Salicylanilide diethyl phosphates as cholinesterases inhibitors

Article abstract of DOI:10.1016/j.bioorg.2014.11.005

Based on the presence of dialkyl phosphate moiety, we evaluated twenty-seven salicylanilide diethyl phosphates (diethyl [2-(phenylcarbamoyl)phenyl] phosphates) for the inhibition of acetylcholinesterase (AChE) from electric eel (Electrophorus electricus L.) and butyrylcholinesterase (BChE) from equine serum. Ellman's spectrophotometric method was used. The inhibitory activity (expressed as IC₅₀ values) was compared with that of the established drugs galantamine and rivastigmine. Salicylanilide diethyl phosphates showed significant activity against both cholinesterases with IC₅₀ values from 0.903 to 86.3 μM. IC₅₀s for BChE were comparatively lower than those obtained for AChE. All of the investigated compounds showed higher inhibition of AChE than rivastigmine, and six of them inhibited BChE more effectively than both rivastigmine and galantamine. In general, derivatives of 4-chlorosalicylic acid showed enhanced activity when compared to derivatives of 5-halogenated salicylic acids, especially against BChE. The most effective inhibitor of AChE was O-{5-chloro-2-[(3-bromophenyl)carbamoyl]phenyl} O,O-diethyl phosphate with IC₅₀ of 35.4 μM, which is also one of the most potent inhibitors of BChE. O-{5-Chloro-2-[(3,4-dichlorophenyl)carbamoyl]phenyl} O,O-diethyl phosphate exhibited in vitro the strongest inhibition of BChE (0.90 μM). Salicylanilide diethyl phosphates act as pseudo-irreversible cholinesterases inhibitors.

Full text of DOI:10.1016/j.bioorg.2014.11.005

Bioorganic Chemistry 58 (2015) 48–52
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Salicylanilide diethyl phosphates as cholinesterases inhibitors
a
b
b
a,
⇑
´
ˇ
ˇ
Martin Krátky , Šárka Štepánková , Katarína Vorcáková , Jarmila Vinšová
a Department of Inorganic and Organic Chemistry, Faculty of Pharmacy, Charles University in Prague, Heyrovského 1203, 500 05 Hradec Králové, Czech Republic
b Department of Biological and Biochemical Sciences, Faculty of Chemical Technology, University of Pardubice, Studentská 573, 532 10 Pardubice, Czech Republic
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 18 August 2014
Available online 13 November 2014
Based on the presence of dialkyl phosphate moiety, we evaluated twenty-seven salicylanilide diethyl
phosphates (diethyl [2-(phenylcarbamoyl)phenyl] phosphates) for the inhibition of acetylcholinesterase
(AChE) from electric eel (Electrophorus electricus L.) and butyrylcholinesterase (BChE) from equine serum.
Ellman’s spectrophotometric method was used. The inhibitory activity (expressed as IC50 values) was
compared with that of the established drugs galantamine and rivastigmine. Salicylanilide diethyl phos-
Keywords:
Acetylcholinesterase
Butyrylcholinesterase
Enzyme inhibition
Organophosphate
phates showed significant activity against both cholinesterases with IC50 values from 0.903 to 86.3 lM.
IC50s for BChE were comparatively lower than those obtained for AChE. All of the investigated compounds
showed higher inhibition of AChE than rivastigmine, and six of them inhibited BChE more effectively than
both rivastigmine and galantamine. In general, derivatives of 4-chlorosalicylic acid showed enhanced
activity when compared to derivatives of 5-halogenated salicylic acids, especially against BChE. The
most effective inhibitor of AChE was O-{5-chloro-2-[(3-bromophenyl)carbamoyl]phenyl} O,O-diethyl
Salicylanilide diethyl phosphate
phosphate with IC50 of 35.4
lM, which is also one of the most potent inhibitors of BChE. O-{5-Chloro-
2-[(3,4-dichlorophenyl)carbamoyl]phenyl} O,O-diethyl phosphate exhibited in vitro the strongest inhibi-
tion of BChE (0.90
inhibitors.
l
M). Salicylanilide diethyl phosphates act as pseudo-irreversible cholinesterases
Ó 2014 Elsevier Inc. All rights reserved.
1. Introduction
The number of patients suffering from AD increased continu-
ously during the last decades. One typical characteristic feature
Acetylcholine (ACh) is a cholinergic neurotransmitter interact-
ing with either nicotinic or muscarinic receptors thereby affecting
function of postsynaptic cells. Signalling action of ACh is termi-
nated by the action of acetylcholinesterase (AChE; E.C. 3.1.1.7)
and butyrylcholinesterase (BChE; E.C. 3.1.1.8), which control ACh
level by its hydrolysis. Both enzymes are widely distributed
throughout the body; however, AChE remains the major cholines-
terase within the human brain. BChE does not possess the same
affinity for ACh as AChE does [1,2].
Inhibitors of cholinesterases (ChE) have been used in the treat-
ment of various diseases, e.g., myasthenia gravis, Alzheimer’s
disease (AD) and some other dementias [3], parasitic infections
[4], glaucoma, obstipation or to antagonize muscle relaxation [5].
Pesticides or chemical warfare nerve agents belong to their other
applications [3,5].
of AD is a decreased level of ACh. The changes result in a cognitive
impairment. Based on cholinergic hypothesis, inhibition of ChE
represents one of the major pharmacological interventions for
AD. During the progress of AD, BChE activity is increased. Drugs
which have been introduced into the treatment of AD are non-
selective (rivastigmine) or AChE-selective inhibitors (galantamine
or donepezil) [1,5]. In addition to the increased level of ACh, recent
findings indicate that cholinesterase inhibitors can attenuate neu-
ronal damage and protect them from cellular death, and therefore
might affect AD pathogenesis and delay the progression. This
mechanism seems to be complex [6].
Compounds based on phosphoric moieties are well-known
anticholinesterases agents including substituted diethyl phenyl
phosphates, e.g., paraoxon. They cause irreversible inhibition of
both AChE and BChE [5,7]. Organophosphorus-based molecules
act as inhibitors of the esteratic part of ChE by interaction with ser-
ine providing stable esters. Organophosphorus compounds create
stable, covalently bound adducts with spontaneous dissociation
once covalently connected with serine hydroxyl [3].
Abbreviations: ACh, acetylcholine; ATCh, acetylthiocholine; AD, Alzheimer’s
disease; AChE, acetylcholinesterase; BChE, butyrylcholinesterase; ChE, cholinester-
ases; DTNB, 5,50-dithiobis-2-nitrobenzoic acid; TNB, 5-thio-2-nitrobenzoic acid.
Organophosphates have been also investigated as potential
insecticides [8–10], herbicides [11], or antifungal agents [12].
⇑
Corresponding author. Fax: +420 495067166.
E-mail address: jarmila.vinsova@faf.cuni.cz (J. Vinšová).
http://dx.doi.org/10.1016/j.bioorg.2014.11.005
0045-2068/Ó 2014 Elsevier Inc. All rights reserved.

´
49
M. Krátky et al. / Bioorganic Chemistry 58 (2015) 48–52
A long-acting irreversible inhibitor of AChE metrifonate (trichlor-
fon) has been evaluated as potential drug for the treatment of AD
[13]. Metrifonate is a prodrug which is activated non-enzymati-
cally into dichlorvos (2,2-dichlorovinyl dimethyl phosphate) [4].
Salicylanilide-like derivatives have exhibited a wide range of
interesting biological activities [14–20]. Previously, two groups of
salicylanilide derivatives have been reported as cholinesterases
inhibitors – salicylanilide N-alkyl carbamates [21] and, more
importantly, O,O-diethyl thiophosphates (phosphorothioates)
which were described as potent inhibitors of both AChE and BChE
with IC50 values in the micromolar range [22]. The fact that orga-
nophosphate pesticides acting via ChE inhibition are more toxic
than their thioforms [3] inspired us to the evaluation of salicylan-
ilide diethyl phosphates (oxygen-isosteres of salicylanilide diethyl
thiophosphates [22]) against both AChE a BChE.
Acetylcholinesterase was obtained from electric eel (Electropho-
rus electricus L.) and butyrylcholinesterase was from equine serum.
Rivastigmine and galantamine were involved as reference drugs.
2.3. Investigation of inhibition type
Three salicylanilide diethyl phosphate derivatives (15, 24, 27;
see Table 1) were used for investigation of mechanism of cholines-
terases inhibition. For each of them three different concentrations
of inhibitor were chosen according to their IC50 values. The purpose
was to observe the effect of inhibitor on enzyme activity (A) in
time. On this basis, it is possible to distinguish reversible and irre-
versible inhibition [27,28].
The enzyme activity was determined using spectrophotometric
Ellman’s method. Pursuant the procedure described in [29], the
determination was performed subsequently: The reaction mixture
containing phosphate buffer, AChE or BChE and chosen salicylani-
lide derivative (in one of the chosen concentrations) was prepared
and intensively stirred. In given times (5, 10, 15, 20, 30, 40, 50, 60,
80, 240 and 1380 min), DTNB and ATCh were added to the sample
withdrawn from reaction mixture, quickly mixed and absorbance
was measured. Consequently the enzyme activity was determined.
Based on knowledge of enzyme activity in absence of inhibitor (i.e.
100% activity), the percentages of residual enzyme activity in pres-
ence of inhibitor were calculated. Then the dependence of
logarithm of percentage of residual enzyme activity (log % A) vs.
time was constructed. Based on these kinetic data, it is possible
to distinguish reversible, pseudo-irreversible and irreversible
inhibition.
Salicylanilide diethyl phosphates (Fig. 1) were reported as
potential antimicrobial agents against both drug-susceptible and
resistant strains of Mycobacterium tuberculosis, atypical mycobac-
teria, Gram-positive bacteria and some fungal species. Addition-
ally, they share alleviated cytotoxicity when compared to parent
salicylanilides [23].
2. Materials and methods
2.1. Chemistry
The synthetic pathway for salicylanilide diethyl phosphates 1–
27 (diethyl [(2-phenylcarbamoyl)phenyl] phosphates) was pub-
lished previously by our group [23]. They were obtained by a quite
simple procedure (Scheme 1). First, salicylanilides were prepared
by the reaction of appropriate salicylic acids with anilines in the
presence of phosphorus trichloride under microwave irradiation
[14]. In the next step, salicylanilide triethyl ammonium salts
generated in situ were esterified with diethyl chlorophosphate at
ambient temperature [23].
3. Results and discussion
3.1. Chemistry
Salicylanilides were obtained with the efficiency about 80–95%.
The general yield of salicylanilide diethyl phosphates 1–27
(Table 1) ranged from 11% up to 78% [23]. Some comparatively
low yields were caused by isolation and purification process, while
the reactions were monitored repeatedly by thin layer chromatog-
raphy till all reactions were complete (until 2 h for all compounds).
2.2. Determination of IC50 for cholinesterases
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 [24]. This
method is widely used for the evaluation of cholinesterase activity
and screening the efficiency of ChE inhibitors. Cholinesterase activ-
ity is measured indirectly by quantifying the concentration of the
5-thio-2-nitrobenzoic acid (TNB) ion formed in the reaction
between the thiol reagent 5,50-dithiobis-2-nitrobenzoic acid
(DTNB) and thiocholine, a product of substrate hydrolysis (i.e.,
acetylthiocholine, ATCh) by cholinesterases [25]. All of the tested
compounds were dissolved in 0.01 M dimethyl sulphoxide and
then diluted in demineralised water to 0.001 M and 0.0001 M.
Ellman’s method was modified slightly according to Zdrazilova
et al. [26].
3.2. In vitro cholinesterases inhibition
The ability of the investigated salicylanilide derivatives 1–27 to
inhibit AChE from electric eel and BChE from equine serum was
screened in vitro using modified Ellman’s method. The effective-
ness of the inhibitors is expressed as IC50, representing the concen-
tration of an inhibitor required for 50% inhibition of the enzyme.
The obtained results were compared with rivastigmine and galan-
thamine (Table 1). These standards were chosen due to their differ-
ent structures. Rivastigmine is an acylating pseudo-irreversible
carbamate inhibitor that inhibits AChE as well as BChE, while
galanthamine acts as a non-acylating competitive reversible inhib-
itor. Furthermore, it modulates allosterically nicotinic ACh
receptors. The choice of these drugs with different mechanism of
action can provide relevant results.
O
R2
N
With respect to the inhibition of cholinesterases, all tested
phosphates 1–27 exhibited good inhibitory activity, with IC50 val-
R1
H
O
P
O
ues from 0.903 to 86.3 lM (Table 1). Salicylanilide diethyl phos-
phates 1–27 could be divided into two groups based on their
substitution and activity. Group 1 includes derivatives of 5-bromo-
salicylic and 5-chlorosalicylic acids 1–18 and Group 2 includes
4-chlorosalicylic acid derivatives 19–27. In general, most of the
tested compounds inhibited butyrylcholinesterase more effectively
than acetylcholinesterase, sometimes by several fold; only for 1 are
O
O
Fig. 1. Salicylanilide diethyl phosphates 1–27 (diethyl [2-(phenylcarba-
moyl)phenyl] phosphates; R1 = 4-Cl, 5-Cl, 4-Br; R2 = 3-Cl, 4-Cl, 3,4-diCl, 3-Br, 4-Br,
3-F, 4-F, 3-CF3, 4-CF3).

´
M. Krátky et al. / Bioorganic Chemistry 58 (2015) 48–52
50
Scheme 1. Synthesis of diethyl [(2-phenylcarbamoyl)phenyl] phosphates 1–27 (Et3N = triethylamine; R1 for esters = 4-Cl, 4-Br, 5-Cl, R2 = 3-Cl, 4-Cl, 3,4-diCl, 3-Br, 4-Br, 3-F,
4-F, 3-CF3, 4-CF3)
Table 1
IC50 values against acetylcholinesterase and butyrylcholinesterase.
Code
R
R1
IC50 for AChE (lM)
IC50 for BChE (lM)
Selectivity to BChE
Group 1
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
4-Br
4-Br
4-Br
4-Br
4-Br
4-Br
4-Br
4-Br
4-Br
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
3-F
3-Cl
4-Cl
3,4-di-Cl
3-CF3
4-CF3
3-Br
4-Br
4-F
3-Cl
4-Cl
3-F
4-F
4-Br
3,4-di-Cl
3-Br
3-CF3
4-CF3
69.8 3.4
79.9 0.3
45.5 1.2
55.2 0.3
46.1 1.9
56.7 0.9
70.3 1.7
50.8 1.2
53.7 0.4
64.3 2.6
60.9 4.4
61.1 2.1
48.5 0.3
59.2 0.7
80.9 2.2
63.3 4.3
51.5 1.8
41.6 0.3
68.3 3.7
24.4 3.3
19.9 0.8
49.6 1.5
14.3 3.7
10.3 0.1
18.0 0.0
13.0 1.7
22.7 1.1
28.1 2.5
42.1 1.0
31.6 1.0
20.5 0.1
40.2 2.4
40.3 4.0
16.9 0.3
24.8 0.7
20.9 1.1
1.0
3.3
2.3
1.1
3.2
5.5
3.9
3.9
2.4
2.3
1.4
1.9
2.4
1.5
2.0
3.7
2.1
2.0
Group 2
19
20
21
22
23
24
25
26
5-Cl
5-Cl
5-Cl
5-Cl
5-Cl
5-Cl
5-Cl
5-Cl
5-Cl
3-Cl
3-Br
3-F
4-F
4-Br
4-Cl
3,4-di-Cl
3-CF3
4-CF3
41.1 0.5
35.4 0.5
60.2 3.5
80.8 5.9
55.1 0.5
60.9 0.8
45.5 4.4
71.9 7.2
86.3 4.9
3.68 0.1
1.77 0.1
11.5 0.3
4.43 0.2
8.76 0.5
5.01 0.5
0.903 0.003
3.53 0.05
9.84 0.06
11.2
20.0
5.2
18.2
6.3
12.2
50.4
20.4
8.8
27
Rivastigmine
Galantamine
501 3.08
19.95 0.20
7.96 0.59
–
–
4
0.13
AChE and BChE inhibition is expressed as the mean SD (n = 3 experiments). The best results for each enzyme are shown in bold.
Selectivity to BChE: IC50 for AChE/IC50 for BChE.
IC50s for both enzymes comparable. Similarly, Kaboudin et al. [30]
found that S-substituted-O,O0-diethyl phosphorothioates behaved
more selectively towards BChE.
Members of Group 2 (5-chloro derivatives synthesized from 4-
chlorosalicylic acid) are more effective inhibitors of BChE (IC50
form 0.9 to 11.5 M) than derivatives of Group 1 (10.3–68.3 M).
s
l
l
The most effective inhibitor of AChE is O-{5-chloro-2-[(3-
bromophenyl)carbamoyl]phenyl} O,O-diethyl phosphate 20 with
Similar to the trend shown for acetylcholinesterase inhibition,
there was no observed effect of salicylic ring substitution by 4-
chlorine or 4-bromine on the power of inhibition. In general, there
is no pronounced effect of aniline substitution by various substitu-
ents at different positions on both cholinesterase inhibitions, only
particular aspects were identified. For both AChE and BChE, esters
containing 4-substituted anilines exhibited higher activity then
isomeric 3-substituted anilines in Group 1. The same relationship
but only for BChE was found in Group 2 with one exception (fluo-
rinated molecules 21 and 22). For inhibition of both ChE in Group
1, anilines bearing trifluoromethyl group confer a slightly better
activity.
an IC50 of 35.4
lM, which is also the second-best inhibitor of BChE
(IC50 = 1.77 M) of all evaluated compounds. O-{5-Chloro-2-[(3,4-
l
dichlorophenyl)carbamoyl]phenyl} O,O-diethyl phosphate 25
exhibited the best in vitro inhibition of BChE, with an IC50 lower
than 1 lM (0.903 lM).
O-(5-Chloro-2-{[(4-trifluoromethyl)phenyl]carbamoyl}phenyl
O,O-diethyl phosphate 27 was assayed as the least potent inhibitor
of AChE (IC50 = 86.3
O-{4-bromo-2-[(3-fluorophenyl)carbamoyl]phenyl}
phosphate 1 (IC50 = 68.3 M).
l
M) and the least effective inhibitor of BChE is
O,O-diethyl
l
The activities of phosphates 1–27 were compared with those of
galantamine and rivastigmine which are clinically used for the
treatment of dementia. All tested compounds 1–27 showed greater
inhibition of AChE than rivastigmine but not galantamine, and six
of them (19, 20, 22, 24–26) inhibited BChE more effectively than
both drugs.
IC50s of all derivatives 1–27 for AChE are within a comparatively
narrow concentration range (35–86 lM). The situation for BChE is
similar although there is a Group 2 which contains more potent
compounds. Based on literature [21] and our experiments
(Section 3.2.1), we presume that this is a consequence of the mech-
anism of action – phosphorylation of serine localized in the active

´
51
M. Krátky et al. / Bioorganic Chemistry 58 (2015) 48–52
2.00
1.90
1.80
1.70
1.60
1.50
30 µM
60 µM
90 µM
0
200
400
600
800
1000
1200
1400
t (min)
Fig. 2. The dependence log % A vs. time. Enzyme: acetylcholinesterase, derivative: 24, concentration of inhibitor: 30
l
M (rhomb), 60 lM (square), 90 lM (triangle).
2.00
2 µM
5 µM
8 µM
1.80
1.60
1.40
1.20
1.00
0
200
400
600
800
1000
1200
1400
t (min)
Fig. 3. The dependence log % A vs. time. Enzyme: butyrylcholinesterase, derivative: 24, concentration of inhibitor: 2 lM (rhomb), 5 lM (square), 8 lM (triangle).
site of enzyme. For this action, the phosphate part of molecule is
the key fragment of salicylanilide diethyl phosphates, while sali-
cylanilide core serves mainly as a carrier of this phosphorylating
moiety. Theoretically, the differences in IC50s may consist in the
fact that salicylanilide moiety influences probably the orientation
and binding of the inhibitor in the active site of enzyme and its
electronic and/or steric effects may have an impact on subsequent
reaction of phosphorus with enzyme hydroxyl group. Keeping in
mind that hydrophobicity favours the entering of inhibitors into
the active site gorge of the enzyme [21], the differences in
lipophilicity may also play some role.
Surprisingly, salicylanilide diethyl phosphates 1–27 showed
significantly weaker inhibition of AChE than their sulphur isosters,
salicylanilide diethyl thiophosphates (phosphorothioates). For
BChE, the average activity was higher in the case of thiophos-
phates, but six phosphates (19, 20, 22, 24, 25, and 26) exhibited
lower IC50 values than all thiophosphates [22].
We used the procedure described in Section 2.3, which enables
to distinguish reversible, pseudo-irreversible and irreversible inhi-
bition on the basis of the changes of enzyme activity in presence of
inhibitor. For reversible inhibition, the activity of enzyme goes
down immediately but the inhibiting molecule is bound to the
enzyme for a short period of time, after which the inhibitor and
enzyme molecules dissociate and enzyme activity is restored. Dur-
ing pseudo-irreversible inhibition, the inhibitor binds to the
enzyme molecule, but the bond is broken down more slowly,
delaying the return of enzymatic activity to initial state. In the case
of irreversible inhibitors, the reaction of enzyme and inhibitor is
not instantaneous. Instead, there is a time-dependent decrease in
enzymatic activity. Irreversible inhibitors decrease enzyme activity
successively and the dependence log % A vs. time in presence of
such inhibitor is linear. These inhibitors bind permanently to the
enzyme, and thus the enzyme does not become available again
[31,32].
Salicylanilide diethyl phosphates 1 demonstrated comparable
or slightly higher IC50 values for AChE than salicylanilide N-alkyl
carbamates [21].
For investigation of cholinesterases type of inhibition we chose
three salicylanilide derivatives mentioned above (15, 24, 27). For
each of them three different concentrations were chosen according
to the value of IC50. The first concentration was lower than IC50, the
3.2.1. Type of cholinesterases inhibition determination
second was close to IC50 and the third was higher than IC50.
We also investigated the type of inhibition for both AChE and
BChE. For this advance experiment, we selected one derivative
with higher inhibitory activity (24; Group 2), one with poorer
IC50s (15; Group 1) and diethyl phosphate 27 (Group 2) as a repre-
sentative of derivatives with an intermediate activity towards
BChE within this series.
All obtained dependences log % A vs. time for three chosen
derivatives and both enzymes show very similar course. First the
dependence log % A vs. time in presence of inhibitor shows
decreasing course. This decreasing of enzyme activity is stopped
after approximately 30 min and longer incubation period does
not lead to further drop of enzyme activity. Moreover, after

´
M. Krátky et al. / Bioorganic Chemistry 58 (2015) 48–52
52
240 min of incubation the activity slightly increases. The depen-
dences log % A vs. time in presence of derivative 24 are presented
in Fig. 2 (inhibition of AChE) and Fig. 3 (inhibition of BChE) as
examples.
The results obtained for remaining two salicylanilide diethyl
phosphates 15 and 27 showed identical trends and curves (data
not shown).
The work was financially supported by the Research project IGA
NT 13346 (2012).
This work was also supported by the Faculty of Chemical Tech-
nology, University of Pardubice and project SGSFChT_2014005.
We want to thank to J. Urbanová, M.A., for the language help.
References
In our opinion, the obtained results showed that three investi-
gated salicylanilide diethyl phosphates do not act as irreversible
nor reversible inhibitors. Both ChE are inhibited by chosen deriva-
tives for longer period, but none of observed dependences log % A
vs. time shows the typical course for irreversible inhibition (i.e. the
linear decrease of log % A in time). Owing to the longer period of
enzyme inhibition, we can conclude that tested salicylanilide
derivatives act as pseudo-irreversible inhibitors.
[1] S. Schwarz, S.D. Lucas, S. Sommerwerk, R. Csuk, Bioorg. Med. Chem. 22 (2014)
3370–3378.
[2] A. Saeed, P.A. Mahesar, S. Zaib, M.S. Khan, A. Matin, M. Shahid, J. Iqbal, Eur. J.
Med. Chem. 78 (2014) 43–53.
[3] M. Pohanka, Biomed. Pap. 155 (2011) 219–229.
[4] R. Heinig, D. Zimmer, S. Yeh, G.J. Krol, J. Chromatogr. B 741 (2000) 257–269.
[5] B. Ibach, E. Haen, Curr. Pharm. Des. 10 (2004) 231–251.
[6] P.T. Francis, A. Nordberg, S.E. Arnold, Trends Pharmacol. Sci. 26 (2005) 104–
111.
[7] J. Bajgar, Adv. Clin. Chem. 38 (2004) 151–216.
[8] L.L. Murdock, T.L. Hopkins, J. Agric. Food Chem. 16 (1968) 954–958.
[9] B. Hetnarski, R.D. O0Brien, J. Agric. Food Chem. 20 (1972) 543–546.
[10] P.P. Mager, E. Seese, Pharmazie 37 (1982) 729–730.
[11] F. Kaschani, S. Nickel, B. Pandey, B.F. Cravatt, M. Kaiser, R.A.L. van der Hoorn,
Bioorg. Med. Chem. 20 (2012) 597–600.
[12] N. Latif, F.M. Asaad, H. Hosni, Liebigs Ann. Chem. 1987 (1987) 495–498.
[13] J.M. Lopez-Arrieta, L. Schneider, Cochrane Database Syst. Rev. 2006 (2006)
(Article No.: CD003155.pub3).
4. Conclusions
A series of twenty-seven salicylanilide diethyl phosphates as
representatives of organophosphorus compounds were evaluated
against acetylcholinesterase and butyrylcholinesterase. All of the
compounds shared significant inhibition of both cholinesterases
with IC50 values in the micromolar range. It was observed that
the activity against butyrylcholinesterase was comparatively
higher than those obtained for acetylcholinesterase. Presented
derivatives showed a clear superiority to rivastigmine against ace-
tylcholinesterase and six of them exhibited more potent inhibition
of butyrylcholinesterase than both rivastigmine and galantamine.
In general, derivatives of 4-chlorosalicylic acid showed better inhi-
bition results. The mechanism of cholinesterases inhibition was
determined experimentally as pseudo-irreversible.
ˇ
[14] M. Krátky´, J. Vinšová, V. Buchta, K. Horvati, S. Bösze, J. Stolaríková, Eur. J. Med.
Chem. 45 (2010) 6106–6113.
[15] A. Imramovsky, M. Pesko, J.M. Ferriz, K. Kralova, J. Vinsova, J. Jampilek, Bioorg.
Med. Chem. Lett. 21 (2011) 4564–4567.
[16] M. Krátky
´, J. Vinšová, Mini-Rev. Med. Chem. 11 (2011) 956–967.
[17] M. Krátky´, J. Vinšová, Curr. Pharm. Des. 17 (2011) 3494–3505.
ˇ
[18] M. Krátky´, J. Vinšová, E. Novotná, V. Wsól, V. Ulmann, J. Stolaríková, S.
Fernandes, S. Bhat, J.O. Liu, Tuberculosis (Edinb). 92 (2012) 434–439.
[19] J. Wang, H. Wu, J.M. Li, Q.L. Xu, G.W. He, G.C. Zhong, Y.C. Zhang, J. Org. Chem.
33 (2013) 1026–1034.
[20] A. Fomovska, R.D. Wood, E. Mui, J.P. Dubey, L.R. Ferreira, M.R. Hickman, P.J. Lee,
S.E. Leed, J.M. Auschwitz, W.J. Welsh, C. Sommerville, S. Woods, C. Roberts, R.
McLeod, J. Med. Chem. 55 (2012) 8375–8391.
[21] A. Imramovsky, S. Stepankova, J. Vanco, K. Pauk, J. Monreal-Ferriz, J. Vinsova, J.
Jampilek, Molecules 17 (2012) 10142–10158.
´
ˇ
ˇ
[22] J. Vinšová, M. Krátky, M. Komlóová, E. Dadapeer, Š. Štepánková, K. Vorcáková, J.
Conflict of interest
ˇ
Stolaríková, Molecules 19 (2014) 7152–7168.
ˇ
[23] J. Vinšová, J. Kozic, M. Krátky´, J. Stolaríková, J. Mandíková, F. Trejtnar, V. Buchta,
The authors declare no conflict of interest.
Bioorg. Med. Chem. 22 (2014) 728–737.
[24] S.Y. Ou, K.C. Kwok, Y. Wang, H.Y. Bao, Food Chem. 88 (2004) 317–320.
[25] G. Sinko, M. Calic, A. Bosak, Z. Kovarik, Anal. Biochem. 370 (2007) 223–227.
[26] P. Zdrazilova, S. Stepankova, K. Komers, K. Ventura, A. Cegan, Z. Naturforsch. C
59 (2004) 293–296.
Acknowledgments
[27] S.E. Szedlacsek, R.G. Duggleby, Method. Enzymol. 249 (1995) 144–180.
[28] J. Singh, R.C. Petter, T.A. Baillie, A. Whitty, Nat. Rev. Drug Discov. 10 (2011)
307–317.
[29] E. Carletti, N. Aubrek, E. Gillon, M. Loiodice, Y. Nicolet, J.C. Fontecilla-Camps, P.
Masson, H. Thiermann, F. Nachon, F. Worek, Biochem. J. 421 (2009) 97–106.
[30] B. Kaboudin, S. Emadi, A. Hadizadeh, Bioorg. Chem. 37 (2009) 101–105.
[31] M.W. Jann, K.L. Shirley, G.W. Small, Clin. Pharmacokinet. 41 (2002) 719–739.
[32] M. Bartolini, V. Cavrini, V. Andrisano, J. Chromatogr. A 1144 (2007) 102–110.
This publication is a result of the project implementation:
‘‘Support of establishment, development, and mobility of quality
research teams at the Charles University’’, project number
CZ.1.07/2.3.00/30.0022, supported by The Education for Competi-
tiveness Operational Programme (ECOP) and co-financed by the
European Social Fund and the state budget of the Czech Republic.