Quaternary salts of 4,3′ and 4,4′ bis-pyridinium monooximes. Part 2: Synthesis and biological activity

Article abstract of DOI:10.1016/j.bmcl.2006.01.065

In continuation of our investigations of unsymmetrical bisquaternary monooximes, we synthesized four new series of compounds bridged by hexyl, heptyl, octyl and nonyl groups. All eight monooximes viz., dibromides of 1-(4-hydroxyiminomethylpyridinium)6-(3/4-carbamoylpyridinium)hexane, 1-(4-hydroxyiminomethylpyridinium)-7-(3/4-carbamoylpyridinium)heptane, 1-(4-hydroxyiminomethylpyridinium)-8-(3/4-carbamoylpyridinium)octane, 1-(4-hydroxyiminomethylpyridinium)-9-(3/4-carbamoylpyridinium)nonane as well as the corresponding bis-oximes were synthesized and characterized by spectral data. Their ability to reactivate tetraethylpyrophosphate (TEPP) inhibited mouse total brain cholinesterase was investigated and compared with the conventional oxime 2-pyridinealdoxime chloride (2-PAM). Mouse brain homogenate was used as the source of acetylcholinesterase. Among all the compounds, tested the compound with the hexylene bridge (6b) and a 3-carbamoyl group on the second pyridine ring was found to be the most active acetylcholinesterase reactivator (72%) which is greater than that of 2-PAM (56%). However, the activity was reversed; as the chain length increased from a heptylene to a nonylene bridge, they potentiated the inhibitory effect of TEPP rather than reactivation. It is interesting to note that compound 6b with a carbamoyl group at the 3rd position of the pyridine ring showed dose dependent reactivation whereas the corresponding compound 6a with the carbamoyl group present at the 4th position of the pyridine ring showed reactivation at lower concentration (30 μM) and potentiation of TEPP inhibition at higher concentrations (100 and 300 μM).

Full text of DOI:10.1016/j.bmcl.2006.01.065

Bioorganic & Medicinal Chemistry Letters 16 (2006) 2134–2138
0
0
Quaternary salts of 4,3 and 4,4 bis-pyridinium monooximes.
Part 2: Synthesis and biological activity
a
a,b,
a
*
Chennamaneni Srinivas Rao, Vobalaboina Venkateswarlu
and Garlapati Achaiah
a
University College of Pharmaceutical Sciences, Kakatiya University, Warangal 506 009, Andhra Pradesh, India
Research & Development, Dr. Reddy’s Laboratories Ltd. Bachupalli, Qutubullapur, R.R. District, 500 072, Andhra Pradesh, India
b
Received 24 October 2005; revised 4 January 2006; accepted 16 January 2006
Available online 14 February 2006
Abstract—In continuation of our investigations of unsymmetrical bisquaternary monooximes, we synthesized four new series of
compounds bridged by hexyl, heptyl, octyl and nonyl groups. All eight monooximes viz., dibromides of 1-(4-hydroxy-
iminomethylpyridinium)6-(3/4-carbamoylpyridinium)hexane, 1-(4-hydroxyiminomethylpyridinium)-7-(3/4-carbamoylpyridinium)-
heptane, 1-(4-hydroxyiminomethylpyridinium)-8-(3/4-carbamoylpyridinium)octane, 1-(4-hydroxyiminomethylpyridinium)-9-(3/4-
carbamoylpyridinium)nonane as well as the corresponding bis-oximes were synthesized and characterized by spectral data. Their
ability to reactivate tetraethylpyrophosphate (TEPP) inhibited mouse total brain cholinesterase was investigated and compared with
the conventional oxime 2-pyridinealdoxime chloride (2-PAM). Mouse brain homogenate was used as the source of acetylcholines-
terase. Among all the compounds, tested the compound with the hexylene bridge (6b) and a 3-carbamoyl group on the second pyr-
idine ring was found to be the most active acetylcholinesterase reactivator (72%) which is greater than that of 2-PAM (56%).
However, the activity was reversed; as the chain length increased from a heptylene to a nonylene bridge, they potentiated the inhib-
itory effect of TEPP rather than reactivation. It is interesting to note that compound 6b with a carbamoyl group at the 3rd position
of the pyridine ring showed dose dependent reactivation whereas the corresponding compound 6a with the carbamoyl group present
at the 4th position of the pyridine ring showed reactivation at lower concentration (30 lM) and potentiation of TEPP inhibition at
higher concentrations (100 and 300 lM).
ꢀ
2006 Elsevier Ltd. All rights reserved.
Deliberate self-poisoning with organophosphorus (OP)
pesticides has become an increasingly common response
to emotional distress in young adults, and it is now one
of the most frequent reasons for emergency hospital
pounds. Current medical protection against the toxicity
of OP compounds consists of a regimen of anticholiner-
gic drugs, such as atropine, that antagonizes the effects
of accumulated acetylcholine and an AChE ‘reactivator’
that restores enzyme activity.
1
admission in India. On average, most patients will re-
quire 5–14 days of intensive care monitoring. Mortality
2
,3
has been reported as between 15% and 36%. Mecha-
nism of reactivation of TEPP inhibited cholinesterase
Pralidoxime is the oxime used worldwide, for the treat-
ment of OP pesticide poisoning and available in three
common forms: pralidoxime chloride (2-PAM, used
world-wide), iodide (used in rural India) and mesylate
4
,5
by the oximes is presented in Scheme 1. Furthermore,
phosphorylated AChE can undergo a fairly rapid pro-
cess of ‘aging’ (loss of one alkyl or alkoxy group) so that
within hours of pesticide poisoning it becomes complete-
ly resistant to the reactivators. Although the highly toxic
nature of OP compounds has been known for many
years, there still exist serious limitations in the antidotal
therapy available against poisoning by these com-
3
,6
(P2S, used in the UK). There is considerable pub-
lished data on the role and choice of available oximes
(like 2-PAM, obidoxime, HI-6 and HL o¨ -7) for the treat-
7
–9
ment of OP pesticide poisoning.
Earlier we reported
the synthesis and biological activity of bis-pyridinium
monooximes and found that compounds with a propyl
or butyl bridge were potent reactivators of TEPP inhib-
1
0
ited AChE in comparison to 2-PAM. Propane and
butane bridged bis-pyridinium monooximes were also
tested on nerve agents (also known as chemical warfare
agents such as sarin, tabun and soman, in mode of
action they are related to OP pesticides but are highly
Keywords: Organophosphate; Pesticide; Tetraethylpyrophosphate;
Acetylcholinesterase; Pralidoxime; Bis-pyridinium monooximes.
*
Corresponding author. Tel.: +91 9391383983; e-mail addresses:
chennamanenir@yahoo.com; vobala@yahoo.com; achaiah_g@
yahoo.com
1
1,12
toxic to mammals) and found more effective.
0
960-894X/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.01.065

CH. Srinivas Rao et al. / Bioorg. Med. Chem. Lett. 16 (2006) 2134–2138
2135
obtained from the Sigma-Aldrich chemicals private
limited, Hyderabad, India. KH PO , NaOH and NaCl
2
4
were purchased from Merck chemicals private limited,
Hyderabad, India. 2-PAM was provided as a gift sam-
ple by the Troika Parenterals private limited, Ahme-
dabad, India.
Solvents were dried or distilled before use. Melting
points were obtained on a Mel-temp apparatus (Shi-
tal Scientifics), Mumbai, India in open capillary tubes
and are uncorrected. Infrared spectra (IR) were
recorded with KBr pellet on a Perkin-Elmer BX Ser-
Scheme 1. Reactivation of acetylcholinesterase inhibited by
tetraethylpyrophosphate.
1
ies, infrared spectrophotometer. H NMR spectra
were recorded on Avance-300 MHz spectrometer in
1
6
DMSO-d6.
The general procedure for the synthesis of compounds
were as follows: (Scheme 2). Selected physicochemical
data of compounds with hexylene, heptylene, octylene
and nonylene bridges are given in Table 1.
Procedure A
Step I: Preparation of oxime intermediate compounds. 4-
Pyridinealdoxime and 1,6-dibromohexane, 1,7-dibromo-
heptane, 1,8-dibromooctane, or 1,9-dibromononane in
1:1.2 molar ratio were added to chloroform and heated
at reflux with stirring for 120 h and then cooled to room
temperature. The product was collected by filtration,
washed with chloroform and crystallized from
acetonitrile.
F
Scheme 2. Synthesis of alkylene-linked bis-pyridinium monooximes.
Step II: Preparation of bis-pyridinium monooximes.
Intermediate compound 6, 7, 8, or 9 and isonicotina-
mide or nicotinamide in 1:1.2 molar ratio were added
to dimethylformamide (DMF). The reaction mixture
was heated at 68–70 ꢁC for 24 h and then cooled to
room temperature. The product was collected by filtra-
tion and crystallized with absolute ethanol.
0
Pang et al. also reported that 1,7-heptylene-bis-N,N -
syn-2-pyridiniumaldoxime is 100 times more potent than
2
-PAM in reactivating hAChE poisoned by isofluro-
1
3
phate. Hence, we continued our investigations, and
synthesized bis-pyridinium monooximes with hexylene
to nonylene bridges and evaluated their effectiveness
for the reactivation of TEPP inhibited enzyme.
The title compounds were also prepared by an alterna-
tive method as follows:
Anaesthetized animals were sacrificed by decapitation
and exsanguinated; the mice brains were removed
and used as a source of AChE after homogenization.
Tetraethylpyrophosphate (TEPP), 5,5 -dithio-bis(2-
nitrobenzoic acid) (DTNB), acetylthiocholine iodide
Procedure B
0
Step I: Preparation of amide intermediate compounds.
Isonicotinamide or nicotinamide and 1,6-dibromohex-
ane, 1,7-dibromoheptane, 1,8-dibromooctane or 1,9-
dibromononane in 1:1.1 molar ratio were added to ace-
tonitrile and heated at 68–70 ꢁC for 20 h. The product
(
Asch), eserine, 4-pyridinealdoxime, 1,6-dibromohex-
ane, 1,7-dibromoheptane, 1,8-dibromooctane, 1,9-dib-
romononane, isonicotinamide and nicotinamide were
Table 1. Physical data of compounds
a
Compound
Conditions of the reaction
Molecular formula
Mp (ꢁC)
Yield (%)
6a
6b
7a
7b
8a
8b
9a
9b
DMF, 68–70 ꢁC, 24 h
DMF, 68–70 ꢁC, 24 h
DMF, 68–70 ꢁC, 24 h
DMF, 68–70 ꢁC, 24 h
DMF, 68–70 ꢁC, 24 h
DMF, 68–70 ꢁC, 24 h
DMF, 68–70 ꢁC, 24 h
DMF, 68–70 ꢁC, 24 h
C
C
C
C
C
C
C
C
18
H
18
H
19
H
19
H
20
H
20
H
21
H
21
H
24
N
24
N
26
N
26
N
28
N
28
N
30
N
30
N
4
4
4
4
4
4
4
4
Br
2
Br
2
Br
2
Br
2
Br
2
Br
2
Br
2
Br
2
O
2
O
2
O
2
O
2
O
2
O
2
O
2
O
2
248–250
246–248
242–244
246–248
252–254
248–250
238–240
242–244
80
74
56
58
67
64
68
66
a
Melting points are uncorrected.

2136
CH. Srinivas Rao et al. / Bioorg. Med. Chem. Lett. 16 (2006) 2134–2138
was collected by filtration, washed with acetonitrile and
crystallized using chloroform.
hexylene bridge with a carbamoyl group at the 3rd posi-
tion, showed 72 ± 3.51 % reactivation compared to
5
6 ± 2.84 % exhibited by 2-PAM. Compound 6a with
Step II: Preparation of bis-pyridinium monooximes. The
isonicotinamide or nicotinamide intermediates (10a,
a heptylene bridge and the carbamoyl group present at
the 4th position showed inhibition of the enzyme instead
of reactivation. The corresponding bis-oxime 6c showed
23 ± 2.46 % reactivation (Fig. 2). Compounds 7a, 7b
and 7c exhibited significant reactivation of the enzyme
(antidotal property) at low concentration (30 lM) but,
at higher concentrations (100 and 300 lM), potentiated
inhibition of the enzyme by TEPP. However, the bis-ox-
ime 7c reactivated AChE even at 100 lM concentration
(14 ± 2.36%). Compounds 8a, 8b, 8c, 9a, 9b and 9c
showed only inhibition of the enzyme at all concentra-
tions tested (Fig. 3). Compounds 7a, 7b and 7c have
shown 15 ± 1.4%, 23 ± 2.6% and 39 ± 2.8% reactivation,
respectively, at 30 lM concentration (Fig. 2). Com-
pound 6b (hexylene bridge) has excellent reactivation
(20 ± 1.95%) at increased (10·) concentration of TEPP
(1 lg), whereas 2-PAM had only 2 ± 0.6% reactivation.
All other compounds did not show reactivation at this
concentration of TEPP.
1
0b, 11a, 11b, 12a, 12b, 13a, or 13b) and 4-pyr-
idinealdoxime in 1:1.2 molar ratio were added to DMF.
The reaction mixture was heated at 68–70 ꢁC for 24 h.
The product was collected by filtration and washed with
acetonitrile. All compoundswerepurified by recrystallisa-
tion with DMF, and then dried under vacuum.
Compounds 6c, 7c, 8c and 9c were synthesized accord-
4
ing to the methods of Wilson and Ginsburg and Pang
1
3
et al. Compounds 6c, 7c, 8c and 9c are known in the
1
literature.
3
Brain AChE activity was determined by the colorimetric
1
4
procedure described by Voss and Sachsse. Physical
data, TLC, IR and H NMR spectra confirmed the
1
structures and purity of the synthesized compounds.
All bis-pyridinium monooxime products decomposed
(
>180 ꢁC) before melting. All the synthesized
compounds were evaluated for their in vitro reactivation
capabilities of TEPP inhibited AChE enzyme by color-
imetry. Compound 6b, the most potent which, has an
All the conventional oximes reported to date differ
from each other by the number of pyridinium rings
present (mono-pyridnium vs bis-pyridinium oximes),
the position of the oxime group on the pyridinium
ring, and, in the case of bis-pyridinium oximes, by
the chemical structure of the bridge between the pyri-
dinium rings (Fig. 1). The bis-pyridinium monooximes
differ from bis-pyridinium oximes both in the length
of the side chain and the position of the substituent
on the second pyridine ring. The bis-pyridinium
monooxime 6b showed higher reactivation (72%) than
the conventional oxime 2-PAM (52%). It is interesting
to note that changing the carbamoyl group from the
3
rd to the 4th position (6a) resulted not only in loss
of reactivating capacity but also a gain in inhibitory
effect. However, the corresponding bis-oxime showed
less potency than 6b and 2-PAM (Fig. 2). Further in-
crease in the chain length from a 6-carbon bridge
(
hexylene) to 7-carbon bridge (heptylene) resulted in
a slight increase in activity at lower concentration
30 lM) but showed inhibition of the enzyme at high-
er concentrations (100 and 300 lM). However, the
Figure 1. Structures of the standard oximes used for the treatment of
OP pesticide poisoning as well as our newly synthesized bis-pyridinium
monooximes.
(
8
6
4
2
0
0
0
0
2
-PAM
6a
6
6
7
7
7
b
c
a
b
c
0
0
50
100
150
200
250
300
350
-
-
-
-
20
40
60
80
Concentration (μM)
Figure 2. Reactivation of mouse brain AChE inhibited with TEPP by 2-PAM and test compounds.

CH. Srinivas Rao et al. / Bioorg. Med. Chem. Lett. 16 (2006) 2134–2138
2137
10
0
0
50
100
150
200
250
300
-
-
-
-
-
-
-
-
10
20
30
40
50
60
70
80
8
a
b
8
8c
9a
9b
9c
Concentration (μM)
Figure 3. Reactivation of mouse brain AChE inhibited with TEPP by test compounds.
compounds with 8 (octylene), or 9-carbon (nonylene)
bridges showed only inhibition of the enzyme instead
of reactivation at all concentrations (Fig. 3). From
our studies, it was observed that the optimal alkylene
side chain length for reactivation of OP inhibited
AChE in bis-pyridinium monooximes is from propyl
to hexyl bridges with a carbamoyl group present at
the 3rd position of the second pyridine ring.
5. Thiermann, H.; Szinicz, L.; Eyer, F.; Worek, F.; Eyer, P.;
Felgenhauer, N.; Zilker, T. Toxicol. Lett. 1999, 107,
2
33.
6
7
. Bismuth, C.; Inns, R. H.; Marrs, T. C. In Clinical and
Experimental Toxicology of Organophosphates and Carba-
mates; Ballantyne, B., Marrs, T. C., Eds.; Butterworth
Heinemann: Oxford, 1992; p 555.
. Worek, F.; Backer, M.; Thiermann, H.; Szinicz, L.; Mast,
U.; Klimmek, R.; Eyer, P. Hum. Exp. Toxicol. 1997, 16,
4
66.
0
Four new series of asymmetrically substituted 4,3 and
0
8. Worek, F.; Kirchner, T.; Backer, M.; Szinicz, L. Arch.
Toxicol. 1996, 70, 497.
4
,4 bis-pyridinium monooximes bridged by hexylene,
9
. Worek, F.; Diepold, C.; Eyer, P. Arch. Toxicol. 1999, 73, 7.
heptylene, octylene and nonylene groups were prepared.
Evaluation of these compounds as antidotes for anti-
AChE intoxication in the mouse brain model revealed
that their effectiveness depends significantly on the
length of the side chain. The bis-pyridinium monooxi-
mes reactivate the inhibited enzyme faster than the
mono-pyridinium and bis-pyridinium oximes. From
our data, it can be concluded that propyl to hexyl bridge
compounds with a carbamoyl group at the 3rd position
of the second pyridine ring were potent reactivators of
AChE inhibited with TEPP. Further studies are required
to establish the mechanism by which a change in the po-
sition of carbamoyl group results in drastic changes in
AChE reactivation.
1
1
1
1
0. Srinivas Rao, C. H.; Venkateswarlu, V.; Achaiah, G.
Bioorg. Med. Chem. Lett. 2005, 15, 3076.
1. Kuca, K.; Bielavsky, J.; Cabal, J.; Bielavska, M. Tetrahe-
dron Lett. 2003, 44, 3123.
2. Kuca, K.; Bielavsky, J.; Cabal, J.; Kassa, J. Bioorg. Med.
Chem. Lett. 2003, 13, 3545.
3. Pang, Y.-P.; Kollmeyar, T. M.; Hong, F.; Lee, J.-c.;
Hammond, P. I.; Haugabouk, S. P.; Brimijoin, S. Chem.
Biol. 2003, 10, 491.
14. Voss, G.; Sachsse, K. Toxicol. Appl. Pharmacol. 1970, 16,
64, The mouse brain was removed, rinsed with cold saline,
7
blotted dry, and weighed. It was homogenized under cold
conditions in a sufficient amount of 0.9% NaCl to give a
final solution of 100 mg brain tissue/mL of saline. The
homogenate was then centrifuged at 3000 rpm for 10 min.
The reactivating efficacy of oximes was evaluated by adding
TEPP (100 ng) to the homogenate for 10 min followed by
tested oximes for 10 min. The solution was incubated at
Acknowledgments
3
0 ꢁC for 10 min after the addition of substrate (i.e.,
The first author thanks CSIR, New Delhi, for providing
financial assistance in the form of a SRF and gives his
heart felt gratitude to retired Professor Krishna Murthy
for his valuable suggestions and to Michael Eddleston
for his review. Thanks are due to Troika Parenterals
Pvt. Ltd. for providing 2-PAM as gift sample.
acetylthiocholine iodide). All assays were carried out in
triplicate. Percentage reactivation was calculated with the
following equation:
is the control enzyme activity at 0 min, E
1
5
ErꢀEi
ꢁ 100. In the above equation, E
0
E ꢀE
0
i
i
is the inhibited
r
enzyme activity, and E is the activity of inhibited enzyme
after incubation with the oxime test compounds.
5. Sikder, A. K.; Ghosh, A. K.; Jaiswal, D. K. J. Pharm. Sci.
1
1
1
6. The new compounds gave satisfactory H NMR Spectra:
993, 82, 258.
1
References and notes
Compound 6a: 1.31m, 4H (CH –CH –CH – CH –CH –
2
2
2
2
2
CH
2 2 2 2 2 2 2
); 1.92 m, 4H (CH –CH –CH – CH –CH –CH ); 4.62–
1
. Gunnell, D.; Eddleston, M. Int. J. Epidemiol. 2003, 32,
02.
4.67 m, 4H (CH –CH –CH – CH –CH –CH ); 8.42–8.44
2
m, 4H (Py); 8.67–8.80 m, 3H (CONH , CH@NOH); 9.26–
9.27 m, 4H (Py).
Compound 6b: 1.33 m, 4H (CH –CH –CH – CH –CH –
2
2
2
2
2
9
2
2
. Lee, P.; Tai, D. Y. Intensive Care Med. 2001, 27, 694.
. Srinivas Rao, C. H.; Venkateswarlu, V.; Surender, T.;
Eddleston, M.; Buckley, N. A. Trop. Med. Int. Health
3
2
2
2
2
2
CH ); 1.91 m, 4H (CH –CH –CH – CH –CH –CH ); 4.59–
2
2
2
2
2
2
2
2
005, 1, 581.
. Wilson, I. B.; Ginsburg, S. Biochim. Biophys. Acta 1955,
8, 168.
2 2 2 2 2 2
4.62 m, 4H (CH –CH –CH – CH –CH –CH ); 8.39–8.44
4
m, 4H (Py); 8.58–8.72 m, 3H (CONH , CH@NOH); 9.23–
2
1
9.35 m, 4H (Py).

2138
CH. Srinivas Rao et al. / Bioorg. Med. Chem. Lett. 16 (2006) 2134–2138
Compound 7b: 1.29 m, 6H (CH
CH –CH ); 1.88–1.90 m, 4H (CH
2
–CH
2
–CH
–CH
2
–CH
2
–CH
2
2
–
–
Compound 8a: 1.26 m, 8H (CH
CH –CH –CH ); 1.90 m, 4H (CH
2
–CH
–CH
2
–CH
–CH
2
– CH
– CH
2
2
–CH
–CH
2
–
–
2
2
2
–CH
2
2
–CH
2
–CH
2
2
2
2
2
2
2
CH –CH ); 4.54–4.59 m, 4H (CH –CH –CH –CH –CH –
2
CH – CH –CH ); 4.59–4.64 m, 4H (CH –CH –CH – CH –
2
2
2
2
2
2
2
2
2
2
2
2
2
CH
(
2
–CH
2
); 8.18–8.23 m, 4H (Py); 8.42–8.67 m, 3H
CH
(CONH
2
–CH
2 2 2
– CH –CH ); 8.41–8.43 m, 4H (Py); 8.67 m, 3H
CONH
2
, CH@NOH); 9.00–9.08 m, 4H (Py).
2
, CH@NOH); 9.22–9.24 m, 4H (Py).