Synthesis and Antimicrobial Activity of Some New Thiadiazoles, Thioamides, 5-Arylazothiazoles and Pyrimido[4,5-d][1,2,4]triazolo[4,3-a]pyrimidines

A novel series of 1,3,4-thiadiazoles, 5-arylazothiazoles and hexahydropyrimido-[4,5-d][1,2,4]triazolo[4,3-a]pyrimidines were synthesized via reaction of hydrazonoyl halides with each of alkyl carbothioates, carbothioamides and 7-thioxo-5,6,7,8-tetrahydropyrimido-[4,5-d]pyrimidine-2,4(1H,3H)-diones in the presence of triethylamine. The structures of the newly synthesized compounds were established based on their spectral data, elemental analyses and alternative synthetic routes whenever possible. Also, the newly synthesized compounds were screened for their antimicrobial activity against various microorganisms.

In further experiments treatment of 5a with 5,6,7, The 1 H-NMR spectrum of 26a showed a triplet at δ 1.18 due to methyl group of ethyl ester, a singlet at δ 2.33 of tolyl methyl group, a singlet at δ 3.33 from the two pyrimidinedione methyl groups, a quartet at δ 4.13 due to the methylene of ethyl ester, a singlet at δ 5.62 that corresponding to the In further experiments treatment of 5a with 5,6,7, In further experiments treatment of 5a with 5,6,7, The 1 H-NMR spectrum of 26a showed a triplet at δ 1.18 due to methyl group of ethyl ester, a singlet at δ 2.33 of tolyl methyl group, a singlet at δ 3.33 from the two pyrimidinedione methyl groups, a quartet at δ 4.13 due to the methylene of ethyl ester, a singlet at δ 5.62 that corresponding to the The 1 H-NMR spectrum of 26a showed a triplet at δ 1.18 due to methyl group of ethyl ester, a singlet at δ 2.33 of tolyl methyl group, a singlet at δ 3.33 from the two pyrimidinedione methyl groups, a quartet at δ 4.13 due to the methylene of ethyl ester, a singlet at δ 5.62 that corresponding to the pyrimidine H-4 and a multiplet at δ 6.90-7.83 that account for 14 aromatic protons and H-5 of pyrazole. In the IR spectrum, an absorption peak at 1650 cm −1 corresponding to conjugated carbonyl groups and an absorption near at 1615 cm −1 could account for the presence of the imino group.
The mechanism outlined in scheme 7 seems to be the most plausible pathway for the formation of 26a from the reaction of 5 with 20: (1) 1,3-addition of the thiol tautomer 21 to the nitrilimine 6a would give the thiohydrazonate ester 22 which could undergo nucleophilic cyclization to yield spiro compounds 23. The latter ring could then open and cyclize to yield 26a with loss of hydrogen sulfide; and (2) 1,3-cycloaddition of nitrilimine 6a to C=S of 20 would directly yield 22 (cf., Scheme 7). Attempts to isolate the thiohydrazonate ester 22 or intermediates 23 and 24 did not succeed even under mild conditions as they readily undergo in situ cyclization followed by elimination of hydrogen sulfide to give the final product 26 in Scheme 7.

Antimicrobial Activity
Twenty seven of the newly synthesized target compounds were evaluated for their in vitro antibacterial activity against Staphylococcus aureus and Bacillus subtilis as examples of Gram-positive bacteria and Pseudomonas aeruginosa and Escherichia coli as examples of Gram-negative bacteria. They were also evaluated for their in vitro antifungal activity against a representative panel of fungal strains i.e., Aspergillus fumigatus, and Candida albicans. Ampicillin, Gentamicin and Amphotericin B are used as reference drugs for in vitro antibacterial activity and for in vitro antifungal activity, respectively, at The Regional Center for Mycology and Biotechnology at Al-Azhar University (Nasr City, Cairo, Egypt). The results of testing for antimicrobial effects are summarized in Tables 1-3. Table 1. Mean zone of inhibition beyond well diameter (6 mm) produced on a range of clinically pathogenic microorganisms using (5 mg/mL) concentration of tested samples.  10a, 13b, 14b, 15f, and 28a when compared to the Gentamicin standard.

Compound
The minimum inhibitory concentration (MIC) is the lowest concentration of an antimicrobial that will inhibit the visible growth of a microorganism after overnight incubation. Minimum inhibitory concentrations are important in diagnostic laboratories to confirm resistance of microorganisms to an antimicrobial agent and also to determine the potency of new antimicrobial agents [34]. A MIC is generally regarded as the most basic laboratory measurement of the activity of an antimicrobial agent against an organism [35]. MIC was determined by the broth micro dilution method using 96-well micro-plates [36,37]. Pseudomonas aeruginosa showed an the same as the MIC value of 12.5 mg/mL of the tested compound 13b suggesting high inhibitory activity compared to that of Gentamicin. Also, compound 10a showed an MIC value of 3.9 against Escherichia coli which was the same MIC value of Gentamicin against Escherichia coli (Table 2). The half maximal inhibitory concentration (IC 50 ) is a measure of the effectiveness of a substance in inhibiting a specific biological or biochemical function. This quantitative measure indicates how much of a particular substance is needed to inhibit a given biological process by half. And here the biological function is the growth of microorganisms Aspergillus fumigatus, Syncephalastrum racemosum, Geotrichum candidum, Candida albicans, Streptococcus pneumoniae, Bacillus subtilis, Pseudomonas aeruginosa, and Escherichia coli (Table 3).

General Information
All melting points were determined on an Electrothermal apparatus (Bibby Sci. Lim. Stone, Staffordshire, UK) and are uncorrected. IR spectra were recorded (KBr discs) on a FT-IR 8201 PC spectrophotometer (Shimadzu, Tokyo, Japan). 1 H-NMR spectra were recorded in CDCl 3 and DMSO-d 6 solutions on a Gemini 300 MHz spectrometer (Varian, Mercury VX-300 NMR spectrometer, Bruker BioSpin GmbH, Rheinstetten, Germany) and chemical shifts are expressed in δ ppm units using TMS as an internal reference. Mass spectra were recorded on a Shimadzu GC-MS QP1000 EX instrument. (Tokyo, Japan) Elemental analyses were carried out at the Microanalytical Center of Cairo university. Hydrazonoyl halides 5a-d were prepared as previously reported [38][39][40][41]. Antimicrobial screening was performed at the Regional Center for Mycology and Biotechnology, Al-Azhar University, Cairo, Egypt.

Method B
Benzenediazonium chloride (5 mmol), which prepared from aniline (0.45 mL, 5 mmol), hydrochloric acid (6 N, 6 mL), and sodium nitrite (0.35 g, 5 mmol), was added dropwise with stirring to a cold solution of 19 (5 mmol) and sodium acetate trihydrate (1.3 g, 10 mmol) in ethanol (50 mL). The reaction mixture was stirred in ice bath for 3 h. The resulting solid was collected and crystallized to give a product identical in all aspects (mp, mixed mp, and spectra) with 18a.