Reactivation potency of fluorinated pyridinium oximes for acetylcholinesterases inhibited by paraoxon organophosphorus agent

Article abstract of DOI:10.1016/j.bmcl.2008.12.070

For the purpose of developing new oxime reactivators of acetylcholinesterases (AChE) that have been inhibited by organophosphorus agents, emphasis was given to the finding that the lipophilic nature of fluorinated compounds is responsible for their enhanc

Full text of DOI:10.1016/j.bmcl.2008.12.070

Bioorganic & Medicinal Chemistry Letters 19 (2009) 1214–1217
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Reactivation potency of fluorinated pyridinium oximes for acetylcholinesterases
inhibited by paraoxon organophosphorus agent
a,b
a
a
b
a,*
Hee Chun Jeong , Nam Sook Kang , No-Joong Park , Eul Kyun Yum , Young-Sik Jung
a
Drug Discovery Division, Korea Research Institute of Chemical Technology, PO Box 107, Yuseong, Daejeon 305-600, Republic of Korea
Department of Chemistry, Chungnam National University, Daejeon 305-764, Republic of Korea
b
a r t i c l e i n f o
a b s t r a c t
Article history:
For the purpose of developing new oxime reactivators of acetylcholinesterases (AChE) that have been
inhibited by organophosphorus agents, emphasis was given to the finding that the lipophilic nature of
fluorinated compounds is responsible for their enhanced transport across the blood brain barrier
Received 10 November 2008
Revised 15 December 2008
Accepted 17 December 2008
Available online 24 December 2008
(BBB). As a result, we have designed and synthesized the fluorinated oxime derivatives, which quantum
mechanical calculations suggest should have a greater lipophilicity and BBB permeability than their non-
fluorinated analogs. Among the compounds explored in this study, 4 was found to have the highest
potency for reactivation of paraoxon-inhibited housefly (HF) AChE.
Keywords:
Organophosphorus agents
Acetylcholinesterase
Fluorinated pyridinium oxime
Oxime reactivators
Ó 2008 Elsevier Ltd. All rights reserved.
A number of organophosphorus compounds that serve as nerve
agents and insecticides (e.g., parathion and malathion) are cholin-
esterase inhibitors and, in particular, acetylcholinesterase (AChE)
inhibitors. Electrophilic phosphorous atoms in these agents are
reactive toward nucleophilic attack by the serine hydroxyl group
in the acetylcholinesterases. The toxicity of these organophospho-
rus agents is a consequence of the fact that the resulting, cova-
lently inactivated phosphorylated enzymes resist hydrolysis. The
inhibition of AChE increases the amount of neurotransmitter ace-
tylcholine at central and peripheral sites of the nervous system
and causes excessive stimulation of muscarinic and nicotinic
NOH
O
NOH
2Cl
CONH2
N
NOH
N
NOH
N
N
N
CH3 Cl
2Cl
O
HI-6
2
-PAM
Obidoxime
Figure 1. Structures of AChE reactivators.
tration remains a key issue in the development of the oxime type
AChE reactivators.
Lipophilicity is a key molecular parameter in medicinal chemis-
try, especially in the development of central nervous system (CNS)
active drugs that require BBB permeability. Biological absorption
and distribution are largely controlled by the ionization state of a
drug that leads to a balance between lipophilicity and hydrophilic-
ity. Enhanced lipophilicity can lead to an increase in the measured
binding free energy by making partitioning between the polar
aqueous solution and the less polar receptor site more favorable.
Fluorinated compounds are frequently encountered in modern
medicinal chemistry and many of these substances are highly
1
receptors. The inhibited AChE can be reactivated by using selec-
2
tive nucleophilic substances, such as pyridinium oximes. Oxime-
induced reactivation is the primary therapeutic antidote for poi-
soning by organophosphorus warfare agents and insecticides. As
a result, the development of oxime reactivators has received great
attention during the last several decades.
6
These efforts, some of which have been carried out in our labo-
3
ratory, have led to the discovery of a variety of pyridinium oximes,
several of which are in clinical use currently (e.g., pralidoxime, obi-
4
doxime and HI-6, Fig. 1). However these oximes are not ideal reac-
tivators of inhibited AChEs, primarily because they have limited
blood brain barrier (BBB) penetration. Even though the mean BBB
penetration of 2-PAM is approximately 10%, determined by using
7
effective drugs. Commonly, fluorine is introduced to block a met-
abolically labile site in the molecule. Another beneficial property
associated with fluorine substitution is an increase in BBB perme-
ability due to changes in lipophilicity. The results of studies prob-
ing the effect of replacing hydrogen by fluorine on lipophilicity
show that a single H/F exchange raises the logD value by approx-
5
an in vivo rat microdialysis technique with HPLC/UV, BBB pene-
*
Corresponding author. Tel.: +82 42 860 7135; fax: +82 42 861 0307.
8
E-mail address: ysjung@krict.re.kr (Y.-S. Jung).
imately 0.25. Thus, we hypothesized that introduction of fluorine
0
960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.12.070

H. C. Jeong et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1214–1217
1215
at heterocyclic ring positions of pyridinium oximes might repre-
sent a viable strategy for the enhancement of liphophilicity, BBB
permeability, and potency for inhibited AChE reactivation.
In order to assist in the design of ideal fluorinated pyridinium
oximes, computer aided calculations of lipophilicity were per-
formed. Ab Initio Hartree–Fock (HF) and density functional B3LYP
calculations using the Gaussian 03 package were carried on pyrid-
inium oximes 1-3 (Fig. 2) first in order to measure and compare
electron densities. The conformations of 1–3 were optimized at
the HF/6-31+G(d) level. The conductor-like polarizable continuum
model (CPCM) SCRF method, which is implemented in the Gauss-
ian 03 package to consider solvation effect, was also used. To de-
rive a hydrophilicity index (Eq. (1)), we used the electron
densities as parameters to assess the degrees to which atoms in
the compound resemble those in water.9
(BBB) as compared to 1. The aqueous solubilities and BBB penetra-
tions for three compounds were also calculated by using Accelrys/
ADME prediction software as shown in Table 1.
The results of the theoretical treatments described above sug-
gest that fluorinated oximes 2 and 3 should have higher lipophilic-
ities and higher BBB permeabilities than non-fluorinated oxime 1.
As a result, we have synthesized oxime 2 along with the related
bis-pyridinium oximes (4–6) that contain the oxime moiety found
in 2 and compared their activities toward reactivation of organo-
phosphorus compound inactivated AChE.
The first step in the preparation of the fluorinated mono-pyrid-
inium oxime 2 involved treatment of 3-fluoro-4-pyridinecarboxal-
2
dehyde 6 with NH OH–HCl in EtOH containing pyridine. This
process produces the aldoxime 7 in 96% yield (Scheme 1). The
oxime 2 was then generated in 24% yield by N-methylation 8 with
ꢀ
ꢁ
3
methyl iodide in CHCl .
X
X
q
q
aHB
H O
2
Hydrophilic index ¼ q
q
a=
q
H
2
O þ q
1 ꢀ
Our interest also focused on fluorinated derivatives of bis-pyrid-
inium oximes (4–6), obidoxime analogs, because the obidoxime is
already clinically in use.
a<
q
H
2
O
a>H
2
O
X
ꢀ
qaHC=
q
H
2
O
ð1Þ
Methods for the preparation of the non-fluorinated oximes HI-6
and obidoxime, bearing a bis-chloromethyl-ether linker, have been
In equation 1,
q
a represents the electron densities of individual
1
0
atoms in the compound,
q
H2O represents the electron density of
published. As a result, the fluorinated analogs (4–6) were pre-
pared by using the established procedures. Accordingly, reaction
of aldoxime 8 with 0.33 equiv of bis-chloromethyl-ether 9 at
water, HB refers to potential hydrogen-bonding atoms (O, N, F)
and to hydrogen atoms bonded to O or N, and HC refers to hydro-
gen atoms bonded to carbon. The electron densities of hydrogen
atoms were compared with the electron density of hydrogen atoms
in water regardless of their origin (0.457) and the electron densi-
ties of O, N, or F atoms were compared with the electron density
of the oxygen atom of water (7.868). By using this relationship,
the hydrophilic indexes of 1–3 were determine to be 2.53, ꢀ0.2
and ꢀ0.33, respectively (Table 1), where the lower value corre-
sponds to lower hydrophilicity and more liphophilicity. These re-
sults suggest that the fluorinated compounds 2 and 3 are more
liphophilic than the non-fluorinated substance 1. Consequently, 2
and 3 should be more able to penetrate the Brain Blood Barrier
45 °C in CHCl yields the bis-pyridinium oxime 4 (15%). In contrast,
3
reaction of aldoxime 10 with 5 equiv of bis-chloromethyl-ether 9
in CHCl3 generates the mono-pyridinium oxime 11 (98%), which
upon reaction with 8 provides the oxime 5 (40%). In the route for
preparation of the bis-pyridinium oxime 6, isonicotinamide 12 is
reacted with 5 equiv of bis-chloromethyl-ether 9 in CHCl3 to give
intermediate 13 (26%), which reacts with aldoxime 8 in DMF to
produce the oxime 6 (63%).
The pyridinium oximes and HI-6 were prepared by using known
3
synthetic methods. 2-PAM was purchased from Sigma–Aldrich.
Diisopropyl fluorophosphates (DFP) and paraoxon are available
from Fluka and Sigma–Aldrich, respectively. AChE, from bovine
red blood cells (RBC), was purchased from Sigma–Aldrich. Known
sequential protocols were used to monitor reactivation of AChE.11
In order to assess the reactivation potencies of the oximes,
paraoxon was used to inactivate AChE since this substance has
structure that is similar to typical nerve gases. As shown in Table
OH
N
OH
N
OH
N
F
2, oxime reactivations were carried out on paraoxon-inhibited HF
N
N
N
F
and RBC AChEs. The potencies of the prepared oximes were com-
pared with the three currently used AChE reactivators, 2-PAM, obi-
doxime and HI-6 (Fig. 3). The importance of fluorine substitution at
pyridinium ring positions is demonstrated by comparison of data
obtained using N-methyl-4-pyridinium oxime 1 and N-methyl-3-
fluoro-4-pyridinium oxime 2 in paraoxon-inhibited HF and RBC
1
2
3
NOH
NOH
F
NOH
NOH
F
CONH2
F
F
1
2
AChEs.
NOH
N
N
N
N
N
O
6
N
Because of structural similarities, the fluorinated oxime 4
should be compared with obidoxime, and the fluorinated oxime
2
Cl
2Cl
2Cl
O
O
6
can be compared with HI-6. The results of these measurements
4
5
show that non-fluorinated oximes have higher reactivation poten-
cies than their fluorinated analogs with paraoxon-inhibited RBC
Figure 2. Structure of bis-pyridinium oximes.
Table 1
QM calculations of electron densities and hydrophilicity of compounds
*
*
Compound
Electron densities
N2
Hydrophilicity index
Aqueous solubility
BBB penetration
N1
O
1
2
3
6.576927
6.604342
6.726558
7.099565
7.113086
7.101621
7.703776
7.683838
7.695776
2.53
ꢀ0.767
ꢀ1.152
ꢀ1.484
ꢀ0.553
ꢀ0.490
ꢀ0.372
ꢀ0.20
ꢀ0.33
*
These values are calculated using Accelrys/ADME prediction software. Lower solubility value represents less soluble in water. Greater BBB value represents more BBB
penetration.

1
216
H. C. Jeong et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1214–1217
NOH
F
F
F
OHC
HON
NH2OH-HCl, Py
EtOH, reflux, 4 h
CH3I
HON
N
F
N
CHCl , 45 ºC, 25 h
3
N
I
9
6%
24%
7
8
2
CHCl3
45 ºC, 10 h
5%
+
Cl
O
Cl
4
N
9
1
8
NOH
NOH
Cl
9
8
5
N
CHCl3, 45 ºC, 10 h
N
O
Cl
DMF, 40 ºC, 34 h
8
2%
40%
1
0
11
O
O
9
H2N
Cl
8
H2N
6
N
O
Cl
N
CHCl3, 60 ºC, 0.5 h
6%
DMF, 60 ºC, 24 h
63%
2
1
2
13
Scheme 1. Synthesis of new pyridinium oximes.
Table 2
Reactivation potency of oximes for paraoxon-inhibited AChEs
xon-inhibited HF AChE. In this case, fluorinated oximes have higher
potencies than the corresponding non-fluorinated derivatives. This
is demonstrated by the finding that 4 is a more potent reactivator
than obidoxime, and 6 is also slightly more potent than HI-6. How-
ever 5 showed lower reactivation potencies compared with other
oxime compounds. Thus, among the substances explored in this
study, 4 has the highest reactivation potency in reactivation of
paraoxon-inhibited HF AChE.
In summary, we have designed and synthesized several
fluorinated oxime compounds in order to probe the effect of fluo-
rine substitution on reactivation of inhibited AChE. The results of
computational studies, indicating that fluorinated oxime 2 is more
hydrophobic than the non-fluorinated oxime 1, stimulated the de-
sign of new oximes with potentially increased BBB permeabilities.
Among the substances explored in this study, 4 has the highest
reactivation potency in reactivation of paraoxon-inhibited HF
AChE. Further investigation will be carried out in our laboratory
to probe the in vivo reactivation activities of the fluorinated oxi-
mes. It is hope that this effort leads to new guidelines for the de-
sign of promising oxime reactivators of inactivated AChE.
Compound
HF AChE
RBC AChE
Meana
Meana
SE
2.47
SE
1
2
4
5
6
7.5
19
50
2.2
22.1
48.4
33
20.5
46.3
40.9
26.9
29.5
62.6
78.1
48.2
2.38
3.36
3.96
2.87
2.99
3.55
7.61
3.94
2.85
8.98
0.85
3.17
11.63
6.47
1.3
2
-PAM
Obidoxime
HI-6
4.6
a
Mean is the average of % reactivation with 4 replications, and SE is the ‘Standard
Error’.
Acknowledgments
Financial support for this work was provided by the Korea
Foundation for International Cooperation of Science & Technology
(
KICOS) through a grant provided by the Korean Ministry of Educa-
tion Science & Technology (No. K20702000710-08B1200-01910).
References and notes
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AChE. The potency order was found to be obidoxime >4, and HI-6
>6. Among the oximes tested, obidoxime was observed to be a
4.
more potent reactivator than either 2-PAM or HI-6. In contrast,
the trends outlined above are reversed in reactivation of parao-

H. C. Jeong et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1214–1217
1217
5.
6.
7.
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12. AChE inhibition and reactivation. DFP and paraoxon were used as
organophosphorus inhibitors and the AChE reactivating capability of 2-
PAM and HI-6 were examined against OP-inhibited HF and RBC AChE,
respectively. AChE was inhibited with the minimum quantity of inhibitor
necessary to inactivate 99% of activity for 10 min at room temperature. The
concentration of DFP was 12.5
lM for HF AChE and 25 lM for RBC AChE,
8
9
.
.
Wildman, S. A.; Crippen, G. M. J. Chem. Inf. Comput. Sci. 1999, 39, 868.
Dwyer, D. S. Structure, Function, and Bioinformatics 2006, 63, 939.
respectively, and 20 M of paraoxon for both AChEs. To remove excess
l
inhibitor after reaction with enzyme, the enzyme solution was mixed with
2-fold volume of hexane and vigorously shaken with a vortex mixer for 1
min. The aqueous phase was separated by centrifugation at 3000g for 10
min at 3 °C. The solution containing phosphorylated AChE was incubated
with various concentration of 2-PAM or HI-6 for various reactivation
periods. To remove small molecules such as reactivator and
1
0. Musilek, K.; Jun, D.; Cabal, J.; Kassa, J.; Gunn-Moore, F.; Kuca, K. J. Med. Chem.
007, 50, 5514.
1. In vitro determination of AChE activity. The enzyme activity was measured in a
6-well microplate using a microplate reader (Benchmark Microplate Reader,
2
1
9
Bio-Rad) at 415 nm and 37 °C with acetylthiocholine (1 mM) as substrate and
DTNB (1 mM) as chromogen in 0.05 M Tris–HCl buffer, pH 7.8. Total reaction
phosphorylated oxime, the reactivating mixture was filtered through a
volume was adjusted to 250
assay method. The enzyme concentrations of both AChEs in 250
l
L with slight modification from Ellman’s AChE
L of final
micro spin-column packed with Sephadex-G50 (Bio-Rad) in a centrifuge at
300g for 1 min at 3 °C. The AChE activity of the filtrate was measured in a
96-well microplate.AChE reactivation with newly synthesized oxime
compounds. The reactivating abilities of the newly synthesized oximes
were evaluated against DFP or paraoxon-inhibited HF or RBC AChE,
respectively. The inhibited AChE was extracted with hexane, and then the
aqueous phase was reacted with 5 mM of each oxime compound for 30 min
for DFP-inhibited AChE and for 1 h for paraoxon-inhibited AChE,
respectively.
l
reacting solutions were 0.05 mg/ml for HF AChE and 0.02 U/mL for RBC AChE,
respectively. For RBC AChE, 1% of Triton X-100 (Sigma–Aldrich) was added in
the Tris–HCl buffer to preserve enzyme activity. AChE activity was measured
by using the change of optical density per minute (OD/min). The percentage of
reactivation of AChE activity was calculated by comparison with the AChE
activity without inhibitor showing 0.2 OD/min.