Amidine-Oximes: Reactivators for organophosphate exposure

Article abstract of DOI:10.1021/jm200054r

Figure Presented. Figure presented. A new class of amidine-oxime reactivators of organophosphate (OP)-inhibited cholinesterases (ChE) were designed, synthesized, and tested. These compounds represent a novel group of oximes with enhanced capabilities of crossing the blood-brain barrier. Lack of brain penetration is a major limitation for currently used oximes as antidotes of OP poisoning. The concept described herein relies on a combination of an amidine residue and oxime functionality whereby the amidine increases the binding affinity to the ChE and the oxime is responsible for reactivation. Amidine-oximes were tested in vitro and reactivation rates for OP-BuChE were greater than pralidoxime (2-PAM) or monoisonitrosoacetone (MINA). Amidine-oxime reactivation rates for OP-AChE were lower compared to 2-PAM but greater compared with MINA. After pretreatment for 30 min with oximes 15c and 15d (145 μmol/kg, ip) mice were challenged with a soman model compound. In addition, 15d was tested in a post-treatment experiment (145 μmol/kg, ip, administration 5 min after sarin model compound exposure). In both cases, amidine-oximes afforded 100% 24 h survival in an animal model of OP exposure.

Full text of DOI:10.1021/jm200054r

ARTICLE
pubs.acs.org/jmc
AmidineꢀOximes: Reactivators for Organophosphate Exposure
Jaroszaw Kalisiak,* Erik C. Ralph, Jun Zhang, and John R. Cashman
Human BioMolecular Research Institute, 5310 Eastgate Mall, San Diego, California 92121, United States
S Supporting Information
b
ABSTRACT: A new class of amidineꢀoxime reactivators of
organophosphate (OP)-inhibited cholinesterases (ChE) were de-
signed, synthesized, and tested. These compounds represent a
novel group of oximes with enhanced capabilities of crossing the
bloodꢀbrain barrier. Lack of brain penetration is a major limita-
tion for currently used oximes as antidotes of OP poisoning. The
concept described herein relies on a combination of an amidine residue and oxime functionality whereby the amidine increases the
binding affinity to the ChE and the oxime is responsible for reactivation. Amidineꢀoximes were tested in vitro and reactivation rates
for OP-BuChE were greater than pralidoxime (2-PAM) or monoisonitrosoacetone (MINA). Amidineꢀoxime reactivation rates for
OP-AChE were lower compared to 2-PAM but greater compared with MINA. After pretreatment for 30 min with oximes 15c and
15d (145 μmol/kg, ip) mice were challenged with a soman model compound. In addition, 15d was tested in a post-treatment
experiment (145 μmol/kg, ip, administration 5 min after sarin model compound exposure). In both cases, amidineꢀoximes
afforded 100% 24 h survival in an animal model of OP exposure.
’ INTRODUCTION
Organophosphate (OP) nerve agents and pesticides could be
used as weapons of mass destruction and represent a potential
threat to military personnel and civilians. In addition, farmers and
agricultural workers handle large amounts of OPs and are
constantly exposed to these toxic materials. Between 150 000
and 300 000 OP-related toxic incidences are reported annually in
the United States.1 OP pesticides (e.g., parathion, chlorpyrifos)
and chemical warfare nerve agents (e.g., sarin, soman, tabun, VX)
Figure 1. Chemical structures of currently available pyridinium oximes.
are potent covalent inhibitors of cholinesterase enzymes (ChE)
in the blood and in the central nervous system (CNS).2 The
resulting OPꢀChE adducts undergo very slow spontaneous
hydrolysis and allow acetylcholine (ACh) to build up in the
synapses. Accumulation of ACh stimulates autonomic receptors
and blocks neuromuscular junction receptors.3 The symptoms
resulting from nerve agent exposure are primarily the conse-
quence of accumulation of excess ACh where ordinarily only
small amounts of ACh are present at nerve junctions.4 Currently,
clinically available treatment of acute OP poisoning includes
combined administration of a ChE reactivator (i.e., oxime), a
muscarinic receptor antagonist (i.e., atropine), and an antic-
onvulsant (i.e., diazepam). Oximes currently in use for treatment
of OP poisoning are functionalized pyridinium salts (Figure 1).
Pralidoxime (2-PAM chloride), discovered in 1955,5 was the first
oxime used clinically for treatment of OP poisoning and cur-
rently it is a standard antidote in the United States.
AChE, HI-6 KD = 24 μM, 2-PAM KD = 210 μM).8 For example,
for sarin-inhibited AChE, HI-6 has a reactivation rate constant
(kr) = 22 000 Mꢀ1 minꢀ1, whereas the kr for 2-PAM = 403 Mꢀ1
minꢀ1.8 However, introduction of a second positive charge into a
pyridinium oxime effectively precludes the compound from entry
into the brain via the bloodꢀbrain barrier (BBB). Because brain
ChEs are the key target for toxic action of OPs, bis-cationic
reactivators possess significant deficiencies in reactivating brain
ChE’s. Treatment of OP poisoning with bis-cationic pyridinium
oximes does not provide complete protection from CNS toxicity,
and consequently, individuals exposed suffer from chronic
neurological side effects. Approximately 20% of the victims of
sarin exposure in the Tokyo subway attack of 1995 had symp-
toms of OP poisoning 1 year after the attack.9 To address the
deficiencies of cationic pyridinium oximes and develop agents
that protect the CNS from toxicity of OP exposure, oximes
capable of crossing the BBB were developed.
Recent efforts in the field of OP reactivation have focused on
development of more effective bis-quaternary oximes6,7 (e.g., HI-
6, MMB-4, obidoxime, Figure 1). Elaboration of an oxime as a
bis-quaternary compound generally improves its reactivation
properties, because the second cation interacts with the periph-
eral binding site of ChE and increases binding affinity (e.g., for
Received: January 19, 2011
Published: March 25, 2011
r
2011 American Chemical Society
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To date, only a few BBB-penetrable oximes have been reported
in the literature. However, none of them have been extensively
developed further because of low functional activity and/or
significant toxicity.10,11 Modification of quaternary oximes to
improve BBB permeability requires introducing lipophilic sub-
stituents to counteract the hydrophilic character of the pyridi-
nium positive charge. The active site serine of ChEs lies at the
bottom of a deep, narrow gorge and one possible disadvantage of
quaternary oximes with large lipophilic groups might be the lack
of space at the active site to accommodate additional bulk.
Another disadvantage of this approach is that lipophilic deriva-
tives of 4-PAM have been described to have significant toxicity
(i.e., LD50 values from 4.2 to 21.9 mg/kg, rats, iv).11 2-PAM has
been delivered into the CNS for protection by relying on
modification of 2-PAM by attaching a glucose molecule. This
approach presumably utilizes glucose transporters for BBB
delivery.12 Recent efforts to reactivate CNS ChEs have relied
on oxime antidotes with increased lipophilicity and BBB perme-
ability compared to 2-PAM-type reactivators. The uncharged
oximes monoisonitrosoacetone (MINA) and diacetylmonoox-
ime (DAM) (Figure 2) are reportedly able to penetrate the BBB
but have a much lower propensity for reactivation of OPꢀChE in
peripheral tissues and blood ChEs compared to 2-PAM and
other quaternary oximes.13,14
An alternative approach relies on the use of a prodrug of
2-PAM that undergoes oxidation within the CNS to produce a
functionally active quaternary oxime (Figure 2).15ꢀ17 However,
this approach suffers from two disadvantages: (1) the synthesis of
the prodrug is inherently difficult,18 and (2) pro-2-PAM is
unstable to autoxidation. Therefore, novel uncharged reactiva-
tors capable of crossing the BBB and reactivating ChEs within the
CNS are needed.
Retrostructural analysis of 2-PAM led us to the general
structure of novel compounds containing a strongly basic amidine
center adjacent to the oxime function (Figure 3).
An amidine group is frequently used in drugs (e.g., dabigatran,
diminazen, pentamidine) because of its nontoxic properties,
pronounced water solubility, and action as an efficient hydro-
gen-bond acceptor. In the case of amidineꢀoxime reactivators
(Figure 3), the basic group readily undergoes protonation under
physiological conditions to form a positively charged center,
similar to a pyridinium quaternary nitrogen. Protonated amidine
groups presumably facilitate efficient binding to ChE’s through
πꢀcation interactions with aromatic amino acid residues at the
ChE active site. The concept of incorporating an amidine group
into small molecules to increase a compound’s binding affinity to
biomolecules was reported in the literature, and amidines have
been used as inhibitors of AChE19,20 and antagonists of the
muscarinic acetylcholine receptor.21
In the approach developed herein, the amidine moiety sup-
plies a pseudopositive charge responsible for efficient binding to
OP-inhibited ChEs, while the proximal oxime group is a nucleo-
phile responsible for reactivation of the phosphylated enzyme. In
addition, the electron-withdrawing character of the protonated
amidine group enhances ionization of the oxime group and
increases the concentration of the functionally active nucleophi-
lic oxime anion (Figure 4).
The pKa of the oxime group in MINA is 8.322 and provides
approximately 12% of the oxime in the deprotonated form at pH
7.4, according to the HendersonꢀHasselbalch equation. On the
basis of the structural similarities between MINA and amidi-
neꢀoximes and taking into consideration the electron-with-
drawing character of the protonated amidine functionality,
amidineꢀoximes herein should be deprotonated at physiological
pH equally if not greater than MINA. Therefore, the amidine
functionality plays a dual role in amidineꢀoximes: first as a
pseudopositive charge to efficiently bind to an OP-inhibited
enzyme and second as a functional group that enhances the
nucleophilicity of the oxime.
Herein, we describe a new class of amidineꢀoxime reactiva-
tors of OPꢀChEs. Compared to the pro-2-PAM/2-PAM system
or MINA, amidineꢀoximes have advantageous properties in-
cluding (1) improved chemical stability (compared to pro-2-
PAM), (2) greatly improved lipophilicity (compared to quatern-
ary oximes), (3) increased in vitro reactivation efficacy of OP-
inhibited BuChE (compared to 2-PAM) and AChE (compared
to MINA), and (3) significant protection from the in vivo toxic
effects of OPs.
’ RESULTS AND CHEMISTRY
Because of the paucity of examples in the literature of
compounds containing an amidine and oxime functionality in
Figure 2. Chemical structures of uncharged oximes MINA and DAM
Figure 4. An amidine functional group plays a dual role by providing a
and a prodrug of 2-PAM that undergoes in vivo oxidation to 2-PAM.
positive charge and increasing the ionization of the oxime oxygen atom.
Figure 3. Retrostructural analysis of 2-PAM led to the structure of an amidineꢀoxime.
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Scheme 1. Unsuccessful Synthesis of AmidineꢀOxime 15b
by Path A
Figure 5. Two retrosynthetic paths for synthesis of amidineꢀoxime 1.
ammonia or alkyl amine MeNH2, EtNH2, PrNH2 to afford
11aꢀd (Scheme 2). Thioamides 12aꢀd were obtained by
treating 11aꢀd with Lawesson’s reagent in good yield. The
triflate salts of 13aꢀd were prepared by S-methylation of 12aꢀd
using MeOTf. The desired amidines 14aꢀd were obtained by
treating 13aꢀd with Me2NH in THF according to a previously
established procedure shown in Scheme 1.
the same molecule, the synthesis of amidineꢀoximes required a
new strategy. Retrosynthetic analysis revealed two possible
approaches (Figure 5). In path A, the amidine 2a was synthesized
first and then a protected aldehyde was converted to an oxime 1.
In path B, the starting material was a protected oxime 4b and then
the amidine 2b was synthesized. Finally, the oxime group was
deprotected and the desired product 1 was obtained. Generally,
both strategies followed the same synthetic approach but differed
in the type of protecting group.
Both approaches (paths A and B, Figure 5) started from
readily available esters 4a,b containing either a protected alde-
hyde or a protected oxime group. The ester function was
subsequently modified into amides 3a,b that served as a starting
point for making amidine 2a,b using a thioimidate interme-
diate.23,24 Application of the Pinner method for synthesis of an
amidine group in an amidineꢀoxime was not considered because
it required a hypothetical cyanoꢀoxime that is predicted to be
very unstable. The desired product 1 was obtained from 2a after
deprotection and subsequent reaction with hydroxylamine or by
deprotection of 2b. In theory, neither of the two methods had a
significant advantage over the other and hence we explored both
synthetic routes in parallel.
In path A, ethyl diethoxyacetate 5 was readily converted to
amide 6 in the presence of MeNH2 and then to thioamide 7 using
Lawesson’s reagent (Scheme 1). Activation of the carbonyl group
through S-methylation of thioamide 7 provided thioimidate 8 in
quantitative yield. An attempt to methylate 7 using MeI in
acetone required elevated temperature and led to starting
material and/or product decomposition. Treatment of 7 with
MeOTf in MeNO2 at room temperature gave 8 in quantitative
yield. Further treatment with Me2NH in THF gave amidine 9
with a satisfactory yield (i.e., 59%). The final step was acid-
catalyzed deprotection of acetal 9. No reaction was observed
upon treatment of 9 with 4 M HCl at room temperature, and
more harsh reaction conditions caused decomposition of the
starting material and/or product.
An initial attempt to remove the benzyl group of 14aꢀd using
Pd/C/H2 was unsuccessful and only starting material was
recovered, even after using an equivalent amount of the catalyst,
probably because of sulfur-based impurities from the previous
step that poisoned the catalyst. Extensive chromatographic
purification of 14aꢀd did not solve this problem. To overcome
this issue, sulfur impurities were removed using in situ prepared
fine powder of silver metal. Amidine hydrochlorides 14aꢀd were
dissolved in H2O and then added to a solution containing an
equivalent amount of AgNO3. The precipitated AgCl quickly
underwent decomposition upon exposure to light and formed a
black silver powder. Metallic silver helped to remove sulfur
impurities from the previous synthetic step and prevented
poisoning of the Pd catalyst in the debenzylation step. Debenzy-
lation of highly purified amidine chlorides 14bꢀd was efficient,
and in the case of methyl-substituted amidine 14b, the desired
amidineꢀoxime 15b was obtained in 97% yield. In the case of
ethyl and propyl amidineꢀoximes 14c,d, debenzylation provided
products 15c,d with much lower yields, probably because of
reduction of the oxime group. Deprotection of unsubstituted
amidineꢀoxime 14a using the method described in Scheme 2
was unsuccessful, and only reduction of the oxime group was
observed. Debenzylation of 14a was not successful using differ-
ent catalysts (i.e., Pd/C, Pd/BaSO4, Pd/CaCO3/5%Pb) and
solvents (EtOH, THF, EtOAc). However, it was observed that
strong Lewis acids such as anhydrous AlCl3 in CH2Cl2 removed
the benzyl group of 14a to afford amidineꢀoxime 15a in good
yield (Scheme 2).
Amidineꢀoxime hydrochlorides 15aꢀd were tested for che-
mical stability and showed no sign of decomposition after 3
months at ꢀ18 °C, as judged by NMR. However, the free base
form of 15aꢀd underwent decomposition in CD2Cl2, and after 5
In path B, benzyl-protected ethyl glyoxylate oxime 10 was
synthesized as described before.25 Ester 10 was treated with
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Scheme 2. Synthesis of N,N0,N00-Trisubstituted AmidineꢀOximes 15aꢀd
Scheme 3. Synthesis of AmidineꢀOximes 15d and 19 Using a PMB Protecting Group
days, significant amounts of impurities were detected by NMR. The
synthetic method was successfully scaled-up to prepare over 200 mg
of amidines 15aꢀd. However, debenzylation of 14bꢀd was
challenging, and extensive purification from sulfur impurities was
always required to obtain satisfactory yields of 15bꢀd. Difficulties in
removing the benzyl group encouraged the search for an alternative
protecting group that could be removed under mild conditions
besides hydrogenation. From a broad panel of protecting groups, p-
methoxybenzyl (PMB) was selected, because it could be removed
using significantly milder conditions. To test the utility of the PMB
group in the synthesis, propyl amidineꢀoxime 15d was chosen, as it
gave a low yield in the debenzylation step. PMB-protected ethyl
Figure 6. pKa determination for 15c.
glyoxylate oxime 16 was converted as above to the amide 17 and
thioamide 18 derivatives (Scheme 3).
Unexpected results were obtained during the S-methylation of
18a, where traces of water likely caused hydrolysis of MeOTf,
forming triflic acid in situ. Under acidic conditions the removal of
the PMB group occurred, and therefore, the crude reaction mixture
was directly used in the amidine synthesis. Using this two-step
procedure (Scheme 3), the final product 15d was obtained in 31%
overall yield compared with 3.2% following the method of Scheme 2.
A similar methodology shown in Scheme 3 was used to prepare the
N-butyl-substituted amidineꢀoxime 19, and in this case the product
was isolated in greater than 58% overall yield.
amidineꢀoxime that exists in the zwitterionic form at physiolo-
gical pH, the pKa values for one representative compound, 15c,
were determined. A series of 50 mM buffer solutions with pH
from 3 to 13.5 and constant ionic strength (300 mM) was
prepared. A solution of 15c was added to each buffer and the UV
spectrum was recorded in the range of 220ꢀ700 nm. The change
in absorbance at 260 nm was plotted versus pH to obtain the pKa
profile (Figure 6).
On the basis of the results for 15c, the pKa for the oxime group
is 8.0 ( 0.1, whereas the pKa for the amidine group is 12.1 ( 0.1.
Therefore, at physiological conditions, pH 7.4, the amidine
group is almost exclusively protonated, whereas the oxime group
is deprotonated to approximately 25 ( 5%, based on the
Determination of the pKa for 15c. To support and verify the
hypothesis that a protonated amidine group decreases the pKa of
the oxime function and also to establish the amount of
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Figure 7. Chemical structures of nerve agent model compounds used in
these studies.28,29
Figure 8. Reactivation of OP-inhibited AChE by amidineꢀoximes 15
and 19, MINA, and 2-PAM. Enzyme was inhibited with OP prior to
incubation with 100 μM oxime, as described in the Experimental
Section. After 60 min, the recovered AChE activity was determined by
measuring AChE esterase functional activity. Data depicted shows the
average and standard deviation (as a fraction of noninhibited control
samples) for AChE activity (n = 2 incubations per oxime). Unless
otherwise indicated, the standard deviation bars were smaller than the
data markers. The broken (dashed) lines indicate the relative activity of
samples incubated with 2-PAM at 100 μM. Relative activity values
of enzyme-inhibited samples incubated in the absence of oxime
were e0.03.
HendersonꢀHasselbalch equation. In comparison, MINA has a
pKa = 8.322 and approximately 12% of the oxime exists in the
deprotonated form. On the basis of the literature, an optimal pKa
for the oxime group should be approximately 7.8,26 or within the
range 7.5ꢀ8.0.27 Oxime reactivators with pKa values lower than
7.5 are too weakly nucleophilic, whereas a pKa above 8.0 does not
provide enough of the oxime anion form at physiological
conditions. Amidineꢀoxime 15c fulfills the requirement of an
optimal pKa for efficient reactivation, and its value is above that of
currently available quaternary oximes, 2-PAM (pKa = 7.68), HI-6
(pKa = 7.28), obidoxime (pKa = 7.78), and MMB-4 (pKa =
7.3).27 The difference between pKa values for 2-PAM and 15c
comes from the greater electron-withdrawing character of the
pyridinium quaternary group compared to the protonated ami-
dine in 15c. Although the pKa of the oxime group in 15c is less
favorable than the pKa of a quaternary oxime, the lack of a per-
manent positive charge makes amidineꢀoximes orders of mag-
nitude more lipophilic, and therefore, it has a greater propensity
to enter the CNS.
In Vitro Biochemical Studies. The ability of oximes 15aꢀd
and 19 to reactivate human AChE and BuChE inhibited by nerve
agent model compounds (i.e., model compounds of sarin, cyclo-
sarin, tabun)28,29 and pesticides (i.e., echothiophate) (Figure 7)
was evaluated by incubating OP-inhibited enzyme with oxime for
20ꢀ60 min and then comparing the enzyme’s esterase activity to
a noninhibited control sample.
During the course of the experiment, enzyme sample was
incubated with oxime, and then an aliquot was diluted 20-fold
into a solution containing substrate to assess the amount of
esterase functional activity. The dilution carried with it free
oxime, yielding a concentration of 5 μM in the esterase functional
activity assay. To ensure that this concentration of oxime was
neither inhibiting enzyme nor nonenzymatically increasing the
apparent esterase activity, oxime concentrations were titrated
over a range of concentrations (i.e., 3ꢀ100 μM) in enzyme
incubations with 1 mM substrate and the observed rates of
turnover were recorded. For all but the most lipophilic com-
pound (i.e., 19), the catalytic activity observed in the presence of
100 μM amidineꢀoxime was still within 20% of the no amidi-
neꢀoxime control sample, and no further analysis was con-
ducted. Amidineꢀoxime 19 inhibited AChE with an IC50 value
of 54 ( 8 μM (see Supporting Information). On the basis of this
value, it is estimated that 5 μM amidineꢀoxime present in the
kinetic assay following AChE reactivation trials would yield less
than 10% inhibition, and this putative inhibitory effect was
therefore disregarded when analyzing reactivation levels. Lack
of inhibition was seen for BuChE with oximes 15aꢀd. Compared
to studies with AChE, oxime 19 bound less tightly to BuChE,
with an IC50 value estimate larger than 300 μM.
Reactivation studies of amidineꢀoximes 15aꢀd and 19 were
evaluated together with two reference compounds (i.e., 2-PAM
and MINA). Compared with uncharged reactivator MINA,
amidineꢀoximes 15aꢀd and 19 were more potent reactivators.
A plot of reactivated ChEs activity (relative to a noninhibited
enzyme sample) versus calculated ClogP showed no significant
dependence on reactivator structure in the presence of Sp-GAC
or Sp-GF-SMe (Figure 8). In the presence of ETP, a slight
but discernible increase in AChE reactivation was observed in
going from hydrogen- (15a) to butyl-substituted (19) amidineꢀ
oximes. In contrast, an inverse relationship was observed for
AChE inactivated with Sp-GBC. Initial studies with AChE
showed less than 10% functional activity was recovered after
20 min incubation time with amidineꢀoximes 15aꢀd and 19
(see Supporting Information), and therefore, the incubation time
was increased to 60 min. The increased duration of incubation
improved the levels of reactivation, and most amidineꢀoximes
yielded less than 60% reactivation (Figure 8). The lowest degree
of reactivation was observed for GF-inhibited enzyme. In the
presence of AChE, the quaternary reactivator 2-PAM was a more
potent reactivator than MINA or amidineꢀoximes 15aꢀd and
19 (Figure 7).
In contrast to AChE, reactivation of BuChE showed a
considerably larger dependence on amidineꢀoxime structure
(Figure 9). A plot of relative percent remaining functional activity
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Table 1. Effect of Vehicle, 2-PAM, or AmidineꢀOximes 15c
and 15d on the Lethality of rac-GDC
rac-GDC
experiment
1
treatmenta
μmol mg/kg μmol survival (24 h)
vehicle
15c (100 mg/kg)b
ꢀ
0.25 0.61
0.25 0.61
2/14
6/6
6/8
6/6
6/6
0/6
4/6
557
2
2-PAM (25 mg/kg) 145
15c (26 mg/kg)b
15d (28 mg/kg)b
3
2-PAM (6.25 mg/kg) 36
15c (6.5 mg/kg)b
0.25 0.61
a Mice were pretreated (ip) with vehicle or an amidineꢀoxime 30 min
prior to rac-GDC. b Oximes 15c and 15d were administered as hydro-
chloride salts in isotonic saline. P values for expt 1 = 0.0007, expt 2 =
0.47, expt 3 = 0.06.
Figure 9. Reactivation of OP-inhibited BuChE by amidineꢀoximes 15
and 19, MINA, and 2-PAM. Enzyme was inhibited with OP prior to
incubation with 100 μM amidineꢀoxime, as described in the Experi-
mental Section. After 20 min, the recovered BuChE functional activity
was determined by measuring BuChE activity. Data depicted the average
and standard deviation (as a fraction of noninhibited control samples)
for BuChE activity (n = 2 incubations per oxime). Unless otherwise
indicated, the standard deviation bars were smaller than the data
markers. The broken (dashed) lines indicate the relative activity of
samples incubated with 2-PAM at 100 μM. Relative activity values of
enzyme-inhibited samples incubated in the absence of oxime were
e0.03 for ETP, Sp-GAC, and Sp-GBC. The corresponding Sp-GFC
sample had a value of 0.06.
organophosphate to reactivate.30,31 Soman-inactivated ChEs
rapidly undergo a PꢀO-dealkylation processes (i.e., aging)
within minutes.32 On a molecular level, this leaves a negatively
charged PꢀOꢀ moiety that is resistant to nucleophilic attack and
reactivation by oximes. None of the currently available oxime
reactivators are able to reactivate an aged ChE, and therefore,
reliable in vitro reactivation studies of somanꢀChEs cannot be
conducted or require elaborated methodology.33 A nerve agent
model of sarin (i.e., Sp-GBC) was utilized because in vitro studies
reported above showed efficient reactivation of AChE (approximately
50%, Figure 8) and BuChE (approximately 80%, Figure 9).
For the in vivo studies using rac-GDC, compounds 15c,d were
selected and three sets of experiments were conducted. Mice
were pretreated (ip injection) with vehicle or amidineꢀoxime
30 min prior to soman model compound (rac-GDC, Figure 7)
administration. In human clinical use, im administration is the
preferred route of administration; however, this method is not
recommended for mouse studies, because of the paucity of
muscle mass in a mouse. The 30 min pretreatment regimen
(i.e., the time between amidineꢀoxime pretreatment and OP
administration) was chosen because this was estimated to be the
time point of peak plasma concentrataion of the amidineꢀoxime.
This estimate was based in part on literature information for the
bis-quaternary oxime K-48 (CLogP = ꢀ8.2).34 Peak plasma
concentration of K-48 was achieved after approximately 15
min. In the animal paradigm used herein, after OP administra-
tion, mice were observed for neurological toxicity symptoms (i.e.,
seizures, convulsions, muscles twitching, salivation, and
rhinorrhea). After 24 h the survival was recorded, and animals
were sacrificed by cervical dislocation. In a preliminary experi-
ment, animals were either pretreated with vehicle or 15c at a dose
of 100 mg/kg (ip) to simultaneously evaluate protection from
OP and possible acute toxicity of 15c. No apparent toxicity was
observed for 15c administered alone at a dose of 100 mg/kg (ip),
and all animals pretreated with 15c (100 mg/kg, ip, 30 min prior
administration) survived 24 h after OP exposure (Table 1). We
estimate that the LD50 of amidineꢀoximes far exceeds 100 mg/
kg, but this point was not thoroughly examined. For vehicle-
treated animals, only 2/14 control animals survived to the 24 h
time point. In a second experiment, animals were pretreated (ip)
with either amidineꢀoximes 15c,d or 2-PAM at the same molar
concentration (i.e., 145 μmol; 26 mg/kg for 15c, 28 mg/kg for
15d, and 25 mg/kg for 2-PAM). In the second experiment,
of BuChE versus ClogP showed a modest increase in Sp-GAC
reactivation in going from a hydrogen- (15a) to an N-butyl-
substituted (19) amidineꢀoxime but a strong dependence on
increasing lipophilicity in the presence of Sp-GBC or Sp-GF-
SMe. In the presence of ETP, a parabolic relationship was
observed between lipophilicity of amidineꢀoximes 15 and 19
and BuChE reactivation. Sp-GBC- and Sp-GF-SMe-inhibited
BuChE recovered over 60% functional activity within a 20 min
reactivation period for the most lipophilic N-butyl amidineꢀ
oxime 19. Amidineꢀoximes 15c, 15d, and 19 all showed com-
parable or greater levels of BuChE reactivation compared to
2-PAM. These results illustrated that amidineꢀoximes with
more lipophilic substituents on the amidine group markedly
increased reactivation of Sp-GBC- and Sp-GF-SMe-inhibited
BuChE. For ETP and Sp-GAC, 2-PAM showed greater reactiva-
tion than 15aꢀd and 19.
The amidineꢀoximes showed reactivation rates for OP-AChE
within 2ꢀ2.5-fold of 2-PAM at equal oxime concentrations (i.e.,
100 μM). However, the amidineꢀoximes are orders of magni-
tude more lipophilic than 2-PAM and are predicted to enter the
brain in a correspondingly improved fashion. The hypothesis is
that improved apparent BBB penetration and greater brain
concentration improved the concentration of amidineꢀoximes
necessary for reactivation compared to 2-PAM, despite lower
rates of reactivation and afford protection from OPs in vivo (see
below).
In Vivo Studies. To test the protection of amidineꢀoximes to
exposure to OPs in in vivo experiments, nerve agent model
compounds of soman and sarin (i.e., rac-GDC and Sp-GB-Am,
Figure 7) were used. A nerve agent model of soman was selected
because it is known in the literature to be a particularly challenging
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Table 2. Effect of Vehicle, MINA, or AmidineꢀOxime 15d on
the Lethality of Sp-GB-Am
CLogP from þ0.5 to þ1.5), had reactivation potency of Sp-
GBC- or Sp-GF-SMe-inhibited BuChE comparable to or greater
than that of 2-PAM (CLogP ꢀ3.7). Despite the fact that the
amidineꢀoximes examined did not afford larger reactivation
rates than 2-PAM for OP-inhibited AChE, two amidineꢀoximes
were efficacious in vivo. For example, pretreatment with amidi-
neꢀoximes 15c,d (i.e., 30 min prior to OP administration, ip)
was able to protect mice from rac-GDC toxicity. In a post-
treatment experiment, animals were exposed to the sarin nerve
agent model compound Sp-GB-Am and treated 5 min later with
15d or MINA. The group of mice treated with the amidi-
neꢀoxime survived 24 h and had only minor signs of CNS
toxicity, whereas in the case of MINA or vehicle only 3/6 or 2/6
animals, respectively, survived. In the latter case, very severe
neurotoxicity symptoms were observed for the vehicle- or
MINA-treated animals. We hypothesize that even though ami-
dineꢀoximes are less potent than 2-PAM in reactivation of OP-
AChEs in vitro, they are orders of magnitude more lipophilic
than 2-PAM and achieve much greater concentrations in the
brain and effectively protect against either pre- or post-treatment
of toxic OPs. Additionally, because both enzymes AChE and
BuChE are present in the brain,35 efficient reactivation of BuChE
by amidineꢀoximes 15c and 15d likely also contributed to the
protection of OP-treated animals. We conclude that treatment of
mice with amidineꢀoximes 15c,d delivered the compound to the
plasma and to the CNS and protected animals from CNS toxicity.
The concept of using amidineꢀoximes as an alternative to
quaternary oximes (e.g., 2-PAM) to more efficiently protect the
brain from toxicity of OPs needs to be examined in additional
animal models.
Sp-GB-Am
experiment
1
treatmenta
vehicle
μmol mg/kg μmol survival (24 h)
ꢀ
0.08 0.305
2/6
3/6
6/6
MINA (12.6 mg/kg) 145
15d (28 mg/kg)b
a Mice were treated (ip) with Sp-GB-Am and 5 min later vehicle,
amidineꢀoxime 15d, or MINA was administered. b Oxime 15d was
administered as a hydrochloride salt in isotonic saline. P values for expt 1
(vehicle vs 15d) = 0.06, (MINA vs 15d) = 0.18.
amidineꢀoximes 15c,d provided complete protection to ani-
mals, compared to 2-PAM, which protected 6/8 mice. In the
third experiment, animals were pretreated (ip) with only 25% of
the previous oxime dose (i.e., 6.5 mg/kg for 15c and 6.25 mg/kg
for 2-PAM). In this experiment, none of the animals from the
2-PAM-treated group survived, whereas 15c protected 4/6 animals
(Table 1).
We sought to extend the protection studies to another nerve
agent model compound to examine the universality of the
protective effects of amidineꢀoximes. In all likelihood, in the
case of a real life situation, the OP-exposed victim will be treated
with oxime antidotes after OP exposure (post-treatment). To
verify whether amidineꢀoximes are able to protect animals from
this more challenging post-treatment situation, mice were ex-
posed to sarin model compound (Sp-GB-Am, Figure 7) and
5 min later the vehicle, MINA (12.6 mg/kg, 145 μmol/kg, i.p.),
or 15d (28 mg/kg, 145 μmol/kg, ip) was administered. Animals
were observed for neurological toxicity symptoms as above, and
after 24 h, survival was recorded (Table 2). MINA was used as a
control because this is currently the only known BBB-penetrating
oxime commercially available to compare efficacy with amidineꢀ
oxime 15d.
’ EXPERIMENTAL SECTION
General. All reagents and solvents were used as received from
commercial sources. Buffers were purchased from VWR Scientific, Inc.
(San Diego, CA) in the highest purity commercially available. Synthetic
products were isolated using a flash column chromatography system
(Teledyne ISCO, CombiFlash Rf) with UV detection at 254 nm. NMR
spectra were recorded at 300 MHz (1H) on a Varian Mercury 300 or at
125 MHz (13C) on a Bruker AMX-500 II (NuMega Resonance Lab, San
Diego, CA) in CDCl3 or DMSO-d6, respectively. Chemical shifts were
reported as ppm (δ) relative to CDCl3 at 7.26 ppm and DMSO-d6 at
2.50 or 39.52 ppm. Low-resolution mass spectra were obtained using an
Hitachi M-8000 mass spectrometer with an ESI source. Elemental
analyses were obtained using a Perkin-Elmer PE2400-Series II, CHNS/O
analyzer (NuMega Resonance Lab, San Diego, CA). Calculated ClogP
values were determined using ChemDraw Ultra 11.0. Melting points
were determined using Mettler Toledo FP62 and were uncorrected.
UVꢀvis spectra were recorded using a Nanodrop 1000 spectrophoto-
meter from Thermo Scientific. The pKa values were calculated from the
correlation of absorbance/pH using GraphPad Prism 5.0.
Purity Determination. Purity of products was determined by a
Hitachi 8000 LCꢀMS (Hitachi, San Jose, CA) using reverse phase
chromatography (C18 column, 50 ꢁ 4.6 mm, 5 μm, Thomson Instru-
ment Co., Oceanside, CA). Compounds were eluted using a gradient
elution of 95/5 to 5/95A/B over 10 min at a flow rate of 1.5 mL/min,
where solvent A was aqueous 0.05% TFA and solvent B was acetonitrile
(0.05% TFA). For purity data, peak area percent for the TIC, at 210 or
254 nm, and retention time (tR in minutes) are provided (see Supporting
Information). Purity of products was g95%. Purity for certain com-
pounds was determined using combustion analysis. Thioimidates 8 and
13aꢀd decompose in protic solvents and were used in the synthesis
without further purification and purity determination.
As shown in Table 2, for vehicle-treated animals 2/6 survived
24 h and significant CNS toxicity was observed (i.e., strong
seizures, lack of spontaneous activity, and no interest in food
consumption). Similar symptoms were observed for the MINA-
treated group, which protected 3/6 animals. After administration
of amidineꢀoxime 15d, all OP-treated mice survived the treat-
ment and showed behavioral activity comparable with healthy,
untreated animals.
’ DISCUSSION AND CONCLUSIONS
In summary, novel amidineꢀoximes were designed and pre-
pared using thioimidate intermediates using two different pro-
tected oxime functionalities. Use of p-methoxybenzyl (PMB)
instead of benzyl (Bn) as a protecting group significantly
improved the synthetic efficiency of amidineꢀoximes. Oximes
15aꢀd and 19 were tested in vitro for reactivation of inhibited
AChE and BuChE in the presence of nerve agent model
compounds and pesticide ETP. On the basis of these results,
the amidineꢀoximes examined had superior functional activity
compared with MINA for reactivation of inhibited AChE and
BuChE. Increase in the lipophilic character of the substituent on
the amidine group of the amidineꢀoximes from hydrogen atom
(for 15a) to N-butyl (for 19) consistently increased the reactiva-
tion potency for Sp-GBC- or Sp-GF-SMe-inhibited BuChE. The
most lipophilic compounds, 15c,d and 19 (i.e., an increase in
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Caution! Nerve agent model compounds used in these studies are
toxic and must be handled with extreme care by well-trained personnel.
Use of these materials has been approved by NIH and DOD. After
reactivation studies, biochemical samples were neutralized by stirring
with 2 M NaOH/EtOH for 12 h. Remaining solutions were brought
back to pH ∼7 and disposed of in chemical waste.
71%). 1H NMR (300 MHz, CDCl3) δ = 7.47 (s, 1H), 7.45ꢀ7.28 (m,
5H), 6.48 (br s, 1H), 5.19 (s, 2H), 3.28 (q, J = 6.9 Hz, 2H), 1.62ꢀ1.51
(m, 2H), 0.94 (t, J = 7.2 Hz, 3H).
2-(Benzyloxyimino)ethanethioamide (12a). A mixture of 11a
(3 g, 16.8 mmol) and Lawesson’s reagent (4.1 g, 10.1 mmol, 0.6 equiv) in
anhydrous THF (150 mL) was stirred at 60 °C 1 h and then evaporated.
The crude material was purified by column chromatography (silica gel,
2,2-Diethoxy-N-methylacetamide (6). A mixture of ethyl
diethoxyacetate (15 g, 85.2 mmol) and MeNH2 (40% in H2O,
15 mL) was stirred at room temperature (rt) overnight. The mixture
was evaporated and a clear oil was used without further purification (13.6
g, 99%). 1H NMR (300 MHz, CDCl3) δ = 6.62 (br s, 1H), 4.79 (s, 1H),
3.72ꢀ3.53 (m, 4H), 2.84 (d, J = 4.8 Hz, 3H), 1.24 (t, J = 7.1 Hz, 6H).
2,2-Diethoxy-N-methylethanethioamide (7). A mixture of 6
(12 g, 74.5 mmol) and Lawesson’s reagent (18.1 g, 44.7 mmol, 0.6
equiv) in anhydrous THF (150 mL) was stirred at 60 °C 1 h and then
evaporated. The residue was extracted using hexanes:EtOAc (9:1, 5 ꢁ
100 mL). Combined organic layers were evaporated, and the residue was
purified by column chromatography (silica gel, hexanesf9:1, hexanes:
EtOAc) to give a yellow oil (6.2 g, 47%). 1H NMR (300 MHz, CDCl3) δ
= 8.41 (br s, 1H), 5.08 (s, 1H), 3.72ꢀ3.58 (m, 4H), 3.19 (d, J = 4.8 Hz,
3H), 1.25 (t, J = 7.1 Hz, 6H).
1
hexanesf9:1, hexanes:EtOAc) to give a yellow solid (3 g, 92%). H
NMR (300 MHz, CDCl3) δ = 7.78 (s, 1H), 7.42ꢀ7.31 (m, 5H), 5.21 (s,
2H).
2-(Benzyloxyimino)-N-methylethanethioamide (12b). The
1
title compound was obtained similarly to 12a (yellow solid, 90%). H
NMR (300 MHz, CDCl3) δ = 8.25 (br s, 1H), 7.84 (s, 1H), 7.41ꢀ7.31
(m, 5H), 5.19 (s, 2H), 3.22 (d, J = 5.1 Hz, 3H).
2-(Benzyloxyimino)-N-ethylethanethioamide (12c). The ti-
1
tle compound was obtained similarly to 12a (yellow solid, 89%). H
NMR (300 MHz, CDCl3) δ = 8.18 (br s, 1H), 7.81 (s, 1H), 7.42ꢀ7.28
(m, 5H), 5.20 (s, 2H), 3.78ꢀ3.67 (m, 2H), 1.31 (t, J = 7.2 Hz, 3H).
2-(Benzyloxyimino)-N-propylethanethioamide (12d). The
1
title compound was obtained similarly to 12a (yellow solid, 85%). H
NMR (300 MHz, CDCl3) δ = 8.19 (br s, 1H), 7.81 (s, 1H), 7.41ꢀ7.27
(m, 5H), 5.19 (s, 2H), 3.69ꢀ3.62 (m, 2H), 1.78ꢀ1.66 (m, 2H), 0.99
(t, J = 7.5 Hz, 3H).
Methyl 2,2-Diethoxy-N-methylethanimidothioate Trifluoro-
methanesulfonate Salt (8). To a solution of 7 (3 g, 17 mmol) in
MeNO2 (50 mL) at rt was added MeOTf (1.9 mL, 2.8 g, 17 mmol). The
mixturewasstirredatrtfor1handthenevaporatedtogiveathickyellow oil
(5.8 g, 100%). The crude product was used in the next step without further
Methyl 2-(Benzyloxyimino)ethanimidothioate Trifluoro-
methanesulfonate Salt (13a). To a solution of 12a (2.6 g, 13.4
mmol) in MeNO2 (30 mL) at rt was added MeOTf (1.6 mL, 2.4 g, 14.7
mmol, 1.1 equiv). The mixture was stirred at rt overnight and then
evaporated to give a dark red solid (4.8 g, quant.). Crude product was
used in the next step without further purification. 1H NMR (300 MHz,
CDCl3) δ = 11.24 (br s, 1H), 10.70 (br s, 1H), 8.25 (s, 1H), 7.41ꢀ7.32
(m, 5H), 5.35 (s, 2H), 2.71 (s, 3H).
1
purification. H NMR (300 MHz, CDCl3) δ = 12.08 (br s, 1H), 5.64
(s, 1H), 3.83ꢀ3.71 (m, 4H), 3.33 (d, J = 5.1 Hz, 3H), 2.98 (s, 3H), 1.29 (t,
J = 7.2 Hz, 6H).
2,2-Diethoxy-N,N,N0-trimethylacetimidamide Hydrochlo-
ride Salt (9). To a solution of 8 (3.6 g, 10.5 mmol) in anhydrous THF
(10.5 mL) was added Me2NH (2 M in THF, 10.5 mL, 21 mmol, 2
equiv). The mixture was stirred at rt overnight and then evaporated.
The crude oil was purified by column chromatography (silica gel,
CH2Cl2f9:1, CH2Cl2:MeOH) to give a brown oil as the triflate salt.
The product was dissolved in a small amount of H2O and passed through
an ion-exchange resin column IRA-400 (Chl). The aqueous layer was
washed with CH2Cl2 (2 ꢁ 50 mL) and evaporated to give a yellow oil
(1.4 g, 59%). 1H NMR (300 MHz, DMSO-d6) δ = 9.02 (br s, 1H), 5.71
(s, 1H), 3.84ꢀ3.63 (m, 4H), 3.25ꢀ3.07 (m, 9H), 1.18 (t, J = 7.1 Hz,
6H). ESI MS (MeOH) [M þ H]þ = 188.67 Da.
Methyl 2-(Benzyloxyimino)-N-methylethanimidothioate
Trifluoromethanesulfonate Salt (13b). The title compound was
1
obtained similarly to 13a (yellow solid, quant.). H NMR (300 MHz,
CDCl3) δ = 12.23 (br s, 1H), 8.29 (s, 1H), 7.40ꢀ7.34 (m, 5H), 5.37
(s, 2H), 3.35 (d, J = 5.1 Hz, 3H), 2.72 (s, 3H).
Methyl 2-(Benzyloxyimino)-N-ethylethanimidothioate Tri-
fluoromethanesulfonate Salt (13c). The title compound was ob-
tained similarly to 13a (red oil, quant.). 1H NMR (300 MHz, CDCl3) δ =
12.26 (br s, 1H), 8.36 (s, 1H), 7.40ꢀ7.29 (m, 5H), 5.36 (s, 2H),
3.76ꢀ3.66 (m, 2H), 2.67 (s, 3H), 1.50ꢀ1.43 (m, 3H).
2-(Benzyloxyimino)acetamide (11a). A mixture of 1025 (2 g,
9.6 mmol) and 25% NH3 ꢁ H2O (10 mL) was stirred at rt overnight.
The reaction was filtered and the product was obtained as a white solid
(1.1 g, 64%). 1H NMR (300 MHz, CDCl3) δ = 7.48ꢀ7.27 (m, 6H), 6.39
(br s, 1H), 5.59 (br s, 1H), 5.20 (s, 2H).
Methyl 2-(Benzyloxyimino)-N-propylethanimidothioate Tri-
fluoromethanesulfonate Salt (13d). The title compound was
1
obtained similarly to 13a (yellow oil, quant.). H NMR (300 MHz,
CDCl3) δ = 12.22 (br s, 1H), 8.37 (s, 1H), 7.41ꢀ7.30 (m, 5H), 5.36
(s, 2H), 3.62 (q, J = 6.6 Hz, 2H), 2.66 (s, 3H), 1.96ꢀ1.82 (m, 2H), 1.02
(t, J = 7.6 Hz, 3H).
2-(Benzyloxyimino)-N-methylacetamide (11b). A mixture of
10 (10 g, 48.3 mmol) and MeNH2 (40% in H2O, 10 mL) was stirred at rt
overnight and then diluted with H2O (50 mL) and filtered. The product
was washed with a small amount of Et2O to give a yellow solid (7 g,
75%). 1H NMR (300 MHz, CDCl3) δ = 7.48 (s, 1H), 7.43ꢀ7.29 (m,
5H), 6.46 (br s, 1H), 5.18 (s, 2H), 2.88 (d, J = 6.8 Hz, 3H).
2-(Benzyloxyimino)-N,N-dimethylacetimidamide Hydro-
chloride Salt (14a). To a solution of 13a (2.1 g, 5.8 mmol) in
anhydrous THF (25 mL) was added Me2NH (2 M in THF, 5.8 mL, 11.6
mmol, 2 equiv). The mixture was stirred at rt for 10 min and then
evaporated. The crude oil was purified by column chromatography
(silica gel, CH2Cl2f9:1, CH2Cl2:MeOH) to give a brown oil of the
triflate salt. The product was dissolved in small amount of hot 5% MeOH
in H2O and passed through an ion-exchange resin column IRA-400 (Cl).
The aqueous layer was washed with Et2O (1 ꢁ 100 mL) and evaporated
to give a brown oil. The product was then stirred with anhydrous THF
2-(Benzyloxyimino)-N-ethylacetamide (11c). A mixture of 10
(5 g, 24 mmol) and EtNH2 (2 M in MeOH, 24 mL, 48 mmol, 2 equiv)
was stirred at 60 °C overnight and then evaporated. The residue was
purified by column chromatography (silica gel, hexanesf7:3, hexanes:
EtOAc) to give a yellow solid (3 g, 60%). 1H NMR (300 MHz, CDCl3)
δ = 7.47 (s, 1H), 7.40ꢀ7.29 (m, 5H), 6.42 (br s, 1H), 5.19 (s, 2H),
3.40ꢀ3.32 (m, 2H), 1.19 (t, J = 7.2 Hz, 3H).
1
and changed into a white solid (980 mg, 69%). H NMR (300 MHz,
DMSO-d6) δ = 9.18 (br s, 1H), 8.85 (br s, 1H), 8.39 (s, 1H), 7.51ꢀ7.30
(m, 5H), 5.30 (s, 2H), 3.21 (s, 3H), 3.13 (s, 3H). 13C NMR (125 MHz,
DMSO-d6) δ = 155.9, 140.1, 136.1, 128.6, 128.5, 128.4, 77.4, 40.6, 33.9.
ESI MS for [M þ H]þ = 205.95 Da.
2-(Benzyloxyimino)-N-propylacetamide (11d). A mixture of
10 (5 g, 24.1 mmol) and PrNH2 (3.9 mL, 2.8 g, 48.2 mmol, 2 equiv) in
anhydrous EtOH (50 mL) was stirred at 60 °C 1 day and then
evaporated. The residue was purified by column chromatography
(silica gel, hexanesf7:3, hexanes:EtOAc) to give a yellow solid (3.8 g,
2-(Benzyloxyimino)-N,N,N0-trimethylacetimidamide Hy-
drochloride Salt (14b). To a solution of 13b (5.7 g, 15.4 mmol)
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in anhydrous THF (15 mL) was added Me2NH (2 M in THF, 15.4 mL,
30.8 mmol, 2 equiv). The mixture was stirred at rt overnight and then
evaporated. The crude oil was suspended in a small amount of hot H2O
and passed through an ion-exchange resin column IRA-400 (Cl). The
aqueous layer was washed with CH2Cl2 (2 ꢁ 50 mL) and evaporated.
The crude 14b (2.6 g, 10.2 mmol) was dissolved in H2O (10 mL) and a
solution of AgNO3 (1.7 g, 10.2 mmol) in H2O (10 mL) was added. The
mixture was stirred at rt exposed to sunlight for 1 h and then filtered and
evaporated. The crude oil was purified by column chromatography
(silica gel, 9:1, CH2Cl2:MeOH) to give a white solid of 14b as the nitrate
salt (1.4 g, 32%). The 14b chloride salt can be made by using an ion-
exchange resin IRA-400 (Chl). For the chloride salt: 1H NMR (300 MHz,
DMSO-d6) δ = 9.47 (s, 1H), 8.27 (s, 1H), 7.42ꢀ7.34 (m, 5H), 5.28
(s, 2H), 3.11 (s, 6H), 2.84 (s, 3H). For the nitrate salt: 1H NMR (300
MHz, DMSO-d6) δ = 9.00 (s, 1H), 8.26 (s, 1H), 7.41ꢀ7.34 (m, 5H),
5.28 (s, 2H), 3.09 (s, 6H), 2.85 (s, 3H). 13C NMR (125 MHz, DMSO-
d6) δ = 156.6, 140.3, 136.4, 128.5, 128.3, 77.0, 41.5, 31.7. ESI MS
(MeOH): [M þ H]þ = 219.80 Da. Anal. Calcd for C12H18N4O4: C,
51.06; H, 6.43; N, 19.85. Found: C, 51.20; H, 6.95; N, 20.01.
gel, CH2Cl2f8:2, CH2Cl2:MeOH). Unreacted starting material was
recovered (100 mg) and the product was obtained as a yellowish solid
1
(250 mg, 97%), mp = 138.9 °C. H NMR (300 MHz, DMSO-d6)
δ = 12.97 (br s, 1H), 9.29 (br s, 1H), 8.07 (s, 1H), 3.15 (s, 6H), 2.93 (s,
3H). 13C NMR (125 MHz, DMSO-d6) δ = 157.3, 138.9, 40.6, 31.4. ESI
MS for [M þ H]þ = 129.7 Da.
N0-Ethyl-2-(hydroxyimino)-N,N-dimethylacetimidamide Hy-
drochloride Salt (15c). A mixture of 14c (700 mg, 2.6 mmol) and 10%
Pd/C (50% wet, 276 mg, 0.13 mmol, 5% mol) in anhydrous THF (50 mL)
was stirred under a H2 atmosphere (balloon) at rt overnight. The reaction
was filtered through Celite, washed with MeOH, and evaporated. The crude
product was purified by column chromatography (silica gel, CH2Cl2f8:2,
CH2Cl2:MeOH) to give a yellow oil which turned into a white solid once
stirred in THF (230 mg, 49%; 15c was obtained in 41% yield when the
reaction was carried out in anhydrous EtOH), mp =155.1 °C (dec). 1H
NMR (300 MHz, DMSO-d6) δ = 12.92 (br s, 1H), 9.38 (br s, 1H), 8.09
(s, 1H), 3.34 (q, J = 7.2 Hz, 2H), 3.14 (s, 6H), 1.13 (t, J = 7.2 Hz, 3H). 13
C
NMR (125 MHz, DMSO-d6) δ = 156.9, 138.8, 41.3, 39.9, 15.3. ESI MS for
[M þ H]þ = 143.88 Da. Anal. Calcd for C6H14ClN3O: C, 40.11; H, 7.85;
N, 23.39. Found: C, 40.07; H, 7.80; N, 23.41.
2-(Benzyloxyimino)-N0-ethyl-N,N-dimethylacetimidamide Hy-
drochloride Salt (14c). To a solution of 13c (14.2 g, 36.8 mmol) in
anhydrous THF (250 mL) cooled to 0ꢀ5 °C (ice-bath) was added
Me2NH (2 M in THF, 36.8 mL, 73.6 mmol, 2 equiv). The mixture was
stirred at 0ꢀ5 °C for 5 min and at rt 40 min and then evaporated. The
crude oil was purified by column chromatography (silica gel,
CH2Cl2f9:1, CH2Cl2:MeOH) to give a yellowish oil. 14c as the triflate
salt was dissolved in small amount of hot 5% MeOH in H2O and passed
through an ion-exchange resin column IRA-400 (Cl). The aqueous layer
was washed with Et2O (1 ꢁ 100 mL) and evaporated. 14c (4.4 g, 16.3
mmol) was dissolved in H2O (50 mL) and a solution of AgNO3 (2.8 g,
16.3 mmol) in H2O (50 mL) was added. The mixture was stirred at rt
exposed to sunlight for 1 h and then filtered, passed through IRA-400
(Chl) column, and evaporated to give a yellowish oil that crystallized after
drying on high vacuum (4.4 g, 44%). 1H NMR (300 MHz, DMSO-d6)
δ = 9.48 (br s, 1H), 8.30 (s, 1H), 7.42ꢀ7.30 (m, 5H), 5.27 (s, 2H),
3.28ꢀ3.04 (m, 8H), 0.99 (t, J = 7.2 Hz, 3H). 13C NMR (125 MHz,
DMSO-d6) δ = 155.8, 140.5, 136.6, 128.4, 128.3, 128.2, 76.8, 41.3, 40.0,
15.2. ESI MS for [M þ H]þ = 233.88 Da.
2-(Hydroxyimino)-N,N-dimethyl-N0-propylacetimidamide
Hydrochloride Salt (15d). From 14d. A mixture of 14d (300 mg,
1.06 mmol) and 10% Pd/C (50% wet, 112 mg, 0.05 mmol, 5% mol) in
anhydrous EtOH (5 mL) was stirred under a H2 atmosphere (balloon)
at rt for 4 h. Then reaction was filtered through Celite and evaporated.
The crude product was purified by column chromatography (silica gel,
CH2Cl2f8:2, CH2Cl2:MeOH) to give a white solid (50 mg, 24%),
mp =135.1 °C (dec).
From 18a (Two-Step Procedure). To a solution of 18a (2.8 g, 10.5
mmol) in MeNO2 (20 mL) at rt was added MeOTf (1.2 mL, 1.7 g, 10.5
mmol). The reaction mixture was stirred at rt overnight and then
evaporated. The crude product from the previous step was dissolved
in anhydrous THF (100 mL) and Me2NH (2 M in THF, 10.5 mL, 21
mmol, 2 equiv) was added. The reaction was carried out at rt for 30 min
and then evaporated. The crude mixture was purified by column
chromatography (silica gel, CH2Cl2f9:1, CH2Cl2:MeOH) to give
15d as the triflate salt. The chloride salt was obtained using the IRA-
400 (Cl) ion-exchange resin (yellow oil, 880 mg, 43% over two steps).
1H NMR (300 MHz, DMSO-d6) δ = 12.93 (br s, 1H), 9.42 (br s, 1H),
8.08 (s, 1H), 3.30ꢀ3.20 (m, 2H, the signal overlaps with residual H2O),
3.15 (s, 6H), 1.59ꢀ1.47 (m, 2H), 0.82 (t, J = 7.5 Hz, 3H). 13C NMR
(125 MHz, DMSO-d6) δ = 157.0, 138.8, 46.3, 41.5, 22.8, 10.7. ESI MS
for [M þ H]þ = 157.75 Da.
2-(Benzyloxyimino)-N,N-dimethyl-N0-propylacetimidamide Hy-
drochloride Salt (14d). The title compound was obtained similarly to 14c,
but the reaction time was 1 h (yellow oil, 23%). 1H NMR (300 MHz, DMSO-
d6) δ = 9.45 (br s, 1H), 8.30 (s, 1H), 7.42ꢀ7.31 (m, 5H), 5.27 (s, 2H),
3.17ꢀ3.06 (m, 8H), 1.44ꢀ1.33 (m, 2H), 0.71 (t, J = 7.5 Hz, 3H). 13C NMR
(125 MHz, DMSO-d6) δ = 156.0, 140.5, 136.6, 128.4, 128.3, 128.2, 76.8, 46.3,
41.5, 22.8, 10.6. ESI MS for [M þ H]þ = 247.88 Da.
Ethyl 2-(4-Methoxybenzyloxyimino)acetate (16). A mixture
of ethyl glyoxylate (50% in toluene, 75.3 g, 369 mmol) and NH2OH ꢁ
HCl (25.6 g, 369 mmol) in CH3CN:H2O (9:1, 300 mL) was stirred at rt
for 5 min and then Et3N (51.7 mL, 37.5 g, 369 mmol) was added
dropwise. The reaction was carried out at rt for 1 h and then evaporated.
The residue was dissolved in H2O (50 mL) and Et2O (300 mL). The
organic layer was washed with saturated NH4Cl (50 mL), dried over
Na2SO4, filtered, and evaporated to give colorless wet crystals of ethyl
2-(hydroxyimino)acetate (41.9 g, 97%). 1H NMR (300 MHz, CDCl3)
δ = 9.52 (br s, 1H), 7.56 (s, 1H), 4.32 (q, J = 7.1 Hz, 2H), 1.34 (t, J = 7.1
Hz, 3H).
To a mixture of NaH (60%, 5.64 g, 141.0 mmol, 1.1 equiv) in
anhydrous DMF (650 mL) cooled to 0ꢀ5 °C was added dropwise a
solution of ethyl 2-(hydroxyimino)acetate (15 g, 128.2 mmol) in
anhydrous DMF (50 mL). After addition, the cooling bath was removed
and the reaction was stirred at rt for 1 h. Finally, a solution of
1-(chloromethyl)-4-methoxybenzene (17.5 mL, 20.1 g, 128.2 mmol)
in anhydrous DMF (50 mL) was added and the reaction was stirred at rt
overnight. After evaporation, the yellow oil was dissolved in H2O
(100 mL) and Et2O (500 mL). The organic layer was washed with
brine (3 ꢁ 50 mL), dried over Na2SO4, filtered, and evaporated. The
2-(Hydroxyimino)-N,N-dimethylacetimidamide Hydrochlo-
ride Salt (15a). To a mixture of 14a (500 mg, 2.1 mmol) in anhydrous
CH2Cl2 (10 mL) was added anhydrous AlCl3 (838 mg, 6.3 mmol, 3
equiv). The mixture was stirred at rt for 1 day then MeOH was carefully
added followed by H2O (1 mL). The mixture was evaporated and the
yellow residue was purified by column chromatography (silica gel,
CH2Cl2f8:2, CH2Cl2:MeOH) to give a white solid. The product
was dissolved in H2O (2 mL), passed through the IRA-400 (Cl) column,
and evaporated to give a white solid (300 mg, 96%), mp = 145.7 °C
(dec). 1H NMR (300 MHz, DMSO-d6) δ = 13.11 (s, 1H), 9.20ꢀ8.75
(m, 2H), 8.21 (s, 1H), 3.22 (s, 3H), 3.14 (s, 3H). 13C NMR (125 MHz,
DMSO-d6) δ = 156.9, 139.0, 40.4, 33.9. ESI MS for [M þ H]þ
=
115.95 Da.
2-(Hydroxyimino)-N,N,N0-trimethylacetimidamide Hydro-
chloride Salt (15b). A mixture of 14b (500 mg, 1.95 mmol) and 10%
Pd/C (50% wet, 413 mg, 0.195 mmol, 10% mol) in anhydrous THF
(20 mL) was stirred under H2 atmosphere (balloon) at rt overnight. The
reaction was filtered through Celite, washed with i-PrOH, and evapo-
rated. The crude product was purified by column chromatography (silica
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Journal of Medicinal Chemistry
ARTICLE
crude product was purified by column chromatography (silica gel,
hexanesf9:1, hexanes:EtOAc) to give a light yellow oil (22 g, 72%).
1H NMR (300 MHz, CDCl3) δ = 7.51 (s, 1H), 7.34ꢀ7.30 (m, 2H),
6.92ꢀ6.88 (m, 2H), 5.23 (s, 2H), 4.32 (q, J = 7.2 Hz, 2H), 3.81 (s, 3H),
1.34 (t, J = 7.2 Hz, 3H).
reaction of the enzyme with nerve agent model compounds afforded
covalent modification of the enzyme identical to that obtained with
authentic nerve agents.28,29 An excess of OP was removed from inhibited
enzyme by filtration through a 10 kDa MWCO filter with a modified
PES membrane (VWR Scientific, Inc., San Diego, CA), followed by two
washes prior to the final resuspension in PBS/BLG (pH 7.4). An excess
of 1000-fold dilution of OP was achieved. Enzyme was then added to
PBS/BLG (pH 7.4) containing amidineꢀoxime, MINA, or 2-PAM (100
μM oxime with 2% DMSO after addition of enzyme) or vehicle. Oxime
samples were equilibrated at 37 °C prior to addition of enzyme. Enzyme
was allowed to reactivate at 37 °C for 20 min (BuChE) or 1 h (AChE), at
which time the catalytic activity was determined using a modified
Ellman’s assay36 in the presence of 1 mM substrate in PBS/BLG (pH
7.4). The Ellman assay involved a 20-fold dilution of enzyme from the
oxime incubation, yielding incubation concentrations in the range of
10ꢀ50 units Lꢀ1 (where 1 unit cleaves 1 μmol of substrate per min in
PBS pH 7.4, room temperature) and 5 μM oxime. Two reactivation
samples were prepared per oxime. The reported data show the average
esterase activity for each oxime divided by the average activity of
noninhibited control samples and the propagated errors of the average
activities.
In Vivo Studies. Adult Swiss Webster female mice (from Taconic)
were housed in groups of four and maintained in a temperature-
controlled environment on a 12 h:12 h light cycle (0600 h on, 1800 h
off) upon arrival to the laboratory. Animals were given free access to
food and water during a 1-week habituation period to the laboratory.
Animals used in the research studies were handled, housed, and killed in
accordance with current IACUC protocol (024ꢀ2008) and NIH guide-
lines regarding the use and care of laboratory animals and all applicable
local, state, and federal regulations and guidelines.
rac-GDC Studies. Separate groups of mice were amidineꢀoximes
(145 μmol/kg), 2-PAM (145 μmol/kg), or vehicle pretreated (ip) 30
min prior to rac-GDC (0.25 mg/kg in 0.25 mL/mouse ip). Each test
compound and 2-PAM as hydrochloride salts were dissolved in isotonic
saline and administered to separate groups in a volume of 0.25 mL/
mouse. The survival was recorded after 24 h from the onset of the
experiment. Animals that survived the 24 h period were then immedi-
ately killed by cervical dislocation.
Sp-GB-Am Studies. At the 0 min time point, mice received Sp-GB-
Am hydrochloride (0.08 mg/kg in 0.25 mL/mouse in saline, ip). Five
minutes later amidineꢀoxime 15d (145 μmol/kg), MINA (145 μmol/
kg), or vehicle was administered ip in isotonic saline (0.25 mL/mouse).
The survival was recorded after 24 h. Animals that survived 24 h were
then immediately killed by cervical dislocation.
2-(4-Methoxybenzyloxyimino)-N-propylacetamide (17a).
The title compound was obtained similarly to 11d (yellowish solid,
89%). 1H NMR (300 MHz, CDCl3) δ = 7.43 (s, 1H), 7.32ꢀ7.27 (m,
2H), 6.93ꢀ6.88 (m, 2H), 6.49 (br s, 1H), 5.11 (s, 2H), 3.81 (s, 3H), 3.28
(q, J = 6.9 Hz, 2H), 1.63ꢀ1.51 (m, 2H), 0.94 (t, J = 7.5 Hz, 3H).
N-Butyl-2-(4-methoxybenzyloxyimino)acetamide (17b).
The title compound was obtained similarly to 11d but using n-BuNH2
1
(white solid, 76%). H NMR (300 MHz, CDCl3) δ = 7.43 (s, 1H),
7.33ꢀ7.27 (m, 2H), 6.94ꢀ6.87 (m, 2H), 6.46 (br s, 1H), 5.11 (s, 2H),
3.82 (s, 3H), 3.35ꢀ3.28 (m, 2H), 1.58ꢀ1.48 (m, 2H), 1.43ꢀ1.30 (m,
2H), 0.94 (t, J = 7.2 Hz, 3H).
2-(4-Methoxybenzyloxyimino)-N-propylethanethio-
amide (18a). The title compound was obtained similarly to 12a
1
(yellow oil, 80%). H NMR (300 MHz, CDCl3) δ = 8.19 (br s, 1H),
7.78 (s, 1H), 7.31ꢀ7.27 (m, 2H), 6.93ꢀ6.88 (m, 2H), 5.12 (s, 2H),
3.82 (s, 3H), 3.69ꢀ3.62 (m, 2H), 1.79ꢀ1.67 (m, 2H), 1.00 (t, J =
7.5 Hz, 3H).
N-Butyl-2-(4-methoxybenzyloxyimino)ethanethioamide
(18b). The title compound was obtained similarly to 12a (yellow oil,
97%). 1H NMR (300 MHz, CDCl3) δ = 8.17 (br s, 1H), 7.77 (s, 1H),
7.31ꢀ7.27 (m, 2H), 6.93ꢀ6.88 (m, 2H), 5.11 (s, 2H), 3.82 (s, 3H),
3.77ꢀ3.65 (m, 2H), 1.73ꢀ1.63 (m, 2H), 1.48ꢀ1.35 (m, 2H), 0.97 (t,
J = 7.2 Hz, 3H).
N0-Butyl-2-(hydroxyimino)-N,N-dimethylacetimidamide
(19). The title compound was obtained similarly to 15d but using 18b
(yellow oil, 79%). 1H NMR (300 MHz, CDCl3) δ = 12.96 (br s, 1H),
9.57 (br s, 1H), 8.08 (s, 1H), 3.30ꢀ3.26 (m, 2H, the signal overlaps
with residual H2O), 3.15 (s, 6H), 1.55ꢀ1.45 (m, 2H), 1.31ꢀ1.18 (m,
2H), 0.84 (t, J = 7.2 Hz, 3H). 13C NMR (125 MHz, DMSO-d6) δ =
157.0, 138.8, 44.3, 41.4, 31.6, 19.0, 13.5. ESI MS for [M þ H]þ
=
171.88 Da.
Determination of pKa. Twenty-two buffers (50 mM, ionic
strength = 300 mM, pH from 3 to 13.5) used in these studies were
prepared according to the procedures from http://www.bioinformatics.
org/JaMBW/5/4/index.html. To a 900 μL buffer solution was added
100 μL of 10 mM solution of 15c in H2O. The UV spectrum for each pH
point was recorded in the range of 220ꢀ700 nm in triplicate, and
differences in absorbance at 260 nm were used for the pKa determina-
tion. pKa values were calculated from the correlation of absorbance/pH
using the equation below
’ ASSOCIATED CONTENT
C2 ꢁ 10ðpH ꢀ pK Þ C3 ꢁ 10ðpH ꢀ pK Þ
1
2
Yobs ¼ C1 þ
þ
1 þ 10ðpH ꢀ pK Þ
1 þ 10ðpH ꢀ pK Þ
1
2
S
Supporting Information. Tables with compounds purity
b
data, binding affinity to AChE and BuChE for 19, and reactiva-
tion of OP-inhibited AChE using 15aꢀd, 19, 2-PAM, and MINA
after 20 min. This material is available free of charge via the
Internet at http://pubs.acs.org.
where C1 is the absorbance at low pH, C2 is the increase in absorbance
resulting from deprotonation of the functional group with a pKa of pK1,
and C2 is the increase in absorbance due to deprotonation of the
functional group with a pKa of pK2.
Enzyme Studies. Recombinant AChE, β-lactoglobulin from bo-
vine milk (BLG), acetylthiocholine (ATC), butyrylthiocholine (BTC),
and 5,50-dithiobis-2-nitrobenzoic acid (DTNB) were purchased from
Sigma Aldrich Chemical Co., (St. Louis, MO). BuChE and ETP were
generous gifts from O. Lockridge of the University of Nebraska Medical
Center. Syntheses and purification methods of OP model compounds
were previously reported.28,29 Reactivation studies were conducted the
same for both AChE and BuChE enzymes. Enzyme was diluted in PBS
(pH 7.4) containing 50 ng/μL BLG (PBS/BLG), and a sample was
taken and set aside for noninhibited controls. The remaining portion was
incubated with a nerve agent model compound or ETP (Figure 7) for
sufficient time to achieve 90% or greater inhibition (i.e., ca. 15 min). The
’ AUTHOR INFORMATION
Corresponding Author
*E-mail: jkalisiak@hbri.org. Tel: 001-(858) 458-9305. Fax:
001-(858) 458-9311.
’ ACKNOWLEDGMENT
The financial support from HBRI is gratefully acknowledged.
We acknowledge the helpful support of Professor Oksana
Lockridge.
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Journal of Medicinal Chemistry
ARTICLE
’ ABBREVIATIONS USED
chloride through the bloodꢀbrain barrier in its dihydropyridine pro-
drug form. J. Med. Chem. 1976, 19, 113–117.
ACh, acetylcholine; AChE, acetylcholinesterase; ATC, acetylthiocho-
line; BBB, bloodꢀbrain barrier; BuChE, butyrylcholinesterase; BTC,
butyrylthiocholine; CNS, central nervous system; ChE, cholinester-
ase enzymes; DAM, diacetylmonooxime; DTNB, 5,50-dithiobis-2-
nitrobenzoic acid; BLG, β-lactoglobulin; PMB, p-methoxybenzyl
protecting group; MeOTf, methyl trifluoromethanesulfonate; MINA,
monoisonitrosoacetone; OP, organophosphate; 2-PAM, pralidoxime
(16) Shek, E.; Higuchi, T.; Bodor, N. Improved delivery through
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methyl-1,6-dihydropyridine-2-carbaldoxime hydrochloride, a pro-drug
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Thangavelu, S. G.; Herdman, C. A.; Leader, H.; Schulz, S. M.; Marek, E.;
Medynets, M. A.; Ku, T. C.; Evans, S. A.; Khan, F. A.; Owens, R. R.;
Nambiar, M. P.; Gordon, R. K. Pro-2-PAM therapy for central and
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carbaldoxime chloride. J. Med. Chem. 1976, 19, 102–107.
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