J Pharm Pharmaceut Sci
(www.cspscanada.org) 8(3):426-429, 2005
Microwave-assisted
synthesis and anti-YFV activity of 2,3-diaryl-1,3-thiazolidin-4-ones
Dharmarajan Sriram*,
Perumal Yogeeswari, TG Ashok Kumar
Medicinal Chemistry Research
Laboratory,
Pharmacy Group, Birla Institute of Technology & Science, Pilani, India
Received December
22, 2004, Revised August 8, 2005 Accepted August 19, 2005, Published September
1, 2005
Corresponding Author:
Dharmarajan Sriram, Medicinal Chemistry Research Laboratory, Pharmacy Group,
Birla Institute of Technology & Science, Pilani-333031, India.
dsriram@bits-pilani.ac.in
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ABSTRACT
Purpose:
The purpose
of this study was to prepare several 1,3-thaizolidin-4-ones bearing variously
substituted diaryl ring at C-2 and N-3 positions and evaluate them for their
anti-YFV activity. Methods: Several
1,3-thaizolidin-4-ones were prepared by reacting substituted benzaldehyde with
equimolar amount of an appropriate substituted aromatic amine in the presence
of an excess of mercaptoacetic acid in toluene utilizing microwave irradiation.
The synthesized compounds were also evaluated for their inhibitory effects on
the replication of YFV in green monkey kidney (Vero) cells (ATCC CCL81), by
means of a cytopathic effect reduction assay. Results: The compound DS1 emerged as the most potent anti-YFV
agent with EC50 of 6.9 µM and CC50 more than 100 µM
making it more potent than ribavirin. Conclusion: 2,3-diaryl-1,3-thiazolidin-4-ones
possess anti-YFV potency.
INTRODUCTION
Yellow fever is a
serious viral infection, transmitted by mosquitoes in tropical regions. Yellow
fever virus [YFV] belongs to the Flaviviridae family [1], The virus is
introduced into the bloodstream via the saliva of the mosquito Aedes egyptii
[2] The virus is permanently prevalent,
with a more or less constant number of sufferers
(i.e. it is endemic) in several tropical regions of Africa and on the continent
of America.
In addition, there is an increasing number of epidemics, in which a large
number of people suddenly develop yellow fever. Every year about 200,000 cases
of yellow fever are recorded, and 30,000 of those infected die, but the figures
are underestimated because of poor record keeping. In total, yellow fever
occurs in 33 countries and 468 million people are at risk of catching the
disease [3]. Once a
mosquito has passed the yellow fever virus to a human, the chance of developing
clinical disease is about 5-20%. Symptoms of the disease [3] include high fever, anemia, as well as liver
inflammation, hepatitis and jaundice. The kidneys are also affected and
bleeding from the mouth, nose and stomach may occur, which leads to blood in
vomit and faeces. Because of this degeneration of the internal
organs (specifically the kidneys, liver, and heart), it results in what is
considered the classic triad of yellow fever symptoms: jaundice, black vomit, and
the dumping of protein into the urine. Jaundice causes the whites of the
patient's eyes and the patient's skin to take on a distinctive yellow color.
This is due to liver damage, and the accumulation of bilirubin, which is
normally processed by a healthy liver. There are no current anti-viral
treatments available to combat the yellow fever virus. The only treatment of
yellow fever involves attempts to relieve its symptoms. Fevers and pain should
be relieved with acetaminophen not aspirin or ibuprofen, both of which could
increase the already-present risk of bleeding. The drugs which are useful for
YFV infection include Ribavirin [4] and a-Interferon [4]. Other compounds which show
anti-YFV activity include 3-deazaneplanocin-A [5], sulfated galactomannans [6]
phosphorothioate oligodeoxyribonucleotides [7] and selenazofurin [8]. From a
random screening program of compounds having anti-HIV activity [9-12], we
identified a series of 1,3-thaizolidin-4-ones bearing variously substituted
diaryl ring at C-2 and N-3 positions as inhibitors of YFV. In this
communication, we describe the efficient synthesis and preliminary anti-YFV
activity of novel compounds having the thiazolidinone skeleton.
EXPERIMENTAL
General Procedure for the synthesis of
2,3-diaryl-1,3-thiazolidin-4-ones (DS1-DS15)
To a stirred solution of aromatic amine (0.01 mole) in
dry toluene (50 mL), 2-mercaptoacetic acid (0.02 mole) and the appropriate
aromatic aldehyde (0.01 mole) were added and irradiated in an unmodified
domestic microwave oven at power setting of 80% with 30 seconds/cycle. The
number of cycles in turn depended on the degree of completion of the reaction,
which was checked by thin layer chromatography (TLC). The reaction timing
varied from 6 to 8 minutes. The solution obtained after the completion of the
reaction was kept at 0°C for 30 minutes. During this time the excess of
unreacted mercaptoacetic acid was frozen out at the bottom and the clear solution
was decanted. After the removal of solvent under reduced pressure, the oily
residue was treated with a mixture of ethanol and diethyl ether to yield solid
title compounds in the yields ranging from 64-82%. Physical and spectral data
of representative compounds are as follows.
DS1: 2-(4’-Chlorophenyl)-3-(4”-fluorophenyl)-1,3-thiazolidin-4-one
M.P.: 104°C; Yield: 68%; 1H-NMR, d ppm: 3.87 (d,
1H, 5-HA), 4.09 (dd, 1H, 5-HB), 7.06-7.35 (m, 9H, ArH and
H-2). Anal. (C15H11NOSClF) C, H, N.
DS6: 3-(2,6-Dimethylphenyl)-2-(4”-nitrophenyl)-1,3-thiazolidin-4-one
M.P.: 95°C; Yield:79%; 1H-NMR, d ppm: 1.1 (s,
6H, CH3), 3.86 (d, 1H, 5-HA), 4.11 (dd, 1H, 5-HB),
7.10-7.36 (m, 8H, ArH and H-2). Anal. (C17H16N2O3S)
C, H, N.
DS8: 2-(4’-Chlorophenyl)-3-(2”-chloro-3”-methylphenyl)-1,3-thiazolidin-4-one
M.P.: 121°C; Yield: 70%; 1H-NMR, d ppm: 2.1 (s,
3H, CH3), 3.86 (d, 1H, 5-HA), 4.18 (dd, 1H, 5-HB),
7.06-7.46 (m, 8H, ArH and H-2). Anal. (C16H13NOSCl2)
C, H, N.
DS13:
2-(2’-Chlorophenyl)-3-pyridin-2-yl-1,3-thiazolidin-4-one
M.P.: 100°C; Yield: 64%; 1H-NMR, d ppm: 3.84 (d,
1H, 5-HA), 4.22 (dd, 1H, 5-HB), 6.86-8.20 (m, 9H, ArH and
H-2). Anal. (C14H11N2OSCl) C, H, N.
DS15: 2-(4’Chlorophenyl)-3-(4”-methylpyridin-2-yl)-1,3-thiazolidin-4-one
M.P.: 184°C; Yield: 65%; 1H-NMR, d ppm: 2.25 (s,
3H, CH3), 3.92 (d, 1H, 5-HA), 4.13 (dd, 1H, 5-HB),
7.05-8.25 (m, 8H, ArH and H-2). Anal. (C15H13N2OSCl)
C, H, N.
Anti-YFV
evaluation
Viruses and cells
Green monkey kidney (Vero) cells (ATCC CCL81) were
grown in minimum essential medium (MEM, Gibco, Paisley, Scotland)
supplemented with 10% inactivated calf serum (FCS, Gibco), 1% L-glutamine and
0.3% bicarbonate. The vaccine strain 17D (StamavilÒ, Pasteur Merieux) was used to infect 75 mL
bottles of Vero cells. After a 5-day incubation period at 37°C, extensive
cytopathic effect was observed; the cultures were freeze-thawed, cell debris
removed by centrifugation and the supernatant aliquoted and stored at -70°C.
Inhibition of virus induced
cytopathogenicity
Serial dilutions of the test compounds were added to
confluent Vero cell cultures in microtitre trays after which the cells were
infected with 10CCID50 (Cell culture infective dose 50%) of virus.
Cultures were further incubated at 37°C. Viral cytopathogenicity was recorded
7-8 days post infection. Cultures were fixed with 70% ethanol, stained with
Giemsa solution (50-fold dilution; 2h staining), washed and air-dried. The
antiviral activity of the compound was expressed as the effective concentration
required to inhibit the viral cytopathic effect by 50% (EC50).
Cytotoxicity
Inhibition of
uninfected host cell growth was assessed as follows: the cells were seeded at a
rate of 3 x 103 cells per well in a volume of 0.1 mL into 96-well
microtitre plates and allowed to proliferate for 24h in MEM containing 20% FCS,
1% L-glutamine and 0.3% sodium bicarbonate. Twenty-four hours later, 0.1 mL MEM
(with 2% FCS, 1% L-glutamine and 0.3% sodium bicarbonate) containing different
concentrations of the test compounds were added to each well. After 3 days of
incubation at 37°C in 5% carbon dioxide, the cell number was determined with
Coulter counter. The minimum cytotoxic concentration was expressed as CC50,
the concentration required to reduce cell growth by 50%.
RESULTS AND
DISCUSSION
Chemistry
The synthesis of the 2,3-diaryl-1,3-thiazolidin-4-ones
(DS1-15) was accomplished by
reacting substituted benzaldehyde with equimolar amount of an appropriate
substituted aromatic amine in the presence of an excess of mercaptoacetic acid
in toluene utilizing microwave irradiation (Fig. 1). Unlike the conventional
methods [9-12] (reaction time 48h and yields of 30-70%), microwave-assisted
reactions were very facile (6-8 min.) and provided very good yields (64-82%).
The purity of the compounds was checked by TLC and elemental analyses. Both
analytical and spectral data (1H-NMR) of all the synthesized
compounds were in full agreement with the proposed structures. The 1H-NMR
spectrum revealed that the 3.86-3.88 for 5-HA (d, 1H), 4.09-4.11 for
5-HB (dd, 1H) and 7.06-7.35 for Ar-H and H-2.
Anti-YFV activity
All these compounds were also evaluated for their
inhibitory effects on the replication of YFV in green monkey kidney (Vero)
cells (ATCC CCL81), by means of a cytopathic effect reduction assay [12]. The
antiviral activity of the compound was expressed as the effective concentration
required to inhibit the viral cytopathic effect by 50% (EC50). The
minimum cytotoxic concentration was expressed as CC50, the
concentration required to reduce cell growth by 50%. EC50 and CC50
values are presented in
Table 1. Six compounds (DS1-3, DS6-7 and DS14)
were found to prevent the YFV infection of the cells at concentrations that had
no effect on cell growth. Four compounds (DS1-3
and DS14) were found to be more
effective than ribavirin (EC50 values of 28.0 μM). The compound
DS1 emerged as the most potent
anti-YFV agent with EC50 of 6.9 μM and CC50 more
than 100 μM.
In the
present study we have discovered 2, 3-diaryl-1, 3-thiazolidin-4-ones as a new
lead in the anti-YFV field. These results need to be refined in terms of active
concentration and toxicity. Further studies to acquire more information about
structure-activity relationships are in progress in our laboratory.
ACKNOWLEDGEMENT
The authors are thankful to Prof. E De
Clercq and Dr. J. Neytes, Rega Institute for Medical Research, Leuven, Belgium
for their support in biological testing.
REFERENCES:
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Monath, T.P., Barrett, A.D., Pathogenesis and
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Lourenco-de-Oliveira, R., Vazeille, M., De
Filippis, A.M., Failloux, A.B., Aedes aegypti in Brazil: genetically differentiated
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Barrett, A.D., Monath, T.P., Epidemiology and
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