Synthesis under Microwave Irradiation of [1,2,4]Triazolo[3,4-b][1,3,4]thiadiazoles and Other Diazoles Bearing Indole Moieties and Their Antimicrobial Evaluation

Microwave-assisted synthesis of some novel compounds, namely, 3-(2-methyl-1H-indol-3-yl)-6-aryl-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazoles 5a,b was accomplished via bromination of 2-methyl-3-[4-(arylideneamino)-5-mercapto-4H-[1,2,4]triazol-3-yl]-1H-indoles 3a,b. Also, new [1,3,4]thiadiazoles 12a,b, [1,2,4]triazoles 15a,b and [1,3,4]oxadiazoles 19a,b, with indole moieties, were prepared by cyclization of 1-[(2-methyl-1H-indole)-3-carbonyl]thiosemicarbazides 8a,b under microwave irradiation using different reaction conditions. Moreover, reaction of acid hydrazide 7 with ethyl 2-(N-phenylhydrazono)-3-oxobutanoate (20) gave the respective phenylhydrazonopyrazole derivative 21 under the reaction conditions employed. The structures of the synthesized compounds were assigned based on elemental analyses and spectral data (IR, 1H-NMR, 13C-NMR, MS). The antifungal and antibacterial activities of the new products were also evaluated.

The synthetic utility of 1-[(2-methyl-1H-indole)-3-carbonyl]thiosemicarbazides 8a,b prepared from acid hydrazide 7 [13,16] and potassium thiocyanate or phenyl isothiocyanate was investigated. Thus, treatment of 8a,b with concentrated sulfuric acid at room temperature (25 °C) gave the respective [1,3,4]thiadiazole derivatives 12a,b. The reaction proceeded via intramolecular nucleophilic attack of thiol group into carbonyl group [17] to give intermediates 11a,b. Dehydration of these intermediates 11a,b under the employed reaction conditions gave the isolated products 12a,b (Scheme 2). Protonation of N-4 (NHR) in the thiosemicarbazide moiety under acidic condition (intermediates 9a,b) reduces its nucleophilicity and increases the nucleophilicity of the thiol group [17]. Changes of the pH of the reaction mixture led to the formation of different ring systems. Thus, reactions of 8a,b with potassium hydroxide, as basic catalyst, led to the formation of [1,2,4]triazoles 15a,b. The reaction started by abstraction of a hydrogen atom from the NH-R group to increase its nucleophilicity [17] (intermediates 13a,b) followed by its attack on the carbonyl group with concurrent dehydration of the non-isolable intermediates 14a,b (cf. Scheme 2) to give isolable products 15a,b. The structures of the isolated products 12a,b, and 15a,b were established on the basis of elemental analyses and spectral data (see Experimental). On the other hand, irradiation of 8a,b with mercuric oxide furnished the corresponding [1,3,4]oxadiazoles 19a,b, via elimination of mercuric sulfide [17] from the non-isolable intermediates 16a,b. Nucleophilic attack of hydroxyl group on the carbonyl carbon gave intermediates 18a,b followed by loss of a water molecule to afford the isolated products 19a,b (Scheme 3). Next, we studied the reactivity of hydrazone derivatives which are widely used as intermediates for the synthesis of a large number of heterocyclic compounds [18]. Thus, treatment of (2-methyl-1Hindol-3-yl)-3-acid hydrazide (7) with ethyl 2-(N-phenylhydrazono)-3-oxobutanoate (20) [19] in DMF under microwave irradiation in the presence of a catalytic amount of HCl afforded the corresponding 3-[(3-methyl-4-phenylhydrazono-5-oxopyrazolin-1-yl)carbonyl]-2-methyl-1H-indole (21) via elimination of ethanol and water molecules (Scheme 4).

Scheme 4. Synthesis of phenylhydrazonopyrazolinone derivative 21.
The structure of the latter product was confirmed by the appearance of the hydrazone NH band at 3358 cm −1 in its IR spectrum [20]. The 1 H-NMR spectrum also revealed two singlet signals at δ 11.20 and 13.11 ppm (D 2 O exchangeable) assignable to the NH of the pyrrole ring and hydrazone group [20], respectively. An alternative synthesis of 21 was carried out as outlined in Scheme 4. Thus, reaction of acid hydrazide 7 with ethyl 3-oxobutanoate under microwave irradiation for 6 min. gave pyrazolinone derivative 22. Coupling of 22 with diazotized aniline in pyridine afforded the corresponding hydrazone derivative 21, typical in all respects (mp., mixed mp., and IR) to that obtained from acid hydrazide 7 and 20 (cf. Scheme 4).

Antimicrobial Activity
The in vitro antimicrobial activities of the synthesized products 3a-e, 5a,b, 8b, 12a,b, 15a,b, 19a,b, and 21 were screened against the Gram-positive bacteria Staphylococcus aureus (S. aureus), Bacillus subtilis (B. subtilis), Gram-negative bacteria Escherichia coli (E. coli) and the fungus Candida albicans (C. albicans) under the same conditions using trimethoprim as reference. The bacteria and fungus were subjected to susceptibility testing on Muller-Hinton agar medium by the disc agar diffusion method [21,22]. The results are summarized in Table 1. The following conclusions may be drawn from the results: a) Compounds 3a, 5a, 5b, and 19b exhibited high inhibitory effect against S. aureus, while compounds 3b, 3c, 3e, 8b, 12a, 15a, and 21 have moderate inhibitory effect against same bacterium. On the other hand, compounds 3d, 12b, 15b, and 19a have no inhibitory effect against S. aureus. b) Compounds 3a, 3d, 3e, 5a, 5b, 8b, and 21 revealed high inhibitory activity against B.

Experimental
Melting points were measured on an Electrothermal IA 9000 series digital melting point apparatus. The IR spectra were recorded in potassium bromide discs on a Pye Unicam SP 3300 and Shimadzu FT IR 8101 PC infrared spectrophotometers. The NMR spectra were recorded on a Varian Mercury VX-300 NMR spectrometer operating at 300 MHz ( 1 H-NMR) or 75 MHz ( 13 C-NMR) and run in deuterated dimethylsulphoxide (DMSO-d 6 ). Chemical shifts were related to that of the solvent. Mass spectra were recorded on a Shimadzu GCMS-QP1000 EX mass spectrometer at 70 eV. Elemental analyses (using a German made Elementar vario LIII CHNS analyzer) and the biological evaluation of the products were carried out at the Microanalytical Centre of Cairo University, Giza, Egypt. All reactions were followed by TLC (silica gel, aluminum Sheets 60 F254, Merck). Irradiation was done in a domestic microwave oven (R-658WM, 2500 MHz, 400 W). The reactions were carried out in a closed Teflon vessel which was placed at the center of the oven for irradiation. [16] and ethyl 2-(N-phenylhydrazono)-3oxobutanoate (20) [19] were prepared as previously reported in the respective literature.

Reaction of 2-methyl-3-[4-amino-5-mercapto-4H-[1,2,4]triazol-3-yl]-1H-indole (1) with aldehydes
A mixture of 1 (0.245 g, 1 mmol), and the appropriate aldehyde (1 mmol) in DMF (5 mL) containing a few drops of concentrated HCl was irradiated by MW at 400 Watt in a closed Teflon vessel until all the starting material was consumed (2-3 min., as monitored by TLC). Excess of solvent was removed under reduced pressure and the reaction mixture was added to crushed ice. The product separated was filtered, washed with water, dried and recrystallized from the proper solvent to give compounds 3a-e.

Reaction of 1-[(2-methyl-1H-indole)-3-carbonyl]thiosemicarbazide derivatives 8a,b with conc. H 2 SO 4
A solution of 8a or 8b (1 mmol) in conc. sulfuric acid (5 mL) was stirred at room temperature for 4 h. The reaction mixture was poured into cold water. The solid was collected by filtration, washed with water, dried and crystallized from DMF to give product 12a or 12b.

Reaction of 1-[(2-methyl-1H-indole)-3-carbonyl]thiosemicarbazide derivatives 8a,b with potassium hydroxide
A suspension of 8a or 8b (1 mmol) in potassium hydroxide solution (5 mL, 7%) in a closed Teflon vessel was irradiated by MW at 400 watt for 5 min. The reaction mixture was cooled then acidified with 10% hydrochloric acid to pH 6. The solid that formed was filtered off, washed with water, dried and finally crystallized from DMF to give product 15a or 15b. [1,2,4]

Synthesis of 3-[(3-methyl-4-phenylhydrazono-5-oxopyrazolin-1-yl)carbonyl]-2-methyl-1H-indole (21)
Method A: To a solution of hydrazide 7 (1.89g, 10 mmol) in DMF (5 mL), ethyl 2-(Nphenylhydrazono)-3-oxobutanoate (20) (10 mmol), a few drops of glacial acetic acid were added. The reaction mixture was irradiated by MW at 400 Watt for 3 min, and then the reaction mixture was evaporated to half its volume and kept at room temperature overnight. The solid product was collected by filtration, then diluted with water, dried, and recrystallized from ethanol to give 21. Step (22): To a cold solution of compound 22 (2.55 g, 10 mmol) in pyridine (30 mL) was added phenyldiazonium salt solution (10 mmol) [prepared by diazotizing aniline (10 mmol) dissolved in 6M hydrochloric acid (6 mL) with a solution of sodium nitrite (0.7 g) in water (5 mL)]. The addition of diazonium salt was carried out with rapid stirring over a period of 30 min. The reaction mixture was stirred for a further 30 min and left to stand in an ice bath for 12 h, The solid that separated was filtered off, washed with water and finally recrystallized from ethanol to give product proved to be identical in all respects (mp, mixed mp and IR spectra) with the phenylhydrazone 21 which obtained from method A.

Preliminary Antimicrobial Screening
Overnight culture was streaked on the surface of Muller-Hinton agar plate. Sterile filter paper disc was saturated with 10 μL of 0.5 mg/mL w/v solution of the compound under investigation in DMF. The plates and discs were then incubated at 37 °C (for bacteria) and at 28 °C (for fungi) for 24 h and examined for inhibition zones to determine the activity of the tested compounds.

Conclusions
A number of heterocyclic azoles, incorporating indole moieties, were synthesized using microwave irradiation and evaluated for their biological activities. The results of the antimicrobial activity of the synthesized products were promising.