Synthesis of novel derivatives of 7,8-dihydro-6H-imidazo[2,1-b][1,3]benzothiazol-5-one and their virus-inhibiting activity against influenza A virus

Article abstract of DOI:10.1002/ardp.201800225

Influenza remains a highly pathogenic and hardly controlled human infection. The ability of selecting drug-resistant variants necessitates the search and development of novel anti-influenza drugs. Herein, we describe the synthesis and evaluation of a seri

Full text of DOI:10.1002/ardp.201800225

Received: 31 July 2018
Revised: 21 October 2018
Accepted: 4 November 2018
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DOI: 10.1002/ardp.201800225
FULL PAPER
Synthesis of novel derivatives of
7,8-dihydro-6H-imidazo[2,1-b][1,3]benzothiazol-5-one and
their virus-inhibiting activity against influenza A virus
Anastasia V. Galochkina1
Rakesh K. Bollikanda2,3
Vladimir V. Zarubaev1
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Dmitry G. Tentler4
Nagaraju Chirra2,3
Irina N. Lavrenteva1
Srinivas Kantevari2,3
Alexander V. Slita1
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1 Pasteur Institute of Epidemiology and
Microbiology, St. Petersburg, Russia
Abstract
2 Fluoro and Agrochemicals Division (Organic
Chemistry Division-II), CSIR − Indian Institute
of Chemical Technology, Hyderabad, India
Influenza remains a highly pathogenic and hardly controlled human infection. The
ability of selecting drug-resistant variants necessitates the search and development of
novel anti-influenza drugs. Herein, we describe the synthesis and evaluation of a series
of novel 2-substituted 7,8-dihydro-6H-imidazo[2,1-b][1,3]benzothiazol-5-ones 3a–k
for their virus-inhibiting activity against influenza A virus. The new analogues 3a–k
prepared in two steps from commercially available cyclohexane-1,3-diones were fully
characterized by their NMR and mass spectral data. Among the new derivatives
screened for cytotoxicity and in vitro antiviral activity against influenza virus A/Puerto
Rico/8/34 (H1N1) in MDCK cells, three analogues 3i–k containing a thiophene unit
were found to exhibit high virus-inhibiting activity (high SI values) and a favorable
toxicity profile. The compound 3j (CC50: >1000 μM, SI = 77) with higher potency is the
best anti-influenza hit analogue for further structural optimization and drug
development. The most active compounds did not inhibit viral neuraminidase and
possess therefore other targets and mechanisms of activity than the currently used
neuraminidase inhibitors.
3 Academy of Scientific and Innovative
Research, CSIR − Indian Institute of Chemical
Technology, Hyderabad, India
4 Institute of Cytology, Russian Academy of
Sciences, Russia
Correspondence
Vladimir V. Zarubaev, Pasteur Institute of
Epidemiology and Microbiology, 14 Mira str.,
197101 St. Petersburg, Russia.
Email: zarubaev@gmail.com
Srinivas Kantevari, Fluoro and Agrochemicals
Division (Organic Chemistry Division-II), CSIR
− Indian Institute of Chemical Technology,
Hyderabad-500007, Telangana, India.
Email: ksrinivas@iict.res.in
Funding information
Russian Foundation for Basic Research,
Grant number: 17-54-45113; Department of
Science and Technology, India, Grant number:
INT/RUS/ RFBR/P-296; Russian Science
Foundation, Grant number: 14-50-00068
K E Y W O R D S
1,3-dicarbonyl, antivirals, antiviral activity, benzothiazole, cytotoxicity, imidazole, influenza
virus
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INTRODUCTION
pandemics, the later are often associated with significant morbidity
and mortality.[3] The pandemic of influenza A (H1N1) pdm09 caused by
Influenza is a highly contagious human disease. Every year, 500 million
people suffer from the flu worldwide with about two million with fatal
outcome.[1,2] The influenza virus causes annual epidemics and
a swine-origin H1N1 virus was characterized by a wide coverage of the
population of the world, severe clinical course and about 300000 lethal
outcomes within 18 months.[4,5] Pregnant women, newborns, aged
people, and persons with pre-existing pathologies (diabetes, kidney
failure) are of high risk.[6] Moreover, in recent years, cases of human
infection with avian influenza viruses H5N1, H7N2, H7N7, and H7N9
Anastasia V. Galochkina and Rakesh K. Bollikanda contributed equally to this paper.
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Arch Pharm Chem Life Sci. 2018;1–8.
wileyonlinelibrary.com/journal/ardp
© 2018 Deutsche Pharmazeutische Gesellschaft
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GALOCHKINA ET AL.
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have been reported.[7,8] Therefore, continuous monitoring and
development of control measures to restrict influenza spreading is
highly actual for medical science and health care.
also reflected in both the number of synthetic methods found to access
them and the possibility for diverse functionalization.[19,20] Hence
imidazo[2,1-b]thiazole is considered as platform of choice for
designing new molecules for our ongoing medicinal chemistry pursuits.
The designed new scaffold (Figure 3) is based on recently
reported antiviral agent III.[22] This compound together with relative
ones were shown to inhibit non-structural protein of hepatitis C
virus (HCV) NS4B. It interacts with other non-structural proteins of
HCV, NS3, NS4A, NS5A and others that are involved in viral RNA
synthesis.[25] In the current study, we describe synthesis and
biological assessment of series of novel imidazo[2,1-b]thiazole-
based compounds as potential inhibitors of another RNA-genome
virus, influenza virus, in vitro. The designer scaffold has imidazo[2,1-
b]thiazole core fused with cyclic ketone system. Variations in the
proposed scaffold can be accomplished with substituted aromatic/
heterocyclic ring units.
Four classes of low-molecular compounds are currently used as
etiotropic anti-influenza drugs (Figure 1).[9] Derivatives of adaman-
tane, amantadine, and rimantadine inhibit the virus-specific protein
M2.[10,11] Neuraminidase inhibitors (NAI), oseltamivir (Tamiflu®),
zanamivir (Relenza®), laninamivir (Inavir®), and peramivir (Rapivab®)
interfere with viral neuraminidase emzymatic activity and prevent the
budding of progeny virions.[12,13] Nucleoside analogs ribavirin and
favipiravir (T-705, Avigan®) induce lethal mutagenesis in the viral
genome.[14] Finally, membrane fusion inhibitor arbidol (Umifenovir®) is
approved in Russia and China for prevention and treatment of
influenza.[15] Recently, structure-based in silico screening of library of
compounds bearing a tetrazole moiety effectively suppressed the
replication of influenza viruses.[16] Owing to error-prone activity and
lack of correction mechanisms, viral polymerase provides high rate of
mutations. This results in fast development and spreading of drug
resistance. All influenza virus isolates are resistant to adamantanes,
and almost 100% resistance to oseltamivir has been reported among
H1N1 viruses.[17] There is therefore an urgent need for new drugs of
high level and broad range of efficacy, high barrier for resistance, and
targeting alternative viral proteins essential for viral life cycle.[18] Here
we describe the synthesis and biological activity of novel derivatives of
7,8-dihydro-6H-imidazo[2,1-b][1,3]benzothiazol-5-one.
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RESULTS AND DISCUSSION
Initiating the synthesis (Scheme 1), 2-amino-5,6-dihydrobenzo[d]-
thiazol-7(4H)-ones 1a,b required was prepared from commercially
available cyclic 1,3-dicarbonyls reacting with bromine followed by in
situ condensation with thiourea. For example, 2-amino-5,6-dihydro-
benzo[d]thiazol-7(4H)-one (1a) was prepared in 85% yield by the
reaction of cyclohexane-1,3-dione with bromine in acetic acid in the
presence of sodium acetate; in situ condensation with thiourea at
100°C. Similarly, 2-amino-5,5-dimethyl-5,6-dihydrobenzo[d]thiazol-
7-(4H)-one (1b) was also prepared from 5,5-dimethylcyclohexane-1,3-
dione in 82% yield. Both the compounds 1a and 1b were fully
characterized by their NMR, IR, and ESI-mass spectral data.[23,24]
To build the desired fused heterocyclic analogues, 2-amino-5,6-
dihydrobenzo[d]thiazol-7(4H)-ones 1a,b were reacted with
Recently fused heterocyclic systems with bridgehead nitrogen
(Figure 2) have gained importance as pharmacophore exhibiting broad
spectrum biological activities.[19] Imidazo[2,1-b]thiazole is one such
fused ring system represented well in the literature.[20] Derivatives of
this scaffold have been shown to possess a wide range of biological
activity, from kinase inhibition,[21] antiviral,[22] anticancer,[20] and
antioxidant activities to their use as potential antitubercular
agents.[23,24] Interest in the biological activity of these compounds is
FIGURE 1 Anti-influenza drugs currently in use

GALOCHKINA ET AL.
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FIGURE 2 Antiviral chemical entities with heterocyclic fused ring system
substituted phenacyl bromides 2a–f refluxing in 2-propanol (Scheme
1; Table 1). For example, 2-amino-5,6-dihydrobenzo[d]thiazol-7(4H)-
one (1a) in 2-proponaol was reacted with p-bromophenacyl bromide
(2a) at reflux to give 2-(4-bromophenyl)-6,7-dihydrobenzo[d]imidazo
[2,1-b]-thiazol-8(5H)-one 3a in 82% yield. Under similar conditions, all
the compounds 3a–k were synthesized and fully characterized by their
1H and 13C NMR, IR, and mass (ESI-MS and HRMS) spectral data. LogP
and ClogP required to assess the lipophilic character of new analogs
were calculated using ChemBioDraw 15.0 (Table 1).
presence of para-substituted benzene moieties appended on
imidazole ring and simultaneous gem-dimethyl modification of the
fused cyclohexane ring, the presence of bromo- or cyano-group in
the para position led to an increase in toxicity, while the introduction
of a methoxy group or a chlorine atom in this position led to its
decrease. In some cases, the toxicity of gem-dimethyl derivatives
was higher than that of hydrogen-bearing analogues. Indeed,
introduction of methyl groups instead of hydrogen resulted in
fivefold (compounds 3a and 3b), 1.5-fold (3c and 3d) increase of
toxicity. However, for methoxy- and chlorine-containing derivatives,
the opposite relationship was observed. In these cases, gem-
dimethyl-modification resulted in 7.5-fold and 20-fold decrease of
toxicity, correspondingly.
The cytotoxicity of the compounds against MDCK cells and
antiviral activity of the compounds 3a–k against influenza virus A/
Puerto Rico/8/34 (H1N1) in cell culture were studied by the technique
described previously.[26,27] Based on the results obtained, 50%
cytotoxic concentration (CC50), 50% inhibiting concentration (IC50
)
All three thiophene-substituted analogues 3i–k were of low
toxicity (CC50's from 679 to >1000 μM). In this study, few such
compounds were tested to conclude reliably the decreasing effect of
thiophene group on toxicity of derivatives, although this trend should
be taken into consideration in further studies.
were calculated for each compound. The selectivity index was
calculated for each compound as a ratio of CC50 to IC50. The
compounds with SI = 10 and higher were considered active entities.
The test results are summarized in Table 1. Rimantadine and
oseltamivir carboxylate were used as reference drugs.
Taken together, these results suggest that the toxicity of imidazo
[2,1-b]thiazole-based compounds is complex, and more examples
should be analyzed for reliable structure-toxicity dependence.
Activity against the influenza A virus showed that 3 out of 11
compounds (27%) possess selectivity indices higher than 10, which
allows speaking in general of the moderate potential of this chemical
library as an anti-influenza means. However, all compounds that had
In the group of 6,7-dihydrobenzo[d]imidazo[2,1-b]thiazol-8(5H)-
one derivatives, eleven compounds 3a–k analyzed had a wide range
of cytotoxicity and their CC50 values varied from 49 µM to more
than 1000 µM. All compounds carried phenyl or thiophene unit on
the imidazole ring of imidazo[2,1-b]thiazole core. Structure–activity
correlations of all new compounds 3a–k revealed that in the
FIGURE 3 Design strategy for new derivatives

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GALOCHKINA ET AL.
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SCHEME 1 Synthesis of novel imidazo[2,1-b][1,3]benzothiazol-5-one derivatives 3a–k
no inhibitory activity against influenza virus (3a–h) contained bulky
phenyl substituents. Compared to reference drugs, all the phenyl
substituted variants 3a–h with or without gem-dimethyl group on
fused cyclohexane ring were either more or less toxic but of no antiviral
activity. A decrease in the volume of the substituent on the imidazole
ring (replacement of the substituted benzene moiety by a less bulky
substituted thiophene group) led to a sharp increase in antiviral activity
in all three analyzed representatives of this group. The thiophene-
substituted analogues 3i–k has high activity in inhibiting influenza virus
and lower toxicity than standard drug rimantadine. Compared to
another standard drug oseltamivir (CC50 > 200 μM), thiophene ana-
logues 3i–k have favorable toxicity profile but exhibited lower antiviral
activity. Importantly, the increase of virus-inhibiting activity (high SIs)
after introducing of thiophene instead of benzene moiety was due to
decrease of IC50, and therefore increase of the affinity to specific
target, rather than due to the decrease of toxicity. Thus, further
optimization of the chemical structure in this direction can lead to the
development of effective drugs against influenza. In particular, the
range of thiophene-substituted compounds will be extended to
identify more active compounds of described scaffold.
neuraminidase and have therefore other mechanism of action. Their
further optimization may result in development of novel antivirals that
are active against NAI-resistant variants of influenza virus.
The initial derivative of imidazo[2,1-b]thiazole was shown to
suppress the activity of HCV-encoded protein NS4B. It was shown to
interact with other non-structural proteins of HCV, NS3, NS4A, NS5A
and others, that together provide correct synthesis of viral RNA.[25]
NS4B also targets the replication complex components to specialized
lipid raft compartments[29] and provides numerous regulating inter-
actions with cellular proteins for benefit of viral replication.[30] As both
HCV and influenza virus in their life cycles depend on specific
membrane structures, and both contain RNA-dependent RNA-
polymerase, we supposed that compounds able to inhibit NS4B,
either its membrane-targeting or RNA-polymerase activity, could
appear active against influenza virus as well. Indeed, three of 11
compounds demonstrated high suppressing activity against influenza
virus. Importantly, all three thiophene-substituted derivatives demon-
strated inhibiting activity against amantadine/rimantadine resistant
virus that has been used in experiments. Adamantine derivatives,
amantadine and rimantadine block the viral proton pump M2 thus
preventing the acidification of the virion interior.[10] We conclude
therefore that despite relatively high anti-viral activity of thiophene-
containing imidazo[2,1-b]thiazole derivatives, they are targeted
against neither neuraminidase nor M2 proton channel and have
therefore other mechanism of action. This confirms indirectly our
initial suggestion that their activity could be based on either inhibition
of activity of influenza virus’ polymerase complex or inhibition of
membrane-linked processes in the viral cycle, like transport of RNPs to
specific sites of plasma membrane or processes associated with
membranes of Golgi complex.[31] Their further optimization may result
In attempt to decipher the mechanism of activity of this group of
compounds, three lead compounds 3i–k have been further tested for
their ability to inhibit influenza virus neuraminidase in luminescent
MUNANA-based assay.[28] Based on the data obtained, the values of
IC50 were calculated that appeared 4.4, 121.9, and 6.9 mM for 3i–k
correspondingly (IC50 for reference compound oseltamivir carboxylate
was 0.36 μM). Thus, no correlation was detected between anti-
neuraminidase activity of compounds and their virus-inhibiting
properties. We conclude therefore that despite relatively high anti-
viral activity of novel compounds, they are not targeted against

GALOCHKINA ET AL.
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TABLE 1 Antiviral activity and cytotoxicity of 3a–k against influenza virus A/Puerto Rico/8/34 (H1N1) in MDCK cells
Compound
3a
R1
R2
Yield (%)
LogP/cLogPa
CC50b, μM
IC50c, μM
SId
H
82
4.15/4.35
>1000
>1000
1
3b
3c
3d
3e
Me
H
79
78
79
72
4.95/5.38
3.36/4.35
4.16/3.96
3.88/4.19
210 16
>111
2
1
2
1
108
73
8
86
37
37
9
5
4
Me
H
4
4
49
3f
3g
3h
3i
H
83
77
76
73
3.20/3.49
4.00/4.53
4.68/5.24
4.82/5.29
133
9
>111
1
Me
Me
H
>1000
>1000
>1000
1
>1000
1
679 41
49
13
46
72
6
3
6
8
14
3j
H
78
72
3.68/4.10
4.02/4.26
>1000
>1000
77
22
3k
Me
Rimantadine
N/A
N/A
319 15
>200
4
Oseltamivir carboxylate
0.2 0.0
>1000
aLogP/cLogP was calculated using ChemBioDraw-2016 software.
bCC50 is the median cytotoxic concentration; i.e., the concentration causing 50% cell death.
cIC50 is the 50% inhibiting concentration; i.e., the concentration causing a 50% decrease in virus replication.
dSI is the selectivity index, the CC50/IC50 ratio.
in development of novel antivirals that are active against drug-resistant
variants of influenza virus.
inhibiting influenza A virus compared to the other evaluated compounds.
The substituent on the imidazole ring was shown to have exceptional
importance in anti-viral activity as the decrease of its volume (replacement
of the substituted benzene moiety by a substituted thiophene group) led
to a sharp increase in antiviral activity. Among all compounds, 3j could be
the best candidate to consider as drug-like hit analogue for further
development of library compounds. The results described here demon-
strate the potential utility of 6,7-dihydrobenzo[d]imidazo[2,1-b]thiazol-8
(5H)-ones, tethered with thiophene unit as inhibitors of influenza A virus
and data presented will help global efforts for identification of new
chemical entities as potent drug-like novel antiviral candidates.
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CONCLUSION
In the current study, we have designed and synthesized a series of novel 2-
substituted 6,7-dihydrobenzo[d]imidazo[2,1-b]thiazol-8(5H)-ones 3a–k
through two-step reaction from readily available cyclohexane-1,3-diones.
All the new analogues 3a–k were fully characterized by their NMR and
mass spectral data. Screening of all the 11 new derivatives against antiviral
activity revealed that compounds 3i–k are the most active agents

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EXPERIMENTAL
J = 6.48 Hz, 2H), 2.37 (qt, J = 6.35 Hz, 2H). 13C NMR (75 MHz, CDCl3):
δ 190.69, 143.48, 142.64, 140.76, 132.08, 131.18, 126.50, 122.78,
120.75, 107.70, 36.81, 22.61, 21.41. IR (KBr) 3422, 2929, 1658, 1476,
1353, 1320, 1128, 1002, 833, 725, 541 cm−1. MS (ESI): m/z: 348.9287
[M+H]+2: HRMS (ESI) calcd. for C15H11BrN2OS: 345.97758, found:
345.97755.
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4.1 Chemistry
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4.1.1 General
The original spectra of the investigated compounds as well as their
InChI codes together with some biological activity data are provided as
Supporting Information.
2-(4-Bromophenyl)-6,6-dimethyl-6,7-dihydrobenzo[d]
imidazo[2,1-b]thiazol-8(5H)-one (3b)
Yield: 79%; mp: 243–245°C. 1H NMR (CDCl3, 500 MHz) δ 7.72–7.67
(m, 2H), 7.63 (s, 1H), 7.56–7.51 (m, 2H), 2.86 (s, 2H), 2.56 (s, 2H), 1.25
(s, 6H). 13C NMR (75 MHz, CDCl3) δ 190.75, 151.24, 148.41, 141.14,
132.14, 131.88, 126.91, 122.68, 121.93, 106.23, 51.38, 37.00, 35.00,
28.50. IR (KBr) 3417, 2953, 1663, 1586, 1477, 1355, 1323, 1123,
1002, 727, 543 cm−1. MS (ESI) m/z 377 [M+H]+2: HRMS (ESI) calcd. for
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4.1.2 General procedure for the preparation of
2-amino-5,6-dihydrobenzo[d]thiazol-7(4H)-ones 1a,b
To a mixture of cyclohexane-1,3-dione (5.0 g, 44.5 mmol) and NaOAc
(3.65 g, 44.5 mmol) in acetic acid (50 mL) was added bromine (3.1 g,
44.5 mmol) dropwise at 15–20°C. After stirring the reaction mixture at
room temperature for 12 h, thiourea (5.43 g, 71.3 mmol) was added in
potions and heated at 100°C for 1 h. The reaction mixture was cooled
to RT, acetic acid was removed under vacuum, the resulting crude
product is treated with water (10 mL), neutralized with aqueous
saturated NaHCO3 solution, and extracted with ethylacetate
(3 × 25 mL). The combined organic layer was dried over anhyd.
Na2SO4, filtered and rotary evaporated under vacuum. The crude
residue is triturated with water to give 2-amino-5,6-dihydrobenzo[d]
thiazol-7(4H)-one (1a) as white solid in 85% yield. Mp: 185–187°C.
1H NMR (DMSO, 300 MHz) δ 7.84 (s, 2H), 2.73 (t, J = 6.0 Hz, 2H), 2.42
(t, J = 6.6 Hz, 2H), 2.07 (qt, J = 6.32 Hz, 2H). 13C NMR (75 MHz, CDCl3):
δ 189.40, 173.64, 167.73, 118.65, 36.63, 26.64, 22.29. IR (KBr) 3287,
2943, 1621, 1512, 1384, 1289, 1185, 1080, 835, 540 cm−1. MS (ESI):
m/z: 169 [M+H]+; HR-MS (ESI) calcd. for C7H9N2OS: 169.04301,
found: 169.04326. Similarly, 2-amino-5,5-dimethyl-5,6-dihydrobenzo
[d]thiazol-7(4H)-one (1b) was also prepared from dimedone in 82%
yield. Mp: 205–207°C. 1H NMR (DMSO, 300 MHz) δ 7.79 (s, 2H), 2.62
(s, 2H), 2.30 (s, 2H), 1.10 (s, 6H). 13C NMR (75 MHz, CDCl3 + DMSO) δ
188.97, 178.98, 166.12, 117.30, 50.67, 40.54, 34.03, 27.88. IR (KBr):
3375, 2924, 1628, 1507, 1366, 1053, 832, 524. MS (ESI) m/z 197 [M
+H]+: HR-MS (ESI) calcd. for C9H13N2OS: 197.07505, found: 197.074.
C17H15BrN2OS: 374.00859, found: 374.00885.
4-(8-Oxo-5,6,7,8-tetrahydrobenzo[d]imidazo[2,1-b]thiazol-2-
yl)benzonitrile (3c)
Yield: 78%; mp: 203–206°C. 1H NMR (CDCl3, 500 MHz) δ 7.73–7.69
(m, 2H), 7.66 (s, 1H), 7.56–7.57 (m, 2H), 3.02 (t, J = 6.23 Hz, 2H), 2.71 (t,
J = 6.48 Hz, 2H), 2.37 (qt, J = 6.35 Hz, 6H). 13C NMR (75 MHz,
CDCl3 + DMSO) δ 189.52, 173.32, 167.71, 167.25, 147.71, 138.09,
131.24, 125.29, 118.70, 104.10, 36.20, 26.22, 21.82. IR (KBr) 3377,
3140, 2929, 1639, 1536, 1339, 1127, 1039, 837, 700, 538 cm−1. MS
(ESI) m/z 294 [M+H]+: HRMS (ESI) calcd. for C16H11N3OS: 293.06227,
found: 293.06228.
4-(6,6-Dimethyl-8-oxo-5,6,7,8-tetrahydrobenzo[d]imidazo[2,1-
b]thiazol-2-yl)benzonitrile (3d)
Yield: 79%; mp: 228°C. 1H NMR (CDCl3, 400 MHz): δ 7.98–7.90 (m,
2H), 7.77 (s, 1H), 7.72–7.67 (m, 1H), 2.90 (s, 2H), 2.60 (s, 2H), 1.27 (s,
6H). 13C NMR (125 MHz, CDCl3) δ 190.73, 151.56, 147.35, 141.08,
137.49, 132.56, 125.72, 123.21, 118.79, 111.09, 107.81, 51.34,
36.93, 35.00, 28.44. IR (KBr) 3294, 2942, 2225, 1622, 1512, 1380,
1287, 1184, 1080, 836, 663, 539 cm−1. MS (ESI) m/z 322 [M+H]+:
HRMS (ESI) calcd. for C18H15N3OS: 321.09634, found: 321.09653.
2-(4-Chlorophenyl)-6,7-dihydrobenzo[d]imidazo[2,1-b]thiazol-
8(5H)-one (3e)
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4.1.3 General procedure for the synthesis of
2-substituted 6,7-dihydrobenzo[d]imidazo[2,1-b]-
thiazol-8(5H)-ones 3a–k
Yield: 72%; mp: 237–239°C. 1H NMR (CDCl3, 400 MHz) δ 7.79–7.68
(m, 2H), 7.65 (s, 1H), 7.56–7.36 (m, 2H), 3.01 (t, J = 6.23 Hz, 2H), 2.71 (t,
J = 6.48 Hz, 2H), 2.37 (qt, J = 6.35 Hz, 2H). 13C NMR (100 MHz, CDCl3)
δ 191.06, 148.50, 142.58, 133.83, 132.07, 131.90, 128.96, 126.97,
126.68, 106.23, 37.33, 23.25, 22.00. IR (KBr) 3289, 2942, 1659, 1622,
1514, 1353, 1186, 1128, 1005, 837, 727, 539 cm−1. MS (ESI) m/z 303
[M+H]+: HRMS (ESI) calcd. for C15H11ClN2OS: 302.02851, found:
302.02806.
To a solution of 1a,b (5.0 mmol) in 2-propanol (10 mL) was added
bromides 2a–f (5.0 mmol) and refluxed at 70°C for 3 h. After
completion (TLC), the reaction mixture was cooled to RT, solvent
was evaporated in vacuum. Crude residue was purified over silica gel
column chromatography eluted with hexane/ethyl acetate (5:1) to give
products 3a–k as pale yellow solids.
(2-(4-Bromophenyl)-6,7-dihydrobenzo[d]imidazo[2,1-b]thiazol-
(5H)-one (3a)
Yield: 82%; mp: 257°C. 1H NMR (CDCl3, 500 MHz) δ 7.78–7.63 (m,
2-(4-Methoxyphenyl)-6,7-dihydrobenzo[d]imidazo[2,1-b]-
thiazol-8(5H)-one (3f)
Yield: 83%; mp: 230–231°C. 1H NMR (CDCl3 + DMSO, 300 MHz) δ
2H), 7.66 (s, 1H), 7.56–7.52 (m, 2H), 3.02 (t, J = 6.23 Hz, 2H), 2.71 (t,
7.37–7.24 (m, 2H), 6.90–6.81 (m, 2H), 6.71 (s, 1H), 3.57 (s, 3H),

GALOCHKINA ET AL.
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2.83 (t, J = 6.23 Hz, 2H), 2.62 (d, J = 6.48 Hz, 2H), 1.92 (qt,
J = 6.35 Hz, 2H). 13C NMR (75 MHz, CDCl3 + DMSO) δ 189.37,
173.65, 168.25, 167.73, 148.02, 138.66, 131.77, 125.76, 118.47,
109.20, 104.58, 36.63, 26.65, 22.29. IR (KBr) 3379, 3113, 2944,
2226, 1640,1540, 1349, 1188, 1042, 838, 665 cm−1. MS (ESI) m/z
299 [M+H]+: HRMS (ESI) calcd. for C16H14N2O2S: 299.94520,
found: 299.94518.
2-(5-Bromothiophen-2-yl)-6,6-dimethyl-6,7-dihydrobenzo[d]
imidazo[2,1-b]thiazol-8(5H)-one (3k)
Yield: 72%; mp: 171–173°C. 1H NMR (CDCl3, 500 MHz) δ 7.50 (s, 1H),
7.12 (d, J = 3.96 Hz, 1H), 7.01 (d, J = 3.96 Hz, 1H), 2.85 (s, 2H), 2.57 (s,
2H), 1.24 (s, 6H). 13C NMR (75 MHz, CDCl3) δ 190.24, 149.77, 142.15,
141.53, 138.01, 130.20, 122.86, 121.44, 110.65, 50.70, 36.01, 34.36,
27.76. IR (KBr): 3298, 2957, 1656, 1585, 1477, 1356, 1325, 1245,
1117,
78,
709,
535 cm−1
.
MS
(ESI)
m/z
382
2-(4-Methoxyphenyl)-6,6-dimethyl-6,7-dihydrobenzo[d]-
imidazo[2,1-b]thiazol-8(5H)-one (3g)
[M+H]+2: HRMS (ESI) calcd. for C15H13BrN2OS2: 379.96538, found:
379.96538.
Yield: 77%; mp: 172–174°C. 1H NMR (CDCl3, 400 MHz) δ 7.80–7.66
(m, 2H), 7.50 (s, 1H), 7.03–6.81 (m, 2H), 3.84 (s, 3H), 2.83 (s, 2H), 2.52
(s, 2H), 1.22 (s, 6H). 13C NMR (75 MHz, CDCl3 + DMSO) δ 178.15,
177.73, 168.28, 158.64, 154.22, 150.96, 146.50, 125.10, 120.87,
119.10, 32.63, 27.88, 21.67, 16.96, 14.42. IR (KBr): 3375, 3124, 2960,
1618, 1508, 1366, 1254, 1053, 951, 838, 526 cm−1. MS (ESI) m/z
327.20 [M+H]+: HRMS (ESI) calcd. for C18H19N2O2S: 327.11685,
found: 327.11618.
|
4.2 Biological assays
|
4.2.1 Virus inhibition assay
The compounds 3a–k were dissolved in 0.1 mL DMSO to prepare
stock solutions, and final solutions (1000.0–4.0 μM) were prepared by
adding MEM with 1 μg/mL trypsin. Compounds were incubated with
MDCK cells for 1 h at 36°C. Each concentration of the compounds was
tested in triplicate. The cell culture was then infected with influenza
virus A/Puerto Rico/8/34 (H1N1) (MOI 0.01) for 24 h at 36°C in the
presence of 5% CO2. A virus titer in the supernatant was determined
by hemagglutination test after cultivating of the virus in MDCK cells for
48 h at 36°C in the presence of 5% CO2. Rimantadine and oseltamivir
carboxylate were used as reference drugs. For calculations, virus titer
was expressed as per cent of the titer in control wells without
2-(4-Chlorophenyl)-6,6-dimethyl-6,7-dihydrobenzo[d]-
imidazo[2,1-b]thiazol-8(5H)-one (3h)
Yield: 76%; mp: 212–214°C. 1H NMR (CDCl3, 500 MHz) δ 7.79–7.75
(M, 2H), 7.64 (s, 1H), 7.41–7.37 (m, 2H), 2.88 (s, 2H), 2.58 (s, 2H),
1.25 (s, 6H). 13C NMR (100 MHz, CDCl3) δ 190.75, 151.24, 148.42,
141.15, 133.76, 131.71, 128.95, 126.63, 122.65, 106.18, 51.39,
37.01, 35.01, 28.50. IR (KBr): 3375, 3113, 2925, 1640, 1501, 1366,
1253, 1144, 1053, 949, 585, 522 cm−1. MS (ESI) m/z 331 [M+H]+:
HRMS (ESI) calcd. for C17H15ClN2OS (M+H)+: 330.05942, found:
330.05936.
compounds. The 50% inhibiting concentrations (IC50
) and the
selectivity index (SI, the ratio of CC50 to IC50) were calculated from
the data obtained.
2-(5-Bromothiophen-2-yl)-6,7-dihydrobenzo[d]imidazo[2,1-b]-
thiazol-8(5H)-one (3i)
|
4.2.2 Cytotoxicity assay
Yield: 73%; mp: 148–151°C. 1H NMR (CDCl3, 400 MHz): δ 7.52 (s,
1H), 7.13 (d, J = 3.91 Hz, 1H), 7.01 (d, J = 3.91 Hz, 1H), 2.99 (t,
J = 6.23 Hz, 2H), 2.71 (t, J = 6.48 Hz, 2H), 2.36 (qt, J = 6.35 Hz, H).
13C NMR (CDCl3 + DMSO, 100 MHz): δ 194.43, 150.69, 139.14,
137.83, 136.60, 136.44, 136.27, 128.05, 127.25, 110.89, 35.24,
30.99, 27.49. IR (KBr): 3436, 3286, 3104, 2925, 1630, 1529, 1425,
The microtetrazolium test (MTT)[26] was used to study the cytotoxicity
of the compounds. Briefly, series of threefold dilutions of each
compound in MEM were prepared. MDCK cells were incubated for
48 h at 36°C in 5% CO2 in the presence of the dissolved substances.
The cells were washed twice with phosphate-buffered saline (PBS),
and a solution of 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium
bromide (ICN Biochemicals Inc. Aurora, Ohio) (0.5 μg/mL) in PBS was
added to the wells. After 1 h incubation, the wells were washed and the
formazan residue was dissolved in DMSO (0.1 mL per well). The optical
1331, 1204, 1017, 791, 688 cm−1. MS (ESI): m/z 355 [M+H]+2
HRMS (ESI): calcd. for C13H14BrN2OS2 (M+H)+2: 358.98898, found:
;
358.98819.
density in the wells was then measured on
a
Victor2 1440
2-(5-Chlorothiophen-2-yl)-6,7-dihydrobenzo[d]imidazo[2,1-b]-
thiazol-8(5H)-one (3j)
multifunctional reader (Perkin Elmer, Finland) at wavelength of
535 nm and plotted against the concentration of compounds. Each
concentration was tested in three parallels. The 50% cytotoxic
concentration (CC50) of each compound was calculated from the
data obtained.
Yield: 78%; mp: 202–204°C. 1H NMR (CDCl3, 400 MHz): δ 7.50 (s,
1H), 7.13 (d, J = 3.79 Hz, 1H), 6.87 (d, J = 3.79 Hz, 1H), 2.99 (t,
J = 6.23 Hz, 2H), 2.70 (t, J = 6.48 Hz, 2H), 2.36 (qt, J = 6.35 Hz,
2H). 13C NMR (CDCl3, 125 MHz): δ 190.94, 150.86, 143.47,
142.47, 135.29, 129.53, 126.86, 124.01, 122.55, 105.32, 37.26,
23.17, 21.95. IR (KBr): 3134, 2931, 1661, 1577, 1478, 1354,
1325, 1119, 1026, 801, 710, 541 cm−1. MS (ESI): m/z 309 [M+H]+;
HRMS (ESI) calcd. for C13H9ClN2OS2 (M+H)+: 307.98484, found:
307.98448.
|
4.2.3 Neuraminidase activity assay
The study of virus susceptibility to 3j was conducted by measurement
of activity of neuraminidase in the reaction with fluorogenic
substrate.[28] For this purpose, serial dilutions of the compounds in

8
GALOCHKINA ET AL.
|
the buffer (32.5 mM MES, pH 6.0, 4 mM CaCl2) were mixed with
fivefold dilutions of the virus in the wells of black 96-well plates
(Corning). Plates were incubated 30 min at 37°C. Then 0.2 mM
substrate solution (4-methylumbelliferyl-α-D-N-acetylneuraminic acid)
was added to the wells and plates were further incubated for 30 min at
room temperature. Reaction was stopped with the stop-solution (25%
ethanol, 0.1 М glycine pH 10.7) following by measurement of
luminescence on multifunctional plate reader Victor 1440 (Ex λ
365 nm, Em λ 450 nm). Oseltamivir carboxylate was used as reference
compound. Based on the data obtained, 50% inhibiting concentrations
of 3j and oseltamivir were calculated.
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by Russian Foundation for Basic Research (grant # 17-54-45113) and
Russian Science Foundation grant number 14-50-00068.
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CONFLICT OF INTEREST
All authors declare no financial or commercial conflicts of interest.
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