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Isolation and StructuralElucidation of Chemical

Constituents from Fumariaindica, Ferula oopoda and

Withania somnifera

Thesis submitted

for

The fulfilment of the Degree of

DOCTOR OF PHILOSOPHYBY

SHAKIL AHMED

%'ieraInternational Centre for Chemical Sciences

H.E.J. Research Institute of ChemistryUniversity of Karachi

Karachi

1998

:

‘Dedicatedto

My Dearest‘Parents

ACKNOWLEDGEMENTS

I take pride in acknowledging my assiduous supervisor Prof. Atta-ur-

Rahmart, H.I., S.I., T.I., Director of H.E.J. Research Institute of

Chemistry for his valuable guidance and constructive comments

during the course of this research work as well as for providing an

environment conducive for research. He has been always a source of

inspiration for me.

There has been a significant contribution of Dr. M. Iqbal

Choudhary, to the evolution of this thesis. I wish to express my

gratitude to him for his kind interest and helpful suggestions.

The task of this work would have been more difficult, without the

help of my colleagues. I sincerely gratitude Dr. Shaiq Alit Mr.

Shahid Malik, Mr. M. Nur-e-Alam, Miss Aniqa Naz, and Mr.

Mohammad Aslam for their co-operation and encouraging

comments. I would also like to thank Mr. Riaz Ali Khan, Dr. Jtfgan

Farooq, Dr. Shahid Badar Usmani, Syed Muddasser Kazmi,

Salma Shehnaz, Humera Naz, Mr. and Mrs. Abdul Jabbar and Dr.

Bakht Baidar Ali for their constant support.

I am deeply indebted to my dear parents, a debt which is impossible

to repay. A special thanks are due to my brothers and sisters for

their continuous encouragement and appreciation.

i

I am most grateful to my better-half Iffat for her patience,

encourangements and understanding.

My little angels Gul and Aeliya deserve a big thanks as they were

sometimes neglected by me during my research work.

Our librarian Mr. Aijaz Ahmed Soofi deserves a special bow in

providing library informations in time. I extend my special thanks to

Mr. Mahmood Alam who took a keen interest in solving my problems

whenever they arose.

A great deal of appreciation is due to the technical and non-technical

staff of the institute especially to Mr. Sarjrazullah for his kind help

and to Mr. Naveed Aslam for his photocopying services.

SHAKEEL AHMED

CONTENTS

I. SUMMARY .1

1.0 GENERAL INTRODUCTION l

1.1 ALKALOIDS

1.1.1 Heterocyclic Alkaloids

1.1.2 Isoquinoline Alkaloids

1.1.3 Pharmacology of Isoquinoline Alkaloids

1.1.4 Biosynthesis of Isoquinoline Alkaloids..

1.1.4.1 Isoquinolines

1.1.4.2 Simple Tetrahydrolsoquinoline Alkaloids .. 27

3

5

10

19

25

25

1.1.4.3 Benzylisoqulnoline Alkaloids

1.1.4.4 Pavine and Isopavine

1.1.4.5 Aporphine Alkaloids

1.1.4.6 Protoberberine Alkaloids

1.1.4.7 Protopine Alkaloids

1.1.4.8 Phthalideisoquinoline Alkaloids

1.1.4.9 Secophthalideisoquinoline Alkaloids

1.1.4.10 Spirobenzylisoquinoline Alkaloids

30

32

35

35

38

39

39

40

2.0 INTRODUCTION OF FUMARIA MDICA. 44

2.1 Literature Survey

2.2 Results and Discussion

44

64

3.2.4 Isolation of 2,3-Dihydrowithaferine-A (297)

3.3 Plant Material

3.3.1 Extraction and Purification

3.3.2 Isolation of Withanone (295)

3.3.3 Isolation of Quresimine (296)

3.3.4 Isolation of Withaferine-A (306)

3.3.5 Isolation of 2,3-Dihydrowithaferine-A (297)

163

170

170

170

172

173

174

4.0 INTRODUCTION OF FERULA OOPODA 176.

4.1 Biosynthesis of Sesquiterpenes

4.2 Results and Discussion

4.2.1 Isolation of Feralleate (303)

4.2.2 Isolation of Guaianolide (304)..

4.2.3 Isolation of Grilactone (305)....

4.3 P’ant Material

4.3.1 Extraction and Purification

4.3.2 Isolation of Feralleate (303)

4.3.3 Isolation of Guaianolide (304)..

4.3.4 Isolation of Grilactone (305) ....

177

182

182

190

194

198

198

200

201

202

5.0 REFERENCES 204

LIST OF PUBLICATIONS 221

2.2.1 Fumaileal (144)..

2.2.2 Fumaileate (149)

2.2.3 Papraline (143) ...

2.2.4 9,10-Methylenedloxy karachine (255)

2.2.5 Vanillin (142)

2.2.6 Cryptopine (61)

2.2.7 (+)-p-Hydrastlne (232)

2.2.8 Corydaldine (147)

2.3 General Experimental

2.3.1 Plant Material

2.3.2 Extration and Purification

2.3.3 Isolation of Fumaileal (144)

2.3.4 Isolation of Fumaileate (149)

2.3.5 Papraline (143)

2.3.6 9,10-Methylenedloxy karachine (255)

2.3.7 Vanillin (142)

2.3.8 Cryptopine (61)

2.3.9 (+)-p-Hydrastine (232)

2.3.10 Coiydaldine (147)

64

67:

73

77

87

89

94

99

101

104

104

109

no111

112

113

114

116

117

3.0 INTRODUCTION OF WITHANIA SOMNIFERA . 119

3.1 Biosynthesis of Withanolides ....

3.2 Results and Discussion

3.2.1 Isolation of Withanone (295) ....

3.2,2 Isolation of Quresimine (296)....

3.2.3 Isolation of Withaferine-A (306)

123

133

133

147

159

SUMMARY

The thesis presents the isolation and structure elucidation of various

classes of natural products which were isolated from three medicinal

plants of Pakistan, i.e. Fumaria indica, Withanta somnifera and Ferula

oopoda. The structures of the compounds were determined on the

basis of sophisticated spectroscopic techniques including one-and

two-dimensional NMR spectroscopic experiments such as COSY 45°,

HOHAHA, HMBC, HMQC etc.

Phytochemical investigations on Fumaria indica have led to the

isolation of four new compounds i.e. papraline (143), 4,5-dimethoxy-

2- hydroxybenzaldehyde (144), fumaileate (149) and 9,10-

methylenedioxy karachine (255) along with the four known

isoquinoline alkaloids, 4-hydroxy-3-methoxybenzaldehyde (142),

cryptopine (61), (+)-(3-hydroastine (232) and corydaldine (147).

o

H3C''ÿ 12CH3

L *Jk-CH36

H'T 56

3

ON

H3c<

H5] JL AH

H C

4 OHCH3318

143 144 149

4. H14 5

P>ÿ\4a 6

vH-N B2 14

r&b HaC< 3 O'HI31 1 83 .0CH3m P\i2» w—ra»

or

Ha HO5 10

1!61OCH3142 11

11

255

-:i:-

5

<O'8a N.

H' CH382* 5

H3C<H- IT 3'

4'I .N.9'

8aOCH3 H3C H5' 88’o

OCH3 o232 147

The next section of the thesis describes the chemical constituents of

Withania somnifera and Includes the isolation of two new

wlthanolides, withanone (295) and quresimine-A (296) along with

two known wlthanolides, withaferine-A (256) and 2,3-

dlhydrowithaferine-A (297).

CH3CH3

CH2-OHCH2OH %HH

H3Cv..UVCH3 ,HH T° ‘oo o

CHH

O:H3 H CH3 H

I IOHH H H

29S H3C< 2980* HH OH

28

CH3 28

CH327

27CHj— OHCHj— OH

H&"o o

H ~—H0 19 fn i!CH3 j H \\ CH3flH 1:1:u

M I5y 149 13.a/T 2V?fio s'

OH H OH[4 3ÿ-Hÿ3 0,

297286 O' HH H

The last section deals with the studies on the chemical constituents

from Ferula oopoda which have led to the isolation of ferulaileate

(303) along with two known sesquiterpene lactones guaianolide

(304) and grilactone (305) for the first time from this species.

H-bVH14

H2<H O

rCH3 i 'z

i

k r 5

6oj— lkÿi.3 6

\ 13L CH34. 15ch3hHF H Il5CH3CH3 z k /xl!0ÿ2/ÿH

* O

lOCH3ÿÿ3>CH3H3

30BH O304303

SECTION A

GENERAL INTRODUCTION

GENERAL INTRODUCTION

1.0 THE HISTORY OF MEDICINE

The history of modem medicine and pharmacy is considered to

begin from Hippocrates, the father of modern Medicine. In his

writings nearly 400 substances were named as medicinal substances.

Theophrastus wrote a book on "The History of Plants", in which he

mentioned 500 herbal drugs. His famous book "Materia Medica" was

first published in Greece, which served as the standard

phytopharmacological handbook for a long time. Following this

period the role of herbal medicine was greatly extended in the Islamic

age.

The most famous physician and philosopher of the early

Muslim era, Ibne-Sina, described 760 herbal drugs in his famous

book "Kanun fl al-Tibb" which was known as "the Canon" in Europe

till the 17th century A.D., and formed the basis of the Greco-Arab

system of medicine known as ’Tibbe-Unani". Indeed, no history of

medical science can be complete without reference to the scholarly

pioneering contributions of Alhazen (Ibn-al-Haitham), Rhazes (Al-

Razi) Jildaki, Al-Khawarazmi, Al-Kindi, Al-Biruni, Al-Farabi, Abual-

Kasim and Al-Zahrwi.

Herbs have been widely used since time immemorial as crude

vegetable drugs or extracts for the treatment of many diseases and to

maintain health [1]. For example an important herb "cinchona" has

been used as an antimalarial drug for thousands of years [2]. Over

-:1:-

the years plants have been shown to be rich In anti-cancer agents

(vinblastine and vincristine), narcotic analgesics (morphine, codeine),

antlmalarlal drugs (quinine, artemisinin), anticholinergic drugs

(atropine, hyoscyamine), adrenolytic agents (reserplne),

antiasthmatic drugs (ephedrine, khellin), liver protective agents

(catechin, silymarin), cardiac drugs (digitalis) etc.

Polysaccharides, proteins, fats and nucleic acids are the

fundamental building blocks of living organisms and are considered

as "primary metabolites". These primary metabolites are used by the

living cells in vivo for various purposes and they also give rise to

secondary metabolites such as alkaloids, terpenes, pigments etc. The

secondary metabolites are synthesized in the plants or living

organisms through enzyme catalysis by using the primary metabolites

as building blocks.

Most of these secondary metabolites play some important but

unknown roles in the life of a plant. Many of these secondary

metabolites have also been found to possess interesting

pharmacological activities and some have served as cures for human

and livestock diseases. Although man’s search to-date to Isolate

therapeutic agents from medicinal plants has yielded only modest

success, we should not forget that with the advent of new high

through-put screening techniques, much better ways of looking for

new plant drugs have become available. Knowledge about the basis of

plant metabolism, plant analysis, biosynthesis in plants and even

plant production has also considerably expanded. It is reasonable to

-:2:-

predict that the most exciting and productive period of medicinal

plant research lies ahead of us.

1.1 THE ALKALOIDS

Alkaloids are secondary metabolites which form one of the

most important classes of plant constituents. The term "alkaloid" or

"alkali-like" was first proposed by the pharmacist, W. Meissner in

1818 [31. Meyer presented the first comprehensive definition of

alkaloids in 1896. According to him "Alkaloids" (plant bases) occur

characteristically in plants, and are frequently distinguished by their

remarkable physiological activity. They contain carbon, hydrogen and

nitrogen, and in most cases oxygen as well. However in 1931 Trier

wrote "As a result of scientific progress, a collective term of this kind

(i.e., alkaloids) will have to be abandoned" [4]

Alkaloidal substances can be defined in a broad sense as

basic nitrogen-containing compounds of either plant or animal

origin, which have complex molecular structures and which can

manifest significant pharmacological activities.

A modem definition of alkaloids was given by S.W. Pelletier

[5]. He defined alkaloids "as cyclic organic compounds containing

nitrogen in a negative oxidation state which are of limited

distribution among living organisms." In the light of these definitions

many nitrogen containing natural products are excluded from the

class of alkaloids. For instance, cholchicine (1) is an N-acetyl

derivative and has a neutral nitrogen atom, while aristolochic acid

(2) is not basic and has no heterocyclic ring.

-:3:-

There is another class of related compounds which are called

"protoalkaloids". This class consists of simple amines in which the

amino acid nitrogen is not in the ring. These are the so-called

"biological amines” e.g., ephedrine (3), mescaline (4) etc.

COOH

<“O-

H3C< o.....NH— c— CH3 NO2H3C< X

OCH3

oOCH3

OCH3

Aristolochlc acid (2)(-)-Colchicine (1)

H3C<

\ H/

HO“ÿC— Cÿ-'CH3H5C6

NH2H3C<NH— CH3

OCH3Mescaline (4)

(-)-Ephedrine (3)

Another group of nitrogen containing compounds are called

"pseudoalkaloids". They are not derived from amino acid precursors.

e.g., steroidal alkaloids such as terminaline (5), solasodine (6) and

purine bases such as caffeine (7).

-A.-

CH3H3Cÿ /

\ CH3CH3

CH3 H

IH

HO*i HOH

Terminaline (5)

HOH3

\ NJlMe

ICH3P CH3 H3ÿN!

oCH3 H

\Ii H

H CH3

HO*

Caffeine (7)(-)-Solasodlne (6)

Alkaloids may be further sub-classified on the basis of their

skeleton and biogenesis.

l.i.i Heterocyclic alkaloids

Alkaloids containing atleast one heterocyclic ring are

classified under this category. Examples are:

**

-:5:-

Pyrrolidine Alkaloidse.g. l-(m-methoxy cinnamoyl) Pyrrolidine (8)

QH

/~SÿTS/

1-m-Methoxoy H

cinnamoyl) Pyrrolidine (8) OCH3

Indole Alkaloidse.g. Tryptamine (9)

(CH2)2r-NH2

Tryptamine (9)

Piperidine Alkaloidse.g. Coniine (10)

aH

(CH2)2— CH3(-)-Coniine (10)

Pyridine Alkaloidse.g. Cantleylne (11) .

•:

OH

O

H3corÿ CH3

NCantleylne (11)

-:6:-

Tropane and Related Basese.g. Tropinone (12)

H3(ÿN,

OTropinone (12)

Histamine Alkaloidse.g. (±)-Glochldine (13)

/Ti>cy°n-H13C6

(±)-Glochldlne (13)

Isoquinoline Alkaloidse.g. Hydrohydrastinlne(14)

N\o- CH3

Hydrohydrastlnlne (14)

Quinoline Alkaloidse.g. Quinoline (15)

ooQuinoline (15)

-:7:-

Izidine Alkaloidse.g. PyrroHzidine (16)

H

CDPyrrolizidine (16)

Alkaloids with an Exocyclic Nitrogene.g. (-)-Cassaine (17)

CH3/CH-CO— O— CH2— CH2— N,

\CH3

CH3"VCH3

H

OHO*

(-)-Cassalne (17)H3C CH3

Putrescine Alkaloidse.g. Paucine (18)

OH

HO.

.O

NH

Paucine (18)

-:8:-

Peptide Alkaloidse.g. Integerrine (19)

HN

NH.

\O

N HN

rkH3<\

H3</

CeH5CH- CH

IN(CH3)2

Integerrine (19)

Diterpene Alkaloidse.g. Veatchine (20)

,CH2\

I

,N" / }

\

HII

I

OH

CH3Veatchine (20)

Steroidal Alkaloidse.g. Funtumine (21)

O. CH3CH3

CH3

iH

Hal

Funtumine (21)

-:9:-

ISOQUINOLINE ALKALOIDS1.1.2

The isoquinoline alkaloids form an important class of

secondary metabolites. The first alkaloid isolated i.e. morphine has

an isoquinoline skeleton [6,7]. The isoquinoline class has many

pharmaceutically important compounds which occur mainly in the

plant families Papaueraceae, Magnoliaceae, Annonaceae, Lauraceae,

Alangiacease, Berberidaceae, Ancistrocladaceae and Menispermaceae

[8].

Phytochemical studies have been mostly carried out on plants

of family Menispermaceae. Many of these are used in folk medicine, or

as a local food such as plants of the genera Tinospora, Stephania,

Cycled, Archangelisia, Fibrauria and Tiliacora.

Isoquinoline alkaloids can be further divided into the

following sub-groups on the basis of their structures and biogenesis

[9].

Simple Isoquinoline Alkaloids

e.g. Noroxyhydrastinine (132) [10)

<°'O',N.

H

O

Noroxyhydrastinine (132)

1-Phenylisoquinoline Alkaloidse.g. (+)-Ciystostyline I (22) [11]

H3C<

NSH3C< CH3

H’

H3C< OCH3

OCH3(+)-Crystostyline I (22)

N-Benzylisoquinoline Alkaloidse.g. N-Methylviguine (23) [12]

CXX)<"

N-Methylvlgulne (23)OCH3

4-Phenylisoquinoline Alkaloidse.g. (+)-Latlflne (24) [13]

OH

OHH

H3C<

N.CH3

(+)-LaUflne (24)

Isoquinolinequinon.ese.g. Renierol (25) [14]

O

H3<

NH3C<

OOH

Renierol (26)

Benzylisoquinoline Alkaloidse.g. (±)-Fumarizine (26) 115]

<°'o- NN

CH3

OCH3o'

•o(±)-Fumarizine (26)

Bisbenzylisoquinoline alkaloids with Aryl Links Onlye.g. Nor-2'-pisopowlaridine (27) [16]

,OH HO,

HSCÿN N.OCH3 H3C< H

VJ H

OCH3 HO'

Nor-2’-pisopowiaridine (27)

12:-

Bisbenzylisquinoline Alkaloids with One Ether Linke.g. Northallbroline (28) [17]

.OCH3 H3C<

RH3C<N HO'OH N HH

OH

Northallbroline (28)

Bisbenzylisoquinoline Alkaloids with One Ether/One Aryl Linke.g. (-)-Cordobimine (29) [18]

,OCH3 H3C<

H3C<N OHH'*" .0

OCH3 HO'

(-)-Cordoblmine (29)

Bisbenzylisoquinoline Alkaloids with Two Ether Linkse.g. Pangkorimine (30) [19]

.OCH3 H3C<

NHRHO'

H-1 O,

OH

Pangkorimine (30)

i u vaiaiiiuiÿ

OCH3

,0HN, NH

NH* O

OCH3 HO'

Pachyovatamlne (31)

Bisbenzylisoquinoline Alkaloids with Three Ether Linkse.g. Kurramine (32) [21]

HO„

ON;

O

oOH

Kurramine (32)

Seco Bisbenzylisoquinoline Alkaloidse.g. (-)-PunJabine (33) [22]

OCH3

,0HN,

CH3H1 O

O

CHOO

HO'(-)-PunJabine (33)

Cularine Alkaloidse.g. (+)-Norcularidine (34) [23]

NHHO' C"" H

WOCH3

(+)-Norcularldlne (34)H3C1

Seco-cularine Alkaloidse.g. Norsecocularidlne (35) [24]

<CHsH

HO‘\

wOCH3

Norsecocularidlne (35)

H3C1

Cancentrine Alkaloidse.g. Dehydrocancentrtne-A (36) [25]

H3C(

NCH3

\H3C< o

HO' \ NO

\J Dehydrocancentrine-A (36)

Quettamine Alkaloidse.g. Secoquettamine (37) (26]

/CH3N\

CH3

H3C( uW

Secoquettamine (37) OH

Dibenzopyrrocoline Alkaloidse.g. (-)-Cryptowoline Iodide (38) [27]

H3C<

+ IÿCH3

HO' H''lO

(-)-Cryptowollne Iodide (38)

Indenobenzazepine Alkaloidse.g. Lahorine (39) (28]

<fo-N— CH3

h

Lahorine (30)

-.16:-

i

Pavine Alkaloidse.g. (-)-Caryachine (40) [29]

.OCH3a.NMe

O' OHj

(-)-Caryachlne (40)

Isopavine Alkaloidse.g. Amurensine (41) [30]

OCH3

<“O'N OH

CH3

Amurensine (41)

Protoberberine Alkaloidse.g. (+)-Corydaline (42) [31]

H3C<

N.H3C<

H1,OCH3

HaC*'"

OCH3(+)-Corydaline (42)

Protopine Alkaloidse.g. Protopine (43) [32]

<: 1 /CH3NCO'

DProtopine (43)

Spirobenzylisoquinoline Alkaloidse.g. (-)-Fumarlciiie (44) [33]_

H3C<

N.CH3H3C< . H

HO"

(-)-FumarlcIne (44)

Benzophenanthridine Alkaloidse.g. Oxynitidine (45) [34]

>H3C<

N.H3C< CH3

oOxynitidine (45)

Phthalideisoquinoline Alkaloidse.g. (-)-Hydrastine (46) [35]

<°'o- N.CH3

OCH3o OCH3

(-)-Hydrastlne (40)

Sec-oberberinee.g. (±)-Hypecorine (47) [36]

<: oCHa

O

1o

(±)-Hypecorlne (47)

Aporphine Alkaloidse.g. Cassyfillne (48) [37]

OCH3

<fO'

NH

N

H3C<

OHCassyfillne (48)

Morphine Alkaloidse.g. Carococcullne (49) [38]

H3C<

HO'

N.H CH3

O' OCH3

OH

Carococcullne (49)

1.1.3 PHARMACOLOGY OF ISOgUINOLINES ALKALOIDS

Pharmacology Is the study of the interactions between drugs

and the biological system. Pharmacology has a long history. The

primitive man believed that disease was caused by evil spirits

inhabiting the body. This belief persisted throughout the early

civilization till the Egyption period of medicine until it was

challenged by Hippocrates in 460 BC [39]. After Hippocrates the

Arabs adopted a scientific approach to the study of medicine.

Plant extracts have been used to cure diseases from the very

beginning of civilization. Over the last century plant extracts have

been shown to be responsible for providing relief against many

diseases due to the presence of some biologically active primary and

secondary metabolites which are produced in them. These

metabolites can be isolated and identified by using different

chromatographic and spectroscopic techniques.

Out of the many diverse classes of secondary metabolites the

isoquinoline alkaloids represent one of the largest groups of

alkaloids. Many alkaloids belonging to this group have shown

interesting pharmacological activities. A number of simple

isoquinolines alkaloids have also shown pharmacological effects on

the nervous system [40].

Many benzylisoquinoline alkaloids have also exhibited

interesting pharmacological activities. For example dioxyline (50) is

used in the form of its phosphate salts as a coronary and peripheral

vasodilator[4l]. Its methylenedioxy analogue, 3-methyl-6,7-

H3C< CH3.CH3

<H3C(

H3C< Vo7OCH33-Methyl-6,7-methylenedloxy-1-plperonytsoqulnollne (81)Dioxyline (80)

-:20:-

methylenedioxy-1-piperoxylisoquineline (51) is effective as a smooth

muscle relaxant [42]. Corydaldine (52) another isoquinolone

alkaloid, showed antirheumatic properties [43].

H3C(

N-HH3C<

oCorydaldine (52)

During pharmacological investigations on bisbenzylisoquino-

line alkaloids (+)-tubocurarine (53) was found to have potent

poisonous properties and was used as a poison by the South

American Indians. They used this alkaloid on the tip of arrows and

when this poison was injected into the blood stream of the victim, it

quickly blocked neuromuscular action, causing immediate death.

H3C< 0ci

?<:o' CH3H' CH2

OH

CH2H O

Hÿ;H3CT |

.OH

Cl® OCH3(+)-Tubocurarine chloride (53)

This alkaloid is also used in abdominal surgery in very small doses as

a complement to a local anesthetic since it causes paralysis of the

abdominal muscles without stopping the natural movement of the

intestines [44], The alkaloid is also an effective musqle relaxant.

Glaziovine (54), a proaporphine alkaloid, showed

antidepressive activity [45].

H3C<

N.H3C< CH3

o(±)-Glaziovine (B4)

The aporphine alkaloids are also important pharmacologically.

Many of them exhibit prominent pharmacological activity. For

example apocodeine (56) has antiemetic activity [46].

Boldine (55), an aporphine alkaloid, was found to have mild

sedative, diuretic and antiparasitic actions and it also increases the

secretions of the liver and salivary glands [47]. Another aprophine

alkaloid, xylopine (57), exhibits sedative and analgesic activity.

-:22

<fO- N-HN.

H3C< CH3HH

H3C<

OMeOH

CH3 (±)-Xyloplne (57)(+)-Boldlne (BB)

H<

HHO,

H3C<(±)-Apocodelne (BB)

Protoberberlne alkaloids have also shown pharmacological

activity. For instance the tetrahydroprotoberberlne alkaloids, which

belong to this class, have shown promising transquilizing properties

[481.

Berberine (58) has found use in the treatment of

gastrointestinal disorders. It is reported to depress intestinal

<: NX

,OCH3

OCH3Berberine (68)

-:23:-

peristalsis and to remove inflammatory congestion of the mucosal

surface of the intestine. Therefore, it is used in the treatment of

diarrhea of infancy and childhood [49].

)H3C<

Nv.CH3H3C<

OCH3(+)-8-Methoxydihydronltidine (59)

The benzophenanthridines alkaloid, 8-methyoxydihydro-

nitidlne (59), has shown anticancer activity. Protopine (43) and

cryptopine (61) are known to stimulate the uterus [50].

H3C<

<°'o-

•CH3,CH3 N.N;

H3C<o'-o-

ODoSs

Cryptopine (61)Protopine (43)

Some pathalideisoquinoline alkaloids also exhibit

pharmacological activity. For example narcotine (62) has antitussive

activity and it can be used to suppress cough [51].

-:24:-

CH3

OCH3,o

OCH3w(-)-a-Narcotlne (62)

OCH3

1.1.4 BIOSYNTHESIS OF ISOgUINOLINES ALKALOIDS

1.1.4.1 Isoquinolines

Biosynthesis is the experimental study of the formation of

secondary metabolites. Biosynthetically most alkaloids are derived

from simple amino acids such as ornithine (63), lysine (64),

phenylalanine (65), tyrosine (66), tryptophan (67), histidine (68) and

anthranilic acid (69).

CO2H

<H2NH2fr * CO2H H NH2l C02HH;

HH

Lysine (64) Phenylalanine (65)Ornithine (63)

CO2H CO2H

<<NH2 NH2HO' N

H

Tryptophan (67)Tyrosine (66)

CO2Hrpc CO2H

H NH2Histidine (68) Anthranilic acid (69)

4-:25:-

The isoquinoline alkaloids are generally derived from

phenylalanine (65) and tyrosine (66). These precursors of

isoquinoline alkaloids are themselves derived from shikimic acid (70)

(Scheme-1.1).

COOH COO H-A

&C

O COOIPO'" oOHHO

i ptOH H

Shlklmate-3-phosphate (71)

OH Phosphenol pyruvate (72)

Shikimic acid (70)

co2 co2

CH2coo

.'OPvly

Oÿ OÿÿCCÿPO"‘‘‘ PO"‘”I IOH 73 OH

5-EnoIpyruvyl shlklmate-3-phosphate (74)C02

CH2

.C.co2O'

iOH Chorlsmate (75)

,co2co2 O,

AffO-I-C,,,Prephenatedehydratase

ChorlsmatemutaseCH2

C -co2-HaOro cdi

OH

Chorlsmate (75)<3

oAco2 Prephenate (76) C02

UH+

O NH3Transamination

Phenyl pyruvate (77) L-Phenylalanlne (78)

1-:26.-

(Scheme-1.1) contd....

,C02co2

Prephenatedehydratase

ChorlsmalemutaseCH2 o2c.vI C

-co2-H2Or 1 ° y co2

Chorlsmate (75)

OH

iOH

Prephenate (76) QQ2

L.Hco2

o NH3Transamination

X

OHOH

p-Hydroxyphenyl pyruvic acid (79) L-Tyroslne (66)

Scheme-1.1

1.1.4.2 Simple Tetrahydroisoquinoline Alkaloids

The biosynthesis of the simple tetrahydroisoquinoline

alkaloids starts from the conversion of tyrosine to dopamine. This is

O-methylated to 4-hydroxy-3-methoxyphenylethylamine (60) which

undergoes oxidation to give 4,5-dihydroxy-3-methoxyphenethylamine

(82) and 5-hydroxy-3,4-dimethoxyphenethylamine (87) [52.53]. This

is the key intermediate in the formation of various

tetrahydroisoquinoline alkaloids such as anhalonidine (84), pellotine

(85), etc. (Scheme-1.2). It was originally assumed that the two-

carbon unit i.e., C-l and C-9, which is incorporated in cactaceae

alkaloids such as anhalonidine (84) and pellotine (85) was derived

from acetic acid. However after feeding acetic acid labelled at C-l to

the Peyote cactus, the pellotine isolated had the activity equally

divided between the C-1 and C-9 carbons.

-:27:-

Leete has shown that tyrosine (66) acts as a precursor of

anhalonidine (84) [53].

COOH HO, ,COOH

NH2H NH2

HO'TVroslne (06) DOPA (80) nH3C(

HO. H3ONH2

HO' NH2 NH2HO' HO'OH Dopamine (81)604.5-Dlhydroxy-3-methyoxy

phenylethylamlne (82)

IH3CI H3C( H30

NH2 NH2NH2H3C< H30HO'

OH 0CH3Mescaline (88)

OCH387 83

IH3OH3c<

N"HH3OH3C< H”iOH CH3

Anhalonidine (84)Anhalamine (88)

IH3OH3C(

N.,N.H3C( CH3H3CI CH3 H'"1

OH CH3Pellotlne (85)Anhalldlne (89)

Scheme-1.2

The biosynthesis of anhalonidine (84) in Laphophora williamsii

(Lemaire) was followed by feeding labelled sodium pyruvate. The C-3

of pyruvate was found to be incorporated into C-9 of anhalonidine

<-:28:~

(84). It was suggested that tetrahydrolsoquinoline alkaloids having a

methyl group at C-l are formed through the intermediacy of acetyl

coenzyme A and N-acetylphenethyl amine (90) (Scheme-1.3) [53].

H3C<CC&Nac=o

.1CHa

Labeled sodium pyruvate

O. II

H3Q-C— S— CoAL. willtamsU

NH2H3C<

OH

75

1H3C*

H3C<N— HIH3CI

H3C<OH

OH "CH3Anhalonldlne (84)

BO

Scheme-1.3

The biosynthesis of lophocerine (96) In the cactus Lophocercus

schottii was investigated by using labelled mevalonic acid (91) as a

precursor and resulted in the formation of tyrosine (66) which can

,OP .OH

T*ik. COOH

93Pyrophosphate (92)Labeled mevalonic acid (91)

Hal OP

V -xxCOOH

NH2

Labelled tyrosine (66)95Leucine (94)

H3C<

L. schottti N,HO' CH3

•CH3H*

CH3Labelled lophocerine (96)

Scheme-1.4

-:29:-

act as a precursor to the phenylelthylamine portion of the

tetrahydroisoquinoline nucleus (Scheme 1.4) (55].

1.1.4.3 Benzylisoquinoline Alkaloids

Winterstein and Trier suggested in 1910 that

benzylisoquinoline alkaloids might be derived in nature from two

molecules of 3,4-dihydroxyphenylalanine DOPA (80) via N-

norlaudanosoline (100) (Scheme-1.5) (54).

.COOH H< , ,COOH

H" NH2NH2

H< H<

(+)-Tyros1ne (66) DOPA (80)

ICOOHHOOCL. .NH2

OHOH

OHOH3,4-Dihydroxy phenyl pyruvic acid (98)

3,4-Dlhydroxy phenethylalanine (97)

IHOH<

OH+

NH2H<

Dopamine (81) OH99

H3ClH<

NH NHH3C<HO' H-fH'-t

.OH .OCH3

OH OCH3N-Norlaudanosoline (100) Papaverine (101)

Scheme-1.5

-:30:-

Biogenetic investigations on benzylisoquinoline alkaloids led

to the conclusion that simple benzylisoquinolines originate from

aromatic amino acids [55,56].

The pathway for the biogenesis of benzylisoquinolines was

confirmed by labelling experiments. When 3,4-

dihydroxyphenylpyruvic acid (98) labelled at C-2 was fed into Papaver

somniferum L, the resulting papaverine (101) was found to be labelled

at C-l and C-3 indicating that papaverine (101) is derived from two

units of tyrosine (66) incorporated in nearly equal amounts

(Scheme-1.6) [57].

, .COOH H3C<P. somniferum

NH2HO' H3C(

OCH3(+)-Tyroslne (66)

Papaverine (101) OCH3Scheme-1.6

(+) N-Norlaudanosoline (100) has been shown to be the

precursor of reticuline in Papaver somniferum. Reticuline is Itself the

precursor for morphine alkaloids (Scheme-1.7) [58].

H< HaC<

HO' H HO' CH3P. somniferum

MorphineAlkaloids

H< H3C(

OH OH

N-Norlaudanosollne (lOO) Reticuline (102)

Scheme-1.7

1.1.4.4 Pavine and Isopavine

A probable biosynthetic sequence of pavine and isopavine

starts from laudanosine (N-methyl tetrahydropapaverine).

Argemonine (108), the initial product, is converted to norargemonine

(110) and bisnorargemonine (109) on successive demethylation [59].

OH )H

M< M<,0Me ,0MeX V

NMe NMeV V/HO' MeO'

(-l-Platyccrtne (106)(-)-Munltaglne (1M)

IA O-coupUng0X

Met M<. Route A Route BNMe Ns. .NMe

HO' CHa CH<HO'

.OH

y y'OMe ‘OMe 'OMe

Imlnlum salt (103) (+)-ReLlculIne (102) M-Laudanldine (106)

p-coupling

Met.OMe .OMe

lNMe .NMe

(-)Blsnorargemonlne (109)

HO' Mt'OH

H-lsonorargemonlne (107)

M« MeO,.OMe ,OMe

,NMe ,NMeHO' MeO'

OMe 'OMe

I)-Argemonine (10©(-)- Norargemonine (110)

Scheme-1.8

The biogenetic connection between (+)-reticuline (102) and (-)-

argemonine (108) was investigated by feeding experiments on mixed

plants of Argemone mexicana and Argemone hispida [60].

-:32:-

A possible biosynthetic route based on the intermediacy of (+)-

reticuline (89) is presented In (Scheme-1.8). (+)-Reticuline (102) is

converted to the corresponding iminium salt by the in vivo oxidation.

This salt can then undergo Intramolecular para and ortho phenolic

coupling to afford (-)-bisnorargemonine (109) and (-) argemonine

(108) (Scheme-1.8).

(+)-Reticuline (102) can be converted, as shown in route B

(Scheme-1.8) to (+)-laudanidine (106) and then, by para-coupling, to

(-)-norargemonine (110).

Since pavine and isopavine alkaloids have the same absolute

configuration, it is therefore supposed that both skeletons are derived

biogenetically from the same precursor (4-hydroxybenzyltetrahydro-

isoquinoline (Scheme-1.9) [61,62].

OH>H

L-HHO,H<O

HaC< CH3CH3H3C< H-H-1 HaC<H3C(

H3C<H3C<Roemecarine-2a-N-oxlde (112)Roemecarine (111)

Scheme-1.9

This was later supported by the isolation of the 4-

hydroxybenzyltetrahydroisoquinoline base roemecarine (111) along

with it corresponding N-oxide (112).

-:33:-

H3CIH3C<

NNHO‘

HO'HH

H3C<H3CiOHOH

Retlculine (102)(+)-Norretlcullne (113)

H3CH3O

CH3+N.N.

CH3CH3 HO'HO'

HIH1

H3OH3CIMagnlflorlne (116)

Isoboldlne (114)

IHad

N.

IS. CH3HO'

H3C

OH

Boldine (116)

H3Ci H3O

N, N.CH3HO' CH3HO'

HH i

//

H3OO

OH(+)-Crotonoslne (118)N-Methylcoclaurlne (117)

Scheme-1.10

-:34:-

1.1.4.5 Aporphines Alkaloids

Aporphine alkaloids are derived from the corresponding

phenolic tetrahydrobenzylisoquinolines by direct oxidative coupling

of dienone derivatives (proaporphines), which then rearrange into

aporphines through dienone-phenol rearrangement (Scheme-1.10).

Feeding experiments using labelled reticuline in Papaver

somnifenim showed that isoboldine (114) was derived from reticuline

(102) by direct ortho-para oxidative coupling [63). Moreover, it has

been demonstrated that (±)-4'-0-methylnorlaudanosine (249), (±)-

reticuline (102) and (±)-norreticuline (113) were effective precursors

for boldine (116) in Litsea glutinosa [64).

H<

N.CH3HO*

HO'

OH 249

Since (+)-reticuline (102) but not (-)-reticuline is a possible

precursor for boldine (116) and since (+)-isoboldine (114) was

specifically incorporated into boldine (116), the postulated

biosynthesis of boldine (116) is believed to proceed as follows:

(+)-Norreticuline (113) -> (+)-reticuline (102) -> (+)-isoboldine

(114) -> (+)-Boldine (116) (Scheme-.1.10).

-:35:-

In investigations on the mode of biosynthesis of isocorydine

(120), labelled norreticuline (113),

norlaudanidine and reticuline (102) were fed to Annona sequimosa,

and It was shown that only reticuline was incorporated (Scheme-

1.11) (65).

norprotosinominine,

H3C< H30

N, N.CH3H< HO' CH3H'1 N,

HH< HO,

HaC. H3OIsocorydine (120)Protoslnomlnlne (119)

Scheme-l.il

Another aporphine base, isothebaine (124), may arise by the

dienolbenzene rearrangement from (+)-orientaline (121) via the

intermediate orientalinone (122) and orientalinol (123) (Scheme-

1.12) (66,67).

H3C< H3C

N, N,HO' CH3 HO'

h„ CHa

H3CI

/H3C(

/O Orientalinone (122)Orientallne (121)

IH3O H3C<

N. N.CH3Hi CH3HO'

"H•"HH3C1

/H3C< /Hi

HO Orientalinol (125)Isothebaine (124)

Scheme-1.12

-:30:-

1.1.4.6 Protoberberine Alkaloids

The protoberberine alkaloids are formed from a

benzyltetrahydroisoqulnoline base by condensation with

formaldehyde [68-70].

Spenser and co-workers have shown that tyrosine is a very

efficient precursor for berberine and is incorporated both into the

"top" and "bottom" parts of the alkaloid (Scheme-1.13) [71].

a N!,C00H

I H'" .OCH3NH3

H(

Tyrosine (66) Berberine (58)OCH3

Scheme-1.13

Barton [72] and Battersby [73] proposed that N-

methyltetrahydroisoquinollne could be converted to a protoberberine

H3C< o

<3 £ÿCH2 HN-CHH< 3H'

.OH O

OCH3 OCH3(+)-ReticulIne (102)

12B

,N*

,OCH3

Berberine (B8) OCH3

Scheme-1.14

-.37-

by oxidative cyclization 125 of the N-methyl function rather than by

a Mannich-type condensation with formaldehyde (Scheme-1.14).

1.1.4.7 Protopine Alkaloids

Reticuline (102) has been shown to be an efficient precursor

in the biosynthesis of protopine (43). Thus when labelled (+)-

reticuline (102) hydrochloride was fed to Dicentra spectabilis it gave

rise to labelled protopine (43) (Scheme-1.15) 174].

H3C( H3C<

N.HO' CH3 HO'

H'.OH ,OH

Reticuline (102) OCH3 OCH3Scoulerlne (126)o CH3

krO-

OoProtopine (43)

Scheme-1.15

(+)-Reticuline (102) labelled at the methoxy carbon was also

significantly Incorporated into protopine (60) and the protoberbertine

alkaloids (Scheme-1.16) [75).

H3C< oo— CH3krN-CH3HO' H-i o''ÿ

.OH ooOCH3 Protoplne (43)Reticuline (102)

Scheme-1.16

-:38:

1.1.4.8 Pthalideisoquinoline Alkaloids

The pthalideisoquinoline alkaloids are formed in nature by

oxidative rearrangement of tetrahydroprotoberberine isoquinoline

alkaloids [70]. Tyrosine (66) and methionine serve as the basic

building units in the biosynthesis of tetrahydroprotoberberine

isoquinoline alkaloids [77,78]. The protoberberine alkaloid scoulerine

(126) is itself derived from reticuline (102) in Papaver somniferum[79].

Experiments with scoulerine (126) stereospeclfically labelled

at C-13 have shown that the 13-pro-S-hydrogen of scoulerine (126)

is removed in its conversion to narcotine (62) in Papaver somniferum,

but its H-14 retains its stereochemistry during the conversion of

scoulerine (126) to narcotine (62). The intermediates between

scoulerine (126) and narcotine (62) have not yet been identified

although it has been suggested that ophiocarpine (127) and (+)-

enganine (128) could be the intermediates [80].

In this connection, it is interesting to note that the alkaloid

(+)-enganine (128), containing a hemiacetal group, has been isolated

from P. somniferum (Scheme-1.17) [81].

1.1.4.9 Secophthalideisoquinoline Alkaloids

The secophthalideisoquinoline alkaloids can be subdivided

into enol lactone keto acids, diketo acids, and ene lactams. A

blogenetic scheme is proposed for the secophthalideisoqulnolines

which includes the following sequence:

-:39:-

H3C< H3C<

N\; ,N,

HO'HO' CH3H'i H',OH .OH

ReUcuIlne (102) Scoulerine (126)OCH3“OCH3 IoO'

% aCH3 N.

H‘ H-1H--J .OCH3HÿlCHsOCO

“O OHOphlocorplne (127)Enganlne (128) £H O-

oJ<J

O— NVCH3

H"H1 j

OCH3NarcoUne (62) O OCH3

Scheme-1.17

Classical phthalideisoquinoline (60) phthalideisoquinoline

N-metho salt (129) -» secophthalide enol lactone (130) ->

secophthalide keto acid (131) -» secophthalide diketo acid (132)

fumariflorine (133) - type alkaloid (Scheme-1.18) [83-85],

1.1.4.10 Spirobenzylisoquinoline Alkaloids

Shamma proposed that phenolic protoberberine N-metho salt

could be the possible biogenetic precursor for spirobenzyliso¬

quinoline alkaloids [85,86],

-:40:-

o<J :!<CH3CH3 CH3

H* H'H"7

O ‘oo o o-

(-)-BIcucullln N-metho salt (129)(-)-Blcucullln (60)

ICH3/

3 /CHa,N.

CH3o CH3oo)OOH

O

O T O

U/Adlumldlcelne (131) 0

__J

I /Ha

o

Aobamedlne (130)

Nv. CH3CH3 /uO"

NsoCOOH CH3

.oO'ooOH

Fumariflorlne (133)Blcuculllnlne (132)

Scheme-1.18

Further biosynthetic divisions could be explained by

considering the substituents present on the aromatic ring of the

skeleton. The protoberberine N-metho salt can undergo cleavage to

the corresponding quinoid intermediates which could then form the

spiro system by an electrocyclic Michael condensation process. A

tautomeric shift would yield phenolic spirobenzylisoquinolines which

could then lead to the corresponding alkaloids via modification of the

oxygenated substituents.

H3C<HaC<

:Ar-CHs .N-CH3

fÿrV H3C(H3C<

c °N

NH; W H H3«

kd OH Vf135Dihydroprotobcrberlne (134)H

\H3C<H3C<

X XX c»3H3c< H3C< CH3

H3< VA-OH \-OHV

137OH O

Spirane (136)

*H3C<

XCH3H3C< XH2I

/\--O

AOchotenslmlne (138)

Scheme-1.19

In a proposed mechanism it was suggested that the N-metho

salt can undergo cleavage to the intermediate in basic medium,

which after electrocycllc ring closure (135) can form the spirane

(136). A tautomeric shift can afford (124) which can be easily

converted Into ochotenslmlne (138) (Scheme-1.19). If an exocyclic

methylene is present on C-13, the precursor of the compound is likely

to be the N-metho salt of the C-13 methylated dihydroprotoberberine

[87].

-:42:-

Those splrobenzylisoquinoline (141) having one or two oxygen

functions in ring "C" are probably derived from oxygenated 13-

oxotetrahydroberberine metho salt (139) via intermediate (140). This

photolytic rearrangement has been proposed by Manske and co¬

workers (Scheme-1.20) [88-90].

o aJ/CH3 *N-CH3CHJ

.OCH3 .OCH3O'

OCH313-Oxotetrahydroberberine metho salt (139)

OCH3140

o___

oCH3%

/> OCH3

0CH3Splrobenzylisoquinoline alkaloid (141)

Scheme-1.20

-:43:-

2.0 INTRODUCTION OF FUMARIA INDICA

F\imaria indica (family Fumariaceae) locally known as "Papra"

Is a small herb which is widely distributed in northern Pakistan

(91,92]. Fumaria indica has been used in the indigenous system of

medicine for the treatment of various diseases [93].

The plant extract is regarded as a laxative, diuretic and

alterative, and is said to be beneficial in dyspepsia and scrofulous

skin infections. The seeds of the plant are also used as a mild

analgesic [94],

The taxonomic delineation of families Papaveraceae and

fumariaceae is not uniform. Fedde [95], Tutin [96] and Melchior [97]

classified the Fumariaceaous plants in Papveraceae, as subfamily

Fiunarioideae, whereas Hutchinson [98] considered the Fumariaceae

to be an independent family.

2.1 LITERATURE SURVEY

The distribution of the alkaloids isolated from Fumaria species

is presented in Table-2.1.

-:44:-

Table-2.1 List of compounds Isolated from various plants of Fumariaceae

Exact8er. M. Formula Name Source Str. Ref.No. No.

[99]4-Hydroxy-3-methoxybenzaldehyde

Fumcuia indlca 142152.047 C8H8O31

Fumaria indica [94]C10H7NO2 Papraline 1432 173.054

C9H10O4 4,5- Fumaria indlca [99]3 182.057 144Dlmethoxy-1-hydroxybenzaldehyde

C10H9NO3 Noroxyhydras-tinlne

Fumaria indica,Fumariaparviflora

145 [100]191.1864

FUmaria indica.FumariaJlabeUata,Fumariaschleicheri

[101,102]205.213 CnHnNOs Oxyhydrasti-nlne

1465

[102]207.093 C11H13NO3 Corydaldine Fumaria

JlabeUata1476

1102,103)N-Methylcorydaldine

148221.255 C12H15NO3 Fumariavailantii,FumariaJlabeUata,Fumaria indica

7

Fumaria indica [99]222.161 C14H22O2 Fumaileate 1408

Fumariflorine [104.97]237.255 C12H15NO4 FumariaparvifloraFumaria indica

1509

Fumariflorineethyl ester

151 [106,107,265.308 C14H19NO4 Fumariaparviflora

10108|

Fumaria indica 152 [104]11 283.128 C17H17NO3 Paprazlne

153 [106,107]285.137 C17H19NO3 Coclaurine Fumariaparviflora

12

154 [107,109]285.137 C17H19NO3 Norjuziphlne Fumariavailantii

13

[110]Juziphlne Fumariavailantii

155299,369 C18H21NO314

156 [104,111]C18H19NO4 N-Feruloyltyra-mlne

Fumaria indica15 313.129

-:4S:-

(Table 2.1) contd.

mm-Ser. M. Formula Name Source Str. Ref.MassNo. No.

16 317.300 CigHi 1NO4 Norsanguina-rine

Fumaria indica.Fumariavailantii

1S7 [112]

320.324 Coptisine17 158Fumaria indica,Fumariadensijlom

[113.114]CI9H14N+04

18 321.100 CigHi5N04 Dlhydrocoptl-sine

Fumaria indica 159 [104,115.116]

19 322.108 Dehydroche-llanthefoline

Fumaria IndicaCI9HI6N+04 160 1113]

20 323.348 (+)-StyloplneCigHi7N04 Fumaria indica,Fumariaparviflora

[107,116]161

323.348 (±)-StylopineC19H i7N04 [102,117,21 Fumariadensijlora,Fumaria

Jlabellata

161118]

323.348 Ci9Hi7N04 (-)-Stylopine22 Fumariaofficinalis,Fumariashcleicheri

[119,1201161

23 325.363 CigHi9N04 Cheilanthifo- FumariaJudaica,Fumariavailantii

[107,115,162line 117)

24 327.379 Ci9H2iN04 Isoboldlne Fumariaparviflora,Fumariavailantii

[106,107|163

25 327.379 CigH2iN04 Lastourvilline Fumaria indica 1109)164

26 327.379 Ci9H2lN04 (+)-Scoulerine [112|165Fumariaparviflora,Fumariavailantii

327.379 Ci9H2iN04 (±)-Scoulerine27 Fumariaofficinalis

[120,121]165

327.379 (-)-Scoulerine28 CigH2iN04 165 [122]Fumariaofficinalis

CigH23N0429 329.395 Reticullne [121,123,Fumariavailantii

166124]

-:46:-

(Tibia 2.1) coDCd.

ExactMass

M. Formula Ref.Name Source Str.Ha. No.

332.335 LahorineC20Hi4N+O4 Fumariaparvljlora

(123,125,30 167126]

Sangulnarlne31 332.335 Fumaria indica,Fumariacapreolata,Fumariaofficinalis

168 [115,1181C20Hl4N+O4

C20H15NO4 dihydrosan-guinarine

32 333.343 Fumariaofficinalis,Fumariauailantii

1106,127)169

8-Oxocoptlsine Fumaria indica.Fumariaparvljlora,Fumariauailantii

33 335.079 C19H13NO5 U28]102

(115,126,129.130]

Berberlne Fumaria kraiikil34 336.367 170C20Hi8N+O4

C2qH2iN04339.390 Canadlne 1115,130.35 Fumariakralikii,Fumariaofficinalis

172131]

339.390 H-Sinactlne36 C2qH2iN04 |117|Fumaria

officinalis171

C2QH2IN04339.390 (±)-Slnactine37 Fumariaojjicinalis

1117,120]171

38 341.406 C2qH23N04 Isocorydine Fumariauailantii

[124,132.173133]

C19H19NO539 341.363 Ledecorine Fumariauailantii

I134|174

C19H19NO5 Norfumaiitlne FUmaria kralikii40 341.363 175 [1351

347.326 C2qH13NO5 Oxysangutna-rlne

Fumaria indica 1127.136]17641

42 348.378 Chelerynthe-rine

(1271C2)Hi8N+04 Fumariaschlicheri

177

1125)43 348.396 LahoramlneC2iHi8N+04 Fumariaparvijlora

178

351.358 C2qH17NO5 Fumarillne (125,137)Fumaria indica.Fumariaofficinalis

44 179

-:47:-

(Tmblo 2.1) cootd....,

Ser. ExactMass

M. Formula Name Source Str. Ref.No. No.

C21H21NO4 Bulgaramlne Fumariaofficinalis

1138)351.401 18045

46 352.409 Palmatine Fumariadensiflora

181 [114.139]C2lH22N+04

353.374 C20H19NO5 Dihydrofuma-rUlne-1

Fumariaojfficinalis

(125,139)47 182

353.374 C20h19no5 Dihydrofuma-rillne-2

(124,140148 Fumariaojfficinalis

192

Parfumlne353.374 C20H19NO5 Fumaria indica,Fumariaparvijlora

(125,141)49 183

50 353.374 C20H19NO5 Protoptne Fumaria indica.Fumariavailantii

[106.115.120,142]

184

51 354.425 N-Methylcana- 185 (1431Fumariaojfficinalis

C2lH24N+04dine

N-Methylslnac- Fumariaojfficinalis

186 (126)52 354.425 C2lH24N+04tine

53 354.382 Stylopinmethhydroxlde

Fumariavailantii

187 (144)c2oH2oN+o5

355.390 C2oH2lN°5 Dlhydropar-fumlne

Fumaria indica.Fumariaparvijlora

[125.128154 188

355.390 C20H21NO5 FUmaria indica.Fumariaojficinalis,Fumariamuralis

[102.125,55 Fumaritine 189137]

Fumaria indica56 355.390 C20H21NO5 Fumarlzine [145]190

355.390 C20H21NO5 Fumariaparvijlora

[146]57 Izmlrlne 191

[147]355.346 C20H21NO5 Papraine Fumaria indica 19858

Fumaria indica [104]355.390 C20H21NO5 Papracinlne 19359

[123]60 355.180 C2lH25N04 Tetrahydropal-matine

FumariaJlabellata

194

1148)357.158 C20H23NO5 Vallantlne Fumariavailantii

19561

-:48:-

(Table 2.1) contd.

Ser. ExactNo. Mass

Ref.M. Formula Name Source Str.No.

8-methoxydl-hydro-sanguinarlne

1149]C21H17NO5 Fumaria indica 196363.36962

[150]C20H15NO6 Densiflorine Fumariadensijlom

197365.34263

Fumaria indica.Fumariaparvijlora

1151]C20H17NO6 (+)-Adlumldlne 20464 367,106

(±)-AIdumldtne [152,153]367.106 C20H17NO6 Fumariavailantii

20565

[154.155]C20H17NO6 t-)-Blcuculline Fumaria indica.Fumariaparvijlora,Fumariavailantii

206367.10666

[1021(+)-Bicuculline F. vailantii367.106 C20H17NO6 20767

[102,120.C20H17NO6 (+)-Blcuculllne FumariavailantiiFumaria

JlabeUata

20868 367,106155]

Parfumldine [125,156]C21H21NO5 Fumariaparvijlora,Fumariaofficinalis,Fumariavailantii

203367.16569

(-)-Capnoldine 1154,155,C20H17NO6 Fumariavailantii

70 367.106 210157)

[158|Fumaria indica,Fumariavailantii

205368.150 N-Methylprotopine

71 C2IH22N+05

[159]C20H19NO6 Fumariavailantii

212369.373 H-Corledine72

Corlumldine [1551369.373 C20H19NO6 Fumariaparviflora

21373

[100.146]369.416 C21H23NO5 Cryptopine Fumariadens{flora,Fumaria kralvii,

Fumariaojfficinalis

22074

[128]C20H19NO6 209369.373 Fumariavailantii

75 Enganlne

-:49:-

(Table 2.1) contd.

Ser. ExactNo. Mass

Ref.M. Formula Name Source Str.No.

(100.160.369.416 C21H23NO5 Fumarlclne Fumaria indlca,Fumariaofficinalis,Fumariaparviflora

22276161]

1162]369.416 c2lH23N°5 Fumaritrldine Fumariarostellata

77 211217

1131]Fumaroflne369.373 C20H19NO6 Fumariamicrocarpa,Fumariaofficinalis

22378

369.373 Fumaria kralikii [128,1631C20H19NO6 Fumarostelltne 23079

fumartlneN-metho salt

370.424 Fli/nariamauralis

219 [164.165]80 C2iH24N+05

[166]C20H21NO6 Fumaria indica,Fumariaofficinalis,Fumaria kralikii

221FumaritineN-oxlde

371.38981

(167.168]82 380.399 Fumaramine Fumariaparviflora.Fumariavailantil

216C2lH20N+2O5

1169]83 381.384 C2qH15NO7 Fumaflorine Fumariadensiflora

217

Adlumidicineenol lactone

11701381.384 C2iHi9N06 21884 Ftimariaschrammii

383.137 (-)-Adlumlne 225 (120,152]85 C21H21NO6 Fumariaparviflora,Fumariarostelala

(+)-Adlumine (1641383.13786 C21H21NO6 Fumariavailantil

226

(131](±)-Adlumine Fumaria indica383.137 C21H21NO6 22787

(1311383.137 C21H21NO6 (-)-Corlumlne 228Fumariaparviflora

88

383.137 (+)-Corlumlne FUmarta indica 229 1164]C21H21NO689

Fumaria herba (162]383.400 C21H21NO6 Fumaritrine 22490

FUmarta indica,Fumariaparviflora

(131.164]383.13791 C21H21NO6 231(-)-p-Hydras-tlne

-:50:-

(Table 2.1) contd.

Ser. ExactMass

M. Formula Ref.Name Source Str.No. No.

1152)C21H21NO6 232FumariavaUantii

383.13792 (+)-P-Hydroas-tlne

1152]C21H21NO6 Fumaria indica 23393 383.137 (+)-P-Hydras-tlne

1154]C21H21NO6 23494 383.137 Fumariaparvijlora.Fumariaschelicheri

(+)-a-Hydras-tlne

FumariavaUantii

[164]C21H21NO6 23595 383.137 (-)-a-Hydras-tine

Fumariaparvijlora,Fumariaschelicheri

[1661C21H21NO6 23696 383.137 (±}'<x-Hydras-tine

1100]C21H23NO6 Fumaria indica 24997 385.152 Raddeantne

Acetodihydro-sangulnarine

[165]C23H19NO5 FumariavaUantii

19998 389.407

[168]Fumaramidine396.442 C22H24N2O5 Fumariaparvijlora

20099

[168]Fumaridine Fumariaparvijlora,FumariavaUantii

396.442 C22H24N2O5 201100

[139,170]397.427 Fumariaschrammii

202C22H23NC)6 Adlumiceineenol lactone

101

[171]397.427 C22H23NO6 Fumarophy-cine

Fumariakralikii,Fumaria

officinalis.Fumariamuralis

214102

1172]397.384 C22H19NO7 Narlumidtne Fumaria indica 237103

[120.173]397.427 C21H23NO6 N-Methyl-hydrastlne

Fumariaparvijlora,FumariavaUantii

238104

1100]Fumaria indicaC21H19NO7 Paprarine 239105 397.113

[174]Fumschlel-cherlne

398.415 Fumariaschleicheri,Fumariaschrammii

240106 C2iH24N+06

-:S1:-

(Table 3.1) conld.,

UaMfs*So.Ref.M. Formula Source Str.Name

No.

(130)N-Methyladlu-mlne

215Fumartavailantii

107 398.415 C2lH24N+06

Fumarta tndica [1751C2iH2iNC>7 Narlumlclne 242399.399108

[120,1701Fumartaschrammli

243C2iH2iNC>7 Adlumldlcelne109 399.399

O-Methylfuma-rophyclne

(153)C23H25N06411.454 Fumartakraukii,Fumariaofficinalis

244110

(119,1661Fumaria tndica,Fumariaschrammit

C21H19NO8 Narceimine 245413.383111

1120,140,246C22H25N07 Adlumlceine FumariaschrammU

415.163112170]

[176.177JNaraceimlclne Fumaria tndica 247C21H21NO8415.399113

[168.1731C22H25n07 N-Methyl-hydrastelne

248415.163 Fumariaparvijlora,Fumariaschleicheri,Fumariavailantii

114

Fumaria indica 255 [1821C25H23NC>5115 417.157 9,10-Methylenedloxykarachlne

Fumaria indica 250 [168,178]116 426.468 C23H26N2C>6 Nareclneimlde

C22h23NOs Bicuculllnldlne 251 [102,174]FumariaschrammiLFumarialabellata

429.426117

paprafumlne 253 [100]C22H23N08 Fumaria inidca119 429.142

254 [1021Fumaria indica457.177 C24H27NO8 Papraclne120

255 [1791515.516 C26H29NOl0 Parvlflorine Fumariaparviflora

121

[1801Fumariadensijlora

C30H34N2O6 Fumadenslne 241122 518.608

-:52:-

NO'

143

R3<

N.RiR2<

o146 Rj = H, R2 + R3 = CH3146 Rj = CH3> R2 + R3 = CH3147 R1 = H, R2 = R3 = CH3148 Rj = R2 = R3 = CH3

CH3N.O

CH3< ORO

O

160 R = H161 R=C2H5

R2.

N.R3 RI

54R:

Re Re

R7163 Ri = R4 = R5 = R7 = Ra = H, R2 = OCH3. R3 = Re = OH164 Rj = R2 = R5 = R7 = Ra = H, R3 = OCH3, R4 = Re = OH166 R! = CH3, R2 = R5 = R7 = Ra = H. Ra = OCH3, R4 = Re = OH166 Rx = CH3. R2 = Re = OCH3> R4 = R7 = Ra = H. R3 = R5 = OH174 Ri = CH3> R2 + R3 = Re + R7 = 0-CH2, R4 = R5 = H, Re = OH190 Ri = CH3, R2 + R3 = Re + R7 = 0-CH2-0. R4 = R5 = H, Ra = OC

-:63:-

R2<

CH3RI«H

R;

R4<

RS

163 Ri = R3 = R4 = H. Ffe = CH3, R5 = OC1104 Ri = R2 = R3 = H, R4 = CH3> R5 = OCl173 Rj = R2 = R4 = H. R3 = CH3, R5 = H

< N RioRi

O

>O159 Ri = R2 = H102 Ri + R2 = O

Ri

+AR2<

OR3

;

OR,158 Rj + R2 — R3 + R4 — CH2160 RL = CH3( R2 = H. Rs + R4 = CH2170 Rj + R2 = CH2l R3 = R4 = CH3181 Ri = R2 = R3 = R4 = CH3

-:54:-

Rl<

N,

R2<H

OR3

OR4

161 Ri + R2 = R3 + R4 = CH2162 Ri = CH3. R2 = H, R3 + R4 = CH2166 R1 = R4 = CH3> R2=R3 = H171 Ri + R2 = CH3, R3 + R4 = CH2172 Ri + R2 = CH2, R3 = R4 = CH3194 Rl = R2 = R3 = R4 = CH3

Ri'

CH3+N:

R2«

OR3R5

OR4187 Ri + R2 = R3 + «4 = CH2, R5 = OH186 Rx + R2 = CH2. R3 = R4 = CH3, R5 = H186 R1 = R2 = CH3>R3+R4 = CH2, R5=H

°>o

N

O

\-i 167

°>O

JKR2< CH3

ORI 168 RI + R2- CH2177 R1 = R2 = CH3

-:55:-

o

>o

N— CH3O\ / H2 Rl\— o

169 Rj = R2 = H176 R] + R2 = 0196 Ri= H. R2 = OCH3199 R, = H. R2 = CH2-CO-CH3

R2'

IÿCH3NC

Hro'

.ORs

OR4184 Rj + R2 = R3 + R4 — CH2191 Rj = CHs, R2 = H. Ra + R4 = CH2196 R] = R2 = H, R3 = R4 = CH3220 Rj = R2 = CH3, Ra + R4 = CH2

O

< + I/ch3+Nÿ-CH3O'

O'

o

Q206

Rl+ N— CH3/)

Hi

167 Rj + Rz = CH2178 R1 = R2 = CH3

-:66:-

H3C(

N— CH3

Ri' ReR2R3 nR4

R7

R5Re

217 Rj = R2 — R3 = R7 = Rg = H, R4 = OCH3, R5 + Re = 0-CH2-0218 Rj = R3 = R4 = Rg = Rg = H, R2 — OCH3, R7 + Rg — 0-CH2-0196 Rx = CH3, R2 = R3 = r5 = Re = H. R4 = OCH3, R7 + Rg = 0-CH2-0

R2«

CH3RIR3

ORg

O OR5

198 RI = R2 = H,R3 = R4 = (JH. R5 + Rg = CH2204 Rj = R2 = CH3, Rs = aH. R4 = pH, Rg + Rg = CH2205 Ri = R2 = CH3> R3 = PH. R4 = aH. Rg + Rg = CH2206 Ri = R2 = CH3, R3=R4 = H>R5 + Rg = CH2207 Ri + R2 = R5 + Rg = CH2, RS = PH, R4 = aH208 R; + R2 = R5 + Rg = CH2, R3 — R4 = H210 Rj + R2 = R5 + Rg = CH2, R3 = R4 = aH212 Rj + R2 = Rg + Rg = CH2> R3 = R4 = PH213 Ri + R2 = R5 + Rg = CH2, R3 = R4 = H225 R! + R2 = R5 + Rg = CH2. R3 = aH, R4 = pH226 Rx = CH3, R2 = H, Rs = aH. R4 = pH, Rg + Rg = CH2227 Rx = H, R2 = CH3, R3 = R4 = PH. Rg + Rg = CH2228 Ri = R2= CH3, R3 = R4 = aH, R5 + Rg = CH2229 Rx = R2 = CH3, R3 = R4 = PH. Rg + Rg » CH2231 Ri = R2 = CH2, Ra = R4 = aH. R5 = Rg = CH3232 Ri + R2 = CH2, R3 = R4 = PH, R5 = Rg = CH3233 Rx + R2 = CH2, R3 = R4 = H, R5 = Rg = CH3234 Ri + R2 = CH2, R3 = R4 = pH, R5 = Rg = CH3235 Rx + R2 = CH2. R3 = R4 = aH, R5 = Rg = CH3236 Ri + R2 - CH2. R3 = R4 = H. R5 = Rg = CH3

-:57:-

<1 N.O CH3

HH.

Q

OH wOH215

H30

CH3

CH3H3C«H

H-

Q

O

Wo241

CH3N:H3C<

CH3

HH3C(

O

o\—o o 218

CH3N,R2<

CH3

R5RI

OR3

O OR4222 Ri + R2 = R3 + R4 = CH2. R5 = H202 Rj = R-j = CH3, R3 + R4 = CH2, Re = H238 Rt + Rz - CH2. R3 - R4 - CH3. Re - H239 Ri + R2 = R3 + R4 = CH2, R5 = OH

-:68:

Ri

NR21 COOR3

O

o

217 R1 = R2 = CH3> R3 = H

R, ,N.

Re

OR7R2(

.O

OR3

IIV0R4

242 R! = R2 = R3 = R4 = CH3, R5 = R6 = CH3. R7 = H

-:59:

CH3NRa<

CH3

HR2<

RIH— N

OR3

O OR4216 Rj = H, R2 + R3 = R4 + R5 = CH2200 R! = H, R2 = R3 = CH3. R4 + R5 = CH2201 Ri = H, R2 + R3 = CH2> R4 = R5 = CH3250 R! = 0CH3, R2 + R3 = CH2, R4 = R5 = CH3

CH3N;o

CH3< .0oH

Q

Ouo o192

CH3N;o

CH3<oOH

Rl— N

ORg

O OR2

240 R! = H, R2 + R3 = CH2 •241 Ri = CH2-CH2-Ph, R2 = R3 = CH3

-.60:-

CH3NR3< s

CH3o

R5 OReR2< R4

RIO-

OR7

ORe

248 Rj = R4 = R5 = Re = H, R2 + R3 = CH2. Re = R7 = CH3243 R2 + R3 = Re + R7 = CH2, RI = R4 = R5 = Re = H245 Ri = Re = H, R2 + R3 = Re + R7 - CH2, R4 + R5 = O246 Rj = R4 = R5 = Re = H, R2 = R3 = CH3. Re + R7 = CH2251 Rj = Re - H, R2 - R3 = CH3, R4 + R5 = O, Re + R7 = CH2252 Ri = Re = H, R2 + R3 = CH2, R4 + R5 — O, Re = R7 = CH3254 Rj = H, R2 + R3 = CH2, R4 + R5 = O, Re = R7 = CH3, R3 = C2Hs

CH3NS

CH3< oOH\\ .ORo

HO'

O

247 R = H253 R = CH3

.61:-

Re R7R3 V/

N

•' Oc'

-7R4 R5 o

R2O OR,

175 R, = R3 = R4 = Re = R7 = H, R2 = CH3, R5 = OH179 R, + R2 = CH2, R3 = CH3, R4 + R5 = O, Re = R7 = H182 R, + R2 = CH2, R3 = CH3> R4 = Re = R7 = H. R5 = OH237 R, + R2 = CH2, R3 = CH3, R4 = R6 = R7 = H183 R, = Re = R7 = H, R2 = R3 = CH3, R4 + R5 = O189 R, = R4 = R7 = H, R2 = = R3 = CH3, R5 = OH188 R, = R5 = R6 = R7 = H. R2 = R3 = CH3. R4 = OH203 R, = R2 = R3 = CH3, R4 + R5 = O. Re = R7 = H222 R, = R2 = R3 = CH3, R4 = R6 = R7 = H, R5 = OH211 R, = R5 = Re = R7 = H, R2 = R2 = R3 = CH3, R4 = OCH3223 R, = R7 = H, R2 = R3 = CH3, R4 + R5 = O. Re = OH214 Ri = R4 = R6 = R7 = H, R2 = R3 = CH3> R5 = OAC249 R, = R2 = R4 = Re = H, R3- CH3> R5 = R7= OH230 R, = R4 = H, R2 = R3 = CH3. R5 = OH. Re + R7 = O224 R, =R2 = R3 = CH3> R4 = OCH3, R5 = Rg = R7 = H244 R, = R2 = R3 = CH3. R4 = Rg = R7 = H, R5 = OAC255 R, = D-glucose, R2 = R3 = CH3, Rj + R5 = O, Rg = R7 = H

CH3/

N

O_JK i> 0 O

O O

197

-:62:-

Ri ,Ra\

CH3/

0--N

O

R3R4 -Jo

H3C0 OH

193 R! = R2 = R3 = R4 = H221 Rj a R2 = R3 = H, R4 = OH

H3C ru

'V3+ N

J 0_yH OH '

H3CO OH

219

OHO

N

HHO'

152 R = H156 R = OCH3

Ri'

N—Rs

R2< ,OR3

r\OR4

180 Rj = R2 = R5 = CH3. R3 + R4 = CH2

-:63:-

SECTION B

ISOLATION AND STRUCTUREELUCIDATION OF FUMARIA INDICA

2.2 RESULTS AND DISCUSSION

Four new and four known compounds have been isolated from

Fumarta indica. Various spectroscopic studies and other experimental

techniques were carried out for the structural determination of these

compounds. The results of these studies are discussed in this

chapter. The extraction and isolation procedures of these compounds

are discussed in detail in Chapter-3 of the "Experimental section".

New Compounds from Fumaria indica

2.2.1 Fumaileal (144)

The chloroform extract obtained by extraction (scheme-2.8,

Experimental) was chromatographed over a silica gel column using

pet.ether : acetone (8:2) as eluent to obtain fraction FN-2 (Scheme-

1.2, Experimental). This fraction was further purified by preparative

TLC on silica gel (GF-254, 0.2 mm) using pet.ether : acetone (8.1:1.5

+ two drops of NH4OH) as the developing solvent. This afforded

Fumaileal (4,5-dimethoxy-2-hydroxy-benzaldehyde,144, 7.2 mg)

which gave a dark blue colour test with ceric sulphate spray.

o6

H3C( 1H5

,24H3C< OH

3

Fumaileal (144)

The HREIMS showed molecular ion at m/z 182.0594

corresponding to the molecular formula C9H10O4 (calcd. 182.0579)

-:64:-

showing five degree of unsaturations in the molecule. The 1R

spectrum of 144 showed an absorption at 1718 cm*1 corresponding

to the carbonyl functionality.

The UV spectrum showed strong absorption at Xmax (MeOH)

250 (log e) (2.873) nm. The spectrum (CDCI3. 400 MHz)

displayed three sharp singlets in the downfield region one of which at

8 9.8 was assigned to the aldehydic proton. Only two aromatic

protons resonated at 8 7.13 and 7.23, from this it was apparent that

the benzene ring was tetra-substituted. Two methoxy groups

resonated as a sharp 6H "singlet" at 83.69.

The 13C-NMR spectrum (broadband) of 144 showed nine

carbon signals. The DEPT spectrum established that the molecule

contained two methyl, two methine and hence five quaternary

carbons atoms (105). The 13C-NMR assignments are shown in Table-

2.2.

Table-2.2. 13C-NMR assignments of 144

8 (DEPT)13C-NMR ChemicalShift (8)

Carbon No

C-l 132.2 -C-02 147.4 -C-03 105.8 CH04 142.2 -C-05 142.1 -C-06 106.7 CH07 56.5 -OCH308 56.6 -OCH3

o09 190.7 II-C— H

-:65:-

The IR spectrum (CHCI3) of 144 showed an absorption at

1718 cm-1 due to the conjugated carbonyl group present in the

molecule. The HREIMS (m/z 182.0579} revealed the molecular

composition, CgHioC>4, which was consistent with five degree of

unsaturations. Four of these were accounted for a benzene ring and

the fifth for an aldehydic carbonyl group. Two oxygen atoms were

present in the two methoxy groups, the third as a carbonyl and

fourth was due to a hydroxy function. The spectrum

showed a signal at 8 190.7 due to the aldehydic carbonyl group. The

*H-NMR spectrum showed a 1H singlet at 5 9.8 for the aldehydic

proton.

The presence of a hydroxyl group at C-2 was inferred from the

IR, *H and 13C-NMR spectroscopic studies. The IR spectrum showed

a strong absorption band at 3340 cm-1 due to the hydroxyl group.

The 13C-NMR spectrum contained a signal at 8 147.0 for the

aromatic carbon bearing the OH group.

The presence of methoxy groups at C-4 and C-5 positions was

established by *H-NMR and 13C-NMR spectra. The *H-NMR spectrum

showed the two methoxy groups as an overlapping 6H signal at 8

3.96. However in the i3C-NMR spectrum, the two methyl signals of

the methoxy groups resonated separately at 8 56.5 and 8 56.6,

establishing the presence of two methoxy groups in molecule.

The structure of (4,5-dimethoxy-2-hydroxy benzaldehyde) 144

was supported by the mass spectrum. The M+ ion in the HREI MS of

our compound 144 was at m/z 182.0513 corresponding to the

-:66:-

molecular formula C9H10O4. Another important fragment showing a

peak at m/z153.0549(C8HgO3) was due to the loss of the aldehydic

group from the molecular ion. The mass fragmentation pattern is

shown in Scheme-2.1.

t

H3C<H

OHH3C( H3C< ‘OH

m/z 153m/z 182

Scheme-2.1

2.2.2 Acetyl-3,4,5-trimethyl-3-propane-4-cyclohexane

(Fumaileate,149)

The chloroform extract obtained by extraction (Scheme-2.8,

Experimental) was loaded on a silica gel column (70-230 mesh.

ASTM), which was eluted with increasing polarities of pet.ether:

acetone. The fraction "FN-3" obtained on elution with pet.ether:

acetone (7:3, 48 mg) contained four minor compounds and one major

compound. The mixture was subjected to preparative TLC on silica

gel precoated plates which were developed in pet.ether : acetone

(7.5:2.5), This afforded a pure compound, fumaileate (149) as a

colourless amorphous solid (7.0 mg).

O

H3CÿO 12

CH3

CH3

6

H"T 5

H C

H3CH3140

-:67:-

The high resolution mass spectrum afforded the molecular ion

peak at m/z 222.1610 corresponding to the molecular formula

C14H22O2 (calcd. 222.1619) leading to the presence of four double

bonds equivalents in the molecule.

The UV spectrum showed absorptions at A,max (MeOH) (log e)

201 (3.169) and 226 (3.365) nm. The IR spectrum showed bands at

2912, 1718, 1254 cnr1 which were due to (C-H), (C=0), (C-O-C)

groups respectively.

The 1H-NMR spectrum (CDCI3, 500 MHz) of 149 showed

resonances for three downfield protons at 8 5.10 m(lH, H-la), 6.13 m

(1H, H-7) and 6.70 dq (1H, H-8). Five signals for the methyl groups

appeared at 8 0.9 s (3H, H-10). 8 1.15 s (3H, H-12), 1.51 s (3H. H-ll),

1.90 dd (3H, H-9) and 2.03 s (3H, COCH3). Four methylene protons

resonated at 8 1.58 dd (1H, H-2a)1.74 d (1H, H-2p), 2.08 dd (1H, H-

6a) and 2.40 d (1H, H-6P).

The 13C-NMR spectrum (CDCI3, 100 MHz), showed resonances

for fourteen carbon atoms in the molecule. The 13C-NMR chemical

shift assignments are presented in Table-2.3. A signal at 8170.59

corresponded to the presence of an ester functionality.

This ester group was further confirmed from the IR spectrum

which showed an absorption at 1718 cm1. The downfield proton at

(8 5.10 m) showed the attachment of the carbon bearing this proton

to an oxygen atom. Two methyl groups resonated as singlets at 8 0.90

and 1.15 showing that they were attached to a quaternary carbon.

-:68:-

Table-2.3. 13C-NMR assignment of 149

Carbon No. iT* *T>y'i

c-i 68.1 -CH-

43.6C-2 -CH2

C-3 35.7 -C-

C-4 140.0 -C-

C-5 127.5 -C-

C-6 37.0 -CH2

C-7 145.8 -CH

C-8 134.4 -CH

C-9 29.0 -CH3

OC-10 170.6

C-ll 21.0 -CH3

C-12 20.6 -CH3:

C-13 29.4 -CH3

C-14 18.2 -CH3

The protons of one methyl group resonated as a double

doublet at 5 1.90 dd (Jit2 = 2.6 Hz, Ji,3 = 6.9 Hz), Two downfield

oleflnic protons 5 6.13 and 6.70 were assigned in a trans relationship

to each other on the basis of "J" value {J = 15.7 Hz).

The HREIMS of 149 showed the M+ at m/z 222.1610

(C i4H2202, calcd. 222.1620). The peak at m/z 161.1329 (C12H17

icalcd. 161.1330) was due to loss of a acetate group from the

-:69:

molecular Ion. A sizable peak at m/z 121.1041 (C9H13 calcd.

121.1017) showed the loss of propene group from m/z 161.1329.

Another peak at m/z 81.0814 (C6H9 calcd. 81.0704) was due to loss

of ethene group and methyl radical from the m/z 121.1041 fragment.

The mass fragments are presented in (Scheme-2.2).

r ;

CH3.CH3- CHiCOOH

H" CH3CH3 H3<CH3CHa

m/z 222loss of H"

.CH3

CH3

m/z 16l'

Scheme-2.2: Mass fragmentation pattern of fumalleate.

The 13C NMR spectra (Broadband decoupled and DEPT ) [181.

182] showed all signals representing fourteen carbon atoms (Table-

2.3). The DEPT and broadband decoupled spectra exhibited the

presence of five methyls, two methylene, three methine and four

quaternary carbons. The most downfield signal at 8170.5 could be

assigned to the C-10 carbonyl functionality. The signals at 8 21.0,

20.6, 29.5 and 18.2 were ascribed to the C-ll, C-12, C-13 and C-14

methyls respectively. The two methylene signals at 8 43.6 and 37.0 were

attributed to the C-2 and C-6 respectively. Three signals observed in

-:70:-

the sp2 chemical shift range were ascribed to the C-l (8 68.07), C-7 (

8145.8) and C-8 (8 134.5) methines. The remaining three signals were

tantatively assigned to the C-3, C-4 and C-5 quaternary carbon

resonating at 8 35.8, 140.0 and 127.5 respectively. It needs further

support of heteronuclear experiment for an unambiguous

assignment.

The HMBC (Heteronuclear Multiple Bond Connectivity) [182]

was performed to unambiguously assign the 13C NMR chemical shift

values. The methyl protons resonating at 8 2.03 showed 2J

heteronuclear interaction with the C-10 (8 170.5) carbonyl carbon.

Another proton at 8 5.10 (H-l) exhibited interaction with the C-

10 (8 170.5) thereby confirming the position of carbonyl in this region

unambiguously, H-l also showed connectivity with the C-3

quaternary carbon resonating at 8 35.8. Methyl protons (H3-I2)

exhibited HMBC interactions with the C-4 (8 140.0). Another methyl

resonating at 8 1.15 (H3-I3) also displayed heteronuclear correlation

with the C-4. The chemical shift assignments in the isolated propene

unit were also confirmed by HMBC experiment. The H-7 (8 6.7)

displayed 2J and heteronuclear correlation with the C-8 (8 134.5)

and C-9 (8 29.0) respectively. These heteronuclear correlations are

presented around the structure, Fig. 2.3.

The COSY-450 spectrum showed homonuclear correlation of

H-l geminal to ester functionality at 8 5.10 with the H-2 a and -p

methylene protons resonating at 8 2.08 and 2.40 respectively. It also

exhibited vicinal interaction with H-6 methylene protons (8 1.74 and

1.70). H-2 a and -P methylene protons also displayed geminal

-:71:-

interactions. Antother proton at 5 6.70 (H-7) showed COSY 45°

interations with H-8 (5 6.13). H-7 and H-8 displayed allylic

interactions with H-9 methyl (8 1.91).

The nOe difference experiment was performed to establish the

stereochemistry of protons and functional groups at various

asymmetric centers. For example the irradiation of H3.I4 methyl at 8

0.99 exhibited enhancement of the H-2(5 at 8 2.40 which in turn

showed 1.8 % nOe with the H-6|3 resonating at 8 1.74. Irradiation of

another proton at 8 2.08 (H-2oc) displayed the 3.2 % nOe interaction

with the H-la thereby confirming the /)-stereochemistry of ester

functionality at this position. The H-la (8 5.10) also showed

interaction with the H-6a (8 1.58). The nOe interactions are shown

around the structure Fig. 2.1.

3.1%

O3.4%

H HH3C o< .CH3

HH 2.1%

o*'' CH3H HH3C'3.2%

CH3H1.8%

Fig. 2.1: nOe difference mearurements on fumaileate (149).

-:72:-

o J<xi 6|j=14.3H2Jio.6a = 4.5. 1.5Hz, dd

1.74 d

H H 12 1.51(s)

CH3H3C 10 o5.10(m) H'

2.40 dH

62.03(s)51

2 1.15(s)4133

CH32.08 dd H 14 H 6.1pdq j3 )= ! ?Hzi J3.j=15.7 Hz

J2o.2P”ÿ*ÿz*-ÿ2a. io=ÿ.8,Hz H3CT

0.99(B)pj

6.70(m)CH3

1.91 dd JI,3=1.7HZJI,J= 6.9 Hz

Fig. 2.2: 1H-NMR assignments of fumaileate (149).

O

1.58di15.1

H170.521.03 11 12 20 ICH3ÿo''10 C37.0O.

6Ha 5ll127.

140/1.152]2.40d

46.63

134.48ÿÿ)2.08d

’•"zZ145.8

8H3C r99(s)8.25

gCH3HI j6.70m

Fig. 2.3: HMBC assignments of fumaileate (149).

2.2.3 Papraline (143)

The ethanolic extract of aerial parts of Fumaria indica was

diluted with distilled water. Then it was deffated with pet.ether and

remaining aqueous portion was shaken with chloroform. The

chloroform layer was concentrated under reduced pressure which

-:73:-

resulted the fraction "FN" . The remaining aqueous layer was treated

with dilute acetic acid (pH~2.5) and was shaken with chloroform and

separated from the aqueous layer. This chloroform extract was

evaporated to a gum and named as fraction "FA". The aqueous layer

was than basified with ammonium hydroxide (pH~9.0) and extracted

with chloroform and the concentrated chloroform extract was coded

"FB".

The fraction "FB" was subjected to silica gel column

chromatography using chloroform-methanol as the eluent system.

The fraction FB-1 was obtained from this column at increasing

polarity of 19:1 (chloroform-methanol) which showed two compounds

(Scheme-2.9, Experimental). This fraction was subjected to

preparative thin layer chromatography (TLC) using pet. ether :

acetone (82:18) which yeilded a colorless compound (143) along with

a yellowish red colored substance (unidentified). The compound 143

has never been isolated so far from the natural source .

5 4

4a.3

I18

143

The LREIMS of 143 displayed the molecular ion peak at m/z

173 Which was further confirmed by HREIMS at m/z 173.0469

(calcd. 173.0474) indicating the eight degrees of unsaturation. The

mass fragmentation pattern of 143 was distinctly similar to the

simple isoquinoline alkaloids [100]. The peak at m/z 144, is

characteristic for isoquinoline alkaloids and was due to the fragment

-:74:-

Ion CgHeNO. Other major fragment observed In the mass spectrum

was at m/z 115 which arose due to the loss of CsHsN. The mass

fragmentation pattern is presented in Scheme-2.3.

The UV spectrum of 143 displayed absorption maximum at

325,300 and 200 nm characteristic of isoquinoline chromophor.

These absorption suggested that the molecule is fully aromatic.

The IR spectrum showed absorptions at 937, 1480, 2738, 2950

indicating the presence of methylenedioxy, aromatic C=C,

conjugated C=N and C-H respectively.

-1cm

The *H-NMR spectrum (CDCI3, 500 MHz) showed six signals

in the downfield region. A two-proton singlet resonating at 6 6.31

could be assigned to the methylenedioxy protons. A set of two

protons resonating as two singlets at 5 7.31 and 7.45 assigned to the

H-5 and H-8 respectively. The lack of any interaction between these

two protons suggested their para disposition. The most downfield

proton resonating at 6 9.61 exhibited close proximaitly to nitrogen

atom and could be assigned to H-l. Two protons resonated as

doublets at 6 8.20 and 7.91 (J=5.7Hz) were assigned to the H-3 and H-

4 based on coupling constant and COSY-450 experiment-

The DEPT and broadband decoupled 13C-NMR spectra

(CDCI3,125 MHz) showed ten carbon atoms (Table-2.4) indicating

the presence of one methylene, five methine carbon and four

quaternary carbons in the molecule. The methylenedioxy carbon

showed a characteristic signal at 6103.5. The two downfield signals at

-:75:-

8 104.7 and 103.3 could be attributed to the C-5 and C-8 respectively.

Another signal integrated for two carbons at 8123.2 was assigned to

the C-l and C-3 adjacent to nitrogen functionality. The remaining

methine carbon at 8 103.3 was ascribed to the C-4. The assignment

of quatemaiy carbons 8135.1 (C-4a), 142.3 (C-6), 146.0 (C-7) and

136.3 (C-8a) were based on HMBC spectrum.

Table-2.4.

Carbon No. Multiplicity13C-NMR Chemical Shift (8)

C-l 123.2 -CH

C-2

123.2C-3 -CH

104.65C-4 -CH

I

TC-4a 135.1

C-5 104.7 -CH

C-6 142.3 -C-

146.0C-7 -C-

C-8 103.3 -CH

C-8a 136.3 -C-

0-CH2-0 103.5 -CH2

Knowing the Basic skeleton of the compound 143, the

assignment of quaternary carbon were confirmed by HMBC

experiment. The most downfield proton at 8 9.16 (H-l) exhibited 2j

and 3jinteraction with the C-8a (8 136.3), C-8 (8 103.3 ) and C-3 (8

123.2). Another proton at 8 7.92 (H-4) showed hetronuclear

correlation with the C-4a quaternary carbon resonating at 8135.1

and also exhibited connectivity with the C-5 methine at 8104.7. The

-:76:-

H-8 (d 7.45) showed heteronulear connectivity with the C-l, C-8a, C-

7 and C-6 resonating at 8 123.2, 136.3, 146.0 and 142.3 respectively.

The HMBC connectivities are shown around the structure 143 ( Fig.

2.4}

H 7.3] H 7.91

J-J 8.20o.104.6142.3 104.7

123.3103.!

146.0JL. 103.3 ii M

/\,H H 9

O

Fig. 2.4: HMBC assignments of papraline (143).

+•

Nr -H0 4j

P

0-s i

m/z 173 m/z 1726Xo

-HCON

b*m/z 144

m/z 115

Scheme-2.3

9,10-Methylenedioxy Karachine (255)2.2.4

The ethanolic extract of the aerial parts of Fumaria indica was

evaporated and the resulting gum trituraled with chloroform (pH 2.5)

-:77:-

to afford a gummy material (15 gm), which was then to column

chromatography. On elution with chloroform : methanol (99.7:0.3) a

semi-pure substance was obtained as fraction FA-3 (Scheme-2.10,

experimental). This was subjected to preparative thin layer

chromatography on precoated silica gel plates using chloroform :

methanol (99.5:0.5) as the developing solvent system (experimental

section) to afford a pure alkaloid 9,10-methylenedioxy karachine

(255) as colourless amorphous solid (7.5 mg).

r°w4a H'O 5HJ

// e ,P\61 14a

h

-NH

Hb13

\12a V/-(8a

Ha,2<c»3 V,v_/ÿ°>2-*10 J11

o255

The HREIMS of (255) showed the molecular ion at m/z

417.1549 corresponding to the molecular formula C25H23NO5 (calcd.

417.1576) indicating the presence of fifteen double bond equivalents

in the molecule. The exact mass of the molecule (255) obtained by

FAB positive mass spectrometry [248,249] was 418.158 (calcd.

418.1654) indicating the fifteen degree of unsaturations.

-:78:-

The peak at m/z 320.091 C19H14NO4 showed the loss of

C6H9O from the molecule. The loss of 97 a.m.u. corresponds to

C6H9O, or more specifically to 2 molecules of acetone minus the

elements of water. The loss of 97 mass units, from the molecular ion

can occur via cleavage a to the nitrogen atom (C14 to Ce bond)

followed by a retro-Diels-Alder process. The remaining

fragmentation pattern of 255 was distinely similar to the

protoberberine isoquinoline alkaloids. The mass fragmentation

pattern is presented in Scheme-2.4.

mass

The UV spectrum showed strong absorptions at Xmax (MeOH)

(loge) 285 nm (3.942) characteristic for tetrahydro coptlsine (158)

[250].

The 1R spectrum (CHCI3) showed an absorption band at 1710

cm'1 due to the presence of a nonconjugated carbonyl group.

The *H-NMR spectrum (CDCI3, 500 MHz) of (255) showed two

singlets for the C-l and C-4 aromatic protons at 8 6.92, and 8 6.19

respectively, along with two doublets at 8 6.38 (J = 7.3 Hz) and 8 6.47

(7.3 Hz) for C-l1 and C-12 respectively. The methylenedioxy protons

were centered at 8 5.87 dd (2H, Jgem = 5.93 Hz) and 5.80 dd (2H, Jgem

= 3.5 Hz). A 1H double doublet at 8 1.12 (Ja-x = 2.5 Hz, Ja-b = 16.6

Hz) was assigned to Ha while another 1H double doublet centered at

8 2.1 dd (Jb-x = 5.0 Hz, Jb-a = 16.6 Hz) corresponded to Hb, the

upheld singlet at 8 0.82 was assigned to the bridgehead methyl group

attached to C-p. Two sets of 2H doublets at 8 2.38/8 2.41 (Jgem =14.0 Hz) and 8 2.62/2.66 were assigned to two sets of gemenal

-:79-

coupled methylene protons and Joining the carbonyl groups. The

methyline (H-8) proton adjacent to the nitrogen resulted at 5 4.01 q

(J8a = 1.8 Hz, Js.ap = 4.01 Hz). The iH-NMR of 255 is shown in Fig.

2.5.

The 13C-NMR spectrum (CDCI3, 125MHz) indicated that there

were 25 carbon signals. The status of various carbons in the

molecules were made by DEPT experiments. From these experiments

it was clearly that there were seven CH2, two of which were at 5

101.03 and 100.79. Due to its characteristic chemical shifts of

methylenedioxy groups,(substituted at C-2/C-3. C-9/C-10). the three

upheld methylenes at 8 52.97, 55.14 and 53.68 were assigned to C-6,

C-e and C-y carbons respectively, while the other two methylenes (C-5

and C-a) resonating at 8 29.36 and 29.71.

DEPT experiments also indicated that there were four

aromatic methine carbon signals and they all have nearly the same

chemical shift i.e., 8 106.49, 105.04, 108.49 and 110.0 due to the C-

1, C-4, C-ll and C-12 respectively. There were nine quaternary

carbon signals appeared in broadband decoupled 13C-NMR spectrum.

The non-oxygenated quaternary carbons C-4a, C-8a, C-12a and C-

14a resonated at 8 126.44, 129.52, 129.80 and 119.87 respectively.

Two positions in each of the benzene rings were oxygenated and these

quaternary carbons appeared at 8 144.42, 143.64, 146.20 and 146.49

which were designated to the C-2, C-3, C-9 and C-10 carbons.

To establish direct !H/13C connectivites, the HMQC

(Heteronuclear Multiple Quantum Coherence) experiment was

-:80:-

performed. The methyl group resonating at 8 31.38 (C-fi CH3)

showed cross-peak with the proton signal at 8 0.84. The protons at 8

5.87 and 5.80 were found to be cross-linked with carbons resonating

at 8 101.03 and 100.79 assigned to the two methylenedioxys. The

methylene protons of C-5 and C-6 at 8 2.40 and 3.09 exhibited

peaks with carbon signals at 8 29.36 and 52.97 respectively. The C-13

proton (8 3.10) showed heteronuclear interaction with the carbon at 8

35.77 (C-13). The iH/13C connectives of all 25 carbons with their

respective protons. The various carbons resolved with the help of

DEPT experiments are given in Table-2.5.

Table-2.5: 13C-NMR Assignment of 255

cross-

MultiplicityCarbon No. 13C-NMR Chemical Shift (8)

106.49 -CHC-l

C-2 144.42 -C-

143.64 -C-C-3

105.4C-4 -CH

C-4a 126.44 -C-

C-5 29.36 -CH2

C-6 52.97 -CH2

C-8 58.80 -CH

C-9 146.20 -C-

-c-c-10 146.49

-CHC-ll 108.49

C-12 110.0 -CH

129.80 -C-C-12a

35.77 -CHC-13

68.45C14 -C-

119.87 -C-C-14a

O-CH2-O* 101.03 -CH2

81:-

(Table 2.0.) contd.

MultiplicityCarbon No. 13C-NMR Chemical Shift (5)

O-CH2-O* 100.79 -CH2

O

weakC

-CH229.71a - CH2

weakP -c31.38 -CH3P-CH353.68 -CH27 - CH2

55.14 -CH2e - CH2

•Interchangeable values

The presence of incorporated ’’acetone units" in 9,10-

methylenedioxy karachine (255) (like its analogue karachine reported

previously) was established mass, !H- and 13C-NMR spectroscopy

studies.

The HERIMS (m/z 417.154) revealed the molecular

composition C25H23NOs, indicating presence of fifteen double bond

equivalents in the molecule. Four oxygens atoms were accounted for

by the two methylenedioxy groups. Twelve double equivalents are

accounted for by the basic protoberberine skeleton having two

methylenedioxy groups. This indicated that the fifth oxygen might be

present as a ketonic carbonyl and there may be two additional rings

in the molecule. The HRE1MS also showed a peak at m/z 320.091

corresponding to the molecular composition C19H14NO4 due to loss

of (C6H9O) unit from the molecule.

-:82:-

The *H-NMR spectrum of (255) showed a 1H double doublet

at 6 1.12 which was assigned to Ha. Another 1H signal double

doublet 8 2.1 corresponded to Hb. An upheld 3H singlet at 8 0.82 was

assigned to the methyl protons of the bridgehead methyl group

attached to C-(3. The compound 255 was compared with LH-NMR

data of 256 which was already isolated from Befberis aristata [251J.

The comparative proton NMR data of both the compounds

(255 and 256) which are arranged in tabular form Table-2.6 showed

the big difference at ring D of 256. In case of 256 the ring D

contains two methoxyl groups which appeared in *H-NMR at 8 3.82

and 3.77, whereas in compound 255 there were no methoxyl signal

found in !H-NMR spectrum. Instead of disappearance of two methoxy

signals, an additional methylene signal at 8 5.80 was appeared as a

doublet having coupling constant 3.5 Hz.

The 13C-NMR spectrum showed the resonance of the a, P, y

and e carbon at 8 29.71, 8 31.38, 8 53.68, 8 55.14 respectively. The p

quaternary carbon and carbonyl carbons were too weak to be detected

In the 13C-NMR spectrum. While the P-CH3 carbon appeared at 8

31.38. Similarly, the same extra methylene signal was appeared at 8

100.79 in carbon spectrum. The status of this signal was determined

by DEPT experiment.

With the help of above discussion it has been concluded that

an additional ring is formed as methylenedioxy. The formation of this

ring was further confirmed with the help of HRMS showing an extra

-:83:-

degree of unsaturation. The remaining signals are closely matched

with the reported data of 256.

The above evidence confirmed that 255 is a derivative of 256

and named as 9,10-methylenedioxy karachine 255.

Q

O 4

4a H'5

// E6

1 14a5

H<7Y N>L WHr»><G

H,

Hb13

\12a )r/=<8aHa/CH3 \

12\_r OCH3

li

OCH3Karachine (256)

These spectral studies and the close correspondence of the

data with that previously reported by us for karachine (256), let to

the assignments of its structures 9,16-methylenedioxy karachine

(255).

-:84:-

Table-2.6. 1H-NMR chemical shift (5) value of compounds 255and 256

;':iv •-?/-

H. No. 256

6.29 sH-l 6.62 s

6.19 sH-4 6.17, s

2.40 mH-5 2.22 - 2.30 m

3.09 mH-6 3.10 m

4.10 q. J = 4.0, 1.8 Hz)H-8 4.12 q, J = 4.0, 1.8 Hz

6.38 d, J = 7.3 Hz 6.52 d. J = 8.2 HzH-l 1

6.47 d. J = 7.3 HzH-l2 6.55 d, J = 8.2 Hz

3.10 sH-13 3.07 s

2.10 d., J = 16.6, 5.0 Hz 2.08 q, J = 12.6, 1.8 HzH-ab

1.12 dd, J = 16.6, 2.5 Hz 1.11 q. J = 12.6, 1.8 HzH-aa

0.84 s 0.82 s(}-Me

2.38 d, J= 14.6 Hz 2.46 d. J= 14.0 HzH-Y

2.41 d, J = 14.6 Hz 2.46 d, J= 14.0 HzH-Y

2.66 d, J= 14.16 Hz 2.70 d, J = 14.3 HzH-e

2.62 d, J = 14.2 Hz 2.72 d, J = 14.3 HzH-e’

5.80 d. J = 3.5 Hz (OCH2O) 3.82 s. (OMe)

3.77 s. (OMe)

5.87 d. J = 5.9 Hz (OCH2O) 5.82 d, J = 1.5 Hz (OCH2O)

-:86:-

Qr v=?5.87 d. Jgem=5.9Hz 6.19 s2.62

H'2.66do H4ÿ1 14avÿ

2.40 m

\ ;o5II 6> 3.09 m

6.92 s S3.28 s-/-N 4.01 q. Jÿl.SHz. J„b=4.0Hz

2. ldd, Jb)(=5.0Hz. Jha=l 6.6Hz

HH JTHV;3.1 s Hb13

##Ha1.12 dd. Jlut=2.5H2. Jab=16.6Hz

0.84& O

8a12a>—1—

6.47 d. J=7.3Hz /12CH3

Vll

\ JQ>

6.38 d. J=7.3Hz5.8d, Jgem =3.5Hz

Fig. 2.5. of 9,10-methylenedioxy karachine (255).

<„< VNt

H3C

m/z 320 OOm/z 417

I

Om/z 174

m/z 148

Scheme 2.4 Mass fragmentation pattern of 9,10-methylenedioxykarachine (255).

-:86:-

Known Compounds Isolated for the First Time from the

Aerial Parts of Fumaria indica

4-Hydroxy-3-Methoxy Benzaldehyde (Vanillin) (142)2.2.5

The CHCI3 extracts (fraction FN-1, (Scheme-2.8,

Experimental), was chromatographed on a silica gel column, elution

being with pet.ether : acetone (9:1) to afford a fraction FN-1 (Scheme-

2, Experimental). Fraction FN-1 was then subjected to preparative

TLC employing pet.ether : acetone (8.5:1.5) as eluent to give a

colourless powder (6.4 mg).

O

H3CO\3H1

4j6

HO'5

142

The UV (MeOH) spectrum of the compound showing

absorptions at Xmax (MeOH) (log e) 195 (2.962), 204 (3.337), 230

(3.291), 278 (3.114), 308 (3.093) [1051 characteristic for vanillin type

compounds.

The IR (CHCI3) spectrum showed absorptions at 3408 cm'1

(OH), 1670 cm'1 ((conjugated C=0) and 1584 cm1 (C=C).

The 1H-NMR spectrum (CDCI3, 500 MHz) indicated the

presence of four downfleld protons, due to the aldehydic proton at 6

-:87:-

9.87 (s), the two aromatic protons resonating at 8 7.40 and 7.01 (C-

2H, C-5H) and the O-H proton which resonated as a broad singlet at

5 6.17. One 3H singlet at 5 3.94 was due to the methoxy protons

substituted at the C-3 position.

The high resolution mass spectrum afforded the molecular ion

peak at m/z 152.0481 leading to the molecular formula CsHsOs and

indicating five double bond equivalents in the molecule. The

molecular ion peaks was further confirmed by FAB positive mass

spectrometry. The ion at m/z 123.0452 (C7H7O2. calcd. 123.0446)

was due to the loss of CHO group from the molecular ion indicating

the presence of an aldehydic functionality.

The DEPT and broadband decoupled 13C-NMR Spectra

(CDCI3, 100MHz) showed eight carbon atoms (Table 2.7) indicating

the presence of one methoxy, three methine and four quaternary

carbons in the molecule. The methoxy carbon showed a characteristic

signal at 8 56.10. The three downfield methine signals at 8 127.5,

114.4 and 108.8 could be attributed to the C-6, C-5 and C-2 aromatic

methine carbons. A quaternary carbon signal at 8 190.8 was

characteristic for carbonyls carbon. The remaining quaternary carbon

signals at 8 129.11, 140.01 and 51.7 were ascribed to C-l, C-3 and C-

4 respectively, the assignment of quaternary carbons were based on

HMBC spectrum.

The 13C-NMR spectrum (CDC13, 125 MHz) of the compound

showed the presence of only eight carbon atoms in the molecule. The

13C-NMR spectrum is presented in Table-2.7.

-:88:-

Table-2.7. 13C-NMR Assignments of 142

Multiplicity (DEPT )Carbon No. 13C-NMR Chemical Shift (5)

129.11 -C-C-l

-CHC-2 108.8

147.01C-3 -C-

151.7C-4 -C-

C-5 -CH114.4

127.50C-6 -CH

OII 190.8 -C-c

56.10 -OCH3-OCH3

By comparing the spectral data ( UV, IR, MS, 13C-NMR) of

142 with those reported earlier compound 142 was identified as 4-

hydroxy-3-methoxy benzaldehyde (Vanillin).

Cryptopine (61)2.2.6

The CHC13 extract (Fraction FA-4, Scheme-2.10,

Experimental) obtained at pH 2.5 was chromatographed on a silica

gel column which was eluted with increasing polarities of chloroform

: methanol. Elution with chloroform : methanol (9.5:0.5) afforded a

fraction FA-4 (52 mg) (experimental, Scheme-2.10) which was

subjected to preparative thin layer chromatography (TLC) by using

pet.ether : acetone (78:28) as a developing solvent system to afford an

alkaloid as a white crystalline solid (18 mg, 9 x 10’5% yield), mp =156-158 °C named "cryptopine (61)”. The substance give positive test

with DragondrofFs reagent.

-:89:-

54

o. 4a6

3

.CH32 N.

O' 14a 814

1 O'8a .OCH313

912a

1012

OCH3n61

The UV (MeOH) spectrum was characteristic of cryptopine and

protopine [94, 106, 146], showing absorptions at Xmax (MeOH) (log E)

286 (2.801), 206 (3.750) and 193 (3.769) nm. The IR spectrum showed

C-H streching vibration at 2898 cm-1, while the band at 1718 cm'1

indicated the presence of a 0=0 group in the molecule.

The *H-NMR spectrum (CDCI3, 500 MHz) afforded signals for

four downfield aromatic protons. The 1H singlets at 6 7.02 and 6.71

were assigned to the protons C-1H and C-4H respectively. Two others

downfield protons resonated as overlapping double doublets at 8 6.71

and 6.82 was assigned to the C-l1 and C-12 respectively ( Ji1,12 = 7.9

Hz). The assignments of 01 are given in Fig. 2.6.

The C-5 and C-6 methylene protons appeared as two methyl

groug of 2H multiplets, centred at 8 2.11 and 2.83 respectively.

Another 2H multiplet centred at 8 3.75 was assigned to C-13H in the

ten membered flexible ring of cryptopine.

-:90:-

The HREIMS of cryptopine exhibited the molecular ion at m/z

369.1560.(C21H23NO5, calcd. 369.1576)suggesting the presence of ten

degree of unsaturations. The M+ was confirmed by fast atom

bombardment (FAB) mass spectrometiy [240].

Other major peaks appeared at m/z 288, 267, 221, 170, 178

and 148. The fragmentation pattern was similar to that reported for

cryptopine (Scheme-2.5).

CH2H3C<H2(CH3

DN;

H3C<O'

D m/z 148

m/z 369

H3C< H3C<

N\

H3C( CH3 H3C< co o

m/z 221 m/z 178

Scheme-2.5

The 13C-NMR spectrum (CDCI3, 100 MHz) of cryptopine (61)

indicated that there were 21 carbon signals. The multiplicity

assignments were made by DEPT experiments which are presented in

Table-2.7. From these experiments it was clear that there were five

CH2, one of which was at 8 101.27 due to its characteristic chemical

shift, it was apparent that this methylene is that of a methylenedloxy

-:91:

group (substituted at C-2/C-3). The two upfield methylenes at 8

31.76 and 46.48 were assigned to C-5 and C-13 carbons respectively.

while the other two methylenes (C-6 and C-8, present a to nitrogen)

have a downfleld chemical shift, resonating at 8 57.85 and 50.85.

DEPT experiment also indicated that there were four aromatic

methlne carbon signals and they all have nearly the same chemical

shifts i.e.. 8 106.75, 110.54, 108.20 and 110.58 due to the C-l, C-4,

C-ll and 012 respectively. There were nine quaternary carbon

signals including one carbonyl carbon 014, which appeared at 8

194.43. The non-oxygenated quaternary carbons 04a, 08a, 012a

and C-14a resonated at 8 136.20, 129.04, 117.95 and 132.82

respectively. The N-CH3 and two OCH3 carbons resonated at 8 41.51,

56.02 and 56.35 respectively. Two positions in each of the benzene

rings were oxygenated and these quatemaiy carbons appeared at 8

146.41, 148.10, 148.90 which were designated to the 02, 03, 09

and OlO carbons respectively.

On the basis of these spectral studies the compound (61) was

identified as cryptopine.

The one-bond iH/ÿC correlation for 61 were determined on

the basis of HMQC experiments (Tablc-2.8).

-.92:-

Table-2.8. 13C-NMR Assignments of 61

Carbon No. 13C-NMR Chemical Shift (8) :

C-l 106.75 -CH

C-2 146.41 -C-

C-3 148.10 -C-

C-4 110.54 -CH

C-4a 136.2 -C-

C-5 31.76 -CH2

C-6 57.85 -CH2

-CH250.85C-8

C-8a 129.04 -C-

C-9 148.90 -C-

146.09C-10 -C-

c-u 108.20 -CH

C-12 110.58 -CH

C-l2a 117.95 -C-

C-13 46.48 -CH2

C-l4 194.43 -C-

C-14a 132.82 -C-

O-CH2-O 101.27 -CH2

-OCH3 56.02 -OCH3

-OCH3 56.35 -OCH3

N-CH3 41.51 -CH3

-:93:-

6.71 s 2.11 m

O 2.83 m5

< 6l4a 2.21 s

.CH35.95 s2 14a N.1o 3.09 m14

7.02 s a 3.92 s

.OCH313 8<3.75 m

9|12a

3.93s

OCH36.71 t. J=7.9Hz

Fig 2.6. LH-NMR spectral data of cryptopine (61).

(+)-p-Hydxastine (232)2.2.7

The chloroform extract (obtained as shown Scheme-2.10) by

extraction at pH ~2.5 was subjected to column chromatography on a

silica gel column using chloroform : methanol (9 :1) as eluent to

afford fraction FA-5 (Scheme-2.10 Experimental). This fraction was

subjected to preparative TLC using chloroform : methanol (9.7:0.3)

saturated with vapours of ammonia as the developing system to

afford (+) P-hydrastine as a colourless amorphous solid 6.9 mg,

[<X)D25 = +48°. the alkaloid gave a red coloured reaction with

dragendroffs reagent.

The UV (MeOH) spectrum was characteristic of

secophthalideisoquinollnes chromophores (173] showing absorptions

at Xmax (MeOH) (log e) 290 (3.889), 264 (3.655), 218 (4.150) nm. The

IR (CHCI3) spectrum showed an absorption at 1740 cm1 which

indicated the presence of a lactone carbonyl group. Other Intense IR

-:94:-

absorptions were at 1610 (C=C aromatic) and 1130 cm'1 (C-O-C)

[170],

5

O. 43/436

7} 8a N.O H CH38

2'

H9 3‘

Q4'

7'9'6'

OCH35’

o 8‘

OCH3(+)-(}-Hydrastine (232)

The •H-NMR spectrum (CDC13, 500 MHz) Indicated the

presence of four downlleld protons resonating at 5 7.11 d (J = 7.96), 8

6.83 (J = 7.96), 6 6.64 s and 8 6.35 s due to the C-3’H, C-2H, C-5H

and C-8H respectively. Protons of methylenedioxy group, C-1H and

C-9H appeared at 8 6.03, 8 4.07 and 8 5.67 respectively.

The presence of 2-methoxy groups was indicated by the two

3H resonances at 8 3.72 and 8 3.76 which must be located at C-4‘

and C-5’, since the other two aromatic protons (C-2H and C-3H) were

in an ortho relationship. The !H-NMR of 232 are shown in Fig 2.7.

The high resolution electron-impact mass spectrum of (+)-(5-

hydrastine (232) showed the molecular ion at m/z 383.1245. in

agreement with the molecular formula C21H21NO6 (calcd. 383.1368.

-.95:-

indicating the presence of eleven degree of unsaturations in the

molecule. Others peaks were found at m/z 190.0644, 163.0403 and

148.0612 corresponding to the formula CiiHi2N02,CgH7N03 and

C9H8O2 respectively. The corresponding fragments are represented in

Scheme-2.6

<: NS

CH3 CH3H

H-O'

OCH3 OCH3O

OCH3 OCH3m/z 353m/z 383

lO a

o O'CH3

m/z 190+m/z 148O

H2G=°*> CH2

OCH3i

o OCH3m/z 135 m/z 163

Scheme-2.6

The 13C-NMR spectrum (CDCI3, 100 MHz) showed the

presence of 21 carbon resonances in the molecule. The multiplicity

-:96:-

assignments were made by carrying out the DEPT pulse sequence

which are presented in Table-2.9.

Table-2.9. 13C-NMR Assignments of 62

MultiplicityCarbon No. 13C-NMR Chemical Shift (5)

66.30 -CHC-l

50.36 -CHC-3

C-4 41.14 -CH2

126.1 -C-C-4a

107.5 -CHC-5

149.6 -C-C-6

145.45 -C-C-7

106.8 -CHC-8

128.4 -C-C-8a

C-9 80.1 -CH

116.2 -C-C-l’

C-2' 117.6 -CH

C-3' 118.1 -CH

C-4' 150.1 -C-

C-5’ 151.2 -C-

-C-C-6' 140.2

COc=o 167.8

101.5 -CH20-CH2-0

-OCH3 54.8 -OCH3

55.6 -OCH3-OCH3

-N-CH3 44.5 -N-CH3

-:97:-

The C-1 methine carbon and C-3 methylene carbon appeared

at 8 66.30 and 50.36. The downfield chemical shifts of these carbons

were due to the adjacent to nitrogen function. The C-9 methine

carbon appeared at 880.1 due to the acetyl function. The signal at 6

101.5 was assigned to the methylenedioxy group at C-6 and C-7

position. The chemical shifts of the aromatic methine carbons C-5,

C-8, C-2' and C-3' were assignes as 6 107.5, 106.8, 117.6 and 118.1

respectively. DEPT spectrum established that there were eight

quaternary carbons in (+)-|5-hydrastin (232). Out of these the four

oxygenated carbons were C-6, C-7, C-4’ and C-5' which resonated at

8 149.6, 145.45, 150.1 and 151.2 respectively, while the rest of the

carbon atoms appeared at 6 125.12, 128.4, 116.2 and 140.2 due to C-

4a, C-8a, C-l' and C-6’ carbons respectively resonating in broadband

13C-NMR spectrum. The lactonic carbonyl carbon appeared at 8

167.8. The N-CH3 carbon appeared at 6 44.5 while the methoxy

carbons resonated at 5 54.8 and 55.6.

2.40 m6.64 s

P 2.74 m

6.03

2.63 s

CH3o H6.35 s 4.07

6.83 d. J=7.96Hz

5.67*1 7.1) d. J=7.96Hz

Q

3.72

OCH3o 3.76

OCH3

Fig 2.7. iH-NMR spectral data of (+)-P-hydrastine (232).

-:98:-

Corydaldine (147)2.2.8

The CHCI3 extract (pH -8.5) (Scheme-2.9) was

chromatographed on a silica gel (70-230 mesh) column with

increasing polarities of chloroform : methanol (8:2) to afford a

fraction FB-3 (Scheme-2.9, Experimental). Fraction FB-3 was seen by

(6.0 mg) and corydaldine (147) (10 mg) was obtained as a colourless

powder.

H3Ca'"*ÿ 4a 4

3

8aH3CO' H8

oCorydaldine (147)

The UV (MeOH) spectrum was typical of isoquinolone bases

[100,186) showing absorption maxima at \max 295 (2.610), 260

(2.730), 220 (3.240) and 208 (3.182) nm.

The IR spectrum showed an absorption at 3672 cm1 which

indicated the presence of an N-H function. Other intense IR

absorptions were at 1710 (C=0) and 1595 (C=C) cm-1.

-:99:-

EXPERIMENTAL

*

2.3 General Experimental

All chemical and instrumental analyses were performed at the

International Centre for Chemical Sciences, H.E.J. Research

Institute of Chemistry, University of Karachi. All solvents used for

thin layer chromatography and for final elution thereafter were

purchased from E. Merck. For other chromatographic techniques

commercially available solvents were used after distillation at their

respective boiling points except were mentioned. Distilled pet. ether

was collected between 64°C and 68°C. Hydrochloric acid, acetic acid

and ammonium hydroxide were purchased from E. Merck.

Instrumental Details

Physical Constants

All melting points were recorded in glass capillary tubes using

Buchi melting point apparatus. Optical rotations were measured on

JASCO DIP-360 digital polarimeter in chloroform.

Spectroscopy

The UV spectra were recorded in methanol on Shimadzu UV-

240 (Shimadzu Corporation, Kyoto, Japan) spectrophotometers.

The IR spectra were recorded in chloroform on Shimadzu IR-

240 (Shimadzu Corporation, Koyoto, Japan) or (Japan Spectroscopic

Co. Ltd.) instruments.

101:-

The Nuclear Magnetic Resonances (NMR) spectra were

recorded in CDCI3, CD3OD and C5D5N using TMS as on internal

standard on Bruker AM-300 FT NMR, AM-400 FT NMR and AM-500

FT NMR spectrometers.

The 13C-NMR spectra were recorded at 75, 100 and 125 MHz

on Bruker AM-300, Am-400 and Am-500 FT NMR respectively.

Mass spectra were recorded on Finnigan MAT-112 and 312

double focusing mass spectrometers connected to IBM-AT Compatible

PC based system. Peak matching, linked scan, field desorption (FD)

and fast atom bombardment (both +ve and -ve FAB) experiments were

performed on MAT-312 or Jeol HX-110 mass spectrometers. FABMS

were recorded in a glycerol-water (1:1) matrix in the presence of KI.

Accurate mass measurements high resolution electron-impact mass

spectra were recorded on a Jeol-JMS H x 110 mass spectrometer.

Chromatography

Column chromatography was performed with silica gel (E.

Merck, type 60, 70-230 mesh). Precoated silica gel GF-254 preparative

plates (20 x 20 cm 0.2 mm thick) (E. Merck) were used for preparative

thin layer chromatography. The purity of the samples was checked on

the precoated plates.

X-ray Diffraction Studies

All the data were gathered using nicolet R3m/v (now Siemens) four

circle diffractometer with graphite monochromated Cu Ka radiation

(\ = 1.54184 A)

-:102:-

Spray Reagents Used During Detections

Ceric Sulphate

Saturated solution of ceric sulphate in 65% sulfuric acid was

used for detection of terpenes which give a pink/blue colouration

after spraying the spots on TLC plates and upon heating for 10-15

min at 120°C.

Dragendorffs Reagent

A mixture of 25 ml acetic acid, 2.6 gm basic bismuth

carbonate and 7 gm sodium iodide was boiled for a few minutes.

The copious precipitates of sodium acetate were filtered

through a sintered glass filter after about 12 h. 20 ml of the clear

red-brown filtrate was mixed with 80 ml ethyl acetate and 0.5 ml

water was added and the material stored in an amber glass bottle.

This was the stock solution. The spray reagent was prepared by

mixing 10 ml stock solution, 100 ml acetic acid and 240 ml ethyl

acetate. Alkaloids and occasionally a number compounds containing

no nitrogen, appear as orange-coloured spots after spraying.

Iodine

A few iodine crystals were placed in a TLC tank and warmed

for a couple of minutes on a water bath (40-50°C). Spots appeared on

the TLC plate when kept inside the tank for a minute.

103:-

Plant Material2.3.1

The plant material (Fresh aerial parts of Fumaria indica) (40

kg) was collected from Dera Ghazi Khan, Punjab (Pakistan). The

plant was identified by taxonomist, Department of Botany at the

Karachi University, Karachi, where the sample specimen was also

deposited. The plant material was air-dried under shade.

Extraction and Purification2.3.2

The air-dried aerial parts (18 kg) of Fumaria indica were

crushed and soaked in ethanol (40 liters) for seven days and then

filtered. The filtrates were concentrated to give a crude gum (815 g)

which was then fractionated into different fractions by solvent-

solvent extraction (Scheme-2.7). The ethanolic extract was

suspended in distilled water and defeated with pet.ether. The defatted

aqueous layer than extract with chloroform. The chloroform soluble

layer (78 gm) was named "FN". The aqueous layer then acidified with

acetic acid upto pH=2.5, was again extracted with CHCI3. The

chloroform soluble portion (36 gm) was named as "FA". The

remaining aqueous layer was basified with NH4OH at pH=8.5 and

extracted with CHCI3. The chloroform soluble portion (54 gm) was

named "FB". The detailed fractionation procedure is summarized in

Scheme-2.7. These extracts were subjected to column

chromatography and preparative thin-layer chromatography to afford

pure compounds (Scheme-2.8, 2.9, 2.10).

Aerial part of air-dried Fumaria indica(18kg)

Powdered and extracted withethanol (40 litre) and concentratedunder vaccuum

Ethanolic gum(815 gm)

Suspended in distilled water 5.0 litre

Aqueous extract

defatted with pet. ether (40-65°C)

(70 litre)

1IAqueous extract (515 gm) Pet. ether extract

(221 g)Extracted with chloroform(10 litre)

1(Chloroform extract

(78 g)Aqueous extract

Extracted withCHC13 at pH -2.5

( tFN

Aqueous extractChloroform extract(36 g)

Extracted withCHCI3 at pH -8.5

( ]FA

Aqueous layerChloroform extract(54 g)

FB

Scheme-2.7

-:105:-

Chloroform extract(78 g)

FN

Loaded on silica gel column(1500 gm) eluted with pet. ether :acetone (0- 100%)

I 1( Pet ether : acetone(7:3)

Pet. ether : acetone(9:1)

Pet ether : acetone(8:2)

</ia

FN-2FN-1er FN-3Ms 3

FN-2 was subjected topreparative TLC usingsolvent system pet etehr :acetone and NH4OH(8.1:1.5 + 2 drops of NH4OH)

FN-3 was subjected topreparative TLC usingsolvent system pet. ether :acetone (7.5:2.5) as eluent

FN-1 was subjected topreparative TLC usingpet ether : aceonte(8.5:1.5) as eluent

to00

' ftt

Acetyl-3,4,5-trimethyl-3-propene-4-cyclohexane (149)

(fumaileate) 7.0 mg

4,5-Dimethoxy-2-hydroxybenzaldehyde (144)(fumaileal) 7.2 mg

4-Hydroxy-3-methoxybenzaldehyde (142)

(vanillin) 6.4 mg1

Chloroform extract(pH ~8.5) (54 g)

FB

Loaded on silica gel column(1200 gm) eluted with chloroform :methanol (0-100%)

I

I

( IW CHCI3 : MeOHCHCI3 : MeOH(19:1)

CHCI3 : MeOH

U (8:2)

FB-3

oP* (9:1)aao FB-1 FB-2>4 ft

bOFB-2 was subjected topreparative TLC usingsolvent system peL ether :acetone (8:2) as eluent

FB-3 was subjected topreparative TLC usingsolvent system pet ether :acetone (7.5:2.5) as eluent

CO FB-1 was subjected topreparative TLC usingpet ether : aceonte(82:18) as eluent

t1 'Fumaramine (216) (6.0 mg)Corydaldine (147) (10 mg)

Protopine (184) (10.0 mg)jParfumihe (183) (7.0 mg)

Oxysanguinarine (176) (7.5 mg)

Papraline (147)i

I

Chloroform extract(pH -2.5) (36 g)

FA

Loaded on silica gel column(900 gm) eluted with chloroform :methanol (0-100%)

\\ ICHCla : MeOH(9.5:0.5)

CHC13: MeOH(9.8:0.2)

CHCI3: MeOH(99.7:0.3)

CHC)3: MeOH1 f (9.9:0.1)

FA-2 FA-4FA-1 FA-31

FA-2 was subjected topreparative TLC usingsolvent system pet. ether :acetone (7.5:1.5 + 2 dropsdiethyl amine) as eluent

(+)-Adlumine (266)

FA-3 was subjected to

preparative TLC usingsolvent system chloroform :methanol (99.5:0.5) as eluent

FA-4 was subjected topreparative TLC usingpeL ether : aceonte(78:28) as eluent

FA-1 was subjected to

preparative TLC usingpet. ether : aceonte(8.0:2.0) as eluent

0)Otf

Bo Cryptopine (61)

(18 mg)9.10-Methylenedtoxy

karachine (265)(7.5 mg)

Oxyhydrastlne (146)(6 4 mg)

00

10Mo

[CHCI3 : MeOH(9.8:0.2)

FA-5

FA-5 was subjected topreparative TLC usingsolvent system chloroform :methanol (9.7:0.3) as eluent

' '(+)-(!-Hydrastine (232)

(6.5 mg)

Isolation of Fumaileal (144)2.3.3

The fraction FN-2 obtained from the silica gel column was

eluted with pet.ether : acetone (8:2). It was concentrated In a round

bottomed flask to give a gum (70 mg). This material was triturated

with chloroform to give chloroform soluble portion (45.0 mg)

containing one compound along with some impurities. Fraction FN-2

was further purified by preparative TLC on silica gel plates (GF-254,

0.2 mm) when were developed with pet.ether : acetone (8.1:1.5) + two

drops of NH4OH, to give a pure UV active band, fumaileal (144) as an

amorphous solid (7.2 mg, yield 4.00 x 10*5%), (Rf = 0.65).

Spectral Data

la]20 D = 0o (c = 0.258, MeOH)

UV (MeOH), nm (log e): Xmax 250 (2.873).

IR (CHCI3), vmax cm 1: 3340 (OH),1718 (C=0), 1598 (C=C).

EIMS m/z: (rel.int.%): 182 (M+. 100), 167 (22). 53 (14), 139 (12), 111

(12), 93 (15), 65 (18) and 53 (11).

HREIMS m/z: (rel. int. %): 182.0594 (C9H10O4. calcd. 182.0579)

(100), 167.0312 (18), (C8H704, calcd. 167.0344, 153.0549 (10),

(C8H903, calcd. 153.05516), 93.0692 (C7H9. calcd. 93.0704), 53.0412

(8), (C4H5, calcd. 53.0391).

-:I09.-

1H-NMR (CDC13, 400 MHz) 5: 9.8 (1H. s, aldehydic proton), 7.23 (1H.

s, H-3), 7.13 (1H, s,H-6) and 3.69 (6H, s, 2OCH3).

13C-NMR (CDCI3, 100 MHz) 8: See Table-2.3.

Fumaileate (149)2.3.4

The fraction FN-3 was obtained from the silica gel column

(containing CHCI3 extract of Fumaria indica) on elution with pet.

ether : acetone (7:3). This fraction was subjected to preparative TLC

using pet.ether : acetone (7.5:2.5) to afford fumaileate (149) as an

amorphous solid (7.0 mg, yield 3.8 x 10_5%), (Rf = 0.62). This spot of

fumaileate give a positive test on TLC with ceric sulphate spray

reagent after heating.

Spectral Data

[a]25D; +51 (c = 0.0322 CHCI3).

UV (MeOH), nm (log e): Xma* 226 (3.365), 201 (3.169).

IR (CHCI3), vmax cm1: 2912 (C-H), 1718 (C=Q) and 1254 (C-O-C).

HREIMS m/z: (rel. int. %): 222.1610 IC14H22O2. calcd. 222.1619,

(10)), 161.1329 IC12H17, calcd. 161.1330, (5)1, 121.1009 [C9H13,

calcd. 121.1017. (70)1 and 69.0701 [C5H9. calcd. 69.07041.

1H-NMR (CDCI3, 500 MHz): 8 2.03 (3H. s, acetyl methyl), 5.10 (1H,

m, H-l), 1.58 (1H, dd, J2a,2p = 11-75 Hz, J2o,la = 4.0 Hz, H-2a). 1.74

(d. J2a.2p = 11.75, H-20), 2.08 (1H, dd, J6a,6p = 10.5, Jea.ia = 3.5 Hz,

H-6a), 2.40 (1H, d, J6a,63 = 10.5 Hz, H-6p), 6.70 (1H, dq, J7.8 = 13.8

Hz. J8,g = 6.9 Hz, H-8), 1.90 (3H. d, Jg,8 = 6.9 Hz, H-9), 0.99 (3H. s,

H-10), 1.51 (3H, s, H-l1) and 1.15 (3H, s, H-12).

1SC-NMR (CDC13, 125 MHz): See Table-2.4.

Isolation of Papraline ( 143)2.3.5

Papraline (143 ) was isolated from fraction FB-1 (Scheme-

2.9). It was obtained by chromatography on a silica gel column (100

g) on elution with chloroform : methanol (19:1). This fraction when

subjected to TLC using pet.ether : acetone (82:18) afforded a

compound which was identified as papraline (143) after spectroscopic

studies, papraline was obtained as an amorphous solid (5.0 mg) (yield

2.7 x 10'5%) (Rf = 0.54).

Spectral Data

[a]25D = 0° (c = 0.04 MeOH).

,

UV (MeOH), nm (log e): Xmax 200 (2.431, 300 (2.221) and 325 (2.143).

IR (CHC13): vmax cm 1: 2915, 2840 (C-H), 2738 (C=N-) and 1518

(C=C).

EIMS m/z: (rel.int.%): 173 (M+, 100), 172 (82), 149 (10), 134 (14), 126

(5), 115 (25), 104 (5), 88 (11), 87 (12), 62 (32), 58 (16) and 57 (13).

-till:-

HREIMS m/z (rel.int.%): 173.0512 (M+, C10H7NO2. calcd. 173.0714,

100), 172.0368 (CIOH6NC>2, calcd. 172.0398, 82), 148.0471 (C9H802,

calcd. 148.0477, 10), 134.0418 (C8H802, calcd. 134.0376, 14) and

104.0197 (C7H4O, calcd. 104.0252, 5).

1H-NMR (CDCI3, 500 MHz) 8: 9.16 (1H, s, H-l), 8.20 (1H, br.s, H-3),

7.91 (1H, br.s, H-4), 7.31 (1H, s, H-5), 7.45 (1H, s, H-8) and 6.31 (2H,

s, O-CH2-O).

13C-NMR (CDCI3, 100 MHz) 8: See Table-2.5.

Isolation of 9,10-Methylenedioxy Karachine (255)2.3.6

Fraction FA-3 was obtained by column chromatography of the

CHCI3 extract of Fumaria indica obtained at pH 2.5 on a silica gel

column on elution with chloroform : methanol (99.7:0.3). This

fraction when purified by preparative TLC using chloroform :

methanol (99.5:0.5) as the solvent system to afford a pure alkaloid

which was identified after spectroscopic studies as 9,10-

methylenedioxy karachi (7.5 mg, 3.5 x 10-5% yield). This compound

give positive test with dragendroffs reagent.

Spectral Data

[a]25D = +250° (c = 0.204, MeOH)

UV (MeOH), nm (log e): Xmax 220 (3.052), 285 (3.942).

i

IR (CHC13): vmax cm-l; 1710 (C=0). 1605 (C=C) and 1450 (CH2

band).

HREIMS m/z (rel. int. %): 417.1549 (C25H23NO5, calcd. 417.1576,

60), 320.0919 (C19H14NO4, calcd. 320.0922, 100). 174.05481

(CIOH8N02, calcd. 174.0554) and 148.0512 (C9H8O2, calcd. 148.0524.

15).

1H-NMR (CDCI3, 500 MHz) 5: 6.92 (1H, s, H-l), 6.19 (1H, s, H-4),

6.38 (1H, d, J = 7.3 Hz. H-ll), 6.47 (1H, d, J = 7.3 Hz, H-12), 5.87

(2H, dd, Jgem = 5.93 Hz, 0-CH2*-0-), 5.80 (2H, dd, Jgem = 3.57 Hz)

1.12 (1H, dd Ja-x = 2.5 Hz, Hb), 0.82 (3H, s, C-P-CH3), 2.38 (1H, d,

Jgem = 14.0 Hz, H-7*), 2.41 (1H, d, Jgem = 14.0, H-y), 2.62 (1H, d, J =14.16 Hz, He*), 2.66 (1H, d, J = 14.16 Hz. H-e*), 4.0 (1H, q, Jxa = 1.8

Hz, Jxb = 4.0 Hz, H-8), 3.05 (1H. s, H-13), 2.62 - 2.29 (2H, m H-5)

and 3.0 - 3.1 (2H, m, H-6).

(CDCI3, 100 MHz) 5: See Table-2.6.

Isolation of Vanillin (142)2.3.7

Fraction FN-1 ( Scheme-2.5, Experimental) was obtained by

column chromatography of the CHCI3 extract of Fumaria indica on a

silica gel column on elution with pet.ether : acetone (9:1). This

fraction then purified by preparative TLC using pet.ether : acetone

(8.5:1.5) as the solvent system to afford a pure compound which was

identified after spectroscopic studies as 4-Hydroxy-3-methyoxy

benzaldeyhde (142, 6.4 mg, 3.3 x 10*5% yield).

;

Spectral Data

[a]25D = 0° (c = 0.305, MeOH)

UV (MeOH), nm (log e): Xmax 195 (2.962), 204(3.337), 230 (3.291), 278

(3.114), 308 (3.093)

m (CHC13) vmax cm'1: 3408 (OH),1670 (0=0) and1584 (C=C).

HREIMS m/z (rel. int. %): 152.0481 (C8H803), calcd. 152.0451 (45),

123.0452 (C7H7O2, calcd. 123.0467, 20),

1H-NMR (CDCI3, 500 MHz) 6: 9.87 (1H, s, aldlhydlc-H), 7.40(2H, m,

H-2 and H-5), 7.01 (1H, dd, J = 1.8Hz, 8.47Hz, H-6), 6.17 (1H, br.s,

OH) and 3.94 (3H. s, OCH3).

13C-NMR (CDCI3, 125 MHz) 5: See Table-2.7.

Isolation of Cryptopine (61)2.3.8

The chloroform extract obtained at pH ~2.5 was loaded on a

silica gel column (900 gm). This column was eluted with chloroform :

methanol on elution with chloroform : methanol mixtures (9.5:0.5)

fraction FA-4 was obtained. It was concentrated to give a gum (52

mg), which was subjected to preparative thin layer chromatography

using pet.ether : acetone (78:28) as the developing solvent system to

afford an alkaloid which gave red colouration with Dragendorffs

reagent and was identified as cryptopine (61) on the basis of the

spectral data.

Spectral Data

M.P.: 156- 158°C

[a]25D : 0° (c = 0.032. MeOH)

UV (MeOH). nm (log e): Xmax 193. (2.063). 206 (3.750) and 286

(2.801).

IR (CHCI3) vmax cm-*: 2898 (C-H).1718 (OO) and1585 (C=C).

EIMS m/z: (rel.int.%): 369 (M+. 12). 288 (5). 267 (7). 221(22). 190 (9).

178 (30). 170 (15).148 (100), 100 (6). 83 (16), 71 (4). 58 (39) and 55

(8).

HREIMS m/z (rel. int. %): 369.166 (M+, C21H23NO5, calcd. 369.1576,

12). 190.0579 (C10H8NO3, calcd. 190.0658. 9). 179.0658 (C10H11O3.

calcd. 179.0708, 22), 148.0609 (C9H8O2, calcd. 148.024, 100) and

58.0642 (C3H8N, calcd. 58.0418, 39). i

iH-NMR (CDCI3, 400 MHz) 5: 7.02 (1H, s, H-l), 6.71 (1H. s, H-4),

2.11 (2H, m, H-5), 2.83 (2H, m, H-6), 3.09 (2H, m, H-8), 6.71 (2H. t,

H-l1, 12), 3.75 (2H, m. H-13). 5.95 (2H. s, 0-CH2-0), 3.93 (3H, s,

OCH3) and 2.21 (3H, s, N-CH3).

13C-NMR (CDCI3, 100 MHz) 5: See Table-2.9.

(+)-p-Hydrastine (232)2.3.9

The fraction FA-5 obtained from the silica gel column was

eluted with chloroform : methanol (9:1). It was concentrated In a

round bottomed flask on rotary evaporator to give a gum (48 mg).

This material was triturated with chloroform to give chloroform

soluble portion (30 mg) containing one compound along with some

minor compounds. Fraction FA-5 was further purified by preparative

TLC on silica gel plates (GF-254, 0.2 mm) and developed with

chloroform : methanol (9.7:0.3) to give a pure UV active band (+)-p-

hydrastlne (232) as a gum (6.5 mg, yield 3.6 x 10-5%). The spot of

(+)-p-hydrastine (232) give red colour with Dragendorffs reagent.

Spectral Data

[a]25D = +48 (c=.0367 CHC13).

UV (MeOH), nm (log e): Xmax 290 (3.889), 264 (3.655) and 218 (4.150).

IR (CHCI3) vmax cm-1: 1740 (lactone C=0), 1610 (C=C aromatic) and

1130 (C-O-C).

EIMS m/z: (rel.int.%): 383 (M+, 5), 353 (19), 281 (10). 267 (10), 237

(5), 163 (45), 148 (100), 91 (12) and 58 (10).

HREIMS (rel. int. %): 383.1245 (C21H21NO6. calcd. 383 .1368,5)

190.0644 (C11H12NO2. calcd. 190.0868, 20), 163.0403 (C9H7NO3,

calcd. 163.0633. 45) and 148.0612 (C9H8O2, calcd. 148.0524, 100).

1H-NMR (CDC13, 500 MHz) 5: 7.11 (1H, d, J = 7.96 Hz. H-3'), 683

(1H, d, J = 7.96. H-2'), 6.64 (1H. s, H-5), 6.35 (1H. s, H-8), 6.03 (2H.

s. O-CH2-O). 4.07 (1H. d, Ji,9 = 2.9 Hz. H-l), 5.67 (1H. d, J9,l = 2.9

Hz, H-9), 3.72 (3H, s, OCH3), 3.76 (3H, s. OCH3), 2.63 (3H. s, N-

CH3). 2.40 (2H. m. H-4) and 2.74 (2H. m. H-3).

13C-NMR (CDCI3, 100 MHz) 5: See Table-2.9.

2.3.10 Corydaldine (147)

The fraction FB-3 (Scheme-2.9) was concentrated to a gum

(400 mg) and subjected to precoated silica gel plates using pet. ether :

acetone (7.5:2.5) to afford (10 mg) of a pure alkaloid, corydaldine

(147) (5.5 x 10-7% yield).

Spectral Data

[aj25D 0° ( c=.867 MeOH)

UV (MeOH). nm (log e): Xmax 295 (2.610), 260 (2.730), 220 (3.240) and

208 (3.182).

m (CHCI3) vmax cm 1: 3672 (N-H), 1710 (C=0), 1595 (C=C).

HREIMS m/z (rel. int. %): 207.089, (C11H13NO3 calcd. 207.081, 40).

178.068 (C10H10O3. calcd. 178.063), 150.069 (C9H10O2. calcd.

150.064) and 104.026 (C7H4O, calcd. 104.026).

1H-NMR (CDCI3, 500 MHz) 8: 7.55 (1H. s, H-8), 6.67 (1H, s, H-5),

6.30 (1H, br. s, N-H), 3.91 (3H. s, OCH3), 3.t>2 (3H, s, OCH3), 3.55

{2H, t, J3>4 = 4.8 Hz, H-3) and 2.93{2H, t, J4,3 = 4.8 Hz, H-4).

SECTION C

ISOLATION AND STRUCTUREELUCIDATION OF

STEROIDAL LACTONES FROM WITHANIA

SOMNIFERA

3.0 INTRODUCTION

Withania somnifera, also called as the "nightingale", belongs to

the family solanaceae. Withania somnifera L. Dunal occurs over a large

area of tropical and sub-tropical region of the world including

Pakistan, India, Sri-Lanka, Mediterranean regions, Canaries, S.

Africa, Iran, Iraq, Syria and Turkey. Economically this family is veiy

important because it affords important foods and drugs. Plants

belonging to this family include potato, tomato, tobacco, red-pepper

etc. [187].

somnifera (Dunal) is locally known as

"Ashwagandha” or "Asgandh" and it is an ancient Ayurvedic drug

used to treat various body disorders [188, 189].

Withania

The family Solanaceae is divided into 84 genera and 3000

species. Out of these 14 genera and 52 species of this family are

found in Pakistan as herbs and shrubs. Withania somnifera is a

shrub, 30-150 cm long with ascending branches and stellate-

tomentosic shoots. The roots are stout and fleshy, whitish brown in

colour. The leaves are simple, ovate and glabrous, flowers are

greenish yellow, the berries are small, globose, orange-red when

amateur, enclosed in a persistent calyx, seeds are yellow and

reniform [189]

The plant finds extensive use in the indigenous medicinal

systems (Ayurvedic and Unani systems). Its leaves are bitter in taste

and used as an antihelmantic. The infusion is given in fever.

-:119:-

Bruised leaves and fruits are locally applied to tumors and

tubercular glands, carbuncles and ulcers [191, 192].

The roots are used as nutrient and health restorative In

pregnant women and old people. The decoction of the root boiled with

milk and "ghee" Is recommended for curing sterility In women. The

roots are also used in doses of 30 grains in constipation, senile

debility, rheumatism, in cases of general debility, nervous

exhaustion, loss of memory, loss of muscular energy and

spermatorrhoea.

The extract of Withania somnifera was found to have

significant activities. For example the methanolic and chloroform

extracts of the aerial parts of this plant showed significant

antimicrobial activity against gram (+}-bacteria [193]. Withania

somnifera is a plant which showed antitumor activity against

urethane-induced lung adenomas in adult male albino mice by

inducing a state of nonspecific increase in resistance and

immunostimulant properties [193, 194].The aqueous extract of the

Withania somnifera .when administered to a dog, showed a slight

soporific action followed by a complete return to normality [195].

The plant also showed biphasic activity in membrane

stabilization when fresh sheep erythrocytes (SRBC) were subjected to

hypotoxic and heat strains [197]. The fruit is diuretic and its

poisonous seeds have shown mild coagulating effects on milk. The

plant has alkaloids with sedative properties [198].The root extract of

Withania somnifera contains an ingredient which has a GABA-:

mimetic activity [199J.The antinflammatory activity and protective

effect against CCLj-induced hepatotoxicity of the alcoholic extract of

leaves of W. sorrmifera have also been assessed. The extract (at lg/kg

dose) was found to be as active as 50 mg/kg of phenylbutazone and

10 mg/kg of hydrocortisone [200].

The fruits of this plant contain a high proportion of free

amino acids as evident by a positive ninhydrin color reaction. The

presence of these amino acids may be explained by the fact that the

proteolytic enzyme chymase is present in the berries of this plant

[201]. The tubers of Withania somnifera are used in psoriasis

bronchitis, ulcers and scabies [198].

Phytochemical studies on this plant have led to the isolation

of a number of steroidal lactones (withanolldes). Withaferin A (306}

is the most important withanolide isolated so far. It has been

receiving a good deal of attention because of its antibiotic and

antitumor activities. Withaferin-A (306), in concentrations of 10

pg/ml, inhibited the growth of various gram-positive bacteria, acid

fast and aerobic bacilli and pathogenic fungi, but was inactive

against gram-negative organisms and anaerobes. It partially inhibited

the activity of glucose-6-phosphatedehydrogenase-A [202].

,CHaOH

H

V.O' o

ST" HO

iH

O’Withaferin-A (250)OH

The reaction between the antibiotic and glutathione in

Bacillus subtiUls results in the inactivation of the antibiotic activity,

indicating that its carbonyl group is largely responsible for its

antibacterial activity of withaferin-A (306) and this enhancement is

reversed by EDTA [247J.

Withanolide-D (267) was found to active against mouse

leukaemia L5178Y cells [2031.

OH

\ o'H

O

SV-HO

IH

O* Wlthanollde-D (257)OH

-:122:-

Several withanolides are Insect antifeedants e.g. withanolide-

E (258) is a potent antifeedant for larvae of Spodoptera lithorates

[204].

iOH

V I/O oHOH

OH

IH OH

O'WlthanoUdes-E (258)

Withanolide-E (258) also showed immunosuppressive activity

on human p and T-lymphocytes, as well as on mice thymocytes [205].

4p-Hydroxywithanolide E showed a considerable long lasting and

enhanced activity against L-1210 leukemia [206]

3.1 Biosynthesis of Withanolides

Withanolides are naturally occurring C-28 steroidal lactones

having an ergostane skeleton in which C-22 and C-26 have been

appropriately oxidized in order to from a 8 lactone ring. The C28

sterols such as compesterol, 24-methyl-cholesta-5,24-diene-3P-ol and

24-methylene cholesterol are regarded as possible steroidal biogenetic

precursor of withanolides. assuming that withanolide biosynthesis

does not diverge from the major sterol biosynthetic pathway before

the A-desmethyl sterol stage. The possible sterol 24-methylene

-:123 -

cholesterol (284) and compesterol were regarded as precursors of

withanolides. This was confirmed by labelling experiments [207].

oo o

H3G— c— SCoA

H3G— C— CH2— C— SCoA

Acetoacetyl CoA (260)cfI oH2C=C— SCoA

/ H3C— C— SCoA259

\ CoA — SH

fHoG— C— SEnz

IHgO— C OH

H2G— C— SCoAEnzSH

H30~C OHI I

H20— COOH H2O— COOH

NADPHr2623-Hydroxy-3-methyl-glutaryl-SCoA (261)

V NADP+OH

I NADPH NADP+H2G— CH— SEnz H2G— CH2OH

H3O— C OHV

__/I

H3»— C OHI I

H20— COOH H2G— COOH

Mevalonic acid (264)263

Scheme-3.1

The biosynthesis of withanolides was first investigated by

Goodwin and co-workers (208] by administration of [28-3H]-24-

methylenecholesterol to young leaves of Withania somnifera, either

directly or via the stem. The second procedure gave a significantly

higher degree of incorporation.

Squalene (275), a linear C-30 compound which was

discovered in 1916, is now known to be the key precursor of

124:-

cholesterol (284). The biosynthesis of squalene (275) starts with

acetyl CoA (259). Two units of acetyl CoA (259) combine to form

acetoacetyl CoA (260) which then produces 3-hydroxy-3-methyl

glutaryl-SCoA [HMG-SCoA] (261). The (HMG-SCoA] (261) on

reduction with NADPH gives rise to the hemithioacetal (263) which

is converted into mevalonic acid (264) after reduction with NADPH

[209] Scheme-3.1.

Only the JR-form of mevalonic acid is utilized by the living

system for producing terpenes, while the S-form is metabolically

inert.

H2O— CHJJOH H2G— CH2OPPATP ADPI IHgO— C OH

H20— COOH

H30—C OH

H20— COOH

265264

2ATP

V\ ADP

H2rOPPHaO-CHÿOPP C02+ P

I IfH3O—1? H3O—

by— OH

CH2OH3-Isopentenyl pyrophosphate UPP) (267)

H20— jj:— Oÿ-H

OCH— CH2— OPPHgO—C

3-Phospho-5-pyrophosphatemevalonic acid (266)

IIsomeraseCH3

3.3-Dlmethylallyl pyrophosphate (268)

Scheme-3.2

The mevalonic acid (264) is phosphorylated in the presence of

ATP to yield mevalonic -5 - phosphate (265) which is then

decarboxylated and dehydrated to yield isopentenyl pyrophosphate

(IPP, 267) by the route shown in Scheme-3.2, IPP (267) is then

isomerized in the presence of an enzyme "isomerase" to produce

dimethyl allyl pyrophosphate (DMAPP, 268, Scheme-3.2).

IPP (267) can condense in a head-to-tail manner with DMAPP

(267) by formally a combined SN2-E2 process to yield geranyl

pyrophosphate (GPP, 270, Scheme-3.3).

OPP

OPPH

1NPP (269)

266

OPP

GPP (270)

Scheme-3.3

Condensation of GPP (270) with IPP (267) gives famesyl

pyrosphosphate (FPP, 271). FPP is the biosynthetic precursor

sesquiterpenes and diterpenes [210].

-:126:-

The coupling of two FPP (271) units in a head-to-head

manner, produces presqualene (272) which on rearrangement gives

rise to squalene (275, Scheme-3.4).

.OPP

CHaOPP CH3<>CH3oOPP H

CH3

CH3CH3

GPP (270)FPP (271)

+

OPPCH3PPO, head-to-head

IWl]

WÿCH3CH3%ÿCH3

CH3CH3 FPP (271)CH3 Enz CH3

CH3 FPP

Presqualene (272)

CH3PPO.

<n,CH3

CH3CH3273

CH31.2 shift

CH3 CH3

,CH3 ,CH3

nCH3 CH3CH3 CH31©©CH3 CH3NADPH NADPCH3 CH3

CH3H3< H3« CH3274Squalene (270)

Scheme-3.4

The squalene thus formed gets oxidized by atmospheric oxygen

catalyzed by NADPH-linked oxidize to form squalene 2,3-epoxide

(276), which then undergoes cyclization to yield lanosterol (278,

Scheme-3.5).

oSqualene (275)

(O)CH3

CH3

CH3CH3

O* SquaIene-2.3-epoxide (270)

IH3< CH3

f CH3I

VH CH3

HO* 277

|H3<CH3H3<

CH3

CHalCH3

CH3

CH3

HO*’CH3 Lanosterol (278)

Schcmc-3.5H3<

-.128:-

H3< CHJ H3< CÿJ

CH3.H 1 :H3LHCH3 CH3

cn3cn3 14 14

10}

X,CH3 CHO

HO4 HO*H 279’CH3H3< ’CH3

Lanoaterol (278)

j101H3< CHJ H3<

CH3L*HCH3 CH3

CH3 OHb

iM H COOH

HO’ HO'281H 280

H3C CH3H3C CH3 IHI

H3( CHI H3< CH>

.H :H3L-HIsCH3 CH3

CHa CHa

I DemeLhylaUonalC-4H H 283

HO’ HO'

1H3C CHÿ 282

H3( CK,

OH3I.HCH3

CHa

H

HO'Cholesterol (284)

Scheme-3.6

The bio-converslon of lanosterol (278) to cholesterol (284)

involves a number of metabolic changes which occur on a cellular

level and which are not interdependent. The sequence of reactions

and intermediates may also differ slightly among different organisms

(Scheme-3.6). Addition of the methylene group at C-24 may occur

:

-:129:-

with S-adenosyl methionine to form 24-methylene cholesterol, (287)

which Is a possible precursor of the withanolides (Scheme-3.7) (211).

H

H

HO*Cholesterol (284)

/H3C—S\

S-Adenosyl methionine

©/

HaT/NUH

IH H 285

H(

CHa— H

CH3 H

CH3

H H 286

HO*CH2

CUa

CH3 H

CH3

CH3

H

HO*24-Methylene cholesterol (287)

Scheme-3.7

130:-

CH2OH

HH3CH3C,

IO]CH3 CH3

CH3CH3

IH

288HO'HO*

24-Methylene cholesterol (287)[O] CH3loi0°H CH3

Hlr CH2OHH3(H3GV OHOHCH3LHHCH3

CH3CH3

1I HH292289

HO1HO'

HCH3

HCH3

COHH3H3Cÿ

CH3OHO'

CH3L*HH

CH3CH3

IH

HO1 293Hi 290

CH3CH3(O)

CH3CH3

H3C,H3C. V.V oO' OHCH3OHs

10)CH3 CH3

iH

HO' 294Scheme-3.8

131:-

24-Methylene cholesterol (287) Is actually one of the

intermediates leading to the 6-lactone (Scheme-3.7). In each case

(288) should undergo C-22 hydroxylation to (289), a well known

reaction in the steroidal biosynthetic pathway (212).

Two alternative hypotheses for withanolide biosynthesis were

mentioned in the literature (Scheme-3.8). The first involves

cyclization of (289) to (290) and then oxidation to (294) while the

second involves oxidation of the allylic isomer followed by cyclization

to form the lactol (191), present in a number of withanolides.

Further oxidation can then lead to the 6-lactone (194).

-:132:-

RESULTS AND DISCUSSION3.2

3.2.1 Isolation of Withanone (295)

The methanolic extract of aerial parts of Withania somniferawas evaporated under vacuum to a gum. The material was partitioned

between distilled water and pet.ether followed by removal of pet. ether

and extraction with chloroform. The chloroform soluble extract

(Scheme-3.12) was loaded on a silica gel column (mesh 70-230

ASTM) and eluted with pet.ether : chloroform. The fraction (WF-1)

was thus obtained on elution with pet.ether : chloroform (2:8). This

fraction (WF-1) was then subjected to column chromatography on

silica gel (mesh 70-230 ASTM) and the column was eluted with

pet.ether : chloroform (2:8) to afford fractions WSF-1 and WSF-2.

The fraction WSF-2 was subjected to preprative TLC elution having

with pet.ether : chloroform (2:8) , to afford a pure compound,

withanone (295) as a colourless amorphous solid (6.5 mg, yield 1.8 x

lO'7)

28

CH3

!4 CH2OH23 25

21 1,22 126

20 i O O18 20 s

CH3 HH

17n X12N

\\ iU'-H13II I "Is I 14

16

15

2

3

Withanone (205)H

-:133:-

The IR spectrum of withanone (295) exhibited bands at 1660

cm-1 due to a, (i-unsaturated lactone, 1690 cm'1 (non conjugated

ketone) and 3340 cm-1 for the hydroxyl group in the molecule [214].

The UV spectrum of withanone (295) showed absorption

maximum at 215 nm characteristic of a six membered a, (3-

unsaturated lactone chromophore [214].

The iH-NMR spectrum (400 MHz, CDCI3) of withanone (295)

showed three singlets each integrating for three protons at 8 0.81,

1.25 and 2.03 which were assigned to the protons of the tertiary

methyl groups. A doublet at 8 1.03 (J21.20 = 7.0 Hz) was due to the

protons of the C-21 secondary methyl group. Two AB doublets at 8

4.31 and 4.40 (J27a,27(l = 10.4 Hz) were due to the gleminally coupled

C-27 hydroxy methylenic protons. A downfield doublet of double

doublets centered at 8 4.65 (J22.20 = 13.3 Hz, J22,23(3 = 3.5 Hz, J22.23a

= 3.5 Hz) was assigned to the C-22 methine proton geminal to the

lactone oxygen. The !H-NMR assignments of (295) are given in

Fig.3.1

The high resolution electron impact mass spectrum afforded

the molecular ion at m/z 456.2801 establishing the molecular

formula as C28H40O5 (calcd. 456.2875 ) indicating nine degrees of

unsaturation in the molecule. The molecular ion was further

confirmed by +ve fabms.

TheiH-iH long range shift correlations were determined by

recording a series of HOHAHA (TOCSY) spectra with variable delays

134:-

(20, 60, 100 ms). The C-21 methine proton displayed interactions

with the C-22 methine and with the C-23a and C-23P protons. C-

22H also exhibited long-range couplings with the C-21 methyl

protons. The C-28 methyl protons showed homoallylic coupling with

the C-27 methylene protons in the HOHAHA spectra.The HOHAHA

interactions of (295) are presented in Flg.3.2

The COSY-450 spectrum of withanone (295) revealed the

presence of several spin systems in the molecule. One of the spin

systems "A" starts with the C-2 methylene protons at 5 1.5 (m) and 5

2.1 (m) which showed connectivity with the C-3 methylene protons (5

1.62 and 1.9). The C-2 and C-3 methylene protons also showed cross-

peaks with the C-4 methylene protons (5 1.15 and 51.31). This set of

3 adjacent methylenes constitutes spin system "A" Fig. No. I.

1.5

,,if L1.62 m H

1.9 m

V.2

4

H / '''H H

t1.31 m 1.15

Spin system "A"

Fig. No. I

Another larger spin system ”B" starts with the vinylic proton

at 5 5.45. This proton showed interactions in the HOHAHA spectra

with the C-7 methylene protons (5 1.6 and 1.9 ). The vinylic proton

also displayed a long-rang coupling with H-8 methine proton at 5

2.21 and H-4 methylene protons at (8 1.15 and 1.31). On the other

hand the H-9 methine proton displayed long-rang interaction with H-

8 methine proton and H-ll methylene protons. The H-7 methylene

proton resonating at (8 1.60, 1.91) also showed interaction with H-9

and H-6 methine protons respectively, The C-12 protons (81.51, 1.40)

did not show any Interaction in the COSY-450 spectrum except with

the C-ll protons, indicating that the H-12 methylene carbon Is

attached to a quaternary carbon (Fig. No. II).

H.x|

Hr>.H

K3Spin system "B"

Fig. No. n

The broad band decoupled 13C-NMR spectrum (CDCI3, 125

MHz) showed resonances for all twenty eight carbon atoms in the

molecule, The DEPT spectrum showing signals for the C-2, C-3 and

C-4 methylene carbons at 8 32.87, 26.25 and 31.06 respectively.

The HMQC spectrum showed that these carbon atoms were

directly attached to the C-2 protons (8 1.5 and 2.1), C-3 protons (8

1.62 and 1.9) and C-4 protons (8 1.15 and 1.31) respectively which

-:136:-

are presented in Table 3.1.The HMQC spectrum also showed the

following respective carbon - proton- one-bond connectives 122.8 (8

5.45 m). 36.52 (5 1.6 and 1.9 m), 42.0 (8 2.21 m), 32.2 8 1.9 m), 22.26

(8 1.41 and 1.45 m) and 23.67 (8 1.4 and 1.5 m) for the C-6, C-7, C-8,

C-9, C-l1 and C-12 carbons and the corresponding attached protons.

The DEPT spectrum indicated that there were four methyls,

ten methylene, six methine and hence seven quaternary carbons in

the molecule [215], This further confirmed that there were 28 carbons

in the molecule. Downfield signals at 8 167.1 and 8 214.7 were

assigned to the a, p-unsaturated lactonic (C-26) and ketonic (C-3)

carbonyls respectively. The remaining four downfield carbons which

resonated at 8 125.1 (C), 8 154.3 (C), 8 122.8 (CH) and 8 146.5 (C)

were assigned to the C-25, C-24, C-6 and C-5 carbons respectively.

The 3H signals at 8 9.39, 14.9, 9.28 and 19.98 were assigned to the

C-l9, C-18, C-21 and C-28 methyls respectively. The chemical shift

assignments of various carbons of compound (295) are presented in

Fig.3.5

The third spin system "C" (Fig. No. in) starts from the C-17

methine proton centered at 8 1.56 which showed interactions in the

HOHAHA spectra with the C-l6 methylene protons (8 1.45 and 1.51

m) as well as with the C-15 methylene protons (8 1.67 and 1.95 m).

The C-17 methine proton also showed connectivity with the C-20

methine proton (8 2.0 m). This methine proton showed coupling with

the C-21 methyl protons centered at 8 1.01 and with the C-22

methine proton at 8 4.5. These protons were further coupled with the

C-23 methylene protons at 8 2.5 and 2.3 m.

-:137:-

[22

Spin system "C"

Fig. No. Ill

The long-range !H/13C interactions in withanone (29B) were

determined by the HMBC experiment. Some of the couplings were

between C-6 (8 5.45) with the C-4 methylene protons(8 31.0) as well

as between C-4 (8 31.0) with the C-6 proton (8 5.45) hereby

interconnecting the spin system "A" and "B". Long-range coupling of

the C-9 methine proton (8 1.9) was also seen with the C-19 methyl

carbon (8 9.39) and with the C-l carbonyl carbon (8 214.7). The C-2

methylene carbon (8 32.87) also showed long-rang connectivity with

the C-l carbonyl carbon (8 214.7). The C-19 methyl protons (8 1.25)

showed connectivity with the C-10 quaternary carbon . On the basis

of these interactions it was possible to combine fragments "A" and

"B" as shown in Fig. No. IV

-:138:-

H Hn/.<rs o

.. Ill!Jr i

+2 9

H"43 H10 f

I 4 JL 7tvH'

3a H123b

n ft!*'CH3

S'H.9

2 10 8H‘

,3 75

6

[A+B]

Fig. No. IV

The structure of another part "C" of the molecule was deduced

as follows: Long-range iH/ÿC correlations of the 015 methylene

protons (8 1.67, 1.95m) with the C-14 oxygen-bearing quatemaiy

carbon (8 85.09) and with the C-8 methine carbon (8 42.0) were seen

in the HMBC spectrums. This region also showed long-range coupling

of the C-17 methine proton (8 1.56) with the 013 quaternary carbon

(8 48.04) as well as with the C-18 methyl carbon (8 14.90). The C-12

methylene protons in fragment "B" also showed connectivity with the

013 quaternary carbon (8 48.04), C-18 methyl and (8 14.90) the C-17

methine carbon (8 50.6). Similarly, the C-18 methyl protons showed

coupling with the C-13 quaternary carbon (8 48.04). The combination

of fragments (A +B) with ”C" led to a larger structural fragment "D"

shown in Fig. V.

-:139:-

12

HJ1- Idri CHJ” hr H,12

*82 10 ft

fÿi<+

.3 75

6

H3d

i3c H

4ÿCH3J>H

H3

12O 19

151

2 10H

8OH

3 75

6 3e

Fig. No. V

The HMBC interactions also showed connectivity of the C-22

methylene protons (8 4.5) with the lactone carbonyl carbon (8 167.1)

and with the C-24 quaternary carbon (8 154.3). The C-28 methyl

protons (8 2.03) also gave HMBC interactions with the C-24

quaternary carbon. On the basis of these HMBC interactions the

proposed structure (295 ) was deduced for withanone Fig. 3.4. The relative

configuration of 295 is given bellow. CH3

,CH2OH

H3Gÿ.

fH3UHo- o

oCHa

OH

205

H

-:140:-

The El mass spectrum of withanone (295) showed the

molecular ion at m/z 456. The exact mass m/z 456.2801 agreed with

the molecular formula (C28H40O5, calcd. 456.2875). The ion at m/z

438.2761 (C28H3804, calcd. 438.2769) was due to the loss of a water

molecule from the molecular ion. The base peak at m/z 141.0561

arose by cleavage of lactone ring. The mass fragmentation patteren of

withanone (295) is shown in Scheme-3.2.

Stereochemical Assignments

(3-Stereochemistry of the 18 and 19 methyls were deduced due

to the fact that withanolides are biosynthesised from

cholesterol with (3-oriented 18 and 19 methyls.

1.

2 The a-stereochemistry of the 14-OH was assigned based on

the reason that ring C/D is tran-fused in cholesterol (a

biogenetic precursor of the withanolides).

The (3-orientation of C-17 side chain was assigned due to the

biogenetic consideration.

3

Hence, we can draw the complete structure of wihanone

(295) with stereochemistry. The COSY-450 spectrum exhibited

coupling interactions which were found to be in full agreement with

the assigned structure (295) are shown in Fig. 3.5.

141:-

5 2.03

CH35 2.30 m

8 2.51 m 9CH2-OH5 4.316 4.40

HJ2i.22=70Hz 6 2.0 m

8 1.01 d HH3c,

H

8 1.42 m \

/W5 1.51 m

5 1.41 m H5 1.45 m 9

V

' oH 5 4.5 ddd J22.20=13-3H2

•ÿ’’6 1.56 mH J22 23(J='ÿ*5HzJ21 23a=ÿ*ÿÿzP 5 1.25 s

CH3H5 2.21 m5 1.5m

6 1,45 mHr

H8 2.1mH 5 1.51 mH

::= 1 HOH H

5 1.62 m zHH 6 1.95 m

8 1.9 m 8 1.67 m

H5 1.9 m

H 6 1.6 mHH

H8 1.15 m 8 1,9 rn5 1.31 m H8 5.45 m

Fig. 3.1. iH-NMR assignments of withanone (295).

8 2.03

CH3y/ 8 2.30 m

5 2.51 m9:! CH2-OH

'*8 4.318 4.40

H> N8 2.0 m

H"-. y- 6 1.51 m g 1.42 my 5 1.41 m H

(h 1.45 m HA?0ÿÿ0\

H! 8 4.5 ddd

pj 5 1.56 m'

H .-•*

Q 8 1.25

CH35 2.21 myr 5 1.5 m

5 2.1m 9 H,

H H 6 1.5 m .I I I"'/’5 1.62 m

HH OH 5 1.67 m

8 1.95 m /C8 1.9 mlV

••18 1.9 m "HH 5 1.6 mH'"

8 1.15 mH

H 5 1.9 m5 1.31 m H. * 5 5,45 m

Fig. 3.2. HOHAHA interactions of withanone (295).

-:142:-

8 2.03

CH3y/ 8 2.30 m

6 2.51 m8 4.318 4.40

CH2-OH\ 8 2.0 m

r

__H>

\ /ÿ8 151 m 8 1.42 m \

<V 8 1.41 m H/s 1.45 m H \ /MS

yH 8 L56

"'H 8 1.5 m .

H

"D8 2.21 myS 8 1.5 m

8 2.1m H8 1.25

CH3| /H-iH

’6 1.62 m

OH HH, H 8 1.67 m

s8 1.95 m

1.9 m8 1.9 m

HH 8 1.6 mH H

H8 1.15 m 8 1.9 m8 1.31 m H-' 8 5.45 m

Fig. 3.3. COSY interactions of wlthanone (295).

5r2H2.3

2.5 m y 57.4

CH2-OHH

18.34 4.318 1.0 8 4.4'

HaS., 79.3 167.1

JVH VO

H HS4.542.6H % CH3£H,

9 \ 8 1.25

\CH3>H8 1.5 82.21 m 48.0

H8 2.1:\

85.H HI 36.58214.7 42ÿ0:53.6 HH,. H OH8 1.67 mH46.3

•*»# 5 1.95 m'"HHo--'H

iL HV

Fig. 3.4. HMBC Interactions of wlthanone (295).

6 19.98

CH36 57.4

CH2-OH6 125.16 38.34

6 9.28 X_T

H3C...6 14.9

CH3

6 79.3 6 167.1

6 42.6' U UH6 23.67

6 5.06

Q 6 9.39 6 22.26CHgL H

5 48.046 23.67

6 85.096 214.7

6 42.0\5 32.87 6 36.526 53.67

H OH6 26.25xTvÿ 6 36.52

631.06 6 122.8

Fig. 3.5. 13C-NMR assignments of withanone (295).

The perspective view of withanone(295) is presented in Fig.

3.6 [217].

21

,.CH3 CH3H 26'

18

CH3 a*HOOH£H3 Ji 13

.1712 H &2HO A®5

8 1610 [92 14NOH HH I H O743 5

6

H

H

Fig. 3.6. The perspective view of withanone (295).

-:144>

CH3

CH2-OH

H3G..V r°- oCH3 CH3

o CH2-OHCH3 H

TH O' o

m/z 438 m/z 141& n\°1

ACH3

CH2-OH

HHac...•v

r°- oCH3

sr»HO

CH3

1 OH

m/z 456<C.

CH3OH

n CH2-OHCH3 to§O

H;.CH3 3 O' o

m/z 138 m/z 167mIV

CH3

oCH3 H

TH OH

m/z 288

VScheme-3.9. Mass fragmentation pattern of withanone (295).

145:-

Table-3.1. HMQC Assignments of Withanone (295)

No 6 13C8 H

C-l 214.7

C-2 1.5, 2.1 M 32.87

C-3 1.62, 1.90 M 26.5

C-4 1.15, 1.31 M 31.06

C-5 146.5

122.8C-6 5.45 m

C-7 1.60, 1.91 m 36.52

2.21 m 42.0C-8

1.9 m 32.2C-9

C-10 53.67

22.26C-ll 1.41, 1.45 m

C-12 1.40, 1.51 m 23.67

C-13 48.04

85.09C-14

C-15 1.67, 1.95 m 36.52

C-l6 1.45, 1.51 m 23.67

1.56 mC-l7 50.6

C-18 0.81 s 14.90

C-19 1.25s 9.39

2.00 mC-20 42.6

C-21 1.01 d 9.28

4.5 dddC-22 79.3

C-23 2.3, 2.5 m 38.34

154.3C-24

C-25 125.1

C-26 167.1

4.31, 4.40 d 57.4CT27

C-28 2.03 s 19.98

146'.'

Quresimine-A (296)3.2.2

The fraction WF-4 (Scheme-3.12, Experimental) was obtained

on eluting the silica gel column loaded with defatted CHCI3 extract

of Withania somnifera with chloroform : methanol (9.5:0.5). This

fraction was subjected to preparative TLC and elution was carried out

with pet. ether : chloroform (2:8) + 3 drops of diethyl amine. This

yielded quresimine-A as a colourless amorphous solid (7.5 mg) yield

2.08 x 10-5%.

28

CH327

24l CH2-OH23 25

21 H

H3CV18 20 =

26

‘ooCH3 H

17

12o 1611 1319

CH3 H159 14

I1102 8

H H H5 7

6Hgcor

o' HOH

Quresimine-A (296)

The UV absorption maximum at 214 nm was characteristic

and an a,P-unsaturated lactone chromophore [218).

147:-

The IR spectrum displayed absorptions at 1683 cm-1 for a, P-unsaturated lactone, at 3455 cm1 for (O-H) and 1590 cm1 (C=C)

[219].

The high resolution mass spectrum of 296 showed the

molecular ion peak at m/z 502.2910 corresponding to the molecular

formula C29H42O7 which indicated the presence of nine double bond

equivalents in the molecule. The peaks appearing at m/z 484.2804

and 470.2667 showed the loss of water and methylene group

respectively. The peak which appeared at m/z 452.2530 again

exhibited the loss of water molecule. The peak at m/z 387.2522

(C24H35O4) and 86 (C4H6O2) resulted due to the cleavage of C-l/C-4

bond which further indicated that four oxygens were present in major

portion. The mass fragment at m/z 334.1979(C2oH3oO<i) formed by

the cleavage of ring C was indicative of the presence of four oxygen

functions and the remaining fragment appeared at m/z 169.0810

(C9H13O3) and indicated the presence of a six-membered lactone

substituent at the C-20 side chain, the prominent peak at m/z

141.571 (C7H9O3) was originated by cleavage of the C-20/C-22

bonds. The complete mass fragmentation is presented in Scheme-

3.10.

The 1H-NMR spectrum (300 MHz, CDCI3) of 296 Fig. 3.7,

showed three 3H singlets for the quaternary methyls at 6 0.65, 5 1.29

and 5 2.02 assigned to C-18, C-19 and C-28 protons respectively,

while a 3H doublet at 5 0.98 (J = 6.6 Hz) was due to the C-21

secondary methyl protons. A doublet of double doublets appeared at 8

4.41 (J1 J2 = 4.46. J3 = 15.2 Hz) and was assigned to the C-22 proton

-:148:

of the lactone moiety [220]. Two AB doublets at 8 4.36 and 8 4.38

(J27a,27(}=13.5 Hz.) centred due to the C-27 methylenic protons. A

broad 1H signal at 8 3.20 was assigned to the C-6a proton of oxirane

ring. A 1H doublet at 8 3.48 (J = 3.18 Hz) was due to the C-4a

proton. The multlplet for the C-3a proton resonating at 8 3.69 was

found to be coupled with the C-4a methine proton while the C-2

methylene protons appeared as as multiplets at 8 2.58 and 8 3.00. A

three proton singlet resonating at 8 3.33 was due to the C-3 methoxy

group.

The COSY-450 spectrum of 296 served to establish the

proton-proton connectivities which were found to be very helpful in

deducing the structure (296). The signal at 8 3.69 (C-3a proton)

showed strong connectivities with the protons at 8 2.58 and 8 3.00

(C-2 methylene protons) as well as with the protons at 8 3.48 (C-4a

methine proton). The proton at 8 3.20 (C-6a) protons showed

connectivities with those at 8 1.30 and 8 2.15 (C-7 methylene

protons).

O

fsoodd II]2.58 dd1 -TH

H

H3CO<

OH

Spin System "A”

Fig. No. I

The COSY-450 spectrum was used to explore the spin systems

in the molecule. The first spin system "A" starts with the C-4

methine proton (8 3.48) which showed connectivities with another

downfield C-3 methine proton (8 3.09). This latter methine was

further coupled with the C-2 methylene protons (8 2.58 and 8 3.00)

shown in Spin System "A" Fig. No. I.

Another spins systems "B" starts with the C-6 proton of the

oxirane ring which resonated at 8 3.20 and was coupled with the C-7

methylene protons (8 1.30 and 8 2.15). These methylene (C-7) protons

showed further connectivities with the C-8 methine proton at 8 1.39.

The C-8 methine proton in turn showed connectivities with the C-9

methine proton(8 2.10) and with C-14 (82.10). The C-9 methine

proton (8 1.20) and with the C-14 methine proton (d 2.10). the C-9

methine proton also showed coupling with the C-ll methylene

protons (8 1.21). The C-ll proton was in turn connected with the C-

12 proton at (8 1.65). The C-14 methine afforded a cross-peak with

the C-15 methylene proton (8 1.28, 1.25) which in turn showed

connectivities with C-16 methylene protons (8 1.58 and 1.63). The C-

16 proton exhibited cross-peaks with the C-17 methine proton at 8

1.09. This proton was further coupled with the C-20 methine proton

(8 1.90) which was in turn coupled to the C-22 methine proton (8

4.40). The latter showed cross-peaks with the C-23 methylene proton 1

(8 1.90 and 2.45). These Interactions let to the deduction of the spin

system shown in Spin System "B" Fig. No. n.

-:150:-

H 2.451.90 IH.o1.90

n 0.97

5 H3C,,,o-H1.21

H H

PH H

1.39 H

5i.2o , y \

C'*'Ho1

3.20

Spin System "B"

Fig. No. H

The 13C-NMR spectra (CDC13, 125 MHz, DEPT and B.B)

indicated that there were five methyls, eight methylenes, nine

methines and seven quaternary carbons in the molecule, which was

further confirmed that there were 29 carbons associated with 42

hydrogens. Downfield signals at d 209.8 and 167.0 were assigned to

the ketonic (C-l) and a,(i-unsaturated lactonic (C-26) carbonyls

respectively. The remaining two downfield carbons which resonated at

8 152.8 and 125.7 were assigned to the C-25 and C-24 quaternary

carbons respectively. The methyl signals at 8 11.6, 15.7, 13.3, 20.0

and 56.8 were ascribed to C-18, C-l9. C-21, C-28 and -OCH3 carbons

151:-

respectively. The chemical shift assignments of various carbons of

compound 296 are presented in Fig. 3.9.

and the structures of theThe spin systems A and B

remaining fragments were investigated with the help of HOHAHA

spectra recorded with the times of 20, 60 and 100 ms.

To establish direct iH/ÿC connectivites, the HMQC

(Heteronuclear Multiple Quantum Coherence) experiment [182, 221]

was performed. The live methyls groups resonating at 8 11.6 (C-18).

15.7 (C-19), 13.3 (C-21), 20.0 (C-28) and 56.8 (-OCH3) showed cross-

peaks with the proton signals at 8 0.65, 1.29, 0.98, 2.02 and 3.33

respectively. The proton at d 4.41, 3.69, 3.48 and 3.20 were showed

cross-linked with the carbons resonating at 8 78.7, 77.7, 75.1 and

60.3 assigned to the C-22, C-3, C-4 and C-6 methine carbons

respectively. The C-27 methylene protons at 8 4.36 and 4.38 exhibited

cross-peaks with the carbon signal at 8 57.4 in the spectrum.The

1H/13C connectivities of all 29 carbons with their respective protons

are given in Table-3.2.

The assembly of structure 296 on the basis of long-range

heteronuclear correlation observed between carbons and protons of

various spin system, in the HMBC spectrum Fig. 3.8. The HMBC

spectrum of quresimine-A showed the correlations of H-4a (8 3.48),

C-13 (8 209.8), C-2 (8 39.13), C-3 (8 77.50), C-5 (8 64.93) and C-8 (8

29.36) which confirmed the assignments of ring A and B. Another

important signal at 8 4.41 (H-22a) showed interactions with C-20 (8

38.7), C-23 (8 29.36), C-26 (8 167.0), C-25 (8 152.8) and C-24 (8

-:162:-

125.7) and confirmed the assignments of the lactone moiety. So the

proposed structure of 296 establish from various sub-structures

obtained from COSY-450 Fig. 3.10 and HOHAHA spectrum Fig.3.11.

Table-3.2. HMQC Assignments of Quresimine-A (296)

No. 5 1H 8 13C

C-l 209.8

2.58m. 3.00 m 39.13C-2

3.69 m 77.50C-3

3.48 d(J = 3.18 Hz) 75.17C-4

64.93C-53.20 br.s 60.37C-6

1.30 m, 2.15 m 31.17C-7

29.36C-8 1.39 m

C-9 1.20 m 42.78

50.43C-101.21 m 21.62C-ll

24.27C-l2 1.65 m

42.68C-l3

2.10m 56.06C-14

C-l5 1.22 m 27.28

39.56C-16 1.61 m

1.09 mC-l7 51.96

0.65 s 11.56C-18

C-19 1.29 s 15.72

C-20 1.90 m 38.77

0.98 d J = 6.6 Hz 13.34C-21

4.41 dd (Ji=J2 = 4.46 Hz, J3 = 15.2 Hz) 78.34C-22

1.90 m, 2.45 m 29.36C-23

C-24 125.7

152.8C-25

167.0C-26

4.36 dd, 4.38 dd J27a,27|J = 13.5 Hz 57.44C-27

2.02 s 20.0C-28

3.33 56.83-OCH3

163:-

CH3

CH2-OH

H3Cÿi O' o

CH3 H(-•ÿÿIII j~J

oCH3 H

IHHH

V.H3CCy

Ho4OH

Proposed structure of 296.

The steroechemical assignments at C-4 and C-6 are based on

the observed coupling constants. Assuming cts-decline type chair

conformations of ring A and B, the C-4 methine proton with p-

oriented C-4 hydroxyl group (i.e. a-orientation of proton) should

show one diaxial couplings with the C-3 methine proton. The

coupling constants (Ja>e = 3.18 Hz) of the H-4 signal, therefore,

convincingly indicates a p-orientation (equatorial) of the hydroxyl

substituent.

The stereochemistry assignment of the oxirane ring is based

on the chemical shift comparison with earlier reported withanolides.

This was further confirmed by recording the nOe spectrum

where no enhancement of the methyl signal at 8 1.29 (CH3-19) was

164:-

observed by irradiating the H-6 signal at 8 3.20 (H-6) which should

otherwise be observed if the oxirane was a-oriented Fig. 3.12.

2.02 s

28

CH31.90 m

2.45 m H 2724z

CH2-OHH4.36 dd.4.38 dd.J27a.27b=13.5Hz

1.90 m 123

0.98 d, J=6.6Hz 211.65 m H3C

1.21 m 8 20 T=/S' H \ f IÿCH3 H 4.41 ddd.J1.2=4.46Hz. J3=15.2Hz

H j/l2 Jj7

o‘Sfta H19 J

9 14

25

Nx>N>1.61 m

13 i1.39 m 1

2.58 m HH,

H H15 1J'H

1.09 m1

7I I*•-. 3.48 d.ÿ

J=3.18Hz

3 H 5

2 H3.69 m 10 1.22 m85 :J-J 2.10 mHH1.20 m

: 1.30 m\H3ca

6""H 2.15 m4

3.33 s O'OH 3.20 br.s

Fig. 3.7. iH-NMR assignments of 296.

'Z.V’2 a

CH3v* 1.90 m/ TT

2.45 m f? 57.4CH2-OHH

KJ4.361.90 m4 38

H13.3 167.078.4H3C,„;

0.65 s 38.7 r=:CH3S H

O o

:O 1298

CH3 42ii2.58 mH H

S.Cÿmi209.8

3.48 a3.69 m 39 i 50.4 i 29.3iH H HH

771.5 =175.7 64.9= 60.37

H3C3.33 a O’ H

OH

Fig. 3.8. HMBC interactions of 296.

20.0

CH3557.4

CH2— OH

152.8

125.7

29.3

13.3 HH3C„. 167.078.7

38.7 = U oHCH324.2 51.9

21.6

9 157

CH342.6 39.5

H H H56.042.7 27.2

:209.839.1 50.4 29.ftHH... H

64.956.8 77.5: 31.1:

H3CO0ÿ603'"H

OH

Fig. 3.9. 13C-NMR assignments of 296.

CH31.90 m

2.45 m

CH2-OH%1.90 m

H0.98 d

1.65 m H3C/,j

TTR 0.6/4.41i.2i m yy[ ! CH3

:H

3.58ÿ9 1.61 m:if1.39 m HCH3 H H "*

1.09 m H•

(72 :

iz v2.10 m3.48 sK H /1.22 mH HH1.20 m**.. z

H 1.30 m:

' H3C HX 2.15 mO' H3.20 br.sOH

Fig, 3.10. COSY interactions of 296.

-:156:-

CHsH

I CH2-OH

H3CVH H / ‘

v(H0

H \:ÿ

f"'2.58 m H

CH33.(ÿm

HH

V.3.69 m 3.48 s

:ÿHH

Fig. 3.11. HOHAHA interactions of 296.

2i

-C«3Hÿ2o 2819

H3C18 PH*P«3 23n [2

1311Hlo

\\\25UHO. O

HO158 H9o !,267l\ 5 I166H CH227 2H2 4

ott3Co, OH3

HFig. 3.12. Perspective view of 296.

Xu

nx—o

XK>

<?*A Ix°''W°n b 1

I

oV)o Clcer i—oA (O XoB X a

05A Xi

CO V'NjII

I Iom/z 502

(C29H4207)2 O m/z 141

(C7H9O3)P n oI

Om uM IX bOi cCD

P

1 tA

XX +uK>o2P n xx —orr X10

o oD X x x

05 n(0 x—oo .o :i>-h ac10o ooto ac acacxo X XCD o%X03 o05 1o £ O 'O

X m/z 170(C9H14O3)

X o oX X m/z 334(C20H30O4)

5*5 m/z 234(CHH18O3 )

m/z 264(C15H2o04)

O05

XV nIV m

Reported Withanolides from Withania somnifera

Withaferine-A (306)3.2.3

The fraction WF-1 (500 mg) (Scheme 3.12, experimental

section), was subjected to chromatography on a small column packed

with silica gel (150 g, 60-120 mesh size) using pet. ether : chloroform

as the packing solvent. Elution was with chloroform and then with

increasing polarities of chloroform : methanol.

The fraction WSF-1 obtained on elution with pet. ether :

chloroform (1:9) (45 mg) was further purified by preparative TLC,

elution being with pet. ether : chloroform (1:9) to give one major UV

active band. This gave an orange colour with Dragendorffs spray

reagent (30 mg, 8.3x 10-5% yield).

The high resolution mass spectrum afforded the molecular ion

peak at m/z 470.2701, leading to the molecular formula C28H38O6

(calcd. 470.2668) indicating ten double bond equivalents in the

molecule.

The UV spectrum showed an absorption at 225 nm indicative

of an a, p-unsaturated lactone [218].

The IR spectrum the absorption bands for the hydroxyl, and a,

p-unsaturated ketone and lactone groups appeared at 3420, 1725,

1685 cm_1 respectively [214].

159:-

28

CH327

CH2-OH

21 HHsQ 122 26L

tV'Ki'o-ÿoH

t H12o 171319 11

CH3 16,149H 15,

I2ÿÿ110 = 8

OH H H*4

A3H

\a HH

Withaferin-A (306).

The iH-NMR spectrum (CDCI3, 500 MHz) of 306, showed a

double doublet at 6 5.90 (J2,3 = 10.4, J2 4 = 2.3 Hz) for the C-2

proton. Another double doublet resonated at 5 6.48 (J2.3 = 10.4 Hz,

J3,4 = 2.2 Hz) and was assigned to the C-3 methlne proton. The LH-

NMR spectrum also showed three 3H singlets at 8 0.64, 1.30 and 2.12

which were assigned to the C-18, C-19 and the C-28 tertiary methyl

protons respectively. A 3H doublet at 8 1.00 was due to the C-21

secondary methyl protons. A multiplet at 8 4.42 was assigned to the

C-22 methine proton. Other proton chemical shifts are assigned on

Fig. 12.

The COSY 45° spectrum showed that the H-22 at 8 4.42 was

coupled with H-20 methine (8 1.79) and H-23 methylene protons (8

-:160:-

1.69, 2.25). The H-4a (8 3.49) was coupled with H-3 olefinic methine

proton (8 6.48).

&ras27

CH2-OH1.79 dJ21,20=5.0HZ

J2i.20-5.OHz 21 l-I

H3C,0.64 m

CH3

J ABd1.0 d 4.3023 25 4.39

, 1 22 26. J27a.27p=10.4Hz\ ><xÿÿs»J27P,27a=10.4Hz

20 * O O3 H1.35 m .„

18U

11 1.60 m ]j3 *

H jA 1.90 mm /

4.42 m1.43 m

M 1.30 s

CH3

5.90 d

J2.3=10.4Hz

J2.4=2.3Hz 1.601419 9H.

I2ÿ11.29 mJO 5 8

OH H H 2.15 m1.95 m

H\63 2.10mH H4

O6.48 dJ3,2=10.4HzJ3,4ÿ2.2Hz

H 1.40m

H 4.09 d

J0O.7H=5OHZ5.10 br.s

Fig. 3.12. iH-NMR assignments of 306.

The 13C-NMR spectrum showed resonances for all twenty

eight carbons. The lH/13C connectivities of compound 306 were

established on the basis of HMQC experiments. The long-range

Interaction in wlthaferin-A (306) were determined by the

HMBC (Heteronuclear Multiple Bond Connectivity) experiment. In

HMBC spectrum the H-2 proton at (8 5.90) showed interaction with

C-l, C-10, C-3 and C-4 resonating at 8 200.0, 49.5, 143.8 and 69.0

respectively. The H-4 proton at 8 5.10 showed coupling with C-5

quaternary carbon at (8 65.0) as well as C-3, C-6 and C-10 resonating

at 5 143.8, 59.2 and 49.5 respectively.

mr57.0

1155.4 CH2-OH30.1

4.301.79£5H 4.39

%c: 168.2

L 4.421.35

1.43Sÿ26- yo\, .60/ 44ÿ'CH3

21.539.2) 1.90

5.90. 1.6015.0H 56.1 7.5128.< 7

Fig. 3.13. HMBC interactions of Withaferin-A (306).

cif5327 57.0

[5ÿ CH2-OH25ÿ90

30A

13.5 21 JJH3Cÿ

Cfta”22176.0 26U68.2

\ i$9.5= O oH

26.0 18 52.5: H1221.5.

Ts 17\H 16/ 39.2

19 11 44.0

CH3j-f 200.a\l

128xm2 49.5

OH

43.0 56.015.0.|9

27.510 = 29.118H H

i (T 6 'H

Fig. 3.14 13C-NMR assignments of 306.

30.2

H

Another downfteld proton resonating at 8 4.42 assigned for H-

22 showed interaction with C-20, 026 and 023 resonating at 8

39.5, 168.2 and 30.6 respectively. The complete HMBC interaction are

shown in Fig. 3.13.

The long-range connectivities were determined from

the HMBC experiment. The 13C-NMR assignments are shown in Fig.

3.14

21 28

CH3 CH323

22,

O HO19 nH o 9H3 fir

13l t 27 IH. CH2262 fa 16To [9 14

4

OH H o3 5 6

HHH

Fig. 3.IB. Perspective view of withaferine-A (306).

By comparing the spectral data (UV, IR, MS, 13C-

NMR) of (256), the compound was identified as withaferin-A [223].

3.2.4 2,3-Dihydrowithaferin-A (297)

The fraction WF-3 (450 mg, Scheme-3.12) was subjected to

chromatography over silica gel (100 g, 60-120 mesh size). Elution was

carried out with increasing polarities of pet. ether : chloroform. The

fraction "WF-5" obtained on elution with pet. ether : chloroform (2:8)

on evaporation afforded a pure compound as white crystals (10 mg).

-:163:

This compound gave light orange colour on spraying the TLC plates

with Dragendorffs spray reagent.

The high resolution mass spectrum afforded the molecular ion

peak at m/z 472.2804 leading to the molecular formula C28H40O6

(calcd. 472.2824) indicating nine double bond equivalents in the

molecule. The molecular ion peak was further confirmed by mass

spectrometry using FAB and FD sources [224].

The UV spectrum showed absorption at 216 nm characteristic

of an a, p-unsaturated lactone [218].

28

CH327

CH2-OH23

21 HH3<\

18

26l22

I20 = O O

CH3 H

IT H12Q 19 171311

CH3 16,149 !5,

2 1

810 =OH H H74

A3

'''/i O' HH

2,3-Dihydrowithaferin-A (297).

The IR spectrum showed absorption at 3480 cm-1 for (OH) and

1685 cm-1 due to a, (i-unsaturated lactone and at 1705 cm-1 for a

ketonlc carbonyl.

-:184:-

28 2.16 s

CH32.35 m

27l.|ÿ m

1.82 d

J2°220=6.7HZ J2i2°H’7HZH3C

CH2-OH4.34 d4.39 d

2?23 25

00 nfi J22a,20p=12.6Hz

\ 12.6Hz

20 - O O1.53 m 1.00 m

H H1.10 m

f CH3• 18 .

HH1.23 m

H 4.42 ddd J22a-2op=3.5Hz

J-J d22a,23P=7- 1

d22a.23o=7•1Hz

112"

0.95 m

o11 1.29 m

1.80CH3 H1.90 m Hf

%1419 9 H15.0.901

o.8oinNH2 810OH H

H 1.53 m0.85 m 75 H2.45 m 1.20m\62.10 mH1 H

H \ O H 1.20 m1.90 m 3.09 br.s

3.41 lJ=3.1Hz

Fig. 3.16 !H-NMR assignments of 297.

The 1H-NMR spectrum (CDCI3, 500 MHz) exhibited three 3H

singlets at 5 0.67, 1.32 and 2.16 which were assigned to the C-18, C-

19 and the C-28 tertiary methyl protons respectively. A doublet at 8

1.02 was due to the C-21 secondary methyl proton. Two AB doublets

at 8 4.34 and 4.39 were due to the C-27 hydroxymethylene protons.

Two multiplets, one at 8 1.85 and the other at 8 2.35, were assigned

to the C-23 methylene protons. A multiplet at 8 4.42 was assigned to

the C-22 methine proton. A triplet at 8 3.41 was due to the C-4

methine proton geminal to a hydroxyl group in ring A. A broad singlet

at 8 3.09 was due to the C-6 methine proton of the oxirane ring.

-:165:-

crir27 55.9

25P5-229.1

2312.9 21 H

H3c„H

22178.5 26LI66.8\ i oO'u.i

cHr 385HM-Hm 17\

20

24.0

20.8ÿ/I2SNNVf 19 fn 42.9

II CHÿ H 16) 38.7212.m} 42.9 553

29.3=i26.925.8 \2 50.5 10 = 8

OH H H31.231.1

O HH

Fig. 3.17 13C-NMR assignments of 297.

The iHÿH connectivities (COSY 45°) showed that H-22 at 8

4.42 was coupled with H-20 methine proton (8 1.82) and with H-23

methylene protons (8 1.85, 2.35). The H-20 methine showed further

couplings with H-21 (8 1.02) and H-22 (8 4.42). Similarly the H-4a (8

3.41) was coupled with H-3a (8 1.90) and H-3p (8 2.45). The iH-NMR

assignments of 297 are shown in Fig. 3.16.

The 13C-NMR spectrum (CDCI3, 125 MHz) exhibited signals

for twenty eight carbons, in agreement with the molecular formula

C28H40O6. There were four methyl, nine methylene, eight methine

and seven quaternary carbons in the molecule. The 13C chemical

shift assignments of 2,3-dihydrowithaferin-A shown on Fig.3.17 were

made with the help of HMQC and HMBC spectra.

-:166:-

21 28CHg CH3

23

<ÿ3 A24\ HOV 27 I

Q,7HO fH3 *

H

13]

H* CH22 26r8 16|9Ho.H/ H O

H[3 5 6

HHH

Fig. 3.18. Perspective view of 2,3-dihydroxywithaferine-A (297).

By comparing the spectral data (UV, 1R, MS, iH-NMR and

13C-NMR) of (297) with those reported in the literature [223] was

identified as 2,3-dihydrowithaferin-A.

Dried aerial parts ofWithania somnifera

(36 kg)

Powdered and extractedwith methanol (70 litres)

Methanolic extract (1.5 kg)

Suspended in distilledwater (4,5 litres)

defatted with pet. ether (10 litres)

Defatted aqueous layer Pet. ether extract(325 g)

Extracted with chloroform

Chloroform extract(154 g)

Aqueous extract

Scheme 3.11.

Chloroform extract of Wilhania somnifera ( 154 (g)

Loaded on to a silica gel column (mesh 70-230 ASTM) (1400 g) andelution with pet. ether : chloroform (0-100%) than with chloroform :methanol (0-100%)

pet. ether : CHCI3 pet. ether : CHCI3 CHC13 : MeOH(9.5:0.5)

CHCI3: MeOHCHCI3(1:9)(2:8) (9:1)

11

WF-1(500 mg)

WF-2(200 mg)

WF-3(450 mg)

WF-5(50 mg)

WF-4(300 mg)

Subjected to preparativeTLC and eluted with

Loaded on smallcolumn and eluted withpet ether: CHCI3 ( l:9)

Loaded on smallcolumn and eluted withpet. ether : CHCI3 (2:8)

Subjected to preparativeTLC and eluted withCHC13: MeOH (9.5:0 5)

Subjected to preparativeTLC and eluted withpet. ether : CHC13 (2:8) +3 drops of diethyl amine

Opet ether : CHCI3+ 2 drops diethyl amineA O

BM(1:9)o>

CD1

CD 2,3-Dihydrowithaferin-A (297) Quresimine-A (296)(10 mg)

Withanolide-A(7.0 mg)

M* 470 (5.0 mg)(7.5 mg)

10

WSF-1(45 mg)

WSF-2(52 mg)

Subjected to preparative

TLC and eluted pet. ether :

CHCI3 (1:9)

Subjected to preparativeTLC and eluted pet. ether :CHCI3 (2:8)

Withaferine-A (306) (30 mg) Withanone (295) (6.5 mg)

1

3.3 Plant Material

The fresh plant (aerial parts) of Withania somnifera (70 kg) was

collected from Karachi (Pakistan), identified by the plant taxonomist

of Botany Department, University of Karachi and dried in the

absence of sun light. The aerial parts of the plant Withania somnifera(36 kg) were powdered and soaked in methanol (70 liter).

Extraction and Purification3.3.1

The methanolic extract was evaporated to afford a gum (1.5

kg). This gum was partitioned between distilled water and (4.5 liter)

pet. ether (10 liter). The aqueous extract was again extracted with

chloroform (7 liter). This chloroform extract was evaporated and then

loaded on a silica gel (mesh 70-230 ASTM) column, Scheme-3.11.

Isolation of Withanone (295)3.3.2

The chloroform extract (154 gm) was loaded on a silica gel

column and the column eluted with increasing polarities of pet. ether

: chloroform. On elution with pet. ether : chloroform (2:8) fraction

WF-1 (500 mg) was obtained. The WF-1 fraction exhibited several

spots on TLC. This fraction (500 mg) was then subjected to

chromatography on a small column (65 gm) elution being with pet.

ether : chloroform (1:9). Two major fractions obtained from this

column were WSF-1 and WSF-2. Fraction WSF-1 was demonstrated

to contain withaferin-A (30 mg, 8.0 x 10-5 yield) after spectroscopic

techniques.

The second fraction WSF-2 (52 mg) also showed one major

compound along with some minor compounds . This fraction

subjected to preparative TLC, elution being carried out with pet. ether

: chloroform (2:8), to afford withanone 295, 6.5 mg, yield 1.8 x 10-?%(Scheme-3.12).

was

Spectral Data

[oc]25D: +17° (c = 0.0892. CHC13)

UV (MeOH), nm (log e): Xmax 215 nm characteristic for cc.p-

unsaturated lactone.

(IR (CHCI3): vmax cm-1: 3440 (O-H). 1690 (a.(J- unsaturated lactone)

and.1615 (C=C).

EIMS m/z: (rel.int.%): 456 (82), 438 (15), 327 (26), 288 (35), 286 (10),

269 (36). 267 (30). 226 (100). 213 (47). 167 (10). 159 (30). 141 (31),

138 (15) and 95 (42).

HREIMS m/z: (rel. int. %): 456.2801 (C28H40O5. calcd. 456.2875)

(15). 438.2790 (100). (C28H3804. calcd. 438.276ÿ 288.2086 (20).

(C19H28O2, calcd. 288.2089 (28), 167.0705 (C9HUO3. calcd.

176.0708). 141.0546 (8)/ (C7H8O3, calcd. 141.0551).

FAB +ve: 457.2879.

iH-NMR (400 MHz. CDCI3) 8: See Fig. 3.1.

13C-NMR (CDCI3. 125 MHz) 8: See Fig. 3.5.

COSY-450 : See Fig. 3,3.

HOHAHA: See Fig. 3.2.

HMBC: See Fig. 3.4.

HMQC: See Table-3.1.

Isolation of Quxesimine-A (296)3.3.3

The chloroform soluble extract (154 gm) (Scheme-3.12) was

loaded on a silica gel (mesh 70-230 ASTM) column (1400 gm) which

was eluted first with chloroform and then with mixtures of methanol

: chloroform. The fraction WF-4 (300 mg) was thus obtained on

elution with chloroform : methanol (9.5:0.5). The fraction WF-4 was

then subjected to preparative TLC, the plates being eluted with pet.

ether : chloroform (2:8) + 3 drops of diethylamine to afford 296 (7.5

mg, yield 2.1 x 10_7%) as a colourless powder.

Spectral Data

[al25D = + 17° (c = 0.075, CHC13)

UV (MeOH), nm (log e) Xmax: 214 (3.45).

IR (CHCI3) vmax cm'1: 3455 (O-H), 1683 (a, p-unsaturated lactone)

and 1590 (C=C).

EIMS m/z (rel. int. %): 502.2910 (M+. 10), 484 (15), 452 (22), 434

(13), 347 (22). 311 (16). 281 (15), 241 (20), 213 (21), 197 (42) and 141

(100).

HREIMS m/z (rel. int. %): 502.2910 (M+,5),484.2804 (70), 470.2667

(20),452.2530 (12), 387.2522 (15), 334.1979 (25), 234.2795 (17),

264.3011(15),169.0810 (70),170.1932 (10) 141.571 (100) and 86.104

(18).

1H-NMR (CDCI3. 300 MHz) 5: See Fig. 3.7.

13C-NMR (CDC13I 125 MHz) 8: See Fig. 3.9.

COSY-45°: See Fig. 3.10.

HOHAHA: See Fig. 3.11.

HMBC: See Fig. 3.8.

HMQC: See Table-3.2.

Isolation of Withaferine-A (306)3.3.4

The chloroform extract (154 gm) was loaded on a silica gel

column and eluted with pet. ether : chloroform (2:8) on elution at

this polarity a fraction WF-1 was obtained (500 mg). This fraction

was subjected to chromatography on a small column packed with

silica gel (50 gm) and eluted with pet.ether : chloroform (1:9) to afford

another fraction WSF-1 (45 mg) which showed several minor

compounds along with a major UV active band on TLC. This fraction

was subjected to preparative TLC using pet.ether : chloroform (1:9) as

eluent to afford a pure compound (Withaferine-Ay30 mg) (8.3 x 10'5,

yield).

Spectral Data

[a]20D + 35o (c = 1.3 CHCI3)

UV (MeOH), nm (log e) Xmax‘ 225 (3.363).

-:173:-

IR (CHCI3) vmax cm'1: 3420, 1725 and 1685.

EIMS: m/z (rel. int. %): 470 [M+ (10)], 452 (10). 347 (15), 299 (8),

124.0 (95) and 95 (100).

HREIMS m/z (rel. int. %): 470.2701 (M+ 21), 452.2306 (15),95.1062(100).

1H-NMR (CDCI3, 500 MHz) 6: See Fig. 3.12.

13C-NMR (CDCI3, 100 MHz) 5: See Fig. 3.14.

Isolation of 2,3-Dihydrowithaferine-A 2973.3.5

The methanolic extract of Withania somnifera (1.5 kg)

suspended in water and defatted with pet. ether. This defatted

aqueous extract was then extract with chloroform after evaporation

of chloroform under vaccum condition afforded (154 g) dry chloroform

extract. This chloroform extract subjected to silica gel column (1400

g) using (mesh: 70-230 ASTM) and eluted with pet.ether : chloroform

and then chloroform : methanol with increasing polarity at pure

chloroform. The fraction WF-3 (450 mg) obtained UV active

compounds on TLC.

This fraction was again subjected to pencil column and eluted

with pet.ether : chloroform, which afforded a pure compound on

elution with pet.ether : chloroform (2:8) which give orange colour

with Dragendorff s spray.

Spectral Data

[a]25D +89° (c = 0.98 CHCI3)

-:174:-

TR (CHCI3) Vmax cm*1: 1705, 1685 and 3480.

EIMS m/z (rel. lnt. %): 472 [M+ (5)], 454 (54), 3474 (10). 283 (20), 197

(33). 141 (100) and 95 (65).

1H-NMR (CDC13, 400 MHz) 5: See Fig. 3.16.

13C-NMR (CDCI3, 125 MHz) 5: See Fig. 3,17.

-:176:-

SECTION D

ISOLATION AND STRUCTUREELUCIDATION OF

FROM FERULA OOPODA

INTRODUCTION4.0

Ferula oopoda belongs to the plant family Umbelliferae. It Is a

perennial herb distributed from the Mediterranean region to Central

Asia. This genus Ferula consists about 140 species [226J. Only 15

species have been identified in Pakistan [227]. These species are:

Ferula assafoetida, F. baluchistanica, F. communis, F. costata, F,

hindukushensis, F. kokonica, F. lehmanii, F. microlabe, F. narthex, F.

oopoda, Fovina, F. reppiea, F. rubicaulis and F. stewartiana. Some of

the species are important as sources of oleogum and are used in

indigenous medicine [228].

Some species of genus Ferula are extensively used in India for

flavouring the curries, sauces and pickles. They stimulate the

intestinal and respiratory tracts and the nervous system. They are

useful in asthma, whooping cough, chronic bronchitis and intestinal

flatulence. Some species are administered in hysteria, epileptic

affections, cholera and are often employed in veterinary medicine

[229-231].

The ethanolic extracts of Ferula sinaica roots inhibited the

spontaneous movements of rat and guinea pig uterine smooth

muscles and also the contractions induced by oxytocin stimulation.

These data suggest that the plant extract has some antioxytocic

potential [232]. The aqueous extract of Ferula ovina showed

anticholinergic and antihistaminic antispasmodic effects [233].

176:-

The essential oils of Ferula narthex, F. ovina and F. oopoda

when tested in liquid media against standard cultures of

Staphylococcus aureus, Escherichia coli, Salmonella typhi, Shigella

dysenteriae and vibrio cholerae showed good inhibitory activity. The

oils of Ferula narthex and F. ovina were more active against

Staphylococcus aureus, while the growth of the pathogens of dysentery

and cholera was inhibited by F. oopoda oil [234], Many sesquiterpene

lactones have been reported from F. oopoda. [235].

4.1 Biosynthesis of Sesquiterpenes

The biosynthesis of sesquiterpene can be divided into two

major parts. The first part is the biosynthesis of the acyclic precursor

(FPP) while second part is the cyclization of the acyclic precursor into

the sesquiterpenoidal skeleton [236,237].

Biosynthesis of Acyclic Precursor (FPP)

J.W. Comforth, in his work on the biosynthesis of steroids,

characterized two active forms of isoprene i.e., isopentenyl

pyrophosphate (IPP, 267) and dimethylallyl pyrophosphate (DMAPP,

268). These intermediates are obligatory for the synthesis of plant

terpenes.

The incorporation of these intermediates into terpenes is

catalyzed by various enzymes. The biosynthesis of the acyclic

precursor is presented in Scheme-4.1. Two molecule of acetyl co¬

enzyme A (259, derived from carbohydrate, fat or protein catabolism),

condense to yield acetoacetyl co-enzyme-A (260). This acetoacetyl co-

-:177:-

enzyme A further condenses with another molecule of 259 by aldol

type reaction to form 3-hydroxy-3-methyl gluteryl co-enzyme A (260).

H+o

oJ oAcetoacetyl-CoAthlolase

C— S.CoAM(

( o — Cr C— S.CoA

O 260©

©H2C— C— S.CoAH2G— C—S.CoA

Acetyl-coenzyme A (259)Acetyl-coenzyme A (259)

O

H2CS.CoA

3-Hydroxy-3-methyl glutaryl-S.CoAHMG-S.CoA (261)

NADPH

NADP+

HOO CH2OH

3R-Mevalonlc acid (264)

3 ATP

OOH

CH2OPPH— O

265

-co2H+

),CH2OPP- HR .CH2OPP

HMeHS

IsopentenylpyrophosphateaPP) (267)

8cheme-4.1. Biosynthesis of sesquiterpenes.

Dlmethylallylpyrophosphate(DMAPP) (268)

-:178:-

3-Hydroxy-3-methyl glutaryl co-enzyme A (261) Is then

irreversibly reduced through the intervention of NADPH to 3R-

mevalonic acid (264) . Only the R form of mevalonic acid is utilized

by organisms for producing terpenes, while the "S" form Is

metabollcally inert.

Phosphorylation of mevalonic acid (264) by ATP (adenosine

triphosphate) leads to mevalonic acid S-pyrophosphate (266). After

decarboxylation, it affords IPP (267).

Me ( OPP OPP

+

Me'HR(DMAPP) (268) HS(IPP) (267)

MeMe OPP Me OPP

HR V

+

Me'

HS(GPP) (270)(IPP) (267)

Me Me Me OPP

(FPP) (271)

OPPOPP

cis trans

272 273

Scheme-4.2.

-:179:-

IPP is then converted by an enzyme-catalyzed prototropy into

an equilibrium with dimethyl allyl pyrophosphate (DMAPP, 268) in

which the latter predominates.

These two intermediates IPP (267) and DMAPP (268) condense

in head-to-tail manner to produced geranyl pyrophosphate, GPP

(270), which then condenses with IPP (267) to afford famesyl

pyrophosphate (FPP, 271). This is the key acyclic precursor in the

biosynthesis of sesquiterpenes (Scheme-4.2).

The carbon skeleton of almost all the known sesquiterpenes

can be derived from trans-famesyl pyrophosphate (273) and the cis

famesyl pyrophosphate (FPP, 272), through appropriate cyclization

and rearrangements. The cfe-famesyl pyrophosphate (272) should be

derived from trans-famesyl pyrophosphate (273) via a reversible

mechanism.

y-Bisabolene (302) is considered to be the precursor of a

number of sesquiterpene systems. (Scheme-4.3).

-:180:-

cOPPOPP

(c(s -FPP) (272) (irons -FPP) (273)

+<'

298

+

301

-H* 299 300

7-Blsabolene (Blsabolane) (302)

Scheme-4.3.

4.2 RESULTS AND DISCUSSION

New Compound from Aerial parts of Ferula oopoda

Feraileate (303)4.2.1

A new compound, feraileate (303), was isolated (fraction F0-

4, Scheme-4.6, experimental section) from the ethanolic extracts of

Ferula oopoda collected from the Quetta district, Pakistan. The

fraction FO-4 (40 mg) (experimental section, Scheme-4.6) was

further purified by preparative TLC on silica gel (GF-254, 0.2 mm)

using pet. ether : acetone (7:3) as the developing solvent. This

afforded a compound which gave a pink colour with Dragendorffs

reagent and dark brown colour with ceric sulphate reagent.

The UV (MeOH) spectrum of feraileate (303) was typical for

aromatic systems [242] showing absorption at 225 (log e = 3.05), 299

(log e = 2.19) and 303 (log e = 3.40) nm.

The IR spectrum showed an absorption at 1690 cm-1 which

indicated the presence of an a, p-unsaturated ester carbonyl group

(238]. Other intense IR absorptions were at 1120 cm*1 (C-O-C), 1600

(C=C) and 2900 (C-H) cm1.

The high resolution electron impact mass spectrum showed

the molecular ion peak at m/z 306.1103, corresponding to the

molecular formula, Ci6His06. indicating eight degrees of

unsaturation in the molecule. The mass fragmentation pattern of

feraileate (303) is presented in Scheme-4.4. The fragment "a" at m/z

i-:182:~

151.021 of composition C8H7O3 resulted from the cleavage of the

CO bond, indicating that the acyclic ketone moiety was probably

attached to C-l of the aromatic skeleton. The fragment "b" at m/z

179.029, C9H7O4 further suggested that the ketone group is directly

attached to the C-l aromatic carbon. The peak at m/z 55.054 (C4H7)

indicated that butene group is present in the molecule (Scheme-4.4).

The spectrum (CDCI3, 400 MHz) revealed the

presence of four methyl groups in the molecule i.e., a 3H doublet at 8

1.54 (J = 7.0 Hz) for the C-3' methyl protons and two methyl doublet

at 8 2.0 (J = 3.1 Hz) and 8 2.1 (J = 3.1 Hz) for C-4" and C-5"

respectively. A singlet at 8 3.9 was assigned to the methoxy protons.

Two downfleld protons resonating at 8 7.1 d (J = 1.5 Hz), and 8 7.3 (J

= 1.5 Hz) were due to C-6H and C-2H respectively. The

spectrum showed that the benzene ring is tetra substituted. The "J"

values of C-6H and C-2H suggested that these proton .were meta (or

possibly para) to each other. The C-3" methine proton resonating at 8

6.1 as a multiplet indicated the presence of a double bond. A 2H

singlets at 8 6.0 was assigned to the methylenedioxy protons. The C-

2' methine proton appeared as a quartet at 8 5.9 (7.0 Hz) establishing

that the methine proton has a vicinal methyl group. The LH-NMR

assignments of feraileate (303) are given in Fig. 4.1.

-:183:-

:

7.1 d. J=1.5Hz

H o1.54 d. J=7.0Hz

>CH3o. 1* 3'2 —•

"'H6.0 sA O 5.9 q. J=7.0Hz

O 4

1 7.3 d.H 1 1

I J=1.5Hz

OCH34"

CH3 2.0 d,J J=. -3-9SH3C 3.1Hz

2.0 d,J = 3.1Hz

H6.1 m

Fig. 4.1. iH-NMR chemical shifts of feraileate (303).

The COSY-450 spectrum of feraileate (303) exhibited coupling

interactions which were found to be in full agreement with the

assigned structure see Fig. 4.2.

H O

17.3102.9 195.0 CH3o 71.0.

1 r 3'

J!129.1 2i'%140.0

H102.4143.0 109.4

1o 4

149.0H 1670

HsC V

20.456.7

15.8

H

Fig. 4.2. 13C-NMR chemical shifts of feraileate (303).

The Me-3’ protons at 5 1.54 showed interactions with the H-2'

methine proton at 5 5.9. The Me-4” protons at 5 2.0 showed coupling

with H-3” the methine proton at 8 6.1. The H-3” methine proton was

;

-:184:-

further coupled with the Me-5" protons at 8 2.1. The aromatic

protons H-6 (8 7.1) and H-2 (8 7.3) also showed connectivity to each

other.

%d o1.54 d

CH3o 1' 3'

< T'"-Hr O 5-9 q

5

o 4

7.3 dd H2.0 dd

OCH3H3C

<r 2.1 dd

6.1 m

Fig. 4.3. COSY-450 connectivity of feralleate (303).

36%

H O

O

< Ho4.O

O63.23%H

35%68.57%*OCH3 .CH3-

H3C66.3!

66.35%

Fig. 4.4. nOe Interactions of feralleate (303).

-:185:-

o;+o3 IIIo

< I oQ < Ioo

OCH30CH3

m/z 179m/z 306

+

<co

o.

o o< I (not observed)O

OCH3

m/z 151

Scheme-4.4. Mass fragmentation of feraileate (303).

!

*

oH

1.54102.91 195.0 71.0ÿCH

67.0,0 59

o6.0 02.4

129.1140.0

o.J43.0l 109.4

o149.0

20.456.7

OCH3ÿ

15.8

H 6.1

Fig. 4.5. HMBC interactions of feraileate (303).

The 13C-NMR spectra (CDC13, 100 MHz, DEPT) indicated that

there were four methyl, one methylene and four methine carbons in

the molecule. The 13C-NMR spectra (B.B) showed sixteen carbon

signals of these sixteen carbon atoms, nine appeared in DEPT spectra

so that there were seven quaternary carbons.

The downfleld signals at 8 167.0 and 195.0 were assigned to

the a, (5-unsaturated carbonyl carbon (C-l"), and the ketonic carbon

(C-T) respectively. Other downfield carbons resonated at 8 149 (C).

109.4 (CH), 129.1 (C), 102.9 (CH), 140 (C), 143 (C) and were assigned

to C-3, C-2, C-l, C-6, C-5 and C-4 respectively.

The methylene carbon at 8 102.4 was to the methylenedioxy

carbon. Other carbon atoms at 8 139 (CH), 127 (C), 71.0 (CH) were

assigned to C-3", C-2" and C-2' respectively.

-:187:-

The chemical shift assignments of various carbons of

feraileate (303) are presented in Fig. 4.2.

The one-bond !H/13C correlations for feraileate (303) were

determined on the basis of HMQC experiments (Table-4.1). The

vinylic C-3" (139.0) was directly correlated with the proton that

resonated at 6 6.1. The methine C-2' (8 71.0). showed a cross-peak

with H-2 at 8 5.9. The C-2 and C-6 carbons resonating at (8 109.4)

and (8 102.9) showed cross-peaks with the protons at 8 7.3 and 8 7.1

(H-2 and H-6 respectively).

The long-range *H/13C interactions in feraileate (303) were

determined by the HMBC experiment see Fig. 4.5. The signal at 8 6.1

(H-3”) showed couplings with C-2" quaternary carbon and C-4” (8 2.0)

methyl carbon. H-2’ (8 5.9) showed coupling with the C-1’ quaternary

carbon (8 195) and with the C-3' methyl carbon at (8 1.54). The H-6 (8

7.1) showed connectivity with C-1 (8 129.0), C-1’ (8 195.0) and C-5 (8

140).

The structure of feraileate (303) was further confirmed by nOe

experiment. nOe difference measurement established the relative

stereochemistry at C-2’ asymmetric centers and positions of the

functionalities in the molecule. Irradiation at 8 5.9 resulted in

63.25% nOe of the C-2 proton and 36% nOe of the C-6 proton.

Irradiation at C-2H caused a 68.57% reciprocal nOe on the C-3

methoxy (8 3.9) proton. This indicated the methoxy group is adjacent

to C-2 proton (i.e., C-3 position). Irradiation at C-3" proton showed

-:188:-

nOe 66.35% with C-5" and C-4” methyl groups. Detailed nOe

measurements are summarized around Fig. 4.4.

Table-4.1. HMQC Assignments of Feraileate (303)

Carbon No. 6lH 8 13C

129.1C-l

7.3 d (J= 1.5 Hz) 109.4C-2

C-3 149.0

C-4 143.0

C-5 140.0

7.1 d (J= 1.5 Hz) 102.9C-6

195.0C-l'

5.9 q (J = 7.0 Hz) 71.0C-2'

1.54 d (J = 7.0 Hz)C-3’ 17.3

C-l” 167.0

C-2" 127.0

6.1 mC-3" 139.0

C-4" 2.0 d, (J= 3.1Hz) 20.4

C-5" 2.1 d (J = 3.1 Hz) 15.8

O-CH2-O 6.0 s 102.4

3.9 sOCH3 56.7

-:189:-:

REPORTED SESQUITERPENE LACTONE ISOLATED

PROM FERULA OOPODA

Guaianolide (304)4.2.2

The pet. ether extract obtained by extraction of the ethanolic

extract of Ferula oopoda (Scheme - 4.6, experimental section), was

loaded on a silica gel column (70-230 mesh, ASTM), which was eluted

with increasing polarities of pet. ether : acetone. The fraction "FO-3"

obtained on elution with pet. ether : acetone (7:3) 48 mg contained

three compounds. The mixture was subjected to preparative TLC on

silica gel precoated plates which were developed in pet. ether :

acetone (9:1), this afforded one major compound which gave a dark

pink colour with ceric sulphate reagent after heating 120°C and was

identified as guaianolide (304), previously as isolated from Ferula

Arrtgonit bocchieri [239].

14

H2C

10 9'21

8]

n'•in," 13

CH3

oFig. 4.6. Structure of guaianolide (304).

-:10O:-

The UV absorption of gualanolide (304) at Xmax 254 (log e

2.516) indicated an a, p-unsaturated carbonyl chromophore [218].

The high resolution mass spectrum afforded the molecular ion

peak at m/z 230.1305 leading to the molecular formula C15H18O2

and indicating seven double bond equivalents in the molecule. The

molecular ion peak was further confirmed by mass spectrometery

using FAB and FD sources [240, 241].

The peak at m/z 215. 1072 (C14H15O2) corresponded to the

loss of "CH3" from the M+ ion. Other prominent peaks in HREIMS

were at m/z 185, 151 and 120. The mass fragmentation pattern of the

compound (304) was characteristic of sesquiterpene lactone type

compound [235].

The IR spectrum displayed an absorptions at 1740 cm'1

attributable to a conjugated y-lactone. The presence of a cabon -

carbon double bond was indicated by an absorption band at 1645

cm'1 [242].

The spectrum {CDCI3, 400 MHz) of compound (304)

showed only one terminal methylene group with the methylene

protons resonating at 8 4.81 and 4.89 as a br. singlets. Another

downfield proton resonated 8 4.57 as a multiplets was assigned to H-

6a. A three-proton singlet at 8 1.69 was assigned to the allylic methyl

group. The resonance at 8 5.50 is assigned to H-3 in trisubstituted

double bond, on the basis of its coupling with the methyl group at C-

15 (8 1.79 br. s) and with a complex group of signals attributable to

the methylene C-2 (8 2.35 - 2.45 m). The latter signals appear to be

coupled also with a complex multlplet at 5 3.07, which is assigned to

H-l, owing to its further coupling with the vinylidene group and H-5.

The value of (Ji,5 = 7.8 Hz) is in accordance with a cis Junctionbetween the cyclopentane and cycloheptane rings, whereas J5,6 = 10.9

Hz confirms the trans relationship between H-5 and H-6. The 1H-

NMR assignments of guaianolide (304) Eire given in Fig. 4.7.

4.81 br.s 9 br.sH

2.35 m

H 3.07 m « HH \\ 10 _f

K fl

2.07 m

2.45 m

?2.48 m:

4.57 dd,

J12=1.08HZ\8 TT

Jl>3=10.8Hzrÿ7H

'23.0 m1

5.50 dt H-

J3,15=1-8Hz

H H 2.87 m111

6 \!2>15

13H3C 2.38 dd*1.79 br.s |° CH3

1.69s1

J5.6=10.9Hz o

Fig. 4.7. assignments of guaianolide (304).

The COSY-450 experiment was performed to determine the

one-bond connectivities. The H-6a at 8 4.57 was found to be

coupled with H-5fJ methine (8 2.38). H-l (8 3.11 m) was coupled with

H-5P (8 2.38 m) and H-2a, H-2b, 8 2.35 (m) smd 2.45 (m) respectively.

The COSY-450 connectivities Eire shown in Fig. 4.8.

-:192:~

4.81 4.89H H

2.35 2.07C

if w\\ H2.45

H2.48

V H4.573.0

H HHV5.50 XH 2.87

\H3C 2.38CH3o.1.79 1.69

o

Fig. 4.8. COSY-450 interactions of guaianolide (304).

The 13c-NMR spectrum (CDC13, 100 MHz) exhibited all fifteen

carbons, which were in complete agreement with the molecular

formula C15H18O2. There were two methyl, four methylene and four

methine carbons (by difference from the DEPT spectrum) and five

quaternary carbons in the molecule. The C-l1 carbonyl appeared at 8

174.0. The two tertiary methyl carbons resonating at 8 8.4 and 18.36

were assigned to C-13 and C-l5 respectively. The downfield carbons

appeared at 8 122, 162, 142, 126.8 and 149.5 which were assigned to

C-12, C-7, C-4, C-3 and C-10 carbons respectively. The 13C-NMR

assignments of guaianolide are presented in Fig. 4.9.

By comparison of the spectra data (UV, IR, MS, !H-NMR, 13C-

NMR) with those reported earlier the compound was identified as

guaianolide (304) [239],

-:193-

H H

'“'112.6

\' 149.534.7 31.11° 9*r2

49.51

H 8) 30.3126.8' 53.7

83.07/162.0142.0

6 \12 >

15 122.013H3C CH3°ÿu

174.0V

18.08.4

o

rig. 4.9. 13C-NMR assignment of guaianolide (304).

Isolation of Grilactone (305)4.2.3

The pet. ether soluble fraction was subjected to silica gel

column using pet. ether : acetone as solvent. The fraction "FO-l" was

obtained in pet. ether : acetone (9:1, Scheme-4.6, Experimental) was

further chromatographed by preparative TLC in pet. ether acetone

(9.5:0.5). This afforded a sesquiterpene lactone. It gave a

characteristic red coloured reaction with ceric sulphate solution and

a yellow colour with Dragendorffs reagent. The sesquiterpene lactone

was identified grilactone after spectroscopic studies [243, 244].

The UV spectrum (MeOH) of the compound (305) showed only

terminal absorption. The IR spectrum indicated the presence of a

flve-membered y-lactone with absorption at 1730 cm1.

-:194:-

]HU 14

CwH10 9>

21

8H

V613

nVj......CH3A i15CH3 =0ÿ12 H

O

Fig. 4.10. Structure of grllactone (305).

The high resolution mass spectrum of compound showed the

molecular ion at m/z 232.1442 corresponding to the molecular

formula C15H20O2 (calcd. 232.1463) indicating six degrees of

unsaturation. A peak at m/z 217 was due to the loss of a methyl

group from the molecular ion. A large peak at m/z 204.1140 was

ascribed the loss of an ethylene fragment from the molecular ion.

Another peak at m/z 188 represented the loss of CO2 (m.u. 44) from

the M+ ion while the peak at m/z 158 resulted from the loss of

lactone ring along with further loss of two protons. The peak at m/z

105.0721 was due to the loss of a fragment C5H7O2 from the M+-

C2H4 ion. Other major peaks were at m/z 176, 119, 91 and 79.

The iH-NMR spectrum (CDCI3, 400 MHz) of compound (305)

showed two methyl signals 5 1.14 d (J = 6.85 Hz) and 8 1.83 d (J =0.6 Hz) for the Me-13 and M-15 protons respectively. The downfield

proton at 8 4.32 dd (J©p,5a = 11.5 Hz, Jep.7p =* 6.5 Hz) for the oxygen

bearing proton H-6 [245, 246]. Other downfteld protons resonated at

8 4.81 (br. s), 4.82 (br. s) and 5 5.45 which were assigned to H-14a,

H-14P and H-3 respectively. The *H-NMR assignments are shown in

Fig. 4.11.

Jla,20=®-3HzJla,5a=®-6Hz 4.81 br.s

4.82 br.s H14/HC2.44 m

H 3.06 dt.u

¥ VH 2.41 m

H,HI/10 ?

2.72 m \ T T

4ÿ2ddH sWH2 36p.5a=l 1-5 Hz

Jep.7p=6.5Hz1

%H 1.87 mN,

H5.45 br peak

7\\ 13 1.14 d. J=6.85Hz

i IV..-'"CH315 1 2.

CH3 £

12 H1.83 d. J=0.6Hz2.70 m

J5o.6p=ll-0Hz O35a.ia=8.8Hz

Fig. 4.11. iH-NMR assignments of grilactone (305).

The 13C-NMR spectrum (CDCI3, 100 MHz) showed a signals at

5 179.30 due to the carbonyl carbon. The two signals at 5 125.44 and

142.40 were due to the C-3 and C-4 carbons. The terminal methylenic

carbon atom appeared at 5 111.90. The 13C-NMR multiplicity

assignments were made by DEPT and GASPE experiment [181, 182].

The 13C-NMR assignments are shown in Fig. 4.12. The

stereochemistry at Cl, C-5, C-6 and C-7 was established by X-ray

crystallography (Fig. 4.13).

198:-i

AV'“H

147.634.5 33.710 9’r2

1 47.6125.4

H H% 7 19 5H-

1 38.01442.4] 6

'ÿH3H rÿcnr°ÿ12179.3 V

H17.3

O

Fig. 4.12. 13C-NMR assignments of grtiactone (305).

C(14)

C(3)

'CI4)

C(2)

,0(1)©cm C(\C(6! ,0(2)©t(12)

C(10) CI7)C(11)

:(15) C(l

30-0fC(9)

C(13)

Fig. 4.13. X-ray strcuture of grilactone (305).

-:197:-

i

4.3 Plant Material

The plant material (fresh aerial parts of Ferula oopoda) (15.0

kg) was collected from Quetta province, Pakistan in the month of

July 1994. The plant was identified by the plant taxonomist of

Botany Department of the Baluchistan University, Quetta, Pakistan.

Extraction and Purification4.3.1

The fresh plant material was dried in the absence of sunlight

of Ferula oopoda (9.5 kg). This dry plant material was crushed,

powdered, soaked in ethanol (25 litters) for one week, and filtered.

The filtrates were concentrated on a rotary evaporator under reduced

pressure at 45°C, which gave a crude gum (408 g).

The crude gum was suspended in water and extracted with

pet. ether, and the petroleum ether layer evaporated to dryness. The

resulting material (154 g) was found to contain terpenes and

designated as "FO” (Scheme-4.5). The resulting gummy material (154

g) was subjected to chromatography on a silica gel (70-230 mesh

ASTM) column (1.5 kg) subsequent TLC experiments afforded pure

compounds. The detailed fraction procedure is summarized in

Scheme-4.6.

The remaining aqueous layer was then extracted with

chloroform. The chloroform extract was evaporated to dryness on a

rotary evaporator to afford a gummy material (15.0 g) which was

found to contain some minor compounds on TLC.

-:108:-

Ferula oopoda<Vr(95kg)

Powdered and extracted withethanol (25 litres) and evaporatedunder vaccuum

Ethanollc extract (390 g)

Add distilled water

Aqueous extract

Extracted with pet. ether

Pet. ether extract (FO)(154 g)

Defatted aqueous extract

Extracted with chloroform

Chloroform extract(15.0 g)

Aqueous extract

Scheme-4.5.

Pet. ether extract of Ferula oopoda(154 g) "FO"

Loaded on to a silica gel column(mesh 70-230 ASTM) (1.5 g) andeluted with pet. ether : acetone(0-100%) then washed with methanol

Pet. ether :acetone(9:1)

Pet. ether :acetone(9:1)

Pet. ether :acetone(9:1)

Pet. ether :acetone(9:1)

FO-2FO-1(5.0 g)

FO-3(48 mg)

FO-4(40 mg)

Scheme-4.6.

4.3.2 Isolation of Feraileate (303)

The pet.ether extract (FO) 154 g was loaded on silica gel

column (1.5 kg) and was eluted with pet.ether : acetone with

increasing polarity. The fraction FO-4 (40 mg) obtained from this

column at pet. ether : acetone (7:3). The fraction FO-4 was than

subjected to preparative TLC using pet. ether : acetone (7:3) as the

developing solvent afford the pure compound feraileate (304) 18 mg,

1.8 x 10'6%). It gives blue colour with ceric sulfate reagent after

heating upto 120°C.

Spectra Data

[ot]25D = 40° (c = 0.002, CHC13)

UV (MeOH) nm (log e) Xmax: 225 (3.05), 299(2.19) and 303 (3.40).

m(CHCl3) Vmaxcm-l: 2900, 1690, 1600 and 1120.

-:200:-

EIMS m/z (rel. int. %): 306 (M+, 14), 179 (100), 151 (8). 83 (18) and

55 (14)HREIMS m/z (rel. int. %): 306.1103 (M+15), 179.029 (100), 151.021 (10)

and 55.054 (15).

1H-NMR (CDC13, 400 MHz) 8: See Fig. 4.1

13C-NMR (CDCI3, 125 MHz) 8: See Fig. 4.2.

COSY-450: See Fig. 4.3.

nOe: See Fig. 4.4.

HM9C: See Table-4.1.

HMBC: See Fig. 4.5.

Isolation of Guaianolide (304)4.3.3

The ethanolic extract (390 g) of Ferula oopoda extract with pet.

ether afforded a gummy material (154 g) named "FO". The fraction FO

loaded on silica gel column (1.5 kg) and was eluted with pet.ether :

acetone. The fraction "FO-3" (48 mg) was obtained at pet.ether :

acetone (7:3, Scheme-4.6). This fraction when subjected to

preparative TLC using Pet.ether: acetone (9:1) afforded a coumpound

identified as guaianolide (304) after spectroscopic studies

guaianolide (304) was obtained as a colourless amorphous solid (8.5

mg, yield 8.9 x 10'7%)

!Spectra Data

-:201:-

[cc]20D = -21° (c = 0.03, CHC13)

UV (MeOH) ran (log e) Xmax- 202 (2.98), 254 (2.51)

IR (CHCI3) vmax cm'1: 13030 (terminal methylene), 2990 (C-H),

1740 (C=0), 1645 (C=C) and 1120 (C-O).

ElMS m/z (rel. int. %): 230 (M+, 90). 215 (40). 185 (50), 121 (100),

105 (90) and 91 (97).

HREIMS m/z (rel. int. %): 230.1305 (M+ 100), 215.1072 (35) and 121.1017 (20).

1H-NMR (CDCI3, 400 MHz) 6: See Fig. 4.7

13C-NMR (CDCI3, 100 MHz) 8: See Fig. 4.8.

COSY-45®: See Fig. 4.9.

Isolation of Grilactone (305)4.3.4

The fraction "FO-1" was obtained by chromatography on the

silica gel column (loaded with extract of Ferula oopoda on elution

with pet. ether : acetone (9:1, Scheme-4.6). This fraction was

subjected to preparative TLC using pet.ether : acetone (9.5:0.5) as the

solvent system to afford grilactone (305, 2.5 g). This gave a dark

yellow colour with Dragendorffs reagent. The compound was

crystallized after evaporation of the solvent at room temperature,

m.p. 89-91°C.

Spectra Data

[al20D = 85° (c = 1.45, CHC13)

-:202:-

UV (MeOH) nm (log £) Xmax: terminal absorption.

IR (CHCI3) Vmax cm 1: 1730 (lactone C=0), 1600 (C=C) and 1090 (C-

O).

EIMS m/z (rel. Int. %): 232 (M+, 20), 217 (5), 204 (90), 188 (20), 176

(15), 159 (70), 158 (100), 119 (40). 91 (60) and 79 (58).

1H-NMR (CDCI3, 400 MHz) 8: See Fig. 4.11.

13C-NMR (CDCI3, 125 MHz) 8: See Fig. 4.12.

X-ray structure: See Fig. 4.13.i

HREIMS m/z (rel. int. %): 232.1442 (M+ 18), 204.1140(100),

105.0721(40)

-:203:-

5.0 REFERENCES

V

/

V.E. Tyler, PlantaMed.. 33(1), 1 (1987).1.

T. Mukheijee, Fitoterapia, LXD(3), 197 (1991).

M. Hesse "Alkaloid chemistry " John Wiley & Sons, New York,

p. 2 (1981).

2.

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LIST OF PUBLICATIONS

Alkaloidal constituents of Fumaria indica, Atta-ur-Rahman,

Shakil Ahmad, M. Khalid Bhattl and M. Iqbal Choudhary,

Phytochemistry. Vol. 40. p.593 (1995).

1.

New bioactive steroidal lactone from W. somnifera Atta-ur-

Rahman, S. Ahmad, F. Akhater and M. Iqbal Choudhary, J.

Nat Prod, (in press).

2.

3. Isoquinoline alkaloids from Fumaria sp. Atta-ur-Rahman, F.

Akhater S. Ahmad, and M. Iqbal Choudhary, Phytochemistry,

(in press).

Non alkaloidal contituents of Fumaria indica Atta-ur-Rahman,

Shakil Ahmad, M. Khalid Bhatti and M. Iqbal Choudhary.

Phytochemistry, (in press).

4.

5. Isolation and structural studies on the alkaloids of Rhazya

stricta, Atta-ur-Rahman, Habib-ur-Rehman, Shakil Ahmad

and M. Iqbal Choudhary, Heterocycles (in press).

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