Synthesis and Antimicrobial Evaluation of Some Novel Bis-α,β-Unsaturated Ketones, Nicotinonitrile, 1,2-Dihydropyridine-3-carbonitrile, Fused Thieno[2,3-b]pyridine and Pyrazolo[3,4-b]pyridine Derivatives

The title compounds were prepared by reaction of 1,1′-(5-methyl-1-phenyl-1H-pyrazole-3,4-diyl)diethanone (1) with different aromatic aldehydes 2a–c, namely Furfural (2a), 4-chlorobenzaldehyde (2b) and 4-methoxybenzaldhyde (2c) to yield the corresponding α,β-unsaturated ketones 3a–c. Compound 3 was reacted with malononitrile, 2-cyanoacetamide or 2-cyanothioacetamide yielded the corresponding bis[2-amino-6-(aryl)nicotinonitrile] 4a–c, bis[6-(2-aryl)-2-oxo-1,2-dihydropyridine-3-carbonitrile] 5a–c or bis[6-(2-aryl)-2-thioxo-1,2-dihydropyridine-3-carbonitrile] 6a,b, respectively. The reaction of compound 6a with each of 2-chloro-N-(4-bromophenyl) acetamide (7a), chloroacetamide (7b) in ethanolic sodium ethoxide solution at room temperature to give the corresponding 4,4′-(5-methyl-1-phenyl-1H-pyrazole-3,4-diyl)bis-6-(2-furyl)thieno[2,3-b]pyridine-2-carboxamide] derivatives 9a,b. While compound 6a reacted with hydrazine hydrate yielded the 4,4′-(5-methyl-1-phenyl-1H-pyrazole-3,4-diyl)bis[6-(2-furyl)-1H-pyrazolo[3,4-b]pyridin-3-amine] 11. The structures of the products were elucidated based on their spectral properties, elemental analyses and, wherever possible, by alternate synthesis. Antimicrobial evaluation of the products was carried out.

On the other hand, many compounds with pyrazole ring are of interest due to their broad spectrum of biological activities against NOS inhibitor [28], monoamine oxidase inhibitor [29], and antibacterial [30], antiamoebic [31]. Moreover, N-phenylpyrazole derivatives play an important role in antitumor screening [32] as well as potent antimicrobial activity [33,34]. Furthermore, a pyrazolo [3,4-b]pyridines have Potential and selective inhibitors of glycogen synthase kinase-3 (GSK-3) [35]. Also 3-Cyano-2(1H)-pyridinethiones [36,37] and their related compounds were found to be very reactive substances for the synthesis of many different heterocyclic systems which exhibited biological activities such as antibacterial and antifungal [38]. In light of these findings, we report here the synthesis of some novel bis-heterocycles containing N-phenylpyrazole as a base unit. In addition, some of the newly synthesized compounds were screened for their antibacterial and antifungal activities.
Next, condensation of compounds 3a-c with malononitrile in the presence of ammonium acetate in n-butanol afforded the corresponding 4,4'-(5-methyl-1-phenyl-1H-pyrazole-3,4-diyl) bis[2-amino-6-(aryl)nicotinonitrile] 4a-c were also prepared in fair yields by applying the aforementioned one pot reaction of the compound 1, the corresponding aldehyde and malononitrile in the presence of ammonium acetate (Scheme 2).  The structures of the newly synthesized compounds were confirmed on the basis of their elemental analysis and IR, 1 HNMR and mass spectral data. The IR spectrum of compound 4b, taken as a typical example of the series prepared, reveled absorption bands at 2210 and 3438, 3350 cm −1 corresponding to nitrile and NH 2 function, respectively. Its 1 HNMR spectrum showed two singlet signals at δ 2.32 and 7.35 due to CH 3 and 2NH 2 protons in addition to an aromatic multiplet in the region δ 7.38-7.61. Their mass spectra revealed in each case the respective molecular ion peak.
But, 3a-b was condensed with 2-cyanoacetamide and 2-cyanothioacetamide in the presence of pipridine in ethanol to yield the bis pyridine carbonitrile derivatives 5a-c in 60%, 35% and 70% yields, respectively and bis pyridnethione derivatives 6a,b in 60% and 55% yields, respectively, as shown in Scheme 2. The structures of the newly synthesized compounds were confirmed based on their elemental analysis, IR, 1 HNMR and mass spectral data (see experimental). Compound 6a was taken as an example reacted with each of 2-chloro-N-(4-bromophenyl) acetamide (7a), chloroacetamide (7b) by stirring in ethanolic sodium ethoxide solution at room temperature to give the corresponding 4,4'- However, treatment of the compound 6a with hydrazine hydrate by refluxing in dioxane afforded the 4,4'- (11) as shown in Scheme 3. The structure of the products 9a,b and 11 was elucidated by considering the data of IR, 1 HNMR, mass spectra and elemental analyses.

General Experimental Procedures
All melting points were measured on an Electrothermal Gallenkamp apparatus (Weiss-Gallenkamp, London, UK). The infrared spectra were recorded in potassium bromide discs on a Pye Unicam SP3300 and Shimadzo FT IR 8101 PC infrared spectrophotometers (Pye Unicam Ltd. Cambridge, England and Shimadzu, Tokyo, Japan, respectively). The 1 H-NMR spectra were recorded on a Varian Mercury VXR-300 spectrometer (300 MHz). The mass spectra were recorded on a GCMS-Q1000-EX Shimadzu and GCMS 5988-A HP spectrometers, the ionizing voltage was 70 eV. Elemental analyses were carried out at the Micro-analytical Center of Cairo University, Giza, Egypt. The biological evaluation of the products was carried out in the Microbiology Division of Micro-analytical Center of Cairo University. The starting material Pyrazole 1 was prepared as previously reported in the literature [39,41]. (1)

Antimicrobial Evaluation
The antibacterial and antifungal activity assays were carried out at the Microbiology Division of Microanalytical Center of Cairo University using the diffusion plate method [43][44][45]. A bottomless cylinder containing a measured quantity (1 mL, mg/mL) of the sample is placed on a plate (9 cm diameter) containing a solid bacterial medium (nutrient agar broth) or fungal medium, which has been heavily seeded with a spore suspension of the test organism. After incubation (24 h for bacteria and 5 days for fungi), the diameter of the clear zone of inhibition surrounding the sample is taken as measure of the inhibitory power of the sample against the particular test organism. The solvent used was DMSO and the concentration of the sample used is 100 μg/mL. The results of antimicrobial activity are summarized in Table 1. Most of the synthesized compounds were evaluated for their antibacterial against a Gram negative bacterium (Escherichia coli anaerobic (EC)), a Gram positive bacterium (Staphylococcus aureus (SA)) and for antifungal activity against Candida albicans (CA) and Aspergillus flavus (AF) by diffusion technique [43][44][45]. As seen from the data present in Table 1, Escherichia coli anaerobic is sensitive to compounds 3a, 4c and 9a; furthermore, Staphylococcus aureus is sensitive to compounds 4b,c, 5a and 9a. Whereas, all tested compounds did not exhibit the antifungal activity against the two tested fungi species Candida albicans and Aspergillus flavus. The activity of 4c and 9a is attributed to the presence of pharmacological active 4-methoxyphenyl at position 6 of the nicotinonitrile and 4-bromophenyl at position N of carboxamide.  3a-c, 4a-c, 5a,c, 6a,b)