Pesticide Synthesis Through Rational Approaches - American

4 The Discovery and Development of Bromethalin, an Acute Rodenticide with a Unique Mode of Action BARRY A. DREIKORN and GEORGE O. P. O'DOHERTY Downloaded by GEORGETOWN UNIV on August 25, 2015 | http://pubs.acs.org Publication Date: June 26, 1984 | doi: 10.1021/bk-1984-0255.ch004

Lilly Research Laboratories, Eli Lilly and Company, Greenfield, IN 46140 Because rodent populations world-wide were becoming resistant to the widely used Warfarin-type anticoagulant poisons, a search was initiated to find a rodenticide with a different mode of action; one that would be effective against these resistant rodents. This search led to the discovery of the toxic nature of a family of diphenyl amines which act as uncouplers of oxidative phosphorylation. A structure-activity relationship (SAR) study was undertaken to choose a derivative that would be both poisonous to rodents but still readily consumed by them. This approach led to the discovery of bromethalin, N-methyl-2,4-dinitro-N (2,4,6-tribromophenyl)-6-(trifluoromethyl)benzeneamine, which had a l l the desired rodenticidal properties. H i s t o r y . For c e n t u r i e s man has recognized that rodent pests destroy h i s h a b i t a t , consume h i s food, and cause the spread o f v i r u l e n t diseases. Throughout the same c e n t u r i e s man has sought to e l i m i n a t e these pests with a v a r i e t y o f poisons such as s t r y c h n i n e , arsenious oxide, and red s q u i l l - a s t e r o i d a l g l y c o s i d e e x t r a c t e d from the bulb o f a l i l y - l i k e plant, Urginea maritima. These m a t e r i a l s , as w e l l as more recent poisons l i k e z i n c phosphide and f l u o r o a c e t i c a c i d , work as acute r o d e n t i c i d e s and can be e f f e c t i v e when a rodent consumes a l e t h a l quantity o f poison i n a s i n g l e dose i n i t s food or drink. Unfortunately, animals o f t e n consume l e s s than a l e t h a l dose, which produces side e f f e c t s which the animals a s s o c i a t e with the b a i t . The r e s u l t i s that they become " b a i t shy" and are l i k e l y to be wary of the same poison b a i t a second time. The discovery o f the anticoagulant p r o p e r t i e s o f dicoumarin (1) l e d t o the development o f the more potent anticoagulant w a r f a r i n (2), (Structure 1). The subsequent discovery that the a n t i c o a g u l a n t s can be s u c c e s s f u l l y used as m u l t i p l e dose

0097-6156/ 84/ 0255-0045506.00/ 0 © 1984 American Chemical Society

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

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Downloaded by GEORGETOWN UNIV on August 25, 2015 | http://pubs.acs.org Publication Date: June 26, 1984 | doi: 10.1021/bk-1984-0255.ch004

r o d e n t i c i d e s ( 3 ) s i g n a l l e d a s h i f t from the development o f acute r o d e n t i c i d e s t o the chronic, a n t i c o a g u l a n t m a t e r i a l s . These compounds a l l work by suppressing the a c t i v i t i e s o f vitamin-K dependent c l o t t i n g f a c t o r s i n the t a r g e t animal's blood which consequently causes the animal t o bleed t o death. The a n t i coagulants avoid the development of b a i t shyness because they don't produce poisoning symptoms that cause feeding to stop. Because they are e f f e c t i v e a t low dosages(0.005-0.25%) they are r e l a t i v e l y non-toxic to domestic animals and man.

CH

I

CO

I

ο I Warfarin

1 Indandiones

Very r a p i d l y , a number of other a n t i c o a g u l a n t s , i n c l u d i n g the indanediones ( 4 ) , ( S t r u c t u r e 2), were developed as r o d e n t i c i d e s . Warfarin f i r s t came i n t o wide usage as a r o d e n t i c i d e i n 1950 and v i r t u a l l y supplanted a l l other m a t e r i a l s then i n use. In the case o f a l l these e a r l y m a t e r i a l s , m u l t i p l e b a i t a p p l i c a t i o n s were needed to c o n t r o l rodent populations which, while making the m a t e r i a l s s a f e r t o use than the a v a i l a b l e acute poisons, c u r t a i l e d t h e i r use i n underdeveloped and l e s s a f f l u e n t c o u n t r i e s because of the l a r g e q u a n t i t i e s of b a i t that must be placed t o destroy the populations of rodents. Within the f i r s t decade a f t e r i t and the other a n t i coagulants were f i r s t s u c c e s s f u l l y employed, reports(_5) were received that Norway r a t s i n a farming area i n Scotland were "remarkably t o l e r a n t " t o w a r f a r i n and other a n t i c o a g u l a n t s . A d d i t i o n a l cases o f r e s i s t a n c e were reported i n Norway, Denmark, England, Wales, Germany, and the N e t h e r l a n d s . ( 6 ) The f i r s t evidence o f a n t i c o a g u l a n t r e s i s t a n c e i n the United States came from a study of Norway r a t s l i v i n g on a farm near Raleigh, North Carolina.(J7) They have s i n c e been found i n a great number o f other s i t e s i n the U.S.(8) Soon r e s i s t a n t mice populations a l s o began t o be discovered.(9) The a n t i c o a g u l a n t r e s i s t a n t populations were found to be c r o s s r e s i s t a n t to the v a r i o u s , chronic r o d e n t i c i d e s .


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Bromethalin, an Acute Rodenticide

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Discovery o f bromethalin I n i t i a l search f o r a r o d e n t i c i d e . I n t e r e s t a t L i l l y i n the d i s c o v e r y o f a r o d e n t i c i d e from among our extensive f i l e o f b i o l o g i c a l l y a c t i v e compounds f i r s t began with N. J . A. Gutteridge, a L i l l y chemist working i n England. When he became aware o f the r e s i s t a n c e problem he s t a r t e d a search f o r some r o d e n t i c i d a l l y a c t i v e m a t e r i a l s that would c o n t r o l r e s i s t a n t rodent populations. He appreciated the f a c t that although t o x i c i t y i s an e s s e n t i a l p r e r e q u i s i t e f o r an e f f e c t i v e r o d e n t i c i d e , i t was not the s o l e c r i t e r i o n upon which an i d e a l r o d e n t i c i d e i s based. He o u t l i n e d , i n one o f the most extensive reviews o f r o d e n t i c i d e s to date(10), other features o f an i d e a l r o d e n t i c i d e as f o l l o w s : (i) (ii) (iii) (iv) (v) (vi) (vii) (viii) (ix)

The t o x i c a c t i o n should be slow t o allow animal t o consume a l e t h a l dose, The poison should not be unpalatable, and should p r e f e r a b l y be o d o r l e s s , Symptoms o f acute poisoning should be absent; no b a i t shyness. The manner o f death should not arouse s u s p i c i o n s i n s u r v i v i n g animals, The poison should be s p e c i f i c t o the species t o be controlled. No d i f f e r e n c e i n s u s c e p t i b i l i t y due t o age, sex, o r s t r a i n should be present. There should be no danger o f secondary poisoning through animals e a t i n g poisoned rodents. No immunity o r build-up o f t o l e r a n c e t o the poison should develop. The chemical compound i n the b a i t should be s t a b l e under v a r i e d environmental c o n d i t i o n s .

Although Gutteridge was u n s u c c e s s f u l i n f i n d i n g a compound that would s a t i s f y these c r i t e r i a , h i s work sparked our i n t e r e s t i n t h i s problem. This i n t e r e s t i n r o d e n t i c i d e s was f u r t h e r increased by the d i s c o v e r y o f the a n t i c o a g u l a n t a c t i v i t y o f a s e r i e s of imidazo(4,5b)pyridines ( S t r u c t u r e 3), which were s t r u c t u r a l l y so d i s s i m i l a r t o the known a n t i c o a g u l a n t s that they were f i r s t thought t o have a d i f f e r e n t mode o f a c t i o n . Subsequent s t u d i e s ( l l ) i n d i c a t e d that i n s p i t e o f the t o t a l l y d i f f e r e n t chemical s t r u c t u r e o f the imidazo(4,5b)pyridines, these compounds suppressed the same f o u r v i t a m i n Κ dependent procoagulants that w a r f a r i n supresses. Further, rodents r e s i s t a n t t o the warfarin-type o f a n t i c o a g u l a n t s were a l s o r e s i s t a n t t o these m a t e r i a l s . T h i s continues t o be true f o r a l l the s o - c a l l e d "second g e n e r a t i o n " a n t i c o a g u l a n t s now a v a i l a b l e .

American Chemical Society Library 1155 16th St. N. w. In Pesticide Synthesis Through Rational Approaches; Washington, 0 . C. 20038Magee, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1984.

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Br 3

N0

2

4


X = Halogen, Trifluoromethy 1 R = Hydrogen, Hydroxy1, O - a l k y l Ρ = Perfluoroalkyl

Bromethalin

The discovery of the a c t i v i t y of the diphenyl amines. At t h i s point there began a sequence of events that f i n a l l y l e d to the the discovery of the diphenyl amine r o d e n t i c i d e bromethalin (Structure 4). Our i n t e r e s t i n the chemistry of the diphenylamines i n i t i a l l y came about because of the discovery that they had f u n g i c i d a l a c t i v i t y against a number of plant diseases (12). A large number of these compounds were synthesized i n order to develop a f u n g i c i d a l 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 . One of the most promising of these f u n g i c i d e s i n greenhouse t e s t s , was t r i c h l o r o - t r i n i t r o diphenyl amine (Structure 5).

CI

N0

2

5 This compound, targeted as a f o l i a r a p p l i e d f u n g i c i d e , was a c t i v e against diseases of grape, e s p e c i a l l y downy mildew, and so a l a r g e sample was prepared f o r p o s s i b l e e v a l u a t i o n under f i e l d conditions. Before a p o t e n t i a l f u n g i c i d e i s t e s t e d on a l a r g e s c a l e i n a f i e l d t e s t program, an e v a l u a t i o n i s c a r r i e d out t o determine i f there i s any p o t e n t i a l danger to the a p p l i c a t o r . While undergoing t h i s routine t o x i c o l o g i c a l hazard e v a l u a t i o n , i t was noted that t h i s compound had an o r a l L D 5 0 of 3.2 mg/kg i n Harlan r a t s . On the b a s i s of t h i s r e s u l t , we decided that compound _5 was too hazardous to be evaluated as a f o l i a r



4.



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fungicide. Instead we submitted the compound to a r o d e n t i c i d e feeding study. I t proved to be t o x i c to r a t s i n t h e i r d i e t at 25 ppm and i t appeared that the onset of t o x i c i t y was delayed. With a t e s t i n g system a l r e a d y i n place from our work with the a n t i c o a g u l a n t s , we began a search f o r other r o d e n t i c i d a l diphenyl amines from among the diphenyl amines synthesized f o r t h e i r fungicidal activity. Since the r e l a t i v e t o x i c i t y of the other analogs made f o r the f u n g i c i d e program was unknown, we set out t o determine what aspects of the s t r u c t u r e of diphenyl amines made the compounds t o x i c . To accomplish t h i s we u t i l i z e d a screen o r i g i n a l l y e s t a b l i s h e d to determine the t o x i c i t y of drug candidates. The t e s t used was one to determine the t o x i c i t y of compounds to mice, i n which groups of three mice were i n j e c t e d i n t r a p e r i t o n e a l l y (I.P.) with an a c a c i a suspension of the t e s t compound u n t i l a l e v e l was reached where no t o x i c i t y was observed. This screen avoids many of the p i t f a l l s of f e e d i n g s t u d i e s s i n c e the t e s t compounds cannot be r e j e c t e d . By means of t h i s t e s t , we were a b l e to semi-quantify the t o x i c i t y of a l a r g e number of diphenyl amines very r a p i d l y . Some of the r e s u l t s obtained can be seen i n Appendix I. Once we determined the t o x i c i t y by t h i s I.P. route, we then t e s t e d the most a c t i v e compounds i n a non-choice f e e d i n g t e s t i n which the t e s t compounds were incorporated i n t o a r a t d i e t and fed to weanling and a d u l t Sprague-Dawley r a t s f o r ten days. This t e s t required f i v e r a t s per dose l e v e l and the l e v e l s were from 200 ppm to 10 ppm. D a i l y feed consumption data as w e l l as necropsy data on the dead and moribund r a t s were c o l l e c t e d . There appeared to be a good c o r r e l a t i o n between the mouse t o x i c i t y data from an I.P. route and the r a t feeding study (Appendix I I ) . With the r e s u l t s from these t e s t s we were a b l e to d e f i n e the "A" r i n g s u b s t i t u t i o n requirements r e q u i r e d to make the t r i n i t r o diphenyl amines t o x i c to r a t s . Since t o x i c i t y i s a necessary but not s u f f i c i e n t q u a l i t y of a r o d e n t i c i d e , the next phase of the t e s t i n g was conducted to determine the a c c e p t a b i l i t y of the t r e a t e d d i e t to r a t s and mice. The t e s t c o n s i s t e d of twenty r a t s per treatment l e v e l with at l e a s t twenty c o n t r o l animals. The t e s t s were run from three to ten days, with the feed consumption data and necropsy data recorded f o r each animal. The d e s i r a b l e candidate compounds should comprise at l e a s t 30% of the t o t a l d i e t of the r a t s and should have a m o r t a l i t y of 90% or more. The most a c t i v e r o d e n t i c i d a l compound turned out to be our i n i t i a l l e a d , 2 , 4 , 6 - t r i c h l o r o - 2 * , 4 , 6 ' - t r i n i t r o d i p h e n y l amine (Structure 5). This was t e s t e d under a v a r i e t y of c o n d i t i o n s with white laboratory r a t s and mice. We were able to determine from these t e s t s that, even g i v e n a choice, l a b o r a t o r y r a t s and mice would consume enough of the t o x i c a n t to cause 80% l e t h a l i t y . To determine how a c t i v i t y against l a b o r a t o r y rodents t r a n s l a t e d i n t o a c t i v i t y against w i l d rodents, we asked Dr. W i l l i a m B. Jackson at the Center f o r Environmental Research f



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and S e r v i c e s , Bowling Green U n i v e r s i t y , to conduct a study using compound 5 on some o f h i s w i l d rodent populations. The t r i a l s he ran on w i l d r a t s and mice p a r a l l e l l e d our own s t u d i e s on laboratory r a t s and mice, but h i s r e s u l t s were q u i t e d i f f e r e n t from ours. In h i s studies with w i l d Norway r a t s (Rattus norvegicus), the r a t s t o t a l l y r e j e c t e d the treated d i e t , a c t u a l l y choosing not t o eat rather than eat the t o x i c a n t . The r e s u l t s with house mouse (Mus musculus) proved e q u a l l y u n s a t i s f a c t o r y . What we had was a very t o x i c m a t e r i a l that was not r e a d i l y acceptable t o w i l d rodents, even i n a non-choice t e s t . We r e a l i z e d that i n order to develop one of these diphenylamines as a r o d e n t i c i d e , we had t o f i n d a way t o get rodents t o eat a t o x i c amount of the t o x i c a n t . We were convinced that the novel r o d e n t i c i d a l mode o f a c t i o n o f these m a t e r i a l s ( i . e . non-anticoagulant) would be v a l u a b l e i f we could f i n d a way to get rodents t o consume them, so we approached the problem as one of e i t h e r f i n d i n g a analog of 5^ that would be more t o x i c o r more acceptable. I f t h i s approach f a i l e d , as a l a s t r e s o r t we could look at ways to make formulations more acceptable. The search f o r a more a c t i v e diphenyl amine concentrated on the s u b s t i t u e n t s on the "B r i n g , s i n c e we were confident that we had d e f i n e d the s u b s t i t u t i o n patterns on the "A" r i n g . A 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 study (SAR) was c a r r i e d out with the r e s u l t s seen i n Appendix I I I . Only one s u b s t i t u t i o n seemed to be s u p e r i o r to the t r i n i t r o ; the s u b s t i t u t i o n of one o f the ortho n i t r o groups with a trifluoromethy 1. A new SAR u s i n g the Mouse T o x i c i t y Test as a screen was conducted, holding "B" r i n g constant with 2 - t r i f luorome thy 1-4, 6 - d i n i t r o phenyl and s u b s t i t u t i n g the "A" r i n g . The r e s u l t i n d i c a t e d that the same s u b s t i t u t i o n pattern that l e d t o the most a c t i v e t r i n i t r o compounds h e l d f o r the 2 , 4 - d i n i t r o - 6 - t r i f l u o r o m e t h y 1 compounds, except that the l a t t e r compounds were more a c t i v e (Appendix I V ) . However, even these more t o x i c compounds were d i s c r i m i n a t e d against i n our c h o i c e - e f f i c a c y s t u d i e s . Before we got involved i n the extensive delays and expense i n c u r r e d i n screening a l a r g e number o f these compounds i n free choice e f f i c a c y t e s t s , t e s t s which might not t r a n s l a t e i n t o e f f i c a c y against w i l d rodents, a means was sought t o generate a more acceptable compound by choosing the most "rodent-acceptable atoms" i n the "A" r i n g from among the most a c t i v e s u b s t i t u e n t s and by s u b s t i t u t i n g on the amine n i t r o g e n . We i n i t i a t e d a l i t e r a t u r e search t o determine the kinds o f f u n c t i o n a l groups that a t t r a c t rodents. The object o f the search was t o t r y t o d e f i n e the aromatic s u b s t i t u e n t s on r i n g "A" that have been shown to have a t t r a c t a n t p r o p e r t i e s . This e f f o r t met w i t h no success. Much has been published on the types o f feed g r a i n s and mixtures thereof that are p r e f e r r e d by rodents but l i t t l e on the types of chemicals. While searching the l i t e r a t u r e f o r such p o s i t i v e information, M


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i t became evident that while the work on chemical a t t r a c t a n t s had been very q u a l i t a t i v e , the search f o r r e p e l l e n t s had been put on a rather formal s c i e n t i f i c b a s i s . Working f o r the F i s h and W i l d l i f e S e r v i c e , U.S. Dept. o f the I n t e r i o r , B e l l a c k and DeWitt(13) developed an equation f o r a r e p e l l e n c y Index, K, whose f i n a l form was: K=100-1/100W(8T +4T +2T +T4)(U +U +2U3+4U4+8X)


1

2

3

1

2

where T^....T4 represents the d a i l y consumption o f t r e a t e d food; U^...U4 represents the d a i l y consumption o f untreated food, X the r e s i d u a l untreated food and W the kilogram body weight of the animals. Many compounds were t e s t e d using t h i s equation a t both the F i s h and W i l d l i f e Service and a t the Army's Natick Laboratory, and the i t s u s e f u l n e s s i n determining r e p e l l e n c y was v e r i f i e d . On examining t h e i r r e s u l t s , we were s t r u c k by the f a c t that, p e r i o d i c a l l y , negative Κ values occurred. Examination o f the equation shows that t h i s can o n l y happen when l a r g e amounts o f the t r e a t e d d i e t were eaten e a r l y and/or untreated m a t e r i a l was untouched. We i n t e r p r e t e d the f a c t that compounds had l a r g e negative Κ values as an i n d i c a t i o n that they might a c t u a l l y a c t as a t t r a c t a n t s . Although a v a r i e t y of s t r u c t u r e s were represented by these negative Κ v a l u e s , among the s u b s t i t u t e d aromatic compounds a p a t t e r n was observed. Whereas p o l y c h l o r i n a t i o n o f t e n was a s s o c i a t e d with the r e p e l l e n c y o f compounds, polybromination appeared t o confer " a n t i r e p e l l e n c y " e f f e c t s . The K-values shown i n Table I i l l u s t r a t e t h i s p o i n t . Although " a n t i r e p e l l e n c y " i s n ' t n e c e s s a r i l y a d i r e c t measure o f a t t r a c t i v e n e s s o f compounds, i t gave us a r a t i o n a l e f o r our choice o f s u b s t i t u e n t s on the "A" r i n g .

Table I Structures w i t h a negative r e p e l l e n c y index (K = negative v a l u e ) COMPOUND Κ Value A c e t i c a c i d , 1,3,6-tribromo-2-naphthalenyl e s t e r -63 2,4-Dibromo-l-(2,4-dibromophenoxy)benzene -213 1,3,5-Tribromo-2-(2,4,6-tribromophenoxy)benzene -137 2-Amino-l,3-dibromo-4-methy1-9,1O-anthracenedione.... -88 9-Bromoanthracene. · · -77 4-Amino-2, 6-dibromopheno 1 -134 We a l s o recognized that compound _5 possessed a very a c i d i c proton on the amine n i t r o g e n and that many known compounds that e x h i b i t odor and t a s t e c o n t a i n a c i d i c SH, OH, o r NH protons. We thought i t d e s i r a b l e t o replace the proton on the amine with an



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a l k y l group to mask the a c i d i t y . We hoped to r e t a i n the t o x i c i t y of the compound while a v o i d i n g the t a s t e and/or odor c h a r a c t e r i s t i c s that we suspected rodents a s s o c i a t e d with these compounds. Because the most a c t i v e diphenyl amines are s u b s t i t u t e d i n the 2,6- and 2 6 p o s i t i o n s , causing a s t e r i c b a r r i e r to replacement of the amine proton, the methylation could not be c a r r i e d out on the already s u b s t i t u t e d diphenyl amine. The synthesis of these m a t e r i a l s required new, stepwise chemical approaches (Scheme I ) . I


I

il SCHEME I



4.



53

In the case o f the p i c r y l d e r i v a t i v e s , the coupling o f 2,4,6trichloro-N-methyl a n i l i n e , 6, with p i c r y l c h l o r i d e , ( F i g u r e 7), gave the d e s i r e d N-methyldiphenyl amine i n the t r i n i t r o s e r i e s (Figure 8). However, the same methylated a n i l i n e , when reacted with 2 - c h l o r o - 3 , 5 - d i n i t r o b e n z o t r i f l u o r i d e , (Figure 9) , c o n s i s t e n t l y demethylated t o the N-H m a t e r i a l , (Figure 10), instead o f g i v i n g the d e s i r e d product, (Figure 11). This was true regardless o f the bases, s o l v e n t s , and c o n d i t i o n s used. Attempts t o use other N-alkylated a n i l i n e s , i n c l u d i n g N-methy1 a n i l i n e i t s e l f a l s o l e d to the d e a l k y l a t e d m a t e r i a l . F i n a l l y an approach was developed whereby 11 could be generated by f i r s t r e a c t i n g 6 with the d e s - n i t r o compound 12 t o form the d e s - n i t r o diphenyl amine JJ3, and then n i t r a t i n g under mild c o n d i t i o n s with ammonium n i t r a t e i n t r i f l u o r o a c e t i c a c i d . Side-by-side comparisons o f the a c c e p t a b i l i t y o f the N-methyl m a t e r i a l 1Λ, versus i t s N-H analog TO, i n d i c a t e d that by b l o c k i n g the f r e e amine we could make the diphenyl amine f a r more acceptable t o r a t s , ( i . e . Table I I ) . Table I I

(a) Treated Diet Males14g Females- l l g Total25g Diet

Untreated Diet 17g 24g 41g

37.9%

62.1%

15g 29$ 44g 62.9%

5g 21g 26g 37.1%

10

CR

.CI

O" "w' C f c H

c

CI

N o

NO,

11

10

5 Males 5 Females Total % o f Diet

vs

11

(b) lOppm 11 lOppm 10 i n Diet i n Diet 63g 30g 5 Males 62g 5 Females 38§ 125g 68g Total 65.4% % o f Diet 35.( >%

(a) Toxicant l e v e l 100 ppm i n treated d i e t (b) The animals were given a choice o f t o x i c a n t s


54


On the b a s i s of the i n f o r m a t i o n we had developed, i t appeared that the compound o f choice should be polybrominated i n the "A r i n g , be s u b s t i t u t e d with a 2-trifluoromethy1-4,6-dinitrophenyl group i n r i n g "B", and have a methyl group on the amine nitrogen. S y n t h e t i c a l l y t h i s was a problem since the a p p r o p r i a t e l y s u b s t i t u t e d N-H diphenyl amine cannot be d i r e c t l y converted t o the N-methy1 analog, and the routes used i n the case of the trichloro-N-methyl m a t e r i a l l e d t o poor r e s u l t s . A s y n t h e t i c route was f i n a l l y developed which resolved most of the problems (Scheme I I ) . A diphenyl amine u n s u b s t i t u t e d i n the A r i n g was synthesized from 2 - c h l o r o - 3 , 5 - d i n i t r o b e n z o t r i f l u o r i d e , 9_ and a n i l i n e , and t h i s was methylated u s i n g very m i l d methylating c o n d i t i o n s ; i . e . dimethyl s u l f a t e and sodium carbonate i n acetone t o form the des-brominated compound, (Structure 15). The bromination of JL5 took place i n two steps. The f i r s t two bromines added r e a d i l y while the t h i r d bromine required the use of N-bromosuccinimide (NBS) i n s u l f u r i c a c i d , t o a f f o r d the d e s i r e d m a t e r i a l 4 i n high y i e l d . ,f


fl

Br

N0

2

4 SCHEME I I


M

4. DREIKORN AND O'DOHERTY


55

F i n a l l y , choice t e s t s were i n i t i a t e d both at L i l l y and at Bowling Green on the a c c e p t a b i l i t y and e f f i c a c y o f t h i s m a t e r i a l as a r o d e n t i c i d e ( J A ) . A l l laboratory t e s t s i n d i c a t e d that compound 4 was r e a d i l y accepted by r a t s and mice (Appendix V ) . In some cases rodents a c t u a l l y seemed to p r e f e r the t r e a t e d d i e t over the b a s a l d i e t . Compound 4 was assigned the L i l l y code EL-614, and, somewhat l a t e r , the generic name bromethalin.


Development o f bromethalin as a r o d e n t i c i d e Once bromethalin had been i d e n t i f i e d as a compound that was both t o x i c t o rodents a t low dose l e v e l s and acceptable to them i n b a i t , i t was necessary to develop information on i t s performance under f i e l d c o n d i t i o n s and t o l e a r n more about the mode o f a c t i o n . F i e l d e v a l u a t i o n of bromethalin. Both indoor and outdoor f i e l d t r i a l s a g a i n s t Norway r a t and house mouse populations were conducted under an EPA experimental use permit i n a number o f geographical l o c a t i o n s i n the U.S.( 1_5,_16) In a l l cases 0.005% bromethalin was used i n a b a i t c o n t a i n i n g 65% corn meal, 25% r o l l e d o a t s , 5% sugar and 5% c o r n o i l ( E . P . A . d i e t ) . Three census techniques were used before and a f t e r bromethalin treatment i n order to estimate c o n t r o l o f the rodent population, followed by snap t r a p p i n g at the end o f the t e s t as an a d d i t i o n a l measure of c o n t r o l . F i e l d r e s u l t s i n d i c a t e that bromethalin b a i t was e x c e p t i o n a l l y e f f e c t i v e against both Norway r a t s and house mouse populations. B a i t acceptance was e x c e l l e n t with no signs o f bait-shyness observed. It was a l s o e s s e n t i a l to determine how e f f e c t i v e bromethalin was a g a i n s t w a r f a r i n - r e s i s t a n t r a t s and mice. Such animals, whose r e s i s t a n c e t o a n t i c o a g u l a n t s had been determined by World Health O r g a n i z a t i o n tests,(l_7) were subjected to a standard EPA choice feeding e f f i c a c y t e s t with bromethalin at 0.005% i n the treated d i e t . The r e s u l t s i n d i c a t e (Appendix 6) that 90% o f the animals were k i l l e d and that consumption patterns were s i m i l a r to those observed i n other choice t e s t s . A f i e l d t r i a l was conducted using 0.005% bromethalin b a i t against a r e s i s t a n t p o p u l a t i o n o f house mice i n a p o u l t r y house. Resistance had been v e r i f i e d by using the standard WHO l a b o r a t o r y procedure. About two-thirds o f the mice t e s t e d were r e s i s t a n t t o warfarin. C o n t r o l was achieved under extremely d i f f i c u l t c o n d i t i o n s ( i . e . , extremely l a r g e p o p u l a t i o n w i t h an abundant food supply i n a complex p h y s i c a l environment). Toxicology o f bromethalin Acute t o x i c i t y of bromethalin. Acute LD5QS were determined i n v a r i o u s t a r g e t and non-target species by gavage a d m i n i s t r a t i o n o f bromethalin s o l u b l i z e d i n p o l y e t h y l e n e - g l y c o l 200. These data


PESTICIDE SYNTHESIS T H R O U G H RATIONAL APPROACHES

56


i n d i c a t e that s i m i l a r doses were r e q u i r e d to produce l e t h a l i t y i n a l l species t e s t e d . Nevertheless, species s e l e c t i v i t y i s obtained with bromethalin b a i t s s i n c e rodents w i l l consume l a r g e r q u a n t i t i e s of food per u n i t body weight than l a r g e r animals. Secondary t o x i c i t y . In a study run to determine i f bromethalin treated r a t s pose any threat to animals that happen to eat the t r e a t e d carcass, r a t s that had been fed bromethalin b a i t f o r 16 hours at a l e v e l of 0.005% i n t h e i r d i e t s u f f i c i e n t to k i l l 95%, were k i l l e d , ground i n t o "ratburger", and fed to dogs c o n d i t i o n e d to e a t i n g r a t meat. The dogs were fed t h i s d i e t f o r two weeks. At the end of t h i s time, none of the dogs showed any signs of t o x i c i t y . It was concluded that dogs consuming r a t s that had consumed a l e t h a l dose of bromethalin d i d not r e c e i v e enough t o x i c a n t to produce any signs of t o x i c i t y . Mode of a c t i o n of bromethalin. Signs of t o x i c i t y observed i n l a b o r a t o r y or f i e l d e f f i c a c y s t u d i e s u s i n g l a r g e , s i n g l e doses i n c l u d e tremors, one or two episodes of c l o n i c convulsions, and p r o s t r a t i o n , with death u s u a l l y o c c u r r i n g w i t h i n 36 hours. In c o n t r a s t , s u b l e t h a l doses from i n g e s t i o n of repeated small q u a n t i t i e s of b a i t or from doses l e s s than the L D 5 0 can produce l e t h a r g y , h i n d - l e g weakness, loss of muscle tone and paralysis.(18) These e f f e c t s have been observed i n laboratory s t u d i e s and have been shown to be r e v e r s i b l e . Experiments on the p h y s i o l o g i c a l and biochemical mechanisms of a c t i o n suggest that bromethalin uncouples o x i d a t i v e phosphorylation i n c e n t r a l nervous system mitocondria(19). This could lead to a decreased production o f ATP, a diminished a c t i v i t y of Na /K ATPase, and a subsequent f l u i d b u i l d up manifested by f l u i d - f i l l e d vacuoles between the myelin sheaths. T h i s vacuole formation i n t u r n leads to an increased c e r e b r o s p i n a l f l u i d pressure and increased pressure on the nerve axons, y i e l d i n g a decrease i n nerve impulse, p a r a l y s i s , and death. At present i t i s not c l e a r whether the same sequence o f a c t i o n i s r e s p o n s i b l e f o r b r o m e t h a l i n s convulsive and p a r a l y t i c a c t i o n s observed at d i f f e r e n t doses; however, i t i s l i k e l y that some areas of the b r a i n would be more s e n s i t i v e to ATP d e p l e t i o n and f l u i d imbalance than o t h e r s . The c e r e b r a l edema produced by s u b l e t h a l doses of bromethalin can be ameliorated by treatment with an osmotic d i u r e t i c and corticosteroids. Pathology from s u b l e t h a l doses, even without treatment have been shown to be r e v e r s i b l e . +

+

1

Conclusion Bromethalin i s a unique, h i g h l y potent r o d e n t i c i d e which s a t i s f i e s most of the c r i t e r i a of an i d e a l r o d e n t i c i d e . I t o f f e r s a d i s t i n c t advantage over c u r r e n t l y used acute and anticoagulant r o d e n t i c i d e s because of i t s unique mode of a c t i o n . Bromethalin


4. DREIKORN AND O'DOHERTY


57


provides a l e t h a l dose t o rodents i n a s i n g l e feeding with death g e n e r a l l y delayed two or three days. Rodents do not d i s c r i m i n a t e against bromethalin b a i t , t h e r e f o r e e x c e l l e n t b a i t acceptance i s achieved. I t has been shown t o have a mode o f a c t i o n d i f f e r e n t from the a n t i c o a g u l a n t s and i s very e f f e c t i v e against a n t i c o a g u l a n t - r e s i s t a n t r a t s and mice. Properly used i t poses no danger t o non-target s p e c i e s . The d i s c o v e r y and development o f bromethalin was the r e s u l t of both s e r e n d i p i t y and the t i m e l y a p p l i c a t i o n o f the s c i e n t i f i c method. The c o n t r i b u t i o n s o f s c i e n t i s t s from many d i s c i p l i n e s were necessary f o r i t s success. Acknowledgments We would l i k e t o thank our L i l l y c o l l e a g u e s Kenneth E. Kramer A l b e r t J . C l i n t o n , Dr. A l v i n M e l l i e r e , Dr. Robert van L i e r , and Steven Spaulding f o r t h e i r many c o n t r i b u t i o n s to the development of bromethalin. We would a l s o l i k e t o recognize the important advisory r o l e played by Dr. W i l l i a m B. Jackson (Bowling Green State U n i v e r s i t y ) .

References 1. 2.

3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.

Stahmann, M. A.; Huebner, C. F . ; Link, K. P. J . Biol. Chem., 1941, 138, 513 Overman, R. S.; Stahman, Μ. Α.; Huebner, W. R.; Sullivan, W. R.; Spero, L . ; Doherty, D. G.; Ikawa, M.; Graf, L . ; Roseman, S.; Link, K. P. J . Biol. Chem.,1944, 153, 5. O'Connor, J . A. Research, 1:334-336, 1948 Kabat, H.; Stohlman, E. F . ; Smith, M. I. J . Pharmacol, 1944, 80, 160 Boyle, M. Nature 4749:519, 1960 Drummond, D. C. Symp. Zool. Soc. Lond., 26:351-367 Jackson, W. B.; Kaukeinen, D. E. Science, 176 1343-1344, 1972 Jackson, W. B.; Ashton, A. D. 8th Steenbock Symp., Vitamin Κ metabolism and Vitamin K-dependent proteins, University of Wisconsin, Madison, 1979 Rowe, F. P.; Redfern, R. J . Hygiene, 63, 417-425, 1965 Gutteridge, N. J . A. Chemical Society Reviews, vol 1(3) 1972 Bang, N. U.; O'Doherty, G. O. P.; Barton, R. D. Clinical Research, 23 no.4, 251 (1975) Dreikorn, Β. Α.; Kramer, Κ. E. U.S.Patent 4,381,312 Bellack, E . ; DeWitt, J . B. Journal of the American Pharmaceutical Association, ν 38, pp 109-112, 1949


58 14. 15. 16. 17.


18. 19.

PESTICIDE SYNTHESIS THROUGH RATIONAL APPROACHES Dreikorn, Β. Α.; O'Doherty, G. O. P.; Clinton, A. J.; Kramer, Κ. E. Proceedings Brit. Crop Protection Conf., 1979 491-498 Jackson, W. B.; Spaulding. S. R.; Dreikorn, Β. Α.; van Lier, R. B. L. Proc. Tenth Vert. Pest. Conf., (R.E. Marsh, Ed.), Univ. of Calif.Press, Davis, Calif. Spaulding, S. R.; Jackson, W. B. Vertebrate Pest and Management Materials (in press) World Health Organization Technical Report Series 443: 140-147, 1970 van Lier, R. B. L., Ottosen, L. D., Hanasono, G. Κ., and Carter, J . L . ; Proceedings of the 19th Annual Meeting of the Society of Toxicology, 1980 van Lier, R. B. L., and Ottinger, L. D. The Toxicologist 1(1), 114, (1981)


4.

DREIKORN A N D O'DOHERTY

59



Appendix I

PPM

S u b s t i t u t i o n on Ring "A"

(a) < > Dose L e v e l s i n Mouse T o x i c i t y

Mono s u b s t i t u t e d C l , Br, F, CN, 1000 300 methyl, e t h y l , n i t r o , methoxy.

3/3

100

30

10

2/3 3/3 1/3 2/3 2/3

0/3 0/3 0/3 0/3 0/3

3/3

3/3

3/3

0/3

3/3

3/3

3/3

3/3

3/3 3/3 3/3 3/3

3/3 0/3 3/3 0/3

3/3

0/3

3/3

0/3

3/3 3/3 3/3 3/3

0/3 3/3 3/3 3/3

3/3 2/3 3/3

0/3 0/3 3/3

Test 3

2,3-, 2,4-,

3/3 3/3

(a).The Mouse t o x i c i t y data i s expressed as the r a t i o o f the number o f dead mice over the t o t a l t e s t e d a t each l e v e l


60



Appendix I I

Substituents on Ring "A"

MTT(mg/kg) Feeding Study(PPM) (300) (300) (1000) (3) (3) (1000) (30) (300) (10) (300) (1000) 1/3 (100) (30)

0/5 0/5 0/5 5/5 5/5 0/5 5/5 0/5 5/5 0/5 0/5 0/5 5/5

(200) (200) (200) (15) (15) (200) (100) (200) (30) (200) (200) (200) (100)

(a)Mouse T o x i c i t y Test expressed as a r a t i o o f the number o f dead mice over the t o t a l t e s t e d at the lowest l e v e l t o x i c i t y was observed. (b)Norway r a t feeding study expressed as a r a t i o o f the number o f dead r a t s over the number t e s t e d a t lowest l e v e l t o x i c i t y was observed.


4.




Appendix I I I

MTT(a)( g/ g) M

N0 N0 CF N0 N0 N0 H N0 N0 CH N0 i -Propyl N0 t-Butyl N0 CN N0 S0 NH S0 K N0 CH N0 S0 NH N0 C0 H N0 C0 Et N0 N0 CI CI N0 CF N0 2

2

2

3

2

2

2

2

3

2

2

2

2

2

2

2

3

3

2

2

2

2

2

2

2

2

2

2

3

2

N0 N0 H H N0 N0 N0 N0 N0 N0 N0 N0 N0 N0 N0 N0 N0

2

2

2

2

2

2

2

2

2

2

2

2

2

2

2

3/3 2/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 0/3 3/3

(3) (1000) (1000) (1000) (1000) (1000) (1000) (1000) (1000) (1000) (1000) (1000) (1000) (1000) (1000) (1000) (3)

K

FEEDING^) (ppm)

5/5 (15) 0/5 (200)

5/5 (10 ppm)

(a)Mouse T o x i c i t y Test expressed as the r a t i o of the number o f dead mice over the t o t a l number of animals at the lowest l e v e l t o x i c i t y was observed. (b)Norway r a t feeding study expressed as d e a d / t o t a l a t the lowest l e v e l i n feed that t o x i c i t y was observed.


61


Appendix IV


N0

a

SUBSTITUENTS ON "A"

3.5- D i c h l o r o 3,4-Dichloro 2,4-Dibromo 2.6-Dibromo 2,4-Difluoro 2.4- D i n i t r o 2.5- D i f l u o r o 3, 5-Di t r i f luorome thy 1 3.4- Dimethyl 2.5-Dimethoxy 2,4,6-Trichloro 2,4,6-Tribromo 2,4,6-Trifluoro 2.6-Dichloro-4-bromo 2-Chloro-4,6-dibromo 2,4,6-Trimethyl 3,4,5-Trimethoxy 2,6-Dibromo-4-trif luorome thy 1

2

MTT< ) (mg/kg)

•

1/3 3/3 2/3 1/3 2/3 2/3 2/3 3/3 0/3 0/3 3/3 1/3 3/3 2/3 2/3 1/3 0/3 2/3

(3) (10) (3) (30) (30) (30) (30) (3) (1000) (1000) (10) (3) (10) (10) (1) (1000) (1000) (10)


4.



63

Appendix V Choice E f f i c a c y Studies with 0.005% Bromethalin


Rattus norvegicus Rattus norvegicus Rattus norvegicus Mus musculus Mus musculus Mus musculus Rattus r a t t u s

£/ £/

Dead Total

Test

Strain

Species

3-day 3-day 1-day 3-day 3-day 1-day 3-day

Wistar Wild Wild ICR Wild Wild Wild

136/140 19/20 20/20 19/20 20/20 19/20 20/20

1/ 3.4 2.8 3.6 3.1 3.3 3.4 2.5

Bait

*/

£/

51.2 48.9 56.2 54.4 45.5 47.5 46.0

3.04 1.79 2.65 7.47 5.10 3.47 2.27

Average days u n t i l death; ]>/ Percent acceptable; Bromethalin consumed mg/kg.

Appendix VI E f f i c a c y of 0.005% Bromethalin Against Warfin-Resistant Rodents Laboratory 3-Day Choice Test With Warfarin-Resistant Wild Norway Roats and House Mice a/ Species Rattus norvegicus Mus Musculus

Bromethalin Consumed Mortality (mg/kg)

EPA Placebo

±1 Bromethalin

10 M 10 F

8.2 21.3

7.0 7.7

10/10 9/10

1.0 1.5

10 M 10 F

3.4 1.6

3.3 1.3

8/10 10/10

5.9 2.6

No. Sex

£/ Average 3-Day Consumption (g) RECEIVED December 23,

1983


Pesticide Synthesis Through Rational Approaches - American

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