Preparation, in vitro screening and molecular modelling of symmetrical bis-quinolinium cholinesterase inhibitors - Implications for early Myasthenia gravis treatment

Article abstract of DOI:10.1016/j.bmcl.2011.02.047

This paper describes the preparation and in vitro evaluation of 18 newly prepared bis-quinolinium inhibitors on human recombinant acetylcholinesterase (AChE) and human plasmatic butyrylcholinesterase (BChE). Their inhibitory (IC₅₀) and was compared to the chosen standards ambenonium dichloride, edrophonium chloride, BW284c51 and ethopropazine hydrochloride. One novel compound was found to be a promising inhibitor of hAChE (in nM range) and was better than edrophonium chloride or BW284c51, but was worse than ambenonium chloride. This compound also showed selectivity towards hAChE and it was confirmed as a non-competitive inhibitor of hAChE by kinetic analysis. A molecular modelling study further confirmed its binding to the peripheral active site of hAChE via apparent π-π or π-cationic interactions.

Full text of DOI:10.1016/j.bmcl.2011.02.047

Bioorganic & Medicinal Chemistry Letters 21 (2011) 2505–2509
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Preparation, in vitro screening and molecular modelling of symmetrical
bis-quinolinium cholinesterase inhibitors—implications for early
Myasthenia gravis treatment
Marketa Komloova a, Kamil Musilek b,c,d, , Anna Horova b, Ondrej Holas a, Vlastimil Dohnal c,
⇑
Frank Gunn-Moore e, Kamil Kuca c,d,f
a Charles University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry and Drug Control, Heyrovskeho 1203, 500 05 Hradec Kralove, Czech Republic
b University of Defence, Faculty of Military Health Sciences, Department of Toxicology, Trebesska 1575, 500 01 Hradec Kralove, Czech Republic
c University of Jan Evangelista Purkynje, Faculty of Science, Department of Chemistry, Ceske mladeze 8, 400 96 Usti nad Labem, Czech Republic
d University Hospital, Sokolska 581, 500 05 Hradec Kralove, Czech Republic
e School of Biology, University of St. Andrews, Medical and Biological Sciences Building, St. Andrews, KY16 9TF Fife, UK
f University of Defence, Faculty of Military Health Sciences, Centre of Advanced Studies, Trebesska 1575, 500 01 Hradec Kralove, Czech Republic
a r t i c l e i n f o
a b s t r a c t
Article history:
This paper describes the preparation and in vitro evaluation of 18 newly prepared bis-quinolinium inhib-
itors on human recombinant acetylcholinesterase (AChE) and human plasmatic butyrylcholinesterase
(BChE). Their inhibitory (IC50) and was compared to the chosen standards ambenonium dichloride, edro-
phonium chloride, BW284c51 and ethopropazine hydrochloride. One novel compound was found to be a
promising inhibitor of hAChE (in nM range) and was better than edrophonium chloride or BW284c51, but
was worse than ambenonium chloride. This compound also showed selectivity towards hAChE and it was
confirmed as a non-competitive inhibitor of hAChE by kinetic analysis. A molecular modelling study fur-
Received 24 January 2011
Revised 10 February 2011
Accepted 12 February 2011
Available online 21 February 2011
Keywords:
Cholinesterase
Inhibitor
Myasthenia gravis
Quaternary
ther confirmed its binding to the peripheral active site of hAChE via apparent
interactions.
p–p or p–cationic
Ó 2011 Elsevier Ltd. All rights reserved.
In vitro
Molecular modelling
Acetylcholinesterase inhibitors are used in the treatment of dis-
orders with impaired cholinergic transmission. The inhibition of
acetylcholinesterase (EC 3.1.1.7; AChE) is a key treatment strategy
for Alzheimer’s disease, the most common form of dementia in the
elderly population.1 In contrast, peripherally acting AChE inhibi-
tors are used in various conditions, such as glaucoma, constipation,
spasmolysis and also to antagonise muscle relaxation in anaesthe-
siology.2,3 In addition, a significant use of peripheral AChE inhibi-
tors is to mitigate the symptoms of early Myasthenia gravis
(MG), an autoimmune disorder resulting from the destruction of
the post-synaptic membrane in the neuromuscular junction.4
In most MG cases, human antibodies are produced to the nico-
tinic acetylcholine receptor (nAChR), but other neuromuscular
junction proteins (e.g., muscle-specific tyrosine kinase, MuSK)
can be also targetted.5,6 these antibodies initiate the autoimmune
attack to the endplate region of neuromuscular junction resulting
in reduced density of nAChR, destruction of the synaptic folds
and the general simplification of the post-synaptic membrane.
The decreased probability of the acetylcholine (ACh)–nAChR inter-
action causing a reduced transmission in the neuromuscular junc-
tion results in a characteristic symptom of MG, that is, weakness of
the striated muscles.7 The AChE inhibitors work by increasing the
concentration of ACh in the synaptic junction and thus enhance
the cholinergic transmission in spite of the nAChR depletion.8
Though the application of AChE inhibitors is only a symptomatic
approach and it does not resolve the original cause of the disease,
it plays a significant role in early/mild MG treatment. The periph-
eral AChE inhibitors are usually the initial drugs for MG treatment
and have been successfully used in the sole treatment of mildly
presenting MG. However, progressive MG forms require additional
immunosuppressive treatment.9
Compounds that are currently commercially available for MG
treatment belong to carbamate drugs, for example, neostigmine
bromide (A; ProstigminÒ, VagostigminÒ, Fig. 1) and the most fre-
quently used drug—pyridostigmine bromide (B; MestinonÒ,
Fig. 1), which is slightly better tolerated than neostigmine.10
Edrophonium chloride (1; TensilonÒ, Fig. 1), is used as a diagnostic
tool for MG. It has a rapid onset and short pharmacological action,
thus it cannot be used for treatment purposes.11 The use of a
⇑
Corresponding author. Tel.: +420 973 255 167; fax: +420 495 518 094.
E-mail address: kamil.musilek@gmail.com (K. Musilek).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.02.047

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M. Komloova et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2505–2509
Table 1
N
Prepared bis-quinolinium salts bearing different linkers
Br
O
Br
O
N
O
O
N
N
(A)
2 X
N
X-(A)-X
X= Cl, Br
N
N
DMF; 70°C
pyridostigmine bromide (B)
neostigmine bromide (A)
5–14 (Br)a
(CH2)3–12
CH2OCH2
(CH2)2O(CH2)2
(E)–CH2CH@CHCH2
(Z)–CH2CH@CHCH2
1,2-Phenylenyl
1,3-Phenylenyl
1,4-Phenylenyl
3,6-Naphtalenyl
15 (Cl)
16 (Br)
17 (Br)
18 (Cl)
19 (Br)
20 (Br)
21 (Br)
22 (Br)
N
Cl
O
H
Cl
N
N
N
H
N
Cl
O
OH
2 Cl
edrophonium chloride (1)
ambenonium dichloride (2)
a
All compounds are described in detail in the Supplementary data.
H
N
O
N
matic part of the inhibitor and the enzyme aromatic residues for
this type of compounds, the bis-quinolinium compounds seem to
bridge the gap between bis-pyridinium and bis-isoquinolinium
series.21 The bisquaternary structure of prepared compounds was
selected to promote a peripheral inhibitory effect. Since there is a
possibility that monoquaternary compounds could penetrate the
BBB, the addition of another charge is predicted to benefit minimal
BBB penetration. In addition, though some compounds from bis-
quinolinium series had been previously prepared, they had not
been evaluated for their cholinesterase inhibition activity.22–24
The novel inhibitors were prepared by quaternisation of quino-
line (4.2 mmol) with a corresponding alkylating agent (1.9 mmol)
in MeCN (30 ml). The solution was stirred at 70 °C for 10–20 h
and then cooled to room temperature. The crude product was col-
lected by filtration, washed with acetone and re-crystallized from
MeCN. NMR, ESI-MS and elemental analysis were used to deter-
mine the structure and purity of all compounds.
The inhibitory ability of the newly prepared compounds was
determined in vitro on human recombinant AChE (hAChE) and hu-
man plasmatic BChE (hBChE) using a modified Ellman’s proce-
dure.25 The IC50 values were compared to those obtained for
edrophonium chloride (1), ambenonium dichloride (2) and selec-
tive AChE/BChE inhibitors BW284c51 (3, Fig. 1) or ethopropazine
hydrochloride (4, Fig. 1). Edrophonium (1) and ambenonium (2)
were chosen as standards that are currently used for MG diagnosis
and treatment, and which are non-carbamate related drugs.
BW284c51 (3) and ethopropazine (4) were determined as selective
AChE/BChE inhibitors to cover selectivity issues.26,27 The selectivity
index was calculated as a ratio between the IC50 of BChE/AChE. The
kinetic experiments on recombinant hAChE were done for chosen
inhibitors of interest. The results are summarised in Table 2.
The in vitro inhibitory ability of the MG standards was con-
Cl
2 Br
N
N
S
BW284c51 (3)
ethopropazine (4)
Figure 1. AChE inhibitors used for MG treatment (A and B, 1 and 2) and selective
cholinesterase inhibitors (3 and 4).
non-carbamate bisquaternary drug with prolonged effect, such as
ambenonium dichloride (MytelaseÒ, Fig. 1) is another treatment
option. However, the application of MG drugs can be followed by
serious side-effects caused by the increased muscarinic activity
(gastrointestinal discomfort, increased salivation, lacrimation and
bronchial secretion).12,13 These side-effects are dose-dependent
and can be treated with anticholinergic drugs (e.g., diphenoxylate
hydrochloride, atropine or loperamide hydrochloride).14 An exces-
sive dosing of MG commercial drugs may also lead to the choliner-
gic crisis, resulting in even greater muscle weakness, diarrhoea,
bradycardia, excessive oropharyngeal and bronchial secretion.15
In order to avoid central nervous system AChE-related side-ef-
fects, it is recommended to treat MG with compounds that do
not penetrate across the blood–brain barrier (BBB). All MG com-
mercial used compounds contain a positive charge in their struc-
ture which should restrict their action to the periphery. However,
some recent studies have suggested that pyridostigmine may affect
centrally mediated AChE due to stress-induced BBB penetration.16
Additionally, compounds designed for MG treatment should be
selective for AChE leaving other non-specific cholinesterases
(butyrylcholinesterase; BChE, EC 3.1.1.8) unaffected. This AChE
selectivity will provide a notably decrease in the appearance of
side-effects and potential dosing difficulties.17
Previously, the preparation and evaluation of the inhibitory ef-
fect of bis-pyridinium and bis-isoquinolinium compounds were
investigated.18,19 Since the AChE esteratic site (Ser203) is located
on the bottom of the narrow gorge and a peripheral anionic/aro-
matic site (PAS) lies in its entrance, the length of the connecting
linker as well as a spatial orientation of the inhibitor molecule
were considered as being the most important factors for the inter-
action between inhibitor and enzyme.20 The bis-quinolinium com-
pounds (Table 1) described in this paper were prepared and
investigated to support these formerly reported findings. Hence,
various aliphatic or aromatic linkers between two quinolinium
moieties were used to study their molecular interaction with cho-
linesterases. A quinolinium moiety was chosen to follow the influ-
ence of the heteroaromatic part of the molecule towards inhibition
of the cholinesterases, because previously bis-isoquinolinium com-
pounds showed a very promising inhibitory ability.19 Assuming
that the enzyme–inhibitor interactions depend largely on the for-
firmed. While 1 (IC50 = 5 lM) was found to be a weak AChE inhib-
itor with high selectivity for AChE, compound 2 (0.7 nM) was the
best inhibitor among all the tested compounds with five orders
of magnitude selectivity for AChE. Moreover, the kinetic experi-
ments for hAChE showed that compound 1 influenced the sub-
strate (acetylthiocholine) decomposition rate, whereas inhibitor 2
was found to be a non-competitive AChE inhibitor. The selective
inhibitors (3, 4) also confirmed older data with a strong inhibition
of cholinesterases (30 nm for hAChE by 3, 1.6 lM for hBChE by 4)
and a high selectivity for tested enzymes. Compound 3 was found
to be a non-competitive hAChE inhibitor, while the selective hBChE
inhibitor 4 influenced the hAChE substrate decomposition rate.
The novel compounds showed various inhibitory capabilities of
the tested enzymes. The lowest inhibitory ability towards hAChE
was observed in compounds bearing an oxygen in the alkylenyl lin-
ker (13, 14) with an IC50 value in the mM or <mM range. Com-
pounds with xylenyl linkers (19–21) did not demonstrate a
mation of
p–p or p–cationic interactions between the heteroaro-

M. Komloova et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2505–2509
2507
M)
Table 2
Screening of standard and prepared hAChE/hBChE inhibitors
d
Inhibitor
AChE IC50 SDa
(
lM)
BChE IC50 SDa
1370 223
(lM)
SIc BChE/AChE
Ki1/Ki2
(l
Edrophonium (1)
Ambenonium (2)
BW-284c51 (3)
Ethopropazine (4)
5
6
7
8
5
1
274
10000
11800
0.002
1.6
0.8
8.5
0.2
0.8/4.8
0.0007 0.0001
0.03 0.006
1020 199
7
1
0.005/0.006
354 58
1.6 0.3
0.01/0.05
25/12100
9
38
4
2
8
0.8
5
13
32
34
5
2
5
6
—
—
—
—
25
0.8
9
0.09 0.02
0.08 0.01
0.08 0.02
0.001 0.0002
0.01 0.002
0.02 0.004
418 82
0.4 0.06
0.7 0.1
0.6 0.1
0.1 0.02
0.5 0.07
0.4 0.07
871 141
4.4
8.8
7.5
100
50
20
2.1
1.4
51
0.4
0.2
0.5
—
—
—
—
10
11
12
13
14
15
16
17
18
19
20
21
22
0.04/0.06
—
—
—
—
—
—
—
35
6
7
1
48
8
308
5
2
35
40
0.4
7
8
0.8 0.1
6
9
20
—
3
—
—
2.3/8.2
b
b
—
0.8 0.2
0.2 0.03
0.3
a
b
c
Mean value of three independent determinations standard deviation (SD).
No inhibition in selected concentration scale.
Selectivity index.
d
Ki1-dissociation constant for enzyme-inhibitor complex; Ki2-dissociation constant for enzyme–inhibitor–substrate complex.
significant inhibitory ability. Though compound 19 was not soluble
in the screening medium, no notable difference in hAChE inhibition
compared to the structurally similar compounds 17 and 18 are pre-
dicted. Compounds with but-2-enyl linkers or with C3–C6 methy-
lenyl units showed only moderate hAChE inhibition (up to 2 lM)
which is similar to the standard compound 1 and above those
oxyanion hole (Fig. 2). First quinolinium moiety was attached by
–cationic interactions to aromatic residues of Tyr124 (3.8 Å),
p
Trp286 (3.7 Å), Phe297 (3.2 Å), Phe338 (3.4 Å) and Tyr341 (3.4 Å),
whereas the second quinolinium moiety displayed only distant
binding to Trp286 (4.6 Å). Similarly, the top-scoring docking pose
of 22 (À9.44 kcal/mol) showed binding to the PAS and was not af-
fected by the catalytic triad (Fig. 2). First quinolinium moiety was
stacked among the aromatic residues of Tyr72 (4.0 Å), Tyr124
(2.9 Å), Trp286 (3.2 Å), Phe297 (3.6 Å), Phe338 (3.5 Å) and Tyr341
achieved by the other standards (2, 3).
The most interesting inhibitory ability was observed for novel
compounds with C7–C12 methylenyl units (9–14) and a napht-
halenyl linker (22). Two compounds (13 and 14; 10–20 nM)
showed comparable hAChE inhibition compared to standard 3,
whereas compound 12 (1 nM) resulted as a hAChE inhibitor one
order of magnitude better than the standard 3. Three compounds
(12–14) also presented a higher selectivity for hAChE, although
their selectivity index (SI) was lower than standards 2, 3. Two
structurally different novel inhibitors (alkylenyl and naphtalenyl)
were further used for kinetic experiments. Both compound 12
bearing a C10 methylenyl linkage and compound 22 with naph-
talenyl linkage, were found to be non-competitive hAChE inhibi-
tors. The in vitro results for these newly prepared compounds
highlighted compound 12 as a promising non-competitive and
selective hAChE inhibitor, and therefore warranted further
investigation.
(3.1 Å). The naphtalenyl linker displayed some weak
p–p interac-
tions with Trp286 (4.2 Å) and Tyr341 (4.0 Å), but the other quino-
linium moiety was found to be at a large distance from PAS Trp286
(6.2 Å). Based on this molecular modelling and concerning the
in vitro data, compound 12 (1 nM) probably resulted as two orders
of magnitude more potent hAChE inhibitor to compound 22
(0.8 lM) because of its spatial and conformational flexibility which
Molecular docking studies were performed on two promising
compounds after the above in vitro screening (12, 22) in order to
rationalise possible interactions within AChE and BChE.28 Specifi-
cally, compound 12 was chosen among the novel inhibitors bearing
polymethylenyl linkers and a sub-lM inhibitory ability (9–14). For
other compounds of similar origin (9–14), a similar binding to cho-
linesterases was hypothesised and docking studies were not per-
formed. In contrast, compound 22 bearing a naphtalenyl linkage
but still with sub-lM inhibitory ability, was chosen for docking
studies to determine additional interactions within the enzymes’
active sites and important SAR features. Three AChE structures
(1b41, 2gyv, 2jez) and one BChE (1p0i) structure were used.29–36
Regarding AChE, the best results were obtained for the hAChE
structure (1b41). The top-scored docking pose of 12 (À8.27 kcal/
mol) displayed binding in the PAS region without penetrating the
Figure 2. Molecular docking results for hAChE interactions with compound 12
(blue) and 22 (magenta).

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M. Komloova et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2505–2509
would help in its accommodation in the hAChE peripheral active
site, when compared to a structurally rigid molecule 22 with a
naphtalenyl linkage.
The molecular modelling results for hBChE are depicted in Fig-
ure 3. Similar to the AChE results, the top-scored docking pose of
compound 12 (À9.22 kcal/mol) displayed important interactions
with aromatic residues. Namely, both quinolinium moieties were
In summary, 18 symmetrical bis-quinolinium compounds were
prepared and their inhibitory abilities were tested in vitro on hAChE
and hBChE. Three compounds showed inhibitory results better or
comparable to the standards of edrophonium chloride and
BW284c51 and in addition one compound (12) also presented selec-
tivityforhAChE. Noneofthepreparedcompoundswereabletoexceed
the ambenonium chloride in hAChE inhibition. The kinetic experi-
ments confirmed non-competitive inhibition of hAChE by two chosen
bis-quinolinium compounds. The binding of novel compounds to the
activesiteSer203wasfurthersuggestedbymolecularmodellingstud-
ies. TheSARfindingswerealsocomparabletopreviouslypreparedbis-
pyridinium and bis-isoquinolinium series of compounds, but high-
lighted thatthebis-quinolinium serieswillbe the focus of futurestud-
ies on the design of non-symmetrical bisquaternary molecules.
Experimental section: All experimental details are listed in the
Supplementary data.
accommodated by
p–cationic interactions between Trp82 (3.5 Å)
and Trp231 (4.1 Å) and was further stabilized by interactions with
Phe329 (3.4 Å), Phe398 (3.8 Å), His438 (3.7 Å), Trp430 (3.6 Å) or
Tyr440 (3.1 Å). The top-scored docking pose of compound 22
(À9.48 kcal/mol) was stabilized differently to compound 12. Its
first quinolinium moiety was attached to Trp82 (3.3 Å) via a p–cat-
ionic interaction and stabilized by additional interactions with aro-
matic residues of His438 (3.7 Å), Trp430 (3.8 Å) or Tyr440 (3.0 Å).
The naphtalenyl linker was sandwiched between Phe329 (3.4 Å)
and Tyr332 (3.4 Å) by p–p interactions, whereas the second quin-
olinium moiety was found to be not interacting with hBChE aro-
Acknowledgements
matic residues. Concerning the in vitro results, both compounds
12 (0.1
l
M) and 22 (0.2
l
M) resulted in similar inhibitory abilities
This work was supported by the Grant Agency of the Czech
Republic (No. 203/09/P130), the Grant Agency of the Charles Uni-
versity (No. 117909/2009/B-CH/FaF), the Grant Agency of the Min-
istry of Education, Youth and Sports Czech Republic (No. SVV-
2010-261-001) and the Ministry of Health of the Czech Republic
(No. MZO00179906).
towards hBChE, although both compounds were found to bind in
different manners to hBChE. A plausible explanation of their simi-
lar inhibitory ability may consist in the accommodation of com-
pound 12 in the hBChE cavity which closes the entrance of the
active site Ser198, while compound 22 closes the narrow entrance
to this hBChE cavity between Phe329 and Tyr332 because it is a ri-
gid molecule.
Supplementary data
The SAR results with the bis-quinolinium series confirmed our
previous findings with bis-pyridinium and bis-isoquinolinium
compounds.18,19 Whereas the compounds bearing connecting link-
ers with heteroatoms, double bond or xylenyl moiety were found
to be inefficient cholinesterase inhibitors, compounds bearing a
methylenyl linkage produced promising AChE inhibitors, especially
compounds with a C7–C12 methylenyl linkage. The best in vitro
inhibitory results towards hAChE were obtained with a C10 linker
(1 nM) that corresponds with the best results for the bis-pyridini-
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.02.047.
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