Benzothiazole carbamates and amides as antiproliferative species

Article abstract of DOI:10.1016/j.ejmech.2018.08.067

A series of new benzothiazole-based carbamates and amides were synthesized and their antiproliferative activity was determined. Derivatives with profound activity were identified and further investigated for their possible mechanism of action. It was found that these compounds induce specific apoptosis, G2/M cell cycle arrest and decrease ROS level in MCF-7 human breast cancer cell line. Moreover, submicromolar antiproliferative activity of examined carbamates against NT2/D1 testicular embryonal carcinoma was shown. The most potent derivatives strongly inhibited NT2/D1 cell migration and invasiveness.

Full text of DOI:10.1016/j.ejmech.2018.08.067

Accepted Manuscript
Benzothiazole carbamates and amides as antiproliferative species
Milica Videnović, Marija Mojsin, Milena Stevanović, Igor Opsenica, Tatjana Srdić-
Rajić, Bogdan Šolaja
PII:
S0223-5234(18)30739-6
10.1016/j.ejmech.2018.08.067
EJMECH 10678
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 2 July 2018
Revised Date: 17 August 2018
Accepted Date: 25 August 2018
Please cite this article as: M. Videnović, M. Mojsin, M. Stevanović, I. Opsenica, T. Srdić-Rajić, B. Šolaja,
Benzothiazole carbamates and amides as antiproliferative species, European Journal of Medicinal
Chemistry (2018), doi: 10.1016/j.ejmech.2018.08.067.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Benzothiazole carbamates and amides as antiproliferative species
Milica Videnović,‡ Marija Mojsin,¶ Milena Stevanović,¶,§,# Igor Opsenica,◊ Tatjana Srdić-
*
*
Rajić,┴, Bogdan Šolaja◊,#,
‡Faculty of Chemistry Innovative Centre, Studentski trg 12-16, 11158 Belgrade, Serbia
¶Institute of Molecular Genetics and Genetic Engineering, University of Belgrade, Vojvode
Stepe 444a, 11010 Belgrade, Serbia
§University of Belgrade – Faculty of Biology, Studentski trg 16, 11158 Belgrade, Serbia
#Serbian Academy of Sciences and Arts, Knez Mihailova 35, 11158 Belgrade, Serbia
◊University of Belgrade – Faculty of Chemistry, Studentski trg 16, P.O. Box 51, 11158
Belgrade, Serbia
┴Institute for Oncology and Radiology of Serbia, Pasterova 14, 11000 Belgrade, Serbia
Abstract
A series of new benzothiazole-based carbamates and amides were synthesized and their
antiproliferative activity was determined. Derivatives with profound activity were identified
and further investigated for their possible mechanism of action. It was found that these
compounds induce specific apoptosis, G2/M cell cycle arrest and decrease ROS level in
MCF-7 human breast cancer cell line. Moreover, submicromolar antiproliferative activity of
examined carbamates against NT2/D1 testicular embryonal carcinoma was shown. The most
potent derivatives strongly inhibited NT2/D1 cell migration and invasiveness.
Keywords: benzothiazoles, antiproliferative, MCF-7, NT2/D1, anoikis
1. Introduction
Cancer is a complex and lethal disease, responsible for more than 8 million deaths p.a.
according to the World Health Organization report.1 One of the main features of tumor cells is
their rapid proliferation, and respective diversity.2 Therefore, the discovery of new drugs that
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would prevent the cell proliferation and induce apoptosis, stop cancer cell mobility and
metastasis, is a long standing task of researchers in the field.
Benzothiazoles3,4 are prominent class of compounds that exert various pharmacological
activities, such as antimicrobial,5 topoisomerase inhibitory activity,6 anti-HIV,7
multifunctional
anti-Alzheimer’s
disease
activity,8
anti-inflammatory
activity,9
immunosuppresive (1, Frentizole),10 anticonvulsive and neuroprotective (2, Riluzole).11
Members of benzothiazole chemotype are also potent anti-infectives – specifically possessing
antimalarial12,13,14 and antileishmanial activity15,16 (compounds 3 and 4, Figure 1).
Benzothiazoles were investigated on several instances for their anti-cancer activity.17-
2617,18,19,20,21,22,23,24,25,26
They were tested against selected number of cell lines, e.g., human
cervical cancer, liver cancer, NSCL, prostate cancer, and human breast cancer exhibiting good
to excellent antiproliferative activity with low toxicity. Former clinical candidate, Phortress
(6), lysylamide prodrug of C(2) and C(6) substituted benzothiazole 5F203 (5), Figure 1)
generates DNA adducts in sensitive cancer cell lines, including MCF-7 breast cancer cell
line.17
F3CO
S
O
S
O
N
O
S
N
HN
N
N
N
H
N
H
H
N
N
H
3
antimalarial12
Cl
2
(Riluzole)
1
(Frentizole)
immunosuppresive10
anticonvulsive
neuroprotective11
O
S
S
N
5: R= H
5F203
F
R
N
H
S
N
6: R= Lisyl Phortress
F
antitumor activity17
4
antileismanial 15
Figure 1. Sample structures of C(2) substituted benzothiazoles and their physiological
properties.10,11,12,15,17
In addition, the research on mechanism of action of individual benzothiazoles was the subject
of few reports involving the induction of apoptosis by C(2)-substituted benzothiazolyl
derivatives 7-10 (Figure 2).21,24,27,28
2

O
O
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S
S
O
Cl
S
S
N
N
N
H
O
N
NH Cl
O
N
N
H
7
8
O
N
S
Cl
F
F
S
N
N
NH
O
F
O
10
9
O
O
Figure 2. Benzothiazole chemotypes that induce apoptosis - compounds 7 and 8: HepG2;21,27
compounds 9 and 10: MCF-7.24,28
The above examples clearly indicate that substitution pattern at C(6) plays an
important role in antiproliferative/anticancer activity of aminobenzothiazoles. We have
chosen to introduce the thioalkyl substituent at C(6) in order to investigate the influence the
electron-donor substituent (S) and respective electron-withdrawal (SO) and (SO2)
counterparts and explore the space around C(6) sulfur by changing the alkyl substituents
while keeping the carbamate moiety at C(2) nitrogen. For comparison, we also prepared
respective C(6) oxygen analogue, as well as few amides.
Therefore, we report the synthesis, activity and investigation on modes of action of
new benzothiazole chemotype against breast cancer cell lines MCF-7 and testicular
embryonal carcinoma NT2/D1. Furthermore, the newly synthesized benzothiazole-based
carbamates were evaluated against NT2/D1 cells in terms of antimigratory activity. To the
best of our knowledge, only a limited number of benzothiazole-based apoptosis inducers in
MCF-7 human breast cancer cell line (compounds 9 and 10, Figure 2) have been reported.
Their proapoptotic activity was confirmed either by ELISA detection of mono- and
oligonucleosomes enrichment after treatment,28 or caspase-9 activity.24 Moreover, there is no
report of benzothiazole-based compounds with antiproliferative and antimigratory potential
against NT2/D1 testicular teratocarcinoma cell line.
2. Chemistry
A series of novel benzothiazole carbamates and amides were synthesized starting with
commercially available 1-chloro-4-nitrobenzene which was converted to 4-(alkylthio)anilines
(13 – 16) by one-pot transformation in a reaction with the appropriate alkyl thiols and KOH in
PEG-600 (Scheme 1).29 Aminobenzothiazole moiety was obtained in reaction of 4-substituted
anilines with NH4SCN using bromine in acetic acid according to modified procedure for
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30
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synthesis of aminobenzothiazole core. After submitting aminobenzothiazoles to further
derivatization with different alkyl chloroformates, the target carbamate derivatives (24 – 26
and 28 – 37) were obtained in 19 – 62% yield.
41: R1= n-Pr, R2= n-Pr, 21%
O
R2
42: R1= n-Bu, R2= n-Pr, 28%
43: R1= n-Bu, R2= OCH3, 45%
44: R1= n-Pr, R2=CH2OCH3, 30%
45: R1= n-Bu, R2= CH2OCH3, 32%
46: R1= n-Pr, R2= OCH3, 23%
R2
O
S
S
N
X
R1
O
S
N
R1
NH
NH
24 - 40
41 - 46
d)
24: X = S, R1= Et, R2= Et, 40% from 12
25: X = S, R1= n-Pr, R2= Et, 21% from 13
26: X = O, R1= Et, R2= n-Pr,25% from 17
27: X = SO2, R1= Et, R2= Et, 55%
28: X = S, R1= n-Pr, R2= n-Pr, 47%
29: X = S, R1= n-Bu, R2= Et, 19% from 14
30: X = S, R1= n-Bu, R2= n-Pr, 29%
31: X = S, R1= n-Pr, R2= n-Bu, 28% from 13
32: X = S, R1= Et, R2= n-Pr, 27% from 12
33: X = S, R1= Pentyl, R2= Et, 21%
34: X = S, R1= Pentyl, R2= n-Pr, 40%
35: X = S, R1= i-Bu, R2= Et, 23%
c)
NH2
NO2
g)
b)
a)
X
S
R1
NH2
N
X
Cl
R1
11
12 - 17
18 - 23
e)
f) h)
18: X = S, R1= n-Et, crude
19: X = S, R1= n-Pr, 82%
20: X = S, R1= n-Bu, 93%
21: X = S, R1= i-Bu, 54%
22: X = S, R1= n-Pentyl,56%
23: X = O, R1= Et, crude
12: X = S, R1= Et as HCl salt, comercial
13: X = S, R1= n-Pr , 63%
14: X = S, R1= n-Bu, 49%
36: X = S, R1= i-Bu, R2= n-Pr, 62%
37: X = S, R1= n-Bu, R2= n-Bu,32%
38: X = SO, R1= Et, R2= Et, 28%
15: X = S, R1= i-Bu, 19%
16: X = S, R1= n-Pentyl, 31%
17: X = O, R1= Et, comercial
39: X = SO, R1= n-Pr, R2= n-Pr, 26%
40: X = SO2, R1= n-Pr, R2= n-Pr,48%
Reagents and conditions: a) KOH, R1SH, PEG-600, 100 oC; b) NH4SCN, Br2, CH3COOH, H2O, r.t. to 75 oC; c) ClCO2R2, Et3N,
benzene, 80 oC; d) alkanoyl chloride, CH2Cl2/benzene, 0oC; e) MCPBA (1 eq), CH2Cl2,r.t; f) MCPBA (4 eq), CH2Cl2, r.t. g) H2O2 (1.2 eq),
CH3CN, CH3OH, K2CO3, 0oC to r.t; h) H2O2 (4 eq), CH3CN, CH3OH, K2CO3, 0oC to r.t.
Scheme 1. Synthesis of aminobenzothiazole derivatives.
Oxidation of sulfide 24 was achieved with one or four equivalents of MCPBA31 at
room temperature in CH2Cl2 to obtain sulfoxy and sulfonyl derivatives 27 and 38,
respectively. To investigate the influence of antioxidant properties of our
aminobenzothiazoles on ROS production, and further impact on antiproliferative activity
against MCF-7 cancer cell line, we have also synthesized 39 and 40, sulfoxy and sulfonyl
analogues of carbamate 28. For this transformation, H2O2 was used32 as MCPBA afforded
only complex reaction mixture. The synthesis of benzothiazole-based amide derivatives (41 –
46) was performed under mild reaction conditions starting from amines 19 and 20 and
synthesized alkanoyl or alkoxyalkanoyl chlorides.
3. Results
3.1. Biological evaluation
3.1.1. In vitro antiproliferative screening and cytotoxicity evaluation
Seventeen selected compounds (24 – 38, 41 and 44) were submitted to NCI-60 Cell Screen
through the Developmental Therapeutics Program (DTP) in National Cancer Institute (NCI).
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Selected compounds were initially tested at a single dose (10µM) against the full NCI 60-cell
panel (data not shown), following the accepted DTP protocol.33 Seven benzothiazole
carbamates (24 – 26, 28 – 30 and 33) satisfied pre-determined inhibition threshold criteria in a
minimum number of cell lines and were evaluated against the 60-cell panel at five
concentrations level. Table 1 reveals mean GI50 (µM) values (MID) for selected compounds
tested against full 60-cell panel after 48 h treatment obtained using SRB assay (full data are
given in Supplementary material).
Table 1. Mean GI50 (µM) values (MID) for selected compounds obtained after 48 h treatment
against a panel of 60 cell lines in vitro a
Compound
MIDb
24
19.9
25
2.13
26
81.2
28
2.14
29
0.74
30
0.58
33
0.83
a Five dose assay was performed against 60 cancer cell lines treated with selected compounds for 48 hours using
SRB procedure
bMID = Mean GI50 values for each compound against full 60-cell panel
The most potent compound was 30 with GI50 values in submicromolar range against
the majority cell lines of 60-cell panel, followed by compounds 29 and 33. Considering
investigated ethyl carbamates (24, 25, 29 and 33), lengthening of the alkyl group linked to S-
C(6) attached to benzothiazole moiety led to enhanced antiproliferative activity according to
MIDs (submicromolar MID GI50 values for butyl and pentyl derivatives 29 and 33 vs. MID
GI50> 2 µM for ethyl and propyl derivatives 24 and 25). Compound 26, containing O-C(6)
substituent instead of S-C(6), with the highest GI50 value (>80 µM), clearly emphasized the
importance of S-C(6) for the antiproliferative activity. Cytotoxic/antiproliferative activities of
synthesized benzothiazole derivatives were further evaluated against four tumor cell lines
(estrogen receptor positive breast adenocarcinoma MCF-7, myelogenous leukemia K562,
melanoma A375, testicular embryonal carcinoma NT2/D1) and human lung fibroblast MRC-5
cell line derived from normal lung tissue. Standard MTT assay was applied after 48h cell
exposure to the tested compounds (25, 26, 28 – 30, 32, 34 – 37, 39 – 46) (Table 2 and
Supplementary material I)
Table 2. IC50 values calculated for selected compounds (25, 28 – 30, 36, 39 – 41, 44, 45).c
MCF-7
A375
K562
NT2/D1
MRC-5
Compound
Structure
(IC50, µM)
(IC50, µM) (IC50, µM)
(IC50, µM)
(IC50, µM)
S
S
S
NH
61.4±4.2
85.0±5.6
> 100
> 100
> 100
>1
-
25
28
N
O
O
S
NH
O
> 300
24.2±3.1
0.2±0.03
N
O
5

S
S
N
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NH
O
> 100
91.6±6.0
> 100
>1
-
29
30
36
39
40
41
44
O
S
S
NH
> 100
45.2±3.4
> 100
>300
0.1±0.01
N
O
O
O
S
O
S
N
> 100
> 100
7.7±2.0
>1
-
-
NH
O
O
S
O
S
N
> 100
-
-
-
-
-
N
H
O
O
S
O
S
N
> 100
-
-
> 300
-
N
O
H
O
S
S
S
N
77.5±4.5 53.2±4.0
>1
>1
30.5±2.5
95.5±5.5
NH
O
O
S
N
> 100
> 100
66.3±4.2
44.2±3.3
NH
O
N
S
S
92.5±5.0
>1
-
45
O
N
H
0.4
-
-
-
2
-
-
-
-
Doxorubicin
Cisplatin
1.11±0.17
cIC50 values were calculated after 48 h treatment of selected cell lines with five concentrations of investigated
compounds using MTT assay. The measurements were performed in triplicate.
Obtained results indicated a high sensitivity of NT2/D1 cell line towards carbamates
28 and 30 with IC50 values 0.2 and 0.1 µM, respectively. The activity of both inhibitors
against NT2/D1 was investigated in detail, vide infra.
The MTT assay indicated that other tumor cell lines (K562, A375 and MCF-7) mostly
keep their metabolic activity after 48 h exposure to examined compounds. Notable IC50 values
(24.2 µM and 30.5 µM) were estimated for carbamate 28 and amide 41 (R1 = R2 = n-Pr)
against MCF-7 cells, while compound 41 was also active against A375 and K562 cells.
Additional methylene group in alkylthio chain induced a complete lack of activity for
compound 42 (Supplementary material). The most active compound against K562 cell line
was iso-butyl derivative 36 with IC50 = 7.7 µM.
High IC50 values (>300 µM) for compounds 28, 30, and 41 against control MRC-5
line, indicated good selectivity of benzothiazole derivatives for cancer cell lines and very low
toxicity against normal human cell line in vitro.
Our newly synthesized aminobenzothiazole derivatives with the observed
antiproliferative activity were investigated for their mechanism of action. We have chosen
two cancer cell lines, moderately sensitive MCF-7 and very sensitive NT2/D1 cells. First we
tested the molecular mechanisms underlying the inhibitory effect of carbamate 28 and amide
41 on the proliferation and viability of MCF-7 cells.
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3.1.2. Treatment of MCF-7 cells with benzothiazole derivatives induced G2/M arrest and
increased expression of cyclin B1
To investigate possible antiproliferative effects we selected IC50 concentration for
compounds 28 and 41 and analyze their effects on cell cycle distribution of MCF-7 at two
time points (24 and 48 h treatments). Applied equipotent concentrations were 25 µM for 28
and 30 µM for 41. Treatment with these compounds exhibited time-dependent effects on
MCF-7 cell cycle progression. Both examined compounds induced the accumulation of cells
in G2/M phase after 24 h treatment. It is followed by gradual increase in apoptotic cell
population (sub-G1 phase) with reduction of cells in both G0/G1 and S cell cycle phases
compared to untreated control cells (Figure 3). Extended 48 h treatment of MCF-7 cells
(Figure 4) resulted in further increase in cell death (sub-G1 phase cells around 50%).
Figure 3. Cell cycle distribution after 24 h treatment (M1 – sub G1, M2 – G0/G1, M3 – S,
M4 – G2/M).
Figure 4. Cell cycle distribution after 48 h treatment (M1 – sub G1, M2 – G0/G1, M3 – S,
M4 – G2/M)
To gain insight into events preceding G2/M arrest of MCF-7 cells, we measured the
expression of cyclin B1, one of the regulatory proteins that control mitosis, during 24 h
treatment with the already applied concentrations of tested compounds. Flow cytometric
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analysis revealed that treatment of MCF-7 cells induced the increase in cyclin B1 expression
(Figure 5), what is in accordance with accumulation of cells in G2/M phase after 24 h.
Figure 5. Level of intracellular cyclin B1 after 24 h treatment of MCF-7 cells with
antiproliferatives 28 and 41
3.1.3. Translocation of cell membrane phosphatidyl serine
The observed apoptosis-inducing effect of investigated compounds was also confirmed by
bivariate Annexin V-FITC/PI flow cytometry. We examined the kinetics of induced apoptosis
at IC50 concentrations of compounds 28 and 41. In line with previous results, 24 h and 48 h
treatments induced significant increase in programmed cell death (Figure 6). Apoptosis
effects were time dependent with the highest number of early apoptotic MCF-7 cells detected
48 h after the treatment (61% and 49% for compounds 28 and 41, respectively). In addition,
low level of late apoptotic/necrotic cells was observed.
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Figure 6. Effects of compounds 28 and 41 on apoptosis in terms of Annexin-V and PI
staining of MCF-7 cells for 24 h (A) and 48 h (B).
3.1.4. Benzothiazole derivatives induced mitochondrial events related to apoptosis in
MCF-7 cells
Data obtained by measuring the translocation of the cell membrane phosphatidyl serine
revealed the increased population of early apoptotic MCF-7 cells upon treatment with
compounds 28 and 41 (Figure 6). Consequently, we examined the observed pro-apoptotic
effects of tested compounds in relation to mitochondrial injury. We performed FACS Rh123
fluorochrome incorporation assay.34 The mitochondrial membrane potential (∆Ψm) changed
slightly after 24 h of MCF-7 cell treatment with both investigated compounds (Figure 7A). In
contrast, a significant loss of ∆Ψm in more than 80% MCF-7 cells, was observed upon 48 h
treatment (Figure 7B) indicating that apoptosis induced by 28 and 41 involved mitochondrial
dysregulation.
Figure 7. Mitochondrial membrane potential (MMP) in MCF-7 cells after 24 h (A) and 48 h
(B) treatment with 28 and 41 compared to control cells’ MMP (violet)
The relative levels of pro-apoptotic proteins such as Bax and anti-apoptotic proteins
such as Bcl-2 determines whether cell death will occur following an apoptotic stimulus. The
Bax/Bcl-2 complex regulates the mitochondrial membrane permeability.35 The overexpression
of the pro-apoptotic Bax induces the loss of the mitochondrial membrane potential that
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initiates the progression of apoptosis. Considering dysregulation of mitochondrial membrane,
we evaluated Bax/Bcl-2 ratio during the treatment of MCF-7 cells with compounds 28 and 41
at single equipotent concentration 24 and 48 h after treatments. FACS analysis performed to
quantify the pattern of Bax and Bcl-2 expressions revealed that incubation with tested
compounds for 24 h resulted in increased protein expression of the mitochondrial pro-
apoptotic protein Bax (Figure 8). On the other hand, exposure to these agents for 24 h did not
significantly modify the expression of Bcl-2. This resulted in slightly increased Bax/Bcl-2
ratio (Figure 8). After 48 h of treatment, protein expression of Bax increased further, while
expression of Bcl-2 decreased, which resulted in significant increase of Bax/Bcl-2 ratio and
the observed apoptosis (Figure 8). Derivative 28 exhibited more than 5-fold increase of
Bax/Bcl-2 ratio compared to untreated cells after 48 h treatment.
Figure 8. Bax/Bcl-2 ratio (fold increase compared to control cells) after 24 and 48 h treatment
of MCF-7 cells with tested benzothiazoles at IC50 concentration. *P < 0.05, **P < 0.01, ***P
< 0.001
To identify the putative involvements of p53 and p73 proteins in pro-apoptotic effect of
benzothiazole derivatives, protein expressions were measured in treated cell line. The
expression of p53 and p73 proteins was measured in MCF-7 cells treated with IC50
concentrations of 28 and 41 using flow cytometry at two time points. Twenty four hour
treatment induced significant p53 protein accumulation and a persistently increased level was
observed up to 48 h (Figure 9A), along with reduction of p73 expression level at both time
points (Figure 9B), implicated that apoptosis induced by examined compounds might be
mediated by p53-dependent pathway.
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Figure 9. p53 (A) and p73 (B) protein level (fold change compared to control) in MCF-7 cells
after benzothiazole treatment.*P < 0.05, **P < 0.01, ***P < 0.001
3.1.5. Benzothiazole derivatives sharply reduced reactive oxygen species (ROS)
production in MCF-7 cell line
Affecting ROS production in tumor cells is one of therapeutic approaches for treating
cancer. Based on the structures of investigated inhibitors with R1S-C(6) possibly rendering
antioxidant properties, we conducted experiments to check whether our compounds scavenge
ROS and would that activity be related to their pro-apoptotic effect. The ROS level was
measured in MCF-7 cells treated with compounds 28 (c = 25 µM) and 41 (c = 30 µM) for 24
and 48 h by flow cytometry using fluorescent dye DCFH-DA. The obtained data
demonstrated that both compounds significantly decrease the ROS level in MCF-7 cells after
24 h treatment (Figure 10) with a persistent reduction of ROS production after 48 h treatment.
In addition, we have tested the effects of sulfoxide and sulfonyl analogues of 28, 39 and 40,
respectively, after 24 h treatment (c = 25 µM). Results demonstrated in Figure 11 show that
these derivatives with higher oxidation state of sulfur exhibited reduction of ROS
accumulation to a lesser extent, which can be ascribed to their reduced antioxidative ability.
Partial ROS suppressing activity of sulfonyl derivative 40 indicated the possibility of thiazole
sulfur’s antioxidative properties. Along with this observation, the RO-C(6) derivative 26 was
checked for its possible antioxidative action (c = 25 µM). This compound, containing only
thiazole sulfur, induced 15% decrease in ROS accumulation compared to control cells after 24
h treatment (Figure 11), thus retaining the potential antioxidative properties. Taken together,
these results showed that ROS affecting activity of examined benzothiazole derivatives is
strongly enhanced with RS-C(6) substituent, however, the thiazole sulfur also contributes to
overall antioxidative action.
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Figure 10. Intercellular ROS level after treatment with 28 and 41 compared to control cells
***P < 0.001
Figure 11. Intracellular ROS level in MCF-7 cells after 24 h treatment with compound 28, its
oxidized analogues 39 and 40, and compound 26 at 25 µM compared to control cells.
In addition, we examined effects on apoptosis after 24 h treatment for sulfoxide and
sulfonyl analogues of 28, the compounds 39 and 40, respectively, at 25 µM (Figure 12). In
spite of the lack of activity shown in vitro in MTT assay (Table 2) derivatives 39 and 40
exhibited significant pro-apoptotic activity against MCF-7 cells. The results indicate that
oxidation of sulfur at RS-C(6) did not affect apoptosis-inducing properties of parent
compound 28.
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Figure 12. Effects of compounds 39 and 40 on apoptosis in terms of Annexin-V and PI
staining of MCF-7 cells upon 24 h treatment
3.1.6. Treatment of NT2/D1 cells with compounds 28 and 30 induced cell detachment
and delayed apoptosis
In order to reveal mechanism of extreme sensitivity of NT2/D1 cells to the compounds
28 and 30 we examined their apoptotic effect using bivariate Annexin V-FITC/PI flow
cytometry. After 24 h treatment with compounds 28 and 30 at IC50 concentration, NT2/D1
apoptotic response was not observed. Interestingly, we observed massive detachment of
NT2/D1 cells seen in cell cultures 24 h after the treatment with both compounds at
concentration of 1 µM (Figure 13).
Figure 13. Detachment of NT2/D1 cells upon 24 h of treatment with 1 µM of compounds 28
and 30
To further investigate the observed massive cell detachment, progression of apoptosis
was analyzed separately in both harvested floating and adherent NT2/D1 cells after 24 h and
48 h treatment with 1 µM of compounds 28 and 30. Time-course of apoptotic events was
similar for both analyzed compounds (Figure 14). In the sub-population of adherent cells, less
than 10% of cells underwent apoptosis in both examined time points (24 h and 48 h
treatments). In contrast, floating cells showed higher apoptotic response. At 24 h time point
~20% of cells were in early apoptosis and ~50% in late apoptosis stage. After 48 h treatment,
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this ratio was increased in favor of late apoptotic cells (~10% early apoptotic cells and ~80%
in late apoptosis) showing that both compounds affect cell adhesion and lead to the rapid
induction of the cell death.
Figure 14. Effects of compounds 28 and 30 on apoptosis of (A) adherent and (B) floating
NT2/D1 cells upon 24 h and 48 h of treatment
Detached cells that underwent apoptosis in later stages, point to the anoikis as a mechanism of
NT2/D1 cell death. To analyze this outcome in more details we preformed trypan blue
exclusion test of cell viability36 and count the number of adherent and floating cells (Table 3).
Experiments carried out with the concentration of 1 µM of both, 28 and 30 compounds,
revealed that a high number of floating cells (42% and 79%, respectively) was present in the
cultures after 24 h of treatment (Table 3); 29% of them were alive after treatment with
compound 28 and even more (61%) after treatment with compound 30. As exposure
proceeded, number of floating cell increased to 69% in 28 treated cultures and up to 91% in
cells treated with compound 30 but number of live floating cells markedly decreased (15% in
28 treatments and 32% in 30 treatments). Table 3. NT2/D1 cell survival and adhesion after
treatment with compounds 28 and 30 counted with trypan blue exclusion assay.
28
30
Live
Live
Time
(h)
Control
Floating
Floating
%
Adherenta Floatinga
floating Adherenta Floatinga
floating
b
b
adherenta
%
c
c
%
%
24
48
52±9
32±10
24±6
23±2
54±4
42
69
29
15
11±3
4±2
42±7
41±5
79
91
61
32
113±13
a number of cells ×104;
b % floating over total cell number;
% live cells over the floating cell number. Data are presented as the mean ± SD of three independent
experiments.
c
These results suggest that tested carbamates 28 and 30 induced NT2/D1 cell growth inhibition
accompanied with detachment from the surface and delayed apoptosis. All these
characteristics present hallmarks of anoikis.37 Anoikis triggered by loss of cell anchorage is of
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great relevance for many physiological processes while anchorage-independency and
resistance to anoikis led to various pathological conditions including metastasis.38
Further examination of antiproliferative effects on NT2/D1 cells showed that 24 h treatment
with 1 µM of compounds 28 and 30 caused accumulation of the cells in G2/M phase
(Supplementary material I). Apoptosis induced in NT2/D1 cells involved mitochondrial
membrane potential reduction and both tested benzothiazole derivatives, 28 and 30,
significantly decreased ROS level after 48 h treatment (Supplementary material I). These
results indicated that carbamate 28 induced very similar pattern of cellular response in both
NT2/D1 and MCF-7 cell lines.
3.1.7. Benzothiazole derivatives significantly inhibited formation and growth of NT2/D1
colonies
Next, we checked the effect of examined compounds and the subsequent loss of
anchorage on malignant capacity of NT2/D1 cells. We investigated the effect of tested
compounds on colony formation and growth, as well as on migration and invasion capacity of
NT2/D1 cells.
Colony assays were performed in order to evaluate the effect of carbamates 28 and 30
on NT2/D1 cells colony formation and on colony growth. In colony forming assay cells were
seeded and immediately treated with compounds at IC50 concentrations, and colonies formed
after seven days were stained and counted. As shown in Figure 15A the treatment with both
compounds led to significant reduction in the number of colonies, as compared with control.
The inhibitory effect of 30 (~70% inhibition) on colony formation of NT2/D1 cells was
higher than that of 28 (~40% inhibition).
In colony growing assay, colonies containing ~10 cells were treated with compounds
28 and 30 and counted when colonies in control untreated NT2/D1 comprised ~50 cells.
Decrease of 45% and 25% in the number of large colonies were detected following the
treatments with compounds 28 and 30, respectively (Figure 15 B). These results showed that
compounds 28 and 30 have inhibitory potential on the formation and growth of NT2/D1 cell
colonies.
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Figure 15. Effects of compounds 28 and 30 on NT2/D1 cells colony formation (A) and
colony growth. (B) Representative colonies are shown. The relative number of colonies was
calculated as a percentage of the number of colonies of untreated NT2/D1 cells that was set as
100%. Results were presented as the means ± SEM of two independent experiments
3.1.8. Cell migration
In order to evaluate the impact of compounds 28 and 30 on the migration potential of
NT2/D1 cells, we used scratch wound healing assay. NT2/D1 cells were grown to near
confluency, wounded and subsequently treated with IC50 concentrations of 28 and 30 or
vehicle control (DMSO). Images of wounds were captured immediately after scratches (0 h)
and 20 h post-wounding to measure the number of cells invading the denuding zone. As
shown in Figure 16, treatments with compounds 28 and 30 decreased migratory potential of
NT2/D1 cells to approximately 60% and 40%, respectively compared to control.
Figure 16. Effects of compounds 28 and 30 on the migratory potential of NT2/D1 cells. The
wound healing assay of NT2/D1 cells treated with vehicle control (NT2/D1), compound 28
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and compound 30. Representative images of the wounds were captured at the indicated time
points. The number of treated cells migrated in the denuded area were presented as the
percentage of the number of untreated NT2/D1 cells that was set as 100%. The error bars
indicate the standard error of the mean. Experiments were performed in triplicate (*P < 0.01).
Scale bar: 100 mm
3.1.9. Invasion potential of NT2/D1 cells after treatment with benzothiazole derivatives
To investigate whether cell detachment induced by compounds 28 and 30 is associated
with metastatic activity of NT2/D1 cells, we examined the invasive potential of NT2/D1 cells
after treatment with tested compounds. NT2/D1 cells were treated with sub-apoptotic IC50
concentrations of 28 and 30 in complete medium for 24 h and seeded to Matrigel coated
transwell inserts in serum-free medium. FBS was used as chemoattractant. Cells were allowed
to invade for 48 h. As shown in Figure 17, both compounds suppressed NT2/D1 cell invasion
reaching 70% inhibition for 28 and even 95% inhibition for compound 30.
Altogether, results obtained by wound healing assay and cell invasion test
demonstrated profound anti-metastatic potential of carbamates 28 and 30 against NT2/D1
cells.
Figure 17. Transwell invasion assay on NT2/D1 cells treated with compounds 28 and 30.
NT2/D1 cells were treated with compounds 28 and 30 in complete culture medium for 24 h
and seeded in serum-free medium to transwell inserts (8 mm pore size) coated with Matrigel.
After 48 h, untreated and treated invading cells were fixed, stained, and counted
microscopically. Representative images of transwell invasion assays were presented. The
relative change in cells invasion was calculated as a percentage of the invasion of untreated
NT2 cells that was set as 100%. Cells were counted from five fields and averages were
calculated. Results were presented as the means ± SEM of two independent experiments.
3.1.10. In vivo NCI acute toxicity determination
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After NCI in vitro screening on 60 cell lines panel, compounds 28 and 30 were
selected for determination of maximum tolerated dose in a nontumored animal toxicity assay.
After i.p. administrated single injection to a female athymic nude mice, examined compounds
proved to be nontoxic at the given doses during observation period of 14 days (Supplementary
material). Compound 28 showed to be nontoxic at 200 mg/kg dose and compound 30 at 400
mg/kg dose.
4. Discussion
Our newly synthesized benzothiazole derivatives showed antiproliferative activity in
vitro against a broad spectrum of human cancer cell lines. Moreover, lack of toxicity of
selected compounds in vitro against MRC-5 cell line as well as in vivo, encouraged us to
investigate the possible mechanistic pathways underlying observed bioactivity. We selected
MCF-7 human breast cell line and NT2/D1 human testicular embryonal carcinoma cells for
further experiments. Parallel investigation was performed for carbamate 28 and amide 41 as
the most potent compounds against selected MCF-7 cell line and for carbamates 28 and 30
against NT2/D1 cells.
The results obtained on MCF-7 cells demonstrate that compounds 28 and 41 caused
the growth inhibition of tumor cells and an apparent block in G2/M cell cycle phase (24 h),
subsequently resulting in massive cell accumulation in sub-G1 phase after 48 h treatment.
Cell cycle progression is mediated by phosphorylation of different substrates by cyclin
dependent kinases (Cdks) at a specific cell cycle phase. Cyclin B1, one of proteins that
regulate cell division is mostly cytoplasmic, and it enters nucleus at the end of G2 phase.39 In
our study the level of cyclin B1 increased after 24 h and the percentage of cells arrested in
G2/M phase was elevated after treatment with two selected compounds, as detected by flow
cytometric analysis.
Cell growth inhibition by early apoptosis is one of the preferred modes of anticancer
action of therapeutics, mostly because of inducing fewer side effects compared to other types
of cell death.40 Presented results indicate that examined benzothiazole derivatives were able to
induce time-dependent apoptosis in the human breast cancer MCF-7 cells. Moreover, insight
into the signaling mechanisms that underlie benzothiazole-induced apoptosis was provided.
Mitochondrial dysfunction could be an early event preceding apoptosis.41 It is characterized
by an increase in mitochondrial membrane permeability and loss of membrane potential.
Indeed, our data revealed that more than 80% of MCF-7 cells upon 48 h treatment with
compounds 28 and 41 exhibited dissipation of mitochondrial membrane potential (Figure 7)
and an increase of Bax/Bcl-2 ratio (Figure 8), confirming the completion of the apoptotic
program upon G2/M arrest.
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It is known, but not yet fully understood, that in a response to a cellular stress p53
molecule plays role in activating the expression and function of numerous pro-apoptotic
genes, typically followed by intrinsic cell death pathway.42 Persistently increased level of p53
up to 48 h in MCF-7 cells treated with examined benzothiazole derivatives may indicate that
apoptosis induced by those compounds is p53 dependent. Unexpectedly, the expression level
of p73, another member of p53 family of transcription factors, decreased compared to control
cells at both measuring time points. We speculate that significant down-regulation of p73
along with remarkable induction of p53 could be explained assuming that some of caspases
cleaved the p73 protein during apoptosis.43 Since MCF-7 cells lack caspase-3,44 the cleavage
of p73 could be mediated by other executioner caspases, -6 or -7.
Production of reactive oxygen species is increased in cancer cells and those species
participate in tumor initiation, progression and maintenance.45 Decreasing intracellular ROS
levels is known method for inhibiting cancer growth, by modulating many physiological
processes that are relevant to cancer growth and ROS are required for those processes.46 In
addition, suppressing ROS production could prevent the ROS from spreading and protect
adjacent healthy cells from oxidative DNA damage, consequently diminishing toxic side
effects of cancer therapy.47 We found that tested compounds significantly decrease the
production of ROS in MCF-7 cells after treatment. A comparative study of 28 and analogues
39 and 40 indicated that RS-C(6) substituent is important for suppressing ROS accumulation
in MCF-7 cells, in addition to thiazole sulfur due to their overall antioxidative contribution.
Considering the obtained results, it is worth to notice that, in spite of functional group
difference between the two test compounds used in this study, carbamate 28 and amide 41,
our findings indicated that both follow the same mechanism of action against MCF-7 cancer
cell line.
The results obtained on NT2/D1 cell line indicated that carbamates 28 and 30 induced
cell growth inhibition and triggered cell detachment from the substrate accompanied with
apoptosis. These sequence of events is characteristic of a particular type of apoptosis,
designated as anoikis.48,49 The occurrence of anoikis is supported by the high number of
floating live cells followed by detected apoptosis.37,49
Cell detachment is probably caused by the aberrant regulation of cell adhesion and
further investigation is needed to elucidate exact mechanism of carbamates’ action used in
this study. Although some differences in the degree of cellular response to compounds 28 and
30 have been detected, it is likely that both compounds engaged the same molecular
mechanisms in NT2/D1 cells. Different cellular responses of MCF-7 and NT2/D1 to
carbamate 28 probably reflects diverse interactions of 28 in different cellular context. It
suggests that cell detachment and delayed apoptosis may be limited to testicular cancer cells
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and does not represent common mechanism in other cancer cells. Nevertheless, tested
compounds induced very similar response in terms of cell cycle arrest, mitochondrial
involvement in apoptosis and ROS affection in both MCF-7 and NT2/D1 cell lines.
Anoikis is potential target for new anticancer therapies approaches as a signal for
cancer cell death induced by cell detachment.50 The only consideration about these
approaches is potential metastatic spread following cell detachment.37 Induction of NT2/D1
cell detachment and observed restriction in cell viability and growth, together with a reduction
in cell invasiveness and ability to migrate, marked compounds 28 and 30 as promising new
compounds that need further validation as potential effective therapeutic agents. Since
NT2/D1 cells showed exceptional sensitivity to the tested carbamates and that compounds 28
and 30 had no cytotoxic effect on control cells and in non-tumored mice, it would be
interesting to analyze the potential synergistic effect of these compounds and cisplatin in
future study. Cisplatin-based chemotherapy is the most efficient treatment of testicular germ
cell tumors51 but its clinical application may be limited due to its toxicity and resistance.52 To
minimize these effects, there is constant search for new combinatorial therapies that could
reduce common side effects of platinum based chemotherapeutic drugs and its effective
dose.53 Further studies would evaluate the potential use of compound 28 and/or 30 in
combinatorial treatment of testicular carcinoma.
In a last few years testicular carcinoma NT2/D1 cells are being considered as cancer
stem cells (CSC), a subpopulation of cancer cells responsible for tumor growth, progression
and its metastatic spreading.54,55 CSCs are considered responsible for tumor resistance to
chemo- and radiotherapies and consequent failure of conventional therapeutic approaches.54
Therefore, targeting unique features of CSCs is another ultimate goal of cancer treatment
research. These approaches include pluripotency restriction, induction of differentiation and
sensitization to particular compound.56 To elucidate whether tested carbamates 28 and 30 may
be candidates for these strategies further investigations are needed.
5. Conclusion
Synthesized benzothiazole derivatives showed good potency against proliferation of various
cancer cell lines in vitro. In this study it was demonstrated that synthesized propyl [6-
(propylsulfanyl)-1,3-benzothiazol-2-yl]carbamate
28
and
N-[6-(propylsulfanyl)-1,3-
benzothiazol-2-yl]pentanamide 41, selected for mechanistic evaluation of activity against
MCF-7, exhibit their antiproliferative activity by blocking cell cycle in G2/M phase and by
promoting apoptosis. Apoptotic cell death was further determined with mitochondrial
membrane potential reduction and increased Bax/Bcl-2 ratio after treatment of MCF-7 cells
with selected benzothiazoles, along with induction of p53. While the most of strategies for
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cancer therapies based on affecting ROS and apoptosis consider induction of ROS
production,57 compounds tested in this study showed significant reduction of ROS
accumulation. In addition, two most potent derivatives, carbamate 28 and propyl [6-
(butylsulfanyl)-1,3-benzothiazol-2-yl]carbamate 30 exhibited significant antiproliferative
activity against NT2/D1 cell line. The massive detachment of the NT2/D1 cells after
treatment with compounds 28 and 30 accompanied with apoptosis and inhibitory activity
against migration and invasiveness of these cells were shown. Moreover, derivatives 28, 30
and 41 were nontoxic against normal MRC-5 cells in vitro, and the most potent compounds
28 and 30 showed no toxicity in non-tumored animal toxicity assay. Further studies are
needed to clarify the precise targets for antiproliferative and antimigratory activity of
aminobenzothiazole derivatives on NT2/D1 cells, as well as new aspects for possible
improvement of current therapies.
6. Experimental section
6.1. General information
Melting points were determined on a Boetius PMHK apparatus and were not corrected. IR
spectra were taken on a Thermo-Scientific Nicolet 6700 FT-IR diamond crystal. NMR: 1H
and 13C NMR spectra were recorded on a Bruker Ultrashield Advance III spectrometer (at
500 and 125 MHz, respectively) in the indicated solvent using TMS as the internal standard.
Chemical shifts are expressed in ppm (δ) values, and coupling constants (J), in Hz. ESI MS
spectra of the synthesized compounds were recorded on an Agilent Technologies 6210 time-
of-flight LC/MS instrument in positive ion mode using CH3CN/H2O = 1/1 with 0.2%
HCOOH as the carrying solvent solution. The samples were dissolved in pure methanol
(HPLC grade). The selected values were as follows: capillary voltage = 4 kV; gas temperature
350 °C; drying gas = 12 L min−1; nebulizer pressure = 45 psig; and fragmentator voltage = 70
V. The flash chromatography was performed on Biotage SP1 system equipped with UV
detector and FLASH 12+, FLASH 25+ or FLASH 40+ columns charged with KP-SIL (40 –
63 µm, pore diameter 60 Å), KP-C18-HS (40 – 63 µm, pore diameter 90 Å) or KP-NH (40 –
63 µm, pore diameter 100 Å) as an adsorbent. Compounds were analyzed for purity (HPLC)
using Agilent 1200 HPLC system equipped with a Quat Pump (G1311B), an injector
(G1329B) 1260 ALS, TCC 1260 ( G1316A) and a detector 1260 DAD VL+ (G1315C). All
tested compounds are fully characterized and the purities were > 95% as determined by HPLC
(Supplementary material). HPLC analysis was performed in two diverse systems for each
compound. Compounds were dissolved in methanol, final concentrations were ~ 1mg/mL.
Applied HPLC methods were as follows.
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Method A. Zorbax Eclipse Plus C18 4.6 × 150mm, 1.8µ, S.N. USWKY01594 was used as
the stationary phase. Eluent was made of the following solvents: 0.2% formic acid in water
(A) and acetonitrile (B). The analysis were performed at 280 nm for compounds 23 – 26 and
29 – 31; at 290 nm for compounds 28, 33, 34 and 41 and at 320 nm for compounds 42 – 46.
Flow rate was 0.5 mL/min.
Method B. Zorbax Eclipse Plus C18 4.6 × 150mm, 1.8µ, S.N. USWKY01594 was used as
the stationary phase. Eluent was made of the following solvents: 0.2% formic acid in water
(A) and methanol (B). The analysis were performed at 280 nm for compounds 24 – 26 and 29
– 31; at 290 nm for compounds 28, 34 and 41 and at 320 nm for compounds 32 and 42 – 46.
Flow rate was 0.5 mL/min.
Method C. Zorbax Eclipse Plus C18 2.1 × 100mm, 1.8µ, S.N. USUXU04444 was used as the
stationary phase. Eluent was made of the following solvents: water (A) and methanol (B). The
analysis were performed at 320 nm for compound 32. Flow rate was 0.5 mL/min.
Method D. Poroshell 120 EC-C18, 4.6 × 50mm, 2.7µ, S.N. USCFU07797 was used as the
stationary phase. Eluent was made of the following solvents: 0.2% formic acid in water (A)
and acetonitrile (B). The analysis were performed at 290 nm for compound 33 and 280 nm for
compounds 27 and 38. Flow rate was 1 mL/min.
Method E. Zorbax Eclipse Plus C18 4.6 × 150mm, 1.8µ, S.N. USWKY01594 was used as
the stationary phase. Eluent was made of the following solvents: water (A) and acetonitrile
(B). The analysis were performed at 280 nm for compound 37 and at 290 nm for compounds
35 and 36. Flow rate was 0.5 mL/min.
Method F. Zorbax Eclipse Plus C18 4.6 × 150mm, 1.8µ, S.N. USWKY01594 was used as the
stationary phase. Eluent was made of the following solvents: water (A) and methanol (B). The
analysis were performed at 290 nm for compounds 35 and 36 and at 280 nm for compound
37. Flow rate was 0.5 mL/min.
Method G. Zorbax Eclipse Plus C18 2.1 × 100mm, 1.8µ, S.N. USUXU04444 was used as the
stationary phase. Eluent was made of the following solvents: 0.2% formic acid in water (A)
and methanol (B). The analysis were performed at 295 nm for compounds 39 and 40. Flow
rate was 0.5 mL/min.
Method H. Zorbax Eclipse Plus C18 2.1 × 100mm, 1.8µ, S.N. USUXU04444 was used as the
stationary phase. Eluent was made of the following solvents: 0.2% formic acid in water (A)
and acetonitrile (B). The analysis were performed at 295 nm for compound 39. Flow rate was
0.5 mL/min.
Method I. Poroshell 120 EC-C18, 4.6 × 50mm, 2.7µ, S.N. USCFU07797 was used as the
stationary phase. Eluent was made of the following solvents: 0.2% formic acid in water (A)
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and methanol (B). The analysis were performed at 280 nm for compounds 27 and 38 and 295
nm for compound 40. Flow rate was 0.5 mL/min.
6.2. Chemical synthesis
6.2.1. General procedure C for synthesis of (6-Substituted-1,3-benzo[d]thiazol-2-
yl)carbamates 24 – 26, 29, 31 and 32
A solution of bromine (1.25 eq) in glacial acetic acid was added to a stirring mixture of an
appropriate 4-substituted aniline (1 eq), ammonium thiocyanate (4 eq), acetic acid and water
at 10 °C. The reaction mixture was stirred at room temperature for 18 h, and then at 80 °C for
3 h. After cooling to room temperature, the reaction mixture was poured onto water and
Na2CO3 was added in order to adjust pH to 5-6. The reaction mixture was extracted with ethyl
acetate, layers were separated and organic layer was washed with brine. Organic layer was
dried over anhydrous Na2SO4, filtered and evaporated to dryness. The crude product 6-
substituted aminobenzothiazole was used in the next reaction step. An appropriate alkyl
chloroformate (1.1 eq) and triethylamine (1.8 eq) were added to a solution of 6-substituted
aminobenzothiazole in benzene. The reaction mixture was stirred at 80°C for 3 h, and then
poured onto cold water and extracted with ethyl acetate. Combined organic layers were dried
over anhydrous Na2SO4, filtered and evaporated to dryness. The crude product was further
purified in a manner provided for each compound.
6.2.1.1. Ethyl [6-(ethylsulfanyl)-1,3-benzothiazol-2-yl]carbamate (24). Yield 40%. M.p. =
(162 – 165) °C. IR (ATR): 3432w, 3400w, 3162m, 3124m, 3079m, 3045m, 2973s, 2924s,
2776m, 1720s, 1599s, 1557s, 1444s, 1364m, 1290s, 1250s, 1119m, 1070m, 1048m, 1021w,
1
820m, 757m cm−1. H NMR (500 MHz, (CD3)2SO, δ): 7.96 (d, J = 1.6 Hz, 1H), 7.61 (d, J =
8.4 Hz, 1H), 7.36 (dd, J1 = 8.4 Hz, J2 = 1.8 Hz, 1H), 4.24 (q, J = 7.1 Hz, 2H), 2.98 (q, J = 7.3
Hz, 2H), 1.28 (t, J = 7.1 Hz, 3H), 1.22 (t, J = 7.3 Hz, 3H). 13C NMR (125 MHz, (CD3)2SO, δ):
159.53, 153.88, 147.84; 132.61, 130.26, 127.58, 121.85, 120.52, 61.94, 27.48, 14.30. (+)ESI-
HRMS: m/z 283.05655 corresponds to molecular formula C12H14N2O2S2H+ (error, -1.38
ppm). HPLC purity, method A: tR = 8.695, area 99.27%. Method B: tR = 9.906, area 96.29%.
6.2.1.2. Ethyl [6-(propylsulfanyl)-1,3-benzothiazol-2-yl]carbamate (25). Yield 21%. M.p.
= (158 – 162) °C. IR (ATR): 3135w, 3076w, 2957m, 2909m, 2869m, 2782m, 1725s, 1597s,
1561s, 1453s, 1428m, 1270s, 1243s, 1110m, 1069m, 1045m, 1022m, 816m, 759m, 707m
cm−1. 1H NMR (500 MHz, (CD3)2SO, δ): 12.00 (bs, 1H), 7.96 (d, J = 1.4 Hz, 1H), 7.60 (d, J =
8.5 Hz, 1 H), 7.36 (dd, J1 = 8.5 Hz, J2 = 1.8 Hz, 1H), 4.24 (q, J = 7.1 Hz, 2H), 2.94 (t, J = 7.1
Hz, 2H), 1.61 – 1.54 (m, 2H), 1.27 (t, J = 7.1 Hz, 3H), 0.96 (t, J = 7.3 Hz, 3H). 13C NMR (50
MHz, (CD3)2SO, δ): 159.85, 154.23, 148.00, 132.89, 130.76, 127.84, 122.09, 120.78, 62.20,
35.57, 22.22, 14.52, 13.34. (+)ESI-HRMS: m/z 297.07191 corresponds to molecular formula
23

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C13H16N2O2S2H (error, -2.30 ppm). HPLC purity, method A: tR = 9.194, area 99.67%.
Method B: tR = 10.528, area 99.44%.
6.2.1.3. Propyl (6-ethoxy-1,3-benzothiazol-2-yl)carbamate (26). Yield 25%. M.p. = (168 –
169) °C. IR (ATR): 3161w, 3081m, 2977s, 2933m, 2802m, 1718s, 1612s, 1562s, 1463s,
1
1391m, 1272s, 1242s, 1212s, 1113m, 1057s, 971w, 941m, 792m, 760m cm−1. H NMR (500
MHz, (CD3)2SO, δ): 11.83 (bs, 1H), 7.56 (d, J = 8.9 Hz, 1H), 7.51 (d, J = 2.5 Hz, 1H), 6.97
(dd, J1 = 8.7 Hz, J2 = 2.5 Hz, 1H), 4.14 (t, J = 6.6 Hz, 1H), 4.04 (q, J = 7.0 Hz, 2H), 1.69 –
1.62 (m, 2H), 1.33 (t, J = 6.9 Hz, 3H), 0.93 (t, J = 7.5 Hz, 3H). 13C NMR (125 MHz,
(CD3)2SO, δ): 157.46, 155.12, 143.28, 132.76, 120.81, 114.97, 105.47, 67.16, 63.62, 21.73,
14.70, 10.13. (+)ESI-HRMS: m/z 281.09541 corresponds to molecular formula
C13H16N2O3SH+ (error, -0.10 ppm). HPLC purity, method A: tR = 8.520, area 99.48%. Method
B: tR = 9.741, area 96.69%.
6.2.1.4. Ethyl [6-(butylsulfanyl)-1,3-benzothiazol-2-yl]carbamate (29). Yield 19%. M.p. =
(138 – 140) °C. IR (ATR): 3139m, 3072s, 2983s, 2954s, 2931s, 2865s, 2794s, 1724s, 1603s,
1570s, 1452s, 1366m, 1340m, 1313m, 1293s, 1274s, 1250s, 1113m, 1070m, 1048m, 1022m,
1
816s, 781m, 760s, 708m cm−1. H NMR (500 MHz, CDCl3, δ): 11.90 (bs, 1H), 7.86 (d, J =
8.6 Hz, 1H), 7.78 (d, J = 1.7 Hz, 1H), 7.40 (dd, J1 = 8.4 Hz, J2 = 1.8 Hz, 1H), 4.41 (q, J = 7.3
Hz, 2H), 2.96 (t, J = 7.5 Hz, 2H), 1.67 – 1.61 (m, 2H), 1.50 – 1.41 (m, 5H), 0.93 (t, J = 7.3
Hz, 3H). 13C NMR (125 MHz, (CD3)2SO, δ): 161.39, 153.81, 147.21, 132.40, 131.82, 128.33,
122.21, 120.81, 62.88, 34.73, 31.28, 21.89, 14.46, 13.63. (+)ESI-HRMS: m/z 311.08801
corresponds to molecular formula C14H18N2O2S2H+ (error, -0.76 ppm).HPLC purity, method
A: tR = 9.757, area 97.08%. Method B: tR = 11.594, area 96.14%.
6.2.1.5. Butyl [6-(propylsulfanyl)-1,3-benzothiazol-2-yl]carbamate (31). Yield 28%. M.p.
= (74 – 80) °C. IR (ATR): 3170m, 3070m, 2961s, 2931s, 2872m, 1721s, 1602s, 1562s,
1456m, 1293s, 1248s, 1074w, 814w, 762w cm−1. 1H NMR (500 MHz, (CD3)2SO, δ): 7.94 (d,
J = 1.6 Hz, 1H), 7.60 (d, J = 8.3 Hz, 1H), 7.35 (dd, J1 = 8.5 Hz, J2 = 1.8 Hz, 1H), 4.19 (t, J =
6.6 Hz, 2H), 2.94 (t, J = 7.1 Hz, 2H), 1.65 – 1.53 (m, 4H), 1.41 – 1.35 (m, 2H), 0.95 (t, J = 7.3
Hz, 3H), 0.91 (t, J = 7.5 Hz, 3H). 13C NMR (125 MHz, (CD3)2SO, δ): 159.60, 154.04, 147.74,
132.63, 130.53, 127.60, 121.84, 120.51, 65.59, 35.43, 30.34, 22.02, 18.50, 13.55, 13.11.
(+)ESI-HRMS: m/z 325.10391 corresponds to molecular formula C15H20N2O2S2H+ (error,
+0.05 ppm). HPLC purity, method A: tR = 10.346, area 99.78%. Method B: tR = 11.916, area
99.36%.
6.2.1.6. Propyl [6-(ethylsulfanyl)-1,3-benzothiazol-2-yl]carbamate (32). Yield 27%. M.p.
= (137-138) °C. IR (ATR): 3062m, 2968s, 2922s, 1717s, 1600m, 1562m, 1445m, 1396w,
1
1287m, 1248m, 1072w, 1044w, 759w cm−1. H NMR (500 MHz, (CD3)2SO, δ): 12.01 (bs,
1H), 7.95 (d, J = 1.8 Hz, 1H), 7.61 (d, J = 8.5 Hz, 1H), 7.36 (dd, J1 = 8.5 Hz, J2 = 1.8 Hz,
24

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1H), 4.15 (t, J = 6.8 Hz, 1H), 2.98 (q, J = 7.3 Hz, 2H), 1.70 – 1.63 (m, 2H), 1.22 (t, J = 7.3
Hz, 3H), 0.93 (t, J = 7.5 Hz, 3H). 13C NMR (125 MHz, (CD3)2SO, δ): 160.13, 154.54, 148.24,
133.06, 130.67, 128.02, 122.27, 120.93, 67.73, 27.93, 22.12, 14.74, 10.56. (+)ESI-HRMS:
m/z 297.07243 corresponds to molecular formula C13H16N2O2S2H+ (error, -0.57 ppm). HPLC
purity, method B: tR = 12.996, area 95.61%. Method C: tR = 14.178, area 95.09%.
6.2.2. General procedure D for synthesis of compounds 28, 30 and 33 – 37
An appropriate alkyl chloroformate (1.1 eq) and triethylamine (1.8 eq) were added to a
solution of corresponding 6-(alkylsulfanyl)-1,3-benzothiazol-2-amine (1 eq) in benzene. After
3 h of stirring at 80 °C the reaction mixture was poured onto water and extracted with ethyl
acetate. Combined organic layers were dried over anhydrous Na2SO4, filtered and evaporated
to dryness. The crude product was subjected to silica gel column chromatography and silica
gel flash chromatography, Biotage SP1, using hexane/ethyl acetate as eluent to afford the final
product.
6.2.2.1. Propyl [6-(propylsulfanyl)-1,3-benzothiazol-2-yl]carbamate (28). Yield 47%. M.p.
= (138 – 140) °C. IR (ATR): 3167m, 3062m, 2960s, 2932m, 2876m, 1724s, 1598s, 1562s,
1
1449m, 1447s, 1308m, 1273s, 1244s, 1047m, 962w, 888w, 805m, 782m, 755m cm−1. H
NMR (500 MHz, (CD3)2SO, δ): 12.01 (bs, 1H), 7.96 (d, J = 1.7 Hz, 1H), 7.60 (d, J = 8.4 Hz,
1H), 7.36 (dd, J1 = 8.4 Hz, J2 = 1.9 Hz, 1H), 4.15 (t, J = 6.7 Hz, 2H), 2.94 (t, J = 7.2 Hz, 2H),
1.70
̶
1.63 (m, 2H), 1.61-1.54 (m, 2H), 0.98-0.92 (m, 6H). 13C NMR (125 MHz, (CD3)2SO,
δ): 159.54, 154.00, 147.74, 132.61, 130.49, 127.58, 121.82, 120.49, 67.28, 35.38, 21.99,
21.68, 13.09, 10.10. (+)ESI-HRMS: m/z 311.08810 corresponds to molecular formula
C14H18N2O2S2H+ (error, -0.47 ppm). HPLC purity, method A: tR = 11.532, area 98.23%.
Method B: tR = 13.159, area 98.53%.
6.2.2.2. Propyl [6-(butylsulfanyl)-1,3-benzothiazol-2-yl]carbamate (30). Yield 29%. M.p.
= 130 °C. IR (ATR): 3169m, 3068m, 2960s, 2926s, 2785m, 1725s, 1601m, 1564m, 1451m,
1
1288m, 1247m, 1070w, 818w, 752w cm−1. H NMR (500 MHz, CDCl3, δ): 11.35 (bs, 1H),
7.82 (d, J = 8.5 Hz, 1H), 7.78 (d, J = 1.4 Hz, 1H), 7.40 (dd, J1 = 8.5 Hz, J2 = 1.8 Hz, 1H), 4.30
(t, J = 6.8 Hz, 2H), 2.96 (t, J = 7.4 Hz, 2H), 1.84
̶ 1.77 (m, 2H), 1.67 – 1.61 (m, 2H), 1.50 –
1.42 (m, 2H), 0.99 (t, J = 7.5 Hz, 3H), 0.92 (t, J = 7.3 Hz, 3H). 13C NMR (125 MHz, CDCl3,
δ): 161.06, 153.80, 147.35, 132.54, 131.83, 128.42, 122.29, 120.83, 68.53, 34.77, 31.31,
22.14, 21.91, 13.65, 10.32. (+)ESI-HRMS: m/z 325.10408 corresponds to molecular formula
C15H20N2O2S2H+ (error, +0.56 ppm). HPLC purity, method A: tR = 10.371, area 96.76%.
Method B: tR = 12.388, area 98.58%.
6.2.2.3. Ethyl [6-(pentylsulfanyl)-1,3-benzothiazol-2-yl]carbamate (33). Yield 21%. M.p. =
(137 – 138) °C. IR (ATR): 3175m, 3152m, 3123m, 3058m, 2956s, 2924s, 2854s, 1724s,
1
1597s, 1550s, 1460s, 1370m, 1296s, 1241s, 1069m, 818m, 766m cm−1. H NMR (500 MHz,
25

ACCEPTED MANUSCRIPT
CDCl3, δ): 11.60 (bs, 1H), 7.84 (d, J = 8.5 Hz, 1H), 7.78 (d, J = 1.6 Hz, 1H), 7.40 (dd, , J1 =
8.5 Hz, J2 = 1.6 Hz, 1H), 4.40 (q, J = 7.1 Hz, 2H), 2.95 (t, J = 7.5 Hz, 2H), 1.69 – 1.63 (m,
2H), 1.45 – 1.39 (m, 5H), 1.37 – 1.29 (m, 2H), 0.89 (t, J = 7.2 Hz, 3H). 13C NMR (125 MHz,
CDCl3, δ): 161.19, 153.75, 147.25, 132.47, 131.85, 128.37, 122.22, 120.82, 62.90, 35.01,
30.93, 28.88, 22.23, 14.46, 13.94. (+)ESI-HRMS: m/z 325.10435 corresponds to molecular
formula C15H20N2O2S2H+ (error, +1.40 ppm). HPLC purity, method A: tR =12.206, area
98.52%. Method D: tR = 4.563, area 98.28%.
6.2.2.4. Propyl [6-(pentylsulfanyl)-1,3-benzothiazol-2-yl]carbamate (34). Yield 40%. M.p.
= (116 – 118) °C. IR (ATR): 3170m, 3127m, 3062m, 2956s, 2923s, 2853s, 2784m, 1725s,
1
1601s, 1562s, 1451m, 1393m, 1309m, 1289s, 1248s, 1069m, 821m, 752m cm−1. H NMR
(500 MHz, CDCl3, δ): 11.72 (bs, 1H), 7.84 (d, J = 8.6 Hz, 1H), 7.78 (d, J = 1.5 Hz, 1H), 7.40
(dd, J1 = 8.4 Hz, J2 = 1.8 Hz, 1H), 4.30 (t, J = 6.8 Hz, 2H), 2.94 (t, J = 7.4 Hz, 2H), 1.84 –
1.77 (m, 2H), 1.69 – 1.63 (m, 2H), 1.45 – 1.39 (m, 2H), 1.36 – 1.29 (m, 2H), 0.99 (t, J = 7.5
Hz, 3H), 0.89 (t, J = 7.2 Hz, 3H). 13C NMR (125 MHz, CDCl3, δ): 161.31, 153.88, 147.26,
132.43, 131.80, 128.37, 122.23, 120.77, 68.50, 35.03, 30.92, 28.87, 22.22, 22.11, 13.93,
10.30. (+)ESI-HRMS: m/z 339.12006 corresponds to molecular formula C16H22N2O2S2H+
(error, +1.51 ppm). HPLC purity, method A: tR = 13.079, area 96.82%. Method B: tR =
14.812, area 95.51%.
6.2.2.5. Ethyl {6-[(2-methylpropyl)sulfanyl]-1,3-benzothiazol-2-yl}carbamate (35). Yield
23%. M.p. = (159 – 160) °C. IR (ATR): 3139m, 3081m, 2968s, 2914s, 2866m, 1722s, 1599s,
1
1560s, 1458m, 1275s, 1246s, 1111m, 1070m, 1049m, 1019m, 820m, 789m, 762m cm−1. H
NMR (500 MHz, CDCl3, δ): 11.47 (bs, 1H), 7.84 (d, J = 8.5 Hz, 1H), 7.77 (d, J = 1.6 Hz,
1H), 7.40 (dd, J1 = 8.5 Hz, J2 = 1.8 Hz, 1H), 4.41 (q, J = 7.1 Hz, 2H), 2.85 (d, J = 6.9 Hz,
2H), 1.88 (sep, J = 6.7 Hz, 1H), 1.42 (t, J = 7.1 Hz, 3H), 1.05 (d, J = 6.6 Hz, 6H). 13C NMR
(125 MHz, CDCl3, δ): 161.25, 153.77, 147.18, 132.44, 132.27, 128.30, 122.13, 62.88, 44.10,
28.32, 22.01, 14.46. (+)ESI-HRMS: m/z 311.08796 corresponds to molecular formula
C14H18N2O2S2H+ (error, -0.92 ppm). HPLC purity, method E: tR = 12.189, area 99.00%.
Method B: tR = 13.873, area 99.59%.
6.2.2.6. Propyl {6-[(2-methylpropyl)sulfanyl]-1,3-benzothiazol-2-yl}carbamate (36). Yield
62%. M.p. = (139 – 141) °C. IR (ATR): 3172m, 3131m, 3074m, 2919s, 2852m, 1722s,
1
1599m, 1565m, 1456m, 1282s, 1245s, 1074m, 818m, 760m cm−1. H NMR (500 MHz,
CDCl3, δ): 11.60 – 11.58 (m, 1H), 7.83 (d, J = 8.4 Hz, 1H), 7.77 (d, J = 1.6 Hz, 1H), 7.40 (dd,
J1 = 8.4 Hz, J2 = 1.8 Hz, 1H), 4.30 (t, J = 6.8 Hz, 2H), 2.85 (d, J = 6.8 Hz, 2H), 1.91 – 1.77
(m, 3H), 1.05 (d, J = 6.6 Hz, 6H), 0.99 (t, J = 7.43 Hz, 3H). 13C NMR (125 MHz, CDCl3, δ):
161.18, 153.85, 147.23, 132.47, 132.25, 128.33, 122.16, 120.79, 68.49, 44.11, 28.31, 22.00,
10.30. (+)ESI-HRMS: m/z 325.10387 corresponds to formula C15H20N2O2S2H+ (error, -0.08
26

ACCEPTED MANUSCRIPT
ppm). HPLC purity, method E: tR = 12.787, area 98.08%. Method F: tR = 14.618, area
99.74%.
6.2.2.7. Butyl [6-(butylsulfanyl)-1,3-benzothiazol-2-yl]carbamate (37). Yield 32%. M.p. =
(120-121) °C. IR (ATR): 3143m, 3077m, 2953s, 2928s, 2866m, 1727s, 1598s, 1452m,
1
1276m, 1246m, 1108w, 1074w, 820m, 782w, 756m, cm−1. H NMR (500 MHz, CDCl3, δ):
11.64 – 11.41 (m, 1H), 7.84 – 7.81 (m, 1H), 7.78 (d, J = 1.5 Hz, 1H), 7.40 (dd, J1 = 8.6 Hz, J2
= 1.7 Hz, 1H), 4.35 (t, J = 6.7 Hz, 2H), 2.96 (t, J = 7.4 Hz, 2H), 1.79 – 1.73 (m, 2H), 1.67 –
1.61 (m, 2H), 1.50 – 1. 38 (m, 4H), 0.97 – 0.91 (m, 6H). 13C NMR (125 MHz, CDCl3, δ):
161.50, 153.95, 147.22, 132.39, 131.77, 128.33, 122.25, 120.76, 66.76, 34.75, 31.28, 30.76,
21.88, 18.99, 13.66. (+)ESI-HRMS: m/z 339.12048 corresponds to molecular formula
C16H22N2O2S2H+ (error, +2.77 ppm). HPLC purity, method E: tR = 12.749, area 95.92%.
Method F: tR =14.577, area 98.25%.
6.2.3. General procedure E for synthesis of N-[6-(Propylsulfanyl)-1,3-benzothiazol-2-
yl]alkanamides 41 – 46
The alkanoyl chlorides were prepared according to known procedures using an appropriate
commercially available acids and thionyl chloride as starting materials.58 A solution of an
appropriate alkanoyl chloride (1.3 eq) in benzene was added dropwise into the solution of
corresponding aminobenzothiazole (1 eq) (19 or 20) in CH2Cl2/benzene (1:1, v/v) at 0 °C. The
reaction mixture was stirred at the same temperature until consumption of starting
aminobenzothiazole (TLC control). The reaction was quenched with cold water. The layers
were separated and the aqueous layer was extracted with CH2Cl2. The combined organic
layers were dried over anhydrous Na2SO4, filtered and evaporated to dryness. The crude
product was subjected to a multiple column chromatography to afford desired compound.
6.2.3.1. N-[6-(propylsulfanyl)-1,3-benzothiazol-2-yl]pentanamide (41). Yield 21%. M.p. =
113 °C. IR (ATR): 3276m, 3178m, 3128m, 3064m, 2960s, 2930m, 2870m, 1660s, 1594s,
1
1538s, 1439m, 1374w, 1345m, 1295m, 1266m, 1192w, 1087w, 815w, 774w cm−1. H NMR
(500 MHz, CDCl3, δ): 10.66 (bs, 1H), 7.81 (d, J = 1.6 Hz, 1H), 7.65 (d, J = 8.5 Hz, 1H), 7.44
(dd, J1 = 8.5 Hz, J2 = 1.8 Hz, 1H), 2.94 (t, J = 7.3 Hz, 2H), 2.46 (t, J = 7.6 Hz, 2H), 1.73
̶
1.66 (m, 4H), 1.37
̶
1.30 (m, 2H), 1.04 (t, J = 7.4 Hz, 3H), 0.88 (t, J = 7.3 Hz, 3H). 13C NMR
(125 MHz, CDCl3, δ): 171.64, 158.91, 146.43, 132.89, 132.38, 128.68, 122.46, 120.58, 36.91,
36.26, 26.97, 22.54, 22.19, 13.64, 13.38. (+)ESI-HRMS: m/z 309.10813 corresponds to
molecular formula C15H20N2OS2H+ (error, -2.76 ppm). HPLC purity, method A: tR = 11.671,
area 97.05%. Method B: tR = 13.245, area 98.01%.
6.2.3.2. N-[6-(butylsulfanyl)-1,3-benzothiazol-2-yl]pentanamide (42). Yield 28%. M.p. =
117 °C. IR (ATR): 3144m, 3116m, 3036m, 2958s, 2927s, 2870s, 1694s, 1590s, 1542s,
1443m, 1380m, 1349m, 1306w, 1269s, 1172m, 1099w, 1052w, 976w, 892w, 810m, 769w
27

−1 1
ACCEPTED MANUSCRIPT
cm . H NMR (500 MHz, CDCl3, δ): 10.15 (bs, 1H), 7.80 (d, J = 1.6 Hz, 1H), 7.65 (d, J = 8.5
Hz, 1H), 7.43 (dd, J1 = 8.5 Hz, J2 = 1.8 Hz, 1H), 2.98 – 2. 95 (m, 2H), 2.49 – 2. 46 (m, 2H),
1.74 – 1.68 (m, 2H), 1.67 – 1.61 (m, 2H), 1.50 – 1.34 (m, 4H), 0.94 – 0.89 (m, 6H). 13C NMR
(125 MHz, CDCl3, δ): 171.61, 158.86, 146.39, 132.87, 132.45, 128.53, 122.31, 120.55, 36.22,
34.53, 31.21, 26.94, 22.16, 21.86, 13.61, 13.59. (+) ESI-HRMS: m/z 323.12407 corresponds
to molecular formula C16H22N2OS2H+ (error, -1.73). HPLC purity, method A: tR = 12.200,
area 96.72%. Method B: tR =13.409, area 98.18%.
6.2.3.3. N-[6-(butylsulfanyl)-1,3-benzothiazol-2-yl]-2-methoxyacetamide (43). Yield 45%.
M.p. = 62 °C. IR (ATR): 3382w, 3207w, 2956m, 2929m, 2871w, 1703m, 1594m, 1537s,
1
1448m, 1272m, 1196w, 1119m, 994w, 817w, 745w cm−1. H NMR (500 MHz, CDCl3, δ):
9.86 (bs, 1H), 7.79 – 7.78 (m, 1H), 7.68 (d, J = 8.5 Hz, 1H), 7.43 (dd, J1 = 8.5 Hz, J2 = 1.8
Hz, 1H), 4.16 (s, 2H), 3.52 (s, 3H), 2.97 – 2.94 (m, 2H), 1.67 – 1.61 (m, 2H), 1.49 – 1.42 (m,
2H), 0.92 (t, J = 7.3 Hz, 3H). 13C NMR (125 MHz, CDCl3, δ): 168.07, 156.52, 146.85,
133.03, 132.65, 128.58, 122.18, 121.26, 71.25, 59.57, 34.55, 31.24, 21.89, 13.61. (+) ESI-
HRMS: m/z 311.08743 corresponds to molecular formula C14H18N2O2S2H+ (error, -2.62
ppm). HPLC purity, method A: tR = 10.999, area 98.02%. Method B: tR = 12.336, area
98.37%.
6.2.3.4. 3-Methoxy-N-[6-(propylsulfanyl)-1,3-benzothiazol-2-yl]propanamide (44). Yield
30%. M.p. = 111 °C. IR (ATR): 3270w, 3118m, 3038m, 2962s, 2922s, 2811m, 1704m, 1591s,
1
1544s, 1447m, 1394m, 1270s, 1174m, 1120m, 1067m, 810m cm−1. H NMR (500 MHz,
CDCl3, δ): 10.26 (bs, 1H), 7.79 (d, J = 1.8 Hz, 1H), 7.69 (d, J = 8.5 Hz, 1H), 7.43 (dd, J1 =
8.5 Hz, J2 = 1.8 Hz, 1H), 3.76 (t, J = 5.5 Hz, 2H), 3.48 (s, 1H), 2.93 (t, J = 7.3 Hz, 2H), 2.77
(t, J = 5.5 Hz, 2H), 1.71
̶
1.64 (m, 2H), 1.03 (t, J = 7.3 Hz, 3H).13C NMR (125 MHz, CDCl3,
δ): 169.86, 157.57, 146.97, 133.08, 132.12, 128.67, 122.39, 121.02, 67.67, 59.20, 36.96,
36.90, 22.56, 13.37. (+)ESI-HRMS: m/z 311.08741 corresponds to molecular formula
C14H18N2O2S2H+ (error, -2.67 ppm). HPLC purity, method A: tR = 10.325, area 98.09%.
Method B: tR = 11.326, area 99.00%.
6.2.3.5. N-[6-(butylsulfanyl)-1,3-benzothiazol-2-yl]-3-methoxypropanamide (45). Yield
32%. M.p. = 99 °C. IR (ATR): 3147s, 3046m, 2953s, 2924s, 2875s, 2814m, 1703s, 1592s,
1536s, 1451m, 1417m, 1395m, 1332m, 1267s, 1160s, 1116s, 1068m, 988w, 960m, 808m,
793m, 757m cm−1. 1H NMR (500 MHz, CDCl3, δ): 10.33 (bs, 1H), 7.78 (d, J = 1.6 Hz, 1H),
7.69 (d, J = 8.5 Hz, 1H), 7.42 (dd, J1 = 8.5 Hz, J2 = 1.8 Hz, 1H), 3.77 – 3.74 (m, 2H), 3.47 (s,
3H), 2.97 – 2.94 (m, 2H), 2.78 – 2.75 (m, 2H), 1.67 – 1.61 (m, 2H), 1.50 – 1.42 (m, 2H), 0.92
(t, J = 7.3 Hz, 3H). 13C NMR (125 MHz, CDCl3, δ): 169.88, 157.62, 146.90, 133.07, 132.24,
128.55, 122.24, 121.00, 67.67, 59.18, 36.90, 34.60, 31.26, 21.89, 13.62. (+) ESI-HRMS: m/z
28