Discovery of a potent non-oxime reactivator of nerve agent inhibited human acetylcholinesterase

Article abstract of DOI:10.1016/j.ejmech.2018.08.016

Organophosphorous (OP) compounds (such as nerve agents) inhibit the enzyme acetylcholinesterase (AChE) by covalent phosphylation of a key serine residue in the active site of the enzyme resulting in severe symptoms and ultimately death. OP intoxications are currently treated by administration of certain oxime compounds. The presently fielded oximes reactivate OP-inhibited AChE by liberating the phosphylated serine. Recent research towards new reactivators was predominantly devoted to design, synthesis and evaluation of new oxime-based compounds dedicated to overcoming some of the major limitations such as their intrinsic toxicity, their permanent charge which thwarts penetration of brain tissues and their inability to effectively reactivate all types of nerve agent inhibited AChEs. However, in over six decades of research only limited success has been achieved, indicating that there is a need for alternative classes of compounds that could reactivate OP-inhibited AChE. Recently, a number of non-oxime compounds was discovered in which the 4-amino-2-((diethylamino)methyl)phenol (ADOC) motif proved to be able to reactivate OP-inhibited AChE to some extent. In this paper several structural derivatives of ADOC were synthesized and screened for their ability to reactivate human AChE (hAChE) inhibited by the nerve agents VX, sarin, tabun, cyclosarin and paraoxon. We here disclose that one of those compounds showed a remarkable ability to reactivate OP-inhibited hAChE in vitro and that it is the most potent non-oxime reported to date.

Full text of DOI:10.1016/j.ejmech.2018.08.016

European Journal of Medicinal Chemistry 157 (2018) 151e160
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
Discovery of a potent non-oxime reactivator of nerve agent inhibited
human acetylcholinesterase
,
Martijn Constantijn de Koning a *, Gabriele Horn b, Franz Worek b, Marco van Grol a
a TNO, Lange Kleiweg 137, 2288, GJ, Rijswijk, the Netherlands
b Bundeswehr Institute of Pharmacology and Toxicology, Neuherbergstrasse 11, 80937, Munich, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Organophosphorous (OP) compounds (such as nerve agents) inhibit the enzyme acetylcholinesterase
(AChE) by covalent phosphylation of a key serine residue in the active site of the enzyme resulting in
severe symptoms and ultimately death. OP intoxications are currently treated by administration of
certain oxime compounds. The presently fielded oximes reactivate OP-inhibited AChE by liberating the
phosphylated serine. Recent research towards new reactivators was predominantly devoted to design,
synthesis and evaluation of new oxime-based compounds dedicated to overcoming some of the major
limitations such as their intrinsic toxicity, their permanent charge which thwarts penetration of brain
tissues and their inability to effectively reactivate all types of nerve agent inhibited AChEs. However, in
over six decades of research only limited success has been achieved, indicating that there is a need for
alternative classes of compounds that could reactivate OP-inhibited AChE. Recently, a number of non-
oxime compounds was discovered in which the 4-amino-2-((diethylamino)methyl)phenol (ADOC)
motif proved to be able to reactivate OP-inhibited AChE to some extent. In this paper several structural
derivatives of ADOC were synthesized and screened for their ability to reactivate human AChE (hAChE)
inhibited by the nerve agents VX, sarin, tabun, cyclosarin and paraoxon. We here disclose that one of
those compounds showed a remarkable ability to reactivate OP-inhibited hAChE in vitro and that it is the
most potent non-oxime reported to date.
Received 19 May 2018
Received in revised form
29 June 2018
Accepted 4 August 2018
Available online 6 August 2018
Keywords:
Acetylcholinesterase
Nerve agents
Chemical warfare agent
Reactivator
Non-oxime
© 2018 Elsevier Masson SAS. All rights reserved.
1. Introduction
over 200.000 deaths annually, and unfortunately, the use of OP
chemical warfare agents (CWA) by state actors as well as by ter-
Organophosphorus (OP) compounds, such as certain pesticides
(e.g., chlorpyrifos) and the nerve agents sarin (GB), soman (GD),
tabun (GA), VX, Russian VX (VR) and cyclosarin (GF) are highly toxic
[1]. Their toxicological target is the enzyme acetylcholinesterase
(AChE) which catalyzes the hydrolysis of the neurotransmitter
acetylcholine (ACh) in cholinergic synapses [2,3]. OPs inhibit AChE
through covalent phosphylation of a serine residue in the active site
of AChE [1]. The inhibition of AChE results in accumulation of ACh
levels which leads to overstimulation of cholinergic receptors,
failure of the cholinergic synaptic transmission, flaccid muscle
paralysis and seizures in the central nervous system. Exposure to
OP pesticides is a worldwide issue in agriculture [4] resulting in
rorists has been documented several times in the last decades [5].
These events underline the existence of a continuous threat to
civilian and military personnel by these compounds and conse-
quently, effective medical treatment strategies are needed.
The treatment of OP-poisoning comprises the administration of
an anticonvulsant drug such as diazepam, atropine as an anti-
muscarinic agent and an oxime. Currently fielded oximes, such as
2-PAM [6] (Fig. 1), liberate the blocked active site serine in AChE by
the nucleophilic displacement of the phosphyl residue, thereby
restoring the activity of the enzyme. However, the currently
employed oximes have several disadvantages, such as a relatively
high toxicity, a lack of broad spectrum efficiency towards all
possible OP-AChE adducts, and limited brain accessibility as a result
of the presence of one or multiple permanent charges. These
problems have been addressed in decades of research towards
more effective oximes, but unfortunately only with limited success
[7e9]. To date, no broad spectrum reactivator has been discovered.
Recent advances have been made in the design and synthesis of
* Corresponding author.
E-mail addresses: m.dekoning@tno.nl (M.C. de Koning), Gabriele-Horn@gmx.de
(G. Horn), franzworek@bundeswehr.org (F. Worek), marco.vangrol@tno.nl (M. van
Grol).
https://doi.org/10.1016/j.ejmech.2018.08.016
0223-5234/© 2018 Elsevier Masson SAS. All rights reserved.

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M.C. de Koning et al. / European Journal of Medicinal Chemistry 157 (2018) 151e160
Fig. 1. Comparison of the structures of a current oxime (2-PAM) and a non-oxime reactivator, including summary of previously reported structure-activity trends.
effective, non-ionic oxime reactivators for enhanced brain perme-
ability [10e23], but most of the reported molecules are structurally
complex and sometimes difficult to synthesize in large quantities,
thwarting in vivo evaluation. Moreover, the lack of charge often
results in reduced reactivity of the oxime functionality, compli-
cating design of effective oxime-based reactivators. Apparently,
there is a need to further expand the chemical space for designing
reactivators beyond that of oximes.
A couple of years ago, Bhattacharjee et al. reported a study in
which in silico pharmacophore modeling and virtual screening
strategies were employed to identify potential reactivators from
large libraries of virtual compounds [24,25]. A number of selected
compounds displayed some ability to in vitro reactivate eel AChE
inhibited by diisopropylfluorophosphate (DIFP), a nerve agent
simulant. In a similar effort, Katz et al. discovered a number of
compounds with reactivation potency by in vitro screening of large
libraries of bioactive compounds and approved drugs [26,27].
Astonishingly, none of the compounds reported by Bhattacharjee or
by Katz possessed the oxime structural motive that has dominated
the past 60 years of reactivator development. While the active
compounds reported by Battacharjee showed little structural
resemblance, the compounds reported by Katz displayed common
structural features such as basic moieties (pyridine, imidazole and
piperazine) [27] or a Mannich phenol [28] (i.e., phenols featuring a
presence and position of the hydroxyl function was essential for
reactivation, that varying the steric bulk on the benzylic amine led
to drastic changes in affinity (IC50), and that the nature of the
benzylic amine and the aniline amine contributed to the affinity of
ADOC for the inhibited enzyme. Unfortunately, none of the com-
pounds generated in that study performed better than ADOC
in vitro reactivation assays. Intriguingly, all deviations to the ADOC
structure led to a (nearly) diminished reactivation potency, indi-
cating a delicate sensitivity of biological activity to structural
changes. In this contribution, the synthesis of ADOC derivatives in
which relatively small adjustments were made to the structure of
the benzylic amine are reported. These compounds were screened
for their ability to reactivate hAChE inhibited by several nerve
agents. We here present some unusual observations in the behavior
of these compounds as well as the discovery of one compound that
clearly out-performed ADOC in the in vitro reactivation of all nerve
agent-inhibited AChE's tested (except for tabun-AChE).
2. Results and discussion
2.1. Synthesis of Mannich phenols
Fig. 2 shows the structures that were targeted for synthesis.
These structures reflect the focus on making relatively small vari-
ations of the lead structure ADOC (compound 3h in Fig. 2). The
synthesis of Mannich phenols 3a-r was accomplished following a
simple, previously reported [31], 2-step general procedure. Acet-
amidophenol (1) was treated overnight in ethanol (or water) with
paraformaldehyde (or formaldehyde when executed in water) and
the respective amine at 90 ꢀC. The resulting monosubstituted in-
termediates 2 were then separated from unreacted material and
the bis-substituted product using automated flash column chro-
matography. The second step comprised treatment of pure 2 with
10% HCl at elevated temperature and monitoring the progress of the
reaction by LC-MS. When complete, the mixture was concentrated
and co-evaporated to give the HCl-salt of the target product, which
did not require further purification. All of the resulting compounds
were characterized by 1H/13C-NMR, LC-MS and HRMS.
The envisaged variations comprised asymmetrical (3a, 3c-g) as
well as symmetrical (3b, 3i) substitution of the benzylic amine with
aliphatic carbon chains. Cyclic amines of varying ring-size (3e6
atoms, 3j-n) were also targeted. The synthesis of derivatives with
the smallest ring-sizes (i.e., aziridine 2j and azetidine 2k) were
unproductive, but 3l-n (5 and 6 membered rings) were successfully
isolated. Further derivatization was attempted by introducing un-
saturated ring systems (3o-r). The synthesis of 2o and 2p suffered
from polymerization of the amine and/or the product and could not
be obtained. The imidazole (3q) and pyrazole (3r) derivatives could
only be isolated in low overall yield, because of low conversion of 1
into 2q and 2r and the need to isolate these intermediates by HPLC
purification.
benzylic amine in the
a-position). In particular, 4-amino-2-
((diethylamino)methyl)phenol (ADOC, Fig. 1) exhibited promising
reactivation potential for paraoxon-ethyl (PXE) and DIFP-inhibited
AChE [26]. The latter discovery triggered subsequent studies, for
instance by Bierwisch et al. who investigated the kinetic in-
teractions of amodiaquine, an ADOC based anti-malaria drug, with
AChE as well as with butyrylcholinesterase (BChE) [29]. Cadieux
et al. reported a study [30] in which they characterized ADOC for
activity against a range of OP nerve agent-inhibited AChEs, using
the standard kinetic model for reactivation to determine kr, which
is the reactivation rate constant that describes the nucleophilic
displacement reaction, and the dissociation constant KD, which
reflects the affinity of the reactivator for the inhibited enzyme. The
results revealed that the kr values for ADOC mediated reactivation
of AChE inhibited by GB, GF, VX and VR were higher than those of 2-
PAM (GD and GA adducts could not be reactivated). However, the
corresponding KD values were less favorable for ADOC. As a
consequence, the overall bimolecular reactivation rate constant
(kr2¼ kr/KD) of ADOC was only significantly better than that of 2-
PAM in the case of VR-inhibited AChE. It was also found that
ADOC was a strong reversible inhibitor of the native enzyme
(IC50 ¼ 17.8
mM). Cadieux et al. also synthesized a series of structural
analogs of ADOC to probe the essential structural elements needed
for reactivation and binding, as the mechanism by which these
compounds act is currently unknown. They varied the bulk, type
and position of various substituents on either the aniline, the hy-
droxyl or the benzylic amine (Fig. 1). The results indicated that the

M.C. de Koning et al. / European Journal of Medicinal Chemistry 157 (2018) 151e160
153
Fig. 2. Structures of all targeted Mannich phenols.
2.2. Inhibition of hAChE
system (kic < kiu) indicating that 3h (Fig. 3A) as well as 3l (Fig. 3B)
are mixed noncompetitive inhibitors of human AChE. The
The 50% inhibitory concentrations (IC50) of compounds 3 were
determined using erythrocyte human acetylcholinesterase
(hAChE). The results are summarized in Table 1. A clear structure-
activity relationship was difficult to establish, but compounds
that displayed (some) affinity had similar bulk (4e6 atoms) around
the benzylic amine (i.e., 3h-q). The configuration and nature of
these atoms strongly influenced the affinity. For instance, ADOC
competitive dissociation constant of 3h (kic ¼ 16.0 0.2
about 2.5 fold higher than reported by Cadieux [30] for recombi-
nant hAChE (6.3 0.9 M) while the noncompetitive dissociation
constant (kiu ¼ 37.7 2.8 M) was virtually the same as previously
determined (kiu ¼ 37.4 2.1 M). The corresponding values for
compound 3l were also determined (kiu ¼ 7.8 0.0 M,
M) confirming the higher overall inhibition po-
mM) was
m
m
m
m
kic ¼ 3.3 0.08
m
exhibited an IC50 of 48.3
ported [30] value of 17.8 M for recombinant hAChE), while its ring-
closed derivative 3l had an IC50 of 6.3 M. Conversely, expansion of
mM (which is comparable with the re-
tency of 3l over 3h.
m
m
2.3. In vitro reactivation screening
the pyrrolidine ring with one methyl group (i.e. 3m) or loss of one
methyl group in 3h (/3c) resulted in significant loss of affinity
(456 and 965 mM respectively).
Measurement of AChE activities at different substrate concen-
trations in the absence and presence of non-oxime compounds
enabled the identification of the inhibition type of 3h and 3l. Direct
Michaelis-Menten graph revealed a decreasing maximum hydro-
lysis rate (vmax) at increasing concentrations of 3h and 3l. All
straight lines of the Lineweaver-Burk plots converged on a common
intersection point within the second quadrant of the coordinate
All compounds were screened for their ability to reactivate OP-
inhibited hAChE. To this end, human erythrocyte AChE (ghosts) was
inhibited with VX, paraoxon-ethyl (PXE), GA, GF and GB and sub-
jected to respectively 10, 100 and 1000 mM concentrations of the
compounds 3 [32]. The increase in enzyme activity (reactivation)
over time was measured using a modified Ellman assay [33,34], and
expressed as the percentage reactivation with respect to the 100%
control. The use of different agents and three concentrations of
each reactivator over a wide concentration range provided a good
indication of the potency of the reactivators in an efficient way. All
experiments were carried out in duplicate. The direct reaction
(‘oximolysis’) [35] between compound 3l and the substrate (ace-
Table 1
The inhibition of native human AChE by non-oxime compounds was tested with 11
tylthiocholine) was measured at 100 mM concentration and its rate
concentrations (1e1000 mM) in duplicate. Data are given as mean SD.
was found to be negligible. All reactivation curves are provided in
the S.I. The results confirmed the potency of ADOC (3h) as a non-
oxime reactivator of nerve agent inhibited hAChE (Fig. 4AeD).
None of the compounds was able to reactivate GA-inhibited hAChE.
All other alkylamine derivatives (i.e., 3a-g and 3i) were (much) less
efficient reactivators than 3h. Most of them showed some reac-
Compound
IC50
(
m
M) SD
Compound
IC50 (mM) SD
3a
3b
3c
3d
3e
3f
333.5 18.1
1536.5 604.6
965.1 134.0
1151.0 53.0
125.2 2.4
3h
3i
3l
3m
3n
3q
3r
48.3 0.2
131.1 1.4
6.3 0.0
456.0 4.0
270.6 0.2
482.5 63.1
844.5 28.0
805.1 68.5
1053.5 28.5
tivation at 1000
mM concentration, but compounds 3f and 3g,
3g
having the longest alkyl chains, exhibited very low reactivation

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M.C. de Koning et al. / European Journal of Medicinal Chemistry 157 (2018) 151e160
Fig. 3. Inhibition assays using 3h (graph A) and 3l (graph B) in varying concentrations as the inhibitor and acetylthiocholine (ATCh) as the substrate.
potency. From the two hexacyclic amines 3m showed little reac-
tivation and 3n not at all. Remarkably, while the pyrazole derivative
3r gave negligible reactivation, the closely related imidazole de-
rivative 3q was able to achieve 40e100% reactivation (depending
under physiological conditions, but it confirms that the formation
of compound 4 may be expected if reactivation proceeds via a
nucleophilic mechanism. The isolated (HPLC) adduct 4 was stable
for at least 24 h in phosphate buffer, in contrast to many phosphyl-
oxime adducts that usually rearrange to form cyanides. The inhib-
on the OP used) at 1000 mM concentration after 60 min. Finally, it
was very gratifying to find that compound 3l, carrying the 5-
membered cyclic pyrrolidine, clearly exhibited superior perfor-
mance over 3h in the reactivation of all OP-inhibited hAChEs (see
Fig. 4EeH and Table 2). For instance, comparison of the clinically
itory constant (ki) was determined to be 1.9 ꢁ 104 Mꢂ1minꢂ1
,
indicating that 4 is a very weak inhibitor of hAChE thus ruling out
that 4 contributes to the observed decrease of enzyme activity in
the reactivation reaction.
relevant 10
m
M concentration curves in Fig. 4 clearly shows that 3h
The decrease in enzyme activity during the reactivation exper-
iment could be explained by exposing the native enzyme to
1000 mM concentration of reactivators 3l or 3h (Fig. 6) which
resulted in a similar decrease in enzyme activity as observed in the
reactivation experiments. This effect was not observable with
gave no or <25% reactivation after 60 min of incubation, while
compound 3l was capable of reactivating 50e100% of the inhibited
hAChE's at the same conditions. Moreover, in a number of cases
reactivation was very fast, as exemplified by the complete recovery
of VX-inhibited hAChE within 20 min with 10
m
M of reactivator and
reactivator concentrations ꢃ100
mM. The structure of 3l in 0.1 M
within approximately 10 min with 100 M reactivator. One striking
m
phosphate buffer pH 7.4 remained intact over the course of 24 h, as
gauged by LC-MS and 1H NMR, indicating that this compound did
not spontaneously rearrange into another high affinity byproduct.
Perhaps the interaction of Mannich phenols with the enzyme
induces denaturation upon prolonged exposure to higher concen-
trations [26]. An alternative explanation is that the enzyme se-
questers any quinone methide intermediate that might be formed
in or near the enzyme, from for instance 3l, resulting in alkylation
of the enzyme (Fig. 7) [36,37].
notion was that the ability to reactivate was strongly dependent on
subtle changes in the structures. For instance, while ADOC (3h)
showed appreciable reactivation (Fig. 4AeD), the ring-closed de-
rivative of ADOC (3l) led to a considerable increase in reactivation
potency (Fig. 4EeH), but the further expansion of the ring-size with
only one methylene group (/3m) led to virtually complete loss of
reactivation potency (Fig. 8AeB and S.I.)
A closer inspection of all of the reactivation curves led to a
number of additional observations. First of all, some of the 1000
m
M
A second intriguing observation was that in some cases reac-
tivation was delayed and commenced after prolonged incubation
times, which could take up to 20 min. Two examples of such curves
for the reactivation of VX- and paraoxon-inhibited hAChE by 3m
reactivation curves showed an abrupt stop of rapid initial reac-
tivation after early time points followed by a further decrease of
enzyme activity after prolonged reaction times (see Fig. 4 and
similar curves in the S.I.). These observations were made with
compounds 3d, 3e, 3h, 3i, 3l and 3m, but not with imidazole de-
rivative 3q, which gave progressive reactivation (up to 100% for
PXE-hAChE) of all OP-AChE adducts. When oximes are employed,
attenuation of reactivation rates over time can sometimes be
ascribed to re-inhibition of the enzyme by the phosphyl-oxime
adduct that accumulates with progressing reactivation. If reac-
tivation with non-oximes 3 proceeds via a nucleophilic mechanism
the formation of non-oxime-phosphyl adduct 4 (Fig. 5) may be
expected. Another mechanism of reactivation has also been pro-
posed [26,27] in which the reactivator recruits water molecules
that hydrolyze the phosphyl-serine bond, which would not give
rise to adduct formation. The adduct 4 was synthesized to verify
whether it could act as an inhibitor of hAChE. To this end 3l was
reacted with paraoxon in MOPS buffer at pH 7.4. However, no
appreciable formation of compound 4 was observed, as gauged by
31P NMR after 16 h. More forcing conditions (0.45 M N-ethyl-
morpholine buffer, pH 11, 4 days) were necessary giving partial
hydrolysis of PXE as well as the formation of 4. This result dem-
onstrates that the nucleophilicity of compound 3l is quite low
(1000 mM) are depicted in Fig. 8. This phenomenon was observed
with several other compounds as well (3b, 3c, 3e, 3i, 3m and 3q, see
S.I.), and seems to occur predominantly with PXE- and VX-inhibited
hAChE and occasionally with GF-inhibited hAChE. At this time no
explanation for this phenomenon can be provided.
3. Conclusions
In summary, a number of novel derivatives (3a-r) of 4-amino-2-
((diethylamino)methyl)phenol (3h) were designed with varying
substituents on the benzylic amine. The majority (13 out of 17) of
the target compounds could be successfully synthesized and iso-
lated. The compounds were screened for their ability to reactivate
OP-inhibited hAChE, using various nerve agents, and a wide range
of reactivator concentrations. The reactivation rates as well as the
inhibitory potencies were strongly affected by subtle changes to the
amine substituents. While most derivatives performed worse than
the previously reported [26] lead compound 3h, the pyrrolidine
derivative 3l clearly outperformed 3h in the reactivation of a broad
range of OP-inhibited hAChEs, even at low concentrations, except

M.C. de Koning et al. / European Journal of Medicinal Chemistry 157 (2018) 151e160
155
Fig. 4. Reactivation curves of 3h (graphs A-D) and of 3l (graphs E-H).

156
M.C. de Koning et al. / European Journal of Medicinal Chemistry 157 (2018) 151e160
Table 2
formed during the reactivation reaction. In some cases reactivation
rates suddenly increased after a certain delay (10e20 min). This
observation cannot be explained at this time. In conclusion, Man-
nich phenol 3l is the most potent non-oxime reactivator known to
date. The results confirm that Mannich phenols may constitute an
alternative research avenue towards reactivators of nerve agent
inhibited hAChE. The uncharged nature of these compounds may
increase their ability to penetrate brain tissues, which further jus-
tifies future studies towards the synthesis of derivatives with even
higher potency. Current research focuses on more detailed char-
acterization of the most active compounds. For instance, the
determination of the kinetic parameters (kr and KD) of compounds 3
will allow a better comparison of their potency with the current
oxime-based reactivators and provide a better understanding of the
crucial features that govern their reactivation potency. In addition,
compound 4 could serve as a reference compound to probe its
formation during reactivation by the LC-MS analysis of reactivation
mixtures. Those aspects should aid the better understanding of the
mechanism of reactivation and provide clues for future design of
further improved reactivators.
The pseudo-first-order rate constants (kobs) at non-oxime concentrations of 10 and
100 M were determined by linear regression analysis using Eq. (2). Data are shown
m
as means SD (n ¼ 2). Ø calculation not possible due to inadequate reactivation of
inhibited AChE.
OP
kobs (minꢂ1
10
)
m
M
100 mM
3 h (ADOC)
3l
3 h (ADOC)
3l
GA
GB
GF
VX
PXE
ø
ø
ø
ø
0.010 0.0
0.002 0.0
0.010 0.001
0.001 0.0
0.020 0.001
0.025 0.001
0.190 0.019
0.025 0.001
0.047 0.0
0.008 0.0
0.114 0.005
0.030 0.0
0.245 0.004
0.205 0.007
0.581 0.026
0.174 0.003
4. Experimental section
Fig. 5. Synthesis of the (putative) adduct 4.
Details on chemicals, equipment, additional descriptions and
results of in vitro work and NMR spectra of compounds can be
found in the supporting information.
for GA-inhibited hAChE, which could not be reactivated by any of
the compounds tested. Several unusual phenomena were observed
in the reactivation experiments. The enzyme integrity is affected by
4.1. General synthetic procedure for the synthesis of 3a-3r
high concentrations (1000
mM) of reactivator, but this effect was
absent at physiologically more relevant concentrations (<100
over the same time-course (60 min). It could be excluded that the
reduced activity was caused by adduct 4, a compound that might be
m
M)
1.52 g (10 mmol) of 4-acetaminophenol was dissolved in 20 mL
ethanol. 0.39 g (13 mmol) of paraformaldehyde was added followed
by addition of 0.13 mmol of the requisite amine. The resulting
Fig. 6. Incubation of native enzyme with 1 mM of 3h (A) or 3l (B) decreased enzyme activity. At lower concentrations this effect was not observed.
Fig. 7. Postulated mechanism of enzyme deactivation observed with some of the compounds 3. A quinone methide intermediate might be formed that is subsequently sequestered
by nucleophilic (Nu) amino acid residues from the enzyme, leading to deactivation of the enzyme.

M.C. de Koning et al. / European Journal of Medicinal Chemistry 157 (2018) 151e160
157
Fig. 8. Examples of delayed reactivation by 3m of VX-hAChE (A) and of PXE-hAChE (B).
mixture was refluxed for 16e20 h and concentrated using a rotary
2d: 1H NMR (400 MHz, Methanol-d4)
d
7.26 (d, J ¼ 2.6 Hz, 1H),
evaporator. The crude product was purified using automated flash
chromatography (Biotage Isolera 4) using a gradient of MeOH in
EtOAc (typically 5e15%), or a gradient of MeOH in DCM (typically
1e5%), in the presence of 2% Et3N. Compounds 2a, 2b, 2q and 2r
were purified using automated preparative HPLC. The intermediate
product (compound 2) was dissolved in 10% HCl and refluxed for
5 h. LC-MS analysis of aliquots of the reaction mixture were taken to
verify completeness of the reaction. If complete, the mixture was
allowed to cool, after which acetonitrile was added and the mixture
was concentrated using a rotary evaporator. The procedure of
adding acetonitrile and concentration was repeated 2e3 times. No
further purification was required. As the compounds 3 were iso-
lated as the HCl-salts quantitative NMR was used to establish the
mass content of a number of representative examples of com-
pounds 3. The results showed that the compounds are isolated as
the double HCl salts.
7.22 (dd, J ¼ 8.6, 2.6 Hz, 1H), 6.69 (d, J ¼ 8.6 Hz, 1H), 3.69 (s, 2H),
2.52e2.43 (m, 2H), 2.29 (s, 3H), 2.10 (s, 3H), 1.61 (h, J ¼ 7.4 Hz, 2H),
0.95 (t, J ¼ 7.4 Hz, 3H). 13C NMR (101 MHz, Methanol-d4)
d 169.91,
154.35, 129.99, 122.19, 121.37, 120.95, 115.18, 60.30, 58.52, 39.95,
22.10, 19.71, 10.53. 3d: 1H NMR (400 MHz, Methanol-d4)
d 7.56 (d,
J ¼ 2.7 Hz, 1H), 7.42 (dd, J ¼ 8.7, 2.8 Hz, 1H), 7.11 (d, J ¼ 8.7 Hz, 1H),
4.41 (dd, J ¼ 107.9, 13.0 Hz, 2H), 3.28e3.07 (m, 2H), 2.84 (s, 3H), 1.89
(dt, J ¼ 8.7, 7.4 Hz, 2H), 1.05 (t, J ¼ 7.4 Hz, 3H). 13C NMR (101 MHz,
Methanol-d4)
d 157.01, 127.20, 126.17, 122.12, 117.79, 116.41, 57.80,
54.03, 39.56, 17.37, 9.78. Overall yield: 30%. HRMS [MþH]þ calc
(found): 195.14919 (195.1488).
2e: 1H NMR (400 MHz, Methanol-d4)
d
7.26 (d, J ¼ 2.6 Hz, 1H),
7.22 (dd, J ¼ 8.6, 2.6 Hz, 1H), 6.69 (d, J ¼ 8.6 Hz, 1H), 3.69 (s, 2H),
2.63e2.45 (m, 2H), 2.29 (s, 3H), 2.10 (s, 3H), 1.64e1.44 (m, 2H), 1.36
(m, J ¼ 13.8, 7.0, 6.5 Hz, 2H), 0.96 (t, J ¼ 7.3 Hz, 3H). 13C NMR
(101 MHz, Methanol-d4)
d 169.90, 154.36, 129.98, 122.20, 121.37,
2a: 1H NMR (400 MHz, D2O)
d
7.62e7.51 (m, 1H), 7.43e7.32 (m,
120.95, 115.18, 60.30, 56.32, 40.00, 28.76, 22.08, 20.01, 12.80. 3e: 1H
NMR (400 MHz, Methanol-d4)
1H), 6.91 (dd, J ¼ 12.0, 8.7 Hz, 1H), 4.15 (s, 2H), 2.68 (d, J ¼ 14.4 Hz,
d
7.55 (d, J ¼ 2.7 Hz, 1H), 7.41 (dd,
3H), 2.12 (d, J ¼ 2.6 Hz, 3H). 13C NMR (101 MHz, Methanol-d4)
J ¼ 8.7, 2.8 Hz, 1H), 7.11 (d, J ¼ 8.7 Hz, 1H), 4.40 (dd, J ¼ 108.5,
13.0 Hz, 2H), 3.31e3.12 (m, 2H), 2.84 (s, 3H), 1.90e1.77 (m, 2H), 1.45
(h, J ¼ 7.4 Hz, 2H), 1.02 (t, J ¼ 7.4 Hz, 3H). 13C NMR (101 MHz,
d
170.22, 152.84, 130.61, 123.93, 123.59, 117.28, 115.02, 48.33,
31.76, 22.11. 3a: 1H NMR (400 MHz, D2O)
d 7.33e7.20 (m, 2H), 7.00
(d, J ¼ 8.3 Hz, 1H), 4.16 (s, 2H), 2.65 (s, 3H). 13C NMR (101 MHz, D2O)
Methanol-d4) d 157.02, 127.16, 126.15, 122.11, 117.83, 116.40, 56.13,
d
155.76, 126.16, 125.94, 121.83, 118.92, 116.75, 47.98, 32.39. Overall
54.08, 39.56, 25.83, 19.48, 12.46. Overall yield: 60%. HRMS [MþH]þ
yield: 20%. HRMS [MþH]þ calc (found): 153.10224 (153.1021).
calc (found): 209.1648 (209.16484).
2b: 1H NMR (400 MHz, Methanol-d4)
d
7.28e7.20 (m, 2H),
2f: 1H NMR (400 MHz, Methanol-d4)
d
7.25 (d, J ¼ 2.5 Hz, 1H),
6.90e6.58 (m, 1H), 3.63 (s, 2H), 2.33 (s, 6H), 2.10 (s, 3H). 13C NMR
(101 MHz, Methanol-d4) 169.91, 154.19, 129.97, 122.26, 121.65,
121.06, 115.13, 61.08, 43.27, 22.09. 3b: 1H NMR (400 MHz,
7.22 (dd, J ¼ 8.5, 2.6 Hz,1H), 6.69 (d, J ¼ 8.5 Hz,1H), 3.68 (s, 2H), 2.49
(dd, J ¼ 8.2, 6.6 Hz, 2H), 2.28 (s, 3H), 2.09 (s, 3H), 1.58 (p, J ¼ 7.4 Hz,
2H), 1.44e1.21 (m, 4H), 0.93 (t, J ¼ 6.9 Hz, 3H). 13C NMR (101 MHz,
d
Methanol-d4)
d
7.55 (d, J ¼ 2.7 Hz, 1H), 7.43 (dd, J ¼ 8.7, 2.8 Hz, 1H),
Methanol-d4) d 169.89, 154.36, 130.00, 122.22, 121.35, 120.93,
7.12 (d, J ¼ 8.7 Hz, 1H), 4.41 (s, 2H), 2.92 (s, 6H). 13C NMR (101 MHz,
115.20, 60.33, 56.57, 40.01, 29.10, 26.28, 22.12, 12.94. 3f: 1H NMR
(400 MHz, Methanol-d4)
Methanol-d4)
d
156.99, 127.08, 126.25, 122.12, 117.86, 116.42, 55.79,
d
7.54 (d, J ¼ 2.7 Hz, 1H), 7.41 (dd, J ¼ 8.7,
42.05. Overall yield: 23%. HRMS [MþH]þ calc (found): 167.11789
2.7 Hz, 1H), 7.11 (d, J ¼ 8.7 Hz, 1H), 4.61e4.16 (m, 2H), 3.21 (dt,
J ¼ 26.2, 8.3 Hz, 2H), 2.84 (s, 3H), 1.97e1.76 (m, 2H), 1.41 (qd, J ¼ 6.8,
2.7 Hz, 4H), 0.98 (t, J ¼ 6.8 Hz, 3H). 13C NMR (101 MHz, Methanol-
(167.1179).
2c: 1H NMR (400 MHz, Methanol-d4)
d
7.26 (d, J ¼ 2.6 Hz, 1H),
7.22 (dd, J ¼ 8.6, 2.6 Hz,1H), 6.69 (d, J ¼ 8.6 Hz,1H), 3.70 (s, 2H), 2.57
d4) d 156.99, 127.12, 126.12, 122.17, 117.84, 116.39, 56.35, 54.09,
(q, J ¼ 7.2 Hz, 2H), 2.31 (s, 3H), 2.10 (s, 3H), 1.16 (t, J ¼ 7.2 Hz, 3H). 13
C
39.57, 28.31, 23.58, 21.82, 12.74. Overall yield: 49%. HRMS [MþH]þ
NMR (101 MHz, Methanol-d4)
d
173.75, 172.01, 157.01, 127.07,
calc (found): 223.18049 (223.1809).
126.13, 122.14, 116.41, 53.68, 51.36, 38.82, 19.09, 8.33. 3c: 1H NMR
(400 MHz, Methanol-d4)
2g: 1H NMR (400 MHz, Methanol-d4)
d
7.26 (d, J ¼ 2.6 Hz, 1H),
d
7.56 (d, J ¼ 2.7 Hz, 1H), 7.42 (dd, J ¼ 8.7,
7.22 (dd, J ¼ 8.6, 2.6 Hz, 1H), 6.70 (d, J ¼ 8.6 Hz, 1H), 3.68 (s, 2H),
2.54e2.45 (m, 2H), 2.28 (s, 3H), 2.10 (s, 3H), 1.57 (q, J ¼ 7.5 Hz, 2H),
1.42e1.21 (m, 6H), 0.99e0.88 (m, 3H). 13C NMR (101 MHz,
2.8 Hz, 1H), 7.11 (d, J ¼ 8.8 Hz, 1H), 4.40 (dd, J ¼ 99.2, 13.1 Hz, 2H),
3.50e3.33 (m, 2H), 2.83 (s, 3H), 1.44 (t, J ¼ 7.3 Hz, 3H). 13C NMR
(101 MHz, Methanol-d4)
d
156.98, 127.31, 126.23, 122.13, 117.73,
Methanol-d4)
115.20, 60.32, 56.57, 40.01, 31.36, 26.56, 26.54, 22.23, 22.11, 12.96.
3g: 1H NMR (400 MHz, Methanol-d4)
7.56 (d, J ¼ 2.7 Hz, 1H), 7.42
d 169.90, 154.36, 129.99, 122.21, 121.36, 120.94,
116.49, 53.47, 51.32, 38.90, 8.53. Overall yield: 50%. HRMS [MþH]þ
calc (found): 181.13354 (181.1334).
d

158
M.C. de Koning et al. / European Journal of Medicinal Chemistry 157 (2018) 151e160
(dd, J ¼ 8.7, 2.7 Hz, 1H), 7.11 (d, J ¼ 8.7 Hz, 1H), 4.59e4.16 (m, 2H),
3.32e3.08 (m, 2H), 2.84 (s, 3H), 1.85 (dd, J ¼ 8.7, 6.4 Hz, 2H),
1.48e1.31 (m, 6H), 1.01e0.87 (m, 3H). 13C NMR (101 MHz,
1H), 5.49 (s, 2H). 13C NMR (101 MHz, Methanol-d4)
d 156.44,135.48,
125.33 (d, J ¼ 8.5 Hz), 122.10, 119.61, 117.15, 116.73, 116.40, 116.01,
55.29. Overall yield: 6%. HRMS [MþH]þ calc (found): 190.09749
(190.0981).
Methanol-d4)
d 156.97, 127.15, 126.12, 122.20, 117.82, 116.40, 56.38,
54.05, 39.57, 30.97, 25.89, 23.86, 12.87. Overall yield: 64%. HRMS
2r: 1H NMR (400 MHz, Methanol-d4)
d
7.72 (d, J ¼ 2.3 Hz, 1H),
[MþH]þ calc (found): 237.19614 (237.1968).
7.57 (d, J ¼ 1.9 Hz, 1H), 7.36 (dd, J ¼ 8.7, 2.6 Hz, 1H), 7.09 (d,
2h: 1H NMR (400 MHz, Methanol-d4)
d
7.27 (d, J ¼ 2.6 Hz, 1H),
J ¼ 2.6 Hz, 1H), 6.80 (d, J ¼ 8.6 Hz,1H), 6.35 (t, J ¼ 2.2 Hz,1H), 5.34 (s,
7.21 (dd, J ¼ 8.6, 2.6 Hz,1H), 6.68 (d, J ¼ 8.6 Hz,1H), 3.80 (s, 2H), 2.67
2H), 2.07 (s, 3H).13C NMR (101 MHz, Methanol-d4)
d 169.98, 151.87,
(q, J ¼ 7.2 Hz, 4H), 2.10 (s, 3H), 1.14 (t, J ¼ 7.2 Hz, 6H). 13C NMR
138.31, 130.71, 130.40, 122.97, 121.95, 121.86, 114.90, 105.35, 50.27,
(101 MHz, Methanol-d4)
d
173.78, 172.02, 157.01, 127.00, 126.02,
22.03. 3r: 1H NMR (400 MHz, Methanol-d4)
d
8.30 (dd, J ¼ 18.1,
122.21, 117.93, 116.43, 50.64, 19.34, 7.74. 3h: 1H NMR (400 MHz,
Methanol-d4)
7.60 (d, J ¼ 2.7 Hz, 1H), 7.43 (dd, J ¼ 8.7, 2.7 Hz, 1H),
2.7 Hz, 2H), 7.45 (d, J ¼ 2.7 Hz, 1H), 7.36 (dd, J ¼ 8.7, 2.8 Hz, 1H), 7.04
d
(d, J ¼ 8.7 Hz, 1H), 6.80 (t, J ¼ 2.8 Hz, 1H), 5.67 (s, 2H). 13C NMR
7.12 (d, J ¼ 8.7 Hz, 1H), 4.41 (s, 2H), 3.34e3.20 (m, 4H), 1.42 (t,
(101 MHz, Methanol-d4) d 156.44, 135.85, 135.18, 125.44, 124.97,
J ¼ 7.3 Hz, 6H). 13C NMR (101 MHz, Methanol-d4)
d
157.01, 127.16,
121.94, 121.12, 116.32, 107.72, 50.28. Overall yield: 6%. LC-MS
126.09, 122.14, 117.86, 116.45, 50.50, 7.84. HRMS [MþH]þ calc
[MþH]þ calc (found): 190.09749 (190.1).
(found): 195.14919 (195.1494). Overall yield: 21%.
4: 61.1 mg (231
m
mol) of 3l$2HCl (MW ¼ 265 g/mol) was dis-
2i: 1H NMR (400 MHz, Methanol-d4)
d
7.26 (d, J ¼ 2.4 Hz, 1H),
solved in 850
added as a base. 150
monitoring of the progress of the reaction by NMR. The mixture
was stirred until 3l was dissolved. Finally, 56 l (259.2 mol) par-
ml water, 66
ml (521.5 mmol) N-ethylmorpholine was
7.21 (dd, J ¼ 8.6, 2.6 Hz, 1H), 6.68 (d, J ¼ 8.6 Hz, 1H), 3.76 (s, 2H),
ml of deuterium oxide was added to enable
2.56e2.45 (m, 4H), 2.10 (s, 3H), 1.67e1.52 (m, 4H), 0.92 (t, J ¼ 7.4 Hz,
6H). 13C NMR (101 MHz, Methanol-d4)
d
169.84, 154.43, 130.03,
m
m
122.46, 121.18, 120.81, 115.24, 57.48, 55.19, 22.19, 19.24, 10.74. 3i: 1H
NMR (400 MHz, Methanol-d4)
aoxon was added and the mixture was transferred to an NMR tube.
The mixture was allowed to react measuring 31P and 1H NMR
spectra at regular intervals. After 4 days 31P NMR indicated com-
plete consumption of PXE and the formation of approximately 60%
of the adduct (based on 31P-content). The product was purified
using automated preparative HPLC, after which the sample was
isolated by lyophilization. Yield: 13.3%. 1H NMR (400 MHz, D2O)
d
7.57 (d, J ¼ 2.7 Hz, 1H), 7.41 (dd,
J ¼ 8.8, 2.7 Hz, 1H), 7.10 (d, J ¼ 8.7 Hz, 1H), 4.43 (s, 2H), 3.29e3.04
(m, 4H), 1.95e1.75 (m, 4H), 1.02 (t, J ¼ 7.3 Hz, 6H). 13C NMR
(101 MHz, Methanol-d4)
d 157.05, 127.14, 126.10, 122.16, 117.89,
116.41, 54.68, 51.50, 16.80, 9.83. Overall yield: 75%. HRMS [MþH]þ
calc (found): 223.18049 (223.1810).
2l: 1H NMR (400 MHz, Methanol-d4)
d
7.29e7.20 (m, 2H), 6.70
(d, J ¼ 8.5 Hz, 1H), 3.73 (s, 2H), 2.62e2.53 (m, 4H), 2.09 (s, 3H),
1.86e1.74 (m, 4H). 13C NMR (101 MHz, Methanol-d4)
169.79,
154.27, 129.93, 122.73, 121.12, 120.88, 115.21, 57.41, 53.03, 23.21,
22.39. 3l: 1H NMR (400 MHz, Methanol-d4)
7.41 (d, J ¼ 2.7 Hz,1H),
7.26 (dd, J ¼ 8.7, 2.7 Hz, 1H), 7.02 (d, J ¼ 8.7 Hz, 1H), 4.41 (s, 2H), 3.41
(s, 4H), 2.13 (s, 4H). 13C NMR (101 MHz, Methanol-d4)
155.12,
d
7.57e7.25 (m, 3H), 4.37 (s, 2H), 4.26 (h, J ¼ 7.7 Hz, 4H), 3.50 (s, 2H),
3.15 (s, 2H), 2.11 (s, 2H), 1.94 (s, 2H), 1.28 (t, J ¼ 7.1 Hz, 6H). 13C NMR
d
(101 MHz, D2O) d 146.47, 133.68, 124.55, 124.19, 123.19, 121.77,
117.81, 114.91, 66.93, 54.13, 51.95, 22.34, 15.29. 31P NMR (162 MHz,
d
D2O)
d
ꢂ5.80. HRMS [MþH]þ calc (found): 329.1625 (329.1635).
d
125.67, 125.15, 124.47, 118.44, 116.30, 53.66, 52.82, 22.54. Overall
4.2. Inhibition of human AChE by non-oximes
yield: 59%. HRMS [MþH]þ calc (found): 193.13354 (193.1333).
2m: 1H NMR (400 MHz, Methanol-d4)
d
7.25 (d, J ¼ 2.5 Hz, 1H),
20
were added to tempered cuvettes (37 ꢀC) containing 1.820
phosphate buffer (0.1 M, pH 7.4), 100 l DTNB (0.5 mM) and 10
native human AChE. After addition of 50
volume 2000 l) AChE activity was measured for 1 min and referred
ml non-oxime compounds (1e1.000 mM final concentration)
7.21 (dd, J ¼ 8.6, 2.6 Hz,1H), 6.69 (d, J ¼ 8.6 Hz,1H), 3.68 (s, 2H), 3.37
(s, 3H), 2.55 (m, 4H), 1.66 (m, J ¼ 5.6 Hz, 4H), 1.54 (m, J ¼ 6.8 Hz, 2H).
m
m
l
l
m
13C NMR (101 MHz, Methanol-d4)
d
169.90, 154.36, 129.96, 121.70,
ml ATCh (0.71 mM) (final
121.44, 120.88, 115.17, 60.96, 53.39, 25.56, 23.61, 22.07. 3m: 1H NMR
(400 MHz, Methanol-d4)
m
d
7.59 (d, J ¼ 2.7 Hz, 1H), 7.39 (dd, J ¼ 8.7,
to control activity. The IC50 values were calculated from semi-
logarithmic plots of the non-oxime compound concentration
versus the % AChE activity.
2.7 Hz, 1H), 7.09 (d, J ¼ 8.7 Hz, 1H), 4.36 (s, 2H), 3.50 (dd, J ¼ 12.1,
3.7 Hz, 2H), 3.08 (td, J ¼ 12.2, 3.5 Hz, 2H), 2.01e1.77 (m, 5H),
1.63e1.49 (m, 1H). 13C NMR (101 MHz, Methanol-d4)
d 157.30,
127.55, 126.06, 121.96, 117.24, 116.48, 54.37, 52.77, 22.51, 21.21.
Overall yield: 29%. HRMS [MþH]þ calc (found): 207.14919
(207.1493).
4.3. Determination of the inhibition type of non-oxime compounds
2n: 1H NMR (400 MHz, Methanol-d4)
d
7.28 (d, J ¼ 2.6 Hz, 1H),
Seven ATCh concentrations (50e1000
were used to determine the inhibition type of 3h (ADOC) and 3l
with human AChE. 50
l ATCh was added to cuvettes (37 ꢀC) filled
with 3000 l phosphate buffer (0.1 M, pH 7.4), 100 DTNB
(0.3 mM), 10 l human native AChE and 5 l 3h (ADOC) (0, 20, 40,
60, 80, 100 M) or 3l (0, 2.5, 5, 10, 15 M) and AChE activity was
measured for 1 min. AChE activity (v (mA/min)) was plotted against
ATCh concentration ([S] ( M)) and analyzed by non-linear regres-
mM final concentration)
7.23 (dd, J ¼ 8.6, 2.6 Hz, 1H), 6.70 (d, J ¼ 8.6 Hz, 1H), 3.72 (t,
J ¼ 4.7 Hz, 4H), 3.67 (s, 2H), 2.54 (t, J ¼ 4.6 Hz, 4H), 2.08 (s, 3H). 13
C
m
NMR (101 MHz, Methanol-d4)
d
171.34, 155.14, 131.65, 123.32,
m
ml
122.74, 122.54, 116.61, 67.86, 61.60, 54.09, 23.49. 3n: 1H NMR
(400 MHz, Methanol-d4)
7.60 (d, J ¼ 2.8 Hz, 1H), 7.39 (dd, J ¼ 8.8,
2.8 Hz, 1H), 7.10 (d, J ¼ 8.7 Hz, 1H), 4.43 (s, 2H), 4.15e3.80 (m, 4H),
3.50e3.20 (m, 4H). 13C NMR (101 MHz, Methanol-d4)
157.30,
m
m
d
m
m
d
m
127.62, 126.21, 122.18, 116.53, 63.37, 54.69, 51.60. Overall yield: 20%.
sion analysis (Michaelis-Menten plot). Double reciprocal
Lineweaver-Burk plots were identified by plotting 1/v against 1/[S]
and performed by linear regression analysis. Two secondary graphs
were derived from the Lineweaver-Burk plot: Slopes or ordinate
intercepts were plotted versus non-oxime compound concentra-
tions and analyzed by linear regression analysis. The dissociation
constants kic (slopes against non-oxime concentration) and kiu
(ordinate intercepts against non-oxime concentration) were
determined from the x-intercepts [38].
HRMS [MþH]þ calc (found): 209.12854 (209.1285).
2q: 1H NMR (400 MHz, Methanol-d4)
d 9.01e8.95 (m, 1H),
7.66e7.59 (m, 2H), 7.55e7.49 (m, 1H), 7.30 (dd, 1H), 6.85 (d,
J ¼ 8.7 Hz, 1H), 5.38 (s, 2H), 2.12 (s, 3H). 13C NMR (101 MHz,
Methanol-d4)
d 170.01, 151.97, 137.18, 130.46, 127.18, 123.12, 122.05,
119.60, 114.88, 61.52, 45.54, 22.05. 3q: 1H NMR (400 MHz,
Methanol-d4)
9.10 (d, J ¼ 1.6 Hz, 1H), 7.68 (t, J ¼ 1.7 Hz, 1H),
7.59e7.54 (m, 2H), 7.36 (dd, J ¼ 8.7, 2.7 Hz, 1H), 7.05 (d, J ¼ 8.7 Hz,
d

M.C. de Koning et al. / European Journal of Medicinal Chemistry 157 (2018) 151e160
159
4.4. Inhibition of human AChE by OPs
https://doi.org/10.1016/j.ejmech.2018.08.016.
Hemoglobin-free erythrocyte membranes ('ghosts') were pre-
pared as previously described [39] (see S.I.). Human erythrocyte
ghosts were incubated with small volumes (ꢃ1% v/v) of tabun,
sarin, cyclosarin, VX or paraoxon for 15 min at 37 ꢀC to achieve an
AChE inhibition of >95% of control activity. Inhibited and control
samples were dialyzed overnight (phosphate buffer, 0.1 M, pH 7.4)
to remove residual inhibitor and to adjust pH 7.4. Absence of re-
sidual inhibitor was verified by incubation of inhibited and control
ghosts (30 min, 37 ꢀC) and determination of AChE activity. Aliquots
were stored at ꢂ80 ꢀC. At the day of experiment thawed erythro-
cyte ghosts were homogenized on ice with a Sonoplus HD 2070
ultrasonic homogenizer (Bandelin electronic, Berlin; Germany)
twice for 5s with a 30s interval to achieve a homogenous matrix for
the kinetic studies.
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ðA1 ꢂ AiÞ
%reac ¼
*100
(1)
ðA0 ꢂ AiÞ
ꢁ
ꢀ
A0 represents control enzyme activity, Ai OP-inhibited enzyme
activity and A1 the activity of reactivated enzyme.
The pseudo-first-order rate constants kobs were calculated for
compounds 3h (ADOC) and 3l at 10
m
M and 100
m
M by linear
regression analysis using Eq. (2) [39,41[39,41].
ðv0 ꢂ vtÞ
ln
¼ ꢂkobsꢄt
(2)
ðv0 ꢂ viÞ
v0 represents the maximum velocity, vi the velocity of inhibited
AChE and vt the velocity of the reactivated enzyme.
Funding
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Chem. Biol. Interact. 203 (2013) 77e80.
This work was supported by Ministry of Defense of the
Netherlands (CBRN Program V1802) and the German Ministry of
Defense.
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Declaration of interest
None.
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