Selected AChE reactivators in different crystalline environment: Salts and enzyme

Article abstract of DOI:10.1007/s11224-009-9575-0

The investigation of relationships between the molecular structure of the compounds capable to reactivate acetylcholinesterase (AChE) inhibited by organophosphorus toxins, such as nerve agents and pesticides, is an important step toward synthesis of more efficient antidota. In the present article, we describe the crystal structures of two new AChE reactivators, which are bromides of (E)-1,4-bis(4-hydroxyiminomethylpyridinium)-but-2-ene (K075) and of 4,4′-bis(hydroxyiminomethyl)-1,1′-(1,4-phenylenedimethyl)-bispyridinium (K114). Their molecular geometry and intermolecular interactions in the crystalline state are compared to those in the crystal structures of the well-known AChE reactivators, obidoxime, and TMB-4. Inspection of hydrogen bonds and other short intermolecular contacts in the crystalline AChE-obidoxime complex revealed their similarity to those observed in the crystal structures of K075 and K114.

Full text of DOI:10.1007/s11224-009-9575-0

Struct Chem (2010) 21:495–501
DOI 10.1007/s11224-009-9575-0
ORIGINAL RESEARCH
Selected AChE reactivators in different crystalline environment:
salts and enzyme
´
Agnieszka Skorska-Stania Magdalena Sliwa
•
•
´
Kamil Musilek Kamil Kuca Josef Jampilek
•
•
•
•
Robert Musiol Barbara J. Oleksyn Jiri Dohnal
•
Received: 11 August 2009 / Accepted: 18 December 2009 / Published online: 7 January 2010
ꢀ Springer Science+Business Media, LLC 2010
Abstract The investigation of relationships between the
molecular structure of the compounds capable to reactivate
acetylcholinesterase (AChE) inhibited by organophospho-
rus toxins, such as nerve agents and pesticides, is an
important step toward synthesis of more efficient antidota.
In the present article, we describe the crystal structures
of two new AChE reactivators, which are bromides of
(E)-1,4-bis(4-hydroxyiminomethylpyridinium)-but-2-ene
(K075) and of 4,40-bis(hydroxyiminomethyl)-1,10-(1,4-
phenylenedimethyl)-bispyridinium (K114). Their molecular
geometry and intermolecular interactions in the crystalline
state are compared to those in the crystal structures of the
well-known AChE reactivators, obidoxime, and TMB-4.
Inspection of hydrogen bonds and other short intermolecu-
lar contacts in the crystalline AChE–obidoxime complex
revealed their similarity to those observed in the crystal
structures of K075 and K114.
´
´
A. Skorska-Stania (&) ꢀ M. Sliwa ꢀ B. J. Oleksyn
Faculty of Chemistry, Jagiellonian University,
´
Ingardena 3, 30-060 Krakow, Poland
e-mail: skorska@chemia.uj.edu.pl
Keywords AChE reactivators ꢀ Oximes ꢀ
Hydrogen bonds ꢀ Crystalline state
K. Musilek
Faculty of Military Health Sciences, Department of Toxicology,
University of Defence, Trebesska 1575, 500 01 Hradec Kralove,
Czech Republic
Introduction
Although there are many natural and synthetic compounds
that inhibit the enzyme acetylcholinesterase (AChE, EC
3.1.1.7), the organophosphorus inhibitors (OPI; e.g., nerve
agents, pesticides, and flame retardants) remain one of
the most dangerous and deleterious series of compounds
developed by man [1]. Namely, the OPI molecule binds
covalently to the hydroxyl group of the serine residue
within the enzyme active site, so that the AChE is not able
to cleave the neuromediator acetylcholine, which causes
the permanent activation of muscarinic and nicotinic
receptors. This leads to a central cholinergic crisis with
symptoms of lacrimation, salivation, and miosis, with
additional neuromuscular and breathing difficulties; death
is caused by suffocation [2].
K. Musilek ꢀ K. Kuca
Faculty of Science, Department of Chemistry,
University of Jan Evangelista Purkyne, Ceske mladeze 8,
400 96 Usti nad Labem, Czech Republic
K. Kuca
Faculty of Military Health Sciences,
Center of Advanced Studies, University of Defence,
Trebesska 1575, 500 01 Hradec Kralove, Czech Republic
J. Jampilek ꢀ J. Dohnal
Zentiva a.s., U kabelovny 130, 102 37 Prague 10,
Czech Republic
J. Jampilek ꢀ J. Dohnal
Faculty of Pharmacy, Department of Chemical Drugs,
University of Veterinary and Pharmaceutical Sciences,
´
Palackeho 1/3, 612 42 Brno, Czech Republic
Various treatments are used to counteract the toxic
effects of OPI produced either by pre- or by post-intoxi-
cation [2, 3]. For example, pre-treatment methods of
potentially threatened persons includes weak AChE
R. Musiol
Institute of Chemistry, University of Silesia,
Szkolna 9, 40-007 Katowice, Poland
123

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Struct Chem (2010) 21:495–501
Fig. 1 Structural formulae of
commercially available
acetylcholinesterase reactivators
OH
N
HO
N
N
OH
N+
Cl-
N+
N+
2 Cl-
CH3
pralidoxime
TMB-4
OH
N
OH
HO
N
N
N+
O
N+
2 Cl-
N+
O
N+
2 Cl-
HO
N
HI-6
obidoxime (toxogonin)
inhibitors (e.g., pyridostigmine) to sequester the enzyme,
other esterases (e.g., human butyrylcholinesterase) to
scavenge OPI or one of oximes (e.g., HI-6) to reactivate
AChE.
counteract its effect [8, 9]. A drawback of these oximes is
their higher toxicity than that of HI-6 [11].
In this study, we present some results concerning crystal
structures of newly synthesized highly active reactivators,
K075, and K114, as well as a discussion of their potential
interactions with the enzyme.
Post-treatment regimens involve oxime reactivators of
AChE in combination with atropine and diazepam [2–4].
Reactivators of AChE (e.g., pralidoxime, HI-6, obidoxime,
and TMB-4; Fig. 1) are commonly used in the treatment
of OPI intoxications [2, 3, 5–7].
Experimental
They contain a nucleophilic oxime moiety (oximate
anion), which is able to cleave the OPI molecule from
AChE and thereby to restore its function. However, the
group of OPI called nerve agents (e.g., tabun, sarin, soman,
and VX; Fig. 2) undergoes a process called ‘‘aging’’, where
some parts of the OPI molecule are cleaved and replaced
by a hydroxyl group with a negative charge [1, 8]. After
this aging process, an oxime is unable to counteract the
inhibited AChE [9].
Compounds K075 and K114 were synthesized according to
the known procedure shown in Scheme 1. The heating of
pyridinium aldoximes with dibromo linkers afforded the
desired products in good yield and purity [12–14].
Single crystals of compounds K075 and K114 were
obtained from water solutions by slow evaporation. The
phase problem was solved by direct methods using
SHELXS program [15]. The structures were refined by
full-matrix least-squares method using SHELXL97 [15]
with anisotropic displacement parameters for non-hydro-
gen atoms. All the hydrogen atoms bonded to the carbon
atoms were located in the difference Fourier maps and
refined isotropically using the riding model. The hydrogen
atoms bonded to the oxygen atoms were located in dif-
ference Fourier maps and included in the refinement
without constraints. The details of the crystal data, data
collection, and refinement are listed in Table 1. The sup-
plementary crystallographic data obtained for K075 and
K114 have been deposited with the Cambridge Crystallo-
graphic Data Centre and allocated the deposition numbers:
CCDC 714420 and CCDC 714421, respectively. These
data can be obtained free of charge at www.ccdc.cam.
ac.uk/conts/retrieving.html [or from the Cambridge
Crystallographic Data Centre (CCDC), 12 Union Road,
Cambridge CB2 1EZ, UK; fax: ?44(0)1223-336033;
email: deposit@ccdc.cam.ac.uk].
While OPI nerve agents were prepared in the first half of
the twentieth century, still no reactivator of AChE able to
counteract the full spectrum of different OPI has been
discovered [2]. Presently, the HI-6 oxime is the most ver-
satile reactivator of AChE for intoxication by nerve agent
with the exception of tabun [10]. Moreover, the changes
induced by tabun into amino acids of AChE lead to a
partial closing of the enzyme active site. As a result, only a
few reactivators, obidoxime, and TMB-4, are able to
N
O
N
EtO
Me
O
S
F
O
O
F
O
O
P
P
P
P
EtO
Me
Me
N
soman
sarin
tabun
VX
Fig. 2 OPI nerve agents—organophosphorus inhibitors with lethal
effects
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Struct Chem (2010) 21:495–501
497
Scheme 1 Synthesis of
investigated AChE reactivators
CH=NOH
(
E
)- BrCH2CH=CHCH2Br
DMF; 100°C; 4h
N
96%
N
2 Br
N
HON=HC
HON=HC
K075
Br
CH=NOH
N
2 Br
Br
86%
N
DMF; 100°C; 6h
CH=NOH
K114
Table 1 Crystal data, the
measurement and calculation
details
Identification code
Empirical formula
K075
K114
C16H22Br2N4O4
C20H20Br2N4O2
508.22
Formula weight (g/mol)
Diffractometer
494.20
Nonius KappaCCD [16]
293(2) K
Nonius KappaCCD [16]
293(2) K
Temperature (K)
Crystal system, space group
Unit cell dimensions
Monoclinic, P21/c
a = 9.1318 (2),
b = 9.0531 (2),
c = 13.0659 (3),
Orthorhombic, Pcab
a = 7.9405 (2),
b = 15.0311 (3),
c = 17.5202 (3),
a = b = c = 90
2091.11 (8)
˚
a, b, c (A)
a, b, c (ꢁ)
a = c = 90, b = 108.979 (1)
1021.45 (4)
3
˚
Volume (A )
Z, calculated density (g/cm3)
Absorption coefficient (mm-1
F(000)
2, 1.607
4, 1.614
)
3.996
3.900
496
1016
Crystal size (mm)
0.60 9 0.35 9 0.25
3.26–30.03
0.25 9 0.20 9 0.15
3.57–27.47
h range for data collection (ꢁ)
Limiting indices
12 B h B 12,
-12 B k B 11,
-18 B l B 18
9115/2975 [R(int) = 0.0347]
99.4
-9 B h B 10,
-18 B k B 19,
-22 B l B 22
Reflections collected/unique
Completeness (%)
15124/2385 [R(int) = 0.0373]
99.6
Absorption correction
Max. and min. transmission
Refinement method
Multi-scan [17]
0.4349 and 0.1978
Full-matrix least-squares on F2
2975/3/127
Multi-scan [17]
0.5923 and 0.4423
Full-matrix least-squares on F2
2385/0/130
Data/restraints/parameters
Goodness-of-fit on F2
Final R indices [I [ 2r(I)]
1.080
1.047
R1 = 0.0350,
wR2 = 0.0769
R1 = 0.0544,
wR2 = 0.0846
0.272 and -0.508
R1 = 0.0331,
wR2 = 0.0750
R1 = 0.0528,
R indices (all data)
wR2 = 0.0856
0.267 and -0.594
Largest diff. peak and hole (e.A-3
)
Results and discussion
K114 consists of four 4,40-bis(hydroxyiminomethyl)-1,10-
(1,4-phenylenedimethyl)-bispyridinium cations and eight
bromide anions. The ORTEP [18] projections of the
structures of both molecules together with water and bro-
mide ions are shown in Figs. 3 and 4, respectively.
The unit cell of K075 contains two (E)-1,4-bis(4-hydro-
xyiminomethylpyridinium)-but-2-ene cations, four bro-
mide anions, and four water molecules. The unit cell of
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Struct Chem (2010) 21:495–501
Br1
torsion angles which determine the mutual orientation of
the pyridine ring and the linker are the same on both sides
of the molecule center, namely: N2-C7-C8-C80 = 121.6ꢁ
in K075 and N2-C7-C8-C9 = 83.7ꢁ and N2-C7-C8-
C10 = 95.6ꢁ in K114, respectively. The corresponding
torsion angle value in obidoxime is 76.5ꢁ, while in TMB-4
two values of the torsion angles are observed: 69.9ꢁ and
66.02ꢁ. The absolute values of pseudo-torsion angles,
N–C…C–N, where C denotes the first and the last atom of
the linker between two pyridine rings, are 180ꢁ for the
centrosymmetric K075 and K114 molecules, while for
obidoxime and TMB-4 they are 135.7ꢁ and 111.6ꢁ,
respectively. The differences in the lengths of the linkers
and in the symmetry of the molecules lead to the differ-
ences in the overall lengths, i.e., the distances between
oxygen atoms of the oxime groups. These lengths vary in
the order: K075 [ K114 [ obidoxime [ TMB-4 and are:
C3
C4
C7
C8
N2
C5
O1w
N1
C2
C1
C6
O1
Br1
Fig. 3 The ORTEP drawing of the K075 salt and two co-crystallising
water molecules
Br1
C5
N2
C6
C7
C10
C8
C1
O1
C2
N1
C9
C4
C3
˚
18.07, 17.69, 14.71, and 14.92 A, respectively.
Br1
Fig. 4 The ORTEP drawing of the K114 salt
Intermolecular interactions
Structure of molecules in comparison with obidoxime
and TMB-4
Hydrogen bonds are the most important intermolecular
interactions in the crystal of K075. Their parameters are
listed in Table 2. For visual interpretation of intermolecu-
lar interactions, the Mercury program was used [21]. The
chains of water molecules hydrogen-bonded with bromide
ions form a sort of channels along y axis (Fig. 5). Each
water molecule is an acceptor of a proton belonging to an
oxime group of one cation and a proton donor to two
bromide anions. A chain with repeating fragment …H1W-
O1W-H2W…Br1…(H1W), formed in this way, can be
described using Etter notation as C11ð4Þ [22]. The same
hydrogen bonds also form a ring R180ð58Þ. Both patterns of
hydrogen bonding are shown in Fig. 5.
The oxime groups in both compounds have E-configura-
tion. Both molecules are of a zigzag shape. The molecule
of K075 has an inversion center, which coincides with the
midpoint of the double bond between aliphatic carbon
atoms, while in K114—with the middle of benzene ring. In
this respect, K075 and K114 differ from the molecules of
the known AChE reactivators, obidoxime [19], and TMB-4
[20]. The symmetry element of obidoxime in its chloride
crystal is the crystallographic twofold axis, which passes
through the central oxygen atom of the linker between two
pyridine rings. The molecule of TMB-4 has an approximate
(non crystallographic) twofold symmetry. Because of the
molecular symmetry of K075 and K114, the values of the
Other short contacts, which may be qualified as O…p
and N…p intermolecular interactions, are shown in
Table 3. These interactions lead to intercalation of each
Table 2 Intermolecular
´
´
˚
´
˚
˚
d(D-H) (A)
D-H…A
D(H…A) (A)
d(D…A) (A)
\(DHA) (ꢁ)
hydrogen-bonding geometry in
the crystal structures of K075,
K114, obidoxime and TBM-4
K075
O1W-H1WꢀꢀꢀBr1#2
O1W-H2WꢀꢀꢀBr1#3
O1-H1OꢀꢀꢀO1W
0.92 (2)
0.91 (2)
0.91 (3)
2.33 (2)
2.36 (2)
1.77 (3)
3.238 (2)
3.267 (2)
2.669 (2)
169 (2)
176 (3)
171 (3)
#2 [x ? 1, y ? 1, z]; #3 [-x ? 2, y ? 1/2, -z ? 1/2]
K114
O1-H1OꢀꢀꢀBr1#2
#2 [x ? 1/2, -y ? 1/2, z];
Obidoxime [19]
O1-H1OꢀꢀꢀCl1
0.81 (4)
2.35 (4)
3.139 (2)
168 (4)
0.98
0.83
2.07
2.35
2.973
3.183
153
TMB-4 [20]
O1-H1OꢀꢀꢀBr1
176.8
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Struct Chem (2010) 21:495–501
499
Fig. 6 Hydrogen-bonding pattern in K114
Fig. 5 Hydrogen-bonding pattern in K075
Table 3 Selected interactions in AChE complex with obidoxime and
in crystal structures of K075 and K114
˚
Shortest contacts (A) Crystal structures of
AChE-obidoxime K075
K114
C(pyridine)
…aromatic ring
N(oxime)
…aromatic ring
O(oxime)
3.27, 3.14
3.14, 3.47
3.44
Not observed 3.69, 3.72
3.23, 3.50
3.39
3.48, 3.59
3.14
…aromatic ring
Fig. 7 Hydrogen-bonding pattern in TMB-4 [20]
pyridinium ring between the oxime group on one side and
the water molecules on the other.
The crystal structure of K114 is stabilized by the
hydrogen bonds between the hydroxyls of oxime groups
and bromide anions (Table 2). ‘‘Quasi’’-stacking of mole-
cules along x axis becomes a very pronounced feature of
the structure most probably due to the lack of water mol-
ecules in this structure (Fig. 6). The distance between the
centers of pyridinium rings of the neighboring molecules,
related by the glide plane ‘‘a’’ perpendicular to y axis, is
The motifs characteristic for K075 and TMB-4 [20]
crystals are the loop-shaped chains linking the molecules in
pairs. They consist of three water molecules and two bro-
mide anions in K075 crystal (Fig. 5). In TMB-4, the loops
are shorter (one water molecule and two bromide ions)
resulting in a more ‘‘open’’ structure (Fig. 7).
The motifs in K114 and obidoxime [19] crystals, shown
in Figs. 6 and 8, respectively, are mainly the result of the
‘‘organizing’’ role of bromide and chloride anions which
form N–O–H…Br and N–O–H…Cl hydrogen bonds.
˚
4.006 A. The angle between the ring planes is 15.27ꢁ.
Comparison of structural motifs in K075, K114, TMB-4
and obidoxime
Inspection of enzyme–reactivator interactions
The type of motifs occurring in the packing of molecules in
the crystal structures of AChE reactivators depends on the
co-crystallising species, i.e., anions and/or water molecules
(Table 2).
Regarding the activity of the investigated compounds as
AChE reactivators, it is worthwhile to compare the inter-
molecular interactions observed in their crystal structures
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Struct Chem (2010) 21:495–501
Fig. 8 Hydrogen-bonding pattern in obidoxime [19]
with those in the oxime complexes that are able to reacti-
vate AChE-tabun conjugates.
The crystal structures of such complexes, formed by the
known reactivators: HI-6, Ortho-7, and obidoxime with
non-phosphorylated AChE, were determined by Ekstrom
et al. [23].
These authors described also the modes of reactivator-
enzyme binding. According to Ekstrom, the most important
interactions with AChE amino acids are formed by such
reactivator fragments as the pyridine and benzene rings,
nitrogen, and oxygen atoms of oxime groups as well as
by water molecules. Thus, in Table 3 we compare the
parameters of selected hydrogen bonds and pyridine con-
tacts, which occur in the crystal structures of K075 and
K114, with those observed in the AChE–obidoxime
complex.
Fig. 9 Fragment of crystal structure of mAChE complex with
˚
obidoxime [23, 24]. The shortest distances (A) between obidoxime
´
In Figs. 9a and b, the surroundings of each of the obi-
doxime pyridinium rings are separately depicted. One of
them, together with the oxime group, is located in a
‘‘sandwich-like’’ way between aromatic rings of Trp-286
and Tyr-72 side chains of AChE in its peripheral anionic
site (PAS), while the oxygen atom, O1, forms a hydrogen
bond with the carboxylic group of Val-282 (Fig. 9a). The
other pyridine moiety of obidoxime interacts with the ring
of Tyr-337 and its oxime group—with Phe-338 as well as
with water molecule through hydrogen bonding; both these
amino acid residues belong to the AChE catalytic site
(Fig. 9b).
and different residues of mAChE are marked with dashed lines.
a Interactions with peripheral anionic site (PAS) of mAChE;
b interactions with catalytic site of mAChE
Inspection of the crystal structure fragments of K075
and K114 (Figs. 10a and b) shows that molecules of the
new AChE reactivators are able to form interactions similar
to those observed in the crystal of AChE complex with
obidoxime. Particularly in K075, interesting contacts
C4…C1 and N1…O1 resemble to those between C(pyri-
dine) of obidoxime and aromatic ring of Trp-286 of AChE
at peripheral anionic site (PAS). In the crystal structure of
K114, the mutual arrangement of molecules is more
complicated than in the case of K075, but there are several
shorter contacts. In particular, distances: N1…C9 and
O1…O6 are similar to those which occur in the AChE–
obidoxime complex.
Fig. 10 Selected intermolecular interactions in the crystal structure
of a K075 and b K114
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Struct Chem (2010) 21:495–501
501
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Conclusions
The single crystal of (E)-1,4-bis(4-hydroxyiminomethy-
lpyridinium)-but-2-ene (K075) and of 4,40-bis(hydroxyim-
inomethyl)-1,10-(1,4-phenylenedimethyl)-bispyridinium
(K114) bromides was obtained and their structures were
determined with the use of X-ray diffraction. K075 is a
dihydrate in which the water molecules are hydrogen-
bonded with oxygen atoms of oxime groups. In K114, the
acceptors of protons of the oxime groups are bromide
anions. The molecules of both compounds are centrosym-
metric and in this respect they differ from the well-known
obidoxime and TMB-4 whose symmetry is twofold and
approximate twofold axis, respectively.
The intermolecular interactions in the crystals of K075
and K114 are similar to those in the crystalline hydrate of
TMB-4 bromide and of obidoxime chloride, respectively.
This similarity, together with the comparable reactivation
potencies of K075 and obidoxime [13], suggests that the
interactions occurring in their crystals may play an
important role in the reactivation activity. This conclusion
is confirmed by the comparison of the interactions
observed in the crystalline AChE–obidoxime complex and
in the investigated crystals.
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Determination of the exact relationship between struc-
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Acknowledgments The authors express their appreciation to Mrs.
M. Hrabinova for her technical assistance. This study was supported
by Grant Agency of Ministry of Education, Youth, and Sports (Czech
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