Novel Organophosphate Ligand O-(2-Fluoroethyl)-O-(p-Nitrophenyl)Methylphosphonate: Synthesis, Hydrolytic Stability and Analysis of the Inhibition and Reactivation of Cholinesterases

Article abstract of DOI:10.1021/acs.chemrestox.6b00160

The organophosphate O-(2-fluoroethyl)-O-(p-nitrophenyl) methyphosphonate 1 is the first-in-class, fluorine-18 radiolabeled organophosphate inhibitor ([¹⁸F]1) of acetylcholinesterase (AChE). In rats, [¹⁸F]1 localizes in AChE rich regions of the brain and other tissues where it likely exists as the (CH₃)(¹⁸FCH₂CH₂O)P(O)-AChE adduct (ChE-1). Characterization of this adduct would define the inhibition mechanism and subsequent postinhibitory pathways and reactivation rates. To validate this adduct, the stability (hydrolysis) of 1 and ChE-1 reactivation rates were determined. Base hydrolysis of 1 yields p-nitrophenol and (CH₃) (FCH₂CH₂O)P(O)OH with pseudo first order rate constants (k_obsd) at pH 7.4 (PBS) of 3.25 × 10^-4 min^-1 (t_1/2 = 35.5 h) at 25 °C and 8.70 × 10^-4 min^-1 (t_1/2 = 13.3 h) at 37 °C. Compound 1 was a potent inhibitor of human acetylcholinesterase (HuAChE; k_i = 7.5 × 10⁵ M^-1 min^-1), electric eel acetylcholinesterase (EEAChE) (k_i = 3.0 × 10⁶ M^-1 min^-1), and human serum butyrylcholinesterase (HuBChE; 1.95 × 10⁵ M^-1 min^-1). Spontaneous and oxime-mediated reactivation rates for the (CH₃) (FCH₂CH₂O)P(O)-serine ChE adducts using 2-PAM (10 μM) were (a) HuAChE 8.8 × 10^-5 min^-1 (t_1/2 = 131.2 h) and 2.41 × 10^-2 min^-1 (t_1/2 = 0.48 h), (b) EEAChE 9.32 × 10^-3 min^-1 (t_1/2 = 1.24 h) and 3.33 × 10^-2 min^-1 (t_1/2 = 0.35 h), and (c) HuBChE 1.16 × 10^-4 min^-1 (t_1/2 = 99.6 h) and 4.19 × 10^-2 min^-1 (t_1/2 = 0.27 h). All ChE-1 adducts undergo rapid and near complete restoration of enzyme activity following addition of 2-PAM (30 min), and no aging was observed for either reactivation process. The fast reactivation rates and absence of aging of ChE-1 adducts are explained on the basis of the electron-withdrawing fluorine group that favors the nucleophilic reactivation processes but disfavors cation-based dealkylation aging mechanisms. Therefore, the likely fate of radiolabeled compound 1 in vivo is the formation of (CH₃)(FCH₂CH₂O)P(O)-serine adducts and monoacid (CH₃)(FCH₂CH₂O)P(O)OH from hydrolysis and reactivation.

Full text of DOI:10.1021/acs.chemrestox.6b00160

Article
pubs.acs.org/crt
Novel Organophosphate Ligand O‑(2-Fluoroethyl)-
O‑(p‑Nitrophenyl)Methylphosphonate: Synthesis, Hydrolytic Stability
and Analysis of the Inhibition and Reactivation of Cholinesterases
Chih-Kai Chao,† S. Kaleem Ahmed,†,‡ John M. Gerdes,†,‡ and Charles M. Thompson*,†
†Department of Biomedical and Pharmaceutical Sciences, University of Montana, Missoula, Montana 59812, United States
‡Center for Neuromolecular Research, Drug Discovery Division, Southern Research Institute, Birmingham, Alabama 35205, United
States
ABSTRACT: The organophosphate O-(2-fluoroethyl)-O-(p-
nitrophenyl) methyphosphonate 1 is the first-in-class, fluorine-
18 radiolabeled organophosphate inhibitor ([18F]1) of
acetylcholinesterase (AChE). In rats, [18F]1 localizes in
AChE rich regions of the brain and other tissues where it
likely exists as the (CH3)(18FCH2CH2O)P(O)-AChE adduct
(ChE-1). Characterization of this adduct would define the
inhibition mechanism and subsequent postinhibitory pathways
and reactivation rates. To validate this adduct, the stability
(hydrolysis) of 1 and ChE-1 reactivation rates were
determined. Base hydrolysis of 1 yields p-nitrophenol and
(CH3) (FCH2CH2O)P(O)OH with pseudo first order rate
constants (kobsd) at pH 7.4 (PBS) of 3.25 × 10−4 min−1 (t1/2 = 35.5 h) at 25 °C and 8.70 × 10−4 min−1 (t1/2 = 13.3 h) at 37 °C.
Compound 1 was a potent inhibitor of human acetylcholinesterase (HuAChE; ki = 7.5 × 105 M−1 min−1), electric eel
acetylcholinesterase (EEAChE) (ki = 3.0 × 106 M−1 min−1), and human serum butyrylcholinesterase (HuBChE; 1.95 × 105 M−1
min−1). Spontaneous and oxime-mediated reactivation rates for the (CH3) (FCH2CH2O)P(O)-serine ChE adducts using 2-PAM
(10 μM) were (a) HuAChE 8.8 × 10−5 min−1 (t1/2 = 131.2 h) and 2.41 × 10−2 min−1 (t1/2 = 0.48 h), (b) EEAChE 9.32 × 10−3
min−1 (t1/2 = 1.24 h) and 3.33 × 10−2 min−1 (t1/2 = 0.35 h), and (c) HuBChE 1.16 × 10−4 min−1 (t1/2 = 99.6 h) and 4.19 × 10−2
min−1 (t1/2 = 0.27 h). All ChE-1 adducts undergo rapid and near complete restoration of enzyme activity following addition of 2-
PAM (30 min), and no aging was observed for either reactivation process. The fast reactivation rates and absence of aging of
ChE-1 adducts are explained on the basis of the electron-withdrawing fluorine group that favors the nucleophilic reactivation
processes but disfavors cation-based dealkylation aging mechanisms. Therefore, the likely fate of radiolabeled compound 1 in vivo
is the formation of (CH3)(FCH2CH2O)P(O)-serine adducts and monoacid (CH3)(FCH2CH2O)P(O)OH from hydrolysis and
reactivation.
thiocholine), as compared with insecticide structures that often
contain O-aryl leaving groups with higher conjugate acid pKa
values.
INTRODUCTION
■
Organophosphate (OP) compounds are primarily recognized
as useful chemicals for crop protection. However, included in
this class are structurally similar OP nerve agents that have been
deployed as nondiscriminating chemical weapons. Both OP
types are toxic to mammals and insects due to the initial
inactivation of acetylcholinesterase (AChE), the enzyme
responsible for the hydrolysis of the neurotransmitter
acetylcholine (ACh) that is found within central and peripheral
nervous tissues and blood.1−7 The difference in toxic action is
largely due to reactivity. Most OP insecticides are thionate
(PS) triesters that must first be converted into the more
reactive oxon form (PO) (Scheme 1; R = alkyl; Y = O-alkyl;
leaving group = Z) to inactivate AChE. Conversely, OP nerve
agents exist in the reactive oxon form and further differ from
insecticide structures in that they are phosphonates that contain
a direct carbon−phosphorus (C−P) bond (e.g., Y = Me,
Scheme 1). Nerve agents typically contain highly labile leaving
groups that lead to rapid inactivation of AChE (e.g., Z = F,
Despite these differences, OP insecticide oxons and OP
nerve agents inhibit AChE and other cholinesterases by the
same mechanism, namely, covalent attachment of the OP to the
AChE active site serine concomitant with the ejection of the
leaving group to form an OP-AChE adduct (Scheme 1).8−10
OP-AChE adducts are relatively stable, but many undergo
secondary reactions by discrete mechanisms depending on the
nature of the remaining groups attached to phosphorus. For
example, OP-adducts containing small alkyl ester groups (R =
Me, Et) are easily cleaved at the phospho-serine bond thereby
reactivating AChE.3,11 Conversely, OP-AChE adducts with
highly branched alkyl ester groups (R = iPr, etc.) derived from
OP nerve agents are less likely to be cleaved at the phospho-
Received: May 9, 2016
© XXXX American Chemical Society
A
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fluoroethoxy (Scheme 1; R = CH2CH2F). Similarly, tracer
[18F]1 when evaluated in vivo within rats is found to localize
within AChE rich central and peripheral nervous system tissues
and in blood, where the majority of measured tissue
radioactivity over the course of 1−2 h post-tracer admin-
istration is presumed to be the 18F-OP-AChE adduct in the
CNS and a mixture of 18F-OP-BChE (butyrylcholinesterase)
and 18F-OP-AChE adducts in blood and elsewhere.
Scheme 1. Structure of OP Insecticides and Nerve Agents,
Their Reaction with Acetylcholinesterase to Afford OP-
AChE Adducts, and the Possible Adduct Fates
From these initial in vitro and tracer PET imaging studies, we
sought to better understand the mechanism of action of the
unique fluorine-containing OPs and the resultant cholinesterase
adducts since the presence of a fluorine atom could influence
the rate and preference of the reactivation or aging processes.
We initially approached the adduct fate inquiry by employing in
vitro kinetic measures using nonradioactive 1 and determining
the rate constants of the OP-AChE and OP-BChE adduct
reactivation processes relative to the competitive aging
mechanism. Because the OP-ChE adducts formed from 1
differs from the OP-ChE adduct from the nerve agent VX only
in the beta-fluorine atom, the role of this electronegative atom
on the reactivation and aging reactions could be effectively
appraised by considering similar in vitro measures previously
conducted with VX.21−23
VX is a 10- to 100-fold more potent anticholinesterase
inhibitor (ki ∼ 107 M−1 min−1) than compound 1 due to the
improved attraction for AChE and reactivity of the beta-
diisopropylamino thiolester leaving group. VX yields an OP-
AChE adduct in which the phosphylated serine bears an O-
ethyl methylphosphonate.18 The O-ethyl methylphosphonate
OP-AChE adduct formed from VX undergoes spontaneous
reactivation in humans (1% per hour over 70 h), and the ethyl
ester moiety predictably undergoes aging very slowly relative to
branched OP adducts derived from other nerve agents.24,25 The
rate constants of oxime-mediated reactivation (kr) of rat brain
homogenates inhibited by VX are 4.7 × 10−2 min−1 and 3.30 ×
10−1 min−1 using at 10−1 to 10−8 M of the nucleophilic
reactivation agents 2-pyridine aldoxime methiodide (2-PAM)
and obidoxime, respectively.22,26 Treatment of VX-inhibited
blood using 2-PAM leads to 55−85% reactivation of AChE
activity within 24 h further demonstrating a preference for
reactivation over aging in vivo (Scheme 1).
serine bond due to steric and electronic effects and are more
susceptible to a dealkylation process known as “aging”.8,12,13
Therefore, the differences between the two OP-adduct
mechanistic fates are manifested in distinct kinetic processes
that are predicated to a large extent on the OP ester
substituents of the OP-AChE adduct. Reactivation14−16 may
be further distinguished as a spontaneous process in which
water acts as a nucleophile (hydrolysis) or more rapidly
induced in an oxime-mediated nucleophilic process, both of
which lead to scission of the AChE serine O−P bond.8,11,13,17
Alternatively, aging is mechanistically aligned with a putative
cationic process (protonation) leading to scission at an alkyl-
oxygen bond affording a toxicologically deleterious AChE-
phosphoanion (AChE-OP(O)(R)(O−) species that is recalci-
trant to reactivate even with oximes.5,7,8,13,15,18 As a result,
branched phosphoesters such as iPrO- and OCH(CH3)C-
(CH3)3 that can better stabilize cations in the transition state
favor aging and proceed via a path alternate to reactivation.
Clearly, reactivation and aging mechanisms are competing
postinhibitory processes whose rates and outcomes are
influenced by substituents with opposing electronic properties.
We recently reported19,20 the first-in-class fluorine-18 (18F)
labeled positron emission tomography (PET) imaging tracer
[18F]1 where the 18F-radionuclide is attached to the beta-
position of an AChE-reactive phosphonate ethyl ester moiety
(Figure 1). The tracer design was based on a hybridized
On the basis of the structural similarity of 1 to VX, the ChE-
1 adduct would be expected to undergo similar spontaneous
and oxime-mediated reactivation rates and show correspond-
ingly slow aging rates. In part, a more detailed evaluation of the
postinhibitory kinetic profile of ChE-1 adducts for acetyl- and
butyrylcholinesterase would support the proposed mechanism
of the in vivo PET tracer in blood and AChE-rich tissues.
Herein, we report the rates of spontaneous reactivation and
oxime-mediated reactivation for the ChE-1 adducts, namely,
(CH3) (FCH2CH2O)P(O)-ChE, resulting from inhibition of
acetyl- and butyrylcholinesterase by compound 1. Also included
is an evaluation of the postinhibitory rates with electric eel
AChE, which exhibits greatly extended solution stability and
can be used to examine the progress of spontaneous
reactivation over several days. To accomplish the kinetic
evaluations, an improved synthesis and the in vitro hydrolytic
stability of 1 are also reported since adequate quantities were
needed, and the solution half-life is an important determinant
in assessing the amount of intact inhibitor for in vitro and in vivo
experiments.
Figure 1. Structures of 2-fluoroethyl 4-nitrophenyl methylphospho-
nate 1 and PET imaging tracer [18F]1.
scaffold of paraoxon, (EtO)2P(O)-OPhNO2, and the nerve
agent VX, in which the resultant OP-cholinesterase adduct
would closely resemble that formed from VX. The non-
radioactive form of 1 possesses excellent activity as an AChE
inhibitor (recombinant human AChE; ki ∼ 106 M−1 min−1) by
in vitro measures,20 suggesting that the structure of the resultant
OP-AChE adduct most likely results from ejection of the p-
nitrophenoxy leaving group with retention of the beta-
B
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standard in the same solution in which 1 mg/mL was equivalent to
1.89 mg/mL of compound 1.
EXPERIMENTAL PROCEDURES
■
General.19,20 Chemicals were obtained from Sigma-Aldrich (St.
Louis, MO) unless otherwise noted. Flash chromatography was
conducted using silica gel (200−300 mesh). Thin-layer chromatog-
raphy (TLC) was visualized by UV and/or staining by 2,6-
Evaluation of Stability of Compound 1. A stock solution of
compound 1 in acetonitrile was diluted in acetonitrile/phosphate-
buffered saline at pH 7.4 (1:9) to afford a concentration of 10.0 μg/
mL. The absorbance at 405 nm was continuously monitored at 25 or
37 °C for 24 h using a BioTek Synergy Mx microplate reader or visible
detection and retention time by HPLC. The observed rate constant
(kobsd) was calculated according to pseudo-first-order kinetics.
Inhibition of HuAChE, EEAChE, and HuBChE by Compound
1. The bimolecular inhibition constant (ki) for compound 1 against
recombinant human (HuAChE), human serum butyrylcholinesterase
(HuBChE), and electric eel acetylcholinesterase (EEAChE) were
determined using previously published methods20 adapted from the
original spectrophotometric assay by Ellman.27
Spontaneous Reactivation of HuAChE, HuBChE, and EE-
AChE Following Inhibition by 1. HuAChE (∼0.01 mg; 0.16 nmol)
or HuBChE (∼0. One mg; 1.6 nmol) was diluted in phosphate-
buffered saline (PBS, pH 7.4) containing 0.76% (w/v) asolectin
(added to stabilize the protein over the course of the reactivation
experiments) and a control rate of substrate hydrolysis (activity)
recorded. The enzyme solution was then incubated with 10 μM of 1 at
25 or 37 °C to achieve >90% inhibition. Aliquots of each sample at
various time points were placed in a 96-well microplate followed by
mixing with the substrate (1.0 mM ATChI or BTChI) and Ellman’s
reagent (0.3 mM DTNB) in PBS. At the 25 or 37 °C temperature
experiments, the absorbance at 412 nm was recorded at various time
points correlating with the production of the thionitrobenzoic acid
anion. The overall recovery and rate constant of reactivation (kr) were
determined for each enzyme according to the following equation:
dibromoquinone-4-chloroimide (DBQ) or iodine. The H-, 19F-, and
1
31P NMR spectra were recorded on a Varian Avance 400-MHz
spectrometer. High resolution mass spectrometry was performed with
a Micromass LCT -Waters 2795 HPLC with a 2487 UV detector using
caffeine as a molecular weight standard. Biochemical reagents were
purchased from Fisher Scientific or Sigma-Aldrich (St. Louis, MO).
Electric eel acetylcholinesterase (EEAChE), human recombinant
acetylcholinesterase (HuAChE), human serum butyrylcholinesterase
(HuBChE), acetylthiocholine iodide (ATChI), S-butyrylthiocholine
iodide (BTChI), 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB), and
asolectin were purchased from Sigma-Aldrich.
One-Pot Synthesis of 2-Fluoroethyl 4-Nitrophenyl Methyl-
phosphonate 1.19,20 A solution of diisopropylamine (0.13 mL, 0.98
mmol) and triethylamine (0.13 mL, 0.98 mmol) in dry THF (5 mL)
was added dropwise to a solution of dichloromethylphosphine (0.095
mL, 1.1 mmol) at −10 °C with stirring (Scheme 2). The reaction was
Scheme 2. Improved, One-Pot Synthesis of 2-Fluoroethyl 4-
Nitrophenyl Methylphosphonate 1
(enzyme activity)t − (enzyme activity)0
(enzyme activity)max − (enzyme activity)0
= 1 − e−k ·t
r
An EEAChE solution in pH 7.4 PBS (50 mU/mL) was reacted with
aliquots of a stock solution of 1 in acetonitrile/phosphate-buffered
saline (1:9, 1 mg/mL) to achieve >90% inhibition. The inhibited
EEAChE mixture was incubated at 25 °C and sampled for enzyme
activity at 0, 0.25, 0.50, 1.0, 2.0, 4.0, 22, 94, 190, 358, 526, and 694 h,
for which percent recovery and the rate constant of reactivation (kr)
were determined for as described above at 25 or 37 °C.
Oxime-Mediated Reactivation of HuAChE, HuBChE, and
EEAChE. Compound 1 was prepared as 100.0, 200.0, 400.0, 800.0, or
1600 nM solutions in acetonitrile, and 20.0 μL aliquots of each
concentration were added to 180.0 μL of enzyme solution in PBS
(HuAChE and HuBChE contained 0.76% w/v asolectin). After
vortexing, each mixture was incubated at 25 °C for 30 min to achieve
>90% reduction in activity based on initial (untreated) velocity. A 2.50
μL aliquot from each inhibition solution was then added to a 250 μL
solution of Ellman’s reagent (1.0 mM ATChI or BTChI; 0.3 M
DTNB) containing 2-pyridine-aldoxime methiodide (2-PAM) (10
μM) and the enzyme activity measured at 25 °C as the change in
absorbance at 412 nm versus time. The rate constant of reactivation
(kr) was calculated as indicated above.
allowed to continue for 1 h at 25 °C whereupon a solution of p-
nitrophenol (0.14 g, 0.98 mmol) and triethylamine (0.13 mL, 0.98
mmol) in dry THF (10 mL) were added at 25 °C with vigorous
stirring over 3 h. Immediate formation of triethylamine hydrochloride
occurred, and the formation of intermediate 4 (31P NMR 128.0 ppm)
was observed along with the protonated intermediate 5 (31P NMR
185.71 ppm). To this reaction mixture was added 2-fluoroethanol
(0.063 mL, 1.1 mmol) in dry THF (5 mL) at 25 °C. The reaction
mixture was stirred for 14 h under argon to form methylphosphonite
6. Subsequently, the flask was opened to an oxygen atmosphere to
form 1, stirred for 5 min, then the reaction was filtered and extracted
with 5 mL CHCl3, and the filtrate was concentrated in vacuo.
Purification was conducted by flash chromatography using 4:6 ethyl
RESULTS AND DISCUSSION
1
■
acetate/hexanes to obtain pure 1 (148 mg, 52%): H NMR (400.18
Synthesis of 2-Fluoroethyl 4-Nitrophenyl Methyl-
phosphonate 1. We previously reported the cold ligand
synthesis of fluoroethoxy phosphonate 1 via carbodiimide
coupling of fluoroethanol with the corresponding phosphonic
acid.20 For preparation of [18F]1, beta-fluoroethyl tosylate was
reacted with MeP(O) (OPNP)O−Cs+ using microwave-assisted
accleration.19 Since neither approach was found to be amenable
to a larger scale, a new experimental protocol was
examined28−34 (Scheme 2). Commercially available dichlor-
omethylphosphine 2 (31P NMR 191.0 ppm) was reacted
sequentially with diisopropylamine and p-nitrophenol to afford
phosphonamidite 431 (31P NMR 128.0 ppm; 185.71 ppm in
protonated form). When 2-fluoroethanol is added, the
MHz, CDCl3) δ/ppm 8.84 (d, J = 9.2 Hz, 2H), 7.41 (d, J = 9.2 Hz,
2H), 4.50−4.67 (m, 2H), 4.24−4.47 (m, 2H), 1.75 (d, J = 17.8 Hz,
3H). 13C NMR (100.63 MHz, CDCl3) δ/ppm 155.14, 144.75, 125.89,
121.11, 82.72, 81.35, 12.21 (d, JCP = 144.15 Hz). 31P NMR (162.0
MHz, CDCl3) δ/ppm 29.33. 19F NMR (376.55 MHz, CDCl3) δ/ppm
−224.47. ESI-MS 264 (M+1), HRMS: 264.0434 [(M+H)+].
Hydrolysis of Compound 1. Compound 1 (5 mg; 0.019 mmol)
was dissolved in acetonitrile (1 mL), and the solution was added to 0.1
M NaOH (99 mL) at 25 °C, where the hydrolysis was monitored for
30 min. The predominant hydrolytic product, p-nitrophenoxide
(PNP), was spectrophotometrically quantified at 405 nm using a
BioTek Synergy Mx microplate reader and covalidated versus the
standard by visible detection and retention time by HPLC. For
quantification, p-nitrophenol (PNP) was used as the reference
C
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Table 1. Hydrolysis of O-(2-Fluoroethyl)-O-(p-nitrophenyl)methylphosphonate 1 (Pseudo-First-Order Kinetics)
a
b
structure
°C (°K)
kobsd (min−1
)
t1/2 (h)
R2
MeP(O)(OCH2CH2F)(OPNP)
25 (303)
37 (310)
37 (310)
3.25 × 10−4 (n = 4; 0.66)
8.70 × 10−4 (n = 4; 1.08)
4.03 × 10−5
35.6
13.3
4.8
0.962
0.998
MeP(O)(OCH2CH3) (OPNP)40
a
b
ln(Ct/C0) vs 1/t where Ct = decrease in 1. t1/2 = 0.693/kobsd
.
a
Table 2. Rate Constants for the Inhibition and Reactivation of HuAChE, HuBChE, and EEAChE by Compound 1
process
HuAChE
HuBChE
EEAChE
b
bimolecular inhibition constant (ki; M−1min−1
)
7.49 ( 1.37) × 105
1.95 ( 0.48) × 105
5.90 ( 0.15) × 106
spontaneous reactivation (kr; min−1
)
9.31 ( 0.07) × 10−5
(t1/2 = 124 h)
1.13 ( 0.02) × 10−4
(t1/2 = 102 h)
8.75 ( 1.89) × 10−3
(t1/2 = 1.3 h)
c
oxime-mediated reactivation (koxime; min−1) 30 min
2.40 ( 0.14) × 10−2
(t1/2 = 0.48 h)
2.47 ( 0.12) × 10−2
(t1/2 = 0.47 h)
1.14 ( 0.34) × 10−2
(t1/2 = 1.0 h)
4.19 ( 0.12) × 10−2
(t1/2 = 0.28 h)
3.37 ( 0.74) × 10−2
(t1/2 = 0.34 h)
postinhibition
c
oxime-mediated reactivation (koxime; min−1) 18 h
nd
postinhibition
a
b
c
Mean SD (n ≥ 3). ki for EEAChE from ref 20. 2-Pyridine aldoxime methiodide (2-PAM). nd = not determined due to negligible inhibition.
intermediate trivalent phosphonate diester 6 is formed and
upon exposure to air for 5 min is converted to the desired oxon
(PO) product 1.
37 °C. The hydrolysis rate of 1 at 37 °C is expectedly slower
than that found for the ethyl (nonfluorinated) compound
(CH3) (CH3CH2O)P(O)OPNP with (khyd) = 2.42 × 10−3
min−1 (t1/2 = 4.8 h) conducted at pH 8.3.40 The hydrolysis rates
indicate that compound 1 is stable for the duration of the
inhibition experiments but would hydrolyze to a large extent at
the latter time points during reactivation measurements, which
would suggest that reinhibition of reactivated enzyme would be
unlikely. As noted, the rate constants for 1 are in general
agreement with those found for triester OP compounds bearing
a p-nitrophenoxy leaving group (e.g., paraoxon), although the
reported hydrolysis conditions differed.41 From the hydrolysis
data, it is concluded that the fluorine atom does not cause
enhanced hydrolytic degradation of compound 1 and is stable
enough for accurate in vitro enzyme assays, in PBS formulations
used in vivo for the tracer [18F]119 and over the course of the
tracer lifetime.
Inhibition of HuAChE, HuBChE, and EEAChE by 1.
Compound 1 was previously shown to be a relatively potent
inhibitor of HuAChE and EEAChE;20 however, the inhibition
of human butyrylcholinesterase (HuBChE) had not been
evaluated. Compound 1 was found to be a relatively potent
inhibitor of HuBChE with ki = 1.95 × 105 M−1min−1 (Table 2),
which is only 7.4-fold weaker than paraoxon as the inhibitor (ki
= 1.44 × 106 M−1min−1). This ratio correlates with data
showing that compound 1 is an 8.4-fold weaker inhibitor (ki =
7.49 × 105 M−1 min−1) than paraoxon (6.28 × 106 M−1 min−1)
toward HuAChE (Table 2).20 The inhibition rate constants for
1 relative to paraoxon indicate that the anticholinesterase
activity does not appear to be affected by the presence of the
fluorine atom. In comparison, work by Chambers and co-
workers demonstrated23,42 that the p-nitrophenoxy analogue of
VX, (Me)(EtO)P(O)(OPNP) and previously reported by
Fukuto,40 is a potent inhibitor (ki ∼ 105 M−1min−1) of various
acetyl- and butyrylcholinesterases and simulates the mechanism
of VX inhibition.23,42
The one-pot, four-step process (Scheme 2) takes 18 h and
affords a 52% yield of desired product, and 1 is characterized by
a
31P NMR shift of 29.3 ppm that is identical when compared
to the authentic material.19 The synthetic sequence relies upon
in situ protonation of the phosphonamidite nitrogen to convert
the diisopropylamine to a better leaving group and permit
reaction with the weakly nucleophilic fluoroethanol. Reaction of
alcohols with N,N-dialkylphosphonamidites is typically con-
ducted in sequence with tetrazole and oxidizing
agents.28−30,35−39 However, in this reaction sequence triethyl-
amine hydrochloride or p-nitrophenol serves as the proton
donor, and the use of oxidizing agents such as m-CPBA and t-
BuOOH to produce 1 from 6 was less effective than air
exposure. Product 1 may also be accessed via purification of 4
by column chromatography followed by the addition of
tetrazole31 and fluoroethanol, and oxidation with t-BuOOH
(∼25% from 4).
Hydrolytic Stability of 1. Hydrolysis rates were
determined for compound 1 since any significant degradation
over the time course of the reactivation or in vivo studies would
influence the mechanistic interpretation. The p-nitrophenoxy
(PNP) leaving group of 1 was specifically installed in the tracer
design to minimize volatility, reduce toxicity relative to nerve
agents, and slow the OP hydrolysis rate relative to a fluorine
leaving group present on nerve agents (e.g., sarin, soman).
However, the PNP group is labile (pKa ∼7.2), and an
assessment of the hydrolysis of 1 under the assay conditions
is warranted. Since the hydrolysis of compound 1 (λmax = 281
nm) forms nitrophenol and (CH3) (FCH2CH2O)P(O)OH,
the nitrophenolate anion (λmax = 405 nm) was used as an
indicator. Aliquots (30 μg/mL) of a 10.8 mM stock solution of
compound 1, prepared from 2.86 mg/mL (1.51 mg/mL PNP)
in CH3CN, were diluted 100-fold with 0.1 M NaOH, and the
phenolate absorbance was measured at 405 nm. Construction
of a standard curve afforded a slope = 0.180 (R2 = 0.99) that
was used to determine an average maximal response of 10.2
0.4 mM of 1. Compound 1 was then formulated in phosphate
buffered saline (PBS) at pH 7.4 and the stability of this solution
determined at 25 and 37 °C (Table 1). The pseudo-first-order
rate constants (kobsd) at pH 7.4 (PBS) were 3.25 × 10−4 min−1
(t1/2 = 35.6 h) at 25 °C and 8.70 × 10−4 min−1 (t1/2 = 13.3 h) at
Enzymatic Reactivation. HuAChE, EEAChE, and
HuBChE were used to evaluate the rate and extent of
spontaneous and oxime-mediated reactivation following in-
hibition by compound 1 to determine the influence and
contribution of the fluorine atom. Human AChE and BChE
were assessed to identify differences in the response of
mammalian cholinesterases to 1, which may advance our
understanding of the previously evaluated tracer interaction
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with AChE- and BChE-rich CNS tissues and blood.19 Electric
eel acetylcholinesterase (EEAChE) was examined because it has
excellent solution stability over an extended experimental time
course that would permit multiday, extended monitoring of the
reactivation experiments.
Because of the greater solution stability of EEAChE, the
spontaneous reactivation was followed over multiple days.
When EEAChE was inhibited >95% by 1−5 nM with
compound 1, complete restoration of the EEAChE activity
was observed within a day, and inhibition using a higher
concentration of 1 (20−100 nM) still led to >98% recovery of
the original activity by 3 weeks. These data suggest that very
little aging occurred, although aging cannot be completely ruled
out for the mammalian enzymes.
Assessment of Spontaneous Reactivation. Spontaneous
reactivation occurs when the phosphoserine bond of an OP-
cholinesterase adduct (Scheme 1) is cleaved without the aid of
reactivating agents.1,2,13,15,22,43 In principle, the resultant ChE,
free of OP, may have part or all of the catalytic activity restored,
although the fraction of ChE that is reactivated varies with the
structure of the OP attached and the cholinesterase subtype
and reactivator used. OP-ChE adducts with small phosphoester
groups such as MeO- or EtO- undergo spontaneous
reactivation more readily with half-lives ranging from a few
minutes to hours.5,13 Conversely, OP-ChE adducts containing
branched esters such as iPrO- or OCH(Me)C(Me)3 are slower
to reactivate and prone to aging. Spontaneous reactivation
occurs at rates far slower (>100×) than reactivation induced by
oxime countermeasures such as 2-pyridine aldoxime methio-
dide (2-PAM). To evaluate spontaneous reactivation, a solution
containing huAChE or huBChE was treated with compound 1
at various concentrations (10−160 nM) leading to greater than
95% inhibition at 30 min. The recovery of enzyme activity at
each concentration was then monitored following a 40-fold
dilution with PBS at pH 7.4 to minimize further inhibition.
The spontaneous reactivation rate constants kr for HuAChE
and HuBChE following inhibition by 1 were 9.31 × 10−5 min−1
(t1/2 = 124 h) and 1.13 × 10−4 min−1 (t1/2 = 102 h) (Table 2).
The spontaneous reactivation rates did not show any evidence
of dependence on the concentration of the inhibitor (10−160
nM). The mammalian reactivation data determined in this
study were in general agreement with spontaneous reactivation
rates found for VX and the p-nitrophenoxy VX surro-
gate.22,25,42,44 However, the spontaneous rates for the
mammalian enzymes were 75-fold slower than that observed
for EEAChE (8.75 × 10−3 min−1; t1/2 = 1.32 h), which
undergoes reactivations rapidly. The spontaneous reactivation
of EEAChE inhibited by 1 is slightly faster than the t1/2 = 2.6 h
reported for sarin-inhibited EEAChE (Scheme 1; R = iPr, Z =
F, Y = Me),45 which is presumably due to the fact that sarin-
modified EEAChE contains an isopropyl ester rather than the
fluoroethoxy. Therefore, sarin-modified EEAChE would be
expected to reactivate slower as a result of the bulkier
substituent that impedes delivery of the hydroxyl/water to
the phosphorus atom in the reactivation mechanism. Paraoxon
and haloxon are related as ethyl and beta-chloro ethyl esters
(Figure 2) that react with bovine red blood cell AChE to afford
OP-AChE adducts bearing a diethoxyphosphyl and di-
(chloroethoxy)phosphyl ester groups, respectively. In this
comparison, the spontaneous reactivation rates for paraoxon
t1/2 = 58 h46 and haloxon t1/2 = 0.3 h47 suggest that beta-
halogen or related withdrawing substituents dramatically
accelerate spontaneous reactivation rates.
Assessment of Oxime-Mediated Reactivation. Quater-
nary oximes such as 2-PAM and MMB-4 are well-recognized
for their use as peripheral acting antidotes for organophosphate
poisoning.2,4,11,13,17,22,26,48−50 2-PAM (pralidoxime) is the only
FDA-approved countermeasure for OP chemical agent
exposure for use in the US. The reaction of the ChE-1
inhibitor complex with 2-PAM was evaluated using the same
concentrations of inhibitor 1 as that used for spontaneous
reactivation. Following incubation of AChE or BChE (0.16
nM) with inhibitor 1 at 25 °C using a concentration range of
10−160 nM for 30 min, greater than 90% reduction in enzyme
activity was observed based on initial (untreated) velocity. At
30 min and then at 18 h, the mixture was treated with 2-PAM
(10 μM), and the rate of ATChI or BTChI hydrolysis activity
was measured (Table 2). At both 30 min and 18 h
postinhibition time points, dramatic accelerations of oxime-
mediated reactivation rates were observed as compared to
spontaneous reactivation. Reactivation rates for huAChE
inhibited by 1 (10−160 nM) ranged from 1.762 × 10−2
min−1 to 2.410 × 10−2 min−1, and huBChE inhibited by 1
varied from 1.080 × 10−2 min−1 to 4.051 × 10−2 min−1
indicating no dependence of reactivation rate on [1]. HuAChE
inhibited by 1 undergoes oxime-mediated reactivation at
statistically equivalent rates of 2.40 × 10−2 min−1 (t1/2 = 0.48
h) and 2.47 × 10−2 min−1 (t1/2 = 0.47 h) at 30 min and 18 h,
respectively. The previous finding that no aging occurred by 4 h
after the rat brain was inhibited by the p-nitrophenoxy VX
surrogate supports reactivation as the major pathway; however,
12% aging was observed at 24 h suggesting partial loss of the
ethoxy group.23 Yet the presence of the fluorine atom in the
AChE-1 adduct may favor greater oxime-mediated reactivation
and less aging accounting for this small difference.
HuBChE inhibited by 1 undergoes oxime-mediated reac-
tivation at a slightly slower rate at 30 min (1.14 × 10−2 min−1;
t1/2 = 1.01 h) than at 18 h (4.19 × 10−2 min−1; t1/2 = 0.27 h),
although both rates are comparable to that found for HuAChE
and producing a similar dephosphylation mechanism. Because
of the rapid spontaneous reactivation of the EEAChE-1 adduct
(∼20 min), an oxime-mediated rate at the 18 h time point was
not possible. The oxime-mediated rate constant at 30 min
correlates with paraoxon-inhibited EEAChE that is reactivated
by 2-PAM (10 μM) with a rate constant kr = 0.176 min−1 or t1/2
= 0.065 h (4 min).51 One explanation for the slightly slower
oxime-mediated reactivation is that the AChE-inhibitor
complex formed from 1 (ChE-1) contains a C−P bond rather
than a C−O−P bond (paraoxon) that poses a greater steric
barrier to nucleophilic attack by the oxime oxyanion.
The fluoroethoxyphosphonate compound 1 was previously
reported as a potent acetylcholinesterase inhibitor in vitro, and
the radiolabeled 18F-form has recently been shown to be a
useful PET imaging tracer in rodents.19,20 In this article, we
have improved the preparation of 1 by reducing the synthetic
process to a one-pot sequence that affords 1 in a greater yield
relative to that of the previous synthetic route reported. We
Figure 2. Structures of paraoxon and haloxon.
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Article
also demonstrated that 1 is hydrolytically stable in vitro at 25 °C
(t1/2 ∼ 35.6 h) and at 37 °C (t1/2 ∼ 13.3 h) in pH 7.4 buffer.
Compound 1 hydrolyzes with the loss of the p-nitrophenoxy
group as the dominant degradation pathway (Scheme 3). The
rates and near complete restoration of activity using 2-PAM
including the restoration of activity at 48 h. The absence of any
significant aging for this adduct is likely due to the unbranched
ethyl ester moiety in combination with the electron-with-
drawing fluorine atom such that both contribute to the
suppression of the putative cation-based aging mechanism.
However, an aging pathway cannot be ruled out in vivo as was
reported for the VX surrogate compound.23
Scheme 3. Compound 1 Hydrolysis, Inhibition, and
Reactivation Rates
Therefore, the presence of the beta-fluorine atom in the
ChE-1 adducts is thought to influence reactivation and aging
rate processes by inductive effects. The reactivation rates are
slightly faster than that observed for VX-modified AChE, and
aging was not found. These in vitro findings with OP-1 provide
support for a similar mechanism of AChE inhibition with the
radiotracer [18F]1 in vivo, in which the corresponding [18F]1-
AChE adduct most likely does not undergo aging over the PET
imaging time course (e.g., 90−120 min) and tracer lifetime
(fluorine-18 half-life, ∼110 min). In that case, a significant
portion of the measured tissue radioactivity by PET imaging
over the course of 2 h post-tracer administration likely
represents an [18F]1−AChE adduct.
It is important to note that studies with recombinant
huAChE, serum huBChE, and EEAChE provide relevant and
useful mechanistic insights that may not be operative in vivo. In
this study, the rate constants of inhibition and reactivation and
proposed mechanisms do not take into account the asymmetry
of 1 at phosphorus, which is known to affect inhibitory and
postinhibitory rate and mechanistic processes.52−56 Preparation
or separation of the individual enantiomers of 1 and evaluation
of their antiesterase properties are the subject of a future study.
On the basis of the studies described here, experiments in
our laboratories are ongoing to quantitatively assess whether
the in vitro spontaneous and/or oxime-induced reactivation
rates are similar to the in vivo rates, where the fate of the [18F]
1−AChE adduct in live tissues prospectively can be observed
and quantified by PET imaging. Results from the in vivo
reactivation studies will be reported in due course.
in vitro hydrolytic stability half-life suggests that a significant
fraction of tracer 1 will survive in vivo dose formulation and
administration, although hydrolytic breakdown in vivo by
enzymes could also play a significant role. This notion is
consistent with the demonstrated in vivo ability of tracer [18F]1
that remains intact within blood and penetrates into the brain.
Conversely, if compound 1 had been found to undergo rapid in
vitro hydrolytic breakdown to the corresponding phosphonic
acid, the product phosphonic acid would ionize at physiologic
pH and would be unlikely to penetrate and localize in the brain.
The reaction of 1 with ChE’s affords beta-fluoroethoxy
methylphosphonate adducts that undergo both spontaneous
and oxime-mediated reactivation. Spontaneous and oxime-
mediated reactivation rates were comparable for both HuAChE
and HuBChE inhibited by 1 with the oxime-mediated rates
100- to 250-fold faster than spontaneous rates. Rate
accelerations of 100-fold when using oximes for VX-inhibited
cholinesterases have been reported.21,22,26 There was little
difference in the oxime-mediated reactivation rates found at 30
min and 18 h, and in general, mammalian enzyme inactivated at
>95% of initial velocity fully recovered activity. EEAChE, used
as a control enzyme for longer analysis time points, underwent
spontaneous reactivation almost 100-times faster than HuAChE
and recovered almost 50% of the original activity within an
hour. No evidence of aging was observed as indicated by
greater than 99% of the original activity being restored at all
time points upon treatment with 2-PAM.
Although it is not surprising that the oxime-mediated
reactivation rates occurred faster than spontaneous reactivation,
most OP-AChE adducts formed from nerve agents (e.g.,
methylphosphonates) do not undergo complete reactivation
due to competing aging processes. The nerve agent VX is an
exception25 that affords an ethoxy methylphosphonate AChE
adduct that is largely reactivatable but undergoes some
aging.17,22−24 The OP-AChE adduct formed from 1 differs
from the AChE-VX adduct in that the former possesses a beta-
fluoro substituent where the rapid reactivation of the EEAChE-
1 adduct is explained on the basis of the electron-withdrawing
fluorine atom favoring the nucleophilic process (water or an
oxime). In this study, no evidence of aging of any of the ChE-1
adducts was observed that is supported by the rapid reactivation
AUTHOR INFORMATION
Corresponding Author
■
*Office SB 477A, Department of Biomedical and Pharmaceut-
ical Sciences, 32 Campus Drive, Skaggs 395, University of
Montana, Missoula, MT 59812-1552. Phone: 406-243-4643. E-
mail: chuck.thompson@mso.umt.edu.
Funding
Supported by the CounterACT Program, National Institutes of
Health Office of the Director (NIH OD), and the National
Institute of Neurological Disorders and Stroke (NINDS),
Grant Award Number U01NS092495. Additional support was
provided by the CounterACT Program, Office of the Director,
National Institutes of Health, and the NINDS, Grant Number
R21 NS072079.
Notes
The content is solely the responsibility of the authors and does
not necessarily represent the official views of the National
Institutes of Health.
The authors declare no competing financial interest.
ABBREVIATIONS
■
ACh, acetylcholine; AChE, acetylcholinesterase; huAChE,
recombinant human acetylcholinesterase; huBChE, human
butyrylcholinesterase; EEAChE, electric eel acetylcholinester-
F
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DOI: 10.1021/acs.chemrestox.6b00160
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