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Investigations into carbamate insecticide selectivity
PESTICIDE
BIOCHEMISTRY
AND
PHYSIOLVQY
Investigations II: Differential AN-HORNG
into Degradation
T,EE,
JAMES
4, 77-85
Carbamate
Insecticide
of Selectophores
R.
SANBORN,
AND
in insect ROBERT
Selectivity and Mammal I,.
1
METCALF
Investigations were made of the effects of various modifications of the ether moiety alkoxyiminomethylphenyl N-met,hylcarbamates on the selective toxicity of the compounds to mouse vs. housefly. The highest mammalian selectivit,y ratio was found with o-(isopropyloxyiminomethyl)-phenyl IV-methylcarbamate which was about 30-fold more toxic to fly than to mouse. The o-(I-propynyloxyiminomethyl)-phenyl iV-methylcarbamate was the most toxic compound to the housefly. The biochemcial basis for selective act,ion was investigated. -
inactive i/l vitro as cholinestcrase inhibit’ors (5), but arc converted in oivo in insects to the toxic parent carbamate. However, in mammals more complete degradation occurs to produce the nontoxic phenol (6).
INTRODUCTION
In a previous paper (1) we investigated the incorporation of functional groups such as ester, nitrile, or amide on the aryl moiety of phenyl N-methylcarbamatcs, hoping to find that differences in rate of degradation in mouse and housefly might provide selectivity. The results were unsatisfactory, although an interesting example of reverse selectivity (a rodenticide of low toxicity to insects) was devised. It was concluded that an activation step to provide a delay factor permitting selective dctoxication is essential for the dcwlopment of highly selective carbamatc ins&icides. The only examples of a suitable delay factor mechanism in t,he carbamate inswticides are the N-acyl carbamates (2) which were extended by the invesbigations of N- (O,O-dialkylphosphoryl) carbamatcs (3) and N-(X-aryl) carbamatrs (3). These N-dcrivatized carbamates are generally i This research was supported by a grant The Rockefeller Foundation for the development selective and biodegradable insect’icides.
MATERIALS
0 1974 by Academic Press, Inc. of reproduction in any form reserved.
METHODS
The carbamatcs ww prepared by the reaction of the phenols with methyl isocyant’e in methylene chloride in t’he presence of a catalytic amount of triethylamine. The phenols for II-X, XII, XIII, and XVIIXXI were prepared by reaction of the appropriate alkoxy, alkenyloxy, or alkynyloxyaminc hydrochloride with hydroxybenzaldehyde or hydroxyacetophenonc. The former, oxyamine, was prepared as described (7). Thus, equal molar quant(itics of 1-propynyloxyamim hydrochloride and salicylaldchyde in ayuwus ethanol in th(k presence of two eyuivalcnt,s of sodium hydroxide n-we refluxcd ovrrnight. After the removal of ethanol under vacuum, t’ht: neutralized ayueous fract’ion n-as worked up with ether and dried over magnesium sulfate. The product was further purihcd
from of
77 Copyright All rights
AND
78
LEE,
SANBORN,
on a silica gel column clutrd wit,h a mixtuw of Skelly B-ether (1: 1 v/v). Crystallizrd o- (l-propyI13rlox~inlil~onl(~t hj.1) phwol from bcnzenc-Skelly B had a m.p. of 6Y”70°C. All other substituted phenols were oils. The phenols for II, XI, and XIV-XVI wrc pwparcd by reaction of equal molar quantitiw of salicylaldrhyd(~, sodium (thylat,e, and alkyl iodide or bromidcl in ethanol. Thr mixture was rpfluxcd overnight, workcad up and purified on a silica gc.1 column cluted with a mixture of Sk~lly B-Bwlzrnr: (17 : 3, v/v). The o- (l-propynyloxyiminomethyl) phenol prepared by this procedure and the other method had identical melting points, infrared and NMR spectra. Cornpound I was propared by the rclact’ion of o-cyanophwyl N-rnrthylcarbamatr~ with five times excess of acetic anhydride in the prescnw of a traw of conwntratc~d sulfuric acid (2). The compounds listctd in Tahl(l 1 w(w purified by either recrystallization from the mixbur~~ of Skrlly B and brnzcne, or column c*hromatography on silica gc>l, or both. The compounds \vere identificbd by infrared and NMR spectroscopy. The NMR spectra for t’hc carbamates were obtained in Ccl,. dcutrrat’ed DMSO, CDCls-DG-DRISO, or CDCl, with tctramethylsilanc as an internal standard at 44°C. Biological act,ivity of the carbamatcs wrc determined as drsrribed (1, 8). Anticholinest,crasc activit.y of thr compounds as cxpresscd by IO-min Iso valuw wwc determined from the li; (9). Thr clxprrimontal procedures were the sam(l as dcscribed previously (8, 10).
RESULTS
Itlsect Toxicity
AND
DISCUSSION
of Carbamates
The data collected in Tabh 2 indicate that thew (alkoxyiminomethyl)pht!nyl Nmethylcarbamatrs possess good inswticidal activity both against 3-4-day-old suscept,ible SNAIDM and RsP female houwflirs. the most toxic compound to thr houseflies, o- (l-propynyloxyiminomethyl)ph-
AND
METCALF
enyl N-mcthylcarbamate, 11, has an I,l>,a of’ 8.7 and 10.3 pg/ g to SNAIUW and RSP, rcspectively. The effective insecticidal ac%ivity of II against the houseflies probably is related t’o the autosyncrgism by thr propynyloxy group (11). Further, aryl propynyloxg ethers such as %,4,5-trichlorophcnyl propynyl clthcr (I?), naphth>rl propynyl cthw (13), and ?,-I,.;,,-t,richlorobcnzaldoximyl propynyl (bth(lr (14) ar(’ all eff c&w synergists for carbamatcb illsecticidcs. In addition, if houwfliw aw pretreated with the propynyl oximth c+,h(tI of benzaldehyde OXinlC (XXIV) or acrtophenone oxime (XXV) at 50 pg/g 1 hr before treatment with carbaryl, the LDnll is reduced to 3.6 pg/g for XXIV and 3.7 rg/‘g for XXV. The syncrgist,ic ratio of 4.3 for II against SNAm>I is the smallest valw for any of the compounds of t’his wries and further suggests that, the good insc&c~idal activity of this compound is wlat,ed t.o the autosynergism. Further (Lxamination of the houwfly toxicity data demonstratw clearly t’hat compounds substituted in the ortho position are decidedly more active t,han those substit,uted in either the meta or para positions (compare II wit#h III and IV; compare VII and VIII ; comparc X and XII). The consistclntly lower toxicities of thp meta and para compounds, as compared t,o the ortho substituted molwulcs, is reflrct,cd yuitc well in t.hft dtcreascd affinitv for awtylcholint~st~rrase. For (Lxamplr, II is about, 6.7 X mow toxic to the housrfly than 111 and II is 12.3 X bett,cr inhibitor of AChE than 111. Another intcrcst,ing aspect of the inwc+ toxicity of these compounds is the c$fwt of carbon chain length. Earlier invrstigations have demonstrated the effect of chain length on the inwcticidal activity of carbamatc insecticides (15, 16). For this wries, increaws in carbon chain lrngth do not, dwreaw the toxicity as dramat,ically as in the o-akloxyphcnyl N-mrthylcarbamatw For example, comparison of the LDjo values against susceptible houseflies of o-n-butoxy-
INVESTIGATIONS
INTO
CARBAMATE
INSECTICIDE
TABLE
Compound
79
the CarbanLates
of
M.P.
II
1
Physical Constants
--__.-
SELECTIVITY.
(“C)
74.5-5.5
NMRQ(T)
2.30-2.95 (m), aromatic, 3H ; 7.51 (s), CH,, 3H.
ppm
4H ; 6.66 (s), NCH+
OC(O)NHCHz CH=NOCH&=CH II
81-2
1.90(s), CH=N, matic, 4H; 4.89(b), 2H; 7.19(d), NCH,,
1H ; 2.20-3.09 (m), aroNH, 1H; 5.31(d), OCH?, 3H; 7.55(t), C=CH, 1H.
67-8
1.95(s), CH=N, 1H ; 2.49-3.01 (m), aromatic, 4H; 4.89(b), NH, 1H; 5.25(d), OCHz, 2H; 7.15(d), NCHI, 3H; 7.50(t), C=CH, 1H.
136-7
1.78(s), CH=X, IH ; 2.28-2.94 (m), aromatic, 4H; 4.91 (b), NH, 1H; 5.26(d), OCH+ 2H; 7.28(d), NCH,, 3H; 7.49(t), C=CH, 1H.
13%3
1.88(s), 3.36(s), 4.92(b), NCHS,
134-5
1-99(s), CH=N, 1H; 2.52-3.17(m), 3H; 3.53(b), NH, 1H; 5.29(d), 6.17(s), OCH,, 3H ; 7.04-7.30(m), 3H; 7.40(t), C=CH, 1H.
OC(O)NHCHx III
OC(O)NHCHz /A
IV u\
cH=NOCH&=CH OC (0)NHCHa CH=NOCHKkCH
1’
CH=?;, 1H; 2.75(s), aromatic, 1H ; aromatic, 1H; 4.03(s), OCHZO, 2H ; NH, 1H; 5.28(d), OCH2, 2H; 7.13(d), 3H; 7.50(t), CrCH, 1H.
0 I CH2--0
(‘II:,0
OC (0)NHCHa
VI CH=NOCH&=CH
aromatic,
OCHz, 2H; NCH,,
OC (0)NHCHz 57-8
1.79(s), CH=N, 1H; 2.09-3.00(m), aromatic, 4H; 3.60-4.29(m), CH=C, 1H; 4.47-4.91(m), ===CH2, NH, 3H ; 5.21-5.45(m), OCH,, 2H; 7.19(d), NCH, 3H.
40-l
1.96(s), CH=N, 1H; 2.52-3.02(m), aromatic, 4H; 3.594.27(m), CH=C, 1H; 4.48-4.91(m), =CH,, NH, 3H; 5.20-5.43(m), OCH,, 2H; 7.14(d), NCH,, 3H.
73-4
1.82 (s), CH=N, 1H ; 2.07-3.01 (m), aromatic, 4H; 4.83(b), NH, 1H; 5.79(q), OCHz, 2H; 7.14(d), NCH,, 3H; 8.69(t), CCHs, 3H.
CH=NOCH,CH=CHX VIL
OC(O)NHCHI
VIII
H=NOCH,CH=CH2 OC (0)NHCHa
IX
(b)
u (s) = singlet, = broad.
(d)
= doublet,
(t)
= triplet,
(q)
= quartet,
(sex)
= sextuplet,
(hept)
= heptuplet,
80
LEE,
SANBORN,
AND
TABLE Compound
l-Continued M.P.
OC (0)NHCHy CH=?JOCH&H&HI
x
METCALF
(“‘C)
il-2
1.81 (s), CH=N, IH; 2.09-3.03(m), 4H ; 4.77(b), NH, 1H ; 5.90(t), 7.20(d), NCHI, 3H; 8.27(sex), 8.89-9.21 (m), CCH3, 3H.
aromatic, OCH,, 2H; CCH,C, 2H:
76-7
1.80(s), CH=N, 1H; 2.09-3.02(m), aromatic, 4H; 4.83(b), NH, 1H ; 5.54(hept), OCH, 1H ; 7.17(d), NCHz, 3H; 8.70(d), (CH&, 6H.
41-z
1.98(s), CH=N, 1H; 2.50-3.06(m), 4H; 4.90(b), NH, 1H; 5.89(t), 7.15(d), NCHI, 3H; 8.25(sex), 8.83-9.20(m), CCH,, 3H.
51-2
1.82 (s), CH=N, 1H ; 2.09-3.02 (m), aromatic, 4H ; 4.82(b), NH, 1H; 5.83(t), OCH2, 2H ; 7.17(d), NCH3, 3H; 8.01-8.81(m), (CH?),, 4H ; 8.81-9.27 (m), CHJ, 3H.
56-7
1.82(s), CH=N, 1H; 2.07-3.01(m), 4H; 4.79(b), NH, 1H; 5.75 (sex), 7.19(d), NCH,, 3H; 8.10-8.61(m), 8.73(d), (CH,), 3H; 8.89-9.23(m),
30-l
1.81 (s), CH=h’, 1H ; 2.09-3.01 (m), aromatic, 4H; 4.89(b), NH, 1H; 5.83(t), OCHz, 2H; 7.15(d), NCHa, 3H; 8.00-8.89(m), (CHz),, 6H ; 8.89-9.33 (m), CH3, 3H.
OC (0)NHCHs
3 /’
XI
CH=NOCH(CH,)z
\
OC (O)NHCHa XII
CH=NOCH,CH /
,.J CH 2
aromst,ic, OCHe, 2H; CCH&, 2H;
OC (O)NHCHs /’
CH=NOCHz(CH&CH3
XIII 3\ OC (O)NHCHa CH=NOCH
(CH&H&H,
XIV
aromatic, OCH, 1H; CHS, 2H; CHI, 3H.
OC (0)NHCHa / Xl’ c\
CH=?TOCH,(CHz),CH3 11 CO (O)NHCHa CH=NOCH?(CH,)&H3
XVI
oil
1.83(s), CH=N, 1H; 2.09-3.17(m), 4H ; 4.77(b), NH, IH ; 5.87(t), 7.17(d), NCH3, 3H; 8.00-8.93(m), 10H ; 8.93-9.33 (m), CH3, 3H.
aromatic, OCH,, 2H; (CHZ)~,
.I C (CHa)=NOCH&kCH XVII
77-8
2.46-3.00(m), aromatic, 4H ; 4.86(b), NH, 1H ; 5.25 (d), OCHz, 2H; 7.17(d), NCHz, 3H; 7.51 (t), CzCH, IH; 7.81(s), C(CHs), 3H.
OC (O)XHCHB XVIII )=NOCH
2:( =CH
65-6
2.50-3.02(m), aromatic, 4H; 4.86(b), NH, 1H; 5.23(d), OCHz, 2H; 7.15(d), NCHI, 3H; 7.53 (t,), C=CH, 1H ; 7.79(s), C(CH& 3H.
OC(O)NHCHr XIX
80-Z
2.24-3.00(m), aromatic, -I-H; 4.85(b), KH, IH ; 5.22 (d), OCHz, 2H; 7.15(d), NCHS, 3H; 7.52(t), C-CH, 1H; 7.79(s), C&Ha), 3H.
65-6
2.49-3.02(m), aromatic, 4H; 3.59-4.25(m), CH=C, 1H ; 4.50-4.94(m), ===CH,, NH, 3H ; 5.23-5.42(m), OCHZ, 2H ; 7.18(d), NCHS, 3H ; 7.80(s), C(CH& 3H.
C (CHa)=NOCH&rCH OC(O)NHCH3 C(CHS)=NOCH&H=CH~ xx
INVEd’~IGATIOXS
IIiTO
CARBAMATE
INSECTICIDE
TABLE Compound
XXII
C (CHs)=KOCH&H=CH,
II
81
I-fZ’&in~ted M.P. (“C)
UC (O)NHCH, XXI
SELECTIVITY.
NMRa(7)
ppm
6778
2.49-3.02(m), aromatic, 4H ; 3.60-4.27 (m), CH+, 1H ; 4.50-4.94(m), =CH,, NH, 3H ; 5.21-5.40(m), OCH2, 2H : 7.17(d), YCHI, 3H ; 7.78(s), C (CH& 3H.
95-6
l.iO(s), CH=N, 1H ; 2.21-2.70(m), aromah, 5H, 3.65(h), NH, 1H; 7.04(d), NCHa, 3H.
XXIII
oil
1.95(s), CH=N, 1H ; 2.33-2.83 (m), aroma&, 5H; 5.87(t.), OCHZ, 2H; 8.28 (sex), CCH&, 2H ; 8.88M.20(m), CH3, 3H.
XXIV
oil
I .91 (s), CH=N, 1H ; 2.3%2.i7(m), aromak, 5H, 5.25(d), CXH2, 2H; 7.51 (t), C=CH, 1H.
XXV
oil
9.23-2.73(m), aromatic, 5H ; 5.21 (d), OCH,, 2H; 7.52(t), C=CH, 1H; 7.75(s), CHa, 3H.
phenyl N-methylcarbamate and XIII demonstrates t,hat the latter is 5.8 X more t,oxic than the rl-butoxy carbamate. Furthermore, the ISo value for XIII is about; 0.055 X that obtained for o-nbutoxyphenyl N-methylcarbamate which correlates satisfactorily with the relative lIDSo values of the two compounds. Clearly, the insertion of HC=N between the oxygen and phenyl ring of o-n-hutoxyphenyl Nmc+hylcarbamat~r~ to give XIII increases thtl affinity of XIII for AChE. The incwasrd interaction with AChE probably plays an important role in t’he higher toxicit,y of these oximr rt’hcr carbamat,es as compared to t,heir alkoxy analogues. The most puzzling aspect of the housefly toxicity data is thtl inactivity of XVII \vhich was dcriwd from o-hydroxyacetol~hcwonc~.A suitahl(> ckxplanation can be found in the comparison of the ISo values of 1.17 X 1OP M for 11 and 97.22 X lo-” M for XVII. The decreased affinity for cholinestcrase of XVII as compared to II, along with th(l incrcasrd susccspt,ibility for tnc+abolism as mr~asur~~dby t,h(t approxinlatc4y sixfold incwaw in thr, synwgistic
value of XVII, provide plausible explanations for the differences in toxicities of II and XVII t,o the housefly. While replacing the aldehydic hydrogen with a methyl group in the ortho position greatly affected the t,oxicity, the same st’ructural modification in the rlleta position (compare III and XVIII) only reduced the housefly toxicity by about twofold. Examination of the t,oxicit,y of this series of compounds to the rest, of t.hc insects demonstrates that these compounds arc‘, on an average, about 0.81 as toxic to 11s~ and three of t’hem, IX, XII, and XVIII, are more toxic to this resistant strain of housefly. None of the compounds tested is toxic t.o the larval stages of mosquitoes at 1.0 ppm, but several of them, I, II, VII, and IX-XI, were toxic t,o the adults of thcw mosquitoes. It may br significant that one of the compounds t,hat is quite actiw against mosquito adults, I, has a very low LD60 to the white mouse of > 1,000 mg/kg. However, it would seem unlikely that this molecule could ever be utilized for rontrol of mosquito adults as the parent compound (I) and, particularly, the phenol haw a
>500.0 8.7 *x0 >500.0 > 500.0 >500.0 11.5 151.8 13.4 17.1 15.9 212.5 30.4 22.4 38.1 104.3 > 500.0 133.2 >500.0 >500.0 >500.0
0.3 4.0
0.6 7.3 1.5 1.1 1.0 13.3 1.6 1.7 4.8 5.6
10.2
f f
f f f f f f f f f f
f
Alone
SNAIDM
p.b.
(dg)
domestica
21.7 f 2.4 2.0 f 0.01 9.7 i 1.0 >500.0 6.7 i 0.9 >500.0 1.5 f 0.3 20.8 f 2.7 1.7 f 0.1 1.9 f 0.2 1.8 f 0.1 17.0 i 1.5 1.6 f 0.1 1.4 f 0.2 2.1 f 0.2 12.9 f 1.8 19.1 f 2.8 1.8 f 0.2 > 500.0 20.7 f 1.5 8.7 f 1.2
With
LD5o
Musca
>24.1 >57.5
7.7 7.3 7.9 9.0 8.8 12.5 19.0 16.0 18.1 8.1 >26.2 74.0
>74.6 -
23.0 4.3 6.0 -
SR”
(L.)
a SK. (Synergistic Ratio) = Ll)so alone/LD50 with p.b. b MSR (Mammalian Selective Ratio) = IMouse LDjO/Housefly
I II III IV V VI VII VIII IX X XI XII XIII XIV xv XVI XVII XVIII XIX xx XXI
Compound
RSP
Activity
>500.0 10.3 165.4 >500.0 >500.0 >500.0 14.4 231.9 12.9 19.9 20.4 179.1 49.4 56.1 67.4 206.6 >500.0 67.8 >500.0 >500.0 >500.0
LDsO.
Biological
zk
2
0.6 12.0
7.1
8.5 127.5 >160.0 >160.0 > 160.0 > 160.0 6.7 > 160.0 5.7 10.2 10.9 >160.0 >160.0 >160.0 >160.0 >160.0 >160.0 >160.0 >160.0 >160.0 >160.0
0.3 6.3
f f f
1.5 1.2 0.2
zt 1.0
f f
Anopheles albimanus Adults LDsa &g/cm?
of the Carbamates
rt 1.1 f 14.3 f 0.6 f 2.3 f 1.8 * 22.9 f 4.3 f 5.4 f 5.1 zk 15.9
f f
TABLE
-
57.5 > 160.0 > 160.0 > 160.0 >160.0 > 160.0 21.8 > 160.0 18.7 30.3 >160.0 >160.0 >160.0 >160.0 >160.0 >160.0 >160.0 >160.0 >160.0 > 160.0 >160.0
5.1
+ 1.3 * 3.1
rt 0.9
*
Culex pipe-n.3 quinquefasciutus Say Adults LJh (wdcm?
> 1000 -50 >500 >300 >300 >500 200-300 >500 15Ck200 300-400 400-500 >500 20-30 300-350 150-325 350-400 >500 >500 > 500 >500 >500
Mouse Oral LD60 (mg/kg)
-
-
>3.8
17.4-26.1 >3.3 11.2-14.9 17.5-23.4 25.1-31.4 >2.4 0.7-1.0 13.4-15.6 3.9-5.9 3.4-3.8 -
,-5.7 >8.b
MSRb
2 L M ;:
ks 3
Y
INVES’I~IGATIONS
very distinctive burned surgar.
INTO
and penetrating
CARBAAMATE
odor
of
Mouse Toxicity The purpose of this investigation and the previous work (1) was the preparation of carbamatc insecticides that were more toxic to the housefly than to the femaIe white mouse. In the earlier investigation, it was demonstrated that the incorporation of potenbial selectophorcs such as an ester, amide, or a nitrile did not enhance the dcsircd selectivity appreciably. Further, it was pointed out that the bis-carbamate of salicylaldoxime had a very unfavorable mammalian selectivity ratio of CO.03 and an LDju to the mouse of lo-20 mgikg. The conclusions drawn from that work indicatcd that, in addition to a selcctophore, a delay factor as is present in the phosphorothionate insecticides, such as malathion, is requisite for selectivity between insects and mammals in t’he carbamate insecticides. The data outlined in the present work appears to confirm the original thesis t.hat, if part,icular functional moieties, in this that if particular functional moieties, in this case the alkoxyiminomethyl group (HC=N-OR) where R = alkyl group are incorporated into carbamat,e insecticides, then more favorable MSR ratios can be obtained. The explanation for the very unfavorable mouse LD50 of the salicylaldoxime his-carbamatc provided a useful lead for the preparation of carbamates with desirable MSR values. The rationalization for the high toxicity of this bis-carbamate to the mouse involved implication of a facile conversion of the o-N-methylcarbamoyloxime into a cyano group in the acid stomach of the mouse. This highly toxic carbamate provided the impetus for substitution of the carbamoyi moiety by an alkyl group which would, on tho basis of physical organic chemistry, not rearrange as rapidly to the nitrile as the carbamoyl derivatized species. In an effort to investigate the relative rates of acid catalyzed rearrangement of an oxime
INSECTICIDE
SELECTIVITY.
II
83
ether and a N-methylcarbamoxyloxime ester, the oximc of benzaldehyde was reacted with propyl bromide under basic conditions to give XXIII and with methyl isocyanate to give XXII. Ultraviolet spectroscopic study indicated that the treatment of XXII with cthanolic hydrochloric acid (0.2 N) at 25% leads to the disappearance of the parent compound (X = 256 rnp) to give benzonitrile (A = 222 rnp) and others with a pseudo first order rate constant k$ = 0.0092 hour-’ and half-life of 75 hr over a period of 120 hr. The benzonitrile constituted approximately 25% of the transformed parent compound under the condition described. The spectrum of XXIII under the same conditions does not change over a time period of 250 hr. Thercfore, if the rates of acid and biochemically catalized Beckmann rearrangements of derivatized oximes can be related, and, if selectivity between mice and insects is a function of the rate rearrangement of a derivatized oxime to the o-cyanophenyl carbamate, then the compounds of this series which have MSR values 100-1,000X greater than the bis-carbamate of salicylaldoxime are selective because of the greatly reduced rate of conversion of the oxime into the nitrile in the stomach of the mouse. Currently, efforts are being directed towards confirmation of this hypothesis by the synthesis of 14C-labeled material and evaluation of the metabolic pathways of the bis-carbamate of salicylaldoxime and one of the (o-alkoxyiminomethyl)phenyl Nmethylcarbamates of this series. One final aspect of the mouse toxicity data is the greatly reduced toxicity of I, the N-acylated derivative of o-cyanophenyl N-methylcarbamate. The carbamate without the N-acyl group had an oral LD,, to mice of about 225-250 mg/kg (1). The toxicity to the white mouse of I of >l,OOO mg/kg appears to confirm the conclusion of the initial work on selective carbamates that both a selectophore, the cyano functional group, and a delay factor, the N-acetyl group are requisite for reduced
LEE,
84
HANBORN,
Anticholincsteras~
AND
Activity of thy Carbamatcs ~__ .-.. ~~~
Compound
ESW
IjO Housefly (IOF
II III IV v VI VII VIII IX x XI XII XIII XIV XV XVI XVII XVIII XIX xx
XXI (Enzyme
head AChEJ Ill)
Bovine
_--7348.41 f 524.25 1.17 f 0.18 144.13 + 12.86 84.27 f 9.29 2.87 f 0.23 114.83 f 7.33 0.97 It 0.08 159.63 f 10.76 0.84 + 0.07 1.26 + 0.09 0.50 f 0.05 136.20 f 10.38 0.66 f 0.06 O..i3 f 0.07 0.29 zt 0.03 0.15 f 0.03 97.22 It 1.69 110.30 f 1.52 152.96 f 14.06 156.39 f 15.21 105.42 f 3.09 ~- .~.
I
” ESR
METCALF
Selectivity
Ratio)
= 150 bovine/Isa
toxicity to the white mouse. The presence of the N-acetyl group probably allows metabolism of the cyano group to polar product’s before the loss of the acetyl group to yield an active toxic anticholin&erasc I?-met.hylcarbamate. Antich.olilzesterase Activity In Table 3 the Is0 values arc collected for inhibition at 25°C of purified housefly head aceytlcholinesterasc and bovine erythrocytc cholinesterase. Again, the generally more efficient inhibition of housefly cholinesterasc than bovine cholinesterase is demonstrated by the enzyme selectivity ratios greater than one. Except for I, which has an ESR value of 0.04, the values range from one for XIX to 2,556 for XV. Attempts to provide a rationale for reduced mouse toxicity as compared to insect using ESR and MSR values were yuit,e unsuccessful. Perhaps t,h(i most, interesting aspect of th(b cholirr-
erythrocyte AChE (10-h M)
3.15
f
0.25
1.31
*
4.12 2.61 1.17 2.72 2.88 5.42 1.27 0.77 3.62 7.57 2.69 4.68 5.88 3.75 4.66 2.66 1.54 4.06 4.24
f f f f f xt + f f f zk f f It f f f f + --
0.14 0.54 0.19 0.08 0.12 0.11 0.11 0.22 0.10 0.44 0.18 0.26 0.60 0.69 0.13 0.38 0.29 0.16 0.14 0.41 ~ __-.
0.04 112.0 2.9 3.1 40.8 ‘2.4 296.9 3.4 151.2 61.1 1124.0
5.6 407.6 883.0 2.556.5 2500.0 4.8 2.4 1.0
2.6 4.0 ____.....~~~_~__~~
housefly.
esterase data was the much greater srnsitivity of housefly head AChE to ortho versus meta or para substitution of the hydroxybenzaldehyde derivatives as compared to bovine crythrocyte AChE. For example, the Ijo values against housefly AChE of II and III differ by 115 X and yet, against bovine erythrocyte AChE, these two compounds differ by only 3.14 X. Comparison of VII and VIII and X and XII yields similar conclusions as to the effect of ring substitution on housefly head AChE inhibition. However, for the compounds derived from hydroxy acetophenonn (XVII-XXI), the 150values for inhibition of housefly head AChE do not change as dramatically (compare XVII and XIX, compare XX and XXI) as those derived from the hydroxy benzaldehydes and arc’ similar to the changes in 150values against bovinc AChE. Perhaps compounds with 15” valucls that, are in the 10e4 M range
INVESTIGATIONS
(l~vII-xxI)
are
just
INTO
not
as
WnSitiW
CARBAMATE
t0
structural changw as those with lo~wr ISo values. The dat,a for the bovine AChE certainly support this conclusion as well as those derived from substituted hydroxy acetophenones.
From the data presented in this paper it is clear t,hat the alkoxyiminomethyl moietJJ / (HC-?J-OR) when incorporated into a phenyl carhamat,c can provide selective biological act’ivity between insects and mammals. The rationale for the synthesis of this series was provided by the outst,anding rodenticidal activity of b&N-methylcarbamoyl salicylaldoxime where t)hc toxicity was explained by implicating a Beckmann rearrangement to the nitrile that took place more rapidly in the mouse than in the ins&. Therefore, by replacement of the N-me?thylcarhamoyl oximtb with a linear or branched alcohol, it was hoped that, the rat,c of rcarrangemcnt, would be reduced and mammalian selcrtivr ratio increased. The substitut,ion of the carbamoyl group with an alkoxy group clearly increased the selectivity as the compounds of this series are 100-1,000 X nl(Jw selective than the his salicylaldoximc carbamate. Perhaps other functional groups can be found that will increase the unfavorable nlSR values of carbamatcx insecticides. The functional groups which seem to provide selectivity for the phosphorothionat(~s, ester, nit,rile, alld amide arc’ IlOt effective for carbamates, but :I somcx\lhat, surprising moichy, the, alkoxyiminomr~thpl (HC-NOR), to provide the desired selectivity.
is able
INSEC’IICIDE
II
85
3. hr. A. H. Fahmy, T. R. Fukuto, 1~. 0. Meyers, and It. B. ;Cfarch, The selective toxicity of new .\:-phosphorylt,hioylcarbamate esters, J. Agr. Food Chent. 18, 793 (1970). 4. A. L. Black, Yi-chang Chiu, RI. A. H. Fahmy, and T. It. Fukuto, Selective t,oxicity of :V-sulfenylated derivatives of insecticideal methylcarbamate est,ers, J. izgr. Food Chrm. 21, 747 (1973). 5. h\lohamed A. H. Fahmy, It. L. Metcalf, T. R. Fukuto, and I>. J. Hennessy, Effects of deuteration, fluorination and other struct,ural modifications on the carbamoyl moiety upon t.he anticholinesterase and insecticidal activit.ies of phenyl S-methylcarbamates, J. Agr. Food Chm. 14, 79 (1966). 6. 11. P. Miskus, T. L. Andrews, and &‘I. L. Look, hletaboliu pathways affecting toxicity of :V-acetyl Zectran, J. Agr. Food Chew 17, 842 (1969). 7. Brit. Patent 889,086 (Chemical Abstracts 57 : 728Sd-7288e). 8. R. L. Metcalf, I. P. Kapoor, and A. S. Hirwe, Biodegradable analogues of I>1 IT, BuU. JFor/d Hdth Org. 44, 363 (1971). 9. W. N. Aldridge, Some properties of specific cholinesterase with particular reference to t,he mechanism of inhibition by diethyl p-nitrophenyl thiophosphate (E60.i) and analogues, Biochem. J. 46, 461 (1930). 10. An-horng Lee and R. I,. Metcalf, In vifro inhibition of acetylcholinesterase by O,O-dimethyl S-aryl phosphorothioates, Pest. RioChern. Physiol. 2, 408 (1973). 11. 11. L. AIetcalf, The role of oxidative reactions in the mode of a&on of insecticides, in “Enzymatic oxidation of toxicants,” 0;. Hodgson, Ed.), p. 151, North Carolina State University, Raleigh, N. C. 1968. 12. J. IX. C&da, nlixed Function oxidase involvement, in the biochemistry of insecticide aynergists, J. Agr. Food Chem. 18, 753 (1970). 1:3. 11. 31. Sacher, It. L. Metcalf, and T. R. Fukuto, Propynyl napthyl et,hers a selective carbamate synergista, J. Agr. Food Chwn. 16,
i79 (1968). 14. 1). J. Henmessy, synergists chemical O’Brien Academic
REFERE?;CES
li. Sanborn, A. Lee and It. L. hfetcalf, into carbamate insecticide Investigations selectivity. I. Evaluation of potential selectophores. Pest.Biochem. Physiol. 4, 67-76. 2. J. Fraser, P. (:. Clinch, and R. C. Reay, ;V-acylation of l\rmethylcarbamate insecticides and it,s effect on biological activity, J. Sci. Food Agr. 16, 615 (1965).
SELECTIVITY.
1. d.
Id.
The potential of carbamate as pest control agents, in “BioToxicology of Insecticides,” (Et. I). and I. Yamamoto, Ed.), p. 105, Press, New York (1970).
K. L. >Ietcalf, T. R. Fukuto, and M. Y. Winton, Alkoxyphenyl ‘V-methylcarbamates as insect,icides, J. Econ. Ent. 53, 828 (1960). 16. It. L. AIetcalf, T. R. Fukuto, and M. Y. Winton, The synergism of substituted phenyl Nmethylcarbamates by piperonyl butoxide, J. Econ.. Ent. 55, 341 (1962).
BIOCHEMISTRY
AND
PHYSIOLVQY
Investigations II: Differential AN-HORNG
into Degradation
T,EE,
JAMES
4, 77-85
Carbamate
Insecticide
of Selectophores
R.
SANBORN,
AND
in insect ROBERT
Selectivity and Mammal I,.
1
METCALF
Investigations were made of the effects of various modifications of the ether moiety alkoxyiminomethylphenyl N-met,hylcarbamates on the selective toxicity of the compounds to mouse vs. housefly. The highest mammalian selectivit,y ratio was found with o-(isopropyloxyiminomethyl)-phenyl IV-methylcarbamate which was about 30-fold more toxic to fly than to mouse. The o-(I-propynyloxyiminomethyl)-phenyl iV-methylcarbamate was the most toxic compound to the housefly. The biochemcial basis for selective act,ion was investigated. -
inactive i/l vitro as cholinestcrase inhibit’ors (5), but arc converted in oivo in insects to the toxic parent carbamate. However, in mammals more complete degradation occurs to produce the nontoxic phenol (6).
INTRODUCTION
In a previous paper (1) we investigated the incorporation of functional groups such as ester, nitrile, or amide on the aryl moiety of phenyl N-methylcarbamatcs, hoping to find that differences in rate of degradation in mouse and housefly might provide selectivity. The results were unsatisfactory, although an interesting example of reverse selectivity (a rodenticide of low toxicity to insects) was devised. It was concluded that an activation step to provide a delay factor permitting selective dctoxication is essential for the dcwlopment of highly selective carbamatc ins&icides. The only examples of a suitable delay factor mechanism in t,he carbamate inswticides are the N-acyl carbamates (2) which were extended by the invesbigations of N- (O,O-dialkylphosphoryl) carbamatcs (3) and N-(X-aryl) carbamatrs (3). These N-dcrivatized carbamates are generally i This research was supported by a grant The Rockefeller Foundation for the development selective and biodegradable insect’icides.
MATERIALS
0 1974 by Academic Press, Inc. of reproduction in any form reserved.
METHODS
The carbamatcs ww prepared by the reaction of the phenols with methyl isocyant’e in methylene chloride in t’he presence of a catalytic amount of triethylamine. The phenols for II-X, XII, XIII, and XVIIXXI were prepared by reaction of the appropriate alkoxy, alkenyloxy, or alkynyloxyaminc hydrochloride with hydroxybenzaldehyde or hydroxyacetophenonc. The former, oxyamine, was prepared as described (7). Thus, equal molar quant(itics of 1-propynyloxyamim hydrochloride and salicylaldchyde in ayuwus ethanol in th(k presence of two eyuivalcnt,s of sodium hydroxide n-we refluxcd ovrrnight. After the removal of ethanol under vacuum, t’ht: neutralized ayueous fract’ion n-as worked up with ether and dried over magnesium sulfate. The product was further purihcd
from of
77 Copyright All rights
AND
78
LEE,
SANBORN,
on a silica gel column clutrd wit,h a mixtuw of Skelly B-ether (1: 1 v/v). Crystallizrd o- (l-propyI13rlox~inlil~onl(~t hj.1) phwol from bcnzenc-Skelly B had a m.p. of 6Y”70°C. All other substituted phenols were oils. The phenols for II, XI, and XIV-XVI wrc pwparcd by reaction of equal molar quantitiw of salicylaldrhyd(~, sodium (thylat,e, and alkyl iodide or bromidcl in ethanol. Thr mixture was rpfluxcd overnight, workcad up and purified on a silica gc.1 column cluted with a mixture of Sk~lly B-Bwlzrnr: (17 : 3, v/v). The o- (l-propynyloxyiminomethyl) phenol prepared by this procedure and the other method had identical melting points, infrared and NMR spectra. Cornpound I was propared by the rclact’ion of o-cyanophwyl N-rnrthylcarbamatr~ with five times excess of acetic anhydride in the prescnw of a traw of conwntratc~d sulfuric acid (2). The compounds listctd in Tahl(l 1 w(w purified by either recrystallization from the mixbur~~ of Skrlly B and brnzcne, or column c*hromatography on silica gc>l, or both. The compounds \vere identificbd by infrared and NMR spectroscopy. The NMR spectra for t’hc carbamates were obtained in Ccl,. dcutrrat’ed DMSO, CDCls-DG-DRISO, or CDCl, with tctramethylsilanc as an internal standard at 44°C. Biological act,ivity of the carbamatcs wrc determined as drsrribed (1, 8). Anticholinest,crasc activit.y of thr compounds as cxpresscd by IO-min Iso valuw wwc determined from the li; (9). Thr clxprrimontal procedures were the sam(l as dcscribed previously (8, 10).
RESULTS
Itlsect Toxicity
AND
DISCUSSION
of Carbamates
The data collected in Tabh 2 indicate that thew (alkoxyiminomethyl)pht!nyl Nmethylcarbamatrs possess good inswticidal activity both against 3-4-day-old suscept,ible SNAIDM and RsP female houwflirs. the most toxic compound to thr houseflies, o- (l-propynyloxyiminomethyl)ph-
AND
METCALF
enyl N-mcthylcarbamate, 11, has an I,l>,a of’ 8.7 and 10.3 pg/ g to SNAIUW and RSP, rcspectively. The effective insecticidal ac%ivity of II against the houseflies probably is related t’o the autosyncrgism by thr propynyloxy group (11). Further, aryl propynyloxg ethers such as %,4,5-trichlorophcnyl propynyl clthcr (I?), naphth>rl propynyl cthw (13), and ?,-I,.;,,-t,richlorobcnzaldoximyl propynyl (bth(lr (14) ar(’ all eff c&w synergists for carbamatcb illsecticidcs. In addition, if houwfliw aw pretreated with the propynyl oximth c+,h(tI of benzaldehyde OXinlC (XXIV) or acrtophenone oxime (XXV) at 50 pg/g 1 hr before treatment with carbaryl, the LDnll is reduced to 3.6 pg/g for XXIV and 3.7 rg/‘g for XXV. The syncrgist,ic ratio of 4.3 for II against SNAm>I is the smallest valw for any of the compounds of t’his wries and further suggests that, the good insc&c~idal activity of this compound is wlat,ed t.o the autosynergism. Further (Lxamination of the houwfly toxicity data demonstratw clearly t’hat compounds substituted in the ortho position are decidedly more active t,han those substit,uted in either the meta or para positions (compare II wit#h III and IV; compare VII and VIII ; comparc X and XII). The consistclntly lower toxicities of thp meta and para compounds, as compared t,o the ortho substituted molwulcs, is reflrct,cd yuitc well in t.hft dtcreascd affinitv for awtylcholint~st~rrase. For (Lxamplr, II is about, 6.7 X mow toxic to the housrfly than 111 and II is 12.3 X bett,cr inhibitor of AChE than 111. Another intcrcst,ing aspect of the inwc+ toxicity of these compounds is the c$fwt of carbon chain length. Earlier invrstigations have demonstrated the effect of chain length on the inwcticidal activity of carbamatc insecticides (15, 16). For this wries, increaws in carbon chain lrngth do not, dwreaw the toxicity as dramat,ically as in the o-akloxyphcnyl N-mrthylcarbamatw For example, comparison of the LDjo values against susceptible houseflies of o-n-butoxy-
INVESTIGATIONS
INTO
CARBAMATE
INSECTICIDE
TABLE
Compound
79
the CarbanLates
of
M.P.
II
1
Physical Constants
--__.-
SELECTIVITY.
(“C)
74.5-5.5
NMRQ(T)
2.30-2.95 (m), aromatic, 3H ; 7.51 (s), CH,, 3H.
ppm
4H ; 6.66 (s), NCH+
OC(O)NHCHz CH=NOCH&=CH II
81-2
1.90(s), CH=N, matic, 4H; 4.89(b), 2H; 7.19(d), NCH,,
1H ; 2.20-3.09 (m), aroNH, 1H; 5.31(d), OCH?, 3H; 7.55(t), C=CH, 1H.
67-8
1.95(s), CH=N, 1H ; 2.49-3.01 (m), aromatic, 4H; 4.89(b), NH, 1H; 5.25(d), OCHz, 2H; 7.15(d), NCHI, 3H; 7.50(t), C=CH, 1H.
136-7
1.78(s), CH=X, IH ; 2.28-2.94 (m), aromatic, 4H; 4.91 (b), NH, 1H; 5.26(d), OCH+ 2H; 7.28(d), NCH,, 3H; 7.49(t), C=CH, 1H.
13%3
1.88(s), 3.36(s), 4.92(b), NCHS,
134-5
1-99(s), CH=N, 1H; 2.52-3.17(m), 3H; 3.53(b), NH, 1H; 5.29(d), 6.17(s), OCH,, 3H ; 7.04-7.30(m), 3H; 7.40(t), C=CH, 1H.
OC(O)NHCHx III
OC(O)NHCHz /A
IV u\
cH=NOCH&=CH OC (0)NHCHa CH=NOCHKkCH
1’
CH=?;, 1H; 2.75(s), aromatic, 1H ; aromatic, 1H; 4.03(s), OCHZO, 2H ; NH, 1H; 5.28(d), OCH2, 2H; 7.13(d), 3H; 7.50(t), CrCH, 1H.
0 I CH2--0
(‘II:,0
OC (0)NHCHa
VI CH=NOCH&=CH
aromatic,
OCHz, 2H; NCH,,
OC (0)NHCHz 57-8
1.79(s), CH=N, 1H; 2.09-3.00(m), aromatic, 4H; 3.60-4.29(m), CH=C, 1H; 4.47-4.91(m), ===CH2, NH, 3H ; 5.21-5.45(m), OCH,, 2H; 7.19(d), NCH, 3H.
40-l
1.96(s), CH=N, 1H; 2.52-3.02(m), aromatic, 4H; 3.594.27(m), CH=C, 1H; 4.48-4.91(m), =CH,, NH, 3H; 5.20-5.43(m), OCH,, 2H; 7.14(d), NCH,, 3H.
73-4
1.82 (s), CH=N, 1H ; 2.07-3.01 (m), aromatic, 4H; 4.83(b), NH, 1H; 5.79(q), OCHz, 2H; 7.14(d), NCH,, 3H; 8.69(t), CCHs, 3H.
CH=NOCH,CH=CHX VIL
OC(O)NHCHI
VIII
H=NOCH,CH=CH2 OC (0)NHCHa
IX
(b)
u (s) = singlet, = broad.
(d)
= doublet,
(t)
= triplet,
(q)
= quartet,
(sex)
= sextuplet,
(hept)
= heptuplet,
80
LEE,
SANBORN,
AND
TABLE Compound
l-Continued M.P.
OC (0)NHCHy CH=?JOCH&H&HI
x
METCALF
(“‘C)
il-2
1.81 (s), CH=N, IH; 2.09-3.03(m), 4H ; 4.77(b), NH, 1H ; 5.90(t), 7.20(d), NCHI, 3H; 8.27(sex), 8.89-9.21 (m), CCH3, 3H.
aromatic, OCH,, 2H; CCH,C, 2H:
76-7
1.80(s), CH=N, 1H; 2.09-3.02(m), aromatic, 4H; 4.83(b), NH, 1H ; 5.54(hept), OCH, 1H ; 7.17(d), NCHz, 3H; 8.70(d), (CH&, 6H.
41-z
1.98(s), CH=N, 1H; 2.50-3.06(m), 4H; 4.90(b), NH, 1H; 5.89(t), 7.15(d), NCHI, 3H; 8.25(sex), 8.83-9.20(m), CCH,, 3H.
51-2
1.82 (s), CH=N, 1H ; 2.09-3.02 (m), aromatic, 4H ; 4.82(b), NH, 1H; 5.83(t), OCH2, 2H ; 7.17(d), NCH3, 3H; 8.01-8.81(m), (CH?),, 4H ; 8.81-9.27 (m), CHJ, 3H.
56-7
1.82(s), CH=N, 1H; 2.07-3.01(m), 4H; 4.79(b), NH, 1H; 5.75 (sex), 7.19(d), NCH,, 3H; 8.10-8.61(m), 8.73(d), (CH,), 3H; 8.89-9.23(m),
30-l
1.81 (s), CH=h’, 1H ; 2.09-3.01 (m), aromatic, 4H; 4.89(b), NH, 1H; 5.83(t), OCHz, 2H; 7.15(d), NCHa, 3H; 8.00-8.89(m), (CHz),, 6H ; 8.89-9.33 (m), CH3, 3H.
OC (0)NHCHs
3 /’
XI
CH=NOCH(CH,)z
\
OC (O)NHCHa XII
CH=NOCH,CH /
,.J CH 2
aromst,ic, OCHe, 2H; CCH&, 2H;
OC (O)NHCHs /’
CH=NOCHz(CH&CH3
XIII 3\ OC (O)NHCHa CH=NOCH
(CH&H&H,
XIV
aromatic, OCH, 1H; CHS, 2H; CHI, 3H.
OC (0)NHCHa / Xl’ c\
CH=?TOCH,(CHz),CH3 11 CO (O)NHCHa CH=NOCH?(CH,)&H3
XVI
oil
1.83(s), CH=N, 1H; 2.09-3.17(m), 4H ; 4.77(b), NH, IH ; 5.87(t), 7.17(d), NCH3, 3H; 8.00-8.93(m), 10H ; 8.93-9.33 (m), CH3, 3H.
aromatic, OCH,, 2H; (CHZ)~,
.I C (CHa)=NOCH&kCH XVII
77-8
2.46-3.00(m), aromatic, 4H ; 4.86(b), NH, 1H ; 5.25 (d), OCHz, 2H; 7.17(d), NCHz, 3H; 7.51 (t), CzCH, IH; 7.81(s), C(CHs), 3H.
OC (O)XHCHB XVIII )=NOCH
2:( =CH
65-6
2.50-3.02(m), aromatic, 4H; 4.86(b), NH, 1H; 5.23(d), OCHz, 2H; 7.15(d), NCHI, 3H; 7.53 (t,), C=CH, 1H ; 7.79(s), C(CH& 3H.
OC(O)NHCHr XIX
80-Z
2.24-3.00(m), aromatic, -I-H; 4.85(b), KH, IH ; 5.22 (d), OCHz, 2H; 7.15(d), NCHS, 3H; 7.52(t), C-CH, 1H; 7.79(s), C&Ha), 3H.
65-6
2.49-3.02(m), aromatic, 4H; 3.59-4.25(m), CH=C, 1H ; 4.50-4.94(m), ===CH,, NH, 3H ; 5.23-5.42(m), OCHZ, 2H ; 7.18(d), NCHS, 3H ; 7.80(s), C(CH& 3H.
C (CHa)=NOCH&rCH OC(O)NHCH3 C(CHS)=NOCH&H=CH~ xx
INVEd’~IGATIOXS
IIiTO
CARBAMATE
INSECTICIDE
TABLE Compound
XXII
C (CHs)=KOCH&H=CH,
II
81
I-fZ’&in~ted M.P. (“C)
UC (O)NHCH, XXI
SELECTIVITY.
NMRa(7)
ppm
6778
2.49-3.02(m), aromatic, 4H ; 3.60-4.27 (m), CH+, 1H ; 4.50-4.94(m), =CH,, NH, 3H ; 5.21-5.40(m), OCH2, 2H : 7.17(d), YCHI, 3H ; 7.78(s), C (CH& 3H.
95-6
l.iO(s), CH=N, 1H ; 2.21-2.70(m), aromah, 5H, 3.65(h), NH, 1H; 7.04(d), NCHa, 3H.
XXIII
oil
1.95(s), CH=N, 1H ; 2.33-2.83 (m), aroma&, 5H; 5.87(t.), OCHZ, 2H; 8.28 (sex), CCH&, 2H ; 8.88M.20(m), CH3, 3H.
XXIV
oil
I .91 (s), CH=N, 1H ; 2.3%2.i7(m), aromak, 5H, 5.25(d), CXH2, 2H; 7.51 (t), C=CH, 1H.
XXV
oil
9.23-2.73(m), aromatic, 5H ; 5.21 (d), OCH,, 2H; 7.52(t), C=CH, 1H; 7.75(s), CHa, 3H.
phenyl N-methylcarbamate and XIII demonstrates t,hat the latter is 5.8 X more t,oxic than the rl-butoxy carbamate. Furthermore, the ISo value for XIII is about; 0.055 X that obtained for o-nbutoxyphenyl N-methylcarbamate which correlates satisfactorily with the relative lIDSo values of the two compounds. Clearly, the insertion of HC=N between the oxygen and phenyl ring of o-n-hutoxyphenyl Nmc+hylcarbamat~r~ to give XIII increases thtl affinity of XIII for AChE. The incwasrd interaction with AChE probably plays an important role in t’he higher toxicit,y of these oximr rt’hcr carbamat,es as compared to t,heir alkoxy analogues. The most puzzling aspect of the housefly toxicity data is thtl inactivity of XVII \vhich was dcriwd from o-hydroxyacetol~hcwonc~.A suitahl(> ckxplanation can be found in the comparison of the ISo values of 1.17 X 1OP M for 11 and 97.22 X lo-” M for XVII. The decreased affinity for cholinestcrase of XVII as compared to II, along with th(l incrcasrd susccspt,ibility for tnc+abolism as mr~asur~~dby t,h(t approxinlatc4y sixfold incwaw in thr, synwgistic
value of XVII, provide plausible explanations for the differences in toxicities of II and XVII t,o the housefly. While replacing the aldehydic hydrogen with a methyl group in the ortho position greatly affected the t,oxicity, the same st’ructural modification in the rlleta position (compare III and XVIII) only reduced the housefly toxicity by about twofold. Examination of the t,oxicit,y of this series of compounds to the rest, of t.hc insects demonstrates that these compounds arc‘, on an average, about 0.81 as toxic to 11s~ and three of t’hem, IX, XII, and XVIII, are more toxic to this resistant strain of housefly. None of the compounds tested is toxic t.o the larval stages of mosquitoes at 1.0 ppm, but several of them, I, II, VII, and IX-XI, were toxic t,o the adults of thcw mosquitoes. It may br significant that one of the compounds t,hat is quite actiw against mosquito adults, I, has a very low LD60 to the white mouse of > 1,000 mg/kg. However, it would seem unlikely that this molecule could ever be utilized for rontrol of mosquito adults as the parent compound (I) and, particularly, the phenol haw a
>500.0 8.7 *x0 >500.0 > 500.0 >500.0 11.5 151.8 13.4 17.1 15.9 212.5 30.4 22.4 38.1 104.3 > 500.0 133.2 >500.0 >500.0 >500.0
0.3 4.0
0.6 7.3 1.5 1.1 1.0 13.3 1.6 1.7 4.8 5.6
10.2
f f
f f f f f f f f f f
f
Alone
SNAIDM
p.b.
(dg)
domestica
21.7 f 2.4 2.0 f 0.01 9.7 i 1.0 >500.0 6.7 i 0.9 >500.0 1.5 f 0.3 20.8 f 2.7 1.7 f 0.1 1.9 f 0.2 1.8 f 0.1 17.0 i 1.5 1.6 f 0.1 1.4 f 0.2 2.1 f 0.2 12.9 f 1.8 19.1 f 2.8 1.8 f 0.2 > 500.0 20.7 f 1.5 8.7 f 1.2
With
LD5o
Musca
>24.1 >57.5
7.7 7.3 7.9 9.0 8.8 12.5 19.0 16.0 18.1 8.1 >26.2 74.0
>74.6 -
23.0 4.3 6.0 -
SR”
(L.)
a SK. (Synergistic Ratio) = Ll)so alone/LD50 with p.b. b MSR (Mammalian Selective Ratio) = IMouse LDjO/Housefly
I II III IV V VI VII VIII IX X XI XII XIII XIV xv XVI XVII XVIII XIX xx XXI
Compound
RSP
Activity
>500.0 10.3 165.4 >500.0 >500.0 >500.0 14.4 231.9 12.9 19.9 20.4 179.1 49.4 56.1 67.4 206.6 >500.0 67.8 >500.0 >500.0 >500.0
LDsO.
Biological
zk
2
0.6 12.0
7.1
8.5 127.5 >160.0 >160.0 > 160.0 > 160.0 6.7 > 160.0 5.7 10.2 10.9 >160.0 >160.0 >160.0 >160.0 >160.0 >160.0 >160.0 >160.0 >160.0 >160.0
0.3 6.3
f f f
1.5 1.2 0.2
zt 1.0
f f
Anopheles albimanus Adults LDsa &g/cm?
of the Carbamates
rt 1.1 f 14.3 f 0.6 f 2.3 f 1.8 * 22.9 f 4.3 f 5.4 f 5.1 zk 15.9
f f
TABLE
-
57.5 > 160.0 > 160.0 > 160.0 >160.0 > 160.0 21.8 > 160.0 18.7 30.3 >160.0 >160.0 >160.0 >160.0 >160.0 >160.0 >160.0 >160.0 >160.0 > 160.0 >160.0
5.1
+ 1.3 * 3.1
rt 0.9
*
Culex pipe-n.3 quinquefasciutus Say Adults LJh (wdcm?
> 1000 -50 >500 >300 >300 >500 200-300 >500 15Ck200 300-400 400-500 >500 20-30 300-350 150-325 350-400 >500 >500 > 500 >500 >500
Mouse Oral LD60 (mg/kg)
-
-
>3.8
17.4-26.1 >3.3 11.2-14.9 17.5-23.4 25.1-31.4 >2.4 0.7-1.0 13.4-15.6 3.9-5.9 3.4-3.8 -
,-5.7 >8.b
MSRb
2 L M ;:
ks 3
Y
INVES’I~IGATIONS
very distinctive burned surgar.
INTO
and penetrating
CARBAAMATE
odor
of
Mouse Toxicity The purpose of this investigation and the previous work (1) was the preparation of carbamatc insecticides that were more toxic to the housefly than to the femaIe white mouse. In the earlier investigation, it was demonstrated that the incorporation of potenbial selectophorcs such as an ester, amide, or a nitrile did not enhance the dcsircd selectivity appreciably. Further, it was pointed out that the bis-carbamate of salicylaldoxime had a very unfavorable mammalian selectivity ratio of CO.03 and an LDju to the mouse of lo-20 mgikg. The conclusions drawn from that work indicatcd that, in addition to a selcctophore, a delay factor as is present in the phosphorothionate insecticides, such as malathion, is requisite for selectivity between insects and mammals in t’he carbamate insecticides. The data outlined in the present work appears to confirm the original thesis t.hat, if part,icular functional moieties, in this that if particular functional moieties, in this case the alkoxyiminomethyl group (HC=N-OR) where R = alkyl group are incorporated into carbamat,e insecticides, then more favorable MSR ratios can be obtained. The explanation for the very unfavorable mouse LD50 of the salicylaldoxime his-carbamatc provided a useful lead for the preparation of carbamates with desirable MSR values. The rationalization for the high toxicity of this bis-carbamate to the mouse involved implication of a facile conversion of the o-N-methylcarbamoyloxime into a cyano group in the acid stomach of the mouse. This highly toxic carbamate provided the impetus for substitution of the carbamoyi moiety by an alkyl group which would, on tho basis of physical organic chemistry, not rearrange as rapidly to the nitrile as the carbamoyl derivatized species. In an effort to investigate the relative rates of acid catalyzed rearrangement of an oxime
INSECTICIDE
SELECTIVITY.
II
83
ether and a N-methylcarbamoxyloxime ester, the oximc of benzaldehyde was reacted with propyl bromide under basic conditions to give XXIII and with methyl isocyanate to give XXII. Ultraviolet spectroscopic study indicated that the treatment of XXII with cthanolic hydrochloric acid (0.2 N) at 25% leads to the disappearance of the parent compound (X = 256 rnp) to give benzonitrile (A = 222 rnp) and others with a pseudo first order rate constant k$ = 0.0092 hour-’ and half-life of 75 hr over a period of 120 hr. The benzonitrile constituted approximately 25% of the transformed parent compound under the condition described. The spectrum of XXIII under the same conditions does not change over a time period of 250 hr. Thercfore, if the rates of acid and biochemically catalized Beckmann rearrangements of derivatized oximes can be related, and, if selectivity between mice and insects is a function of the rate rearrangement of a derivatized oxime to the o-cyanophenyl carbamate, then the compounds of this series which have MSR values 100-1,000X greater than the bis-carbamate of salicylaldoxime are selective because of the greatly reduced rate of conversion of the oxime into the nitrile in the stomach of the mouse. Currently, efforts are being directed towards confirmation of this hypothesis by the synthesis of 14C-labeled material and evaluation of the metabolic pathways of the bis-carbamate of salicylaldoxime and one of the (o-alkoxyiminomethyl)phenyl Nmethylcarbamates of this series. One final aspect of the mouse toxicity data is the greatly reduced toxicity of I, the N-acylated derivative of o-cyanophenyl N-methylcarbamate. The carbamate without the N-acyl group had an oral LD,, to mice of about 225-250 mg/kg (1). The toxicity to the white mouse of I of >l,OOO mg/kg appears to confirm the conclusion of the initial work on selective carbamates that both a selectophore, the cyano functional group, and a delay factor, the N-acetyl group are requisite for reduced
LEE,
84
HANBORN,
Anticholincsteras~
AND
Activity of thy Carbamatcs ~__ .-.. ~~~
Compound
ESW
IjO Housefly (IOF
II III IV v VI VII VIII IX x XI XII XIII XIV XV XVI XVII XVIII XIX xx
XXI (Enzyme
head AChEJ Ill)
Bovine
_--7348.41 f 524.25 1.17 f 0.18 144.13 + 12.86 84.27 f 9.29 2.87 f 0.23 114.83 f 7.33 0.97 It 0.08 159.63 f 10.76 0.84 + 0.07 1.26 + 0.09 0.50 f 0.05 136.20 f 10.38 0.66 f 0.06 O..i3 f 0.07 0.29 zt 0.03 0.15 f 0.03 97.22 It 1.69 110.30 f 1.52 152.96 f 14.06 156.39 f 15.21 105.42 f 3.09 ~- .~.
I
” ESR
METCALF
Selectivity
Ratio)
= 150 bovine/Isa
toxicity to the white mouse. The presence of the N-acetyl group probably allows metabolism of the cyano group to polar product’s before the loss of the acetyl group to yield an active toxic anticholin&erasc I?-met.hylcarbamate. Antich.olilzesterase Activity In Table 3 the Is0 values arc collected for inhibition at 25°C of purified housefly head aceytlcholinesterasc and bovine erythrocytc cholinesterase. Again, the generally more efficient inhibition of housefly cholinesterasc than bovine cholinesterase is demonstrated by the enzyme selectivity ratios greater than one. Except for I, which has an ESR value of 0.04, the values range from one for XIX to 2,556 for XV. Attempts to provide a rationale for reduced mouse toxicity as compared to insect using ESR and MSR values were yuit,e unsuccessful. Perhaps t,h(i most, interesting aspect of th(b cholirr-
erythrocyte AChE (10-h M)
3.15
f
0.25
1.31
*
4.12 2.61 1.17 2.72 2.88 5.42 1.27 0.77 3.62 7.57 2.69 4.68 5.88 3.75 4.66 2.66 1.54 4.06 4.24
f f f f f xt + f f f zk f f It f f f f + --
0.14 0.54 0.19 0.08 0.12 0.11 0.11 0.22 0.10 0.44 0.18 0.26 0.60 0.69 0.13 0.38 0.29 0.16 0.14 0.41 ~ __-.
0.04 112.0 2.9 3.1 40.8 ‘2.4 296.9 3.4 151.2 61.1 1124.0
5.6 407.6 883.0 2.556.5 2500.0 4.8 2.4 1.0
2.6 4.0 ____.....~~~_~__~~
housefly.
esterase data was the much greater srnsitivity of housefly head AChE to ortho versus meta or para substitution of the hydroxybenzaldehyde derivatives as compared to bovine crythrocyte AChE. For example, the Ijo values against housefly AChE of II and III differ by 115 X and yet, against bovine erythrocyte AChE, these two compounds differ by only 3.14 X. Comparison of VII and VIII and X and XII yields similar conclusions as to the effect of ring substitution on housefly head AChE inhibition. However, for the compounds derived from hydroxy acetophenonn (XVII-XXI), the 150values for inhibition of housefly head AChE do not change as dramatically (compare XVII and XIX, compare XX and XXI) as those derived from the hydroxy benzaldehydes and arc’ similar to the changes in 150values against bovinc AChE. Perhaps compounds with 15” valucls that, are in the 10e4 M range
INVESTIGATIONS
(l~vII-xxI)
are
just
INTO
not
as
WnSitiW
CARBAMATE
t0
structural changw as those with lo~wr ISo values. The dat,a for the bovine AChE certainly support this conclusion as well as those derived from substituted hydroxy acetophenones.
From the data presented in this paper it is clear t,hat the alkoxyiminomethyl moietJJ / (HC-?J-OR) when incorporated into a phenyl carhamat,c can provide selective biological act’ivity between insects and mammals. The rationale for the synthesis of this series was provided by the outst,anding rodenticidal activity of b&N-methylcarbamoyl salicylaldoxime where t)hc toxicity was explained by implicating a Beckmann rearrangement to the nitrile that took place more rapidly in the mouse than in the ins&. Therefore, by replacement of the N-me?thylcarhamoyl oximtb with a linear or branched alcohol, it was hoped that, the rat,c of rcarrangemcnt, would be reduced and mammalian selcrtivr ratio increased. The substitut,ion of the carbamoyl group with an alkoxy group clearly increased the selectivity as the compounds of this series are 100-1,000 X nl(Jw selective than the his salicylaldoximc carbamate. Perhaps other functional groups can be found that will increase the unfavorable nlSR values of carbamatcx insecticides. The functional groups which seem to provide selectivity for the phosphorothionat(~s, ester, nit,rile, alld amide arc’ IlOt effective for carbamates, but :I somcx\lhat, surprising moichy, the, alkoxyiminomr~thpl (HC-NOR), to provide the desired selectivity.
is able
INSEC’IICIDE
II
85
3. hr. A. H. Fahmy, T. R. Fukuto, 1~. 0. Meyers, and It. B. ;Cfarch, The selective toxicity of new .\:-phosphorylt,hioylcarbamate esters, J. Agr. Food Chent. 18, 793 (1970). 4. A. L. Black, Yi-chang Chiu, RI. A. H. Fahmy, and T. It. Fukuto, Selective t,oxicity of :V-sulfenylated derivatives of insecticideal methylcarbamate est,ers, J. izgr. Food Chrm. 21, 747 (1973). 5. h\lohamed A. H. Fahmy, It. L. Metcalf, T. R. Fukuto, and I>. J. Hennessy, Effects of deuteration, fluorination and other struct,ural modifications on the carbamoyl moiety upon t.he anticholinesterase and insecticidal activit.ies of phenyl S-methylcarbamates, J. Agr. Food Chm. 14, 79 (1966). 6. 11. P. Miskus, T. L. Andrews, and &‘I. L. Look, hletaboliu pathways affecting toxicity of :V-acetyl Zectran, J. Agr. Food Chew 17, 842 (1969). 7. Brit. Patent 889,086 (Chemical Abstracts 57 : 728Sd-7288e). 8. R. L. Metcalf, I. P. Kapoor, and A. S. Hirwe, Biodegradable analogues of I>1 IT, BuU. JFor/d Hdth Org. 44, 363 (1971). 9. W. N. Aldridge, Some properties of specific cholinesterase with particular reference to t,he mechanism of inhibition by diethyl p-nitrophenyl thiophosphate (E60.i) and analogues, Biochem. J. 46, 461 (1930). 10. An-horng Lee and R. I,. Metcalf, In vifro inhibition of acetylcholinesterase by O,O-dimethyl S-aryl phosphorothioates, Pest. RioChern. Physiol. 2, 408 (1973). 11. 11. L. AIetcalf, The role of oxidative reactions in the mode of a&on of insecticides, in “Enzymatic oxidation of toxicants,” 0;. Hodgson, Ed.), p. 151, North Carolina State University, Raleigh, N. C. 1968. 12. J. IX. C&da, nlixed Function oxidase involvement, in the biochemistry of insecticide aynergists, J. Agr. Food Chem. 18, 753 (1970). 1:3. 11. 31. Sacher, It. L. Metcalf, and T. R. Fukuto, Propynyl napthyl et,hers a selective carbamate synergista, J. Agr. Food Chwn. 16,
i79 (1968). 14. 1). J. Henmessy, synergists chemical O’Brien Academic
REFERE?;CES
li. Sanborn, A. Lee and It. L. hfetcalf, into carbamate insecticide Investigations selectivity. I. Evaluation of potential selectophores. Pest.Biochem. Physiol. 4, 67-76. 2. J. Fraser, P. (:. Clinch, and R. C. Reay, ;V-acylation of l\rmethylcarbamate insecticides and it,s effect on biological activity, J. Sci. Food Agr. 16, 615 (1965).
SELECTIVITY.
1. d.
Id.
The potential of carbamate as pest control agents, in “BioToxicology of Insecticides,” (Et. I). and I. Yamamoto, Ed.), p. 105, Press, New York (1970).
K. L. >Ietcalf, T. R. Fukuto, and M. Y. Winton, Alkoxyphenyl ‘V-methylcarbamates as insect,icides, J. Econ. Ent. 53, 828 (1960). 16. It. L. AIetcalf, T. R. Fukuto, and M. Y. Winton, The synergism of substituted phenyl Nmethylcarbamates by piperonyl butoxide, J. Econ.. Ent. 55, 341 (1962).