Synthesis and in vitro evaluation of neutral aryloximes as reactivators of Electrophorus eel acetylcholinesterase inhibited by NEMP, a VX surrogate

Article abstract of DOI:10.1016/j.cbi.2019.05.048

Casualties caused by nerve agents, potent acetylcholinesterase inhibitors, have attracted attention from media recently. Poisoning with these chemicals may be fatal if not correctly addressed. Therefore, research on novel antidotes is clearly warranted. Pyridinium oximes are the only clinically available compounds, but poor penetration into the blood-brain barrier hampers efficient enzyme reactivation at the central nervous system. In searching for structural factors that may be explored in SAR studies, we synthesized and evaluated neutral aryloximes as reactivators for acetylcholinesterase inhibited by NEMP, a VX surrogate. Although few tested compounds reached comparable reactivation results with clinical standards, they may be considered as leads for further optimization.

Full text of DOI:10.1016/j.cbi.2019.05.048

Accepted Manuscript
Synthesis and in vitro evaluation of neutral aryloximes as reactivators of
Electrophorus eel acetylcholinesterase inhibited by NEMP, a VX surrogate
Samir F. de A. Cavalcante, Daniel A.S. Kitagawa, Rafael B. Rodrigues, Leandro B.
Bernardo, Thiago N. da Silva, Wellington V. dos Santos, Ana Beatriz de A. Correa,
Joyce S.F.D. de Almeida, Tanos C.C. França, Kamil Kuča, Alessandro B.C. Simas
PII:
S0009-2797(19)30231-5
https://doi.org/10.1016/j.cbi.2019.05.048
CBI 8682
DOI:
Reference:
To appear in: Chemico-Biological Interactions
Received Date: 27 February 2019
Revised Date: 23 April 2019
Accepted Date: 27 May 2019
Please cite this article as: S.F. de A. Cavalcante, D.A.S. Kitagawa, R.B. Rodrigues, L.B. Bernardo, T.N.
da Silva, W.V. dos Santos, A.B. de A. Correa, J.S.F.D. de Almeida, T.C.C. França, K. Kuča, A.B.C.
Simas, Synthesis and in vitro evaluation of neutral aryloximes as reactivators of Electrophorus eel
acetylcholinesterase inhibited by NEMP, a VX surrogate, Chemico-Biological Interactions (2019), doi:
https://doi.org/10.1016/j.cbi.2019.05.048.
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ACCEPTED MANUSCRIPT
Synthesis and in vitro evaluation of neutral aryloximes as reactivators of Electrophorus eel
acetylcholinesterase inhibited by NEMP, a VX surrogate
Samir F. de A. Cavalcantea,b,c,d,*, Daniel A. S. Kitagawae, Rafael B. Rodriguesa, Leandro B. Bernardoa,
Thiago N. da Silvac, Wellington V. dos Santosf,g , Ana Beatriz de A. Correaa, Joyce S. F. D. de Almeidae,
Tanos C.C. Françad,e, Kamil Kučad, and Alessandro B. C. Simasb,*
a Brazilian Army CBRN Defense Institute (IDQBRN), Avenida das Américas 28705, Rio de Janeiro, 23020-470,
Brazil.
b Walter Mors Institute of Research on Natural Products (IPPN), Federal University of Rio de Janeiro (UFRJ), CCS,
Bloco H, Rio de Janeiro, 21941-902, Brazil.
c University Castelo Branco (UCB), School of Pharmacy, Avenida Santa Cruz 1631, Rio de Janeiro, 21710-255,
Brazil.
d Department of Chemistry, Faculty of Science, University of Hradec Králové, Rokitanskeho 62, 50003 Hradec
Králové, Czech Republic.
e Laboratory of Molecular Modelling Applied to Chemical and Biological Defense (LMACDB), Praça General
Tibúrcio 80, Rio de Janeiro, 22290-270, Brazil.
f Emergency and Rescue Department (DSE), Rio de Janeiro State Fire Department (CBMERJ), Praça São Salvador 4,
Rio de Janeiro, 22231-170, Brazil.
g
University Universus Veritas (UNIVERITAS), School of Biomedicine, Rua Marquês de Abrantes 55, Rio de
Janeiro, 22230-060, Brazil.
1

Abstract
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Casualties caused by nerve agents, potent acetylcholinesterase inhibitors, have attracted attention from media
recently. Poisoning with these chemicals may be fatal if not correctly addressed. Therefore, research on novel
antidotes is clearly warranted. Pyridinium oximes are the only clinically available compounds, but poor penetration
into the blood-brain barrier hampers efficient enzyme reactivation at the central nervous system. In searching for
structural factors that may be explored in SAR studies, we synthesized and evaluated neutral aryloximes as
reactivators for acetylcholinesterase inhibited by NEMP, a VX surrogate. Although few tested compounds reached
comparable reactivation results with clinical standards, they may be considered as leads for further optimization.
Keywords: Acetylcholinesterase reactivators; Nerve agents’ surrogates; Neutral oximes; Drug screening; Chemical
defence.
* Corresponding authors: Dr. Samir F. de A. Cavalcante, e-mail: samir.cavalcante@eb.mil.br; Dr. Alessandro B. C.
Simas, e-mail: abcsimas@nppn.ufrj.br.
1. Introduction
Acetylcholinesterase (AChE, EC 3.1.1.7, 1) is a serine-estearase, with pivotal role in the parasympathetic
neurotransmission, ending the potential action at the post-synaptic cleft by hydrolysis of neurotransmitter
acetylcholine (ACh, 2, Scheme 1) into its two precursors, choline (3) and acetate (4). AChE is mainly located in
brain, but it can also be found in erythrocytes and muscles.1-7
Scheme 1. Hydrolysis of ACh by AChE.
2

The hydrolysis of ACh takes place at the estearic site of AChE, which has a catalytic triad composed by residues
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of serine, histidine and glutamate, which are highly conserved throughout species. In case of organophosphorus
poisoning, phosphorylation of the serine hydroxyl group in the active site leads to AChE inhibition, bringing about
ACh accumulation, which may be fatal depending on the dose, due to overstimulation of cholinergic innervations.8-12
This could be avoided by the action of an AChE reactivator, i.e. a molecule capable of reverting the serine
phosphorylation and restoring the enzyme functions. Such compounds can be discovered through kinetic studies on
the isolated and purified enzyme.13-15 In spite of interspecies differences that may occur related to affinity and
reactivity towards inhibitors,16 and the use of different isoforms, AChE from electric eel (Electrophorus electricus)
EeAChE, is largely reported in studies on novel reactivators against inhibitors or active compounds to address
neurodegenerative diseases, being considered a trustable and affordable model.17,18
Nerve agents are strictly regulated by the Chemical Weapons Convention (CWC) and its international watchdog,
the Organization for the Prohibition of Chemical Weapons (OPCW), which oversees the CWC implementation and
compliance.13 Figure 1 depicts examples of such toxic chemicals, the nerve agents sarin (5), soman (6), tabun (7), VX
(8), the recently confirmed Novichok agents, e.g., A-234 (9), and the pesticide paraoxon (10).
Novichok agents, a class of compounds developed to circumvent CWC and North Atlantic Treaty Organization
(NATO) standard protective gear and detection systems, have been involved in poisonings that took place in the
United Kingdom. According to different sources, diverse structures have been proposed for these compounds. These
structures have been considered to enter in the official documents, as CWC, and databases, as the OPCW Central
Analytical Database (OCAD).20-26
Figure 1. Examples of toxic organophosphorus compounds.
3

Development of antidotes towards nerve agents is essential, as there is no “universal antidote” available. The
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production, transfer and use of the so-called “live agents” for must be done in accordance to CWC.19 Therefore, they
are under strict control. To overcome these limitations, surrogates may be employed. They possess lower volatility
and toxicity in comparison to actual agents, being easier to manipulate. Nonetheless, they afford the same enzymatic
adduct, allowing the search for novel antidotes, among other studies.23,24 An example of such compounds is O-(4-
nitrophenyl) O-ethyl methylphosphonate (NEMP, 11), a VX surrogate, which yields the same O-ethyl
methylphosphonyl adduct.23-25 Figure 2 depicts VX and NEMP structures, with similarities shown in bold red.
Figure 2. Structures of VX and NEMP.
As depicted in the Scheme 2, phosphonylation of the AChE catalytic serine residue (12) caused by NEMP leads to
accumulation of ACh (2), triggering the SLUDGEM syndrome (salivation, lacrimation, urination, defecation,
gastrointestinal disturbance, emesis, miosis and muscle spasms).9,20 Similarly to VX, NEMP does not cause
significant AChE aging, i.e., disproportion of the O-ethyl moiety that generates a phosphonate anion (13, Scheme
3).24 Aged AChE cannot be reactivated by any of the clinically available antidotes, pyridinium oximes, due to ionic
interactions with the imidazolium ring of the histidine moiety in the catalytic triad.25,26
Scheme 2. AChE inhibition by NEMP.
4

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Scheme 3. Aging of O-ethyl methylphosphonyl-AChE adduct.
Pralidoxime (2-PAM, 14), obidoxime (OBD, 15), trimedoxime (TMB, 16), HI-6 (17) and HLö-7 (18) are
examples of therapeutic compounds (Figure 3).27-30 Under physiological conditions, they yield oximates that
reactivate AChE by displacing the organophosphorus moiety from the serine residue via nucleophilic attack.
Although they reactivate AChE well at neuromuscular junctions, their positive charge reduces permeability through
the blood-brain barrier, thereby decreasing effectiveness at the central nervous system (CNS). These limitations
warrant the development of novel AChE reactivators with enhanced pharmacokinetics.9,31-33
Figure 3. Clinically available antidotes.
Along with other research groups working on tackling these issues involving pyridinium oximes,32-39 we set out to
investigate the action of neutral aryloximes as reactivators for NEMP-inhibited AChE. For that, we employed the
Ellman’s spectrophotometric assay and commercial Electrophorus eel as source of AChE. Although aryloximes
lacking a cationic nitrogen atom are matter-of-factly expected to perform worse than the pyridinium analogues, the in
vitro assays with selected neutral oximes might disclose further structural features to be exploited in the design of
novel AChE reactivators.
5

We reckoned that the electronic effects and other properties bestowed by electron-withdrawing or donating groups
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in different positions, might provide useful insights in that work. Besides the expected gain in the mentioned
pharmacokinetics properties due to better permeation into the brain, additional interactions by the neutral aryl moiety
could compensate the absence of the cationic nitrogen (present in clinically available compounds) in the estearic site.
9,38,42
2. Experimental
2.1. General information
Acetylthiocholine iodide (ATCI), 5,5’-dithiobis-(2-nitrobenzoic) acid (DTNB), lyophilized acetylcholinesterase
from Electrophorus eel (EeAChE, 1000U per mg protein, type V-S, C2888), O,O-diethyl methylphosphonate,
pralidoxime iodide (2-PAM), N,N-dimethylformamide (DMF, dry, oxygen-free sealed bottle), dimethylsulfoxide
(DMSO, biological grade, dry, oxygen-free sealed bottle), triethylamine (TEA, dry, oxygen-free sealed bottle),
absolute 2-propanol, paraformaldehyde, dichloromethane (dried over 4 Å molecular sieves before use), toluene (dried
over 4 Å molecular sieves before use), sodium hydroxide (pellets), anhydrous sodium sulphate, anhydrous sodium
chloride, sodium phosphate monobasic hydrate and sodium phosphate dibasic dihydrate were purchased from Sigma-
Aldrich (São Paulo, Brazil). Absolute ethanol was purchased from Tedia (Rio de Janeiro, Brazil). Hydrochloric acid
(37% p/p, 1.19 g/mL), chlorosulphonic acid, sodium hypochlorite, ethyl acetate, hexane and 4-nitrophenol (PNP)
were purchased from Vetec (Rio de Janeiro, Brazil). Oxalyl chloride was purchased from TCI Chemicals (J1L1
Eireli, Rio de Janeiro, Brazil). Deuterated solvents (CDCl3 and DMSO-d6) containing tetramethylsilane as internal
standard were purchased from Cambridge Isotopes Laboratories (Tewksbury, Massachusetts, USA). Purified water
was obtained from Millipore Milli-Q system (18.2 MΩ cm at 25°C, Millipore Brazil, São Paulo, Brazil). Biotage
Initiator+ Eight and Isolera ACI Chromatography System (Charlotte, North Carolina, USA) were used for synthesis
of all oximes and flash chromatography, respectively. Sealed tubes (Q-Tube) were purchased from Q-Labtech (East
Lyme, Connecticut, USA). TLC (Thin Layer Chromatography) aluminium plates coated with silica gel F254 were
purchased from Merck Brazil (São Paulo, Brazil). Camag TLC-MS (Thin Layer Chromatography – Mass
Spectrometry) interface was used to follow up reactions (CAMAG, Muttenz, Switzerland). NMR spectra were
obtained from Varian Unity 400MHz (Santa Clara, California, USA) and Bruker Avance 400MHz (Palo Alto,
California, USA) and referred to tetramethylsilane for 1H and 13C-NMR spectra. GC-MS (Gas Chromatography-Mass
6

Spectrometry) data were obtained from Agilent 6890 GC system equipped with 5975C mass spectrometer detector
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(Billerica, Massachusetts, USA). LCMS (Liquid Chromatography – Mass Spectrometry) data were obtained from
Agilent 1210 LC system equipped with 6410B triple quadrupole mass spectrometer detector (Billerica,
Massachusetts, USA). Melting points were obtained from SRS OptiMelt 100 (Stanford, CA, USA) using open
capillary method and were uncorrected. SpectraMax Plus 384 microplate reader (Molecular Devices, San Jose,
California, USA) was used in all assays. 96-wells microplates were purchased from Kasvi Brasil (São José dos
Pinhais, Paraná, Brazil). Gilson single channel pipettes were purchased from Gilson Inc. (Middleton, Wisconsin,
USA) and Eppendorf 8-channel pipettes were acquired from Eppendorf Brasil (São Paulo, Brazil). Ellman´s tests
were performed in triplicate, in three different assays, by at least three different operators, measured at 24 ± 2 °C.
Microsoft Excel® 2010 was used for all calculations. All disposable materials and glassware in contact with
organophosphorus compounds were decontaminated with aqueous solution containing 10% w/v NaOH and 10% w/v
NaClO for 48 h at room temperature before correct destination. Estimations of pKa and logP for clinical antidotes
and test compounds were obtained from ChemAxon Online Suite. Agilent Chemstation E.02.02.1431 was used for
area integration of GC data for purity calculations. O-(4-nitrophenyl) O-ethyl methylphosphonate (NEMP) was
synthesized in accordance to literature.23
2.2. Synthesis of neutral aryloximes
Scheme 4 illustrates the synthetic procedure for the oximes employed in this work. Their preparation was
performed as follows: a microwave vial equipped with a magnetic stirrer was charged with 1 mmol of carbonyl
compound, 2 mmol of hydroxylamine hydrochloride and 1.5 mL of 50% v/v water/ethanol. The system was sealed
and stirred for 60 s at room temperature. Then the vial was irradiated at 120ºC with automatic power control for 1h.
After completion of reaction, the solid products were filtered off and washed with cold water. If desired, the solids
may be recrystallized from ethanol-water mixtures, but they usually are very pure compounds, as checked by TLC
and GC. If no solid was formed, extraction with ethyl acetate (15 mL) and washing of organic layer with water (2 x
15 mL) were performed. The organic layer was collected, dried over anhydrous sodium sulphate, filtered and
concentrated in the rotary evaporator to give oily products in high purity, in accordance to TLC and GC methods. If
desired, oily products may be subjected to flash chromatography, using 10 to 40% ethyl acetate/hexane mixture.
7

Table 1 depicts all synthesized compounds, yields and relevant estimated physicochemical properties. Spectroscopic
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data for all synthesised compounds are provided in Supplementary Material.
Scheme 4. Synthetic procedure for oximes.
2.3. Synthesis of bis-chloromethyl ether
Bis-chloromethyl ether (21) was synthesized using a slightly modified procedure from literature (Scheme 5).43 In a
50 mL, 3-neck-round-bottomed flask equipped with a magnetic stirrer, addition funnel and thermometer, 5.25g
(0.175 mmol) of paraformaldehyde was added and cooled to 0ºC. Then, 5 mL of HCl 37% p/p (pre-cooled to 0ºC)
were added in one portion. The slurry was vigorously stirred and cooled to 0ºC. 7.5 mL of chlorosulphonic acid were
added in such a rate that internal temperature was kept at 0-5ºC (full addition took 20 min). The suspension was then
left to reach room temperature and stirred for additional 24 h. After that time, stirring was interrupted and the
reaction medium separated in two layers. A yellow bottom layer was collected and stirred by 15 min with anhydrous
sodium chloride to remove moisture and carefully filtered with mild vacuum. The product was pure enough for the
intended purposes (9 g, 90%) and must be handled with CAUTION, as it is a known carcinogenic.
Scheme 5. Synthetic procedure for bis-chloromethyl ether.
2.4. Synthesis of standard antidotes
Obidoxime (OBD) dichloride (15) and trimedoxime (TMB) dibromide (16) were synthesized using a slightly
modified procedure (Scheme 6).44,45 In a sealed tube, pyridine-4-aldoxime (1 mmol, 19ad) was dissolved in dry DMF
(0.8 mol/L) and bis-chloromethyl ether (0.45 mmol, 21) or 1,3-dibromopropane (22) was added in one portion.
8

Reaction was heated at 80 ºC for 6 h. Then, the reaction mixture was cooled to room temperature and cold acetone
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was added (5 mL per mmol of aldoxime) to full precipitation of white solids. These were filtered and washed with
cold acetone, yielding 50% of OBD dichloride (77 mg) and 40% of TMB dibromide (65mg).
Scheme 6. Synthetic routes for standard antidotes used in this work.
2.5. Ellman’s spectrophotometric assays
Ellman’s assays were performed in accordance to our previously published procedure,48 using 96-wells
microplates, with maximum volume of 200 µL. Briefly, for AChE inhibition, we set the microplate reader to 412 nm
and pipetted 70µL of EeAChE (2.14 U/mL, prepared from commercial lyophilized), 80µL of DTNB 0.4 mg/mL, 20
µL of phosphate buffer solution (PBS, pH 7.60 ± 0.10), 10 µL of inhibitor (positive control, Ai; CAUTION as NEMP
is a toxic organophosphorus compound) or 10 µL of PBS (negative control, A0), and waited for 10 min for inhibition
reaction. Then, we added 20 µL of 1 mmol/L of ATCI and read the absorbance in different times to calculate the
enzyme inhibition. AChE inhibition percent was given by Equation 1. Slight modifications were put forth for aging
and spontaneous hydrolysis assays. We set as inhibition reaction time 15 and 75 min for aging and 10 min and 12 h
for spontaneous hydrolysis.
ꢁ ꢃꢁ
ꢂ
ꢄ
%ꢀ = 100 ×
(1)
ꢁ
ꢂ
For AChE reactivation, we set the microplate reader to 412 nm and pipetted 70 µL of EeAChE (2.14 U/mL,
prepared from commercial lyophilized), 80 µL of 0.4 mg/mL DTNB, 10 µL of inhibitor (positive control) or 10 µL
of PBS (negative control), and waited for 10 min for inhibition reaction. Following this time, we added 20 µL
standard antidotes or test molecules in different concentrations (1000, 100 and 10 µmol/L as final concentrations) and
waited for 30 min for reactivation reaction. At last, added 20 µL of 1 mmol/L of ATCI and read the absorbance (Ar)
in different times to calculate the enzyme reactivation. AChE reactivation percent was given by Equation 2.
9

ꢁ ꢃꢁ
ꢆ
ꢄ
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%ꢅ = 100 ×
(2)
ꢁ ꢃꢁ
ꢂ
ꢄ
3. Results and Discussion
All oximes synthesized were evaluated as NEMP-inhibited EeAChE reactivators in three different concentrations,
in accordance to our procedure.48 Table 1 shows the calculated physicochemical properties of assayed compounds
and standard antidotes. NEMP solution was freshly prepared from crude compound in absolute ethanol at
approximately 20 µmol/L, (0.5 mg in 100 mL, 1 µmol/L in each well after all dilutions). At this concentration,
NEMP caused inhibition of 93 ± 1% of EeAChE after 10 min of incubation in our Ellman’s assay conditions. Major
impurity, 4-nitrophenol, did not interfere in absorbance measurements.29,30 Results were determined after 30 minutes
of reactivation reaction. Results for the most active compounds are shown in Table 2 (full results on the EeAChE
reactivation are shown in the Supplementary Material).
We also evaluated if NEMP, in our Ellman inhibition assay’s conditions, would cause EeAChE aging. We
performed two sets of experiments, modifying the enzyme-surrogate incubation time to 15 and 75 min, respectively.
Then, we added 2-PAM 100 µmol/L and let the reactivation reaction occurs for 30 min before addition of substrate
(ATCI) and read the absorbance. EeAChE inhibition level by crude NEMP solution was similar for both incubation
times, 95 ± 1%, and reactivation rates were virtually the same, 56% and 59%, respectively. These results (shown in
Table 3) put forward the idea that NEMP indeed does not age significantly EeAChE under our test conditions. As a
complimentary study, we also measured spontaneous enzyme reactivation using NEMP as inhibitor. After incubating
EeAChE with crude NEMP solution for 10 min and 12 h at room temperature, we added ATCI and read the
absorbance. Enzyme inhibition level after 10 min of incubation with NEMP was 93 ± 1%, while after 12 h inhibition
was 55 ± 3% (see Table 4). This result suggests that spontaneous hydrolysis of O-ethyl methylphosphonyl moiety
from serine residue at the estearic site might be occurred, rendering some enzyme activity. This result also supports
our hypothesis that NEMP does not significantly age EeAChE.23,24
The synthesised compounds, as expected, displayed better lipophilicity (as indicated by estimated logP values,
Table 1) than standard antidotes (entries 34-36), thus, potentially enabling more effective passage through the
hematoencephalic barrier. Higher concentration of reactivator in CNS is necessary for AChE reactivation in the
brain.41,42 Moreover, most calculated pKa values compare well with the standard antidotes.
10

All selected oximes were evaluated as NEMP-inhibited EeAChE reactivators at three different concentrations via
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Ellman´s procedure.48 Few compounds retrieved comparable reactivation results in comparison to standard antidotes.
Nonetheless, some structural factors identified could be included in further investigations on novel cholinesterase
reactivators. Considering that 100 µmol/L is the maximum attainable concentration in vivo for clinically available
reactivators,49,50 we decided to also assay our compounds at this condition. This would provide comparable results.
We employed 2-PAM (13) as main antidote reference and OBD (14) and TMB (15), under the same assay conditions,
to confirm the current knowledge that bispyridinium compounds are substantially more efficient in AChE
reactivation.32 Care was taken to discount the absorbance of the tested compounds, avoiding calculation errors.
As neutral aryloximes are devoid of some structural features for complementary interactions inside enzymatic
gorge, we decided to assay all compounds at 1000 µmol/L as well. Such experiments might be useful in disclosing
other relevant molecular moieties for further development of reactivators with improved pharmacokinetics.
Reactivation percent was calculated by equation 2, with absorbance read after 30 min of incubation of test
compounds with NEMP-inhibited EeAChE. We set as threshold 10 ± 1% of reactivation of NEMP-inhibited EeAChE
for selection of promising candidates, in accordance to literature.48
Table 1.Oximes 20a-ag synthesized for assay in this work
Entry
Code
20a
20b
20c
20d
20e
20f
Oxime
pKa
logP
1.39
Yield %
85
6.61 (OH= 9.99)
1
2
3
4
5
6
7
2-hydroxybenzaldoxime
3-hydroxybenzaldoxime
7.09 (OH= 9.74)
1.39
1.39
1.54
1.54
1.54
2.46
95
4-hydroxybenzaldoxime
7.57 (OH= 10.15)
93
6.69
7.20
7.69
6.96
2-methoxybenzaldoxime
3-methoxybenzaldoxime
91
92
4-methoxybenzaldoxime
2-bromobenzaldoxime
91
20g
94
11

3-bromobenzaldoxime
6.69
2.46
2.30
2.30
2.30
1.84
1.84
1.84
2.57
2.57
2.57
2.21
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8
9
20h
20i
89
95
4-bromobenzaldoxime
6.80
7.26
6.67
6.82
7.11
6.80
5.52
6.13
6.29
8.08
10
11
12
13
14
15
16
17
18
Entry
19
20
21
22
23
24
25
26
20j
2-chlorobenzaldoxime
3-chlorobenzaldoxime
94
20k
20l
90
4-chlorobenzaldoxime
96
20m
20n
20o
20p
20q
20r
2-fluorobenzaldoxime
3-fluorobenzaldoxime
86
90
4-fluorobenzaldoxime
81
2-trifluoromethylbenzaldoxime
3-trifluoromethylbenzaldoxime
88
88
4-trifluoromethylbenzaldoxime
94
Code
20s
20t
Oxime
pKa
7.97
logP
2.21
Yield %
83
2-methylbenzaldoxime
3-methylbenzaldoxime
4-methylbenzaldoxime
8.14
2.21
2.94
1.64
1.64
1.80
2.52
1.23
84
8.21
20u
20v
20w
20x
20y
20z
87
3-nitrobenzaldoxime
5.83
5.80
90
4-nitrobenzaldoxime
93
4-isopropylbenzaldoxime
4-(N,N-dimethylamino)benzaldoxime
4-(N,N-diethylamino)benzaldoxime
8.71
95
8.80
90
7.18 (OH= 10.60)
94
12

6.69
2.46
1.23
1.23
0.48
1.15
0.96
2.55
-3.26
-6.93
-7.04
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27
28
29
30
31
32
33
34
35
36
20aa
20ab
20ac
20ad
20ae
20af
20ag
13
Vanillin oxime
90
90
90
98
98
98
93
6.21 (OH= 10.47)
7.18 (OH= 10.14)
10.21
Isovanillin oxime
Orthovanillin oxime
Pyridine-4-aldoxime
Pyridine-2-aldoxime
Isatin 3-oxime
9.02
7.13 (NH= 15.51)
7.31
N-benzylisatin 3-oxime
Pralidoxime (2-PAM)
Obidoxime (OBD)
Trimedoxime (TMB)
a
7.63
-
7.51, 8.11
8.63, 9.24
14
50
40
15
a Commercial source
Table 2 shows the EeAChE reactivation percent for the most active compounds. Under our Ellman’s conditions,
we observed that the best results for reactivation of NEMP-inhibited EeAChE were at 1000 µmol/L, the highest
concentration assayed (entries 1-7). Reference antidote 2-PAM and bispyridinium oxime TMB, also exhibited the
same profile (entries 8 and 10, respectively), differently from the bispyridinium oxime OBD (entry 9) which showed
opposite results, being more active at 100 µmol/L. These results may suggest that, for the neutral or pyridinium
monooximes, reactivation potency is concentration-dependent, and corroborates with the literature findings, that
oximes present different reactivation profiles for each inhibitor, being capable of even acting as AChE inhibitors.51
All tested neutral oximes showed essentially no reactivation at 10 µmol/L. At 100 µmol/L, four compounds (20q,
3-trifluoromethyl analogue; 20r, 4-trifluoromethyl analogue; 20af, isatin 3-oxime, and 20ag, N-benzylisatin 3-oxime)
reactivated NEMP-inhibited EeAChE approximately at 60% of the 2-PAM reactivation, indicating that these are
interesting compounds for further structural optimization.
13

From results obtained at 1000 µmol/L, we could identify 3 additional compounds. 2-Chlorobenzaldoxime (20j),
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result in agreement with data that showed that 2-chloroaryloximes successfully reactivates AChE inhibited by
different nerve agents);52 2-trifluoromethylbenzaldoxime (20p) and orthovanillin oxime (20ac) could all reactivate
NEMP-inhibited EeAChE. The activity of the trifluoromethyl derivatives may be related, at least in part, to polar
hydrophobicity effects.53,54 Isatin-derived oximes stood out in the high-concentration experiments, even surpassing 2-
PAM and OBD as reactivators. Isatin derivatives, diversely available, have also been reported as cholinesterase
inhibitors.56-59 and are clearly a choice for further development. 60,61
Table 2.Percent of EeAChE reactivation by the most active oximes
Reactivator Concentration (µmol/L)
Entry
Oxime
1000
100
10
1
2
3
4
5
6
7
20j
20p
20q
20r
36 ± 2
10 ± 1
32 ± 2
32 ± 3
13 ± 1
34 ± 1
45 ± 5
1 ± 0
0
1 ± 0
0
13 ± 1
3 ± 0
1 ± 0
1 ± 0
2 ± 0
1 ± 0
13 ± 1
20ac
20af
20ag
4 ± 1
13 ± 1
10 ± 1
Reactivator Concentration (µmol/L)
Entry
Oxime
1000
29 ± 2
26 ± 1
57 ± 2
100
10
8
9
2-PAM (13)
OBD (14)
TMB (15)
21 ± 1
46 ± 1
39 ± 1
4 ± 0
15 ±1
17 ±1
10
Table 3.Percent of NEMP-inhibited EeAChE aging
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Inhibiton time (min)
15
%EeAChE Inhibitiona
%EeAChE Reactivationb
95 ± 1
59 ± 3
56 ± 5
3
95 ± 1
75
∆%EeAChE Reactivation
a Crude NEMP 1 µmol/L, b 2-PAM 100 µmol/L
Table 4.Percent of NEMP-inhibited EeAChE spontaneous hydrolysis
Inhibiton time
10 min
%EeAChE Inhibitiona
93 ± 1
55 ± 3
12 h
a Crude NEMP 1 µmol/L
4. Conclusion
In conclusion, using NEMP, a VX surrogate, as acetylcholinesterase inhibitor, we assayed a series of simple,
neutral oximes, aiming at identifying scaffolds for further drug development. Based on the reactivation profile shown
in our assays, we were able to identify seven active compounds which are now under structural optimization for
development of novel cholinesterase reactivators. All active compounds were concentration-dependent as reactivators
for NEMP-inhibited EeAChE. One of the compounds identified from the study (an isatin derivative) has been
modified and successfully afforded new, active cholinesterase reactivators for organophosphorus-inhibited
cholinesterases. As it is also known that oximes may act as reversible inhibitors of cholinesterases, our molecules are
also being investigated in order to identify scaffolds that could act as AChE inhibitors, useful against
neurodegenerative diseases.
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Acknowledgments
Authors would like to thank Dr. Maria do Carmo Pinto (IPPN) for donation of aldehydes, Central Analítica do
IPPN and Núcleo de Competência para o Desenvolvimento de Tecnologias de Carbono – NCDTC/CTEx for NMR
spectra. S.F.A.C. is grateful to OPCW for research grant (L/ICA/ICB/201062/15), Brazilian Army for infrastructure,
University of Hradec Kralove (UHK) for travel grant and OPCW & Spiez Laboratory (Switzerland) for Fellowship in
Organic Synthesis (Synthesis of CWC-Related Chemicals, 2012). This work was also supported by the long-term
development plan UHK. S.F.A.C. would like also to dedicate this work to the beloved memory of Professor Antonio
Ventura Pinto (IPPN).
Conflict of Interests
None.
Authors’ Information
ORCID numbers for authors: Samir F. de A. Cavalcante: 0000-0002-3478-2747; Joyce S. F. D. de Almeida: 0000-
0002-1426-6681; Tanos C.C. França: 0000-0002-6048-8103; Kamil Kuča: 0000-0001-9664-1109; Alessandro B. C.
Simas: 0000-0002-0470-3024.
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Supplementary Material
Supplementary data (NMR and MS spectra of synthesized compounds, full results on EeAChE reactivation for all
synthesised compounds) can be found in the online version.
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Highlights:
•
•
•
•
A practical method for synthesis of neutral aryloximes is presented;
A VX surrogate was used as inhibitor for Electrophorus eel Acetylcholinesterase;
Neutral aryloximes retrieved promising predicted properties for use as reactivators;
Seven compounds were identified as potential leads for further optimization.

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Declaration of interests
☒ The authors declare that they have no known competing financial interests or personal relationships
that could have appeared to influence the work reported in this paper.
☐The authors declare the following financial interests/personal relationships which may be considered
as potential competing interests: