Acetylcholinesterase-inhibiting activity of salicylanilide N-alkylcarbamates and their molecular docking

Article abstract of DOI:10.3390/molecules170910142

A series of twenty-five novel salicylanilide N-alkylcarbamates were investigated as potential acetylcholinesterase inhibitors. The compounds were tested for their ability to inhibit acetylcholinesterase (AChE) from electric eel (Electrophorus electricus L.). Experimental lipophilicity was determined, and the structure-activity relationships are discussed. The mode of binding in the active site of AChE was investigated by molecular docking. All the discussed compounds expressed significantly higher AChE inhibitory activity than rivastigmine and slightly lower than galanthamine. Disubstitution by chlorine in C'_(3,4) of the aniline ring and the optimal length of hexyl-undecyl alkyl chains in the carbamate moiety provided the most active AChE inhibitors. Monochlorination in C'₍₄₎ exhibited slightly more effective AChE inhibitors than in C'₍₃₎. Generally it can be stated that compounds with higher lipophilicity showed higher inhibition, and the activity of the compounds is strongly dependent on the length of the N-alkyl chain.

Full text of DOI:10.3390/molecules170910142

Molecules 2012, 17, 10142-10158; doi:10.3390/molecules170910142
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molecules
ISSN 1420-3049
www.mdpi.org/molecules
Article
Acetylcholinesterase-Inhibiting Activity of Salicylanilide
N-Alkylcarbamates and Their Molecular Docking
Ales Imramovsky 1,*, Sarka Stepankova 2, Jan Vanco 3, Karel Pauk 1, Juana Monreal-Ferriz 4,
Jarmila Vinsova 4 and Josef Jampilek 3,*
1
Institute of Organic Chemistry and Technology, Faculty of Chemical Technology,
University of Pardubice, Studentska 573, 532 10 Pardubice, Czech Republic
2
Department of Biological and Biochemical Sciences, Faculty of Chemical Technology,
University of Pardubice, Studentska 573, 532 10 Pardubice, Czech Republic
3
Department of Chemical Drugs, Faculty of Pharmacy, University of Veterinary and Pharmaceutical
Sciences Brno, Palackeho 1/3, 612 42 Brno, Czech Republic
4
Department of Inorganic and Organic Chemistry, Faculty of Pharmacy in Hradec Kralove,
Charles University in Prague, Heyrovskeho 1203, 500 05 Hradec Kralove, Czech Republic
* Authors to whom correspondence should be addressed; E-Mails: ales.imramovsky@upce.cz (A.I.);
josef.jampilek@gmail.com (J.J.); Tel.: +420-466-037-739 (A.I.); Fax: +420-466-038-004 (A.I.);
Tel.: +420-541-562-925 (J.J.); Fax: +420-541-240-607 (J.J.).
Received: 30 July2012; in revised form: 1 August 2012 / Accepted: 10 August 2012 /
Published: 24 August 2012
Abstract: A series of twenty-five novel salicylanilide N-alkylcarbamates were investigated
as potential acetylcholinesterase inhibitors. The compounds were tested for their ability to
inhibit acetylcholinesterase (AChE) from electric eel (Electrophorus electricus L.).
Experimental lipophilicity was determined, and the structure-activity relationships are
discussed. The mode of binding in the active site of AChE was investigated by molecular
docking. All the discussed compounds expressed significantly higher AChE inhibitory
activity than rivastigmine and slightly lower than galanthamine. Disubstitution by chlorine
in C'(3,4) of the aniline ring and the optimal length of hexyl-undecyl alkyl chains in the
carbamate moiety provided the most active AChE inhibitors. Monochlorination in C'(4)
exhibited slightly more effective AChE inhibitors than in C'(3). Generally it can be stated
that compounds with higher lipophilicity showed higher inhibition, and the activity of the
compounds is strongly dependent on the length of the N-alkyl chain.

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Keywords: 4-chloro-2-(chlorophenylcarbamoyl)phenyl alkylcarbamates; in vitro acetyl-
cholinesterase inhibition; lipophilicity; molecular docking
1. Introduction
It is well-known that salicyl-like compounds show ability to inhibit cyclooxygenase (COX) [1].
Various carbamates are also known as reversible cholinesterase inhibitors [1]. Salicylanilides
(2-hydroxy-N-phenylbenzamides) and/or their O-substituted derivatives show diverse pharmacological
activities [2–8]. The presence of the amide group (-NHCO-) together with the carbamate group
(-NHCOO-) in the structure of phenylcarbamoylphenyl N-alkylcarbamates can cause interaction with
various enzymes or enzymatic systems, e.g. both amide and carbamate moieties are characteristic for a
number of herbicides acting as photosynthetic electron inhibitors [7–9], and the carbamate moiety or
its bioisosteres are typical for a number of potential acetylcholinesterase inhibitors [10–19].
Acetylcholinesterase (AChE, EC 3.1.1.7) plays a vital role in the central and peripheral nervous
systems, where it catalyzes the hydrolysis of the neurotransmitter acetylcholine (ACh) in cholinergic
synapses, and subsequently it can affect a number of pathogenic processes [16]. AChE inhibitors are
used in treatment of various neuromuscular disorders and have provided the first generation of drugs
for treatment of Alzheimer’s disease (AD) [20], which is a progressive physical disorder that causes
increasingly severe impairment in the cognitive and functional ability of individuals suffering from the
disease [21]. The progression of this neurodegenerative disease leads to dementia [22]. The cause for
most Alzheimer’s cases is still essentially unknown. Several competing hypotheses exist trying to
explain the cause of the AD [23]. The oldest, on which most currently available drug therapies are
based, is the cholinergic hypothesis that proposes that AD is caused by reduced synthesis of the
neurotransmitter ACh [24]. This cholinergic hypothesis has not maintained widespread support,
nevertheless it can be stated that the cholinergic deficit is responsible for the symptomatology of AD [23].
The inhibition of AChE causes an increase in the concentration of ACh in cholinergic synapses, which
results in alleviation of the disease. For this reason, new and potent AChE inhibitors may be helpful in
the treatment of this disease.
N-Substituted carbamates are characterized as pseudosubstrate inhibitors or active site-directed
irreversible inhibitors of AChE that, in the presence of substrate, compete for the AChE active site. A
proposed simplified mechanism for AChE inhibition by carbamates consists in rapid formation of the
covalent enzyme-inhibitor tetrahedral intermediates via a nucleophilic attack of the hydroxyl moiety in
Ser200 of the enzyme to the carbamate carbonyl and following slow generation of a carbamylated-enzyme
intermediate from the tetrahedral intermediate. The hydroxyl-moiety of the carbamate ester can be
consequently recovered at a very slow rate or blocked irreversibly depending on the character of the
used N-substituted carbamates [1,14–19].
Reaching the inhibitory site of action and binding to the active site gorge would be influenced by
hydro/lipophilic properties of the compounds; therefore the lipophilicity of the target compounds was
experimentally determined using RP-HPLC. Structure-activity relationships between the chemical

Molecules 2012, 17
10144
structure, physical properties and biological activities of the evaluated compounds are discussed. The
specific orientation of the inhibitors in the AChE binding site was suggested using molecular docking.
2. Result and Discussion
2.1. Chemistry
The synthesis of the discussed carbamates is shown in Scheme 1. The synthetic pathway was
discussed recently [6,25]. The starting salicylanilides 1–3 were routinely prepared by the reaction of
5-chlorosalicylic acid with the appropriate aniline and PCl3 in chlorobenzene using a microwave
reactor [3]. For the synthesis of the corresponding N-alkylcarbamates 4a–6i, a suspension of
salicylanilides 1–3 in acetonitrile (ACN) was treated with equivalent of triethylamine (TEA), followed
by adding of the appropriate isocyanate. This reaction was performed at ambient temperature due to
thermal instability of the products. The prepared N-alkylcarbamates 4a–6i belong to three groups:
those having chlorine at positions C'(3), C'(4) and C'(3,4) of the anilide ring; the prepared compounds are
summarized in Table 1. The yields of the synthesized compounds varied in the interval of 35–80%.
A small library of discussed compounds 4a–6i was characterised by means of IR, NMR spectroscopy,
CHN analysis and published recently [6,8]. The chemical stability (carbamate bond hydrolysis and the
release of the parent salicylanilide) of the discussed compounds were studied in aqueous buffer
solutions at pH 3–8 at 37 °C by HPLC. The compounds were stable at acidic pH values, but they were
decomposed in alkaline environment, approx. T1/2 64 h at pH 7.0 and approx. T1/2 43 h at pH 7.4 were
shown [6].
Scheme 1. Synthesis of target 4-chloro-2-(chlorophenylcarbamoyl)phenyl alkylcarbamates
4, 5 and 6.
O
O
1
1
R
R
2
Cl
R
Cl
a
N
N
H
+ O C N
H
OH
O
1
2
1
2
R = 3-Cl = comp. 1
R = alkyl: C - C
2 11
R = 3-Cl = comp. 4
R
O
N
H
1
1
R = 4-Cl = comp. 2
(except alkyl C )
3
R = 4-Cl = comp. 5
1
1
R = 3,4-diCl = comp. 3
R = 3,4-diCl = comp. 6
Reagents and conditions: (a) TEA, ACN, ambient temperature [5,6,25].
2.2. Lipophilicity
Lipophilicity is a property that has a major effect on absorption, distribution, metabolism, excretion
and toxicity properties as well as pharmacological activity, because drugs cross biological membranes
through the passive transport, which strongly depends on their lipophilicity. Lipophilicity has been
studied and applied as an important drug property for decades. Different lipophilicity descriptors such
as log kw, log P, log D, etc. are used for structure-activity relations description and prediction.
However, the algorithms used in their calculation do not take into account configuration specificity and
sometimes even intramolecular interactions of these N-substituted carbamoyl derivatives.

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Table 1. IC50 Values of AChE inhibition in comparison with standards rivastigmine (RIV)
and galanthamine (GLT) and lipophilicity values (log k, log P). AChE inhibition is
expressed as mean ± SD (n = 3 experiments), (N.D. = not determined; * calculated by
ACD/LogP (ver. 1.0), Advanced Chemistry Development Inc., Toronto, Canada).
AChE IC50
Comp.
4a
R 1
R 2
log k
log P *
(μmol/L)
N.D.
3-Cl
3-Cl
3-Cl
3-Cl
3-Cl
3-Cl
3-Cl
3-Cl
3-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
3,4-Cl
3,4-Cl
3,4-Cl
3,4-Cl
3,4-Cl
3,4-Cl
3,4-Cl
3,4-Cl
3,4-Cl
–
C2H5
C4H9
0.7523
0.7611
0.7732
0.7758
0.7772
0.7831
0.7837
0.7873
0.7902
0.7695
0.7759
0.7838
0.7859
0.7872
0.7899
0.7919
0.7942
0.7947
1.0234
1.0457
1.0601
1.0611
1.0667
1.0845
1.0880
1.0894
1.0903
–
4.02 ± 0.40
5.08 ± 0.40
5.61 ± 0.40
6.14 ± 0.40
6.67 ± 0.40
7.20 ± 0.40
7.74 ± 0.40
8.27 ± 0.40
8.80 ± 0.40
3.98 ± 0.39
5.04 ± 0.39
5.57 ± 0.39
6.10 ± 0.39
6.63 ± 0.39
7.16 ± 0.39
7.70 ± 0.39
8.23 ± 0.39
8.76 ± 0.39
4.88 ± 0.41
5.94 ± 0.41
6.47 ± 0.41
7.01 ± 0.41
7.54 ± 0.41
8.07 ± 0.41
8.60 ± 0.41
9.13 ± 0.41
9.66 ± 0.41
2.14 ± 0.27
1.59 ± 0.45
4b
4c
4d
4e
4f
N.D.
C5H11
C6H13
C7H15
C8H17
C9H19
C10H21
C11H23
C2H5
50.4 ± 0.48
35.1 ± 0.31
61.7 ± 0.50
69.9 ± 0.49
34.2 ± 0.26
52.7 ± 0.45
56.9 ± 0.48
59.1 ± 0.33
48.1 ± 0.31
41.7 ± 0.29
31.0 ± 0.29
49.4 ± 0.31
62.5 ± 0.41
52.2 ± 0.39
64.6 ± 0.41
44.3 ± 0.33
67.3 ± 0.52
64.7 ± 0.79
56.0 ± 0.42
41.2 ± 0.36
36.7 ± 0.28
32.1 ± 0.29
43.9 ± 0.32
21.8 ± 0.20
29.9 ± 0.22
501 ± 3.08
4.0 ± 0.13
4g
4h
4i
5a
5b
5c
5d
5e
5f
C4H9
C5H11
C6H13
C7H15
C8H17
C9H19
C10H21
C11H23
C2H5
5g
5h
5i
6a
6b
6c
6d
6e
6f
C4H9
C5H11
C6H13
C7H15
C8H17
C9H19
C10H21
C11H23
–
6g
6h
6i
RIV
GLT
–
–
–
Calculation of log P/Clog P by CS ChemOffice Ultra ver. 10.0 is based on references [26–31], not
in order while calculation of log P by ACD/LogP ver. 1.0 is based on Hansch and Leo [32]. The
program ChemOffice did not resolve various lipophilicity values of individual positional isomers, in
particular, the same log P/Clog P data were calculated for series 3-Cl (compounds 4a–i) and series
4-Cl (compounds 5a–i), therefore these data are not included in Table 1. Log k data specify
lipophilicity within the series of the discussed compounds, and all the chiral compounds were
measured several times with the same results. The determined differences in log k parameters for
individual R/S-enantiomers cannot be explained on the basis of the results presented here.

Molecules 2012, 17
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2.3. In Vitro Evaluation of AChE-Inhibiting Activity
The discussed compounds were tested for their ability to inhibit AChE from electric eel
(Electrophorus electricus L.) using Ellman’s method [12,13], and they expressed good activity
compared with the drugs in clinical practice rivastigmine (Exelon®) and galanthamine (Reminyl®). All
the discussed carbamate-like compounds expressed significantly higher AChE inhibitory activity than
the standard rivastigmine and slightly lower inhibitory activity than the standard galanthamine. The
IC50 values are summarized in Table 1.
The compounds under investigation could be divided into three groups based on their substitution:
Group 1 includes 3-Cl substituted carbamates 4a–i; Group 2 contains 4-Cl derivatives 5a–i; and Group 3
is composed of 3,4-Cl disubstituted compounds 6a–i, see Table 1. The most potent AChE inhibition of
the isolated enzyme was observed for Group 3 (C'(3,4) series) that provided the most active inhibitors:
compounds 6h (R2 = decyl, IC50 = 21.8 µmol/L, 23-fold higher than RIV) and 6i (R2 = undecyl,
IC50 = 29.9 µmol/L, 16-fold higher than RIV). Monochlorination of C'(3) provided less effective
inhibitors than C'(4) monosubstitution: compound 5d (R2 = hexyl, IC50 = 31.0 µmol/L). These results
demonstrated that 4-chloro-2-(3,4-chlorophenylcarbamoyl)phenyl long-chain-N-alkylcarbamates can
interact with all the binding AChE sites more tightly than the monochlorinated compounds and block
the AChE gorge.
The dependences of AChE inhibition (1/IC50) on R2 substitution (the length of the alkyl chain) of
the discussed compounds are illustrated in Figure 1. A similar trend as in Figure 1 can be observed for
dependences of AChE inhibition on compound lipophilicity expressed as log k, see Figures 2 and 3.
Generally it can be stated that compounds with higher lipophilicity, that is caused by 3,4-Cl
disubstitution, are more effective inhibitors, Group 3 (6a–i) >>> Group 2 (5a–i) > Group 1 (4c–i).
Within monochlorinated compounds different dependences compared with dichlorinated carbamates
can be found, see Figures 2 and 3. Thus it is evident that activity is dependent both on the length of the
alkyl chain (R2 substituent) and on lipophilicity.
Figure 1. Dependence of AChE inhibition (log 1/IC50 [mol/L]) on long alkyl chain of R2
substitution within individual Groups 1–3. (Et = ethyl, Bu = butyl, Pen = pentyl, Hex = hexyl,
Hep = heptyl, Oct = octyl, Non = nonyl, Dec = decyl, Undc = undecyl).
4.7
4.6
4.5
4.4
4.3
4.2
4.1
4.0
3.9
Et (a)
Bu (b)
Pen (c) Hex (d) Hep (e)
Compounds
Oct (f)
Non (g) Dec (h) Undc (i)
Group 1 (4c-i) Group 2 (5a-i) Group 3 (6a-i)

Molecules 2012, 17
10147
Figure 2. General dependences of AChE inhibition (log 1/IC50 [mol/L]) on lipophilicity
of compounds 4 and 5 expressed as log k. Compounds 4e, 4f and 5i were excluded/are
not plotted.
4.6
3c
5d
4.5
4.4
4.3
4.2
4.1
4g
b
4d
R = 0.8239
5c
5e
4h
R = 0.6753
5b
5g
4c
4i
5a
5h
5f
0.765
0.770
0.775
0.780
0.785
0.790
0.795
0.800
log k
4c-i (3-Cl) 5a-i (4-Cl)
Figure 3. Dependence of AChE inhibition (log 1/IC50 [mol/L]) on lipophilicity of
compounds 6 expressed as log k.
4.7
6h
4.6
6i
6f
4.5
6e
4.4
6d
R = 0.8386
6g
4.3
4.2
4.1
6c
6b
6a
1.01
1.02
1.03
1.04
1.05
1.06
1.07
1.08
1.09
1.10
log k
The shapes of biological response curves shown in Figure 1 are typical dependences of n-alkyl
homologous series, when activity can alternate with the number of carbon atoms (with a change of a
long side/straight chain) resulting in a “zigzag” pattern. Inhibition ability of carbamates (binding to the
active site of AChE) seems to be influenced by the length of alkyl substituents due to the difficulty of
larger inhibitors to enter the narrow enzyme active site gorge as well as strictly balanced
hydro/lipophilic properties of the inhibitors, taking into account the fact that hydrophobic substituents
accelerate the inhibitors entering the active site gorge of the enzyme [15]. Further, Davis et al. also
described that long-chain N-alkylcarbamate AChE inhibitors are much more stable than their short-chain
homologues [33].

Molecules 2012, 17
10148
A number of papers refer to the fact that an increase of N-alkyl chain length resulted in
AChE inhibition, especially to N-hexyl. A further increase of the chain length resulted in a great
variation in carbamylation rates. This fact is in compliance with observation that the introduction
of bulky hydrophobic groups at the carbamoyl nitrogen led to compounds with better AChE
inhibition [15,17,19,34]. It was also observed that within the series of phenyl N-methyl carbamates
activity increases with increasing electron-withdrawing effect [10]. From these facts it can be
concluded that AChE inhibition can be correlated with hydrophobicity, electronic, inductive or polar
properties and steric effects [15].
Monochlorinated carbamates C'(3) (Group 1, 4) or C'(4) (Group 2, 5) expressed different
dependences in comparison with C'(3,4) dichlorinated carbamates (Group 3, 6). Series 4 and 5 possess
similar log k values (differ from each other by about 1%) compared with C'(3,4) series 6. Group 1
differs from Group 2 only by chlorine position in the anilide part; therefore some comparable
trends/shapes can be observed. In both cases AChE inhibition grows exponentially to derivatives 4d,
5d (R2 = hexyl, IC50 = 35.1, 31.0 µmol/L), then decreases to compounds 4f, 5f (R2 = octyl, IC50 = 69.9,
62.5 µmol/L), after that increases to carbamates 4g, 5g (R2 = nonyl, IC50 = 34.2 or 52.2 µmol/L) and
subsequently decreases again. Only in Group 2 (C'(4)) activity increases insignificantly to compound 5i
(R2 = undecyl, IC50 = 44.3 µmol/L). This fact can be connected with probably different fitting of C'(4)
chloro substitution in comparison with carbamates group C'(3) substituted with chlorine, but in general
it can be stated that both series expressed similar AChE inhibition. Two compounds 4d and 4g showed
similar activities, their lipophilicity (log k) is approaching to the log k value of compound 5d, and
further lipophilicity increase (alkyl chain prolongation) did not have any influence on AChE inhibition
increase. Therefore lipophilicity seems to be an important parameter. These results can be explained by
the fact that the optimal lipo/hydrophilic ratio for monosubstituted N-alkylcarbamates is about 0.786,
and hexyl or nonyl chains are flexible and both can sterically fit the active site of the AChE gorge.
When from the set of 16 tested compounds of Group 1 and Group 2 the least active
carbamates/outlier, i.e., 4f, 4e and 5i, were eliminated, the dependences of AChE inhibition
(log 1/IC50, [mol/L]) on log k can be presented in Figure 2. In the first case (to log k ca 0.786, C6 for 5d or
C9 for 4g) the dependence of activity on lipophilicity is linear with correlation coefficient R = 0.8239.
As discussed above, the activity showed a “zigzag” pattern, and the range of individual inhibition
values is narrow, therefore in the second case the dependence of AChE inhibition of significantly less
effective carbamates 4h, 4i, 5e, 5f, 5g and 5h on lipophilicity (log k from ca 0.787) does not seem to be
so clear, but generally, for Group 1 and Group 2, it can be stated that AChE inhibition dramatically
decreases with lipophilicity increase. This observation supported the above mentioned fact that activity
is dependent both on the length of the alkyl chain and on lipophilicity (substitution of anilide ring),
then on the balanced lipo/hydrophilic properties (lipophilicity/polarity) of the individual discussed
carbamates due to their optimal interaction with the active site of the AChE gorge.
Different AChE inhibition results can be observed for Group 3. In general, the whole series 6
showed higher activity than both monochlorinated series 4 and 5. The lipophilicity of the compounds
within Group 3 is much higher than within Group 1 and Group 2, as mentioned above, and the optimal
lipo/hydrophilic ratio for disubstituted N-alkylcarbamates is about 1.089. Inhibition capability grows
exponentially to the local maximum, compound 6f (R2 = octyl, IC50 = 32.1 µmol/L), which is similar
to the above mentioned most effective chlorine monosubstituted compounds 4d, 4g and 5d. After that

Molecules 2012, 17
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it decreases to derivative 6g (R2 = nonyl, IC50 = 43.9 µmol/L), then significantly grows to the
most active compound 6h (R2 = decyl, IC50 = 21.8 µmol/L) and slightly decreases to carbamate 6i
(R2 = undecyl, IC50 = 29.9 µmol/L), which is the second effective compound among all the discussed
N-alkylcarbamates. The dependence of AChE inhibition (log 1/IC50, [mol/L]) on log k of compounds
from Group 3 is illustrated in Figure 3. The approximately linear dependence with correlation
coefficient R = 0.8386 of AChE inhibition on lipophilicity of compounds 6 can be observed.
This different AChE-inhibiting behaviour of C'(3,4) dichlorinated series 6 can be probably explained
based on the easier entrance to the gorge and slightly different fitting of these carbamates to the active
site of AChE (different docking to the active site due to both chlorine atoms in the anilide phenyl).
Higher inhibition ability of C'(3,4) dichlorinated derivatives may be also partly caused by the increase of
electron-withdrawing effect (Hammett’s σ parameters) of individual substituents in the anilide part of
the molecule [35]. As discussed above, inhibition potential of the carbamates is also affected by the
bulk parameter. The variations in the chain length exhibited an obvious effect on AChE inhibition.
Generally, within monosubstituted Groups 1 and 2 C6 and/or C9 expressed the highest AChE
inhibitory activity, C8 derivative showed the lowest inhibition, and the activities of N-alkylated C5, C7,
C10 and C11 carbamates were almost equal but lower in comparison with disubstituted Group 3, where
C10, C11 and C8 expressed the highest AChE inhibitory activity, and inhibition of N-alkylated C6, C7
and C9 carbamates was almost the same but lower.
2.4. Molecular Docking
Structural analysis of AChE revealed that the active site is placed near the bottom of a narrow gorge
imbedded halfway into the protein and 14 aromatic residues lining a substantial portion of the surface
of the gorge. This cavity was named the ‘active site gorge’ and, further at the gorge mouth a peripheral
anionic binding site (PAS) was found. The active site of AChE contains: (1) an esteratic site (ES)
comprising the catalytic triad Ser200-His440-Glu327, which is located at the bottom of the gorge;
(2) an oxyanion hole (OAH) that stabilizes the tetrahedral intermediate binding of the carbamate
carbonyl group; (3) an acyl binding site (ABS) that binds the acetyl group of ACh or the alkyl moiety
of carbamate inhibitors; (4) an anionic substrate binding site (AS) that contains a small number of
negative charges but many aromatic residues, where the quaternary ammonium pole of ACh and of
various active site ligands binds through a preferential interaction of quarternary nitrogens or a partial
positive charge generated by electron-withdrawing moieties.
Structural analysis of the Torpedo californica AChE demonstrated an active site near the bottom of
a 20 Å deep and narrow gorge imbedded halfway into the protein and 14 aromatic residues lining a
substantial portion of the surface of the gorge. This cavity was named the ‘active site gorge’, and at the
gorge mouth a peripheral anionic binding site (PAS) was found. The active site of AChE contains:
(1) an esteratic site (ES) comprising the catalytic triad Ser-His-Glu, which is located at the bottom of
the gorge; (2) an oxyanion hole (OAH) that stabilizes the tetrahedral intermediate binding of the
carbamate carbonyl group; (3) an acyl binding site (ABS) that binds the acetyl group of ACh or the
alkyl moiety of carbamate inhibitors [36]; (4) an anionic substrate binding site (AS) that contains a
small number of negative charges but many aromatic residues, where the quaternary ammonium pole
of ACh and of various active site ligands binds through a preferential interaction of quarternary

Molecules 2012, 17
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nitrogens or a partial positive charge generated by electron-withdrawing moieties. The distance
between the ES and the AS is 5 Å [11,15,37].
Due to these facts, carbamate inhibitors would be the most efficient if the distance between the ES
and the AS is also 5 Å, therefore meta-carbamates are more advantageous. Nevertheless Sundberg et al.
stated that compounds with a non-rigid skeleton (cycles linked with flexible chains) could adapt to
the binding site and are not necessarily constrained to the extended structure present in the crystal,
e.g., various ortho-carbamate inhibitors were described [11,14,38,39], or the differently substituted
N-alkylcarbamylphloroglucinols with high rotational freedom of long-chain substituents were
described in the recent paper of Lin et al. [36]. In addition to the majority of carbamate inhibitors
binding to the active site of AChE and blocking the hydroxyl moiety in Ser in the ES, dual-site binding
inhibitors of AChE were described recently, e.g. donepezil. These dual-site binding inhibitors bind to
both the active and the peripheral sites, especially Trp in the PAS [40–43]. Further different dual or
multiple-site inhibitors of AChE were prepared and showed stacking interaction with Trp or Tyr in the
PAS or with Tyr and Phe that existed in the middle of the gorge [11,44–46].
Molecular docking within the study was carried out using Gold 5.0 (CCDC, Cambridge, UK) to
produce the optimal conformations for the docked ligands. The set of best ranked conformations of the
discussed compounds can be characterized, similarly as by Paz et al. [46], by low direct interaction
with the catalytic triad. On the other hand, they bind to the peripheral part of the gorge by means of the
hydrogen bonds between the amide nitrogen and the Ser125 side chain (O-N = 3.226–3.465 Å) and
between the amide oxygen and the Ser125 side chain (O-O = 3.969–4.222 Å) or Gly126 nitrogen
(N-O = 3.897 Å). Interactions of the carbamate nitrogen with the Tyr337 OH moiety
(N-O = 2.553–2.711 Å) and π-π interaction between the trisubstituted phenyl ring and the Trp86 side
chain (centroid-centroid = 3.875–3.957 Å) were approved as real. The interactions Cl··H-N/O that
were identified in the molecular docking experiments could also play an important role in positioning
of the ligands inside the active site gorge. Interestingly, in accordance with the recent paper from
Lin et al. [36], the conformation of carbamate residue observed for the most active inhibitors was
found to be in cis-arrangement which allowed the molecules to form the “hairpin”-like structure. On
the other hand, in comparison with Lin et al. [36], the carbamate residue in the tested compounds is not
involved in the interactions with the catalytic triad of the active site gorge. This could be due to the
differences in constitution of the derivatives described by Lin et al. [36] and the compounds presented
in our manuscript, while the first ones contain only one part of higher rigidity (i.e., the aromatic system
of differently substituted benzene ring) and all other parts have high freedom of rotation. The
compounds described in our manuscript, on the other hand, contain semi-rigid residue of two
substituted benzene ring connected by amide group and only one freely rotable alkyl-carbamate chain.
The representative example of interaction of the active AChE-inhibitor 6i and the amino acid residues
within the active site gorge is depicted at Figure 4. The low-energy conformations of the investigated
compounds (especially the most active derivatives involving the long alkyl-chain substitution)
confirmed that the molecule polar parts are oriented to the catalytic gorge and non-polar parts, to the
PAS. The graphical model of the AChE molecule involving three most active AChE inhibitors (6h, 6i
and 5d) and demonstrating the conformations of docked ligands is shown in Figure 5. This inhibitor
orientation can cause a competitive reaction with native ionic ligand, considerable ACh repulsion and
resulting effective AChE blocade.

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Figure 4. (A) Representation of three-dimensional structure of acetylcholinesterase (shown
as solvent accessible surface), showing gorge and specific parts of active site (catalytic
triad: blue, OAH: yellow, PAS: orange, ABS: green). (B) Example of interactions between
docked molecule of 6i and amino acid residues within active site gorge. The interacting
atoms and amino acid residues are depicted; other parts of the active site gorge are omitted
for clarity.
4A
4B
Figure 5. Three-dimensional view from within active site gorge (using same colour
scheme for active site as in Figure 4A), containing the overlaid highest-ranked
conformations of all docked ligands, represented by solvent accessible surface coloured by
element appearance (chlorine-green, carbon and hydrogen-brown or white) or lone pair
presence (purple colour), showing regions with preferred orientation of different residues
(especially hydrophobic region near ABS site, regions containing high occurrence of lone-
pair interactions near OAH and PAS, and regions with higher occurrence of chlorine-
substituted phenyl residues near catalytic triad).
In case of the studied protein, the gorge of AChE contains the catalytic triad (Ser203, His447,
Glu334), marked blue in Figure 4A and other parts that are used to correct the orientation of the

Molecules 2012, 17
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normal substrate (ionized acetylcholine) within the gorge like “oxanion hole” (OAH) formed by
Ala204, Gly121 and Gly122 (indicated by yellow colour in Figure 4A), which is used to correct the
orientation of the ester group of acetylcholine. Another specific site is called “peripheral anionic
binding site” (PAS), serves to orient the cationic part of acetylcholine, is located in the peripheral part of
the gorge and is composed of Trp86, Tyr337, Trp286 and Tyr72 (marked by orange colour in Figure 4A).
The space for acetyl function is provided by “acyl binding site” (ABS), which is located near the OAH
and is formed by Trp236, Phe338, Phe295, Phe297 and Gly122 (marked by green colour in Figure 4A).
The molecular docking yielded a series of conformations of ligands, see Figure 5, that represented
quite different modes of interactions, dependent on their structure. The derivatives involving short
lipophilic substitution usually folded deep within the gorge, near the catalytic triad and OAH. On the
other hand, the highly active derivatives involving longer lipophilic substitution (6 to 11 carbon long
alkyl chain) interacted mainly near the OAH and folded throughout the gorge, exiting it with the linear
alkyl chain near the most lipophilic part of the gorge (near the ABS), see Figure 6. On the basis of such
orientation and the set of interactions, which were identified between the ligands and the protein
residues within the gorge, it can be hypothesized that the studied compounds could act as “bulky”-blockers
of the normal ionic substrate (ACh) entrance into the active site gorge.
Figure 6. Graphical representation of AChE structure (solvent accessible surface, PDB ref.
2WHQ) with highlighted active site and three best ranked conformations of AChE
inhibitors (6h, 6i and 5d, marked by brown color).

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3. Experimental
3.1. Chemistry
Synthesis and characterization of the discussed 4-chloro-2-{[(3-chlorophenyl)amino]carbonyl}
phenyl alkyl-carbamates (4a–i), 4-chloro-2-{[(4-dichlorophenyl)amino]carbonyl}phenyl alkyl carbamates
(5a–i) and 4-chloro-2-{[(3,4-dichlorophenyl)amino]carbonyl}phenyl alkylcarbamates (6a–i) were
described by Ferriz et al. [6] and Imramovsky et al. [8,25] and characterization of all the prepared
compounds was recently reported [6].
3.2. Lipophilicity Determination by HPLC (Capacity Factor k/Calculated log k)
A Waters Alliance 2695 XE HPLC separation module and a Waters Photodiode Array Detector
2996 (Waters Corp., Milford, MA, USA) were used. A Symmetry® C18 5 μm, 4.6 × 250 mm
chromatographic column, Part No. WAT054275 (Waters Corp., Milford, MA, USA) was used. The
HPLC separation process was monitored by Empower™ 2 Chromatography Data Software, Waters
2009 (Waters Corp., Milford, MA, USA). A mixture of MeOH p.a. (70%) and H2O-HPLC–Mili-Q
Grade (30%) was used as a mobile phase. The total flow of the column was 1.0 mL/min, injection
volume, 30 μL, column temperature, 45 °C and sample temperature, 10 °C. The detection wavelength
of 210 nm was chosen. The KI methanolic solution was used for the dead time (tD) determination. The
retention times (tR) were measured in minutes. The capacity factors k were calculated using the
Empower™ 2 Chromatography Data Software according to formula k = (tR − tD)/tD, where tR is the
retention time of the solute, whereas tD denotes the dead time obtained using an unretained analyte.
Log k, calculated from the capacity factor k, is used as the lipophilicity index converted to log P scale.
The log k values of the individual compounds are shown in Table 1.
3.3. Calculation of Lipophilicity
Log P, i.e., the logarithm of the partition coefficient for n-octanol/water, was calculated using the
programs CS ChemOffice Ultra ver. 10.0 (CambridgeSoft, Cambridge, MA, USA) and ACD/LogP
ver. 1.0 (Advanced Chemistry Development Inc., Toronto, Canada). Clog p values (the logarithm of
n-octanol/water partition coefficient based on established chemical interactions) were generated by
means of CS ChemOffice Ultra ver. 10.0 (CambridgeSoft, Cambridge, MA, USA) software. The
values of the individual compounds are shown in Table 1.
3.4. In Vitro Evaluation of AChE-Inhibiting Activity
The ability of the tested compounds to inhibit acetylcholinesterase (purchased from Sigma) from
electric eel (Electrophorus electricus L.) was tested. The effectiveness of the inhibitor could be
described by the 50% inhibitory concentration IC50. The IC50, or the half maximal inhibitory
concentration, represents the concentration of an inhibitor that is required for 50% inhibition of the
enzyme (sometimes it is referred to as the negative logarithm of the molar concentration inhibiting the
enzyme activity by 50%, pI50 = log 1/IC50). The IC50 values were determined by the spectrophotometric
Ellman’s method.

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The IC50 values were determined using the spectrophotometric Ellman’s method, which is a simple,
rapid and direct method to determine the SH and -S-S- group content in proteins [47]. This method is
widely used for measuring of cholinesterase activity and effectivity of cholinesterase inhibitors.
Cholinesterase activity is measured indirectly by quantifying the concentration of 5-thio-2-
nitrobenzoic acid (TNB) ion formed in the reaction between the thiol reagent 5,5′-dithiobis-2-
nitrobenzoic acid (DTNB) and thiocholine, a product of substrate (i.e., acetylthiocholine, ATCh)
hydrolysis by cholinesterase [48]. All tested compounds were dissolved in dioxane (concentration 0.01 M)
and then diluted in demineralised water (concentration 0.001 M and 0.0001 M). The procedure of
determination of IC50 is a slightly modified Ellman’s method according to Zdrazilova and described in
detail in [49]. For determination of IC50 values the inhibition was determined for 25 different
compound concentrations with three replicates. The obtained results are summarized in Table 1.
3.5. Molecular Docking
The three-dimensional structure of acetylcholinesterase, phosphonylated with sarin and complexed
with the reactivator HI-6 [50], was obtained from Protein Databank (www.pdb.org, PDB code 2whq).
This specific structure has been chosen due to high quality (i.e., resolution of 2.15 Å), its mammalian
origin (i.e., mouse enzyme expressed in human cells) and the dimensions and chemical nature
(i.e., carbamate) of complexed inhibitor HI-6 to obtain the initial structure, reflecting the possible
conformational changes, changes in the volume of the active site or its accessibility. For the molecular
docking calculations, the molecules of water, molecules of crystallization and the inhibitor HI-6 were
deleted, and Ser203 was dephosphonylated.
To carry out the molecular docking, GOLD 5.0 (CCDC, Cambridge, UK) in Linux version was
used. The usual procedure consisting of the automatic substitution of all hydrogen atoms for the
GOLD-optimized ones and the standard settings for flexible docking (free rotation and flipping of
specific functional groups) were used to produce the set of optimal conformations of both the ligand
and the protein. The standard GOLD scoring function was used. Prior to docking, the ligands were
optimized by MM2 force field with RMS Gradient set to 0.0001 using the ChemBio3D Ultra
12.0.2.1076 (a part of ChemBioOffice Ultra 2010 Package, ver. 12.0.2.1076, CambridgeSoft,
Cambridge, USA). The interaction site was defined as a cavity around Ser203 with 15 Å diameter,
comprising 1566 atoms, see Figure 4A. All molecular graphics material was prepared using the
Discovery Studio 3.1 Client (ver. 3.1.1.11157, Accelrys Software Inc., San Diego, CA, USA).
4. Conclusions
A series of twenty-five novel salicylanilide N-alkylcarbamates were tested for their ability to inhibit
acetylcholinesterase (AChE) from electric eel (Electrophorus electricus L.). All the discussed
compounds expressed significantly higher AChE inhibitory activity than rivastigmine and slightly
lower than galanthamine. Experimental lipophilicity and other molecular descriptors of the studied
compounds were determined, and the structure-activity relationships were discussed. Disubstitution by
chlorine in C'(3,4) of the aniline ring and the optimal length of hexyl-undecyl alkyl chains in the
carbamate moiety provided the most active AChE inhibitors. Monochlorination in C'(4) exhibited
slightly more effective AChE inhibitors than in C'(3). Generally it can be stated that compounds with

Molecules 2012, 17
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higher lipophilicity showed higher inhibition, and the activity of the compounds is strongly dependent
on the length of the N-alkyl chain. The mode of binding in the active site of AChE was investigated by
molecular docking. The molecular docking yielded a series of conformations of ligands that
represented quite different modes of interactions, dependent on their structure. On the basis of such
orientation and the set of interactions, which were identified between the ligands and the protein
residues within the gorge, it can be hypothesized that the studied compounds could act as “bulky”-blockers
of the normal ionic substrate (ACh) entrance into the active site gorge.
Acknowledgments
This study was supported by the Ministry of Education, Youth and Sports of the Czech Republic
(institutional support of Faculty of Chemical Technology, University of Pardubice) and by IGA VFU
Brno, Project No. 96/2012/FaF.
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© 2012 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article
distributed under the terms and conditions of the Creative Commons Attribution license
(http://creativecommons.org/licenses/by/3.0/).