Cholinesterase reactivators derived from pyridine-2-carbaldoxime

Article abstract of DOI:10.1135/cccc19980199

Monoquaternary reactivators of cholinesterase 3a-3c were prepared by quaternization of substituted pyridine-2-carbaldoximes with an ester (1a, 1b) or carbamoyl (2) function in position 4. A new bisquaternary reactivator, 4-carbamoyl-2′-[(hydroxyimino)methyl]-1,1′-(but-2-ene-1,4-diyl) bispyridinium salt (5), was derived from the unsubstituted pyridine-2-carbaldoxime. The compounds were characterized by UV spectra and ionization constants. The substances exhibit antidote effects in intoxications with organophosphate cholinesterase inhibitors.

Full text of DOI:10.1135/cccc19980199

Cholinesterase Reactivators
199
CHOLINESTERASE REACTIVATORS DERIVED FROM
PYRIDINE-2-CARBALDOXIME
a1
a2
a
b
Jiri BIELAVSKY , Jiri KASSA , Iva ELSNEROVA and Lubos DEJMEK
a J. E. Purkyne Military Medical Academy, 500 01 Hradec Kralove, Czech Republic;
e-mail: 1 bielavsky@post.cz, 2 kassa@pmfhk.cz
b Research Institute for Pharmacy and Biochemistry, 130 60 Prague 3, Czech Republic
Received February 27, 1997
Accepted October 20, 1997
Monoquaternary reactivators of cholinesterase 3a–3c were prepared by quaternization of substituted
pyridine-2-carbaldoximes with an ester (1a, 1b) or carbamoyl (2) function in position 4. A new bis-
quaternary reactivator, 4-carbamoyl-2′-[(hydroxyimino)methyl]-1,1′-(but-2-ene-1,4-diyl)bispyridinium
salt (5), was derived from the unsubstituted pyridine-2-carbaldoxime. The compounds were charac-
terized by UV spectra and ionization constants. The substances exhibit antidote effects in intoxica-
tions with organophosphate cholinesterase inhibitors.
Key words: Reactivators; Cholinesterase; Pyridines; Pyridinium salts; Organophosphates.
The currently used efficient cholinesterase reactivators are based on quaternized unsub-
stituted pyridine-2- or -4-carbaldoximes1. The application of substituted pyridine-2-
carbaldoximes has not attracted attention as yet, although their interesting
pharmacological properties can be expected. This fact may be due to a difficult prepa-
ration of substituted pyridine-2-carbaldehydes. A route to 4-substituted pyridine-2-
carbaldehydes consists in the reduction of esters of pyridine-2,4-dicarboxylic acid with
LiAlH4 or with diisobutyl aluminium hydride using the procedure of Queguiner and
Pastour2, where only the ester function in position 2 is reduced to the aldehyde. Alter-
natively, corresponding 2-methylpyridines can be reacted with dimethyl sulfoxide and
iodine3. The synthesis of a 4-substituted pyridine-2-carbaldehyde via the 2-pyridyl-
lithium intermediate has been recently reported4.
In the present work, the Queguiner–Pastour approach was applied to obtain pyridine-
2-carbaldoximes with the ester function in position 4 (1a, 1b), which served as the
intermediates in the preparation of the carbamoyl derivative of pyridine-2-carbaldox-
ime 2, which has not been described until now. The aldoximes 2 were then employed
in the synthesis of monoquaternary compounds 3a–3c.
The most efficient reactivators were found in the group of aldoxime derivatives of
bispyridinium salts with a –(CH2)3– or –CH2–O–CH2– bridge, where the aldoxime
group need not to be present at both pyridinium rings. The trans-but-2-ene-1,4-diyl
bridge between the pyridinium rings of bisquaternary reactivators has already been em-
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200
Bielavsky, Kassa, Elsnerova, Dejmek:
ployed. In comparison with analogous compounds containing the –CH2OCH2– bridge,
the butenediyl bridge gives compounds which are somewhat more stable but also more
toxic5. The new reactivator 5 with the bridge described in this paper is an analog of the
efficient reactivator HI-6 (4-carbamoyl-2′-[(hydroxyimino)methyl]-1,1′-(oxydimethylene)-
bispyridinium dichloride)6.
In the experiments involving reduction of lutidinates, better results were achieved
with rather aged LiAlH4 preparations having lower hydride contents. The crude alde-
hydes obtained were oximated with hydroxylamine at pH 8 without isolation yielding
corresponding aldoxime esters 1a and 1b.
COR
COOR
CONH
2
I
N
CH NOH
N
CH NOH
N
CH NOH
CH
3
2
R
CH
1a
1b
3
R
C H
2
5
3a OCH
3
3b
3c
3d
OC H
2
5
NH
2
OH
CONH
N
2
N
CH NOH
N
CH NOH
2 X
2
Br
CH CH CHCH Br
CH CH CHCH
2
2
2
4
X = Br
X = Cl
5a,
5b,
The conversion of the esters 1a and 1b to the amide 2 proceeded easily by the action
of concentrated aqueous ammonia on the solid esters at room temperature. Methanolic am-
monia can be employed as well.
The quaternization of substituted pyridinealdoximes was more tedious than that of
the unsubstituted pyridine-2-carbaldoxime.
The bisquaternary monoaldoxime 5 was prepared by a two-step procedure. In the
first step, the monoquaternary compound 4 was prepared using an excess of 1,4-
dibromobut-2-ene. The intermediate 4 is not very stable, and hence it was used without
purification in the subsequent reaction with isonicotinamide giving dibromide 5a,
which was converted to dichloride 5b using an ion exchanger.
Spectral properties and ionization constants of the compounds are given in Table I.
As found by UV spectroscopy and confirmed by HPLC, monoquaternary substances 3
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Cholinesterase Reactivators
201
decompose in 0.1 M NaOH to give compound 3d, with the carboxy group presumably
being in position 4. The decomposition is the fastest for methyl ester 3a.
Compounds 3a, 3c, and 5a were tested in vivo for their therapeutic effect by investi-
gating their influence on the mean lethal dose LD50 of the relatively toxic organophos-
phate insecticide fosdrin (Mevinphos, 2-(methoxycarbonyl)-1-methylvinyl dimethyl
phosphate) in mice. The oximes were administered intramuscularly in doses corre-
sponding to 5% of the LD50 values found 1 180, 1 290, and 266 mg/kg for compounds
3a, 3c, and 5a, respectively, also administered intramuscularly. The therapeutic effect
is expressed by the therapeutic protection index, which is the LD50 ratio for the treated
and untreated animals (Table II).
If monoquaternary oximes 3a and 3c were administered 2 min after the intramuscular
intoxication, they nearly doubled the LD50 value of fosdrin. This relatively low efficacy
TABLE I
UV spectra and pKa values of aldoximes 1–3 and 5a
λmax (log ε)
Compound
pKa1
pKa2
MeOH
0.05 M HCl
0.05 M borax
1a
1b
2
299 (3.69)
–
–
1.92 ± 0.02
1.94 ± 0.04
2.23 ± 0.02
7.60 ± 0.04
7.53 ± 0.02
7.57 ± 0.02
7.92 ± 0.05
7.63 ± 0.02
9.89 ± 0.05
300 (3.65)
–
–
9.90 ± 0.05
292 (3.65)
–
–
9.91 ± 0.02
3a
3b
3c
3d
5a
–
–
–
–
–
313 (3.99)
315 (3.98)
308 (4.03)
309 (3.98)a
295 (4.04)
361 (4.08)
355 (4.08)
355 (4.13)
347 (4.09)
342 (4.17)
–
–
–
–
–
a pH 6.0, λmax (log ε): 303 (4.00).
TABLE II
Therapeutic protection index of aldoximes 3a, 3c, and 5a
Time of administration after intoxication
1 min
Compound
30 s
2 min
3a
3c
5a
9.0
11.1
4.5
7.4
1.8
1.8
1.8
8.2
3.75
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Bielavsky, Kassa, Elsnerova, Dejmek:
was due to the intoxication symptoms fully developed at the time of administration. If
the time period between the intoxication and treatment was shortened to 30 s, bis-
quaternary oxime 5a increased the LD50 value of fosdrin 4.5 times, which is consider-
ably less than with monoquaternary oximes 3a and 3c bringing about a 9-fold and
11-fold increase, respectively. The increase was statistically significant (p < 0.05) irre-
spective of the time of administration and the oxime chosen. The higher efficacy of the
monoquaternary oximes 3a and 3c towards fosdrin as compared to bisquaternary oxime
5a is apparently due to the appreciably lower toxicity of the former oximes.
EXPERIMENTAL
Melting points were determined on a Boetius apparatus and are not corrected. IR spectra were
scanned on a PU 8706 spectrophotometer, and UV spectra on an HP 8453 spectrophotometer. NMR
spectra (δ, ppm; J, Hz) were run on a Bruker DPX 250 instrument. Ionization constants were deter-
mined spectrophotometrically. TLC of aldoximes 1 and 2 was performed on Kavalier Silufol UV 254
plates using the benzene–acetone 3 : 1 mixture and detection with UV radiation and with a 1% sol-
ution of (NH4)2FeSO4 6 H2O (intense, very stable red to violet spots). The quaternary salts were
chromatographed on Merck Cellulose F using the butanol–acetic acid–water 5 : 1 : 2 mixture, UV
detection, and the Draggendorff reagent7. HPLC was accomplished on a Spectra Physics instrument
equipped with a UV 1000 detector, SP 4000 integrator, and Tessek SGX C-18 column (150 mm long,
3.3 mm i.d.). A 70 : 30 (v/v) mixture of acetate buffer (pH 4.8) and methanol containing sodium octane-
sulfonate and tetramethylammonium chloride served as a mobile phase.
Methyl 2-[(Hydroxyimino)methyl]pyridine-4-carboxylate (1a)
Dimethyl pyridine-2,4-dicarboxylate (19.5 g, 0.1 mol) in tetrahydrofuran (600 ml), cooled under ni-
trogen to –60 °C, was reduced with 5 × 1.6 g of LiAlH4 within 20 min. The reaction temperature was
then held constant for another hour. The reaction was terminated by adding a mixture of tetrahydro-
furan (100 ml), water (40 ml), and acetic acid (60 ml), the reaction mixture was evaporated to dry-
ness in vacuum, and the residue was digested with 3 × 150 ml of ether. The extract was evaporated
and the residue was mixed with a solution of hydroxylamine hydrochloride (12 g) in water (50 ml)
neutralized with Na2CO3. The mixture was allowed to stand in a refrigerator to give 7.5 g (40%) of
the crude product, m.p. 156–170 °C. After double crystallization from 50% aqueous ethanol, the
melting point was 179–183 °C (ref.3: 184–185 °C; ref.4: 178 °C). For C8H8N2O3 (180.2) calculated:
1
53.35% C, 4.48% H, 15.55% N; found: 53.35% C, 4.54% H, 15.34% N. H NMR spectrum: 11.72 s,
1 H (OH); 8.75 dd, 1 H, J = 0.9 and 5.0 (H-6); 8.17 bs, 2 H (H-3, CH aldoxime); 7.75 dd, 1 H, J = 1.6 and
5.0 (H-5); 3.93 s, 3 H (CH3).
Ethyl 2-[(Hydroxyimino)methyl]pyridine-4-carboxylate (1b)
The compound was prepared analogously to 1a; yield 45%, melting point after double crystallization
from ethanol 160–162 °C (ref.4: 149 °C). For C9H10N2O3 (194.2) calculated: 55.67% C, 5.19% H,
14.43% N; found: 55.35% C, 5.12% H, 14.75% N. 1H NMR spectrum: 11.74 s, 1 H (OH); 8.78 dd,
1 H, J = 1.0 and 4.7 (H-6); 8.20 dd, J = 1.0 and 1.6 + 8.19 s, 2 H (H-3, CH aldoxime); 7.79 dd, 1 H,
J = 1.6 and 4.7 (H-5); 4.40 q, 2 H, J = 7.2 (CH2); 1.37 t, 3 H, J = 7.2 (CH3).
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2-[(Hydroxyimino)methyl]pyridine-4-carboxamide (2)
A suspension of ester 1a (1.12 g, 6.15 mmol) in 25% aqueous ammonia (5.6 ml) was stirred at room
temperature for 24 h. The product was filtered off (0.89 g, 93%) and crystallized from 50% aqueous
ethanol; m.p. 241–243 °C. For C7H7N3O2 (165.2) calculated: 50.91% C, 4.27% H, 25.44% N; found:
50.62% C, 4.33% H, 25.28% N. 1H NMR spectrum: 11.31 s, 1 H (OH); 8.67 dd, 1 H, J = 0.6 and
5.0 (H-6); 8.16 s, 1 H + 8.13 dd, J = 0.6 and 1.6, 1 H (H-3, CH aldoxime); 7.70 dd, 1 H, J = 1.6
and 5.0 (H-5); 7.58 bs, 2 H (NH2).
2-[(Hydroxyimino)methyl]-4-methoxycarbonyl-1-methylpyridinium Iodide (3a)
A solution of oxime 1a (0.5 g, 3.3 mmol) and methyl iodide (1.5 g, 12 mmol) in dimethylformamide
(5 ml) was heated in an ampoule at 60 °C for 12 h. Then the reaction mixture was evaporated in
vacuum, and the residue was double crystallized from ethanol. Yield 0.63 (70%) of a yellow sub-
stance, m.p. 212–214 °C (dec.). For C9H11IN2O3 (322.1) calculated: 33.56% C, 3.44% H, 8.70% N,
1
39.40% I; found: 33.83% C, 3.24% H, 8.55% N, 39.11% I. H NMR spectrum: 9.18 d, 1 H, J = 6.5
(H-6); 8.73 s, 1 H (CH aldoxime); 8.64 d, 1 H, J = 1.9 (H-3); 8.36 dd, 1 H, J = 1.9 and 6.5 (H-5);
4.48 s, 3 H (NCH3); 4.02 s, 3 H (OCH3).
2-[(Hydroxyimino)methyl]-4-ethoxycarbonyl-1-methylpyridinium Iodide (3b)
The compound was prepared analogously to 3a; yield 70%. Crystallization from ethanol gave a yellow
substance, m.p. 191.5–194 °C (dec.). For C10H13IN2O3 (336.1) calculated: 35.73% C, 3.90% H,
8.33% N, 37.75% I; found: 35.35% C, 4.19% H, 8.11% N, 37.43% I. 1H NMR spectrum: 13.18 bs,
1 H (OH); 9.21 d, 1 H, J = 6.3 (H-6); 8.76 s, 1 H (CH aldoxime); 8.65 d, 1 H, J = 2.2 (H-3); 8.41 dd,
1 H, J = 2.2 and 6.3 (H-5); 4.49 s, 3 H (NCH3); 4.48 q, 2 H, and 1.40 t, 3 H, J = 7.2 (CH2CH3).
4-Carbamoyl-2-[(hydroxyimino)methyl]-1-methylpyridinium Iodide (3c)
The compound was prepared analogously to 3a; yield 65%. Crystallization from ethanol gave a yel-
low substance, m.p. 214–219 °C (dec.). For C8H10IN3O2 (307.1) calculated: 31.29% C, 3.28% H,
13.68% N, 41.32% I; found: 30.96% C, 3.35% H, 13.52% N, 41.08% I. 1H NMR spectrum: 12.85 bs, 1 H
(OH); 9.13 d, 1 H, J = 6.5 (H-6); 8.69 s, 1 H (CH aldoxime); 8.63 d, 1 H, J = 1.9 (H-3); 8.32 dd, 1 H,
J = 1.9 and 6.5 (H-5); 8.11 bs, 2 H (NH2); 4.41 s, 3 H (CH3).
1-(4-Bromobut-2-enyl)-2-[(hydroxyimino)methyl]pyridinium Bromide (4)
A mixture of pyridine-2-carbaldoxime (12.2 g, 0.1 mol) and 1,4-dibromobut-2-ene (42.5 g, 0.2 mol)
in acetone (400 ml) was allowed to stand at room temperature for 6 days. The product separated as
a grey substance (11.5 g, 34%), m.p. 121–127 °C (dec.).
4-Carbamoyl-2′-[(hydroxyimino)methyl]-1,1′-(but-2-ene-1,4-diyl)bispyridinium Dibromide (5a)
A solution of crude monoquaternary salt 4 (11.5 g, 0.034 mol) and isonicotinamide (5 g, 0.041 mol)
in dimethylformamide (100 ml) was allowed to stand for 2 days to obtain 13.44 g of the crude pro-
duct, m.p. 190–199 °C (dec.). This was crystallized from ethanol (160 ml) and water (20 ml) to obtain
12.85 g (81%) of the hemihydrate, m.p. 117–123 °C. For C16H18Br2N4O2 0.5 H2O (467.2) calcu-
lated: 41.14% C, 4.10% H, 11.99% N, 34.21% Br; found: 40.98% C, 4.26% H, 12.13% N, 34.01% Br.
1H NMR spectrum: 13.09 s, 1 H (OH); 9.29 d, 2 H, J = 6.6 (H-2′, H-6′); 9.21 d, 1 H, J = 6.0 (H-6); 8.79 bs,
1 H + 8.31 bs, 1 H (NH2); 8.71 s, 1 H (CH aldoxime); 8.66 t, 1 H, J = 8.5 (H-4); 8.51 d, 2 H, J =
Collect. Czech. Chem. Commun. (Vol. 63) (1998)

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Bielavsky, Kassa, Elsnerova, Dejmek:
6.6 (H-3′, H-5′); 8.45 dd, 1 H, J = 1.3 and 8.5 (H-3); 8.19 m, 1 H (H-5); 6.39 dt, 1 H, J = 5.0 and
15.7 (CH butene); 6.04 dt, 1 H, J = 6.6 and 15.7 (CH butene); 5.63 d, 2 H, J = 5.0 (CH2); 5.47 d, 2 H,
J = 6.6 (CH2).
4-Carbamoyl-2′-[(hydroxyimino)methyl]-1,1′-(but-2-ene-1,4-diyl)bispyridinium Dichloride (5b)
Solution of bromide 5a (4.67 g, 0.01 mol) in water (100 ml) was passed through a column of Am-
berlite IRA-401 (50 ml) in the chloride form. The eluate was evaporated to dryness in vacuum, and
the residue was crystallized from a mixture of 40 ml of ethanol and 8 ml of water to obtain 2.5 g
(66%) of the product, m.p. 220–226 °C (dec.). For C16H18Cl2N4O2 H2O (387.3) calculated: 18.31% Cl,
4.65% H2O; found: 18.45% Cl, 4.20% H2O. The spectral properties of this compound were identical
with those of compound 5a.
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