Quinuclidinium-imidazolium compounds: Synthesis, mode of interaction with acetylcholinesterase and effect upon Soman intoxicated mice

Article abstract of DOI:10.1007/s002040050504

Four compounds were prepared: 3-oxo-1-methylquinuclidinium iodide (I), 2-hydroxyiminomethyl-1,3-dimethylimidazolium iodide (II) and two conjugates of I and II linked by -(CH₂)₃- (III) and -CH₂-O-CH₂- (IV). The aim was to evaluate separately the properties of I and II as opposed to III and IV, which contain both moieties in the same molecule. All four compounds were reversible inhibitors of acetylcholinesterase (AChE; EC 3.1.1.7). The enzyme/inhibitor dissociation constants for the catalytic site ranged from 0.073 mM (II) to 1.6 mM (I). The dissociation constant of I for the allosteric (substrate inhibition) site was 4.8 mM. Possible binding of the other compounds to the allosteric site could not be measured because II, III and IV reacted with the substrate acetylthiocholine (ATCh) and at high ATCh concentrations the non-enzymic reaction interfered with the enzymic hydrolysis of ATCh. The rate constants for the non-enzymic ATCh hydrolysis were between 23 and 37 l/mol per min. All four compounds protected AChE against phosphorylation by Soman and VX. The protective index (PI) of I (calculated from binding of I to both, catalytic and allosteric sites in AChE) agreed with the measured PI; this confirms that allosteric binding contributes to the decrease of phosphorylation rates. The PI values obtained with III and IV were higher than those predicted by the assumption of their binding to the AChE catalytic site only. The toxicity (i.p. LD₅₀) of compounds I, II, III and IV for mice was 0.21, 0.68, 0.49 and 0.77 mmol/kg body wt. respectively. All four compounds protected mice against Soman when given (i.p.) together with atropine 1 min after Soman (s.c.). One-quarter of the LD₅₀ dose fully protected mice (survival of all animals) against 2.52 (IV), 2.00 (I and III) and 1.58 (II) LD₅₀ doses of Soman.

Full text of DOI:10.1007/s002040050504

Arch Toxicol (1998) 72: 289±295
Ó Springer-Verlag 1998
ORGAN TOXICITY AND MECHANISMS
Vera Simeon-Rudolf á Elsa Reiner á Mira Skrinjaric-Spoljar
Bozica Radic á Ana Lucic á Ines Primozic
Srdanka Tomic
Quinuclidinium-imidazolium compounds:
synthesis, mode of interaction with acetylcholinesterase
and effect upon Soman intoxicated mice
Received: 20 October 1997 / Accepted: 14 January 1998
Abstract Four compounds were prepared: 3-oxo-1- IV for mice was 0.21, 0.68, 0.49 and 0.77 mmol/kg body
methylquinuclidinium iodide (I), 2-hydroxyiminometh- wt. respectively. All four compounds protected mice
yl-1,3-dimethylimidazolium iodide (II) and two conju- against Soman when given (i.p.) together with atropine 1
gates of I and II linked by -(CH2)3- (III) and -CH2-O- min after Soman (s.c.). One-quarter of the LD50 dose
CH2- (IV). The aim was to evaluate separately the fully protected mice (survival of all animals) against 2.52
properties of I and II as opposed to III and IV, which (IV), 2.00 (I and III) and 1.58 (II) LD50 doses of Soman.
contain both moieties in the same molecule. All four
compounds were reversible inhibitors of acetylcholine- Key words Acetylcholinesterase á Quinuclidine-
sterase (AChE; EC 3.1.1.7). The enzyme/inhibitor dis- imidazolium compounds á Reversible inhibition and
sociation constants for the catalytic site ranged from protection of acetylcholinesterase against
0.073 mM (II) to 1.6 mM (I). The dissociation constant phosphorylation á Antidotal e€ect against Soman
of I for the allosteric (substrate inhibition) site was poisoned mice á Soman
4.8 mM. Possible binding of the other compounds to the
allosteric site could not be measured because II, III and
IV reacted with the substrate acetylthiocholine (ATCh)
and at high ATCh concentrations the non-enzymic re-
action interfered with the enzymic hydrolysis of ATCh.
The rate constants for the non-enzymic ATCh hydro-
lysis were between 23 and 37 l/mol per min. All four
Abbreviations DTNB 5,5¢-Dithiobis (2-nitrobenzoic
acid) á Soman O-1,2,2-trimethylpropyl
methylphosphono¯uoridate á VX O-ethyl S-2-
diisopropylaminoethyl methylphosphonothioate
Introduction
compounds protected AChE against phosphorylation by
Soman and VX. The protective index (PI) of I (calcu-
lated from binding of I to both, catalytic and allosteric
sites in AChE) agreed with the measured PI; this con-
®rms that allosteric binding contributes to the decrease
of phosphorylation rates. The PI values obtained with
III and IV were higher than those predicted by the as-
sumption of their binding to the AChE catalytic site
only. The toxicity (i.p. LD50) of compounds I, II, III and
Oximes are reactivators of phosphorylated acetylcho-
linesterase (AChE; EC 3.1.1.7) and are therefore used in
the therapy of poisoning by organophosphorus (OP)
compounds. AChE has two binding sites for many re-
versible ligands: the catalytic site and an allosteric site.
Several imidazolium and pyridinium oximes have been
shown to bind reversibly to both of these sites (cf. Reiner
et al. 1996). The substrates, acetylcholine and acetyl-
thiocholine also bind to both sites; binding of substrates
to the allosteric site causes substrate inhibition (cf.
Aldridge and Reiner 1972; Radic et al. 1991).
V. Simeon-Rudolf (&) á E. Reiner á M. Skrinjaric-Spoljar
Laboratory of Biochemistry,
Institute for Medical Research and Occupational Health,
Ksaverska cesta 2, 10001 Zagreb, Croatia
Reversible ligands protect the catalytic site against
phosphorylation due to a direct competition between the
ligand and the OP compound. Protection of the catalytic
site may, however, also be achieved with ligands which
bind to the allosteric site only. This was proved for the
allosteric ligands, propidium and one coumarin deriva-
tive (Aldridge and Reiner 1969, 1972; Radic et al. 1984,
1991; Taylor and Lappi 1975), and suggested for two
bis-imidazolium oximes (Franciskovic et al. 1993).
B. Radic á A Lucic
Laboratory of Toxicology,
Institute for Medical Research and Occupational Health,
Ksaverska cesta 2, 10001 Zagreb, Croatia
I. Primozic á S. Tomic
Department of Chemistry,
Faculty of Science and Mathematics, University of Zagreb,
Strossmayerov trg 14, 10000 Zagreb, Croatia

290
sorbance was read at 412 nm, at 15 s intervals, against a blank
which contained the erythrocytes suspended in bu€er and DTNB.
Enzyme activities were corrected for substrate hydrolysis due to the
reaction of acetylthiocholine with the oxime. At 5 and 10 mM
substrate, activities were also corrected for spontaneous substrate
hydrolysis.
The reaction of acetylthiocholine with the oxime (compounds
II, III and IV) was measured in a medium which contained 0.1 M
phosphate bu€er, DTNB, substrate and oxime. The increase in
absorbance was read for 1 min against a blank which contained
bu€er and DTNB. The oximes did not react with DTNB and blank
samples therefore did not contain the oximes. Concentrations of
the oximes and of the substrate are given in Table 2. Two to four
measurements were done with each substrate/oxime concentration
pair. The spontaneous substrate hydrolysis was measured in a
medium which contained bu€er, DTNB and substrate; the blank
contained bu€er and DTNB.
Reversible inhibition of AChE was measured in a medium
which contained the erythrocytes suspended in bu€er, DTNB, the
studied compound and the substrate. Control samples contained no
studied compound. The increase in absorbance was read for 1 min.
The concentrations of the studied compounds and of the substrate
are given in Table 3. Two to four measurements were done with
each substrate/compound concentration pair. Protection of AChE
against phosphorylation was measured in a medium which con-
tained the erythrocytes suspended in bu€er, DTNB, the studied
compound and the OP compound. After a given time of inhibition
(up to 4 min) the substrate was added (®nal concentration 1.0 mM)
and the increase in absorbance read for 1 min. Control samples
contained no studied compound.
Four compounds are described in this paper
(Table 1): a 3-oxoquinuclidinium derivative (I), an
imidazolium oxime (II) and two conjugates of 3-oxo-
quinuclidinium and the imidazolium oxime (III and IV).
Compound I was shown by Sterling et al. (1988) to
protect rats against the toxicity of Soman, when ad-
ministered in conjunction with atropine and N-methyl-
2-hydroxyiminomethylpyridinium chloride (2-PAM).
Compound II was shown by Galosi et al. (1988) to be an
inhibitor of AChE and reactivator of Sarin-phosphory-
lated AChE. It was further shown that some conjugates
of 3-substituted quinuclidinium derivatives with pyr-
idinium oximes, prepared by Amitai et al. (1987), were
antidotes against Tabun and Soman poisoning in dogs
and monkeys (Amitai et al. 1995).
In the present paper, conjugates of the 3-oxo-
quinuclidinium derivative (I) with an imidazolium oxime
(II) were prepared and studied for their mode of binding
to AChE, their e€ect upon phosphorylation of AChE by
Soman and VX, and their e€ect upon Soman intoxicated
mice. These compounds (III and IV) have not been
previously described. The aim was to evaluate separately
the properties of compounds I and II, as opposed to
compounds III and IV, which contain both moieties in
the same molecule. None of the four compounds has
been investigated to date for their mode of interaction
with AChE.
Studies in vivo
Male BALB-C mice (18±25 g body wt.) were fed on a standard diet,
which was withdrawn 24 h prior to treatment. Animals had free
access to water and were kept in a room maintained by thermostat
at 21 °C in Macralone cages under a light/dark cycle of 12:12 h.
The acute i.p. toxicity (LD50) was based upon 24 h mortality rates
calculated according to Thompson (1947) and Weil (1952). Each
LD50 was evaluated from results obtained with 4±6 doses of a given
compound (dissolved in water); six animals were injected per dose;
whenever the results of the experiment allowed, the 95% con®dence
limits were estimated from the tables (Thompson 1947; Weil 1952).
The therapeutic e€ect against Soman intoxication was tested by
giving the studied compounds (1/4 of their LD50 dose) together
with atropine sulphate (10 mg/kg), or atropine sulphate only, im-
mediately after Soman. Compounds I±IV and atropine sulphate
were given i.p.; Soman was given s.c. The ecacy of compounds
was expressed as therapeutic factor (TF) and therapeutic dose
(TD). The therapeutic factor was calculated from:(LD50 of Soman
plus antidote)/(LD50 of Soman only). The therapeutic dose is the
highest multiple of the LD50 of Soman, which was fully counter-
acted (survival of all animals) by the antidotes.
Materials and methods
Synthesis of compounds
Formylation of 1-methylimidazole was performed by the method of
Iversen and Lund (1966). The aldehyde was converted to the oxime
derivative in a standard fashion. 3-Oxo-1-(chloromethoxymeth-
yl)quinuclidinium chloride (1), 3-oxo-1-(3-iodopropyl)quinuclidi-
nium iodide (2) and 3-oxo-1-(3-bromopropyl)quinuclidinium
bromide (3) were prepared by a modi®ed method of Amitai et al.
(1987). 3-Oxo-l-methylquinuclidinium iodide (compound I) and
2-hydroxyiminomethyl-1,3-dimethylimidazolium iodide (com-
pound II) were synthesized according to the procedure described by
Galosi et al. (1988) and Grifantini et al. (1972).
3-Oxo-1-[3-(2-hydroxyiminomethyl-3-methyl-l-imidazolio)pro-
pyl]-quinuclidinium diiodide (compound IIIa) and dibromide
(compound IIIb), and 3-oxo-1-[3-(2-hydroxyiminomethyl-3-methyl
-l-imidazolio)-2-oxapropyl]quinuclidinium dichloride (compound
IV) were synthesized by the reaction of (1), (2) or (3) with one
equivalent of 1-methylimidazole-2-aldoxime in dry dimethyl for-
mamide DMF at room temperature for 24 h. The crystals were
separated by ®ltration and washed with ether. Analytical and
spectral data of compounds I±IV are given in Table 1. Compound
III was prepared as an iodide (a) and bromide (b). Studies in vitro
were done with IIIa and in vivo with IIIb.
Results and discussion
Reaction of acetylthiocholine
with the imidazolium oximes
It has been shown earlier that oximes react with ATCh
whereby thiocholine is one of the reaction products (cf.
Skrinjaric-Spoljar et al. 1992). It has further been shown
that the reaction occurs on a 1:1 molar basis (Skrinjaric-
Spoljar et al. 1992).
Studies in vitro
The source of AChE was native human erythrocytes; the ®nal di-
lution during enzyme assay was 400-fold. The substrate was ace-
tylthiocholine iodide (ATCh). All experiments were done in 0.1 M
phosphate bu€er, pH 7.4, at 37 °C. The enzyme activity was
measured by the spectrophotometric method of Ellman et al. (1961)
with the thiol reagent DTNB (®nal conc. 0.33 mM). The reaction
All three studied oximes reacted with ATCh. The
second-order rate constant of the reaction (k) was cal-
was performed in a total volume of 3.0 ml. The increase in ab- culated according to:

291

292
Table 2 Second-order rate constants (k ‹ SE) for reaction of the
substrate acetylthiocholine with the indicated compounds
Kapp is the apparent enzyme/compound dissociation
constant at a given substrate concentration (s), vo and vi
are the enzyme activities measured at a given substrate
concentration in the absence and in the presence res-
pectively of a given inhibitor concentration (i). Km is the
Michaelis constant for the substrate. The Ka values ob-
tained are given in Table 3. Compound II has the
highest anity (lowest Ka) and compound I the lowest
anity. The two 3-oxoquinuclidinium-imidazolium ox-
imes (III and IV) have Ka values between those of
compounds I and II.
Substrate
(mM)
Compound
(mM)
k ‹ SE
(l/mol per min)
0.25±1.0
0.05±1.0
0.05±1.0
II 0.08±0.25
III 0.20±1.0
IV 0.10±1.0
37 ‹ 4
27 ‹ 4
23 ‹ 2
Constants were calculated according to Eq. 1 (see text). Each
constant was obtained from measurements done with 6-11 sub-
strate/compound concentration pairs
3-Oxoquinuclidinium (I) does not react with acetyl-
thiocholine, and inhibition by this compound could
therefore be studied over a wide concentration range. At
high substrate concentrations, the kinetics of AChE in-
hibition with compound I deviated from Eq. 2. This was
attributed to allosteric binding of the quinuclidinium
moiety. The corresponding dissociation constant for the
allosteric site (Ki) was calculated from the linear rela-
tionship between Kapp and substrate concentrations be-
tween 1 and 10 mM (Table 3). For allosteric binding, the
intercept of the line (Kapp vs s) with the ordinate corre-
sponds to Ki and the intercept on the abscissa to Kss, the
substrate inhibition constant (Aldridge and Reiner 1969;
Radic et al. 1991). Possible binding of II, III and IV to
the allosteric site in AChE could not be studied, because
at high substrate concentrations all three oximes induced
the hydrolysis of ATCh at a rate which was equal to, or
even higher than the rates of the enzymic hydrolysis, and
thus interfered with the studied reaction.
The Kss constant derived from the kinetics of inhibi-
tion with compound I (Table 3) was within the order
known for the AChE/ATCh reaction (cf. Aldridge and
Reiner 1972; Radic et al. 1991; Simeon 1974) thus sup-
porting the conclusion that compound I binds to the
allosteric site. The Km values, derived from the kinetics
of inhibition with all four compounds at low substrate
concentrations, ranged from 0.073 to 0.45 mM
(Table 3); these values are close to the published Km for
ATCh covering a range from 0.05 to 0.14 mM (cf.
Aldridge and Reiner 1972; Radic et al. 1991; Simeon
1974). This supports the conclusion that binding con-
stants derived from inhibition studies at low substrate
concentrations correspond to binding of the inhibitors
to the catalytic site of AChE.
Table 3 Reversible inhibition of acetylcholinesterase by the in-
dicated compounds measured with acetylthiocholine as substrate
Compound
(mM)
Substrate
(mM)
Ka
(mM)
Km
(mM)
I
1±10
0.10±1.0
1.6 ‹ 0.2
0.45 ‹ 0.05
II 0.1±0.5
III 0.5, 1.0
IV 0.5±2.0
0.05±0.50
0.05±0.50
0.05±0.25
0.073 ‹ 0.011 0.34 ‹ 0.08
0.24 ‹ 0.05 0.13 ‹ 0.03
0.25 ‹ 0.04 0.073 ‹ 0.012
Ki
(mM)
Kss
(mM)
I 5, 10
1, 5, 10
4.8 ‹ 0.1
22 ‹ 7
Constants ‹ SE were calculated as described in the text. Each
constant was derived from measurements done with 6±8 substrate/
compound concentration pairs
c=t ˆ k Á s Á i
ꢀ1†
where c is the released thiocholine concentration, t is the
time of reaction, and s and i are the initial substrate and
oxime concentrations. Initial concentrations were used,
because the time of reaction was short and the decrease
in concentration did not need to be taken into account.
It was shown by means of one-way analysis of vari-
ance and multiple-range Sche€e test (at the 95% con®-
dence level) that the imidazolium oxime (II) reacted
signi®cantly faster with ATCh than its conjugates with
3-oxoquinuclidinium (III and IV); the latter two reacted
about equally fast (Table 2). All three rate constants
were of the same order as those of 2-PAM, Toxogonin
and six other imidazolium or pyridinium oximes
(Skrinjaric-Spoljar et al. 1992).
Protection of acetylcholinesterase against
phosphorylation by Soman and VX
Reversible binding of compounds
to acetylcholinesterase
All four compounds protected the enzyme against
phosphorylation. Protection was expressed in terms of
the protective index (PI) which corresponds to the ratio:
All four compounds were reversible inhibitors of AChE.
The enzyme/compound dissociation constants for
binding to the catalytic site (Ka) were evaluated from the
kinetics of enzyme inhibition at low substrate concen-
trations and calculated according to Aldridge and Re-
iner (1969, 1972) and Radic et al. (1984, 1991):
PI ˆ ka=ka0
ꢀ3†
where ka and ka¢ are the ®rst-order rate constants of
phosphorylation in the absence and in the presence of
the reversible inhibitor respectively. The measured PI
values are given in Table 4. For each OP compound, PI
Kapp ˆ ꢀvi Á i†=ꢀvo ꢁ vi† ˆ Ka‰1 ‡ ꢀs=Km†Š
ꢀ2†

293
Table 4 Protection of AChE
against phosphorylation by
Soman (5 nM) and VX (20 nM)
Compound
(mM)
Organo-
phosphate
PImeasured
PIcalculated
Eq. 4
1.9
Eq. 5
2.2
Eq. 6
2.4
I 1.4
I 6.0
Soman
VX
Soman
VX
1.9
2.0
9.0
7.0
4.8
6.0
10.7
II 0.08
II 0.16
Soman
VX
Soman
VX
2.1
2.0
3.0
3.0
2.1
3.2
III 0.2
III 0.4
Soman
VX
Soman
VX
2.0
1.9
3.2
4.1
1.8
2.7
IV 0.1
IV 0.3
Soman
VX
Soman
VX
2.0
2.1
3.8
4.2
1.4
2.2
The protective indices are mean values of two experiments, except for IV where single experiments
were done. The protective indices PIcalculated were calculated according to the indicated equations using
Ka and Ki values from Table 3; PIcalculated value applies to any acylating inhibitor, because it is a ratio
of acylating rate constants
was measured at two concentrations of the reversible higher than those calculated from Eq. 4. The di€erence
inhibitor.
between measured PI and those calculated from Eq. 4
When a reversible inhibitor binds only to the catalytic was more pronounced at higher concentrations of the
site, the PI value is (Reiner 1986):
reversible ligand. The higher PI values suggest that al-
losteric binding of the 3-oxoquinuclidinium derivative
contributed towards the enzyme protection. Equations 5
and 6 could be veri®ed only for compound I (Table 4).
The measured PI values were higher than predicted from
Eq. 5, but lower than predicted from Eq. 6. From this
result one may only conclude that allosteric binding
contributed to the enzyme protection, but the result was
PI ˆ 1 ‡ i=Ka
ꢀ4†
When a reversible inhibitor binds to the catalytic and
allosteric site, the PI value is either:
PI ˆ 1 ‡ i=Ka ‡ i=Ki
or:
ꢀ5†
Table 5 Acute toxicity (i.p. LD50) of the tested compounds in male
mice
PI ˆ ꢀ1 ‡ i=Ka† Á ꢀ1 ‡ i=Ki†
ꢀ6†
Eq. 5 holds for compounds which form two binary
complexes with the enzyme (one at the catalytic site and
the other at the allosteric site), while Eq. 6 holds for
compounds which in addition form a ternary complex
where both sites on the enzyme are simultaneously oc-
cupied by the reversible inhibitor (Reiner 1986).
The measured PI values for compound II agreed with
PI values calculated from Eq. 4. For compounds I, III
and IV, the measured PI values were the same as, or
Compound
LD50 (95% con®dence limits)
(mg/kg)
(mmol/kg)
I
II
III
IV
56a
182 (154±217)
0.21a
0.68 (0.58±0.81)
225a
0.49a
283a
0.77a
a
95% con®dence limits could not be calculated from the tables in
Thompson (1947) and Weil (1952)
Table 6 E€ect of the tested
Antidotes
Soman LD50 (lg/kg)
(95% con®dence limits)
TFb
TDc
compounds combined with
atropine upon (s.c.) Soman
toxicity in male mice
(TF Therapeutic factor, TD
therapeutic dose)
None
220 (183±264)
247 (204±298)
495a
438 (372±516)
525 (468±588)
622a
±
1.12 (0.93±1.36)
±
Atropine only
I+atropine
II+atropine
III+atropine
IV+atropine
0.79
2.00
1.58
2.00
2.52
2.25a
1.99 (1.69±2.35)
2.38 (2.13±2.67)
2.83a
a
95% con®dence limits could not be calculated from the tables in Thompson (1947) and Weil (1952)
Calculated as: LD50 (Soman+antidote)/LD50 (Soman)
Highest multiple LD50 of Soman fully counteracted by an antidote (survival of all animals)
b
c

294
the NMR measurements. The technical assistance of Mrs Andelka
Buntic and Mr A. Fajdetic is gratefully acknowledged. This work
was supported partly by the Ministry of Science and Technology of
the Republic of Croatia.
ambiguous concerning which of the two equations is
more applicable.
E€ect of compounds on mice poisoned by Soman
All four compounds were toxic to mice (Table 5).
Symptoms of poisoning (tremor, ataxia, dyspnoea, im-
paired movement) showed up 3±5 min after the i.p. ad-
ministration. Animals died after 5±7 min or recovered
after c. 20±30 min. Expressed on a molar basis, com-
pound I was the most toxic and compound IV the least
toxic. The four compounds, plus atropine, were tested as
antidotes against Soman (Table 6). The compounds
were administered at equitoxic doses and were given
(i.p.) immediately after Soman (given s.c.). Animals de-
veloped mild central, nicotinic and muscarinic symp-
toms within 30±60 min. After that time all symptoms
(salivation, fasciculation, ataxia, dyspnoea) became
more pronounced and persisted for up to 3 h; tremor
could be provoked for up to 24 h.
The therapeutic factors ranged from 1.99 to 2.83
LD50 of Soman. The LD50 of Soman (220 lg/kg) given
alone was slightly lower than the LD50 of Soman with
atropine (247 lg/kg; Table 6). TF values calculated on
the basis of LD50 of Soman and atropine (not given in
Table 6), were therefore negligibly lower than those
based on the LD50 of Soman without atropine. The
therapeutic dose (TD) of the tested compounds was
between 1.58 and 2.52 LD50 of Soman while the thera-
peutic dose of atropine alone was 0.79 LD50 of Soman
(174 lg/kg). All animals given Soman with no antidote
survived at 0.72 LD50 of Soman (159 lg/kg). All four
compounds had very similar antidotal e€ects (Table 6).
However, compound II tended to be the least e€ective
and compound IV was slightly more ecient than the
other studied compounds.
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sulted in better antidotal properties than the -(CH2)3-
link (III). The oxime group seems not to be required for
an antidotal e€ect. Compound I, with no oxime group,
had the same TD as III (Table 6) and also the same TD
as the 3-hydroxyimino-1-methylquinuclidinium, which
was tested under the same experimental conditions
(Lucic et al. 1997).
Some oximes seem to have pharmacological e€ects,
which are not related to the reactivation of phosphory-
lated AChE (cf. Van Helden et al. 1996). Several qua-
ternary and tertiary 3-substituted quinuclidinium
derivatives were shown to inhibit choline uptake into the
neuron, hence in¯uencing the acetylcholine synthesis
(Sterling et al. 1991, 1993). The antidotal e€ect of
3-substituted quinuclidinium derivatives against OP
poisoning has not yet been elucidated.
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Acknowledgements We thank Mrs Biserka Metelko, Mr Z Marinic
and Mr B. Sokac from the Rugjer Boskovic Institute, Zagreb, for

295
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