Synthesis and characterization of new inhibitors of cholinesterases based on N-phenylcarbamates: In vitro study of inhibitory effect, type of inhibition, lipophilicity and molecular docking

Article abstract of DOI:10.1016/j.bioorg.2018.03.012

Based on current treatment of Alzheimer's disease, where the carbamate inhibitor Rivastigmine is used, two series of carbamate derivatives were prepared: (i) N-phenylcarbamates with additional carbamate group (1–12) and (ii) N-phenylcarbamates with monosaccharide moiety (13–24). All compounds were tested for the inhibitory effect on both of the cholinesterases, electric eel acetylcholinesterase (eeAChE) and butyrylcholinesterase from equine serum (eqBChE) and the inhibitory activity (expressed as IC₅₀ values) was compared with that of the established drugs Galanthamine and Rivastigmine. The compounds with two carbamate groups 1–12 revealed higher inhibitory efficiency on both cholinesterases in compared with monosaccharide derived carbamates 13–24 and with Rivastigmine. The significant decrease of inhibitory efficiency on eqBChE (also for eeAChE but in less manner) was observed after deacetalization of monosaccharide. Moreover, the type of inhibitory mechanism of five chosen compounds was studied. It was found, that compounds with two carbamate groups act presumably via a mixed inhibitory mechanism and the compounds with monosaccharide moiety act as non-competitive inhibitors. The lipophilicity of tested compounds was determined using partition coefficient. Specific positions of the inhibitors in the binding sites of cholinesterases were determined using molecular modeling and the results indicate the importance of phenylcarbamate orientation in the catalytic gorges of both enzymes.

Full text of DOI:10.1016/j.bioorg.2018.03.012

Accepted Manuscript
Synthesis and characterization of new inhibitors of cholinesterases based on N-
phenylcarbamates: In vitro study of inhibitory effect, type of inhibition, lipo-
philicity and molecular docking
Katarína Vorč áková, Magdaléna Májeková, Eva Horáková, Pavel Drabina,
Miloš Sedlák, Š árka Štěpánková
PII:
S0045-2068(18)30020-8
DOI:
https://doi.org/10.1016/j.bioorg.2018.03.012
YBIOO 2302
Reference:
Bioorganic Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
9 January 2018
9 March 2018
12 March 2018
Please cite this article as: K. Vorč áková, M. Májeková, E. Horáková, P. Drabina, M. Sedlák, S. Štěpánková,
Synthesis and characterization of new inhibitors of cholinesterases based on N-phenylcarbamates: In vitro study of
inhibitory effect, type of inhibition, lipophilicity and molecular docking, Bioorganic Chemistry (2018), doi: https://
doi.org/10.1016/j.bioorg.2018.03.012
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Synthesis and characterization of new inhibitors of cholinesterases based on N-
phenylcarbamates: In vitro study of inhibitory effect, type of inhibition, lipophilicity and
molecular docking
Katarína Vorčáková1, Magdaléna Májeková2, Eva Horáková3, Pavel Drabina3, Miloš Sedlák3,
Šárka Štěpánková1,*
1
Department of Biological and Biochemical Sciences, Faculty of Chemical Technology,
University of Pardubice, Pardubice, Czech Republic
2
Department of Biochemical Pharmacology, Institute of Experimental Pharmacology &
Toxicology, Slovak Academy of Science, Bratislava, Slovak Republic
3
Institute of Organic Chemistry and Technology, Faculty of Chemical Technology,
University of Pardubice, Pardubice, Czech Republic
* Corresponding author: email: sarka.stepankova@upce.cz, Studentská 573, 53210 Pardubice,
Czech Republic. Telephone: +421 466 037 294, fax number: +421 466 037 068
Keywords
Carbamates; Monosaccharide derivatives; Cholinesterases; IC50; Molecular Modeling; Water
Bridge

Abstract
Based on current treatment of Alzheimer's disease, where the carbamate inhibitor
Rivastigmine is used, two series of carbamate derivatives were prepared: i) N-
phenylcarbamates with additional carbamate group (1-12) and ii) N-phenylcarbamates with
monosaccharide moiety (13-24). All compounds were tested for the inhibitory effect on both
of the cholinesterases, electric ell acetylcholinesterase (eeAChE) and butyrylcholinesterase
from equine serum (eqBChE) and the inhibitory activity (expressed as IC50 values) was
compared with that of the established drugs Galanthamine and Rivastigmine. The compounds
with two carbamate groups 1-12 revealed higher inhibitory efficiency on both cholinesterases
in compared with monosaccharide derived carbamates 13-24 and with Rivastigmine. The
significant decrease of inhibitory efficiency on eqBChE (also for eeAChE but in less manner)
was observed after deacetalization of monosaccharide. Moreover, the type of inhibitory
mechanism of five chosen compounds was studied. It was found, that compounds with two
carbamate groups act presumably via a mixed inhibitory mechanism and the compounds with
monosaccharide moiety act as non-competitive inhibitors. The lipophilicity of tested
compounds was determined using partition coefficient. Specific positions of the inhibitors in
the binding sites of cholinesterases were determined using molecular modeling and the results
indicate the importance of phenylcarbamate orientation in the catalytic gorges of both
enzymes.

1. Introduction
Alzheimer's disease represent the most common type of dementia with characteristic
symptoms, i.e. changes in levels of ChEs, enhanced production and accumulation of β-
amyloid peptide, formation of neurofibrillary tangles inside nerve cell bodies etc. In process
of development of AD, the levels of ChEs are altered in different manner. In early stages of
AD, the level of AChE is increased at a much higher rate than level of BChE. On the other
hand, in later stages of AD, decrease of level AChE and rapid increase of level of BChE in
brain occurs. In this stage, BChE substitutes the function of AChE – the hydrolysis of
acetylcholine (ACh). Generally, the inferior level of ACh during AD is obvious and therefore
inhibition of ChEs represents one of the major pharmacological interventions for this disease
[1].
Inhibitors of cholinesterases (ChEIs) represent a wide-range group of different chemical
substances with application in two areas. The first one involves the pharmaceutical
employment (as cholinergic drugs) for treatment of various diseases such as Alzheimer's
disease and other dementias, Myasthenia gravis and glaucoma. The second one includes their
using as pesticides or chemical warfare nerve agents [2]. Generally, carbamate inhibitors,
such as Alzheimer's disease drug Rivastigmine ((S)-N-ethyl-3-[(1-dimethylamino)ethyl] N-
methylphenylcarbamate), are classified as the pseudo-irreversible inhibitors, which form a
carbamoylated complex with the serine residue of the catalytic triad [3,4]. It was found out,
that Rivastigmine exhibits tenfold greater affinity for brain AChE than for the peripheral one.
This selectivity for the brain enzyme and inhibitory activity against AChE and BChE makes
Rivastigmine a drug with a good pharmacological profile [5]. Another member of ChEIs –
Cymserine (or more potent and selective bisnorcymserine), represents a BChE selective
carbamate inhibitor, which is currently pursued for clinical development as an Alzheimer

therapeutics. The structure of Cymserine was designed on the basis of the structure of the
alkaloid physostigmine [6,7].
Currently, utilization of carbamate functional group incorporated to the structure of
inhibitor plays an important role in modern drug discovery and medicinal chemistry.
Carbamate functional group can be found in many of approved therapeutic agents, besides
Rivastigmine, i.e. Felbamate (antiepileptic drug), Flupirtine (nonopioid analgesic),
Albendazole (anthelmintic drug), Efavirenz (inhibitor of reverse transcriptase), Ritonavir
(inhibitor of HIV-1 and HIV-2 protease) etc. [8]. Using two carbamate groups in molecule
(biscarbamate) also represent a potential area. In the past, we can found using biscarbamates
in synthesis antitumor compounds [9,10] and also as inhibitors of acetylcholinesterase [11].
Nowadays, group of researcher Bosak et al. intensively work on synthesis new biscarbamate
compounds as butyrylcholinesterase inhibitors [12,13].
The compounds containing halogen atom could allow the formation of specific type of
non-covalent interaction – halogen bonding – formed between a halogenated compound as
inhibitor and protein backbone of cholinesterase enzymes [14–24]. There are a number of
experimental studies, where the effect of halogen substitution on binding affinity has been
systematically evaluated [25–27]. In some cases, present halogen atom, which enables the
formation of halogen bonding, led to the significant increase of binding affinity. The
explanation of this effect lays in the structure of catalytic triad of ChEs constituted from three
aminoacids His-Ser-Glu, in which nitrogen atoms of histidine participate in this type of
interaction. In addition, histidine can form another types of interactions, e.g. hydrogen bonds,
charge−charge or charge-dipole interactions, π−π-contacts, or complexes with metal ions [19].
This fact can be also considered in designing of new ChEs inhibitors.

Furthermore, one of the most popular halogen-containing functional groups in drug
molecules is the trifluoromethyl moiety (CF3) [28]. Many of familiar drugs contain
trifluoromethyl group, e.g. antidepressants (Prozac and Luvox), anaesthetics (Halothane, ) or
antimalarial drug Lariam [29]. Martins et al. published a series of trifluoromethylated
isoxazoles exhibiting antimicrobial activity [30]. Finally, also several ChEs inhibitors
containing CF3 moiety were previously described [31,32].
The compounds with monosaccharide moiety could possess a high level of ability to pass
through blood-brain barrier via GLUT-family sugar/polyol transport facilitators into
intracellular space of CNS, what can lead to their enhanced inhibitory activity [33–36].
Moreover, monosaccharide moiety is generally used in designing of structure of new drugs in
order to improve their water solubility or serum stability [34]. Phenolic glycosides can be a
part of some natural products [37]. Our research group previously published the study in
which N-(2-phenylcyclopropyl)carbamates with monosaccharide moiety were tested as
potential cholinesterase inhibitors. Some of them were evaluated as quite potent in inhibition
of BChE with IC50 in micromolar range [38].
Nowadays, many researchers are focused on studying the role of butyrylcholinesterase (as
the second cholinesterase (ChE)) in human body, especially in the brain of patients with
Alzheimer's disease (AD) during the process of degeneration of nerve cells. In addition, other
functions of this enzyme are also investigated. With regard to the fact, that BChE is present in
plasma (where its average concentration is 5 mg/L) and numerous vertebrate tissues (liver,
intestine and lung), some studies discuss the noncholinergic functions of BChE in
detoxification of numerous drugs and toxins [39,40]. For example, purified BChE was used
for treatment of succinylcholine-induced apnea in humans. Further, it is known that BChE to
protect rodents from the toxic effects of cocaine [41,42]. The therapeutic potential of BChE

relies on its ability to stay in the circulation for a prolonged period, which in turn depends on
maintaining its tetrameric quaternary configuration. BChE occured in the neuromuscular
junctions and the central nervous system is anchored to membranes via interactions with
ColQ (AChE-associated collagen tail protein) and PRiMA (proline-rich membrane anchor)
proteins containing proline-rich domains [43,44].
The above-mentioned findings inspired us for synthesis and characterization of twenty-four
differently substituted N-phenylcarbamates, whose inhibitory activity against ChEs and
lipophility should be evaluated. In the case of the most promising compounds, we also
focused on study of mechanism of inhibitory effect using the molecular modeling.
2. Results and discussion
2.1 Chemistry
The preparation of target carbamate derivatives 1-12 was performed by two-step synthesis
described previously (Scheme 1). The synthesis started from 3-aminophenol as the structural
core of all carbamates 1-12. It was transformed to corresponding N-(3-
hydroxyphenyl)carbamates by the action of methyl, ethyl or butyl chloroformate. Two
equivalents of 3-aminophenol were used with regard to the formation of its unreactive
hydrochloride during the reaction course. The application of different bases, e.g.
triethylamine, led to undesired by-products and less yields of the reaction, caused by
formation of phenolate anion in equilibrium and subsequent O-acylation. The yields of this
first step were satisfactory (50–75%). The structure of these monocarbamate intermediates
1
was confirmed by means of H NMR spectroscopy. The second reaction step included the
formation of further carbamate group by the addition of N-(3-hydroxyphenyl)carbamates into
slight excess of phenyl isocyanate derivatives in the presence of triethylamine as base
catalyst. Beside non-substituted, 4-methylphenyl and 4-methoxyphenyl isocyanate also 4-

chlorophenyl and 3,4-dichlorophenyl derivatives were chosen, based on above mentioned data
in introduction. The urea possibly formed as a by-product was removed by purification of
products by means of column chromatography. Yields of these transformations were almost
quantitative [45].
Scheme 1. Preparation of N-phenylcarbamate derivatives 1–12 (The first group).
The second group of studied carbamate derivatives 13–24 contained monosaccharide
moiety in their structure. The preparation of carbamates 13–18 was performed by simple
treatment of the protected monosaccharide with corresponding phenyl isocyanates (Scheme
2). This addition reaction was catalyzed by triethylamine. In this case, the reaction needed
high temperature (reflux in 1,4-dioxane) and relatively long reaction time to achieve
satisfactory yields (31–67%). Unreacted monosaccharide as well as isocyanate was removed
by means of column chromatography. Furthermore, the group of carbamate derivatives 19–24
was obtained by deprotection of compounds 13–18 (deacetalization of monosaccharide
moiety) performed in aqueous trifluoroacetic acid (TFA) (1/1) [46]. The deprotection
proceeded with high conversion at set condition (24 h, r.t.). The products were obtained as a
mixture of both anomers, which were detected in NMR spectra as two sets of signals.
1
Completion of deprotection process can be monitored by means of H NMR spectroscopy as
marked disappearing of characteristic signals of methyls belonging to protecting groups in
1
13
area 1.30–1.60 ppm. All carbamates 13-24 were characterized by melting point, H and C
NMR spectroscopy and high resolution mass spectroscopy.

Scheme 2. Preparation of N-phenylcarbamate derivatives 13–24 (The second group).
2.2 In vitro acetylcholinesterase and butyrylcholinesterase inhibition
The ability of the investigated N-phenylcarbamate derivatives 1–24 to inhibit eeAChE and
eqBChE was screened in vitro using modified Ellman's method. The effectiveness of the
inhibitors is expressed as IC50, representing the concentration of an inhibitor required for 50%
inhibition of the enzyme. The obtained results were compared with those obtained for
Rivastigmine (acylating pseudo-irreversible carbamate inhibitor that inhibits AChE as well as
BChE) and Galanthamine (non-acylating competitive reversible inhibitor) (Table 1).
Tab. 1
The first group of tested substituted N-phenylcarbamates 1-12 represents moderate or quite
potent inhibitors on both of cholinesterases what correlate with results from molecular
modeling. This group of inhibitors shows more inhibition potency for eqBChE. The values of
IC50 are in the range from 7.7 to 20.9 µM for eeAChE and from 6.1 to 22.0 µM for eqBChE.

In general, it is possible to conclude that better effectiveness was achieved when the halogen
substitution (chloro) was present. Inhibitors with two atoms of chlorine (N-3,4-dichlorophenyl
derivatives 10-12) have lower values of IC50 and were identified as more effective eqBChE
(also eeAChE) inhibitors in contrast with compounds without chlorine.
The second group of tested substituted N-phenylcarbamates 13-24 exhibits inhibition
efficiency in wide range for both ChEs and significant difference between protected
monosaccharide derivatives and their deprotected version is also obvious. For eeAChE is the
range of inhibitory concentration from 46.9 to 103.5 µM and the inhibitors with protected
monosaccharide moiety have inhibitory concentration approximately two times lower as the
inhibitors with deprotected monosaccharide moiety. In case of eqBChE, for the compounds
13, 14, 17 and 18 the lower inhibitory concentration in comparison of eeAChE was observed.
The other compounds from this group possessed very low inhibitory activity (> 200 µM). In
some cases, the introduction of CF3 group into phenol slightly increases IC50 values for both
ChEs.
The inhibitory activities of N-phenylcarbamate derivatives 1-24 were compared with those
of Galanthamine and Rivastigmine. All of the tested compounds 1-24 showed a worse
inhibition of eeAChE and eqBChE than Galanthamine. The first group of tested compounds
1-12 exhibited better inhibition efficiency for both of ChEs in comparison with Rivastigmine.
In case of the second group of tested compounds 13-24, the higher inhibitory concentration
for eeAChE was determined. For eqBChE only the compounds 13, 14, 17 and 18 had lower
inhibitory concentration in comparison of Rivastigmine.
Considering the selectivity for AChE vs. BChE (see Tab. 1) all tested compounds (except
15-17 and 19-24) showed a balanced inhibition of both ChEs (AChE/BChE ratio ˂2).

The kinetic parameters Km and Vm for tested compounds 1, 10, 12, 13 and 18 were
calculated from the Lineweaver-Burk plot and based on results the type of inhibition was
evaluated. The kinetic parameters, for reactions in the presence of the tested compounds 1, 10
and 12, were changed in different extent compared to the value for the reaction in their
absence. These results indicated that group of inhibitors 1-12 inhibits BChE in a varied
manner and it could be concluded that this group of compounds presumably acts via a mixed
inhibitory mechanism. On the other hand, the compounds with monosaccharide moiety 13-18,
presumably act via a non-competitive inhibitory mechanism. The Vm values obtained for the
inhibited reactions were lower than Vm for the reaction in the absence of the inhibitor, the Km
values did not change in the presence or absence of the inhibitor. The example of kinetic
analysis for compound 18 is shown in Figure 1.
Fig. 1
2.3 Partition coefficient
The ability of tested inhibitors to pass across the blood-brain barrier (BBB) was estimated
by traditional shake-flask method. The optimal values of partition coefficient are in range 4-5.
This property of compounds represents better transport of the active molecules into
intracellular space of CNS. The results of partition coefficient are presented in Table 1.
Almost all of tested compounds have value of partition coefficient in range 3.3-6.32 (except
15, 19-24). Compounds with deprotected monosaccharide moiety had the values of partition
coefficient lower than 3.1, as the result of deacetalization of monosaccharide moiety when the
compounds became more hydrophilic.
2.4 Molecular modeling

The compounds studied are poor to moderate inhibitors of both AChE and BChE, with the
exception of compounds 10, 11 and 12. For this reason we concentrated our in silico study on
these derivatives. Compound 10, the most effective inhibitor of both enzymes, is firmly set in
the active site of BChE by H-bond with His438 and water mediated H-bond with Ser198, two
members of the catalytic triad Ser198, Glu325 and His338. The position is further stabilized
by π-π interaction of the phenyl group with His438, H-bond with Tyr440 and hydrophobic
interaction with Gly117 (Figure 2A, left). The whole complex is fixed by a compact water
network over di-Cl-phenyl part of molecule as it can be easy visible from the space over the
gorge of BChE (Figure 2A, right). Changing methyl substituted phenylcarbamate moiety by
sugar (α-D-galactopyranose) in compound 19 caused turning of the whole molecule (probably
due changing the orientation of a lipole – a lipophilic dipole) out of the position obtained for
compound 10. The sugar moiety is kept by Glu197 and Gly115 and water does not create a
systematic network as for compound 10. No interaction with the member of catalytic triad
was observed (Figure 2B).
Fig. 2
A similar situation as for BChE was found for compound 10 in AChE, however, in this case
compound 10 interacts with Ser203 of the catalytic triad and the gap to the active site is
narrower (Figure 3).
Fig. 3
3. Conclusions
A series of twenty-four N-phenylcarbamates 1-24 were evaluated for their inhibition
against AChE from electric eel and BChE from equine serum. Inhibitory concentration was in
micromolar range. It was observed that derivatives containing chlorine atoms possess higher
inhibitory efficiency. On the other hand, the substitution of benzene ring by CF3 increases the

inhibitory concentration. The compounds with monosaccharide moiety 13-24 showed lower
inhibitory efficiency in comparison with compounds with two carbamate groups 1-12. This
fact is probably related to recently observed trend of molecules with higher number of OH
groups to behave as PAINS (the compounds giving moderate or poor interactions with many
proteins, through manifold OH-mediated H-bonds). Such interactions result in a poor enzyme
selectivity, therefore the compounds 13-24 do not act as selective inhibitors of AChE and
BChE.
Based on results of type of inhibition, the compounds 1-12 presumably belong to the
group of inhibitors with mixed type of inhibition and the compounds 13-24 presumably act as
non-competitive inhibitors. Owing to the obtained results of partition coefficient, it is possible
to conclude that all of the tested phenylcarbamates should pass across the blood-brain barrier.
4. Materials and methods
4.1 Chemistry
4.1.1 General
All reagents and solvents were purchased from Penta Chemicals or Sigma Aldrich. The
starting chemicals (including phenyl, 3-methylphenyl, 4-methoxyphenyl, 4-chlorophenyl,
3,4-dichlorophenyl and 3-trifluoromethylphenyl isocyanate) were obtained from Sigma-
Aldrich or TCI Chemicals and used without further purification. The synthetic method for
preparation of compounds 1-12 was published by Bergon et al. [47]. The compounds 1–4 and
8–11 were described by Boroschewski et al. [48], purity and characterization data were
verified.
Column chromatography was performed using 60 Å (60–200 μm) silica gel. All solvents
were reagent-grade and when necessary, were purified and dried by standard methods. TLC
was performed on aluminium-backed silica gel plates (Merck DC, Alufolien Kieselgel 60
F254) with spots visualized by UV light. The melting point temperatures are uncorrected. FT-
1
IR spectra were measured on a Nicolet iS50 instrument. H NMR spectra were recorded on a

Bruker Avance 400 instrument. Chemical shifts  were referenced to the residual peak of the
CDCl3 at 7.26 ppm. The 13C NMR spectra were calibrated with respect to the middle signal in
the triplet of CDCl3 (δ = 77.23 ppm) or the multiplet of CD3OD (δ = 49.00 ppm) and DMSO-
d6 (δ = 39.51 ppm). High-resolution mass spectra were measured on the Thermo Fisher
Scientific MALDI LTQ Orbitrap instrument. The used matrix was 0.2M solution of 2,5-
dihydroxybenzoic acid (DHB) in MeCN/H2O (95/5). Spectra were calibrated with respect to
used matrix.
4.1.2 Synthesis of compounds
4.1.2.1 Synthesis of substituted carbamates 1–12 [47]
The carbamates 1–12 were prepared by a two-steps process. Methyl, ethyl or butyl
chloroformate (0.025 mol) was added dropwise to 3-aminophenol (0.05 mol) dissolved in dry
THF (50 mL) at room temperature. The mixture was stirred for 2 h and the hydrochloride of
3-aminophenol formed was filtered off. The filtrate was evaporated to dryness to give
corresponding N-(3-hydroxyphenyl)carbamate. This carbamate was converted to
bis(carbamate) by reaction with phenylisocyanate derivative (0.010 mol) in dry benzene (50
mL) in presence of catalytic amount of TEA (few drops) under reflux for 30 min. Solvent
was removed under reduced pressure and the residue was purified by column chromatography
on silica gel (EtOAc/n-hexane (1/2)).
O-3-[(Butoxycarbonyl)amino]phenyl-N-(3-methylphenyl)carbamate 5
White crystalline solid; yield: 63%; mp 115–116 °C; FT-IR (cm–1): υ 3290; 1699; 1538; 1426;
1
1218; 1042; 778; 688. H NMR (400 MHz, CDCl3): δ 7.36 (bs, 1H, NH), 7.28 (bs, 1H, NH),
3
7.13 (m, 5H, Ph), 6.87 (m, 3H, Ph), 4.16 (t, J = 6.8 Hz, 2H, OCH2), 2.32 (s, 3H, CH3), 1.64
3
13
(m, 2H, CH2), 1.40 (m, 2H, CH2), 0.94 (t, J = 7.2 Hz, 3H, CH3); C NMR (100 MHz,
CDCl3): δ 153.8, 153.5, 151.2, 139.3, 139.2, 137.4, 129.7, 129.1, 128.9, 124.9, 124.3, 121.0,
117.4, 116.6, 65.4, 31.1, 21.6, 19.2, 13.9. HR-MALDI-MS (DHB): calcd for C19H22N2O4 m/z
365.14718 ([M+Na]+), found 365.14736.
O-3-[(Ethoxycarbonyl)amino]phenyl-N-(4-methoxyphenyl)carbamate 6
White crystalline solid; yield: 59%; mp 160–161 °C; FT-IR (cm–1): υ 3324; 1709; 1534; 1435;
1
1220; 1022; 865; 644. H NMR (400 MHz, DMSO-d6): δ 10.05 (bs, 1H, NH), 9.80 (bs, 1H,
3
NH), 7.43 (m, 3H, Ph), 7.32 (m, 2H, Ph), 6.92 (m, 2H, Ph), 6.85 (m, 1H, Ph), 4.13 (q, J = 7.2
3
13
Hz, 2H, OCH2), 3.71 (s, 3H, OCH3), 1.24 (t, J = 7.2 Hz, 3H, CH3); C NMR (100 MHz,

DMSO-d6): δ 154.8, 153.1, 151.3, 150.5, 139.9, 131.2, 129.0, 119.7, 119.5, 115.3, 114.5,
113.7, 113.5, 111.1, 59.9, 54.7, 14.1. HR-MALDI-MS (DHB): calcd for C19H22N2O5 m/z
353.11080 ([M+Na]+), found 353.11149.
O-3-[(Butoxycarbonyl)amino]phenyl-N-(4-methoxyphenyl)carbamate 7
White crystalline solid; yield: 67%; mp 131–132 °C; FT-IR (cm–1): υ 3329; 1709; 1533; 1432;
1
1219; 1020; 863; 642. H NMR (400 MHz, DMSO-d6): δ 10.08 (bs, 1H, NH), 9.83 (bs, 1H,
NH), 7.39 (m, 3H, Ph), 7.29 (m, 2H, Ph), 6.90 (m, 2H, Ph), 6.83 (m, 1H, Ph), 4.08 (t, 3J = 6.8
Hz, 2H, OCH2), 3.72 (s, 3H, OCH3), 1.60 (m, 2H, CH2), 1.37 (m, 2H, CH2), 0.91 (t, 3J = 7.4
13
Hz, 3H, CH3); C NMR (100 MHz, DMSO-d6): δ 155.2, 153.6, 151.7, 150.9, 140.3, 131.6,
129.5, 120.1, 115.7, 115.0, 114.1, 111.6, 64.0, 55.2, 30.6, 18.6, 13.7. HR-MALDI-MS
(DHB): calcd for C19H22N2O5 m/z 381.14209 ([M+Na]+), found 381.14222.
O-3-[(Butoxycarbonyl)amino]phenyl-N-(3,4-dichlorophenyl)carbamate 12
White crystalline solid; yield: 71%; mp 137–138 °C; FT-IR (cm–1): υ 3269; 1700; 1591; 1539;
1476; 1214; 1078; 1019; 868; 765; 683; 609.1H NMR (400 MHz, DMSO-d6): δ 10.57 (bs, 1H,
NH), 9.80 (bs, 1H, NH), 7.80 (m, 1H, Ph), 7.59 (m, 1H, Ph), 7.44 (m, 2H, Ph), 7.32 (m, 2H,
Ph), 6.88–6.85 (m, 1H, Ph), 4.08 (m, 2H, OCH2), 1.60 (m, 2H, CH2), 1.37 (m, 2H, CH2), 0.91
3
13
(t, J = 7.6 Hz, 3H, CH3); C NMR (100 MHz, DMSO-d6): δ 153.6, 151.5, 150.5, 140.4,
138.9, 131.2, 130.9, 129.5, 124.5, 119.6, 118.5, 115.6, 115.3, 111.4, 64.0, 30.6, 18.6, 13.6.
HR-MALDI-MS (DHB): calcd for C18H18Cl2N2O4 m/z 419.05358 ([M+Na]+), found
419.05435.
4.1.2.2 Synthesis of O-glycosyl-N-(3,4-dichlorophenyl) and O-glycosyl-N-(3-trifluorophenyl)
carbamates 13-18
To the solution of substituted phenylisocyanate (1.33 mmol) in dry 1,4-dioxane (5 mL)
was added corresponding protected monosaccharide (1.60 mmol) and TEA (1.60 mmol). The
mixture was heated to reflux for 72 h, and then, the solvent was removed under reduced
pressure. The crude products were purified by column chromatography on silica gel
(EtOAc/n-hexane (1/1)).
O-(1,2:3,4-Di-O-isopropylidene-α-D-galactopyranose-6-yl)-N-(3,4-dichlorophenyl)carbamate
13
White crystalline solid; yield: 67%; mp 63–65 °C; FT-IR (cm–1): υ 3314; 2987; 1761; 1592;
1
1523; 1478; 1380; 1210; 1116; 1003; 894; 689; 511. H NMR (400 MHz, CDCl3): δ 7.61 (s,
1H, Ph), 7.34 (d, 3J = 8.8 Hz, 1H, Ph), 7.19 (dd, 3J = 8.8, 2.8 Hz, 1H, Ph), 6.89 (bs, 1H, NH),

5.57 (m, 1H, CH), 4.64 (m, 1H, CH), 4.37 (m, 2H, 2xCH), 4.28 (m, 2H, OCH2), 4.08 (m, 1H,
13
CH), 1.52 (s, 3H, CH3), 1.47 (s, 3H, CH3), 1.36 (s, 3H, CH3), 1.34 (s, 3H, CH3); C NMR
(100 MHz, CDCl3): δ 153.1, 137.6, 133.1, 130.7, 126.8, 120.4, 118.0, 110.0, 109.1, 96.5,
71.3, 70.9, 70.5, 66.4, 64.9, 26.2, 26.2, 25.1, 24.7. HR-MALDI-MS (DHB): calcd for
C19H23Cl2NO7 m/z 470.07438 ([M+Na]+), found 470.07551.
O-(1,2:3,4-Di-O-isopropylidene-α-D-galactopyranose-6-yl)-N-(3-
(trifluoromethyl)phenyl)carbamate 14
White crystalline solid; yield: 66%; mp 51–53 °C; FT-IR (cm–1): υ 3321; 2988; 1717; 1550;
1335; 1210; 1066; 1000; 891; 765; 698; 510.1H NMR (400 MHz, CDCl3): δ 7.68 (bs, 1H,
NH), 7.52 (m, 1H, Ph), 7.35 (m, 1H, Ph), 7.25 (s, 2H, Ph), 5.53 (m, 1H, CH), 4.60 (m, 1H,
CH), 4.37 (m, 1H, CH), 4.33 (m, 1H, CH), 4.25 (m, 2H, OCH2), 4.08 (m, 1H, CH), 1.48 (s,
13
3H, CH3), 1.42 (s, 3H, CH3), 1.31 (s, 3H, CH3), 1.29 (s, 3H, CH3); C NMR (100 MHz,
CDCl3): δ 153.4, 138.7, 132.0, 131.7, 131.4, 131.0, 129.7, 128.1, 125.4, 122.7, 121.8, 120.1,
120.0, 115.5, 109.9, 109.0, 96.5, 71.2, 70.9, 70.5, 66.4, 64.7, 26.1, 26.1, 25.0, 24.6. HR-
MALDI-MS (DHB): calcd for C20H24F3NO7 m/z 470.13971 ([M+Na]+), found 470.14033.
O-(1,2:5,6-Di-O-isopropylidene-α-D-glucofuranose-3-yl)-N-(3,4-dichlorophenyl)carbamate 15
White crystalline solid; yield: 65%; mp 102–104 °C; FT-IR (cm–1): υ 3298; 2988; 1737; 1593;
1
1529; 1471; 1383; 1210; 1054; 1019; 875; 670. H NMR (400 MHz, CDCl3): δ 7.61 (s, 1H,
Ph), 7.35 (d, 3J = 8.8 Hz, 1H, Ph), 7.18 (dd, 3J = 8.8, 2.8 Hz, 1H, Ph), 7.04 (bs, 1H, NH), 5.88
(m, 1H, CH), 5.24 (m, 1H, CH), 4.63 (m, 1H, CH), 4.27 (m, 1H, CH), 4.21 (m, 1H, CH), 4.05
(m, 2H, OCH2), 1.53 (s, 3H, CH3), 1.43 (s, 3H, CH3), 1.32 (s, 3H, CH3), 1.31 (s, 3H, CH3);
13C NMR (100 MHz, CDCl3): δ 151.8, 137.2, 133.1, 130.7, 127.1, 120.3, 117.9, 112.7, 110.0,
105.1, 83.5, 79.8, 77.6, 72.5, 67.6, 27.1, 26.9, 26.3, 25.4. HR-MALDI-MS (DHB): calcd for
C19H23Cl2NO7 m/z 470.07438 ([M+Na]+), found 470.07499.
O-(1,2:5,6-Di-O-isopropylidene-α-D-glucofuranose-3-yl)-N-(3-
(trifluoromethyl)phenyl)carbamate 16
White crystalline solid; yield: 44%; mp 51–53 °C; ; FT-IR (cm–1): υ 3314; 2988; 1742; 1551;
1
1334; 1207; 1068; 1017; 838; 698. H NMR (400 MHz, CDCl3): δ 7.69 (bs, 1H, NH), 7.54
(m, 1H, Ph), 7.36 (m, 3H, Ph), 5.90 (m, 1H, CH), 5.29 (m, 1H, CH), 4.66 (m, 1H, CH), 4.35
(m, 1H, CH), 4.25 (m, 1H, CH), 4.11 (m, 2H, OCH2), 1.55 (s, 3H, CH3), 1.45 (s, 3H, CH3),
13
1.34 (s, 3H, CH3), 1.32 (s, 3H, CH3); C NMR (100 MHz, CDCl3): δ 151.9, 138.3, 132.2,
131.8, 131.5, 131.2, 129.8, 128.0, 125.3, 122.6, 121.6, 120.5, 120.5, 115.3, 112.7, 110.0,

105.2, 83.6, 79.9, 77.4, 72.5, 67.6, 27.1, 26.9, 26.3, 25.4. HR-MALDI-MS (DHB): calcd for
C20H24F3NO7 m/z 470.13971 ([M+Na]+), found 470.14062.
O-(2,3:4,5-Di-O-isopropylidene-β-D-fructopyranose-1-yl)-N-(3,4-dichlorophenyl)carbamate
17
White crystalline solid; yield: 57%; mp 163–165 °C; FT-IR (cm–1): υ 3411; 2998; 1729; 1510;
1380; 1209; 1068; 870; 579. 1H NMR (400 MHz, CDCl3): δ 7.60 (s, 1H, Ph), 7.32 (d, 3J = 8.8
Hz, 1H, Ph), 7.18 (dd, 3J = 8.8, 2.8 Hz, 1H, Ph), 6.83 (bs, 1H, NH), 4.59 (m, 1H, CH), 4.48
(m, 1H, CH), 4.27 (m, 1H, CH), 4.30 (m, 1H, CH), 4.17 (m, 1H, CH), 3.88 (m, 1H, OCH2),
3.77 (m, 1H, OCH2), 1.53 (s, 3H, CH3), 1.48 (s, 3H, CH3), 1.37 (s, 3H, CH3), 1.33 (s, 3H,
CH3); 13C NMR (100 MHz, CDCl3): δ 152.7, 137.3, 133.1, 130.7, 127.0, 120.5, 118.1, 109.3,
109.0, 101.6, 71.1, 70.8, 70.2, 67.1, 61.5, 26.5, 26.1, 25.3, 24.2. HR-MALDI-MS (DHB):
calcd for C19H23Cl2NO7 m/z 470.07438 ([M+Na]+), found 470.07514.
O-(2,3:4,5-Di-O-isopropylidene-β-D-fructopyranose-1-yl)-N-(3-
(trifluoromethyl)phenyl)carbamate 18
White crystalline solid; yield: 31%; mp 47–49 °C; FT-IR (cm–1): υ 3350; 2986; 1744; 1533;
1448; 1220; 1182; 1068; 1047; 883; 696.1H NMR (400 MHz, CDCl3): δ 7.73 (bs, 1H, NH),
7.58 (m, 1H, Ph), 7.41 (m, 1H, Ph), 7.30 (m, 2H, Ph), 4.62 (m, 1H, CH), 4.54 (m, 1H, CH),
4.33 (m, 1H, CH), 4.26 (m, 1H, CH), 4.20 (m, 1H, CH), 3.92 (m, 1H, OCH2), 3.79 (m, 1H,
OCH2), 1.55 (s, 3H, CH3), 1.50 (s, 3H, CH3), 1.40 (s, 3H, CH3), 1.35 (s, 3H, CH3); 13C NMR
(100 MHz, CDCl3): δ 152.9, 138.6, 132.0, 131.7, 131.4, 131.1, 129.7, 128.0, 125.3, 122.6,
121.9, 120.2, 119.9, 115.6, 109.3, 108.9, 101.7, 71.0, 70.8, 70.1, 66.7, 60.6, 26.5, 26.0, 25.2,
24.1. HR-MALDI-MS (DHB): calcd for C20H24F3NO7 m/z 470.13971 ([M+Na]+), found
470.14036.
4.1.2.3 General procedure for deprotection of carbamates 13-18
The solution of carbamate 13-18 (0.5 mmol) in the mixture of H2O/TFA (1/1; v/v; 5 mL)
was stirred at room temperature for 24 h. The solvents were removed under reduced pressure
and the residue was dissolved in MeOH (10 mL) and the solvent was distilled off. This
procedure was repeated at least fivefold. The crude products were purified by column
chromatography on silica gel (CH2Cl2/MeOH (1/1)).
O-(α/β-D-Galactopyranose-6-yl)-N-(3,4-dichlorophenyl)carbamate 19

White crystalline solid; yield: 83%; decomposition 117–120 °C; FT-IR (cm–1): υ 3291; 1669;
1
1595; 1524; 1201; 1134; 1075; 1047; 1017; 801; 516. H NMR (400 MHz, CD3OD): δ 7.68
(m, 2H, Ph), 7.33 (m, 2H, Ph), 7.26 (m, 2H, Ph), 5.13 (m, 1H, CH), 4.43 (m, 1H, CH), 4.26
(m, 5H, 5xCH), 3.90 (m, 1H, CH), 3.83 (m, 1H, CH), 3.76 (m, 2H, OCH2), 3.47 (m, 2H,
13
OCH2), 3.27 (m, 1H, CH); C NMR (100 MHz, CD3OD): δ 155.3, 155.3, 140.3, 133.3,
131.5, 126.6, 120.9, 119.2, 98.6, 94.2, 74.7, 74.1, 73.6, 71.0, 70.3, 70.2, 69.7, 69.4, 65.6, 65.4.
HR-MALDI-MS (DHB): calcd for C13H15Cl2NO7 m/z 390.01178 ([M+Na]+), found
390.01254.
O-(α/β-D-Galactopyranose-6-yl)-N-(3-(trifluoromethyl)phenyl)carbamate 20
White crystalline solid; yield: 85%; decomposition 109–112 °C; FT-IR (cm–1): υ 3303; 1670;
1
1548; 1453; 1335; 1202; 1100; 1067; 696; 519. H NMR (400 MHz, CD3OD): δ 7.81 (s, 2H,
Ph), 7.57 (d, 3J = 8.0 Hz, 2H, Ph), 7.40 (t, 3J = 8.0 Hz, 2H, Ph), 7.24 (d, 3J = 8.0 Hz, 2H, Ph),
5.16 (m, 1H, CH), 4.46 (m, 1H, CH), 4.27 (m, 5H, 5xCH), 3.93 (m, 1H, CH), 3.87 (m, 1H,
13
CH), 3.79 (m, 2H, OCH2), 3.49 (m, 2H, OCH2); C NMR (100 MHz, CD3OD): δ 155.5,
155.5, 141.0, 132.6, 132.3, 131.9, 131.6, 130.6, 129.5, 126.8, 124.1, 122.8, 121.4, 120.2,
115.9, 98.6, 94.1, 74.7, 74.1, 73.6, 71.0, 70.9, 70.3, 70.2, 69.4, 65.5, 65.4. HR-MALDI-MS
(DHB): calcd for C14H16F3NO7 m/z 390.07711 ([M+Na]+), found 390.07790.
O-(α/β-Glucopyranose-3-yl)-N-(3,4-dichlorophenyl)carbamate 21
White crystalline solid; yield: 97%; decomposition 167–170 °C; FT-IR (cm–1): υ 3277; 1677;
1
1535; 1247; 1113; 1097; 1027; 561. H NMR (400 MHz, CD3OD): δ 7.72 (m, 2H, Ph), 7.31
(m, 4H, Ph), 5.11 (m, 2H, 2xCH), 4.56 (m, 1H, CH), 3.86–3.63 (m, 5H, 5xCH), 3.50 (m, 3H,
13
1xOCH2, CH), 3.36 (m, 1H, CH), 3.27 (m, 2H, OCH2); C NMR (100 MHz, CD3OD): δ
155.8, 155.5, 140.5, 133.3, 131.4, 126.4, 121.0, 119.3, 98.1, 93.9, 80.0, 78.0, 77.8, 74.7, 72.9,
72.2, 69.9, 69.8, 62.5, 62.3. HR-MALDI-MS (DHB): calcd for C13H15Cl2NO7 m/z 390.01178
([M+Na]+), found 390.01259.
O-(α/β-D-Glucopyranose-3-yl)-N-(3-(trifluoromethyl)phenyl)carbamate 22
White crystalline solid; yield: 67%; decomposition 125–128 °C; FT-IR (cm–1): υ 3296; 1679;
1544; 1332; 1240; 1116; 1017; 725. 1H NMR (400 MHz, CD3OD): δ 7.84 (s, 2H, Ph), 7.60 (d,
3J = 8.0 Hz, 2H, Ph), 7.39 (t, 3J = 8.0 Hz, 2H, Ph), 7.23 (d, 3J = 8.0 Hz, 2H, Ph), 5.16 (m, 1H,
1xCH), 5.12 (m, 1H, 1xCH), 4.60 (m, 1H, CH), 3.90–3,67 (m, 5H, 5xCH), 3.56 (m, 3H,
13
1xOCH2, CH), 3.35 (m, 3H,1xOCH2, CH); C NMR (100 MHz, CD3OD): δ 156.0, 155.8,
141.1, 132.5, 132.2, 131.9, 131.5, 130.5, 129.5, 126.8, 124.1, 122.9, 121.4, 120.1 116.0, 98.1,

93.9, 80.0, 77.9, 77.7, 74.6, 72.9, 72.1, 69.9, 69.7, 62.4, 62.2. HR-MALDI-MS (DHB): calcd
for C14H16F3NO7 m/z 390.07711 ([M+Na]+), found 390.07787.
O-(α/β-D-Fructofuranose-1-yl)-N-(3,4-dichlorophenyl)carbamate 23
White crystalline solid; yield: 89%; decomposition 171–173 °C; FT-IR (cm–1): υ 3278; 1715;
1
1592; 1538; 1226; 1115; 1067; 785; 417. H NMR (400 MHz, CD3OD): δ 7.74 (m, 2H, Ph),
7.38 (m, 2H, Ph), 7.31 (m, 2H, Ph), 4.27–4.02 (m, 7H, 5xCH, OCH2), 3.88 (m, 1H, OCH2),
13
3.83 (m, 1H, OCH2), 3.75 (m, 3H, CH, CH2), 3.65 (m, 2H, CH2); C NMR (100 MHz,
CD3OD): δ 155.2, 155.2, 140.3, 133.3, 131.5, 126.6, 120.9, 119.2, 101.6, 98.3, 83.3, 78.3,
76.4, 71.4, 71.0, 69.5, 67.1, 66.1, 64.7, 64.1. HR-MALDI-MS (DHB): calcd for
C13H15Cl2NO7 m/z 390.01178 ([M+Na]+), found 390.01244.
O-(α/β-D-Fructofuranose-1-yl)-N-(3-(trifluoromethyl)phenyl)carbamate 24
White crystalline solid; yield: 67%; decomposition 151–153 °C; FT-IR (cm–1): υ 3274; 1718;
1
1565; 1336; 1229; 1094; 1067; 761; 522. H NMR (400 MHz, CD3OD): δ 7.86 (s, 2H, Ph),
7.63 (m, 2H, Ph), 7.46 (t, 3J = 8.0 Hz, 2H, Ph), 7.29 (d, 3J = 8.0 Hz, 2H, Ph), 4.28–4.02 (m,
7H, 5xCH, OCH2), 3.88 (m, 1H, OCH2), 3.83 (m, 1H, CH, OCH2), 3.75 (m, 3H, CH, CH2),
3.64 (m, 2H, CH2); 13C NMR (100 MHz, CD3OD): δ 155.4, 155.2, 141.2, 132.7, 132.3, 132.0,
131.7, 130.7, 129.6, 126.9, 124.2, 122.8, 121.6, 120.2, 115.9, 101.6, 98.3, 83.4, 78.4, 76.4,
71.4, 71.0, 69.6, 67.1, 66.1, 64.7, 64.2. HR-MALDI-MS (DHB): calcd for C14H16F3NO7 m/z
390.07711 ([M+Na]+), found 390.07797.
4.2 Biology
4.2.1 Determination of actual enzyme activity
For determination of enzyme actual activity modified spectrophotometric Ellman's method
(ELM) was used. Ellman's method is a simple, rapid and direct method to determine the SH
and -S-S- groups contained in proteins [49]. 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 and thiocholine (TCh), a product
of substrate (i.e. acetylthiocholine, ATCh, or butyrylthiocholine, BTCh) hydrolysis catalyzed
by cholinesterase [50].

Cholinesterase activity was determined using ELM at 25 °C in presence of 0.1 mol/L
phosphate buffered saline (PBS, pH 7.4) in glass cuvette with 1 cm optical path. Product of
given enzymatic reaction, TNB, was detected by spectrophotometer with diode array Hewlett-
Packard 8453 at the wavelength of 412 nm against comparative solution. The dependence of
absorbance A (412 nm) vs. time (t) was observed for 70 s and actual activity of enzyme was
calculated. Measurement of activity was carried out in triplicate at least. The average value of
daily determined catalytic activity of given enzyme was used to calculate its suitable volume
which had to be added into initial reaction mixture for determination of inhibitory effect to
achieve the final activity in reaction mixture 0.2 U/mL.
4.2.2 Determination of cholinesterase inhibition in presence of substituted N-
phenylcarbamates
All tested compounds (Table 1) were dissolved in DMSO to concentration 0.01 M and then
diluted in demineralized water as necessary. The inhibitory efficiency of tested substituted N-
phenylcarbamates was evaluated by the value of IC50 (50% inhibitory concentration, i.e. the
concentration of inhibitor that is necessary for reduction of enzyme activity or reaction rate to
50%). For determination of IC50 the previously published measuring procedure (modified
Ellman's method) was used [46]. The enzyme activity in total reaction mixture (2000 μL) was
0.2 U/mL, concentration of ATCh (or BTCh) 40 μM and concentration of DTNB 0.1 mM for
all reactions.
For all tested compounds and standards (Rivastigmine and Galanthamine) four or five
different concentrations of inhibitor (or standard) were used. Measurements were carried out
in duplicate at least, and the average value of reaction rate (vi) was calculated. Then the
dependence v0/vi vs. concentration of inhibitor was constructed and IC50 was calculated from
obtained equation of regression curve (based on the definition of IC50).

4.2.3 Investigation of inhibition type
For the determination of the inhibition mechanism a Lineweaver-Burk plot was used [51]
and the measuring procedure was similar to that for determination of IC50. Five
N-phenylcarbamates (1, 10, 12, 13 and 18: see Table 1) and four different concentrations of
substrate (20, 40, 60 and 80 μM) were used for investigation of cholinesterase inhibitory
mechanism. For each of substrate concentration four or five different concentrations of
inhibitor were chosen according to their IC50 values. Each measurement was performed in
duplicate. The dependence of absorbance (412 nm) on time was measured, and the reaction
rate was calculated for uninhibited and inhibited reactions. Then, a Lineweaver-Burk plot was
constructed, and Michaelis constant and maximum rate were calculated. The purpose was to
observe the effect of inhibitor on kinetic parameters.
4.2.4 Determination of partition coefficient in system n-octanol/water
The partition coefficient serves as a quantitative descriptor of lipophilicity and is one of the
key determinants of pharmacokinetic properties. The partition coefficient Pow
(n-octanol/water) indicates potential of the drug for crossing the blood-brain barrier for direct
inhibition of brain cholinesterases. The classical and most reliable method of partition
coefficient determination is the „shake-flask method“, which consists of dissolving some of
the solute in question in volume of n-octanol and water, then measuring the concentration of
the solute in each solvent [52]. The most common method of measuring the distribution of the
solute is by UV/VIS spectroscopy.
In first step, into the test tube 2000 µL of n-octanol were placed. Then 15 µL of n-octanol
solution of chosen inhibitor (0.01 M) were added. Mixture was intensively shaken for 15 min.
Then 1000 µL of this mixture was placed into the cuvette and its absorbance at the
wavelength of absorption maximum was measured. Comparative solution was n-octanol. The

value of absorbance which is corresponding to 100% of chosen inhibitor in n-octanol was
obtained.
In second step, 15 µL of n-octanol solution of chosen inhibitor (0.01 M) were added into
the mixture of n-octanol (2000 µL) and demineralized water (2000 µL). Mixture was
intensively shaken for 15 min and then centrifuged (3000 rpm, 10 min). Consequently, 1000
µL of the n-octanol layer was put into the cuvette and its absorbance at the wavelength of
absorption maximum was measured. Comparative solution was n-octanol again. Percentage
content of chosen inhibitor in the n-octanol layer (%)1 was obtained. Then the percentage
content of inhibitor in the water layer was calculated [100 - (%)1]. Partition coefficients were
calculated according to eq. 1, where c1 and c2 are molar concentrations of the chosen inhibitor
in n-octanol and water.
(1)
4.2.5 Molecular modeling of ligand-enzyme interaction
The starting structures of compounds 1-24 were obtained by equilibrium conformer
systematic search (MMFF94) in the program SPARTAN’08 (Wavefunction, Inc., Irvine, CA,
2009). The crystallographic structures of human enzymes were taken from the Brookhaven
protein databank: the structure with pdb code 4m0f for AChE and 5k5e for BChE. The
protonation state corresponding to the experimental pH 7.4 was taken into account. The
standard docking protocol and YAMBER3 force field included in program YASARA were
used. The maximum number of the first five clusters was then searched for the minimum
value of the binding energy Ebin within the optimization protocol em_run.mcr, which allows
optimizing the whole complex by a combination of gradient optimization, simulated
annealing and single molecular dynamics calculation in water. The optimal geometries of the
complexes were then used for analysing the key interactions.

Declaration of interest
The authors declare no conflict of interest.
Acknowledgements
This work was supported by the Faculty of Chemical Technology, University of Pardubice
and VEGA 2/0033/14.
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Tab. 1 List of tested N-phenylcarbamates and their inhibitory activity against eeAChE and
eqBChE expressed as IC50 (µM) in comparison with standards Rivastigmine and
Galanthamine, selectivity and partition coefficient expressed as log Pow.
IC50 (µM)
AChE/BChE
ratio
R1
R2
No.
log Pow
eeAChE
20.6 ± 0.3
12.9 ± 0.2
15.7 ± 0.2
18.3 ± 0.4
20.9 ± 1.0
15.1 ± 0.7
12.4 ± 1.4
14.1 ± 0.1
15.0 ± 0.8
7.7 ± 1.2
eqBChE
Me
Et
-H
15.7 ± 1.3
11.4 ± 0.3
11.2 ± 0.6
10.2 ± 0.2
12.2 ± 0.5
11.1 ± 0.7
12.4 ± 0.4
22.0 ± 0.2
14.5 ±0.4
6.1 ± 0.02
9.6 ± 0.03
10.5 ± 0.04
29.4 ± 0.03
36.7 ± 0.2
> 200
1.3
1.1
1.4
1.8
1.7
1.4
1.0
0.6
1.0
1.3
0.9
0.9
1.6
1.7
---
3.31 ± 0.3
3.9 ± 0.4
5.1 ± 0.7
4.6 ± 0.7
5.3 ± 0.4
3.5 ± 0.2
5.0 ± 0.5
4.8 ± 0.5
5.4 ± 0.6
4.9 ± 0.3
5.3 ± 0.5
6.3 ± 0.6
6.2 ± 0.6
5.0 ± 0.3
7.3 ± 0.7
5.9 ± 0.5
4.9 ± 0.4
6.7 ± 0.5
2.0 ± 0.1
2.03 ± 0.18
3.1 ± 0.3
1.9 ± 0.1
3.0 ± 0.4
3.0 ± 0.3
2.2 ± 0.2
1.6 ± 0.1
1
2
-H
nBu
-H
3
Et
3-Me
4
nBu
3-Me
5
Et
4-MeO
4-MeO
4-Cl
6
nBu
7
Et
8
nBu
4-Cl
9
Me
3,4-di(Cl)
3,4-di(Cl)
3,4-di(Cl)
3,4-di(Cl)
3-CF3
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
Et
8.6 ± 0.2
nBu
9.9 ± 0.8
PG-galactosyl
PG-galactosyl
PG-glucosyl
PG-glucosyl
PG-fructosyl
PG-fructosyl
Galactosyl
Galactosyl
Glucosyl
Glucosyl
Fructosyl
Fructosyl
46.9 ± 0.3
61. 4 ± 0.8
69.4 ± 1.4
70.0 ± 0.9
76.3 ± 2.2
76.5 ± 0.03
94.3 ± 0.1
99.2 ± 2.2
89.1 ± 2.6
103.4 ± 1.3
98.0 ± 2.4
103.5 ± 1.0
56.1 ± 1.4
1.5 ± 0.02
3,4-di(Cl)
3-CF3
> 200
---
3,4-di(Cl)
3-CF3
35.3 ± 0.8
40.1 ± 0.3
> 200
2.2
1.9
---
3,4-di(Cl)
3-CF3
> 200
---
3,4-di(Cl)
3-CF3
> 200
---
> 200
---
3,4-di(Cl)
3-CF3
> 200
---
> 200
---
Rivastigmine
Galantamine
66.3 ± 5.3
4.0 ± 0.02
0.9
0.4
ChE inhibition and partition coefficient are expressed as the mean ± SD (n = 2 experiments).

310
260
210
160
110
60
c (I) = 25 µM
c (I) = 20 µM
c (I) = 10 µM
c (I) = 5 µM
c (I) = 0 µM
10
-5.00E+03 5.00E+03 1.50E+04 2.50E+04 3.50E+04 4.50E+04 5.50E+04
-40
1/[BTCh] (M)
Fig. 1 The example of kinetic analysis of compound 18 for combination BTCh-eqBChE.
The measurements were done in duplicate and the points are express as average values.

Fig. 2 Molecular modeling for compound 10 (2A) and 19 (2B) in active site of BChE (PDB
code: 5k5e).

Fig. 3 Molecular modeling for compound 10 in active site of AChE (PDB code: 4m0f).