Synthesis and Biological Evaluation of 2-Aminobenzothiazole and Benzimidazole Analogs Based on the Clathrodin Structure

Article abstract of DOI:10.1002/ardp.201500365

A series of 2-aminobenzothiazole and benzimidazole analogs based on the clathrodin scaffold was synthesized and investigated for their antimicrobial and antiproliferative activities as well as for their effects in hepatitis C virus (HCV) replicon model. Compound 7, derived from 2-aminobenzothiazole, exhibited moderate antimicrobial activity only against the Gram-positive bacterium, Enterococcus faecalis. In the antiviral assay, compounds 4d and 7 were found to suppress the HCV replicon by >70%, but also to exhibit cytotoxicity against the host cells (35 and 44%, respectively). Compounds 4a and 7 demonstrated good activity in the antiproliferative assays on the human melanoma cell line A-375. To assess the selectivity of the effects between cancerous and noncancerous cells, a mouse fibroblast cell line was used. The IC₅₀ values for compound 7 against the melanoma cell line A-375 and the fibroblast cell line BALB/c 3T3 were 16 and 71 μM, respectively, yielding fourfold selectivity toward the cancer cell line. These results suggest that compound 7 should be studied further in order to fully explore its potential for drug development. A series of novel 2-aminobenzothiazole and benzimidazole derivatives based on the clathrodin structure was synthesized and evaluated for antimicrobial, antiproliferative and antiviral activities. (9H-Fluoren-9-yl)-methyl [(2-aminobenzo[d]thiazol-6-yl)methyl]carbamate 7 exhibited antiproliferative activity against the melanoma cell line A-375 (IC₅₀ = 16 μM) with 4-fold selectivity between cancerous (A-375) and noncancerous (BALB/c 3T3) cells.

Full text of DOI:10.1002/ardp.201500365

RCHH HARM
A P
Arch. Pharm. Chem. Life Sci. 2016, 349, 137–149
Archiv der Pharmazie
Full Paper
Synthesis and Biological Evaluation of 2-Aminobenzothiazole and
Benzimidazole Analogs Based on the Clathrodin Structure
1
2
2
1
2
~
Sofia Montalvao , Teppo O. Leino , Paula S. Kiuru , Katja-Emilia Lillsunde , Jari Yli-Kauhaluoma ,
1
€
and Paivi Tammela
1
Centre for Drug Research, Division of Pharmaceutical Biosciences, Faculty of Pharmacy, University of
Helsinki, Helsinki, Finland
Division of Pharmaceutical Chemistry and Technology, Faculty of Pharmacy, University of Helsinki,
2
Helsinki, Finland
A series of 2-aminobenzothiazole and benzimidazole analogs based on the clathrodin scaffold was
synthesized and investigated for their antimicrobial and antiproliferative activities as well as for their
effects in hepatitis C virus (HCV) replicon model. Compound 7, derived from 2-aminobenzothiazole,
exhibited moderate antimicrobial activity only against the Gram-positive bacterium, Enterococcus
faecalis. In the antiviral assay, compounds 4d and 7 were found to suppress the HCV replicon by
>70%, but also to exhibit cytotoxicity against the host cells (35 and 44%, respectively). Compounds
4a and 7 demonstrated good activity in the antiproliferative assays on the human melanoma cell line
A-375. To assess the selectivity of the effects between cancerous and noncancerous cells, a mouse
fibroblast cell line was used. The IC50 values for compound 7 against the melanoma cell line A-375
and the fibroblast cell line BALB/c 3T3 were 16 and 71 mM, respectively, yielding fourfold selectivity
toward the cancer cell line. These results suggest that compound 7 should be studied further in order
to fully explore its potential for drug development.
Keywords: Antimicrobial activity / Antiproliferative assay / Cytotoxic activity / Synthesis
Received: October 7, 2015; Revised: November 24, 2015; Accepted: November 26, 2015
DOI 10.1002/ardp.201500365
Additional supporting information may be found in the online version of this article at the publisher’s web-site.
:
however, marine pharmacognosy is still considered a novel
field comparing to terrestrial one.
Introduction
Seas and oceans offer a wide diversity of marine organisms,
chemical conditions, and other parameters such as tempera-
ture, pressure, and light. This resource is nowadays recog-
nized to have great potential for developing novel
applications and products. Particularly, the discovery of
new bioactive compounds from marine sources for therapeu-
tic applications gains a lot of attention [1]. As of now,
Lately, there has been a great interest in the synthesis of
bioactive molecules using marine natural compounds as an
inspiration. Marine sponges, belonging to phylum Porifera,
are very potential sources of bioactive compounds. These
organisms are primitive multicellular animals, which have
existed for more than 1 billion years [2]. For instance, Agelas sp.
are potential sources of novel biologically active compounds.
Clathrodin (Fig. 1), a 2-aminoimidazole alkaloid originally
isolated from a Caribbean sea sponge, Agelas clathrodes [3],
wasusedinthisstudyasaversatilestartingpoint.Clathrodinper
se has previously been reported to possess cytotoxic effects [3],
but more interestingly its analogs have shown promising
antimicrobial [4], proapoptotic [5] and voltage-gated sodium
channel blocking [6] effects.
€
Correspondence: Dr. Paivi Tammela, Centre for Drug Research,
Division of Pharmaceutical Biosciences, Faculty of Pharmacy, P.O.
Box 56 (Viikinkaari 5 E), FI-00014 University of Helsinki, Helsinki,
Finland.
E-mail: paivi.tammela@helsinki.fi
Fax: þ358 2941 59138
Benzo-fused heterocyclic compounds, such as benzimid-
azole and 2-aminobenzothiazole, are common scaffolds in
ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com
137

RCHH HARM
A P
Arch. Pharm. Chem. Life Sci. 2016, 349, 137–149
S. Montalvao et al.
~
Archiv der Pharmazie
with the corresponding carboxylic acid, acyl chloride, or
2-(trichloroacetyl)pyrrole (Scheme 1). The N-(4-nitrobenzyl)
amides 2a–e were then hydrogenated in the presence of
palladium on carbon to give the N-(4-aminobenzyl) amides
3a–e in excellent yield. The anilines 3a–e were treated with
ammonium thiocyanate in glacial acetic acid followed by
addition of bromine [22]. After treating the resulting mixture
with aqueous solution of ammonia, the 2-aminobenzothia-
zoles 4a–d were produced in low to moderate yields.
Figure 1. Chemical structure of clathrodin.
drug discovery. 2-Aminobenzothiazole, a bicyclic analog for
2-aminoimidazole moiety of clathrodin, was chosen as a core
structure since it is rare among natural products. In marine
compounds, the benzothiazole unit can be found in
violatinctamine, isolated from the marine tunicate Cystodytes
cf. violatinctus [7]. Benzothiazole-containing compounds
have recently been reviewed and have been found to have
antibacterial, antifungal, anticancer, anti-inflammatory,
antioxidant, and antiparasitic activity [8]. Benzimidazole, an
important heterocyclic fragment in medicinal chemistry, is
well known by its wide range of biological activities [9, 10],
especially antimicrobial [11–14], anticancer [15–17], antivi-
ral [18, 19], and anti-inflammatory [20]. In the last decades,
numerous benzimidazole derivatives have been synthesized,
due to their various biological activities and favorable
selectivity indices, and the benzimidazole scaffold can
be considered as a useful and privileged building block [21].
The aim of this study was to examine the antimicrobial,
antiviral, and antiproliferative activity of 14 new clathrodin-
inspired 2-aminobenzothiazole and benzimidazole deriva-
tives as well as their synthetic intermediates.
The last step of a three-step synthetic route (Scheme 1) for
the preparation of 2-aminobenzothiazole analogs gave very
low yields (10%) for pyrrole 4b and indole 4d as well as no
product in case of the target compound with an
N-methylpyrrole moiety. This is why a more convenient
four-step route was developed (Scheme 2). This route starts
with a fluorenylmethyloxycarbonyl (Fmoc) protection [23] of
4-aminobenzylamine 5 due to the acidic conditions in the
following step in which the 2-aminobenzothiazole ring was
formed when the Fmoc-protected aniline 6 was treated with
ammonium thiocyanate and bromine. The piperidine-
mediated deprotection of 7 gave benzylic amine 8, which
was converted to the target amides with pyrrole 4b, indole 4d,
and furan 4f moieties in reasonable yields.
Synthesis of benzimidazole analogs of clathrodin started
from 3,4-diaminobenzonitrile 9 (Scheme 3). It was cyclized in
the presence of formic acid to give 1H-benz[d]imidazole-5-
carbonitrile 10. Reduction of 10 with Raney-Ni in a solution of
ammonia in methanol gave the corresponding benzylic amine
11 in excellent yield. Compound 11 was treated without
further purification with 2-trichloroacetylpyrrole or the
corresponding acyl chloride or carboxylic acid to give the
target amides 12a–c in moderate yields.
2-(N,N-Dimethylamino)benzimidazole-5-carbonitrile
15
Results and discussion
was obtained from the reaction between 9 and phosgene
iminium chloride (Scheme 4) [24]. Rest of the route was
analogous to the related benzimidazole derivatives
(Scheme 3). Finally, a benzofuran analog N-(benzofuran-5-
Chemistry
The synthesis of 2-aminobenzothiazole analogs of clathrodin
(4a–d) commenced with amidation of 4-nitrobenzylamine
Scheme 1. a) Benzoic acid, EDC ꢀ HCl, DMAP, DMF, 17 h (2a); b) 2-(trichloroacetyl)pyrrole, Et3N, DMF, 23 h (2b); 2-thiophenecarbonyl
chloride, Et3N, THF, 27 h (2c); indole-2-carboxylic acid, EDC ꢀ HCl, DMAP, DMF, 5 h (2d); or 1-methyl-2-pyrrolecarboxylic acid, EDC ꢀ HCl,
DMAP, DMF, 21 h (2e), b) H2, 10% Pd/C, EtOH-EtOAc, 4 h, c) 1. NH4SCN, Br2, AcOH, 10–15°C, 2. 25% NH3 (aq).
ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com
138

RCHH HARM
A P
Arch. Pharm. Chem. Life Sci. 2016, 349, 137–149
2-Aminobenzothiazole and Benzimidazole Analogs
Archiv der Pharmazie
Scheme 2. a) Fmoc-OSu, Et3N, MeCN, DMF, b) 1. NH4SCN, Br2, AcOH, 10–15°C, 2. 25% NH3 (aq), c) piperidine, DMF, 1.5 h,
d) 2-(trichloroacetyl)pyrrole, Et3N, DMF, 23 h (4b); indole-2-carboxylic acid, EDC ꢀ HCl, DMAP, DMF, 17 h (4d); or furan-2-carboxylic
acid, EDC ꢀ HCl, DMAP, DMF, 18 h (4f).
Scheme 3. a) HCOOH, reflux, 2 h, b) Raney-Ni, 7 M NH3 in MeOH, 19 h, c) 2-(trichloroacetyl)pyrrole, Na2CO3, DMF, 23 h (12a);
2-thiophenecarbonyl chloride, Et3N, THF, 27 h (12b); indole-2-carboxylic acid, EDC ꢀ HCl, DMAP, DMF, 5 h (12c).
Scheme 4. a) Phosgene iminium chloride, DCM, reflux, 3.5 h, b) Raney-Ni, 7 M NH3 in MeOH, 24 h, c) 2-(trichloroacetyl)pyrrole,
Na2CO3, DMF, 5 h.
ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com
139

RCHH HARM
A P
Arch. Pharm. Chem. Life Sci. 2016, 349, 137–149
S. Montalvao et al.
~
Archiv der Pharmazie
Scheme 5. a) Raney-Ni, 7 M NH3 in MeOH, 23 h, b) 2-(trichloroacetyl)pyrrole, Na2CO3, DMF, 48 h.
ylmethyl)-1H-pyrrole-2-carboxamide 18 was synthesized simi-
larly as the benzimidazole derivative 12a but starting from 1-
benzofuran-5-carbonitrile 16 (Scheme 5).
Susceptibility Testing (EUCAST) [25] and Clinical and Labora-
tory Standards Institute (CLSI) [26]. Antibacterial and antifun-
gal activities detected in the primary screen are summarized
as bioactivity profiles in Table 1. Only 2-aminobenzothiazole
compound 7 showed potential antibacterial activity against
Gram-positive bacteria in the initial testing, that is, 56%
inhibition against E. faecalis. However, the results from dose–
response assays with concentrations ranging from 6.25 to
150 mM, showed only moderate inhibition below 50% at the
highest concentration tested.
In order to evaluate the potential of the compounds to inhibit
hepatitis C virus (HCV) replication, we used a replicon cell line in
which a HCV replicon is co-expressed in Huh-7 human hepatoma
cells with a marker protein, firefly luciferase [27]. As seen from
Table 1, compounds 4d and 7showed the most promising activity,
Biological activity
Antimicrobial activities of the final compounds 2b–18 and
some intermediates were evaluated in vitro by using standard
bacterial strains recommended for clinical susceptibility
testing, Gram-positive Staphylococcus aureus (ATCC 25923)
and Enterococcus faecalis (ATCC 29212), and Gram-negative
Escherichia coli (ATCC 25922). A fungal strain, Candida
albicans (ATCC 90028), was also used in this study. Primary
antimicrobial screening at 50 mM concentration was carried
out by using broth microdilution assay according to the
guidelines of European Committee on Antimicrobial
Table 1. Bioactivity profiles of compounds 2b–18.
Suppression
of HCV
replicon (%)
Antiproliferative
activity (%)
Antimicrobial activity (%)
Compound
C. albicans
E. faecalis
S. aureus
E. coli
HCV replicon
A-375
2b
3d
3e
4a
4b
4c
4d
4f
7
12a
12b
12c
15
18
0
4
0
10
46
16
22
0
1
0
0
24
0
0
0
0
8
0
20
0
0
0
0
5
8
9
12
4
5
6
2
20
3
10
36
0
0
3
4
52
34
0
47
4
78
0
0
0
19
29
33
49
0
0
0
0
0
0
0
26
73
0
86
13
0
8
4
0
0
56
13
19
27
0
9
13
14
11
10
0
0
0
0
0
<0%
0–20%
20–50%
>50%
Ciprofloxacin was used as a positive control in the antibacterial assays [minimum inhibitory concentration (MIC90) values for
E. faecalis ATCC 29212, S. aureus ATCC 25923, and E. coli ATCC 25922 were 3.0, 1.5, and 0.048 mM, respectively] and amphotericin
B in the antifungal assays against C. albicans ATCC 90028 (MIC90: 0.5 mM). Ribavirin (guanosine analog) was used as a positive
control in the antiviral assays (IC50: 100 mM) and polymyxin B sulphate in the antiproliferative assays (IC50: 15000 IU/mL).
Compounds were tested at final concentration of 50 mM. Results are expressed as average % (n ¼ 3).
ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com
140

RCHH HARM
A P
Arch. Pharm. Chem. Life Sci. 2016, 349, 137–149
2-Aminobenzothiazole and Benzimidazole Analogs
Archiv der Pharmazie
inhibiting the HCV replicon by 73 and 86%, respectively.
Therefore these compounds were taken for cytotoxicity studies,
in order to exclude possible false positive results caused by toxicity
toward the host cell. ATP quantitation was used to determine the
viability of the cells after treatment with the compounds, as the
amount of ATP is directly proportional to cell viability. Both
compounds 4d and 7 turned out to have cytotoxic effects on the
HCV replicon host cells (35 and 44%, respectively) and due to lack
ofselectivereplicon-inhibitingactivity, thestudiesontheantiviral
properties of these compounds were not continued.
The antiproliferative effects of compounds 2b–18 and some
intermediates were primarily evaluated against melanoma
cell line A-375 by using the MTT assay [28]. As shown in
Table 1, 4a and 7 exhibited cytotoxicity >50% (52 and 78%,
respectively) and were further evaluated in confirmatory
dose–response experiments with concentrations from 6.25 to
100 mM to determine IC50 (concentration required for 50%
inhibition of cell viability). Compound 4a showed only weak
toxicity in the cells, IC50 level was not reached even at the
highest test concentration (100 mM). On the other hand, the
cytotoxic effect of compound 7 was clear, showing an effect
higher than 50% even at 25 mM. The IC50 obtained (16 mM)
demonstrates that this compound has an antiproliferative
effect on the human melanoma cell line. Measurement of cell
membrane damage (release of intracellular lactate dehydro-
genase, LDH) after compound exposure demonstrated that
compound 7 at concentrations 6.25–100 mM caused 1.4 to 2.1-
fold increase in LDH leakage compared to vehicle-treated
control (Supporting Information Fig. S1). For comparison,
the positive control polymyxin B sulphate caused 3.5-fold
increase at concentration of 15000 IU/mL. Compound 4a did
not cause significant leakage of LDH even at the highest test
concentration 100 mM.
Figure 2. Dose–response curves of compound 7 against human
melanoma cell line A-375 and mouse fibroblast cell line BALB/c
3T3, expressed as percentage of cytotoxicity.
were synthesized and their antimicrobial, antiviral, and
antiproliferative activities were tested. In addition, experi-
ments with four intermediates (2b, 3d, 3e, and 7) were also
carried out. For antibacterial activity, the results were not very
encouraging. Compound
7 (fluorenyl intermediate) was
To assess selectivity of the effects between cancerous and
noncancerous cells, the antiproliferative activities of deriva-
tives 4a and 7 were also tested on noncancerous, embryonic
mouse fibroblast cell line (BALB/c 3T3). According to MTT
assay, compound 7 showed a cytotoxic effect also against
these cells, but with significantly higher IC50 (71 mM) than
against the cancerous cell line (Fig. 2). The trend was similar
also in the results from the LDH leakage assays; increased
leakage was only observed at concentrations >75 mM for
compound 7 (Supporting Information Fig. S2). The positive
control, polymyxin B sulphate at concentration of 20000 IU/mL
caused a 2.1-fold increase in LDH leakage.
The SI (selectivity index) is one of the parameters to be
considered when evaluating the potential of compounds for
further development. The data obtained are generally used to
calculate the SI of a compound as an estimate of a therapeutic
window. In this case, the SI between a normal cell line (BALB/c
3T3) and a cancerous cell line (A-375) was 4.44. This value
shows that compound 7 exhibited a moderate degree of
selectivity toward cancer cells.
showing 56% inhibition against E. faecalis at 50 mM concen-
tration and none of the compounds showed over 50%
inhibition against E. coli, S. aureus, or C. albicans. Compounds
7 and 4d (indole derivative) inhibited HCV replicon by 86 and
73%, respectively. However, these effects are most likely
accentuated due to host cell toxicity. Compound 7 was again
the best for the antiproliferative activity against A-375 cells
with 78%, followed by 4a (phenyl derivative) with 52%
activity. Benzimidazole and benzofuran derivatives showed
no antiproliferative activity. Contrary to our expectations, the
most active compound 7 in all assays was an intermediate and
not the final amide. It may indicate that the length of the
linker chain is not optimal and/or bulkier lipophilic groups
than pyrrole are favored on the right hand side of the
molecule. This is in line with our previous antimicrobial results
where bulky indole-based derivatives of 4-phenyl-2-amino-
imidazoles were the most active [4].
Conclusions
In this preliminary study, five 2-aminobenzothiazole (4a–d,
f), three benzimidazole (12a–c), one 2-(N,N-dimethylamino)-
benzimidazole 15 and benzofuran 18 analogs of clathrodin
By using a marine alkaloid, clathrodin, as a scaffold, we
synthesized its 2-aminobenzothiazole and benzimidazole
ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com
141

RCHH HARM
A P
Arch. Pharm. Chem. Life Sci. 2016, 349, 137–149
S. Montalvao et al.
~
Archiv der Pharmazie
analogs as well as evaluated their antimicrobial, antiviral, and
without further purification. 1H NMR (300 MHz, CDCl3) d 8.22–
8.18 (m, 2H), 7.84–7.80 (m, 2H), 7.57–7.43 (m, 5H), 6.61 (s, 1H),
4.76 (d, J ¼ 6.0 Hz, 2H).
antiproliferative activities. 2-Aminobenzothiazole
7 was
found to be the most potent compound of the set, displaying
an antiproliferative effect on melanoma cells with an IC50
value of 16 mM and fourfold selectivity between cancerous
and noncancerous cells. Therefore, using the marine scaffold,
N-(4-Nitrobenzyl)-1H-pyrrole-2-carboxamide 2b
4-Nitrobenzylamine hydrochloride (0.50 g, 2.65 mmol) and
2-(trichloroacetyl)pyrrole (0.56 g, 2.65 mmol) were dissolved
in dry DMF (25 mL) under argon. Triethylamine (0.74 mL,
5.30 mmol, 2 eq) was added to form a white suspension, which
was stirred at rt for 22 h. The suspension was concentrated in
vacuo and the residue was dissolved in EtOAc (50 mL) and the
resulting solution was washed with H2O (4 ꢁ 25 mL), dried
over anhydrous Na2SO4, filtered, and evaporated to provide a
brown solid. The crude product was purified by flash
chromatography on silica gel (elution with EtOAc/Hex 25:75
! 60:40) to give a yellow solid (0.44 g, 68%). For assaying
biological activity, part of the product was recrystallized
from chloroform to give white crystals. 1H NMR (300 MHz,
d6-acetone) d 10.79 (s, 1H), 8.22–8.17 (m, 2H), 8.08 (s, 1H), 7.63–
7.59 (m, 2H), 6.99–6.97 (m, 1H), 6.84–6.82 (m, 1H), 6.18–6.15
(m, 1H), 4.68 (d, J ¼ 6.3 Hz, 2H); 13C NMR (75 MHz, d6-acetone)
d 161.9, 149.0, 147.9, 129.1, 127.0, 124.3, 122.5, 110.4, 109.9,
42.8. LC-MS: [MþH]þ, m/z 246 (tr ¼ 4.0 min), >99%. FT-IR
(prism, n/cmꢂ1) 3316, 1613, 1556, 1504, 1346, 850, 738; mp
177–178°C. Rf ¼ 0.43 (EtOAc/Hex 1:1).
clathrodin, as
a starting point for synthesis of novel
compounds can be considered a promising approach for
further development of anticancer compounds.
Experimental
Chemistry
Commercial reagents were used as received without addi-
tional purification. Tetrahydrofuran (THF) was distilled over
sodium/benzophenone ketyl. The reactions were monitored
by thin-layer chromatography on silica gel 60-F254 plates
acquired from Merck (Darmstadt, Germany) and detected by
UV (254 nm). The products were purified by flash chromatog-
raphy on silica gel with a Biotage SP1 purification system
(Uppsala, Sweden) using SNAP 10-g, 25-g, or 50-g cartridges,
detection at 254 nm. Melting points were measured using an
IA9100 digital melting point apparatus (Electrothermal
Engineering, Essex, UK) and are uncorrected. IR spectra were
recorded on a Bruker Vertex 70 FT-IR spectrometer (Ettlingen,
Germany) with ATR technique. The synthesized compounds
were analyzed by NMR on a Varian Mercury 300 MHz
spectrometer (Varian, Palo Alto, CA). 1H and 13C NMR spectra
were recorded as solutions in CDCl3, d6-DMSO, d6-acetone, or
CD3OD. Chemical shifts (d) are given in parts per million (ppm)
relative to the NMR solvent signals (CDCl3 7.26 and 77.16 ppm,
d6-DMSO 2.50 and 39.51 ppm, d6-acetone 2.05 and 29.84 ppm,
CD3OD 3.31 and 49.00 ppm, for 1H and 13C NMR, respectively).
LC-MS analyses were performed by the use of an HP1100
instrument with UV detector (l 210 nm) and an Esquire LC
spectrometer (Bruker Daltonik, Bremen, Germany) with ESI
ion source. Signal separation was carried out by the use of a
Waters XBridge C18 column (2.1 mm ꢁ 500 mm, 2.1 mm) with a
Waters XBridge C18 guard column (2.1 mm ꢁ 10 mm, 2.5 mm).
The eluent of the gradient run (80:20 ! 5:95) consisted of
water (þ0.1% HCO2H) and acetonitrile (þ0.1% HCO2H). Purity
of all tested compounds was >95%. High resolution mass
spectra (HRMS) were measured on a Synapt G2 HDMS Q-TOF-
instrument (Milford, MA, USA) with positive ESI.
N-(4-Nitrobenzyl)thiophene-2-carboxamide 2c
2-Thiophenecarbonyl chloride (0.43 g, 0.31 mL, 2.92 mmol, 1.1
eq) and triethylamine (0.92 mL, 6.60 mmol, 2.5 eq) were
added under argon to a suspension of 4-nitrobenzylamine
hydrochloride (0.50 g, 2.65 mmol) in dry THF (40 mL). White
suspension was stirred at rt for 27 h and concentrated
in vacuo. The residue was dissolved in EtOAc (30 mL) and
the resulting solution was washed with 0.5 M aqueous
solution of HCl (15 mL) and H2O (15 mL), dried over anhydrous
Na2SO4, filtered and evaporated to provide an orange solid.
The crude product was purified by flash chromatography on
silica gel (elution with EtOAc/Hex 25:75 ! 75:25) to yield a
white solid (0.37 g, 53%). 1H NMR (300 MHz, CDCl3) d 8.21–
8.16 (m, 2H), 7.56 (dd, J ¼ 3.6, 1.2 Hz, 1H), 7.53–7.49 (m, 3H),
7.11–7.08 (m, 1H), 6.49 (s, 1H), 4.72 (d, J ¼ 6.3 Hz, 2H); 13C NMR
(75 MHz, CDCl3) d 162.2, 147.6, 145.8, 138.1, 130.7, 128.8,
128.5, 128.0, 124.1, 43.4.
N-(4-Nitrobenzyl)-1H-indole-2-carboxamide 2d
4-Nitrobenzylamine hydrochloride (0.50 g, 2.65 mmol), in-
dole-2-carboxylic acid (0.43 g, 2.65 mmol), EDC ꢀ HCl (0.50 g,
2.61 mmol), and 4-DMAP (0.65 g, 5.30 mmol, 2 eq) were
dissolved in dry DMF (25 mL) under argon. The yellow solution
was stirred at rt for 5 h and concentrated in vacuo. The residue
was dissolved in EtOAc (50 mL) and the solution was washed
with H2O (4 ꢁ 25 mL), dried over anhydrous Na2SO4, filtered,
and evaporated to provide a yellow solid. The crude product
was purified by flash chromatography on silica gel (elution
with EtOAc/Hex 50:50 ! 75:25) to give a white solid (0.65 g,
83%). 1H NMR (300 MHz, d6-acetone) d 10.90 (s, 1H), 8.51
N-(4-Nitrobenzyl)benzamide 2a
4-Nitrobenzylamine hydrochloride (0.50 g, 2.65 mmol), ben-
zoic acid (0.32 g, 2.65 mmol), EDC ꢀ HCl (0.50 g, 2.61 mmol), and
4-DMAP (0.65 g, 5.30 mmol, 2 eq) were dissolved in dry DMF
(20 mL) under argon. The yellow solution was stirred at rt for
17 h and concentrated in vacuo. The residue was dissolved in
EtOAc (50 mL) and the solution was washed with 1 M aqueous
solution of HCl (25 mL) and H2O (3 ꢁ 25 mL), dried over
anhydrous Na2SO4, filtered and evaporated to provide a
white solid (0.62 g, 91%), which was used in the next step
ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com
142

RCHH HARM
A P
Arch. Pharm. Chem. Life Sci. 2016, 349, 137–149
2-Aminobenzothiazole and Benzimidazole Analogs
Archiv der Pharmazie
(s, 1H), 8.23–8.18 (m, 2H), 7.69–7.61 (m, 3H), 7.54–7.50 (m, 1H),
7.25–7.19 (m, 2H), 7.10–7.05 (m, 1H), 4.78 (d, J ¼ 6.3 Hz, 2H);
13C NMR (75 MHz, d6-acetone) d 162.6, 148.5, 148.0, 137.9,
132.2, 129.2, 128.7, 124.7, 124.3, 122.6, 121.0, 113.1, 103.5,
43.1. FT-IR (prism, n/cmꢂ1) 3394, 3268, 3072, 1643, 1548, 1510,
1340, 746, 736. Rf ¼ 0.64 (EtOAc/Hex 1:1).
(prism, n/cmꢂ1) 3387, 1622, 1559, 1514, 1318, 738; mp 162–163°C.
Rf ¼ 0.17 (EtOAc/Hex 1:1).
N-(4-Aminobenzyl)thiophene-2-carboxamide 3c
N-(4-Nitrobenzyl)thiophene-2-carboxamide (0.20 g, 0.76 mmol)
was dissolved in EtOH (10 mL, 96%). Pd/C (0.02 g, 10 wt%) was
added and the resulting mixture was stirred under H2
atmosphere at rt for 4 h. The reaction mixture was filtered
through a small pad of Celite and concentrated in vacuo to
provide a yellowish solid. The crude product was purified by flash
chromatography on silica gel (elution with EtOAc/Hex 50:50 !
75:25) to give an orange solid (0.12 g, 65%). 1H NMR (300 MHz,
CDCl3) d 7.47–7.45 (m, 2H), 7.17–7.13 (m, 2H), 7.06 (dd, J ¼ 4.5,
3.6 Hz, 1H), 6.69–6.64 (m, 2H), 6.11 (s, 1H), 4.50 (d, J¼ 5.4 Hz, 2H),
3.70 (s, 2H); 13C NMR (75 MHz, CDCl3) d 161.7, 146.2, 139.1, 129.9,
129.5, 128.1, 127.9, 127.7, 115.4, 43.9.
1-Methyl-N-(4-nitrobenzyl)-1H-pyrrole-2-carboxamide 2e
4-Nitrobenzylamine hydrochloride (0.50 g, 2.65 mmol),
1-methylpyrrole-2-carboxylic acid (0.33 g, 2.65 mmol), EDC ꢀ
HCl (0.50 g, 2.61 mmol), and 4-DMAP (0.65 g, 5.30 mmol, 2 eq)
were dissolved in dry DMF (25 mL) under argon. The orange
solution was stirred at rt for 21 h and concentrated in vacuo.
The residue was dissolved in EtOAc (50 mL) and the solution
was washed with H2O (4 ꢁ 25 mL), dried over anhydrous
Na2SO4, filtered, and evaporated to provide an orange solid.
The crude product was purified by flash chromatography on
silica gel (elution with EtOAc/Hex 1:2 ! 1:1) to yield a
yellowish solid (0.55 g, 80%). 1H NMR (300 MHz, CDCl3) d
8.21–8.17 (m, 2H), 7.51–7.47 (m, 2H), 6.76 (t, J ¼ 2.0 Hz, 1H),
6.60 (dd, J ¼ 3.9, 1.8 Hz, 1H), 6.31 (s, 1H), 6.11 (dd, J ¼ 3.9,
2.7 Hz, 1H), 4.67 (d, J ¼ 6.3 Hz, 2H), 3.96 (s, 3H); 13C NMR
(75 MHz, CDCl3) d 162.1, 147.3, 146.7, 128.6, 128.1, 125.1,
123.9, 112.2, 107.6, 42.6, 36.9. FT-IR (prism, n/cmꢂ1) 3293,
1631, 1550, 1507, 1344, 1328, 736. Rf ¼ 0.59 (EtOAc/Hex 1:1).
HRMS (ESIþ): calculated 260.1035 (C13H14N3O3), found
260.1035.
N-(4-Aminobenzyl)-1H-indole-2-carboxamide 3d
N-(4-Nitrobenzyl)-1H-indole-2-carboxamide (0.40 g,
1.35 mmol) was dissolved in EtOH (40 mL, 96%) and EtOAc
(20 mL). Pd/C (0.04 g, 10 wt%) was added and the resulting
mixture was stirred under H2 atmosphere at rt for 4 h. The
reaction mixture was filtered through a small pad of Celite
and concentrated in vacuo to give a yellowish solid (0.34 g,
95%), which was used in the next step without further
purification. For assaying biological activity, part of the
product was recrystallized from chloroform to give white
crystals. 1H NMR (300 MHz, d6-acetone) d 10.74 (s, 1H), 7.99
(s, 1H), 7.59 (d, J ¼ 8.1 Hz, 1H), 7.54 (dd, J ¼ 8.3, 1.1 Hz, 1H),
7.23–7.18 (m, 1H), 7.12–7.02 (m, 4H), 6.64–6.60 (m, 2H), 4.53
(s, 2H), 4.45 (d, J ¼ 6.0 Hz); 13C NMR (75 MHz, d6-acetone) d
161.9, 148.5, 137.7, 133.0, 129.6, 128.9, 128.3, 124.4, 122.5,
120.8, 115.1, 113.1, 102.8, 43.4. LC-MS: [MþH]þ, m/z 266
(tr ¼ 1.7 min), >99%. FT-IR (prism, n/cmꢂ1) 3360, 3258, 1627,
1535, 1245, 1212, 747; mp 212–214°C. Rf ¼ 0.28 (EtOAc/Hex
1:1).
N-(4-Aminobenzyl)benzamide 3a
N-(4-Nitrobenzyl)benzamide (0.50 g, 1.95 mmol) was dis-
solved in EtOH (20 mL, 96%) and EtOAc (10 mL). Pd/C
(0.05 g, 10 wt%) was added and the resulting mixture was
stirred under H2 atmosphere at rt for 19 h. The reaction
mixture was filtered through a small pad of Celite and
concentrated in vacuo to provide a pink solid. The crude
product was purified by flash chromatography on silica gel
(elution with EtOAc/Hex 1:2 ! 3:1) to give a pink solid (0.38 g,
86%). 1H NMR (300 MHz, d6-acetone) d 7.94–7.91 (m, 3H),
7.53–7.40 (m, 3H), 7.09–7.06 (m, 2H), 6.63–6.59 (m, 2H), 4.51
(s, 2H), 4.43 (d, J ¼ 5.7 Hz, 2H).
N-(4-Aminobenzyl)-1-methyl-1H-pyrrole-2-carboxamide 3e
1-Methyl-N-(4-nitrobenzyl)-1H-pyrrole-2-carboxamide (0.45 g,
1.74 mmol) was dissolved in EtOH (30mL, 96%) and EtOAc
(15 mL). Pd/C (0.045 g, 10wt %) was added and the resulting
mixture was stirred at rt under H2 atmosphere for 4 h. The
reaction mixture was filtered through a small pad of Celite and
concentrated in vacuo to provide a pale white solid (0.28 g,
91%), which was used in the next step without further
purification. For assaying biological activity, part oftheproduct
was recrystallized from chloroform to give off-white crystals.
1H NMR (300 MHz, d6-acetone) d 7.50 (s, 1H), 7.06–7.01 (m, 2H),
6.80(t, J ¼ 2.3 Hz, 1H), 6.75(dd, J ¼ 3.8, 1.7 Hz, 1H), 6.62–6.58(m,
2H), 5.98 (dd, J ¼ 4.1, 2.6 Hz, 1H), 4.52 (s, 2H), 4.33 (d, J ¼ 6.0 Hz,
2H), 3.92 (s, 3H); 13C NMR (75 MHz, d6-acetone) d 162.2, 148.2,
129.5, 128.9, 128.2, 127.1, 115.0, 112.4, 107.5, 42.8, 36.7. LC-MS:
N-(4-Aminobenzyl)-1H-pyrrole-2-carboxamide 3b
N-(4-Nitrobenzyl)-1H-pyrrole-2-carboxamide (0.35g, 1.43mmol)
was dissolved in EtOH (30 mL, 96%) and EtOAc (15 mL). Pd/C
(0.035 g, 10 wt%) was added and the resulting mixture was
stirred under H2 atmosphere at rt for 4 h. The reaction mixture
was filtered through a small pad of Celite and concentrated
in vacuo to provide an off-white solid (0.28 g, 91%), which was
used in the next step without further purification. For assaying
biological activity, part of the product was recrystallized from
acetone and chloroform to yield white crystals. 1H NMR
(300 MHz, d6-acetone) d 7.57 (s, 1H), 7.05–7.00 (m, 2H), 6.95–
6.92 (m, 1H), 6.79–6.76 (m, 1H), 6.62–6.57 (m, 2H), 6.13–6.10 (m,
1H), 4.53 (s, 2H), 4.36 (d, J ¼ 6.0 Hz, 2H); 13C NMR (75 MHz, d6-
acetone) d 161.4, 148.3, 129.5, 128.7, 127.7, 121.8, 115.0, 109.8,
109.6, 43.0. LC-MS: [MþH]þ, m/z 216 (tr ¼ 0.58 min), >99%. FT-IR
[MþH]þ, m/z 230 (tr ¼ 0.69min), >99%. FT-IR (prism, n/cmꢂ1
)
3367, 3258, 1612, 1541, 1506, 1317, 1257, 729; mp. 151°C.
Rf ¼ 0.26 (EtOAc/Hex 1:1). HRMS (ESIþ): calculated 230.1293
(C13H16N3O), found 230.1296.
ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com
143

RCHH HARM
A P
Arch. Pharm. Chem. Life Sci. 2016, 349, 137–149
S. Montalvao et al.
~
Archiv der Pharmazie
N-((2-Aminobenzo[d]thiazol-6-yl)methyl)benzamide 4a
N-(4-Aminobenzyl)thiophene-2-carboxamide (0.10 g,
0.44 mmol) and NH4SCN (0.07 g, 0.88 mmol, 2 eq) were
dissolved in glacial acetic acid (1 mL) in a water bath (10–
15°C). After 5-min stirring, Br2 (0.10 g, 0.03 mL, 0.65 mmol, 1.5
eq) in glacial acetic acid (0.3 mL) was added dropwise to the
reaction mixture over 10 min. The red reaction mixture was
stirred in a water bath (10–15°C) for 2 h and it was allowed to
warm up to rt and stirred for 16 h. A black precipitate was
formed. Water (5 mL) was added to the reaction mixture and
the mixture was made basic (pH 9–10) with 25% aqueous
solution of NH3. The resulting white precipitate was extracted
with EtOAc (3 ꢁ 15 mL). EtOAc phases were combined, dried
over anhydrous Na2SO4, filtered and evaporated to provide a
white solid. The crude product was purified by flash
chromatography on silica gel (elution with EtOAc/Hex 1:1
! 1:0) to give a yellowish solid (0.08 g, 63%). Recrystallization
from acetone gave white crystals (0.04 g, 32%). 1H NMR
(300 MHz, d6-DMSO) d 9.01 (t, J ¼ 5.9 Hz, 1H), 7.91–7.88 (m,
2H), 7.58 (d, J ¼ 1.2 Hz, 1H), 7.55–7.40 (m, 5H), 7.28 (d,
J ¼ 8.1 Hz, 1H), 7.17 (dd, J ¼ 8.1, 1.5 Hz, 1H), 4.48 (d, J ¼ 6.0 Hz,
2H); 13C NMR (75 MHz, d6-DMSO) d 166.3, 166.1, 151.8, 134.4,
132.4, 131.2, 131.0, 128.3, 127.3, 125.1, 119.7, 117.4, 42.6. LC-
MS: [MþH]þ, m/z 284 (tr ¼ 1.1 min), >99%. FT-IR (prism,
n/cmꢂ1) 3367, 3053, 1624, 1526, 1463, 1288, 733; mp 230°C.
Rf ¼ 0.60 (EtOAc 100%). HRMS (ESIþ): calculated 284.0858
(C15H14N3OS), found 284.0862.
min), >99%. FT-IR (prism, n/cmꢂ1) 3421, 3147, 1615, 1528,
1461, 727; mp 225–226°C. Rf ¼ 0.59 (EtOAc 100%).
(9H-Fluoren-9-yl)methyl(4-aminobenzyl)carbamate 6
Dry DMF (1 mL) and triethylamine (1.14 mL, 8.18 mmol) were
added to
a solution of 4-aminobenzylamine (1.00 g,
8.18 mmol) in MeCN (10 mL) under argon. Fmoc-OSu (2.76 g,
8.18 mmol) in MeCN (20 mL) was added to the reaction
mixture dropwise over 20 min, which caused a clear solution
to turn into a white suspension. The white suspension was
stirred at rt for 1 h and it was concentrated in vacuo. The
residue was dissolved in EtOAc (80 mL), and the resulting
solution was washed with H2O (2 ꢁ 25 mL) and brine (25 mL),
dried over anhydrous Na2SO4, filtered and evaporated to
provide a reddish white solid. The crude product was purified
by flash chromatography on silica gel (elution with EtOAc/Hex
20:80 ! 75:25) to yield an off-white solid (1.72 g, 61%).
1H NMR (300 MHz, d6-acetone) d 7.85 (d, J ¼ 7.2 Hz, 2H), 7.70
(d, J ¼ 7.2 Hz, 2H), 7.40 (t, J ¼ 7.5 Hz, 2H), 7.33–7.28 (m, 2H),
7.01 (d, J ¼ 7.8 Hz, 2H), 6.74 (s, 1H), 6.61 (d, J ¼ 8.4 Hz, 2H), 4.55
(s, 2H), 4.34 (d, J ¼ 7.2 Hz, 2H), 4.23 (d, J ¼ 6.9 Hz, 1H), 4.18 (d,
J ¼ 5.7 Hz, 2H); 13C NMR (75 MHz, d6-acetone) d 157.2, 148.4,
145.2, 142.1, 129.3, 128.6, 128.5, 127.9, 126.1, 120.8, 115.1,
66.8, 48.1, 45.0. HRMS (ESIþ): calculated 345.1603
(C22H21N2O2), found 345.1610.
(9H-Fluoren-9-yl)methyl[(2-aminobenzo[d]thiazol-6-yl)-
methyl]carbamate 7
N-[(2-Aminobenzo[d]thiazol-5-yl)methyl]thiophene-2-
carboxamide 4c
(9H-Fluoren-9-yl)methyl-4-aminobenzylcarbamate (1.50 g,
4.36 mmol) and NH4SCN (0.66 g, 8.72 mmol, 2 eq) were
dissolved in glacial acetic acid (15 mL) on water bath (10–
15°C). After 20-min stirring, Br2 (1.05 g, 0.34 mL, 6.54 mmol,
1.5 eq) in glacial acetic acid (3.75 mL) was added dropwise to
the yellow suspension over 20 min. The resulting purple
solution was stirred on water bath (10–15°C) for 1 h and a
black solid was formed. The reaction mixture was allowed to
warm up to rt. After stirring 17 h, water (20 mL) was added to
the reaction mixture and it was made basic (pH 9–10) with
25% aqueous solution of NH3 (60 mL). The light-yellow
precipitate was extracted with EtOAc (6 ꢁ 50 mL). The organic
phases were combined, dried over anhydrous Na2SO4,
filtered and evaporated to provide a yellowish solid. The
crude product was purified by flash chromatography on
silica gel (elution with EtOAc/Hex 1:1 ! 1:0) to give an
off-white solid (1.18 g, 67%). For assaying biological activity,
part of the product was recrystallized from acetone and
chloroform to give white crystals. 1H NMR (300 MHz,
d6-acetone) d 7.86 (d, J ¼ 7.5 Hz, 2H), 7.70 (d, J ¼ 7.2 Hz, 2H),
7.56 (d, J ¼ 0.9 Hz, 1H), 7.43–7.38 (m, 2H), 7.35–7.28 (m, 3H),
7.19 (dd, J ¼ 8.1, 1.2 Hz, 1H), 6.95 (s, 1H), 6.74 (s, 2H), 4.39–4.35
(m, 4H), 4.24 (t, J ¼ 6.9 Hz, 1H); 13C NMR (75 MHz, d6-acetone) d
167.1, 157.3, 153.3, 145.2, 142.1, 134.0, 132.7, 128.5, 127.9,
126.1, 120.8, 120.6, 119.1, 66.8, 48.1, 45.2. LC-MS: [MþH]þ, m/z
402 (tr ¼ 5.9 min), >99%. FT-IR (prism, n/cmꢂ1) 3398, 1692,
1632, 1529, 1467, 739; mp 170–171°C. Rf ¼ 0.75 (EtOAc 100%).
N-(4-Aminobenzyl)thiophene-2-carboxamide (0.10 g,
0.43 mmol) and NH4SCN (0.07 g, 0.86 mmol, 2 eq) were
dissolved in glacial acetic acid (1 mL) on water bath (10–
15°C). After 5-min stirring, Br2 (0.10 g, 0.03 mL, 0.65 mmol, 1.5
eq) in glacial acetic acid (0.3 mL) was added dropwise to the
orange reaction mixture over 15 min on water bath (10–15°C).
The reaction mixture was stirred at rt for 2 h and it was
allowed to stand in the reaction flask overnight without
stirring and a brown precipitate formed. The reaction flask
was flushed twice with a mixture of water (1 mL) and glacial
acetic acid (2 mL). Flushes were combined, water (15 mL) was
added and the solution was made basic (pH 9–10) with 25%
aqueous solution of NH3. The resulting orange precipitate was
extracted with DCM (2 ꢁ 20 mL). DCM-phases were combined,
dried over anhydrous Na2SO4, filtered and evaporated to
provide a yellow solid. The crude product was purified by flash
chromatography on silica gel (elution with DCM/MeOH 5%)
to give a yellowish solid (0.08 g, 65%). Recrystallization from
acetone and chloroform gave yellowish crystals (0.04 g, 28%).
1H NMR (300 MHz, d6-DMSO) d 8.98 (t, J ¼ 6.0 Hz, 1H), 7.79 (dd,
J ¼ 3.6, 1.2 Hz, 1H), 7.75 (dd, J ¼ 5.1, 1.2 Hz, 1H), 7.57 (d,
J ¼ 1.2 Hz, 1H), 7.39 (s, 2H), 7.28 (d, J ¼ 8.1 Hz, 1H), 7.18–7.13
(m, 2H), 4.44 (d, J ¼ 6.3 Hz, 2H); 13C NMR (75 MHz, d6-DMSO) d
166.3, 161.0, 151.8, 139.9, 132.1, 131.0, 130.7, 128.0, 127.8,
125.1, 119.7, 117.4, 42.4. LC-MS: [MþH]þ, m/z 290 (tr ¼ 0.92
ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com
144

RCHH HARM
A P
Arch. Pharm. Chem. Life Sci. 2016, 349, 137–149
2-Aminobenzothiazole and Benzimidazole Analogs
Archiv der Pharmazie
HRMS (ESIþ): calculated 402.1276 (C23H20N3O2S), found
402.1281.
J ¼ 6.3 Hz, 2H); 13C NMR (75 MHz, d6-acetone) d 167.1, 162.0,
153.3, 137.7, 133.7, 132.8, 132.7, 128.8, 126.3, 124.5, 122.5,
120.8, 120.8, 119.1, 113.1, 103.0, 43.5. LC-MS: [MþH]þ, m/z
323 (tr ¼ 2.9 min), >97%. FT-IR (prism, n/cmꢂ1) 3241, 1626,
1533, 1463, 747. Rf ¼ 0.65 (EtOAc 100%). HRMS (ESIþ):
calculated 323.0966 (C17H15N4OS), found 323.0970.
6-(Aminomethyl)benzo[d]thiazol-2-amine 8
Carbamate 7 (1.15 g, 2.86 mmol) was dissolved in dry DMF
(9.2 mL) under argon. Piperidine (2.3 mL) was added and the
resulting yellow solution was stirred at rt for 1.5 h. The
solvents were evaporated in vacuo and the residue was
washed with hexane (4 ꢁ 20 mL). The resulting yellowish solid
(quantitative yield) was used in the next step without further
purification. 1H NMR (300 MHz, CD3OD) d 7.61 (d, J ¼ 1.2 Hz,
1H), 7.36 (d, J ¼ 8.1 Hz, 1H), 7.26 (dd, J ¼ 8.4 Hz, 1.8 Hz, 1H),
3.90 (s, 2H).
N-[(2-Aminobenzo[d]thiazol-6-yl)methyl]furan-2-
carboxamide 4f
6-(Aminomethyl)benzo[d]thiazol-2-amine
8 (0.15 g,
0.84 mmol), furan-2-carboxylic acid (0.09 g, 0.84 mmol),
EDC ꢀ HCl (0.16 g, 0.83 mmol), and 4-DMAP (0.10 g,
0.84 mmol) were dissolved in dry DMF (25 mL) under argon.
The resulting light-yellow suspension was stirred at rt for
18 h. The solution was concentrated in vacuo and the residue
was dissolved in EtOAc (50 mL), washed with 0.5 M aqueous
solution of NaOH (2 ꢁ 25 mL), H2O (2 ꢁ 25 mL), and brine
(25 mL), dried over anhydrous Na2SO4, filtered and evapo-
rated to provide yellow oil. The crude product was
purified by flash chromatography on silica gel (elution with
EtOAc/Hex 1:1 ! 1:0) to yield an off-white solid solid (0.11 g,
54% from 7). 1H NMR (300 MHz, d6-acetone) d 8.05 (s, 1H),
7.65 (dd, J ¼ 1.8, 0.6 Hz, 1H), 7.63 (d, J ¼ 1.2 Hz, 1H), 7.34 (d,
J ¼ 7.8 Hz, 1H), 7.25 (dd, J ¼ 7.8, 1.8 Hz, 1H), 7.06 (dd, J ¼ 3.3,
0.9 Hz, 1H), 6.74 (s, 2H), 6.58–6.56 (m, 1H), 4.57 (d, J ¼ 6.0 Hz,
2H); 13C NMR (75 MHz, d6-acetone) d 167.1, 158.6, 153.3,
149.6, 145.2, 133.7, 132.7, 126.4, 120.9, 119.1, 114.1, 112.6,
43.1. LC-MS: [MþH]þ, m/z 274 (tr ¼ 0.66 min), ꢃ98%. FT-IR
(prism, n/cmꢂ1) 3418, 3137, 1626, 1524, 1461, 1015, 765.
Rf ¼ 0.50 (EtOAc 100%). HRMS (ESIþ): calculated 274.0650
(C13H12N3O2S), found 274.0659.
N-[(2-Aminobenzo[d]thiazol-6-yl)methyl]-1H-pyrrole-2-
carboxamide 4b
2-(Trichloroacetyl)pyrrole (0.12 g, 0.56 mmol) and triethyl-
amine (0.12 mL, 0.84 mmol, 1.5 eq) were added to a solution
of 8 (0.10 g, 0.56 mmol) in dry DMF (2 mL) under argon. The
yellow solution was stirred at rt for 23 h. The solution was
concentrated in vacuo and the residue was dissolved in EtOAc
(40 mL), washed with 5% aqueous solution of NH3 (2 ꢁ 20 mL),
H2O (2 ꢁ 20 mL), and brine (20 mL), dried over anhydrous
Na2SO4, filtered and evaporated to provide orange oil. The
crude product was purified by flash chromatography on silica
gel (elution with EtOAc/Hex 1:1 ! 1:0) to yield an off-white
solid (0.05 g, 36% from 7). Recrystallization from acetonitrile
and methanol gave light-brown crystals (0.02 g). 1H NMR
(300 MHz, d6-acetone) d 10.68 (s, 1H), 7.79 (s, 1H), 7.60 (d,
J ¼ 1.8 Hz, 1H), 7.33 (d, J ¼ 8.1 Hz, 1H), 6.97–6.95 (m, 1H), 6.81–
6.78 (m, 1H), 6.73 (s, 2H), 6.14–6.11 (m, 1H), 4.55 (d, J ¼ 6.3 Hz,
2H); 13C NMR (75 MHz, d6-acetone) d 167.1, 161.6, 134.2, 132.7,
127.5, 126.3, 122.0, 120.7, 119.1, 110.0, 109.7, 43.2. LC-MS:
1H-Benz[d]imidazole-5-carbonitrile 10
[MþH]þ, m/z 273 (tr ¼ 0.69 min), ꢃ98%. FT-IR (prism, n/cmꢂ1
)
3,4-Diaminobenzonitrile 9 (1.00 g, 7.50 mmol) was dissolved in
formic acid (3 mL) and the mixture was stirred under reflux for
2 h. The solution was neutralized with 10% aqueous solution
of NaOH and extracted with EtOAc (2 ꢁ 15 mL). The combined
organic phases were washed with brine (10 mL) and dried over
anhydrous Na2SO4, filtered and evaporated to yield 1.06 g of
dark solid. Recrystallization from EtOAc/MeOH gave 10 as off-
white crystals (0.74 g, 69%). 1H NMR (300 MHz, d6-DMSO) d
8.46 (d, J ¼ 1.1 Hz, 1H), 8.15 (d, J ¼ 1.5 Hz, 1H), 7.75 (dq, J ¼ 0.7,
8.4 Hz, 1H), 7.58 (dt, J ¼ 1.4, 8.3 Hz, 1H).
3290, 1624, 1531, 1463, 737; mp 229–231°C. Rf ¼ 0.54 (EtOAc
100%). HRMS (ESIþ): calculated 273.0810 (C13H13N4OS), found
273.0818.
N-[(2-Aminobenzo[d]thiazol-6-yl)methyl]-1H-indole-2-
carboxamide 4d
6-(Aminomethyl)benzo[d]thiazol-2-amine
8 (0.15 g,
0.84 mmol), indole-2-carboxylic acid (0.14 g, 0.84 mmol),
EDC ꢀ HCl (0.16 g, 0.83 mmol), and 4-DMAP (0.10 g,
0.84 mmol) were dissolved in dry DMF (25 mL) under argon.
The resulting light-yellow suspension was stirred at rt for
17 h. The solution was concentrated in vacuo and the residue
was dissolved in EtOAc (50 mL), washed with 5% aqueous
solution of NH3 (2 ꢁ 25 mL), H2O (2 ꢁ 25 mL), and brine
(25 mL), dried over anhydrous Na2SO4, filtered and evapo-
rated to provide yellow oil. The crude product was purified
by flash chromatography on silica gel (elution with EtOAc/
Hex 1:1 ! 1:0) to give an off-white solid (0.13 g, 54% from 7).
1H NMR (300 MHz, d6-acetone) d 10.78 (s, 1H), 8.24 (s, 1H),
7.65 (d, J ¼ 1.8 Hz, 1H), 7.62–7.53 (m, 2H), 7.35 (d, J ¼ 8.1 Hz,
1H), 7.26 (dd, J ¼ 8.4, 1.8 Hz, 1H), 7.24–7.19 (m, 1H), 7.15 (dd,
J ¼ 2.1, 0.6 Hz, 1H), 7.08–7.03 (m, 1H), 6.74 (s, 2H), 4.63 (d,
(1H-Benz[d]imidazol-5-yl)methanamine 11
6-Cyanobenzimidazole 10 (0.74 g, 5.12 mmol) was dissolved in
a 7 M solution of ammonia in MeOH (40 mL) and added to a
flask with Raney-Ni (0.74 g) and stirred for 19 h under H2
atmosphere at rt. The reaction mixture was filtered through a
pad of Celite and washed with methanol and evaporated to
dryness. A light brown solid was obtained with a quantitative
yield and used for the next step without purification. 1H NMR
(300 MHz, CD3OD) d 8.56 (d, 1H), 8.12 (d, 1H), 7.57 (d,
J ¼ 8.0 Hz, 2H), 7.26 (d, J ¼ 8.0 Hz, 1H), 3.91 (s, 2H); 13C NMR
(75 MHz, CD3OD) d 170.36, 142.78, 138.31, 123.81, 116.39,
114.80, 46.94.
ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com
145

RCHH HARM
A P
Arch. Pharm. Chem. Life Sci. 2016, 349, 137–149
S. Montalvao et al.
~
Archiv der Pharmazie
N-[(1H-Benz[d]imidazol-5-yl)methyl]-1H-pyrrole-2-
carboxamide 12a
evaporated. The crude product was purified by flash
chromatography on silica gel, eluting with DCM/MeOH
6-Methyleneaminebenzimidazole 11 (0.10 g, 0.68 mmol) was
dissolved in dry THF (4 mL) under argon. 2-(Trichloroacetyl)-
pyrrole (0.14 g, 0.68 mmol) and anhydrous Na2CO3 (0.72 g,
0.68 mmol) were added. The reaction mixture was stirred at rt
for 3 h, concentrated and the residue partitioned between
EtOAc (40 mL), water (20 mL), and 1 M aqueous solution of
NaOH (5 mL). The organic phase was washed with water
(2 ꢁ 20 mL) and brine (20 mL). The organic phase was dried
over anhydrous Na2SO4, filtered and evaporated. The crude
product was purified by flash chromatography on silica gel
eluting with DCM/MeOH (10%)/MeOH ! gradient 0–10% to
yield 12a (0.09 g, 56%). Recrystallization from DCM/MeOH
yielded 12a as white crystals (0.03 g). 1H NMR (300 MHz, d6-
DMSO) d 12.30 (bs, 1H), 11.41 (s, 1H), 8.50 (t, J ¼ 6.1 Hz, 1H),
8.15 (s, 1H), 7.52 (d, J ¼ 11.5 Hz, 2H), 7.16 (d, J ¼ 8.2 Hz, 1H),
6.84 (m, 2H), 6.08 (dt, J ¼ 2.4, 3.6 Hz, 1H), 4.54 (d, J ¼ 6.1 Hz,
2H), 4.06 (s, 1H), 3.17 (s, 1H). 13C NMR (75 MHz, d6-DMSO) d
160.55, 141.99, 134.41, 133.13, 126.26, 122.05, 121.28, 118.46,
117.60, 111.34, 109.91, 108.51, 42.12. LC-MS: [MþH]þ, m/z 241
(tr ¼ 0.57 min), >97%. HRMS (ESIþ): calculated 241.1089
(C13H13N4O), found 241.1095.
(10%)/MeOH
!
gradient 0–10%. Recrystallization from
CHCl3/MeOH gave white crystals (0.05 g, 24%); mp 244–
245°C. 1H NMR (300 MHz, d6-DMSO) d 12.35 (s, 1H), 11.59 (s,
1H), 9.03 (s, 1H), 8.17 (s, 1H), 7.70–7.32 (m, 4H), 7.26–6.99 (m,
4H), 4.62 (d, J ¼ 5.7 Hz, 2H); 13C NMR (75 MHz, d6-DMSO) d
160.99, 142.21, 142.01, 136.41, 133.81, 131.72, 127.08, 123.21,
122.10, 121.43, 121.02, 119.64, 118.71, 112.24, 110.05, 102.50,
42.46. LC-MS: [MþH]þ, m/z 291 (tr ¼ 1.56 min), >97%. HRMS
(ESIþ): calculated 291.1250 (C17H15N4O), found 291.1246.
2-(Dimethylamino)-1H-benz[d]imidazole-5-carbonitrile 13
A mixture of 3,4-diaminobenzonitrile 10 (0.50 g, 3.76 mmol)
and phosgene iminium chloride (0.73 g, 4.51 mmol, 1.2 eq) in
dry DCM (30 mL) was refluxed for 3.5 h. Water (20 mL) was
added to the mixture and it was made alkaline with 2 M
aqueous solution of NaOH. The formed precipitate was
filtered to give a beige solid (0.51 g, 73%), which was used for
the next step without further purification. 1H NMR (300 MHz,
d6-DMSO) d 11.68 (s, 1H), 7.51 (s, 1H), 7.34–7.24 (m, 2H), 3.09
(s, 6H).
5-(Aminomethyl)-N,N-dimethyl-1H-benz[d]imidazol-2-
amine 14
N-[(1H-Benz[d]imidazol-5-yl)methyl]thiophene-2-
carboxamide 12b
The compound 13 (0.40 g, 2.15 mmol) was dissolved in a 7 M
solution of ammonia in MeOH (20 mL) and added to a flask
with Raney-Ni (0.4 g) and stirred for 24 h under H2 atmosphere
at rt. The reaction mixture was filtered through a pad of
Celite, washed with MeOH, and evaporated to dryness. A
light-brown solid was obtained (78% yield) and used for the
next step without further purification. 1H NMR (300 MHz,
CD3OD) d 7.30–7.04 (m, 2H), 6.86 (s, 1H), 4.94 (s, 2H), 3.06 (s,
6H).
6-Methyleneaminebenzimidazole 11 (0.05 g, 0.34 mmol) was
dissolved in dry THF (7 mL) under argon. 2-Thiophenecarbonyl
chloride (0.06 g, 0.34 mmol) and triethylamine (0.07 mL,
0.51 mmol, 1.5 eq) were added. The reaction mixture was
stirred at rt for 2 h and concentrated. The residue was
partitioned between EtOAc (20 mL) and water (15 mL). The
aqueous layer was extracted with EtOAc (2 ꢁ 20 mL), the
organic layers were combined, washed with brine (15 mL),
dried with anhydrous Na2SO4, filtered and evaporated. The
crude product was purified by flash chromatography on silica
gel, eluting with DCM/MeOH (5%)/MeOH ! gradient 0–40%.
Recrystallization from CHCl3 gave white crystals (0.03 g, 34%).
1H NMR (300 MHz, CDCl3) d 7.98 (s, 1H), 7.62–7.40 (m, 3H),
7.29–7.17 (m, 2H), 7.03 (dd, J ¼ 3.7, 5.0 Hz, 1H), 6.87 (bs, 1H),
4.69 (d, J ¼ 5.7 Hz, 2H). 13C NMR (75 MHz, CDCl3) d 162.05,
141.48, 139.15, 132.89, 130.12, 128.29, 127.76 (2C), 122.88
(2C), 116.29, 114.42, 44.41. LC-MS: [MþH]þ, m/z 258 (tr ¼ 0.65
min), >97%. HRMS (ESIþ): calculated 258.0701 (C13H12N3OS),
found 258.0703.
N-[(2-(Dimethylamino)-1H-benz[d]imidazol-5-yl)methyl]-
1H-pyrrole-2-carboxamide 15
A mixture of 2-(N,N-dimethylamino)-6-methyleneamineben-
zimidazole 14 (0.09 g, 0.49 mmol), anhydrous Na2CO3 (0.05 g,
0.49 mmol), and 2-(trichloroacetyl)pyrrole (0.10 g, 0.49 mmol)
in dry DMF (20 mL) was stirred at rt and under argon
atmosphere for 5 h. The reaction mixture was partitioned
between EtOAc (20 mL) and water (20 mL) and extracted with
EtOAc (3 ꢁ 20 mL). The combined organic layers were washed
with water (3 ꢁ 20 mL), dried with anhydrous Na2SO4, filtered
and evaporated. The crude product was purified by flash
chromatography on silica gel eluting with DCM/MeOH (10%).
Recrystallization from CHCl3/MeOH gave white crystals
(0.03 g, 35%); mp 152–154°C. 1H NMR (300 MHz, d6-DMSO)
d 11.44 (s, 1H), 11.15 (bs, 1H), 8.45 (t, J ¼ 6.1 Hz, 1H), 7.14–7.00
(m, 2H), 6.88–6.80 (m, 3H), 6.07 (q, J ¼ 2.8 Hz, 1H), 4.44 (d,
J ¼ 6.0 Hz, 2H), 3.02 (s, 6H); 13C NMR (75 MHz, d6-DMSO) d
160.49, 156.91, 130.95, 126.38, 121.22, 118.92, 109.84, 108.52,
79.19, 42.24, 38.00. LC-MS: [MþH]þ, m/z 284 (tr ¼ 0.67 min),
>97%. HRMS (ESIþ): calculated 284.1512 (C15H18N5O), found
284.1511.
N-[(1H-Benz[d]imidazol-5-yl)methyl]-1H-indole-2-
carboxamide 12c
6-Methyleneaminebenzimidazole 11 (0.10 g, 0.68 mmol), in-
dole-2-carboxylic acid (0.11 g, 0.68 mmol), triethylamine
(0.07 mL, 0.51 mmol, 1.2 eq) and 4-DMAP (0.08 g, 0.68 mmol)
were dissolved in dry DMF (7 mL) under argon. The reaction
mixture was stirred at rt for 5 h and concentrated. Water
(20 mL) was added to the residue and extracted with EtOAc
(3 ꢁ 20 mL). The combined organic layers were washed with
water (15 mL), dried with anhydrous Na2SO4, filtered and
ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com
146

RCHH HARM
A P
Arch. Pharm. Chem. Life Sci. 2016, 349, 137–149
2-Aminobenzothiazole and Benzimidazole Analogs
Archiv der Pharmazie
1-Benzofuran-5-ylmethylamine 17
of 620 nm, previously calibrated against plate counts. A
suspension of Candida was diluted into RPMI-1640 media
[with L-glutamine, w/o NaHCO3, and supplemented with 2%
glucose and 0.165 M morpholinepropanesulfonic acid
(MOPS), buffered to pH 7; Lonza, Switzerland] to obtain a
final inoculum of 2.5 ꢁ 103 CFU/mL in the assay. Assays were
performed in clear 96-well microplates (Thermo Fisher
Scientific, Finland). Compounds were first diluted into the
assay media and then mixed with the bacteria/fungi suspen-
sion on the assay plates. Plates were incubated at 37°C for 24 h
(bacteria) or at 28°C for 48 h (fungi), at 500 rpm. Absorbance
values, at a wavelength of 620 nm, were measured at 0, 4, and
24 h for bacterial species and 0, 24, and 48 h for Candida, and
were used for assessing the inhibition of growth. Antimicro-
bial activity of the compounds was calculated by comparing to
vehicle-treated controls and expressed as % of inhibition.
Initially, all compounds were tested at a final concentration of
50 mM (n ¼ 3). For compounds that showed >50% inhibition,
dose–response experiments with concentrations ranging
from 6.25 to 150 mM were carried out.
Raney-Ni slurry in water (0.25 g) was weighed into a two-
necked flask, which was placed under hydrogen atmosphere.
1-Benzofuran-5-carbonitrile 16 (0.25 g, 1.71 mmol) was dis-
solved in a 7 M solution of NH3 in MeOH (10 mL) and the
resulting solution was added to the flask. The reaction
mixture was stirred at rt for 23 h. The solution was filtered
through
a pad of Celite, washed with methanol, and
evaporated to give a white solid in quantitative yield. It
was used for the next step without further purification.
1H NMR (300 MHz, d6-DMSO) d 7.97 (d, 1H), 7.52 (m, 2H), 7.24
(m, 1H), 6.92 (s, 1H), 3.84 (bs, 2H).
N-(Benzofuran-5-ylmethyl)-1H-pyrrole-2-carboxamide 18
A
mixture of 1-benzofuran-5-ylmethylamine 17 (0.10 g,
0.68 mmol), anhydrous Na2CO3 (0.07 g, 0.68 mmol), and
2-(trichloroacetyl)pyrrole (0.14 g, 0.68 mmol) in dry THF
(10 mL) was stirred at rt and under argon atmosphere for
48 h. THF was evaporated and the residue diluted with EtOAc
(20 mL). The solution was washed with water (3 ꢁ 15 mL),
dried with anhydrous Na2SO4, filtered and evaporated. The
crude product was purified by flash chromatography on silica
gel, eluting with DCM/MeOH (1%). Recrystallization from
hexane/acetone gave light-yellow crystals (0.06 g, 40%); mp
Evaluation of HCV replicon suppression
The Huh-7 cell line expressing a HCV genotype 1b replicon has
been described by Vrojlik and co-workers [27] and was kindly
provided by Prof. Ralf Bartenschlager (University of Heidel-
berg, Germany). Under constant G418 selection, this cell line
expresses a subgenomic replicon of the HCV genome and a
firefly luciferase marker. Cells were cultured in Dulbecco’s
modified Eagle’s medium (DMEM) supplemented with 10%
FBS (Gibco, USA), 100 IU/mL penicillin, 100 mg/mL streptomy-
cin, 292 mg/mL L-glutamine, 1% non-essential amino acids,
and 250 mg/mL of G418. Cells were incubated at 37°C in a
humidified, 5% CO2 atmosphere, and routinely passaged 2–3
times a week.
1
126–127°C. H NMR (300 MHz, d6-DMSO) d 11.44 (s, 1H), 8.54
(t, J ¼ 6.1 Hz, 1H), 7.96 (d, J ¼ 2.2 Hz, 1H), 7.61–7.49 (m, 2H),
7.27 (dd, J ¼ 1.8, 8.5 Hz, 1H), 6.94 (dd, J ¼ 0.9, 2.2 Hz, 1H), 6.84
(m, 2H), 6.09 (dt, J ¼ 2.4, 3.6 Hz, 1H), 4.52 (d, J ¼ 6.1 Hz, 2H);
13C NMR (75 MHz, d6-DMSO) d 160.83, 153.64, 146.46, 134.97,
127.41, 126.45, 124.13, 121.58, 119.92, 111.13, 110.20, 108.79,
106.88, 42.11. LC-MS: [MþH]þ, m/z 241 (tr ¼ 4.8 min), >97%.
HRMS (ESIþ): calculated 241.0977 (C14H13N2O2), found
241.0976.
Biological methods
For the experiment, Huh-7 cells carrying the HCV replicon
were seeded on opaque-white and clear-bottomed 96-well
microplates (PerkinElmer Inc., USA) with a cell density of
30000 cells/well. After incubation at 37°C for 24 h, the cells
were exposed to the compounds. Stock solutions of the
compounds were diluted into assay medium (growth medium
with 5% FBS). After 24-h exposure, luciferase expression was
measured using a Firefly luciferase assay kit (Promega, USA),
according to the manufacturer’s instructions. The lumines-
cence signal was recorded using a Varioskan Flash plate
reader (Thermo Fisher Scientific, Finland) with a measurement
time of 1 s and automatic dynamic range settings. The % of
suppression of the viral replicon was calculated by comparing
the sample signal to the maximum signal (from vehicle-
treated control).
Viability of the HCV replicon-containing Huh-7 cells was
determined after exposure to compounds to exclude possible
false positives identified in the antireplicon assay. Test
conditions were identical to those of the antireplicon assay
described above. After 24-h exposure to the compounds, cell
viability was determined by ATP quantitation using a CellTiter
GLO1 Luminescent Cell Viability Assay Kit (Promega, USA).
Evaluation of antimicrobial activity
Bacterial strains Staphylococcus aureus (Gram-positive, ATCC
25923), Enterococcus faecalis (Gram-positive, ATCC 29212),
Escherichia coli (Gram-negative, ATCC 25922), and a fungal
strain Candida albicans (ATCC 90028), obtained from Micro-
biologics Inc. (USA), were used for assessing antimicrobial
activity. Bacterial stock cultures were maintained on Mueller-
Hinton agar (MHA, BD, USA) and fungal culture on Saboraud
dextrose agar (SDA, BD, USA), and stored at 4°C. Before the
assay, bacterial suspensions were inoculated into Mueller-
Hinton broth (MHB, BD, USA) from a stock culture and
incubated at 37°C for 16–20 h at 100 rpm. Candida was
inoculated on SDA plates, incubated at 28°C for 18–24 h and
suspended into sterile 0.9% NaCl (Sigma–Aldrich, USA) for the
assay.
Antimicrobial activity was tested using broth microdilution
method according to EUCAST [25] and CLSI [26] guidelines.
From overnight suspension cultures of bacteria, a dilution
yielding a final inoculum of 5 ꢁ 105 colony-forming units
(CFU)/mL in the assay was prepared into MHB. CFU/mL was
determined on the basis of absorbance values at a wavelength
ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com
147

RCHH HARM
A P
Arch. Pharm. Chem. Life Sci. 2016, 349, 137–149
S. Montalvao et al.
~
Archiv der Pharmazie
Luminescence measurements were performed using a Vari-
oskan Flash plate reader. The cytotoxic effect of the
compounds was determined as % using the maximum signal
(vehicle-treated cells) as the reference and wells with reagent
only as the background value.
compounds that showed a high percent of LDH leakage from
A-375 cell assays were tested against BALB/c 3T3. At the end of
the exposure, 50 mL of supernatant from the cells was
transferred into white, clear bottom 96-well plates (Perki-
nElmer Inc., USA) and incubated at room temperature in dark
for 20–30 min. After the incubation period, 50 mL of CytoTox-
ONE reaction mixture was added and the plates were shaken
for 10 min at rt. After 30 min, reactions were stopped with
25 mL of CytoTox-ONE stop solution and the plate was shaken
for 10 s. LDH activity was determined by fluorescence
measurement using a Varioskan Flash plate reader with lEX
560 nm and lEM 655 nm. Vehicle-treated cells were used as
normalization control in the calculation of the percentage of
LDH leakage.
Evaluation of antiproliferative activity against mammalian
cells
Human malignant melanoma cell line A-375 was kindly
provided by Prof. Marikki Laiho (University of Helsinki,
Finland) and BALB/c 3T3 mouse embryonic fibroblast cell line
was obtained from the European Collection of Cell Cultures
(ECACC, UK). A-375 cells were cultured in Glutamax DMEM
with 4.5 g/L D-glucose (Gibco, USA), supplemented with 10%
fetal bovine serum (FBS, Gibco, USA), 100 IU/mL penicillin
(Gibco, USA), and 100 mg/mL streptomycin (Gibco, USA). BALB/c
3T3 cells were grown in DMEM (Sigma–Aldrich, USA), supple-
mented with 5% FBS (Gibco, USA), 5% newborn calf serum
(NCS, Gibco, USA), 292 mg/mL L-glutamine, 100 IU/mL penicillin,
and 100 mg/mL streptomycin. Cells were incubated in standard
cell cultureflasks ina humidifiedatmospherewith5% ofCO2 at
37°C. Cells were subcultured approximately every 2–3 days
when cultures had reached 70–80% confluency.
Cells from a stock culture were plated with the density of
20000 cells/well into transparent and clear-bottomed 96-well
microplates and allowed to attach overnight. Cells were then
exposed to the compounds for 24 h. Stock solutions of test
compounds and a positive control (polymyxin B sulphate,
Sigma–Aldrich, Denmark) were prepared and diluted into
assay media (growth media with 5% FBS). After removing the
old media, 150 mL of sample solution per well was added to
the cells. Control wells (nontreated and vehicle-treated cells)
and blank wells (no cells, medium and vehicle-containing
medium) were included on each assay plate. The primary test
concentration used was 50 mM.
Data analysis
Origin Graphing and Analysis, version 8.6 (OriginLab, USA)
was used for determination of IC50 values (concentrations that
produced 50% inhibitory effect). SI was calculated as a ratio of
IC50 values between BALB/c 3T3 fibroblasts and A-375
melanoma cells.
The research leading to these results has received funding
from the European Union Seventh Framework Programme
(FP7/2007-2013) under grant agreement no. FP7-KBBE-2009-
3-245137 (MAREX) as well as from the Academy of Finland
(grant nos. 277001 and 265481). Technical assistance of Nora
Jokinen in the primary HCV replicon screening is gratefully
acknowledged.
The authors have declared no conflicts of interest.
References
MTT assay: The cytotoxicity effect of the compounds on A-
375 cells was determined with an MTT assay [28]. The same
method was used with BALB/c 3T3 cells, but only the
compounds that were cytotoxic against the melanoma cells
were tested. After 24 h compound exposure, 10 mL of 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide
(MTT) stock solution (Sigma–Aldrich, USA; 5 mg/mL in sterile
phosphate-buffered saline) was added to the wells and the
plates were incubated for 3 h in a humidified atmosphere at
37°C and 5% CO2. After incubation, the medium was removed
and 200 mL of DMSO was added to the wells and the plates
were agitated until the blue crystals, for example, formazan,
had completely dissolved. Absorbance was measured with
Multiskan1 GO UV/Vis spectrophotometer (Thermo Fisher
Scientific, Finland) at 550 and 655 nm (background). The
cytotoxicity (%) was calculated by normalizing background-
subtracted absorbance values against the values from vehicle-
treated control.
[1] P. Kiuru, M. V. D’Auria, C. D. Muller, P. Tammela,
H. Vuorela, J. Yli-Kauhaluoma, Planta Med. 2014, 80,
1234–1246.
[2] S. R. Haseley, H. J. Vermeer, J. P. Kamerling,
J. F. G. Vliegenthart, Proc. Natl. Acad. Sci. USA 2001,
98, 9419–9424.
ꢀ
[3] J. J. Morales, A. D. Rodrıguez, J. Nat. Prod. 1991, 54,
629–631.
ꢁ
ꢁ
~
[4] N. Zidar, S. Montalvao, Z. Hodnik, D. A. Nawrot, A. Zula,
ꢁ
ꢁ ꢁ
J. Ilas, D. Kikelj, P. Tammela, L. Peterlin Masic, Mar. Drugs
2014, 12, 940–963.
ꢁ
ꢁ ꢁ
[5] T. Tomasic, D. Nabergoj, S. Vrbek, N. Zidar, Z. Jakopin,
ꢁ
ꢁ
ꢁ
ꢁ
A. Zula, Z. Hodnik, M. Jukic, M. Anderluh, J. Ilas,
ꢀ
M. S. Dolenc, J. Peluso, G. Ubeaud-Sequier, C. D. Muller,
ꢁ ꢁ
L. Peterlin Masic, D. Kikelj, Med. Chem. Commun. 2015, 6,
105–110.
ꢁ ꢀ
[6] T. Tomasic, B. Hartzoulakis, N. Zidar, F. Chan, R. W. Kirby,
D. J. Madge, S. Peigneur, J. Tytgat, D. Kikelj, J. Chem. Inf.
Model. 2013, 53, 3223–3232.
[7] L. Chill, A. Rudi, Y. Benayahu, Y. Kashman, Tetrahedron
Lett. 2004, 45, 7925–7928.
LDH assay: Release of LDH from compound-treated A-375
cells was measured with CytoTox-ONE-Homogeneous Integ-
rity Assay Kit (Promega, USA). As in the MTT assay, only the
ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com
148

RCHH HARM
A P
Arch. Pharm. Chem. Life Sci. 2016, 349, 137–149
2-Aminobenzothiazole and Benzimidazole Analogs
Archiv der Pharmazie
[8] R. S. Keri, M. R. Patil, S. A. Patil, S. Budagumpi, Eur. J.
Med. Chem. 2015, 89, 207–251.
[9] Y. Bansal, O. Silakari, Bioorg. Med. Chem. 2012, 20,
6208–6236.
[10] G. Yadav, S. Ganguly, Eur. J. Med. Chem. 2015, 97,
419–443.
[11] I. Kerimov, G. Ayhan-Kilcigil, B. Can-Eke, N. Altanlar,
M. Iscan, J. Enzyme Inhib. Med. Chem. 2007, 22,
696–701.
[12] B. V. S. Kumar, S. D. Vaidya, R. V. Kumar, S. B. Bhirud,
R. B. Mane, Eur. J. Med. Chem. 2006, 41, 599–604.
[13] D. Seenaiah, P. R. Reddy, G. M. Reddy, A. Padmaja,
V. Padmavathi, N. S. Krishna, Eur. J. Med. Chem. 2014, 77,
1–7.
[19] D. Sharma, B. Narasimhan, P. Kumar, V. Judge,
R. Narang, E. De Clercq, J. Balzarini, J. Enzyme Inhib.
Med. Chem. 2009, 24, 1161–1168.
[20] K. C. S. Achar, K. M. Hosamani, H. R. Seetharamareddy,
Eur. J. Med. Chem. 2010, 45, 2048–2054.
[21] F. Fei, Z. Zhou, Expert Opin. Ther. Pat. 2013, 23,
1157–1179.
[22] R. V. Patel, P. K. Patel, P. Kumari, D. P. Rajani,
K. H. Chikhalia, Eur. J. Med. Chem. 2012, 53, 41–51.
[23] R. Behrendt, M. Schenk, H. J. Musiol, L. Moroder, J. Pept.
Sci. 1999, 5, 519–529.
[24] A. J. Carpenter, K. A. Al-Barazanji, K. K. Barvian,
M. J. Bishop, C. S. Britt, J. P. Cooper, A. S. Goetz,
M. K. Grizzle, D. L. Hertzog, D. M. Ignar, R. O. Morgan,
G. E. Peckham, J. D. Speake, W. R. Swain, Bioorg. Med.
Chem. Lett. 2006, 16, 4994–5000.
[14] K. F. Ansari, C. Lal, Eur. J. Med. Chem. 2009, 44,
4028–4033.
[15] Y. Tong, J. J. Bouska, P. A. Ellis, E. F. Johnson, J. Leverson,
X. Liu, P. A. Marcotte, A. M. Olson, D. J. Osterling,
M. Przytulinska, L. E. Rodriguez, Y. Shi, N. Soni,
J. Stavropoulos, S. Thomas, C. K. Donawho, D. J. Frost,
Y. Luo, V. L. Giranda, T. D. Penning, J. Med. Chem. 2009,
52, 6803–6813.
[16] H. M. Refaat, Eur. J. Med. Chem. 2010, 45, 2949–2956.
[17] S.-T. Huang, I.-J. Hsei, C. Chen, Bioorg. Med. Chem. 2006,
14, 6106–6119.
[25] G. Kahlmeter, D. F. J. Brown, F. W. Goldstein,
A. P. MacGowan, J. W. Mouton, I. Odenholt,
A. Rodloff, C.-J. Soussy, M. Steinbakk, F. Soriano,
O. Stetsiouk, Clin. Microbiol. Infect. 2006, 12, 501–503.
[26] M. A. Wikler, F. R. Cockerill, W. A. Craig, M. N. Dudley,
G. M. Eliopoulos, D. W. Hecht, J. F. Hindler, M. J. Ferraro,
J. M. Swenson, D. E. Low, D. J. Sheehan, F. C. Tenover,
J. D. Turnidge, M. P. Weinstein, B. L. Zimmer, CLSI 2006,
26, 1–35.
[18] M. Tonelli, M. Simone, B. Tasso, F. Novelli, V. Boido,
F. Sparatore, G. Paglietti, S. Pricl, G. Giliberti, S. Blois,
C. Ibba, G. Sanna, R. Loddo, P. La Colla, Bioorg. Med.
Chem. 2010, 18, 2937–2953.
[27] J. M. Vrolijk, A. Kaul, B. E. Hansen, V. Lohmann,
B. L. Haagmans, S. W. Schalm, R. Bartenschlager,
J. Virol. Methods 2003, 110, 201–209.
[28] T. Mosmann, J. Immunol. Methods 1983, 65, 55–63.
ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com
149