compared with the other bicyclic systems studied. The rigid planar nature of this system, combined Lvith the presence of two oxygen atoms: is considered of major importance in deterrnining the biological activity. Sulfur can replace oxygen in the system with only a slight decrease in activity . Synergistic activity of the esters of piperonyl alcohol is considerably modified by the nature and relative position of the side chain attached to the alphacarbon. This apparently results from such factors as lipoid solubility, steric hindrance, and binding on the active surface of the target enzyme(s). Minimal requirernents for synergistic activity are the presence of the methylenedioxyphenyl nucleus in combination with a simple group such as methyl substituted in the phenyl ring. In such simple compounds,, nuclear methoxy and nitro groups gxatly enhance synergistic activity. The best synergi:,t studied, a-methylpiperonyl benzoate (XIX), had synergistic ratios of 128, 56, and 3.9 with Sevin, 3,4-dimetho:ryphenyl ,\-methylcarbamate, and Z’ectran. This compared very favorably Lvith the synergistic ratios a t 5 to 1 for piperonyl butoxide of 75, 37.5, and 2.6: respectively.
Literature Cited (1) Adams. R.. Levine. I.. J . Am. Chem. Soc. 45, 2373 (19:!3). (2) Adams. R.. Montgomery, E.? Ibid., 46, 1518 (1924). (3) Adolphi. H., Blum, D., Duffer. P., Hertel. 0..Pasedach. H . , Stunmeyer, H. (to Badische Anilin und Soda Fabrik): German Patent 1,058,306 W a y 27. 1959); C. ,4. 55, 3916f ( 1961). (4) Arnold: R. T.. J . .Im. Chem. Soc. 61, 1405 (1939). (5) Arnold. R . T.. McCool: J.? Ibid., 64, 1315 (1942). (6) Barthel. \V. F.. .4lexander, B. H.,
U. S. Dept. Agr. ARS-33-42 (September 1957). (7) Barthel. W. F., Gertler! S. I.. Ibid., ARS-33-77 (July 1956). (8) Beroza, M., Barthel, W.F., J. AGR. FOODCHEM.5,855 (1957). (9) Birch, S. F., Dean, R . A , , Whitehead, E. V., J . Inst. Petrol. 40, 76 (1954). (10) Boeseken, J . ? Greup, J., Rec. Trav. Chim. 58, 528 (1939). (11) Boeseken, J., Sloof. G . , Proc. dcad. Sci.Amsterdam 35, 1250 (1932); C. A. 27, 3457 (1933). (12) Campbell, K. N., Hopper: P. F., Campbell. B. K.? J . Org. C h f m . 16, 1736 (1951). (13) Casida. J. E.:Ann. Reu. Eniomol. 8 , 39 (1963). (14) Casida. J. E.:Report of Conference of International Atomic Energy Agency. Vienna. 1963. (15) Chatelus. G., Cagniant, P . . Compt. Rend. 224, 1777 (1947). (16) Clark. P. H . . Cole, M. M.. U. S. Dept. Agr. ARS-33-62 (October 1960). (17) Dallacker, F.. Binsack. R.. -2fonatsh. 92, 492 (1961); C. A . 55, 27180d (1961). (18) Davidson. D . , LYeiss. 51., Org. Slnthesis, Coll. Vol. 2, 590 (1947). (19) Dorough. H. \V.> Casida. J. E.. J. AGR.FOODCHEM.1 2 , 2 9 4 (1964). (20) Dorough. H. LV.. Leeling. S . C., Casida. J . E.? Science 140, 170 (1963). (21) Drabek, J.. Chem. Zuesti lQ, 357 (1956); C. A . 50, 16023 (1956). (22) Fukuto. T. R . , Metcalf. R . L., \Yinton, hf. Y.:Roberts. P. .4.> J. Econ. Entomol. 5 5 , 341 (1962). (23) , . Gatterman. L.. i l n n . Chem. Justus Liebigs 347, 347 (1906). (24’1 Georghiou. G . P.. Metcalf. R . L.. J . Econ. Entomol. 55. 125 11962). (25) Georghiou: G. P . . Metcalf. R . L.. March. R . B.. Ibid.,54, 132 (1961). (26) Haller. H. L.. La Forge. F. B., Sullivan: \V. N.: J . Org. Chem. 7, 185 (1942). (27) Haller: H. L., McGovran. E. R.! Goodhue, L. D.. Sullivan, \V. N.! Ibid.,7, 183 (1942). (28) Hewlett. P. S..Adaan. Pest Control Res. 3, 27 (1960). (29) Hickinbottom, I V . J.. “Reactions
of Organic Compounds,” 2nd ed., Longmans, Green, London, New York, and Toronto, 1948. (30) Howard, J. W., J . A m . Chem. SOC. 57, 2317 (1935). (31) Kerr, R. I,\:.. Bull. Commonwealth Sci. Ind. Res. Org., Australia, No. 261 (1951). (32) Metcalf, R. L., “Organic Insecticides,” Interscience, New York-London, 1955. (33) Metcalf, R. L.. Fukuto, T. R., iVinton, M. Y.? J . Econ. Entomol. 53, 828 (1960). (34) Ibid., 55, 345 (1962). (35) Metcalf. R . L.. March. R. B., Ibid., 46, 288 (1953). (36) hliller, E., Read, R. R.. J . .Im. Chem. SOC.55, 1224 (1933). (37) Moore. B. P.. Heivlett. P. S., J . Sci. Food 4 g r . 9, 666 (1958). (38) hloorefield. H. H.. Coritribs. B o p Thompson Inst. 19, 501 i1958). (39) Moorefield. H . H.. .\fisc. Publ. Entomol. Soc. .4m.2, 145 (1960). (40) hfoorefield. H . H.. LVeiden. M. H . J.. Contribs. Bolce Thombson Insl. 22, 425 11964). (41) Mosettig. E.. hlozingo. R.. Org. Reactions 4. 362 (1948). (42) Nystrom. R.‘ F.. Brolvn, \I7. G., J . A m . Chem. Soc. 69, 119’ (1947). (43) Pasedach. H.? Seefelder. 51. (to Badische Anilin und Soda Fabrik A. G . ) . German Patent 1,042,602 (Tu’ov. 6. 1958). (44) Perkin. \V. H.. Trikojus. \-. 5,1.> J . Chem. Soc. 128, 2925 (1926). (45) Philleo, \V. I\:.. Schonbrod. R. .%.. Terriere. L. J. AGR. FOODCHEM. 13, 113 (1965). (46) Prill. E. .2.. Smith. !V. R.. Contrihs. Bojce ~ ’ h o m p s o Inst. n 18, 187 (1955). ( $ 7 ) Stoermer. R.. Kahlert. B.. Ber. Deut. Chem. Ges. 34 ( 2 ) . 1806 (1901). (48) Wilkinson. C . F.. hletcalf. R. L., Fukuto, T. R.. unpublished data. Receiced f o r reciezc March 19: 1965. Accepted September 27> 7965. From a dissertation submitted by the senior author in January 1965 to the Graduate Diz,ision, Unizersr‘ly of California, Rirerside. in partial fuiuifillment of the requirements for the degree doctor of philosophy. Supported in part by a grant f r o m the U. S. Public Health Sercice, .Vo. CC-00038-08. Paper AYo. 16.78, Citrus ResParch Center and Agricultural Experiment Station.
c..
S T R U C T U R E AND A C T I V I T Y
Effects of Deuteration, Fluorination, and Other Structural Modifications of the Carbamyl Moiety upon t h e Anticholinesterase and Insecticidal Activities of Phenyl N-Met hylcarbamat es
T
PROCESSES of intoxication and detoxication of the aryl .\--methylcarbamate insecticides are under in-
HE
Present address. University of .ksiut, Egypt, U.A.R. 9 Present address. ~ ~ cniversity, ~ New York, N. Y.
MOHAMED A. H. FAHMY,‘ R. 1. METCALF, T. R. FUKUTO, and D. J. HENNESSY Department of Entomology, University of California, Riverside, Calif.
tensive study in a number of laboratories. There is: hoLvever. no general agreement that the ability of these compounds to inhibit cholinesterase (ChE) is either competitive or the of noncompetitive carbamylation of the esteratic site d h ~ ~ of the enzyme ( 7 7 ) or that detoxication VOL.
occurs through hydroxylation of the aromatic ring or of the .\--methyl group, or by hydrolysis (5.7 7 ) . It appeared that additional evidence regarding both processes might be obtained b!- investigating the comparative anticholinesterase activitl-, insecticidal action. and
1 4, N O . 1, J A N . - F E B .
1966
79
The anticholinesterase and insecticidal activity of aryl NHCD3, NHCF3, NH2, NHSi[CHJ3,
CH2 and N < ~ carbamates are described. CH2
These alterations of the normal NHCHS carba-
mate insecticides resulted in both decreased anticholinesterase and insecticidal activity to the housefly and mosquito larva, except for the NHCD3 carbamate which showed no deuterium isotope effect in either cholinesterase inhibition or toxicity. The NHCF3 and NH2carbamates showed substantial synergism when evaluated in admixture with piperonyl butoxide. Since these compounds cannot be detoxified by NHCHzOH formation, this synergism suggests that the primary attack is hydroxylation of the aromatic ring followed b y hydrolysis of the carbamate ester.
synergism by piperonyl butoxide of carbamates with modified ,$--substituents such as 3-isopropylphenyl NHCD3 carbamate and 3-isopropylphenyl NHCF3 carbamate. These are isosteres of the highly insecticidal 3-isopropylphenyl NHCH; carbamate but differ in the stability of the C-X bonds of the .\-methyl group. Thus, if conversion of "CHI to NHCHzOH is the primary pathway of detoxication by microsomal oxidation, which can be inhibited by piperonyl butoxide. then both the toxicity and degree of synergism of the three compounds should be measurably different. Other alterations in the groups attached to the carbamate ,\-atom are known to affect activity greatly. For example. the .\-..\-dimethylcarbamates are only about 0.01 to 0.1 as active as the corresponding ,\ - methylcarbamates either as anticholinesterases and as insecticides ( 7 3 ) and .\-ethyl, .\-benzyl, and A7-phen)-lcarbamates are virtually inactive ( 9 ) . To obtain additional information on the role of the ,I--methyl group in biological activity, a series of substituted phenyl carbamates and phenyl .\7-trimethylsilylcarbamates was prepared and evaluated along with rnisopropJ-lphen!-l aziridinylcarbamate which has a cyclic ethyleneimine ring. Additional data are included on a corresponding group of L\-acylcarbamates.
Table 1.
I1
Materials and Methods. Deuterated 3-isopropylphenyl .\-d,d.d.-methylcarbamate was prepared from nitromethane interchanged with heavy water and reduced to methylamine hydrochloride which was added to m-isopropylphenylchloroformate prepared from the sodium salt of the phenol and phosgene. The deuterocarbamate was recrystallized several times from petroleum ether and had a m.p. of 69-72' C. as compared
ii
\-TRlFLJCRCMET~Y-C4RBe"nA-E
'iiYY.
350C
3000
25C0
LMC
l9C3
l80C
1700 *bd-
I600 N and isopropyl proton a t 3.3 to 3.5 p.p.m. The spectra indicated a purity of >95% for the deutero-compound.
€xperirnental
3-Isopropylphenyl ~Y-inethylcarbainate 3-Isopropylphenvl
M.P.,
C.
Analysis Found
Theory
72-3
( 74)
...
69-72
...
Musca domesfica Pg./GrOm Fly Degree of A B ( I : 5 Synergism Aftinify falone) p.b./" A/B
-LD60,
160
Fly ChE
LCjo Culex pipiens 5-fasciofus I . , P.P.M.
4 . 5 X 10-7
450
90
9.5
9.5
0,038
4 . 5 X IO-'
450
90
9.5
9.5
0.045
.\--d.d,d-inethylcarbamate
111 IV
3-Isopropylpheny! .Y-trifluoromethvlcarbainate Phenyl .Y-methylcarbaniate
b.p. 88-90/3 Inm.
85-6
Phenyl .Y-trifluoroinethyl84-8 carbamate (' Piperonrl butoxide synergist. Calculated for 60% carbamate.
V
80
C = 53.44 C H = 4.85 H
J. A G R . F O O D C H E M .
= =
63.5 6.73
(7 4
C = 46.83
H
=
C = 46.67 2 . 9 3 H = 2.71
0.66
3 X
2
x
10-4
> l . 6 X 10-3 (10%)
1 500
500 >500
1605
>3.1
38
13
>500
1.0
>IO >10 >10
Table 11.
Properties and Biological Activities of Carbamates, N-Trimethylsilylcarbamates, and Other Modifications Musca domestico Pg./Gram
Anolvsis Compound
VI Phenclcarbalnate VI1 3-Isopropyl. phenylcarbamate VI11 3-Isopropvlphenyl S .VdimethylcarbamatI X 3-Isopropylphen) laziridinylcarba-
M.P.,
C.
Theory
A
n
Found
135-9 126-7
130
>i.o
C H
= =
67.04 C = 67.39 7.25 H = 7.15
= =
x
10-3
3 . 7 X lo-'
Affinify
70.24 C = 70.00 7.28 H = 7.43
lolone)
5oo
0.6
>500
5 . 0 X 10-5
( 73)
C H
Fly ChE
4
450
l C j o Culex Degree of prpiens Synergism 5-fosciatur
B(J :5
A/B
p.b.)
l.,P.P.M.
10
>500 77.5
>
>10
6.4
1.17
5.0
0.25
90
> 1 . 1 X 10-3 (10%)
500
>SO0
1.0
>1 X
500
>500
1.0
8.2
7.0
3.6
0.3
>500
27.0
>18.5
24
12.5
2.1
135
3.7
>IO
mate
X 3-Isopropylb p., loo"/ phenyl .V,.l-0 3"mm. diethylcarbamate X I 2-Isopropouyphenyl S methvlcarbamate XI1 2-Isopropoxy113-18 phenvlcarbamate X I I I 4-Methylthio118-20 3,5-\vlen!,1 .Vmethvlcarbamate X I V 4-Methylthio117-21 3,5-xvlenvlcarbamate X V 1-Naphthyl -V142 methylcarbamate XVI 1-Naphthyl175-7 carbamate XVII 3-Ethoxyphenyl 55-6 ,\--met hvlcarbamate XVI I I 3-Ethoxyphenyl113-1 15 carbamate X I X 3-Isopropyl. 55-7 phenyl .Y-trimethylsilvlcarbamate XX 2-Isopropoxy64-70 phenyl *Y-trimet hylsilvlcarbamatr X X I 1 -Kaphthyl >V93-6 trimethvlsiIvlcar6a-
C = 71.49 C = 71.86 H = 8.94 H = 8 . 9 3
6 . 9 X 10-7
( 7 4
C H
= =
61.54 C 6.65 H
= =
61.94 6.53
Mesurol (Bayer 37344)b
C = 56.87 C H = 6.15 H
= =
57.33 6.27
Carbary-1or Sevin
( 74)
c
59.67 6.08 C = 62.15 H = 8.36 = =
1 . 2 X 10-6
>1.6 X (2070) 9.0
C = 70.53 C2 = 70.72 H = 4 . 8 0 H = 5.07
H
1 . 0 X 10-3
C = 60.03 H = 5.8 C = 62.49 H = 7.55
x
290
0.2 165
1 X
500 900
>500 75
12.5
>10 0.23
>10 1 .o
72
>500
10
1.0
14.5
5.2
3
>500
>500
1.0
>10
2 . 3 X lo-'
0.88
120
40
3.0
>10
57.99 7.48
1 . 2 X 10-3
0.17
500
44
11.5
>10
64.86 C = 65.31 6.56 H = 6 . 4 7
> 1 . 2 X 10-3
500
ca. 500
1.0
>10
X X I I 4-Methylthio84-9 C = 55.12 C = 55.56 > 1 . 1 X 3,j-xylenyl H 7.43 H = 7.76 .\--trimethylsilvlcarbamate X S I I I Phenyl 'V-tri54-9 C = 57.42 C = 57.93 >1 X metliylsil~ylH = 7.17 H = 7.07 carbamate X X I V 3-Ethoxyphenyl 55-8 C = 56.92 C: = 57.17 >1 X &T-trime t h y 1H = 7.51 H = 7.60 silvlcarbamate X X V 3-Isopropylb.p. 136-40/ ( 79) 4 . 0 X 10-5 2 mm. phenyl -I'\ (Boots R.D. methyl, I f 14838) acetylcarbamate X X V I 3-Isopropylb.p. 146-8/2 ( 79) 1.1 X phenyl A mm. methyl, z'bT(Boots R.D. propionyl14990) carbamai e X X V I I 3-Isopropylb.p. 140-4/ ( 79) 4.4 x 10-5 phenyl h1 . 5 mm. methyl (Boots R.D. butyryl15914~) carbamate " Purification of this compound was effected by several washings with ligroin (75). * Sample provided by Farbenfabriken Bayer, Leverkusen, Germany. Samples provided by Boots Pure Drug, Nottingham, England.
500
190
2.6
>10
500
>500
1.0
>10
500
>500
1.0
>10
5
235
21
11.2
1.8
150
14
10.7
4.5
85
14
6.1
C = 58.43 C H = 7.86 H
C H
= =
= =
>
mate
VOL. 1 4 , N O . 1, JAN.-FEB.
1966
0.028
0.034
0.018
81
Trifluoromethyl isocyanate, b.p. - 35', was prepared according to Motornyi, Kirenskaya, and Yarovenko (16). With phenol this gave phenyl ,V-trifluoromethylcarbamate, m.p. 84-8' (lit. 88'). LYith 3-isopropylphenol a product was obtained, b.p. 88-90' a t 3 mm. This did not give a satisfactory analysis; hokvever, comparisons of the infrared spectra of this and the pure phenyl Strifluorometh>-lcarbamate (Figure 1) showed that the product contained 60% of the desired carbamate, and the remainder was unreacted m-isopropylphenol. An attempt to wash out the phenol with O.lyO sodium hydroxide solution almost completely hydrolyzed the carbamate within 1 minute. The other carbamates \vith altered ,\'-methyl groups were prepared from the appropriate phenylchloroformate (20) by reaction with ammonia ( 2 ) , ethyleneimine (8),or other amines and are shoivn in Table 11. .\'-trimethylsilylcarbamates \sere prepared according to the method of Pump and i2;annagut from the appropriate phenylchloroformate and hexamethyldisilazane (Table 11). These carbamates were evaluated for activity as inhibitors of fly head cholinesterase (ChE) and as toxicants to the female housefly (Musca domestica S A I D M strain) and to Culex pipiens quinpefasciatus mosquito larvae by methods previously described (70).
Discussion of Resuhs
NHCD3 Carbamate. T h e anticholinesterase and insecticidal activity of 3-isopropy1phen)-l 2V-methylcarbamate (I) and 3-isopropylphenyl *\--d.d,d?-methylcarbamate (11) are shown in Table I. The tivo compounds had identical activities. and the values obtained for the former were consistent with those previously reported (Id). These data show the lack of any deuterium isotope effect in regard to reaction Lvith ChE, intoxication of and detoxication in insects, or effect of methylenedioxyphenyl synergist on detoxication. Neither I nor I1 was toxic at 500 pg. per gram to the resistant strain of houseflies, RYIp, selected with (I) (6). Both I and I1 were equally toxic to the larvae of the salt marsh caterpillar, Estigimene acrea. The chemical reactivity of carbondeuterium (C-D) bonds is generally lo\yer than that of carbon-hydrogen (C-H) bonds, and the ratio of kc--o or the deuterium isotope effect has become an important tool for investigations of chemical mechanisms. This isotope effect? which is primarily the result of a difference in the zero-point energy between C-D and C-H, is maximal when the bond to hydrogen or deuterium is cleaved in the formation of the activated complex, and decreases to a limiting value, djor 1.4, with increasing binding in the activated complex (23). Thus, studies of the comparative toxicity of D D T and a-deutero D D T to the housefly have shown deuterium
82
J. A G R . F O O D C H E M .
isotope effects of 1.25 to 1.5 ( I , 75):and this is in accord with the \vel1 kno\vn detoxication pathway for D D T of dehydrochlorination, an Es-type elimination (3),mediated by the enzyme DDTdehydrochlorinase. The data obtained here xvith the SHCD3 carbamate suggest that the N-CH3 group is not directly involved in reaction with ChE and that the formation of NHCHpOH is not the primary mechanism in the detoxication of 3-isopropylphenyl S-methylcarbamate by the housefly. Alternatively, it may be that slow A\'-oxideformation is the rate-determining step in detoxication with rapid rearrangement to NHCHeOH, in which case no isotope effect Jvould be observed. NHCF3 Carbamates, As shown in Table I ? neither phenyl (\') nor 3-isopropylphenyl (111) A!2-trifluoromethylcarbamates were highly active. Compound I11 had a n affinity for ChE of
