Synthesis and Antimicrobial Activity of Some New Pyrazole, Fused Pyrazolo[3,4-d]-pyrimidine and Pyrazolo[4,3-e][1,2,4]-triazolo[1,5-c]pyrimidine Derivatives

Hydrazonyl bromides 2a,b reacted with active methylene compounds (dibenzoylmethane, acetylacetone, ethyl acetoacetate, phenacyl cyanide, acetoacetanilide, ethyl cyanoacetate, cyanoacetamide and malononitrile) to afford the corresponding 1,3,4,5-tetrasubstituted pyrazole derivatives 5-12a,b. Reaction of 12a,b with formamide, formic acid and triethyl orthoformate give the pyrazolo[3,4-d]pyrimidine, pyrazolo[3,4-d]pyrimidin-4(3H)one and 5-ethoxymethylene-aminopyrazole-4-carbo-nitrile derivatives 13-15a,b, respectively. Compounds 15a,b reacted with benzhydrazide and hydrazine hydrate to afford pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidine and [4-iminopyrazolo-[3,4-d]pyrimidin-5-yl]amine derivatives 16a,b and 17a,b. Reactions of compounds 17a,b with triethyl orthoformate and carbon disulfide give the corresponding pyrazolo[4,3-e]-[1,2,4]triazolo[1,5-c]pyrimidine derivatives 18a,b and 19a,b, respectively.


Results and Discussion
Reaction of hydrazonyl bromides 2a,b with dibenzoylmethane in sodium ethoxide solution at room temperature gave, as the sole separable product, 3-aryl-4-benzoyl-1-(4-nitrophenyl)-5-phenylpyrazoles 5a,b (Scheme 1). The assignments of the structures of products 5a,b were based on their correct elemental analyses and spectroscopic data. Their 1 H-NMR spectra revealed only the expected aromatic proton multiplet signals at δ 7.1-8.4 ppm. The IR spectra of each product showed two characteristic absorption bands at 1644-1650 cm -1 and 1593-1594 cm -1 , assignable to a conjugated benzoyl carbonyl and C=N groups, respectively.

Scheme 1
Compound 5a was also obtained when the nitrilimine 3a [generated in situ from the reaction of triethylamine with hydrazonyl bromide 2a] was reacted with dibenzoylmethane in tetrahydrofuran. This finding supports the mechanism suggested earlier for a similar reaction [29], whereby the carbanion -acting as a base -reacts with the hydrazonyl bromide to form a nitrilimine dipole 3, which adds to the enol tautomers of the active methylene compounds used to form 4, which then loses one molecule of water to give the pyrazole product.

Scheme 3
The condensations of compounds 12a,b with triethyl orthoformate at reflux afforded the corresponding 5-ethoxymethyleneaminopyrazole-4-carbonitrile derivatives 15a,b, respectively (Scheme 3). The confirmation of these structures was based on their analytical ( Table 2) and spectroscopic data ( Table 3). The structures of 15a,b were further confirmed by their reactions with benzhydrazide and hydrazine hydrate. Thus, treatment with benzhydrazide in tetrahydrofuran at reflux temperature afforded 16a,b (Scheme 4). The IR spectra of the products confirmed the disappearance of the nitrile absorption bands. The 1 H-NMR spectra showed in each case a signal at δ 8.5 ppm, corresponding to the pyrimidine proton. Furthermore, when compounds 15a,b were reacted with hydrazine hydrate in tetrahydrofuran at ambient temperature they produced the corresponding [3-aryl- heat heat heat 4-imino-1-(4-nitrophenyl)-1,4-dihydro-5H-pyrazolo[3,4-d]pyrimidin-5-yl]amine derivatives 17a,b (Scheme 4). The structures proposed for these products were established from their correct elemental analyses (Table 2) and spectroscopic data (Table 3). Their IR spectra revealed the absence of nitrile absorption frequencies. The 1 H-NMR spectra for 17a,b showed signals at δ 8.4 ppm, corresponding to the pyrimidine proton, and 5.0 ppm, assignable to the NH 2 protons. The 13 C-NMR spectrum of 17b was also compatible with the proposed structure.
Further confirmation of the structures of 17a,b was achieved from their reactions with triethyl orthoformate and carbon disulfide. Thus, refluxing compounds 17a,b in an excess of triethyl orthoformate gave products 18a,b, which were identified on the basis of correct elemental analyses and spectroscopic data as 9-aryl-7-(4-nitrophenyl)pyrazolo[4,3-e][1,2,4]-triazolo[1,5-c]pyrimidine derivatives (Scheme 4). The IR spectra of 18a,b displayed no absorption bands for the NH and NH 2 groups, which were observable in compounds 17a,b. The 1 H-NMR spectra showed signals near δ 9.7-9.8 and 9.4-9.5 ppm, assignable to the pyrimidine and the triazole proton, respectively. In addition, refluxing compounds 17a,b with carbon disulfide and potassium hydroxide in ethanol gave 19a,b, which were identified as 9-aryl-7-(4-nitrophenyl)pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidine-2thione derivatives (Scheme 4). The structures of the compounds produced were established by spectroscopic data. The IR spectra revealed absorption bands corresponding to the NH and C=S functions near 3421.1 or 3422.4 and 1240 cm -1 , respectively. The 1 H-NMR spectra exhibited two singlets at δ 9.2 and 9.8 ppm, representing the protons of the pyrimidine and NH. Also, the mass spectra of all prepared compounds were compatible with the proposed structures (Table 3).  (namely 2a,b, 6a, 9b, 17b and 18b) were screened for antibacterial activity (in nutrient agar broth) and antifungal activity (in Dox's medium and Saboured's agar) by the agar diffusion method [31,32] at a concentration 20 mg/mL using DMSO as solvent and blank. The compounds were tested for their activities against Gram +ve bacteria (Staphylococcus aureus) and Gram -ve bacteria (Escherichia coli), in addition to the pathogenic fungi Aspergillus flavus and Candida albicans. The antimicrobial screening results were measured by the average diameter of the inhibition zones, expressed in mm, and are presented in Table 1. As shown in the results, all the tested compounds displayed significant activities against E. coli, S. aureus and C. albicans, while only compound 2a was very active against A. flavus and showed almost the same activity when compared with the usually used antifungal agents at the same concentration. Also, it was observed that the hydrazonoyl bromide 2a has shown the highest activity toward all the tested organisms among all the tested compounds. Table 1. Antimicrobial activities of the tested compounds

Conclusions
In this report, the synthesis of new pyrazole derivatives 5-12 by the reaction of some active methylene compounds with hydrazonoyl bromides 2a,b is reported. Also, the reaction of 12 with formamide, formic acid and triethyl orthoformate afforded the corresponding pyrazolo [3,4-

Reaction of hydrazonyl bromides with active methylene compounds: General method for the synthesis of 4,5-disubstituted-3-aryl-1-(4-nitrophenyl)pyrazoles 5-12
The appropriate active methylene compound (dibenzoylmethane, acetylacetone, ethyl acetoacetate, phenacyl cyanide, acetoacetanilide, ethyl cyanoacetate, cyanoacetamide, malononitrile, 0.005 mol) was added with stirring to an ethanolic sodium ethoxide solution [20 mL, prepared from sodium metal (0.11 g, 0.005 mol) and absolute ethanol]. To the resulting solution the appropriate hydrazonyl bromide 2a,b (0.005 mol) was added at room temperature. The mixture was stirred for 24 h, during which the bromide dissolved and the crude pyrazole precipitated. The latter was collected, washed with water, dried and crystallized from the indicated solvent.