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DOI: 10.1080/14756366.2016.1212193
N-(2-phenylcyclopropyl)carbamates as acetyl- and butyrylcholinesterase inhibitors
3
inhibitor in the total reaction mixture) and certain volume of Lipophilicity
PBS (to achieve a total volume of reaction mixture 2 ml after
The values of partition coefficient were determined using
traditional shake-flask method51,52,54. The obtained results are
shown in Table 1. All tested compounds could be divided into two
groups: Group 1 includes compounds 3a–i (R ¼ 9 alkyls) and
Group 2 includes compounds 3j–o (R ¼ 6 glycosyls). All com-
pounds in the Group 1 fulfill in fact the condition of Lipinski’s
rule of five (except trans( )3c with logPow ¼ 6.63). The lowest
value of logPow in the Group 1 was obtained for compound
trans( )3d. But there is no significant trend in increasing/
decreasing of logPow values depending on chain length. The
compounds in Group 2 containing protecting isopropylidene
groups show high lipophilicity, the logPow values are in range
from 4.10 for trans( )3k to 5.87 for (1S,2S)-cis(–)3j.
adding of enzyme) were placed. The enzymatic reaction was
started by adding of the enzyme. The dependence of absorb-
ance (ꢀ ¼ 412 nm) versus time was observed for 70 s (reference
solution was the same as for uninhibited reaction) and then
reaction rate (vi) was calculated. Five different concentration of
inhibitor were used and each measurement was performed in
duplicate at least.
Finally, the dependence v0/vi versus concentration of inhibitor
was constructed and IC50 was calculated from obtained equation
of regression curve for y ¼ 2 (coming out from the definition of
IC50). The obtained IC50 values of all tested compounds are
shown in Table 1. The comparison of both IC50 values (for AChE
and BChE) of each compounds 3a–o was statistically analyzed
with GraphPad Prism (version 5.00; La Jolla, CA) statistical
software using unpaired t-test: p ꢂ 0.01 or p 5 0.05.
Inhibition studies
The ability of all prepared carbamates to inhibit AChE from
electric eel (Electrophorus electricus) and BChE from equine
serum was determined in vitro using modified Ellman’s method.
The effectiveness of the inhibitors was expressed as IC50 value
In vitro cytotoxicity assay
Cell lines
The human T-cell acute lymphoblastic leukemia cell line Jurkat representing the concentration of an inhibitor which is necessary
was purchased from European Collection of Cell Cultures (UK). for reduction of enzyme activity (or reaction rate) to 50%.
These cells were cultured in Roswell Park Memorial Institute The obtained results are shown in Table 1.
1640 medium (Life Technologies, Carlsbad, CA) supplemented
with 10% fetal bovine serum, 1% non-essential amino acid, 1% In vitro cytotoxicity assay
penicillin/streptomycin, 2 mM L-glutamine, 1 mM sodium pyru-
The cytotoxicity of all prepared compounds was screened using a
vate and 10 mM 4-(2-hydroxyethyl)-piperazine-1-ethanesulfonic
standard tetrazolium salt XTT cytotoxicity assay after 48 h of
acid buffer (all supplements from Life Technologies) in a
treatment. As shown in Table 2, most of the evaluated compounds
humidified atmosphere containing 5% CO2 at 37 ꢁC.
of Group 1 exhibited cytotoxic activity against Jurkat cells at
concentrations ꢅ100 mM. Contrary thereto, trans( )3g at all
Cytotoxicity measurement
tested concentrations and trans( )3d at concentrations ꢂ 250 mM
do not significantly affect cell viability of Jurkat cells. Similarly,
All compounds were tested using a standard colorimetric method
measuring a tetrazolium salt reduction via mitochondrial dehydro-
also the majority of the compounds in Group 2 exhibited
cytotoxic activity against Jurkat cells at concentrations
genase activity in Jurkat cells. The cells were seeded in density
133 000 cells per well in a 96-well plate. The cells were treated
ꢅ100 mM (Table 2). Exceptions are trans( )3m with statistically
insignificant
effect
on
Jurkat
cell
viability
at
with each of the tested substances dissolved in DMSO. All
compounds were prepared in six incubation concentrations
(1–500 mM) in triplicates. Also, the vehiculum controls (0.2%
DMSO) were prepared in triplicates. After 48 h incubation, cell
survival was determined using Cell Proliferation Kit II (XTT,
Roche, Germany) according to manufacturer’s instructions. In
brief, XTT reagent (2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-
2H-tetrazolium-5-carboxanilide) was added to each well and
incubated for 3 h at 37 ꢁC; absorbance was then measured at
470 nm using a 96-multiwell microplate reader Tecan Infinite
concentrations ꢂ 250 mM and trans( )3n together with
trans(ꢃ)3j with statistically insignificant effect on Jurkat cell
survival at concentrations ꢂ 100 mM.
Discussion
The key intermediate for the synthesis of carbamate derivatives
3a–l was 2-phenylcyclopropanecarboxylic acid 2. It was prepared
by standard two-step synthesis, including at first cyclopropane
cycle formation by the action of ethyl diazoacetate onto styrene
afforded cyclopropanecarboxylate 1 and subsequent hydrolysis of
ester group under basic condition (Scheme 1).
¨
M200 (Tecan Group Ltd., Mannedorf, Switzerland). Viability was
calculated as described in the paper by Havelek et al. using the
following formula: (%) viability ¼ (A470sample ꢃ A470blank)/
(A470control ꢃ A470blank) ꢄ 100, where A470 is the absorbance
of utilized XTT formazan measured at 470 nm53. Data were
statistically analyzed with GraphPad Prism (version 5.00; La
Jolla, CA) statistical software using unpaired t-test: p ꢂ 0.01.
The cyclopropanation leading to racemic trans/cis ( )-1 was
conducted at high temperature (120–130 ꢁC) without the presence
of any catalyst with the yield of 62%55. The individual isomers
formed trans( )-1 and cis( )-1 were not separated, the minor cis
isomer was removed from the mixture by recrystallization after its
hydrolysis to the corresponding acid trans/cis ( )-2. The optically
pure forms of ester 1 were prepared according to the protocol
described by Evans et al.56 employing the enantioselective catalyst
based on copper(I) complex of commercially available chiral
bisoxazoline derivative. This method can be considered as the most
convenient for the preparation of all of the isomers of 1, because,
according to the original paper, it enables the formation of the
esters 1 with excellent optical purity (95–98% ee) and with
diastereomeric ratio 77/23 (trans/cis), i.e. relatively favorable ratio
for less thermodynamically stable cis form. In our case, we
observed optical purity 96% ee, the ratio of trans/cis, we found
75/25 and isolated yields were 79–85%. The asymmetric
Results
Chemistry
The serie of N-(2-phenylcyclopropyl)carbamate derivatives 3a–o
was prepared by three-step synthesis with good yields. All of the
newly prepared carbamates 3a–o were characterized by means of
melting point, 1H and 13C NMR spectroscopy and high-resolution
mass spectroscopy. The optical purity of non-racemic derivatives
was determined by means of chiral HPLC and their optical
rotatory power was measured. The purity of all compounds was
verified by means of elemental analysis.
Horáková, Eva
