Mechanism of Action Carbamates and Phosphor A Dos
Mechanism of Action Carbamates and Phosphor A Dos
Mechanism of Action Carbamates and Phosphor A Dos
245-254, 1990
Introduction
The toxicity of insecticidally active organophosphorus and carbamate esters to animals is attributed to their ability to inhibit acetylcholinesterase (AChE, choline hydrolase, EC 3.1.1.7), which is a class of enzymes that catalyzes the hydrolysis of the neurotransmitting agent acetylcholine (ACh). The inhibition of AChE and related esterases, often referred to as serine hydrolases, has been clearly demonstrated to be the result of an actual chemical reaction between the enzyme and the organophosphate or carbamate ester (1-3). The phosphorylated or carbamylated enzyme is no longer capable of effecting the hydrolysis of ACh; this results in a buildup of the neurotransmitter at a nerve synapse or neuromuscular junction. The organophosphorus and carbamate insecticides are represented by a wide variety of chemical structures having different chemical and physical properties. The toxicity of these materials to insects and mammals is determined by a number of factors that may affect the insecticides as they are absorbed, translocated to the target site, and as they inactivate the target, leading to poisoning. This review is concerned with the mode of action of organophosphorus and carbamate esters, with emphasis placed on structural requirements necessary for inhibition of AChE.
reptiles, and insects (4). It is responsible for the rapid hydrolytic degradation of the neurotransmitter ACh into the inactive products choline and acetic acid, as indicated by Eq. (1). Acetylcholine is one of a number of
CH3-N-CH2CH20CCH3 I
+:
CH3
AChE H20
ACh (acetylcholine)
CH3
CH3-N-CH2CH20H + CH3C-OH
acetic acid (1) physiologically important neurotransmitting agents and is involved in the transmission of nerve impulses to effector cells at cholinergic, synaptic, and neuromuscular junctions. The presence of AChE has been demonstrated in a variety of animal tissues and enzymes from a number of different sources, including fish electric organs, mammalian erythrocyte, insect and mammalian brain, and other tissues. These enzymes have been purified and characterized (4-8). AChE is virtually a ubiquitous enzyme in vertebrates and invertebrates, and in mammals, it is localized in certain areas of the central nervous system and in organs and glands that are controlled by the parasympathetic division of the autonomic nervous system. The role of AChE in cholinergic transmission at a synaptic or cholinergic junction is depicted in the eleCH3 choline
Acetylcholinesterase
Before entering into any discussion on the inhibition of AChE by organophosphorus and carbamate esters, it is appropriate first to provide a brief review of the enzyme. AChE is present and has been isolated from a wide range of animals, including mammals, birds, fish,
*Department of Entomology, University of California, Riverside,
CA 92521.
246
T. R. FUKUTO
site in postsynaptic
ACh
membrane
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,
(E S)
()CH.
I-~CH
P
OH
1
H
Vi-
HOHC2
(E )
( E)
mentary scheme given in Figure 1. When a nerve impulse moves down a parasympathetic neuron and reaches a nerve ending, the ACh stored in vesicles in the ending is released into the synaptic or neuromuscular junction. Within 2 to 3 msec the released ACh interacts with the ACh receptor site on the postsynaptic membrane, causing stimulation of the nerve fiber or muscle. AChE serves as a regulating agent of nervous transmission by reducing the concentration of ACh in the junction through AChE catalyzed hydrolysis of ACh into choline (Ch) and acetic acid (A). These products do not stimulate the postsynaptic membrane. In the scheme En denotes the enzyme AChE; AChi En- is the enzyme-substrate complex formed prior to hydrolysis of ACh into choline and acetic acid. When AChE is inactivated, e.g., by an organophosphorus or carbamate ester, the enzyme is no longer able to hydrolyze ACh; the concentration of ACh in the junction remains high, and continuous stimulation of the muscle or nerve fiber occurs, resulting eventually in exhaustion and tetany. Based on a number of studies (2), a plausible mechanism for AChE-catalyzed hydrolysis of ACh is pre-
sented in Figure 2. ACh is drawn to the active site of the enzyme by electrostatic attraction between the positive charge on the ACh nitrogen atom and negative charge in the anionic site (structure E + S) resulting in the enzyme-substrate complex (ES). Acetylation of a serine hydroxyl (OH) in the esteratic site is catalyzed by the basic imidazole moiety B (histidine) and acidic moiety AH (tyrosine hydroxyl), leading to the acetylated enzyme EA. Deacetylation then takes place very fast, resulting in the free enzyme (E) within milliseconds. As presented, ACh hydrolysis by AChE has the elements of an acid-base catalyzed reaction, including both the acetylation and deacetylation reaction. The negative charge at the anionic site is attributed to the carboxylate anion of aspartic or glutamic acid. The reaction steps given in Figure 2 provide an elementary presentation of the AChE active site and a reasonable mechanism for the hydrolysis of ACh. The enzyme is in reality a highly complex protein, having in addition to the esteratic and anionic sites, a number of peripheral sites and hydrophobic areas (5). While the preceding discussion has focused on AChE, it should be pointed out that there is at least one other type of cholinesterase enzyme beside AChE, namely pseudocholinesterase. AChE has the highest specificity for ACh of any other choline ester and pseudocholinesterase has the highest specificity for butyrylcholine. The physiological role of pseudocholinesterase in animals is not as well defined as that of AChE (4). However, both enzymes are inhibited by organophosphorus and carbamate esters.
Organophosphorus Insecticides
Mechanism of Inhibition
The inhibition of AChE by an organophosphorus ester (Fig. 3) takes place via a chemical reaction in which the serine hydroxyl moiety in the enzyme active site is phosphorylated in a manner analogous to the acetylation of AChE (Fig. 2, EA). In contrast to the acetylated enzyme, which rapidly breaks down to give acetic acid and the regenerated enzyme, the phosphorylated enzyme (Fig. 4) is highly stable, and in some cases, depending on the groups attached to the phosphorus atom (R and R'), it is irreversibly inhibited (9). The serine hydroxyl group, blocked by a phosphoryl moiety, is no longer able to participate in the hydrolysis of ACh. The inhi-
247
En\
EtC-. (-C
8
o
EtO 4 EtO
NC
0
EtO
En-OP(OEt)2
pNO2
bition reaction takes place in a two-step process, as indicated by Eq. (2). In this equation
Kd
kp
(En-OH-R-P-X]:
'
En-OH + R-P-X
Re
II
En-O-P-R + X
= (complex)
kj
Ioo
RI
(2)
En-OH represents AChE in which the serine hydroxyl moiety (-OH) is emphasized, R and R' are a variety of different groups (alkoxy, alkyl, amino, thioalkyl, etc.), X is the leaving group, Kd is the dissociation constant between the enzyme-inhibitor complex and reactants, kp is the phosphorylation constant, and ki is the bimolecular rate constant for inhibition and is equal to k/IKd (10). Since Kd provides a measure of the dissociation of the enzyme-inhibitor complex, it is regarded as an estimate for binding and is dependent on the structural and steric properties of the molecule. In contrast, the phosphorylation constant kp is regarded as an estimate of the reactivity of the organophosphorus ester. The bimolecular rate constant ki is dependent on the values of Kd and kp and is generally regarded as the most useful parameter for the estimation of the inhibitory potency of an organophosphorus (and carbamate) anticholinesterase. According to Eq. (2), the moiety X is displaced from the phosphorus atom by the serine hydroxyl of the enzyme and is, therefore, referred to as the leaving group.
'I
H A
e~~
FIGURE 4. Phosphorylated, and irreversibly inhibited, AChE (see text and Figure 2 for details).
is apparent that the single most important property required in an organophosphate for anticholinesterase activity is chemical reactivity, i.e., the phosphorus ester must be reactive to the extent that the serine hydroxyl moiety in the enzyme is phosphorylated. Structure-activity studies have revealed a direct relationship between anticholinesterase activity and reactivity of the phosphorus atom. The inhibition of erythrocyte AChE by diethyl p-nitrophenyl phosphate (paraoxon) and some of its substituted phenyl analogs was found to proceed with pseudo first-order kinetics (1)t The bimolecular rate constant for inhibition paralleled the rates of alkaline hydrolysis of these phosphates. It was subsequently demonstrated with a larger series of diethyl substituted phenyl phosphates that the inhibition of fly-head AChE by paraoxon analogs was related to the effect of the substituent on the liability of the P-Ophenyl bond as estimated by Hammett's sigma constants, shifts in P-O-phenyl infrared stretching frequencies, and hydrolysis rates (11). A plot of the log of anticholinesterase activity against Hammett's sigma constant (also P-O-phenyl stretching frequency) resulted in a good linear relationship with activity directly related to the electron-withdrawing properties of the substituents. The effect of an electron-withdrawing substituent on the reactivity of a diethyl substituted phenyl phosphate is demonstrated in Figure 4 with paraoxon. Because of the strong electron-withdrawing property of the nitro group, electrons are attracted away from the phosphorus atom, creating an electron-deficient center that facilitates a nucleophilic attack by the enzyme serine hydroxyl moiety on the phosphorus atom with simultaneous expulsion of the nitrophenoxide leaving group. Diethyl-substituted phenyl phosphates with weak electron-withdrawing or electron-donating substituents were weak inhibitors or were devoid of activity. While chemical reactivity of the phosphorus atom is of prime importance for anticholinesterase activity, steric properties sometimes have a strong effect on the anticholinesterase activity of an organophosphorus ester. This became apparent from examining a series of ethyl p-nitrophenyl alkylphosphonates for anticholinesterase activity and alkaline hydrolysis rates where the alkyl group was varied (12). The rate of hydrolysis of the phosphonate ester to ethyl alkylphosphonic acid and p-nitrophenol, in general, decreased with an in-
248
T. R. FUKUTO
crease in alkyl chain length, and it decreased strongly with branching in the 1 and 2 positions of the alkyl moiety. Determination of housefly-head anticholinesterase activities of these compounds revealed the relationship shown in Figure 5 between bimolecular rate constants for inhibition of housefly-head AChE (ki) and pseudo first-order hydrolysis constants (khyd, pH 8.3). Although the plot between log ki and log khyd showed a general trend toward linearity, close examination of the data revealed that many of the points followed a sigmoidal relationship, particularly those compounds with a straight chain alkyl. For example, as the chain length increased from three to six carbon atoms, the rate of inhibition of AChE dropped rapidly even though hydrolysis rates remained relatively constant. The plot reveals the influence of steric effects in the inhibition of AChE by these compounds, i.e., the inhibition rates are reduced as the alkyl moiety attached to the phosphorus atom becomes bulkier. The importance of steric effects in AChE inhibition was subsequently pointed out by Hansch and Deutsch (13) who showed that anticholinesterase activities for the compounds in Figure 5 were related to Taft's steric substituent constant, E., according to Eq. (3) where n is the number of compounds, s is the standard deviation, and r is the correlation coefficient. log ki = 3.738ES + 7.539 n = 13, s = 0.749, r = 0.901 (3) While this study provided fundamental information on the effect of the P-alkyl moiety on the reactivity of
4
-
alkylphosphonate esters, it also revealed that the Palkyl moiety should be kept small (methyl or ethyl) in designing potential insecticides from phosphonate esters.
C2H 5
3
3 -
n - C3H74
i- C H1 5 1
#*n--C4H.
i
CtH13
,0
*phenyl
2-
4,4-dimethylpentyl
i - CH
4 9
IC
0
1
-
-i
cyi -Ch3H7
cyclohexyl
RO0
R'
0(S)
X
En-O-P -OR
LOG (KhYd x
105 )
(A)
(B)
FIGURE 5. Relationship between log acetylcholinesterase inhibition constant ki and log hydrolysis constant khyd for ethyl p-nitrophenyl alkylphosphonates. Data from Fukuto and Metcalf (12).
FIGURE 6. General structure of organophosphorus insecticides (A) and phosphorylated AChE (B). R is methyl or ethyl; R' is either methoxy, ethoxy, ethyl, phenyl, amino, substituted amino or aklylthio: X is an appropriate leaving group and En is AChE.
249
CH3Q,
CH30
0S
ON02
C2H50
C2HAQ + p O. des
, C C3H7-j
C2H5Q1,
C2H5
^ S
-0%%Ib\
C2H5Q,
<D
S
FN2
H2
H2N
(R'
(R'
d0
C2H50,
nn -C3H7S
i-C3H7-N
H
0
Br
C1
nemacur
(R'
subst'd amino)
X is a moiety that is metabolically activated to give a labile P-X bond. In most cases X is a substituted phenoxy or aromatic group containing hetero atoms, substituted thioalkyl, or substituted alkoxy. Additional examples of prominent organophosphorus insecticides with variation in the leaving group, X, are given in Figure 8. The large number of organophorus insecticides that have attained commercial importance is attributable primarily to the large number of leaving groups possible. In no other class of insecticides has it been possible to have broader variation in structure and in spectrum of insecticidal activity.
CH3
C1
(C2H50)2P-OC1
Cl
(CH30) 2P-0
dfSCH3
(CH30)2PSCH2-N
CH200Et
11
(CH3O)2P-SCHC0OEt
0
Metabolic Activation
More than half of the compounds in Figures 7 and 8 P-S moiety. Phosphorothionate esters (P=S) are generally poor anticholinesterases, yet all of the compounds given in Figures 7 and 8 are potent insecticides. The poor anticholinesterase activity of P-S esters is explained on the basis of their relatively low reactivity, attributed to the smaller extent to which the P-S bond is polarized compared to the P=O, owing to the lower electronegativity of sulfur compared to oxygen. Polarization ofthe P=O linkage (Fig. 9) results in a more electropositive phosphorus atom, which facilitates attack on phosphorus by nucleophilic agents, e.g., the serine hydroxyl of AChE. Organophosphorus esters containing the P-S moiety are less reactive and more stable to hydrolytic degradation than the corresponding P=O ester. Investigations on the metabolism and mode of action of organophosphorus insecticides revealed that the toxicity of a P-S ester is attributed to the corresponding
contain the
malathion (thioalkyl)
S
(CH30) 2PSCH2CNHCH3
(C2H50) 2P-SCH2CH2SCH2CH3
dimethoate (thioalkyl)
disulfoton
(thioalkyl)
101
(CH30) 2P-0-CH=CHCOC2H5
(CH30) 2P0CH=CCl2
250
T. R. FUKUTO
I
-P = 0'
+ I
-P- O
Table 1. Toxicological data (11) for diethyl p-methylthio-, pmethylsulfinyl-, and p-methylsulfonyphenyl phosphates.
x
Ki/M/min 1.4 x 103
1.5 x 104 1.5 x 105 Housefly LD6, ,ug/g 2.5 1.5 2.0
P=O ester, formed by metabolic oxidation of P=S to P=O (16,17). This metabolic reaction is believed to be mediated by the mixed function oxidases (MFO), a ubiquitous enzyme system responsible for oxidation of foreign compounds in animals (18). A classical example of this activation reaction is found in the conversion of parathion (a poor anticholinesterase) to paraoxon (a strong anticholinesterase) [Eq. (4)].
(CH30) 2P\
.s
OH hydrolase
or MFO
#0
(CH30) 2P
OH
(C2H50) P-O )t NO
MFO
I
HO "
[0]
NO2
MFO
*
(CH30) 2P
(CH30) 2P-O
I
hydrolase
or
GSH transferase
HON #0
I I
hydrolase
N02
hydrolase
or GSH transferase
paraoxon (4) (strong anticholinesterase) Another example of the effect of metabolic activation is evident in the toxicological data in Table 1 (11). The essentially identical housefly toxicity of these compounds in spite of the 10-fold differences in their bimolecular rate constant for inhibition (ki) is readily explained on the basis of the metabolic activation of the thiomethyl moiety to the sulfoxide [-S(O)CH3], which in turn is metabolized to the sulfone [-S(0)2CH3]. Thioether groups are highly susceptible to metabolic activation that proceeds through the sulfoxide and eventually to the sulfone. The metabolic oxidation of the thioether moiety in organophosphorus insecticides was first demonstrated in plants and animals with the demeton isomers, as indicated in Eq. (5) with the thiol
CH30
O 0j
N02
CH30
0N.
N02
FIGURE 10. Reactions showing the metabolic degradation of methyl paraoxon and its activation product methyl paraoxon.
in Table 1, demeton sulfoxide and sulfone were substantially stronger anticholinesterases than demeton, although all three compounds were equally effective insecticides. The addition of electronegative oxygen atoms to the thioether sulfur increases the electron withdrawing ability of this moiety, resulting in greater reactivity of the phosphorus atom.
Metabolic Degradation
Anticholinesterase organophosphorus insecticides are, without exception, tertiary esters, and as tertiary esters they are susceptible to hydrolytic degradation, resulting in detoxication products. Detoxication of an organophosphorus insecticide may occur in a number of different ways. Cleavage of any bond attached to the phosphorus atom will lead to a detoxication product, e.g., by enzymatic or chemical hydrolysis (20). Enzymatic hydrolysis is mediated by a number of different esterases, which are generally referred to as hydrolases or phosphotriester hydrolases (17). Typical reactions for hydrolase catalyzed degradation of an organophosphorus insecticide are presented in Figure 10 in which methyl parathion is used as an example. Metabolic degradation or detoxication may take place at a site away from the phosphorus center. The classical
(C2H50) PSCH2CH2FCH2CH3
0
(5)
251
example of this is found in the carboxylecsterase catalyzed hydrolysis of malathion to its nontox;ic carboxylic acid derivatives, as shown in Eq. (6). Deltoxication to the monoacids occurs at a rate faster thain that of the P=S to P=O activation reaction, and the,refore malathion with a rat LD50 of about 3000 mg/kg is relatively safe to mammals. Malathion is toxic to inseZcts owing to generally low concentrations of carboxylestterases in insects. Because of their susceptibility to hy drolytic degradation, organophosphorus insecticides arre nonpersistent in the environment and in biological s,ystems.
carboxyl jj esterase (CH30) 2P-SCHCOC2H5 (CH30) 2P-X3CHCOH
CH2gjOC2H5
0
CH2joC2H5
0
malathion
ma lathion
aOS- monoacid
(CH30) 2P-SCHCOC2H5
carbamylation rate constant (from complex to carbamylated enzyme), and Kd is the equilibrium constant for the complex dissociating back to reactants. In contrast to Eq. (2) for an organophosphorus ester, Eq. (7) contains a regeneration step (kr) in which the carbamylated (inhibited) enzyme spontaneously regenerates to active enzyme, methylamine, and carbon dioxide. Although the equations depicting the inhibition of AChE by a carbamate and organophosphorus ester are similar, there are distinct differences in the reaction between the enzyme and the two classes of compounds. First, while appropriate chemical reactivity is essential for high anticholinesterase activity for an organophosphorus ester, a good fit of the carbamate on the enzyme active site is essential for high anticholinesterase activity by a carbamate ester. This material will be discussed at greater length in the next section. Second, spontaneous regeneration of the carbamylated enzyme to active or original enzyme is relatively fast compared to spontaneous regeneration of a phosphorylated enzyme. For example, the half-life for recovery of N-methylcarbamylated AChE is approximately 30 min, while that for an organophosphorus ester ranges from several hours to days, depending upon the nature of the groups attached to the phosphorus atom [R and R' in Eq. (2)]. In some cases AChE that is inhibited by certain types of organophosphorus esters is irreversibly phosphorylated and spontaneous regeneration does not occur.
CH2JOH
0
malathion
t-monoacid
Carbamate Insecticides
Mechanism of Inhibition
The inhibition of AChE by a carbamate insecticide occurs by a mechanism virtually identical to that described earlier for an organophosphorus ester. The first step in the inhibition process involves the formation of the enzyme-inhibitor complex with subsequent carbamylation of the seine hydroxyl [Eq. (7)] resulting in inhibition of the enzyme (21).
En-OH + X-CNHCH3 [ En-OH *X_CNHCH3 ]---
17
Kd
kc En_OCNHCH3
+ X
enzyme-inhibitor
complex
kr
(7)
_CN
CH3
N
X = aryloxy or oxime moiety
As in the case of inhibition by an organophosphorus ester [Eq. (2)], the bimolecular inhibition constant ki [not shown in Eq. (7)] is equal to kC/Kd where kc is the
925
T. R. FUKUTO
CH3
0
ICNHCH3
.CH2
NHCH3
CH2 0
CH3-N-CH CH3
CH3-N-CH3
CH3
H-C-CH3
CH3
acetylcholine
ki
2.8 X
107
ki
4.99 X 104
101 /C\
9 /0
ZCs
0
1I
NHCH3
NHCH3
H-C-CH3
CH3-C-CH3
H-C-CH3
H
CH3
CH3
ki
5.64 X
105
ki
4 .43 X
105
ki
3.27 X
103
sC
9z0
NHCH3
C\NHCH3
0 II
01
C,
Q 0
NHCH3
H-i-H
CH3
CH3 ki = 4.6 X 10
ki
4.7 X
103
ki
5.0 X 102
FIGURE 12. Structure and anticholinesterase activities of different substituted phenyl methylcarbamates. ki values/M/min are for bovine erythrocyte AChE. Data from Nishioka et al. (22) and Reiner and Aldridge (23). Table 2. Dissociation (Kd) and rate constants (k1 and kr) for the inhibition of bovine erythrocyte AChE by substituted phenyl methylcarbamates.'
Kd,M Ring substituents kc, min' ki, M'lminn 2.8 x 107 3-Trimethylammonium 1.98 4.99 x 104 3.97 x lo-6 2-Isopropoxy 3.27 x 103 0.53 1.62 x 10-4 2-Ethoxy 1.42 4.43 x 105 3.20 x 10-6 3-tert-Butyl 3.13 5.64 x 105 5.55 x 10-6 3-Isopropyl 3.43 x 104 2.22 6.45 x 10-4 3-Ethyl 3.72 x 103 1.40 3.76 x 10-3 3-Methyl 2.84 x 102 0.86 3.02 x 10-3 Unsubstituted aThe Kd, kc, and ki values for all ring substitutes except 3-trimethylammonium are from Nishioka et al. (22) and the ki value for 3-trimethylammonium from Reiner and Aldridge (23).
cholinesterase activity. Evidence in support of this is found in the values of Kd, kc and ki detemined for a wide spectrum of substituted phenyl methylcarbamates
with representative values given in Table 2 (22,23). According to the data, the overall bimolecular inhibition constant ki for these methylcarbamates is almost totally dependent on Kd, the equilibrium constant for dissociation of the enzyme-inhibitor complex. Kd and kc data were not available for the 3-trimethylammonium analog. 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 strong anticholinesterases, and those with large Kd were poor anticholinesterases. In contrast to Kd, which was highly variable in value, kc, the rate constant for the carbamylation step [Eq. (7)], showed relatively little variation, indicating similar levels of reactivity for the different methylcarbamates. Methylcarbamates of substituted phenols and oximes with good complementary fit to the enzyme active site are generally strong inhibitors of AChE (21). Com-
253
pounds that structurally resemble acetylcholine, the natural substrate for AChE, invariably are strong inhibitors. This is made apparent by the structures of the substituted phenyl methylcarbamates in Table 2 and their anticholinesterase activities as presented in Figure 12. The structure of acetylcholine is included to show spatial similarities between it and carbamates with strong anticholinesterase activities. Noteworthy is the approximately 10-fold increase in anticholinesterase activity as the three-substituent is increased in size from hydrogen, methyl, ethyl to isopropyl > t-butyl. Evidently maximum hydrophobic interaction of the ring substituent with the enzyme active site is reached when two methyl groups are attached to the central carbon atom. Moreover, replacement of the central carbon atom with a positively charged quaternary nitrogen atom resulted in about a 50-fold increase in anticholinesterase activity (compare 1 with 4 in Table 2), attributable to electrostatic attraction between the positive nitrogen atom and the negative charge in the anionic site. It should be added that results similar to those given in Table 2 were also observed for the inhibition of insect AChE (24). Oxime methylcarbamates, represented by aldicarb and methomyl (Fig. 13), are structurally similar to acetylcholine, good inhibitors of AChE, and also potent insecticides. Another important methylcarbamate insecticide, carbofuran, is structurally closely related to 2-isopropoxyphenyl methylcarbamate (propoxur, compound 2 in Fig. 12) but where the isopropoxy moiety is bridged to the ring by a methylene. In this case the gem-dimethyl group is rigidly fixed at an optimum distance from the carbamate moiety, allowing maximum interaction with the enzyme active site. Carbofuran is about 10-fold more potent in inhibiting AChE than propoxur and is one of the most effective carbamate insecticides.
CHn3 CH3
thiodicarb (LARVIN, LEPRICON)
= C-SCH3
iH3
101
O-C-N-S-N(n-Bu)2
(CH3)
C3
N-S-N-C-0-Bu-n
(CH3)
CH3 Cr13
furathiocarb
FIGURE 14. Structures of procarbamate insecticides.
also highly susceptible to alkaline hydrolysis but are relatively stable to neutral or acidic conditions (27). Methylcarbamate insecticides are, on the whole, relatively toxic to mammals. One of the reasons for their high acute toxicity is that they are direct inhibitors of AChE, and metabolic activation is not required as in the case of many safe organophosphorus insecticides, e.g. malathion. In order to improve the toxicological properties of methylcarbamate insecticides, a number of these compounds have been converted to derivatives by replacement of the hydrogen atom on the carbamate nitrogen with an appropriate functional group (28). The reaction leading to derivatized methylcarbamates is illustrated by Eq. (8) using methomyl as the starting carbamate.
Z-X
fH3
0N
CH3
oNHCH3
CH3 ,- AS I K~
.
NHCH3 NCH
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-
254
T. R. FUKUTO 12. Fukuto, T. R., and Metcalf, R. L. The effect of structure on the reactivity of alkylphosphonate esters. J. Amer. Chem. Soc. 81: 372-377 (1959). 13. Hansch, C., and Deutsch, E. W. The use of substituent constants in the study of structure-activity relationships in cholinesterase inhibitors. Biochem. Biophys. Acta 126: 117-128 (1966). 14. Worthing, C. R., and Walker, S. B. The Pesticide Manual. British Crop Protection Council, Croyden, 1987. 15. Kao, T. S., and Fukuto, T. R. Metabolism of O,S-dimethyl-propionyl- and hexanoylphosphoramidothioate in the house fly and white mouse. Pestic. Biochem. Physiol. 7: 83-95 (1977). 16. Gage, J. C. A cholinesterase inhibitor derived from 0, 0-diethyl O-p-nitrophenyl thiophosphate in vivo. Biochem. J. 4: 426-430
17. Dauterman, W. C. Biological and nonbiological modifications of organophosphorus compound. Bull. WHO 44: 133-150 (1971). 18. Jacoby, N. B. Enzymatic Basis of Detoxication, Vol. 1. Academic Press, New York, 1980. 19. Fukuto, T. R., Wolf, J. P., III, Metcalf, R. L., and March, R. B. Identification of the sulfoxide and sulfone plant metabolites of the thiol isomer of systox. J. Econ. Entomol. 49: 147-151 (1956). 20. Eto, M. Organophosphorus Pesticides. CRC, Cleveland, OH, 1974. 21. Metcalf, R. L. Structure-activity relationships for insecticidal carbamates. Bull. WHO 44: 43-78 (1971). 22. Nishioka, T., Fujita, T., Kamoshita, K., and Nakajima, M. Mechanism of inhibition reaction of acetylcholinesterase by phenvy Nmethylcarbamates. Pestic. Biochem. Physiol. 7: 107-121 (1977). 23. Reiner, E., and Aldridge, A. N. Effect of pH on inhibition and spontaneous reactivation of acetylcholinesterase treated with esters of phosphorus acids and of carbamic acids. Biochem. J. 107: 171-179 (1967). 24. Kamoshita, K., Ohno, I., Fujita, T., Nishioka, T., and Nakajima, M. Quantitative structure-activity relationships of phenyl Nmethylcarbamates against house fly and its acetylcholinesterase. Pestic. Biochem. Physiol. 11: 83-103 (1979). 25. Knack, J. B. Biological and nonbiological modifications of carbamates. Bull. World Health Organization 44: 121-131 (1971). 26. Fukuto, T. R. Metabolism of carbamate insecticides. Drug Metab. Rev. 1: 117-151 (1972). 27. Aly, 0. M., and El-Dib, M. A. Persistence of some carbamate insecticides in the aquatic environment. In: Fate of Organic Pesticides in the Environment (S. D. Faust and J. V. Hunter, Eds.), Advances in Chemistry Series III, American Chemical Society, Washington, DC, 1972, pp. 210-243. 28. Fukuto, T. R. Propesticides. Pesticide Synthesis through Rational Approaches (P. S. Magee, G. K. Kohn, and J. J. Menn. Eds.), ACS Symposium Series 255, American Chemical Society, Washington, DC. 1984, pp. 87-101.
ment of hydrogen with Z usually results in dramatic reduction in anticholinesterase activity along with substantial improvement in acute mammalian toxicity, which is attributed to the delayed factor provided by Z that allows alternative routes for detoxication in mammals. In insects, the N-Z bond is rapidly broken, leading to in vivo generation of the parent methylcarbamate and intoxication of the insect. These derivatives are referred to as procarbamate insecticides and are represented by structures given in Figure 14.
REFERENCES
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