New safe method for preparation of sarin-exposed human erythrocytes acetylcholinesterase using non-toxic and stable sarin analogue isopropyl p-nitrophenyl methylphosphonate and its application to evaluation of nerve agent antidotes

Article abstract of DOI:10.1007/s11095-006-9123-1

Introduction. A non-toxic and stable sarin analogue, isopropyl p-nitrophenyl methylphosphonate (INMP), was synthesized for safe preparation of sarin-exposed acetylcholinesterase (AChE). Results and Discussion. This agent was stable for years, able to be handled in an ordinary laboratory without special care, and its 50% inhibitory concentration (IC₅₀) on 0.04 U/ml human erythrocytes AChE was 15 nM. This reagent was thought to be especially useful since it enables experiments that require sarin-inhibited AChE, such as the development of antidotes for sarin, in a usual laboratory. To demonstrate the usefulness of this method, 40 known and novel pyridinealdoxime methiodide (PAM)-type oxime antidotes were synthesized, and their reactivation activities to INMP-exposed AChE and structure-activities correlation were studied. Conclusion. Among the antidotes tested in this experiment except for 2-PAM, the compound found to have the highest reactivation activity, was the novel hydrophobic 2-PAM-type compound, 2-[(hydroxyimino)methyl]-1-[4-(tert- butyl)benzyl] pyridinium bromide.

Full text of DOI:10.1007/s11095-006-9123-1

#
Pharmaceutical Research, Vol. 23, No. 12, December 2006 ( 2006)
DOI: 10.1007/s11095-006-9123-1
Research Paper
New Safe Method for Preparation of Sarin-Exposed Human Erythrocytes
Acetylcholinesterase Using Non-Toxic and Stable Sarin Analogue
Isopropyl p-Nitrophenyl Methylphosphonate and its Application
to Evaluation of Nerve Agent Antidotes
Hikoto Ohta,1,6 Takeshi Ohmori,4 Shinichi Suzuki,5 Hiroshi Ikegaya,2 Koichi Sakurada,2 and Takehiko Takatori3
Received May 31, 2006; accepted July 14, 2006; published online November 10, 2006
Introduction. A non-toxic and stable sarin analogue, isopropyl p-nitrophenyl methylphosphonate
(INMP), was synthesized for safe preparation of sarin-exposed acetylcholinesterase (AChE).
Results and Discussion. This agent was stable for years, able to be handled in an ordinary laboratory
without special care, and its 50% inhibitory concentration (IC50) on 0.04 U/ml human erythrocytes
AChE was 15 nM. This reagent was thought to be especially useful since it enables experiments that
require sarin-inhibited AChE, such as the development of antidotes for sarin, in a usual laboratory. To
demonstrate the usefulness of this method, 40 known and novel pyridinealdoxime methiodide (PAM)-
type oxime antidotes were synthesized, and their reactivation activities to INMP-exposed AChE and
structureYactivities correlation were studied.
Conclusion. Among the antidotes tested in this experiment except for 2-PAM, the compound found to
have the highest reactivation activity, was the novel hydrophobic 2-PAM-type compound, 2-
[(hydroxyimino)methyl]-1-[4-(tert-butyl)benzyl] pyridinium bromide.
KEY WORDS: acetylcholinesterase; antidote; isopropyl p-nitrophenyl methylphosphonate (INMP);
PAM; sarin.
INTRODUCTION
nerve agent, sarin (isopropyl methylphosphonofluoridate, 1a
in Fig. 1).
In these attacks, 19 people were killed and over 5,000
In Japan, in 1994 and 1995 ordinary citizens suffered
from two horrifying terrorist attacks with an extremely toxic injured (1Y3), and we previously reported a new method for
detection of the hydrolysis products of sarin from the blood
1 Toxicology Section, Department of Forensic Chemistry, National
and brain of victims (4Y6). In Japan, synthesis and use of
sarin are strictly controlled by the Chemical Warfare
Convention, even for basic studies. This strongly prevents
Research Institute of Police Science, 6-3-1, Kashiwa-no-ha,
Kashiwa, Chiba, 277-0882, Japan.
2 Department of Forensic Biology, National Research Institute of
Police Science, 6-3-1, Kashiwa-no-ha, Kashiwa, Chiba, 277-0882, general researchers from studying the pathophysiology of
Japan.
3 National Research Institute of Police Science, 6-3-1, Kashiwa-no-ha,
Kashiwa, Chiba, 277-0882, Japan.
4 Chemical Warfare Section, Department of Forensic Chemistry,
National Research Institute of Police Science, 6-3-1, Kashiwa-no-ha,
Kashiwa, Chiba, 277-0882, Japan.
5 Physical Evidence Section, Department of Forensic Chemistry,
National Research Institute of Police Science, 6-3-1, Kashiwa-no-ha,
Kashiwa, Chiba, 277-0882, Japan.
6 To whom correspondence should be addressed. (e–mail:ohta@
nrips.go.jp)
sarin poisoning and developing antidotes for nerve agents.
We have already reported a preparation method for sarin-
exposed human erythrocytes by using bis(isopropyl methyl-
phosphonate; BIMP) instead of extremely dangerous sarin
itself (4). Unfortunately, BIMP is still toxic, and readily
hydrolyzed during storage; thus, it must be freshly prepared
for experimental use. In this paper, we report a new, safe and
convenient method for preparation of sarin-exposed human
erythrocytes acetylcholinesterase (AChE) using a stable and
non-toxic sarin analogue, isopropyl p-nitrophenyl methyl-
phosphonate (INMP, 1b in Fig. 1).
According to our previous work, at the 1995 sarin attack
in the Tokyo subway, there were several cases of victims
dying by sarin intoxication after the antidote 2-pyridineal-
doxime methiodide (2-PAM, 2a in Fig. 1) was administered
(5). Their plasma AChE activities had been recovered by 2-
PAM administration, but brain AChE activities could not be
recovered (5), even though 2-PAM is reported to penetrate
the bloodYbrain barrier (BBB) somewhat (7). On the other
ABBREVIATIONS: AChE, acetylcholinesterase; BBB, bloodYbrain
barrier; BIMP, bis(isopropyl methylphosphonate); CH2Cl2,
dichloromethane; DCC, dicyclohexylcarbodiimide; DCU,
dicyclohexylurea; DMAP, dimethylaminopyridine; IC50, 50%
inhibitory concentration; IMPA, methylphosphonic acid monoisopropyl
ester; INMP, isopropyl p-nitrophenyl methylphosphonate; LCYESI-MS,
liquid chromatographyYelectrospray mass spectrometry; NMR, nuclear
magnetic resonance; PAM, pyridinealdoxime methiodide; pKa,
dissociation constant; RT, room temperature; THF, tetrahydrofuran;
TLC, thin-layer chromatography.
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2006 Springer Science + Business Media, Inc.

2828
Ohta et al.
monitored by thin-layer chromatography (TLC) using Merck
Kieselgel F254 silica-gel plate (0.25 mm thickness) as the
stationary phase, n-hexane-ethyl acetate (2:1) as the mobile
phase, and ultraviolet absorption at 254 nm as the detecting
method. The Rf values of INMP and p-nitrophenol were 0.4
and 0.8, respectively. The mixture was further stirred for 48
h at RT until the spot of p-nitrophenol on TLC had mostly
disappeared. The mixture was concentrated by evaporation
in vacuo and re-dissolved in 2 ml of CH2Cl2. Most of the
dicyclohexylurea (DCU) that formed in the reaction depos-
ited as a thick oil and was easily removed by filtration with
filter paper, but still remained in the filtrate. The CH2Cl2
filtrate was left for 12 h, and then further deposited DCU was
filtered again. The final filtrate was evaporated in vacuo and
carefully chromatographed on a silica-gel column (n-hexane-
ethyl acetate) to give 339 mg of INMP (90.3% yield) as a pale
O
P
F
O
P
OH
N
CH3
O
CH3
O
N
I-
O
Me
1a
2a
1b
NO2
Fig. 1. The chemical structures of sarin (1a), INMP (1b) and 2-PAM (2a).
hand, it is known that some 4-PAM homologues with long
aliphatic hydrocarbon chains and high lipophilicities can
permeate the BBB and reactivate sarin-inhibited brain
AChE (8). Therefore, a basic study of BBB-permeable PAM
analogues seems necessary to protect ordinary citizens from
another such terrorist attack. In this paper, 23 known and
novel homologues of 2-, 3- and 4-PAM were synthesized, and
their reactivation activities to sarin-exposed human erythro-
cytes AChE prepared by using INMP were examined. On the
other hand, some N-benzyl PAM analogues with lipophilic
structures are also known to have high reactivation activities
(9). Thus, 17 known and novel alkylated benzyl and homo-
benzyl analogues of PAM were also synthesized and examined
as reactivators for sarin-inhibited AChE. On the basis of all
the results of the experiments described above, the structur-
eYactivity correlation of PAM analogues was also discussed.
1
yellow oil. Six hundred megahertz H NMR: d 1.28 (d, 3 H,
J = 6.0 Hz, isopropyl CH3), 1.37 (d, 3 H, J = 6.0 Hz, isopropyl
2
CH3), 1.68 (d, 3 H, JPYCYH = 17.6 Hz, P-CH3), (dsept, 1 H,
J = 6.0 Hz, isopropyl CH), 7.37Y7.41 (m, 2H, 2,6-CH of 4-
nitrophenoxy group), 8.22Y8.26 (m, 2H, 3,5-CH of 4-
nitrophenoxy group). One hundred fifty-one megahertz 13C
NMR: d 12.0 (isopropyl CH3), 13.0 (isopropyl CH3), 24.0 (d,
1JPYC = 21.9 Hz, P-CH3), 72.3 (isopropyl CH), 121.1 (2,6-C of
4-nitrophenoxy group), 125.7 (3,5-C of 4-nitrophenoxy
group), 144.6 (4-C of 4-nitrophenoxy group), 155.7 (1-C of
4-nitrophenoxy group).
Neat INMP was stable for years at j20-C in a
refrigerator. For the AChE inhibition experiment, a freshly
prepared stock solution of INMP of various concentrations in
methanol stored at j20-C was diluted with 0.1 M phosphate
buffer (pH 7.0) and used as the inhibitor reagent.
MATERIALS AND METHODS
Chemicals and Reagents
Preparation of 2-PAM Analogue
Human erythrocyte AChE and 2-PAM were purchased
from Sigma (St. Louis, MO, USA). All other reagents were
of the highest grade available commercially.
PAM analogues listed in Table I except 2-PAM (2a)
itself were synthesized as follows: To a stirred solution of
1.00 g (8.19 mmol) of 2-, 3- or 4-pyridinealdoxime in 5 ml of
tetrahydrofuran (THF), two molar equivalents (16.4 mmol)
of corresponding alkyl bromides or four molar equivalents
Instruments
Measurements of 600 MHz 1H and 151 MHz 13C nuclear (32.8 mmol) of chlorides or iodides were added dropwise at
magnetic resonance (NMR) were performed with JEOL RT. The reaction was monitored by TLC using Merck
ECP600 (JEOL Ltd., Tokyo, Japan). Analyses by low- and Kieselgel F254 silica-gel plate (0.25 mm thickness) as the
high-resolution liquid chromatographyYelectrospray mass stationary phase, ethyl acetate as the mobile phase, and
spectrometry (LCYESI-MS) were performed with JEOL ultraviolet absorption at 254 nm as the detecting method.
LCmate (JEOL Ltd.).
The Rf values of 2-, 3- and 4-pyridinealdoxime were 0.5Y0.6.
The mixtures were refluxed until the spot of pyridinealdox-
imes on TLC disappeared. If the reaction was too slow, it was
stopped at 12 h. Typical reaction times were 8Y12 h for alkyl
Preparation of INMP
Methylphosphonic acid monoisopropyl ester (IMPA) halides and 15 min for benzyl bromides. Formed 2-PAM
was prepared as we previously reported (10). To a stirred analogues that had side chains longer than n-butyl group
solution of 309 mg of dicyclohexylcarbodiimide (DCC) in 2 deposited as thick oils during the reaction. In these cases, the
ml of dry dichloromethane (CH2Cl2), a solution of 200 mg supernatants were removed after cooling to RT, 5 ml of fresh
(1.45 mmol) of IMPA in 2 ml of CH2Cl2 was added at room THF was added and the mixtures were refluxed with
temperature (RT). To this was added a solution of 17.7 mg vigorous stirring for 1 h. This process was repeated three
(0.145 mmol) of dimethylaminopyridine (DMAP) in 1 ml of times to remove excess reagents completely. After super-
CH2Cl2 dropwise at RT. A large amount of white precipitate natants were finally removed, the remaining thick oils were
formed. To this was added a solution of 222 mg (1.59 mmol) allowed to cool to RT and left until they crystallized. After
of p-nitrophenol in 10 ml of CH2Cl2 dropwise to give a crystallization, they were recrystallized from THF-ethanol or
yellowish emulsion. To dissolve 222 mg of p-nitrophenol, n-hexane-THF. Other reaction products precipitated as
more than 7Y8 ml of CH2Cl2 was required. The reaction was powders. They were collected by filtration and recrystallized

Preparation of Sarin-Exposed AChE Using Stable Sarin INMP
Table I. Synthesized PAM Analogues
2829
Compound Type
Original 2-PAM
No.
R
X
I
I
Br
Br
Br
Br
Br
Yield (%)
2a
2b
2c
2d
2e
2f
2g
2h
2i
methyl (2-PAM)
-
ethyl
18
n-butyl
8
OH
Linear side chain
type
n-hexyl
6
N
n-octyl
5
n-decyl
1
N
n-octadecyl
isoamyl
1
R
X-
Branched side chain
type
Br
Br
Br
Br
Br
Br
Br
Cl
I
Br
Br
Br
Br
Br
Br
Br
Br
Br
Br
Br
Br
Cl
I
Br
Br
Br
Br
Br
Br
Br
Br
Br
Br
Br
Br
Cl
6
2-ethylhexyl
benzyl (Benzyl-P2A)
p-methylbenzyl
p-tert-butylbenzyl
2-phenylethyl
3-phenylpropyl
4-phenylbutyl
ethyl
no reaction
2-PAM analogues
2j
2k
2l
12
Benzyl type
1
17
5
2m
2n
2o
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n
3o
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
Homobenzyl type
1
no reaction
98
83
93
100
61
24
46
9
n-butyl
Linear side chain
type
n-hexyl
OH
n-octyl
N
n-decyl
n-octadecyl
isoamyl
2-ethylhexyl
benzyl
p-methylbenzyl
p-tert-butylbenzyl
2-phenylethyl
3-phenylpropyl
4-phenylbutyl
ethyl
Branched side chain
type
N
R
X-
83
99
47
88
27
5
96
99
91
76
89
16
38
7
75
82
81
77
57
2
3-PAM analogues
Benzyl type
Homobenzyl type
n-butyl
n-hexyl
Linear side chain
type
n-octyl (OPAB, SPK-3)
n-decyl
n-octadecyl
isoamyl
2-ethylhexyl
benzyl
p-methylbenzyl
p-tert-butylbenzyl
2-phenylethyl
3-phenylpropyl
4-phenylbutyl
OH
N
N
Branched side chain
type
R
X-
4-PAM analogues
Benzyl type
4m
4n
4o
Homobenzyl type
from THF-ethanol or ethanol. All the 2-PAM analogues 4g was insoluble in waterYethanol (1:1) and could not be used
synthesized were characterized by 600 MHz 1H and 151 MHz for this experiment.
13C NMR, and low- and high-resolution LCYESI-MS. The
yields are also shown in Table I.
In Table I, the low yields of 2-PAM analogues were
due to their very low reaction speed, which might be due
Inhibitory Effect of INMP on AChE
Twenty-five microliters of 0.2, 2, 20, 200, 2,000 and
to the steric hindrance of the 2-hydroxyiminomethyl group 20,000 nM INMP (for inhibition) or 0.1 M phosphate buffer
of 2-pyridinealdoxime. The yields of 3g and 4g were also (pH 7.0, for positive control) were added to 25 ml of 0.02 U
low; this could be caused by their high solubilities in THF. human erythrocytes AChE in 0.1 M phosphate buffer (pH
The low yields of 3i and 4i seemed to be due to steric 7.0). This mixture was incubated at 25-C for 30 min. Then,
hindrance of 2-ethylhexyl bromide. Synthesized PAM ana- 50 ml of distilled water was added, and the remaining AChE
logues were used for reactivation experiments as an aqueous activities were measured by the method of Ellman et al. (11).
solution. Some water-insoluble analogues were initially AChE activities relative to the positive control at the various
dissolved in ethanol and diluted with water. Unfortunately, INMP concentrations are shown in Fig. 2. From Fig. 2, the

2830
Ohta et al.
In Table II, 29 of 39 PAM analogues showed AChE
reactivation activities and the activities varied from 3 to 92%
of the activity of 2-PAM. Concerning to branched side chain
analogues, alkylbenzyl analogues and benzyl homologues,
13 of 22 analogues showed AChE reactivation activities,
seven of them showed the similar activities (95 to 117%) and
three of them showed higher activities (164 to 278%) to the
linear side chain analogues that have the same or similar
carbon numbers in the side chain. To discuss about the result
and the structureYactivity correlation in detail, the result was
separated by compound types and studied as follows.
100
80
60
IC50
40
20
0
Reactivation Activities of PAM Analogues with Linear Side
Chain to INMP-Inhibited AChE
15
AChE reactivation activities of PAM analogues with
linear aliphatic side chains (2aY2g, 3bY3g, 4bY4f) relative to 2-
PAM in Table II are extracted and shown in Fig. 3. In Fig. 3,
2-PAM itself showed the best recovery activity, and the
activities of 2-PAM analogues were reduced by elongation of
the side chain. This could be explained as a result of the
decrease of the dissociation constant (pKa) with the increase
of lipophilicity of analogues (12) or the increase in steric
hindrance due to the side chain which may prevent the reac-
tion of oxime group and inhibited AChE. All the 3-PAM
analogues showed low recovery activities, consistent with a
prior report (13). Recovery activities of all the 4-PAM ana-
logues were more than moderate and increased by side chain
elongation. This is also consistent with prior reports on in vitro
or in vivo experiments (8,12Y14). 4e, as known as BSPK-3^ (8)
or BOPAB^ (14), is a well-known antidote able to reactivate
sarin-inhibited brain AChE (8) and also known as potent
decontamination agent for organophosphorus compounds by
its micellar property (13). It is reported that 4e showed mostly
equal reactivation activities to 2-PAM for blood AChE of
sarin-inhibited mice (8); by contrast, it showed 46% relative
activity to 2-PAM in Fig. 3. This probably reflects the
difference between the in vivo and in vitro experiments, and
the difference in experimental conditions of reactivation.
0.1
1
10
100
1000
10000
INMP Concentration (nM)
Fig. 2. Inhibitory effect of INMP to human erythrocyte AChE.
50% inhibitory concentration (IC50) of INMP to 0.04 U/ml
human erythrocytes AChE was determined to be 15 nM.
Reactivation of Inhibited AChE with 2-PAM
Twenty-five microliters of 30 nM INMP was added to
25 ml of 0.02 U human erythrocyte AChE and incubated at
25-C for 30 min to form 50%-inhibited AChE solution. This
was used as a negative control in the subsequent experiments.
To this was added 50 ml of aqueous solution of 2-PAM at
various concentrations, and the mixture was incubated at
25-C for 15 min; then the recovered AChE activities were
measured by the method of Ellman et al. (11). When the final
concentration of 2-PAM solution was 150 mM, 50%-inhibited
AChE was reactivated to 75% activity in relation to the
positive control. Thus, the final concentrations of other PAM
analogues were also fixed to 150 mM for comparison with
AChE activities in response to 2-PAM.
Reactivation Activities of PAM Analogues with Branched
Side Chain to INMP-Inhibited AChE
Reactivation of Inhibited AChE with PAM Analogues
Twenty-five microliters of 30 nM INMP was added to
Relative AChE reactivation activities of PAM ana-
25 ml of 0.02 U human erythrocyte AChE and incubated at logues with branched aliphatic side chains (2h, 3hY3i, 4hY4i)
25-C for 30 min. To this was added 50 ml of 300 mM solutions of in Table II are extracted and shown in Fig. 4. The results of
PAM analogues, and the mixture was incubated at 25-C for PAM analogues with linear side chains are also shown in Fig.
15 min; then the recovered AChE activities were measured by 4 for comparison (error bars omitted). A novel compound, 2-
the method of Ellman et al. (11). Three samples were assayed [(hydroxyimino)methyl]-1-isoamylpyridinium bromide (2h),
for each data point for calculation of standard deviations.
showed intermediate recovery activity between 2c and 2d,
and its behavior was just the same as that expected for an n-
amyl analogue. 2-[(hydroxyimino)methyl]-1-[2-ethylhexyl]
pyridinium bromide (4i), which is also novel compound,
RESULTS AND DISCUSSION
AChE reactivation activities of the synthesized 39 PAM showed the same activity as 4e. In these cases, the recovery
analogues are shown in Table II. The activities are shown as activities to sarin-inhibited AChE seemed to depend on the
the relative activities relative to 2-PAM (2-PAM: 100%). To number of carbons and lipophilicities of the side chains, not
evaluate the branched side chain analogues, alkylbenzyl the length of the side chains. For unknown reasons, novel
analogues and benzyl homologues, the relative activities of compounds 3h, 3i and 4h did not show recovery activities.
those compounds to the common linear side chain analogues Considering the low yields of 2h, 3hY3i, 4hY4i in Table I,
that have the same or similar carbon numbers in the side PAM analogues with branched side chains were thought to
chain are also shown in Table II.
have no advantages over linear side chain analogues.

Preparation of Sarin-Exposed AChE Using Stable Sarin INMP
Table II. AChE reactivation activities of synthesized PAM Analogues
2831
Relative
activity
to 2-PAM
(%)
Relative activity
to linear side
chain analogue
(%)a
Carbons
in side
chain
Compound
type
S.D.
(%, n = 3)
No.
R
X
Linear side
chain type
2b
Ethyl
I
2
92
9.5
2c
2d
2e
2f
n-butyl
n-hexyl
n-octyl
n-decyl
Br
Br
Br
Br
Br
Br
4
6
58
39
34
23
Yb
48
1.5
2.2
14
0.8
Y
8
10
18
5
2g
2h
n-octadecyl
Isoamyl
2-PAM
Branched side
chain type
8.0
99c
analogues
2j
Benzyl
Br
Br
Br
Br
Br
7
8
39
55
64
5.3
Y
0.7
6.6
1.1
2.5
Y
108d
164
278e
16
Benzyl type
2k
2l
2m
2n
p-methylbenzyl
p-tert-butylbenzyl
2-phenylethyl
3-phenylpropyl
11
8
Homobenzyl type
9
Y
3b
3c
3d
Ethyl
n-butyl
n-hexyl
I
2
4
6
11
8.4
11
0.5
1.8
3.2
Br
Br
Linear side
chain type
3e
3f
n-octyl
n-decyl
Br
Br
Br
Br
8
10
18
5
5.4
3.6
Y
7.2
3.2
Y
3g
3h
n-octadecyl
Isoamyl
3-PAM
Branched side
chain type
Y
Y
Y
analogues
3i
2-ethylhexyl
Benzyl
Br
Br
Br
Br
Br
Br
Cl
8
7
Y
Y
9.0
15
Y
Y
Y
3.9
3.7
Y
Y
Y
3j
Benzyl type
3k
3l
p-methylbenzyl
p-tert-butylbenzyl
2-phenylethyl
3-phenylpropyl
4-phenylbutyl
8
167
4.2e
Y
Y
117
11
8
Homobenzyl type
3m
3n
3o
9
10
Y
4.2
Y
11
4b
4c
Ethyl
n-butyl
I
Br
2
4
29
30
11
8.9
Linear side
chain type
4d
4e
4f
n-hexyl
n-octyl
n-decyl
Isoamyl
Br
Br
Br
Br
6
8
43
46
59
Y
6.3
0.4
1.2
Y
10
5
4-PAM
Branched side
chain type
4h
Y
analogues
4i
2-ethylhexyl
Benzyl
Br
Br
Br
Br
Br
8
7
47
Y
48
30
44
5.6
Y
5.2
1.5
4.6
101
Y
104
51e
95
Benzyl type
4j
4k
4l
p-methylbenzyl
p-tert-butylbenzyl
2-phenylethyl
8
11
8
Homobenzyl
type
4m
4n
4o
3-phenylpropyl
4-phenylbutyl
Br
Cl
9
10
Y
3.1
Y
2.0
Y
5.3
a Compared with the activities of linear side chain analogues that have the same number of carbons in the side chains.
b FY_ No AChE reactivation activities were observed.
c Compared with average of the activities of 2c and 2d.
d Compared with average of the activities of 2d and 2e.
e Compared with the activities of 2f, 3f and 4f, respectively.
Reactivation Activities of N-alkylbenzyl PAM Analogues
to INMP-Inhibited AChE
and shown in Fig. 5. The results of PAM analogues with linear
side chains are also shown in Fig. 5 for comparison (error
bars omitted). 2j, 2-[(hydroxyimino)methyl]-1-benzylpyridi-
Relative AChE reactivation activities of N-alkylbenzyl nium bromide, known as BBenzyl-P2A,^ is a well-known anti-
PAM analogues (2jY2l, 3jY3l, 4jY4l) in Table II are extracted dote for N,N-dimethyl O-ethyl phosphoramidecyanidate

2832
Ohta et al.
2a
2b
2a
2-PAM Homologues
3-PAM Homologues
4-PAM Homologues
2-PAM AlkylBenzyl Analogues
3-PAM AlkylBenzyl Analogues
4-PAM AlkylBenzyl Analogues
100
80
60
40
20
0
100
2-PAM Homologues
3-PAM Homologues
4-PAM Homologues
2b
80
60
40
20
0
2l
2c
2c
4f
2k
3k
4f
4e
4k
4e
4d
2d
4d
2d
2e
2j
4c
4b
3b
2f
4b
3b
4l
3l
2f
3f
4c
3c
3d
2g
3g
3f
3e
3j
4j
3d
3c
0 1 2 3 4 5 6 7 8 9 10 12 14 16 18 20
Number of carbons in side chain
0
1
2
3
4
5
6
7
8
9
10 11 12
Number of carbons in side chain
Fig. 3. Relative AChE reactivation activities of PAM analogues with
linear alkyl side chains to 2-PAM.
Fig. 5. Relative AChE reactivation activities of N-alkylbenzyl PAM
analogues to 2-PAM.
(tabun) (9). In this experiment, 2j showed moderate recovery difference between sarin and tabun, or in vitro and in vivo
activity in Fig. 5. 2k, 2l and 4k showed higher reactivation experiments. In Fig. 5, 3-PAM analogues 3jY3l showed low
activities than the known compounds 2j (BBenzyl-P2A^) and reactivation activities, as in Figs. 3 and 4. 3j and 4j showed
4e (BOPAB,^ BSPK-3^). 2k, known as less potent activator almost no reactivation activities, and this was also reported in
for tabun-exposed bovine AChE than 2j (9), unexpectedly the prior report. Generally, the results in Fig. 5 showed
showed a stronger recovery effect for INMP-inhibited AChE similarity to those of the prior reactivation experiment on
than 2j in Fig. 5. A novel compound, 2-[(hydroxyimino)- tabun-inhibited bovine AChE (9).
methyl]-1-[4-(tert-butyl)benzyl] pyridinium bromide (2l), was
found to have the highest reactivation activity among the Reactivation Activities of N-benzyl PAM Homologues
antidotes tested in this experiment except for 2-PAM itself. to INMP-Inhibited AChE
As demonstrated in this case, the INMP method enables
rapid assay of reactivation activities of a large number of
Relative AChE reactivation activities of N-benzyl PAM
newly synthesized compounds, and helps rapid screening and homologues (2mY2n, 3mY3o, 4mY4o) in Table II are ex-
evaluation of antidotes. The difference between this result tracted and shown in Fig. 6. The results of PAM analogues
and that of the prior report (9) might be based on the with linear side chains are also shown in Fig. 5 for
2a
2a
2-PAM Homologues
3-PAM Homologues
4-PAM Homologues
2-PAM Homologues
3-PAM Homologues
4-PAM Homologues
2-PAM Benzyl Homologues
3-PAM Benzyl Homologues
4-PAM Benzyl Homologues
100
80
60
40
20
0
100
80
60
40
20
0
2b
2b
2-PAIA
2h
3-PAIA
3h
4-PAIA
4h
3-PAEH
3i
4f
4-PAEH
4i
4f
2c
2c
4i
4e
2h
4c
4d
2d
4d
4e
2e
4m
2d
2e
4b
3b
4b
3b
2f
2f
4c
3c
2n
3n
4n
3d
3h 4h
3c
3d
3o
4o
2g
3g
2m
3e
3f
3i
3m
0 1 2 3 4 5 6 7 8 9 10 12 14 16 18 20
Number of carbons in side chain
0
1
2
3
4
5
6
7
8
9
10 11
Number of carbons in side chain
Fig. 4. Relative AChE reactivation activities of PAM analogues with Fig. 6. Relative AChE reactivation activities of N-benzyl PAM
branched alkyl side chains to 2-PAM.
homologues to 2-PAM.

Preparation of Sarin-Exposed AChE Using Stable Sarin INMP
2833
comparison (error bars omitted). 2m showed low reactivation ACKNOWLEDGMENT
activities, and this result is similar to that of the tabun-
inhibited bovine AChE experiment (9). In this series, only
This study was supported in part by a Grant-in-Aid for
the novel compound 4m showed equal reactivation activity Scientific Research (B) from the Ministry of Education,
to the known antidote 4e (BOPAB,^ BSPK-3^). In this case, Culture, Sports, Science and Technology of Japan.
similar to 2h and 4i in Fig. 4, the number of carbons in the
side chain seemed to be more important for reactivation
activity than chain length, and it seemed not to be so
important whether the substituents were aliphatic or
aromatic.
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CONCLUSION
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A non-toxic sarin analogue INMP was found to enable
safe and easy preparation of sarin-exposed AChE. This agent
could be easily handled, and its IC50 on 0.04 U/ml human
erythrocytes AChE was 15 nM. To demonstrate the useful-
ness of this reagent, 40 hydrophobic PAM-type oxime anti-
dotes were synthesized, and their reactivation activities to
sarin-exposed AChE were examined in order to demonstrate
their abilities for easy development of BBB-permeable
antidotes for sarin poisoning in normal laboratories. In
general, the results of the recovery experiment were similar
to those of the experiments previously reported using real
sarin or tabun. StructureYactivity correlation studies showed
some interesting properties of PAM analogues. 2-PAM
showed the best recovery activity; the activities of its
homologues were reduced by side chain elongation. In
contrast, 4-PAM showed moderate reactivation activities,
and the activities of its homologues increased by side chain
elongation. All the 3-PAM analogues showed poor reactiva-
tion activities. Regarding the side chains of PAM analogues,
linear hydrocarbons seemed to be more suitable than
branched hydrocarbons in terms of both activity and
availability. All the alkylbenzyl analogues of 2-PAM and
some alkylbenzyl analogues of 4-PAM tested showed strong
reactivation activities. In the N-benzyl PAM homologues
tested, only the 4-phenethyl analogue showed strong activity.
According to the collective results, the reactivation activities
of the oximes tested seemed to depend on side chain carbon
number and lipophilicity, not the chain length. Among the
antidotes tested in this experiment except for 2-PAM, the
novel hydrophobic 2-PAM-type compound 2-[(hydroxyimi-
no)methyl]-1-[4-(tert-butyl)benzyl] pyridinium bromide was
found to have the highest reactivation activity. As demon-
strated in this experiment, the INMP method enabled rapid
assay of reactivation activities of a large number of newly
synthesized compounds, and helped rapid screening and
evaluation of antidotes.
14. D. Knezevic, V. Tadic, and S. Cetkovic. The efficacy of
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