Why further investigation of brazilian Strychnos?

W H Y F U R T H E R I N V E S T I G A T I O N O F B R A Z I L I A N S T R Y C H N O S ? (*)

L . A n g e n o t (**)

S U M M A R Y

Vuning thz paòt (i^tczn yza/u>, AfinÀcan S t t y c f m o i bpzcizo have b e e n pharmacologi-cally and chzmicalty àdAe&n&d by &omz European UniveJiòitieA, iomztimeJ, With thz col labonati.on of, òom& A^Kican ImtituXek . StAychnoh uAambaAZnòií, will be d-L&cuAied hzn.z b e c a u s e ofa oai intzneòt in i t , a(ten. the. diòcovzmy o$ iti, U Ò Z in the. pnzpan.cution of, an oMiOW poiton with ciLhaAizinq activity.

The. European òtudieA have íhown that thz A^nican ipzcizi o £ Sttufchnoi chemically one ueAy &imil.cm to centain South-AmznXcan òpzcizi. Incized, thz occuAAencz in thz A(nÁ.cayi &pzcizi oft cun.aAA.zing quatennan.y bai,£4, and in the AmeAican ipzcieA oft tztaniz-ing tZAÀiany baizò, éuch cu> akageninz, zmpha&izzi, thz unity 0(, g&nui St/Lgchnof, in tznmi ofi i£f> alkaloidò. Many biological actioitizò [antimicAobial, hypotensive, hpatmotytic, cwozbicidal...} have b e e n dzttctzd.

Finally, the. antimitotic piopzAtiu ojj òomz alkaloidi will b e pnuentzd. Sz&ides known antitumor dnugà, e . g . al&toninz and zltipticim, Kfanican òpzcieA po&AZAi nzw antimitotic pnodactò, e . g . i>tjiychnopzntãmine pfoeAznt in Stnychrw& uAombcVtznhii,.

Secaiwe o{ thz&z ^indingf,, the. main Azaòonò why (unthzA investigate. Bnazilian StJiychnoA bpzciei one. the. lack o(, knowlzdgz about:

- thz diAtAibutÂon o j thz ipzcizò in thz Aouthznn pant o( thz gnzat Amazon Benin. Acconding to K/mkofâ, i t ii> poòiiblz that n e w òpzcizò may bz ficund thznz. - thz diitAA-bution o& alkaloids in many o{, known ipzeiza. I n d e e d , thznz it tittle

in&onmaticn on thz variation in composition ofj thz alkaloidal mixtunej, in thz difâeAznt pãJlti of, thz plant*,.

- thz biological activities o{ alkaloids. I n thz past, intzMJít dai be.cn focused too ncMAowly on poiòiblz cuncutizing pwpentxei.

Why should Biazilian spzcieA not contain alkaloids with pnopeAtizS similan. tothobZ faound in thz A^nican spzeizs?

(*) L e c t u r e g i v e n at the V I I I S y m p o s i u m of M e d i c i n a l P l a n t s of B r a z i l M a n a u s -S e p t e m b e r 1 9 8 4 . (**) L a b o r a t o i r e d e P h a r m a c o g n o s i e et d e C h i m i e s t r u c t u r a l e o r g a n i q u e , I n s t i t u t d e P h a r m a c i e - F a c u l t e d e M ê d e c i n e d e 1 ' U n i v e r e i t é d e L í ê g e - R u e F u s c h , 5 - B - 4 0 0 0 L I E G E ( B é l g i c a ) . SlíPL. A C T A A M A Z O N I C A , 18(1-2) : 2 ^ 1 - 2 5 ^ - 1 9 8 8 . 2Ί1

I H T R O D Ü C T I O N

The g e n u s S t r y c h n o s is p a n t r o p i c a l a n d c o m p r i s e s a b o u t 200 s p e c i e s , w h i c h may be s u b d i v i d e d into t h r e e g e o g r a p h i c a l l y s e p a r a t e d g r o u p s : o n e in A s i a w i t h s p e c i e s , one in A m e r i c a w i t h a b o u t 70 s p e c i e s a n d o n e in A f r i c a n w i t h 75 s p e c i e s .

A l t h o u g h i n v e s t i g a t i o n s into the g e n u s S t r y c h n o s h a v e b e e n g o i n g o n for a long t i m e , the A f r i c a n m e m b e r s s u f f e r e d a long p e r i o d o f n e g l e c t . T h e f i r s t s y s t e m a t i c studies w e r e u n d e r t a k e n in L i e g e by the late P r o f e s s o r Denoel o n m a t e r i a l s f r o m the Belgian C o n g o (Zaire) (Denoel e t a l , , 1 9 5 3 ) . O t h e r w o r k s i ncl ude the t a x o n o m i c r e v i s ion of the genus by L e e u w e n b e r g ( 1 9 6 9 ) , the e t h n o b o t a n y { B i s s e t , 1970) a n d a p r e l i m i n a r y s c r e e n i n g for a l k a l o i d s by Bisset a n d P h i l l i p s o n ( 1 9 7 ' ) · D u r i n g the p a s t f i f t e e n y e a r s , h& African s p e c i e s h a v e been s c r e e n e d by the S w e d i s h U n i v e r s i t y of Uppsa1 a f or m u s e 1 e - r e l a x a n t and/ o r c o n v u l s a n t a c t i v i t y . In r e c e n t y e a r s , o t h e r c h e m i c a l a n d b i o l o g i c a l s t u d i e s have been u n d e r t a k e n in m a n y U n i v e r s i t i e s o f E u r o p e ( G r e a t - 8 r i t a i η , F r a n c e , I t a l y , Sweden, N e t h e r l a n d s and B e l g i u m ) . A r e v i e w o f the p h y t o c h e m i s t r y o f A f r i c a n S t r y c h n o s species and their biological a c t i v i t i e s h a s b e e n r e c e n t l y p r e s e n t e d in the J o u r n a l o f Ethnophar m a c o l o g y (Ohiri e t a l . , 1 9 8 3 ) .

STUDY O F A F R I C A N L O G A N I A C E A E A T T H E U N I V E R S I T Y O F L I E G E

The r e s e a r c h p r o g r a m m e of the U n i v e r s i t y o f L i e g e o n S t r y c h n o s a l k a l o i d s is also a n o f f s h o o t of a n i n v e n t o r y o f m e d i c i n a l a n d t o x i c p l a n t s in the E a s t e r n part of Rwanda c a r r i e d o u t d u r i n g the y e a r s 1969-70 ( A n g e n o t , 1 9 7 0 ) .

A m o n g the t o x i c p l a n t s , t h o s e s p e c i e s w h i c h a r e i n g r e d i e n t s of a r r o w p o i s o n s , are really w o r t h s t u d y i n g . A n i n t r i g u i n g a c c o u n t o f a h u n t e r n a m e d K a h i j a m a , g a m e k e e p e r in the National Park of A k a g e r a b e c a m e a special i n s p i r a t i o n to the i n i t i a t i o n of our p r o g r a m m e , K a h i j a m a b e l o n g s to the t r i b e o f B a n y a m b o : it is a small t r i b e living along the A k a g e r a river o n the border b e t w e e n R w a n d a a n d T a n z a n i a . T h i s t r i b e p r e p a r e d in f r o n t of us a n a r r o w p o i s o n ( A n g e n o t , 1 9 7 1 ) .

The m a i n i n g r e d i e n t s of this p r e v i o u s l y u n k n o w n p o i s o n w e r e S . u s a m b a r e n s i s leaves a n d r o o t s , w e had f i r s t e x a m i n e d .

So the o b j e c t i v e s o f o u r p r o g r a m m e o n S t r y c h n o s w e r e : the i n c r e a s e of the limited k n o w l e d g e of the A f r i c a n s p e c i e s , m a i n l y a c h e m o - a n d p h a r m a c o - t a x o n o m i c e v a l u a t i o n and the d i s c o v e r y of some p h a r m a c o l o g i c a l l y a c t i v e c o m p o u n d s .

The m o s t a c t i v e i n g r e d i e n t s of S t r y c h n o s are indole a l k a l o i d s . T h e y b e l o n g to the s o - c a l l e d C o r y n a n t h e / S t r y c h n o s t y p e of t e r p e n o i d s indole a l k a l o i d s . C o n d e n s a t i o n of t r y p t a m i n e a n d s e c o l o g a n i n u n i t s leads to g l u c o - a l k a l o i d s f r o m w h i c h the v a r i o u s types o f indole a l k a l o i d s are o b t a i n e d by f u r t h e r r e a c t i o n s .

Let us e x a m i n e in d e t a i l s S t r y c h n o s u s a m b a r e n s i s . It is a c l i m b i n g s h r u b in West A f r i c a , w h i l e East a n d S o u t h A f r i c a n s p e c i m e n s a r e small t r e e s ,

T h e root b a r k s , leaves a n d f r u i t s o f S. u s a m b a r e n s i s c o l l e c t e d in R w a n d a a n d Tan z a n i a , h a v e been e x a m i n e d a b o u t t e r t i a r y a n d q u a t e r n a r y a l k a l o i d s .

isolated t w e n t y - o n e a l k a l o i d s f r o m root b a r k s . T h e s e a l k a l o i d s w e r e d i v i d e d into t h r e e g r o u p s a c c o r d i n g to t h e i r p o l a r i t y a n d the c h a r a c t e r i s t i c s o f their a m i n e f u n c t i o n s . T h e i d e n t i f i c a t i o n a n d the s t r u c t u r a l d e t e r m i n a t i o n of t h e s e c o m p o u n d s w e r e a c h i e v e d by s p e c t r o s c o p y a n d s o m e t i m e s X Ray a n a l y s i s .

T h e first g r o u p c o n t a i n s t e r t i a r y a l k a l o i d s . A p a r t f r o m h a r m a n w h i c h w a s f o u n d for the f i r s t t i m e in L o g a n i a c e a e , w e h a v e i s o l a t e d t h r e e new a l k a l o i d s .

T w o o f t h e m ( u s a m b a r e n s i n e a n d d i h y d r o d e r i v a t i v e ) a r e b i s i n d o l e type b i o g e n e t i cally d e r i v e d from two t r y p t a m i n e a n d o n e m o n o t e r p e n e u n i t s as some al kaloids of A p o c y -n a c e a e (Ochrosia) a -n d R u b i a c e a e (U-ncaria) , two f a m i l i e s very c l o s e to L o g a -n i a c e a e from a c n e m o t a x o n o m y p o i n t of v i e w . T h e y a r e u n s y m m e t r i c a l d i m e r s . The i r s t r u c t u r e a n d c o n f i g ^ ration w e r e p r o v e d e i t h e r by X - R a y a n a l y s i s for u s a m b a r e n s i n e (Fig. 1) or by s y n t h e s i s for d i h y d r o - u s a m b a r e n s i n e (Fig. 2 ) .

U s a m b a r e n s i n e o f f e r s a t r o p i n e - ! I k e a n d s p a s m o l y t i c a c t i v i t i e s (on the smooth m u s c l e s ) . 0 i h y d r o - u s a m b a r e n s i n e h a s a m o e b i c i d e p r o p e r t i e s .

The t h i r d a l k a l o i d o f the first g r o u p is a l s o n e w ; a k a g e r i ne(Fig .3) is a tetracyclic indole a l k a l o i d p o s s e s s i n g a p e r h y d r o - a z e p i n e ring c o u p l e d to t e t r a h y d r o- u c a r b o l i n e . b y an o r i g i n a l - C j ^ b o n d (Angenot e t a ) . , 1 9 7 5 ) . A k a g e r i n e s h o u l d be a p r e c u r s o r of some s o p h i s t i c a t e d a l k a l o i d s ( b i s - i n d o l e a n d h e p t a c y c l i c ) that w o u l d be o b t a i n e d by react ion with t r y p t a m i n e . A k a g e r i n e a n d d e r i v a t i v e s h a v e been r e c e n t l y r e c o v e r e d in s o m e A f r i c a n and S o u t h A m e r i c a n s p e c i e s s t u d i e d by the U n i v e r s i t i e s of U p p s a l a (Sweden) a n d Roma (Italy) { M a r i n i - B e t t o l o e t a l . , 1 9 8 0 ) . A k a g e r i n e h a s e x h i b i t e d t e t a n i z i n g p r o p e r t i e s , but is a h u n d r e d times less a c t i v e than s t r y c h n i n e .

T h e s e c o n d g r o u p is r e p r e s e n t e d by an a n h y d r o n i u m b a s e ( 6 , 7 d i h y d r o f I a v o p e r e i r i -ne) a n d m a i n l y by two s u b s t a n c e s c o n t a i n i n g s i m u l t a n e o u s l y a q u a t e r n a r y a m m o n i u m a n d a tertiary a m i n e f u n c t i o n . T h e y a r e thus h y b r i d a l k a l o i d s w i t h p a r t i c u l a r p r o p e r t i e s o f solubility a n d e x t r a c t i o n . I n d e e d , t h e s e p r o d u c t s r e m a i n b e t w e e n the a q u e o u s p h a s e a n d the o r g a n i c p h a s e d u r i n g the e x t r a c t i o n p r o c e d u r e . T h e last g r o u p c o n t a i n s q u a t e r n a r y a m m o n i u m c o m p o u n d s M o n o m e r i c a 1 ka 1 o i d s include: m e t i n o n i n e F ( C a p r a s s e e t a l . , 1 9 8 3 a ) , f l u o r o c u r a r i n e (Fig. 4) ( C a p r a s s e e t al ., 1981 b)and macusine Β , three p r o d u c t s f o r m e r l y isolated from A m e r i c a n S t r y c h n o s a n d c u r a r e . M a c u s i n e Β (Fig. 5) has been s h o w n to p o s s e s s a d r e n o l y t i c p r o p e r t i e s . T h e s e last y e a r s , w e h a v e also o b t a i n e d N^-methyl a n t i r r h i n e p r e v i o u s l y f o u n d in S. camptonetira a n d m a l i n d i n e ( F i g . 6} first isolated from S . d e c u s s a t e (Caprasse e t a l . , 1 9 4 8 a ) . T h r e e n e w d e r i v a t i v e s

0-íethyl-macusine B , O - m e t h y l - 1 9 , 20 d í h y d r o - m a c u s i n e Β a n d i s o m a l i n d i n e w e r e still d e ­ tected in t h i s g r o u p .

D i m e r l c q u a t e r n a r y c o m p o u n d s b e l o n g m a i n l y to S t r y c h n o s t y p e . T h e symmetrical dimers a r e v e r y a c t i v e p r o d u c t s . T h r e e of t h e m ( c u r a r i n e , c a l e b a s s i n e , dihydrotoxifer_i_ ne) (Fig. 7) w e r e p r e v i o u s l y i s o l a t e d f r o m C a l a b a s h - C u r a r e a n d s o m e S o u t h A m e r i c a n Strychnos. T h e y a r e f o u n d a n d i s o l a t e d for the first t i m e in a S t r y c h n o s species growing outside of A m e r i c a . T h e s e d i q u a t e r n a r y a m m o n i u m c o m p o u n d s o f f e r i n t e r e s t i n g a n d p o t e n t neuromuscular b l o c k i n g p r o p e r t i e s , a n d w e m a y c o n c l u d e that the lethal a c t ion of the arrow son p r e p a r e d by the B a n y a m b o h u n t e r s is c e r t a i n l y d u e to the c u r a r izing p r o p e r t i e s o f

t h e s e p r o d u c t s ( A n g e n o t e t a l . , 1 9 7 5 a ) .

T h e last d i m e r i c c o m p o u n d is a n e w p r o d u c t t h a t w e h a v e c a l l e d a f r o c u r a r i n e (Fig, 8 ) . It is a n u n s y m m e t r i c a l b i s i n d o l e a l k a l o i d less c u r a r í z í n g t h a n t h e s y m m e t r i c a ! d i m e r s ( C a p r a s s e e t a l . , 1 9 8 4 b ) .

T h e l e a v e s o f the s a m e S t r y c h n o s w e r e t h e r e f o r e s t u d i e d . T h e y contain sixteen a l k a l o i d s , that a r e all b i s i n d o l e a l k a l o i d s of C o r y n a n t h e t y p e a n d d i f f e r e n t f r o m these p r e s e n t in the r o o t s . T h e y a r e n e w a n d p o s s e s s the usambarane s k e l e t o n b u t w i t h a shift o f the d o u b l e b o n d f r o m C j g - C2Q to C j g - C ^ o r by l a c k i n g o f t h i s b o n d . ( C a p r a s s e et a l . , 1 9 8 3 b ) . M o r e o v e r , m a n y a r e p h e n o l i c a n d a m o n g s t t h e m , s t r y c h n o p e n t a m i n e a n d isomers a r e c u r i o u s a l k a l o i d s w i t h f i v e n i t r o g e n a t o m s ; a m e t h y l p y r r o l i d i n e g r o u p is j o i n e d to the b e n z e n e ring o f the Corynane p a r t of the m o l e c u l e s . O x i n d o l e al k a l o i d s fetrychnofoline, s t r y c h n o p h y l 1 i n e a n d isomers) a r e a l s o f o u n d for the f i r s t t i m e in S t r y c h n o s g e n u s .

F r u i t s o f S . u s a n b a r e n s i s s u c h a s c h e r r i e s h a v e c a u s e d the p o i s o n i n g of c h i l d r e n in A f r i c a . W e h a v e then i s o l a t e d t h e i r m a i n a l k a l o i d a n d i d e n t i f i e d it as d e s c a r b o m e -t h o x y d i h y d r o g a m b i r -t a n n i n e ; -tha-t a l k a l o i d h a d b e e n n e v e r f o u n d in -the f a m i l y o f L o g a n i a ­ c e a e but w a s p r e v i o u s l y d i s c o v e r e d in O c h r o s i a g e n u s ( A p o c y n a c e a e ) .

A m o n g the o t h e r S t r y c h n o s r e c e n t l y s t u d i e d a t L i e g e , w e will h o l d the a t t e n t i o n o n :

- S . v a r i a b i l i s (small t r e e e n d e m i c a r o u n d K i n s h a s a in Z a i r e ) . T h e g o l d e n fruit p u l p is d e l i c i o u s l y s w e e t b u t the root b a r k is a v i o l e n t p o i s o n from w h i c h t w e n t y a l k a ­ loids h a v e b e e n i s o l a t e d a n d e s p e c i a l l y t w o p a i r s of d i a s t e r e o m e r i c e q u i l i b r a t i n g alka­ loids that c o u l d be the b i o g e n e t i c s t a r t i n g p o i n t of two s e r i e s of m o n o - a n d d i m e r i c a l k a loids w h i c h h a v e a d i f f e r e n t s t e r e o c h e m i s t r y for C j ^ : the r e t u l i n e s e r i e s a n d t h e e p i m e r i s o r e t u l i n e s e r i e s (Tits e t a l . , 1 9 8 0 ) . T h e s e a l k a l o i d s still c o n t a i n a n e w g r o u p of S t r y c h n o s u n s y m m e t r i c a l d i m e r s : the s t r y c h n o b i 1 i n e s , c h a r a c t e r i z e d by a carbi nolami tie e t h e r g r o u p in a h e x a c y c l i c r i n g s i m i l a r to t h a t o f g e i s s o s p e r m i n e (Tits e t a l . , 1 9 8 3 a , b ) . M o r e o v e r , the q u a t e r n a r y a l k a l o i d s - m a v a c u r i n e a n d f l u o r o c u r i n e - ( F i g . 9) have b e e n e x t r a c t e d f r o m this A f r i c a n s p e c i e s (Tits e t a l . , 1 9 8 1 ) . - S . i c a j a u s e d a s o r d e a l a n d a r r o w poi s o n I n t h e N o r t h E a s t o f Z a i r e a n d in G a b o o n . T h e root b a r k s s o m e t i m e s c o n t a i n u p to f i f t e e n p e r c e n t o f a l k a l o i d s , o u t o f w h i c h s t r y c h n i n e is the m a i n c o n s t i t u e n t b u t a g a i n w e h a v e f o u n d a n e w d i m e r i c p r o d u c t that w e h a v e c a l l e d s u n g u c i n e ( L a m o t t e e t a l . , 1 9 7 9 ) . - S . g o s s w e i l e r i v e r y rich in a n e w g l u c o a l k a l o i d : d o l i c h a n t o s i d e a n d in q u a t e r n a r y a l k a l o i d s ( d i p l o c e l i n e ( F i g . 10) w i t h a n t i m i c r o b i a l a c t i v i t y ) a n d anhydroniurn b a s e s like a l s t o n i n e a n d a n e w o n e : s t r y c h n o x a n t h i n e ( C o u n e e t a l . , 1 9 8 4 ) . S . s c h e f f l e r i f r o m Z a i r e w i t h a l k a l o i d s r e l a t e d to the t a b a s c a n i n e a n d s p e r m o s -t r y c h n i n e -type b a s e s , a l r e a d y k n o w n f r o m -the A m e r i c a n S . b r a s i l i e n s i s a n d S . -t a b a s c a n a ( C a p r a s s e a n d A n g e n o t , I38la). j - a c e t y l - 0 - m e t h y l s t r y c h n o s p l e n d i n e , m a i n a l k a l o i d in the l e a v e s , w a s n o w f o u n d to h a v e s t r o n g m u s c l e - r e l a x a n t a c t i v i t y ( W e e r a t u n g a e t a l . , 1 9 8 4 ) . D u r i n g o u r p h y t o c h e m i c a l s c r e e n i n g o n m e d i c i n a l p l a n t s o f C e n t r a l A f r i c a , w e h a v e 244 A n g e n o t

o b t a i n e d s a m p l e s of an o t h e r s p e c i e s of L o g a n i a c e a e ; A n t h o c l e r s t a g r a n d ! f l o r a is a tree (5-35 m high) u n a r m e d a n d c a l l e d " c a b b a g e - t r e e " or " f e v e r - t r e e " b e c a u s e used against fever. T h i s s p e c i e s n e v e r c o n t a i n s a l k a l o i d s but a l w a y s s e c o i r i d o i d g l y c o s i d e s . The main c o n s t i t u e n t of the b a r k is s w e r o s i d e , w h o s e f e b r i f u g e a c t i v i t y w a s a l s o p e r f o r m e d during this s t u d y ( C h a p e l l e , 1 9 7 6 ) . M o r e r e c e n t l y , an a n t i - a m o e b i c a c t i v i t y h a s been demon^ strated for this c o m p o u n d (Van Beek e t a l . , 1 9 8 4 ) . A c c o r d i n g to these r e s u l t s . w e think that the b i o l o g i c a l a c t i v i t y o f the i r i d o i d s r e l a t e d to loganin a n d q u i t e c o m m o n in the S t r y c h n o s a s they take part in the b i o g e n e s i s of a l k a l o i d s , h a s to be investigated in the f u t u r e .

R E A S O N S F O R T H E S T U D Y O F S T R Y C H N O S IN T H E FIELD OF T U M O R INHIBITORS

It s e e m s that two f u n d a m e n t a l a p p r o a c h e s c a n be f o l l o w e d to i d e n t i f y p l a n t s havi ng these proper t i e s :

R a n d o m s e l e c t i o n s c r e e n i n g

It is the m e t h o d m a i n l y p r a c t i s e d by National C a n c e r I n s t i t u t e ( U . S . A . ) . A c c o r d i n g to S u f f n e s s a n d D o u r o s (1982) , NCI s c r e e n s a b o u t 10,000 n e w s y n t h e t i c c o m p o u n d s a n d 400 pure natural p r o d u c t s per y e a r p l u s a b o u t 14,000 c r u d e natural extracts(8,000 from ferrren tat ion ; 5 ,000 f r o m p l a n t a n d 1,000 f r o m mar ine a n i mal) . A b o u t 6-8 c o m p o u n d s e n t e r cl in_i_ cal t r i a l s e a c h y e a r . S l i g h t y less than half of them are natural p r o d u c t s . The cost of bioassay p r o c e d u r e s b e c o m e s g r e a t e r w i t h increasing n u m b e r o f plant p a r t s tested,but this program o f f e r s a n a d v a n t a g e in that it is p r o v i d i n g s o m e n e w s t r u c t u r e s — p r e v i o u s l y u n a s s o c i a t e d w i t h tumor i n h i b i t i n g a c t i v i t y — , that s e r v e a s model for s y n t h e s i s of d e r i v a t i v e s w i t h c o u l d be m o r e a c t i v e , a n d h o p e f u l l y h a v e fewer s i d e - e f f e c t s , than the parent natural c o m p o u n d ( F a r n s w o r t h a n d K a a s , 1 9 8 1 ) .

S c r e e n i n g o f m e d i c i n a l a n d t o x i c p l a n t s

The a p p r o a c h of t e s t i n g p l a n t s w i t h a n a c c o u n t o f use for any c o n d i t i o n in t r a ­ ditional m e d i c i n e has been m o s t successful up to n o w a n d a t a lower c o s t . One recent study d e s c r i b e s an a t t e m p t to c o r r e l a t e results o b t a i n e d f r o m the NCI p l a n t a n t i t u m o r screening p r o g r a m w i t h s e l e c t e d t y p e s of f o l k l o r i c u s e s for the same p l a n t s .

F r o m these d a t a , it w o u l d a p p e a r that o n e w o u l d increase by a factor o f two the number o f p l a n t s s p e c i e s that w o u l d show e x p e r i m e n t a l in v i t r o or In v i v o c y t o t o x i c i t y or a n t i t u m o r a c t i v i t y if the p l a n t s w e r e s e l e c t e d on the b a s i s o f a l l e g e d use a s a n t i ­ cancer r e m e d i e s , a n d by a factor o f a b o u t f i v e , for the p l a n t s a l l e g e d to be useful as arrow poi sons ( F a r n s w o r t h a n d K a a s , 1 9 8 1 ) . N o w , the g e n u s S t r y c h n o s i s wel 1 k n o w n in the world a s welt a s a n a r r o w a n d dart p o i s o n (curare in S o u t h America,Ipoh in A s i a , 8 a n y a m b o!s gift in A f r i c a , a s a n ordeal p o i s o n ( M ' B o u n d o u in A f r i c a . . . ) .

M o r e o v e r , w e have c o n s i d e r e d the p u b l i s h e d e x p e r i m e n t a l data indicating antitumor activity for c r u d e e x t r a c t s f r o m S t r y c h n o s :

- the a l k a l o i d a l e x t r a c t s of S . h e n n i n g s i i w e r e shown to be a c t i v e a g a i n s t c a n c e r cells ( H o k a n s o n , 1975) .

- the a q u e o u s e x t r a c t s of S . e l a e o c a r p a a n d S . l u c e n s s t e m b a r k , a n d the c h l o r o f o r m extract of S. u s a m b a r e n s l s leaves h a v e all been shown to be a c t i v e a g a i n s t lymphatic

l e u k e m i a P-388 in v i v o in m i c e (personnel c o m m u n i c a t i o n o f the l a t e M . K u p c h a n t o W, R o l f s e n (1980). S i n c e I98I w e h a v e c a r r i e d o u t e x p e r i m e n t s a b o u t c y t o t o x i c a n d a n t i t u m o r proper­ t i e s o f a l k a l o i d s i s o l a t e d f r o m S t r y c h n o s u s a m b a r e n s i s , s i n c e t h i s s p e c i e s is u s e d as a r r o w p o i s o n a n d h a s s h o w n a n a n t i t u m o r a c t i v i t y f r o m a c r u d e e x t r a c t . M E T H O D S C y t o t o x i c i t y A n t i m i t o t i c a c t i v i t y t e s t s h a v e b e e n c a r r i e d o u t o n c u l t u r e d : - h e p a t o m a c e l l s d e r i v e d f r o m H W 165 h e p a t o m a o f W i s t a r r a t s ; - Β 16 m e l a n o m a c e l l s d e r i v e d f r o m C 57 Bl m o u s e m e l a n o m a ; - E h r l i c h a s c i t e s c e l l s (line ELT) d e r i v e d f r o m a m o u s e m a m m a r y g l a n d c a r c i n o m a a n d t r a n s p l a n t e d into C 57 Bl m o u s e p e r i t o n e a l c a v i t y . T h e c e l l s a r e c u l t u r e d in a l i q u i d n u t r i e n t (MEM G i b c o m e d i u m 9 0 ¾ c o m p l e m e n t e d with 10¾ foetal c a l f s e r u m ) a n d 100 U/ml p e n i c i l l i n . T h e a g e n t to be t e s t e d Is a d d e d to the c u l t u r e m e d i u m a t v a r i o u s c o n c e n t r a t i o n s a n d for a m a x i m u n o f 4 8 h .

A f t e r the t r e a t m e n t , the c e l l s a r e f i x e d a n d s t a i n e d by F e u l g e n r e a c t i o n f o r cyto logical a n a l y s i s (light m i c r o s c o p y ) . M i t o t i c a c t i v i t y o r d e g r e e o f cell d e a t h is ex p r e s s e d as m i t o t i c or p y c n o t i c index ( n u m b e r o f c e l l s in m i t o s i s o f p y c n o s í s for 1,000 c e l l s ) . In e a c h c a s e , 5 to 10,000 c e l l s a r e a n a l y s e d . T h e p e r c e n t a g e s of mitotic phases a r e c a l c u l a t e d in v i e w of d e t e c t i n g p o s s i b l e m i t o t i c d i s t u r b a n c e s . M e a n index are c o m p a r e d w i t h S t u d e n t t t e s t . A n t i t u m o r a c t i v i t y t e s t s "in v i v o " C 57 Bl 6 J m a l e m i c e w e r e i n j e c t e d i.p. w i t h 10 E h r l i c h a s c i t e s c e l l s . T h e tumor w a s a l l o w e d to g r o w for k d a y s a n d a t that t i m e , the m i c e r e c e i v e d the d r u g i.p. e i t h e r in a s i n g l e shot or in r e p e a t e d i n j e c t i o n s a t a d a y interval for 3 d a y s . V a r i o u s d o s a g e s of the d r u g w e r e u s e d . M i c e w e r e s a c r i f i c e d by e x s a n g u i n a t i o n . P e r i t o n e a l w a s h i n g s w e r e p e r f o r m e d to r e m o v e all t u m o r c e l l s a n d cell c o u n t w a s d o n e wi th h e m o c y t o m e t e r or C o u l t e r c o u n t e r model B. T e s t s w e r e c o n t r o l l e d e a c h t i m e o n 10 m i c e . F I R S T E X P E R I M E N T S ON A L K A L O I D S I S O L A T E D FROM S . U S A M B A R E N S I S - S e l e c t i o n b a s e d o n s t r u c t u r e - a n t í m i t o t i c a c t i v i t y r e l a t i o n s h i p s : a) T w o a l k a l o i d s of the r o o t s ( m e l í n o n i n e F a n d d i h y d r o f 1 a v o p e r e i r i n e ) p o s s e s s a t r i c y c l i c β c a r b o l i n i u m ring a n d a r e v e r y c l o s e to p e n t a c y c l i c a n h y d r o n i u m b a s e s s u c h a s a l s t o n l n e , s e r p e n t i n e ( F i g . 11) a n d s e m p e r v i r i n e w h o s e p a t e n t a s a n t i t u m o r d r u g is p e n d i n g (Beljanski a n d B u g i e i , 1 9 7 9 ) . A n o t h e r r e p r e s e n t a t i v e c o m p o u n d s o f t h i s f a m i l y o f p l a n a r a l k a l o i d s a r e e l l i p t i c i n e ( F i g . 12) f o u n d n o t o n l y in O c h r o s i a s p . ( A p o c y n a -c e a e ) but a l s o in S t r y -c h n o s d i n k l a g e i (Mi-chel e t al ., 19 8 0 ) - a n d m a i n l y 9- h y d r o x y - 2 methyl el 1ipticiniurn ( F i g . 13)· T h i s q u a t e r n a r y c o m p o u n d is less t o x i c t h a n e l l i p t i c i n e

and used as anticancer drug in Europe.

Quaternary ammonium salts of a n h y d r o n i u m bases thus seem attractive candidates

as potential a g e n t s . Various cytotoxic effects have been detected by cytological methods

and compared to sempervirine (Table 1) (Bass I eer e t a I . , 1982, 1983 and 198*0.

Concomitantly with tests of biological a c t i v i t y , it appeared useful to Investi

gate the interaction of these products with DNA, which has most frequently been found as

[he main target of many similar drugs in the c e l l . The differences between the behaviours

of the three alkaloids so far investigated can be interpreted on the basis of different

tent of penetration of the chromophore ring into the DNA helix (Caprasse and H o u s s i e r ,

I

1983 and 1984) .

b) Three alkaloids of the leaves (strychnofoline, dίhydro-usambarine and s t r y c h n £

pentamine) possessing the usambarane skeleton were first tested. They are structurally

Similar to emetine (Fig. ]k) and may be regarded as indole a n a l o g u e s . Emetine acts by

means of ρ rote i η synthes is inhibit! on and has been eva luated c l i n i c a l l y a s a n a n t i m i t o t i c

The structural similarities prompted us to evaluate the cytotoxic properties of

these alkaloids. Strychnofoline and d i h y d r o - u s a m b a r i n e showed a certain degree of

totic activity at relatively high doses [10 yg/ml and mainly 50 μ/ml (Bassleer e t

al., 1982)]. St rychnopen tami ne is about ten tImes more powerfu1 than strychnofoli ne

and 18,19 dihydro-usambarine as an antimitotic agent in animal tumor cell cu 1 tures,sinee

[t inhibits 50¾ of the cells in mitosis at a concentration of I ug/ml ( 1 . 8 p m ) , The com

pari son of the ir molecular structures induces us to think that the presence of a Ν methyl

-pyrrolidine group increases the antimitotic activity of these alkaloids {Table 2) (Tits

1984) .

Considering this a n t i m i t o t i c a c t i v i t y , we have undertaken preliminary e x p e r i ­

ments to search for a possible antitumor activity of this molecule in the a n i m a l . Our

experiments have been carried out on Ehrlich ascites tumor celts in male mice (Tits et

al., 1984). When they received strychnopentamine in a single injection of 1 m g , there

was a med i um decrea se of cells number (about 5 0 ¾ ) . At a s i n g l e d o s e o f 2mg ,a v e r y st rong

decrease of tumor cell number was observed (more than 8 0 ¾ ) . The very rapid regression

isobvious, but m i c e rapidly show signs of intoxication, since four mice out of ten died

very quickly after the injection. In the comi ng futu re , s Ϊ m i 1 ar test s wi Π a) so be carried

out with isomers of strychnopentarnine and other derived alkaloids at various dosages in

view of establ i sh i ng a closer structure-activity r e l a t i o n s h i p s , and to look for less

toxic compounds .

WHY FURTHER INVESTI GAT I ON OF BRAZILIAN STRYCHNOS?

The ma in reasons why we shou Id f u r t h e r ] η vestigate Brazilian Strychnos spec ies are :

- the lack of knowledge concerning the distribution of alkaloids among many of them

(Marini-Bettolo and B i s s e t , 1 9 7 2 ; B i s s e t , 1 9 7 7 ) . M o r e o v e r , according to K r u k o f f , the

distribution of species in Southern part of the great Amazon Basin — particularly from

the upper Rio Xingu and Rios Tapajós and Madeira — is poorly known and it is possible

Ifty further investigation ... 247

I

Why

that new species may be found there (Krukoff , 1 9 7 2 ) . Indeed, there is little informatfcn

ava i 1 able not on I y on the var iat ion in compos i t ion of the a) kaloi da 1 ml xture i η the d iffer

ent parts of the plants (e.g. the l e a v e s ) , but also on their biological activities too

focused in the past on curar ΐ ζing act i νΪ ty.

- the new results in the antimitotic field from A f r i c a n spec ies of

Strychnos

,which

chemically are very similar to certain South American s p e c i e s . Indeed, the occurrence

in the A f r i c a n species of guaternary bases and in the A m e r i c a n spec i es of ter t i ary bases

such as akagerine emphasizes the unity of the genus

S t r y c h n o s

in terms of i ts a \ kaloids,

Besides known antitumor d r u g s , e.g. alstonine and e 1 1 i pt i c i ne ,Af r i can

S t r y c h n o s

species

possess m a i n t y new antimitotic p r o d u c t s , e.g. st rychnopentam i ne . Why should Brazilian

species not contain alkaloids with similar properties? As far as I a m concerned,! would

be grateful to anyone who w o u l d accept to cooperate in this f i e l d .

According to many r e s u l t s , it is a clear that research into arrow poi sons and their

ingredients remains closely connected w i t h current activities in the never-end i ng search

for new and better d r u g s , both naturally-occurring and s y n t h e t i c . Natural products have

previously been of great benefit in providing molecular structures for study by organic

chemists and as models for study by p h a r m a c o l o g i s t s . It is a part they still have ttj

play at our present time.

F I G . 8 . A f r o c u r a r i n e .

EMETINE

Antiamoebic and antitumor activities FIG. 14. Emetine

1 8 , 1 9 à\hydro-usambarine S t r y c h n o f o l i n e S t r y c h n o p e n t a m i n e a O n Β 16 M e l a n o m a A f t e r 7 2 h o u r s A f t e r 72 h o u r s A f t e r 2k hours E D5 0 > 10 u g / m l E D5 0 > 10 u g / m l E D ^ = 1 ug/ml < 50 ug/m! < 50 ug/nil b O n A s c i t e s A f t e r 2k h o u r s A f t e r 2<4 h o u r s A f t e r 24 h o u r s E h r l i c h c e l l s E D ^ = 10 pg/ml E D ^ := 10 ug/ml E D ^ = 3 pg/ml c O n H W 165 H e p a t o m a A f t e r 7 2 h o u r s A f t e r 24 hours E D5 0 > 10 ug/ml E D ^ = 3 U g / m ] < 50 ug/ml < 1 ug/ml T a b l e 2 R e f e r e n c e s A n g e n o t , L . - 1 9 7 0 . E s s a i s p h y t o c h i m i q u e s p r é l i m i n a i r e s s u r q u e l q u e s plantes medicinales d u R w a n d a o r i e n t a l . P l a n t e s M e d i c i n a l e s e t P h y t o t b ê r a p i e , 4 ( 4 ) : 2 6 3 - 2 7 8 . A n g e n o t , L . - 1 9 7 1 , D e 1 ' e x i s t e n c e e n A f r i q u e C e n t r a l e d ' u n p o i s o n d e f l ê c h e c u r a r i s a n t i s s u d u S t r y c h n o s u s a m b a r e n s i s G I L G . A n n a t e s Pharmaceutiqu.es F r a n ç a is e s ,29 (5-6): 353-3 6 4 . A n g e n o t , L . ; D u b o i s , M . 5 G i n i o n , C ; V a n D o r s e r , W . ; D r e s s e , A . - 1 9 7 5 a . C h e m i c a l S t r u c t u r e a n d P h a r m a c o l o g i c a l ( c u r a r i z i n g ) P r o p e r t i e s of V a r i o u s I n d o l e A l k a l o i d s E x t r a c t e d f r o m a n A f r i c a n S t r y c h n o s . A r c h i v e s i n t e r n a t i o n a l e s d e P h a r m a c o d y n a m i c et d e T h ê r a p i e , 2 1 5 ( 2 ) : 2 4 6 - 2 5 8 . A n g e n o t , L . ; D i d e b e r g , 0.; D u p o n t , L . - 1 9 7 5 b . I s o l a t i o n a n d S t r u c t u r e of A k a g e r i n e : a n e w type of I n d o l e A l k a l o i d . T e t r a h e d r o n L e t t e r s , 1 6 : 1 3 5 7 - 1 3 5 8 . B a s s l e e r , R. ; D e p a u w - G i l l e t , Μ . C ; M a s s a r t , B . ; M a r n e t t e , J . Μ . ; W i l i q u e t , P.jCaprasse, M . ; A n g e n o t , L . 1 9 8 2 . E f f e t s d e t r o i s a l c a l ó i d e s e x t r a i t s d u S t r y c h n o s u s a m b a r e n -s i -s -s u r d e -s c e l l u l e -s c a n c é r e u -s e -s e n c u l t u r e . P l a n t a M e d i c a , 4 5 : 1 2 3 - 1 2 6 . B a s s l e e r , R . ; M a r n e t t e , J . M . ; W i l i q u e t , P . ; D e p a u w - G i l l e t , M . C . ; C a p r a s s e , M . ; A n g e n o t , L . - 1 9 8 3 . E t u d e c o m p l ê m e n t a i r e d e la c y t o t o x i c i t y d e l a m e l í n o n í n e F , a l c a l ó i d e d e r i v e d e l a β - c a r b o l i n e . P l a n t a M e d i c a , 4 9 : 1 5 8 - 1 6 1 .

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H o k a n s o n , G . C . - 1 9 7 5 , P o t e n t i a l a n t i t u m o r a g e n t s f r o m h i g h e r p l a n t s ; P a r t , n o s h e n n i n g s i i . P a r t . 2: C e n t a u r e a s o l s t i t i a l i s . P a r t 3 : C r o t o n t e x e n s i s . T h e s i s . U n i v e r s i t y of P u r d u e ( U . S . A . ) . I: Strych P h . D. K r u k o f f , B . A . - 1 9 7 2 . A m e r i c a n s p e c i e s of S t r y c h n o s . L l o y d i a , 3 5 , 1 9 3 . L e e u w e n b e r g , A . J . Μ . 1 9 6 9 . T h e L o g a n i a c e a e o f A f r i c a V I I I , S t r y c h n o s I I I . M e d e d e -l i n g e n L a n d b o u w h o g e s c h o o -l , W a g e n i n g e n , 6 9 : 1 - 3 3 6 . L a m o t t e , J . ; D u p o n t , L . ; D i d e b e r g , 0.; K a m b u , K . ; A n g e n o t , L . - 1 9 7 9 .- I s o l a t i o n and S t r u c t u r e o f s u n g u c i n e : a n e w t y p e of b i s i n d o l i n e a l k a l o i d . T e t r a h e d r o n L e t t e r s (43) 4 2 2 7 - 4 2 2 8 . M a r i n i - B e t t o l o , G . B . & B i s s e t , Ν . G . - 1 9 7 2 . C h e m i c a l s t u d i e s o n the a l k a l o i d s of A m e r i c a n S t r y c h n o s s p e c i e s . L l o y d i a , 3 5 , 1 9 5 . M a r i n i - B e t t o l o , G . B . ; M e s s a n a , I.; N i c o l e t t i , M . ; P a t a m i a , Μ . ; G a l e f f i , C . - 1 9 8 0 . On t h e a l k a l o i d s of S t r y c h n o s X X X V . T h e o c c u r r e n c e o f a k a g e r i n e i n S o u t h A m e r i c a n Stry_ c h n o s . J o u r n a l o f N a t u r a l P r o d u c t s , 4 3 , 7 1 7 . M i c h e l , S . ; T i l l e q u i n , F . ; K o c h , N . ; A k e - A s s i , L . - 1 9 8 0 . L ' e l l i p t i c i n e . a l c a l ó i d e majeur d e s é c o r c e s de S t r y c h n o s d i n k l a g e i . J o u r n a l of N a t u r a l P r o d u c t s , 4 3 , 2 9 4 . O h i r i , F . C.j V e r p o o r t e , R . B a e r h e i m - S v e n d s e n , A . - 1 9 8 3 . T h e A f r i c a n S t r y c h n o s species a n d t h e i r a l k a l o i d s : a r e v i e w . J o u r n a l o f E t h n o p h a r m a c o l o g y , 9 : 1 6 7 - 2 2 3 . R o l f s e n , U . - 1 9 8 0 . P h y t o c h e m i s t r y a n d p h a r m a c o l o g y of s o m e A f r i c a n S t r y c h n o s s p e c i e s . P h . D. T h e s i s . U n i v e r s i t y o f U p p s a l a ( S w e d e n ) . S u f f n e s s , M . a n d D o u r o s , J . - 1 9 8 2 . C u r r e n t s t a t u s o f the N C I p l a n t a n d a n i m a l p r o d u c t p r o g r a m . J o u r n a l o f N a t u r a l P r o d u c t s , 4 5 , 1. T i t s , M . ; A n g e n o t , L . ; T a v e r n i e r , D. - 1 9 8 0 . N e w i n d o l e a l d e h y d i c a l k a l o i d s of S t r y c h n o s v a r i a b i l i s . T e t r a h e d r o n L e t t e r s , 21 ( 2 5 ) , 2 4 3 9 - 2 4 4 2 . T i t s , M . ; F r a n z , M . ; T a v e r n i e r , D . ; A n g e n o t , L . - 1 9 8 1 . L e s a l c a l ó i d e s q u a t e r n a i r e s m a j o r i t a i r e s d u S t r y c h n o s v a r i a b i l i s d u Z a i r e . P l a n t a M e d i c a , 4 2 , 3 7 1 - 3 7 4 . T i t s , Μ . ; A n g e n o t , L . ; T a v e r n i e r , D. - 1 9 8 3 a . D i d e h y d r o - 1 6 ,17 i s o s t r y c h n o b i l i n e .nouvel a l c a l ó i d e b i s i n d o l i n i q u e a s y m ê t r i q u e d u S t r y c h n o s v a r i a b i l i s . J o u r n a l d e P h a r m a c i e d e B e l g i q u e , 3 8 ( 5 ) : 2 4 1 - 2 4 5 . T i t s , M . ; A n g e n o t , L . ; T a v e r n i e r , D . - 1 9 8 3 b . H y d r o x y s t r y c h n o b i l i n e , n o u v e l a l c a l ó i d e b i s i n d o l i n i q u e d u S t r y c h n o s v a r i a b i l i s . J o u r n a l o f N a t u r a l P r o d u c t s , 4 6 , 5 , 6 3 8 - 6 4 5 . T i t s , M . ; D e s a i v e , C.J M a r n e t t e , J . M . ; B a s s l e e r , R . ; A n g e n o t , L . - 1 9 8 4 . A n t i m i t o t i c a c t i v i t y of s t r y c h n o p e n t a m i n e , a b i s i n d o l i c a l k a l o i d . J o u r n a l o f E t h n o p h a r m a c o l o g y . U n d e r p r e s s . V a n B e e k , Τ . Α . ; D e e l d e r , A . M . ; V e r p o o r t e , R . ; B a e r h e i m - S v e n d s e n , A . - 1 9 8 4 . A n t i ­ m i c r o b i a l , a n t i a m o e b i c a n d a n t i v i r a l s c r e e n i n g o f s o m e T a b e m a e m o n t a n a s p e c i e s . P l a n t a H e d i c a , 5 0 , 1 8 0 . W e e r a t u n g a , G . ; G o o n e t i l e k e , Α . ; R o l f s e n , W . ; B o h l i n , L . ; S a n d b e r g , F . - 1 9 8 4 . A l k a l o i d s in S t r y c h n o s a c u l e a t a . A c t a P h a r m a c e u t i c a S u e c i c a , 2 1 , 1 3 5 .