Novel irreversible butyrylcholinesterase inhibitors: 2-chloro-1-(substituted-phenyl)ethylphosphonic acids.

Article abstract of DOI:10.1016/S0968-0896(01)00391-1

2-Chloroethylphosphonic acid (ethephon) as the dianion phosphorylates butyrylcholinesterase (BChE) at its active site. In contrast, the classical organophosphorus esterase inhibitors include substituted-phenyl dialkylphosphates (e.g., paraoxon) with electron-withdrawing aryl substituents. The chloroethyl and substituted-phenyl moieties are combined in this study as 2-chloro-1-(substituted-phenyl)ethylphosphonic acids (1) to define the structure--activity relationships and mechanism of BChE inhibition by ethephon and its analogues. Phenyl substituents considered are 3- and 4-nitro, 3- and 4-dimethylamino, and 3- and 4-trimethylammonium. Phosphonic acids were synthesized via the corresponding O,O-diethyl phosphonate precursors followed by deprotection with trimethylsilyl bromide. They decompose under basic conditions about 100-fold faster than ethephon to yield the corresponding styrene derivatives. Electron-withdrawing substituents on the phenyl ring decrease the hydrolysis rate while electron-donating substituents increase the rate. The 4-trimethylammonium analogue has the highest affinity (K(i)=180 microM) and potency (IC(50)=19 microM) in first binding reversibly at the substrate site (possibly with stabilization in a dianion--monoanion environment) and then progressively and irreversibly inhibiting the enzyme activity. These observations suggest dissociation of chloride as the first and rate-limiting step both in the hydrolysis and by analogy in phosphorylation of BChE by bound at the active site.

Full text of DOI:10.1016/S0968-0896(01)00391-1

Bioorganic & Medicinal Chemistry 10 (2002) 1281–1290
Novel Irreversible Butyrylcholinesterase Inhibitors:
2-Chloro-1-(substituted-phenyl)ethylphosphonic Acids
Nanjing Zhang and John E. Casida*
Environmental Chemistry and Toxicology Laboratory, Department of Environmental Science, Policy and Management,
University of California, Berkeley, CA 94720-3112, USA
Received 7 September 2001; accepted 1 November 2001
Abstract—2-Chloroethylphosphonic acid (ethephon) as the dianion phosphorylates butyrylcholinesterase (BChE) at its active site.
In contrast, the classical organophosphorus esterase inhibitors include substituted-phenyl dialkylphosphates (e.g., paraoxon) with
electron-withdrawing aryl substituents. The chloroethyl and substituted-phenyl moieties are combined in this study as 2-chloro-1-
(substituted-phenyl)ethylphosphonic acids (1) to define the structure–activity relationships and mechanism of BChE inhibition by
ethephon and its analogues. Phenyl substituents considered are 3- and 4-nitro, 3- and 4-dimethylamino, and 3- and 4-trimethyl-
ammonium. Phosphonic acids 1 were synthesized via the corresponding O,O-diethyl phosphonate precursors followed by depro-
tection with trimethylsilyl bromide. They decompose under basic conditions about 100-fold faster than ethephon to yield the
corresponding styrene derivatives. Electron-withdrawing substituents on the phenyl ring decrease the hydrolysis rate while electron-
donating substituents increase the rate. The 4-trimethylammonium analogue has the highest affinity (Ki=180 mM) and potency
(IC50=19 mM) in first binding reversibly at the substrate site (possibly with stabilization in a dianion–monoanion environment) and
then progressively and irreversibly inhibiting the enzyme activity. These observations suggest dissociation of chloride as the first and
rate-limiting step both in the hydrolysis and by analogy in phosphorylation of BChE by 1 bound at the active site. # 2002 Elsevier
Science Ltd. All rights reserved.
Introduction
Many ethephon analogues have been prepared with
Butyrylcholinesterase (BChE; EC 3.1.1.8) activity is
progressively and irreversibly inhibited on phosphoryl-
ation at Ser-198 by the classical triester phosphates such
as paraoxon1ꢀ3 and surprisingly also by 2-chloro-
ethylphosphonic acid (ethephon) dianion.4ꢀ6 Ethephon
is one of the most important plant growth regulators
serving as a source of the phytohormone ethylene on
spontaneous decomposition at physiological pH.7 The
irreversible inhibition of BChE activity by ethephon and
its close analogues is unique among phosphonic acids
since they are usually not reactive species and act
instead as reversible enzyme inhibitors.8 In order to
better understand the mechanisms involved, the present
investigation considers the design, synthesis and prop-
erties of novel irreversible BChE inhibitors based on
ethephon as the model.
variations at the 1-or 2 position and enhanced reactiv-
ity but not greatly increased BChE inhibitory potency.
2-Substituted ethephon analogues have very short half-
life times in neutral and basic aqueous conditions9 and
2-butylethephon is a poor BChE inhibitor possibly due
to rapid hydrolysis (t1/2 < 5 min) under the enzyme
assay conditions (pH 7.4).6 Thus, only the 1-position
was considered as a variable in the present studies. On
the other hand, organophosphorus (OP) triester choli-
nesterase (ChE) inhibitors like paraoxon contain a good
leaving group, usually a substituted-phenyl moiety with
an electron-withdrawing substituent. This type of struc-
ture provides high affinity at the binding site and
enhanced reactivity, most probably through interactions
with the aromatic residue-rich area in the active site
gorge.10 Accordingly, 2-chloro-1-(substituted-phenyl)-
ethylphosphonic acids (1) are particularly interesting
since they combine the structural features of the para-
oxon-and ethephont-ype compounds (Fig. 1). Phenyl
substituents that proved useful in studying the structure–
activity relationships of paraoxon-type inhibitors (spe-
cifically nitro, dimethylamino and trimethylammonium
*Corresponding author. Tel.: +1-510-642-5424; fax: +1-510-642-
6497; e-mail: ectl@nature.berkeley.edu
0968-0896/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(01)00391-1

1282
N. Zhang, J. E. Casida / Bioorg. Med. Chem. 10 (2002) 1281–1290
groups),1ꢀ3 both 3-and 4p-ositions, were selected for
study in the 1 series to identify structural features con-
ferring high potency as BChE inhibitors and to probe
the inhibitory mechanism. We show in this investigation
that substitution at the 4-position with electron-with-
drawing groups provides improved BChE inhibitory
potency, not directly related to enhanced hydrolysis
rate, and that phosphonic acids 1 first bind competi-
tively then inhibit progressively and irreversibly. On this
basis, we propose that BChE phosphorylation by bound
1 involves the reactive species produced during sponta-
neous chloride dissociation as the first and rate-limiting
step.
Scheme 1. (a) TMSBr, MeCN, 60 C, overnight; (b) MeOH/H2O, rt, 1 h.
mediate 4, which was reduced by borane at room tem-
perature to give the corresponding alcohol (5).18 The
alcohol was converted to chloride 2 by reaction with
Ph3P and CCl4.
Results
Syntheses of 2d and 2e were achieved from 2b and 2c by
catalytic hydrogenation to convert the nitro groups to
amino groups, which were further transformed by
reductive alkylation19 with formaldehyde and
NaCNBH3 to 2d and 2e, respectively (Scheme 3). Reac-
tion of 2d and 2e with MeI provided the corresponding
Synthesis of2-chloro-1-(substituted-phenyl)ethylphos-
phonic acids (1a–1g)
The strategy required consideration of the known labil-
ity of 2-haloethylphosphonic acids under neutral and
basic aqueous conditions11,12 and difficulties in purify-
ing the acids. Accordingly, in a two-step procedure,
diethyl phosphonates (2) were prepared as precursors
and purified by chromatography, then converted to the
target phosphonic acids (1) by McKenna reaction
(Scheme 1).13,14 Treatment of 2a–2g with trimethylsilyl
bromide (TMSBr) followed by hydrolysis with MeOH/
water provided phosphonic acids 1a–1g in essentially
pure form and quantitative yields (byproducts were
removed completely under reduced pressure). These
phosphonic acids were used for chemical reactivity
studies and enzyme assay.
trimethylammonium-substituted
precursors 2f and 2g.
diethylphosphonate
Relation ofstructure to hydrolysis rate
Hydrolysis of1a–1g at pH 7.4. Phosphonic acids 1a–1g
decompose at pH 7.4 where the dianion is the major
form observed by 31P NMR. The decomposition pro-
ducts were identified as the corresponding styrene ana-
1
logues by H NMR and as phosphate by 31P NMR in
comparison with an authentic standard (Scheme 4). The
formation of styrene analogues allowed the reaction to
be easily followed by UV absorption change (Fig. 2).
Kinetic analysis showed a first-order reaction, that is,
the logarithm value of the decreasing concentration of
phosphonic acid was linear with reaction time.
Precursor diethyl 2-chloro-1-phenylethylphosphonate
(2a) can be considered as the parent compound and was
prepared as shown in Scheme 2. Diethyl benzylphos-
phonate was deprotonated by n-BuLi and reacted with
paraformaldehyde to provide alcohol 3, which was
converted to the chloride 2a by the general method with
triphenylphosphine (Ph3P) and CCl4.
Compared with ethephon, analogues 1a–1g decompose
50-to 163f-old faster (Table 1). The substituent on the
phenyl ring affects the decomposition to a much less
extent than the substituent at the 1-position, following
the order of 1d > 1e > 1a > 1b ꢁ 1c ꢁ 1f ꢁ 1g. Com-
pounds with electron-donating groups decompose fas-
This procedure was not suitable to synthesize 2b and 2c,
possibly due to the lower reactivity of the generated
anion to form the corresponding alcohol when treated
with paraformaldehyde. An alternative and more gen-
eral route (Scheme 3) was explored using readily avail-
able starting materials. The key step is a coupling
reaction of the phosphonate anion and substituted
iodobenzene,15,16 with copper(I) iodide as the catalyst.
Thus triethyl phosphonoacetate was deprotonated by
NaH and reacted with 3-nitro- or 4-nitroiodobenzene,
in the presence of copper(I) iodide,17 to produce inter-
Table 1. Hammett’s s constants and hydrolysis rates at pH 7.4
Compound
No.
Ratea
X
sb
k (minꢀ1) ꢂ 103
t1/2 (min)
1a
1b
1c
1d
1e
1f
1g
H
4-NO2
3-NO2
4-NMe2
3-NMe2
4-N+Me3
3-N+Me3
—
0
33
21
22
65
42
22
20
0.4
21
32
32
11
16
31
34
1735
0.81
0.71
ꢀ0.63
ꢀ0.10
0.82
0.88
—
Ethephon
aDetermined by rate of formation of styrene derivatives in pH 7.4
phosphate buffer (100 mM) by UV absorption (see Fig. 2) or of
phosphate in 2,4,6-collidine (0.7 M)–HCl buffer (pH 7.4) by 31P NMR
(for ethephon only), both at 23 ꢃC.
Figure 1. Structures of ethephon (the plant growth regulator) as the
dianion, paraoxon (a classical organophosphorus esterase inhibitor),
and 2-chloro-1-(substituted-phenyl)ethylphosphonic acids (1) which
combine the structural features of the first two compounds.
bFrom ref 20.

N. Zhang, J. E. Casida / Bioorg. Med. Chem. 10 (2002) 1281–1290
1283
Scheme 2. (a) n-BuLi, THF, ꢀ78 ꢃC, 15 min; (b) (HCHO)n, ꢀ78 ꢃC to
rt; (c) H+/H2O; (d) PPh3, CCl4, reflux, 1 h.
Scheme 4. Hydrolysis and methanolysis of 2-chloro-1-(substituted-
phenyl)ethylphosphonic acids.
Scheme 3. (a) NaH, DMF, rt, 10 min; (b) ArI, CuI; (c) BH3–THF,
rt, 2 days; (d) PPh3, CCl4, reflux, 1 h; (e) H2/Pd/C, MeOH, rt, 2 h;
(f) HCHO, NaCNBH3, MeCN, HOAc, rt, 2.5 h; (g) MeI, MeNO2, rt,
2 days.
Figure 2. Time course of spectral change for decomposition of phos-
phonic acid 1d (lmax 248 nm, 0.1 mM) to 4-dimethylaminostyrene
(lmax 280 nm) in pH 7.4 100 mM phosphate buffer. Curves recorded at
1 min intervals.
ter. The highest rate is for compound 1d (X=4-NMe2)
with a half-life time of 11 min. Compounds with elec-
tron withdrawing groups, for example, 1b, 1c, 1f, and
1g, have slower and almost the same decomposition
rates.
the enzyme activity decreases as the incubation time
increases. As an example, 1a (0.3 mM) showed an
activity loss of 20% after 60 min and 65% after 360 min
(Fig. 3). Interestingly, the irreversible inhibition con-
tinues to increase even at a time of up to 20-fold the t1/2
when almost all of the phosphonate dianion in solution
is gone. Although this could conceivably be due to
inhibition by the decomposition products rather than
the parent compound, this was not the case. When
BChE was incubated with the decomposition products,
little enzyme inhibition was observed, for example, IC50
> 1000 mM with 24 h preincubation of the most potent
1b or 1f in buffer prior to addition of BChE. The irre-
versible manner of the inhibition was also indicated by
the lack of recovery of enzyme activity when the inhi-
bitor (1a) was removed from the solution.
Phosphorylation ofMeOH. In a solution of MeOH with
K2CO3, phosphonic acid 1a decomposes to the styrene
derivative with phosphorylation of MeOH to methyl
phosphate. Similarly, 1a in 50% aq MeOH with KOH
gives methyl phosphate and phosphate (molar ratio 1:4)
(Scheme 4).
Relation ofstructure to BChE inhibition
IC50. Potencies as IC50 values were determined after 90
min incubation of BChE with the inhibitor as a com-
promise between the relatively short half-lives of the
compounds (see above) and slow inhibition rates (con-
sidered below). The IC50 values ranged from 19 to 468
mM (Table 2). The 4-trimethylammonium compound
(1f) was most potent, 14-fold better than ethephon, and
the 4-nitro compound was second in activity. The least
potent were 1e, with a 3-dimethylamino group, and the
parent 1a. Obviously, an electron-withdrawing group at
the 4-position greatly increases the potency (using com-
pound 1a as the standard for comparison). Introduction
of an electron-withdrawing group at the 3- position also
increases the potency but to a much less extent based on
comparing 1b with 1c and 1f with 1g.
Table 2. IC50 and Ki values
Compound
No.
IC50 (mM)
Ki (mM)
X
1a
1b
1c
1d
1e
1f
1g
H
434ꢄ88
42ꢄ2
1443
616
1030
593
1076
180
561
–
4-NO2
3-NO2
4-Nme2
3-Nme2
4-N+Me3
3-N+Me3
—
124ꢄ20
179ꢄ11
468ꢄ6
19ꢄ1
159ꢄ25
267ꢄ2a
Progressive and irreversible inhibition. Phosphonic acids
1a–1g (at micromolar concentrations) inhibit BChE (at
an extremely low assay level) in a progressive manner;
Ethephon
aFrom ref 6.

1284
N. Zhang, J. E. Casida / Bioorg. Med. Chem. 10 (2002) 1281–1290
Competitive inhibition. The relatively slow irreversible
deactivation by 1a–1g made it possible to determine the
binding type and Ki between BChE and inhibitor. On a
short time scale, compounds 1a–1g are competitive
inhibitors, that is, as inhibitor concentration [I] increa-
ses, Km(app) increases but Vmax remains constant (Fig.
4). Ki varies with the substituents on the phenyl ring.
Generally, substitution at the 4-position gives higher
affinity than at the 3-position with increasing Ki in the
order of 1b < 1c, 1d < 1e and 1f < 1g (Table 2).
Discussion
Mechanism ofhydrolysis
Spontaneous decomposition of 1a–1g under physio-
logical conditions involves the unusual property of
2-haloethylphosphonic acids to break the phosphorus–
carbon bond. An early suggested mechanism21 involved
intramolecular attack of the phosphonate oxygen anion
on the b-carbon to form a ‘b-phostone’ intermediate but
this reaction mechanism was not consistent with later
kinetic studies.22,23 Three other proposed mechanisms
are considered here for ethephon and its analogues (Fig.
5). Mechanism (A) is similar to the hydrolysis of most
OP triesters by attack of a nucleophile (H2O or serine
hydroxyl in BChE) on phosphorus (through a penta-
coordinated phosphorus transition state or direct dis-
placement to break the phosphorus–carbon bond)
coupled to the release of ethylene and chloride.11,24 Two
observations rule against this mechanism. First, the
Hammett correlation20 for phosphonic acids 1 shows
that an electron-withdrawing group on the phenyl ring
decreases the hydrolysis rate and an electron-donating
group increases the rate with a negative r value (ꢀ0.32;
Fig. 6), supporting an electron-deficient center in the
rate-limiting step. This is opposite to the Hammett cor-
relation in the paraoxon series (r > 0)3 with an estab-
lished mechanism by direct attack of a nucleophile on
phosphorus, indicating different mechanisms for the
hydrolysis of these two types of compounds.25 Second,
considering the steric effect of the substituted-phenyl
group, a hydrolysis rate lower than that of ethephon
would be expected for phosphonic acids 1 by mechan-
ism (A), contrary to the experimental results.
Figure 3. Effect of 1a concentration on rate of inhibition of BChE
activity. Enzyme activity as the logarithm value is plotted as a function
of incubation time. In the absence of inhibitor, no deactivation of
enzyme was observed during the time scale shown in the plot.
Figure 4. Determination of Ki of phosphonic acid 1g with BChE at
assay times of 0.5–1 min. For each inhibitor concentration, the inverse
of the velocity is plotted as a function of the inverse of substrate con-
centration. Ki=[I]/[Km(app)/Kmꢀ1]. Km(app) determined with inhi-
bitor and BTCh and Km with BTCh alone.
Figure 5. Proposed mechanisms for hydrolysis of ethephon and by analogy for phosphorylation of BChE (serine hydroxyl replaces hydroxyl anion);
(A) nucleophilic displacement at phosphorus; (B) concerted dissociation via metaphosphate; (C) dissociation of chloride as first and rate-limiting
step.

N. Zhang, J. E. Casida / Bioorg. Med. Chem. 10 (2002) 1281–1290
1285
The other two mechanisms are based on dissociation of
the phosphonic acid dianion. Mechanism (B) involves a
concerted dissociation process to metaphosphate as the
highly reactive intermediate,11,23 which is trapped
instantly by solvent (H2O) or other nucleophile (e.g.,
serine hydroxyl) to give phosphorylated products.
Mechanism (C) instead involves stepwise dissociation of
chloride as the first and rate-limiting step so that the
intermediate is a partially dissociated species rather than
free metaphosphate.9 Both mechanisms (B) and (C) are
consistent with the observed substituent effects due to
the developing positive charge from chloride dissocia-
tion. The phenyl group can stabilize the positive charge
through s–p conjugation and thus phosphonic acids 1
have much higher hydrolysis rates (about 100-fold) than
ethephon. An electron-donating group on the phenyl
ring can provide extra stabilization of the positive
charge center resulting in a relatively higher reactivity.
However, substitution at the 2-position of ethephon has
a much greater effect (ꢅ104-fold)9,23 than substitution
at the 1-position (this study), suggesting that the posi-
tive charge is located at the 2-carbon not the 1-carbon in
the rate-limiting step. In addition, ethephon reacts with
water at a very different rate than with isopropanol or
tert-butanol,26 implying that the intermediate is still
sensitive to steric factors. In this respect, pathway (C),
in which dissociation of chloride is the first and rate-
limiting step and a partially dissociated intermediate
serves as the reactive species, appears more consistent
with the kinetic behavior.
Comparison ofBChE inhibition by 1a–1g and triester
OPs
Inhibition of ChE by triester OPs involves a two-stage
process: reversible binding of inhibitor (I) with enzyme
(E) to form the Michaelis complex (EI)R then phos-
phorylation of the enzyme to give a poorly reversible or
irreversible derivative (E-I)I (Scheme 5).1,27 BChE inhi-
bition by phosphonic acids 1a–1g follows the same
course with reversible binding as competitive inhibitors
on a short time scale and irreversible inhibition on a
long time scale. Compared with the OP triesters, the
irreversible inhibition by 1a–1g and ethephon requires
hours rather than minutes.
Competitive inhibition of butyrylthiocholine (BTCh)
hydrolysis by phosphonic acids 1 shows that they share
a common binding site on BChE. No correlation was
found between the electronic effects of the phenyl sub-
stituents and Ki values. However, higher affinities were
observed for 4-substituted than the corresponding
3-substituted compounds.
Inhibitor potency determined as IC50 represents the
final result of irreversible inhibition as opposed to Ki
values for reversible binding. Much enhanced potency
Figure 6. Hammett correlation of the hydrolysis of 1 at pH 7.4. The
slope is r=ꢀ0.32, indicating a positive charge developing at the reac-
tion center in the activated complex. See Table 1 for data.
Scheme 5. Two-step process for ChE inhibition by triester OP.
Figure 7. Postulated model for inhibition of BChE by phosphonic acid 1f showing stabilization of the dianion as a tetrahedral intermediate analogue
at the active site (a), undergoing slow dissociation of chloride (b), as the first and rate-limiting step in the phosphorylation (c), and with liberation of
the styrene derivative (d).

1286
N. Zhang, J. E. Casida / Bioorg. Med. Chem. 10 (2002) 1281–1290
for 1b and 1f fulfills our expectation that electron-with-
drawing substituents at the phenyl ring can increase the
potency as observed also in the triester OP inhibitor
series; however, the higher potency is not related to a
higher hydrolysis rate as opposed to the OP triester
inhibitors.3 Compound 1f with the 4-trimethylammo-
nium group has the highest potency (IC50=19 mM) but
the lowest hydrolysis rate. The high potency is pre-
sumably due to the high affinity for ammonium cation-
active site interactions as observed for many reversible
and irreversible inhibitors.2,3 Compounds 1c and 1g also
with strong electron-withdrawing groups but at the
3-position have much lower potency, consistent with
the profile of the observed Ki values. It appears that the
steric effect of the substituent plays an important role in
determining potency. Substitution at the 3-position
could make the molecule more bulky and then more
difficult to access the enzyme active center for irrever-
sible inhibition.
moiety and a reactive center to generate an irreversible
inhibitor for BChE, in a manner which may also be
applicable to other enzyme systems.
Experimental
Chemicals and general methods
Chemicals were obtained from commercial sources and
used without further purification, unless otherwise
noted. THF was distilled from sodium-benzophenone
ketyl. Column chromatography was performed on silica
1
gel. H, 13C and 31P NMR spectra were recorded on a
Bruker AMX-300 spectrometer at 300, 75, and
121.5 MHz, respectively. Chemical shifts for 1H and 13
C
are relative to the solvent chemical shifts and for 31P is
relative to 85% H3PO4 (d=0 ppm). Fast atom bom-
bardment (FAB)-high resolution mass spectrometry
(HRMS) was performed by the University of Notre
Dame Mass Spectrometry Facility (Notre Dame, IN,
USA). Melting points were measured on a Fisher–Johns
melting point apparatus without correction. Spectro-
photometric determinations of BChE activity and the
hydrolysis rate of 1 were made with a Hewlett Packard
8452A diode array spectrometer.
Possible model for interaction between BChE and
phosphonic acids 1a–1g
The irreversible inhibition of BChE is presumably
through phosphorylation as suggested by postlabeling
experiments with a labeled triester OP6 and established
in studies with [33P]ethephon.28 Similar to hydrolytic
decomposition, the phosphonic acid bound at the
enzyme active site may produce a highly reactive species
serving as the phosphorylating agent for the serine
hydroxyl or solvent molecule. The unique inhibition
behavior of phosphonic acids 1, in which inhibition
continues to increase even when almost all of the phos-
phonic acid dianion in solution is gone, suggests some
mechanism for localized stabilization of the dianion
(Fig. 7). The main chain nitrogens in the oxyanion hole
might stabilize the negatively charged phosphonic dia-
nion by hydrogen bonding as they do for a tetrahedral
intermediate.29 The phosphonic acid could also exist in
its monoanion form due to its pKa change in non-aqu-
eous environment23,30 or the pH of the enzyme micro-
environment is different from the solution pH. The
monoanion form is much more stable than the dianion
form and serves as a potential long-term phosphorylat-
ing agent and irreversible inhibitor.
Synthesis
Diethyl 2-hydroxy-1-phenylethylphosphonate (3). A solu-
tion of n-BuLi (1.6 M, 5 mL) in hexane was added
dropwise into diethyl benzylphosphonate (1.27 g, 5.6
mmol) in THF (12.0 mL) at ꢀ78 ꢃC under N2. The
solution was stirred at this temperature for 15 min until
a bright yellow color appeared. Paraformaldehyde (0.4
g, 13.3 mmol) was then added and the cooling bath was
removed. The reaction was continued at room tem-
perature with stirring for 30 min until the yellow color
disappeared completely. The solution was acidified by
HCl (2 N ꢂ 5 mL), extracted with CH2Cl2 (3 ꢂ 50 mL)
and dried with anhydrous Na2SO4. Removal of the sol-
vent followed by chromatography with CHCl3 and
MeOH (25:1) gave 3 (1.09 g, 76%). 31P NMR (CDCl3):
d +27.2; 1H NMR (CDCl3): d 7.34–7.30 (m, 5H), 4.22–
3.82 (m, 6H), 3.32 (dt, 1H, J=6.7, 21.5 Hz), 1.29 (t, 3H,
J=7.2 Hz), 1.13 (t, 3H, J=7.2 Hz); 13C NMR (CDCl3):
d 134.2 (d, J=6.7 Hz), 129.1 (d, J=6.7 Hz), 128.6,
127.5, 62.8 (d, J=6.7 Hz), 62.5, 62.1 (d, J=6.7 Hz),
47.6 (d, J=149.8 Hz), 16.3 (d, J=7.8 Hz), 16.2 (d,
J=7.8 Hz); FAB-HRMS calcd for C12H20O4P (MH+):
259.1099, found: 259.1107.
Conclusions
A series of 2-chloro-1-(substituted-phenyl)ethylphos-
phonic acids 1 has been designed and synthesized as
candidate irreversible BChE inhibitors. These phos-
phonic acids have the features of transition state ana-
logues for reversible binding and triester OPs for
irreversible phosphorylation. BChE inhibition by 1 is a
two-stage process with reversible binding at the sub-
strate site followed by irreversible phosphorylation,
which is presumably through the same mechanism as it
hydrolyzes in aqueous media involving dissociation of
chloride as the first and rate-limiting step. At physio-
logical pH, phosphonic acids 1 appear to be stabilized
by the microenvironment of the enzyme active site.
These findings are an example of combining a binding
Diethyl 2-chloro-1-phenylethylphosphonate (2a). Into a
solution of phosphonate 3 (0.9 g, 3.5 mmol) in CCl4 (10
mL) was added Ph3P (1.3 g, 5.0 mmol). The solution
was refluxed for 1 h and then cooled to room tempera-
ture. Hexane (5 mL) was added and the solution was
filtered. The residue was washed by a mixture of CCl4
and hexane (10 mL). Chromatography of the residue
from evaporation of the combined filtrate gave 2a (0.48
g, 50%). 31P NMR (CDCl3) d +24.4; 1H NMR
(CDCl3) d 7.38–7.37 (m, 5H), 4.23–3.87 (m, 4H), 3.76–
3.67 (m, 2H), 3.42 (ddd, 1H, J=4.1, 11.3, 23.1 Hz), 1.31

N. Zhang, J. E. Casida / Bioorg. Med. Chem. 10 (2002) 1281–1290
1287
(t, 3H,=6.9 Hz), 1.10 (t, 3H, J=6.9 Hz); 13C NMR
(CDCl3) d 133.6, 129.2 (d, J=6.7 Hz), 128.6, 127.9, 63.1
(d, J=6.7 Hz), 62.3 (d, J=6.7 Hz), 48.2 (d, J=132.5
Hz), 43.6 (d, J=6.7 Hz), 16.4, 16.1; FAB-HRMS calcd
for C12H19ClO3P (MH+): 277.0760, found: 277.0766.
(dd, 1H, J=7.7, 8.2 Hz), 4.31–3.96 (m, 6H), 3.45 (dt,
1H, J=6.7, 22.6 Hz), 1.32 (t, 3H, J=7.2 Hz), 1.21 (t,
3H, J=7.2 Hz); 13C NMR (CDCl3) d 148.3, 136.9, 135.4,
129.5, 124.1 (d, J=6.7 Hz), 122.5, 62.9 (d, J=6.7 Hz),
62.6 (d, J=6.7 Hz), 62.1, 47.2 (d, J=134.8 Hz), 16.3 (d,
J=6.7 Hz), 16.2 (d, J=6.7 Hz); FAB-HRMS calcd for
C12H19NO6P (MH+): 304.0950, found: 304.0955.
Ethyl ꢀ - diethoxyphosphinyl - ꢀ - (4 - nitrophenyl)acetate
(4b).17 To a suspension of NaH (80% dispersion in
mineral oil, 0.66 g, 22 mmol) in DMF (10 mL) was
added a solution of triethyl phosphonoacetate (4.48 g,
20 mmol) at room temperature. After stirring for 10
min, 1-iodo-4-nitrobenzene (1.95 g, 10 mmol) and cop-
per(I) iodide (4.4 g, 23 mmol) were added in turn. The
mixture was stirred at 100 ꢃC overnight, after which the
reaction was quenched by 10% HCl. The solution was
filtered and extracted with EtOAc. The organic layer
was combined, dried with Na2SO4 and evaporated in
vacuum. The residue was subjected to chromatography
with CHCl3 and EtOAc (6:1) to give 4b (0.91 g, 35%).
Diethyl 2-chloro-1-(4-nitrophenyl)ethylphosphonate (2b).
Reaction of 5b (0.66 g, 2.2 mmol) with Ph3P (0.86 g,
3.3 mmol) in CCl4 (10 mL) followed by workup and
chromatography with CHCl3 and MeOH (30:1) gave 2b
(0.53 g) in 76% yield. Mp 98–100 ꢃC. 31P NMR (CDCl3)
1
d +22.4; H NMR (CDCl3) d 8.24 (d, 2H, J=8.2 Hz),
7.55 (dd, 2H, J=2.1, 8.7 Hz), 4.24–3.85 (m, 6H), 3.56
(ddd, 1H, J=4.1, 11.8, 23.6 Hz), 1.32 (t, 3H, J=7.2
Hz), 1.17 (t, 3H, J=7.2 Hz); 13C NMR (CDCl3) d
147.6, 141.3 (d, J=6.7 Hz), 130.2 (d, J=4.5 Hz), 123.7,
63.3 (d, J=6.7 Hz), 62.8 (d, J=6.7 Hz), 48.2 (d,
J=130.3 Hz), 16.4 (d, J=6.7 Hz); FAB-HRMS calcd
for C12H18ClNO5P (MH+): 322.0611, found: 322.0614.
1
31P NMR (CDCl3) d +17.9; H NMR (CDCl3) d 8.20
(d, 2H, J=8.2 Hz), 7.71 (dd, 2H, J=2.0, 8.7 Hz), 4.36
(d, 1H, J=24.1 Hz), 4.27–4.02 (m, 6H), 1.29 (t, 3H,
J=7.2 Hz), 1.28 (t, 3H, J=6.9 Hz), 1.22 (t, 3H, J=6.9
Hz); 13C NMR (CDCl3) d 167.2 (d, J=4.7 Hz), 148.2,
139.2, 131.3 (d, J=4.8 Hz), 124.2, 64.3 (d, J=5.9 Hz),
64.1 (d, J=6.9 Hz), 63.0, 52.8 (d, J=131.3 Hz), 16.9,
14.7; FAB-HRMS calcd for C14H21NO7P (MH+):
346.1056, found: 346.1066.
Diethyl 2-chloro-1-(3-nitrophenyl)ethylphosphonate (2c).
Reaction of 5c (1.60 g, 5.3 mmol) with Ph3P (2.10 g,
8.0 mmol) in CCl4 (15 mL) followed by workup and
chromatography with CH2Cl2 and MeOH (30:1) gave
2c (1.36 g) in 80% yield. 31P NMR (CDCl3) d +22.6;
1H NMR (CDCl3) d 8.23 (d, 1H, J=2.0 Hz), 8.21 (d,
1H, J=8.7 Hz), 7.73 (d, 1H, J=6.7 Hz), 7.58 (dd, 1H,
J=6.7, 8.7 Hz), 4.26–3.87 (m, 6H), 3.56 (ddd, 1H,
J=4.1, 11.8, 23.6 Hz), 1.32 (t, 3H, J=7.2 Hz), 1.20 (t,
3H, J=7.2 Hz); 13C NMR (CDCl3) d 148.4, 135.9,
135.3, 129.6, 124.2 (d, J=6.7 Hz), 122.9, 63.2 (d, J=6.7
Hz), 62.8 (d, J=6.7 Hz), 47.9 (d, J=130.3 Hz), 43.0,
16.4 (d, J=6.7 Hz), 16.3 (d, J=6.7 Hz); FAB-HRMS
calcd for C12H18ClNO5P (MH+): 322.0611, found:
322.0601.
Ethyl ꢀ - diethoxyphosphinyl - ꢀ - (3 - nitrophenyl)acetate
(4c).17 Reaction of triethyl phosphonoacetate with 1-
iodo-3-nitrobenzene by the above method gave 5 in
67% yield. 31P NMR (CDCl3) d +18.0; 1H NMR
(CDCl3) d 8.39 (s, 1H), 8.18 (d, 1H, J=8.2 Hz), 7.90 (d,
1H, J=7.7 Hz), 7.54 (dd, 1H, J=7.7, 8.2 Hz), 4.36 (d,
1H, J=24.1 Hz), 4.29–4.07 (m, 6H), 1.33–1.22 (m, 9H);
13C NMR (CDCl3) d 166.7, 148.2, 135.8, 133.4, 129.3,
124.7 (d, J=6.7 Hz), 122.9, 63.6, 63.4 (d, J=6.7 Hz), 62.3,
51.8 (d, J=132.5 Hz), 16.2, 14.0; FAB-HRMS calcd for
C14H21NO7P (MH+): 346.1056, found: 346.1055.
Diethyl 1 - (4 - aminophenyl) - 2 - chloroethylphosphonate
(6b). A solution of 2b (0.52 g, 1.6 mmol) in MeOH was
hydrogenated with 10% Pd/C (100 mg) at room tem-
perature for 2 h, after which the solution was filtered
and concentrated, providing 6b as a residue (0.48 g,
Diethyl 2 - hydroxy - 1 - (4 - nitrophenyl)ethylphosphonate
(5b). Into 4b (0.91 g, 2.6 mmol) was added a solution
of borane in THF (1.0 M ꢂ 10 mL). The solution was
stirred at room temperature for 2 days, then MeOH (10
mL) was added. The solution was concentrated after the
evolution of hydrogen stopped. Chromatography of the
residue with CHCl3 and EtOAc (25:1) gave 5b (0.66 g)
100%). Mp 97–98 ꢃC; 31P NMR (CDCl3) d +25.0; H
1
NMR (CDCl3) d 7.15 (dd, 2H, J=2.1, 8.7 Hz), 6.69 (d,
2H, J=8.2 Hz), 4.18–3.36 (m, 6H), 3.29 (ddd, 1H,
J=4.1, 12.3, 23.6 Hz), 1.31 (t, 3H, J=6.9 Hz), 1.13 (t,
3H, J=6.9 Hz); 13C NMR (CDCl3) d 146.0, 130.1 (d,
J=6.7 Hz), 123.0 (d, J=6.7 Hz), 115.4, 63.1 (d, J=6.7
Hz), 62.2 (d, J=9.0 Hz), 47.2 (d, J=132.5 Hz), 44.0 (d,
J=9.0 Hz), 16.3 (d, J=9.0 Hz), 16.2 (d, J=6.7 Hz);
FAB-HRMS calcd for C12H20ClNO3P (MH+):
292.0869, found: 292.0874.
1
in 87% yield. 31P NMR (CDCl3) d +25.7; H NMR
(CDCl3) d 8.21 (d, 2H, J=8.2 Hz), 7.55 (dd, 2H, J=2.1,
8.7 Hz), 4.26–3.94 (m, 6H), 3.45 (dt, 1H, J=6.4, 22.5
Hz), 1.32 (t, 3H, J=7.2 Hz), 1.19 (t, 3H, J=7.2 Hz); 13C
NMR (CDCl3) d 147.4, 142.5, 130.1 (d, J=4.5 Hz), 123.7,
63.1 (d, J=6.7 Hz), 62.6 (d, J=6.7 Hz), 62.2, 47.6 (d,
J=134.8 Hz), 16.4 (d, J=6.7 Hz); FAB-HRMS calcd
for C12H19NO6P (MH+): 304.0950, found: 304.0960.
Diethyl 1 - (3 - aminophenyl) - 2 - chloroethylphosphonate
(6c). Hydrogenation of 2c (1.34 g, 4.2 mmol) in MeOH
in the presence of 10% Pd/C (200 mg) gave 6c (1.17 g)
1
Diethyl 2 - hydroxy - 1 - (3 - nitrophenyl)ethylphosphonate
(5c). Reduction of 4c (2.2 g, 6.4 mmol) with borane
(1.0 M ꢂ 15 mL) at room temperature for 2 days affor-
ded 5c (1.61 g) in 83% yield. 31P NMR (CDCl3) d
in 97% yield. 31P NMR (CDCl3) d +24.6; H NMR
(CDCl3) d 7.14 (t, 1H, J=7.7 Hz), 6.74 (d, 1H, J=7.2
Hz), 6.73 (d, 1H, J=3.6 Hz), 6.65 (d, 1H, J=7.2 Hz),
4.17–3.67 (m, 6H), 3.30 (ddd, 1H, J=4.9, 11.3, 23.6
Hz), 1.31 (t, 3H, J=7.2 Hz), 1.11 (t, 3H, J=7.2 Hz); 13C
NMR (CDCl3) d 146.2, 134.4, 129.5, 119.7, 115.9 (d,
1
+25.6; H NMR (CDCl3) d 8.23 (d, 1H, J=2.0 Hz),
8.16 (d, 1H, J=8.2 Hz), 7.74 (d, 1H, J=7.7 Hz), 7.53

1288
N. Zhang, J. E. Casida / Bioorg. Med. Chem. 10 (2002) 1281–1290
1
J=6.7 Hz), 115.0, 63.2 (d, J=6.7 Hz), 62.2 (d, J=6.7
Hz), 48.0 (d, J=130.3 Hz), 43.7, 16.4, 16.2; FAB-
HRMS calcd for C12H20ClNO3P (MH+): 292.0869,
found: 292.0860.
NMR (CD3OD) d +24.3; H NMR (CD3OD) d 7.99
(br s, 1H), 7.92 (m, 1H), 7.69–7.67 (m, 2H), 4.22–3.98
(m, 7H), 3.72 (s, 9H), 1.32 (t, 3H, J=7.2 Hz), 1.22 (t,
3H, J=7.2 Hz); 13C NMR (CD3OD) d 148.9, 138.6,
132.7 (d, J=4.5 Hz), 131.9, 122.8 (d, J=6.7 Hz), 120.6,
65.0 (d, J=9.0 Hz), 64.7 (d, J=6.7 Hz), 58.1, 45.6 (d,
J=128.0 Hz), 16.8 (d, J=4.5 Hz), 16.7 (d, J=6.7 Hz);
FAB-HRMS calcd for C15H26ClNO3P (MꢀI)+:
334.1339, found: 334.1326.
Diethyl 2 - chloro - 1 -(4 - dimethylaminophenyl)ethylphos-
phonate (2d). Into a solution of 6b (0.29 g, 1.0 mmol),
formaldehyde (37% aqueous solution, 1.0 mL) and
NaCNBH3 (0.2 g, 3.2 mmol) in MeCN (5.0 mL) was
added HOAc (0.1 mL) dropwise. The mixture was stir-
red at room temperature for 2 h before the addition of
another portion of HOAc (0.1 mL) and an additional
stirring for 30 min. Ether (15 mL) was then added and
the mixture was washed with saturated Na2CO3 (5 mL
ꢂ 3) and brine (10 mL). The organic layer was dried
with anhydrous Na2SO4 and solvents were removed by
rotary evaporator. The residue was subjected to chro-
matography with CHCl3 and MeOH (12:1) to give 2d
Conversion ofphosphonate precursors 2a–2g to phospho-
nic acids 1a–1g. General procedure. Into a solution of
phosphonate 2 (ꢅ 2 mmol) in MeCN (10 mL) was
added TMSBr (2.0 mL). The solution was stirred over-
night at 60 ꢃC followed by treatment with MeOH and
water (5.0 mL) at room temperature for 1 h. Removal of
the solvents gave phosphonic acids 1 as pure products
with no pyrophosphonate derivatives as established by
31P NMR.
1
(0.30 g, 94%). 31P NMR (CDCl3) d +25.2; H NMR
(CDCl3) d 7.22 (dd, 2H, J=2.1, 8.7 Hz), 6.72 (d, 2H,
J=8.7 Hz), 4.19–3.87 (m, 5H), 3.76–3.67 (m, 1H), 3.31
(ddd, 1H, J=4.1, 11.3, 23.6 Hz), 2.95 (s, 6H), 1.31 (t,
3H, J=7.2 Hz), 1.13 (t, 3H, J=6.9 Hz) 13C NMR
(CDCl3) d 146.0, 130.1 (d, J=6.7 Hz), 123.0 (d, J=6.7
Hz), 115.4, 63.1 (d, J=6.7 Hz), 62.2 (d, J=9.0 Hz), 47.2
(d, J=132.5 Hz), 44.0 (d, J=9.0 Hz), 16.3 (d, J=9.0
Hz), 16.2 (d, J=6.7 Hz); FAB-HRMS calcd for
C14H23ClNO3P (M+): 319.1104, found: 319.1110.
2-Chloro-1-phenylethylphosphonic acid (1a). 31P NMR
(CD3OD) d +24.1; H NMR (CD3OCD3) d 7.46–7.29
(m, 5H), 4.32–4.25 (m, 1H), 4.14 (dt, 1H, J=5.1, 11.3
Hz), 3.67 (ddd, 1H, J=3.6, 11.3, 24.1 Hz); 13C NMR
(CD3OD) d 134.9, 130.2 (d, J=6.7 Hz), 129.2, 128.3,
49.0 (d, J=128.0 Hz), 44.3. FAB-HRMS calcd for
C8H11ClO3P (MH+) 221.0134, found: 221.0142.
1
2-Chloro-1-(4-nitrophenyl)ethylphosphonic acid (1b). 31P
NMR (CD3OD) d +19.8; 1H NMR (CD3OD) d 8.22 (d,
2H, J=8.2 Hz), 7.62 (dd, 2H, J=2.1, 8.7 Hz), 4.24–3.85
(m, 2H), 3.56 (ddd, 1H, J=4.1, 11.8, 23.6 Hz); 13C
NMR (DMSO-d6) d 146.5, 144.2 (d, J=6.7 Hz), 130.4
(d, J=4.5 Hz), 123.0, 49.0 (d, J=123.6 Hz), 44.3 (d,
J=4.5 Hz); FAB-HRMS calcd for C8H10ClNO5P
(MH+): 265.9985, found: 265.9989.
Diethyl 2 - chloro - 1 - (3 - dimethylaminophenyl)ethylpho-
sphonate (2e). Reductive alkylation of 6c (1.15 g, 3.9
mmol) with a similar procedure for 2d gave 2e in 63%
1
yield. 31P NMR (CDCl3) d +25.0; H NMR (CDCl3) d
7.21 (t, 1H, J=8.2 Hz), 6.69 (m, 3H), 4.21–3.88 (m, 4H),
3.76–3.68 (m, 2H), 3.35 (ddd, 1H, J=4.6, 11.3, 23.6
Hz), 2.96 (s, 6H), 1.32 (t, 3H, J=6.7 Hz), 1.12 (t, 3H,
J=6.7 Hz); 13C NMR (CDCl3) d 150.7, 134.1 (d, J=6.7
Hz), 129.2, 117.2 (d, J=4.5 Hz), 113.4 (d, J=6.7 Hz),
112.1, 63.1 (d, J=6.7 Hz), 62.2 (d, J=6.7 Hz), 48.5 (d,
J=130.3 Hz), 40.5, 16.4 (d, J=6.7 Hz), 16.2 (d, J=6.7
Hz); FAB-HRMS calcd for C14H23ClNO3P (M+):
319.1104, found: 319.1112.
2-Chloro-1-(3-nitrophenyl)ethylphosphonic acid (1c). 31P
1
NMR (CDCl3) d +20.3; H NMR (CDCl3) d 8.29 (d,
1H, J=2.1 Hz), 8.19 (br d, 1H, J=7.7 Hz), 7.80 (br d,
1H, J=7.7 Hz), 7.62 (t, 1H, J=8.2 Hz), 4.30–4.08 (m,
2H), 3.63 (ddd, 1H, J=3.6, 11.8, 23.6 Hz); 13C NMR
(DMSO-d6) d 147.7, 138.4 (d, J=6.7 Hz), 136.0 (d,
J=4.5 Hz), 123.6 (d, J=4.5 Hz), 121.9, 48.3 (d,
J=123.6 Hz), 44.3 (d, J=4.5 Hz); FAB-HRMS calcd
for C8H10ClNO5P (MH+): 265.9985, found: 265.9973.
Diethyl 2-chloro-1-(4-trimethylammoniumphenyl)ethyl-
phosphonate iodide (2f). Into a solution of 2d (0.3 g,
0.94 mmol) in MeNO2 (5.0 mL) was added MeI (0.4
mL). The solution was stirred at room temperature for
60 h and then evaporated. The residue was washed with
ether (3 ꢂ 10 mL) and then recrystallized from acetone
and ether to give 2f (0.30 g, 70%) as crystals. 31P NMR
2-Chloro-1-(4-dimethylaminophenyl)ethylphosphonic acid
1
(1d). 31P NMR (CD3OD) d +20.6; H NMR (CD3OD)
d 7.70 (d, 2H, J=8.2 Hz), 7.61 (dd, 2H, J=2.1, 8.7 Hz),
4.27–4.05 (m, 2H), 3.55 (ddd, 1H, J=3.6, 11.8, 23.6
Hz), 3.33 (s, 6H); 13C NMR (DMSO-d6) d 142.2, 136.1,
130.5, 119.7, 48.4 (d, J=123.6 Hz), 45.2, 44.6 (d, J=6.7
Hz); FAB-HRMS calcd for C10H16ClNO3P (MH+):
264.0556; found: 264.0541.
1
(CD3OD) d +24.3; H NMR (CD3OD) d 7.97 (d, 2H,
J=8.7 Hz), 7.70 (dd, 2H, J=2.6, 9.2 Hz), 4.20–3.97 (m,
6H), 3.72 (s, 9H), 1.32 (t, 3H, J=7.2 Hz), 1.22 (t, 3H,
J=7.2 Hz); 13C NMR (DMSO-d6) d 147.1, 137.0, 131.8,
121.0, 64.3 (d, J=9.0 Hz), 64.1 (d, J=9.0 Hz), 57.6, 46.5
(d, J=128.0 Hz), 43.8, 16.9, 16.8. FAB-HRMS calcd
for C15H26ClNO3P (MꢀI)+: 334.1339, found: 334.1335.
2-Chloro-1-(3-dimethylaminophenyl)ethylphosphonic acid
1
(1e). 31P NMR (CD3OD) d +20.6; H NMR (CD3OD)
Diethyl 2-chloro-1-(3-trimethylammoniumphenyl)ethyl-
phosphonate iodide (2g). Reaction of 2e (0.36 g, 1.1
mmol) with MeI (0.4 mL) in MeNO2 (10 mL) at room
temperature for 60 h gave 2g (0.42 g, 80%) by recrys-
tallization from acetone and ether. Mp 122–123 ꢃC. 31P
d 7.72–7.60 (m, 4H), 4.27–4.09 (m, 2H), 3.59 (ddd, 1H,
J=4.1, 11.3, 23.6 Hz), 3.33 (s, 6H); 13C NMR (DMSO-d6)
d 143.4, 138.2, 129.6, 129.1, 120.5, 118.4, 48.8 (d, J=123.6
Hz), 45.3, 44.4 (d, J=6.7 Hz); FAB-HRMS calcd for
C10H16ClNO3P (MH+): 264.0556, found: 264.0570.

N. Zhang, J. E. Casida / Bioorg. Med. Chem. 10 (2002) 1281–1290
1289
2-Chloro-1-(4-trimethylammoniumphenyl)ethylphospho-
nic acid iodide (1f). 31P NMR (CDCl3) d +20.6; 1H
NMR (CDCl3) d 7.93 (d, 2H, J=8.7 Hz), 7.66 (dd,
2H, J=2.6, 9.2 Hz), 4.27–4.05 (m, 2H), 3.70 (s,
9H), 3.65–3.52 (ddd, 1H, J=4.6, 10.7, 23.6 Hz);
13C NMR (DMSO-d6) d 145.8, 138.1, 130.5, 120.0,
56.4, 48.3 (d, J=123.6 Hz), 44.5 (d, J=4.5 Hz); FAB-
HRMS calcd for C12H19ClO3P (MꢀI)+: 278.0713,
found: 278.0715.
DMSO rather than in pH 7.4 phosphate buffer due to
their hydrolytic instability.
Determination ofIC 50. BChE activity was determined
spectrophotometrically by the Ellman method.31 BChE
(0.1 units) was incubated with phosphonic acid 1 in pH
7.4 100 mM phosphate buffer (1.0 mL) for 90 min at
23 ꢃC before addition of BTCh (0.3 mM) and DTNB
(1.0 mM). Optical density was monitored at 412 nm for
5 min. Inhibitors were tested with a concentration series of
10, 33, 100, 333, 1000 mM in comparison with a control.
IC50 values are reported as the average and standard error
of three experiments. They were determined by plotting
inhibitor concentration on a logarithm scale versus percent
enzyme activity using iterative nonlinear least-squares
regression with the Sigmaplot program (Jandel Scientific
Software, San Rafael, CA, USA).
2-Chloro-1-(3-trimethylammoniumphenyl)ethylphospho-
1
nic acid iodide (1g). 31P NMR (CDCl3) d +20.6; H
NMR (CDCl3) d 7.94 (br s, 1H), 7.89–7.88 (m, 1H),
7.68–7.63 (m, 2H), 4.26–4.15 (m, 2H), 3.71 (s, 9H), 3.65
(ddd, 1H, J=4.6, 10.7, 23.6 Hz); 13C NMR (DMSO-d6)
d 147.0, 138.4 (d, J=6.7 Hz), 130.6, 129.5, 121.2 (d,
J=6.7 Hz), 118.5, 56.4, 48.8 (d, J=123.6 Hz), 44.3 (d,
J=4.5 Hz); FAB-HRMS calcd for C12H19ClO3P (MꢀI)+:
278.0713, found: 278.0715.
Effect ofincubation time. A solution of BChE (2 units)
in phosphate buffer (1.0 mL) was incubated with phos-
phonic acids 1 (100–1000 mM). Enzyme activity was
measured by mixing an aliquot (10 mL) of the incuba-
tion mixture with DTNB (1.0 mM in 100 mM phos-
phate buffer, 1.0 mL) and BTCh (0.3 mM in 100 mM
phosphate buffer, 1.0 mL). The reaction was followed at
412 nm for 5 min.
Hydrolysis of1a–1g at pH 7.4
Identification ofdecomposition products in pH 7.4 bu-f
fer. A solution of 1a (12 mg) in pH 7.4 phosphate buf-
fer (100 mM, 2.0 mL) was kept at room temperature for
2 h and then extracted with CDCl3. The styrene hydro-
1
lysis product in CDCl3 was identified by H NMR. The
phosphorus-containing product from incubation of 1a
(2 mg) in pH 7.4 2,4,6-collidine–HCl buffer (0.7 M, 1.0
mL) for 2 h was identified as phosphate by 31P NMR
comparison with authentic standard.
Determination ofirreversible inhibition. A solution of
BChE (2 units) in phosphate buffer (0.5 mL) was incu-
bated with phosphonic acid 1a (1000 mM) for 90 min
and then passed through a desalting column (Bio-Rad,
Hercules, CA, USA) to remove inhibitor. Enzyme activity
of the eluent (1.0 mL) was measured as above.
Determination ofhydrolysis rate. The hydrolysis rate of
ethephon was determined by 31P NMR in 2,4,6-collidine
(0.7 M)–HCl buffer (pH 7.4). Hydrolysis rates of 1 were
measured spectrophotometrically. A solution of 1 (10 mM
in DMSO, 10 mL) was mixed with pH 7.4 phosphate buffer
(100 mM, 1.0 mL). The reaction was followed by the
absorbance change at 248 nm (1a, 1d, 1e, 1f, 1g) or
320 nm (1b) or 242 nm (1c) for 30ꢅ60 min. Kinetic
constants for these reactions were calculated according
to Kezdy-Swinbourne25 by plotting At versus At+t, where
At is the absorbance at time t, At+t is the absorbance at
time (t+t) and t is a constant time interval, set to
1ꢅ1.5 t1/2 for greatest accuracy. Kinetic constants were
calculated as k=ln(slope)/t.
Determination of Ki and Km. Ki was determined by
measuring the enzyme activity at various substrate
levels in the presence of a known concentration of inhi-
bitor. BChE (0.4 units) in DTNB solution (as above)
was mixed with BTCh solution (0.3–1.0 mM in phos-
phate buffer, 1.0 mL) and inhibitor 1 (10 mL). The
reaction was immediately followed for 0.5–1 min. The
inhibition type was determined by a Lineweaver–Burk
plot and Km (without inhibitor) and Km(app) (with inhi-
bitor) were obtained in the same way.
Acknowledgements
Phosphorylation ofMeOH. A solution of 1a (10 mg) in
CD3OD or MeOH (0.5 mL) was treated with excess
solid K2CO3 (20 mg) and monitored by 1H or 31P
NMR, respectively. This reaction was repeated using
MeOH/water (1:1) (1.0 mL) with KOH (20 mg). The
molar ratio of methyl phosphate and phosphate formed
after 2 h was determined by integration of the appro-
priate peak areas in the 31P NMR spectra.
This work was supported by grant R01 ES08762 from
the National Institute of Environmental Health Sciences
(NIEHS), NIH, and its contents are solely the respon-
sibility of the authors and do not necessarily represent
the official views of NIEHS, NIH. We are greatly
indebted to our laboratory colleagues J. Eric Haux,
Gary Quistad, Yoffi Segall, and Nilantha Sirisoma for
valuable suggestions.
Enzyme studies
Materials. BChE from horse serum (highly purified,
lyophilized powder, item C 1057), BTCh and 5,50-
dithiobis(2-nitrobenzoic acid) (DTNB) were obtained
from Sigma Chemical Co (St. Louis, MO, USA). Stock
solutions of phosphonic acids 1 were prepared in
References and Notes
1. Aldridge, W. N.; Reiner, E. Enzyme Inhibitors as Substrates:
Interactions of Esterases with Esters of Organophosphorus and
Carbamic Acids; North-Holland: Amsterdam, 1972.

1290
N. Zhang, J. E. Casida / Bioorg. Med. Chem. 10 (2002) 1281–1290
2. Eto, M. Organophosphorus Pesticides: Organic and Biologi-
cal Chemistry; CRC: Cleveland, OH, 1974.
14. McKenna, C. E.; Schmidhauser, J. J. Chem. Soc., Chem.
Comm. 1979, 739.
3. Fukuto, T. R.; Metcalf, R. L. J. Agric. Food Chem. 1956, 4,
930.
15. Tarasenko, E. A.; Lukashev, N. V.; Beletskaya, I. P. Tetra-
hedron Lett. 2000, 41, 1611.
4. Hennighausen, G.; Tiefenbach, B.; Lohs, K. Pharmazie
1977, 32, 181.
5. Hennighausen, G.; Tiefenbach, B. Arch. Exper. Vet. Med.
1978, 32, 609.
6. Haux, J. E.; Quistad, G. B.; Casida, J. E. Chem. Res. Toxi-
col. 2000, 13, 646.
7. Tomlin, C. D. S. The Pesticide Manual, 12th ed.; British
Crop Protection Council: Farnham, UK, 2000.
8. For recent examples with a variety of enzymes, see (a) Lu,
H.; Berkman, C. E. Bioorg. Med. Chem. 2001, 9, 395. (b)
Cermak, D. M.; Wiemer, D. F.; Lewis, K.; Hohl, R. J. Bioorg.
Med. Chem. 2000, 8, 2729. (c) Eummer, J. T.; Gibbs, B. S.;
Zahn, T. J.; Sebolt-Leopold, J. S.; Gibbs, R. A. Bioorg. Med.
Chem. 1999, 7, 241. (d) Cushman, M.; Mihalic, J. T.; Kis, K.;
Bacher, A. J. Org. Chem. 1999, 64, 3838. (e) Groves, M. R.;
Yao, Z.-J.; Roller, P. P.; Burke, T. R., Jr; Barford, D. Bio-
chemistry 1998, 37, 17773.
16. Qiu, W.; Burton, D. J. Tetrahedron Lett. 1996, 37, 2745.
17. Minami, T.; Isonaka, T.; Okada, Y.; Ichikawa, J. J. Org.
Chem. 1993, 58, 7009.
18. Lane, C. F. Chem. Rev. 1976, 76, 773.
19. Borch, R. F.; Hassid, A. I. J. Org. Chem. 1972, 37, 1673.
20. Smith, M. B.; March, J. March’s Advanced Organic
Chemistry. Reactions, Mechanisms, and Structure, 5th ed.;
Wiley: New York, 2001.
21. Conant, J. B.; Jackson, E. L. J. Am. Chem. Soc. 1921, 43,
1665.
22. Conant, J. B.; Jackson, E. L. J. Am. Chem. Soc. 1924, 46,
1003.
23. Calvo, K. C.; Westheimer, F. H. J. Am. Chem. Soc. 1984,
106, 4205.
24. Segall, Y.; Toia, R. F.; Casida, J. E. Phosphorus, Sulfur
Silicon 1993, 75, 191.
25. Espenson, J. H. Chemical Kinetics and Reaction Mechan-
isms; McGraw-Hill: New York, 1981.
26. Segall, Y. Personal communication.
9. Gregory, M. J.; Higgins, G. M. C. J. Chem. Soc. Perkin
Trans. 2 1974, 711.
10. Sussman, J. L.; Harel, M.; Frolow, F.; Oefner, C.; Gold-
man, A.; Toker, L.; Silman, I. Science 1991, 253, 872.
11. Maynard, J. A.; Swan, J. M. Aust. J. Chem. 1963, 16,
596.
12. Maynard, J. A.; Swan, J. M. Aust. J. Chem. 1963, 16,
609.
27. Aldridge, W. N. Biochem. J. 1950, 46, 451.
28. Haux, J. E. and Casida, J. E. Personal communication.
29. Harel, M.; Quinn, D. M.; Nair, H. K.; Silman, I.; Suss-
man, J. L. J. Am. Chem. Soc. 1996, 118, 2340.
30. Semiokhin, I. A.; Andreen, Y. P. Russ. J. Phys. Chem.
(Engl. Transl.) 1966, 40, 1161.
13. McKenna, C. E.; Higa, M. T.; Cheung, N. H.; McKenna,
M.-C. Tetrahedron Lett. 1977, 155.
31. Ellman, G. L.; Courtney, K. D.; Andres, V., Jr; Feather-
stone, R. M. Biochem. Pharmacol. 1961, 7, 88.