10 Zearalenone and Related Compounds S. V. PATHRE and C. J. MIROCHA Department of Plant Pathology, University of Minnesota, St. Paul, Minn. 55101
Zearalenone [6-(10-hydroxy-6-oxo-trans-1-undecenyl)-β-resorcylic acid-μ-lactone] is a secondary metabolite of species of Fusarium and is notable because of its estrogenic and anabolic activity in animals. Mass spectral, NMR, fluores cence, and x-ray diffraction properties are summarized with emphasis on fragmentation patterns resulting from analyses by mass spectrometry. Use of mass spectrometry in struc ture elucidation is illustrated. Zearalenone is biosynthesized via the acetate-malonyl-CoA pathway and can be metabo lized to the two isomers of 8'-hydroxyzearalenone. Struc ture-activity relationships indicate that the most active derivative of the series is 7'-carboxyzearalane which is one -tenth as active as diethylstilbestrol and 100 times more active than the parent zearalenone. The naturally occurring macrolides related to zearalenone are radicicol, lasiodiplodin, and curvularin. The chemistry and total synthesis of this molecule are described and discussed.
Zearalenone is a secondary metabolite produced by Fusarium roseum, Fusarium tricinctum, Fusarium oxysporum, Fusarium culmorum, and Fusarium moniliforme. It is usually produced on maize and barley in storage, and when fed to animals, particularly swine, it causes hyperestrogenism. A thorough treatment of this subject can be found in reviews by Mirocha et al. (1) and Mirocha and Christensen (2). Hyperestrogenism was first noted in swine by Buxton in 1927 (3) and Legenhausen in 1928 in herds in Iowa (4). Although they did not know its cause, they described symptoms in young gilts of the swelling and eversion of the vagina until, in some cases, the cervix was visible. Legenhausen also described a swelling of the prepuce in males. McNutt was the first to associate this disease with the consumption of moldy maize and to reproduce the estrogenic syndrome (5). 178
10.
P A T H R E
A N D
M I R O C H A
and Related Compounds
Zearalenone
Stob et a l . ( 6 ) isolated an active principle from cultures of zeae (Fusarium
roseum,
179
GUbberella
G r a m i n e a r u m ) i n 1962 f o l l o w e d b y C h r i s t e n s e n
et a l . ( 7 ) i n 1965. T h i s m e t a b o l i t e h a d m a r k e d a n a b o l i c a n d u t e r o t r o p h i c a c t i v i t y w h e n a d m i n i s t e r e d t o m i c e , a p p e a r e d t o i n c i t e t h e estrogenic s y n d r o m e , a n d w a s c a l l e d F - 2 ( 7 ) . U r r y e t a l . (8) n a m e d t h e a c t i v e p r i n c i p l e zearalenone. Z e a r a l e n o n e acts as a h o r m o n e i n Fusarium
roseum w h e r e i t regulates
the p r o d u c t i o n o f t h e s e x u a l stage—Le., f o r m a t i o n o f p e r i t h e c i a ( 9 ) . I n the f u n g u s system i t acts i n c o n c e r t w i t h c y c l i c 3'-5'-adenosine p h o s p h a t e t o r e g u l a t e p e r i t h e c i a p r o d u c t i o n (10).
mono
A l t h o u g h i t regulates
t h e s e x u a l stage i n o t h e r genera o f f u n g i as w e l l , i t has b e e n f o u n d o n l y w i t h i n t h e genus Fusanum
(11).
Z e a r a l e n o n e b e l o n g s t o a g r o u p o f n a t u r a l p r o d u c t s c a l l e d resorcylates. I n this p a p e r t h e c h e m i s t r y o f z e a r a l e n o n e , its d e r i v a t i v e s , a n d other r e l a t e d n a t u r a l p r o d u c t s
are r e v i e w e d to summarize the more
i n t e r e s t i n g a n d u n u s u a l features o f m a c r o c y c l i c resorcylates. A n a t t e m p t is m a d e t o treat those aspects o f z e a r a l e n o n e o f s p e c i a l interest t o t h e o r g a n i c c h e m i s t a n d t o t h e b i o l o g i s t as w e l l . T h e estrogenic m a c r o l i d e w a s d e s i g n a t e d z e a r a l e
Nomenclature.
none, a n enone d e r i v a t i v e o f r e s o r c y l i c a c i d l a c t o n e i s o l a t e d f r o m Gibberella
zeae (8). Z e a r a l a n e is a p a r e n t c o m p o u n d o f zearalenone. T h e
n u m b e r i n g system u s e d t h r o u g h o u t this p a p e r f o r t h e z e a r a l a n e system is s h o w n i n F i g u r e 1. T h e c h e m i c a l n a m e c o m m o n l y u s e d f o r z e a r a l a n e is 6 - ( 1 0 - h y d r o x y - l - u n d e c y l ) - ^ - r e s o r c y l i c z e a r a l e n o n e is n a m e d
a c i d - μ-lactone.
Accordingly,
6-(10-hydroxy-6-oxo-imfw-l-undecenyl)-^-resorcylic
a c i d μ-lactone.
Figure
1. Structure and number ing system for zearalane
Zearalenone Structural E l u c i d a t i o n . S t r u c t u r e 1 of z e a r a l e n o n e
(C18H22O5) w a s
e l u c i d a t e d i n 1966 b y U r r y e t a l . ( δ ) w h o f o u n d t h a t i t a b s o r b e d h y d r o g e n i n t h e presence o f p l a t i n u m t o g i v e a d i h y d r o p r o d u c t 2
(C18H24O5);
h o w e v e r , hydrogénation o v e r R a n e y n i c k e l a t 5 0 p s i g a v e a m i x t u r e o f t w o d i a s t e r e o m e r i c alcohols 3
(Ci8H2e05).
C h e m i c a l degradation of zearale-
180
M Y C O T O X I N S
2 4
Ci H 0 R = C H 8
2 4
5
R^=H
3
C18H26O5
3
none (Scheme I ) v i a B e c k m a n n rearrangement y i e l d e d a product i d e n t i fied
as 2 ( 5 - c a r b o x y
pentyl)-4,6-dimethoxybenzoic
acid (5)
determined
o n t h e basis o f N M R d a t a . O z o n o l y s i s o f t h e d i m e t h y l ether o f z e a r a l e n o n e gave 2 , 4 - d i m e t h o x y - 6 - f o r m y l b e n z o i c fluxed
acid.
Zearalenone, w h e n r e -
8 h r i n 1 0 % sodium bicarbonate solution, yields a n alcohol 6
(C17H24O4)
after a c i d i f i c a t i o n . T h e s e studies e s t a b l i s h e d t h e positions
of t h e olefin a n d ketone i n t h e a l i c y c l i c lactone r i n g . A l s o N M R s p e c t r a l d a t a (see
S p e c t r a l P r o p e r t i e s ) i n d i c a t e d t h e p r e s e n c e o f trans olefinic
protons ( / = 16 H z ) a n d a s e c o n d a r y m e t h y l g r o u p . T h u s t h e s t r u c t u r e of 1 is consistent w i t h t h e d a t a o b t a i n e d f r o m t h e studies l i s t e d a b o v e (see S c h e m e I ) . A b s o l u t e C o n f i g u r a t i o n . B e c a u s e o f a n a s y m m e t r i c center a t C I O ' , naturally
occurring
zearalenone
( [«] 546 — - 1 7 0 . 5 ° , c = 25
exhibits
optical
activity a n d purity
1 . 0 M M e O H ) . T h e absolute c o n f i g u r a t i o n o f
this n a t u r a l e n a n t i o m o r p h w a s d e t e r m i n e d b y K u o et a l . (12) m e t h o d b a s e d o n t h e k i n e t i c r e s o l u t i o n t e c h n i q u e (13) 15,16)
using a
of H o r e o u
(14,
t o d e t e r m i n e t h e configurations o f s e c o n d a r y alcohols. T h e y c o n
v e r t e d t h e n a t u r a l z e a r a l e n o n e to t h e d i h y d r o s e c o a c i d k e t a l 7, f o r m e d t h e m e t h y l ester 8, a n d t h e n a l l o w e d i t to react w i t h ( ± ) - a - p h e n y l b u t y r i c a n h y d r i d e i n p y r i d i n e . T h e excess o f a - p h e n y l b u t y r i c a c i d w a s r e c o v e r e d , a n d its r o t a t i o n w a s d e t e r m i n e d . T h e r e c o v e r e d a c i d e x h i b i t e d a n e g a t i v e r o t a t i o n ( c o r r e s p o n d s to R ( — ) α - p h e n y l b u t y r i c a c i d )
thereby
denoting
10.
an
P A T H R E
A N D
M I R O C H A
S-configuration
at the
Zearalenone
and
SCHEME
I
optically active
Related
Compounds
ΙΟ'-center of
181
zearalenone.
A l t e r n a t i v e l y t h e l a c t o n e 9 d e r i v e d f r o m t h e exhaustive o x i d a t i o n
of
z e a r a l e n o n e h a d a n i d e n t i c a l o p t i c a l r o t a t i o n to t h a t of t h e S ( — ) e n a n t i o m e r of 5 - h y d r o x y h e x a n o i c a c i d lactone.
Naturally Occurring Derivatives of Zearalenone M i r o c h a et a l . ( 1 ) r e p o r t e d the n a t u r a l o c c u r r e n c e of at least s e v e n d e r i v a t i v e s of z e a r a l e n o n e f r o m Fusarium CH 0
t
3
8
C H
3
roseum
growing i n culture on
.H
9
182
MYCOTOXINS
( 8'R,10'S ) -8'Hydroxyzearalerione Figure 2. (A) Packing of molecules in a crystal of 8'-dihydroxyzearalenone (F-5-3) based on x-ray crystallography analysis. Note the intermohcular association arising from water of hydration. (Courtesy of I. F. Taylor.) (B) Stereochemistry of 8 -dihydroxy zearalenone. f
10.
P A T H R E
A N D
M I R O C H A
Zearalenone
183
and Related Compounds
c o r n , a n d d e s i g n a t e d t h e m as F - 5 - 0 t h r o u g h F - 5 - 7 . T h e m e t a b o l i t e s w e r e partially characterized b y their u v absorption spectra a n d separation b y thin layer and g a s - l i q u i d chromatography.
T h e m o s t a b u n d a n t o f these
d e r i v a t i v e s w e r e d e s i g n a t e d as F - 5 - 3 ( m p 1 9 8 ° - 1 9 9 ° C ) a n d F - 5 - 4
(mp
1 6 8 ° - 1 6 9 ° C ) , b o t h of w h i c h h a v e a mass of 334 a n d t h e e m p i r i c a l f o r mula C i H 3 0 . 8
2
6
T h e s t r u c t u r e w a s i n c o r r e c t l y r e p o r t e d as 3 ' - h y d r o x y -
z e a r a l e n o n e ( a l p h a a n d b e t a i s o m e r s ) b y M i r o c h a et a l . ( 1 ) . studies b y J a c k s o n et a l . (17)
Subsequent
r e v e a l e d t h a t the O H g r o u p of b o t h isomers
is i n the 8' p o s i t i o n . I n d e p e n d e n t l y , B o l l i g e r a n d T a m m (18) two
isomeric hydroxyzearalenones
which when
isolated
oxidized with
Jones
reagent ( C r 0 - H S 0 i n a c e t o n e ) y i e l d e d i d e n t i c a l diketones ( ία] 3
2
=
24
Ώ
4
—93.2° a n d —92.8° ) i n d i c a t i n g t h e e p i m e r i c n a t u r e of the h y d r o x y g r o u p . M a s s s p e c t r a l d a t a i n t e r p r e t a t i o n r e v e a l e d t h a t these i s o m e r i c h y d r o x y zearalenones w e r e e p i m e r i c 8 ' - h y d r o x y z e a r a l e n o n e s . i n t o [ S ] - z e a r a l a n o n e , J a c k s o n et a l . (17)
B y converting them
also s h o w e d t h a t F - 5 - 3 a n d
F - 5 - 4 are e p i m e r i c . R e c e n t l y F - 5 - 3 has b e e n s h o w n (19)
to b e ( 8 ' R , 1 0 ' S ) -
hydroxyzearalenone, b y x-ray crystallography ( F i g u r e 2 ) . B o l l i g e r a n d T a m m (18)
also r e p o r t e d t h e o c c u r r e n c e of 5 - f o r m y l -
z e a r a l e n o n e 11 a n d 7 ' - d e h y d r o z e a r a l e n o n e (Table I).
roseum
Steele (20)
12 i n c u l t u r e s of
isolated a compound
Fusanum
from
Fusarium
roseum g r o w i n g o n a s o l i d m e d i u m of s h r e d d e d w h e a t w h i c h w a s i d e n t i c a l w i t h 6 ' , 8 ' - d i h y d r o x y z e a r a l e n e 13, N a B H - r e d u c t i o n p r o d u c t of F - 5 - 3 . 4
H e also i s o l a t e d 7 - d e h y d r o z e a r a l e n o n e 12 w h e n t h e c h l o r o f o r m extract of /
the c u l t u r e s w a s p a r t i t i o n e d w i t h 2 N K C 0 . H o w e v e r 12 w a s n o t d e 2
3
t e c t e d w h e n t h e base p a r t i t i o n w a s e h m i n a t e d .
Table I.
N a t u r a l l y O c c u r r i n g Derivatives of Zearalenone
HO'
R 10 11 12 13
8'-hydroxyzearalenone 5-formylzearalenone 7 -dehydrozearalenone 6',8'-dihydroxyzearalene ,
— — — —
H CHO H H
2
= 0 = 0 = 0 —OH
R
R
3
= H = H — H = H
2 2
2
— = — —
4
OH H H OH 2
184
MYCOTOXINS
Reactions of Zearalenone R i n g Stability. A l t h o u g h zearalenone is a l a c t o n e , i t is f a i r l y stable i n c o l d a l k a l i ; b u t p r o l o n g e d exposure e x t e n s i v e l y h y d r o l y z e s t h e l a c t o n e r i n g . T h e l a c t o n e f u n c t i o n o f 1 is easily h y d r o l y z e d w i t h s o d i u m h y d r o x i d e i n refluxing aqueous d i m e t h y l sulfoxide
(DMSO)
r a c e m i z e d seco a c i d 14 i n a l m o s t q u a n t i t a t i v e y i e l d (21).
a n d yields
a
T h e racemiza-
t i o n o f t h e seco a c i d is a t t r i b u t e d to i n t e r n a l d i s p r o p o r t i o n a t i o n a r i s i n g f r o m t h e presence of t h e 6' k e t o n e ( 15 * ± 16 ) since t h e e t h y l e n e k e t a l 17 of z e a r a l e n o n e u n d e r g o e s t h e o p e n i n g o f t h e lactone w i t h o u t r a c e m i z a t i o n (21).
Peters a n d H u r d
(22)
u s e d this p r i n c i p l e i n o p e n i n g t h e
l a c t o n e r i n g of t h e n a t u r a l l y o c c u r r i n g ( S ) - z e a r a l e n o n e to p r e p a r e ( R ) zearalanone, a n d they have inverted the configuration at C 1 C v i a the p - t o l u e n e s u l f o n i c ester of t h e seco a c i d .
Aromatic Substitution.
W i n d h o l z a n d B r o w n (23)
examined sub
s t i t u t i o n o f t h e c a r b o x y l a n d f o r m y l groups i n t o t h e a r o m a t i c p o r t i o n o f z e a r a l e n o n e . W h e n 1 is h e a t e d a t 175° w i t h a n h y d r o u s p o t a s s i u m c a r b o n a t e at 8 0 0 p s i C 0
2
f o r 3 h r , i t is c a r b o x y l a t e d at t h e C 3 p o s i t i o n ;
h o w e v e r i f t h e r e a c t i o n t i m e is i n c r e a s e d to 5 h r , t h e c a r b o x y l a t i o n o c c u r s a t C - 5 (24).
T h e s e results are consistent w i t h t h e c o n c l u s i o n s ( 2 5 ) t h a t
s u c h c a r b o x y l a t i o n is r e v e r s i b l e a n d t h a t , k m e t i c a l l y , s u b s t i t u t i o n i n t h e
10.
P A T H R E
A N D
Zearalenone
M I R O C H A
OR
l a or
185
and Related Compounds C H
3
OH—DMSO
lb
RO' 0 ± 1 4
15
R =
H or C H
3
17
16
o r t h o p o s i t i o n is f a v o r e d , b u t , t h e r m o d y n a m i c a l l y , t h e p a r a p o s i t i o n is favored.
T h i s is l o g i c a l since the greater s e p a r a t i o n of the t w o n e g a t i v e
charges
(phenoxide
and
carboxylate)
gives
greater
thermodynamic
stability. Z e a r a l e n o n e is f o r m y l a t e d at the C 3 p o s i t i o n b y a l l o w i n g e t h y l f o r m a t e to react w i t h a l u m i n u m c h l o r i d e ( F r i e d e l - C r a f t f o r m y l a t i o n ) a n d b y t h e R i e m e r - T i e m a n n a n d G a t t e r m a n n reactions (18).
(23)
(Reimer-
T i e m a n n r e a c t i o n is u s e d to f o r m y l a t e sensitive a r o m a t i c r i n g s ; f o r m y l a t i o n o c c u r s i n c h l o r o f o r m i n t h e p r e s e n c e of a s t r o n g base s u c h as potas sium hydroxide
R e f . 26).
(see
f o r m y l a t e phenols (see
T h e G a t t e r m a n n r e a c t i o n is u s e d
Refs. 27 a n d 28).
to
I n these reactions f o r m y l a t i o n
y i e l d e d a m i x t u r e of 3 - f o r m y l a n d 5 - f o r m y l zearalenone. T h e G a t t e r m a n n r e a c t i o n gave 8 9 %
of the f o r m y l a t e d p r o d u c t i n w h i c h t h e 3 a n d 5
f o r m y l d e r i v a t i v e s w e r e i n the r a t i o of 2 : 1 , r e s p e c t i v e l y . N i t r a t i o n of d i m e t h y l ether z e a r a l e n o n e l b w i t h a 5 0 / 5 0 m i x t u r e of c o n c e n t r a t e d s u l f u r i c a c i d a n d n i t r i c a c i d gives a m i x t u r e of 3,5-dinitro a n d 5-nitro d e r i v a t i v e s ( 2 9 ) .
These nitro derivatives can be transformed
i n t o the c o r r e s p o n d i n g a m i n o d e r i v a t i v e s b y r e d u c t i o n w i t h i r o n p o w d e r i n a 5 0 % m e t h a n o l i c s o l u t i o n c o n t a i n i n g h y d r o c h l o r i c a c i d (0.25 e q u i v ) . J o h n s t o n et a l . (30)
r e p o r t e d the c o n v e r s i o n of 5 - a m i n o z e a r a l e n o n e 18 to
5-hydroxyzearalenone
20 b y o x i d i z i n g t h e a m i n o d e r i v a t i v e w i t h s i l v e r
o x i d e - a m m o n i u m h y d r o x i d e to a 4 - h y d r o x y q u i n o n e d e r i v a t i v e 19 w h i c h y i e l d e d 20 w h e n t r e a t e d w i t h a q u e o u s s o d i u m thiosulfate.
186
MYCOTOXINS
20 H a l o g e n - s u b s t i t u t e d d e r i v a t i v e s of z e a r a l e n o n e s u c h as m o n o - a n d diiodozearalenone a n d monobromozearalenone have been reported (31). H a l o m e r c u r i c - a n d arsonic a c i d - s u b s t i t u t e d d e r i v a t i v e s of
zearalenone
h a v e also b e e n p r e p a r e d a n d s h o w a n t i b a c t e r i a l a n d a n t i f u n g a l a c t i v i t y (32). Diels-Alder Adducts. prepared (Figure 3)
S e v e r a l a d d u c t s of z e a r a l e n o n e h a v e b e e n
to d e v e l o p n e w g r o w t h - p r o m o t i n g agents
(33).
M a l e i c a n h y d r i d e , s u l f u r d i o x i d e , p - b e n z o q u i n o n e , 1,4-naphthoquinone, nitroethylene,
and
acrolein
can
easily
form
the
5-2 -adduct
zearalenone.
Figure 3.
Diels-Alder
adduct of
zearalenone
/
with
10.
P A T H R E
A N D
M I R O C H A
Birch Reduction.
Zearalenone
and Related Compounds
B i r c h r e d u c t i o n of zearalenone
s t u d i e d b y W i n d h o l z a n d B r o w n (23).
derivatives
187 was
T h e ethylene k e t a l of the d i m e t h y l
ether of zearalenone 21, w h e n treated w i t h 4 e q u i v of s o d i u m i n l i q u i d a m m o n i a a n d tert-butyl
alcohol yielded two rather unstable products
a n d 23), w h i c h o n t r e a t m e n t w i t h C r 0
3
(22
i n p y r i d i n e a r o m a t i z e d t o the
d e o x y d e r i v a t i v e s 24 a n d 25, r e s p e c t i v e l y . T h e m e c h a n i s t i c i n t e r p r e t a t i o n of these reactions is s h o w n i n S c h e m e II ( 2 3 ) .
T h e deoxy product
26
w a s i s o m e r i z e d to 23 because of t r e a t m e n t w i t h a base d u r i n g i s o l a t i o n .
188
MYCOTOXINS
Scheme I I
26 Hydrogénation and Hydrogenolysis.
T h e c o n d i t i o n s u n d e r which
1 is h y d r o g e n a t e d to 2 a n d to the d i a s t e r e o m e r i c z e a r a l a n o l s 3 h a v e b e e n n o t e d i n o u r s t r u c t u r a l e l u c i d a t i o n of z e a r a l e n o n e . T h e c o m p l e t e s a t u r a t i o n of the a r o m a t i c r i n g 27 ( c o n c o m i t a n t w i t h t h e r e d u c t i o n of t h e olefin a n d t h e 6'-keto g r o u p s )
occurs w h e n hydrogénation is c a r r i e d o u t i n
m e t h a n o l u n d e r d r a s t i c c o n d i t i o n s (34).
T h e perhydrozearalanol deriva
t i v e has b e e n s h o w n to h a v e a n t i - i n f l a m m a t o r y a c t i v i t y ( 3 5 ) .
10.
P A T H R E
A N D
Zearalenone
M I R O C H A
R
189
and Related Compounds
— Η or C H
3
27 H y d r o g e n o l y s i s of t h e p h e n o l i c h y d r o x y l s i n zearalenone has b e e n r e p o r t e d b y J o h n s t o n et a l . ( 3 0 )
and Wehrmeister and Robertson
(36).
I t i n v o l v e s p r e p a r a t i o n of the l - p h e n y l - 5 - t e t r a z o l y l 28 or b e n z y x a z o l y l 29 ethers a n d the subsequent h y d r o g e n o l y s i s w i t h 5 %
P d - C w h i c h also
reduces the Γ-olefln. ΟβΗδ I
O x i d a t i o n . Z e a r a l e n o n e or its d i m e t h y l ether, w h i l e r e f l u x i n g i n 1 4 % n i t r i c a c i d , oxidizes extensively to y i e l d g l u t a r i c , s u c c i n i c , a n d o x a l i c acids. T h e n i t r a t e d a r o m a t i c f r a g m e n t c a n b e i s o l a t e d o n l y b y o x i d i z i n g the d i m e t h y l ether d e r i v a t i v e ( 8 , 3 7 ) .
T h e a d v a n t a g e of s u c h d e g r a d a t i o n
w a s t a k e n to d e t e r m i n e the d i s t r i b u t i o n of isolated from acetate
cultures of Fusarium
1 4
C i n zearalenone ( F i g u r e 4 )
roseum
inoculated w i t h
1 4
(37).
T h e a l l y l i c o x i d a t i o n of the ether of zearalenone reagent
[1- C]-
( C r 0 - p y r i d i n e complex) 3
30 w i t h Sarett
gives a 3-keto d e r i v a t i v e 31
(38).
T h e o x i d a t i o n appears to b e sensitive to the t y p e of s u b s t i t u e n t at the 6' p o s i t i o n . T h e y i e l d of t h e 3-keto d e r i v a t i v e o b t a i n e d f r o m the other 30 w a s less t h a n 1 5 % ; h o w e v e r the e t h y l e n e k e t a l 32 a n d t h e
epimeric
acetates 33 afforded the c o r r e s p o n d i n g 3-keto d e r i v a t i v e s i n m o r e t h a n 7 0 % y i e l d . A c c o r d i n g to Jensen et a l . ( 3 8 ) the c h a n g e i n h y b r i d i z a t i o n of C 6 ' (sp
2
-» sp ) 3
a l l o w s a f a v o r a b l e r i n g c o n f o r m a t i o n , a n d t h e presence
of o x y g e n a t t a c h e d to sp
3
C 6 ' assists the attack at 0 3 ' .
O s m i u m tetroxide reacts s m o o t h l y w i t h the d o u b l e
bond
of
d i b e n z y l ether of zearalenone 34 to p r o d u c e a m i x t u r e of e p i m e r i c
the Γ,2'-
190
MYCOTOXINS
17,9,13
•co
2
Figure 4.
Chemical degradation of C-labelled zearalenone. The filled circles mark the location of C atoms derived from 1- C-acetate. 14
(·)
14
14
31 d i o l 35 w h i c h i n t h e presence of a n a c i d rearranges to g i v e 36 p r o b a b l y v i a a c i d - c a t a l y z e d o p e n i n g s of t h e l a c t o n e 35 to f o r m 3 7 .
(38), The
2 ' , 1 0 ' - d i o l 37 t h e n undergoes a n i n t r a m o l e c u l a r k e t a l f o r m a t i o n w i t h t h e 6'-ketone.
10.
P A T H R E
A N D
OR
Ο
Zearalenone
M I R O C H A
I
Η
OR Os0
RO'
^
^Ιι
191
and Related Compounds Q
I
Η
4
Ί
RO" S
O
HO'
36
37
Modification of the Lactone R i n g . R e a c t i o n s i n v o l v i n g the a l i p h a t i c p o r t i o n of z e a r a l e n o n e h a v e b e e n e x a m i n e d extensively either to m o d i f y the estrogenic a c i v i t y ( 3 9 )
o r to s y n t h e s i z e n a t u r a l l y o c c u r r i n g d e r i v a
tives of z e a r a l e n o n e ; n a m e l y , 7 ' - d i h y d r o z e a r a l e n o n e 12 a n d 8 ' - h y d r o x y z e a r a l e n o n e 10
(40).
J e n s e n et a l . (38) ( a t 5' a n d 7')
r e p o r t e d t h a t t h e reactions of a c t i v a t e d methylenes
of z e a r a l e n o n e s h o w c o n s i d e r a b l e ' r e g i o s e l e c t i v i t y "
(see
R e f . 41 f o r the d e f i n i t i o n ) . F o r m y l a t i o n o f zearalenone ethers i n b e n z e n e u n d e r the c o n d i t i o n s of s o d i u m h y d r i d e a n d
terf-butyl
a l c o h o l gave p r e
d o m i n a n t l y t h e C 7 ' p r o d u c t 38 ( < 7 0 % ). T h i s p r e f e r e n c e for C 7 ' f o r m y l a t i o n w a s also n o t i c e d i n t h e z e a r a l a n o n e d e r i v a t i v e s . R e a c t i o n of zearalenone-2-4-diacetate 39 w i t h i s o p r o p e n y l acetate i n the presence of p - t o l u e n e s u l f o n i c a c i d g a v e t h e e n o l acetate i n w h i c h the m a j o r p r o d u c t i s o l a t e d w a s 4 0 ( e n o l i z a t i o n i n C 5 ' d i r e c t i o n ) i n 6 1 % y i e l d . T h e other isomer
(enolization i n C 7 ' direction)
w a s i s o l a t e d i n ca.
4%
yield.
W h e t h e r s u c h s e l e c t i v i t y is k i n e t i c o r t h e r m o d y n a m i c has n o t y e t b e e n d e t e r m i n e d . O n e p o s t u l a t i o n is t h a t t h e a c i d - c a t a l y z e d e n o l i z a t i o n p r i o r to e n o l acetate f o r m a t i o n y i e l d s the i s o m e r m o s t f a v o r e d t h e r m o d y n a m i cally w h i l e base-catalyzed alkylation favors that product resulting from r e a c t i o n at t h e least s t e r i c a l l y h i n d e r e d p o s i t i o n . S o m e p r e h m i n a r y obser vations o n d e u t e r i u m e x c h a n g e e x p e r i m e n t s w i t h zearalenone i n s o d i u m
192
MYCOTOXINS
PTS =
p-toluenesulfonic
acid
m e t h o x i d e - m e t h a n o l i n d i c a t e d t h a t e x c h a n g e occurs r a p i d l y at t h e C 7 ' position
(42).
T h e selective i n t r o d u c t i o n of f o r m y l g r o u p at C 7 ' p o s i t i o n is u s e f u l since i t serves as a p o t e n t i a l p r e c u r s o r i n the synthesis of v a r i o u s z e a r a l e none derivatives. J e n s e n et a l . (38)
Synthesis of i s o m e r i c z e a r a l e n o n e
was reported
i n w h i c h the h y d r o x y m e t h y l e n e d e r i v a t i v e 38a
by was
c o n v e r t e d i n t o z e a r a l a n - 7 - o n e ( S c h e m e I I I ) . Synthesis of l ' , 7 ' - z e a r a l d i e /
none (7'-dehydrozearalenone)
was attempted i n w h i c h 7'-formylzearale-
n o n e - 2 , 4 - d i m e t h y l ether 38b w a s u s e d as a n i n t e r m e d i a t e (40).
Bromina-
t i o n of this ether w a s e x p e c t e d to y i e l d the 7 - b r o m o d e r i v a t i v e 4 1 w h i c h /
c o u l d b e d e h y d r o h a l o g e n a t e d to the d i e n o n e . H o w e v e r the c r u d e b r o m i -
10.
P A T H R E
A N D
M I R O C H A
Zearalenone SCHEME
and Related Compounds
193
III
7'-Zearalanone n a t i o n p r o d u c t w a s a m i x t u r e of m o n o b r o m o 41 a n d d i b r o m o d e r i v a t i v e s 4 2 w h i c h o n t h i n l a y e r c h r o m a t o g r a p h y d e f o r m y l a t e d extensively w i t h o u t y i e l d i n g a satisfactory s e p a r a t i o n .
D e h y d r o b r o m i n a t i o n of this p r o d u c t
w i t h c o l l i d i n e g a v e l ' , 7 ' - z e a r a l d i e n o n e i n s m a l l y i e l d as d e t e c t e d b y c o m b i n e d gas c h r o m a t o g r a p h y - m a s s s p e c t r o m e t r y ( G C - M S ) . T h e d o u b l e b o n d i n t h e m a c r o c y c l i c l a c t o n e is q u i t e resistant to bromination (40), epoxidation, and hydroboration (38).
T h i s is p r o b a b l y
a r e s u l t of t h e e l e c t r o n w i t h d r a w i n g effect of the o r t h o c a r b o x y l g r o u p w h i c h m a k e s the olefin e l e c t r o n d e f i c i e n t .
T r e a t i n g zearalenone
with
N - b r o m o s u c c i n i m i d e f a i l e d to p r o d u c e t h e a l l y l i c b r o m i d e ; i n s t e a d t h e
41 42
H Br
194
MYCOTOXINS
Table II.
Reaction Conditions for Forming Various Ethers of Zearalenone
R 0' 2
Ri
R
Conditions
2
H CH CH
C H H CH
3
3
3
Ref
excess of CH N i n ether ( C H ) S 0 in 10% N a O H excess of ( C H ) S 0 i n 1 0 % N a O H or C H I i n acetone i n the presence of K C 0 at reflux C H C H C 1 i n a n h y d r o u s M e O H i n the presence of K C 0 a t reflux {see Scheme I V ) C 6 H , C H C l i n acetone i n the presence of K C 0 at reflux for 5 d a y s 1) N a H i n D M F a t 0 ° ; 2) C H O C H C l i n D M F a t 0 ° / \ 2
3
3
2
4
3
2
4
8 8 8
3
2
—CH C H 2
6
H
5
6
3
5
2
—CH C H 2
6
—CH C H
5
2
6
5
r
2
3
—CH,OCH
3
3
3
H
^ \
ÇH 6
5
I
f V Ν—Ν
C H I 6
5
v V Ν—Ν Ν \
3
80
2
2
—CH OCH
18
2
2
38 38 30
l-phenyl-5-chlorotetrazole i n refluxing a n h y d r o u s acetone i n the presence ,
,
K
A
2-chlorobenzoxazole presence of 24 h r
i n acetone i n the for
K2CO3 a t reflux
86
b r o m i n a t e d zearalenone i s o l a t e d h a d b r o m i n e s u b s t i t u t e d o n the a r o m a t i c ring
(40). Ether Formation. B e c a u s e of its t w o p h e n o l i c h y d r o x y l s , z e a r a l e n o n e
forms a v a r i e t y of ethers; h o w e v e r these t w o h y d r o x y l s differ i n r e a c t i v i t y . T h e p h e n o l i c p r o t o n at the C 2 p o s i t i o n is h y d r o g e n b o n d e d to the p e r i c a r b o n y l of t h e l a c t o n e f u n c t i o n . T h e r e f o r e i t is m o r e a c i d i c , a n d r e a c tions of this h y d r o x y l d e m a n d h i g h steric r e q u i r e m e n t s . T a b l e II shows the r e a c t i o n c o n d i t i o n s for p r e p a r i n g different ethers of z e a r a l e n o n e w i t h c o r r e s p o n d i n g references.
A l t h o u g h t h e C 2 O H p r o t o n is m o r e
acidic
10.
P A T H R E
A N D
Zearalenone
M I R O C H A
and Related Compounds
195
SCHEME IV HO
Ο
| ^ H
1. Q ) Ο L
2. l-phenyl-5-chlorotetrazoIe 3 . 5% P d / C
H*
2. C6H5CH2CI, K2CO3
3. H 4. l-phenyl-5-chlorotetrazole 5. 5% P d / C , H +
2
1. l-phenyl-5-chlorotetrazole 2. 5% P d / C
than the 4 0 H proton, methylation with diazomethane occurs at 4 0 H . This can be attributed to the strong intramolecular Η bonding w h i c h precludes the transfer of the bonded proton ( C 2 O H ) to diazomethane.
196
MYCOTOXINS
H o w e v e r , i n t h e presence of a l k a l i t h e C 2 p h e n o x i d e c a n b e s e l e c t i v e l y f o r m e d to effect t h e c o r r e s p o n d i n g
e t h e r i f i c a t i o n b y metathesis
when
steric r e q u i r e m e n t s are n o t h i g h . S u c h selective etherification has b e e n u s e d (SO) to m a k e the 2- or 4-deoxy d e r i v a t i v e of z e a r a l e n o n e as s h o w n i n S c h e m e I V . T h e k e y r e a c t i o n i n this s e q u e n c e is the selective m o n o t e t r a h y d r o p y r a n y l ether f o r m a t i o n at C 4 i n g o o d y i e l d . C i s - t r a n s I s o m e r i z a t i o n . T h e g e o m e t r i c a l i s o m e r i z a t i o n of z e a r a l e n e d e r i v a t i v e s is a c c o m p l i s h e d p h o t o c h e m i c a l l y .
This can be brought about
b y l i g h t of a w a v e l e n g t h t h a t is a b s o r b e d b y t h e olefinic system ( u s u a l l y uv light).
S i n c e energy
is a b s o r b e d ,
this process does n o t e s t a b l i s h
t h e r m a l e q u i l i b r i u m . H o w e v e r a steady state m a y b e r e a c h e d
which
g e n e r a l l y corresponds to a p r e d o m i n a n c e of the less stable isomer.
Thus
i r r a d i a t i o n of frans-zearalenone w i t h u v l i g h t l e d t o a m i x t u r e c o n t a i n i n g 8 8 % d s - z e a r a l e n o n e 43
(43).
Miscellaneous Reactions.
S e v e r a l reactions of 6' ketone h a v e
been
r e p o r t e d , s u c h as: G r i g n a r d reactions ( 4 4 ) , a d d i t i o n of h y d r o g e n c y a n i d e (45), Reformatsky reaction (46), chlorination w i t h phosphorus
penta-
c h l o r i d e ( 4 7 ) , t h i o k e t a l f o r m a t i o n ( 4 8 ) , a n d a d d i t i o n of a c e t y l e n e Related
Natural
(49).
Macrolides
C o m p o u n d s s i m i l a r i n m o l e c u l a r f o r m to z e a r a l e n o n e , b u t p e r h a p s not i n biological activity, have reportedly been synthesized b y fungi. Radicicol (Monorden).
T h i s c o m p o u n d , a n a n t i b i o t i c , w a s first i s o
l a t e d i n 1953 f r o m Monosporium
honor den
(50)
a n d was named monor
d e n (51 ). T e n years later M c C a p r a et a l . (51 ) a n d M i r r i n g t o n et a l . ( 5 2 ) i n d e p e n d e n t l y p r o v e d t h a t the s t r u c t u r e of this c o m p o u n d w a s 4 4 . c o m p o u n d w a s c a l l e d r a d i c i c o l since i t w a s i s o l a t e d f r o m Nectri (52).
The
radicicola
I m p o r t a n t c h e m i c a l studies w e r e c a r r i e d o u t o n r a d i c i c o l b y M i r
r i n g t o n et a l . ( 5 3 ) w h o f o u n d t h a t r a d i c i c o l e x h i b i t e d m a r k e d i n s t a b i l i t y toward alkali.
W h e n i t w a s t r e a t e d w i t h aqueous
ethanolic potassium
h y d r o x i d e , the s o l u t i o n i n s t a n t a n e o u s l y t u r n e d b r i g h t r e d . T h e r e a c t i o n w a s r e v e r s e d o n l y i f the a l k a l i n e s o l u t i o n w a s q u i c k l y q u e n c h e d w i t h a n
10.
P A T H R E
A N D
Zearalenone
M I R O C H A
and Related Compounds
197
HO
OCH a c i d . H o w e v e r t h e t e t r a h y d r o d e r i v a t i v e 45 o b t a i n e d b y c a t a l y t i c h y d r o génation of r a d i c i c o l d i d n o t s h o w s u c h d e c o m p o s i t i o n .
The
comparison
of the u v a n d N M R spectra of r a d i c i c o l w i t h those of t e t r a h y d r o r a d i c i c o l indicated
the
presence
of
an
isolated,
linear
conjugated
dienone
Ο ( — C — C H = C H — C H = C H — ) system. A l s o the i s o l a t i o n o f a d i p i c a c i d
198
MYCOTOXINS
46 from t h e c h r o m i c a c i d o x i d a t i o n p r o d u c t s of the t e t r a h y d r o d e r i v a t i v e c o n f i r m e d the presence of the d i e n o n e s y s t e m . A series of c h e m i c a l transformations ( 4 5 - » 4 8 ) s h o w e d t h e i n v o l v e m e n t of a n o x i r a n e r i n g w h i c h w a s a s s i g n e d the trans c o n f i g u r a t i o n o n t h e basis of f o r m a t i o n of t h e trans olefin 4 9 f r o m 4 5 . It was mentioned earlier that r a d i c i c o l undergoes degradation under alkaline conditions.
S u c h d e g r a d a t i o n is i n i t i a t e d b y t h e f o r m a t i o n
of
t h e enolate a n i o n 50 a n d gives rise t o t w o i s o l a b l e p r o d u c t s : a p h t h a l i d e 51 a n d a n i s o c o u m a r i n d e r i v a t i v e 52. T e t r a h y d r o r a d i c i c o l u n d e r a l k a l i n e c o n d i t i o n s also y i e l d s a n i s o c o u m a r i n d e r i v a t i v e . Lasiodiplodin and De-O-methyllasiodiplodin. O-methyllasiodiplodin
are
produced
by
L a s i o d i p l o d i n a n d de-
Lasiodiplodium
theobromae. H
OCH; 51
+
10.
P A T U R E
A N D
M I R O C H A
Zearalenone
lasiodiplodin
and
R = C H
Related
3
de-O-methyllasiodiplodin
OH
R^=H
Ο
OH
curvularin Figure 5.
199
Compounds
0
—dehydrocurvularin Naturally occurring
macrolides
B a s e d o n the o x i d a t i o n a n d s p e c t r a l studies, A l d r i d g e et a l . (54) t h e s t r u c t u r e of these m a c r o l i d e s .
reported
O x i d a t i o n of l a s i o d i p l o d i n w i t h Jones
reagent gave the quinone i n d i c a t i n g a resorcylic a c i d derivative. F u r t h e r , t h e c o m p a r i s o n of t h e r e l a t e d s y n t h e t i c m a c r o l i d e 76 w i t h d e - O - m e t h y l l a s i o d i p l o d i n c o n f i r m e d t h e s t r u c t u r e of L a s i o d i p l o d i n ( F i g u r e 5 ) . C u r v u l a r i n . M u s g r a v e i n 1956 (55) f r o m species of Curvnilaria.
isolated two related metabolites
T h e m e t a b o l i t e , C i H o 0 , w h i c h appears to 6
be a major metabolite, was n a m e d curvularin. C i H i 0 , w a s a n α,^-dehydrocurvularin ( 5 6 ) . 6
8
5
B i r c h et a l . (58) et a l . (58)
2
5
T h e minor metabolite, M u s g r a v e (55, 57)
p r o v i d e d t h e p r o o f for the s t r u c t u r e of c u r v u l a r i n .
and Birch
also d e m o n s t r a t e d t h a t c u r v u l a r i n is d e r i v e d f r o m the h e a d t o
t a i l c o n d e n s a t i o n of e i g h t acetate u n i t s . N a t u r a l l y O c c u r r i n g 3-4-Dehydroisocoumarins.
Since zearalenone
is a m e m b e r of a class of n a t u r a l p r o d u c t s c a l l e d t h e β-resorcylates, the naturally occurring dihydroisocoumarin derivatives, although not macro lides, are u s u a l l y c o n s i d e r e d to b e r e l a t e d to zearalenone.
T a b l e III lists
s o m e of these d i h y d r o i s o c o u m a r i n s a n d a p p r o p r i a t e references.
Synthetic Approaches to Macrolides (Zearalenone and Its Derivatives and Related Macrolides) T h e first t o t a l synthesis of n a t u r a l l y o c c u r r i n g zearalenone
together
200
MYCOTOXINS
Table III.
Naturally Occurring
Compound 3,4-Dihydro-6-methoxy,8-hydroxy-3-methylisocoumarin 3,4-Dihydro-6,8-dihydroxy-3-methylisocoumarin 3 , 4 - D i h y d r o - 6 , 8 - d i h y d r o x y - 3 - [β- ( 4 - m e t h o x y p h e n y l ) e t h y l ] i s o c o u m a r i n 3,4-Dihydro-6,8-dihydroxy-3,4,5-trimethylisocoumarin 3,4-Dihydro-6,8-dihydroxy-3,4,5-trimethylisocoumarin-7-carboxylic acid Reticulol Cladosporin
w i t h its o p t i c a l r e s o l u t i o n w a s a c c o m p l i s h e d b y a t e a m of M e r c k chemists (21).
T h e k e y feature i n v o l v e d i n t h e synthesis ( S c h e m e V ) w a s t h e W i t
t i n g c o n d e n s a t i o n of a n a p p r o p r i a t e l y s u b s t i t u t e d a r o m a t i c n u c l e u s 53 w i t h a n a l i p h a t i c c o m p o n e n t 54.
N o t e w o r t h y i n this synthesis are the
c r i t i c a l steps u s e d to construct the a l i p h a t i c m o i e t y 54 i n w h i c h t h e C 6 a n d C I O f u n c t i o n a l i t i e s are m u t u a l l y m a s k e d v i a i n t e r n a l k e t a l f o r m a t i o n .
Girotra and Wendler
(65,
66)
w e r e successful i n c o n d e n s i n g t h e
a l i p h a t i c i n t e r m e d i a t e 56 w i t h d i m e t h o x y h o m o p h t h a l i c a n h y d r i d e 57 to g i v e l a c t o n i c a c i d 58 w h i c h u n d e r g o e s d e c a r b o x y l a t i o n to y i e l d t h e seco a c i d 14. C y c l i z a t i o n of t h e seco a c i d u s i n g t r i f l u o r o a c e t i c a c i d a n h y d r i d e i n benzene a n d subsequent demethylation w i t h boron tribromide y i e l d e d ( db )-zearalenone. (21)
A n a p p r o a c h at synthesis s i m i l a r to t h a t of T a u b et a l .
w a s m a d e b y V l a t t a s et a l . ( 6 7 ) ; h o w e v e r t h e a r o m a t i c 59 a n d
a l i p h a t i c 60 portions w e r e c o n s t r u c t e d i n a different m a n n e r
(Scheme
V I ) . N o t e t h a t t h e specific c l e a v a g e o f t h e k e t a l at C I O o f t h e side c h a i n of t h e a c i d 61 i n a q u e o u s acetone c o n t a i n i n g p - t o l u e n e s u l f o n i c
acid
10.
P A T H R E
A N D
Zearalenone
M I R O C H A
and Related Compounds
201
6,8-Dihydroxyisocoumarin Derivatives
Ri
R%
R
Η Η Η Η Η Η Η
Η Η Η Η COOH OCHs Η
CH Η Η Η Η Η Η
Rs
s
3
Η Η Η CH CHs Η Η 3
Η Η Η CHs CHs Η Η
Ref
Re
59 60
CHs CH., CUT G CHsCH CHs CHs CHs
OCH3 61 64
-CH CH(CH ) CH 2
2
3
CH p r o c e e d e d i n 8 5 % y i e l d to g i v e t h e r e q u i r e d m o n o k e t a l 62.
3
However
the c o r r e s p o n d i n g ester of the a c i d w a s c l e a v e d to a m i x t u r e of m o n o k e t a l s and diketone. W e h r m e i s t e r a n d R o b e r t s o n ( 3 6 ) r e p o r t e d the synthesis of d i d e o x y z e a r a l a n e 65,
a simple macrocyclic
lactone h a v i n g t h e same s k e l e t a l
s t r u c t u r e as zearalenone. T h e y s y n t h e s i z e d the b a s i c skeleton 63 r e q u i r e d for m a k i n g the a p p r o p r i a t e h y d r o x y a c i d 64 b y c o n d e n s a t i o n
of
10-
u n d e c e n o i c a n h y d r i d e w i t h p h t h a l i c a n h y d r i d e i n the presence of s o d i u m acetate.
T h e h y d r o x y a c i d 64 w a s e x p e c t e d to l a c t o n i z e to the d e s i r e d
c o m p o u n d 65; h o w e v e r the c y c l i z a t i o n of the h y d r o x y a c i d 64 to d i d e o x y z e a r a l a n e , w h i c h lacks zearalenone's d o u b l e b o n d , ketone, a n d a r o matic hydroxyls, was unexpectedly
difficult.
S i m i l a r difficulties
were
r e p o r t e d i n attempts to p r e p a r e d i - o - m e t h y l c u r v u l a r i n b y c y c l i z a t i o n of t h e h y d r o x y a c i d 66
57
(68, 6 9 ) .
S e v e r a l t e c h n i q u e s , i n c l u d i n g use
58
of
202
MYCOTOXINS
SCHEME OCH
V
3
J ^ C O O N a
ΓΓΐΓ
+
?
J
- [ D M S O ] — ± 1 4
(C H )aP^^^t5g;
C H a O - ^ ^ C H O 53
e
5
54
T h e W i t t i n g - C o u p l i n g w i t h s o d i u m s a l t of the a c i d 53 proceeded r a p i d l y i n good y i e l d .
[(CF CO) 0 3
1. BCI3
^ D L - lb
2
·(—)-zearalenone
2. R e s o l v e d 3. B B r 3
S y n t h e s i s of t h e a r o m a t i c nucleus
53:
Q
CH3O
C H Li(0-iBut) AlH T H F , 20° -
3
O
OCH
3
.COOH
Χ
3
CH3O
OCHT
OCH3
1
CHO
Η
53
S y n t h e s i s of t h e a l i p h a t i c c o m p o n e n t :
1. CH CO(CH ) COOH 3
2
H ^
3
B
H
4
^ ^ ^
^v^s^-MgBrJ EtîO, - 1 5 °
2. d i s t i l 1. l2. -13. 4. 5. OCHo
1. HC1, MeOH 2. 0 , MeOH, -60° 3
55
56
NaBH PTSC1, P y . NaBr |*54 (C H ) P NaH, DMSO 4
e
5
3
10.
P A T H B E
A N D M I R O C H A
Zearalenone
and Related
203
Compounds
SCHEME VI OCH
3
X . C O O E t JL
Jl
CH3O' ^
CHO
+
(CeHsUP'
59
