Molecular Design and Target Site Analysis in Fungicide Development

Chapter 14

Molecular Design and Target Site Analysis in Fungicide Development 1

2

Hugh D. Sisler and Nancy N . Ragsdale

Downloaded by UNIV LAVAL on July 11, 2016 | http://pubs.acs.org Publication Date: November 14, 1989 | doi: 10.1021/bk-1989-0413.ch014

1Department of Botany, University of Maryland, College Park, MD 20705 2Cooperative State Research Service, U.S. Department of Agriculture, Washington, DC 20250-2200 Antifungal compounds that specifically block sterol biosynthesis, microtubule assembly, succinate oxidation or polyketide melanin biosynthesis are discussed. These compounds are considered in respect to structure-activity relationships as they are influenced by target site characteristics and cellular biochemistry. Most fungicides developed for plant disease control before 1965 are multisite biochemical inhibitors that lack the chemical properties and biological specificity required for internal therapeutic action in higher plants. This group of plant protectants include such compounds as the inorganic copper fungicides, captan, chlorothalonil, dithiocarbamates and chlorinated quinones. Although these compounds are usually less effective than some of the newer systemic fungicides, they are inexpensive and have encountered very few problems with fungal resistance; therefore, they continue to play an important role in crop protection. They are particularly valuable for use in programs designed to prevent or manage problems of fungal resistance to biochemically specific fungicides. Since 1965 most of the new fungicides adopted for practical use are biochemically specific compounds that have local or systemic internal therapeutic activity. This development has extended the disease control spectrum of available fungicides and has made possible the use of a curative as well as a preventative strategy in disease control programs. However, not all has gone well because serious problems have been encountered with the development of fungal resistance to several of these fungicide mechanism groups (1). Various studies have indicated that most systemic fungicides act at a single target site and that most cases of fungicide resistance result from mutations that lead to loss of target affinity (2). There has been intense interest in devising ways to 0097-6156/89/0413-0198S06.00/0 © 1989 American Chemical Society

Magee et al.; Probing Bioactive Mechanisms ACS Symposium Series; American Chemical Society: Washington, DC, 1989.


14.

SISLER & RAGSDALE

Molecular Design and Target Site Analysis

c o u n t e r t h e s e r e s i s t a n c e problems. W h i l e u s i n g m i x t u r e s o r a l t e r n a t i n g d i f f e r e n t f u n g i c i d e mechanism groups a r e i m p o r t a n t measures f o r r e d u c i n g t h e r i s k o f r e s i s t a n c e development, t h e d e s i g n o f c h e m i c a l s t r u c t u r e s t o f i t t h e mutated t a r g e t s i t e s i n the r e s i s t a n t organisms and a c c e l e r a t e d development o f f u n g i c i d e s w i t h new modes o f a c t i o n a r e ways i n w h i c h t h e o r g a n i c c h e m i s t s , f u n g a l p h y s i o l o g i s t s , b i o c h e m i s t s and e n z y m o l o g i s t s c a n h e l p t o combat t h e f u n g i c i d e r e s i s t a n c e problems and improve c h e m i c a l c o n t r o l o f fungal diseases i n other respects. For e x i s t i n g f u n g i c i d e s , i t i s i m p o r t a n t t o know and c h a r a c t e r i z e t h e i r t a r g e t s i t e s n o t o n l y because t h i s knowledge c a n h e l p i n t h e d e s i g n o f new a n a l o g u e s t o c o u n t e r r e s i s t a n c e problems, b u t b e c a u s e i t may p r o v i d e fundamental i n f o r m a t i o n about t h e m o l e c u l a r d e s i g n n e c e s s a r y t o broaden the a n t i f u n g a l spectrum o f a f u n g i t o x i c mechanism group. The i d e n t i f i c a t i o n and c h a r a c t e r i z a t i o n o f t a r g e t s i t e s a r e c r i t i c a l l y i m p o r t a n t f o r programs aimed a t t h e d e l i b e r a t e d e s i g n o f new f u n g i c i d e mechanism groups because t h e s e programs a r e l i k e l y t o depend on m i m i c k i n g t h e s t r u c t u r e and c o n f o r m a t i o n o f a s u b s t r a t e , a h i g h energy o r t r a n s i t i o n s t a t e i n t e r m e d i a t e o r an a l l o s t e r i c regulator. S e l e c t i o n o f c a n d i d a t e t a r g e t s on w h i c h s e l e c t i v e t o x i c i t y i s t o be b a s e d c a n be a r i s k y endeavour b e c a u s e f a c t o r s such as u p t a k e , metabolism, and compensation c a n r e n d e r an e x c e l l e n t i n h i b i t o r a t the s u b c e l l u l a r t a r g e t s i t e i n e f f e c t i v e i n the i n t a c t t a r g e t organism. A r e a c t i o n o r pathway c h a r a c t e r i s t i c o f t h e f u n g a l pathogen b u t n o t o f t h e p l a n t h o s t o r mammals would o r d i n a r i l y be o f g r e a t i n t e r e s t i n t h e development o f s e l e c t i v e fungicides. C h i t i n b i o s y n t h e s i s i s o f t e n s u g g e s t e d as an example o f s u c h a pathway. On t h e o t h e r hand, most t a r g e t s o f e x i s t i n g f u n g i c i d e s a l s o o c c u r i n n o n - t a r g e t organisms and s t i l l , good selectivity exists. The f o l l o w i n g d i s c u s s i o n c o n c e r n i n g t h e mode o f a c t i o n o f s e l e c t e d f u n g i c i d e s now i n u s e w i l l s e r v e t o i l l u s t r a t e t h e importance o f some o f t h e p o i n t s mentioned above i n g u i d i n g t h e development o f new f u n g i c i d e s . Ergosterol Biosynthesis

Inhibitors

The t r i t e r p e n o i d pathway l e a d i n g t o t h e b i o s y n t h e s i s o f e r g o s t e r o l ( F i g u r e 1) i s t h e t a r g e t o f 3 f u n g i c i d e mechanism groups u s e d t o c o n t r o l f u n g a l pathogens o f p l a n t s and a n i m a l s . These groups consist of: (A) s q u a l e n e e p o x i d a s e i n h i b i t o r s ( a l l y l a m i n e s and t h i o c a r b a n i l a t e s ) which b l o c k c o n v e r s i o n o f s q u a l e n e t o 2,3oxidosqualene; (B) s t e r o l C-14 demethylase i n h i b i t o r s ( p i p e r a z i n e s , p y r i d i n e s , p y r i m i d i n e s , i m i d a z o l e s and t r i a z o l e s ) w h i c h b l o c k c o n v e r s i o n o f i n t e r m e d i a t e b t o c ( F i g u r e 1 ) ; and (C) sterol A — > A isomerase i n h i b i t o r s and/or s t e r o l A reductase i n h i b i t o r s ( m o r p h o l i n e s ) which b l o c k c o n v e r s i o n o f i n t e r m e d i a t e c t o d and/or f t o g ( F i g u r e 1 ) . I t i s a n i n t e r e s t i n g phenomenon t h a t t h e s e 3 f u n g i c i d e mechanism groups d i s c o v e r e d b y r o u t i n e s c r e e n i n g p r o c e d u r e s s h o u l d a l l p r o v e t o be i n h i b i t o r s o f t h e t r i t e r p e n o i d pathway t o e r g o s t e r o l . T h i s would s u g g e s t t h a t o t h e r enzymes i n t h e pathway l i k e 2 , 3 - o x i d o s q u a l e n e c y c l a s e o r s t e r o l C-4 demethylase might a l s o be good p r o s p e c t i v e t a r g e t s f o r f u n g i c i d e development.


199

PROBING BIOACTIVE MECHANISMS


200


14.

SISLER

& RAGSDALE


Thus f a r , compounds i n mechanism group A have n o t b e e n a d o p t e d f o r p r a c t i c a l use i n p l a n t p r o t e c t i o n , b u t a r e u s e d f o r the c o n t r o l o f f u n g a l pathogens o f humans ( 3 , 4 ) . On the o t h e r hand, compounds o f group B a r e u s e d t o c o n t r o l a v a r i e t y o f f u n g a l pathogens o f b o t h p l a n t s and a n i m a l s . Compounds i n group C a r e u s e d o n l y f o r the c o n t r o l o f p l a n t pathogens; however, t h e i r p r a c t i c a l d i s e a s e c o n t r o l s p e c t r u m i s n a r r o w e r t h a n t h a t o f the compounds i n group B. I n h i b i t o r s o f S t e r o l C-14 D e m e t h y l a t i o n . The g r e a t e s t v a r i e t y o f f u n g i c i d e s i n a s i n g l e mechanism group u s e d t o c o n t r o l pathogens o f p l a n t s and a n i m a l s a r e i n h i b i t o r s o f s t e r o l C-14 d e m e t h y l a s e , a cytochrome P-450 ( P - 4 5 0 - ^ ) enzyme. T y p i c a l l y , these s t e r o l d e m e t h y l a t i o n i n h i b i t o r s (DMI) c o n s i s t o f a p y r i m i d i n e , imidazole, t r i a z o l e or p y r i d i n e h e t e r o c y c l e attached to a s i n g l e l i p o p h i l i c s u b s t i t u e n t (5-2). An e x c e p t i o n t o t h i s g e n e r a l s t r u c t u r e i s t h a t o f the p i p e r a z i n e , t r i f o r i n e . Structures of representative compounds o f t h i s mechanism group a r e shown i n F i g u r e 2.


DM

These i n h i b i t o r s p r o d u c e type I I d i f f e r e n c e s p e c t r a w i t h the cytochrome P-450 monooxygenase enzymes w h i c h i n d i c a t e b i n d i n g o f an N atom o f the h e t e r o c y c l e a t the 6 t h c o o r d i n a t i o n p o s i t i o n o f the i r o n atom o f the P-450 heme p r o s t h e t i c group, the p o s i t i o n a t w h i c h 0 o r CO would n o r m a l l y b i n d ( F i g u r e 3 ) . The l i p o p h i l i c s u b s t i t u e n t o f the f u n g i c i d e b i n d s c o n c u r r e n t l y t o a n e a r b y l i p o p h i l i c r e g i o n o f the apoenzyme n o r m a l l y o c c u p i e d a t l e a s t i n p a r t by a 14a-methyl s t e r o l (8) ( F i g u r e 3 ) . Consequently, a f f i n i t y o f t h e s e i n h i b i t o r s f o r the P-450 enzyme i s d e t e r m i n e d by the b i n d i n g o f the N atom t o the heme i r o n and the l i p o p h i l i c s u b s t i t u e n t t o the apoenzyme ( 9 ) . Extensive s t r u c t u r a l - a c t i v i t y d a t a i n d i c a t e t h a t the l i p o p h i l i c s u b s t i t u e n t i s p r i m a r i l y r e s p o n s i b l e f o r the s e l e c t i v i t y o f DMI fungicides. The low s p e c i f i c i t y and open n a t u r e o f the P - 4 5 0 ^ apoenzyme i n the v i c i n i t y o f the heme p r o s t h e t i c group v e r y l i k e l y e x p l a i n s why i t i s p o s s i b l e t o have so much s t r u c t u r a l v a r i a b i l i t y among the many p y r i m i d i n e s , t r i a z o l e s , i m i d a z o l e s and p y r i d i n e s t h a t have b e e n s u c c e s s f u l l y t a r g e t e d on t h i s enzyme i n f u n g i . Structural s t u d i e s i n d i c a t e t h a t the environment and t o p o g r a p h y I n the v i c i n i t y o f the Oo b i n d i n g s i t e i s v e r y s i m i l a r i n a l l cytochrome P-450 enzymes ( 1 0 ) . I t may seem s u r p r i s i n g , t h e r e f o r e , t h a t P^"*®14DM f H i s much l e s s s e n s i t i v e t o k e t o c o n a z o l e and m i c o n a z o l e t h a n the same enzyme from y e a s t ( 1 1 ) . Some d e t a i l e d d i f f e r e n c e s o b v i o u s l y a c c o u n t f o r f u n g i c i d e s e l e c t i v i t y between t h e s e enzymes o f the same c l a s s and f u n c t i o n . The s e l e c t i v i t y o f DMI f u n g i c i d e s f o r the t a r g e t enzyme i n a n o n t a r g e t s p e c i e s has b e e n d e m o n s t r a t e d w i t h maize s t e r o l C-14 d e m e t h y l a s e . The DMI f u n g i c i d e s t r i a d i m e f o n and p r o p i c o n a z o l e a r e moderate ( I C ^ Q 8 uH) o r good ( I C C Q 2 /iM) i n h i b i t o r s o f maize P - 4 5 0 - j ^ , whereas the e x p e r i m e n t a l DMI f u n g i c i d e s l a b 158241F and l a b 170250F a r e h i g h l y p o t e n t i n h i b i t o r s o f t h i s enzyme w i t h r e s p e c t i v e I C v a l u e s o f 0.3 and 0.05 /iM (12.). These o b s e r v a t i o n s i n d i c a t e t h a t w h i l e t h e r e may be much s i m i l a r i t y i n v a r i o u s cytochrome P-450 enzymes, s m a l l d i f f e r e n c e s i n s t r u c t u r a l d e s i g n s o f DMI c a n be u s e d t o e x p l o i t t h o s e t h a t do e x i s t f o r p u r p o s e s o f f u n g a l d i s e a s e c o n t r o l . However, s i d e e f f e c t s a r e known t o o c c u r w i t h the use o f t h e s e fungicides. One commonly o b s e r v e d i n c o n t r o l l i n g p l a n t d i s e a s e s 2

D M

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D M

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Imidazole

:


Cl-^

^CH-0-CH -CH=CH 2

C 12^25

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c=o I N—!J

Imazalil

l-Dodecylimidazole

Prochloraz

F

Triazolec-Qo

CH

F

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-Q_ ? i

C H

,

Triadimenol

Propiconazole n I

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Flusilazol

1

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CI —

CH, f~\

r~\

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^ N - C H - C H

2

CH,

Myclobutanil I traconazole :

Pyrimidine, Pyridine, Piperazine

OCH,

CHO

CHO

NH Nv^N

Triarimol F i g u r e 2.

N

V

^ N

Fenarimol

6

CC C HL- N

^~CH

Pyrifenox Triforine

S t r u c t u r e s o f s t e r o l C-14 d e m e t h y l a t i o n i n h i b i t o r s .



14.

SISLER & RAGSDALE

Molecular Design andTarget Site Analysis

w i t h DMI i s the growth r e g u l a t o r e f f e c t (13) t h a t r e s u l t s from i n h i b i t i o n o f the P-450 enzyme i n v o l v e d i n o x y g e n a t i o n o f the C-19 m e t h y l group o f kaur-16-ene (14), a p r e c u r s o r o f g i b b e r e l l i c a c i d . When h i g h l e v e l s o f k e t o c o n a z o l e a r e u s e d m e d i c i n a l l y , t h e r e may be i n t e r f e r e n c e w i t h the cytochrome P-450 1 7 , 2 0 - l y a s e i n v o l v e d i n the pathway o f c o n v e r s i o n o f 17a-hydroxy p r o g e s t e r o n e t o t e s t o s t e r o n e (15). W i t h c a r e f u l s e l e c t i o n o f f u n g i c i d e s t r u c t u r e and p r o p e r r e g u l a t i o n o f dosage, s u c h s i d e e f f e c t s c a n l a r g e l y be a v o i d e d . U s i n g s t r u c t u r e - a c t i v i t y c o r r e l a t i o n s and a n a l y s e s o f s t e r o c h e m i s t r y , l i p o p h i l i c i t y , c o n f o r m a t i o n and o t h e r p h y s i o c h e m i c a l p a r a m e t e r s , v a r i o u s i n v e s t i g a t o r s (16-19) have c o n s t r u c t e d cytochrome P-450 b i n d i n g - s i t e models and have a t t e m p t e d to p r e d i c t i n h i b i t o r s t r u c t u r e s with optimal f u n g i c i d a l a c t i v i t y . I t i s a p p a r e n t from e x a m i n i n g s t r u c t u r e s o f s t e r o l C-14 d e m e t h y l a t i o n i n h i b i t o r s ( F i g u r e 2) t h a t many would n o t f u l l y occupy the s t e r o l b i n d i n g s i t e on the apoenzyme and t h a t some s u c h as i t r a c o n a z o l e , p r o b a b l y e x t e n d o u t s i d e the s t e r o l b i n d i n g s i t e . Moreover, the r e g i o n s o f c o n t a c t w i t h the apoenzyme must d i f f e r a p p r e c i a b l y among t h e s e d e r i v a t i v e s . S t e r i c f i t comparisons by computer g r a p h i c s o f d i n i c o n a z o l e w i t h l a n o s t e r o l when the N-4 atom o f the f u n g i c i d e i s i n c l o s e p r o x i m i t y o f the l a n o s t e r o l C-14 m e t h y l group, show o v e r l a p o f t h e s e m o l e c u l e s m a i n l y i n the r e g i o n o f the "D" r i n g and s i d e c h a i n o f the s t e r o l ( 1 8 ) . U s i n g computer g r a p h i c p r o c e d u r e s i t has been p o s s i b l e t o a l i g n the f u n g i c i d e RRp a c l o b u t r a z o l so t h a t i t has good o v e r l a p w i t h the 24-methylene24,25-dihydrolanosterol s u b s t r a t e (17). Fuj imoto e t a l . (19) have r e c e n t l y c o n s t r u c t e d a f o l d e d c o n f o r m a t i o n model o f m y c l o b u t a n i l t h a t resembles a p o r t i o n o f the a c t i v e c o n f o r m a t i o n o f the l a n o s t e r o l m o l e c u l e . Advancement o f t h e s e t e c h n i q u e s may e v e n t u a l l y g u i d e s y n t h e s i s o f e n t i r e l y new f u n g i c i d e s r a t h e r t h a n the m o d i f i c a t i o n o r improvement o f f u n g i c i d e s a l r e a d y d i s c o v e r e d as i s now the c a s e . As mentioned e a r l i e r , the apoenzyme r e g i o n s u r r o u n d i n g the heme p r o s t h e t i c group o f cytochrome P-450 enzymes i s one o f r e l a t i v e l y low s p e c i f i c i t y . However, t h e r e a r e a p p a r e n t l y significant differences i n P - 4 5 0 ^ even among v a r i o u s f u n g i as s u g g e s t e d by the o p t i m a l i n h i b i t o r y s t r u c t u r e o f DMI f o r d i f f e r e n t species. W h i l e most s i n g l e gene m u t a t i o n s a f f e c t i n g t h i s r e g i o n o f the enzyme would p r o b a b l y n o t r e s u l t i n d r a s t i c l o s s o f f u n g i c i d e e f f e c t i v e n e s s and s t i l l a l l o w the o r g a n i s m t o r e t a i n good f i t n e s s , the change i n s e n s i t i v i t y might be as g r e a t as t h a t p r e s e n t l y e x i s t i n g among c e r t a i n f u n g a l s p e c i e s . I t seems l i k e l y , t h e r e f o r e , t h a t i f a t a r g e t change l e a d s t o d e c r e a s e d s e n s i t i v i t y , t h i s c o u l d be c o u n t e r e d by a DMI f u n g i c i d e p r e s e n t l y a v a i l a b l e o r one t h a t c o u l d be r e a d i l y s y n t h e s i z e d . Quantitative structure a c t i v i t y r e l a t i o n s h i p s s h o u l d be h e l p f u l i n d e s i g n i n g the p r o p e r d e r i v a t i v e s t o combat s u c h t a r g e t s i t e r e s i s t a n c e . Development o f r e s i s t a n c e t o DMI has b e e n l e s s r a p i d t h a n e x p e c t e d , p o s s i b l y b e c a u s e o f the c h a r a c t e r i s t i c s o f the P-450 t a r g e t s i t e d e s c r i b e d above. With t h e s e f u n g i c i d e s , i t may be the e f f l u x system d e s c r i b e d by DeWaard and Van N i s t e l r o o y (20) o r some o t h e r mechanism r a t h e r t h a n t a r g e t s i t e change t h a t w i l l p r e s e n t the g r e a t e s t h a z a r d f o r p r a c t i c a l resistance. D M


203

204


Inhibitors of Sterol A — > A Isomerization. I n i t i a l mode o f a c t i o n s t u d i e s i n d i c a t e d that the morpholine f u n g i c i d e tridemorph blocked s t e r o l A ---> A i s o m e r i z a t i o n (21-21) o r s t e r o l A r e d u c t i o n (24). S u b s e q u e n t l y , i t was shown t h a t m o r p h o l i n e s may b l o c k b o t h r e a c t i o n s o r o n l y t h e former, d e p e n d i n g on t h e o r g a n i s m and m o r p h o l i n e d e r i v a t i v e i n v o l v e d ( 2 1 ) . D e t a i l e d mode o f a c t i o n s t u d i e s have f o c u s e d m a i n l y on i n h i b i t i o n o f t h e A - --> A i s o m e r i z a t i o n by tridemorph. An a n a l y s i s o f t h e mechanism o f sterol A — > A i s o m e r i z a t i o n (21) i n d i c a t e s t h a t t h e e n z y m a t i c r e a c t i o n i n v o l v e s f i r s t an a - p r o t o n a t i o n o f t h e A d o u b l e bond l e a d i n g t o a h i g h energy i n t e r m e d i a t e (HEI) w i t h a c a r b o c a t i o n a t C-8, f o l l o w e d b y a n e l i m i n a t i o n o f t h e C-7 p r o t o n t o g i v e a A d o u b l e bond ( F i g u r e 4 ) . Studies i n the l a b o r a t o r y o f Benveniste (27-29) have l e d t o t h e p r o p o s a l t h a t s i n c e t h e pKa o f N - s u b s t i t u t e d m o r p h o l i n e s l i k e t r i d e m o r p h i s between 7 and 8, some m o r p h o l i n i u m c a t i o n s e x i s t a t p h y s i o l o g i c a l pH v a l u e s and t h e s e c a n a c t as an i n h i b i t o r y c a r b o c a t i o n i c mimics o f t h e s t e r o l HEI i n v o l v e d i n t h e isomerase r e a c t i o n . T h i s r e a s o n i n g c a n be under s t o o d b y comparing t h e s t r u c t u r e o f t h e s t e r o l c a r b o c a t i o n i c HEI w i t h t r i d e m o r p h and an 8 - a z a d e c a l i n l i k e t h e d e r i v a t i v e shown i n 7


7

F i g u r e 4 which i s a potent i n h i b i t o r o f A > A isomerase (21). T a t o n e t a l . (28) have p o i n t e d o u t t h a t s e v e r a l r e a c t i o n s i n the s t e r o l b i o s y n t h e t i c pathway i n v o l v e c a t i o n i c HE ( h i g h e n e r g y ) o r t r a n s i t i o n s t a t e (TS) i n t e r m e d i a t e s w i t h a h i g h d i p o l e moment. Some o f t h e s e a r e i n d i c a t e d i n T a b l e I . These i n t e r m e d i a t e s c a n s e r v e as models f o r d e s i g n o f s t a b l e a n a l o g u e s t h a t might be effective fungicides. Table I.

Target

Enzymes, TS o r HE I n t e r m e d i a t e s and C o r r e s p o n d i n g I n h i b i t o r Analogues

Target Enzyme

Intermediate

2,3-0xidosqualene cyclase

TS e l e c t r o n deficient C-2 s q u a l e n e

Sterol

HE C-8 c a r b o c a t i o n i c sterol

J

--->

7

A isomerase

Sterol C-24 m e t h y l transferase

HE C-25 carbocationic sterol

Inhibitor Analogue 2-amino, 2N-oxide(22) and 2,3-imino(30) squalene Cationic

morpholines(21,21) and 8 a z a d e c a l i n s (21) C-25 c a t i o n i c s t e r o l s (amine, sulfonium, arsonium groups) (22)

Sterol A reductase

HE C-15 carbocationic sterol

Cationic morpholines(31)



14.

SISLER & RAGSDALE


F i g u r e 3. Diagrams p o r t r a y i n g t h e i n t e r a c t i o n o f 14a-methyl s t e r o l (A) and a s t e r o l C-14 d e m e t h y l a t i o n i n h i b i t o r (B) w i t h cytochrome - 4 5 G ^ ™ i n t h e r e g i o n o f t h e heme p r o s t h e t i c group. An atom o f oxygen has a l r e a d y been i n s e r t e d i n t o t h e s t e r o l C-14 m e t h y l group ( A ) . A n i t r o g e n atom o f t h e f u n g i c i d e h e t e r o c y c l e i s shown i n t e r a c t i n g w i t h t h e heme p r o s t h e t i c group o f t h e enzyme ( B ) . p

Tridemorph

8-Azcidecalin

F i g u r e 4. Pathway showing mechanism o f s t e r o l A > A i s o m e r i z a t i o n and s t r u c t u r a l s i m i l a r i t y o f p r o t o n a t e d t r i d e m o r p h and an 8 - a z a d e c a l i n t o t h e C-8 c a r b o c a t i o n i c HEI s t e r o l .


205

206



The 2 , 3 - o x i d o s q u a l e n e c y c l i z a t i o n r e a c t i o n i s one o f p a r t i c u l a r i n t e r e s t as a f u n g i c i d e t a r g e t s i n c e i t i s q u i t e c o m p l i c a t e d and i n v o l v e s more t h a n one TS o r HE i n t e r m e d i a t e . I t i s s u r p r i s i n g t h a t more i n h i b i t o r s o f t h e enzyme c a t a l y z i n g t h i s r e a c t i o n have n o t been s y n t h e s i z e d . A point o f i n t e r e s t i s the u n u s u a l o v e r l a p o f s p e c i f i c i t y o f 1 - d o d e c y c l i m i d a z o l e ( F i g u r e 2) as an i n h i b i t o r o f s t e r o l C-14 d e m e t h y l a t i o n and 2 , 3 - o x i d o s q u a l e n e c y c l i z a t i o n (12,32). The s i m p l e a l i p h a t i c s u b s t i t u e n t o f t h i s i m i d a z o l e may a l l o w t h e compound t o a c t a s a TS a n a l o g u e o f 2,3o x i d o s q u a l e n e and a l s o t o b i n d t o t h e s t e r o l C-14 demethylase and i n t e r a c t w i t h t h e heme i r o n . A s i m i l a r o v e r l a p o f s p e c i f i c i t y apparently e x i s t s a l s o with the morpholines f o r s t e r o l A > A i s o m e r a s e and s t e r o l A reductase (21). B e n z i m i d a z o l e s and Phenylcarbamates B e n z i m i d a z o l e s came i n t o use as a g r i c u l t u r a l f u n g i c i d e s i n t h e e a r l y 1970's. These compounds c o n t r o l a r e l a t i v e l y b r o a d spectrum o f f u n g i , b u t have e n c o u n t e r e d many s e r i o u s problems w i t h t h e development o f f u n g a l r e s i s t a n c e . Benomyl, c a r b e n d a z i m and t h i a b e n d a z o l e ( F i g u r e 5) have been t h e main f u n g i c i d e s u s e d f o r p l a n t d i s e a s e c o n t r o l . S i n c e t h e a c t i v i t y o f benomyl c a n be a t t r i b u t e d t o t h e c a r b e n d a z i m formed as a r e s u l t o f t h e l o s s o f t h e b u t y l c a r b a m o y l m o i e t y (33), c a r b e n d a z i m i s t h e s t r u c t u r e o f b a s i c i n t e r e s t i n regard to target s i t e i n t e r a c t i o n . Benzimidazoles s p e c i f i c a l l y i n t e r f e r e with the formation o f m i c r o t u b u l e s which f u n c t i o n i n a v a r i e t y o f c e l l u l a r p r o c e s s e s i n c l u d i n g m i t o s i s ( 3 4 ) . M i c r o t u b u l e s a r e formed under a p p r o p r i a t e c o n d i t i o n s b y assembly o f t u b u l i n , a h e t e r o d i m e r i c p r o t e i n o f which the s u b u n i t s , a r e u s u a l l y d e s i g n a t e d as a- and tubulin. B i o c h e m i c a l and g e n e t i c s t u d i e s have c l e a r l y shown t h a t t h e t a r g e t s i t e o f c a r b e n d a z i m and r e l a t e d b e n z i m i d a z o l e f u n g i c i d e s i s t h e c o l c h i c i n e b i n d i n g s i t e on t u b u l i n , and t h a t t h e p-tubulin subunit i s u s u a l l y the primary determinant o f b i n d i n g a f f i n i t y (34). I n regard t o t a r g e t s i t e s e l e c t i v i t y o f benzimidazoles, carbendazim i s a t l e a s t 300 times more e f f e c t i v e i n p r e v e n t i n g assembly o f y e a s t t u b u l i n t h a n p i g b r a i n t u b u l i n ( H ) . On t h e o t h e r hand, s u b s t i t u t i o n o f t h e benzene r i n g o f c a r b e n d a z i m as i n n o c o d a z o l e l e a d s t o about a 200 f o l d i n c r e a s e i n e f f e c t i v e n e s s f o r b l o c k i n g assembly o f p i g b r a i n t u b u l i n b u t o n l y a 4 - f o l d i n c r e a s e f o r b l o c k i n g assembly o f y e a s t t u b u l i n . Thus, t h i s s u b s t i t u e n t l a r g e l y e l i m i n a t e s t h e s e l e c t i v i t y shown b y c a r b e n d a z i m f o r t h e y e a s t and the mammalian t a r g e t s i t e s . W i t h t h e emergence o f f u n g a l r e s i s t a n c e t o b e n z i m i d a z o l e s , t h e r e h a s been i n t e n s e i n t e r e s t i n ways t o c o u n t e r t h i s problem. S i n c e most c a s e s o f b e n z i m i d a z o l e r e s i s t a n c e a r e b e l i e v e d t o be due t o changes i n t u b u l i n a f f i n i t y f o r t h e s e f u n g i c i d e s , one a p p r o a c h to combatting r e s i s t a n c e i s t o design analogues w i t h h i g h a f f i n i t y f o r t h e mutated t a r g e t s i t e s . The p o t e n t i a l f o r s u c c e s s o f t h i s a p p r o a c h was s u g g e s t e d by t h e o b s e r v a t i o n s o f L e r o u x and G r e d t (36) t h a t b e n z i m i d a z o l e r e s i s t a n t mutants o f Botrytis cinerea and Penicillium expansum a r e much more s e n s i t i v e t o p h e n y l carbamate h e r b i c i d e s than the benzimidazole s e n s i t i v e s t r a i n s o f these f u n g i . F o l l o w i n g t h i s o b s e r v a t i o n , i t was shown t h a t m e t h y l N-(3,5-



14.

SISLER & RAGSDALE


d i c h l o r o p h e n y l ) carbamate (MDPC) and i s o p r o p y l N-(3,4d i e t h o x y p h e n y l ) carbamate ( d i e t h o f e n c a r b ) a r e h i g h l y t o x i c t o b e n z i m i d a z o l e carbamate r e s i s t a n t s t r a i n s o f B. cinerea, but not to b e n z i m i d a z o l e carbamate s e n s i t i v e s t r a i n s (12,18). These compounds ( F i g u r e 5) a l s o show l i t t l e p h y t o t o x i c i t y . M i x t u r e s o f b e n z i m i d a z o l e carbamates and d i e t h o f e n c a r b a r e p r e s e n t l y b e i n g u s e d i n F r a n c e t o c o n t r o l Botrytis d i s e a s e o f grape i n r e g i o n s where b e n z i m i d a z o l e carbamates a l o n e a r e no l o n g e r e f f e c t i v e . The e x t e n t t o w h i c h t h i s a p p r o a c h c a n be u s e d t o c o n t r o l b e n z i m i d a z o l e resistance i n other fungal species i s unclear. I t i s known, f o r example, t h e MDPC does n o t c o n t r o l some b e n z i m i d a z o l e r e s i s t a n t s t r a i n s o f Cercospora beticola (39). Whether a p r a c t i c a l p r o b l e m from d o u b l e r e s i s t a n c e t o t h e p a i r e d i n h i b i t o r s o r d i f f i c u l t i e s w i t h t o x i c i t y t o n o n t a r g e t organisms p r o v e t o be l i m i t i n g f a c t o r s must a w a i t f u r t h e r e x p e r i e n c e . R e c e n t s t u d i e s have shown t h a t a s t r a i n o f Neurospora crassa r e s i s t a n t t o b e n z i m i d a z o l e carbamates b u t s e n s i t i v e t o t h e p h e n y l c a r b a m a t e s , MDPC and d i e t h o f e n c a r b , r e s u l t e d from a m u t a t i o n a l change i n t h e t u b u l i n gene (4Q). The change o f a s i n g l e amino a c i d from g l u t a m i c a c i d t o g l y c i n e i n tubulin a p p a r e n t l y a c c o u n t e d f o r b e n z i m i d a z o l e r e s i s t a n c e i n t h i s mutant strain. R e v e r t a n t s o f t h i s mutant were r e s i s t a n t t o d i e t h o f e n c a r b b u t e x h i b i t e d t h e same b e n z i m i d a z o l e s e n s i t i v i t y as t h e w i l d t y p e . E x t e n s i v e a n a l y s e s o f s t r u c t u r e a c t i v i t y r e l a t i o n s h i p s (SAR) o f f u n g i c i d a l m e t h y l N-phenyl carbamates i n d i c a t e t h a t r e c e p t o r r e g i o n s c o r r e s p o n d i n g t o t h e 0 and M - s u b s t i t u e n t s on t h e benzene r i n g a r e h i g h l y h y d r o p h o b i c whereas t h a t f o r t h e P s u b s t i t u e n t i s moderately hydrophobic (38). S t e r i c i n t e r a c t i o n o f t h e £fs u b s t i t u e n t w i t h t h e r e c e p t o r as w e l l as h y d r o g e n b o n d i n g b y t h e P s u b s t i t u e n t a p p e a r e d t o be n e c e s s a r y f o r h i g h a c t i v i t y . A p p r e c i a b l e m o d i f i c a t i o n o f p o t e n c y o f p h e n y l c a r b a m a t e s c o u l d be p r o d u c e d by c h a n g i n g t h e n a t u r e o f t h e a l c o h o l o f t h e e s t e r m o i e t y . The i s o p r o p y l e s t e r f o r example was h i g h l y f u n g i t o x i c whereas t h e i s o b u t y l e s t e r showed v e r y low t o x i c i t y ( 4 1 ) . S t u d i e s o f t h e SAR o f b e n z i m i d a z o l e s and p h e n y l c a r b a m a t e s have shown t h a t t a r g e t s i t e s e l e c t i v i t y between f u n g a l s t r a i n s and among f u n g a l s p e c i e s , h i g h e r p l a n t s and mammals c a n be a f f e c t e d b y r a t h e r s i m p l e s t r u c t u r a l changes. This not only points out opportunities f o r c o m b a t t i n g f u n g a l r e s i s t a n c e t o t h e s e d e r i v a t i v e s and f o r s e l e c t i v e c o n t r o l o f various pests, b u t a l s o i n d i c a t e s the p o t e n t i a l h a z a r d f o r n o n t a r g e t s p e c i e s t h a t might r e s u l t b e c a u s e o f the c l o s e s i m i l a r i t y o f t u b u l i n among d i v e r s e e u k a r y o t i c o r g a n i s m s . Carboxamides Carboxamides a r e a group o f f u n g i c i d e s t h a t c o n t r o l d i s e a s e s c a u s e d by B a s i d i o m y c e t e type f u n g i ( 4 2 ) . The b e s t known member o f t h i s group i s c a r b o x i n ( F i g u r e 6 ) . Carboxamides s p e c i f i c a l l y b l o c k membrane bound s u c c i n a t e - u b i q u i n o n e oxidoreductase a c t i v i t y i n the m i t o c h o n d r i a l e l e c t r o n t r a n s p o r t c h a i n (43,44). The c a r b o x i n r e c e p t o r i n t h e s u c c i n i c dehydrogenase complex (SDC) i s b e l i e v e d t o be t h e i r o n - s u l f u r c l u s t e r S^ complexed w i t h s m a l l coenzyme Q b i n d i n g p o l y p e p t i d e ( s ) i n a p h o s p h o l i p i d environment (45,46).


207


208


fi Carbendazim

i

C

Nocodazole NH-(CH ) CH 2

3

H

2 5° Diethofencarb

3

o=c

Benomyl

Thiabendazole

MDPC

F i g u r e 5. S t r u c t u r e s o f b e n z i m i d a z o l e and p h e n y l c a r b a m a t e fungicides.

0 Carboxin

F i g u r e 6.

Structure of carboxin.


C H

3


14.

SISLER & RAGSDALE

Molecular Design andTarget Site Analyst

Extensive studies o f s t r u c t u r a l - a c t i v i t y r e l a t i o n s h i p s o f carboxamides have been made, p a r t i c u l a r l y i n r e g a r d t o t h e m o l e c u l a r d e s i g n s t h a t i n h i b i t c a r b o x i n r e s i s t a n t mutants o f Ustilago maydis (45,47,48). These i n v e s t i g a t i o n s have shown t h a t i t i s p o s s i b l e t o produce a n a l o g s o f c a r b o x i n w h i c h a r e good i n h i b i t o r s o f t h e mutated s u c c i n i c dehydrogenase complex (SDC) i n c a r b o x i n r e s i s t a n t s t r a i n s t h a t a r e a l s o good i n h i b i t o r s o f growth o f t h e s e s t r a i n s . There a r e i n d i c a t i o n s t h a t w i t h p r o p e r s t r u c t u r a l d e s i g n , s u c c i n i c dehydrogenase i n h i b i t o r s (SDI) c a n be p r o d u c e d w h i c h c o n t r o l f u n g a l pathogens i n m y c o l o g i c a l groups o t h e r t h a n B a s i d i o m y c e t e s (49,50). I t i s p u z z l i n g , t h e r e f o r e , why t h e p r a c t i c a l u s e f u l n e s s o f c a r b o x a n i l i d e s remains e s s e n t i a l l y c o n f i n e d t o t h e c o n t r o l o f B a s i d i o m y c e t e type f u n g i . Whether t h i s i s due t o inadequate t a r g e t s i t e a f f i n i t y , c e l l u l a r p e r m e a b i l i t y , metabolism o r o t h e r f a c t o r s i s n o t known. There a p p e a r s t o be a n i n t e r e s t i n g and p e r h a p s a n e c o n o m i c a l l y r e w a r d i n g c h a l l e n g e t o d e s i g n e f f e c t i v e SDI f o r n o n B a s i d i o m y c e t e f u n g i , b u t i t i s p o s s i b l e t h a t b l o c k i n g o f the SDC complex may n o t have t h e same consequences i n t h e s e f u n g i as i n t h e s e n s i t i v e B a s i d i o m y c e t e s . While i t i s b e l i e v e d t h a t p r i m a r y c e l l u l a r t o x i c i t y o f SDC i n h i b i t o r s r e s u l t s from f a i l u r e o f the c i t r i c a c i d c y c l e t o o p e r a t e t o produce ATP a n d b i o s y n t h e t i c i n t e r m e d i a t e s (51), t h e d e s t r u c t i o n o f m i t o c h o n d r i a l membranes and enzymes b y r e a c t i v e oxygen r a d i c a l s r e s u l t i n g from t h e b l o c k i n g o f the SDC c o u l d a c t u a l l y be t h e p r i m a r y t o x i c mechanism i n s e n s i t i v e Basidiomycetes. I f t h i s i s the case, then f u n g i o f other m y c o l o g i c a l groups may be l e s s s e n s i t i v e because t h e y a r e more c a p a b l e t h a n B a s i d i o m y c e t e s o f e l i m i n a t i n g t h e s e r e a c t i v e oxygen products. E v i d e n c e f o r a r a d i c a l type o f t o x i c mechanism i s s u g g e s t e d by t h e o b s e r v a t i o n t h a t c a r b o x i n t r e a t m e n t l e a d s t o s t r u c t u r a l damage t o m i t o c h o n d r i a (52) a n d d e s t r u c t i o n o f t h e Sg c e n t e r o f t h e SDC ( 4 4 ) . M e l a n i n B i o s y n t h e s i s I n h i b i t o r s . Among t h e most d e s i r a b l e t a r g e t s f o r s e l e c t i v e f u n g i t o x i c a c t i o n are those u n i q u e l y a s s o c i a t e d w i t h f u n g a l p a t h o g e n i c i t y such as t h e p o l y k e t i d e pathway t o m e l a n i n i n the fungus Pyricularia oryzae which c a u s e s r i c e b l a s t d i s e a s e . T h i s pathway o f s e c o n d a r y m e t a b o l i s m ( F i g u r e 7 ) , w h i c h i s n o t r e q u i r e d f o r growth o f t h e fungus as a s a p r o p h y t e , i s i n d u c e d i n the a p p r e s s o r i a ( p e n e t r a t i o n s t r u c t u r e s ) formed b y germ tubes o f spores on a p l a n t epidermal s u r f a c e o r other b a r r i e r s . This pathway l e a d s t o a p p r e s s o r i a l w a l l m e l a n i z a t i o n a n d i s n e c e s s a r y f o r a p p r e s s o r i a l p e n e t r a t i o n o f p l a n t e p i d e r m a l w a l l s b y P. o r y z a e (53.54) and Colletotrichum s p e c i e s (55). Genetic o r chemical b l o c k s i n t h e pathway t h a t r e s u l t i n t h e i n h i b i t i o n o f m e l a n i n biosynthesis, prevent a p p r e s s o r i a l penetration o f p l a n t epidermal barriers. A g e n e t i c o r a c h e m i c a l b l o c k (by c e r u l e n i n ) p r i o r t o pentaketide c y c l i z a t i o n leads to albino appressoria that f a i l t o p e n e t r a t e e p i d e r m a l o r c e l l u l o s i c b a r r i e r s (56.57). Melanization and p e n e t r a t i o n c a p a c i t y i n P. oryzae c a n be r e s t o r e d i n t h e s e a p p r e s s o r i a b y a d d i n g t h e m e l a n i n p r e c u r s o r s s c y t a l o n e , vermelone o r 1 , 8 - d i h y d r o x y n a p h t h a l e n e (1,8-DHN) ( 5 7 ) . The compounds shown i n F i g u r e 8, t h a t have been d e v e l o p e d f o r t h e c o n t r o l o f r i c e b l a s t disease, b l o c k NADPH dependent r e d u c t a s e r e a c t i o n s i n t h e pathway a t s i t e s i n d i c a t e d b y a s t e r i s k s i n F i g u r e 7. Y e l l o w o r r e d d i s h


209

210


OH

0

0

OH


Acetate

2 - H J

Pentaketide

1,3,6,8-THN

1,3,8-THN

Scytalone

1,8-DHN

Vermetone

Melanin

0 Flaviolin

OH

OH

4-Hydroxy scytalone

4,6,8-DTN

F i g u r e 7. P o l y k e t i d e pathway o f f u n g a l m e l a n i n b i o s y n t h e s i s showing b r a n c h pathways l e a d i n g t o s y n t h e s i s o f 2 - h y d r o x y j u g l o n e (2HJ), f l a v i o l i n and o t h e r shunt m e t a b o l i t e s .

CI

CI CI,

CH OH 2

CI

CI

CH,

CI CI

Fthalide

PCBA

Chlobenthiazone

CH

0 Tricyclazole

Pyroquilon

C H

PP 3 8 9

F i g u r e 8. S t r u c t u r e s o f compounds t h a t b l o c k r e d u c t a s e r e a c t i o n s i n t h e p o l y k e t i d e pathway t o m e l a n i n a t p o i n t s i n d i c a t e d b y a s t e r i s k s i n F i g u r e 7.


3


14.

SISLER & R A G S D A L E


brown pigments a r e formed i n s t e a d o f b l a c k m e l a n i n . Vermelone and 1,8-DHN, b u t n o t s c y t a l o n e , c a n r e s t o r e m e l a n i z a t i o n and p a r t i a l l y r e s t o r e p e n e t r a t i o n c a p a c i t y i n a p p r e s s o r i a t r e a t e d w i t h these compounds ( 5 7 ) . U t i l i z i n g knowledge c o n c e r n i n g t h e s t r u c t u r a l r e l a t i o n s h i p o f known m e l a n i n b i o s y n t h e s i s i n h i b i t o r s (MBI) t o 1,3,8trihydroxynaphthalene (1,3,8-THN), t h e s u b s t r a t e o f a t a r g e t enzyme i n t h i s pathway, Omata e t a l . (58) have r e c e n t l y d e s i g n e d p h t h a l a z i n e d e r i v a t i v e s t h a t a r e good MBI and r i c e b l a s t c o n t r o l agents. W h i l e i t i s known t h a t b l o c k i n g o f t h e m e l a n i n b i o s y n t h e t i c pathway i n P. oryzae by a n t i p e n e t r a n t s l i k e t r i c y c l a z o l e , f t h a l i d e , p y r o q u i l o n and c e r u l e n i n , r e s u l t s i n f a i l u r e o f a p p r e s s o r i a l p e n e t r a t i o n , i t remains u n c l e a r why t h i s i s s o . Among t h e v a r i o u s c a u s e s t h a t have been s u g g e s t e d a r e l a c k o f a p p r e s s o r i a l w a l l r i g i d i t y (59), p o o r a d h e s i o n (60) o r t h e a c c u m u l a t i o n o f t o x i c p o l y k e t i d e shunt p r o d u c t s such as 2 - h y d r o x y j u g l o n e (2HJ) ( 6 1 ) . C o n t r o l o f a major f u n g a l d i s e a s e o f p l a n t s by MBI i l l u s t r a t e s how s p e c i f i c knowledge c o n c e r n i n g f u n g a l p a t h o g e n i c i t y c a n be e x p l o i t e d f o r plant protection. A b e t t e r understanding o f fungal p a t h o g e n i c i t y mechanisms and o f h o s t - p a t h o g e n i n t e r a c t i o n s w i l l no doubt r e v e a l o t h e r s p e c i f i c t a r g e t s t h a t c a n be u s e d t o c o n t r o l fungal diseases. However, t h e r e may be l i m i t e d economic v a l u e o f i n h i b i t o r s of a p a r t i c u l a r target i f i t i s r e s t r i c t e d to a single s p e c i e s o r a v e r y narrow m y c o l o g i c a l spectrum. T h i s i s the case w i t h t h e MBI. The p o l y k e t i d e pathway t o m e l a n i n a p p e a r s t o be c r i t i c a l o n l y f o r p a t h o g e n i c i t y o f P. oryzae and Colletotrichum s p e c i e s and a t p r e s e n t , t h e MBI a r e u s e d o n l y f o r c o n t r o l o f r i c e b l a s t d i s e a s e c a u s e d by P. oryzae. Conclusions We have examined i n f o r m a t i o n on m o l e c u l a r d e s i g n i n r e l a t i o n t o t a r g e t s i t e f i t and a n t i f u n g a l a c t i v i t y . T h i s knowledge has a i d e d i n t h e development o f d e r i v a t i v e s t h a t b r o a d e n t h e a n t i f u n g a l s p e c t r u m o f e x i s t i n g f u n g i c i d e groups t o i n c l u d e s p e c i e s o r r e s i s t a n t s t r a i n s n o t a d e q u a t e l y c o n t r o l l e d by t h e o r i g i n a l compounds d i s c o v e r e d by c o n v e n t i o n a l s c r e e n i n g p r o c e d u r e s . A c c e l e r a t e d development o f f u n g i c i d e s b a s e d on r a t i o n a l d e s i g n s w i l l be h i g h l y dependent on t h e a b i l i t y t o i d e n t i f y and p r e c i s e l y analyze the s t r u c t u r a l f e a t u r e s o f promising t a r g e t sites. H i g h p o t e n c y i n t h e l a b o r a t o r y c a n o n l y be s u g g e s t i v e o f p r a c t i c a l success. These f u n g i c i d e s w i l l need t o e x h i b i t p r o p e r t i e s o f s t a b i l i t y , s e l e c t i v i t y and m o b i l i t y i n t h e p l a n t s i m i l a r t o t h o s e o f s u c c e s s f u l f u n g i c i d e s d i s c o v e r e d by conventional proecedures.

Literature Cited 1. 2.

Dekker, J . In Modern Selective Fungicides; Lyr, H . , Ed.; Longman: London, 1987; pp 39-52. Sisler, H. D. In Fungicide Resistance in North America; Delp, C. J., Ed.; Amer. Phytopathological Soc. Press: St. Paul, 1988; pp 6-8.


211

212


3. 4. 5. 6. 7. 8.


9. 10. 11. 12. 13. 14. 15. 16. 17.

18.

19. 20. 21. 22. 23. 24. 25.

Ryder, N. S. Pestic. Sci. 1987, 21, 281-288. Ryder, N. S.; Frank, I.; Dupont, M. C. Antimicrob. Agents Chemother. 1986, 29, 858-860. Sisler, H. D.; Ragsdale, N. N. In Mode of Action of Antifungal Agents; Trinci, A. J. P.; Ryley, J . F . , Eds.; Cambridge Univ. Press: London, 1984; pp 257-282. Scheinpflug, H.; Kuck, K. H. In Modern Selective Fungicides; Lyr, H. Ed.; Longman: London, 1987; pp 173-204. Buchenauer, H.; In Modern Selective Fungicides; Lyr, H . , Ed.; Longman: London, 1987; pp 205-231. Gadher, P.; Mercer, E. I.; Baldwin, B. C.; Wiggins, T. E. Pestic. Biochem. Physiol. 1983, 19, 1-20. Vanden Bossche, H. In Recent Trends in the Discovery. Development and Evaluation of Antifungal Agents; Fromtling, R. A . , Ed.; J. R. Prous: Barcelona, 1987; pp 207-221. Poulos, T. L. In Cytochrome P-450: Structure. Mechanism and Biochemistry; Oritz deMontellano, P. R., Ed.; Plenum: New York, 1986; pp 505-523. Isaacon, D. M.; Tolman, E. L.; Tobia, A. J.; Rosenthale, M. E . ; McGuire, J. L . ; Vanden Bossche, H.; Janssen, P. A. J. J . Antimicrob. Chemother. 1988, 21, 333-343. Taton, M.; Ullmann, P.; Benveniste, P.; Rahier, A. Pestic. Biochem. Physiol. 1988, 30, 178-189. Shive, J . B.; Sisler, H. D. Plant Physiol. 1976, 57, 640-644. Coolbaugh, R. C.; Swanson, D. I.; West, C. A. Plant Physiol. 1982, 69, 707-711. Santen, R. J.; Vanden Bossche, H.; Symoens, J.; Brugmans, J.; Decoster, R. J . Clin. Endocrinol. Metab. 1983, 57, 732-736. Marchington, A. F . ; Lambros, S. A. In Modern Selective Fungicides; Lyr, H . , Ed.; Longman: London, 1987; pp 325-336. Worthington, P. A. In Synthesis and Chemistry of Agrochemicals; Baker, D. R.; Fenyes, J . G.; Moberg, W. K.; Cross, B., Eds. ACS Symposium Series No. 355; American Chemical Society, Washington, DC, 1987; pp 302-317. Katagi, T.; Mikami, N.; Matsuda, T . ; Miyamoto, J . In Synthesis and Chemistry of Agrochemicals; Baker, D. R.; Fenyes, J . G.; Moberg, W. K.; Cross, B., Eds.; ACS Symposium Series No. 355; American Chemical Society, Washington, DC, 1987; pp 340-352. Fujimoto, T. T.; Quinn, J. A.; Egan, A. R.; Shaber, S. H . ; Ross, R. R. Pestic. Biochem. Physiol. 1988. 30, 199-213. DeWaard, M. A.; Van Nistelrooy, J . G. M. Experimental Mycol. 1987, 11, 1-10. Kato, T.; Shoami, M.; Kawase, Y. J . Pestic. Sci. 1980, 5, 6979. Leroux, P.; Gredt, M. Agronomie 1983, 3, 123-130. Berg, L. R.; Patterson, G. W.; Lusby, W. R. Lipids 1983, 18, 448-452. Kerkenaar, A.; Uchiyama, M.; Versluis, G. G. Pestic. Biochem. Physiol. 1981, 16, 97-104. Baloch, R. I.; Mercer, E. I.; Wiggins, T. E. and Baldwin, B. C. 1984. Proc. Brit. Crop Prot. Conf. Pests D i s . 1984, 3, 893-898.


14.

SISLER & RAGSDALE

26. 27. 28. 29.


30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51. 52.


Wilton, D. C.; Rahimtula, A. D.; Akhtar, M. Biochem. J . 1969, 114. 71-73. Rahier, A.; Taton, M.; Bouvier-Navé, P.; Benveniste, P.; Schuber, F . ; Narula, A. S.; Cattel, L . ; Anding, C.; Place, P. Lipids 1986, 21, 52-62. Taton, M.; Benveniste, P.; Rahier, A. Pure and Appl. Chem. 1987, 59, 287-294. Rahier, A.; Schmitt, P.; Huss, B.; Benveniste, P.; Pommer, E. H. Pestic. Biochem. Physiol. 1986, 25, 112-124. Pinto, W. J.; Nes, W. R. J . Biol. Chem. 1983, 258. 4472-4476. Kerkenaar, A. Recent Trends in The Discovery and Evaluation of Antifungal Agents; Fromtling, R. A . , Ed.; J. R. Prous: Barcelona, 1987 p 523-542. Henry, M. J.; Sisler, H. D. Antimicrob. Agents Chemother. 1979, 15, 603-607. Clemons, G. P.; Sisler, H. D. Phytopathology 1969, 59, 705706. Davidse, L. C. Annu. Rev. Phytopathology 1986, 24, 43-65. Kilmartin, J . V. Biochemistry 1981, 20, 3629-3633. Leroux, P.; Gredt, M. C. R. Acad. Sci. 1979, 289. 691-693. Suzuki, K.; Kato, T . ; Takahishi, J.; Kamoshita, K. J . Pestic. Sci. 1984, 9, 497-501. Takahishi, J.; Kirino, O.; Takayama, C.; Nakamura, S.; Noguchi, H.; Kato, T . , Kamoshita, K. Pestic. Biochem. Physiol. 1988, 10, 262-271. Demakopoulou, M. G.; Georgopoulos, S. G. Abstracts. 6th Int. Congr. Pesticide Chem. 1986, 3E-05. Fujimura, M.; Oeda, K.; Inoue, H . ; Kato, T. Abstracts. 5th Int. Congr. Plant Pathol. 1988, IX-3-4, p 308. Takahishi, J.; Nakamura, S.; Noguchi, H . ; Kato, T . ; Kamoshita, K. J . Pestic. Sci. 1988, 13, 63-69. Kulka, M.; Von Schmeling, B. In Modern Selective Fungicides; Lyr, H . , Ed.; Longman: London, 1987; pp 119-131. Ulrich, J . T.; Mathre, D. E. J . Bacteriol. 1972, 110. 628632. Mowery, P. C.; Ackrell, B. A. C . ; Singer, T. P.; White, G. A.; Thorne, G. D. Biochem. Biophys. Res. Commun. 1976, 71, 354361. White, G. A.; Thome, G. D.; Georgopoulos, S. G. Pestic. Biochem. Physiol. 1978, 9, 165-182. Schewe, T.; Lyr, H. In Modern Selective Fungicides; Lyr, H . , Ed.; Longman: London, 1987; pp 133-142. White, G. A.; Thome, G. D. Pestic. Biochem. Physiol. 1980, 14, 26-40. White, S. G. Pestic. Biochem. Physiol. 1988, 31, 129-145. Edgington, L. V . ; Barron, G. L. Phytopathology 1967, 57, 1256-1257. White, G. A.; Georgopoulos, S. G. Pestic. Biochem. Physiol. 1986, 25, 188-204. Ragsdale, N. N.; Sisler, H. D. Phytopathology 1970, 60, 14221427. Lyr, H . ; Ritter, G.; Casperson, G. Z. Allg. Mikrobiol. 1972, 12, 271-280.


213

214 53. 54. 55. 56. 57. 58.


59. 60. 61.


Woloshuk, C. P.; Sisler, H. D.; Tokousbalides, M. C.; Dutky, S. R. Pestic. Biochem. Physiol. 1980, 14, 256-264. Woloshuk, C. P.; Sisler, H. D. J . Pestic. Sci. 1982, 7, 161166. Kubo, Y . ; Suzuki, K.; Furusawa, I.; Ishida, N.; Yamamoto, M. Phytopathology 1982, 72, 498-501. Kubo, Y . ; Katoh, M.; Furusawa, I.; Shishiyama, J . Exp. Mycol. 1986, 10, 301-306. Chida, T . ; Sisler, H. D. J . Pestic. Sci. 1987, 12, 49-55. Omata, K.; Tomita, H . ; Nakajima, T . ; Natsume, B. Abstract 103. Div. Agrochemicals. 196th National Meeting. Amer. Chemical Soc., 1988, Los Angeles. Woloshuk, C. P.; Sisler, H. D . ; V i g i l , E. L. Physiol. Plant Pathol. 1983, 22, 245-259. Inoue, S.; Kato, T . ; Jordan, V. W. L.; Brent, K. J . Pestic. Sci. 1987, 19, 145-152. Yamaguchi, I.; Sekido, S.; Seto, H.; and Misato, T. J . Pestic. Sci. 1983, 8, 545-550.

RECEIVED June 28, 1989


Molecular Design and Target Site Analysis in Fungicide Development

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