The preparation, in vitro screening and molecular docking of symmetrical bisquaternary cholinesterase inhibitors containing a but-(2E)-en-1,4-diyl connecting linkage

Article abstract of DOI:10.3109/14756366.2010.496362

Carbamate inhibitors (e.g. pyridostigmine bromide) are used as a pre-treatment for the prevention of organophosphorus poisoning. They work by blocking the native function of acetylcholinesterases (AChE) and thus protect AChE against irreversible inhibitio

Full text of DOI:10.3109/14756366.2010.496362

Journal of Enzyme Inhibition and Medicinal Chemistry, 2011; 26(2): 245–253
© 2011 Informa UK, Ltd.
ISSN 1475-6366 print/ISSN 1475-6374 online
DOI: 10.3109/14756366.2010.496362
rESEarCH artICLE
e preparation, in vitro screening and molecular docking
of symmetrical bisquaternary cholinesterase inhibitors
containing a but-(2E)-en-1,4-diyl connecting linkage
Kamil Musilek1,4, Ruzena Pavlikova1, Jan Marek1, Marketa Komloova2, Ondrej Holas2,
Martina Hrabinova3, Miroslav Pohanka3, Vlastimil Dohnal4, Martin Dolezal2, Frank Gunn-Moore5,
and Kamil Kuca3,4
1University of Defence, Faculty of Military Health Sciences, Department of Toxicology, Trebesska 1575, Hradec Kralove,
Czech Republic, 2Charles University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry and Drug Control,
Heyrovskeho 1203, Hradec Kralove, Czech Republic, 3University of Defence, Faculty of Military Health Sciences, Centre
of Advanced Studies, Trebesska 1575, Hradec Kralove, Czech Republic, 4University of Jan Evangelista Purkynje, Faculty
of Science, Department of Chemistry, Ceske mladeze 8, Usti nad Labem, Czech Republic, and 5University of St. Andrews,
School of Biology, Bute Building, St. Andrews, Fife, UK
abstract
Carbamateinhibitors(e.g.pyridostigminebromide)areusedasapre-treatmentforthepreventionoforganophosphorus
poisoning. They work by blocking the native function of acetylcholinesterases (AChE) and thus protect AChE against
irreversible inhibition by organophosphorus compounds. However, carbamate inhibitors are known for their many
undesirable side effects related to the carbamylation of AChE. In this paper, we describe 17 novel bisquaternary
compounds and have analysed their effect on AChE inhibition. The newly prepared compounds were evaluated in
vitro using both human erythrocyte AChE and human plasmatic butyrylcholinesterase. Their inhibitory ability was
expressed as the half maximal inhibitory concentration (IC50) and then compared to the standard carbamate drugs
and two AChE reactivators. One of these novel compounds showed promising AChE inhibition in vitro (nM range) and
was better than the currently used standards. Additionally, a kinetic assay confirmed the non-competitive inhibition
of hAChE by this novel compound. Consequently, the docking results confirmed the apparent π-π or π-cationic
interactions with the key amino acid residues of hAChE and the binding of the chosen compound at the enzyme
catalytic site.
Keywords: Cholinesterase, inhibitor, pre-treatment, myasthenia gravis, quaternary, in vitro, docking
Introduction
accumulates on the synaptic junctions and this excess
Acetylcholinesterase (AChE; E.C. 3.1.1.7) inhibitors
are compounds which have been of interest for several
decades. ey are used for the treatment of neurodegen-
erative diseases such as Alzheimer’s disease or myasthe-
nia gravis [1–2]. In addition, some AChE inhibitors are
used as pre-exposure drugs to protect against organo-
phosphorus poisonings, such as nerve agents [3].
Generally, the organophosphorus (OP) compounds (e.g.
nerve agents and pesticides) are lethal, causing irrevers-
ible inhibition of AChE [4]. Consequently, acetylcholine
causes permanent stimulation of the muscarinic or nico-
tinic receptors. Gradually, the stimulation of the receptors
leads to a cholinergic crisis and death by malfunction of
the muscles associated with breathing control [5]. Among
the OP compounds, nerve agents are among the most
toxic compounds that have been developed by man. ere
are several methods to counteract the toxicity of nerve
agents [5] either involving a pre-exposure or post-exposure
approach. Reversible AChE inhibitors, stoichiometric and
catalytic scavengers can be used as a pre-exposure strategy
Address for Correspondence: Kamil Musilek, Department of Toxicology, Faculty of Military Health Sciences, Trebesska 1575, Hradec
Kralove, Czech Republic. E-mail: musilek@pmfhk.cz; Tel: +420-973-251-523; Fax: +420-495-518-094
(Received 08 February 2010; revised 18 May 2010; accepted 24 May 2010)
245

246 Kamil Musilek et al.
[6], whilst a post-exposure treatment usually consists of a
combination of an anticholinergic drug, AChE reactivator
and an anticonvulsant such as diazepam [5].
K203; 3–4), especially those bearing the same but-
(2E)-en-1,4-diyl linkage (4), that had been previously
found to protect against OP exposure both in vitro and
in vivo [13–15]. Essentially, the core part of the newly
prepared molecules remained consistent with former
With regard to the pre-exposure strategies, the only
method currently allowed for human use is based on
reversible AChE inhibitors (usually carbamates, e.g. pyri-
dostigmine bromide, neostigmine bromide; 1–2; Figure
1) in combination with anticholinergic drugs (e.g. ben-
actyzine, trihexyphenidyl) to antagonise the effects of the
accumulated acetylcholine [7]. e carbamates reversibly
inhibit AChE, protect the enzyme against inhibition by
OP and are spontaneously decarbamylated with normal
restoration of AChE activity [8]. However, although car-
bamates are widely used, they are known for many side
effects including gastrointestinal effects (nausea, intesti-
nal obstruction), increased bronchial secretion, cardiac
arrhythmia or a cholinergic crisis [9–10].
AChE reactivators including
a but-(2E)-en-1,4-diyl
linkage and two pyridinium rings. e oxime moieties
on both pyridinium rings were changed and a broad
variety of functional groups were added. e functional
groups were chosen as representatives of lipophilic
(Me, Ph) or hydrophilic (OH, COOH) moieties to assess
the potential interactions with the enzyme’s active site
[16]. Specifically these were placed on the 4-position
on the pyridinium ring as this was the most suitable for
potential interactions within the narrow AChE cleft and
so would hypothetically increase its inhibitory ability
towards AChE [17].
e most widely used carbamate for OP pre-treatment
is pyridostigmine (1) [7]. Its charged molecule has poor
penetration of the blood-brain barrier (BBB) and probably
cannot protect brain AChE, but the pre-treatment effect is
ensured by use of anticholinergic drugs (benactyzine, tri-
hexyphenidyl) [11]. On account of its serious side effects,
a carbamate inhibitor 1 in combined pre-treatment strat-
egies, can be replaced by other reversible inhibitors (e.g.
hupezine A) [7]. e advantage of such reversible cho-
linesterase inhibitors should be its selectivity for AChE,
where butyrylcholinesterase (BChE; EC 3.1.1.8) can act
as a physiological scavenger of OP. Decreasing the side
effects of a new reversible inhibitor would also be an
advantage if it does not directly affect the AChE active site
(S203). However, there remains the problem of BBB pen-
etration that can be overcome by anticholinergic drugs
(benactyzine, trihexyphenidyl) thereby without increas-
ing toxicity from the reversible AChE inhibitor.
Due to these necessary improvements for protect-
ing against OP, a series of symmetrical bisquaternary
compounds bearing (2E)-buten-1,4-diyl linkage were
synthesised to potentially replace pyridostigmine for
the combined pre-exposure strategy. e quaternary
compounds (e.g. AChE reactivators) are also known to
be receptor acting drugs [12]. Many of them were found
to be potent activators, competitive antagonists or non-
competitive blockers of muscarinic or nicotinic receptors.
However, their effects on receptors cannot be accurately
predicted and the quaternary origin of these novel com-
pounds might help to increase protection against OP [12].
e design of novel compounds (5–21; Figure 2)
originated from AChE reactivators (e.g. pralidoxime,
All the new compounds were prepared using a stan-
dard synthetic strategy [18]. e solution containing the
pyridine derivative (4.7 mmol) and 1,4-dibromobut-(2E)-
ene (2.3 mmol) in dimethylformamide (DMF) (10ml)
was stirred at 70°C for 2 to 48 hours. Subsequently, the
reaction mixture was cooled to room temperature. It was
then portioned with acetone (50ml) and again cooled in a
refrigerator (5°C) overnight. e crystalline or amorphous
crude product was collected by filtration, washed with ace-
tone (3×20ml) and re-crystallised from MeCN [19]. NMR,
ESI-MS and elemental analysis were used to determine the
purity of all the compounds.
material and methods
Chemistry
Solvents (acetone, DMF, MeCN) and reagents were
purchased from Fluka (Prague, Czech Republic) and
Sigma-Aldrich (Prague, Czech Republic) and used with-
out further purification. Reactions were monitored by
TLC using DC-Alufolien Cellulose F (Darmstadt, Merck,
Germany) and a mobile phase of BuOH-CH3COOH-
H2O 5:1:2, detection by solution of Dragendorff reagent
(solution containing 10 mL CH3COOH, 50 mL H2O and
5 mL of basic solution prepared by mixing of two frac-
tions – fraction A: 850 mg Bi(NO3)3, 40 mL H2O, 10 mL
CH3COOH; fraction B: 8 g KI, 20 mL H2O). Melting points
were measured on a micro heating stage PHMK 05 (VEB
Kombinat Nagema, Radebeul, Germany) and were
uncorrected.
NMR spectra were generally recorded using a Varian
Gemini 300 (1H 300 MHz, 13C 75 MHz, Palo Alto CA, USA).
O
NH2
N
Br
Cl
Br
N
N
N
O
N
O
NOH
HON
2 Br
O
N
O
N
neostigmine(2)
pyridostigmine(1)
pralidoxime(3)
K203(4)
Figure 1. Selected acetylcholinesterase inhibitors and reactivators.
Journal of Enzyme Inhibition and Medicinal Chemistry

Symmetrical bisquaternary cholinesterase inhibitors with but-(2E)-en-1,4-diyl linker 247
R
EA: calculated 48.02% C, 5.04% H, 7% N; found 48.44% C,
4.87% H, 5.24% N.
Br
N
R
Br
N
N
DMF;70°C
2 Br
but-(2E)-en-1,4-diyl-1,1’-bis(4-ethylpyridinium) dibromide (6)
Mp 227–228°C. Yield 49%. 1H NMR (300 MHz, DMSO d6):
δ (ppm) 8.55 (d, 4H, J = 6.2 Hz, H–2,2’,6,6’), 7.96 (d, 4H, J
= 6.2 Hz, H–3,3’,5,5’), 6.28–6.24 (m, 2H, =CH-), 5.30–5.25
(m, 4H, -CH2-), 2.98 (m, 4H, -CH2-CH3), 1.31 (t, 6H, J = 7.4
Hz, -CH3). 13C NMR (75 MHz, DMSO d6): δ (ppm) 142.78,
129.31, 127.08, 60.23, 27.93, 12.15. ESI-MS: m/z 268.1
[M2+] (calculated for [C18H24N22+] 268.19). EA: calculated
50.49% C, 5.65% H, 6.54% N; found 50.12% C, 5.56% H,
6.76% N.
R
5, R = Me
6, R = Et
7, R = tert.-Bu
8, R = Ph
9, R = Bn
10, R = (CH2)3Ph
11, R = 4-NO2-Bn
12, R = OH
but-(2E)-en-1,4-diyl-1,1’-bis(4-tert-butylpyridinium)
dibromide (7)
13, R = CH2OH
14, R = (CH2)3OH
15, R = N(CH3)2
16, R = CH=NOH
17, R = COCH3
18, R = COOH
19, R = COOCH3
20, R = CONH2
21, R = CN
Mp 235–237°C. Yield 66%. 1H NMR (300 MHz, DMSO d6):
δ (ppm) 8.74 (d, 4H, J = 5.8 Hz, H–2,2’,6,6’), 8.12 (d, 4H, J
= 5.8 Hz, H–3,3’,5,5’), 6.29–6.25 (m, 2H, =CH-), 5.31–5.27
(m, 4H, -CH2-), 1.39 (s, 18H, CH3-). 13C NMR (75 MHz,
DMSO d6): δ (ppm) 142.82, 129.33, 125.02, 60.07, 35.46,
28.55. ESI-MS: m/z 324.1 [M2+] (calculated for [C22H32N2
]
2+
324.26). EA: calculated 54.56% C, 6.66% H, 5.78% N; found
54.3% C, 6.76% H, 5.84% N.
but-(2E)-en-1,4-diyl-1,1’-bis(4-phenylpyridinium)
dibromide (8)
Mp 259–261°C. Yield 55%. 1H NMR (300 MHz, DMSO d6):
δ (ppm) 8.78–8.65 (m, 4H, H–2,2’,6,6’), 8.31–8.17 (m, 4H,
H–3,3’,5,5’), 7.94–7.79 (m, 4H, Ph–2,2’,6,6’), 7.63–7.44 (m,
6H, Ph–3,3’,4,4’,5,5’), 6.23–6.12 (m, 2H, =CH-), 5.23–5.11
(m, 4H, -CH2-). 13C NMR (75 MHz, DMSO d6): δ (ppm)
157.65, 157.63, 145.52, 134.73, 133.86, 131.25, 129.37,
126.19, 61.98. ESI-MS: m/z 364.2 [M2+] (calculated for
[C26H24N22+] 364.19). EA: calculated 59.56% C, 4.61% H,
5.34% N; found 59.85% C, 4.4% H, 4.9% N.
Figure 2. Prepared bisquaternary pyridinium salts.
In all cases, the chemical shift values for 1H spectra were
reported in ppm (δ) relative to residual CHD2SO2CD3
(δ 2.5) or D2O (δ 4.79), shift values for 13C spectra are
reported in ppm (δ) relative to solvent peak dimethylsul-
phoxide - d6 δ 39.43. Signals are quoted as s (singlet), d
(doublet), t (triplet) and m (multiplet).
e mass spectra (MS respectively MSn) were mea-
sured on a LCQ FLEET ion trap and evaluated using
Xcalibur v 2.5.0 software (both ermo Fisher Scientific,
San Jose, CA, USA). e sample was dissolved in
deionised water (Goro, Prague, Czech Republic) and
injected continuously (8 ul/ min) by Hamilton syringe
into the electrospray ion source. e parameters for the
electrospray were set up as follows: sheath gas flow rate
20 arbitrary units, aux gas flow rate 5 arbitrary units,
sweep gas flow rate 0 arbitrary units, spray voltage 5 kV,
capillary temperature 275°C, capillary voltage 13 V, tube
lens 100 V.
but-(2E)-en-1,4-diyl-1,1’-bis(4-phenylmethylpyridinium)
dibromide (9)
Mp 212–214°C. Yield 4%. 1H NMR (300 MHz, DMSO
d6): δ (ppm) 9.32 (d, 2H, J = 6.1 Hz, H–2,6), 8.98 (d,
2H, J = 6.1 Hz, H–2’,6’), 8.41 (d, 2H, J = 6.1 Hz, H–3,5),
8.08 (d, 2H, J = 6.1 Hz, H–3’,5’), 7.94–7.77 (m, 3H, Ph),
7.72–7.58 (m, 2H, Ph), 7.44–7.23 (m, 5H, Ph), 6.43–6.12
(m, 2H, =CH-), 5.58–5.19 (m, 4H, N-CH2-), 4.3 (s, 4H,
Ph-CH2-). 13C NMR (75 MHz, DMSO d6): δ (ppm) 151.98,
145.85, 144.48, 137.5, 134.84, 134.04, 130.24, 130.09,
129.97, 129.11, 129.05, 128.88, 127.67, 127.03, 126.87,
60.99, 60.13. ESI-MS: m/z 391 [M2+-H] (calculated for
[C28H28N22+-H] 391.22). EA: calculated 60.89% C, 5.11%
H, 5.07% N; found 60.28% C, 5.57% H, 5.67% N.
Prepared bisquaternary salts
but-(2E)-en-1,4-diyl-1,1’-bis(4-methylpyridinium) dibromide (5)
1
Mp 256–257°C. Yield 54%. H NMR (300MHz, DMSO d6):
but-(2E)-en-1,4-diyl-1,1’-bis(4-(3-phenylpropyl)-pyridinium)
dibromide (10)
δ (ppm) 8.71–8.67 (m, 4H, H–2,2,’6,6’), 8.08–8.04 (m, 4H,
H–3,3,’5,5’), 6.17–6.12 (m, 2H, =CH-), 5.17–5.14 (m, 4H,
-CH2-), 3.65 (s, 6H, N-CH3). 13C NMR (75MHz, DMSO
d6): δ (ppm) 150.26, 147.04, 145.84, 131.25, 126.01, 62.45.
ESI-MS: m/z239.9 [M2+] (calculated for [C16H20N22+]240.16).
Mp 204–206°C. Yield 43%. 1H NMR (300 MHz, DMSO d6):
δ (ppm) 8.58 (d, 4H, J = 6.1 Hz, H–2,2’,6,6’), 7.79 (d, 4H, J =
6.1 Hz, H–3,3’,5,5’), 7.27–7.11 (m, 10H, Ph), 6.18–6.14 (m,
2H, =CH-), 5.23–5.19 (m, 4H, -CH2-), 2.89 (t, 4H, J = 7.3
© 2011 Informa UK, Ltd.

248 Kamil Musilek et al.
Hz, Pyr-CH2-), 2.63 (t, 4H, J = 7.3 Hz, Ph-CH2-), 2.06–1.94
(m, 4H, Pyr-CH2-CH2-). 13C NMR (75 MHz, DMSO d6):
δ (ppm) 142.7, 141.24, 129.39, 128.28, 128.08, 127.72,
125.65, 60.3, 34.22, 33.94, 29.79. ESI-MS: m/z 447.9 [M2+]
(calculated for [C32H36N22+] 448.29). EA: calculated 63.17%
C, 5.96% H, 4.6% N; found 63.16% C, 6.22% H, 4.61% N.
6.82 (d, 4H, J = 6.7 Hz, H–3,3’,5,5’), 5.88–5.84 (m, 2H,
=CH-), 4.7–4.66 (m, 4H, -CH2-), 3.07 (s, 12H, N-CH3).
13C NMR (75 MHz, DMSO d6): δ (ppm) 157.08, 142.53,
130.57, 108.77, 58.57, 40.75. ESI-MS: m/z 297.9 [M2+]
(calculated for [C18H26N42+] 298.21). EA: calculated
47.18% C, 5.72% H, 12.23% N; found 47.12% C, 6.04%
H, 12.15% N.
but-(2E)-en-1,4-diyl-1,1’-bis(4-(4-nitrobenzyl)-pyridinium)
dibromide (11)
but-(2E)-en-1,4-diyl-1,1’-bis(4-
1
Mp 248–249°C. Yield 81 %. H NMR (300 MHz, DMSO
hydroxyiminomethylpyridinium) dibromide (16)
Mp 232–234°C. Yield 95%. Further physic-chemical data
are consistent with the literature [20].
d6): δ (ppm) 8.62 (d, 4H, J = 6.1 Hz, H–2,2’,6,6’), 7.99–7.89
(m, 4H, H–3,3’,5,5’), 7.79 (d, 4H, J = 7 Hz, Ph–2,2’,6,6’),
7.37 (d, 4H, J = 7 Hz, Ph–3,3’,5,5’), 6.1–6.04 (m, 2H,
=CH-), 5.18–5.03 (m, 4H, N-CH2-), 4.28 (s, 4H, Ph-CH2-).
13C NMR (75 MHz, DMSO d6): δ (ppm) 147.85, 146.03,
but-(2E)-en-1,4-diyl-1,1’-bis(4-methylcarbonylpyridinium)
dibromide (17)
Mp 228–230°C. Yield 56 %. 1H NMR (300 MHz, DMSO d6):
δ (ppm) 9.36 (d, 4H, J= 5.6 Hz, H–2,2’,6,6’), 8.54 (d, 4H,
J= 5.8 Hz, H–3,3’,5,5’), 6.32–6.26 (m, 2H, =CH-), 5.53–5.47
(m, 4H, -CH2-), 2.76 (s, 6H, -CH3). 13C NMR (75 MHz,
DMSO d6): δ (ppm) 195.67, 148.63, 146.39, 130.21, 125.92,
60.87, 27.49. ESI-MS: m/z 296.0 [M2+] (calculated for
[C18H20N2O22+] 296.15). EA: calculated 47.39% C, 4.42% H,
6.14% N; found 47.51% C, 4.66% H, 5.97% N.
145.34, 131.82, 131.19, 129.63, 125.34, 62.22, 41.49.
ESI-MS: m/z 482.3 [M2+] (calculated for [C28H26N4O4
]
2+
482.19). EA: calculated 52.36% C, 4.08% H, 8.72% N;
found 52.53% C, 4.37% H, 8.36% N.
but-(2E)-en-1,4-diyl-1,1’-bis(4-hydroxypyridinium) dibromide
(12)
1
Mp 258–260°C. Yield 53 %. H NMR (300 MHz, DMSO
d6): δ (ppm) 8.48 (d, 4H, J = 6.4 Hz, H–2,2’,6,6’), 7.24
(d, 4H, J = 6.4 Hz, H–3,3’,5,5’), 6.24–6.12 (m, 2H, =CH-),
5.12–5.03 (m, 4H, -CH2-). 13C NMR (75 MHz, DMSO
d6): δ (ppm) 144.6, 128.96, 114.34, 58.81. ESI-MS: m/z
244.1 [M2+] (calculated for [C14H16N2O22+] 244.12). EA:
calculated 41.61% C, 3.99% H, 6.93% N; found 41.45% C,
4.35% H, 7.07% N.
but-(2E)-en-1,4-diyl-1,1’-bis(4-carboxypyridinium) dibromide
(18)
Mp 274–276°C. Yield 35 %. 1H NMR (300 MHz, DMSO d6):
δ (ppm) 9.27 (d, 4H, J= 5.9 Hz, H–2,2’,6,6’), 8.51 (d, 4H,
J= 5.9 Hz, H–3,3’,5,5’), 6.27–6.22 (m, 2H, =CH-), 5.5–5.45
(m, 4H, -CH2-). 13C NMR (75 MHz, DMSO d6): δ (ppm)
146.3, 145.61, 130.18, 127.31, 61.03. ESI-MS: m/z 299.7
[M2+] (calculated for [C16H16N2O42+] 300.11). EA: calcu-
lated 41.77% C, 3.5% H, 6.09% N; found 41.44% C, 3.8%
H, 6.29% N.
but-(2E)-en-1,4-diyl-1,1’-bis(4-hydroxymethylpyridinium)
dibromide (13)
1
Mp 210–212°C. Yield 48 %. H NMR (300 MHz, D2O d6):
δ (ppm) 8.76 (d, 4H, J= 5.5 Hz, H–2,2’,6,6’), 8.03 (d, 4H,
J= 5.8 Hz, H–3,3’,5,5’), 6.27–6.22 (m, 2H, =CH-), 5.33–5.27
(m, 4H, -CH2-), 4.96 (s, 4H, -CH2-OH). 13C NMR (75 MHz,
D2O): δ (ppm) 162.11, 143.39, 129.5, 124.42, 61.1, 60.65.
ESI-MS: m/z 271 [M2+-H] (calculated for [C16H20N2O22+-H]
271.15.). EA: calculated 44.47% C, 4.66% H, 6.48% N;
found 44.62% C, 4.9% H, 6.3% N.
but-(2E)-en-1,4-diyl-1,1’-bis(4-methyloxycarbonylpyridinium)
dibromide (19)
Mp 179–181°C. Yield 80 %. 1H NMR (300 MHz, DMSO d6):
δ (ppm) 9.33 (d, 4H, J= 5.8 Hz, H–2,2’,6,6’), 8.54 (d, 4H,
J= 5.6 Hz, H–3,3’,5,5’), 6.29–6.24 (m, 2H, =CH-), 5.54–5.47
(m, 4H, N-CH2-), 4.45 (q, 4H, J= 7 Hz, O-CH2-), 1.37 (t,
6H, J= 7 Hz, O-CH2-CH3). 13C NMR (75 MHz, DMSO d6): δ
but-(2E)-en-1,4-diyl-1,1’-bis(4-(3-hydroxypropyl)pyridinium)
dibromide (14)
(ppm) 161.95, 146.43, 144.12, 130.18, 127.15, 62.93, 61.07,
13.81. ESI-MS: m/z 328 [M2+] (calculated for [C18H20N2O4
]
2+
Mp 171–173°C. Yield 69 %. 1H NMR (300 MHz, DMSO d6):
δ (ppm) 8.96 (d, 4H, J= 5.8 Hz, H–2,2’,6,6’), 8.06 (d, 4H,
J= 5.8 Hz, H–3,3’,5,5’), 6.21–6.16 (m, 2H, =CH-), 5.32–5.28
(m, 4H, -CH2-), 3.43 (t, 4H, J= 6.2 Hz, -CH2-OH), 2.9 (t,
4H, J= 7.6 Hz, -CH2-N), 1.81 (m, 4H, -CH2-CH2-N). 13C
NMR (75 MHz, DMSO d6): δ (ppm) 143.98, 130.07, 127.7,
59.94, 59.53. ESI-MS: m/z 327.9 [M2+] (calculated for
[C20H28N2O22+] 328.21). EA: calculated 49.2% C, 5.78% H,
5.74% N; found 48.87% C, 6% H, 6.01% N.
328.14). EA: calculated 44.29% C, 4.13% H, 5.74% N; found
44.45% C, 3.93% H, 5.55% N.
but-(2E)-en-1,4-diyl-1,1’-bis(4-carbamoylpyridinium)
dibromide (20)
1
Mp 268–270°C. Yield 90 %. H NMR (300 MHz, DMSO
d6): δ (ppm) 9.28 (d, 4H, J = 5.5 Hz, H–2,2’,6,6’), 8.75 (s,
2H, -CONH2), 8.48 (d, 4H, J = 5.6 Hz, H–3,3’,5,5’), 8.31 (s,
2H, -CONH2), 6.28–6.21 (m, 2H, =CH-), 5.48–5.39 (m, 4H,
-CH2-). 13C NMR (75 MHz, DMSO d6): δ (ppm) 163.19,
148.39, 145.89, 130.1, 125.86, 60.82. ESI-MS: m/z 298
[M2+] (calculated for [C16H18N4O22+] 298.14). EA: calcu-
lated 41.95% C, 3.96% H, 12.23% N; found 41.85% C, 3.8%
H, 12.32% N.
but-(2E)-en-1,4-diyl-1,1’-bis(4-(N,N-dimethylamino)-
pyridinium) dibromide (15)
Mp decomp 314°C. Yield 68 %. 1H NMR (300 MHz,
DMSO d6): δ (ppm) 7.94 (d, 4H, J = 6.7 Hz, H–2,2’,6,6’),
Journal of Enzyme Inhibition and Medicinal Chemistry

Symmetrical bisquaternary cholinesterase inhibitors with but-(2E)-en-1,4-diyl linker 249
but-(2E)-en-1,4-diyl-1,1’-bis(4-carbonitrilpyridinium)
dibromide (21)
concentration (0.1 mM–1M) of ATChCl in PBS (100 μL).
e yellow colour produced by the 5-thio-2-nitrobenzoic
acid was measured at 412nm against a control blank (mix-
ture of DTNB and ATChCl in given concentrations). e
spontaneous interaction between the tested inhibitor and
DTNB was excluded after incubation of the whole reaction
mixture without AChE that was replaced by PBS (25 μL).
e inhibition was evaluated by a Lineweaver-Burk plot for
all inhibitor concentrations (10−8–10−2 mol/L) and ATChCl
concentrations (10−5–10−1 mol/L). e measurements were
carried out in triplicate and the average value was used for
the plot construction.
Mp 251–253°C. Yield 46 %. 1H NMR (300 MHz, DMSO d6):
δ (ppm) 9.02 (d, 4H, J = 6.1 Hz, H–2,2’,6,6’), 8.38 (d, 4H,
J = 6.1 Hz, H–3,3’,5,5’), 6.25–6.21 (m, 2H, =CH-), 5.32–5.28
(m, 4H, -CH2-). 13C NMR (75 MHz, DMSO d6): δ (ppm)
147.17, 132.54, 131.39, 129.63, 115.56, 63.78. ESI-MS:
m/z 262 [M2+] (calculated for [C16H14N42+] 262.12). EA:
calculated 45.53% C, 3.34% H, 13.27% N; found 45.63% C,
3.26% H, 13.24% N.
In vitro assay
A multichannel Sunrise spectrophotometer (Tecan,
Salzburg, Austria) was used for all measurements of
cholinesterases activity. An optimised Ellman’s proce-
dure was slightly adapted in order to estimate the anti-
cholinergic properties [21]. Some 96-well photometric
microplates made from polystyrene (Nunc, Rockilde,
Denmark) were used for measuring purposes. Human
erythrocyte AChE or human plasmatic BChE (Prague,
Aldrich; commercially purified by affinity chromatogra-
phy) were suspended into phosphate buffer (pH 7.4) up
to a final activity of 0.002 U/μL. Cholinesterase (5 μL),
freshly mixed solution of 0.4 mg/mL 5,5’-dithio-bis(2-
nitrobenzoic) acid (40 μL), 1 mM acetylthiocholine chlo-
ride in phosphate buffer (20 μL) and the appropriate
concentration of the inhibitor (1 mM–0.1 nM; 5 μL) were
injected into each well. e absorbance was measured
at 412 nm after 5 minutes incubation using automated
shaking of the microplate. All measurements were per-
formed in triplicate.
e obtained data were processed by Origin 8.0 soft-
ware (Northampton, MA, USA). e constants were
calculated from the enzyme kinetics using a Lineweaver-
Burk plot (double reciprocal plot). e AChE dissociation
constant for the enzyme-inhibitor complex (Ki1) and the
dissociation constant for the enzyme–inhibitor–sub-
strate complex (Ki2) were calculated using the following
equations:
E I
[ ][ ]
Ki1
=
EI
[
]
ES I
[ ][ ]
=
Ki2
ESI
[
]
Molecular docking
e docking calculations were carried out using Autodock
4.0.1 [23]. e structure of mus musculus AChE and human
AChE were prepared from the crystal structure (pdb code
2jez, 2jf0 and 1b41) using the Autodock Tools 1.5.2 [23].
e 3D affinity grid box was designed to include both the
full active and the peripheral site of AChE. e number
of grid points in the x-, y- and z-axes was 110, 110 and
110 with grid points separated by 0.253 Å. e molecular
models of ligands were built using ChemDraw 11.1 and
minimised with UCSF Chimera 1.3 (Amber Force field)
in the charged form [24]. Docking calculations were set
to 50 runs. A population of 150 individuals and 2 500 000
function evaluations were used. e structure optimisa-
tion was performed for 27 000 generations using a genetic
algorithm. e maximum root mean square tolerance for
the conformational cluster analysis was 2 Å. At the end of
calculation, the Autodock performed cluster analysis was
utilised. e visualisations of the enzyme-ligand interac-
tions (Figure 3 and Figure 4) were prepared using Pymol
1.1 [25].
e percentage inhibition (I) was calculated from the
measured data as follows:
∆A
i
I = 1−
∆A0
where ΔAi indicates the absorbance change provided
by cholinesterase exposed to an anticholinergic com-
pound. ΔA0 indicates the absorbance change caused by
the intact cholinesterase, where phosphate buffer was
applied instead of the anticholinergic compound.
e IC50 was determined using the software package
Origin 6.1 (Northampton, MA, USA). e percentage
inhibition was calculated by a Hill plot (n = 1). e other
plot variants were not optimal and their correlation coef-
ficients were lower compared to the chosen method.
Subsequently, the IC50 was computed.
Enzyme kinetics assay
Human erythrocyte AChE (Aldrich) was used throughout
the experiments. e adapted photometrical Ellman’s
method was used to evaluate the AChE activity [22]. A
polystyrene cuvette was filled with 0.4mg/ml DTNB
(0.4ml), AChE solution with an overall activity of 0.5 μkat
(μmol/s) in phosphate buffered saline (PBS; 25 μL), tested
inhibitor (25 μL), and PBS (450 μL). e mixture was gently
shaken and the reaction was started by addition of varying
results and discussion
In vitro results
e bisquaternary compounds were assayed for their
inhibitory ability using a standard inhibition test utilis-
ing human erythrocyte AChE (hAChE) and human plas-
matic BChE (hBChE) [21]. e hAChE was chosen as the
main target for the OP pre-treatment [11]. Additionally,
© 2011 Informa UK, Ltd.

250 Kamil Musilek et al.
hBChE was chosen as a member of the cholinesterase
family, which is usually affected by all the compounds
that interact with AChE [26]. e IC50 values of all com-
pounds are listed in Table I.
Among the newly prepared compounds (3–19), the
inhibitory ability towards hAChE greatly varied within
the whole series. One novel compound (7) was not sol-
uble in the screening solution and consequently could
not be evaluated. Two compounds (16, 18) showed
negligible inhibition on a mM scale. Some compounds
(4, 6, 9–12, 14–15, 17, 19) showed inhibition on a less
than mM scale, but did not exceed the standard carbam-
ate inhibitor 1. More interestingly, four compounds (3,
5, 8, 13) presented an inhibitory ability comparable (5)
or better (3, 8, 13) than the standard carbamate 1. In
addition, one newly prepared compound (8) showed
an inhibition of hAChE on a nM scale and exceeded
both carbamate inhibitors 1 and 2.
Based on the inhibition of hAChE, the commercial
compound 1 presented a satisfactory inhibition of
AChE in the µM range. In contrast, compound 2 was a
stronger inhibitor of hAChE and had an IC50 approxi-
mately three orders of magnitude lower compared to
1. From these in vitro results, compound 2 appears
to be a potentially more valuable inhibitor of hAChE
between these two tested carbamate compounds. e
AChE reactivators are also known for their inhibitory
ability towards AChE [27] as some of them were found
to be potent AChE reactivators/inhibitors in vitro [28].
However, both the tested reactivators (3, 4) presented
only a minor inhibition of hAChE that probably limits
their use in pre-exposure treatment as functional AChE
inhibitors. erefore their potential receptor mediated
protection against OP should be further studied.
For hBChE, the commercial compound 1 was found to
be a very poor inhibitor. Interestingly, a second carbam-
ate compound (2) showed a strong inhibition of hBChE
on a less than µM scale and about 5 orders of magnitude
H287
Y124
S203
W286
P338
W86
Y341
Figure 4. Penetration of compound 10 into the the hAChE active site
Figure 3. Docking results for compound 10 with hAChE.
gorge.
Table 1. e IC50 values of the tested compounds (a; no inhibition observed; b; not soluble in screening medium).
Compound
AChE IC50 SD (µM)
40 7.8
BChE IC50 SD (µM)
Ki1/Ki2 (µM)
4.94/2.52
pyridostigmine (1)
neostigmine(2)
pralidoxime (3)
K203 (4)
5
16 000 260.8
0.8 0.13
a
0.1 0.02
0.44/0.07
878 171
---
---
566 110
3 130 510
a
0.9 0.2
---
6
7
212 41
850 138
169 28
---
40
8
---
8
9
71 14
b
18
3
---
b
---
10
11
12
13
14
15
16
17
18
19
20
21
0.05 0.01
73 14
22
18
4
3
0.003/0.009
---
509 99
799 155
54 11
a
---
4 510 735
1 440 234
---
---
1.2 0.2
189 37
139 27
3 870 754
401 78
1 200 234
70 14
17
3
---
1 620 264
---
324 53
---
a
---
a
---
a
---
514 84
---
Journal of Enzyme Inhibition and Medicinal Chemistry

Symmetrical bisquaternary cholinesterase inhibitors with but-(2E)-en-1,4-diyl linker 251
better than 1. e AChE reactivators (3, 4) were negli-
display any interactions with the hAChE catalytic site
(S203) as might have been hypothesised from its chemi-
cal structure. Inhibitor 8 also showed a good penetra-
tion of the one 4-(3-phenylpropyl)-pyridinium moiety
into the hAChE active site (Figure 4).
e SAR results of the novel synthesised compounds
originated from the docking study and the in vitro data
[32]. Since the but-(2E)-en-1,4-diyl-bispyridinium struc-
ture remained the same for all the novel compounds, the
main part of the molecule influencing the SAR from the
various functional groups attached to the pyridinium
moieties were at the 4-position.
gible inhibitors of hBChE. Among the newly prepared
compounds (3–19), the inhibitory ability towards hBChE
greatly varied within the whole series. One novel com-
pound (7) was not soluble in the screening solution and
consequently could not be further evaluated. Several
novel compounds (3, 10, 16–18) showed no inhibition of
hBChE activity. ree compounds (11–12, 14) were neg-
ligible inhibitors at the mM scale and four compounds
(4–5, 15, 19) were poor inhibitors on a less than mM
scale. More interestingly, several newly prepared com-
pounds (6, 8–9, 13) showed inhibition at the µM scale.
However, the novel compounds were not able to exceed
the carbamate 2 at hBChE inhibition.
e IC50 screening method provides a complex
comparison between inhibitors because of their
different inhibition mechanisms, and so the kinet-
ics data were measured for specific compounds of
interest. e dissociation constants (Ki1 and Ki2) were
calculated for 2 carbamate standards (1, 2) and for
the most promising compound (8) interacting with
hAChE [22]. In all cases, a non-competitive mecha-
nism of inhibition was confirmed by the shape of the
Lineweaver-Burk plot. For pyridostigmine (1), the inhi-
bition constants differed approximately two fold (Ki1 =
4.94 μmol/l; Ki2 = 2.52 μmol/l), where the substrate
presence increased the inhibition of AChE (decreased
the inhibition constant). e kinetic constants for neo-
stigmine (2) differed about six fold (Ki1 = 0.44 μmol/l;
Ki2 = 0.07 μmol/l) and the presence of the substrate
again increased the inhibition of AChE. e inhibition
constants of compound 8 (Ki1 = 0.003 μmol/l; Ki2 = 0.009
μmol/l) differed only slightly, where the presence of the
substrate decreased the inhibitory ability of compound
8 towards AChE. Importantly, both Ki values of com-
pound 8 were found to be at least one order of magni-
tude lower compared to the tested standards (1, 2).
For hAChE, the lipophilic moieties (3–9) suggested
that they are very promising inhibitors. The aliphatic
functional groups (3–5) showed interesting differences
in the inhibitory ability, where the methyl moiety (3)
was superior to tert.-butyl (5) or the ethyl (4). Among
the aromatic functional groups (6–9), 3-phenylpropyl
compound (8) was found to be the best inhibitor
from the prepared series, superior to phenyl (6) or
4-nitrobenzyl (9), on account of the valuable π-π or
cation-π interactions with the aromatic residues of
AChE (i.e. His, Phe, Trp, Tyr) [33]. The benzyl com-
pound (7) remained insoluble, but introduction of a
hydrophilic 4-nitro moiety, resulting in 4-nitrobenzyl
(9), showed good solubility and inhibitory ability
to the same level as the phenyl compound (6). The
hydrophilic moieties (10–19) also showed apparent
differences in their inhibitory ability. Among them,
3-hydroxypropyl (12), N,N-dimethylamino (13) and
4-carbonitril (19) were superior to the other hydroxy
(10, 11) and carbonyl compounds (14–18). Though
the N,N-dimethylamino compound (13) tested as the
best hydrophilic moiety, its inhibitory ability was still
two orders of magnitude lower as compared to the
3-phenylpropyl compound (8).
For hBChE, the SAR extensively changed. Among the
lipophilic molecules (3–9), the phenyl (6), 3-phenyl-
propyl (8) and 4-nitrobenzyl (9) compounds showed
a similar inhibitory ability. e benzyl compound (7)
remained insoluble, but introduction of a hydrophilic
4-nitro moiety, resulting in 4-nitrobenzyl (9), showed
good solubility and an inhibitory ability similar to the
phenyl compound (6). e aliphatic moieties (3–5) pre-
sented minor inhibition of hBChE. In contrast to hAChE,
the methyl compound (3) did not inhibit hBChE. e
hydrophilic moieties (10–19) also showed differences
in their inhibitory ability towards hBChE. e hydroxy
(10), carboxy (16), methyloxycarbonyl (17) and car-
bamoyl (18) moieties did not show inhibition of hBChE.
e N,N-dimethylamino compound (13) was found to
be superior to all the other hydroxy (11, 12) and carbo-
nyl compounds (14, 15, 19), and its inhibitory ability
towards hBChE was comparable to the best lipophilic
moieties (6, 8, 9).
Molecular docking and SAR results
e docking study was performed on the most promis-
ing compound after the in vitro screening (8) in order
to rationalise the possible interactions within the main
enzyme of interest (AChE) [29]. ree crystal structures
were used for the docking calculations (hAChE – 1b41,
mAChE – 2jez, 2jf0) and the best results were obtained
for a hAChE model (1b41) [30]. It was found that the
top scoring docking pose of compound 8 (−9.66 kcal/
mol) showed apparent interactions with the aromatic
residues of an internal anionic site (IAS) and a periph-
eral anionic site (PAS) (Figure 3) [31]. Specifically, a π-π
interaction occurred between one phenyl moiety and
Trp86 (3.9 Å) from the IAS. One pyridinium ring showed
T-stacking both with Tyr124 (3.9 Å) and Phe338 (4.1 Å)
and a weak π-π or cation-π interaction with Tyr341 (4.5
Å) from PAS. A second pyridinium ring displayed a long
distance from the Trp286 (5.3 Å), but the second phenyl
moiety presented a π-π interaction with His287 (4.1 Å).
Besides the aromatic interactions, inhibitor 8 did not
e selectivity between AChE and BChE is another
necessary factor for suitable future SAR determination
and further development. In this study, two compounds
© 2011 Informa UK, Ltd.

252 Kamil Musilek et al.
(5, 10) were found to be greater than three orders of
magnitude more potent inhibitors of AChE. ough their
moieties are different, it can be hypothesised why such
moieties tested so well. e selectivity of compound 5
with a methyl moiety was probably caused by its small
spatial size, thereby helping this compound to be accom-
modated in the spatially restricted AChE active site.
Regarding BChE, compound 5 was probably unable to
interact with its active site as it does not have the aromatic
residues similar to the PAS of AChE [34]. e selectivity
of compound 10 with a 3-phenylpropyl moiety towards
AChE was confirmed by the docking results, where many
π-π or cation-π interactions were possible. In the case
of BChE, many aromatic residues were absent and so
compound 10 could not interact with this enzyme on a
similar basis as with AChE.
e valuable SAR features for quaternary
cholinesterase inhibitors can be defined. e bispyri-
dinium origin of these novel compounds established
their high affinity for cholinesterases. In addition, spa-
tially narrow molecules (e.g. containing a (2E)-buten-1-
,4-diyl linker) might help access to the AChE active site.
However, these interactions also depend upon the other
moieties carried on the pyridinium ring: small moieties
(e.g. methyl) were found to be suitable for the selective
inhibition of AChE due to its small spatial size. Similarly,
the aromatic moieties (e.g. 3-phenylpropyl) were found
to be convenient and selective inhibitors due to their
π-electron interactions with the AChE active site.
and by the Grant Agency of the Charles University No.
117909/2009/B-CH/FaF.
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is work was supported by the Grant Agency of the
MinistryofDefenceCzechRepublicNo.OVUOFVZ200805
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