Investigation of salicylanilide and 4-chlorophenol-based N-monosubstituted carbamates as potential inhibitors of acetyl- and butyrylcholinesterase

Article abstract of DOI:10.1016/j.bioorg.2018.07.017

Based on the presence of carbamate moiety, twenty salicylanilide N-monosubstituted carbamates concomitantly with their parent salicylanilides and five newly prepared 4-chlorophenyl carbamates obtained from isocyanates were investigated using Ellman's method for their in vitro inhibitory activity against acetylcholinesterase (AChE) from electric eel and butyrylcholinesterase (BChE) from equine serum. The carbamates and salicylanilides exhibited mostly a moderate inhibition of both cholinesterase enzymes with IC₅₀ values ranging from 5 to 235 μM. IC₅₀ values for AChE were in a narrower concentration range when compared to BChE, but many of the compounds produced a balanced inhibition of both cholinesterases. The derivatives were comparable or superior to rivastigmine for AChE inhibition, but only a few of carbamates also for BChE. Several structure-activity relationships were identified, e.g., N-phenethylcarbamates produce clearly favourable BChE inhibition. The compounds also share convenient physicochemical properties for CNS penetration.

Full text of DOI:10.1016/j.bioorg.2018.07.017

Accepted Manuscript
Investigation of Salicylanilide and 4-Chlorophenol-based N-Monosubstituted
Carbamates as Potential Inhibitors of Acetyl- and Butyrylcholinesterase
Martin Krátký, Š árka Štěpánková, Katarína Vorč áková, Jarmila Vinšová
PII:
S0045-2068(18)30572-8
DOI:
https://doi.org/10.1016/j.bioorg.2018.07.017
YBIOO 2436
Reference:
Bioorganic Chemistry
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Revised Date:
Accepted Date:
11 June 2018
13 July 2018
15 July 2018
Please cite this article as: M. Krátký, S. Štěpánková, K. Vorč áková, J. Vinšová, Investigation of Salicylanilide and
4-Chlorophenol-based N-Monosubstituted Carbamates as Potential Inhibitors of Acetyl- and Butyrylcholinesterase,
Bioorganic Chemistry (2018), doi: https://doi.org/10.1016/j.bioorg.2018.07.017
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Investigation
of
Salicylanilide
and
4-Chlorophenol-based
N-
Monosubstituted Carbamates as Potential Inhibitors of Acetyl- and
Butyrylcholinesterase
Martin Krátký a,*, Šárka Štěpánková b, Katarína Vorčáková b, and Jarmila Vinšová a
a Department of Organic and Bioorganic Chemistry, Faculty of Pharmacy, Charles University,
Akademika 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
*Corresponding author. Akademika Heyrovského 1203, 500 05 Hradec Králové, Czech
Republic. E-mail address: martin.kratky@faf.cuni.cz, tel.: +420-495067343, fax: +420-
495067166.
Abstract
Based on the presence of carbamate moiety, twenty salicylanilide N-monosubstituted
carbamates concomitantly with their parent salicylanilides and five newly prepared 4-
chlorophenyl carbamates obtained from isocyanates were investigated using Ellman’s method
for their in vitro inhibitory activity against acetylcholinesterase (AChE) from electric eel and
butyrylcholinesterase (BChE) from equine serum. The carbamates and salicylanilides
exhibited mostly a moderate inhibition of both cholinesterase enzymes with IC50 values
ranging from 5 to 235 µM. IC50 values for AChE were in a narrower concentration range
when compared to BChE, but many of the compounds produced a balanced inhibition of both
cholinesterases. The derivatives were comparable or superior to rivastigmine for AChE
inhibition, but only a few carbamates also for BChE. Several structure-activity relationships
were identified, e.g., N-phenethylcarbamates produce clearly favourable BChE inhibition. The
compounds also share convenient physico-chemical properties for CNS penetration.
Keywords
acetylcholinesterase, butyrylcholinesterase, carbamate, enzyme inhibition, in vitro inhibition,
salicylanilide*
*
Abbreviations: ACh: acetylcholine; AD: Alzheimer’s disease; AChE: acetylcholinesterase; BBB: blood brain barrier; BChE:
butyrylcholinesterase; CNS: central nervous system; PSA: polar surface area.
1

1. Introduction
Acetylcholine (ACh) represents the neurotransmitter capable to interact with muscarinic or
nicotinic receptors. The action of ACh in synapses is terminated by the action of the enzyme
acetylcholinesterase (AChE; EC 3.1.1.7) and butyrylcholinesterase (BChE, EC 3.1.1.8).
AChE remains the major cholinesterase within the human brain although BChE can substitute
for AChE constitutively. The esteratic site, where ACh is hydrolysed into choline and acetate,
contains the catalytic triad of three amino acids: serine, histidine and glutamate [1,2].
Degeneration of the cholinergic projection from the nucleus basalis Meynerti and the
deficiency of ACh belongs to the pathological features related to Alzheimer’s disease (AD)
[2], the most frequent dementia. AD is characterized basically by a progressive deterioration
in cognition, in particular the memory domain, daily functions and behaviour. The incidence
rate increases exponentially with age. Unfortunately, the global spread and incidence of this
disease as well as other dementias has been increased continuously worldwide during the last
period [3].
Based on cholinergic hypothesis, inhibition of cholinesterases in central nervous system
(CNS) represents the keystone of current therapy of patients suffering from AD. The
cholinergic augmentation improves the symptoms [4]. Current drugs approved for the
treatment
of
AD
are
non-selective
(carbamate
rivastigmine,
(S)-3-[1-
(dimethylamino)ethyl]phenyl ethyl(methyl)carbamate) or AChE-selective inhibitors
(galantamine, donepezil) [1,5] and also an adamantane derivative memantine with ACh-
independent mechanism of action (N-methyl-D-aspartate receptor antagonist). In early stages
of AD, the level of AChE is increased at a much higher rate than level of BChE. On the other
hand, as AD progresses level of AChE declines in the brain, but BChE level remains
unchanged or even it is increased. Moreover, in the later stages of AD, BChE substitutes the
function of AChE (i.e., hydrolysis of ACh) [6,7]. That is why BChE inhibition in addition to
AChE may provide a therapeutic benefit not only in Alzheimer’s dementia [1,8]. Furthermore,
selective inhibitors of BChE do not share the adverse cholinergic effects typical for AChE
inhibitors and that is why potent and/or selective BChE inhibitors represents a promising
trend in drug research [9]. Up-to-date, the administration of cholinesterases inhibitors is not
considered to be a causative therapy. However, recent in vitro findings indicate that AChE
inhibitors can attenuate neuronal damage and delay their cellular death, thus affecting also
pathogenesis [10].
It is beyond that searching for novel drugs including AChE and BChE inhibitors is an
imperative research topic of a high healthcare and socio-economic significance [9,11].
Moreover, AChE and BChE inhibitors can be used for the treatment of other diseases, not
only AD dementias [9].
Carbamate functional group can be found in many of approved therapeutic agents used as
HIV-protease inhibitors (ritonavir), antiepileptic drugs (felbamate), anthelmintic
(mebendazole), anti-cancer (entinostat) and non-opioid analgesic drugs (flupirtine) [12],
illustratively.
Carbamate moiety has been proved as a promising pharmacophore for the inhibition of both
cholinesterases. The majority of carbamates behave as substrate analogues of ACh. They
enter the gorge of AChE where they carbamoylate the serine hydroxyl group of the esteratic
site providing a covalent bond being more stable than those resulted from reaction with
natural substrate ACh. This carbamoyl-serine complex can be hydrolysed spontaneously
2

slowly (i.e., pseudo-irreversible inhibition). However, carbamates can produce also non-
covalent competitive inhibition [2,13-16].
Importantly, novel carbamates of various structures have been proposed and studied as
inhibitors of both AChE and BChE with the activity in low micromolar range [9,17,18]. O-
Substituted salicylanilide derivatives have been reported as inhibitors of cholinesterases: N-n-
alkylcarbamates (IC50 for AChE ≥21.8 μM) [16], N,N-disubstituted carbamates and
thiocarbamates (IC50 ≥1.6 μM) [19], diethyl thiophosphates [20] and diethyl phosphates [21]
(IC50 ≥8.0 μM, concomitantly the most effective salicylanilide-based AChE inhibitors
reported so far, and ≥0.90 μM, respectively).
In accordance with these data, we evaluated our antimicrobial salicylanilide N-cycloalkyl,
phenyl and phenylalkyl carbamates [22] 1-4, their parent salicylanilides SAL-1-SAL-4 and
newly synthesized analogous 4-chlorophenyl carbamates 5 for their ability to inhibit AChE
and BChE.
2. Materials and Methods
2.1 Chemistry
2.1.1 General methods
All the reagents and solvents were purchased from Sigma-Aldrich (Darmstadt, Germany) or
Penta Chemicals (Prague, Czech Republic) and used as received. The reactions and the purity
of the products were monitored by thin-layer chromatography using a mixture with a ratio of
toluene to ethyl acetate of 4:1 (v/v) as the eluent. The plates were coated with 0.2-mm Merck
60 F254 silica gel (Merck, Darmstadt, Germany) and were visualised by UV irradiation (254
nm). The melting points were determined on a Büchi Melting Point B-540 apparatus (BÜCHI,
Flawil, Switzerland) using open capillaries. The reported values are uncorrected. Infrared
spectra (ATR) were recorded on a FT-IR spectrometer (Nicolet 6700 FT-IR, Thermo Fisher
Scientific, Waltham, MA, USA) in the range of 650–4 000 cm-1. The NMR spectra were
measured in CDCl3 at ambient temperature using a Varian Mercury-Vxbb 300 (300 MHz for
1H and 75.5 MHz for 13C; Varian, Inc., Palo Alto, CA, USA). The chemical shifts δ are given
in ppm with respect to tetramethylsilane as an internal standard. The coupling constants (J)
are reported in Hz. Elemental analysis was performed on an automatic microanalyser CHNS-
O CE instrument (FISONS EA 1110, Milano, Italy).
2.1.2 Synthesis
The microwave-assisted synthesis of salicylanilides SAL-1-SAL-4 and the preparation of N-
monosubstituted salicylanilide carbamates 1-4 was described previously [22].
An equivalent of 4-chlorophenol (1 mmol) was dissolved in dry acetonitrile (MeCN; 5 mL).
Then 1 mmol of triethylamine (139.4 μL) was added in one portion followed by an
appropriate isocyanate (1.1 of equivalents). Isocyanates were added in two portions, portion
after 15 min, and the mixture was stirred at room temperature for additional 2 h (cyclohexyl,
phenyl, benzyl and phenethyl isocyanates) or 12 h (adamantan-1-yl isocyanate). The progress
of the reaction was monitored using TLC. After this time, the reaction mixture was
evaporated partly to initiate the crystallization and then, it was stored at +4 °C for 24 h. The
resulting precipitate was collected by filtration and dried. If necessary, products were
recrystallized from ethyl acetate.
3

The identity of the known compounds (i.e., 5a, and 5b) was established using NMR and IR
spectroscopy by the comparison with previously reported data. Additionally, their purity was
checked by melting points measurement and elemental analysis.
4-Chlorophenyl cyclohexylcarbamate (5a) [23]. White solid; yield 95%; mp 165-167 ºC (lit.
172-174 °C [23]). Anal. Calcd. for C13H16ClNO2 (253.73): C, 61.54; H, 6.36; N, 5.52. Found:
C, 61.42; H, 6.60; N, 5.66.
4-Chlorophenyl phenylcarbamate (5b) [24]. White solid; yield 85%; mp 143-143.5 ºC (lit.
146-148 °C [24]). Anal. Calcd. for C13H10ClNO2 (247.68): C, 63.04; H, 4.07; N, 5.66. Found:
C, 62.95; H, 4.14; N, 5.87.
4-Chlorophenyl benzylcarbamate (5c) [25]. White solid; yield 96%; mp 140-141.5 ºC (lit.
143.6-144.5 °C [25]). Anal. Calcd. for C14H12ClNO2 (261.71): C, 64.25; H, 4.62; N, 5.35.
Found: C, 64.48; H, 4.70; N, 5.11.
4-Chlorophenyl (2-phenylethyl)carbamate (5d) [26]. White solid; yield 93%; mp 131-132 ºC
(lit. 130-132 °C [26]). Anal. Calcd. for C15H14ClNO2 (275.73): C, 65.34; H, 5.12; N, 5.08.
Found: C, 65.09; H, 5.30; N, 5.21.
4-Chlorophenyl (adamantan-1-yl)carbamate (5e). White solid; yield 88%; mp 173.5–175.5 ºC.
IR (ATR): 3357 (N-H), 2910, 2848, 1738, 1720 (C=O), 1623, 1558, 1520, 1486, 1445, 1361,
1346, 1296, 1279, 1201, 1162, 1088, 1007, 973, 915, 855, 836, 813, 761, 741, 693, 681 cm-1.
1H NMR (300 MHz, CDCl3): δ 7.33-7.27 (2H, m, H2, H6), 7.09-7.02 (2H, m, H3, H5), 4.89
(1H, s, NH), 2.13-2.07 (3H, m, CH), 2.01-1.96 (6H, m, C-CH2), 1.70-1.66 (6H, m, CH-CH2).
13C NMR (75 MHz, CDCl3): δ 151.80, 149.43, 130.23, 129.12, 123.04, 51.28, 41.61, 36.18,
29.37. Anal. Calcd. for C17H20ClNO2 (305.80): C, 66.77; H, 6.59; N, 4.58. Found: C, 66.64;
H, 6.47; N, 4.73.
2.2 Determination of physico-chemical parameters
The logP values, molecular weights and topological polar surface areas were determined
using the CS ChemOffice Ultra program (version 16.0, CambridgeSoft, Cambridge, MA,
USA).
2.3 Determination of cholinesterases inhibition
The IC50 values were determined using the spectrophotometric Ellman’s method modified
according to Zdražilová et al. [27], which is a simple, rapid and direct method to determine
the SH and -S-S- group content in proteins [28]. This method is widely used for the screening
of the efficiency of cholinesterases inhibitors. The enzymatic activity is measured indirectly
by quantifying the concentration of the 5-thio-2-nitrobenzoic acid ion formed in the reaction
between the thiol reagent 5,5’-dithio-bis(2-nitrobenzoic acid) and thiocholine, a product of
substrate hydrolysis (i.e., acetylthiocholine) by cholinesterases [29]. The tested compounds were
dissolved in dimethyl sulfoxide and then diluted in demineralised water to 1 µM.
Acetylcholinesterase was obtained from electric eel (Electrophorus electricus L.) and
butyrylcholinesterase was from equine serum. Rivastigmine was used as a reference drug. All
of the enzymes and rivastigmine were purchased from Sigma-Aldrich (Prague, Czech
Republic).
3. Results and Discussion
3.1 Chemistry
4

The synthesis, yields, physical and spectral characteristics of the carbamates 1-4 were
reported in depth by Krátký and Vinšová [22]. Briefly, the synthesis pathway consists in two-
step procedure. First, salicylanilides SAL-1-SAL-4 are obtained using PCl3-mediated
microwave (MW) reaction (yields >85 %) [30]. Then, they are converted to carbamates by the
treatment with a stoichiometric amount of a tertiary base (triethylamine or N,N-
diisopropylethylamine, i.e., Hünig’s base) and an appropriate isocyanate in a mild excess (1.1
of equivalents, Scheme 1) in a suitable solvent (usually acetonitrile, acetone or
tetrahydrofuran). The final carbamates 1-4 were obtained with 50-95% yields [22].
Scheme 1. Synthesis of salicylanilide N-monosubstituted carbamates [22] (R1 = H, Cl; R2 =
H, F, Cl, Br; R3 = cyclohexyl, phenyl, benzyl, phenethyl, adamantan-1-yl)
To evaluate the importance of salicylanilide core and carbamate bond for the biological
activity, we synthesized also five “simplified” analogous 4-chlorophenol-based carbamates 5,
i.e., derivatives without 2-[N-(substituted phenyl)carbamoyl] structural fragment. The
synthesis was similar as used for the carbamates 1-4 (Scheme 2). The reaction was carried in
acetonitrile (MeCN) for 2 hours at ambient temperature. The yields of 5 ranged from 85 to
96 %.
Scheme 2. Synthesis of 4-chlorophenyl N-monosubstituted carbamates (R3 = cyclohexyl, 5a;
phenyl, 5b; benzyl, 5c; phenethyl, 5d; adamantan-1-yl, 5e)
New compounds were characterised by melting points, IR and NMR spectra; their purity was
checked by thin-layer chromatography and elemental analysis. In the IR spectra of 4-
chlorophenyl carbamates 5, sharp and strong bands appear at around 1699–1720 cm-1
(carbamic C=O). The derivatives displayed also well-marked N–H stretch bands (3303–3357
cm-1).
The structures of twenty-five carbamates 1-5 are summarised in Table 1.
3.2 In vitro inhibition of acetylcholinesterase and butyrylcholinesterase
Twenty-five carbamates 1-5 and four salicylanilides SAL-1-SAL-4 were evaluated to affect
the function of AChE from electric eel and BChE from equine serum using modified Ellman’s
spectrophotometric method [28]. The efficacy of inhibitors is expressed as IC50 values, i.e.,
the concentrations required for 50% inhibition of the function of the enzymes. We also
calculated selectivity indices (SI) as a ratio of IC50 value for AChE/IC50 value for BChE to
5

distinguish the selectivity for each enzyme quantitatively. The results obtained were
compared with the activities of rivastigmine, an established carbamate drug used for the
treatment of Alzheimer’s and Parkinson’s dementias. Rivastigmine acts as an acylating
pseudo-irreversible inhibitor of both cholinesterases uniquely.
All the salicylanilides SAL as well as the carbamates 1-5 inhibited significantly both
cholinesterase enzymes with IC50 values from 5.34 to 235.28 µM (Table 1).
Table 1. Structures of salicylanilides SAL-1-SAL-4 and carbamates 1-5 and their IC50 values
for acetylcholinesterase and butyrylcholinesterase
IC50 (AChE)
µM
IC50 (BChE)
µM
Selectivity
AChE/BChE
0.24
Code
R1 R2
R3
H
H
H
H
H
H
H
H
H
H
H
F
F
F
F
F
-
Cyclohexyl
Phenyl
49.71±3.73
49.97±0.93
44.12±1.62
44.00±0.08
57.33±2.71
43.33±0.78
48.27±8.90
49.77±4.32
50.96±0.85
43.88±0.69
61.00±4.33
60.46±0.36
76.53±2.17
45.51±0.62
48.99±1.53
57.71±0.32
51.50±0.54
55.72±1.23
58.25±4.87
40.54±0.78
37.40±2.93
46.17±0.39
46.42±0.29
49.75±2.75
53.71±0.42
45.24±1.33
58.80±1.82
57.37±0.57
57.63±3.02
56.10±1.41
205.73±12.15
89.96±4.63
122.31±12.90
69.33±0.06
72.59±4.08
122.25±1.11
132.99±15.81
80.13±3.02
235.28±15.57
125.13±2.54
23.15±0.68
170.03±2.45
169.60±3.09
70.75±2.73
143.00±13.61
129.68±1.24
5.34±0.21
104.48±2.06
118.74±6.65
55.49±4.50
78.67±0.22
68.34±1.29
43.84±0.98
71.55±1.47
77.94±1.77
137.38±6.99
170.03±7.96
145.05±0.27
208.78±0.24
38.40±1.97
SAL-1
0.56
0.36
0.63
0.79
0.35
0.36
0.62
0.22
0.35
2.63
0.36
0.45
0.64
0.34
0.45
9.64
0.53
0.49
0.73
0.48
0.68
1.06
0.70
0.69
0.33
0.35
0.40
0.28
SC-1A 1a
SC-1B 1b
SC-1C 1c
SC-1D 1d
SC-1E 1e
SAL-2
SC-4A 2a
SC-4B 2b
SC-4C 2c
SC-4D 2d
SC-4E 2e
SAL-3
SC-2A 3a
SC-2B 3b
SC-2C 3c
SC-2D 3d
SC-2E 3e
SAL-4
SC-3A 4a
SC-3B 4b
SC-3C 4c
SC-3D 4d
SC-3E 4e
SCP-A 5a
SCP-B 5b
SCP-C 5c
SCP-D 5d
SCP-E 5e
Benzyl
Phenethyl
Adamantan-1-yl
-
Cyclohexyl
Phenyl
H
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Benzyl
Phenethyl
Adamantan-1-yl
-
Cyclohexyl
Phenyl
F
Cl
Cl
Cl
Cl
Cl
Cl
Benzyl
Phenethyl
Adamantan-1-yl
-
Cyclohexyl
Phenyl
Cl Br
Cl Br
Cl Br
Cl Br
Cl Br
Cl Br
Benzyl
Phenethyl
Adamantan-1-yl
Cyclohexyl
Phenyl
Benzyl
Phenethyl
Adamantan-1-yl
-
-
-
-
-
-
-
-
-
-
1.46
Rivastigmine
AChE and BChE inhibition is expressed as the mean ± SD (n = 3 experiments). The best results and the most selective inhibition for each
enzyme are shown in bold.
6

Focusing on in vitro potency of the salicylanilides SAL against both enzymes, it was observed
that they inhibited AChE with IC50 at the concentration of 48.27-76.53 μM. Unsubstituted
salicylanilide
(2-hydroxy-N-phenylbenzamide;
SAL-1)
concomitantly
with
fluorosalicylanilide SAL-2 produced the greatest inhibition (ca 49 μM), followed by the
bromine derivative SAL-4 (58.25 μM). The function of BChE was affected at markedly
higher concentrations (IC50 values of 118.74-205.73 μM). In this case, a halogenation of the
salicylanilide core improved the inhibitory activity. The order of substituents on the anilide
part contributing to the lower IC50 is as follows: Br > F > Cl. Regarding these data, the parent
salicylanilides SAL-1-SAL-4 are stronger inhibitors of AChE (SI ≤0.49 with non-halogenated
SAL-1 being the most selective one).
Focusing on the salicylanilide carbamates 1-4, the IC50 values for AChE are in a concentration
range of 37.40-61.00 M. In general, it is possible to conclude that a better effectiveness was
achieved by the derivatives of N-(4-bromophenyl)-5-chloro-2-hydroxybenzamide (SAL-4; all
IC50 values <50 M). Two the best AChE inhibitors in this study belong to this group: 2-[(4-
bromophenyl)carbamoyl]-4-chlorophenyl phenylcarbamate 4b was found to be the most
potent AChE inhibitor (IC50 = 37.40±2.93 M), closely followed by its cyclohexyl analogue
4a (IC50 = 40.54±0.78 M). On the other hand, the least effective inhibitor is 4-chloro-2-[(4-
fluorophenyl)carbamoyl]phenyl phenethylcarbamate 2d. However, the differences in IC50
values are not sharply differentiated. The introduction of the carbamate bond led to a
uniformly improved AChE inhibition in the case of the salicylanilides with a higher molecular
weight (SAL-3, SAL-4), but not for “halogen-free” salicylanilide SAL-1 and the
fluoroderivative SAL-2 (variable results). Similarly, there was no clear trend observed for the
influence of substituent R3 on the action. We demonstrated that halogenation of salicylanilide
scaffold is not necessary for the efficient inhibition of AChE. Importantly, only one
compound (SAL-3) exhibited a significantly lower activity than rivastigmine (56.14 µM),
other were fully comparable or superior to this widely used drug.
In contrast to AChE, IC50 found for BChE are in a broader range of 5.34-235.28 M. We can
conclude again that brominated derivatives 4 achieved the best results for inhibition of BChE
because their IC50 values do not exceed 80 M. Nevertheless, highlighting the most effective
molecules, the best inhibition of BChE was determined for 4-chloro-2-[(4-
chlorophenyl)carbamoyl]phenyl phenethylcarbamate 3d (IC50 = 5.34±0.21 M), followed by
4-chloro-2-[(4-fluorophenyl)carbamoyl]phenyl phenethylcarbamate 2d (23.15±0.68 M). In
contrast, 4-chloro-2-[(4-fluorophenyl)carbamoyl]phenyl phenylcarbamate 2b was identified
as the least potent compound (IC50 = 235.28±15.57 M). The study of structure-activity
relationship (SAR) revealed that the carbamate bond formation improves the inhibitory
properties of the non-substituted molecule SAL-1 and two halogenosalicylanilides (SAL-3,
SAL-4) collectively up to 32 times (SAL-3 vs. 3d). The carbamoylation of 5-chloro-N-(4-
fluorophenyl)-2-hydroxybenzamide SAL-2 provided the derivatives with a lower or an
enhanced activity (2b and 2e vs. 2a, 2c, and 2d) ambiguously. The combination of the
halogens with the carbamate group in one molecule decreases IC50 values in case of N-
cyclohexyl a and N-phenethyl d carbamates but not for N-phenyl b, N-benzyl c (even an
opposite behaviour) and adamantyl d derivatives. In contrast to AChE inhibition, it is possible
to identify clear SAR for the R3 part of the final structures 1-4. The general increment in
lowering IC50 values for halogenated BChE inhibitors is in the following order: phenethyl d >
cyclohexyl a > benzyl c > adamantan-1-yl e > phenyl b.
7

Unlike AChE and rivastigmine, only two carbamates (2d and 3d) inhibited BChE at lower
concentrations than this dual inhibitor; an additional phenethylcarbamate 4d produced a
comparable in vitro inhibition.
Considering the selectivity for AChE versus BChE (Table 1), most of the N-monosubstituted
carbamates 1-4 showed a balanced inhibition of both cholinesterases (selectivity within the
range of 0.5 to 2.0). Only two derivatives (2d and especially 3d) exhibited stronger BChE
inhibition (SI values of 2.63 and 9.64, respectively) but in contrast, eight derivatives displayed
a more intense inhibition of AChE (SI <0.5; 1b, 1e, 2c, 2e, 3b, 3c, 4b, and particularly 2b
with SI of 0.22). It is possible to postulate that N-phenylcarbamate moiety favours selectivity
to AChE, whereas N-phenethyl fragment tends to be more potent towards BChE.
To evaluate a potential contribution of 2-[N-(substituted phenyl)carbamoyl] moiety for the
enzyme inhibition, we synthesized and evaluated five analogous carbamates of 4-
chlorophenol (cycloxexyl a, phenyl b, benzyl c, phenethyl d, adamantan-1-yl e). The results
led to the conclusion that the tested 4-chlorophenol-based carbamates are quite potent AChE
inhibitors with 4-chlorophenyl phenylcarbamate 5b superiority (45.24 M). On the other
hand, the benzylcarbamate 5c exhibited the highest IC50 value of 58.80 M. Nevertheless,
their IC50 values in a narrow concentration range do not differ significantly and they are fully
comparable to those obtained for the salicylanilide carbamates 1-4. The situation changes
when focusing on less potent BChE inhibition. First, the IC50 were found in a wider range
(77.94-208.78 M) with 4-chlorophenyl cyclohexylcarbamate 5a superiority. The inhibitory
potencies of other phenolic carbamates were lower markedly up to the adamantane-derived
molecule 5e (208.78 M). Regarding benzyl-, phenethyl- and adamantan-1-yl-carbamates 5c-
e, the presence of the both halogenated and non-substituted 2-(N-phenyl)carbamoyl fragment
improved the inhibitory potency significantly (4e vs. 5e, or even more than 27 times for the
pair 3d and 5d). Universally, an introduction of 2-[N-(4-bromophenyl)carbamoyl] moiety into
the structure of phenolic carbamates led to decreased IC50 values for BChE. With the
exceptions of the brominated molecules, related salicylanilide and 4-chlorophenyl
cyclohexylcarbamates a share a comparable activity as well as the majority of N-
phenylcarbamates b. Unexpectedly, 4-chlorophenyl phenylcarbamate 5b was superior to its
fluorinated salicylanilide counterpart 2b (137.38 μM vs. 235.28 μM). Together, 4-
chlorophenyl carbamates 5 are more potent inhibitors of AChE than BChE with the selectivity
ratio (AChE/BChE) ranging from 0.28 (5e) to 0.69 (5a). They are comparable to rivastigmine
for AChE inhibition, but BChE is inhibited more effectively by this drug.
Importantly, it was demonstrated that dual AChE-BChE inhibition may afford additional
therapeutic benefit in the treatment of dementia [11,31].
Previously, Imramovsky et al. [16] investigated the inhibition of AChE by salicylanilide N-n-
alkyl carbamates. Their reported data and our new results are similar. The activity towards
BChE was not investigated. When compared to salicylanilide N,N-disubstituted carbamates
and thiocarbamates [19] as well as diethyl phosphates [21], they produced a comparable
inhibition of AChE again but they are more potent BChE inhibitors generally. Contrarily,
diethyl thiophosphates produced consistently lower IC50 values for both cholinesterases [20].
Lipinski’s rule of five defines four physicochemical parameter ranges (molecular weight
≤500, logP ≤5, number of H-bond donors and acceptors of ≤5 and ≤10, respectively) that are
associated with drug-like properties of potential drugs for oral administration [32]. Table 2
reports these calculated parameters and topological polar surface areas (PSA). All of the
8

derivatives meet the criteria for the presence of H-bond donors and acceptors and with one
exception (adamantane derivative 4e) also fit limit for MW. Salicylanilides SAL, 4-
chlorophenyl carbamates 5 concomitantly with non-halogenated 1 and majority of fluorinated
carbamates 2 showed logP values lower than five. On the other hand, lipophilicity of all O-
derivatives of SAL-3 and SAL-4 exceeded slightly the borderline. It is worth mentioning that
these escalated logP values may help to overcome a poor bioavailability of the parent
salicylanilides SAL that is low despite fitting Lipinski’s rule of five [33]. More lipophilic
compounds should be also able to cross the biomembranes and blood-brain-barrier (BBB) via
a passive diffusion.
Table 2. Lipinski’s rule of five parameters and PSA values of the derivatives SAL and 1-5
logP
(calc.)
2.45
3.88
4.0
4.07
4.35
4.16
3.17
4.6
4.71
4.78
5.06
4.88
3.57
5.0
5.11
5.18
5.46
5.28
3.84
5.27
5.38
5.45
5.73
5.55
3.63
3.75
3.82
4.1
Number of H-
bond donors
Number of H-
bond acceptors
Number of tPSA
violation
Code
R1 R2
R3
MW
[Å2]
H
H
H
H
H
H
H
H
H
H
H
F
F
F
F
F
-
213.24
338.41
332.36
346.39
360.41
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
1
1
1
1
1
3
5
5
5
5
5
3
5
5
5
5
5
3
5
5
5
5
5
3
5
5
5
5
5
3
3
3
3
3
0
0
0
0
0
0
0
0
0
0
1
0
0
0
1
1
1
1
0
1
1
1
1
2
0
0
0
0
0
49.33
67.43
67.43
67.43
67.43
67.43
49.33
67.43
67.43
67.43
67.43
67.43
49.33
67.43
67.43
67.43
67.43
67.43
49.33
67.43
67.43
67.43
67.43
67.43
38.33
38.33
38.33
38.33
38.33
SAL-1
1a
1b
1c
1d
1e
SAL-2
2a
2b
2c
2d
2e
SAL-3
3a
3b
3c
3d
3e
SAL-4
4a
Cyclohexyl
Phenyl
Benzyl
Phenethyl
Adamantan-1-yl 390.47
H
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
-
265.67
390.84
384.79
398.82
412.85
Cyclohexyl
Phenyl
Benzyl
Phenethyl
Adamantan-1-yl 442.92
F
Cl
Cl
Cl
Cl
Cl
Cl
-
282.12
407.29
401.24
415.27
429.30
Cyclohexyl
Phenyl
Benzyl
Phenethyl
Adamantan-1-yl 459.37
Cl Br
Cl Br
Cl Br
Cl Br
Cl Br
Cl Br
-
326.57
451.75
445.70
459.72
473.75
Cyclohexyl
Phenyl
Benzyl
Phenethyl
Adamantan-1-yl 503.82
Cyclohexyl
Phenyl
Benzyl
Phenethyl
Adamantan-1-yl 305.80
4b
4c
4d
4e
5a
5b
5c
5d
-
-
-
-
-
-
-
-
-
-
253.73
247.68
261.71
275.73
3.91
5e
In addition to lipophilicity, PSA was proposed as another crucial factor for drugs targeting
CNS [34]. PSA is defined as a sum of surfaces of polar atoms in the molecule (usually
nitrogen and oxygen atoms and hydrogens bound to them). A PSA value of less than 70 Å2
should be preferred for increasing the probability of BBB permeability [32,34]. All the
presented derivatives share sufficiently low PSA values.
4. Conclusions
We synthesized five 4-chlorophenyl N-substituted carbamates 5 from isocyanates in excellent
yields. Together with previously prepared salicylanilide N-monosubstituted carbamates 1-4
9

and salicylanilides SAL-1-SAL-4, they were investigated in vitro as potential inhibitors of
acetylcholinesterase and butyrylcholinesterase.
All twenty-five derivatives exhibited from weak to moderate inhibition of both
cholinesterases in micromolar and low micromolar concentrations starting from 5 μM. Some
of them were superior to the established drug rivastigmine. IC50 values for BChE were within
a broader concentration range than those obtained for AChE. This inhibition is not selective;
several derivatives are more potent inhibitors of AChE and a few affect BChE more
effectively.
Several important structure-activity relationships were identified. To point out, neither the
halogenation of the salicylanilide nor the presence of 2-phenylcarbamoyl moiety is required
for the potent inhibition of AChE. The derivatives of N-(4-bromophenyl)-5-chloro-2-
hydroxybenzamide caused a more efficient inhibition of both cholinesterases. N-
Phenethylcarbamates showed lower IC50 values for BChE than other N-substituents.
The presented data can stimulate a follow-up study in order to find more potent N-
monosubstituted carbamates with even lower IC50. Especially N-phenethylcarbamates of
halogenated salicylanilides seem to be promising hits as starting compounds for further
optimization.
Acknowledgements
This work was supported by the Czech Science Foundation [grant number 17-27514Y] and by
the project EFSA-CDN [grant No. CZ.02.1.01/0.0/0.0/16_019/0000841] co-funded by ERDF.
The authors wish to acknowledge financial support from the University of Pardubice, Faculty
of Chemical Technology.
We thank to J. Urbanová for the language help provided.
Declaration of Interest
Declarations of interest: none.
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Highlights
 N-Monosubstituted phenolic carbamates inhibit cholinesterases.
 IC50 values from 5.34 µM.
 AChE inhibition comparable or superior to rivastigmine.
 N-Phenethylcarbamates produced favourable BChE inhibition.
 Carbamates share convenient physico-chemical properties for targeting CNS.
13

Graphical abstract
14