Synthesis and in vitro evaluation of novel non-oximes for the reactivation of nerve agent inhibited human acetylcholinesterase

Article abstract of DOI:10.1016/j.cbi.2020.109139

Since several decades oximes have been used as part of treatment of nerve agent intoxication with the aim to restore the biological function of the enzyme acetylcholinesterase after its covalent inhibition by organophosphorus compounds such as pesticides and nerve agents. Recent findings have illustrated that, besides oximes, certain Mannich phenols can reactivate the inhibited enzyme very effectively, and may therefore represent an attractive complementary class of reactivators. In this paper we further probe the effect of structural variation on the in vitro efficacy of Mannich phenol based reactivators. Thus, we present the synthesis of 14 compounds that are close variants of the previously reported 4-amino-2-(1-pyrrolidinylmethyl)-phenol, a very effective non-oxime reactivator, and 3 dimeric Mannich phenols. All compounds were assessed for their ability to reactivate human acetylcholinesterase inhibited by the nerve agents VX, tabun, sarin, cyclosarin and paraoxon in vitro. It was confirmed that the potency of the compounds is highly sensitive to small structural changes, leading to diminished reactivation potency in many cases. However, the presence of 4-substituted alkylamine substituents (as exemplified with the 4-benzylamine-variant) was tolerated. More surprisingly, the dimeric compounds demonstrated non-typical behavior and displayed some reactivation potency as well. Both findings may open up new avenues for designing more effective non-oxime reactivators.

Full text of DOI:10.1016/j.cbi.2020.109139

Journal Pre-proof
Synthesis and in vitro evaluation of novel non-oximes for the reactivation of nerve
agent inhibited human acetylcholinesterase
Martijn C. de Koning, Gabriele Horn, Franz Worek, Marco van Grol
PII:
S0009-2797(20)30663-3
https://doi.org/10.1016/j.cbi.2020.109139
CBI 109139
DOI:
Reference:
To appear in: Chemico-Biological Interactions
Received Date: 20 April 2020
Revised Date: 6 May 2020
Accepted Date: 19 May 2020
Please cite this article as: M.C. de Koning, G. Horn, F. Worek, M. van Grol, Synthesis and in vitro
evaluation of novel non-oximes for the reactivation of nerve agent inhibited human acetylcholinesterase,
Chemico-Biological Interactions (2020), doi: https://doi.org/10.1016/j.cbi.2020.109139.
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Credit Author Statement
De Koning: Conceptualization, Methodology, Validation, Formal analysis, Writing - Original
Draft, Visualization, Supervision, Project administration; Horn: Methodology, Validation,
Formal analysis, Investigation, Writing - Review & Editing, Visualization; Worek: Writing -
Review & Editing, Visualization; Supervision, Project administration; van Grol: Methodology,
Validation, Formal analysis, Investigation.

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Synthesis and in Vitro Evaluation of Novel Non-Oximes for the
Reactivation of Nerve Agent Inhibited Human
Acetylcholinesterase
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Martijn C. de Koning, a,* Gabriele Horn,b Franz Worek,b Marco van Grol,a
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aTNO, Lange Kleiweg 137, 2288 GJ Rijswijk, The Netherlands. bBundeswehr Institute of
Pharmacology and Toxicology, Neuherbergstrasse 11, 80937 Munich, Germany.
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*) corresponding author: m.dekoning@tno.nl
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Abstract
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Since several decades oximes have been used as part of treatment of nerve agent intoxication
with the aim to restore the biological function of the enzyme acetylcholinesterase after its
covalent inhibition by organophosphorus compounds such as pesticides and nerve agents.
Recent findings have illustrated that, besides oximes, certain Mannich phenols can reactivate
the inhibited enzyme very effectively, and may therefore represent an attractive
complementary class of reactivators. In this paper we further probe the effect of structural
variation on the in vitro efficacy of Mannich phenol based reactivators. Thus, we present the
synthesis of 14 compounds that are close variants of the previously reported 4-amino-2-(1-
pyrrolidinylmethyl)-phenol, a very effective non-oxime reactivator, and 3 dimeric Mannich
phenols. All compounds were assessed for their ability to reactivate human
acetylcholinesterase inhibited by the nerve agents VX, tabun, sarin, cyclosarin and paraoxon
in vitro. It was confirmed that the potency of the compounds is highly sensitive to small
structural changes, leading to diminished reactivation potency in many cases. However, the
presence of 4-substituted alkylamine substituents (as exemplified with the 4-benzylamine-
variant) was tolerated. More surprisingly, the dimeric compounds demonstrated non-typical
behavior and displayed some reactivation potency as well. Both findings may open up new
avenues for designing more effective non-oxime reactivators.

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Keywords
Acetylcholinesterase, non-oxime, nerve agents, reactivation
1. Introduction
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Nerve agents and a number of pesticides belong to a family of organophosphorus (OP)
compounds that exert their toxicity through the covalent inhibition of the enzyme
acetylcholinesterase (AChE).[1,2] Examples of OP’s are the commonly used pesticides (e.g.,
chlorpyrifos, malathion, paraoxon), but also nerve agents (e.g., VX, soman, sarin and tabun).
AChE is present on synaptic neurons, and catalyzes the hydrolysis of the neurotransmitter
acetylcholine (ACh) in the synaptic clefts.[2,3] Inhibition of the enzyme by OP’s occurs
through the formation of a covalent bond between the active site serine-203 residue and the
OP, resulting in the permanent phosphylation. This blockage leads to accumulation of ACh,
over-stimulation of the ACh receptors, which in turn leads to severe symptoms and death.[4]
Part of the treatment of OP poisoning involves the administration of compounds that contain
a nucleophilic oxime functionality, which reactivates the enzyme by attack of the oximate
anion on the phosphorus atom of the blocking phosphyl residue.[5,6] The most well-known
examples of these reactivators of OP-inhibited AChE are the clinically employed HI-6,
Obidoxime and 2-PAM (Figure 1), that were all developed in the fifties/sixties of the last
century. From a structural point of view, all of these compounds have a pyridinium aldoxime
group in common. The charged pyridinium moiety lowers the pKa of the oxime, necessary
for the generation of a sufficient fraction of reactive oximate anions under physiological
conditions, and contributes to the requisite affinity of the compound for the inhibited enzyme.
The presence of charge(s), however, also results in limited brain accessibility of the
oximes.[7] Another limitation is the lack of broad-spectrum activity: not one oxime is capable

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of reactivating AChE inhibited by one of the many OP’s. Hence, a tremendous effort has
been devoted over the last six decades to research focusing on the design, synthesis and
evaluation of novel compounds with the aim to improve brain permeability and increased
spectrum activity.[8–11] The design of preferably low molecular weight compounds that lack
charge (or possess overall neutrality, such as zwitterionic compounds), but have sufficient
affinity for the enzyme and are at the same time reactive enough (appropriate pKa) for the
intended phosphyl displacement reaction, is still an enormous challenge. While a great
variety of chemical structures have been reported over the years, the use of an oxime
functionality as the nucleophilic moiety has been virtually ubiquitous. Despite all of these
efforts, none of these compounds has reached the clinic to date. Clearly, a complementary
design strategy leading to fundamentally different compounds would be highly welcome.
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Figure 1. Structures of currently employed oximes and recently discovered non-oximes for
the reactivation of nerve agent inhibited acetylcholinesterase. While the oximes work via
direct nucleophilic attack on the phosphyl moiety the mechanism of reactivation by non-
oximes is still unclear.

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Several years ago, Katz reported that a Mannich phenol (ADOC, 1a in Figure 1) was capable
of reactivating OP-inhibited AChE in vitro.[12] Although the reactivation rates were far
inferior to those of the current oximes, the discovery of a reactivator that lacked the oxime
group (hence the expression ‘non-oxime’) was surprising and appealing, as this discovery
pointed at the possibility of developing reactivators that were fundamentally different from
current reactivator design. Moreover, the absence of charge, its relatively low molecular
weight and its short route of synthesis are equally attractive features from a medicinal
chemistry point of view.[13] The mechanism of reactivation of OP-inhibited AChE by
ADOC remains to be unraveled, but both a nucleophilic mechanism (arguably through the
phenolate) as well as an acid-base mechanism (generation of reactive hydroxyls by
reactivator-mediated deprotonation of water) have been hypothesized (Figure 1).[14,15] Not
long afterwards, Cadieux presented a number of ADOC derivatives in which the chemical
space around ADOC was explored, by varying number, type and bulk of substituents and
substituent positions, and their effect on the reactivation potency.[16] Unfortunately, none of
these new derivatives showed any potency as a reactivator. We have recently reported a
systematic investigation that focused solely on minor structural variations around the
benzylic amine group.[17] Thus, a set of aliphatic, cyclic and unsaturated benzylic amine
derivatives of ADOC were prepared and tested in vitro. Although most of the structures had
no significant potency as a reactivator, we were happy to find that the ring-closed derivative
of ADOC (1b or PADOC in Figure 1) was at least ten times more potent than ADOC in the
reactivation of human AChE (hAChE), inhibited by various OP’s.[18] Moreover, for VX and
paraoxon (POX) inhibited hAChE its potency was equal or superior to that of HI-6, one of
the best reactivators know to date. Another intriguing finding was that the potency of these
non-oximes was very strongly influenced by small structural changes, thwarting analyses of
structure-activity relationships. For instance, expansion of the pyrrolidine ring in PADOC

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with just a single methylene group (to give 1c in Figure 1) resulted in a dramatic decrease of
reactivation potency. Determination of the reactivation parameters kr (reactivity rate constant)
and KD (apparent dissociation constant) revealed that the reactivation potency of PADOC
mainly stems from a high affinity for the inhibited enzyme. In line with the previous work,
we here report 17 novel, non-oxime compounds (Figure 2) and their in vitro biological
evaluation.
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Figure 2. Non-oxime compounds synthesized in this study
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Compounds 2 and 3 are close variants of the current lead compound 1b and were selected on
the basis of results previously reported by Hadad and co-workers. They found that 2-
methylpyrrolidine variants of structurally related compounds, designed for re-alkylation of
aged OP-inhibited AChE, exhibited an increased affinity for AChE. These compounds lacked

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the 4-amine in 1, and featured a pyridine instead of a benzene moiety.[10] In an analogous
fashion, the pyridine feature was adopted in compounds 3.
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Compounds 4 represent structures in which the substituent at the 4-position of 1b was varied.
These substituents featured electron withdrawing groups, electron donating groups and
moieties that may be able to exert further interaction (e.g., H-bonding) with enzyme residues.
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Finally, we synthesized three dimeric compounds, in which two Mannich phenol units were
connected through spacers of varying length. This design was inspired by the suggestion that
interaction of two ADOC molecules to the enzyme at the same time may be required for
efficient reactivation.[12] The chemical linkage of these molecules could possibly result in
more efficient binding of the two non-oxime moieties compared to 2 separate molecules. In
the absence of in silico models the design of compounds 5 was merely based on synthetic
accessibility.
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2. Materials and Methods
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The synthetic procedures for all compounds are provided in this section. General information
(chemicals, equipment, suppliers, etc) are provided in the supporting information. The
biological experiments (determination of IC50, reactivation screening, and reactivation
kinetics) were carried out as previously described.[18] Brief descriptions are provided below,
while details are provided in the supporting information.2.12-Step synthetic procedure
compound 2a/2b
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4-acetamidophenol (1.8g, 11.9mmol; 1.2eq) and paraformaldehyde (0.3g, 10.2 mmol; 1eq)
were dissolved in ethanol (20 ml). (R)-(-)-2-methylpyrrolidine (or the S-isomer, 0.84ml, 7.7
mmol; 0.76 eq), was added and the mixture was refluxed at 80oC for 24h. After concentration
in vacuo, the residue was dissolved in MeOH (2 ml) and applied on a silica gel column,

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which was subsequently eluted with a gradiënt of 0-10% MeOH in EtOAc. Yield 0.33g;
1.33mmol (17%). 1H-NMR (400MHz, MeOH-d4) δ 7.29(s, CH); 7.22(d, CH); 6.69(d, CH);
4.22(d, CH2); 3.44(dt, CH2); 2.67(q, CH2); 2.37(q, CH2); 2.10(s, CH3); 1.81(p, CH2); 1.54(m,
CH2); 1.24(d, CH2). 13C-NMR (101MHz, MeOH-d4) δ 169.90(C=O); 154,20(C-OH);
129.93(CH); 122,62(C); 121.20(C); 120,91(CH); 115.13(CH); 59,98(CH2); 55,56(CH3);
53,45(CH2); 32,33(CH2); 17,63(CH3). [M+H]+: 248 Da.
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The product from the previous step (0.33g, 1.33mmol; 1eq) was dissolved in 10% HCl (10
ml). After 6h reflux at 110oC, the mixture was concentrated and coevaporated with
acetonitrile (2-3x). Yield: 0.38g; 1.52mmol; 19.8% (2 steps). 1H NMR (400 MHz, Methanol-
d4) δ 7.55 (s, 1H), 7.39 (d, J = 8.7 Hz, 1H), 7.09 (d, J = 8.7 Hz, 1H), 4.62 (d, J = 12.9 Hz,
1H), 4.24 (d, J = 13.0 Hz, 1H), 3.72 – 3.61 (m, 1H), 3.52 – 3.44 (m, 1H), 3.35 (d, J = 16.1 Hz,
7H), 2.39 (dd, J = 12.9, 5.0 Hz, 1H), 2.14 (dd, J = 15.2, 6.4 Hz, 1H), 2.02 (s, 1H), 1.81 (dd, J
= 13.0, 8.7 Hz, 1H), 1.50 (d, J = 6.4 Hz, 3H). 13C NMR (Methanol-d4, 101 MHz): δ (ppm)
156.85, 127.04, 126.05, 122.05, 118.54, 116.44, 64.66, 53.91, 51.25, 31.23, 21.05, 15.30.
[M+H] +: 206 Da.
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2.2
2-Step synthetic procedure compound 3a
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5-hydroxy-2-pivalamidopyridine (180 mg, 927 µmol; 1 eq) was dissolved in ethanol (5 ml).
Formaldehyde solution (37% in water, 64 µl (0.93 eq)) and diethylamine (89 µl, 0.93 eq)
were added. The mixture was refluxed for 240h at 95 °C. After concentration in vacuo, the
mixture was purified by automated column chromatography (Biotage Isolera 4, using a
gradient of 0-90 % EtOAc in heptane, containing 3% triethylamine). Yield: 80 mg, 285 µmol
(33%) 1H NMR (400 MHz, Methanol-d4) δ 7.77 (d, J = 8.8 Hz, 1H), 7.12 (d, J = 8.7 Hz, 1H),
3.91 (s, 2H), 2.71 (q, J = 7.2 Hz, 4H), 1.31 (s, 9H), 1.15 (t, J = 7.2 Hz, 6H). 13C NMR (101

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MHz, Methanol-d4) δ 177.77, 151.94, 142.55, 140.52, 124.98, 115.18, 57.77, 46.90, 39.10,
26.36, 10.15.
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3-hydroxy-6-pivalamido-2-(diethylaminomethyl)pyridine was dissolved in 10% HCl and
refluxed for 96h at 110 °C. Progress was monitored by LC-MS. After complete conversion
the mixture was concentrated in vacuo and co-evaporated with acetonitrile (2-3 x).
Quantitative yield. 1H NMR (400 MHz, Deuterium Oxide) δ 7.63 (d, J = 9.5 Hz, 1H), 7.03 (d,
J = 9.5 Hz, 1H), 4.36 (s, 2H), 3.24 (q, J = 7.3 Hz, 4H), 1.28 (t, J = 7.3 Hz, 6H). 13C NMR
(101 MHz, Deuterium Oxide) δ 149.11, 146.01, 135.28, 122.38, 117.28, 48.70, 48.14, 8.11.
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2-Step synthetic procedure compound 3b
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5-hydroxy-2-pivalamidopyridine (61 mg, 312 µmol, 1 eq) was dissolved in ethanol (5 ml).
Paraformaldehyde (9 mg, 300 µmol, 0.96 eq) and pyrrolidine 23.4 µl (280 µmol, 0.90 eq)
were added. The mixture was refluxed for 168h at 95 °C and concentrated in vacuo. The
product was purified by automated column chromatography using a gradiënt of 0-90 %
EtOAc in heptane, containing 3% Triethylamine. Yield: 54 mg, 195 µmol (70%). 1H NMR
(400 MHz, Methanol-d4) δ 7.76 (d, J = 8.8 Hz, 1H), 7.15 (d, J = 8.8 Hz, 1H), 3.96 (s, 2H),
2.82 – 2.72 (m, 4H), 1.97 – 1.85 (m, 4H), 1.31 (s, 9H). 13C NMR (101 MHz, Methanol-d4) δ
177.87, 151.62, 142.31, 140.61, 124.95, 115.34, 58.45, 53.37, 39.09, 26.30, 23.12.
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3-hydroxy-6-pivalamido-2-(pyrrolidinomethyl)pyridine was dissolved in 10% HCl and
refluxed for 96h at 110 °C. Progress was monitored by LC-MS. After complete conversion
the mixture was concentrated in vacuo and co-evaporated with acetonitrile (2-3 x).
Quantitative yield. 1H NMR (400 MHz, Methanol-d4) δ 7.74 (d, J = 9.5 Hz, 1H), 7.16 (d, J =
9.4 Hz, 1H), 4.59 (s, 2H), 3.54 (m, 4H), 2.17 (m, 4H). 13C NMR (101 MHz, Methanol-d4) δ
146.14, 135.07, 117.09, 54.35, 49.36, 22.57.

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2.4
Synthetic procedure compound 4a
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Nitrophenol (5,28g, 37mmol, 1.2eq) and paraformaldehyde (0.95g, 31.5mmol, 1eq) were
dissolved in THF. Pyrrolidine (2.5ml, 31.5mmol; 1eq) was added and the mixture was
refluxed at 70oC for 3h. After concentration purification was accomplished using column
chromatography (0-15% MeOH in EtOAc). Yield 3.91g, 17.6mmol; (56%). 1H NMR (400
MHz, DMSO-d6) δ 8.04 (s, 1H), 7.96 (d, J = 9.1 Hz, 1H), 6.57 (d, J = 9.1 Hz, 1H), 3.96 (s,
3H), 2.85 (s, 4H), 1.85 (s, 4H). 13C NMR(DMSO, 101 MHz): δ (ppm) 170.54, 135.22,
126.43, 126.33, 122.62, 117.41, 56.03, 53.33, 23.51. [M+H]+: 222 Da.
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2.5
Synthetic procedure compound 4b
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4-hydroxybenzylalcohol (2.48g, 20mmol; 1.2eq) and paraformaldehyde (0.53g, 16.6mmol;
1eq) were dissolved in MeOH (13.3ml) MeOH. Pyrrolidine (1.39ml, 16.6mmol; 1eq) was
added and the mixture was refluxed for 3h at 70 oC. After concentration the mixture was
purified by column chromatography (0-15% MeOH in EtOAc). Yield: 2.84g; 13.7mmol;
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(82%). H NMR (400 MHz, DMSO-d6) δ 7.03 (s, 2H), 6.75 – 6.61 (m, 1H), 4.37 (s, 2H),
3.73 (s, 2H), 2.54 (s, 4H), 1.76 (s, 4H). 13C NMR (DMSO, 101 MHz): δ (ppm) 156.31,
132.97, 127.64, 127.06, 123.20, 115.24, 63.30, 57.24, 53.53, 23.69. [M+H]+: 207 Da.
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2.6
Synthetic procedure compound 4c
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4-methoxyphenol (2.48g, 20mmol; 1.2eq) and paraformaldehyde (0.60g, 16.6mmol; 1eq)
were dissolved in toluene (13.3 mL). Pyrrolidine (3.9ml, 16.6mmol; 1eq) was added. After
refluxing (19h at 110 oC) and concentration the product was purified by column

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chromatography (20-0% pentaan in EtOAc. Yield: 2.6g; 12.6mmol; (76%). 1H NMR (400
MHz, DMSO-d6) δ 6.68 (s, 1H), 6.66 (d, J = 2.9 Hz, 1H), 6.63 (d, J = 9.1 Hz, 1H), 3.69 (s,
2H), 3.66 (s, 3H), 2.51 (s, 8H), 1.75 (s, 4H). 13C NMR(DMSO, 101 MHz): δ (ppm) 152.28,
151.03, 124.56, 116.00, 114.56, 113.39, 57.03, 55.74, 53.56, 23.67. [M+H]+: 207.
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2.7
Synthetic procedure compound 4d
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4-hydroxybenzamide (1.74g, 12.7mmol; 1,2eq) and paraformaldehyde (0.30g, 10.0mmol;
1eq) were dissolved in MeOH (8mL). Pyrrolidine (0.83ml, 10mmol; 1eq) was added and the
mixture was refluxed for 16h at 70 oC. The mixture was concentrated in vacuo and purified
by column chromatography, using a gradient of 0-15% MeOH in EtOAc. Yield 1.64g;
7.45mmol (60%). 1H NMR (400 MHz, Methanol-d4) δ 7.71 (d, J = 8.5 Hz, 1H), 7.66 (s, 1H),
6.76 (d, J = 8.4 Hz, 1H), 3.96 (s, 2H), 3.37 (s, 2H), 3.33 (s, 5H), 2.80 (s, 4H), 1.93 (s, 4H).
13C NMR(Methanol-d4, 101 MHz): δ (ppm) 170.94, 163.19, 128.75, 128.63, 122.58, 121.74,
115.66, 57.13, 52.91, 23.06. [M+H]+: 220 Da.
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2.8
Synthetic procedure compound 4e/4f
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4-cyanophenol (3.60g, 30mmol, 1.2eq) and paraformaldehyde (0. 79g, 25mmol, 1eq) were
dissolved in toluene (20 mL). Pyrrolidine (2.08ml, 25mmol; 1eq) was added and the mixture
was refluxed for 3h at 110 oC. After completion of the reaction the mixture was concentrated
in vacuo and purified using column chromatography with an eluent of 0-15% MeOH in
EtOAc. Yield (4e): 3.11g; 15.4mmol (62%). 1H NMR (400 MHz, DMSO-d6) δ 7.54 (d, J =
6.6 Hz, 2H), 6.84 (d, J = 9.0 Hz, 1H), 3.78 (s, 2H), 2.57 (s, 4H), 2.51 (s, 4H), 1.77 (s, 4H).
13C NMR(DMSO, 101 MHz): δ (ppm) 162.46, 133.12, 125.35, 120.14, 116.87, 100.44,
55.70, 53.43, 23.65. [M+H]+: 202 Da.

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2,6-(dipyrrolidinomethyl)-4-cyanophenol (4f) was isolated as well from the column
purification. 1H NMR (400 MHz, DMSO-d6) δ 7.45 (s, 2H), 5.67 (s, 4H), 3.72 (s, 4H), 2.54
(s, 9H), 1.74 (s, 8H). 13C NMR(DMSO, 101 MHz): δ (ppm) 161.94, 131.81, 125.39, 120.34,
99.46, 55.08, 53.51, 23.67. [M+H]+: 285 Da.
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2.9
Synthetic procedure compound 4g
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2,5-dihydroxybenzaldehyde (2.09g, 15mmol; 1eq) was dissolved in 1,2-dichloroethane
(39.5ml) and stirred at room temperature for 5 minutes. Next, pyrrolidine (1.02ml, 18mmol;
1.2eq) and acetic acid (0.857ml, 15mmol; 1eq) were added. The mixture was cooled to 0 oC
and sodium triacetoxyborohydride (4.45g, 21.0mmol; 1.4eq) was added. The ice bath was
removed and the mixture was stirred for 16h. The solids were filtered and dissolved in water.
Then 10% HCl was added until pH 2. Dichloromethane was added and the organic layer was
separated from the water layer. The water fraction was brought to pH 10 using 5M NaOH
solution, followed by extraction with EtOAc (2x). The combined EtOAc layers were dried
with magnesium sulfate, and filtered. After concentration (in vacuo) the product was
obtained. Yield: 1.99g, 10.3mmol (69%). 1H NMR (400 MHz, DMSO-d6) δ 6.51 (s, 3H),
3.63 (s, 2H), 2.51 (s, 5H), 1.74 (s, 4H). 13C NMR(DMSO, 101 MHz): δ (ppm) 149.91,
149.64, 124.31, 115.98, 115.61, 114.66, 57.09, 53.55, 23.66. [M+H]+: 193 Da.
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2.10 Synthetic procedure compound 4h
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LiAlH4 (0.35g. 9mmol, 6eq) was dissolved in THF (2 ml). To this solution was carefully
added a solution of 2-(pyrrolidinomethyl)-4-cyanophenol 4e (0.32g, 1.5mmol,1eq) in THF (2
ml). After complete addition, the mixture was refluxed for 24h at 70oC. The mixture was
cooled to 0oC and water (6ml) was added. Next, 4M NaOH (3 ml) was added as well and the

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resulting mixture was stirred for 15min at room temperature. Next, MgSO4 was added and the
mixture was stirred for another 15min at room temperature. The solids were filtered and
washed with MeOH. The combined filtrates were concentrated in vacuo. Purification was
accomplished by silica gel column chromatography using a gradient of 0-15% MeOH in
EtOAc in the presence of 1% ammoniumhydroxide. Yield: 0.3g, 1.45mmol (97%). 1H NMR
(400 MHz, Methanol-d4) δ 7.10 (d, J = 8.2 Hz, 1H), 7.04 (s, 1H), 6.71 (d, J = 8.0 Hz, 1H),
3.82 (s, 2H), 3.68 (s, 2H), 2.65 (s, 4H), 1.87 (s, 4H). 13C NMR (Methanol-d4, 101 MHz): δ
(ppm) 156.44, 132.65, 127.64, 127.35, 122.68, 115.22, 57.31, 53.01, 44.83, 23.15. [M+H]+:
206 Da.
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2.11 2-Step synthetic procedure compound 4i
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2-(pyrrolidinomethyl)-4-cyanophenol 4e (0.17g, 0.86 mmol) was dissolved in a cooled (ice
bath) solution of HCl in MeOH (3M, 2 ml). Acetylchloride (3.12ml, 43.7mmol, 51.4eq) was
added. After 15 min of stirring the ice bath was removed and the mixture was refluxed for
24h at 75 oC. After cooling the precipitate was collected by filtration and washed with EtOAc.
Yield: 0.18g, 0.79mmol (92.8%). 1H NMR (400 MHz, Methanol-d4) δ 8.04 (s, 1H), 7.89 (d, J
= 8.4 Hz, 1H), 7.14 – 6.96 (m, 1H), 4.44 (s, 2H), 3.55 (s, 2H), 2.19 (s, 2H), 2.06 (s, 2H). 13C
NMR (Methanol-d4, 101 MHz): δ (ppm) 170.01, 159.55, 132.21, 131.14, 125.11, 117.19,
115.02, 53.72, 53.20, 22.59. [M+H]+: 235 Da.
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274
The product from the previous step (0.1g, 0.42mmol, 1eq) was dissolved in ammonia (7N, 3
ml) and stirred for 24h at room temperature. Acetonitrile was added and the mixture was
concentrated in vacuo. The addition of acetonitrile and concentration was repeated until
dryness. 1H NMR (400 MHz, DMSO-d6) δ 9.15 (d, J = 60.2 Hz, 4H), 8.14 (s, 1H), 7.77 (d, J
= 8.6 Hz, 1H), 7.20 (d, J = 8.6 Hz, 1H), 4.26 (s, 2H), 3.18 (s, 4H), 1.93 (s, 4H). 13C NMR

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(DMSO, 101 MHz): δ (ppm) 165.29, 161.98, 133.65, 131.15, 119.07, 118.12, 116.29, 53.29,
52.05, 23.14. [M+H]+: 219 Da.
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2.12 4-Step synthetic procedure compound 4j
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287
4-aminophenol (1.891g, 17.3mmol; 1,15eq) was dissolved in MeOH (20ml). After addition of
benzaldehyde (1.528ml, 15.0mmol, 1eq) the mixture was refluxed for 20min at 90 °C. After
cooling till about 40 °C, NaBH3CN (1.3g) was added and the mixture was refluxed for
another 2h. After completion of the reaction, the mixture was concentrated in vacuo, and the
product was isolated by column chromatography, using a gradient of 10-100 % EtOAc in
toluene. Yield: 2.7g, 13.6mmol (90.2%). 1H NMR (400 MHz, Acetone-d6) δ 7.40 (d, J = 7.6
Hz, 2H), 7.31 (t, J = 7.5 Hz, 2H), 7.22 (t, J = 7.3 Hz, 1H), 6.63 (d, J = 8.8 Hz, 2H), 6.55 (d, J
= 8.8 Hz, 2H), 4.28 (s, 2H). 13C NMR (101 MHz, Acetone-d6) δ 148.94, 142.18, 140.85,
128.21, 127.30, 126.55, 115.65, 113.94, 48.40.
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293
4-(benzylamino)phenol (179mg, 898 µmol, 1 eq) and di-tert-butyl dicarbonate (239mg, 1.1
mmol, 1.2 eq) were added in water (4ml). The mixture was stirred vigorously. After the
release of gas ceased (approximately 30 min) the mixture was concentrated in vacuo. Yield:
269mg, 898µmol (quant). 1H NMR (400 MHz, Acetone-d6) δ 7.20 – 7.07 (m, 5H), 6.83 (d, J
= 7.8 Hz, 2H), 6.60 (d, J = 8.7 Hz, 2H), 4.66 (s, 2H), 1.27 (s, 9H). 13C NMR (101 MHz,
Acetone-d6) δ 155.37, 139.09, 128.19, 127.64, 126.87, 121.22, 114.98, 79.17, 53.59, 27.57.
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295
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298
N-Boc-4-(N-benzyl)aminophenol (218mg, 728µmol, 1.3 eq) was dissolved in MeOH (20 ml).
Pyrrolidine (47µl, 558µmol, 1.0 eq) and 37%-formaldehyde (42 µl, 564µmol, 1.01 eq) were
added. The mixture was refluxed for 72h at 75°C. The mixture was concentrated in vacuo,
and the title compound was purified by column chromatography, using a gradient of 0-10 %
MeOH in EtOAc. Yield: 66mg, 173µmol (31% yield). 1H NMR (400 MHz, Methanol-d4) δ

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7.23 – 7.02 (m, 5H), 6.69 (d, J = 33.1 Hz, 2H), 6.53 (d, J = 8.6 Hz, 1H), 4.62 (s, 2H), 3.58 (s,
2H), 2.45 (s, 4H), 1.69 (s, 4H), 1.30 (s, 9H). 13C NMR (101 MHz, Methanol-d4) δ 155.98,
155.72, 138.37, 133.36, 128.01, 127.68, 127.58, 127.07, 126.88, 122.85, 115.31, 80.19,
56.75, 53.79, 52.94, 27.29, 23.08.
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304
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306
307
308
309
310
4-(N-benzyl-N-Boc)-amino-2-(pyrrolidinomethyl)phenol (66 mg, 0.17 mmol) was dissolved
in a solution of HCl in MeOH (1M). The mixture was refluxed for 5h at 85°C. After
completion (LC-MS) the mixture was concentrated in vacuo and coevaporated 2-3 times with
MeOH to yield the title compound (quantitative yield). 1H NMR (400 MHz, Methanol-d4) δ
7.45 (s, 1H), 7.38 – 7.29 (m, 5H), 7.25 (d, J = 8.7 Hz, 1H), 6.93 (d, J = 8.7 Hz, 1H), 4.49 (s,
2H), 4.28 (s, 2H), 3.36 (s, 2H), 3.08 (s, 2H), 2.01 (d, J = 50.1 Hz, 4H). 13C NMR (101 MHz,
Methanol-d4) δ 157.25, 130.52, 130.26, 129.43, 128.79, 127.10, 126.22, 126.08, 118.75,
116.34, 55.61, 53.71, 52.53, 22.52.
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312
2.13 General 2-step procedure for the synthesis of compounds 5a-c
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319
4-N-Acetamidophenol (0.75g, 5mmol) was dissolved in ethanol (20 ml). Paraformaldehyde
(0.15g, 5mmol) and the requisite diamine (2.5 mmol) were added. The mixture was refluxed
for 20h and concentrated. The residue was purified by silica gel column chromatography
using a gradient of 5-15% MeOH in EtOAc. The product was dissolved in 10% HCl and
refluxed for 5h. Upon completion (monitoring by LC-MS) acetonitrile was added and the
mixture was concentrated. The residue was coevaporated several times with acetonitrile to
yield the product.
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321
322
5a: 1H NMR (400 MHz, Deuterium Oxide) δ 7.29 (d, J = 7.6 Hz, 4H), 7.02 – 6.89 (m, 2H),
4.47 – 4.20 (m, 4H), 2.79 (s, 3H). 13C NMR (101 MHz, Deuterium Oxide) δ 155.98, 126.60,
126.33, 122.05, 117.89, 116.70, 55.86, 40.81. [M+H]+: 273.88.

323
324
325
326
5b: 1H NMR (400 MHz, Methanol-d4) δ 7.66 (d, J = 2.7 Hz, 2H), 7.44 (dd, J = 8.7, 2.7 Hz,
2H), 7.14 (d, J = 8.8 Hz, 2H), 4.56 (d, J = 39.6 Hz, 4H), 3.91 (d, J = 18.7 Hz, 4H), 2.96 (s,
6H). 13C NMR (101 MHz, Methanol-d4) δ 157.11, 127.59, 126.54, 122.18, 116.88, 116.66,
54.63 (d, J = 7.9 Hz), 49.91, 39.73. [M+H]+: 331.06.
327
328
329
330
5c: 1H NMR (400 MHz, Methanol-d4) δ 7.62 (d, J = 2.7 Hz, 2H), 7.43 (dd, J = 8.7, 2.7 Hz,
2H), 7.13 (d, J = 8.7 Hz, 2H), 4.49 (d, J = 67.5 Hz, 4H), 3.38 (s, 4H), 2.92 (s, 6H), 2.58 –
2.39 (m, 2H). 13C NMR (101 MHz, Methanol-d4) δ 157.06, 127.44, 126.30, 122.16, 117.39,
116.52, 54.09, 52.75, 39.52, 19.27. [M+H]+: 345.15.
331
332
2.14 IC50 determination
333
334
335
336
Native hAChE was incubated with several concentrations (1 -1000 µM) of non-oxime and the
enzyme activity was measured using the Ellman assay. Measurements were conducted in
duplicate. The IC50 values were calculated from semi-logarithmic plots of the compound
concentration versus the hAChE activity.
337
338
2.15 Enzyme inhibition
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340
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342
Human erythrocyte (ghosts) were incubated with an inhibitor (paraxon, or one of the nerve
agents) to achieve ≥95% of inhibition. Both the inhibited and the control samples were
dialyzed overnight at 4oC to remove residual inhibitor. Aliquots of inhibited enzyme were
stored -80 oC.
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344
2.16 Reactivation of OP-inhibited hAChE

345
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347
348
OP-inhibited hAChE was incubated with several concentrations of non-oxime. The fraction
of reactivated enzyme (with respect to the 100% control) was determined by taking aliquots
at several time points, during a total incubation time of 60 minutes, and measure enzyme
activity using the Ellman assay. Experiments were carried out in duplicate.
349
350
351
3. Results and Discussion
3.1. Chemistry
352
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356
357
358
359
360
361
362
363
364
365
366
367
368
The synthesis routes of all compounds are depicted in Figure 3. Synthesis of
methylpyrrolidine derivatives 2 (Panel A) was accomplished by treating the commercially
available compound 6a with formaldehyde and the requisite pyrrolidine (7b or 7c).[19] After
purification, the resulting intermediate was subjected to acid to remove the acetyl group,
which gave the target compounds as the ammonium salts. In a similar sequence of reactions
pyridine derivatives 3a and 3b were prepared (Panel E). In these cases, the commercially
available N-(4-Hydroxyphenyl) Pivalamide 6b was used as the starting compound.
Compounds 4a-4e (Panel B) were all accessible via Betti reactions (using formaldehyde and
7a) on their respective 4-substituted phenol precursors (6c-g). The conversion of cyanide 6g
to 4e gave a double-substituted byproduct (4f) that was also isolated. Compound 4e also
served as an intermediate for further derivatization (Panel D). Thus, 4e was reduced to give
aminomethyl-substituted compound 4h, and converted to 4i in a two-step process via 8.
Compound 4g was (Panel C) accessible via reductive amination of dihydroxybenzaldehyde
6h and pyrrolidine (7a), under the influence of NaBH(OAc)3. The synthesis of N-Benzyl
substituted compound 4j (Panel F) proved to be more challenging. The first step entailed
reductive amination of benzaldehyde with p-aminophenol (6h) giving 9. This compound
proved to be unstable, so it was used directly in subsequent reactions after its purification by

369
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371
372
373
374
375
376
377
378
379
380
381
382
383
384
385
column chromatography. Direct conversion of 9 to target compound 4j was not productive as
the benzylic amine in 9 competed with pyrrolidine 7a in the Mannich reaction. Therefore, the
amine in 9 was protected with a benzyloxycarbonyl (Boc) group. Installation of the Boc
group was initially attempted using Boc2O in dichloromethane or THF, in the absence and in
the presence of triethylamine. This reaction suffered from a preference for the formation of
the tert-butoxycarbonate (Boc protection of the phenol group in 9), leading to a mixture of O-
tert-butyloxycarbonyl-4-benzylaminophenol and O,N-di-(tert-butyloxycarbonyl)-4-
benzylaminophenol as the major products. However, a selective and quantitative conversion
of 9 into the desired compound 10 could be accomplished by treatment of 9 with Boc2O in
water, with vigorous stirring.[20,21] The subsequent Betti reaction was carried out in
refluxing MeOH, instead of refluxing EtOH, as the latter led to premature loss of the Boc
group (thermal deprotection). Finally, Mannich product 11 was treated with HCl in MeOH to
furnish the desired compound 4j. The dimeric compounds 5a-c (Panel G) were synthesized
from 6a using a Betti reaction in the presence of methylamine (‰5a), or in the presence of
N,N’-dimethylethylenediamine (‰5b) or in the presence of N,N’-dimethyl-1,3-
propanediamine (‰ 5c). All compounds were satisfactorily characterized with 1H-NMR ,13C-
NMR and mass spectrometry.
386

387
388
389
390
391
Figure 3. Synthesis routes for the various compounds
3.2. In vitro biological evaluation
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395
The 50% inhibitory concentration (IC50) was measured by exposing human erythrocyte
AChE to various concentrations (1-1000 µM) of all compounds and recording the enzyme
activity using the well-known Ellman assay.[22,23] Details are provided in SI, paragraph 2.6.
The results are summarized in Table 1.

396
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398
Table 1. The inhibition of native human AChE by non-oxime compounds was tested with 11
concentrations (1-1000 µM) in duplicate. Data are given as mean ± SD.
Compound
IC50 (µM)
Compound
IC50 (µM)
6.3 ± 0.0 [17]
9.21 ± 0.03
42.7 ± 0.8
973 ± 27
135 ± 4.5
300.5 ± 14.9
>1000
>1000
>1000
1b
2a
2b
3a
3b
4a
4b
4c
4d
4e
4f
19.9 ± 0.5
348 ± 9
>1000
4g
4h
4i
196 ± 5.3
>1000
4j
5a
5b
5c
>1000
838 ± 76
368 ± 5.1
>1000
399
400
401
402
403
404
405
406
407
408
409
Next we used an in vitro reactivation assay to assess the ability of all compounds to reactivate
OP-inhibited hAChE.[24] To this end, human erythrocyte AChE (ghosts) was inhibited with
VX, paraoxon-ethyl (PXE), tabun (GA), cyclosarin (GF) and sarin (GB) and subjected to 3-5
concentrations (10-1000 µM) of each reactivator. The enzymatic activity was measured at
various time points within 60 minutes. The enzyme activity at each time point was corrected
for the blank (no reactivator) and expressed as the percentage reactivation with respect to the
100% control. All experiments were carried out in duplicate. The reactivation curves of
selected compounds are provided in the supporting information. Table 2 shows the
percentage reactivation achieved after 60 min incubation with 10, 100 and 1000 µM

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412
413
414
415
416
417
418
419
concentrations of each reactivator / OP-hAChE combination. The potencies of compounds
3a, 4b and 4c were negligible and were therefore omitted from Table 2. None of the
compounds reactivated GA-inhibited hAChE and therefore the column for GA is lacking in
Table 2 as well. The red/italic values in Table 2 were expected to be higher (judging from
reactivation percentages at lower concentrations), and were most probably attenuated by
reversible inhibition or denaturation of the enzyme at higher reactivator concentrations (vide
infra). Note, that cells in Table 2 were left blank when reactivation was less than 5%.
Consequently, Table 2 allows for a quick but rough review of the performance of the
reactivators, because compounds with a higher number of reactivation percentages
(especially at lower concentrations) have, in general, a higher potency.
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422
Table 2. Reactivation-%, after 60 min. incubation with 10, 100 and 1000 µM concentrations
of reactivator of OP-inhibited hAChE
Compound Concentration PXE VX
(µM)
GF
GB
1000
100
10
<5% <5% <5% <5%
<5% 38
11
22
6
2a
2b
3b
7
13
16
62
10
36
1000
100
10
21
53
34
8
10
6
34
21
26
1000
100
29
6

10
1000
100
10
5
4a
4d
4e
4f
1000
100
10
7
6
1000
100
10
9
10
7
5
1000
100
10
1000
100
10
28
16
20
10
5
4g
4h
4i
1000
100
10
8
21
16
1000
100
10
10
7
1000
100
10
22
8
69
52
26
19
42
10
4j

423
1000
100
10
33
73
34
6
6
13
5a
5b
5c
424
425
1000
100
10
13
6
48
25
18
26
17
9
6
6
9
8
426
427
1000
100
10
16
6
6
428
7
429
430
431
432
433
434
435
436
437
438
439
440
441
442
443
Judging from the amount of numbers and their values in the corresponding columns, GF-
hAChE was the most difficult to reactivate, while VX-hAChE was the easiest to reactivate. It
is also evident that compounds 2, 4j and 5 were the most active from this series. A selection
of the reactivation curves of those compounds are displayed in Figure 4, and from some of
these compounds the reactivation parameters kr (reactivation rate) and KD (dissociation
constant) were determined (Table 3). The parameters for 1b, the most potent non-oxime
reported to date, were added for comparison in Table 3. Compound 2b appreciably
reactivated VX-, PXE- and GB-inhibited hAChE. At 500 and 1000 µM concentrations the
reactivation of OP-hAChE by 2a and 2b was attenuated, after an initial steep increase in
enzyme activity, and followed by a progressing decline in activity over time. This

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447
448
449
450
451
452
453
454
455
456
457
phenomenon is in line with previous observations for a number of PADOC derivatives[17],
and was postulated to be due to a combination of reversible inhibition (compare IC50’s in
Table 1) and by denaturation or covalent modification[17] of the liberated AChE by the
reactivator at higher reactivator concentrations (500 and 1000 µM). The possibility of re-
inhibition of the enzyme by a putative OP-inhibitor adduct formed during reactivation (see
the nucleophilic reactivation mechanism in Figure 1), cannot be excluded but was deemed
less likely, as a synthetic PADOC-OP conjugate was not an inhibitor of hAChE and the
decline in enzyme activity was also observed with native AChE incubated with 1a or 1b [17].
The previously observed sensitivity towards small structural changes was corroborated by the
nearly 5-fold lower IC50 of 2a compared to its isomer 2b, as well as the approximately 50-
fold loss of potency toward VX-AChE reactivation when compared to 1b, which lacks the
methyl substituent on the pyrrolidine ring. The pyridine-pyrrolidine compound 3b was not
very potent, but it was more potent than the diethyl substituted variant 3a, which corresponds
to the higher potency achieved by the same substitution in ADOC.[17]
458

459
460
461
Figure 4. Selection of reactivation curves (all other curves are provided in the supporting
information).
462
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465
466
467
468
469
470
471
472
473
From the 4-substituted compounds 4j had a mediocre and relatively broad spectrum activity
(except for tabun) and it was the only compound from the 4-substituted series that displayed
some potency, suggesting that a 4-N-alkyl substituent may be better tolerated in these designs
than all other substitutions. The latter was confirmed by Cadieux and co-workers who
reported that the 4-N-ethyl-derivative of 1a (Figure 1), had a higher potency than 1a itself
(but lower than 1b).[15] Another interesting observation was that no decline in enzymatic
activity over time occurred with this compound when incubated with the enzyme at high
concentrations. Whether this resulted from the structure (4-N-alkyl substituent) or simply
because of a moderate affinity (IC50 196 µM) of the compound for the enzyme remains to be
investigated.

474
475
476
477
478
479
480
481
482
483
484
485
486
487
In light of the rather strong response of the reactivation potency towards small structural
changes in the compounds reported here and in previous accounts [16,17], the observed
potency of compounds 5 was rather surprising. Whereas in most cases the presence of
substituents to the benzylic amine, in particular bulky ones[16,17], leads to a diminished
potency, compounds 5 were actually capable of reactivating VX-inhibited hAChE. Moreover,
compound 5a, having the bulk more closely located around the central benzylic amine is
more active than 5b and 5c, that have their substituents at more remote distances,
respectively. Another peculiar observation was that the affinity for the native enzyme (IC50)
decreased going from 5a to 5c (>1000, 838 and 368 µM, respectively), while their potencies
respectively decreased. Although the IC50 value cannot be used to predict the KD or the
overall potency, it is not uncommon that compounds that exhibit a low IC50 have a lower KD
as well, which increases their chances of exhibiting some reactivation potency (e.g., compare
3a/3b, 4b-f (IC50>1000 and no or low potency), or 1a/1b [17]). In the case of compounds 5
this ‘trend’ is reversed.
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490
Table 3. Kinetic parameters for the reactivation of OP-inhibited hAChE for some compounds
and OPs.
Compound Agent kr (min-1)
KD (µM)
kr2 (min-1mM-1)
VX
VX
VX
GB
VX
0.93 ± 0.00
0.02 ± 0.00
0.07 ± 0.01
0.04 ± 0.00
0.09 ± 0.00
45 ± 1
20.67 ± 0.10 (ref. 18)
0.46 ± 0.02 (this work)
0.35 ± 0.06 (this work)
1b
2a
2b
4j
41 ± 1
209 ± 55
1164 ± 129 0.03 ± 0.00 (this work)
516 ± 11 0.17 ± 0.01 (this work)
4j
491

492
4. Conclusion
493
494
495
496
497
498
499
500
501
502
503
504
505
506
507
508
509
510
The goal of this work was to further explore the chemical space for Mannich phenol based
non-oximes in relation to their reactivation potency of nerve agent inhibited hAChE. To this
end two isomers of 2-methylpyrrolidine derivatives of 1b (‰2a/2b), two pyridine derivatives
of 1a and 1b (‰3a/3b), nine 4-substituted derivatives of 1b (‰4a-j) and three dimeric
Mannich phenols (5a-c) were synthesized. These 17 compounds were subjected to in vitro
assays to determine their inhibitory potencies (IC50) towards native hAChE and their
reactivation potencies towards a number of OP-inhibited hAChE. Although none of the
compounds had a potency that exceeded that of the lead non-oxime 1b, some interesting
features were revealed, that may guide future design of novel non-oximes. For instance, the
4-N-benzylamine substituted compound 4j showed reasonable and relatively broad spectrum
reactivation potency, without denaturation of the enzyme, and this result motivates us to
further explore other N-alkylamine substituents at the 4-position. The Mannich phenol dimers
5, in particular 5a, also showed some potency for reactivation. These compounds are trend
breaking, considering that the introduction of bulky substituents on the Mannich phenol
system usually leads to diminished reactivation potency. Secondly, the correlation of higher
potency with lower IC50 is usually not observed. Future quantum mechanics calculations and
molecular modeling (QM/MM) may provide clues to explain these observations and aid the
design of future dimers.
511
512
Acknowledgements
513
514
The authors thank Vincent Berkien (BSc), Isabelle van Buuren (BSc) and Danny van der
Meer (BSc) for their synthetic contributions as part of their graduation internships. Funding:

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This work was supported by the Dutch Ministry of Defense (as part of the program RDO
Impulsfinanciering), and the German Ministry of Defense.
517
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520
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