New thiopyraNo [ 2 , 3-d ] [ 1 , 3 ] thiazole derivatives as poteNtial aNtiviral ageNts

A series of novel thiopyrano[2,3-d][1,3]thiazole derivatives were synthesized and evaluated for their antiviral activity in vitro within AAcF NIAID programme. compounds were studied towards Dengue Virus Type 2, Venezuelan equine encephalitis Virus, Respiratory syncytial Virus, sARs coronavirus, Rift Valley Fever Virus, Tacaribe Virus, Influenza Virus Types A and B. Among the tested thiopyrano[2,3-d][1,3]thiazoles, alkyl rel-(5R,5aR,11bS)-2,6-dioxo-3,5a,6,11b-tetrahydro-2Н,5H-chromeno[4’,3’:4,5]thiopyrano[2,3-d] [1,3]thiazole-5-carboxylates 8 and 11 were found to be the most active and showed significant antiviral activity against Influenza Virus Types A H3N2 and H5N1.

In the last two decades, there has been an increase in the number of studies on 4-thiazolidinone derivatives as potential antiviral agents 8- 13 .Thiopyrano[2,3-d][1,3]thiazoles could be of special interest as cyclic mimetics of biologically active (including antiviral) 4-thiazolidinones.The aim of present study was to estimate the antiviral activity of new thiopyrano[2,3-d][1,3]thiazole derivatives.

Fig. 2. compounds tested for antiviral activity
Antiviral assay.Antiviral assay was performed at a virus's panel with a protocol of the NIAID's antimicrobial acquisition and coordinating 17 .Results for each tested compound were reported as virusinhibitory concentration; 50% endpoint (EC 50 ) and cell-inhibitory concentration, 50% endpoint (CC 50 ) were determined.A general selectivity index (SI) was calculated as a ration of (EC 50 )/(CC 50 ).An SI of 3 or greater indicates that confirmatory testing is needed.
Inhibition of Viral Cytopathic Effect (CPE).This test, run in 96 well flat-bottomed microplates, was used for the initial antiviral evaluation of compounds.In this CPE inhibition test, four log10 dilutions of each test compound (e.g.1000, 100, 10, 1 µg/ml) were added to 3 cups containing the cell monolayer; within 5 min.On the next step, the virus was added and the plate was sealed and incubated at 37°C.CPE read microscopically when untreated infected controls develop a 3 to 4+ CPE (approximately 72 to 120 hr).A known positive control drug Ribavirin was evaluated in parallel with test drugs in each test.The data are expressed as 50% effective concentrations (EC 50 ).
Increase in Neutral Red (NR) Dye uptake.This test was run to validate the CPE inhibition seen in the initial test, and utilized the same 96-well micro plates after the CPE has been read.When neutral red was added to the medium cells that were not damaged by virus take up a greater amount of dye, which is desplayed on a computerized microplate autoreader.An EC 50 was determined from this dye uptake.
cytotoxicity.In the CPE inhibition tests, two wells of uninfected cells treated with each concentration of tested compounds was run in parallel with the infected, treated wells.At the time CPE was determined microscopically.The toxicity control cells were also examined microscopically for any changes in cell appearance compared to normal control cells run in the same plate.These changes may be enlargement, granularity, cells with ragged edges, filmy appearance, rounding, detachment from the surface of the well, or other changes.These changes were given a designation of T (100% toxic), PVH (partially toxic-very heavy -80%), PH (partially toxic-heavy -60%), P (partially toxic -40%), Ps (partially toxic-slight-20%), or 0 (no toxicity -0%), conforming to the degree of cytotoxicity seen.A 50% cell inhibitory (cytotoxic) concentration (CC 50 ) was determined by regression analysis of these data.

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The screening of 24 thiopyrano[2,3-d][1,3] thiazole derivatives against a wide range of viruses has been carried out.Some compounds possess mode rate levels of the antiviral activity.However, the preliminary results of antiviral activity allowed to identify the active compounds 8 and 11, which have shown the significant antiviral activity against Influen za Virus Type A H 3 N 2 (Perth strain) and Influenza Virus Type A H 5 N 1 (Vietnam strain).
Thus, derivatives bearing thiopyrano[2,3-d] [1,3]thiazole fragment could be considered as promising basis for further modification in searching for new antiviral agents.

acknowledgments
The authors are grateful to Dr. Chris Tseng, from Division of Microbiology and Infectious Diseases NIAID/NIH, Bethesda, USA, for in vitro evalua tion of antiviral activity.Thank you for Cedars Sinai Medical Center's International Research and Innovation in Medicine Program, the Association for Regional Cooperation in the Fields of Health, Science and Technology (RECOOP HST Association) for their support of our organization as participating Cedars -Sinai Medical Center -RECOOP Research Centers (CRRC).
a Compounds 2