350 Chem. Res. Toxicol.

2003, 16, 350-356

Major Intermediates in Organophosphate Synthesis

(PCl3, POCl3, PSCl3, and Their Diethyl Esters) Are
Anticholinesterase Agents Directly or on Activation
Yoffi Segall,† Gary B. Quistad, Susan E. Sparks, and John E. Casida*
Environmental Chemistry and Toxicology Laboratory, Department of Environmental Science,
Policy and Management, University of California, Berkeley, California 94720-3112

Received October 17, 2002

Three phosphotrichlorides [phosphorus trichloride (PCl3), phosphorus oxychloride (POCl3),

and thiophosphoryl chloride (PSCl3)] with an annual U.S. production of >500 000 000 pounds
and their diethyl esters are intermediates in the production of organophosphorus pesticides,
plastics, flame retardants, and hydraulic fluids. They are classified as highly toxic to mammals
based on acute oral and inhalation data with rats. This study considers their mechanisms of
toxicity. PCl3 and POCl3 inhibit acetylcholinesterase (AChE) and butyrylcholinesterase (BChE)
from several species with in vitro IC50 values of 5-36 and 88-1200 µM, respectively; PSCl3 is
a less potent inhibitor. These phosphotrichlorides have high vapor toxicity to houseflies with
in vivo inhibition of brain AChE activity correlating with mortality. PCl3 and POCl3 produce
cholinergic poisoning signs on ip administration to mice, and all three phosphotrichlorides
give marked in vivo inhibition of serum BChE but not brain AChE activity. PCl3 is a direct
acting AChE inhibitor. Our earlier proposed activation of POCl3 is confirmed here by preparing
pure Cl2P(O)OH and its potassium and dicyclohexylamine salts that reproduce the action of
POCl3 as in vitro AChE inhibitors and toxicants in mice. PSCl3 on hydrolysis yields Cl2P(O)-
SH [which oxidizes with peracid to Cl2P(O)SOH] as the proposed activation product. Vapors
of (EtO)2PCl, (EtO)2P(O)Cl, and (EtO)2P(S)Cl are lethal to houseflies as in vivo AChE inhibitors,
the first two acting directly and the last one on oxidative activation to (EtO)2P(O)Cl (possibly
by P450) or (EtO)2P(O)SCl (a phosphorylating agent in a peracid oxidation system). Thus PCl3,
(EtO)2PCl, and (EtO)2P(O)Cl act directly as AChE inhibitors whereas POCl3 and PSCl3 undergo
hydrolytic activation and (EtO)2P(S)Cl undergoes oxidative activation. In contrast, the toxicity
to mice of phosphofluorides [FP(O)Cl2, F(Cl)P(O)OH, and F2P(O)OH; studied as model
compounds for comparison] may be due to liberating fluoride ion.

Introduction Table 1. Phosphotrichloride and

Diethoxyphosphochloride Toxicity to Mammals
The OP1 compounds of greatest toxicological concern
oral LD50 (mg/kg) inhalation LC50
are the insecticides and chemical warfare agents, which
compd hazard classa rat mouse (ppm 4 h, rat)
are esters acting as AChE inhibitors. Much less attention
has been given to intermediates for manufacturing OP PCl3 HT 550b 104b
pesticides, plastics, flame retardants, and hydraulic fluids POCl3 HT 380b 48c
PSCl3 HT 660d, 750e 700d 20d
including PCl3, POCl3, PSCl3, and their diethyl esters. (EtO)2PCl COR, FL
More than 500 million pounds of PCl3 (420 million (EtO)2P(O)Cl HT 11b 100d
pounds) (1), POCl3, and PSCl3 are produced annually in (EtO)2P(S)Cl HT 1000b 800d 20b
the U.S. Diethyl ester derivatives of these phospho- a Ref 2: HT, highly toxicsoral LD
50 rat < 50 mg/kg or LC50 4
trichlorides are synthetic intermediates for pesticides h inhalation < 200 ppm; COR, corrosive; FL, flammable liquid.
b Ref 4. c Ref 1. d Aldrich MSDS. e Ref 5.
[(EtO)2P(O)Cl and (EtO)2P(S)Cl] or versatile phosphory-
lating agents [(EtO)2PCl] (2, 3).
The three phosphotrichlorides and two of their diethyl to PCl3 causes corrosive damage to the skin and mucous
esters are categorized as highly toxic to mammals both membranes and reduced pulmonary function (1, 6). The
orally and by inhalation (Table 1), and the third diethyl phosphotrichlorides and diethyl phosphochloridates might
ester is also toxic as considered later. Human exposure act at AChE or some other target and either directly or
indirectly following hydrolytic or oxidative activation,
* To whom correspondence should be addressed. Tel: 510-642-5424.
Fax: 510-642-6497. E-mail: ectl@nature.berkeley.edu.
e.g., POCl3 is converted to Cl2P(O)OH, which, although
† Present address: Israel Institute for Biological Research, P.O. Box not isolated pure, is proposed to be the AChE inhibitor
19, Ness-Ziona 74100, Israel. (7). The present study considers the chemistry and
1 Abbreviations: AChE, acetylcholinesterase; BChE, butyrylcho-

linesterase; Bu4N+F-, tetra-n-butylammonium fluoride; ChE, cho- toxicology of PCl3, POCl3, PSCl3, their diethyl esters, and
linesterase; DCHA, dicyclohexylamine; IC50, concentration for 50% the corresponding hydrolysis and oxidation products
inhibition; KD, knockdown; MCPBA, m-chloroperoxybenzoic acid; OP,
organophosphorus; PB, piperonyl butoxide; TEPP, [(EtO)2P(O)]2O, (Figure 1) as AChE inhibitors and toxicants in houseflies
tetraethyl pyrophosphate. and mice.
10.1021/tx020094l CCC: $25.00 © 2003 American Chemical Society
Published on Web 02/08/2003
Organophosphate Anticholinesterase Agents
Figure 1. Hydrolytic and oxidative reactions of PCl3, POCl3,
PSCl3, and their diethyl esters. The proposed ultimate AChE
inhibitors shown in rectangles are the parent PCl3, (EtO)2PCl
and (EtO)2P(O)Cl, hydrolysis products Cl2P(O)OH and Cl2P(O)-
SH, and oxidation products (EtO)2P(O)Cl and possibly (EtO)2P-
(O)SCl. The major pathway with (EtO)2P(S)Cl to (EtO)2P(O)SCl
or (EtO)2P(O)Cl may depend on the oxidant and medium, which
differ with the P450 and peracid systems. Brackets designate
possible products not observed by NMR (see text).
Scheme 1. Preparation and Reactions of
Cl2P(O)OH, Cl2P(O)SH, and Potassium and DCHA
Salts Thereof Starting from POCl3 and PSCl3a
a MHz relative to the center 13C line of CDCl3 (at 77.23).
The first intermediate from PSCl3 may also be Cl2P(S)OC(O)OH.
The chemical studies require preparation of pure Cl2P-
(O)OH, Cl2P(O)SH, and salts thereof from POCl3 and
PSCl3, respectively. Monohydrolytic processes leading to
analytically pure phospho- or thiophosphodichloridic
acids are usually difficult to achieve. There are multiple
side reactions that produce mixtures comprising mono-,
di-, and trihydrolysis products as well as pyrophosphates
and combined pyrophosphate-phosphite byproducts.
Therefore, access to those monoacids requires aprotic
synthesis pathways in which further hydrolysis is elimi-
nated. Earlier aprotic preparations of these phosphorod-
ichloridic acids gave their formamidine (8) and quater-
nary ammonium salts (9). We now describe simple,
selective, and quantitative methods to prepare Cl2P(O)-
OH and Cl2P(O)SH using dry KHCO3 or K2CO3 in an
aprotic solvent such as acetone with isolation as pure
potassium or DCHA salts (Scheme 1). We find that this
reaction is specific in that only one P-Cl bond undergoes
the Cl/OH exchange even with a large excess of KHCO3
and that a P-F bond is not affected. The diethyl esters
Chem. Res. Toxicol., Vol. 16, No. 3, 2003 351
are considered as mechanisms of hydrolysis and biomi-
metic oxidation reactions with MCPBA (10) to evaluate
possible active intermediates.
The toxicological studies involve several aspects for the
phosphotrichlorides and their diethyl esters. The anti-
cholinesterase activity is first compared for the test
compounds in vitro. Then, housefly (Musca domestica)
adults are used as a model for studying AChE inhibiton
in vivo. This system has several advantages. First,
housefly brain is a convenient AChE source with high
activity and sensitivity to OP inhibitors (11). Second, in
vivo inhibition of enzymatic activity correlates with
poisoning signs and toxicity for a variety of OPs (12).
Third, compounds can be tested in the vapor state (7)
thereby minimizing exposure to moisture. Furthermore,
the extensive network of tracheoles (13) allows direct
passage of vapor toxicants into the housefly brain without
the requirement for transport in aqueous medium. Mice
are then studied to relate toxicity with brain AChE and
serum BChE inhibition. Finally, an attempt is made to
identify the actual AChE inhibitors responsible for the
toxicity of PCl3, POCl3, PSCl3, and their diethyl esters.
Experimental Section
Caution: PCl3, POCl3, PSCl3, and their diethyl esters are
volatile, corrosive, and highly toxic and therefore must be used
under containment conditions.
General Procedures. (1) Chemicals and Spectroscopy.
Chemicals were from the following sources: PCl3 (98%), POCl3
(99%), PSCl3 (98%), P(OH)3 (99%), (EtO)2PCl (98%), (EtO)2P-
(O)Cl (97%), (EtO)2P(S)Cl (97%), EtOP(S)Cl2 (95%), EtOP(O)-
Cl2 (98%), FP(O)(OH)2 (70% in H2O), (EtO)2P(S)SH (90%),
(EtO)3P (98%), (EtO)2P(O)H [tautomeric with (EtO)2POH] (98%),
and DCHA (99%) from Aldrich Chemical (Milwaukee, WI);
(EtO)3PS from K + K Labs (Plainview, NY); F2P(O)OH [hemi-
hydrate, technical grade with 1:1 FP(O)(OH)2] from Strem
Chemicals (Newburyport, MA). Syntheses for other phosphorus
compounds studied are given in the Supporting Information.
NMR spectra were recorded for CDCl3 solutions (unless indi-
cated otherwise) on a Bruker AM-300 spectrometer. Hydrolyti-
cally sensitive products were analyzed in the original reaction
solvent using 10% C6D6 as the field frequency locking medium.
Chemical shifts (δ ppm) are reported as follows: 1H at 300 MHz
relative to tetramethylsilane; 31P at 121.5 MHz relative to
external (CH3O)3PO (5% solution in acetone-D6); 13C at 75.5
(2) Hydrolytic Stability and Peracid Oxidations. Hy-
drolytic stability was determined by 31P NMR monitoring loss
of the parent compound in D2O as a function of time at 25 °C.
MCPBA (80-85%) from Aldrich was upgraded to 100% by
washing with a phosphate buffer at pH 7.5 (14) and stored at
-20 °C. Peracid oxidations were usually carried out in dry
acetone or CDCl3 and monitored by 1H or 31P NMR directly in
an NMR tube.
Preparation of Phosphochloridic and Phosphofluoridic
Acids and Derivatives Thereof. (1) Cl2P(O)H (Figure 1).
The only procedure found to give Cl2P(O)H (tautomeric with Cl2-
POH) (the monohydrolysis product of PCl3) in a definable
mixture was the reaction of P(OH)3 with acetyl chloride (see
Supporting Information). Thus, P(OH)3 (215 mg, 2.56 mmol) in
EtOAc (10 mL) was added dropwise to a cooled (0 °C) solution
of acetyl chloride (400 mg, 5.12 mmol) in EtOAc (10 mL). The
mixture was then stirred for 30 min at room temperature. 31P
NMR (10% C6D6/EtOAc) indicated the presence of P(OH)3
(-0.67, 59%) and Cl2P(O)H (-12.39, 41%) with the latter
confirmed on its reaction with EtOH and Et3N to give (EtO)2P-
(O)H. This P(OH)3/Cl2P(O)H mixture was assayed directly
considering that P(OH)3 is not active in the housefly AChE
system examined.
352 Chem. Res. Toxicol., Vol. 16, No. 3, 2003
(2) Cl2P(O)OH (Scheme 1). To prepare Cl2P(O)OH, a slurry
of POCl3 (304 mg, 2 mmol) and KHCO3 (200 mg, 2 mmol, oven
dried) in dry acetone (5 mL) was vigorously stirred for 10 min
at room temperature. The reaction is slightly exothermic, and
CO2 is evolved. 31P NMR (10% C6D6 in acetone) indicates two
peaks at -3.85 (POCl3) and -8.00 [Cl2P(O)OH] after 5 min but
only a single peak at -8.00 ppm after 10 min. Cl2P(O)OK was
obtained with POCl3 and 2 mol equiv of KHCO3 and gave 31P
NMR (10% C6D6 in acetone) -16.79. Presumably, the hydroxyl
in Cl2P(O)OH and the oxygen in Cl2P(O)OK are donated by
KHCO3 followed by loss of CO2 with concomitant formation of
KCl (Scheme 1); no water molecule is involved in this process,
and thus, side reactions are eliminated. The pure slightly
hygroscopic salt was isolated as white crystals on filtration
through glass wool covered with Celite, evaporation of 95% of
the acetone, addition of dry ether, collection of the precipitate
by filtration, and drying with an oil pump. To prepare
Cl2P(O)O-DCHA+, the KCl slurry from preparation of Cl2P(O)-
OH (2 mmol) was filtered through glass wool and Celite and
further washed with dry acetone as above. Dry ether was added
to the clear solution followed by a 3-fold excess of DCHA. The
white crystals were washed with ether to give a quantitative
yield of pure Cl2P(O)O-DCHA+, mp 155-158 °C. 1H NMR: 8.27
(2H, br s), 3.04 (2H, br septet), 2.06 (4H, br d), 1.85 (4H, br s),
1.68 (4H, br s), 1.55 (4H, br d), 1.29 (4H, br s). 31P NMR: -12.68.
To establish that the product is a monoacid, Cl2P(O)OH was
prepared in acetone, excess CH2N2 in ether (predried on KOH
pellets) was added, and the solvents were evaporated after 2
min. 1H NMR: 4.02 (3H, d, JP-H ) 16.9 Hz), identical to Cl2P-
(O)OMe synthesized independently on reacting 1:1 equiv of
POCl3 and MeOH in the presence of Et3N. To verify that two
chlorines are attached to the phosphorus, Cl2P(O)O-DCHA+
(31.5 mg, 0.1 mmol in 2 mL of CDCl3) was further reacted with
Bu4N+F- [250 µL of 1 M solution in THF, 0.25 mmol] for 10 h
at room temperature. 31P NMR (10% C6D6 in acetone, -22.15,
t, JF-P ) 951.2 Hz) indicated 95% conversion to F2P(O)O-DCHA+
(Scheme 1, X ) O) [see below for its preparation from F2P(O)-
OH].
(3) Cl2P(O)SH (Scheme 1). To prepare Cl2P(O)SH, PSCl3
(184 mg, 1 mmol) and KHCO3 (100 mg, 1 mmol) were vigorously
stirred for 16 h in dry acetone (5 mL) at room temperature in a
sealed flask. KCl was filtered off to leave an acetone solution of
pure Cl2P(O)SH. 31P NMR (10% C6D6 in acetone): 37.13. Cl2P-
(O)SK was obtained utilizing 2 mol equiv of KHCO3 and 3 h of
reaction time. 31P NMR (10% C6D6 in acetone): 32.57. To
prepare Cl2P(O)S-DCHA+, the acetone solution of pure Cl2P-
(O)SH was partially evaporated, ether was added, and the
solution was refiltered. While it was stirred, DCHA (2 mol
excess) was added to give a heavy precipitate, which was filtered
and washed with ether to give pure Cl2P(O)S-DCHA+ (99%),
mp 245-248 °C (dec). 1H NMR: 3.16 (t of triplets, 2H), 2.14
(m, 4H), 1.87 (m, 4H), 1.63 (m, 8H), 1.28 (m, 6H). 31P NMR:
38.26. Treatment of an acetone solution of Cl2P(O)SH with
excess CH2N2 as above gave Cl2P(O)SMe as the major product
(>80%). 31P NMR: 27.67 (q, JH-P ) 20.4 Hz). Two minor
products were also obtained, one of which was identified as
ClCH2P(O)(SCH3)Cl.
(4) F(Cl)P(O)OH. Pure FP(O)Cl2 was prepared according to
Fluck and Steck (15) and distilled at 54-55 °C and atmospheric
pressure (lit. 54 °C). 31P NMR: -5.84 (d, JF-P ) 1195.3 Hz).
F(Cl)P(O)OH and F(Cl)P(O)OK were the only products from FP-
(O)Cl2 and KHCO3 at 1:1 and 1:2 equiv, respectively, in acetone
with 20 min of reaction time. In all of these reactions, only one
chlorine was exchanged with hydroxyl while the fluorine
remained intact. 31P NMR (10% C6D6 in acetone): F(Cl)P(O)-
OH, -14.61 (d, JF-P ) 1047.8 Hz); F(Cl)P(O)OK, -16.20 (d, JF-P
) 1027.4 Hz). F(Cl)P(O)O-DCHA+ was synthesized quantita-
tively from F(Cl)P(O)OH utilizing similar conditions as for
Cl2P(O)O-DCHA+, mp 170-180 °C (dec). 31P NMR: -15.41 (d,
JF-P ) 1037.6 Hz).
(5) F2P(O)OH. F2P(O)OK and F2P(O)O-DCHA+ were pre-
pared by treating F2P(O)OH‚1/2H2O with KHCO3 (2 equiv) in
Segall et al.
acetone or DCHA (1 equiv) in ether to obtain the salts in 90-
94% yield after washing with ether. 31P NMR (10% C6D6 in
acetone): F2P(O)OK, -22.69 (t, JF-P ) 946.1 Hz); F2P(O)O--
DCHA+, -24.99 (t, JF-P ) 961.3 Hz).
Properties of Phosphochloridic Acids. (1) Stability. In
pure form, the stability at room temperature is as follows: Cl2P-
(O)OH , Cl2P(O)OK < Cl2P(O)O-DCHA+ (see Supporting
Information). Both Cl2P(O)OK and Cl2P(O)O-DCHA+ react with
Me2SO (either dry or wet) with a half-life of 50 min presumably
by chlorination of the solvent (16); the expected products ClP-
(O)(OH)2 and ClCH2SCH3 were not observed by 31P NMR and
13C.
(2) Thio Oxidation. In contrast to the ease of MCPBA
oxidation of thiophosphoryl triesters (17, 18) or S-alkyl phos-
phorothioates (19, 20) or thiophosphorus acids (21), PSCl3 is
stable to MCPBA oxidation for at least 18 h in acetone (Figure
1). The thiophosphorus substituent is also retained in vivo in
mice based on observing thiophosphoric acid as a urinary
metabolite of PSCl3 (see Supplementary Information). On the
other hand, reacting 1:1 equiv of Cl2P(O)SK (31P NMR 32.15)
and MCPBA in acetone at -60 °C yields a new product (31P
NMR: 10.30), tentatively assigned as Cl2P(O)SO-K+, which
rearranges to Cl2P(O)OS-K+ (31P NMR: 9.30). Excess MCPBA
oxidation (4:1) of Cl2P(O)SK leads ultimately, via the same
intermediate, to Cl2P(O)OH. Therefore, the overall candidate
in vivo reactions of PSCl3 based on these models may involve
hydrolysis as the initial step and further oxidation to Cl2P(O)-
SOH and Cl2P(O)OSH, only the former one being a phospho-
rylating agent.
(3) Methyl and Fluoro Analogues. The reactivity with
KHCO3 in acetone was also examined for analogues with methyl
or fluorine replacing chlorine, i.e., CH3P(O)Cl2 and CH3P(O)F2
(see Supporting Information). The expected CH3P(O)(OH)Cl
from CH3P(O)Cl2 is not stable as compared with Cl2P(O)OH
from POCl3. No reaction occurred with CH3P(O)F2 indicating
that the P-F bond is stable to KHCO3 in acetone both in the
phosphate and in the methylphosphonate series.
Reactions of Ethyl Phosphochloridates. (1) Stability of
(EtO)2PCl. Hydrolysis of (EtO)2PCl in D2O yields initially
(EtO)2P(O)D, which proceeds to give EtOP(O)(OD)D, (DO)2P-
(O)D, and (DO)3PO. (EtO)2PCl in the vapor phase (under the
housefly bioassay conditions given below) is partially hydrolyzed
at 4 min and completely at 10 min to (EtO)2P(O)H but not
oxidized to (EtO)2P(O)Cl. (EtO)2PF is slightly more stable in
aqueous solution as compared to (EtO)2PCl (see Supporting
Information).
(2) Peracid Oxidation of Thio Analogues. The rate of
MCPBA oxidation increases as chlorines are replaced with
ethoxy substituents, i.e., PSCl3 , EtOP(S)Cl2 < (EtO)2P(S)Cl
< (EtO)3P(S). EtOP(S)Cl2 (31P NMR: 54.80) is oxidized by
equimolar MCPBA to give EtOP(O)Cl2 (31P NMR: -2.34),
together with 2-3% H3PO4. In contrast, (EtO)2P(S)Cl (31P
NMR: 60.45) with equimolar MCPBA gives primarily (EtO)2P-
(O)SCl (31P NMR: 18.25), the structure of which was unequivo-
cally elucidated by independent synthesis (22) and 31P NMR
peak matching (Scheme 2). The half-life of (EtO)2P(S)Cl with
1:1 MCPBA (2.5 M in acetone, room temperature) is 12 min.
The mechanisms, pathways, and products obtained on bio-
mimetic MCPBA oxidation of (EtO)2P(S)Cl may have toxicologi-
cal relevance (Scheme 2). Initial oxidation at sulfur is followed
by internal rearrangement to (EtO)2P(O)SCl via a proposed ion
pair intermediate and back attack by chloride on sulfur. If the
MCPBA oxidation reaction is carried out in EtOH, the only
product obtained is (EtO)3P(O). The initial step comprises
formation of (EtO)2P(O)SCl, as monitored by 31P NMR, which
further reacts with the solvent leading ultimately to (EtO)3P-
(O), which is not formed via (EtO)3P(S); that is, (EtO)2P(O)SCl
is a phosphorylating agent. When examined more directly, the
reaction of (EtO)2P(O)SCl with MeOH or EtOH leads exclusively
to (EtO)2P(O)OMe and (EtO)3P(O), respectively, with concomi-
tant formation of elemental sulfur (see Supporting Information).
Unlike in MeOH or EtOH, (EtO)2P(O)SCl in water (studied at
Organophosphate Anticholinesterase Agents
Scheme 2. Possible Bioactivation and
Detoxification Mechanisms for (EtO)2P(S)Cl
Shown as Peracid Oxidation and Rearrangement
Pathways Leading to (EtO)2P(O)SCl Followed by
Alcohol Phosphorylation (R ) Me or Et) or
Hydrolysisa
a The starting material and phosphorylating agents formed are
shown in rectangles.
0.01 M in D2O) gives within 24 h a 1:1 molar ratio of (EtO)2P-
(O)OH (31P NMR: -5.53) and (EtO)2P(O)SH (31P NMR: 57.48);
the intermediate (EtO)2P(O)SOH (31P NMR: 18.61) is evident
at 30 min. Conversion of (EtO)2P(O)SOH [from MCPBA oxida-
tion of (EtO)2P(O)SH] to (EtO)2P(O)SH, on loss of oxygen, was
observed earlier in this laboratory (21). Under the model
conditions, direct hydrolysis of (EtO)2P(S)Cl to (EtO)2P(O)SH
is a very slow reaction as compared with the other pathways,
and oxidation to (EtO)2P(O)SOH yields an additional phospho-
rylating agent. If formed in the vicinity of esterases, both
(EtO)2P(O)SCl and (EtO)2P(O)SOH may cause their inhibition
thereby serving as activation pathways. Otherwise, (EtO)2P-
(O)SOH may rearrange to (EtO)2P(O)OSH (21), which, on
further oxidation followed by hydrolysis, leads to the ultimate,
biologically inactive (EtO)2P(O)OH, which is overall a detoxifi-
cation pathway (Scheme 2).
Toxicological Procedures. (1) Esterase Assays and In
Vitro Inhibition. Assays of AChE and BChE inhibition used
the 5,5′-dithio-bis(2-nitrobenzoic acid) (Ellman’s reagent) method
(23) with acetyl- and butyrylthiocholine as substrates, respec-
tively (7). Inhibitors were introduced in 5 µL of dry THF (or
other carrier as noted) to 100 mM phosphate (pH 7.4, total
volume 500 µL) with 15 min incubation prior to ChE assay at
25 °C. Similar inhibitor potency assays were carried out with
carboxylesterase and R-chymotrypsin (7). The IC50 was derived
generally from 3-fold concentration differentials giving 15-85%
inhibition, n ) 3-4. Controls established little or no effects of
the carrier solvents alone. Me2SO is not a suitable solvent
because it reacts with many of the test compounds (see above).
The inhibitors under study presumably cause progressive
phosphorylation of the active site serine, producing irreversible
inhibition. Some of the compounds have half-lives of only a few
minutes. A fixed period of time for inhibition was selected as a
compromise between inhibitor instability and enzyme phospho-
rylation. The IC50 values are based on preincubation of the
enzyme and inhibitor for 15 min before esterase activity assays.
The inhibited AChE is an “aged” or “doubly-aged” enzyme, i.e.,
ethylphosphoryl- or phosphoryl-AChE and, therefore, unlikely
to undergo spontaneous reactivation during the assay (7).
(2) Vapor Toxicity and In Vivo AChE Inhibition in
Houseflies. Adult houseflies cultured in this laboratory were
exposed to vapors of a candidate toxicant in a 120 mL glass
chamber as described previously (7). The time for 100% KD was
observed as the inability to walk or fly. After 15 min, mortality
(no movement) was determined and the insects were frozen on
dry ice and the heads were removed for AChE assay (7) (n ) 3,
each with >20 flies).
(3) ChE Inhibition and Toxicity in Mice. Male Swiss-
Webster mice (22-30 g, Harlan Laboratories, Indianapolis, IN)
Chem. Res. Toxicol., Vol. 16, No. 3, 2003 353
Table 2. PCl3, POCl3, and PSCl3 as Inhibitors of ChEs and
Other Serine Hydrolases in Vitro
IC50 (µM)b
enzymea PCl3 POCl3c PSCl3
AChE
housefly 4.8 ( 1.1d 12 ( 2 96 ( 16e
bovine erythrocyte 14 ( 3 36 ( 4
electric eel 16 ( 1 33 ( 6
mouse 30 ( 15 32 ( 7 >200 (29 ( 5)f
BChE
mouse 220 ( 7 88 ( 23 >200 (39 ( 11)f
dog 360 ( 19 160 ( 14
human >500 1200 ( 120
carboxylesterase 350 ( 20 140 ( 11
R-chymotrypsin >500 (0,0)f 2000
a Enzymes from plasma (BChE), housefly head or mouse brain
homogenate (AChE), or Sigma (St. Louis, MO) (purified prepara-
tions) (see ref 7). b Mean ( SE (n ) 3). c Ref 7. d The hydrolysis
product Cl2POH is less active, i.e., IC50 > 530 (44 ( 9). e The
hydrolysis product Cl2P(O)SK interferes with the Ellman assay,
and its oxidation product Cl2P(O)SOK is less active with IC50 >
200 (23 ( 9). f Percent inhibition for triplicates or individual values
at indicated concentration.
were treated ip with candidate toxicants in suitable carrier
solvents (50 µL) as noted with appropriate controls. Methyl
oleate was found to be a particularly convenient, nonreactive,
and low toxicity carrier solvent. After 1 h or at the time of death,
brain and blood were removed for AChE and serum BChE
assays, with adequate enzyme specificity using acetylthiocholine
and butyrylthiocholine, respectively, as substrates (7). Other
mice were observed for poisoning signs, and mortality was
recorded at 24 h (n ) 3-6 per dose).
(4) Hydrolytic Stability Determined by Ellman’s Re-
agent and AChE Assays. The half-life of PSCl3 (200 µM) in
100 mM phosphate (pH 7.4) containing Ellman’s reagent (23)
(2 mM) was determined over a period of 60 min as thiol release
calculated by plotting log % PSCl3 remaining vs time. Hydrolysis
rates for the other OPs were estimated using residual AChE
activity. Hence, (EtO)2PCl, (EtO)2P(O)Cl, [(EtO)2P]2O, and TEPP
in 5 µL of THF were added to 100 mM phosphate buffer (495
µL, pH 7.4, 25 °C) (final concentrations 200, 20, 20, and 2 nM,
respectively). After 0, 5, and 15 min, housefly head homogenate
was added and residual AChE activity was determined after
an additional 15 min of incubation.
Results and Discussion
PCl3, POCl3, and PSCl3. (1) Inhibitors of ChEs and
Other Serine Hydrolases In Vitro (Table 2). PCl3 is
equipotent to 3-fold more potent than POCl3 on AChE
but generally less potent on the other esterases. POCl3
inhibits AChE and BChE with IC50 values of 12-36 and
88-1200 µM, respectively, and it also inhibits carboxyl-
esterase and R-chymotrypsin in the 140-2000 µM IC50
range (7). PSCl3 is much less active than the other
phosphotrichlorides on housefly and mouse AChE and
mouse BChE. The most sensitive enzyme is always
housefly AChE, and with this assay, the hydrolysis
product Cl2POH is >100-fold less active than PCl3 and
the hydrolysis/oxidation product Cl2P(O)SOK is less
active than PSCl3.
(2) Vapor Toxicity and In Vivo AChE Inhibition
in Houseflies. POCl3 as a vapor toxicant gives 50%
mortality (ED50) at 6-20 mg/L and 50% brain AChE
inhibition at 6 mg/L with total mortality associated with
>90% AChE inhibition (7). PCl3 and PSCl3 give es-
sentially the same result as POCl3 (Figure 2), indicating
in each case that AChE inhibition is the cause of death.
The equal potency of PCl3, POCl3, and PSCl3 as in vivo
354 Chem. Res. Toxicol., Vol. 16, No. 3, 2003
Figure 2. Vapor toxicity of PCl3, POCl3, and PSCl3 to houseflies
correlates with brain AChE inhibition. Data for POCl3 from ref
7. The curves are fitted by Sigma Plot for the composite data of
PCl3, POCl3, and PSCl3.
Table 3. Phosphotrichlorides and Phosphodichloridic
Acids as AChE Inhibitors In Vitro and In Vivo, Serum
BChE Inhibitors In Vivo, and Toxicants in Mice
in vitro IC50 in vivo ED50 (mg/kg)
(µM)a AChE BChE
compd inhibition inhibition mortalitya
PCl3 30 ( 15 (A)b 14c >100e,f (D)b
POCl3 32 ( 7 (A)b 12d 30-60e,f (D)b
Cl2P(O)OH 25 ( 5 (B)b
Cl2P(O)OK 37 ( 6 (C)b 15-30f (C)b
Cl2P(O)O-DCHA+ 37 ( 6 (B)b
PSCl3 >200 (29 ( 5)g (A)b 150c >300e,f (C)b
Cl2P(O)SK 30c 100-300f (C)b
a Mean ( SE or range (n ) 3 or 4). b The following carrier
solvents or dosing vehicles were used with appropriate controls:
A, THF; B, acetone; C, propylene glycol; D, corn oil. c Dose for 50%
BChE inhibition calculated from the following data (mg/kg, %
inhibition ( SE, n ) 3-6 per dose level). For PCl3: (100, 76 ( 6),
(30, 68 ( 8), (10, 41 ( 14); for PSCl3: (300, 78 ( 6), (100, 44 ( 6),
(30, 5 ( 3); for Cl2P(O)SK: (100, 78 ( 5), (30, 47 ( 6), (10, 19 (
7). d Ref 7. e No brain AChE inhibition at highest indicated dose
1 h posttreatment. f Poisoning signs as follows: PCl3, POCl3, and
Cl2P(O)OK give tremors and muscle fasciculation; PSCl3 and
Cl2P(O)SK give respiratory failure but no acute cholinergic
syndrome. g Percent inhibition at indicated concentration.
AChE inhibitors is not predicted from their effects in vitro
with IC50 values of 4.8, 12, and 96 µM, respectively (Table
2), suggesting possible activation of the latter two
compounds. However, the P450 inhibitor PB as a possible
synergist (250 µg/g, 2 h pretreatment) does not affect
PSCl3 toxicity as a vapor or after topical application (data
not shown), implying lack of oxidative activation but
consistent with hydrolysis to Cl2P(O)SH as the active
toxicant.
(3) ChE Inhibition and Toxicity in Mice. PCl3,
POCl3, PSCl3, and selected hydrolysis products were
examined as brain AChE inhibitors in vitro and in vivo,
serum BChE inhibitors in vivo, and toxicants (Table 3).
PCl3 and POCl3 are similar in in vitro potency (IC50 )
30-32 µM) whereas PSCl3 is >6-fold less active. POCl3
is equal in potency to Cl2P(O)OH and salts thereof (IC50
) 25-37 µM), indicating that ionized Cl2P(O)OH is the
actual inhibitor. AChE in the brain of mice treated ip
with PCl3, POCl3, or PSCl3 does not undergo inhibition
whereas serum BChE is inhibited, indicating instability
and/or lack of penetration of the brain by the parent
toxicants and the ionized hydrolysis products. POCl3 is
similar to PCl3 but much more effective than PSCl3 as
an in vivo BChE inhibitor with ED50 values of 12, 14,
and 150 mg/kg, respectively. Cl2P(O)SK is 5-fold more
Segall et al.
potent than PSCl3 for inhibition of BChE in blood.
Mortality in mice is consistent with poisoning by the
hydrolytic products Cl2P(O)OH from POCl3 and Cl2P(O)-
SH from PSCl3; in agreement, the toxicity of PSCl3 is not
affected by the oxidase inhibitor PB (data not shown).
More generally, the poisoning signs are those of acute
cholinergic syndrome (including tremors, muscle fascicu-
lation, and lachrymation) with PCl3 and POCl3 but a
different mechanism (possibly thiol related) for PSCl3
(Table 3).
The ip administration to mice of PCl3 (not detailed
here) and PSCl3 (see Supporting Information) yields HP-
(O)(OH)2 and HSP(O)(OH)2, respectively, as major uri-
nary metabolites identified by 31P NMR; that is, the P-H
and P-SH linkages are retained in the excreted acids.
(4) Possible Activation by Hydrolysis or Oxida-
tion. PCl3, POCl3, and PSCl3 are hydrolytically unstable
and might act directly or possibly as activation products.
PCl3 hydrolyzes quickly to Cl2POH and POCl3 to Cl2P-
(O)OH, but the dichlorides are very transient except in
anhydrous solvents or as salts. PCl3 probably acts directly
as it undergoes no oxidation under the assay conditions
and its hydrolysis product Cl2POH is inactive. With
POCl3, the activated inhibitor and toxicant is Cl2P(O)-
OH with activity equivalent to POCl3 (Table 3). PSCl3 is
a more complex case with possible hydrolysis or oxidation
or both in the course of poisoning. The half-life of PSCl3
at pH 7.4 is 13 min with formation of Cl2P(O)SH as the
sole product. Cl2P(O)SH is moderately stable toward
further hydrolysis and more active than PSCl3 as a BChE
inhibitor in mice (Table 3). The oxidative chemistry was
examined using MCPBA in chloroform with the finding
that PSCl3 is not converted to POCl3 (or does not undergo
oxidation) whereas Cl2P(O)SH is readily converted to
Cl2P(O)SOH, which is less active.
Ethyl Phosphites, Phosphates, and Thiophos-
phates. (1) Inhibitors of Brain AChE and Vapor
Toxicants in Houseflies (Table 4). Fourteen ethyl
esters related to PCl3, POCl3, and PSCl3 were examined
for inhibition of housefly AChE in vitro. [(EtO)2P]2O is
the most potent ethyl phosphite (IC50 ) 12 nM) while
(EtO)2PCl is only 2-fold less active. The 13-fold lower
activity of (EtO)2PF than (EtO)2PCl is surprising as noted
below. Mono- and disubstitution of ethoxy in PCl3 (IC50
) 4800 nM; Table 2) increases potency by 12- and 200-
fold, respectively. Tri- and diethyl phosphites lacking
chlorine are essentially inactive. In the ethyl phosphate
series, the expected high potency of TEPP is affirmed.
(EtO)2P(O)Cl is highly active (IC50 ) 11 nM) relative to
EtOP(O)Cl2 (IC50 ) 290 nM) and particularly POCl3 (IC50
) 12 000 nM; Table 2). In general, ethyl thiophosphates
are much less active. (EtO)2P(O)SCl [the potential oxida-
tive activation product of (EtO)2P(S)Cl] is the most potent
ethyl thiophosphate (IC50 ) 140 nM). Mono- and disub-
stitution of ethoxy in PSCl3 (IC50 ) 96 000 nM; Table 2)
increases the potency by 21- and 74-fold, respectively.
Diethyl phosphorothioic acids are inactive, or in the case
of (EtO)2P(S)SH, activity may be due to impurities, which
are difficult to remove. In summary, the potency order
is generally diethyl esters > monoethyl esters > phos-
photrichlorides and phosphates > phosphites > thiophos-
phates.
The compounds were examined as vapor toxicants in
houseflies. KD and mortality from vapor correlate to
brain AChE inhibition (mortality of 100, 77-87, and 0%
corresponded to inhibition of 84-98, 63-73, and 0-38%,
respectively), indicating this as the mode of action. With
Organophosphate Anticholinesterase Agents Chem. Res. Toxicol., Vol. 16, No. 3, 2003 355

Table 4. Ethyl Phosphites, Phosphates, and Thiophosphates as Inhibitors of Brain AChE and Vapor Toxicants in
Houseflies
vapor toxicity (40 µL/L)
effect at 15 min (%)
compd IC50 (nM)a KD (min) mortality AChE inhibitiona
ethyl phosphites
[(EtO)2P]2O 12 ( 2 >15 0 38 ( 10
(EtO)2PCl 24 ( 3 9 78 73 ( 5
(EtO)2PF 300 ( 60 1 100 98 ( 1
EtOPCl2 400 ( 10 >15 0 19 ( 16
(EtO)3P 51 000 ( 8000 >15 0 24 ( 11
(EtO)2POH >200 000 (0, 0)b >15 0 11 ( 13
ethyl phosphates
TEPP 1.2 ( 0.1 >15 0 0(0
(EtO)2P(O)Cl 11 ( 2 2 100 98 ( 1
EtOP(O)Cl2 290 ( 70 5 77 66 ( 17
ethyl thiophosphates
(EtO)2P(O)SCl 140 ( 40 8 87 63 ( 12
(EtO)2P(S)Cl 1300 ( 200 7 100 85 ( 16
(EtO)2P(S)SH 200-2000 >15 0 0(1
EtOP(S)Cl2 4500 ( 700 6 100 84 ( 10
(EtO)2P(O)SH >200 000 (42, 32)b >15 0 0
a Mean ( SE (n ) 3-4). b Percent inhibition for duplicates at indicated dose.

Table 5. Ethyl Phosphites as ChE Inhibitors and Toxicants in Mice

(EtO)2PClb [(EtO)2P]2Ob
percent percent
dose inhibitionc mortality inhibitionc mortality
(mg/kg)a AChEd BChEd percent (n) AChEd BChEd percent (n)
3 (0, 0) (66, 63) 0 (2)
10 51 ( 7 0 (3) 3(0 77 ( 5 0 (5)
30 (4, 6) 78 ( 2 0 (3) 59 ( 4 97 ( 1 100 (4)
100 27 ( 7 84 ( 1 0 (3) 100 (1)
300 72 ( 4 96 ( 2 100 (3) 100 (1)
a Methyl oleate used as carrier solvent to circumvent oxidation of (EtO) PCl, which was observed in corn oil. b Assays at 1 h posttreatment
2
or death, whichever was first. c Mean ( SE (n ) 3) or individual percent values in parentheses. d In vitro IC50 values (nM): (EtO)2PCl
with AChE, 650 ( 41, and BChE, 170 ( 8; [(EtO)2P]2O with AChE, 2100 ( 1000, and BChE, 36 ( 21.

nine compounds, the in vitro potency for AChE inhibition
predicts the toxicity and the five exceptions are for
analogues with special features, i.e., three low volatility
compounds [[(EtO)2P]2O, TEPP, and the very unstable
EtOPCl2] and two phosphorothionates [(EtO)2P(S)Cl and
EtOP(S)Cl2]. The lower in vitro potency of (EtO)2PF as
compared to (EtO)2PCl may be compensated for by higher
volatility, leading to increased vapor toxicity. The similar
vapor toxicity of (EtO)2P(O)Cl and (EtO)2P(S)Cl, even
though (EtO)2P(O)Cl is >100-fold more active in vitro,
suggests possible bioactivation of (EtO)2P(S)Cl. In con-
firmation, pretreatment of houseflies with PB to mini-
mize P450 metabolism reduces mortality from 83 ( 2 to
22 ( 2% after topical application of (EtO)2P(S)Cl (10 µg/
fly).
(2) Ethyl Phosphites as ChE Inhibitors and Toxi-
cants in Mice (Table 5). Two phosphites, found to be
potent AChE inhibitors in houseflies, were examined
more extensively in mice. (EtO)2PCl and [(EtO)2P]2O are
less active in vitro inhibitors with mouse brain AChE and
serum BChE than with housefly AChE (Tables 4 and 5).
Mice treated with (EtO)2PCl show cholinergic poisoning
signs and dose-dependent inhibition of AChE (ED50 )
100-300 mg/kg) correlating with mortality; BChE inhibi-
tion (ED50 ) 10 mg/kg) is a more sensitive marker of
exposure. [(EtO)2P]2O is 10-fold more toxic to mice than
(EtO)2PCl, and again, mortality is related to inhibition
of brain AChE (ED50 ) 10-30 mg/kg) with BChE inhibi-
tion at a lower dose (ED50 < 3 mg/kg).
(3) Possible Activation by Oxidation or Pyro-
phosphate Formation. Studies were designed to de-
termine if the trivalent derivatives (EtO)2PCl and
[(EtO)2P]2O and the pentavalent compounds (EtO)2P(O)-
Cl and (EtO)2P(S)Cl act directly or following oxidation
or pyrophosphate formation.The phosphites (EtO)2PCl
and [(EtO)2P]2O are of special interest: they are potent
inhibitors of housefly AChE, but little data are available
for AChE inhibition or mortality in mammals; the
inhibited AChE would initially be the diethylphosphinyl
[(EtO)2POAChE] instead of the usual diethylphosphoryl
[(EtO)2P(O)OAChE] derivative; both (EtO)2PCl and
[(EtO)2P]2O are important in organic synthesis with
(EtO)2PCl considered less hazardous than other reagents
used for similar synthetic procedures (3). (EtO)2PCl
hydrolyzes mainly to (EtO)2POH [i.e., (EtO)2P(O)H],
which is inactive with AChE. (EtO)2PCl in phosphate
buffer (pH 7.4) mostly hydrolyzes in a few minutes; a
solution giving 98 ( 1% inhibition at zero time gave 58
( 1 and 23 ( 2% inhibition at 5 and 15 min, respectively.
[(EtO)2P]2O is much more stable to hydrolysis and is not
observed as an impurity in (EtO)2PCl (31P NMR, <1%).
Both of these ethyl phosphites appear to act directly as
AChE inhibitors and toxicants.
(EtO)2P(O)Cl is a known AChE inhibitor with chloride
as the suggested leaving group (24). Under certain
conditions, it is also a potential precursor for TEPP. Our
results confirm in two ways that the more hydrolytically
unstable (EtO)2P(O)Cl acts directly without requirement
for the intermediacy of TEPP. First, there is a manyfold
decrease in inhibitory potency for (EtO)2P(O)Cl but not
TEPP in buffer with 15 min preincubation before assay.
Second, no TEPP was observed by 31P NMR as a reaction
356 Chem. Res. Toxicol., Vol. 16, No. 3, 2003
Table 6. Phosphofluorides and Phosphofluoridic Acids as
AChE Inhibitors and Toxicants in Mice
in vitro IC50 (µM) mortality
compd AChE inhibitiona ED50 (mg/kg)b
FP(O)Cl2 300 ( 17 90-120
F(Cl)P(O)OH 272 ( 24
F(Cl)P(O)OK 144 ( 22 120-180
F2P(O)OH + FP(O)(OH)2 (1:1) >300 (0,0)
F2P(O)O-DCHA+ 188 ( 30
KF‚2H2O >300 (0,0) 60-90
a Mean ( SE (n ) 3) or individual percent values in parenthe-
ses. b Poisoning signs involve depressed activity in each case plus
tremors with F(Cl)P(O)OK and labored respiration with KF‚2H2O
(n ) 3-5 per dose).
product under any assay conditions. (EtO)2P(S)Cl hy-
drolysis gives (EtO)2P(O)SH (inactive) while peracid
oxidation gives (EtO)2P(O)SCl [but not (EtO)2P(O)Cl],
which inhibits housefly AChE (IC50 ) 140 nM) and is
toxic. Mice dosed with (EtO)2P(S)Cl (300 mg/kg in methyl
oleate) display tremors, which are minimized by pre-
treatment with PB (250 mg/kg, intraperitoneal 1 h),
suggesting oxidative bioactivation.
Phosphofluorides and Phosphofluoridic Acids.
Replacement of chlorine by fluorine greatly changes the
level and type of toxicity. FP(O)Cl2 and its hydrolysis
product [tested as F(Cl)P(O)OH and F(Cl)P(O)OK] are
much less potent AChE inhibitors than POCl3 and Cl2P-
(O)OH (Tables 3 and 6). The phosphodifluorides are also
less effective than the phosphodichlorides. Mortality in
mice from FP(O)Cl2 and F(Cl)P(O)OK occurs at similar
dose levels (90-180 mg/kg) but is not clearly associated
with acute cholinergic syndrome and may be due to
fluoride intoxication, which is also implied by the toxic
level of KF in this study (Table 6).
Conclusions
Six major intermediates in OP synthesis are examined
as anticholinesterase agents directly or on activation
using housefly brain AChE and mouse brain AChE and
serum BChE both in vitro and in vivo. An aprotic process
is reported for preparation of pure phosphorodichloridic,
phosphorochlorofluoridic, and phosphorothioic acids for
chemical and toxicological studies. The proposed AChE
inhibitors and toxicants are PCl3, (EtO)2PCl, and (EtO)2P-
(O)Cl acting directly; POCl3 and PSCl3 acting as Cl2P-
(O)OH and Cl2P(O)SH, respectively; and (EtO)2P(S)Cl
acting as (EtO)2P(O)Cl or possibly (EtO)2P(O)SCl (based
on evidence from PB as a P450 oxidase inhibitor and
peracid reactions, respectively; Figure 1 and Scheme 2).
Acknowledgment. The project described was sup-
ported by Grant R01 ES08762 from the National Insti-
tute of Environmental Health Sciences (NIEHS), NIH,
and its contents are solely the responsibility of the
authors and do not necessarily represent the official
views of the NIEHS, NIH. We thank our laboratory
colleagues James Mun for assistance in the housefly
assays and Nanjing Zhang and Daniel Nomura for
helpful suggestions.
Supporting Information Available: Syntheses and prop-
erties as indicated in the text relative to phosphochloridic acids,
phosphofluorides and phosphofluoridic acids, diethyl esters,
diethyl thiophosphates, and methylphosphonates. This material
is available free of charge via the Internet at http://pubs.acs.org.
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