Preparation and in vitro screening of symmetrical bis-isoquinolinium cholinesterase inhibitors bearing various connecting linkage - Implications for early Myasthenia gravis treatment

Article abstract of DOI:10.1016/j.ejmech.2010.12.011

Inhibitors of acetylcholinesterase are compounds widely used in the treatment of various diseases, such as Alzheimer's disease, glaucoma and Myasthenia gravis (MG). Compounds used in the therapy of MG posses a positive charge in the molecule to ensure peripheral effect of action and minimal blood-brain barrier penetration. The most prescribed carbamate inhibitors are however known for many severe side effects related to the carbamylation of AChE. This paper describes preparation and in vitro evaluation of 20 newly prepared bis-isoquinolinium inhibitors of potential concern for MG. The newly prepared compounds were evaluated in vitro on human recombinant AChE and human plasmatic butyrylcholinesterase (BChE). Their inhibitory ability was expressed as IC ₅₀ and compared to chosen standards ambenonium dichloride, edrophonium chloride, BW284c51 and ethopropazine hydrochloride. Three novel compounds presented promising inhibition (in nM range) of both enzymes in vitro better or similar to edrophonium and BW284c51, but worse to ambenonium. The novel inhibitors did not present higher selectivity toward AChE or BChE. The kinetic assay confirmed non-competitive inhibition of hAChE by two selected promising novel compounds. Two newly prepared compounds were also chosen for docking studies that confirmed apparent π-π or π-cationic interactions aside the cholinesterases catalytic sites. The SAR findings were discussed.

Full text of DOI:10.1016/j.ejmech.2010.12.011

European Journal of Medicinal Chemistry 46 (2011) 811e818
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
Preparation and in vitro screening of symmetrical bis-isoquinolinium
cholinesterase inhibitors bearing various connecting linkage e Implications
for early Myasthenia gravis treatment
Kamil Musilek a , Marketa Komloova , Ondrej Holas , Martina Hrabinova , Miroslav Pohanka ,
,
d
,
b
b
c
b
*
d
e
b
a,c
Vlastimil Dohnal , Florian Nachon , Martin Dolezal , Kamil Kuca
a
University of Defence, Faculty of Military Health Sciences, Department of Toxicology, Trebesska 1575, 500 01 Hradec Kralove, Czech Republic
Charles University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry and Drug Control, Heyrovskeho 1203, 500 05 Hradec Kralove, Czech Republic
University of Defence, Faculty of Military Health Sciences, Centre of Advanced Studies, Trebesska 1575, 500 01 Hradec Kralove, Czech Republic
University of Jan Evangelista Purkynje, Faculty of Science, Department of Chemistry, Ceske mladeze 8, 400 96 Usti nad Labem, Czech Republic
Toxicology Department, Enzymology Unit, Centre de Recherches du Service de Sante des Armees, BP 87, 38702 La Tronche Cedex, France
b
c
d
e
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 2 August 2010
Received in revised form
Inhibitors of acetylcholinesterase are compounds widely used in the treatment of various diseases, such
as Alzheimer’s disease, glaucoma and Myasthenia gravis (MG). Compounds used in the therapy of MG
posses a positive charge in the molecule to ensure peripheral effect of action and minimal bloodebrain
barrier penetration. The most prescribed carbamate inhibitors are however known for many severe side
effects related to the carbamylation of AChE. This paper describes preparation and in vitro evaluation of
5
December 2010
Accepted 10 December 2010
Available online 21 December 2010
20 newly prepared bis-isoquinolinium inhibitors of potential concern for MG.
The newly prepared compounds were evaluated in vitro on human recombinant AChE and human
Keywords:
plasmatic butyrylcholinesterase (BChE). Their inhibitory ability was expressed as IC50 and compared to
chosen standards ambenonium dichloride, edrophonium chloride, BW284c51 and ethopropazine
hydrochloride. Three novel compounds presented promising inhibition (in nM range) of both enzymes in
vitro better or similar to edrophonium and BW284c51, but worse to ambenonium. The novel inhibitors
did not present higher selectivity toward AChE or BChE. The kinetic assay confirmed non-competitive
inhibition of hAChE by two selected promising novel compounds. Two newly prepared compounds were
Cholinesterase
Inhibitor
Myasthenia gravis
In vitro
Molecular docking
SAR
also chosen for docking studies that confirmed apparent
pep or pecationic interactions aside the
cholinesterases catalytic sites. The SAR findings were discussed.
Ó 2010 Elsevier Masson SAS. All rights reserved.
1
. Introduction
where there are still sufficient AChRs presented [2]. They enhance
cholinergic transmission by inhibiting the hydrolysis of acetylcho-
line (ACh) and thus increasing its concentration in the synaptic cleft.
Current early MG treatment involves AChE carbamate inhibitors
such as pyridostigmine bromide or neostigmine bromide (Fig. 1).
Pyridostigmine bromide is better tolerated and more widely used
compared to neostigmine [3]. However, the usage of both carbamate
compounds often leads to development of serious gastrointestinal
side effects (nausea, diarrhea, abdominal cramping), increased
bronchial secretion and cardiac arrhythmia [4]. Additionally, high
concentration of these drugs may evolve into a cholinergic crisis,
characterized by even more severe weakness [5]. Mentioned side
effects of carbamates are related to the carbamylation of AChE. This
type of inhibition is common for carbamate inhibitors that bind to
serine oxygen in the AChE active site [6]. The resulting carbamylated
enzyme intermediate inhibits AChE activity until a water molecule
attacks the carbonyl to reactivate enzyme and produces a carbamic
Myasthenia gravis (MG) is an autoimmune disorder which is
caused by antibody-mediated destruction of postsynaptic membrane
in neuromuscular junction. In most cases antibodies target the
nicotinic acetylcholine receptors (nAChRs), although other compo-
nents of postsynaptic membrane can be affected as well. Impaired
neuromuscular transmission results in a painless weakness and
fatigue of striated muscles [1].
Peripheral inhibitors of acetylcholinesterase (EC 3.1.1.7; AChE)
are a first-line treatment in early stages and in the mild forms of MG,
*
Corresponding author. University of Defence, Faculty of Military Health
Sciences, Department of Toxicology, Trebesska 1575, 500 01 Hradec Kralove, Czech
Republic. Tel.: þ420 973 251 523; fax: þ420 495 518 094.
E-mail address: kamil.musilek@gmail.com (K. Musilek).
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.12.011

812
K. Musilek et al. / European Journal of Medicinal Chemistry 46 (2011) 811e818
Table 1
Newly prepared AChE/BChE inhibitors.
Fig. 1. Carbamate inhibitors used for MG treatment.
(A)
CH
CH
Compound (anion)
(
2
)
1e12
3e14 (Br)
15 (Cl)
16 (Br)
17 (Br)
18 (Cl)
acid derivative. The spontaneous regeneration of carbamylated
enzyme proceeds in the range of minutes.
2
OCH
O(CH
2
(
(
(
CH
E)-CH
Z)-CH
2
)
2
2
)
2
2
CH]CHCH
CH]CHCH
2
Importantly for MG treatment, the AChE inhibitors are not
required to penetrate through the bloodebrain barrier (BBB) to
decrease their central side effects [7]. Thus, both carbamate drugs
contain quaternary nitrogen. This structural feature is important for
increased peripheral effect of mentioned drugs, where the charged
compounds are penetrating in minor ratio [8]. The BBB crossing of
pyridostigmine or neostigmine is limited, but may proceed through
disruptive mechanisms [9]. Hence, these compounds may still
exhibit central side effects related to carbamylation of brain AChE
that could result in cholinergic crisis [6].
Additionally, structurally dissimilar compounds from above
mentioned carbamates are used for the MG treatment. Bisquaternary
inhibitor ambenonium dichloride (1; Fig. 2) is favored with one of the
highest known inhibitory ability toward AChE (in sub-nM range) [10].
Its outstanding potency is unique for the compound that does not
form any covalent bond with the active site of the enzyme. Docking
studies on AChEeambenonium complex showed that 1 is able to
establish highly favorable contacts with amino acids of the catalytic
and the peripheral AChE sites [11]. Ambenonium produces fewer
muscarinic side effects compared to carbamates and it is advanta-
geous because of prolonged action that results in a greater thera-
peutic effect during the night and awakening in contrast to short-
acting anti-AChE compounds. Moreover, its bisquaternary structure
better prevents the passage through the BBB after conventional oral
or intravenous route of administration [11]. Otherdrug, edrophonium
chloride (2; Fig. 2), is used as diagnostic tool for MG. It has rapid onset
and short pharmacologic action, thus it cannot be used for treatment
purposes [12].
2
2
19 (Br)
20 (Br)
21 (Br)
22 (Br)
isoquinolinium AChE inhibitors (3e22; Table 1). Some of these
compounds (3e14) were previously evaluated as inhibitors of rat
brain homogenate with moderate results [17]. The further evalua-
tion of these compounds on the source of human AChE and their
selectivity toward AChE/BChE was considered to be important for
future design of novel compounds. Some experimental data were
added to better characterize the synthesized compounds.
Though the synthesis of such compounds is trivial, their struc-
tural differences were found important for comprehension of key
factors influencing inhibition of AChE and/or BChE [18]. Namely,
presented compounds differ in the structure of the connecting
linker. The key length and/or spatial orientation of connecting
linker are highly important factors for molecular interactions
among the enzyme active sites. Subsequently, the optimal structure
of the linker depends not only on the length, but is usually related
There is a huge variety of compounds reversibly inhibiting AChE
that might be used in the MG treatment [13]. In contrast to carba-
mates, their effect should originate in competitive reversible
inhibition of AChE aside the active serine (S203 for human AChE)
14]. The selective AChE inhibition instead of dual AChE/BChE
butyrylcholinesterase; EC 3.1.1.8) inhibition might keep the non-
[
(
to presented
p-electrons (double bond or aromatic residues) or
heteroatom (hydrogen-bonding interactions), too [19]. The bis-
isoquinolinium part of the molecule was chosen as an extension of
previously prepared bispyridinium compounds [16]. While the
lipophilicity of the best AChE inhibitor from bispyridinium series
with decylenyl linker was very low (log P ꢀ1.97), similar compound
from bis-isoquinolinium series was calculated to have still low
lipophilicity (log P 0.40) to cross BBB and thus may act as peripheral
AChE inhibitor. Presumably, bis-isoquinolinium compounds may
specific esterase (BChE) active for other toxic substrates [15]. Our
approach originates from design, synthesis, in vitro screening and
docking studies of selected bisquaternary bis-isoquinolinium
compounds connected by various linkages.
2
. Design and synthesis bis-isoquinolinium compounds
Formerly, the bispyridinium compounds bearing various linkers
extend the pep or pecationic interactions with aromatic residues
were prepared and evaluated as AChE and BChE inhibitors [16].
This paper describes the preparation of 20 symmetrical bis-
(His, Trp, Tyr, Phe) of the enzyme active sites found for bispyr-
idinium ones. These amino-acid residues are well known for their
principal function in the enzyme active sites via non-covalent
interactions (AChE or BChE) [20]. Moreover, the isoquinolinium,
quinolinium or acridinium compounds were formerly found to be
very effective inhibitors of AChE [21]. Hence, higher AChE and/or
BChE affinity was hypothesized for bis-isoquinolinium moiety.
Moreover, the related bispyridinium compounds were found to be
non-competitive inhibitors of both enzymes aside the active serine
(S203) in contrast to carbamate compounds [16]. Additionally, the
Fig. 2. Non-covalent AChE inhibitors used for MG treatment or diagnosis.
peripheral effect of AChE inhibitors is preferred for MG treatment in

K. Musilek et al. / European Journal of Medicinal Chemistry 46 (2011) 811e818
813
order to decrease central side effects caused by interactions with
Table 2
Inhibitory potency of tested compounds toward cholinesterases.
the brain AChE. For this reason, the charged molecules were
chosen. Moreover, the monoquaternary compounds were pene-
trating BBB at least in 10%, whereas the bisquaternary compounds
were previously found to penetrate the bloodebrain barrier (BBB)
in less than 2% [8,22,23]. For these reasons, the bisquaternary
molecules were designed to maintain the peripheral inhibitory
activity against AChE.
Compound
AChE IC50 ꢃ SD
BChE IC50 ꢃ SD
SI BChE/
AChE
i1 i2
K /K
(mM)
a
a
(
m
M)
(
m
M)
Ambenonium
0.0007 ꢃ 0.0001
5.17 ꢃ 1.0
6.82 ꢃ 1.11
9743
0.005/
0.006
0.79/4.80
(
1)
Edrophonium
2)
1370 ꢃ 223
1400 ꢃ 228
265
(
3
4
654 ꢃ 128
2.1
e
e
b
b
The new inhibitors (3e22; Fig. 3) were prepared via standard
synthetic strategy [24,25]. The solution of isoquinoline (1.6 g,
e
e
e
5
6
7
8
9
10
1
1
1
1
1
1
446 ꢃ 87
36 ꢃ 7
2600 ꢃ 424
40 ꢃ 6.5
0.7 ꢃ 0.1
2 ꢃ 0.3
5.8
1.1
0.05
6.7
6.0
80
15
32
90
0.0007
77
0.04
9.1
1.5
0.002
0.1
70
13
11800
0.002
e
e
1
2.4 mmol) and corresponding alkylating agent (5.6 mmol) in DMF
13 ꢃ 2.5
e
ꢁ
(
10 ml) was stirred at 70 C. The reaction mixture remained at the
0.3 ꢃ 0.06
0.5 ꢃ 0.1
0.005 ꢃ 0.001
0.04 ꢃ 0.008
0.05 ꢃ 0.01
0.1 ꢃ 0.02
137 ꢃ 33
2 ꢃ 0.5
e
room temperature. It was portioned with acetone (50 ml) and cooled
3 ꢃ 0.6
e
ꢁ
in refrigerator (5 C) overnight. The crystalline or amorphous crude
0.4 ꢃ 0.06
0.6 ꢃ 0.09
1.6 ꢃ 0.3
9 ꢃ 1.5
0.62/0.98
1
2
3
4
5
6
e
product was collected by filtration, washed with acetone (3 ꢂ 20 ml)
and recrystallized from MeCN. NMR, ESI-MS and elemental analysis
determined the entity and purity of all prepared compounds.
e
e
0.1 ꢃ 0.02
154 ꢃ 25
51 ꢃ 8
e
e
1310 ꢃ 255
7 ꢃ 1.5
e
3
. AChE and BChE inhibition results
17
64 ꢃ 10
25 ꢃ 4
e
e
1
1
2
2
2
8
9
0
1
2
17 ꢃ 3
250 ꢃ 49
8 ꢃ 2
0.6 ꢃ 0.1
1 ꢃ 0.2
e
The bisquaternary bis-isoquinolinium compounds (3e22) were
e
assayed for their inhibitory ability in standard inhibition test in vitro
using human recombinant AChE (hAChE) and human plasmatic
BChE (hBChE) [26]. The IC50 values and selectivity index for IC50
BChE/IC50 AChE ratio (SI) of all newly prepared compounds along
with the standards 1e2 are listed in Table 2. The selective AChE
inhibitor BW284c51 (23; 1,5-bis(4-allyldimethylammoniumphenyl)
pentan-3-one dibromide; Fig. 4) and selective BChE inhibitor etho-
propazine hydrochloride (24) were also determined to better depict
the selectivity issues [27,28].
12 ꢃ 2
840 ꢃ 137
4 ꢃ 0.7
e
0.3 ꢃ 0.06
0.03 ꢃ 0.006
1020 ꢃ 199
1.25/0.75
0.01/0.05
24.7/
12100
BW284c51 (23)
Ethopropazine
354 ꢃ 58
1.6 ꢃ 0.3
(24)
a
Mean value of three independent determinations.
Not soluble in screening medium.
b
and BChE. Though compound 4 was not soluble in screening medium
not even by addition of DMSO), inhibition of both enzymes similar
to compounds 3 or 5 was supposed. The other group of compounds
6, 7,15,17,18, 20, 21) displayed inhibition of AChE in M scale. Again,
compounds 7 and 20 inhibited BChE more than AChE, though the SI
was lower when compared to compound 14. The third group of
prepared compounds (8e13, 22) was able to inhibit AChE on nM
scale, although none of these compounds exceeded the inhibitory
ability of MG standard 1. Among them, compound 10 with an
aliphatic linker presented the best inhibition results toward AChE
from the newly prepared compounds; however, the SI for hAChE
remained poor. Interestingly, compound 22 also resulted as potent
inhibitor of AChE, while the structure of its connecting linker was
The standard compounds used for MG treatment or diagnosis
e2 were found to be potent hAChE inhibitors, where compound
exhibited inhibitory ability on sub-nM range (0.7 nM). Both
compounds also presented very high selectivity toward AChE
required for therapeutic use. Additionally, the kinetic experiment
confirmed their inhibitory ability and showed that the presence of
substrate did not affect non-competitive inhibition by compound 1
(
1
1
(
m
[
29]. Thus compound 1 was supposed to bind aside the AChE active
serine, whereas inhibition by compound 2 was affected by presence
of the substrate. Compound 2 was hypothesized to bind competi-
tively closely to active serine and consequently influenced by the
substrate [30,31]. The selective standard compounds 23e24 were
found to be potent and also selective AChE or BChE inhibitors.
Compound 23 showed high and selective inhibition of hAChE
completely different. In this case, the different weak (
pep or
pecationic) interactions were supposed to be crucial for the inhib-
(
30 nM), whereas kinetic experiments confirmed its binding to
itory ability of such compound [16]. Unfortunately, the most potent
compounds 10e12 displayed only poor selectivity between AChE
and BChE. This lack of selectivity may limit their further in vivo
evaluation and possible use due to interference with BChE that is
responsible for unspecific esterase activity in the organism [15].
Though BChE has not direct effect on physiological functions, its
pharmacological and toxicological importance have been formerly
recognized. Namely, BChE is involved in degradation of numerous
drugs and poisons [33]. While many drugs are mixed inhibitors of
AChE peripheral active site without influence of substrate to inhi-
bition kinetics. Compound 24 resulted as potent and selective
inhibitor of hBChE (1.6 mM) [32]. The kinetic experiments for hAChE
displayed strong competition of inhibitor 24 and substrate during
enzymatic reaction.
Concerning the newly prepared compounds, there may be seen at
least three trends of inhibitoryability within the whole series. Firstly,
some compounds (3, 5, 14, 16, 19) showed only minor inhibition of
AChE in mM scale. Surprisingly, some of these compounds (14,16,19)
presented better inhibition of BChE than AChE, while compound
14 remained the most selective BChE inhibitor among all tested
compounds with 4 orders of magnitude difference between AChE
Fig. 3. Preparation of bis-isoquinolinium salts.
Fig. 4. Chosen selective AChE/BChE inhibitors.

814
K. Musilek et al. / European Journal of Medicinal Chemistry 46 (2011) 811e818
AChE and BChE, truly selective AChE inhibitors are compounds of
interest to not affect non-specific function of BChE [34].
4
. Molecular docking results and SAR discussion
Regarding the AChE, top-scored docking pose of compound 10
(
ꢀ11.09 kcal/mol) showed apparent interactions with aromatic
residues of peripheral anionic site (PAS) (Fig. 5) [35e38]. Namely,
strong or catione interactions between both isoquinolinium
pep
p
moieties and Trp286 (3.7 and 3.8 Å) occurred. First isoquinolinium
moiety was sandwiched among Tyr72 (3.9 Å), Tyr124 (3.5 Å), Trp286
(
3.8 Å) and Phe297 (3.5 Å). Besides Trp286, second isoquinolinium
moiety displayed some interaction with His287 (4.2 Å). Compound
0 did not penetrate closely to active Ser203 and blocked the AChE
1
gorge entrance. Similarly to inhibitor 10, compound 22 (ꢀ12.04 kcal/
mol) presented sandwiching of one isoquinolinium moiety among
Tyr72 (4.3 Å), Tyr124 (3.5 Å), Trp286 (3.4 Å) and Phe297 (3.3 Å).
Second isoquinolinium moiety was again attached to Trp286 (4.0 Å)
and His287 (4.2 Å). Differently from inhibitor 10, the naphtylenyl
Fig. 6. Molecular docking for hBChE with compound 10 (blue) and 22 (magenta). (For
interpretation of the references to color in this figure legend, the reader is referred to
the web version of this article.)
moiety displayed strong pep interaction with Trp286 (3.0 Å).
Concerning BChE, top-scored docking pose of compound 10
ꢀ9.49 kcal/mol) showed interactions with aromatic Phe, Trp and
(
p
ecationic interactions with aromatic residues of ChE active sites
Tyr residues (Fig. 6). The
pep or cationep interaction with Trp82
and the results obtained for analogous bispyridinium compounds
were confirmed. Additionally, the isoquinolinium compounds were
found to be more potent inhibitors of hAChE compared to former
pyridinium ones. Since the pyridinium moiety was previously
successfully used, the quinolinium compounds will be the aim of
further interest [16,17].
Secondly, the used linkage highly influenced the inhibitory
ability of prepared compounds. The length of the connecting
linkage remained the most important factor within whole series of
compounds. The compounds with 6e11 methylene units (8e13)
revealed the best inhibition of hAChE. These results apparently
correlate with the distance of important aromatic residues of
hAChE and thus with interactions of isoquinolinium moiety. The
situation changed for hBChE, where less aromatic residues are
presented. Compounds 10e14 were plausibly stacked between two
Trp residues and consequently resulted as potent BChE inhibitors.
Compounds bearing shorter linkers (3e6) were found inefficient
for both enzymes. The similar results were previously obtained for
bispyridinium series [16].
(
3.9 Å) and the T-stacking with Trp231 (3.6 Å) occurred. Moreover,
one isoquinolinium ring presented additional T-stacking with
His438 (3.6 Å), Phe329 (3.5 Å) and Phe398 (3.7 Å). Similarly to
inhibitor 10, compound 22 (ꢀ10.18 kcal/mol) presented the
catione interaction with Trp82 (3.5 Å). The same isoquinolinium
moiety was also T-stacked toward His438 (3.7 Å). The naphtylenyl
moiety showed strong interaction with Phe329 (3.1 Å). Differ-
pep or
p
pep
ently to compound 10, the second isoquinolinium moiety was not
attached to aromatic residue and remained close to Pro285 (3.7 Å).
Some SAR, which originated from the docking studies and in
vitro data, can be demonstrated [39]. Firstly, the isoquinolinium
moiety showed its importance from the point of view of pep and
Differently, heteroatom (15e16), double bond (17e18), xylene
(19e21) and naphtylene linkage (22) were introduced to find
possible additional interactions [27]. Not surprisingly, most of these
compounds (15e21) displayed only minor inhibitory activity of both
enzymes (mM range). The linkage length of such compounds varied
from 4 to 6 carbonecarbon (CeC) bonds and thus they were found
insufficient to interact similarly as the potent inhibitors 8e13. Due to
the poor invitro results of 15e21, the plausible interactions with both
enzymes were not further studied. More interestingly, compound 22
with naphtylenyl linkage exhibited the promising inhibitory ability
for both used enzymes. Its linker was similar length to compound 9
(7 CeC bonds) and thus displayed the similar binding to AChE or
BChE [16]. Though the compound 22 was not the best inhibitor in the
tested series, it presented important interactions of its linker with
aromatic residues. Such interactions may help in further design of
more selective AChE inhibitors suitable for MG treatment.
The docking studies further improved the in vitro findings and
confirmed the SAR conclusions. Both chosen compounds 10 and 22
presented similar binding toward AChE aside active site (S203)
and the docking results were confirmed by kinetic experiments,
where the presence of substrate did not affect inhibition by these
compounds. The higher IC50 of compound 22 might be explained by
conformational and spatial rigidity of the naphtylenyl linkage as its
Fig. 5. Molecular docking for mAChE with compound 10 (blue) and 22 (magenta). (For
interpretation of the references to color in this figure legend, the reader is referred to
the web version of this article.)

K. Musilek et al. / European Journal of Medicinal Chemistry 46 (2011) 811e818
815
accommodation in the narrow AChE gorge was difficult. Though the
binding of compounds 10 and 22 was different for BChE, their IC50
resulted only with one order of magnitude difference. This finding
might be explained by closing of BChE gorge, where compound 10
better protect the active site (S198) from accommodation of the
substrate.
calculated 52.81% C, 3.73% H, 6.48% N; found 52.42% C, 4.15% H,
6.38% N.
1,2-Bis(isoquinolinium)-eth-1,2-diyl dibromide (4) [17]. M.p.
ꢁ
1
0
d ppm 9.70 (s, 2H, H-1,1 ),
269e271 C. H NMR (300 MHz, D
2
O)
0
0
0
0
0
8.49e8.43 (m, 4H, H-3,3 ,8,8 ), 8.33e8.24 (m, 6H, H-4,4 ,5,5 ,7,7 ),
0
13
8.08e8.01 (m, 2H, H-6,6 ), 5.56 (s, 4H, NeCH
O) ppm 159.35, 147.76, 147.45, 143.11, 141.54, 139.73, 137.19,
136.94, 136.90, 69.61. ESI-MS: m/z 143.1 [M /2] (calculated for
2
e). C NMR (75 MHz,
D
2
d
2
þ
5
. Conclusion
2
þ
[C H N /2] 143.08). EA: calculated 53.84% C, 4.07% H, 6.28% N;
20 18 2
In summary, 20 novel bis-isoquinolinium cholinesterase inhibi-
found 54.06% C, 4.42% H, 5.88% N.
tors were prepared in effort to compare their in vitro ability to stan-
dard MG drugs. At least three compounds (10e12) showed promising
inhibitory (IC50) ability toward hAChE on nM scale better or similar to
standards edrophonium and BW284c51, but did not present high
selectivity between hAChE and hBChE. None of prepared compounds
was able to exceed ambenonium in hAChE inhibition. The kinetic
experiments confirmed non-competitive inhibition of hAChE by two
chosen novel compounds. Consequently, docking studies confirmed
their binding of two selected inhibitors aside the hAChE active site via
1,3-Bis(isoquinolinium)-prop-1,3-diyl dibromide (5) [17]. M.p.
ꢁ
1
0
244e246 C. H NMR (300 MHz, D
2
O)
d
ppm 9.74 (s, 2H, H-1,1 ),
0
0
0
8.55 (d, 2H, J ¼ 6.7 Hz, H-3,3 ), 8.36e8.27 (m, 4H, H-4,4 ,8,8 ),
0
0
0
8.19e8.08 (m, 4H, H-5,5 ,7,7 ), 7.98 (t, 2H, J ¼ 7.3 Hz, H-6,6 ), 5.04 (t,
13
4H, J ¼ 7.0 Hz, NeCH
(75 MHz, D O) 148.66, 137.07, 136.86, 133.0, 131.19, 129.39, 126.92,
126.66, 126.29, 57.96, 29.70. ESI-MS: m/z 150.1 [M /2] (calculated
2 2 2
e), 3.13e2.98 (m, 2H, NeCH eCH e). C NMR
2
2þ
2
þ
for [C21H
20
N
2
/2] 150.08). EA: calculated 54.81% C, 4.38% H, 6.09%
N; found 54.79% C, 4.32% H, 5.81% N.
p
e
p
or
p
ecationic interactions. The SAR findings were determined.
1,4-Bis(isoquinolinium)-but-1,4-diyl dibromide (6) [17]. M.p.
ꢁ
1
0
Though the bis-isoquinolinium cholinesterase inhibitors exceeded
the formerly prepared bispyridinium compounds, the bis-quinoli-
nium compounds will be the aim of further interest.
263e265 C. H NMR (300 MHz, D
2
O)
d
ppm 9.72 (s, 2H, H-1,1 ),
0
0
0
8.54e8.45 (d, 2H, J ¼ 6.4 Hz, H-3,3 ), 8.39e8.33 (m, 4H, H-4,4 ,8,8 ),
0
0
0
8.22e8.16 (m, 4H, H-5,5 ,7,7 ), 8.04e7.97 (m, 2H, H-6,6 ), 4.87e4.82
13
(
m, 4H, eNeCH
2 2 2
e), 2.31e2.24 (m, 4H, NeCH eCH e). C NMR
6
. Experimental section
(75 MHz, D O) 148.55, 136.97, 136.72, 133.96, 129.49, 127.08, 126.69,
2
2
þ
1
26.07, 60.04, 26.69. ESI-MS: m/z 157.1 [M /2] (calculated for
2
þ
6.1. Chemical preparation
[C22
22
H N
2
/2] 157.09). EA: calculated 55.72% C, 4.68% H, 5.91% N;
found 55.85% C, 5.11% H, 5.56% N.
Solvents(acetone, DMF, MeCN)and reagentswere purchasedfrom
Fluka and SigmaeAldrich (Czech Republic) and used without further
purification. Reactions were monitored by TLC using DC-Alufolien
1,5-Bis(isoquinolinium)-pent-1,5-diyl dibromide (7) [17]. M.p.
ꢁ
1
0
207e209 C. H NMR (300 MHz, D
2
O)
d
ppm 9.61 (s, 2H, H-1,1 ),
0
0
0
8.42 (d, 2H, J ¼ 7.0 Hz, H-3,3 ), 8.30e8.24 (m, 4H, H-4,4 ,8,8 ),
0
0
0
Cellulose F (Merck, Germany) and mobile phase BuOHeCH
COOHeH O 5:1:2, detection by solution of Dragendorff reagent
solution containing 10 ml CH COOH, 50 ml H O and 5 ml of basic
solution prepared by mixing of two fractions e fraction A: 850 mg Bi
NO , 40 ml H O, 10 ml CH COOH; fraction B: 8 g KI, 20 ml H O).
3-
8.16e8.11 (m, 4H, H-5,5 ,7,7 ), 7.99e7.92 (m, 2H, H-6,6 ), 7.72e4.68
2
(m, 4H, NeCH
2H, eNe(CH
2
e), 2.17e2.10 (m, 4H, eNeCH
2
eCH
2
e), 1.40e1.27 (m,
ppm 148.46,
13
(
3
2
2
)
2 2
eCH e). C NMR (75 MHz, D O) d
2
136.83, 136.75, 133.35, 131.04, 129.43, 126.92, 126.73, 125.97, 60.57,
2
þ
2þ
24 2
(
)
3 3
2
3
2
29.08, 21.29. ESI-MS: m/z 164.2 [M /2] (calculated for [C23
H
N
/
Melting points were measured on micro heating stage PHMK 05 (VEB
Kombinat Nagema, Radebeul, Germany) and were uncorrected.
2] 164.10). EA: calculated 56.58% C, 4.95% H, 5.74% N; found 56.12%
C, 5.55% H, 5.43% N.
1
NMR spectra were generally recorded at Varian Gemini 300 ( H
1,6-Bis(isoquinolinium)-hex-1,6-diyl dibromide (8) [17,24]. M.p.
00 MHz, 13C 75 MHz, Palo Alto CA, USA). In all cases, the chemical
233e235 C. H NMR (300 MHz, D
ꢁ
1
O)
d
ppm 9.66 (s, 2H, H-1,1 ),
0
0
3
2
1
0
0
shift values for H spectra are reported in ppm (
d
) relative to residual
8.47 (d, 2H, J ¼ 6.4 Hz, H-3,3 ), 8.39e8.30 (m, 4H, H-4,4 ,8,8 ),
13
0
0
0
CHD
reported in ppm (
39.43. Signals are quoted as s (singlet), d (doublet), t (triplet) and m
SO CD
2 2 3
(d
2
2.50) or D O (d
4.79), shift values for C spectra are
8.24e8.12 (m, 4H, H-5,5 ,7,7 ), 8.05e7.93 (m, 2H, H-6,6 ), 4.73 (t, 4H,
J ¼ 7.3 Hz, NeCH e), 2.17e2.03 (m, 4H, eNeCH eCH e), 1.49e1.39
(m, 4H, eNe(CH e). C NMR (75 MHz, D O) ppm 148.47,
136.95, 136.65, 133.45, 130.96, 129.53, 127.10, 126.75, 125.98, 61.09,
d
) relative to solvent peak dimethylsulfoxide-d
6
2
2
2
13
d
2
)
2
eCH
2
2
d
(
multiplet).
2
þ
2þ
26 2
The mass spectra (MS respectively MSn) were measured on an
29.70, 24.58. ESI-MS: m/z 171.2 [M /2] (calculated for [C24
H
N
/
LCQ FLEET ion trap and evaluated using Xcalibur v 2.5.0 software
both Thermo Fisher Scientific, San Jose, CA, USA). The sample was
dissolved in deionized water (Goro, s.r.o., Prague, Czech Republic),
and injected continuously (8 l/min) by Hamilton syringe into
electrospray ion source. The parameters of electrospray were set up
as follows: sheath gas flow rate 20 arbitrary units, aux gas flow rate
2] 171.11). EA: calculated 57.39% C, 5.22% H, 5.58% N; found 57.55% C,
5.67% H, 5.12% N.
(
1,7-Bis(isoquinolinium)-hept-1,7-diyl dibromide (9) [17]. M.p.
ꢁ
1
0
0
m
205e207 C. H NMR (300 MHz, D
2
O)
d
ppm 9.66 (s, 2H, H-1,1 ),
0
0
8.47 (d, 2H, J ¼ 6.4 Hz, H-3,3 ), 8.33 (t, 4H, J ¼ 7.3 Hz, H-4,4 ,8,8 ),
0
0
0
8.18e8.08 (m, 4H, H-5,5 ,7,7 ), 7.98e7.91 (m, 2H, H-6,6 ), 4.72 (t, 4H,
J ¼ 7.3 Hz, NeCH e), 2.13e2.02 (m, 4H, NeCH eCH e), 1.50e1.30
(m, 6H, Ne(CH e). C NMR (75 MHz, D O) ppm 148.32,
136.81, 136.41, 133.37, 130.76, 129.38, 126.98, 126.58, 125.86, 60.97,
5
arbitrary units, sweep gas flow rate 0 arbitrary units, spray
2
2
2
ꢁ
13
voltage 5 kV, capillary temperature 275 C, capillary voltage 13 V,
tube lens 100 V.
2
)
2
e(CH
2
)
2
2
d
2
þ
2þ
28 2
N /
2
6.92, 24.45. ESI-MS: m/z 178.2 [M /2] (calculated for [C25
H
6.2. Prepared bisquaternary salts
2] 178.11). EA: calculated 58.16% C, 5.47% H, 5.43% N; found 58.41%
C, 5.86% H, 4.97% N.
1
,1-Bis(isoquinolinium)-meth-1,1-diyl dibromide (3) [17]. M.p.
1,8-Bis(isoquinolinium)-oct-1,8-diyl dibromide (10) [17]. M.p.
ꢁ
1
0
ꢁ
1
0
2
8
8
2
57e258 C. H NMR (300 MHz, D
2
O)
d
ppm 10.34 (s, 2H, H-1,1 ),
221e223 C. H NMR (300 MHz, D
2
O)
d
ppm9.67(s,2H, H-1,1 ), 8.48(d,
0
0
0
0
0
0
.90 (d, 2H, J ¼ 6.7 Hz, H-3,3 ), 8.59 (d, 4H, J ¼ 7.0 Hz, H-4,4 ,8,8 ),
2H, J ¼ 6.7 Hz, H-3,3 ), 8.40e8.33 (m, 4H, H-4, 4 ,8,8 ), 8.23e8.12 (m,
0
0
0
0
0
0
.36e8.31 (m, 4H, H-5,5 ,7,7 ), 8.17e8.09 (m, 2H, H-6,6 ), 7.66 (s,
4H, H-5,5 ,7,7 ), 8.03e7.96 (m, 2H, H-6,6 ), 4.72 (t, 4H, J ¼ 7.3 Hz,
NeCH e), 2.13e2.00 (m, 4H, NeCH eCH e), 1.38e1.25 (m, 8H, eNe
(CH e). C NMR (75 MHz, D O) ppm 148.35, 136.85,
136.44,133.37,130.78,129.39,126.61,125.84, 61.06, 29.63, 27.23, 24.55.
13
H, NeCH
2
e). C NMR (75 MHz, D
2
O)
d
ppm 150.68, 139.18,
2
2
2
13
1
38.57, 136.55, 132.64, 132.05, 130.99, 127.31, 127.24, 77.56. ESI-MS:
m/z 136.1 [M /2] (calculated for [C19
2
)
2
e(CH
)
2 2
2
d
2
þ
2þ
16 2
H
N
/2] 136.07). EA:

816
K. Musilek et al. / European Journal of Medicinal Chemistry 46 (2011) 811e818
ESI-MS: m/z 185.2 [M2þ/2] (calculated for [C26
calculated 58.88% C, 5.70% H, 5.28% N; found 58.59% C, 6.18% H, 4.76% N.
,9-Bis(isoquinolinium)-non-1,9-diyl dibromide (11) [17]. M.p.
H
30
N
2þ
/2] 185.12). EA:
J ¼ 7.2 Hz, H-7,7 ), 8.09 (t, 2H, J ¼ 7.2 Hz, H-6,6 ), 6.36e6.32 (m, 2H, ]
0
0
2
13
CHe), 5.51e5.48 (m, 4H, eCH
(ppm) 150.26, 137.03, 134.86, 131.20, 130.43, 130.10, 127.23, 127.12,
125.82, 60.83. ESI-MS: m/z 156.1 [M /2] (calculated for [C22
156.08). EA: calculated 55.96% C, 4.27% H, 5.93% N; found 55.83% C,
4.35% H, 5.94% N.
2 6
e). C NMR (75 MHz, DMSO d ):
1
d
ꢁ
1
0
2þ
2þ
20 2
N /2]
1
(
(
57e159 C. H NMR (300 MHz, D
2
O)
d
ppm 9.67 (s, 2H, H-1,1 ), 8.48
H
0
0
0
d, 2H, J ¼ 6.7 Hz, H-3,3 ), 8.39e8.33 (m, 4H, H-4, 4 ,8,8 ), 8.22e8.11
0
0
0
m, 4H, H-5,5 ,7,7 ), 8.01e7.95 (m, 2H, H-6,6 ), 4.70 (t, 4H, J ¼ 7.3 Hz,
NeCH
CH
2
e), 2.12e1.98 (m, 4H, NeCH
2
eCH
2
e), 1.38e1.21 (m, 10H, Ne
O) ppm 148.21, 136.73,
1,4-Bis(isoquinolinium)-but-(2Z)-ene-1,4-diyldichloride(18). M.p.
229e230 C. H NMR (300 MHz, DMSO d
13
ꢁ
1
(
2
)
2
e(CH
2
)
5
e). C NMR (75 MHz, D
2
d
6
):
d
(ppm) 10.51 (s, 2H, H-
0
0
0
1
2
36.33, 133.31, 130.68, 129.30, 126.91, 126.51, 125.75, 60.98, 29.61,
7.40, 27.17, 24.47. ESI-MS: m/z 192.2 [M /2] (calculated for
/2] 192.13). EA: calculated 59.57% C, 5.93% H, 5.15% N;
found 59.07% C, 6.12% H, 4.97% N.
,10-Bis(isoquinolinium)-dec-1,10-diyl dibromide (12) [17,24].
1,1 ), 9.05 (d, 2H, J ¼ 6.7 Hz, H-3,3 ), 8.66 (d, 2H, J ¼ 6.7 Hz, H-4,4 ), 8.61
2
þ
0
0
(d, 2H, J ¼ 7.8 Hz, H-8,8 ), 8.39 (d, 2H, J ¼ 7.8 Hz, H-5,5 ), 8.28 (t, 2H,
2
þ
0
0
[C
27 32
H N
2
J ¼ 7.1 Hz, H-7,7 ), 8.10 (t, 2H, J ¼ 7.1 Hz, H-6,6 ), 6.33e6.21 (m,
13
2H, ¼ CHe), 5.94e5.85 (m, 4H, eCH
2 6
e). C NMR (75 MHz, DMSO d ):
1
d
(ppm) 150.53, 137.02, 136.94, 135.16, 131.11, 130.51, 128.76, 127.24,
ꢁ
1
0
0
2þ
2þ
20 2
N ]
M.p. 222e224 C. H NMR (300 MHz, D
2
O)
d
ppm 9.68 (s, 2H, H-1,1 ),
125.80, 57.24. ESI-MS: m/z 311.1 [M ] (calculated for [C22
312.16). EA: calculated 68.93% C, 5.26% H, 7.31% N; found 68.86% C,
7.04% H, 7.64% N.
1,1 -Bis(isoquinolinium)-1,2-phenyldimethylenyl dibromide (19).
M.p. 288e289 C. H NMR (300 MHz, DMSO d
H
0
0
8
8
.49 (d, 2H, J ¼ 6.2 Hz, H-3,3 ), 8.44e8.34 (m, 4H, H-4, 4 ,8,8 ),
0
0
0
.26e8.13 (m, 4H, H-5,5 ,7,7 ), 8.08e7.95 (m, 2H, H-6,6 ), 4.70 (t, 4H,
e), 2.11e1.96 (m, 4H, NeCH eCH e),1.37e1.10 (m,
O) ppm 148.37,
0
J ¼ 7.3 Hz, NeCH
2
2
2
13
ꢁ
1
1
2H, Ne(CH e(CH
2
)
2
)
2 6
e). C NMR (75 MHz, D
2
d
6
):
d
(ppm) 9.14 (s, 2H,
0
0
0
1
2
36.90, 136.46, 133.42, 130.81, 129.43, 127.06, 126.64, 125.87, 61.10,
9.70, 27.60, 27.34, 24.58. ESI-MS: m/z 199.2 [M /2] (calculated for
/2] 199.14). EA: calculated 60.23% C, 6.14% H, 5.02% N;
found 60.40% C, 6.48% H, 4.68% N.
,11-Bis(isoquinolinium)-undec-1,11-diyl dibromide (13) [17].
H-1,1 ), 8.34 (d, 2H, J ¼ 6.7 Hz, H-3,3 ), 8.02 (d, 2H, J ¼ 6.6 Hz, H-4,4 ),
2þ
0 0
7.97e7.89 (m, 4H, H-5,5 ,8,8 ), 7.88e7.85 (m, 4H, Ph), 7.81e7.70 (m,
2
þ
0
0
13
[C
28
H
34
N
2
4H, H-6,6 ,7,7 ), 6.17 (s, 4H, eCH
(ppm) 146.73, 137.32, 136.08, 134.27, 132.45, 131.61, 131.30, 130.43,
128.95, 126.45, 125.95, 125.88, 61.06. ESI-MS: m/z 363.2 [M
(calculated for [C26
5.36% N; found 59.59% C, 4.17% H, 5.66% N.
1,1 -Bis(isoquinolinium)-1,3-phenyldimethylenyl
(20). M.p. 286e288 C. H NMR (300 MHz, DMSO d
2
e). C NMR (75 MHz, DMSO d
6
):
d
2þ
1
]
ꢁ
1
0
0
2þ
M.p.180e182 C. H NMR (300 MHz, D
2
O)
d
ppm 9.69 (s, 2H, H-1,1 ),
22
H N
2
] 362.18). EA: calculated 59.79% C, 4.25% H,
0
0
8
8
.49 (d, 2H, J ¼ 6.7 Hz, H-3,3 ), 8.40e8.34 (m, 4H, H-4,4 ,8,8 ),
0
0
0
0
.22e8.11 (m, 4H, H-5,5 ,7,7 ), 8.03e7.94 (m, 2H, H-6,6 ), 4.70 (t, 4H,
dibromide
(ppm) 9.73
ꢁ
1
J ¼ 7.1 Hz, NeCH
2
e), 2.08e1.96 (m, 4H, NeCH
2
eCH
2
e), 1.33e0.99
O) ppm
6
):
d
13
0
0
(
m, 14H, Ne(CH
2
)
2
e(CH
2
)
7
e). C NMR (75 MHz, D
48.23, 136.74, 136.41, 133.35, 130.77, 129.34, 126.91, 126.56, 125.79,
1.01, 29.64, 27.62, 27.31, 24.48. ESI-MS: m/z 206.2 [M /2] (calcu-
/2] 206.15). EA: calculated 60.85% C, 6.34% H,
.89% N; found 60.43% C, 6.68% H, 4.67% N.
,12-Bis(isoquinolinium)-dodec-1,12-diyl dibromide (14) [17,24].
2
d
(s, 2H, H-1,1 ), 8.45 (d, 2H, J ¼ 6.5 Hz, H-3,3 ), 8.33e8.26 (m, 4H, H-
0
0
0
0
0
1
6
4,4 ,8,8 ), 8.23e8.13 (m, 4H, H-5,5 ,7,7 ), 8.04e7.95 (m, 2H, H-6,6 ),
2
þ
13
7.62e7.58 (m, 3H, Ph), 7.27 (s, 1H, Ph), 5.96 (s, 4H, eCH
(75 MHz, DMSO d ): (ppm) 149.11, 137.31, 137.24, 134.43, 133.72,
131.38, 130.31, 126.83, 139.66, 127.74, 127.23, 126.99, 126.31, 63.44.
ESI-MS: m/z 361.8 [M ] (calculated for [C26
calculated 59.79% C, 4.25% H, 5.36% N; found 59.32% C, 4.31% H,
5.46% N.
2
e). C NMR
2
þ
lated for [C29
36
H N
2
6
d
4
2
þ
2þ
22 2
N ] 362.18). EA:
1
H
ꢁ
1
0
0
M.p. 225e227 C. H NMR (300 MHz, D
2
O)
d
ppm 9.71 (s, 2H, H-1,1 ),
0
0
8
8
.50 (d, 2H, J ¼ 6.7 Hz, H-3,3 ), 8.43e8.34 (m, 4H, H-4,4 ,8,8 ),
0
0
0
0
.24e8.12 (m, 4H, H-5,5 ,7,7 ), 8.03e7.94 (m, 2H, H-6,6 ), 4.71 (t, 4H,
1,1 -Bis(isoquinolinium)-1,4-phenyldimethylenyl dibromide (21).
M.p. decomp. 316 C. H NMR (300 MHz, DMSO d
ꢁ
1
J ¼ 7.0 Hz, NeCH
2
e), 2.07e1.94 (m, 4H, NeCH
2
eCH e), 1.28e0.90
2
6
):
d (ppm) 10.37 (s,
13
0
0
(
m,16H, Ne(CH
2
)
2
2
e(CH )
8
e). C NMR (75 MHz, D O) d ppm 148.26,
2
2H, H-1,1 ), 8.85 (d, 2H, J ¼ 6.7 Hz, H-3,3 ), 8.60 (d, 2H, J ¼ 6.8 Hz, H-
0
0
0
1
2
36.78, 136.48, 133.36, 130.83, 129.37, 126.93, 126.60, 125.83, 61.03,
9.65, 27.71, 27.69, 27.33, 24.51. ESI-MS: m/z 213.2 [M /2] (calcu-
4,4 ), 8.52 (d, 2H, J ¼ 8.2 Hz, H-8,8 ), 8.34 (d, 2H, J ¼ 8.1 Hz, H-5,5 ),
2þ
0
0
8.27 (t, 2H, J ¼ 7.1 Hz, H-7,7 ), 8.08 (t, 2H, J ¼ 7.2 Hz, H-6,6 ), 7.70 (s, 4H,
2
þ
13
lated for [C30
H
38
N
2
/2] 213.15). EA: calculated 61.44% C, 6.53% H,
Ph), 6.01 (s, 4H, eCH
2 6
e). C NMR (75 MHz, DMSO d ): d (ppm)
4
.78% N; found 61.25% C, 6.62% H, 4.85% N.
,3-Bis(isoquinolinium)-2-oxaprop-1,3-diyl dichloride (15). M.p.
150.25, 137.12, 136.95, 135.25, 134.68, 131.29, 130.51, 129.59, 127.26,
2þ
1
127.20, 126.23, 62.60. ESI-MS: m/z 361.9 [M ] (calculated for
ꢁ
1
2þ
158e160 C. H NMR (300 MHz, DMSO d
6
):
d
(ppm) 9.85 (s, 2H, H-
[C26
H
22
N
2
] 362.18). EA: calculated 59.79% C, 4.25% H, 5.36% N;
0
0
0
0
1,1 ), 8.63 (d, 2H, J ¼ 6.7 Hz, H-3,3 ), 8.32e8.27 (m, 4H, H-4,4 ,8,8 ),
8
found 59.58% C, 4.34% H, 5.50% N.
0
0
0
.20e8.14 (m, 2H, H-7,7 ), 8.07 (d, 2H, J ¼ 7.9 Hz, H-5,5 ), 8.01e7.94
1,1 -Bis(isoquinolinium)-naphtyl-3,6-dimethylenyl dibromide
0
13
ꢁ
1
(
m, 2H, H-6,6 ), 6.49 (s, 4H, eCH
2
eO). C NMR (75 MHz, DMSO d
6
):
(22). M.p. 241e243 C. H NMR (300 MHz, DMSO d
6
):
d (ppm)
0
0
d
(ppm) 148.29, 138.32, 137.79, 131.89, 131.73, 130.00, 126.90,
10.21 (s, 2H, H-1,1 ), 8.77 (d, 2H, J ¼ 6.4 Hz, H-3,3 ), 8.57e8.47 (m,
2þ
0
0
0
0
0
126.48, 126.34, 87.85. ESI-MS: m/z 302.0 [M ] (calculated for
4H, H-4,4 ,8,8 ), 8.33e8.21 (m, 6H, H-5,5 ,7,7 , Nph), 8.11e7.97 (m,
2
þ
13
[C
20
18
H N
2
O
] 302.14). EA: calculated 64.35% C, 4.86% H, 7.50% N;
found 63.91% C, 5.20% H, 7.08% N.
,5-Bis(isoquinolinium)-3-oxapent-1,5-diyl dibromide (16). M.p.
4H, H-6,6 , Nph) 7.71 (d, J ¼ 7.5 Hz, Nph), 6.20 (s, 4H, eCH
2
e).
C
6
NMR (75 MHz, DMSO d ): d (ppm) 151.35, 139.24, 138.75, 135.76,
1
135.15, 134.65, 133.48, 132.83, 131.89, 130.69, 130.56, 129.34,
ꢁ
1
2þ
2
03e205 C. H NMR (300 MHz, DMSO, d
6
):
d
(ppm) 9.39 (s, 2H, H-1,
128.65, 128.10, 127.91, 65.48. ESI-MS: m/z 412.0 [M ] (calculated
0
0
0
2þ
1
2
), 8.26 (d, 2H, J ¼ 6.7 Hz, H-3,3 ), 8.14e8.06 (m, 2H, H-4,4 ), 8.02 (d,
for [C30
H
24
N
2
] 412.19). EA: calculated 62.96% C, 4.23% H, 4.89% N;
0
0
0
H, J ¼ 7.2 Hz, H-8,8 ), 7.97e7.89 (m, 4H, H-5,5 ,7,7 ), 7.88e7.81 (m,
found 62.59% C, 4.30% H, 4.63% N.
0
2
H, H-6,6 ), 4.88e4.82 (m, 4H, eCH
2
eO), 4.19e4.12 (m, 4H,
13
NeCH
2
e). C NMR (75 MHz, DMSO d
6
):
d
(ppm) 148.69, 137.30,
6.3. In vitro reactivation assay
1
36.62, 133.28, 131.41, 129.35, 126.96, 126.37, 125.46, 67.58, 60.15.
2
þ
2þ
ESI-MS: m/z 329.1 [M ] (calculated for [C22
calculated 53.90% C, 4.52% H, 5.71% N; found 53.98% C, 4.94% H,
.47% N.
,4-Bis(isoquinolinium)-but-(2E)-ene-1,4-diyl dibromide (17). M.p.
H
22
N
2
O
] 330.17). EA:
Multichannel spectrophotometer Sunrise (Tecan, Salzburg,
Austria) was used for all measurements of cholinesterases activity.
ꢁ
5
Standard conditions for temperature and pressure (SATP; 25 C,
1
100 kPa) were used. Previously optimized Ellman’s procedure was
slightly adopted in order to estimate anticholinergic properties [26].
96-wells photometric microplates made from polystyrene (Nunc,
Rockilde, Denmark) were used for measuring purposes. Human
ꢁ
1
275e277 C. H NMR (300 MHz, DMSO d
6
):
d
(ppm) 10.17 (s, 2H, H-
0
0
0
1
,1 ), 8.78 (d, 2H, J ¼ 6.7 Hz, H-3,3 ), 8.63 (d, 2H, J ¼ 6.7 Hz, H-4,4 ), 8.24
0
0
(d, 2H, J ¼ 7.9 Hz, H-8,8 ), 8.37 (d, 2H, J ¼ 7.9 Hz, H-5,5 ), 8.28 (t, 2H,

K. Musilek et al. / European Journal of Medicinal Chemistry 46 (2011) 811e818
817
erythrocyte AChE or human plasmatic BChE (Aldrich; commercially
purified byaffinity chromatography) was suspended into phosphate
(40.09 kcal/mol) were set up. Docking calculations were set to
50 runs. At the end of calculation, Autodock performed cluster
analysis.
buffer (pH 7.4) up to final activity 0.002 U/
cholinesterase (5 l) was dissolved in buffer (20
mixed solution of 0.4 mg/ml 5,5 -dithio-bis(2-nitrobenzoic) acid
ml. Protein solution of
m
ml) and freshly
The structure of mus musculus AChE and human BChE was
prepared using Pymol 1.1 from crystal structure (pdb code 2gyv and
1p0i) using Autodock Tools 1.5.2 [36e38]. The 3D affinity grid box
was designed to include the full active and peripheral site of AChE.
The number of grid points in the x-, y- and z-axes was 110, 110 and
110 with grid points separated by 0.253 Å. The molecular models of
ligands were built using ChemDraw 11.1 and minimized with UCSF
Chimera 1.3 (Amber Force field) in charged form [36]. The
maximum root mean square tolerance for conformational cluster
analysis was 2.0 Å. The visualization of enzymeeligand interactions
(Figs. 5 and 6) was prepared using Pymol 1.1 [37].
0
(
DTNB; 20
ml) was added. Appropriate concentration of inhibitor
(
10 mMe1 nM; 5
m
l) was injected per well and mixture was incu-
bated for 5 min 1 mM acetylthiocholine chloride (ATChCl) in
phosphate buffer (20 l) was added. After additional 5 min incu-
m
bation, absorbance was measured at 412 nm using automatic
shaking of the microplate.
Percentage of inhibition (I) was calculated from the measured
data as follows:
ꢀ
ꢁ
D
Ai
I ¼ 1 ꢀ D
ꢂ 100
A0
Conflict of interest
Authors declare that there are no conflicts of interest.
DA
i
indicates absorbance change provided by cholinesterase
exposed to anticholinergic compound. indicates absorbance
DA
0
Acknowledgements
change caused by intact cholinesterase, where phosphate buffer
was applied in the same way as the anticholinergic compound.
IC50 was determined using Origin 6.1 (Northampton, MA, USA).
Percentage of inhibition was calculated by Hill plot (n ¼ 1). The
other plot variants were not optimal and the correlation coefficient
was lower compared to chosen method. Subsequently, IC50 was
computed.
This work was supported by the Grant Agency of the Czech
Republic No. 203/09/P130 and by the Grant Agency of the Charles
University No. 117909/2009/B-CH/FaF.
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