OStudies on organophosphorus insecticides 3. On the properties of soma alpha-hydroxy phosphonates and related compounds.

Full text of DOI:10.1515/znb-1968-1116

Studies on Organophosphorus Insecticides III
On the Properties of Some a-Hydroxy Phosphonates and Related Compounds
S. M. A. D. Z a y e d , A. H a s s a n , and I. M. I. F a k h r
National Research Centre, Dokki, Cairo and Department of Biology,
Atomic Energy Establishment, Cairo
(Z. Naturforschg. 23 b, 1517— 1521 [1968] ; eingegangen am 20. Mai 1968)
In search for effective organophosphorus esters against the larva of the cotton leaf worm
(Prodenia litura F), some a-hydroxy phosphonic acid dimethyl esters and related compounds have
been synthesized. The effect of substitution on the antiesterase activity and toxicity towards
Prodenia larvae has been investigated.
The infra-red spectra of the prepared phosphorus esters are discussed.
of sodium methoxide as a catalyst.
Recently, it has been reported that 0,0-dimethyl-
2,2,2-trichloro-l-methoxy ethylphosphonate
( l b )
HsCO
0
R
possesses almost the same toxicity as Dipterex ( l a)
towards the larvae of the cotton leaf worm Prodenia
litura F. Moreover, lb proved to be more stable than
Dipterex (0,0-dimethyl-2,2,2-trichloro-l -hydroxy
ethylphosphonate) and possesses lower anticholin-
esterase activity1. In search for organophosphorus
esters with low anticholinesterase activity but
\ll I
P - C - R '
/ I
H3CO
OH
2a, R = 1I; R' = CH3
b, R = H; R' = C6H4Cl-o
c, R = H; R' = C6H4Cl-p
d, R = H; R' = C6H4Br-p
e, R = H; R' = C6H4N02-p
f, R = H; R' = C6H3Cl-2, N02-5
g, R = R' = CHS
HsCO
O
\!l
P —CH —CC13
/
I
H3CO
OR
h 3co
0
CH3
\ll I
HsCO H-N-R
3 a, R = C4H9-n.
b, R = CH2C6H5.
la , R = H
P -C -C H g
b, R = CH3
c, R = CONHC2H5
/ I
are at the same time effective against Prodenia
larvae, studies were continued in the series of a-
hydroxy phosphonates and related compounds.
For this purpose the a-hydroxyphosphonic acid
dimethyl esters 2a_g , the ^-substituted a-amino-
phosphonates 3a_b and the carbamate l c have been
prepared. Their infra-red spectra as well as their
antiesterase activity were studied.
Some aromatic a-hydroxy phosphonates, e. g.,
2 b_d could be prepared directly from the aldehyde,
phosphorus trichloride and methanol by a single
vessel reaction in a manner similar to that used for
the preparation of Dipterex 1.
The a-hydroxy phosphonates are quite stable
towards dilute mineral acids. They suffer only a
Of the a-hydroxy phosphonates 2a2, 2e3, and
2g2 have been previously described and are listed
here for comparison. 2a_g are readily obtained by
condensation of dimethylhydrogen phosphite with
the appropriate aldehyde (or ketone) in the presence
slight decomposition when heated with
n
hydro-
chloric acid. Using paperchromatographic analysis,
monomethyl- and dimethyl phosphate could be
identified as decomposition products. This is in line
1 S. M. A. D. Zayed, A. Hassan, and I. M. I. Fakhr, Z. Natur-
forschg. 20 b, 786 [1965].
3 V. S. Abramov, Zhur Obschtschei Khim. 27, 169 [1957] ;
C. A. 51,12878 [1957].
2 V. S. Abramov and L. P. Semenova, Sbornik Statei Ob-
schtschei. Khim. Akad. Nauk S.S.S.R. 1, 393 [1953] ; C. A.
49, 838 [1955].
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A
similar internal bonding may occur also in a-
with previous findings, that most organophosphorus
esters are fairly stable in acid medium 4.
amino phosphonates. In dilute chloroform solution,
3a does not show a free N-H absorption. A broad
absorption band at 2740 cm-1 may be attributed
to a strongly chelated NH group. 2f_g and 3a show
absorption bands in the regions 1265+ 5 cm-1 and
at 1212+ 8 cm-1 presumably due to free and
associated P = 0 group, respectively; the former
band being near the usual phosphonate absorption
region12. In nujol the band for the associated
(P—>0) is the dominent one.
In alkaline medium, 2a_ g are rapidly decomposed.
When treated with cold
n sodium hydroxide, the
a-hydroxy phosphonates readily liberate the original
aldehyde or ketone, which could be identified as its
2,4-dinitrophenylhydrazone.
The a-amino phosphonates 3a_b, were obtained
by the addition of dimethyl hydrogen phosphite to
the S c h i f f ’ s bases acetone-n-butylimine and
acetone-benzylimine, respectively 5’ 6’ 7. These com-
pounds are fairly stable towards mineral acids or
alkalies and undergo only slight degradation when
heated with these reagents. 0,0-Dimethyl-2,2,2-tri-
chloro-1 (V-ethyl) carbamoyl ethylphosphonate ( l c)
was readily obtained in good yield by condensation
of Dipterex with ethylisocyanate in presence of
triethyl amine. l c has been previously prepared by
using dibutyltin dilaurate as a catalyst8. It is easily
hydrolyzed by acids and alkalies. Cautious hydro-
In chloroform, l c shows an NH-absorption around
3400 cm-1. The streching frequency of the free
P = 0 group appears as a sharp band at 1250 cm-1.
In the region of carbonyl absorption l c shows a
strong band at 1750 cm-1. This is in accordance
with the usual carbonyl absorption of the carbamate
ester group13. The strong absorption at 1290 cm-1
may be attributed, as in esters14, to the C —0
stretching vibration.
lysis with cold
formation of l a.
n
hydrochloric acid leads to the
Anti-esterase activity and Toxicity
The prepared organophosphates were tested for
their anticholinesterase activity in an effort to corre-
late the findings with the toxicological effects. Fig. 1
and table 1 clearly demonstrate that non of the
prepared compounds is active as the parent sub-
stance Dipterex. Aromatic or aliphatic substitution
of the trichloro radical results in a decreased activity.
Also, the replacement of the hydroxy group by a
substituted amine does not seem to provide any
increase in activity. However, the introduction of
a carbamoyl group to Dipterex seems to reduce its
anticholinesterase property only slightly. It is known
that the reaction of organophosphates with cholin-
esterase involves a binding (or affinity) constant
and a phosphorylation constant15. Since the phos-
phorus moiety of all the compounds tested is the
In chloroform solution, the infra-red spectra of
the a-hydroxy phosphonates 2b _d and 2 f_ g show a
strong absorption in the form of a broadend band
in the range of 3265+ 15 cm-1. Such an associated
OH group has been observed in the case of Dipterex
( l a) 9,10. In very dilute solution the spectra show,
in addition to the bonded OH-absorption, another
band of lower intensity around 3530+ 10cm_1 due
to a free OH-valency vibration. The results strongly
suggest the presence of an intramolecular type of
hydrogen bonding. Such internal bonding was re-
ported to occur in certain a-hydroxy phosphonates
(4 )n , where R can be aromatic or aliphatic.
/ \
0
0
R'0\ II
I
/ H3CO\ 9
same I
\
)P ------CH— R
R'CK
_
I , it is believed that it is the
4
\ H3COX
I
4 R. Mühlmann and G. Schrader, Z. Naturforschg, 12b, 196
[1957].
10 W. Lorenz, A. Henglein, and G. Schrader, J. Amer. chem.
Soc. 77, 2554 [1955].
5 A. N. Pudovik, Doklady Akad. Nauk S.S.S.R. 83, 865
[1952]; C. A. 47,4300 [1953].
11 C. D. M ille r, R. C. M ille r, and W. Rogers, jr., J. Amer.
chem. Soc. 80,1562 [1958].
6 A. N. Pudovik and M . V. Korchemkina, Izvest. Akad. Nauk
S.S.S.R., Otdel Khim. Nauk 940 [1952]; C. A. 47, 10468
[1953].
7 E. K. Field, J. Amer. chem. Soc. 74, 1528 [1952].
8 G. K. Kohn, U. S. 3, 069, 312 [1962]; C. A. 59, 666
[1963].
12 H. I. Jacobson, M . J. G riffin , S. Preis, and E. V. Jensen,
J. Amer. chem. Soc. 79, 2608 [1957].
13 N. Shachat and J. J. B agn ell, jr., J. org. Chemistry 28,
991 [1963].
14 L. J. Bellam y, The Infra-red Spectra of Complex mole-
cules, 2nd edition, John Wiley
1959, p. 188.
& Sons, INC. New York
9 W. F. B arth el, B. H. Alexander, P. A. Giang, and S. A .
H a ll, J. Amer. diem. Soc. 77, 2424 [1955].
15 A. R. Main and F. Iverson, Biochem. J. 100, 525 [1966].
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Fig. 1. Inhibition of acetylcholinesterase.
Inhibitor concentration [1] expressed as
Moles/liter.
"3C0^0
p -c h c c i
3
h3co/
oconhc hs
2
^
20
0
3:
§
o
1
Ul
Ct
o
S
100
o
80
o!
log[I ]
equal to or exceeds the rate of phosphorylation of
the enzyme.
Percentage Inhibition*
Compound Rat-brain
Acetyl-
Rat-liver
Aliesterase
The toxicological results show no definite pattern,
so far as substitution is concerned (Table 2), and
again Dipterex seems to be the best organophos-
Cholinesterase
100
80.0
26.0
50.0
12.5
22.5
10.6
11.0
13.0
0.0
la
lb
lc
2a
2b
2C
2d
2e
2f
40.0
94.5
55.1
27.3
23.4
18.0
49.4
39.3
27.3
27.6
23.5
Mortality*
Compound Dose [>g]
20
20
20
20
36
36
20
36
72
36
36
36
75
75
12.5
10
5
10
10
15
5
la
lb
lc
2a
2b
2c
2d
22.0
19.8
23.0
2g
3a
3b
2e
2f
Table 1. Anti-esterase activity of the prepared a-hydroxy- and
a-amino phosphonate derivatives. Inhibitor concentration
12
8
10
2g
3a
3b
*
I
O
“
* m .
affinity constant (£ a) rather than the phosphory-
lation constant (£ p) which determines the degree of
inhibition (as influenced by the “ leaving” group).
Table 2. Toxicity of the investigated a-hydroxy- and a-amino
phosphonate derivatives. * Results are mean of three different
experiments.
The inhibition of rat liver aliesterase by these
compounds shows again the same general trend
(Table 1). An important feature of this inhibition
phonate. Of the parameters investigated to asses
the potency of the prepared organophosphonates,
the anticholinesterase activity is the most important.
is that it is not progressive, i. e., the inhibitor com- Recently, cholinesterase has been reported to be
present in the nerve cord of the Prodenia larva, and
bines reversibly with the enzyme. In this case, it
may be assumed that the dephosphorylation rate is
evidence has been introduced that the organophos-
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Action of hydrochloric acid on 2a-g
phonate Dipterex is incapable of penetrating to the
target enzyme, through the nerve sheath 16. It seems,,
therefore, feasible to assume that the toxic effect of
the organophosphonates is primarily due to in-
hibition of the hemolymph esterases (and probably
also to non-specific inhibition of other enzyme
systems) and that cholinesterase is not involved.
It also explains the low toxicity of Dipterex itself 16
as well as its derivatives. This assumption is strongly
supported by the fact that 0,0-dimethyl-2,2,2-tri-
chloro-l-methoxyethyl phosphonate (lb) -though
possessing much lower anticholinesterase activity
than Dipterexshowed the same toxicity as the parent
compound1. In fact, it was the latter observation
that stimulated the search for compounds of the type
described in this paper.
50 mg. of the compound were heated with 5 ml. 1 N
hydrochloric acid for 30 minutes at 100°. After re-
moval of the unchanged material with chloroform, the
aqueous solution was paper chromatographed on
Schleicher and Schüll 2043 b using isopropanol-water-
ammonia (75:24:l ) 17 and isopropanol-ammonia (75:
25)17. The spots were made visible by spraying with
H a n e s and Is h e r w o o d reagent18 using U.V. light as a
reducing agent. Monomethyl- and dimethyl phosphates
could be identified as decomposition products (Rf of
monomethyl phosphate 0.11 and 0.05; Rf of dimethyl
phosphate 0.6 and 0.50 in the first and second systems,
respectively). The recovered unchanged substance
ranged from 60 —80 per cent.
Action of dilute sodium hydroxide on 2a-g
50 mg. of the compound were treated with 10 ml.
1 N sodium hydroxide solution. The parent carbonyl
compound was readily liberated. After 2 minutes the
solution was acidified with dilute hydrochloric acid and
the separated carbonyl compound was identified as its
2,4-dinitrophenyl hydrazone.
Experimental
Melting points are uncorrected. The infra-red spec-
tra have been carried out on UR 10, Zeiss, Jena, Infra-
red spectrophotometer.
Preparation of the aminophosphonates 3a-b
Preparation of the a-hydroxy phosphonates 2
A
mixture of 0.1 mole acetone, 0.12 mole of the
appropriate amine (n-butylamine or benzylamine) and
2 g. anhydrous potassium carbonate was heated on a
water bath for 30 minutes. Benzene was then added
General procedure
Freshly distilled dimethyl hydrogen phosphite (0.1
mole) was mixed with an equivalent amount of the ap- and the organic layer rapidly separated, dried over
propriate aldehyde or ketone. 1N sodium methoxide
solution (0.5 ml.) was added and the mixture was
shaken for 10 minutes. After the originaly evolved heat
had ceased, the reaction mixture was cooled and the
product collected. It was then purified by crystalliza-
tion from the appropriate solvent. The analytical data
of the new a-hydroxy phosphonates are listed in
table 3.
potassium hydroxide pellets and filtered. The imine
obtained after removal of benzene under reduced pres-
sure, was used directly for the preparation of the
amino phosphonate derivatives.
Acetone-butylimine (0.05 mole) was mixed with the
equivalent amount of dimethyl hydrogen phosphite
while shaking. The reaction mixture was allowed to
stand till no more heat was evolved. Excess dimethyl
Compd.
m. p.°
*
Formula
[Mol.Wt.]
Analysis
H
Halogen
C
N
P
__
—
—
Calcd.
Found
8 8 -9
a, c
43.13
43.21
4.82
4.69
14.14
14.18
12.36
12.21
c 9h 12c i-o 4p
(250.635)
2b
2C
2d
2f
8 0 -1
b
Calcd.
Found
43.13
42.98
4.82
4.71
14.14
13.98
12.36
12.29
C9H 12CIO4P
(250.635)
—
—
—
8 7 -8
a
Calcd.
Found
36.63
36.70
4.09
3.97
27.08
26.98
10.49
10.36
C9H i2Br04P
(295.095)
154-5
a, b
Calcd.
Found
36.56
—
3.75
—
11.99
11.78
4.73
4.86
10.47
10.38
C9H n C lN 06P
(295.635)
Table 3. Analytical data of the a-hydroxy phosphonates 2. * Solvent of crystalization. a) Ether, b) Benzene, c) Benzene-Pet.
ether (b.p. 40—60°).
16 S. M. A. D. Z a y e d and A. H assan, Z . angew. Entomol., in
18 C. S. H an es and F. A. Is h e rw o o d , Nature [London] 164,
press.
1107 [1949],
17 F. W. P la p p and J. E. C a sid a , Analytic. Chem. 30, 1622
[1958].
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Action of hydrochloric acid on l c
hydrogen phosphite was then removed under vacuum
(in vacuum desiccator for 4 days) ; whereupon the
oily product solidifies.
50
mg. of the carbamate was treated with 10 ml. 1 N
hydrochlorid acid and the mixture was left for one
hour at room temperature. The mixture was then ex-
tracted with chloroform and the chloroform layer was
analyzed by paper chromatography in isopropanol-
water-ammonia (75:24:l ) 17, and n-butanol-pyridine-
water (12:8:6)19. The chromatograms showed the pre-
3a gave colourless crystals from methyl alcohol —
ether mixture, m.p. 205 —206°; yield 70 per cent.
Analysis
Calcd. for C9H22N03P (223.26)
C 48.41 H 9.93 N 6.27 P 13.87,
Found: C 48.45 H 9.89 N 6.25 P 13.81.
sence of Dipterex alone (Rf
= 0.79 and 0.95 in the
first and second systems, respectively).
3b gave colourless crystals from a mixture of ethanol
and ether, m.p. 212 —213°; yield 80 per cent.
Determination of Acetylcholinesterase activity
Analysis
Calcd. for C12H20NO3P (257.28)
C 56.02 H 7.83 N 5.44 P 12.04,
Found: C 56.10 H 7.81 N 5.39 P 12.11.
The anticholinesterase activity has been determined
according to the method of H e s t r in 20. A 10% rat-brain
homogenate was used as a source of acetylcholin-
esterase. The reaction mixture had the following com-
position: 0.9 ml. 0.2 m. phsphate buffer pH 7.2; 0.3 ml.
1.0 m. magnesium chloride solution; 0.3 ml. 1.0 m. so-
dium chloride solution; 0.5 ml. 10% rat-brain homo-
genate in isotonic potassium chloride; 0.5 ml. distilled
water or variable concentrations of the tested compound
(inhibitor) in distilled water (final concentration
10~2m. — 10~7m .) . The reaction mixture was incubated
for 30 minutes at 37°. 0.5 ml. 48 millimolar acetyl-
choline chloride in 0.001 m. sodium acetate (final con-
centration 8 millimolar) was then added to stop the
E-I reaction and the residual acetylcholine was assayed
after 30 minutes.
50 mg. of 3a were heated with 1N hydrochloric
acid as previously described. It proved to be fairly
stable.
3a (50 mg.) was heated in 1N sodium hydroxide
solution for 20 minutes at 100°. The cooled mixture
was acidified with dilute hydrochloric acid and ana-
lyzed by paper chromatography in isopropanol-water-
ammonia (75:24:1)17. In addition to the unchanged
material (R f
=
0.75), smaller spots appeared with
i?/-values 0.1 and 0.58 due to monomethyl- and di-
methyl phosphate, respectively. The percentage of de-
composition was less than 30 per cent.
0,0-Dimethyl-2,2,2-trichloro-l -( N-ethyl) carbamoyl
Determination of Aliesterase activity
ethyl phosphonate l c
Rat-liver homogenate in isotonic KC1 (10%) served
as the enzyme source. The enzyme was assayed titri-
metrically, according to the procedure of H a r r e r and
King 21; using a freshly prepared saturated solution
of ethyl butyrate as a substrate. The organophosphate
was preincubated with the enzyme for 30 minutes, fol-
lowed by the addition of the substrate. In some ex-
periments the inhibitor and the substrate were in-
cubated simulatneously with the liver homogenate.
To a solution of Dipterex (0.01 mole) in dry ben-
zene (20 ml.), ethyl isocyanate (0.015 mole) was ad-
ded followed by addition of 0.5 ml. of triethyl amine.
The reaction mixture was refluxed for five hours and
then cooled. After acidification with 1 N hydrochloric
acid, the product was extracted with benzene. The
solvent was removed in a vacuum and the residue cry-
stallized from benzene-petroleum ether (b.p. 40 —50°).
l c formed colourless needles, m.p. 96 —97°, Lit. m.p.
97 —98°)8.
Toxicological studies
Analysis
Calcd. for C7H13C13N05P (328.55)
C 25.59 H 3.98 Cl 32.37 N 4.26 P 9.43,
Found: C 25.61 H 4.00 Cl 32.21 N 4.16 P 9.28.
For the evaluation of the prepared organophospho-
nates as to their effectiveness against Prodenia larvae,
the third larval instar was treated topically with the
organophosphonate in acetone. Each larva received
1 (A. acetone containing the desired amount of the
toxicant. Control larvae received acetone alone. Fifty
larvae were used for each determination and mortality
counts were taken 24 hours after treatment.
19 S. M. A. D. Zayed and A. Hassan, Canad. J. Biochem. Phy-
siol. 43, 1257 [1965].
20 S. Hestrin, J. biol. Chemistry 180, 249 [1949].
21 C. J. H a rre r and C. G. King, J. biol. Chemistry 138, 111
[1941].
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