Discovery, Isolation, and Structure Elucidation of a Family of

Chapter 20

Discovery, Isolation, and Structure Elucidation of a Family of Structurally Unique, Fermentation-Derived Tetracyclic Macrolides 1

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Downloaded by UNIV LAVAL on July 8, 2014 | http://pubs.acs.org Publication Date: September 22, 1992 | doi: 10.1021/bk-1992-0504.ch020

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Herbert A. Kirst , Karl H. Michel , Jon S. Mynderase , Eddie H. Chio , Raymond C. Yao , Walter M. Nakasukasa , LaVerne D. Boeck , John L. Occlowitz , Jonathon W. Paschal , Jack B. Deeter , and Gary D. Thompson 1

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Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, IN 46285 Discovery Research, DowElanco Research Laboratories, Greenfield, IN 46140

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S c r e e n i n g of f e r m e n t a t i o n b r o t h s f o r m o s q u i t o l a r v i c i d a l activity y i e l d e d a n active culture d e n o t e d a s A 8 3 5 4 3 . Nine active, structurally-related f a c t o r s w e r e isolated a n d purified by extractive a n d c h r o m a t o g r a p h i c procedures. Their structures w e r e elucidated b y a c o m b i n a t i o n of s p e c t r o s c o p i c ( N M R , M S , UV, IR) a n d X-ray crystallographic m e t h o d s . Each factor p o s s e s s e d t h e c o r e structure of a 5,6,5-cis-anti-transtricyclic ring s y s t e m f u s e d t o a 1 2 - m e m b e r e d l a c t o n e . In a d d i t i o n , a n a m i n o s u g a r ( f o r o s a m i n e ) a n d a neutral s u g a r (2,3,4-tri-O-methylrhamnose) w e r e glycosidically linked t o t h e tetracyclic framework. Absolute stereochemistry w a s e s t a b l i s h e d b y c o m p a r i n g s a m p l e s of f o r o s a m i n e o b t a i n e d f r o m a c i d i c h y d r o l y s e s of A 8 3 5 4 3 A a n d s p i r a m y c i n . T h e novel t e t r a c y c l i c s t r u c t u r e of A 8 3 5 4 3 s u g g e s t s t h a t u n i q u e features are involved in its biosynthesis. T h e purified factors exhibited potent mosquito larvicidal activity, but lacked antibiotic activity. After s e v e r a l d e c a d e s of intense investigation, s c r e e n i n g of f e r m e n t a t i o n b r o t h s still r e m a i n s a v i a b l e m e t h o d f o r d i s c o v e r y of s t r u c t u r a l l y novel c o m p o u n d s p o s s e s s i n g biological activity (7, 2). T h e ability of physicians to t r e a t m o s t i n f e c t i o u s d i s e a s e s is l a r g e l y a result of t h e s u c c e s s f u l d e v e l o p m e n t of f e r m e n t a t i o n - d e r i v e d antibiotics a n d t h e i r s e m i - s y n t h e t i c d e r i v a t i v e s . T h i s a p p r o a c h is n o w b e i n g m o r e e x t e n s i v e l y a p p l i e d t o d i s c o v e r c o m p o u n d s t h a t exhibit activity in a w i d e v a r i e t y of n o n - a n t i infective applications in both h u m a n a n d veterinary medicine (2 - 5). A m o n g t h e n u m e r o u s c l a s s e s of k n o w n f e r m e n t a t i o n p r o d u c t s , t h e c l a s s of m a c r o l i d e s (or, m o r e b r o a d l y , m a c r o c y c l i c l a c t o n e s ) h a s b e e n e s p e c i a l l y p r o m i n e n t in b o t h s t r u c t u r a l a n d b i o l o g i c a l d i v e r s i t y of its m e m b e r s {6, 7). T h e discovery of A 8 3 5 4 3 a d d s a n o t h e r novel, structurally 0097-6156/92/0504-Ό214$06.00/0 © 1992 American Chemical Society

In Synthesis and Chemistry of Agrochemicals III; Baker, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1992.

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Fermentation-Derived Tetracyclic Macrolides

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u n i q u e f a m i l y of c o m p o u n d s t o t h e rapidly g r o w i n g c l a s s of m a c r o c y c l i c lactones p o s s e s s i n g non-antibiotic biological activity.


S c r e e n i n g of Culture B r o t h s a n d Discovery of an Active

Culture

T h e d i s c o v e r y of n e w i n s e c t i c i d e s is a n i m p o r t a n t r e s e a r c h o b j e c t i v e b e c a u s e of t h e c o n t i n u i n g d e v e l o p m e n t of r e s i s t a n c e t o e x i s t i n g insecticides a n d t h e desire for agents with less environmental a n d m a m m a l i a n t o x i c i t y (8, 9). It is particularly d e s i r a b l e t o d i s c o v e r n e w c h e m i c a l classes of insecticides which o p e r a t e by different m o d e s of action a n d , consequently, lack cross-resistance with currently e m p l o y e d insecticides. In t h i s r e s p e c t , s c r e e n i n g f e r m e n t a t i o n b r o t h s c a n offer u n i q u e a d v a n t a g e s , s i n c e this a p p r o a c h c a n u n c o v e r c o m p l e t e l y novel s t r u c t u r e s unlike a n y t h a t h a v e p r e v i o u s l y b e e n s y n t h e s i z e d , o r e v e n c o n c e i v e d , by o r g a n i c c h e m i s t s . R e c e n t e x a m p l e s of novel f e r m e n t a t i o n products which have revolutionized a thereapeutic area include a v e r m e c t i n (anthelmintic), c y c l o s p o r i n a n d F K - 5 0 6 ( i m m u n o s u p p r e s s i v e ) , a n d mevinolin a n d c o m p a c t i n (cholesterol-lowering). M a n y p r e v i o u s studies have d e m o n s t r a t e d that insecticides c a n be s u c c e s s f u l l y i s o l a t e d f r o m f e r m e n t a t i o n b r o t h s (10 - 16). F u r t h e r m o r e , f e r m e n t a t i o n products m a y be useful a s starting materials for the preparation of s e m i - s y n t h e t i c derivatives p o s s e s s i n g i m p r o v e d insecticidal activity. O n e s u c h recent e x a m p l e is t h e c o n v e r s i o n of a v e r m e c t i n to MK2 4 3 , w h i c h i m p r o v e d t h e insecticidal activity a g a i n s t v a r i o u s s p e c i e s by s e v e r a l h u n d r e d - f o l d ( 7 7 - 19). B a s e d o n literature p r e c e d e n t s s u c h a s t h e s e , a p r o g r a m of s c r e e n i n g f e r m e n t a t i o n broths f o r novel c o m p o u n d s with insecticidal activity is w a r r a n t e d . D e v e l o p m e n t of a s c r e e n that is both relevant f o r t h e target activity a n d a m e n a b l e to c o m p l e x culture broths (i.e., both sensitive a n d selective) is a k e y p r e r e q u i s i t e f o r t h e d i s c o v e r y of f e r m e n t a t i o n p r o d u c t s . Historically, inhibition of microbial g r o w t h a r o u n d a disk o n an a g a r plate w a s j u s t s u c h a highly s e l e c t i v e a n d s e n s i t i v e a s s a y s y s t e m w h i c h , for m o r e t h a n f o u r d e c a d e s , l e d t o t h e d i s c o v e r y of t h e c u r r e n t l y k n o w n m u l t i t u d e of a n t i m i c r o b i a l c o m p o u n d s . T h u s , both t h e c h o i c e a n d t h e d e v e l o p m e n t of a s c r e e n are critical issues w h i c h must be a d d r e s s e d a n d s o l v e d if a fermentation products discovery p r o g r a m is to s u c c e e d . Mosquito larvicidal activity has b e e n e m p l o y e d for m a n y years a s an i n d i c a t o r a s s a y f o r o t h e r i n s e c t i c i d a l a c t i v i t i e s (20, 21). M o r e recent a p p l i c a t i o n s of t h i s m e t h o d o l o g y h a v e b e e n f o u n d in s c r e e n i n g c u l t u r e b r o t h s or s t u d y i n g v a r i o u s f e r m e n t a t i o n - d e r i v e d natural p r o d u c t s (22, 23). In t h e c o u r s e of o u r s c r e e n i n g culture b r o t h s f r o m t h e f e r m e n t a t i o n of r a n d o m soil m i c r o o r g a n i s m s , a broth w a s f o u n d t h a t e x h i b i t e d activity against larvae of t h e mosquito, Aedes aegypti. This activity w a s p r o d u c e d b y a m i c r o o r g a n i s m , d e s i g n a t e d a s A 8 3 5 4 3 , that h a d b e e n isolated f r o m a soil s a m p l e collected in t h e Virgin Islands. S u b s e q u e n t t a x o n o m i c studies i n d i c a t e d t h a t t h i s o r g a n i s m w a s a n e w s p e c i e s w i t h i n t h e rare g e n u s Saccharopolyspora. It h a s n o w b e e n classified a s Saccharopolyspora spinosa (24). T h i s g e n u s h a s not y e t b e e n e x t e n s i v e l y e x p l o r e d with


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respect to its potential for t h e production of s e c o n d a r y m e t a b o l i t e s during f e r m e n t a t i o n (25).


Isolation of the Active C o m p o n e n t s f r o m A83543 Culture

Broths

T h e active c o m p o n e n t s within the A 8 3 5 4 3 c o m p l e x w e r e isolated f r o m the f e r m e n t a t i o n b r o t h a n d p u r i f i e d by a c o m b i n a t i o n of e x t r a c t i v e a n d c h r o m a t o g r a p h i c procedures. T h e broth w a s initially filtered with the use of a filter aid (Hyflo, 1%), a n d the s e p a r a t e d b i o m a s s w a s w a s h e d with water. T h e b i o m a s s w a s t h e n agitated with m e t h a n o l for a p p r o x i m a t e l y o n e hour to release a n d dissolve the d e s i r e d product. T h e mixture w a s filtered a n d t h e filtrate w a s c o n c e n t r a t e d a n d t h e n e x t r a c t e d t h r e e t i m e s with e q u a l v o l u m e s of diethyl ether. T h e c o m b i n e d ether extracts w e r e c o n c e n t r a t e d a n d c h r o m a t o g r a p h e d (in p o r t i o n s ) b y r e v e r s e d - p h a s e H P L C o n an " A u t o p r e p " a u t o m a t e d H P L C s y s t e m (Rainin Instrument C o ) , using a Lobar R P - 8 c o l u m n (size Β; Ε. M. Industries, Inc.) a n d UV d e t e c t i o n of active f r a c t i o n s at 2 5 0 n m ( I S C O - V 4 a b s o r b a n c e d e t e c t o r ) . T h e c o l u m n w a s e l u t e d with a solvent mixture of methanol-acetonitrile-water (49:49:2), with a flow rate of 8 m L / m i n a n d a c y c l e t i m e of 2 8 m i n . T h e a p p r o p r i a t e fractions w e r e automatically c o m b i n e d f r o m e a c h of t w e l v e c y c l e s t h r o u g h t h e A u t o p r e p s y s t e m to y i e l d s e v e r a l p o o l e d f r a c t i o n s w h i c h w e r e e x a m i n e d by m o s q u i t o larvicidal a s s a y a n d analytical H P L C . T h e analytical c h r o m a t o g r a p h y s y s t e m ( L D C Gradient M a s t e r III with a C o n s t a m e t r i s III p u m p a n d S p e c t r o m o n i t o r III U V detector) e m p l o y e d a r e v e r s e d - p h a s e c o l u m n ( N o v a 4 μ C 1 8 , 8 χ 100 m m , W a t e r s A s s o c i a t e s ) which was eluted with acetonitrile-methanol-water (45:45:10) containing a m m o n i u m acetate (0.05%) at 4 m L / m i n a n d U V detection at 2 5 0 n m . T h e active fractions w e r e appropriately c o m b i n e d a c c o r d i n g to the results from t h e m o s q u i t o larvicidal a s s a y s a n d H P L C p r o f i l e s . T h e s e c o m b i n e d f r a c t i o n s w e r e f u r t h e r p u r i f i e d o n t h e A u t o p r e p s y s t e m , u s i n g a high r e s o l u t i o n r e v e r s e d - p h a s e p r e p a r a t i v e c o l u m n (8 μ C 1 8 , 2.14 χ 2 5 c m , Rainin D y n a m a x ) . Elution with m e t h a n o l - a c e t o n i t r i l e - w a t e r m i x t u r e s t h e n yielded the purified factors A 8 3 5 4 3 A , B, C, a n d D. A n a l t e r n a t i v e p r o c e d u r e w a s also d e v e l o p e d for isolation of the A83543 compounds. T h e m e t h a n o l i c e x t r a c t of t h e b i o m a s s w a s c o n c e n t r a t e d a n d t h e n a p p l i e d t o H P - 2 0 resin ( M i t s u b i s h i C h e m i c a l Industries) s u s p e n d e d in water. T h e resin mixture w a s stirred for 1 hr a n d t h e n p o u r e d into a c o l u m n , d r a i n e d , a n d eluted with m e t h a n o l - w a t e r ( 1 : 1 , then 7:3). Elution with methanol subsequently yielded the desired m a t e r i a l . A f t e r e v a p o r a t i o n of s o l v e n t , t h e r e s i d u e w a s d i s s o l v e d in m e t h a n o l - T H F (4:1) a n d p r e c i p i t a t e d by a d d i t i o n to a c e t o n i t r i l e (10 v o l u m e s ) . T h e m i x t u r e w a s filtered a n d t h e filtrate w a s e v a p o r a t e d to d r y n e s s . T h e residue w a s d i s s o l v e d in m e t h a n o l a n d applied to a c o l u m n of S e p h a d e x L H - 2 0 (5.5 χ 90 c m , P h a r m a c i a L K B B i o t e c h n o l o g y , Inc.) p a c k e d in m e t h a n o l . T h e d e s i r e d p r o d u c t s w e r e e l u t e d with m e t h a n o l , a n d a p p r o p r i a t e fractions w e r e c o m b i n e d a c c o r d i n g to b i o a s s a y a n d analytical H P L C results a n d e v a p o r a t e d . T h e residue, d i s s o l v e d in m e t h a n o l , w a s applied to a preparative r e v e r s e d - p h a s e H P L C c o l u m n (8 μ C 1 8 , 4.11 χ 25


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c m , R a i n i n D y n a m a x ) w h i c h h a d b e e n c o n d i t i o n e d in m e t h a n o l a c e t o n i t r i l e - w a t e r ( 3 7 . 5 : 3 7 . 5 : 2 5 ) . T h e c o l u m n w a s e l u t e d w i t h a linear g r a d i e n t of m e t h a n o l - a c e t o n i t r i l e - w a t e r , r u n n i n g f r o m 3 7 . 5 : 3 7 . 5 : 2 5 to 45:45:10. Fractions containing pure A 8 3 5 4 3 A were c o m b i n e d , e v a p o r a t e d , d i s s o l v e d in f-butanol, a n d lyophilized. Fractions c o n t a i n i n g A 8 3 5 4 3 D w e r e c o m b i n e d , c o n c e n t r a t e d , a n d c h r o m a t o g r a p h e d a s before, eluting with a linear gradient of m e t h a n o l - a c e t o n i t r i l e - w a t e r running f r o m 4 0 : 4 0 : 2 0 to 9 5 : 9 5 : 1 0 . A p p r o p r i a t e f r a c t i o n s c o n t a i n i n g p u r e A 8 3 5 4 3 D w e r e c o m b i n e d , e v a p o r a t e d , d i s s o l v e d in f-butanol, a n d lyophilized. Both A 8 3 5 4 3 A a n d A 8 3 5 4 3 D crystallized f r o m a q u e o u s e t h a n o l .


Physical

Chemical

Characterization

of A83543

Factors

T h e m a j o r factor of t h e c o m p l e x , A 8 3 5 4 3 A , w a s d e t e r m i n e d to have t h e molecular f o r m u l a of C ^ h ^ N O - j o a n d a molecular weight of 731 by high resolution mass spectrometry. T h e s e c o n d most a b u n d a n t factor, A 8 3 5 4 3 D , h a d t h e f o r m u l a of C42H67NO10 ( M W 7 4 5 ) . A m o n g t h e minor factors, A 8 3 5 4 3 G w a s isomeric with A 8 3 5 4 3 A , while factors Β, E, F, H, a n d J possessed the formula C 4 o H g N 0 ( M W 717) and A83543C had the formula Ο 9 Η Ν Ο ( M W 7 0 3 ) . B y t h e u s e of M S / M S t e c h n i q u e s , f a c t o r s A , D, E, F, H, a n d J w e r e f o u n d t o c o n t a i n t h e a m i n o s u g a r f o r o s a m i n e , previously k n o w n in t h e macrolide antibiotic, s p i r a m y c i n (26). A 8 3 5 4 3 G c o n t a i n e d a different a m i n o s u g a r , o s s a m i n e , previously k n o w n in t h e f e r m e n t a t i o n - d e r i v e d product, o s s a m y c i n (27). Also isolated w a s a p s e u d o a g l y c o n e , lacking f o r o s a m i n e but containing t h e other s a c c h a r i d e substituent of A 8 3 5 4 3 A , w h i c h h a d t h e f o r m u l a C33H50O9 ( M W 5 9 0 ) . 3

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6 1

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1 0

A 8 3 5 4 3 A p o s s e s s e d a c h r o m o p h o r e at 2 4 3 n m (ε = 9 0 0 0 ) in e t h a n o l s o l u t i o n ; no c h a n g e s w e r e o b s e r v e d u p o n a c i d i f i c a t i o n or alkalization. Its o n l y t i t r a t a b l e g r o u p w a s t h e a m i n o g r o u p of t h e a m i n o s u g a r ( p K a = 7.8 in 6 6 % a q u e o u s D M F ) . It w a s s o l u b l e in most organic solvents, but poorly soluble in w a t e r at p H v a l u e s a b o v e 7. It w a s soluble in dilute a q u e o u s acid d u e to salt f o r m a t i o n ; however, strong acids h y d r o l y z e d t h e acid-labile 2 - d e o x y s u g a r , f o r o s a m i n e . All of t h e o t h e r factors p o s s e s s e d a n a l o g o u s properties. Most of t h e individual f a c t o r s w e r e s e p a r a t e d o n t h e a n a l y t i c a l H P L C s y s t e m d e s c r i b e d a b o v e , w i t h t h e f o l l o w i n g r e t e n t i o n t i m e s (in minutes): Pseudoaglycone (2.55), A 8 3 5 4 3 C (2.62), A 8 3 5 4 3 B (4.22), A 8 3 5 4 3 H (5.29), A 8 3 5 4 3 G (6.49), A 8 3 5 4 3 E (6.57), A 8 3 5 4 3 A (8.97) a n d A 8 3 5 4 3 D ( 1 1 . 6 2 ) . A 8 3 5 4 3 F w a s not well r e s o l v e d f r o m A 8 3 5 4 3 H a n d a p p e a r e d a s a poorly resolved s h o u l d e r eluting after A 8 3 5 4 3 H . Elucidation of Structure of A83543A T h e g r o s s structural f r a m e w o r k of A 8 3 5 4 3 A a n d its p s e u d o a g l y c o n e w a s d e t e r m i n e d f r o m detailed analyses of t h e physical c h e m i c a l d a t a d e s c r i b e d a b o v e , m a s s s p e c t r a l f r a g m e n t a t i o n p a t t e r n s , a n d e x t e n s i v e NMR investigations, including H homonuclear decouplings, C DEPT experiments, 2D one-bond heteronaclear correlations, and 2D long-range 1

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SYNTHESIS AND CHEMISTRY O F A G R O C H E M I C A L S III

h e t e r o n u c l e a r c o r r e l a t i o n s ( F U L C O U P ) . T h e relative s t e r e o c h e m i s t r y of t h e p r o t o n s at c a r b o n a t o m s 3, 4, 7 , 1 1 , a n d 12 w e r e e s t a b l i s h e d f r o m their c o u p l i n g c o n s t a n t s a n d from difference N O E e x p e r i m e n t s . B o t h A 8 3 5 4 3 A a n d its p s e u d o a g l y c o n e c r y s t a l l i z e d f r o m e t h a n o l w a t e r to give w h i t e c r y s t a l s that w e r e a m e n a b l e to s i n g l e c r y s t a l X - r a y diffraction s t u d i e s . T h e c r y s t a l l o g r a p h i c results c o n f i r m e d t h e structural features that had been d e d u c e d from mass spectrometry and Ν MR a s s i g n m e n t s a n d e s t a b l i s h e d t h e r e l a t i v e s t e r e o c h e m i s t r y of t h e substituents at c a r b o n a t o m s 9 , 1 6 , 1 7 , a n d 2 1 . The absolute stereochemistry w a s d e t e r m i n e d by c o m p a r i n g s a m p l e s of f o r o s a m i n e o b t a i n e d f r o m acidic h y d r o l y s i s of A 8 3 5 4 3 A a n d s p i r a m y c i n . T h e t w o s a m p l e s w e r e identical in all respects, including the sign a n d m a g n i t u d e of optical rotation. C o n s e q u e n t l y , A 8 3 5 4 3 A c o n t a i n e d D-(+)-forosamine, as previously d e t e r m i n e d for spiramycin (28). C o m b i n i n g this information with the X-ray results, the absolute c o n f i g u r a t i o n of A 8 3 5 4 3 A w a s d e t e r m i n e d t o be that d e p i c t e d in Figure 1 (29). S e a r c h e s of t h e literature f a i l e d t o u n c o v e r a n y c l o s e l y r e l a t e d c o m p o u n d s , indicating that A 8 3 5 4 3 p o s s e s s e s a v e r y u n i q u e s t r u c t u r e . T h e tetracyclic f r a m e w o r k is c o m p o s e d of a 5,6,5-c/s-anf/-irans-tricyclic ring s y s t e m ( o c t a h y d r o - a s - i n d a c e n e ) f u s e d to a 1 2 - m e m b e r e d macrolide ring. A n α , β - u n s a t u r a t e d k e t o n e a n d a n isolated d o u b l e b o n d are e m b e d d e d within t h e tetracyclic f r a m e w o r k . T h e tetracyclic moiety is s u b s t i t u t e d at o p p o s i t e e n d s by t w o hydroxyl g r o u p s , w h i c h a r e g l y c o s y l a t e d w i t h an a m i n o s u g a r (forosamine) and a neutral sugar (tri-O-methylrhamnose), respectively. The most closely related, previously known c o m p o u n d s f o u n d in t h e literature are s p i r a m y c i n (which also c o n t a i n s f o r o s a m i n e ) , i k a r u g a m y c i n , a n d c a p s i m y c i n (see Figure 2). A l t h o u g h i k a r u g a m y c i n also c o n t a i n s a 5 , 6 , 5 - c / s - a n f / - f r a n s - t r i c y c l i c r i n g s y s t e m , its a b s o l u t e s t e r e o c h e m i s t r y is o p p o s i t e to that of A 8 3 5 4 3 (30). Furthermore, the tricyclic rings of i k a r u g a m y c i n are f u s e d onto a 1 6 - m e m b e r e d l a c t a m into w h i c h a t e t r a m i c a c i d moiety is e m b e d d e d , w h e r e a s the tricyclic rings of A 8 3 5 4 3 are f u s e d to a 1 2 - m e m b e r e d lactone. In addition, the pattern a n d t y p e of s u b s t i t u e n t s l o c a t e d a r o u n d t h e t r i c y c l i c r i n g s y s t e m s of i k a r u g a m y c i n a n d A 8 3 5 4 3 are c o m p l e t e l y d i f f e r e n t . Finally, A 8 3 5 4 3 p o s s e s s e s t w o s a c c h a r i d e substituents while i k a r u g a m y c i n c o n t a i n s none. T h e s t r u c t u r e of c a p s i m y c i n has b e e n r e p o r t e d to be s i m i l a r to that of i k a r u g a m y c i n (31). E l u c i d a t i o n of S t r u c t u r e of O t h e r

Factors

After t h e s t r u c t u r e of A 8 3 5 4 3 A h a d b e e n e s t a b l i s h e d , s t r u c t u r e s for the r e m a i n i n g factors w e r e readily a s s i g n e d by c o m p a r a t i v e a n a l y s e s of N M R a n d m a s s spectral d a t a . It w a s d e t e r m i n e d f r o m their empirical f o r m u l a s a n d N-methyl N M R r e s o n a n c e s that A 8 3 5 4 3 B a n d A 8 3 5 4 3 C differed f r o m A 8 3 5 4 3 A d u e to t h e a b s e n c e of e i t h e r o n e o r b o t h N - m e t h y l g r o u p s , respectively (29). A 8 3 5 4 3 D differed from factor A d u e to the p r e s e n c e of a methyl s u b s t i t u e n t a t t a c h e d at c a r b o n 6 o n t h e isolated d o u b l e b o n d (29). A 8 3 5 4 3 E d i f f e r e d f r o m A 8 3 5 4 3 A d u e to a m e t h y l r a t h e r t h a n an ethyl


20.

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Figure 1 . Absolute configuration a n d n u m b e r i n g of A 8 3 5 4 3 A

cr Figure 2. Structures of spiramycin I, i k a r u g a m y c i n , a n d c a p s i m y c i n


219


220

SYNTHESIS AND CHEMISTRY OF A G R O C H E M I C A L S III

substituent at c a r b o n 2 1 , while A 8 3 5 4 3 F differed f r o m A 8 3 5 4 3 A d u e to the a b s e n c e of the methyl g r o u p at c a r b o n 16. T h e structures of factors D, E, a n d F a r e r e a d i l y e x p l a i n e d f r o m c o n s i d e r a t i o n s of t h e b i o s y n t h e t i c p a t h w a y , d i s c u s s e d below. A 8 3 5 4 3 G d i f f e r e d in t h a t t h e 1 7 - h y d r o x y l g r o u p of t h e p s e u d o a g l y c o n e w a s g l y c o s i d a t e d by o s s a m i n e rather t h a n f o r o s a m i n e . A 8 3 5 4 3 H a n d A 8 3 5 4 3 J w e r e d e t e r m i n e d t o be t h e 2 ' - 0 - d e m e t h y l a n d 3'O - d e m e t h y l d e r i v a t i v e s of A 8 3 5 4 3 A , r e s p e c t i v e l y . Factors H and J p r o b a b l y arise f r o m i n c o m p l e t e O - m e t h y l a t i o n of t h e neutral s a c c h a r i d e m o i e t y at s o m e s t e p in the biosynthetic p a t h w a y . T h e exact s e q u e n c e of glycosidation a n d methylation of t h e t w o s a c c h a r i d e s is a subject of further investigation. T h e structures of t h e s e nine factors are illustrated in Figure 3. From t h e v a r i a t i o n s in s t r u c t u r e of t h e s e f a c t o r s , t h e y r e p r e s e n t a v a r i e t y of modifications of N-, C-, a n d O-methylation in the structure of A 8 3 5 4 3 A . Biological

Activity

T h e purified c o m p o n e n t s of t h e A 8 3 5 4 3 c o m p l e x w e r e t e s t e d for activity against fourth instar larvae of the mosquito, Aedes aegypti. Mortality after 2 4 hr at a d o s e of 0 . 3 1 2 p p m w a s 6 0 % f o r A 8 3 5 4 3 A , A 8 3 5 4 3 B , a n d A 8 3 5 4 3 C ; 3 0 % for A 8 3 5 4 3 D ; and 8 0 % for A 8 3 5 4 3 E . A83543F and A 8 3 5 4 3 G w e r e not active at this c o n c e n t r a t i o n . A 8 3 5 4 3 H a n d A 8 3 5 4 3 J w e r e also less active t h a n A 8 3 5 4 3 A . Titration of t h e d o s e of A 8 3 5 4 3 A required to kill first instar mosquito larvae r e v e a l e d a m i n i m u m larvicidal c o n c e n t r a t i o n of 0.016 p p m for this c o m p o u n d . T h i s level of activity satisfactorily a c c o u n t e d for t h e activity o b s e r v e d in the c r u d e f e r m e n t a t i o n broths, indicating that t h e individual c o m p o n e n t s r e s p o n s i b l e f o r t h e initial activity h a d b e e n s u c c e s s f u l l y identified a n d purified. T h e s t r o n g m o s q u i t o l a r v i c i d a l a c t i v i t y of t h e A 8 3 5 4 3 f a c t o r s distinguishes t h e m f r o m t h e m o r e c o n v e n t i o n a l macrolides s u c h a s tylosin, s p i r a m y c i n , a n d e r y t h r o m y c i n , w h i c h lack insecticidal activity, but p o s s e s s p o t e n t a n t i m i c r o b i a l a c t i v i t y {32). In f u r t h e r c o n t r a s t to t h e s e latter m a c r o l i d e a n t i b i o t i c s , t h e A 8 3 5 4 3 f a c t o r s d i d not inhibit t h e g r o w t h of G r a m - p o s i t i v e b a c t e r i a (Staphylococcus, Streptococcus). Inhibition of G r a m - n e g a t i v e b a c t e r i a (Enterobacteriaceae, Pseudomonas) w a s also not o b s e r v e d . O t h e r biological activities a s s o c i a t e d with t h e s e c o m p o u n d s will be reported e l s e w h e r e . Biosynthesis

of

A83543

B a s e d u p o n incorporation studies using C - l a b e l e d a c e t a t e , p r o p i o n a t e , butyrate, a n d isobutyrate, the biosynthesis of A 8 3 5 4 3 is consistent with the initial f o r m a t i o n of a long chain fatty acid v i a a polyketide-derived p a t h w a y (33). Butyrate w a s apparently c l e a v e d to acetate since it s h o w e d the s a m e i n c o r p o r a t i o n pattern a s t h e latter. No e n r i c h m e n t w a s o b s e r v e d with isobutyrate. T h e O - a n d N - m e t h y l g r o u p s of t h e s a c c h a r i d e s w e r e 1 3


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Figure 3. Structures of individual factors in t h e A 8 3 5 4 3 c o m p l e x


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Figure 4. P r o p o s e d biosynthetic pathway for A 8 3 5 4 3 A


20.

KIRST E T A L .


223

exclusively labeled by m e t h y l - C - m e t h i o n i n e , probably v i a S-adenosyl methionine. Initial f o r m a t i o n of a long c h a i n fatty a c i d is a n a l o g o u s to t h e well established biosynthetic p a t h w a y f o r m o n o c y c l i c macrolide antibiotics s u c h as erythromycin a n d tylosin (34). This p a t h w a y readily a c c o u n t s for factors D, E, a n d F. A 8 3 5 4 3 D arises f r o m t h e substitution of propionate for acetate at t h e a p p r o p r i a t e point d u r i n g c h a i n - e l o n g a t i o n o n t h e p o l y k e t i d e s y n t h a s e , a n d A 8 3 5 4 3 F arises w h e n a c e t a t e substitutes f o r p r o p i o n a t e at a n o t h e r point in t h e cycle. A 8 3 5 4 3 E results w h e n t h e starter unit f o r t h e polyketide s y n t h a s e is acetate rather t h a n propionate. In c o n t r a s t t o t h e m o n o c y c l i c macrolide antibiotics, b i o s y n t h e s i s of t h e A 8 3 5 4 3 t e t r a c y c l i c ring s y s t e m also r e q u i r e s t h e f o r m a t i o n of t h r e e i n t r a m o l e c u l a r c a r b o n - c a r b o n b o n d s in a d d i t i o n t o l a c t o n i z a t i o n . T w o of t h e s e c a r b o n - c a r b o n b o n d s a r e likely t o b e f o r m e d by a n i n t r a m o l e c u l a r Diels-Alder reaction a n d t h e third by a n intramolecular Aldol c o n d e n s a t i o n a n d s u b s e q u e n t d e h y d r a t i o n a n d r e d u c t i o n ( s e e F i g u r e 4 ) . T h e exact s e q u e n c e of t h e s e three ring-forming reactions (lactonization, Diels-Alder, Aldol) h a s not yet b e e n e s t a b l i s h e d . Intramolecular Diels-Alder reactions h a v e p r e v i o u s l y b e e n p r o p o s e d in t h e b i o s y n t h e s i s of o t h e r p o l y k e t i d e d e r i v e d f e r m e n t a t i o n products s u c h a s i k a r u g a m y c i n (30) a n d nargenicin (35). F u r t h e r s t u d i e s a r e n o w in p r o g r e s s t o d e t e r m i n e t h e s t e p s in t h e b i o s y n t h e s i s of A 8 3 5 4 3 that o c c u r after f o r m a t i o n of t h e long c h a i n fatty acid (36).


1 3

Acknowledgments T h e a u t h o r s e x t e n d their d e e p appreciation t o their n u m e r o u s c o l l e a g u e s within t h e r e s e a r c h laboratories of Eli Lilly a n d C o m p a n y a n d D o w E l a n c o w h o h e l p e d in t h e c o u r s e of t h i s project. W e e s p e c i a l l y t h a n k D. K. B a i s d e n , P. J . Baker, D. M. Berry, L. W . C r a n d a l l , L. C . C r e e m e r , V. M. Daupert, T. E. Eaton, T. K. Elzey, O. W . Godfrey, N. D. J o n e s , J . W . Martin, F. P. Mertz, a n d D. W . N o r t o n a n d t h e i r a s s o c i a t e s f o r t h e i r e x c e l l e n t technical assistance a n d support.

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R E C E I V E D April 27,

1992


Discovery, Isolation, and Structure Elucidation of a Family of

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