Structure-Activity Relationships for Insecticidal Carbamates*
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Bull. Org. mond. Sante' 1971, 4, 43-78 Bull. Wld Hith Org. Structure-Activity Relationships for Insecticidal Carbamates* ROBERT L. METCALF 1 Carbamate insecticides are biologically active because of their structural complemen- tarity to the active site of acetylcholinesterase (AChE) and their consequent action as substrates with very low turnover numbers. Carbamates behave as synthetic neurohor- mones that produce their toxic action by interrupting the normal action of AChE so that acetylcholine accumulates at synaptic junctions. The necessary properties for a suitable insecticidal carbamate are lipid solubility, suitable structural complementarity to AChE, and sufficient stability to multifunction-oxidase detoxification. The relationships between the structure and the activity of a large number of synthetic carbamates are analysed in detail, with particular attention to the second of these properties. Carbamate insecticides have their roots in the gical properties (for a review, see Stemple & Aeschli- witchcraft of West Africa, where the Calabar bean, mann, 1956). Physostigma venenosum (BaIlf.), was used as an Gysin (1954) announced the results of a compre- ordeal poison. Extracts of the bean containing the hensive study of the insecticidal properties of di- alkaloid physostigmine or eserine were found to be methylcarbamates of a series of alcohols such as 5,5- miotic as early as 1862 and Robertson (1863) dimethyldihydroresorcinol (Dimetan t) and 1-iso- suggested the use of the drug in ophthalmology. propyl-3-methylpyrazole (Isolan t), and these, toge- The pioneering researches of Stedman & Barger ther with Dimetilan,t have undergone considerable (1925) led to the elucidation of the structure of evaluation and have been used to some extent as physostigmine as the methylcarbamate of a substi- commercial insecticides (see Annex Table 1). All of tuted indole derivative. Physostigmine appears to be the compounds discussed in this important work the only natural product with the N-methylcarba- were N,N-disubstituted carbamates. The lack of moyl group and Stedman (1926) prepared a number critical quantitative data on toxicity for insects and of synthetic analogues with strong cholinergic on anticholinesterase activity precludes a detailed activity, of which m-trimethylammoniumphenyl discussion of the relationships between structure and methylcarbamate was outstanding and had obvious activity of these compounds. However, all of them usefulness in medicine for the treatment of glaucoma are potent anticholinesterase agents, and Roan and myasthenia gravis. However, this compound & Maeda (1954) have determined the I50 values for was unstable in aqueous solution, which interfered AChE from three species of fruit fly (Dacus dorsalis, with its medicinal use, and the corresponding Dacus cucurbitae, and Ceratitis capitata) for a dimethylcarbamate (neostigmine) was prepared by number of them. The values obtained were very Aeschlimann & Reinert (1931) and found to be not constant among the three species and ranged from only an effective miotic but much more stable. It has 5-6 x 10-7 M for Dimetant to 7-10 x 10-8 M for had substantial medical use. Thousands of related Isolan.t The most active inhibitor was 1-phenyl-3- carbamates have been investigated for pharmacolo- methylpyrazolyl dimethylcarbamate (I50 = 2-8 x 10-8 M). * A contribution from the WHO International Insecticide Our laboratory began investigations of carbamate Reference Centre, Departments of Entomology and Zoology, University of Illinois, Urbana-Champaign, Ill., USA. Sup- insecticides in order to determine why physostigmine ported in part by a grant from the World Health Organiza- and other charged compounds, such as the dimethyl- tion and by US Public Health Service Grant No. FD00271 from the Food and Drug Administration. 1 Professor of Zoology and Entomology, University of t Names against which this symbol appears are identified Illinois, Urbana-Champaign, Ill., USA. in the Glossary on pages 445-446. 2615 43- 4 44 R. L. METCALF Fig. 1 OCNHCH3 OCHNHCH3 OCNHCH3 OCNHCH3 CH3 CH3 +N c Si 3 CH3 3CCH3 33 CHL 3 physostigmine m-trimethylammoniumphenyl m - tet - butylphenyl m-trimethylsilylphenyl methylcarbamate methylcarbamate methylcarbamate carbamic acid ester of 3-hydroxy-2-pyridylmethyl ticides. Most of the mammalian toxicity data were dimethylamine, which inhibited fly AChE at 10-8 M, obtained during the course of this Programme.' It is were totally inactive as contact insecticides (Metcalf evident that the different carbamate insecticides vary & March, 1950). We decided to modify the struc- considerably in their toxicity to insects and to the tures of simple phenyl methylcarbamates to increase rat or mouse. The mammalian selectivity ratios, or their lipid solubility and found (Kolbezen et al., 1954) LD50 rat/LD50 housefly, range from > 100 for that phenyl methylcarbamate was insecticidal and butacarb to about 0.2 for aldicarb. An interesting that substitution in the ortho or the meta position comparison can be made between the low toxicity with an alkyl group or a halogen increased both of carbaryl to the rat and the 100-fold higher toxicity anticholinesterase and insecticidal activity. Maxi- of carbofuran; both of these compounds have mum activity was found with m-tert-butylphenyl approximately the same selectivity ratio of about 1.0. methylcarbamate, which is the uncharged isostere Since 1950 our laboratory has synthesized and of Stedman's original m-trimethylammoniumphenyl evaluated quantitatively the anticholinesterase and methylcarbamate (Fig. 1). This work focused atten- insecticidal activity of more than 600 carbamates. tion on the importance of structural complementarity These compounds can be thought of not only as to the active surface of AChE for producing highly candidate insecticides but also as highly precise active enzyme inhibitors and on the necessity for " tools " for exploring the microenvironment of the uncharged lipid-partitioning molecules to penetrate active site of the AChE molecule. The information to the site of action in the insect nerve. The impor- obtained forms the basis of this paper, emphasis tance of molecular complementarity has been further being given to information obtained since the emphasized by investigation of the three isomeric summary of Metcalf & Fukuto (1965b). trimethylsilylphenyl methylcarbamates (Metcalf & Fukuto, 1965a). The m-isomer ('50 = 7 x 10-7 M), INHIBITION OF CHOLINESTERASE BY CARBAMATES whose structural resemblance to the m-tert-butyl- Carbamate insecticides are synthetic analogues of phenyl methylcarbamate is obvious, was almost as the neurohormone acetylcholine (ACh), the synaptic effective as an insecticide and anticholinesterase mediator of nerve impulses in the central nervous agent as the latter, and was much more active than system of insects and in the parasympathetic nervous the o-isomer (I50 = 1.3 x 10-5 M) or the p-isomer system of vertebrates. As such they are attracted (I50 = 1.55 x 10-5 M). in 1: 1 ratio to the active site of the enzyme acetyl- During the past 20 years thousands of carbamates cholinesterase (AChE), where they act as substrates have been investigated as insecticides and a sub- for the enzymes that have very low turnover num- stantial number have emerged as commercial bers. Thus, Goldstein & Hamlesch (1952) showed products. Annex Table 1 includes nearly all of the that the rates of hydrolysis of neostigmine and compounds that have been used commercially or physostigmine were only about 5.5 x 10-7 and 1.2 x have undergone wide-scale investigation. The 10-7, respectively, that of ACh. At the active site of quantitative data on their insecticidal behaviour have ' World Health Organization (1968) Evaluation of been obtained in our laboratory under the WHO insecticides for vector control, part I, Geneva (mimeographed Programme for Evaluating and Testing New Insec- document WHO/VBC/68.66). STRUCTURE-ACTIVITY RELATIONSHIPS FOR CARBAMATES 45 AChE carbamate insecticides mimic the normal Using t = 10 min for the time of reaction of the hydrolysis of ACh (Eq. 1). carbamate with AChE, we find reasonable agreement ki k2 k3 E + I El El' --E + P; Ka = k ,/k, (1) Ki However, the carbamylated enzyme El' is perhaps between Ki values calculated from '50 and those a million-fold more stable than the acetylated enzyme measured directly (see the accompanying table). transitorily produced during the hydrolysis of ACh, and step 3 becomes rate-limiting. The resulting Comparison of calculated and measured Ki values inhibition of AChE produces an accumulation of ACh at the synaptic junctions (Booth & Metcalf, Kj (litre-mol-I min-) for: 1970) resulting in the typical cholinergic responses to Methylcarbamate bovine AChE b intoxication-synaptic facilitation and eventually fly ACh Ea synaptic block. These are manifested at the organ- value reference c ismic level by the sequence of symptoms of irri- tability, tremors, inco-ordination, convulsions, para- phenyl 3.4 x 102 5.4 x 102 1 lysis, and death. 2.9 x102 2 This brief resume of carbamate intoxication m-isopropylphenyl 2 x 105 4.56 x 105 2 suggests that the active carbamate molecules should 5.4 x 3 have some structural resemblance to ACh both in 105 stereochemistry and in reactivity, and should be 3,5-diisopropylphenyl 2 x 106 4.07 x 105 1 complementary in structure to the surface features 1-naphthyl 7.7 x 104 1.25 x 105 1 of AChE, so as to facilitate formation of the El 2.18 x 104 2 complex and subsequent carbamylation of the 2.06 x serine hydroxyl at the active