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P e r g a m o n 0 0 3 1 - 9 4 2 2 9 4 ) 0 0 7 6 2 - 4
P h y t o c h e m i s t r y
Vol. 38, No. 4, pp. 853-857, 1995
Cop yr igh t © 1995 Elsevier Science Ltd
Pr inted in Great Br itain. All r ights reserved
0031 9422/95 9.50 + 0.00
15 HYDROPEROXYDEHYDROABIETIC ACID A CONTACT ALLERGEN IN
COLOPHONY FROM P N U S SPECIES
L IP . SHA O, ELISABETH G,~FVERT, ULRIKA NILSSON,* ANN-THERESE KARLBERG an d J . LARS G. NILSSON'~
De partm ent of Occupatio nal Dermatology, Na tional Institute of Occupational Health, S-17I 84 Solna, Stockholm, Sweden;
*Department of Ana lytical Chemistry, Nation al Institute of Occu pationa l Health, S-171 84 Solna, Sweden; tDe partm ent of Organic
Pharmaceutical Chemistry, Upps ala University, S-751 23 Uppsala, Sweden
(Received in revisedJorm 23 August 1994)
K e y W o r d l n d e x - - P i n u s ; P i n a c e a e ; c o l o p h o n y ; r o s i n ; g u m r o s i n ; t a l l o i l r o s i n ; c o n t a c t a l l e r g e n ;
s y n t h e s i s ; d e h y d r o a b i e t i c a c i d ; h y d r o p e r o x i d e .
A b s t r a c t - - A n e w h y d r o p e r o x i d e , 1 5 - h y d r o p e r o x y d e h y d r o a b i e t i c a c i d (1 5 - H P D A ) , w i t h c o n t a c t a l l e rg e n i c p r o p e r t i e s
h a s b e e n d e t e c t e d i n r o s i n o b t a i n e d f r o m Pinus s p e ci e s. D e t e c t i o n w a s f a c i l i t a t e d u s i n g a s y n t h e t i c p r e p a r a t i o n o f 1 5 -
H P D A f o r re f er e n ce p u r p o se s . T h e s y n t h e si s a n d t h e d e t e c ti o n ( H P L C a n d G C ) o f 1 5 - H P D A i n r o s in a r e d e s cr i b ed .
T h e a l l e r g e n i c a c t i v i t y o f 1 5 - H P D A w a s s t u d i e d i n a n e x p e r i m e n t a l s e n s i t i z a t i o n t e s t o n g u i n e a - p i g s .
INTRODUCTION
I n o u r r e s e a r c h o n n a t u r a l l y o c c u r r i n g a l l e r g e n ic s u b -
s t a n c e s w e h a v e e s p e c i a l l y s t u d i e d t h e a l l e r g e n ic a c t i v i t y
o f c o m p o u n d s f o u n d i n c o l o p h o n y ( g u m r o si n ), th e
d i s t i l l a t i o n r e s i d u e o f o l e o r e s i n f r o m Pinus pa lus t r is M il l .
a n d o t h e r Pinus s p e c i es [1 , 2 ]. C o l o p h o n y i s a m o n g t h e 1 0
m o s t c o m m o n c a u s es o f d e l a y e d h y p e r se n s i ti v i ty [ 3 - 5 ] .
G u m r o s i n a n d t a l l o i l r o s i n , a b y - p r o d u c t i n t h e p u l p
i n d u s t r y , a r e u s e d a l m o s t i n t e r c h a n g e a b l y i n te c h n i c a l
p r o d u c t s a n d w o r l d p r o d u c t i o n i s a b o u t o n e m i l l i o n t o n s
a y e a r . C o l o p h o n y i s a c o m p l e x m i x t u r e o f d i t e r p e n e r e si n
a c i d s ( 8 5 - 9 0 ) , d i t e r p e n e a l c o h o l s , a l d e h y d e s a n d h y d r o -
c a r b o n s . T h e m a j o r r e s i n a c i d s a r e a b i e t i c a c i d ( 1 ) a n d
d e h y d r o a b i e t i c a c i d ( 3 ) [ 6 ] . I n p a r t i c u l a r , a b i e t i c a c i d i s
e a s i l y o x i d i z e d b y a t m o s p h e r i c o x y g e n . I n n a t u r e t h e
o x i d a t i o n g i v e s a n a n t i m i c r o b i a l e f fe c t t o t h e e x u d a t e
w h e n t h i s c o m e s in c o n t a c t w i t h a i r o w i n g t o w o u n d i n g o f
t h e t r e e . T h e t r e e i s t h e n p r o t e c t e d f r o m m i c r o b e s u n t i l
hea led .
I n p r e v i o u s s t u d i e s w e h a v e i s o l a t e d o x i d a t i o n p r o -
d u c t s o f a b i e t i c a c i d a n d d e h y d r o a b i e t i c a c i d a n d i d e n t i -
f i ed t h e m a s c o n t a c t a l le r g e n s [ 7 - 1 0 ] . 1 5 - H y d r o p e r o x y -
a b i e t i c a c i d ( 1 5 - H P A ) ( 2 ) i n g u m r o s i n a n d t a l l o i l r o s i n
h a s b e e n i d e n t i f ie d a s a m a j o r c o n t a c t a l l e r g e n a m o n g t h e
o x i d a t i o n p r o d u c t s [ 7 ] . H o w e v e r , t h e c o r r e s p o n d i n g
h y d r o p e r o x i d e o f d e h y d r o a b i e t i c a c i d ( 1 5 - H P D A ) ( 4 ) h a s
n o t b e e n p r e v i o u s l y i d e n t i f i e d . S i n c e d i d e h y d r o a b i e t i c
a c i d p e r o x i d e ( D e A - O O - D e A ) (5 ) i s a n o t h e r o f t h e i d e n t i -
f i ed a l le r g e n i c c o m p o u n d s [ 1 0 ] , i t is r e a s o n a b l e t o a s s u m e
t h a t 1 5 - H P D A c o u l d b e a n i n te r m e d i a t e in th e f o r m a t i o n
o f t h i s p e r o x i d e . O w i n g t o t h e s t r u c t u r a l s i m i l a r i t y b e -
t w e e n 1 5 - H P D A a n d t h e e a r l i e r i d e n t i f i e d 1 5 - H P A i t i s
p r o b a b l e t h a t 1 5 - H P D A c o u l d b e a c o n t a c t a l l er g e n w i t h
a p o t e n c y s i m i l a r t o t h a t o f 1 5 - H P A . W e , t h e r e f o r e ,
d e c i d e d to d e v e l o p a s y n t h e t i c r o u t e t o 1 5 - H P D A a n d u s e
t h i s c o m p o u n d a s a r e f e r e n ce m a t e r i a l t o f a c i l i t a te t h e
d e t e c ti o n o f 1 5 - H P D A i n c o l o p h o n y . I n t h e p r e s e n t p a p e r
w e d e s c r i b e t h e s y n t h e s i s o f 1 5 - H P D A a n d i t s s u b s e q u e n t
i d e n t i f i c a t i o n i n m e t h y l e s t e r if i e d P o r t u g u e s e c o l o p h o n y
a n d S w e d i s h t a l l o i l r os i n . T h e s e n s i ti z i n g p o t e n t i a l o f 1 5 -
H P D A w a s a l so s t u d i e d in g u i n e a -p i g s. T h e h y d r o p e r o x -
i d e o f d e h y d r o a b i e t i c a c i d w a s s y n t h e s i z e d , d e t e c t e d i n
r o s i n a n d s t u d i e d i n g u i n e a - p i g s a s i ts m e t h y l e s t er . W e
u s e t h e a b b r e v i a t i o n 1 5 - H P D A f o r t h e m e t h y l e s t e r a l s o .
RESULTS AND DISCUSSION
F o r t h e s y n t h e s i s o f t h e h y d r o p e r o x i d e 4 a t h e s y n t h e t i c
r o u t e d e p i c t e d in S c h e m e 1 w a s f o l lo w e d . D e h y d r o a b i e t i c
a c i d m e t h y l e s t e r ( 3 a) w a s u s e d a s s t a r t i n g m a t e r i a l . T h i s
c o m p o u n d w a s d e h y d r o g e n a t e d t o 6 u s in g 2 , 3 - d ic h l o ro -
5 , 6 - d i c y a n o - l ,4 - b e n z o q u i n o n e (D D Q ) . T h e 1 5 - h y d r o x y-
d e h y d r o a b i e t a t e ( 7) w a s o b t a i n e d in 8 0 y i e l d b y o x y -
m e r c u r a t i o n f o l l o w e d b y r e d u c t i o n u s i n g a l k a l i n e s o d i u m
b o r o h y d r i d e . W h i l e t h i s p a p e r w a s i n p r e p a r a t i o n 7 w a s
r e p o r t e d [ 1 1 ] p r e p a r e d e s s e n t i a l l y a c c o r d i n g t o t h e sa m e
p r o c e d u r e. I n o u r s y n th e s is t h e h y d r o x y c o m p o u n d 7 w a s
t r a n s f e r r e d t o t h e c o r r e s p o n d i n g h y d r o p e r o x i d e 4 a. u s i n g
e x c e ss a q u e o u s h y d r o g e n p e r o x i d e a n d a c i d c a t a l y s i s. T h e
n e w c o m p o u n d s w e r e i d en t if i ed w i t h I R , N M R a n d m a s s
s p e c t r o m e t r y .
P o r t u g u e s e c o l o p h o n y ( g u m r o s i n ) a n d S w e d i s h t a ll o il
r o s i n w er e a n a l y s e d b y m e a n s o f H P L C . T h e s y n t h es i ze d
1 5 - H P D A w a s u s e d a s r e f e r e n c e s u b s t a n c e . T a l l o i l ro s i n
c o n t a i n e d a p e a k e l u t i n g a t t h e s a m e r e t e n t i o n t i m e a s 1 5 -
H P D A ( F i g . 1). T h i s p e a k w a s i s o l a t e d fo r s u b s e q u e n t
853
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854 LI P. SHAO
e t a l .
1 R I = R 2 = H , 2 R I = O O H , R 2 - H ,
l a R I = H , R 2 = C H 3 2 a R 1 - O O H , R 2 = C H 3
3 R I = R 2 = H , 4 R I = O O H , R 2 = H ,
3 a R I = H , R 2 = C H 3 4 a R I = O O H , R 2 = C H 3
C O O C H 3
3a ~ Hg OAc)2
~ ~ H 2 S O
4 ~ ° C O O C H 3 C O O C H 3
4 a 7
a n a l y s i s w i t h m a s s s p e c t r o m e t r y . O w i n g t o t h e t h e r m a l
i n s t a b i l i t y o f 1 5 - H P D A , t h e s a m p l e w a s i n t r o d u c e d t o t h e
m a s s s p e c t r o m e t e r w i t h a d i r e c t i n s e r t i o n p r o b e D I P ) .
T h e m a s s sp e c t r u m o b t a i n e d f r o m t h e c o m p o u n d i n th e
c o l l e c t e d H P L C f r a c t i o n s h o w e d a n i d e n t i c a l f r a g m e n t a -
t i o n p a t t e r n t o , a n d t h e s a m e m o l e c u l a r i o n a s t h a t o f t h e
s y n t h e s i z e d r e f e re n c e c o m p o u n d . T h i s c o n f i r m s t h a t 1 5-
H P D A i s p r e s e n t i n S w e d i s h t a l l o i l r o s i n. 1 5 - H P D A w a s
n o t d e t e c t e d i n t h e P o r t u g u e s e g u m r o s i n . T h e o r e t i c a l l y ,
1 5 - H P D A s h o u l d b e p r e s e n t , s i n c e t h e d i d e h y d r o a b i e t i c
a c i d p e r o x i d e h a s b e e n i d e n t i fi e d i n g u m r o s i n [ 1 0 ] . T h i s
p e r o x i d e is p r o b a b l y f o r m e d f ro m 1 5 - H P D A .
I t is n o t l i k e l y t h a t 1 5 - H P D A i s a n a r t e f a c t f ro m t h e
d i s t i l l a t i o n p r o c e s s. T h e h i g h t e m p e r a t u r e w i ll r a t h e r
d e c r e a s e t h e le v e l o f h y d r o p e r o x i d e s p r e s en t . 1 5 - H P D A
h a s n o t b e e n r e p o r t e d i n p i n e e x u d a t e , b u t s e v e r a l
d e h y d r o a b i e t i c a c i d d e r iv a t i v e s w i t h a 1 5 - O H g r o u p h a v e
2 . ~ o 6 . ~ o
I ~ H P D A
1 0 . 0 0 1 4 . ) 0 1 8 . 0 0 2 2 . 0 0 m i n )
Fig. 1. HP LC -chro ma togram of tall oil rosin. Peak eluting at 18.9 was identified as 15-HPD A with M S first
eluting peak in the magnified section). For HP LC conditions, see Experimental.
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Hyd roper oxide of dehydroabietic acid in coloph ony 855
b e e n f o u n d [ 1 2 ] . T h e s e a l c o h o l s d e t e c t e d m a y h a v e b e e n
f o r m e d f r o m t h e c o r r e s p o n d i n g h y d r o p e r o x i d e s . T h e
i s o l a ti o n p r o c e ss i n v o lv e s th e u s e o f a q u e o u s N a O H t o
s e p a r a t e a c i d s f r o m n e u t r a l m a t t e r . S t r o n g b a s e s w i l l
p r o b a b l y d e s t r o y a n y h y d r o p e r o x i d e s p r e se n t . M o r e o v e r ,
G C a n a l y s e s h a v e o f t e n b e e n u s e d fo r t h e a n a l y s i s o f
e x u d a t e s [ 1 3 , 1 4 ]. A c c o r d i n g t o o u r e x p e r i e n c e i t i s n o t
p o s s i b l e to d e t e c t h y d r o p e r o x i d e s o f d i t e r p e n e s u s i n g t h e
G C - t e c h n i q u e , s in c e t h e h y d r o p e r o x i d e s w i ll d e c o m p o s e
a t t h e h i g h t e m p e r a t u r e o f t h e g a s c h r o m a t o g r a p h . T h u s ,
i t i s l i k e l y t h a t 1 5 - H P D A i s a t r u e p l a n t p r o d u c t , a l t h o u g h
p r e s e n t i n s m a l l a m o u n t s .
1 5 - H P D A w a s s h o w n t o b e a s e n s i ti z e r o f t h e s a m e
m a g n i t u d e a s 1 5 - H P A [ 7 ] . T h e a n i m a l s r e a c t e d s i g n i f i c -
a n t l y t o 1 a n d 5 1 5 - H P D A in p e t r o l a t u m (p e t. ). H o w -
e v e r , a n o n - s i g n i f i c a n t r e s p o n s e w a s o b s e r v e d f o r g u m
ros in (Tab le 1 ) .
1 5 - H P D A i s p r e s e n t i n fr e e a c i d f o r m i n n a t u r a l r o s i n
b u t w a s t e s t e d h e r e a s t h e m e t h y l e s t e r . T h e c h a n g e i n
s t r u c t u r e o w i n g t o m e t h y l e s t e r i f ic a t i o n h a s l i t t le e ff e ct o n
the a l l e rgen ic ac t iv i ty o f ro s in a l l e rgens I -7 , 8 , 15 ] .
T h e l o w r e a c t iv i t y t o g u m r o s i n a m o n g t h e 1 5 - H P D A -
s e n s i t i z e d a n i m a l s r e f l e c t s t h e l o w c o n c e n t r a t i o n o f 1 5 -
H P D A i n ro s in , w h e r e th e m i n u t e a m o u n t s m a y b e t o o
l o w t o i n d u c e s e n s i t iv i t y . T h i s w o u l d a l s o e x p l a i n t h e
a b s e n c e o f r e a c t i o n s t o 1 5 - H P D A w h e n t e s t e d i n r o s in -
a l l e r g ic i n d i v i d u a l s [ 1 6 ] . H o w e v e r , i t c a n n o t b e e x c l u d e d
t h a t 1 5 - H P D A m a y b e p r e s e n t i n l a r g e r a m o u n t s i n o t h e r
t y p e s / b a t c h e s o f r o s i n s i n c e t h e c o m p o s i t i o n v a r i e s c o n -
s i d e r a b l y w i t h o r i g i n, p r o d u c t i o n a n d h a n d l i n g o f t h e
m a t e r i a l [ 1 7 2 0 ] a n d th u s 1 5 - H P D A m a y c o n t r i b u t e to
t h e a l l e r g e n ic p o t e n t i a l o f r o s in .
EXPERIMENTAL
D e h y d r o a b i e t i c ac i d w a s p r e p a r e d f r o m P o r t u g u e s e
c o l o p h o n y a c c o r d i n g t o p r o c e d u r e s d e s c r i b e d i n t h e
l i t e ra t u r e [2 1 ] . D i az o g e n ® a n d D D Q w e r e o b t a i n e d fr o m
J a n s s e n C h i m i c a , B e e r s e . B e l g i u m . P o r t u g u e s e c o l o -
p h o n y o f t h e g u m r o s i n ty p e w a s o b t a i n e d f r o m S o c e r -
C o m e r c i o e t I n d u s t r i a d e R e s in a s , SA , P o m b a l , P o r t u g a l .
T a l l o i l r o s i n w a s o b t a i n e d f r o m B e r g v i k K e m i A B ,
S 6 d e r h a m n , S w e d en . A M e r c k o q u a n t 1 0 0 1 1 P e r o x i d e
T e s t , M e r c k , w a s u s e d f o r h y d r o p e r o x i d e s c r e e n i n g .
I R : P e r k i n - E l m e r 29 8 i n s tr u m e n t . 1 H a n d 1 3 C N M R :
J e o l E X 2 7 0 i n s t r u m e n t u s i n g C D C I 3 s o l n s w i t h T M S a s
i n t. s t a n d a r d . H P L C a n a l y se s o f r o s i n w e re p e r f o r m e d o n
a c y a n o p r o p y l m o d i f i e d si l i c a c o l u m n ( 2 50 × 4 r a m , 5 / ~ m ,
L i C h r o s o r b , M e r c k , G e r m a n y ) w i t h U V d e t e c t i o n
( 25 4 n m ) . T h e m o b i l e p h a s e w a s h e x a n e - E t O H ( 4 9 9 : 1 )
w i t h a f lo w r a t e o f 2. 0 m l m i n - 1. A n H P L C f r a c t i o n o f t a l l
o i l r o s i n w a s c o l l e c t ed a t t h e r e t e n t i o n t i m e c o r r e s p o n d -
i n g to t h a t o f t h e r e f er e n c e s u b s t a n c e m e t h y l 1 5 - h y d r o p e r -
o x y d e h y d r o a b i e t a t e ( 1 5 - H P D A ) . T h e f r a c ti o n w a s a n a -
l y s ed w i th D I P - M S . A s p e c t r u m o f t h e c o m p o u n d s h o w -
e d a f r a g m e n t a t i o n p a t t e r n a n d m o l e c u l a r i o n , [ M ] + 34 6,
i d e n t i c a l t o t h o s e o f t h e s y n t h e s i z e d re f e r e nc e c o m p o u n d
i d e n t i fi e d a s 1 5 - H P D A .
M S : t h e m e t h y l a t e d c o m p o u n d s e x c e p t f o r 1 5 - H P D A
w e r e a n a l y s e d w i t h G C - M S u s i ng a D B - 5 c a p i ll a r y
c o l u m n a n d o n - c o l u m n i n je c t io n . D u r i n g i n j e c t io n t h e
c o l u m n t e m p e r a t u r e w a s h e l d a t 8 5 ° f o r 1 m i n . I t w a s t h e n
inc rea s ed l ine a r ly a t a r a t e o f 10° ra in - ' up to 295 ° . Th e
t e m p . o f G C - M S i n t e r fa c e w a s h e l d a t 3 10 ° . O w i n g t o i t s
t h e r m a l i n s t a b i li t y 1 5 - H P D A c o u l d n o t b e a n a l y s e d w i t h
G C - M S , b u t w a s i n t ro d u c e d w i t h a d i r ec t i n s e r ti o n
p r o b e ( D I P ) t o t h e i o n s o u r c e a t a t e m p e r a t u r e o f 5 0 ° . T h e
t e m p e r a t u r e w a s i n c r e a s e d l i n e a r l y a t a r a t e o f 3 0 ° r a i n -
up to 350 ° . Th e s can range w as m/z 5 0 -5 0 0. F o r G C - M S
the s can cy c le t ime w as 0 .6 s ec ; fo r D IP -M S 2 .5 sec . Th e
i o n s o u r c e w a s h e l d a t a t e m p . o f 1 5 0 ° a n d t h e e l e c t r o n
ene rgy was s e t t o 70 eV .
Methyl dehydroabietate
( 3a ). D e h y d r o a b i e t i c a c i d w a s
e s t e r i f i e d q u a n t i t a t i v e l y w i t h d i a z o m e t h a n e g e n e r a t e d
f r o m D i a z o g e n ® .
Methyl 8 ,11 ,13 ,15 -ab ie t a t e t raen -18-oa te (6 ). Th i s c om -
p o u n d w a s p r e p a r e d u s i n g a s y n t h e t i c r o u t e d e s c r i b e d i n
r ef . [ 2 2 ]. A m i x t u r e o f m e t h y l d e h y d r o a b i e t a t e (3 a )
(1.05 g , 3.34 mmo l ) and D D Q (0.95 g , 4 .19 mm ol ) in 1 ,4 -
d i o x a n e ( 2 0 m l ) w a s r e f l e x e d f o r 5 h r . T h e m i x t u r e w a s
p a s s e d t h r o u g h a n a l u m i n a c o l u m n e l u t e d w i t h t o l u e n e
( 10 0 m l ) . C o m p o u n d 6 w a s o b t a i n e d in 7 0 y i e l d a s a n
o i l a f te r p u r i f i c a t i o n w i t h C C o n s i l ic a g e l e l u t e d w i t h
h e x a n e - E t 2 0 ( 9 : 1 ). I R v c m - l : 1 71 5, 16 25 ( e st e r) .
Table. 1. Sensitizing capacity of methyl 15-hydrop eroxydehy droabietate (15-HP DA )
according to Freund's complete adjuvant test (FCAT)
Exposed group Control group
(n = 15)* (n = 15)
Positive Positive
Challenge m ateria l Conc. ( ) reactions P(exp/co) reactions
15-HP DA 5 14 < 0.001 0
15-HP DA 1 13 < 0.001 0
Gum rosin 10 2 NS t 0
Vehicle contro l (pet.) 1 NS 0
*Induction: 15-HPDA 5 (w/w) (0.14 mm ol g- 1) intradermally.
tNS, Not significant.
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856 LI P. SHAOet al.
1H NM R : 6 7 .05 -6 .93 (3H, m , Ar -H) , 5 .17 (1 H , br s, H-16),
4 .88 ( IH , br s , H-16), 3 .61 (3H, s , H-21) , 2 .08 (3H, br s , H-
17), 1 .22 (3H, s , H-19) , 1 .19 (3H, s , H-20) , 13C NM R :
6179.0 (C-18), 142 .0 (C-15), 111.5 (C-16), 149.1 , 134.8 ,
126.7, 125 .9 , 123 .9 , 122 .8 (a rom a t i c c a rbons ) . G C - EI -M S
7 0 e V , m / z (rel . in t .) : 312 [M ] + (18) , 297 [M - M e] + (8),
237 [M - 75 ] + (100). Thes e s pec t ra l da ta a re con s i s t en t
wi th the s t ruc tu re o f 6.
Methy l 15 -hydr oxydehydr oabie ta te ( 7 ) . F o r t h i s s y n -
the s i s , a p rocedure de s c r ibed in re f . [23 ] was us ed .
Hg(O Ac) 2 (0.98 g , 3.1 mm ol ) was d i s s o lved in 3 ml H 20
and then 0 .95 g (3 .0 mm ol ) o f 6 in 3 ml T H F was adde d .
T h e r e a c t i o n m i x t u r e w a s s t ir r e d a t r o o m t e m p . u n d e r N 2
f o r 3 h r . T h e n 3 m l o f 3 .0 M N a O H w a s a d d e d , f o l l o w e d
b y 3 m l o f a s o l n o f 0 .5 M N a B H 4 i n 3. 0 M N a O H . T h e
m i x t u r e w a s s t i r r e d f o r 3 h r . T h e m e r c u r y w a s a l l o w e d t o
s e t tl e a n d N a C I w a s a d d e d t o s a t u r a t e t h e H 2 0 l a y e r. T h e
u p p e r l a y e r o f T H F w a s e v a p d a n d p a s s e d t h r o u g h a n
a l u m i n a c o l u m n e l u t e d w i t h 3 0 E t O A c in h e x a n e. T h i s
y ie lded 2 .4 g (80 ) o f an o i l w i th the fo l lowing s pec t ra l
d a t a : IR v c m - l : 3 45 0 ( O H ) , 1 73 0 ( C- ~- O) . 1 H N M R :
67.2 6-7. 15 (3H, m, Ar-H ), 3 .66 (3H, s , H-21) , 1 .56 (6H, s ,
H-16, H-17), 1 .2 8 (3H, s , H-19) , 1 .21 (3H, s , H-20) ,.
13C NM R : 6179 .1 (C -18), 148 .0 , 146 .1 , 134 .8 , 124.9,
124.2, 122 .0 (a rom a t i c c a rbons ) , 72 .3 (C -15). G C - EI -M S
70 eV, m / z (re l . in t. ): 330 [M ] + (18) , 315 [M - M e] + (87),
312 [M - H 20 ] + (17 ) , 297 [M - H 20 - M e] + (11) , 255
[M - 75 ] + (100), 237 [M - 93 ] + (75 ) . The s pec t ra l da ta
a re cons i s t en t w i th the s t ruc tu r e o f 7 .
M e t h y l 1 5 - h y d r o p e r o x y d e h y d r o a b i e t a t e ( 1 5 - H P D A )
(4a) . The s yn the s i s o f 4a f rom 7 was b a s ed on a p roc edu re
d e s c r i b e d i n re f. [ 2 4 ] . T h e s o l u t i o n o f m e t h y l 1 5 - h y d r o x y -
deh yd roa b ie t a t e (7 ) (0 .4 g, 1 .2 mm ol ) in TH F (15 ml ) was
s t i r red w i th H 20 2 (10 ml , 35 , 97 .2 re too l ) and conc
H 2 S O 4 ( 3 d r o p s ) . T h e m i x t u r e w a s s t i r r e d a t r o o m t e m p .
u n d e r N 2 f o r 3 h r . T h e s o l n w a s s a t d w i t h N a C I a n d
e x t r a c te d w i t h E t O A c . A f t er c h r o m a t o g r a p h y t h e m e t h y l
1 5 h y d r o p e r o x y d e h y d r o a b i e t a t e ( 1 5 - H P D A ) ( 4a ) w a s o b -
t a i n e d i n 2 6 y i e ld . T h e c o m p o u n d g a v e a p o s i t i v e
p e r o x i d e t e st r es u l t. IR v c m - l : 3 50 5 ( O O H ) , 1 73 0
(C = O ) . IH NM R : 67 .28 -6 .98 (3H, m, Ar -H ) , 3 .69 (3H, s,
H-21), 1 .59 (6H, s , H-16, H-17), 1 .29 (3H, s , H-19) , 1 .22
(3H, s, H-20). 13C NM R: 6179.1 (C-18), 148 .8 , 141.3 ,
135.2, 126 .0 , 124 .6 , 122 .9 (a ro ma t i c c a rbons ) , 83 .8 (C -
1 5) . D I P - E I - M S 7 0 e V , m / z (rel. int.): 346 [M ] + (2), 313
[M - O O H ] + (68 ) , 297 [M - 49 ] + (8 ) , 255 [M - 91 ] ~
(38), 237 [ M - 109] + (100). The s i gnals a t m / z 315 and
330, r e s pec t ive ly , p r ob ab ly o r ig in a te f rom 15-
h y d r o x y d e h y d r o a b i e t a t e ( 7 ) . T h i s c o m p o u n d i s m o s t
l i ke l y f o r m e d b y d e g r a d a t i o n o f 1 5 - H P D A w h e n t h e
p r o b e i s h e a t e d . T h e s p e c t r a l d a t a a r e c o n s i s t e n t w i t h
s t ruc tu re 4a .
Sens i t i z in9 capac i t y o f 1 5 -HPD A accor din9 to Fr eund s
comple te ad juvan t t e s t. T h e e x p e r i m e n t w a s p e r f o r m e d a s
d e s c r i b e d b y K l e c a k b u t s l i g h tl y m o d i f i e d a c c o r d i n g t o
ea r l i e r expe r i en ce [25 , 26 ] . Of 30 gu ine a -p igs 15 (g rou p I )
w e r e i n t r a d e r m a l l y i n j e c te d w i t h 5 1 5 - H P D A ( c o r r es -
p o n d i n g t o 0 .1 4 m m o l g - 1 ) i n F C A - H 2 0 - e m u l s i o n . T h e
o t h e r 1 5 a n i m a l s ( g r o u p I I ) w e r e t h e c o n t r o l g r o u p a n d
w e r e i n j e c te d w i t h n e a t F C A H 2 0 - e m u l s i o n . T h e c o n -
c e n t r a t io n o f 1 5 - H P D A w a s c h o se n t o c o r r e s p o n d t o t h e
m o l a r c o n c e n t r a t i o n o f 1 5 - H P A a c c o r d i n g t o p r e v i o u s
e x p e r i e n c e [ 7 ]. T h e a n i m a l s w e r e c h a l le n g e - t e s t e d w i t h 5
a n d 1 1 5 - H P D A i n p e t r o l a t u m ( c o r r e s p o n d i n g t o 0 .1 4
a n d 0 . 0 3 m m o l g 1). T h e t e s t c o n c e n t r a t i o n s o f 1 5-
H P D A w e re s ho w n i n a p re t e s t o n F C A - t r e a t e d a n i m a l s
t o b e n o n - i r r i t a t in g . T h e a n i m a l s w e r e a l s o t e st e d w i t h
P o r t u g u e s e g u m r o s i n 1 0 i n p e t r o l a t u m .
A c k n o w l e d g e m e n t s - - W e w o u l d l i k e t o t h a n k M r s G u n n e l
H a g e l t h o r n a n d M r P e t e r F e r n s t r 6 m f o r s k i lf u l a s s i s t a n c e
w i t h t h e a n i m a l t e s t in g . T h e w o r k w a s s u p p o r t e d b y a
g r a n t f r o m t h e S w e d i s h W o r k E n v i r o n m e n t F u n d .
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PHY 38:4 E
