkyl, mono-, di- and trisubstituted benzyl hydrogen succinates (1-60)

in good yield (Fig. 2). The TLC results and 1H-NMR data of the crude reaction mixture proved

that the esterification gave single product with reasonable high selectivity.

The structure of 1-60, 1a-60a, 1b-60b compounds were established by different spectroscopic

(UV, IR, NMR, and MS) techniques. The physical and spectroscopic data of compounds are

presented (Tables 1-22). The antimicrobial activities of monoesters 1-29 are presented in tables

23 and 24.

2.1. Characterization Monoester 1-56

Monoester 1 was obtained as colorless amorphous solid, m.p. 55-57 °C in 71% yield. The UV

spectrum of 1 displayed a peak at 253 nm (3.5) due to the presence of an aryl chromophore. The

IR spectrum displayed a broad band at 3412 for O-H, 1729 &1717 for C=O and 1210 cm-1

for C-

O. Presence of an aromatic moiety in 1 was evident from the peaks observed at 3023 cm-1

and

1495 cm-1

for Ar-H at and C=C, respectively.

1H-NMR spectrum displayed multiplet in the low-field region, at δ 6.93-7.22 due to aromatic

protons (Table 3). Two peaks at δ 2.85 (d, J = 6.8 Hz, 2H) and δ 2.75 (d, J = 6.8 Hz, 2H) were

assigned to methylene groups between acid and ester functions. Two singlets at δ 3.81 and δ 5.06

were due to protons of OCH3 and methylene (O-CH2) respectively. Presence of the carboxylic

acid functionality was also verified by peak at δ 12.41 which disappeared on addition of D2O.

13C-NMR spectrum of compound 1 displayed twelve peaks for twelve carbon atoms

corresponding to molecular formula C12H14O5 (Table 3). DEPT 13

C-NMR displayed one CH3,

three CH2, two C (carbonyl), four CH (aromatic) and two C atom (aromatic). Further, the

19

chemical shift of three carbons showed that these are attached to three oxygen atom. The 2D-

NMR (1H-

1H) spectra of 1 (COSY-45° and HOHAHA) disclosed two spin systems i.e., i) CH-CH

and ii) CH2-CH2 and were verified by the HMQC technique (Fig. 6).

Esterification in 1 was further confirmed by HMBC (Fig. 6) in which the CH2 resonating at δ

5.06 and the CH2 at δ 2.75 displayed interactions with aryl C-1,2 and the carbonyl group of ester

function (δ 172.7), respectively. In the same spectrum there was another interaction between the

methylene proton (δ 2.85) and the acidic carbon (δ 173.9) (Fig. 6). In the NOE 2D-NMR (1H-

1H)

spectrum, interactions were observed between two CH2 protons resonating at δ 2.75 adjacent to

the C=O group. Interaction between CH2 (δ 2.75) and C=O (δ 172.7) was also observed. The

mass fragmentation pattern of 1 is presented in Fig. 10. In the light of all the spectral evidence,

the formation of 1 was confirmed. Similarly, the structures of compounds (2-56) were established

in this way.

Final structural proof was obtained from elemental analysis that gave molecular formula

C12H14O5 (238.24): C, 60.50%; H, 5.92%; Found: C, 60.34%; H, 5.70%.

2.2. Characterization of Monoester 30

Monoester 30 was obtained as colorless, amorphous solid, m.p. 71-73 °C and yield 60% (3.33 g).

The UV spectrum of 30 showed a peak at 259 nm indicating the presence of an aryl

chromophore. In IR spectrum, the 30 displayed a broad band at 3386 cm-1

for OH. Presence of

aromatic moiety was evident by Ar-H peaks in the aromatic region at 3083 cm-1

. Peaks 1510 and

1473 cm-1

were assigned to aromatic C=C bonds. The significant and most important peak at

1722 cm-1

was assigned to carboxylic group (COOH) while peak at 1773 cm-1

was attributed to

ester group. Peaks at 1247, 1123 and 1022 cm-1

were due to C-O stretching. Presence of C-Cl

bond was revealed by the peaks near 723 cm-1

.

20

The 1

H-NMR spectrum of compound 30 displayed a multiplet in the low-field region, at δ 7.36-

7.87 for three aromatic protons (Table 4). Two peaks at δ 2.93 (d, J = 6.8 Hz, 2H) and δ 2.82 (d,

J = 6.8 Hz, 2H) were assigned to methylene groups. Singlet at δ 5.24 for 2H was due to protons

of oxymethylene (O-CH2). Presence of the carboxylic acid protons was also verified by proton

resonating at δ 11.82 which disappeared on addition of D2O. 13

C-NMR spectrum displayed

eleven peaks for eleven carbon atoms corresponding to molecular formula C11H10Cl2O4 (Table 8).

Broad band (BB) and DEPT 13

C-NMR displayed three CH2, two C (carbonyl), four CH

(aromatic) and four C atom. 13

C-NMR spectrum, of 30 displayed two low field singlets at δ 171.3

and 176.9 and were assigned to carboxylic (C=O) and ester (C=O) carbons, respectively. In the

same NMR two triplets at δ 32.2 and 33.2 were assigned to two methylene groups flanked by

ester and acid functions whereas CH2O carbon gave a triplet as δ 65.8. 13

C-NMR spectrum also

displayed three singlets at δ 137.6, 135.8, 131.4, two doublets at δ131.4d, 128.9 and a singlet at δ

134.1 were due to six aromatic carbons.

COSY-45° and HOHAHA revealed spin systems; i) CH-CH and ii) CH2-CH2 verified by the

HMQC technique (Fig. 7). The molecular formula was confirmed by observing (M+) ion peak in

HR-MS at m/z 277.10. Analytical data afforded C, 47.73%; H, 3.42%, Cl, 25.66% for molecular

formula C11H10Cl2O4. Calculated % are C, 47.68%; H, 3.64 % and Cl 25.59%.

In HMBC (Fig. 7) CH2 resonating at δ 2.93 and δ 2.82 displayed interactions with acid (C=O)

and ester (C=O). A oxymethylene group (CH2O) resonated at δ 5.24 showed interaction with aryl

C-1 and CO2 (δ 171.3) (Fig. 7). In the spectrum, interactions were observed between two CH2

protons resonated at δ 2.82. All above mentioned facts proved the formation of 30. Similarly, the

structures of compounds (2-56) were established in this way.

In the DEPT (13

C-NMR) spectrum, compound 30 displayed eleven peaks for eleven carbon atoms

21

corresponding to molecular formula C11H10Cl2O4. DEPT 13

C-NMR displayed three CH2, three

CH (aromatic) and five C atoms.

Further, the chemical shift of two carbons showed that these are attached to two oxygen atoms.

The 2D-NMR (1H-

1H) spectra of 30 (COSY-45° and HOHAHA) disclosed two fragments: i) CH-

CH and ii) CH2-CH2 and were verified by the HMQC technique (Fig. 7). Esterification was

further confirmed by HMBC in which the CH2 resonating at δH 5.24 and the CH2 at δH 2.82

displayed interactions with aryl C-1,2 and the carbonyl group of ester function (δC 171.3) ,

respectively. In the same spectrum there was another interaction between the methylene proton

(δH 2.85) and the acidic carbon (δC 176.9) (Fig. 7). In the ROESY 2D-NMR (1H-

1H) spectrum,

interactions were observed between two CH2 protons resonating at δH 2.82 adjacent to the C=O

group. Interaction between CH2 (δH 2.82) and C=O (δC 171.3) was also observed. In the light of

all the spectral evidence, the formation of 30 was confirmed. The structures of synthesized (31-

56) compounds were established in this way.

2.3. Characterization of Chlorides 1a-56a

In the present study sixty (1a-60a) acid halides were prepared from sixty monoesters (1-60).

Their formation was confirmed by taking physical data and spectroscopic data analysis (Tables 1-

22). Compounds 1a, 30a and 57a were considered as representatives of all the acid halides and

their structural characterization is discussed in detail.

2.3.1. Characterization of 1a

The principal change on transforming 11a is the replacement of hydroxyl group (OH) by a

chloride (Cl). Therefore, the absence of OH peak in the IR spectrum of 1a was the strong

evidence supporting the formation of 1a. The IR spectrum of 1a showed no OH peak for the

initial monoester 1. The two new peaks observed (723 cm-1

and 1792 cm-1

) were assigned to C-Cl

22

and Cl-C=O, respectively, confirming the formation of 1a. In the 13

C-NMR spectra, a peak at δ

175.6 supported the formation of 1a; this was also verified by HMBC spectrum, in which the

proton resonating at δ 2.74, displayed interactions with carbon resonating at δ 175.6, was

attributed to COCl (Fig. 2). Others 1H- and

13C-NMR assignments were also in accordance with

the structure of 1a (Table 10 and 12). In the high resolution electron impact MS (HRMS)

spectrum, 1a displayed a molecular ion peak [M+] at m/z 256.6823 (calc. 256.6813)

corresponding to C12H13ClO4. Chemical analysis data of 1a supported the molecular formula

C12H13ClO4.

2.3.2. Characterization of 30a

The compound 30a was obtained as viscous oil in 78% yield (1.91 g). Its HR-MS spectrum

indicated [M+] at m/z 295.54 corresponding to molecular formula C11H9Cl3O3. Its UV spectrum

in ethanol absorbed at λmax 257.9 nm (log ε 3.7). The main difference in the IR spectra of 30 and

30a was the absence of OH peak for the initial monoester 30. Three aromatic protons (Ar-H, 3H)

displayed in the IR spectra a peak at 3086. Two peaks at 1721 and 1777 cm-1

in the IR spectra of

30a were assigned to ester (C=O) and acid chloride (1777, Cl-C=O), respectively. IR spectrum

30a showed peaks for double bonds of benzyl ring at 1610, 1478 cm-1

(C=C). Ether linkage was

confirmed by the presence of bands at 1262, 1144, 1021 cm-1

(C-O). The C-Cl bond formation

was evident from a peak at 720 cm-1

, indicating the formation of C-Cl bond.

Formation of 30a was verified by 1H-NMR and

13C-NMR (Table 11 and 13). In the

13C-NMR

spectra, a peak at δ 176.3 was indicative Cl-C=O bond. In its 1H-NMR spectra compound 30a

displayed four peaks due to four different groups of protons. Peaks at δ 2.91(t, J=6.8 Hz) and

2.67 (t, J= 6.8 Hz) were attributed to two methylene groups. A singlet at δ 5.06 was due to CH2O

group. In 13

C-NMR 30a revealed eleven peaks for eleven different carbons (Table 13). The peaks

23

at δ 138.2, 132.3, 131.4, 133.2, 126.3 and 128.5 were for aromatic carbons. Two peaks at δ 38.5

and 28.4 were due to two methylene groups between ester and acid chloride. The assignment was

also verified by HMBC spectrum, in which the proton resonating at δ 2.67, displayed interactions

with carbon resonating at δ 176.3, was attributed to COCl. In the high resolution electron impact

HR-EIMS spectrum, 30a displayed a molecular ion peak at (295.55), calculated for C11H9Cl3O3

(295.55): C, 44.70%; H, 3.07%; Cl, 35.99%; Found: C, 44.68%; H, 3.11%; Cl 35.88%. The

structure of the remaining compounds (2a-56a) was established in the same way.

2.3.3. Characterization of 1b-56b

Our γ-ketoesters differ from those previously reported, mainly in the nature of R, R1, and R

2 (Fig.

1c). In almost all reported examples, R3 is an alkyl or an un-substituted benzyl

(Fig. 1c), whereas

in our case R is ethyl, R1 and R

2 are H, and R

3 is alkyl, mono-, di- or tri-substituted benzyl group

(1b-56b) (Figs. 1 and 2), isopropyl (57b), isobutyl (58b), isopentyl (59b) and benzyl (60b). The

carbon atoms in monoesters (1-60), acid chlorides (1a-60a) and ketoesters (1b-60b) are

numbered for spectral data assignment (Fig. 3).

Based on mechanism suggested by Bansal (1996) for reaction of dimethyl cadmium with acid

halides, we proposed the mechanism of ketohexanoates formation from the reaction of diethyl

cadmium with acid chlorides. Both dimethyl and diethyl cadmium have similarity in structure

and reactivity. This mechanism is further supported by Roberts and Caserio (1964), who reported

the formation of a tetrahedral intermediate from the reaction of RCdCl and CH3COCl (Fig. 4).

Proposed mechanism for the reaction of diethyl cadmium with acid halide is given in Fig. 5.

Conversion of 1a into 1b was established by the HR-EIMS displaying a molecular ion peak at

m/z 250.2905 for C14H18O4 (calc. 250.2903). 1D (1H-NMR and

13C-NMR) spectra similarly

supported the predicted formation of 1b (Fig. 1, Tables 2, 14 and 16). Its formation was further

24

verified by the presence of an ethyl moiety by displaying peaks at δ 1.05 (t, 3H, J = 7.4 Hz, C-6)

and 2.55 (q, 2H, J = 7.4 Hz, C-5) and 13

C-NMR peaks at 7.7 (C-6) and 36.1 (C-5). Formation of

1b was further established by 2D NMR techniques (Figs. 8).

2.3.4. Characterization of 30b

Compound 30b was obtained as viscous oil (yield 78%, 1.91 g). Its HR-EIMS showed molecular

ion (M+) peak at m/z 289.15 with relative intensity 33%. It exhibited UV-VIS absorption band in

ethanol solvent at λmax 258.5 nm (log ε 3.72) and was assigned to aromatic chromophore.

The compound 30b in IR displayed bands at 3076 (Ar-H), 1728 (CO2Ar), 1767 (C=O), 1615,

1479 (C=C), 1267, 1148, 1023 (C-O), 722 (C-Cl) cm-1

. The assignments are compatible with the

structure of 30b. Two additional peaks other than 30a at 1.05 (t, 7.4 Hz), 2.46 (q, 7.4 Hz) and

corresponding peaks in 13

C-NMR at 7.8 and 36.3 were assigned to newly introduced ethyl group

(Tables 15 and 17). Two signals in the 1H-NMR (Table 15) of 30b at δ 2.46 (q, 7.4 Hz) and 2.91

(t, 6.8 Hz) were assigned to two methylenes. A low field singlet at δ 5.06 was assigned to CH2O

protons. In the 13

C-NMR spectra (Table 17) of 30b, the other assignments are; δ 209.4 (C-4, C=O

of ketone, δ 32.3 (C-3, CH2), δ 29.1 (C-2, CH2), δ 171.8 (C-1, C=O of ester), δ 62.9 (C-1ʹʹ,

CH2O), δ 137.5 (C-1ʹ), δ 131.2 (C-2 ʹ), δ 129.3 (C-3 ʹ), δ 133.6 (C-4ʹ), δ 126.8 (C-5 ʹ) and δ 128.6

(C-6ʹ). Finally the structure of 30b was also confirmed by analytical data that led to the

calculation of molecular formula C13H14Cl2O3 (289.15): C, 54.00; H, 4.88; Cl, 24.52; Found: C,

54.05; H, 4.89; Cl, 24.51%. Formation of 30b was also confirmed by 2D NMR techniques (Fig.

9).

2.4. Characterization of 57-60, 57a-60a and 57b-60b

Reaction of succinic anhydride with isopropyl, isobutyl, isopentyl and benzyl alcohols led to

alkyl hydrogen succinates 57-60 which upon treatment with thionylchloride afforded the

25

corresponding 4-alkoxy-4-ketobutanoyl chlorides 57a-60a (Fig. 2). Reaction of 57a-60a with

diethyl cadmium, ethyl magnesium bromide or the corresponding Gilman reagent converted these

compounds to alkyl γ-ketohexanoates 57b-60b. The comparison of the results of the three

organometallic reagents is illustrated in Table-20; the yield was the highest utilizing the cadmium

reagent as compared to others and no side products were formed. The EI-MS spectrum of the

compound showed peak at m/z 57 and was attributed to the loss of keto group from the parent

molecule. The 1H-NMR spectrum of 57b displayed two new peaks at δ 1.08 (t, J = 7.3 Hz, 3H,

Me) and at δ 2.49 (q, J = 7.3 Hz, 2H, CH2CO). Its 13

C-NMR spectrum exhibited two extra peaks

as compared to 57a that were identified as CH3 and CH2 by DEPT-135 13

C-NMR. The 2D NMR

also confirmed the formation of γ-ketoester (57b). The physical, spectroscopic and the literature

data revealed that isobutyl hydrogen succinate (58) and isopentyl hydrogen succinate (59) have

been identified in wine. Benzyl hydrogen succinate (60b), mp 117-119 ºC has been synthesized

in 52% yield by exposure of benzyl β-formyl propionate to air for 30 days. In the series 57b-60b

only benzyl γ-ketohexanoate (60b) is known (Matsunami et al., 2007). This compound was

prepared in low yield (24%) by reaction of benzyl acrylate with excess propanoyl chloride in the

presence of Mg and in DMF as solvent. Our methodology gave 60b in 71% overall yield. The

spectroscopic data of 60b are identical with that reported in the literature (Matsunami et al.,

2007).

2.5. Biological Activity and Structure Activity Relationship 1-29

The results in vitro biological activity of the monoesters 1-29 against three fungi and three

bacteria are denoted in Tables 23 and 24. All of the monoesters were subjected to biological

activities using fungi and bacteria and the compounds showed antifungal/antibacterial activity

against Colletotrichum gloeosporioides, Alternaria brassicicola, Colletotrichum capsici,

26

Klebsiella pneumonia, Escherichia coli and Staphylococcus aureus except nitro substituted (9-

11). So far as structure activity relationships is concerned noticeable low activities of the

compounds having substituents linked through oxygen and having substituent at three position of

benzene ring (Tables 23 and 24). Higher activities were observed for compounds with substituent

at 2 and 4 position of benzene ring. Highest activity was revealed by halogenated monoesters

(12-20) in general and iodinated monoesters in particular (18-20) and was close to ketoconazole

and chloramphenicol standards.

27

CHAPTER-3

EXPERIMENTAL

28

3. EXPERIMENTAL

3.1. General Experimental

All the required chemicals (aryl alcohols, anhydride, p-toluenesulphonic acid anhydrous, toluene,

hexane, benzene, methanol, ethanol, ethyl acetate, diethyl ether, chloroform, sodium sulphate and

sodium bicarbonate) were purchased from Sigma-Aldrich (St. Louis, New York, USA). All the

reagents were of analytical grade and used as such, except toluene. The toluene was dried and

then stored over sodium metal before use. Purity of alcohols and succinic anhydride was checked

by taking IR and NMR spectra. The melting points were determined using Gallenkamp digital

melting point apparatus and are uncorrected.

The UV spectra were recorded in absolute MeOH employing IRMECO UV/VIS Model U-2020

spectrophotometer. IR spectra were obtained on a TENSOR 27 FT-IR spectrophotometer

supplied by Bruker, Ettiligen, Germany. 1H-NMR and

13C-NMR (1D-, 2D-NMR) spectra were

procured in CDCl3 at (1H) 300 MHz, (

13C) 75 MHz using Bruker Biospin, AMX 300 MHz FT

NMR spectrometer; trimethylsilane (TMS) was used as an internal reference. EI-MS were

acquired with a direct insertion probe on a double-focusing Finnigan MAT 112 at 70 eV. HR-

EIMS measurements were made on a JEOL HX 110 spectrometer. Column chromatography was

carried out using silica gel (PF254, mesh size 60-70), E. Merck, Darmstadt Germany; thin layer

chromatography was performed on pre-coated silica gel plate (20 × 20 cm, 0.2 mm thickness)

with UV fluorescence indicator (PF254), E. Merck, Darmstadt, Germany.

3.1.1. Fungal strains

The fungal agents investigated in this study were supplied from Laboratório de Antibiotics,

Universidad Federal de Santa Catarina and included Colletotrichum gloeosporioides, Alternaria

29

brassicicola, Colletotrichum capsici. Ketoconazole was used as standard drug and was

purchased from m/s SMS Pharmaceuticals Ltd., Hyderabad.

3.1.2. Bacterial strains

Bacterial strains included Klebsiella pneumonia NCTC 11228, Escherichia coli ATCC 25922

and Staphylococcus aureus ATCC 25923. Chloramphenicol from Sigma St. Lious, USA was

used as standards.

3.2. General Procedure for the Preparation of Monoesters 1-29

The mono- and disubstitutedbenzyl hydrogen succinates (1-29) were synthesized by adding 15

mmol of corresponding alcohol to succinic anhydride (1.5 g, 15 mmol), anhydrous p-

toluenesulfonic acid (12.5 mg, 0.06 mmol) and toluene (13 g, 15 mL) under nitrogen in a single-

necked round-bottom flask (100 mL), equipped with magnetic stirrer, Dean-Stark trap and a

reflux condenser. The solution was refluxed for 14 h and allowed to cool up to 25 °C. After

cooling, it was poured into saturated aqueous NaHCO3 solution (12.5 mL) and the organic layer

was extracted with hexane (3 × 25 mL). The organic phase was then washed with brine (10 mL),

dried over anhydrous Na2SO4 and the excess of the solvent was removed under vacuum to give a

resinous product. It was then subjected to column chromatography to get pure monoesters (1-29).

3.3. General Procedure for the Preparation of Monoesters 30-56

Monoesters 30-56 were synthesized by above procedure briefly, by mixing 20 mmol of alcohol to

succinic anhydride (20 mmol), anhydrous p-toluenesulfonic acid (0.08 mmol) and toluene (20

mL) under nitrogen atmosphere. The mixture was refluxed for variable times and allowed to cool

up to 25 °C (Table 1). After cooling, it was poured into saturated aqueous NaHCO3 solution (12.5

mL) and the organic layer was extracted with hexane (3 × 25 mL). The organic phase was then

washed with brine (10 mL), dried over anhydrous Na2SO4 and excess of the solvent was removed

30

under vacuum to give a resinous product. The mixture obtained was subjected to separation on

column chromatography using mixture of n-hexane-ethyl acetate (1:0→0:1) to get thirty fractions

(1-30). The fractions (17-22) were combined and re-chromatographed on preparative thin layer

chromatography using n-hexane-ethyl acetate (4:6) as an eluent which yielded colorless

amorphous solid pure compounds (30-56)

3.4. General Procedure for the Preparation of Monoesters (57-60)

Monoesters 57-60 were by adding together succinic anhydride (3.0 g, 30 mmol), anhydrous p-

toluene sulfonic acid (25 mg) and toluene (15 mL) and 30 mmol of the alcohols under nitrogen

were added. After 24 h at reflux, the solution was left to cool at 25 °C then poured into a

saturated aqueous NaHCO3 solution (25 mL) and the aqueous layer was extracted with hexane (3

× 25 mL). The combined organic (toluene and hexanes) layers were washed with brine (20 mL),

dried over anhydrous Na2SO4 and the solvent was removed under vacuum to give a resinous

product, which was subjected to column chromatography to afford the pure monoesters (57-60).

3.5. General Procedure for Preparation of Mono-/di-/tri-benzyl) 4-Chloroketobutanoates

(1a-56a)

Acid chlorides (1a-56a) were prepared by following a standard procedure (Von Rudloff, 1958).

In brief, the required monoester (1-56; 8 mmol) and thionyl chloride (1.64 mL, 22.5 mmol) were

mixed in a round-bottom reaction flask (100 mL) fitted with a reflux condenser. The mixture was

heated to 30-40 °C for different lengths of time (Table 1) and the excess of thionyl chloride was

removed by heating at reduced pressure. The residue was subjected to open column

chromatography on silica gel using a mixture of hexane/ethyl acetate (φr = 4: 1 → 1: 4) as an

eluent to obtain pure compounds 1a-56a.

31

3.6. General Procedure for Alkyl 4-Chloro-4-Ketobutanoates (57a-60a)

Compound 57 (3.6 g, 22.5 mmol) was mixed with thionyl chloride (3.26 mL, 45 mmol), and

warmed at 30-40 °C for 3 hrs. Then the excess of thionyl chloride was removed on a steam bath

under reduced pressure to afford a colorless resin (57a) which was purified by distillation at

reduced pressure.

3.7. Preparation of Diethyl Cadmium Reagent

The diethyl cadmium reagent was prepared from a freshly prepared Grignard solution by

following a standard protocol (Cason, 1946). Briefly, dry CdCl2 (2.15 g, 11.8 mmol) was added

to the Grignard reagent [prepared from ethyl bromide (1.06 g, 11.8 mmol) and magnesium metal

(0.26 g, 11.8 mmol) cooled to 0 °C] in a dry diethyl ether (60 mL), over a period of 10 min with

vigorous stirring. Stirring was continued further for an hour and was used as freshly prepared.

3.8. General Procedure for Preparation of Benzyl γ-Ketohexanoates (1b-56b)

γ-Ketohexanoates (1b-56b) were prepared from acid chlorides (1a-56a), by following a standard

methodology (Cason, 1946). Briefly, 4-benzyloxy-4-ketobutanoyl chloride (5 mmol) was slowly

added to the diethyl cadmium solution. After maintaining reflux for different lengths of time

(Table 2), the mixture was allowed to stand overnight. The material was then poured into a

beaker containing ice and aq. H2SO4 (1 M, 30 mL). The organic layer was extracted with diethyl

ether (3 × 20 mL), washed with an aq. NaHCO3 solution (20 mL) and dried over anhydrous

Na2SO4. After removal of the solvent, the obtained material was subjected to open column

chromatography on silica gel and elution was made with the mixture of hexane/ethyl acetate (φr =

4: 1 → 1: 4) to get pure compounds (1b-56b).

32

3.9. General Procedure for Alkyl γ-ketohexanoates (57b-60b) Preparation

Compound 57b was synthesized by addition of 10 mmol of 57a (1.78 g) over a period of 10

minutes to a solution of diethyl cadmium in diethyl ether. The mixture was refluxed for six hours

and allowed to stand overnight stirring. The reaction mixture was then poured into a beaker (500

mL) containing crushed ice (150 g) and 10% aqueous H2SO4 (30 mL). It was vigorously stirred

for 5 minutes more. The organic layer was extracted with diethyl ether (3 × 20 mL), washed with

50% aqueous NaHCO3 solution (40 mL) and dried over anhydrous Na2SO4. After removal of the

solvent, the obtained material was subjected to column chromatography using an ethyl

acetate/hexane [(1:9) → (2:8) → (3:7)] as eluent. The desired product 57b was obtained after

elution with ethyl acetate/hexane (3:7). Similarly, compounds 58b-60b were prepared by

following the above mentioned procedure.

3.10. Biological Activity

3.10.1. Measurement of zones of inhibition

Biological activity of the monoesters (1-29) in terms of inhibition zones (mm) was determined by

following the disk diffusion method (EUCAST, 2012). Shortly, Mueller-Hinton pre-poured plates

were used for measuring inhibition zones of 1-29 against bacteria and Mueller-Hinton Himedia

(Mueller-Hinton complemented with 5% defibrinated horse blood and 20 mg/L β-NAD) pre-

poured plates against fungi. The 24 hour pre-cultured bacteria and fungi was taken into sterile

saline (0.85-0.90%, 2mL) contained in six test tubes (5 mL) pre-incubated and sterilized at 120

°C for 30 minutes. By adding the saline or inoculum, the densities of the prepared suspensions

was visually adjusted to McFarland standard (0.5) to obtain the suspension having 1-2×108

CFU/mL. It was further diluted to 1:10 to the concentration of 1-2×107 CFU/mL. The inoculum

was applied to pre-poured plates with the help of a sterilized cotton swab. Monoesters (1-29,

33

500µg each) were dissolved in 500 µL of DMSO in six vials. Disk (6mm) were loaded with 200

µg (200µL/disk) of the solution of the monoesters and placed on inoculated petri plates to

allowed to dry for 10 minutes. The plates were then incubated over night at 28 ± 2 °C for fungi

and 35± 2 for bacteria. Diameter was measured using a caliper. Experiment was replicated twice.

3.10.2. Measurement of MICs

Minimum inhibitory concentration (mg/L=μg/mL) of the monoesters (1-29) was determined by

Agar dilution method (EUCAST, 2012). Mueller-Hinton Agar (Oxoid) was used for

antibacterial while Mueller-Hinton Fastidious (Oxoid) for fungi. First of all fourteen sterilized

containers (50 mL) were labeled 128, 56, 32, 16, 8, 4, 2, 1, 0.5, 0.25, a, b, c and d mg/L or

µg/mL; containers a-d were for controls. Three stock solutions A, B, and C of monoesters (1-29)

were then prepared in order to get working solutions in the concentration range of 128-0.25

mg/L.

3.10.2.1. Preparation of stock solutions

Stock solution A was prepared by dissolving monoester 20 mg in 2 mL of diluent DMSO to

obtain over all concentration 10,000 mg/L = 10,000µg/mL.

Stock solution B

To get a stock solution B, with concentration of 1000 mg/L = 1mg/mL = 1000µg/mL, a 100 µL

stock A was added to 900 µL of DMSO.

Stock solution C

To get a stock solution C, with concentration of 100 mg/L=0.1 mg/mL=100µL/mL, a 100 µL

from stock B was added to 900 µL of DMSO

34

3.10.3. Solutions of working concentration

From stock A, 256 µL were transferred to a container labeled 128, 128 µL to the container

labeled 64, 64 µL to the container labeled 32 and 32 µL to container the labeled 16. From stock

solution B, 160 µL were added to the container labeled 8, 80 µL to the container labeled 4, 40 µL

to the container labeled 2. From the stock solution C were moved 200 µL to the container labeled

1, 100 µL to the container labeled 0.5 and 50 µL to the container labeled 0.25.

3.10.4. Controls

Following control containers a-d were included.

a. No antibiotic was added to container labeled “a” it served as antibiotic free growth control.

b. Control “b” was set to ensure that the observed activity was not due to DMSO. In the presence

of DMSO when no monoester was added the fungi and bacteria `showed full growth. Therefore,

any antibacterial or antifungal effect due to DMSO was negated.

c. Control “c” was for chloramphenicol standard antibiotic drug.

d. Control “d” was for ketoconazole standard antifungal drug.

Muller-Hinton nutrient agar (Oxoid, ≈20 mL/container) was prepared in sterilized water

according to manufacturer’s recommendations, cooled to 50 °C and added to all containers

labeled 128-1 and control containers a-d to make the final volume 20 mL in each container. The

concentration of 1-29 in the containers labeled 128-1 then became 128-1 mg/L. Contents were

poured into 90 mm petri dishes, allowed to dry at room temperature for 5-10 minutes. The

inoculua of bacteria and fungi were prepared to have approximately 1-2×108 CFU/mL by

adjusting the turbidity of inoculum to McFarland standard (0.5). One drop of inoculum was

applied to each prepared plate within 15 minutes. The plates were incubated overnight in air at

35±2 °C for bacteria and 28±2 °C for fungi. Least concentration (mg/L or µg/mL) of the

35

monoester that inhibited more than 95% growth of overnight cultured bacteria or fungi was taken

as MIC.

3.12. Physical and Spectroscopic Data of Monoesters 1-60

Physical data; Yield (%), physical state, mps/bps, UU-VIS, IR and Mass/anlytical data for

compounds 1-56 are given below. 1H-, and

13C-NMR data of 1-56 is given in tables 3-9.

3.12.1. 2-Methoxybenzyl hydrogen succinate (1)

Colorless amorphous solid m.p. 55-57 °C; Yield 71%, 2.54 g (10.66

mmol); UV (EtOH) λmax nm (log ε): 253 (3.5); FT-IR (Neat) υmax

(cm-1

): 3412 (OH), 3023 (Ar-H), 1729 (C=O), 1717 (C=O), 1595,

1546, 1429 (C=C), 1239, 1127, 1026 (C-O); 1H-, and

13C-NMR (Table 3 and 6, respectively).

Analysis calculated for C12H14O5 (238.24): C, 60.50%; H, 5.92%; Found: C, 60.34%; H, 5.70%.


Colorless amorphous, solid m.p. 49-51 °C; Yield 68%, 2.43 g (10.28

mmol); UV (EtOH) λmaxnm (log ε): 257.3 (3.3); FT-IR (Neat) υmax

(cm-1

): 3300 (OH), 3123 (Ar-H), 1729 (C=O), 1717 (C=O), 1595,

1546 1429 (C=C, Ar-H), 1239, 1127, 1026 (C-O); 1H-, and

13C-NMR data are given in tables 3

and 6, respectively; Analysis calculated for C12H14O5 (238.24): C, 60.50%; H, 5.92%; Found: C,

60.36%; H, 5.75%.


Colorless amorphous solid m.p. 56-58 °C; Yield 74%, 2.64 g (11.09


(cm-1

): 3366 (OH) 3054 (Ar-H), 1729 (C=O), 1717 (C=O), 1595,

1546, 1429 (C=C, Ar-H), 1239, 1127, 1026 (C-O); 1H-, and

13C-NMR data is given in tables 3

36


60.23%; H, 5.99%.

3.12.4. 2,3-Dimethoxybenzyl hydrogen succinate (4)

Pale yellow, amorphous solid, m.p. 87-89 °C; Yield 77%, 3.1 g

(11.54 mmol); UV (EtOH) λ max nm (log ε): 255 (3.5);

FT-IR (Neat) υmax (cm-1

): 3355 (OH) 3068 (Ar-H), 2850 1729

(C=O), 1717 (C=O), 1592, 1538, 1435 (C=C, Ar-H), 1237, 1133, 1041 (C-O); 1H-, and

13C-NMR

data is given in tables 3 and 6, respectively; Analysis calculated for C13H16O6 (268.26): C,

58.20%; H, 6.01%; Found: C, 58.44%; H, 5.81%.

3.12.5. 2, 4-Dimethoxybenzyl hydrogen succinate (5)

Pale yellow, amorphous solid, m.p. 77-79 °C; Yield 78%,

3.14 g (11.69 mmol); UV (EtOH) λ max nm (log ε): 258 (3.5);


): 3355 (OH), 3090 (Ar-H), 1729

(C=O), 1717 (C=O), 1592, 1538, 1435, (C=C, Ar-H), 1237, 1133, 1041; 1H-, and

13C-NMR data

is given in tables 3 and 6, respectively; Analysis calculated for C13H16O6 (268.26): C, 58.20%; H,

6.01%; Found: C, 58.35%; H, 6.12%.


Pale yellow, amorphous solid, m.p. 81-84 °C; Yield 74%, 2.97

g (11.09 mmol); UV (EtOH) λmax nm (log ε): 253 (3.5);


): 3358 (OH) and 3030 (Ar-H), 2850,

1729 (C=O), 1717 (C=O), 1592, 1538, 1435, (C=C, Ar-H), 1237, 1133, 1041; 1H-, and

13C-NMR

data is given in tables 3 and 6, respectively; Analysis calculated for C13H16O6 (268.26): C,

58.20%; H, 6.01%; Found: C, 58.47%; H, 5.96%.

37



2.93 g (10.94 mmol); UV (EtOH) λmax nm (log ε): 259 (3.5);


): 3358 (OH), 3042 (Ar-H), 2850,

1729 (C=O), 1717 (C=O), 1592, 1538, 1435 (C=C, Ar-H), 1237, 1133, 1041; 1H-, and

13C-NMR

data is given in tables 3 and 6, respectively. Analysis calculated for C13H16O6 (268.26): C,

58.20%; H, 6.01%; Found: C, C, 58.18%; H, 6.23%.





): 3375 (OH), 3095 (Ar-H), 2850,

1729 (C=O), 1717 (C=O), 1592, 1538, 1435 (C=C, Ar-H),

1237, 1133, 1041(C-O); 1H-, and

13C-NMR data is given in tables 3 and 6, respectively; Analysis

calculated for C13H16O6 (268.26): C, 58.20%; H, 6.01%; Found: C, 58.37%; H, 6.09%.

3.12.9. 2-Nirobenzyl hydrogen succinate (9)

Yellow, amorphous solid m.p. 100-102 °C; Yield 55%, 2.09 g (8.25

mmol); UV (EtOH) λmax nm (log ε): 252 (3.5);


): 3400 (OH), 3056 (Ar-H), 2860, 1743

(C=O), 1723 (C=O), 1596, 1530, 1441 (C=C, Ar-H), 1375 (NO2), 1244, 1139, 1045; 1H-, and

13C-NMR data is given in tables 3 and 6, respectively; Analysis calculated for C11H11NO6

(253.21): C, 52.18%; H, 4.38, N, 5.33%; Found: C, 52.31%; H, 4.22, N, 5.27%.

38


Yellow, amorphous solid, m.p. 65-68 °C; Yield 59%, 2.24 g

(8.85 mmol); UV (EtOH) λmax nm (log ε): 253 (3.5);


): 3402 (OH), 3080 (Ar-H), 1739

(C=O), 1721 (C=O), 1597, 1539, 1440, (C=C, Ar-H), 1374 (NO2), 1249, 1137, 1044; 1H-, and


(253.21): C, 52.18%; H, 4.38, N, 5.33%; Found: C, 52.31%; H, 4.22, N, 5.27%.





): 3400 (OH), 3072 (Ar-H), 1740

(C=O), 1722 (C=O), 1595, 1535, 1440, (C=C, Ar-H), 1372 (NO2), 12408, 1136, 1045; 1H-, and


(253.21): C, 52.18%; H, 4.38, N, 5.33%; Found: C, 52.24%; H, 4.29, N, 5.21%.

3.12.12. 2-Chlorobenzyl hydrogen succinate (12)

Colorless, amorphous solid, m.p. 69-71 °C; Yield 58%, 2.11 g (8.7



): 3410 (OH), 3060 (Ar-H), 1740 (CH2C=O),

1715 (C=O), 1597, 1529, 1497, 1456, (C=C, Ar-H), 1387, 1251, 1237, 1134, 1208, 1094, 1047,

1015 (C-O), 723 (C-Cl); 1H-, and

13C-NMR data is given in tables 3 and 6, respectively;

Analysis calculated for C11H11ClO4 (242.66): C, 54.45%; H, 4.57%; Found: C, 54.77%; H,

4.64%.

39


Colorless, amorphous solid, m.p. 58-59 °C; Yield 58%, 2.11 g



): 3415 (OH), 3085 (Ar-H), 1739 (C=O),

1720 (C=O), 1599, 1525, 1452, (C=C, Ar-H), 1248, 1133, 1049 (C-O), 722 (C-Cl); 1H-, and

13C-

NMR data is given in tables 3 and 6, respectively; Analysis calculated for C11H11ClO4 (242.66):

C, 54.45%; H, 4.57%; Found: C, 54.68%; H, 4.54%.





): 3345 (OH), 3048 (Ar-H), 1738 (C=O),

1720 (C=O), 1597, 1529, 1456 (C=C, Ar-H), 1383, 1251, 1134, 1047 (C-O), 723 (C-Cl); 1H-, and

13C-NMR data is given in tables 3 and 6, respectively; Analysis calculated for C11H11ClO4

(242.66): C, 54.45%; H, 4.57%; Found: C, 54.71%; H, 4.58%.

3.12.15. 2-Bromobenzyl hydrogen succinate (15)

Pale brown, amorphous solid, m.p. 100-103 °C; Yield 67%, 2.39 g



): 3345 (OH), 3084 (Ar-H), 1736 (C=O),

1715 (C=O), 1596, 1527, 1453 (C=C, Ar-H), 1413, 1277, 1254, 1144, 1054 (C-O), 521 (C-Br);

1H-, and

13C-NMR data is given in tables 3 and 7, respectively; Analysis calculated for

C11H11BrO4 (287.11): C, 46.02%; H, 3.86%; Found: C, 46.23%; H, 3.96%.

40


Pale brown, amorphous solid, m.p. 77-79 °C; Yield 66%, 2.84 g



): 3345 (OH), 3033 (Ar-H), 1738 (C=O), 1717 (C=O), 1598, 1525, 1457,

(C=C, Ar-H), 1250, 1141, 1059, 522 (C-Br); 1H-, and

13C-NMR data is given in tables 3 and 7,

respectively; Analysis calculated for C11H11BrO4 (287.11): C, 46.02%; H, 3.86%; Found: C,

46.12%; H, 3.78%.


Pale brown, amorphous solid, m.p. 99-102 °C; Yield 70%, 3.02

g (10.5 mmol); UV (EtOH) λmax nm (log ε): 252 (3.5);


): 3377 (OH), 3059 (Ar-H), 1737 (C=O),

1714 (CH2C=O), 1598, 1519, 1457, (C=C, Ar-H), 1250, 1144, 1051 (C-O), 521 (C-Br); 1H-, and

13C-NMR data is given in tables 3 and 7, respectively; Analysis calculated for C11H11BrO4

(287.11): C, 46.02%; H, 3.86%; Found: C, 46.14%; H, 3.66%.

3.12.18. 2-Iodobenzyl hydrogen succinate (18)

Brown, amorphous solid, m.p. 120-124 °C; Yield 61%, 3.06 g (9.15



): 3377 (OH), 3024 (Ar-H), 1733 (C=O),

1715 (C=O), 1599, 1521, 1460 (C=C, Ar-H), 1255, 1153, 1054, 487 (C-I); 1H-, and

13C-NMR

data is given in tables 3 and 7, respectively; Analysis calculated for C11H11IO4 (334.11): C,

39.54%; H, 3.32%; Found: C, 39.34%; H, 3.21%.

41


Brown, amorphous solid, m.p. 92-94 °C; Yield 62%, 3.11 g (9.31



): 3376 (OH), 3059 (Ar-H), 1734

(CH2C=O), 1714 (C=O), 1601, 1524 1462, (C=C, Ar-H), 1253, 1152, 1052 (C-O), 486 (C-I). 1H-,

and 13

C-NMR data is given in tables 3 and 7, respectively; Analysis calculated for C11H11IO4

(334.11): C, 39.54%; H, 3.32%; Found: C, 39.28%; H, 3.27%.


Brown, amorphous solid, m.p. 120-131 °C; Yield 65%, 3.26 g



): 3376 (OH), 3071 (Ar-H), 1739 (C=O),

1718 (C=O), 1600, 1523, 1461 (C=C, Ar-H), 1253, 1151, 1050 (C-O), 486 (C-I); 1H-, and

13C-

NMR data is given in tables 3 and 7, respectively; Analysis calculated for C11H11IO4 (334.11): C,

39.54%; H, 3.32%; Found: C, 39.66%; H, 3.55%.

3.12.21. 2-Methylbenzyl hydrogen succinate (21)

Colorless amorphous solid, m.p. 51-53 °C; Yield 71%, 2.37 g (10.66

mmol); UV (EtOH) λmax nm (log ε): 253 (3.5); FT-IR (Neat) υmax (cm-

1): 3412 (OH), 3023 (Ar-H), 1729 (C=O), 1717 (C=O), 1595, 1546,

1429 (C=C), 1239, 1127, 1026 (C-O); 1H-, and


respectively; Analysis calculated for C12H14O4 (222.24): C, 64.85%; H, 6.35%; Found: C,

64.95%; H, 6.22%.

42


Colorless amorphous solid, m.p. 49-51 °C; Yield 68%, 2.28 g

(10.28 mmol); UV (EtOH) λmax nm (log ε): 257.3 (3.3);


): 3300 (OH), 3123 (Ar-H), 1729

(C=O), 1717 (C=O), 1595, 1546 1429 (C=C, Ar-H), 1239, 1127, 1026 (C-O); 1H-, and

13C-NMR

data is given in tables 4 and 7, respectively; Analysis calculated for C12H14O4 (222.24)

: C, 64.85%; H, 6.35%; Found: C, 64.74%; H, 6.46%.


Colorless amorphous solid, m.p. 54.56 °C; Yield 74%, 2.44 g

(11.09 mmol); UV (EtOH) λ max nm (log ε): 256 (3.5);


): 3366 (OH) 3054 (Ar-H), 1729 (C=O),

1717 (C=O), 1595, 1546, 1429 (C=C, Ar-H), 1239, 1127, 1026 (C-O); 1H-, and

13C-NMR data is

given in tables 4 and 7, respectively; Analysis calculated for C12H14O4 (222.24): C, 64.85%; H,

6.35%; Found: C, 64.67%; H, 6.55%.

3.12.24. 2-Hydroxybenzyl hydrogen succinate (24)

Pale yellow, amorphous solid, m.p. 121-123 °C; Yield 55%, 1.87 g



): 3400-3120 (OH), 3056 (Ar-H), 2860, 1743

(C=O), 1723 (C=O), 1596, 1530, 1441 (C=C, Ar-H), 1244, 1139, 1045; 1H-, and

13C-NMR data


5.39%; Found: C, 58.47%; H, 5.77%.

43





): 3402-3110 (OH), 3080 (Ar-H), 1739

(C=O), 1721 (C=O), 1597, 1539, 1440, (C=C, Ar-H), 1249, 1137, 1044; 1H-, and

13C-NMR data


5.39%; Found: C, 58.66%; H, 5.88%.





): 3400-3100 (OH), 3072 (Ar-H), 1740 (C=O), 1722 (C=O), 1595, 1535,

1440 (C=C, Ar-H), 12408, 1136, 1045; 1H-, and


respectively; Analysis calculated for C11H12O5 (224.21): C, 58.93%; H, 5.39%; Found: C,

58.81%; H, 5.44%.

3.12.27. 2-Aminobenzyl hydrogen succinate (27)

Yellow, amorphous solid, m.p. 115-117 °C; Yield 58%, 1.94 g (8.7



): 3410 (NH2 and OH), 3060 (Ar-H), 1740

(CH2CO), 1715 (CO), 1597, 1529, 1497, 1456, (C=C, Ar-H), 1387 (C-N), 1251, 1237, 1134,

1208, 1094, 1047, 1015 (C-O); 1H-, and

13C-NMR data is given in tables 4 and 7, respectively;

Analysis calculated for C11H13O4 (223.23): C, 59.19%; H, 5.87, N, 6.27%; Found: C, 59.29%; H,

5.76, N, 6.18%.

44





): 3415 (Broad, NH2 and OH), 3085

(Ar-H), 1739 (C=O), 1720 (C=O), 1599, 1525, 1452, (C=C, Ar-H), 1393 (C-N), 1248, 1133,

1049 (C-O); 1H-, and

13C-NMR data is given in tables 4 and 7, respectively; Analysis calculated

for C11H13O4 (223.23): C, 59.19%; H, 5.87, N, 6.27%; Found: C, 59.51; H, 5.81, N, 6.12%.





): 3345 (Broad, intense, NH2 and OH),

3048 (Ar-H), 1738 (C=O), 1720 (C=O), 1597, 1529, 1456 (C=C, Ar-H), 1383 (C-N), 1251, 1134,

1047 (C-O); 1H-, and

13C-NMR data is given in tables 4 and 8, respectively; Analysis calculated

for C11H13O4 (223.23): C, 59.19%; H, 5.87, N, 6.27%; Found: C, 59.46%; H, 5.64, N, 6.48%.

3.12.30. 2,4-Dichlorobenzyl hydrogen succinate (30)




): 3386 (OH), 3063 (Ar-H), 1722 (C=O),

1773 (C=O), 1510, 1473 (C=C), 1247, 1123, 1022 (C-O), 723 (C-Cl); 1H-, and

13C-NMR data is

given in tables 4 and 8, respectively; Analysis calculated for C11H10Cl2O4 (277.10): C, 47.68%;

H, 3.64 %, Cl 25.59%; Found: C, 47.37%; H, 3.42%, Cl, 25.66%.

45





): 3382 (OH), 3060 (Ar-H), 1725 (C=O),

1774 (C=O), 1561, 1423 (C=C), 1247, 1123, 1022 (C-O), 720 (C-Cl); 1H-, and

13C-NMR data is


H, 3.64 %, Cl 25.59; Found: C, 47.44%; H, 3.32%, Cl, 25.64%.




(cm-1

): 3385 (OH), 3023 (Ar-H), 1771 (C=O), 1726 (C=O), 1512,

1441 (C=C), 1249, 1111, 1014 (C-O), 724 (C-Cl); 1H-, and


and 8, respectively; Analysis calculated for C11H10Cl2O4 (277.10): C, 47.68%; H, 3.64 %, Cl

25.59; Found: C, 47.35%; H, 3.46%, Cl, 25.75%.



(13.2 mmol); UV (EtOH) λmax nm (log ε): 259 (3.6); FT-IR

(Neat) υmax (cm-1

): 3375 (OH), 3058 (Ar-H), 1778 (C=O), 1725

(C=O), 1594, 1415 (C=C), 1247, 1123, 1022 (C-O), 721 (C-Cl); 1H-, and

13C-NMR data is given

in tables 4 and 8, respectively; Analysis calculated for C11H10Cl2O4 (277.10): C, 47.68%; H, 3.64

%, Cl 25.59; Found: C, 47.49%; H, 3.71%, Cl, 25.45%.

46





): 3379 (OH), 3055 (Ar-H), 1764 (C=O),

1721 (C=O), 1529, 1432 (C=C), 1247, 1123, 1022 (C-O), 723 (C-Cl); 1H-, and

13C-NMR data is


H, 3.64 %, Cl, 25.59; Found: C, 47.53%; H, 3.36%, Cl, 25.61%.

3.12.35. 2,3-Difluorobenzyl hydrogen succinate (35)



(cm-1

): 3410 (OH), 3074 (Ar-H), 1770 (C=O), 1729 (C=O), 1601,

1408 (C=C), 1246, 1123, 1014 (C-O), 1155 (C-F); 1H-, and


and 8, respectively; Analysis calculated for C11H10F2O4 (244.19): C, 54.10%; H, 4.13; F, 15.56

%; Found: C, 54.23%; H, 4.25; F, 15.36%.





): 3315 (OH), 3069 (Ar-H), 1765 (C=O),

1728 (C=O), 1524, 1471 (C=C), 1236, 1115, 1020 (C-O) 1158 (C-F); 1H-, and

13C-NMR data is

given in tables 4 and 8, respectively; Analysis calculated for C11H10F2O4 (244.19): C, 54.10%; H,

4.13; F, 15.56 %; Found: C, 53.99%; H, 4.24; F, 15.32%.

47



(13.4 mmol); UV (EtOH) λmax nm (log ε): 256 (3.4); FT-IR (Neat)

υmax (cm-1

): 3422 (OH), 3068 (Ar-H), 1766 (C=O), 1726 (C=O),

1499, 1481 (C=C), 1252, 1121, 1025 (C-O), 1159 (C-F); 1H-, and

13C-NMR data is given in

tables 4 and 8, respectively; Analysis calculated for C11H10F2O4 (244.19): C, 54.10%; H, 4.13; F,

15.56 %; Found: C, 53.98%; H, 4.22; F, 15.44%.




(cm-1

): 3425 (OH), 3066 (Ar-H), 1764 (C=O), 1727 (C=O), 1499,

1402 (C=C), 1248, 1125, 1008 (C-O), 1155 (C-F); 1H-, and


and 8, respectively; Analysis calculated for C11H10F2O4 (244.19): C, 54.10%; H, 4.13; F, 15.56

%; Found: C, 54.18%; H, 4.18; F, 15.54%.

3.12.39. 2,4-Dimethylbenzyl hydrogen succinate (39)




): 3366 (OH), 3037 (Ar-H), 1756 (C=O),

1730 (C=O), 1505, 1411 (C=C), 1250, 1123, 1009 (C-O); 1H-, and


tables 4 and 8, respectively; Analysis calculated for C13H16O4 (236.26): C, 66.09%; H, 6.83%;

Found: C, 66.22%; H, 6.75%.

48





): 3360 (OH), 3040 (Ar-H), 1758 (C=O),

1721 (C=O), 1502, 1485 (C=C), 1245, 1135, 1014 (C-O); 1H-, and



Found: C, 66.25%; H, 6.74%.




(Neat) υmax (cm-1

): 3369 (OH), 3036 (Ar-H), 1756 (C=O), 1720

(C=O), 1501, 1477 (C=C), 1255, 1110, 1030 (C-O); 1H-, and



66.16%; H, 6.81%.


Colorless amorphous solid, m.p. 87-86 °C;Yield 69%, 3.26 g


(Neat) υmax (cm-1

): 3372 (OH), 3033 (Ar-H), 1755 (C=O),

1722 (C=O), 1554, 1491 (C=C), 1251, 1120, 1015 (C-O); 1H-, and



Found: C, 66.20%; H, 6.65%.

49

3.12.43. 3,4-Dinitrobenzyl hydrogen succinate (43)




): 3475 (OH), 3085 (Ar-H), 1776

(C=O), 1733 (C=O), 1519, 1452 (C=C), 1369 (NO2), 1246, 1123, 1022 (C-O); 1H-, and

13C-

NMR data is given in tables 5 and 9, respectively; Analysis calculated for C11H10N2O8 (298.21):

C, 44.30%; H, 3.38%, N, 9.39; Found: C, 44.46%; H, 3.32%, N, 9.36%.

3.12.44. 3,5-Dinitrobenzyl hydrogen succinate (44)




): 3466 (OH), 3087 (Ar-H), 1775

(C=O), 1731 (C=O), 1604, 1580, 1410 (C=C), 1378 (NO2), 1249, 1123, 1020 (C-O); 1H-, and

13C-NMR data is given in tables 5 and 9, respectively; Analysis calculated for C11H10N2O8

(298.21): C, 44.30%; H, 3.38%, N, 9.39; Found: C, 44.47%; H, 3.31%, N, 9.37%.

3.12.45. 2,6-Diethoxybenzyl hydrogen succinate (45)




): 3455 (OH), 3086 (Ar-H), 1776 (C=O),

1732 (C=O), 1505, 1496 (C=C), 1250, 1124, 1022 (C-O); 1H-, and



Found: C, 60.74%; H, 6.65%.

50

3.12.46. 3,4-Diethoxybenzyl hydrogen succinate (46)




): 3454 (OH), 3085 (Ar-H), 1775

(C=O), 1731 (C=O), 1506, 1495 (C=C), 1252, 1120, 1021 (C-O); 1H-, and

13C-NMR data is


6.80%; Found: C, 60.77%; H, 6.68%.

3.12.47. 3,5-Dihydroxybenzyl hydrogen succinate (47)



(Neat) υmax (cm-1

): 3450 (OH), 3082 (Ar-H), 1774 (C=O), 1730

(C=O), 1507, 1494 (C=C), 1265, 1132, 1025 (C-O); 1H-, and



55.16%; H, 4.97%.

3.12.48. 2,3,4-Trimethoxybenzyl hydrogen succinate (48)




): 3450 (OH), 3082 (Ar-H), 1774

(C=O), 1730 (C=O), 1508, 1498 (C=C), 1262, 1135, 1015 (C-O); 1H-, and

13C-NMR data is


6.08%; Found: C, 56.25%; H, 6.18%.

51

3.12.49. 2,4,5-Trimethoxbenzyl hydrogen succinate (49)



(Neat) υmax (cm-1

): 3445 (OH), 3078 (Ar-H), 1777 (C=O),

1733 (C=O), 1504, 1498 (C=C), 1260, 1150, 1023 (C-O); 1H-, and



Found: C, 56.27%; H, 6.17%.

3.12.50. 3,4,5-Trimethoxybenzyl hydrogen succinate (50)



(Neat) υmax (cm-1

): 3447 (OH), 3079 (Ar-H), 1778 (C=O),

1732 (C=O), 1501, 1492 (C=C), 1256, 1152, 1024 (C-O); 1H-, and



Found: C, 56.26%; H, 6.16%.

3.12.51. 3-Methoxy-4-nitrobenzyl hydrogen succinate (51)

Light yellow, amorphous solid, m.p. 108-110 °C; Yield 65%,



): 3398 (OH), 3069 (Ar-H), 1779

(C=O), 1730 (C=O), 1601, 1576, 1390 (C=C), 1377 (NO2), 1255, 1135, 1020 (C-O); 1H-, and


(283.07): C, 50.89%; H, 4.63%; N, 4.95%; Found: C, 50.77%; H, 4.73%; N, 4.4.85%.

52





): 3396 (OH), 3067 (Ar-H), 1777

(C=O), 1728 (C=O), 1607, 1585, 1388 (C=C), 1373 (NO2), 1253, 1133, 1018 (C-O); 1H-, and


(283.07): C, 50.89%; H, 4.63%; N, 4.95%; Found: C, 50.75%; H, 4.78%; N, 4.84%.





): 3393 (OH), 3064 (Ar-H), 1774

(C=O), 1725 (C=O), 1503, 1484 (C=C), 1372 (NO2), 1249, 1128, 1014 (C-O); 1H-, and

13C-

NMR data is given in tables 5 and 9, respectively; Analysis calculated for C12H13NO7 (283.07):

C, 50.89%; H, 4.63%; N, 4.95%; Found: C, 50.97%; H, 4.80%; N, 4.81%.

3.12.54. 2,4,6-Trichlorobenzyl hydrogen succinate (54)



(Neat) υmax (cm-1

): 3390 (OH), 3061 (Ar-H), 1779 (C=O), 1728

(C=O), 1612-1390 (C=C), 1248, 1125, 1010 (C-O), 729 (C-Cl); 1H-, and


in tables 5 and 9, respectively; Analysis calculated for C11H9Cl3O4 (311.55): C, 42.41%; H,

2.91%; Cl, 34.14%; Found: C, 42.49%; H, 2.86%; Cl, 34.22%.

53

3.12.55. 2,4,5-Tribromobenzyl hydrogen succinate (55)




): 3386 (OH), 3078 (Ar-H), 1778 (C=O),

1729 (C=O), 1514, 1491 (C=C), 1257, 1132, 1015 (C-O), 527 (C-Br); 1H-, and

13C-NMR data is

given in tables 5 and 9, respectively; Analysis calculated for C11H9Br3O4 (444.90): C, 29.70%; H,

2.04%; Br, 53.88%; Found: C, 29.62%; H, 2.22%; Br, 53.76%.

3.12.56. 2,4,6-Trimethylbenzyl hydrogen succinate (56)




): 3376 (OH), 3070 (Ar-H), 1775

(C=O), 1727 (C=O), 1612-1392 (C=C), 1254, 1133, 1016 (C-O); 1H-, and


in tables 5 and 9, respectively; Analysis calculated for C14H18O4 (250.29): C, 67.18%; H, 7.25%;

Found: C, 67.34%; H, 7.11%.

3.12.57. Isopropyl hydrogen succinate (57)

Physical data, yield (%), UU-VIS, IR, Mass/anlytical, 1H-, and

13C-NMR

of 57 is given in tables 19 and 21.

3.12.58. Isobutyl hydrogen succinate (58)


13C-

NMR of 58 is given in tables 19 and 21.

3.12.59. Isopentyl hydrogen succinate (59)


13C-


54

3.12.60. Benzyl hydrogen succinate (60)


13C-


3.13. Physical characteristics of acid halides 1a-60a

Yield (%), UU-VIS, IR and Mass/anlytical data for compounds 1a-56a are given below. Physical

characteristics of 1a-20a are given in table 1. 1H-, and

13C-NMR data of 57a-60a is given in

tables 19-21.

3.13.1. 2-Methoxybenzyl 4-chloro-4-ketobutanoate (1a)

Physical characteristics, yields (%) and analytical data are given in

table 1; UV-VIS (MeOH), λmax/nm (log ɛ): 278 (3.7); MS, m/z (Ir/%):

256.68 (11) (M+); FT-IR (Neat) υmax (cm

-1): 3056 (Ar-H), 1792 (Cl-

C=O), 1737 (C=O), 1595, 1546, (C=C, Ar-H), 1026 (C-O), 723 (C-Cl); 1H-, and

13C-NMR (300

MHz, 75 MHz, CDCl3): (Tables 10 and 12).




256.68 (13) (M+); IR, ~ /cm

-1: 3057 (Ar-H), 1793 (Cl-C=O), 1738

(C=O), 1592, 1541, (C=C, Ar-H), 1021 (C-O), 721 (C-Cl); 1H-, and

13C-NMR (300 MHz, 75

MHz, CDCl3): (Tables 10 and 12).


Physical characteristics, yields (%) and analytical data are given

in table 1; UV-VIS (MeOH), λmax/nm (log ɛ): 278 (3.7);

MS, m/z (Ir/%): 256.68 (16) (M+); FT-IR (Neat) υmax (cm

-1):

55

3057 (Ar-H), 1793 (Cl-C=O), 1738 (C=O), 1593, 1542, (C=C, Ar-H), 1023 (C-O), 722 (C-Cl);

1H-, and

13C-NMR (300 MHz, 75 MHz, CDCl3): (Tables 10 and 12).

3.13.4. 2,3-Dimethoxybenzyl 4-chloro-4-ketobutanoate (4a)




-1): 3065 (Ar-H), 1797 (Cl-

C=O), 1741 (C=O), 1598, 1547, (C=C), 1027 (C-O), 725 (C-Cl); 1H-, and

13C-NMR (300 MHz,

75 MHz, CDCl3): (Tables 10 and 12).





-1):

3067 (Ar-H), 1799 (Cl-C=O), 1743 (C=O), 1591, 1544, 1431 (C=C), Ar-H), 1245, 1135, 1029

(C-O), 727 (C-Cl); 1H-, and






-1):

3069 (Ar-H), 1790 (Cl-C=O), 1747 (C=O), 1594, 1541, 1433 (C=C), Ar-H), 1249, 1131, 1022

(C-O), 724 (C-Cl); 1H-, and





56


-1): 3077 (Ar-H), 1799 (Cl-C=O), 1738

(C=O), 1598, 1547, 1431 (C=C), Ar-H), 1241, 1129, 1021 (C-O), 725 (C-Cl); 1H-, and

13C-NMR

(300 MHz, 75 MHz, CDCl3): (Tables 10 and 12).





-1):

3046 (Ar-H), 1789 (Cl-C=O), 1733 (C=O), 1601, 1548, 1432 (C=C), Ar-H), 1243, 1127, 1019

(C-O), 726 (C-Cl); 1H-, and


3.13.9. 2-Nitrobenzyl 4-chloro-4-ketobutanoate (9a)




-1): 3085 (Ar-H), 1788 (Cl-

C=O), 1734 (C=O), 1602, 1549, 1433 (C=C), Ar-H), 1365 (NO2), 1244, 1128, 1018 (C-O), 724

(C-Cl); 1

H-, and 13

C-NMR (300 MHz, 75 MHz, CDCl3): (Tables 10 and 12).



table 1; UV-VIS (MeOH), λmax/nm (log ɛ): 238 (3.1);


-1): 3087

(Ar-H), 1786 (Cl-C=O), 1735 (C=O), 1603, 1559, 1443 (C=C), Ar-H), 1371 (NO2), 1255, 1138,

1028 (C-O), 722 (C-Cl); 1H-, and


57




MS, m/z (Ir/%): 271.66 (14) (M+);


): 3083 (Ar-H), 1785 (Cl-C=O), 1734 (C=O), 1601, 1558, and 1441

(C=C), Ar-H), 1375 (NO2), 1251, 1134, and 1025 (C-O), 727 (C-Cl); 1H-, and

13C-NMR (300


3.13.12. 2-Chlorobenzyl 4-chloro-4-ketobutanoate (12a)




-1): 3053 (Ar-H), 1787 (Cl-C=O), 1738

(C=O), 1603, 1551, 1439 (C=C), Ar-H), 1257, 1138, 1021 (C-O), 721 (C-Cl); 1H-, and

13C-NMR

(300 MHz, 75 MHz, CDCl3): (Tables 10 and 12).





-1): 3059 (Ar-

H), 1791 (Cl-C=O), 1736 (C=O), 1605, 1548, 1432 (C=C), Ar-H), 1251, 1132, 1019 (C-O), 726

(C-Cl); 1H-, and

13C-NMR data (Tables 10 and 12).





-1): 3061

58

(Ar-H), 1789 (Cl-C=O), 1737 (C=O), 1606, 1549, 1436 (C=C), Ar-H), 1252, 1131, 1018 (C-O),

724 (C-Cl); 1H-, and


3.13.15. 2-Bromobenzyl 4-chloro-4-ketobutanoate (15a)




-1): 3063 (Ar-

H), 1788 (Cl-C=O), 1739 (C=O), 1602, 1575, 1431 (C=C), Ar-H), 1258, 1139, 1028 (C-O), 722

(C-Cl), 521 (C-Br); 1H-, and






-1): 3064 (Ar-

H), 1786 (Cl-C=O), 1729 (C=O), 1599, 1567, 1443, (C=C), Ar-H), 1253, 1144, 1024, 723 (C-

Cl), 523 (C-Br); 1H-, and






-1): 3061

(Ar-H), 1787 (Cl-C=O), 1731 (C=O), 1598, 1569, 1441 (C=C), Ar-H), 1257, 1142, 1021, 725 (C-

Cl), 525 (C-Br); 1H-, and


3.13.18. 2-Iodobenzyl 4-chloro-4-ketobutanoate (18a)



59


-1): 3075 (Ar-H), 1791 (Cl-C=O), 1736

(C=O), 1593, 1561, 1446 (C=C), Ar-H), 1261, 1147, 1025, 723 (C-Cl), 487 (C-I); 1H-, and

13C-

NMR (300 MHz, 75 MHz, CDCl3): (Tables 10 and 12).





-1): 3077 (Ar-

H), 1793 (Cl-C=O), 1738 (C=O), 1595, 1562, 1448 (C=C), Ar-H), 1263, 1148, 1026, 723 (C-Cl),

485 (C-I); 1H-, and






-1): 3064 (Ar-H), 1786 (Cl-C=O), 1729

(C=O), 1599, 1567, 1443, (C=C), Ar-H), 1253, 1144, 1024, 723 (C-Cl), 523 (C-Br); 1H-, and

13C-


3.13.21. 2-Methylbenzyl 4-chloro-4-ketobutanoate (21a)

Yield 76 %, 1.82 g (6.16 mmol), viscous oil; MS, m/z (Ir/%): 240 (22)

(M+); UV-VIS (MeOH), λmax/nm (log ɛ): 258.5. (3.64); FT-IR (Neat)

υmax (cm-1

): 3083 (Ar-H), 1731 (C=O), 1779 (Cl-C=O), 1596, 1543,

1422 (C=C), 1243, 1122, 1020 (C-O); 1H-, and

13C-NMR (300 MHz, 75 MHz, CDCl3): (Tables

10 and 12); Analysis calculated for C12H13ClO3 (240.68): C, 59.88%; H, 5.44%; Cl, 14.73%;

Found: C, 59.67%; H, 5.53%; Cl14.78%.

60


Yield 75 %, 1.46 g (6.08 mmol), viscous oil; MS, m/z (Ir/%): 240.87

(22) (M+); UV-VIS (EtOH), λmax nm (log ε): 259.9 (3.47);


): 3099 (Ar-H), 1732 (C=O), 1782 (Cl-C=O),

1599, 1556 1435 (C=C, Ar-H), 1243, 1134, 1022 (C-O); 1H-, and

13C-NMR (300 MHz, 75 MHz,

CDCl3): (Tables 10 and 12); Analysis calculated for C12H13ClO3 (240.68): C, 59.88%; H, 5.44%;

Cl, 14.73%; Found: C, 59.65%; H, 5.59%; Cl 14.77%.


Yield 77 %, 1.50 g (6.24 mmol), viscous oil;

MS, m/z (Ir/%): 240.67 (22) (M+); UV-VIS (EtOH) λ max nm (log

ε): 263.4 (3.68); FT-IR (Neat) υmax (cm-1

): 3076 (Ar-H), 1728 (C=O), 1786 (Cl-C=O), 1598,

1547, 1434 (C=C, Ar-H), 1243, 1132, 1022 (C-O); 1H-, and



Cl, 14.73%; Found: C, 59.78%; H, 5.55%; Cl 14.71%.

3.13.24. 2-Hydroxybenzyl 4-chloro-4-ketobutanoate (24a)

Yield 75 %, 1.47 g (6.08 mmol), viscous oil;

MS, m/z (Ir/%): 242.66 (23) (M+);

UV-VIS (EtOH) λmax nm (log ε):

257.8 (3.63); FT-IR (Neat) υmax (cm-1

): 3365-3122 (OH), 3076 (Ar-

H), 1745 (C=O), 1791 (Cl-C=O), 1599, 1538, 1443 (C=C, Ar-H), 1240, 1133 (C-O); 1H-, and

13C-NMR (300 MHz, 75 MHz, CDCl3): (Tables 10 and 12); Analysis calculated for C11H11ClO4

(242.65): C, 54.45%; H, 4.57%; Cl, 14.61%; Found: C, 54.57%; H, 4.66%; Cl 14.55%.

61



(23) (M+); UV-VIS (EtOH) λmax nm (log ε): 259.5 (3.45);


): 3376-3118 (OH), 3096 (Ar-H), 1738 (C=O),

1783 (CO-Cl), 1618, 1554, 1448, (C=C, Ar-H), 1242, 1134, 1041 (C-O); 1H-, and

13C-NMR (300

MHz, 75 MHz, CDCl3): (Tables 10 and 12); Analysis calculated for C11H11ClO4 (242.65): C,

54.45%; H, 4.57%; Cl, 14.61%; Found: C, 54.56%; H, 4.64%; Cl 14.56%.


Yield 77 %, 1.51 g (6.24 mmol), viscous oil; MS, m/z (Ir/%):

242.64 (23) (M+); UV-VIS (EtOH) λmax nm (log ε): 256.7 (3.72);


): 3355-3120 (OH), 3078 (Ar-H), 1748 (C=O), 1781 (Cl-C=O), 1614,

1545, 1446 (C=C, Ar-H), 1224, 1133, 1040 (C-O); 1H-, and



Cl, 14.61%; Found: C, 54.59%; H, 4.67%; Cl 14.63%.

3.13.27. 2-Aminobenzyl 4-chloro-4-ketobutanoate (27a)

Yield 72%, 1.41 g (5.84 mmol), viscous oil; MS, m/z (Ir/%): 241.66

(21) (M+); UV-VIS (EtOH) λmax nm (log ε): 257.8 (3.92); FT-IR

(Neat) υmax (cm-1

): 3215 (NH2), 3088 (Ar-H), 1742 (C=O), 1788 (Cl-

C=O), 1607, 1532, 1499, 1458, (C=C, Ar-H), 1384 (C-N), 1257, 1231, 1130, 1202, 1098, 1042,

1012 (C-O); 1H-, and

13C-NMR (300 MHz, 75 MHz, CDCl3): (Tables 10 and 12); Analysis

calculated for C11H12ClNO3 (241.67): C, 54.67%; H, 5.00%; Cl, 14.67%; N, 5.80; Found: C,

54.64%; H, 4.92%; Cl 14.68%; N, 5.78%.

62





): 3215 (Broad, NH2), 3097 (Ar-H), 1738

(C=O), 1787 (Cl-C=O), 1592, 1520, 1442, (C=C, Ar-H), 1391 (C-N), 1246, 1135, 1045 (C-O);

1H-, and

13C-NMR (300 MHz, 75 MHz, CDCl3): (Tables 10 and 12); Analysis calculated for

C11H12ClNO3 (241.67): C, 54.67%; H, 5.00%; Cl, 14.67%; N, 5.80; Found: C, 54.65%; H,

4.91%; Cl 14.69%; N, 5.77%.


Yield 78%, 1.53 g (6.32 mmol), viscous oil; MS, m/z (Ir/%):



): 3232 (Broad, intense, NH2), 3085 (Ar-

H), 1739 (C=O), 1779 (Cl-C=O), 1607, 1592, 1458 (C=C, Ar-H), 1382 (C-N), 1263, 1154, 1020

(C-O); 1H-, and

13C-NMR (300 MHz, 75 MHz, CDCl3): (Tables 11 and 13); Analysis calculated

for C11H12ClNO3 (241.67): C, 54.67%; H, 5.00%; Cl, 14.67%; N, 5.80; Found: C, 54.65%; H,

4.91%; Cl 14.69%; N, 5.77%.

3.13.30. 2,4-Dichlorobenzyl 4-chloro-4-ketobutanoate (30a)




): 3086 (Ar-H), 1721 (C=O), 1777 (Cl-

C=O), 1610, 1478 (C=C), 1262, 1144, 1021 (C-O), 720 (C-Cl); 1H-, and

13C-NMR (300 MHz, 75

MHz, CDCl3): (Tables 11 and 13); Analysis calculated for C11H9Cl3O3 (295.55): C, 44.70%; H,

3.07%; Cl, 35.99%; Found: C, 44.68%; H, 3.11%; Cl 35.88%.

63





): 3087 (Ar-H), 1724 (C=O), 1776 (Cl-

C=O), 1609, 1433 (C=C), 1266, 1138, 1027 (C-O), 721 (C-Cl); 1H-, and

13C-NMR (300 MHz, 75

MHz, CDCl3): (Tables 11 and 13); Analysis calculated for C11H9Cl3O3 (295.55): C, 44.70%; H,

3.07%; Cl, 35.99%; Found: C, 44.69%; H, 3.03%; Cl 35.89%.





): 3083 (Ar-H), 1778 (Cl-C=O), 1727 (C=O),

1607, 1442 (C=C), 1248, 1121, 1011 (C-O), 726 (C-Cl); 1H-, and


CDCl3): (Tables 11 and 13); Analysis calculated for C11H9Cl3O3 (295.55): C, 44.70%; H, 3.07%;

Cl, 35.99%; Found: C, 44.68%; H, 3.02%; Cl 35.91%.





): 3066 (Ar-H), 1777 (Cl-C=O), 1726

(C=O), 1611, 1444 (C=C), 1255, 1132, 1029 (C-O), 724 (C-Cl); 1H-, and

13C-NMR (300 MHz,

75 MHz, CDCl3): (Tables 11 and 13); Analysis calculated for C11H9Cl3O3 (295.55): C, 44.70%;

H, 3.07%; Cl, 35.99%; Found: C, 44.67%; H, 3.09%; Cl 35.92%.

64





): 3095 (Ar-H), 1765 (Cl-C=O), 1722

(C=O), 1608, 1444 (C=C), 1248, 1129, 1022 (C-O), 727 (C-Cl); 1H-, and

13C-NMR (300 MHz,


H, 3.07%; Cl, 35.99%; Found: C, 44.71%; H, 3.05%; Cl 35.93%.

3.13.35. 2,3-Difluorobenzyl 4-chloro-4-ketobutanoate (35a)




): 3088 (Ar-H), 1776 (Cl-C=O), 1728 (C=O),

1606, 1478 (C=C), 1252, 1126, 1010 (C-O), 1150 (C-F); 1H-, and


CDCl3): (Tables 11 and 13); Analysis calculated for C11H9ClF2O3 (262.64): C, 50.30%; H,

3.45%; Cl, 13.50%; F, 14.47; Found: C, 50.28%; H, 3.47%; Cl, 13.52%; F, 14.44%.





): 3098 (Ar-H), 1766 (Cl-C=O), 1729

(C=O), 1594, 1476 (C=C), 1239, 1119, 1026 (C-O) 1151 (C-F); 1H-, and

13C-NMR (300 MHz,

75 MHz, CDCl3): (Tables 11 and 13); Analysis calculated for C11H9ClF2O3 (262.64): C, 50.30%;

H, 3.45%; Cl, 13.50%; F, 14.47; Found: C, 50.29%; H, 3.46%; Cl, 13.49%; F, 14.46%.

65





): 3099 (Ar-H), 1767 (Cl-C=O), 1727

(C=O), 1599, 1486 (C=C), 1258, 1122, 1025 (C-O), 1153 (C-F); 1H-, and

13C-NMR (300 MHz,

75 MHz, CDCl3): (Tables 11 and 13); Analysis calculated for C11H9ClF2O3 (262.64): C, 50.30%;

H, 3.45%; Cl, 13.50%; F, 14.47; Found: C, 50.28%; H, 3.47%; Cl, 13.48%; F, 14.49%.





): 3088 (Ar-H), 1765 (Cl-C=O), 1726 (C=O),

1597, 1406 (C=C), 1245, 1120, 1008 (C-O), 1157 (C-F); 1H-, and


CDCl3): (Tables 11 and 13); Analysis calculated for C11H9ClF2O3 (262.64): C, 50.30%; H,

3.45%; Cl, 13.50%; F, 14.47; Found: C, 50.31%; H, 3.47%; Cl, 13.49%; F, 14.46%.

3.13.39. 2,4-Dimethylbenzyl 4-chloro-4-ketobutanoate (39a)




): 3097 (Ar-H), 1775 (Cl-C=O), 1731

(C=O), 1605, 1435 (C=C), 1254, 1123, 1008 (C-O); 1H-, and



Cl, 13.92%; Found: C, 61.28%; H, 5.95%; Cl, 13.94%.

66





): 3088 (Ar-H), 1767 (Cl-C=O), 1722

(C=O), 1602, 1487 (C=C), 1244, 1136, 1012 (C-O); 1H-, and



Cl, 13.92%; Found: C, 61.31%; H, 5.98%; Cl, 13.97%.





): 3088 (Ar-H), 1775 (Cl-C=O), 1721

(C=O), 1601, 1478 (C=C), 1259, 1125, 1010 (C-O); 1H-, and



Cl, 13.92%; Found: C, 61.29%; H, 5.93%; Cl, 13.93%.





): 3098 (Ar-H), 1774 (Cl-C=O), 1724

(C=O), 1632, 1494 (C=C), 1256, 1123, 1018 (C-O); 1H-, and



Cl, 13.92%; Found: C, 61.32%; H, 5.93%; Cl, 13.92%.

3.13.43. 3,4-Dinitrobenzyl 4-chloro-4-ketobutanoate (43a)

Yield 63%, 1.59 g (5.04 mmol), pale yellow viscous oil;

67

MS, m/z (Ir/%): 316.64 (29) (M+); UV-VIS (EtOH) λmax nm (log ε): 272.3 (3.5); FT-IR (Neat)

υmax (cm-1

): 3114 (Ar-H), 1779 (Cl-C=O), 1732 (C=O), 1517, 1450 (C=C), 1364 (NO2), 1242,

1120, 1020 (C-O); 1H-, and


calculated for C11H9ClN2O7 (316.65): C, 41.72; H, 2.86; Cl, 11.20; N, 8.85; Found: C, 41.71; H,

2.87; Cl, 11.21; N, 8.84%.

3.13.44. 3,5-Dinitrobenzyl 4-chloro-4-ketobutanoate (44a)


MS, m/z (Ir/%): 316.66 (27) (M+); UV-VIS (EtOH) λmax nm (log

ε): 271.8 (3.7); FT-IR (Neat) υmax (cm-1

): 3104 (Ar-H), 1776 (Cl-

C=O), 1730 (C=O), 1605, 1582, 1413 (C=C), 1373 (NO2), 1248, 1122, 1025 (C-O);

1H-, and


C11H9ClN2O7 (316.65): C, 41.72; H, 2.86; Cl, 11.20; N, 8.85; Found: C, 41.73; H, 2.87; Cl,

11.19; N, 8.87%.

3.13.45. 2,6-Diethoxybenzyl 4-chloro-4-ketobutanoate (45a)



(Neat) υmax (cm-1

): 3105 (Ar-H), 1778 (Cl-C=O), 1733 (C=O), 1612,

1491 (C=C), 1253, 1125, 1020 (C-O); 1H-, and


11 and 13); Analysis calculated for C15H19ClO5 (314.76): C, 57.24; H, 6.08; Cl, 11.26; Found: C,

57.25; H, 6.03; Cl, 11.25%.

3.13.46. 3,4-Diethoxybenzyl 4-chloro-4-ketobutanoate (46a)



68


): 3354 (OH), 3077 (Ar-H), 1777 (Cl-C=O), 1732 (C=O), 1606, 1475

(C=C), 1249, 1124, 1022 (C-O); 1H-, and

13C-NMR (300 MHz, 75 MHz, CDCl3): (Tables 11 and

13); Analysis calculated for C15H19ClO5 (314.76): C, 57.24; H, 6.08; Cl, 11.26; Found: C, 57.23;

H, 6.06; Cl, 11.27.

3.13.47. 3,5-Dihydroxybenzyl 4-chloro-4-ketobutanoate (47a)




): 3250 (OH), 3066 (Ar-H), 1779 (Cl-

C=O), 1731 (C=O), 1515, 1490 (C=C), 1261, 1130, 1021 (C-O); 1H-, and

13C-NMR (300 MHz,

75 MHz, CDCl3): (Tables 11 and 13); Analysis calculated for C11H11ClO5 (258.66): C, 51.08; H,

4.29; Cl, 13.71; Found: C, 51.08; H, 4.29; Cl, 13.71%.

3.13.48. 2,3,4-Trimethoxybenzyl 4-chloro-4-ketobutanoate (48a)


316.72 (25) (M+); UV-VIS (EtOH) λmax nm (log ε): 275.3

(3.98); FT-IR (Neat) υmax (cm-1

): 3097 (Ar-H), 1774 (Cl-C=O),

1731 (C=O), 1618, 1499 (C=C), 1267, 1131, 1012 (C-O); 1H-, and

13C-NMR (300 MHz, 75

MHz, CDCl3): (Tables 11 and 13); Analysis calculated for C14H17ClO6 (316.73): C, 53.09; H,

5.41; Cl, 11.19; Found: C, 53.10; H, 5.40; Cl, 11.20%.

3.13.49. 2,4,5-Trimethoxbenzyl 4-chloro-4-ketobutanoate (49a)




): 3087 (Ar-H), 1778 (Cl-C=O),

1731 (C=O), 1604, 1493 (C=C), 1266, 1154, 1020 (C-O); 1H-, and

13C-NMR (300 MHz, 75

69


5.41; Cl, 11.19; Found: C, 53.11; H, 5.42; Cl, 11.18%.

3.13.50. 3,4,5-Trimethoxybenzyl 4-chloro-4-ketobutanoate (50a)




): 3076 (Ar-H), 1777 (Cl-C=O),

1731 (C=O), 1504, 1488 (C=C), 1257, 1155, 1023 (C-O); 1H-, and

13C-NMR (300 MHz, 75


5.41; Cl, 11.19; Found: C, 53.08; H, 5.39; Cl, 11.21%.

3.13.51. 3-Methoxy-4-nitrobenzyl 4-chloro-4-ketobutanoate (51a)

Yield 66%, 1.61 g (5.36 mmol), light yellow viscous oil; MS,

m/z (Ir/%): 301.69 (29) (M+); UV-VIS (EtOH) λmax nm (log ε):

268.7 (3.86); FT-IR (Neat) υmax (cm-1

): 3083 (Ar-H), 1778 (Cl-

C=O), 1730 (C=O), 1621, 1575, 1392 (C=C), 1374 (NO2), 1250, 1134, 1022 (C-O); 1H-, and

13C-

NMR (300 MHz, 75 MHz, CDCl3): (Tables 11 and 13); Analysis calculated for C12H12ClNO6

(301.68): C, 47.78; H, 4.01; Cl, 11.75; N, 4.64; Found: C, 47.77; H, 4.03; Cl, 11.74; N, 4.65%.


Yield 64%, 1.54 g (5.12 mmol), light yellow viscous oil;

MS, m/z (Ir/%): 301.69 (25) (M+); UV-VIS (EtOH) λmax nm

(log ε): 269.8 (3.84); FT-IR (Neat) υmax (cm-1

): 3077 (Ar-H),

1783 (Cl-C=O), 1726 (C=O), 1618, 1584, 1386 (C=C), 1372 (NO2), 1251, 1132, 1016 (C-O); 1H-

, and 13

C-NMR (300 MHz, 75 MHz, CDCl3): (Tables 11 and 13); Analysis calculated for

70

C12H12ClNO6 (301.68): C, 47.78; H, 4.01; Cl, 11.75; N, 4.64; Found: C, 47.76; H, 3.99; Cl,

11.79; N, 4.63%.


Yield 68%, 1.64 g (5.44 mmol), light yellow viscous oil; MS,


268.7 (3.96); FT-IR (Neat) υmax (cm-1

): 3069 (Ar-H), 1785 (Cl-

C=O), 1724 (C=O), 1512, 1482 (C=C), 1370 (NO2), 1245, 1123, 1010 (C-O); 1H-, and

13C-NMR

(300 MHz, 75 MHz, CDCl3): (Tables 11 and 13); Analysis calculated for C12H12ClNO6 (301.68):

C, 47.78; H, 4.01; Cl, 11.75; N, 4.64; Found: C, 47.79; H, 3.98; Cl, 11.76; N, 4.65%.

3.13.54. 2,4,6-Trichlorobenzyl 4-chloro-4-ketobutanoate (54a)




): 3086 (Ar-H), 1779 (Cl-C=O), 1729 (C=O), 1616-1395 (C=C), 1250,

1126, 1015 (C-O), 733 (C-Cl); 1H-, and

13C-NMR (300 MHz, 75 MHz, CDCl3): (Tables 11 and

13); Analysis calculated for C11H8Cl4O3 (329.99): C, 40.04; H, 2.44; Cl, 42.97; Found: C, 40.08;

H, 2.43; Cl, 42.96%.

3.13.55. 2,4,5-Tribromobenzyl 4-chloro-4-ketobutanoate (55a)




): 3090 (Ar-H), 1781 (Cl-C=O), 1728

(C=O), 1516, 1493 (C=C), 1256, 1132, 1018 (C-O), 529 (C-Br); 1H-, and

13C-NMR (300 MHz,

75 MHz, CDCl3): (Tables 11 and 13); Analysis calculated for C11H8Br3ClO3 (463.34): C, 28.51;

H, 1.74; Br, 51.74; Cl, 7.65; Found: C, 28.52; H, 1.73; Br, 51.76; Cl, 7.63%.

71

3.13.56. 2,4,6-Trimethylbenzyl 4-chloro-4-ketobutanoate (56a)

Viscous oil: Yield 71%, 1.52 g (5.68 mmol): MS, m/z (Ir/%):



): 3084 (Ar-H), 1778 (Cl-C=O), 1726

(C=O), 1614-1397 (C=C), 1258, 1135, 1010 (C-O); 1H-, and


CDCl3): (Tables 11 and 13); Analysis calculated for C14H17ClO3 (268.74): C, 62.57; H, 6.38; Cl,

13.19; Found: C, 62.58; H, 6.37; Cl, 13.20%.

3.13.57. Isopropyl 4-chloro-4-ketobutanoate (57a)

Physical data, yield (%), UV-VIS, IR, mass/anlytical, 1H-, and

13C-NMR

data for compound 57a is given in tables 19 and 21.

3.13.58. Isobutyl 4-chloro-4-ketobutanoate (58a)

Physical data, yield (%), UU-VIS, IR, mass/anlytical, 1H-, and

13C-

NMR data for compound 58a is given in tables 19 and 21.

3.13.59. Isopentyl 4-chloro-4-ketobutanoate (59a)


13C-


3.13.60. Benzyyl 4-chloro-4-ketobutanoate (60a)


13C-


3.14. Physical Characteristics of γ-Ketoesters 1b-56b

Yield (%), UU-VIS, IR and Mass/anlytical data for compounds 1b-56b are given below. Some

of the physical characteristics of 1b-20b are given in Table 2. 1H-, and

13C-NMR data of 1b-56b

is given in tables 14-18.

72

3.14.1. 2-Methoxybenzyl γ-ketohexanoate (1b)

Physical characteristics are given in Table 2; UV-VIS (MeOH),

λmax/nm (log ɛ): 271.3 (3.3); MS, m/z (Ir/%): 250.29 (23) (M+)


): 3057 (Ar-H), 1749 (C=O), 1717 (CO2Ar),

1595, 1546, 1429, (C=C), Ar-H), 1239, 1127, 1026 (C-O); 1H-, and

13C-NMR (300 MHz, 75




λmax/nm (log ɛ): 271.6 (3.5). MS, m/z (Ir/%): 250.29 (29) (M+); FT-IR

(Neat) υmax (cm-1

): 3065 (Ar-H), 1725 (C=O), 1714 (CO2Ar), 1592,

1545, 1431 (C=C), 1230, 1128, 1025 (C-O); 1H-, and

13C-NMR (300 MHz, 75 MHz, CDCl3):

(Tables 14 and 16).


Physical characteristics are given in Table 2;

UV-VIS (MeOH), λmax/nm (log ɛ): 271.1 (3.6);

MS, m/z (Ir/%): 250.29 (25) (M+). FT-IR (Neat) υmax (cm

-1):

3069 (Ar-H), 1747 (C=O), 1722 (CO2Ar), 1596, 1543, 1430 (C=C), 1243, 1129, and 1023 (C-O);

1H-, and


3.14.4. 2,3-Dimethoxybenzyl γ-ketohexanoate (4b)


UV-VIS (MeOH), λmax/nm (log ɛ): 278.8 (3.7); MS, m/z (Ir/%):


-1): 3085 (Ar-H), 2850,

73

1757 (C=O), 1723 (CO2Ar), 1592, 1538, 1435 (C=C), 1237, 1133, 1041 (C-O); 1H-, and

13C-




UV-VIS (MeOH), λmax/nm (log ɛ): 278.6 (3.87) ;


-1)

3054 (Ar-H), 1754 (C=O), 1726 (CO2Ar), 1600, 1540, 1436 (C=C), 1290, 1135(C-O); 1H-, and




UV-VIS (MeOH), λmax/ nm (log ɛ): 282.5 (3.6);


-1):

3092 (Ar-H), 1753 (C=O), 1722 (ArO-C=O), 1605, 1595, 1435 (C=C), 1245, 1125 (C-O); 1H-,

and 13



Physical characteristics are given in Table 2; UV-VIS

(MeOH), λmax/nm (log ɛ): 279.7 (3.8). MS, m/z (Ir/%): 280.11

(18) (M+). FT-IR (Neat) υmax (cm

-1): 3086 (Ar-H), 1755

(C=O), 1721 (CO2Ar), 1603, 1592, 1433 (C=C), 1247, 1128 (C-O); 1H-, and

13C-NMR (300




(MeOH), λmax/nm (log ɛ): 279.9 (3.8); MS, m/z (Ir/%): 280.11

74

(22) (M+); FT-IR (Neat) υmax (cm

-1): 1751 (C=O), 1726 (CO2Ar), 1592, 1538, 1515, 1435 (C=C),

1235, 1155, 1141, 1133, 1041, 1022 (C-O); 1H-, and


(Tables 14 and 16).

3.14.9. 2-Nitrobenzyl γ-ketohexanoate (9b)


λmax/nm (log ɛ): 267 (4.0); MS, m/z (Ir/%): 265.12 (16) (M+); FT-IR

(Neat) υmax (cm-1

): 3092 (Ar-H), 1746 (C=O), 1723 (CO2R), 1596,

1530, 1441 (C=C, Ar-H), 1375 (NO2), 1244, 1139, 1045 (C-O); 1H-, and

13C-NMR (300 MHz,




λmax/nm (log ɛ): 267.5 (4.0). MS, m/z (Ir/%): 265.12 (19) (M+); FT-

IR (Neat) υmax (cm-1

): 3086 (Ar-H), 1757 (C=O), 1721 (CO2R),

1597, 1539, 1440 (C=C), 1374 (NO2), 1249, 1137, 1044 (C-O); 1H-, and

13C-NMR (300 MHz, 75




(MeOH), λmax/nm (log ɛ): 267.6 (4.2); MS, m/z (Ir/%): 265.12

(14) (M+); FT-IR (Neat) υmax (cm

-1): 3071 (Ar-H), 1753

(C=O), 1722 (CO2Ar), 1595, 1535, 1440 (C=C), 1372 (NO2), 1248, 1136, 1045(C-O); 1H-, and


3.14.12. 2-Chlorobenzyl γ-ketohexanoate (12b)


75



-1): 3077 (Ar-H), 1748 (C=O), 1721

(CO2Ar), 1597, 1529, 1497, 1456 (C=C), 1251, 1237, 1134, 1094, 1047, 1015 (C-O), 723 (C-Cl);

1H-, and






-1):

3079 (Ar-H), 1739 (C=O), 1722 (CO2Ar), 1599, 1525, 1452 (C=C), 1248, 1133 (C-O), 727 (C-

Cl); 1H-, and






-1):

3093 (Ar-H), 1754 (C=O), 1720 (CO2Ar), 1597, 1529 (C=C), 1383, 1251, 1047, 721 (C-Cl); 1H-,

and 13


3.14.15. 2-Bromrobenzyl γ-ketohexanoate (15b)



-1): 3085

(Ar-H), 1736 (C=O), 1715 (CO2Ar), 1596, 1527(C=C), 1413, 1254,

1054, 535 (C-Br); 1H-, and




76



-1): 3085 (Ar-H), 1739 (C=O), 1718

(CO2Ar), 1591, 1522, 1451 (C=C), 1417, 1271, 1252, 1140, 1051 (C-O), 528 (C-Br); 1H-, and




λmax/nm (log ɛ): 263.9 (3.9); MS, m/z (Ir/%): 299.11 (22) (M+);


): 3083 (Ar-H), 1743 (C=O), 1717

(CO2Ar), 1598, 1532, 1455 (C=C), 1419, 1266, 531 (C-Br); 1H-, and

13C-NMR (300 MHz, 75


3.14.18. 2-Iodobenzyl γ-ketohexanoate (18b)




): 3046 (Ar-H), 1735 (C=O), 1718 (CO2Ar),

1592, 1524, 1456 (C=C), 1258, 1156, 488 (C-I); 1H-, and


(Tables 14 and 16).





): 3042 (Ar-H), 1732 (C=O), 1719

(CO2Ar), 1591, 1529, 1467 (C=C), 1251, 1161, 1058, 492 (C-I); 1H-, and

13C-NMR (300 MHz,


77





): 3048 (Ar-H), 1733 (C=O), 1715

(CO2Ar), 1599, 1521, 1460 (C=C) 1255, 1151, 1054, 483(C-I); 1H-, and

13C-NMR (300 MHz, 75


3.14.21. 2-Methylbenzyl γ-ketohexanoate (21b)


(28) (M+). UV-VIS (MeOH), λmax/nm (log ɛ): 272.1 (3.35); FT-IR

(Neat) υmax (cm-1

): 3084 (Ar-H), 1755 (C=O), 1719 (CO2Ar), 1606,

1554, 1424 (C=C), 1228, 1120, 1025 (C-O); 1H-, and


(Tables 14 and 17); Analysis calculated for C14H18O3 (234.29): C, 71.77; H, 7.74; Found: C,

71.73; H, 7.76.



234.29 (26) (M+); UV-VIS (EtOH), λmax nm (log ε): 263.9

(3.66). FT-IR (Neat) υmax (cm-1

): 3094 (Ar-H), 1735 (CO2Ar),

1756 (C=O), 1622, 1550 1438 (C=C, Ar-H), 1255, 1136, 1028 (C-O); 1H-, and

13C-NMR (300

MHz, 75 MHz, CDCl3): (Tables 14 and 17); Analysis calculated for C14H18O3 (234.29): C,

71.77%; H, 7.74%; Found: C, 71.75%; H, 7.75%.



234.29 (26) (M+); UV-VIS (EtOH) λ max nm (log ε): 266.3

78


): 3076 (Ar-H), 1731 (CO2Ar), 1757 (C=O), 1596, 1557, 1444

(C=C, Ar-H), 1256, 1136, 1015 (C-O); 1H-, and


14 and 17); Analysis calculated for C14H18O3 (234.29): C, 71.77%; H, 7.74%; Found: C, 71.76%;

H, 7.74%.

3.14.24. 2-Hydroxybenzyl γ-ketohexanoate (24b)




): 3225-3187 (OH), 3088 (Ar-H), 1739

(CO2Ar), 1763 (C=O), 1608, 1532, 1440 (C=C, Ar-H), 1252, 1123 (C-O); 1H-, and

13C-NMR

(300 MHz, 75 MHz, CDCl3): (Tables 14 and 17); Analysis calculated for C13H16O4 (236.26): C,

66.09%; H, 6.83%; Found: C, 66.12%; H, 6.84%.





): 3276-3145 (OH), 3098 (Ar-

H), 1736 (CO2Ar), 1768 (C=O), 1619, 1551, 1442, (C=C, Ar-H), 1247, 1138, 1044 (C-O); 1H-,

and 13

C-NMR (300 MHz, 75 MHz, CDCl3): (Tables 14 and 17); Analysis calculated for C13H16O4

(236.26): C, 66.09%; H, 6.83%; Found: C, 66.10%; H; 6.82%.




(3.76); FT-IR (Neat) υmax (cm

-1): 3255-3120 (OH), 3095 (Ar-H),

1749 (CO2Ar), 1757 (C=O), 1617, 1546, 1443 (C=C, Ar-H), 1229, 1130, 1044 (C-O); 1H-, and

79

13C-NMR (300 MHz, 75 MHz, CDCl3): (Tables 14 and 17); Analysis calculated for C13H16O4

(236.26): C, 66.09%; H, 6.83%; Found: C, 66.08%; H; 6.85%.

3.14.27. 2-Aminobenzyl γ-ketohexanoate (27b)


(24) (M+); UV-VIS (EtOH) λmax nm (log ε): 262.9 (3.95). FT-IR

(Neat) υmax (cm-1

): 3235 (NH2), 3089 (Ar-H), 1745 (CO2Ar), 1764

(C=O), 1603, 1537, 1492, 1453, (C=C, Ar-H), 1381 (C-N), 1252, 1230, 1135, 1041, 1002 (C-O);

1H-, and


C13H17NO3 (235.28): C, 66.36%; H, 7.28%; N, 5.95%; Found: C, 66.38; H, 7.27; N, 5.94%.





): 3230 (Broad, NH2), 3096

(Ar-H), 1739 (CO2Ar), 1757 (C=O), 1598, 1533, 1456, (C=C, Ar-H), 1393 (C-N), 1241, 1136,

1042 (C-O); 1H-, and


calculated for C13H17NO3 (235.28): C, 66.36%; H, 7.28%; N, 5.95%; Found: C, 66.34; H, 7.29;

N, 5.96%.





): 3236 (Broad, intense, NH2),

3095 (Ar-H), 1738 (CO2Ar), 1767 (C=O), 1611, 1595, 1459 (C=C, Ar-H), 1387 (C-N), 1268,

1153, 1026 (C-O); 1H-, and

13C-NMR (300 MHz, 75 MHz, CDCl3): (Tables 15 and 17). Analysis

80

calculated for C13H17NO3 (235.28): C, 66.36%; H, 7.28%; N, 5.95%; Found: C, 66.35; H, 7.27;

N, 5.97%.

3.14.30. 2,4-Dichlorobenzyl γ-ketohexanoate (30b)




): 3076 (Ar-H), 1728 (CO2Ar), 1767

(C=O), 1615, 1479 (C=C), 1267, 1148, 1023 (C-O), 722 (C-Cl); 1H-, and

13C-NMR (300 MHz,

75 MHz, CDCl3): (Tables 15 and 17); Analysis calculated for C13H14Cl2O3 (289.15): C, 54.00; H,

4.88; Cl, 24.52; Found: C, 54.05; H, 4.89; Cl, 24.51%.



(31) (M+); UV-VIS (EtOH) λmax nm (log ε): 264.2 (3.57);FT-IR

(Neat) υmax (cm-1

): 3097 (Ar-H), 1726 (CO2Ar), 1768 (C=O), 1619,

1439 (C=C), 1268, 1131, 1024 (C-O), 725 (C-Cl);1H-, and


CDCl3): (Tables 15 and 17); Analysis calculated for C13H14Cl2O3 (289.15): C, 54.00; H, 4.88; Cl,

24.52; Found: C, 54.09; H, 4.87; Cl, 24.53%.



289.15 (34) (M+). UV-VIS (EtOH) λmax nm (log ε): 264.2 (3.57);


): 3099 (Ar-H), 1729 (CO2Ar), 1764

(C=O), 1622, 1428 (C=C), 1262, 1135, 1020 (C-O), 725 (C-Cl); 1H-, and

13C-NMR (300 MHz,

75 MHz, CDCl3): (Tables 15 and 17); Analysis calculated for C13H14Cl2O3 (289.15): C, 54.00; H,

4.88; Cl, 24.52; Found: C, 54.10; H, 4.85; Cl, 24.51%.

81




(3.88). FT-IR (Neat) υmax (cm-1): 3087 (Ar-H), 1759 (C=O),

1728 (CO2Ar), 1619, 1447 (C=C), 1258, 1135, 1028 (C-O), 727 (C-Cl); 1H-, and 13C-NMR

(300 MHz, 75 MHz, CDCl3): (Tables 15 and 17); Analysis calculated for C13H14Cl2O3 (289.15):

C, 54.00%; H, 4.88%; Cl, 24.52%; Found: C, 54.08%; H, 4.89%; Cl, 24.53%.





): 3066 (Ar-H), 1767 (C=O),

1725 (CO2Ar), 1618, 1438 (C=C), 1249, 1127, 1020 (C-O), 732

(C-Cl); 1H-, and

13C-NMR (300 MHz, 75 MHz, CDCl3): (Tables 15 and 17); Analysis calculated

for C13H14Cl2O3 (289.15): C, 54.00; H, 4.88; Cl, 24.52; Found: C, 54.07; H, 4.87; Cl, 24.51%.

3.14.35. 2,3-Difluorobenzyl γ-ketohexanoate (35b)




): 3076 (Ar-H), 1765 (C=O), 1729

(CO2Ar), 1608, 1483 (C=C), 1258, 1128, 1015 (C-O), 1155 (C-F); 1H-, and

13C-NMR (300 MHz,

75 MHz, CDCl3): (Tables 15 and 17); Analysis calculated for C13H14F2O3 (256.25): C, 60.93; H,

5.51; F, 14.83; Found: C, 60.92; H, 5.52; F, 14.82%.

82




(3.98); FT-IR (Neat) υmax (cm-1): 3094 (Ar-H), 1767 (C=O),

1731 (CO2Ar), 1597, 1471 (C=C), 1245, 1114, 1021 (C-O) 1158 (C-F); 1H-, and 13C-NMR (300

MHz, 75 MHz, CDCl3): (Tables 15 and 17); Analysis calculated for C13H14F2O3 (256.25): C,

60.93; H, 5.51; F, 14.83; Found: C, 60.94; H, 5.50; F, 14.84%.




(Neat) υmax (cm-1

): 3092 (Ar-H), 1768 (C=O), 1728 (CO2Ar), 1594,

1489 (C=C), 1250, 1130, 1026 (C-O), 1149 (C-F); 1H-, and

13C-

NMR (300 MHz, 75 MHz, CDCl3): (Tables 15 and 17); Analysis calculated for C13H14F2O3

(256.25): C, 60.93; H, 5.51; F, 14.83; Found: C, 60.95; H, 5.52; F, 14.82%.



256.25 (34) (M+); UV-VIS (EtOH) λmax nm (log ε): 267.6 (3.95); FT-

IR (Neat) υmax (cm-1

): 3075 (Ar-H), 1766 (C=O), 1732 (CO2Ar),

1598, 1405 (C=C), 1235, 1129, 1011 (C-O), 1159 (C-F); 1H-, and


CDCl3): (Tables 15 and 17); Analysis calculated for C13H14F2O3 (256.25): C, 60.93; H, 5.51; F,

14.83; Found: C, 60.94; H, 5.53; F, 14.85%.

83

3.14.39. 2,4-Dimethylbenzyl γ-ketohexanoate (39b)




): 3094 (Ar-H), 1772 (C=O),

1731 (CO2Ar), 1609, 1437 (C=C), 1259, 1122, 1018 (C-O); 1H-, and

13C-NMR (300 MHz, 75

MHz, CDCl3): (Tables 15 and 18); Analysis calculated for C15H20O3 (248.32): C, 72.55%; H,

8.12%; Found: C, 72.54%; H, 8.13%.




(Neat) υmax (cm-1

): 3074(Ar-H), 1765 (C=O), 1723 (CO2Ar), 1607,

1485 (C=C), 1246, 1138, 1013 (C-O); 1H-, and


15 and 18); Analysis calculated for C15H20O3 (248.32): C, 72.55%; H, 8.12%; Found: C, 72.56%;

H, 8.11%.




(3.84);


): 3089 (Ar-H), 1772 (C=O), 1724 (C=O), 1608, 1476 (C=C), 1255,

1124, 1016 (C-O); 1H-, and


calculated for C15H20O3 (248.32): C, 72.55%; H, 8.12%; Found: C, 72.56%; H, 8.13%.

84




(3.76);


): 3087 (Ar-H), 1773 (C=O), 1726

(CO2Ar), 1621, 1492 (C=C), 1251, 1124, 1017 (C-O); 1H-, and


CDCl3): (Tables 15 and 18); Analysis calculated for C15H20O3 (248.32): C, 72.55%; H, 8.12%;

Found: C, 72.57%; H, 8.10%.

3.14.43. 3,4-Dinitrobenzyl γ-ketohexanoate (43b)

Yield 68%, 1.05 g (3.40 mmol), pale yellow viscous oil; MS,


271.2 (3.56); FT-IR (Neat) υmax (cm-1

): 3117 (Ar-H), 1778

(C=O), 1734 (CO2Ar), 1518, 1456 (C=C), 1368 (NO2), 1245, 1123, 1024 (C-O); 1H-, and

13C-

NMR (300 MHz, 75 MHz, CDCl3): (Tables 15 and 18); Analysis calculated for C13H14N2O7

(310.26): C, 50.33%; H, 4.55%; N, 9.03%; Found: C, 50.34%; H, 4.56%; N, 9.05%.

3.14.44. 3,5-Dinitrobenzyl γ-ketohexanoate (44b)




): 3107 (Ar-H),

1771 (C=O), 1729 (CO2Ar), 1612, 1581, 1412 (C=C), 1372 (NO2), 1249, 1124, 1022 (C-O). 1H-,

and 13

C-NMR (300 MHz, 75 MHz, CDCl3): (Tables 15 and 18); Analysis calculated for

C13H14N2O7 (310.26): C, 50.33%; H, 4.55%; N, 9.03%; Found: C, 50.32%; H, 4.56%; N, 9.08%.

85

3.14.45. 2,6-Diethoxybenzyl γ-ketohexanoate (45b)




): 3123 (Ar-H), 1775 (CO-Cl), 1734 (C=O),

1615, 1494 (C=C), 1255, 1124, 1022 (C-O); 1H-, and


(Tables 15 and 18); Analysis calculated for C17H24O5 (308.37): C, 66.21%; H, 7.84%; Found: C,

66.22%; H, 7.83%.

3.14.46. 3,4-Diethoxybenzyl γ-ketohexanoate (46b)




): 3323 (OH), 3074 (Ar-H),

1776 (C=O), 1731 (CO2Ar), 1608, 1472 (C=C), 1246, 1122, 1026 (C-O); 1H-, and

13C-NMR (300

MHz, 75 MHz, CDCl3): (Tables 15 and 18); Analysis calculated for C17H24O5 (308.37): C, 66.21;

H, 7.84; Found: C, 66.20; H, 7.85%.

3.14.47. 3,5-Dihydroxybenzyl γ-ketohexanoate (47b)




): 3250 (OH), 3064 (Ar-H),

1774 (C=O), 1732 (CO2Ar), 1516, 1493 (C=C), 1268, 1136, 1027 (C-O); 1H-, and

13C-NMR (300

MHz, 75 MHz, CDCl3): (Tables 15 and 18); Analysis calculated for C13H16O5 (252.26): C, 61.90;

H, 6.39; Found: C, 61.91; H, 6.38;%.

86

3.14.48. 2,3,4-Trimetxoxybenzyl γ-ketohexanoate (48b)


310.34 (31) (M+); UV-VIS (EtOH) λmax nm (log ε): 276.5.


): 3091 (Ar-H), 1772 (C=O),

1736 (CO2Ar), 1616, 1490 (C=C), 1262, 1133, 1014 (C-O); 1H-, and

13C-NMR (300 MHz, 75

MHz, CDCl3): (Tables 15 and 18); Analysis calculated for C16H22O6 (310.34): C, 61.92; H, 7.15;

Found: C, 61.91; H, 7.16%.

3.14.49. 2,4,5-Trimethoxbenzyl γ-ketohexanoate (49b)




):3088 (Ar-H), 1777 (C=O),

1730 (CO2Ar), 1606, 1482 (C=C), 1268, 1155, 1028 (C-O); 1H-, and

13C-NMR (300 MHz, 75


7.15%. Found: C, 61.93%; H, 7.14%.

3.14.50. 3,4,5-Trimethoxybenzyl γ-ketohexanoate (50b)




): 3078 (Ar-H), 1776 (C=O),

1732 (C=O), 1508, 1485 (C=C), 1252, 1157, 1023 (C-O); 1

H-, and 13

C-NMR (300 MHz, 75


7.15%; Found: C, 61.91%; H, 7.16%.

3.14.51. 3-Methoxy-4-nitrobenzyl γ-ketohexanoate (51b)


87

MS, m/z (Ir/%): 295.29 (25) (M+); UV-VIS (EtOH) λmax nm (log ε): 269.8 (3.87); FT-IR (Neat)

υmax (cm-1

): 3084 (Ar-H), 1774 (C=O), 1733 (CO2Ar), 1625, 1572, 1390 (C=C), 1373 (NO2),

1252, 1131, 1027 (C-O);1H-, and

13C-NMR (300 MHz, 75 MHz, CDCl3): (Tables 15 and 18);

Analysis calculated for C14H17NO6 (295.29): C, 56.94%; H, 5.80%; N, 4.74%; Found: C,

56.95%; H, 5.81%; N, 4.75%.





): 3094 (Ar-H),

1779 (C=O), 1728 (CO2Ar), 1622, 1575, 1383 (C=C), 1371 (NO2), 1254, 1133, 1015 (C-O);

1H-, and


C14H17NO6 (295.29): C, 56.94%; H, 5.80%; N, 4.74%; Found: C, 56.93%; H, 5.82%; N, 4.76%.



MS, m/z (Ir/%): 295.29 (22) (M+); UV-VIS (EtOH) λmax nm (log ε):

269.4 (3.92); FT-IR (Neat) υmax (cm-1

): 3066 (Ar-H), 1782 (C=O),

1725 (CO2Ar), 1516, 1488 (C=C), 1373 (NO2), 1248, 1124, 1011 (C-O); 1H-, and

13C-NMR (300

MHz, 75 MHz, CDCl3): (Tables 15 and 18); Analysis calculated for C14H17NO6 (295.29): C,

56.94%; H, 5.80%; N, 4.74%; Found: C, 56.96%; H, 5.81%; N, 4.75%.

3.14.54. 2,4,6-Trichlorobenzyl γ-ketohexanoate (54b)




): 3084 (Ar-H), 1776 (C=O), 1728

88

(CO2Ar), 1615-1394 (C=C), 1248, 1127, 1012 (C-O), 732 (C-Cl); 1H-, and

13C-NMR (300 MHz,


H, 4.05%; Cl, 32.87%; Found: C, 48.24%; H, 4.08%; Cl, 32.86%.

3.14.55. 2,4,5-Tribromobenzyl γ-ketohexanoate (55b)




): 3092 (Ar-H), 1776 (C=O), 1727

(CO2Ar), 1518, 1494 (C=C), 1250, 1137, 1016 (C-O), 526 (C-Br); 1H-, and

13C-NMR (300 MHz,

75 MHz, CDCl3): (Tables 15 and 18); Analysis calculated for C13H13Br3O3 (456.95): C, 34.17%;

H, 2.87%; Br, 52.46%; Found: C, 34.17%; H, 2.87%; Br, 52.46%.

3.14.56. 2,4,6-Trimethylbenzyl γ-ketohexanoate (56b)




): 3087 (Ar-H), 1774 (C=O),

1725 (CO2Ar), 1616-1394 (C=C), 1256, 1138, 1014 (C-O); 1H-, and

13C-NMR (300 MHz, 75


8.45%; Found: C, 73.26%; H, 8.44%.

3.14.57. Isopropyl γ-ketohexanoate (57b)

Yield, b.p. and elemental analysis of 57b have been provided in Table

19. Spectroscopic data (UV, IR, MS, 1H-, and

13C-NMR) of 57b is

given in Table 21. In table 20 yields of 57b-60b from other

89

organometallic reagents are compared.

3.14.58. Isobutyl γ-ketohexanoate (58b)

Yield, b.p. and elemental analysis of 58b have been provided in

Table 19. Spectroscopic data (UV, IR, MS, 1H-, and

13C-NMR) of

58b is given in Table 21.

3.14.59. Isopentyl γ-ketohexanoate (59b)


table 19. Spectroscopic data (UV, IR, MS, 1H-, and

13C-NMR) of


3.14.60. Benzyl γ-ketohexanoate (60)


table 19. Spectroscopic data (UV, IR, MS, 1H-, and 13C-NMR) of


90

TABLES

91

Table 1. Physical characteristics of 1a-20a

Entry Ara

Time Yield Formula Mr Physical state wi(calc.)/%

wi(found)/%

Min % C H N Cl Br I

1a 2-MeO-C6H4 165 75 C12H13ClO4 256.68 Viscous oil 56.15

56.23

5.10

4.78

- 13.81

13.94

- -


56.34

5.10

5.22

- 13.81

13.33

- -


56.33

5.10

5.23

- 13.81

13.86

- -

4a 2,3-(MeO)2-C6H3 150 79 C13H15ClO5 286.70 Viscous oil 54.46

54.37

5.27

5.31

- 12.37

12.66

- -


54.64

5.27

5.11

- 12.37

12.56

- -


54.55

5.27

5.13

- 12.37

12.14

- -


54.61

5.27

5.16

- 12.37

12.48

- -


54.59

5.27

5.16

- 12.37

12.22

- -

9a 2-NO2-C6H4 180 70 C11H10ClNO5 271.66 Viscous oil 48.63

48.86

3.71

3.65

5.16

5.31

13.05

12.84

- -


48.33

3.71

3.58

5.16

5.10

13.05

12.97

- -

92

a) Aromatic substituent (Fig. 2)


48.42

3.71

3.88

5.16

5.23

13.05

12.82

- -

12a 2-Cl-C6H4 210 63 C11H10Cl2O3 260.95 Viscous oil 50.60

50.76

3.86

3.65

- 27.16

27.33

- -


50.47

3.86

3.79

- 27.16

27.32

- -


50.67

3.86

3.68

- 27.16

26.98

- -

15a 2-Br-C6H4 225 68 C11H10BrClO3 305.76 Viscous oil 43.24

43.16

3.30

3.45

- 11.60

11.26

26.15

26.45

-


43.35

3.30

3.21

- 11.60

11.53

26.15

26.24

-


43.17

3.30

3.26

- 11.60

11.76

26.15

26.08

-

18a 2-I-C6H4 210 63 C11H10ClIO3 352.65 Viscous oil 37.47

37.42

2.86

2.77

- 10.06

10.32

- 36.00

35.98


37.56

2.86

2.67

- 10.06

9.97

- 36.00

36.23


37.41

2.86

2.98

- 10.06

9.98

- 36.00

35.76

93

Table 2. Physical characteristics of 1b-20b

Entry Ar Time Yield Formula Mr Physical state wi(calc.)/%

wi(found)/%

H % C H N Cl Br I

1b 2-MeO-C6H4 13 69 C14H18O4 250.29 Viscous oil 67.18

67.43

7.25

7.16

- - - -


67.32

7.25

7.13

- - - -


67.13

7.25

7.36

- - - -

4b 2,3-(MeO)2-

C6H3

11 74 C15H20O5 280.11 Viscous oil 64.27

64.12

7.19

7.14

- - - -

5b 2,4-(MeO)2-

C6H3

11 79 C15H20O5 280.11 Viscous oil 64.27

64.18

7.19

7.23

- - - -

6b 2,5-(MeO)2-

C6H3

13 75 C15H20O5 280.11 Viscous oil 64.27

64.23

7.19

7.11

- - - -

7b 3,4-(MeO)2-

C6H3

14 74 C15H20O5 280.11 Viscous oil 64.27

64.34

7.19

7.11

- - - -

8b 3,5-(MeO)2-

C6H3

12 71 C15H20O5 280.11 Viscous oil 64.27

64.19

7.19

7.35

- - - -

9b 2-NO2-C6H4 13 68 C13H15NO5 265.12 Viscous oil 58.86

58.95

5.70

5.56

5.28

5.32

- - -

94


56.73

5.70

5.67

5.28

5.38

- - -


56.95

5.70

5.66

5.28

5.45

- - -

12b 2-Cl-C6H4 12 59 C13H15ClO3 254.71 Viscous oil 61.30

61.28

5.94

5.77

- 13.92

13.99

- -


61.19

5.94

5.83

- 13.92

13.87

- -


61.55

5.94

5.45

- 13.92

13.88

- -

15b 2-Br-C6H4 12 63 C13H15BrO3 299.11 Viscous oil 52.19

52.23

5.05

5.12

- - 26.71

25.72

-


52.33

5.05

4.87

- - 26.71

26.68

-


52.21

5.05

5.02

- - 26.71

26.85

-

18b 2-I-C6H4 15 59 C13H15IO3 346.09 Viscous oil 45.11

45.31

4.37

4.16

- - - 36.66

36.55

19B 3-I-C6H4 16 67 C13H15IO3 346.09 Viscous oil 45.11

44.87

4.37

4.46

- - - 36.66

36.45

20b 4-I-C6H4 14 66 C13H15IO3 346.09 Viscous oil 45.11

45.24

4.37

4.15

- - - 36.66

36.67

95

Table 3. 1H-NMR (300 MHz, CDCl3, δ, multiplicity, J in Hz) of 1-20

Cmpd OH 3 2 1ʹʹ Ar-H R Rʹ

1 12.41, br, s 2.85, t, 6.8 Hz 2.75, t, 6.8 Hz 5.06, s 6.93-7.22 m, 4H 3.81, s

2 12.33, br, s 2.80, t, 6.8 Hz 2.73, t, 6.8 Hz 5.23, s 6.86-7.25, m, 4H 3.75, s

3 12.40, br, s 2.81, t, 6.8 Hz 2.72, t, 6.8 Hz 5.09, s 6.90-7.20, m, 4H 3.77, s

4 12.37, br, s 2.82, t, 6.8 Hz 2.73, t, 6.8 Hz 5.06, s 6.93-7.19, m, 3H 3.82, s 3.75, s

5 12.36, br, s 2.84, t, 6.8 Hz 2.73, t, 6.8 Hz 5.04, s 6.53-7.29, m, 3H 3.85, s 3.79, s

6 12.25, br, s 2.84, t, 6.8 Hz 2.73, t, 6.8 Hz 5.06, s 6.68-7.10, m, 3H 3.78, s 3.79, s

7 12.32, br, s 2.83, t, 6.8 Hz 2.73, t, 6.8 Hz 5.11, s 6.81-7.10, m, 3H 3.82, s 3.74, s

8 12.31, br, s 2.81, t, 6.8 Hz 2.73, t, 6.8 Hz, 5.14, s 6.41-6.81, m, 3H 3.75, s 3.75, s

9 12.12, br, s 2.84, t, 6.8 Hz 2.73, t, 6.8 Hz 5.18, s 7.46-8.05, m, 4H

10 12.50, br, s 2.81, t, 6.8 Hz 2.73, t, 6.8 Hz 5.25, s 7.46-8.22, m, 4H

11 12.36, br, s 2.84, t, 6.8 Hz 2.72, t, 6.8 Hz 5.19, s 7.40-8.06, m, 4H

12 12.22, br, s 2.82, t, 6.8 Hz, 2.72, t, 6.8 Hz 5.17, s 7.27-7.54, m, 4H

13 12.44, br, s 2.83, t, 6.8 Hz 2.75, t, 6.8 Hz 5.19, s 7.15-7.41, m, 4H

14 12.37, br, s 2.84, t, 6.8 Hz 2.72, t, 6.8 Hz 5.16, s 7.41-7.56, m, 4H

15 12.28, br, s 2.83, t, 6.8 Hz 2.73, t, 6.8 Hz 5.17, s 6.96-7.36, m, 4H

16 12.43, br, s 2.84, t, 6.8 Hz 2.73, t, 6.8 Hz 5.20, s 7.10-7.30, m, 4H

17 12.36, br, s 2.86, t, 6.8 Hz 2.70, t, 6.8 Hz 5.16, s 7.23-7.32, m, 4H

18 12.31, br, s 2.87, t, 6.8 Hz 2.77, t, 6.8 Hz, 5.20, s 7.17-7.38, m, 4H

19 12.39, br, s 2.82, t, 6.8 Hz 2.72, t, 6.8 Hz 5.20, s 6.99-7.41, m, 4H

20 12.27, br, s 2.79, t, 6.8 Hz 2.72, t, 6.8 Hz 5.70, s 7.32-7.46, m, 4H

96



21 12.27, br, s 2.90, t, 6.8 Hz 2.78, t, 6.8 Hz 5.10, s 7.15-7.29, m, 4H 2.36, s

22 12.22, br, s 2.91, t, 6.8 Hz 2.79, t, 6.8 Hz 5.22, s 7.11-7.55, m, 4H 2.37, s

23 12.32, br, s 2.92, t, 6.8 Hz 2.75, t, 6.8 Hz 5.22, s 7.14-7.35, m, 4H 2.39, s

24 11.98, br, s 2.84, t, 6.8 Hz 2.76, t, 6.8 Hz 5.26, s 6.98-7.44, m, 4H 13.51, br, s

25 11.96, br, s 2.85, t, 6.8 Hz 2.77, t, 6.8 Hz 5.19, s 6.98-7.35,m, 4H 13.55, br, s

26 11.94, br, s 2.88, t, 6.8 Hz 2.78, t, 6.8 Hz 5.19, s 6.88-7.10, m, 4H 12.58 br, s




30 11.82, br, s 2.93, t, 6.9 Hz 2.82, t, 6.9 Hz 5.24, s 7.36-7.87, m

31 11.82, br, s 2.93, t, 6.9 Hz 2.82, t, 6.9 Hz 5.24, s 7.30-7.77, m

32 11.75, br, s 2.95, t, 6.9 Hz 2.85, t, 6.9 Hz 5.26, s 7.69-7.71, m

33 11.68, br, s 2.93, t, 6.9 Hz 2.84, t, 6.9 Hz 5.22, s 7.30-7.79, m

34 11.89, br, s 2.93, t, 6.9 Hz 2.84, t, 6.9 Hz 5.22, s 7.56-7.74, m

35 11.98, br, s 2.91, t, 6.9 Hz 2.77, t, 6.9 Hz 5.15, s 7.21-7.26, m

36 11.81, br, s 2.94, t, 6.9 Hz 2.81, t, 6.9 Hz 5.12, s 6.70-7.42, m

37 11.98, br, s 2.95, t, 6.9 Hz 2.84, t, 6.9 Hz 5.16, s 6.98-7.27, m

38 11.90, br, s 2.96, t, 6.9 Hz 2.82, t, 6.9 Hz 5.10, s 7.41-7.84, m

39 11.99, br, s 2.87, t, 6.9 Hz 2.76, 6.9 Hz 5.10, s 7.04-7.20, m 2.42, s 2.42, s

40 11.91, br, s 2.87, t, 6.9 Hz 2.74, t, 6.9 Hz 5.20, s 7.10-7.24, m 2.39, s 2.39, s

97



41 11.85, br, s 2.87, t, 6.9 Hz 2.74, t, 6.9 Hz 5.23, s 6.99-7.22, m 2.38, s 2.38, s

42 11.72, br, s 2.86, t, 6.9 Hz 2.75, t, 6.9 Hz 5.24, s 7.10-7.38, m 2.40, s 2.40, s

43 11.99, br, s 2.96, t, 6.9 Hz 2.83, t, 6.9 Hz 5.25 s 8.15-8.65, m

44 11.96, br, s 2.97, t, 6.9 Hz 2.81, t, 6.9 Hz 5.21, s 8.69-8.73, m

45 11.55, br, s 2.86, t, 6.9 Hz 2.69, t, 6.9 Hz 5.12, s 6.57-7.25, m 1.38, t, 7.6 Hz,

2CH3

4.15, q, 7.6

Hz,2CH2

46 11.57, br, s 2.88, t, 6.9 Hz 2.70, t, 6.9 Hz 5.21, s 6.66-7.21, m 1.37, t, 7.6 Hz,

2CH3)

4.16, q, 7.6 Hz,

2CH2

47 11.75, br, s 2.88, t, 6.9 Hz 2.70, t, 6.9 Hz 5.21, s 6.36-6.75, m

48 11.74, br, s 2.89, t, 6.9 Hz 2.71, t, 6.9 Hz 5.27, s 6.30-6.75, m 3.87, s, 3×C H3

49 11.80, br, s 2.88, t, 6.9 Hz 2.71, t, 6.9 Hz 5.27, s 6.38-6.96, 2s 3.86, s, 3× CH3

50 11.80, br, s 2.90, t, 6.9 Hz 2.72, t, 6.9 Hz 5.21, s 6.59, s 3.86, s, 3×CH3)

51 11.89, br, s 2.87 t, 6.9 Hz 2.71 (t, 6.9 Hz 5.22, s 7.22-8.12, m 3.85, s, CH3)

52 11.96, br, s 2.88 t, 6.9 Hz 2.71 t, 6.9 Hz 5.22, s 7.22-8.18, m 3.86, s, CH3

53 11.97, br, s 2.87, t, 6.9 Hz 2.74, t, 6.9 Hz 5.26, s 7.20-8.21, m 3.84, s, CH3

54 11.96, br, s 2.89, t, 6.9 Hz 2.75, t, 6.9 Hz 5.12, s 7.71 s, 2H

55 11.99, br, s 2.85, t, 6.9 Hz 2.70, t, 6.9 Hz 5.12, s 7.90, s

56 12.02, br, s 2.85, t, 6.9 Hz 2.69, t, 6.9 Hz 5.12, s 6.97, s, 2H 2.46, s, 3×CH3)

98

Table 6. 13

C-NMR (75 MHz, CDCl3, δ, DEPT, multiplicity) of 1-14

Cmpd 4 3 2 1 1ʹʹ 1ʹ 2ʹ 3ʹ 4ʹ 5ʹ 6ʹ Rʹ Rʹ

1 172.7, s 31.3,

t

29.5,

t

173.9, s 59.9, t 131.1 147.7 113.4 130.3 118.8 127.5 55.8, q, OMe

2 172.5, s 30.9,

t

29.0,

t

173.9, s 65.6, t 139.2 112.2 159.4 113.3 129.3 118.3 55.9, q, OMe

3 173.2, s 32.5,

t

30.0,

t

174.2, s 65.1, t 129.2 129.3 113.6 159.1 113.6 129.3 55.0, q, OMe

4 173.7, s 32.2,

t

29.3,

t

175.0, s 60.3, t 130.6 139.1 153.1 116.6 124.4 119.2 60.2, q, OMe 57.1, q, OMe

5 172.2, s 33.3,

t

31.5,

t

173.7, s 59.8,

T

123.1 153.1 98.7 162.6 108.5 128.8 55.7, q, OMe 55.2, q, OMe

6 173.1, s 32.2,

t

29.3,

t

174.2, s 60.2, t 130.5 139.1 113.1 118.5 154.5 110.5 55.9, q, OMe 55.6, q, OMe

7 172.5, s 33.2,

t

31.1,

t

174.0, s 65.5, t 130.6 110.8 149.3 149.2 112.7 120.5 55.7, q, OMe 55.5, q, OMe

8 172.6, s 32.6,

t

30.2,

t

173.4, s 66.3, t 137.5 104.3 160.9 102.3 160.9 104.3 55.3, q, 2 × OMe

9 171.2, s 33.0,

t

30.1,

t

172.7, s 61.5, t 133.9 144.6 124.7 130.4 131.7 130.3

10 173.2, s 33.7,

t

31.9,

t

174.1, s 68.4, t 130.1 121.4 147.8 123.8 130.6 124.6

11 172.2, s 36.2,

t

29.3,

t

173.5, s 68.4, t 1431 130.0 123.8 147.4 123.8 130.0

12 172.7, s 34.0,

t

32.1,

t

174.2, s 63.7, t 132.5 129.6 129.2 131.2 127.4 127.1

13 173.5, s 32.8,

t

29.3,

t

174.1, s 65.6, t 138.1 125.8 136.3 128.1 130.3 124.1

14 173.6, s 33.7,

t

32.0,

t

175.3, s 65.3, t 137.9 127.5 128.3 133.8 128.3 127.5

99

Table 7. 13



15 173.0, s 32.9, t 30.9, t 174.1, s 66.6, t 140.3 120.8 131.8 128.9 128.8 127.1

16 173.1, s 33.5, t 31.5, t 174.2, s 66.6, t 138.5 128.7 122.2 130.7 129.9 126.1

17 173.3, s 32.7, t 30.2, t 175.1, s 65.5, t 136.8 128.8 131.6 121.7 131.6 128.8

18 173.1, s 32.8, t 30.2, t 176.0, s 71.8, t 142.6 95.7 138.2 129.3 127.7 126.7

19 173.6, s 35.1, t 33.5, t 175.3, s 66.8, t 139.2 135.1 94.2 137.1 129.7 127.3

20 173.2, s 34.0, t 31.1, t 175.2, s 65.1, t 137.7 128.6 137.4 93.7 137.4 128.6

21 173.4, s 30.7, t 28.9, t 175.3, s 66.5, t 141.9 133.4 128.2 128.1 124.5 129.1 16.9, q

22 173.7, s 30.8, t 29.8, t 175.8, s 67.6, t 137.7 128.9 138.2 128.1 128.4 123.8 21.6, q

23 174.3, s 30.7, t 29.4, t 175.3, s 67.3, t 136.9 128.9 130.8 138.1 130.8 128.9 20.9, q

24 170.2, s 32.5, t 29.7, t 172.6, s 65.8, t 127.6 155.4 117.2 128.7 122.9 130.6

25 170.6, s 32.4, t 29.8, t 172.7, s 65.7, t 138.8 113.5 158.7 113.8 132.9 120.1

26 170.2, s 32.5, t 29.4, t 172.8, s 66.1, t 127.9 130.1 115.8 156.8 115.8 130.1

27 173.7, s 32.1, t 29.4, t 175.2, s 63.5, t 139.6 143.5 117.0 128.6 125.3 126.4

28 173.8, s 32.2, t 29.5, t 175.3, s 66.8, t 138.6 115.4 145.5 112.8 128.1 11.8

100

Table 8. 13



29 173.9, s 32.3, t 29.7, t 175.4, s 66.9, t 125.8 130.1 116.4 148.6 116.4 130.1

30 171.3, s 33.2, t 32.2, t 176.9 s 65.8, t 137.6, s 135.8, s 131.4, d 134.1,s 128.9

,d

129.7,

d

31 171.4, s 33.1, t 32.6, t 177.9, s 66.5, t 138.3, s 129.4, s 133.3, d 133.6, d 131.2

, s

128.6,

d

32 171.7, s 32.5, t 32.1, t 176.8, s 59.8, t 142.3, s 132.7, s 128.4, d 130.5, d 128.4

, d

132.7,

s

33 171.4, s 33.1, t 32.6, t 177.9, s 66.8, t 138.3, s 129.6, d 133.3, s 133.6, s 131.5

, d

128.7,

d

34 171.4, s 33.1, t 32.6, t 177.9, s 66.1, t 146.2, s 126.3, d 136.5, s 129.8, d 136.5

, s

126.3,

d

35 172.9, s 33.2, t 32.4, t 178.7, s 62.6, t 130.7, s 138.6, s 150.6, s 117.5, d 127.5

, d

125.4,

d

36 171.7, s 32.9, t 31.4, t 177.2, s 62.1, t 124.6, s 162.3, s 105.4, d 162.2, s 112.2

, d

133.6,

d

37 172.1, s 32.2, t 31.7, t 179.7, s 62.4, t 130.6, s 156.8, s 118.3, d 117.2, d 159.1

, s

116.9,

d

38 171.4, s 33.1, t 32.6, t 177.9, s 56.5, t 115.4, s 162.6, s 113.6, d 129.9, d 113.6

, d

162.6,

s

39 171.4, s 33.1, t 32.6, t 177.9, s 66.3, t 140.6, s 138.7, s 132.2, d 139.2, s 127.6

, d

128.4,

d

19.8,

q, CH3

22.8,

q, CH3

40 171.9, s 33.1, t 32.6, t 177.2, s 66.3, t 143.6, s 132.6, s 131.5, d 129.8, d 136.3

, s

129.6,

d

19.7, q,

CH3

22.8,

q, CH3

41 171.7, s 33.3, t 32.5, t 177.6, s 67.2, t 134.6, s 129.7, d 138.3, s 136.2, s 131.6

, d

125.4,

d

19.2, q,

CH3

20.3,

q, CH3

42 171.3, s 33.4, t 32.2, t 177.9, s 67.4, t 136.4, s 126.3, d 139.6, s 130.2, d 139.6

, s

126.3,

d

22.4, q,

CH3

22.4,

q, CH3

101

Table 9. 13



43 171.9, s 32.8, t 31.6, t 179.7, s 66.3, t 149.8, s 125.6, d 150.3, s 144.9, s 126.

4, d

135.

5, d

44 171.7, s 33.6, t 32.4, t 179.4, s 65.6, t 144.2, s 130.1, d 150.4, s 118.4, d 150.

4, s

130.

1, d

45 172.1, s 32.6, t 31.4, t 175.9, s 56.6, t 119.7, s 155.5, s 105.7, d 130.1, d 105.

7, d

155.

5, s

15.7, q,

2CH3

65.7, t,

2CH2

46 172.4, s 32.9, t 31.3, t 175.8 s 67.8, t 124.6, s 115.5, d 150.3, s 151.2, s 113.

7, d

122.

3, d

15.5, q,

2CH3

65.8, t,

2CH2

47 172.2, s 33.3, t 31.5, t 175.9, s 67.7, t 139.2, s 103.1, d 157.9, s 101.7, d 157.

9, s

103.

1, d

48 172.6,s 33.3, t 31.1 t 175.9, s 63.7, t 121.2, s 150.5, s 141.5, s 154.2, s 105.

7, d

122.

3, d

57.5

3×CH3

49 172.8, s 33.2, t 31.7, t 176.3, s 63.6, t 121.3, s 152.5, s 96.9, d 150.4 (s 143.

0, s

112.

3, d

57.3, s,

3×CH3

50 172.8, s 33.2, t 31.7, t 176.3 s 63.6, t 131.3, s 105.2, d 154.5, s 137.5, s 154.

5, s

105.

2, d

57.3, q,

2CH3

61.3, q,

CH3

51 171.8, s 33.5, t 31.5, t 177.6, s 67.3, t 143.9, s 113.5, d 155.4, s 140.3, s 126.

7, d

121.

3, d

55.6, q,

CH3

52 171.6, s 33.8, t 31.1, t 177.7, s 66.7, t 135.4, s 125.6, d 142.1, s 151.4, s 113.

7, d

136.

5, d

55.6, q,

CH3

53 171.3, s 33.4, t 31.7, t 177.6, s 66.7, t 129.3, s 163.2, s 113.4, d 125.1, d 141.

1, s

125.

6, d

56.9, q,

CH3

54 172.1,

s

33.2, t 31.8, t 178.7, s 59.3, t 140.2, s 136.3, s 129.1, d 135.9, s 129.

1, d

136.

3, s

55 173.2,

s

33.7, t 31.2, t 178.7, s 64.3, t 140.7, 124.6, s 136.8, d 127.9, s 123.

9, s

136.

8, d

56 171.2, s 33.9, t 31.2, t 176.5, s 63.9, t 133.7, s 135.2, s 130.1, d 136.9, s 130.

1, d

135.

2, s

19.9 q,

2CH3

22., q,

CH3

102

Table 10. 1

H-NMR (300 MHz, CDCl3, δ, multiplicity, J in Hz) of 1a-28a

Cmpd 3 2 1ʹʹ Ar-H R Rʹ

1a 2.74, t, 6.5 Hz 2.60, t, 6.5 Hz 5.14, s 6.96-7.80, m, 4H 3.89, s

2a 2.74, t, 6.5 Hz 2.60, t, 6.5 Hz 5.14, s 6.96-7.80, m, 4H 3.89, s

3a 2.76, t, 6.5 Hz 2.61, t, 6.5 Hz 5.18, s 6.97-7.25, m, 4H 3.82, s

4a 2.79, t, 6.5 Hz 2.64, t, 6.5 Hz 5.23, s 6.96-7.31, m, 3H 3.87, s 3.87, s

5a 2.78, t, 6.5 Hz 2.66, t, 6.5 Hz 5.23, s 6.73-7.18, m, 3H 3.82, s 3.82, s

6a 2.74, t, 6.5 Hz. 2.60, t, 6.5 Hz 5.14, s 6.96-7.80, m, 3H 3.89, s, 2×OMe

7a 2.93, t, 6.5 Hz 2.79, t, 6.5 Hz 5.19, s 6.66-7.40, m, 3H 3.84, s, 2 × OMe

8a 2.87, t, 6.5 Hz 2.67, t, 6.5 Hz 5.16, s 6.92-6.53, m, 3H 3.83, s, OMe 3.84, s, OMe

9a 2.98, t, 6.5 Hz 2.81, t, 6.5 Hz 5.14, s 7.54-7.92, m, 4H

10a 2.99, t, 6.5 Hz 2.83, t, 6.5 Hz 5.21, s 7.45-7.99, m, 4H

11a 2.98, t, 6.5 Hz 2.78, t, 6.5 Hz 5.19, s 7.57-8.05, m, 4H

12a 2.85, t, 6.5 Hz, 2.67, t, 6.5 Hz 5.11, s 7.22-7.65, m, 4H

13a 2.87, t, 6.5 Hz 2.68, t, 6.5 Hz 5.19, s 7.31-7.59, m, 4H

14a 2.88, t, 6.5 Hz 2.72, t, 6.5 Hz 5.21, s 7.28-7.39, m, 4H

15a 2.83, t, 6.5 Hz 2.75, t, 6.5 Hz 5.12, s 7.23-7.55, m, 4H

16a 2.84, t, 6.5 Hz 2.73, t, 6.5 Hz 5.18, s 7.25-7.54, m, 4H

17a 2.82, t, 6.5 Hz 2.73, t, 6.5 Hz 5.22, s 7.21-7.86, m, 4H

18a 2.85, t, 6.5 Hz 2.76, t, 6.5 Hz 5.20, s 7.11-7.81, m, 4H

19a 2.88, t, 6.5 Hz 2.77, t, 6.5 Hz 5.25, s 7.16-7.72, m, 4H

20a 2.84, t, 6.5 Hz 2.73, t, 6.5 Hz 5.18, s 7.25-7.54, m, 4H

21a 2.92, t, 6.5 Hz 2.77, t, 6.5 Hz 5.11, s 7.22-7.27, m, 4H 2.35, s

22a 2.92, t, 6.5 Hz 2.76, t, 6.5 Hz 5.21, s 7.13-7.52, m, 4H 2.36, s

23a 2.96, t, 6.5 Hz 2.81, t, 6.5 Hz 5.21, s 7.15-7.21, m, 4H 2.37, s

24a 2.87, t, 6.5 Hz 2.74, t, 6.5 Hz 5.24, s 6.78-7.32, m, 4H 13.41, br, s

25a 2.92, t, 6.5Hz 2.75, t, 6.5 Hz 5.22, s 6.93-7.36,m, 4H 13.24, br, s

26a 2.93, t, 6.5 Hz 2.77, t, 6.5 Hz 5.23, s 6.88-7.15, m, 4H 12.78 br, s

27a 2.90, t, 6.5Hz 2.69, t, 6.5 Hz 5.10, s 6.87-7.32, m, 4H 12.32, br, s

28a 2.91, t, 6.5 Hz 2.70, t, 6.5 Hz 5.12, s 6.82-7.31, m, 4H 12.33, br, s

103

Table 11. 1H-NMR (300 MHz, CDCl3, δ, multiplicity, J in Hz) of 29a-56a

Cmpd 3 2 1ʹʹ Ar-H R Rʹ

29a 2.92, t, 6.5 Hz 2.71, t, 6.5 Hz 5.18, s 6.76-7.27, m, 4H 12.31, br, s

30a 2.91, t, 6.8 Hz 2.67, t, 6.8 Hz 5.06, s 7.18-7.87, m, 3H

31a 2.90, t, 6.8 Hz 2.71, t, 6.8 Hz 5.05, s 7.32-7.74, m, 3H

32a 2.92, t, 6.8 Hz 2.68, t, 6.8 Hz 5.07, s 7.46-7.66, m, 3H

33a 2.93, t, 6.8 Hz 2.69, t, 6.8 Hz 5.18, s 7.25-7.77, m, 3H

34a 2.91, t, 6.8 Hz 2.68, t, 6.8 Hz 5.22, s 7.41-7.79, m, 3H

35a 2.92, t, 6.8 Hz 2.69, t, 6.8 Hz 5.06, s 7.05-7.22, m, 3H

36a 2.93, t, 6.8 Hz 2.68, t, 6.8 Hz 5.07, s 6.58-7.46, m, 3H

37a 2.91, t, 6.8 Hz 2.70, t, 6.8 Hz 5.07, s 6.76-7.24, m, 3H

38a 2.92, t, 6.8 Hz 2.71, t, 6.8 Hz 5.11, s 7.22-7.83, m, 3H

39a 2.90, t, 6.8 Hz 2.68, t, 6.8 Hz 5.04, s 6.88-7.22, m, 3H 2.38, s, 2×Me

40a 2.92, t, 6.8 Hz 2.69, t, 6.8 Hz 5.05, s 6.98-7.19, m, 3H 2.39, s, 2×Me

41a 2.91, t, 6.8 Hz 2.68, t, 6.8 Hz 5.22, s 6.86-7.16 m, 3H 2.37, s, 2×Me

42a 2.90, t, 6.8 Hz 2.65, t, 6.8 Hz 5.21, s 7.12-7.34, m, 3H 2.38, s, 2×Me

43a 2.93, t, 6.8 Hz 2.66, t, 6.8 Hz 5.23, s 7.97-8.58, m, 3H

44b 2.92, t, 6.8 Hz 2.67, t, 6.8 Hz 5.23, s 8.55-8.72, m, 3H

45a 2.88, t, 6.8 Hz 2.66, t, 6.8 Hz 5.06, s 6.42-7.24, m, 3H 1.26, t; 4.25, q, 7.6 Hz

46a 2.89, t, 6.8 Hz 2.68, t, 6.8 Hz 5.19, s 6.62-7.09, m, 3H 1.25, t; 4.28, q, 7.6 Hz

47a 2.88, t, 6.8 Hz 2.69, t, 6.8 Hz 5.22, s 6.27-6.75, m, 3H 11.5 br, s

48a 2.87, t, 6.8 Hz 2.67, t, 6.8 Hz 5.26, s 6.39-6.84, m, 2H 3.98, s, 3×OMe

49a 2.88, t, 6.8 Hz 2.69, t, 6.8 Hz 5.26, s 6.44-6.95,2s, 2H 3.94, s, 3×OMe

50a 2.87, t, 6.8 Hz 2.71, t, 6.8 Hz 5.22, s 6.66, s, 2H 3.95, s, 3×OMe

51a 2.91, t, 6.8 Hz 2.71, t, 6.8 Hz 5.21, s 7.22-8.15, m, 3H 3.91, s, OMe

52a 2.89, t, 6.8 Hz 2.69, t, 6.8 Hz 5.19, s 7.24-8.23, m, 3H 3.97, s, OMe

53a 2.91, t, 6.8 Hz 2.68, t, 6.8 Hz 5.26, s 7.25-8.18, m, 3H 3.97, s, OMe

54a 2.92, t, 6.8 Hz 2.67, t, 6.8 Hz 5.03, s 7.77, s, 2H

55a 2.91, t, 6.8 Hz 2.66, t, 6.8 Hz 5.05, s 7.33-7.98, m, 2H

56a 2.93, t, 6.8 Hz 2.69, t, 6.8 Hz 5.12, s 6.92, s, 2H 2.47, s, OMe

104

Table 12. 13

C-NMR (75 MHz, CDCl3, δ) of 1a-28a

Cmpd 4 3 2 1 1ʹʹ 1ʹ 2ʹ 3ʹ 4ʹ 5ʹ 6ʹ R Rʹ

1a 175.6 36.6 31.5 169.9 67.8 121.2 159.1 113.8 128.1 120.1 128.9 56.6, OMe

2a 175.3 36.2 30.9 169.1 67.5 137.6 112.5 115.6 112.2 128.3 118.4 56.7

3a 176.5 37.6 30.1 168.9 67.8 127.2 127.3 112.5 157.5 112.4 126.4 55.5

4a 177.4 36.7 30.5 169.9 67.7 127.2 156.5 143.8 112.3 120.7 120.1 56.1 58.6,OMe

5a 176.7 36.3 29.4 169.2 67.2 115.8 158.6 99.8 159.6 105.9 128.2 56.3, 2×OMe

6a 179.7 36.1 29.6 169.9 64.8 121.1 159.2 113.3 128.8 128.8 120.1 56.6, 2× OMe

7a 176.6 36.3 29.5 167.4 68.4 123.5 113.3 149.1 147.8 111.7 120.7 55.2 (2 × OMe)

8a 173.3 36.1 29.2 168.5 68.9 136.7 104.6 160.7 98.2 160.7 104.6 57.4 (2 × OMe)

9a 172.3 35.5 30.2 169.7 64.7 135.3 148.3 126.2 129.5 131.1 113.7

10a 173.6 36.7 29.8 167.8 66.7 141.2 123.6 147.2 121.9 128.9 132.3

11a 173.2 35.8 29.3 169.5 66.9 141.6 127.1 123.2 145.7 123.2 127.1

12a 174.3 35.6 29.7 169.9 66.7 137.8 129.8 128.1 128.3 126.2 127.2

13a 174.4 35.5 29.7 168.5 64.4 137.6 125.4 133.0 126.2 128.8 123.7

14a 174.5 35.7 29.6 168.9 65.9 133.7 129.2 128.5 132.7 128.5 129.2

15a 174.4 35.5 29.3 168.8 63.9 136.6 125.2 128.0 128.3 126.2 130.2

16a 173.5 36.6 29.8 167.6 66.8 141.8 130.3 121.7 128.9 128.1 124.5

17a 173.6 36.5 29.4 167.8 66.2 136.3 128.7 130.6 121.4 130.6 128.7

18a 173.3 36.6 29.1 167.7 66.9 139.3 94.5 138.5 128.6 126.7 127.5

19a 173.5 36.2 29.3 167.4 66.5 139.4 138.5 94.6 135.8 129.6 126.7

20a 173.5 36.6 29.8 167.6 66.8 141.8 130.3 121.7 128.9 128.1 124.5

21a 175.2 37.5 28.9 168.9 66.3 140.8 132.3 122.5 122.3 124.2 129.4 20.6

22a 174.2 37.4 28.3 168.9 66.4 135.3 128.2 137.8 128.2 126.9 125.4 22.5

23a 174.9 37.1 28.2 168.8 66.1 136.4 127.6 128.5 136.4 128.5 127.6 22.4

24a 174.8 37.5 28.1 168.7 63.2 127.8 154.3 115.2 127.8 122.7 131.3

25a 174.7 38.1 28.5 168.8 66.3 136.4 113.2 158.3 116.1 131.4 120.2

26a 175.3 37.4 28.5 168.6 66.2 127.8 130.2 115.8 156.5 115.8 130.2

27a 174.9 38.2 28.4 168.7 63.5 142.4 115.6 127.3 125.2 126.3 138.8

28a 174.7 37.9 28.8 168.8 65.9 136.5 115.3 145.4 114.6 128.5 118.4

105

Table 13. 13

C-NMR (75 MHz, CDCl3, δ) of 29a-56a

Cmpd 4 3 2 1 1ʹʹ 1ʹ 2ʹ 3ʹ 4ʹ 5ʹ 6ʹ R

29a 175.1 37.6 28.2 168.3 66.1 125.6 128.5 114.4 146.6 114.4 128.5

30a 176.3 38.5 28.4 168.2 62.8 138.2 132.3 131.4 133.2 126.3 128.5

31a 175.9 38.7 28.8 168.7 62.9 138.8 127.6 129.3 128.5 131.6 127.8

32a 175.8 38.4 28.6 169.4 57.5 142.5 134.5 128.3 130.6 28.3 134.5

33a 175.7 38.4 28.8 169.2 66.2 138.3 129.4 133.7 133.7 131.6 128.4

34a 175.6 38.6 28.7 169.6 66.3 145.0 126.5 136.7 129.4 136.7 126.5

35a 174.7 37.9 28.3 170.2 62.3 130.4 138.8 148.3 115.4 125.6 123.6

36a 174.3 37.5 28.6 170.4 62.5 122.5 162.4 105.8 162.7 113.4 132.6

37a 173.9 37.6 28.9 170.3 61.8 130.8 156.4 116.7 115.6 159.5 114.7

38a 174.5 38.3 28.1 170.5 54.9 115.7 160.5 112.4 129.7 112.4 160.5

39a 175.6 38.0 28.4 169.9 65.4 138.6 136.7 132.1 136.7 125.6 125.9 20.6, 22.4

40a 174.7 37.8 28.2 170.4 65.8 141.2 130.1 129.0 126.7 134.5 127.8 19.3, 22.8

41a 174.0 38.4 28.3 171.2 66.9 132.3 127.9 136.7 134.5 129.4 123.1 19.4, 20.5

42a 175.2 38.6 28.1 171.3 67.2 134.5 125.2 137.5 128.9 137.5 125.2 22.6, 2C

43a 176.2 39.3 29.8 171.4 66.8 147.8 123.4 149.0 142.3 124.1 133.4

44a 176.3 39.4 29.6 171.5 64.7 142.2 128.7 148.9 116.7 148.9 128.7

45a 175.8 38.6 28.5 171.7 55.8 117.8 153.2 103.4 127.6 103.4 153.2 65.4, 15.9 (4C)

46a 174.9 38.3 29.4 171.6 66.8 122.3 112.4 145.8 146.7 111.8 119.4 65.5, 15.8 (4C)

47a 175.2 38.4 29.3 170.7 67.1 137.8 106.8 157.4 104.6 157.4 106.8

48a 175.5 39.2 29.5 171.3 62.5 119.8 147.5 140.4 153.6 105.7 119.8 61.5, 2×OMe

49a 174.4 38.8 29.7 172.9 63.1 119.6 149.8 96.9 148.5 141.3 112.6 57.2, 2×OMe

50a 175.1 38.9 29.1 171.2 67.5 129.2 108.4 154.6 137.9 154.6 108.4 61.2, 57.3, 3×OMe

51a 174.7 38.5 29.6 171.4 65.9 143.4 113.8 153.2 136.5 124.7 118.9 55.3, OMe

52a 175.7 37.6 28.9 171.3 65.7 133.2 123.8 139.6 149.5 112.4 133.7 55.4, OMe

53a 174.6 37.6 28.4 172.8 61.8 127.4 157.5 112.7 123.6 140.7 125.8 57.4, OMe

54a 174.3 39.2 29.4 171.6 57.8 138.4 134.9 127.5 134.9 127.5 134.9

55a 175.8 38.7 28.5 171.9 63.7 136.2 122.5 136.7 125.8 124.6 135.4

56a 175.2 37.9 28.6 171.4 63.5 135.8 132.6 127.4 132.6 127.4 132.6 20.3, 22.6 (3OMe)

106

Table 14. 1

H-NMR (300 MHz, CDCl3, δ, multiplicity, J in Hz) of 1b-27b

Cmpd 6 5 3 2 1ʹʹ Ar-H R Rʹʹ

1b 1.05, t, 7.4 Hz 2.55, q, 7.4 Hz 2.82, t, 6.8 Hz 2.72, t, 6.8 Hz 5.22, s 6.81-7.26, m, 4H 3.86, s



4b 1.02, t, 7.4 Hz 2.61, q, 7.4 Hz 2.85, t, 6.8 Hz 2.72, t, 6.8 Hz 5.13, s 6.67-6.99, m, 3H 3.79, s 3.82, s

5b 1.05, t, 7.4 Hz 2.62, q,7.4 Hz 2.85, t, 6.8 Hz 2.79, t, 6.8 Hz 5.21, s 6.63-7.41, m, 3H 3.78, s 3.85, s

6b 1.04, t, 7.4 Hz 2.49, q, 7.4 Hz 2.86, t, 6.8 Hz 2.73, t, 6.8 Hz 5.21, s 6.82-7.33, m, 3H 3.82, s, 2 × OMe

7b 1.03, t, 7.4 Hz 2.48, q, 7.4 Hz 2.85, t, 6.8 Hz 2.75, t, 6.8 Hz 5.17, s 6.43-7.21, m, 3H 3.81, s, 2 × OMe

8b 1.03, t,7.4 Hz 2.44, q, 7.4 Hz 2.81, t, 6.8 Hz 2.71, t, 6.8 Hz 5.19, s 6.45-6.99, m, 3H 3.84, s, 2 × OMe

9b 1.06, t, 7.4 Hz 2.57, q, 7.4 Hz 2.85, t, 6.8 Hz 2.72, t, 6.8 Hz 5.19, s 7.62-8.26, m, 4H




13b 1.03, t, 3H, Hz 2.42, q, 7.4 Hz 2.81, t, 6.8 Hz 2.72, t, 6.8 Hz 5.19, s 7.33-7.59, m, 4H



16b 1.03, t, 7.4 Hz 2.43, q, 7.4 Hz 2.83, t, 6.8 Hz 2.75, t, 6.8 Hz 5.20,s 7.29-7.55, m, 4H








24b 1.05, t, 7.4 Hz 2.43, q, 7.4 Hz 2.87, t, 6.8 Hz 2.66, t, 6.8 Hz 5.23, s 6.82-7.25, m, 4H 11.2 br, s


26b 1.05, t, 7.4 Hz 2.51, q, 7.4 Hz 2.91, t, 6.8Hz 2.69, t, 6.8 Hz 5.18, s 6.72-7.02, m, 4H 11.3 br, s

27b 1.04, t, 7.4 Hz 2.49, q, 7.4 Hz 2.88, t, 6.8Hz 2.68, t, 6.8 Hz 5.05, s 6.68-7.18, m, 4H 11.6 br, s

107

Table 15. 1H-NMR (300 MHz, CDCl3, δ, multiplicity, J in Hz) of 28b-56b

Cmpd 6 5 3 2 1ʹʹ Ar-H R Rʹʹ












39b 1.03, t, 7.4 Hz 2.42, q, 7.4 Hz 2.90, t, 6.8 Hz 2.68, t, 6.8 Hz 5.04, s 6.88-7.22, m, 3H 2.38, s, 2×Me

40b 1.03, t, 7.4 Hz 2.43, q, 7.4 Hz 2.92, t, 6.8 Hz 2.69, t, 6.8 Hz 5.05, s 6.98-7.19, m, 3H 2.39, s, 2×Me

41b 1.04, t, 7.4 Hz 2.42, q, 7.4 Hz 2.91, t, 6.8 Hz 2.68, t, 6.8 Hz 5.22, s 6.86-7.16 m, 3H 2.37, s, 2×Me

42b 1.05, t, 7.4, Hz 2.44, q, 7.4 Hz 2.90, t, 6.8 Hz 2.65, t, 6.8 Hz 5.21, s 7.12-7.34, m, 3H 2.38, s, 2×Me



45b 1.04, t, 7.4 Hz 2.43, q, 7.4 Hz 2.88, t, 6.8 Hz 2.66, t, 6.8 Hz 5.06, s 6.42-7.24, m, 3H 1.26, t; 4.25, q, 7.6 Hz

46b 1.03, t, 7.4 Hz 2.42, q, 7.4 Hz 2.89, t, 6.8 Hz 2.68, t, 6.8 Hz 5.19, s 6.62-7.09, m, 3H 1.25, t; 4.28, q, 7.6 Hz


48b 1.04, t, 7.4 Hz 2.43, q, 7.4 Hz 2.87, t, 6.8 Hz 2.67, t, 6.8 Hz 5.26, s 6.39-6.84, m, 2H 3.98, s, 3×OMe

49b 1.05, t, 7.4 Hz 2.42, q, 7.4 Hz 2.88, t, 6.8 Hz 2.69, t, 6.8 Hz 5.26, s 6.44-6.95,2s, 2H 3.94, s, 3×OMe

50b 1.04, t, 7.4 Hz 2.43, q, 7.4 Hz 2.87, t, 6.8 Hz 2.71, t, 6.8 Hz 5.22, s 6.66, s, 2H 3.95, s, 3×OMe

51b 1.05, t, 7.4 Hz 2.41, q, 7.4 Hz 2.91, t, 6.8 Hz 2.71, t, 6.8 Hz 5.21, s 7.22-8.15, m, 3H 3.91, s, OMe



54b 1.03, t, 7.4 Hz 2.41, q, 7.4 Hz 2.92, t, 6.8 Hz 2.67, t, 6.8 Hz 5.03, s 7.77, s, 2H


56b 1.05, t, 7.4 Hz 2.42, q, 7.4 Hz 2.93, t, 6.8 Hz 2.69, t, 6.8 Hz 5.12, s 6.92, s, 2H 2.47, s, OMe

108

Table 16. 13

C-NMR (75 MHz, CDCl3, δ) of 1b-19b

Cmpd 6 5 4 3 2 1 1ʹʹ 1ʹ 2ʹ 3ʹ 4ʹ 5ʹ 6ʹ R Rʹ

1b 7.7 36.1 212.1 34.2 28.3 172.2 62.8 124.7 155.6 129.3 128.9 123.7 127.5 56.3, OMe

2b 7.7 36.1 212.1 31.3 29.3 172.2 65.4 138.8 112.2 159.4 113.3 129.3 118.3 54.9, OMe

3b 7.7 34.2 212.1 33.4 27.8 172.2 66.2 129.2 129.3 113.6 159.1 113.6 129.3 55.4, OMe

4b 7.7 34.9 213.1 32.4 27.3 174.2 62.0 127.6 138.9 147.2 115.5 124.2 119.1 55.6, OMe 59.7, OMe

5b 7.7 36.1 214.1 33.2 29.3 171.2 62.8 116.1 158.3 100.4 162.6 108.5 128.8 55.4, OMe 56.5, OMe

6b 7.7 35.3 214.7 32.6 29.6 171.4 63.4 128.7 148.5 124.6 115.8 155.5 114.8 56.3, 2 × OMe

7b 7.7 36.1 214.4 35.8 31.2 171.8 66.3 124.7 147.3 148.7 146.3 111.4 121.2 55.7, 2 × OMe

8b 7.7 36.4 215.4 32.7 27.2 171.1 66.7 137.2 105.1 158.4 98.7 158.4 105.1 55.6, s, 2 × OMe

9b 7.7 36.7 213.2 32.6 29.1 171.2 65.1 134.3 147.4 124.7 129.2 129.9 134.3

10b 7.7 36.2 213.8 36.1 29.3 171.4 65.6 143.2 123.3 147.8 123.8 130.6 132.4

11b 7.7 36.6 214.2 31.4 27.8 171.6 66.3 143.4 129.9 123.7 147.4 123.7 129.9

12b 7.7 36.1 213.2 30.6 28.5 174.2 62.2 138.4 129.6 129.2 131.2 127.4 127.1

13b 7.7 36.2 212.1 36.1 29.3 172.0 65.7 138.6 125.8 133.5 128.1 130.1 124.1

14b 7.7 35.7 213.0 32.3 28.7 171.7 66.5 133.9 128.8 128.6 133.8 128.6 128.8

15b 7.7 37.1 214.2 32.2 27.7 173.2 66.3 139.7 123.7 128.6 128.7 127.5 131.2

16b 7.7 37.2 214.4 32.3 27.8 173.4 66.3 143.6 131.7 123.5 130.3 129.1 125.8

17b 7.7 37.4 214.7 32.7 27.3 173.8 66.5 134.6 139.7 132.3 121.6 132.3 139.7

18b 7.7 35.4 208.5 31.7 29.2 169.2 67.6 142.7 92.5 138.2 129.3 127.7 126.8

19b 7.7 35.6 208.1 31.3 29.4 169.7 65.1 142.2 136.1 94.2 137.1 129.7 127.3

109

Table 17. 13


Cmpd 6 5 4 3 2 1 1ʹʹ 1ʹ 2ʹ 3ʹ 4ʹ 5ʹ 6ʹ R Rʹ

20b 7.7 35.8 208.9 31.7 29.0 169.3 66.1 134.2 129.6 137.9 92.3 137.9 129.6

21b 7.6 36.2 209.1 32.3 29.4 170.2 66.3 144.2 135.8 128.4 128.7 126.2 129.4 19.9

22b 7.8 36.4 209.1 32.2 29.4 170.3 67.1 137.1 128.4 136.8 129.3 127.2 125.5 22.6

23b 7.6 36.3 208.6 31.6 29.3 171.2 66.9 135.8 126.5 128.8 138.6 128.8 126.5 22.2

24b 7.7 37.1 209.2 31.4 29.1 171.4 62.2 129.3 155.8 117.1 128.3 125.4 131.2

25b 7.8 36.9 208.8 31.3 29.2 171.5 66.2 138.4 111.9 156.6 113.5 129.7 118.8

26b 7.8 36.4 208.7 31.5 29.5 171.6 66.7 129.5 130.4 117.6 154.5 117.6 130.4

27b 7.7 36.7 208.5 31.2 29.6 171.4 63.6 141.3 143.7 115.8 127.2 123.4 124.8

28b 7.8 36.5 208.4 31.7 29.1 172.2 66.8 136.2 115.2 127.4 112.1 146.9 113.8

29b 7.6 36.2 209.3 32.3 29.4 172.3 66.7 125.2 127.4 116.2 145.6 116.2 127.4

30b 7.8 36.3 209.4 32.3 29.1 171.8 62.9 137.5 131.2 129.3 133.6 126.8 128.6

31b 7.7 36.1 209.1 32.4 29.5 172.2 61.9 138.5 127.4 129.6 128.3 131.7 128.9

32b 7.7 36.3 209.1 32.4 29.0 171.8 57.9 139.8 134.3 128.5 126.8 128.5 134.3

33b 7.8 36.4 208.6 32.1 29.2 172.1 66.3 137.2 129.4 133.5 133.6 131.6 128.4

34b 7.8 36.1 208.2 32.4 29.4 172.3 65.9 145.8 126.4 136.8 127.9 136.8 126.4

35b 7.8 36.2 209.3 32.6 29.5 172.4 62.3 130.2 136.3 150.2 117.5 127.4 125.5

36b 7.8 36.7 208.8 31.9 29.1 172.1 62.1 122.4 159.8 105.4 158.4 112.3 129.6

37b 7.8 36.3 209.1 31.2 29.2 172.6 61.9 127.5 153.2 116.4 114.2 156.3 114.8

38b 7.7 36.4 208.9 33.3 29.4 172.2 55.2 113.4 162.4 112.6 129.7 112.6 162.4

110

Table 18. 13


Cmpd 6 5 4 3 2 1 1ʹʹ 1ʹ 2ʹ 3ʹ 4ʹ 5ʹ 6ʹ R

39b 7.8 36.5 208.4 33.2 29.1 172.3 66.2 140.2 138.1 132.5 137.6 127.2 127.8 20.5, 22.6

40b 7.7 37.1 209.4 32.8 30.1 172.4 66.3 141.5 132.2 131.6 128.5 138.4 127.8 19.8, 22.4

41b 7.8 36.9 208.9 33.3 30.2 172.2 66.9 134.7 129.4 138.0 136.2 131.2 124.5 19.3, 20.4

42b 7.7 37.1 209.2 32.9 29.8 171.9 67.3 136.7 127.2 139.5 128.9 139.5 127.2 22.6 2×Me

43b 7.8 36.7 209.3 32.5 29.1 172.2 65.8 149.5 123.7 151.6 145.3 125.8 134.9

44b 7.8 36.5 208.8 32.2 29.7 171.8 64.8 144.2 130.2 148.5 116.4 148.5 130.2

45b 7.7 36.1 209.5 33.1 29.2 171.2 55.7 117.2 155.6 106.5 127.6 106.5 155.6 15.7, 65.4

46b 7.8 37.3 209.4 33.0 29.4 172.2 67.1 124.5 112.6 149.8 151.2 113.5 122.4 15.6, 65.5

47b 7.8 36.9 209.3 33.2 29.5 171.7 67.5 137.4 106.5 157.8 104.3 157.8 106.5

48b 7.8 36.8 209.6 32.9 29.3 172.4 62.9 119.6 148.5 143.6 153.2 106.3 123.7 62.2, 62.6, 57.2

49b 7.7 36.4 208.9 32.7 29.2 172.3 62.8 119.7 152.2 98.4 151.3 141.2 112.6 57.3 3×OMe

50b 7.8 37.2 209.1 32.1 29.4 172.2 67.6 132.6 108.3 154.7 139.6 154.7 108.3 62.5, 57.4 (2C)

51b 7.7 36.5 208.6 32.6 29.2 171.9 67.2 145.2 113.6 155.3 141.6 126.2 121.4

52b 7.8 36.5 208.7 32.3 29.5 172.1 65.8 135.6 125.8 142.3 153.2 115.4 136.7 55.2

53b 7.7 36.1 208.5 32.9 29.1 172.5 62.2 129.5 163.5 112.4 126.5 142.3 126.7 56.8

54b 7.8 36.2 208.6 32.8 29.2 172.6 58.4 140.7 136.6 129.8 137.9 129.8 136.6

55b 7.8 36.3 208.4 32.4 29.1 172.4 64.4 141.2 124.5 138.6 128.3 126.8 137.4

56b 7.4 37.1 208.7 33.4 29.4 172.3 63.7 135.2 135.8 129.4 138.5 129.4 135.8 20.3, 22.7

111

Table 19. Yield, mps/bps and Elemental Analysis of 57-60 and 57a-60a

Cmpd

Yield

(%)

mp.

(°C)

bp. /18mm

(°C)

Elemental Analysis (Found)

C H

57 76 64-65 ………. 52.49 (52.52) 7.55 (7.56)

58 77 69-71 ………. 55.16 (55.10) 8.10 (8.11)

59 76 75-76 ………. 57.43 (57.45) 8.57 (8.58)

60 76 115-116

(Lit.117-119)

………. 63.45 (63.47) 5.81 (5.75)

57a 78 … 94-95 ………. ……….

58a 78 … 101-103 ………. ……….

59a 72 … 108-109 ………. ……….

60a 71 … 110-112 ………. ……….

112

Table 20. Yield and bps of 57b-60b for comparison

Cmpd

Yield

(%)

bp. /18mm

(°C)

Diethyl cadmium Gilman reagent Grignard reagent

57b 77 29 22 106-107

58b 74 28 21 112-113

59b 72 27 18 118-119

60b 71 23 18 116-117

113

Table 21. Spectroscopic data of 57-60, 57a-60a and 57b-60b

Cmpd

IR

(cm-1

)

1H-NMR

(δ)

13C-NMR (DEPT)

(δ)

HR-EI-MS (m/z)

Calcd.

Found

57 3417 (OH)

1728 (CO2H)

1755 (CO2R)

2.55 (t, 6.7)

2.72 (t, 6.7)

4.45 (septet, 7.5)

1.1 (d, 7.5)

173.3 (s), 28.2 (t)

36.6 (t), 180 (s)

68.3 (d) 21.2 (q)

160.17

C7H12O4

160.15

58 3438 (OH)

1725 (CO2H)

1759 (CO2R

2.55 (t, 6.7), 2.72 (t, 6.7)

4.01 (d, 7.5)

1.18 (septet, 7.5)

1.1 (d, 7.5)

173.3 (s), 28.2 (t)

36.6 (t),180 (s)

68.3 (t), 26.7 (d)

21.2 (q)

174.19

C8H14O4

174.18

59 3396 (OH)

1722 (CO2H)

1765 (CO2R)

2.55 (t, 6.7), 2.72 (t, 6.7)

4.45 (d, 7.5), 1.48 (m)

1.77 (m), 1.1 (d, 7.5)

173.3 (s), 28.2 (t)

36.6 (t), 180 (s)

68.3 (t), 36.7 (t)

23.6 (d), 21.2 (q)

188.22

C9H16O4

188.24

60 3324 (OH)

3014 (Ar-H)

1748 (CO2R)

1708 (CO)

2.57 (t, 6.7), 2.69 (t, 6.7)

5.2 (s), 7.2-7.3 (Ph)

171.7 (s), 28.3 (t)

35.5 (t), 203.2 (s)

79.1 (t), 128-137

(1C, s, 5C, d)

208.21

C11H12O4

208.19

57a 1810 (COCl)

1755 (CO2R)

741 (C-Cl)

2.53 (t, 6.7), 2.73 (t, 6.7)

4.5 (septet, 7.5)

1.12 (d, 7.5)

171.4 (s), 28.4 (t)

37.5 (t), 172.6 (s)

66.2 (d), 20.1 (q)

178.61

C7H11ClO3

178.62

58a 1802 (COCl)

1740 (CO2R)

734 (C-Cl)

2.56 (t, 6.7), 2.70 (t, 6.7)

4.05 (d, 7.5), 1.47 (m)

1.16 (d, 7.5)

172.2 (s), 27.0 (t)

37.8 (t), 205.2 (s)

64.5 (t), 31.7 (d)

22.1 (q)

192.64

C8H13ClO3

192.65

59a 1807 (COCl)

1739 (CO2R)

739 (C-Cl)

2.58 (t, 6.7), 2.74 (t, 6.7)

4.1 (t, 7.5), 1.6 (dt, 7.5)

1.5 (m), 1.1 (d, 7.5)

171.1 (s), 27.5 (t)

36.9 (t), 172.3 (s)

59.9 (t), 37.2 (t)

25.5 (d), 22.2 (q)

206.67

C11H20O3

206.66

60a 3094 (Ar-H)

1787 (COCl)

1748 (CO2R)

734 (C-Cl)

2.57 (t, 6.7), 2.69 (t, 6.7),

5.2 (s), 7.2-7.3 (Ph)

171.7 (s), 28.3 (t)

35.5 (t), 176.7 (s)

79.1(t), 128-137

(1C, s. 5C, d)

226.66

C11H11ClO3

226.67

57b 1745 (CO2R)

1729 (CO)

1210 (C-O)

2.57 (t, 6.7), 2.71 (t, 6.7)

2.49 (t, 7.3), 1.08 (t, 7.3)

4.52 (septet,7.5)

1.15 (d, 5)

171.2 (s), 27.9 (t)

35.6 (t), 204.5 (s)

34.7 (t), 7.9 (q)

63.7 (d), 22.6 (q)

172.22

C9H16O3

172.21

58b 1740 (CO2R)

1722 (C=O)

1220 (C-O)

2.56 (t, 6.7), 2.70 (t, 6.7)

2.47 (t, 7.3), 1.07 (t, 7.3)

4.05 (d, 7.5), 1.47(m)

1.16 (d, 7.5)

172.3 (s), 27.0 (t)

37.8 (t), 205.2 (s)

35.2 (t), 8.3 (q)

64.5 (t), 31.7 (d)

186.23

C10H18O3

186.24

114

Table 22. Spectroscopic data of 59b-60b

Cmpd IR (cm-1

) 1H-NMR (δ)

13C-NMR (DEPT)

(δ)

HR-EI-MS (m/z)

Calcd. Found

59b 1739 (CO2R)

1717 (C=O)

1225 (C-O)

2.58 (t, 6.7), 2.74 (t, 6.7)

2.51 (q, 7.3), 1.09 (t, 7.3)

4.1 (t, 7.5), 1.6 (dt, 7.5)

1.5 (septet, 7.5)

1.1 (d, 7.5)

173.2 (s), 27.5 (t)

36.9 (t), 202.3 (s)

35.9 (t), 7.7 (q)

59.9 (t), 37.2 (t)

25.5 (d), 22.2 (q)

200.27

C11H20O3

200. 26

60b 1736 (CO2R)

1718 (C=O)

1230 (C-O)

1.05 (t, J = 7.3 Hz, 3 H)

2.48 (q, J = 7.3 Hz, 2 H)

2.65 (t, J = 6.7 Hz, 2 H)

2.74 (t, J = 6.7 Hz, 2 H)

5.15 (s, 2 H)

7.33-7.35 (m, 5 H)

171.7 (s), 28.3 (t)

35.5 (t), 208.2 (s)

36.1 (t), 7.9 (q)

66.1 (t)

128-137

(1C, s, 5C, d)

220.26

C13H16O3

220.24

115

Table 23. Inhibition zones of monoesters 1-29 against fungi and bacteria

Organism

/compound

Antifungal activity

Zone of inhibition (mm)

Antibacterial activity

Zone of inhibition (mm)

C.

gloeosporioides

A.

brassicicola

C.

capsici

K.

pneumonia

E. coli Staph.

Aureus

1 11.72 12.43 13.47 13.14 12.10 13.74

2 9.96 11.24 10.19 11.11 10.71 10.54

3 13.24 15.55 13.22 13.66 15.82 13.11

4 9.66 8.64 9.45 11.22 10. 55 10.56

5 8.98 9.43 8.45 9.81 10.17 10.66

6 10.50 9.24 6.86 9.29 7.83 8.77

7 8.66 8.89 6.74 7.77 8.37 9.66

8 8.44 9.33 6.72 10.75 9.71 9.77

9 - - - - - -

10 - - - - - -

11 - - - - - -

12 14.33 16.34 17.37 12.35 13.53 14.36

13 12.35 14.38 13.39 11.37 9.39 10.38

14 14.33 15.36 16.36 13.33 12.38 15.36

15 13.27 14.31 16.28 12.27 11.44 13.28

16 12.39 11.35 12.36 11.65 13.32 13.33

17 21.88 22.88 19.28 9.32 11.31 8.43

18 22.15 22.11 23.11 16.19 17.18 16.23

19 16.45 18.50 16.15 14.43 15.29 16.25

20 22.21 23.13 23.12 22.12 22.13 20.12

21 12.25 12.34 11.22 11.34 12.31 11.45

22 12.71 11.23 10.23 9.23 8.65 10.33

23 11.36 12.24 10.35 9.53 11.32 10.34

24 12.68 12.64 12.09 12.66 12.26 12.76

25 12.72 12.17 12.47 12.11 12.70 12.73

26 12.66 12.14 12.36 12.56 12.67 12.64

27 12.48 12.54 12.58 12.33 12.53 12.59

28 12.64 12.63 12.56 12.36 12.61 12.64

29 12.24 12.54 12.48 12.56 12.54 12.49

Chloramph

enicol

26.67 25.11 26.15 - - -

Ketoconazo

le

- - - 25.25 24.13 23.56

116

Table 24. MICs of monoesters 1-29 against fungi and bacteria

Organism

/compound

Antifungal activity

MIC mg/L=µg/Ml

Antibacterial activity

MIC mg/L=µg/mL

C.

gloeosporioides

A.

brassicicola

C. capsici K.

pneumonia

E. coli Staph. Aureus

1 4.7 0 4.21 4.71 4.73 4.26 4.75

2 5.2 9 5.31 5.61 5.51 5.76 5.41

3 5.61 4.49 4.51 4.7 1 3.31 3.61

4 4.89 7.58 6.89 6.81 6.41 5.81

5 5.58 6.19 7.68 7.59 7.14 6.68

6 4.81 6.39 6.78 6.89 6.37 7.78

7 4.78 6.18 6.87 7.77 7.38 6.63

8 6.68 6.29 6.71 6.61 7.29 7.72

9 - - - - - -

10 - - - - - -

11 - - - - - -

12 2.31 2.41 2.71 2.51 2.54 2.65

13 2.51 2.81 2.91 2.71 2.9 1 2.86

14 2. 31 2.61 2.61 2. 31 2.80 2.64

15 2.71 2.14 2.86 2.71 2.43 2.80

16 2.91 2.51 2.65 2.81 2.26 2.36

17 2.92 2.88 2.87 2.29 2.12 2.48

18 2.45 2. 50 2.55 2.21 2.91 2.41

19 2.95 2.97 2.94 2.96 2.88 2.78

20 2.12 2.13 2.12 2.22 2.32 2.22

21 3.25 3.40 3.71 3.40 3.11 4.51

22 3.30 3.21 3.41 3.29 3.08 3.31

23 3.63 3.31 3.51 3.56 3.29 3.46

24 6.86 6.41 6.91 6.62 6.21 6.71

25 7.28 7.11 7.41 7.10 7.01 7.32

26 5.64 5.11 6.31 6.53 6.78 6.48

27 4.88 5.41 5.81 5.71 5.32 5.68

28 6.41 6.31 6.51 6.36 6.12 6.51

29 4.91 5.41 4.81 5.61 5.21 4.90

Chloramp

henicol

1.22 1.12 1.12 - - -

Ketoconaz

ole

- - - 1.15 1.32 1.22

117

FIGURES

118

Fig. 1. General structures for various ketoesters

119

Fig. 2. Preparation of monoesters (1-60), acid chlorides (1a-60a) and γ-ketoesters (1b-60b)

Ar Compound Ar Compound

2-(OMe)C6H4 1, 1a, 1b 2,5-(Cl2)C6H3 31, 31a, 31b

3-(OMe)C6H4 2, 2a, 2b 2,6-(Cl2)C6H3 32, 32a, 32b

4-(OMe)C6H4 3, 3a, 3b 3,4(Cl2)C6H3 33, 33a, 33b

2,3-(OMe)2C6H3 4,4a,4b 3,5-(Cl2)C6H3 34, 34a, 34b

2,4-(OMe)2C6H3 5,5a,5b 2,3-(F2)C6H3 35, 35a, 35b

2,5-(OMe)2C6H3 6, 6a, 6b 2,4-(F2)C6H3 36, 36a, 36b

3,4-(OMe)2C6H3 7, 7a, 7b 2,5-(F2)C6H3 37, 37a, 37b

3,5-(OMe)2C6H3 8, 8a, 8b 2,6-(F2)C6H3 38, 38a, 38b

120

2-NO2-C6H4 9, 9a, 9b 2,4-(Me2)C6H3 39, 39a, 39b

3-NO2-C6H4 10, 10a, 10b 2,5-(Me2)C6H3 40, 40a, 40b

4-NO2-C6H4 11, 11a, 11b 3,4-(Me2)C6H3 41, 41a, 41b

2-Cl-C6H4 12, 12a, 12b 3,5-(Me2)C6H3 42, 42a, 42b

3-Cl-C6H4 13, 13a, 13b 3,4-(NO2)2C6H3 43, 43a, 43b

4-Cl-C6H4 14, 14a, 14b 3,5-(NO2)2C6H3 44, 44a, 44b

2-Br-C6H4 15, 15a, 15b 2,6-(EtO)2C6H3 45, 45a, 45b

3-Br-C6H4 16, 16a, 16b 3,4-(EtO)2C6H3 46, 46a, 46b

4-Br-C6H4 17, 17a, 17b 3,5-(HO)2C6H3 47, 47a, 47b

2-I-C6H4 18, 18a, 18b 2,3,4-(MeO)3C6H2 48, 48a, 48b

3-I-C6H4 19, 19a 19b 2,4,5-(MeO)3C6H2 49, 49a, 49b

4-I-C6H4 20, 20a, 20b 3,4,5-(MeO)3C6H2 50, 50a, 50b

2-Me-C6H4 21, 21a, 21b 3-MeO-4-NO2-C6H3 51, 51a, 51b

3-Me-C6H4 22, 22a 22b 4-MeO-3-NO2-C6H3 52, 52a, 52b

4-Me-C6H4 23, 23a, 23b 2-MeO-5-NO2-C6H3 53, 53a, 53b

2-OH-C6H4 24, 24a, 24b 2,4,6-(Cl)3C6H2 54, 54a, 54b

3-OH-C6H4 25, 25a, 25b 2,4,5-(Br)3C6H2 55, 55a, 55b

4-OH-C6H4 26, 26a, 26b 2,4,6-(Me3)C6H2 56, 56a, 56b

2-NH2-C6H4 27, 27a, 27b (CH3)2CH 57, 57a, 57b

3-NH2-C6H4 28, 28a, 28b (CH3)2CH-CH2 58, 58a, 58b

4-NH2-C6H4 29, 29a, 29b (CH3)2CH- CH2-CH2 59, 59a, 59b

2,4-(Cl2)C6H3 30, 30a, 30b C6H5 60, 60a, 60b

121

Fig. 3. Atom numbering in compounds for assignment of spectral data

122

Fig. 4. Tetrahedral intermediate from reaction of RCdCl with CH3COCl

123

Fig. 5. Proposed mechanism for ketone formation from acid chlorides and diethyl cadmium

124

Fig. 6.

1H (


0 and HOHAHA Bold-bond (ii) HMBC

(iii) NOESY (iv) HMQC for compound 1.

125

Fig. 7. Presentation of 2D NMR interactions in 30

126

1H (


0 and HOHAHA Bold-bond (ii) HMBC

(iii) NOESY (iv) HMQC

Fig. 8. Presentation of 2D NMR interactions in 1b

127

Fig. 9. Presentation of 2D NMR in 30b

128

Fig. 10. Fragmentation pattern of compound 1

129

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130

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141

PART-II

PHYTOCHEMICAL STUDIES

OF

BIOACTIVE COMPOUNDS

FROM

CORIANDRUM SATIVUM LINN.

UMBELLIFERAE

142

CHAPTER-1

INTRODUCTION

143

1. INTRODUCTION

1.1. General Introduction

Importance of the plants like, fig, olive, date palm, pomegranates, onion, garlic, lentils, zinger,

colocynth and basil has been referred in “AL-QURAN and Hadith” (Surah No. 16, Verse No. 68,

69; Marwat et al., 2009; Janick, 2007).

Almost all medicines in use have directly or indirectly origin from plants (Marwat et al., 2009).

Hence plants can be considered as the major source of medicines and drugs (Kainsa et al., 2012;

Lu et al., 2012; Shoeb, 2006; Li et al., 2003; Nirmala et al., 2011). In this connection some of the

drugs/medicines origin from plants with uses are listed in Table 1. The importance of Natural

Products can be highlighted from the fact that most of the anticancer drugs have plants origin and

still the work is going on to explore such type of drugs (Marwat et al., 2009; Newman and Cragg,

2007; Ali et al., 2008).

It has therefore, become mandatory to explore the plants for their phytochemicals constituents

and evaluate their bioactivity. Luckily, Indopak subcontinent is very rich in medicinal plants and

a number of medicinal plants families like the Berberidaceae, the Euphorbiaceae, the

Caesalpiniaceae, the Solanaccac etc are in abundance (Chen et al., 2002; Juan et al., 2008;

Maghraby and Hassan, 2005). However, upto now only 10% plants has been explored and there

is a need to extract, isolate and identify bio-active constituents of the plants. The prevailing

situation prompterd us to undertake phytochemical study on the Coriandrum sativum Linn, a

species of family the Umbelliferae.

144

Photo: Coriandrum sativum Linn, with shoots, leaves and inflorescence

145

1.2. The Umbelliferae

The Umbelliferae family comprises of 455 genera and 3600-3751 species and is distributed all

over the world (Pimenov and Leonov, 1993; Menglan, 2005; Tenni et al., 1998; Suguna et al.,

1996). A major number of plants of the family are native of hilly and plain regions of Indo-Pak

subcontinent (Kljuykov and Ukrainskaja, 2010). Pakistan is a habitat of approximately 56 genera

and 167 species of the Umbelliferae (Perveen and Qaiser, 2006).

Coriandrum sativum grow in hilly and plain parts of Pakistan and is widely used in folk

medicines (Perveen and Qaiser, 2006; Pimenov and Leonov, 2004; Khan and Khatoon, 2008;

Khan et al., 2011). Coriandrum sativum is a rich source of spices and is termed as plant of

economical significance ( Koppula and Choi, 2011). Plants of the Umbelliferae have diversity in

chemical constituents (Sriti et al., 2010; Radulovic et al., 2012; Siripornvisal et al., 2011; Singh

et al., 2012; James and Dubery, 2009; Olle and Bender, 2010; Pimenov and Leonov 2004; Amin

and Mousa, 2007; Vetter, 2004). Food, perfumery and medicine industries make use of these

Plants (Olle and Bender, 2010; Pimenov and Leonov, 2004; Gui-Sheng et al., 2012).

The Umbelliferae family is enrich with essential oils having antioxidant, antibacterial, antifungal

and insecticidal properties, and are also used against mosquito slaying (Kilama, 2009; Nickavar

and Abolhasani, 2009; Orhan et al., 2012; Singh et al., 2012).

The genus Coriandrum of the Umbelliferae has two species: Coriandrum tordylium Fenzl (Hedge

and Lamond, 1972; Mouterde, 1986) and Coriandrum sativum Linn.

Coriandrum sativum Linn is an annual green herb with an approximate height of 250-1450 cm. It

is cultivated all over the world almost in all seasons. Stem of the plant is erect, glabrous with

several side branches at the basal node and root is taproot. Leaves are alternate and each branch

ends with an inflorescence called umbel.

146

The botanical taxonomy of Coriandrum sativum Linn is given below

Kingdom Eukaryota

Division Spermatophyta

Subdivision Angiosperm

Class Dicotyledon

Sub class Rosidae

Order Umbellales

Family Umbelliferae

Genus Coriander

Species Coriandrum sativum Linn

Coriandrum sativum has been subject of an active research of a large number of researchers

(Pandey et al., 2011; Mouterde, 1986; Hedge and Lamond, 1972; Burdock and Carabin, 2009;

Burt, 2004; Wangensteen et al., 2004; Cantore et al., 2004; Ramadan et al., 2003; Barrtta et al.,

1998; Sriti et al., 2011, 2012; Msaada, 2007; Sreelatha et al., 2009; Emamghoreishi et al., 2005)

From the seeds of coriandrum sativum glucosinolates and sinapine have been isolated (Matthäus

and Angelini, 2005, Chen and Viljoen, 2010; Bakkali et al., 2008; Wangensteen et al., 2004;

Zhou et al., 2011).

Unfortunately, all of the research that has been carried out up to now on C. sativum was limited

to the essential oils (Bhuiyan et al., 2009; Wangensteen et al., 2004; Cantore et al., 2004;

Ramadan et al., 2003; Burt, 2004). But no proper attention has been concentrated to isolate other

natural products from the species (Ramadan and Morsel, 2003).

These properties prompted us to explore Coriandum sativum Linn for some more bioactive

compounds and study their biological activity against bacteria and fungi.

Present investigation on C. sativum Linn, led us to quite encouraging results and several new

oleane type pentacyclic triterpenoids have been isolated and characterized with the help of

modern spectroscopic techniques.

147

CHAPTER-2

RESULTS AND DISCUSSION

148

2. RESULTS AND DISCUSSION

2.1. Essential Oil

After hydrodistillation of C. sativum seeds the obtained EO was subjected to gas chromatography

which displayed seventeen peaks. The peaks were identified by running the parallel authentic

samples and determining retention time as well as Rf values. In addition, functional groups

present in the essential oil were identified by recording IR spectrum. Alcoholic, aldehydic, and

ester groups were identified (Fig. 1). The results revealed that the essential oil is a rich source of

β-Linalool (79.86%) as displayed in Table 2.

149

2.2. Isolates

2.2.1. 1-Oxo-11β, 21β-dihydroxyoleane (1)

The whole plant methanolic extract was suspended in water and re-extracted, with various

organic solvents (Experimental section 3.6.1). Ethyl acetate soluble portion was subjected to

column chromatography on silica gel, elution was carried out with mixture of petroleum

ether/ethyl acetate to yield eight (1-8) fractions. The active fractions 3-4 containing more than

two components were subjected to separation on the same silica gel column and eluted with

petroleum ether/acetone (7/3), to get semi-pure triterpenes. Further purification was carried out

on preparative HPLC and eluted with MeOH-H2O (80/20) to get compounds (1) (31.3 mg), (tR =

7.9 min) (2), (tR = 8.1 min) (25.5 mg), (tR = 8.4 min) (3) (31.7 mg), (tR = 8.7 min) (4) (15.8 mg)

and (tR = 9.3 min) (5) (37.6 mg).

Powdered fresh shade dried seeds of C. sativum L. were extracted with methanol using the

procedure as described in experimental section (3.6.5). Ethyl acetate soluble fraction was

150

subjected to purification on preparative HPLC, using MeCN/ H2O, gradient scheme (A = H2O;

B = MeCN/H2O 88/ 12; 5 min A, linear gradient to B in 20 min, 5 min B, linear gradient back to

A in 2 min at 2 mL/min flow rate), as a mobile phase, to afford pure compounds (6) (131.3 mg),

(7) (125.3 mg), (8) (130.1. mg), (9) (151.5 mg), (10) (127.4 mg) (11) (137.6 mg), and 12 (155.3

mg), and 13 (152.5 mg), respectively. The pure compounds were subjected to spectroscopic

study for structure elucidation. After spectral analysis, compounds 6-13 were identified (Tables

11,13 ).

Compound 1, obtained as solid needles, m.p. 255-258 ºC; [α]25

D + 5.43° and displayed an [M]+ ion

peak at m/z= 458 in HR-EIMS for [C30H50O3]+

. EIMS gave peaks at m/z= 430 [M-28-CO]+, 440

[M-18- H2O]+ and m/z= 412 [M-36-2H2O]

+. In IR spectrum bands were at 1689 cm

-1 (keto) and

3404, 3532 (OH) cm-1

. Its UV spectrum displayed an absorption band at 216 nm (1.56) for an

isolated cyclic ketone (Gershenzon and Dudareva, 2007). Its 1H-NMR (Table 3) displayed eight

singlets three protons each at δ 0.97, 1.07, 1.13, 1.16, 1.17, 1.55,1.66, 1.38 and were due to eight

methyl groups in the basic skeleton of the molecule. Out of these four were gem-methyl that

resonated at 1.66, 1.07 (Me-23,Me-24) at C-4, 1.38, 0.97 (Me-29, Me-30) at C-20 were identified

by HMBC (Fig. 2). In the same spectrum there were nine methylene groups appeared as a

complex multiplicity. Peaks appeared at δ 2.22 ddd (J=13, 5.2, 3.5 Hz), 1.97 dd (J=13, 5.2 Hz)

were assigned to CH2-2 & CH2-3. Similarly other peaks integrated for two protons were at δ 1.83

d (J= 11.5 Hz), 2.17 dd (J= 14, 6.5 Hz) for CH2-6 and CH2-7 and peaks at δ 2.44 ddd (J=13.5,

13.5, 3.0 Hz), 1.46 ddd (J=13.5, 13.5, 3.1 Hz), 2.53 ddd (J= 13.5, 13.5, 3.0 Hz), 1.56 ddd

(J=13.5, 13.5, 3.0 Hz) were assigned to α β methylene protons of C-15,16 (Table 3). In addition

there was another set of methylene protons resonated at δ 1.88 ddd (J= 14.1, 4.5, 12.2 Hz, CH2-

12), δ 1.71 ddd (J=14.5, 11.5, 4.0 Hz, H-19α), δ 1.17 dd (J=14.5, 4.0 Hz, H-19β), δ 2.91 ddd

151

(J=13.5, 13.5, 2.5 Hz, H-22α), δ 1.91 dd (J=13.5, 13.5 Hz, H-22β) (Table 3). 1H-NMR spectrum

of compound 1 revealed six methine protons. Out of these two were in the middle region of NMR

(δ 3.29 ddd (J=14, 6.5, 4.2 Hz), δ 3.46 ddd (J= 13.5, 13.5, 3.0 Hz) for hydroxy methine moieties

at C-11 and C-21 by NOE irradiation. Remaining four methine protons were identified by peaks

at δ 1.83 d (J=11.5 Hz), 1.64 dd (J=14, 6.5 Hz), 2.01 dd (J=12.2, 4.5 Hz), 1.78 ddd (J=12.5, 12.5,

3.5 Hz). 13

C-NMR of compound 1 displayed 30 peaks for thirty carbons (Table 4). In 13

C-NMR

spectrum peak appeared at δ 214.7 due to carbonyl group and its Position was located with the

help of HMBC experiments (Fig. 2).

Various protons bearing fragments were identified with the help of COSY-45º and HOHAHA

experiments (Fig. 2). These fragments were connected with each other with the help of HMBC

technique (Fig. 2).

The BB and DEPT 13

C-NMR spectrum of 1 showed 30 carbon signals (Table 4), 8Me, 9CH2,

6CH and 7C. The relative stereochemistry of OH groups at C-11 and C-21 in 1 was deduced by

NOESY spectrum (Fig. 3). In this spectrum interactions were observed among α-oriented H-8, H-

13, H-18, Me-23 and Me-27. In the light of these experimental results it was concluded thad

compound 1 has basic skeleton of triterpene as reported earlier (Sule et al., 2011; Abreu et al.,

2011). Hence, structure for 1 was confirmed as 1-oxo-11β, 21β-dihydroxy-oleane.

152

2.2.2. 1-Oxo-11β-hydroxy-21β-O-acetyloleane (2)

Compound 2 was acetyl derivative of 1 as in HR-EIMS molecular ion peak was at m/z= 500.7515

for C32H52O4. 1H-NMR spectrum of 2 was very close to 1 except presence of an extra peak at δ

2.29 for three protons. Presence of acetyl moiety in 2 was proved by 13

C-NMR spectrum. In this

spectrum in addition to other peaks there were two more peaks present at δ 176.2 and δ 22.3 was

assigned to acetyl group. Position of the acetyl group was located at C-21 with the help of HMBC

and NOE experiments (Fig. 2, 3). The DEPT 13

C-NMR showed 32 carbon signals, 9Me, 9CH2,

6CH and 8C. Hence the proposed structure of 2 was 1-Oxo-11β-hydroxy-21β-O-acetyloleane.

153

2.2.3. 1-Oxo-11β-hydroxy-21β-O-angeloyloleane (3)

Compound 3 was obtained as white solid, crystalline, m.p. 258-260 ºC; [α]25

D + 5.43°. In UV

specrum displayed λmax at 308 (4.28) and 214 (4.26), 267 (3.7) nm indicated presence of α,β-

unsaturated ester as a chromophore.

Compound 3 showed molecular ion [M+] peak m/z=540.8143 in HR-EIMS corresponding to

C35H56O4. Therefore, it was proposed an angeloyl derivative of 1. 1H-,

13C-NMR spectra of 3 was

similar to 1 but there were peaks for angelate moiety: H-3 (δ 6.13, 1H, qq, J=6.6 Hz), CH3-4 (δ

2.01, 3H, dq, J=6.5, 2.5 Hz), and CH3-5 at δ 1.93 [3H, dqm, J= 6.5, 2.2 Hz]; δ 168.3 (C-1), 128.0

(C-2), 137.3 (C-3), 15.9 (C-4), and δ 20.6 (C-5). Therefore, 3 was considered as angeloyl

derivative of 1. Position of angelate was inferred at C-21 by HMBC and NOE interaction

between H-21 and an angeloyl proton H-3. The DEPT 13

C-NMR spectrum of 3 showed 35 carbon

signals, 10Me, 9CH2, 7CH and 9C. On the basis of these results structure of 3 was concluded as

1-oxo-11β-hydroxy-21β-O-angeloyloleane.

154

2.2.4. 1-Oxo-11β-O-angeloyl-21β-O-acetyloleane (4)

Compound 4, colorless, amorphous, m.p. 267-268 ºC; [α]25

D + 6.43°. UV spectrum showed

compound is uv active by displaying λmax at 347 (4.55), 284 (4.56), 253 (4.22) 234 (2.7) nm

typical for angelic moiety. IR spectrum showed absence of OH groups in the compound. There

were peaks at νmax 1723, 1706 cm-1

for ester carbonyl groups.

Molecular formula C37H58O5 of compound 4 was established with the help of HR-EIMS;

displaying molecular ion peak at m/z= 582. In EIMS fragmentation peak appeared at m/z = 522

[M+-60- AcOH] indicated presence of an acetate. Other peak at m/z= 482 was due to loss of

angelic acid from the parent molecule. Therefore, it was considered as diester of acetic and

angelic acid of 1

NMR (1H- and

13C-) spectra of 4 was almost same as 3 except presence of an extra acetyl moiety.

It was also proved by DEPT 13

C-NMR spectrum which showed 37 carbon signals 11Me, 9CH2,

155

7CH and 10C. Position of the acetate moiety was deduced at C-21 by HMBC and NOE

interaction. Thus structure of 4 was concluded as 1-oxo-11β-O-acetyl-21β-O-angeloyloleane.

2.2.5. 1-Oxo-11β, 21β-O-dibenzoyloleane (5)

Fractions 6-8 were combined and chromatographed over silica gel column. Active fraction was

applied to a reversed-phase preparative HPLC afforded a pure compound 5 (tR = 9.3 min)

displayed in Scheme 1(Experimental).

It was white solid, m.p. 264-268 ºC, optically active displayed [α]25

D + 5.43° in polarimeter.

Presence of chromophore was revealed by displaying λmax peaks at 349 (4.55), 286 (4.56), 254

(4.22), 247 (1.7) nm in UV. Presence of aromatic moiety was indicated by IR spectrum. Peak at

νmax 3133 (Ar-H stretching) and peaks at 806, 791, 760 cm-1

suggested monosubstituted benzene

ring. Presence of the ester carbonyl was proved by peaks at 1745, 1725 cm-1

.

156

Molecular composition C44H58O5 of 5 was proposed by HR positive ion FAB-MS in which it

indicated an [M]+ ion peak at m/z= 667. EIMS, fragmentation pattern revealed presence of two

benzoic acid species by displaying peaks at m/z = 544 [M-122-benzoic acid]+ and 422 [M-2

×122- 2 ×benzoic acid]+. The

1H,

13C-NMR spectra of 5 was close to 1 except presence of

(double) peaks in aromatic region at δ 8.04 (2H, d, J=7.2 Hz), 7.41 (2H, t, J=7.2 Hz), 7.53 (1H,

dt, J=7.2, 2.5 Hz) δ 164.6 [2 × CO s]), 2 × 5CH (δ 129.7 d, 130.0 d, 130.0 d, 128.4 d, 128.4 d), 2

× C (133.7). Therefore, it was suggested as diester of benzoic acid of 1.

Positions of the ester moieties were deduced by HMBC interactions. The H-11 (δ 4.29) displayed

interaction with carbonyl carbon of ester (δ 164.6) similarly H-21(δ 4.46) displayed interaction

with other carbonyl (δ 164.4) in HMBC spectrum (Fig. 2). Therefore, ester moieties were

suggested at C-11 and C-21, respectively.

On this basis, compound 5 was declared as 1-oxo-11β, 21β-O-dibenzoyloleane. The DEPT 13

C-

NMR spectrum of 5 showed 44 carbon signals 8Me, 9CH2, 16CH and 11C. On this basis,

compound 5 was declared as 1-oxo-11β, 21β-O-dibenzoyloleane. According to our knowledge,

since compounds 1-5 have not been reported earlier from plant kingdom and hence can be

declared as new entities.

157

2.2.6. 28-β-D-Glucopyranosyl, 2α, 3α, 19α, 24-tetrahydroxy-12-ene-oleaenoate (6)

Compound 6 was obtained as an amorphous powder and its positive ion FAB-MS indicated an

[M + Na]+ ion peak at m/z= 689 and molecular formula of 6 was found to be C36H58O11.

The 1H-NMR spectrum of 6 was close to triterpenenoid arjunglucoside I isolated from

Terminalia tropophylla (Cao et al., 2010). On the basis of COSY-45° and HOHAHA (Fig . 5);

and other 2D-NMR (1H,

13C) data suggested that compounds 6-13 were saponins of olea-12-en-

28-oic acid β-D-glucopyranose. Compounds 6-13 has same basic skeleton which is triterpene

oleanes (Cao et al., 2010). They are different from each other on the basis of position and

stereochemistry of OH groups. Therefore structure elucidation of 6 will be discussed in detail.

Six singlets were observed in 1H-NMR for three protons each at δ 1.66, 1.09, 1.17, 1.54, 1.14,

0.98 and were due to six methyl in the basic skeleton of the molecule. Out of these two were

gem-methyl that resonated at δ 1.14, 0.98 (Me-29,Me-30) at C-20 were identified by HMBC (Fig.

4). The methyl group numbered 27 present at quaternary allylic carbon (C-14) displayed signals

at δ 1.54 in 1H-NMR spectrum. Two CH2 were oxidized into alcohol and carboxylic acid. In the

158

same spectrum there were peaks in the middle region of the NMR resonated as α β proton on the

same carbon (δ 4.13 d, J=11.5 Hz and δ 3.85 d, 11.5 Hz) were assigned to hydroxyl methylene

present at C-4. There were other eight methylene groups appeared as a complex. Peaks at δ 2.01

(ddd, 12.5, 3.5, 3.5) and 1.41 (dd, 12.5, 3.5) were due to H-C1α), H-C1β. 1HNMR revealed that

C-2,3,5,9,12, 18 and 19 as methine carbons (Table 11).

The DEPT 13

C-NMR spectrum of 6 showed 36 carbon signals, comprising of one ester carbonyl

carbon (δ= 177.3), one trisubstituted double bond system (δ= 144.4, 123.8), three oxygenated

methine carbons (δ= 81.1, 74.2, 66.3), one oxygenated methylene carbon (δ= 65.2), and one

glucopyranosyl group (δ= 95.9, 74.4, 79.3, 71.2, 79.2, 62.3). These NMR signals were confirmed

using 2D NMR techniques (HMQC, HMBC) in 6. Position of the ester group was determined by

NOE and HMBC interactions (Fig. 4). Relative stereochemistry of various stereogenic centers

was confirmed by recording ROESY spectrum (Fig. 5).

In the ROESY spectrum of 6, key NOEs were observed between H-2 and H3-25, and H-5 and H3-

23. In this compound H-2β showed interaction with H-3, therefore OH group at C-3 was placed

at α-position. There was also interaction between H-19 with H-2β and H-3β. Results of these

experiments concluded that OH groups at C-2, C-3 and C-19 were at α-position. In the light of

these spectral evidences the structure of 6 was therefore concluded to be 2α, 3α, 19α, 24-

tetrahydroxyolea-12-en-28-oic acid β-D-glucopyranosyl ester.

Compounds 7-10 were found to be stereoisomers of 6. In FAB-MS, IR and NMR data was very

close to 6. But there was a little difference in the stereochemistry of OH groups attached at C-2,

C-3, C-19 which was evident in NMR (1H,

13C) and ROESY interactions observed in these

spectra.

159

2.2.7. 28-β-D-Glucopyranosyl, 2β, 3α, 19α, 24-tetrahydroxy-12-ene-oleaenoate (7)

There was no interaction between H-2 and H-3 while H-19 displayed interaction with H-3;

therefore OH at C-2 concluded at β-position. Structure of 7 was therefore suggested as 28-β-D-

glucopyranosyl 2β,3α,19α,24-tetrahydroxy-12-ene-oleaenoate.

On the basis of spectroscopic and mass measurement data 8 was assigned a structure of 28-β-D-

glucopyranosyl 2α,3β,19α,24-tetrahydroxy-12-ene-oleaenoate (8). Similarly, structures 28-β-D-

glucopyranosyl 2β,3β,19α,24-tetrahydroxy-12-ene-oleaenoate and 28-β-D-glucopyranosyl 2β,3β,

19β,24-tetrahydroxy-12-ene-oleaenoate were given to 9 and 10, respectively.

160

2.2.8. 28-β-D-Glucopyranosyl 2α, 3β, 19α, 24-tetrahydroxy-12-ene-oleaenoate (8)

161

2.2.9. 28-β-D-Glucopyranosyl 2β, 3β, 19α, 24-tetrahydroxy-12-ene-oleaenoate (9)

162

2.2.10. 28-β-D-Glucopyranosyl 2β, 3β, 19β, 24-tetrahydroxy-12-ene-oleaenoate (10)

163

2.2.11. 28-β-D-Glucopyranosyl 2α-O-acetyl, 3α, 19α, 24-trihydroxy-12-eneoleaenoate (11)

Compounds 11-13 were acetyl derivatives of 6. Compounds were obtained as an amorphous

powder and their positive ion FAB-MS indicated an [M + Na]+ ion peak at m/z= 731. The

molecular formula of 11 was found to be C38H60O12 by using HR positive ion FAB-MS. In EIMS

peak at m/z 671 was due to loss of acetic acid from parent molecule.

In 1H-NMR presence of a peak at 2.23 (s) was attributed to acetyl moiety in the molecule. The

DEPT 13

C-NMR spectrum of 11 showed two additional carbons (38 carbons) signals which were

similar to 6 except absence of one methine attached with OH group due to replacement by acetyl

group. Position of acetyl group was located by 1H-NMR as well as

13C-NMR by displaying H-2

and C-2 peaks in low field region of NMR as compared to 6 as shown in NMR data (Tables 11,

12 and 13). Therefore acetyl was suggested at C-2 in 11.

The same assignment was also confirmed with the help of HMBC and NOE interactions between

H-2 and acetyl carbon as well as H-2 with acetyl methyl group. On the basis of these spectral

164

evidences the structure of 12 was determined as 28- β-D-glucopyranosyl 2α-O-acetyl-3α,19α,24-

trihydroxyo-12-ene-olenoate.

165

2.2.12. 28-β-D-Glucopyranosyl 3α-O-acetyl, 2α, 19α, 24-triahydroxy-12-eneoleaenoate (12)

166

2.2.13. 28-β-D-Glucopyranosyl 19α O-acetyl, 2α, 3α, 24-trihydroxy-12-eneoleaenoate (13)

167

2.3. Biological Activity

2.3.1. Antibacterial and antifungal activity

Essential oil and seed hexane fraction (SHF), seed ethyl acetate fraction (SEF), seed butanol

fraction (SBF) and seed water fraction (SWF) displayed activity against fungi and bacteria. Zones

of inhibition of essential oil (EO) and various crude extracts against fungi and bacteria are

presented in the form of histograms 1 and 2, respectively, which were in the order of T. rubrum>

C. albicans = M. audouini> C. neoformans> T. mentagrophytes> E. floccosum.

The MIC values of EO and fractions against C. albicans and C. neoformans displayed highest

activity. The results have been shown in histograms 3 and 4.

The antibacterial and antifungal activity of compounds 1-5 against the employed bacteria and

fungi was qualitatively and quantitatively assessed by measuring the inhibition zones (Tables 5

and 6), minimum inhibition concentration (MIC) (Tables 7 and 8), minimum bactericidal

concentration (MBC) (Table 9), and minimum fungicidal concentration (MFC) (Table

10).Compounds 1-5 did not show noticeable activity against tested microorganisms as compared

to control that was in accordance with the literature (Caldwell et al., 2000; Hichri et al., 2003;

Sun et al., 2006).

2.3.2. Anticancer Activity

Literature revealed that saponins anti-cancer agents (Li et al., 2009; Mbaze et al., 2007; Chen et

al., 2002). Therefore, it was decided to investigate anticancer activity of isolates 6-13 against HL-

60, SMMC-7721, A-549, MCF-7 and SW-480 cancer cell lines. Results in this connection are

displayed in Table 24. The results revealed that compounds 6-13 were significantly active against

HL-60 and displayed IC50= 2.14, 2.19, 2.17, 3.16, 2.57, 2.87, 2.84 and 3.96 µM, respectively,

whereas the standard (cisplatin) showed IC50 1.04 µM. Activity was found dependent over the

168

stereochemistry of OH group. Compounds 6 displayed maximum activity (IC50= 2.14) in which

all the three OH groups were α-oriented (Mbaze et al., 2007; Chen et al., 2002

169

CHAPTER-3

EXPERIMENTAL

170

3. EXPERIMENTAL

3.1. General Experimental Procedure

All chemicals & pure essential oil were purchased from Sigma St. Louis, USA. Bio-chemicals,

strains and culture media were obtained from, Darmstadt, Germany and Sigma St. Louis, USA.

Uncorrected melting points were determined by a Gallenkamp (U.K) melting point apparatus.

Optical rotation was measured in CHCl3 using digital polarimeter supplied by OSK OGAWA

Seiki Co. Ltd, Tokyo, Japan. The UV spectra were taken in absolute MeOH using IRMECO

UV/VIS Model U-2020 spectrophotometer, Germany. The IR spectra were recorded over

TENSOR 27 FT-IR spectrophotometer supplied by Bruker, Switzerland. The 1H-NMR and

13C-

NMR, spectra were recorded in CDCl3 on a Bruker Biospin, AMX 300 MHz FT NMR (300 MHz

for 1H, 75 MHz for

13C) spectrometer.

1H and

13C-NMR chemical shifts were referenced to

residual CHCl3 (δH = 7.26) and the central peak of the CDCl3 (δC = 77.0), respectively. EI-MS

were acquired on a double-focusing Finnigan MAT 112 at 70 eV. HR-EIMS measurements were

made on JEOL HX 110 spectrometer. Analysis of Essential Oil was carried out on GCMS-

QP2010, Shimadzu, Japan. Column chromatography was carried out with Sephadex LH-20,

Chromatorex ODS chromatography and silica gel (PF254). HPLC (Perkin Elmer, USA), equipped

with RP-18 column was used and UV detector by employing solvent gradient technique. TLC

was performed on HPTLC plates, E. Merck, Germany. The compounds were visualized, by

spraying with 10% sulfuric acid followed by heating. The final separation of the crude triterpene

fractions was performed through HPLC (Perkin Elmer). All chemicals used in the present study

were of analytical reagent quality.

171

3.2. Microorganisms

3.2.1. Fungi and bacteria

The fungi used in this study were Candida albicans ATCC 10231, Cryptococcus neoformans

TIMM 0362, Microsporum audouinii ATCC 10008, Trichophyton rubrum BCRC 32805,

Trichophyton mentagrophytes BCRC 32066, Epidermophyton Floccosum BCRC 30531,

Microsporum canis BCRC 30541 and Aspergillus niger F2723.

Bacteria employed for determination of activity were Escherichia coli MTCC-8739, Proteus

mirabilis ATCC 14153, Pseudomonas aeruginosa MTCC-2435, Micrococcus luteus MTCC-106,

Entero coli, Bacillus cereus ATCC 14603, Staphylococcus aureus MTCC-2940 and Klebsiella

pneumoniae ATCC 700603. Tested microbes were obtained from the Korea Food and Drug

Administration (KFDA), Daegu, Republic of Korea. Cultures of each fungal strain were maintained

on Luria-Bertani (LB) agar medium at 4 ºC. The bacteria were maintained and tested on trypticase

soya agar (TSA) and the yeast on Sabouraud-dextrose agar (SDA). Miconazole and Imipenem

(Sigma-Aldrich Co., St. Louis, MO, USA) were used as control.

3.2.2. Cancer cells

Human tumor cell lines HL-60, SMMC-7721, A-549, MCF-7 and SW-480 and DDP (cisplatin)

were purchased from Sigma, St. Louis, MO, USA (Sigma), maintained in RPMI-1640 medium

supplemented with 10% foetal bovine serum, 100 IU/mL penicillin (Sigma) and 100 μg/mL

streptomycin (Sigma). Experiments were performed in 96-well plates in triplicate.

3.3. Collection of Plant Material

C. sativum Linn was grown in the botanical garden, Rakh Bibi Campus Gomal University, Dera

Ismail Khan, KPK, Pakistan. The plants were identified by Professor Hamidullah Khan, Head of

Pharmacognosy Department, Faculty of Pharmacy Gomal University Dera Ismail Khan. A

172

specimen number CS 25 was retained in the herbarium. The seeds, the leaves and the whole parts

of the plant were collected at appropriate time and investigated for its essential oil, bioactive

constituents and bioactivity.

3.4. Hydrodistillation of Essential Oil from Seeds

The healthy Coriander seeds (1050 g) were placed in a locally made stainless steel sieve and

washed with distilled water to remove the dust and without letting them dry, subjected to

hydrodistillation for 10 continuous hours using Clavenger-type apparatus. The obtained EO was

dried by addition of anhydrous sodium sulphate (0.5 g) to get light yellow oil 3.5 g (yield=

0.33%).

3.5. GC Analysis of Essential Oil of C. sativum

Analysis of Essential Oil was carried out on GCMS using the following parameters; Column

oven temp: 40 °C; Capillary column length 30 m × 0.25 mm; Injection temp: 240 °C; Injection

mode: Split; Flow control mode: Linear velocity; Pressure: 68.9 KPa; Total Flow: 72.9 mL/min;

Column Flow: 1.27 mL/min; Linear Velocity: 40.7 cm/sec; Purge Flow: 8.0 mL/min; Split Ratio:

50.0;High Pressure Injection Pressure: 150.0 KPa; Oven temp rate: 40 °C to 90 °C, 2 °C/min and

from 90 °C t0 200 °C, 3 °C/min.

3.6. Extraction

The whole dried plant material (roots, stems, leaves and seeds) of C. sativum L. was powdered

using grinding machine. The grinded material (5000 g) was extracted with MeOH (7.5 L× 3) at

room temperature. Methanolic solution was filtered using whatman #1 filter paper and

concentrated at reduced temperature and pressure to get dark brown extract 500 g 10 % yield on

the basis of dry plant weight which was considered as 100%.

173

3.6.1. Isolation of compounds

The methanol extract (480 g) was suspended in water (1000 mL) and re-extracted with n-hexane

(3 × 300 mL) chloroform (3 × 250 mL), ethyl acetate (3 × 250 mL) and n-butanol (3 × 50 mL).

Each fraction was dried over anhydrous sodium sulphate and evaporated to dryness to yield n-

hexane fraction (122.4 g, 25.5%), chloroform fraction (134.3 g, 27.98%), ethyl acetate fraction

(124.3 g, 25.89%), n-butanol fraction (72.3 g, 15.06%) and aqueous fraction (25.4 g, 5.29%).

Biological activity against used microbes was evaluated and the result showed that ethyl acetate

fraction was most active among the tested fractions results are shown in histograms 1-4.

Therefore, the ethyl acetate soluble portion was further proceeded to isolate biologically active

constituents as indicated in scheme 1.

The ethyl acetate soluble fraction (20 g) was subjected to chromatograph on silica gel column

(192 × 5.4 cm). It was eluted with petroleum ether/ethyl acetate mixture (6/4, 25000 mL) with

flow rate of 2 mL/min, to yield twenty (1-20) fractions each 250 mL and evaporated to dryness in

vacuo. Fractions 3–5 (1.4g), containing triterpenoids constituents were combined, owing to their

TLC similarities, and subsequently purified by semipreparative HPLC using 15%

water:acetonitrile as eluent, yielding 1 (7 mg) and an inseparable mixture of 2 (6 mg) and 3 (5

mg). Fractions 11-13 (1.2 g) after combination was subjected to rechromatography on same

column of silica gel. The column was eluted using increasing concentrations of ethyl acetate in

petroleum ether to end up with nine (1.1-1.9) pooled fractions.

Fractions 1.4 & 1.5 were combined together (1.3 g) based on TLC behaviour and was

chromatographed over a silica gel column. The column was eluted using chloroform, 5%

methanol in chloroform and 10% methanol in chloroform, successively. The 5% methanol in

174

chloroform elution resulted in three (1.4.1–1.4.3) fractions. Repeated column chromatography of

these fractions resulted in the isolation of compound 4 (23 mg) and 5 ( 15 mg).

Fractions 6-8 (1.7 g) were combined and chromatographed over silica gel column using

chloroform as eluent and the polarity was increased by methanol. Ten (6.1-6.10) fractions were

collected. Fraction 6.1.4-6.1.7 (350 mg) was applied to a reversed-phase preparative HPLC on a

LiChrosorb RP-18 column (7 μm , 2 × 30 cm i.d, Merck), eluted with MeOH-H2O (80/20) at the

rate of 10 mL/min. (tR = 7.5 min), to afford pure compounds (1) (31.3 mg), (tR = 7.9 min) (2), (tR

= 8.1 min) (25.5 mg), (tR = 8.4 min) (3) (31.7 mg), (tR = 8.7 min) (4) (15.8 mg) and (tR = 9.3 min)

(5) (37.6 mg), respectively displayed in Scheme 1.

175

Scheme 1. Extraction of compounds 1-5

176

Scheme 2. Extraction of compounds 6-13 and solvent fractionation of MeOH extract

177

3.6.2. Crude seed extract, solvent fractionation and isolation of 6-13

The air dried and pulverized seeds of C. sativum (5000 g), were macerated at room temperature

(25 °C) in methanol (30 L) for 24 hours and extracted three times. The combined methanolic

extracts were concentrated to dryness (( 400 g). The concentrated brownish methanolic extract

(350 g) was dissolved in distilled water (1 L). It was first defatted with n-hexane (3 × 250 mL,

SHF, 77.28 g) followed by ethyl acetate (3 × 250 mL, SEF, 91.04 g), n-butanol (3 × 50 mL,

SBF, 90.56 g) and water fraction (SWF, 114.48 g) as displayed in Scheme 2. These fractions

were subjected to biological (antibacterial and antifungal) activities (Histograms 1-4).

The ethyl acetate extract (91.04 g) was found to be active against the fungal strains C. albicans C.

neoformans, M. audouinii, T. rubrum, T. mentagrophytes, E. Floccosum, M. canis and A niger.

Bacteria employed for determination of activity were E. coli, P. mirabilis, P. aeruginosa, M.

luteus, E. coli, B. cereus, S. aureus and K. pneumoniae so it was subjected to vacuum liquid

chromatography (VLC) (hexane: chloroform and chloroform: methanol) on silica gel (900 g, 60

PF254 Merck) followed by column chromatography on silica gel (70±230 mesh size). The column

was eluted with a chloroform and methanol mixture with increasing amounts of methanol to

afford twenty (1-20) fractions 250 mL each. Fraction 4 eluted with CHCl3: MeOH (90:10)

afforded compound 1 (49.8 mg), Fraction 6 after elution with CHCl3: MeOH (85:15) yielded

compound 2 (30.1 mg) while on elution with CHCl3: MeOH (80:20) gave compound 3 (79.6 mg).

Fractions 9-14 were combined together (2.38 g) using solvent system Pet. Ether:CHCl3: MeOH

(5:60:25) based on TLC behavior and was subjected to purification on preparative HPLC Perkin

Elmer RP-18 column (250 × 2.5 cm), using MeCN/ H2O, gradient scheme (A = H2O; B =

MeCN/H2O 88/ 12; 5 min A, linear gradient to B in 20 min, 5 min B, linear gradient back to A in

2 min at 2 mL/min flow rate), as a mobile phase, to afford pure compounds with tR 5.6 min. (6,

178

131.3 mg), with tR 6.8 min. (7, 125.3 mg), with tR 7.2 min. (8, 130.1. mg), with tR 7.9 min. (9,

151.5 mg), with tR 8.7 min. (10, 127.4 mg), with tR 9.6 min. (11,137.6 mg), and with tR 10.6 min.

(12, 155.3 mg), and with tR 11.3 min. (13,152.5 mg), respectively. The pure compounds were

subjected to spectroscopic study for structure elucidation. After spectral analysis, compounds 6-

13 displayed were identified (Scheme 2).

3.7. Physical and Spectroscopic Data of Isolates

3.7.1. 1-Oxo-11β,21β-dihydroxyoleane (1)

White solid needles, crystals, m.p. 255-258 ºC; [α]25

D + 5.43 (c 1.4, CHCl3); UV (MeOH) λmax

(log ) 216 (1.56) nm; IR (dry) νmax 3532, 3404, 2940, 2870, 1620, 1450, 1250, 760 cm-1

; 1

H-,

13C-NMR (300 MHz, 75 MHz, C5D5N) Tables 3 and 4; EI-MS: m/z= 458 (M

+, 3, C30H50O3),

440 (12, M+-H2O), 430 (27, M

+-CO), 422 (15, M

+-2H2O), 291 (10, C19H31O2

+), 263 (11,

C18H31O+), 249 (23, C16H25O2

+), 207 (31, C14H23O

+), (100, ), 189 (43, C14H21

+), 135 (42,

C9H11O+), 109 (34, C7H9O

+), 95 (44, C7H11

+); HR-EI-MS: m/z=458.7162, calcd. for C30H50O3;

458.7158 observed.

3.7.2. 1-Oxo-11β-hydroxy-21β-O-acetyloleane (2)

White solid, crystals, m.p. 291-293 ºC; [α]25

D + 4.43 (c 1.4, CHCl3); UV (MeOH) λmax (log)

247 (1.7) nm; IR (dry) νmax 3436, 2965, 2879, 1728, 1712, 1458, 1266, 779 cm-1

; 1

H-, 13

C-NMR

(300 MHz, 75 MHz, C5D5N) Tables 3 and 4; EI-MS: m/z= 500 (M

+, 3, C32H52O4), 482 (15,

M+-H2O), 472 (21, M

+-CO), 440 (15, M

+-MeCO2H), 291 (10,), 263 (11, C18H31O

+), 249 (23,

C16H25O2+), 207 (31, C14H23O

+), (100, C5H8O2

+), 189 (43, C14H21

+), 135 (42, C9H11O

+), 109

(34, C7H9O+), 95 (44, C7H11

+), 43 (46); HR-EI-MS: m/z= 500.7529, calcd. for C32H52O4;

500.7515 observed.

179

3.7.3. 1-Oxo-11β-hydroxy-21β-O-angeloyloleane (3)

White solid, crystalline, m.p. 258-260 ºC; [α]25

D + 5.43 (c 1.4, CHCl3); UV (MeOH) λmax (log )

308 (4.28) and 214 (4.26), 267 (3.7) nm; IR (dry) νmax 3348 (OH), 2947, 2877, , 1723 (C=C-

CO2), 1707 (CO), 1599, 1534 (C=C), 1451, 1256, 767 cm-1

; 1

H-, 13

C-NMR (300 MHz, 75 MHz,

C5D5N) Tables 3 and 4; EI-MS: m/z = 540 (M+, 3, C35H56O4), 518 (12, M

+-H2O), 512 (27, M

+-

CO), 440 (15, M+-C5H8O2), 291 (10, C19H31O2

+), 263 (11, C18H31O

+), 249 (23, C16H25O2

+), 207

(31, C14H23O+), (100, C5H8O2

+), 189 (43, C14H21

+), 135 (42, C9H11O

+), 109 (34, C7H9O

+), 95

(44, C7H11+), 83 (45), 82 (34); HR-EI-MS: m/z= 540.8167, calcd. for C35H56O4; 540.8143

observed.

3.7.4. 1-Oxo-11β-O-angeloyl-21β-O-acetyloleane (4)

White solid, amorphous, m.p. 267-268 ºC; [α]25


347 (4.55), 284 (4.56), 253 (4.22) 234 (2.7) nm; IR (dry) νmax 2955, 2885, 1738, 1690, 1635,

1593, 1465, 1260, 926, 807, 764 cm-1

; 1H-,

13C-NMR (300 MHz, 75 MHz, C5D5N) Tables 3 and

4; EI-MS: m/z = 582 (M+, 3, C37H58O5), 564 (12, M

+-H2O), 554 (27, M

+-CO), 522 (15, M

+-

MeCO2H), 464 (15, M+-C5H8O2), 291 (10, C19H31O2

+), 263 (11, C18H31O

+), 249 (23,

C16H25O2+), 207 (31, C14H23O

+), (100, C5H8O2

+), 189 (43, C14H21

+), 135 (42, C9H11O

+), 109 (34,

C7H9O+), 95 (44, C7H11

+), 83 (45), 82 (56), 43 (78); HR-EI-MS: m/z= 582.8334, calcd. for

C37H58O5; 582.8343, observed.

3.7.5. 1-Oxo-11β,21β-O-dibenzoyloleane (5)

White solid, crystals, m.p. 264-268 ºC; [α]25


349 (4.55), 286 (4.56), 254 (4.22), 247 (1.7) nm; IR (dry) νmax 3133, 2940, 2870, 1745, 1620,

1527, 1450, 1250, 923, 806, 791, 760 cm-1

; 1

H-, 13

C-NMR (300 MHz, 75 MHz, C5D5N) Tables

3 and 4; EI-MS: m/z= 666 (M

+, 3, C44H54O5), 440 (12, M

+-H2O), 430 (27, M

+-CO), 422 (15,

180

M+-2H2O), 291 (10, C19H31O2

+), 263 (11, C18H31O

+), 249 (23, C16H25O2

+), 207 (31, C14H23O

+),

(100, ), 189 (43, C14H21+), 135 (42, C9H11O

+), 105 (34, C7H5O

+), 109 (34, C7H9O

+), 95 (44,

C7H11+), 77 (23, C6H5

+), 65 (23, C5H5

+); HR-EI-MS: m/z= 666.9283, calcd. for C44H54O5;

666.9273 observed.

3.7.6. 28-β-D-Glucopyranosyl 2α,3α,19α,24-tetrahydroxy-12-ene-oleaenoate (6).

Amorphous powder, m.p. 248-250 °C; [α] + 2.1º (c = 1.5, C5H5N); IR νmax (dry) cm-1

: 3453,

3452, 3303 (OH), 3021, 2918, 2859, 1748 (CO), 1727 (CO2), 1653, 1641 (C=C), , 1531, 1471,

1434, 1383, 1217, 1021, 923, 847; 1H-,

13C-NMR (300 MHz, 75 MHz, C5D5N) Tables 11 and

13; Positive ion FAB-MS m/z= 689 [M + Na]+; HR positive ion FAB-MS m/z= 689.3877

(Calcd. for C36H58O11: 689.3877).

3.7.7. 28-β-D-Glucopyranosyl 2β,3α,19α,24-tetrahydroxy-12-ene-oleaenoate (7)

Amorphous powder, m.p. 250-252 °C; [α] + 1.9º (c = 1.5, C5H5N); IR νmax (dry) cm

-1: 3445,

3431, 3316 (OH), 3024, 2914, 2853, 1747, 1723, 1646, 1531, 1476 (C=C), 1438, 1356, 1221,

1025, 929, 845; 1H-,

13C-NMR (300 MHz, 75 MHz, C5D5N) Tables 11 and 13; Positive ion

FAB-MS m/z= 689 [M + Na]+; HR positive ion FAB-MS m/z= 689.3877 (Calcd. for C36H58O11:

689.3877).

3.7.8. 28-β-D-Glucopyranosyl 2α,3β,19α,24-tetrahydroxy-12-ene-oleaenoate (8)

Amorphous powder, m.p. 249-252 °C; [α]D 25 + 2.1º (c = 1.5, C5H5N); IR νmax (dry) cm-1

: 3466,

3462, 3311 (OH), 3021, 2918, 2859, 1744, 1721, 1649, 1538, 1477 (C=C), 1434, 1355, 1216,

1022, 924, 849; 1H-, 13

C-NMR (C5D5N, 300 MHz, 75 MHz) Tables 11 and 13; Positive ion

FAB-MS m/z= 689 [M + Na]+; HR positive ion FAB-MS m/z= 689.3877 (Calcd. for C36H58O11:

689.3877).

181

3.7.9. 28-β-D-Glucopyranosyl 2β,3β,19α,24-tetrahydroxy-12-ene-oleaenoate (9)


: 3460,

3443, 3333, (OH), 1738, 1724, 1531 (C=C), 1219, 1044, 923, 846; 1H-,

13C-NMR (C5D5N, 300

MHz, 75 MHz) Tables 11 and 13; Positive ion FAB-MS m/z= 689 [M + Na]+; HR positive ion

FAB-MS m/z= 689.3877 (Calcd. for C36H58O11: 689.3877).

3.7.10. 28-β-D-Glucopyranosyl 2β,3β,19α,24-tetrahydroxy-12-ene-oleaenoate (10)


: 3466,

3399, 3277, (OH), 1748, 1727, 1591 (C=C), 1219, 1025, 923, 845; 1H-,

13C-NMR (C5D5N, 300



3.7.11. 28-β-D-Glucopyranosyl 2α-O-acetyl, 3α, 19α, 24-tetrahydroxy-12-ene-oleaenoate (11)


: 3455,

3305 (OH), 1744, 1715( CO), 1585, (C=C), 1217, 1021, 923, 847; 1H-,

13C-NMR (C5D5N, 300



3.7.12. 28-β-D-Glucopyranosyl 3α-O-acetyl, 2α, 19α, 24-tetrahydroxy-12-ene-oleaenoate (12)


: 3471,

3321 (OH), 1746, 1735, 1587 (C=C), 1217, 1021, 923, 847; 1H-,

13C-NMR (C5D5N, 300 MHz,

75 MHz) Tables 12 and 13; Positive ion FAB-MS m/z= 731 [M + Na]+; HR positive ion FAB-

MS m/z= 731.3877 (Calcd. for C38H60O12: 731.3877).

182

3.7.13. 28-β-D-Glucopyranosyl 19α O-acetyl,2α,3α,24-tetrahydroxy-12-ene-oleaenoate (13)


: 3453,

3303 (OH), 1755, 1724, 1598 (C=C), 1223, 1026, 928, 844; 1

H-, 13

C-NMR (C5D5N, 300 MHz, 75

MHz) Tables 12 and 13; Positive ion FAB-MS m/z=731 [M + Na]+; HR positive ion FAB-MS

m/z= 731.3877 (Calcd. for C38H60O12: 731.3877).

3.8. Biological Activities

3.8.1. Antifungal and antibacterial activity

Antifungal/antibacterial activities of pure compounds essential, seed hexane fraction (SHF) seed

ethyl acetate fraction (SEF), seed butanol fraction (SBF)) and seed water fraction (SWF) were

determined according to a standard protocol (Chandrasekaran and Venkatesalu, 2004). For

antibacterial activity solutions of extracts (200µg/disc) were laid on top of the inoculated agar

plates and incubated for 36 hours at 28° C for fungi and 24 hours at 37 °C for bacteria. The

standard drugs Miconazole and Imipenem 30 µg/disc was used as a positive control. Zones of

inhibition for essential oil and extracts were measured in millimeters. Each assay in this

experiment was replicated three times.

Mninimum inhibitory concentrations (MICs) of essential oils and crude extracts (SHF, SEF, SBF

and SWF) were measured by following standard protocols (EUCAST Definitive Document 2000,

2012). Stock solutions of essential oil, seed hexane fraction (SHF) seed ethyl acetate fraction

(SEF), seed butanol fraction (SBF)) and seed water fraction (SWF) having concentrations of

10000 mg/L were prepared in DMSO (2 mL). From the stock solutions concentration ranges of

512-0.125 mg/L were prepared in 25 mL containers by serial dilution technique. Molten agar

183

(≈19 mL; total volume in each container was 20 mL) was then added to each container and the

mixture were thoroughly mixed and poured into pre-labeled 9 cm sterile petri dishes and let dry.

The inoculum was prepared by emulsifying overnight colonies (4-5 morphologically similar)

from a potato dextrose agar (Oxoid) at 28 °C for fungal strains and a Mueller-Hinton agar (Oxid,

Hamshire, UK) at 37 ºC for bacterial strain. A 0.5 McFarland was used as a standard to get a

suspension having a density equivalent to 108 CFU/mL. The plates were inoculated within 30

min of standardizing the inoculum. The inoculum 1µL (104 CFU/spot) was transferred to each

plate employing replicator pins 2.5 mm in diameter. The plates were incubated for antifungal

activity at 28 °C and for antibacterial at 37 °C in air for 18 h and the MICs values were

determined by noticing the minimum concentration of essential oil and fractions that on plates

had inhibited the fungal/bacterial growth approximately more than 90%.

Antimicrobial activity of 1-5 was procured by following standard method. Briefly, LB medium

20 mL was poured in petri plates and after solidification standard inoculum (100 µL)

bacteria/fungi concentration 107 CFU/mL suspension was poured and dried for 5 min. Filter

paper discs (50 µg/disc, 6 mm diameter) were impregnated with 1-5 solution obtained in 5%

DMSO that was used as negative control, and standard drugs (Miconazole, and Imipenem (10

µg/disc) as a positive control) were incubated at 37 ºC for 24 h and zone of inhibition was

measured.

The MIC of compounds 1-5 was determined by following standard twofold serial dilution method

(Chandrasekaran and Venkatesalu, 2004). Solution of tested compounds (in 5% DMSO) were

prepared, and incorporated into LB broth medium to get 500 µg/mL and serially diluted to

achieve 62.5, 31.25, 15.62, 7.81, 3.95 and 1.97 µg/mL. A suspension (10 µL) of each tested

organism (107 CFU/mL) was transferred to each tube (compound containing tubes and control

184

tubes), were incubated at 37 ºC for 24 h and the lowest concentration was determined and

expressed as µg/mL. Concentration that inhibit complete growth of bacteria/fungi was identified,

and 50 µL solution of each culture broth was transferred onto the agar plates, incubated at 37 ºC

for 24 h and complete absence of growth on the agar surface was defined as MBC and MFC.

3.8.2. Cytotoxicity

All tumor cells were cultured in RPMI-1640 or DMEM medium (Hyclone, Logan, UT),

supplemented with 10% fetal bovine serum (Hyclone) at 37 ºC in a humidified atmosphere with

5% CO2. Cell viability was assessed by conducting colorimetric measurements of the amount of

insoluble formazan formed in living cells based on the reduction of 3-(4, 5-dimethylthiazol-2-yl)-

2,5-diphenyltetrazolium bromide (MTT) (Sigma, St. Louis, MO) (Hu et al., 2005; Monks et al.,

1991). Briefly, 100 mL of adherent cells was seeded into each well of a 96-well cell culture plate

and allowed to adhere for 12 h before drug addition, while suspended cells were seeded just

before drug addition, both with an initial density of 1 × 105 cells/ mL in 100 mL of medium. Each

tumor cell line was exposed to test various plant extracts at various concentrations in triplicate for

48 h, with cisplatin (Sigma) as positive controls. After the incubation, MTT (100 mg) was added

to each well, and the incubation continued for 4 h at 37 ºC. The cells were lysed with 100 mL of

20% SDS/50% DMF after removal of 100 mL of medium. The optical density of the lysate was

measured at 595 nm in a 96-well microtiter plate reader (Bio-Rad 680). The IC50 value of each

fraction was calculated by Reed and Muench’s method. Cytotoxicity of the pure isolated

compounds was also evaluated by adaption of above procedure (Table 14).

3.9. Statistical analysis

The collected data was analyzed, the IC50 values were determined, and their 95% confidence

intervals (CI 95%) were obtained through nonlinear regression using Finney software.

185

TABLES

186

Table 1. Natural product as anticancer drugs

S.No Name of Drug Source Major use Ref.

1 Allicin Allium sativum

Anticancer and antioxidant Kainsa et al., 2012

2 Chelidonine Chelidonium

majus

Anticancer Kainsa et al., 2012

3 Glycyrrhetic

acid

Glycyrrhiza

glabra


4 Vinblastine Catharanthus roseus

Antineoplastic Kainsa et al., 2012

5 Vincristine Catharanthus

roseus

Antineoplastic Kainsa et al., 2012

6 Taxol Taxus

baccata Taxus

brevifolia

Nutt


7 Cannabinoids Cannabis sativa

Lung-adenocarcinoma Kainsa et al., 2012

8 Salvicine Salvia

prionitis

Malignant tumors Kainsa et al., 2012

9 Berberine Rhizoma

coptidis

Anticancer, epilepsy, diarrhea, antiinflammatory,

antibacterial, antidiabetes, antiulcer, expansion of blood vessels, inhibition of platelet aggregation, hepatoprotective

Lu et al., 2012

10 Evodiamine Evodia

rutaecarpa

Antianxiety, antiobese, antinociceptive, antiinflammatory,

antiallergic,and anticancer

Lu et al., 2012

11 Matrine Sophora

flavescens Ait.

Antibacterial, antiviral, antiinflammatory, antiasthmatic,

antiobesity, anticancer, diuretic, choleretic, hepatoprotective, nephroprotective, and cardioprotective

Lu et al., 2012

12 Piperine Piper nigrum

and

Piper longum

Antioxidant, antiinflammatory, antidiarrheal,

anticonvulsant, antimutagenic, hypolipidemic, promoting

bile secretion, and Antitumor

Lu et al., 2012

13 Sanguinarine Sanguinaria canadensis L.

and

Chelidonium majus L.

Anticancer, antibacterial, antifungal, antischistosomal, antiplatelet, and anti-inflammatory

Lu et al., 2012

14 Tetrandrine Stephania

tetrandra

Anticancer, immunomodulating, antihepatofibrogenetic,

antiinflammatory, antiarrhythmic, antiportal hypertension,

and neuroprotective activities [

Lu et al., 2012

14 Paclitaxel

(Taxol)

Taxus

brevifolia

Ovarian cancer, advanced breast cancer Shoeb, 2006

15 Topotecan Semisynthetic derivative of

Camptothecin

obtained from Camptotheca

acuminate

Ovarian, lung and colo-rectal cancers Shoeb, 2006

16 3-Oxo-

19α,23,24-trihydroxyurs-

12-en-28-oic

acid

Symplocos

chinensis

Anticancer Li et al., 2003

17 Podophyllotoxin Podophyllum species

Podophyllum

peltatum L.

Anticancr

18 Betulinic acid Betula alba Anticancer Nirmala et al., 2011

191

Table 2. Quantitative analysis of EO of C. sativum Linn

ID# Name R.Time Area Conc.(%)

2 α-Pinene 8.674 354491 4.28

3 Camphene 9.342 1851 0.02

5 β-Pinene 10.531 31276 0.38

10 o-Cymene 12.726 30140 0.36

11 D-Limonene 12.919 7552 0.09

17 γ-Terpinene 14.320 20498 0.25

19 α-methyl-α[4-methyl-3-pentyl]

oxiranemethanol

15.779 13699 0.17

20 β-Linalool 16.575 6617739 79.86

23 Alcanfor 18.311 3966 0.05

25 1-Terpinen-4-ol 19.418 5217 0.06

27 p-Menth-1-en-8-ol 19.914 27330 0.33

28 n-Decaldehyde 20.130 13209 0,16

30 cis-Geraniol 20.925 2563 0.03

35 α-Citral 21.598 4050 0,05

44 Nerol acetate 23.284 9358 0.11

45 Geraniol acetate 23.620 1142423 13.79

52 α-Limonene diepoxide 25.233 1465 0.02

192

Table 3.1H-NMR (C5D5N, 300 MHz) of compounds 1-5 isolated from C. sativum L. Seeds

C# 1 2 3 4 5

2 2.22 ddd (13, 5.2,

3.5)

1.88 ddd (13, 5.2,

3.5)

1.87 ddd (13, 5.2, 3.5) 1.86 ddd (13, 5.2, 3.5) 1.87 ddd (13, 5.2, 3.5)

3 1.97 dd (13, 5.2) 2.24 dd (5, 12) 2.25 dd (4.5, 12) 2.24 dd (4.5, 12) 4.35 dd (4.5, 12)

5 1.83 d (11.5) 1.72 d (11.5) 1.74 d (11.5) 1.73 d (11.5) 1.76 d (11.5)

6 2.17 dd (14, 6.5) 2.19 dd (6.5, 14) 2.17 dd (6.5, 14) 2.18 dd (6.5, 14) 2.17 dd (6.5, 14)

7α 2.89 ddd (16.5, 12.5,

6.5)

2.87 ddd (16.5,

12.5, 6.5)

2.87 ddd (16.5, 12.5,

6.5)

2.84 ddd (16.5, 12.5,

6.5)

2.83 ddd (16.5, 12.5,

6.5)

7β 2.74 dd (16.5, 5.0) 2.71 dd (16.5, 5.0) 2.69 dd (16.5, 5.0) 2.68 dd (16.5, 5.0) 2.69 dd (16.5, 5.0)

9 1.64 dd (14, 6.5) 1.65 dd (14, 6.5) 1.66 dd (14, 6.5) 1.62 dd (14, 6.5) 1.63 dd (14, 6.5)

11 3.29 ddd (14, 6.5,

4.2)

4.23 ddd (3.0, 13.5,

13.5)

4.47 ddd (3.0, 13.5,

13.5)

4.29 ddd (3.0, 13.5,

13.5)

4.29 ddd (3.0, 13.5,

13.5)

12 1.88 ddd (14.1, 4.5,

12.2)

5.79 ddd (14.1, 4.5,

12.2)

5.77 ddd (14.1, 4.5,

12.2)

5.77 ddd (14.1, 4.5,

12.2)

5.78 ddd (14.1, 4.5,

12.2)

13 2.01 dd (12.2, 4.5) 2.02 dd (12.2, 4.5) 2.05 dd (12.2, 4.5) 2.06 dd (12.2, 4.5) 2.07 dd (12.2, 4.5)

15α 2.44 ddd (13.5, 13.5,

3.0)

2.43 ddd (13.5,

13.5, 3.0)

2.44 ddd (13.5, 13.5,

3.0)

2.45 ddd (13.5, 13.5,

3.0)

2.44 ddd (13.5, 13.5,

3.0)

15β 1.46 ddd (13.5, 13.5,

3.1)

1.47 ddd (13.5,

13.5, 3.1)

1.46 ddd (13.5, 13.5,

3.1)

1.45 ddd (13.5, 13.5,

3.1)

1.46 ddd (13.5, 13.5,

3.1)

16α 2.53 ddd (13.5, 13.5,

3.0)

2.55 ddd (13.5,

13.5, 3.0)

2.52 ddd (13.5, 13.5,

3.0)

2.51 ddd (13.5, 13.5,

3.0)

2.54 ddd (13.5, 13.5,

3.0)

16β 1.56 ddd (13.5, 13.5,

3.0)

2.57 ddd (13.5,

13.5, 3.0)

1.56 ddd (13.5, 13.5,

3.0)

1.55 ddd (13.5, 13.5,

3.0)

1.56 ddd (13.5, 13.5,

3.0)

18 1.78 ddd (12.5, 12.5,

3.5)

1.77 ddd (12.5,

12.5, 3.5)

1.78 ddd (12.5, 12.5,

3.5)

1.79 ddd (12.5, 12.5,

3.5)

1.78 ddd (12.5, 12.5,

3.5)

19α 1.71 ddd (14.5, 11.5,

4.0)

1.70 ddd (14.5,

11.5, 4.0)

1.71 ddd (14.5, 11.5,

4.0)

1.72 ddd (14.5, 11.5,

4.0)

1.73 ddd (14.5, 11.5,

4.0)

19β 1.17 dd (14.5, 4.0) 1.16 dd (14.5, 4.0) 1.19 dd (14.5, 4.0) 1.18 dd (14.5, 4.0) 1.17 dd (14.5, 4.0)

21 3.46 ddd (13.5, 13.5,

3.0)

4.46 ddd (13.5,

13.5, 3.0)

4.57 ddd (13.5, 13.5,

3.0)

4.29 ddd (13.5, 13.5,

3.0)

4.46 ddd (13.5, 13.5,

3.0)

22α 2.91 ddd (13.5, 13.5,

2.5)

2.90 ddd (13.5,

13.5, 2.5)

2.93 ddd (13.5, 13.5,

2.5)

2.92 ddd (13.5, 13.5,

2.5)

2.97 ddd (13.5, 13.5,

2.5)

22β 1.91 dd (13.5, 13.5) 1.92 dd (13.5, 13.5) 1.95 dd (13.5, 13.5) 2.89 dd (13.5, 13.5) 1.90 dd (13.5, 13.5)

23 1.66 s 1.67 s 1.65 s 1.66 s 1.65 s

24 1.07 s 1.08 s 1.09 s 1.09 s 1.10 s

25 1.13 s 1.12 s 1.09 s 1.08 s 1.14 s

26 1.16 s 1.17 s 1.18 s 1.15 s 1.19 s

27 1.55 s 1.56 s 1.58 s 1.61 s 1.60 s

28 1.17 s 1.15 s 1.14 s 1.18 s 1.17 s

29 1.38 s 1.37 s 1.36 s 1.39 s 1.37 s

30 0.97 s 0.98 s 0.97s 0.96 s 0.99 s

1H-NMR (300 MHz, C5D5N) data of acid residues in 2-5

Ac Ang Bz

1 2.29 s

2 8.04, 1H, d, J=7.2 Hz

3 6.13, 1H, qq, J=6.6 Hz 7.41, 1H, t, J=7.2 Hz

4 2.01, 3H, dq, J=6.5, 2.5 Hz 7.53, 1H, dt, J=7.2, 2.5 Hz

5 1.93, 3H, dqm, J= 6.5, 2.2 Hz 7.41, 1H, t, J=7.2 Hz

6 8.04, 1H, d, J=7.2 Hz

193

Table 4.13

C-NMR (DEPT, C5D5N, 75 MHz) of compounds 1-5 isolated from C. sativum L. Seeds

C# 1 2 3 4 5

1 214.7 s

214.5 s 214.2 s 214.8 s 214.9 s

2 68.2 t 67.9 t 68.4 t 68.3 t 68.5 t

3 47.9 t 47.8 t 47.7 t 47.6 t 47.5 t

4 45.7 s 45.5 s 45.8 s 45.9 s 46.1 s

5 56.7 d 56.6 d 56.9 d 56.6 d 56.7 d

6 19.5 t 19.6 t 19.4 t 19.3 t 19.8 t

7 35.5 t 35.4 t 35.5 t 35.5 t 35.6 t

8 40.4 s 40.3 s 40.3 s 40.2 s 40.3 s

9 50.6 d 50.6 d 50.6 d 50.6 d 50.6 d

10 38.8 s 38.8 s 38.7 s 38.8 s 38.9 s

11 71.4 d 77.4 d 78.1 d 77.9 d 77.8 d

12 43.7 t 44.6 t 44.6 t 44.5 t 44.6 t 13 51.3 d 54.4 d 54.8 d 54.7 d 54.5 d

14 42.1 s 42.4 s 42.3 s 42.7 s 42.6 s

15 29.1 t 29.0 t 29.2 t 29.3 t 29.1 t

16 27.9 t 28.0 t 28.4 t 27.9 t 28.3 t

17 46.5 s 46.6 s 46.7 s 46.4 s 46.5 s

18 44.6 d 44.8 d 44.5 d 44.6 d 44.9 d

19 27.1 t 27.3 t 27.8 t 27.6 t 27.5 t

20 35.7 s 35.5 s 35.4 s 35.5 s 35.5 s

21 72.8 d 78.8 d 78.8 d 81.8 d 81.8 d

22 42.9 t 43.0 t 43.5 t 43.6 t 44.1 t

23 24.1 q 24.2 s 24.0 s 24.1 s 24.2 s

24 23.5 q 23.7 s 23.8 s 23.8 s 23.5 s

25 17.5 q 17.4 s 17.6 s 17.5 s 17.4 s

26 17.4 q 17.3 s 17.3 s 17.1 s 17.4 s

27 24.9 q 24.7 s 24.8 s 24.8 s 24.9 s

28 25.1 q 25.5 s 25.6 s 25.7 s 25.5 s

29 28.8 q 28.7 s 28.6 s 28.8 s 28.9 s

30 24.6 q 24.8 s 24.9 s 24.6 s 24.8 s 13

C-NMR (75 MHz, C5D5N) data of acid residues in 2-5

Ac Ang Bz

1 176.2 168.3 164.6 s

2 22.3 128.0 129.7 d

3 137.3 130.0 d

4 15.9 128.4 d

5 20.6 133.7 d

6 128.4 d

7 130.0 d

194

Table 5. Inhibition zones of 1-5 isolated from C. sativum against bacteria

Zone of inhibition (mm) of 1-5

Comp/Organism 1 2 3 4 5 Std*

E. coli 7.1±1.2 22±2 17±2 8±2 8±2 28±1.2

S. aureus 3.6±1.2 23±2 16±2 8±2 18±2 31±1.2

P. mirabilis 2.4±1.2 22±2 19±2 21±2 10±2 29±1.2

P. aeruginosa 2.2±1.2 29±2 21±2 29±2 19±2 33±1.2

B. cereus 2.2±1.2 23±2 22±2 8±2 8±2 27±1.2

K. pneumonia 0 0 17±2 8±2 8±2 29±1.2

M. luteus 0 0 8±2 8±2 17±2 37±1.2

Entero Coli 0 0 0 0 0 34±1.2

* Imipenem was used as standard

195

Table 6. Inhibition zones of 1-5 isolated from C. sativum against fungi

* Miconazole was used as standard

Zone of inhibition (mm) of 1-5

Comp/Organism 1 2 3 4 5 Std*

T. rubrum 9.1±2 7±2 3±2 2±2 1±2 29±2

C. albicans 7.6±2 5±2 3±2 2±2 2±2 31±2

M. audouinii 5.4±2 6±2 2±2 2±2 1±2 24±2

C. neoformans 6.2±2 6±2 2±2 3±2 1±2 26±2

T. mentagrophytes 9.±2 5±2 3±2 2±2 1±2 29±2

E. floccosum 6.3±2 6±2 3±2 1±2 2±2 27±2

M. canis 7.6±2 3±2 3±2 3±2 1±2 28±2

A. niger 2.3±2 0 3±2 3±2 1±2 28±2

196

Table 7. MICs of 1-5 isolated from C. sativum against bacterial strains

MIC (µg/mL) of compounds 1-5 against Bacterial strains

Compd E. coli P.

mirabilis

P.

aeruginosa

M.

luteus

Entero

Coli

K.

pneumonia

B.

cereus

S.

aureus

1 4±0.5 8±1.5 8±1.5 4±0.5 3±0.5 4±0.5 4±0.5 4±0.5

2 4±0.5 8±1.5 8±1.5 4±0.5 4±0.5 8±1.5 8±1.5 4±0.5

3 4±0.5 16±2.0 8±1.5 8±1.5 5±0.5 16±1.5 16±1.5 4±0.5

4 8±0.5 16±1.5 8±1.0 5±0.5 4±0.5 16±1.5 4±0.5 4±0.5

5 64±2.0 32±2.0 32±2.0 4±0.5 4±0.5 64±2.0 64±2.0 64±2.0

197

Table 8. MICs of 1-5 from C. sativum against fungal strains

MIC (µg/mL) of compounds 1-5 against Fungal strains

Comp/

Organism

C.

albicans

C.

neoformans

M.

audouinii

A.

niger

T.

mentagrophytes

E.

floccosum

M.

canis

T.

rubrum

1 4±0.5 4±0.5 8±0.5 0 8±0.5 16±0.5 0 16±1.5

2 4±0.5 8±1.5 16±1.5 0 16±0.5 16±0.5 0 16±1.5

3 4±0.5 8±1.5 16±1.5 0 16±0.5 32±0.5 16±1.5 32±2.0

4 8±1.5 8±1.5 16±1.5 0 64±2.5 32±1.5 16±1.5 16±1.5

5 16±1.5 64±2.0 16±1.5 0 32±1.5 32±2.0 16±1.5 8±1.5

198

Table 9. MBCs of 1-5 from C. sativum against bacterial strains

MIC (µg/mL) of compounds 1-5 against Bacterial strains

Compd/

Organism

E. coli P.

mirabilis

P.

aeruginosa

M.

luteus

Entero

Coli

K.

pneumoniae

B.

cereus

S. aureus

1 8±1.5 16±1.5 16±1.5 8±1.5 0 8±1.5 9±1.5 8 ±1.5

2 8±1.5 16±1.5 16±1.5 9±1.5 0 16±1.5 16±1.5 8±1.5

3 8±1.5 33±1.5 16±1.5 16±1.5 0 32±1.5 33±1.5 8±1.5

4 17±1.5 32±1.5 16±1.5 0 0 33±1.5 9±1.5 9±1.5

5 123±1.5 65±1.5 65±1.5 0 0 124±1.5 125±1.5 124±1.5

.

199

Table 10. MFCs of 1-5 from C. sativum Seeds against fungal strains

MIC (µg/mL) of compounds 1-5 against Fungal strains

Cmpd C.

albicans

C.

neoformans

M.

audouinii

A.

niger

T.

mentagrophytes

E.

floccosum

M.

canis

T.

rubrum

1 9±1.5 9±1.5 17±1.5 0 17±1.5 31±1.5 0 31±1.5

2 9±1.5 16±1.5 31±1.5 0 31 31 0 31±1.5

3 9±1.5 16±1.5 31±1.5 0 31±1.5 63±1.5 32±1.5 63±1.5

4 16±1.5 16±1.5 31±1.5 0 122±1.5 61±1.5 3±1.52 31±1.5

5 32±1.5 112±1.5 31±1.5 0 62±1.5 61±1.5 32±1.5 16±1.5

200

Table 11. 1H-NMR (300 MHz, C5D5N) data of 6-9 isolated from C. sativum

Aglycone 6 7 8 9

H-C(1α) 2.01 ddd, (12.5, 3.5, 3.5) 2.08 ddd, (12.5, 3.5, 3.5) 2.09 ddd, (12.5, 3.5, 3.5) 2.02 ddd, (12.5, 3.5, 3.5)

H-C(1β) 1.41 dd, (12.5, 3.5) 1.43 dd, (12.5, 3.5) 1.44 dd, (12.5, 3.5) 1.40 dd, (12.5, 3.5)

H-2 4.46 ddd (3.0, 3.0, 12.0) 3.56 ddd (3.0, 3.0, 12.0) 4.45 ddd (3.0, 3.0, 12.0) 4.46 ddd (3.0, 3.0, 12.0)

H-3 4.58 d (3.0) 4.59 d (3.0) 4.65 d (3.0) 4.58 d (3.0)

H-5 1.87 d (12.0) 1.88 d (12.0) 1.86 d (12.0) 1.87 d (12.0)

H-C(6α) 1.56 dddd, (12.6, 5.5, 3.5,

3.5)

1.61 dddd, (12.5, 5.5, 3.5,

3.5)

1.58 dddd, (12.6, 5.5, 3.5,

3.5)

1.58 dddd, (12.6, 5.5, 3.5,

3.5)

H-C(6β) 1.37 ddd, (12.6, 5.5, 3.5) 1.33 ddd, (12.6, 5.3, 3.5) 1.39 ddd, (12.6, 5.4, 3.5) 1.33 ddd, (12.6, 5.5, 3.7)


H-C(7β) 1.49 dd, (12.5, 5.5) 1.46 dd, (12.5, 5.5) 1.48 dd, (12.5, 5.5) 1.47 dd, (12.5, 5.5)

H-C(9) 1.64 dd, (13.5, 6.5) 1.66 dd, (13.5, 6.5) 1.65 dd, (13.5, 6.5) 1.67 dd, (13.5, 6.5)


H-C(11β) 1.88 dd, (13.5, 4.5) 1.85 dd, (13.3, 4.5) 1.86 dd, (13.4, 4.5) 1.85 dd, (13.5, 4.4)

H-12 5.50 d (3.0) 5.49 d (3.0) 5.51 d (3.0) 5.50 d (3.0)

H-15 2.36 ddd (3.0, 13.5, 13.5) 2.36 ddd (3.0, 13.5, 13.5) 2.36 ddd (3.0, 13.5, 13.5) 2.36 ddd (3.0, 13.5, 13.5)

H-16 2.80 ddd (3.0, 13.5, 13.5) 2.82 ddd (3.0, 13.5, 13.5) 2.81 ddd (3.0, 13.5, 13.5) 2.78 ddd (3.0, 13.5, 13.5)

H-17 3.07 ddd (4.5, 13.5, 13.5) 3.08 ddd (4.5, 13.5, 13.5) 3.09 ddd (4.5, 13.5, 13.5) 3.10 ddd (4.5, 13.5, 13.5)

H-18 3.12 br s 3.12 br s 3.12 br s 3.12 br s

H-19 3.65 d (4.0) 4.54 d (4.0) 3.58 d (4.0) 4.62 d (4.0)

H-C(21α) 2.11 dddd, (13.2, 13.2,

4.5, 4.2)

2.14 dddd, (13.3, 13.1, 4.5,

4.2)

2.15 dddd, (13.3, 13.2,

4.5, 4.2)

2.12 dddd, (13.3, 13.2, 4.5,

4.2)

H-C(21β)

1.77 ddd, (13.4, 13.2, 3.3) 1.73 ddd, (13.5, 13.2, 3.5) 1.74 ddd, (13.5, 13.2, 3.7) 1.79 ddd,(13.5, 13.5, 3.5)

H-C

(22α)

2.10 ddd, (13.5, 13.5, 3.5) 2.06 ddd,(13.5, 13.5, 3.5) 2.05 ddd, (13.5, 13.5, 3.5) 2.12 ddd, (13.5, 13.5, 3.5)

H-C(22β) 1.51 dd, (13.5, 13.5) 1.45 dd, (13.5, 13.4) 1.41 dd, (13.5, 13.3) 1.48 dd, (13.5, 13.2)

H-23 1.66 s 1.64 s 1.67 s 1.69 s

Hα -24 4.13 d (11.5) 4.10 d (11.5) 4.15 d (11.5) 4.14 d (11.5)

Hβ -24 3.85 d (11.5) 3.85 d (11.5) 3.85 d (11.5) 3.85 d (11.5)

H-25 1.09 s 1.10 s 1.08 s 1.07 s

H-26 1.17 s 1.18 s 1.16 s 1.17 s

H-27 1.54 s 1.56 s 1.55 s 1.53 s

H-29 1.14 s 1.13 s 1.18 s 1.15 s

H-30 0.98 s 0.99 s 0.97 s 0.96 s

Glc

H-1 6.36 d (8.5) 6.37 d (8.5) 6.35 d (8.5) 6.34 d (8.5)

H-2 4.21 dd (8.5, 9.0) 4.20 dd (8.5, 9.0) 4.22 dd (8.5, 9.0) 4.19 dd (8.5, 9.0)

H-3 4.29 dd (9.0, 9.0) 4.28 dd (9.0, 9.0) 4.30 dd (9.0, 9.0) 4.31 dd (9.0, 9.0)

H-4 4.36 dd (9.0, 9.0) 4.37 dd (9.0, 9.0) 4.39 dd (9.0, 9.0) 4.34 dd (9.0, 9.0)

H-5 4.02 ddd (2.5, 4.5, 9.0) 4.03 ddd (2.5, 4.5, 9.0) 4.01 ddd (2.5, 4.5, 9.0) 4.04 ddd (2.5, 4.5, 9.0)

Hα -6 4.45 dd (2.5, 12.0) 4.48 dd (2.5, 12.0) 4.47 dd (2.5, 12.0) 4.49 dd (2.5, 12.0)

Hβ -6 4.39 dd (4.5, 12.0) 4.40 dd (4.5, 12.0) 4.38 dd (4.5, 12.0) 4.41 dd (4.5, 12.0)

201

Table 12. 1H-NMR (300 MHz, C5D5N) data of 10-13 isolated from C. sativum

Aglycone 10 11 12 13


H-C(1β) 1.39 dd, (12.5, 3.5) 1.42 dd, (12.5, 3.5) 1.49 dd, (12.5, 3.5) 1.38 dd, (12.5, 3.5)

H-2 4.76 ddd (3.0, 3.0, 12.0) 4.96 ddd (3.0, 3.0, 12.0) 4.45 ddd (3.0, 3.0, 12.0) 4.46 ddd (3.0, 3.0, 12.0)

H-3 4.68 d (3.0) 4.58 d (3.0) 4.98 d (3.0) 4.58 d (3.0)

H-5 1.87 d (12.0) 1.87 d (12.0) 1.87 d (12.0) 1.87 d (12.0)

H-C(6α) 1.63 dddd, (12.6, 5.5, 3.5,

3.5)

1.62 dddd, (12.5, 5.5, 3.5,

3.5)

1.55 dddd, (12.6, 5.5, 3.5,

3.5)

1.56 dddd, (12.6, 5.5, 3.5,

3.5)

H-C(6β) 1.34 ddd, (12.6, 5.5, 3.5) 1.36 ddd, (12.6, 5.3, 3.5) 1.37 ddd, (12.6, 5.4, 3.5) 1.35 ddd, (12.6, 5.5, 3.7)


H-C(7β) 1.48 dd, (12.5, 5.5) 1.44 dd, (12.5, 5.5) 1.47 dd, (12.5, 5.5) 1.47 dd, (12.5, 5.5)

H-C(9) 1.66 dd, (13.5, 6.5) 1.67 dd, (13.5, 6.5) 1.64 dd, (13.5, 6.5) 1.63 dd, (13.5, 6.5)

H-(11α) 2.09 ddd, (8.3, 4.4, 2.5) 2.08 ddd, (8.5, 4.3, 2.5) 2.05 ddd, (8.5, 4.4, 2.5) 2.06 ddd, (8.5, 4.5, 2.5)

H-C(11β) 1.89 dd, (13.5, 4.5) 1.83 dd, (13.3, 4.5) 1.82 dd, (13.4, 4.5) 1.86 dd, (13.5, 4.4)

H-12 5.50 d (3.0) 5.50 d (3.0) 5.53 d (3.0) 5.53 d (3.0)

H-15 2.36 ddd (3.0, 13.5, 13.5) 2.36 ddd (3.0, 13.5, 13.5) 2.36 ddd (3.0, 13.5, 13.5) 2.36 ddd (3.0, 13.5, 13.5)

H-16 2.80 ddd (3.0, 13.5, 13.5) 2.79 ddd (3.0, 13.5, 13.5) 2.81 ddd (3.0, 13.5, 13.5) 2.82 ddd (3.0, 13.5, 13.5)

H-17 3.07 ddd (4.5, 13.5, 13.5) 3.09 ddd (4.5, 13.5, 13.5) 3.10 ddd (4.5, 13.5, 13.5) 3.05 ddd (4.5, 13.5, 13.5)

H-18 3.12 br s 3.12 br s 3.12 br s 3.12 br s

H-19 3.57 d (4.0) 4.81 d (11.4) 4.79 d (11.5) 4.75 d (11.8.0)

H-C(21α) 2.07 dddd, (13.2, 13.2, 4.5,

4.2)

2.04 dddd, (13.3, 13.1, 4.5,

4.2)

2.05 dddd, (13.3, 13.2, 4.5,

4.2)

2.09 dddd, (13.3, 13.2, 4.5,

4.2)

H-C(21β)

1.75 ddd, (13.4, 13.2, 3.3) 1.77 ddd, (13.5, 13.2, 3.5) 1.75 ddd, (13.5, 13.2, 3.7) 1.74 ddd, (13.5, 13.5, 3.5)

H-(22α)

2.09 ddd, (13.5, 13.5, 3.5) 2.08 ddd, (13.5, 13.5, 3.5) 2.09 ddd, (13.5, 13.5, 3.5) 2.07 ddd, (13.5, 13.5, 3.5)

H-C(22β) 1.50 dd, (13.5, 13.5) 1.48 dd (13.5, 13.4) 1.44 dd (13.5, 13.3) 1.46 dd (13.5, 13.2)

H-23 1.66 s 1.67 s 1.69 s 1.70 s

Hα -24 4.13 d (11.5) 4.14 d (11.5) 4.16 d (11.5) 4.11 d (11.5)

Hβ -24 3.85 d (11.5) 3.85 d (11.5) 3.85 d (11.5) 3.85 d (11.5)

H-25 1.09 s 1.08 s 1.07 s 1.04 s

H-26 1.17 s 1.14 s 1.15 s 1.18 s

H-27 1.54 s 1.52 s 1.55 s 1.53 s

H-29 1.14 s 1.16 s 1.13 s 1.12 s

H-30 0.98 s 0.99 s 0.96 s 0.97 s

Me-CO 2.23 s 2.22 s 2.22 s

Glc

H-1 6.36 d (8.5) 6.33 d (8.5) 6.35 d (8.5) 6.41 d (8.5),

H-2 4.21 dd (8.5, 9.0) 4.26 dd (8.5, 9.0) 4.27 dd (8.5, 9.0) 4.28 dd (8.5, 9.0)

H-3 4.29 dd (9.0, 9.0) 4.31 dd (9.0, 9.0) 4.32 dd (9.0, 9.0) 4.30 dd (9.0, 9.0)

H-4 4.36 dd (9.0, 9.0) 4.34 dd (9.0, 9.0) 4.39 dd (9.0, 9.0) 4.33 dd (9.0, 9.0)

H-5 4.02 ddd (2.5, 4.5, 9.0) 4.05 ddd (2.5, 4.5, 9.0) 4.07 ddd (2.5, 4.5, 9.0) 4.01 ddd (2.5, 4.5, 9.0)

Hα -6 4.45 dd (2.5, 12.0) 4.43 dd (2.5, 12.0) 4.46 dd (2.5, 12.0) 4.48 dd (2.5, 12.0)

Hβ -6 4.39 dd (4.5, 12.0) 4.40 dd (4.5, 12.0 4.38 dd (4.5, 12.0) 4.37 dd (4.5, 12.0)

202

Table 13. 13

C-NMR (75 MHz, C5D5N) data of 6-13 isolated from C. sativum

C# 6 7 8 9 10 11 12 13

C-1 43.3 43.5 43.7 43.4 43.0 43.2 44.0 43.8

C-2 66.3 74.3 66.7 66.9 68.3 69.3 66.4 66.5

C-3 74.2 71.3 76.2 74.2 79.2 74.2 76.2 74.2

C-4 45.2 45.1 45.4 45.6 45.7 45.2 45.3 45.2

C-5 49.7 50.1 49.9 49.7 49.6 49.7 49.9 49.8

C-6 19.2 19.3 19.1 19.4 18.9 18.8 19.5 19.0

C-7 33.8 33.7 33.9 33.7 34.1 334.2 33.6 40.0

C-8 40.6 40.8 40.5 40.7 40.9 40.6 40.4 41.0

C-9 48.5 48.8 48.7 48.6 48.2 48.9 48.3 48.4

C-10 38.9 39.1 39.2 39.0 38.8 38.7 38.6 38.5

C-11 24.5 24.7 24.8 24.6 24.9 24.5 25.1 25.0

C-12 123.8 123.9 123.7 123.6 123.4 124.0 124.1 123.5

C-13 144.4 143.8 144.5 144.3 144.6 144.7 144.6 144.8

C-14 42.2 42.3 42.5 42.4 42.7 42.8 42.2 42.6

C-15 29.0 29.1 29.5 29.4 29.3 29.6 29.8 29.2

C-16 28.0 28.9 28.6 28.5 28.3 28.0 28.2 28.1

C-17 46.5 46.8 46.4 46.8 46.9 46.3 46.2 46.6

C-18 44.6 44.4 44.7 44.0 44.9 44.7 44.8 44.2

C-19 81.1 78.4 81.2 81.3 81.4 81.7 81.3 84.1

C-20 35.6 35.7 36.1 35.9 35.8 36.1 36.8 35.8

C-21 29.0 29.1 29.2 29.3 29.0 28.9 28.8 29.5

C-22 33.1 33.2 33.8 33.7 33.6 33.4 33.5 33.4

C-23 23.8 23.9 23.5 24.0 24.1 23.7 23.6 23.2

C-24 65.2 65.7 65.4 65.5 65.9 65.3 65.6 65.0

C-25 17.1 17.2 17.3 17.7 17.6 17.4 17.8 16.9

C-26 17.1 17.3 17.4 17.6 17.5 17.5 17.7 17.0

C-27 24.9 25.1 24.8 24.7 24.6 24.4 25.2 24.5

C-28 177.3 177.7 177.6 177.8 177.9 177.1 177.2 177.0

C-29 28.8 28.9 28.7 28.6 28.5 28.7 29.2 29.3

C-30 16.7 16.8 16.5 16.4 16.6 16.9 16.5 16.7

Me-CO 22.3 22.1 22.2

Me-CO 176.2 176.4 176.5

Glc

C-1 95.9 95.8 95.7 95.4 95.6 96.1 96.2 96.0

C-2 74.4 74.5 74.6 74.7 74.8 74.9 74.3 74.2

C-3 79.3 79.7 79.8 79.5 79.6 80.3 80.2 80.1

C-4 71.2 71.3 71.4 71.6 71.9 71.7 71.8 71.4

C-5 79.2 79.4 79.3 79.5 79.6 79.1 79.7 78.9

C-6 62.3 62.2 62.5 62.8 62.7 62.1 62.0 62.4

203

Table 14. IC50 values (µM) of Compounds 1-13 isolated from C. sativum L. for various cell lines

Com HL-60 SMMC-7721 A-549 MCF-7 SW-480

6 2.14± 0.08 10.27± 0.07 11.63± 0.02 12.73± 0.04 14.03± 0.02

7 2.19± 0.05 14.34± 0.02 16.69± 0.12 18.14± 0.06 21.48± 0.02

8 2.17± 0.04 15.06± 0.07 13.62± 0.02 17.68± 0.05 20.04± 0.02

9 3.16± 0.02 18.38± 0.02 20.73± 0.02 27.37± 0.04 24.73± 0.02

10 2.57± 0.12 19.62± 0.11 28.28± 0.05 24.84± 0.06 23.19± 0.02

11 2.87± 0.08 19.93± 0.02 38.19± 0.04 27.49± 0.05 25.79± 0.02

12 2.84± 0.07 22.63± 0.02 31.26± 0.07 26.06± 0.03 27.14± 0.02

13 3.96± 0.06 19.45± 0.05 19.93± 0.03 21.83± 0.04 29.43± 0.08

Std 1.04± 0.05 14.75± 0.04 13.61± 0.03 16.95± 0.07 19.68± 0.02

DDP ((MW300) cisplatin) was used a positive control.

204

FIGURES

205

Fig. 1. FTIR of essential oils obtained from C. sativum

206

Fig. 2. (i) COSY-450 and HOHAHA Bold-bond (ii) HMBC Interaction in 1-5

207

Fig. 3. NOESY Interaction in 1-5

208

Fig. 4. (i) COSY-450 and HOHAHA Bold-bond (ii) HMBC Interaction in 6-13

209

Fig. 5. ROESY interaction in 6-13

210

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211

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Published Papers

1. Muhammad Iqbal, Imam Bakhsh Baloch and Musa Kaleem Baloch. Synthesis and

Structural Characterization of Novel Monoesters of Succinic Anhydride with Aryl

Alcohols. Chemistry Journal, 2 (1), 2-19 (2012).

Available online at www.scientific-journals.co.uk

2. Muhammad Iqbal, Imam Bakhsh Baloch and Musa Kaleem Baloch. Preparation of

Novel Monoesters of Succinic Acid from Succinic Anhydride using p-Toluensulphonic

Acid as a Catalyst. Chemistry Journal, 2 (2), 87-94 (2012).

Available online at www.scientific-journals.co.uk

3. Muhammad Iqbal, Imam Bakhsh Baloch, Musa K. Baloch. An efficient method for the

preparation of benzyl γ-ketohexanoates. Chemical Papers, 67 (4) 444–455 (2013).

4. Muhammad Iqbal, Imam Bakhsh Baloch, Musa Kaleem Baloch, Muhammad Ayaz

Khan, Adnan Amin and Muhammad Akram. Bio-active Compounds from Coriandrum

sativum L. Seeds. Wulfenia Journal, 20 (1), 105-120, (2013).

5. Muhammad Iqbal, Imam Bakhsh Baloch and Musa Kaleem Baloch. Synthesis of Novel

γ-Ketoesters from Succinic Anhydride. Asian Journal of Chemistry, 25 (17), 9701-9703

(2013).

6. Muhammad Iqbal, Imam Bakhsh Baloch and Musa Kaleem Baloch. Evaluation of

antifungal and antibacterial activities of monoesters of succinic anhydride. Bull. Chem.

Soc. Ethiop., 28 (1), 131-136 (2014).

http://www.scientific-journals.co.uk/

http://www.scientific-journals.co.uk/

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