Drugs From Natrual Products-plant Sources

8/11/2019 Drugs From Natrual Products-plant Sources

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Drugs from Natural ProductsPlant SourcesS . M O R R I S K U P C H A N

U n i v e r s i t y of Vi r g i n i a , C h a r l o t t e s v i l l e , Va. 22901

The plant kingdom has served as one of man s oldest sourcesof useful drugs. The history of classic plant-derived medic-inals, such as morphine and quinine, illustrates the origin of

the older medicinals as major and relatively easily isolatedconstituents of folk remedies. More recently discoveredagents, such as reserpine and vincaleukoblastine, have beenminor constituents of complex mixtures, whose isolation wasguided by pharmacological assay. A model for futuresearches for plant derived medicinals is illustrated by theisolation and characterization of the tumor inhibitors ver-

nolepin and jatrophone. Screening of many hundreds of

crude extracts yielded a significant number of active extracts,

and fractionations guided by biological assays have yielded afascinating array of novel biologically active plant products.

^T^he use i n m e d i c i n e of d r u g s d e r i v e d f r o m p l a n t s goes b a c k to a n t i q u i t y . W h e n on e c o n s i d e r s the t h e r a p e u t i c i m p a c t of m o r p h i n e , q u i n i n e ,

d i g i t a l i s , e rg o t , a t r o p i n e , c o c a i n e , r e s e r p i n e , a n d v i n c a l e u k o b l a s t i n e , ton a m e bu t a fe w, it is e v i d e n t h o w great is the d e b t of m e d i c i n e to p l a n t -d e r i v e d d r u g s e v e n t o d a y. If one a d d s the s y n t h e t i c d e r i v a t i v e s and

v a r i a n t s of p l a n t - d e r i v e d p r o d u c t s , th e r o l e of n a t u r a l p r o d u c t s f r o mp l a n t s o u r c e s has b e e n m o s t i m p r e s s i v e .

T h e m o s t i m p o r t a n t p l a n t - d e r i v e d d r u g s w e r e d e v e l o p e d b e t w e e n1 8 0 0 a n d 1950. T h e p a s t f e w decades h a v e w i t n e s s e d an u n q u e s t i o n a b l ed i m i n u t i o n i n the n u m b e r of s u c h c o m p o u n d s i n t r o d u c e d i n t o m e d i c i n e .These facts h a v e le d to s u g g e s t i o n s t h a t the in t e n s i v e i n v e s t i g a t i o n s oft h e p a s t c e n t u r y h a v e n e a r l y e x h a u s t e d th e p l a n t k i n g d o m as a p o t e n t i a ls o u r c e for new d r u g s and t h a t f u t u r e w o r k i n t h i s a r e a is u n l i k e l y to ber e w a r d i n g . I a d d r e s s m y s e l f to the c o n t r a r y thesis viz., t h a t the p l a n t

k i n g d o m c o n t i n u e s to o f f e r a r i c h an d v i r t u a l l y i n e x h a u s t i b l e s u p p l y ofn e w p o t e n t ia l d r u g s . H o w e v e r , the success i n t a p p i n g t h i s s o u r c ew i l l d e p e n d u p o n the extent to w h i c h n e w e r a p p r o a c h e s to the s t u d y ofb i o l o g i c a l l y - a c t i v e p l a n t c o n s t i t u e n t s ar e u s e d .

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d e d b y U M I C H O A C A N A S A N N I C O L A S

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In Drug Discovery; Bloom, B., et al.;Advances in Chemistry; American Chemical Society: Washington, DC, 1971.


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2 D R U G D I S C O V E R Y

The Fast M orphi ne and Qui nine

A s we l o o k to the p a s t, m o r p h i n e an d q u i n i n e r e p r e s e n t c l a s s i c ex

a m p l e s of e a r l y p l a n t - d e r i v e d m e d i c in a l s . O p i u m , the s u n - d r i e d l a t e x oft h e u n r i p e f r u i t of Papaver somniferum, is b e l i e v e d to h a v e b e e n u s e dbefore h i s t o r y wa s r e c o r d e d . T h e f i r s t u n d i s p u t e d reference to p o p p yj u i c e is f o u n d in the w r i t i n g s of T h e o p h r a s t u s i n the t h i r d c e n t u r y B. C.D i o s c o r i d e s i n the firs t c e n t u r y A . D . was f u l l y a c q u a i n t e d w i t h the m e t h o df o r c o l l e c t i n g a n d p r e p a r i n g o p i u m , an d his d i r e c t i o n s for p r e p a r i n g s y r u po f p o p p y ar e e s s e n t ia l ly u n c h a n g e d i n m o d e r n p h a r m a c o p ei a s . A r a b i a np h y s i c i a n s w e r e w e l l v e r s e d i n the uses of o p i u m . T h i s d r u g was i n t r od u c e d to the O r i e n t an d C h i n a b y A r a b i a n t ra d e rs . T h e s p r e a d of the

o p i u m h a b i t t h r o u g h o u t C h i n a d i d not o c c u r u n t i l the l a t t e r p a r t of thee i g h t e e n t h c e n t u r y w h e n the P o r t u g u e s e a n d l a t e r the E n g l i s h s t a r t e dt o e x p l o i t the n a t i v e s i n t h i s r e g a r d . Th e war a g a i n s t o p i u m has c o n t i n u e di n th e O r i e n t a n d e l s e w h e r e e v e r s i n c e .

Figure 1 . Morphine

F r o m the s i x t e e n t h c e n t u r y an d w e l l i n t o th e n i n e t e e n t h c e n t u r y theuses of o p i u m for its a n a l g e s i c a nd h y p n o t i c p r o p e r t ie s w e r e f a i r l y w e l lu n d e r s t o o d i n E u r o p e . In 1805 a y o u n g G e r m a n p h a rm a c i s t i n H a n o v e r

n a m e d S e r t u rn e r i s o l a te d an d d e s c r i b e d m o r p h i n e . T h i s e p o c h a l findingw e n t u n n o t i c e d u n t i l hi s l a te r p u b l i c a t i o n i n 1816. S e r t u r n e r a l m o s t l o s th i s l i f e b y e x p e r i m e n t i n g w i t h m o r p h i n e on h i m s e l f . T h e d i s c o v e r y ofo t h e r a l k a l o i d s i n o p i u m q u i c k l y f o l l o w e d t h at of m o r p h i n e , a n d the useo f p u r e a l k a l o i d s r a t h e r t h a n c r u d e p r e p a r a t i o n s s o o n s p r e a d t h r o u g h o u tt h e m e d i c a l w o r l d . E x t e n s i v e s t r u c t u r a l s t u d i e s le d to e l u c i d a t i o n of m o rp h i n e ' s s t r u c t u r e b y G u l l a n d an d R o b i n s o n i n 1925 ( I ) , an d t o t a l s y ntheses b y G a t e s a n d T s c h u d i (2) an d b y E l a d a n d G i n s b u r g ( 3) c o nf i r m e d c o m p l e t e l y the s t r u c t u r e a n d s t e r e o c h e m i s t r y of the m o l e c u l e

( F i g u r e 1 ). It is n o t e w o r t h y t h a t m o r p h i n e is the m a j o r a l k a l o i d of o p i u m ;i n a g o o d g r a d e of o p i u m i t averages 1 0 % , a l t h o u g h s a m p l e s c o n t a i n i n go v e r 2 0 % h a v e b e e n r e p o r t e d .

Q u i n i n e is the c h i e f a l k a l o i d of c i n c h o n a , the b a r k of the c i n c h o n atree i n d i g e n o u s to c e r t a i n r e g i o n s of S o u t h A m e r i c a . T h e f i r s t w r i t t e n

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1 . C H A N Fiant Sources 3

r e c o r d o f t h e u se o f c i n c h o n a o c c u r s i n a r e l i g i o u s b o o k w r i t t e n i n 1 6 33a n d p u b l i s h e d i n S p a i n i n 1 63 9. A v a r i e t y o f c o l o r f u l a n d f a n c i f u l v e rs i o n s o f t h e d i s c o v e r y o f t h e fe v e r b a r k e xi s t. A p o p u l a r a n d p e r s i s t e n t

v e r s i o n i s t h a t t h e b a r k w a s u s e d i n 1 6 3 8 t o treat C o u n t e s s A n n a d e lC h i n c h o n , w i f e o f t h e v i c e r o y t o P e r u , a n d t h a t h e r m i r a c u l o u s c u r e r es u l t e d i n t h e i n t r o d u c t i o n o f c i n c h o n a i n t o S p a i n i n 1 6 39 f o r t h e t r e a t m e n to f a g u e . B y 1 64 0 , t h e d r u g w a s b e i n g u s e d f o r f e v e r s i n E u r o p e . T h et e r m c i n c h o n a w a s c h o s e n b y L i n n ( w h o a c c i d e n t a l l y m i s s p e l l e d i t ) f o rt h e species o f p l a n t s y i e l d i n g t h e d r u g . Jesuit p r i e s t s w e r e t h e m a i n i mp o r t er s a n d d i s t r i b u t o r s o f c i n c h o n a i n E u r o p e , a n d t h e n a m e Jesuit b a r ks o o n became a t t a c h e d t o t h e d r u g .

F o r a l m o s t t w o c e n t u r i e s , t h e b a r k w a s u s e d i n m e d i c i n e as a p o w d e r ,extract, o r i n f u s i o n . I n 1 82 0 P e l l e t i e r a n d C a v e n t o u i s o l a t e d q u i n i n e a n dc i n c h o n i n e f r o m c i n c h o n a , a n d t h e u s e o f t h e a l k a l o i d s a s s u c h g a i n e df a v o r r a p i d l y. E x t e n s i v e a n d c l a s s i c s t u d i e s l e d t o e l u c i d a t i o n o f t h es t r u c t u re o f q u i n i n e ( F i g u r e 2 ) 4) a n d t o i t s t o t a l s y n t h e s i s i n 1 9 4 4 ( 5 ) .C i n c h o n a c o n t a i n s 2 5 c l o s e l y r e l a t e d a l k a l o i d s , o f w h i c h t h e m o s t i m p o rt a n t a r e q u i n i n e , q u i n i d i n e , c i n c h o n i n e , a n d c i n c h o n i d i n e . T h e a v e r a g ey i e l d o f a l k a l o i d is a b o u t 6 - 7 % , o f w h i c h o n e - h a l f t o t w o - t h i r d s is q u i n i n e .I t h a s b e e n s a i d t h a t q u i n i n e o w e s i t s d o m i n a n t p o s i t i o n i n t h e t r e a t m e n t

o f m a l a r i a o n l y t o th e fact t h a t i t w a s t h e firs t a l k a l o i d i s o l a t e d f r o mc i n c h o n a , a n d th a t there is l i t t l e a m o n g t h e f o u r m a j o r a l k a l o i d s t o choosef r o m i n t r e a t i n g t h i s d i s e a s e 6).

Figure 2. Quinine

T h e h i s t o r y o f m o r p h i n e a n d q u i n i n e , l i k e t h a t o f m o s t c la s s i c p l a n t -d e r i v e d m e d i c i n a l s , r e v e a l s t h a t t h e c o m p o u n d s r e p r e s e n t e d m a j o r a n dr e l a t i v e l y e a s i l y i s o l a t e d p l a n t c o n s t i t u e n t s . T h e r e a d y a c c e s s i b i l i t y o f t h ec o m p o u n d s p l a y e d a m a j o r r o l e i n t h e i r c h a r a c t e r i z a t i o n a s t h e a c t i v ep r i n c i p l e s o f t h e p l a n t s .

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h e Pr esent Reser pi ne a n d Vi ncaleukobl ast i ne

R e s e r p i n e a n d v i n c a l e u k o b l a s t i n e r e p r e s en t t h e m o s t i m p o r t a n t p l a n t -

d e r i v e d m e d i c i n a l s i n t r o d u c e d i n t o m e d i c i n e b y o u r g e n e r a t i o n , a n d i t isi n s t r u c t i v e t o c o m p a r e t h e i r h i s t o r y w i t h those o f m o r p h i n e a n d q u i n i n e .D e s c r i p t i o n s o f t h e u s e o f extracts o f p l a n t s r e s e m b l i n g Rauwolfia m a yb e t r a c e d b a c k t o a n c i e n t H i n d u a y u r v e d i c w r i t i n g s . T h e y w e r e u s e d i np r i m i t i v e H i n d u m e d i c i n e f o r a v a r i e t y o f d i s e a s e s , i n c l u d i n g s n a k e b i t e ,h y p e r t e n s i o n , i n s o m n i a , a n d i n s a n i t y. T h e e a r l y r e m e d i e s w e r e u s e d f o rv a r i o u s o t h e r p u r p o s e s , b u t i t seems c l e a r n o w t h a t o u r p r e s e n t d a ya p p l i c a t i o n o f Rauwolfia a l k a l o i d s i n t r e a t i n g h y p e r t e n s i o n a n d m e n t a ld i s e a s e w a s f o r e s h a d o w e d i n t h e f o l k m e d i c i n e o f t h e E a s t e r n p e o p l e s .

A l t h o u g h Rauwolfia w a s i n v e s t i g a t e d i n t h e n i n e t e e n t h c e n t u r y a n dt h e presence o f a l k a l o i d s i n d i c a t e d , a s y s t e m a t i c i n v e s t i g a t i o n o f Rauwolfiaw a s o n l y s t a r t e d b y S i d d i q u i a n d S i d d i q u i i n 1 9 31 ( 7 ) . F i v e a l k a l o i d sw e r e i s o l a t e d a t t h a t t i m e , a n d d e s p i t e t h e fact t h a t o n e a l k a l o i d (serp e n t i n e ) h a d a b l o o d - p r e s s u r e r e d u c i n g effect, n o n e o f t h e fiv e s h o w e d

t h e c h a r a c t e r i s t i c s w h i c h w e r e l a t e r c a l l e d r e s e r p i n e l i k e 8) . C h o p r aa n d o th e rs c o n c l u d e d t h a t a d d i t i o n a l p h a r m a c o l o g i c a l l y a c t i v e m a t e r i a lm u s t b e p r e s e n t i n t h e w h o l e r o o t f o r w h i c h t h e c r y s t a l l i n e a l k a l o i d sa v a i l a b l e a t t h e t i m e c o u l d n o t a c c o u n t ( 9 , 10). T h e Rauwolfia p r o b l e mr e c e i v e d a great s t i m u l u s f r o m t h e 1 9 49 p a p e r b y V a k i l i n t h e BritishHeart Journal o n t h e a n t i h y p e r t e n s i v e effects o f Rauwolfia extracts i n m a n( I I ) . I n t h e n e w e r s t u d i e s , s y s t e m a t i c f r a c t i o n a t i o n a n d i s o l a t i o n w e r ec o u p l e d w i t h p h a r m a c o l o g i c a l e v a l u a t i o n , a n d i t became a p p a r e n t t h a tt h e h y p o t e n s i v e a n d a l k a l o i d a l m a t e r i a l w a s c o n c e n t r a t e d i n t o t h e o l e o -

r e s i n f r a c t i o n . R e s e r p i n e , t h e m o s t i m p o r t a n t Rauwolfia a l k a l o i d , w a si s o l a t e d f r o m t h e o l e o r e s i n f r a c t i o n i n 1 9 52 ( 1 2 ) , a n d s h o r t l y a f t e r w a r di t w a s s h o w n t o b e r e s p o n s i b l e f o r m o s t o f t h e t r a n q u i l i z i n g a n d h y p ot e n s i v e effects o f Rauwolfia extracts. T h e e l u c i d a t i o n o f i ts s t r u c t u r e( F i g u r e 3 ) 13) a n d a n e l e g a n t t o t a l s y n t h e s i s 14,15) c o n s t i t u t e d m a j o r

O M e

Figure 3. Reserpine

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1 . K U P C H N Fiant Sources 5

a c h i e v e m e n t s i n a l k a l o i d c h e m i s t r y . R e s e r p i n e is on e of o v e r 50 a l k a l o i d si s o l a t e d f r o m v a r i o u s Rauwolfia s p e c i e s .

T h e b e n e f i c i a l p r o p e r t i e s of the p e r i w i n k l e p la n t , Vinca rosea L i n n . ,

h a v e b e e n d e s c r i b e d i n m e d i c i n a l f o l k l o r e f o r m a n y y e a rs i n v a r i o u s p a r t so f t he w o r l d . A n a l l e g e d a c t i v i ty as a n o r a l h y p o g l y c e m i c agent p r o m p t e dit s p h y t o c h e m i c a l e x a m i n a t i o n i n t w o d i f fe r e n t l a b o r a to r i e s i n d e p e n d e n t l y .W h i l e n e i t h e r g r o u p c o u l d s u b s t a n t i a t e t h i s r e p o r t e d a c t i v i t y i n e i t h e rn o r m a l or e x p e r i m e n t a l l y - i n d u c e d h y p e r g l y c e m i c r a b b it s , t he C a n a d i a ng r o u p of N o b l e , Beer, a n d C u t t s o b s e r v e d a p e r i p h e r a l g r a n u l o c y t o p e n i aa n d b o n e m a r r o w d e p r e s s i o n i n rats a s s o c i a t e d w i t h c e r t a i n f r a c t i o n s

16). These effects g u i d e d the e x t r a c t i o n a n d p u r i f i c a t i o n of an a c t i v ea l k a l o i d , t e r m e d v i n c a l e u k o b l a s t i n e . T h e L i l l y g r o u p , w h i c h i n c l u d e dJ o h n s o n , S v o b o d a , a n d o t h e rs , d e m o n s t r a t e d t h a t c e r t a i n a l k a l o i d a l f r a ct i o n s i n h i b i t e d the g r o w t h of an acute l y m p h o c y t i c l e u k e m i a i n m i c e .F r a c t i o n a t i o n , f o l l o w e d b y assay i n the l e u k e m i c m i c e , y i e l d e d v i n c a l e uk o b l a s t i n e , v i n c r i s t i n e , a n d t w o o t h e r a c t i v e d i m e r i c a l k a l o i d s 17, 18).V i n c a l e u k o b l a s t i n e a n d v i n c r i s t i n e a re n ow a m o n g the m o s t i m p o r t a n td r u g s for the t r e a t m e n t of acute l e u k e m i a of c h i l d h o o d a n d o t h e r n e op l a s m s 19). T h e m o l e c u l a r s t r u c t u re s of v i n c a l e u k o b l a s t i n e a n d v i nc r i s t i n e w e r e d e t e r m i n e d b y c h e m i c a l s t u d ie s i n 1964 ( F i g u r e 4) 20),a n d the c o m p l e t e s t e r e o c h e m i s t r y a n d a b s o l u t e c o n f i g u r a t i o n w e r e e l uc i d a t e d b y x - r a y c r y s t a l l o g r a p h i c a n a l y s i s i n 1965 21). V i n c a l e u k ob l a s t i n e is on e of m o r e t h a n 50 a l k a l o i d s is o l a t e d f r o m Vinca rosea.

Figure 4. Vincaleukoblastine

T h e r e v i e w of the h i s t o r y of r e s e r p i n e a n d v i n c a l e u k o b l a s t i n e r e v e a l st h a t e a c h a l k a l o i d w a s a m i n o r c o n s t i tu e n t of a c o m p l e x m i x t u r e a n d t h a ti ts i s o l a t i o n f r o m the m i x t u r e w a s g u i d e d i n e a c h case b y assay f o r c h a ra c t er i s ti c p h a r m a c o l o g i c a l p r o p e r t i e s . It is l i k e l y t h a t, h a d t he i n v e s t i g a -

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1 , 1 9 7 1 | d o i : 1

0 . 1 0 2 1 / b a -

1 9 7 1 - 0

1 0 8 . c h

0 0 1



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t i o n s o f Rauwolfia serpentina a n d Vinca rosea p r o c e e d e d a l o n g c l a s s i c a lp h y t o c h e m i c a l l in e s , w i t h o u t p h a r m a c o l o g i c a l g u i d a n c e , th e d i s c o v e r yo f r e s e r p i n e a n d o f v i n c a l e u k o b l a s t i n e w o u l d h a v e b e e n p o s t p o n e d b ym a n y y e a r s .

h e Fut ure Vernol epi n a n d Jat rophone

T h e p a s t a n d p r e s e n t states o f a n y field a r e f a r s i m p l e r t o c o m m e n tu p o n t h a n t h e f u t u r e . O n t h e o t h e r h a n d , t h e absence o f c l e a r - c u t g u i d el i n e s p r o v i d e s t h e w r i t e r c o n s i d e r a b l e l a t i t u d e i n d i s c u s s i n g t h e f u t u r e .

B e l o w I o u t l i n e b r i e f l y s o m e recent fin din g s i n m y l a b o r a t o r y i n ap r o g r a m d i r e c t e d a t t u m o r i n h i b i t o r s o f p l a n t o r i g i n . T h i s p r o g r a m , w h i c hh a s a l r e a d y l e d t o t h e i s o l a t i o n o f t h e a c t i v e p r i n c i p l e s o f m o r e t h a n 8 0t u m o r - i n h i b i t o r y extracts, h a s b e e n t h e subject o f t w o recent r e v i e w s

22). F o r t h i s d i s c u s s i o n o f t h e f u t u r e o f p l a n t - d e r i v e d d r u g s , t h e s t or i eso f v e r n o l e p i n a n d j a t r o p h o n e w i l l e x e m p l i f y o n e i m p o r t a n t a p p r o a c h .

Powdered leaves

A (1.5 Kg)

Concentrated chloroform extract

87.0g)

Partition

1 11 0 Aqueous methanol Interfacial solid Petroleum ether

extract, C 5l.0g) D I0.6g) extract, (23.8 g)

Silica ge l chromatography

Solvent: 1 5 acetone in chloroform

Vernomenin (6.5 g)

Mixture (6.5 g)

Vernolepin (5.5 g)

Figure 5. Fractionation of tumor-inhibitory extract from Ve r n o n i a h y m e n o l e p i s

I n o u r p r o g r a m , t h e fr a c t i o n a t i o n a n d i s o l a t i o n s t u d ie s a r e g u i d e da t e v e r y stage b y b i o l o g i c a l a s s a y s . T h e s y s t e m a t i c f r a c t i o n a t i o n h a s m a d e

p o s s i b l e t h e i s o l a t i o n o f i m p o r t a n t m i n o r c o n s t i t u e n t s w h i c h w o u l d m o s tp r o b a b l y h a v e b e e n m i s s e d i n t h e c l a s s i c a l a p p r o a c h . D u r i n g t h e s c r e e ni n g p r o g r a m s p o n s o r e d b y t h e C a n c e r C h e m o t h e r a p y N a t i o n a l S e r v i c eC e n t e r , a n extract o f Vernonia hymenolepis, A. rich, w a s f o u n d t o s h o ws i g n i f i c a n t a n d r e p r o d u c i b l e c y t o t o x i c i t y a g a i n s t th e K B t i s s u e c u l t u r e

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H G O o n

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b e r

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0 1 4 | h t t p :

/ / p u b s . a c s . o r g

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0 . 1 0 2 1 / b a -

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1 . K U P C H A N Fiant Sources 7

o f h u m a n c a r c i n o m a of the n a s o p h a r y n x . F i g u r e 5 s u m m a r i z e s t he f r a ct i o n a t i o n p r o c e d u r e that l e d to the i s o l a t i o n of the c y t o t o x i c p r i n c i p l e s ,v e r n o l e p i n , a n d v e r n o m e n i n . A l t h o u g h the c o m p o u n d s w e r e c o n c e n t r a t e d

a n d i s o l a t e d s o l e l y on the b a s i s of in vitro c y t o t o x i c i t y , v e r n o l e p i n wass u b s e q u e n t l y f o u n d to s h o w s i g n i f i ca n t in vivo t u m o r i n h i b i t o r y a c t i v i t ya g a i n s t the W a l k e r 256 c a r c i n o s a r c o m a i n the rat. Ve r n o l e p i n a n d itsi s o m e r , v e r n o m e n i n , w e r e i n t e r r e l a t e d b y c o n v e r s i o n to a c o m m o n m e t ha n o l a d d u c t . A c o m b i n a t i o n of d e g r a d a t i v e , s p e c t r a l , a n d x - r a y c r y s t a l l o -g r a p h i c s t u d i e s r e s u l t e d i n a s s i g n m e n t of the b i o g e n e t i c a l l y n o v e l ,e l e m a n o l i d e d i l a c t o n e s t r u ct u r e s s h o w n i n F i g u r e 6 (23, 24).

Figure 6. Structures of vernolepin (upper left, R=H) and vernomenin (upperright, R=H)

S e v e r a l recent o b s e r v a t i o n s h a v e f o c u s e d a t t e n t i o n o n the i m p o r t a n c eo f the c o n j u g a t e d - m e t h y l e n e l a c t o n e f u n c t i o n for the b i o l o g i c a l a c t i v i t yo f v e r n o l e p i n a n d o t h e r s e s q u i t er p e n e l a c to n e s . F u r t h e r m o r e , the r e s u l t ss u p p o r t the v i e w that the - m e t h y l e n e lactones ma y exert t h e i r effects onc e l l s b y i n t e r a c t i n g w i t h s u l f h y d r y l e n z y m e s that r e g u l a t e c e l l u l a r g r o w t h .F o r i n s t a n c e , v e r n o l e p i n is a p o t e n t i n h i b i t o r of the e x te n s io n g r o w t h ofw h e a t c o l e o p t i l e s e c t i o n s -25); t h i s i n h i b i t o r y effect is b l o c k e d c o m p l e t e l yb y a d d i n g s u l f h y d r y l c o m p o u n d s s u c h as m e r c a p t o e th a n o l to the m e d i u m .

S e c o n d , v e r n o l e p i n a n d o t h e r s e s q u i t e r p e n e lactones c an i n h i b i t p h o s p h o -f r u c t o k i n a s e b y r e a c t i n g w i t h the e n z y m e ' s s u l f h y d r y l g r o u p s (26).T h i r d , as s h o w n i n F i g u r e 7, the c y t o t o x i c i t y of v e r n o l e p i n d e r i v a t i v e sa p p e a r s to be r e l a t e d d i r e c t l y to the presence of free c o n j u g a t e d - m e t hy l e n e l a c t o n e f u n c t i o n s . T h u s , s e l e c t iv e r e d u c t i o n of the e t h y l i d e n e

D o w n l o a


H G O o n

S e p t e m

b e r

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/ / p u b s . a c s . o r g

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0 0 ' 0 '

VERNOLEPIN

ED 5 0 2.0 /

DIHYDROVERNOLEPIN TETRAHYDROVERNOLEPIN

2 . 0 / 1 9 . 0 /

'

OC H 3

HEXAHYDROVERNOLEPIN

> 1 0 0 /

ACIDIC METHANOLYSIS

PRODUCT

2 6 /

Figure 7. Cytotoxicity of vernolepin derivatives

d o u b l e b o n d d o e s n o t a p p e a r to affect t h e c y t o t ox i c i t y . H o w e v e r , m o d i

f icat ion of th e a

- m e t h y l e n e - y - l a c t o n e ( b y t r a n s - e s t e r i f i c a t io n t o t h e m e t ha n o l a d d u c t o r b y h y d r o g n a t i o n ) r e s u l t s i n a 1 0 - f o l d d i m i n u t i o n i nc y t o t o x i c i t y . M o d i f i c a t i o n o f b o t h - m e t h y l e n e l a c t o n e s y s t e m s , as i nh e x a h y d r o v e r n o l e p i n , l e a d s t o a d e r i v a t i v e w h i c h i s e s s e n t i a l l y i n a c t i v e .[ T h e s y n t h e s i s o f d i h y d r o v e r n o l e p i n e x e m p l i fi e s a n e w b l o c k i n g sequencef o r t h e p r o t e c t i o n o f t h e h i g h l y r e a c t i v e c o n j u g a t e d - m e t h y l e n e g r o u p so f l a c to n e s ( F i g u r e 8 ) . V e r n o l e p i n w a s t r e a t e d w i t h excess n - p r o p y l t h i o la t p H 9 .2 t o g i v e a b i s t h i o l a d d u c t . H y d r o g n a t i o n o f t h e b i s t h i o l a d d u c t( w i t h o n e m o l e e q u i v a l e n t o f h y d r o g e n ) , f o l l o w e d b y m e t h y l i o d i d e

m e t h y l a t i o n a n d s o d i u m b i c a r b o n a t e - c a t a l y z e d e l i m i n a t i o n , g a v e d i h y d r ov e r n o l e p i n (-27).] R e c e n t l y w e s t u d i e d t h e r e a c t i o n s o f s e v e r a l c o nj u g a t e d - m e t h y l e n e l a c t o n e s w i t h m o d e l b i o l o g i c a l n u c l e o p h i l e s , s u c h a sc y s t e in e , l y s i n e , a n d g u a n i n e 28). T h i o l s s u c h a s c y s t e i n e w e r e t h e m o s tr e a c t i v e , a n d t h e rate o f r e a c t i o n w a s o f t h e same o r d e r a s t h a t o f c y s t e i n ew i t h i o d o a c e t a t e , a c o m m o n l y u s e d s u l f h y d r y l r e a g e n t ( F i g u r e 9 ) . T h eb i s c y s t e i n e a d d u c t s , i n a c c o r d w i t h e x p e c t a t i o n s , w e r e e s s e n t i a l l y i n a c t i v e .

E x t r a c t s o f Jatropha gossypiifolia L . a n d r e l a t e d s p e c ie s h a v e b e e nu s e d f o r m a n y y e a rs i n C o s t a R i c a to treat c a n c e r o u s g r o w t h s . A n a l c o

h o l i c e x t r a c t o f t h e r o o t s o f Jatropha gossypiifolia s u p p l i e d b y J A . S a e n zR e n a u l d o f t h e U n i v e r s i t y o f C o s t a R i c a ) w a s f o u n d b y C C N S C t o s h o wi n h i b i t o r y a c t i v i t y a g a i n s t f o u r s t a n d a r d a n i m a l t u m o r s y s t em s ( s a r c o m a1 8 0 , L e w i s l u n g c a r c i n o m a , a n d l y m p h o c y t i c l e u k e m i a P-388 i n t h e m o u s e ,a n d t h e W a l k e r 2 56 i n t r a m u s c u l a r c a r c i n o s a r c o m a i n t h e r a t ) a n d in vitro

D o w n l o a


H G O o n

S e p t e m

b e r

2 3 , 2

0 1 4 | h t t p :

/ / p u b s . a c s . o r g

P u b l i c a t

i o n

D a t e :

J u n e

1 , 1 9 7 1 | d o i : 1

0 . 1 0 2 1 / b a -

1 9 7 1 - 0

1 0 8 . c h

0 0 1



9/13

1. C H A N Fhnt Sources 9

a g a i n s t c e ll s d e r i v e d f r o m h u m a n c a r c i n o m a of the n a s o p h a r y n x K B ) .F i g u r e 10 s u m m a r i z e s the f r a c t i o n a t i o n p r o c e d u r e that le d to the i s o l at i o n of the c y t o t o x i c p r i n c i p l e , j a t r o p h o n e . A f t e r i s o l a t i o n on the basiso f f r a c t i o n a t i o n g u i d e d b y assay a g a i n s t K B c e l l c u l t u r e , j a t r o p h o n es h o w e d r e p r o d u c i b l e i n h i b i t o r y a c t i v i t y a g a i n s t the P-388 l y m p h o c y t i c

Dihydrovernolepin ^

Figure 8. Dihydrovernolepin

NH 3

RSH= CH-CH 2 -SH/ C 02

Figure 9. Reaction of vernolepin with ^cysteine at 25C, pH 7.4. Initialreaction rate: k 2 = 12,000 liters/mole/min.

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H G O o n

S e p t e m

b e r

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11/13

1. K U P C H A N font Sources 11

uate w h e t h e r j a t r o p h o n e a n d o t h e r p l a n t - d e r i v e d t u m o r i n h i b i t o r s m a ya c t b y s e le c t i v e a l k y l a t i o n o f s u l f h y d r y l e n z y m e s t h a t r e g u l a t e c e l l u l a rg r o w t h .

T h i s d i s c u s s i o n o f v e r n o l e p i n a n d j a t r o p h o n e i l lu s t r a t e s o u r a p p r o a c ht o t h e i s o l a t i o n f r o m p l a n t s o f n e w, n o v e l , a n d b i o l o g i c a l l y a c t i v e n a t u r a lp r o d u c t s . B o t h e x a m p l e s i n v o l v e d b i o l o g i c a l assays f o r g r o w t h - i n h i b i t o r ya c t i v i t y , b u t a n y o t h e r s a t is f a c to r y b i o l o g i c a l assay c o u l d b e u s e d i n as e a r c h f o r o t h e r t y p e s of c o m p o u n d s . T o t h e i n v e s t i g a t o r w h o w i l l u n d e rt a k e s u c h a s y s t e m a t i c a p p r o a c h to b i o l o g i c a l l y a c t i v e n a t u r a l p r o d u c t s ,

Figure 12 . Transannular conjugate additions of Br

t h e p l a n t k i n g d o m r e p r es e n t s a v i r t u a l l y u n t a p p e d r e s o u r c e . O u r b o t a n i s tc o ll e a g u e s e s t im a t e t h a t a p p r o x i m a t e l y 500,000 species o f p l a n t s o c c u r ,a n d t h a t p e r h a p s 1 0 % h a v e b e e n i n v e s t i g a t e d p h y t o c h e m i c a l l y . O f th es m a l l m i n o r i t y w h i c h h a v e r e c e i v e d c h e m i c a l a t te n t i on , o n l y a t i n y p r op o r t i o n h a v e e v e r b e e n a t t a c k e d w i t h a s y s t e m a t i c a p p r o a c h i n v o l v i n ga n y t y p e o f b i o l o g i c a l a s s a y. D i f f e r e n t b i o a s s a y s w i l l p r o v i d e h a n d l e sf o r i s o l a t i n g n e w c o m p o u n d s f r o m those f e w p la n t s w h i c h h a v e a l r e a d yb e e n e x a m i n e d w i t h t h e g u i d a n c e o f o n e assay s y s t e m . I n d e e d , w e h a v ea l r e a d y w i t n e s s e d t h e effectiveness o f t h i s a p p r o a c h i n t h e p l a n t t u m o r -

i n h i b i t o r p r o g r a m . P e r i o d i c r e e x a m i n a t i o n o f p l a n t r e c o l le c t i o n s w i t hn e w s c r e e n i n g s y s t e m s c o n t i n u e s to y i e l d n e w a c t i v e s . A s o n e l o o k s t ot h e f u t u r e , p l a n t s o u r ce s m a y p r o v e t o b e i n e x h a u s t i b l e s i n c e p e r i o d i cr e e x a m i n a t i o n o f extracts o f t he same p l a n t w i l l b e d e s i r a b l e a s n e w a n ds e n s i t i v e b i o a s s a y p r o c e d u r e s f o r d i f f e r e n t t y p e s o f a c t i v i t y a r e d i s c o v e r e d .

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S e p t e m

b e r

2 3 , 2

0 1 4 | h t t p :

/ / p u b s . a c s . o r g

P u b l i c a t

i o n

D a t e :

J u n e

1 , 1 9 7 1 | d o i : 1

0 . 1 0 2 1 / b a -

1 9 7 1 - 0

1 0 8 . c h

0 0 1



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O n e i m p o r t a n t q u e s t i o n r e m a i n s : w h e r e w i l l t h e n e w l e a d s c o m ef r o m ? S o m a n y o f t h e o l d e r p l a n t - d e r i v e d m e d i c i n a l s h a v e s p r u n g f r o mf o l k r e m e d i e s t h a t m a n y r e a d e r s a s s u m e t h a t o n l y p l a n t s r e p o r t e d t o b e

t h e r a p e u t i c a l ly u s e f u l s h o u l d b e e x a m i n e d f o r p h a r m a c o l o g i c a l p r o p e rt i e s . S e l e c t e d i n d i v i d u a l f o l k r e m e d i e s w h o s e r e p o r t e d p h a r m a c o l o g i c a lp r o p e r t i e s a r e detectable i n o b j e c t i v e assays w i l l c o n t i n u e t o p r o v i d e s o m el e a d s . H o w e v e r , l o g i s t i c c o n s i d e r a t i o n s w i l l r e q u i r e t h a t t h e m a j o r i t y o ff u t u r e le a d s i n t h i s a r e a w i l l emerge f r o m l a r g e scale r a n d o m s c r e e n i n go f p l a n t extracts. I t i s s i g n i f i ca n t t h a t o f t he f ir st 40,000 c r u d e extractsp r e p a r e d f r o m p l a n t s c o l l e c t e d b y t h e C C N S C r a n d o m l y f r o m a r o u n dt h e w o r l d a p p r o x i m a t e l y 3 % s h o w e d r e p r o d u c i b l e a c t i v i t y i n o n e or a no t h e r o f t h e t u m o r s y s t e m s u s e d ( 3 0 ) . T h e d e m o n s t r a t e d effectiveness o fr a n d o m s c r e e n i n g o f p l a n t extracts i n u n c o v e r i n g s i g n i f i c a n t l e a d s f o rs y s t e m a t i c i s o l a t i o n o f g r o w t h - i n h i b i t o r y c o m p o u n d s s u p p o r t s c o n f i d e n c ei n t h i s a p p r o a c h to n e w c o m p o u n d s w i t h o t he r d e s i r e d p h a r m a c o l o g i c a lp r o p e r t i e s .

O u r s m a l l i n c u r s i o n i n t o t h e s e a r c h f o r p l a n t - d e r i v e d t u m o r i n h i b i t o r sd u r i n g th e p a s t d e c a d e h a s y i e l d e d a f a s c i n a t i n g a r r a y o f n o v e l b i ol o g i c a l l y a c ti v e n a t u r a l p r o d u c t s . T h e p l a n t k i n g d o m w i l l c o n t i n u e t oy i e l d n o v e l d r u g s t o those w h o w i l l u s e b i o l o g i c a l assay a s a d i v i n i n g

r o d i n t h e i r h i g h l y s i g n i f i ca n t a n d c h a l l e n g i n g e x p l o r a t i o n s .

Literature ited

(1) Gulland, J. M . , Robinson, R., Mem. Proc Manchester Lit. Phil Soc.(1925) 69 79.

(2) Gates, M., Tschudi, G., J. Am. Chem Soc. (1952) 74, 1109; (1956) 78,1380.

(3) Elad, D ., Ginsburg, D., J. Chem Soc. (1954) 3052.(4) Turner, R. B., Woodward, R. B., The Alkaloids (1953) 3 1.

(5) Woodward, R. B., von E. Doering, W., J. Am. Chem Soc. (1944) 66849; (1945) 67, 860.(6) Russell, P. B ., in Medicinal Chemistry, A . Burger, Ed ., p. 821, 2nd ed.,

Interscience, New York, 1960.(7) Siddiqui, S., Siddiqui, R. H ., J. Indian Chem Soc. (1931) 8, 667; (1932)

9 539; (1935) 12, 37.(8) Woodson, R. E., Jr., Youngken, H . W. , Schlittler, E . , Schneider, J. .,

Rauwolfia, Little , Brown, & Co ., Boston, 1957.(9) Chopra, R . N. , Chakravarti, M . , Indian J. M. Res. (1941) 29, 763.

(10) Chopra, R . N. , Gupta, J. C., Bose, B. C., Chopra, I., Indian J. M. Res.(1943) 31, 71.

(11) Vakil, R. J., British Heart J. (1949) 11, 350.(12) Mueller, J. M . , Schlittler, E., Bein, H. J., Experientia (1952) 8, 338.(13) Dorfman, L . A. et al. Helv Chim Acta (1954) 37, 59.(14) Woodward, R. B., Bader, F. E., Bickel, H., Frey, A. J., Kierstad, R. W.,

J. Am. Chem Soc. (1956) 78, 2023, 2657.(15) Woodward, R. B., Bader, F. E., Bickel, H., Frey, A. J., Kierstad, R. W.,

Tetrahedron (1958) 2, 1.

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RECEIVED November 5, 1970.

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D a t e :

J u n e

1 , 1 9 7 1 | d o i : 1

0 . 1 0 2 1 / b a -

1 9 7 1 - 0

1 0 8 . c h

0 0 1

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