Novel piperidinium and pyridinium agents as water-soluble acetylcholinesterase inhibitors for the reversal of neuromuscular blockade

Article abstract of DOI:10.1016/S0960-894X(02)00483-3

A series of piperidinium and pyridinium agents containing a common structural fragment of 5,6-dimethoxybenzothiophene have been synthesised as water-soluble acetylcholinesterase inhibitors. Several compounds, for example 42 (AChE IC₅₀ 0.03 μM) have been found to reverse the neuromuscular blockade induced by vecuronium bromide in vitro and in vivo. Coupled with their high water solubility (up to 30-60 mg/mL), these compounds are potentially useful as intravenous reversal agents of neuromuscular blocking agents in surgical anaesthesia.

Full text of DOI:10.1016/S0960-894X(02)00483-3

Bioorganic & Medicinal Chemistry Letters 12 (2002) 2569–2572
Novel Piperidinium and Pyridinium Agents as Water-Soluble
Acetylcholinesterase Inhibitors for the Reversal of
Neuromuscular Blockade
Ronald Palin,a,* John K. Clark,a Phill Cowley,a Alan W. Muir,b Eleanor Pow,b
Alan B. Prosser,a Robert Taylora and Ming-Qiang Zhanga,y
aDepartment of Medicinal Chemistry, Organon Laboratories Ltd., Newhouse, Lanarkshire ML1 5SH, UK
bDepartment of Pharmacology, Organon Laboratories Ltd., Newhouse, Lanarkshire ML1 5SH, UK
Received 30 April 2002; revised 14 June 2002; accepted 21 June 2002
Abstract—A series of piperidinium and pyridinium agents containing a common structural fragment of 5,6-dimethoxy-
benzothiophene have been synthesised as water-soluble acetylcholinesterase inhibitors. Several compounds, for example 42 (AChE
IC50 0.03 mM) have been found to reverse the neuromuscular blockade induced by vecuronium bromide in vitro and in vivo.
Coupled with their high water solubility (up to 30–60 mg/mL), these compounds are potentially useful as intravenous reversal
agents of neuromuscular blocking agents in surgical anaesthesia. # 2002 Elsevier Science Ltd. All rights reserved.
Acetylcholinesterase (AChE) inhibitors1 continue to
have widespread use as therapeutic agents in disease
areas2 such as Alzheimer’s disease, myasthnia gravis
and glaucoma. In addition, they are also widely used in
anaesthetic practice to reverse the skeletal muscle
relaxation induced by non-depolarising neuromuscular
blocking agents (NMBA).3 By inhibiting the hydrolysis
of acetylcholine (ACh), AChE inhibitors increase the
levels of ACh in the neuromuscular junction facilitating
cholinergic neurotransmission and hence recovery of
muscle functions.
identified a series of piperidinium containing com-
pounds, for example the quaternary salt of E2020 (1,
IC50=0.25 mM) as lead compounds.4 Subsequent opti-
misation of the dimethoxy-1-indanone moiety of 1 led
to the discovery of benzothiophene 2 which had a
marked improvement in AChE inhibition (IC50=0.008
mM) and one less asymmetric center than the lead 1.
In this paper, we report our attempt to optimise 2
aiming at maintaining AChE inhibitory potency,
increasing water solubility and removing the potential
problems associated with stereoisomers of the quaternary
salt.
The initial study began by optimising the linker unit
(W-X-Y-Z) between the benzothiophene and the piper-
idinium ring (Table 1). The synthesis of benzothiophene
analogues 2, 6, 7, and 8 is outlined in Scheme 1. Stan-
dard aldol conditions with 2-acetyl-5,6-dimethoxy-1-
benzothiophene 3 and the appropriate aldehyde affor-
ded the aldol adducts 4, which upon quaternisation with
methyl bromide afforded 6 and 7. Dehydration of the
aldol products with HCl in methanol gave the alkenes,
which under hydrogenation conditions afforded the
saturated analogues 5, which after quaternisation gave 2
and 8. Quaternisation of these piperidines with methyl
bromide afforded mixtures of isomers in favour of the
In our program to develop potent, water soluble rever-
sal agents without cardiovascular liabilities, we have
*Corresponding author. E-mail: r.palin@organon.nhe.akzonobel.nl
yPresent address: Shire BioChem Inc., 275 Armand-Frappier Blvd,
Laval, Canada H7V 4A.
1
syn methyl isomer (as determined by H NMR). The
0960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(02)00483-3

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R. Palin et al. / Bioorg. Med. Chem. Lett. 12 (2002) 2569–2572
Table 1. In vitro activity and water solubility of benzothiophene-containing piperidiniums
Compd
Quaternary isomer ratio
(Me, syn/anti)
W
X-Y-Z
AChE inhibition IC50
(mM)a
Rev. EC50
(mM)
Solubility
(mg/mL)b
2
6
7
8
95:5
97:3
20:1
95:5
5:95
85:15
95:5
90:10
95:5
—
CO
CO
C(O)CH2
C(O)CH2
CO
CH2CH2–CH–CH2
CH2C(OH)–CH–CH2
C(OH)CH2CH2–CH–CH2
CH2CH2CH2–CH–CH2
CH2C(OH)–CH–CH2
CH2C(OCH3)–CH–CH2
CH2C(OH)–CH–CH2 (+)-Isomer
CH2C(OH)–CH–CH2 (ꢀ)-Isomer
CH2–C(OH)–CH2
0.008
0.043
0.38
0.11
0.19
0.019
0.17
6.6
>10
—
2
10
2.5
1
10
1.5
10
9
10
11
12
14
17
18
21
23
25
CO
CO
CO
CO
CO
CO
—
—
0.12
0.65
0.219
0.665
0.09
0.52
0.75
19.58
2.67
10–50
No data
No data
9.43
8
1.25
25–30
20–25
2–3
1–2
3
HC=C–CH2
CH2–CH=CH
C(OH)–CH–CH2
HC=C–CH2
CH2–CH–CH2
7.67
—
>30
>30
>30
>30
85:15
—
86:14
—
aMeasured by the colorimetric method according to Ellman et al.5 in 96-well microtitre plate format using ACh as the substrate and human
recombinant AChE as the enzyme source. Values are means of three experiments.
bMeasured in water.
ratios of these quaternary salt isomers vary from com-
pound to compound and are listed in Table 1.
of the hydroxyl resulted in 10 with even further decreased
AChE inhibitory activity. The enantiomers of the aldol
product (11 and 12) were separated by chiral HPLC and
showed slight enantioselectivity in AChE inhibition in
favour of the (+)-isomer 11. However, compared with
the alkane derivative 2, the gain in water solubility in
these OH-containing compounds is counterbalanced by
the loss in their inhibitory potency against AChE.
To evaluate the difference between isomers of the qua-
ternary salts in AChE inhibition, we prepared 9 enriched
with the methyl anti isomer (Table 1). This was prepared
in a similar manner as outlined in Scheme 1 but starting
with 1-methyl-4-formyl piperidine (R=Me) as the alde-
hyde in the aldol reaction. After quaternisation with ben-
zyl bromide, a 1:1 mixture of quaternary isomers was
obtained. Extensive purification using silica gel chroma-
tography followed by crystallisation from dichloro-
methane/ethanol gave 9, which has a methyl anti/syn ratio
of 95:5. Compound 9 showed approximately 4-fold
decrease in AChE inhibition compared with 6 in which the
syn methyl quaternary isomer is dominant, indicating the
syn methyl isomer has the preferred conformation for
AChE inhibition. As far as water solubility is concerned,
the hydroxyl containing compounds 6 and 9 are more
water soluble (10 mg/mL) than the alkane 2, but >5–20-
fold less active in AChE inhibition than 2. Methylation
The linker (W-X-Y-Z) was made shorter as outlined in
Schemes 2 and 3. Treating the enolate of 3 with 1-benzyl-
4-piperidone gave the spiro alcohol intermediate 13,
which upon quaternisation with methyl bromide affor-
ded 14 (Scheme 2). Elimination of the hydroxyl unit
resulted in two regioisomers, in which the double bond was
either exo 15 or endocyclic 16. These were efficiently sepa-
rated using silica gel chromatography and quaternised
with methyl bromide to give 17 (exo) and 18 (endo).
Scheme 1. (a) Aldehyde, LDA, THF, DIPEA, ꢀ78 ꢁC; (b) HCl gas,
Scheme 2. (a) 1-Benzyl-4-piperidone, LDA, THF, DIPEA, ꢀ78 ꢁC;
MeOH, 60 ꢁC; (c) H2, 10% Pd/C, 25 ꢁC; (d) CH3Br, CH3CN, 25 ꢁC.
(b) HCl gas, MeOH, 60 ꢁC; (c) CH3Br, CH3CN, 25 ꢁC.

R. Palin et al. / Bioorg. Med. Chem. Lett. 12 (2002) 2569–2572
2571
Compound 14 showed a 2-fold decrease in AChE inhi-
bition compared to 6. Both exo and endo compounds
(17 and 18) were shown to have poor AChE inhibition
(0.52 and 0.75 mM, respectively) but surprisingly greater
water solubility.
Table 2. In vitro activity and water solubility of various N-alkylated
benzothiophene-containing piperidinium salts
Further shortening the length of the linker (Scheme 3)
by reacting the anion of 5,6-dimethoxy-1-benzothio-
phene 19 with 1-benzyl-4-piperidine acetaldehyde gave
the secondary alcohol 20, which upon quaternisation
resulted in 21. Elimination of the hydroxyl unit to the
alkene 22 and subsequent quaternisation gave 23.
Reduction of the double bond gave 24, which after
quaternisation with methyl bromide resulted in 25.
Me Syn/Anti
2/1
R
AChE inhibb IC50 Solubility
(mM)a
(mg/mL)b
26
27
28
29
30
31
32
0.053
60
54/46
1/1
0.054
0.032
0.028
0.079
0.39
60
0.6
1.5
1.5
0.9
2
Shortening the linker further caused dramatic reduction
in AChE inhibition, although lack of a carbonyl capable
of forming hydrogen bond may also be a reason.6 All
these compounds are less active than 2 and 6, suggesting
the propanone is the optimum linker.
65/35
5/1
We went on to investigate the possibility of increasing
water solubility by replacing the lipophilic benzyl at the
quaternary nitrogen with smaller and/or more polar
groups. Table 2 lists the results of some of these
modifications.
3/2
All the groups investigated gave varying mixtures of
quaternary isomers with the methyl predominately syn.
Of the groups studied the N-methoxyethyl and N-car-
boxymethyl derivatives (26 and 27) gave excellent water
solubility (60 mg/mL) whilst retaining reasonable AChE
inhibitory potency, IC50=0.054 and 0.53 mM, respec-
tively. The 2-nitrofuran 28 and tetrahydropyran 29
derivatives gave the best AChE inhibition (IC50= 0.032
and 0.02 mM, respectively), although water solubility
was less than 2 mg/mL in both cases. Phenoxyethyl
derivative 31 and cyanomethyl derivative 32 showed
decreased AChE inhibition and poor water solubility.
70/30
>1.0
aMeasured by the colorimetric method according to Ellman et al.5 in
96-well microtitre plate format using ACh as the substrate and human
recombinant AChE as the enzyme source. Values are means of three
experiments.
bMeasured in water.
We then decided to replace the piperidine moiety with a
pyridinium group so to maintain the water solubility
and avoid the difficulty in separating the quaternary
isomers of piperidiniums.
By using different small and/or polar alkylating agents,
we prepared a series of pyridinium salts (Table 3). The
key intermediate 5,6-dimethoxy-2-(3-pyridin-4-yl)pro-
pionylbenzothiophene 34 was prepared in a similar way
as described above for the piperidiniums but starting
with pyridine-4-carboxaldehyde as the aldehyde in the
aldol reaction with 3 (Scheme 4). The alcohol inter-
mediate 33 was mesylated to facilitate the elimination of
the hydroxyl unit. Subsequent reduction gave the key
Scheme 3. (a) 1-Benzyl-4-piperidine acetoaldehyde, LDA, THF,
DIPEA, ꢀ78 ꢁC; (b) HCl gas, MeOH, 60 ꢁC; (c) H2, 10% Pd/C, 25 ꢁC;
(d) CH3Br, CH3CN, 25 ꢁC.
Scheme 4. (a) Pyridine-4-carboxaldehyde, LDA, THF, DIPEA,
ꢀ78 ꢁC; (b) MsCl, Et3N, CH2Cl2; (c) H2, 10% Pd/C, MeOH-THF
25 ꢁC; (d) RCH2Br, CH3CN, 60 ꢁC.

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R. Palin et al. / Bioorg. Med. Chem. Lett. 12 (2002) 2569–2572
Table 3. In vitro activity and water solubility of various N-alkylated
benzothiophene-containing pyridinium salts
Introduction of a choline motif, for example, 42 and 43,
improved the AChE inhibition (IC50= 0.03 mM and
0.007 mM, respectively) and dramatically increased the
water solubility (33 and 50 mg/mL, respectively). The in
vitro reversal potency of 42 (only measured for this
compound) was also improved in comparison with the
alkyl analogues (36–41).
R1
AChE inhib.
IC50 (mM)a
Rev EC50
(mM)
Solubility
(mg/mL)b
N-Benzyl pyridinium 44 (the equivalent of 2 in the
piperidinium series) showed high potency in AChE
inhibition (IC50=0.0046 mM) and excellent in vitro
reversal activity against vecuronium-induced block in
isolated guinea pig hemi-diaphragm (EC50=0.099 mM).
However, its water solubility is low at 2 mg/mL. Para-
substitution on the benzene ring with a fluorine 45
enhanced the AChE inhibition but reduced the in vitro
reversal activity probably due to low water solubility.
Para-carboxyl substitution 46 improved slightly the
water solubility but reduced both AChE inhibitory and
in vitro reversal activities. Heteroaromatics such as the
thiophene 47 and 2-nitrofuran 48 gave excellent AChE
inhibition (IC50=0.006 and 0.007 mM, respectively),
but their in vitro reversal potency proved disappointing.
36
37
Me
0.9
2.5
3
0.28
>3.0
25
38
39
40
41
42
43
44
45
46
47
48
0.54
2.57
>3.0
No data
>3.0
1.72
25
16
15
6
0.11
0.04
In conclusion, we have identified a series of novel piper-
idiniums and pyridiniums that are potent inhibitors of
AChE and as such reverse vecuronium-induced neuro-
muscular block in vitro and in vivo. Several of these
compounds have good levels of water solubility (up to
30–50 mg/mL) and are compatible with intravenous
formulation. The pyridiniums, specially those with a
choline motif at the pyridine nitrogen, for example, 42
and 43, have the additional advantage of being achiral in
comparison with the piperidiniums. They are therefore
devoid of potential difficulties of isomeric purification
and are feasible for large scale production.
0.03
0.29
33
50
2
0.007
0.0046
0.0026
1.0
No data
0.099
1.65
1–2
3.5
2
1.67
Acknowledgements
0.006
0.0045
2.7
We would like to thank K. Anderson, F. Hope and R.
Mason for biological testing of the compounds and our
colleagues in the Department of Analytical Chemistry
for NMR, MS, HPLC measurements.
2.67
35
aMeasured by the colorimetric method according to Ellman et al.5 in
96-well microtitre plate format using ACh as the substrate and human
recombinant AChE as the enzyme source. Values are means of three
experiments.
References and Notes
bMeasured in water.
1. Brufani, M.; Filocam, L.; Lappa, S.; Maggi, A. Drugs
Future 1997, 397.
2. Millard, C. B.; Broomfield, C. A. J. Neurochem. 1995, 64, 1909.
3. Bevan, D. R.; Donati, F.; Kopman, A. F. Anaesthesiology
1992, 77, 785.
4. Clark, J. K.; Cowley, P.; Muir, A. W.; Palin, R.; Pau, E.;
Prosser, A. B.; Taylor, R.; Zhang, M.-Q. Bioorg. Med. Chem.
Lett. 2002, 12, 2565.
5. Ellman, G. L.; Courtney, K. D.; Andres, V., Jr.; Feath-
erstone, R. M. Biochem. Pharmacol. 1961, 7, 88.
6. Kaur, J.; Zhang, M.-Q. Current Med. Chem. 2000, 7, 273.
intermediate 34. Quaternisation was conducted in
acetonitrile at 60 ꢁC to afford the pyridinium salt 35.
As shown in Table 3, simple alkyl groups such as methyl
36, ethyl 37 and allyl 38 have decreased AChE inhibition
and in particular the in vitro reversal activity against
vecuronium-induced block in guinea pig hemi-diaphragm.