Synthesis and in vitro anti-HIV activity of N-1,3-benzo[d]thiazol-2-yl-2- (2-oxo-2H-chromen-4-yl)acetamide derivatives using MTT method

Article abstract of DOI:10.1016/j.bmcl.2011.03.105

A series of novel N-1,3-benzo[d]thiazol-2-yl-2-(2-oxo-2H-chromen-4-yl) acetamide derivatives has been synthesized. All the newly synthesized compounds were evaluated for their anti-HIV activity using MTT method. Most of these compounds showed moderate to potent activity against wild-type HIV-1 with an EC₅₀ ranging from >7 EC₅₀ [μg/ml] to <100 EC ₅₀ [μg/ml]. Among them, N-1,3-benzo[d]thiazol-2-yl-2-(2-oxo-2H- chromen-4-yl)acetamide 6v was identified as the most promising compound (EC ₅₀ = <7 μg/ml). Among all the compounds, three compounds 6m, 6v and 6u have been exhibits potent anti-HIV activity against MT-4 cells.

Full text of DOI:10.1016/j.bmcl.2011.03.105

Bioorganic & Medicinal Chemistry Letters 21 (2011) 3443–3446
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and in vitro anti-HIV activity of N-1,3-benzo[d]thiazol-2-yl-2-(2-oxo-
2H-chromen-4-yl)acetamide derivatives using MTT method
Dhairya Bhavsar a, Jalpa Trivedi a, Shrey Parekh a, Mahesh Savant a, Shailesh Thakrar a, Abhay Bavishi a,
Ashish Radadiya a, Hardevsinh Vala a, Jignesh Lunagariya a, Manisha Parmar a, Ladwa Paresh a,
Roberta Loddo b, Anamik Shah a,
⇑
a Department of Chemistry, Saurashtra University, Rajkot 360005, India
b University of Cagliari, Department of Biomedical Sciences and Technologies, Division of General Microbiology and Virology and Microbial Biotechnology, 500 09042 Monserrato, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel N-1,3-benzo[d]thiazol-2-yl-2-(2-oxo-2H-chromen-4-yl)acetamide derivatives has been
synthesized. All the newly synthesized compounds were evaluated for their anti-HIV activity using MTT
method. Most of these compounds showed moderate to potent activity against wild-type HIV-1 with an
Received 3 March 2011
Revised 24 March 2011
Accepted 28 March 2011
Available online 5 April 2011
EC50 ranging from >7 EC50
[
lg/ml] to <100 EC50
[
lg/ml]. Among them, N-1,3-benzo[d]thiazol-2-yl-2-(2-
oxo-2H-chromen-4-yl)acetamide 6v was identified as the most promising compound (EC50 = <7
lg/ml).
Among all the compounds, three compounds 6m, 6v and 6u have been exhibits potent anti-HIV activity
against MT-4 cells.
Keywords:
Benzothiazole chromene derivatives
Anti-HIV activity
MT-4 cells
Ó 2011 Elsevier Ltd. All rights reserved.
MTT method
In the present era, acquired HIV (Human Immunodeficiency
Virus) is the most fatal disorder for which no completely successful
chemotherapy has been developed so far. A variety of anti-HIV
agents rely on inhibition of key enzymes involved with the virus life
cycle. Over 20 anti-retroviral medications have now been approved
for the treatment of HIV-infected individuals.1 The current therapy
against the human immunodeficiency virus type 1 (HIV-1), which
is the etiological agent of acquired immunodeficiency syndrome
(AIDS), is based on six of categories drugs: nucleoside/nucleotide re-
verse transcriptase inhibitors (NRTIs/NtRTIs); nonnucleoside re-
verse transcriptase inhibitors (NNRTIs); protease inhibitors (PIs);
cell entry inhibitors [fusion inhibitors (FIs) and co-receptor inhibi-
tors (CRIs)]; and integrase inhibitors (INIs).2 HAART (Highly active
anti-retroviral therapy) has demonstrated that targeting multiple
enzymes provides one means of circumventing this problem, with
combination therapies directed against both the HIV reverse trans-
criptase and the protease enzymes showing good results in reducing
viral loads in HIV-1 infected patients.3 However, the benefits of
HAART as the standard AIDS chemotherapy are compromised by
high cost and severe toxicity,4,5 both resulting from using multiple
drugs.
transcriptase.6 NNRTIs7–11 bind to the nonnucleoside binding site
(NNBS), an allosteric hydrophobic pocket located about 10 Å far
from the polymerase active site. However, the above inhibitors
failed in monotherapy, combination protocols were designed with
both RTIs and PRIs in order to increase the clinical efficacy and re-
duce the emergence of resistant variants. Rapid development of
drug resistance and toxicity problems make urgent the need to
investigate new targets in the replicative cycle of HIV-1 to develop
inhibitors different from RTIs and PRIs.12,13 Apart from RT and PR,
another target useful for chemotherapeutic intervention is the
HIV-1 integrase (IN), an enzyme which catalyzes the insertion of
the viral DNA into the genome of the host cell through a multistep
process.14,15 However, these drugs have only limited or transient
clinical benefit due to their side effects and the development of
drug-resistant viral strains.16 Therefore, current searches for new
anti-HIV agents are focused on discovering compounds with novel
structures and different mechanisms of action.
Numerous different classes of compounds17,18 have been re-
ported as potent anti-HIV-1 agent. Coumarins (2-oxo-2H-chromen)
have been found to exhibit a wide range of biological and controlled
therapeutic activities in view of their extensive occurrence in nature
and wide range of toxicity. (+)-Calanolide,19,20 a natural dipyrano-
coumarin, currently undergoing anti-AIDS clinical trials, has also
proven to be an effective anti-mycobacterial against drug-sensible
and drug-resistant Mycobacterium tuberculosis strains. Several het-
erocyclic substituted and fused coumarin derivatives are widely
used in drugs. The anti-HIV-1 compounds such as 4-phenyl
The nonnucleoside reverse transcriptase inhibitors (NNRTIs) are
a structurally and chemically dissimilar group of anti-retrovirals
that are potent and highly selective inhibitors of HIV-1 reverse
⇑
Corresponding author.
E-mail address: anamik_shah@hotmail.com (A. Shah).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.03.105

3444
D. Bhavsar et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3443–3446
Figure 1. Biologically active anti-HIV molecules.
coumarins (Fig. 1, 1-2), which could serve as lead compounds for the
development of additional therapeutic approaches against AIDS.
Benzothiazole containing five member heterocyclic compounds
and several benzothiazole derivatives show considerable biological
activities. Moreover, benzothiazoles (Fig. 1, 3) showed inhibitors of
HIV-1 protease with improved potency and anti-viral activities.21
In our efforts to discover novel anti-HIV agents22–25 and
synthesis of various biologically active heterocycles,26–29 we have
synthesized diverse N-1,3-benzo[d]thiazol-2-yl-2-(2-oxo-2H-chro-
men-4-yl)acetamides by the condensation of coumarin-4-acetic
acids and respected benzothiazoles (scheme 1). All the synthesi-
zed N-1,3-benzo[d]thiazol-2-yl-2-(2-oxo-2H-chromen-4-yl)aceta-
mides were screened for their in vitro anti-HIV activity against
human HIV cell line. These compounds required to achieve 50%
protection of MT-4 cells from the HIV-1-induced cytopathogenici-
ty, as determined by the MTT method.
To synthesize substituted coumarin-4-acetic acid (scheme 1) a
mixture of citric acid monohydrate (0.1 M) and 28 ml of concd
sulphuric acid was stirred at room temperature for 60 min and
then slowly heated to 70 °C. After 30 min, the evolution of carbon
monoxide had slackened and the clear yellow colored solution was
rapidly cooled to 0 °C. To this stirred solution, phenol (0.08 M) was
added and 11.2 ml of concd sulphuric acid, each in three equal por-
tions, at a rate that the temperature does not exceed 10 °C. The
resulting solution was stored at 0 °C for 16 h, poured into ice and
the resulting crystalline precipitate was filtered off and washed
with water. It was then treated with 10% sodium bicarbonate solu-
tion and filtered out. The filtrate, on acidification gave substituted
coumarin-4-acetic acid. The physical data of all the synthesized
compounds are cited in Table 1.
limiting dilution method (dilution 1:2, four replica wells per dilu-
tion) in 96-well plates. The infectious virus titer was determined
by light microscope scoring of syncytia after four days of incuba-
tion. Virus titers were expressed as CCID50/mL.
A 20 ml of MTT (7.5 mg/ml) solution (warmed to 37 °C) was
added to each well of the microtiter plates using the titertek multi-
drop dispenser. The trays were incubated at 37 °C in a CO2 incubator
for 1 h. A constant volume of medium (e.g., 150 ml) was removed
from each cup using a multichannel pipette or a microplate washer
without disturbing the MT-4 cell clusters containing the formazan
crystals. The fomazan crystals were solubilized by addition 100 ml
of the acidified triton X-100 isopropanol solution to each cup using
the microplate washer. It was dissolved completely using vibrating
platform shaker for 10 min. The absorbance was examined in an
eight-channel computer-controlled multiskan ascent reader and
stacker at two wavelengths (540 and 690 nm). The absorbance mea-
sured at 690 nm from the absorbance at 540 nm was subtracted to
eliminate the effects of scattering by cell debris.
CC50 is the concentration of compound that reduced the absor-
bance (OD540) of the mock-infected control sample by 50%. The
concentration (lg/ml) required to achieve 50% protection in HIV-
infected cells was calculated by the following formula.
ðODTÞHIV ꢀ ðODCÞHIV
EC50
¼
ðODCÞMOCK ꢀ ðODCÞHIV ꢁ 100
where (ODT)HIV is the OD measured with a given concentration of
the test compound in the HIV-infected cells; (ODC)HIV is the OD
measured for the control untreated, HIV-infected cells, which
stands for 100% infection-related CPE; and (ODC)mock is the OD
measured for the control untreated, mock-infected cells, which
stands for 0% infection-related CPE. A strict scheme of cell cultiva-
tion (subcultivating the cells every 3–4 d, seeding at 6–105 cells
per ml for MT-4 cells) was necessary to avoid overgrowth of the
uninfected cells and decrease of cellular viability after five day
incubation.
MT-4 cells were obtained from institutions such as MRC and
NIH. ‘Complete medium’: RPMI-1640 medium with 20 mM HEPES
buffer, supplemented with 10% (v/v) heat-inactivated FCS, 2 mM
L-glutamine, 0.1% sodium bicarbonate and 20 mg/ml gentamicin.
Titration of HIV was performed in MT-4 cells by the standard
Scheme 1. Synthesis of various N-1, 3-benzo[d]thiazol-2-yl-2-(2-oxo-2H-chromen-4-yl)acetamides. Reagents and condition: (a) concd sulfuric acid; (b) KCNS, Br2, CH3COOH,
0 °C; (c) THF, pyridine, 0-5 °C, SOCl2.

D. Bhavsar et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3443–3446
3445
Table 1
Physical and activity data of functionalized N-1,3-benzo[d]thiazol-2-yl-2-(2-oxo-2H-chromen-4-yl)acetamide 6a–z
Entry
Substitution
Melting range (°C)
Yield (%)
HIV-1a EC50
(lg/ml)
MT-4b CC50
(lg/ml)
R1
R2
R3
R4
R5
R6
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
6m
6n
6o
6p
6q
6r
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
CH3
CH3
CH3
CH3
H
H
H
H
CH3
CH3
CH3
CH3
H
H
H
H
H
H
H
H
H
CH3
CH3
CH3
CH3
H
H
H
H
CH3
CH3
CH3
CH3
H
H
H
H
OH
OH
OH
OH
CH3
CH3
CH3
CH3
H
H
H
H
H
H
H
H
Cl
H
H
H
Cl
H
H
H
Cl
H
H
H
Cl
H
H
H
Cl
H
H
H
Cl
H
H
NO2
Cl
F
F
NO2
Cl
F
F
NO2
Cl
F
F
NO2
Cl
F
F
NO2
Cl
F
F
NO2
Cl
130–132
160–162
106–108
140–142
192–194
118–120
136–138
188–190
110–112
118–120
108–110
116–118
124–126
152–154
148–150
142–144
112–114
96–98
136–138
104–106
186–188
184–186
188–190
208–210
122–124
136–138
54
58
61
63
58
56
51
48
43
63
61
59
54
58
60
61
63
56
58
49
55
52
56
58
67
74
12
21
100
58
49
44
44
17
32
27
20
12
9
60
24
97
100
65
38
16
8
>12
>21
>100
>58
>49
>44
>44
>17
>32
>27
>20
>12
>9
>60
>24
>97
>100
>65
>38
>16
>8
H
H
H
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
H
6s
6t
6u
6v
6w
6x
6y
6z
AZT
H
H
H
7
>7
F
F
21
100
100
100
>21
>100
>100
>100
65.40
–Benzo–
OCH3
OCH3
–Benzo–
H
H
0.0012 0.003
a
Compd concd (
l
g/ml) required to achieve 50% protection of MT-4 cells from the HIV-1-induced cytopathogenicity, as determined by the MTT method.
b
Compd concd (lg/ml) required to reduce the viability of mock-infected MT-4 (CD4+ Human T-cells containing an integrated HTLV-1 genome) cells by 50%, as determined
by the colorimetric MTT method.
The OD ratio, defined as (ODC)MOCK/(ODC)HIV. The OD ratio was
low when the cells were not in optimal condition or not enough
viruses were added. Reference compounds were chosen by consid-
eration their spectrum of activity, for example, the nucleoside re-
verse transcriptase inhibitor (NRTI) AZT is equipotent in blocking
the HIV-1 replication in vitro, whereas nevirapine, an NNRTI, has
a spectrum that is limited to HIV-1. AZT was used as a control on
the cytotoxicity.
MTT method. Among them, three compounds 6m, 6u and 6v have
been found potent active against wild-type HIV-1 cells with EC50 9,
8 and 7, respectively. However, few compounds were demon-
strated moderate in vitro anti-HIV activity against wild-type HIV-
1 cell line. The significance of this process is good yield and easy
work up process for biological active molecules.
Acknowledgments
Three groups of the compounds were studied for anti-HIV activi-
tiesaccordingto theEC50 valuesmeasuredonthewild-type HIV-1 cell
The authors are thankful to FIST-DST and SAP-UGC for their
generous financial and instrumentation support. Special thanks
are due to ‘National Facility for Drug Discovery through New
Chemical Entities (NCE’s) Development & Instrumentation Support
to Small Manufacturing Pharma Enterprises’. Programme under
Drug & Pharma Research Support (DPRS) jointly funded by Depart-
ment of Science & Technology, New Delhi, Government of Gujarat
Industries Commissionerate & Saurashtra University, Rajkot.
line: strong anti-HIV effect 0 < EC50 < 10
6m, 6u and 6v), moderate anti-HIV effect 10 < EC50 < 50
compounds, Table 1) and no anti-proliferative effect where EC50
>50 g/ml was found (Table 1). The lowest EC50 value was found
<7 g/ml (6v) while some compounds were ineffective even at the
applied 50 g/ml concentration (i.e., 6c, 6q, 6x–z).
lg/ml (three compounds:
lg/ml (13
l
l
l
Structure–activity reveals that –OH substitution increase the
potency thus –OH group plays a vital role in hydrogen bonding.
While methyl substitution at R2 and R4 position (6q– s) lowers
the biological potential so bulky substitution at both R2 and R4
group does not favorable and this explains that the inactivity of
compounds having benzo ring attached at R1 and R2 position in
6z molecule and R3 and R4 position in 6y molecule.
Supplementary data
Supplementary data (general procedure of 2-amino benzothia-
zole
and
N-1,3-benzo[d]thiazol-2-yl-2-(2-oxo-2H-chromen-
In summary, we have developed a facile and efficient synthesis
of N-1,3-benzo[d]thiazol-2-yl-2-(2-oxo-2H-chromen-4-yl)acetamide
derivatives and exhibits there in vitro anti-HIV activity using
4-yl)acetamide deri-vatives 6a–z, experimental details, compound
characterization 1H NMR, MS and IR analysis for final products and
assay methods. This material is available free of charge via the

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D. Bhavsar et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3443–3446
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