Isolation and Characterization of Bioactive Diterpenoid Alkaloids from Aconitum Heterophyllum Wall and Related Species

ISOLATION AND CHARACTERIZATION OF BIOACTIVE DITERPENOID ALKALOIDS FROM ACONITUM HETEROPHYLLUM WALL AND RELATED SPECIES

Mr. HANIF AHMAD

DOCTOR OF PHILOSPHY (PhD)

CHEMISTRY

DEPARTMENT OF CHEMISTRY UNIVERSITY OF MALAKAND 2016 ISOLATION AND CHARACTERIZATION OF BIOACTIVE DITERPENOID ALKALOIDS FROM ACONITUM HETEROPHYLLUM WALL AND RELATED SPECIES

Mr. HANIF AHMAD

Thesis submitted to the Department of Chemistry,

University of Malakand for the partial fulfillment of the requirements for the degree of

DOCTOR OF PHILOSOPHY (PhD)

CHEMISTRY

DEPARTMENT OF CHEMISTRY UNIVERSITY OF MALAKAND 2016 Declaration

I declare that the thesis “ISOLATION AND CHARACTERIZTION OF BIOACTIVE DITERPENOID ALKALOIDS FROM ACONITUM HETROPHYLLUM WALL. AND RELATED SPECIES” is my original work and has never been presented for the award of any degree at any other university before and that all the information sources I have used and or quoted have been indicated and acknowledged by means of complete reference.

Hanif Ahmad

Certificate

It is recommended that thesis submitted by Mr. HANIF AHMAD entitled “ISOLATION AND CHARACTERIZTION OF BIOACTIVE DITERPENOID ALKALOIDS FROM ACONITUM HETROPHYLLUM WALL. AND RELATED SPECIES” be accepted as fulfilling this part of the requirements for the degree of Doctor of Philosophy (PhD) in Chemistry.

______

SUPERVISOR CO-SUPERVISOR Dr. Manzoor Ahmad Prof. Dr. Farzana Shaheen Associate Professor HEJ Research Institute of Chemistry ICCBS, University of Karachi

______

EXTERNAL EXAMINER CHAIRMAN Department of Chemistry University of Malaknd

Dedicated

To My

Beloved Mother

Contents

ACKNOWLEDGMENTS ...... i

Aims of the Study ...... iii

SUMMARY ...... iv

List of Tables ...... ix

List of Figures ...... xi

List of Schemes ...... xiv

List of Abbreviations ...... xv

INTRODUCTION ...... 1

1.1 Plants as a source of medicine ...... 1

1.2 Strategies in the search of new natural compounds ...... 3

1.3 Ranunculaceae ...... 5

1.3.1 The Genus Aconitum ...... 5

1.3.2 Aconitum heterophyllum Wall...... 6

1.3.3 The Aconitum laeve Royle ...... 6

1.3.4 Systematic Position ...... 7

1.3.5 Traditional importance of Aconitum Species ...... 7

1.3.6 Chemistry of the Genus Aconitum ...... 8

1.3.7 Literature survey on A. heterophyllum Wall and A. laeve Royle...... 8

1.4 The Genus Delphinium ...... 9

1.4.1 Systematic Position ...... 10

1.4.2 Delphinium denudatum Wall...... 10

1.4.3 Ethnobotanical importance of the Genus Delphinium ...... 11 1.4.4 Literature survey on Delphinium denudatum Wall...... 12

1.5 Diterpenoid alkaloids ...... 27

1.6 Classification of diterpenoid alkaloids...... 27

1.6.1 C20 diterpene alkaloids ...... 27

1.6.2 C19-diterpenoid alkaloids ...... 29

1.6.3 C18 diterpene alkaloids ...... 31

1.7 Biosynthesis of diterpenoid alkaloids ...... 31

EXPERIMENTAL...... 36

2.1 General Experimental Conditions ...... 36

2.1.1 Physical constants ...... 36

2.1.2 Spectroscopic techniques ...... 36

2.1.3 Column chromatography ...... 36

2.1.4 Materials for chromatography ...... 36

2.1.5 Solvents ...... 37

2.2 Plant materials ...... 37

2.2.1 Extraction ...... 37

2.2.2 Crude alkaloids fractionation ...... 38

2.2.3 Isolation of diterpenoids from A. heterophyllum Wall...... 40

2.2.4 Isolation of diterpenoids from A. laeve Royle ...... 41

2.2.5 Isolation of diterpenoids from D. denudatum Wall ...... 43

2.3 X-Rays Diffraction Studies ...... 45

2.3.1 DFT Calculations ...... 45

2.4 Cholinesterase inhibition assay and determination of IC50 ...... 45 2.5 Experimental data of new diterpenoids from A. heterophyllum Wall...... 46

2.5.1 1α, 6β-dimethoxy, 8,9β-dihydroxy heteratisine (1) ...... 46

2.5.2 1,11,13α, Trihydroxyl atisine (2) ...... 47

2.5.3 2α, 6β Dihydroxy atisine (3) ...... 47

2.6 Experimental data of new diterpenoids from A. laeve Royle...... 48

2.6.1 Swatinine-C (4) ...... 48

2.6.2 Swatinine-D (5) ...... 48

2.6.3 Methyl 2 acetamidobenzoate (6) ...... 49

2.6.4 Methyl 4-[2-(methoxycarbonyl)anilino]-4-oxobutanoate (7) ...... 49

2.7 Experimental data of new diterpenoids from D. denudatum Wall...... 50

2.7.1 1β-hydroxy, 14β-acetyl condelphine (8) ...... 50

2.7.2 1,14α, 8,10β-Tetrahydroxy, 16,18β dimethoxy-N-ethyl- aconitane (9) ...... 50

2.7.3 1α,8,16,18β-Tetramethoxy, 14α-hydroxy-N-ethyl-5-aconitene (10) ...... 51

2.8 Experimental data of known diterpenoids from A. heterophyllum Wall...... 52

2.8.1 Isoatisine (11) ...... 52

2.8.2 19-Epiisoatisine (12) ...... 52

2.8.3 Atidine (13) ...... 53

2.8.4 Heteratisine (14) ...... 53

2.8.5 Hetisinone (15) ...... 54

2.9 Experimental data of known diterpenoids from A. leave Royle...... 55

2.9.1 Aconorine (16) ...... 55

2.9.2 Lappaconitine (17) ...... 55

2.10 Experimental data of known diterpenoids from D. denudatum Wall...... 56 2.10.1 Isotalatizidine hydrate (18) ...... 56

2.10.2 Dihydropentagynine (19) ...... 57

3.1 Diterpenoids isolated from A. heterophyllum Wall...... 58

3.2 New Diterpenoids from A. heterophyllum Wall...... 59

3.2.1 1α, 6β-dimethoxy, 8,9β-dihydroxy heteratisine (1) ...... 59

3.2.2 1,11,13α, Trihydroxyl atisine (2) ...... 63

3.2.3 2α, 6β Dihydroxy atisine (3) ...... 66

3.3 New diterpenoids from A. laeve Royle ...... 69

3.3.1 Swatinine-C (4) ...... 69

3.3.2 Swatinine-D (5) ...... 74

3.3.3 Methyl 2-acetamidobenzoate (6) ...... 78

3.3.4 Methyl 4-[2-(methoxycarbonyl)anilino]-4-oxobutanoate (7) ...... 80

3.4 New diterpenoids from D. denudatum Wall...... 83

3.4.1 1β-hydroxy, 14β-acetyl condelphine (8) ...... 83

3.4.2 HOMO-LUMO energy gap of 1β-hydroxy, 14β-acetyl condelphine (8) ..... 90

3.4.3 1,14α, 8,10β-Tetrahydroxy, 16,18β dimethoxy-N-ethyl- aconitane (9) ...... 91

3.4.4 1α,8,16,18β-tetramethoxy, 14α-hydroxy-N-ethyl- 5-aconitene (10) ...... 94

3.5 Known diterpenoids from A. heterophyllum Wall...... 98

3.5.1 Isoatisine (11) ...... 98

3.5.2 19-Epiisoatisine (12) ...... 100

3.5.3 Atidine (13) ...... 102

3.5.4 Heteratisine (14) ...... 103

3.5.5 HOMO-LUMO energy gap of heteratisine (14) ...... 106 3.5.5 Hetisinone (15) ...... 106

3.6 Known diterpenoids from A. laeve Royle...... 109

3.6.1 Aconorine (16) ...... 109

3.6.2 Lappaconitine (17) ...... 111

3.7 Known diterpenoids from D. denudatum Wall ...... 113

3.7.1 Isotalatizidine hydrate (18) ...... 113

3.7.2 HOMO-LUMO energy gap of Isotalatizidine hydrate (18) ...... 117

3.7.3 Dihydropentagynine (19) ...... 117

3.8 Enzyme inhibition activity of isolated diterpenoids ...... 119

3.8.1 Cholinesterase inhibition of diterpenoid from A. heterophyllum Wall ...... 119

3.8.2 Cholinesterase inhibition of diterpenoids from A. laeve Royle ...... 121

3.8.3 Cholinesterase inhibition of diterpenoidfrom D.denudatum Wall ...... 126

References ...... 128

ACKNOWLEDGMENTS

I wish to express my appreciation and sincere gratitude to Prof. Dr. Rashid Ahmad;

Chairman, Department of Chemistry, University of Malakand whose administrative assistance and guidance proved immensely valuable to me at every step. He was always there to provide all possible necessities with smiling face.

I would also like to express my deep thanks and great appreciation to my supervisor; Dr.

Manzoor Ahmad, Associate Professor, Department of Chemistry, University of Malakand, whose infectious enthusiasm for natural product chemistry had hovered over me for the entire period of my studies. I am thankful for his helpful and friendly discussion, his teaching, research skills and above all his endless fascination for chemistry. His guidance helped me in all movements of research and also of writing this thesis.

I would like to acknowledge the co-operation of my co-supervisor Prof. Dr. Farzana

Shaheen HEJ Research Institute of Chemistry, ICCBS, University of Karachi. I am thankful to her for giving me so much of his precious time.

I am also thankful to the faculty members of the Department of Chemistry, University of

Malakand, Dr. Sultan Alam, Dr. Khalid Saeed, Dr. Najib ur Rahman, Dr. Ezzat Khan, Dr. Mumtaz

Ali, Dr. Naveed Umar, Dr. Mian Muhammad, Dr. Muhammad Sadiq, Dr. Muhmmad Zahoor, Dr.

Behisht Ara, Dr. Maria Sadia, Mr. Abdul Waheed Kamran and Ms. Sumaira Naz for their motivation and support.

I would like to appreciate the support of Mr. Qaisar Khan, Mr. Sultan Ali, Mr. Younus Ali and

Salman Ahmad (non-teaching staff) of the Department of Chemistry University of Malakand.

My thanks are extended to research collaborators; Dr. Syed Adnan Ali Shah, Dr. Humera

Naz, Faculty of Pharmacy, Universiti Teknologi MARA Selangor D. E., Malaysia, Prof. Dr. Nawaz

Tahir, Department of Physics, University of Sargodah and Dr. Hidayatullah Khan, Department of

Biotechnology, University of Science and Technology Bannu for their help in instrumental analysis and biological activities.

I am grateful to my lab. Mates and best friends; Mr. Shujaat Ahmad, Dr. Adnan Shahzad,

Mr. Shujat Ali, Mr. Misal Bacha, Mr. Sultan Muhammad, Mr. Umar Ali Khan, Mr. Idrees Khan,

Mr. Alam Khan, Mr. Najibullah, Mr. Nasib Khan, Mr. Zarif Gul, Mr. Mr. Noor Zada, Mr. Tariq

Shah and Mr. Sajid Ali for having made the lab feel like a home. It was a wonderful time with them.

I would like to thank those whom I deeply love, respect, admire and whom I am dedicating this work to my family, especially to my beloved mother, my sisters and my brothers whose infinite prayers, unwavering support kept my morale high during difficult times. Thanks to my wife, without her support, I could not have finished this research work and because of the happiness you brought me, I can go to the place which I never even know.

Hanif Ahmad

Aims of the Study

Several species of genus Aconitum and Delphinium found at high altitude of Malakand

Division which have been poorly examined phytochemically, or not at all. The main goals of my research work were to:

 Collect plants material of certain Aconitum and Delphinium species of Malakand Division.

 Gain chemotaxonomically valuable information concerning the Ranunculaceae family.

 Examine the alkaloid contents of the collected species.

 Select species with rich and complex alkaloid contents.

 Carry out preparative work to isolate pure diterpene alkaloids from the selected species.

 Elucidate the structures of isolated pure compounds by using spectroscopic methods.

 Evaluate the isolated compounds for their biological activities

 Identify diterpene alkaloids which are potential objects or tools for drug development.

iii

SUMMARY

This PhD dissertation describes the phytochemicals and biological exploration of three plants species belonging to family Ranunculaceae from Malakand Division.

We report herein the isolation, structure elucidation, selective AChE and BChE enzyme inhibition activities of C19 and C20 diterpenoid alkaloids from the basic (pH = 8-10) chloroform soluble fraction of A. heterophyllum Wall, A. laeve Royle and D. denudatum Wall. Ten new, 1α,

6β-dimethoxy, 8,9β-dihydroxy heteratisine (1), 1, 11,13α, trihydroxyl atisine (2), 2α, 6β dihydroxy atisine (3), swatinine-C (4), swatinine-D (5), methyl 2-acetamidobnzoate (6), methyl 4-[2-

(methoxycarbonyl)anilino]-4-oxobutanoate (7), 1β-hydroxy, 14β-acetyl condelphine (8), 1,14α,

8,10β-tetrahydroxy, 16,18β-dimethoxy-N-ethyl aconitane (9), 1α,8,16,18β-tetramethoxy, 14α- hydroxy-N-ethyl-5-aconitene (10) and nine known, isoatisine (11), 19-epi-isoatisine (12), atidine

(13), heteratisine (14), hetisinone (15), aconorine (16), lappaconitine (17), isotalatizidine hydrate

(18) and dihydropentagynine (19), diterpenoid and nor diterpenoid alkaloids have been isolated from aforesaid species.

The title compounds (1-19) were characterized by using modern spectroscopic techniques

(EI-MS, 1H-NMR, 13C-NMR, and 2D-NMR) and single X-rays crystallography. All the isolated compounds were screened for their enzyme inhibition activities (AChE & BChE). Our present findings indicate an interest in C19 and C20 diterpenoid alkaloids as potent AChE and BChE inhibotrs present in A. heterophyllum Wall, A. laeve Royle and D. denudatum Wall. They may contribute towards naturally accessible inhibitors used for the treatment of Alzheimer’s disease.

HO 13 O 16 13 HO 17 12 CH H CO 12 2 3 17 14 C O OH 11 20 16 OH 14 1 10 15 15 11 9 1 2 8 9 3 N 5 2 10 8 7 OH N 4 6 3 5 7 4 19 OCH3 6 18 18 19 (1) (2)

17 OCH3 CH2 12 16 16 OH 12 13 14 20 11 OCOCH 14 17 3 15 H HO 10 1 9 1 9 15 2 10 8 2 11 N 8 N 3 5 3 5 7 7 O 4 4 6 H H O 18 HO 19 18 19 O HO

(3) (4)

OCH3 CH3 13 16 OCH3 12 14 17 OCH3 O O H H 1 10 9 15 2 11 1 N 8 N 3 5 O 7 O 6 4 6 2 H O 18 5 19 3 H3C HO 4

(5) (6)

OCH3 16 CH OH 13 3 12 14 17 H OCOCH3 O O H 15 1 10 9 2 11 N 8 H 3 5 N 4 7 OH 1 3' 6 6 2 4' 2' H 19 5 3 O 1' 18 4 O H3CO OCH3 (7) (8)

OCH OCH 3 3 13 OH 13 16 OCH3 12 16 17 12 14 OH 17 14 OH OH 15 15 10 H 1 10 9 H 1 9 2 11 2 11 N 8 N 8 3 5 3 5 4 6 7 OH 4 6 7 OCH3 H 19 18 19 18 H CO 3 H3CO (9) (10)

13 13 17 17 12 CH 12 CH 16 2 16 2 11 11 20 20 14 14 15 15 1 9 OH 1 9 OH 2 8 21 10 21 2 10 8 N N 3 5 7 3 22 4 22 5 7 6 4 6 O O 19 18 19 18

(11) (12)

13 17 16 CH2 13 O 12 11 16 H CO 12 20 3 17 14 14C O 15 1 9 OH 1 10 9 15 2 10 8 2 11 N N 8 3 5 7 3 5 4 7 OH 4 6 O 6 OH 19 19 18 18HO (13) (14)

OCH 3 OCH3 13 14 16 17 12 OH HO 1 10 13 2 11 9 17 8 15 12 N HO CH 3 5 7 2 4 6 OH 16 20 11 14 15 19 O 1 9 O 2 10 8 N C O 3 5 7 4 6 NHCOCH3

19 18

(15) (16)

vii

OCH3

OCH3 OCH3 OCH3 OH 13 OH 12 16 17 14 OH H N 15 OH 1 10 9 H H 2 11 N 8 O 3 5 4 7 OH C O 6 H .H2O 19 18 NHCOCH3 H3CO

(17) (18)

OCH3 13 16 OH 17 12 14 OH H 15 H 1 10 9 2 11 N 8 3 5 OH 4 6 7 H 19 18 OCH3 (19)

viii

LIST OF TABLES

No Title Page No

Table 1.1. Summary of the previous phytochemical studies on A. heterophyllum Wall. and

A. laeve Royle...... 13

Table 1.2 Summary of the previous phytochemical studies on Delphinium denudatum Wall...... 24

Table 3.1. 1H & 13C-NMR (500 & 125 MHz) data of compound (1) ...... 62

Table 3.2. 1H & 13C-NMR (600 & 150 MHz) data of compound (2) ...... 65

Table 3.3. 1H & 13C-NMR (500 & 125 MHz) data compound (3) ...... 68

Table 3.4. 1H & 13C-NMR (600 & 150 MHz) data of swatinine-C (4) ...... 73

Table 3.5. 1H and 13C-NMR (600 & 150MHz) data of swatinine-D (5) ...... 77

Table 3.6. 1H- (300 MHz) &13C- (75 MHz) NMR data of compound (6) ...... 80

Table 3.7. 1H- (300 MHz) &13C- (75 MHz) NMR data of compound (7) ...... 83

Table 3.8. 1H- (500 MHz) and 13C-NMR (125 MHz) data of compound (8) ...... 88

Table 3.9. X-ray data and structure refinements of compound (8) ...... 89

Table 3.10. List of HOMO-LUMO energy, ionization energy (IE), electron affinity (EA) global hardness (η), chemical potential (μ) and global electrophilicity (ω) of

(8) ...... 90

Table 3.11. 1H- (600 MHz) and 13C-NMR (150 MHz) data of compound (9) ...... 93

Table 3.12. 1H- (500 MHz) and 13C-NMR (125 MHz) data of compound (10) ...... 97

Table 3.13. Crystal data and structure refinements of compound (11) ...... 100

Table 3.14. Crystal data and structure refinements of compound (14) ...... 105

Table 3.15. List of HOMO-LUMO energy, ionization energy (IE), electron affinity (EA) global hardness (η), chemical potential (μ) and global electrophilicity (ω) of

(14) ...... 106

Table 3.16. Crystal data and structure refinements of compound (15) ...... 108

Table 3.17. X-ray data and structure refinements of isotalatizidine hydrate (18) ...... 115

Table 3.18. List of HOMO-LUMO energy, ionization energy (IE), electron affinity (EA) global hardness (η), chemical potential (μ) and global electrophilicity (ω) of

(18) ...... 117

Table 3.19. AChE and BChE inhibitory activities of alkaloids from A. heterophyllum 120

Table 3.20. AChE and BChE inhibitory activities of diterpenoids from A.laeve ...... 121

Table 3.21. AChE and BChE inhibitory activities of alkaloids from D. denudatum ..... 127

LIST OF FIGURES

No Title Page No

Figure 1.1. Examples of plant-derived anticancer drugs ...... 2

Figure 1.2. Basic skeletons of C20 diterpenoid alkaloids ...... 29

Figure 1.3. Basic skeletons of C19 diterpenoid alkaloids ...... 30

Figure 1.4. Delcosine (R= H) & acetyl delcosine (R= Acetyl) ...... 34

Figure 1.5. Hetidine ...... 35

Figure 3.1. 1α, 6β-dimethoxy, 8,9β-dihydroxy heteratisine (1) ...... 59

Figure 3.2. Key HMBC correlation in 1α, 6β-dimethoxy, 8,9β-dihydroxy heteratisine (1)

...... 60

Figure 3.3. Key COSY correlation in 1α, 6β-dimethoxy, 8,9β-dihydroxy heteratisine (1)

...... 61

Figure 3.4. 1, 11,13α, Trihydroxyl atisine (2) ...... 63

Figure 3.5. Key HMBC correlation in 1, 11,13α, trihydroxyl Atisine (2) ...... 64

Figure 3.6. Key COSY correlation in 1, 11,13α, Trihydroxyl atisine (2) ...... 65

Figure 3.7. 2α, 6β Dihydroxy atisine (3) ...... 66

Figure 3.8. Key HMBC correlation in 2α, 6β dihydroxy atisine (3) ...... 68

Figure 3.9. Swatinine-C (4) ...... 70

Figure 3.10. Key HMBC correlation in swatinine-C (4) ...... 71

Figure 3.11. Key COSY interaction in swatinine-C (4) ...... 72

Figure 3.12. Swatinine-D (5) ...... 74

Figure 3.13. Key HMBC correlation in swatinine-D (5) ...... 75

Figure 3.14. Key COSY correlation in Swatinine-D (5) ...... 76

Figure 3.15. Methyl 2-acetamidobenzoate (6)...... 78

Figure 3.16. Key HMBC correlation in Methyl 2-acetamidobenzoate (6) ...... 79

Figure 3.17. Methyl 4-[2-(methoxycarbonyl)anilino]-4-oxobutanoate (7) ...... 81

Figure 3.18. Methyl 4-[2-(methoxycarbonyl)anilino]-4-oxobutanoate (7) ...... 82

Figure 3.19. 1β-hydroxy, 14β-acetyl condelphine (8) ...... 84

Figure 3.20. Key HMBC interaction in1β-hydroxy, 14β-acetyl condelphine (8) ...... 85

Figure 3.21. Key COSY correlation in 1β-hydroxy, 14β-acetyl condelphine (8) ...... 86

Figure 3.22. Structural representation of 8, with 50 % probability of thermal ellopsiodes

and hydrogen atoms are omitted for clarity...... 87

Figure 3.23. 1,14α, 8,10β-Tetrahydroxy, 16,18β dimethoxy-N-ethyl-aconitane (9) ...... 91

Figure 3.24. Key HMBC correlation in compound (9) ...... 92

Figure 3.25. 1α,8,16,18β-Tetramethoxy, 14α-hydroxy-N-ethyl-5-aconitene (10) ...... 94

Figure 3.26. Key HMBC interaction in compound (10) ...... 96

Figure 3.27. Isoatisine (11) ...... 98

Figure 3.28. Structural representation of compound 11, with 50 % probability of thermal

ellopsiodes and hydrogen atoms are omitted for clarity...... 99

Figure 3.29. 19-epiisoatisine (12) ...... 101

Figure 3.30. Atidine (13) ...... 102

Figure 3.31. Heteratisine (14) ...... 103

xii

Figure 3.32. Structural representation of compound 14, with 50 % probability of thermal

ellopsiodes and hydrogen atoms are omitted for clarity...... 105

Figure 3.33. Hetisinone (15) ...... 107

Figure 3.34. Structural representation of compound 15, with 50 % probability of thermal

ellopsiodes and hydrogen atoms are omitted for clarity...... 108

Figure 3.35. Aconorine (16) ...... 110

Figure 3.36. Lappaconitine (17) ...... 111

Figure 3.37. Isotalatizidine Hydarte 18 ...... 113

Figure 3.38. Structural representation of compound 18, with 50 % probability of thermal

ellopsiodes and hydrogen atoms are omitted for clarity...... 115

Figure 3.39. Dihydropentagynine (19) ...... 118

xiii

LIST OF SCHEMES

No Title Page No

Scheme 1.1. General procedure for obtaining active metabolites from plants ...... 4

Scheme 1.2. Possible biogenesis of delphenine ...... 32

Scheme 1.3. Possible biogenesis of C20 diterpenoid alkaloids ...... 35

Scheme 1.4. Proposed biogenetic pathway to miyaconitine which is supposed to be an intermediate in transformation of atisine to hetisine ...... 36

Scheme 2.1. Fractionation of A. heterophyllum, A. laeve & D. denudatum ...... 39

Scheme 2. 2. Isolation of alkaloids from A. heterophyllum Wall...... 41

Scheme 2.3. Isolation of alkaloids from A. laeve Royle ...... 43

Scheme 2.4. Isolation of diterpenoids alkaloids from D. denudtum Wall ...... 44

xiv

LIST OF ABBREVIATIONS

BB Broad (decoupled) band

CC Column chromatography

COSY Correlated spectroscopy

DEPT Distortionless enhancement by polarization transfer

EI-MS Electron impact mass spectrum

HMBC Heteronuclear multiple bond connectivity

HMQC Heteronuclear multiple quantum coherence

HR-EIMS High resolution electron impact mass spectrum

IR Infrared spectrophotometry

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

NMR Nuclear magnetic resonance

TLC Thin layer chromatography

UV Ultraviolet

XRD X-ray diffraction

CDCl3 Deutreated Chloroform

Chloroform CHCl3

ACE Acetone

DEA Diethyl Amine

Hex n- Hexane

AChE Acetyl Cholinesterase

BChE Butyryl Cholinesterase

NCEs New Chemical Entities

MeOH Methanol

MeOD Deutreated Methanol

GGPP Geranylgeraniol pyrophosphate

xvi

CHAPTER-1

INTRODUCTION

Chapter-1 Introduction

INTRODUCTION

1.1 Plants as a source of medicine

Medicinal plants have served humankind as a source of medicinal agents throughout history. It is believed that 50% of all drugs used in clinical treatment are natural products, and its derivatives [1]. Natural products obtained from plants can be used in new drug development of the pharmaceutical industry [2]. The chemical and biological explorations of medicinal plants have yielded varieties of drugs which are used for treating diseases in humans. Additionally, it opens a window for the growth of modern synthetic organic chemistry and the development of medicinal chemistry as a key route for the innovation of novel and more effective therapeutic agents [3].

Various types of extraction techniques have been used to isolate and purify pharmacologically active natural products that can be used as a remedy for the treatment of different diseases in human beings as well as in livestock [4-6]. Natural compounds obtained from plants have much complicated structural formulae and remain an important source for new drugs, new chemical entities (NCEs) and new drug leads [7-9].

According to a survey report (2001-2002), nearly one fourth of the world’s best marketed drugs were either natural products or their derivatives [7]. About 28% of NCEs from 1981 to 2002 were products obtained from plants or their derivatives (Figur 1.1). In another survey which was carried out during this period, it has been reported that 20% of NCEs were synthetic products derived from natural products [9]. According to these reports research on natural products accounts for nearly 48% of the NCEs reported from 1981 to 2002. Moreover the documented natural products obtained from plants have been used as starting material in various organic syntheses with different structures and multiple stereo centers. [10-13].The different structural features (e.g., aromatic rings, chiral centers, complex ring system, and number ratio of hetero atoms) present in

1 Chapter-1 Introduction natural products have proved to be vital for the efforts of drug discovery [14-16]. Many synthetic and medicinal chemists have shown keen interest in the development of natural products and its derivatives which resembles the structural characteristics of natural products with the compound- generating potential of combinatorial chemistry [17-21]. Many new natural products obtained from plants not only act as new drugs but can be converted into useful medicine after processing and further necessary modifications. Sometimes already known compounds show new pharmacological activities and also give important drug directions [22]. All these findings clearly show that plant-derived products are still important as therapeutic agents and hence will be used for drug designing, synthesis, and semi-synthesis of novel drugs for treating different disorders.

Etoposide: R = CH H 3 Teniposide: R= R O S O O OH N HO O O OH O O N O O Camptothecin O O

H3CO OCH3 N OH HO O OH N AcO H N H3COOC

PhCOHN O OH

H CO N OCOCH O 3 3 O H Ph AcO OH Vincristine: R= CHO R OCOCH3 OOCPh OH Viblastine: R= CH3 Taxol

Figure 1.1. Examples of plant-derived anticancer drugs

2 Chapter-1 Introduction

1.2 Strategies in the search of new natural compounds

To carry out the phytochemicals and biological evaluation of a particular medicinal plant, it is extremely necessary to know that which plant is to be selected and for what type of biological activity it will be screened. The selection criteria of plants that contain potent and new phytochemicals consist of five principle approaches i.e (a) the random approach, (b) the taxonomic approach, (c) the phytochemical approach, (d) the ethnomedicinal approach and (e) the information-managed approach. In random approach, all accessible species of plants are collected, without any previous scientific knowledge and experience. In taxonomic approach, plants of a specific genus or family are selected and sought out from their natural habitat of diverse location.

In the phytochemical (chemo-taxonomic) approach, plant is selected on the basis of particular class of compounds (targeted isolation). Plants expected to produce related class of compounds are collected. In view of the above approaches, it can be stated that the taxonomic and the phytochemical approaches are closely related and cannot be obviously divided into two. In the ethnomedicinal approach, plant for scientific research is to be preferred on the basis of information obtained for the medicinal use of the plant in the given locality [23].

Most of the traditionally used medicines clearly represent real medicinal properties.

Actually, 74% of plant based medicines were investigated as a result of empirical use [24]. Field work carried out on the use of traditional medicines should be significant supplementary tool in the selection of plants for further studies. Most of the medicinal plants used as remedy are not well described concerning their phytochemical composition and their biological properties [25].

Information-managed plant selection organizes taxonomic, ethnomedicinal, biological and phytochemical information to launch a useful plants list for the purpose of collection of specific plants. The data obtained is compiled through computerized databases such as NAPRALERT

3 Chapter-1 Introduction

(Natural Products Alert) and a specific relational database on natural products, based on systematic literature searches [26].

For each of the approaches, the most useful method of proceeding involves evaluating the collected plant material in a variety of bioassays [23]. Depending on the objective of the study, a specific assay either for a certain activity or a general screening is accomplished. The active fractions obtained were subjected to bioassay guided fractionation over column silica gel in the column chromatography which on repeated flash column chromatography give active components.

Plants, besides their use in the medical field as a source for pure, chemically active component, also find their application as active extracts containing a wide range of chemical constitutents [27]

(Scheme 1.1).

Random Taxanomic Ethnomedical Phytochemical Information-managed approach approach approach approach approach

Plant Material Toxicology

Extraction

Plant Extract Bioassays Separation Bioassays

Pure Compound

Synthesis Strucutre Elucidation Structure Modification Bioassays

Toxicology

Scheme 1.1. General procedure for obtaining active metabolites from plants

4 Chapter-1 Plant Introduction

1.3 Ranunculaceae

Ranunculaceae is a group of flowering plants, also called buttercup or crow foot family.

The family comprises of 50 genera and about 2000 species, the most important genera are

Delphinium, Consolida, Aconitum Thalictrum, and Clematis [28]. In Pakistan, this family is comprised of 22 genera and 114 species [29]. The flowers are generally radially symmetrical and cross-sexual, yet Aconitum and Delphinium are respectively symmetric whereas Thalictrum has unisexual flowers. The calyx and corolla are poorly differentiated, but by no means always. The quantity of perianth parts shifts from 3 to numerous and can be isolated from one another. The gynaecium comprises of few (3-7) to numerous straight forward carpels. Actaea has a gynoecium of 2 united carpels, with parietal placentation; its natural product is a berry. Nigella, a developed species has a gynoecium of 5 united carpels, and structures a container at development. The ovary is always superior [30].

1.3.1 The Genus Aconitum

Depending on the taxonomic approach, the Aconitum genus of the Ranunculaceae family is divided into 60–350 species [31]. Aconitum is a circumboreal arctic and alpine genus that extends into lower latitudes where there is suitable mesic habitat at high altitude along the North-

South chains of mountains. Aconitum species are mostly distributed in Asia, with some species also found in North America and Europe [32].

The species are perennials, with stout leafy stem and tuberous stock with brown, fragile roots. The leaves are alternate and palmately or pedately divided, and the segments are dentate or lobed. The flowers are zygomorphic, in a terminal raceme or racemose panicle. The posterior parts of the 5 petaloid perianth-segments form a large erect hood (helmet). The posterior pair of the 2–

5 Chapter-1 Plant Introduction

10 honey-leaves is included in the helmet with long claws and limbs prolonged into nectar- secreting spurs; others are very small or absent. The stamens are numerous; the 2-5 follicles are free or shortly connate at the base [33]. A great deal of variation occurs in all species, perhaps as a result of hybridization. They do not in general fall into a recognizable morphological or geographical pattern, though many local populations can be recognized and have been given specific or sub specific ranks. Species belong to different parts may be different morphologically, but linked by a series of intermediate races. The Aconitum genus consists of about 7 species which are found at elevation of 2800-4000 m above sea level in the Northern area of Pakistan and

Kashmir [34]. The plant materials of A. heterophyllum Wall and A. laeve Royle were collected during flowering season from their natural habitate for the present research work.

1.3.2 Aconitum heterophyllum Wall.

It is one of the representative specie of family Runanculaceae and is found at the height of

2800-4000 m above ocean level. The plant is perenial or biennial herbs disseminated around the world. Because of the cardiotonic potency of the plant, it is locally known as "Sarba waley". The tubers are utilized for treating fever, gout and cough. It is likewise utilized as a tonic, antiperiodic, astringent, anthelmintic, diarrheal, epigastric pain and cold [35].

1.3.3 The Aconitum laeve Royle

It is one of the well-known specie of the genus Aconitum, native to North West Himalaya.

It is distributed from Chitral to Kashmir and then to North India. The plant usually grows to a height of 1.5 m, with elongated roots and erect stem. The inflorescence is composed of branched racemes up to 50 cm with leafy apices. The whole plant is considered to be toxic [36]. The symptoms after ingestion are increased salivation, vomiting, nausea, diarrhea and abdominal pain

6 Chapter-1 Plant Introduction as well as blurred vision, weakness and dizziness. Cardiovascular symptoms include cardiac arrhythmia and paralysis of the heart [37-40].

1.3.4 Systematic Position

Kingdom: Plantae

Subkingdom: Tracheobionta

Superdivision: Spermatophyta

Division: Magnoliophyta

Class: Magnoliopsida

Subclass: Magnoliidae

Order: Ranunculales

Family: Ranunculaceae

Genus: Aconitum

Species: Aconitum heterophyllum Wall and A. laeve Royle [41-43].

1.3.5 Traditional importance of Aconitum Species

Some species of Aconitum are commonly used in traditional medicines, especially after processing, to avoid its toxic effects. These processed drugs are used as analgesics and cardiotonics. The unprocessed tubors are toxic and are used only externally as local anesthetics

[44-45]. The conventional Japanese medicine bushi (processed roots of certain Aconitum species) is used for relieving muscular pain [46]. In Korea, the roots of A. koreanum are used for muscular pain [47]. In Vietnam the unprocessed tubers of A. fortunei are used externally for its analgesic

7 Chapter-1 Plant Introduction effect [48]. Vatsnabhi is an Ayurvedic medicinal product contains the processed tuberous roots of different Aconitum species and is used as a diuretic, diaphoretic, antidiabetic, antiphlogistic and antipyretic [49]. .A. heterophyllum is comparatively less toxic among various Aconitum species and is used in Ayurvedic as well as in Tibetan medicine to treat abdominal pain, fever and cough

[50]. In European countries extracts of Aconitum species were used externally as poisons for killing lices and parasites [51].

1.3.6 Chemistry of the Genus Aconitum

The phytochemicals assessment of Aconitum species were planned in search of compounds, particulary diterpenoids with significant physiological effects. Previous experimental works demonstrate that the significant biological activity of the plants is attributable to their alkaloids content. Aconitine was the first isolated and identified alkaloid from Aconitium by Geiger in 1833 [52]. Jacobs in 1963 worked on alkaloids of Aconitum and published more than 20 research articles on Aconitum [53]. The complete structure of aconitine, including the absolute configuration at the asymmetric centres was determined by means of X-ray crystallography [54].

Careful chemical analysis demonstrated that the Aconitum species accumulate mainly alkaloids with a diterpene structure.

1.3.7 Literature survey on A. heterophyllum Wall and A. laeve Royle.

Previously, a large number of diterpenoid alkaloids were isolated from plants of higher altitude. Plants of genus Aconitum, Consolida, and Delphinium belong to the family

Ranunculaceae, have proven to be the richest sources of diterpenoid alkaloids. Aconitum species produce diterpenoid and nor-diterpenoid alkaloids that are generally of the aconitine and lycoctonine-types [55]. The roots of A. heterophyllum are used for the treatment of rheumatic and neuralgic pain [56]. Lycaconitine, isolated from different Aconitum species such as A was used as

8 Chapter-1 Plant Introduction an effective remedy against multi-drug resistant cancers. Aconitum plants are extensively used in

Indian and Chinese traditional systems of medicine [57].

Previous phytochemical research on A. heterophyllum Wall and A. laeve Royle resulted in the isolation of several nor-diterpenoid and diterpenoid alkaloids, including 6- dehydroacetylsepaconitine, 13-hydroxylappaconitine, lycoctonine, delphatine and lappaconitine

[58], heterophyllinine-A, heterophyllinine-B [57], isoatisne, heteratisine [59], hetisinone [60]. 6- benzoyl heteratisine, 20α atisine, 20β atisine, atidine, heterophyllisine, heterophyllinine [61-63].

14,18-dimethoxygadesine, α-methoxyanthranoyllycoctonine [64]. Swatinin-I [65]. 8- methyllycaconitine, 14-demethyllycaconitine, N-deethyllycaconitine-N-aldehyde, lappaconitine, lapaconidine and lycoctonine (Table 1.1) [66].

1.4 The Genus Delphinium

The name of the genus Delphinium (Ranunculaceae) was derived from the fanciful resemblance of the unopened flower bud to a little dolphin. Delphinium is a large genus of family

Ranunculaceae, which represents a group of very beautiful annual, rarely biennial and perennial plants, commonly called as Larkspur. The genus has about 270 species, which are found in the

North temperate region. The original or wild types are natives of California, Siberia, Syria and

India. The genus is represented by 16 species in Pakistan, with no known critically threatened species [67].

9 Chapter-1 Plant Introduction

1.4.1 Systematic Position

Kingdom: Plantae

Subkingdom: Tracheobionta

Superdivision: Spermatophyta

Division: Magnoliophyta

Class: Magnoliopsida

Subclass: Magnoliidae

Order: Ranunculales

Family: Ranunculaceae

Genus: Delphinium

Species: Delphinium denudatum Wall. [43].

1.4.2 Delphinium denudatum Wall.

The plant is 50–80 cm long found at the height of 1500-2600 m in Northern zones of

Pakistan. The stem is quite expanded, subglabrous to strigose or spreading pubescent in the upper part. Petioles of lower surrenders over to 15 cm, upper ones much shorter, leaf cutting edge of basal leaves 5-15 mm wide, adjusted, 3-5 separated into comprehensively obovate fragments, portions pinnately and divertically laciniate into oval flaps or teeth 2-3 mm wide. Cauline leaves comparative yet littler. Inflorescence paniculate, of few-bloomed racemes. Bracts 5-15 mm, direct.

Pedicels 10-40 mm, climbing, bracteoles joined close to the center of the pedicel. Sepals blue to violet, upper sepal 12-13 x 6-7 mm, praise, intense, pubescent, goad 14-15 mm long, 3.5 mm wide

10 Chapter-1 Plant Introduction at the base, parallel sepal 13 x 7-8 mm, elliptical applaud, adjusted, pubescent on the midline, lower sepals 14-15 x 7 mm, elongated obovate, adjusted, and pubescent. Upper petal white with pale blue zenith, appendage 8-9 mm, glabrous, 2-dentate, sideways, goad 13-15 mm, lower petal blue or violet, 6 mm long, extensively elliptic, adjusted, parted nearly to the center, paw 5 mm.

Stamens 5-6 mm. Follicles 3, 10-16 x 3-3.5 mm, inadequately strigose or subglabrous, style 2-3 mm. Seed obpyramidal, 1 mm long, dull, scales unpredictably masterminded, generally long [68].

1.4.3 Ethnobotanical importance of the Genus Delphinium

Plants belong to the genus Delphinium contain bio-active and structurally complex diterpenoid and norditerpenoid alkaloids [69-73]. Many species of Delphinium have been reported to be toxic to cattle. It is estimated that 12% of the cattle death in the Western United State is caused by Larkspur plants [74]. It is believed that the relative toxicity among varieties of larkspurs depends on the contents of the diterpenoid alkaloids [75]. Plants bearing norditerpenoid alkaloids are reported to be used as cardiotonic, sedatives, febrifuges and analgesics [76]. Majority of the species belong to Delphinium genus are utilized in the production of herbal medicine that can be used as therapeutic agent in different diseases. Some of the species of Delphinium are reported to be used as insecticides, antirheumatic and for the treatment of sciatica. In Turkey, various plants of genus Delphinium are applied externally for rheumatic pain and against body lice [77]. It is considered as one of the essential medicine used in indigenous medicinal system of India. The entire plant has been recognized to be useful in a variety of ailments such aspiles and toothache, brain diseases, fungal asthma, aconite poisoning, infection, as analgesic and astringent [78]. Some species of Delphinium have been used for their antifungal, antioxidant and anti-epileptic activities since long time [79-80]. Anticonvulsant activities of FS-1 fraction obtained from Delphinium species have been reported by various research groups [81-83].

11 Chapter-1 Plant Introduction

1.4.4 Literature survey on Delphinium denudatum Wall.

The plant D. denudatum Wall., selected for this phytochemical and biological investigation belongs to the family Ranunculaceae. The common name of the genus Delphinium is “Jadwar” which is widely distributed in Dir, Swat, Chitral, Azad Kashmir, and Baluchistan [84-85], in the

Himalayan and Garhwal area of India [86]. It is widely used as cardiotonic and sedative in the local systems of medicine [76].

Phytochemicals exploration on D. denudatum Wall., have yielded various C19 and C20 diterpenoid alkaloids, delphatambine, 8-acetylheterophylisine [79]. Denudatin, panicutine, condelphine, isotalatizidine and delnudine [87-89] (Table 1.2).

12 Chapter-1 Plants Introduction

Table 1.1. Summary of the previous phytochemical studies on A. heterophyllum Wall. and A. laeve Royle.

Mol. Formula/ Sr. No. Name of Compounds Structure References Mol. Mass

CH2 C22H33NO2/

1 Heterophyllinine-A OH 57 N 343

CH2 C24H35NO4/

2 Heterophyllinine-B OCOCH3 57 N 568

O HO

13 Chapter-1 Plants Introduction

OCH3 OCH3 OH OCH3 C32H42N2O10/ OH N OH H 3 6-Dehydroacetylsepaconitine O O 614 58 C O H N CH3

OH OCH OCH 3 3 OCH 3 C31H43N2O9/ OH 13-Hydroxylappaconitine N 4 OH 58 H H O 587 C O H N CH3 O

OH Lycoctonine OCH C25H41NO8/ OCH 3 3 OCH 3 H 5 N 58 583 OH OH OCH3 HO

14 Chapter-1 Plants Introduction

OCH3 OCH3 OCH3 H C26H43NO8/ 6 Delphatine N 58

OH OH 497 OCH3 H3CO

OCH3

OCH3 OCH3 OH C32H44N2O8/ N OH 7 Lappaconitine H 58, 65 O 584 C O

NHCOCH3

15 Chapter-1 Plants Introduction

CH2

C22H33NO2/ Isoatisne 8 OH 59

N 343

OCH3 C O C22H33NO5/

9 Heteratisine 59 N OH 391

HO CH2 C20H25NO3/

10 Hetisinone O 60 N 327

16 Chapter-1 Plants Introduction

OCH3 C O

C29H27NO6/ N OH 11 6-benzoylheteratisine 61 o C O 495

CH O H 2 C22H33NO2/ 20

12 20α-Atisine OH 61 N 343

CH O H 2 C22H33NO2/

13 20β-Atisine OH 61 N 343

17 Chapter-1 Plants Introduction

CH2 C22H33NO3/

15 14 Atidine OH 62 N 359

O HO

O C21H31NO4/ OH C O

15 Heterophyllisine 63 N 361 OH

O C22H33NO4/ OCH3 C O

16 Heterophyllinine 63 N 375 OH

18 Chapter-1 Plants Introduction

OCH3

OCH3 C25H39NO7/ 14,18-dimethoxygadesine 17 N 465 64 (Swatinine-A) OH

O OH OCH3 H3CO

OCH3 OCH3 OCH3

N C32H46N2O8/ α-methoxyanthranoyl OH H OH 18 lycoctonine OCH3 586 64 O C O (Swatinine-B) NH2

OCH OCH 3 3 OH

Foresticine C24H39NO6/ N 19 OH 437 64 H OH H3CO

19 Chapter-1 Plants Introduction

OCH OH 3 OH C24H39NO6/

20 Neoline N 437 64 OH H OCH3 H3CO

OCH OH 3 OCH3

OCH3 C32H46N2O8/ Delvestine H OH 21 OCH3 586 64 O C O

NH2

OH OCH3 OCH 3 C25H39NO6/

22 Chasmanine N 451 64 OH

H OCH3 H3CO

20 Chapter-1 Plants Introduction

OCH3 OCH3 OCH3 OH C25H41NO8/

23 Swatinine-1 N 483 65 OH H OH OCH3 HO

OCH3 OCH3 C26H43NO8/ OCH3 H 24 Delphatine N 497 65 OH OH OCH3 H3CO

OCH3

OCH3 17 H OCH3 OH N C32H44N2O9/ 25 Puberanine H OH 65 O OH C O 585

NHCOCH3

21 Chapter-1 Plants Introduction

OCH3

OCH3 OH OCH3 OH

N C32H44N2O9/ 26 N-acetylsepaconitine H OH 65 O C O 600

NHCOCH3

CH3

O O

4-[2-(methoxycarbonyl) H C12H13NO5/ 27 65 N anilino]-4-oxobutanoic acid 251

O O OH

22 Chapter-1 Plants Introduction

OCH3 OCH3 H OCH3 O H N N-Deethyllycaconitine-N- H OH H C35H44N2O11/668 28 aldehyde O OH 66 OCH3

C O O

23 Chapter-1 Plants Introduction

Table 1.2 Summary of the previous phytochemical studies on Delphinium denudatum Wall. S. Mol. Formula/ Name of compound Structure References No. Mol. Mass

CH2 C23H27NO5/

H3COCO 397 1 Delphatambine 79 N

O O

O OCH 3 C O C28H29NO5/ 79 2 8-Acetylheterophylisine 470 N OCOCH3

24 Chapter-1 Plants Introduction

HO CH2 C22H35NO2/

3 Denudatine 86 OH N 345

O CH 2 C25H37NO4/ Panicutine H3COCO 4 87 N 415

OCH3 OH OCOCH3 C25H39NO6/

5 Condelphine N 88 OH H 449

H3CO

25 Chapter-1 Plants Introduction

OCH3 OH OH C23H37N2O5/ Isotalatizidine

6 N 88 OH 407 H

H3CO

O C6H6O/

3-Hydroxy-2-Methyl-4-Pyrone OH 7 88 126

O CH3

C20H25NO3/ CH2

8 Delnudine HO 89 327 N

26 Chapter-1 Diterpenoid Alkaloids

1.5 Diterpenoid alkaloids

Genus Aconitum, Delphinium and Consolida are considered as rich sources of C19 and C20 diterpenoid alkaloids having valuable biological activities [90-92]. The biogenesis of diterpene alkaloids has not been completely clarified; they are possibly derived bases from tetra or penta cyclic diterpenes having nitrogen atom of ethylamine, methylamine or β-aminoethanol linked to

C-17 and C-19 in the C19 diterpene alkaloids and to C-19 and C-20 in the C20 diterpene alkaloids, forming a substituted piperidine ring [93]. Diterpene alkaloids belong to the class of the pseudo alkaloids, since they appear to be derivatives of the amination of nitrogen-free terpenes. Rarely, the nitrogen atom is not a member of the ring system (e.g. Erythrophleum alkaloids with the nitrogen atom present in an aliphatic chain) [94]. On the basis of number of carbon atoms in the basic skeleton, the N-heterocyclic diterpene alkaloids are grouped into C20, C19 and C18 diterpene alkaloids.

1.6 Classification of diterpenoid alkaloids

Diterpene alkaloids are generally divided into three classes:

 A group of relatively simple C20–alkaloids having little oxygenation.

 A group of C19–alkaloids substituted by many hydroxyl or methoxyl groups where some

of the hydroxyl groups are esterified.

 A group of C18 alkaloids, whose carbon framework contains 18 carbon atoms due to

absence of C-18.

1.6.1 C20 diterpene alkaloids

C20 diterpene alkaloids are not extensively oxygenated; they usually contain 2–5 oxygen functions which are present in some alkaloids as monoester of acetic acid or benzoic acid. In most

27 Chapter-1 Diterpenoid Alkaloids

cases, methoxy group is absent. The presence of an exocyclic methylene is characteristic, many of

which have a secondary hydroxy function in an allylic position. It is hypothesized that C20

diterpene alkaloids are derived biogenetically from pimaradienes via rearrangements [95]. As

compared with the C19 diterpene alkaloids, the structural diversity of the C20 diterpene alkaloids is

more distinctive. The C20 diterpenoid alkaloids often occur as esters (acetate, benzoate) and are

relatively non-toxic. Structurally they are further subdivided into veatichin, atisine and delnudine

(Figure 1.2). Very few alkaloids are based on the parent ring system, since many skeletal

variations are known.

I. Atisine-type

These are comparatively simple and non-toxic alkaloids, which are not extensively

oxygenated and contain one methoxyl group [96].

II. Veatchine-type

Veatchine-type alkaloids possess veatchine-type skeleton, which differs from the atisine-

type skeleton in the formation of ring D and ring E. A striking similarity is found in the chemistry

of atisine and veatchine-type alkaloids inspite of the fact that they belong to two different families

[97-99].

III. Delnudine-type

Delnudine-type alkaloids are based on the hetisine skeleton and may possibly be

derivatives of hetisine or veatchine, in which further modification of the ring system has occurred

[100]. It was proposed that N-C-6 bond formation is based on the structures of the natural

alkaloids; miyaconitine and miyaconitinone [101].

28 Chapter-1 Diterpenoid Alkaloids

13 17 CH 12 13 17 17 2 CH CH2 12 11 2 11 12 11 13 20 16 20 16 20 16 14 14 14 HO 15 15 1 9 15 1 9 1 9 2 10 8 2 10 8 2 10 8 R N N R N 3 5 7 3 7 3 5 7 4 6 4 4 6 5 6 19 19 18 18 19 18 OH Atisine-type Veatchine-type Delnudine-type

Figure 1.2. Basic skeletons of C20 diterpenoid alkaloids

1.6.2 C19-diterpenoid alkaloids

C19 diterpene (norditerpene) alkaloids are based on hexacyclic carbon skeleton. These compounds are usually, highly oxygenated, characteristically with at least 5 oxygen functions, 1 or 2 of which are rarely esterified with aromatic acid or acetic acid [102]. These alkaloids may be regarded as aconitane (9) derivatives. It is hypothesized that the biosynthesis of C19 diterpene alkaloids begins from C20 precursors (atisine or napelline) by rearrangement or Baeyer-Villiger oxidation. These are the highly toxic alkaloids and 2-5 mg may prove to be lethal in human. These alkaloids have been divided into four categories as defined below [103].

I. Aconitine-type

This group of alkaloids possesses aconitine skeleton (Figure-1.3) in which the C-7 position is not substituted by any other group except hydrogen, for example, aconitine, delphinine and condelphine.

29 Chapter-1 Diterpenoid Alkaloids

II. Lycoctonine-type

This group of alkaloids possesses lycoctonine skeleton, in which the C-7 position is always oxygenated, e.g. lycoctonine, browniine, delcosine and dictyocarpine.

III. Pyrodelphinine-type

This group of alkaloids possesses skeleton in which there is a double bond (C=C) between

C-8 and C-15, e.g falaconitine and mithaconitine.

IV. Heteratisine-type

This group of alkaloids possesses heteratisine skeleton (Figure 1.3), in which lactone functionality is observed in the ring C of the molecule, e.g. heteratisine and heterophylline [103].

16 13 13 16 12 17 14 17 12 14

10 10 1 9 1 9 2 11 15 2 11 15 N 8 N 8 3 5 3 5 7 7 4 4 6 OH 6 OH

19 18 19 18 OH Aconitine-type Lycoctonine-type 16 16 13 13 O 12 17 14 12 17 14 O 10 9 10 1 1 9 15 2 11 N 15 2 11 8 N 8 3 5 5 7 3 4 4 7 6 6

19 19 18 18 Pyrodelphinine-type Heteratisine-type

Figure 1.3. Basic skeletons of C19 diterpenoid alkaloids

30 Chapter-1 Diterpenoid Alkaloids

1.6.3 C18 diterpene alkaloids

The C18 diterpene (bisnorditerpene) alkaloids [e.g. lappaconitine] are derivatives of the C19 diterpenoids, contain 18 carbon atoms skeleton due to the absence of C-18. In these compounds,

C-4 is substituted with hydrogen atom, or an ester group or a 3, 4-epoxide group. It is assumed that the removal of C-18 occurs due to Baeyer Villiger oxidation type reaction. Another possible mechanism of biosynthesis is hydroxylation of the methyl group at C-18, oxidative conversion of the hydroxymethyl group into a carboxyl group with successive decarboxylation, and also of 3-4 double bonds epoxidation [95].

1.7 Biosynthesis of diterpenoid alkaloids

Biogenetically diterpenoid alkaloids are derived from diterpenoids. It is therefore important to understand the biogenesis of parent diterpenoids skeleton. Diterpenoids are biogenetically derived from geranyl geraniol pyrophosphate (GGPP), an important precursor in the isoprenoid biosynthetic pathway. The cyclization of GGPP leads to the formation of mono-, bi-, or tri-cyclic systems depending on the conformation of the substrate, which is determined by the enzyme cyclase. The absolute stereochemistry of the cyclic diterpenes is also dictated by the nature of the enzyme, cyclase.

31 Chapter-1 Diterpenoid Alkaloids

O OH HO OH OPP [2-14C] Mevalonic acid DMAPP OCH OCH3 3

OCH3 OCH3 N + N O O H O OH OH

OCH3 OCH3 OCH3 N O O OH Delpheline

Scheme 1.2 Possible biogenesis of delphenine Mevalonic acid contains six carbon atoms, one of which must be lost to form the iso- pentane unit. Phosphorylation of mevalonic acid first produces mevalonic acid-5 phosphate, which is followed by a second phosphorylation to give mevalonic acid-5-pyrophosphate. The biosynthetic incorporation of suitably labeled mevalonic acid into a natural product is frequently utilized as a tool to identify whether the substance is of terpenoidal origin or not. However, biosynthesis of the diterpenoid alkaloid, delpheline (Scheme 1.2) emphasizes the need of caution in conducting the experiments with mevalonic acid.

In the biosynthetic studies of diterpenoid alkaloids, [2-14C] mevalonic acid was applied to the cut stems and cut ends of leaf stalks of Delphinium elatum. It was found that [2-14C]-mevalonic

14 acid was not significantly incorporated into the alkaloid, delpheline, whereas L-[ CH3] methionine was incorporated (0.025%) into delpheline under the same application of the techniques using intact plants at the same stage of the growth (Scheme 1.2). 88% percent of the

32 Chapter-1 Diterpenoid Alkaloids label entered into the methoxy groups, 11% into the N-ethyl group and less than 5% was found in the methylenedioxy group [104].

Similarly, when [1-14C]- and [2-14C]-acetates and [2-14C] mevalonate were administered to the stem of intact D. brownii prior to blooming, the label was seen to be poorly incorporated into the alkaloids lycoctonine and browniine to the extent of only 0.002, 0.003 and 0.006%, respectively. The distributions of the radioactive label in other alkaloids were not determined. It was suspected that the primary site of alkaloid biosynthesis in D. brownii is the roots. The failure of Herbert and Kirby to observe the incorporation of mevalonic acid into the delpheline in D. elatum could probably be due to the transport problems (cell-membrane impermeability).

Moreover the incorporation of labeled methionine into delpheline reflects the existence of a metabolic equilibrium between the methylated and demethylated alkaloids in the leaves.

Alternatively, it is possible that the methylation occurs in the leaves, while the alkaloid is biosynthesized elsewhere in the plant [105].

The role of [2-14C]-glycine and [2-14C]-mevalonate as the biosynthetic precursors was investigated by administering them directly into the internodes of the plants of D. Ajacis. The observed incorporation of [2-14C]-glycine into delcosine (Figure 1.4) was found to be 0.09%, while that of [2-14C]-mevalonate was 0.04%. The incorporation of mevalonate into the diterpenoid alkaloids supported the hypothesis that the isoprenoid biosynthetic pathway is involved in the formation of diterpenoid alkaloids. Glycine is presumably incorporated intact into the preformed oxygenated diterpene skeleton to yield the N-ethyl side chain. Glycine could afford either acetate, by well-established biochemical reactions, or serine, which could be incorporated to the methyl groups of delcosine and O-acetyldelcosine [106].

33 Chapter-1 Diterpenoid Alkaloids

OCH3 HO OR

N OH OH OCH3 H3CO

Figure 1.4. Delcosine (R= H) & acetyl delcosine (R= Acetyl)

The possible pathway by which the nitrogen atom enters the skeleton remains obscure. It may be presumed that a di-aldehydic precursor accepts an ammonia molecule from a glutamine or lysine residue, as appears to be the case for the triterpenoid steroidal alkaloids, in a reductive amination process [107].

It has been suggested that the N-6 bridge found in hetisine, delnudine, miyaconitine etc. is probably formed by the displacement of a 6-pyrophosphate group. The facile formation of the bridge has already been demonstrated in ajaconine chemistry [108]. The mobile equilibrium between 6-keto-derivatives and the corresponding carbinolamines in hetidine (Figure 1.5) (A. heterophyllum), delnudine (D. denudatum) and kobusine methiodide proved the strain-free nature of the hexacyclic skeleton.

34 Chapter-1 Diterpenoid Alkaloids

CH2

Figure 1.5. Hetidine

The seeds of D. denudatum have afforded another skeletal variant, delnudine. It was postulated (Scheme 1.3) that the delnudine might be biogenetically derived by the concerted rearrangement of hetisine followed by oxidation to the corresponding carbinol amine [100].

H O OH CH2 HO CH2 HO HO N N

Delnudine Hetisine

Scheme 1.3. Possible biogenesis of C20 diterpenoid alkaloids

Miyaconitine is the major alkaloid isolated from Aconitum miyabei. The location of the carbonyl group on the bicyclo [2, 2, 2] octane system of A is of biosynthetic interest. It has been reported that alkaloid A may be regarded as a biosynthetic intermediate in the transformation from the atisine skeleton (C20H31N) to the hetisine skeleton (C20H27N) in Aconitum alkaoids

(Scheme1.4) [109].

35 Chapter-1 Diterpenoid Alkaloids

CH CH 2 2 O OH OH O

+ N N

O O (B H (A O O CH2 CH2 HO HO OH N N OH

Scheme 1.4. Proposed biogenetic pathway to miyaconitine which is supposed to be an intermediate in transformation of atisine to hetisine

N-Methylation via S-adenosyl methionine is a common biochemical reaction, whereas the corresponding N-ethylation reaction is still largely un-established. It is postulated that C2 unit might be transferred in the form of acetaldehyde to a secondary amine with subsequent elimination of water and reduction of the resulting imminium ion. In the alternative pathway, the N-ethyl group might arise from alanine, whereas the N-hydroxyethyl side chain seen in salts of veatchine, garrine and atisine, could similarly be obtained from the serine. Definitive proof of these ideas is, however, still lacking.

CHAPTER-2

EXPERIMENTAL

Chapter-2 Experimental

EXPERIMENTAL

2.1 General Experimental Conditions

2.1.1 Physical constants

Melting points (corrected and uncorrected) were determined in glass capillary tubes using melting point and an electro thermal melting point instrument, Model BioCote stuart-SMP10

Japan. Optical rotations were noted on Schmidt Haensch Polartronic D polarimeter.

2.1.2 Spectroscopic techniques

All the isolated compounds were characterized using different spectroscopic techniques like IR, MS (EIMS), 1D-NMR (1H, 13C and DEPT) and 2D-NMR (HSQC, HMBC, COSY and

NOSY). Infra-Red (IR) spectra were recorded on а Jаsco-320-A spectrophotometer in KBr. NMR analysis of isolated compounds was conducted using 300, 500 & 600 MHz Bruker spectrometer.

Mass spectra were obtained using MS (EIMS).

2.1.3 Column chromatography

Based on TLC profile of basic alkaloid crude (fraction B) with retrosine standard (Sigma,

CAS number 480-54-6) and Dragendorff’s reagent test, open Column Chromatography (CC) of fraction B was performed to isolate C19 and C20 diterpenoid alkaloids from of A. heterophyllum

Wall, A.laeve Royle and D. denudatum Wall.

2.1.4 Materials for chromatography

(i) TLC was performed on pre coated silica gel 60 F-254 glass plates, 5x 10 cm, 0.25 mm thick (E. Merck, Germany). The solvent systems used were: n-hexane- Acetone-DEA

(8:2:10), (9:1:10), (7:3:10) and (6:4:10), unless otherwise indicated.

Chapter-2 Experimental

(ii) Flash Column Chromatography (FCC) was carried out using silica gel HF-254, particle size 0.04-0.063 mm, 230-400 mesh, (Fluka AG, Buchs SG).

2.1.5 Solvents

The commercially available solvents such as n-hexane, chloroform, ethyl acetate, acetone and methanol were used after distillation for extraction processes, chromatography and crystallization whereas spectroscopic grade solvents were used for spectral measurements.

Deuterated solvents (Aldrich) were used for NMR analyses.

2.2 Plant materials

The plants of A. heterophyllum Wall (8 kg, aerial parts), A. laeve Royle (3 kg, tubers) and

D. denudatum Wall (5 kg, aerial parts) were collected during flowering seaon from District Swat and Lowari Top Dir (U) of Khyber Pakhtunkhwa, Pakistan. The plants were identified by Prof.

Mehboob Ur Rahman, Chairman Department of Botany, Govt: Afzal Khan Lala Post Gradute

College Matta District Swat. Voucher specimens (H.UOM.BG-158, H.UOM.BG-159 &

H.UOM.BG-160) were deposited in the herbarium, University of Malaknd, Chakdara Dir (L).

Plants materials were shade dried for two weeks and exact weight of the dried plants were recorded. The dry plants materials were grinded to powder with the help of grinder.

2.2.1 Extraction

Each shade dried plant; of A. heterophyllum (8 kg), A. laeve (3 kg) and D. denudatum (5 kg) was macerated with MeOH (3 x10 L) at room temperature. The combined MeOH extract was concentrated by rotary evaporator to get thick gummy crude of MeOH fractions (720 g, 300 g &

600g) respectively.

Chapter-2 Experimental

2.2.2 Crude alkaloids fractionation

The crude MeOH fractions (720 g , 300 g & 600 g of A. heterophyllum, A. laeve and D. denudatum) respectively were dissolved in 1.5 liter of 0.5 N H2SO4 solution, shake for one hour, filtered, and yielded acidic aqueous fraction (pH= 1-2). The acidified fraction was extracted with chloroform for removing non-alkaloidal materials. The extraction process was repeated several times with chloroform (3x2L) until the organic layer became clear. Organic layer was collected, concentrated by using rotary evaporator to produce non-alkaloid acidic chloroform fraction

(Fraction A, 220 g, 80 g & 115 g) which showed negative result with Dragendorff’s reagent.

Acidic aqueous fraction was basified to pH 8-10 by adding and stirring 10 % KOH solution using pH meter. The basic aqueous fraction was extracted with equal volume of chloroform (3x2L) many times until it became transparent. The basic organic layers obtained, were concentrated using rotary evaporator to produce basic alkaloidal fractions (Fraction B, 22g, 13.2g, 8.0g) which give positive Dragendorff’s reagent test. The remaining aqueous portion was neutralized to pH 7 with

0.5 N H2SO4. The neutral aqueous fraction was further extracted with ethyl acetate to obtain the ethyl acetate fraction (fraction C, 18 g & 10 g, 11 g). Finally, aqueous fraction was stored in bottle

(Scheme 2.1).

Chapter-2 Experimental

Chopped Plant Materials A. heterophylum (8 kg) A. laeve (3 kg) D. denudatum (5kg) Soaked in Methanol (3x10L) Filtered

Filtrate (Me-OH extract) Residues Evaporated Me-OH at 40 0C Dried and Stored

Acidified with 0.5 N H2SO4 (pH: 1-2) Partitioned with CHCl3 (3x2L)

Organic Phase-I Aqueous Phase-I Acidic Fraction-A (stored)

Basified with 10 % KOH (pH: 8-10) Partition with CHCl3 (3x2L)

Aqueous Phase-II Organic Phase-II 0 CHCl3 Evaporated at 40 C Neutralized (pH = 7) Crude alkaloids Tested for alkaloids with (Basic fraction-B) Dragendorff's reagent

Separated & purified using chromatographic technique Aqueous Phase-III structure identification by spectroscopic technique Water soluble materials Ethyl acetate Bio-assays Pure alkaloids fraction-C

Scheme 2.1. Fractionation of A. heterophyllum, A. laeve & D. denudatum 39

Chapter-2 Experimental

2.2.3 Isolation of diterpenoids from A. heterophyllum Wall.

The crude basic fraction-B (22 g) was further fractionated on column chromatography

(CC), using silica gel (440 g) with elution started from n-hexane followed by increasing its polarity, using n-Hexane-chloroform gradients. Finally the column exhausted by gradual increase in polarity of the mobile phase up to 20% methanol-chloroform gradient that afforded fifteen sub- fractions (A1-A15). From the TLC analysis, fractions were combined according to their separation profile. Four sub-fractions (AH1-AH4) were obtained. All the sub-fractions on repeated flash column chromatography (FCC) using solvent system n-hexane- acetone-diethyl amine; afforded eight diterpenoid alkaloids; (Scheme 2.2).

The spectral and literature data revealed that four of the alkaloids were confirmed known compounds i.e isoatisine, heteratisine, atidine and hetisinone, while 19-Epi-isoatisine is ew source alkaloid. However three new alkaloids are characterized as 1α, 6β-dimethoxy, 8,9β-dihydroxy heteratisine (1), 1,11,13α, trihydroxyl atisine (2) and 2α, 6β dihydroxy atisine (3). Among the isolated compounds, the known alklaoids atidine, isoatisine and heteratisine compounds were found to be the major alkaloids of A. heterophyllum followed by lesser amount of compound 1, 2 and 3. To identify and establish the structures of all the isolated compounds, physico-chemical, spectral and single X-ray analysis were conducted and the results are discussed here in the next chapter.

Chapter-2 Experimental

Chloroform Basic Fraction B (22 g)

Chromatographic resolution on silica gel using n-hex, CHCl3, M-OH as solvent system

AH AH AH2 3 1 AH4 100 % CHCl3 0-50 % CHCl3 70-90 % CHCl3 10-20 %M-OH 5 % M-OH

n-hex:Ace:DEA 98:2:10 Drp n-hex:Ace:DEA 8:2:10Drp

Isoatisine 11 19-Epiisoatisine 12 (80 mg) (30 mg)

Hetisinone 15 Compound 2 Compound 3 n-hex:Ace:DEA (10 mg) 9:1:10 Drp (20 mg) (7 mg)

Heteratisine 14 Atidine 13 Compound 1 (15 mg) (50 mg) (30 mg)

Scheme 2. 2 Isolation of alkaloids from A. heterophyllum Wall.

2.2.4 Isolation of diterpenoids from A. laeve Royle

The crude methanolic extract was suspended in water, acidified (pH 1-2) with 0.5N H2SO4 and was then extracted with CHCl3 (3 x 2L) to obtain acidic fraction (120 g). The acidic aqueous solution was basified (pH 8–10) by using 10% KOH (aq) and extracted with CHCl3 (3 x 2 L) to

Chapter-2 Experimental yield 13.2 g of alkaloid fraction. The crude basic fraction was loaded on column chromatography, using silica gel (160 g) and eluted with n-Hexane-chloroform gradients. Finally the column exhausted by gradual increased in polarity of the mobile phase upto 20% methanol-chloroform gradient that afforded fifteen sub-fractions (AL1-AL15). From the TLC analysis, fractions were combined according to their separation profile and four sub fractions (AR1-AR4) were obtained.

Fraction AR2 (600 mg) was subjected to FCC, using solvent system n-hexane- acetone (9:1:10 drops DEA/100 ml) afforded compound 6 (11 mg), 7 (18 mg) along with two known compounds

16 & 17. Fraction AR3 (300 mg) afforded swatinine-C (4) (13 mg) and swatinine-D (5) (10 mg) by FCC, using solvent system n-hexane-acetone (8:2:10 drops DEA/ 100 ml). The structures of known compounds were established by comparing their physical and spectral data with those available in literature (Scheme 2.3).

Chapter-2 Experimental

CHCl3 fraction AL (13.2 g)

using Silica gel (260 g),

AR , 0.2 g AR1, 1.5 g AR2, 0.6 g AR3, 0.3 g 4

RFCC, 10%Acetone, hexane Repeated Flash Column Chromatography +10 drops of diethyl amine (RFCC), 2:8, acetone Hex. DEA in 100ml

ALR5 (4) 13 mg ALR3 (6) (11 mg) ALR (5) ALR1(16), 6 (16 mg) 10 mg ALR4 (7) ALR2 (17) (18 mg) (23 mg)

Scheme 2.3. Isolation of alkaloids from A. laeve Royle 2.2.5 Isolation of diterpenoids from D. denudatum Wall

Dried and powdered aerial parts (5 kg) of the plant was extracted thoroughly with methanol (20 L). The filtrate was evaporated by rotary evaporator at 40 oC to yield a gummy solid crude methanolic extract (600 g). The residue was acidified to pH= 1-2 by the addition of 0.5N H2SO4 and extracted with CHCl3 (5 L) to obtain acidic fraction (115 g). The aqueous acidic fraction was then basified by adding 10% KOH solution (pH 8-10) and extracted with CHCl3 (12 L) to obtain basic fraction (8 g). The basic fraction was

Chapter-2 Experimental adsorbed on silica gel (240 g, E. Merck, type 60, 70-230 mesh) and eluted with gradients of n-hexane, chloroform and methanol. As a result, ten sub-fractions were obtained. On repeated FCC of sub frictions D-4, D-6, D-7 and D-8, using solvent systems acetone/n- hexane/DEA [(5:95), (1:9), (2:8) containing 10 drops of DEA per 100 ml each], afforded,1β-hydroxy, 14β-acetyl condelphine (8) (18 mg), 1,14α, 8,10β-tetrahydroxy,

16,18β dimethoxy-N-ethyl aconitane (9) (10 mg) 1α,8,16,18β-tetramethoxy, 14α-hydroxy-

N-ethyl-5-aconitene (10) (8 mg), Isotalitazidine hydrate (18), Dihydopentagynine (19) (15 mg) (Scheme 2.4).

CHCl3 fraction (8.0g)

Column chromatography (CC) (240g), hex., hex.-chlo., chlo., chlo.-MeOH

D-1 D-2 D-3 D-4 D-5 D-6 D-7 D-8 D-9 D-10 CC, 5%Acetone,hexane CC, 2:8 +10 drops of DEA CC, 1:9, ace. Hex. DEA ace. Hex. DEA

CC, 2:8 HA-4 (9) ace. Hex. DEA 10 mg

HA-1 (8) HA-2 (18) 18 mg 25 mg HA-5 (10) 8 mg HA-3 (19) 15 mg

Scheme 2.4. Isolation of diterpenoids alkaloids from D. denudtum Wall

Chapter-2 Experimental

2.3 X-Rays Diffraction Studies

XRD (X-ray diffraction) crystal structure analysis was performed by using a STOE-

IPDS II fitted with low-temperature unit of a Bruker kappa APEXII CCD diffractometer using Mo-Kα radiation (λ = 0.71073 Å) and graphite-monochromator at room temperature.

Crystal structure determination and refinements were accomplished by SIR97 [110],

SHELXL97 [111] and WinGX [112].

2.3.1 DFT Calculations

Crystallographic data was used to get optimized ground state geometry of isotalatizidine hydrate using DFT method following B3LYP-631G model of theory [113-117]. Electronic properties such as Frontier molecular orbital (HOMO-LUMO) energies, optimized geometries gap, global hardness, ionization potential, electron affinity and global electrophilicity of the compounds were calculated with same methods [113]. GAUSSIAN-03 program and Gauss-view molecule visualizer were used for calculation of the above mentioned data.

2.4 Cholinesterase inhibition assay and determination of IC50

Acetylcholinesterase (Electric-eel EC 3.1.1.7), butyrylcholinesterase (horse-serum E.C

3.1.1.8), acetylthiocholine iodide, butyrylthiocholine chloride, 5,5´-dithiobis[2-nitrobenzoic-acid]

(DTNB) and galanthamine were purchased from Sigma Aldrich and used without any further processing. All solvents used during the course of isolation and purification were of analytical grade and were used without further purification. Enzyme (AChE/BChE) inhibition activities were measured by spectrophotometric method as described in literature [118]. Reported protocol and assay conditions were followed throughout [119].

Chapter-2 Experimental

ACh-iodide and BCh-chloride substrates were used to assay AChE and BChE, respectively. The reagent 5,5´-Dithiobis [2-nitrobenzoic-acid] (DTNB) was used for the measurement of cholinesterase activity. Solution containing DTNB (0.2mM) in 62 mM sodium phosphate buffer (pH 8.0, 880 μL), test compound solution (40 μL) and acetylcholinesterase or butyrylcholinesterase solution (40 μL) were mixed and incubated for 15 minutes (25 ºC). ACh or

BCh (40 μL), were added to initiate the respective solution. The hydrolysis of ACh and BCh were monitored by the formation of yellow colored 5-thio-2-nitrobenzoate anionat a wavelength of 412 nm (15 min). Reactions were performed in triplicate in a BMS spectrophotometer (USA) and the results presented are average values. The concentrations of the isolated compounds that inhibited the hydrolysis of substrates (as mentioned above) by 50% (IC50) were determined as a function of increasing concentration of the compound in the assays on the inhibition values. The final DMSO concentration in the reaction mixture was 6%.

2.5 Experimental data of new diterpenoids from A. heterophyllum Wall.

2.5.1 1α, 6β-dimethoxy, 8,9β-dihydroxy heteratisine (1)

Physical state: Amorphous powder

Yield: 30 mg

Melting point: 272-275 oC

Rf: 0.27 [n-hexane, acetone, diethyl amine (7:3:10 drops)]

30 [α]D 0.00 (c = 1, CHCl3)

-1 IR υmax cm : 3450, 3405 (OH), 1740 (α-lactone)

EI-MS m/z: 421 (C23H35NO6, calcd. 421.5340)

Chapter-2 Experimental

EI-MS m/z (peak %): 421 (9), 404 (75), 388 (49), 358(27), 328 (4), 314 (32), 300 (6)

1 13 H & C-NMR (500 & 125 MHz, CDCl3): Given in table 3.1, (Results and discussion)

2.5.2 1,11,13α, Trihydroxyl atisine (2)

Physical state: Amorphous powder

Yield: 10 mg

Melting point: 259-261 oC

Rf: 0.19 [CHCl3: Me-OH (9:1)]

30 o [α]D -25 (c = 0.8, Me-OH)

-1 IR υmax cm : 3437, 3420. 3401 (OH), 3030, 1620 (C=CH2)

EI-MS m/z: 329 (C20H27NO3, calcd:329.4322)

EI-MS m/z (peak %): 329 (12), 312 (84), 296 (30), 280 (15), 266 (4), 253 (15)

1H & 13C-NMR (600 & 150 MHz, MeOD): Given in table 3.2, (Results and discussion)

2.5.3 2α, 6β Dihydroxy atisine (3)

Physical state: Amorphous powder

Yield: 07 mg

Melting point: 248-251 oC

Rf: 0.21 [CHCl3: Me-OH (9:1)]

30 o [α]D -29 (c = 0.5, Me-OH)

-1 IR υmax cm : 3440, (OH), 3018, 1626 (C=CH2)

Chapter-2 Experimental

EI-MS m/z: 313 (C20H27NO2,calcd: 313.4410)

EI-MS m/z (peak %): 313 (36), 296 (60), 296 (30), 280 (20), 266 (14), 253(12)

1H & 13C-NMR (500 & 125 MHz, MeOD): Given in table 3.3, (Results and discussion)

2.6 Experimental data of new diterpenoids from A. laeve Royle.

2.6.1 Swatinine-C (4)

Physical state: Yellowish amorphous powder

Yield: 13 mg

Melting point: 204-207 oC

Rf: 0.29 [n-hexane/acetone/DEA (7:3:10 drops)]

30 o [α]D -37.5 (c = 1, CHCl3)

-1 IR υmax cm : 3487, 3440 (OH), 1735 (C=O, seven member ring), 1116 & 1050

(C-O-C), 1690 cm-1 (C = O, carbonyl)

HREI-MS m/z: 477.2407 (C25H35NO8, calcd: 477.5459)

EI-MS m/z (peak %): 477 (16), 446 (36), 417 (25), 405 (65), 391 (10), 333 (40)

1 13 H & C-NMR (600 & 150 MHz, CDCl3): Given in table 3.4, (Results and discussion)

2.6.2 Swatinine-D (5)

Physical state: Amorphous powder

Yield: 10 mg

Melting point: 178-180 oC

Chapter-2 Experimental

Rf: 0.30 [n-hexane/acetone/DEA (7:3:10 drops)]

30 o [α]D -55 (c = 1, CHCl3)

-1 IR υmax cm : 3492 (OH), 1119 and 1064 (simple ether bonds)

HREI-MS m/z: 449.2854 (C25H39NO6, calcd: 449.5787 )

EI-MS m/z (peak %): 449 (4), 418 (90), 375 (18), 365 (15), 349 (13)

1 13 H & C-NMR (600 & 150 MHz, CDCl3): Given in table 3.5, (Results and discussion)

2.6.3 Methyl 2 acetamidobenzoate (6)

Physical state: White crystals

Yield: 11 mg

Melting point: 84-87 oC

Rf: 0.48 [n-hexane/acetone/DEA (8:2:10 drops)]

-1 IR υmax cm : 3460 (NH) amide, 1710 (C = O ester), 1625 &1585 (C=C) Ar

HREI-MS m/z: 193 ( C10H11NO3, calcd:193.1988)

EI-MS m/z (peak %):193 (28), 151 (62), 119 (33), 92 (52), 78 (8), 65 (35)

1 13 H & C-NMR (300 & 75 MHz, CDCl3): Given in table 3.6, (Results and discussion)

2.6.4 Methyl 4-[2-(methoxycarbonyl)anilino]-4-oxobutanoate (7)

Physical state: White crystals

Yield: 18 mg

Melting point: 113-115 oC

Chapter-2 Experimental

Rf: 0.41 [n-hexane/acetone/diethyl amine (8:2:10 drops)]

-1 IR υmax cm : 3460 (NH) amide, 1730 (C = O, ester), 1605 and 1595 (C = C)

EI-MS m/z: 265 (C13H15NO5, calcd:265.2613)

EI-MS m/z (peak %): 265 (65), 234 (21), 202 (32), 174 (15), 151 (90), 146 (25), 132 (4),

119 (78), 115 (42), 90 (10), 82 (7), 77 (3), 65 (2), 55 (13)

1 13 H & C-NMR (300 & 75 MHz, CDCl3): Given in table 3.7, (Results and discussion)

2.7 Experimental data of new diterpenoids from D. denudatum Wall.

2.7.1 1β-hydroxy, 14β-acetyl condelphine (8)

Physical state: White transparent crystals.

Yield: 18 mg

Melting point: 157-159 oC

Rf: 0.46 [n-hexane, acetone, DEA (8:2, 10 drops)]

25 o [] D +30.0 (c = 1, CHCl3)

-1 IRmax cm : 3500 (OH), 3155 (H-bonded OH), 1734 and 1222 (C=O, OAc)

EI-MS (m/z): 449 (observed), C25H39NO6, 449.5880 (calcd.)

EI-MS m/z (peak %): 449 (8), 432 (95), 416 (18), 400 (10), 378 (4), 362 (13), 58 (3)

1 13 H & C-NMR (500 & 125 MHz, CDCl3): Given in table 3.8, (Results and discussion)

2.7.2 1,14α, 8,10β-Tetrahydroxy, 16,18β dimethoxy-N-ethyl- aconitine (9)

Physical state: Amorphous powder

Chapter-2 Experimental

Yield: 10 mg

Melting point: 182-185 oC

Rf: 0.32 [n-hexane/ acetone/ DEA (7:3:10 drops)]

-1 -1 -1 IRmax cm : 3550, 3487 cm (OH), 1073 cm (C-O).

EI-MS (m/z): 423 (observed), C23H37NO6, 423.5415 (calcd.)

EI-MS m/z (peak %): 423 (12), 406 (87), 376 (16), 360 (6), 344 (4), 330 (15), 300 (10), 91 (40),

58 (5), 44 (7)

1 13 H & C-NMR (600 & 150 MHz, CDCl3): Given in table 3.10, (Results and discussion)

2.7.3 1α,8,16,18β-Tetramethoxy, 14α-hydroxy-N-ethyl-5-aconitene (10)

Physical state: Amorphous powder

Yield: 8mg

Melting point: 140-145 oC

Rf: 0.27 [n-hexane/acetone/DEA (8:2, 5 drops)]

25 o [] D: +20.0 (c = 0.30, CHCl3)

-1 -1 IRmax cm : 3466 cm (OH), 3015 cm-1 (C=C), 1097 cm-1 (C-O),

EI-MS (m/z): 433 (observed), C25H41NO7, 433.5939(calcd.)

EI-MS m/z (peak %): 433 (34), 402 (75), 386 (13), 356 (8), 330 (18), 300 (32), 286 (26), 57

(25), 43 (21)

1 13 H & C-NMR (500 & 125 MHz, CDCl3): Given in table 3.11,(Results and discussion)

Chapter-2 Experimental

2.8 Experimental data of known diterpenoids from A. heterophyllum Wall.

2.8.1 Isoatisine (11)

Physical state: White crystalline

Yield: 80 mg,

Rf: 0.47 [n-hexane/acetone/DEA (9:1:10 drops)]

30 [α]D -22.4 (c = 1, CHCl3)

-1 IR υmax cm : 3356 (OH), 3012, 1656 (C=CH2)

HREI-MS m/z: 343.5110 (C22H33NO2,calcd: 343.4412)

1 H-NMR (600 MHz, CDCl3): δ 5.1/5.03 (1H, s, H-17), 3.97 (1H, br s, H-19), 3.85 (2H, m, H-22),

3.79 (1H, br s, H-15), 2.99 (2H, d, J = 2.2 Hz, H-20), 2.39 (2H, m,

H-21), 2.1 (1H, m, H-13), 1.08 (3H, s, H-18).

2.8.2 19-Epiisoatisine (12)

Physical state: White crystalline

Yield: 30 mg,

Rf: 0.48 [n-hexane, acetone, diethyl amine (9:1:10 drops)]

30 o [α]D - 23.1 (c = 1.25, CHCl3)

-1 IR υmax cm : 3356 (OH), 3012, 1656 (C=CH2)

HREI-MS m/z: 343.5110 (C22H33NO2,calcd: 343.4412)

Chapter-2 Experimental

1 H-NMR (600 MHz, CDCl3): δ 5.09/5.03 (1H, s, H-17), 3.97 (1H, br s, H-19), 3.83 (2H, m, H-

22), 3.79 (1H, br s, H-15), 2.85 (2H, d, J = 2.25 Hz, H-20), 2.39 (2H,

m, H-21), 2.1 (1H, m, H-13), 1.09/0.9(3H, s, H-18).

2.8.3 Atidine (13)

Physical state: White crystalline solid

Yield: 50 mg

30 o [α]D - 47 (c = 1.7, CHCl3)

Rf: 0.38 [n-hexane/acetone/diethyl amine (8:2: 10 drops)]

-1 -I IR υmax cm : 3534 & 3444 (OH), 3084 & 1660 cm (C = CH2), 1690 (C = O) and 1379

-I cm (CCH3)

EI-MS m/z: 359 (C22H33NO3, calcd: 359.51)

1 H-NMR (500 MHz, CDCl3): δ 5.2, 5.07 (each 1H, dd, J = 3.8 Hz, H-17), 4.56 (1H, br s, H-15),

3.70 (2H, t, J = 5.5 Hz, H-22), 2.97 (1H, t, J = 11.25 Hz, H-21),

2.79-2.77 (1H, dd, J = 9 Hz, H-6) 2.34-2.25 (1H, dd, J = 2.25 Hz,

H-19), 2.34-2.31 (2H, dd, J = 4 Hz, H-20), 0.79 (3H, s, H-18).

2.8.4 Heteratisine (14)

Physical state: White crystals

Yield: 15 mg

Rf: 0.27 [n-hexane/acetone/diethyl amine (7:3:10 drops)]

30 o [α]D + 40 (c = 1, CHCl3)

Chapter-2 Experimental

-1 -I IR υmax cm : 3462, 3410 (OH), 1742 (lactone) and 1386 cm (CCH3)

EI-MS m/z: 391(C22H33NO5, calcd:391.5080)

1 H-NMR (500 MHz, CDCl3): δ 4.86 (2H, s, H-13), 4.45 (1H, d, J = 7.3 Hz, H-6), 3.88 1H, d, J =

7.6 Hz, H-19) 3.55 (1H, s, H-17), 3.26 (1H, s, H-1), 1.09 (3H, t, J =

7.15 Hz, NCH2CH3), 0.99 ( 3H, s, H-18), 2.64 (1H, d, J = 11.76 Hz,

H-5)

2.8.5 Hetisinone (15)

Physical state: White crystalline solid

Yield: 20 mg,

Rf: 0.42 [chloroform/methanol (8:2]

Melting point : 193-196 o C

25 o [] D +40.0 (c = 0.9, Me-OH)

-1 -1 IR max cm : 3575 (OH), 1725 (C = O), 1655 and 910 (C = CH2) cm .

EI-MS (m/z): 327 (calcd: 327.4240,C20H25NO3)

1H-NMR (500 MHz, MeOD):  4.88 & 4.70 (1H, s, H-17), 4.25 (1H, d, J = 8.9 Hz H-11), 4.12

(1H, t, J = 2.6 Hz, H-13), 3.01 (1H, s, H-20), 3.33(2H, OH), 3.56

(2H, d, J = 14.65 Hz, H-1), 3.38 (2H, s, H-19), 3.38 (1H, s, H-6),

1.18 (3H, s, H-18).

Chapter-2 Experimental

2.9 Experimental data of known diterpenoids from A. leave Royle.

2.9.1 Aconorine (16)

Physical state: White powder

Yield: 16 mg,

Rf: 0.37 [n-hexane/acetone/diethyl amine (8:2: drops)]

Melting point : 235-237 oC

-1 -1 -1 IR max cm : 1710 cm (ester carbonyl), 1600, 1280, 1250,& 750 cm (Ar).

HREI-MS (m/z): 568.241(calcd: 568.711), C32H44N2O7,

1 H-NMR (CDCl3, 400 MHz):  11.01 (1H, br s, NH), 8.66 and 7.90 (each 1H, d, J = 8.4 Hz Ar-

H), 7.48 and 7.02 (each 1H, t, J = 7.6 Hz, Ar-H), 3.69 (1H, s, H-14),

3.52 (1H, d, J = 11.2 Hz, H-19), 3.39, 3.30 and 3.27 (each 3H, s, 3

x OMe), 3.20 (2H, m, H-16), 2.91 (1H, s, H-17), 2.20 (3H, s,

COCH3), 1.09 (3H, t, J = 6.8 Hz, NCH2CH3).

2.9.2 Lappaconitine (17)

Physical state: White amorphous powder

Yield: 23 mg,

Rf: 0.35 [n-hexane/acetone/diethyl amine (8:2: drops)]

25 o [] D: +27.0 (c = 0.22, CHCl3)

-1 -1 IR max cm : 3500, 3250 (OH, NH), 1680 (CO), 1080 (C-O, ether) cm .

HREI-MS (m/z): 584.3096 (calcd: 584.3079), C32H44N2O8, 55

Chapter-2 Experimental

1 H-NMR (CDCl3, 400 MHz):  11.01 (1H, br s, NH), 8.65 and 7.90 (each 1H, d, J = 7.2 Hz Ar-

H), 7.47 and 7.01 (each 1H, t, J = 7.0 Hz, Ar-H), 3.56 (1H, s, H-19),

3.53 (1H, d, J = 6.0 Hz, H-14), 3.38, 3.28 and 3.27 (each 3H, s, 3

x OMe), ), 1.11 (3H, t, J = 7.0 Hz, NCH2CH3).

2.10 Experimental data of known diterpenoids from D. denudatum Wall.

2.10.1 Isotalatizidine hydrate (18)

Physical state: White crystals

Yield: 25 mg

Rf: 0.39 [n-hexane, acetone, DEA (8:2, 10 drops)]

Melting point : 119-121 oC

25 o [] D +15.0 (c = 0.55, CHCl3)

-1 IR max, cm : 3550, 3414, 3630, and 3370

EI-MS (m/z): 425 (observed), C23H39NO6, 425.5660 (calcd.)

1 H-NMR (CDCl3, 600 MHz):  4.25 (1H, t, J = 5.1 Hz, H-14), 3.74 (1H, s, H-1), 3.42 (1H, m,

H-16), 3.36 and 3.33 (each, s, 2x OMe), 3.17 and 3.03 (2H, dd, J =

8.76 Hz, H-18), 2.81 (1H, s, H-17), 2.39 (2H, m, H-19), 2.24 (1H, t,

J = 5.64 Hz, H-7) and 1.14 (3H, t, J = 7.14 Hz, NCH2CH3).

Chapter-2 Experimental

2.10.2 Dihydropentagynine (19)

Physical state: White crystals

Yield: 15 mg

Rf: 0.36[n-hexane/acetone/ diethyl amine (8:2, 10 drops)]

Melting point: 152-155 oC

25  : -20.0o (c = 1.1, MeOH) D

-1 IR maxcm : 3440, 3414 (OH).

EI-MS (m/z): 407.0 (observed), C23H44NO5, 407.2672 (calcd.)

1 H-NMR (CDCl3, 500 MHz):  4.16 (1H, t, J = 4.7 Hz, H-14), 3.33 and 3.26 (3H, s, 2x

OMe), 3.90 (1H, br s, H-1), 2.57 (1H, s, H-17), 3.31 (1H, m,

H-16), 1.08 (3H, t, J = 7.15 Hz, NCH2CH3).

CHAPTER-3

RESULTS AND DISCUSSION

Chapter-3 Results and discussion

RESULTS AND DISCUSSION

3.1 Diterpenoids isolated from A. heterophyllum WALL.

The phytochemical evaluation of A. heterophyllum Wall (Ranunculaceae) has resulted in the isolation of new and known diterpenoid alkaloids from the basic CHCl3 soluble part of the methanolic extract. The structures of the isolated compounds were established on the basis of spectroscopic technique like EI-MS, HREI-MS, 1H-NMR, 13C-NMR, DEPT, and 2D NMR, including 1H-1H COSY, HMQC, and HMBC. Enzyme inhibition activities of isolated compounds were also studied.

The aerial parts of A. heterophyllum Wall were extracted with methanol. The methanolic extract was partition in CHCl3 at different pH (Scheme 2.1). The CHCl3-soluble fraction obtained at pH

8-10 was subjected to column chromatography which yielded four sub-fractions (AH1-AH4). On repeated flash column chromatography (FCC) of sub-fractions resulted in the isolation of three new compounds (1-3) and five known diterpenoid alkaloids (11-15) (Scheme 2.2).

Chapter-3 Results and discussion

3.2 New Diterpenoids from A. heterophyllum Wall.

3.2.1 1α, 6β-dimethoxy, 8,9β-dihydroxy heteratisine (1)

Compound 1 was isolated from the sub-fraction AH2 on flash column chromatography using 10 % acetone/n-hexane (1:9) containing 10 drops of DEA per 100 ml (Figure 3.1).

16 13 O

12 H CO 3 17 14 C O

10 OH 1 9 15 2 11 N 8 3 5 OH 7 4 6

OCH 19 18 3

Figure 3.1. 1α, 6β-dimethoxy, 8,9β-dihydroxy heteratisine (1)

The molecular formula C23H35NO6 (m/z 421, cald: 421.5340) of compound 1 was established with the help of mass spectrum (EI-MS) and nuclear magnetic resonance (NMR) spectral data.

The infrared (IR) spectrum of 1 showed absorption bands for hydroxyl groups at 3450 and 3405 cm-1 whereas α-lactone functionalities showed strong absorption band at 1740 cm-1.

The 1H-NMR spectrum of compound 1 (Table 3.1) showed signals for N-ethyl, methoxy, methylene and several methine protons. A three protons triplet at δ 1.07 (J = 7.1 Hz) was assigned to the methyl of N-ethyl group while a singlet of three proton integration at δ 0.98 was assigned to the methyl group at C-18 position of compound 1. A sharp singlet each of three proton integration was abserverd at δ 3.29 due to methoxyl group at C-1 and C-6. The characteristic H-13 and H-17 59

Chapter-3 Results and discussion methine proton singlet was appeared down field at δ 4.51 and δ 3.53 in the same 1H-NMR spectrum. The proton attached to methoxy substituted carbon, produced a triplet signal at δ 3.18

(J = 9.7 Hz). Similerly the H-19 proton of compound 1 exbibted a doublet of two protons integration at δ 2.61(J = 11.9 Hz).

The 13C-NMR (BB & DEPT) spectrum (Table 3.1) showed twenty three signals, including two methoxy, two methyl, seven methylene, seven methine, and five quaternary carbons. The signal at δ 174.8 was assigned to the carbonyl of the lactone skeleton while the signals in upfield region at δ 75.3 and 100.4 were assigned to quaternary carbons bearing hydroxyl group at C-8 and

C-9. The peaks at δ 49.2, 29.6 and 26.2 were due to C-7, C-16 and C-18. The characteristic sharp signals at δ 83.2, 62.3, 58.3, 57.7 and 55.2 were due to the resonane of C-1, C-17, and C-19 and

two methoxyl carbon repectively.”

H H

16 H 13 O

H3CO H 12 O 17 14 C

10 OH H 1 9 15 2 11 H N 8 3 5 OH 7 4 6 H H 19 18 H3CO

Figure 3.2. Key HMBC correlation in 1α, 6β-dimethoxy, 8,9β-dihydroxy heteratisine (1)

Chapter-3 Results and discussion

The 1H-13C correlations were determined by the HMQC spectrum (Fig. 3.2) while the long- range 1H-13C connectivities were obtained through the HMBC technique as H-17 at δ 3.53 showed correlation with C-11 (δ 49.5), and C-10 (δ 42.7). The H-7 proton was connected to C-6 (δ 75.9),

C-8 (δ 73.5) and C-15 (δ 29.2) while H-5 showed cross peaks in the same spectrum with C-6 (δ

75.9), C-4 (δ 34.7) and C-11 (δ 49.5) respectively. The alkaloid contains two methoxy, two hydroxy groups, as concluded from the NMR spectrum. One hydroxyl was placed at C-8 ( 73.5), second at C-9 ( 100.4). The β oriented methoxy groups was placed at C-6 ( 75.9) on the basis of the chemical shift and biogenetic considerations.

The proton connectivities were confirmed by 1H-1H-COSY correlations (Fig. 3.3) in the basic skeleton. H-6 methine proton at (δ 4.51) showed cross peak with C-7 methine proton at δ

(4.00) and H-5 (δ 2.08). Similarly, the H-13 ( 4.77) proton showed coupling with H-12 ( 1.9), which in turn showed coupling with H-10 ( 2.3ppm).

H H

16 H H 13 O

H3CO H 12 17 H 14 C O

10 OH H 1 9 15 2 11 H N 8 3 5 OH 7 4 186 H H CO H 3 H 19

Figure 3.3. Key COSY correlation in 1α, 6β-dimethoxy, 8,9β-dihydroxy heteratisine (1) 61

Chapter-3 Results and discussion

On the basis of various spectroscopic techniques, structure of compound 1 was deduced as

1α, 6β-dimethoxy, 8,9β-dihydroxy heteratisine (1).

Table 3.1. 1H & 13C-NMR (500 & 125 MHz) data of compound 1

C. No 1H- δ (J Hz) 13C- (δ) Multiplicity HMBC Correlations 1 3.18, t, J = 9.7 Hz, 83.2 CH

2 26.8 CH2

3 36.5 CH2 4 34.7 C 5 2.08, m 50.8 CH 5, 4, 6, 11 6 4.51, br s, 75.9 CH 6, 7, 8, 15 7 4.00, d, J = 7.6 Hz 49.2 CH 8 73.5 C 9 100.4 C 8, 10, 12, 14 10 2.3, m 42.7 CH 11 49.5 C

12 1.9 m 33.4 CH2 13 4.77, t, J = 6.1 Hz 75.3 CH 13, 12, 16, 14 14 174.8 C=O

15 1.37, d, J= 6 Hz 29.2 CH2

16 1.7, m 29.6 CH2 17 δ 3.53, s 62.3 CH 17, 11, 10

18 0.98, s 26.2 CH3

19 2.61, d, J = 11.9 Hz, 58.3 CH2

N 2.6, m 48.9 CH2

CH2 1.07, t, J = 7.1 Hz 13.4 CH3

CH3

OCH3 3.29, , 3.25, s 57.7,55.2 OCH3

Chapter-3 Results and discussion

3.2.2 1,11,13α, Trihydroxyl atisine (2)

Compound 2 was isolated as transparent gummy solid, from sub-fraction AH3 on subjecting to flash column chromatography, using 20 % acetone/n-hexane (2:8) containing 10 drops of DEA per 100 ml (Figure 3.4).

13 HO 17 CH2 12 OH 11 16 20 14 15 1 9 2 10 8 N 3 5 7 4 6 18 19

Figure 3.4. 1, 11,13α, Trihydroxyl atisine (2)

The molecular formula C20H27NO3 (m/z 329, calcd:329.4322) was given to compound 2 on the basis of mass spectrum (EI-MS) and nuclear magnetic Resonance (NMR) assigned spectral data. The IR spectrum of compound 2 showed absorption bands at 3437, 3420 and 3401 cm-1 for

-1 –OH, 3030 and 1320 cm for C=CH2 bond stretching.

The 1H-NMR spectrum of compound 2 (Table 3.2) showed signals for one methyl, six methylene and several methine protons. The H-17 terminal methylene protons resonated as singlet at δ 4.70. The methyl protons at C-18 resonated as a singlet of three protons at δ 1.18 respectively.

The proton at C-13 showed a triplet of one proton integration at δ 4.12 whereas the H-11 proton resonated as doublet signal at δ 4.26 (J= 9 Hz) which confirmed the presence of hydroxyl

Chapter-3 Results and discussion substituent at C-11 and C-13 of 2. The characteristic sharp peak at δ 3.04 was due to the resonance of H-1 proton. All other signals in the 1H-NMR spectrum of 2 are in their expected range which are common for all C20 diterpenoid alkaloids.

The 13C-NMR (BB & DEPT) spectra (Table 3.2) showed twenty signals, one methyl, six methylene, nine methine and four quaternary carbons. In the downfield region, the signal at δ 106.5 and δ 146.6 were due to C-17 and C-16 respectively”. The carbon bearing hydroxyl group resonated at δ 71.7 and δ 74. 7. Similarly the peaks at δ 69.8, 65.1, 63.8 and 54.6 were due to C-1,

C-20, C-19 and C-9.

13 H HO CH2 16 HO H 14 15 1 9 2 H 10 8 N 3 5 7 4 H H H 19 18

Figure 3.5. Key HMBC correlation in 1, 11,13α, trihydroxyl Atisine (2)

Further, H-C connectivities were made from HMBC spectrum as H-11 at δ 4.2 showed correlation with C-13 (δ 71.03), C-12 (δ 59.8) and C-10 (δ 50.8). In the same way the H-1 at δ

3.04 proton was connected to C-1 (δ 69.8) and C-20 (δ 65.1) while H-3 at δ 2.72 showed cross peaks in the same spectrum with C-1 (δ 69.8), C-5 (δ 55.3 and C-18 (δ 27.4) (Fig. 3.5). The H-13 methine proton at δ (4.1) exhibited cross peak with C-14 methine proton at δ (2.3). Similarly the

Chapter-3 Results and discussion

H-14 methine proton showed correlation with H-9 methine proton. The remaining correlations among H-7/H-6, H-6/H-5 were confirmed from the same spectrum of compound 2 (Figure 3.6).

HO H 13

HO 17 CH2 H H 16 OH 14 15 1 9 2 10 8 N 3 5 7 4 H H H

Figure 3.6. Key COSY correlation in 1, 11,13α, Trihydroxyl atisine 2

On the basis of various spectroscopic techniques, structure of 2 was characterized as 1,

11,13α, trihydroxyl atisine 2.

1 13 Table 3.2. H & C-NMR (600 & 150 MHz) data of compound 2

C. No 1H- δ (J Hz) 13C- (δ) Multiplicity HMBC Correlations 1 3.04, br s 69.8 CH 1, 20

2 2.18, m 49.1 CH2

3 2.72, m 44.2 CH2 1, 5, 18 4 41.9 C 5 2.15, d, J = 3 Hz 55.3 CH 6 2.13, d, J= 2 Hz 65.1 CH 5,10

7 2.28, t, J=2.5 Hz 33.2 CH2 15, 18, 6

Chapter-3 Results and discussion

8 41.9 C 9 2.15, d, J= 3 Hz 54.6 CH 10 50.8 C 11 4.2, d,J= 9 Hz 74.6 CH 13, 12, 10, 16 12 2.17, m 59.8 CH 13 4.1, t, J= 2.04Hz 71.03 CH 14 2.3,d, J= 2.64 Hz 51.6 CH

15 1.75 and 2.60, m 35.5 CH2 16 146.6 C

17 4.87, 4.70 s 106.5 CH2 15,12

18 1.18, s 27.4 CH3

19 2.20, s 63.8 CH2

20 3.40, s 65.1 CH

3.2.3 2α, 6β Dihydroxy atisine (3)

Compound 3 was isolated from sub-fraction AH3 on repeated flash column chromatography using 20% acetone/n-hexane (2:8) gradient containing 10 drops of DEA per 100 ml. It was visualized at 254 nm under UV light on pre-coated silica gel TLC plates.

13 17 CH 12 2 11 16 20 14 HO 15 1 9 2 10 8 N 3 5 7 4 H HO

Figure 3.7. 2α, 6β Dihydroxy atisine (3)

Chapter-3 Results and discussion

The IR spectrum of compound 3 demonstrated absorption bands at 3458 and 3440 cm-1due to the presence of hydroxyl group (OH). Similarly the same spectrum provided a strong absorption bands 3018 and 1626 cm-1due to C=C bond stretching. The mass spectrum (EI-MS) of compound

+ 3 showed the molecular ion [M] at m/z 313 assigning molecular formula C20H27NO2 (calcd:

313.4410).

The 1H-NMR spectrum (Table 3.3) of compound 3 revealed a singlet of one proton integration each at  5.12 and 5.08 for terminal methylene group. A sharp singlet of one proton appeared downfield at  3.72 due to the H-20 proton. The H-2 proton of 3 resonated in the downfield region, showing a multiplet signal at  4.13 respectively.The C-18 methyl proton of compound 3 resonated as triplet of three protons at  1.10. All other methylene and methine proton of compound 3 displayed signals in their respective range.

The 13 C-NMR (Table 3.3) of 3 showed the presence of twenty carbons while their multiplicity was determined by DEPT which indicated one methyl, eight methylene, six methine, and five quaternary carbon atoms. The terminal olefinic carbons resonated downfield at  154.01

(C - 16) and 109.3 (C -17) whereas the C-6 quaternary carbon was found to resonate at  100.04.

The C-2 methine carbon, substituted by hydroxyl group was found to occur at  63.5. Other signals in the same spectrum were found to appear at  74.4, 57.1, 48.2, 46.08 and 23.6 due to C-20, C-

19, C-3, C-10 and C-18 repectively.

The 1H- 13C correlations were accomplished with the help of the HMQC spectrum, while the long-range connectivities were determined through the HMBC spectrum (Figure 3.8). In the

HMBC spectrum (Figure 3.8), H-1 ( 1.77) showed correlation with C-2 ( 63.5), C-9 ( 50.02)

Chapter-3 Results and discussion and C-10 ( 46.08). Similarly, H-7 ( 1.38) showed HMBC interactions with C-8 ( 37.1), C-5 (

59.2) and C-6 ( 100.04) showed the exact attachment of hydroxyl group at C-6 position of the compound 3. Furthermore, H-15 ( 2.43) showed correlation with C-16 ( 154.01), C-8 ( 37.1) and C-17 ( 109.3).

13 17 CH H 12 2 11 16 H 20 14 HO 15 1 9 H 2 10 8 H N 3 5 7 4 H H H OH

Figure 3.8. Key HMBC correlation in 2α, 6β dihydroxy atisine (3)

Hence, on the basis of aforementioned spectroscopic data, the structure of compound 3 was deduced as 2α, 6β dihydroxy atisine.

Table 3.3. 1H & 13C-NMR (500 & 125 MHz) data compound (3)

C. No 1H NMR δ (J Hz) 13C (δ) Multiplicity HMBC Correlations

1 1.51, d, J= 8Hz 39.3 CH2 2, 10 2 4.13, m 63.5 CH 3 1.58, d, J= 7.0Hz 48.2 CH2 3, 4, 18 4 33.02 C

Chapter-3 Results and discussion

5 3.6, s 59.2 CH 6 100.04 C 7 1.38, brd s 44.6 CH2 4, 6, 8 8 37.1 C 9 1.47, d, J= 6Hz 50.02 CH 10, 8, 7 10 46.08 C

11 1.87, t, J= 7Hz 27.4 CH 9, 12, 16

12 2.19, m 40.5 CH 11, 13, 16, 17

13 2.03, d,J= 7.5Hz 30.1 CH2

14 1.6,t, J=8.7Hz 48.05 CH 12, 8, 20

15 2.43, s 35.5 CH2 8, 16, 17

16 154.01 C

17 4.96, 4.54, s 109.3 CH2

18 1.1, s 23.6 CH3

19 4.01, m 57.1 CH2

20 3.72, s 74.4 CH

3.3 New diterpenoids from A. laeve Royle

3.3.1 Swatinine-C (4)

Compound 4 was isolated from the sub-fraction AR3 on flash column chromatography using solvent system n-hexane/acetone (8:2:10 drops DEA/100 ml).

Chapter-3 Results and discussion

OCH3

13 16 OH 12 14 17 OCOCH3 H 1 10 9 2 11 15 N 8 3 5 7 O 4 6 H O 18 19 O HO

Figure 3.9. Swatinine-C (4)

The molecular formula C25H35NO8 (m/z 477.2407 calcd. 477.5459) of compound 4 was established with the help of mass spectrum (EI-MS & HREI-MS) and nuclear magnetic resonance

(NMR) spectral data which is characteristic of lycoctonine type alkaloids [120]. The infrared (IR)

-1 spectrum of 4 showed absorption bands for hydroxyl groups at 3487, 3440 cm ether linkage at

1116 & 1050 cm-1 and carbonyl functionalities at 1735 cm-1 in the molecule.

The 1H-NMR spectrum of compound 4 showed signals for N-ethyl, methoxy, acetoxy, methylenedioxy and methine protons. A three protons triplet at δ 0.954 (J = 7 Hz,) was assigned to the methylof N-ethyl group while two singlets each of one proton integration at δ 5.15 & 4.92 were assigned to methylenedioxy group (OCH2-O) at position 7 & 8. Two sharp singlets, each of three protons integration at δ 3.32 and δ 2.08 was due to the methoxy at C-16 and acetoxy mehyl

protons at C-14 respectively”. The characteristic H-14 and H-16 methine proton triplet was observed down field at δ 3.56 (J = 5 Hz) and δ 3.69 (d, J = 9.6 Hz).

Chapter-3 Results and discussion

The 13C-NMR (BB & DEPT) spectrum (Table 3.4) of 4 showed twenty five signals, including one methoxy, one acetyl, one methyl, eight methylene, eight methine, and six quaternary carbons. The signals at δ 210.4 and 171.5 were assigned to the carbonyl carbon of 4 while the signal at δ 91.4 was assigned to methylene carbon bonded to two oxygen atoms. The peaks at δ

89.7, 86.1, 72.1, 82.0, and 71.9 were assigned to C-7, C-8, C-14, C-16 and C-18. The overall spectral data of compound 4 was identical to that of ilidine except the presence of hydroxyl groups at C-1 & C-18 instead of methoxy group. The acetoxy group at C-14 also differentiate it from ilidine where a hydroxyl group exists at C-14 [121]. The alkaloid contains one methoxy, one acetoxy and two hydroxyl groups, as decided from the NMR spectra, one hydroxyl was placed at

C-1 ( 72.0) and second at C-18 ( 71.9). The acetyl and methoxy groups were placed at C-14

(72.1) and C-16 ( 82.0), on the basis of the chemical shift and biogenetic considerations.

H H3CO H 16 13 12 OH H 14 OCOCH3

17 10 1 9 H

2 11 15 N 8 3 5 O 7 4 6 H C H 19 O 18 O H HO

Figure 3.10. Key HMBC correlation in swatinine-C (4)

Chapter-3 Results and discussion

Further structural assignments were made by using 2D-NMR experiments. The 1H-13C correlations were determined by the HMQC spectrum, while the long-range 1H-13C connectivities were obtained through the HMBC technique as H-5 at δ 2.31 showed 2J correlation with C-6 (δ

210.4), 3J correlations C-11 (δ 48.6) and C-7 (δ 89.7). The methylenedioxy protons were connected to C-7 (δ 89.7) and C-8 (δ 86.1) while H-9 showed cross peaks in the same spectrum with C-8 (δ

86.1), C-10 (δ 36.1), C-13 (δ 42.3) and C14 (δ 72.1) (Fig. 3.10).

The proton connectivities were confirmed by 1H-1H-COSY correlations in the basic skeleton. H-13 methine proton at δ (1.66) showed cross peak with C-14 methine proton at δ (3.56).

Similarly, the H-9 ( 2.07) proton showed coupling with H-14 ( 3.56), which in turn showed coupling with H-13 ( 2.40 ppm), supporting the presence of acetoxy group at C-14 ( 72.1).

H OCH3 16 H 13 H 12 14 OCOCH3 HO 17 10 H 9 H 2 11 15 N 8 3 5 H 7 4 O 6 H O 19 18 O HO

Figure 3.11. Key COSY interaction in swatinine-C (4)

Chapter-3 Results and discussion

The structure of compound 4 was established from 1H-1H COSY, 1H-13C, HMBC and

HMQC experiment. Thus the structure of compound 4 was deduced as 1α, 18β-hydroxy, 14α- acetyl, 16β-methoxy, 7β, 8β, methylenedioxy-N-ethyl lycoctonine (Swatinine-C).

Table 3.4. 1H & 13C-NMR (600 & 150 MHz) data of swatinine-C

C. No 1H- δ (J Hz) 13C- (δ) Multiplicity HMBC Correlations 1 3.34, t, J = 8 Hz, 72.0 CH 2, 17, 10

2 1.6, m 29.3 CH2

3 1.4, m 29.8 CH2 4 40.0 C 5 2.50, br s 52.3 CH 3, 4, 6, 10,11, 17, 6 210.4 C = O 7 89.7 C 8 86.1 C 9 2.07, d, J= 5.4 Hz 48.9 CH 8, 10, 12, 14 10 36.1 CH 11 48.6 C

12 1.59 m 26.4 CH2 13 2.40, m 42.3 CH 12, 10, 16 14 3.56, t, J = 5 Hz, 72.1 CH 14, 8, 15

15 1.50, d, J = 10 Hz 40.03 CH2 16, 8, 7 16 3.69, t, J = 9.6 Hz 82.0 CH 13, 15, 16 17 2.73, s 64.3 CH 5, 6, 10, 11

18 3.11 and 3.54 71.9 CH2

19 54.1 CH2

N- CH2 2.48, 2.35, m 51.3 CH2

CH3 0.95, t, J = 7.2 Hz, 13.0 CH3

OCH3 3.32, s 57.3 OCH3

Chapter-3 Results and discussion

OCH2O 5.15, 4.92, s, 91.4 OCH2O

COCH3 2.08, s 29.6 CH3 C =O 171.5 C =O

3.3.2 Swatinine-D (5)

Compound 5 (Figure 3.12) was purified as amorphous powder (11 mg) from the sub- fraction AR3 on flash column chromatography using solvent system n-hexane/acetone (8:2:10 drops DEA/100 ml). The molecular formula C25H39NO6 (m/z 449.2854 calcd. 449.5787) was assigned to compound 5 on the basis of mass spectrum (HREI-MS) and nuclear magnetic resonance (NMR) spectral data which is characteristic of those alkaloids with a lycoctonine skeleton [120]. The IR spectrum of 5 showed absorption bands at 3492 cm-1 for OH, 1119 and

1064 cm-1 for simple ether bond.

OCH3 16 13 OCH3 12 14 17 OCH3 H 10 1 9 15 2 11 N 8 3 5 7 O 4 6 H O 18 19 HO

Figure 3.12. Swatinine-D (5)

Chapter-3 Results and discussion

The 1H-NMR spectrum (Table 3.5) of compound 5 showed signals for N-ethyl group, one methoxy and several methine protons. The H-14 methine proton resonated as triplet at δ 4.69 (J =

6.6 Hz, 1H). The methyl protons of N-ethyl group resonated as a triplet of three protons at δ 1.006

(J = 7.2 Hz, 3H) while the protons of methylenedioxy (OCH2-O) group appeared as singlets each at δ 5.17 and 5.06 respectively. A singlet of nine protons integration were observed at δ 3.45 due to the methoxy groups present at C-1, C-14 and C-16 in the molecule.

The 13C-NMR (BB & DEPT) spectra (Table 3.5) showed twenty five signals, three methoxy, one methyl, eight methylene, nine methine, and four quaternary carbons. The signal at δ

93.0 was assigned to methylene carbon of the methylenedioxy group (OCH2-O). The peaks at δ

78.2, 80.5 & 82.3 were assigned to C-1, C-14 and C-16 carbon atom of the compound 5.

H OCH3 H 13 16 12 14 OCH OCH3 H 3 17 1 10 9 H 2 11 15 N 8 3 5 O 7 4 6 H C H 19 O 18 H HO

Figure 3.13. Key HMBC correlation in swatinine-D (5)

The H-13 methine proton at δ (2.21) exhibited cross peak with C-14 methine proton at δ (4.69).

Similarly the H-14 methine proton shows correlation with H-9 methine proton. The remaining 75

Chapter-3 Results and discussion correlations among H-13/H-16, H-16/H-15 were confirmed from the same spectrum of compound

5. Further 1H-13C connectivities were made from HMBC spectrum (Figure 3.13) as H-9 at δ 2.04 showed 2J correlation with C-8 (δ 85.3), 3J correlations C-14 (δ 80.5) and C-10 (δ 35). In the same way the methylenedioxy protons were connected to C-7 (δ 90) and C-8 (δ 85.3) while H-14 showed cross peaks in the same spectrum with C-9 (δ 48.1) and C-13 (δ 44.3).

H OCH3

13 H 16 H 12 14 H OCH3 H3CO 17 10 H 9 H 2 11 15 N 8 3 5 H 4 7 O 6 H 19 18 O HO

Figure 3.14. Key COSY correlation in Swatinine-D 5 The proton connectivities were confirmed by 1H-1H-COSY (Figure 3.14) correlations among H-13/H-14, 14/H-9, H-13/H-16, H-16/H-15.The structure of compound 5 was established from 1H-1H COSY, 1H-13C, HMBC and HMQC experiment. Thus the structure of 5 was deduced as 1α, 14α, 16β-trimethoxy, 18β hydroxyl, 7β, 8β, methylenedioxy-N-ethyl lycoctonine

(Swatinine-D).

Chapter-3 Results and discussion

Table 3.5. 1H and 13C-NMR (600 & 150MHz) data of swatinine-D (5)

C. No 1H- δ (J Hz) 13C- (δ) Multiplicity HMBC Correlations 1 3.41, m 78.2 CH 2, 17, 11

2 2.2, m 25.1 CH2

3 26.8 CH2 4 46 C 5 2.2, t, J = 4.2 Hz 42.7 CH 4, 6, 7, 18, 17, 6 1.98, m 28.6 CH 4, 5, 7, 11, 7 90.0 C 8 85.3 C 9 2.04, d, J = 9.6 Hz, 48.1 CH 8, 10,11,13, 10 35 CH 11 51 C

12 1.57 and 1.75, m 27.1 CH2 13 2.54, br s 44.3 CH 12, 9, 16 14 4.69, d, J =6.6 Hz, 80.5 CH 13, 9, 8

15 1.75 and 2.60, m 34.1 CH2 16 3.59, d, J= 7.8Hz 82.3 CH 13, 15, 16 17 2.54, s 66.7 CH 5, 6,10, 11

18 3.38 and 3.41, m 72.0 CH2

19 2.47, s 52.8 CH2

N 2.29, 2.35, m 49.2 CH2 CH2 CH3 1.006, t, J=7.2 Hz 13.9 CH3

OCH3 3.45, s 57.7 OCH3

OCH2O 5.17, 5.06, s 93

Chapter-3 Results and discussion

3.3.3 Methyl 2-acetamidobenzoate (6)

Compound 6 was obtained from the sub-fraction AR2 on flash column chromatography using solvent system n-hexane/acetone (9:1) containing 10 drops of DEA per 100 ml.

CH3

O O

H 1 N CH3 6 2 O 5 3 4

Figure 3.15. Methyl 2-acetamidobenzoate 6

Compound 6 as white crystalline substance (17 mg) having molecular formula C10H11NO3 with molecular mass (EIMS) 193 (cald. 193.0750). In the IR spectrum of 6, absorption band at

3460 cm-1 confirmed the presence of an amide (NH) group in the molecule. The absorption signal at 1710 cm-1 showed the presence of (C = O) stretching of the ester group. The value at 1625 and

1585 cm-1 were due to the (C = C) aromatic ring present in 6.

The 1H-NMR spectrum (Table 3.6) of 6 showed signals for aromatic, acetyl, methoxy and amide protons. In the down field region of the spectrum, two doublets and two triplets of one proton integration at δ 8.02 (J = 8.1 Hz), δ 8.69 (J = 8.4 Hz), δ 7.55 (J = 8.4 Hz) and δ 7.08 (J =

7.8 Hz) were observed which are characteristic for aromatic protons of 6. A broad singlet of one proton integration at  11.03 was assigned to the NH. The 1H-NMR spectrum also displayed two

Chapter-3 Results and discussion singlets, each of three protons integration at δ 2.22 and δ 3.91 due to the methyl and methoxyl group directly linked to carbonyl functionality in the compound 6.

The 13C-NMR (BB & DEPT) spectrum (Table 3.6) of compound 6 showed ten signals, including one methoxy, one methyl, four methine and four quaternary carbons. Signals in the downfield region at  168.7 and 169.1 were assigned to the carbonylic carbons. Similarly, signals at  114.9, 141.5, 120.3, 134.6, 122.4 and 130.7 were due to the aromatic carbons. In the upfield region signals at  25.1 and  52.3 was due to the methyl and methoxyl carbon. The 1H- 13C correlations were obtained with the help of the HMQC spectrum, while the long-range connectivities were determined through the HMBC spectrum. In the HMBC spectrum (Fig 3.16),

N-H ( 11.03) showed correlation with C-1 ( 114.9) and C-3 ( 120.3). Similarly, H-4 ( 7.08) showed HMBC interactions with C-2 ( 141.5), C-3 ( 120.3) and C-6 ( 130.7).

CH3

O O H

N CH3 1 6 2 3 5 4 O

Figure 3.16. Key HMBC correlation in Methyl 2-acetamidobenzoate (6)

Chapter-3 Results and discussion

On the basis of spectral data, the structure of compound 6 was deduced as Methyl2- acetamidobenzoate which has been found as a C-18 side chain in many of the norditerpenoid alkaloids [122].

Table 3.6. 1H- (300 MHz) &13C- (75 MHz) NMR data of compound (6)

C. No 1H NMR δ (J Hz) 13C (δ) Multiplicity HMBC Correlations 1 114.9 C 2 141.5 C 3 8.6, d, J = 8.4 Hz 120.3 CH 4 7.08, d, J = 7.8 Hz 134.6 CH 2, 3, 6 5 7.55, d, J= 8.4Hz 122.4 CH 6 8.02, d, J= 8.1Hz 130.7 CH NH 11.03, br s 1,3 C=O C=O 169.1 C

OCH3 3.91, s 52.3 CH3 C=O 168.7 C

CH3 2.22, s 25.1

3.3.4 Methyl 4-[2-(methoxycarbonyl)anilino]-4-oxobutanoate (7)

Compound 7 was isolated as white crystalline solid from sub-fraction AR2 on flash column chromatography using solvent system n-hexane/acetone (9:1) containing 10 drops of DEA per 100 ml.

Chapter-3 Results and discussion

CH3

O O

H N 1 3' 4' 2' 6 2 5 3 O 1' 4 O OCH3

Figure 3.17. Methyl 4-[2-(methoxycarbonyl)anilino]-4-oxobutanoate (7)

“The IR spectrum of 7 showed absorption band at 3460 for amide (NH). The absorption band at 1730 was due to C = O stretching of ester while at 1605 and 1595 was due to (C = C) stretching of aromatic ring. The electron impact mass spectrum (EI-MS) of 7 showed the

+ molecular ion [M] at m/z 265 consistent with the molecular formula C13H15NO5 (Calcd.

265.2613). The 1H-NMR spectral data (Table 3.7) of compound 7 exhibited signals for NH, two methoxy group, two methylene and four aromatic methine protons. The 1H-NMR spectrum displayed two singlets, each of three protons integration at  3.69 and 3.91 due to the methoxyl group of 7. A broad singlet of one proton integration at  11.16 was assigned to the NH. A multiplet of four protons integration at  2.78 was assigned to the C-2 and C-3 methylene protons while,

1 multiplets in the range  7.07-8.01 in the H-NMR spectrum were due the aromatic protons.”

The 13C-NMR (BB & DEPT) spectrum (Table 3.7) of 7 showed thirteen signals, including two methoxy, two methylene, four methine and five quaternary carbons. The downfield region signals at  170.2 and 168.7 were assigned to the carbonylic carbons. Similarly, signals at  114.8

(C-1), 141.4 (C-2), 120.3 (C-3), 134.6 (C-4), 122.4 (C-5) and 130.7 (C-6) were due to the aromatic

Chapter-3 Results and discussion carbons. In the upfield region signals at  32.4 and 29.1 were assigned to the C-2′ and C-3′ methylene carbons, whereas, the signal at  52.3 and 51.6 was due to the methoxyl carbon. The

1H- 13C correlations were obtained with the help of the HMQC spectrum, while the long-range connectivities were determined through the HMBC spectrum.

In the HMBC spectrum (Figure3.18), N-H ( 11.16) showed correlation with C-2 (

141.4), C-4′ ( 170.2), and C-3 ( 120.3). Similarly, H-3′ ( 2.78) showed HMBC interactions with

C-2′ ( 32.6), C-4′ ( 170.2) and C-1′ ( 170.2). Furthermore, H-4 ( 7.52) showed correlation with C-2 ( 141.4), C-3 ( 120.3), and C-6 ( 130.7).

CH3

O O

H H

N 3' 1 4' 6 2 2' 5 3 1' 4 O 1 O OCH3 H

Figure 3.18. Methyl 4-[2-(methoxycarbonyl)anilino]-4-oxobutanoate 7

On the basis of spectral data, the structure of 7 was deduced as methyl 4-[2-

(methoxycarbonyl)anilino]-4-oxobutanoate which has been found as a C-18 side chain in many of the norditerpenoid alkaloids [122].

Chapter-3 Results and discussion

Table 3.7. 1H- (300 MHz) &13C- (75 MHz) NMR data of compound (7)

C. No 1H NMR δ (J Hz) 13C (δ) Multiplicity HMBC Correlations

1 114.8 C 2 141.4 C 3 8.01, m 120.3 CH 1, 5, 4, C=O

4 7.52, m 134.6 CH 2, 3, 6

5 7.07, m 122.4 CH 1, 2, 3, 6

6 7.99, m 130.7 CH 2, 5, 4

1′ 170.2 C

2′ 2.78, m 32.4 CH2 1′, 3′, 4

3′ 2.78, m 29.1 CH2 1′, 2′, 4′

4′ 170.2 C

C=O 168.7

OCH3 3.91, s 52.3 CH3

OCH3 3.69, s 51.6 CH3 NH 11.16, br s 1, 3, 4′

3.4 New diterpenoids from D. denudatum Wall.

3.4.1 1β-hydroxy, 14β-acetyl condelphine (8)

Compound 8 (Figure 3.19) was purified from sub-fraction D4, obtained from chloroform soluble part of crude methanolic extract, using 5% acetone/n-hexane (5:95) containing 10 drops of

DEA per 100 ml. The IR spectrum of 8 displayed absorptions band at 3500 (OH), 3155 cm-1 (H- bonded OH), 1734 & 1222 cm-1 (C=O, OAc). The molecular formula of 8 was established as

C25H39NO6 from the EI-MS spectrum, m/z: 449 (calcd: 449.5880)

Chapter-3 Results and discussion

OCH3

13 16 OH 12 14 OCOCH 17 H 3 H 1 10 9 15 2 11 N 8 3 5 OH 4 6 7 H 19 18

H3CO

Figure 3.19. 1β-hydroxy, 14β-acetyl condelphine (8)

The 1H-NMR spectrum (Table 3.8) of 8 showed a broad singlet of one proton at δ 7.15 which was assigned to hydroxyl group at C-1. The H-14 proton with α orientation resonated as a triplet of one proton integration at δ 4.89 (J = 4.75 Hz) while the singlet appeared at δ 2.08 was due the methyl of the acetyl group with β configuration at C-14. The 1H-NMR spectrum of 8 also exhibited two singlets separately at δ 3.34 and δ 3.28 for methoxyl groups at C-16 and C-18 respectively. A broad singlet of three protons integration at  1.15 was due to the methyl of N- ethyl group. The H-1 proton with α orientation also resonated as singlet at  3.78 while the H-16 proton resonated as doublet at δ 3.04 (J = 8.75Hz). The 1H-NMR spectrum of 8 also displayed a doublet of two proton integration at  3.16 (J = 9.75 Hz) due to H-18.

13 The C-NMR (BB) spectrum (Table 3.8) aided by DEPT of 8 in CDCl3 exhibited twenty five signals, including one acetoxy, two methoxy, one methyl, eight methylene, nine methine, and three quaternary carbons. The two methoxyl carbons resonated at δ 59.45 and δ 56.49 while the carbonyl carbon of acetyl group resonated at δ 170.42. In the downfield region of the spectrum,

Chapter-3 Results and discussion the signal at δ 82.00, 78.98, 76.99, 74.73 and 72.08 were assign to the substituted carbons (C-16,

C-18, C-14, C-8 and C-1) respectively. In the upfield region, the signals for other carbons are in their expected range. H-13C connectivities were established from HMBC spectrum (Figure 3.20) as H-6 proton showed correlation with C-7 (45.54), C-5 (41.43) and C-11 (48.97). In the same way, H-14α revealed 3J correlation with C-8 (74.73), C-9 (44.78), C-13 (36.57) and C-10 (43.32) respectively. Further H-C connectivities were made from the same HMBC spectrum as H-9 at δ

2.02 showed correlations with C-8 (δ 74.73) and C-10 (δ 43.32). The H-13 showed cross peaks with C-12 (δ 26.66) and C-16 (δ 82). Further H-13C connectivities were made from the same

HMBC spectrum (Figure 3.20).

H OCH3 H 13 H 16 12 OH H 14 OCOCH3 17 10 1 9 H 2 11 15 N 8 3 5 OH 7 4 6 19 18 H HO

Figure 3.20. Key HMBC interaction in1β-hydroxy, 14β-acetyl condelphine (8)

The protons connectivities were confirmed by 1H-1H-COSY correlations in the basic skeleton. H-13 methine proton at δ 2.3 showed cross peak with C-14 methine proton at δ 3.56.

Chapter-3 Results and discussion

Similarly, the H-9 ( 2.02) proton showed coupling with H-14 ( 4.89), which in turn showed coupling with H-13 ( 2.3), supporting the presence of acetyl group at C-14 ( 76.99).

Further 1H-1H-COSY (Figure 3.21) correlations were established between H-5, H-6, H-7 and

H-17 proton from the same spectrum. The overall spectral data of 8 exhibited close resemblance to the known compound condelphine, previously isolated from D. roylei Munz [88], with the difference in configuration at C-1 and C-14 positions.

OCH H 3 H H 16 12 13 OH H 14 OCOCH3 H 17 H 1 10 9 2 11 15 N 8 H 3 5 OH 7 H 4 6 H H 19 H 18 HO

. Figure 3.21. Key COSY correlation in 1β-hydroxy, 14β-acetyl condelphine (8)

The single-crystal X-ray crystallographic study of 8 was also carried out for the first time.

The compound 8 was crystallized in monoclinic unit of crystal system with space group of P21.

The structure of 8 is given in figure 3.22 and the data permitted to crystal structure refinement and geometric parameters of compound 8 is given in table 3.9. All the bond length and angles are in expected range [112]. The compound 8 is highly rigid consist of six main rings attaining chair

Chapter-3 Results and discussion configuration in which eight member ring is more twisted. The structure and relative α, β configuration was unambiguously deduced on the basis of X-ray diffraction studies (Figure 3.22).

The hydroxyl group (δ 7.15) at C-1 (δ 72.08) is β-oriented in the new compound 8 whereas in condelphine, it was α-oriented. Similarly the acetyl group at C-14 (δ 76.99) is beta (β) in the new compound 8 while in condelphine; it was α- oriented.

Figure 3.22. Structural representation of 8, with 50 % probability of thermal ellopsiodes and hydrogen atoms are omitted for clarity.

On the basis of the X-ray diffraction studies (Figure 3.22) &13C-NMR spectral data, the structure of 8 has been established as 1-β hydroxy, 14-β acetyl condelphine.

Chapter-3 Results and discussion

1 13 Table 3.8. H- (500 MHz) and C-NMR (125 MHz) data of compound (8)

C. No 1H- δ (J Hz) 13C- (δ) Multiplicity HMBC Correlations 1 3.78, brd s 72.08 CH 2, 17, 11

2 1.9, m 29.07 CH2

3 29.69 CH2 4 37.26 C 5 41.43 CH 6 1.95, m 25.02 CH 5, 7, 11, 7 45.54 CH 8 74.73 C 9 2.02, m 44.78 CH 8, 10,11,13, 10 43.32 CH 11 48.97 C

12 1.64 and 1.87 26.66 CH2 13 2.3, t, J = 9.1 Hz 36.57 CH 12,16 14 4.89, t, J = 4.75 Hz 76.99 CH 13, 9, 8

15 1.91, m 42.61 CH2 16 3.04, d, J= 8.7 Hz 82.00 CH 13, 15, 16 17 2.77, s 63.60 CH 10, 11

18 3.16, d, J= 9.75 Hz 78.98 CH2

19 2.12, s 56.08 CH2

N- CH2 2.35, 2.30, m 48.52 CH2

CH3 1.15, brd s 12.99 CH3

2xOCH3 3.34, 3.28, s 59.45, 56.49 2xOCH3

COCH3 2.08, s 170.4, 21.3 -C=O,CH3

Chapter-3 Results and discussion

Table 3.9. X-ray data and structure refinements of compound 8

CRYSTAL DATA

-3 C25H39NO6 Dx = 1.283 Mg m

Mr = 449.56 Mo Kα radiation

Monoclinic, P2 λ = 0.71073 Å a =8.9365 (4) Å Cell parameters from 4280 reflections b = 13.1841 (7) Å θ = 2.8-25.5o c = 9.8757 (5) Å μ = 0.09 mm-1

β = 93.633 (3)° T = 0 K

V = 1161.21 (10) Å3 Colorless Solid

Z = 2 0.75 x 0.20 x 0.19 mm

DATA COLLECTION

Bruker kappa APEXII CCD diffracto-meter 4280 independent reflections

ω scans 3741 reflections with I> 2σ(I)

SIR97 [110], SHELXL97 [111] and WinGX Rint = 0.128 [112].

15678 measured reflections θmax = 25.5°

h = -10 → 10

k = -15→ 15

l = -11 → 11

REFINEMENT

2 2 2 2 Refinement on F w = 1/[σ (Fo ) + (0.0776P) + 0.5763P]

R[F2> 2σ(F2)] = 0.062 where

Chapter-3 Results and discussion wR(F2) = 0.161

2 2 S = 1.05 P = (Fo + 2Fc )/3

4280 reflections (∆/σ)max= 0.216

−3 290 parameters ∆ρmax = 0.40 e Å

−3 H atoms treated by a mixture of independent ∆ρmin= −0.38 e Å and constrained refinement

3.4.2 HOMO-LUMO energy gap of 1β-hydroxy, 14β-acetyl condelphine (8)

The energy gaps between HOMO-LUMO are 0.167 for compound 8 which shows the stability of LUMO due to the electron accepting properties. The low band gap is due to electron donating groups and other parameters of the compound are responsible for its high reactivity and low stability [123]. The calculated energy parameters of 8 are given in table 3.10

Table 3.10. List of HOMO-LUMO energy, ionization energy (IE), electron affinity (EA) global hardness (η), chemical potential (μ) and global electrophilicity (ω) of 8

ЄHOMO (eV) -0.160

ЄLUMO (eV) 0.007

ΔЄ = (ЄLUMO-ЄHOMO) (eV) 0.167

IE= -ЄHUMO (eV) 0.160

EA= -ЄLUMO (eV) -0.007

Global Hardness(η) = 1/2 (ЄLOMO-ЄHOMO) 0.083

Chemical Potential μ = 1/2 (ЄHOMO+ЄLUMO) -0.076

Global Electrophilicity ω = μ2/2η 0.034

Chapter-3 Results and discussion

3.4.3 1,14α, 8,10β-Tetrahydroxy, 16,18β dimethoxy-N-ethyl- aconitine (9)

Compound 9 was isolated from the sub-fraction D7 of the basic chloroform fraction using solvent system n-hexane/acetone (8:2) containing 10 drops of DEA. Compound 9 was assigned the molecular formula C23H37NO6 on the basis of its molecular ion peak at m/z 423 (calcd. for

423.5415) from electron impact mass spectrum (EI-MS). The infrared (IR) spectrum of 9 showed absorption bands at 3550, 3487, 1073 cm-1 for hydroxyl and C-O bond respectively.

OCH3 13 16 OH 12 17 14 OH OH H 1 10 9 2 11 15 N 8 3 5 4 7 6 OH H 19 18

H3CO

Figure 3.23. 1,14α, 8,10β-Tetrahydroxy, 16,18β dimethoxy-N-ethyl-aconitine (9)

The 1H-NMR spectrum of 9 exhibited triplet signals in the upfiled region for methyl of N- ethyl group at δ 1.07 (3H, t, J= 7.14 Hz). In the down field region of the spectrum, a triplet of one proton integration at δ 4.29 (J = 4.8 Hz) was observed which is characteristic for H-14β methine proton. A broad singlet of one proton integration was observed at δ 3.89 for H-1 proton of 9. Two singlet peaks at δ 3.36 & δ 3.32, each of three protons integration was assigned to two methoxyl groups at C-16 and C-18 of 9. The H-18 methylene protons resonated as triplet of doublet at δ 3.13

(J= 8.04 Hz) and 3.05 (J= 9.06 Hz). A sharp singlet of one proton integration at δ 2.59 was due to

Chapter-3 Results and discussion the resonance of H-17 proton.The overall spectral data of 9 was similar to that of isotalatizidine

[88], except the presence of an additional hydroxyl group at C-10 position.

The 13C-NMR (Table-3.10) spectrum (BB & DEPT) showed twenty three signals, including two methoxy, one methyl, eight methylene, eight methine, and four quaternary carbons.

The 13C-NMR spectrum of 9 displayed that the methyl carbon of N-ethyl group resonated at δ 13.5 while the two methoxyl carbons resonated at δ 59.5 and 56.3 respectively. In the downfield region of the spectrum, the signals at δ 82.2, 79.4, 77.1, 75.8, 73.8, and 68.7 were due to the resonance of

C-16, C-18, C-10, C-14, C-8 and C-1 carbons. The quaternary carbons C-4 and C-11 resonated at

δ 41.4 and 49.4 respectively. All the other methine and methylene carbons resonated in their expected range as confirmed from the same 13C-NMR (BB) spectrum.

H H3CO H 13 H 16 12 H 14 OH OH OH 17 10 1 9 H 2 11 15 N 8 3 5 OH 7 4 6 19 CH H 182 H3CO

Figure 3.24. Key HMBC correlation in compound 9

The 1H-13C correlations were determined by HMQC spectrum, while the long-range 1H-

13C connectivity was obtained through the HMBC technique (Figure-3.24). H-13 proton showed

Chapter-3 Results and discussion correlations with C-16 (δ 82.2), C-10 (δ 77.1), C-12 (δ 30.5) and C-14 (δ 75.8), whereas the H-16 proton showed interactions with C-13 (δ 39.3) and C-15 (δ 44.6) respectively. Similarly the HMBC interactions of H-18 were observed with C-3 (δ 27.1), C-4 (δ 41.4), C-5 (δ 38.5) and C-19 (δ 53.2).

Further1H-13C connectivity was established from the same spectrum as H-6 exhibited interaction with C-5 (38.5), C-7 (46.7) and C-8 (73.8) respectively. Thus, on the basis of the above spectral evidences the structure of 9 was deduced as 1,14α, 8,10β-tetrahydroxy, 16,18β dimethoxy-N-ethyl- aconitine.

1 13 Table 3.11. H- (600 MHz) and C-NMR (150 MHz) data of compound (9)

C. No 1H- δ (J Hz) 13C- (δ) Multiplicity HMBC Correlations

1 3.89, brd s 68.7 CH 2, 17, 11 2 1.91, m 28.9 CH2 3 1.22, m 27.1 CH2 4 41.4 C 5 38.5 CH 6 1.6, s 24.7 CH 5, 7, 11, 7 2.28, t, J = 5.94 Hz 46.7 CH 8 73.8 C 9 2.05, d, J= 8.16 Hz 41.6 CH 8, 10,11,13, 10 77.1 C 11 49.4 C

12 1.7, m 30.5 CH2 13 2.3, m 39.3 CH 10, 12, 14, 16 14 4.29,t, J = 4.8 Hz, 75.8 CH 13, 9, 8 15 1.98, m 44.6 CH2 16 3.33, s 82.2 CH 13, 15, 16

Chapter-3 Results and discussion

17 2.59, s 63.9 CH 10, 11 18 3.13,3.05, dd, J= 8.05, 9.06 Hz, 79.4 CH2 3, 4, 5, 19 19 2.52, s 53.2 CH2 N- CH2 2.40, 2.38, m 49.04 CH2 1.07 t, J=7.14 Hz CH CH3 13.5 3 59.5 OCH 3.32, 3.36 s 2xOCH3 3 56.3

3.4.4 1α,8,16,18β-tetramethoxy, 14α-hydroxy-N-ethyl- 5-aconitene (10)

Compound 10 was obtained from sub-fraction D8 (70 mg) on repeated flash column chromatography, using n-hexane/acetone (8:2) containing 10 drops of DEA. The molecular formula C25H39NO5 and its structure were established on the basis of mass spectrum (EIMS) and nuclear magnetic resonance (NMR) spectral assignments. The infrared (IR) spectrum of 10 showed absorption bands at 3466 cm-1 and 3404 cm-1 (OH) group, 3015 cm-1 (C=C) and 1097 cm-1 (C-O) respectively.

OCH3

13 16 12 OCH3 17 14 OH

H 1 10 9 2 11 15 N 8 5 3 OCH 4 6 7 3

19 18

H3CO

Figure 3.25. 1α,8,16,18β-Tetramethoxy, 14α-hydroxy-N-ethyl-5-aconitene 10 94

Chapter-3 Results and discussion

The 1H-NMR spectrum (Table 3.11) of 10 revealed signals for various protons including N- ethyl group, four methoxy groups, and several methine protons. In the down field region, one proton doublet at δ 5.37 (J = 4.1 Hz) was observed for H-6 proton which indicate the presence of

C=C bond at C-5 & C-6 position of the molecule. Similarly, singlet of one proton integration at δ

4.04 was observed for H-1 proton in the molecule. The signal for H-14β in the 1H NMR spectrum was observed as triplet at δ 4.30 (J= 5 Hz). In the up field region, a triplet of three proton integration at δ 0.96 (J= 7.7 Hz) was assigned to the methyl of N-ethyl. The 1H NMR spectrum also displayed four singlets at δ 3.47, 3.39, 3.36, and δ 3.32 corresponding to four methoxy groups at C-18, C-1,

C-16 and C-8 positions respectively. The H-18 protons resonated as multiplet of two protons integration at δ 3.17 while the H-16 proton resonated as one proton doublet at δ 3.42 (J= 8.4 Hz) ppm. All the other methine and methylenic protons have demonstrated signals in their respective range, common to all aconitane type of C19 diterpenoid alkaloids.

The 13C-NMR (BB and DEPT) spectrum (Table 3.11) of 10 in chloroform exhibited twenty five signals, including four methoxy, one methyl, seven methylene, nine methine, and four quaternary carbons. The signals of C-1 at δ 87.5 and C-2 at δ 24.3 ppm supports the presence of a methoxy group at C-1 with β configuration, since norditerpenoid alkaloids with C-1 β- methoxy group exhibited a sharp increase in the intensity of the M+ -15 peak as compared with their α counterpart [64]. The four methoxyl groups of 10 resonated at δ 59.7, 58.0, 57.3, and 56.2 while the C-4, C-8 and C-11 quaternary carbons gives signal at δ 42.0, 86.73 and 52.92 respectively. In the downfield region the signals at δ 148.03 and 130.1 were due to the resonance of olefinic carbons. All the other methane and methylene carbons resonated in their expected range.

Chapter-3 Results and discussion

The HMQC and HMBC technique were used to establish 1H-13C correlations in compound

10 (Figure 3.26). H-6 proton showed correlations with C-5 (δ 148.0), C-6 (130), C-7 (δ 45.2) and

C-11 (52.92) whereas H-14 proton showed interactions with C-9 (δ 47.4) and C-13 (δ 43.3).

Similarly the HMBC interactions of H-16 were observed with C-15 (δ 41.09) and C-13 (δ 43.3) respectively.

H OCH3 H 13 H 16 12 14 OH OCH3 17 1 10 9 H 2 11 15 N 8 3 5 OCH3 7 4 6

19 H CH2 18 H3CO

Figure 3.26. Key HMBC interaction in compound 10

Henece, on the basis of aforementioned spectral data, the structure of compound 10 was deduced as 1α, 8, 16,18β-tetramethoxy, 14α-hydroxy, 5-Aconitene 10.

Chapter-3 Results and discussion

1 13 Table 3.12. H- (500 MHz) and C-NMR (125 MHz) data of compound (10)

C. No 1H- δ (J Hz) 13C- (δ) Multiplicity HMBC Correlations 1 4.04, brd s 87.5 CH

2 1.81, br s 24.3 CH2

3 1.32, m 22.89 CH2 4 42 C 5 148.03 CH 6 5.37, d, J= 4.1 Hz 130.10 CH 5, 7, 11, 7 2.34, m 45.21 C 8 86.73 C 9 2.03, d, J= 6.25 Hz 47.40 CH 8, 10,14, 10 32.50 CH 11 52.92 C

12 1.30, m 27.14 CH2 13 43.30 CH 12,14, 16 14 4.30, t, J = 5 Hz, 75.00 CH 13, 9, 8

15 1.98, d,J= 5.15 Hz 41.09 CH2 16 3.42, d, J= 8.4 Hz 80.01 CH 13, 15, 16 17 2.78, d, J= 7.7 Hz 63.21 CH

18 3.17, m 79.02 CH2

19 2.39, d, J= 6.9Hz 53.14 CH2

N- CH2 2.33, m 49.02 CH2 0.96, t, J = 7.7 Hz 13.9 CH CH3 3

OCH3 3.47, 3.39, 3.36, 3.32 s 59.7, 58.0 4xOCH3 57.3, 56.2

Chapter-3 Results and discussion

3.5 Known diterpenoids from A. heterophyllum Wall.

3.5.1 Isoatisine (11)

Compound 11 was purified from sub-fraction AH1, obtained from the basic chloroform soluble fraction of crude methanolic extrac. The IR spectrum of 11 showed absorptions bands at

-1 -1 3356 cm (OH) and 3012, 1656 cm (C=CH2). The molecular formula (C22H33NO2) of compound was established on the basis of electron ionization mass spectrum (EI-MS) with molecular ion peak [M]+ at m/z 343 (calcd: 343.5110).

13 17 12 CH2 11 16 20 14 15 1 9 OH 21 2 10 8 N 3 22 5 7 4 6

O 19 18

Figure 3.27. Isoatisine 11

The 1H-NMR spectrum of compound 11 displayed two singlets each of one proton integration at  5.1 and 5.03 due to the presence of terminal methylen group in the molecule. A singlet of three protons integration at  1.08 indicated the presence of methyl groups in compound

11. Two multiplets, each of two protons integration at  3.85 and 2.39 in the 1H-NMR spectrum of 11 was assigned to C-22 and C-21 proton respectively. A broad singlet of one protons integration at δ 3.97 was assigned to C-19 protons. 98

Chapter-3 Results and discussion

The twenty-two signals in the broad band 13C-NMR spectrum were resolved into one methyl, twelve methylene, five methine and four quaternary carbons. In the 13C NMR spectrum of

11, the signals at δ 156.6 and 109.5 was due to the resonsnce of C-16 and C-17 carbons. A sharp signal at δ 98.5 in the same spectrum was assigned to C-19.

The single-crystal X-ray crystallographic study of 11 was also carried out. The compound

11 is crystallized in orthorhombic unit of crystal system with space group of P212121. The structure of the 11 is given in figure 3.28 and the data permitted to crystal structure refinement and geometric parameters of 11 is given in table 3.13.

Figure 3.28. Structural representation of compound 11, with 50 % probability of thermal ellopsiodes and hydrogen atoms are omitted for clarity.

Chapter-3 Results and discussion

Table 3.13. Crystal data and structure refinements of compound 11

Crystal Parameter 11 Crystal Parameter 11

3 3 Empirical formula C22H33NO2 Volume Å 1874.20 (18) Å

Formula weight 343.4412 μ (mm-1) 0.08

Temperature (K) 296 Z 2

Wavelength (Å) 0.71073 Density (Mg m-3) 1.217

Crystal system Orthorhombic (h, k, l) min (-16, -13, -17)

Space group P212121 (h, k, l) max (16, 13, 16)

A 13.1842 (7) Å Theta (max) 27.2

B 10.4009 (6) Å R (reflection) 0.062(4140)

C 13.6676 (7) Å wR2 0.189

3.5.2 19-Epiisoatisine (12)

Compound 12 was isolated from the sub fraction AH1 on repeated flash column chromatgarpy, using n-hexane/acetone (98:2) containing 10 drops of DEA per 100 ml. The IR

-1 -1 spectrum of 12 exhibited absorption band at 3356 cm (OH) and 3012, 1656 cm (C=CH2). The high resolution mass spectrum (HREI-MS) 12 showed the molecular ion [M]+ at m/z 343.421, with molecular formula C22H33NO2 (calcd:343.5110).

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Chapter-3 Results and discussion

13 17 12 CH2 11 16 20 14 15 1 9 OH 21 2 10 8 N 3 22 5 7 4 6

O 19 18

Figure 3.29. 19-epiisoatisine (12)

The 1H-NMR spectrum of 12 has singlets signals each of one proton integrations at δ 5.09 and 5.03 were due to terminal methylene group. The broad singlet signal appeared at δ 2.85 was assigend to the H-20 proton of the compound 12. Similerly the H-18 protons of the methyl group resonated at δ 1.09/.09 where as sevral multiplet signal at δ 3.83, 2.39 and 2.1 was observerd for

H-22, H-21 and H-13 respectively. The 13C-NMR spectrum (BB & DEPT) experiment showed twenty two signals, including one methyl, twelve methylen, five methane and four quaternary carbons. The signals at δ 156.7 and 109.7 were assigned to the C-16 and terminal methylen group of 12 while the signal at δ 96.3 was assigned to C-19 methine carbon bonded to oxygen atoms.

The peak at δ 76.7 was due to C-15 bearing a beta hydroxyl group.

The overall spectral data of 12 was compared with that of the known compound available in literature [59] and hence the structure of 12 was deduced as 19-Epiisoatisine.

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Chapter-3 Results and discussion

3.5.3 Atidine (13)

The sub-fraction AH2 was purified by flash column chromatography and eluted with n- hexane/acetone (9:1) containing 10 drops of DEA per 100 ml to afford a white crystalline compound, atidine 13 (Figure 3.30).

17 CH2 11 12 20 16 14 15 1 9 OH 2 10 8 N 3 5 7 4 6 O HO 19

Figure 3.30. Atidine (13)

The IR spectrum of 13 had an absorption bands at 3540, 3555 cm-1 (OH), 3088, 1658 and

-1 900 cm (C = CH2, terminal methylene) and is in good agreement with the reported values. The mass spectrum (EI-MS) of 13 showed the molecular ion [M]+ at m/z 359.16 consistent with the

1 molecular formula C22H33NO3 (calcd:359.5010 ). The H-NMR spectrum displayed two doublets each of one proton integration at  5.2 and 5.07 (J = 3.8 Hz) due to the protons of terminal methylene.

The 1H-NMR spectrum of 13 indicated a broad singlet of one proton integration at  4.56 which was assigned to the proton of C-15 in the molecule.Two triplets signals of two protons integration at  3.70 (J = 5.5Hz) and  2.97 (J = 11.25Hz) were assigned to H-22 and H-21 protons

102

Chapter-3 Results and discussion respevtively. The 13C-NMR of 13 showed twenty-two signals which were resolved through DEPT experiment as one methyl, twelve methylene, four methine and five quaternary carbons.

All the structural assignments in the IR and 1H-NMR spectrum were further supported by the 13C-NMR values. In the 13C-NMR spectrum of 13, a downfield signal at  216.7 was assingn to the ketonic carbon while the signals at  151.7 and 109.8 were due to the resonance of C-16 and

C-17 (terminal methylene). The parameters of all carbon signals were in good agreement with those for the literature values of atidine (13). On the basis of the above spectral evidences and comparison with the reported data [124], the structure of 13 was elucidated as atidine.

3.5.4 Heteratisine (14)

The sub-fraction AH2 was loaded on flash column chromatography and eluted by n- hexane/acetone (9:1) containing 10 DEA per 100 ml to afford a white crystaline compound, heteratisine 14 (Figure 3.31).

13 O 16 12 H3CO 17 14 C O

1 10 9 15 2 11 N 8 3 5 7 OH 4 6

19 18 HO

Figure 3.31. Heteratisine (14)

The IR spectrum of 14 displayed strong absorption bands at 3460, 3400 cm-1due to OH,

-1 and 1738 cm (α- lactone) respectively. The compound 14 showed molecular formula C22H33NO5

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Chapter-3 Results and discussion with a molecular weight of 391 (calcd: 391.5080) as determined from the EI-MS.In the 1H-NMR spectrum of compound 14, a sharp singlet of two protons integration at  4.86 was assigned to the

H-13 proton. A doublet of one proton at  4.45 was assigned to H-6 proton of 14.

The 1H-NMR spectrum also displayed singlet of three protons integration at  3.33 due to methoxyl group. In the same way the H-1 proton also resonated as singlet of one proton integration at  3.26 where as the H-18 proton resonated as singlet at  0.99 respectively. All the assignments in the IR and 1H-NMR spectra were further supported by the 13C-NMR values. The twenty-two signals in the broad band of the 13C-NMR spectrum were resolved into three methyl, nine methylene, five methine and five quaternary carbons. In the downfield region of 13C-NMR spectrum, signal at  175.5 was assigned to carbonylcarbons while the C-1 substituetd carbon resonated at  83.2 respectively. The parameters of all carbons signal were in good agreement with those for the literature values of heteratisne.

The single-crystal X-ray crystallographic study of compound 14 was also carried out. The compound 14 is crystallized in orthorhombic unit of crystal system with space group of P212121.

The structure of the compound 14 is given in figure 3.32 and the data permitted to crystal structure refinement and geometric parameter of compound 14 is given in table 3.13.

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Chapter-3 Results and discussion

Figure 3.32. Structural representation of compound 14, with 50 % probability of thermal ellopsiodes and hydrogen atoms are omitted for clarity.

Table 3.14. Crystal data and structure refinements of compound 14 Crystal Parameter 14 Crystal Parameter 14

3 3 Empirical formula C22H33NO5 Volume Å 989.23 (16) Å Formula weight 391.5130 μ (mm-1) 0.10 Temperature (K) 296 Z 2 Wavelength (Å) 0.71073 Density (Mg m-3) 1.604 Crystal system Orthorhombic (h, k, l) min (-11, -8, -12)

Space group P212121 (h, k, l) max (11, 7, 8) A 9.1526 (9) Å Theta (max) 27.1 B 10.6636 (9) Å R (reflection) 0.051(2707)

C 10.1453 (9) Å wR2 0.145

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Chapter-3 Results and discussion

By comparison of the spectral and crystal data with literature clearly indicate that the structure of 14 is heteratisne, previously reported from the same plant [59].

3.5.5 HOMO-LUMO energy gap of heteratisine (14)

The energy gaps between HOMO-LUMO are 0.185 for compound 14 which shows the stability of LUMO due to the electron accepting properties. The low band gap is due to electron donating groups and other parameters of the compound are responsible for its high reactivity and low stability [123]. The calculated energy parameters of the compound 14 are given in table 3.15

Table 3.15. List of HOMO-LUMO energy, ionization energy (IE), electron affinity (EA) global hardness (η), chemical potential (μ) and global electrophilicity (ω) of 14

ЄHOMO (eV) -0.180

ЄLUMO (eV) 0.005

ΔЄ = (ЄLUMO-ЄHOMO) (eV) 0.185

IE= -ЄHUMO (eV) 0.180

EA= -ЄLUMO (eV) -0.005

Global Hardness(η) = 1/2 (ЄLOMO-ЄHOMO) 0.092

Chemical Potential μ = 1/2 (ЄHOMO+ЄLUMO) -0.087

Global Electrophilicity ω = μ2/2η 0.041

3.5.5 Hetisinone (15)

On repeated flash column chromatography of sub-fraction AH3, using n-hexane/acetone

(8:2) containing 10 drops of DEA per 100 ml, compound 15 was isolated as white crystalline solid.

It was visualized at 254 nm under UV light on the pre-coated silica gel TLC plates. In the IR spectrum of 15, absorption bands exhibited at 3575 cm-1, 1725 cm-1, 1655 and 910 cm-1 were due

106

Chapter-3 Results and discussion to OH, C=O and C = CH2 stretching vibartions. The molecular formula C20H25NO3 of 15 was constituted from mass spectrum (EI-MS) with molecular ion [M]+ at m/z 327.

13 12 17 HO CH2 11 16 20 14 O 15 1 9 2 10 8 N 3 5 7 4 6

19 18

Figure 3.33. Hetisinone (15)

The 1H-NMR spectrum of compound 15 exhibited singlets of one proton integration at 

4.88 and 4.70 due to terminal methylene group. Similerly a doublt of one proton at  4.25 (J = 8.9

Hz) was observed due to reosnance of hydroxyl substituted H-11 proton. The H-13 proton of 15 produced a triplet of triplet at  4.12 (J = 2.6 Hz) in the same spectrum. All the assignments in the

IR and 1H-NMR spectra were further supported by the 13C-NMR values.

The twenty signals in the broad band of the 13C-NMR spectrum were resolved into one methyl, six methylene, eight methine and five quaternary carbons. In the downfield region of 13C-

NMR spectrum, signal at  214.7 was assigned to ketonic functionality whereas the the terminal methylene carbon resonated at  106.4 respectively. Similerly the quaternary carbons C-4, C-8, C-

11 and C-16 showed signals at  41.9, 44.2, 74.7 and 146.6 respectively. The values of all other carbons signals were in good agreement with those available in literature of hetisinone.

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Chapter-3 Results and discussion

The single-crystal X-ray crystallographic study of 15 was also conducted using single XRD machine. The compound 15 is crystallized in monoclinic unit of crystal system with space group of P21. The structure of the compound 15 is given in figure 3.34 and crystal structure refinement and geometric parameter of 15 is given in table 3.16.

Figure 3.34. Structural representation of compound 15, with 50 % probability of thermal ellopsiodes and hydrogen atoms are omitted for clarity.

Table 3.16. Crystal data and structure refinements of compound 15

Crystal Parameter 15 Crystal Parameter 15

3 3 Empirical formula C20H25NO3.H2O Volume Å 864.10 (10) Å

Formula weight 345.439 μ (mm-1) 0.09

Temperature (K) 296 Z 2

Wavelength (Å) 0.71073 Density (Mg m-3) 1.328

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Chapter-3 Results and discussion

Crystal system Orthorhombic (h, k, l) min (-8, -22, -10)

Space group P212121 (h, k, l) max (7, 21, 10)

A 6.3776 (3) Å Theta (max) 27.5

B 17.0424(13) Å R (reflection) 0.045 (3430)

C 8.1655 (6) Å wR2 0.127

By comparison of the spectral and crystal data available in literaturethe structure of compound 15 is deduced as hetisinone, previously reported from A. heterophyllum and A. naviculare [60].

3.6 Known diterpenoids from A. laeve Royle.

3.6.1 Aconorine (16)

The crude alkaloidal mixture obtained at pH 8-10 was fractionated over silica gel using

CC. Compound 16 was obtained from the sub-fraction AR2 on flash column chromatography using solvent system n-hexane/acetone (8:2:10 drops DEA/100 ml).

The IR spectrum of 16 had an absorption bands at 1710 cm-1 (ester carbonyl), 1600, 1280,

1250, and 750 cm-1 (1, 2-substituted aromatic ring), and in good agreement with the reported values. The high-resolution mass spectrum (HREI-MS) of 16 showed the molecular ion [M]+ at m/z 568.241 consistent with the molecular formula C32H44N2O7 (calcd: 568.711 ).

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Chapter-3 Results and discussion

OCH3

OCH 13 16 3 12 14 17 OH

1 10 9 2 11 15 N 8 3 5 7 4 6 OH H

19 O O

NHCOCH3

Figure 3.35. Aconorine (16)

The 1H-NMR spectrum displayed three singlets each of three protons integration at  3.39,

3.30, and 3.27 due to the protons of methoxyl groups. A triplet of three protons at  1.09 (J = 6.8

Hz) was assigned to the methyl of an N-ethyl group. In the 1H-NMR spectrum of 16 there was a broad singlet of one proton integration at  11.0 which was assigned to the proton of amino group in the molecule. A doublet of two proton at  3.52 (J = 11.2 Hz) was assigned to H-19.

The 13C-NMR of 16 showed thirty-two signals which were resolved through DEPT experiment as two methyl, three methoxyl, seven methylene, eleven methine and nine quaternary carbons. All the assignments in the IR and 1H-NMR were further supported by the 13C-NMR values. In the 13C-NMR spectrum of 16 three methoxyl signals at  57.7, and 56.52 were attached

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Chapter-3 Results and discussion to C-1, and C-16, respectively. The parameters of all carbons signals were in good agreement with those reported values in the literature of aconirine.

On the basis of the above spectral evidences and comparison with the reported data [125], the structure of 16 was elucidated as aconorine.

3.6.2 Lappaconitine (17)

The crude alkaloidal mixture obtained at pH 8-10 was fractionated over silica gel column.

Compound 17 was obtained from the sub-fraction AR2 on flash column chromatography using solvent system n-hexane/acetone (8:2: 10 drops DEA/100 ml)

OCH3

OCH 13 16 3 12 14 17 OCH3

1 10 9 OH 2 11 15 N 8 3 5 7 4 6 OH H

19 O O

NHCOCH3

Figure 3.36. Lappaconitine (17)

The IR spectrum of 17 displayed strong absorption bands at 3500, 3250 cm-1 due to OH, and NH groups respectively. The absorption at 1680 cm-1 showed the carbonyl function in the

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Chapter-3 Results and discussion molecule while the absorption band at 1080 cm-1 was because of the ether group. The compound

17 showed molecular formula C32H44N2O8 with a molecular weight of 585.678 (584.708 calcd.) as determined from the HREI-MS.

In the 1H-NMR spectrum of compound 17 a broad singlet of one proton integration at 

11.01 was assigned to the proton of amino group in the molecule. A doublet of one proton at 

3.56 was assigned to H-19. The 1H-NMR spectrum also displayed three singlets each of three protons at  3.38, 3.28, and 3.27 due to methoxyl groups. A singlet of three protons integration at

 2.19 was assigned to the methyl of acetyl group. A triplet of three protons at  1.11 (J = 6.8 Hz) was assigned to the proton of the methyl of an N-ethyl group. All the assignments in the IR and

1H-NMR spectra were further supported by the 13C-NMR values.

The thirty-two signals in the broad band of the 13C-NMR spectrum were resolved into five methyl, seven methylene, thirteen methine and seven quaternary carbons. In the 13C-NMR spectrum of compound 17, three signals at  57.9, 56.5, and 56.1 were assigned to methoxyl carbons attached to C-14, C-1 and C-16, respectively. The parameters of all carbons signals were in good agreement with those for the literature values of lappaconitine.

On the basis of the above spectral evidences and comparison with reported data [81-83], the structure of compound 17 was deduced as lappaconitine.

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Chapter-3 Results and discussion

3.7 Known diterpenoids from D. denudatum Wall

3.7.1 Isotalatizidine hydrate (18)

Compound 18 was obtained from the crude 80% methanolic extract of D. denudatum Wall.

The crude alkaloidal mixture obtained at pH 8-10 was fractionated over silica gel column.

Compound 18 was purified as white crystal from sub-fraction D4. The IR spectrum of compound

18 exhibited a strong absorption bands at 3350, 3414 cm-1 due to hydroxyl. The compound 18 has the molecular formula C23H39NO6, with a molecular weight of 425.5660 (calcd.), established on the basis of single X-ray analysis.

OCH3 13 16 12 OH 17 14 OH H H 1 10 9 15 2 11 N 8 3 5 OH 4 6 7 H .H2O 19 18

H3CO

Figure 3.37. Isotalatizidine Hydarte 18

In the 1H-NMR spectrum of 18, a triplet of three protons integration at  1.14 (J = 7.14 Hz) for a methyl of an N-ethyl group, and two singlets each of three protons at  3.33 and 3.38 for two methoxyl groups. The 1H-NMR spectrum also displayed signals at  3.04 and  3.17 attributed to an oxygenated C-18 methylene proton, and singlet signals at  3.74 was assigned to oxygenated methines protons at C-1. The β-oriented H-14 proton showed a triplet signal at  4.25 (J= 5.1Hz)

113

Chapter-3 Results and discussion respectively. The multiplet signal at  3.42 were due to H-16 proton where as a sharp singlet of one proton integration at  2.81 was assign to H-17 proton in the same 1H-NMR spectrum.

The twenty-three signals in the broad band 13C-NMR spectrum were resolved into three methyl, eight methylene, nine methine and three quaternary carbons. In 13C-NMR spectrum of 18 two signals at  56.3 and  59.4 were assigned to the methoxyl carbons attached to C-16 and C-

18, respectively. The parameters of all carbons signal were in good agreement with those available in literature for isotaltizidine previously isolated from D. roylei [88].

The single-crystal X-ray crystallographic study of 18 was also carried out for the first time.

The compound 18 is crystallized in hexagonal unit of crystal system with space group of P65. The structure of 18 is given in figure 3.38 and the data permitted to crystal structure refinement and geometric parameters of 18 is given in Table 3.17. All the bond length and angles are in expected range. All other rings twisting and structural parameter are similar to compound 18.

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Chapter-3 Results and discussion

Figure 3.38. Structural representation of compound 18, with 50 % probability of thermal ellopsiodes and hydrogen atoms are omitted for clarity.

Table 3.17. X-ray data and structure refinements of isotalatizidine hydrate (18)

Crystal data

-3 C23H39NO6 Dx = 1.247Mg m

Mr = 425.01 Mo Kα radiation

Hexagonal, P65 λ = 0.71073 Å

a= 10.700 (3) Å Cell parameters from 4536 reflections

c= 34.28 (1) Å θ = 3.7-27.6o

β = 102.744 (2)o μ = 0.09 mm-1

V = 3399 (2) Å3 T = 296 K

Z = 4 Block, colorless

0.37 x 0.30 x 0.28mm

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Chapter-3 Results and discussion

Data collection

Bruker kappa APEXII CCD diffracto- 4536 independent reflections

meter 3134 reflections with I> 2σ(I) ω scans Rint = 0.055 SIR97 [110], SHELXL97 [111] and θmax = 27.6° WinGX [112]. h = -12 → 13 15455 measured reflections k = -13 → 10

l = -35 → 44

Refinement

2 2 2 2 Refinement on F w = 1/[σ (Fo ) + (0.1075P) ] 2 2 R[F2> 2σ (F2)] = 0.062 where P = (Fo + 2Fc )/3

wR(F2) = 0.170 (∆/σ)max = 0.001 -3 S = 0.99 ∆ρmax = 0.24 e Å -3 4536 reflections ∆ρmin = -0.30 e Å

283 parameters

H atoms treated by a mixture of

independent and constrained refinement

On the basis of the above spectral evidences [88] and single-crystal X-ray crystallographic study, the structure of 18 was elucidated as isotalatizidine hydrate.

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Chapter-3 Results and discussion

3.7.2 HOMO-LUMO energy gap of Isotalatizidine hydrate 18

The energy gaps between HOMO-LUMO are 0.220 for compound 18 which shows the stability of LUMO due to the electron accepting properties. The low band gap is due to electron donating groups and other parameters of the compound are responsible for its high reactivity and low stability [123]. The calculated energy parameters of the compound 18 are given in table 3.18

Table 3.18. List of HOMO-LUMO energy, ionization energy (IE), electron affinity (EA) global hardness (η), chemical potential (μ) and global electrophilicity (ω) of 18

ЄHOMO (eV) -0.166

ЄLUMO (eV) 0.054

ΔЄ = (ЄLUMO-ЄHOMO) (eV) 0.220

IE= -ЄHUMO (eV) 0.166

EA= -ЄLUMO (eV) -0.054

Global Hardness(η) = 1/2 (ЄLOMO-ЄHOMO) 0.11

Chemical Potential μ = 1/2 (ЄHOMO+ЄLUMO) -0.056

Global Electrophilicity ω = μ2/2η 0.014

3.7.3 Dihydropentagynine (19)

The crude Me-OH extract of D. denudatum Wall yielded compound 19. The IR spectrum of 19 exhibited strong bands at 3440 and 3414 cm-1 due to hydroxyl. The compound 19 has the molecular formula C23H37NO5 with a molecular weight of 407 determined from the EI-MS.

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Chapter-3 Results and discussion

OCH3 13 12 16 OH 17 14 OH H 15 H 1 10 9 2 11 N 8 3 5 OH 4 6 7 H 19 18 OCH3

Figure 3.39. Dihydropentagynine (19)

In the 1H-NMR spectrum of 19 showed a triplet of three proton integration at  1.07 (J =

7.15 Hz) was assigned to methyl of an N-ethyl group and two singlet each of three protons at 

3.33 and 3.27 for two O-methyl groups. The 1H-NMR spectrum also displayed two signals at 

3.03 and  3.18 assigned to oxygenated C-18 methylene, a downfield one proton broad singlet at

 3.90, and a triplet at  4.16 (J = 4.75 Hz) due to oxygenated methines, were assigned to H-1 and

H-14, respectively.

The twenty-three signals in the 13C-NMR spectrum (BB & DEPT) were resolved into four methyl, six methylene, ten methine and three quaternary carbons. The parameters of all carbons signal were in good agreement with those for the literature values of dihydropentagynine [126].

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Chapter-3 Results and discussion

3.8 Enzyme inhibition activity of isolated diterpenoids

3.8.1 Cholinesterase inhibition of diterpenoid from A. heterophyllum Wall

“Several serine hydrolases acts as catalase enzymes which include both acetylcholinestrase

(AChE, EC 3.1.1.7) and butyrylcholinesterase (BChE, 3.1.1.8). AChE has a key role in the catalysis of breakdown of acetylcholine; a neurotransmitter responsible for cholinergic actions found in brain. Being a member of carboxylesterase family, the primary role of AChE is to terminate the signals transmission of acetylcholine by hydrolyzing it which results in the delay of neurogenic information causing various brain diseases including dementia [127]. The aim of AChE inhibiters is to inhibit AChE from hydrolyzing acetylcholine thus boosting its level and duration in the brain of Alzheimer’s disease patients and herby, to boost cholinergic neurotransmission.

BChE is a gherlin regulator enzyme, made in liver, occurs in blood plasma and is considered to be a non-specific cholinesterase enzyme [128-129]. One of the aims of our study is to find new drug like candidate as cholinesterase. Alkaloids such as atropine and or huperzine-A, had experimentally been identified as potent cholinesterase inhibitors in previous studies [130]. BChE is produced in the liver and enriched in the circulation. In addition it is also present in the intestine, smooth muscle cells, in the white matter of the brain and many other tissues [131].”

We are interested to identify small molecules with the required potency to inhibit AChE,

BChE and possibly other such or different enzymes. The identification targets are usually medicinal plants, and the search is always bioassay guided. All the compounds isolated from A. heterophyllum Wall, A. laeve Royle and D. denudatum Wall were tested against these two enzymes, AChE and BChE. The activities of the isolated compounds were encouraging and the plant extract or the compound in its pure form can be good candidate(s) as medicine for the

119

Chapter-3 Results and discussion treatment of Alzheimer’s disease [131]. The inhibition of the above mentioned enzymes by the isolated compounds were initially determined at a concentration range 0.1 mM. As per practice the inhibition of the compounds were greater than 50%, were subsequently assayed for the determination of IC50 value. The compounds were found effective against AChE and BChE as compared to standard drug. Among the isolated compounds, 1 and 2 showed significant inhibitor effect on acetylcholinesterase (AChE) and butylcholinesterase (BChE) with IC50 were 5.41 ± 0.01

μM and 6.52 ± 0.09μM aginst AChE while 8.63 ± 0.27 μM and 9.31± 0.73μM against BChe respectively (Table 3.19). These exciting results highlight the interest in isolation and importance of this class of secondary metabolites present in A. heterophyllum Wall.

Table 3.19. AChE and BChE inhibitory activities of alkaloids from A. heterophyllum

SNo Compounds AChE ± SEMa BChE ± SEMa Type of inhibition (μM) (μM) 1 Compound (1) 5.41 ± 0.01 8.63 ± 0.27 Non competitive

2 Compound (2) 6.52 ± 0.09 9.31± 0.73 competitive

3 Compound (3) 12.81 ± 0.11 16.22± 0.4 Non competitive

4 Isoatisne (11) 8.44± 0.51 13.10± 0.4 Non competitive

5 19-Epiisoatisine (12) 10.32± 0.13 14.71± 0.35 Non competitive

6 Atidine (13) 14.69± 0.53 18.10± 0.07 competitive

7 Heteratisine (14) 6.57 ± 0.21 10.44± 0.15 Non competitive

8 Hetisinone (15) 10.12 ± 1.30 15.67± 0.88 Non Competitive

9 Allanzanthane A 4.2 ± 0.18 10.12 ± 0.1

10 Galanthamineb 8.03 ±0.01 12.03 ± 0.04

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Chapter-3 Results and discussion

3.8.2 Cholinesterase inhibition of diterpenoids from A. laeve Royle

All the isolated compounds from A. laeve Royle were tested against both AChE and BChE, which represent the most attractive target for drug design. Swatinine-C (4), swatinine-D (5), aconorine (16) and lappaconotine (17) showed most potent inhibition against AChE and BChE as compare to the standard compounds. The IC50 values of swatinine-C (4) and aconorine (16) against

AChE were determined to be 3.7 μMand 2.51 μM, while against BChE, 12.23 and 8.72 μM respectively. The IC50 values of swatinine-D (5) and lappaconotine (17) against AChE were noted as 4.53 and 6.13 μM, while against BChE were measured as 9.94 and 11.24 μM respectively. The remaining compounds 6 and 7 pronounced weak inhibition (Table 3.20).

Table 3.20. AChE and BChE inhibitory activities of diterpenoids from A.laeve Royle

SNo Compounds AChE ± SEMa BChE ± SEMa Type of inhibition (μM) (μM) 1 Swatinine-C (4) 3.7± 0.085 12.23± 0.014 Competative

2 Swatinine-D (5) 4.53± 0.062 9.94± 0.073 Competative

3 Benzene Derivative (6) 17.35± 0.021 26.14± 0.013 Non Competative

4 Benzene Derivative (7) 10.52± 0.011 18.17± 0.091 Non Competative

5 Aconorine (16) 2.51± 0.037 8.72± 0.023 Non Competative

6 Lappaconotine (17) 6.13± 0.019 11.24± 0.12 Non Competative

7 Allanzanthane A 4.12± 0.017 9.87 ± 0.034

8 Galanthamine b 3.26 ±0.021 10.13 ± 0.05 aStandard error of mean of five assays b Positive control used in the assays. Data shown are values from triplicate experiments.

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Chapter-3 Results and discussion

Mechanism-based kinetic study revealed that compound swatinine-C (4) and swatinine-D

(5) is competitive inhibitors of AChE and BChE. Lineweaver-Burk, Dixon plots and their replots indicated pure competitive type of inhibition for compounds swatinine-C (4) and swatinine-D (5) against acetylcholinstrase and butyrylcholinstrase enzyme, as there was increase in Vmax while decreasing the affinity (Km values) of the AChE and BChE towards the ACh and BCh respectively.

In other words we can say that compounds swatinine-C (4) and swatinine-D (5) and acetylthiocholin and butyrylthiocholin bind randomly and independently at the active sites of

AChE and BChE. The graphical analysis of steady state inhibition data for compounds swatinine-

C (4) and swatinine-D (5) AChE and BChE has been presented in figure 3.40-41.

Similerly, Lineweaver-Burk, Dixon plots and their replots indicated pure non-competitive type of inhibition for compounds aconorine (16) and lappaconotine (17) against AChE and BChE enzyme, as there was decrease in Vmax without affecting the affinity (Km values) of the AChE and

BChE towards the ACh and BCh respectively. In other words we can say that compounds aconorine (16) and lappaconotine (17) and acetylthiocholin and butyrylthiocholin bind randomly and independently at the different sites of AChE and BChE respectively. It indicates that inhibition depends only on the concentration of compounds aconorine (16), lappaconotine (17) and dissociation constant (Ki). Mechanism-based kinetic study revealed that compounds aconorine

(16) and lappaconotine (17) is a non-competitive inhibitor of AChE and BChE, it also showed that the structure of under study compound doesn’t suit the active site spanning mode. The high inhibition potency of compounds aconorine (16) and lappaconotine (17) makes it a lead candidate

for further pharmacological investigation to treat AChE and BChE associated pathologies.”

122

Chapter-3 Results and discussion

(A) “AChE inhibition by swatinine-C(1) is the Lineweaver–Burk plot of reciprocal of initial velocities versus reciprocal of four fixed substrate concentrations in absence (×) and presence of 100 µM (▲), 75 µM (■)), 50 µM (●) of swatinine-C (1)” (B) “BChE inhibition by Swatinine-C (1)is the Lineweaver–Burk plot of reciprocal of initial velocities versus reciprocal of four fixed substrate concentrations in absence (×) and presence of 100 µM (▲), 75 µM (■)), 50 µM (●) swatinine-C (1)”

123

Chapter-3 Results and discussion

Figure 3.40.L ineweaver–Bur k plot of Aconorine (16)

(A) “AChE inhibition by aconorine (16) is the Lineweaver–Burk plot of reciprocal of initial velocities versus reciprocal of four fixed substrate concentrations in absence (×) and

presence of 100 µM (▲), 75 µM (■)), 50 µM (●) of aconorine (16)”

(B) “BChE inhibition by aconorine (16) is the Lineweaver–Burk plot of reciprocal of initial velocities versus reciprocal of four fixed substrate concentrations in absence (×) and

presence of 100 µM (▲), 75 µM (■)), 50 µM (●) of aconorine (16)”

124

Chapter-3 Results and discussion

3.8.3 Cholinesterase inhibition of diterpenoidfrom D.denudatum Wall

All the isolated compounds from D. denudatum Wall were tested against both the aforementioned enzymes, which showed promising inhibition and could be targeted drugs for the treatment of Alzheimer’s disease [132]. All the compounds showed effective inhibition against

AChE and BChE as compared to the standard drugs in a dose dependent manner. The determined

IC50 value of compound 8, 9, 10, 18 and 19 against AChE was 19.84± 0.11, 9.24± 0.12 μM, 16.82±

0.31μM, 12.13± 0.43 and 11.27± 0.23 μM while against BChE the same value was 31.53± 0.58

μM, 34.72± 0.26 μM, 19.61± 0.72 μM, 21.41± 0.23 μM and 22.25± 0.33 μM respectively (Table

3.21). These exciting results highlight the importance of this particular class of secondary metabolites present in D. denudatum Wall. Compounds isolated during the course of this study were found to be extremely efficient against the two enzymes as compared to our recently studied compounds. These findings will exponentially increase the interest of this type of compounds found in species of genus Aconitum and Delphinium and may prompt chemists towards total synthesis of these compounds for possible commercialization as medicine against Alzheimer’s disease [132].

126

Chapter-3 Results and discussion

Table 3.21. AChE and BChE inhibitory activities of alkaloids from D. denudatum

SNo Compounds AChE ± SEMa BChE ± SEMa Type of inhibition (μM) (μM)

1 Compound (8) 19.84± 0.11 31.53± 0.58 Non competitive

2 Compound (9) 9.24± 0.12 19.61± 0.72 Competitive

3 Compound (10) 16.82± 0.31 34.72± 0.26 Non competitive

4 Compound (18) 12.13± 0.43 21.41± 0.23 Competitive

5 Compound (19) 11.27± 0.23 22.25± 0.33 Competitive

6 Allanzanthane A 8.23± 0.01 18 ± 0.06

7 Galanthamine b 10.12 ±0.06 20.62 ± 0.08 a Standard error of mean of five assays b Positive control used in the assays. Data shown are values from triplicate experiments.

127

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