Synthesis and in vitro evaluation of novel N-cycloalkylcarbamates as potential cholinesterase inhibitors

Article abstract of DOI:10.1007/s00706-017-2026-5

Abstract: This present paper describes the preparation and characterization of a series of O-substituted N-cycloalkylcarbamate derivatives. These compounds were tested as inhibitors of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). All studied carbamates exhibited moderate inhibitory activity of both cholinesterases with values of IC₅₀ in the range of 36.1–78.6?μM for AChE and 9.8–215.4?μM for BChE, respectively. These values are comparable with those values of inhibition obtained with the established drug rivastigmine. The cytotoxicity of all carbamates was evaluated using standard in vitro test with Jurkat cells. Many of the studied carbamates can be considered as promising compounds for potential medicinal applications with regard to their inhibitory activity as well as negligible cytotoxicity.

Full text of DOI:10.1007/s00706-017-2026-5

Monatsh Chem
DOI 10.1007/s00706-017-2026-5
ORIGINAL PAPER
Synthesis and in vitro evaluation of novel N-cycloalkylcarbamates
as potential cholinesterase inhibitors
1
1
2
2
•
Eva Hor a´ kov a´
•
Pavel Drabina
2
•
Lenka Br u˚ cˇ kov a´
•
ˇ ˇ
S a´ rka St eˇ p a´ nkov a´
1
Katar ´ı na Vor cˇ a´ kov a´
•
Milo sˇ Sedl a´ k
Received: 28 April 2017 / Accepted: 14 June 2017
Ó Springer-Verlag GmbH Austria 2017
Abstract This present paper describes the preparation and
characterization of a series of O-substituted N-cycloalkylcar-
bamate derivatives. These compoundswere tested as inhibitors
of acetylcholinesterase (AChE) and butyrylcholinesterase
Keywords Bioorganic chemistry Á Drug research Á
Enzymes Á Carbamates Á Cholinesterase inhibitors Á
In vitro cytotoxicity
(BChE). All studied carbamates exhibited moderate inhibitory
activity of both cholinesterases with values of IC in the range
5
Introduction
0
of 36.1–78.6 lM for AChE and 9.8–215.4 lM for BChE,
respectively. These values are comparable with those values of
inhibition obtained with the established drug rivastigmine. The
cytotoxicity of all carbamates was evaluated using standard
in vitro test with Jurkat cells. Many of the studied carbamates
can be considered as promising compounds for potential
medicinal applications with regard to their inhibitory activity
as well as negligible cytotoxicity.
Alzheimer’s disease (AD) belongs among the progressive
neurodegenerative brain disorders accompanied with
characteristic clinical and pathological symptoms [1–3].
AD causes formation of dementia in ca. 60–70% cases
diagnosed in the elderly population and it is supposed that
about 6% of the population worldwide aged over 65 years
is affected by AD [1–4]. The origin of AD is still unclear
up to the present day [5]. One of the possible explanations
of AD manifestation is provided by the so-called cholin-
ergic hypothesis [6, 7], which proposes that the
pathophysiology of AD is related to the low level of the
neurotransmitter acetylcholine in cholinergic synapses [8].
The low levels of acetylcholine can be attributed to the
increased activity of cholinesterase enzymes (AChE and
BChE, respectively). Therefore, inhibition of cholines-
terases is considered as a promising strategy for the therapy
of AD [6, 7]. With regard to these findings, many of the
pharmaceutical substances currently used to treat the cog-
nitive problems of AD are based on reversible
cholinesterase inhibitors: rivastigmine, galantamine, and
donepezil [8]. Unfortunately, these drugs exhibit several
disadvantages, for instance, poor selectivity [9], lower
bioavailability [9], and adverse side effects on peripheral
Graphical abstract
Dedicated to Prof. Jarom ´ı r Kav a´ lek on the occasion of his 80th
birthday.
&
Pavel Drabina
pavel.drabina@upce.cz
1
Institute of Organic Chemistry and Technology, Faculty of
Chemical Technology, University of Pardubice, Studentska´
nervous system [10] and liver [11, 12]. The rivastigmine
Ò
(sold under the trademark Exelon ) used in the therapy of
5
73, 532 10 Pardubice, Czech Republic
patients in the early or middle stage of AD contains the
carbamate group in the structure, which is among others
responsible for the inhibitory effect [13]. Therefore,
2
Department of Biological and Biochemical Sciences, Faculty
of Chemical Technology, University of Pardubice,
Studentsk a´ 573, 532 10 Pardubice, Czech Republic
123

E. Hor a´ kov a´ et al.
differently substituted carbamate derivatives become the
object of interest in terms of the study of their inhibitory
activity against AChE and/or BChE [14–25]. Moreover, the
AChE and BChE inhibitors, respectively, can be applied in
the therapy of other diseases, including myasthenia gravis,
some other dementias, parasitic infections, glaucoma,
obstipation, or to antagonize muscle relaxation [26–28].
In our previous paper, we prepared and studied biolog-
modifications in the area of N-(2-phenylcyclopropyl) sub-
stituent including 1-phenyl isomers, non-substituted
cyclopropyl derivative or extension of cyclopropane into
cyclobutane derivatives were considered. Hence, these
corresponding carbamate derivatives were also prepared
and their inhibitory activity of cholinesterases as well as
cytotoxicity was determined.
ical
activity
of
the
series
of
N-(2-
phenylcyclopropyl)carbamate derivatives [29]. It was
found that these compounds exhibited moderate inhibitory
activity of cholinesterases (54.8–94.4 lM for AChE and up
to 5.8 lM for BChE, respectively). While the inhibitory
activity of AChE of this class of carbamates did not change
with O-substitution dramatically, their cytotoxicity signif-
icantly depended on the type of O-alkyl group. Generally,
lower cytotoxicity against Jurkat cells showed the deriva-
tives bearing bulky alkyl groups, for instance tert-butyl,
cyclohexyl and groups derived from monosaccharides or
monoterpenes. With regard to these findings, we decided to
extend this study with new N-(2-phenylcyclopropyl)car-
bamates derived from some other monoterpenes and
monosaccharides. A further aim of this work was the
preparation and study of the biological activity of N-(2-
phenylcyclopropyl)carbamates containing halogen atom in
their structure, which could allow the formation of an
additional type of non-covalent interaction—halogen
bonding—between the inhibitor and protein backbone of
cholinesterase enzymes [30–44]. Moreover, the structural
Results and discussion
Chemistry
The survey of prepared carbamate derivatives 1–16 is
summarized in Fig. 1. The compounds 1–13 were synthe-
sized from individual carboxylic acid derivatives. While
1-phenylcyclopropanecarboxylic acid and 1-phenylcy-
clobutanecarboxylic acid, respectively, were commercially
available, 2-phenylcyclopropanecarboxylic acid (21) and
its 4-halogeno analogues 22–25 were synthesized accord-
ing to previously described methods [45–49]. In the case of
the carboxylic acids 22–24, a standard two-step synthesis
was used, including at first cyclopropane cycle formation
by the action of ethyl diazoacetate onto the corresponding
styrene derivative catalysed by a copper(I) salt that affor-
ded ethyl 2-phenylcyclopropanecarboxylates 17–20 as a
mixture of the cis/trans form. The major stereoisomers
with trans arrangement were separated by column chro-
matography with satisfactory yields (50–70%). The
Fig. 1 Survey of the prepared carbamates 1–16
1
23

Synthesis and in vitro evaluation of novel N-cycloalkylcarbamates as potential…
Scheme 1
subsequent hydrolysis of the ester group under basic con-
dition gave the acids 22–24 with high yields (75–99%).
Differently, 4-iodo derivative 25 was prepared by direct
iodination of trans-2-phenylcyclopropanecarboxylic acid
introduced by the action of di-tert-butyl dicarbonate onto
primary amines [i.e. 3-phenylpropylamine, cyclopropy-
lamine, and 3-phenylcyclobutylamine (28)] [53]. The
compound 28 was available by three-step synthesis,
including at first 3-phenylcyclobutanone (26) formation
(46% yield) via [2 ? 2] cycloaddition of styrene with
keteniminium salt [54]. Subsequently, ketone 26 was
transformed into the corresponding oxime 27 with a good
yield of 85% and, finally, the reduction of oxime 27 by
(
21) with a moderate yield of 42% [49]. Finally, the car-
boxylic acids 21–25 were transformed to carbamate
derivatives 1–13 by the treatment of diphenylphosphoryl
azide in the presence of triethylamine and the corre-
sponding hydroxyl derivative [50–52] (Scheme 1). The
solvent used for this reaction depended on the type of
individual alcohols. The reacting tert-butyl alcohol was
used as solvent in the case of preparation of the carbamates
LiAlH in THF afforded the required amine 28 with a
4
moderate yield of 49% as a mixture of cis/trans form
[55, 56]. Unfortunately, the separation of the individual
stereoisomers of amine 28 as well as its carbamate
derivative 16 by column chromatography was
unsuccessful.
1
–4, 9, and 12, whereas the carbamates 5–7 and 10 derived
from higher alcohols were prepared in dry toluene.
Alternatively, for more polar alcohols (e.g. protected
monosaccharides) dry 1,4-dioxane was a more convenient
solvent (used for carbamates 8, 11, and 13). The yields of
this transformation, including Curtius rearrangement and
the subsequent addition of alcohol onto the isocyanate
intermediate, varied significantly with the individual alco-
hol used from 40 to 86%.
All newly prepared carbamates 1–16 were character-
1 13
ized by means of melting point, H and C NMR
spectroscopy and high-resolution mass spectroscopy. In
1
3
some cases, two sets of signals were observed in the
C
NMR spectra (e.g. compounds 9 and 10), which belong to
different rotamers. The measurement of their spectra at
higher temperature (347 K) gave only one set of signals.
The purity of compounds was verified by means of ele-
mental analysis.
The alternative synthetic strategy was used for the
preparation of the carbamates 14–16 containing O-tert-
butyl group. In this case, the carbamate group was
123

E. Hor a´ kov a´ et al.
Biology
all of the tested carbamates 1–16 showed a considerable
inhibitory activity of both choline esterases. The IC50
values for AChE inhibition varied in the range of
36.1–78.6 lM and for BChE inhibition in the range of
9.8–215.4 lM. Markedly, the IC50 values for BChE are in
a significantly broader range than for AChE.
The lipophilicity of the prepared carbamates 1–16 was
studied as one of the important physicochemical parame-
ters of potential drugs. Generally, the lipophilicity strongly
affects passive transport of drug across biological mem-
branes, and in the case of the cholinesterase inhibitors
designed for AD therapy, it is necessary that they possess
high ability to cross the blood–brain barrier. For evaluation
of this ability, the partition coefficient in the system n-
octanol:water expressed as Pow or log Pow may be used
In comparison with the results of the inhibitory activity
of carbamate derivatives tested in our previous study [29],
it is obvious that the introduction of the halogen atom at
position 4 of the phenyl cycle does not change the inhibi-
tory activity of AChE (62.9 lM (non-substituted) vs.
44.7–78.6 lM for 1–4), whereas the inhibitory activity of
1–4 against BChE decreased [21.1 lM (non-substituted)
vs. 89.4–215.4 lM for 1–4]. On the other hand, halo-
genated derivatives exhibited low cytotoxicity against
blood-originated Jurkat cell line (Table 2). O-Benzyl-N-(2-
phenylcyclopropyl)carbamate (5) was also significantly
less cytotoxic compared with O-2-phenylethyl as well as
O-3-phenylpropyl homologues tested in a previous study
[29], while the inhibitory activity remained unchanged. On
the contrary, the geranyl derivative 7 was found to be the
most cytotoxic derivative in the studied series of carba-
mates, although its inhibitory activity was comparable. The
protected fructopyranosyl derivative 8 showed better inhi-
bitory activity (36.8 lM) of AChE than the previously
tested monosaccharide derivatives (i.e. galactopyranosyl,
[
57, 58]. The partition coefficient serves as a quantitative
descriptor of lipophilicity and is one of the key determi-
nants of the pharmacokinetic properties. The log Pow
values of carbamates 1–16 varied in the range from 2.07 to
5.48 (Table 1), wherein only two derivatives (7 and 11)
slightly exceeded the Lipinski’s rule of five [59]. There-
fore, it is possible to conclude that all tested carbamates 1–
16 possess adequate lipophilicity and could cross the
blood–brain barrier.
The carbamates 1–16 were consequently investigated
for their inhibitory activity against AChE from electric eel
and BChE from equine serum using modified Ellman’s
method [60]. The results were compared with that previ-
ously determined for the established drug rivastigmine
[
61]. From the obtained results (Table 1), it is obvious that
Table 1 The inhibitory activity of carbamate derivatives 1–16 against AChE and BChE expressed as IC50; lipophilicity log Pow and selectivity
to BChE
a
b
a
Compound
IC50/lM
Selectivity to BChE
log Pow
AChE
BChE
1
2
3
4
5
6
7
8
9
44.67 ± 3.82
78.63 ± 4.63
50.43 ± 1.09
73.17 ± 4.24
54.93 ± 6.33
49.39 ± 1.50
36.07 – 2.33
36.76 ± 5.34
59.49 ± 5.29
84.93 ± 9.77
54.89 ± 4.81
75.45 ± 9.19
45.00 ± 2.79
41.89 ± 6.93
44.09 ± 5.94
53.18 ± 3.89
56.10 ± 1.41
96.13 ± 2.81
168.58 ± 3.40
215.39 ± 2.40
89.44 ± 2.23
33.29 ± 0.01
15.85 ± 0.64
47.45 ± 1.48
120.61 ± 2.09
53.33 ± 5.73
179.50 ± 3.86
9.77 – 0.32
45.66 ± 0.73
47.38 ± 0.74
30.65 ± 1.44
106.64 ± 0.36
97.20 ± 0.45
38.40 ± 1.97
0.46
0.47
0.23
0.82
1.65
3.12
0.76
0.30
1.12
0.47
5.60
1.65
0.95
1.36
0.41
0.55
1.46
3.02 ± 0.11
4.27 ± 0.55
3.91 ± 0.30
3.96 ± 0.18
3.57 ± 0.20
4.52 ± 0.51
5.33 ± 0.43
3.50 ± 0.35
3.37 ± 0.27
4.20 ± 0.27
5.48 ± 0.62
3.59 ± 0.27
3.38 ± 0.22
3.25 ± 0.25
2.07 ± 0.24
3.16 ± 0.25
–
10
11
12
13
14
15
16
Rivastigmine [61]
The best values of IC50 for each enzyme and selectivity are shown in bold
a
Data are presented as mean values ± SD from two independent experiments
b
Selectivity to BChE: IC50 for AChE/IC50 for BChE
1
23

Synthesis and in vitro evaluation of novel N-cycloalkylcarbamates as potential…
Table 2 Cell viability of Jurkat cells measured using XTT cytotoxicity assay after treatment with different concentrations of the tested
compounds
a
Compound
Cell viability/% of control
0
.2% DMSO
5 lM
10 lM
50 lM
100 lM
250 lM
500 lM
1
2
3
4
5
6
7
8
9
100.02 ± 2.45
100.00 ± 3.17
100.02 ± 1.63
100.36 ± 3.69
100.12 ± 3.27
100.18 ± 1.07
100.01 ± 1.59
100.06 ± 5.72
100.38 ± 2.05
100.11 ± 0.15
100.43 ± 2.94
100.38 ± 0.29
100.45 ± 1.54
100.14 ± 3.96
100.17 – 4.21
100.33 ± 1.43
99.30 ± 1.62
83.59 ± 5.79
99.12 ± 2.27
99.83 ± 0.92
95.94 ± 2.11
99.88 ± 1.29
99.15 ± 1.43
99.95 ± 2.06
98.43 ± 2.91
98.79 ± 2.60
100.71 ± 2.25
94.74 ± 1.52
100.07 ± 2.47
98.45 ± 2.15
99.80 – 1.63
97.42 ± 2.62
98.40 ± 1.22
85.85 ± 6.14
97.01 ± 4.10
96.33 ± 2.15
91.08 ± 3.70
98.91 ± 3.79
97.03 ± 0.94
96.26 ± 0.63
94.82 ± 6.35
95.23 ± 0.44
97.91 ± 1.75
84.68 ± 3.30
96.32 ± 1.31
94.41 ± 2.47
99.43 – 1.49
92.98 ± 1.22
92.96 ± 1.23
85.53 ± 4.97
90.44 ± 4.66
93.05 ± 0.71
88.47 ± 2.57
78.63 ± 1.87
81.56 ± 0.60
96.46 ± 1.07
95.23 ± 3.89
92.92 ± 2.76
98.43 ± 2.03
71.60 ± 1.81
94.68 ± 1.50
76.92 ± 2.56
97.76 – 1.37
89.19 ± 2.78
91.05 ± 2.91
81.95 ± 4.79
87.97 ± 3.15
91.50 ± 0.54
85.41 ± 4.38
45.74 ± 2.74
33.30 ± 3.24
95.45 ± 0.87
87.57 ± 1.94
91.29 ± 1.10
95.70 ± 1.04
67.55 ± 3.59
95.63 ± 3.80
64.40 ± 3.36
95.30 – 0.45
86.83 ± 1.85
86.61 ± 0.97
74.02 ± 2.35
88.90 ± 4.66
86.34 ± 2.36
77.03 ± 4.23
25.24 ± 0.57
4.15 ± 1.36
91.43 ± 1.68
56.81 ± 2.34
88.76 ± 0.87
91.45 ± 4.26
54.30 ± 0.92
92.33 ± 1.93
47.38 ± 2.43
94.30 – 0.82
74.33 ± 2.52
65.78 ± 3.74
68.32 ± 5.04
79.52 ± 1.08
83.41 ± 0.75
40.93 ± 4.06
8.57 ± 0.62
0.73 ± 0.23
88.81 ± 2.28
46.94 ± 2.11
80.27 ± 3.06
84.94 ± 1.41
45.52 ± 2.19
88.25 ± 6.11
32.03 ± 0.79
93.14 – 0.71
52.39 ± 0.92
10
11
12
13
14
15
16
Treatment for 48 h. Viability is referred to cells treated with DMSO 0.2% (control). Negative control (NC) was treated with the vehicle (DMSO
.2%) used for diluting the tested substance
0
The values of Cell viability for the carbamate with the lowest cytotoxicity are shown in bold
a
Data are presented as mean values ± SD from three independent experiments
glucofuranosyl, and pinitolyl) (54.8–94.4 lM) [29]; how-
ever, the obtained inhibition of BChE was inferior.
Conclusion
All N-(1-phenylcyclopropyl) derivatives 9–11, N-(1-
phenylcyclobutyl) derivatives 12, 13, and N-(3-phenylcy-
clobutyl) derivative 14 exhibited favourable cytotoxicity,
especially the carbamates derived from protected galac-
topyranose 11 and 13. Moreover, the compound 11
produced the most powerful inhibition of BChE (9.8 lM)
and AChE/BChE selectivity (5.6). With regard to the fact
that the ratio of AChE/BChE changes dramatically in the
cortical regions affected by AD from a value of 0.2 up to
as much as value 11 [62], the selective BChE inhibitors
are currently attractive candidates for AD therapy.
Moreover, selective BChE inhibitors do not exhibit
adverse cholinergic effects typical for AChE inhibitors
In summary, the series of 16 N-cycloalkylcarbamate
derivatives 1–16 were prepared by simple synthetic pro-
cedures. These compounds were subsequently tested for
their inhibitory activity of AChE and BChE, respectively.
All studied carbamates showed moderate inhibitory activ-
ity against AChE and weak to moderate inhibitory activity
against BChE, where the most effective inhibitor for BChE
was found to be derivative 11. This compound also
exhibited the highest selectivity to BChE. Generally, the
inhibitory activity of the carbamates 1–16 investigated in
this work can be considered as comparable with the pre-
viously
studied
N-(2-phenylcyclopropyl)carbamate
derivatives [29]. On the other hand, the carbamates 1–16
possess lower cytotoxicity against Jurkat cells, except
derivatives 7 and 8. In this regard, the derivative 15 can be
evaluated as the promising AChE and BChE inhibitor due
to its potent activity and concurrently very low cytotoxic-
ity. Moreover, this compound as well as many of the others
can be easily synthesized and its structure does not involve
cis/trans isomerism and chirality. From this point of view,
this research contributed new candidates, which can be
utilized as inhibitors for AChE and BChE. Therefore, these
compounds deserve further attention with the aim of
studying their biological activity in detail.
[
63, 64].
The structural modification of previously studied O-tert-
butyl-N-(2-phenylcyclopropyl)carbamate [29] into the
carbamates 15 (without the phenyl cycle) and 16 (propyl
group instead of the cyclopropyl cycle) led to only slight
changes of inhibitory activities (increasing for AChE and
decreasing for BChE, respectively). However, the deriva-
tive 15 can be considered as the most promising compound
for further study due to its very low cytotoxicity and
concurrently moderate inhibitory activity against
cholinesterases.
123

E. Hor a´ kov a´ et al.
Experimental
Synthesis of (± )-trans-2-(4-halogenophenyl)cyclopropane-
1
-carboxylic acids 22–24
To the cooled solution of 1 g NaOH (25 mmol) in 4 cm
EtOH/H O (85/15; v/v) was added the solution of esters 18–
3
The starting chemicals (including styrene derivatives,
1
-phenylcyclopropanecarboxylic acid, 1-phenylcyclobu-
tanecarboxylic acid, cyclopropylamine,
-phenylpropylamine) were obtained from Sigma-Aldrich
or TCI Chemicals and used without further purification.
-Phenylcyclopropanecarboxylic acid (21) (and its corre-
2
3
20 (2.21 mmol) in 9 cm EtOH. The reaction mixture was
stirred at room temperature for 5 h and then the solventswere
removed under reduced pressure. The residue was dissolved
3
3
3
2
in 30 cm water and washed with 2 9 15 cm CH
Cl . An
2 2
sponding ethyl ester 17) were prepared according to the
earlier described procedure [29]. Column chromatography
aqueous layer was acidified with concentrated HCl and
3
extracted with 3 9 15 cm CH
Cl . The organic layers were
2 2
˚
combined and dried over Na SO and evaporated to dryness.
4
was performed using 60 A (60–200 lm) silica gel. All
2
solvents were of reagent grade and when necessary purified
and dried by standard methods. TLC was performed on
aluminium-backed silica gel plates (Merck DC, Alufolien
(
± )-trans-2-(4-Fluorophenyl)cyclopropane-1-carboxylic
acid (22)
White solid; yield: 382 mg (96%). M.p.: 104–106 °C; the
H NMR spectrum was found to be identical with the one
1
Kieselgel 60 F254) with spots visualized by UV light. H
1
NMR spectra were recorded on a Bruker Avance 400
described in Ref. [47].
instrument. Chemical shifts d were referenced to the
residual peak of CDCl at 7.26 ppm. The C NMR spectra
1
3
(± )-trans-2-(4-Chlorophenyl)cyclopropane-1-carboxylic
acid (23)
3
were calibrated with respect to the middle signal in the
triplet of CDCl3 (d = 77.36 ppm) or the multiplet of
White solid; yield: 326 mg (75%). M.p.: 116–117 °C; the
H NMR spectrum was found to be identical with the one
1
DMSO-d (d = 39.51 ppm). The elemental analyses were
6
performed on an apparatus of FISONS Instruments, EA
described in Ref. [47].
1
108 CHNS. High-resolution mass spectra were measured
(
± )-trans-2-(4-Bromophenyl)cyclopropane-1-carboxylic
on the Thermo Fisher Scientific MALDI LTQ Orbitrap
instrument. The used matrix was 0.2 M solution of 2,5-
acid (24)
White solid; yield: 528 mg (99%). M.p.: 119–121 °C (Ref.
dihydroxybenzoic acid (DHB) in MeCN/H O (95/5).
2
1
[48] 118.5–122.4 °C); the H NMR spectrum was found to
be identical with the one described in Ref. [48].
Synthesis of (± )-trans-ethyl 2-(4-halogenophenyl)cyclo-
propane-1-carboxylates 18–20 [45]
Synthesis of (± )-trans-2-(4-iodophenyl)cyclopropane-1-
carboxylic acid (25) [49]
A solution of 8.5 mg CuBF Á4MeCN (27 lmol) and
4
3
-halogenostyrene (5.4 mmol) in dry 9 cm CH Cl was
4
3
To a solution of 1 cm H SO (10% aq.) in 4 cm AcOH,
3
2
2
2
4
stirred under argon atmosphere at room temperature. Then,
3
a solution of 0.31 g ethyl diazoacetate (2.7 mmol) in 3 cm
0
.2 g 21 (1.23 mmol) [29], 173 mg iodine (0.68 mmol),
and 0.61 g KIO (0.28 mmol) were added. The reaction
3
CH Cl was added dropwise over 5 h. Further, the reaction
2
2
mixture was heated to reflux for a period of 4 h, and
mixture was stirred for 17 h, concentrated under reduced
pressure and the residue was purified by column chro-
matography on silica/pentane:EtOAc (10:1, v/v).
3
0 cm AcOH was added in five portions of 4 cm to rinse
3
2
iodine from the condenser. After cooling to room temper-
3
ature, the reaction was quenched with 100 cm 1 M aq.
(
± )-trans-Ethyl
2-(4-fluorophenyl)cyclopropane-1-car-
solution of Na
2
S
2
O
4
. The mixture was extracted with
3 9 100 cm AcOEt; the combined extracts were washed
with brine, dried over Na SO , and concentrated under
3
boxylate (18)
1
Colourless oil; yield: 280 mg (50%). The H NMR spectrum
was found to be identical with the one described in Ref. [46].
2
4
reduced pressure. The crude product was recrystallized
from heptane and afforded 149 mg (42%) 25 (white solid).
1
(
± )-trans-Ethyl
2-(4-chlorophenyl)cyclopropane-1-car-
M.p.: 155–156 °C; the H NMR spectrum was found to be
identical with the one described in Ref. [49].
boxylate (19)
1
Colourless oil; yield: 420 mg (69%). The H NMR
spectrum was found to be identical with the one described
in Ref. [46].
Synthesis of O-alkyl-N-(2-phenylcyclopropyl)carbamates
1–8 [50–52]
To a solution of the corresponding (± )-trans-2-phenylcy-
3
clopropane-1-carboxylic acid, 21–25 (2.0 mmol) in 5 cm
(
± )-trans-Ethyl
2-(4-bromophenyl)cyclopropane-1-car-
boxylate (20)
3
dry tert-butyl alcohol (for 1–4) or 5 cm dry toluene (for 5–
1
Colourless oil; yield: 510 mg (70%). The H NMR
spectrum was found to be identical with the one described
in Ref. [46].
3
) or 5 cm dry 1,4-dioxane (for 8) corresponding alcohol
7
3
2.4 mmol) (except for 1–4), 0.43 cm diphenylphosphoryl
(
1
23

Synthesis and in vitro evaluation of novel N-cycloalkylcarbamates as potential…
3
azide (2.0 mmol) and 0.33 cm TEA (2.4 mmol) were
added. The mixture was heated to reflux for 24 h, and then
the solvent was removed under reduced pressure. The
128.7, 130.0, 136.6, 140.6, 157.0 ppm; HR-MALDI-MS
?
([M?Cs] )
(DHB): m/z calcd for C H CsNO
2
1
7
17
400.03083, found 400.03092.
± )-trans-O-(4-Bromobenzyl)-N-(2-phenylcyclopropyl)-
carbamate (6, C H BrNO )
3
residue was dissolved in 20 cm CH Cl and the solution
2
2
(
3
was washed with 2 9 25 cm saturated sodium bicarbon-
3
ate and 2 9 25 cm brine. The organic layer was dried
17 16
2
White crystalline solid; yield: 428 mg (62%). M.p.:
over Na SO , concentrated under reduced pressure and
4
2
1
8
2
1
0–82 °C; H NMR (400 MHz, CDCl ): d = 1.19 (m,
3
purified by column chromatography on silica gel [EtOAc/
H), 2.06 (m, 1H), 2.75 (m, 1H), 5.05 (s, 2H), 5.17 (bs,
1
H), 7.20 (m, 7H), 7.45 (m, 2H) ppm;
n-hexane (1/1)].
3
C NMR
(
± )-trans-O-tert-Butyl-N-(2-(4-fluorophenyl)cyclopropyl)-
(100 MHz, CDCl ): d = 16.4, 25.2, 32.8, 66.1, 122.3,
3
carbamate (1)
White crystalline solid, yield: 201 mg (40%). M.p.:
126.4, 126.7, 128.6, 130.0, 131.8, 135.6, 140.5, 156.8 ppm;
HR-MALDI-MS (DHB): m/z calcd for C H BrCsNO
2
1
7
16
1
?
([M?Cs] ) 477.94134, found 477.94147.
1
04–105 °C; the H NMR spectrum was found to be
identical with the one described in Ref. [65]. C NMR
1
3
(
± )-trans-O-Geranyl-N-(2-phenylcyclopropyl)carbamate
7, C H NO )
(
100 MHz, CDCl ): d = 16.2, 24.8, 28.7, 32.5, 80.0,
3
(
2
0
27
2
1
1
15.3, 115.5, 128.5, 128.5, 136.6, 136.6, 156.7, 160.5,
1
Colourless oil; yield: 439 mg (70%). H NMR (400 MHz,
62.9 ppm; HR-MALDI-MS (DHB): m/z calcd for C
?
1
4-
CDCl ): d = 1.18 (m, 2H), 1.60 (s, 3H), 1.70 (d,
3
H CsFNO ([M?Cs] ) 384.03706, found 384.03712.
2
1
8
J = 7.6 Hz, 6H), 2.08 (m, 5H), 2.75 (m, 1H), 4.62 (m,
(
± )-trans-O-tert-Butyl-N-(2-(4-chlorophenyl)cyclopropyl)-
2H), 5.08 (m, 2H), 5.36 (bs, 1H), 7.16 (m, 3H), 7.26 (m,
1
3
carbamate (2)
White crystalline solid; yield: 321 mg (60%). M.p.:
2H) ppm; C NMR (100 MHz, CDCl ): d = 16.4, 16.8,
3
18.0, 25.4, 26.0, 26.6, 32.9, 62.1, 119.0, 124.1, 126.4,
1
13
132–135 °C (Ref. [66] 122–123 °C); the H and
NMR spectra were found to be identical with the one
C
126.9, 128.6, 132.1, 140.8, 142.2, 157.5 ppm.
(
± )-trans-O-(2,3:4,5-Di-O-isopropylidene-b-D-fructopy-
ranose-1-yl)-N-(2-phenylcyclopropyl)carbamate
8, C H NO )
described in Ref. [66]. HR-MALDI-MS (DHB): m/z calcd
?
([M?Cs] ) 400.00751, found
for C H ClCsNO
2
1
4
18
(
2
2
29
7
4
00.00765.
White crystalline solid; yield: 461 mg (55%). M.p.:
1
(
± )-trans-O-tert-Butyl-N-(2-(4-bromophenyl)cyclopropyl)-
75–77 °C; H NMR (400 MHz, CDCl ): d = 1.20 (m,
3
carbamate (3)
White crystalline solid; yield: 268 mg (43%). M.p.:
2H), 1.34 (s, 3H), 1.38 (s, 3H), 1.49 (s, 3H), 1.54 (s, 3H),
2.08 (m, 1H), 2.77 (m, 1H), 3.76 (m, 1H), 3.89 (m, 1H),
4.11 (m, 1H), 4.23 (m, 1H), 4.29 (m, 1H), 4.41 (m, 1H),
4.59 (m, 1H), 5.12 (bs, 1H), 7.15 (m, 3H), 7.26 (m, 2H)
1
1
28–130 °C; the H NMR spectrum was found to be
identical with the one described in Ref. [65]. C NMR
1
3
1
3
(
100 MHz, CDCl ): d = 16.4, 24.9, 28.7, 32.7, 80.0,
ppm; C NMR (100 MHz, CDCl ): d = 16.5, 24.4, 25.5,
3
3
1
19.9, 128.6, 131.6, 140.1, 156.6 ppm; HR-MALDI-MS
26.2, 26.7, 32.9, 61.6, 66.6, 70.4, 71.7, 102.0, 108.9, 109.4,
?
DHB): m/z calcd for C H BrCsNO ([M?Cs] )
(
126.5, 126.8, 128.7, 140.6, 156.4 ppm; HR-MALDI-MS
?
([M?Cs] )
1
4
18
2
4
43.95699, found 443.95711.
(DHB): m/z calcd for C H CsNO
7
2
2
29
552.09930, found 552.09923.
(
± )-trans-O-tert-Butyl-N-(2-(4-iodophenyl)cyclopropyl)-
carbamate (4)
White crystalline solid; yield: 395 mg (55%). M.p.:
Synthesis of O-alkyl-N-(1-phenylcyclopropyl)carbamates
9–11
1
1
23–125 °C; H NMR spectrum was found to be identical
with the one described in Ref. [67]. C NMR (100 MHz,
The carbamates 9–11 were prepared according to the above
described procedure for the synthesis of 1–8. For the
preparation of 9, tert-butyl alcohol was used; for the
preparation of 10, dry toluene; and for the preparation of
11, 1,4-dioxane was used as the solvent.
1
3
CDCl ): d = 16.5, 25.1, 28.7, 32.8, 80.1, 91.2, 129.0, 137.6,
3
1
40.8, 156.6 ppm; HR-MALDI-MS (DHB): m/z calcd for
?
C H Cs INO ([M?Cs] ) 491.94312, found 491.94327.
1
4
18
2
(
± )-trans-O-Benzyl-N-(2-phenylcyclopropyl)carbamate
5)
White crystalline solid; yield: 385 mg (72%). M.p.:
O-tert-Butyl-N-(1-phenylcyclopropyl)carbamate (9)
White crystalline solid; yield: 350 mg (75%). M.p.:
(
1
13
70–71 °C; (Ref. [68] 77–78 °C); the H and C NMR
spectra were found to be identical with the one
described in Ref. [68]. HR-MALDI-MS (DHB): m/
1
6
2
1
5–66 °C; H NMR (400 MHz, CDCl ): d = 1.19 (m,
3
H), 2.05 (m, 1H), 2.75 (m, 1H), 5.11 (s, 2H), 5.16 (bs,
1
H), 7.25 (m, 10H) ppm; C NMR (100 MHz, CDCl3):
3
?
z calcd for C H CsNO ([M?Cs] ) 366.04648, found
14
19
2
d = 16.3, 25.2, 32.8, 67.0, 126.3, 126.7, 128.3, 128.5,
366.04661.
123

E. Hor a´ kov a´ et al.
O-(2-Phenylethyl)-N-(1-phenylcyclopropyl)carbamate
10, C H NO )
White crystalline solid; yield: 388 mg (69%). M.p.:
Synthesis of cis/trans-3-phenylcyclobutylamine 28 3-
phenylcyclobutanone 26 [54]
To a solution of 3.3 g N,N-dimethylacetamide (38 mmol)
(
1
8
29
2
1
3
3
9
4
5
3
1
2–94 °C; H NMR (400 MHz, CDCl ): d = 1.21 (m,
in 100 cm 1,2-dichloroethane, 7.6 cm triflic anhydride
(45 mmol) was added dropwise under stirring at 5 °C. The
mixture was stirred at 5 °C for 30 min and then a solution
3
H), 2.89 (t, J = 8.0 Hz, 2H), 4.27 (t, J = 8.0 Hz, 2H),
1
3
.48 (bs, 1H), 7.20 (m, 10H) ppm; C NMR (100 MHz,
47 K, DMSO): d = 17.7, 34.4, 34.8, 63.9, 124.5, 125.3,
25.9, 127.7, 128.0, 128.6, 138.0, 143.7, 155.8 ppm; HR-
3
3
of 17.2 cm styrene (150 mmol) and 6 cm 2,4,6-
3
trimethylpyridine (45 mmol) in 50 cm 1,2-dichloroethane
MALDI-MS (DHB): m/z calcd for C H CsNO
2
was added dropwise. The reaction mixture was refluxed for
18 h, then 1,2-dichloroethane was removed in vacuo and
1
8
29
?
[M?Cs] ) 414.04648, found 414.04663.
(
3
3
the residue was treated with 50 cm water and 50 cm
CH Cl . The obtained mixture was refluxed for 18 h, and
O-(1,2:3,4-Di-O-isopropylidene-a-D-galactopyranose-6-
yl)-N-(1-phenylcyclopropyl)carbamate (11, C H NO )
White crystalline solid; yield: 722 mg (86%). M.p.:
2
2
2
2
29
7
after cooling the water layer was extracted with
3
3
9 30 cm CH Cl . The organic layers were combined,
2 2
1
1
4
4
5
28–129 °C; H NMR (400 MHz, CDCl ): d = 1.25 (m,
3
dried over Na SO , concentrated under reduced pressure
2
4
H), 1.31 (s, 6H), 1.43 (s, 3H), 1.46 (s, 3H), 4.05 (m, 2H),
and purified by column chromatography on silica gel
pentane and then EtOAc/n-hexane (1/1)]. It afforded
.21 (m, 1H), 4.32 (m, 2H), 4.59 (m, 1H), 5.53 (m, 1H),
1
.69 (bs, 1H), 7.24 (m, 5H) ppm; C NMR (100 MHz,
[
3
1
2
.56 g (46%) 26 as a colourless oil. The H NMR spectrum
was found to be identical with the one described in Ref.
54].
CDCl ): d = 17.9, 24.4, 24.7, 25.8, 25.9, 35.2, 63.9, 66.3,
3
7
0.3, 70.6, 71.0, 96.2, 108.6, 109.5, 125.3, 126.2, 128.2,
[
1
42.6, 155.8 ppm; HR-MALDI-MS (DHB): m/z calcd for
?
C H CsNO ([M?Cs] ) 552.09930, found 552.09937.
Synthesis of 3-Phenylcyclobutanone oxime (27) [55, 56]
3
To a solution of 0.5 g 26 (3.42 mmol) in 17.5 cm 96%
3
ethanol, 2.08 cm hydroxylamine (50% aq. solution,
2
2
29
7
Synthesis of O-alkyl-N-(1-phenylcyclobutyl)carbamates
2 and 13
1
33.9 mmol) was added and the reaction mixture was
The carbamates 12 and 13 were prepared according to the
above described procedure for synthesis of 1–8. For the
preparation of 12, tert-butyl alcohol was used and for the
preparation of 13, 1,4-dioxane was used as the solvent.
stirred for 16 h at room temperature. The solution was
3
3
diluted with 25 cm EtOAc and washed with 2 9 50 cm
3
water and 2 9 50 cm brine. The organic layer was dried
over Na SO and concentrated under reduced pressure. The
2
4
O-tert-Butyl-N-(1-phenylcyclobutyl)carbamate (12)
White crystalline solid; yield: 223 mg (45%). M.p.:
purification of the crude product by flash chromatography
(n-hexane and then EtOAc/n-hexane (1/1)) afforded
1
469 mg (85%) 27 as a colourless oil. H NMR
1
5
9
1
5–57 °C; H NMR (400 MHz, CDCl ): d = 1.35 (s,
3
H), 1.84 (m, 1H), 2.08 (m, 1H), 2.56 (m, 4H), 5.10 (bs,
1
(400 MHz, CDCl ): d = 3.04 (m, 2H), 3.35 (m, 1H),
3
3
H), 7.23 (m, 1H), 7.33 (m, 2H), 7.42 (m, 2H) ppm;
C
3.46 (m, 1H), 3.61 (m, 1H), 7.26 (m, 5H), 9.07 (bs, 1H)
1
3
NMR (100 MHz, CDCl ): d = 15.4, 28.7, 34.3, 59.4, 79.7,
ppm; C NMR (100 MHz, CDCl ): d = 33.1, 38.5, 39.6,
3
3
1
25.6, 126.7, 128.3, 146.4, 154.7 ppm; HR-MALDI-MS
?
([M?Cs] )
126.7, 126.9, 128.9, 144.2, 156.8 ppm.
(
DHB): m/z calcd for C H CsNO
2
1
5
21
Synthesis of cis/trans-3-Phenylcyclobutylamine
3
80.06213, found 380.06222.
(
28) [55, 56]
To a solution of 330 mg 27 (2 mmol) in 10 cm dry THF,
3
O-(1,2:3,4-Di-O-isopropylidene-a-D-galactopyranose-6-
yl)-N-(1-phenylcyclobutyl)carbamate (13, C H NO )
White crystalline solid; yield: 579 mg (69%). M.p.:
3
4.41 cm 1 M solution of LiAlH in THF (4.41 mmol) was
2
3
31
7
4
added dropwise over a period of 3 min. The reaction was
3
heated to reflux for 4 h. After cooling, 2 cm 0.5 M
1
1
3
2
4
7
15–116 °C; H NMR (400 MHz, CDCl ): d = 1.31 (s,
3
H), 1.32 (s, 3H), 1.43 (s, 3H), 1.45 (s, 3H), 1.84 (m, 1H),
.10 (m, 1H), 2.55 (m, 4H), 4.02 (m, 2H), 4.20 (m, 1H),
.30 (m, 2H), 4.58 (m, 1H), 5.37 (bs, 1H), 5.53 (m, 1H),
solution of NaOH was added and the mixture was stirred
for 30 min. The suspension was filtered, and the solid was
3
washed with 3 9 5 cm THF. The filtrate was concentrated
1
3
.23 (m, 1H), 7.33 (m, 2H), 7.42 (m, 2H) ppm; C NMR
under reduced pressure and the residue was dissolved in
3
25 cm 1 M HCl. The resulting solution was washed with
(
100 MHz, CDCl ): d = 15.6, 24.8, 25.3, 26.3, 26.3, 34.2,
3
3
3 9 15 cm diethyl ether; the water layer was neutralized
5
9.6, 64.1, 66.9, 70.8, 71.0, 71.5, 96.7, 109.1, 109.9, 126.0,
3
with 1 M NaHCO and extracted with 3 9 20 cm CH
1
27.1, 128.6, 145.7, 154.8 ppm; HR-MALDI-MS (DHB):
3
2-
?
m/z calcd for C H CsNO ([M?Cs] ) 566.11495, found
Cl . The organic layers were combined, dried over Na SO
2 2 4
2
3
31
7
5
66.11495.
and concentrated in vacuo. Colourless oil; yield: 144 mg
1
23

Synthesis and in vitro evaluation of novel N-cycloalkylcarbamates as potential…
1
49%). H NMR (400 MHz, CDCl ): trans isomer:
3
1 cm of this mixture was placed in a quartz cuvette and its
(
3
d = 1.68 (bs, 2 H), 1.78 (m, 2H), 2.73 (m, 2H), 3.05 (m,
H), 3.39 (m, 1H), 7.23 (m, 5H) ppm; cis isomer: d = 1.68
absorbance at the absorption maximum wavelength was
measured. Thereby, the value of absorbance corresponding
to 100% of the tested compound in n-octanol was obtained.
The reference solution was n-octanol. Into the other test
1
(
(
bs, 2 H), 2.17 (m, 2H), 2.46 (m, 2H), 3.59 (m, 1H), 3.67
1
m, 1H), 7.23 (m, 5H) ppm. The H NMR spectrum was
3
3
found to be similar to the one described for the corre-
tube, 1.5 cm n-octanol, 1.5 cm water and n-octanol
3
solution of the tested compound (10 mm ; 0.01 M) were
sponding hydrochloride of 28 in Ref. [54].
placed. This mixture was intensively shaken for 15 min
3
and then centrifuged (3000 rpm, 10 min). Then, 1 cm of
Synthesis of O-tert-butyl-N-alkylcarbamates from substi-
tuted amines [53]
this mixture was placed in the quartz cuvette and its
absorbance at the absorption maximum wavelength was
measured. The reference solution was n-octanol again.
Thereby, the percentage content of the tested compound in
the n-octanol layer (%) was obtained. Subsequently, the log
Pow (log Pow = log (c /c ), where c and c are molar
To a solution of cyclopropylamine, 3-phenylpropylamine,
3
or 28 (5 mmol) in 15 cm dry CH Cl , a solution of
2
2
9
82 mg di-tert-butyl dicarbonate (4.5 mmol) was added
3
dropwise in 10 cm CH Cl . The mixture was stirred at
2
2
room temperature for 2 h. The solvent was removed under
reduced pressure and the residue was purified by flash
chromatography on silica gel [EtOAc/n-hexane (1/1)].
1
2
1
2
concentrations of the tested compound in n-octanol and
water), was calculated. For each compound, at least two
determinations were performed. The obtained results are
summarized in Table 1.
O-tert-Butyl-N-(3-phenylpropyl)carbamate (14)
Colourless oil; yield: 741 mg (70%). The H NMR
1
spectrum was found to be identical with the one described
3
1
in Ref. [69]. C NMR (100 MHz, CDCl ): d = 28.6, 31.9,
Inhibition of cholinesterases
3
3
3.3, 40.3, 79.1, 126.0, 128.5, 128.5, 141.7, 156.2 ppm.
´
AChE, BChE, acetylthiocholine (ATCh), 5,5-dithiobis-2-
O-tert-Butyl-N-(cyclopropyl)carbamate (15)
White crystalline solid; yield: 693 mg (98%). M.p.:
nitrobenzoic acid (DTNB), and n-octanol were purchased
from Sigma-Aldrich. All tested compounds were dissolved
in DMSO (0.01 M) and diluted in demineralized water to
1
5
4–55 °C (Ref. [53] 61–63 °C); the H NMR spectrum
was found to be identical with the one described in Ref.
0.001 M. The ability of the tested compounds to inhibit
1
3
[
69]. C NMR (100 MHz, CDCl ): d = 7.0, 23.2, 28.7,
3
AChE (from electric eel) and BChE (from equine serum)
was determined using modified Ellman’s method at 25 °C
in the presence of phosphate-buffered saline (PBS, 0.1 M,
pH 7.4) in a glass cuvette with 1 cm optical path. The
3
1.5, 79.7, 157.0 ppm; HR-MALDI-MS (DHB): m/z calcd
?
for C H CsNO ([M?Cs] ) 290.01518, found 290.01531.
8
15
2
cis/trans-O-tert-Butyl-N-(3-phenylcyclobutyl)carbamate
16)
White crystalline solid; yield: 1.09 g (98%). M.p.:
3
(
enzyme activity in the total reaction mixture (2 cm ) was
3
0.2 U/cm , the concentration of ATCh 40 lM and the
concentration of DTNB 0.1 mM for all reactions. The IC50
1
6
2–85 °C; H NMR (400 MHz, CDCl ): trans isomer:
3
d = 1.52 (s, 9H), 2.00 (m, 2H), 2.83 (m, 2H), 3.20 (m, 1H),
.19 (m, 1H), 4.81 (bs, 1H), 7.31 (m, 5H) ppm; cis isomer:
d = 1.51 (s, 9H), 2.40 (m, 2H), 2.59 (m, 2H), 3.61 (m, 1H),
value was obtained from the dependence v /v vs. concen-
0
i
4
tration of the tested compound (inhibitor), where v is the
0
reaction rate of uninhibited reaction and v is the reaction
i
1
3
4
.34 (m, 1H), 4.97 (bs, 1H), 7.31 (m, 5H) ppm; C NMR
rate of the inhibited reaction (for a given concentration of
the inhibitor). For each compound, at least two determi-
nations were performed. The obtained IC50 values of all
tested compounds are shown in Table 1.
(
100 MHz, CDCl ): d = 28.7, 33.0, 34.5, 37.3, 39.1, 42.2,
3
4
1
4.4, 79.6, 126.2, 126.3, 126.7, 126.8, 128.6, 128.7, 144.8,
45.3, 155.22 ppm; HR-MALDI-MS (DHB): m/z calcd for
?
C H CsNO ([M?Cs] ) 380.06210, found 380.06255.
1
5
21
2
In vitro cytotoxicity evaluation
Lipophilicity
The human T cell acute lymphoblastic leukaemia cell line
Jurkat was purchased from European Collection of Cell
Cultures (ECACC, UK). These cells were cultured in
RPMI 1640 medium (Life Technologies, USA) supple-
mented with 10% foetal bovine serum, 1% non-essential
amino acid, 1% penicillin/streptomycin, 2 mM L-glu-
tamine, 1 mM sodium pyruvate, and 10 mM HEPES (all
supplements from Life Technologies, USA) in a humidified
The traditional shake flask method was used for determi-
nation of log Pow values [70, 71]. First, the two solvents
were mutually saturated at the temperature of the experi-
ment in a way described in literature [71]. Then the
determination log P
was performed subsequently:
ow
3
.5 cm n-octanol and n-octanol solution of the tested
1
3
compound (10 mm ; 0.01 M) were placed in a test tube.
This mixture was intensively shaken for 15 min. Then
atmosphere containing 5% CO at 37 °C.
2
123

E. Hor a´ kov a´ et al.
All compounds were tested using a standard colorimet-
ric method measuring a tetrazolium salt reduction via
mitochondrial dehydrogenase activity in Jurkat cells. The
cells were seeded in density 133,000 cells per well in a
13. Bacalhau P, San Juan AA, Goth A, Caldeira AT, Martins R,
Burke AJ (2016) Bioorg Chem 67:105
1
4. Sawatzky E, Wehle S, Kling B, Wendrich J, Bringmann G,
Sotriffer CA, Heilmann J, Decker M (2016) J Med Chem 59:2067
15. Liew K-F, Chan K-L, Lee C-Y (2015) Eur J Med Chem 94:195
ne M, Dhilly M,
16. Bohn P, Gourand F, Papamica e¨ l C, Ibazize
Gembus V, Alix F, Tintas M-L, Marsais F, Barr e´ L, Levacher V
2015) ACS Chem Neurosci 6:737
`
9
6-well plate. The cells were treated with each of the tested
substances dissolved in DMSO. All compounds were pre-
pared in six incubation concentrations (1–500 lM) in
triplicate. Also, the vehicle controls (0.2% DMSO) were
prepared in triplicate. After 48 h of incubation, cell sur-
vival was determined using Cell Proliferation Kit II (XTT,
Roche, Germany) according to the manufacturer’s
instructions. In brief, the XTT reagent (2,3-bis-(2-meth-
oxy-4-nitro-5-sulphophenyl)-2H-tetrazolium-5-carbox-
anilide) was added to each well and incubated for 3 h at
(
ˇ
ˇ ˇ
1
7. Sedl a´ k M, Hanusek J, Drabina P, St eˇ p a´ nkov a´ S, Cegan A (2009)
Arkivoc vii:1
ˇ ˇ
A, Pejchal V, St eˇ p a´ nkov a´ S, Vor cˇ a´ kov a´ K, Jamp ´ı lek
18. Imramovsky
´
ˇ
J, Van cˇ o J, Sim u˚ nek P, Kr a´ lovec K, Br u˚ cˇ kov a´ L, Mand ´ı kov a´ J,
Trejtnar F (2013) Bioorg Med Chem 21:1735
ˇ
ˇ
1
9. Vor cˇ a´ kov a´ K, St eˇ p a´ nkov a´ S, Sedl a´ k M, Vyt rˇ as K (2015) Che-
mosensors 3:274
20. Saxena J, Meloni D, Huang M-T, Heck DE, Laskin JD, Heindel
ND, Young SC (2015) Bioorg Med Chem Lett 25:5609
2
1. Camerino E, Wong DM, Tong F, K o¨ rber F, Gross AD, Islam R,
Viayna E, Mutunga JM, Li J, Totrov MM, Bloomquist JR, Carlier
PR (2015) Bioorg Med Chem Lett 25:4405
3
7 °C; the absorbance was then measured at 470 nm using
a 96-multiwell microplate reader Tecan Infinite M200
Tecan Group Ltd., M a¨ nnedorf, Switzerland). The viability
(
22. Verma A, Wong DM, Islam R, Tong F, Ghavami M, Mutunga
JM, Slebodnick C, Li J, Viayna E, Lam PC-H, Totrov MM,
Bloomquist JR, Carlier PR (2015) Bioorg Med Chem 23:1321
was calculated as described in the paper by Havelek and
colleagues using the following formula:
2
2
2
3. Anand P, Singh B (2013) Med Chem 9:694
4. Anand P, Singh B (2013) Med Chem Res 22:1648
5. Ma H-J, Xie R-L, Zhao Q-F, Mei X-D, Ning J (2010) J Agric
Food Chem 58:12817
Viability ð%Þ ¼ ½ðA470 sample À A470 blankÞ=ðA470 control
À A470 blankފ  100;
2
2
6. Pohanka M (2011) Biomed Pap Med Fac Univ Palacky Olomouc
Czech Repub 155:219
7. Heinig R, Zimmer D, Yeh S, Krol GJ (2000) J Chromatogr B
where A470 is the absorbance of utilized XTT formazan
measured at 470 nm [72].
7
41:257
8. Ibach B, Haen E (2004) Curr Pharm Des 10:231
2
2
Acknowledgements The authors acknowledge the financial support
of the Ministry of Education, Youth and Sports of the Czech
Republic.
ˇ
ˇ
9. Hor a´ kov a´ E, Drabina P, Bro zˇ B, St eˇ p a´ nkov a´ S, Vor cˇ a´ kov a´ K,
Kr a´ lovec K, Havelek R, Sedl a´ k M (2016) J Enzym Inhib Med
Chem 31:173
3
0. Hardegger LA, Kuhn B, Spinnler B, Anselm L, Ecabert R, Stihle
M, Gsell B, Thoma R, Diez J, Benz J, Plancher J-M, Hartmann G,
Banner DW, Haap W, Diederich F (2011) Angew Chem Int Ed
References
5
0:314
1
. Alzheimer’s Association (2014) Alzheimer’s Association report:
31. Riley KE, Hobza P (2011) Cryst Growth Des 11:4272
32. Hardegger LA, Kuhn B, Spinnler B, Anselm L, Ecabert R, Stihle
M, Gsell B, Thoma R, Diez J, Benz J, Plancher J-M, Hartmann G,
Isshiki Y, Morikami K, Shimma N, Haap W, Banner DW, Die-
derich F (2011) ChemMedChem 6:2048
33. Zhou P, Huang J, Tian F (2012) Curr Med Chem 19:226
34. Lu YX, Liu YT, Xu ZJ, Li HY, Liu HL, Zhu WL (2012) Expert
Opin Drug Discov 7:375
2
1
014 Alzheimer’s disease facts and figures. Alzheimers Dement
0:e47
2
3
4
5
. Lu LC, Bludau JH (2001) Alzheimer’s disease. Greenwood
Publishing Group, Santa Barbara
. Ballard C, Gauthier S, Corbett A, Brayne C, Aarsland D, Jones E
(
2011) Lancet 377:1019
. Amemori T, Jendelova P, Ruzicka J, Urdzikova LM, Sykova E
2015) Int J Mol Sci 16:26417
. Alzheimer’s Association (2015) Alzheimer’s Association report:
(
35. Wilcken R, Zimmermann MO, Lange A, Zahn S, Boeckler FM
(2012) J Comput Aided Mol Des 26:935
2
1
015 Alzheimer’s disease facts and figures. Alzheimers Dement
1:332
36. Scholfield MR, Vander Zanden CM, Carter M, Ho PS (2013)
Protein Sci 22:139
6
7
. Contestabile A (2011) Behav Brain Res 221:334
. Francis PT, Palmer AM, Snape M, Wilcock GK (1999) J Neurol
Neurosurg Psychiatry 66:137
. Colovi c´ MB, Krsti c´ DZ, Lazarevi c´ -Pa sˇ ti TD, Bond zˇ i c´ AM, Vasi c´
VM (2013) Curr Neuropharmacol 11:315
37. Wilcken R, Zimmermann MO, Lange A, Joerger AC, Boeckler
FM (2013) J Med Chem 56:1363
38. Sirimulla S, Bailey JB, Vegesna R, Narayan M (2013) J Chem Inf
Model 53:2781
39. Xu ZJ, Yang Z, Liu YT, Lu YX, Chen KX, Zhu WL (2014) J
Chem Inf Model 54:69
40. Ho PS (2015) Biomolecular halogen bonds. In: Metrangolo P,
Resnati G (eds) Halogen bonding I: impact on materials chemistry
and life sciences. Topic in current chemistry. Springer, Berlin
41. Persch E, Dumele O, Diederich F (2015) Angew Chem Int Ed
54:3290
42. Yang Z, Liu YT, Chen ZQ, Xu ZJ, Shi JY, Chen KX, Zhu WL
(2015) J Mol Model 21:138
43. Cavallo G, Metrangolo P, Milani R, Pilati T, Priimagi A, Resnati
G, Terraneo G (2016) Chem Rev 116:2478
8
9
. Khan MA, Fazal-ur-Rehman S, Hameed A, Kousar S, Dalvandi
K, Yousuf S, Choudhary MI, Basha FZ (2015) RSC Adv 5:59240
1
0. Toda N, Tago K, Marumoto S, Takami K, Ori M, Yamada N,
Koyama K, Naruto S, Abe K, Yamazaki R, Hara T, Aoyagi A,
Abe Y, Kaneko T, Kogen H (2003) Bioorg Med Chem 11:1935
1. Zheng X-Y, Zhang Z-J, Chou G-X, Wu T, Cheng X-M, Wang
C-H, Wang Z-T (2009) Arch Pharmacol Res 32:1245
2. Savini L, Campiani G, Gaeta A, Pellerano C, Fattorusso C,
Chiasserini L, Fedorko JM, Saxena A (2001) Bioorg Med Chem
Lett 11:1779
1
1
1
23

Synthesis and in vitro evaluation of novel N-cycloalkylcarbamates as potential…
ˇ
ˇ
4
4
4. Jiang SQ, Zhang LJ, Cui DB, Yao ZQ, Gao B, Lin JP, Wei DZ
2016) Sci Rep 6:34750
5. Evans DA, Woerpel KA, Hinman MM, Faul MM (1991) J Am
Chem Soc 113:726
6. Chen Y, Zhang XP (2007) J Org Chem 72:5931
7. Concell o´ n JM, Rodruguez-Solla H, Simal C (2007) Org Lett
61. Kr a´ tk y´ M, St eˇ p a´ nkov a´ S, Vor cˇ a´ kov a´ K, Navr a´ tilov a´ L, Trejtnar
F, Stola rˇ ´ı kov a´ J, Vin sˇ ov a´ J (2017) Bioorg Chem 71:244
62. Greig NH, Utsuki T, Ingram DK, Wang Y, Pepeu G, Scali C, Yu
QS, Mamczarz J, Holloway HW, Giordano T, Chen D, Furukawa
K, Sambamurti K, Brossi A, Lahiri DK (2005) Proc Natl Acad
Sci 102:17213
(
4
4
9
:2685
63. Giacobini E (2004) Pharmacol Res 50:433
4
4
8. Gee YS, Goertz NJM, Gardiner MG, Hyland CJT (2016) Org
Biomol Chem 14:2498
9. Christiansen E, Urban C, Grundmann M, Due-Hansen ME,
Hagesaether E, Schmidt J, Pardo L, Ullrich S, Kostenis E, Kas-
sack M, Ulven T (2011) J Med Chem 54:6691
64. Brus B, Ko sˇ ak U, Turk S, Pi sˇ lar A, Coquelle N, Kos J, Stojan J,
Colletier JP, Gobec S (2014) J Med Chem 57:8167
65. Benelkebir H, Hodgkinson C, Duriez PJ, Hayden AL, Bulleid
RA, Crabb SJ, Packham G, Ganesan A (2011) Bioorg Med Chem
19:3709
5
5
0. Rappopot Z (ed) (1987) The chemistry of the cyclopropyl group,
vol 1–2. Wiley-VCH, New York
1. Tedeschi RE (1961) Monoamine oxidase inhibition. US Patent
2,997,422, 22 Aug 1961
2. Tedeschi RE (1962) Chem Abstr 56:7554
66. Wurz RP, Charette AB (2004) J Org Chem 69:1262
67. Vianello P, Botrugno OA, Cappa A, Dal Zuffo R, Dessanti P, Mai
A, Marrocco B, Mattevi A, Meroni G, Minucci S, Stazi G, Thaler
F, Trifiro P, Valente S, Villa M, Varasi M, Mercurio C (2016) J
Med Chem 59:1501
5
5
5
3. Bridges AJ, Sanchez JP (1990) J Heterocycl Chem 27:1527
4. Chernykh AV, Radchenko DS, Chernykh AV, Kondratov IS,
Tolmachova NA, Datsenko OP, Kurkunov MA, Zozulya SX,
Kheylik YP, Bartels K, Daniliuc CG, Haufe G (2015) Eur J Org
Chem 2015:6466
5. Burgess LE (2009) 6-Substituted phenoxychroman carboxylic
acid derivatives. WO Patent WO 2009158426 A1, 30 Dec 2009
6. Burgess LE (2009) Chem Abstr 152:119424
7. Leo A, Hansch C, Elkins D (1971) Chem Rev 71:525
8. Lipinski CA, Lombardo F, Dominy BW, Feeney PJ (2001) Adv
Drug Deliv Rev 46:3
9. Lipinski CA (2004) Drug Discov Today Technol 1:337
0. Ellman GL, Courtney KD, Andres V, Feather-Stone RM (1961)
Biochem Pharmacol 7:88
68. Woodroofe CC, Zhong B, Lu X, Silverman RB (2000) J Chem
Soc Perkin Trans 2:55
69. Tsui GC, Menard F, Lautens M (2010) Org Lett 12:2456
70. Sangster J (1989) Phys Chem Ref Data 18:1111
71. OECD Guidelines for the testing of chemicals, section 1, books/
OECD guidelines for the testing of chemicals, section 1/test no.
107: partition coefficient (n-octanol/water): shake flask method.
http://www.oecd-ilibrary.org/environment/test-no-107-partition-
coefficient-n-octanol-water-shake-flask-method_978926406962
6-en. Accessed 20 Feb 2017
5
5
5
5
72. Havelek R, Siman P, Cmielova J, Stoklasova A, Vavrova J,
Vinklarek J, Knizek J, Rezacova M (2012) Med Chem 8:615
5
6
123