ISOLATION AND CHARACTERIZATION OF ALKALOIDS: STUDY OF DELPHINIUM CHITRALENSE AND RELATED MEDICINAL PLANTS

BY

Mr. SHUJAAT AHMAD

DOCTOR OF PHILOSOPHY (PhD) IN CHEMISTRY

DEPARTMENT OF CHEMISTRY UNIVERSITY OF MALAKAND 2016

ISOLATION AND CHARACTERIZATION OF ALKALOIDS: STUDY OF DELPHINIUM CHITRALENSE AND RELATED MEDICINAL PLANTS

BY

Mr. SHUJAAT 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) IN

CHEMISTRY

DEPARTMENT OF CHEMISTRY

UNIVERSITY OF MALAKAND

2016

Declaration

I declare that the thesis “ISOLATION AND CHARACTERIZATION OF ALKALOIDS: STUDY OF DELPHINIUM CHITRALENSE AND RELATED MEDICINAL PLANTS” 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 references.

Shujaat Ahmad

Certificate

It is recommended that this thesis submitted by Mr. SHUJAAT AHMAD entitled “ISOLATION AND CHARACTERIZATION OF ALKALOIDS: STUDY OF DELPHINIUM CHITRALENSE AND RELATED MEDICINAL PLANTS” be accepted as fulfilling this part of the requirements for the degree of Doctor of Philosophy (PhD) in Chemistry.

______

SUPERVISOR EXTERNAL EXAMINER Dr. Manzoor Ahmad Associate Professor

______

CHAIRMAN DEPARTMENT OF CHEMISTRY

Dedicated

To ALL PEACE AND

KNOWLEDGE LOVING

PEOPLE

Contents

ACKNOWLEDGMENTS ...... i

SUMMARY ...... iii

List of Table ...... vii

List of Figures ...... viii

List of Scheme ...... x

List of abbreviations ...... xi

Chapter-1 ...... 1

1.1 Introduction ...... 1

1.2 Plants as drugs sources ...... 2

1.3 Medicinal plants and Islamic literature ...... 3

1.4 Plants as traditional medicine and drugs discovery ...... 6

1.5 Alkaloids...... 6

1.5.1 Diterpene alkaloids ...... 7

1.5.2 C20 diterpene alkaloids ...... 7

1.5.3 C19 diterpene alkaloids ...... 9

1.5.4 C18 diterpene alkaloids ...... 11

1.6 Therapeutic potential of diterpene alkaloids ...... 11

1.7 The Genus Delphinium ...... 13

1.7.1 Systematic position ...... 14

1.7.2 Delphinium chitralense ...... 14

1.7.3 Delphinium denudatum Wall...... 15

1.7.4 Literature survey of genus Delphinium ...... 16

Chapter-2 EXPERIMENTAL ...... 20

2.1 General experimental conditions ...... 20

2.1.1 Physical constants ...... 20

2.1.2 Spectroscopic techniques ...... 20

2.1.3 Column chromatography (CC) ...... 20

2.1.4 Solvents ...... 20

2.1.5 Detection of alkaloids on chromatographic plates ...... 21

2.2. Plant materials ...... 21

2.2.1 Plants materials pretreatment ...... 21

2.2.2 Extraction ...... 21

2.2.3 Fractionation ...... 22

2.2.4 Isolation of alkaloids from D. chitralense ...... 24

2.2.5 Isolation of alkaloids from D. denudatum Wall...... 25

2.3 X- rays diffraction studies ...... 26

2.4 Assay for enzymatic inhibition and IC50 value determination ...... 26

2.5 Physical and spectroscopic data of new compounds from D. chitralense ...... 28

2.5.1 Chitralinine-A (1) ...... 28

2.5.2 Chitralinine-B (2)...... 28

2.5.3 Chitralinine-C (3)...... 29

2.5.4 Chitraline-A (4) ...... 29

2.5.5 Chitraline-B (5) ...... 30

2.5.6 Chitraline-C (6) ...... 31

2.5.7 Chitraline-D (7) ...... 31

2.6 Physical and spectroscopic data of new compounds from D. denudatum Wall. .... 32

2.6.1 Compound (8) ...... 32

2.6.2 Compound (9) ...... 33

2.7 Physical and spectroscopic data of known compounds from D. Chitralense...... 33

2.7.1 Ajaconine (10) ...... 33

2.7.2 Delectinine (11) ...... 34

2.8 Physical and spectroscopic data of known compounds from D. denudatum Wall. 34

2.8.1 Dihydropentagynine (12) ...... 34

Chapter-3 RESULTS AND DISCUSSION ...... 36

3.1 Present work ...... 36

3.2 New diterpenoids from Delphinium chitralense ...... 36

3.2.1 Chitralinine-A (1) ...... 36

3.2.2 Chitralinine-B (2)...... 43

3.2.3 Chitralinine-C (3)...... 47

3.2.4 Chitraline-A (4) ...... 51

3.2.5 Chitraline-B (5) ...... 55

3.2.6 Chitraline-C (6) ...... 59

3.2.7 Chitraline-D (7) ...... 63

3.3 New diterpenoids from D. denudatum wall...... 67

3.3.1 Compound (8) ...... 67

3.3.2 Compound (9) ...... 71

3.4 Known diterpenoids from D. chitralense ...... 74

3.4.1 Ajaconine (10) ...... 74

3.4.2 Delectinine (11) ...... 77

3.5 Known diterpenoids from D. denudatum Wall ...... 80

3.5.1 Dihydropentagynine (12) ...... 80

3.8 Enzyme inhibitory activities of isolated compounds ...... 81

3.8.1 Cholinesterase inhibition of diterpene alkaloids from D. chitralense & D.

denudatum ...... 81

REFERENCES...... 84

ACKNOWLEDGMENTS

Firstly, I would like to thank my supervisor, Dr. Manzoor Ahmad, Associate Professor

University of Malakand, whose infectious enthusiasm for natural products 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 of writing this thesis.

I am also indebted 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 am also thankful to 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 of the Department of Chemistry, University of Malakand 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 i

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 Mr. Hanif Ahmad, Mr. Shujat Ali, Mr. Misal Bacha, Mr,

Sultan Muhammad, Mr. Idrees Khan, Mr. Adnan Shahzad, Mr. Nasib Khan, Mr. Zarif Gul, Mr.

Umar Ali Khan, Mr. Noor Zada, Mr. Sajid Ali and Mr.Alam Khan 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 my whole family including my elders and especially to my beloved mother whose infinite prayers, unwavering support kept my morale high during difficult times.

Shujaat Ahmad

ii

SUMMARY

The present Ph.D thesis describes the phytochemical and biological exploration of two species belong to family Ranunculaceae of District Dir (U), Khyber Pakhtunkhwa Pakistan.

The main goal of our research was the isolation, structure elucidation, selective AChE and BChE enzyme inhibition of C19 and C20 diterpene alkaloids from the basic chloroform soluble fraction of D. chitralense and D. denudatum Wall. Seven new compounds; Chitralinine-A (1),

Chitralinine-B (2), Chitralinine-C (3), Chitraline-A (4), Chitraline-B (5), Chitraline-C (6),

Chitraline-D (7) and two known, Ajaconine (10) and Delectinine (11) have been isolated from the aerial part of D. chitralense. Two new Jadwarine-A (8) and Jadwarine-B (9) and one known dihydropentagynine (12) have been isolated for the first time from the aerial parts of D. denudatum Wall.

All the isolated compounds (1-12) were characterized by various spectroscopic techniques and screened for enzyme inhibition activities (AChE & BChE). The isolated compounds were found to be potent AChE and BChE inhibitors. Our present findings indicate the interest of C19 and C20 diterpenoids as potent AChE and BChE inhibitors present in the investigated two delphinium species. They may contribute towards naturally accessible inhibitors used for the treatment of

Alzheimer’s disease.

iii

New Compounds

12 17 OH 16 CH2 17 HO CH 11 12 16 2 OH 13 O 20 HO 11 OH 13 O 14 20 HO 15 HO 14 1 9 HO 15 1 9 2 10 8 2 10 8 H3C N H3C N 3 5 7 3 5 7 4 6 4 6

O 19 O 19 18 18 HO

Chitralinine-A (1) Chitralinine-B (2)

OH OH OCH HO 17 3 12 16 CH2 13 16 11 OH 13 12 20 HO H3CO 17 14 OCH3 14 HO HO 15 1 9 1 10 9 2 10 8 2 11 15 H3C N N 8 3 5 7 3 5 4 6 4 7 OH 6 H 19 O 18 19 18 OCH3 HO

Chitralinine-C (3) Chitraline-A (4)

OCH3 H OCH3 H H 16 13 13 16 12 12 14 OCH3 14 OCH OCH3 OCH3H 3 17 17 10 1 9 1 10 9 H 11 2 15 2 11 8 15 N N 8 3 5 OCOCH 3 3 5 OH 4 7 6 4 7 H 6 19 OCH H 18 3 19 OH H CO 18 H3CO 3 H3CO

Chitraline-B (5) Chitraline-C (6)

iv

OCH 3 OCH3 13 16 12 13 H3COCO 17 14 OCH3 OH 12 16 17 14 OH OH 15 1 10 9 2 11 15 1 10 9 H N 8 2 11 3 5 8 7 OH N 4 6 3 5 OH 4 6 7 OH 19 18 OCH3 H H3CO 19 18 H3CO Chitraline-D (7) Jadwarine-A (8)

OCH3 OCH 13 16 137 12 14 OH 15 1 10 9 H 2 11 N 8 3 5 4 6 7 OCH3

19 18 H3CO

Jadwarine-B (9)

v

Known Compounds

CH2 OCH 17 3 16 12 13 16 20 11 13 12 H3CO 17 14 OH H 14 1 9 15 1 10 H3C 9 HO 2 10 O 8 2 11 15 N OH N 8 5 3 5 3 7 7 4 6 4 6 OH H H OH 19 19 18 18 OCH3 HO

Ajaconine (10) Delectinine (11)

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

Dihydropentagynine (12)

vi

LIST OF TABLE

No Title Page No

Table 1. 1. Some Plants Cited in Quran and Hadith ...... 4

Table 1. 2. List of diterpenoids from Genus Delphinium ...... 17

1 13 Table 3.1. Н- (600 MН z) and С -NMR (150 MHz) data of chitralinine-A (1) in CDCl3

...... 41

Table 3. 2. Crystal data and structure refinement of chitralinine-A ...... 42

1 13 Table 3.3. H- (500 MHz) and C-NMR (125 MHz) data of chitralinine-B (2) in CDCl3

...... 45

1 13 Table 3. 4. H- (500 MHz) and C-NMR (125 MHz) data of chitralinine-C (3) in CDCl3

...... 49

Table 3. 5. 1H- (500 MНz) and 13C-NMR (125 MHz) data of chitraline-A (4) ...... 54

1 13 Table 3. 6. H- (500 MHz) and C-NMR (125 MHz) data of chitraline-B (5) in CDCl3 58

Table 3. 7. 1H- (500 MHz) and 13C-NMR (125 MHz) data of chitraline-C (6) in CDCl3 62

1 13 Table 3.8. H- (600 MHz) and С-NMR (150 MHz) data of chitraline-D (7) in CDCl3. 66

1 13 Table 3. 9. H- (500 MНz) and C-NMR (125 MHz) data of Compound (8) in СDCl3 . 70

1 13 Table 3. 10. H- (500 MНz) and C-NMR (125 MНz) data of 9 in СDCl3 ...... 73

Table 3.11. Crystal data and structure refinement of ajaconine...... 76

Table 3.12. Hydrogen bond geometry (Å, °) ...... 77

Table 3. 13. Crystal data and structure refinement of Delectinine ...... 80

Table 3. 14. Enzyme inhibitory activities of D. chetralense and D. danudatum ...... 82

vii

LIST OF FIGURES

Figure 3. 1. Structure of chitralinine-A (1) ...... 37

Figure 3. 2. COSY interaction in chitralinine-A (1) ...... 38

Figure 3. 3. HMBC interaction in chitralinine-A (1) ...... 39

Figure 3. 4. Crystal Structure of compound 1, with 50 % probability of thermal ellipsoids

and hydrogen atoms are omitted for clarity...... 40

Figure 3. 5. Structure of chitralinine-B (2) ...... 43

Figure 3. 6. HMBC interaction in chitralinine-B (2) ...... 45

Figure 3. 7. Structure of chitralinine-C (3) ...... 47

Figure 3. 8. HMBC interaction in chitralinine-C (3) ...... 49

Figure 3. 9. Structure of chitralin-A (4) ...... 51

Figure 3. 10. HMBC interaction in chitraline-A (4) ...... 53

Figure 3. 11. Structure of chitraline-B (5) ...... 55

Figure 3. 12. Key HMBC interaction in chitralin-B (5) ...... 57

Figure 3. 13. Structure of chitraline-C (6) ...... 60

Figure 3. 14. Key HMBC interaction in chitraline-C (6) ...... 61

Figure 3. 15. Structure of chitraline-D (7) ...... 64

Figure 3. 16. Key HMBC interaction in chitraline-D (7) ...... 65

Figure 3. 17. Structure of compound 8 ...... 68

Figure 3. 18. Key HMBC interaction in compound 8 ...... 69

Figure 3. 19. Structure of compound 9 ...... 71

Figure 3. 20. Key HMBC correlation in 9 ...... 73

viii

Figure 3. 21. Structure of ajaconine 10 ...... 74

Figure 3. 22. ORTEP plot of ajaconine (10), thermal ellipsoid is drawn at 50 %

probability, hydrogen atoms are omitted for clarity, ...... 76

Figure 3. 23. Structure of delectinine (11) ...... 78

Figure 3. 24. ORTEP representation of delectinine with 50% thermal ellipsoids,

hydrogen atoms are omitted for clarity ...... 79

Figure 3. 25. Structure of dihydrpentagynine (12) ...... 81

ix

LIST OF SCHEME

Scheme 2. 1. Fractionation of D. chitralense & D. denudatum Wall ...... 23

Scheme 2. 2. Isolation of alkaloids from D. chitralense ...... 25

Scheme 2. 3. Isolation of alkaloids from D. denudatum Wall ...... 26

x

LIST OF ABBREVIATIONS

BB Broad (decoupled) band

CC Column chromatography

COSY Correlated spectroscopy

DAs Diterpenoid Alkaloids

DEPT Distortionless enhancement by polarization transfer

EI-MS Electron impact mass spectrum

FCC Flash column chromatography

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

CHCl3 Chloroform

ACE Acetone

DEA Diethyl Amine

xi

Hex n- Hexane

AChE Acetyl Cholinesterase

BChE Butyryl Cholinesterase

NCEs New Chemical Entities

NDLs New Dru Leads

MeOH Methanol

xii

Chapter-1 Introduction

CHAPTER-1

1.1 Introduction

Flora of Pakistan is rich in medicinal plants due to its unique environmental conditions.

In Pakistan about 6000 species of flowering plants are available in which 400-600 have pharmacological importance [1-2]. The history of using medicinal plants by humans is very old for health care. It is revealed that medicinal plants contain active chemical compounds, which show important pharmacological activities. Morphine was the first natural bio-active medicinal product isolated from opium in 19th century. Drugs discovery from plants started when drugs like digoxin, quinine, cocaine and codeine were isolated primarily. Some of the early isolated drugs such as morphine is still used as therapeutic agent [3-5]. Scientists have been involved worldwide in isolating and characterizing the therapeutically active compounds from plants. The significance of plants in solving health complications is gaining importance gradually. History showed that the origin of most potent available drugs is plants, either in the raw or finished form

[6].

Drugs discovery from plants involves a lot of research and investigational procedures.

The first step in research toward drug discovery is the identification of desired plant by a botanist or plant taxonomist, followed by the collection from its natural habitat [7]. After collection series of steps are involved (e.g. shade drying, grinding or crushing, soaking etc.) for extracting crude drug from the selected medicinal herb or plant based on its ethno-botanical and ethno-medicinal knowledge. The crude extracts are screened for their desired biological activities and then in the next step the extracts of interest are subject to isolation and purification for getting pure

1

Chapter-1 Introduction compounds or phytochemicals. The entire process is termed as targeted isolation or bioassay guided isolation.

1.2 Plants as drugs sources

Various Isolation methods have been employed for getting bio-active agents that can act as drugs for various ailments. These procedures contain isolation from natural sources, synthetic, combinatorial chemistry and molecular modeling [8-10]. Though there is massive research work in the fields of molecular modelling, combinatorial and synthetic chemistry skills which has been financially supported by pharmaceutical firms and organizations, natural products having difficult structural formulae always remain key source for new drugs development, new chemical entities and new drugs leads based on a survey report in 2001 & 2002 around one quarter of best marketed drugs are obtained from natural sources or their derivatives [4, 11].

According to a report from 1981 to 2002 about 28 percent new chemical entities were obtained directly from natural products or derived from them while another survey during the same period showed that about 20 percent of new chemical entities were synthetic compounds derived from natural products [11]. Keeping in view this report it is deduced that natural compounds accounts for around 48 percent of reported new chemical entities form year 1981 to year 2002. Furthermore, it has been acknowledged that natural compounds act as preliminary material for laboratory scale preparation having varied structures and multiple stereo centers that can be challenging synthetically [12-15]. Natural compounds display a lot of common structural features which proved to be an important effort for discovery of new drugs [12, 16 and 17].

Synthetic chemists are working for designing natural products and natural products like libraries that shows similarities with structural features of natural products with the compound-generating

2

Chapter-1 Introduction potential of combinatorial chemistry [18-22]. Isolated compounds can act as drugs and their activity can be enhanced by modifying their structures by synthetic chemists using modern techniques [23].

1.3 Medicinal plants and Islamic literature

The significance of medicinal plants has thoroughly been discussed in the doctrines of

Islamic theological tradition. The tradition of Islamic medicine began with the advent of first man on earth, Adam (Peace be upon Him) and continued till the last Prophet Muhammad (Peace be upon Him). The holiest scripture in Islam, the Holy Quran, started revealing 1400 years ago with 6600 verses entwined with every dimension of religious life. In all these verses, 1000 verses are of scientific nature in which 900 verses deals with the advent of new scientific discoveries

[24]. There are twenty-two distinguished species in plants belonging to seventeen families of plants in Quran [25], which include Olea europea, Phoenix dactylifera, Punica granatum, Vitis vinifera, Zingiber officinale, Ocimum basilicum, Ficus carica, brassica nigra, Tamarix salvadora Persia, Citrulus colocynthi , Zizyphus spina-christi, Allium sativum, Cucumis sativus,

Lens esculents, Allium cepa, Musa sapientum, Triticum vulgare and Hordeum vilgare .

In Islamic doctrines, the knowledge of plant based medicine is called Tibb. Tibb is associated with the use of plants to cope with hygiene and health problems of human beings.

Some fruit plants, namely, figs, dates, pomegranates and olives are exclusively discussed in plants. Other plants including basil, lentils, garlic, camphor, colocynth and onions are also discussed in the Holy Quran [25]. The Prophet (Peace be upon Him) has prescribed many herbs as a remedy for some common ailments [26]. The herbs mentioned in Quran and sayings of the

Prophet (Peace be upon Him) have been discussed extensively and compiled by many prominent

3

Chapter-1 Introduction

religious and other scholars over the years. The history of Islamic medicine began in the 2nd

century of the Islamic calendar. In this regard the famous books, which have been written are:

Haddi Kabeen, Tib-e-Nabvi, Kamal-ul- Sannaf” [27-28]. The herbs discussed in these books

have significantly been utilized in the treatment of various diseases as the primary source of

drugs.

A recent research regarding the herbs discussed in the Islamic scriptures confirmed 32

medicinal plants belong to different families in the plant kingdom [29]. The scientific names,

Arabic names, plant families and the use of these herbs with reference to literature from Islamic

tradition is provided in Table 1.1.

Table 1. 1. Some Plants Cited in Quran and Hadith

S.No Plant Uses Ref

Arabic names Species Family

1. Basal Allium cepa Alliaceae Hepatitis, cholera, piles [30-33]

and influenza.

2 Soom Allium sativum Alliaceae Dog bites, asthma, [30-34]

paralysis, cough,

tuberculosis, hysteria.

3 Kafoor Cinnamomum Lauraceae Parkinson, tetanus, [30-33]

camphora tuberculosis, inner

wounds, breast pain.

4 Teen Ficus carica Moraceae Remove urinary [30,31]

4

Chapter-1 Introduction

bladder and kidney

stone and dyspepsia.

5 Shair Hordeum Poaceae Weakness, Fever, [31,33,

vulgare kidney pain, heart 35]

diseases, jaundice.

6 Yakteen, Daba Lagenaria Cucurbitaceae Piles, arthritis, liver and [30,31,

siceraria kidney diseases. 33,34]

Standl

7 Adas Lens culinaris Papilionaceae Measles, common cold, [30, 36]

Medic and eye infection.

8 Rehan Ocimum Lamiaceae Cough, fever, baldness, [30,31,

basilicum asthma, hepatitis 33,35]

9 Zaiytoon Olea europea Oleaceae Strengthen muscles, [30,31,

slow down aging, and 34,35]

tuberculosis

10 Tammar, Rutab Phoenix Arecaceae Intestinal pain, skin and [30,34]

dactylifera heart diseases

11 Rumman Punica Punicaceae Muscle pain, dental [30,33]

granatum problems, diarrhea and

oral diseases.

5

Chapter-1 Introduction

1.4 Plants as traditional medicine and drugs discovery

The practice of medicinal plants for health care in China and India is very old [37]. The use of medicinal plants has been reported to be still practiced in various “Traditional medicine systems of other culture”. According to WHO report from 1959-1980 at least ‘119’ important drugs were derived from 90 medicinal herbs and are in use till date [38]. The review of Newman and Cragg propose that “from the year 1940-2007 about 73 percent of 155 molecules are natural with 47 % being natural products or their derivatives”. With advancement in field of medicinal chemistry research in 1990 the percentage of natural products based drug drops to 50 %. In US

13 new natural products derived based drugs were approved from 2005-2007[39].

Quinine was obtained from the bark of Cinchona plant having anti-malarial activity by

French pharmacists in 1820. Later on, in early 1600 the bark was introduced in Europe for treating malaria. Quinine produced two potent anti-malarial derivatives (Chloroquine and

Mefloquine) on further studies and research. Artimisnin another anti-malarial drug was isolated from Artemisia annua. The derivatives of artimisnin are artemeter and artether both having useful activity against resistant plasmodium strains [40].

1.5 Alkaloids

Alkaloids are natural organic compounds mostly found in plants and in rare cases in animals. Mostly they are found in plants having seeds and are found mainly in bark, berries, fruits, leaves and seeds. They are basic in nature that’s why they are known as alkaloids (alkali- like). Amongst secondary metabolites alkaloids are the most potent pharmacologically active substances obtained from plants [41]. There are about 5500 known alkaloids having one or more than one Nitrogen atoms, commonly in combination as part of a cyclic structure [42]. Strychnine, 6

Chapter-1 Introduction atropine, caffeine, morphine, codeine, nicotine and cocaine are some of the alkaloids obtained from nature. As alkaloids have medicinal and physiological properties for that reason they have great interest for humans. Alkaloids in the form of extracts from plants have been used as poisons and medicines in all cultures since the life started on earth. Due to their potent physiological nature, they act as muscle relaxants, analgesics, stimulants, antimicrobial, antiepileptic, anti-hypertensive, anti-oxidants, anti-diabetic, anti-HIV and anti-cancer. Morphine was the example of first isolated alkaloid from the P. somniferum in 1805.More than 10000 alkaloids have been isolated and discovered from various sources [43]

1.5.1 Diterpene alkaloids

Aconitum, Consolida and Delphinium genera of family Rananculaceae are rich sources of toxic diterpenoid alkaloids and most of them show a broad spectrum of biological activities in therapeutic amounts [44-46]. The secretes of biosynthesis of diterpenoids is still a mystery for scientists; they are believed to be bases resulting from penta or tetracyclic diterpenes having N atom of methylamine, ethylamine or β-aminoethanol connected to Ϲ-17 and Ϲ-19 in Ϲ19 diterpenoids, and to С-20 and С-19 in С-20 diterpenoids, forming a substituted piperidine ring

[47]. Another view about their biosynthesis is that they belong to pseudoalkaloids class and they seem to be derived from the amination of nitrogen-free terpenes. In few cases N atom is absent in ring system such as Erythrophleum alkaloids [48]. On phytochemical basis diterpenoids can be classified into three major groups.

1.5.2 C20 diterpene alkaloids

C20 diterpene alkaloids are usually found as monoesters of benzoic or acetic acid in plants. The existence of an exocyclic methylene is characteristic; most possess a secondary

7

Chapter-1 Introduction hydroxyl group in an allylic position. These alkaloids are believed to be derived from pimaradienes via rearrangement reactions or Baeyer Villiger oxidation [49]. As compare to norditerpenoid alkaloids these alkaloids have more diverse structural diversity. These alkaloids are mostly found as esters of acetate or benzoate and are said to be less poisonous comparatively.

On the basis of structure, they are of three types (Figure-1.1).

I. Atisine-type

They are relatively simple and having less toxicity. They are not extensively oxygenated and most of them possess a single methoxyl group [50].

II. Veatchine-type

These alkaloids differ from atisine types because of construction of E and D rings in their basic veatchine type skeleton. Much resemblance is found in their chemistry although they both belong to different families [51-53].

III. Delnudine-type

They contain hetisine skeleton and are considered as derivatives of either hetisine or veatchine having alteration in the ring system [54]. It is suggested N-C-6 bond is formed as a result of the structures of miyaconitinone and miyaconitine alkaloids [55].

8

Chapter-1 Introduction

13 17 12 17 17 CH2 13 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 1.1. Basic skeletons of C20 diterpenoid alkaloids

1.5.3 C19 diterpene alkaloids

C19 diterpene alkaloids are commonly highly oxygenated and are thus very toxic, rarely with the presence of at least five oxygen atoms, connected to aromatic or aliphatic acid functionalities or in hydroxyl forms [56]. Biosynthetically, С20 DAs acts as precursor towards the bio-synthesis of NDAs with a probable mechanism of rearrangement through Baeyer-Villiger oxidation. They are of four types. [57]

I. Aconitine-type

They have aconitine nucleus (Figure 1.2) in which the C-7 position is not substituted by oxygen substituents and contains a hydroxyl or ester group on C-8 position. In most of the known diterpenoid alkaloids more than 50 % contain aconitine skeleton. Aconitine was the first

NDAs isolated in 1821 by Pescheir from Aconitum nepellus. Condelphine and delphinine are the examples.

9

Chapter-1 Introduction

II. Lycoctonine-type

They contain lycoctonine skeleton and always having substituted oxygen at C-7, e.g. delcosine and lycoctonine.

III. Pyrodelphinine-type

These alkaloids contain the skeleton of the pyrolysis product of delphinine and possess double bond btw C-8 and C-15 e.g. mithaconitine and falaconitine.

IV. Heteratisine-type

They contain heteratisine nucleus (Figure-1.2), which possess a lactone moiety in ring C, e.g. heterophyllin and the skeleton of the pyrolysis of heteratisine [57].

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 1.2. Basic skeletons of C19 diterpenoid alkaloids

10

Chapter-1 Introduction

1.5.4 C18 diterpene alkaloids

These alkaloids are derived from C19 norditerpenoid alkaloids. The basic skeleton of this type possesses 18 carbons in their basic skeleton while lacking 18th carbon atom along with replacement of C-4 by either 3,4-epoxide group, ester group or only hydrogen atom. An exclusion of terminal C-18 is proposed to take place due to Beyer-Villiger oxidation mechanism or alternatively due to the oxidation of hydroxyl methyl group towards carboxy miotey, subsequently proceeded towards decorboxylation and epoxidation of the 3-4 alkenic bond [49].

1.6 Therapeutic potential of diterpene alkaloids

I. Analgesic and anti-inflammatory activities:

Diterpene alkaloids possess analgesic anti-inflammatory activities in vitro mainly due to the presence of tertiary amine group in their basic skeleton. [58]. The structural activity relationship reveals that an acetoxy group at C-8 along with aromatic ester and saturated D ring greatly enhance the analgesic activity of these alkaloids. Aconitum and their derivatives have been reported to interfere with Na+ channels, inhibiting neuronal conduction or blocking of Na+ channels as in the case of paconitine [59]. These alkaloids also report analgesic effects in acetic acid induced rats when in vivo tested [60]. Lappaconine and puberanine possess promising anti- inflammatory activities even more than other non-steroidal drugs [61].

II. Anti-hypertensive activities:

C19 diterpenoids are well known for their cardiovascular activities especially lappoaconitin, pyrodelphinin and pyroaconitin derivatives are k their benzoyl derivative has been reported to exhibit more potent anti-arrhythmic action than the reference drugs, quinidine and

11

Chapter-1 Introduction novocainamide [62]. Diterpene alkaloidal bases have been developed as therapeutic agents for the treatment of arrhythmia in China [63].

III. Insecticidal and anti-parasitic activities:

Diterpenes alkaloids have been found to inhibit growth of various parasites and show strong insecticidal activities against Spodoptera species due to the presence of hydroxyl groups and acetate functionalities in molecules [64]. Hestatine strongly inhibits Tribollium casteneum when used as insect repelling agent. These alkaloids are better insecticides even on a dosage level of less than 1 µgcm-1. Several atisine type alkaloids have demonstrated anti-proliferative effects against parasites in vitro with the same doses as reference drugs. Azitin derivatives inhibit the growth of Trypnosoma cruzi very strongly when tested again [65].

IV. Anti-epilaptic activity:

Mesoaconitine exhibit anti-epileptic form activity in vivo which is supported from its mechanism of action in hypocampus, mediated through α-receptors in selected mice model.

These alkaloids can inhibit continued repetition firing in hippocampal neurons in voltage dependent manner which is the basic mechanism of all the anti-epileptic drugs [66].

V. Anticancer activity:

Diterpene alkaloids have showed remarkable anticancer activities against various human cancer line cells like Pgp-MDR, SW480, HeLa and SkMe125. C-19 alkaloids are more potent than C-20 in suppression of tumor cell lines such as HeLa and Raji [65]. Lycoaconitine and benzoyl aconnine derivatives exhibit potent inhibitory activities against PgP-MDR cells [67].

Neolin, pubesenine, 14-decetylajadine and ajadelphinine exhibit selective effects in cancerous cells.

12

Chapter-1 Introduction

VI. Cholinergic action:

Various diterpenoid alkaloids have been found to possess both concentration dependent and voltage dependent antagonist effects which are more enhanced when a side chain, preferably benzoyl group at position 18, is present which ultimately lead to potent nicotine acetylene receptors effect. [68]. Methyllycaconitine have been confirmed to possess α7-nAChR antagonistic activities and is useful tool in neurobiological research with in vivo neuroprotective properties. [69]. Due to their α7-nAChR antagonist properties, these diterpenoid alkaloids are interesting cholinergic agents [70].

VII. Antimicrobial activity:

Several diterpenoid alkaloids have been found to possess antibacterial, antifungal and antiprotozoal activities. Atisine type alkaloids (C-19 and C-20) have been reported to inhibit the growth of various Trypanosoma species with a very low toxicity profile [71]. Delphinium alkaloids have shown strong antifungal effects, specially panicutine, 8-acetylheterophyllisine and vilmorrianone [72].

1.7 The Genus Delphinium

Delphinium is a large genus belongs to family Rananculaceae (Butter cup family).

Flowers of delphinium resemble with little dolphin that is why this genus is named as

Delphinium. Delphinium genus comprises of 270 species, occurring throughout the northern temperate region, mostly biennials and perennials herbs commonly known as Larkspur. In

Pakistan, sixteen species have been reported to be found and are almost in the red list [73].

13

Chapter-1 Introduction

1.7.1 Systematic position

Kingdom: Plantae

Subkingdom: Tracheobionta

Superdivision: Spermatophyta

Division: Magnoliophyta

Class: Magnoliopsida

Subclass: QAS

Order: Ranunculales

Family: Ranunculaceae

Genus: Delphinium

Species: D. chitralense and D. denudatum [74].

1.7.2 Delphinium chitralense

Delphinium chitralense is an herb found at altitude of 1520 to 1830 m. The size of the stem varies from 40-60cm or more, which is branched vigorously, having hairs on the stem, strigulose, covered with leaves except the upper part. Leaves are petiolate. Petiole of the leaf is

10-13 cm, strigulose, blade 4 cm wide, subreniform in shape, tender below, 3-parted for about

3/4 to 4/5 of its length, segments 2-3-lobed, obovate-cuneate. Leaves are attached to the stem directly (cauline), palmately lobed, alternate phytolloxy, slightly dissected. The leaves of the

14

Chapter-1 Introduction upper and middle portion are 3-5 parted, entire or 3-lobed. Inflorescence is racemes type, composed of one or several. Flowers are violet, showy and irregular. The scale leaves below sepals of the flower are linear, 2.5-12 x 0.5-1 mm. Pedicels 9-25 mm, divaricated or ascending in manner. Bracteoles are minute, which are attached in the middle of petals. Sepals pale bluish or violet, upper sepals are 6-8 x 3.5-4 mm, strigulose outside. The size of the spur varies 9-10 mm in length, 1.7-2(-3) mm wide at base, strigulose, sometime straight, lateral sepals are 7-8.5 x 4 mm, elliptic-ovate, obtuse, sometime rounded in shape, strigulose along the midline, lower sepal

8-9 x 3.5 mm, oblong-elliptic, apiculate, strigulose all over. Petals pale yellow to whitish. Limb of upper petal is smooth, having size 6-7 mm, having two shallow lobes, lobulated of the size less than l mm in lenght, spur 8-9 mm, limb of lower petal 3.5-4 mm long and wide, rounded, oblique, bilobed to middle or more, bearded on the entire outer surface, claw 4-4.5 mm. Stamens are about 5 mm and are smooth. Carpals are smooth, scabridulous along the veins. Fruit is not observed in the plant. Distribution: Endemic [75]

1.7.3 Delphinium denudatum WALL.

The Delphinium denudatum is an annual or perennial herb, having beautiful flowers and toxic in young condition. The height of the plant ranges from 50 - 80 cm, found at altitude of

1500 to 2600 m in Northern regions of Pakistan on the grassy slopes. The stem is branched, quite expanded, covered with short, stiff and pressed hairs or spreading tender in the upper portion of the stem. Stalk of leaves surrenders above to 15.0 cm. The upper petiole is too shorter and the base of the leaf is 5-15 mm broad, which is obovately divided into 3-5 fragments, portions are pinnately compound and oval flaps or teeth 2-3 mm broad. Leaves are cauline, pinnately lobed, petiolate narrowly, having distinguishing long petiole, comparative yet littler. Inflorescence is

15

Chapter-1 Introduction recemose type of few flower, often paniculate. Bracts are 5-15 mm. The size of the pedicle is from 10.0-40 mm, climbing, floral leaves lies near to center of pedicel. The color of sepals ranges from blue color to violet. The upper sepal having size of 13-14 x 6-7 mm, tender, delicate, goad 14-15 mm long, c. 3.5 mm wide at the base, parallel sepal 13 x 7-8 mm, elliptical applaud, adjusted, pubescent on the midline, lower sepals ranges 14-15 x 7 mm in size, elongated obovate in shape, adjusted, tender. Upper petals are white having light bluish color at the top. The appendages are 8-9 mm in size, smooth, bidentate, present in one side, goad 11-16mm, the lower petal having bluish or violet color, and 0.6 mm long, extensively oval in shape, centrally parted, the paw is 5.0 mm. Stamens ranges from 5-6 mm in length. Follicles are 3, having size, ranges from 3-3.35x10-16 mm, inadequately haired or having too much less hair, style 2-3 mm. the shape of the Seed is obpyramidal, which is 1 mm long, dull in color, long in size and endospermic [76].

1.7.4 Literature survey of genus Delphinium

Two species of genus delphinium (D. chitralense and D. denudatum) belong to the family

Ranunculaceae were selected for the present research work. Jadwar is the common name of genus Delphinium [77]. Both plants are extensively found in Chitral, Swat, Dir, Azad Kashmir,

Baluchistan and in the Himalaya and Garrhwall area of India [78-79]. In local system of medicine, it is used as cardiotonic and sedative [80]. According to literature, no phytochemical work has been conducted on D. chitralensi whereas the D. denudatum was partially explored.

The available phytochemical literature on Genus Delphinium is given in table 1.2.

16

Chapter-1 Introduction

Table 1. 2. List of diterpenoids from Genus Delphinium

S.No M. Mass Formula Name Source Ref.

1 417.5384 C24H35NO5 8- Acetylheterophyllisine D. denudatum Wall 72

2 435.1834 C29H25NO3 Delnudine D. denudatum Wall 72

3 383.2197 C23H29NO4 Panicutine D. denudatum Wall 72

4 343.5029 C22H33NO2 Denudatine D. denudatum Wall 72

5 407.5435 C23H37NO5 Isotalatizidine D. denudatum Wall 72

6 313.2111 C20H27NO2 Davisine D. davisii Munz 81

7 386.2384 C27H32NO 18-benzoyldavisinol D. davisii Munz 81

8 313.2108 C20H27NO2 Davisinol D. davisii Munz 81

9 313.4338 C20H27NO2 Kobusine D. davisii Munz 81

10 377.5176 C22H35NO4 Karakoline D. davisii Munz 81

11 329.4332 C20H27NO3 Hetisine D. davisii Munz 81

12 327.4174 C20H25NO3 Hetisinone D. davisii Munz 81

13 391.5011 C22H33NO5 Tianshanisine D. tianshanicum 82

14 423.5429 C23H37NO6 Tianshanine D. tianshanicum 82

15 571.5530 C34H37NO7 Tianshanidine D. tianshanicum 82

16 449.2927 C25H39NO6 Kohatenine 1 D.kohatense Munz. 83

17 435.5521 C24H37NO6 Winkleriline D. winklerianum 84

18 423.5411 C23H37NO6 Winkleridine D. winklerianum 84

19 339.1834 C21H25NO3 Carduchoron D.carduchorum 85

20 341.1990 C21H27NO3 Delcarduchol D.carduchorum 85

17

Chapter-1 Introduction

21 491.2883 C27H41NO7 8-acetylcondelphine D. pyrimadale 86

22 664.3432 C37H48N2O9 Yunnanenseine A D. yunnanense 87

23 499.2565 C28H37NO7 Yunnanenseine B D. yunnanense 87

24 457.2539 C26H35NO6 Yunnanenseine C D. yunnanense 87

25 783.2961 C43H45NO13 Tatsienenseine A D. tatsienense 88

26 397.2331 C24H31NO4 Tatsienenseine B D. tatsienense 88

27 381.2378 C24H31NO3 Tatsienenseine C D. tatsienense 88

28 453.2726 C24H39NO7 Gigactonine D. schmalhausenii 89

29 467.5955 C25H41NO7 Lycoctonine D. schmalhausenii 89

30 671.8308 C37H53NO10 Delsemine A D. schmalhausenii 89

31 671.8308 C37H53NO10 Delsemine B D. schmalhausenii 89

32 467.2883 C25H41NO7 Delphinifl exine D. flexuosum 90

33 642.7630 C35H48NO10 Ajadine D. flexuosum 90

34 423.2351 C22H33NO7 Tiantaishansine D. tiantaishanense 91

35 484.3301 C26H46NO7 Tiantaishannine D. tiantaishanense 91

36 461.2473 C25H35NO7 Tiantaishanmine D. tiantaishanense 91

37 476.2439 C29H34NO5 Tiantaishandine D. tiantaishanense 91

38 711.2581 C40H41NO11 Delphigraciline D. gracile 92

39 359.1754 C20H25NO5 15-Hydroxyhetisinone N- D. gracile 92

oxide

40 391.2713 C23H37NO4 8-methoxykarakoline D. gracile 92

41 479.2045 C26H41NO7 Cardiopetamine D. cardiopetalum 93

18

Chapter-1 Introduction

42 599.567 C33H45NO9 15-acetylcardiopetamine D. cardiopetalum 93

43 361.5695 C21H31NO4 Neoline D.pentagynum 94

44 363.517 C21H33NO4 Karakolin D. pentagynum 94

45 409.5429 C22H35NO6 Dihydrogadesine D. pentagynum 94

46 435.5521 C24H37NO6 Pentagynine D. pentagynum 94

47 449.6853 C24H35NO7 Occidentalidine D. occidentale 95

48 449.6163 C25H39NO6 Deltaline D. occidentale 95

49 453.6429 C24H39NO7 Delphisine D. staphisagria 96

50 449.1621 C25H37NO6 Nordhagenine A D. nordhageni 97

51 493.2676 C26H39NO8 Nordhagenine B D. nordhageni 97

52 493.5897 C27H39NO8 Nordhagenine C D. nordhageni 97

53 467.2883 C25H41NO7 Deltatsine D. crispulum 98

54 407.2671 C23H37NO5 Crispulidine D. crispulum 98

55 511.2934 C30H41NO6 Delphicrispuline D. crispulum 98

19

Chapter-2 Experimental Introduction

CHAPTER-2 EXPERIMENTAL

2.1 General experimental conditions

2.1.1 Physical constants

Melting points and optical rotations of the isolated compounds were measured with Buchi melting point apparatus model 535 and P-200 JASCO Digital Polarimter respectively.

2.1.2 Spectroscopic techniques

Structures of all pure isolated compounds were established by using spectrometric and spectroscopic procedures including Ultraviolet (UV), Infra-Red (IR), MS (EIMS), 1D- and 2D-

NMR.

2.1.3 Column chromatography

For column chromatography, silica gel (70-230 mesh) and for thin layer chromatography, aluminum sheets “20 x 20 cm silica gel 60 F254” were used which were purchased from Merck,

Darmstadt, Germany. The solvent systems used were “Ace: Hex: DEA (8-2-10), (1-9-10), (7-3-

10) and (4-6-10)”, otherwise specified. Flash column chromatography was executed using “silica gel HF-254, having particle size 0.04-0.063 millimeters, 230-400 mesh, (Fluka AG, Buchs SG)”.

2.1.4 Solvents

Commercial grade solvents including methanol, ethyl acetate, acetone, chloroform and n-hexane were used during whole research work. For NMR analysis, deuterated solvents

(Aldrich) were used.

20 Chapter-2 Experimental Introduction

2.1.5 Detection of alkaloids on chromatographic plates

UV light 254 nm and 366 nm were used for visualizing spots on TLC plates.

Dragendorff’s reagent was also employed for detecting reddish spots of alkaloids.

2.2. Plant materials

Delphinium chitralense and Delphinium denudatum Wall were collected from Kumrat and Doogdara valleys district Dir (Upper) of Khyber Pakhtunkhwa, Pakistan in May-June 2013.

Botanical identification was done by Dr. Ali Hazrat, Head of the Department of Botany SBBU

Sheringal. Voucher specimens (H.UOM.BG-160 & H.UOM.BG-161) of both plants were deposited at the Herbarium of the Botany Department, University of Malakand.

2.2.1 Plants materials pretreatment

The aerial parts of shade dried plants, D. chitralense and D. denudatum Wall were crushed into powder with the help of a mechanical grinder. Fine powdered plant materials were further processed for extraction and isolation of pure compounds.

2.2.2 Extraction

Dried and powdered aerial parts (10 kg, & 8 kg each) of both selected plants were extracted with 80% commercial grade methanol (3 x 20 L) at room temperature. After extracting, the filtrates (Menstrum) were filtered through a nylon cloth for removing the undissolved materials (marc). The methanolic extracts of both plants were evaporated under reduced pressure using rotary evaporator to yield dense gummy crude methanol fractions 800 g & 630 g respectively.

21 Chapter-2 Experimental Introduction

2.2.3 Fractionation

Crude methanol extracts of both plants (800 g & 630 g each) were dissolved separately in

1.5 liters distilled water with continuous stirring and shaking. HCl (1N) solution is added to both dissolved extracts with constant stirring to acidify the solutions having pH range from 1-2. The acidic fractions were then treated with chloroform in separating funnel for removing non- alkaloid constituents. The solvent solvent extraction technique was repeated using chloroform

(3x2 L) to get maximum extraction. Collected chloroform layers were concentrated using rotary evaporator yielded 210 g and 166 g acidic fractions.

The remaining aqueous fractions were basified with 10 percent sodium hydroxide solution having final pH ranging from 8-10. Both basic aqueous fractions were then extracted using chloroform (3x2 L) until chloroform layer become transparent and clear. Chloroform is evaporated using rotary evaporator which yield basic alkaloid fractions (Fraction B, 18 g and 13 g). Both basic fractions showed positive results with Dragendorff reagent. (Scheme 2.1).

22 Chapter-2 Experimental Introduction

Crushed Plant Materials D. chitralense (10kg) D. denudatum Wall (8kg)

Maceration with Me-OH

Crude (Me-OH extract) Residues Evaporated at 40 0C Stored

Acidified (1N HCl, pH: 1-2) Extracted with CHCl3 (3x1.5L)

Organic layer-I Aqueous layer-I Acidic (stored)

Basified (10 % NaOH, pH: 8-10) Extracted with CHCl3 (3x1.5L)

Organic layer-II Aqueous layer-II CHCl3 ( Evaporated) Water soluble substances

Crude alkaloids Tested with Dragendorff

Purification through Chromatographic technique Structure elucidation through spectroscopic technique

Bio-assays Pure alkaloids

Scheme 2. 1. Fractionation of D. chitralense & D. denudatum Wall

23 Chapter-2 Experimental Introduction

2.2.4 Isolation of alkaloids from D. chitralense

The 18 g basic fraction was adsorbed on silica gel for making slurry which was loaded in a suitable size glass column having 360 g silica gel (E. Merck; type 230, 70 and 60 mesh). The column was eluted with gradients of n-hexane, chloroform and methanol to get 14 sub-fractions

(C1- C14). After comparative TLC the fractions were combined and the resulting fractions were reduced to six (DX1-DX6). All six-sub fractions on repetitive FCC, using n-hexane: acetone:

DEA solvent system resulted, six new and three known alkaloids for the first time from D. chitralense.

Amongst isolated compounds, the known alkaloids ajaconine (10) and delectinine (11) were major alkaloids while the other compounds were isolated in lesser amount from D. chitralense. Structures of all isolated compounds were elucidated from their spectroscopic and

X-ray data (Scheme 2.2).

24 Chapter-2 Experimental Introduction

Fraction-B (18 g) (CC) (360g), hex., hex.-chlo., chlo., chlo.-MeOH

DX-1 DX-2 DX-3 DX-4 DX-5 DX-6 CC, 5%Acetone,hexane CC, 2:8, +5 drops of diethyl amine CC, 1:9, CC,2:8 ace. Hex. DEA ace. Hex. in 100ml ace. Hex. DEA DEA

SH-1 (20 mg) SH-9 8 mg SH-2 (27 mg) SH-6 (13 mg) SH-3 (20 mg) SH-5 (10 mg)

SH-4 SH-7 SH-8 (10 mg) (17 mg) (10 mg)

Scheme 2. 2. Isolation of alkaloids from D. chitralense

2.2.5 Isolation of alkaloids from D. denudatum Wall.

Same procedure was used for the isolation and purification of compounds from D. denudatum as the one used for the isolation and purification from D. chitralense. As a result, two new and one known compounds were isolated (Scheme 2.3).

25 Chapter-2 Experimental Introduction

Alkaloidal Fraction-B (13 g) (CC) (260g), hex., hex.-chlo., chlo., chlo.-MeOH

DD-1 DD-2 DD-3 DD-4 DD-5 CC, 5%Acetone,hex +5 drops of diethyl amine CC,2:8 in 100ml ace. Hex. DEA

T-1 T-2 T-3 (10 mg) (8mg) (15 mg)

Scheme 2. 3. Isolation of alkaloids from D. denudatum Wall

2.3 X- Rays diffraction studies

XRD for all isolated crystals was carried out with the help of STSE-IPDS II fitted with low temperature unit of a Brukar kappa APEXII СCD diffractometer using Mo-Kα radiation (λ =

0.71073 Å) and graphite-monochromator at room temperature. Crystal structures & refinements were accomplished by SIR97 [99], SHELXL97 [100] and WinGX [101].

2.4 Assay for enzymatic inhibition and ic50 value determination

Acetylcholinesterase Electric-eel E.С 3.1.1.7, butyrylcholinesterase horse-serum E.С

3.1.1.8, acetylthiocholine iodide, butyrylthiocholine chloride, galanthamine and 5,5´-dithiobis[2- nitrobenzoic-acid] were purchased from Sigma Aldrich and used as such. Commercial grade solvents were employed for isolation as well as purification of compounds. Spectrophotometric

26 Chapter-2 Experimental Introduction method was used for measuring acetylcholinesterase and butyrylcholinesterase enzymes inhibitory activities according to procedure mentioned in literature [102]. Defined practice and evaluation conditions were applied during whole assay [103].

Acetylthiocholine iodide and butyrylthiocholine chloride substrates for the assay of acetylcholinesterase and butyrylcholinesterase respectively were used. DTNB reagent was employed for measuring activity of cholinesterases. A solution having 0.2 mM DTNB in 62 mM sodium phosphate buffer having basic pH 8, test compound solution 40 μL and BChE or AChE solution (40 μL) both properly mixed, incubated at 25 ºС for 15.0 minutes. Acetylthiocholine or butyrylthiocholine 40 μL, were added for starting the respective solution Hydrolysis of acetylthiocholine iodide or butyrylthiocholine chloride by the respective enzymes and formation of 5-2-Nitrobenzoate anion followed by formation of complex DTNB to give a compound with yellow color. The final yellow colored compound was detected with the help of BMS spectrophotometer. Reactions were performed in triplicate and the results presented were average values. The concentrations of the isolated compounds that inhibited the hydrolysis of substrates

(as mentioned above) by 50% (IС50) were determined as function of increasing concentration of the compound in the assays on the inhibition values. The concentration of DMSO was 6% in reaction mixture.

27 Chapter-2 Experimental Introduction

2.5 Physical and spectroscopic data of new compounds from D. chitralense

2.5.1 Chitralinine-A (1)

Physical state: White crystal

Yield: 17 mg

Rf: 0.31 [Acetone: n-Hexane: DEA (3:7:10 drops)]

Melting point: 224-227 0C

Solubility: Chloroform and Methanol

30 0 [α]D : -35 (c = 1, CHCl3)

-1 IR υmax cm : 1720 & 1650 (C=O), 988 (C=CH2), 3460, 3422 (OH)

EI-MS m/z : 373.2 (C21H27NO5, cal. 373.4417)

EI-MS (m/z, peak %): 373 (6), 357 (65), 340 (5), 324 (24), 310 (6), 298 (8)

1 13 H-NMR & C (600 & 150 MHz / CDCl3): See Table 3.1

2.5.2 Chitralinine-B (2)

Physical state: White powder

Yield: 10 mg

Rf: 0.23 [Acetone: n-Hexane: DEA (3:7:10 drops)]

Melting point: 252-255 0C

Solubility: Chloroform and Methanol

30 0 [α]D : -25 (c = 1, CHCl3)

28 Chapter-2 Experimental Introduction

-1 IR υmax cm : 1660, 1450 (C=O), 3480, 3360, 3280 (OH) 1030 (C=CH2)

EI-MS m/z : 421 (C21H27NO8, cal. 421.4396)

EI-MS (m/z, peak %): 421 (3), 404 (95), 388 (38), 372 (25), 358 (20), 342 (14), 326 (6)

1 13 H-NMR & C (500 & 125 MHz / CDCl3): See Table 3.3

2.5.3 Chitralinine-C (3)

Physical state: Amorphous powder

Yield: 8 mg

Rf: 0.23 [Acetone: n-Hexane: DEA (4:6:10 drops)]

Melting point: 233-238 0C

Solubility: Chloroform and Methanol

30 0 [α]D : -23 (c = 1, CHCl3)

-1 IR υmax cm : 1658, 1446 (C=O), 3500, 3350 (br OH), 1032 (C=CH2)

EI-MS m/z: 407 (C21H29NO7, cal. 407.4563)

EI-MS (m/z, peak %): 407 (7), 390 (90), 376 (55), 360 (30), 344 (12), 328 (8), 312 (5)

1 13 H-NMR & C (600 & 150 MHz / CDCl3): See Table 3.4

2.5.4 Chitraline-A (4)

Physical state: Amorphous powder

Yield: 10 mg

Rf: 0.27[Acetone: n-Hexane: DEA (2:8:10 drops)]

29 Chapter-2 Experimental Introduction

Melting point: 176-179 0C

UV activity: Active on TLC

Solubility: Chloroform and Methanol

30 0 [α]D : -31 (c = 0.9, CHCl3)

-1 IR υmax cm : 3560, 3400, 3450 (OH), 1110 & 1091 (OCH3)

EI-MS m/z: 467.1 (C25H41NO7, cal. 467.5939)

EI-MS (m/z, peak %): 467.1(6), 450 (78), 434(55), 418 (36), 388 (25), 358 (20), 328 (15), 298

(12), 284 (6), 58 (4)

1 13 H-NMR & C (500 & 125 MHz / CDCl3): See Table 3.5

2.5.5 Chitraline-B (5)

Physical state: Amorphous powder

Yield: 10 mg

Rf: 0.33 [Acetone: n-Hexane: DEA (2:8:10 drops)]

Melting point: 149-153 0C

UV activity: Active on TLC

Solubility: Chloroform and Methanol

30 [α]D : -40(c = 1, CHCl3)

-1 IR υmax cm : 1740, 1690 (С=O), 1220 & 1110 (OСH3)

HREI-MS m/z: 537.1150 (C29H47NO8, cal. 537.6835)

30 Chapter-2 Experimental Introduction

HREI-MS (m/z, peak %): 537.11 (8), 506 (75), 476 (55), 446 (25), 358 (25), 416 (18), 386

(15), 356 (10), 342 (7), 298 (5) 57 (3)

1 13 H-NMR & C (500 & 125 MHz / CDCl3): Table 3.6

2.5.6 Chitraline-C (6)

Physical state: White amorphous powder

Yield: 13 mg

Rf: 0.37 [Acetone: n-Hexane: DEA (2:8:10 drops)]

Melting point: 161-164 0C

UV activity: Active on TLC

Solubility: Chloroform and Methanol

30 0 [α]D : 20 (c = 0.5, СHCl3)

-1 IR υmax cm : 3500, 3450 (br OH)

EI-MS m/z: 451.3 (C25H41NO6, cal. 451.5945)

EI-MS (m/z, peak %): 451.3 (8), 420 (66), 390 (40), 374 (28), 358 (25), 328 (15), 314 (6), 271

(12), 284 (5), 43 (3)

1 13 H-NMR & C (500 & 125 MHz / CDCl3): See Table 3.7

2.5.7 Chitraline-D (7)

Physical state: Amorphous powder

Yield: 20 mg

Rf: 0.42 [Acetone: n-Hexane: DEA (2:8:10 drops)]

31 Chapter-2 Experimental Introduction

Melting point: 113-118 0C

UV activity: Active on TLC

Solubility: Chloroform and Methanol

30 0 [α]D : -36 (c = 1, CHCl3)

-1 IR υmax cm : 1765, 1710 (С=O)

EI-MS m/z: 509.0 (C27H43NO8, cal. 509.6305)

HREI-MS (m/z, peak %): 509 (3), 478 (78), 448 (60), 432 (24), 426 (8), 386 (4), 47 (2)

1 13 H-NMR & C (600 & 150 MHz / CDCl3): See Table 3.8

2.6 Physical and spectroscopic data of new compounds from D. denudatum wall.

2.6.1 Compound (8)

Physical state: Amorphous powder

Yield: 10 mg

Rf: 0.32 [Acetone: n-Hexane: DEA (2:8:10 drops)]

Solubility: Chloroform and Methanol

UV activity: Active

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

EI-MS (m/z): 423.16, (C23H37NO6, cal.423.55)

1 13 H-NMR & C (600 & 150 MHz / CDCl3): See Table 3.9

32 Chapter-2 Experimental Introduction

2.6.2 Compound (9)

Physical state: Amorphous powder

Yield: 8 mg

Rf: 0.27 [Acetone: n-Hexane: DEA (2:8:10 drops)]

Solubility: Chloroform and Methanol

UV activity: Active

25 o [] D: +20.00 (c = 0.30, СHCl3)

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

EI-MS (m/z): 433.19, (C25H39NO5, cal.433.5890)

1 13 H-NMR & C (500 & 125 MHz / CDCl3): See Table 3.12

2.7 Physical and spectroscopic data of known compounds from D. chitralense.

2.7.1 Ajaconine (10)

Physical state: White crystals

Yield: 20 mg

Melting point: 173-175 oC

Rf: 0.43 [Acetone: n-Hexane: DEA (2:8:10 drops)]

Solubility: Chloroform and Methanol

UV activity: Active on TLC

25 o [] D: -53.00 (c = 1, СHCl3)

-1 IR max, cm : 1118 (С-O ether), 1660 & 935 (С= СH2), 3370, 3285 (OH)

33 Chapter-2 Experimental Introduction

EI-MS (m/z): 359.0 (С22H33NO3, cal. 359.5010)

1 H-NMR (600 MHz/ СDCl3): Available in results and discussion

2.7.2 Delectinine (11)

Physical state: White crystals

Yield: 27 mg

Melting point: 119-121 oC

Rf: 0.42 [Acetone: n-Hexane: DEA (2:8:10 drops)]

Solubility: Chloroform and Methanol

UV activity: Active

25 o. [] D:::. +15.0 (c = 0.55, СHCl3)

-1 IR max, cm (СHCl3): 3550, 3414, 3630 and 3370

EI-MS (m/z): 453.0(C24H39NO7, cal 453.5370 )

1 H-NMR (600 MHz/ СDCl3): Available in result and discussion

2.8 Physical and spectroscopic data of known compounds from D. denudatum wall.

2.8.1 Dihydropentagynine (12)

Physical state: White crystals

Yield: 15 mg

Rf: 0.36 [Acetone: n-Hexane: DEA (2:8:10 drops)]

Melting point: 151-156 oС

UV activity: Active on TLC

34 Chapter-2 Experimental Introduction

Solubility: Chloroform and Methanol

25  : -20.0o (c = 1.1, MeOH) D

-1 IR max cm : 3440, 3414. (OH).

EI-MS (m/z): 407.0 (С23H37NO5, cal. 407.2672)

1 H-NMR (600 MHz/ CDCl3): Available in result and discussion

35 Chapter-3 Results and Discussion

CHAPTER-3 RESULTS AND DISCUSSION

3.1 Present work

During research, we isolated pure alkaloids from the basic chloroform fractions of both investigated species using column chromatography. Structures of pure alkaloids were elucidated using spectroscopic data. Enzymatic inhibitory activities were also studied for the new and known isolated compounds.

The aerial parts of both plants were macerated with methanol for extraction. The crude extract was partitioned with chloroform at varied pH levels. (Scheme 2.1). Basic chloroform soluble fractions obtained at varied pH levels were subjected to column chromatography which yielded six sub-fractions (DX-1 to DX-6 from D. chitralensi) and five sub-fractions (DD-1 to

DD-5 from D. denudatum Wall). On subject to Column Chromatography and repeated Flash column chromatography sub-fractions from both plants resulted in the isolation of fifteen compounds from both plants out of which nine (seven new and two known but new source) were isolated from D.chitralense and three (two new and one known) from D.danudatum Wall.

3.2 New diterpenoids from Delphinium chitralense

3.2.1 Chitralinine-A (1)

Chitralinine-A (Figure 3.1) was isolated from crude basic fraction (Scheme 2.1). On repeated FCC, by solvent system 20% acetone, 80% n-hexane with 10 drops of DEA/ 100 ml.

36 Chapter-3 Results and Discussion

12 17 CH 16 2 11 OH 13 O 20 HO 14 HO 15 1 9 2 10 8 H3C N 3 5 7 4 6

O 19 18

Figure 3. 1. Structure of chitralinine-A (1)

IR spectrum of chitralinine -A (1) exhibited absorption at 3460 and 3422 cm-1 (OH

-1 -1 groups), 1720 & 1650 cm (C=O) and 988 cm (C=CH2). A molecular formula C21H27NO5 was established on the basis of its molecular peak in EI-MS at m/z 373.2(cal.373.4417). Other

+ + + eminent fragments were at m/z 356 [M-OH] (60), 340 (6) [M -OH] and 324 (14). Chitraline-A

(1) mass fragmentation clearly showed that it is similar to alkaloids having atisine skeleton.

Chitralinine-A (1) 1H-NMR spectrum of displayed signals for six methylene, two methyl and some methine protons. The signal of terminal methylene protons in the 1H-NMR spectrum of chitralinine-A (1) was observed downfield at  5.07 and 4.8 respectively. Similarly, two singlets each having a single proton integration at  4.22 and 3.59 were allocated, to H-1 and H-20 in downfield region of spectrum. Triplet of one proton integration at  4.67 (J = 4.7 Hz) typical for

C-2 methine bearing hydroxyl group. Similarly, the three other triplets each of one proton integration at  2.08 (J = 5.8 Hz) and 2.66 (J = 11 Hz) were given to Н-9 and Н-12 protons. In

37 Chapter-3 Results and Discussion the up-field region, a singlet of three protons integration at  1.17 and two singlets each with one proton integration at  2.44 and 2.25 were observed due to the resonance of Н -18, Н -5 and Н -7 in the structure of 1, while a doublet of one proton integration at  2.19 (J = 3 Hz) was due to the

Н -14 methine proton.

13C-NMR (DEPT & BB) spectrum (Table-3.1) of chitralinine-A (1) presented twenty- one signals which includes six methylene, two methyl, six methine and seven quaternary carbons. The 1Н -13С correlati0ns were determined from HMQC spectrum, whereas long-range

1H-13С connectivity were obtained through the HMBC. 13C-NMR spectrum displayed signals in the downfield region at  209.4, 142, 110.3, 78.6, 78.4 and 75.9, respectively, were due to the resonance of Ϲ-6, Ϲ-13, Ϲ-16, Ϲ-17, Ϲ-9, Ϲ-1 and Ϲ-2. Similarly, the signal at  48 and 27 were the indicative of the two methyl groups present in chitralinine-A (1).

H 12 H 17 16 CH2 11 13 HO O H OH20 14 HO 15 H 1 9 2 10 8 H3C N 3 5 7 H 4 6 H

O 19 18

Figure 3. 2. COSY interaction in chitralinine-A (1)

38 Chapter-3 Results and Discussion

The compound contains three hydroxyl groups and two ketonic functionalities, as deduced from NMR spectrum, one hydroxyl was placed at Ϲ-1 ( 78.4), second at Ϲ-2 ( 75.9) and third at C9 (78.6) on the basis of the chemical shift and biogenetic considerations.

In the 1H-1H COSY 45o spectrum (Figure 3.2), H-1 showed correlations with the C-2 methine proton at  4.67 (J = 4.74 Hz) as well as H-3 ( 1.82) indicating the position of two hydroxyl groups to be at Ϲ-1 and С-2. Similarly, H-11 ( 2.31) showed coupling with Н-12 (

2.66 ppm), supporting the presence of carbonyl functionality at Ϲ-13 ( 209.4). In the HMBC spectrum (Figure-3.3), H-17 ( 5.07) showed correlation with C-16 ( 142), C-12 ( 53.4) and

C-15 ( 34.9). Similarly, H-20 ( 3.59) showed interactions with Ϲ-10 ( 39.7), Ϲ-8 ( 44.) and

Ϲ-13 ( 209.4). Furthermore, H-20 ( 3.59) showed correlation with C-10 ( 39.7), C-9 ( 78.6), and C-1 ( 78.4), while the H-5 ( 2.44) showed interaction with C-4 ( 36.7), C-10 (39.7), C-9

( 78.6) and C-6 ( 209.4).

12 17 16 CH2 H HO 11H OH 13 O 20 14 HO 15 1 9 2 10 8 H3C N 3 5 7 4 6 H H O 19 18

Figure 3. 3. HMBC interaction in chitralinine-A (1)

39 Chapter-3 Results and Discussion

Single-crystal X-ray crystallographic analysis of chitralinine-A was also performed.

Chitralinine-A was crystallized as monoclinic unit of crystal system with C2 space group. Its crystal structure is given in (Figure 3.4) and other important crystal data in (Table 3.2).

Figure 3. 4. Crystal Structure of compound 1, with 50 % probability of thermal ellipsoids and hydrogen atoms are omitted for clarity.

40 Chapter-3 Results and Discussion

1 13 Table 3.1. Н- (600 MН z) and С -NMR (150 MHz) data of chitralinine-A (1) in CDCl3

Ϲ. No. 1Н - δ (J Hz) 13 С - (δ) Multiplicity HMBϹ Correlations

1. 4.22, s 78.4 ϹH 2, 3, 10

2. 4.67, t, J =4.74 Hz 75.9 CH

3 1.82, m 40.7 CH2

4 36.7 C

5 2.44, s 59.9 CH 4, 6, 10, 9

6 209.4 C

7 2.25, s 49.7 CH2 6, 8

8 44 C

9 2.08, t, J= 5.8 Hz 78.6 CH

10 39.7 C

11 2.31, t, J= 2.1 Hz 32.3 CH2 8, 9, 12, 16

12 2.66, t, J= 11 Hz 53.4 CH

13 209.4 CH

14 2.19, d, J= 3. Нz 57.9 ϹH

15 2.59, d, J= 4 Нz 34.9 CH2

16 142 C

17 5.07, 4.8, s 110.3 CH2 12, 15, 16

41 Chapter-3 Results and Discussion

18 1.17, s 27 CH3

19 3.04, s 63.1 CH2 3, 4, 18

20 3.59, br s 70.3 CH 10, 1, 9

21 2.25, s 48 CH3

Table 3. 2. Crystal data and structure refinement of chitralinine-A

Crystal.Parameter 1 Crystal. Parameter 1

3 3 Empirical formula. C21H27NO5 Volume. Å 2150.4 (6) Å

Formula weight. 373.43 μ (m.m-1) 0.0.8

Temperature(K) 29.6 Z 4

Wavelength. (Å) 0.71073 Density. (Mg m-3) 1.153

Crystal system. Monoclinic (h, k, l) min (-31, -5, -15)

Space group C2 (h, k, l.) max (31, 9, 15)

A 25.726 (5) Å Theta (max) 26.0

B 7.5766 (12) Å R (reflection) 0.053(2408)

C 119.326 (7)Å wR2 0.185

The structure of chitralinine-A was deduced from its spectral and crystal data. Thus the structure of chitralinine-A was assumed as 1α, 2β, 9α-trihydroxy diatisinone (Chitralinine-A).

42 Chapter-3 Results and Discussion

3.2.2 Chitralinine-B (2)

Chitralinine-B (Figure 3.5) was obtained from crude basic alkaloidal sub fraction DX4

(Scheme 2.2). On repetitive FCC, via solvent system 20% acetone, 80% hexane with 10 drops of

DEA /100 ml.

OH

HO 17 CH 12 16 2 11 OH 13 O 20 HO 14 HO 15 1 9 2 10 8 H3C N 3 5 7 4 6

19 O 18 HO

Figure 3. 5. Structure of chitralinine-B (2)

The IR (KBr) spectrum, of chitralinine-B (2) showed absorption at 3480, 3360 and 3280 cm-1 which were tentatively assigned to OH group while 1660, 1450 and 1030 cm-1 carbonyl and terminal methylene group.

Electron impact mass measurement on the molecular ion afforded the exact mass to be

1 m/z 421.0 (cal. for C21H27NO8, 421.4396). The Н-NMR spectrum (Table-3.3) of 2 exhibited

43 Chapter-3 Results and Discussion signals for two methyl, four methylene and several methine protons. In the downfield region of the spectrum, a singlet of proton integration each at  5.02 and 4.85 were assigned to terminal methylinic protons. The methyl protons at C-18 resonated in the up field region as a singlet of three protons at δ 1.16. The proton at С-14 showed a doublet of one proton integration at δ 2.26

(J = 2.5 Hz) whereas the H-15 proton singlet appeared at δ 1.89. The characteristic sharp triplet peak at δ 4.64 (J = 4.9 Hz) was due to the resonance of Н-2 proton. The three singlet signals in the same spectrum δ 5, 3.59, and 2.46 were due to the resonance of H-19, H-20 and H-21 protons respectively. All other signals in the 1Н-NMR spectrum of 2 were in their expected range which are common for all Ϲ20 diterpene alkaloids.

The 13Ϲ-NMR spectrum (Table-3.3) (BB & DEPT) of chitralinine-B (2) showed twenty- one signals, including two methyl, four methylene, seven methine and eight quaternary carbons.

The 1H-13Ϲ correlations were determined by the НMQϹ spectrum, while the long-range 1Н-1Ϲ connectivity were obtained through the НMBϹ technique. The 13Ϲ-NMR spectrum displayed signals at  209.4, 209.5, 141.9, 110.4, 89.3, 44, 38.7 and 49.7 were assigned to C-6, C-13, C-16,

C-17,C-12, C-10, C-21 and C-5 respectively. Similarly, the signals at  94.2, 89.3, 80.7, 78.3,

75.4, and 73.3 were due the resonance of carbons bearing hydroxyl group. The two signals observed in the upfield at  38.7 and 13.4 were due to Ϲ-21 and Ϲ-18 methyl carbons respectively.

In the НMBϹ spectrum (Figure 3.6), Н-1 ( 4.16) showed correlation with Ϲ-2 ( 73.3) and Ϲ-10 ( 44). Similarly, Ϲ-7 ( 2.05) showed НMBϹ interactions with Ϲ-8 ( 36.7), Ϲ-5

(49.7) and Ϲ-6 ( 209.4) showed the exact position of ketonic functionality at Ϲ-6 of chitralinine-B. Furthermore, Н-15 ( 1.89) showed correlation with Ϲ-16 ( 141.9), Ϲ-8 ( 36.7)

44 Chapter-3 Results and Discussion and Ϲ-17 ( 110.4). The overall spectral data of chitralinine-B (2) was identical to chitralinine-A

(1) except the presence of additional hydroxyl groups at Ϲ-11, Ϲ-12 and Ϲ-19.

OH

HO 17 CH 12 16 2 11 H13 O HO H 20 14 15 H HO 1 9 H 2 10 OH 8 H3C N 3 5 7 H 4 6 H

19 O 18 HO

Figure 3. 6. HMBC interaction in chitralinine-B (2)

On the basis of physical and spectroscopic data, the structure of chitralinine-B (2), was deduced as 1, 2,9,11,12,19 hexahydoxy di-atisinone.

1 13 Table 3.3. H- (500 MHz) and C-NMR (125 MHz) data of chitralinine-B (2) in CDCl3

Ϲ. No 1Н- δ (J Нz) 13Ϲ- (δ) Multiplicity НMBϹ Correlations

1. 4.16, s 75.4 CH 2, 3, 10

2 4.64, t, J=4.9 Hz 73.3 CH

3 1.92, m 34 CH2 2, 3, 4, 19

4 32.5 C

45 Chapter-3 Results and Discussion

5 2.84, s 49.7 CH 4, 6, 10, 9

6 209.4 C

7 2.05, s 44 CH2 5, 6, 8

8 36.7 C

9 78.3 C

10 43.6 C

11 3.36, s 80.7 CH 8, 9, 12, 16

12 89.3 C

13 209.5 Ϲ

14 2.26, d, J= 2.5 Hz 48.7 CH 13, 20, 8

15 1.89, s 30.8 CH2 8, 16, 17

16 141.9 C

17 5.02, 4.85, s 110.4 CH2 12, 15, 16

18 1.16, s 13.4 CH3

19 5, s 94.2 CH 3, 4, 18

20 3.59, br s 70.1 CH

21 2.46, s 38.7 CH3

46 Chapter-3 Results and Discussion

3.2.3 Chitralinine-C (3)

Chitralinine-C (Figure 3.7) was isolated from sub-fraction (DX-5) of the crude alkaloidal mixture (Scheme 2.2). On repeated FCC, using solvent system 30% acetone, 70% hexane with

10 drops of DEA / 100 ml.

OH

HO 17 CH 12 16 2 11 OH 13 20 HO 14 HO 15 1 9 2 10 8 H3C N 3 5 7 4 6

19 O 18 HO

Figure 3. 7. Structure of chitralinine-C (3)

The IR (KBr) spectrum, of chitralinine-C (3) presented absorption at 3500 and 3350/ cm

-1 which were allocated to OH group while 1658 and 1446 cm (C=O), and 1032 (C=CH2) respectively. Chatralinine-C has assigned molecular formula C21H29NO7 on the basis of electron impact mass measurement [m/z 407, calcd. 407.4563].

The 13C-NMR spectrum (Table-3.4) of chitralinine-C (3) showed signals for seven methionine and two methyl protons. In the downfield region of the 1H-NMR spectrum, a singlet of proton integration, each at  5.02 and 4.85 were due to terminal methylinic protons. The

47 Chapter-3 Results and Discussion methyl protons at C-4 resonated in the up field region as a singlet of three protons at δ 1.15. A multiplet of one proton integration at δ 1.63 was observed due to H-14 methine proton whereas one other multiplet δ 1.91 was due to the up field resonance of H-3 of two protons integration.

The characteristic two broad singlet signals at δ 4.97 and 3.57 were due to the downfield resonance of H-19 and H-20 methine protons, while the doublet of doublet signal at δ 1.95 and

1.91 (J= 2 Hz) were due to the H-13 methylinic protons with integration ratio of two. The prominent signals were observed for the rest of the methine and methylinic protons in their expected range.

The 13C-NMR spectrum (Table-3.4) of chitralinine-C (3) presented twenty-one signals, including five methylene, two methyl, seven methine and seven quaternary carbons. The 1H-13С correlations were determined from HMQС spectrum, while long range 1H-13С connectivities were obtained from HMBС spectrum. The 13С-NMR spectrum displayed signals in the downfield region at  209.6, 142, 110.4, 94.2, 83.8, 80.7, 78.3, 75.7 and 73.3 were due to С-6, C-

16, С-17, С-19, C-9, С-11, С-12, С-1 and С-2 of chitralinine-C (3). The two signals observed in the up field at  38.7 and 13.5 were due to С-21 and С-18 methyl carbons respectively.

In the HMBC spectrum (Figure 3.8), H-13 ( 1.95) showed correlation with С-12 (

78.3) and С-14 ( 40.7). Similarly, H-17 ( 5.0, 4.85) showed HMBC interactions with С-16 (

142.0), С-12 ( 78.3) and С-15 ( 26.9) showed the exact position of olifinic double bond in chitralinine-C. Furthermore, H-3 ( 1.91) showed correlation with С-3 ( 34.0), С-4 ( 32.6) and

С-19 ( 94.2).

48 Chapter-3 Results and Discussion

OH

HO 17 CH 12 16 2 H 11 H13 HO H 20 H 14 15 H HO 1 9 H 2 10 OH 8 H3C N 3 5 7 H 4 6 H

19 O 18 HO

Figure 3. 8. HMBC interaction in chitralinine-C (3)

The overall spectral data of chitralinine-C (3) was identical to chitralinine-A (1) except the absence of ketonic functionality at С-13. From the overall above spectroscopic data, the structure of chitralinine-C (3), was assumed as 1,2,9,11,12,19 hexahydoxy atisinone.

1 13 Table 3. 4. H- (500 MHz) and C-NMR (125 MHz) data of chitralinine-C (3) in CDCl3

1 13 C. No Н - δ (J Нz) С- (δ) Multiplicity НMBС Correlations

1 4.19, s 75.7 СH 2, 1, 10, 20

2 4.64, t, J=4.86 Hz 73.3 CH2

3 1.91, m 34.0 CH2 2, 3, 4, 19

4 32.6 C

49 Chapter-3 Results and Discussion

5 2.24, s 48.9 CH 4, 5, 10, 6

6 209.6 C

7 2.30, s 44.2 CH2 5, 6, 8

8 36.7 C

9 83.8 C

10 44.0 C

11 3.27, s 80.7 CH2

12 78.3 CH

13 1.95, 1.93, dd, J= 2 34.8 C 20, 8, 13

Hz

14 1.63, m 40.7 CH

15 1.15, s 26.9 CH2 8, 16, 17

16 142 C

17 5.02, 4.85, s 110.4 CH2 12, 16, 15

18 1.15, s 13.5 CH3

19 4.97, s 94.2 CH2

20 3.57, br. S 70.1 CH

21 2.45, s 38.7 CH3

50 Chapter-3 Results and Discussion

3.2.4 Chitraline-A (4)

Chitraline-A (Figure 3.9) was obtained from sub-fraction (DX-3) of the crude basic alkaloidal fraction (Scheme 2.2). On repeated FCC, using solvent system 10% acetone, 90% hexane with 10 drops of DEA/100 ml

OH OCH3

13 16 12 H3CO 17 14 OCH3 HO

1 10 9 2 11 15 N 8 3 5 OH 4 7 6 H 19 18 OCH3

Figure 3. 9. Structure of chitralin-A (4)

The IR spectrum of chitraline-A showed absorption bands at 3560, 3400, 3450 (OH groups), 1110 & 1091 (OCH3). A molecular formula C25H41NO7 was established on the basis of its molecular peak in EI-MS at m/z 467.1(cal. 467.5939). The other prominent fragments were at m/z 450 (78), 434(55), 418 (36), 388 (25), 358 (20), 328 (15), 298 (12), 284 (6), 58 (4).

The 1H-NMR spectrum (Table 3.5) of chitraline-A (4) presented signals for N.-ethyl, four methoxy and several methylene-methine protons. The signal of H-14 β in the 1H-NMR spectrum of chitraline-A (4) was observed as singlet at  3.87. In the upfield region a triplet of three protons integration at  1.07 (J = 6.5 Hz) and quartet with two protons integration at  2.63

(J = 9 Hz) were indicative of N-ethyl group in the structure of chitraline-A (4). In the downfield

51 Chapter-3 Results and Discussion region of the spectrum four singlets each of three protons integration at  3.49, 3.37, 3.32, and

3.27 were given to the methoxyl groups attached to С-1, С-6, С-14, and С-16 respectively. The triplet with one proton integration at  3.08 (J = 4.9 Hz) was assigned to H-1 proton. The two doublets at  3.23 (J = 7.2 Hz) and  2.35 (J = 4.7 Hz) was due to the resonance of H-6 and H-7 protons respectively. Similarly, other two doublet signals with two protons integration each at 

1.88 (J = 7.6 Hz) and 1.92 (J = 5.2 Hz) were due the resonance of H-12 and H-15 respectively.

The 13C-NMR spectrum (Table 3.5) of chitraline-A (4) exhibited twenty-five signals, which include two methyl, four methoxy, six methylene, eight methine and five quaternary carbons. The 1H-13C correlations were determined by the HMQC spectrum, while the long-range

1H-13C connectivities were obtained through the HMBС technique. The 13C-NMR spectrum displayed four signals at  56.2, 58.8, 57.9 and 57.8 were due to the four methoxyl groups.

Similarly, the signals at  13.2 and 48.9 were the indicative of the N-ethyl group in the chitraline-

A (4).

The alkaloid contains four methoxy, three hydroxy groups, as decided from the NMR spectra. The hydroxyl groups were placed at С-8 ( 80.4), С-9 ( 77.5) and С-13 ( 76.7). The first of the methoxy groups was placed at С-1 ( 86.9), on the basis of the chemical shift and biogenetic considerations. The second methoxyl group was found be present at С-16 ( 83.9), the third methoxyl was placed at С-14 ( 90.5) while the fourth methoxyl was placed at С-6 ( 84.1).

52 Chapter-3 Results and Discussion

OCH OH 3

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

Figure 3. 10. HMBC interaction in chitraline-A (4)

In the HMBC spectrum (Figure 3.10), the H-5 ( 1.70) showed correlation with C-6 (

84.1), C-4 ( 31.04), C-11 ( 43.2), as well as correlations with C-10 ( 28.7). Similarly, H-6 (

3.23) showed interactions with C-7 ( 46), C-5 ( 55.7) and C-8 ( 80.4). Furthermore, H-16 (

3.63) showed correlation with C-15 ( 38.09), while the H-17 ( 3.79) showed interaction with

C-10 ( 28.7) and C-11 ( 43.2). The H-14 ( 3.87) showed correlation with С-8 ( 80.4), С-9 (

77.5) and С-13 ( 76.7). The structure of chitraline-A (4) was recognized from 1H-1H СOSY, 1H-

13С, HMBC and HMQC data. Thus, the structure of chitraline-A (4) was deduced as 1α, 6β, 14α,

16β- tetramethoxy -8β, 9β, 13β-trihydroxy-N-ethyl aconitane.

53 Chapter-3 Results and Discussion

Table 3. 5. 1H- (500 MНz) and 13C-NMR (125 MHz) data of chitraline-A (4)

C. No. 1 Н - δ (J Hz) 13С-(δ) Multiplicity HMBС Correlations

1 3.08, t, J = 4.9 Нz 86.9 СH 2, 11, 17

2 2.16, m 25.9 СH2

3 2.11, m 38.5 СH2

4 31.04 С

5 1.70, d, J = 6 Hz 55.7 CH 4, 6, 11, 10

6 3.23. d, J =7.2 Нz 84.1 СH

7 2.35, d, J = 4.7 Нz 46 CH 5, 7, 8

8 80.4 C

9 77.5 C

10 1.68, s 28.7 CH

11 43.2 C

12 1.88,d, J = 7.6 Hz 33.6 CH2

13 76.7 C

14 3.87, s 90.5 CH 8, 9, 13

15 1.92, d, J = 5.2 Hz 38.09 CH2 13, 15, 16

16 3.63, m 83.9 CH

17 3.79, s 67.7 CH 11, 10, 1

54 Chapter-3 Results and Discussion

18 1.53, s 24.7 CH3

19 2.83, s 64.8 CH2 3, 4, 5, 19

N 2.63, q, J = 9 Hz 48.9 CН2 CH2 CH3 CН3 1.07, t, J = 6.5 Hz 13.2

OCН3 3.27, s 58.8 CH3

OCН3 3.32, s 57.9 CH3

OCН3 3.37, s 57.8 CH3

OCН3 3.49, s 56.2 CH3

3.2.5 Chitraline-B (5)

Chitraline-B (Figure 3.11) was obtained from sub-fraction (DX-3) of the crude basic alkaloidal fraction (Scheme 2.2). On repeated FCC, using solvent system 10% acetone, 90% hexane with 10 drops of DEA / 100 ml.

OCH3 H 16 13 12 14 OCH OCH3 3 17 1 10 9 2 11 15 N 8 3 5 OCOCH3 7 4 6 H 19 18 OCH3 H CO H3CO 3

Figure 3. 11. Structure of chitraline-B (5)

55 Chapter-3 Results and Discussion

The IR spectrum of chitraline-B (5) showed absorption bands at 1740, 1690 cm-1 (C=O),

-1 1220 and 1110 cm (OCH3) respectively. A molecular formula C29H47NO8 was established on the basis of its molecular peak in EI-MS at m/z 537.1150 (cal. 537.6835). The other prominent fragments were at m/z 450 (78), 434(55), 418 (36), 388 (25), 358 (20), 328 (15), 298 (12), 284

(6), 58 (4).

The 1H-NMR spectrum (Table 3.6) of chitraline-B (5) indicated norditerpenoids skeleton and presented signals for N-ethyl, one acetoxy, six methoxy groups, and several methylene and methine protons. The H–14 β proton showed triplet at  4.78 (J = 4.9 Hz) while the H-16 triplet signal was observed at  3.10 (J = 6.3 Hz). In the upfield region a triplet of three protons integration at  0.95 (J = 7.4 Hz) and a quartet with two protons integration at  2.96 (J = 6 Hz) were indicative of N-ethyl group in the structure of chitraline-B (5). In the downfield region of the spectrum six singlets each of three protons integration at  3.50, 3.47, 3.42, 3.37, 3.35 and

3.30 were assigned to the methoxyl groups attached to С-1, С-6, С-7, С-14, С-16 and С-18. The triplet with one proton integration at  3.93 (J = 5.5 Hz) was assigned to H-1 proton while the

H-9 methine proton showed another triplet of one proton integration at  2 (J = 6 Hz). The doublet with one proton integration at  3.66 (J = 3.2 Hz) was due to the resonance of H-6 methine proton.

The 13C-NMR spectrum of chitraline-B (5) exhibited signal for methyl carbon of N-ethyl group resonated at δ 14.1 whereas the six methoxy carbons resonated at δ 58, 57.9, 57.8, 56.3,

56.2 and 55.7. The signals in the downfield region at δ 91.3, 87.4, 83.9, 82.3, 77.2 and 75.5 were found due to the resonance of С-1, С-7, С-6, С-16, С-8 and С-14 carbons respectively. All other methylene and methine carbons showed 13C signals in estimated range as established from 13C-

NMR spectrum.

56 Chapter-3 Results and Discussion

H OCH3

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

OCH3 19 18H3CO H

H3CO

Figure 3. 12. Key HMBC interaction in chitralin-B (5)

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

13C connectivity was obtained through the HMBС technique (Figure 3.12). H-6 proton showed correlations with С-5 (δ 43.2), С-6 (δ 83.9), С-7 (δ 87.4) and С-8 (δ 77.2), whereas the H-9 proton showed interactions with С-8 (δ 77.2) and С-14 (δ 75.5) respectively. Further1H-13С connectivity was established from the same spectrum as H-17 exhibited interaction with С-11 (δ

43.2), С-10 (δ 31.9) and С-1 (δ 91.3) respectively. Thus, on the basis of spectral evidences the structure of chitraline-B (5) was deduced as 1,14α, 8,10β-tetrahydroxy, 16,18β-dimethoxy aconitane.

57 Chapter-3 Results and Discussion

1 13 Table 3. 6. H- (500 MHz) and C-NMR (125 MHz) data of chitraline-B (5) in CDCl3

C. No 1Н- δ (J Нz) 13С- (δ) Multiplicity НMBС Correlations

1 3.93, t, J= 5.5 Нz 91.3 СH 2, 11, 17

2 1.57, m 24.3 СH2

3 1.66, m 29.6 СH2

4 67.9 С

5 2.03, m 43.2 СH

6 3.66, d, J = 3.2 Hz 83.9 СH

7 87.4 C 7, 8, 5, 11

8 77.2 C

9 2.00, t, J = 6 Hz 42.5 CH 8, 9, 14, 13

10 1.70, m 31.9 CH

11 43.2 C

12 1.91, m 29.6 CH2

13 2.45, t, J = 3.7 Hz 38.5 СH 12, 10, 16

14 4.78, t, J = 4.9 Hz 75.5 СH 13, 9

15 1.78, m 33.7 CH2 15, 16, 13

16 3.10, t, J = 6.3 Hz 82.3 CH

17 3.89, s 66.4 CH 1, 11, 10

58 Chapter-3 Results and Discussion

18 3.16 79 CH2

19 3.18, d, J = 3 Hz 63.5 CH2

N 2.96, q, J = 6 Hz 51.1 CН2-CН3 CH2 CH3 0.95, t, J = 7.4 Hz 14.1

OСH3 3.50 58.0 CH3

OСH3 3.47, s 57.9 CH3

OСH3 3.42, s 57.8 CH3

OСH3 3.37, s 56.3 СH3

OСH3 3.35, s 56.2 СH3

OСH3 3.30, s 55.7 СH3

OCOCH3 2.09, s 174.1 OCOCH3

21.5

3.2.6 Chitraline-C (6)

Chitraline-C (Figure 3.13) was obtained as amorphous powder from the crude sub- fraction (DX-3) (Scheme 2.2) on repeated FCC using solvent system 10 acetone : 90% n-hexane:

10 drops DEA/100 ml).

59 Chapter-3 Results and Discussion

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

Figure 3. 13. Structure of chitraline-C (6)

A molecular formula C25H41NO6 was established on the basis of its molecular peak in EI-

MS at m/z 451.3(cal.451.5945) 6 mass fragmentation clearly showed that it is similar to alkaloids having atisine skeleton [81]. IR spectrum of 6 exhibited absorptions at 3500 and 3450 cm-1 for (OH group). Chitraline-C, 1H-NMR spectrum (Table 3.7) displayed signals for N-ethyl group, four methoxy and some methine protons. The H-14 methine proton resonated as singlet at

δ 3.87. The two methyl protons resonated as a singlet of three protons each at δ 1.07 and 1.28 while another singlet of two protons integration was observed at δ 2.90. The other four signals observed downfield at δ 3.48, 3.42, 3.34, and 3.32 were due to α and β oriented methoxyl groups at С-1, С-6, С-14 and С-16 respectively. The H-5 and H-6 methine proton resonated as doublet at δ 2.33 (J = 3 Hz) and 3.24 (J= 3 Hz). The other methylene and methine proton of the 6 showed

1H-NMR signal in their expected range. The 13 С-NMR (BB and DEPT) spectra (Table 3.7) showed twenty-five signals, four methoxy, two methyl, nine methine, six methylene, and four quaternary carbons. The downfield signals at δ 90.6, 88.3, 83.9, 80.4, and 77.1 were due to the resonance of С-14, С-7, С-1, С-6 and С-8 respectively. The two methyl carbons of 6

60 Chapter-3 Results and Discussion demonstrated signal at δ 13.1 (NCH3) and 24.7 (C18) whereas the four methoxyl carbons displayed 13C-NMR signal at δ 58.8, 57.9, 57.6, and 55.7.

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

Figure 3. 14. Key HMBC interaction in chitraline-C (6)

The H-13 proton at δ 2.27 displayed correlation with С-14 methine proton at δ 3.86.

Similarly, the H-14 (3.86) proton showed correlation with H-9 methine proton (Figure 3.14).

The remaining correlations among H-16/H-15, H-13/H-16, were confirmed from the same spectrum of 6. Further H-C connectivities were made from HMBС spectrum as H-9 at δ 1.66 showed correlation with С-8 at δ 77.1, С-14 at δ 90.6 and С-10 at δ 33.6. In the same way the H-

6 methine proton was connected to С-5 at δ 51 and С-7 at δ 88.3 and С-8 at δ 77.1 while H-14 showed cross peaks in the same spectrum with С-9 at δ 46.1 and С-13 at δ 42.5. The proton connectivities were confirmed by 1H-1H- СOSY correlations among H-13/H-14, 14/H-9, H-

13/H-16, H-16/H-15. The structure of 6 was established from 1H-1H СOSY, 1H-13С, HMBС and

HMQС experiment. Thus the structure of 6 is assumed as 1α, 14α, 6,16β-tetramethoxy, 7,8β- dihydroxy, 7β, 8β, N-methyl lycoctonine.

61 Chapter-3 Results and Discussion

Table 3. 7. 1H- (500 MHz) and 13C-NMR (125 MHz) data of chitraline-C (6) in CDCl3

С. No 1H- δ (J Hz) 13С- (δ) Multiplicity HMBС Correlations

3.08, m 83.9 СH 2, 11, 17

1.90, m 28.8 СH2

1.82, m 43.3 СH2

38.5 С

5 2.33, d, J = 3 Hz 51.0 СH 4, 6, 11, 10

6 3.24. d, J = 3 Hz 80.4 CH

7 88.3 C 5, 7, 8

8 77.1 C

9 1.66, d, J = 6.4 Hz 46.1 CH

10 1.56, d, J = 4.8 Hz 33.6 CH

11 48.3 C

12 2.35, t, J = 37.7 CH2

4.3Hz

13 2.27, m 42.5 CH

14 3.86, br s 90.6 CH 8, 9, 13

15 1.92, d, J = 5.2 Hz 38.1 CH2 13, 15, 16

16 3.63, m 82.5 CH

62 Chapter-3 Results and Discussion

17 3.37, s 67.8 CH 11, 10, 1

18 1.28, s 24.7 CH3

19 2.90, s 64.7 CH2 3, 4, 5, 19

N-CH3 2.80, s 44.8 CH3

3.48, s 58.8 СH3

3.42, s 57.9 СH3

3.34, s 57.6 СH3

3.32, s 55.7 СH3

3.2.7 Chitraline-D (7)

Chitraline-D (Figure 3.15) was obtained as powder from the crude sub-fraction (DX-1)

(Scheme 2.2) on FCC using solvent system 20% acetone / 80% n-hexane: 10 drops DEA/100 ml).

IR spectrum of chitraline-D (7) (Figure 3.15) showed absorption at 3500 and 3155 cm-1

(OH groups) and 1734 and 1222 (C=O, OAc) bond respectively. A molecular formula

C25H39NO6 was established on the basis of its molecular peak in EI-MS at m/z 449 (cal.

449.5743).

63 Chapter-3 Results and Discussion

OCH3 13 16 12 H3COCO 17 14 OCH3

1 10 9 2 11 15 N 8 3 5 4 7 OH 6 OH 19 18 OCH3 H3CO

Figure 3. 15. Structure of chitraline-D (7)

The 1H-NMR spectrum (Table 3.8) of chitraline-D (7) displayed triplet signals for methyl of N-ethyl group at δ 1.06 (3H, t, J= 7.08 Hz) in the up field region. In the down field region of the spectrum, a triplet of one proton integration at δ 3.61 (J = 4.5 Hz) was detected which is characteristic for H-14β methine proton. Four singlets, each of three protons integration were observed downfield at δ 3.45, 3.42, 3.34 and 3.26 due to the methoxyl groups of chitraline-

D (7). The H-18 methylene protons resonated as singlet at δ 3.37. A broad singlet of one proton integration at δ 3.32 was due to the resonance of H-17 proton. The C-13 and C-16 methine proton resonated as triplets at δ 2.35 (J = 4.6 Hz) and 3.23 (J = 8.1 Hz) respectively.

The 13С-NMR (Table-3.8) spectrum showed twenty-five signals, including four methoxy, two methyl, seven methylene, seven methine, and five quaternary carbons. The 13С-NMR spectrum of chitraline-D (7) displayed that the methyl carbon of N-ethyl group resonated at δ

14.1 whereas the four methoxyl carbons resonated at δ 57.9, 57.8, 56.2 and 55.8 respectively. In the downfield region of the spectrum, the signals at δ 90.6, 88.4, 84.3, 83.9, 82.6, 77.5 and 77.3

64 Chapter-3 Results and Discussion were due to the resonance of С-14, С-7, С-1, С-6, С-16, С-18 and С-8 carbons respectively. All the other methine and methylene carbons resonated in their expected range as confirmed from the same 13С-NMR spectrum.

H OCH3

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

Figure 3. 16. Key HMBC interaction in chitraline-D (7)

The 1H-13С correlations were determined by HMQС spectrum, while the long-range 1H-

13С connectivity was obtained through the HMBС technique (Figure-3.16). H-13 proton showed correlations with С-16 (δ 82.6), С-10 (δ 67.7), С-12 (δ 31.6) and С-14 (δ 90.6), whereas the H-

16 proton showed interactions with С-13 (δ 45.7) and С-15 (δ 42.6) respectively. Further1H-13С connectivity was established from the same spectrum as H-5 exhibited interaction with С-11

(48.8), С-4 (43.2) and С-8 (77.3) respectively. Thus, on the basis of spectral evidences the structure of 7 is deduced as 1,14α, 8,10β-tetrahydroxy, 16,18β dimethoxy aconitane.

65 Chapter-3 Results and Discussion

1 13 Table 3.8. H- (600 MHz) and С-NMR (150 MHz) data of chitraline-D (7) in CDCl3

С. No 1H- δ (J Hz) 13С- (δ) Multiplicity HMBС Correlations

1 2.91, m 84.3 СH

2 1.94, m 26.1 СH

3 1.59, m 28.7 СH2

4 43.2 С

5 1.92, d, J = 5.2 Hz 46.1 СH 4, 11, 6

6 3.23. d, J =7.2 Hz 83.9 СH

7 88.4 С

8 77.3 С

9 1.82, t, J = 7.5 Hz 49.6 СH 8, 9, 14

10 1.69, m 33.6 СH

11 48.8 С

12 1.66, t, J = 6 Hz 31.6 СH2

13 2.35, t, J = 4.6 Hz 45.7 СH 10, 12, 14, 16

14 3.61, t, J = 4.5 Hz 90.6 СH

15 1.84, m 42.6 СH2

66 Chapter-3 Results and Discussion

16 3.23, t, J = 8.1 Hz 82.6 СH 8, 13, 15, 16

17 3.32, s 67.7 СH 1, 10, 11

18 3.37, s 77.5 СH3

19 2.62, d, J = 4 Hz 64.8 СH2

N 282, q, J = 6.7 Hz 51.1 СH2 CH2 CH3 1.06, t, J = 7.08 Hz 14.1 СH3

3.45, s 57.9 OCH3 СH3

3.42, s 57.8 СH3

3.34, s 56.2 СH3

3.26, s 55.8 СH3

OCOCH3 2.09, s 171.9

21.5

3.3 New diterpenoids from D. denudatum Wall.

3.3.1 Compound (8)

The sub-fraction DD-2 (Scheme 2.3) obtained from chloroform soluble fraction was put into series of FCC separations with solvent system 10% acetone: 90% n-hexane: 10 drops

DEA/100 ml yielded compound 8 (Figure 3.17) as white amorphous powder which was found as

UV active on TLC.

67 Chapter-3 Results and Discussion

A molecular formula C23H37NO6 was established on the basis of its molecular peak in EI-MS at m/z 423.16 (cal.423.5415). Almost all the functionalities present in 8 were detected in its IR spectrum; notably at 3550 (hydroxyl group) and 1073 cm-1 (C-O-C groups) respectively.

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

H3CO

Figure 3. 17. Structure of compound 8

1 The H-NMR spectrum in CDCl3 of 8 displayed signals for almost all the protons in the molecule. The N-ethyl side chain showed pronounced signals at δ 1.07 (3H, t, J=7.14 Hz) for methyl group while another triplet of one proton integration at δ 4.29 (1H, J= 4.8 Hz) was assigned to the H-14β methine proton. The methylenic protons (CH2-18) yielded a separate doublet at δ 3.13 (J =8.04) and at δ 3.05, (J =9.06 Hz), followed by the methoxy group protons

(OCH3) signals (attached to C-16, C-18) which appeared as separate singlets at δ 3.36, 3.32 (3H intensity each) and further proceeded by another distinct singlet at δ 3.89 assigned to H-1 proton in compound 8. A singlet at δ 2.59 was assigned to proton of C-17 while all the other 1H-NMR signals showed similar pattern to those of isotalatizidine [72] with a major difference of the existence of an additional hydroxyl group (OH) at position 10 (C-10) in 8.

68 Chapter-3 Results and Discussion

13С-NMR spectrum (BB) exhibited signals for all the twenty- three carbon atoms which were distinguished by its DEPT 45, 90 and 135 spectra into eight methine, eight methylene, three methyl and four quaternary carbons. The methyl carbon of N-ethyl moiety resonated at δ 13.5, two methoxy carbons at δ 56.3 and 59.5 followed by the signals for C-1 (δ 68.7), С-8 (δ 73.8), С-

14 (δ 75.8), С-10 (δ 77.1), С-18 (δ 79.4) and С-16 (δ 82.2) respectively. The quaternary carbon atoms (C-4, C-11) were identified from their signals at δ 41.4 and δ 49.4 while the other methylenic and methine carbons were observed to yield signals at expected chemical shifts values (Table 3.9).

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

Figure 3. 18. Key HMBC interaction in compound 8

HMQC and HMBC spectra were used to further validate the 1H-13C connectivity (3J and

2J) for finalizing the structural assignments (Figur 3.18) as proton of H-13 showed 3J cross peaks with С-.10 (δ 77.1), С-.16 (δ 82.2), С-.14 (δ 75.8) and С-.12 (δ 30.5) and while the H-.16 proton was connected to С-.13 (δ 39.3) and С-15 (δ 44.6) in the same spectrum. Further HMBC correlations were detected as H-18 connected to C-3, 4, 5 and 19 (δ 27.1, 41.4, 38.5, and 53.2) while H-6 connected to C-5, 7, 8 (38.5, 46.7 and 73.8) respectively. The spectral evidence and

69 Chapter-3 Results and Discussion chemical features of 8 are helpful in elucidation of its structure as 1,14α, 8,10β-tetrahydroxy,

16,18β-dimethoxy aconitane.

1 13 Table 3. 9. H- (500 MНz) and C-NMR (125 MHz) data of Compound (8) in СDCl3

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

1 3.89, 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 C 8 73.8 C 9 2.05, d, J= 8.16 Hz 41.6 CH 8, 10,11,13, 10 77.1 CH 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 17 2.59, s 63.9 CH 10, 11 18 3.13,3.05, td, J= 8.05, 9.06 Hz, 79.4 CH2 3, 4, 5, 19 19 2.52, d, J= 11.34 Hz 53.2 CH2 N- CH 2 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

70 Chapter-3 Results and Discussion

3.3.2 Compound (9)

Repeated FCC of the sub-fraction (DD-2) (Scheme 2.3) on silica gel with the solvent system of 90% hexane: 10% acetone: 10 drops DEA/100ml yielded compound 9 (Figure 3.19) as amorphous powder which was assigned the molecular formula C25H39NO5 (cal. 433.5939) from its molecular ion peak at m/z 433.01 from EI-MS and other spectroscopic data. IR spectrum for 9 exhibited pronounced absorptions for hydroxyl functionality at 3466 and 3404 cm-1, 3015 cm-olifenic group and for ether function group at 1097 cm-1 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. 19. Structure of compound 9

Compound 9 (Figure 3.19) was also subjected for 1H-NMR spectroscopic measurements and its spectrum showed signals for all the protons including N-ethyl group, methoxy protons and methane protons including olifenic and bridged protons (Table 3.10).

The presence of olefinic functional group was confirmed by the signals for H-6 methine proton resonated at δ 5.37 (1H, d, J = 4.1 Hz), and further HMBC correlations with C-6 while methane proton (H-1) resonated as singlet separately at δ 4.04 followed by the signals assigned to H-14β at δ 4.30 (1H, t, J= 4.30 Hz). The triplet appeared at δ 0.96 (J= 7.7Hz) was assigned to

71 Chapter-3 Results and Discussion the methyl group of N-ethyl moiety and twelve methyl singlet appeared at 3.47, 3.39, 3.36, 3.32, for methoxy groups present at (С-18), (С-1), (С-16) and (С-8) positions respectively were assigned with the help of HMQС spectral data. Methine proton at С-16 appeared downfield as doublet at δ 3.42 (J= 8.4Hz) while other methine/methylenic protons appeared at various chemical shifts values in their respective ranges, specified in case of aconitine type C-19 diterpenes skeleton (Table-3.10)

13C-NMR (BB) together with (DEPT) (45, 90, 135) spectra were used to deduce the number and types of carbon atoms in 9 as total of twenty-five carbon atoms including five methyl, seven methlene, nine methine and four quaternary. C-1 carbon resonated at δ 87.5, indicating the attachment of methoxy group, preferably in β orientation stereochemically, which was supported by the increased chemical shift values of C-2 at δ 24.30 and [M]+-15 indicative for nor-diterpene alkaloids having a β methoxy group at position one [104]. Signals for the four methoxy carbons appeared at δ 56.2, 57.3, 58.0 and 59.7 while the quaternary carbon atoms resonated at δ 42 (С-4), 86.73 (С-8) and 52.92 (С-11). Vinyllic carbons appeared at δ 130.1 and

148.03 while all the other carbon atoms resonated in their expected range.

Further structural assignments and 1H-13C connectivity in compound 9 were established by using HMQC, HMBC techniques shown in Fig. 3.20. In HMBC spectrum, proton H-6 showed cross peaks with С-5, С-6, С-7 and С-11 (δ148.0, 130.0, 45.2 and 52.9 respectively), H-

14 was coupled to C-9 and С-13 (δ 47.4, 43.3) while of H-16 was found connected to С-15, С-13

(δ 41.0, 43.3).

72 Chapter-3 Results and Discussion

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. 20. Key HMBC correlation in 9

The spectral evidence and chemical features of compumd 9 are helpful in elucidation of its structure as 1α, 8, 16,18β-.tetramethoxy, 14α-hydroxy, 5-aconitene.

1 13 Table 3. 10. H- (500 MНz) and C-NMR (125 MНz) data of 9 in СDCl3

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

2 1.81, brd s 24.3 CH2

3 1.32, m 22.8 CH2 4 42 C 5 148.03 CH 6 5.37, d, J= 4.1 Hz 130.1 CH 5, 7, 11, 7 2.34, m 45.2 C 8 86.7 C 9 2.03, d, J= 6.25 Hz 47.4 CH 8, 10,14, 10 32.5 CH 11 52.9 C

73 Chapter-3 Results and Discussion

12 1.30, m 27.1 CH2 13 43.3 CH 12,14, 16 14 4.30, t, J = 5 Hz, 75 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= 6.9 Hz 53.1 CH2

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

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

3.4 Known diterpenoids from D. chitralense

3.4.1 Ajaconine (10)

The known compound, ajaconine (Figure 3.21) was purified as white crystals from sub- fraction DX-1 (Scheme 2.2), obtained from alkaloidal mixture (pH 8-10) from D. chitralense.

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

Figure 3. 21. Structure of ajaconine 10

74 Chapter-3 Results and Discussion

The IR spectrum of ajaconine (10) contains absorption bands at 1118 cm-1(C-O), 1660 &

-1 935 (C = CH2), 3370 and 3285cm (OH). Ajaconine afforded the molecular formula C22H33NO3 in its HREI-MS by exhibiting molecular ion peak at m/z 359, while its calculated molecular mass was measured as 359.5010.

In 1H-NMR spectrum, ajconine (10) provided over all similar atisine type chemical shift values as: H-17 resonated at  5.06 (2H, singlet), H-20 at  4.79 (3H-s), H-18 at  0.75 8 (3H, s), H-15 at  3.93 (3H, s) and H-22 at  3.83 (2H, m). The 13С-NMR of ajaconine (10) exhibited twenty- two signals, which were resolved by DEPT experiment corresponded to the presence of one methyl, eleven methylene, six methine and four quaternary carbon atoms. Some major chemical shift values obtained were at  146.8,  110.4, and  90.3, and  75.8 were due to С-20. С-17, С-

16 and С-7 respectively.

Confirmation of structural assignments for ajaconin (10) was established from its X-ray diffraction analysis. The molecule of polycyclic ajaconine (10) contains six rings (A-F). In the basic skeleton of ajaconine (Figure 3.22), the junctions of rings A/E [С4-C5- С10- С20 =

63.2 (3)°] and rings B/ С [С7- С8- С9- С11 = 155.2 (2)°] are trans-fused, while rings A/B [С4-

С5- С10- С1 = -58.9 (3)°] and rings E/F [С4- С5- С10- С-9 = -178.5 (2)°] are cis-fused. Crystal data (Table 3.11 & 3.12) demonstrated that all angles and bond lengths in ajaconine (10) are found to be in their estimated range [105]. С-15 hydroxyl group is found to be β-oriented. All rings from (A-F) adopt chair (A & F rings), boat (ring С) and half boat conformation (B and F) respectively. The geometry around all the bridged carbons C-17, C-15, C-14, C-13, C-6, C-5, C-

4 and C-3 are tetrahedral as expected.

75 Chapter-3 Results and Discussion

Figure 3. 22. ORTEP plot of ajaconine (10), thermal ellipsoid is drawn at 50 %

probability, hydrogen atoms are omitted for clarity,

Table 3.11. Crystal data and structure refinement of ajaconine.

Crystal Parameter 11 Crystal Parameter 11

3 Formula C22H33NO3 Volume Å 1936.4(2)

Formulae weight 359.490 μ /mm 0.08

Temperatures (K) 296.00 Z 4

Wave lengths (Å) 00.71073 Density (Mg/ m-3) 1.233

Crystal system Orthorhombic (l, k, h) min (-25, -15, -10)

Space group. P212121 (l, k, h) max (25, 10, 5)

76 Chapter-3 Results and Discussion

A 8.111(5) Theta (max) 27.5

B 12.032(9) R (reflection) 0.050(4272)

C 19.839(14) wR2 0.119

Ajaconine form a supra-molecular structure stabilized by strong intramolecular and intermolecular hydrogen bonds H----OH with an average distance of (1.940 Ǻ).

Table 3.12. Hydrogen bond geometry (Å, °)

D—H····A D—H Н····A D····A D-H H····A

O1-H1····Ο 0.82 1.87 2.644 (3) 156.4

Ο3-H3A····O1 0.82 1.94 2.758 (3) 175.0

Ϲ12-Н12B··O3 0.97 2.46 3.125 (4) 125.7

Symmetry code: (i) x+1/2, –y+1/2,-z+1

From spectral and crystal data of 10 was identified as ajaconine [106].

3.4.2 Delectinine (11)

Delectinine (Figure 3.23) was obtained as white crystalline solid having melting point

119-121 °C. Its IR spectrum (KBr) was used to identify main functionality, hydroxyl group (OH) as its absorption was noted at 3550, 3414, 3630 and 3370 cm-1. The molecular formula for delectinine was established from its EI-MS spectrum at m/z 453 as C24H39NO7 (Calc. for

453.5370).

77 Chapter-3 Results and Discussion

OCH3

13 16 12 OCH317 H 14 OH

10 H H3C 1 9 2 11 15 N 8 3 5 7 4 6 OH H OH 19 H CO 18 3 HO

Figure 3. 23. Structure of delectinine (11)

Its 1H-NMR spectrum exhibited various characteristic signals such as a methyl triplet 

1.14 (J = 7.14 Hz, Me of N-ethyl group), methoxy groups singlets at  3.36, 3.33, and 3.20, followed by the presence of broad singlet at  4.25 due to H-14β. 13C-NMR spectrum was conclusive in establishing the skeletal assignments within delectinine between carbons and protons through BB decoupled, DEPT and HMQC techniques. Twenty-three signals were detected for carbon atoms, which were than resolved into four methyl, seven methylene, nine methine, and four quaternry carbon atoms. The 13С-NMR spectrum displayed five signals at 

91.3, 89.40, 85.7, 75.3, 57.7, 56.3, and 55.8 due to С-6, С-7, С-1, С-14 and three methoxyl groups. Similarly, the signals at  13.80 and 48.19 were indicative of the N-ethyl group in delectinine.

The crystal structure of delectinine is given in Figure 3.24, the crystal data related to crystal structure resolve and refinements are given in table 3.13. The title compound 11 having six rings of different size (three are six members, two are five member rings and one of the seven membered respectively). In the elementary skeleton of Delectinine, the intersections of

78 Chapter-3 Results and Discussion rings rings B/C [С8- С9-C- С10- С12 = -82.8 (4)°] and A/E [С4- С5- С11- С17 = -75.7 (4)°] are cis-fused, while rings E/F [С4- С5- С11- С10 = 167.9 (3)°] and rings A/B [C6-C5-C11-C1 =

162.2 (3)°] are trans-fused. All of the six membered rings of the title compound (A, D & E) assume chair, distorted chair-boat and boat conformations, the rings C & F, are in envelope conformations. However, the ring is in the form of twisted-boat conformation. Delectinine having three hydroxyl groups are β-oriented at C-7, C-8 and C-18, while, the hydroxyl group at

C-7 in ring C is α-oriented compared to the β-methoxy group, at С-16. The -OСH3 group at С-1 is α-oriented where the С-6 is –OСH3 is β-oriented. Delectinine form one dimensional extended structure via C···O and O···O π interactions.

Figure 3. 24. ORTEP representation of delectinine with 50% thermal ellipsoids, hydrogen atoms are omitted for clarity

From above spectral evidences and comparison with available literature the structure is established as delectinine [107].

79 Chapter-3 Results and Discussion

Table 3. 13. Crystal data and structure refinement of Delectinine

Crystal Parameter 11 Crystal Parameter 11

3 Empirical formulae C24H40NO8 Volume Å 2387.3(4)

Formulae weight 470.57 μ (mm-1) 0.10

Temperatures (K) 296 Z 4

Wavelengths (Å) 0.71073 Density (Mg/ m) 1.309

Crystal system Orthorhombic (h, k, l) min (-9, -12, -26)

Space group P212121 (h, k, l) max (11, 4, 36)

A 8.893(6) Theta (max) 27.5

B 9.621(10) R (reflection) 0.061(5356)

C 27.899(2) wR2 0.156

3.5 Known diterpenoids from D. denudatum Wall

3.5.1 Dihydropentagynine (12)

Compound 12 (Figure 3.25) showed molecular ion peak at 407 in EI-MS spectrum which yielded its formula as C23H37NO5 while its IR spectrum revealed the presence of strong absorption for hydroxyl group at 3440 and 3414 cm-1.

80 Chapter-3 Results and Discussion

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

Figure 3. 25. Structure of dihydrpentagynine (12) In the spectrum of 1H-NMR a methyl group of N-ethyl side chain resonated as triplet at 

1.07 (J = 7.15 Hz), followed by two distinct singlets for two methoxy groups separately at 3.33 and 3.27. Oxygenated methylenes were assigned on the basis of their chemical shift values at 

3.03 and  3.18 (singlets) while two oxygenated methines were confirmed from the values at 

3.90 (H-1, s) and  4.16 (H-14, t, J = 4.75 Hz)

13С-NMR spectrum showed all the twenty-three carbon atoms signals (BB and DEPT), which were resolved into six methylene, four methyl, ten methine and three quaternary carbons. The data coincided with literature for dihydropentagynine [108].

3.8 Enzyme inhibitory activities of isolated compounds

3.8.1 Cholinesterase inhibition of diterpene alkaloids from D. chitralense & D. denudatum

The main goal of current research project was to find such new compounds that possess the ability of inhibiting acetylcholine esterase and butrylcholine esterase as well as such other similar types of enzymes. Usually bioassay guided isolation procedure on medicinal plants are used for such research activities. All isolated compounds obtained from D. chitralensi and D.

81 Chapter-3 Results and Discussion denudatum were examined for their acetylcholine esterase and butrylcholine esterase activities respectively. All compounds showed encouraging results and therefore can be used as drugs for treating Alzheimer disease [109]. Initially at a concentration of 0.1 mM the compounds were analyzed for their inhibition against the above-mentioned enzymes. As per procedure the inhibitory action of the compounds were above 50 percent and were then tested for their IC50 value determination. All the isolated compounds from both Delphinium species showed promising effectiveness against both acetylcholine esterase and butrylcholine esterase. Amongst isolated compounds; chitraline-1 and delectinine from D. chitralense and compound 8 and compound 12 from D. danudatum showed significant acetylcholine esterase and butrylcholine esterase inhibitory activities respectively. Results of all isolated compounds from both investigated species are given in (Table 3.14).

The stirring results clearly highlight the importance and interest in the purification and isolation of this class of compounds present in D. chitralense and D. danudatum.

Table 3. 14. Enzyme inhibitory activities of D. chetralense and D. danudatum

S.No. Compounds AChE±SEMa BChE ± SEMa Type of inhibition

(μM) (μM)

1 Chitralinine A 13.86± 0.35 28.17± 0.92 Competitive

2 Chitralinine B 11.64 ± 0.08 24.31± 0.33 Competitive

3 Chitralinine-C 12.11 ± 0.82 26.35± 0.06 Competitive

4 Chitraline-A 10.44± 0.71 20.10± 0.88 Non competitive

5 Chitraline-B 17.86± 0.21 30.93± 0.33 Non competitive

82 Chapter-3 Results and Discussion

6 Chitraline-C 10.51± 0.06 25.13± 0.04 Non competitive

7 Chitraline-D 10.71± 0.32 23.41± 0.51 Competitive

8 Ajaconine 12.61 ± 0.05 10.18± 0.91 Competitive

9 Delectinine 5.04± 0.09 9.21± 0.06 Noncompetitive.

10 Compound 8 9.24± 0.12 19.61± 0.72 Competitive .

11 Compound 9 16.82± 0.31 34.72± 0.26 Noncompetitive .

12 Compound 12 11.27± 0.23 22.25± 0.33 Competitive .

13 Allanzanthane A 8.23± 0.01 18 ± 0.06

14 Galanthamine b 10.12 ±0.06 20.62 ± 0.08

83 Chapter-4 References

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