Environmental Health PerspectivesVol. 87, pp. 245-254, 1990

Mechanism of Action of Organophosphorusand Carbamate Insecticidesby T. Roy Fukuto*

Organophosphorus and carbamate insecticides are toxic to insects and mammals by virtue of their abilityto inactivate the enzyme acetylcholinesterase. This review addresses the mechanism of inhibition ofacetylcholinesterase by organophosphorus and carbamate esters, focusing on structural requirementsnecessary for anticholinesterase activity. The inhibition of acetylcholinesterase by these compounds isdiscussed in terms of reactivity and steric effects. The role of metabolic activation or degradation in theoverall intoxication process is also discussed.

IntroductionThe toxicity of insecticidally active organophosphorus

and carbamate esters to animals is attributed to theirability to inhibit acetylcholinesterase (AChE, cholinehydrolase, EC 3.1.1.7), which is a class of enzymes thatcatalyzes the hydrolysis of the neurotransmitting agentacetylcholine (ACh). The inhibition ofAChE and relatedesterases, often referred to as serine hydrolases, hasbeen clearly demonstrated to be the result of an actualchemical reaction between the enzyme and the organo-phosphate or carbamate ester (1-3). The phosphoryl-ated or carbamylated enzyme is no longer capable ofeffecting the hydrolysis ofACh; this results in a buildupof the neurotransmitter at a nerve synapse or neuro-muscular junction.The organophosphorus and carbamate insecticides

are represented by a wide variety ofchemical structureshaving different chemical and physical properties. Thetoxicity of these materials to insects and mammals isdetermined by a number of factors that may affect theinsecticides as they are absorbed, translocated to thetarget site, and as they inactivate the target, leadingto poisoning. This review is concerned with the modeof action of organophosphorus and carbamate esters,with emphasis placed on structural requirements nec-essary for inhibition of AChE.

AcetylcholinesteraseBefore entering into any discussion on the inhibition

of AChE by organophosphorus and carbamate esters,it is appropriate first to provide a brief review of theenzyme. AChE is present and has been isolated from awide range of animals, including mammals, birds, fish,

*Department of Entomology, University of California, Riverside,CA 92521.

reptiles, and insects (4). It is responsible for the rapidhydrolytic degradation of the neurotransmitter AChinto the inactive products choline and acetic acid, asindicated by Eq. (1). Acetylcholine is one of a numberof

CH3+: AChE

CH3-N-CH2CH20CCH3 -I H20

ACh (acetylcholine)

CH3

CH3-N-CH2CH20H + CH3C-OH

CH3choline acetic acid (1)

physiologically important neurotransmitting agents andis involved in the transmission of nerve impulses toeffector cells at cholinergic, synaptic, and neuromus-cular junctions. The presence of AChE has been dem-onstrated in a variety of animal tissues and enzymesfrom a number of different sources, including fish elec-tric organs, mammalian erythrocyte, insect and mam-malian brain, and other tissues. These enzymes havebeen purified and characterized (4-8). AChE is virtuallya ubiquitous enzyme in vertebrates and invertebrates,and in mammals, it is localized in certain areas of thecentral nervous system and in organs and glands thatare controlled by the parasympathetic division of theautonomic nervous system.The role of AChE in cholinergic transmission at a

synaptic or cholinergic junction is depicted in the ele-

T. R. FUKUTO

AChsite in postsynapticmembrane

ACh. En

A Ch

FIGURE 1. Scheme showing the role of AChE and ACh in cholinergic transmission.

j~CH 10

(B OC+~C)t,CH3~CH' "CHHN,

esterotic G nOiOc (E S)site (E +S) site I

()CH.

I-~CH OH _ H HOHC2P 1 Vi-

(E ) ( E)

FIGURE 2. Mechanism of hydrolysis of acetylcholine by acetylcho-linesterase (see text for details).

mentary scheme given in Figure 1. When a nerve im-pulse moves down a parasympathetic neuron andreaches a nerve ending, the ACh stored in vesicles inthe ending is released into the synaptic or neuromus-cular junction. Within 2 to 3 msec the released AChinteracts with the ACh receptor site on the postsynapticmembrane, causing stimulation of the nerve fiber ormuscle. AChE serves as a regulating agent of nervoustransmission by reducing the concentration of ACh inthe junction through AChE catalyzed hydrolysis of AChinto choline (Ch) and acetic acid (A). These products donot stimulate the postsynaptic membrane. In thescheme En denotes the enzyme AChE; AChi En- isthe enzyme-substrate complex formed prior to hydrol-ysis of ACh into choline and acetic acid. When AChEis inactivated, e.g., by an organophosphorus or carba-mate ester, the enzyme is no longer able to hydrolyzeACh; the concentration of ACh in the junction remainshigh, and continuous stimulation of the muscle or nervefiber occurs, resulting eventually in exhaustion and te-tany.Based on a number of studies (2), a plausible mech-

anism for AChE-catalyzed hydrolysis of ACh is pre-

sented in Figure 2. ACh is drawn to the active site ofthe enzyme by electrostatic attraction between the pos-itive charge on the ACh nitrogen atom and negativecharge in the anionic site (structure E + S) resultingin the enzyme-substrate complex (ES). Acetylation ofa serine hydroxyl (OH) in the esteratic site is catalyzedby the basic imidazole moiety B (histidine) and acidicmoiety AH (tyrosine hydroxyl), leading to the acety-lated enzyme EA. Deacetylation then takes place veryfast, resulting in the free enzyme (E) within millise-conds. As presented, ACh hydrolysis by AChE has theelements of an acid-base catalyzed reaction, includingboth the acetylation and deacetylation reaction. Thenegative charge at the anionic site is attributed to thecarboxylate anion of aspartic or glutamic acid. The re-action steps given in Figure 2 provide an elementarypresentation of the AChE active site and a reasonablemechanism for the hydrolysis of ACh. The enzyme is inreality a highly complex protein, having in addition tothe esteratic and anionic sites, a number of peripheralsites and hydrophobic areas (5).While the preceding discussion has focused on AChE,

it should be pointed out that there is at least one othertype of cholinesterase enzyme beside AChE, namelypseudocholinesterase. AChE has the highest specificityfor ACh of any other choline ester and pseudocholin-esterase has the highest specificity for butyrylcholine.The physiological role of pseudocholinesterase in ani-mals is not as well defined as that of AChE (4). How-ever, both enzymes are inhibited by organophosphorusand carbamate esters.

Organophosphorus InsecticidesMechanism of InhibitionThe inhibition ofAChE by an organophosphorus ester

(Fig. 3) takes place via a chemical reaction in which theserine hydroxyl moiety in the enzyme active site is phos-phorylated in a manner analogous to the acetylation ofAChE (Fig. 2, EA). In contrast to the acetylated en-zyme, which rapidly breaks down to give acetic acid andthe regenerated enzyme, the phosphorylated enzyme(Fig. 4) is highly stable, and in some cases, dependingon the groups attached to the phosphorus atom (R andR'), it is irreversibly inhibited (9). The serine hydroxylgroup, blocked by a phosphoryl moiety, is no longerable to participate in the hydrolysis of ACh. The inhi-

246

MECHANISMS OF ORGANOPHOSPHORUS AND CARBAMATE INSECTICIDES

En\ 8I EtC-. (-C o

EtO

EtO 4 O NCEtO 0

0

En-OP(OEt)2 + O pNO2

FIGURE 3. The effect of an electron-withdrawing substituent on the reactivity of paraoxon.

bition reaction takes place in a two-step process, asindicated by Eq. (2). In this equation

Kd kpEn-OH + R-P-X (En-OH-R-P-X]: ' En-O-P-R + X

I IIRe R= Ioo RI (2)(complex)

kj

En-OH represents AChE in which the serine hydroxylmoiety (-OH) is emphasized, R and R' are a varietyof different groups (alkoxy, alkyl, amino, thioalkyl,etc.), X is the leaving group, Kd is the dissociation con-stant between the enzyme-inhibitor complex and reac-tants, kp is the phosphorylation constant, and ki is thebimolecular rate constant for inhibition and is equal tok/IKd (10). Since Kd provides a measure of the disso-ciation of the enzyme-inhibitor complex, it is regardedas an estimate for binding and is dependent on the struc-tural and steric properties of the molecule. In contrast,the phosphorylation constant kp is regarded as an es-timate of the reactivity of the organophosphorus ester.The bimolecular rate constant ki is dependent on thevalues of Kd and kp and is generally regarded as themost useful parameter for the estimation of the inhib-itory potency of an organophosphorus (and carbamate)anticholinesterase. According to Eq. (2), the moiety Xis displaced from the phosphorus atom by the serinehydroxyl of the enzyme and is, therefore, referred toas the leaving group.

Reactivity and Steric PropertiesBased on systematic investigations ofthe relationship

between chemical structure and inhibition of AChE, it

R

0 HA'I e~~

FIGURE 4. Phosphorylated, and irreversibly inhibited, AChE (seetext and Figure 2 for details).

is apparent that the single most important propertyrequired in an organophosphate for anticholinesteraseactivity is chemical reactivity, i.e., the phosphorus estermust be reactive to the extent that the serine hydroxylmoiety in the enzyme is phosphorylated. Structure-ac-tivity studies have revealed a direct relationship be-tween anticholinesterase activity and reactivity of thephosphorus atom. The inhibition of erythrocyte AChEby diethyl p-nitrophenyl phosphate (paraoxon) andsome of its substituted phenyl analogs was found toproceed with pseudo first-order kinetics (1)t The bi-molecular rate constant for inhibition paralleled therates of alkaline hydrolysis of these phosphates. It wassubsequently demonstrated with a larger series of di-ethyl substituted phenyl phosphates that the inhibitionof fly-head AChE by paraoxon analogs was related tothe effect of the substituent on the liability of the P-O-phenyl bond as estimated by Hammett's sigma con-stants, shifts in P-O-phenyl infrared stretching fre-quencies, and hydrolysis rates (11). A plot of the log ofanticholinesterase activity against Hammett's sigmaconstant (also P-O-phenyl stretching frequency) re-sulted in a good linear relationship with activity directlyrelated to the electron-withdrawing properties of thesubstituents. The effect ofan electron-withdrawing sub-stituent on the reactivity of a diethyl substituted phenylphosphate is demonstrated in Figure 4 with paraoxon.Because of the strong electron-withdrawing propertyof the nitro group, electrons are attracted away fromthe phosphorus atom, creating an electron-deficient cen-ter that facilitates a nucleophilic attack by the enzymeserine hydroxyl moiety on the phosphorus atom withsimultaneous expulsion of the nitrophenoxide leavinggroup. Diethyl-substituted phenyl phosphates withweak electron-withdrawing or electron-donating sub-stituents were weak inhibitors or were devoid of activ-ity.While chemical reactivity of the phosphorus atom is

of prime importance for anticholinesterase activity,steric properties sometimes have a strong effect on theanticholinesterase activity of an organophosphorus es-ter. This became apparent from examining a series ofethyl p-nitrophenyl alkylphosphonates for anticholin-esterase activity and alkaline hydrolysis rates wherethe alkyl group was varied (12). The rate of hydrolysisof the phosphonate ester to ethyl alkylphosphonic acidand p-nitrophenol, in general, decreased with an in-

247

T. R. FUKUTO

crease in alkyl chain length, and it decreased stronglywith branching in the 1 and 2 positions of the alkylmoiety. Determination of housefly-head anticholinest-erase activities of these compounds revealed the rela-tionship shown in Figure 5 between bimolecular rateconstants for inhibition of housefly-head AChE (ki) andpseudo first-order hydrolysis constants (khyd, pH 8.3).Although the plot between log ki and log khyd showeda general trend toward linearity, close examination ofthe data revealed that many of the points followed asigmoidal relationship, particularly those compoundswith a straight chain alkyl. For example, as the chainlength increased from three to six carbon atoms, therate of inhibition of AChE dropped rapidly even thoughhydrolysis rates remained relatively constant. The plotreveals the influence of steric effects in the inhibitionof AChE by these compounds, i.e., the inhibition ratesare reduced as the alkyl moiety attached to the phos-phorus atom becomes bulkier. The importance of stericeffects in AChE inhibition was subsequently pointed outby Hansch and Deutsch (13) who showed that anti-cholinesterase activities for the compounds in Figure 5were related to Taft's steric substituent constant, E.,according to Eq. (3) where n is the number of com-pounds, s is the standard deviation, and r is the cor-relation coefficient.

log ki = 3.738ES + 7.539n = 13, s = 0.749, r = 0.901 (3)

While this study provided fundamental information onthe effect of the P-alkyl moiety on the reactivity of

4 -

3 -

2-

1 -

0

* C2H 5n - C3H74

i- CH15 1 #*n--C4H.

3

i CtH13 *phenyl

n - C6H 5nC5H1 1613# ,0

4,4-dimethylpentyl (CH ) Cli - CH 2

4 9

cyi-Ch3H7cyclohexyl

alkylphosphonate esters, it also revealed that the P-alkyl moiety should be kept small (methyl or ethyl) indesigning potential insecticides from phosphonate es-ters.

Structure of OrganophosphorusAnticholinesterase InsecticidesApproximately 100 different organophosphorus in-

secticides have reached the stage of commercial devel-opment during the past four decades (14). Examinationof these insecticides reveals a variety of different struc-tures with virtually all ofthem falling within the generalstructure depicted in Figure 6, where R is methyl orethyl; R' is either methoxy, ethoxy, ethyl, phenyl,amino, substituted amino or alkylthio; and X is an ap-propriate leaving group. As indicated, the double-bonded oxygen may be replaced with sulfur. Examplesof organophosphorus insecticides having each of the dif-ferent R' groups are given in Figure 7. Although R'and RO for most of the organophosphorus insecticidesare the same, i.e., R'=RO=CH30 or C2H50, as in thecase of methyl parathion and diazinon, the examplesgiven previously indicate the range of groups found inR'. Compounds where R'=RO and R is either propylor isopropyl are less effective insecticides compared tothose where R is methyl or ethyl and, therefore, theyhave generally not been developed as insecticides. Sincethe previous examples are all effective insecticides (andnematicide in the case of nemacur), it may be assumedthat each compound or a corresponding metabolic prod-uct is able to phosphorylate AChE to give the phos-phorylated enzyme as indicated in Figure 6, where Rand R' are the same as in the examples in Figure 7, orR' is converted metabolically into another functionalgroup [for example the acetamido moiety in acephateto amino in methamidophos (15)].The leaving group is represented by a much broader

range of structures, and the largest number of varia-tions in the structure of an organophosphorus insecti-cide is found in X. The ability of X to leave the phos-phorus atom is governed by the chemical nature of Xand that of the other groups attached to phosphorus.For organophosphorus esters of high insecticidal activ-ity, X is an electronegative group or a group containingan electronegative substituent leading to a compoundwith a reactive or labile P-X bond. In some instances

RO0 0(S)

R ' X

En-O-P -OR

0 2 3

LOG (KhYd x 105 )

FIGURE 5. Relationship between log acetylcholinesterase inhibitionconstant ki and log hydrolysis constant khyd for ethyl p-nitrophenylalkylphosphonates. Data from Fukuto and Metcalf (12).

(A) (B)

FIGURE 6. General structure of organophosphorus insecticides (A)and phosphorylated AChE (B). R is methyl or ethyl; R' is eithermethoxy, ethoxy, ethyl, phenyl, amino, substituted amino or ak-lylthio: X is an appropriate leaving group and En is AChE.

o

x

IC0-i

248

I

MECHANISMS OF ORGANOPHOSPHORUS AND CARBAMATE INSECTICIDES

CH3Q, 0S

CH30 ON02

methyl parathion(R' = methoxy)

C2HAQ + S , C3H7-jdesp O.

C

C2H50

CH3diazinon(R' = ethoxy)

C2H5Q1, ^ S

-0%%Ib\C2H5 S

dyfonate(R' = ethyl)

CH30 0

H2 pH2N SCH3

CH30 0CC p%.C

CH3C-N SCH3

EPN(R' = phenyl)

C2H50. #0 CH3

i-C3H7-N 0 )SCHH

nemacur

(R' = subst'd amino)

methamidophos(R' = amino)

C2H50, d0

;3 nn

acephate(R' = subst'd amino)

-C3H7S 0 C1

Brprofenofos

(R' = propylthio)

FIGURE 7. Different organophosphorus compounds showing variation in R'.

X is a moiety that is metabolically activated to give alabile P-X bond. In most cases X is a substituted phen-oxy or aromatic group containing hetero atoms, sub-stituted thioalkyl, or substituted alkoxy. Additional ex-amples of prominent organophosphorus insecticideswith variation in the leaving group, X, are given inFigure 8. The large number of organophorus insecti-cides that have attained commercial importance is at-tributable primarily to the large number of leavinggroups possible. In no other class of insecticides has itbeen possible to have broader variation in structure andin spectrum of insecticidal activity.

Metabolic ActivationMore than half of the compounds in Figures 7 and 8

contain the P-S moiety. Phosphorothionate esters(P=S) are generally poor anticholinesterases, yet all ofthe compounds given in Figures 7 and 8 are potentinsecticides. The poor anticholinesterase activity ofP-S esters is explained on the basis of their relativelylow reactivity, attributed to the smaller extent to whichthe P-S bond is polarized compared to the P=O, owingto the lower electronegativity of sulfur compared tooxygen. Polarization ofthe P=O linkage (Fig. 9) resultsin a more electropositive phosphorus atom, which fa-cilitates attack on phosphorus by nucleophilic agents,e.g., the serine hydroxyl of AChE. Organophosphorusesters containing the P-S moiety are less reactive andmore stable to hydrolytic degradation than the corre-sponding P=O ester.

Investigations on the metabolism and mode of actionof organophosphorus insecticides revealed that the tox-icity of a P-S ester is attributed to the corresponding

CH3

(CH30) 2P-0 dfSCH3

fenthion(substituted phenoxy)

S N

11 I

(CH30)2PSCH2-N0

guthion(hetero aromatic )

S 0

(CH30) 2PSCH2CNHCH3

dimethoate

(thioalkyl)

0

(CH30) 2P-0-CH=CHCOC2H5

CH3

mevinphos(alkoxy)

C1

(C2H50)2P-OC1

Cl

chloropyrifos(hetero aromatic)

S

(CH3O)2P-SCHC0OEt

CH200Et

malathion(thioalkyl)

S

(C2H50) 2P-SCH2CH2SCH2CH3

disulfoton

(thioalkyl)

0101(CH30) 2P0CH=CCl2

dichlorvos

(alkoxy)

FIGURE 8. Different organophosphorus insecticides showing varia-tion in the leaving group X.

C2H5Q, S

<D °FN°2

249

T. R. FUKUTO

I + I-P = 0' -P-O

FIGURE 9. Polarization of the P=O linkage in organophosphorusesters.

P=O ester, formed by metabolic oxidation of P=S toP=O (16,17). This metabolic reaction is believed to bemediated by the mixed function oxidases (MFO), a ubiq-uitous enzyme system responsible for oxidation of for-eign compounds in animals (18). A classical example ofthis activation reaction is found in the conversion ofparathion (a poor anticholinesterase) to paraoxon (astrong anticholinesterase) [Eq. (4)].

(C2H50)P-O )t NO I

MFO

parathion(poor anticholinesterase)

0II(C2H50) 2P- NO2

paraoxon(strong anticholinesterase) (4)

Another example of the effect of metabolic activationis evident in the toxicological data in Table 1 (11). Theessentially identical housefly toxicity of these com-pounds in spite of the 10-fold differences in their bi-molecular rate constant for inhibition (ki) is readily ex-plained on the basis of the metabolic activation of thethiomethyl moiety to the sulfoxide [-S(O)CH3], whichin turn is metabolized to the sulfone [-S(0)2CH3].Thioether groups are highly susceptible to metabolicactivation that proceeds through the sulfoxide and even-tually to the sulfone. The metabolic oxidation of thethioether moiety in organophosphorus insecticides wasfirst demonstrated in plants and animals with the de-meton isomers, as indicated in Eq. (5) with the thiolisomer of demeton (17,19).

0 [0](C2H50) PSCH2CH2SCH2CH3

8l 1 ([0](C2H50) PSCH2CH2SCH2CH3

0 0

(C2H50) PSCH2CH2FCH2CH30 (5)

As in the case of the diethyl p-methylthiophenyl analogs

Table 1. Toxicological data (11) for diethyl p-methylthio-, p-methylsulfinyl-, and p-methylsulfonyphenyl phosphates.

(C2H50) 2P- x

Fly-head AchE Housefly LD6,X Ki/M/min ,ug/g

-SCH3 1.4 x 103 2.5-S(O)CH3 1.5 x 104 1.5-S(O)2CH3 1.5 x 105 2.0

.s(CH30) 2P\

OH

I#0

(CH30) 2POH

hydrolaseor

MFO

hydrolase

S [0] 0

(CH30)2P ° NO2 * (CH30) 2P-O N02MFO I

hydrolaseor

GSH transferase

HO"

S

CH30 O 0j N02

hydrolaseor

GSH transferaseIHON #0

CH30 0N. N02

FIGURE 10. Reactions showing the metabolic degradation of methylparaoxon and its activation product methyl paraoxon.

in Table 1, demeton sulfoxide and sulfone were sub-stantially stronger anticholinesterases than demeton,although all three compounds were equally effective in-secticides. The addition of electronegative oxygen at-oms to the thioether sulfur increases the electron with-drawing ability of this moiety, resulting in greaterreactivity of the phosphorus atom.

Metabolic DegradationAnticholinesterase organophosphorus insecticides

are, without exception, tertiary esters, and as tertiaryesters they are susceptible to hydrolytic degradation,resulting in detoxication products. Detoxication of anorganophosphorus insecticide may occur in a number ofdifferent ways. Cleavage of any bond attached to thephosphorus atom will lead to a detoxication product,e.g., by enzymatic or chemical hydrolysis (20). Enzy-matic hydrolysis is mediated by a number of differentesterases, which are generally referred to as hydrolasesor phosphotriester hydrolases (17). Typical reactions forhydrolase catalyzed degradation of an organophospho-rus insecticide are presented in Figure 10 in whichmethyl parathion is used as an example.

Metabolic degradation or detoxication may take placeat a site away from the phosphorus center. The classical

250

I

MECHANISMS OF ORGANOPHOSPHORUS AND CARBAMATE INSECTICIDES

example of this is found in the carboxyleclyzed hydrolysis of malathion to its nontoxacid derivatives, as shown in Eq. (6). Delthe monoacids occurs at a rate faster thaiP=S to P=O activation reaction, and thethion with a rat LD50 of about 3000 mg/kgsafe to mammals. Malathion is toxic to insegenerally low concentrations of carboxylestsects. Because of their susceptibility to hyradation, organophosphorus insecticides artent in the environment and in biological s

carboxylesterase jj

(CH30) 2P-SCHCOC2H5 (CH30) 2P-XICH2gjOC2H5

0

malathion maaOS-

i(CH30) 2P-SCHCOC2H5

CH2JOH0

malathion

t-monoacid (6)

Carbamate InsecticidesMechanism of InhibitionThe inhibition of AChE by a carbamate insecticide

occurs by a mechanism virtually identical to that de-scribed earlier for an organophosphorus ester. The firststep in the inhibition process involves the formation ofthe enzyme-inhibitor complex with subsequent carba-mylation of the seine hydroxyl [Eq. (7)] resulting ininhibition of the enzyme (21).

17 KdEn-OH + X-CNHCH3

8 kc +[ En-OH *X_CNHCH3 ]--- En_OCNHCH3 + X

sterase cata- carbamylation rate constant (from complex to carba-;ic carboxylic mylated enzyme), and Kd is the equilibrium constanttoxication to for the complex dissociating back to reactants. In con-n that of the trast to Eq. (2) for an organophosphorus ester, Eq. (7),refore mala- contains a regeneration step (kr) in which the carba-is relatively mylated (inhibited) enzyme spontaneously regeneratesZcts owing to to active enzyme, methylamine, and carbon dioxide.terases in in- Although the equations depicting the inhibition ofdrolytic deg- AChE by a carbamate and organophosphorus ester arere nonpersis- similar, there are distinct differences in the reaction,ystems. between the enzyme and the two classes of compounds.

First, while appropriate chemical reactivity is essentialfor high anticholinesterase activity for an organophos-phorus ester, a good fit of the carbamate on the enzyme

3CHCOH + active site is essential for high anticholinesterase activ-ity by a carbamate ester. This material will be discussedat greater length in the next section. Second, sponta-

CH2joC2H5 neous regeneration of the carbamylated enzyme to ac-0 tive or original enzyme is relatively fast compared to

spontaneous regeneration of a phosphorylated enzyme.For example, the half-life for recovery of N-methylcar-

lathion bamylated AChE is approximately 30 min, while thatmonoacid for an organophosphorus ester ranges from several

hours to days, depending upon the nature of the groupsattached to the phosphorus atom [R and R' in Eq. (2)].In some cases AChE that is inhibited by certain typesof organophosphorus esters is irreversibly phosphoryl-ated and spontaneous regeneration does not occur.

Reactivity and Steric PropertiesMost carbamate insecticides are derivatives of meth-

ylcarbamic acid and, therefore, are referred to as meth-ylcarbamates. Insecticidal methylcarbamates are estersof substituted phenols and oximes (Fig. 11). Substitutedphenols and oximes, each with pKa values in the regionof 10, give methylcarbamates that are reactive to thedegree that they are able to carbamylate the serinehydroxyl of AChE. Owing to the intrinsic reactivity ofmethylcarbamate esters, an electron-withdrawing sub-stituent is not required in the aryl moiety for high an-ticholinesterase activity. In fact, the introduction of anitro substituent into the phenyl ring of phenyl meth-ylcarbamate results in a compound of such high reac-tivity that it is hydrolytically degraded before it has anopportunity to inhibit the enzyme (22). It should bepointed out that methylcarbamate esters of alcohols(PKa approximately 16) are intrinsically unreactive and,therefore, they are generally poor anticholinesterases.A good fit of a methylcarbamate ester in the enzyme

active site, i.e., formation of the enzyme-inhibitor com-plex prior to carbamylation, is crucial for high anti-

enzyme-inhibitorcomplex / kr

En-OH + CH3NH2 + Co2 (7)

As in the case of inhibition by an organophosphorusester [Eq. (2)], the bimolecular inhibition constant ki[not shown in Eq. (7)] is equal to kC/Kd where kc is the

0

_CN CH3X N X = aryloxy or oxime moiety

FIGURE 11. General structure of insecticidal methylcarbamates.

251

T. R. FUKUTO

I CH3.CH2

CH2+ I

CH3-N-CH

CH3

acetylcholine

H-C-CH3

CH3

ki = 5.64 X 105

sC9z0 NHCH3

H-i-H

CH3ki = 4.6 X 10

IC-0 NHCH3

CH3-N-CH3

CH3

ki = 2.8 X 107

101/C\

9 /0 NHCH3

CH3-C-CH3

CH3

ki = 4 .43 X 105

0II

/C\9 NHCH3

CH3

ki = 4.7 X 103

0 NHCH3

0

H-C-CH3

CH3

ki = 4.99 X 104

01I

ZCs0 NHCH3

H-C-CH3

H

ki = 3.27 X 103

01C,

Q 0 NHCH3

ki = 5.0 X 102

FIGURE 12. Structure and anticholinesterase activities of different substituted phenyl methylcarbamates. ki values/M/min are for bovineerythrocyte AChE. Data from Nishioka et al. (22) and Reiner and Aldridge (23).

Table 2. Dissociation (Kd) and rate constants (k1 and kr) for theinhibition of bovine erythrocyte AChE by substituted phenyl

methylcarbamates.'

Ring substituents Kd,M kc, min' ki, M'lminn3-Trimethylammonium 2.8 x 1072-Isopropoxy 3.97 x lo-6 1.98 4.99 x 1042-Ethoxy 1.62 x 10-4 0.53 3.27 x 1033-tert-Butyl 3.20 x 10-6 1.42 4.43 x 1053-Isopropyl 5.55 x 10-6 3.13 5.64 x 1053-Ethyl 6.45 x 10-4 2.22 3.43 x 1043-Methyl 3.76 x 10-3 1.40 3.72 x 103Unsubstituted 3.02 x 10-3 0.86 2.84 x 102aThe Kd, kc, and ki values for all ring substitutes except 3-trimethyl-

ammonium are from Nishioka et al. (22) and the ki value for 3-trime-thylammonium from Reiner and Aldridge (23).

cholinesterase activity. Evidence in support of this isfound in the values of Kd, kc and ki detemined for awide spectrum of substituted phenyl methylcarbamates

with representative values given in Table 2 (22,23). Ac-cording to the data, the overall bimolecular inhibitionconstant ki for these methylcarbamates is almost totallydependent on Kd, the equilibrium constant for dissocia-tion of the enzyme-inhibitor complex. Kd and kc datawere not available for the 3-trimethylammonium ana-log. Kd may be regarded as an affinity constant, i.e.,the smaller the value of Kd, the tighter the complex.Carbamate esters with small values for Kd were stronganticholinesterases, and those with large Kd were pooranticholinesterases. In contrast to Kd, which was highlyvariable in value, kc, the rate constant for the carba-mylation step [Eq. (7)], showed relatively little varia-tion, indicating similar levels of reactivity for the dif-ferent methylcarbamates.Methylcarbamates of substituted phenols and oximes

with good complementary fit to the enzyme active siteare generally strong inhibitors of AChE (21). Com-

925

MECHANISMS OF ORGANOPHOSPHORUS AND CARBAMATE INSECTICIDES

pounds that structurally resemble acetylcholine, thenatural substrate for AChE, invariably are strong in-hibitors. This is made apparent by the structures of thesubstituted phenyl methylcarbamates in Table 2 andtheir anticholinesterase activities as presented in Fig-ure 12. The structure of acetylcholine is included toshow spatial similarities between it and carbamateswith strong anticholinesterase activities. Noteworthy isthe approximately 10-fold increase in anticholinesteraseactivity as the three-substituent is increased in size fromhydrogen, methyl, ethyl to isopropyl > t-butyl. Evi-dently maximum hydrophobic interaction of the ringsubstituent with the enzyme active site is reached whentwo methyl groups are attached to the central carbonatom. Moreover, replacement of the central carbonatom with a positively charged quaternary nitrogenatom resulted in about a 50-fold increase in anticholin-esterase activity (compare 1 with 4 in Table 2), attrib-utable to electrostatic attraction between the positivenitrogen atom and the negative charge in the anionicsite. It should be added that results similar to thosegiven in Table 2 were also observed for the inhibitionof insect AChE (24).Oxime methylcarbamates, represented by aldicarb

and methomyl (Fig. 13), are structurally similar to ace-tylcholine, good inhibitors of AChE, and also potentinsecticides. Another important methylcarbamate in-secticide, carbofuran, is structurally closely related to2-isopropoxyphenyl methylcarbamate (propoxur, com-pound 2 in Fig. 12) but where the isopropoxy moiety isbridged to the ring by a methylene. In this case thegem-dimethyl group is rigidly fixed at an optimum dis-tance from the carbamate moiety, allowing maximuminteraction with the enzyme active site. Carbofuran isabout 10-fold more potent in inhibiting AChE than pro-poxur and is one of the most effective carbamate insec-ticides.

Activation and DegradationThe principal route of metabolism of carbamate in-

secticides in animals is oxidative in nature and is gen-erally associated with the MFO enzymes. Typical ox-ygenation reactions in which an oxygen atom isintroduced into the molecule include hydroxylation ofaromatic rings, O-dealkylation of ethers, N-methyl hy-droxylation and demethylation, oxidation of aliphaticside chains, and thioether oxidation (25,26). However,the metabolism of aldicarb to its sulfoxide is believedto be an activation reaction, since aldicarb sulfoxide isapproximately 10-fold more potent as an anticholinest-erase than aldicarb. Methylcarbamate insecticides are

CH3

CH3SC - N-O-C-N-S-N-C-O-NI ICHn3 CH3

iH3= C-SCH3

thiodicarb (LARVIN, LEPRICON)

101O-C-N-S-N(n-Bu)2

(CH3) V C3

carbosulfan (MARSHAL, ADVANTAGE)

N-S-N-C-0-Bu-nI ICH3 Cr13

furathiocarbFIGURE 14. Structures of procarbamate insecticides.

also highly susceptible to alkaline hydrolysis but arerelatively stable to neutral or acidic conditions (27).Methylcarbamate insecticides are, on the whole, rel-

atively toxic to mammals. One of the reasons for theirhigh acute toxicity is that they are direct inhibitors ofAChE, and metabolic activation is not required as inthe case of many safe organophosphorus insecticides,e.g. malathion. In order to improve the toxicologicalproperties of methylcarbamate insecticides, a numberof these compounds have been converted to derivativesby replacement of the hydrogen atom on the carbamatenitrogen with an appropriate functional group (28). Thereaction leading to derivatized methylcarbamates is il-lustrated by Eq. (8) using methomyl as the startingcarbamate.

CH3

CH3S-C = N-O-C-N-H + Z-XICH3

(CH3)

fH3 -0N oNHCH3CH3S-CI-CHNN

CH3 °aldicarb

CH3 AS NHCH3I ,- K~ . NCHCE2S-C=N C

0met hornyl

FIGURE 13. Structure of the oxime methylcarbamates aldicarb andmethomyl.

CH3

CH3S-C = N-O-C-N-Z + HX

CH3 (8)Z may be represented by a wide variety of nucleophiles,many having a nucleophilic sulfur atom. The replace-

253

254 T. R. FUKUTO

ment of hydrogen with Z usually results in dramaticreduction in anticholinesterase activity along with sub-stantial improvement in acute mammalian toxicity,which is attributed to the delayed factor provided by Zthat allows alternative routes for detoxication in mam-mals. In insects, the N-Z bond is rapidly broken, lead-ing to in vivo generation of the parent methylcarbamateand intoxication of the insect. These derivatives arereferred to as procarbamate insecticides and are rep-resented by structures given in Figure 14.

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