Oxyaniliniums as acetylcholinesterase inhibitors for the reversal of neuromuscular block

Article abstract of DOI:10.1016/S0960-894X(01)00703-X

A series of oxyanilinium-based AChE inhibitors have been synthesised and tested for the reversal of vecuronium-induced neuromuscular block. Several compounds, for example 2-hydroxy- and 2-methoxy-N,N-dimethyl-N-allylanilinium bromide (3 and 6) showed comparable reversal potencies to edrophonium and clean in vivo cardiovascular profiles.

Full text of DOI:10.1016/S0960-894X(01)00703-X

Bioorganic & Medicinal Chemistry Letters 12 (2002) 193–196
Oxyaniliniums as Acetylcholinesterase Inhibitors for the
Reversal of Neuromuscular Block
Simon J. A. Grove,a Jasmit Kaur,a Alan W. Muir,b Eleanor Pow,b
Gary J. Tarvera and Ming-Qiang Zhanga,*
aDepartment of Medicinal Chemistry, Organon Laboratories Ltd., Newhouse, Lanarkshire ML1 5SH, Scotland, UK
bDepartment of Pharmacology, Organon Laboratories Ltd., Newhouse, Lanarkshire ML1 5SH, Scotland, UK
Received 20 July 2001; revised 12 October 2001; accepted 19 October 2001
Abstract—A series of oxyanilinium-based AChE inhibitors have been synthesised and tested for the reversal of vecuronium-induced
neuromuscular block. Several compounds, for example 2-hydroxy- and 2-methoxy-N,N-dimethyl-N-allylanilinium bromide (3 and
6) showed comparable reversal potencies to edrophonium and clean in vivo cardiovascular profiles. # 2002 Elsevier Science Ltd.
All rights reserved.
As well as being useful therapeutic agents for Alzhei-
mer’s disease, myasthenia gravis, glaucoma and so on,
acetylcholinesterase (AChE) inhibitors are important
drugs widely used in anaesthetic practice to reverse the
skeletal muscle relaxation induced by nondepolarising
neuromuscular blocking agents (NMBA).1 By inhibiting
the breakdown of acetylcholine (ACh), AChE inhibitors
such as neostigmine (1) and edrophonium (2) (Fig. 1)
increase the levels of ACh in the synaptic clefts so to
facilitate cholinergic neurotransmission in the neuro-
muscular junction and hence the recovery of muscle
function.
effects that are not related to AChE inhibition, for
example, direct activation of cardiac muscarinic ACh
receptors, are at least in part responsible for the more
severe bradycardiac side effects of neostigmine (1).2
Edrophonium (2), an AChE inhibitor with no direct
effects on ACh receptors, has been found to cause much
less CV side effects. Edrophonium is, however, less
potent than neostigmine as a NMB reversal agent and
therefore is less widely used.
Within our program of finding a cardiovascularly clea-
ner NMB reversal agent, we were interested in water-
soluble, fast-onset AChE inhibitors with potency similar
to or better than neostigmine but CV side effects at least
not more than those of the combination of neostigmine
and glycopyrrolate.
However, the use of AChE-based NMB reversal agents
is often associated with bradycardiac side effects due to
non-selective activation of muscarinic receptors in the
cardiac parasympathetic pathway by the elevated ACh
levels. In clinical practice, NMB reversal agents have to
be used in combination with muscarinic receptor
antagonists such as atropine and glycopyrrolate to
minimise cardiovascular (CV) side effects. Ironically,
muscarinic receptor antagonists themselves can cause
CV side effects such as tachycardia.
In screening a commercial library of 240 quaternary
amines (Microsource), we found a lead 3 (Table 1) that
showed an IC50 of 1.85 mM against human recombinant
AChE in vitro and an ED50 of 0.50 mmol/kg in reversing
vecuronium-induced neuromuscular block in cats in
vivo. Although compound 3 is less potent than neo-
stigmine in reversing vecuronium-induced block, it has a
Although many believe CV side effects of NMB reversal
agents are inevitable because of their mechanism of
action, there is compelling evidence suggesting that
*Corresponding author. Fax: +44-1698-736187; e-mail: m.zhang@
organon.nhe.akzonobel.nl
Figure 1. Structures of neostigmine (1) and edrophonium (2).
0960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(01)00703-X

194
S. J. A. Grove et al. / Bioorg. Med. Chem. Lett. 12 (2002) 193–196
Table 1. In vitro AChE inhibitory activities of oxyphenyl- and oxybenzyl-ammonium bromides
Compd
Structure
R0
AChE inhibition
IC50 (mM)a
In vitro reversal
GPHD, EC50 (mM)b
R
Y
n
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
H
H
H
CH3
CH3
CH3
Ac
Ac
H
H
H
CH3
CH3
H
CH2CH¼CH2
CH2CH¼CH2
CH2CH¼CH2
CH2CH¼CH2
CH2CH¼CH2
CH2CH¼CH2
CH2CH¼CH2
CH2CH¼CH2
CH2C(CH3)¼CH2
CH2C(CH3)¼CH2
CH2C(CH3)¼CH2
CH2C(CH3)¼CH2
CH2C(CH3)¼CH2
CH2Ph
2
3
4
2
3
4
3
4
2
3
4
2
3
2
3
4
2
3
4
2
3
4
2
3
3
3
3
3
3
3
3
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
1
1
1
1
1
0
0
1.85
ꢀ3
74
13
>10
1–3
>3
2.89
>3
>30
3.87
>3
>3
>3
>3
>3
>3
>3
>3
>3
>3
>3
>3
>3
>3
>3
>10
>3
>3
n.t.
n.t.
>3
n.t.
2.77
0.028
>10
>10
1–3
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
ꢀ10
>10
>10
>10
>10
>10
>10
3.96
0.002
H
H
CH2Ph
CH2Ph
CH2Ph
CH2Ph
CH2Ph
CH2Ph
CH2Ph
CH2Ph
CH3
CH3
CH3
Ac
Ac
Ac
H
H
CH3
H
CH3
H
CH2CH2CH¼CH2
CH2CH¼CH2
CH2CH¼CH2
CH2Ph
29
30
CH2Ph
CH3
CH3
CH2CH3
CH3
31
Edrophonium
Neostigmine
CH3
H
(CH3)2NCO
aMeasured by the colorimetric method according to Ellman et al.4 in 96-well microtitre plate formate using ACh as the substrate and human
recombinant AChE as the enzyme source. Values are means of three experiments, standard deviation is given in parentheses.
bn.t., not tested.
much cleaner CV profile than neostigmine with max-
imum changes of <10% in all hemodynamic para-
meters. In this paper, we would like to report our
attempt to optimise this lead aiming at increasing the
reversal potency whilst maintaining its clean CV profile.
lead 3 but showed higher in vitro potency in reversing
vecuronium-induced block in guinea pig hemidiaphram.
From the X-ray crystal structure of Torpedo AChE–
edrophonium complex,5 it was known that edropho-
nium only interacts with the bottom part of the 20-A
long active site gorge of AChE, that is the quaternary N
interacting with the indole of Trp-84 and its m-hydroxy
showing bifurcated H-bonding to two members of the
catalytic triad, Ser-200 and His-440. The upper half of
the active site gorge is not occupied by edrophonium.
Since the structures of the current series are very similar
Compounds in Table 1 were synthesised by reductive
methylation of a corresponding aniline or benzylamine,
followed by quaternisation with an alkyl bromide
(Scheme 1). Most of starting anilines and benzylamines
are commercially available. 2-Methoxy-N,N-dimethyla-
niline, the intermediate for the preparation of com-
pounds 6, 14 and 19, was prepared by direct amination
of veratrole with lithium dimethylamide in THF.3 The
yield (not optimised) was about 22%. 3-Hydroxy-N,N-
dimethylbenzylamine, the starting material for com-
pounds 26, 28 and 30, was prepared by reductive ami-
nation of 3-hydroxybenzaldehyde with dimethylamine
and NaBH(OAc)3 (yield: ꢀ96%).
As shown in Table 1, the SAR of this series is very tight
and does not allow much structural manipulation. The
only interesting compound of this series is 6 which
exhibited similar AChE inhibitory potency to that of the
Scheme 1. (a) HCHO, NaBH4, CH3OH, rt, yields: 72–96%; (b) AcCl,
Hunigs base, DCM, rt, yields: >90%; (c) R0Br, CH3CN (for the phe-
nols) or refluxing acetone or neat (for the rest), yields: 11–98%, all
quats were isolated by simple filtration.

S. J. A. Grove et al. / Bioorg. Med. Chem. Lett. 12 (2002) 193–196
195
to that of edrophonium, it is not unreasonable to
believe that they, too, bind to the bottom part of the
enzyme active site. We reckoned that the AChE inhibi-
tory activity could be increased if we could increase the
size of the inhibitor so to have full occupancy of the
active site.
As shown in Table 2, most benzyl quaternised com-
pounds are more active than methyl quaternised anal-
ogues, indicating the importance of molecular size. One
compound 34 showed >5.5-fold increased AChE inhi-
bitory potency as compared with the lead 3 and it was
also slightly more potent than edrophonium in reversing
vecuronium-induced block in guinea pig hemi-
diaphgram in vitro.
Donepezil (32) is one of the most potent AChE inhibi-
tors in clinical use (IC50 ꢀ6 nM). Several computer
modelling studies have indicated that the molecule binds
to the active site gorge in its extended conformation
with full occupancy of the narrow active site gorge.6 The
protonated N of donepezil, like that of edrophonium,
forms a cation–p interaction with Trp-84 at the bottom
of the gorge and the indanone interacts with aromatic
residues near the entrance of the gorge, for example
Trp-279.6 The two methoxyls of donepezil are exposed
to water. This mode of interaction was later supported
by the X-ray crystal structure of donepezil–AChE com-
plex although Phe-330 seemed more important than
Trp-84 in forming the interaction with the protonated
N.7
When tested in anaesthetised cats,8 6 showed slightly
improved reversal potency (ED50 0.22 mmol/kg) as
compared to the lead 3 (ED50 0.50 mmol/kg). More
importantly, both 3 and 6, at their maximum reversal
doses (2.10 and 1.28 mmol/kg, respectively), caused
much less changes in haemodynamic parameters than
either the combination of neostigmine plus glycopyrro-
late or edrophonium plus atropine (Table 3). Com-
pound 34, on the other hand caused more pronounced
CV effects than the standard combinations at 2.56 mmol/
kg, the dose that gave maximum reversal.
In conclusion, we have identified two quaternary anili-
niums 3 and 6 that reverse vecuronium-induced neuro-
muscular block in vitro and in vivo via the inhibition of
AChE. In the in vivo cat model, both compounds
showed cleaner CV profiles than the standard combina-
tions of AChE inhibitors plus mAChR antagonists,
which suggest they may potentially be useful NMB
reversal agents.
We therefore further modified this series of oxyanili-
niums by substituting the benzene ring with a group
that is similar in length to the dimethoxy-indanone-2-
methyl of donepezil, that is 3,4-dimethoxy-phenyl-
ethoxy or -phenylpropyl (33–41, Table 2). As illustrated
in Schemes 2 and 3, these compounds were synthesised
in two different ways. The ether derivatives 33–37
were prepared by alkylation of dimethylaminophe-
nols with a-bromo-3,4-dimethoxy-acetophenone in
acetone, followed by quaternisation (Scheme 2) and
their methylene analogues 38–41 were synthesised by
Aldol condensation between 3,4-dimethoxyaceto-
phenone and an appropriately substituted benzalde-
hyde, followed by hydrogenation and quaternisation
(Scheme 3).
Scheme 2. (a) a-Bromo-3,4-dimethoxyacetophenone, K2CO3, acetone,
reflux, 4 h, yields: 26–40%; (b) R0Br, CH3CN, rt, overnight, yields: 40–
88%.
Table 2. In vitro AChE inhibitory activities of extended anilinium
bromides
Compd
Structure
R0
AChE inhib In vitro reversal
(IC50, mM)
(EC50, mM)
X
Y
33
34
35
36
37
38
39
40
41
O
O
O
O
O
CH2
CH2
CH2
CH2
CH3
CH2Ph
CH3
3
3
4
4
4
3
3
4
4
ꢀ10
0.33
7.08
1.69
9.8
19.4
0.5
n.t.
>3
2
ꢀ10
CH2Ph
CH2CH¼CH2
CH3
ꢀ10
ꢀ1.0
>10
1.04ꢁ0.01
Scheme 3. (a) 3,4-Dimethoxyacetophenone, aq NaOH, EtOH, rt,
overnight, yields: 60–68%; (b) H2, PtO2, EtOH/THF, yields: 78–90%;
(c) HCHO, NaBH(OAc)3, THF, yield: 90%; (d) R0Br, CH3CN, rt,
overnight, yields: 11–37%.
CH2Ph
CH3
CH2Ph
3–10
1.3ꢁ0.04
4.11

196
S. J. A. Grove et al. / Bioorg. Med. Chem. Lett. 12 (2002) 193–196
Table 3. Haemodynamic effects in anaesthetised cats of the test compounds at doses producing full reversal of vecuronium-induced neuromuscular
block
Compd
Dose (mmol/kg)
Haemodynamic changes (%)
MAP
HR
LVP
LV dp/dt
Vagus
Nic Memb
3 (4)
6 (1)
34 (1)
Neo/Gly (2)
Edro/atr (3)
2.1ꢁ0.7
1.28
2.56
0.076/0.011
1.026/0.089
2ꢁ7
ꢂ24
ꢂ1ꢁ1
ꢂ2
12ꢁ5
ꢂ9
8ꢁ3
ꢂ9
5ꢁ11
41
5
ꢂ5ꢁ2
ꢂ2
56
8
63
82
34
ꢂ6ꢁ2
ꢂ7ꢁ1
ꢂ22ꢁ1
22ꢁ7/ꢂ12ꢁ6
4ꢁ5
2ꢁ2
ꢂ14ꢁ1
23ꢁ6/ꢂ6ꢁ5
ꢂ12ꢁ1
20ꢁ6/ꢂ5ꢁ4
62ꢁ2
ꢂ67ꢁ15
Number of experiments in parentheses beside compound. Compounds 3, 6 and 34 were administered by cumulative bolus dosing to produce com-
plete reversal of neuromuscular block, dose increments being administered immediately following the maximum effects from the preceding dose.
Neo/Gly and Edr/atr were administered as a single bolus dose sufficient to produce full reversal of neuromuscular block.
Acknowledgements
5. Harel, M.; Schalk, I.; Ehret-Sabatier, L.; Bouet, F.; Goeld-
ner, M.; Hirth, C.; Axelsen, P. H.; Silman, I.; Sussman, J. L.
Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 9031.
6. Kaur, J.; Zhang, M.-Q. Curr. Med. Chem. 2000, 7, 273.
7. Kryge, G.; Silnab, I.; Sussman, J. L. J. Physiology (Paris)
1998, 92, 191.
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. Dr. P. Cowley
was involved in the purchase of the Microsource qua-
ternary amine library.
8. Reversal experiments were carried out in female cats (1.9–
3.0 kg) anaesthetised with
a
mixture of a-chloralose
80 mg kgꢂ1 and sodium pentobarbitone 5 mg kgꢂ1 ip. Neuro-
muscular block was induced using a bolus followed by an
infusion of vecuronium. The infusion rate was adjusted to
provide a stable 85–95% depression of tibialis anterior mus-
cle twitch height. After 10–15 min stable block, the infusion
was switched-off and the twitches allowed to recover. Two
hours after full recovery, stable block was induced again
as above. Immediately after switching-off the infusion, test
drug was administered and the effects on neuromuscular
(twitch), autonomic vagal and nictitating membrane, arterial
pressure, heart rate, LV pressure and LV dp/dt responses
were recorded.
References and Notes
1. Mirakhur, R. K. Acta. Anaesthesiol. Scand. Suppl. 1995,
106, 62.
2. Endou, M.; Tanito, Y.; Okumura, F. J. Pharmacol. Exp.
Ther. 1997, 282, 1480, and references cited therein.
3. Ten Hoeve, W.; Kruse, C. G.; Luteyn, J. M.; Thiecke,
J. R. G.; Wynberg, H. J. Org. Chem. 1993, 58, 5101.
4. Ellman, G. L.; Courtney, K. D.; Andres, V., Jr.; Feather-
stone, R. M. Biochem. Pharmacol. 1961, 7, 88.