Synthesis and in vitro evaluation of new derivatives of 2-substituted-6-fluorobenzo[d]thiazoles as cholinesterase inhibitors

Article abstract of DOI:10.1016/j.bmc.2013.01.052

A series of novel cholinesterase inhibitors based on 2-substituted 6-fluorobenzo[d]thiazole were synthesised and characterised by IR, ¹H, ¹³C and ¹⁹F NMR spectroscopy and HRMS. Purity was checked by elemental analyses. The novel carbamates were tested for their ability to inhibit acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). The toxicity of the most active compounds was investigated using a standard in vitro test with HepG2 cells, and the ratio between biological activity and toxicity was determined. In addition, the toxicity of the most active compounds was evaluated against MCF7 cells using the xCELLigence system. Structure-activity relationships reflecting the dependence of cholinesterase inhibitors on the lipophilicity of the compounds as well as on the Taft polar and steric substituent constants are discussed. The specific orientation of the inhibitors in the binding site of acetylcholinesterase was determined using molecular docking of the most active compound.

Full text of DOI:10.1016/j.bmc.2013.01.052

Bioorganic & Medicinal Chemistry 21 (2013) 1735–1748
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and in vitro evaluation of new derivatives of 2-substituted-
6-fluorobenzo[d]thiazoles as cholinesterase inhibitors
a,
a
b
c
d
⇑
´
ˇ
ˇ
Aleš Imramovsky , Vladimír Pejchal , Šárka Štepánková , Katarína Vorcáková , Josef Jampílek ,
d
a
b
b
e
e
ˇ
ˇ
Ján Vanco , Petr Šimu˚ nek , Karel Královec , Lenka Bru˚ cková , Jana Mandíková , František Trejtnar
a Institute of Organic Chemistry and Technology, Faculty of Chemical Technology, University of Pardubice, Studentská 573, CZ-532 10 Pardubice, Czech Republic
b Department of Biological and Biochemical Sciences, Faculty of Chemical Technology, University of Pardubice, Studentská 573, Pardubice 53210, Czech Republic
c Department of Analytical Chemistry, Faculty of Chemical Technology, University of Pardubice, Studentská 573, Pardubice 53210, Czech Republic
d Department of Chemical Drugs, Faculty of Pharmacy, University of Veterinary and Pharmaceutical Sciences, Palackeho 1/3, 612 42 Brno, Czech Republic
e Department of Pharmacology and Toxicology, Faculty of Pharmacy, Charles University, Heyrovského 1203, 500 05 Hradec Králové, Czech Republic
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel cholinesterase inhibitors based on 2-substituted 6-fluorobenzo[d]thiazole were synthes-
ised and characterised by IR, 1H, 13C and 19F NMR spectroscopy and HRMS. Purity was checked by ele-
mental analyses. The novel carbamates were tested for their ability to inhibit acetylcholinesterase
(AChE) and butyrylcholinesterase (BChE). The toxicity of the most active compounds was investigated
using a standard in vitro test with HepG2 cells, and the ratio between biological activity and toxicity
was determined. In addition, the toxicity of the most active compounds was evaluated against MCF7 cells
using the xCELLigence system. Structure–activity relationships reflecting the dependence of cholinester-
ase inhibitors on the lipophilicity of the compounds as well as on the Taft polar and steric substituent
constants are discussed. The specific orientation of the inhibitors in the binding site of acetylcholinester-
ase was determined using molecular docking of the most active compound.
Received 3 December 2012
Revised 22 January 2013
Accepted 23 January 2013
Available online 1 February 2013
Keywords:
6-Fluorobenzo[d]thiazole
Lipophilicity
Acetylcholinesterase
Butyrylcholinesterase
Cholinesterase inhibition
In vitro cytotoxicity
Molecular docking
Ó 2013 Elsevier Ltd. All rights reserved.
Structure–activity relationships
1. Introduction
toms of these diseases. Thus, research on new ChEIs may be valu-
able for further progress in the treatment of these diseases.
Two important enzymes from the group of serine hydrolases,
acetylcholinesterase (AChE, EC 3.1.1.7) and butyrylcholinesterase
(BChE, EC 3.1.1.8; also known as pseudocholinesterase), are usually
defined as cholinesterases (ChEs). Structurally, these serine hydro-
lases belong to the class of proteins known as the esterase/lipase
(R)-1-(6-Fluorobenzo[d]thiazol-2-yl)ethanamine is a basic scaf-
fold for antimicrobials, herbicides, plant desiccants and defoliant
compounds.14 Generally, benzothiazoles have been reported as a
class of compounds with a wide range of biological activities,
including antibacterial and antimicrobial,15 anticancer,16–18 antitu-
mour,19 antiviral,20 anthelmintic,21 antiparasitic,22 anti-inflamma-
tory,23 fungicidal24 and antioxidant25 activity. The benzothiazole
scaffold has provided several promising candidates, which were
investigated as a clinical antitumor candidates.26 Also other inter-
esting bioactivities of these compounds were described in recent
years, such as protein inhibitors27,28 or biosensors.29 The benzothi-
azole ring is also present in various natural marine and terrestrial
compounds which have interesting biological activities.30–32 The
moiety of these compounds can serve as a unique and versatile
scaffold for experimental drug design. Benzothiazole has an impor-
tant place in research, especially in synthetic and pharmaceutical
chemistry, due to its potent pharmacological activities. Studies
on structure–activity relationships have interestingly revealed that
a change in the structure of the substituent group at the C(2) posi-
tion commonly results in a change in bioactivity. Such derivatives
family within the a
/b-hydrolase fold superfamily.1 The major role
of AChE is to catalyse the hydrolysis of acetylcholine (ACh) in cho-
linergic synapses, whereas the function of BChE is less clearly de-
fined because it can hydrolyse ACh as well as other esters.2,3 The
inhibition of these enzymes causes an increase in the concentra-
tion of ACh in cholinergic synapses and can subsequently affect a
number of pathogenic processes.2 ChE inhibitors (ChEIs) are used
in the treatment of various neuromuscular disorders and have pro-
vided the first generation of drugs for the treatment of Alzheimer’s
disease,4–9 myasthenia gravis10,11 and glaucoma.12,13 An increase in
the concentration of ACh can result in an alleviation of the symp-
⇑
Corresponding author. Tel.: +420 466037739; fax: +420 466038004.
E-mail address: Ales.Imramovsky@upce.cz (A. Imramovsky´).
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.01.052

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A. Imramovsky et al. / Bioorg. Med. Chem. 21 (2013) 1735–1748
1736
have received considerable attention due to their potential biolog-
ical activity.33
3. Liphophilicity
Substituted carbamates are characterised as pseudosubstrate
inhibitors or active site-directed irreversible inhibitors of ChEs
that, in the presence of the substrate, compete for the AChE active
site via nucleophilic attack of the hydroxyl moiety of Ser residues
in the enzyme;2,34–37 nevertheless; non-covalent ChEIs have also
been described.38–40 This publication is a follow-up paper to our
previous articles40,41 dealing with the synthesis and cholinesterase
inhibiting ability of novel 2-substituted-6-fluorobenzo[d]thiazole
derivatives. In the context of research on benzothiazoles previously
described by us, new modifications of the 6-fluorobenzothiazole
moiety that can trigger interesting biological activity were investi-
gated. Reaching the inhibitory site of action and binding to the ac-
tive site cleft are influenced by the hydro/lipophilic properties of
compounds; therefore, the lipophilicity of the target compounds
was experimentally determined using RP-HPLC. The structure–
activity relationships between the chemical structure, physical
properties and biological activities of the evaluated compounds
are discussed. The specific orientation of the inhibitors in the AChE
binding site was determined using molecular docking.
The calculated hydrophobicity (logP/ClogP values) of com-
pounds 6a–w was obtained using two commercial programs, and
their experimental lipophilicities were additionally measured by
RP-HPLC to determine their capacity factors (k) with the subse-
quent calculation of their logk values. This involved the measure-
ment of compound retention times using isocratic conditions
with methanol as an organic modifier in the mobile phase. The
capacity factor k was calculated using a non-polar C18 stationary
RP column and the corresponding log k value was used as a lipo-
philicity index converted to the logP scale.43 The results are shown
in Table 1. A graphical illustration and comparison of the experi-
mentally determined and calculated lipophilicity values is shown
and commented on in detail in the Supplementary data associated
with this article.
The compounds under investigation could be divided into two
groups based on their chemical structure: Group 1 includes
R1 = n-propyl substituted compounds 6a–k and Group 2 contains
R1 = isopropyl substituted compounds 6l–w. Generally, it can be
stated that the compounds with a branched alkyl substituent
showed lower lipophilicity, that is, Group 1 possessed higher lipo-
philicity than Group 2, and within the individual series, the isopro-
pyl or isobutyl derivatives 6d,f,o,q showed less lipophilicity than
the unbranched derivatives 6c,e,n,p. The substitution of the alkyl
chain by fluorine atoms is noteworthy. According to the assump-
tion, the –CF3 substituted compounds 6i,u possessed less lipophil-
icity than the –CCl3 derivatives 6h,t. The order of all the fluorine
substituted compounds is the following: –CH2CF2CHF2 (6j,v) < –
CH2CF3 (6i,u) < –CH2CF2CF2CF3 (6k,w). Thus, it can be concluded
that the log k data specify lipophilicity within the series of
compounds.
One of the main goals in drug discovery is to evaluate the safety
of potential therapeutic agents. Cytotoxicity assays are among the
first in vitro methods used to predict the toxicity of novel com-
pounds. In vitro cytotoxicity assays can also be used to predict hu-
man toxicity and for the general (scheme 1) screening of
chemicals.42 For this reason, the studied compounds were also
evaluated using two standardised in vitro cytotoxicity assays.
2. Chemistry
The synthesis of the desired compounds can be described as a
step-by-step synthesis. The first part of synthetic pathway is the
two-step preparation of activated 2-[(alkoxycarbonyl)amino] car-
boxylic acid. At this stage, the amino acid is combined with a se-
lected chloroformate, and the obtained acid is then transformed
to a reactive derivate using isobutyl chloroformate. This reactive
intermediate reacts with (R)-1-(6-fluorobenzo[d]thiazol-2-yl) eth-
anamine, which is released from its p-toluene sulphonate salt
in situ. All synthetic steps are described in detail in the following
paragraphs.
One of the advantages of the applied method of synthesis is the
simple preparation of all intermediates. The desired products 6a–w
were obtained by filtration or vacuum concentration of the organic
phase. On the other hand, the isolation of compounds 3a or 4w was
possible and not problematic. Finally, the method is applicable to
various amino acids and a number of chloroformates.
4. In vitro evaluation of cholinesterase/anticholinesterase
activity
All the prepared compounds were tested for their inhibition po-
tency using AChE from Electrophorus electricus (Sigma–Aldrich) and
BChE from equine serum (Sigma–Aldrich). The activities of the
compounds were compared with the internal standards rivastig-
mine (RIV, ExelonÒ) and galanthamine (GLT, ReminylÒ) (Table 1).
These standards were chosen due to their different structures.
While rivastigmine is a classical acylating pseudo-reversible carba-
mate cholinesterase inhibitor that inhibits both AChE and BChE,
galanthamine is a non-acylating competitive reversible cholines-
terase inhibitor as well as an allosteric ligand at nicotinic acetyl-
choline receptors. The choice of these reference drugs with
different mechanisms of action can provide relevant results. The
results are summarised in Table 1 and are expressed as 50% inhib-
itory concentration (IC50 [lmol/L]). The IC50 represents the concen-
tration of inhibitor required for 50% inhibition of the mentioned
enzymes.
R1
O
R1
O
Based on these results (see Table 1), it is evident that effective
AChE inhibitors are different from BChE inhibitors. Both ChEs differ
from each other by the distribution of amino acids in the cavity and
the peripheral sites of the enzymes. Some aromatic amino acids
typical for AChE (e.g., phenylalanine) are replaced in the structure
of BChE with aliphatic amino acids (e.g., valine, leucine), and the
active site of both ChEs is thus sterically accessible to substrates
differing in terms of bulkiness or the ability to interact with amino
acids within the peripheral site.2,3,45–48
R1
OR2
O
a, b, c
d
O
O
HO
HN
HO
+
NH2
C
O
NH
OR2
C
O
NH
OR2
C
O
Cl
O
3
4
1
2
+
N
S
N
S
e
PTS
O
NH2
F
F
NHCOOR2
5
6a-w
R1
All the discussed carbamate-like compounds demonstrated
activity comparable with or higher than that of the standard riv-
astigmine and slightly lower than that of the standard galantha-
mine. In general, Group 1 showed higher activity, particularly in
Scheme 1. Synthesis of reactive intermediates 4a–4w. Reagents and conditions: (a)
NaOH, T <10 °C, 30 min; (b) parallel addition of chloroformate and NaOH, T <10 °C,
60 min; (c) HCl, pH 7; (d) TOL, DMBA, isobutyl chloroformate, T <10 °C, 15 min,
T = RT, 45 min; (e) NaOH, T = RT, 6 h.

´
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A. Imramovsky et al. / Bioorg. Med. Chem. 21 (2013) 1735–1748
Table 1
List of prepared derivates 6a–6w and their inhibitory activity against AChE and BChE expressed as IC50
galanthamine (GLT)
[
lmol/L] in comparison with standards rivastigmine (RIV) and
N
S
R2
O
(
R
)
O
O
HN
F
) NH
S
(
R1
R1
R2
IC50
(
lmol/L)
logk
logP/ClogP
logP
r⁄44
ES
44
Compd
AChE
BChE
ChemOffice
ACD/LogP
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
6m
6n
6o
6p
6q
6r
(S)-(CH2)2CH3
(S)-(CH2)2CH3
(S)-(CH2)2CH3
(S)-(CH2)2CH3
(S)-(CH2)2CH3
(S)-(CH2)2CH3
(S)-(CH2)2CH3
(S)-(CH2)2CH3
(S)-(CH2)2CH3
(S)-(CH2)2CH3
(S)-(CH2)2CH3
(S)-CH(CH3)2
(S)-CH(CH3)2
(S)-CH(CH3)2
(S)-CH(CH3)2
(S)-CH(CH3)2
(S)-CH(CH3)2
(S)-CH(CH3)2
(S)-CH(CH3)2
(S)-CH(CH3)2
(S)-CH(CH3)2
(S)-CH(CH3)2
(S)-CH(CH3)2
RIVastigmine (ExelonÒ
–CH3
–C2H5
48.80 0.35
34.77 0.40
28.54 0.25
21.31 0.20
28.00 0.21
20.22 0.25
35.38 0.32
28.59 0.27
29.26 0.27
30.71 0.32
46.01 0.45
91.23 0.89
153.0 1.52
108.6 1.15
95.04 0.99
68.05 0.66
49.26 0.52
47.50 0.42
47.15 0.40
30.30 0.32
49.40 0.45
28.97 0.25
85.51 0.77
501 3.08
20.42 0.20
14.10 0.13
19.25 0.18
32.20 0.29
19.58 0.18
26.13 0.23
54.11 0.55
62.69 0.63
59.00 0.48
43.95 0.38
59.75 0.55
32.00 0.29
35.60 0.33
70.45 0.64
41.10 0.35
62.50 0.58
41.10 0.45
54.20 0.62
ND
0.4166
0.4873
0.5873
0.5752
0.7231
0.7063
0.5140
0.8297
0.5825
0.5415
0.9463
0.4113
0.4834
0.5851
0.5725
0.7135
0.7041
0.5014
0.5418
0.8043
0.5643
0.5237
0.9218
3.19/2.64611
3.52/3.17511
4.01/3.70411
3.84/3.48411
4.43/4.23311
4.41/4.10311
3.90/3.23811
4.91/4.95411
4.16/3.43911
4.11/3.73611
5.37/4.89911
3.17/2.51611
3.51/3.04511
3.99/3.57411
3.82/3.35411
4.41/4.10311
4.39/3.97311
3.88/3.10811
4.00/3.24811
4.89/4.82411
4.15/3.30911
4.09/3.60611
5.35/4.76911
2.36/2.099
3.20 0.69
3.73 0.69
4.26 0.69
4.08 0.69
4.80 0.69
4.61 0.69
4.00 0.70
5.77 0.77
4.50 0.87
4.61 0.91
6.54 1.07
3.02 0.69
3.55 0.69
4.08 0.69
3.90 0.69
4.61 0.69
4.43 0.69
3.82 0.70
4.00 0.69
5.59 0.77
4.32 0.88
4.43 0.91
4.62 1.06
0
0
ꢀ0.10
ꢀ0.115
ꢀ0.19
ꢀ0.13
ꢀ0.125
0.385
NF
NF
NF
NF
0
ꢀ0.10
ꢀ0.115
ꢀ0.19
ꢀ0.13
ꢀ0.125
0.385
NF
NF
NF
NF
NF
ꢀ0.07
ꢀ0.36
ꢀ0.47
ꢀ0.39
ꢀ0.93
NF
NF
NF
NF
NF
–(CH2)2CH3
–CH(CH3)2
–(CH2)3CH3
–CH2CH(CH3)2
–CH2CH2Cl
–CH2CCl3
–CH2CF3
–CH2CF2CHF2
–CH2CF2CF2CF3
–CH3
0
–C2H5
ꢀ0.07
ꢀ0.36
ꢀ0.47
ꢀ0.39
ꢀ0.93
NF
NF
NF
NF
NF
–(CH2)2CH3
–CH(CH3)2
–(CH2)3CH3
–CH2CH(CH3)2
–CH2CH2Cl
–CH2CH2Br
–CH2CCl3
–CH2CF3
–CH2CF2CHF2
–CH2CF2CF2CF3
6s
6t
6u
6v
6w
26.56 0.20
72.00 0.80
33.80 0.32
83.20 0.75
19.95 0.20
7.96 0.59
NF
)
GaLanThamine (ReminylÒ
)
4
0.13
1.41/1.025
ChE inhibitions are expressed as mean SD (n = 3 experiments). Comparison of calculated lipophilicities (logP/ClogP) with experimentally determined logk values, Taft polar
substituent constants (r⁄) and Taft steric substituent constants (ES).ND—not determined, NF—not found.
case of non-halogenated R2 substituents in comparison with Group
2. Based on this fact, it can be assumed that R1 substitution by the
unbranched n-propyl chain plays a crucial role for AChE/BChE inhi-
bition (see Figs. 1 and 2). Whereas the n-propyl chain in position R1
is necessary for both types of AChE/BChE inhibitors, for higher
AChE-inhibiting activity, R2 branched long-chain alkyl substitution
of the carbamate ester seems to be preferred (see Fig. 1) in contrast
to BChE inhibitors, where a short unbranched alkyl chain at posi-
tion R2 is more favoured (see Fig. 2). Within a halogenated R2 chain,
R1 substitution seems to be not so important. Based on the docking
study (see below), it can be stated that the studied compounds
could act as ‘bulky’ blockers of entrance into the active site cleft;
this observation agrees with the above-mentioned fact that, due
to different distribution of amino acids in the peripheral site of
both ChEs, BChE is resistant to bulky inhibitors. Isobutyl-(S)-1-
[(R)-1-(6-fluorobenzo[d]thiazol-2-yl)ethylcarbamoyl]butyl-carba-
mate (6f) and isopropyl-(S)-1-[(R)-1-(6-fluorodrobenzo[d]thiazol-
2-yl)ethylcarbamoyl]butyl carbamate (6d) were the most active
AChE-inhibiting compounds, while ethyl-(S)-1-[(R)-1-(6-fluoro
benzo[d]thiazol-2-yl)ethylcarbamoyl]butylcarbamate (6b) was
the most active BChE inhibitor.
The shapes of the biological response curves shown in Figures 1
and 2 are typical of n-alkyl homologous series, when activity can
alternate with the number of carbon atoms (with a change in a
long side/straight chain). The inhibition ability of carbamates
(binding to the active site of AChE/BChE) is influenced by the bulk-
iness of the substituents, due to the difficulty of larger inhibitors to
enter the narrow enzyme active site cleft, as well as the strictly bal-
anced hydro/lipophilic properties of the inhibitors, taking into ac-
count the fact that hydrophobic substituents accelerate inhibitor
entry into the active site cleft of the enzyme.6 The rate of carbamyl-
ation depends on a delicate balance between the length of the res-
idue and its degree of freedom of rotation.4
A number of papers refer to the fact that an increase in N-alkyl
chain length results in ChE inhibition. This fact is in compliance
with the observation that the introduction of bulky hydrophobic
4.9
4.6
4.3
4.0
3.7
Me
Et
Pr
iPr
Bu
iBu
C2-Cl
C2-Cl3
C2-F3
C-CF2-
CHF2
C-CF2-
CF2-CF3
Compounds
iPr (6l-w) Pr (6a-k)
Figure 1. Dependence of AChE inhibition (log1/IC50 [mol/L]) on long alkyl chain of R2 substitution within Group 1 (Pr) and Group 2 (iPr).

´
A. Imramovsky et al. / Bioorg. Med. Chem. 21 (2013) 1735–1748
1738
4.9
4.6
4.3
4.0
3.7
Me
Et
Pr
iPr
Bu
iBu
C2-Cl
C2-Cl3
C2-F3
C-CF2-
CHF2
C-CF2-
CF2-CF3
Compounds
iPr (6l-w) Pr (6a-k)
Figure 2. Dependence of BChE inhibition (log1/IC50 [mol/L]) on long alkyl chain of R2 substitution within Group 1 (Pr) and Group 2 (iPr).
groups at the carbamoyl nitrogen leads to compounds with better
AChE inhibition2,34,37,49 and, vice versa, to compounds with low
BChE inhibition.46 It was also observed that activity increases with
increasing electron-withdrawing effect.50 From these facts, it can
be concluded that ChE inhibition correlates with lipophilicity, elec-
tronic, inductive or polar properties and steric effects.43
AChE inhibition dramatically decreased with decreasing bulkiness.
The AChE inhibition activity of these compounds was also posi-
tively influenced by a negative polar parameter r⁄
.
The dependences of AChE inhibition (log1/IC50 [mol/L]) on the
Taft steric substituent constant ES is illustrated in Figure 4. It is evi-
dent that AChE inhibition of compounds 6a–f was positively influ-
enced by a negative polar parameter r⁄ as well as by a bulky
substituent. AChE inhibition of compounds 6l–q was again posi-
tively influenced by a bulky substituent (Fig. 4B), but the effect
of the r⁄ parameter seemed to be only secondary.
As discussed above, different BChE inhibition results were ob-
served for the two groups. The dependence of BChE inhibition
(log1/IC50 [mol/L]) on the Taft polar and steric parameters r⁄ and
ES within Group 1 is illustrated in Figure 5A and B; the results for
Group 2 are illustrated in Figure 6A and B. Figure 5A and B show
the bilinear dependence of BChE inhibiting activity on both Taft
parameters with a preferred polarity value of ꢀ0.10 (compound
6b) and bulkiness expressed as ES = ꢀ0.07 (ethyl, compound 6b).
Within Group 2, the least active carbamates/outliers, that is, 6n
and 6p, were eliminated; the dependence of BChE inhibition
(log1/IC50, [mol/L]) on r⁄ or ES is presented in Figure 6A and B. In
both cases, the dependence of activity on the Taft polar and steric
parameters was linear with the correlation coefficient R = 0.9198
or R = 0.8610, respectively. Therefore, it can be stated that within
the R2 substituents, minimal steric (Fig. 6B) and polar (Fig. 6A) ef-
fects seem to be important for the inhibition of BChE.
The dependence of AChE inhibition (log1/IC50 [mol/L]) on logk
within Group 1 is illustrated in Figure 3A. In the set of 11 tested al-
kyl- and/or halogeno-substituted compounds, a quasi-parabolic
dependence was found. It can be concluded that, for optimum
activity, the range of lipophilicity expressed as logk 0.6–0.7 and
the branched alkyl chain are advantageous. Also, a multihalogenat-
ed C2 or C3 chain seems to be advantageous. Within Group 2, the
dependence of AChE inhibition on logk did not seem to be as clear
(lipophilicity was only a secondary parameter) but, generally,
AChE-inhibiting activity increases with prolongation of the chain
and an increasing number of halogens in the chain (see Table 1).
The substitution with a branched alkyl at the R2 position positively
affected the activity as well. Universal advantageous R2 substitu-
ents for both groups were -CH2CCl3 and -CH2CF2CHF2, because
6h/t, 6j/v expressed similar activity (see Table 1). From the data
shown in Table 1, it follows that lipophilicity for both groups
was only a secondary parameter for BChE inhibition. Nevertheless,
in both cases, activity decreased with increasing lipophilicity and,
similarly to AChE inhibition, halogenated R2 substituents seemed
to be more advantageous in Group 2 than in Group 1.
ChE inhibition activity was also correlated with the Taft polar
and steric substituent constants r⁄ and ES. Values r⁄ represent
field, inductive and resonance contributions of the substituent
and ES values represent the bulkiness of substituent. Both Taft
parameters are listed in Table 1. These parameters were not found
in the literature for this series of halogenated substituents. From
the data in Table 1, it is evident that AChE inhibition activity within
both groups was particularly influenced by the bulkiness of the
substituent, that is, the Taft steric substituent constant ES, which
is illustrated Figure 4. For Group 1 and Group 2, it can be stated that
Different ChE inhibition results were observed for halogenated
compounds. In general, it can be concluded that halogenated R2
substituents showed fewer differences between AChE/BChE activ-
ity in comparison with non-halogenated alkyl chain substituents,
and in the context of these facts, the highly electronegative –CCl3
moiety (compounds 6h,t) showed high AChE and BChE inhibition
activity within Groups 1 and 2.
5. Molecular docking
Structural analysis of AChE revealed that the active site is
placed near the bottom of a narrow gorge imbedded halfway into
the protein and 14 aromatic residues lining a substantial portion
of the surface of the gorge. This cavity was named the ‘active site
gorge’ and, further at the gorge mouth a peripheral anionic binding
site (PAS) was found. The active site of AChE contains: (i) an ester-
atic site (ES) comprising the catalytic triad Ser-His-Glu, which is lo-
cated at the bottom of the gorge; (ii) an oxyanion hole (OAH) that
stabilizes the tetrahedral intermediate binding of the carbamate
carbonyl group; (iii) an acyl binding site (ABS) that binds the acetyl
group of ACh or the alkyl moiety of carbamate inhibitors; and (iv)
an anionic substrate binding site (AS) that contains a small number
of negative charges but many aromatic residues, where the quater-
nary ammonium pole of ACh and of various active site ligands
binds through a preferential interaction of quarternary nitrogens
4.8
4.7
4.6
4.5
4.4
4.3
4.2
6f
6d
6c
6j
6b
6e
6i
6h
6g
6a
6k
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
log k
Figure 3. Dependence of AChE inhibition (log1/IC50 [mol/L]) on the lipophilicity of
compounds 6a–k.

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A. Imramovsky et al. / Bioorg. Med. Chem. 21 (2013) 1735–1748
4.8
4.7
4.6
4.5
4.4
4.3
4.2
4.4
A
B
6q
4.3
6d
6f
4.2
6e
6p
6c
4.1
6l
6o
6b
4.0
6n
3.9
6m
3.8
6a
3.7
-1.0
-0.8
-0.6
-0.4
-0.2
0.
-1.0
-0.8
-0.6
-0.4
-0.2
0.0
ES
ES
Figure 4. Dependence of AChE inhibition (log1/IC50 [mol/L]) on Taft steric substituent constants expressed as ES: Figure 4A—compounds 6a–f; Fig. 4B—compounds 6l–q.
A
4.9
4.8
4.7
4.6
4.5
4.4
B
4.9
4.8
4.7
4.6
4.5
4.4
6b
6b
6c
6e
6e
6c
6a
6a
6f
6f
6d
6d
-0.20
-0.15
-0.10
-0.05
0.00
-1.0
-0.8
-0.6
-0.4
-0.2
0.0
ES
σ*
Figure 5. Dependence of BChE inhibition (log1/IC50 [mol/L]) of compounds 6a–f on the Taft polar r⁄ (5A) and steric ES (5B) substituent constants.
A
4.6
B
4.6
4.5
4.4
4.3
4.2
6l
4.5
6m
6l
6m
6o
6q
4.4
4.3
4.2
6q
6o
-0.20
-0.15
-0.10
σ*
-0.05
0.00
-1.0
-0.8
-0.6
-0.4
-0.2
0.0
ES
Figure 6. Dependence of BChE inhibition (log1/IC50 [mol/L]) of compounds 6l–q on the Taft polar r⁄ (6A) and steric ES (6B) substituent constants. Compounds 6n and 6p were
excluded and are not plotted.
or a partial positive charge generated by electron-withdrawing
moieties.35,38,51
atom) and the Tyr121 side chain (O–O = 2.576–3.924 Å, or N–
O = 2.632–3.696 Å) and between the amide oxygen and the
Tyr334 side chain (O–O = 2.917–2.945 Å). Moreover, the possible
interactions of the carbamate moiety with the Tyr70 OH moiety
(O–O = 3.816–3.881 Å) or the amide nitrogen from Phe288 (O–
N = 2.913–2.973 Å) were considered as probable. Other interac-
Molecular docking was carried out by Gold 5.0.1 (CCDC, UK)
using the primary GoldScore function and ASP (Astex Statistical Po-
tential) function for re-scoring the docking results to produce the
optimal conformations for the docked ligands. Additionally, the
GoldScore values were used to formulate the QSAR theory between
the results of AChE inhibition studies and the structural parame-
ters. The majority of best ranked conformations for the applied li-
gands (18 out of 22, while other 2 results comply with the
proposed arrangement of ligands when using the second best
ASP score) are based on the same arrangement of the molecules
within the active site gorge characterized, similarly as reported
by Paz et al.,52 by insignificant direct interaction with the catalytic
triad. On the other hand, they are able to bind in the middle part of
the peripheral anionic site (PAS) by means of hydrogen bonds be-
tween the amide moiety (either by the NH group or the oxygen
tions, like p–p interaction between the benzothiazole moiety and
the Trp84 side chain (centroid–centroid ꢁ3.384 Å) and F–H–N/O
hydrogen bond formation, which was identified in our molecular
docking experiments between the fluorine heterocyclic substitu-
tion and Glu199 (F–O = 2.558–3.315 Å), Tyr130 (F–O = 2.626–
3.618 Å) or the NH moiety from the backbone of Ser124
(F–O = 2.880–3.408 Å), see Figure 7, could also play an important
role in positioning and stabilizing the ligands inside the active site
gorge. The low-energy conformations of all investigated com-
pounds confirmed that the non-polar parts of the molecules are
oriented to the outer part of the active site gorge. The graphical

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A. Imramovsky et al. / Bioorg. Med. Chem. 21 (2013) 1735–1748
Table 2
Cytotoxicity of tested compounds in HepG2 and MCF7 cells
Compd
IC50 (lmol/L)
CellTiter96 (HepG2)
RTCA SP (MCF7)
6d
6f
273
275
1213
612
6n
6m
1734
>5000
4393
>4393
determined for compound 6m, because the course of the toxicity
curve did not allow for a valid calculation of the IC50 value. A
graphical representation of this assay is shown in the supplemen-
tary data for this paper.
Figure 7. Example of interactions between docked molecule of 6c (coloured in
yellow and shown with solvent accessible surface) and amino acid residues from
active site gorge, within range of 3 Å. Interacting atoms from ligand and amino acid
residues are depicted and corresponding distances between non-hydrogen atoms
are shown.
The xCELLigence RTCA SP system was used for real-time and
time-dependent analysis of the cellular response of MCF7 cells. A
parameter termed Cell Index (CI) was derived to represent cell sta-
tus based on the measured electrical impedance. It was observed
that a reduction in the CI value occurred immediately after treat-
ment with 1000 and 2500
suggest that 6d, 6f, 6n and 6m, at the concentrations 1000 and
2500 mol/L, inhibit the proliferation of MCF7 cells. However,
treatment with 1000 mol/L of 6n and 6m and 2500 mol/L of
6m initially inhibited MCF7 cell proliferation, but the cells eventu-
ally recovered to the level of the vehicle control treated cells. MCF7
lmol/L of 6d, 6f, 6n and 6m. These data
model of the AChE molecule, involving the AChE inhibitors and
demonstrating the conformations of docked ligands within the ac-
tive site gorge, is shown in Figure 8. On the basis of such orienta-
tion and the set of interactions, which were identified between
the ligands and the protein residues within the gorge, it can be
hypothesized that the studied compounds could act as ‘bulky’-
blockers the entrance of the normal ionic substrate (ACh) into
the active site gorge.
l
l
l
cells treated at lower concentrations (0.01–100 lmol/L) of investi-
gated compounds 6d, 6f, 6n and 6m proliferated in parallel to cells
treated with the vehicle control, as indicated by an increase in the
CI values. These results correlate with the data obtained from the
MTS assay. However, the IC50 values of MCF7 cells (Table 2) were
higher than those of HepG2 cells, suggesting the selective cytotox-
icity of 6d, 6f, 6n and 6m towards HepG2 cells. The course of a
measurement for each compound is shown in the separate part
of the Supplementary data section.
6. In vitro cytotoxicity assay
To evaluate the potential harmful effects of the tested com-
pounds on human cells, a standard in vitro method using the hu-
man liver cell line HepG2 was used. Representative members of
the investigated group (6d, 6f, 6m and 6n) were tested for their
in vitro cytotoxicity. It was observed that 6d and 6f at concentra-
7. Conclusion
tions of >10
only at concentrations of >1000
the growth of HepG2 cells. Compounds 6d and 6f at concentrations
ranging from 0.001 to 10 mol/L did not exhibit a dose-dependent
growth inhibitory effect. Similarly, 6n at concentrations
<100 mol/L and 6m even at concentrations <1000 mol/L showed
l
mol/L, 6n at concentrations >100
lmol/L and 6m
lmol/L were effective in inhibiting
In this study, twenty-two original derivates of (R)-1-(6-fluoro
benzo[d]thiazol-2-yl)ethanamine were designed, synthesised and
fully characterised. The synthetic pathway was quick and effective.
All the compounds were evaluated in vitro for their ability to inhi-
bit acetylcholinesterase and butyrylcholinesterase. The anticholin-
esterase activities were compared with those of rivastigmine and
galanthamine as the standards. Isobutyl-(S)-1-[(R)-1-(6-fluor-
obenzo[d]thiazol-2-yl)ethylcarbamoyl]butylcarbamate (6f) and
isopropyl-(S)-1-[(R)-1-(6-fluoro benzo[d]thiazol-2-yl)ethylcarba-
moyl]butylcarbamate (6d) expressed the highest AChE-inhibiting
l
l
l
dramatically decreased efficacy against the growth of HepG2 cells.
To evaluate the effect of 6d, 6f, 6n and 6m on the cell viability of
HepG2 cells, the MTS assay was performed. Cytotoxicity was also
expressed as IC50, that is, the concentration at which the viability
of cells decreased to 50% of the maximal viability (Table 2). The
IC50 was determined for 6d, 6f and 6n. Only an estimate could be
Figure 8. Schematic visualization of basic parts of AChE active site gorge, showing catalytic triad (green), oxanion hole (yellow), acyl binding site (orange), anionic substrate
binding site (blue) and highest ranked conformations of studied compounds with solvent-accessible surface (grey). Insert (on the right) contains enlarged view at active site
part of AChE molecule.

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A. Imramovsky et al. / Bioorg. Med. Chem. 21 (2013) 1735–1748
activity, while ethyl-(S)-1-[(R)-1-(6-fluoro benzo[d]thiazol-2-
yl)ethylcarbamoyl]butylcarbamate (6b) showed the highest BChE
inhibition. The cytotoxicity of the selected compounds was deter-
mined using HepG2 and MCF7 cells. The tested compounds
showed low cytotoxicity. It can be assumed that R1 substitution
with an n-propyl chain is more advantageous for high AChE/BChE
inhibition. R2 substitution with a branched long-chain alkyl is cru-
cial for potent AChE inhibition, while R2 substitution by a short un-
branched alkyl chain seems to be preferred for potent BChE
inhibition. AChE/BChE inhibition was especially dependent on the
Taft constants. In the case of halogenated R2 chains, R1 substitution
seemed to be not as important, and it seemed that the CCl3 moiety
was a universal non-selective substituent of carbamate esters for
AChE/BChE inhibition. Based on the comprehensive docking study,
it can be proposed that the studied compounds could act as ‘bulky’
blockers of ACh entry into the active site cleft. Although primary
screening of the discussed compounds confirmed medium or
rather moderate activity, these noteworthy compounds surely de-
serve further attention, because their chemical analogues and
alterations of their physico-chemical properties may confer some
positive changes in terms of biological effect.
reaction mixture. Temperature was kept between 4 and 12 °C, and
addition of a base was accomplished over a 30 min period. Reac-
tion 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. An appropriate chlo-
roformate was obtained by fractional distillation as a colourless
liquid.
8.1.3. Characterization of prepared chloroformates
8.1.3.1. Characterization data of 2,2,2-trifluoroethyl chlorofor-
mate 2a.
Colourless liquid; yield 76%; bp 75–76 °C. 1H NMR
(CDCl3) d = 4.63 (q, 2H, 3J(1H,19F) = 8). 13C NMR (CDCl3) d = 150.6,
121.9 (q, 1J(19F,13C) = 280.8 Hz), 65.5 (q, 2J(19F,13C) = 37.8 Hz). 19F
NMR (CDCl3) d = ꢀ74.7.
8.1.3.2. Characterization data of 2,2,3,3-tetrafluoropropyl chlo-
roformate 2b.
Colourless liquid; yield 78.0%; bp 112–113 °C.
1H NMR (CDCl3) d = 5.92 (tt, 1H, 2J(1H,19F) = 53.2, 3J(1H,19F) = 3.2, H-
3), 4.67 (tt, 2H, 3J(1H,19F) = 12.4, 4J(1H,19F) = 1.2, H-1). 13C NMR
(CDCl3) d = 150.5, 113.3 (tt, 1J(19F,13C) = 250.2 Hz, 2J(19F,13C) =
28.5 Hz), 109.1 (tt, 1J(19F,13C) = 251.5 Hz, 2J(19F,13C) = 38.1 Hz),
64.8 (t, 2J(19F,13C) = 29.3 Hz). 19F NMR (CDCl3) d = ꢀ123.7 (t,
3J(19F,19F) = 3.8, F-2), -137.3 (t, 3J(19F,19F) = 3.8, F-3).
8. Experimental
8.1. Chemistry
8.1.1. Instrumentation and chemicals
8.1.3.3. Characterization data of 2,2,3,3,4,4,4-heptafluorobutyl
All reagents and solvents were purchased from commercial
sources (Sigma–Aldrich, Merck, Acros Organics). Phosgene was
purchased from Synthesia (Pardubice, Czech Republic). Fluorinated
alcohols were purchased from Fluorochem UK. Commercial grade
reagents were used without further purification. Reactions were
monitored by thin layer chromatography plates coated with
0.2 mm silica gel 60 F254 (Merck). TLC plates were visualized by
the UV irradiation (254 nm).
chloroformate 2a.
Colourless liquid; yield 82%; bp 108–
109 °C. 1H NMR (CDCl3) d = 4.75 (tt, 3J(1H,19F) = 12.8, 4J(1H,19F) =
1.2). 13C NMR (CDCl3) d = 150.6, 117.4 (qt, 1J(19F,13C) = 287.0 Hz,
2J(19F, 13C) = 32.6 Hz), 113.1 (tt, 1J(19F,13C) = 258.7 Hz, 2J(19F,
13C) = 30.4 Hz), 108.5 (tq, 1J(19F, 13C) = 263.3 Hz, 2J(19F,13C) =
38.4 Hz), 64.4 (t, 2J(19F, 13C) = 26.5 Hz). 19F NMR (CDCl3) d = ꢀ81.0
(t, 4J(19F, 19F) = 11.3, F-4), ꢀ120.7–120.89 (m, F-2), ꢀ127.7–127.8
(m, F-3).
All melting points were determined on a Melting Point B-545
apparatus (Buchi, Switzerland) and are uncorrected. Infrared spec-
tra (KBr pellets) were recorded on FT-IR spectrometer Nicolet 6700
FT-IR in the range of 400–4000 cmꢀ1. The NMR spectra were mea-
sured in DMSO-d6 solutions at ambient temperature on a Bruker
Avance III 400 spectrometer (400 MHz for 1H, 100 MHz for 13C
and 376.5 MHz for 19F). Coupling constants are expressed in Hz.
Proton chemical shifts in CDCl3 are related to the internal TMS
(d = 0.0) and in DMSO-d6 to the middle of the solvent multiplet
(d = 2.50). 13C NMR spectra were measured using APT pulse se-
8.1.4. General experimental procedure for the synthesis of
compounds 6a-6w
Amino acid 1 (8.65 mmol) was dissolved in 2.1 mL of distilled
water and 2.4 g of 23% water solution of NaOH. The mixture was
stirred for 30 min, and during this time it was cooled under
10 °C. Chloroformate 2 (13.63 mmol) and 2.4 g of 23% water solu-
tion of NaOH were added concurrently to the amino acid sodium
salt solution for 15 min. Reaction mixture was then stirred for
45 min at 10 °C. After that, the pH of the reaction mixture was ad-
justed with HCl (approx. 2.4 g of 10% water solution). Product 3
was formed, and it was used directly in the next step of synthesis.
Toluene (20 mL) and dimethyl benzylamine (DMBA,
0.155 mmol) were added to the reaction mixture at temperature
under 10 °C. Isobutyl chloroformate (8.60 mmoL) was added to
the reaction mixture for 15 min. Reaction mixture was then heated
to 25 °C and stirred for 45 min. Distilled water (35 mL) was added
to the reaction mixture, and the toluene layer was separated. Prod-
uct 4 was formed in the organic layer.
quence optimized to (
1J 13C, 1H) = 145 Hz. Carbon chemical shifts
are referenced to the middle of the solvent’s multiplet (d = 77.0
or 39.5 in CDCl3 or DMSO-d6, respectively). Fluorine-19 NMR spec-
tra were measured using Waltz-16 proton decoupling and were
standardised against fluorobenzene as the secondary external
standard (d = ꢀ113.1 against CFCl3 as the primary standard44) td
64 k zero filled to 128 k. Chemical shifts for the AB systems were
calculated as weighed average of the line positions weighted by
the peak intensities. Elemental analyses (C, H, N) were performed
on an automatic microanalyser EA1110CE (Fisons Instruments,
Milano, Italy). High resolution mass spectra were measured using
a LTQ Orbitrap Hybrid Mass Spectrometer (Thermo Electron Corpo-
ration, USA) with direct injection into APCI source (400 °C) in the
positive mode.
To the separated organic phase toluene (20 mL) and p-toluene
sulfonate salt of (R)-1-(6-fluorobenzo[d]thiazole)ethanamine
5
(8.60 mmol) were added. A solution of sodium hydroxide was
added dropwise to the reaction mixture to reach pH 9.7 (approx.
4.5 g of 10% solution). Reaction mixture was stirred for 5 h at room
temperature. The pH was maintained on 9.7 with addition of a so-
dium hydroxide solution.
8.1.2. General experimental procedure for synthesis of
chloroformates 242
Phosgene (0.5 mol) was condensed in anhydrous diethyl ether
(150 mL) at ice bath temperature (T <10 °C). A selected alcohol
(0.2 mol) was added to diethyl ether solution for 15 min. An anhy-
drous triethylamine or DIEA (0.22 mol) was added dropwise to the
After that the isolation of final product 6 followed. Two isolation
techniques were used for isolation of desired products:
8.1.4.1. Isolation method A.
This method was used for exper-
iments where a solid compound precipitated during stirring of the

´
A. Imramovsky et al. / Bioorg. Med. Chem. 21 (2013) 1735–1748
1742
reaction mixture. The crude product was filtered off by vacuum fil-
tration. Crystals were dissolved in hot chloroform, and non-soluble
impurities (PTS) were filtered off by simple hot filtration. The clear
solution was evaporated till dryness. The residue was crystallized
using a chloroform/n-hexane mixture.
1H) = 9.1 Hz, H5), 7.22 (1H, d, J = 8.2 Hz, NH-COOMe), 5.24 (1H,
kv, J = 7.2 Hz, H8), 4.05 (1H, m, H10), 3.90 (2H, t, J = 6.3 Hz, H14),
1.56 (7H, m, H9,H11, H15), 1.34 (2H, m, H12), 0.88 (6H, m, H13,
H16). 13C NMR (100.62 MHz, DMSO-d6):
d
175.5 (d, 4J(19F,
13C) = 3.2 Hz), 172.3, 159.5 (d, 1J(19F,13C) = 240.9 Hz), 156.2, 149.6,
135.9 (d, 3J(19F, 13C) = 11.9 Hz), 123.7 (d, 3J(19F, 13C) = 9.5 Hz),
114.5 (d, 2J(19F,13C) = 24.9 Hz), 108.6 (d, 2J(19F,13C) = 27.3 Hz),
65.4, 54.3, 47.4, 34.0, 22.0, 20.1, 18.8, 13.6, 10.3. 19F NMR
(376.46 MHz, DMSO-d6): d -116.6. IR (ATR): 3282, 2949, 2927,
1680 (CO—carbamate), 1649 (CO—amide), 1537, 1455, 1381,
8.1.4.2. Isolation method B.
This method was used for exper-
iments where a solid compound did not precipitated from the reac-
tion mixture. Distilled water was added to the reaction mixture
and heated to 60–70 °C, then the toluene layer was separated,
cooled down and dried with sodium sulphate. The Na2SO4 was col-
lected by filtration, toluene was partially evaporated under vac-
uum, and the mixture was left for crystallization at 4 °C
overnight. Products were filtered off and dried under vacuum at
room temperature. The residue was crystallized using a chloro-
form/n-hexane mixture
1276, 1259, 1136, 1066, 926, 907, 854, 821, 780, 745, 677 cmꢀ1
.
Anal. Calcd for C18H24FN3O3S (381.15): C, 56.67; H, 6.34; N,
11.02. Found: C, 56.77; H, 6.43; N, 11.15. HR-MS: for C18H25FN3O3S
[M+H+] calcd 382.1595 m/z, found 382.1591 m/z.
8.1.5.4. Isopropyl-(S)-1-[(R)-1-(6-fluorobenzo[d]thiazole)ethylc-
arbamoyl] butylcarbamate 6d.
lation method A); mp 178–178.5 °C. 1H NMR (400.13 MHz,
DMSO-d6): 8.80 (1H, d, J = 7.8 Hz, NH-CO), 7.99 (1H, dd,
White solid; Yield 88.1% (iso-
8.1.5. Characterization data of prepared compounds 6a–6w
8.1.5.1. Methyl-(S)-1-[(R)-1-(6-fluorobenzo[d]thiazole)ethylcar-
d
bamoyl]butylcarbamate 6a.
(isolation method A); mp 180–181 °C. 1H NMR (400.13 MHz,
DMSO-d6): 8.85 (1H, d, J = 7.6 Hz, NH-CO), 7.99 (1H, dd,
White solid; yield 45.7%
4J = 2.6 Hz, 3J(19F, 1H) = 8.9 Hz, H7), 7.97 (1H, dd, J = 8.9 Hz, 4J(19F,
1H) = 4.7 Hz, H4), 7.36 (1H, dt, J = 2.6 Hz, J = 9.1 Hz, 3J(19F,
1H) = 9.1 Hz, H5), 7.11 (1H, d, J = 8.2 Hz, NH-COOMe), 5.24 (1H,
kv, J = 7.2 Hz, H8), 4.74 (1H, m, J = 6.3 Hz, H14), 4.03 (1H, m,
H10), 1.59 (2H, m, H11), 1.55 (3H, d, J = 7.2 Hz, H9), 1.34 (2H, m,
H12), 1.16 (6H, d, J = 6.2 Hz, H15), 0.88 (3H, m, H13). 13C NMR
(100.62 MHz, DMSO-d6): d 175.7 (d, 4J(19F, 13C) = 3.5 Hz), 172.3,
159.5 (d, 1J(19F,13C) = 240.9 Hz), 155.7, 149.6, 135.9 (d, 3J(19F,
13C) = 11.7 Hz), 123.7 (d, 3J(19F, 13C) = 9.6 Hz), 114.5 (d,
2J(19F,13C) = 24.8 Hz), 108.6 (d, 2J(19F,13C) = 26.9 Hz), 67.0, 54.3,
47.4, 34.0, 22.0, 20.1, 18.8, 13.6. 19F NMR (376.46 MHz, DMSO-
d6): d -116.6. IR (ATR): 3300, 3267, 2971, 2940, 1678 (CO—carba-
mate), 1644 (CO—amide), 1568, 1537, 1457, 1385, 1277, 1263,
1239, 1218, 1195, 1180, 1112, 1062, 1041, 1024, 960, 847,
820 cmꢀ1. Anal. Calcd for C18H24FN3O3S (381.152): C, 56.67; H,
6.34; N, 11.02. Found: C, 56.80; H, 6.57; N, 11.31. HR-MS: for
d
4J = 2.6 Hz, 3J(19F, 1H) = 8.9 Hz, H7), 7.97 (1H, dd, J = 8.9 Hz, 4J(19F,
1H) = 4.9 Hz, H4), 7.36 (1H, dt, J = 2.6 Hz, J = 9.1 Hz, 3J(19F,
1H) = 9.1 Hz, H5), 7.30 (1H, d, J = 8.3 Hz, NH-COOMe), 5.23 (1H,
kv, J = 7.2 Hz, H8), 4.05 (1H, m, H10), 3.53 (3H, s, H14), 1.59 (2H,
m, H11), 1.55 (3H, d, J = 7.2 Hz, H9), 1.34 (2H, m, H12), 0.88 (3H,
m, H13). 13C NMR (100.62 MHz, DMSO-d6): d 175.7 (d, 4J(19F,
13C) = 3.2 Hz), 172.2, 159.5 (d, 1J(19F,13C) = 240.9 Hz), 156.5, 149.6
(d 5J(19F, 13C) = 1.5 Hz), 135.9 (d, 3J(19F, 13C) = 11.9 Hz), 123.7 (d,
3J(19F, 13C) = 9.3 Hz), 114.5 (d, 2J(19F,13C) = 25.4 Hz), 108.6 (d,
2J(19F,13C) = 27.1 Hz), 54.4, 51.5, 47.4, 34.1, 20.6, 18.7, 13.7. 19F
NMR (376.46 MHz, DMSO-d6): d ꢀ116.6. IR (ATR): 3273, 3287,
3006, 2928, 1691 (CO—carbamate), 1647 (CO—amide), 1541,
1457, 1264, 1236, 1196, 1059, 965, 918, 850, 824, 797, 755, 701,
689 cmꢀ1. Anal. Calcd for C16H20FN3O3S (353.121): C, 54.38; H,
5.70; N, 11.89. Found: C, 54.69; H, 6.02; N, 11.91. HR-MS: for
C
18H25FN3O3S [M+H+] calcd 382.1595 m/z, found 382.1594 m/z.
C
16H21FN3O3S [M+H+] calcd 354.1282 m/z, found 354.1282 m/z.
8.1.5.5. Butyl (S)-1-[(R)-1-(6-fluorobenzo[d]thiazole)ethylcarba-
moyl] butylcarbamate 6e. White solid; yield 72.3% (isolation
8.1.5.2. Ethyl-(S)-1-[(R)-1-(6-fluorobenzo[d]thiazole)ethylcar-
bamoyl] butylcarbamate 6b. White solid; yield 56.7% (isola-
method A); mp 177.5–178.5 °C. 1H NMR (400.13 MHz, DMSO-d6): d
8.82 (1H, d, J = 7.5 Hz, NH-CO), 7.99 (1H, dd, 4J = 2.6 Hz, 3J(19F,
1H) = 9.1 Hz, H7), 7.97 (1H, dd, J = 9.1 Hz, 4J(19F, 1H) = 4.9 Hz, H4),
7.37 (1H, dt, J = 2.7 Hz, J = 9.1 Hz, 3J(19F, 1H) = 9.1 Hz, H5), 7.22
(1H, d, J = 8.2 Hz, NH-COOMe), 5.24 (1H, kv, J = 7.2 Hz, H8), 4.05
(1H, m, H10), 3.90 (2H, t, J = 6.3 Hz, H14), 1.56 (7H, m, H9,H11,
H15), 1.34 (2H, m, H12), 0.88 (6H, m, H13, H16). 13C NMR
(100.62 MHz, DMSO-d6): d 175.5 (d, 4J(19F, 13C) = 3.2 Hz), 172.3,
159.5 (d, 1J(19F,13C) = 240.9 Hz), 156.2, 149.6, 135.9 (d, 3J(19F,
13C) = 11.9 Hz), 123.7 (d, 3J(19F, 13C) = 9.5 Hz), 114.5 (d,
2J(19F,13C) = 24.9 Hz), 108.6 (d, 2J(19F,13C) = 27.3 Hz), 65.4, 54.3,
47.4, 34.0, 22.0, 20.1, 18.8, 13.6, 10.3. 19F NMR (376.46 MHz,
DMSO-d6): d ꢀ116.6. IR (ATR): 3282, 2949, 2927, 1680 (CO—carba-
mate), 1649 (CO—amide), 1537, 1455, 1381, 1276, 1259, 1136,
1066, 926, 907, 854, 821, 780, 745, 677 cmꢀ1. Anal. Calcd for
tion method A); mp 182–184 °C. 1H NMR (400.13 MHz, DMSO-d6):
d 8.82 (1H, d, J = 7.7 Hz, NH-CO), 7.99 (1H, dd, 4J = 2.6 Hz, 3J(19F,
1H) = 8.9 Hz, H7), 7.97 (1H, dd, J = 8.9 Hz, 4J(19F, 1H) = 4.9 Hz, H4),
7.36 (1H, dt, J = 2.8 Hz, J = 9.1 Hz, 3J(19F, 1H) = 9.1 Hz, H5), 7.22
(1H, d, J = 8.1 Hz, NH-COOMe), 5.23 (1H, kv, J = 7.2 Hz, H8), 4.00
(3H, m, H10, H14), 1.59 (2H, m, H11), 1.55 (3H, d, J = 7.2 Hz, H9),
1.51 (2H, m, H15), 1.34 (2H, m, H12), 0.88 (3H, m, H13). 13C
NMR (100.62 MHz, DMSO-d6): d 175.7 (d, 4J(19F, 13C) = 3.4 Hz),
172.3, 159.5 (d, 1J(19F,13C) = 242.7 Hz), 156.1, 149.6, 135.9 (d,
3J(19F, 13C) = 11.6 Hz), 123.7 (d, 3J(19F, 13C) = 9.6 Hz), 114.5 (d,
2J(19F,13C) = 24.8 Hz), 108.6 (d, 2J(19F,13C) = 27.4 Hz), 59.8, 54.3,
47.4, 34.0, 20.1, 18.8, 14.6, 13.6. 19F NMR (376.46 MHz, DMSO-
d6): d ꢀ116.6. IR (ATR): 3274, 3255, 1684 (CO—carbamate), 1648
(CO—amide), 1540, 1457, 1373, 1276, 1259, 1238, 1150, 1019,
898, 846, 821, 668 cmꢀ1. Anal. Calcd for C17H22FN3O3S (367.137):
C, 55.57; H, 6.03; N, 11.44. Found: C, 55.69; H, 6.08; N, 11.78.
C
18H24FN3O3S (381.15): C, 56.67; H, 6.34; N, 11.02. Found: C,
56.77; H, 6.43; N, 11.15. HR-MS: for C18H25FN3O3S [M+H+] calcd
382.1595 m/z, found 382.1591 m/z.
HR-MS: for
368.1440 m/z.
C
17H23FN3O3S [M+H+] calcd 368.1439 m/z, found
8.1.5.6. Isobutyl- (S)-1-[(R)-1-(6-fluorobenzo[d]thiazole)ethylc-
arbamoyl] butylcarbamate 6f.
White solid; Yield 52.1%
8.1.5.3. Propyl-(S)-1-[(R)-1-(6-fluorobenzo[d]thiazole)ethylcar-
bamoyl] butylcarbamate 6c. White solid; yield 61.8%
(isolation method A); mp 177.5–178.5 °C. 1H NMR (400.13 MHz,
(isolation method A); mp 134-135 °C. 1H NMR (400.13 MHz,
DMSO-d6):
d 8.81 (1H, d, J = 7.6 Hz, NH-CO), 7.99 (1H, dd,
4
J = 2.7 Hz, 3J(19F, 1H) = 9.1 Hz, H7), 7.97 (1H, dd, J = 8.9 Hz, 4J(19F,
1H) = 5.0 Hz, H4), 7.36 (1H, dt, J = 2.7 Hz, J = 9.1 Hz, 3J(19F,
1H) = 9.1 Hz, H5), 7.22 (1H, d, J = 8.2 Hz, NH-COOMe), 5.23 (1H,
kv, J = 7.2 Hz, H8), 4.02 (1H, m, H10), 3.72 (2H, d, J = 6.3 Hz, H14),
DMSO-d6):
d 8.82 (1H, d, J = 7.5 Hz, NH-CO), 7.99 (1H, dd,
4J = 2.6 Hz, 3J(19F, 1H) = 9.1 Hz, H7), 7.97 (1H, dd, J = 9.1 Hz, 4J(19F,
1H) = 4.9 Hz, H4), 7.37 (1H, dt, J = 2.7 Hz, J = 9.1 Hz, 3J(19F,

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A. Imramovsky et al. / Bioorg. Med. Chem. 21 (2013) 1735–1748
1.82 (2H, m, J = 6.6 Hz, H15), 1.58 (2H, m, H11), 1.54 (3H, d,
J = 7.2 Hz, H9), 1.33 (2H, m, H12), 0.87 (9H, m, H13, H16). 13C
NMR (100.62 MHz, DMSO-d6): d 175.7 (d, 4J(19F, 13C) = 3.5 Hz),
172.3, 159.5 (d, 1J(19F,13C) = 242.6 Hz), 156.2, 149.6, 135.9 (d,
3J(19F, 13C) = 11.7 Hz), 123.7 (d, 3J(19F, 13C) = 9.8 Hz), 114.5 (d,
2J(19F,13C) = 25.0 Hz), 108.6 (d, 2J(19F,13C) = 27.0 Hz), 69.8, 54.3,
47.4, 34.0, 27.7, 20.1, 19.9, 18.8, 13.6. 19F NMR (376.46 MHz,
DMSO-d6): d -116.6 IR (ATR): 3297, 3233, 1683 (CO—carbamate),
1648 (CO—amide), 1542, 1397, 1275, 1363, 1237, 1148, 1068,
985, 917, 845, 785, 667 cmꢀ1. Anal. Calcd for C19H26FN3O3S
(395.168): C, 57.70; H, 6.63; N, 10.62. Found: C, 57.87; H, 6.43;
N, 10.80. HR-MS: for C19H27FN3O3S [M+H+] calcd 396.1752 m/z,
found 396.1752 m/z.
J = 2.6 Hz, J = 9.1 Hz, 3J(19F, 1H) = 9.1 Hz, H5), 5.24 (1H, kv,
J = 7.2 Hz, H8), 4.65 (2H, m, H14), 4.07 (1H, m, H10), 1.62 (2H, m,
H11), 1.56 (3H, d, J = 7.0 Hz, H9), 1.33 (2H, m, H12), 0.90 (3H, t,
J = 7.2 Hz, H13). 13C NMR (100.62 MHz, DMSO-d6): d 175.6 (d,
4J(19F, 13C) = 2.8 Hz), 171.7, 159.5 (d, 1J(19F,13C) = 242.2 Hz), 154.3,
149.6,
135.9
(d,
3J(19F,
13C) = 11.4 Hz),
123.8
(tt,
1J(19F,13C) = 277.9 Hz), 123.7 (d, 3J(19F, 13C) = 9.4 Hz), 114.5 (d,
2J(19F,13C) = 24.5 Hz), 108.6 (d, 2J(19F,13C) = 27.0 Hz), 59.7 (q,
2J(19F,13C) = 34.4 Hz), 54.6, 47.4, 33.8, 20.1, 18.8, 13.5. 19F NMR
(376.46 MHz, DMSO-d6): d ꢀ72.7 (s, CF3), -116.6 (s, Ar-F). IR
(ATR): 3306, 3063, 2950, 1698 (CO—carbamate), 1651 (CO—
amide), 1541, 1459, 1283, 1256, 1237, 1168, 957, 858, 822, 771,
683 cmꢀ1. Anal. Calcd for C17H19F4N3O3S (421,108): C, 48.45; H,
4.54; N, 9.97. Found: C, 48.76; H, 4.60; N, 10.16. HR-MS: for
C
17H20F4N3O3S [M+H+] calcd 422.1157 m/z, found 422.1157 m/z.
8.1.5.7.
2-Chloroethyl-(S)-1-[(R)-1-(6-fluorobenzo[d]thia-
White solid; yield
zole)ethylcarbamoyl] butylcarbamate 6g.
32.8% (isolation method B); mp 162.5–163 °C. 1H NMR
(400.13 MHz, DMSO-d6): d 8.83 (1H, d, J = 7.6 Hz, NH-CO), 7.98
(1H, dd, 4J = 2.6 Hz, 3J(19F, 1H) = 9.1 Hz, H7), 7.97 (1H, dd,
J = 9.1 Hz, 4J(19F, 1H) = 4.7 Hz, H4), 7.48 (1H, d, J = 8.3 Hz, NH-
COOMe), 7.36 (1H, dt, J = 2.7 Hz, J = 8.9 Hz, 3J(19F, 1H) = 8.9 Hz,
H5), 5.24 (1H, kv, J = 7.2 Hz, H8), 4.21 (2H, m, H15), 4.05 (1H, m,
H10), 3.78 (2H, t, J = 5.2 Hz, H14), 1.60 (2H, m, H11), 1.56 (3H, d,
J = 7.2 Hz, H9), 1.34 (2H, m, H12), 0.90 (3H, t, J = 7.3 Hz, H13). 13C
NMR (100.62 MHz, DMSO-d6): d 175.7 (d, 4J(19F, 13C) = 3.5 Hz),
172.1, 159.5 (d, 1J(19F,13C) = 240.1 Hz), 155.6, 149.6, 135.9 (d,
3J(19F, 13C) = 11.6 Hz), 123.7 (d, 3J(19F, 13C) = 9.5 Hz), 114.5 (d,
2J(19F,13C) = 24.8 Hz), 108.5 (d, 2J(19F,13C) = 27.0 Hz), 64.2, 54.4,
47.4, 43.1, 33.9, 20.1, 18.8, 13.6. 19F NMR (376.46 MHz, DMSO-
d6): d -116.6. IR (ATR): 3271, 2965, 1689 (CO—carbamate), 1646
(CO—amide), 1539, 1483, 1399, 1376, 1276, 1238, 1197, 1149,
1132, 1108, 1050, 918, 853, 823, 685, 665 cmꢀ1. Anal. Calcd for
8.1.5.10.
obenzo[d]thiazole)ethylcarbamoyl]butylcarbamate
6j. White solid; yield 58% (isolation method A); mp 163–
2,2,3,3-Tetrafluoropropyl
(S)-1-[(R)-1-(6-fluor-
165 °C. 1H NMR (400.13 MHz, DMSO-d6): d 8.9 (1H, d, J = 7.7 Hz,
NH-CO), 8.0 (1H, dd, J = 8,8 Hz, 4J(19F, 1H) = 4.7 Hz, H4), 7.98 (H,
dd, 4J = 3.0 Hz, 3J(19F, 1H) = 8.8 Hz, H7), 7.8 (1H, d, J = 8.2 Hz, NH-
COOMe),7.37 (1H, dt, J = 2.6 Hz, J = 9.0 Hz, 3J(19F, 1H) = 9.0 Hz,
H5), 6.5 (1H, t, 2J(19F, 1H) = 52.3 Hz, 3J(19F, 1H) = 5.3 Hz, H15), 5.2
(1H, kv, J = 7.2 Hz, H8), 4.5 (2H, m, 3J(19F, 1H) = 14.1 Hz, H14), 4.1
(1H, m, H10), 1.6 (2H, m, H11), 1.5 (3H, d, J = 7.2 Hz, H9), 1.3
(2H, m, H12), 0.9 (3H, t, J = 7.3 Hz, H13). 13C NMR (100.62 MHz,
DMSO-d6):
d
175.6 (d, 4J(19F, 13C) = 2.8 Hz), 171.7, 159.5 (d,
1J(19F,13C) = 242.0 Hz), 154.5, 149.6, 135.9 (d, 3J(19F, 13C) =
11.7 Hz), 123.7 (d, 3J(19F, 13C) = 9.6 Hz), 114.8 (tt, 1J(19F,13C) =
249.3 Hz, 2J(19F,13C) = 25.1 Hz), 114.6 (d, 2J(19F,13C) = 24.8 Hz),
109.3 (1J(19F,13C) = 248.4 Hz, 2J(19F,13C) = 33.1 Hz), 108.5 (d,
2J(19F,13C) = 27.1 Hz), 59.5 (t, 2J(19F,13C) = 26.7 Hz), 54.6, 47.4,
33.9, 20.1, 18.7, 13.5. 19F NMR (376.46 MHz, DMSO-d6): d ꢀ116.6,
ꢀ124.9 (2F,dt, 2J(19F, 1H) = 40.7 Hz, 3J(19F, 1H) = 5.5 Hz)), ꢀ138.8
(2F,dt, 2J(19F, 1H) = 22.7 Hz, 3J(19F, 1H) = 5.5 Hz)). IR (ATR): 3306,
3064, 2950, 1698 (CO—carbamate), 1646 (CO—amide), 1607,
1551, 1456, 1394, 1274, 1239, 1110, 993, 825, cmꢀ1. Anal. Calcd
for C18H20F5N3O3S (453,4267): C, 47.68; H, 4.45; N, 9.27. Found:
C, 47.76; H, 4.47; N, 9.30. HR-MS: for C18H21F5N3O3S [M+H+] calcd
454.1218 m/z, found 454.1221 m/z.
C
17H21ClFN3O3S (401.098): C, 50.81; H, 5.27; N, 10.46. Found: C,
51.05; H, 5.39; N, 10.57. HR-MS: for C17H22ClFN3O3S [M+H+] calcd
402.1049 m/z, found 402.1048 m/z.
8.1.5.8. 2,2,2-Trichloroethyl-(S)-1-[(R)-1-(6-fluorobenzo[d]thia-
zole)ethylcarbamoyl]butylcarbamate 6h.
White solid; yield
41.2% (isolation method A); mp 116.4–117.0 °C. 1H NMR
(400.13 MHz, DMSO-d6): d 8.89 (1H, d, J = 7.3 Hz, NH-CO), 7.99
(1H, dd, 4J = 2.5 Hz, 3J(19F, 1H) = 8.9 Hz, H7), 7.97 (1H, dd,
J = 8.9 Hz, 4J(19F, 1H) = 4.7 Hz, H4), 7.91 (1H, d, J = 8.2 Hz, NH-
COOMe), 7.37 (1H, dt, J = 2.6 Hz, J = 8.9 Hz, 3J(19F, 1H) = 8.9 Hz,
H5), 5.24 (1H, kv, J = 7.2 Hz, H8), 4.82 (2H, m, H14), 4.09 (1H, m,
H10), 1.62 (2H, m, H11), 1.56 (3H, d, J = 7.2 Hz, H9), 1.36 (2H, m,
H12), 0.89 (3H, t, J = 7.3 Hz, H13). 13C NMR (100.62 MHz, DMSO-
8.1.5.11. 2,2,3,3,4,4,4-Heptafluorobutyl (S)-1-[(R)-1-(6-fluor-
obenzo[d]thiazole)ethylcarbamoyl]butylcarbamate
6k.
White solid; yield 68% (isolation method A); mp 125–
126 °C. 1H NMR (400.13 MHz, DMSO-d6): d 8.91 (1H, d, J = 7.6 Hz,
NH-CO), 7.98 (1H, dd, J = 9,2 Hz, 4J(19F, 1H) = 4.8 Hz, H4), 7.96 (H,
dd, 4J = 2.9 Hz, 3J(19F, 1H) = 8.9 Hz, H7), 7.93 (1H, d, J = 8.2 Hz, NH-
COOMe),7.36 (1H, dt, J = 2.7 Hz, J = 9.1 Hz, 3J(19F, 1H) = 9.1 Hz,
H5), 5.24 (1H, kv, J = 7.2 Hz, H8), 4.78 (2H, m, 3J(19F,
1H) = 15.6 Hz, H14), 4.09 (1H, m, H10), 1.62 (2H, m, H11), 1.56
(3H, d, J = 7.0 Hz, H9), 1.35 (2H, m, H12), 0.90 (3H, t, J = 7.5 Hz,
d6):
d
175.5 (d, 4J(19F, 13C) = 3.0 Hz), 171.7, 159.5 (d,
154.4, 149.6, 135.9 (d,
3J(19F,
1J(19F,13C) = 242.6 Hz),
13C) = 11.7 Hz), 123.7 (d, 3J(19F, 13C) = 9.4 Hz), 114.5 (d,
2J(19F,13C) = 24.8 Hz), 108.5 (d, 2J(19F,13C) = 27.0 Hz), 96.3, 73.5,
54.7, 47.4, 33.8, 20.1, 18.8, 13.3. 19F NMR (376.46 MHz, DMSO-
d6): d -116.6. IR (ATR): 3336, 3311, 1710 (CO—carbamate), 1647
(CO—amide), 1566, 1528, 1453, 1375, 1251, 1226, 1197, 1162,
1133, 1108, 1086, 1046, 1022, 952, 911, 859, 822, 788, 767, 752,
H13). 13C NMR (100.62 MHz, DMSO-d6):
d
175.6 (d, 4J(19F,
13C) = 2.9 Hz), 171.7, 159.5 (d, 1J(19F,13C) = 241.4 Hz), 154.2, 149.6,
135.9 (d, 3J(19F, 13C) = 11.6 Hz), 123.7 (d, 3J(19F, 13C) = 9.6 Hz),
117.3 (qt, 1J(19F,13C) = 286.4 Hz, 2J(19F,13C) = 33.7 Hz), 114.5 (d,
2J(19F,13C) = 25.0 Hz), 114.4 (tt, 1J(19F,13C) = 254.1 Hz, 2J(19F,13C) =
30.0 Hz), 108.5 (d, 2J(19F,13C) = 27.2 Hz), 108.0 (tq, 1J(19F,13C) =
275.8.0 Hz, 2J(19F,13C) = 37.8 Hz), 58.7 (t, 2J(19F,13C) = 25.6 Hz),
54.7, 47.4, 33.7, 20.1, 18.7, 13.5. 19F NMR (376.46 MHz, DMSO-
d6): d ꢀ80.5 (t, 4J(19F,19F) = 9.5, CF3), ꢀ116.7 (s, Ar-F), ꢀ119.9
(brq, 3J(19F,19F) = 9.5, CH2CF2), ꢀ127.4 (s CF2). IR (ATR): 3312,
3269, 2966, 1706 (CO—carbamate), 1659 (CO—amide), 1543,
1456, 1234, 1216, 1175, 1150, 1020, 923, 846, 818, 761,
709 cmꢀ1. Anal. Calcd for C19H19F8N3O3S (521,102): C, 43.77; H,
716, 682 cmꢀ1
43.37; H, 4.07; N, 8.93. Found: C, 43.42; H, 4.20; N, 9.05. HR-MS:
for
17H20Cl3FN3O3S [M+H+] calcd 470.0269 m/z, found
. Anal. Calcd for C17H19Cl3FN3O3S (469.02): C,
C
470.0273 m/z.
8.1.5.9. 2,2,2-Trifluoroethyl (S)-1-[(R)-1-(6-fluorobenzo[d]thia-
zole)ethylcarbamoyl] butylcarbamate 6i.
White solid; yield
59% (isolation method A); mp 171–173 °C. 1H NMR (400.13 MHz,
DMSO-d6):
d 8.91 (1H, d, J = 7.7 Hz, NH-CO), 7.99 (1H, dd,
J = 9,0 Hz, 4J(19F, 1H) = 4.5 Hz, H4), 7.97 (H, dd, 4J = 2.4 Hz, 3J(19F,
1H) = 8.9 Hz, H7), 7.90 (1H, d, J = 8.1 Hz, NH-COOMe),7.37 (1H, dt,

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A. Imramovsky et al. / Bioorg. Med. Chem. 21 (2013) 1735–1748
1744
3.67; N, 8.06. Found: C, 44.00; H, 3.88; N, 8.24. HR-MS: for
19H20F8N3O3S [M+H+] calcd 522.1083 m/z, found 522.1096 m/z.
8.1.5.15. Isopropyl (S)-1-[(R)-1-(6-fluorobenzo[d]thiazol-2-yl)
ethylcarbamoyl]-2-methylpropylcarbamate 6o. White solid;
yield 93% (isolation method A); mp 166.9–168.0 °C. 1H NMR
(400.13 MHz, DMSO-d6): d 8.83 (1H, d, J = 7.2 Hz, NH-CO), 7.98
(1H, dd, J = 8.9 Hz, 4J(19F, 1H) = 4.8 Hz, H4),7.96 (1H, dd,
4J = 2.6 Hz, 3J(19F, 1H) = 8.9 Hz, H7), 7.36 (1H, dt, J = 2.6 Hz,
J = 9.1 Hz, 3J(19F, 1H) = 9.1 Hz, H5), 7.02 (1H, d, J = 8.9 Hz, NH-COO-
iPr), 5.27 (1H, kv, J = 7.2 Hz, H8), 4.75 (1H, m, J = 6.2 Hz, H11), 3.88
(1H, m, H10), 1.96 (1H, m, H13), 1.55 (3H, d, J = 7.2 Hz, H9), 1.16
(6H, d, J = 6.2 Hz, 2H, H14), 0.89 (3H, d, J = 6.5 Hz, H12), 0.87 (3H,
d, J = 6.5 Hz, H12). 13C NMR (100.62 MHz, DMSO-d6): d 175.5 (d,
4J(19F, 13C) = 3.0 Hz), 171.5, 159.5 (d, 1J(19F,13C) = 242.1 Hz), 155.9,
149.6, 135.9 (d, 3J(19F, 13C) = 11.9 Hz), 123.7 (d, 3J(19F,
13C) = 9.5 Hz), 114.5 (d, 2J(19F,13C) = 24.9 Hz), 108.5 (d,
2J(19F,13C) = 27.1 Hz), 67.0, 60.2, 47.2, 30.3, 22.0, 22.1, 20.1, 19.2,
18.4. 19F NMR (376.46 MHz, DMSO-d6): d ꢀ116.5. IR (ATR): 3288,
3264, 3061, 2975, 2928, 2871, 1686 (CO—carbamate), 1647 (CO—
amide), 1567, 1537, 1456, 1383, 1372, 1344, 1287, 1247, 1225,
1194, 1178, 1111, 1073, 1037, 919, 841, 824, 815, 801, 710, 679,
650, 603, 557, 538, 472, 431 cmꢀ1. Anal. Calcd for C18H24FN3O3S
(381.15): C, 56.67; H, 6.34; N, 11.02. Found: C, 56.91; H, 6.20; N,
10.95. HR-MS: for C18H25FN3O3S [M+H+] calcd 382.1595 m/z, found
382.1597 m/z.
C
8.1.5.12. Methyl (S)-1-[(R)-1-(6-fluorobenzo[d]thiazole)ethylc-
arbamoyl]-2-methylpropylcarbamate 6l. White solid; yield
26.1% (isolation method B); mp 188.8–189.1 °C. 1H NMR
(400.13 MHz, DMSO-d6): d 8.87 (1H, d, J = 7.2 Hz, NH-CO), 8.00
(1H, dd, 4J = 2.5 Hz, 3J(19F, 1H) = 8.8 Hz, H7), 7.97 (1H, dd,
3J = 8.9 Hz, 4J(19F, 1H) = 4.6 Hz, H4), 7.36 (1H, dt, 4J = 2.5 Hz,
3J = 9.1 Hz, 3J(19F, 1H) = 9.1 Hz, H5), 7.20 (1H, d, J = 8.9 Hz, NH-
COOMe), 5.28 (1H, kv, J = 6.8 Hz, H8), 3.91 (1H, t, J = 8.0 Hz, H10),
3.55 (3H, s, H14), 1.98 (1H, m, H11), 1.56 (3H, d, J = 7.2 Hz, H9),
0.89 (6H, d, H12). 13C NMR (100.62 MHz, DMSO-d6): d 177.5 (d,
4J(19F, 13C) = 3.4 Hz), 171.4, 159.5 (d, 1J(19F,13C) = 239.0 Hz), 156.7,
149.6 (d, 5J(19F, 13C) = 1.6 Hz), 135.9 (d, 3J(19F, 13C) = 11.8 Hz),
123.6 (d, 3J(19F, 13C) = 9.6 Hz), 114.5 (d, 2J(19F,13C) = 24.9 Hz),
108.6 (d, 2J(19F, 13C)) = 27.4 Hz), 60.2, 51.5, 47.2, 20.1, 19.2, 18.4.
19F NMR (376.46 MHz, DMSO-d6): d -116.56. IR (ATR): 3269,
3068, 2962, 2872, 1693 (CO—carbamate), 1650 (CO—amide),
1567, 1537, 1456, 1250, 1197, 1044 cmꢀ1
. Anal. Calcd for
C
16H20FN3O3S (353.121): C, 54.38; H, 5.70; N, 11.89. Found: C,
54.64; H, 5.78; N, 11.99. HR-MS: for C16H20FN3O3S [M+H+] calcd
354.1283 m/z, found 354.1282.
8.1.5.13. Ethyl (S)-1-[(R)-1-(6-fluorobenzo[d]thiazole)ethylcar-
8.1.5.16. Butyl (S)-1-[(R)-1-(6-fluorobenzo[d]thiazole)ethylcar-
bamoyl]-2-methylpropylcarbamate 6m.
White solid; yield
bamoyl]-2-methylpropylcarbamate 6p.
White solid; yield
35.8% (isolation method B); mp 188.8–189.1 °C. 1H NMR
(400.13 MHz, DMSO- d6): d 8.84 (1H, d, J = 7.6 Hz, NH-CO), 8.00
(1H dd, 4J = 2.6 Hz, 3J(19F, 1H) = 8.8 Hz, H7), 7.99 (1H, dd,
3J = 8.8 Hz, 4J(19F, 1H) = 4.9 Hz, H4), 7.37 (1H, dt, 4J = 2.5 Hz,
3J = 9.1 Hz, 3J(19F, 1H) = 9.1 Hz, H5), 7.12 (1H, d, J = 9.2 Hz, NH-
COOMe), 5.28 (1H, kv, J = 7.2 Hz, H8), 4.00 (2H, q, J = 7.2 Hz, H14),
3.90 (1H, t, J = 7.8 Hz, H10), 1.98 (1H, m, H11), 1.56 (3H, d,
J = 7.2 Hz, H9), 1.17 (3H, t, J = 7.2 Hz, H15), 0.89 (6H, t, H12). 13C
NMR (100.62 MHz, DMSO-d6): d 175.5 (d, 4J(19F, 13C) = 3.4 Hz),
171.4, 159.5 (d, 1J(19F,13C) = 242.0 Hz), 156.3, 149.6, 136.0 (d,
3J(19F, 13C) = 11.8 Hz), 123.7 (d, 3J(19F, 13C) = 9.5 Hz), 114.6 (d,
2J(19F,13C) = 24.8 Hz), 108.6 (d, 2J(19F, 13C)) = 27.0 Hz), 60.2, 59.9,
47.3, 30.3, 20.1, 19.2, 18.4, 14.7. 19F NMR (376.46 MHz, DMSO-
d6): d ꢀ116.57. IR (ATR): 3289, 3256, 3064, 2981, 2962, 2931,
2909, 2871, 1683 (CO carbamate), 1645 (CO amide), 1541, 1456,
27.8% (isolation method B); mp 152.4–153.1 °C. 1H NMR
(400.13 MHz, DMSO-d6): d 8.85 (1H, d, J = 7.6 Hz, NH-CO), 7.99
(1H, dd, 4J = 2.5 Hz, 3J(19F, 1H) = 8.9 Hz, H7), 7.97 (1H, dd,
3J = 8.8 Hz, 4J(19F, 1H) = 4.3 Hz, H4), 7.36 (1H, dt, 4J = 2.6 Hz,
3J = 9.1 Hz, 3J(19F, 1H) = 9.1 Hz, H5), 7.12 (1H, d, J = 8.8 Hz, NH-
COOMe), 5.28 (1H, kv, J = 7.2 Hz, H8), 3.96 (2H, t, J = 6.4 Hz, H14),
3.90 (1H, t, J = 8.4 Hz, H10), 1.99 (1H, m, H11), 1.56 (3H, d,
J = 7.2 Hz, H9), 1.52 (2H, m, H15), 1.33 (2H, m, H16), 0.88 (9H, m,
H12, H17). 13C NMR (100.62 MHz, DMSO-d6): d 177.5 (d, 4J(19F,
13C) = 3.3 Hz), 171.4, 159.5 (d, 1J(19F,13C) = 242.1 Hz), 156.4, 149.6,
135.9 (d, 3J(19F, 13C) = 11.7 Hz), 123.7 (d, 3J(19F, 13C) = 9.5 Hz),
114.5 (d, 2J(19F,13C) = 24.8 Hz), 108.6 (d, 2J(19F, 13C)) = 27.1 Hz),
63.6, 60.2, 47.3, 30.8, 30.3, 20.1, 19.2, 18.6, 18.4, 13.6. 19F NMR
(376.46 MHz, DMSO-d6): d = ꢀ116.58. IR (ATR): 3288, 3067, 2962,
2931, 2871, 2359, 1686 (CO—carbamate), 1646 (CO—amide),
1244, 1227, 1196, 1044 cmꢀ1
.
Anal. Calcd for C17H22FN3O3S
1539, 1458, 1246, 1229, 1196, 1046 cmꢀ1
. Anal. Calcd for
(367.137): C, 55.57; H, 6.03; N, 11.44. Found: C, 55.39; H, 5.94;
N, 10.57. HR-MS: for C17H22FN3O3S [M+H+] calcd 368.1439 m/z,
found 368.1438 m/z.
C19H26FN3O3S (395.168): C, 57.70; H, 6.63; N, 10.62. Found: C,
57.82; H, 6.83; N, 10.90. HR-MS: for C19H26FN3O3S [M+H+] calcd
396.1752 m/z, found 396.1751 m/z.
8.1.5.14. Propyl (S)-1-[(R)-1-(6-fluorobenzo[d]thiazole)ethylcar-
8.1.5.17. Isobutyl (S)-1-[(R)-1-(6-fluorobenzo[d]thiazole)ethylc-
bamoyl]-2-methylpropylcarbamate 6n.
White solid; yield
arbamoyl]-2-methylpropylcarbamate 6q.
White solid; yield
33.2% (isolation method B); mp 175.8–176.2 °C. 1H NMR
(400.13 MHz, DMSO-d6): d 8.83 (1H, d, J = 7.6 Hz, NH-CO), 7.99
(1H, dd, 4J = 2.7 Hz, 3J(19F, 1H) = 8.9 Hz, H7), 7.97 (1H, dd,
3J = 8.8 Hz, 4J(19F, 1H) = 5.0 Hz, H4), 7.37 (1H, dt, 4J = 2.6 Hz,
3J = 9.1 Hz, 3J(19F, 1H) = 9.1 Hz, H5), 7.12 (1H, d, J = 8.9 Hz, NH-
COOMe), 5.27 (1H, kv, J = 7.2 Hz, H8), 3.90 (3H, m, H10, H14),
1.98 (1H, m, H11), 1.56 (5H, m, H9, H15), 0.89 (9H, m, H12, H16).
10.0% (isolation method B); mp 198.9–199.2 °C. 1H NMR
(400.13 MHz, DMSO-d6): d 8.86 (1H, d, J = 7.2 Hz, NH-CO), 7.98
(2H, m, H4, H7), 7.37 (1H, dt, J = 2.4 Hz, J = 8.8 Hz, 3J(19F,
1H) = 8.8 Hz, H5), 7.14 (1H, d, J = 8.8 Hz, NH-COOMe), 5.28 (1H,
kv, J = 7.2 Hz, H8), 3.90 (1H, t, J = 8.4 Hz, H10), 3.76 (2H, d,
J = 6.8 Hz, H14), 1.99 (1H, m, H11), 1.84 (1H, m, H15), 1.56 (3H,
d, J = 7.2 Hz, H9), 0.88 (12H, m, H12, H16). 13C NMR (100.62 MHz,
13C NMR (100.62 MHz, DMSO-d6):
d
175.5 (d, 4J(19F,
DMSO-d6):
d
175.5 (d, 4J(19F, 13C) = 3.4 Hz), 171.4, 159.6 (d,
156.4, 149.6, 135.9 (d,
3J(19F,
13C) = 2.9 Hz), 171.4, 159.5 (d, 1J(19F,13C) = 242.0 Hz), 156.4, 149.6,
135.9 (d, 3J(19F, 13C) = 11.6 Hz), 123.7 (d, 3J(19F, 13C) = 9.8 Hz),
114.6 (d, 2J(19F,13C) = 24.6 Hz), 108.6 (d, 2J(19F, 13C)) = 27.1 Hz),
65.4, 60.2, 47.3, 30.3, 22.1, 20.1, 19.2, 18.4, 10.2. 19F NMR
(376.46 MHz, DMSO-d6): d ꢀ116.56. IR (ATR): 3280, 2959, 2932,
1685 (CO—carbamate), 1643 (CO—amide), 1605, 1535, 1455,
1376, 1287, 1244, 1226, 1193, 1151, 1047, 917, 845, 824, 800,
669 cmꢀ1. Anal. Calcd for C18H24FN3O3S (381.152): C, 56.67; H,
6.34; N, 11.02. Found: C, 56.72; H, 6.40; N, 11.11. HR-MS: for
1J(19F,13C) = 241.8 Hz),
13C) = 11.9 Hz), 123.7 (d, 3J(19F, 13C) = 9.6 Hz), 114.6 (d,
2J(19F,13C) = 24.9 Hz), 108.6 (d, 2J(19F, 13C)) = 27.2 Hz), 69.9, 60.3,
47.2, 30.3, 27.7, 20.1, 19.2, 18.9, 18.8, 18.4. 19F NMR
(376.46 MHz, DMSO-d6): d ꢀ116.55. IR (ATR): 3283, 3241, 2959,
1685 (CO—carbamate), 1644 (CO—amide), 1605, 1533, 1457,
1379, 1287, 1245, 1196, 1150, 1129, 1108, 1041, 917, 844, 798,
777, 668 cmꢀ1. Anal. Calcd for C19H26FN3O3S (395.168): C, 57.70;
H, 6.63; N, 10.62. Found: C, 57.84; H, 6.72; N, 10.77. HR-MS: for
C
18H24FN3O3S [M+H+] calcd 382.1595 m/z, found 382.1595 m/z.
C
19H26FN3O3S [M+H+] calcd 396.1752 m/z, found 396.1751 m/z.

´
1745
A. Imramovsky et al. / Bioorg. Med. Chem. 21 (2013) 1735–1748
8.1.5.18.
2-Chloroethyl
(S)-1-[(R)-1-(6-fluorobenzo[d]thia-
White
8.1.5.21. 2,2,2-Trifluoroethyl (S)-1-[(R)-1-(6-fluorobenzo[d]thia-
zole)ethylcarbamoyl]-2-methylpropylcarbamate 6u. White
zole)ethylcarbamoyl]-2-methylpropylcarbamate 6r.
solid; yield 37.0% (isolation method B); mp 193.4–193.6 °C. 1H
NMR (400.13 MHz, DMSO-d6): d 8.85 (1H, d, J = 7.6 Hz, NH-CO),
7.99 (1H, dd, 4J = 2.6 Hz, 3J(19F, 1H) = 8.9 Hz, H7), 7.97 (1H, dd,
3J = 8.9 Hz, 4J(19F, 1H) = 4.3 Hz, H4), 7.40 (1H, d, J = 9.0 Hz, NH-
COOMe), 7.36 (1H, dt, 4J = 2.5 Hz, 3J = 9.1 Hz, 3J(19F, 1H) = 9.1 Hz,
H5), 5.28 (1H, kv, J = 7.2 Hz, H8), 4.24 (2H, t, J = 5.4 Hz, H15), 3.90
(1H, t, J = 8.0 Hz, H10), 3.80 (2H, t, J = 5.0 Hz, H14), 2.00 (1H, m,
J = 6.9 H11), 1.57 (3H, d, J = 7.2 Hz, H9), 0.90 (6H, m, H12). 13C
NMR (100.62 MHz, DMSO-d6): d 175.5 (d, 4J(19F, 13C) = 3.7 Hz),
171.2, 159.5 (d, 1J(19F,13C) = 242.1 Hz), 155.8, 149.6, 135.9 (d,
3J(19F, 13C) = 11.6 Hz), 123.7 (d, 3J(19F, 13C) = 9.5 Hz), 114.6 (d,
2J(19F,13C) = 24.9 Hz), 108.6 (d, 2J(19F, 13C)) = 27.0 Hz), 64.2, 60.4,
47.3, 43.1, 30.2, 20.1, 19.2, 18.6. 19F NMR (376.46 MHz, DMSO-
d6): d ꢀ116.56. IR (ATR): 3283, 3242, 2960, 1690 (CO—carbamate),
1641 (CO—amide), 1605, 1542, 1455, 1396, 1375, 1286, 1244,
1227, 1193, 1153, 1080, 1032, 916, 844, 822, 794, 756, 708, 687,
667 cmꢀ1. Anal. Calcd for C17H21ClFN3O3S (401.098): C, 50.81; H,
5.27; N, 10.46. Found: C, 50.91; H, 5.52; N, 10.56. HR-MS: for
solid; yield 52% (isolation method A); mp 202.9–203.8 °C. 1H
NMR (400.13 MHz, DMSO-d6): d 8.93 (1H, d, J = 7.6 Hz, NH-CO),
7.98 (2H, m, H4, H7), 7.83 (1H, d, J = 8.8 Hz, NH-COOMe), 7.37
(1H, dt, J = 2.4 Hz, J = 9.2 Hz, 3J(19F, 1H) = 9.2 Hz, H5), 5.28 (1H, kv,
J = 7.2 Hz, H8), 4.67 (2H, m, H14), 3.91 (1H, t, J = 8.4 Hz, H10),
2.02 (1H, m, CH-(CH3)2), 1.56 (3H, d, J = 7.2 Hz, H9), 0.92 (6H, d,
J = 6.4 Hz, H9). 13C NMR (100.62 MHz, DMSO-d6): d 175.4 (d,
4J(19F, 13C) = 3.2 Hz), 170.9, 159.6 (d, 1J(19F,13C) = 242.4 Hz), 154.5,
149.6,
135.9
(d,
3J(19F,
13C) = 11.8 Hz),
123.8
(d,
1J(19F,13C) = 278.1 Hz), 123.7 (d, 3J(19F, 13C) = 9.6 Hz), 114.6 (d,
2J(19F,13C) = 24.8 Hz), 108.6 (d, 2J(19F, 13C)) = 27.1 Hz), 60.7, 59.8
(q, 2J(19F,13C) = 34.5 Hz), 47.3, 30.2, 20.1, 19.2, 18.6. 19F NMR
(376.46 MHz, DMSO-d6): d ꢀ116.6 (F-6), -72.8 (CF3). IR (ATR):
3295, 3068, 2969, 1708 (CO—carbamate), 1650 (CO—amide),
1541, 1456, 1241, 1173, 1062 cmꢀ1. Anal. Calcd for C17H19F4N3O3S
(421.11): C, 48.45; H, 4.54; N, 9.97. Found: C, 48.55; H, 4.59; N,
10.15. HR-MS: for C17H19Cl3FN3O3S [M+H+] calcd 422.1173 m/z,
found 422.1157 m/z.
C
16H20FN3O3S [M+H+] calcd 402.1049 m/z, found 402.1048 m/z.
8.1.5.22.
obenzo[d]thiazole)ethylcarbamoyl]-2-methylpropylcarbamate
6v. White solid; yield 55% (isolation method A); mp 179.2–
180.8 °C. 1H NMR (400.13 MHz, DMSO-d6):
8.94 (1H, d,
2,2,3,3,-Tetrafluoropropyl
(S)-1-[(R)-1-(6-fluor-
8.1.5.19.
2-Bromoethyl
(S)-1-[(R)-1-(6-fluorobenzo[d]thia-
White
zole)ethylcarbamoyl]-2-methylpropylcarbamate 6s.
solid; yield 32.0% (isolation method B); mp 174.9–175.2 °C. 1H
NMR (400.13 MHz, DMSO-d6): d 8.86 (1H, d, J = 7.6 Hz, NH-CO),
7.99 (1H, dd, 4J = 2.7 Hz, 3J(19F, 1H) = 8.9 Hz, H7), 7.97 (1H, dd,
3J = 8.9 Hz, 4J(19F, 1H) = 4.7 Hz, H4), 7.40 (1H, d, J = 9.6 Hz, NH-
COOMe), 7.36 (1H, dt, J = 2.5 Hz, J = 9.2 Hz, 3J(19F, 1H) = 9.2 Hz,
H5), 5.28 (1H, kv, J = 6.8 Hz, H8), 4.30 (2H, t, J = 5.6 Hz, H15), 3.90
(1H, t, J = 8.4 Hz, H10), 3.65 (2H, t, J = 5.6 Hz, H14), 1.99 (1H, m,
H11), 1.57 (3H, d, J = 7.2 Hz, H9), 0.91 (3H, d, J = 6.5 Hz, H12),
0.89 (3H, d, J = 6.5 Hz, H12). 13C NMR (100.62 MHz, DMSO-d6): d
175.5 (d, 4J(19F, 13C) = 3.7 Hz), 171.2, 159.5 (d, 1J(19F,13C) =
242.2 Hz), 155.8, 149.6, 135.9 (d, 3J(19F, 13C) = 11.6 Hz), 123.7 (d,
3J(19F, 13C) = 9.6 Hz), 114.6 (d, 2J(19F,13C) = 24.9 Hz), 108.6 (d,
2J(19F, 13C)) = 27.0 Hz), 64.0, 60.4, 47.3, 31.4, 30.2, 20.1, 19.2, 18.9.
19F NMR (376.46 MHz, DMSO-d6): d ꢀ116.55. IR (ATR): 3284,
3242, 2968, 2930, 1691 (CO—carbamate), 1642 (CO—amide),
1605, 1565, 1541, 1455, 1395, 1375, 1291, 1226, 1245, 1193,
1154, 1133, 1075, 1006, 997, 916, 905, 844, 828, 819, 713,
675 cmꢀ1. Anal. Calcd for C17H21BrFN3O3S (445.047): C, 45.75; H,
4.74; N, 9.41. Found: C, 45.95; H, 4.80; N, 9.45. HR-MS: for
d
J = 7.4 Hz, NH-CO), 7.98 (1H, dd, J = 8,8 Hz, 4J(19F, 1H) = 4.9 Hz,
H4), 7.96 (H, dd, 4J = 2.4 Hz, 3J(19F, 1H) = 8.8 Hz, H7), 7.72 (1H, d,
J = 8.9 Hz, NH-COOMe), 7.37 (1H, dt, J = 2.6 Hz, J = 9.1 Hz, 3J(19F,
1H) = 9.1 Hz, H5), 6.55 (1H, tt, 2J(19F, 1H) = 52.1 Hz, 3J(19F,
1H) = 5.5 Hz, H15), 5.28 (1H, kv, J = 7.2 Hz, H8), 4.54 (2H, m, H14),
3.91 (1H, t, J = 8.2 Hz, H10), 2.00 (1H, m, J = 7.0 Hz CH-(CH3)2),
1.55 (3H, d, J = 7.0 Hz, H9), 0.90 (6H, d, J = 6.4 Hz, H9). 13C NMR
(100.62 MHz, DMSO-d6): d 175.4 (d, 4J(19F, 13C) = 3.0 Hz), 170.9,
159.5 (d, 1J(19F,13C) = 242.5 Hz), 154.7, 149.6, 135.9 (d, 3J(19F,
13C) = 11.8 Hz), 123.7 (d, 3J(19F, 13C) = 9.4 Hz), 114.7 (tt,
1J(19F,13C) = 252.5 Hz, 2J(19F,13C) = 25.0 Hz), 114.5 (d, 2J(19F,13C) =
24.7 Hz), 109.3 (1J(19F,13C) = 248.3 Hz, 2J(19F,13C) = 29.5 Hz), 108.6
(d, 2J(19F, 13C)) = 27.0 Hz), 60.5, 59.5 (t, 2J(19F,13C) = 26.5 Hz), 47.3,
30.3, 20.1, 19.1, 18.4. 19F NMR (376.46 MHz, DMSO-d6): d -116.6,
(arom.F), ꢀ139.0 (AB system, 2J(19F,19F) = 303.0, 3J(19F,19F) = 5.5,
CFaFb), ꢀ138.9 (AB system, 2J(19F,19F) = 303.0, 3J(19F,19F) = 5.4,
CFaFb), ꢀ125.1 (AB system, 2J(19F,19F) = 269.0, 3J(19F,19F) = 5.5,
CHFaFb), ꢀ124.9 (AB system, 2J(19F,19F) = 269.0, 3J(19F,19F) = 5.4,
CHFaFb). IR (ATR): 3310, 3300, 2966, 1703 (CO—carbamate), 1655
(CO—amide), 1541, 1450, 1247, 1207, 1128, 918, 797, 818,
710 cmꢀ1. Anal. Calcd for C18H20F5N3O3S (453,4267): C, 47.68; H,
4.45; N, 9.27. Found: C, 47.75; H, 4.46; N, 9.30. HR-MS: for
C
16H20FN3O3S [M+H+] calcd 446.0544 m/z, found 446.0544 m/z.
8.1.5.20. 2,2,2-Trichloroethyl (S)-1-[(R)-1-(6-fluorobenzo[d]thi-
azole)ethylcarbamoyl]-2-methylpropylcarbamate
6t.
White solid; yield 25.1% (isolation method B); mp 107.8–
8.93 (1H, d,
C
18H21F5N3O3S [M+H+] calcd 454.1218 m/z, found 454,1220 m/z.
108.5 °C. 1H NMR (400.13 MHz, DMSO-d6):
d
J = 7.5 Hz, NH-CO), 7.99 (1H, dd, 4J = 2.2 Hz, 3J(19F, 1H) = 9.1 Hz,
H7), 7.97 (1H, dd, 3J = 9.1 Hz, 4J(19F, 1H) = 4.7 Hz, H4), 7.85 (1H, d,
J = 8.9 Hz, NH-COOMe), 7.37 (1H, dt, 4J = 2.6 Hz, 3J = 9.1 Hz, 3J(19F,
1H) = 9.1 Hz, H5), 5.28 (1H, kv, J = 7.2 Hz, H8), 4.84 (1H, H14),
4.83 (1H, H14), 3.92 (1H, t, J = 8.5 Hz, H10), 2.02 (1H, m, H11),
1.56 (3H, d, J = 7.2 Hz, H9), 0.92 (6H, d, J = 7.0 Hz, H12). 13C NMR
(100.62 MHz, DMSO-d6): d 175.4 (d, 4J(19F, 13C) = 3.0 Hz), 170.9,
159.6 (d, 1J(19F,13C) = 239.0 Hz), 154.5, 149.6, 135.9 (d, 3J(19F,
13C) = 11.7 Hz), 123.7 (d, 3J(19F, 13C) = 9.6 Hz), 114.6 (d,
2J(19F,13C) = 24.6 Hz), 108.6 (d, 2J(19F, 13C)) = 27.2 Hz), 96.3, 73.5,
60.8, 47.3, 30.2, 20.1, 19.2, 18.6. 19F NMR (376.46 MHz, DMSO-
d6): d ꢀ116.52. IR (ATR): 3265, 2962, 2938, 1682 (CO—carbamate),
1648 (CO—amide), 1598, 1537, 1453, 1382, 1288, 1249, 1238,
8.1.5.23. 2,2,3,3,4,4,4-Heptafluorobutyl (S)-1-[(R)-1-(6-fluor-
obenzo[d]thiazole)ethylcarbamoyl]-2-methylpropylcarbamate
6w.
White solid; yield 62.0% (isolation method A); mp 162.1–
162.6 °C. 1H NMR (400.13 MHz, DMSO-d6): d 8.93 (1H, d, J = 7.6 Hz,
NH-CO), 7.98 (1H, dd, J = 8,9 Hz, 4J(19F, 1H) = 4.8 Hz, H4), 7.96 (H,
dd, 4J = 2.3 Hz, 3J(19F, 1H) = 8.7 Hz, H7), 7.87 (1H, d, J = 8.8 Hz, NH-
COOMe), 7.36 (1H, dt, 4J = 2.5 Hz, J = 9.1 Hz, 3J(19F, 1H) = 9.1 Hz,
H5), 5.28 (1H, kv, J = 7.2 Hz, H8), 4.80 (2H, m, H14), 3.92 (1H, t,
J = 8.4 Hz, H10), 2.01 (1H, m, H11), 1.57 (3H, d, J = 6.8 Hz, H1),
0.91 (3H, d, J = 6.6 Hz, H12), 0.90 (3H, d, J = 6.6 Hz, H12). 13C
NMR (100.62 MHz, DMSO-d6): d 175.4 (d, 4J(19F, 13C) = 2.8 Hz),
170.9, 159.5 (d, 1J(19F,13C) = 242.3 Hz), 154.4, 149.6 (d, 5J(19F,
13C) = 1.7 Hz), 135.9 (d, 3J(19F, 13C) = 11.7 Hz), 123.7 (d, 3J(19F,
13C) = 9.6 Hz), 117.3 (m, 1J(19F,13C) = 288.4 Hz, 2J(19F,13C) =
33.8 Hz), 114.6 (d, 2J(19F,13C) = 24.6 Hz), 114.5 (m, 1J(19F,13C) =
255.0 Hz, 2J(19F,13C) = 30.7 Hz), 108.7 (d, 2J(19F, 13C)) = 27.2 Hz),
1222, 1201, 1194, 1148, 1050, 916, 845, 833, 825, 806, 671 cmꢀ1
.
Anal. Calcd for C17H19Cl3FN3O3S (469.02): C, 43.37; H, 4.07; N,
8.93. Found: C, 43.42; H, 4.15; N, 9.15. HR-MS: for C17H19Cl3FN3O3S
[M+H+] calcd 470.0269 m/z, found 470.0269 m/z.

´
A. Imramovsky et al. / Bioorg. Med. Chem. 21 (2013) 1735–1748
1746
108.6 (q, 1J(19F,13C) = 267.0 Hz), 2J(19F,13C) = 33.0 Hz), 60.7, 58.7 (t,
3J(19F, 13C) = 60.8 Hz), 47.3, 30.2, 20.1, 19.1, 18.4. 19F NMR
8.3.2. Cytotoxicity measurement
8.3.2.1. MTS assay.
The most active compounds 6d, 6f, 6n and
(376.46 MHz, DMSO-d6):
ꢀ116.6 (s, F-6),
d
ꢀ80.5 (t, 4J(19F,19F) = 7.5, F-17),
6m were tested on in vitro cytotoxicity in human liver cell line
HepG2 using a standard colorimetric method measuring a tetrazo-
lium salt reduction (CellTiter 96 AQueous One Solution Cell Prolif-
eration Assay,56 Promega, USA). The cells were seeded in density
10,000 cells per well in a 96-well plate. Next day, the cells were
treated with each of the tested substances dissolved in DMSO.
Compound 6f was prepared in 12 incubation concentrations
ꢀ119.9
(dq,
4J(19F15,19F17) = 7.5,
3J(19F15,19F16) = 3.7, F-15), ꢀ127.4 (br s, F-16). IR (ATR): 3307,
3261, 1707(CO—carbamate), 1654 (CO—amide), 1558, 1541,
1457, 1232, 1219, 1198, 1172, 1149, 1131, 1021 cmꢀ1. Anal. Calcd
for C19H19F8N3O3S (521.102): C, 43.77; H, 3.67; N, 8.06. Found: C,
43.95; H, 3.78; N, 8.26. HR-MS: for C19H19F8N3O3S [M+H+] calcd
522.1093 m/z, found 522.1089 m/z.
(0.001–5000
lmol/L) in triplicates. Compounds 6d, 6m, 6n were
prepared in 12 incubation concentrations (0.001–2500
lmol/L) in
triplicates. The following controls were incubated concomitantly:
100% cell viability, 0% cell viability (the cells treated by 10% DMSO),
no cell control and vehiculum control. After 24 h incubation in a
humidified atmosphere containing 5% CO2 at 37 °C, reagent of
the used kit was added into the wells. After 3.5 h incubation at
37 °C, the absorbance was recorded at 490 nm using a plate reader.
A standard toxicological parameter IC50 (50% inhibitory concentra-
tion) was calculated in each of the tested substances using Graph-
Pad Prism software (version 5.02).
8.2. In vitro evaluation of prepared compounds
All reagents and solvents were purchased from commercial
sources (Sigma–Aldrich, Lach-Ner, Penta). The IC50 values were
determined using the spectrophotometric Ellman’s method, which
is a simple, rapid and direct method to determine the SH and –S–S–
group content in53 This method is widely used for measuring of
cholinesterase activity and effectivity of cholinesterase inhibitors.
Cholinesterase activity is measured indirectly by quantifying the
concentration of 5-thio-2-nitrobenzoic acid (TNB) ion formed in
the reaction between the thiol reagent 5,50-dithiobis-2-nitroben-
zoic acid (DTNB) and thiocholine, a product of substrate (i.e., acet-
ylthiocholine, ATCH) hydrolysis by cholinesterase.54 All tested
compounds were dissolved in dioxane (concentration 0.02 M)
and then diluted in demineralised water (concentration 0.002
and 0.0002 M). The procedure of determination of IC50 is a slightly
modified Ellman’s method according to Zdrazilova and described in
detail in.55 Into the glass test tubes eight different concentrations
of tested compound were placed. All solutions were filled up to
1 mL with phosphate buffer (0.1 M, pH 8) and 0.5 mL of the
enzyme preparation (AChE from Electrophorus electricus or BChE
from equine serum diluted in phosphate buffer). The catalytic
activity of enzyme in final reaction mixtures was 0.14 U/mL. The
reaction mixtures were homogenized and incubated at 25 °C for
5 min. Then 0.25 mL of indicating solution (0.5 mM 5,50-dithi-
obis(2-nitrobenzoic acid), DTNB, purchased from Sigma-Aldrich)
and 0.25 mL of substrate (0.25 mM acetylthiocholine dissolved in
demineralised water, purchased from Sigma–Aldrich) was added
to all test tubes, the mixtures were homogenized and incubated
at 25 °C for 10 min. Then the absorbance at 412 nm of all mixtures
was measured (in comparative solution the enzyme was replaced
by demineralised water). From dependence inhibition in percent-
ages (%I) versus concentration of inhibitor (IC) the IC50 value was
determined. For determination of IC50 values the inhibition was
determined for 23 different compound concentrations with 3
replicates.
8.3.2.2. Real-time cellular toxicity assay.
Real time growth
kinetics of MCF7 cells was examined by the impedance-based
xCELLigence RTCA (Real-Time Cell Analysis) SP (Single plate) sys-
tem (Roche Diagnostic, Mannheim, Germany). The system moni-
tors cellular events in real time without the incorporation of
labels. The principle of the system is to monitor the changes in
electrode impedance induced by the interaction between testing
cells and electrodes.57,58 The impedance measurement provides
quantitative information about the biological status of the cells,
including cell number, viability, and morphology. The xCELLigence
System was connected and tested by Resistor Plate Verification be-
fore the RTCA SP Station was placed inside the incubator at 37 °C
and 5% CO2. Cell growth was recorded as cell index (CI), which cor-
responds to the electrical impedance of a well measured by the
xCELLigence system. Cells were seeded at a density of 75,000 cells
per well in E-plate 96. Cell proliferation was dynamically moni-
tored at 1 h interval. When the cells entered logarithmic growth
phase, they were treated with 10
lL of 6d, 6f, 6n and 6m dissolved
in DMSO at concentrations from 0.01 to 2500
l
mol/L in triplicates.
Growth curves were normalized to the CI at the last measured time
point before compound addition for each well. Cells treated with
0.1% of DMSO were used as a vehicle control and 10% DMSO trea-
ted cells were used as a negative control (i.e., 0% viability). Upon
incubation with tested compounds the cell status and the cytotoxic
effect were visualized using cell characteristic index-time profile.
The results were also expressed as IC50 after 24 h of incubation
with the tested compounds. Evaluations were performed using
the RTCA 1.2.1 Software.
8.3. In vitro cytotoxicity assay
8.4. Lipophilicity
8.3.1. Cell lines
The human hepatocellular liver carcinoma cell line HepG2 (p13)
purchased from Health Protection Agency Culture Collections
(ECACC, Salisbury, UK) was routinely cultured in MEM (Minimum
Essentials Eagle Medium) (Sigma Aldrich) supplemented with
8.4.1. HPLC determination of liphophilicity (capacity factor k/
calculated logk)
A Waters Alliance 2695 XE HPLC separation module and a
Waters Photodiode Array Detector 2996 (Waters Corp., Milford,
MA, USA) were used. A SymmetryÒ C18
5
l
m, 4.6 ꢂ 250 mm, Part
10% foetal bovine serum (PAA), 1% L-Glutamine solution (Sigma Al-
drich) and non-essential amino acid solution (Sigma Aldrich) in a
humidified atmosphere containing 5% CO2 at 37 °C. The human
breast adenocarcinoma cell line MCF7 was a kind gift from Dr.
Francoise Farace (Institut Gustave Roussy, Translational Research
Laboratory, Villejuif, France). MCF7 (p37) cells were cultured in
Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Carlsbad,
CA, USA) supplemented with 10% foetal calf serum (Gibco), 1% pen-
icillin-streptomycin antibiotics (Gibco) and 0.01 mg/mL insuline
(Gibco) at 37 °C in a humidified 5% CO2 atmosphere.
No. WAT054275 (Waters Corp., Milford, MA, USA) chromato-
graphic column was used. The HPLC separation process was mon-
itored by Empower™ 2 Chromatography Data Software, Waters
2009 (Waters Corp., Milford, MA, USA). A mixture of MeOH p.a.
(70%) and H2O-HPLC – Mili-Q Grade (30%) was used as a mobile
phase. The total flow of the column was 1.0 mL/min, injection vol-
ume 30 lL, column temperature 45 °C, and sample temperature
10 °C. The detection wavelength of 210 nm was chosen. The KI
methanolic solution was used for dead time (tD) determination.

´
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Retention times (tR) were measured in minutes. The capacity fac-
tors k were calculated using the Empower™ 2 Chromatography
Data Software according to formula k = (tRꢀtD)/tD, where tR is the
retention time of the solute, whereas tD denotes the dead time ob-
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8.5. Molecular docking
The three-dimensional structure of acetylcholine esterase from
Torpedo californica, non-aged and conjugated with soman59, was
obtained from Protein Databank (www.pdb.org, PDB code 2wg2).
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activity studies). For molecular docking calculations, the molecules
of water, other molecules of crystallization, for example, alpha-l-
fucose, N-acetyl-l-glucosamine, di(hydroxyethyl)ether or tetraeth-
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was dephosphonylated.
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automatic substitution of all hydrogen atoms for GOLD-optimized
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primary docking procedure and then the results were rescored
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Acknowledgments
Authors wish to acknowledge for financial support of Faculty of
Chemistry and Chemical Technology, University of Pardubice to
Czech Ministry of Education Youth and Sports and student project
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