Novel bifunctional hybrid small molecule scavengers for mitigating nerve agents toxicity

Article abstract of DOI:10.1016/j.cbi.2016.04.036

The antidotal treatment of organophosphates (OP) nerve agents (NA) poisoning is based on anticholinergics (e.g. atropine) combined with oxime reactivators (e.g. 2PAM) of acetylcholinesterase (AChE). This treatment is symptomatic and does not degrade the OP. New small-molecule OP scavengers were developed as bifunctional hybrids. Their molecular design was based on combining a nucleophile that directly degrades OP with a moiety that reactivates OP-inhibited AChE. The OP degrading moiety is either benzhydroxamic acid (BHA) or 4-pyridinehydroxamic acid (4PHA) coupled via [sbnd](CH₂)_n[sbnd], (n = 1 or 3) to 2PAM. Three newly synthesized oxime-hydroxamate hybrids: 2PAMPr4PHA, 2PAMMeBHA and 2,4-DiPAMMeBHA were found to detoxify sarin, cyclosarin and soman in solution at 3–10-fold faster rate than 2PAM and to reactivate OP-AChE in vitro. 2PAMPr4PHA displayed 18-fold faster reactivation than 2-PAM of cyclosarin-inhibited HuAChE (k_r = 3.6 × 10² vs. 0.2 × 10² M^?1min^?1, respectively, 37 °C). These hybrids inhibited AChE reversibly, IC₅₀ = 16–48 μM, thereby decreasing the inhibition rates by OPs. The LD₅₀ (im) of 2PAMPr4PHA, 2PAMMeBHA and 2,4DiPAMMeBHA are >568, 508 and >506 μmol/kg in rats and 144, 203 and >506 μmol/kg in guinea pigs. The rate of blood ChE recovery by the hybrids administered either pre- or post-exposure to 0.8xLD₅₀ sarin was comparable or faster than 2PAM. Antidotal efficacy of 2PAMPr4PHA, 2PAMMeBHA and 2,4DiPAMMeBHA administered with atropine, as pre-treatment to sarin in rats (im), yielded protection ratios (PR) 11.6, 11.5 and 4.7, respectively, vs. 5.5 with 2PAM. Post-treatment against various OPs in rats and guinea-pigs yielded PRs higher or similar to that of 2 PAM. Our in vivo data indicates that some hybrids may serve as efficient small molecule scavengers for mitigating the toxicity of OP NAs.

Full text of DOI:10.1016/j.cbi.2016.04.036

Chemico-Biological Interactions xxx (2016) 1e18
Contents lists available at ScienceDirect
Chemico-Biological Interactions
journal homepage: www.elsevier.com/locate/chembioint
Novel bifunctional hybrid small molecule scavengers for mitigating
nerve agents toxicity
,
Gabriel Amitai a *, Rellie Gez a, Lily Raveh a, Nira Bar-Ner b, Ettie Grauer a,
Shira Chapman a
a Department of Pharmacology, Division of Medicinal Chemistry, Israel Institute for Biological Research, PO Box 19, Ness Ziona 74100, Israel
b Department of Organic Chemistry, Division of Medicinal Chemistry, Israel Institute for Biological Research, PO Box 19, Ness Ziona 74100, Israel
a r t i c l e i n f o
a b s t r a c t
Article history:
The antidotal treatment of organophosphates (OP) nerve agents (NA) poisoning is based on anticho-
linergics (e.g. atropine) combined with oxime reactivators (e.g. 2PAM) of acetylcholinesterase (AChE).
This treatment is symptomatic and does not degrade the OP. New small-molecule OP scavengers were
developed as bifunctional hybrids. Their molecular design was based on combining a nucleophile that
directly degrades OP with a moiety that reactivates OP-inhibited AChE. The OP degrading moiety is either
benzhydroxamic acid (BHA) or 4-pyridinehydroxamic acid (4PHA) coupled via e(CH2)ne, (n ¼ 1 or 3) to
2PAM. Three newly synthesized oxime-hydroxamate hybrids: 2PAMPr4PHA, 2PAMMeBHA and 2,4-
DiPAMMeBHA were found to detoxify sarin, cyclosarin and soman in solution at 3e10-fold faster rate
than 2PAM and to reactivate OP-AChE in vitro. 2PAMPr4PHA displayed 18-fold faster reactivation than 2-
Received 21 January 2016
Received in revised form
22 April 2016
Accepted 25 April 2016
Available online xxx
Keywords:
Acetylcholinesterase
Oxime
Benzhydroxamic acid
4-Pyridine hydroxamic acid
2-Pyridine aldoxime methyl chloride
Organophosphates
Sarin
Cyclosarin
Soman
VX
Bifunctional
Small molecule
Scavengers
PAM of cyclosarin-inhibited HuAChE (kr ¼ 3.6 ꢀ 102 vs. 0.2 ꢀ 102
M
ꢁ1minꢁ1, respectively, 37 ꢂC). These
hybrids inhibited AChE reversibly, IC50 ¼ 16e48
m
M, thereby decreasing the inhibition rates by OPs. The
mol/kg in rats
mol/kg in guinea pigs. The rate of blood ChE recovery by the hybrids admin-
LD50 (im) of 2PAMPr4PHA, 2PAMMeBHA and 2,4DiPAMMeBHA are >568, 508 and >506
and 144, 203 and >506
m
m
istered either pre- or post-exposure to 0.8xLD50 sarin was comparable or faster than 2PAM. Antidotal
efficacy of 2PAMPr4PHA, 2PAMMeBHA and 2,4DiPAMMeBHA administered with atropine, as pre-
treatment to sarin in rats (im), yielded protection ratios (PR) 11.6, 11.5 and 4.7, respectively, vs. 5.5
with 2PAM. Post-treatment against various OPs in rats and guinea-pigs yielded PRs higher or similar to
that of 2 PAM. Our in vivo data indicates that some hybrids may serve as efficient small molecule
scavengers for mitigating the toxicity of OP NAs.
Nerve agents
Detoxification
Rat
© 2016 Elsevier Ireland Ltd. All rights reserved.
Guinea pig
1. Introduction
agricultural OP pesticides, causes excessive accumulation of
acetylcholine (ACh) in cholinergic synapses. If left untreated, OP
The inhibition of acetylcholinesterase (AChE) by organophos-
phorus (OP) chemical warfare (CW) nerve agents (NAs) and
poisoning leads to dose-dependent toxic signs including tremor,
hyper secretion, seizures, respiratory depression and eventually
death. Various quaternary oxime reactivators such as 2PAM and
toxogonin (obidoxime), combined with anticholinergics such as
atropine and scopolamine have been used to treat acute poisoning
by OP nerve agents [1,2]. Seizures are often controlled by the
administration of GABA receptor agonists such as diazepam or
midazolam [3].
The currently available combination of antidotes alleviate
significantly the symptoms of OP intoxication but are not able to
degrade OPs and thereby incapable to diminish their level in blood
and tissues. Consequently, the re-appearance of toxic signs in
Abbreviations: AChE, acetylcholinesterase; BChE, butyrylcholinesterase; OP, or-
ganophosphates; NA, nerve agents; PR, protection ratio; NP, no protection; 2-PAM,
2-pyridine aldoxime methochloride; 4PHA, 4-pyridine hydroxamic acid; BHA,
benzhydroxamic acid; i.m, intra muscular.
* Corresponding author. Current address: The Wohl Drug Discovery Institute, The
Grand Israel National Center for Personalized Medicine (G-INCPM), Weizmann
Institute of Science, PO Box 26, Rehovot 760001, Israel.
E-mail addresses: amitai.gabi36@gmail.com (G. Amitai), rellieg@gmail.com
(R. Gez), raveh.lily@gmail.com (L. Raveh), nirab@iibr.gov.il (N. Bar-Ner), ettieg@
iibr.gov.il (E. Grauer), shirac@iibr.gov.il (S. Chapman).
http://dx.doi.org/10.1016/j.cbi.2016.04.036
0009-2797/© 2016 Elsevier Ireland Ltd. All rights reserved.
Please cite this article in press as: G. Amitai, et al., Novel bifunctional hybrid small molecule scavengers for mitigating nerve agents toxicity,
Chemico-Biological Interactions (2016), http://dx.doi.org/10.1016/j.cbi.2016.04.036

2
G. Amitai et al. / Chemico-Biological Interactions xxx (2016) 1e18
severe OP intoxications requires continuous medical support such
as respiratory ventilation and repeated administration of antidotes.
Such continuous medical attendance and treatment of battlefield
CW casualties as well as mess casualties during a civilian chemical
terror act may create a heavy logistic burden on the medical
personnel [4]. Various bio-scavengers that could sequester toxic
OPs thus preventing their blood ChE inhibitions, were developed
during the last three decades. The physiologically disrupting irre-
versible inhibition of AChE by OPs was utilized to develop AChE
[5,6] as well as its non-physiological isoenzyme butyr-
ylcholinesterase (BChE) [7], as effective stoichiometric bio-
scavengers of OP NAs in vivo. However, using stoichiometric
enzyme scavengers constitutes an excessive pharmaceutical
payload. For instance, AChE with molecular weight (MW) of 80 kD
(D ¼ Dalton) and BChE with MW 360 kD were used to sequester
low molecular weight OPs such as sarin or VX of MW 140D or 267D,
respectively.
Consequently, enzymes that could catalytically hydrolyze OPs
were developed as catalytic bioscavengers. These phospho-
triesterases (PTEs) include OP acid anhydrolases (OPAA) from bac-
terial sources [8,9], OP hydrolases (OPH) from bacteria [10] and
mammalian sources e.g. serum paraoxonase1 PON1 [11], and dii-
sopropyl fluorophosphatase (DFPase) isolated from the squid giant
axon (Loligo vulgaris) [12,13]. Some PON1 variants developed by
directed evolution displayed marked hydrolytic activity on NAs
bearing a PeF bond (e.g. sarin, cyclosarin) [14]. Further, using
computational libraries combined with enhanced evolution, a more
than three orders of magnitude improvement in the rate of hy-
drolysis of V-type NAs by bacterial PTE was demonstrated [15]. The
outstanding achievements in the enhancement of the detoxifica-
tion kinetics by catalytic bioscavengers measured in vitro were also
corroborated by successful in vivo antidotal efficacy in OP NA-
challenged animals [16,17]. However, some bio-pharmaceutical is-
sues concerning protein therapy remained unresolved. Catalytic
protein bio-scavengers are limited in their route of administration
(no oral preparations), they still may create excessive payload by
their large mass per volume of administered solution with MW
~40 KD, and may induce immunological response upon repeated
administration unless chemically modified. Small molecule scav-
engers may be used to overcome most of these disadvantages and
be pharmaceutically compatible with current small molecule
therapy.
Quaternary pyridinium oxime reactivators of OP-inhibited AChE
that were developed in the early 1950's [18] provided the first
efficient reactivation of diisopropyl fluorophosphate (DFP)-and
Tetraethyl pyrophosphate (TEPP)-inhibited AChE by 2-pyridine
aldoxime methiodide (2PAM, Scheme 1). Efficient reactivation of
sarin inhibited AChE by 2PAM was later demonstrated [19]. The first
bisquaternary pyridinium oximes TMB-4 (trimedoxime) (Scheme
1) and some of its congeners such as MMB-4 (methoxime)
(Scheme 1) demonstrated a faster rate of reactivation of sarin-
inhibited AChE than 2PAM [20,21]. The second generation of bis-
quaternary oximes termed H-oximes [22], such as HI-6 (Scheme 1)
demonstrated high antidotal efficacy in the treatment of non-
human primates exposed to sarin, soman and tabun [23,24]. The
development of mono- and bis-quaternary oximes and their effi-
cacy in the treatment of OP poisoning was reviewed extensively by
Worek and Thiermann [2].
It was suggested that 2PAM and other oximes could be used for
both reactivation of OP-AChE conjugates by displacing the OP
moiety from the enzyme active site as well as reactive nucleophiles
directly reacting with OPs [25]. Mono- and bis-pyridinium oximes
(e.g. 2PAM, Toxogonin and HI-6) were indeed examined kinetically
as direct scavengers of OP NAs in vitro [26,27]. However, the rate of
the direct reaction of these oximes with OP NAs were found too
slow for significant detoxification of OP poisoning in vivo [26].
Notably, Benzhydroxamic acid (BHA), N-methyl 4-Pyridine
hydroxamic acid (NeMe 4-PHA), N-methyl 2-PHA and some of
their structural analogues were found to be more active than their
respective pyridinium oximes as nucleophiles toward OP NAs
causing their degradation under physiological conditions [28e30].
By contrast, hydroxamic acid combined with quaternary pyr-
idinium, the structural homologs of 2-PAM and 4-PAM, were very
poor reactivators of sarin-inhibited AChE compared to their oxime
analogues [31,32].
The efficacious reactivation property of quaternary oximes, as
well as the relatively rapid rate of direct reaction of aromatic
hydroxamates with OPs, served as a basis for our design of new
hybrid molecules. Our synthetic approach was based on linking
either BHA or 4PHA, capable of hydrolyzing directly the OP agent,
to 2PAM moiety via short hydrocarbon bridge (CH2)n (n ¼ 1 or 3)
(Scheme 2). These double headed hybrids were expected to elicit
bifunctional activity that will both diminish the level of toxic OP
NAs in blood with concomitant reactivation of OP-inhibited AChE
in situ. Since these hybrid compounds include a positively charged
quaternary pyridinium moiety (Scheme 2), it may enhance the
affinity of the whole molecule to AChE by guiding the hybrid
molecule to the negatively charged active-site region [1]. One
important advantage of covalently combined moieties compared
to a mixture of two separate compounds, is the unitary pharma-
cokinetics of these two complementary activities (scavenging and
reactivation) in vivo. Uniform rates of absorption, distribution,
metabolism and elimination (ADME) of a hybrid compound are
preferable to two distinct active pharmaceutical ingredients, each
displaying its own ADME pattern. This bifunctional drug concept
has recently been applied to novel therapy of chronic obstructive
pulmonary disease (COPD) that is presently under clinical trials
[33]. The hybrid drug combines b2-adrenergic receptor agonist
with muscarinic receptor antagonist in one small molecule. One of
the benefits of this approach includes avoiding the problem of
formulating two different drugs in one pill or inhaler [33]. In the
course of our studies 22 new oxime-hydroxamate hybrids were
synthesized of which three compounds were selected for further
in vivo studies: 2PAMPr4PHA, 2PAMMeBHA and 2,4DiPAMMeBHA
(Scheme 2).
Scheme 1. Chemical structure of quaternary oximes 2-PAM, HI-6, TMB-4 (trimedox-
ime) and MMB-4 (methoxime).
Please cite this article in press as: G. Amitai, et al., Novel bifunctional hybrid small molecule scavengers for mitigating nerve agents toxicity,
Chemico-Biological Interactions (2016), http://dx.doi.org/10.1016/j.cbi.2016.04.036

G. Amitai et al. / Chemico-Biological Interactions xxx (2016) 1e18
3
Scheme 2. The chemical structure of the oxime-hydroxamate hybrids 2PAMPr4PHA, 2PAMMeBHA and 2,4DiPAMMeBHA.
2. Materials and methods
(75e500 mM) and 2PAM (0.5e2.5 mM). The activity of whole blood
ChE sampled from rats and guinea pigs in vivo was measured by
using the radioactive assay with [3H]acetylcholine as substrate
[35].
2.1. Materials
Acetylthiocholine iodide (ATC), 5,5-dithio bis 3- nitrobenzoic
acid (DTNB), pyridostigmine bromide, atropine sulfate, 2-pyridine
aldoxime, 2-pyridine aldoxime methchloride (2-PAM Cl-), benz-
hydroxamic acid (BHA), Na2HPO4, Na2HPO4, sodium chloride,
anhydrous dimethylsufoxide (DMSO) were products of Aldrich-
Sigma, Israel. 2,4-pyridine dialdoxime was a product of Accel
Pharmtech, East Brunswick, NJ, USA. Purified recombinant Human
acetylcholinesterase (HuAChE) was a kind gift of Dr. A. Shafferman,
IIBR. A solution of Tritium-labeled acetylcholine iodide ([3H]ACh)
[acetyl-3H]] 1 mCi (37 MBq) 1 Ci/mmol was purchased from Perkin
Elmer, UK. The radioactive [3H]ACh was used in the evaluation of
whole blood ChE activity. The organophosphorus compounds sarin,
cyclosarin, soman and VX were prepared at >95% purity, at the
Department of Organic Chemistry, IIBR.
2.4.2. Kinetics of detoxification of OP compounds using the AChE
rescue assay
Sarin, cyclosarin, soman and VX (10ꢁ6 M) were incubated either
in buffer (10 mM phosphate, 0.1 M NaCl, pH 7.4) for spontaneous
hydrolysis or in the presence of a hybrid small molecule scavengers
(10ꢁ3 M) in buffer at 37 ꢂC. 2PAM served as a reference compound.
Samples were drawn from the reaction mixture and diluted
50e100-fold into AChE solution for 3 min incubation with AChE to
allow inhibition by residual OP that was not degraded yet during
the hydrolysis reaction. Aliquots of 20
tions were then added to 1 ml cuvette containing ATC/DTNB for
measuring the enzyme residual activity by the increase in OD412
ml OP-AChE conjugate solu-
/
min during 0.25e0.5 min. The rate of OP detoxification was linearly
correlated with the increase in residual AChE during the hydrolysis
reaction. The increase in OD412 with time was measured using Cary
3 spectrophotometer, Varian, USA.
2.2. Synthesis of oxime-hydroxamate hybrids
The synthetic procedures for preparing the hybrids 2PAMPr4-
PHA, 2PAMMeBHA and 2,4DiPAMMeBHA (Scheme 2) are described
in detail in Appendix A.
2.4.3. Reversible inhibition of AChE
AChE (1.5 Units/ml, 20 ml) was incubated for 10 min either in
phosphate buffer containing 1 mM DTNB, pH ¼ 7.4 or with the
reversible inhibitor i.e. quaternary pyridinium oximes or oxime-
2.3. Animals
hydroxamate
hybrids,
at
various
concentrations
(1e1000 ꢀ 10ꢁ6 M) in 140
ml DTNB containing buffer in 96-well
Sprague Dawley male Rats 250e350 gr and Hartley male guinea
pigs 300e450 gr were supplied by Charles River, UK. Protocols of
experiments with animals were approved by the IIBR Institutional
Animal Care and Use Committee (IACUC) and all processes adhered
to the NIH guidelines for animal care and experiments.
plate. Then 20
ml of acetylthiocholine (ATC) solution was added
and the increase of OD412 with time was measured for 1 min at
23 ꢂC, using Tecan F200 Optical Reader, Switzerland.
2.4.4. In vivo toxicity of drugs and OP agents
The toxicity of, 2PAMPr4PHA, 2PAMMeBHA and 2,4 DiPAM-
MeBHA in rats and guinea pigs was evaluated by intramuscular (im)
administration of 50e250 mg/ml solutions of oxime-hydroxamates
in either saline or double distilled water or a mixture of 70% DMSO
and 30% double distilled water for dissolving the less soluble
oxime-hydroxamate hybrid 2,4DiPAMMeBHA at 200 mg/ml. The
survival and general behavior of animals were monitored up to 7
days. The LD50 values for the oxime-hydroxamate hybrids were
determined using the up-and-down method [36]. Toxicity of OP
nerve agents was determined by im administration of OPs solutions
in saline and the LD50 of the OP agents in rats and guinea pigs was
also evaluated by the up and down method [36].
2.4. Methods
2.4.1. AChE assay for OP detoxification and reactivation of OP-
inhibited AChE
Human recombinant AChE (HuAChE) activity was measured by
the Ellman assay [34] using acetylthiocholine (ATC) 1.5 mM and the
Ellman reagent DTNB 2 mM in 50 mM phosphate buffer pH ¼ 7.4.
Reactivation kinetics of OP-inhibited AChE were performed by
inhibiting AChE with sarin, cyclosarin or VX to a steady state level of
92e95% AChE inhibition. The OP-AChE conjugates were then
diluted 50e100 fold into either phosphate buffer or oxime solution
at specified concentrations, pH ¼ 7.4, 37 ꢂC. Reactivation kinetics
with a single concentration of 2PAM or oxime-hydroxamate were
performed either with 0.5 mM (sarin- and cyclosarin-inhibited
AChE) or 0.375 mM (VX-inhibited-AChE). Reactivation of
cyclosarin-inhibited AChE was performed in the presence of the
following concentrations of reactivators based on their reactivation
2.4.5. In vivo regeneration of blood ChE following exposure to sub-
lethal sarin
In order to evaluate the efficacy of the hybrid compounds as ChE
regenerators in vivo we have performed pharmacodynamic (PD)
studies in rats and guinea pigs exposed to sub-lethal dose of sarin
potency: HI-6 (1e2.5
mM), ICD-585 (2.5e7.5 mM), 2PAMPr4PHA
Please cite this article in press as: G. Amitai, et al., Novel bifunctional hybrid small molecule scavengers for mitigating nerve agents toxicity,
Chemico-Biological Interactions (2016), http://dx.doi.org/10.1016/j.cbi.2016.04.036

4
G. Amitai et al. / Chemico-Biological Interactions xxx (2016) 1e18
(0.8xLD50). Animals were administered with 0.8xLD50 of sarin
(80 g/kg in rats, im, 30 g/kg in guinea pigs, im) either 1 min
before or 15 min after administration of hybrids or 2PAM in
conjunction with atropine. The doses of hybrids and 2PAM were 42,
that in the last step 4-Bromomethyl benzhydroxamic acid was
reacted with 2,4 pyridine dialdoxime (for further details see
Appendix A).
m
m
71, 83 (in rats and guinea pigs, im), and 142
im) and the doses of atropine were 3 mol/kg, im in rats and
mol/kg im in guinea pigs. Blood samples were drawn at specified
m
mol/kg (in rats only,
3.2. Detoxification of OP nerve agents by oxime-hydroxamate
hybrids
m
7
m
time intervals following sarin poisoning (0, 0.5, 1, 3 and 6 h) and
stored at ꢁ70 ꢂC until ChE activity was measured using the radio-
active assay with [3H] acetylcholine [35].
The rates of hydrolysis of OP NAs sarin, cyclosarin, soman and
VX (1
mM) was studied in the presence of 2PAMMeBHA,
2,4DiPAMMeBHA and 2PAMPr4PHA hybrid oxime-hydroxamates
(1 mM) in 10 mM phosphate, 0.1 M NaCl, pH ¼ 7.4, 37 ꢂC. The
2.4.6. In vivo antidotal efficacy studies
concentration of NAs used in the detoxification reaction (1 mM) was
The antidotal efficacy studies were performed with the oxime-
hydroxamate hybrids 2PAMMeBHA, 2PAMPr4PHA and 2,4DiPAM-
MeBHA and 2PAM (as reference oxime), in conjunction with atro-
pine, against poisoning by sarin, cyclosarin, VX and soman. All
antidotes and OP NAs were administered intra-muscularly (im). In
the pre-treatment regimen, the tested hybrid compounds and
atropine were administered im 15 min before challenging the an-
imals with OP NAs. In the case of post-treatment of soman
poisoning, pretreatment with pyridostigmine was employed by
administration of 0.1 mg/kg, im, 15 min before soman challenge in
rats and guinea pigs.
The acute toxicity of NAs with and without treatment was
evaluated using the up-and-down method [36] and animals were
observed for toxic signs and survival up to 24 h after im adminis-
tration of the NAs. The LD50 values obtained after antidotal treat-
ment were calculated according to the Spearman-Kaerber
statistical protocol [37]. The dose of oxime-hydroxamate hybrids
selected based on expected blood levels during acute poisoning by
multiples of LD50 dose of sarin in humans (LD50 of sarin estimated
in humans is 14 mg/kg, iv). The apparent first order kinetic constants
(kobs, minꢁ1) and half-life time (minutes) of hydrolysis of OP agents
by oxime-hydroxamate hybrids, 2PAM and BHA are summarized in
Table 1. The decreasing level of OP (%OPt) during the detoxification
reaction was determined by measuring its residual potency as AChE
inhibitor (AChE “rescue assay” as described in methods) and was
calculated according to the following equation:
% OPt ¼ [1ꢁ(EtꢁEi)/E0ꢁEi)] ꢀ 100
where: OPt ¼ concentration of OP agent at time t, E0 ¼ AChE activity
at time ¼ 0 (control enzyme activity), EI ¼ AChE residual activity in
the presence of non-hydrolyzed OP (measured before starting the
OP hydrolysis, AChE inhibition time of 3 min using 1e3 ꢀ 10ꢁ8
M
final non-hydrolyzed OP concentration with AChE depending on
the OP agent), Et e AChE activity measured at time t during OP
hydrolysis. The fastest detoxification rate was obtained with
2PAMMeBHA reacting with sarin cyclosarin and soman (kobs ¼ 340,
355, and 190 ꢀ 10ꢁ3 minꢁ1, respectively, Table 1) compared to
slower detoxification rates exerted by the oxime 2PAM and
hydroxamate BHA (23 and 138 ꢀ 10ꢁ3 minꢁ1, respectively, Table 1).
Detoxification of sarin by the hybrid 2,4DiPAMMeBHA displayed a
biphasic kinetics (not shown). In the first fast phase 50% of sarin
was detoxified in less than 1 min followed by a second slower phase
(kobs ¼ 17 ꢀ 10ꢁ3 minꢁ1, Table 1). The biphasic kinetic pattern may
arise from an initial combined effect of one of the two oxime groups
located at 2 and 4 position on the pyridine ring together with the
relatively active hydroxamate group of BHA. The slower phase may
stem from putative formation of phosphoryl-oxime intermediate
due to reaction of sarin with the 2,4-dioxime pyridine moiety,
especially with the oxime at position 4 on the pyridine ring that
may form a relatively stable phosphoryl-4-pyrinium-oxime inter-
mediate that could re-inhibit AChE during the bio-assay [39]. The
hybrid 2PAMPr4PHA detoxified sarin, cyclosarin, and soman with
the following rates kobs ¼ 112, 88 and 51 ꢀ 10ꢁ3 minꢁ1, respectively
(Table 1). These rates were moderate but significantly faster than
and 2PAM, 142 mmol/kg, was determined based on the in vivo ChE
reactivation data following exposure to sarin (0.8xLD50) in rats and
guinea pigs (vide infra). The dose of atropine used in the antidotal
efficacy studies in rats (30 mmol/kg, im) and guinea pigs (45 mmol/
kg, im) were determined by preliminary dose-range finding studies
and based on our previous experience in the treatment of OP
intoxicated rats and guinea pigs with atropine only. The dose-range
finding studies with atropine in rats and guinea pigs (not shown)
were based on creating a therapeutic window for each dose of the
new hybrids and 2PAM that will minimize the masking effect of
atropine palliative contribution. For instance, the dose of atropine
used in rats against VX poisoning was 7.5 mmol/kg (lower than what
was used for the treatment of G-Agents) since higher atropine
doses yielded very similar PRs against VX for all tested hybrid
compounds. The atropine doses employed in our studies were in
very good agreement with those previously used against OP NAs
poisoning in rats and guinea pigs [38].
3. Results and discussion
3.1. Syntheses of oxime-hydroxamate hybrids
those observed with 2PAM (kobs ¼ 23, 37, 7, and 12.8 ꢀ 10ꢁ3 minꢁ1
,
The synthetic procedures of the oxime-hydroxamate hybrids are
described in detail in Appendix A. In short, the oxime-hydroxamate
hybrid compound 2PAMPr4PHA was synthesized by starting with
tertiary 2-pyridine aldoxime that was N-alkylated with 1,3 dibro-
mopropane to yield 3-bromo-propyl-2PAM that was then coupled
by N-alkylation to the 4-pyridine hydroxamate (4PHA). 2PAM-
MeBHA was prepared by a three step synthesis. In the first step 4-
bromomethyl benzoyl chloride was prepared from 4-
bromomethylbenzoic acid. Then 4-bromomethyl benzhydroxamic
acid was prepared by reacting 4-bromomethyl benzoylchloride
with hydroxylamine. The last step was based on the N-alkylation of
2-pyridine aldoxime with 4-bromomethyl benzhydroxamic acid to
yield 2PAMMeBHA. The first two steps in the synthesis of 2PAM-
MeBHA were also used for the synthesis of 2,4DiPAMMeBHA except
respectively, Table 1). The difference in rates indicates the contri-
bution of the hydroxamate nucleophile in 4-pyridine hydroxamate
(4-PHA) as demonstrated previously [30]. All oxime-hydroxamate
hybrids, 2PAM and BHA were not active toward VX yielding very
slow rates (kobs ¼ 2e7 ꢀ 10ꢁ3 minꢁ1, Table 1). The slow reaction of
hydroxamates with VX stems probably from a lower electrophilic
potency of the phosphorus atom bound to sulfur and a relatively
stable PeS bond.
3.3. Reactivation of OP-inhibited AChE conjugates by hybrids
The time-course of reactivation by oxime-hydroxamate hybrids
of AChE inhibited by sarin, VX and cyclosarin are described in Fig. 1,
respectively. The reactivation rate parameters (kobs, minꢁ1 and %
Please cite this article in press as: G. Amitai, et al., Novel bifunctional hybrid small molecule scavengers for mitigating nerve agents toxicity,
Chemico-Biological Interactions (2016), http://dx.doi.org/10.1016/j.cbi.2016.04.036

G. Amitai et al. / Chemico-Biological Interactions xxx (2016) 1e18
5
Table 1
Kinetic parameters for detoxification of organophosphate nerve agents (OP NAs) by oxime-hydroxamate hybrid compounds. The OP NAs sarin, cyclosarin, soman and VX were
used at 1 mM, oxime (2PAM), hydroxamate (BHA) and oxime hydroxamates (2PAMMeBHA, 2,4DiPAMMeBHA and 2PAMPr4PHA) were used at 1 mM, in 10 mM phosphate, 0.1 M
NaCl, pH ¼ 7.4, 37 ꢂC.
Nerve agent
Compound
Sarin
Cyclosarin
Soman
VX
kobs ꢀ 10ꢁ3 (minꢁ1
)
t1/2 (min)
kobs ꢀ 10ꢁ3 (minꢁ1
)
t1/2 (min)
kobs ꢀ 10ꢁ3 (minꢁ1
)
t1/2 (min)
kobs ꢀ 10ꢁ3 (minꢁ1
)
t1/2 (min)
BHA
2PAM
2PAMMeBHA
2,4DiPAMMeBHA
2PAMPr4PHA
Bufferd
138 16
23 0.5
340 55
17 5c
5
n.d.a
37.7
355 20
108 10
e
58
4
12
12.8
3.6
46
13.5
246
n.a.b
2.0 0.1
7.0 0.2
n.d.
e
345
99
34.5
2.0
41
6.2
300
18.4
1.9
6.4
7.8
138
54 10
190 25
15 5.0c
51
2.8 0.6
e
112
7
88
5
9
1
4
2.5 0.01
289
>720d
2.3 0.6
n.d.a
a
n.d. e not determined.
n.a. e not active.
b
c
Biphasic kinetics, the first fast phase ending with 45e50% sarin or soman hydrolysis by 2,4-DiPAMMeBHA took less than 1 min (estimated kobs >0.69 minꢁ1). The second
phase was significantly slower with t1/2 ¼ 41 and 46 min (kobs ¼ 17 ꢀ 10ꢁ3 and 15 ꢀ 10ꢁ3 minꢁ1) for sarin and soman, respectively.
d
10 mM phosphate, 0.1 M NaCl, pH ¼ 7.4, 37 ꢂC, VX was relatively stable in this buffer and the half-life time (t1/2) of its hydrolysis under these conditions was estimated to be
>12 h.
Table 2
Kinetic parameters of reactivation of OP-Inhibited AChE by oxime-hydroxamate
hybrids and 2PAM e kobs (minꢁ1) mean
tivation (in brackets) (reactivator concentration 0.5 mM with sarin- and cyclosarin-
AChE and 0.375 mM with VX-AChE conjugate, 50 mM phosphate pH ¼ 7.4, 0.03%
BSA, 37 ꢂC).
s.e.m., n ¼ 3, and % maximal reac-
Reactivator
OP agent
Sarin
VX
Cyclosarin
(kobs, minꢁ1
)
(kobs, minꢁ1
)
(kobs, minꢁ1
)
2PAMPr4PHA
2PAMMeBHA
2,4DiPAMMeBHA
2PAM
0.079 0.006
(70%)
0.142 0.007
(60%)
0.152 0.013
(69%)
0.476 0.025
(90%)
0.040 0.006
(55%)
0.084 0.014
(56%)
0.045 0.007
(71%)
0.067 0.015
(94%)
0.302 0.02
(70%)
0.053 0.004 (60%)
0.078 0.008
(60%)
0.059 0.006 (65%)
(0.5 mM). The most potent reactivator of sarin-inhibited AChE was
2PAM (kobs ¼ 0.476 minꢁ1) compared to 2PAMMeBHA 2,4DiPAM-
MeBHA and 2PAMPr4PHA (kobs ¼ 0.151, 0.142 and 0.128 minꢁ1
,
respectively, Table 2). The maximal reactivation level obtained with
2PAM 90% was higher compared to 60e70% reactivation obtained
with all oxime-hydroxamate hybrids (Fig. 1A, Table 2). Less than 5%
spontaneous reactivation was observed in buffer after 90 min
(Fig. 1A).
3.3.2. Reactivation of VX-inhibited AChE
Fig. 1B describes the time-course of reactivation of VX-inhibited
AChE by oxime-hydroxamate hybrids and 2PAM (0.375 mM).
Generally, all compounds displayed significantly slower kinetics of
reactivation of VX-inhibited AChE than those measured for sarin-
AChE (Table 2). The most potent reactivator of VX-inhibited AChE
was 2PAMMeBHA (kobs ¼ 0.084 minꢁ1) compared to 2PAM,
2,4DiPAMMeBHA 2PAMPr4PHA and (kobs ¼ 0.067, 0.045 and
0.040 minꢁ1, respectively, Table 2). However, the difference in kobs
values obtained for 2PAM and 2PAMMeBHA (0.067 and
0.084 minꢁ1) is statistically insignificant (based on the s.e.m values
in Table 2). In addition, the maximal reactivation level obtained
with 2PAM 94% was higher compared to 55e71% reactivation ob-
tained at steady state (measured between 100 and 240 min) with
the oxime-hydroxamate hybrids. The spontaneous reactivation of
VX-AChE was 10% after 4.5 h in buffer at pH ¼ 7.4, 37 ꢂC (Fig. 1B).
Fig. 1. Time-course of reactivation of sarin (A), VX (B) and cyclosarin (C) - inhibited
HuAChE by oxime-hydroxamate hybrids and 2PAM (0.5 mM reactivator, 50 mM
phosphate, pH ¼ 7.4, 0.03% BSA, 37 ꢂC) (mean
s.e.m. n ¼ 3, the kobs values are
summarized in Table 2, the kinetic plots presented here were taken from single ex-
periments that represent three repeats for each OP inhibitor).
maximal reactivation levels) are summarized in Table 2. The oxime-
hydroxamate hybrids 2PAMMeBHA, 2,4DiPAMMeBHA and
2PAMPr4PHA and the oxime 2PAM, that served as a reference
reactivator, were incubated at 0.5 mM with 50-fold diluted OP-
AChE conjugates after reaching a steady state of 92e95% AChE in-
hibition. All measurements were performed in 50 mM phosphate
pH ¼ 7.4, 0.03% BSA, 37 ꢂC.
3.3.1. Reactivation of sarin-inhibited AChE
Fig. 1A describes the time-course of reactivation of sarin-
inhibited AChE by oxime-hydroxamate hybrids and 2PAM
3.3.3. Reactivation of cyclosarin-inhibited AChE
Fig. 1C, describes the time-course of reactivation of cyclosarin-
Please cite this article in press as: G. Amitai, et al., Novel bifunctional hybrid small molecule scavengers for mitigating nerve agents toxicity,
Chemico-Biological Interactions (2016), http://dx.doi.org/10.1016/j.cbi.2016.04.036

6
G. Amitai et al. / Chemico-Biological Interactions xxx (2016) 1e18
inhibited AChE by oxime-hydroxamate hybrids and 2PAM
(0.5 mM). The most potent oxime-hydroxamate hybrid reac-
tivator of cyclosarin-inhibited AChE was 2PAMPr4PHA
(kobs ¼ 0.302 minꢁ1) compared to 2,4DiPAMMeBHA, 2PAM and
2PAMMeBHA (kobs ¼ 0.078, 0.059 and 0.053 minꢁ1, respectively,
Fig. 1C, Table 2). The maximal reactivation level obtained after
60 min with 2PAM was comparable to that obtained with the
oxime-hydroxamate hybrids i.e. 60e70% reactivation at pH ¼ 7.4,
37 ꢂC (Fig. 1C, Table 2). Spontaneous reactivation of AChE
following inhibition by cyclosarin was <5% after 2 h (not shown).
Notably, the oxime-hydroxamate 2PAMPr4PHA resembles the
structure of the bispyiridinium oxime ICD-585 (Scheme 3) shown
previously to be an efficacious reactivator of AChE inhibited by
OP NAs [40]. ICD585 includes in its structure an amide moiety in
position 4 of the pyridine ring (isonicotinamide) instead of
hydroxamic acid as in 2PAMPr4PHA (Scheme 3). Pertinently, ICD-
585 is a close structural analogue of HI-6 and was considered by
the US Military to substitute 2PAM as the oxime antidote due to
its marked reactivation potency and antidotal efficacy toward OP
NAs [40]. Therefore, we have compared the rate of reactivation
rate exerted by 2PAMPr4PHA to those elicited by ICD585 and HI-
reactivator with 1.5-fold higher rate than HI-6 (kr ¼ 24 and
16.5 ꢀ 103 Mꢁ1 minꢁ1, respectively, Table 3) and the oxime-
hydroxamate hybrid 2PAMPr4PHA (kr ¼ 0.32 ꢀ 103 Mꢁ1 minꢁ1
)
was 67- and 46-fold less potent than ICD585 and HI-6, respectively,
but 20-fold more potent than 2PAM (kr ¼ 0.018 ꢀ 103 Mꢁ1 minꢁ1
,
Table 3). Interestingly, the 4PAM hybrid analogues of 2PAMMeBHA
and 2PAMPr4PHA, that are not included in this report, were
significantly less potent as reactivators of sarin-AChE and
cyclosarin-AChE conjugates but more potent than their 2PAM
hybrid congeners toward Tabun-inhibited AChE (not shown).
3.4. Reversible inhibition of AChE by oxime-hydroxamate hybrids
All three oxime-hydroxamate hybrids include in their structure
a quaternary 2-pyridinium aldoxime (2PAM) moiety (Scheme 2).
The positively charged 2PAM moiety could direct the hybrid
molecule into the negatively charged active site of AChE and thus
increase their affinity to the enzyme. The anionic site inside the
active site, the acyl pocket and the peripheral anionic site amino
acid residues of AChE have been previously described as important
loci for the formation of electrostatic and hydrophobic interactions
with quaternary pyridinium oximes [41]. Therefore, we have esti-
mated the affinity of the newly synthesized oxime-hydroxamate
hybrids to AChE by measuring the reversible inhibition of AChE
by these hybrids.
Fig. 3 describes the concentration-dependence of reversible
inhibition of HuAChE by the oxime-hydroxamates 2PAMMeBHA,
2,4DiPAMMeBHA, 2PAMPr4PHA and 2PAM (Fig. 3A) and by the
oximes HI-6, HLo-7, TMB-4, MMB-4, Toxogonin and 4PAM (Fig. 3B).
The IC50 values calculated by a non-linear fit to the experimental
data are summarized in Table 4. The IC50 values obtained for all
6
toward sarin- cyclosarin- and VXeinhibited AChE. The
concentration-dependent reactivation kinetics of sarin- VX- and
cyclosarin-inhibited AChE by these oximes and 2PAMPr4PHA
were studied. However, only the kinetic plots obtained with
cyclosarin-AChE conjugate are demonstrated in Fig. 2. Table 3
summarizes the bimolecular rate constants obtained for AChE
reactivation following inhibition by the NAs: sarin-, cyclosarin-
and VX-AChE conjugates by HI-6, ICD585, 2PAMPr4PHA and
2PAM. The descending ranking order of reactivation potency
toward sarin-AChE conjugate based on the bimolecular
rate
6 > ICD585 > 2PAM > 2PAMPr4PHA (kr ¼ 5.5, 1.49, 0.34 and
0.08 Table 3). The ranking order for
103 Mꢁ1 minꢁ1
reactivation of VX-AChE conjugate was HI-
constant
of
reactivation
(kr)
was:
HI-
oxime-hydroxamate hybrids range at 16e48 mM (Fig. 3A, Table 4).
The apparent IC50 values estimated from the non-linear fit to the
experimental data obtained for the oximes 2-PAM, 4-PAM, MMB-4,
ꢀ
,
6 > ICD585 > 2PAM > 2PAMPr4PHA (kr ¼ 3.5, 0.77, 0.18 and
0.15 ꢀ 103 Mꢁ1 minꢁ1, Table 3). The ranking order for reactivation
of cyclosarin-AChE was ICD-585 > HI-6 > 2PAMPr4PHA > 2PAM
(Table 3). All tested reactivators demonstrated lower reactivation
potency toward VX-inhibited AChE than toward sarin-inhibited
AChE (Table 3).
Fig. 2 demonstrates the concentration-dependent kinetics of
reactivation of cyclosarin-inhibited AChE by 2PAMPr4PHA, ICD585,
HI-6 and 2PAM at the specified concentrations. The ranking order of
TMB-4 and Toxogonin are 34e90
mM (Table 4). However, these
IC50's were based on 25e50% maximal inhibition of AChE activity at
their highest tested concentration (1 mM, Fig. 3B). Therefore, the
IC50 values for these oximes were listed in a separate group of
weaker AChE inhibitors. Notably, HI-6 is the only oxime that
inhibited AChE at relatively low IC50 value (11
bition at 1 mM (Fig. 3B, Table 4). HLo-7 inhibited 85% of AChE ac-
tivity at 1 mM and its estimated IC50 (104 M, Table 4 and Fig. 3B)
was higher than that of HI-6. Based on the IC50 values obtained for
those compounds that inhibited 70e90% AChE activity at 1 mM, the
descending ranking order of potency of AChE reversible inhibition
was: HI-6 > 2PAMMeBHA > 2PAMPr4PHA > 2,4DiPAMBHA > HLo-
7 (Table 4). All other oximes displayed significantly lower inhibition
potency toward AChE reaching only 25e50% inhibition at 1 mM
mM) with 90% inhi-
m
reactivation potency of cyclosarin-AChE is: ICD-585
>
HI-
6
>
2PAMPr4PHA 2PAM (kr
>
¼
24.0, 16.5, 0.356 and
0.018 ꢀ 103 Mꢁ1 minꢁ1, Table 3). However, the maximal level of
AChE reactivation obtained with 2PAMPr4PHA and ICD585 was
90e95% within 30 min compared to 60% maximal reactivation
obtained with HI-6 and 2PAM (Fig. 2). In the case of cyclosarin-
inhibited AChE, the oxime ICD585 was the most potent
with
the
following
ranking
order:
TMB-
4 > 2PAM > toxogonin > 4PAM > MMB-4.
Scheme 3. The resemblance in chemical structure of the oxime-hydroxamate 2PAMPr4PHA (center) to known bisquaternary oximes, ICD-585 (right) and HI-6 (left).
Please cite this article in press as: G. Amitai, et al., Novel bifunctional hybrid small molecule scavengers for mitigating nerve agents toxicity,
Chemico-Biological Interactions (2016), http://dx.doi.org/10.1016/j.cbi.2016.04.036

G. Amitai et al. / Chemico-Biological Interactions xxx (2016) 1e18
7
Fig. 2. Reactivation of cyclosarin-inhibited HuAChE by 2PAMPr4PHA, ICD-585, HI-6 and 2PAM at specified concentrations of reactivators shown on top right of each graph. HuAChE
was inhibited by cyclosarin at 92e95% inhibition at steady state before reactivation was started by 50-fold dilution of OP-AChE conjugate into the reactivator solution (50 mM
phosphate, pH ¼ 7.4, 0.03% BSA, 37 ꢂC) (mean sem, n ¼ 3, values of the bimolecular rate constants of reactivation kr, Mꢁ1min-1 are summarized in Table 3, the kinetic plots
presented here demonstrate a single representative experiment out of the three repeats for each reactivator).
Table 3
The bimolecular rate constants of reactivation of Sarin- Cyclosarin- and VX-inhibited AChE by HI-6, ICD-585, 2PAMPr4PHA, and 2PAM (bimolecular rate constants
kr ꢀ 103 Mꢁ1 minꢁ1, 50 mM phosphate, pH ¼ 7.4, 0.03% BSA, 37 ꢂC) (the bimolecular rate constants of reactivation are presented as mean s.e.m, n ¼ 3).
Reactivator
OP-AChE
Sarin-AChE
Cyclosarin-AChE
VX-AChE
kr ꢀ 103 s.e.m. Mꢁ1 minꢁ1
kr ꢀ 103 s.e.m. Mꢁ1 minꢁ1
kr ꢀ 103 s.e.m. Mꢁ1 minꢁ1
HI-6
5.20 0.22
1.49 0.08
0.08 0.03
0.34 0.08
16.5 0.9
24.0 1.4
0.356 0.068
0.018 0.008
3.50 0.16
0.77 0.10
0.15 0.04
0.18 0.04
ICD-585
2PAMPr4PHA
2PAM
3.5. The effect of reversible inhibition by the hybrids on the rate of
phosphorylation of AChE by OP nerve agents
the kp values obtained with 2PAMPr4PHA and 2PAM as protectants
against the irreversible inhibition by all tested OP NAs were
significantly lower (0.3e5 ꢀ 10ꢁ5 Mxmin) than those obtained for
2PAMMeBHA and 2,4DiPAMMeBHA (Table 5). The slowdown in
AChE phosphorylation by the OP agents in the presence of hybrids
may have apparently been interpreted also by direct degradation
effect of the hybrids toward the OPs. However, the concentrations
of hybrids (2.5e10 ꢀ 10ꢁ5 M) and OPs (1e2 ꢀ 10ꢁ8 M) in these
experiments are significantly lower than those used in the direct
detoxification studies (10ꢁ3 M hybrids and 10ꢁ6 M OPs, Table 1).
The relatively low concentrations of the reactants together with a
short time period of measuring the anti-phosphorylation effect by
AChE reversible inhibition, rule out a direct degradation effect by
the hybrids under these conditions. Interestingly, the hybrid
2PAMPr4PHA exerted the lowest protection of AChE against inhi-
bition by the OP agents as demonstrated by the similarity of inhi-
bition rates by OPs in the presence of various concentrations of
2PAMPr4PHA, expressed by its relatively low kp values (Table 5).
Further, when cyclosarin was used as an irreversible inhibitor after
pre-treatment with 2PAMPr4PHA, there was a dose-dependent
recovery in AChE activity starting 10 min after AChE inhibition by
cyclosarin (not shown). which indicates reactivation of cyclosarin-
AChE exerted by 2PAMPr4PHA. Indeed, this partial recovery in
AChE activity is consistent with the relatively high reactivation rate
obtained with 2PAMPr4PHA toward cyclosarin-inhibited AChE
(Table 2).
Based on the reversible inhibition of AChE by the oxime-
hydroxamate hybrids (Fig. 3, Table 4), we explored the effect of
pre-treatment with these hybrids on the rate of AChE irreversible
inhibition by OP NAs. Fig. 4 demonstrates the kinetics of AChE
irreversible inhibition by sarin (Fig. 4A), cyclosarin (Fig. 4B), soman
(Fig. 4C) and VX (Fig. 4D) in the presence of 0, 2.5, 5 and
10 ꢀ 10ꢁ5 M of 2PAMMeBHA, and 2,4DiPAMMeBHA (the kinetics of
inhibition by OP NAs in the presence of 2PAMPr4PHA and 2PAM are
not shown). A double reciprocal plot of 1/kobs vs. 1/[hybrid] enabled
a quantitative evaluation of the anti-phosphorylation effect of the
hybrids and 2PAM (not shown). Table 5 summarizes the slopes of
the double reciprocal plots defined as protection coefficients (kp,
Mxmin), calculated by linear regression of 1/kobs vs. 1/[hybrid]. The
kp values served as a measure for the retardation of AChE phos-
phorylation rate induced by the hybrids and 2PAM.
As demonstrated in Fig. 4, the hybrids 2PAMMeBHA and
2,4DiPAMMeBHA elicited a pronounced anti-phosphorylation ef-
fect for all tested OP NAs (Fig. 4AeD), whereas 2PAMPr4PHA and
2PAM were less potent (not shown in Fig. 4 but their respective kp
values appear in Table 5). The protection coefficient (kp) calculated
for 2PAMMeBHA, 2,4DiPAMMeBHA, against irreversible inhibition
by VX, sarin, cyclosarin and soman were 23 and 31, 52 and 25.5, 12
and 38,18.5 and 36 ꢀ 10ꢁ5 Mxmin, respectively (Table 5). Generally,
Please cite this article in press as: G. Amitai, et al., Novel bifunctional hybrid small molecule scavengers for mitigating nerve agents toxicity,
Chemico-Biological Interactions (2016), http://dx.doi.org/10.1016/j.cbi.2016.04.036

8
G. Amitai et al. / Chemico-Biological Interactions xxx (2016) 1e18
from mg/ml to mM concentration (i.e. dividing mg/ml by the mo-
lecular weights of 2PAMPr4PHA, 2,4DiPAMMeBHA and 2PAM-
MeBHA, 556, 395 and 352, respectively) the maximal
concentrations of the hybrids 2PAMPr4PHA, 2,4DiPAMMeBHA and
2PAMMeBHA (corresponding to the low and high injected doses 25
and 50 mg/kg) are 0.65, 1.3, 0.9,1.8, 1, 2 mM, respectively. Usually,
the drug bioavailability following im injection is about 20% of the
drug concentration adsorbed by iv administration. Thus, the cor-
rected peak blood concentration of 2PAMPr4PHA, 2,4DiPAM-
MeBHA and 2PAMMeBHA following im injection may reach the
maximal level of ca. 130, 260, or 180 (for 25 mg/kg, im) or 360 and
200 or 400 mM (for 50 mg/kg, im), respectively. These estimated
in vivo blood levels of hybrids are significantly higher than the IC50
values measured for AChE inhibition by 2PAMPr4PHA, 2,4DiPAM-
MeBHA and 2PAMMeBHA, 29, 48 and 16 mM, respectively, (Table 4).
Therefore, it is conceivable that significant reversible AChE inhibi-
tion by the oxime-hydroxamate hybrids may indeed occur during
the in vivo antidotal studies using either 25 or 50 mg/kg, im as
injected doses.
Consequently, reversible inhibition of AChE by oxime-
hydroxamate hybrids may indicate an additional mechanism of
action by these hybrids, expressed by protection of AChE that slows
down the irreversible inhibition by OP NAs. This putative mecha-
nism resembles the effect of pyridostigmine, used as pre-treatment
of NA poisoning [47]. This “pyridostigmine-like” effect could slow
down the rate of irreversible ChE inhibition in vivo during the early
critical stage of poisoning by OP NAs (as demonstrated by the
in vitro data in Fig. 4, Table 5). Maintaining partial AChE activity in
the cholinergic synapses at the initial period of intoxication may
assist in overcoming the massive cholinergic intoxication crisis
caused by irreversible inhibition of AChE by OP NAs. The protection
of AChE from irreversible phosphorylation provides another anti-
dotal mechanism, especially in the pre-treatment drug regimen. It
thus provides an added value to both direct scavenging of OP NAs
and reactivation of OP-inhibited AChE exerted by the oxime-
hydroxamate hybrids as indeed demonstrated by the in vivo effi-
cacy data (section 3.8).
Fig. 3. Concentration-dependence of reversible HuAChE inhibition by oxime-
hydroxamates and 2PAM (A) and the quaternary pyridinium oximes HI-6, HLo-7,
Toxognin, TMB-4, MMB-4 and 4PAM (B) (mean s.e.m., n ¼ 3, some s.e.m. values are
smaller than the size of the graphic symbols).
Table 4
Summary of IC50 values for the reversible inhibition of AChE by oxime-hydroxamate
hybrids, mono-pyridinium oximes and bis-pyridinium oximes.
Compound
IC50 (95% confidence intervals) (mM)
2PAMMeBHA
2PAMPr4PHA
2,4DiPAMMeBHA
2PAM
HI-6
HLo-7
TMB-4
MMB-4
Toxogonin
4PAM
16 (11e24)
29 (24e35)
48 (35e66)
34 (19e59)a
11 (3e40)
104 (21e215)
26 (13e52)a
90 (26e309)a
71 (45e112)a
88 (36e211)a
3.6. In vivo toxicity of oxime-hydroxamate hybrids in rats and
guinea pigs
Table 6 summarizes the toxicity (LD50, mmol/kg and mg/kg, im)
of the oxime-hydroxamate hybrids in rats and guinea pigs. Table 7
summarizes the acute toxicity of known bisquaternary oximes HLo-
7, HI-6, MMB-4, Toxogonin (obidoxime) and TMB-4. Since 2PAM
moiety is part of the hybrids chemical structure, the LD50 of 2PAM
was added to Table 6 [42e44] and used as a reference oxime in the
antidotal efficacy studies with the 2PAM-based oxime-
hydroxamate hybrids. Following im injection of specified doses of
the tested compounds, animals were observed for signs of
intoxication and 24 h survival rate was monitored for calculating
the LD50 values. Body weights and general behavior of animals
were monitored until 14 days after drug administration (not
shown). Each drug was first administered at a dose of 50 mg/kg
(im) followed by the up-and-down process. Effects of toxicity and
mortality rate were observed 24 h post dosing. Surviving animals
demonstrated normal general behavior and weight gain was very
similar to control animals during the 14-day observation period
(not shown). The hybrid 2PAMPr4PHA displayed no toxic signs
when administered up to 200 mg/kg (im). We did not increase the
injected dose above 200 mg/kg since it was suspected that im in-
jection of compounds at hyper-osmomolar concentration, will
cause local muscular damage at the site of injection. Therefore, to
avoid unnecessary pain the estimated LD50 of 2PAMPr4PHA im in
rats was >200 mg/kg (Table 6). The up-and-down procedure with
a
These oximes caused less than 50% AChE inhibition at the highest tested con-
centration (1 mM), therefore, their apparent IC50 values are related to a reduced
inhibition scale.
In order to evaluate the relevance of the IC50 values of AChE
inhibition by oxime-hydroxamate to in vivo scenarios, we esti-
mated the expected peak blood concentrations of oxime-
hydroxamate hybrids during treatment. Based on the LD50 value
of both 2PAMPr4PHA and 2,4DiPAMMeBHA in rats >200 mg/kg,
(im) (section 3.6), and the LD50 of 2PAMMeBHA e 179 mg/kg, (im)
(section 3.6), we selected 50 and 25 mg/kg as representative doses
for the antidotal studies in rats (section 3.8). The total blood volume
of rats that weigh 350 gr is ~24.5 ml (~7% of body weight), and the
doses of 25 and 50 mg/kg were transformed to 8.75 and 17.5 mg per
rat, respectively. Assuming that the injected volume of drug solu-
tion was completely absorbed in blood like in a bolus iv adminis-
tration, then the anticipated maximal blood concentrations due to
iv injection are 0.36 and 0.72 mg/ml, respectively. After changing
Please cite this article in press as: G. Amitai, et al., Novel bifunctional hybrid small molecule scavengers for mitigating nerve agents toxicity,
Chemico-Biological Interactions (2016), http://dx.doi.org/10.1016/j.cbi.2016.04.036

G. Amitai et al. / Chemico-Biological Interactions xxx (2016) 1e18
9
Fig. 4. Time-course of HuAChE inhibition by sarin (A), cyclosarin (B), soman (C) and VX (D) in the presence of various concentrations of 2PAMMeBHA and 2,4DiPAMMeBHA. HuAChE
(1.5 U/ml) was pre-incubated for 1 min in buffer (OP NA only) or with the hybrids at various concentrations (2.5, 5 and 10 ꢀ 10ꢁ5 M)) and then the OP inhibitor was added (at final
concentration of 20 nM except cyclosarin 10 nM) (50 mM phosphate, pH ¼ 7.4, 23 ꢂC) (mean s.e.m, n ¼ 3).
Table 5
Protection coefficients (kp, M*min) calculated from the slopes of the double reciprocal plots of 1/kobs vs. 1/[hybrid] (not shown) processed from the concentration dependence
of the rate of AChE inhibition by VX, sarin, cyclosarin and soman in the presence of oxime-hydroxamate hybrid and 2PAM. Data of AChE protection by the hybrids 2PAMMeBHA
and 2,4DiPAMMeBHA against sarin, cyclosarin soman and VX originated from Fig. 4AeD, respectively (50 mM phosphate, pH ¼ 7.4, 23 ꢂC).
OP agent
kp
2PAMMeBHA
2,4DiPAMMeBHA
2PAMPr4PHA
2PAM
[ꢀ10ꢁ5 (M*min)]
[ꢀ10ꢁ5 (M*min)]
[ꢀ10ꢁ5 (M*min)]
[ꢀ10ꢁ5 (M*min)]
VX
Sarin
Cyclosarin
Soman
23
52
12
31
25.5
38
5.3
1.6
0.3
1.8
4.0
1.3
2.3
3.5
18.5
36
2PAMPr4PHA in guinea pigs provided LD50 value of 80 mg/kg
(Table 6). The LD50 values obtained for the hybrid 2PAMMeBHA
179 mg/kg in rats and 113 mg/kg in guinea pigs indicate lower
toxicity of this hybrid compared to 2PAM in rats and higher toxicity
than 2PAM in guinea pigs (Table 6). Notably, the LD50 values in rats
obtained for both 2PAMMeBHA and 2PAMPr4PHA are higher than
Please cite this article in press as: G. Amitai, et al., Novel bifunctional hybrid small molecule scavengers for mitigating nerve agents toxicity,
Chemico-Biological Interactions (2016), http://dx.doi.org/10.1016/j.cbi.2016.04.036

10
G. Amitai et al. / Chemico-Biological Interactions xxx (2016) 1e18
Table 6
pretreatment was dose-dependent. Thus, blood ChE levels were 25,
37 and 42% caused by 2PAMPr4PHA at 42, 84 and 142 mol/kg,
respectively (3 h post sarin exposure Fig. 5A). The hybrid 2PAM-
MeBHA (142 mol/kg) caused 22% regeneration level of blood ChE
within 3 h, that was lower than 2PAMPr4PHA (42%) but higher than
that induced by 2PAM (15%) at equimolar dose (Fig. 5A).
The time-course of in vivo ChE regeneration in the post-
treatment regimen is described in Fig. 5B. Regeneration of blood
ChE activity by 2PAMPr4PHA was most pronounced at a dose of
LD50 values of the oxime-hydroxamate hybrids in rats and guinea pigs determined
by the up and down method [36] (mg/kg, im).
m
Compound
LD50, Rats, im
LD50, Guinea pigs, im
mg/kg, (mmole/kg)
m
mg/kg, (
m
mole/kg)
2PAMPr4PHA
2PAMMeBHA
2,4DiPAMMeBHA
2PAM
>200
(>568)
179
(508)
>200
(>506)
80
(144)
113
(203)
>200
(>506)
168 (43); 185 (44)
(974, 1072)
142
20% caused either by 2PAMMeBHA or 2PAM at equimolar dose
(142 mol/kg). The higher regeneration rate induced by
mmol/kg (45e50% reactivation between 1 and 6 h) compared to
150 (42)
(869)
m
2PAMPr4PHA in the post-treatment regimen could be ascribed to
its higher reactivation rate of sarin-inhibited rat blood ChE. By
contrast, our in vitro reactivation data with sarin-inhibited human
AChE displayed slower reactivation of sarin-HuAChE by 2PAMPr4-
PHA than 2PAM. (Tables 2 and 3). This apparent discrepancy may
reflect different reactivation rates of human AChE (used in our
in vitro studies) compared to whole blood ChE (containing both
AChE and BChE) in rats and guinea pigs [48].
The hybrid-induced regeneration of whole blood ChE following
sarin poisoning in vivo could be ascribed to both reactivation of
sarin-inhibited ChE as well as direct degradation of sarin by the
hydroxamate moiety of the hybrid. Transient protection of AChE
active sites by oxime-hydroxamate hybrids from irreversible inhi-
bition by sarin (as was shown by the in vitro data in Figs. 3A and 4A,
Tables 4 and 5) may also contribute to slower inhibition by sarin
in vivo and may therefore elicit a more rapid recovery in blood ChE
activity during the initial period of poisoning.
Table 7
Acute toxicity of known oximes in rats (LD50, im).
Oxime
LD50, im
(mg/kg) (ref)
HLo-7 DMS
HI-6 2Clꢁ
845 [45a]
816 [23,45a]
642 [45d]
172 [45a,b,c]
123 [45b,c]
MMB-4 2Clꢁ
Toxogonin 2Clꢁ
TMB-4 2Brꢁ
those obtained in guinea pigs. By contrast, the same relative low
toxicity was observed for 2,4DiPAMMeBHA in both rats and guinea
pigs (Table 6). In addition, the LD50 of the hybrids 2-PAMPr4PHA
and 2PAMMeBHA in rats are equal or higher than toxogonin, 2-PAM
and TMB-4 but lower than the LD50 of the oximes MMB-4, HLo-7
and HI-6 (Tables 6 and 7). The relatively low toxicity of the hybrids
2PAMMeBHA and 2,4DiPAMMeBHA in rats and guinea pigs are
consistent with previously reported low toxicity of benzhy-
droxamic acid (BHA) derivatives [46].
3.8. Antidotal efficacy against nerve agents poisoning
The antidotal efficacy data in rats are summarized in
Tables 8e11 and Fig. 6 and the efficacy data in guinea pigs are
summarized in Tables 12e15, and Fig. 7. All treatments with the
hybrid compounds 2PAMMeBHA, 2PAMPr4PHA and 2,4DiPAM-
MeBHA and 2PAM (used as reference oxime) were performed in
3.7. In vivo regeneration of whole blood ChE following sarin
poisoning
conjunction with atropine (30
mmol/kg, im, except for VX treatment
Whole blood ChE was used as a biomarker to study the phar-
macodynamics (PD) of the oxime-hydroxamate hybrids and 2PAM
by measuring their efficacy to regenerate ChE activity following
exposure to a sub-lethal dose of sarin (0.8xLD50). These experi-
ments were also used for dose-range finding of compounds for the
subsequent antidotal efficacy studies (section 3.8). This experi-
mental setup, using 0.8xLD50 sarin, was developed in order to
enable monitoring the efficacy of the hybrid compounds in
regenerating blood ChE activity in surviving animals within 24 h.
All experiments were performed in conjunction with atropine
7.5 mol/kg in rats, Tables 8e11, and 45 m
m
mol/kg in guinea pigs,
Tables 12e15), when given either as pre-treatment (15 min before
OP challenge) or post-treatment (1 min after OP challenge) against
poisoning by the NAs sarin, cyclosarin, VX and soman. In the post-
exposure treatment regimen against soman poisoning, pyridostig-
mine bromide (0.1 mg/kg, im) was used as pre-treatment in rats
and guinea pigs.
2PAM was used as
a reference oxime since all oxime-
hydroxamate hybrids include 2PAM moiety in their structure.
Thus, it was of interest to study the added value for the antidotal
efficacy contributed by the hydroxamate moiety covalently coupled
to 2PAM. In addition, the main goal of our antidotal studies was to
compare the efficacy of the hybrids 2PAMMeBHA, 2PAMPr4PHA
and 2,4DiPAMMeBHA between themselves and relatively to 2PAM
rather than differentiate their efficacy from that of atropine alone.
Thus, the doses of atropine used in rats and guinea pigs were
selected based on preliminary dose-range finding experiments (not
shown) in such a manner that the antidotal efficacy of atropine
alone will not mask the effect of the hybrids and 2PAM. Previous
studies in our laboratory using atropine alone at equivalent doses,
indeed yielded protection ratios (PR) that were significantly lower
than those presented in Tables 8e15 for the treatment of OP NAs-
poisoned rats and guinea pigs using the oxime-hydroxamate hy-
brids and 2PAM in combination with atropine. For instance, the
(3
m
mol/kg, im).
Fig. 5 demonstrates the time-course of blood ChE regeneration
following pretreatment (Fig. 5A) and post-treatment (Fig. 5B) by
2PAMPr4PHA (42, 84 and 142 mol/kg, im), 2PAMMeBHA (71 and
142 mol/kg im) and 2PAM (71 and 142 mol/kg, im) in rats
m
m
m
exposed to 0.8xLD50 sarin. A significantly higher ChE activity of
25e47% was regained within 0.5e1 h, following pretreatment with
2PAMPr4PHA (42, 84 and 142
mmol/kg) compared to 2PAM (5e10%,
142 mol/kg) (Fig. 5A). Notably, a higher regeneration level of ChE
m
activity induced by 2PAMPr4PHA relative to 2PAM was achieved
0.5e3 h post sarin exposure at a 3-fold lower dose than 2-PAM
(42
ChE level (42%) 3 h following pretreatment by 2PAMPr4PHA at
142 mol/kg was ~3-fold higher than that obtained with pretreat-
ment by 2PAM (142 mole, 15%) (Fig. 5A). The spontaneous
mmol/kg vs. 142 mmol/kg, respectively, Fig. 5A). Further, blood
m
m
treatment with atropine only (50 mmol/kg, im,1.67-fold higher than
regeneration of blood ChE in the presence of atropine alone was 5%
within 6 h (Fig. 5A). ChE regeneration by 2PAMPr4PHA given as
the dose employed in the studies reported here) provided post-
treatment protection against exposure to 1.1e1.2xLD50 of sarin
Please cite this article in press as: G. Amitai, et al., Novel bifunctional hybrid small molecule scavengers for mitigating nerve agents toxicity,
Chemico-Biological Interactions (2016), http://dx.doi.org/10.1016/j.cbi.2016.04.036

G. Amitai et al. / Chemico-Biological Interactions xxx (2016) 1e18
11
Fig. 5. Time-course of in vivo regeneration of blood ChE in rats following pretreatment with 2PAMPr4PHA (142, 83 and 42
mmol/kg) 2PAMMeBHA (83 and 42
mmol/kg), and 2PAM
(143 and 72 mmol/kg) in conjunction with atropine (3 mmol/kg) administered im in rats15 min before [A] and 1 min post-exposure [B] to sarin (0.8xLD50, 80 mg/kg, i.m.). Fractional
ChE activity is % of control whole blood ChE activity at time ¼ 0 measured for each animal before starting the experiment. Relative ChE activity is presented as mean s.e.m., n ¼ 6
per group.
Table 8
LD50 values of sarin and protection ratios (PR) of 2PAM, 2PAMMeBHA, 2PAMPr4PHA and 2,4DiPAMMeBHA in pre-treatment (PRE) and post-treatment (POST) regimens against
sarin poisoning in rats. All compounds were administered at 142
without treatment.
mmol/kg in conjunction with atropine 30
m
mol/kg, i.m. Protection ratio (PR) ¼ LD50 with treatment/LD50
a,b
LD50 of Sarin (
(In conjunction with atropine 30
(Values in parentheses are 95% confidence limits)
m
g/kg, im) with various treatments in rats
m
mol/kg, im)
Hybrid/oxime
PREa
2PAM
2PAMMeBHA
2PAMPr4PHA
2,4DiPAMMeBHA
604
1261
1278
512
(428e855) n ¼ 14
(993e1601)
n ¼ 8
(942e1734)
n ¼ 8
(441e594)
n ¼ 14
4.7
PR
5.5
11.5
11.6
(3.9e7.8)
288
(9e14.6)
302
(8.6e15.8)
290
(4.0e5.4)
POSTb
192b
(268e309)
n ¼ 17
3.2
(279e327)
n ¼ 11
3.4
(267e314)
n ¼ 9
(173e214)
n ¼ 10
2.1
PR
3.2
(3.0e3.4)
(3.1e3.6)
(3.0e3.4)
(1.9e2.4)
a
Sarin LD50 ¼ 110
m
g/kg (im) (weight of rats used in pre-treatment 310e360 gr).
b
Sarin LD50 ¼ 90
mg/kg (im) (weight of rats used in post-treatment 380e420 gr).
and cyclosarin in rats (GA personal communication).
3.4 and 3.2, respectively, whereas 2,4DiPAMMEBHA yielded a PR
value of 2.1 (Table 8, Fig. 6). These in vivo antidotal PRs against
sarin poisoning obtained for 2PAM and the hybrids are in good
agreement with the in vivo regeneration rates of whole blood
3.8.1. Antidotal efficacy against nerve agents poisoning in rats
3.8.1.1. Sarin. Table 8 and Fig. 6 demonstrate the LD50 values of
sarin and the protection ratios (PR) obtained with treatment by
2PAM, 2PAMMeBHA, 2PAMPr4PHA and 2,4DiPAMMeBHA in rats.
The most efficacious hybrids in the pre-treatment regimen were
2PAMMeBHA and 2PAMPr4PHA with PR values 11.5 and 11.6,
respectively. These PR values were significantly higher than
those obtained with 2PAM and 2,4DiPAMMeBHA, 5.5 and 4.7,
respectively (Table 8, Fig. 6). In the post-treatment regimen
2PAM, 2PAMMeBHA and 2PAMPr4PHA yielded similar PRs, 3.2,
ChE in rats exposed to sub-lethal dose of sarin (0.8xLD50
(Fig. 5).
)
3.8.1.2. Cyclosarin. Using the pre-treatment mode against cyclo-
sarin poisoning, the highest PR value 4.6 was obtained for
2PAMPr4PHA (Table 9, Fig. 6). The relatively large PR value obtained
for 2PAMPr4PHA against cyclosarin poisoning in rats correlates
well with its superior reactivation rate of cyclosarin-inhibited
Please cite this article in press as: G. Amitai, et al., Novel bifunctional hybrid small molecule scavengers for mitigating nerve agents toxicity,
Chemico-Biological Interactions (2016), http://dx.doi.org/10.1016/j.cbi.2016.04.036

12
G. Amitai et al. / Chemico-Biological Interactions xxx (2016) 1e18
Table 9
LD50 values of cyclosarin and protection ratios (PR) of 2PAM, 2PAMMeBHA, 2PAMPr4PHA and 2,4DiPAMMeBHA in pre-treatment (PRE) and post-treatment (POST) regimens
against cyclosarin poisoning in rats. All compounds were administered at 142
treatment/LD50 without treatment.
m
mol/kg in conjunction with atropine 30
mmol/kg, i.m. Protection ratio (PR) ¼ LD50 with
LD50 of cyclosarin (
(In conjunction with atropine 30
(Values in parentheses are 95% confidence limits)
m
g/kg, im) with various treatments in ratsa
mol/kg, im)
m
Hybrid/oxime
PRE
2PAM
2PAMMeBHA
2PAMPr4PHA
621
2,4DiPAMMeBHA
171
231
255
(160e182)
n ¼ 10
1.3
(210e252)
n ¼ 8
(475e812) n ¼ 20
(233e279)
n ¼ 11
1.9
PR
1.7
4.6
(1.2e1.4)
184
(1.6e1.9)
255
(3.5e6.1)
266
(1.7e2.1)
195
POST
(160e211)
n ¼ 8
(233e279)
n ¼ 12
1.9
(232e307)
n ¼ 12
2.0
(166e229)
n ¼ 10
1.5
PR
1.4
(1.2e1.6)
(1.7e2.1)
(1.7e2.3)
(1.2e1.7)
a
LD50 of Cyclosarin in rats (i.m.) ¼ 134
mg/kg.
Table 10
LD50 values of VX and protection ratios (PR) of 2PAM, 2PAMMeBHA, 2PAMPr4PHA and 2,4DiPAMMeBHA in pre-treatment (PRE) and post-treatment (POST) regimens against
VX poisoning in rats. All compounds were administered at 142
treatment.
mmol/kg in conjunction with atropine 7.5
mmol/kg, i.m. Protection ratio (PR) ¼ LD50 with treatment/LD50 without
LD50 of VX (
(In conjunction with atropine 7.5
(Values in parentheses are 95% confidence limits)
m
g/kg, im) with various treatments in ratsa
mol/kg, im)
m
Hybrid/oxime
PRE
2PAM
2PAMMeBHA
2PAMPr4PHA
2,4DiPAMMeBHA
185
216
310
114
(155e222)
n ¼ 16
(186e250)
n ¼ 14
14
(277e347)
n ¼ 14
19
(104e124)
n ¼ 8
7
PR
12
(10e14)
124
(12e16)
104
(17e22)
115
(7e8)
60
POST
(100e154) n ¼ 8
(82e132)
n ¼ 16
7
(85e155)
n ¼ 12
7
(22e160)
n ¼ 16
4
PR
8
(6e10)
(5e8)
(5e10)
(1e10)
a
LD50 of VX without treatment (im): 15 mg/kg.
Table 11
LD50 values of soman and protection ratios (PR) of 2PAM, 2PAMMeBHA, 2PAMPr4PHA and 2,4DiPAMMeBHA in pre-treatment (PRE) and post-treatment (POST) regimens
against soman poisoning in rats. All compounds were administered at 142
LD50 without treatment.
mmol/kg in conjunction with atropine 30
m
mol/kg, i.m. Protections ratio (PR) ¼ LD50 with treatment/
LD50 of Soman (
(In conjunction with atropine 30
(Values in parentheses are 95% confidence limits)
m
g/kg, im) with various treatments in ratsa
mol/kg, im)
m
Hybrid/oxime
2PAM
2PAMMeBHA
2PAMPr4PHA
2,4DiPAMMeBHA
PRE
PR
N.P.b
178
N.P.
178
N.P.
264
N.P.
180
POSTc
(161e197) n ¼ 7
(161e197)
n ¼ 7
1.8
(216e323)
n ¼ 11
2.7
(145e224)
n ¼ 6
1.8
PR
1.8
(1.6e2.0)
(1.6e2.0)
(2.2.-3.3)
(1.5e2.3)
a
LD50 of soman without treatment (im): 99 mg/kg.
b
c
N.P. ¼ No protection was observed in the pretreatment regimen against soman poisoning in rats (15 min before soman challenge without any post-treatment).
Pre-treatment with Pyridostigmine, 0.1 mg/kg (im) was administered 15 min before soman challenge followed by a post-challenge antidotal treatment.
HuAChE in vitro as demonstrated in Fig. 2 and Table 2. The oxime-
hydroxamate hybrids 2PAMMeBHA, 2PAMPr4PHA and 2,4DiPAM-
MeBHA exhibited a statistically significant larger PR than 2PAM
against cyclosarin poisoning in rats in the pre-treatment regimen:
PR ¼ 1.7, 4.6 and 1.9 vs. 1.3, respectively (Table 9, Fig. 6). The hybrid
compounds 2PAMMeBHA and 2PAMPr4PHA displayed a higher PR
value than 2PAM against cyclosarin poisoning in the post-
(Table 9, Fig. 6). The PR obtained with 2,4DiPAMMeBHA (1.5) in
post-treatment against cyclosarin was similar to 2PAM (PR ¼ 1.4,
Table 9). Notably, the hybrid 2PAMPr4PHA yielded the highest an-
tidotal PR values both in the pre-treatment (4.6) and post-
treatment regimen (2.0) against cyclosarin poisoning in rats
(Table 9, Fig. 6).
treatment regimen, with PR
¼
1.9, 2.0 vs. 1.4, respectively
3.8.1.3. VX. The PR values of the hybrid compounds and 2PAM
Please cite this article in press as: G. Amitai, et al., Novel bifunctional hybrid small molecule scavengers for mitigating nerve agents toxicity,
Chemico-Biological Interactions (2016), http://dx.doi.org/10.1016/j.cbi.2016.04.036

G. Amitai et al. / Chemico-Biological Interactions xxx (2016) 1e18
13
Fig. 6. Summary of PR values obtained for the antidotal treatment by 2PAMMeBHA, 2PAMPr4PHA and 2,4DiPAMMeBHA and 2PAM (142
mmol/kg, im) against cyclosarin, sarin VX
and soman poisoning in rats, in conjunction with atropine (30 mmol/kg, except for VX poisoning 7.5 mmol/kg, im). Pyridostigmine (0.1 mg/kg, im) was administered 15 min before
soman challenge when antidotes were evaluated in the post-treatment regimen.
Table 12
efficacy of most oximes stem from rapid aging of soman-inhibited
AChE that leads to the formation reactivation-resistant somnayl-
AChE. The addition of pyridostigmine as pre-treatment in cases of
soman poisoning contributed significantly to the antidotal efficacy
of oximes and atropine as indeed was noted previously by Von
Bredow et al. and other laboratories [47]. Neither of the new
hybrids nor 2PAM displayed any antidotal efficacy above 1e1.2
LD50 of soman when given with atropine as pre-treatment against
soman poisoning in rats (Table 11). However, when used as post-
treatment with atropine combined with pre-treatment by pyri-
dostigmine (0.1 mg/kg, im), all compounds displayed moderate
antidotal efficacy. The PRs for 2PAMMeBHA, 2PAMPr4PHA,
2,4DiPAMMeBHA and 2PAM were 1.8, 2.7, 1.8 and 1.8, respectively
(Table 11, Fig. 6). Again, the hybrid 2PAMPr4PHA yielded the
highest PR value (2.7) among all tested compounds. The PR values
obtained for 2PAMPr4PHA in rats poisoned by sarin, cyclosarin,
VX and soman that are significantly higher than those of 2PAM at
equimolar doses, indicate that this hybrid may serve as efficient
antidote against poisoning by these NAs especially in the pre-
treatment regimen.
LD50 values of sarin and protection ratios (PR) values of 2PAM, 2PAMMeBHA,
2PAMPr4PHA and 2,4DiPAMMeBHA against sarin poisoning in guinea pigs. All
compounds were administered at 86
mmol/kg except for 2PAMPrPHA at 50 mmol/kg,
in conjunction with atropine 45 mol/kg, im.
m
Sarin challenge (
g/kg, im)a preceded or followed by antidotal treatments, with
hybrid compounds and 2PAM PRE-pretreatment, POST- post treatment,
(Values in parentheses are 95% confidence limits)
m
Hybrid/oxime 2PAM
2PAMMeBHA 2PAMPr4PHA 2,4DiPAMMeBHA
PRE
>700
n ¼ 10
361
505
<400
n ¼ 8
(335e389)
n ¼ 10
9
(442e576)
n ¼ 12
13
PR
>17.5
<10
(9.0e9.7)
155
(11e14)
168
POST
185
118
(167e204) (142e170)
(156e181)
n ¼ 8
(105e131)
n ¼ 10
3.0
n ¼ 12
n ¼ 6
PR
4.6
3.9
4.2
(4.2e5.1)
(3.6e4.3)
(3.9e4.5)
(2.6e3.3)
a
LD50 of sarin without treatment in guinea pigs: 40 mg/kg (im).
against VX poisoning in rats are summarized in Table 10 and Fig. 6.
The highest PR value in the pre-treatment regimen was obtained
for 2PAMPr4PHA, PR ¼ 19, compared to 14, 7 and 12 obtained for
2PAMMeBHA, 2.4DiPAMeBHA and 2PAM, respectively (Table 10).
Although all antidotes yielded relatively high PRs compared to the
antidotal efficacy toward sarin and cyclosarin (Tables 8 and 9) the
PR obtained for 2PAMPr4PHA is exceptionally high (PR ¼ 19). In the
post-treatment regimen 2PAMMeBHA, 2PAMPr4PHA, 2,4DiPAM-
MeBHA and 2PAM yielded similar PRs: 7, 7, 4 and 8, respectively,
with overlapping 95% confidence limits (Table 10).
3.8.2. Antidotal efficacy against nerve agents poisoning in guinea
pigs
The antidotal studies in guinea pig were performed in either
pre-treatment or post-treatment regimen using the following
doses of hybrids: 2PAMMeBHA, 2,4DiPAMMeBHA, 2PAMPr4PHA
and 2PAM 85, 87, 50 and 86
used in guinea pigs are lower than those employed for the anti-
dotal studies in rats (142 mol/kg) due to their relatively higher
toxicity in guinea pigs (Table 6). In particular, a lower dose of
2PAMPr4PHA (50 mol/kg) was used in guinea pigs estimated as
its sign-free-dose in guinea pigs (not shown). The LD50 values of
sarin, cyclosarin, VX and soman: 40, 45, 10 and 25 g/kg, respec-
mmol/kg, im, respectively. The doses
m
m
3.8.1.4. Soman. The PR values for the antidotal efficacy of the
hybrids and 2PAM against soman poisoning in rats are summa-
rized in Table 11 and Fig. 6. It was previously established that
antidotal efficacy of most oximes except for HI-6 and HLo-7
against soman poisoning are limited [2]. The poor antidotal
m
tively, in guinea pigs were determined by im administration using
the up-and-down method [36]. The LD50 values of all NAs were
validated by the up-and-down method (n ¼ 3e4) prior to each
Please cite this article in press as: G. Amitai, et al., Novel bifunctional hybrid small molecule scavengers for mitigating nerve agents toxicity,
Chemico-Biological Interactions (2016), http://dx.doi.org/10.1016/j.cbi.2016.04.036

14
G. Amitai et al. / Chemico-Biological Interactions xxx (2016) 1e18
Table 13
LD50 values of cyclosarin and protection ratios (PR) values of 2PAM, 2PAMMeBHA, 2PAMPr4PHA and 2,4DiPAMMeBHA against cyclosarin poisoning in guinea pigs. All
compounds were administered at 86 mol/kg except for 2PAMPrPHA at 50 mol/kg, in conjunction with atropine 45 mol/kg, im.
m
m
m
Cyclosarin challenge (
g/kg, im)a preceded or followed by antidotal treatments,
m
PRE-pretreatment, POST- post treatment with hybrid compounds and 2PAM
(Values in parentheses are 95% confidence limits)
Hybrid/Oxime
PRE
2PAM
2PAMMeBHA
2PAMPr4PHA
2,4DiPAMMeBHA
70
165
153
119
(65e75)
n ¼ 18
1.6
(124e219)
n ¼ 18
3.7
(126e186)
n ¼ 16
3.4
(94e157)
n ¼ 10
2.6
PR
(1.4e1.7)
90
(2.8e4.9)
70
(2.8e4.1)
149
(2.1e3.5)
84
POST
(78e210) n ¼ 6
(58e85)
n ¼ 8
(110e201)
n ¼ 19
3.3
(70e102)
n ¼ 6
PR
2.0
1.6
1.9
(1.7e4.1)
(1.3e1.9)
(2.4e4.5)
(1.6e2.3)
a
LD50 of cyclosarin without treatment in guinea pigs: 45 mg/kg (im).
Table 14
LD50 values of VX and protection ratios (PR) values of 2PAM, 2PAMMeBHA, 2PAMPr4PHA and 2,4DiPAMMeBHA against VX poisoning in guinea pigs. All compounds were
administered at 86 mol/kg except for 2PAMPrPHA at 50 mol/kg, in conjunction with atropine 45 mol/kg, im.
m
m
m
VX challenge (
g/kg, im)a preceded or followed by antidotal treatments, with hybrid compounds and 2PAM PRE-pretreatment, POST- post-treatment,
m
(Values in parentheses are 95% confidence limits)
Hybrid/oxime
PRE
2PAM
2PAMMeBHA
2PAMPr4PHA
2,4DiPAMMeBHA
196
111
46
79
(140e276) n ¼ 10
(105e117) n ¼ 10
(35e62)
n ¼ 8
5
(57e111)
n ¼ 8
8
PR
20
11
(14e28)
183
(11e12)
112
(4e6)
79
(6e11)
116
POST
(135e246) n ¼ 8
(87e145)
n ¼ 6
11
(61e104)
n ¼ 4
8
(79e172)
n ¼ 6
12
PR
18
(14e25)
(9e15)
(6e10)
(8e17)
a
LD50 of VX in guinea pigs:10 mg/kg (im).
Table 15
LD50 values of soman and protection ratios (PR) values of 2PAM, 2PAMMeBHA, 2PAMPr4PHA and 2,4DiPAMMeBHA against soman poisoning in guinea pigs. All compounds
were administered at 86 mol/kg except for 2PAMPrPHA at 50 mol/kg, in conjunction with atropine 45 mol/kg, im.
m
m
m
Soman challenge (
g/kg, im)a preceded or followed by antidotal treatments, with hybrid compounds and 2PAM PRE-pretreatment, POST- post treatment, (Values in
m
parentheses are 95% confidence limits)
Hybrid/oxime
PRE
2PAM
2PAMMeBHA
2PAMPr4PHA
2,4DiPAMMeBHA
57
52
58
73
(51e63)
n ¼ 11
(46e59)
n ¼ 12
(43e59)
n ¼ 15
2.3
(55.6e95.7)
n ¼ 13
2.9
PR
2.3
2.1
(2.0e2.5)
94
(1.9e2.4)
83
(1.7e2.4)
105
(2.2e3.8)
105
POSTb
(63e140) n ¼ 12
(63e110) n ¼ 12
(91e121)
n ¼ 12
4.2
(91e121)
n ¼ 12
4.2
PR
3.8
3.3
(2.5e5.6)
(2.5e4.4)
(3.6e4.8)
(3.6e4.8)
Hybrid/Oxime
POST
2PAM
2PAMMeBHA
2PAMPr4PHA
2,4DiPAMMeBHA
e
e
59
e
(43.9e78.7)
n ¼ 10
2.3
PR
N.P.c
N.P.c
N.P.c
(1.8e3.1)
a
LD50 of soman without treatment in guinea pigs was 25
m
g/kg (im).
All antidotal post-treatments were carried out with pre-treatment by pyridostigmine 0.1 mg/kg, im administered 15 min before soman challenge.
N.P. ¼ No Protection was observed when animals were exposed to 1.5e1.6xLD50 soman (35e40 g/kg) when the post-treatment with 2PAM, 2PAMMeBHA 2,4DiPAM-
b
c
m
MeBHA in conjunction with atropine was given without pre-treatment by pyridostigmine.
antidotal experiment. The dose of atropine (45
conjunction with oxime-hydroxamates in guinea pigs was deter-
mined experimentally by a dose-range finding study (not shown).
m
mol/kg) used in
Pertinently, it was reported by other laboratories that atropine
used against NAs in guinea pigs is administered at relatively
higher doses than in other species [38]. All PR mean values with
Please cite this article in press as: G. Amitai, et al., Novel bifunctional hybrid small molecule scavengers for mitigating nerve agents toxicity,
Chemico-Biological Interactions (2016), http://dx.doi.org/10.1016/j.cbi.2016.04.036

G. Amitai et al. / Chemico-Biological Interactions xxx (2016) 1e18
15
Fig. 7. Compilation of PR values obtained for the antidotal treatment by 2PAMMeBHA, 2,4DiPAMMeBHA and 2PAM (86
mmol/kg, im) and 2PAMPr4PHA (50
mmol/kg, im) against
cyclosarin, sarin VX and soman poisoning in guinea pigs, in conjunction with atropine (45
challenge when antidotes were evaluated in the post-treatment regimen.
mmol/kg, im). Pyridostigmine (0.1 mg/kg, im) was administered 15 min before soman
their respective 95% confidence limits obtained for the tested
compounds against sarin, cyclosarin, VX and soman poisoning in
guinea pigs are summarized in Tables 12e15 and Fig. 7. Statisti-
cally significant PR values were defined as those with no overlap
in their 95% confidence limits [37].
2PAMPr4PHA and 2,4DiPAMMeBHA in conjunction with atropine
against cyclosarin poisoning in guinea pigs. The PR values in pre-
treatment mode were: 1.6, 3.7, 3.4 and 2.6, respectively,
compared to 2.0, 1.6, 3.3 and 1.9, respectively, in the post treatment
mode (Table 13, Fig. 7). Notably, the highest PR values 3.7 and 3.4
were obtained for 2PAMMeBHA and 2PAMPr4PHA, respectively, in
the pre-treatment mode (Table 13, Fig. 7). The relatively high PR
obtained for 2PAMMeBHA (3.7) in the pre-treatment mode could
be corroborated by our previous finding of high detoxification rate
in vitro demonstrated by 2PAMMeBHA toward cyclosarin
(kobs ¼ 0.244 minꢁ1, Table 1). Noteworthy, the only compound that
yielded relatively high PR against cyclosarin in the post-treatment
mode (PR ¼ 3.3) was 2PAMPr4PHA (Table 13, Fig. 7). This result may
stem from high reactivation potency by 2PAMPr4PHA toward
cyclosarin-inhibited AChE as indeed demonstrated in Table 2. In
addition, the lower PR values obtained against cyclosarin poisoning
in guinea pigs compared to those in rats (Table 9) may stem from
using significantly lower doses of hybrids in guinea pigs than those
in rats. The relatively high PR obtained with 2PAMPr4PHA in post-
treatment against cyclosarin (PR ¼ 3.3, Table 13), in spite of its
3.8.2.1. Sarin. Table 12 and Fig. 7 summarize the LD50 values and
protection ratios (PR) obtained for 2PAM, 2PAMMeBHA,
2PAMPr4PHA and 2,4DiPAMMeBHA, in conjunction with atropine,
against sarin poisoning in guinea pigs. All test compounds were
significantly more effective in pre-treatment than in post-
treatment mode. The PR values in pre-treatment were: >17.5, 9,
13 and >10, respectively, compared to post treatment mode PRs 4.6,
3.9, 4.2 and 3.0, respectively (Table 12, Fig. 7). The highest PR values
>17.5 (pre) and 4.6 (post) were obtained for 2PAM (Table 12, Fig. 7).
These results may be attributed to relatively high reactivation po-
tency of 2PAM compared to the hybrids toward sarin-inhibited
AChE in vitro (Table 2). Relatively high PR values i.e. 9, 13 and
>10, were obtained for 2PAMMeBHA, 2PAMPr4PHA and
2,4DiPAMMeBHA in the pre-treatment mode (Table 12), indicating
that the direct nucleophilic attack of these compounds on sarin
before it reached ChE in tissues was probably more prevalent than
reactivation of sarin-AChE. The superior PRs obtained by pre-
treatment compared to post-treatment may also indicate a pro-
tective effect on AChE against sarin inhibition (see the relatively
high protection coefficients (kp) of 2PAMMeBHA and 2,4DiPAM-
MeBHA against inhibition by sarin in Table 5). All PR values ob-
tained in this study for the hybrids as pre-treatment against sarin
poisoning in guinea pigs are similar to those obtained in rats
(Tables 8 and 12).
lower administered dose (50 mmol/kg), emphasizes its unique an-
tidotal efficacy against cyclosarin poisoning in guinea pigs. Gener-
ally, the efficacy of all tested hybrids against cyclosarin were lower
than those obtained against sarin poisoning in the pre-and post-
treatment modes in guinea pigs (Tables 12 and 13). These results
indicate that antidotal treatment of cyclosarin poisoning is prob-
ably more challenging than that of sarin poisoning. Notably, all the
PR values obtained for the hybrids 2PAMMeBHA, 2PAMPr4PHA and
2,4DiPAMMeBHA in the pretreatment mode (PR ¼ 3.7 (2.8e4.9), 3.4
(2.8e4.1) and 2.6 (2.1e3.5), were significantly higher than 2PAM
(PR ¼ 1.56 (1.4e1.7) (Table 13).
3.8.2.2. Cyclosarin. Table 13 and Fig. 7 summarize the LD50 values
of cyclosarin and PRs obtained for 2PAM, 2PAMMeBHA,
3.8.2.3. VX. Table 14 and Fig. 7 summarize the LD50 values of VX
Please cite this article in press as: G. Amitai, et al., Novel bifunctional hybrid small molecule scavengers for mitigating nerve agents toxicity,
Chemico-Biological Interactions (2016), http://dx.doi.org/10.1016/j.cbi.2016.04.036

16
G. Amitai et al. / Chemico-Biological Interactions xxx (2016) 1e18
and PRs obtained for 2PAM, 2PAMMeBHA, 2PAMPr4PHA and
2,4DiPAMMeBHA in conjunction with atropine against VX
poisoning in guinea pigs. The PR values were: 20, 11, 5 and 8,
respectively, in pre-treatment mode and 18, 11, 8 and 12, respec-
tively, in the post-treatment mode (Table 14, Fig. 7). Notably, the
PR values obtained for the hybrid compounds as post-treatment
against VX poisoning were similar or higher than those obtained
in the pre-treatment mode. This result may arise from a relatively
long onset time of toxic signs (few hours) caused by VX poisoning
compared to a shorter onset elicited by the G-agents sarin and
cyclosarin (minutes). In summary, all hybrids yielded relatively
high PR values (PRs range at 5e12) against VX poisoning in guinea
pigs in both pre- and post-treatment regimens (Table 14).
Transparency document
Transparency document related to this article can be found
online at http://dx.doi.org/10.1016/j.cbi.2016.04.036.
Appendix A. Syntheses
This supplementary material appendix contains the details of
synthetic procedures of the oxime-hydroxamate hybrids. The
starting materials 2-pyridine aldoxime, 1,3-dibromopropane, 4-
bromomethylbenzoic acid and hydroxylamine hydrochloride
were purchased from Sigma-Aldrich. All organic solvents were of
the highest available purity and purchased from Merck.
3.8.2.4. Soman. Table 15 and Fig. 7 summarize the LD50 values of
soman and PR values obtained for the oxime-hydroxamate hybrids
and 2PAM. All hybrids displayed PR values that are larger than 2
when applied as pre-treatment before soman exposure. These re-
sults are in contrast to no antidotal efficacy obtained with all tested
compounds in the pre-treatment regimen against soman poisoning
in rats (Table 11). Since reactivation of somanyl-AChE by the oxime
moiety of the tested hybrids is expected to be marginal, it is
conceivable that some hybrids acted like pyridostigmine by tran-
siently protecting AChE from being inhibited by soman during
in vivo poisoning in guinea pigs (see Fig. 4C demonstrating the
slowdown in AChE inhibition by soman in the presence of 2PAM-
MeBHA and 2,4DiPAMMeBHA). The PRs against soman poisoning in
the post-treatment regimen were obtained only when pyridostig-
mine was used as pre-treatment (0.1 mg/kg, im) administered
15 min before soman challenge (Table 15). Interestingly, a PR of 2.3
was obtained with 2PAMPr4PHA in conjunction with atropine as
post-treatment, even without pre-treatment by pyridostigmine
(see Table 15 extension at the bottom). The hybrids 2PAMMeBHA,
2,4DiPAMMeBHA and the reference oxime 2PAM did not yield any
antidotal efficacy without pyridostigmine as pre-treatment of so-
man poisoning in guinea pigs (designated as No Protection (N.P.) in
Table 15 footnote c).
1. Synthesis of the oxime-hydroxamate hybrid 2PAMPr4PHA (2)
(Scheme A2)
1-(2-Hydroxyiminomethylpyridinium)-3-(4-hydroxycarbamoyl
pyridinium) propyl dibromide (2PAMPr4PHA, (2).
Step 1:
1-(3-Bromopropyl)-2-hydroxyiminomethylpyridinum bromide
(3Br-Pr-2PAM, (1)) (Scheme A1).
A stirred solution of 2-pyridinealdoxime (8 gr, 65.5 mmol) and
1,3-dibromopropane (33.4 ml, 327 mmol) in acetone (60 ml) was
refluxed for 24 h. The reaction mixture was cooled to room tem-
perature and the precipitate was collected by filtration and washed
with acetone (3 ꢀ 20 ml). Acetonitrile was added to the crude
product (100 mle1 gr solid) and the boiling solution was filtered.
The filtrate was evaporated under reduced pressure to give the title
compound (1, Scheme A1) (1.3 gr, 6% yield). 1H NMR (300 MHz,
DMSO-d6)
d
13.21 (s, 1H, OH), 9.11 (d, J ¼ 5.4 Hz, 1H, aromatic H),
8.83 (s, 1H, CH]N), 8.61 (t, J ¼ 8.1 Hz, 1H, aromatic H), 8.44 (dd,
J ¼ 8.4, 1.2 Hz, 1H, aromatic H), 8.16 (app dt, J ¼ 6.0, 1.2 Hz, 1H,
aromatic H), 4.87 (t, J ¼ 7.4 Hz, 2H, NCH2), 3.67 (t, J ¼ 6.6 Hz, 2H,
CH2Br), 2.48 (m, 2H, CH2CH2Br) ppm; 13C NMR (300 MHz, DMSO-
d6)
d 148.0, 147.1, 146.4, 142.4, 128.5, 126.9, 57.6, 33.9, 31.1 ppm.
In summary, the in vivo antidotal efficacy data indicate the
advantage of the hybrid 2PAMPr4PHA as antidote over 2PAM-
MeBHA, 2,4DiPAMMeBHA and 2PAM in the pre- and post-
treatment of poisoning by sarin, cyclosarin and soman, in rats
and guinea pigs. Particularly noteworthy is the PR ¼ 2.3 obtained
in post-treatment of soman poisoning with 2PAMPr4PHA and
atropine without pre-treatment by pyridostigmine in guinea pigs
(Table 15). Further, the relatively high PR value 2.7, obtained for
2PAMPr4PHA against soman in rats following pre-treatment by
pyridostigmine compared to PR ¼ 1.8 for all other compounds
(Table 11), generates an advantage to this hybrid compared to all
other tested compounds. In VX poisoning, 2PAMPr4PHA, 2PAM-
MeBHA and 2,4DiPAMMeBHA yielded relatively high PR values
i.e. PR ¼ 5e11 in pre-treatment and 8e12 in post-treatment
regimen in guinea pigs (Table 14), and 7e19 and 4e7, respec-
tively, in rats (Table 10). These PR values are higher than those
obtained against poisoning by the G-agents sarin, cyclosarin and
soman in guinea pigs (Fig. 7). Apparently, 2PAM yielded higher
PRs than the hybrids toward VX poisoning in guinea pigs but
these differences in the post-treatment mode were statistically
insignificant (Fig. 7).
Scheme A1. Chemical structure of the intermediate compound 1-(3-Bromopropyl)-2-
hydroxyiminomethylpyridinum bromide (3Br-Pr-2PAM) (1).
Step 2:
A
solution
of
1-(3-bromopropyl)-2-
hydroxyiminomethylpyridinum bromide (1) (1.1 gr, 3.4 mmol)
and 4-pyridinehydroxamic acid (0.51 gr, 4.2 mmol) in acetonitrile
(80 ml) was refluxed 6 h. The crystalline crude product was
collected by filtration and washed with boiling acetonitrile
(100 ml). The title compound (2) (Scheme A2) was obtained as a
white solid (80 mg). 1H NMR (500 MHz, DMSO-d6)
d 13.28 (s, 1H,
C]NOH), 12.92 (s, 1H, OH), 9.08 (m, 3H, aromatic H), 8.72 (s, 1H,
N]CH), 8.61 (t, J ¼ 7.8 Hz, 1H, aromatic H), 8.49e8.47 (m, 2H, N]
OH and aromatic H), 8.31 (d, J ¼ 6.6 Hz, 2H, aromatic H), 8.17 (t,
J ¼ 6.8 Hz, 1H, aromatic H), 4.87 (t, J ¼ 7.5 Hz, 2H, CH2), 4.77 (t,
J ¼ 7.4 Hz, 2H, CH2), 2.57 (m, 2H, CH2) ppm. 13C NMR (300 MHz,
Funding
DMSO-d6)
d 149.6, 148.2, 146.8, 146.4, 146.1, 142.4, 128.5, 126.7,
Supported by US DoD DTRA HDTRA1-C-12-0008.
125.3, 58.0, 55.6, 32.2 ppm.
Please cite this article in press as: G. Amitai, et al., Novel bifunctional hybrid small molecule scavengers for mitigating nerve agents toxicity,
Chemico-Biological Interactions (2016), http://dx.doi.org/10.1016/j.cbi.2016.04.036

G. Amitai et al. / Chemico-Biological Interactions xxx (2016) 1e18
17
Scheme A2. The chemical structure of 1-(2-hydroxyiminomethylpyridinium)-3-(4-
hydroxycarbamoylpyridinium)propyl dibromide 4-aminopyridine salt (2-PAMPr4-
PHA, (2)).
Scheme A4. The chemical structure of 1-(4-Hydroxycarbamoyl-benzyl)-2-(hydrox-
yimino-methyl)-pyridinium bromide (2PAMMeBHA (6)).
Step 3:
1-(4-Hydroxycarbamoyl-benzyl)-2-(hydroxyimino-methyl)-
pyridinium bromide (2PAMMeBHA (6) (Scheme A4): A mixture of
4-Bromomethylbenz-hydroxamic acid (5) (Scheme A3), 1.6 gr,
6.96 mmol) and 2-pyridine aldoxime (SigmaeAldrich, USA)
(0.85 gr, 6.96 mmol) in acetone (30 ml) was refluxed for 6.5 h,
diluted with acetone (100 ml) and filtered. The obtained solid was
washed with hot acetone to give the title compound (6, Scheme A4)
2. Synthesis of the oxime-hydroxamate hybrid 2PAMMeBHA (6,
Scheme A4)
(0.177 gr). 1H NMR (500 MHz, DMSO-d6)
d 13.16 (s, 1H, ]NOH),
11.32 (s,1H, OH), 9.26 (d, J ¼ 6 Hz,1H, aromatic H), 9.15 (br NH), 8.73
(s, 1H, CH]N), 8.68 (app t, J ¼ 7.75 Hz, 1H, aromatic H), 8.47 (d,
J ¼ 8 Hz, 1H, aromatic H), 8.24 (app t, J ¼ 6.75 Hz, 1H, aromatic H),
7.80 (d, J ¼ 8 Hz, 2H, aromatic H), 7.35 (d, J ¼ 8 Hz, 2H, aromatic H),
6.19 (s, 2H, CH2) ppm. 13C NMR (500 MHz, DMSO-d6)
d 164.4, 148.2,
147.6, 147.1, 142.4, 137.8, 134.1, 129.0, 128.7, 128.2, 127.1, 60.9 ppm.
3. Synthesis of the oxime-hydroxamate hybrid 2,4DiPAMMeBHA (8,
Scheme A5)
1-(4-Hydroxycarbamoyl-benzyl)-2,4-
bis(hydroxyiminomethyl)-pyridinium bromide (2,4DiPAMMeBHA
(8))
A mixture of 4-bromomethylbenzhydroxamic acid (5, Scheme
A5) (Scheme A5), 139 mg, 0.61 mmol), 2,4-pyridinedialdoxime
(Accel Pharmtec, USA) (7, Scheme A5) (Scheme A5) (100 mg,
0.61 mmol) THF (5 ml) and acetonitrile (2.5 ML) was heated to
100 ꢂC in a pressure flask for 32 h. The obtained solid was filtered to
give the title compound (8, Scheme A5) (64 mg). 1H NMR (300 MHz,
Scheme A3. Chemical structure of starting material (3) and intermediates (4) and (5)
for the synthesis of 2PAMMeBHA (6).
Step 1:
a) 4-Bromomethylbenzoyl chloride (4, Scheme A3): Thionyl chlo-
ride (12.5 ml) was added under nitrogen atmosphere to 4-
bromomethylbenzoic acid (3, Scheme A3) at room tempera-
ture. The stirred suspension was refluxed for 4.5 h. Excess thi-
onyl chloride was removed by vacuum distillation. The obtained
oil was dissolved in toluene (25 ml) and the solvent was
removed under vacuum to give the title compound as an off
white solid. The acid chloride was used for the next step without
further purification.
DMSO-d6) d 13.18 (br s, ]NOH),11.33 (br s, OH), 9.16 (s, NH), 9.11 (d,
J ¼ 6.6 Hz, 1H, aromatic H), 8.71 (s, 1H, CH]N), 8.55 (s, 1H, CH]N),
8.54 (d, J ¼ 1.8 Hz, 1H, aromatic H), 8.32 (dd, J ¼ 6.6, 1.8 Hz, 1H,
aromatic H), 7.80 (d, J ¼ 8.4 Hz, 2H, aromatic H), 7.36 (d, J ¼ 8.4 Hz,
2H, aromatic H), 6.12 (s, 2H, CH2) ppm. 13C NMR (300 MHz, CD3OD)
d
152.4, 150.6, 148.4, 146.5, 142.7, 139.0, 135.4, 130.2, 129.5, 125.3,
Step 2:
125.0, 62.5 ppm.
b) 4-Bromomethylbenzhydroxamic acid (5, Scheme A3): Into a
stirred solution of hydroxylamine hydrochloride (2.34 gr,
33.7 mmol) in 2 N NaOH (17 ml) was slowly added (15 min) a
solution 4-Bromomethylbenzoyl chloride (from step a) in ether
(100 ml) at 0ꢂ C. The reaction mixture was stirred at room
temperature for 30 min, then cooled to 0 ꢂC and 2 N HCl (14 ml)
was added. The obtained solid was filtered, washed with water
and dried in vacuum to give the title compound (3.08 gr, 88%
yield). 1H NMR (500 MHz, DMSO-d6)
d 11.28 (s, 1H, OH), 9.11 (br
s, 1H, NH), 7.76 (d, J ¼ 8 Hz, 2H, aromatic H), 7.55 (d, J ¼ 8 Hz, 2H,
aromatic H), 4.77 (s, 2H, CH2) ppm.13C NMR (500 MHz, DMSO-
d6)
d 164.7, 142.0, 133.6, 130.3, 128.2, 34.5 ppm.
Scheme A5. The synthetic pathway for preparing 1-(4-Hydroxycarbamoyl-benzyl)-
2,4-bis(hydroxyiminomethyl)-pyridinium bromide (2,4DiPAMMeBHA (8)).
Please cite this article in press as: G. Amitai, et al., Novel bifunctional hybrid small molecule scavengers for mitigating nerve agents toxicity,
Chemico-Biological Interactions (2016), http://dx.doi.org/10.1016/j.cbi.2016.04.036

18
G. Amitai et al. / Chemico-Biological Interactions xxx (2016) 1e18
[24] G. Amitai, I. Rabinovitz, G. Zomber, R. Chen, G. Cohen, R. Adani, L. Raveh,
References
Antidotal efficacy of the bisquaternary oximes: AB-8, AB-13, toxognin, HI-6
and HLo-7 against tabun and soman poisoning, in: Proceedings 5th Interna-
tional Symposium on Protection against Chemical and Biological Warfare
Agents, Stockholm, Sweden, 11e16 June 1995, pp. 247e254.
[1] P. Taylor, Anticholinesterase agents, in: A. Goodman Gilman, L.S. Goodman,
T.W. Rall, F. Murad (Eds.), Goodman & Gilman the Pharmacological Basis of
Therapeutics, seventh ed., Macmillan Publishing Company, NY, NY 10022,
1985, pp. 110e129.
[2] F. Worek, H. Thiermann, The value of novel oximes for treatment of poisoning
by organophosphorus compounds, Pharmacol. Ther. 139 (2013) 249e259.
[3] T.-M. Shih, T.C. Rowland, J.H. McDonough, Anticonvulsants for nerve agent-
induced seizures: the influence of the therapeutic dose of atropine,
J. Pharmacol. Exp. Ther. 320 (2007) 154e161.
[4] Y. Seto, The Sarin Gas Attack in Japan and the Related Forensic Investigation,
Organization for the prohibition of chemical weapons (OPCW) e website
newsletter, 2001, https://www.opcw.org/news/article/the-sarin-gas-attack-
in-japan-and-the-related-forensic-investigation/.
[5] A. Ordentlich, D. Barak, C. Kronman, Y. Flashner, M. Leitner, Y. Segall, N. Arieli,
S. Cohen, B. Velan, A. Shafferman, Dissection of the human acetylcholines-
terase active center determinants of substrate specificity. Identification of
residues constituting the anionic site, the hydrophobic site, and the acyl
pocket, J. Biol. Chem. 268 (1993) 17083e17095.
[25] D.R. Davies, A.L. Green, The chemotherapy of poisoning by organophosphate
anticholinesterase, Brit. J. Ind. Med. 16 (1959) 128.
[26] G. Becker, A. Kawan, D. Gutzeit, F. Worek, L. Szinicz, Direct reaction of oximes
with crotylsarin, cyclosarin, or VX in vitro, Arch. Toxicol. 81 (2007) 415e420.
[27] F. Terrier, P. Rdrigeuz-Dafnte, E. Le Guevel, G. Moutiers, Org. Biomol. Chem. 4
(2006) 4352e4363.
[28] R. Swidler, G.M. Steinberg, The kinetics of the reaction of Isopropyl methyl-
phosphonofluoridate (Sarin) with benzohydroxamic acid, J. Am. Chem. Soc. 78
(1956) 3594e3598.
[29] J. Epstein, P.L. Cannon, H.O. Michel, B.E. Hackley, W.A. Mosher, The reaction of
organophosphate with alpha nucleophiles hydroxamates, J. Am. Chem. Soc. 89
(1967) 2937e2943.
[30] L. Louise Leriche, E. Paunesco, G. Saint-Andre, R. Baati, A. Romieu, A. Wagner,
P.-Y. Renard, A HTS assay for the detection of organophosphorus nerve agent
scavengers, Chem. Eur. J. 16 (2010) 3510e3523.
[31] G. Gilbert, T. Wagner-Jauregg, G.M. Steinberg, Hydroxamic acids: relationship
between structure and ability to reactivate phosphonate-inhibited acetyl-
cholinesterase, Arch. Biochem. Biophys. 93 (1961) 469e475.
[32] A.F. Childs, D.R. Davies, A.L. Green, J.P. Rutland, The reactivation by oximes and
hydroxamic acids of cholinesterase inhibited by organophosphorus com-
pounds, Brit. J. Pharmacol. 10 (1955) 462e465.
[6] C. Kronman, T. Chitlaru, E. Elhanany, B. Velan, A. Shafferman, Hierarchy of
post-translational modifications involved in the circulatory longevity of gly-
coproteins. Demonstration of concerted contributions of glycan sialylation
and subunit assembly to the pharmacokinetic behavior of bovine acetylcho-
linesterase, J. Biol. Chem. 275 (2000) 29488e29502.
[7] L. Raveh, J. Grunwald, D. Marcus, Y. Papier, E. Cohen, Y. Ashani, Human
[33] Y. Mushtaq, The COPD pipeline, Nat. Rev. Drug Discov. 13 (2014) 253e254.
[34] G.L. Ellman, K.D. Courtney, V. Andres Jr., R.M. Featherstone, A new and rapid
colorimetric determination of acetylcholinesterase activity, Biochem. Phar-
macol. 7 (1961) 88e95.
butyrylcholinesterase as
a general prophylactic antidote for nerve agent
toxicity. In vitro and in vivo quantitative characterization, Biochem. Phar-
macol. 45 (12) (1993) 2465e2474.
[8] T-c Cheng, V.K. Rastogi, J.J. DeFrank, G.P. Sawiris, Enzyme Engineering XIV, vol.
864, Annals of the New York Academy of Sciences, 1998, p. 253.
[9] L. Raveh, Y. Segall, H. Leader, N. Rothschild, D. Levanon, Y. Henis, Y. Ashani,
Protection against tabun toxicity in mice by prophylaxis with an enzyme
hydrolyzing organophosphate esters, Biochem. Pharmacol. 44 (1992)
397e400.
[10] D.P. Dumas, H.D. Durst, W.G. Landis, F.M. Raushel, J.R. Wild, Inactivation of
organophosphorus nerve agents by the phosphotriesterase from Pseudomonas
diminuta, Arch. Biochem. Biophys. 277 (1990) 155e159.
[11] G. Amitai, L. Gaidukov, R. Adani, S. Yishay, G. Yacov, M. Kushnir, S. Teitlboim,
M. Lindenbaum, P. Bel, O. Khersonsky, D.S. Tawfik, H. Meshulam, Enhanced
stereoselective hydrolysis of toxic organophosphates by directly evolved
variants of mammalian serum paraoxonase, FEBS J. 273 (2006) 1906e1919.
[12] D.D. Gay, H.F.C.G. Hoskin, Stereospecificity and active site requirements in a
diisopropylphosphorofluoridate hydrolyzing enzyme, Biochem. Pharmacol. 28
(1979) 1259e1261.
[35] C.D. Johnson, R.L. Russell, A rapid simple radiometric assay for cholinesterase,
suitable for multiple determinations, Anal. Biochem. 64 (1975) 229e238.
[36] W.J. Dixon, The up-and-down method for small samples, J. Am. Stat. Assoc. 60
(1965) 967e978.
[37] J. Finney, in: Statistical Methods in Biological Assays, Griffin and Co, London,
1952, pp. 524e530 (Chapter 20.6).
[38] J. Kassa, Review of oximes in the antidotal treatment of organophsophorus
nerve agents poisoning, J. Toxicol. Clin. Toxicol. 40 (2002) 803e816.
[39] F. Worek, T. Wille, M. Koller, H. Thiermann, Structural requirements for
effective oximes e evaluation of kinetic in vitro data with phosphylated hu-
man AChE and structurally different oximes, Biochem. Pharmacol. 83 (2012)
1700e1706.
[40] Ts-M. Shih, J.W. Skovira, J.C. O'Donnell, J.H. McDonough, Evaluation of nine
oximes on in vivo reactivation of blood, brain, and tissue cholinesterase ac-
tivity inhibited by organophosphorus nerve agents at lethal dose, Toxicol.
Mech. Meth. 19 (6e7) (2009) 386e400.
€
[13] M. Melzer, J.C. Chen, A. Heidenreich, J. Gab, M. Koller, K. Kehe, M.M. Blum,
[41] H. Grosfeld, D. Barak, A. Ordentlich, B. Velan, A. Shafferman, Interactions of
Oxime reactivators with diethyiphosphoryl adducts of human acetyicholi-
nesterase and its mutant derivatives, Mol. Pharmacol. 50 (1996) 639e649.
[42] Pralidoxime Chloride Injection e Meridian Medical Technologies Inc. http://
dailymed.nlm.nih.gov/dailymed/archives/fdsDrugInfo.cfm?archiveid¼252.
[43] J.H. Fleisher, L.W. Harris, G.R. Miller, N.C. Thomas, W.J. Cliff, Antagonism of
sarin poisoning in rats and guinea pigs by atropine, oximes, and mecamyl-
amine, Toxicol. Appl. Pharmacol. 16 (1970) 40e47.
[44] T.C. Marrs, Toxicology of oximes used in treatment of organophosphate
poisoning, Adverse Drug React. Toxicol. Rev. 10 (1991) 61e72.
[45] a) G. Amitai, I. Rabinovitz, R. Chen, G. Cohen, G. Zomber, R. Adani,
B. Manistersky, H. Leader, L. Raveh, Antidotal efficacy of bisquaternary oximes
against soman and tabun poisoning in various species, Med. Def. Biosci. Rev. 2
(1993) 527e533;
Reversed enantioselectivity of diisopropyl fluorophosphatase against organ-
ophosphorus nerve agents by rational design, J. Am. Chem. Soc. 131 (47)
(2009) 17226e17732.
[14] R.D. Gupta, M. Goldsmith, Y. Ashani, Y. Simo, G. Mullokandov, H. Bar, M. Ben-
David, H. Leader, R. Margalit, I. Silman, J. Sussman, D.S. Tawfik, Directed
evolution of hydrolases for prevention of G-type nerve agent intoxication,
Nat. Chem. Biol. 7 (2011) 120e125.
[15] I. Cherny, P. Greisen Jr., Y. Ashani, S.D. Khare, G. Oberdorfer, H. Leader,
D. Baker, D.S. Tawfik, Engineering V-type nerve agents detoxifying enzymes
using computationally focused libraries, ACS Chem. Biol. 8 (2013) 2394e2403.
[16] F. Worek, T. Seeger, M. Goldsmith, Y. Ashani, H. Leader, J.L. Sussman,
D.S. Tawfik, H. Thiermann, T. Wille, Efficacy of the rePON1 mutant IIG1 to
prevent cyclosarin toxicity in vivo and to detoxify structurally different nerve
agents in vitro, Arch. Toxicol. 88 (2014a) 1257e1266.
[17] F. Worek, T. Seeger, G. Reiter, M. Goldsmith, Y. Ashani, H. Leader, J.L. Sussman,
N. Aggarwal, H. Thiermann, D.S. Tawfik, Post-exposure treatment of VX
poisoned guinea pigs with the engineered phosphotriesterase mutant C23: a
proof-of-concept study, Toxicol. Lett. 231 (2014b) 45e54.
[18] I.B. Wilson, S. Ginsburg, A powerful reactivator of alkylphosphate-inhibited
acetylcholinesterase, Biochim. Biophys. Acta 18 (1955) 168e170.
[19] A.L. Green, H.J. Smith, The reactivation of cholinesterase inhibited with
organophosphorus compounds, Biochem. J. 68 (1958) 32e35.
[20] E.J. Poziomek, B.E. Hackley, G.M. Steinberg, Pyridinium aldoximes, J. Org.
Chem. 23 (1958) 714e717.
[21] F. Hobbiger, D.C. O'Sullivan, P.W. Sadler, New potent reactivators of ace-
tocholinesterase inhibited by tetraethyl pyrophosphate, Nature 182 (1958)
1498e1499.
[22] P. Eyer, The role of oximes in the management of organophosphorus pesticide
poisoning, Toxicol. Rev. 22 (3) (2003) 165e190.
[23] P.M. Lundy, L. Raveh, G. Amitai, Development of the bisquaternary oxime HI-6
toward clinical use in the treatment of organophosphate nerve agent
poisoning, Toxicol. Rev. 25 (2006) 231e243.
b) B. Antonijevic, M.P. Stojiljkovic, Unequal efficacy of pyridinium oximes in
acute organophosphate poisoning, Clin. Med. Res. 6 (2007) 71e82;
c) J.G. Clement, Toxicology and pharmacology of bis-pyridinium oximes e
insight into the mechanism of action vs soman poisoning in vivo, Fundam.
Appl. Toxicol. 1 (1981) 193e202;
d) L. Sevelova, J. Vachek, Effect of methoxime combined with anticholinergic,
anticonvulsant or anti-HCN drugs In tabun-poisoned mice, Acta Medica
(Hradec Kralove) 46 (2003) 109e112.
[46] B. van’t Riet, G.L. Wampler, H.L. Elford, Synthesis of hydroxyl and amino-
substituted benzohydroxamic acids: inhibition of ribonucleotide reductase
and antitumor activity, J. Med. Chem. 22 (1979) 589e592.
[47] J. Von Bredow, Effectiveness of oral pyridostigmine pretreatment and
cholinolytic-oxime therapy against soman intoxication in non-human pri-
mates, Fund. Appl. Toxicol. 17 (1991) 761e770.
[48] C. Luo, M. Tong, N. Chilukuri, K. Brecht, D.M. Maxwell, A. Saxena, An in vitro
comparative study on the reactivation of nerve agent-inhibited guinea pig and
human acetylcholinesterases by oximes, Biochemistry 46 (2007)
11771e11779.
Please cite this article in press as: G. Amitai, et al., Novel bifunctional hybrid small molecule scavengers for mitigating nerve agents toxicity,
Chemico-Biological Interactions (2016), http://dx.doi.org/10.1016/j.cbi.2016.04.036