Synthesis and Biological Properties of Novel Triazole-Thiol and Thiadiazole Derivatives of the 1,2,4-Triazole-3(5)-one Class

2,2'-(4,4'(Butane-1,4-diyl/hexane-1,6-diyl)bis(3-methyl-5-oxo-4,5-dihydro-1,2,4-triazole-4,1-diyl)) diacetohydrazides 3a,b were obtained via the formation of diethyl 2,2'-(4,4'(butane-1,4-diyl/hexane-1,6-diyl)bis(3-methyl-5-oxo-4,5-dihydro-1,2,4-triazole-4,1-diyl))diacetates 2a,b, obtained starting from di-[3(methyl-2-yl-methyl)-4,5-dihydro-1H-[1,2,4]-triazole-5-one-4yl]-n-alkanes 1a,b in two steps. The synthesis of the compounds 7a,b–9a,b incorporating the 1,3,4-thiadiazole, and 10a,b–11a,b with a 1,2,4-triazole-thiol nucleus as the second heterocycle, was performed by the acidic or basic treatment of compounds 4a,b–6a,b which were obtained from the reaction of 3a,b with several isothiocyanates. Newly synthesized compounds were screened for antimicrobial activities and their antioxidant properties by the 1,1-diphenyl-2-picryl hydrazyl (DPPH) radical scavenging method. Compounds 4a,b, 5a,b, and 6a,b were found to possess good antioxidant properties. Almost all compounds have significant antimicrobial activities.

Heterocycles containing a 1,2,4-triazole or 1,3,4-thiadiazole moiety, and the compounds consisting of 1,2,4-triazole and 1,3,4-thiadiazole condensed nucleus systems constitute a class of compounds possessing a wide spectrum of biological activities such as anti-inflammatory, antiviral and antimicrobial and antitumoral properties [12][13][14][15][16][17][18]. It was reported that more efficacious antibacterial compounds can be designed by joining two or more biologically active heterocyclic systems together in a single molecular framework. Keeping this observation in mind, this paper has presented the synthesis of new triazole thiadiazole derivatives incorporating different pharmacophores as hybrid molecules possessing antioxidant and antimicrobial activities [19].
Analytical and spectroscopic data of compounds 2a,b confirmed this reaction by the additional signals derived from the -CH 2 CO 2 Et group at the expected chemical shift values. Moreover, compounds 2a,b gave a stable M+1 ion peak. The 1 H-NMR spectra of compounds 3a,b displayed no signals belonging to the -OCH 2 CH 3 group; instead, new signals derived from the hydrazide structure appeared at 3.38-3.57 ppm (-NHNH 2 ) and 9.16-9.17 ppm (-NHNH 2 ) integrating for two protons and one proton, respectively (D 2 O exchange). Furthermore, compounds 3a,b gave relatively stable M+1 ion peaks.

General Information
Melting points were measured on an electrothermal apparatus and are uncorrected. 1

General Method for the Synthesis of Compounds 2
The corresponding compound 1 (0.01 mol) was refluxed with an equivalent amount of sodium in absolute ethanol for 2 h. Then, ethyl bromoacetate (0.01 mol) was added and the mixture was refluxed for an additional 5 h. After evaporation of the solvent under reduced pressure at 35-40 °C, a solid appeared. This was recrystallized from 1:1 ethanol/water to afford the desired product.

General Method for the Synthesis of Compounds 3
A solution of the corresponding compound 2 (10 mmol) in n-butanol was refluxed with hydrazine hydrate (25 mmol) for 4 h. After cooling it to room temperature, a white solid appeared. This was recrystallized from 1:2 ethanol-water to obtain the desired compound.

General Method for the Synthesis of Compounds 4-6
A mixture of corresponding compound 3 (10 mmol) and 4-fluorophenylisothiocyanate (for compounds 4), 4-bromophenylisothiocyanate (for compounds 5) or p-tolylisothiocyanate (for compounds 6) (15 mmol) was refluxed in ethanol for 4 h. The solution was cooled and a white solid appeared. This was filtered and recrystallized from ethanol to afford the desired product.

General Method for the Synthesis of Compounds 7-9
A mixture of corresponding thiosemicarbazides 4-6 (10 mmol) in cold concentrated sulfuric acid (30 mL) was stirred for 10 min then, the mixture was allowed to reach room temperature. After stirring for an additional 30 min, the resulting solution was poured into ice cold water and made alkaline to pH 8 with ammonia. The precipitated product was filtered, washed with water and recrystallized from ethanol to afford the pure compounds.    -1,4-diyl)bis(2-((5-(4-bromophenylamino)1,3,4-thiadiazole-2-yl)

General Method for the Synthesis of Compounds 10 and 11
A solution of corresponding carbothioamide (4-6) (10 mmol) inequivalent amount of 2 N NaOH solution was refluxed for 3 h. The resulting solution was cooled to room temperature and acidified topH 3-4 with 37% HCl. The precipitate formed was filtered, washedwith water and recrystallized from dimethyl sulfoxide/water (1:1) to afford the desired compound.

Antioxidant Activity
DPPH assay: The hydrogen atoms or electrons donation ability of the samples was measured from the bleaching of purple coloured methanol solution of DPPH. This spectrophotometric assay uses stable radical 2,2'-diphenylpicrylhydrazyl (DPPH) as a reagent [21,22]. Fifty microliters of various concentrations of the samples in methanol was added to a 0.004% methanol solution of DPPH (5 mL). After a 30 min incubation period at room temperature the absorbance was read against a blank at 517 nm. Inhibition free radical DPPH in percent (I %) was calculated in following way: I %: (A blank − A sample /A blank ) × 100 where A blank is the absorbance of the control reaction (containing all reagents except the test sample), and A sample is the absorbance of the test compound. Sample concentration providing 50% inhibition (IC 50 ) was calculated form the graph plotted inhibition percentage against extract concentration. Tests were carried out in triplicate. Butylated hydroxytoluene (BHT) was used as positive control. The results are shown in the Table 1 and Figure 1.  of compounds 4a,b, 5a,b, 6a,b, 10a,b and 11a 40 ± 0.9 11a 36± 0.9 11b 10 ± 0.7 BHT (Positive control) 19.8 ± 0.5 Figure 1. The graphical representation of antioxidant activities compounds.

Conclusions
In this study, a series of new triazole derivatives having carbohydrazide, thiosemicarbazide, thiadiazole and triazole-thiol moieties, respectively, at the 1-position was synthesized, and their antioxidant and antimicrobial activities were evaluated. It was observed that in vitro the newly synthesized triazole-thiosemicarbazides 4a,b-6a,b possess highly potent antioxidant properties and triazole/triazole-thiol derivatives 10a,b-11a,b possess moderate potent antioxidant properties .  Compounds 7a,b, 8a,b and 9a,b, containing triazole-thiadiazole and triazole/triazole-thiol moieties didn't show antioxidant properties. All newly synthesized compounds were screened for their antibacterial and antifungal activities by the inhibition zones (mm) method. Almost all the synthesized compounds showed significant activity against bacteria, while no compounds showed activity against fungi (Aspergillus, Fusarium, Rhizopus). In particular the 1,2,4-triazole-possessing thiosemicarbazides 4a,b-6a,b show both antibacterial and antifungal activities.