First Symposium on Chemical-Biological Correlation, May 26-27, 1950 (1951)

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EFFECT OF STRUCTURAL CHANGES IN PLANT INSECTICIDES AND RELATED SYNTHETIC COMPOUNDS ON THEIR TOXICITY TO INSECTS by H.L. Haller Bureau of Entomology and Plant Quarantine Agricultural Research Administration U.S. Department of Agriculture vVashin^ton, D. C.

246 The spectacular results obtained in recent years in the development of new synthetic- organic insecticides and the wide publicity accorded them have tended to obscure the value of the insecticides derived from plants. Valuable as the chlorinated hydrocarbon and organic phosphorus-containing insecticides are, they have not completely displaced the naturally occurring organic plant products. Pyrethrum, the rotenone-containing plants derris and cube, and nicotine from tobacco, which are the best known plant insecticides, are still used in sizable quantitites. A few others, such as ryania, quassia, sabadilla, and hellebore, find utilization for specific purposes and in limited localities. Others, such as Heliopsis , Spilanthes, and pellitory are still in the experimental stages of investigation. Although the insecticides derived from plants do not have the long residual action of some of the synthetic organic insecticides, they possess other characteristics that merit their use in controlling agricultural and household insects. In contrast to the chlorinated hydrocarbon insecticides rotenone and pyrethrum present no health hazard from spray residues. Nicotine, although a toxic alkaloid, is very soluble in water and hence, when employed against aphids, mealy bugs, and thrips, is readily removed and therefore presents little or no spray-residue problem. Pyrethrum can be used safely in food establishments, households and dairy barns without concern for toxic residues. Moreover, its rapid paralytic, or knockdown, effect is especially important in the control of disease-carrying insects. Derris and cube are still the only effective insecticides for controlling cattle grubs, a pest that annually causes losses of one hundred million dollars to our livestock industry. Plants have been used as insecticides for several hundred years, but chemical investiga- tions of their active principles are relatively recent. Nicotine was first isolated from tobacco in 1828,°® but sixty-five years elapsed before its structural formula was established. Rotenone, the principal insecticidal constituent of derris and cube, has been known since 1892, '" but it was not until 1932 that its structural formula was determined. 58,59 Structural formulas for the pyrethrins, the insecticidal constituents of pyrethrum, were first proposed in 1924, 80 buj not until 1946 were the detailed structures of these complicated esters established with certainty."" Nicotine, rotenone, and the pyrethrins all have complicated structural formulas, and a comparison of them reveals no common grouping of atoms to which insecticidal action might be ascribed. Numerous derivatives of each of these compounds and closely related products have been prepared and tested for insecticidal properties, but only a few are equal to the natural products. TOBACCO Nicotine and Related Alkaloids The earlier efforts to correlate the chemical structure of plant insecticides with insect toxicity were devoted chiefly to derivatives and analogs of nicotine, partly because its less complicated structure, compared to the pyrethrins and rotenone, facilitated the syntheses of related compounds. Moreover, the availability of two closely related alkaloids, nornicotine" and anabasine,°4 made further comparisons possible. Nicotine (I) is 1-methyl-2-(3-pyridyl)- pyrrolidine, nornicotine (II) is 2-(3-pyridyl)pyrrolidine, and anabasine (III) is 2,3'-pyridyl piperidine.

247 CH3 H^N H : H2 III All three occur naturally in levorotatory form. It is natural to inquire whether insect toxicity is due to the molecule as a whole or to some special grouping of its component parts. The point of attachment of the two rings, optical activity, and other questions present themselves. When tested against Aphis rumicis L. natural levo- nicotine and levo-anabasine were much more toxic than the dextro forms; on the other hand, the natural levo-nornicotine was only slightly more toxic to this insect than the dextro isomer. Both dl-nicotine (I) and dl-nornicotine (II) were much more toxic to Aphis rumicis than the corresponding alpha forms, l-methyl-2-(2-pyridyl)pyrrolidine and 2-(2-pyridyl)pyrrolidine. Tests were also made of dipyridyls, 73, 76 dipiperidyls, 7?* '9 pyridyl piperidines, "*, 79 and pyridyl pyrrolidines,'• ^ as well as of simpler derivatives of pyridine^ and pyrrolidine. 53 with the exception of neonicotine (III), all were distinctly inferior to the three natural alkaloids. Neonicotine, obtained as one of the reaction products of pyridine and sodium, is the optically inactive form of anabasine. Table I summarizes some of the entomological results obtained with nicotine and the more important related compounds. The data were taken from a table compiled by Metcalf. " The toxicity values were obtained from articles by several different authors and therefore represent order of magnitude of relative toxicity rather than precise comparisons. TABLE 1 RELATIVE TOXICITY OF NICOTINE AND ITS DERIVATIVES AND ANALOGS TO APHIS RUMICIS Compound Median lethal concentration Levo-nicotine 1 Dextro -nicotine 5 di- Nicotine 2 d_l- a -Nicotine 31 Levo-nornicotine 0. 5 Dextro -nornicotine 0.7 dl-Nornicotine I

248 TABLE 1 (Cont. ) Compound Median lethal concentration dl-a-Nornicotine Le vo- ana ba sine dl-Anabasine (neonicotine) 2, 3'-Dipyridyl 3, 2'-Pyridyl-piperidine 2, 3'-Dipiperidyl 2-n-Tolyl pyrrolidine Pyrrolidine Pyridine Piperidine 31 0.1 5 100 50 100 50 20 125 25 DERRIS AND CUBE Rote none and Rotenoids These compounds are the principal insecticidal constituents of a number of leguminous fish-poison plants, of which derris and cube are the most important commercially. In the roots of these two plants the rotenone content usually constitutes one-third to one-half of the total ether extractives, the remainder being rotenoids. Rotenoids is the name given to a group of compounds closely related structurally to rotenone. Rotenone5® has the structural formula (IV), and the principal rotenoids - deguelin, ' tephrosin, °- 39 and toxicarol' - the formula (V). IV R = H, R1 = H Deguelin R = OH. R1 = H Tephrosin R = H, R' = OH Toxicarol

249 In rotenone ring E is a substituted dihydrofuran ring, whereas in the three rotenoids it is a substituted pyran. The chemistry of the rotenoids has been summarized by Haller et al. 33 Numerous derivatives of rotenone were obtained in the course of determining its structure. Some of them were tested by Gersdorff, 21 , 23, 24, 25 using goldfish as the test animal in a pro- cedure developed by him. ^ The results obtained are summarized in Table 2. TABLE 2 RELATIVE TOXICITY OF ROTENONE AND SOME OF ITS DERIVATIVES TO GOLDFISH Compound Approximate Relative Toxicity Rotenone Isorotenone Dihydrorotenone Acetyl rotenone Acetyl dihydrorotenone Rotenolone Acetyl rotenolone Dihydrorotenolone Acetyl dihydrorotenolone 1 0. 23 1.4 0. 55 0. 5 0.1 0. 11 0. 2 0. 13 Dihydrorotenone and the other dihydro derivatives differ from the parent compound in that the side chain of ring E (formula IV) has been saturated. In isorotenone the double bond of the side chain has been shifted into the five-membered ring. Acetyl rotenone is an enol acetate (VI), and rotenolone is a hydroxyrotenone, as shown in VII. O OCCH, VI VII

250 With the exception of dihydrorotenone, which was about 1.4 times as toxic to goldfish as rotenone, all the derivatives were much less toxic than the parent compound. However, dihydro- rotenone is no more toxic to insects than rotenone and is less toxic to some. GersdorffZS points out that "each change in chemical constitution effects a characteristic change in toxicity independ- ent of the effect of any other change. " Gersdorff also compared three rotenoids with rotenone in toxicity to goldfish. In these tests all three rotenoids were optically inactive isomers and the rotenone was optically active. At the time.optically active rotenoids were not available and rotenone has not yet been obtained in inactive form. The results are as follows: TABLE 3 RELATIVE TOXICITY OF ROTENONE AND SOME ROTENOIDS TO GOLDFISH Compound Approximate Relative Toxicity Rotenone 1. 0 Deguelin 0.56 Tephrosin 0.23 Toxicarol 0.65 Rotenone and the rotenoids contain asymmetric carbon atoms and optically active isomers of all except deguelin have been isolated in crystalline form from the plant extracts. Evidence has been presented^ that deguelin occurs in the plants as a levo-rotatory isomer, but all attempts to obtain it in crystalline form have resulted in failure. The relative toxicity of rotenone and the optically active and inactive forms of three rotenoids against several insects are summar- ized in Table 4. TABLE 4 RELATIVE TOXICITY OF ROTENONE AND RELATED COMPOUNDS TO INSECTS Compound Approximate Relative Toxicity Test Insect and Reference Rotenone Dihydrorotenone (active) 100 70 >30 House fly (Musca domestica L. ) (82) Silkworm (gpmbyx mori L. ) (75)

251 TABLE 4 (Cont. ) Compound Approximate Relative Toxicity Test Insect and Reference Deguelin (inactive) 10 Bean aphid (Aphis rumicis L,. ) (12) 10 House fly (82) 30 Silkworm (75) Oeguelin concentrate (active) 50 House fly (82) Dihydrodeguelin (active) 50 House fly (82) Dihydrodeguelin (inactive) 13 House fly (82) Tephrosin (inactive) 2 Bean aphid (12) 10 Silkworm (75) Toxic,: rial (active) 7 Bean aphid (12) Toxicarol (inactive) <1 Bean aphid (12) Dehydrorotenone 0 Imported cabbage worm (Ascia rapae L. ) (13) With mosquito larvae as the test insects. Fink and Haller'7 determined the relative toxicity of rotenone, deguelin, and the optically active and inactive forms of isorotenone and di- hydrodeguelin. The results are illustrated graphically in Figure 1. As in the tests by GersdorffZS with goldfish, the optically active compounds were more toxic than the corresponding optically inactive compounds at all concentrations tested. A ACTiVE OiHYOROOCGUELiN A iNACTIVE DIHfOROliKUCl IN O ACTiVE iSOROTENONE • iNACTiVE ISOROTENONE + ROTENONE OEGUELiN CONClNTMTiOn PCH LiTER Fig. 1 Relative toxicity of rotenone, deguelin, and the optically active and inactive forms of isorotenone and dihydrodeguelin

252 It will be noted that in this series of compounds, as with nicotine and related compounds, optical activity plays an important role in insecticidal action. PYRETHRUM Pyrethrins and Cinerins The insecticidal action of pyrethrum is due to a mixture of several closely related esters. From their classical pioneering studies Staudinger and Ruzicka^O concluded that two compounds, designated pyrethrin I and pyrethrin II, were the principal active ingredients. However, studies by LaForge and 1'..i r1in.i '"'.''''.'"' have demonstrated the presence of two other insecticidal esters, which have been designated cinerin I and cinerin II. Both the pyrethrins and the cinerins are high-boiling oils, and all four compounds are closely related in chemical structure. Pyrethrin I and cinerin I are esters of chrysanthemum monocarboxylic acid (VIII) and pyrethrin II and cinerin II are esters of chrysanthemum dicarboxylic acid monomethyl ester (IX). Hs /COOH HK /COOH H VIII IX CH3 CH3 Hol " l H2C - C = 0 HO X XI XII H2 H H H XIa R = -6 — C—C—£— CH2 H2 H H Xlb R = -i—C—C—CH3 The alcoholic component of both pyrethrins is known as pyrethrolone and that of the cinerins as cinerolone. The structural formula originally proposed for pyrethrolone is given in X. More recent studies by LaForge and his associates in this country and Gillam and West in England have necessitated a revision and modification of formula X to that given in XIa. The studies lead- ing to this structure, which is in accord with all the known facts, are excellently summarized by Harper.40 The structural formula of cinerolone ,s given in Xlb. Pyrethrolone and cinerolone differ only in that the side chain of the former is a doubly unsaturated five-carbon straight chain while that of cinerolone is a singly unsaturated four-carbon chain. The accepted structural formulas for the pyrethrins and the cinerins are shown in XIII.

253 Pyrethrin I Pyrethrin II Cinerin I Cinerin III R = CH3 R = CH3OOC R = CH3 R = CH3OOC H2 H H H R' = -C— C—C— C— CH2 R- = -C—C —C-CH, Comparative toxicities of the pyrethrins and the 'cinerins to house flies" are shown in Table 5. Each compound was obtained by esterification of the appropriate naturally occurring dextrorotatory keto alcohol with the proper naturally occurring dextro acid. The esters obtained from the optically inactive keto alcohols and the optically active acids did not differ in toxicity from the compounds with both components optically active". Also with cinerin I, in which the side chain of the keto alcohol has been shown to have a cis configuration, "' no difference in toxicity was noted when the sid* chain had a trans configuration. 27 TABLE 5 RELATIVE TOXICITY OF PYRETHRINS AND CINERINS TO HOUSE FLIES Compound Relative Toxicity Pyrethrin I Pyrethrin II 1 0.25 Cinerin I 0. 69 Cinerin II 0. 17 Both the keto alcohols and the acids of the pyrethrins are unsaturated. Staudinger and Ruzicka^ have shown that most of the insecticidal action of the pyrethrins is destroyed by minor changes in their molecules. Thus, the catalytic hydrogenation of a crude pyrethrin concentrate yielded a product that was insecticidally inactive. In these experiments the cockroach, probably Blattglla germanica L. , was used as the test insect. Similar results were obtained by Haller and Sullivan^® in experiments with house flies. They found that on mild catalytic hydrogenation of of pyrethrum concentrates, containing 63. 5 per cent of pyrethrin I, 6. 5 per cent of pyrethrin II, and the remainder inert constituents, not only the toxicity but also the knockdown was for the greater part destroyed. A pyrethrum concentrate containing 83. 7 per cent of pyrethrin II and 5.4 per cent of pyrethrin I with the remainder inert, although having good knockdown properties, caused so little mortality before hydrogenation that a marked reduction would not normally be

Z54 expected. More recent data^ on the effect of hydrogenation of the doable bonds in pyrethrin I and cinerin I are giren in Table 6. TABLE 6 RELATIVE TOXIC1TY OF SOME HYDROGENATED DERIVATIVES OF THE PYRETHRINS AND CINERINS TO HOUSE FLIES Compound Relative Toxic ity Pyrethrin I 1 Tetrahydropyrethrin I 0. 06 Isodihydropy rethrm I 0. 5 Cinerin I 0.69 Dihydrocinerin I 0. 08 Isodihydrocinerin I 0.35 Tetrahydropyrethrin I was prepared by catalytic hydrogenation of natural pyrethrin I. All attempts to prepare a dihydro compound have thus far failed. " Hydrogenation of the side chain not only reduces toxicity considerably but also destroys the characteristic knockdown effect of the pyrethrins. The dihydrocinerin I was obtained from optically inactive keto alcohol and the optically active dextro acid. " In the preparation of the isodihydro compounds both the keto alcohols and dihydro acid were optically active. SYNTHETIC PYRETHRIN-LIKE ESTERS The destruction of most of the toxicity of both the pyrethrins and the cinerins on satura- tion of the side chains in the keto alcohol portions of their molecules made it of interest to determine the effect of other changes in the side chains, such as changes in length, branching of the chain, position of the unsaturated bond, and stereochemical effects. Methods for the prep- aration of such compounds have recently been described. The excellent method developed by Schechter et al. ^ for the synthesis of cyclopentenolones makes possible the preparation of a wide variety of keto alcohols analagous to cinerolone. The keto alcohols on esterification with chrysanthemum monocarboxylic acid, for which an improved method of preparation has recently been published4, yield the suitably substituted esters. Six substituted cyclopentenolones were prepared. They are represented by the general formula XI, in which R indicates different side chains, which are shown in Table 7. These keto alcohols were esterified with the natural d-trans-chrysanthemum monocarboxylic acid. Two of the keto alcohols were also esterified with the synthetic racemic (dl) cis and trans forms of this acid. Thus there were ten compounds closely related to cinerin I available for comparison of

255 toxicity.2' Their comparative toxicities to house flies are given in Table 7. TABLE 7 RELATIVE TOXICITY OF ANALOGS OF CINERIN I TO HOUSE FLIES No. Cyc lope ntenol one Side Chain Chrysanthemum Monocarboxylic Acid Relative Toxicity 1 -CH2CH = CHCH=CH2 (Pyrethrolone) d-trans- (natural) 1 2 -CH2CH=CHCH3 (Cinerolone) ii 0. 7 3 -CH2CH=CHCH3 M 0. 7 4 -CH2CH=CH2 ii 3. 3 5 -CH2C(CH3)=CH2 n 1. 7 6 -CH2CH2CH=CH2 it 0. 3 7 -CH2CH=C(CH3)2 n 0. 1 8 -CH2CH2CH2CH3 n 0.08 9 -CH2CH=rCHCH3 dl-tifi- 0. 2 10 -CH2CH=CHCH3 dl-trans- 0. 2 11 -CH2CH = CH2 dl-cia- .9 12 -CH2CH=CH2 dl -trans - .9 Compounds 2 and 3, which differ only in that the side chain in 2 has a cis configuration while that of 3 is trans, are about equally toxic. Compound 4 when esterified with the natural dextro acid is more than three times as toxic as compounds 11 and 12 obtained from the optically inactive acids. Esterification of the synthetic trans cinerolone with the optically inactive acids {compounds 9 and 10) also caused reductions in mortality to about one-third of the values obtained when natural acid is used. A mixture of compounds 11 and 12 will shortly be commercially available. By the synthesis developed by Schechter et al. ?4 it is also possible to prepare a cyclo- pentenolone in which the methyl group in the three position has been replaced with other groups. The substitution of the phenyl group for the methyl group in the allyl ester decreased the toxicity to one-fifteenth that of the allyl homolog of cinerin I. 27 Gersdorff has also tested on house flies an uncyclized compound (formula XII where R = allyl) esterified with natural chrysanthemum acid. It was less than one-seventieth as toxic as the corresponding cyclized compound (compound 4 in Table 7).

256 In addition to the esters mentioned above, the synthesis developed by Schechter et al. '4 opened up a whole new field for further investigation. Many other modifications can be made in the keto-alcohol portion of the molecule and such alcohols can then be esterified not only with chrysanthemum monocarboxylic acidt but with other substituted cyclopropanecarboxylic acids or with entirely different types of acids. Studies in which the acid component of pyrethrin I and cinerin I, chrysanthemum mono- carboxylic acid (VIII), has been esterified with alcohols other than pyrethrolone or cinerolone have also been reported. Staudinger and Ruzicka8l tested the reaction products of chrysanthemum acid chloride with various alcohols and phenols against cockroaches. In none of the experiments was the reaction product isolated, the assumption being that the reaction had proceeded with the formation of the desired ester. None of the products tested was sufficiently toxic to warrant further studies. Harvill44 esterified the acid with an homologous series of eighteen aliphatic alcohols ranging from ethyl to cetyl and tested them against aphids and cockroaches. The lauryl, myristyl, and cetyl esters showed some toxicity to the aphids, but none of the esters had the pyrethrin-like action on cockroaches. With the improved method of obtaining chrysanthemum acid* now available, other esters are worthy of further detailed studies. SYNERGISTS A recent development in the utilization of pyrethrum which is of considerable practical importance is the discovery that certain synthetic organic compounds can replace part of the pyrethrins without reducing either their paralytic or their killing power. In many instances an increased effectiveness is obtained with the combination. Compounds of this type have been loosely referred to as synergists, activators, and intensifiers, and the belief has developed that all these compounds are effective only in the presence of pyrethrins, that all are equally useful with pyrethrum, and that all are nontoxic to warm-blooded animals. These assumptions are not valid. As with other synthetic organic compounds, the toxicity of these products to a wide variety of insect pests, as well as their effect on warm-blooded animals, must be ascertained before their general value in economic entomology can be stated. One of the first compounds shown to increase the effectiveness of the pyrethrins is N-isobutylundecyleneamide (XIV). 88 CH2=CH(CH2)8CONHCH2CH(CH3)2 XIV When tested in kerosene solution as a spray against house flies, the amide alone was of little value. A spray containing 40 mg. of pyrethrins and 420 mg. of the amide per 100 ml. was superior to the standard spray solution containing 100 mg. of pyrethrins per 100 ml. An investigation of the effect of N-isobutylundecyleneamide and the pyrethrins on house flies has been made by Hartzell and Scudder. 42 in histopathological studies they found that each chemical shows rather distinct and characteristic effects upon the central nervous system and associated tissues of the adult house fly. Pyrethrum has a widespread clumping effect on the chromatin of the cell nuclei, while the amide seems to cause a chromatolysis or dissolution of the chromatin. A combination of the two products shows a histological picture that is a summa- tion of the effects of each.

257 Another unsaturated isobutylamide that increases the effect of the pyrethrins is fagaramide (N-isobutyl-3,4-methylenedioxycinnamamide) (XV), occurring in the root bark of Zanthoxylum senegalense DC. and Z. macrophyllum Oliver. 31, 86 H H =C-CONHCH2CH(CH3)2 xv Fagaramide, at 2 mg. per ml. plus 0. 5 mg. pyrethrins per ml. , killed as many house flies as a solution containing twice as much pyrethrins but without the fagaramide. Z' Several insecticidal N-isobutylamides of aliphatic unsaturated acids have been isolated from plant materials - namely, spilanthol (N-isobutyl-4, 6-decadienamide) (XVI) from the flower heads of Spilanthes oleraceae Jacquin' • 19 and S. acmella Murr. , ^O pellitorine (N-isobutyl-2, 6- decadienamide) (XVII) from the roots of Anacyclus pyrethrum DC. ,32.48 N-isobutyl-2, 6, 8- decatrienamide (XVIII) from the roots of Heliopsis longipes (A. Gray) Blake, 51 herculin (N-iso- butyl-2, 8-dodecadienamide) (XIX) from the bark of Zanthoxy1um clavaherculis L. ,47 and scabrin [(N-isobutyl-2, 4,8, 10, 14-octadecapentaenamide) (XXa) or (N-isobutyl-2, 4, 8, 12, 14- octadecapentaenamide) (XXb)]. *' CH3(CH2)2CH=CHCH=CH(CH2)2COR XVI CH3(CH2)2CH=CH(CH2)2CH=CHCOR XVII CH3CH=CHCH=CH(CH2)2CH=CHCOR XVIII CH3(CH2)2CH=CH(CH2)4CH=CHCOR XIX CH3(CH2)2CH=CH(CH2)2CH=CHCH=CH(CH2)2CH=CHCH=CHCOR XXa CH3(CH2)2CH=CHCH=CH(CH2)2CH=CH(CH2)2CH = CHCH = CHCOR XXb CH3(CH2)4CH=CHCH=CHCOR XXI CH3(CH2)8CH=CHCOR XXII where R is NHCH2CH(CH3)2 Compound XVI has been reported"'. °6 to be an effective mosquito larvicide. Compound XVII, which differs from XVI only in the position of one of the double bonds, shows toxicity to house flies somewhat greater than one-half that of pyrethrins. *8 Compound XVIII, having one double bond more than XVI and XVII, is somewhat more toxiq than pyrethrins to house flies. 51 Compound XIX has approximately the same order of toxicity to houseflies as the pyrethrins. 47 Scabrin (XXa or b) is appreciably more toxic than the pyrethrins to house flies. ^° Compounds XVI to XX all show the rapid paralytic or knockdown action characteristic of the pyrethrins. However, saturation of the double bonds gives compounds that are completely devoid of insect- icidal activity, although N-isobutyllauramide (hydrogenated XIX) shows some synergism with pyrethrins. Geometrical configuration about the double bonds plays a large part in the toxicity of the unsaturated isobutylamides. For example, the cis-trans and trans-trans isomers of compound XVII were synthesized and found to be nontoxic to house flies. 11, 50 N-Isobutyl-2, 4-decadienamide (XXI), differing from XVI and XVII only in the position of one double bond, and N-isobutyl-2-dodecenamide (XXII), differing from XDC only by the lack of a double bond in position 8, were both synthesized. They showed rapid paralytic or knockdown

258 action but gave very low mortality of house Hies. Another group of compounds that have been found to be especially useful with pyrethrum are certain piperonyl or methylene dioxyphenyl derivatives. Interest in this class of compounds resulted in a large measure from an observation of Eaglesonl" in the course of testing pyrethrum solutions in admixture with a number of vegetable and fish oils against house flies. Eagleson found that sesame oil, to the exclusion of all other oils, markedly increased the effectiveness of the pyrethrins. That the increase in toxicity was due to a synergistic or activator effect, and not to the addition of another insecticide, was shown by the failure of sesame oil alone to kill flies. By means of a hypnotic-dose technique developed by him, Eagleson'5 followed the recovery of house flies that had been sprayed with a pyrethrum insecticide to which various percentages of sesame oil had been added. The results, given in time-torpor curves, are shown in Figure 2. 5% TiUE-MOUMS Fig. 2 Recovery of house flies sprayed with pyrethrum insecticide to which various percentages of sesame oil had been added Each point on the curves represents the mean torpor, at successive intervals after spraying, for five replications on approximately fifty-five flies. With the pyrethrum solutions to which no sesame oil had been added, 92 per cent of the flies recovered. When 5 per cent of sesame oil was added to the pyrethrum solution only 12 per cent of the flies sprayed recovered. By his method Eaglesonl* had previously shown that few flies that are still affected six hours after the spraying ever recover. At the suggestion of Eagleson a chemical study of sesame oil was undertaken by Haller and co-workers. 37 Sesame oil was separated into four fractions by means of high-vacuum distillation. Each fraction was separately added to pyrethrum extract in refined kerosene and tested against house flies by the turntable method. * The results are shown in Table 8. From the combined first and second fractions a crystalline solid was isolated and shown to be sesamin. When it was added to pyrethrins in a refined kerosene-acetone mixture, the effectiveness against flies was greatly increased (Table 9). (Ten per cent of acetone in.the kerosene is necessary to dissolve the sesamin. ) It was not possible to obtain from the noncrystalline active fraction any crystalline compound other than sesamin.

259 TABLE 8 EFFECTIVENESS AGAINST HOUSE FLIES OF VARIOUS FRACTIONS OF SESAME OIL, WITH AND WITHOUT PYRETHRUM, IN REFINED KEROSENE (2 tests with about 150 flies each; concn. of pyrethrins 1 mg. , and of sesame oil and its fractions 10 mg. /cc. ) Material Knockdown Mortality in 48 hr. . % in 10 in iD , % Sesame oil 0 2 Pyrethrins 99 21 Pyrethrins + sesame oil 100 57 Pyrethrins + fraction I 100 100 Pyrethrins + fraction II 100 91 Pyrethrins + fraction III 100 21 Pyrethrins + fraction IV 100 29 TABLE 9 EFFECTIVENESS AGAINST HOUSE FLIES OF FRACTIONS OF SESAME OIL, WITH AND WITHOUT PYRETHRUM, IN REFINED KEROSENE PLUS 10% OF ACETONE (2 tests with 150 flies each; concn. of pyrethrins 1 nig. , and of sesame oil fractions 2. 5 mg. /cc. ) Material Knockdown in 10 Min. , Mortality in 24 hr. , Pyrethrins 100 20 Sesamin (crystalline fraction) 0 5 Pyrethrins + sesamin (crystal- line fraction) 100 85 Pyrethrins + noncrystalline res idue 100 89

260 Sesamin has the following structural formula: CH^O It is a bicyclodihydrofuran substituted symmetrically with two methylenedioxyphenyl groups.Z'" It has four asymmetric carbon atoms, and natural sesamin is dextrorotatory. Hartzell and Wexler*3 have studied the histological effects of pyrethrum and sesamin on the central nervous system and muscles of the house fly. Flies rendered moribund by pyrethrum showed clear spaces in the brain tissue and dissolution of the fiber tracts, and sesamin caused vacuolation around the large nerve cells. When sesamin and pyrethrum were combined, not only were the nerve fibers destroyed but the larger nerve cells were highly vacuolated until almost complete lysis of the tissue resulted. An excellent summary of the physiological studies of pyrethrum and the various activators or synergists that have been studied is given by Metcalf. ^ A number of plant materials have been shown to contain compounds related to sesamin. ^° Among these compounds are asarinin, found in various oriental plants and in the bark of American prickly-ash; pinoresinol, a constituent of the exudate of spruce and related species; and eudesamin, a constituent of kino gum from eucalyptus. Their relation to sesamin is shown in the formula XXIII. XXIII R,R' = O2CH2 (methylenedioxy) for sesamin and asarinin R = OH and R1 = OCH3 for pinoresinol R.R' = OCH3 for eudesamin Asarinin is levorotatory and is the optical antipode of isosesamin, which is obtained on treatment of sesamin with alcoholic hydrochloric acid. As some of these compounds were available, they were tested for their synergistic effect with the pyrethrins. The diacetyl derivative of pinoresinol was also included. Isosesamin and asarinin were as effective as sesamin, but pinoresinol dimethylether, the optical antipode of eudesamin, was without appreci- able synergistic action, as were pinoresinol itself and its diacetyl derivative. The results are summarized in Table 10.

261 TABLE 10 EFFECT OF SESAMIN AND RELATED COMPOUNDS ON THE INSECTIC1DAL ACTION OF PYRETHRINS AGAINST HOUSE FLIES (3 tests using about 150 flies per test; solvent, refined kerosene plus 10% of acetone where needed to increase solubility) No. Material Concentration * Average Mortality After 24 hrs. % Sesamin and its isomers: 1 Sesamin 0.2 4 2 Sesamin + pyrethrins 0.2 + 0.05 84 3 Isosesamin 0.2 5 4 Isosesamin + pyrethrins 0. 2 .t 0.05 87 5 Asarinin 0.2 14 6 Asarinin t pyrethrins 0.2 + 0. 05 88 7 Pyrethrins (control) 0.05 25 Pinoresinol and derivatives: 8 Pinoresinol 0. 18 1 9 Pinoresinol + pyrethrins 0. 18 + 0. 05 12 10 Dimethyl pinoresinol 0. 2 1 11 Dimethyl pinoresinol t pyrethrins 0. 2 + 0.05 17 12 Diacetyl pinoresinol 0.03 2 13 Di.ii riyi pinoresinol + pyrethrins 0.03 + 0. 05 11 14 Pyrethrins (control) 0.05 19 From these findings it was concluded that the methylene dioxyphenyl grouping was an important one for compounds useful as synergists with pyrethrum. They have led to the prepara- tion and testing of a large number of different types of compounds containing the grouping. Many of the compounds that have been prepared are derived from safrole because it is the most ready source of the methylene dioxyphenyl group. Workers at the Boyce Thompson Institute have been testing products obtained by the addition of various aldehydes,?0 mercaptans, 69 maleic acid esters, " and various other compounds, "5 to the double bond of safrole and isosafrole. Deriva- tives of piperonal, ? 1 . 83 piperonylic acid, 28 piperine and related compounds ^ . 4.>'**4 have also been tested. From the research in this field three products - piperonyl cyclonene, piperonyl butoxide. and jl-propyl isome - have been developed that have found commercial acceptance. Technical piperonyl cyclonene obtained on condensation of alkyl-3, 4-methylene dioxystyryl ketones with ethyl acetoacetate^, ®? contains 80 per cent of a mixture of compounds XXIV and

262 XXV in addition to higher molecular condensation products. CHj-O -! T «.--3 -jr y 2 Alkyl - CX. ^CO Alkyl - Ox ^*-.^ ^* H2C CH-COOC2H5 H2C C C H XXIV XXV Piperonyl butoxide^Z- 87 [s obtained as a technical product containing 80 per cent of XXVI. CH2CH2CH3 CH2OCH2CH2OCH2CH2OC4H9 XXVI n-Propyl i.somr, ' * prepared by a Diels-Alder type condensation of isosafroLe with n-propyl malt.ate, has the structure shown in XXVII. XXVII

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267 DISCUSSION DR. D. F. MARSH (University of West Virginia. Morgantown, West Virginia): I think since the time of Cushny's work with the cocaines and epinephrine, pharmacologists in general have been interested in the very intriguing problem of the effect which optical isomerism may have on the biological activity. I do not mean by this that I think the biological activity is the direct result of rotation of plane polarized light; it is just that these are two interesting properties which are possessed by the same structures in the molecule. Perhaps 1 misunderstood the data on the slide but, if the data is as I understood it, it has some very interesting aspects in relation to this problem of synergism or potentiation or antagonism, or whatever you call it. In regard to the alkyl analogs of cinerin, the activity of the d-material was given as 3. 3, I believe. If you take one part of the e)-isomer and one part of the talc or other inactive material, presumably the activity of that mixture would be about 1. 7. According to the data given on the slide, the dl-mixture was given as having an activity of only 0. 9. I presume that this might be due to the technique of testing. Perhaps Dr. Haller will elaborate on this although it is quite a wide margin. I wonder if it could mean that the I -isomer is acting not only in a noncontributory manner, but is acting, shall we say, to protect the house fly, as it were, against the d-isomer. If that is the case, does it mean, then, that we have here a sort of p-aminobenzoic acid - sulfanilamide mutual antagonism between the two? There was another example of this which happened to strike my eye earlier. Perhaps, again, I misunderstood the data but levo anabasine was listed as having a toxicity of 10. 1. If it does, then the d_l-material was given as requiring five times as much to be toxic. In this case, the ^1-anabasine is removing the activity of about twenty-four times as much levo anabasine, if this is the true situation. The dl-mixture ought to be about 0. 2 instead of being 'way up there at 5. I do not know; maybe there is a very simple explanation of this but, in any event, the figures are very intriguing from a pharmacological view. DR. HALLER: Thank you, Dr. Marsh. Perhaps I did not make it clear enough that the figures I gave are to indicate order of magnitude, and they were not all done in the same laboratory at the same time. If we take anabasine, for example, and I could cite some of the other figures, too, you will find the same thing - where you may actually appear to have antagonism. I do not believe that is so. You will recall that I said we need more work in this field, because too much of it has been done in different laboratories. My slides are a compilation of results from different places, trying to bring out the effect of optical activity and the effect of configuration rather than the precise data. I sometimes feel it might be well worth while, from the standpoint of studies in this field, to start from scratch, making these compounds in pure form, and repeating some of that early work. That is no reflection upon the early work at all. We are going ahead with the studies of the pyrethrin-like esters and I must say that we have a man in our organization, Gersdorff, who has really done some very careful work in making the comparisons of which you speak, not only in this field, with house flies, but the earlier work which I showed, with rotenone on goldfish. Some of his papers are well worth studying, for any of you who may be specifically interested.

Z68 I notice Mr. Schechter coming up. I might say to you that he has done some very fine synthetic work on the pyrethrins and cinerins working with Dr. LaForge. MR. SCHECHTER: Thank you. Dr. Haller. I have some later information on this particular point which might shed a little more light on the question Dr. Marsh raised. We have recently tested the allyl-substituted cyclopentenolone esterified with the J_-:s omer of chrysanthemum monocarboxylic acid and find that the ester has only about one-fiftieth of the activity of the ester with the d-form of the acid: that is. it is relatively nontoxic. In this series of tests, the ester with the d^-acid (which is the ester in commercial use) has a relative toxicity of about 1. 5 or 1. 6, as I remember it. This fits in closely with the fact that the ester with the .l.-acid is relatively nontoxic since the ester with the d_-acid has the highest toxicity and the combinauon appears to be approximately half as toxic as the ester with the d-acid In the case of the individual dl-c_is and dl-trans acids, we had only very small amounts available which were sent to us by Dr. Harper of Kings' College, England. We had so little that we could not check on their purity so that the figures obtained with esters of these acids might perhaps be due to the lower degree of purity of the acids employed. This should be checked up more closely and we may do so in the future. DR. ALFRED BURGER (University of Virginia, University, Virginia): 1 was particularly intrigued with Dr. Haller's remarks, that the pyrethrins cause such rapid paralysis. These compounds are alpha-beta unsaturated ketones, and they may have some relation to those anti- biotics which, as Ceiger and Conn have pointed out, add mercaptan groups. Even though the reaction of mercapto groups with enzyme systems does not explain all the antibiotic properties of a compound, one could reason that if the pyrethrins would be capable of adding to sulfhydryl groups, the deactivation of many enzyme systems in the insect might be due to this reaction. A thought coming to mind in seeing the formula of sesamin is the possible overlapping with that of biotin. Biotin has a thiophane ring condensed with a ureyl group and carries a butyric acid side chain. The formula of sesamin with its condensed hydrofuran rings conveys a sirr.ilar sterical picture at least of one portion of the molecule. I would like to ask Dr. Haller whether anybody has ever tested biotin antagonists, of which there are quite a few, as insecticides. DP HALLER: So far as I know they have not been tested. DR. BURGER: The second point concerns the toxicity of nicotine and anabasine. In these compounds, Dr. Haller has pointed out the insecticidal toxicity drops every time you remove the pyrrolidine or the piperidine portion from the beta to the alpha position. In 1942, Nieman and Hays of the California Institute of Technology, pointed out that, if you put onto a pyridine r'ng an ethylamine side chain in the alpha position, you get histaminic activity. They related it at that time with possible chelation of the two nitrogen atoms in the ionic form of the compound. If you write the molecule of histamine or anabasine in the ionic form, you cannot possibly construct such a chelated formula but, if you construct the alpha isomer of nicotine and anabasine then, particularly in the case of anabasine where there is no N-mcthyl group to introduce any steric factors, you have the possibility of constructing a chelated ring. I want to ask Dr. Haller whether anybody has ever considered that nicotine toxicity to insects might be in any way connected with histamine-type activity. DR. HALLER: Here, again, the question, so far as I can answer it, is no. I do not know of anything which has been done along those lines. Dr. Burger. Probably that is one of the biggest weaknesses we have in our work, in that, we are working with exceedingly minute organisms, and it is not easy to bring out some of these points you have mentioned. 1 do not know specifically that anyone has given that particular angle or problem any thought, nor that tests of that sort have been made. A big field is there, as I said.