PERSISTENT and BIOLOGICALLY DEGRADABLE ANALOGUES of DDT Bull

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SESSION IX PERSISTENT AND BIOLOGICALLY DEGRADABLE ANALOGUES OF DDT Bull. Org. mond. Sant 1971, 44, 355-362 Bull. Wld Hith Org.

Rational Design of Insecticides

G. HOLAN 1

A steric model of diaryl insecticides, which includes the structure of DDT and its analogles, was usedfor the synthesis ofnew highly active insecticides. The new compounds show low toxicity to mammals and their insecticidal activity can be potentiated by microsomal oxidase inhibitors. There is evidence that they readily undergo biological degradation. However, a spontaneous controlled chemical degradation was also established for the diaryl oxetanes, which form one group of a new series of insecticides. The theoretical model was further investigated using X-ray crystallographty to establish one accurate structure. Biologically, the model is supported by studies of the correlation between insect mortalities and temperature and by measurements of nerve impulses at fly chemoreceptors after treatment with selected compounds.

For the past several years we have been engaged in tuents. The size limitation for this part of the molecule the rational synthesis of broad-spectrum insecticides was found to be less restrictive than that for the apex. having low toxicity for mammals. This paper briefly An hypothesis that assigned definite roles to the outlines several facets of our work in both the two parts of the theoretical model was developed chemical and the biological fields. from the known biological effects of this type of There are good reasons for using a theoretical insecticide in whole organisms and from more model rather than the more common approach of detailed studies of their mode of action in nerve the random synthesis of biologically active com- axons (Narahashi & Haas, 1968). It was postulated pounds. If the model is valid, it becomes possible to that (1) lipid-soluble insecticides distribute themselves correct past omissions in the syntheses of related at the protein-lipid interface ofa nerve membrane and analogues; it also becomes feasible to develop new the base containing the phenyl rings locks itself by structures that could not be obtained by intuition. complex-formation with the aromatic rings into the overlying protein; (2) the apex keeps open a molecu- THE MODEL lar spring, site, or channel for sodium ions, thus in- ducing a leakage of these ions into the nerve axon; Our model for the synthesis of aryl insecticides was and (3) this action causes the delay in the falling phase developed from a series of diaryl halocyclopropane of the action potential and results in the characteristic analogues of DDT (Holan, 1969). The three- multiple spikes of generated nerve impulses. dimensional model, which also accommodates previ- Since this model was formulated we have obtained ously known DDT analogues, was obtained by com- additional evidence for the validity of its proposed paring projections of steric atomic models with biological role. Complex-formation between the accurate insect mortality data for active compounds. insecticide and nerve protein is consistent with The shape that was obtained in this way resembled the well-known phenomenon of negative correlation a "molecular wedge" with two distinct features. between insect mortality and temperature. This First, the influence of the apex of the "wedge" effect has been observed in the hydrocarbon series (e.g., the part containing the -CCI3 group of DDT) only for insecticides containing an aryl ring (Barker, on the insecticidal activity was found to be indepen- 1957; Hoffman & Lindquist, 1949; Guthrie, 1950). dent of the chemical composition of the group form- The equilibrium constants of charge-transfer com- ing it, provided one kept to strict dimensional plexes are similarly temperature-related (Briegleb, requirements. Second, the two phenyl rings compris- 1961) and their stability is directly dependent on the ing the "base" had to have electron-donating substi- polarization of the aromatic rings. Fig. 1, which is based on previously synthesized halocyclopropanes I Division of Applied Chemistry, CSIRO, Box 4331 G.P.O. Melbourne, Australia. and on several of the new insecticides, shows that the 2648 -355- 356 G. HOLAN

Fig. 1 Temperature-mortality curves for five different diaryl insecticides *

__j _ _ _ _L L_ _ __ i- _ __ __- 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 3 Teniperiture °C

CH3 R - - c -CH3 cIi R 1X R CH3 CI

NO R -- - - - CH -C R CL2-CH3 R \ ci CI

Cl - CH-c-Cl

]~ ~ ~ ~ ~ o2i RZcl

R = OC Hs RI = Cl

* The fact that the lines are almost parallel (i.e., that their slopes are almost identical) indicates an effect of suitable substitution in the phenyl rings of otherwise dissimilar structures. This negative correlation is related to similar temperature effects of 7r-charge-transfer-complex equilibrium constants and is believed to involve the formation of a complex between the phenyl rings of the insecticides and the overlaying protein of nerve membranes. RATIONAL DESIGN OF INSECTICIDES 357

Fig. 2 Projection of an atomic model of 1,1-bis(p-ethoxyphenyl)-2,2-dimethylpropane, obtained from X-ray crystallographic data

illot ops

O hydrogen atoms O carbon atoms * oxygen atoms carbon atoms in the neopentane skeleton negative correlation between temperature and mor- DESIGN OF COMPOUNDS tality is the same for all the phenyl-ring-substituted insecticides, provided they contain an electron-donor The validity of the model was further confirmed by group in thepara-position. This effect is independent the predictive synthesis of active structures. In the of the magnitude of the insecticidal activity and of study of numerous compounds in the fluorohydro- the chemical nature of the molecule. From an carbon, phosphorus, heterocyclic, alicyclic, and organic chemist's point of view there is no relation- hydrocarbon series we did not find a single excep- ship between the dichlorocyclopropane moiety and tion to the defined dimension of a projected van der a tert-butyl group or between this and a nitroalkane, Waals diameter of the apex of the molecules. This except for their dimensional similarity. Therefore must be within the limits of 0.6 0.05 nm, and it has the complex-formation must reside in the suitably been found that the LD50 of compounds that con- substituted aromatic rings of the insecticides. form to this limitation for a DDT-susceptible strain The biological role of the apex of the molecule, of the housefly (WHO/IN/Musca domestica/l) is less which was claimed to have a size related to a hydra- than 5.0 ,ug/insect. A few selected new structures ted sodium ion, was more difficult to prove and the that illustrate this design are listed in the accom- detailed work will be reported elsewhere. Briefly, panying table. however, it was found that the insecticide-induced In the nitroalkane series the p-ethoxyphenyl ana- multiplicity of single spikes in the train of impulses logues of the insecticides 1,1-bis(p-chlorophenyl)-2- measured at a salt-stimulated labellar taste receptor nitropropane (Prolan) and 1 ,1-bis(p-chlorophenyl)-2- of a susceptible housefly (Barton-Browne & Kerr, nitrobutane (Bulan, compound XI 1) were not 1967) is independent of concentration. In struc- known. This is understandable since, in the absence turally similar size-graded halocyclopropane insec- of a predictive model, the lack of activity of the easily ticides this effect can be related both to the diametre 1 Compounds referred to by roman numerals are those ofthe apex ofthe molecule and to the insect mortality. listed in the accompanying table. 358 G. HOLAN

Toxicity of new insecticides to the mouse and to a susceptible strain of the housefly* GENERAL FORMULA

Rl-4R3 -CR-2

LDso (,ug/insect) Compound for Musca domestica a Synergistic LDso (mg/kg) R3 substituent No. Other substituents ratio for the mouse c LUnsyn- Syn - ergized ergized b

ci 0.24 0.25 CH-C-Cl/I R1 = R2 = Cl (DDT) 0 570 ci

CH3 11 R' = R2 = C2H50 1.92 0.32 5.5 5 200 CH-C-CH3 / CH3 III RI = R2 = CH30 3.2 1.7 1.9 CH3 CH-CH2-c-CH3 IV RI = R2 = C2H50 2.0 0.36 5.5 / CH3 iv CH3 /-CH, V R1 = R2 = Cl 12.0

Vi RI = R2 = CH30 >20

vii R' = R2 = C2H50 0.48 0.065 7.4 1 150

CH-CH-CH3 Vill R' = C2H5S 0.16 0.015 10.7 1 040 N02 R2= C2HsO 360 d

RI = 3,4-(-O-CH2-O-) -2 000 ix 0.14 0.019 7.4 R2= C2H50 -2 000 d

CH3 CH-C-NO2 x R' = R2 = C2HsO 1.01 0.053 10.6 CH3

Xi R' = R2 = Cl (Bulan) 1.69 1.73 0

Xii R' = R2 = C2HsO 0.55 0.061 9.0 1 160 980 d CH-CH-CH2-CH3 /I Xiii R' = C2H5S 0.11 0.04 2.8 N02 R2 = C2H50

Xiv R' = 3,4-(-O-CH2-O-) 0.12 0.028 4.3 R2= C2H50 RATIONAL DESIGN OF INSECTICIDES 359

Toxicity of new insecticides to the mouse and to a susceptible strain of the housefly (concluded) v GENERAL FORMULA

LD50 (,jg/insect) R3 Compound LDso substituent No. Other substituents forUsnMusca domesticaSy- a Synergisticratio for the(mg/kg)mouse c ergized ergized b

XV R1 = R2 = Cl 1.27 0.66 1.9 3 500 R4 = R5 = CH3 R6 = H

XVI RI = R2 = Cl >100 >100 _ _ R4 = R6 = CH3 R5= H

XVII RI = R2 = C2HsO 0.52 0.01 52 1 200 R4= R5 = CH3 380 d Re = H

XVIII R1 = R2 = C2H50 >100 >100 - _ R = Rs= R6 = CH3

XIX R' = C2HsO 1.43 0.086 17.9 - R4 = CH30 R4= R5 = CH3 Re = H

* All tests and calculations of LD50 values were carried out according to a previously described method (Holan, 1969). a Using a DDT-susceptible strain of the housefly (WHO/IN/Musca domestica/1). b In potentiation experiments, 0.5 microlitre of a 1 % solution of sesamex in acetone was applied immediately after the application of the dose of insecticide. c The compounds, in olive oil, were injected intraperitoneally into female albino Swiss mice. The average weight of the mice was 20 g and 5 mice were used for each dosage level. The median lethal dose was calculated from the mortalities obtained in 5 days. d Toxicity when synergized with sesamex (1: 1 insecticide: sesamex ratio). prepared 1,1-bis(p-methoxyphenyl) analogue (VI) latter compound is of particular interest because of and the 1-p-ethoxyphenyl-1-o-ethoxyphenyl ana- its relationship to the dimethylcyclopropane struc- logue (Jacob et al., 1951) would discourage workers tural part of pyrethrin insecticides. from undertaking the extensive chemical investiga- The synthesis of 1,1-bis(p-ethoxyphenyl)-2,2-dime- tions that we found were necessary to solve the thylpropane (II) gave us an opportunity to obtain difficult synthesis of the bis(p-ethoxyphenyl) deriva- from X-ray crystallographic data the first exact tives VII & XII. Moreover, the activity of the new structure of a DDT-type insecticide (Fig. 2). This will tertiary nitroalkane insecticide X could not have shortly be followed by the solution of the structure of been predicted without the use of the model. DDT itself. In the rigidly structured surrounding In the hydrocarbon series, the 3,3-dimethyl- of the nerve membrane (Hechter, 1965) where the butane derivative IV and the 2,2-dimethylcyclo- insecticides are thought to act, the compounds are propane derivative V were prepared after the better represented by structures obtained in a solid examination of their projected atomic models. The crystal lattice than by their conformation in solution. 360 G. HOLAN

Fig. 3 Projected maximum van der Waals diameters of p-chlorophenyl-substituted 3,3-dimethyl oxetane (left) and trans-3,4-dimethyl oxetane (right) rings*

Cl Cl Cl

1CH3 ICH33 \ I \ 01 ICH3 / I .C- I I H CH31

;,0.7 nm _-.

* The LD5o values (iug/insect) for a susceptible strain of the housefly were 1.27 for the 3,3-dimethyl compound and >200 for the 3,4-dimethyl compound.

However, the best illustration of the design is and, when potentiated, the bis(p-ethoxyphenyl) provided by the formation of the dimethyl oxetanes. derivative XVII shows an insecticidal activity In a search for a non-persistent insecticide we looked 25 times that of DDT. The 3,4-dimethyl and for a semistable heterocyclic ring that would conform 3,3,4-trimethyl derivatives XVI and XVIII do not to the stated dimensions. From projections of conform and are completely inactive. The initial atomic models it was deduced that the correct size selection of the oxetane ring was also based on would be obtained by the minor change of one previous reports (Margerum et al., 1959) that methyl group between the 3,3-dimethyl and the indicated that the compounds would readily undergo 3,4-dimethyl oxetanes; similarly, the addition of a degradation by electrocyclic fission to formaldehyde methyl group at the 4-position of the 3,3-dimethyl and the biologically inactive 1,1-bis(aryl)-2,2-dimethyl- oxetanes would alter unfavourably the down- ethylenes (Fig. 4). We have found that this chemical ward projection of the model (Fig. 3). The 3,3- degradation can be controlled by the removal or dimethyl oxetanes have the correct dimensions addition of free-radical initiators.

Fig. 4

II + H CHO H3C 1 C HC ~~~~H3C CH3

A-O :9:113 RATIONAL DESIGN OF INSECTICIDES 361

POTENTIATION OF INSECTICIDAL ACTIVITY The addition of synergists to overcome resistance In the alkoxyphenyl or alkylthiophenyl deriva- in insects or to increase the activity of insecticides is tives it is possible to assume the formation of not an established method in public-health and quinonic resonance structures.' These enhance agricultural pest control. Therefore we attempted the reactivity of the molecules and their ability the synthesis of " self-potentiated" compounds. to form n-charge-transfer complexes. The reson- This approach was based on the observation that a ance structures would also contribute to a highly lack of symmetry of substituents in the phenyl rings increased susceptibility to electrophilic oxidative does not negate the activity of the compounds. Com- attack at the benzylic carbon. That this takes place pounds IX and XIV contain the 3,4-methylenedioxy- is strongly supported by the effect of microsomal phenyl group, which is a major substituent of mixed-function oxidase inhibitors (e.g., sesamex) on several insecticide synergists (Wilkinson, 1967). the activity of the alkoxy and alkylthio insecticides Keeping within the dimensional limits of our model, (see the accompanying table). With the p-chloro- we substituted a p-ethoxy group in the other phenyl phenyl-substituted insecticides, such as Bulan (XI) or ring. The non-synergized insecticidal activity of DDT (1), no potentiation is observed. Furthermore, these unsymmetrical compounds reaches nearly the comparison of the synergistic ratios of the p-chloro- level of the synergized activity of their bis(p-ethoxyphenyl-substituted and p-ethoxyphenyl-substituted phenyl) analogues VII and XII. A similar increase oxetanes XV and XVII demonstrates the contribu- in activity is found for the thiophenyl derivatives VIII tion of the ethoxy group to the ease of oxidative and XIII. We have not as yet determined whether degradation of the insecticides. If the assumption is the compounds actively inhibit the oxidase system or valid that the intrinsic insecticidal activity is observed only present a substrate that is more resistant to the only when the compounds are potentiated (i.e., when enzymatic oxidation. This we hope to resolve by the full dose of the non-degraded compound reaches further synthesis and the testing of combinations of the active site), then several of the alkoxyphenyl the insecticides. compounds are much more active than their halogen The high activity and rapid degradation of some analogues. The potentiation of the alkoxyphenyl of the new compounds, together with their low acute compounds does not discriminate to any extent be- toxicity for mammals (see the table) could make them tween the synergists reported to be inhibitors of the a valuable alternative to some existing insecticides. mixed-function oxidase in insects (Wilkinson, 1967). However, the lack of persistence of the new insecti- When synergized with a 1.0% solution of piperonyl cides is unsatisfactory under present practice of butoxide, the nitrobutane XII showed an LD,0 of control of insect pests. Consequently, we are con- 0.05 pg/insect for the housefly, whereas it showed tinuing our research and hope to obtain in the future an LD50 of 0.12 jg/insect when synergized with a structure with a correct balance of activity and a 0.5 ' solution of 4-dimethylamino-5-nitro-1,2- persistence. methylenedioxybenzene (Wilkinson, 1967). The results of studies of the chemistry of the com- Potentiation would be an attractive method for the pounds reported herein, of X-ray determinations of control of insects in particular, since the data in the their structure, and of investigations of their degrada- accompanying table show that, for several compounds tion and of their activity against other insects will be (VIII, IX, XII, and XVII), potentiation takes place published elsewhere. predominantly in the insect rather than the mammal. The new insecticides are covered by patents assigned to the Commonwealth Scientific and I These have been referred to as " quinone-methide" resonance structures. Industrial Research Organization.

ACKNOWLEDGEMENTS

The author thanks Dr E. Shipp, Entomology Depart- Mr D. O'Keefe, Mr R. Eibl, and Mr R. Walser for ment, School of Life Sciences, University of New South assistance in the chemical investigations. Studies of Wales, for supervising the entomological work; Dr C. L. toxicities for mammals were carried out in part at the Kennard and Mr T. Delacy for X-ray structure analysis; Department of Pharmacology, University of Melbourne, Mrs J. Johnston, Mrs T. Ejmont, Mr F. Romer, and and at the Commonwealth Serum Laboratories, Mel- Miss N. Ali for assistance in the biological studies; and bourne. 362 G. HOLAN

REFERENCES

Barker, R. J. (1957) J. econ. Ent., 50, 446-450 Holan, G. (1969) Nature (Lond.), 221, 1025-1029 Barton-Browne, L. & Kerr, R. W. (1967) Ent. exp. appl., Jacob, T. A., Bachman, G. B. & Haas, H. B. (1951) 10, 337-346 J. org. Chem., 16, 1572-1574 Briegleb, G. (1961). In: Electron-Donator-Acceptor Kom- Margerum, J. D., Pitts, J. N., Rutgers, J. G. & Searle, plexe, Berlin, Springer, pp. 140-146 S. S. (1959) J. Amer. chem. Soc., 81, 1549-1551 Guthrie, F. E. (1950) J. econ. Ent., 43, 559-560 Narahashi, T. & Haas, H. G. (1968) J. gen. Physiol., 51, Hechter, 0. (1965) Ann. N. Y. Acad. Sci., 125, 625-646 177-198 Hoffman, R. A. & Lindquist, A. W. (1949) J. econ. Ent., Wilkinson, C. F. (1967) J. agric. Food Chem., 15, 139- 42, 891-893 147

DISCUSSION

WRIGHT: How stable are these compounds in relation to implications for health. Do you have any information the existing chlorinated hydrocarbons? on whether your compounds induce microsomal enzymes? HOLAN: They are less stable. HOLAN: No. The inducement of oxidative enzymes HAYES: The term " persistent " applied to pesticides may follows from the model I have proposed. A " molecular be misleading. All organic compounds undergo degrada- wedge " should affect membranes generally and should tion at a slower or a faster rate. The serious fault of release bound enzymes from mitochondrial surfaces. DDT is not merely that it is persistent but that it accumul- Our compounds are less stable in biological environ- ates to a high degree in all steps of at least a few food ments than is DDT, and therefore such action is less chains. likely. HOLAN: We are attempting to alter the stability of our DONNINGER: I do not fully understand your reply to compounds to make them more persistent. Several, Dr Dauterman's question. Have any long-term mam- such as the oxetanes, would be expected to be stable on malian feeding studies been carried out and if so is any dry surfaces but possibly to hydrolyse in an aqueous liver enlargement observed? environment. HOLAN: We have not carried out any chronic toxicity DAUTERMAN: A number of chlorinated-hydrocarbon tests. In a short study of a few of the new compounds, insecticides induce microsomal enzymes, which has Dr Barnes demonstrated no cumulative toxicity.