Molecular Sciences Synthesis and Antimicrobial Evaluation of Some Novel Thiazole, Pyridone, Pyrazole, Chromene, Hydrazone Derivatives Bearing a Biologically Active Sulfonamide Moiety

This study aimed for the synthesis of new heterocyclic compounds incorporating sulfamoyl moiety suitable for use as antimicrobial agents via a versatile, readily accessible N-[4-(aminosulfonyl)phenyl]-2-cyanoacetamide (3). The 2-pyridone derivatives were obtained via reaction of cyanoacetamide with acetylacetone or arylidenes malononitrile. Cycloaddition reaction of cyanoacetamide with salicyaldehyde furnished chromene derivatives. Diazotization of 3 with the desired diazonium chloride gave the hydrazone derivatives 13a–e. Also, the reactivity of the hydrazone towards hydrazine hydrate to give Pyrazole derivatives was studied. In addition, treatment of 3 with elemental sulfur and phenyl isothiocyanate or malononitrile furnished thiazole and thiophene derivatives respectively. Reaction of 3 with phenyl isothiocyanate and KOH in DMF afforded the intermediate salt 17 which reacted in situ with 3-(2-bromoacetyl)-2H-chromen-2-one and methyl iodide afforded the thiazole and ketene N,S-acetal derivatives respectively. Finally, reaction of 3 with carbon disulfide and 1,3-dibromopropane afforded the N-[4-(aminosulfonyl)


Introduction
Cyanoacetamides and their related heterocyclic derivatives have generated a great deal of attention due to their interesting biological and therapeutic value; their pharmaceutical activities include: antimicrobial [1,2], antifungal [3], insulin releasing [4], carbonic anhydrase inhibitory [5], anti-inflammatory [6], and antitumor properties [7]. Some active sulfonamides as antibacterials are also known for their immunmodifying effects [8,9]. In addition, several thiazole derivatives possess important pharmacological activities and therefore they are useful materials in drug research. Over the past few decades, the literature has been enriched with progressive findings about the anticonvulsant activities of various substituted thiazole derivatives [10][11][12][13][14] that are of interest as potential neuroprotective agents [15,16]. Some 2-pyridones are also reported to possess antitumor [17], antibacterial [18] and other biological activities [19,20]. In view of these facts and as a continuation of our research program on the chemistry of butanamide [21,22], the present investigation aimed to synthesize and characterize newer hydrazones, pyridones, acrylamide, Pyrazole, thiadiazole, and thiophene incorporating sulfonamide moiety. It was found that N- [4-(aminosulfonyl)phenyl]-2-cyanoacetamide (3) is an excellent building block for the synthesis of the target objectives.

Results and Discussion
One reason of our interest in amines is related to the conversion possibility of their NH 2 group to the NHCOCH 2 CN group, which leads to cyanoacetamide with further useful functionalization at this position [23,24]. Cyanoacetamide 3 was synthesized by cyanoacetylation of 1 with 3,5-dimetyl-1-cyanoacetyl Pyrazole (2) [25] as previously described (Scheme 1). Thus, the Knoevengel condensation of the cyanoacetamide 3 with aromatic aldehydes namely benzaldehyde, p-anisaldehyde, and p-chlorobenzaldehyde furnished the corresponding arylidene derivatives 5a-c (Scheme 2). The IR spectrum of compound 5a, taken as a typical example of the series prepared, revealed absorption bands at 1680, 2220 and 3362 cm −1 corresponding to carbonyl, nitrile and NH functions, respectively. Its 1 H-NMR spectrum showed signals at δ 8.32 and 10.70 (D 2 O-exchangeable) due to CH and NH protons in addition to two aromatic protons at δ 7.81-7.83. Its mass spectrum showed a molecular ion peak at m/z 327. Pyridin-2(1H)-ones 7a-c was obtained through the reaction of the arylidene derivatives 5a-c with malononitrile in dioxane containing piperidine as catalyst. One-pot reactions of the cyanoacetamide derivative 3 with malononitrile and the same aldehydes (1:1:1 molar ratio) at reflux temperature in the presence of piperidine afforded the 2-pyridone derivatives 7a-c. Spectroscopic data as well as elemental analyses of the obtained products were in complete agreement with the assigned structures 7a-c. In addition, when the cyanoacetamide 3 was reacted with acetylacetone in dioxane in the presence of a catalytic amount of triethylamine, the cyclocondensation reaction occurred and the 2-pyridinone derivatives 10 were smoothly afforded. It can be postulated that the reaction initially proceeds via a nucleophilic attack to form the Michael adduct which in turn cyclized and eliminated two water molecules, affording the final product (Scheme 2). Similarly, cyclocondensation of cyanoacetamide 3 with salicyaldehyde in dioxane in the presence of a catalytic amount of piperidine afforded 2-iminochromene 11 in high yield. On the other hand, by interaction of 3 with salicyaldehyde in the presence of AcOH/AcONa, chromenone 12 was obtained in reasonably good yield. The structure of compound 12 was further confirmed through its synthesis upon hydrolysis of 11 with ethanolic HCl. (Scheme 3). The IR spectrum of the reaction product 11 revealed the disappearance of cyano absorption band and showed absorption bands at 1680, 3244 and 3318 cm −1 corresponding to carbonyl and two NH functions, respectively. Its 1 H-NMR spectrum showed two D 2 O-exchangeable signal at δ 9.29 and 13.11 due to two NH protons, in addition to an aromatic multiplet in the region 7.58-7.83. It mass spectrum showed a molecular ion peak at m/z 343 while 1 H-NMR spectrum of 12 showed one D 2 O-exchangeable signal at δ 10.88 due to one NH proton. Next, we studied the reactivity of the active methylene group present in compound 3 towards diazonium salts. Thus, cyanoacetamide 3 coupled with diazonium salts, derived from the appropriate aromatic amines (4-methylaniline, 4-methoxyaniline, aniline, 4-chloroaniline, and methyl anthranilate) in pyridine to afford the respective hydrazones 13a-e (Scheme 4). Analytical and spectral data of the latter reaction products are all consistent with the proposed structures.

13a-e
Further elucidation of the structure of 13a,b came from the reaction with hydrazine hydrate to furnish the Pyrazole products 14a,b. The structures of compounds 14a,b were confirmed based on elemental analysis and spectral data (see Experimental section).
In view of the growing biological importance of thiazole derivatives, it was considered of interest to synthesize some new derivative of thiazole. Thus, the reaction of compound 3 with phenyl isothiocyanate and elemental sulfur gave the thiazole-2-thione derivative 15. The reaction of cyanoacetamide 3 with elemental sulfur and malononitrile gave the thiophene derivative 16 (Scheme 5). Analytical and spectral data of the products are in agreement with the proposed structure (see Experimental section).   [26][27][28]. The fungicide Amphotericin B and the bactericides Ampicillin and Gentamicin were used as references to evaluate the potency of the tested compounds under the same conditions. The results are depicted in Table 1. As seen from the data present in Table 1, Streptococcus pneumoniae and Bacillis subtilis are sensitive to all tested compounds except compounds 11 and 16; furthermore, Pseudomonas aeruginosa is sensitive to compounds 5a, 7c and 15, while Escherichia coli is sensitive to 5a, 7b, 7c, 10, 13b, 14a, 15, 18, 19 and 22 except compounds 11 and 16. All tested compounds except compound 16 exhibit antifungal activity against the three tested fungi species Aspergillus fumigatus, Syncephalastrum racemosum and Geotricum candidum. Also the Candida albicans strain is sensitive to compounds 5a, 5c, 7c, 10, 11. The high activity of 5a, 7c and 10 is attributed to the presence of pharmacological active arylidene moiety in compound 5a and pyridone ring in 7c and 10. The inactivity of compound 16 against the tested bacteria and fungi is due to the presence of a thiophene ring.

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, UK and Shimadzu, Tokyo, Japan, respectively). The 1 H-NMR spectra were recorded on a Varian Mercury VXR-300 spectrometer (300 MHz, Vernon Hills, IL, USA). The mass spectra were recorded on a GCMS-Q1000-EX Shimadzu and GCMS 5988-A HP spectrometers (Kyoto, Japan), 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 Medical Mycology Laboratory of the Regional Center for Mycology and Biotechnology of Al-Azhar University, Cairo, Egypt. The starting material 3-(2-bromoacetyl)-2H-chromen-2-one was prepared as previously reported in the literature [29,30].

N-[4-(Aminosulfonyl)phenyl]-3-aryl-2-cyanoacrylamide (5a-c)
General Procedure: To a solution of cyanoacetanilide 3 (0.239 g, 1 mmol) and the appropriate aromatic aldhyeds (1 mmol) in dioxane (20 mL), was added few drops of piperidine and the reaction mixture was refluxed for 6 h. The solid product so formed was filtered off, washed with EtOH and then recrystallized from proper solvent to give 5a-c.

Synthesis of Pyridines 7a-c
Method A: A mixture of 5 (10 mmol) and malononitrile (0.66 g, 10 mmol) in ethanol (30 mL) containing piperidine (0.5 mL) was heated under reflux for 3 h. After cooling, the precipitate was filtered off, washed with ethanol and then recrystallized from the proper solvent to give 7a-c.

Coupling of N-[4-(Aminosulfonyl)phenyl]-2-cyanoacetamide (3) with the Appropriate Diazonium Salt of Aromatic Amines
General procedure: To a cold solution of cyanoacetanilide 3 (1.20 g, 5 mmol) in pyridine (20 mL), was added the appropriate diazonium salt of aromatic amine (4-methylaniline or 4-methoxyaniline or aniline or 4-chloroaniline or methyl 2-aminobenzoate) (5 mmol) [prepared according to literature procedures] [32]. The addition was carried out portion wise with stirring at 0-5 °C over a period of 30 min. After complete addition, the reaction mixture was stirred for a further 4 h then kept in an ice chest for 12 h and finally diluted with water. The precipitated solid was collected by filtration, washed with water, dried and finally recrystallized from the proper solvent to afford the corresponding coupling products 13a-e.  (22) To a stirred suspension of finely powdered potassium hydroxide (0.26 g, 5 mmole) in dry DMF (20 mL) cyanoacetamide 3 (1.20 g, 5 mmole) was added, the resulted mixture was cooled at 10 °C in an ice bath, then carbon disulfide (5 mmol) was added slowly over the course of 10 min. After addition was complete, stirring of the reaction mixture was continued for additional 2 h. Then dibromopropane (5 mmol) was added to the mixture while cooling (~15 °C) and stirring for 1 h. The mixture was then poured into crushed ice and the resulting precipitate was filtrated off, dried and crystallized from the proper solvent to

Antimicrobial Evaluation
The antibacterial and antifungal activity assays were carried out in the Medical Mycology Laboratory of the Regional Center for Mycology and Biotechnology of Al-Azhar University, Cairo, Egypt using the diffusion plate method [26][27][28] as follows: a bottomless cylinder containing a measured quantity (1 mL, 5 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.

Conclusions
In conclusion, the reactivity of N- [4-(aminosulfonyl)phenyl]-2-cyanoacetamide (3) was investigated as a versatile and readily accessible building block for the synthesis of new heterocycles incorporating a sulfamoyl moiety of biological and pharmaceutical importance.