One pot synthesis and reactions of novel 5-amino[1,3]thiazolo[3,2-b ][1,2,4]triazoles

5-Mercapto-3-phenyl-1,2,4-triazole 8 was reacted with a variety of cyano compounds containing active methylene group such as ethyl cyanoacetate, cyanoacetamide and malonitrile in boiling acetic acid in the presence of concentrated sulfuric acid, to give the corresponding 5-amino-2-phenyl[1,3]thiazolo[3,2-b ][1,2,4]triazole-6-carboxylate 10a . While on using cyanoacetamide or malonitrile the 5-amino-2-phenyl[1,3]thiazolo[3,2-b ][1,2,4]triazole-6-carboxamide 10b was obtained in one step reaction. Reaction of 10b with triethyl orthoformate, acetic anhydride, benzaldehyde, benzoyl chloride and/or carbon disulfide gave the corresponding 2-phenyl[1,2,4]triazolo[2 ' ,3 ' :3,2][1,3]thiazolo[4,5-d ]pyrimidinones 15-18 in good yield. Upon treatment of 5-mercapto-3-phenyl-1,2,4-triazole 8 with chloroacetonitrile and benzaldehyde in boiling acidified acetic acid afforded 6-benzylidene-2-phenyl[1,3]thiazolo[3,2-b ][1,2,4]triazol-5(6 H )-one 19 rather than the isomeric product 6-benzylidene-3-phenyl[1,3]thiazolo[2,3-c ][1,2,4]triazol-5(6 H )-one 20 in one pot reaction. The mechanism of the reactions is under investigation and the structures of all new compounds were elucidated using IR, 1 H-NMR, 13 C-NMR, mass spectral data and elemental analysis. The biological activity of selected compounds was investigated and summarized.

The structures of compounds 10a,b were confirmed on the basis of their elemental analysis, IR, 1 H-NMR and mass spectral analysis.The IR spectra of compounds 10a showed bands at 3400-3300 cm -1 (NH2) and 1680 cm -1 (C=O ester), while 10b showed bands at 3400-3190 cm -1 (NH2) and 1660 cm -1 (C=O amide) besides the expected bands.The 1 H-NMR spectra of compounds 10a,b were characterized by the appearance of multiple signals at δ 7.4-8.1 attributed to the aromatic protons, in addition the compound 10a showed a triplet signal at δ 1.1 and a quartet signal at δ 4.2 attributed to ethyl group.The mass spectra of 10a,b showed the molecular ion peaks at m/z 288.7 (100%) and 259.1 (100%) respectively.Further, the structure of compound 10b was confirmed by unequivocal synthesis via the reaction of 8 with bromocyanoacetamide in an aqueous solution of potassium hydroxide (Scheme 2).Scheme 2. Reaction of 5-mercapto-3-phenyl-s-triazole 8 with bromocyanoacetamide.
The proposed reaction mechanism is summarized in Scheme 1.It may proceed via the formation of dimeric disulfide 11 followed by nucleophilic attack by the imine form on the dimeric disulfide to give the carbonium ion 12, which undergoes intramolecular cyclization to produce the cyclized imino structures 13.Protonation of 13 in the presence of acid medium gives the cyclized carbonium ion 14 followed by deportation to yield the cyclized compounds 10a,b as shown in the following (Scheme 3).

Scheme 3. Reaction mechanism of formation of compounds 10a,b.
The suggested mechanism 45 is supported by formation of disulfide 11 in 81% yield on refluxing the 5-mercapto-3-phenyl-s-triazole 8 in acetic acid in the presence of concentrated sulfuric acid in the absence of an active methylene compound. 44The formation of compound 10b showed that the cyano group of 10c undergoes hydrolysis to the corresponding amide.
The formation of 19 can be explained by S-alkylation of 8 followed by intramolecular cyclization via nucleophilic attack of NH to the cyano group to form the cyclized imine derivative 22, which undergoes hydrolysis to the ketone 23 followed by condensation with benzaldehyde to give the product.The intermiediacy of ketone 23 was verified by refluxing 8 with chloroacetic acid and benzaldehyde or by refluxing 2-[(5-phenyl [1,2,4]triazolo-3yl)thio]acetonitrile/acetic acid 21a,b, which were prepared by the reaction of 3-phenyl-5mercapto-1,2,4-triazole 8 with chloroacetic acid or chloroacetonitrile in alcoholic potassium hydroxide solution, and benzaldehyde under the same reaction conditions, both of which also gave product 19 (Scheme 6).Based on the previously reported studies using similar compounds, 46,47 the higher nucleophilicity of N-2 than N-4 due to the alpha N effect.Molecular modeling calculations (MM2) indicated that isomer 19 is more stable than 20, while the E-isomer (HF = 137.64kcal/mol) of 19 is more favorable than the Z-isomer (137.65 Kcal/mol), Figure 2. 47

Biological activity
One of the purposes of the present work was to synthesize new heterocyclic compounds which might be of certain biological interest.Some of the newly synthesized compounds were screened for their antibacterial and antifungal activity.For antibacterial studies microorganisms employed were Serratia marcescens, Pseudomonas aeruginosa, Staphylococcus aureus, Bacillus cereus and Escherichia coli.For antifungals, Candida albicans, Geotrichum candidum, Aspergillus flavus, Trichophyton rubrum, Scopulariopsis bervicaulis and Fusarium oxysporum were used.Both microbial studies were assessed by minimum inhibitory concentration (MIC) by serial dilution method. 48For this the compound whose MIC has to be deretmined was dissolved in serially diluted DMSO, then a standard drop of the culture prepared for the assay was added to each of the dilutions and incubated for 16-18 h at 37 °C (Tables 1 and 2).

Experimental Section
General.Melting points were determined using Gallen Camp melting point apparatus and are uncorrected.IR spectra were measured on a Shimatzu-470 spectrometer using KBr techniques. 1 H-NMR spectra were measured on a Varian EM-390, 90MHz spectrometer (Spectral Unit, Assiut University, Egypt) or a Bruker DX 400-MHz spectrometer (Department of Physical Chemistry, Geneva) using CDCl3 or DMSO-d6 as a solvent and TMS as internal standard. 13C NMR spectra were measured on a Bruker DX 400-MHz spectrometer.Mass spectra were recorded on Jeol-Jms-600H spectrometer using the direct inlet system.The elemental analyses were performed using Perkin-Elmer elemental analyzer 240-C.
[(5-Phenyl-1,2,4-triazolo-3-yl)thio]acetonitrile/acetic acid (21a,b).General procedure To a solution of 5-mercapto-3-phenyl-1,2,4-triazole 8 (0.88 g, 0.005 mol) in absolute EtOH (15 mL) and KOH (0.56 g, 0.01 mol), a solution of chloroacetonitrile or chloroacetic acid (0.0055 mol) in absolute EtOH (5 mL) was added dropwise with stirring.The reaction mixture was stirred at room temperature for a further 30 min, followed by refluxing for 1 h.The precipitated KCl was filtered off.The excess solvent was removed from the mixture by evaporation under vacuum and the residue was treated with H2O.The crude product thus formed was collected by filtration and crystallized from (EtOH) to give 21a,b as colorless needles.

Table 1 .
The antibacterial activity (inhibition zone in (mm) and MICs given in brackets) of some selected compounds *CHL = Chloramphenicol as standard.

Table 2 .
The antifungal activity (inhibition zone in (mm) and MICs given in brackets) of some selected compounds *CLO = Clotrimazole as standard.