Synthesis, structural characterization, docking, lipophilicity and cytotoxicity of 1-[(1R)-1-(6-fluoro-1,3-benzothiazol-2-yl)ethyl]-3-alkyl carbamates, novel acetylcholinesterase and butyrylcholinesterase pseudo-irreversible inhibitors

Article abstract of DOI:10.1016/j.bmc.2016.02.033

In the current study, sixteen novel derivatives of (R)-1-(6-fluorobenzo[d]thiazol-2-yl)ethanamine were synthesized as acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitors. Chemical structures together with purity of the synthesized compounds were substantiated by IR, ¹H, ¹³C, ¹⁹F NMR, high resolution mass spectrometry and elemental analysis. The optical activities were confirmed by optical rotation measurements. The synthesized compounds were evaluated for their AChE and BChE inhibitory activities. In addition, the cytotoxicity of the most active compounds was investigated against human cell lines employing XTT tetrazolium salt reduction assay and xCELLigence system allowing a label-free assessment of the cells proliferation. Our results demonstrated that the inhibitory mechanism was confirmed to be pseudo-irreversible, in line with previous studies on carbamates. Compounds indicated as 3b, 3d, 3l and 3n showed the best AChE inhibitory activity of all the evaluated compounds and were up to tenfold more potent than standard drug rivastigmine. The binding mode was determined using state-of-the-art covalent docking and scoring methodology. The obtained data clearly demonstrated that 3b, 3d, 3l and 3n benzothiazole carbamates possess high inhibitory activity against AChE and BChE and concurrently negligible cytotoxicity. In conclusion, our results indicate, that these derivatives could be promising in an effective therapeutic intervention for Alzheimer's disease.

Full text of DOI:10.1016/j.bmc.2016.02.033

Accepted Manuscript
Synthesis, structural characterisation, docking, lipophilicity and cytotoxicity of
1
-[(1R)-1-(6-fluoro-1,3-benzothiazol-2-yl)ethyl]-3-alkyl carbamates, novel
acetylcholinesterase and butyrylcholinesterase pseudo-irreversible inhibitors
Vladimír Pejchal, Š árka Štěpánková, Marcela Pejchalová, Karel Královec,
Radim Havelek, Zdeňka Rů žičková, Haresh Ajani, Rabindranath Lo, Martin
Lepš ík
PII:
S0968-0896(16)30124-9
DOI:
Reference:
http://dx.doi.org/10.1016/j.bmc.2016.02.033
BMC 12835
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
3 December 2015
29 January 2016
24 February 2016
Please cite this article as: Pejchal, V., Štěpánková, S., Pejchalová, M., Královec, K., Havelek, R., Rů žičková, Z.,
Ajani, H., Lo, R., Lepš ík, M., Synthesis, structural characterisation, docking, lipophilicity and cytotoxicity of 1-
[(1R)-1-(6-fluoro-1,3-benzothiazol-2-yl)ethyl]-3-alkyl
carbamates,
novel
acetylcholinesterase
and
butyrylcholinesterase pseudo-irreversible inhibitors, Bioorganic & Medicinal Chemistry (2016), doi: http://
dx.doi.org/10.1016/j.bmc.2016.02.033
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Synthesis, structural characterisation, docking, lipophilicity and
cytotoxicity of 1-[(1R)-1-(6-fluoro-1,3-benzothiazol-2-yl)ethyl]-3-alkyl
carbamates, novel acetylcholinesterase and butyrylcholinesterase
pseudo-irreversible inhibitors
a
b
b
b
Vladimír Pejchal *, Šárka Štěpánková , Marcela Pejchalová , Karel Královec , Radim
b
c
d,e
d
d
Havelek , Zdeňka Růžičková , Haresh Ajani, Rabindranath Lo, Martin Lepšík
a
Institute of Organic Chemistry and Technology, Faculty of Chemical Technology, University of Pardubice, Studentska 573, 532 10
Pardubice, Czech Republic
Department of Biological and Biochemical Sciences, Faculty of Chemical Technology, University of Pardubice, Studentska 573, Pardubice
b
5
32 10, Czech Republic
c
Department of General and Inorganic Chemistry, Faculty of Chemical Technology, University of Pardubice, Studentska 573, 532 10
Pardubice, Czech Republic
d
Institute of Organic Chemistry and Biochemistry v.v.i and Gilead Sciences and IOCB Research Center, Academy of Sciences of the Czech
Republic, Flemingovo nam. 2, 166 10 Prague 6, Czech Republic
e
Regional Center of Advanced Technologies and Materials, Department of Physical Chemistry, Palacký University, Olomouc, 771 46
Olomouc, Czech Republic
*
5
Corresponding author: Email: vladimir.pejchal@upce.cz, Address: Faculty of Chemical Technology, University of Pardubice, Studentska
73, 532 10 Pardubice, Czech Republic. Tel.: +420 4660637740
Abstract:
In the current study, sixteen novel derivatives of (R)-1-(6-fluorobenzo[d]thiazol-2-yl)ethanamine were synthesized as
acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitors. Chemical structures together with purity of the
1
13
19
synthesized compounds were substantiated by IR, H, C, F NMR, high resolution mass spectrometry and elemental
analysis. The optical activities were confirmed by optical rotation measurements. The synthesized compounds were evaluated
for their AChE and BChE inhibitory activities. In addition, the cytotoxicity of the most active compounds was investigated
against human cell lines employing XTT tetrazolium salt reduction assay and xCELLigence system allowing a label-free
assessment of the cells proliferation. Our results demonstrated that the inhibitory mechanism was confirmed to be pseudo-
irreversible, in line with previous studies on carbamates. Compounds indicated as 3b, 3d, 3l and 3n showed the best AChE
inhibitory activity of all the evaluated compounds and were up to tenfold more potent than standard drug rivastigmine. The
binding mode was determined using state-of-the-art covalent docking and scoring methodology. The obtained data clearly
demonstrated that 3b, 3d, 3l and 3n benzothiazole carbamates possess high inhibitory activity against AChE and BChE and
concurrently negligible cytotoxicity. In conclusion, our results indicate, that these derivatives could be promising in an
effective therapeutic intervention for Alzheimer's disease.
Keywords: halogenated benzothiazole, carbamates, acetylcholinesterase, butyrylcholinesterase inhibition, pseudo-irreversible
mechanism, covalent docking
1
. Introduction
The search for new and potent cholinesterase inhibitors (ChEIs) is an ongoing quest mobilizing many organic
1
chemistry and virtual screening groups around the world. It was previously shown that these molecules can be
2
used to treat the late symptoms of Alzheimer´s disease (AD) and could also act as neuroprotecting agents.
Cholinesterases (ChEs) belong to the group of serine hydrolases which are responsible for the breakdown of
3
acetylcholine (ACh) and butyrylcholine (BCh). The loss of cholinergic neurotransmission in the brain of
patients with AD is accompanied by a reduced concentration of ACh within synaptic clefts and contributes to the
2
salient cognitive and behavioural disturbances characteristic of AD. Application of ChEIs increases the amount
of ACh available for neuronal and neuromuscular transmission through their ability to reversibly or irreversibly
4
inhibit the activity of ChEs. Therefore, the application of ChEIs has become the main approach to the
5
symptomatic treatment of AD. There are two major forms of cholinesterases in vertebrates –
acetylcholinesterase (AChE, EC 3.1.1.7) and butyrylcholinesterase (BChE, EC 3.1.1.8), also called
pseudocholinesterase or nonspecific cholinesterase. The latter name comes from the ability of BChE to cleave6
various substrates, including acetylcholine, butyrylcholine, succinylcholine, organophosphates, and cocaine.
The characteristic structural feature of these enzymes is a 20 Å-deep gorge, at the bottom of which the active site
7
,8
is situated (Fig. 1). Four functional sub-domains of the gorge where inhibitors bind are called (from outside to
the inside of the enzymes): i) peripheral anionic site (PAS) located at the rim of the gorge, ii) the acyl binding
1

pocket, iii) the choline binding site and iv) the Ser-His-Glu catalytic triad in the catalytic active site (CAS). The
9
acyl-binding pocket of BChE is larger than its counterpart in AChE because six out of 14 aromatic residues
which line the gorge of AChE are replaced by aliphatic residues in BChE, resulting in the lack of the PAS.
1
0
11
Figure 1. Structure of the active-site gorge of AChE and BChE. A) AChE (PDB code 1AMN) and B) BChE (PDB code 4XII). The
conserved amino acids between AChE and BChE are: the catalytic triad Ser200 (198), His440 (438) and Glu327 (325) as well as Glu199
(197), Trp84 (82) and Trp233 (231). Amino acid numbering comes from AChE (BChE). In the choline binding site, AChE has Phe330,
while BChE has Ala328 (orange). In the acyl pocket, AChE has Phe288 and Phe290, while BChE has Leu286 and Val288 (yellow). In PAS,
AChE has Tyr70, Tyr121 and Trp279, while BChE has Asn68, Gln121 and Ala277 (cyan).
Rivastigmine is a pseudo-irreversible inhibitor of the TcAChE (AChE from Torpedo californica) with the k
of 2.0 M min . It is assumed that a noncovalent AChE-rivastigmine complex is formed first, followed by a
i
-
1
-1 12
nucleophilic attack of the catalytic Ser200 at the rivastigmine carbamate core. The Ser200 gets carbamoylated
12
and the leaving group is cleaved off. This state was confirmed by the crystal structure. A reversal of the
reaction occurs in the active site.
The compounds containing the 2-substituted-1,3-benzothiazole scaffold have received enormous attention
13,14
over the past few decades for their potent antimicrobial, herbicidal and plant growth regulatory activities.
Isopropyl [(S)-1-[(R)-1-(6-fluorobenzothiazole-2-yl)ethylcarbamoyl]-2-methylpropyl] carbamate, also known
with common name benthiavalicarb-isopropyl, has excellent long lasting fungicidal activity against Oomycete
1
5
diseases and is frequently used to protect leaves, stems and tubers of agricultural crops. The inhibititory activity
of ureas containing 2-substituted-1,3-benzothiazole against AChE and BChE was tested in our previous work.
1
6
Preliminary bioassay indicated that the target ureas displayed excellent AChE and BChE inhibitory activity.
The in vitro inhibitory activity of other new prepared 1,3-substituted imidazolidine-2,4,5-triones and
1
7
carbamates containing 2-substituted-1,3-benzothiazole against AChE and BChE was described by our team.
The inhibitory activity against AChE was significantly higher than that of the AD therapeutics drug rivastigmine.
The above-mentioned compounds are also promising inhibitors of BChE, as some of the prepared compounds
1
7,18
inhibit BChE better than the well characterized ChEIs rivastigmine.
The binding mode of the most active inhibitor from the 2R-6-fluorobenzo[d]thiazole series was studied
previously by classical noncovalent molecular docking by us and was found to bind only in the PAS due to its
1
8
large size. Another class of AChE and BChE inhibitors from our laboratory based on salicylanilide diethyl
19
phosphates were found to act as pseudo-irreversible cholinesterase inhibitors. Carbamoylating inhibitors of
AChE and BChE generally use the same mechanism of binding and as such should be evaluated with the
2
0,21
recently developed computational tool – covalent docking.
Passage through the blood-brain barrier is key issue for central nervous system-acting drugs. Although drug
entry into the brain is a complex phenomenon which is affected by diverse physicochemical factors, it depends
strongly on drug lipophilicity, therefore the experimental log P (or log Kow) n-octanol/water partition coefficients
were determined. The log Pow can also partly explain the permeation through the blood-brain barrier using
passive transport. It is a very important parameter for anti-psychotic drugs which exert their action in the central
2

nervous system. Structure-activity relationships between the chemical structure, physical properties and
17, 18
biological activities of the evaluated compounds are discussed in our previous papers.
This paper is a follow-up of our paper on the synthesis, characterization, cholinesterase inhibition and
cytotoxicity of novel substituted ureas, imidazolidine-2,4,5-triones and carbamates containing 2-substituted-1,3-
1
6,17,18
benzothiazoles.
Its highlights are the new chemical structures, the determination of the pseudo-irreversible
mechanism of inhibition and covalent docking and scoring.
2
. Results and discussion
.1. Chemistry
2
The starting compound (R)-1-(6-fluorobenzo[d]thiazol-2-yl)ethanamine 2 was prepared as the corresponding
2
2
p-toluenesulfonate salt (PTS) according to Scheme 1. The synthesis of substituted 6-fluorobenzo[d]thiazole
carbamates 3a–p was carried out according to Scheme 2. The desired compounds 3a–p were synthesized in
toluene, where the starting (R)-1-(6-fluorobenzo[d]thiazol-2-yl)ethanamine liberated from its p-toluene sulfonate
salt and subsequently reacts with corresponding alkyl chloroformates in reaction mixture using an aqueous
solution of sodium hydroxide. Reaction of alkyl chloroformates with in situ liberated amine (from 2) form target
molecules 3a-p (Scheme 2). The products were precipitated by cooling of the toluene solution in high yields 80-
1
9
0%. Summary of products is shown in Table 1. Compounds 3a–p were characterized by melting points, IR, H,
1
3
19
C, F NMR spectroscopy, high resolution mass spectrometry and elemental analyses (CHN). The optical
activities were confirmed by optical rotation measurements. The IR spectral peaks of compounds 3a–p were
-
recognized for C=O of CONH from 1675 to 1710; NH of CONH from 1520 to 1539 and from 3289 to 3322 cm
.
1
1
H NMR spectra of compounds 3a-p showed the presence OCONH carbamate peaks as doublets due to
coupling to CH group. The signal for CH group bound to the carbamate function appears as a quintet due to the
1
interaction with CH
in Scheme 1 and given in the experimental section. The C NMR spectra display two peaks in the alkyl region
indicating the presence of the CH -CH- group. Other seven peaks appearing as doublets (split by an interaction
3
group and OCONH protons. The assignment and numbering of H NMR shifts is illustrated
13
3
1
9
with F nuclei) were found in the aromatic part of spectra and assigned to substituted benzothiazole group.
Scheme 1. Synthetic route to compound 2.
Scheme 2. Synthetic route to compound 3.
2
.2. Crystallography
Based on the extensive search within the Cambridge Structural Database there are no structural analogs of
2
3-26
methoxyethyl group substituted carbamates. Only similarities can be found for a couple of cyclic
carbohydrate derivatives
or a
2
7,28
decorated with methoxyethyl or similar moieties. On the other hand, the literature
data in the area of structural investigations of benzothiazole derivatives seems to be very extensive with about
2
2, 29-
1
500 examples. A couple of directly related structures to 3p have been determined in the past three decades.
3
1
A high degree π-electron density conjugation of the S, N-heterocyclic moiety of the benzothiazole similarly as
1
7 22, 30-35
the carbamate part of the molecule is detected (for selected interatomic distances see Fig. 1 caption).
3

1 1 1
Globally, the compound 3p crystallizes in the chiral orthorhombic space group P2 2 2 with three
structurally/symmetry independent molecules. The centers of chirality are located at C8, C108 and C208 atoms
being R isomers. All three independent molecules within the unit cell are very similar one to each other. The
extensive hydrogen bonding via the N-H···O=C connections of neighboring molecules give rise of the infinite
stairs-like one dimensional chains on the supramolecular level. This particular connection is probably also
responsible for short contacts through a non-classical H-bond between methine C-H and the formal C=N part of
the benzothiazole ring of the adjacent molecule. Moreover, neighboring chains are interconnected by the
methylene-ether contacts making thus a pool of the U-shaped oligoether domain (Fig. 2) interconnected by
perpendicular S (from benzothiazole) to methyl contacts.
Figure 2. ORTEP (50% probability level) view for compound 3p another two independent molecules are omitted for clarity. Selected
interatomic distances [Å] and angles [°], appropriate distances and angles for remaining two independent molecules are given in parenthesis:
S1-C3 1.740(7) (1.737(7), 1.729(7)), S1-C7 1.753(6) (1.750(7), 1.749(6)), N2-C7 1.294(9) (1.283(9), 1.286(9)), N2-C4 1.392(9) (1.411(9),
1
1
1
1
.392(9)), C3-C4 1.390(9) (1.402(10), 1.405(9)), C7-C8 1.505(9) (1.513(10), 1.505(10)), N1-C8 1.456(9) (1.445(9), 1.458(9)), N1-C10
.333(9) (1.318(9), 1.342(9)), O4-C10 1.212(8) (1.214(9), 1.213(8)), O5-C10 1.336(9) (1.354(9), 1.344(9)), O5-C11 1.451(9) (1.435(9),
.441(9)); C3-S1-C7 89.1(3) (88.8(4), 89.1(3)), C4-N2-C7 109.9(5) (110.6(6), 110.9(6)), S1-C7-N2 116.0(5) (116.6(6), 116.0(5)), N1-C8-C7
09.5(5) (111.1(6), 109.6(5)), C8-N1-C10 121.1(6) (122.8(6), 119.9(6)), N1-C10-O4 124.6(7) (125.4(8), 125.0(7)), N1-C10-O5 111.9(6)
(111.3(6), 110.9(6)), C10-O5-C11 117.0(6) (116.3(6), 114.8(6)).
Figure 3. Crystal packing of 3p with intermolecular N-H···O and C-H···N interactions view along x-axis.
4

2
.3. Lipophilicity
Lipophilicity is a property that has a major effect on the absorption, distribution, metabolism, excretion and
toxicity properties, as well as pharmacological activity, because many drugs cross biological membranes through
passive transport, which strongly depends on their lipophilicity. Lipophilicity has been studied and applied as an
3
6,37
important drug property for decades. According to Lipinski's rule of 5, log Pow values should not exceed 5.
The experimental partition coefficient log Kow (n-octanol/water) was determined using the traditional shake-flask
38,39
method.
The n-octanol/water partition coefficient P (also referred to as Kow) is a measure of the propensity of
a neutral compound to differentially dissolve in these immiscible phases. The partition coefficient log Kow (n-
octanol/water) indicates potential for crossing the blood-brain barrier (except for the cases where receptors are
4
0
used for transport) for direct inhibition of brain cholinesterases. The results are shown in Table 1.
Compounds 3j and 3k showed the lowest lipophilicity, while compounds 3b-g demonstrated the highest. The
obtained results indicate the values of lipophilicity increase with long alkyl chain. The effect of branched alkyl
chain could be observed as well. In the group of carbamates with branched alkyl chain (without halogen) the
highest value of log Po/w was achieved for compound 3e. But there is no significant trend in
increasing/decreasing of values of log Po/w for carbamates with halogen in unbranched alkyl chain with the same
number of carbon atoms (e.g. 3h, 3i). The highest value of log Po/w in the group of carbamates with halogen was
achieved for compound 3o.
2
.4. Inhibition of Cholinergic Enzymes
All the prepared carbamates 3a-p were tested for their inhibition of AChE and BChE. The activities of the
compounds were compared with the reference drug rivastigmine (RIV, Exelon®) Rivastigmine is a classical
acylating pseudo-irreversible carbamate cholinesterase inhibitor that inhibits both acetylcholinesterase and
4
1
butyrylcholinesterase. The results are summarized in Table 1 and expressed as apparent 50% inhibitory
concentration (IC50 [μM]), or the concentration of inhibitor required for 50% inhibition of the mentioned
4
2
enzymes. The value of IC50 significantly depends on reaction conditions (e.g. pH, temperature, ionic strength,
enzyme activity).
The activity of the prepared derivatives 3a–p against AChE was comparable or better than that of
rivastigmine (IC50 = 56.1 μM). A trend could be observed in which large branched halogenated substituents
(
such as compounds 3k, 3m and 3o) lent the compounds weaker activity (40.5 - 92.2 μM), whereas linear
aliphatic chains (such as compounds 3a-3d) made the inhibitors strongest of the series (6.1 - 13.6 μM). This 2D
structure-activity relationship has its grounds in the inhibitor binding modes, as will be shown below (section
2
(
.5.). The BChE inhibition by the prepared compounds is comparable to the BChE inhibition by rivastigmine
IC50 = 38.4 μM). Only compound 3k (IC50 = 8.3 μM) showed higher BChE inhibition than rivastigmine. The
compounds 3a, 3m, 3o and 3p showed lower inhibition as compared to rivastigmine.
2
.4.1. Type of cholinesterases inhibition determination
We also investigated the type of inhibition for AChE. For this advanced experiment, we selected three
derivatives with higher inhibitory activity (3b, 3l and 3n). We used the procedure described in Section 9.5.
which enables to distinguish reversible, pseudo-irreversible and irreversible inhibition on the basis of the
changes of enzyme activity in the presence of inhibitor during prolonged incubation. For reversible inhibition,
the activity of enzyme goes down immediately but the inhibiting molecule is bound to the enzyme for a short
period of time, after which the inhibitor and enzyme molecules dissociate and the enzyme activity is restored.
During pseudo-irreversible inhibition, the inhibitor binds to the enzyme molecule covalently but the bond is
slowly broken down, delaying the return of enzymatic activity to the initial state. In the case of irreversible
inhibitors, the reaction of enzyme and inhibitor is not instantaneous due to the reaction barrier which has to be
overcome. Instead, there is a time-dependent decrease in enzymatic activity. Irreversible inhibitors decrease
enzyme activity successively and the dependence log % A vs. time in presence of such inhibitor is linear. These
43,44
inhibitors bind covalently and permanently to the enzyme, and thus the enzyme gets inactivated.
1
9.
The procedure of measurement and data evaluation is described in reference. The data illustrating
dependences log % A vs. time in presence of derivatives applied at different concentrations are presented in Fig.
4
A for 3b, in Fig. 4B for 3l and in Fig. 4C for 3n as examples. Our results assert the pseudo-irreversible
12, 44
inhibition mechanism of the carbamate compounds, which is in line with previous findings.
5

Figure 4. The dependence log % A vs. time. Enzyme: acetylcholinesterase, A) derivative 3b, concentration of inhibitor: 4 M (rhomb), 6
 (square), 8  (triangle). B) derivative 3l, concentration of inhibitor: 6 M (rhomb), 8  (square), 10  (triangle). C) derivative 3n,

concentration of inhibitor: 5 M (rhomb), 7  (square), 9  (triangle).
Table 1 The IC50,log PO/W and AChE/BChE selectivity of tested carbamates 3a-p
IC50 (μM)
a
Compound
R
AChE/BChE
Selectivity
log PO/W
AChE
BChE
3
3
a
-CH
3
13.62 ± 0.13
6.06 ± 0.02
90.32 ± 0.99
20.11 ± 0.08
0.15
0.30
2.50
4.20
b
-CH
2
CH
3
3
c
-CH
2
CH
2
CH
3
13.11 ± 0.09
9.26 ± 0.04
34.61 ± 0.74
40.94 ± 0.09
0.38
0.23
4.56
4.82
3
d
-CH
2
CH
2
CH
2
CH
3
3
e
-CH(CH
3
)
2
16.52 ± 0.10
12.90 ± 0.11
32.71 ± 0.08
40.21 ± 0.11
0.51
0.32
5.33
4.24
3
f
2 3 2
-CH CH(CH )
3
3
g
-C(CH
3
)
3
10.51 ± 0.22
12.71 ± 0.13
23.09 ± 0.08
41.19 ± 0.20
0.46
0.31
4.41
3.09
h
2 2
-CH CH Cl
6

3
3
i
CH
2
CH
2
Br
11.40 ± 0.08
80.52 ± 0.36
92.20 ± 0.23
8.14 ± 0.04
40.51 ± 0.15
38.38 ± 0.19
38.81 ± 0.05
8.31 ± 0.03
25.62 ± 0.04
63.42 ± 0.17
0.30
2.07
11.10
0.32
0.64
3.17
1.92
2.34
2.82
2.69
j
2 2 2
CH CH CH Cl
3
k
2 3
-CH CCl
3
l
2 2
-CH CClF
3
m
2 3
-CH CF
3
n
-CH
2
CF
2
CHF
2
6.75 ± 0.02
25.00 ± 0.04
74.51 ± 0.70
0.27
1.10
2.92
3.20
3
3
o
-CH
2
CF
2
CF
2
CF
3
81.92 ± 0.52
p
-CH
2
CH
2
OCH
3
56.21± 0.32
56.10 ± 1.41
66.33± 0.15
38.42 ± 1.97
0.85
1.46
3.35
-nd
Rivastigmine
ChE inhibitions are expressed as mean ± SD (n = 3 experiments).
2
.5. Inhibitor binding modes to AChE and BChE
The binding modes of the evaluated compounds to AChE and BChE were first studied using classical non-
covalent docking using Glide (see Methods). The compounds 3a-j and 3p were positioned with their carbamoyl
and benzothiazole moieties in the CAS site. The C10 carbon of the carbamoyl group was positioned suitably for
the nucleophilic attack by the catalytic Ser200 OG oxygen (Scheme 3). In contrast, the compounds 3k-o possess
branched halogenated substituents which precluded similar binding modes. Instead, these compounds bound
45
noncovalently in the PAS site (Fig. 5A).
Scheme 3: Nucleophilic addition reaction between AChE and benzothiazole carbamate pseudo-irreversible inhibitors.
7

As the next step, we performed the state-of-the-art covalent docking using CovDock (see Methods). The
covalently docked poses of the CAS-fitting 3a-j and 3p carbamate subseries formed the covalent bond with
Ser200. The carbamoyl moiety was oriented with the carbonyl O4 oxygen atom pointing into the oxyanion hole
toward Gly119 and Ala201, and the amino moiety toward Phe288 and Phe290 in the acyl pocket (Fig 5B). The
benzathiazole ring does not have an interaction with W84 (contrary to an analogous group of rivastigmin, see
below).
This covalent binding mode of the benzothiazole subseries was compared to that of the reference compound
rivastigmine. There, the carbamoyl moiety also makes the covalent bond with Ser200 but the leaving group
points to the opposite side. The carbamoyl moiety is oriented toward Phe288 and Phe290 in the acyl pocket and
the substituted phenol has a C-H…π stacking interaction with Trp84 at a distance of ~5.35 Å (Fig. 5C).
Figure 5. Structures of AChE-inhibitor complexes as obtained from noncovalent (A) and covalent (B, C) docking into the TcAChE protein
1
0
(
PDB code: 1AMNA). The non-covalent binding mode of 3o benzothiazole compound in the PAS of AChE. B) The covalent binding mode
of the benzothiazole compound 3b. (C) The covalent binding mode of rivastigmine. Note that the leaving group of the benzothiazoles and
rivastigmine points in an opposite direction. All distances are in Å.
2
.6. Covalent Score of Inhibitor Activity
4
6
In line with our previous studies aimed at quantifying the inhibitory activity of covalent protein inhibitors
4
2
we explore here even faster approaches based on CovDock methodology and scoring. For this we use only the
benzothiazole carbamate subseries (compounds 3a-j, 3p) which were shown above to be able to undergo the
8

42
covalent bond formation. Plotting the apparent affinity score versus the experimental binding free energies
2
(
ΔGapp) yields a high correlation of R =0.75 (Graph 1). These results need to be taken on a semiquantitative basis
42
due to the approximations embodied in such an approach and also as the range of the experimental apparent
affinites is quite narrow. Nevertheless, they clearly distinguish between the weakest compounds 3j and 3p, on
one hand and the others on the other hand.
4
0
Graph 1: The apparent affinity score plotted versus the experimental binding free energies (ΔGapp = RT lnIC50). Only benzothiazole
carbamate subseries (compounds 3a-j and 3p) which was shown above to be able to undergo the covalent bond formation is considered.
2
.7. AChE/BChE Selectivity
Having studied the AChE inhibition in detail, we now turn our attention to the inhibition of BChE and the
AChE/BChE selectivity. We have performed the same covalent docking in BChE and the docked poses of the
compounds differ in comparison to those of AChE with the exception of the covalent bond with Ser200 residue.
The major difference is the binding of the benzothiazole core. In AChE, the benzothiazole core is held by
interaction with Phe330 and Tyr121 hydrophobic amino acids which stabilize the benzothiazole ring in CAS. In
contrast, in BChE, the core is directed toward the PAS site because Tyr121 and Phe330 of AChE are mutated
into Gln121 and Ala328, respectively, in BChE (Fig. 6)
9

Figure 6. Comparison of covalently docked poses of compound 3b in TcAChE (purple) and BChE (orange). The differing orientation of the
benzothiazole core can be clearly seen.
2
.8. In vitro cytotoxicity assay
The cytotoxicity of the 3b, 3d, 3l and 3n was analysed using xCELLigence system on the SK-BR-3 cells. It
was observed that SK-BR-3 cells treated with 1 µM, 10 µM, 50 µM (data not shown), 100 µM and 250 µM of
b (Fig. 7A), 3l (Fig. 7C) and 3n (Fig. 7D) were proliferating in parallel to cells treated with vehicle control,
3
although treatment with 250 µM of 3n caused a slight reduction in CI value. Likewise, SK-BR-3 cells treated
with 1 µM, 10 µM, 50 µM (data not shown), and 100 µM of 3d showed no reduction in CI values. In contrast,
treatment with 250 µM of 3d initially exhibited increase in CI value followed by time-dependent decrease in cell
proliferation as indicated with decrease in CI values (Fig. 7B). Collectively, these data suggest that the toxicity
of 3b, 3d, 3l and 3n is very low even at concentrations substantially higher than inhibitory concentration. The
IC50, i.e. the concentration reducing cell viability to 50% compared to control cannot be determined. These
results well correlate with the data obtained by the end-point tetrazolium salt XTT cytotoxicity assay. As shown
in Table 2 only 3d at concentrations of 100 µM and 250 µM and 3n at concentration 250 µM significantly
(p≤0.01) decrease viability of Jurkat cells.
Figure 7. Dynamic monitoring of cytotoxic response to different concentrations of the compound 3b (A), 3d (B), 3l (C) and 3n (D).
Normalized Cell Index (CI) measured for 91 hours on human breast adenocarcinoma cell line SK-BR-3. Cells treated with 0.1% of DMSO
was used as vehicle control and 5% DMSO treated cells were used as positive control. Plotted CI values were normalized to the to the time
point of treatment.
Table. 2 Cell viability of Jurkat cells measured by using XTT cytotoxicity assay after treatment with different
concentrations of the tested compounds 3b, 3d, 3l and 3n. Treatment for 48 h. Viability is referred to cells
treated with DMSO 0.1% (control). Negative control (NC) was treated with the vehicle (DMSO 0.1 %) used for
diluting the tested substance. Data are presented as mean values ± SD from three independent experiments. * -
significantly different to control (p≤0.01), t-test.
Compound
Cell viability (% of control)
10 M 50 M
0
.1 % DMSO
1 M
100 M
250 M
1
0

3
3
b
d
100.00 ± 6.24
100.00 ± 6.24
100.00 ± 6.24
100.00 ± 6.24
87.36 ± 2.88
90.34 ± 7.51
91.10 ± 3.97
93.00 ± 5.26
87.59 ± 3.01
80.16 ± 5.77
86.61 ± 5.98
85.32 ± 5.10
87.92 ± 1.95
88.71 ± 4.38
89.57 ± 2.33
89.31 ± 6.11
85.64 ± 4.71
66.84 ± 4.85*
85.67 ± 3.66
82.87 ± 2.75
77.04 ± 6.71
49.50 ± 3.94*
74.00 ± 6.71*
80.28 ± 4.20
3
l
3
n
3
. Conclusion
In this study, sixteen original derivates of (R)-1-(6-fluorobenzo[d]thiazol-2-yl)ethanamine were designed,
synthesised and fully characterized as agents for Alzheimer’s disease therapy. The synthetic pathway was quick
and effective. All the compounds were evaluated in vitro for their ability to inhibit AChE and BChE. The
anticholinesterase activities were compared with those of rivastigmine as the standard. The pseudo-irreversible
inhibition mechanism was confirmed by biochemical studies. Compounds 3b, 3l and 3n expressed the highest
AChE-inhibiting activity, while compound 3k showed the highest BChE inhibition. Most of the compounds
exhibited selectivity on AChE excepting compounds 3j, 3k and 3o which showed better activity on BChE. The
binding mode was evaluated using state-of-the-art covalent docking and scoring methodology. The cytotoxicity
of the most promising drug candidates with potent both AChE and BChE inhibitory activity (3b, 3d, 3l and 3n)
was determined in vitro using two human cell lines. The two independent methods of proliferation and viability
analysis showed negligible cytotoxicity at concentrations substantially higher than corresponding 50%
cholinesterase inhibitory concentrations evaluated for tested compounds. Taken together, these results indicate
that compounds 3b, 3d, 3l and 3n are potential candidates for Alzheimer's disease therapeutic agents.
4
4
4
. Experimental and computational methods
.1. Chemistry
.1.1. Instrumentation and chemicals
All reagents and solvents were purchased from commercial sources (Sigma-Aldrich, USA; Merck, Germany;
Acros Organics, Belgium). Phosgene was purchased from Synthesia (Pardubice, Czech Republic). Reactions
were monitored by thin layer chromatography plates coated with 0.2 mm silica gel 60 F254 (Merck, Germany).
TLC plates were visualized by the UV irradiation (254 nm). All melting points were determined on Melting
Point B-545 apparatus (Buchi, Germany) and are uncorrected. Infrared spectra (ZnSe ATR experiments) were
−1
recorded on a FT-IR spectrometer (Perkin Elmer, USA) in the range of 600–4000 cm . The NMR spectra were
measured in CDCl and DMSO-d solutions at ambient temperature on a Bruker Avance III 400 (400.13 MHz
3
6
1
13
19
for H, 100.62 MHz for C and 376.46 MHz for F). Coupling constants are presented in Hz. Proton chemical
shifts in CDCl are related to the internal TMS ( = 0.0) and in DMSO-d to the middle of the solvent multiplet
 = 2.50). C NMR spectra were measured using APT pulse sequence optimized to J( C, H) = 145 Hz.
Carbon chemical shifts are referenced to the middle of the solvent’s multiplet ( = 77.0 or 39.5 in CDCl or
, respectively). F-NMR spectra were measured using waltz-16 proton decoupling and were
3
6
13
1
13
1
(
3
1
9
DMSO-d
6
standardised against fluorobenzene as the secondary external standard (δ = −113.1) against CFCl3 as the primary
standard. The optical rotation was measured on a Perkin–Elmer 341 instrument, concentration is given in g/100
mL. Elemental analysis (C, H, N) were performed on an automatic microanalyser CE instruments EA 1110 CHN
elemental analyser (Fisons instruments, UK). Mass spectra were measured using high resolution MALDI mass
spectrometer LTQ Orbitrap XL (Thermo Fisher Scientific, Bremen, Germany) via “dried droplet” method. The
LTQ Orbitrap instrument equipped with nitrogen UV laser (337 nm, 60 Hz) was operated in positive-ion or
negative-ion mode over a normal mass range (m/z 50 – 2000) with resolution 100,000 at m/z = 400. Predefined
spiral plate motion patterns were set for the choice of laser shot position. The used matrices were 0.2M solutions
2
of 2,5-dihydroxybenzoic acid (DHB) in MeCN:H O (95:5) or 2-[(2E)-3-(4-tert-butylphenyl)-2-methylprop-2-
enylidene]malononitrile (DCTB) in MeCN. The matrix: sample molar ratio was approx. 40:1. For all measured
samples, the mass spectra were averaged over the whole MS record.
4
.1.2. Experimental procedure for synthesis of chloroformates
The compounds 2,2,2-trifluoroethyl chloroformate, 2,2,3,3-tetrafluoropropyl chloroformate and 2,2,3,3,4,4,4-
heptaflorobutyl chloroformate were prepared by reaction of phosgene with corresponding alcohols according to
18
paper previously published by our group.
1
1

4
.1.2.1. 2-chloro-2,2-difluoroethyl chloroformate
Phosgene (0.5 mol) was condensed in anhydrous diethyl ether (150 mL) at ice bath temperature (T <10 ºC). A
-chloro-2,2-difluoroethanol (0.2 mol) was added to diethyl ether solution for 15 min. An anhydrous
2
triethylamine (0.22 mol) was added dropwise to the reaction mixture. Temperature was kept between 5 and 10
ºC, and addition of a base was accomplished over a 30 min period. Reaction mixture was stirred for 3 h at room
temperature. After that the excess of phosgene was removed from the reaction mixture with a fine stream of
argon. The outgoing gas was discharged through the water solution of sodium hydroxide. The precipitated
hydrochloride salt was collected by filtration. 2-chloro-2,2-difluoroethyl chloroformate was obtained by
fractional distillation as a colourless liquid. Phosgene is extremely poisonous by inhalation, skin and eye and the
relevant safety rules must be obeyed.
4
4
.1.3. Characterization of prepared chloroformate
.1.3.1. 2-chloro-2,2-difluoroethyl chloroformate
1
3
Colourless liquid; Yield 76%; bp 103-104 ºC; H NMR (400.13 MHz, CDCl
3
): 4.73 (2H, t, JF-H = 10.4
1
3
1
2
19
Hz); C NMR (100.62 MHz, CDCl
3
):  150.4, 124.0 (t, JF-C = 289.6 Hz), 124.0 (t, JF-C = 31.0 Hz); F NMR
(
3
376.46 MHz, CDCl ):  -62.6.
4
.1.4. General experimental procedure and characterization data of prepared carbamate compounds 3a-3p.
To the mixture of toluene (70 mL) and water (30 mL) was added sulfonate salt of (R)-1-(6-
fluorobenzo[d]thiazol-2-yl)ethanamine 2 (5.0 mmol) as a grey powder and substituted alkyl chloroformate (7.5
mmol). Solution of sodium hydroxide was added dropwise to the reaction mixture to change pH to 9 - 10
(
approx. 4.5 g of 10% solution). Reaction mixture was stirred 5 h at room temperature. The pH was maintained
within 9 – 10 with addition of sodium hydroxide solution. Toluene layer was concentrated by vacuum distillation
and cooled down to the 0-5 degrees Celsius. Finally the product was isolated by crystallization.
22
4
.1.5. Characterization of synthesized compounds 3a-m.
1
Scheme 4. Proton numbering for assignment of H NMR shifts
4
.1.5.1. Methyl [(1R)-1-(6-fluoro-1,3-benzothiazol-2-yl)ethyl]carbamate (3a)
2
0
-1
White solid; Yield 85.0 %; m.p.127-129 °C; []
D
= +80.1 (acetone c = 1); IR (max, cm ): 3303, 1532 (NH
1
of CONH), 1699 (CO of CONH), 1456, 1303, 1260, 1199, 1047, 816; H NMR (400.13 MHz, DMSO-d
6
):
3
4
3
3
4
8
4
(
1
1
1
.19 (1H, d, J = 7.4 Hz, H-1), 7.98 (1H, dd, J = 2.6 Hz, JF-H = 8.8 Hz, H-6), 7.96 (1H, dd, J = 8.9 Hz, JF-H =
4 3 3 3
.7 Hz, H-4), 7.36 (1H, dt, J = 2.6 Hz, J = 9.2 Hz, JF-H = 9.2 Hz, H-5), 5.02 (1H, quin, J = 7.2 Hz, H-3), 3.60
3
13
4
3H, s, H-1´), 1.55 (3H, d, J = 7.2 Hz, H-2); C NMR (100.62 MHz, DMSO-d6):  176.6 (d, JF-C = 3.2 Hz),
1 5 3 3
59.5 (d, JF-C = 242.5 Hz), 156.4, 149.8 (d, JF-C = 1.3 Hz), 135.7 (d, JF-C = 11.4 Hz), 123.7 (d, JF-C = 9.3 Hz),
2 2 19
14.5 (d, JF-C = 24.8 Hz), 108.6 (d, JF-C = 27.1 Hz), 51.7, 49.5, 20.3; F NMR (376.46 MHz, DMSO-d
6
):  -
16.6; Anal. Calcd for C11
N, 11.00; S. 12.66. HR-MS: for C11
H
11FN
2
O
H
2
S (254.28): C, 51.96; H, 4.36; N, 11.02; S, 12.61; Found: C, 52.12; H, 4.22;
+
11FN
2
O
2
S [M+H ] Calcd 255.05980 m/z, Found 255.05991 m/z.
4
.1.5.2. Ethyl [(1R)-1-(6-fluoro-1,3-benzothiazol-2-yl)ethyl]carbamate (3b)
2
0
-1
White solid; Yield 88 %; m.p. 102-103 °C; []
D
= +66.8 (acetone c = 1); IR (max, cm ): 3308, 1528 (NH of
1
CONH), 1682 (CO of CONH), 1451, 1300, 1252 1018, 812, 644; H NMR (400.13 MHz, DMSO-d
1H, d, J = 7.3 Hz, H-1), 7.99 (1H, dd, J = 2.6 Hz, JF-H = 9.0 Hz, H-6), 7.98 (1H, dd, J = 9.0 Hz, JF-H = 4.9
6
):  8.16
3
4
3
3
4
(
1
2

4
3
3
3
Hz, H-4), 7.36 (1H, dt, J = 2.7 Hz, J = 9.1 Hz, JF-H = 9.1 Hz, H-5), 5.00 (1H, quin, J = 7.2 Hz, H-3), 4.04 (2H,
3
3
3
13
q, J = 7.2 Hz, H-1´), 1.54 (3H, d, J = 7.2 Hz, H-2), 1.20 (3H, t, J = 7.2 Hz, H-2´); C NMR (100.62 MHz,
DMSO-d6):  176.4 (d, JF-C = 3.3 Hz), 159.5 (d, JF-C = 243.0 Hz), 155.9, 149.8 d, JF-C = 1.7 Hz), 135.7 (d,
F-C = 11.2 Hz), 123.7 (d, JF-C = 9.4 Hz), 114.5 (d, JF-C = 24.8 Hz), 108.6 (d, JF-C = 27.0 Hz), 60.2, 49.4, 20.3,
4.6; F NMR (376.46 MHz, DMSO-d
.88; N, 10.44; S, 11.95; Found: C, 53.66; H, 5.00; N, 10.32; S, 12.03. HR-MS: for C12
4
1
5
3
3
2
2
J
19
1
6
2 2
):  -116.6; Anal. Calcd for C12H13FN O S (268.31): C, 53.72; H,
+
4
2 2
H13FN O S [M+H ]
Calcd 269.07545 m/z, Found 269.07568 m/z.
4
.1.5.3. Propyl [(1R)-1-(6-fluoro-1,3-benzothiazol-2-yl)ethyl]carbamate (3c)
2
0
-1
White solid; Yield 87 %; m.p. 96-98 °C; []
D
= +60.5 (acetone c = 1); IR (max, cm ): 3305, 1524 (NH of
1
CONH), 1684 (CO of CONH), 1450, 1304, 1242, 1020, 817, 666; H NMR (400.13 MHz, DMSO-d
6
):  8.17
4
3
4
3
3
(
1H, d, J = 7.6 Hz, H-1), 7.98 (1H, dd, J = 2.6 Hz, JF-H = 9.0 Hz, H-6), 7.96 (1H, dd, J = 9.0 Hz, JF-H = 5.0
4 3 3 3
Hz, H-4), 7.36 (1H, dt, J = 2.6 Hz, J = 9.1 Hz, JF-H = 9.1 Hz, H-5), 5.00 (1H, quin, J = 7.2 Hz, H-3), 3.97 (2H,
3
3
13
m, H 1´), 1.59 (2H, m, H-2´), 1.55 (3H, d, J = 7.2 Hz, H-2), 0.91 (3H, t, J = 7.2 Hz, H-3´); C NMR (100.62
MHz, DMSO-d6):  176.8 (d, JF-C = 3.1 Hz), 159.5 (d, JF-C = 241.6 Hz), 156.0, 149.8 d, JF-C = 1.8 Hz), 135.7
d, JF-C = 12.1 Hz), 123.6 (d, JF-C = 9.6 Hz), 114.5 (d, JF-C = 24.8 Hz), 108.6 (d, JF-C = 27.3 Hz), 65.7, 49.4,
2.0, 20.2, 10.2; F NMR (376.46 MHz, DMSO-d ):  -116.7; Anal. Calcd C13H15FN O S (282.33): C, 55.30;
H, 5.36; N, 9.92; S, 11.36; Found: C, 55.15; H, 5.22; N, 10.01; S, 11.25. HR-MS: for C13
Calcd 283.09110 m/z, Found 283.09094 m/z.
4
1
5
3
3
2
2
(
2
19
6
2
2
+
2 2
H15FN O S [M+H ]
4
.1.5.4. Butyl [(1R)-1-(6-fluoro-1,3-benzothiazol-2-yl)ethyl]carbamate (3d)
2
0
White solid; Yield 85.0 %; m.p.90-92 °C; []
D
= +54.5 (acetone c = 1); IR 3310, 1537 (NH of CONH),
1 3
1
692 (CO of CONH), 1456, 1242, 1021, 815, 652; H NMR (400.13 MHz, DMSO-d ): 8.16 (1H, d, J = 7.6
6
4 3 3 4
Hz, H-1), 7.99 (1H, dd, J = 2.6 Hz, JF-H = 9.0 Hz, H-6), 7.96 (1H, dd, J = 9.0 Hz, JF-H = 4.9 Hz, H-4), 7.36
3
3
3
3
(
(
(
1H, dt, J = 2.7 Hz, J = 9.0 Hz, JF-H = 9.0 Hz, H-5), 5.00 (1H, quin, J = 7.2 Hz, H-3), 4.00 (2H, m, H-1´), 1.57
3
3
1
3
2H, m, H-2´), 1.54 (3H, d, J = 7.2 Hz, H-2),1.36 (2H, m, H-3´), 0.90 (3H, t, J = 7.2 Hz, H-4´); C NMR
4
1
5
100.62 MHz, DMSO-d6):  176.8 (d, JF-C = 3.1 Hz), 159.5 (d, JF-C = 241.6 Hz), 156.0, 149.8 d, JF-C = 1.3
Hz), 135.7 (d, JF-C = 12.0 Hz), 123.6 (d, JF-C = 9.5 Hz), 114.5 (d, JF-C = 25.0 Hz), 108.6 (d, JF-C = 27.1 Hz),
3.9, 49.4, 30.7, 20.3, 18.6, 13.6; F NMR (376.46 MHz, DMSO-d
296.36): C, 56.74; H, 5.78; N, 9.45; S, 10.82; Found: C, 56.55; H, 5.62; N, 9.31; S, 10.71. HR-MS: for
3
3
2
2
19
6
(
6
2 2
):  -116.7; Anal. Calcd C14H17FN O S
+
14 2 2
C H17FN O S [M+H ] Calcd 297.10675 m/z, Found 297.10642 m/z.
4
.1.5.5. Propan-2-yl [(1R)-1-(6-fluoro-1,3-benzothiazol-2-yl)ethyl]carbamate (3e)
2
0
White solid; Yield 81.0 %; m.p.135-137 °C; []
D
= +61.2 (acetone c = 1); IR 3289, 1534 (NH of CONH),
1
3
1
=
7
688 (CO of CONH), 1449, 1295, 1257, 1056, 810, 677; H NMR (400.13 MHz, DMSO-d
6
): 8.11 (1H, d, J
4
3
3
4
7.4 Hz, H-1), 7.98 (1H, dd, J = 2.6 Hz, JF-H = 8.9 Hz, H-6), 7.96 (1H, dd, J = 8.9 Hz, JF-H = 5.0 Hz, H-4),
4 3 3 3
.36 (1H, dt, J = 2.6 Hz, J = 9.1 Hz, JF-H = 9.1 Hz, H-5), 5.00 (1H, quin, J = 7.2 Hz, H-3), 4.80 (1H, m, H-1´),
3 3 13
1
.53 (3H, d, J = 7.2 Hz, H-2), 1.20 (6H, d, J = 6.2 Hz, H-2´); C NMR (100.62 MHz, DMSO-d6): 177.0 (d,
1 5 3
4
J
J
F-C = 3.1 Hz), 159.5 (d, JF-C = 241.7 Hz), 155.5, 149.8 d, JF-C = 1.6 Hz), 135.7 (d, JF-C = 11.7 Hz), 123.6 (d,
3
2
2
1
9
F-C = 9.5 Hz), 114.5 (d, JF-C = 25.1 Hz), 108.6 (d, JF-C = 26.8 Hz), 67.4, 49.5, 22.1, 22.0, 20.2; F NMR
(
376.46 MHz, DMSO-d ):  -116.7; Anal. Calcd C13 S (282.33): C, 55.30; H, 5.36; N, 9.92; S, 11.36;
6
2 2
H15FN O
+
2 2
Found: C, 55.41; H, 5.48; N, 9.86; S, 11.17. HR-MS: for C13H15FN O S [M+H ] Calcd 283.09110 m/z, Found
2
83.09131 m/z.
4
.1.5.6. 2-methylpropyl [(1R)-1-(6-fluoro-1,3-benzothiazol-2-yl)ethyl]carbamate (3f)
2
0
White solid; Yield 82.0 %; m.p. 98-100 °C; []1
688 (CO of CONH), 1454, 1252, 1063, 813, 670; H NMR (400.13 MHz, DMSO-d ): 8.16 (1H, d, J = 7.5
4
D
= +56.0 (acetone c = 1); IR 3293, 1526 (NH of CONH),
3
1
6
4
3
3
Hz, H-1), 7.99 (1H, dd, J = 2.7 Hz, JF-H = 8.8 Hz, H-6), 7.96 (1H, dd, J = 9.0 Hz, JF-H = 4.9 Hz, H-4), 7.36
4
3
3
3
(
(
1H, dt, J = 2.7 Hz, J = 9.1 Hz, JF-H = 9.1 Hz, H-5), 5.01 (1H, quin, J = 7.2 Hz, H-3), 3.79 (2H, m, H-1´), 1.87
3 3 13
1H, m, H-2´), 1.55 (3H, d, J = 7.2 Hz, H-2), 0.90 (6H, t, J = 6.7 Hz, H-3´); C NMR (100.62 MHz, DMSO-
4
1
5
3
d6): 176.9 (d, JF-C = 3.0 Hz), 159.5 (d, JF-C = 242.1 Hz), 156.1, 149.8 d, JF-C = 1.3 Hz), 135.7 (d, JF-C
1.9 Hz), 123.6 (d, JF-C = 9.5 Hz), 114.5 (d, JF-C = 24.8 Hz), 108.6 (d, JF-C = 27.1 Hz), 70.1, 49.4, 27.6, 20.2,
8.9; F NMR (376.46 MHz, DMSO-d ):  -116.7; Anal. Calcd C14H17FN O S (296.36): C, 56.74; H, 5.78; N,
2 2
=
3
2
2
1
1
19
6
1
3

+
9
2
.45; S, 10.82; Found: C, 56.62; H, 5.88; N, 9.15; S, 10.99. HR-MS: for C14
97.10675 m/z, Found 297.10713 m/z.
2 2
H17FN O S [M+H ] Calcd
4
.1.5.7. Tert-butyl [(1R)-1-(6-fluoro-1,3-benzothiazol-2-yl)ethyl]carbamate (3g)
2
0
White solid; Yield 84.0 %; m.p.102-104 °C; []
D
= +61.5 (acetone c = 1); IR 3300, 1535 (NH of CONH),
1
1
675 (CO of CONH), 1456, 1248, 1060, 810,698; H NMR (400.13 MHz, DMSO-d ): 7.95 (3H, m, H-1,H-4,
H-6), 7.34 (1H, dt, J = 2.7 Hz, J = 9.0 Hz, JF-H = 9.0 Hz, H-5), 4.96 (1H, quin, J = 7.2 Hz, H-3), 1.53 (3H, d,
J = 7.2 Hz, H-2), 1.42 (9H, s, H-2´); C NMR (100.62 MHz, DMSO-d6): 177.4 (d, JF-C = 3.5 Hz), 159.5 (d,
F-C = 242.3 Hz), 155.2, 149.9, 135.7 (d, JF-C = 11.5 Hz), 123.6 (d, JF-C = 9.8 Hz), 114.5 (d, JF-C = 24.7 Hz),
6
4
3
3
3
3
1
13
4
3
3
2
J
2
1
9
1
C
08.6 (d, JF-C = 27.3 Hz), 78.6, 49.1, 28.2, 20.2; F NMR (376.46 MHz, DMSO-d
6
):  -116.8; Anal. Calcd
14
H
17FN
2
O
H
2
S (296.36): C, 56.74; H, 5.78; N, 9.45; S, 10.82; Found: C, 56.82; H, 5.86; N, 9.38; S, 11.00. HR-
+
MS: for C14
17FN
2
O
2
S [M+H ] Calcd 297.10675 m/z, Found 297.10687 m/z.
4
.1.5.8. 2-chloroethyl [(1R)-1-(6-fluoro-1,3-benzothiazol-2-yl)ethyl]carbamate (3h)
2
0
White solid; Yield 84.0 %; m.p. 121-123 °C; []1
688 (CO of CONH), 1453, 1252, 1090, 818, 667; H NMR (400.13 MHz, DMSO-d ): 8.40 (1H, d, J = 7.5
4
D
= +53.3 (acetone c = 1); IR 3300, 1537 (NH of CONH),
3
1
6
4
3
3
Hz, H-1), 7.99 (1H, dd, J = 2.8 Hz, JF-H = 8.8 Hz, H-6), 7.98 (1H, dd, J = 8.8 Hz, JF-H = 5.2 Hz, H-4), 7.36
4
3
3
3
3
(
1H, dt, J = 2.6 Hz, J = 8.9 Hz, JF-H = 8.9 Hz, H-5), 5.03 (1H, quin, J = 7.2 Hz, H-3), 4.28 (2H, t, J = 5.2 Hz,
3 3 13
H-2´), 3.83 (2H, t, J = 5.2 Hz, H-1´), 1.56 (3H, d, J = 7.2 Hz, H-2); C NMR (100.62 MHz, DMSO-d6):
4
1
5
3

176.4 (d, JF-C = 3.2 Hz), 159.5 (d, JF-C = 241.9 Hz), 155.5, 149.8 (d, JF-C = 1.2 Hz), 135.7 (d, JF-C = 11.6
3 2 2 19
Hz), 123.7 (d, JF-C = 9.6 Hz), 114.5 (d, JF-C = 24.9 Hz), 108.6 (d, JF-C = 27.1 Hz), 64.4, 49.6, 43.1, 20.2;
F
NMR (376.46 MHz, DMSO-d 12ClFN S (302.75): C, 47.61; H, 4.00; N, 9.25;
6
):  -116.6; Anal. Calcd C12
H
2 2
O
+
S, 10.59; Found: C, 47.82; H, 4.04; N, 9.38; S, 10.48. HR-MS: for C12
m/z, Found 303.03661 m/z.
2 2
H12ClFN O S [M+H ] Calcd 303.03648
4
.1.5.9. 2-bromoethyl [(1R)-1-(6-fluoro-1,3-benzothiazol-2-yl)ethyl]carbamate (3i)
2
0
White solid; Yield 82.0 %; m.p. 101-103 °C; []
D
= +46.2 (acetone c = 1); IR 3290, 1534 (NH of CONH),
1
3
1
=
7
690 (CO of CONH), 1449, 1240, 1195, 1020, 818, 668; H NMR (400.13 MHz, DMSO-d
6
): 8.40 (1H, d, J
4
3
3
4
7.8 Hz, H-1), 7.99 (1H, dd, J = 2.6 Hz, JF-H = 8.8 Hz, H-6), 7.98 (1H, dd, J = 8.8 Hz, JF-H = 5.0 Hz, H-4),
4 3 3 3 3
.36 (1H, dt, J = 2.6 Hz, J = 9.0 Hz, JF-H = 9.0 Hz, H-5), 5.03 (1H, quin, J = 7.2 Hz, H-3), 4.33 (2H, t, J = 5.4
3 3 13
Hz, H-2´), 3.68 (2H, t, J = 5.4 Hz, H-1´), 1.56 (3H, d, J = 7.2 Hz, H-2); C NMR (100.62 MHz, DMSO-d6):
4
1
3
3

9
176.4 (d, JF-C = 3.3 Hz), 159.5 (d, JF-C = 242.3 Hz), 155.4, 149.8, 135.7 (d, JF-C = 11.7 Hz), 123.7 (d, JF-C =
2
2
1
9
.7 Hz), 114.5 (d, JF-C = 24.9 Hz), 108.6 (d, JF-C = 27.3 Hz), 64.2, 49.6, 31.4, 20.2; F NMR (376.46 MHz,
):  -116.6; Anal. Calcd C12 12BrFN S (347.20): C, 41.51; H, 3.48; N, 8.07; S, 9.24; Found: C,
1.63; H, 3.44; N, 7.89; S, 9.02. HR-MS: for C12
m/z.
DMSO-d
6
H
2 2
O
+
4
2 2
H12BrFN O S [M+H ] Calcd 346.98596 m/z, Found 346.98648
4
.1.5.10. 3-chloropropyl [(1R)-1-(6-fluoro-1,3-benzothiazol-2-yl)ethyl]carbamate (3j)
2
0
White solid; Yield 87.0 %; m.p. 83-84 °C; []
D
= +50.1 (acetone c = 1); IR 3294, 1534 (NH of CONH),
1
3
1
=
7
688 (CO of CONH), 1450, 1295, 1255, 1080, 814, 673; H NMR (400.13 MHz, DMSO-d
6
): 8.24 (1H, d, J
4
3
3
4
7.5 Hz, H-1), 7.97 (1H, dd, J = 2.6 Hz, JF-H = 8.9 Hz, H-6), 7.95 (1H, dd, J = 8.9 Hz, JF-H = 4.9 Hz, H-4),
4 3 3 3 3
.35 (1H, dt, J = 2.6 Hz, J = 9.1 Hz, JF-H = 9.1 Hz, H-5), 5.03 (1H, quin, J = 7.2 Hz, H-3), 4.14 (2H, t, J = 6.3
3 3 3 13
Hz, H-3´), 3.72 (2H, t, J = 6.3 Hz, H-1´), 2.05 (2H, quin, J = 6.3 Hz, H-2´), 1.56 (3H, d, J = 7.2 Hz, H-2); C
4
1
5
NMR (100.62 MHz, DMSO-d6): 176.5 (d, JF-C = 3.1 Hz), 159.5 (d, JF-C = 242.2 Hz), 155.8, 149.8 d, JF-C
.2 Hz), 135.7 (d, JF-C = 11.5 Hz), 123.6 (d, JF-C = 9.8 Hz), 114.5 (d, JF-C = 24.8 Hz), 108.6 (d, JF-C = 26.9 Hz),
1.2, 49.5, 41.8, 31.6, 20.2; F NMR (376.46 MHz, DMSO-d ):  -116.6; Anal. Calcd C13H14ClFN O S
2 2
316.78): C, 49.29; H, 4.45; N, 8.84; S, 10.12; Found: C, 49.19; H, 4.25; N, 8.64; S, 9.91. HR-MS: for
=
3
3
2
2
1
6
(
19
6
+
13 2 2
C H14ClFN O S [M+H ] Calcd 317.05213 m/z, Found 317.05201 m/z.
4
.1.5.11. 2,2,2-trichloroethyl [(1R)-1-(6-fluoro-1,3-benzothiazol-2-yl)ethyl]carbamate (3k)
2
0
White solid; Yield 82.0 %; m.p. 76-78 °C; []
D
= +46.0 (acetone c = 1); IR 3322, 1520 (NH of CONH),
1
1
708 (CO of CONH), 1449, 1290, 1249, 1098, 818, 715; H NMR (400.13 MHz, DMSO-d
6
): 8.80 (1H, d,
1
4

3
4
3
3
4
J = 7.6 Hz, H-1), 7.99 (1H, dd, J = 2.7 Hz, JF-H = 9.0 Hz, H-6), 7.98 (1H, dd, J = 9.0 Hz, JF-H = 5.2 Hz, H-4),
4
3
3
3
7
1
=
.36 (1H, dt, J = 2.5 Hz, J = 9.2 Hz, JF-H = 9.2 Hz, H-5), 5.08 (1H, quin, J = 7.2 Hz, H-3), 4.87 (2H, m, H-1´),
3
1
3
4
1
.59 (3H, d, J = 7.2 Hz, H-2); C NMR (100.62 MHz, DMSO-d6): 175.7 (d, JF-C = 3.1 Hz), 159.5 (d, JF-C
5
3
3
2
243.3 Hz), 154.2, 149.7 d, JF-C = 1.6 Hz), 135.7 (d, JF-C = 11.8 Hz), 123.7 (d, JF-C = 9.6 Hz), 114.6 (d, JF-C
=
2
19
2
4.9 Hz), 108.7 (d, JF-C = 27.1 Hz), 96.0, 73.6, 49.9, 20.1; F NMR (376.46 MHz, DMSO-d
Calcd C12 FN
HR-MS: for C12
6
):  -116.5; Anal.
H10Cl
3
2 2
O S (371.64): C, 38.78; H, 2.71; N, 7.54; S, 8.63; Found: C, 38.89; H, 2.77; N, 7.42; S, 8.51.
10Cl FN O S [M+H ] Calcd 370.95854 m/z, Found 370.95902 m/z.
3 2 2
+
H
4
.1.5.12. 2-chloro-2,2-difluoroethyl [(1R)-1-(6-fluoro-1,3-benzothiazol-2-yl)ethyl]carbamate (3l)
2
0
White solid; Yield 82.0 %; m.p. 99-101 °C; []
D
= +62.0 (acetone c = 1); IR 3299, 1532 (NH of CONH),
1
1
(
699 (CO of CONH), 1452, 1286, 1255, 1166. 1092, 815, 699; H NMR (400.13 MHz, DMSO-d
6
): 8.77
3
4
3
3
4
1H, d, J = 7.6 Hz, H-1), 7.99 (1H, dd, J = 2.7 Hz, JF-H = 8.9 Hz, H-6), 7.97 (1H, dd, J = 9.0 Hz, JF-H = 5.0
4 3 3 3
Hz, H-4), 7.37 (1H, dt, J = 2.7 Hz, J = 9.1 Hz, JF-H = 9.1 Hz, H-5), 5.07 (1H, quin, J = 7.2 Hz, H-3), 4.79 (2H,
3
13
4
m, H-1´), 1.58 (3H, d, J = 7.2 Hz, H-2); C NMR (100.62 MHz, DMSO-d6): 175.7 (d, JF-C = 3.1 Hz),
1
5
3
1
1
1
2
59.5 (d, JF-C = 242.6 Hz), 154.1, 149.7 d, JF-C = 1.4 Hz), 135.7 (d, JF-C = 11.9 Hz), 126.3 (t, JF-C = 292.5 Hz,),
3 2 2 3
23.7 (d, JF-C = 9.6 Hz), 114.6 (d, JF-C = 24.7 Hz), 108.6 (d, JF-C = 27.2 Hz), 68.0 (t, JF-C = 29.0 Hz), 49.8,
19
0.1; F NMR (376.46 MHz, DMSO-d ):  -116.5; Anal. Calcd C12H10ClF N O S (338.83): C, 42.55;
H, 2.98; N, 8.27; S, 9.47; Found: C, 42.35; H, 2.77; N, 8.04; S, 9.25. HR-MS: for C12
Calcd 339.01764 m/z, Found 339.01806 m/z.
6
3 2 2
+
3 2 2
H10ClF N O S [M+H ]
4
.1.5.13. 2,2,2,-trifluoroethyl [(1R)-1-(6-fluoro-1,3-benzothiazol-2-yl)ethyl]carbamate (3m)
2
0
White solid; Yield 81.0 %; m.p. 107-108 °C; []
D
= +67.2 (acetone c = 1); IR 3298, 1535 (NH of CONH),
1
1
(
5
710 (CO of CONH), 1451, 1289, 1248, 1164, 1095, 820, 640; H NMR (400.13 MHz, DMSO-d
1H, d, J = 7.6 Hz, H-1), 7.99 (2H, m, H-4, H-6), 7.36 (1H, dt, J = 2.5 Hz, J = 9.0 Hz, JF-H = 9.0 Hz, H-5),
6
): 8.74
3
4
3
3
3
3
13
.06 (1H, quin, J = 7.2 Hz, H-3), 4.72 (2H, m, H-1´), 1.58 (3H, d, J = 7.2 Hz, H-2); C NMR (100.62 MHz,
4
1
5
DMSO-d6): 175.6 (d, JF-C = 3.2 Hz), 159.5 (d, JF-C = 242.3 Hz), 154.1, 149.7 d, JF-C = 1.5 Hz), 135.7 (d,
3
3
1
2
2
J
F-C = 12.0 Hz), 123.8 (d, JF-C = 9.5 Hz), 123.7 (q, JF-C = 276.1 Hz), 114.6 (d, JF-C = 24.4 Hz), 108.6 (d, JF-C
=
2
19
2
7.2 Hz), 59.9 (d, JF-C = 35.0 Hz), 49.8, 20.1; F NMR (376.46 MHz, DMSO-d
Calcd C12
HR-MS: for C12
6
): - -116.5; Anal.
H F N O S (322.28): C, 44.72; H, 3.13; N, 8.69; S, 9.95; Found: C, 44.83; H, 3.00; N, 8.72; S, 10.00.
10 4 2 2
+
10 4 2 2
H F N O S [M+H ] Calcd 323.04719 m/z, Found 323.04733 m/z.
4
.1.5.14. 2,2,3,3-tetrafluoropropyl [(1R)-1-(6-fluoro-1,3-benzothiazol-2-yl)ethyl]carbamate (3n)
2
0
White solid; Yield 81.0 %; m.p. 92-93 °C; []
D
= +55.8 (acetone c = 1); IR 3321, 1527 (NH of CONH),
1
3
1
=
7
5
684 (CO of CONH), 1452, 1290, 1252, 1022, 820, 708; H NMR (400.13 MHz, DMSO-d
7.6 Hz, H-1), 7.99 (1H, dd, J = 2.7 Hz, JF-H = 8.9 Hz, H-6), 7.97 (1H, dd, J = 9.0 Hz, JF-H = 5.0 Hz, H-4),
6
): 8.67 (1H, d, J
4
3
3
4
4
3
3
2
3
.37 (1H, dt, J = 2.7 Hz, J = 9.1 Hz, JF-H = 9.1 Hz, H-5), 6.57 (1H, tt, JF-H = 52.1 Hz, JF-H = 5.0 Hz, H-3´),
3
3
1
3
.06 (1H, quin, J = 7.2 Hz, H-3), 4.61 (2H, m, H-1´), 1.58 (3H, d, J = 7.2 Hz, H-2); C NMR (100.62 MHz,
4
1
5
DMSO-d6): 175.7 (d, JF-C = 3.0 Hz), 159.5 (d, JF-C = 242.0 Hz), 154.5, 149.7 d, JF-C = 1.2 Hz), 135.7 (d,
3
3
1
2
2
J
F-C = 11.7 Hz), 123.7 (d, JF-C = 9.6 Hz), 115.0 (tt, JF-C = 247.5 Hz, JF-C = 25.8 Hz), 114.6 (d, JF-C = 24.8 Hz),
1 2 2 3 19
1
09.4 (tt, JF-C = 249.9 Hz, JF-C = 36.1 Hz), 108.6 (d, JF-C = 27.2 Hz), 59.6 (t, JF-C = 25.6 Hz), 49.8, 20.2;
NMR (376.46 MHz, DMSO-d ):  -116.5, -124.7, -138.4; Anal. Calcd C13 S (354.30): C, 44.07; H,
.13; N, 7.91; S, 9.05; Found: C, 43.85; H, 3.00; N, 7.70; S, 8.87. HR-MS: for C13
55.05342 m/z, Found 355.05393 m/z.
F
6
11 5 2 2
H F N O
+
3
3
11 5 2 2
H F N O S [M+H ] Calcd
4
.1.5.15. 2,2,3,3,4,4,4-heptafluorobutyl [(1R)-1-(6-fluoro-1,3-benzothiazol-2-yl)ethyl]carbamate (3o)
2
0
White solid; Yield 82.0 %; m.p. 84-86 °C; []
D
= +45.0 (acetone c = 1); IR 3319, 1539 (NH of CONH),
1
3
1
=
7
1
=
2
=
698 (CO of CONH), 1454, 1295, 1221, 1115, 817, 696; H NMR (400.13 MHz, DMSO-d
7.6 Hz, H-1), 7.99 (1H, dd, J = 2.6 Hz, JF-H = 8.8 Hz, H-6), 7.96 (1H, dd, J = 8.8 Hz, JF-H = 5.0 Hz, H-4),
6
): 8.77 (1H, d, J
4
3
3
4
4
3
3
3
.36 (1H, dt, J = 2.6 Hz, J = 9.0 Hz, JF-H = 9.0 Hz, H-5), 5.07 (1H, quin, J = 7.2 Hz, H-3), 4.84 (2H, m, H-1´),
3
1
3
4
1
.58 (3H, d, J = 7.2 Hz, H-2); C NMR (100.62 MHz, DMSO-d6): 175.5 (d, JF-C = 3.1 Hz), 159.5 (d, JF-C
5
3
3
1
242.0 Hz), 154.1, 149.7 d, JF-C = 1.5 Hz), 135.8 (d, JF-C = 11.8 Hz), 123.7 (d, JF-C = 9.6 Hz), 117.2 (qt, JF-C
=
2
2
1
2
2
87.7 Hz, JF-C = 32.7 Hz), 114.6 (d, JF-C = 24.0 Hz), 114.5 (tt, JF-C = 255.0 Hz, JF-C = 30.5 Hz), 108.7 (d, JF-C
1 2 3 19
26.2 Hz), 108.6 (tq, JF-C = 270.0 Hz), JF-C = 37.1 Hz), 58.9 (t, JF-C = 25.8 Hz), 49.8, 20.1; F NMR (376.46
):  -116.5, -119.9, -127.4; Anal. Calcd C14 S (422.29): C, 39.82; H, 2.39; N,
MHz, DMSO-d
6
10 8 2 2
H F N O
1
5

+
6
.63; S, 7.59; Found: C, 39.98; H, 2.44; N, 6.52; S, 7.66. HR-MS: for C14
H
10
F
8
N
2
O
2
S [M+H ] Calcd 423.04080
m/z, Found 423.04145 m/z.
4
.1.5.16. 2-methoxyethyl [(1R)-1-(6-fluoro-1,3-benzothiazol-2-yl)ethyl]carbamate (3p)
2
0
White solid; Yield 81.0 %; m.p. 67-68 °C; []
D
= +49.6 (acetone c = 1); IR 3301, 1531 (NH of CONH),
1
3
1
=
7
3
698 (CO of CONH), 1456, 1262, 1200, 1068, 817, 712; H NMR (400.13 MHz, DMSO-d
7.5 Hz, H-1), 7.98 (1H, dd, J = 2.6 Hz, JF-C = 9.1 Hz, H-6), 7.96 (1H, dd, J = 9.0 Hz, JF-H = 4.9 Hz, H-4),
6
): 8.29 (1H, d, J
4
3
3
4
4
3
3
3
.36 (1H, dt, J = 2.7 Hz, J = 9.0 Hz, JF-H = 9.0 Hz, H-5), 5.01 (1H, quin, J = 7.2 Hz, H-3), 4.12 (2H, m, H-1´),
3
3
1
3
3
.52 (2H, t, , J = 4.4 Hz, H-2´), 3.27 (3H, s, O-CH ) 1.55 (3H, d, J = 7.2 Hz, H-2); C NMR (100.62 MHz,
1 5
4
DMSO-d6): 176.6 (d, JF-C = 3.4 Hz), 159.5 (d, JF-C = 242.4 Hz), 155.9, 149.8 d, JF-C = 1.2 Hz), 135.7 (d,
3
3
2
2
J
F-C = 11.6 Hz), 123.6 (d, JF-C = 9.4 Hz), 114.5 (d, JF-C = 24.9 Hz), 108.6 (d, JF-C = 27.1 Hz), 70.2. 63.5, 58.0,
19
4
5
2
9.5, 20.3; F NMR (376.46 MHz, DMSO-d
6
): -116.6; Anal. Calcd C13
.07; N, 9.39; S, 10.75; Found: C, 52.22; H, 5.11; N, 9.49; S, 10.69. HR-MS: for C13
99.08602 m/z, Found 299.08652 m/z.
H15FN
2
O
H
3
S (298.33): C, 52.34; H,
15FN O S [M+H ] Calcd
2 3
+
4
.2. Crystallographic details
The X-Ray data for colourless crystal of compound 3p, were obtained at 150 K using Oxford Cryostream
low-temperature device on a Nonius KappaCCD diffractometer with MoKα radiation (λ = 0.71073 Å), a graphite
4
7
monochromator and the φ and χ scan mode. Data reductions were performed with DENZO-SMN. The
4
8
49
absorption was corrected by integration methods. Structures were solved by direct methods (Sir92) and
5
0
refined by full matrix least-square based on F2 (SHELXL97). Hydrogen atoms were mostly localized on a
difference Fourier map, however to ensure uniformity of the treatment of the crystal, all hydrogen atoms were
recalculated into idealized positions (riding model) and assigned temperature factors Hiso(H) = 1.2 Ueq(pivot
atom) or of 1.5 Ueq for the methyl moiety with C–H = 0.96, 0.97, 0.98 and 0.93 Å , and 0.86 Å for N-H bond.
Crystallographic data for 3p: C13
4.6477(14), c = 39.240(3) Å, Z = 12, V = 4241.9(6) Å , D
.920/0.956; -7 ≤ h ≤ 9, -18 ≤ k ≤ 19, -50 ≤ l ≤ 46; 19088 reflections measured (max = 27 °), 19057 independent
int = 0.0475), 7958 with I > 2(I), 541 parameters, S = 1.128, R1(obs. data) = 0.0709, wR2(all data) = 0.1270;
H15FN O S, M = 298.33, orthorhombic, P2 2 2 , a = 7.3800(6), b =
2 3 1 1 1
3 -3 -1
1
0
c
= 1.401 g.cm ,  = 0.249 mm , Tmin/Tmax =
(
R
-
3
max., min. residual electron density = 0.499, -0.385 eǺ .
2
2
2
2
2 2
½
R
int = F
c
o
- Fo,mean /F
o
, GOF = [(w(F
o
- F
- F
c
) )/(Ndiffrs - Nparams)] for all data, R(F) = F  -
o
2
2
2 2
2 2
½
F
/F
o
for observed data, wR(F ) = [(w(F
o
c
o
) )/(w(F ) )] for all data.
Crystallographic data for structural analysis have been deposited with the Cambridge Crystallographic Data
Centre (deposition number CCDC 1437739). Copies of this information may be obtained free of charge from the
Director, CCDC, 12 Union Road, Cambridge CB2 1EY, UK (fax: +44-1223-336033; e-mail:
deposit@ccdc.cam.ac.uk or http://www.ccdc.cam.ac.uk).
4
.3. Determination of n-octanol/water partition coefficient Kow
Before the partition coefficient is determined, the two solvents are mutually saturated at the temperature of the
experiment. To do this, it is practical to shake two large stock bottles, one containing n-octanol and a sufficient
quantity of water, and the other containing water and a sufficient quantity of n-octanol, for 24 hours on a
mechanical shaker and then to let them stand long enough to allow the phases to separate. The procedure of
determination was the following: n-octanol (2 mL) was placed in a test tube. Then an octanol solution of the
chosen inhibitor (15 μL, 0.01 M) was added. Mixture was intensively shaken for 15 min. This mixture (1 mL)
was placed into the cell, and its absorbance at the absorption maximum wavelength was measured. The reference
solution was n-octanol. The value of absorbance corresponding to 100% of the chosen inhibitor in n-octanol was
obtained. An n-octanol solution of the chosen inhibitor (0.01 M, 15 μL) was added into the mixture of n-octanol
and water (1:1, total volume 4 mL). The mixture was intensively shaken for 15 min and then centrifuged (3000
rpm, 10 min). One mL of the n-octanol layer was put into the cell, and its absorbance at the wavelength of
absorption maximum was measured. The comparative solution was n-octanol again. The percentage content of
chosen inhibitor in the octanol layer (%) was obtained. The n-octanol/water partition coefficient is defined as
K
ow = c /c , where c and c are molar concentrations of tested compounds in n-octanol and water. For each
1 2 1 2
38,39
compound, at least three determinations were performed.
are shown in Table 1.
The log Kow values of the individual compounds
1
6

4
.4. In vitro evaluation of AChE- and BChE-inhibiting activity
The ability of all tested compounds to inhibit acetylcholinesterase from electric eel (Electrophorus electricus
L.) and butyrylcholinesterase from equine serum (both purchased from Sigma-Aldrich, USA) was tested. The
effectiveness of the inhibitor could be described by the 50% inhibitory concentration IC50. The IC50, or the half
maximal inhibitory concentration, represents the concentration of an inhibitor that is required for 50% inhibition
of the enzyme (sometimes it is referred to as the negative logarithm of the molar concentration inhibiting the
enzyme activity by 50%, pI50 = log 1/IC50). The IC50 values were determined by the spectrophotometric Ellman’s
51
method. The Ellman’s method is a simple, rapid and direct method to determine the SH and –S–S– group
5
2,53
content in proteins.
All tested compounds were dissolved in dioxane (concentration 0.01 M) and then diluted
in demineralized water (concentration 0.001 M and 0.0001 M). The procedure of determination of IC50 is in
5
4
detail described in reference. For determination of IC50 values the inhibition was determined at 12 different
compound concentrations with three replicates. The obtained results are summarized in Table 1.
4
.5. Investigation of inhibition mechanism
Three carbamate derivatives (3a, 3l and 3n; see Table 1) were used for investigation of mechanism of
acetylcholinesterase inhibition. For each of them three different concentrations of inhibitor were chosen
according to their IC50 values. The purpose was to observe the effect of inhibitor on enzyme activity (A) in time.
55,56
On this basis, it is possible to distinguish reversible and irreversible inhibition.
The enzyme activity was
determined using spectrophotometric Ellman’s method. The course of determination was in detail described
1
9
previously in reference.
4
.6. Covalent docking
Protein and Ligand Preparation. The in silico experiments were carried out using the Schrödinger Suite
5
7
2
015-3 molecular modelling software package. All the ligands were prepared with Ligprep module in Maestro
5
6
5
8
v10.2 visualisation program. The Epik module was used to generate tautomeric states; the one with the lowest
ionization penalty was chosen. The ChE proteins were prepared from the X-ray structures of their complexes: for
1
0
Torpedo californica AChE, the complex with a transition-state analog was used (PDB code 1AMN). and for
human BChE its complex with 5-nitroquinolin-8-ol based inhibitor (PDB code 4XII). The Protein Preparation
Wizard of Schrödinger Suite 2015-3 was used to add missing side chains and hydrogen atoms and optimize H-
1
1
59
bonding networks.
60
61,62
Covalent Docking. The Schrödinger CovDock algorithm uses both Glide and Prime.
The Schrödinger
CovDock program was used to mimick the covalent ligand binding. First, the pre-reaction form of the ligand was
6
0
positioned in the binding site close to the receptor reactive Ser200 residue using Glide docking. A maximum of
0 poses per ligand reaction site are kept and subject to post docking minimization. All poses within 2.5
1
kcal/mol of the minimum are retained, and poses with a distance greater than 5 Å to Cβ atom of the receptor
reactive residue are rejected. In the following step, the rotamers of Ser200 were sampled using the Prime
6
1
63
VSGB2.0 energy model with the OPLS2005 force field. Ligand poses that place the reacting pair of atoms
within 5 Å were kept, and a covalent bond was formed according to the nucleophilic addition reaction (Scheme
A).
Apparent Affinity Scoring. As a rough estimate of the apparent affinity scoring that estimates apparent affinity
4
1
of a covalent inhibitor without including a direct calculation of the bond formation energy. An apparent
binding affinity score is defined as the averaged Glide Score taken at two steps in the simulated reaction process:
the first is the Glide score of the binding mode of the prereactive ligand species and the second is approximate
Glide score of the ligand in covalent complex after a formation of the covalent bond.
4
4
.7. In vitro cytotoxicity assay
.7.1. Cell lines
The human breast adenocarcinoma cell line SK-BR-3 was purchased from European Collection of Cell
Cultures (ECACC, Salisbury, UK). SK-BR-3 cells were cultured in McCoy's 5A medium (Life Technologies,
USA) supplemented with 10 % foetal bovine serum and 1% penicillin/streptomycin (all reagents and
1
7

supplements from Life Technologies, USA) in a humidified atmosphere containing 5% CO2 at 37 °C. The
human T-cell acute lymphoblastic leukemia cell line Jurkat was purchased European Collection of Cell Cultures
(
1
ECACC, UK). These cells were cultured in RPMI 1640 medium (Life Technologies, USA) supplemented with
0 % foetal bovine serum, 1% non-essential amino acid, 1% penicillin/streptomycin, 2 mM L-glutamine, 1 mM
sodium pyruvate and 10 mM HEPES (all supplements from Life Technologies, USA) in a humidified
atmosphere containing 5% CO2 at 37 °C.
4
4
.7.2. Cytotoxicity measurement
.7.2.1. Real-time cytotoxicity assay
The cytotoxicity of the most active compounds 3b, 3d, 3l and 3n was assessed against SK-BR-3 cells using
the xCELLigence RTCA (Real-Time Cell Analysis) SP (Single plate) system (Roche Diagnostic, Germany),
allowing label-free, dynamic monitoring of cell events in real-time. The principle of the system is to monitor the
64
changes in electrode impedance induced by the interaction between testing cells and electrodes.
A
dimensionless parameter termed Cell Index (CI) was derived to represent cell status based on the electrical
impedance measured. The xCELLigence system was connected and tested by Resistor Plate Verification before
the RTCA SP station was placed inside the incubator at 37 °C and 5% CO . Background measurements were
2
taken by adding 100 µl of appropriate medium to the wells of the E-Plate 96. Cell suspension (90 µl) at cell
density of 40,000 cells per well was added to each well of the E-plate 96. The SK-BR-3 cell proliferation was
dynamically monitored at 30 min. interval. When the cells entered logarithmic growth phase, they were treated
with 10 µL of 3b, 3d, 3l and 3n dissolved in DMSO at concentrations range of 1 µM to 250 µM in triplicates.
Cells treated with 0.1% of DMSO was used as vehicle control while cells treated with 5% DMSO were used as
positive control. After 91 hours of incubation with tested compounds the cell status and the cytotoxic effect were
plotted using characteristic cell index-time profile. Growth curves were normalized to the time point of
treatment. Evaluations were performed using the RTCA 1.2.1 software.
4
.7.2.2. XTT assay
The most active compounds 3b, 3d, 3l and 3n were tested using a standard colorimetric method measuring a
tetrazolium salt reduction via mitochondrial dehydrogenase activity in Jurkat cells. The cells were seeded in
density 190 000 cells per well in a 96-well plate. The cells were treated with each of the tested substances
dissolved in DMSO. All compounds were prepared in 5 incubation concentrations (1 - 250 µM) in triplicates.
Also, the vehiculum controls (0.1% DMSO) were prepared in triplicates. After 48 hours 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 hours at 37°C; absorbance was then measured at 470 nm using a 96-multiwell
microplate reader Tecan Infinite M200 (Tecan Group Ltd., Männedorf, Switzerland). Viability was calculated as
6
5
described elsewhere in the literature using the following formula: (%) viability = (A470sample - A470blank) /
A470control - A470blank) x 100, where A470 is the absorbance of utilized XTT formazan measured at 470 nm.
Data were statistically analysed with GraphPad Prism (version 5.00) statistical software using unpaired t-test: p ≤
.01.
(
0
Acknowledgements
HA, RL and ML thank for support to research projects RVO 61388963 awarded by the Institute of Organic
Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, v.v.i., Prague and to the Czech
Science Foundation (grant number P208/12/G016).
References
1
.
Berg, L.; Andersson, C. D.; Artursson, E.; Hörnberg, A.; Tunemalm, A. K.; Linusson, A.; Ekström, F.
Plos One 2011, 11, e26039.
2
3
.
.
Marcelo, F.; Silva, F. V. M. Bioorg. Med. Chem.2009, 17, 5016.
Massoulié, J.; Pezzementi, L.; Bon, S.; Krejci. E.; Vallette, F. M. Prog. Neurobiol. 1993, 41, 31.
1
8

4
5
6
7
.
.
.
.
Stepankova, S.; Komers, K. Current Enzyme Inhibition 2008, 4, 160.
Grutzendler. J.; Morris, J.C. Drugs 2001, 61, 41.
Massoulié, J.; Bon S. Annu. Rev. Neurosci.. 1982, 5, 57.
Sussman, J. L.; Harel, M.; Frolow, F.; Oefner, C.; Goldman, A.; Toker, L.; Silman, I. Science 1991, 23,
8
72.
Nicolet, Y.; Lockridge, O.; Masson, P.; Fontecilla-Camps, J.C.; Nachon, F. J.Biol.Chem. 2003, 278,
1141.
8
9
.
.
4
Harel, M.; Sussman, J. L.; Krejci, E.; Bon, S.; Chanal, P.; Massoulio, J.; Silman, I. Proc. Natl. Acad.
Sci. 1992, 89, 10827.
1
1
0. Harel, M.; Quinn, D. M.; Nair, H. K.; Silman, I.; Sussman, J. L. J. Am. Chem. Soc. 1996, 118, 2340.
1. Knez, D; Brus, B; Coquelle, N; Sosic, I; Sink, R; Brazzolotto, X; Mravljak, J; Colletier, JP; Gobec, S.
Bioorg. Med. Chem. 2015, 23, 4442.
1
2. Bar-On, P.; Millard, C. B.; Harel, M.; Dvir, H.; Enz, A.; Sussman, J. L.; Silman, I. Biochemistry 2002,
4
1, 3555.
1
1
3. Patel, N. B.; Rathod, R. D. Int. J. Chem. Sci. 2006, 4, 569.
4. Menges, M.; Hamprecht, G.; Menke, O.; Reinhard, R.; Schafer, P.; Zagar, C. Substituted 2-
(benzoaryl)pyridines, WO/1999/006394 A1 (PCT/EP1998/003833), Feb 11, 1999.
1
1
1
1
5. Reuveni, M. Eur. J. Plant. Pathol. 2003, 109, 243.
6. Pejchal, V.; Stepankova, S.; Drabina, P. J. Heterocycl. Chem. 2011, 48, 57.
7. Pejchal, V.; Stepankova, S.; Padelkova, Z.; Imramovsky, A.; Jampilek, J. Molecules 2011, 16, 7565.
8. Imramovsky. A.; Pejchal, V.; Stepankova, S.; Vorcakova, K.; Jampilek, J.; Vanco, J.; Simunek, P.;
Kralovec, K.; Bruckova, L.; Mandikova, J.; Trejtnar, F. Bioorg. Med. Chem. 2013, 21, 1735.
9. Kratky M.; Stepankova, S.; Vorcakova , K.; Vinšova, J. Bioorganic Chemistry 2015, 58, 48.
0. Warshavik, D. T.; Golan, G.; Borrelli, K. W.; Zhu, K.; Kalid, O. J. Chem. Inf. Model. 2014, 54, 1941.
1. Ouyang, X.; Zhou, S.; Su, Ch. T. T.; Ge, Z.; Li, R.; Kwoh, C. K. J. Comput. Chem. 2013, 34, 326.
2. Pejchal, V.; Pejchalova, M.; Ruzickova Z. Med. Chem. Res. 2015, 24, 3660.
3. Suarez-Castillo, O. R.;.Bautista-Hernandez, C. I.; Sanchez-Zavala, M.; Melendez-Rodriguez, .M.;
Sierra-Zenteno, A. ; Morales-Rios, M. S.; Joseph-Nathan P. Heterocycles 2012, 85, 2147.
4. Engler, T. A.; Letavic, M. A.; Iyengar, R.; LaTessa, K. O.; Reddy, J. P. J. Org. Chem. 1999, 64, 2391.
5. Lussmann, J.; Hoppe, D.; Jones, P. G.; Fittscher, C.; Sheldrick, G. M. Tetrahedron Lett. 1986, 27, 3595.
6. Harris, L.; Mee, S. P. H.; Furneaux, R. H.; Gainsford, G. J.; Luxenburger, A. J. Org. Chem. 2011, 76,
1
2
2
2
2
2
2
2
3
58.
2
2
7. Hager,C.; Miethchen, R.; Reinke, H. (2000) J. Fluorine Chem. 2000, 104, 135.
8. Kopf, J.; Sheldrick, G. M.; Reinke, H.; Rentsch, D.; Miethchen, R. Acta Crystallogr. Sect. C: Cryst.
Struct. Commun. 1995, 51, 1198.
2
3
9. Brandenburg, K. L.; Heeg, M. J.; Abrahamson, H. B. Inorg. Chem. 1987, 26, 1064.
0. Kello, E.; Kettman, V.; Miertus, S.; Vrabel, V.; Konecny, V. Collect. Czech. Chem. Commun. 1986, 51,
1
864.
3
3
1. Guo-Bao, C.; Qiang-Jin, W.; Xiao-Ying, H. Jiegou Huaxue (Chin. J. Struct. Chem.), 1995, 4, 47.
2. Allen, F.H.; Kennard, O.; Watson, D. G.; Brammer, L.; Orpen, A. G.; Taylor, R. J. Chem. Soc., Perkin
Trans. 2 1987, S1.
3
3
3. Pindinelli, E.; Pilati, T.; Troisi, L. Eur. J. Org. Chem., 2007, 5926.
4. Yong, Z.; Bi-lin, Z.; Shi-lei, Z.; Yuan, Q.; Xian-you, X. Acta Crystallogr., Sect. E: Struct. Rep. Online,
2
009, 65, o1674.
5. Karagiannidis, L.E. ; Gale, P.A.; Light, M.E.; Massi, M.; Ogden, M.I. Dalton Transactions 2011, 40,
2097.
6. Kerns, E. H.; Li, D. Drug-like Properties: Concept, Structure Design and Methods; Elsevier: San
Diego, CA, USA 2008.
3
3
1
3
3
3
7. Lipinski, C. A. Drug Discovery Today: Technologies 2004, 1, 337.
8. Sangster J. J. Phys. Chem. Ref. Data 1989, 18, 1111.
9. Berthod, A,; Carda-Broch, S. Journal of Chromatography A (2004), 1037, 3.
1
9

4
4
4
0. Darvesh, S.; McDonald, R. S.; Darvesh, K. V.; Mataija, D.; Conrad, S.; Gomes G.; Walsk, R.; Martin E.
Bioorg. Med. Chem. 2007, 15, 6367.
1. Luo, W.; Yu, Q.; Kulkarni,S. S.; Parrish, D. A.; Holloway, H. W.; Tweedie, D.; Shafferman, A.; Lahiri,
D. K.; Brossi, A.; Greig, N. H. J. Med. Chem. 2006, 49, 2174.
2. Zhu, K.; Borrelli, K. W.; Greenwood, J. R.; Day, T.; Abel, R.; Farid, R. S.; Harder, E. J. Chem. Inf.
Model. 2014, 54,1932.
4
4
4
3. Jann, M. W.; Shirley, K. L.; Small, G. W. Clin. Pharmacokinet. 2002, 41, 719.
4. Bartolini, M.; Cavrini, V.; Andrisano, V. J. Chromatogr. A 2007, 1144, 102.
5. Fink A. L. In Enzyme Mechanisms; Page, M. I., Williams A., Eds; Royal Society of Chemistry;
London, 1987; pp 159.
4
6. Fanfrlík, J.; Brahmkshatriya, P. S.; Řezáč, J.; Jílková, A.; Horn, M.; Mareš, M.; Hobza, P.; Lepšík, M.
J. Phys. Chem. B 2013, 117, 14973.
4
4
7. Otwinowski, Z.; Minor, W. Methods in Enzymology 1997, 276, 307.
8. Ahmed, F. R.; Hall, S. R.; Huber, C. P. Crystallographic Computing; Munksgaard: Copenhagen,
Denmark 1970.
4
5
5
5
5
5
5
5
5
5
5
9. Altomare, A.; Cascarano, G.; Giacovazzo, C.; Guagliardi, A. J. Appl. Crystallogr.1993, 23, 343.
0. Sheldrick, G.M. SHELXL-97; University of Göttingen: Göttingen, Germany, 1997.
1. Ellman, G. L.; Courtney, K. D.; Andres, V.; Featherstone F. M. Biochem. Pharmacol. 1961, 7, 88.
2. Kwok, S. O.; Wang, K. C.; Kwok, H. B. Food Chem. 2004, 88, 317.
3. Sinko, G.; Calic, M.; Bosak, A.; Kovarik, Z. Anal. Biochem. 2007, 370, 223.
4. Zdrazilova, P.; Stepankova, P.; Komers, K.; Ventura, K.; Cegan, A. Z. Naturforschung 2004, 59, 293.
5. Szedlacsek, S. E.; Duggleby, R. G. Method. Enzymol. 1995, 249, 144.
6. Singh, J.; Petter, R. C.; Baillie, T. A.; Whitty, A. Nat. Rev. Drug Discov. 2011, 10, 307.
7. Schrödinger Release 2015-3: Maestro, version 10.3, Schrödinger, LLC, New York, NY, 2015.
8. Greenwood, J. R.; Calkins, D.; Sullivan, A. P.; Shelley, J. C. J. Comput. Aid. Mol. Des., 2010, 24, 591.
9. Sastry, G.M.; Adzhigirey, M.; Day, T.; Annabhimoju, R.; Sherman, W. J. Comput. Aid. Mol.
Des, 2013, 27, 221.
6
6
0. Friesner, R. A.; Banks, J. L.; Murphy, R. B.; Halgren, T. A.; Klicic, J. J.; Mainz, D. T.; Repasky, M. P.;
Knoll, E. H.; Shelley, M.; Perry, J. K.; Shaw, D. E.; Francis, P.; Shenkin, P. S. J. Med. Chem. 2004, 47,
1
739.
1. Halgren, T. A.; Murphy, R. B.; Friesner, R. A.; Beard, H. S.; Frye, L. L.; Pollard, W. T.; Banks, J. L. J.
Med. Chem. 2004, 47, 1750.
6
6
2. Li, J.; Abel, R.; Zhu, K.; Cao, Y.; Zhao, S.; Friesner, R. A. Proteins 2011, 79, 2794.
3. Shivakumar, D.; Williams, J.; Wu, Y.; Damm, W.; Shelley, J.; Sherman, W. J. Chem. Theory Comput.
2
010, 6, 1509.
4. Xing, J. Z.; Zhu, L.; Jackson, J. A.; Gabos, S.; Sun, X. J.; Wang, X. B.; Xu, X. Chem. Res. Toxicol.
005, 18, 154.
5. Havelek, R.; Siman, P.; Cmielova, J.; Stoklasova.A.; Vavrova J..;Vinklarek, J. Med. Chem. 2012, 8,
15.
6
6
2
6
2
0

Graphical Abstract
Synthesis, structural characterisation, docking, lipophilicity and cytotoxicity of 1-[(1R)-
1
-(6-fluoro-1,3-benzothiazol-2-yl)ethyl]-3-alkyl carbamates, novel acetylcholinesterase
and butyrylcholinesterase pseudo-irreversible inhibitors
a
b
b
b
b
Vladimír Pejchal *, Šárka Štěpánková , Marcela Pejchalová , Karel Královec , Radim Havelek ,
c
d,e
d
d
Zdeňka Růžičková , Haresh Ajani, Rabindranath Lo, Martin Lepšík
a
Institute of Organic Chemistry and Technology, Faculty of Chemical Technology, University of
Pardubice, Studentska 573, 532 10 Pardubice, Czech Republic
b
Department of Biological and Biochemical Sciences, Faculty of Chemical Technology, University of
Pardubice, Studentska 573, Pardubice 532 10, Czech Republic
c
Department of General and Inorganic Chemistry, Faculty of Chemical Technology,
University of Pardubice, Studentska 573, 532 10 Pardubice, Czech Republic
d
Institute of Organic Chemistry and Biochemistry v.v.i and Gilead Sciences and IOCB Research
Center, Academy of Sciences of the Czech Republic, Flemingovo nam. 2, 166 10 Prague 6, Czech
Republic
e
Regional Center of Advanced Technologies and Materials, Department of Physical Chemistry,
Palacký University, Olomouc, 771 46 Olomouc, Czech Republic
a
Institute of Organic Chemistry and Technology, Faculty of Chemical Technology, University of Pardubice, Studentska 573, 532 10
Pardubice, Czech Republic
b
Department of Biological and Biochemical Sciences, Faculty of Chemical Technology, University of Pardubice, Studentska 573, Pardubice
5
32 10, Czech Republic
c
Department of General and Inorganic Chemistry, Faculty of Chemical Technology,
University of Pardubice, Studentska 573, 532 10 Pardubice, Czech Republic
d
Institute of Organic Chemistry and Biochemistry v.v.i and Gilead Sciences and IOCB Research Center, Academy of Sciences of the Czech
Republic, Flemingovo nam. 2, 166 10 Prague 6, Czech Republic
e
Regional Center of Advanced Technologies and Materials, Department of Physical Chemistry, Palacký University, Olomouc, 771 46
Olomouc, Czech Republic
2
1