SYNTHESIS AND BIOLOGICAL ACTIVITIES OF 3 , 6-DISUBSTITUTED-1 , 2 , 4-TRIAZOLO-1 , 3 , 4-THIADIAZOLE DERIVATIVES

Twelve novel triazolothiadiazole derivatives were synthesized from 4-amino-5-substituted-4H1,2,4-triazole-3-thiols with various aromatic carboxylic acids by cyclization in the presence of phosphorous oxychloride. All the newly synthesized compounds were characterized by FTIR, H NMR, mass spectroscopy and elemental analysis. The antimicrobial activities of the title compounds were examined by disc diffusion method against Escherichia coli, Staphylococcus aureus, Pyricularia oryzae and Rhizoctnia solani. The bioassay indicated all synthesized triazolothiadiazole derivatives possessed moderate to good antibacterial and antifungal activities against the tested organisms. Especially, compounds 2e and 2k exhibited excellent antibacterial and antifungal activities among these triazolothiadiazole derivatives.

Therefore, with the purpose of broadening the class of compounds exhibiting good antimicrobial activity, we introduce the fluorophenyl, chlorophenyl moieties into the triazole ring and the phenyl, nitrophenyl, tert-butylphenyl moieties into the thiadiazole ring to investigate their antimicrobial activities.Herein we report the synthesis and biological activities of some new 3,6-disubstituted-1,2,4-triazolo-1,3,4-thiadiazole derivatives.Structures and properties of the obtained compounds were demonstrated by means of elemental analysis, FT-IR, 1 H NMR, ESI-MS spectroscopy.

General
Melting points were determined using X-4 digital melting-point apparatus and are uncorrected.Elemental analysis (C, H, N) was performed with a Perkin Elmer 2400 elemental analyzer.Infrared spectra were recorded on a Nicolet FTIR 5700 spectrophotometer with KBr pellets.
Electrospray ionization mass spectra (ESI-MS) were performed with a Finnigan LCQ Advantage Max spectrometer. 1 H NMR spectra were measured on an Avance III TM 300 MHz NB Digital NMR spectrometer in CDCl 3 or DMSO-d 6 solution with TMS as internal standard.Substituted benzoyl hydrazines were synthesized by our group according to the method reported in the literature [17].Hydrazine hydrate, phosphorus oxychloride and substituted benzoic acid were purchased from Shanghai Chemical Reagent Company Ltd. (Shanghai, China).Other reagents were of analytical grade purity and used without further purification.

Biological activity tests
The newly synthesized compounds (2a2l) were tested for their in vitro antibacterial activity against Escherichia coli and Staphylococcus aureus and antifungal activity against Pyricularia oryzae and Rhizoctnia solani by the disc diffusion method [18,19] at a concentration of 50 mg L 1 .Muller-Hinton agar (Hi-Media) was employed as culture medium and DMSO was used as solvent control for antimicrobial activity.Norfloxacin and Triadimefon were used as standard for antibacterial and antifungal activities, respectively.The inhibition zones were measured in mm at the end of an incubation period of 24 h at 37 o C for bacteria and 72 h at 24 o C for fungi.Generally, the results were taken in duplicate.Results with difference higher than 5% were neglected and repeated.In addition, the relative inhibition percentage of the tested compounds with respect to standard drug was calculated according the formula RI = D t / D s  100%, where RI is the relative inhibition percentage, D t is the diameter of inhibition zone for the tested compounds, D s is the diameter of inhibition zone for the standard drug.
Scheme 1. Synthetic pathway for the title compounds 2a2l.
The newly synthesized compounds were confirmed by elemental analysis, FTIR, 1 H NMR, and mass spectral data.In the IR spectra, the relatively strong peaks at 33263226 cm 1 were attributed to the NH 2 stretching vibrations and the weak single peaks at 26222631 cm 1 due to the SH stretching vibrations for the triazole compounds 1a1d.However, in the title compounds 2a2l, their IR spectra did not exhibited the characteristic absorptions of the NH 2 and SH stretching vibrations of parent compounds 1a1d, which clearly indicated the fusing between compounds 1a1d and substituted aromatic carboxylic acids [20].The 1 H NMR spectra of the triazole compounds 1a1d showed two characteristic two proton signals at 11.9310.75and 5.024.82ppm, which were attributed to the protons of SH and NH 2 group for the triazoles, respectively.But these two proton signals were disappeared in the 1 H NMR spectra of the title compounds 2a2l.The chemical shifts at 8.447.22ppm were assigned to aromatic protons of compounds 2a2l.Here, we chose the compound 2l as an example to discuss their chemical shifts.The 1 H NMR spectrum of the compound 2l was shown in Figure 1.From Figure 1, the multiple peak at  8.448.40ppm was assigned to H a and H a' and the two doublet peaks (J = 6.7 Hz) at  7.87 and 7.58 ppm were due to H c and H c' and H d and H d' , respectively.The protons H b and H b' exhibited a triplet peak (J = 8.7 Hz) at  7.25 ppm.The nine-proton singlet peak at  1.38 ppm was attributed to the protons of the tert-butyl group.These results also confirmed that the triazoles 1a1d had converted to target triazolothiadiazole compounds 2a2l by reacting with substituted aromatic carboxylic acids in phosphorus oxychloride.
The ESI mass spectra of the all synthesized compounds 1a1d and 2a2l were compared to confirm elemental compositions.Their molecular ion peaks (M + ) for these compounds were observed in accordance with the Nitrogen Rule.For example, the electrospray ionization mass spectrum (Figure 2) of the compound 2l displayed a peak at m/z 353.18 [M+1] + , which was due to its molecular ion peak.

Antimicrobial activity
In the bioassay screening, the triazolothiadiazole compounds 2a2l were tested for their antimicrobial activity against Escherichia coli, Staphylococcus aureus, Pyricularia oryzae and Rhizoctnia solani.The inhibition zones and relative inhibition percentage of these compounds against bacteria and fungi are listed in Table 1.The results showed that all tested compounds exhibited moderate to good antibacterial and antifungal activities in DMSO.Comparatively, compounds 2e (R 1 = 2-fluorophenyl, R 2 = 4-nitrophenyl), and 2k (R 1 = 4-fluorophenyl, R 2 = 4nitrophenyl) revealed much significant antimicrobial activities than the other triazolothiadiazole derivatives, which could be due to the presence of fluorophenyl and nitrophenyl moieties in triazolothiadiazole.However, compounds 2c (R 1 = 2-chlorophenyl, R 2 = 4-tert-butylphenyl), 2f (R 1 = 2-fluorophenyl, R 2 = 4-tert-butylphenyl) and 2i (R 1 = 4-chlorophenyl, R 2 = 4-tertbutylphenyl) revealed much lower activities against the tested microorganisms among all synthesized triazolothiadiazole derivatives, which probably assigned to tert-butylphenyl moiety in triazolothiadiazole.By the preliminary structure-activity relationship analysis, it was concluded that the introduction of the electron-withdrawing group in triazolothiadiazole can distinctly improve their antimicrobial activities and the introduction of the electron-donating group leads to the decrease of their antimicrobial activities.Especially, the triazolothiadiazole derivatives with fluorophenyl and nitrophenyl moieties at 3,6-position of the triazolothiadiazole ring showed potent antimicrobial activities against the tested microorganisms.As a result, it indicated that the electronic nature of the substituent groups at 3,6-positions in the triazolothiadiazole ring played a significant role in antimicrobial activities.

CONCLUSION
In summary, some new 3,6-disubstituted-1,2,4-triazolo-1,3,4-thiadiazole derivatives were synthesized.Their structures were confirmed by elemental analysis, IR, 1 H NMR, and mass spectroscopy.The biological activities of these compounds were evaluated against Escherichia coli, Staphylococcus aureus, Pyricularia oryzae and Rhizoctnia solani by disc diffusion method.The results showed that these triazolothiadiazole derivatives exhibited moderate to good antibacterial and antifungal activities.Especially, compounds 2e and 2k displayed much higher antibacterial and antifungal activities in all the synthesized triazolothiadiazole derivatives.It demonstrated that the triazolothiadiazole derivatives with fluorophenyl and nitrophenyl moieties at 3,6-position in the triazolothiadiazole ring showed potent antimicrobial activities.Therefore, it is helpful for further structural modification of the triazolothiadiazole derivatives to improve their antimicrobial activities.

Figure 1 .
Figure 1.The 1 H NMR spectrum of the compound 2l.