J.S.F.A., 1 , May, 1950 CAMPBELL A N D MITCHELL-POLYMERIZED PYRETHRINS 137

AN EXAMINATION OF POLYMERIZED PYRETHRINS

By A. CAMPBELL and Wm. MITCHELL

The resinous material formed by polymerization of a highly purified pyrethrum concentrate has been examined. It was in- soluble in cold light petroleum or deodorized kerosene, and gave high values for apparent pyrethrins by the Seil or mercury- reduction methods, although it was devoid of insecticidal effect on houseflies. In these respects it resembled the additional ' pyrethrins ' recently shown' to be extracted by chloroform or by warm light petroleum from pyrethrum flowefs, differing only in its higher content of apparent 'pyrethrin 1.

On hydrolysis of this material, pure chrysanthemum mono- and di-carboxylic acids were isolated in yields of some 75% of the quantities calculated from the analysis. These acids were iden- tical with those derived from normal pyrethrum extracts. Hence it appears that polymerization is confined to the keto-alcoholic constituents of the esters, the acids not being involved. The chemical assay methods at present available do not distinguish such polymerized material from true pyrethrins : but the modi- fication described by Mitchell and Tresadernl largely prevents the extraction of polymerized material.

The need for confirming all chemical assays by biolugical tests is emphasized. A portion of the monocarboxylic acid was esteri- fied with DL-allylcyclopentenolone ; thc ester possessed the expected insecticidal activity. The action of this ester on house- flies is discussed and contrasted with that of the pyrethrins.

an examination of polymerized pyrethrins, and this work forms the subject of the present paper.

A highly purified pyrethrum concentrate (100 g.), prepared by the nitromethane method of Barthel, Haller and LaForge,2 was dissolved in light petroleum and exposed to daylight for 3 months, while being maintained at 30' c. The pale yellow solution gradually deposited an amber-coloured resin. By dilution with a considerable volume of cold light petroleum (b.p. 40' to 60" c.) the mixture was then separated into two portions, one soluble and the other insoluble in this solvent. The soluble material (41 g.) was an amber-coloured viscous oil and the insoluble material (58 g.) an amber-coloured glassy thermoplastic solid resembling colophony. The analyses and biological tests of these fractions, and of the original con- centrate, are given in Tables I and 11.

These results showed that the light petroleum-insoluble fraction (which was also insoluble in deodorized kerosene) was devoid of insecticidal action, though it gave a high chemical test of ' pyrethrins I and I1 ' ; the figures for ' pyrethrin I ' were practically identical both by the Seil and mercury- reduction methods. Apart from its higher relative content of pyrethrin I,' it was thus similar in its properties to the material extracted by chloroform or by warm light petroleum in the earlier work. The light petroleum-soluble portion was similar to the original pyrethrum concentrate. The total

Table I Coniparatiue assays of a pyrethrum concentrate and of the fractions soluble and insoluble in cold light petroleum (b.p. 40' to 60' c.)

after prolonged exposacre of the concewtraie at 30' c. to dayligkt 3Iaterial Seil method Mercury-reduction method

Pyrethrins, 76 Pyrethrins, % I I1 Total I I1 Total

Original Concentrate . . .. .. .. .. .. .. . 40'4 32.6 73'0 47.8 324 80.4

Light petroleum-insoluble fraction . . .. . . . . .. .. 32'9 30'9 63.8 32.8 30'3 63.1 Light petroleum-soluble fraction . . .. .. .. .. .. 35'2 26.8 62.0 39'7 26.9 66.6

Table I1 Biological tests of a pyrethrum concentrate arid of fhe fractions soluble and insoluble in cold light petroleum (b.p. 40" lo 60" c.) after proloaged exposure of the concentrate at 30" c. to daylight. T h e tests are by a modified Peet-Grady procedure. against housefiies.

(The results in each case are the mean of three tests) Insecticide solution (0.1% w/v total

pyrethrins on Seil basis) 30 sec. Official Test insecticide .. .. .. .. .. 88 Original concentrate in deodorized kerosene . . .. 86 Light petroleum-soluble fraction in deodorized kerosene . . 80 Light petroleum-insoluble fraction in deodorized kerosene

containing 40% v/v of acetone .. .. .. .. 25 Deodorized kerosene containing 40% v/v of acetone (solvents

only) . . . . . . .. .. .. . . . . 20

In a recent paper from this laboratory' it was shown that all the insecticidally active material is extracted from pyrethrum flowers by cold light petroleum. Warm solvent was found to extract significant additional quantities of ' pyrethrins,' the amount extracted varying with the grade of the solvent used ; warm chloroform extracted such material in even greater amount. These 'pyrethrins,' which were shown to be devoid of insecticidal properties, could be estimated by the usual Seil or mercury-reduction methods of assay, being thus shown mainly as ' pyrethrin 11.' It was noted that the mercury-reduction figures for ' pyrethrin I ' were the same as, or even lower than, those obtained by the Seil process ; this was unusual, as the mercury-reduction figures for ' pyrethrin I ' are usually some 15% higher.

Since both assay methods incorporate a preliminary puri- fication stage, designed to eliminate fatty acids, it appeared likely that the acids finally determined were in fact chrysan- themum mono- and di-carboxylic acids, or closely related acids. It seemed of interest to confirm this suggestion by

' Knock-down ' ( X ) of flies in Flies dead after 9 SCC. 2 min. 4 min. 10 min. 48 hr., yo

46 49 44

- - - 100 I00 - - - - - 95 100

50 60 85 98 4

40 55 80 95 3

weight of the two fractions (99 g.) showed practically no loss of material, but the assays disclosed an over-all loss of apparent pyrethrins, based on the original concentrate (Table 111).

The light petroleum-soluble portion was not investigated further, but the insoluble fraction was hydrolysed and the liberated acids isolated by a method similar to that used in the mercury-reduction assay. During the hydrolysis, the amber-coloured material gradually altered into an insoluble,

Table 111

Recovery of apparent pyrethrins from the fractions of a polymerized pyrethrum extract. T h e figures are calculated f r o m the Seil analyses of

the fractions and of the original concentrate Light petroleum- Light petroleum- Total

soluble insoluble recoveries, % fraction, yo fraction, yo

Pyrethrin I . . 35.7 47'2 82.9 Pyrethrin IT . . 33.7 55'0 88.7 Total pyrethrins 34.8 50'7 85'5

138 CAMPBELL A N D MITCHELL-POLYMERIZED PYRETHRINS J.S.F.A., 1 , May, 1950

brown, opaque mass which ultimately was isolated as a brittle resin that could be powdered. This behaviour is not charac- teristic of normal pyrethrum extracts ; however, traces of a similar resin are sometimes observed on hydrolysis of such extracts and are no doubt derived from small amounts of altered material present in them.

The monocarboxylic acid obtained, in a yield of 15'5% on the light petroleum-insoluble fraction, was a slightly yellow oil. By direct titration with sodium hydroxide (phenol- phthalein) it showed a purity of 89.8y0, whereas a mercury- reduction assay showed 83.3%. On the basis of the latter figure the yield of pure acid on the light petroleum-insoluble material was 12.9%, a recovery of 76.3% of the quantity calculated from the original analysis. Purified by distilla- tion in vacuo, the acid was obtained as a low-melting solid showing 100% purity by the mercury-reduction assay (in which the normal range of colours characteristic of chrysan- themum monocarboxylic acid developed, though perhaps more slowly than usual). Apart from a somewhat lower optical rotation, suggesting slight racemization, the acid appeared to be identical in all respects with chrysanthemum mono- carboxylic acid similarly prepared from a normal, unpurified pyrethruni extract. The amides and p-phenylphenacyl esters of the two acids were also identical.

A quantity of this acid was steam-distilled under the con- ditions of the Seil test. Titration of the light petroleum extract of the aqueous distillate showed 85.4% recovery of the acid used. Titration of the ether extracts of this dis- tillate and of the distillation residue showed respectively I O . O ~ ~ and 4'7% calculated as chrysanthemum monocarb- oxylic acid. These recoveries presumably represented the less steam-volatile acid suggested by Mitchell et al.3 to be a hydration product of chrysanthemum monocarboxylic acid ; the results thus showed the loss typical of the Seil assay. Since this acid had already been shown to be 100')'~ pure by the mercury-reduction method, and was isolated by a hydro- lytic method closely similar to those used in both assay methods, it is difficult to account for the fact that the original material from which it was derived gave the same results for ' pyrethrin I ' by both methods ; this phenomenon had been already noted' with similar polymerized material extracted as such from pyrethrum.

The pure crystalline dicarboxylic acid was obtained in a yield of 13.0% on the light petroleum-insoluble material, representing a recovery of 75*oy0 of the quantity calculated from the original analysis. It was identical in all respects, apart again from a slightly lower optical rotation, with chrysanthemum dicarboxylic acid similarly prepared from a normal, unpurified pyrethrum extract. . The P-phenylphenacyl esters of the two acids were also identical.

It is known that the ketwalcohols pyrethrolone and cinerolone are very prone to polymerization, as might be expected from their highly unsaturated structures. The same behaviour is also characteristic of their simple esters, the pyrethrins and cinerins, which lose their insecticidal action on polymerization. Although the chrysanthemum acids are themselves unsaturated, it appears quite clear from the present results that polymerization of the pyrethrins and cinerins is a change confined to the keto-alcoholic portions of the molecules ; the esterified acids remain attached to the polymers and are capable of recovery by hydrolysis in amounts approaching those present in the unpolymerized esters.

These results indicate very clearly that the chemical assay methods available at present do not distinguish between true pyrethrins and inert (polymerized) material. In order to obtain correct results for active pyrethrins it is necessary to modify the chemical assay methods to avoid the extraction of such polymerized material from the flowers ; this can be

done by using cold light petroleuni for the extraction, as described by Mitchell and Tresadern.l The need for con- firming all such analyses by biological tests is emphasized, particularly in assessing the insecticidal value of old or badly stored specimens of pyrethrum or pyrethrum preparations.

Although apparently identical in other respects with normal D-trans-chrysanthemum monocarboxylic acid, the slightly lower optical rotation of the acid recovered from the poly- merized material suggested the possibility that it might not be fully active when re-esterified with pyrethrolone. The latter not being readily obtainable, it was decided to esterify the acid with DL-allylcyclopentenolone, made by the method of LaForge et aZ.* A comparison of the resultant ester with that made from normal D-trans-chrysanthemum monocarb- oxylic acid showed no significant difference in their insecticidal activities towards houseflies (Table IV).

Gersdorff,6 using the turntable method of Campbell and Sullivan,6 has reported the uL-allylcyclopentenolone esters of D-trans- and DL-cis-trans-chrysanthemum mono-carboxylic acids as having respectively six and three times the activity of the natural mixture of pyrethrins and cinerins. Stoddard and Dove' have tested the ester of the m-cis-trans acid by the Peet-Grady method and reported its activity as approxi- mately equal to that of natural pyrethrins. The figures in Table I V are typical of our results with the ester of the D-trans- acid which we find to have nearly twice the activity of the natural mixture of pyrethrins to houseflies. Allowing for the differences in results reported by the two methods of testing, this would appear to be in line with Gersdorff's results for the D-trans ester. We have observed that the 'knock- down ' behaviour is quite different from that due to pyrethrum. Initially it is slower, though substantially complete in three minutes. After six minutes the flies become highly activated ; although their legs remain paralysed, they appear to recover the normal use of their wings. Their attempts to fly cause them to spin round on their backs and to move about erratic- ally on the floor of the test chamber ; this activity persists for a considerable period. Although a similar behaviour can often be observed momentarily in isolated individual flies after spraying with pyrethrum, it cannot be compared with that caused by this synthetic ester. The ' knock-down ' effect of the latter on houseflies, though superior to that of most other synthetic insecticides, is definitely poorer than that of pyrethrum ; and the subsequent characteristic activa- tion of the insects does not appear to be suppressed by the use of higher dosages of the ester.

Experimental (Micro-analyses are by Drs. Weiler and Strauss, Oxford.

Melting points are corrected.) Separation of the polymerized pyrethrum concentrate.-A

purified pyrethrum concentrate (100 g.) in light petroleum (b.p. 40" to 60" c. ; 200 ml.) was maintained in a closed vessel at 30" c. for 3 months, and exposed to direct daylight. The mixture was then diluted with light petroleum (b.p. 40" to 60"c.; 500 ml.) and agitated. When the supernatant liquid had become clear it was decanted. The residue was repeatedly extracted with the solvent until the washings were colourless. The solvent was recovered, finally in vaczco, from the united extracts to give an amber-coloured oil (41 g.). The residue from the .extraction was freed from solvent by warming a t 30" c. in vacuo. It was an amber-coloured, thermoplastic resin (58 g.) resembling colophony in appearance.

Hydrolysis of the light petroleum-insoluble fraction.-The material (20 g.) was boiled under reflux for 4 hr. with potas- sium hydroxide (7.5 g.) in 95% v!v alcohol (150 ml.). The clear yellow resin did not dissolve but gradually changed to a brown, opaque solid. After cooling, the liquid was poured

J.S.F.A., 1 , May, 1950 CAMPBELL A N D MITCHELL-POLYMERIZED P Y R E T H R I N S 139

Table IV Biological tests of DL-all~~~lcyclopente,lolone ~- t rans-~hr~~d~thdnLate Prepared (a) from normal chrvranlhemuin tnonocarboxyltc acid and (b) from the actd recovered from a polyineriztd pyrethrum concentrate, usuig a modtfied PeetCrady test on houseflies.

(The reszrlts are tn earh case the mean of three tests) Insecticide solution

Official Test Insecticide . . . . . . Ester (a), od5"/; w/v in deodorized kerosene Ester (b), 0.060i w/v in deodorized kerosene

m.p. of acid . . .. .. [a:ozoo of acid in chloroform . . [(r]DZoo of acid in absolute alcohol

nzQo of acid . . .. . . m.p. of amide . . .. . . m.p. of p-phenylphenacyl ester

m.p. of acid .. .. .. [ l x I D z 6 O of acid in chloroform . . [ ( r ] ~ ~ ~ ' of acid in absolute alcohol

m.p. of p-phenylphenacyl ester . .

. .

. .

..

..

..

..

.. .. ..

..

..

..

..

. .

. .

' Knock-down ' (76) of flies in 30 sec. go sec. 2 min. 4 min.

- .. .. 91 99 I00 .. .. 70 90 96 I00 . . . . 60 96 98 I00

Table V From polymerized From normal pyrethrum extract pyrethrum extract

.. .. 18.5" c. 18.5" c.

. . . . + 25.6' (2.54% w/v) + 28.5" (3.08" w/v)

.. . . + 1 5 . 3 ~ (2.29% w/v) + 19.4' (2.894,; W/V)

.. .. 1.4790 1'4779

.. . . 65' c. 65" c.

.. .. 130-131" C. 130-131" C.

(mixed m.p.'s showed no depression)

Table VI From polymerized From normal pyrethrum extract pyrethrum extract

.. .. 167-168' c. 167-168' c.

.. .. + 75.0" (2.4% w/v) + 76.4' (239% W/V)

.. .. -6 147.5' (2'0% w/V) -!- 149.0' ('2.07; W / V )

.. .. 153-154' C. 153-154' C. (mixed m.p.'s showed no depression)

off, and the residual solid was washed with 95% v/v alcohol ( 3 portions, each of 10 ml.). After drying, the solid was brittle and could be powdered ; it was not examined further. The united alcoholic extracts were distilled at atmospheric pressure until the volume was reduced to about 30 ml., when the residue was poured into water (1500 ml.). Barium chloride (7 g.) in water (70 ml.) was added ; this sufficed for complete precipitation. After standing for I hr. the mixture was suction-filtered through a bed of kieselguhr on paper. The clear filtrate was acidified (Congo red) with hydrochloric acid and extracted with light petroleum (b.p. 40' to 60" c., 4 por- tions, each of IOO ml.) ; the aqueous layer (A) was reserved. The united light petroleum solutions were extracted with x-potassium hydroxide (30, 10, 10 ml.), and discarded. The combined alkaline extracts were diluted with water (zoo ml.), acidified (Congo red) with hydrochloric acid, extracted with light petroleum (b.p. 40" to 60" c., 4 portions, each of 50 ml.), and then discarded. The united light petroleum extracts were dried (sodium sulphate). Recovery of the solvent yielded 3.1 g. of a pale yellow oil containing chrysanthemum mono- carboxylic acid, S y S % by direct titration (phenolphthalein), or 83-3y0 by the mercury-reduction method ; the figure of 83.3% corresponded to a yield of 12.9% of pure acid. The acid (2.j g.) was distilled at 0.1 mm. pressure, the first fraction of 0.6 g. being rejected. The main fraction (1.5 g.) formed a crystalline solid on chilling. (Found: C, 71.5 ; H, 9.6. Calc. for C,,,H,,O, : C, 71.4 ; H, 9*5y0.) The physical proper- ties of the acid and two derivatives were compared with those of materials similarly prepared from a normal pyrethrum extract (see Table V).

The reserved liquid (A) was made alkaline with sodium bicarbonate and'concentrated i q z vacuo (250 ml.). It was then acidified (Congo red) with hydrochloric acid, extracted with ether (6 portions, each of IOO ml.), and discarded. The com-

Flies dead after

51 54 57

10 min. 48 hr., % - - -

References

17-ZI'C. (Campbell and Harper8)

+ 20.10 (Staudinger and RuzickaB)

-

-k I4.?' (Campbell and Harper')

131" c. (Staudinger and KuzickaB)

6 j3 C. (Wilcoxonlo)

References

168' c. (Wilcoxonlo)

f 72.8" (Staudinger and RuzickaO)

154" c. (WilcoxonLO)

-

bined ethereal extracts were dried over sodium sulphate. Recovery of the solvent left an oily, crystalline mass (3.1 g.) ; by fractional extraction with boiling water and concentration of the extracts a total quantity of 2.6 g. of crystalline material was obtained. Calc. for CloHlaOa : C, 60.6 ; H, 7.1y0.) The physical properties of the acid and its p-phenylphenacyl ester were compared with those of materials similarly prepared from a normal pyrethrum extract (see Table VI).

Biological tests.-These were made according to the modified Peet-Grady procedure described in an earlier paper.

(Found : C, 60.5 ; H, 7.0.

Acknowledgments We wish to thank Mrs. Beryl 0. Stroud for performing the

biological tests, Mr. F. H. Tresadern for other analytical work, and the Directors of Stafford Allen & Sons Limited for permission to publish this work.

Stafford Allen & Sons Limited Wharf Road, London, N.I

Received March 9, 1950

References

68, 221 Mitchell, Wm. and Tresadern, F. H., J . Sac. cliein. Ind. , 1949,

Barthel, W. F., IIaller, H. L. and LaForge, I;. B., Soap, 1944, 20

3 Mitchell, Wm., Tresadern, F. H. and Wood, S. A,, Analysl, 1948,

4 Schechter, M. S., Green, K. and LaForge, F. U., J . Anier. chent.

s Campbell, F. I>. and Sullivan, W. N., ibitl., 1938, 14 (6), 119 7 Stoddard, R. B. and Dove, IV. E., ibitl., 1949, 25 (10). I I Y * Campbell, I. G. M. and Harper, S. H., J . client Sac., 1945, 285

Staudinger, H. and Ruzicka, L., Helu. ckim. Acta, 1924, 7, 201

lo Wilcoxon. F., Conlr. Boyce l'kornpson Inst., 1936, 8 , 175

(719 121

73, 484

Soc., 1949. 71, 3165 Gersdorff, W. A,, Soap, 1949, 25 (11). 129.

*