ORIGINAL PAPER

Research paper

Journal of Environmental Immunology and Toxicology 1:1, 35-40; January/February/March 2014; 2014 STM Publishing

Pralidoxime and Obidoxime: Phosphylationinduced Changes in logP (partition coefficient)

Georg A Petroianu1, Dietrich E Lorke1, Gagani Athauda1, Ferenc Darvas1,2, Huba Kalasz3
Department of Cellular Biology and Pharmacology, Herbert Wertheim College of Medicine, Florida International University, Miami, USA; 2Thales Nano,
Budapest, Hungary; 3Department of Pharmacology and Pharmacotherapy, Semmelweis University, Budapest, Hungary

Abstract

The acute toxicity of organophosphorus esters (organophosphates and organophosphonates) is due to inhibition of the enzyme
acetylcholinesterase (AChE), which metabolizes the neurotransmitter acetylcholine (ACh). Esterase inhibition results from
phosphylation (i.e. either phosphorylation or phosphonylation) of the serine hydroxyl group in the active center of the enzyme and
translates into an endogenous acetylcholine poisoning.
The therapy of cholinesterase poisoning by organophosphorus inhibitors includes the use of oximes (such as pralidoxime in
the US and obidoxime in Europe). Oximes reactivate the inhibited enzyme by dephosphylating it, thereby becoming themselves
phosphylated. The phosphylated oxime thus generated can be itself a potent inhibitor of cholinesterases, which may reduce the
efficacy of the reactivation attempt.
The present study estimates logP values of phosphylated pralidoxime and obidoxime after in-silico exposure to a number of
organophosphorus esters [ethyl-paraoxon, methyl-paraoxon, diisopropyl-fluoro-phosphate, VX, soman, tabun, sarin, cyclosarin],
compares them with the logP of native oximes and discusses possible therapeutic relevance. Our data indicate that phosphylation
of oximes generally increases their lipophilicity, facilitating penetration into the brain where they can inhibit or re-inhibit enzymes.
Estimated logP values of phosphylated pralidoxime increase on average by 0.6 compared to native pralidoxime, while for obidoxime
the increase is on average 0.9. For both oximes, phosphylation by tabun shows the most significant effect on logP (increase by 0.9 and
1.9 respectively). Possible consequences with regard to blood-brain-barrier passage are discussed.
Journal of Environmental Immunology and Toxicology 2014; 1:35-40

Key words

cholinesterase; obidoxime; organophosphorus esters; oxime; pralidoxime; phosphorylation; phosphonylation; logP

Introduction
The acute toxicity of organophosphorus esters (organophosphates
and organophosphonates) is due to inhibition of the
enzyme acetylcholinesterase (AChE), which inactivates the
neurotransmitter acetylcholine (ACh). The inhibition of esterases
results from phosphylation. Phosphylation is the umbrella
term used to describe phosphorylation (when the enzyme is
inhibited by an organophosphate insecticide, such as paraoxon)
or phosphonilation (when inhibited by an organophosphonate
nerve gas).
Phosphylation of the serine hydroxyl group in the active
center of the enzyme translates into an endogenous acetylcholine
poisoning. The therapy of organophosphorus inhibitors of
cholinesterase poisoning is known by the acronym A FLOP =
Atropine, FLuids, Oxygen, Pralidoxime,1 reflecting the sometimes
disappointing therapeutic results.
The history of the development of organophosphorus inhibitors
Correspondence to: Georg A Petroianu, Department of Cellular Biology and
Pharmacology, Herbert Wertheim College of Medicine, Florida International
University, Miami, USA; Email: georg.petroianu@fiu.edu
Submitted: 10/10/2013; Revised: 01/11/2013; Accepted: 29/11/2013
DOI: 10.7178/jeit.8

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of cholinesterase has been described.2-4 Phosphylated AChE can be

reactivated by oximes; all reactivators in clinical use are pyridinium
oximes.5 Using 2-methyl-pyridine as a starting point, Wilson and
Ginsburg working at Columbia University in the laboratory of
David Nachmansohn synthesized a number of pyridine oximes.
Among those was also the first aldoxime cholinesterase reactivator
of clinical relevance, pralidoxime (2-pyridinium aldoxime or
2-PAM). Such research has also been conducted independently in
Britain by Davies and Green [for a review see ref.5]. Obidoxime,
developed by Luettringhaus and Hagedorn in Germany was
initially known by the acronym LueH-6.6
The mode of action of pyridinium oximes has been recently
reviewed. By interacting with the anionic site of the enzyme, the
pyridinium moiety favors an optimal orientation of the reactivator
at the catalytic site of the enzyme, thereby increasing efficacy.7,8
Whereas emergency treatment of nerve gas exposure with oximes
is accepted doctrine, the therapeutic value of oximes in human
organophosphate pesticide poisoning is doubtful.9,10
A possible reason for the questionable efficacy may be the
generation of phosphylated oximes during AChE reactivation.10,11
Phosphylated oximes are themselves potent inhibitors of
cholinesterase.12
The properties of the phosphylated oximes have therefore to
be taken into account when evaluating kinetic studies on the

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Research paper

reactivation and aging of organophosphate-AChE conjugates.13

One way to characterize biologically active compounds is the
octanol-water partition coefficient [logP]. Partition of substances
between oil and water, a concept introduced over a century ago
by Berthelot [according to refs.14], is often correlated with their
biological activities.15-18
Begley reviewed various mechanisms by which substances cross
the blood-brain barrier. A strong correlation was identified for
lipid solubility and BBB permeability; the importance of good
solubility in the respective compartments (plasma and CSF) was
also emphasized. He writes There is well-established relationship
between lipid solubility, either calculated or determined as an oilwater partition coefficient, with brain penetration, which increases
with increasing lipid solubility.19
Purpose of the study
The present work estimates logP values of phosphylated
pralidoxime and obidoxime after in-silico exposure to a number
of organophosphorus esters [ethyl-paraoxon, methyl-paraoxon,
diisopropyl-fluoro-phosphate (DFP), VX, soman, tabun, sarin,
cyclosarin], compares them with the logP of native oximes and
discusses possible therapeutic relevance.
Material and method
Chemical structures of all compounds were drawn using
ChemDraw Ultra 12.0 (CambridgeSoft Software, PerkinElmer
Inc. Waltham, Massachusetts). LogP values of organophosphorus
esters, oximes, phosphylated pralidoxime and phosphylated
obidoxime were estimated using the PrologP module of the Pallas
3413 software (CompuDrug Inc., Sedona, AZ, USA). Details of
the algorithm used for calculations are given by.20 The program
takes into account all lipophilic and hydrophilic fragments of
a specific compound and makes minor corrections based on
octanol-water partition data, as available from the literature.
The authors emphasize that their neural network-based method
(pseudo-linear algorithms) combines the precision of non-linear
approaches with the transparency of the early linear methods. The
logP value of a substance is most relevant for neutral substances
and is also useful as a general reference point to help compare
overall hydrophobicity trends of compounds.
Results
Structure and logP values of organophosphorus pesticides (ethylparaoxon, methyl-paraoxon), diisopropyl-fluoro-phosphate and
nerve gases (sarin, cyclosarin, soman, tabun, VX) are listed in table
1. Their logP values range from -0.02 (tabun) to =2.18 (ethylparaoxon). All compounds except tabun are therefore lipophilic
(positive logP values).
Table 2 shows the chemical formulas and logP values of
pralidoxime [2-PAM] and of pralidoxime phosphylated by various
organophosphates (ethyl-paraoxon, methyl-paraoxon, sarin,
cyclosarin, soman, tabun, VX). The logP value of pralidoxime
is -1.960.19 reflecting its hydrophilicity. Phosphylation of
pralidoxime significantly reduces hydrophilicity to values between
36

-1.700.01 (phosphylation by ethyl-paraoxon) and -1.020.01

(phosphylation by tabun), the most significant increase in logP.
Phosphylated pralidoxime derivatives are thus less hydrophilic
than unphosphylated pralidoxime.
Chemical formulas and logP values of obidoxime and of
obidoxime phosphylated by various organophospates are listed
in table 3. Having a logP value of -3.400.26, obidoxime is
markedly more hydrophilic than pralidoxime. As observed for
pralidoxime, phosphylation of obidoxime significantly reduces
its hydrophilicity, except for obidoxime phosphylation by
soman. LogP values of phosphylated obidoxime range between
=-3.630.01 (phosphylation by soman) and =-1.470.01
(phosphylation by tabun).
Discussion
Oximes in general have disappointed clinically.9 Although,
theoretically, their mode of action is reasonably well understood,
their practical value remains uncertain.21 Oximes are polar
molecules with a negative logP, indicating hydrophilicity. Since
the blood brain barrier generally only allows free passage of
small lipophilic compounds, oximes have only a limited brain
penetration.22 For the monopyridinium aldoxime pralidoxime, the
brain concentration is only 10% of the blood concentration, while
penetration of bis-pyridinium aldoximes, such as obidoxime, is
one order of magnitude lower.23,24
Among the fundamental questions still unanswered is the
relationship between oxime efficacy and brain penetration: is the
limited efficacy of oximes due to their limited brain penetration?
Would an increase in brain penetration translate into superior
efficacy? It appears that initially the answer must have been yes,
as evidenced by Nicholas Bodors attempt to develop pro-2-PAM,
a pro-drug dihydropyridine derivative of pralidoxime (2-PAM)
penetrating the brain.25 Although pralidoxime brain levels were
considerably higher when using the brain penetrating pro-drug,
overall results were disappointing.26 In a recent review, it was
concluded that Increasing the BBB penetration by oximes does
not actually lead to significant benefits of survival rate, but certainly
amplifies the neurotoxic risks.27
Our own animal work suggests that a good predictor for low
oxime toxicity (as assessed by survival) is a high negative logP
(strongly hydrophilic), which would imply that limited brain
penetration is actually desirable.17,18 A putative explanation for these
surprising findings is the intrinsic toxicity of phosphylated oximes
generated by the reaction of oximes with organophosphorusinhibited enzymes.11
Phosphylated oximes are themselves potent inhibitors of
AChE, sometimes much more potent than the initial offending
organophosphate or organophosphonate (organophosphates
do not contain a direct phosphorus-carbon link, while
organophosphonates contain one), which may translate into high
toxicity.7,11,13,28,29
The group of Worek from Muenchen and others had some
success in the attempt at synthesizing phosphylated oximes.
For example they isolated isolated mono(diethylphosphoryl)
obidoxime (DEP-obidoxime) showing that the compound
displayed remarkable stability under physiological conditions.30

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Table 1. LogP values of organophosphorus esters [phosphylating agents]. All compounds with the exception of tabun are lipophilic (positive logP values).
logP=2.18
Ethyl-paraoxon
O
P

O
+

logP=1.51

Methyl-paraoxon
O
O

O
N+

logP=1.08

Di-isopropyl-fluoro-phosphate

O
P

logP=0.84

Sarin

O
P

Cyclosarin

logP=1.78

O
P

logP=1.89

Soman

O
P

logP=-0.02

Tabun
N

N
C

VX

logP=2.10
O

N
S

P
O

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Table 2. LogP values of pralidoxime [2-PAM] and of the phosphylated-pralidoxime. Pralidoxime is a hydrophilic compound; phosphylation significantly reduces
hydrophilicity [on average by 0.6]. Phosphylation by tabun shows the most significant effect on logP
2-PAM
logP=-1.960.19
OH
N
N+

Phospylated Oxime

logP phosphylated Oxime

Structure

2-PAM phosphylated by ethylparaoxon

logP=-1.700.01
logP=0.26

O
P

N
O

N+

2-PAM phosphylated by methylparaoxon

logP=-1.070.01
logP=0.89

O
P

N
O

N+

logP=-1.350.01
logP=0.61

2-PAM phosphylated by diisopropyl-fluoro-phosphate

O
P

N
O

N+

logP=-1.570.01
logP=0.39

2-PAM phosphylated by sarin

O
P

O
N

N+

logP=-1.220.01
logP=0.74

2-PAM phosphylated by
cyclosarin
O
P

O
N

N+

logP=-1.50.01
logP=0.46

2-PAM phosphylated by soman

O
P

O
N

N+

logP=-1.020.01
logP=0.94

2-PAM phosphylated by tabun

N
P

N
O

N+

2-PAM phosphylated by VX
O
O

logP=-1.360.01
logP=0.6

N
N+

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Table 3. LogP values of obidoxime and of phosphylated-obidoxime. Obidoxime is a highly hydrophilic compound; phosphylation significantly reduces
hydrophilicity [on average by 0.9]. Oxime phosphylation by tabun shows the most significant effect on logP.
Obidoxime
logP=-3.400.26
N+

N+

HO

Phospylated Oxime
Obidoxime
phosphylated by ethylparaoxon

OH

Structure
N+

logP phosphylated
Oxime
logP=-2.510.01
logP=0.89

N+

O
N

HO

Obidoxime
phosphylated by
methyl-paraoxon

logP=-1.820.01
logP=1.58

O
N

N
HO

Obidoxime
phosphylated by
di-isopropyl-fluorophosphate

N+

logP=-2.810.01
logP=0.59

N+

HO

logP=-2.600.01
logP=0.8

Obidoxime
phosphylated by sarin
N+

N+

O
N

N
HO

P
O

Obidoxime
phosphylated by
cyclosarin
N+

logP=-3.000.01
logP=0.4

N+
O
N

N
HO

P
O

Obidoxime
phosphylated by soman

N+

logP=-3.630.01
logP=0.23

N+
O
N

N
HO

P
O

Obidoxime
phosphylated by tabun

N+

logP=-1.470.01
logP=1.93

N+
N

HO

Obidoxime
phosphylated by VX

N+
N

logP=-2.200.01
logP=1.2

N+
N

HO

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4-pyridinium aldoximes (MMB-4 [methoxime], TMB-4

[trimedoxime] and LuH-6 [obidoxime]), have phosphylated forms
that decompose slower than those of 2-pyridinium aldoximes (2PAM [pralidoxime] and HI-6 [asoxime]).7,31 While some of the
compounds have indeed short t1/2 some others dont; considering
the vast number of possible phosphylated oximes any attempt at
generalization is bound to have serious limitations.
LogP: Pyridinium oximes are hydrophilic compounds (large
negative value of LogP) with very limited CNS penetration.22,23
Phosphylation results in a significant reduction in the absolute
value of LogP, corresponding to a reduction in hydrophilicity, i.e.
increase in lipophylicity. This decrease in hydrophilicity favors
penetration into the brain, where phosphylated oximes might
phosphylate AChE, thereby becoming trapped. For pralidoxime,
phosphylation on average results in a change towards lipophilicity
of 0.6 (which indicates a six-fold increase), with the maximum
noticed for phosphylation induced by tabun. For obidoxime an
average change towards lipophilicity of 0.9 was estimated (which
indicates a nine-fold increase), with the maximum noticed for
phosphylation induced by tabun.
Numerous other parameters such as logD have been correlated
with BBB penetration. LogD does not appear to have advantages
over log P in the context analyzed. A thorough analysis is offered
by Vilar.32
Conclusion
Based on preliminary data derived in silico we conclude that the
ideal oxime must not only be non-toxic itself, but should also
yield non- toxic products after phosphylation, since phosphylation
generally increases brain penetration. Any such conclusions is in
need of experimental confirmation.
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