Synthesis and antiviral activity of novel [1,2,4]triazolo[3,4-b ][1,3,4]thiadiazoles, [1,2,4]triazolo[3,4-b ] [1,3,4]thiadiazines and [1,2,4]triazolo[3,4-b ][1,3,4] thiadiazepines

Starting from the 4-amino-3-(1,3-diphenyl-1 H -pyrazol-4-yl)-4,5-dihydro-[1,2,4] triazole-5(1 H )- thione 2 , a series of new [1,3,4]thiadiazoles 3, 4, 7 and [1,3,4] thiadiazines 8, 10, 11, 12, 14 were prepared. Also, [1,3,4]thiadiazepines 16-18 could be synthesized. Some of the newly prepared compounds were evaluated for their antiviral potential .


Results and Discussion
The starting material 4-amino-3-(1,3-diphenyl-1H-pyrazol-4-yl)-4,5-dihydro- [1,2,4]triazole-5(1H)-thione 2 was prepared in good yield: by the reaction of the oxadiazole thione 1 with hydrazine hydrate.The latter compound is useful intermediate for the synthesis of triazolothiadiazoles, triazolothiadiazines and triazolothiadiazepines.The amino and mercapto groups are ready-made nucleophilic centers for the synthesis of condensed heterocyclic rings. 1 Thus, an attempt to prepare the oxo-analog of 4 by treatment of 2 with ethyl chloroformate in dry benzene was unsuccessful and the product obtained was identified as the S-substituted derivative 5 which could not be cyclized into 3 on boiling in pyridine.However, compound 3 could be obtained via the interaction of amino thione 2 with 1,1`-carbonyldiimidazole (CDI) in dry dioxane.Treatment of 3 with P 2 S 5 in dry pyridine resulted in the formation of the thione derivative 4.This latter compound could be obtained directly by the reaction of 2 with carbon disulphide in ethanolic KOH (Scheme 1).

ARKAT
Treatment of triazolethione 2 with acetyl-or benzoyl chloride in refluxing dioxane did not afford the bicyclic structure triazolothiadiazoles 7a,b but gave the acetyl-or benzoyl amino derivatives 6a,b respectively.Compounds 6a,b could be smoothly cyclidehydrated by boiling in phosphoryl chloride affording the [1,2,4]triazolo [3,4-b] [1,3,4]thiadiazoles 7a,b in good yield.It is worthy to note that compound 7a could be obtained, in a rather better yield: (82 %), directly by the reaction of 2 with acetic acid in boiling phosphoryl chloride.The IR spectrum of the latter compound showed no NH bands and the 1 H NMR spectrum showed a singlet at 2.71 ppm corresponding to the methyl substituent at position 6 (Scheme 2).From the above reactions we could conclude that the cyclidehydration could only occur in the presence of phosphoryl chloride; the acid chlorides under the used reaction conditions could only give the corresponding actyl(benzoyl)amino derivatives.Treatment of the amino thione 2 with ethyl chloroacetate gave a product whose NMR spectrum showed a signal corresponding to the amino group at 4.48 ppm, a quartet corresponding to (CH 2 CH 3 ) at 4.18 ppm, a singlet assigned to (CH 2 ) at 3.97 ppm, and a triplet assigned to (CH 2 CH 3 ) at 1.27 ppm.This proves the structure 10 and rules out the possible formation of 12.An attempt to cyclize the amino triazole 10 into the corresponding triazolothiadiazinone 12 by heating in refluxing pyridine was unsuccessful.When choroacetic acid was used instead of its ester in the former reaction, the product was identified as the amino acid 11 which could be cyclized into triazolothiadiazinone 12 in boiling phosphoryl chloride (Scheme 4).Furthermore, treatment of the amino thione 2 with oxalyl chloride in dry benzene afforded the corresponding triazolothiadiazine-6,7-dione 13.Also, its interaction with bromo diethylmalonate led to the formation of the thiadiazine-7-carboxylic acid ethyl ester 14.Whereas, reaction of compound 2 with methyl-2-bromopropionate gave the corresponding S-substituted amino triazole 15 (Scheme 5).

Pharmacological activity assessment and results
Compounds were tested for antiviral activity and cytotoxicity in various viral test systems, according to previously published procedures 23,24 .The results of in vitro evaluation are summarized in Tables 1-3.None of the compounds inhibited the cytopathicity induced by vesicular stomatitis virus, Coxsackie virus B4, respiratory syncytial virus, parainfluenza-3 virus, reovirus-1, Sindbis virus and Punta Toro virus, herpes simplex virus-1 (KOS) or herpes simplex virus-2 (G), and vaccinia virus at subtoxic concentrations in HeLa, Vero or E 6 SM cell cultures, respectively (Tables 1-3).

General procedure for the synthesis of compounds (7a,b). Method a
After removing the excess of phosphoryl chloride under reduced pressure, an icewater mixture was added to the residue with vigorous stirring.The reaction mixture was neutralized with ammonium hydroxide and the solid precipitate formed was filtered and recrystallized from ethanol to give 7a,b.Method b.A mixture of 2 (334 mg, 1 mmol) and acetic acid (60 mg, 1 mmol) in phosphoryl chloride (10 mL) was heated under reflux for 2 h.After cooling, the solvent was removed under reduced pressure and an ice-water mixture was added to the residue with stirring.The reaction mixture was neutralized with ammonium hydroxide and the solid precipitate formed was filtered and recrystallized from ethanol to give 7a.mp. and mixed mp.with a sample obtained by the above procedure are identical.

Table 1 .
Cytotoxicity and antiviral activity in HEL cell culturesMinimum inhibitory concentration b (µg/ml) Required to cause a microscopically dateable alteration of normal cell morphology.b Required to reduce virus-induced cytopathogenicity by 50%.

Table 2 .
Cytotoxicity and antiviral activity in HeLa cell culturesMinimum inhibitory concentration b (µg/ml) Required to cause a microscopically dateable alteration of normal cell morphology.b Required to reduce virus-induced cytopathogenicity by 50%. a