Pesticide Synthesis Through Rational Approaches - ACS Publications

1 The Discovery of Ivermectin and Other Avermectins W. C. CAMPBELL, R. W. BURG, M. H. FISHER, and R. A. DYBAS Downloaded by ST ANDREWS UNIV LIB on January 16, 2014 | http://pubs.acs.org Publication Date: June 26, 1984 | doi: 10.1021/bk-1984-0255.ch001

Merck Institute for Therapeutic Research, Rahway, NJ 07065

The avermectins were initially detected in a program in which thousands of microbial fermentation products were tested in mice for activity against the nematode, Nematospiroides dubius. Among the few preparations showing activity in this assay, was the product of a microorganism isolated from soil by workers at The Kitasato Institute. The microorganism was classified as a new species of actinomycete, Streptomyces avermitilis. Its anthelmintic activity was shown to reside in 8 closely related macrocyclic lactones, named avermectins, which were also found to possess activity against free-living and parasitic arthropods. One of the natural components, avermectin B , is now being evaluated as a pesticide for the control of mites of citrus and cotton crops and control of the Red Imported Fire Ant. A chemical derivative, 22,23-dihydroavermectin B , or ivermectin, has been developed as an antiparasitic agent. It is being marketed for use in cattle, horses and sheep and is expected to become available for swine and dogs. 1

1

The t i t l e of t h i s s e s s i o n , Guided and Serendipitous Discovery (within the symposium, Approaches to R a t i o n a l Synthesis o f P e s t i c i d e s ) focuses a t t e n t i o n on the nature of the d i s c o v e r y , r a t h e r than the outcome. The d i s c o v e r y of the avermectin f a m i l y of compounds was by no means s e r e n d i p i t o u s ; those who were seeking found what they sought. I t i s the purpose of t h i s paper to r e c o r d the manner of the seeking and the manner

0097-6156/ 84/ 0255-0005S06.00/ 0 © 1984 American Chemical Society

In Pesticide Synthesis Through Rational Approaches; Magee, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1984.

Downloaded by ST ANDREWS UNIV LIB on January 16, 2014 | http://pubs.acs.org Publication Date: June 26, 1984 | doi: 10.1021/bk-1984-0255.ch001

6

PESTICIDE SYNTHESIS THROUGH RATIONAL APPROACHES

of assessing and enhancing what was found. The discovery of i v e r m e c t i n i s part of the l a r g e r s t o r y of the discovery of the avermectin family of compounds: the avermectins are produced by a microorganism, and ivermectin i s a chemical m o d i f i c a t i o n of one of those substances. The i n i t i a l o b j e c t i v e of the search was an anthelmintic with p r o p e r t i e s r a d i c a l l y d i f f e r e n t from those of known anthelmintics. What was found was an a n t h e l m i n t i c whose p r o p e r t i e s were indeed markedly d i f f e r e n t — not only i n terms of chemical s t r u c t u r e and e f f i c a c y against helminths, but a l s o i n the extension of the p o t e n t i a l u t i l i t y of the c l a s s to the c o n t r o l of arthropod p a r a s i t e s of animals. Further, the compounds turned out to have s t r i k i n g a c t i v i t y against a v a r i e t y of f r e e - l i v i n g and p l a n t - p a r a s i t i c nematodes and arthropods — and so i t has come about that a new l i v e s t o c k a n t h e l m i n t i c has become an agenda item i n t h i s symposium on agricultural pesticides. Primary Screening

and

Microbiology

Several i n v i t r o assays f o r d e t e c t i n g fermentation products w i t h a n t h e l m i n t i c a c t i v i t y had been run without success, p r i m a r i l y because of the l a r g e number of t o x i c compounds which had to be e l i m i n a t e d . F i n a l l y , the d e c i s i o n was made to use an i n v i v o assay i n mice with the hope that the mice would screen out the n o n - s p e c i f i c t o x i c compounds. The nematode, Nematospiroides dubius, was s e l e c t e d f o r the assay. Infected mice were fed f o r s i x days with m i l l e d Purina Lab Chow which had been mixed with the fermentation product to be t e s t e d . The mice were then fed a normal d i e t and, at 14 days p o s t i n f e c t i o n , f e c a l p e l l e t s were examined f o r the presence of eggs. I f eggs were absent on three successive days, the mice were s a c r i f i c e d and t h e i r small i n t e s t i n e s examined f o r the presence of worms. This assay was r e l a t i v e l y s u c c e s s f u l i n achieving the goal of e l i m i n a t i n g n o n s p e c i f i c t o x i c a c t i v i t i e s . Among the many thousands of c u l t u r e s tested i n t h i s assay, about 1% were a c t i v e i n the f i r s t t e s t . A l l a c t i v e c u l t u r e s were regrown, and about 20% of the c u l t u r e s i n i t i a l l y s e l e c t e d were a c t i v e . Among the c u l t u r e s confirmed a c t i v e , most were s t i l l extremely t o x i c and were not pursued f u r t h e r . However, during the t e s t i n g of a l a r g e c o l l e c t i o n of c u l t u r e s which had been r e c e i v e d from The K i t a s a t o I n s t i t u t e of Tokyo, Japan, the choice of the i n v i v o assay was f i n a l l y j u s t i f i e d . Among one



1.

C A M P B E L L ET AL.

Discovery of Ivermectin

group of 50 K i t a s a t o c u l t u r e s submitted f o r assay was a c u l t u r e bearing the number 0S-3153. The r e s u l t s of the e a r l y assays of t h i s c u l t u r e are summarized i n Table I. I t was scored as f u l l y a c t i v e (no eggs, no worms) i n the f i r s t t e s t . Marked r e d u c t i o n i n mouse body weight i n d i c a t e d that the fermentation product was e i t h e r unpalatable or t o x i c . C u l t u r e 0S-3153 was regrown on the o r i g i n a l medium (designated KH) and on a second, u n r e l a t e d medium (JH) (Table I, Experiment No. 2). The c u l t u r e grown on the second medium was very t o x i c , r e s u l t i n g i n host death on the day that the f i r s t f e c a l p e l l e t was examined f o r eggs. That grown on the o r i g i n a l medium was a l s o t o x i c , causing severe suppression of weight gains; but, again, there were no eggs or worms. In the t h i r d t e s t , the a c t i v i t y was t i t r a t e d u s i n g s e r i a l 2 - f o l d d i l u t i o n s . A l l four l e v e l s were f u l l y a c t i v e , and t h i s time there was l i t t l e s i g n of t o x i c i t y . A few c u l t u r e s had been confirmed a c t i v e p r e v i o u s l y , but none had e x h i b i t e d a s e p a r a t i o n of t o x i c i t y from a c t i v i t y , l e t alone a c t i v i t y over at l e a s t an 8 - f o l d range. A f t e r an i n a u s p i c i o u s s t a r t , the a c t i v i t y of c u l t u r e OS-3153 had been f i r m l y e s t a b l i s h e d . Success had been achieved i n the quest f o r a fermentation product with a n t h e l m i n t i c a c t i v i t y . In f a c t , as events would soon prove, the newly found product had an even broader a c t i v i t y than had been a n t i c i p a t e d . The c u l t u r e , now bearing the product number C-076 and the Merck c u l t u r e c o l l e c t i o n number MA-4680, was submitted f o r taxonomic s t u d i e s . I t s c h a r a c t e r i s t i c s , i n c l u d i n g a brownishgray spore mass c o l o r , smooth spore s u r f a c e , s p i r a l sporophores born as side branches on the a e r i a l mycelia and the p r o d u c t i o n of melanoid pigments, were u n l i k e those of any p r e v i o u s l y d e s c r i b e d species of Streptomyces. The c u l t u r e was named Streptomyces a v e r m i t i l i s , the Streptomyces "capable of separating from worms". Based on a c o r r e l a t i o n of a n t h e l m i n t i c a c t i v i t y and HPLC a n a l y s i s of the t o t a l avermectin complex, i t was estimated that the t h i r d fermentation contained a minimum of 9 yg/ml. Improvement of the medium increased the y i e l d by the o r i g i n a l c u l t u r e (MA-4680) to 120 yg/ml. A high-producing i s o l a t e (MA-4848) obtained from t h i s c u l t u r e produced n e a r l y 500 yg/ml of t o t a l avermectins. Thus, t h i s c u l t u r e y i e l d e d r e a d i l y to medium improvement and i s o l a t e s e l e c t i o n to produce r e l a t i v e l y l a r g e amounts of the avermectins. Accounts of the e a r l y fermentation s t u d i e s and taxonomy have been p u b l i s h e d (1-2).


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PESTICIDE SYNTHESIS T H R O U G H RATIONAL APPROACHES


8

Table I .

Expt. No. 1 2 3

Summary of the F i r s t Assays of C u l t u r e OS-3153.

Medium

Dose

KH KH JH KH

50 50 50 50 25 12 6 50 25 12

JH

» Ml of broth per

'

Feed Eaten (g) 13 25 13 25 25 25 25 25 25 25

Mouse Weight (g) 14 15

22 25 29 28 22 29 —

Activity A A Dead A A A A SA SA ΝΑ

25 g of feed.

(2) A, a c t i v e - no eggs or worms; SA, s l i g h t l y eggs, worms present; NA, not a c t i v e .

a c t i v e - no


1.

9


C A M P B E L L ET A L .

Chemistry I s o l a t i o n . The avermectin complex, c o n s i s t i n g of f o u r major components designated A^ , A , ^ l a * ^2a * ^ l° homologs designated A ^ , A ^, ^ i b * ^2b* ^ with acetone from the mycelia of Streptomyces a v e r m i t i l i s (3). Solvent p a r t i t i o n and a d s o r p t i o n on granular carbon produced an o i l y residue c o n t a i n i n g 5% 16% A , 20% Β and 15% Β · Separation of the A components from the Β components was achieved by p a r t i t i o n chromatography with hexane-methylene chloride-methanol (10:10:1) over Sephadex LH-20. A^ was separated from A u s i n g Sephadex LH-20 and a s o l v e n t system c o n t a i n i n g hexane-toluene-methanol (6:1:1). B^ was separated from Β2 e i t h e r by c r y s t a l l i z a t i o n from ethylene g l y c o l or by Sephadex LH-20 chromatography using hexane-toluenemethanol (3:1:1). S i m i l a r chromatography was a l s o used to separate the lower homologs and p u r i t y was e s t a b l i s h e d by reverse-phase HPLC a n a l y s i s . a n c

a

o u r

w e r

2 a

w

a

s

e x t r a c t e (

2


2

χ

2

2

S t r u c t u r e Determination. The s t r u c t u r e s of a new group of p e s t i c i d a l sixteen-membered lactones named milbemycin B^, B , and Ββ were d e s c r i b e d i n 1975 (4) on the b a s i s of X-ray a n a l y s i s . A l a t e r p u b l i c a t i o n (5) gave d e t a i l s of i s o l a t i o n and s t r u c t u r e s of t h i r t e e n milbemycins, with s p e c t r a l data. The avermectins were discovered i n 1975 and p a r t - s t r u c t u r e s , 1ο deduced from proton and C NMR s p e c t r a and t h e i r mass s p e c t r a l fragmentation p a t t e r n s , suggested a c l o s e r e l a t i o n s h i p with the milbemycins ( 6 ) . Methanolysis of aver mectin A gave an aglycone and a 6:1 mixture of -and -methyl-Loleandroside. The recovery of t h i s g l y c o s i d e i n more than 100 mol. % y i e l d demonstrated the presence of two i d e n t i c a l sugars i n the molecule. Further s p e c t r a l exami n a t i o n i n d i c a t e d the attachment of an a-L-oleandrosyl-ot-Loleandrosyloxy d i s a c c h a r i d e to the 13-ct-position of the macrolide r i n g . A chemical proof f o r the p o i n t of attachment and i d e n t i t y of the d i s a c c h a r i d e was provided by o z o n o l y s i s and i s o l a t i o n of the d i s a c c h a r i d e attached to the fragment l l through 1 4 . The s t r u c t u r e s of the e i g h t components are shown i n F i g . 1 and a l l c o n t a i n the same d i s a c c h a r i d e s u b s t i t u e n t at the 13-α-position. They vary at C-5 with hydroxy or methoxy groups, at C-23 with an a x i a l M i y d r o x y group on a 22,23-olefin and a t C-25 with i s o p r o p y l or s e c - b u t y l groups i n c o n t r a s t to the methyl and e t h y l s u b s t i t u e n t s at the 2 5 - p o s i t i o n of the milbemycins. X-ray a n a l y s i s (7) of B aglycone and B^ both 2

J

2

C

C

2 a


a

In Pesticide Synthesis Through Rational Approaches; Magee, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1984. 3

F i g u r e 1.

5

R = Η

5

R = CH

Δ,

Β,

3

3

(1 atm) ( 0 P ) 3

3

RhCI

2

2

}

5

B

A 2

2

S t r u c t u r e s of the Avermectins.

22, 23-Dihydroavermectin B ivermectin

4' °v H

C

3

3

= CH(CH )

= CH(CH )C H

0 C H , 25°, 18 h 8 5 %

2

2 5

b series R

H

2 5

α series R

QCH,

5

5

R = H

R = CH 3


η χ m in

no

>

25 δ > r

H a: Ο C ο X

Η Χ m

a m

•v m H

1.

CAMPBELL E T A L .


11

confirmed the s t r u c t u r e and, through the L-oleandrose, e s t a b l i s h e d the absolute stereochemistry.


Parasitological Evaluation Following the demonstration of e f f i c a c y i n the Nematospiroides-mouse assay, and the a s s o c i a t e d m i c r o b i o l o g i c a l and chemical research d e s c r i b e d above, much work was needed to determine the r e l a t i v e a n t h e l m i n t i c e f f i c a c y of both the n a t u r a l avermectin components and the d e r i v a t i v e s . For t h i s purpose two types of bioassay were employed. In one, compounds were t e s t e d i n s m a l l l a b o r a t o r y animals i n f e c t e d with nematodes other than N. dubius. For example, t e s t m a t e r i a l s were given to j i r d s (Meriones unguiculatus) i n f e c t e d with T r i c h o s t r o n g y l u s c o l u b r i f o r m i s , and the animals were subsequently k i l l e d f o r determination of worm burden ( 8 ) . The use of that h o s t - p a r a s i t e combination f o r a n t h e l m i n t i c t e s t i n g had been reported by P a n i t z and Shum (9) and has proved u s e f u l i n the e v a l u a t i o n of a v a r i e t y of a n t h e l m i n t i c s , i n c l u d i n g the avermectins. In the other, compounds were tested a g a i n s t a v a r i e t y of nematodes i n sheep. These were s m a l l s c a l e t e s t s , done i n c o n j u n c t i o n with the small-animal t e s t i n g and p r o v i d i n g important i n f o r m a t i o n on the e f f i c a c y of the t e s t substances i n a ruminant host. Compounds of s p e c i a l i n t e r e s t were s i m i l a r l y t e s t e d against helminths i n sheep, and o c c a s i o n a l l y i n other h o s t s , u s i n g l a r g e r numbers of t e s t animals (10-12). As the remarkable potency and unique s t r u c t u r e of the avermectins became apparent, t e s t i n g was extended to organisms other than helminths. The f i r s t t e s t against an i n s e c t was done using the Confused F l o u r B e e t l e , T r i b o l i u m confusum, and the i n c o r p o r a t i o n of the t e s t substance i n t o the f l o u r i n which the b e e t l e s l i v e d . In t h i s f a s h i o n the i n s e c t i c i d a l a c t i v i t y of the avermectins was demonstrated (13) and was followed independently and almost immediately by the demonstration of e f f i c a c y against a p a r a s i t i c i n s e c t (14). E f f i c a c y against p a r a s i t i c i n s e c t s was f u r t h e r e s t a b l i s h e d by t e s t s u s i n g the rodent bot, Cuterebra sp., i n mice (D. A. O s t l i n d , unpublished) and was shown to extend to some p a r a s i t i c a c a r i n e s (15-16). Tests against the trematodes Schistosoma mansoni and F a s c i o l a h e p a t i c a and the cestode Hymenolepis diminuta i n l a b o r a t o r y animals f a i l e d to show e f f i c a c y (D. A. O s t l i n d , unpublished d a t a ) . T h i s i s i n accord w i t h r e p o r t s that the avermectins d i s r u p t GABA-mediated nerve


PESTICIDE SYNTHESIS T H R O U G H RATIONAL APPROACHES

transmission i n nematodes and arthropods and that f l u k e s and tapeworms do not employ GABA as a neurotransmitter (17).


A g r i c u l t u r a l Chemical E v a l u a t i o n The r e s u l t s (above) against the Confused F l o u r B e e t l e ( T r i b o l i u m confusum), rodent bot (Cuterebra spp.), and the e c t o p a r a s i t i c l a r v a of the sheep b l o w f l y ( L u c i l i a cuprina) were s u f f i c i e n t l y encouraging to suggest that the avermectins may possess general b i o l o g i c a l a c t i v i t y against arthropod pests and, i n p a r t i c u l a r , those of importance i n crop p r o t e c t i o n (18). To i n v e s t i g a t e t h i s p o t e n t i a l and to expand our in-house c a p a b i l i t i e s , a research program was e s t a b l i s h e d w i t h the Boyce Thompson I n s t i t u t e f o r P l a n t Research to t e s t the avermectin d e r i v a t i v e s i n t h e i r m i t i c i d e and i n s e c t i c i d e screens. In a l l , n e a r l y s e v e n t y - f i v e r e l a t e d s t r u c t u r e s , n a t u r a l products and semisynthetic d e r i v a t i v e s , were evaluated i n the greenhouse f o r t o x i c i t y t o a spectrum of arthropod p e s t s . Of these, avermectin B^, the major component of the fermentation process, was determined to be the most promising candidate as an a g r i c u l t u r a l p e s t i c i d e . R e s u l t s from these l a b o r a t o r y s t u d i e s demonstrated that avermectin B^ had high t o x i c i t y f o r the twospotted s p i d e r mite (Tetranychus u r t i c a e ) on bean p l a n t s . When a p p l i e d i n s o l u t i o n d i r e c t l y onto adult and nymphal s p i d e r mite populations on f o l i a g e , avermectin B^ was shown to be 50-200 times as potent as commercially a v a i l a b l e a c a r i c i d e s , with an LC of 0.02-0.03 ppm. A d d i t i o n a l t e s t s on f o l i a g e with i n s e c t s i n the order Lepidoptera, Coleoptera, Homoptera, Orthoptera, D i p t e r a , Isoptera and Hymenoptera confirmed the broad spectrum a c t i v i t y and potency of the avermectin f a m i l y of compounds and avermectin B^ i n p a r t i c u l a r . Table II prov i d e s LC(JQ values f o r avermectin B^ f o r the c o n t r o l o f l a r v a l forms of s e v e r a l of these i n s e c t s i n f o l i a r residue assays (18). 9 0


1.

C A M P B E L L ET AL.

T a b l e II.

13


E f f i c a c y of F o l i a r Residues of Avermectin B^ Against Adult mites and L a r v a l Insects. a


Insect Twospotted s p i d e r mite Tomato hornworm Colorado potato b e e t l e Mexican bean b e e t l e Cabbage looper Southern armyworm

L C

m

9 0 (PP ) 0.02-0.03 0.02 0.03 0.2 0.75-1.2 6.0

On the b a s i s of the e f f i c a c y demonstrated i n the greenhouse and l a b o r a t o r y s t u d i e s avermectin was s e l e c t e d f o r development and assigned the Merck development code number MK-936. Avermectin B^ has been evaluated worldwide f o r e f f i c a c y against mites and i n s e c t s a f f e c t i n g a number of a g r i c u l t u r a l crops i n c l u d i n g c i t r u s , c o t t o n , apples, pears, vegetables, potatoes, t r e e nuts, and grapes. Under f i e l d use c o n d i t i o n s i t has been observed that e x c e l l e n t c o n t r o l of a number of economically important pests i n c l u d i n g the c i t r u s rust and red mite, twospotted s p i d e r mite, broad mite, Colorado potato b e e t l e , diamond back moth, pear p s y l l a , and Liriomyza leafminers can be achieved at extremely low a p p l i c a t i o n r a t e s of MK-936 i n the range of 0.005-0.03 l b per acre (5.5 - 33 g per h e c t a r e ) . For f o l i a g e a p p l i c a t i o n s a 0.15 EC (1.8% w/v) e m u l s i f i a b l e concentrate f o r m u l a t i o n has been developed. F i e l d s t u d i e s have shown that the f o r m u l a t i o n i s non-phytotoxic to a l l target crops on which i t has been evaluated i n c l u d i n g many v a r i e t i e s of s e n s i t i v e ornamental plants. During the course of the development program, samples of avermectin B^ were provided to a number of outside agencies f o r e v a l u a t i o n i n s p e c i a l i z e d assays. As a consequence, i t was discovered i n t e s t i n g conducted by the USDA l a b o r a t o r y f o r Insects A f f e c t i n g Man and Animals, G a i n e s v i l l e , F l o r i d a , that the red imported f i r e ant (Solenopsis i n v i c t a ) i s among the most s u s c e p t i b l e species of i n s e c t s to the t o x i c a c t i o n of avermectin B ^ . When a p p l i e d i n a corn g r i t b a i t , avermectin


14



B^ at r a t e s as low as i n 25 to 50 mg per acre has been e f f e c t i v e i n c o n t r o l l i n g f i r e ant i n f e s t a t i o n s i n large s c a l e t r i a l s i n the southern United S t a t e s . A submission f o r r e g i s t r a t i o n of MK-936 f o r t h i s a p p l i c a t i o n has been made. The avermectin n a t u r a l products are p e s t i c i d e s possessing novel chemistry and mode of a c t i o n . C r o s s - r e s i s t a n c e has not been observed i n l a b o r a t o r y or f i e l d s t u d i e s with mites andinsects t o l e r a n t to commercially a v a i l a b l e organophosphate, carbamate, c h l o r i n a t e d hydrocarbon and p y r e t h r o i d p e s t i c i d e s . S y n t h e t i c Program 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 . Compounds of the Β s e r i e s were g e n e r a l l y more potent than those of the A s e r i e s . Thus an u n s u b s t i t u t e d hydroxy group at the 5 - p o s i t i o n i s a c t i v i t y enhancing (19). D i f f e r e n c e s i n potency between the 1- and 2s e r i e s v a r i e d among p a r a s i t e s , but i n most instances the 1s e r i e s was more potent. Reduction of the 22,23-olefin had l i t t l e e f f e c t on a c t i v i t y but f u r t h e r r e d u c t i o n caused a s u b s t a n t i a l decrease i n a c t i v i t y . The monosaccharides were two- to f o u r f o l d l e s s a c t i v e than the parent compounds w h i l e the aglycones were more than t h i r t y f o l d l e s s a c t i v e , Table I I I . A c e t y l a t i o n at the 4 " - p o s i t i o n caused no change i n a c t i v i t y whereas a c e t y l a t i o n at the 5- or 23- p o s i t i o n caused a c o n s i d e r a b l e decrease i n a c t i v i t y . D i a c e t a t e s and t r i a c e t a t e s showed s i m i l a r l y reduced a c t i v i t y , Table IV (20). Ivermectin. E a r l y b i o l o g i c a l s t u d i e s demonstrated that while avermectin B^ was more a c t i v e than avermectin B by o r a l a d m i n i s t r a t i o n , the converse was true when the compounds were given p a r e n t e r a l l y . Furthermore, avermectin B^ was much l e s s e f f e c t i v e against Cooperia species when given p a r e n t e r a l l y than by o r a l treatment. Avermectin B had g e n e r a l l y lower a c t i v i t y against Haemonchus s p e c i e s . Examination of the B^ and B s t r u c t u r e s revealed that the d i f f e r e n c e s centered on the 22,23-position. Avermectin B^ i s a 22,23-olefin whereas i n avermectin B t h i s bond i s hydrated with the hydroxyl group at the 2 3 - p o s i t i o n . The conformation of the r i n g bearing these f u n c t i o n a l i t i e s i s d i f f e r e n t and i t was reasoned that b i o a c t i v i t y might be l i n k e d to conformation. It therefore became an important o b j e c t i v e to s y n t h e s i z e 22,23dihydroavermectin B^ which r e q u i r e d f o r i t s s y n t h e s i s the s e l e c t i v e r e d u c t i o n of one of f i v e o l e f i n s . However, only the 2

2

2

2


1. CAMPBELL ET AL.

15



Table I I I . A c t i v i t y of Avermectin D e r i v a t i v e s against Adult G a s t r o i n t e s t i n a l Helminths of Experimentally Infected Sheep on O r a l A d m i n i s t r a t i o n .

Efficacy Structure

Al A Bl B

0.1 0.1

H2A1

0.3

H Bi BiMS B MS H2B MS H BiAG

0.1

2

0.15

2 3 3 3 2 3 2

0.2

1

1

0.3 3.0 0.2

3

3 2 0

0.1

2

2

1

2

H4B1

H.c. ° O.c. T.a. T.c.° C. spp. O.c.

2 3 3 0 3 3 2

0.05

2

a

Dose, mg/kg

a

1

0

0 3 3 3 0 3 3 3 3 3 1

0 3 3 3 1

3 3 3 3 3 0

2 0 3 3 0 3 3 3 2 1

0

0 = < 50%, 1 = 5 0 - 7 4 % , 2 = 7 5 -•90%, 3 = > 90% e f f i c a c y . Abbreviations used: H.c. Haemonchus con t o r t u s ; O . c , O s t e r t a g i a c i r c u m c i n c t a ; T.a., T r i c h o s t r o n g y l u s a x e i ; T . c , T r i c h o s t r o n g y l u s c o l u b r i f o r m i s ; C. spp., Cooperia spp.; O . c , Oesophagostomum columbianum. *MS = monosaccharide, AG = aglycon, H = 22,23-dihydro d e r i v a t i v e , H 4 = 3,4,22,23-tetrahydro d e r i v a t i v e . 2

Benzimidazole r e s i s t a n t .


0 3 3 3 3 3 0 3 3 3 3


16

Table IV. D e r i v a t i v e s of Avermectin A~ and B^ and Anthelmintic A c t i v i t y against T r i c h o s t r o n g y l u s c o l u b r i f o r m i s i n G e r b i l s Downloaded by ST ANDREWS UNIV LIB on January 16, 2014 | http://pubs.acs.org Publication Date: June 26, 1984 | doi: 10.1021/bk-1984-0255.ch001

a

H

H H CH3CO

H

CH3CO CH3CO

R CH H CH CH CH CH CO 5

3

3 3 3

3

R23 H H H CH CO CH CO CH CO 3 3

3

0R

5

anthelmintic acta 0.05 0.0125 0.0625 0.25 0.5 0.5

aMinimal dose(mg/kg) needed to remove > 83% of the worm burden.



1. CAMPBELL ET AL.

17


22, 2 3 - o l e f i n i s c i s - s u b s t i t u t e d , suggesting the use of Wilkinson's homogenous c a t a l y s t (Ph^P^RhCl known to be very s e n s i t i v e to the s t e r i c environment of an o l e f i n . Hydrogénat i o n of avermectin B^ f o r 20 hours using Wilkinson's c a t a l y s t i n benzene or toluene at 25°C under one atmosphere of hydrogen gave 22,23-dihydroavermectin B^ i n 85% y i e l d (Figure 1). T h i s compound was s e l e c t e d f o r development as a broadspectrum a n t i p a r a s i t i c agent f o r animals on the b a s i s of i t s o v e r a l l e f f i c a c y by o r a l and p a r e n t e r a l routes and f o r i t s improved s a f e t y p r o f i l e (19-21). 22,23-Dihydroavermectin B^, c o n t a i n i n g at l e a s t 80% of 22,23-dihydroavermectin B and not more than 20% of 22,23-dihydroavermectin B ^ has been assigned the non-proprietary name i v e r m e c t i n . The compound was subjected to a l a r g e i n t e r n a t i o n a l program of development, which l i e s beyond the scope of t h i s paper, and which included e f f i c a c y t r i a l s and s a f e t y assessment i n sheep, c a t t l e , horses, swine and dogs. T h i s development program r e s u l t e d i n the i n t r o d u c t i o n of ivermectin as a commercial a n t i p a r a s i t i c agent i n 1981. For c a t t l e , sheep and horses, the dosage recommended f o r general a n t i p a r a s i t i c use i s 0.2 mg/kg; f o r swine the dosage i s 0.3 mg/kg. The compound i s used both o r a l l y and p a r e n t e r a l l y — the formulation and route of a d m i n i s t r a t i o n depending on the host species being t r e a t e d . l a

Discussion The d i s c o v e r y of the avermectins, by v i r t u e of the wide spectrum of the compounds, and t h e i r extreme potency and novel mode of a c t i o n , met the i n i t i a l o b j e c t i v e of f i n d i n g an a n t h e l m i n t i c with r a d i c a l l y d i f f e r e n t c h a r a c t e r i s t i c s . The avermectins are not a c t i v e against a l l groups of helminths — they have not been reported a c t i v e against f l u k e s or tapeworms — but they are a c t i v e against a l l nematode groups that have been t e s t e d , and indeed there i s no c l e a r evidence that any species of any genus of nematode i s r e f r a c t o r y to the a c t i o n of i v e r m e c t i n . In at l e a s t one instance (adult D i r o f i l a r i a immitis) a p a r t i c u l a r l i f e c y c l e stage i s r e f r a c t o r y while other stages of the same species are s u s c e p t i b l e . The occurrance of antinematodal and antiarthropod a c t i v i t y i n a s i n g l e chemical c l a s s , i s not e n t i r e l y unprecedented. The organophosphates are a c t i v e against p a r a s i t e s of both groups, but t h e i r spectrum of a c t i v i t y against nematodes i s r e l a t i v e l y narrow. The s a l i c y l a n i l i d e compounds are a c t i v e against c e r t a i n nematodes and arthropods but are used p r i m a r i l y against f l u k e s .




The discovery of avermectins r e s u l t e d from the d e l i b e r a t e choice of fermentation products as the prime source of substances to be submitted f o r anthelmintic screening. Many f a c t o r s were c r i t i c a l t o the success of the venture, i n c l u d i n g the submission of novel actinomycte i s o l a t e s by workers at The K i t a s a t o I n s t i t u t e i n Japan, the s e l e c t i o n of the i n v i v o screen, the r a p i d i s o l a t i o n and i d e n t i f i c a t i o n of the a c t i v e p r i n c i p l e , the assessment of a n t i p a r a s i t i c p r o p e r t i e s , and the enhancement of b i o l o g i c a l p r o p e r t i e s by s y n t h e t i c chemical m o d i f i c a t i o n o f the s t r u c t u r e . The names of those r e s p o n s i b l e f o r these c o n t r i b u t i o n s may be found i n the e a r l y papers published on the s u b j e c t , and are l i s t e d elsewhere according to s c i e n t i f i c d i s c i p l i n e (2). The discovery of the avermectins thus rested on e m p i r i c a l t e s t i n g — as d i d the d i s c o v e r y of a l l other s u c c e s s f u l a n t h e l m i n t i c s and ectoparasiticides. Such d i s c o v e r i e s are nevertheless a t t r i b u t a b l e t o d e l i b e r a t e and f a r - f r o m - a r b i t r a r y choices made during the i n i t i a l conception and subsequent operation of the screening program. The i d e n t i f i c a t i o n of a biochemical mode of a c t i o n that appears to d i f f e r profoundly from that of previous a n t i p a r a s i t i c agents has provided a t o o l f o r new approaches to understanding and e x p l o i t i n g the b a s i c biochemical pathways of animal and p l a n t p a r a s i t e s . The b i o l o g i c a l p r o p e r t i e s of the avermectins have opened new p o s s i b i l i t i e s f o r the study of low-dose drug d e l i v e r y systems, and many aspects of nematodology, entomology and acarology, as w e l l as c o n t r i b u t i n g d i r e c t l y t o the c o n t r o l of many l i v e s t o c k p a r a s i t e s and a g r i c u l t u r a l pests.

Literature Cited 1.

2.

Burg, R.W., Miller, B.M., Baker, E . E . , Birnbaum, J., Currie, S.A., Hartman, R., Kong, Y-L., Monaghan, R . L . , Olson, G . , Putter, I . , Tunac, J . B . , Wallick, Η., Stapley, E.O., Oiwa, R., and Omura, S. Antimicrob. Agents Chemother. 1979, 15, 361-7. Stapley, E.O. and Woodruff, H.B., in "Proceedings, An International Conference on Trends in Antibiotic Research"; Umezawa, H . , Demain, A . L . , Hata, T . , and Hutchinson, C.R., Eds.; Japan Antibiotics Research Association, Tokyo, 1982; pp. 154-170.


CAMPBELL ET AL.



3.

19

Miller, T.W., Chaiet, L., Cole, D . J . , Cole, L.J., Flor, J . E . , Goegelman, R.T., Gullo, V.P., Kempf, A.J., Krellwitz, W.R., Monaghan, R.L., Ormond, R.E., Wilson, Κ.Ε., Albers-Schonberg, G., and Putter, I. Antimicrob. Agents Chemother. 1979, 15, 368. 5. Takiguchi, Y . , Mishima, H . , Okuda, M., Terao, M., Aoki, Α., and Fukuda, R. J . Antibiot. 1980, 33, 1120. 6. Albers-Schonberg, G., Arison, B.H., Chabala, J . C . , Douglas, A.W., Eskola, P., Fisher, M.H., Lusi, Α., Mrozik, H . , Smith, J.L., and Tolman, R.L. J . Am. Chem. Soc.1981, 103 4216. 7. Springer, J . P . , Arison, B.H., Hirshfield, J.M., and Hoogsteen, K. J . Am. Chem. Soc. 1981, 103, 4221. 8. Ostlind, D.A., and C i f e l l i , S. Research in Veterinary Science, 1981, 31, 255-6. 9. Panitz, E . , and Shum, K.L. J . Parasit. 1981, 67, 135-6. 10. Egerton, J . R . , Ostlind, D.A., Blair, L . S . , Eary, C.H., Suhayda, D., C i f e l l i , S., Riek, R.F., and Campbell, W.C. Antimicrob. Agents Chemother. 1979, 15, 372-8. 11. Blair, L . S . , and Campbell, W.C. J . Helm. 1978, 52, 305307. 12. Blair, L . S . , and Campbell, W.C. Journal of Parasitology, 1978, 64(6), 1032-4. 13. Ostlind, D.A., C i f e l l i , S., and Lang, R. Vet. Rec., 1979, 105, 168. 14. James, P.S., Picton, J., and Riek, R.F. Vet. Rec. 1980, 106, 59. 15. Wilkins, C.A., Conroy, J . A . , Ho, P., O'Shanny, W.J., Malatesta, P.F., and Egerton, J.R. Am. J . Vet. Res. 1980, 41, 2112-13. 16. Wilkins, C.A., Conroy, J., Ho. P., and O'Shanny, W.J. Proc. 25th Annual Mtg. Am. Assoc. Vet. Parasitol., Washington, 1980, p. 18. 17. Wang, C.C., and Pong, S.C. Progress in Clinical and Biological Research 1981, 97, 373-95 18. Putter, I . , MacConnell, J . G . , Preiser, F.Α., Haidri, A.A., Ristich, S.S. and Dybas, R.A. Experientia 1981, 37, 963-964. 19. Chabala, J . C . , Mrozik, H . , Tolman, R . L . , Eskola, P., Lusi, Α . , Peterson, L . H . , Woods, M.F., Fisher, M.H., Campbell, W.C., Egerton, J.R., and Ostlind, D.A. J . Med. Chem. 1980, 23, 1134.


PESTICIDE SYNTHESIS THROUGH RATIONAL APPROACHES 20. 21.

Mrozik, Eskola, P., Fisher, M.H., Egerton, J.R., C i f e l l i , S., and Ostlind, D.A. J . Med. Chem. 1982, 25, 658. Egerton, J.R., Birnbaum, J., Blair, L . S . , Chabala, J . C . , Conroy, J., Fisher, M.H., Mrozik, Η., Ostlind, D.A., Wilkins, C.A., and Campbell, W.C. Br. Vet. J . 1980, 136, 88-97.


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