Synthesis and Evaluation of the Anti-Microbial Activity of New Heterocycles Containing the 1,3,4-Thiadiazole Moiety

A new series of thiadiazole-enaminones 4 were synthesized via reactions of 5-acetyl-1,3,4-thiadiazoles 3 with dimethylformamide-dimethylacetal (DMF-DMA). The simple phenyl substituted thiadiazole-enaminone 4f was used as a synthetic precursor for the preparation of a wide variety of new heterocyclic compounds, including the 5-substituted-1,3,4-thiadiazole derivatives 5, 6, 11, 12 and 13, which were obtained via reactions of 4f with nitrogen nucleophiles. Also, reactions of enaminone 4f with carbon nucleophiles afforded the respective 1,3,4-thiadiazoles 8a–d. In addition, the results of the antimicrobial activities of thiadiazole-enaminones 4 and their precursors 2 and 3 indicate that some members of this series display promising activities against all tested microorganisms.


EtOH
Reaction of enaminone 4f with hydrazine hydrate in ethanol under reflux led to formation of the thiadiazole-pyrazole linked product 5 (Scheme 2) whose structure was assigned using spectroscopic and elemental analysis methods. For example, the IR spectrum of this compound contains a carbonyl band at 1,607 cm −1 attributed to the benzamide group and it does not contain an enaminone groupassociated carbonyl band. Also, no olefinic or methyl proton resonances were observed in the 1 H-NMR spectrum of 5, which did contain a singlet at δ 9.21 ppm due to the pyrazolyl-NH proton. In a related manner, reaction of enaminone 4f with hydroxylamine hydrochloride in ethanol in the presence of potassium carbonate led to formation of the thiadiazole-isoxazole 6. The structure of the latter compounds was also established based on both elemental and spectral data (see Experimental). The reactivity of enaminone 4f towards several C-nucleophiles was explored next. Compound 4f reacts with active methylene compounds in acetic acid in the presence of ammonium acetate under reflux to afford products that could have either of the regioisomeric linked thiadiazole-pyridine structures represented by either 8 or 10. Two pathways are outlined in Scheme 3 for this reaction. The reaction may proceed by initial Michael addition (route A) of the active methylene compound to the activated double bond of 4f to give the Michael adduct 7 followed by tandem elimination of dimethylamine and condensation with ammonia to give product 8 or the other suggested pathway (route B) may proceed by initial condensation of active methylene compound with the carbonyl group of 4f which leads to formation of intermediate 9 that cyclizes in the presence of ammonium acetate to give 10. The latter product 10 was discarded however based on its 1 H-NMR spectral data. For example, the 1 H-NMR spectrum of compound 8a revealed two singlet signals at δ 2.49 and 2.63 ppm assigned to the methyl and acetyl protons, in addition to two doublets at δ 8.04 and 8.39 ppm (J = 7-8 Hz) assigned to pyridine H-3 and H-4. Such value of coupling constant J is characteristic for pyridine H-3 and H-4 and much higher than that for H-2 and H-3 (J = 4-6 Hz) [18,19] in structure 10 (Scheme 3).

Biological Screening Anti-Microbial Activities
In vitro anti-microbial screening of the compounds 2, 3 and 4 prepared in this study was carried out using four fungal strains, including Aspergillus fumigatus RCMB 002003 (AF), Geotrichum candidum RB 052006 (GC), Candida albicans RCMB 005002 (CA) and Syncephalastrum racemosum RCMB 005003 (SR), and four bacterial species, including the Gram positive bacteria Staphylococcus aureus RCMB 000106 (SA) and Bacillus subtilis RCMB 000107 (BS), and the Gram negative bacteria Pseudomonas aeruginosa RCMB 000102 (PA) and Escherichia coli RCMB 000103 (EC). The results of the investigations with the thiadiazole derivatives 2a-h (Tables 1 and 2) showed that 2b displays high activities against all the tested microorganisms. This finding suggests that the presence of an electron-donating C-4 methyl group in the phenyl ring linked to the 1,3,4-thiadiazole moiety promotes increased biological activity. In addition, compound 3c showed high activities against all tested microorganisms, especially AF, when compared to the standard fungicides itraconazole and clotrimazole. In addition, 3c showed high activity against all tested bacteria species, especially BS, when compared with the standard bactericides penicillin G and streptomycin. The data obtained by probing the antimicrobial activities of enaminones 4 are given in Tables 3, 4 and 5. The results indicate that 4c is highly potent against all tested microorganisms. Based on these results, we can conclude that the presence of a bromine substituent at the C-4 of the phenyl group linked to the 1,3,4-thiadiazole moiety causes increased antimicrobial activity.

General
Melting points were determined uisng an Electrothermal Gallenkamp apparatus and are reported uncorrected. IR spectra were recorded in KBr using PyUnicam SP-1000 Spectrometer. 1 H-NMR spectra were recorded using CDCl 3 and DMSO-d 6 solutions using a Varian Em-300 MHz Spectrometer and chemical shifts are reported in ppm relative to that of TMS, which was used as an internal standard. Mass spectra were recorded using a AEI MS 30 mass spectrometer operating at 70 eV. Elemental analyses were carried out by the Microanalytical Center of Cairo University, Giza, Egypt. Antimicrobial activities were carried out at the Regional Center for Mycology and Biotechnology at Al-Azhar University, Cairo, Egypt.

Synthesis of Compounds 2a-h and 3a-h
Compounds 2a-h and 3a-h were prepared using previously described methods [22]. Compounds 2c and 3c are newly prepared and their physical constants, together with spectral and elemental analysis are shown below:

Conclusions
In the investigation described above, a new series of 1,3,4-thiadiazole derivatives was prepared. In addition, 1,3,4-thiadiazole derivatives substituted at position-5 with heterocyclic rings were synthesized via reaction of enaminone 4f with C-and N-nucleophiles. The antimicrobial properties of some of the prepared compounds were evaluated. The results demonstrate that selected members of this series, including 2b, 3c and 4c, show excellent activities against all tested microorganisms compared with the standard fungicides itraconazole and clotrimazole and bactericides penicillin G and streptomycin.