8

£ Jl A P E_ JR _ __11

C H E M I S T R Y O F P I P E R P E E P U L O I D E S

I N T R O D U C T I O N T O G E N U S P I P E R

8

The genus Piper belonging to family Piperaceae which5includes about 4 genera and 2000 species is found throughout

the tropical and subtropical regions of the world. Of the total number of Piper species in the world* at least 50 species are recorded in India* of which P. nigrum* P. betel* P. lonqum are widely cultivated. The various species of Piper are mostly climbing shrubs* rarely herbs and trees.

The generic name Piper has been either derived from/r *7

the Sanskrit word "Pippali" or from Latin "Pepper" * which8refers to the hot tasting and aromatic berry of P, nigrum .

The use of Piper species goes far back to antiquity*Besides commercial and economic importance* various species

9—11of the genus have been recorded to be of medicinal importance They are generally aromatic and a number of them possess carminative* antiseptic* pungent and stimulant properties.Many species of Piper have been mentioned in Ayurvedic and unani system of medicine.

The various species of Piper possessing medicinal properties are as followsz-

P. betel

It is a perennial dioecious creeper* a native of Malaysia and is cultivated in India* for its leaves* which are used for chewing. Chewing of betel leaves with various adjuncts is an ancient practice in India and other countries of Eastern Asia. As a masticatory* it is found to be carminative# stimulant*aromatic and digestive. The whole

[)

plant is credited with various medicinal properties* Betelleaves are useful in catarrhal and pulmonary affections* Theoil of betel# is used for the treatment of respiratorycatarrhs/ and in moderate doses appears to have antispasmodicaction of involuntary muscle tissue, inhibiting excessiveperistaltic movement of intestines. It is a CNS depressantand in lethal doses produces deep narcosis leading to death

12within a few hours . In Orrisa, the roots of the plant arereported to prevent child-bearing^'The extract of theleaf stalk reported to possess the mild progestational activity,

15might be responsible for the antifertility effect • Moreover, the essential oil and extracts of the leaves possess activity against gram +ve and gram —ve bacteria.

P. nigrum

nigrum commonly known as "black pepper" is one of the most common spicea valued for its pungent taste, aroma, and is considered to be the ‘king of spices'. Dried berries of P. nigrum constitute the black pepper of commerce. According to various reports the wealth derived by trade in pepper helped in building Venice and Genoa, and, it was the quest for pepper that lead to the discovery of sea route to India by Vascode Gama towards the 15th Century. Pepper is used in the Ayurvedic system of medicine as a stimulant, carminative, aid to digestion and as a cure for diarrhoea, cholera and arthritis among other ailments. Besides its medicinal properties, it us used as a preservative, in flavouring of

foods/ sausages, certain liquors, beverages, soups etc. Oil174J-* nigrum has exhibited antifungal activity . The world

■1 produces 75,000 tonnes of pepper annually and about 15-20 tonnes of its oil are consumed every year.

P. longum17longum or long pepper has been mentioned as

£eepal* in one of the oldest repositories of human knowledge - the Atharva Veda. Being one of the essential basic ingredients in most of the compound preparations, it enjoys an excellent repute in the Ayurvedic system of medicine. In 'Macer Floridus (lOth century A.D.) the Latin Book of verses, mention ' is made of black and white long pepper. 'Macropiper1 has been

mentioned by Simon of Genoa, who was the physician of Pope Nicholas, IV (1288-1308 A.D), after travelling in the east for the study of medicinal plants. The roots and thicker parts of stem are cut, dried and used as an important drug "Piplamul" in the Ayurvedic and Unani system of medicine. Piplamul is attributed with various medicinal properties. It is used as an astringent, in gastric, intestinal, abdominal, pulmonary and respiratory infections, besides being used as a wormicide and laxative as is evident from the following Sanskrit citation

11

The fruits are used as spice, and also in picJdes etc. Pungentpepper like taste produces nurtiOriess and salivation of the

12mouth . India imports a large quantity of long pepper fruits from Singapore and Malaysia.

P. methysticum

It is widely known as intoxicating or narcotic pepper.A drink known as nKava-kavan is prepared by soaking thepulverized roots of the plant to produce an amber, cloudy liquid.

o n 21 9 9This saponfxc, sedative and hypnotic beverage * hasbeen apparently used by Polynesians and the Pacific Island people, for centuries, and is Closely connected with their religious, social and economic life. The native use of this intoxicating drink was first described by Captain James Cook in an account of his voyage to South seas. However, recent reports have described it as an astringent, refreshing drink which produces nothing more than a tingling sensation of mucous membrane of the mouth and shortlived numbness of the tongue•

P. peepuloides

The extract of this species has been found to cause 23convulsions in experimental animals# It has insecticidal

activity equivalent to 1/5th of pyrethrins and is non—cumulative*Petroleum ether extract of the fruits at 0.125 and 0.625%concentrations has been shown to exhibit both insecticidaland larvicidal properties against adults to Musca domestica

24nebulo and larvae of Aedes aeqypti . When chewed# thefruits have pungency, but exhibit strong sialogogue action,

12followed by numbness and tingling sensation on the tongue •12 25The stems and roots are used as a medicine in leprosy '

in the Khasi and Jaintia hills (India). The fruiting spikes are sold either pure or adultered with the spikes of P. lonqum, and the total product is estimated be about 80,000 kg per annum.

Some other species of Piper which are also attributed with certain medicinal properties are as follows

-j n1. Piper auranti cum .

272• Piper novae-ho1landae123. Piper sylvaticum

124. Piper thomsQni285. Piper cannmum

REVIEW

Genus Piper has attracted the attention of research workers throughout the world, because the various species of

the genus have enjoyed considerable importance in Ayurvedic medicine for curing numerous ailments. Recently# attention is directed towards the evaluation of pharmacological and chemical properties# and an extensive study of the different constituents of various species has been made in the last two decades* A galaxy of chemically active constituents which include hydrocarbons# steroids# pyrones and chalcones like kawain and flavokawain# C-allyl compounds like chavicol and chavibetol# lignans such as cubebin# sesamin# eudesmin# diaeudesmin# clusin# epieudesmin# piperidine amides like piperine and piperettine# cyclohexane epoxides like pipoxide and crotepoxide# isobutyl amides# pyrrolidine amides etc. besides some essential oil constituents.

29 30Mention may be made of three unusual cyclohexane * derivatives - crotepoxide# senepoxide and pipoxide belonging to a family of highly oxygenated 1#3-diepoxides exhibiting antileukemic# antibiotic and tumorinhibitory activity.

Among an array of physiologically active compoundsfrom the fruits of Piperaceae plants# which form a groupof secondary metabolites# there is a group of unsaturatedamides. Piperine - a commonly occurring amide was shownby Harvill# Hartzell and Arthur to be more toxic than pyre—thrum against houseflies. It has displayed CNS depressant#antipyretic# analgesic activities# thereby justifying theuse of P. nigrum in which piperine is found in good amount

175in folklore medicine and as a food additive . Experiments

13

conducted to evaluate the scientific basis of the use of "trikatu" group of acrids (P. longum, P. nigrum and ginger) which enjoy a very prestigious position in Ayurvedic formulat-ions have revealed, that they enhanced the drug bioavailabilityHowever, studies on the mode of action of these herbals andtheir pure constituents have established, that it is purepiperine, the active principle of P. longum and P. nigrumthat affects the bioavailability of drugs, with which it iscompounded to a larger extent than "t r i k a t u " ^ ^ .Jacobson (1953) demonstrated the paralyzing effect of Pellitorin -one of the components of black pepper. Its lethal activitywas one-half of that of pyrethrins. Crude and purifiedextracts of black pepper caused high rate of mortality in bothrice weevils and boll weevils on topical application. Piper-cide, dihydropipercide and guineensine from the fruits ofP. nigrum have been found to be insecticidal. They showedremarkable lethal and knock-down activity against adzuki beanweevils, the activity getting enhanced when all the three

87compounds were administered together . Besides, anti- tubercular and local anaesthetic properties were exhibited by isobutylamides from the various species. Extensive work on Piper amides is in progress. Some of the important piperaceae amides are given in Pig.2a & 2b.

Keeping in view the sedative and hypnotic properties7 20 22 26of P. methysticum * * , oxytocic properties of P. auranticum ,

local anaesthetic and antitubercular properties of P. longum

.14

31

32 29 22and P. peepuloides antitumor activity of crotepoxide *

from P. hookeri and P. brachystachyum^ ^ ^ and progestational13 14activity of leaf stalks of P. betel ' , an extensive

chemical study of P. peepuloides Roxb. was undertaken, so that some new chemically active compounds could be isolated and identified , which would initiate further pharmacological studies or at least provide some substantial basis for the same. Various chemical compounds isolated from different Piper species are tabulated belows

TABLE - I

P. acutifolium O^-pinene, sesquiterpene,"" r ’ ” O 0dillapiole, dill-isoapiole

P. anqustifolium Matico camphor, dillapiole,apiole, cis-asaron, limonene, cadiaene, caryophyllene, palmiticacid, phenol, cineole, borneol

. 36camphor .37Dillapiole, peritone ; 2',6'-

dihydroxy-4'-methoxy-dihydrochal—38 cone .

Trans-trans-piperine, trans-cis-. . . 39piperme, cis-trans-pipenne •

40Ishwarol

3,4-dimethoxyphenyl propionic acid, 3,4-dimethoxy phenyl propyl

P. aduncum

P. album

P. amalaqo

P. arboricola

i r>amine41

attenuatum Piperine, piperlonguminine#guineensine, N-isobutyl—deca-trans-

422-trans-4-dienamide •

auranticum Friedelin, epifriedelanol#|B>-sitosterol^? aurantiamide,

44aurantiamideacetate ; piperine,45piperet-tine, sylvatme ? vanillic

acid, dl-N-benzoyl phenyl alanine^; auranamide^; aurantiamide^?stearic acid, linoleic acid,tricontane# cholestanol, cholesterol49

banksii

bartinqianum

bette

betel

Elemicin, dillapiole, N-isobutyl- trans—2-trans-4-octadienamide~^.

51Dihydropiplartme •

Phenolic and non-phenolic substances,chavicol, chavibetol, allyl

52pyrocatechol •53Hydroxychavicol j leucine# phenyl

alanine, alanine# arginine, threonine# serine# aspartic acid# glutamic acid, valine, methionine# tyrosine#“'ft— aminobutyric acid# glycine# proline# histidine, ascorbic, malic#

P. boehmerifolium

P. brachystachyum

P. callosum

P* camphor!cum

P* chaba

P. clusii

P. cubeba

P. fadyenii

P. futokadsura

oxalic acids, maltose, fructose,54glucose, glucuronic acid y chavicol

diastase, tannin, sesquiterpenes,55phenols ; stearic acid, carviol,

hentriacontane, n-triacontanol,C c*7pentatriaeontane ; 0 -sitosterol ;

I

stigmasterol^®•. 59Pipenne .

Crotepoxide, apiole, triacontanol, tricontane, |.J-si to sterol ;

/T *1caryophyllene oxide ; sylvetin,. . 62sesamxn , asannm •

Pipervatine, pipercallosine, pipercallosidine^.

Terpene, borneol and sesquiterpenealcohol, d-camphor, phenol and

. ,36acid •

Piperine,^-sitosterol, piplartine^; amino acids, monosaccharides^.

66Sesamm, pipenne .

Cubebin, hinokinin, dihydroclusin, cubebinin^ •

68Z and E fadyenolide

Futoxide^; futoquinol^®;71 72crotepoxide ; piperenone ;

17

18

isofutoquinol A, isofutoquinol B isodihydrofutoquinol A, isodihydro-

73 isoasarone,futoquinol B74piperenone ;0\-pinene/ camphene,

75p-pinene, sabinene, limonene

P. quineense

P. hispidum

Sesamin# aschatin, yangambin,2,6-bis (2/3,4-tri- methoxyphenyl)-3/7-dioxabicyclo ^3,3,°] octane/

• 76 ... , , . . 77piperine j dihydrocubebm ?N-iso—butyl octadeca-trans-2-trans- -4-dienamide^8; sylvatine, -dihydropiperine/ trichostachine/

. ./\ -dihydropiperlongumxnxne/ v . 79 . . 8 0dihydrocubebm ; wisanme y

■X \* Q Af\ —dihydrowasanine 7 4,5—dihydro-

822‘-methoxy piperine ? wisanidine.N-isobutyl-trans-2-trans-4-piperine

84-dihydrowisanidine ? okalosm N-piperidyl-5- (2-methoxy-4/ 5-

8385.

methylenedioxy phenyl)-trans-2-cis—4-pantadienamide^; phellandrene^ •

4-(5'E-n-hexadecenyl) phenol/21/31-dihydroxy—41 * 61-dimethoxy/2'- hydroxy-3 1,4'/6'-trimethoxy chalcones/ 6-hydroxy-5/7-dimethoxy-

hookeri

j aborandi

kadsura

lacunosum

lonqum

marginatum

8.,11,5,7-dimethoxy and 5,7,8-88trimethoxy flavanones

89Crotepoxide ; p —sitosterol,triacontanol, tricontane, 1-phenyl

90ethanol benzoate ; 1-benzoyloxy methyl-2-benzoyl-3-hydroxy-l, 6-

Qepoxycyclohex-4-ene (pipoxide) ?61caryophyllene oxide .

92 93Jaborandine ? jaborandin-HCl .94Germacrene D .

95Cubebxn

Piplartine^; piperlongumine^;98 99sesamin ? sylvatin, diaeudesmin y

piperionguminine, piperine, methyl,3,4,5-trimethoxy cinnamate^^^;*»sp -sitosterol, triacontanol,

hentriacontane, hentriacontane- 10116-one ; N-isobutyldeca-trans-

1022-trans-4-dienamide ; 1-undecyl-.103.

1043, 4-methylenedioxy benzene pipemonaline, piperundecalidine

105Vitexin, marginatoside ; safrole, piperonal, stearic acid, 3,4-methyl­enedioxy- 2 -hydroxy- 4 ,5-methylene- dioxy-; 2-methoxy-4,5—methylenedioxy

2 0

P. methysticum

P* nepalense

propiophenone s •

Methysticum/ dihydromethysticum ?108dihydrokawain/ dihydromethysticin ?

dl-methysticin/ dl-dihydromethom-109 110ethysticin ? yangonin/ kawain ?

21-hydroxy-4/41,6 *-trimethoxychaleone/2'-hydroxy-4 * 16'-dimethoxy

111chalcone ; tetrahydroyangonin,demethoxy yangonin/ flavokawain A,

112flavokawaxn B ; 1-cinnamoylpyrrolidine/ 1-(m-methoxy-cinnamoyl)

114pyrrolidine ; n-caproic/ 4-oxo-n- nonanoic acid; benzyl alcohol/

115phenyl acetic acid/salicyclic acid ;dihydrokawain-5-o1 ; 11-hydroxy-12-methoxydihydrokawain/ 11,12-

117dxmethoxy dihydrokawain ;f lavokawainC^8? 5/6-dihydro-7/8-

119dihydromethysticin ; 2/4'-dihydroxy-1204/6—dimethoxy chalcone ; pipermeth-

121ystine ? cxnnamylxdene acetone/3/4-methylenedioxy cinnamylidine

122 113acetone 4 11-methoxy yangonin »

Caryophyllene oxide/ triacontanol/ -sitosterol/ N-(2-methyl propyl)

>

deca-trans-2-trans-4-dienamide.

107

pipenne, piperlongumimne .124 ~-jPipenne ; cx -pmene, p -pinene,

D-limonene, 'p-caryophyllene ;f/,-thujene, sabinene, 3-carene,myrcene, -phellandrene, limonene,

— phellandrene, C -terpineno,t

- 126p -cymene, terpinolene ? cis-p-methen-l-ol, cis-p-2,8—menthadien-

127l-ol; trans pino carveol ; chavicine, isopiperine, isochavicin^^ p-cymen-8-ol Me ether, j?-pinone,1 /1 / 4-trimethy lcyc lohepta-2 , 4—dien-6-one, l-terpinen-5-ol, 3,8(9)-p- menthadien-l-ol, 1(7),2-p-menthadien-4—ol, 1 (7)—2-p-methadien-6-ol/

199N-formylpiperidine y 5/10(15)—cadi-130nen—4-01, sesquisabinene III ?

N-isobutyl eicosa—trans—2-trans-4-dienamide ; hentriacontane, ^-sito-

132sterol, hentriacontanone-16 ;133pipercide ? N-isobutyl-11-(3,4—

methylenedioxy phehyl)-2E, 4E, 10E-134undecatrienoic amide ; kaempferol,

rhamnetin, quercetin, isorhamnetin^^ ? (E,E)-N— (2-methylpropyl)-2,4-decadie namide, (E,E,E)-13-(l,3-benzodioxol-5-yl) —N- (2*»methylpropy 1) 2,4712-tri-

123

novae Hollandiae

officinarum

peepuloides

decatrienamide, (E,E,E)-11-(l,3-benzo-dioxol-5-yl)-N-(2-methyl-propyl-2,4,10-undecatrienamide ; N-trans-feruloyltyramine, N-5-(4-hydroxyphenyl)

1372E, 4E-pentadinoylpiperidine ? N-trans-feruloyl piperidine,

138feruperine, dihydroferuperine ?guineensine, pellitorine, N-isobutyl- 2E, 4E, 8Z-eicosa-trienarru.de,N-isobutyl—2E,4E-4E-octa-decadiena-

139 87mide ; dihydropipericide •

Piperine, chavicine, 3 / S —dihydro­piperine, fragramide, piper longuminine, 3-4-methylenedioxy cinnamoyl piperidine, N-isobutyl- trans-2-trans-4-octa-dienamide,N—isobutyl, deca-trans-2-trans-4- dienamide^4^.

141Methyl piperate ; N-isobutyltrideca*-13-(3,4—methylenedioxy phenyl)-2,4,

142 14312-trienamide ; filfiline ;159dihydropiperine ; isobutylamide

144of eicosa-2,4,8-trienoic acid ;. 145piperine •

N-isobutyldeca-trans—2-trans-4- 102dienamide ; pi pa ta line, sesamm,

retrofractum

sanctum

sylvaticum

. 146 , . 147piperine ? dieeudesmin ;-i A Opeepuloidin ; 5-hydroxy-3', 7 ' -

trimethoxy flavone, 5-hydroxy—41,1497-dimethoxy flavone ? N-isobutyl—

146dodeca-trans-2-tran s-4-di enamide ?2—methoxy—4/ 5-methylenedioxy-cis- cinnamoylpiperidide^^ .

151Piperine

Piperolide, methylenedioxy piperol—ide^^; 1,8-oxide of piperolide^^^;4,5-dimethoxy-6-(3,4-methylenedioxy-styryl)-2-pyrone, its tetrahydro

154derivative ? cepharadione A,155cepharadione B ? 5-acetoxy-6—

methoxykawain ; (5s, 6s) — (+)-5-hydroxy-4,6-dimcthoxy-6-trans-

157styryl-5,6-dihydro-2H-pyran—2-one glycolytic piperolidethreo-(lZ)— (-)—5-(2,3—dihydroxy-l-methoxy—3-phenyl propylidene)-4-methoxy—2(5H) — furanone^^*

1 finSylvatine ? diaeudesmin, pipataline, 5-hydroxy-31,4'-7- trimethoxyflavone, 5-hydroxy-7-methoxyflavone^^^; sesamin

162 163aurantiamide acetate ? alkamide

24

P» t r ic h o s ta c h y o n

P . tu b e rc u 1 a turn

P . v o l k e n s i i

sy lv a m id e '* '^ ^ ; s y l v a t e s m i n , 3 ' / 5 -1 o 5d i h y d r o x y - 4 ' - 7 - d i m e t h o x y f l a v o n e

t 166s y l v o n e •

T r i c h o n i n e ^ ^ ; c y c l o s t a c h i n e A ^ ^ ;

c y c l o s t a c h i n e B , c y c l o p i p e r s t a c h -

i n e 1 6 9 ; t r i c h o l e i n 1 ^ 0 ? 1 - p i p e r e t y l

171 172p y r r o l id in e ; t n c h o s t a c h i n e •

P i p l a r t i n e , p i j o l a r t i n e d im e r A ,

1733 / 4 ,5 - t r i m e t h o x y c in n a m ic a c i d •

B i s a b o l e n e , g e r a n y l a c e t a t e ,

3 6c i t r o n e l l o l , s a f r o l , liom onene •

Pi per iov\0 am n une.

N

F l< i-2 bkJ

O FS T R U C T U R E E L U C I D A T I O N

C O N S T I T U E N T S

It is a slender, glabrous climber or an erect shrub about three meters in height, found in tropical Himalayas from Nepal to Bhutan and in Assam (Lushai and Khasi hills) ascending to a height of 900 meters.

Leaves 5.0 - 12.5 cm long and 2.5 <- 5.0 cm wide, ovate,oblong, accuminate, sometimes minutely cordate base;petiole 2.5 - 5.0 cm. long? male spikes slender, clothedwith pelate bracts; female spikes cylindric; fruit 2 mm in

12diameter, not very firmly held on the axis .

^OTRACTION AND ISOLATION

Finely powdered leaves and stem of Piper peepuloide s were extracted in a soxhlet extractor with light petroleum ether (60-80°) for 90 hours. The pet. ether extract was concentrated and subjected to repeated column chromatography over silica gel. Following compounds were obtained in the increasing order of their polarity s-

1. PP-1 - mriacontanol, m.p. 84-5°2. PP-2 - Sesamin, m.p. 122°

DESCRIPTION PLANT 2

3. PP-3 - |2>-sitosterol, m.p. 1364. PP-4 - Dieudesmin, m.p. 157-8

JO°o

5. PP- 5 - 2-methoxy, 4,5-methylenedioxy cinnamoylpiperidide, m.p. 98°

6 . PP- 6 - (Z)-l- 11- (l,3-benzodioxol-5-yl)1-oxo-3-undecenyl pyrrolidine, m.p. 172

7. PP-7

9.8 . PP- 8

PP-9

10. PP-10 - Cyclobutane-2-(1/3-benzodioxol-5-methoxy-6—y 1) -4- (1 , 3-benzodioxo'l-4,5-dimethoxy-6-y 1)

11* PP-11 - Cyclobutane-2 ,4-bis-(l/3-benzodioxol-5-methoxy-6-yD-l, 3-dicarboxapyrrolidide, m.p. 258°

All the above compounds/ except PP.-6 , PP-_7/ PP _9, PPand PP-1_1 have already been reported from Piper peepuloidesA thorough survey of literature reveals that five compoundsare new, and have not been so far isolated from the plantkingdom. An interesting thing about PP-9 is that/ thisparticular amide is with a cis geometry of the double bondin conjugation with the carbonyl group# which is quite rafeSo far there is only one report of a Piper amide with

1 50cis geometry at the double bond . PP—10 and PP-11 are dimers-pyrrolidine cinnamoylamides/ and PP- 6 and PP-7 are long chain pyrrolidine amides.

1 /3-dicarboxapyrrolidide, m.p. 265o

12. PP-12 ^ -sitosterol glucoside, m.p. 285°

1. Compound PP-9 (m.p. 118°) analysed for C15H17°4N issoluble in benzene, chloroform, ethyl' acetate andinsoluble in water, 10% NaOH solution, 5% ^£00^, thusfalling into the category of nM" class of compounds

179according to Shriner, Fuson and Curtin •

2* It was analyzed for following functional groups and elements s-

(a) It shows the presence of nitrogen in Lassaigne's test and absence of sulfur and halogens.

(b) It responds to Labat test for methylenedioxy grouping.

(c) PP-9 responds to unsaturation-' feest with tetranit- romethane and potassium permanganate solutions.

(d) It gives a positive spot test for the methoxyl grouping*

From the preliminary investigation, it is concluded that PP-9 contains the following functionaliti

i. a methylenedioxy groupii. one or more double bonds iii. a methoxyl group.

SPECTRAL DATA OF PP-9

!• UV SPECTRUMMeOHThe UV spectrum showed a strong absorption ^ max

at 336, 275 nm, indicating the presence of an -,Js t P>

STRUCTURE ELUCIDATION OF P P -9

30

unsaturated system in the molecule.

2. IR SPECTRUM

In the IR spectrum (KBr) bands at 1650 ( cx / -unsat­urated amide carbonyl), 1635 (C-N stretching of amide linkage), 1260, 1040 and 930 cm~^ (methylenedioxy grouping) were observed. However/ no bands corresponding to -NH grouping could be observed, which clearly indicates, the presence of nitrogen could be in the form of tertiary amide.

3. 1H NMR SPECTRUMiThe H NMR spectrum of PP—9 is recorded in table II.

The compound displayed a characteristic pair of AB doublets (J=l2.6Hz) each integrating for one proton at q 5.85 and K 6.8 8,assigned to protons at -X and p>

positions of an unsaturated carbonyl system. Thecomparison of chemical shifts and J values of these protons, with the known piperidide derivative of the compound in discussion, suggested the nature of the doublebond to be cis. On the contrary, the same protons

f' r 150with trans geometry appeared at q 6 . 8 and o 7.8 •Two triplets each integrating for two protons at 3.24and k. 3.46 could be due to the methylenes adjacent tonitrogen —N , which can be obtained if

Xthere is restricted rotation across N-C=0, thereby/

making the two methylenes non-equivalent. These two

<v£*Sf ry; Uk<

7.<r: j

methylenes gave further clue to the cis-geometry at the double bond. However# in case of trans geometry# instead two multiplets for the two methylenes would have been obtained at the same position due to the free rotation across ^N—C=0. A multiplet at £ 1.82 may be assigned to other two methylenes of the pyrrolidine ring. A singlet (3H) at Ct 3.88 was due to the aromatic methoxyl and another sharp singlet (2H) at 5 5*86 is attributed to the methylenedioxy grouping. Two singlets each integrating for one proton at & 6.4 and £> 7.04 could be assigned to two aromatic protons. The nature of these signals as sharp singlets# suggested the placement of these two protons para to each other. The presence of only two signals in the aromatic region indicated the benzene ring to be tetrasubstituted there being the presence of one methoxyl function# one methyloaedioxy group (involving two aromatic protons) and one side chain.

cn

32

TABLE I I

values No, of Multiplicity Protonprotons assignment

1.82 4 m H-3",3.24 2 t H-2"3.46 2 t H-5"3.88 3 s O-CH3

5.85 1 d (J=12.6Hz) H—2 *5.86 2 s' H-26.46 1 s H—76 . 8 8 1 d (J=12.6Hz) H-3*7.04 1 s H—4

4. MASS SPECTRUMMass spectrum was in close agreement wL th the

molecular formula C^^-H^O^N proposed for PP-9. It showed a molecular ion fM]+ at m/z 275, and the loss of fragment m/z 70 by giving a prominent peak at m/z 205 (M**’-70), ’his fragment may be due to the pyrrolidine moiety (C HqN) presumed to be present in PP-9. Other characteristic fragments (Fig. 3) were obtained at 246 (35%), 205 (80%), 190 (60%), 175 (30%), 162 (20%) and 28 (100%).

5. 13C NMRThe NMR (table III) of PP-9 further confirmed

the presence of methoxyl function ( a 57.0, q) , methylene- dioxy group ( ^101.5, t) alongwlth other signals as are given in the following tablet

33

m/z Z 1 S

" 1.0

R q - 3

3 413C chemical shift assignments of PP-9 (CDC1„> TMS=0/JEOL-FX 90Q).

TABLE III

Chemical shift 13C multiplicity

24.0 3" t30.0 4« 'C

46.0 2" t47.0 5'* Jc

57.0 Ar-OMe q95.0 7 d101.5 2 t109.0 2* d117.0 5 s1 2 2 . 0 4 d129.5 3* d141.0 9 s147.5 8 s153.0 6 s165.5 \: = o -

Hydrogenation of PP-9

Upon catalytic hydrogenation over Pd/C (10%)/ PP-9 furnished a dihydroderivative, analysed for C15H19°4N' (m.p. 1 0 2°) absorbing only one mole of hydrogoa, thereby indicating the presence of only one double bond in the

molecule. H NMR of the hydrogenated product showed no peaks corresponding toCA and protons* Instead* two multiplets (each of 2H) appeared at S 2*82 and >)2.62/ thereby clearly confirming the presence of double bond in the molecule. The rest of the spectrum (table IV) was almost consistent with that of the parent compound.

TABLE IV

1

values No. of protons

Multiplicity Protonassignment

6.64 1 s H—46.44 1 s H-75.84 2 s H-23.68 3 s Ar-OMe3.40 4 s H-2M, H-5"2.82 2 m H-l*2.62 2 m H-2 *1.82 4 m H-3", H-4®

From the above chemical and spectral studies* PP-9is assigned the structure I (Fig.4). However* in orderto confirm the proposed structure I for PP—9, its synthesis

150was carried out by a novel route (Fig.5).

Esculetin, (_l) was converted into Ayapin (2) using dioiodomethane in presence of a base. 4,5-methylenedioxy coumarin (ayapin) upon simultaneous ring opening with sodium

n (*O I)

hydride in presence of THF and methylation by methyliodide, resulted in the formation of cis and trans cinnamic acids (3a, 3b), 3a being the major product. The mixture was converted into acid chlorides with thionyl chloride, and finally into corresponding amides#by condensing the acid chlorides with pyrrolidine. The pure cis isomer (I) (m.p. 118°)was separated by column chromatography. The synthesized product I showed an identical m.p., NMR, and superimposable IR with that of the natural amide.

13There were certain ambiguities in the assignment of C values especially the chemical shifts of jk and carbon atoms (C—21, C-l1). Therefore, it was thought worthwhile to synthesize two model compounds 8a and 8b.

Coumarin (5) was converted into Z-2-ethoxy cinnamic acid(6) (Fig.6) by simultaneous ring opening with sodium hydroxide

177in presence of DMSO and ethylation with ethyl iodide .The Z-acid on treatment with thionyl chloride yielded the acid chloride, which upon condensation with pyrrolidine furnished a mixture of l-oxo-3-(5-ethoxy-phenyl)—2-Z- propeny1-1-pyrrolidine 8a (m.p. 86°) and l-oxo-3-(5-ethoxy- phenyl)-2-E-propenyl-1-pyrrolidine 8b (m.p. 120°)•

1 OThe C chemical shift values of synthesized amides 8a and 8b are recorded below (table V).

8 8

^ C chemical shift, assignments of 8a and 8b (CDCl^, FX-100 & FX-200).

TABLE V

Chemical shift 13C multiplicity

8a 8b15.4 14.8 Ar-0-CH2-CH3 q24.9 26.1 C~4« t26.7 24.2 C—3' t46.4 46.4 C-5* ■t47.6 45.8 C—2 1 t64.2 63.8 Ar-0-CH2-CH3 •t111.8 111.9 C—2 d121.3 119.0 C—6 d122.9 120.3 C-8 d124.2 129.3 0-4 s129.7 136.5 C-3 d130.5 129.6 C—9 d131.0 130.4 C-7 d153.2 157.7 C-5

\ = 0 /

s163.0 164.4

From the above studies# it has been confirmed, that13in case of Z and E isomers the C chemical shift values of

carbons to carbonyl group is almost same ( n, 111.9), but there is a considerable shift in the position of -carbons.

3o/,3b

R q - 5 -w

NoPH/DMSO SOCU ------&>

Fu^-G

X f e t 8a,

13 ”>The observed C chemical shift values for p -carbon in thesynthesized Z--isomer 63a is £>129.7 while as in the E isomer8b, it is £ 136,5. Thus cA an<3- chemical shifts of

13PP-9 were in full agreement with the q( and p C chemicalwithshifts of model compound, / Z geometry at the double bond.

Another interesting feature of the HNMR spectrum of the Z and E amides, is the geometry of the double bond in conjugation with the carbonyl group. The presence of multiplets instead of a broad singlet for the tw6 methylenes adjacent to nitrogen in the Z-isomer is, because the structure is extremely hindered. The splitting of these two methylenes into multiplets can be attributed to the restricted rotationof >1—0=0 bond, thereby making them non-equivalent* However,f/in case of E-isomer, there is free rotation across -N-C=0 bond, because the pyrrolidine moiety takes a position away from the benzene ring, thereby making the two methylenes in question equivalent which consequently give a broad singlet.

CONCLUSIONFrom the above studies structure I assigned to PP-9

has been confirmed, and it has been identified as Z-pyrrolidine-1 j 3— (6-methoxy-l,3-benzodioxol—5-yl)^-l-oxo -2-propenyl. It is the first rerx>rt of its occurrence from nature.

40

1. Compound PP-10 (m.p. 265°) analysed for C3]_H36W2°9'soluble in chloroform* benzene, ethylacetate and insoluble in water and petrol; It falls into the category of 11M" class of compounds*

2* It shows the presence of nitrogen and absence ofsulfur and halogens in the Lassaigne"s test.

3. It gives a positive Labat and spot test for the methyl- enedioxy grouping.

4. It responds to the spot test for methoxyl grouping.

5. It shows the absence of phenolic* alcoholic hydroxyls* sterols4 unsaturation other than aromatic* and does not respond to acetylation thereby ruling out the possibility of hydroxyls in the molecule

Acid Hydrolysis of PP-10

On acid hydrolysis with alcoholic HC1 in a sealed tube7 compound PP-12 yielded a base as its chloride* which was identified as pyrrolidine hydrochloride (m.p. 213°) by m.m.p. and co-TLC. However* no acid could be identified* because the residue appeared as a tarry mass.

SPECTRAL DATA OF PP-10

i. UV SPECTRUMThe UV spectrum of PP-10 showed absorption bands

- Me OH 24Q 3Q5 and 3 33/A max

41

STRUCTURE ELUCIDATION OF PP-lO

2 . IR SPECTRUM

3.

In the IR spectrum (KBr) a band at 1635 cm was observed..which indicated the presence of an amide group. Lack ofN-H stretching showed it to be a tertiary amide. Bands

— 1at 1595 and 1605 cm showed the substance to be a benzenoid,—1Besides/ bands at 1380, 1275 and 930 cm were assigned

to possible methylenedioxy groupings.

1HNMR SPECTRUM

— 1

The HNMR spectrum of PP-10 is recorded in table VI.

TABLE VI

values No. of protons

Multiplicity Protonassignment

1*60

3.2

3.68 ) )

3.84 ) )

8

10

m

Twooverlappedmultiplets

3xs

2 x N/ CH2“?S2CH2-iH2

^CH2-CH2 2 x N “M + 'v'CH2-CH21,3H

3xAr-OCH.

On the basis of IR PP-10 showed its nature as tertiaryQ i

amide* and the presence of -d-N-. HNMR indicated thepresence of pyrrolidine moiety* besides showing the presence

established the absence of an olefinic bond* ruling out the

be saturated or may have some functionalities. The side chain doesn't have -OH function (indicated by non formation of acetylation product). The spectrum in addition to these

(embedded in the signals of methylenes attached to nitrogen)• These signals were quite different from the signals of the amide discussed so far. The element analysis* and spectral data* coupled with high melting point gave a fading clue about the dimerization across —C=C-§- bond* making cyclobutane ring system in the molecule. Such type of dimerizations are very common in nature and facilitated by photons. Mechani­stically* there is no possibility of ring formation across the double bond, other than cyclobutane system* which is supported by the MS with \Mj as 580. Besides signals for

of methylene dioxy grouping (s*^5.84) and aromatic methoxyJs (3s,A 3.68, 3.84* \4.0) in the molecule. It clearly

possibility of P' -unsaturated carbonyl system in thesystem may

signals showed a triplet at^ 5.02 and a multiplet at £,3,2

protons H-3" * H-6'/ H-6M at ^6.4* 6.6 and 7.1 were observed.

The stereochemistry of the dimer PP—10 can either betruxillic or truxinic (Fig.7). Truxinic structure iscompletely ruled out, because no fragments at m/z 222 and 358could be obtained in the mass spectrum. So# the dimer has atruxillic structure. Presence of a triplet at ^5*02 (2H, 2and 4-H of the cyclobutane ring) confers an -truxillicgeometry on PP-10. These findings are in tune with the

1 7 8observations made by Montando and Caccamese while discussing the stereochemistry of similar cyclobutane systems.

4. MASS SPECTRUMThe mass spectrum of PP-10 was in complete agreement

with "the molecular formula ^3^35N2°9 ProP°sec f°r compound. It gave molecular ion j" M J* at m/z 580 clearly indicating its dimeric nature. Two principal fragments at m/z 275 and 305 (Fig*7) indicated an unsymmetrical nature of the dimer* The two fragments at m/z 275 and 305 are due to the m/z peaks for each monomer which contribute to the dimer. Since the two fragments differ from each other by 30# there is a probability that out of the three methoxyls present in the compound,two may be present in one monomer and the remaining methoxyl in the other monomer, rest of the monomer portions being identical. A dominating feature of the spectrum is the presence of peaks at m/z 274 and m/z 244 (99%) which can be obtained after the elimination of the methoxyl

44

Stereochemistry of PP-10

OGHU ! £

ynisL 275 '

i ^ CH3

VTVZ Z7£

2-2.1.

FRUXIN1C

CKV V ^ 7.m /z ~3o5N

V^ToV1'\0

F c g - a

group (M+ 31) from the fragment at 305 and 275. Such base peaks are indicative of the fact that one methoxyl is in the ortho position1*^ to side chain in each monomer. The other important peaks appeared at m/z 549 (21%), 235 (40%),205 (38%) 98 and 70 (Fig.8).

CONCLUSION

From the above spectral and chemical data PP-10 was assigned the structure II (Fig.9). It is an unsymmetrical dimer of two different pyrrolidides, one being, 3-(l,3-bemso- dioxol-4,5-dimethoxy-6-yl)-2-propenoyl pyrrolidide (peepuloidin) and the other 3— (l,3—benzodioxol, 5-methoxy—6-yl)—2-propenoyl pyrrolidide. Therefore PP-10 has been identified as cyclobutane-2— (l, 3-benzodioxol—5-methoxy—6-yl) -4- (.1,3-benzo— dioxol-4,5-dimethoxy-6-yl) 1,3—dicarboxapyrrolidide. It is the first report of a plant dimer of its kind from the plant kingdom.

4B

STRUCTURE ELUCIDATION OF PP-U

1. Compound PP-11 (m.p. 258°),was analysed for G3oH34N2°8*2. It shows a very similar chemical behaviour with PP-10.

Various chemical tests indicated, that it contains nitrogen, methoxyl and methylenedioxy groupings.

SPECTRAL DATA OF PP-11

1. The UV and IR spectra were almost identical with that of PP-10.

2. 1HNMR SPECTRUMThe HNMR spectrum of PP-11 showed signals which did not correspond exactly to PP-10. Only one signal integrating for six protons texAr-OCH^) was observed at *\ 3.84. Besides other signals, the -two aromatic protons absorbed at 5 6 . 4 4 and 5 7.02. (table - VII) .

50

TABLE V II

\ valuesu No. of protons

Multiplicity Protonassignment

1.60 8 mrOH„—CH„

2 X N CH

3.2 '10 Two2 —2

^ q i 7-ch2 x z +

3.84 3x2

overlappedmultiplets

s

\ c h 2-4h 21# 3H

2xAr-OCH35.02 2 t 2,4-H5.84 4 s

--02 x .CH_

6.44 2 s—-0 2

2x3* H7.1 2 s 2x6' H

1From HNMR data PP-11 was also proposed to be a dimer.

3* MASS SPECTRUMThe mass spectrum of PP-11 was in agreement with the

molecular formula C3oH34N2°8* ^ gave molecular ion [M]+at m/z 550 clearly indicating its dimeric nature. Besides# oi±uer important peaks were observed at m/z 244 (elimination of methoxyl group), 205, 98 and 70 (Fig. 10). So/ the mass spectrum suggest s' PP-11 is a dimer of two similar cinnamoyl amides-both the monomer units of PP-11 being identical. Truxinic structure has been ruled out and truxillic structure assigned to PP-11 because no fragments at m/z 328 and 222 (Fig. 11) could be observed, thereby confirming O^—truxillic structure for PP-11.

m j y i o i'<nlx?n5" 51

Pu^-dLO

v/x 275-

vn/2.2.75

Compound PP-li was synthesized by photochemical conversion* Compound PP-9 was dissolved in chloroform and irradiated with sunlight for 30 hours, whereupon a tarry mass was obtained. This mass was subjected to column chromatography over silica gel. One of the fractions with same Rf as PP-11 was collected and crystallised to yield a crystalline solid (m*p» 258°)•The synthesized photodimer showed an identical TIC, melting point, ^HNMR, and superimposable IR with that of natural PP-11 and showed no depression in m.m.p. with PP-11»

CONCLUSION

From the chemical and spectral data PP-11 was assigned the structure III (Fig.12). It is a symmetrical dimer of two identical cinnamoyl amide moieties. It was identified as cyclobutane-2,4-bis-(l,3-benzodioxol-5-methoxy -6-yl)-1,3- dicarboxapyrrolidide. It has been reported for the first time from the plant kingdom.

SYNTHESIS OF PP-11

53

F i g -12,

1. The compound PP-6, (m.p. 172°) analysed for C22H2^N02is soluble in benzene, chloroform/ ethyl acetate and alcohol and insoluble in water, 5% and 10% NaOH solutions,thus falling into "M" class of compounds according to Shriner,Curtin and Fuson.

2. After following the usual procedure for the detection of elements and various functional groups, it was concluded that PP-6 has a tertiary amide function with some unsatur­ation, along with a benzenoid system carrying a methylenedioxy group.

SPECTRAL DATA OF PP-6

1* UV SPECTRUMCompound PP-6 showed absorption bands A at/ ' max

232 and 285 nm*

2. IR SPECTRUMThe infrared spectrum of PP-6 in KBr showed char­

acteristic bands at 1660 cm’"’ , indicating an amide carbonyl grouping, besides characteristic bands at 1260,1045 and 930 cm were observed for methylenedioxy grouping.

3. 1HNMR SPECTRUM■ HNMR of Pp-6 (table VIII) indicated the nature of

the compound t.o be aromatic, with a side chain possess­ing an isolated double bond (cis) by showing signal of

STRUCTURE ELUCIDATION OF PP-6

a triplet (2H) at 5.3. A multiplet (2H) centred atK 2.4 could be assigned to methylene adjacent to doublebond and another doublet (2H) at 2.9 could be due tothe methylene adjacent to -R- group. The presence andnature of carbonyl as amide is already confirmed by a band

—1m IR at 1660 cm • The nature of amxde as pyrrolidide is confirmed by the loss of fragment of m/z 70 in mass spectrum. A triplet (2H) at \ 2.8 may be due to the methylene adjacent to benzene ring (benzylic)• The app­earance of this particular methylene suggested, that the double bond is not in conjugation with the benzene ring. This fact is further supported by a broad singlet for three aromatic protons at 6.4. However, in case 'the double bond was in conjugation with the aromatic ring the three aromatic protons would result in two separate singlets - one (2H) at ^ 6.8 and another (lH) at K 6.9. ^HNMR further showed the presence of five methylenes (lOH) andmethylenedioxy group (2H) in the molecule by recording

ia broad singlet at 1.2 and a sharp singlet at ■>> 5.8 respectively. The methylenes of the pyrrolidine ring were observed at ^ 1.56 (m, 4H) and ,C 3.4 (m, 4H).

TABLE V I II

values No* of Multiplicity Protonprotons assignment

1*2 10 m -(CH2)5-X-CH -CH

1*56 4 m -N* >CH2-CH2

2*4 2 m - (CH)2-allylicmethylene

02.9 2 b.d -Oi2_C-2*8 2 t -CH2-(benzylic)

3.4 4 m —NCH2-CH2

5.3 2 t -CH=CH-~~0

5.8 2 s CH..-0

6.4 3 bs aromaticprotons

4. MASS SPECTRUMIn MS, PP-6 showed (mQ+ at m/z 357 supporting the

molecular formula C^H^NO^. Loss of a fragment at m/z 70 to give a peak at 287# supported the nature of the amide as pyrrolidide. Besides# other peaks were observed at m/z 288 (92%)# 205 (38%)# 192 (31%)# 177 (8%)# 163 (8%), 149 (31%)# 135 (92%) and 112 (15%) (Fig.13).

Hydrogenation of PP-6

Upon catalytic hydrogenation over Pd/C# compound PP-6 absorbed only one mole of hydrogen# thereby confirming the

presence of only one double bond in the side chain. HNMRof the hydrogenated product (m.p. 166°) gave a broad singletat 1.3 integrating for sixteen methylene protons. A multi-plet (4H) at £> 1.5 and another multiplet at \,2.8 (2H) were

.. CH -CH ftattributed to -N'C '• and —CH9-c- respectively.

ch2- ch2

Besides another multiplet at ^3.4 was assigned to the methylenes adjacent to nitrogen in the pyrrolidine ring. A triplet at <^2.9 indicated the presence of benzylic protons. A sharp

singlet at ^5.9 is characteristic of methylenedioxy group.A broad singlet at <£ 6.5 (3H) was due to the three aromatic protons•

The exact position of the double bond could not be located in PP-6 due to paucity of the compound, which was obtained in extremely low yield after purification through HPLC. However, mass fragmentation pattern clearly indicated the presence of the double bond at position 3.

CONCLUSION

1

From the above .chemical and spectral studies PP—6 was assigned the structure IV (Fig.14) and identified as (Z)-l-^11-(1,3-benzodioxo 1-5-v 1)-1 -oxo-3-undeceny 1 pyrrolidine.

58

1. The compound PP-7 (m.p. 187°) was analysed for C22HggN04.2. When subjected to various chemical tests, PP-7 showed that,it was a benzenoid system carrying a methoxyl, and methylenedioxygroupings, besides having a tertiary amide function.

SPECTRAL DATA OF PP-7

1. UV and IR spectrum did not show any particular difference when compared with that of PP-6.

2. 1HNMR SPECTRUM*1Compound PP-7 recorded signals in HNMR (table IX)

almost at the identical positions as were observed in PP-6 except for a few additional signals. A singlet(3H) at £> 3.8 was attributed to the aromatic methoxylgroup. Instead of a broad singlet integrating for three protons of the aromatic ring in PP-6, two sharp singlets at A, 6.4 (lH) and ^ 7.04 (lH) were observed and assigned to the two aromatic protons para to each other in

STRUCTURE ELUCIDATION OF PP -7

TABLE IX

-Nvalues

1.2

1.58

2.4

2 . 8

2.9

3.4

3.85.3

5.8

6.47.04

No. of protons

Multiplicity

10

4

2

22

32

11

m

m

m

td

m

ss

Protonassignment

- (ch2 ) 5-

-N/.CH2-CH2

i

'CH2-CH2- (CH2)-aHylic methylene- (CH9) benzylic

l 0-ch2-c-

—n:^ H 2-CH2-c h2-ch2

Ar-0CH3-CH=CH- *~0 ■Ar-HAr-H

3. MASS SPECTRUMIn MS, | MJ** at m/z 387 was observed which supported

the molecular formula C2 3H3 3 N04 proposed for PP-7» Mass fragmentation pattern proposed for PP-7 is given in Fig.l5. Important fragments in MS were observed at m/z 318 (20%), 235 (46%)# 220 (61%), 165 (10%), 152 (61%) and 112 (15%).

Compound PP-7 was hydrogenated in a similar manner as PP-6 over 10% Pd/C* whereupon it absorbed only one mole ofhydrogen confirming the presence of a single double bond in

"1 oPP-7, HNMR spectrum of the hydrogenated product (m.p. 180 )gave signals at the same positions as the hydrogenation productof PP-6 with an additional signal at £) 3.8 (3H) assigned tothe aromatic methoxyl group. Besides two singlets eachintegrating for one proton at X, 6.4 and >> 7.04 were due tothe two aromatic protons para to each other.

The exact position of double bond could not be located in PP—7 because the compound was obtained in extremely low yield after purification through HPDC.

(

CONCLUSION

Prom the above chemical and spectral studies# PP-7 was assigned the structure V# and identified as (Z)- 1- Q li-(1,3- benzodioxol-6—methoxy—5-yl)—1-oxo—3-undecenvl^ pyrrolidine (Fig.15)

Both PP-6 ard PP-7 are also being repojrfced. fox’ the fSc&t. time in nature»

(51Hydrogenation of PP-7

V I

W i i vV \ *5 ^ /

31^ o

pL0- 15*

NM

F ig - 16

PP-1 was obtained as a colourless/ crystalline compound (m.p. 84° ) . It is soluble in benzene, ether, slightly soluble in hot alcohol and insoluble in water. Various chemical tests indicated the presence of alcoholic hydroxyl group and absence of other functional groups. It was confirmed to be Triacontanol by co—T1G, m.m.p and superimposable IR with an authentic sample.

STRUCTURE ELUCIDATION OF PP-2

PP-2, (m.p.\ZZ°) analysed for c2oHi8°6/Was soluble in pet. ether, benzene, chloroform and insoluble in water.

2. The compound was tested for various functional groups chemically. It gave negative tests for nitrogen, sulfur, halogens, sterols, free carboxylic, methoxy, sterols, phenolic and alcoholic hydroxyl groups.

3. It responded to Labat test for methylenedioxy grouping.

4. UV spectrum . EtOHUV spectrum showed strong absorption at 286

and 235 nm.

5. IR spectrumIR spectrum of PP-2 showed peaks at

(aromatic system), 1071, 913 (cyclic diether) and 718,925 cm (methylenedioxy grouping).

STRUCTURE ELUCIDATION OF PP-1

CC1,V ' 1 6 0 0 /max

6. HNMR spectrum^HNMR of PP-3 established its nature as 3,7-dioxa-

bicyclo 1 3,3,0joctane with two phenyl rings orientedin r/. position by giving a doublet (4H, J=4Hz) at^ 3*70assigned to H-2 and H-6, a multiplet (2H) at ^ 3*03assigned to H-l and H-5, a multiplet (2H) at £ 4.00 -3.80 due to two axial protons at C-4 and C~8 and anothermultiplet (2H) at ^ 4.4 - 4.2 was due to two equitorial

*1protons at C—4 and C—8, HNMR clearly establishedsymmetrical nature of this compound. Both the phenyl rings possessed methylenedioxy grouping ( 5*90, 4H, s). Six aromatic protons were spread over >^6*8 — 7*0 region.

7 * Mass spectrumThe mass spectrum showed Mj at m/z 354 which was in

complete agreement with the molecular formula C20H18°6*

From the above spectral and chemical data, PP-2 was identified as Sesamin and confirmed by m*m.p and co-TLC with an authentic sample of Sesamin.

STRUCTURE ELUCIDATION OF PP-3

PP-3 (m.p. 136°) obtained as colourless needles, analysed for C29H5Q0, is soluble in benzene, chloroform, ethylacetate and insoluble in water. It gave a positive Libermann Burchard test for sterols. Its melting point, m*m.p. and co-TLC with an authenic sample identified it as jp>-sitostero 1 *

1

Compound PP-4 (m.p. 157°) analysed for C22H26°6soluble in chloroform ethyl acetate and insoluble in lightpetrariL and water. Chemical tests indicated the presence ofmethoxyl group or groups and absence of nitrogen/ sulfur,halogens,hydroxyl, carboxyl, : groups etc. In UV spectrum

EtOHPP—4 showed characteristic bands A at 286 and 235 nm.— ' max VDpIR spectrum of PP—4 showed peaks at \/ cm 1055, 1085 ---- v max(cyclic diether), 1245, 1600 (aromatic system)•

PP-4 was identified as (+) -Diaeudesmin by superimposable IR, undepressed m.m.p and co—TLC with an authentic sample of (+)-Dieudesmin•

STRUCTURE ELUCIDATION OF PP-5

1. The compound PP-5, (m.p. 98°) analysed for C16H19°4N'was soluble in benzene, ethyl acetate, chloroform and insoluble in water.

Various chemical tests indicated the presence of nitrogen, unsaturation, methylenedioxy and methoxyl groupings, and absence of sulfur, halogens, carboxyl, alcoholic or phenolic hydroxyl group in PP-5.

2 * UV spectrumUV spectrum of PP-5 showed absorption bands at

333, 275, and 240 nm.3. IR spectrum

CC1In the IR spectrum of PP-5 showed peaks at \r 411 11 max

STRUCTURE ELUCIDATION OF PP-4

1160, 1550, 1200, 1000, 930 and 970 cm-1.1HNMR spectrum

iThe HNMR spectrum of PP-5 showed a di aracteristicpair of doublets each integrating for one proton at

*^5.95 and q 6.86. The position and J value (12.3 Hz)of the two doublets, clearly indicated cis geometry ofthe double bond. Contrary to it, two doublets at £ 6.7and (^7.8 with J=l5.8Hz would have been obtained in case

150there was trans geometry of the double bond . A broad singlet at 1.20 - 1.90 (6H) was assigned to the three methylenes of the piperidine ring (-N- (OH^Cf^) 2CS2^' and two triplets at f, 3.30 ard 3.55 due to the methylenes adjacent to nitrogen, gave separate signals because of non-equivalent nature, due to the restricted rotation of the double bond in conjugation with the carbonyl group# It is a characteristic feature of the spectrum. Besides, two singlets at £3*90 (3H) and £ 5.95 (2H) were obtained for the aromatic methoxyl and methylenedioxy groupings respectively* Two singlets each integrating for one proton at c 6.5 and £>.6*9 were assigned to the two aromatic protons*

Prom the above chemical and spectral data PP—5 was identified as 2-methoxy—4,5-methylenedioxy-Z- cinnamoylpiperidide. The final confirmation was obtained by co-TLC, m.rrup and a superimposable IR with an authentic sample*

1. Compound PP-8 (m.p. 149-36)® analysed for c16Hi9°5N/ was soluble in benzene, chloroform/ ethyl acetate and insoluble in water and hot alcohol.

Chemical tests indicated the presence of nitrogen/ unsaturation, methylenedioxy and methoxyl groupings/ and absence of other functionalities.

2. UV spectrum- EtOH .

UV spectrum of PP-8 showed bands /\ max '305 and 240 nm.

3. IR spectrumIn IR spectrum PP-8 showed peaks at 1642/ 1380/ 1275,

997/ 932/ 1605 and 1595 cm"^.

4. ^HNMR spectrumIn the HlNMR spectrum PP-8 gave a multiplet at ^ 1.65

2*2 (4H) which could be assigned to the methylenes of^ CH -CH

the p y r r o l i d i n e r i n g (-N. i ) /Whose p r e s e n c e was^H2 —2

indicated by the mass spectrum. Another multiplet at f'ry 3.65 (4H) was due to the methylenes adjacent to nitrogen in the pyrrolidine ring. Two doublets at

6.68 and ^7,9 each integrating for one proton were due to the and |3 protons. The position of the doublets and coupling constants (J=16Hz) indicated E geometry across the double bond which is supported by the presence of a multiplet at 3*65/ instead of

STRUCTURE ELUCIDATION OF PP-8

( 18

two clear and distinct multiplets for the methylenes of the pyrrolidine ring adjacent to nitrogen as in caseof Z geometry. Three singlets integrating for three

— -0protons (Ar—OMe) , two protons ( '.CH0) and oneprotons (Ar-H) were obtained at .\ 4.10, Vs 5.98 and v 6.68 respectively.

5• Mass spectrum

Mass spectrum of PP-8 indicated J ~ M a t 305*Other fragments were obtained at 275, 274, 259, 235, 220, 205, 193, 178, 157, 98 and 70.

Prom the above chemical and spectral data PP-8 was identified as Peepuloidin.

E X P E R I M E N T A L

EXTRACTION AMD ISOLATION PROCEDURE

The coarsely, powdered stems and leaves of Piper peepuloides (1.5 kg) were extracted exhaustively in a soxhlet extractor with light petroleum ether (60-80°) for 90 hrs. The extract was concentrated underreduced pressure to 3/4th of its volume, and on keeping in a refrigerator for a few days, it did not yield any solid. The solvent was completely removed from the extract, to furnish a dark brown viscous residue (45 g). The residue (20 g) was subjected to column chromato­graphy over silica gel (l.5 kg) and eluted with different solvents in order of their increasing polarity. In all 360 fractions each of 50 ml. were collected,and the various compounds obtained from different fractions are given in table X

7 0

TABLE X

Fractions Solvent Compound

1 - 24 Petroleum ether -25 - 30 Pet. ether : EtOAc (5%) PP-131 - 36 it (.796) PP-1 + PP-237 - 47 ii (10%) PP-248 - 55 it (15%) PP-2 + PP-356 - 76 n (20%) PP-377 - 87 ii (25%) PP-3 + PP—488 - 128 h (30%) PP-4129 - 135 ii (35%) PP—4 + PP-5136 - 154 n (40%) PP-5155 - 175 it (45%) PP-5 + PP-6176 - 186 ii (50%) PP-6 + PP-7187 - 210 n (55%) PP-8211 - 230 n (60%) PP-8 + PP—9231 - 255 f! (65%) PP—9256 - 276 it (70%) PP—9 + PP-10277 - 297 n (80%) PP-10298 - 320 n (90%) -321 - 331 EtOAc -332 - 342 EtOAc : MeOH (5%) PP-10 + PP-11343 350 (10%) PP—12

71Fractions 1-24

These fractions were obtained as a yellow mixture which solidified on cooling. They were discarded, being considered a mixture of fats and oils.

ISOLATION OF PP-1

Fractions 25-30These fractions furnished a waxy compound which showed

an identical spot on TLC. Crystallization from pet. ether : Ethyl acetate furnished a TLC pure colourless compound PP-1 (300 mg), C^qH ^ 0* m.p. 84°* (Rf 0.8/ Benzene - ethyl acetate, 3s2). The compound PP-1 was identified as Triacontanol by superimposable IR, undepressed mixed melting point and co-TLC with an authentic sample of Triacontanol.

ISOLATION OF PP-2

Fractions 37-47These fractions obtained by elution with 10% Ethyl

acetate in pet. ether, after showing an identical spot on TIC were suitably pooled and distilled. Removal of solvent furnished a colourless/ waxy solid. This solid upon repeated crystallization from ethyl alcohol gave TLC pure, colourless crystals of PP-2 (70 mg)/ C20Hi8°6 m-P* 122°/ (Rf 0.4, Pet. ether - Ethyl acetate, 1 s 1) . It was identified as Sesamin by an undepressed mixed melting point, and co-TLC with an authentic sample of sesamin.

ISOLATION OF PP-3

Fractions 56-76

These fractions showed an identical spot on TLC plate.So after pooling and removal of solvent, they furnished a white crystalline solid, which upon crystallization from benzene - ethyl acetate gave white needles of PP-3 (550 mg), C^gHgQO, m.p. 136°/ (Rf. 0.4, Benzene - EtOAC, os2). PP.3 was identified as p-sitosterol by superimposable IR, undepressed mixed melting point and co-TLC with an authentic sample.

ISOLATION OF PP-4

Fractions 83-128Upon TLC examination, these fractions gave a single

and identical spot. They were suitably pooled and concentrated after which dirty white needles were obtained. Repeated crystallization from ethyl acetate - pet. ether furnished pure needles of PP-4 (80 mg), <-'22 26 6# 157—8° (Rf. 0.5,Ethyl acetate — benzene, 2 j5). It was identified as Diaeudesmin by superimposable IR, undepressed mixed melting point and co-TLC with an authentic sample*

ISOLATION OF PP-5

Fractions 136-154

These fractions were pooled, after giving an identical and single spot on TLC plate* After concentration, a dirty

white solid was obtained# which upon repeated crystallization from ethyl acetate — pet. ether furnished white crystals of

PP-5 (100 mg), c16Hi9°4N m,P* 98° R£* °*7' Benzene - ethyl acetate# 3 si). It was identified as 2-methoxy# 4 # 5-methylenedioxy cinnamoyl piperidide by chemical and spectral data.

ISOLATION OF PP-6 AMD PP-7

Fractions 176-186

These fractions# eluted with 50% pet. ether - ethyl acetate showed an identical TIC. After .pooling and concen­tration light green viscous residue which showed two spots on TLC was obtained. The residue was subjected to repeated column chromatography over silica gel# but isolation could not be achieved. Finally the mixture was separated by HPLC (WATER ASSOCIATES# Model 440) using C-18 Bonda Pack column (Flow rates 1.5 ml/mirj# Chart speeds 1 cm/min.# elution with lsl# MeOHswater). PP-»6 (25 mg)/ C22H31N03 Founds C#72.86 ; H# 8.59; N, 3.80/ Requireds C# 73.91?H#8.73; N# 3.91% m.p. 172° (Rf. 0.6, benzene - ethyl acetate/ 3 si) was identified as (Z)-l- 11- (l, 3-benzodioxol—5-yl—1-oxo—3-undecenyl | pyrrolidine and PP—7 (30 mg)/ C23H33N04' Founds C/ 71.15; H# 8.40; N/ 3.20, Required? C, 71.28;H/8.58; N, 3.61% m.p. 187° (Rf. 0.8/ benzene - ethyl acetate,3 si) was identified as (z)-l-j 11-(l,3-benzodioxol-6-methoxy

i-5-yl)-l~oxo-3-undecenyl j pyrrolidine.

JHydrogenation of PP-6 and PP-7

The compound PP-6 (20 mg) was dissolved in methanol

(40 ml), and 10% Pd/C (30 mg) added to it. The reaction was complete af'cer 4 hours absorbing, only mole of hydrogen. The catalyst was removed by filtration. Removal of solvent furnished an oily residue, which after chromatography and crystallization furnished a white solid (m.p. 166°). ^HNMR study of the hydrogenated product gave full proof for the presence of a double bond.

Compound PP-7 was hydrogenated in a similar manner.The dihydro product was crystallized to furnish a white solid (m.p. 180°)•

ISOLATION OF PP-8

Fractions 187-210Compound PP-8 was obtained as a dirty yellowish waxy

solid from the fractions eluted with 55% pet. ether - ethyl acetate. This yellowish waxy solid on treatment with benzene and subsequent cooling furnished a crystalline white solid, which produced upon crystallization from ethyl acetate, TLC pure, white crystals of PP-8 (300 mg) C^H^O,-, m.p. 149-50° (Rf. 0.5, benzene — ethyl acetate, 3 si). It was identified as Peepuloidin after thorough, chemical and spectral studies*

ISOLATION OF PP-9

Fractions 231-255

Upon TLC examination the above fractions showed an identical and single spot, which was UV flourescent. After pooling the fractions and distilling off the solvent, a dirty

white waxy solid was obtained, The solid upon column chrom- ato'graphy over silica gel and elution with pure benzene furnished white flakes of PP-9 (100 mg), c^5Hi7 °4N^Founds C, 65.38; H, 6.20; N, 5*02, Required;; C, 65.44;H, 6.22; N, 5.08%. m.p. 118° (Rf — 0.6, benzene - ethyl acetate, 6;4) . I t was identified as Z-pyrrolidine-l|^ 3- (6-methoxy-I,3-benzodioxol-5-yl)^j -l-oxo-2-propenyl by chemical and spectral studies.

Hydrogenation of PP-9

Compound PP-9 (50 mg) was dissolved in methanol (30 ml).10% Pd/C (70 mg) was added to the mixture. Absorption of hydrogen was complete after 3 f hours, when the TLC examination of the product showed the reaction to be complete. The catalyst was filtered and the solution concentrated, yielding a dark brown waxy solid, which upon purification furnished a white crystalline solid (m.p. 102°). ^HNMR spectrum of the product confirmed the presence of only one double bond in the molecule*

SYNTHESIS OF PP-9

Synthesis of 6,7-methylenedioxy coumarin

Esculetin (2 gm) was dissolved in dry acetone (200 ml) and N,N—dimethylformamide (20 ml) added to it. To the above solution, anhydrous potassium carbonate (50 gm) and methylene iodide (20 ml) was added. After refluxing the mixture on a water bath for 50 hours, it was filtered off* and potassium

carbonate washed with hot acetone. Acetone was partially distilled off, water added and the residue (800 mg) obtained was filtered. It was washed with hot water, dried and crystallized from ethyl acetate *- pet. ether mixture, when white solid of ayapin was obtained# m»p* 231°, cioHe V 1HNMR (CDC13)j £ 6.13 (2H# s, C H ^ ) , 6.33 (1H, d, J=9.5Hz,—CH=CH—C0-0-), 7.63 (lH, d, J=9.5Hz# -CH=CH-C0-0)# and 6.90 (2H, s# Ar-H).

Synthesis of 3-(6-methoxy-l,3-benzodioxol-5-yl)—2-propenoic acid

Ayapin (450 mg) was dissolved in dry THF (20 ml)• To this was added (60%) NaH (500 mg) dispersed in oil, slowly with constant stirring maintaining the temperature of the mixture at 25°C. Once the addition of sodium hydride was complete, methyliodide (3 ml) was added and stirring continued for two hours. After cooling the mixture, and the addition of 5N HC1 (40 ml), it was extracted with ethyl acetate. The organic layer after several washings with distilled water# was dried over anhydrous sodium sulfate# filtered and concentrated when a mixture of cis and trans 3-(6-methoxy, l#3-benzodioxol~5-yl)-2-propenoic acids (200 mg) was obtained.

Synthesis of Z-pyrrolidine—1^3-(6-methoxy-l, 3-benzodioxol— -5-yl)^j -l-oxo-2-propenyl«

The amide was synthesized from the mixture of Z and E# propenoic acids. The mixture of cinnamic acids (200 mg)

77

in dry benzene was converted into a mixture of Z and E acid chloridesby reacting with thionyl chloride in presence of a little pyridine. The acid chlorides were dissolved in dry benzene and pyrrolidine (1,5 ml) added to it. The mixture was warmed on a water bath for 0.5 hr. After cooling the benzene solution/ it was washed with dilute H31 to remove unreacted pyrrolidine. The Z isomer was separated from the mixture by repeated crystallization from ethyl acetate - pet. ether, and obtained as '.white needles (50 mg) / m.p. 118°, (Rf. 0.6/ Benzene ** ethyl acetate, 6;4). The synthesized amide showed a superimposable IR, undepressed mixed melting point and co-TIC with that of natural PP-9.

FURTHER STUDIES ON PP-9

Synthesis of l«oxo-3-(5-ethoxy-phenyl)-2-Z-propenyl-l- pyrrolidine and l-oxo-3 (5-ethoxy phenyl)-2-£-propeny1-1- pyrrolidine .

Synthesis of (Z)-2-Ethoxy cinnamic acid

Coumarin (2 g) was dissolved in dime thy lsuE&xide (40 ml) and sodium hydroxide (4 g.) added to it. The mixture was stirred for ten hours/ followed by the addition of ethyl iodide (l ml). After twenty hours the reaction was complete. Water was added, and after acidification with dilute HC1, the mixture was extracted with ethyl acetate. The extract was washed with water several times, dried over anhydrous sodium sulfate, filtered and concentrated when dirty yellow crystals of Z-cinnamic acid were obtained. The yellow

crystals upon repeated crystallization from ethyl acetate -pet. ether mixture afforded white crystals of the Z- acid(1.5 g.)# C.. o0_, m.p. 94° (Rf. 0.2# benzene - ethyl acetate#11 12 33:1). 1HNMR (CC14) s Q 1.4 (3H# t*Ar~OCHjCH3) , 4.01 (2H, q/Ar-OCH2-CH3)# 5.9 (lH, s# J=l2.5Hz/ -CH-CH-C0-), 6.6 - 7.4 (4H# m) and 7.6 (lH# s# J=12.5Hz# -CH-CH-C0-).

Synthesis of acid chloride of (Z)-2-Ethoxy cinnamic acid

Z- cinnamic acid (200 mg) was dissolved in dry benzene(30 ml). Thionyl chloride (2-3 drops) and pyridine (4 drops)

iwere added to the mixture# and the mixture kept at room temperature (l8°C) for four hours. Pyrrolidine (2 ml) was added to the mixture of Z and E acid chloridesand after shaking for about 0.5 hr.# the solvent was removed. The waxy solid was extracted with ethyl acetate and given a thorough washing with dilute HC1 to remove excess pyrrolidine. The ethyl acetate extract after drying over anhydrous sodium sulfate and concentration provided a mixture of 25 and E amides which were separated by column chromatography over silica gel using benzene as eluant. Both Z and E amides were obtained as white crystals which after crystallization from ethyl acetate - pet* ether furnished l-oxo-3-(5-ethoxy phenyl)-2-1 tZ-prupeny1-1-pyrrolidine 8a# (75 mg)# m.p. 86° (Rf. 0.6#Benzene - ethyl acetate, 7:3)# and l-oxo-3-(5-ethoxy phenyl) -2-E—propenyl-l-prrolidine 8b# (50 mg)# m.p. 120° (Rf. 0.7 Benzene - ethyl acetate# 7:3).

-HNMR (CC14) of 8a: £ 7.5 (lH, dt H-3, J=12Hz) , 7.2 (4H,m, H—6,7,8/9)/ 5.8 (lH, d, H-2, J=12Hz), 4.1 (2H, q, Ar-OCH2-CH 3.4 (2H, t, H-2'), 3.1 (2H, t, H-5'), 1.6 (4H, m, H-31, 4') and 1.4 (3H, t, Ar-OCH2-CH3).

XHNMR (CC14) of 8bs £ 8.1 (lH, H-3# J=l6Hz), 7.2 (4H, m, H-6,7,8,9), 6.4 (lH, d, H-2)/ 4.1 (q, 2H/ Ar-0-CH2-CH3),3.6 (m, 4H/ H-21/ 5'), 2.0 (m, 4H, H-31/ 4*'), 1.4 (3H/ t, Ar-0-CH2-CH3).

ISOLATION OF PP-jO

Fractions 277—297

These fractions yielded a dirty yellow solid which showed the presence of a major component on TLC examination besides a few very minor components. The fractions were pooled, concentrated and subjected to column chromatography over neutral alumina using benzene - ethyl acetate as eluants. Fractions obtained with 5% benzene - ethyl acetate mixture furnished a dirty white crystalline solid, which upon purifi­cation with acetone and petrol furnished a crystalline white solid PP-10 (50 mg), C31H36N2°9# fr°und" C' 64.10; H, 6.1?N/ 4.2? Required:: C, 64.12; H, 6.2 and N/ 4.8%)/ m.p.265-267° (Rf. 0.5, Benzene - ethyl acetate, 8s2). It was identified as cyclobutane—2— (1/3-benzodioxol—5-methoxy-6—yl)- 4- (1/ 3-benzodioxol-4,5-dimethoxv-6-vl) 1, 3-dicarboxapyrrolidjde by spectral and chemical studies.

PP—lQ (20 mg)/ was added 10% alcoholic KC1 (2 ml)>The mixture was heated for 85 hours in a sealed tube on a steam bath. After diluting the contents with water (2 ml)/ alcohol was removed, the residue made just alkaline with KOFI

solution/ extracted with chloroform and washed with water*The washings were mixed with aqueous extract. The chloroform extract was extracted with dilute HC1 (10 ml). The acid aqueous extract was evaporated/ to yield a white residue which on crystallization from ethyl acetate afforded shining crystals/ identified as pyrrolidine hydrochloride (m.p. 213°) by co-TLC and undepressed mixed melting point with an authentic sample*

A tarry mass was obtained, when the aqueous alkaline extract was acidified with dilute HCl and extracted with chloroform. It could not be identified,as purification was not possible. However, it was found to behave like an acid from the chemical tests*

ISOLATION OF PP-11

Fractions 332-342

These fractions showed a mixture of two compounds on TLC plate. Upon cooling and concentration they were rechromatographed over silica gel and eluted with various solvents, but the two compounds could not be separated.So after a number of trials/ the mixture was separated

80Acid Hydrolysis of PP-10

81into two compounds PP-10 and PP—11 by HPLC using MeOH : water (50s50). Compound PP-11 was obtained as a white crystalline solid (60 mg)/ C30H34N2°8 ^ oun< : c• 65.35; Hf 6.1; N/ 5.02. Required? C, 65.44; H/ 6.2; N, 5.08%), m.p. 258-61°. (Rf. 0.4, Benzene - EtOAC/ 8s2). It was identified as cyclobutane-2/4- bis-(1 /3-benzodioxol-5-methoxy -6-vl)-l#3-dicarboxapyrrolidide.

SYNTHESIS OF PP-11

Photochemical conversion of PP-9 Into PP-11

Compound PP-9 (200 mg) was dissolved in chloroform (5 ml) and irradiated with sunlight for 30 hours whereupon a brown tarry mass was obtained. This mass upon column chromatography furnished a white solid/which gave an identical melting point (258°), mixed melting point/ co-TLC with the natural dimer PP-11.

ISOLATION OF PP-12

Fractions 343-350

These fractions were pooled after showing an identicalspot on TLC plate. Upon concentration/ a white residue wasobtained/which was soluble in chloroform — methanol mixture.After crystallization/ white solid of PP-12 (300 mg)/C.^H^O,,, m.p. 285° was obtained. It was identified as 35 60 6

K -sitosterol glucoside by co-TLC, mixed melting point and melting point with an authentic sample.

Compound PP-12 (200 mg) was hydrolysed with 1% at 80°. Usual work up afforded the aglycone and sugar.. The aglycone was identified as K -sitosterol (m.p. 136°) and the sugar moiety as D-glucose# by paper chromatography / on Whatman _1 using butanol : acetic acid % water (4sls5) as solvent system, and aniline hydrogen phthalate as spray reagent.

Acid hydrolysis of PP-12

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