Design and synthesis of 2-(2,6-dibromophenyl)-3-heteroaryl-1,3-thiazolidin-4-ones as anti-HIV agents

Article abstract of DOI:10.1016/j.ejmech.2007.12.015

A series of 2-(2,6-dibromophenyl)-3-heteroaryl-1,3-thiazolidin-4-ones were designed, synthesized and evaluated as selective human immunodeficiency virus type-1 reverse transcriptase (HIV-1 RT) enzyme inhibitors. The results of the HIV-1 RT kit and in vitro cell based assay showed that eight compounds effectively inhibited HIV-1 replication at 20-320 nM concentrations with minimal cytotoxicity in MT-4 as well as in CEM cells.

Full text of DOI:10.1016/j.ejmech.2007.12.015

Available online at www.sciencedirect.com
European Journal of Medicinal Chemistry 43 (2008) 2800e2806
http://www.elsevier.com/locate/ejmech
Original article
Design and synthesis of 2-(2,6-dibromophenyl)-3-heteroaryl-1,
3-thiazolidin-4-ones as anti-HIV agents
Ravindra K. Rawal a, Rajkamal Tripathi b, S.B. Katti a,
Christophe Pannecouque c, Erik De Clercq c
*
,
a Medicinal and Process Chemistry Division, Central Drug Research Institute, Lucknow 226 001, India
b Microbiology Division, Central Drug Research Institute, Lucknow 226 001, India
c Rega Institute for Medical Research, Katholieke Universiteit Leuven, 10 Minderbroedersstraat, B-3000 Leuven, Belgium
Received 9 August 2007; received in revised form 6 December 2007; accepted 11 December 2007
Available online 27 December 2007
Abstract
A series of 2-(2,6-dibromophenyl)-3-heteroaryl-1,3-thiazolidin-4-ones were designed, synthesized and evaluated as selective human immu-
nodeficiency virus type-1 reverse transcriptase (HIV-1 RT) enzyme inhibitors. The results of the HIV-1 RT kit and in vitro cell based assay
showed that eight compounds effectively inhibited HIV-1 replication at 20e320 nM concentrations with minimal cytotoxicity in MT-4 as
well as in CEM cells.
Ó 2007 Elsevier Masson SAS. All rights reserved.
Keywords: 4-Thiazolidinones; Anti-HIV activity; HIV-1RT; NNRTIs
1. Introduction
are strongly dependent on the nature of the substituent at C-2
and N-3 of the thiazolidin-4-one ring. In particular, a high
activity level was observed for compounds possessing a 2,6-
dihalophenyl group at C-2 and heteroaryl with an appropriate
substitution at N-3. In majority of the compounds, the 2,6-
dichlorophenyl substituted derivatives at C-2 are more active
than the corresponding 2,6-difluorophenyl substituted ones,
and the favourable effect of 2,6-dichloro derivative is confirmed
by the finding that 2-chloro-6-fluorophenyl derivatives
possessed intermediate activity between 2,6-dichloro and 2,6-
difluoro analogues [23e26]. Based on this data it was surmised
that there is a positive correlation between size of the halogen
substituent and HIV-RT inhibitory activity and by extension
of this logic it seemed appropriate to explore 2,6-dibromo-
phenyl substituted 4-thiazolidinone for HIV-RT inhibitory ac-
tivity. In the light of the above mentioned findings and as
continuation of our effort to identify new candidate that may
be of value in designing potent, selective and less toxic anti-
HIV agents, we report in the present work the synthesis and
anti-HIV activity of some new 2-(2,6-dibromophenyl)-3-heter-
oaryl-1,3-thiazolidin-4-one derivatives (Fig. 1).
4-Thiazolidinone scaffold has been gaining prominence due
to the fact that its derivatives have been known to possess wide
spectrum of activities like antibacterial [1,2], antifungal [3,4],
anticonvulsant [5,6], COX-1 inhibitor [7], antituberculosis
[8e10], antihistaminic [11] and anticancer [12]. Furthermore,
Barreca et al. and our group have reported 2,3-diaryl-1,3-thiazo-
lidin-4-one scaffold as human immunodeficiency virus-1 (HIV-1)
non-nucleoside reverse transcriptase inhibitor (NNRTI)
[13e19]. Non-nucleoside reverse transcriptase inhibitors are
selective inhibitors of reverse transcriptase enzyme (RT) of
HIV-1, which do not inhibit HIV-2 or human cellular DNA poly-
merases [20e22]. NNRTIs are indicated for the treatment of
HIV/AIDS in combination with other antiretroviral agents.
It is inferred from the previous literature [13e19] that the
anti-HIVactivity of 2,3-diaryl-1,3-thiazolidin-4-one derivatives
* Corresponding author. Tel.: þ91 522 2212412x4280; fax: þ91 522
223405/938.
E-mail address: setu_katti@yahoo.com (S.B. Katti).
0223-5234/$ - see front matter Ó 2007 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2007.12.015

R.K. Rawal et al. / European Journal of Medicinal Chemistry 43 (2008) 2800e2806
2801
HIV activity by determining their ability to inhibit the replica-
tion of HIV-1IIIB and HIV-2ROD reverse transcriptase enzyme in
acutely infected MT-4 and CEM cell lines and the data are
shown in Tables 1 and 2, respectively. The in vitro anti-HIV
activities in MT-4 and CEM cell lines were also in agreement
with the HIV-1 RT inhibition assay which was performed by
using RTassay kit. Those compounds showing inhibitory activ-
ity against RT in HIV-1 RT kit also show anti-HIV-1 activity in
both cell lines (Tables 1 and 2). Compound induced cytotoxic-
ity was also measured in MT-4 and CEM cells in parallel with
the HIV-RT inhibitory activity [31,32] (Tables 1 and 2).
The results of the present study show that our approach has
led to the development of potent anti-HIVagents, up to 14-fold
more active than the corresponding TBZ lead compound. This
is in conformity with the earlier observation namely, nature of
the substituent at C-2 and N-3 of the thiazolidin-4-one ring
and butterfly conformation collectively facilitate better inter-
action with the allosteric binding pocket residues of HIV-1
RT enzyme. This has a direct bearing on the anti-HIV activity.
Structure activity relationship studies (SARs) suggest that
the anti-HIV activity of this series was strongly enhanced by
introducing different heteroaryl moiety with/or without an
appropriate lipophilic substitution at the N-3 atom of the thia-
zolidinone ring and in particular by 2,6-dibromophenyl ring at
C-2. Compounds 6f, 6j, 6k and 6n prevented the cytopathic
effect of HIV-1IIIB with an EC50 value ranging from 20 to
90 nM and were minimally toxic to MT-4 cells resulting in
remarkably high selectivity indices up to 7123. It was worth
noting that eight compounds 6c, 6e, 6f, 6iel and 6n of this
series were found most active 1- to 17.5-fold as compared to
the lead compound TBZ-1 in MT-4 cells. Similarly, these eight
compounds exhibited the most promising activity at nanomo-
lar concentrations in CEM cells whereas compounds 6aec,
6eef, 6iel and 6n were found 1.04- to 30-fold most active
as compared to the lead compound TBZ-2 in MT-4 cells.
Compounds 6j and 6k were found 1.25- to 2.5-fold more
active than nevirapine in MT-4 cells. While none of the com-
pounds were more effective than nevirapine in CEM cells but
the compounds 6f, 6ie6k and 6n were having more selectivity
indices as compared to nevirapine. As observed for other clas-
ses of NNRTIs, none of the compounds inhibited the replica-
tion of HIV-2ROD in MT-4 as well as in CEM cells at subtoxic
concentrations.
Fig. 1. Structures of TBZs (I), Nevirapine (II) and 2-(2,6-dibromophenyl)-3-
heteroaryl-1,3-thiazolidin-4-one (III).
2. Results and discussion
2.1. Chemistry
2,6-Dibromobenzaldehyde (3) was synthesized following
the literature procedure [27] as shown in Scheme 1. In this
procedure, 2,6-dibromoaniline (1) was diazotized by using
H2SO4 and NaNO2 at ꢀ5 ꢁC, then the cold solution of diazo-
nium salt was neutralized by NaOAc and treated with formal-
doxime solution followed by reflux in 50% H2SO4 in water to
obtain the aldehyde (3). The commercially unavailable heter-
oaromatic amines namely, 4-methy-6-trifluoromethylpyrimi-
din-2-ylamine (4l), 4-methyl-6-phenylpyrimidin-2-ylamine
(4m) and 4,5,6-trimethylpyrimidin-2-ylamine (4n) were syn-
thesized in good yields by refluxing free base guanidine solu-
tion in ethanol with corresponding 1,3-dicarbonyl compounds
according to the reported procedure [28] whereas the synthesis
of 4-amino-2,6-dimethylpyrimidine was carried out by heating
the mixture of acetonitrile and NaOMe at 150 ꢁC in a glass
sealed tube [29]. The synthesis of a new series of 2-(2,6-dibro-
mophenyl)-3-heteroaryl-1,3-thiazolidin-4-ones (6aeo) were
carried out according to the reported procedure [14], by react-
ing a suitably substituted heteroaromatic amine (4aeo) with
2,6-dibromobenzaldehyde (3) in the presence of an excess of
mercaptoacetic acid (5) in reflux toluene for 24 h (Scheme 2).
After completion of the reaction, desired final products were
obtained by conventional work-up procedure followed by flash
column chromatography purification on silica gel 230e400
mesh size in moderate to good yields (6aeo). All the synthe-
sized compounds were well characterized by spectroscopic
methods and elemental analysis.
2.2. Biological activity
All compounds 6aeo were evaluated for HIV-1 RT inhibi-
tory activity by determining their percentage inhibition of
HIV-1 RT activity in HIV-1 RT kit [30]. Further, compounds
6aeo, TBZs (I) and nevirapine (II) were evaluated for anti-
3. Conclusion
In conclusion, novel 2-(2,6-dibromophenyl)-3-heteroaryl-
1,3-thiazolidin-4-one
series
were
synthesized
and
Scheme 1. Preparation of 2,6-dibromobenzaldehyde.

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R.K. Rawal et al. / European Journal of Medicinal Chemistry 43 (2008) 2800e2806
Scheme 2. Synthesis of 2-(2,6-dibromophenyl)-3-heteroaryl-1,3-thiazolidin-4-ones (6aeo).
characterized. Eight compounds were found to be the most
promising of this series and proved to be potent anti-HIV-1
agents, inhibiting virus replication at 20e320 nM concentra-
tions with minimal cytotoxicity and high selectivity index up
to 7123 in MT-4 and 6726 in CEM cells, respectively. Further-
more these compounds were found to be 1- to 17.5-fold more
active in MT-4 cells and 1.7- to 36.7-fold more effective in
CEM cells than TBZ-1, respectively. This study suggests
that only possible position for modulating anti-HIV activity
in the case of 4-thiazolidinone skeleton is N-3 position of
this privileged scaffold. It may be inferred that 4-thiazolidi-
none holds promise for further activity optimization studies.
Table 1
Anti-HIV-1 activity, cytotoxicity and selectivity index in MT-4 cells and HIV-1 RT kit assay for compounds 6aeo
Compound
% Yield
Anti-HIV-1 activity
In vitro % inhibition
(HIV-RT kit assay)
100 mg/ml
a
EC50 (mM)
b
CC50 (mM)
SIc
6a
6b
62
64
58
56
50
48
49
37
68
61
42
36
28
25
59
0.50 ꢂ 0.14
0.58 ꢂ 0.21
0.21 ꢂ 0.06
0.84 ꢂ 0.02
0.22 ꢂ 0.02
0.09 ꢂ 0.06
4.56 ꢂ 0.14
1.03 ꢂ 0.41
0.24 ꢂ 0.02
0.04 ꢂ 0.00
0.02 ꢂ 0.01
0.32 ꢂ 0.02
>8.65
108.56 ꢂ 120.20
34.63 ꢂ 0.37
36.39 ꢂ 1.65
173.61 ꢂ 10.42
13.06 ꢂ 8.11
233.22 ꢂ 19.53
29.97 ꢂ 4.00
97.98 ꢂ 25.22
172.13 ꢂ 28.36
33.86 ꢂ 8.18
184.76 ꢂ 42.22
32.59 ꢂ 1.53
183.78 ꢂ 13.16
227.48
218
59
69.50
84.30
99.00
97.60
85.20
79.50
23.87
61.36
100
6c
6d
171
209
60
6e
6f
6g
2663
7
6h
6i
96
687
757
7123
99
6j
6k
100
100
6l
6m
85.37
17.75
100
21
6n
6o
0.06 ꢂ 0.00
5.21 ꢂ 1.35
0.35 ꢂ 0.14
0.60 ꢂ 0.09
0.05
3721
6
33.35 ꢂ 1.80
19.20 ꢂ 2.80
9.40 ꢂ 6.50
66.36
e
e
100
TBZ-1
TBZ-2
Nevirapine
a
54.50
16
>50.00
>1000
Concentration required to reduce HIV-1 induced cytopathic effect by 50% in MT-4 cells.
Concentration required to reduce MT-4 cell viability by 50%.
b
c
Selectivity index ratio CC50/EC50
.

R.K. Rawal et al. / European Journal of Medicinal Chemistry 43 (2008) 2800e2806
2803
Table 2
Anti-HIV-1 activity, cytotoxicity and selectivity index of selected compounds
in CEM cells
ethylacetate. The organic layer was washed with brine and
concentrated under vacuum. The crude product was purified
by using 20% ethylacetate:hexane as eluent through column
chromatography. The structure of 2,6-dibromobenzaldehyde
(3) was characterized by means of TLC, IR, FAB-MS, H
NMR and 13C NMR.
Compound
Anti-HIV-1 activity
a
EC50 (mM)
b
CC50 (mM)
SIc
1
6a
6b
0.56 ꢂ 0.14
0.60 ꢂ 0.14
0.18 ꢂ 0.07
0.63 ꢂ 0.35
0.27 ꢂ 0.07
0.08 ꢂ 0.04
3.96 ꢂ 1.06
1.83 ꢂ 1.44
0.23 ꢂ 0.04
0.04 ꢂ 0.01
0.03 ꢂ 0.01
0.36 ꢂ 0.08
0.06 ꢂ 0.01
4.67 ꢂ 1.31
1.10 ꢂ 0.32
0.01 ꢂ 0.01
108.56 ꢂ 120.20
35.60 ꢂ 2.19
36.63 ꢂ 1.53
117.46 ꢂ 12.50
17.07 ꢂ 11.37
233.22 ꢂ 19.53
29.79 ꢂ 3.48
97.98 ꢂ 25.22
178.70 ꢂ 28.62
36.26 ꢂ 8.90
186.12 ꢂ 36.69
59.30 ꢂ 59.74
>273.42
194
59
6c
6d
200
283
66
4.2.1. 2,6-Dibromobenzaldehyde (3)
This compound was obtained as solid in 40% yield, mp
;
90e91 ꢁC; IR (KBr): nmax C]O 1696 cmꢀ1 1H NMR
(200 MHz, CDCl3): d 7.41 (m, 2H, H3ePh and H5ePh),
7.49 (m, 1H, H4ePh), 10.48 (s, 1H, eCHO); 13C NMR
(50 MHz, CDCl3): d 189.15, 142.81, 137.23, 133.99 (2C),
130.16 (2C); FAB-MS: m/z 265 [M þ 1]þ. Anal. Calcd for
C7H4Br2O: C, 31.86; H, 1.53. Found: C, 32.08; H, 1.35.
6e
6f
6g
2954
8
6h
6i
53
769
930
6726
161
>5020
7
6j
6k
6l
6n
6o
TBZ-1
Nevirapine
33.53 ꢂ 1.62
50.00 ꢂ 3.20
>4.00
4.3. General synthetic procedure for compounds 6aeo
45
>370
The synthesis of compounds 6aeo was performed according
to the previously reported procedure [14]. The appropriate (het-
ero)aromatic amine (1.0 mmol) and 2,6-dibromo-benzaldehyde
3 (1.2 mmol) were stirred in dry toluene under reflux condition
followed by addition of mercaptoacetic acid (2.0 mmol). The
reaction mixture was refluxed under stirring for an additional
24e48 h till the complete consumption of the amine compo-
nent. The reaction mixture was concentrated to dryness under
reduced pressure and the residue was taken up in ethyl acetate.
The organic layer was successively washed with 5% aq. citric
acid, water, 5% aq. sodium hydrogen carbonate and then finally
with brine. The organic layer was dried over sodium sulphate
and solvent was removed under reduced pressure to get a crude
product that was purified by column chromatography on silica
gel using hexaneeethyl acetate as eluent. The structures of final
compounds were characterized by means of TLC, IR, FAB-MS,
1H NMR, 13C NMR and elemental analysis.
a
Concentration required to reduce HIV-1 induced cytopathic effect by 50%
in CEM cells.
b
Concentration required to reduce CEM cell viability by 50%.
Selectivity index ratio CC50/EC50
c
.
4. Experimental protocols
4.1. General experimental procedures
Melting points (mp) were determined on a Complab melt-
ing point apparatus and are uncorrected. Infrared (IR) spectra
were recorded on an FT-IR PerkineElmer 621 spectrometer.
The 1H NMR spectra were recorded on a DPX-200 and
DPX-300 Bruker FT-NMR spectrometer using CDCl3 as a sol-
vent. The chemical shifts are reported as parts per million (d
ppm) from (CH3)4Si (TMS) as an internal standard. The 13C
NMR spectra were recorded on a DPX-300 Bruker FT-NMR
(75 MHz) spectrometer. Mass spectra were obtained using
glycerol or m-nitrobenzyl alcohol as matrix either by fast
atom bombardment (FAB positive) or by electrospray ioniza-
tion (ESI-MS) technique. Elemental analyses were carried
out on CARLO-ERBA EA1108 C, H, N elemental analyzer
and values were in the acceptable limits of the calculated
values. Column chromatography separations were performed
on silica gel (230e400 mesh).
4.3.1. 20-(2,6-Dibromophenyl)-[2,3]bithiazolyl-40-one (6a)
This compound was obtained as solid in 62% yield, mp
;
158e162 ꢁC; IR (KBr): nmax C]O 1693 cmꢀ1 1H NMR
(300 MHz, CDCl3): d 3.9 (dd, J ¼ 2.0 and 16.0 Hz, 1H, 5-
HA), 4.2 (dd, J ¼ 2.2 and 16.0 Hz, 1H, 5-HB), 7.0 (d,
J ¼ 3.4 Hz, 1H, H5ethiazole), 7.0e7.1 (m, 1H, H4ePh),
7.1e7.3 (m, 1H, H3ePh), 7.3 (d, J ¼ 3.3 Hz, 1H, H4ethia-
zole), 7.4e7.6 (m, 2H, H2ePh and H5ePh); 13C NMR
(75 MHz, CDCl3): d 169.4, 155.2, 136.5, 133.4, 131.5,
130.9, 129.9, 128.5, 128.1, 112.8, 61.7, 33.7; ESI-MS: m/z
421 [M þ 1]þ. Anal. Calcd for C12H8Br2N2OS2: C, 34.30;
H, 1.92; N, 6.67. Found: C, 34.28; H, 2.02; N, 6.61.
4.2. General synthetic procedure for 2,6-
dibromobenzaldehyde (3)
2,6-Dibromoaniline 1 (1 mmol) was diazotized by using
H2SO4 (1.5 mmol) and NaNO2 (1.2 mmol) at ꢀ5 ꢁC, then
the cold solution of diazonium salt was neutralized by NaOAc
and treated with catalytic amount of CuSO4, NaSO3 and
formaldoxime (1.5 mmol) solution. The reaction mixture was
stirred at room temperature for 1 h and then stirred at 80 ꢁC
for 4 h. After 4 h 50 ml of 50% H2SO4 was added and refluxed
for 8 h at 150 ꢁC. After completion of the reaction, the reac-
tion mixture was neutralized with NaHCO3 and extracted in
4.3.2. 2-(2,6-Dibromophenyl)-3-(thiophen-
2-ylmethyl)thiazolidin-4-one (6b)
This compound was obtained as semisolid in 64% yield, IR
1
(Neat): nmax C]O 1682 cmꢀ1; H NMR (200 MHz, CDCl3):
d 3.6 (d, J ¼ 15.3 Hz, 1H, CH2 thiophene-2yl-methyl), 3.7 (d,
J ¼ 14.9 Hz, 1H, HA), 3.9 (d, J ¼ 14.9 Hz, 1H, HB), 5.1 (d,
J ¼ 15.3 Hz, 1H, CH2 thiophene-2yl-methyl), 5.8 (s, 1H, H-
2), 6.9e7.0 (m, 3H, H3ePh and H5ePh and H4ethiophene),

2804
R.K. Rawal et al. / European Journal of Medicinal Chemistry 43 (2008) 2800e2806
7.1 (d, J ¼ 3.0 Hz, 1H, H3ethiophene), 7.4 (d, J ¼ 4.7 Hz, 1H,
H5ethiophene), 7.6 (t, 1H, H4ePh); 13C NMR (75 MHz,
CDCl3): d 168.9, 142.3, 134.1, 132.2, 129.8, 126.6, 126.3,
125.6, 125.4, 125.1, 124.8, 56.8, 39.8, 31.2; ESI-MS m/z 434
[M þ 1]þ. Anal. Calcd for C14H11Br2NOS2: C, 38.82; H,
2.56; N, 3.23. Found: C, 38.78; H, 2.44; N, 3.20.
J ¼ 7.9 Hz, H5ePy), 7.5 (m, 2H, H3ePh and H5ePh), 7.6
(d, 1H, J ¼ 2.8 Hz, H-2), 7.9 (dd, 1H, J ¼ 3.2 and 8.3 Hz,
H3ePy); 13C NMR (75 MHz, CDCl3): d 170.0, 155.2, 148.5,
136.6 (2C), 133.4, 131.1, 128.5, 125.2, 118.8 (2C), 112.1,
62.2, 34.6, 22.3; ESI-MS: m/z 429 [M þ 1]þ. Anal. Calcd
for C15H12Br2N2OS: C, 42.08; H, 2.83; N, 6.54; Found: C,
42.01; H, 2.65; N, 6.34.
4.3.3. 2-(2,6-Dibromophenyl)-3-(furan-2-ylmethyl)
thiazolidin-4-one (6c)
This compound was obtained as semisolid in 58% yield, IR
4.3.7. 2-(2,6-Dibromophenyl)-3-(6-trifluoromethylpyridin-2-yl)
thiazolidin-4-one (6g)
This compound was obtained as solid in 49% yield, mp
1
(Neat): nmax C]O 1684 cmꢀ1; H NMR (200 MHz, CDCl3):
d 3.6 (d, J ¼ 15.6 Hz, 1H, CH2eFu, 3.7 (d, J ¼ 15.8 Hz, 1H,
HA), 3.8 (d, J ¼ 15.8 Hz, 1H, HB), 4.9 (d, J ¼ 15.6 Hz, 1H,
CH2eFu), 6.1 (s, 1H, H-2), 6.3 (d, J ¼ 3.1 Hz, 1H, H3eFu),
6.5 (m, 1H, H4eFu), 7.1 (m, 1H, H4ePh), 7.4 (d,
J ¼ 7.3 Hz, 1H, H5eFu), 7.5 (m, 2H, H3ePh and H5ePh);
13C NMR (75 MHz, CDCl3): d 170.1, 146.9, 141.6, 131.7,
131.0, 129.6, 129.3, 128.9, 127.6, 109.2, 108.4, 60.6, 38.1,
33.2; FAB-MS m/z 418 [M þ 1]þ. Anal. Calcd for
C14H11Br2NO2S: C, 40.31; H, 2.66; N, 3.36; S, 7.69. Found:
C, 40.55; H, 2.64; N, 3.21; S, 7.79.
150e152 ꢁC; IR (KBr): nmax C]O 1691 cmꢀ1 1H NMR
;
(300 MHz, CDCl3): d 4.0 (dd, J ¼ 2.7 and 16.0 Hz, 1H, 5-
HA), 4.2 (dd, J ¼ 1.8 and 16.0 Hz, 1H, 5-HB), 6.9 (m, 1H,
H5ePy), 7.1 (m, 1H, H4ePh), 7.3e7.7 (m, 3H, H-2 and
H3ePh and H5ePh), 7.9 (t, 1H, H4ePy), 8.5 (t, 1H, H3e
Py); 13C NMR (75 MHz, CDCl3): d 170.1, 158.0, 149.3,
137.9 (2C), 133.2, 131.4, 130.8, 129.7, 128.8, 128.0, 117.7,
115.5, 62.1, 34.3; ESI-MS: m/z 483 [M þ 1]þ.
4.3.8. 3-(5-Bromo-6-methylpyridin-2-yl)-
2-(2,6-dibromophenyl)thiazolidin-4-one (6h)
This compound was obtained as solid in 37% yield, mp
4.3.4. 2-(2,6-Dibromophenyl)-3-(pyridin-
2-ylmethyl)thiazolidin-4-one (6d)
This compound was obtained as white solid in 56% yield,
148e152 ꢁC; IR (KBr): nmax C]O 1692 cmꢀ1 1H NMR
;
(200 MHz, CDCl3): d 2.4 (s, 3H, CH3 at C6ePy), 3.9 (dd,
J ¼ 4.6 and 15.9 Hz, 1H, 5-HA), 4.1 (dd, J ¼ 1.9 and
15.9 Hz, 1H, 5-HB), 6.9 (m, 2H, H3ePh and H5ePh), 7.2
(m, 1H, H4ePh), 7.5 (d, J ¼ 8.9 Hz, 1H, H3ePy), 7.7 (d,
J ¼ 8.8 Hz, 1H, H4ePy), 7.9 (s, 1H, H-2); 13C NMR
(75 MHz, CDCl3): d 170.1, 153.8, 147.2, 140.0, 133.6,
131.2, 128.7, 128.3, 125.0, 120.2, 115.5, 113.6, 62.0, 34.5,
22.6; ESI-MS: m/z 508 [M þ 1]þ. Anal. Calcd for
C15H11Br3N2OS: C, 35.53; H, 2.19; N, 5.52. Found: C,
35.48; H, 2.10; N, 5.39.
mp 184e187 ꢁC; IR (KBr): nmax C]O 1694 cmꢀ1 1H
;
NMR (200 MHz, CDCl3): d 3.7 (d, J ¼ 15.2 Hz, 1H, CH2e
Py), 3.8 (d, J ¼ 16.9 Hz, 1H, HA), 3.9 (dd, J ¼ 2.2 and
16.9 Hz, 1H, HB), 5.1 (d, J ¼ 15.2 Hz, 1H, CH2ePy), 6.6 (d,
J ¼ 2.0 Hz, 1H, H-2), 7.1e7.4 (m, 5H, H3ePh, H4ePh, H5e
Ph, H3ePy and H5ePy), 7.6 (t, 1H, H4ePy), 8.5 (d, 1H,
J ¼ 3.8 Hz, H6ePy); ESI-MS m/z 429 [M þ 1]þ. Anal. Calcd
for C15H12Br2N2OS: C, 42.08; H, 2.83; N, 6.54; Found: C,
42.04; H, 2.74; N, 6.43.
4.3.5. 2-(2,6-Dibromophenyl)-3-(pyridin-2-yl)thiazolidin-
4-one (6e)
This compound was obtained as solid in 50% yield, mp
4.3.9. 2-(2,6-Dibromophenyl)-3-(pyrimidin-2-yl)thiazolidin-
4-one (6i)
This compound was obtained as solid in 68% yield, mp
;
180e184 ꢁC; IR (KBr): nmax C]O 1703 cmꢀ1 1H NMR
145e149 ꢁC; IR (KBr): nmax C]O 1692 cmꢀ1
;
1H NMR
(200 MHz, CDCl3): d 3.9 (dd, J ¼ 4.9 and 15.8 Hz, 1H, 5-
HA), 4.1 (dd, J ¼ 1.7 and 15.8 Hz, 1H, 5-HB), 6.9
(d, J ¼ 8.1 Hz, 1H, H5ePy), 7.0 (m, 2H, H3ePh and H5e
Ph), 7.4 (m, 1H, H4ePh), 7.5 (dd, J ¼ 7.8 and 9.0 Hz, 1H,
H4ePy), 8.1 (d, J ¼ 8.5 Hz, 1H, H3ePy), 8.2 (d, J ¼ 3.0 Hz,
1H, H-2); 13C NMR (75 MHz, CDCl3): d 170.2, 149.3,
146.1, 136.3, 133.5, 131.3, 129.9, 128.6, 128.2, 119.4,
115.6, 115.3, 62.1, 34.5; ESI-MS: m/z 415 [M þ 1]þ. Anal.
Calcd for C14H10Br2N2OS: C, 40.60; H, 2.43; N, 6.76; Found:
C, 40.54; H, 2.45; N, 6.74.
(300 MHz, CDCl3): d 3.9 (dd, J ¼ 1.6 and 15.9 Hz, 1H, 5-
HA), 4.2 (dd, J ¼ 1.9 and 15.9 Hz, 1H, 5-HB), 6.9 (t, 1H,
H4ePh), 7.0 (m, 1H, H5ePym), 7.0 (s, 1H, H-2), 7.4 (m,
2H, H3ePh and H5ePh), 8.6 (d, J ¼ 4.8 Hz, 2H, H4ePh and
H6ePym); ESI-MS: m/z 416 [M þ 1]þ. Anal. Calcd for
C13H9Br2N3OS: C, 37.61; H, 2.19; N, 10.12. Found: C,
37.44; H, 2.29; N, 9.98.
4.3.10. 2-(2,6-Dibromophenyl)-3-(4-methylpyrimidin-2-yl)
thiazolidin-4-one (6j)
This compound was obtained as solid in 61% yield, mp
;
170e174 ꢁC; IR (KBr): nmax C]O 1718 cmꢀ1 1H NMR
(200 MHz, CDCl3): d 2.4 (s, 3H, CH3), 3.9 (d, J ¼ 15.8 Hz,
1H, 5-HA), 4.2 (dd, J ¼ 1.9 and 15.9 Hz, 1H, 5-HB), 6.8e7.0
(m, 3H, H3, H4ePh and H5ePh), 7.4 (d, J ¼ 4.8 Hz, 1H,
H5ePym), 7.5 (d, J ¼ 1.8 Hz, 1H, H-2), 8.5 (d, J ¼ 5.0 Hz,
1H, H6ePym); 13C NMR (75 MHz, CDCl3): d 169.2, 167.0,
156.4, 134.7, 133.6, 131.1, 128.7, 128.4, 125.0, 120.7,
4.3.6. 2-(2,6-Dibromophenyl)-3-(6-methylpyridin-2-yl)
thiazolidin-4-one (6f)
This compound was obtained as solid in 48% yield, mp
;
173e175 ꢁC; IR (KBr): nmax C]O 1692 cmꢀ1 1H NMR
(300 MHz, CDCl3): d 2.3 (s, 3H, CH3 at C6ePy), 3.9 (d,
J ¼ 15.8 Hz, 1H, 5-HA), 4.1 (dd, J ¼ 2.2 and 15.8 Hz, 1H, 5-
HB), 6.8 (m, 2H, H4ePy and H4ePh), 7.4 (d, 1H,

R.K. Rawal et al. / European Journal of Medicinal Chemistry 43 (2008) 2800e2806
2805
116.1, 62.0, 34.3, 22.4; ESI-MS: m/z 430 [M þ 1]þ. Anal.
Calcd for C14H11Br2N3OS: C, 39.18; H, 2.58; N, 9.79; S,
7.47. Found: C, 39.24; H, 2.59; N, 9.91; S, 7.62.
[M þ 1]þ. Anal. Calcd for C16H15Br2N3OS: C, 42.03; H,
3.31; N, 9.19; Found: C, 42.00; H, 3.16; N, 9.01.
4.3.15. 2-(2,6-Dibromophenyl)-3-(2,6-dimethylpyrimidin-
4-yl)thiazolidin-4-one (6o)
This compound was obtained as solid in 59% yield, mp
4.3.11. 2-(2,6-Dibromophenyl)-3-(4,6-dimethylpyrimidin-
2-yl)thiazolidin-4-one (6k)
This compound was obtained as solid in 42% yield, mp
122e126 ꢁC; IR (KBr): nmax C]O 1708 cmꢀ1 1H NMR
;
>220 ꢁC; IR (KBr): nmax C]O 1717 cmꢀ1
;
1H NMR
(200 MHz, CDCl3): d 2.4 (s, 3H, CH3 at C6epym), 2.5 (s,
3H, CH3 at C2epym), 3.9 (d, J ¼ 16.1 Hz, 1H, 5-HA), 4.1
(dd, J ¼ 1.9 and 16.2 Hz, 1H, 5-HB), 6.9 (m, 1H, H4ePh),
7.4 (s, 1H, H-2), 7.4e7.6 (m, 2H, H3ePh and H5ePh), 8.0
(s, 1H, H5epym); 13C NMR (75 MHz, CDCl3): d 171.1,
167.2, 165.2, 155.3, 134.3, 133.5, 131.8, 129.2, 128.0,
127.2, 105.8, 57.3, 34.5, 23.9, 23.3; ESI-MS: m/z 444
[M þ 1]þ. Anal. Calcd for C15H13Br2N3OS: C, 40.65; H,
2.96; N, 9.48; Found: C, 40.54; H, 2.74; N, 9.39.
(200 MHz, CDCl3): d 2.4 (s, 6H, 2CH3 at C4ePym and C6e
Pym), 3.9 (d, J ¼ 15.7 Hz, 1H, 5-HA), 4.1 (d, J ¼ 15.7 Hz,
1H, 5-HB), 6.7 (s, 1H, H5ePym), 6.9 (t, 1H, H4ePh), 7.4
(m, 2H, H3ePh and H5ePh), 7.5 (s, 1H, H-2); 13C NMR
(75 MHz, CDCl3): d 169.2, 166.7 (2C), 155.0, 134.7, 133.6,
131.1, 128.7, 125.1, 121.0, 115.8, 62.0, 34.3, 22.5 (2C);
ESI-MS: m/z 444 [M þ 1]þ. Anal. Calcd for C15H13Br2N3OS:
C, 40.65; H, 2.96; N, 9.48; S, 7.24; Found: C, 40.85; H, 3.01;
N, 9.58; S, 7.09.
4.4. Anti-HIV activity evaluation
4.3.12. 2-(2,6-Dibromophenyl)-3-(4-methyl-
6-trifluoromethylpyrimidin-2-yl)thiazolidin-4-one (6l)
This compound was obtained as solid in 36% yield, mp
4.4.1. In vitro HIV-RT kit assay [30]
The HIV-RT inhibition assay was performed by using an
RT assay kit (Roche), and the procedure for assaying RT inhi-
bition was performed as described in the kit protocol. Briefly,
the reaction mixture consists of template/primer complex, 20-
deoxy-nucleotide-50-triphosphates (dNTPs) and reverse tran-
scriptase (RT) enzyme in the lysis buffer with or without
inhibitors. After 1 h incubation at 37 ꢁC the reaction mixture
was transferred to streptavidine-coated microtitre plate
(MTP). The biotin labeled dNTPs that are incorporated in
the template due to activity of RT were bound to streptavidine.
The unbound dNTPs were washed using wash buffer and anti-
digoxigenin-peroxidase (DIG-POD) was added in MTP. The
DIG-labeled dNTPs incorporated in the template was bound
to anti-DIG-POD antibody. The unbound anti-DIG-POD was
washed and the peroxide substrate (ABST) was added to the
MTP. A colored reaction product was produced during the
cleavage of the substrate catalyses by a peroxide enzyme.
The absorbance of the sample was determined at OD
405 nM using microtiter plate ELISA reader. The resulting
color intensity is directly proportional to the actual RT activity.
The percentage inhibitory activity of RT inhibitors was calcu-
lated by comparing to a sample that does not contain an inhib-
itor. The percentage inhibition was calculated by formula as
given below
133e137 ꢁC; IR (KBr): nmax C]O 1724 cmꢀ1 1H NMR
;
(300 MHz, CDCl3): d 2.6 (s, 3H, CH3 at C4ePym), 4.0 (dd,
J ¼ 2.4 and 16.0 Hz, 1H, 5-HA), 4.2 (dd, J ¼ 1.8 and
16.0 Hz, 1H, 5-HB), 6.9e7.0 (m, 1H, H3ePh), 7.2 (s, 1H,
H5ePym), 7.4e7.5 (m, 1H, H5ePh), 7.5 (m, 1H, H4ePh),
7.6 (d, J ¼ 1.08 Hz, 1H, H-2); 13C NMR (75 MHz, CDCl3):
d 170.8, 169.0, 133.5, 131.3, 130.7, 129.9, 128.9, 128.6,
127.8, 125.3, 120.4, 111.6, 61.8, 34.0, 23.4; ESI-MS: m/z
498 [M þ 1]þ.
4.3.13. 2-(2,6-Dibromophenyl)-3-(4-methyl-
6-phenylpyrimidin-2-yl)thiazolidin-4-one (6m)
This compound was obtained as solid in 28% yield, mp
;
192e194 ꢁC; IR (KBr): nmax C]O 1718 cmꢀ1 1H NMR
(200 MHz, CDCl3): d 2.5 (s, 3H, CH3 at C4ePym), 3.9 (d,
J ¼ 15.8 Hz, 1H, 5-HA), 4.2 (d, J ¼ 15.5 Hz, 1H, 5-HB), 6.7
(s, 1H, H5ePym), 6.9 (t, 1H, H4ePh), 7.4 (m, 2H, H3ePh
and H5ePh), 7.5 (m, 3H, AreH), 7.9 (s, 1H, H-2), 7.9e8.0
(dd, J ¼ 2.7 and 7.8 Hz, 2H, AreH); 13C NMR (75 MHz,
CDCl3): d 169.0, 168.2, 164.5 (2C), 157.7, 152.3, 134.3,
133.3, 133.1, 129.3, 127.9, 127.0, 122.8, 117.3, 111.3, 110.5
(2C), 57.7, 34.2, 21.0; ESI-MS: m/z 506 [M þ 1]þ. Anal.
Calcd for C20H15Br2N3OS: C, 47.55; H, 2.99; N, 8.32; S,
6.35. Found: C, 47.77; H, 2.89; N, 8.44; S, 6.22.
ꢀ
ꢁ
OD 405 nm with inhibitor
% Inhibition ¼ 100 ꢀ
ꢃ 100
OD 405 nm without inhibitor
4.3.14. 2-(2,6-Dibromophenyl)-3-(4,5,6-trimethylpyrimidin-
2-yl)thiazolidin-4-one (6n)
This compound was obtained as solid in 25% yield, mp
>220 ꢁC; IR (KBr): nmax C]O 1720 cmꢀ1
;
1H NMR
4.4.2. In vitro anti-HIV assay
(300 MHz, CDCl3): d 2.1 (s, 3H, CH3 at C5epym), 2.4 (s,
6H, 2CH3 at C4ePym and C6ePym), 3.9 (d, J ¼ 15.7 Hz,
1H, 5-HA), 4.1 (dd, J ¼ 1.9 and 15.7 Hz, 1H, 5-HB), 7.0 (t,
1H, H4ePh), 7.4e7.5 (m, 2H, H3ePh and H5ePh), 7.5 (d,
J ¼ 1.2 Hz, 1H, H-2); 13C NMR (75 MHz, CDCl3): d 169.0,
164.4 (2C), 152.3, 134.6, 133.3, 133.1, 128.6, 127.9, 127.0,
122.8, 62.0, 34.3, 20.9 (2C), 12.3; ESI-MS: m/z 458
The methodology of the anti-HIV assays has been previ-
ously described [31]. Briefly, MT-4 or CEM cells were
infected with HIV-1IIIB and HIV-2ROD at 100 times the
CCID50 (50% cell culture infective dose) per milliliter of
cell suspension. Then, 100 ml of the infected cell suspension
were transferred to microtiter plate wells, mixed with 100 ml
of the appropriate dilutions of test compounds, and further

2806
R.K. Rawal et al. / European Journal of Medicinal Chemistry 43 (2008) 2800e2806
incubated at 37 ꢁC. In CEM cells, after 4 days of incubation,
HIV-1-induced syncytium formation was recorded. The 50%
effective concentration (EC50) was defined as the compound
concentration required inhibiting virus-induced syncytium
formation by 50% [32]. After 5 days of incubation of MT-4
cells, the number of viable cells was determined. The concen-
tration achieving 50% protection against the cytopathic effect
of the virus in infected cells was defined as the 50% effective
concentration (EC50) in MT-4 cells. The cytotoxic concentra-
tion CC50 was determined as the concentration of compound
to inhibit by 50% the number of viable cells in mock-infected
MT-4 and CEM cell cultures.
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Acknowledgements
The authors thank the Director, CDRI for the support and
the SAIF for the spectral data. C.D.R.I. Communication No.
7284.
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