Quaternary salts of E2020 analogues as acetylcholinesterase inhibitors for the reversal of neuromuscular block

Article abstract of DOI:10.1016/S0960-894X(02)00482-1

A series benzylpiperidinium and benzylpyridinium quaternary salts have been synthesised and tested for inhibition of acetylcholinesterase and reversal of neuromuscular block induced by vecuronium. Several potent reversal agents have been identified and their haemodynamic effects measured.

Full text of DOI:10.1016/S0960-894X(02)00482-1

Bioorganic & Medicinal Chemistry Letters 12 (2002) 2565–2568
Quaternary Salts of E2020 Analogues as Acetylcholinesterase
Inhibitors for the Reversal of Neuromuscular Block
John K. Clark,a,* Phill Cowley,a Alan W. Muir,b Ronald Palin,a 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 benzylpiperidinium and benzylpyridinium quaternary salts have been synthesised and tested for inhibition
of acetylcholinesterase and reversal of neuromuscular block induced by vecuronium. Several potent reversal agents have been
identified and their haemodynamic effects measured. # 2002 Elsevier Science Ltd. All rights reserved.
Despite the continued widespread use of neostigmine as
a reversal agent for neuromuscular block in surgical
anaesthesia, there exists a need for an equally effective
reversal agent devoid of cardiovascular (CV) side effects
associated with neostigmine.1 As a part of our pro-
gramme2 to develop water soluble reversal agents with
reduced cardiovascular side effects, we were interested
in quaternary amines as acetylcholinesterase (AChE)
inhibitors. The advantages of such a permanently
charged quaternary amino compound are that it is more
water soluble and less likely to penetrate to the CNS
than the corresponding tertiary amine. We started our
lead finding by comparing a diverse range of known
AChE inhibitors3 with their N-methyl quaternised ana-
logues. As shown in Table 1, all N-methyl quaternary
derivatives are more potent than their corresponding
tertiary amine counterparts at reversing vecuronium-
induced neuromuscular block in anaesthetised cats4 (N-
methyl huperazine A was not tested due to the lack of
materials). Ease of synthesis and greater scope for opti-
mization led to the the N-methyl derivative of E2020
(aricept, donepezil)5 being chosen for further investiga-
tions.6 In this paper, we report the lead optimization
that has resulted in several potent and water-soluble
reversal agents.
En-route to E2020, we were able to isolate the inter-
mediate aldol 1 which proved to be a potent reversal
agent against vecuronium-induced neuromuscular block
in anaesthetised cats (ED50 0.21 mmol/kg, maximum
reversal 109% at 1.28 mmol/kg) with the following CV
side effects (maximum changes in MAP ꢀ22%, HR
ꢀ12%, Vagus +18%). However, when tested in anaes-
thetized monkeys, 1 gave only partial reversal against
vecuronium.
Due to its cleaner CV profile, especially in vagal poten-
tiation, than the lead N-methyl E2020 (Table 1), we
further investigated the structure–activity relationship
of the hydroxyl group in compound 1. Separation of the
diastereoisomers 2 and 3 demonstrated that the anti iso-
mer 3 was slightly more potent than the syn isomer 2
(Table 2) in reversing vecuronium-induced block in-vitro
in guinea pig (GP) hemi-diaphragm. Since the indanone
chiral center is prone to racemization, no attempt was
made to resolve further enantiomers. In addition to the
two chiral centers in the indanone methanol part of the
molecule, the piperidinium part also gives rise to two
isomeric forms of the quaternary amine, in which
N-benzyl favours the anti isomer (equatorial benzyl
group) by ꢁ9:1.
*Corresponding author. Tel.: +44-1698-736130; fax: +44-1698-
736187; e-mail: jk.clark@organon.co.uk
yPresent address: Shire BioChem Inc., 275 Armand-Frappier Blvd,
Laval, Canada H7V 4A.
0960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(02)00482-1

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J. K. Clark et al. / Bioorg. Med. Chem. Lett. 12 (2002) 2565–2568
Table 1. Activities of some known AChE inhibitors and the corresponding N-methyl quaternary salts
AChE inhiba
(IC50, mM)
In-vitro GPb
(EC50, mM)
In-vivo catc
(ED50, mmol/kg)
Max rev
(%)
MAPd
(Á%)
HRd
(Á%)
Vagusd
(Á%)
E2020
E2020 Quat
Gallanthamine
Gallanthamine Quat
Anseculin
Anseculin Quat
Huperazine
Huperazine Quat
CP 118,954
0.018
0.025
ꢁ0.5
ꢁ0.5
0.085
<0.3
0.03
>100
<0.5
<0.5
<0.5
<0.5
0.043
0.1
1.1
0.63
1.45
0.53
ꢁ0.06
n.t.
0.1
9.7
0.14
0.08
n.t.
0.12
0.023
3.7
106
131
n.t.
130
61
105
156
n.t.
140
176
68
ꢀ12
ꢀ22
n.t.
+16
ꢀ12
ꢀ20
ꢀ17
n.t.
ꢀ12
+11
n.t.
ꢀ5
+14
+37
n.t.
+80
ꢀ14
+25
+102
n.t.
ꢀ9
0.136
0.019
>10
ꢀ4
ꢀ8
n.t.
ꢀ6
0.0047
0.039
1.14
ꢀ7
+10
+15
+8
CP 118,954 Quat
TAK 147
TAK 147 Quat
ꢀ11
ꢀ11
ꢀ6
ꢀ4
ꢀ5
0.331
0.15
140
+4
+10
Quaternary salts were prepared by reaction with methyl bromide or iodide (huperazine was di-methylated, then quaternised). n.t., not tested.
aMethod according to Ellman et al.7 using human recombinant AChE as enzyme source.
bGP diaphragm.
cChloaralose anaesthetised cat.4
d% change in cardiovascular parameters at ED50; MAP, mean arterial pressure; HR, heart rate; Vagus, change in response to vagal stimulation.
Table 2. Stereoselectivity and effects of reducing number of
asymmetric centers in dimethoxyindanone series
Structure
GP reversal (EC50, mM)
1
2
3
4
5
6
7
8
1.44
2.7
0.89
43.5
1.88
0.012
0.1
Scheme 1. Synthesis of dimethoxyindanone benzylpyridinium salts:
(a) 4-pyridinecarboxaldehyde, LDA ꢀ78 ꢂC; (b) HCl gas; (c) H2, Pd;
(d) benzyl bromide.
Dehydration of the aldol to the corresponding en-one
derivative 6 resulted in a marked increase in in-vitro
reversal potency. Hydrogenation of the exocyclic double
bond 7, however, reduced the in-vitro activity compared
with 6. The results of these structural manipulations
seem to suggest that the indanone methanol moiety
functions as a ‘linker’ to connect the two recognition
fragments of dimethoxybenzene and piperidinium.
0.019
Literature data have already demonstrated the impor-
tance of the indanone carbonyl to the AChE inhibitory
activities of E2020 analogues,8 probably by forming a
hydrogen bond within the enzyme active site. Since the
indanone methanol moiety plays only the linker role to
connect the dimethoxybenzene and piperidinium moi-
eties, we decided to replace the indanone carbonyl by an
exocyclic ketone, thus eliminating this chiral center.
The problem of quaternary amine isomers can be tack-
led by using identical alkyl groups or by replacing the
piperidinium with pyridinium. When both piperidinium
N-substituents are benzyl, the resulting compound 4 is a
much weaker reversal agent, but when the piperidinium
is replaced by pyridinium, the resulting compound 5
(Scheme 1) retains the reversal potency (Table 2).
A series of piperidiniums were synthesized from readily
available methylarylketones (Scheme 2) by Aldol con-
densation with 1-benzylpiperidine-4-carboxaldehyde,

J. K. Clark et al. / Bioorg. Med. Chem. Lett. 12 (2002) 2565–2568
2567
Our investigations were then extended to the synthesis
of a variety of readily accessible aromatic components
having an exocyclic ketone group in combination with
the piperidinium salts linked by the three carbon chain
unit (Table 3) (9–16) (Scheme 2) This series of com-
pounds confirmed the general finding that those com-
pounds with the saturated linker retained the best
activity profiles. Water solubility was dependent on the
lipophilicity of the aromatic component. Surprisingly,
the di-oxygenated analogues 9,10 were less water solu-
ble than the equivalent dihydrobenzofuran 11. The
minor syn quaternary isomer 12 was isolated by chro-
matography and found to be much more water soluble
(>10.0 mg/mL vs 1.5), but with a 4-fold loss in AChE
inhibition. As expected, the naphthalene derivative 14 was
poorly water soluble, while the N-methyl indole 15 and the
1-phenylpyrazole 16 analogues showed modest water
solubility while retaining good AChE inhibition (0.022
mM), in the case of 16.
Scheme 2. Synthesis of benzylpiperidinium quaternary salts: (a) 1-
benzyl-4-formylpiperidine, nBuLi ꢀ78 ꢂC; (b) HCl gas; (c) H2, Pd;
(d) CH3I or CH3Br.
In conclusion, we have prepared a diverse series of
benzylpiperidinium quaternary salts with nano molar
inhibition of AChE and potent reversal of vecuronium-
induced neuromuscular block (in-vitro GP, in-vivo
anaesthetised GP, and in-vivo anaesthetised cat).
followed by dehydration, hydrogenation and quaternisa-
tion. From these piperidiniums with an exocyclic ketone
group, the dimethoxybenzothiophene 8 was identified as a
potent AChE inhibitor (IC50 0.008 mM).
Table 3. Benzylpiperidinium quaternary salts with alternative aromatic substituents
Structure
AChE inhib (IC50, mM)
In-vitro GP (EC50, mM)
H2O solubility (mg/mL)
2.0
8
0.008
0.019
9
0.24
1.7
>3.0
0.09
n.t.
0.5
<0.5
1.5
10
11
12
13
14
15
16
0.1
0.017
0.066
0.139
0.05
>10.0
5.0
n.t.
0.53
1.6
<1.0
1.2
0.12
0.022
0.43
2.0

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J. K. Clark et al. / Bioorg. Med. Chem. Lett. 12 (2002) 2565–2568
The dimethoxybenzthiophene derivative 8 remained the
most promising lead and was subsequently studied in
more detail, as reported in the following paper.9
was adjusted to provide stable 85–90% block of the tibialis
anterior twitch height. After 10–15 min stable block, the infu-
sion was switched off and the twitches allowed to recover. Two
h after full recovery, stable block was again induced as above.
Immediately after switching off the vecuronium infusion, the
test substance was administered and the effects on recovery of
NM function, heart rate, arterial pressure and autonomic
vagal responses were recorded.
Acknowledgements
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, HPLC and MS data and interpretation.
5. (a) Sugimoto, H.; Iimuro, Y.; Yamanishi, Y.; Yamatsu, K.
Bioorg. Med. Chem. Lett. 1992, 2, 871. (b) Kawakami, Y.;
Inoue, A.; Kawai, T.; Wakita, M.; Sugimoto, H.; Hopfinger,
A. J. Bioorg. Med. Chem. 1996, 4, 1429. (c) Inoue, A.; Kawai,
T.; Wakita, M.; Iimura, Y.; Sugimoto, H.; Kawakami, Y. J.
Med. Chem. 1996, 39, 4460. (d) Sugimoto, H.; Yamanishi, Y.;
Iimura, Y.; Kawakami, Y. Current Med. Chem. 2000, 7, 303.
6. During the preparation of this manuscript, benzylpyr-
idinium salts of E2020 (inhibition of acetylcholinesterase in rat
brain homogenate) were disclosed in an Eisai patent, WO-
00178728.
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