Synthesis and Biological Evaluation of Some Novel 5-[(3-Aralkyl Amido/Imidoalkyl) Phenyl]-1,2,4-Triazolo[3,4-b]-1,3,4-Thiadiazines as Antiviral Agents

A series of novel 4-amino-5-mercapto-3-[(3-aralkyl amido/imidoalkyl) phenyl]-1,2,4-triazoles (5a-d) were obtained by treating m-(aralkyl amido/imidoalkyl) benzoic acid hydrazides (3a-d) with carbon disulphide in alcoholic KOH and hydrazine hydrate, respectively. These triazole derivatives were employed in the synthesis of 5-[(3′-aralkyl amido/imidoalkyl) phenyl]-1,2,4-triazolo[3,4-b]-1,3,4-thiadiazines (6a-d). The newly synthesized compounds were evaluated for their antiviral activity against two animal viruses, namely, Japanese encephalitis virus (JEV) strain P20778 and herpes simplex virus-1 (HSV-1) strain 753166.


Conclusions
Compound 6c, having R = H and R 1 = 2-phenyl-3-methylquinazolin (3H) 4-one, displayed moderate anti-JEV activity while the other three compounds, namely, 6a, 6b, and 6d containing phthalimido, phthalimidomethyl, and nicotinamido substituents, respectively, were found insignificantly active at the same dose level. It is quite predictable that a larger substituent like 2-phenyl-3-methyl-quinazolin (3H) 4one is mainly responsible for exerting anti-JEV activity. On the contrary, three compounds, namely, 6b, 6c, and 6d displayed some activity against HSV-I. The activity percentage ranged from 10 to 23%. From the available biological activity data of the compounds, it can be concluded that unless a large number of such compounds with greater number of variations at two positions are synthesized their potentialities cannot be predicted with great certainty (Scheme 1).

Maintenance of Japanese Encephalitis Virus (JEV).
It was properly maintained by intracerebral passages in 1-3 day(s) old suckling albino Swiss mice. The brains of the infected mice with specific paralytic symptoms were triturated and a 10% homogenate (m/V) was made in phosphate buffered saline (PBS) of pH 7.2. The mean lethal dose (LD 50 ) of the virus in mice was calculated before each experiment.

Maintenance of Herpes Simplex Virus-1 (HSV-1).
It was maintained in 5-6 g albino Swiss mice following the same route as for JEV; a 10% virus homogenate (m/V) was prepared and LD 50 was calculated as for JEV.

Maintenance of Cells.
Vero cells were maintained in minimum essential medium (MEM) (Sigma, USA) with 10% foetal bovine serum (FBS) (Gibco, USA); 100 units of penicillin, 100 μg of streptomycin, and 40 μg of gentamycin were added per mL of the medium.

Cytotoxicity Test and Antiviral Assay
In Vitro. Cytotoxicity and antiviral assays of the compounds were performed by the standard method [12]. The experiments were performed in 96-well tissue-culture plates. Equal volumes of maintenance medium and compound solution were poured into each well; concentration of 500 μg mL −1 of the compound tested was applied into the first well. Successive dilution by factor 2 was performed in further wells: the compound concentration in the 8th well was 1.9 μg mL −1 . The treated cultures were incubated for a period of 24 h at 37 • C and then observed microscopically for evidence of cytotoxicity, such as distortion, swelling, and sloughing of cells [13][14][15][16]. For the antiviral activity, 0.1 mL of the virus (10TC ID 50 mL −1 , that is, the dilution previous to 1TC ID 50 , which is the virus dilution that shows 50% cytopathic effect, where TC ID 50 is 50% tissue-culture infectious dose) was allowed to adsorb onto cell monolayers for 90 min at 37 • C [17]. The unadsorbed virus was removed by washing with 0.1 mL of MEM and then 0.1 mL of MEM with 2.5% FBS was filled into each well. Nontoxic concentration of the compound tested, ranging from 3.6 to 125 μg mL −1 of the compound, was added into each well. Each dilution was tested in duplicate, keeping separate the virus control and cell control (containing only MEM with 2.5% serum). The culture plates were incubated at 37 • C for 72 h and examined microscopically for evidence of cytopathogenicity caused by the virus and its inhibition by the examined compound.

Experimental Section
Melting points were determined in open capillaries using a Toshniwal melting point apparatus (Japan), and the values recorded are therefore uncorrected. IR spectra in KBr were recorded in the 4000-400 cm −1 range using KBr discs on FTR IR 8201 VC Perkin Elmer Spectrophotometer model 337(USA). The NMR spectra were recorded on a Varian 60 D instrument (200 MHz) (USA) using MeOH/DMSOd 6 . TMS was used as internal standard (δ in ppm). Mass spectra of compounds were run on a Hitachi-Elmer RMV-7 spectrometer at 70 eV and FAB mass spectra were recorded on JEOL SX 102/DA-600 Mass Spectrometer/Data System using Argon/Xenon (6 KV, 10 mA) as the FAB gas.   was checked by TLC on silica gel plates. All reagents were purchased from Merck and Ranbaxy. Synthesis of amido alcohols [18] and polyphosphoric acid [8,19] was accomplished according to the previously reported literature procedures. Table 1). This is an example of C-amido/imidoalkylation reaction [20,21]. A mixture of benzoic acid and an amido/imido alcohol (0.2 mol) each in minimum quantity of concentrated sulphuric acid (20 mL) was stirred for 3 h and subsequently left overnight under refrigeration. Having poured this mixture into ice-cooled water, (100 mL), the precipitate formed was filtered off, washed repeatedly with water, and treated with 10% aqueous sodium bicarbonate solution. It was stirred till the effervescence ceased, filtered, and the filtrate was neutralized with HCl resulting in the precipitation of the amidoalkylated product. Solid was filtered off, dried over calcium chloride in vacuum, and crystallized from appropriate solvent to obtain the desired product.

m-(Aralkyl Amido/Imidoalkyl) Benzoyl Chlorides, 2.
A mixture of m-(aralkyl amido/imidoalkyl) benzoic acid 1 (0.1 mol) and phosphorus pentachloride (0.15 mol) in anhydrous benzene (50 mL) was refluxed for 2 h under anhydrous condition. Benzene was distilled off under diminished pressure and the acid chloride thus obtained was used for further reaction without any purification [21]. Table 1). m-(Aralkyl amido/imidoalkyl) benzoyl chloride 2 (0.05 mol) was cooled to 0 • C and cold hydrazine hydrate (0.1 mol) was added to it dropwise with slow stirring. On entire addition, the reaction mixture was stirred vigorously for 30 min and poured into water. The solid obtained was filtered off, washed sequentially with water, and dried over calcium chloride in vacuum. The crude product was recrystallized from ethanol which afforded the desired acid hydrazides [21].

m-(Aralkyl Amido/Imidoalkyl) Benzoyl-Potassium Dithiocarbazinates, 4.
A mixture of m-(aralkyl amido/imidoalkyl) benzoic acid hydrazide 3 (0.03 mol), potassium hydroxide (0.09 mol), and carbon disulphide (0.09 mol) in absolute ethanol (50 mL) was stirred for 24 h at room temperature. The ensuing reaction mixture was diluted with hexane to double of its volume and was used as such for further reaction.