Melting points were determined in a capillary tube using Electrothermal C14500 apparatus and are uncorrected. NMR spectra were recorded on a Bruker AC spectrometer (400 MHz-¹H and 100.0 MHz-¹³C) in DMSO-d₆. Chemical shifts in ppm are given as δ values against tetramethylsilane as the internal standard. MS analyses were performed on Thermo Fisher Scientific Trace DSQ II, Mass spectrometer at Central Research Lab, Faculty of Pharmacy, Mansoura University. Elemental Microanalyses were performed by the microanalytical centre, Cairo University. Reactions were monitored by TLC performed on silica gel plates (Merck 60 F₂₄₅) and using ethyl acetate: petroleum ether (1:1) as eluent, the spots were visualized by U.V (366, 245 nm). Compounds 5 [31Sohda, T; Mizuno, K; Imamiya, E; Sugiyama, Y; Fujita, T; Kawamasu, Y Studies on Antidiabetic Agents. II. Synthesis of 5-[4-(1-Methylcyclohexylmethoxy)-benzyl]thiazolidine-2,4-dione (ADD-3878) and its derivatives Chem. Pharm. Bull, 1982, 30, 3580-3600.], 6a-6d [32Ha, YM; Park, YJ; Kim, J; Park, D; Park, J Y; Lee, H J; Lee, J Y; Moon, H R; Chung, H Y Design and synthesis of 5-(substituted benzylidene)thiazolidine-2,4-dione derivatives as novel tyrosinase inhibitors Eur. J. Med. Chem, 2012, 49, 245-252.] were prepared according to the reported procedures.

Chloroacetyl chloride (8 mL) was added slowly over a period of 30 min to a solution of sulfanilamide (17.2 g, 100 mmol) in DMF (50 mL). Stirring was continued for 1 h. The reaction mixture was quenched with cold water (150 mL). The formed precipitate was collected by filtration, washed with cold water, dried and recrystallized from methanol to afford pure product. Colorless crystals, yield 85%, mp 218-220 °C [33Jacobs, W A; Heidelberger, M J On amides, uramino compounds, and ureides containing an aromatic nucleus J. Am. Chem. Soc, 1917, 39, 2418-2443.
[http://dx.doi.org/10.1021/ja02256a021] ].

A mixture of 1 (2.48 g, 10 mmol) and NH₄SCN (1.52 g, 20 mmol) in acetone (25 mL) was refluxed for 2 h. The formed precipitate was filtered and dried. The dried solid product was then heated with DMF (20 mL) at 120-130 °C for 1 h. the reaction mixture was cooled to rt and poured over crushed ice to afford white crystalline product, which was filtered, dried and recrystallized from ethanol to afford pure product. White crystals, yield 70%, mp 258-260 °C (reported mp 258 °C) [34Taniyama, H; Takemura, S Studies on thiazole derivatives. I. Synthesis of 4-thiazolidones Yakugaku Zasshi, 1953, 73, 164-165.].

The antimicrobial activity of the synthesized compounds has been studied using the cup-diffusion technique [35Wayne, P Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically, 5^th ed., Approved standard M7- A5. National Committee for Clinical Laboratory Standards, vol. 20, 2000.]. The tested compounds were screened against Staphylococcus aureus and Bacillus subtilis as examples for Gram-positive bacteria, Escherichia coli and Pseudomonas aeruginosa as examples for Gram-negative bacteria and Candida albicans and Candida tropicalis as example for fungi.

In this technique pores were made using a sterile cork borer in the solidified agar medium and an aliquot of 0.05 mL of 1000 μg/mL of the tested substance is placed in each pore in the nutrient agar medium on which a culture of the tested bacteria has been spread to produce uniform growth. After 24 h incubation at 37 °C, the diameter of inhibition zone is measured in mm.

Compounds that exhibited good anti-Gram-positive bacteria were assayed in vitro against Staphylococcus aureus and Bacillus subtilis. The organisms were obtained from the Microbiology Laboratory, Department of Microbiology, Faculty of Pharmacy, University of Mansoura. The antibacterial assay was carried out using NCCLS broth dilution method [36Pelczar, M J; Chan, E C S; Krieg, N R Microbiology Concepts and Applications, 6th ed; McGraw-Hill Inc: New York, 1999. , 37Collee, J G; Fraser, A G; Marmion, B P; Simmons, A Mackie and McCartney In: Practical medical microbiology, 14th ed; Churchill Livingstone: London, 1996. ]. The minimum inhibitory concentrations (MICs) were determined using 96-well microtitre plates. The bacterial suspension was adjusted with sterile saline to a concentration of 1.0x10⁵ c.f.u./mL. Compounds to be investigated were dissolved in DMSO. The microplates were incubated for 24 h at 37 °C. In order to ensure that the solvent had no effect on bacterial growth, a control test was performed containing broth supplemented with only DMSO. The lowest concentrations without visible growth were defined as concentrations that completely inhibited bacterial growth (MICs).

Ampicillin was used as a reference standard to compare the antibacterial activity. All experiments were performed in duplicate and repeated three times. The MIC values were expressed in µg/mL and the results of the most active compounds are shown in Table 1.

Table 1

Minimum inhibitory concentrations (MICs) in (µg/mL) of the most active compounds.

Data obtained from animal experiments were expressed as mean ± standard error (S.E.M.). The statistical significance of difference between groups were determined by means of analysis of variance (ANOVA) followed by Dunnet’s test. The results of the anti-inflammatory studies have been presented in Table 2.

Table 2

Results of anti-inflammatory Screening.

The synthetic methods used for preparation of target compounds are depicted in Schemes 1-5. Compound 1, the precursor of the starting compound was prepared by chloroacetylation of sulfanilamide using chloroacetyl chloride in DMF as a solvent. The starting compound, 3 was prepared through a two-steps reaction. The first step is a nucleophilic substitution of NH₄SCN with 1 using acetone as a solvent to afford the intermediate N-(4-sulfamoylphenyl)-2-thiocyanatoacetamide 2, followed by thermal cyclization using DMF as a polar aprotic solvent. The postulated mechanism for heterocyclic cyclization is illustrated (Scheme 1). The synthesis of compound 3 was reported earlier to proceed in a one-step reaction using ethanol or acetone as solvents, but it was observed that the purity of the product was better when using the two-steps synthesis (Scheme 2).

Scheme 1 Postulated mechanism for formation of compound 3.

Scheme 2 Synthesis of compound 3. Reagents and conditions: i) DMF, stirr, rt, 1 h; ii) NH4SCN, acetone, reflux, 2 h; iii) DMF, 120-130 °C, 1 h.

Scheme 3 Synthesis of target compounds 4a-4d. Reagents and conditions: i) method 1: ArCHO, xylene, pyridine, reflux, 6 h; method 2: ArCHO, AcOH, AcONa, reflux, 3 h.

Scheme 4 Unsuccessful trials for synthesis of compound 4. Reagents and conditions: i) method 1: acetophenone, xylene, pyridine, reflux, NR; method 2: acetophenone, AcOH, AcONa, NR.

Scheme 5 Synthesis of target compounds 7a-7d. Reagents and conditions: i) AcOH, AcONa, reflux, 12 h; ii) 1, K2CO3, DMF, rt.

Synthesis of target compounds, 4a-4d was achieved through Knoevenagel condensation of 3 with the different aromatic aldehydes. The reaction was carried out in two different solvents; in xylene using catalytic amount of pyridine (method 1) and in glacial acetic acid using sodium acetate as catalyst (method 2). Concerning the yield of the two methods, no significant difference was observed, however the purity of the products using method 2 was found to be better (Scheme 3).

Trial for obtaining Knoevenagel condensation product 4, using acetophenone as a ketone, was found unsuccessful, using both methods at different reflux times (Scheme 4).

The benzylidenethiazolidine-2,4-diones (6a-6d) were prepared by condensing thiazolidine-2,4-dione (5) [31Sohda, T; Mizuno, K; Imamiya, E; Sugiyama, Y; Fujita, T; Kawamasu, Y Studies on Antidiabetic Agents. II. Synthesis of 5-[4-(1-Methylcyclohexylmethoxy)-benzyl]thiazolidine-2,4-dione (ADD-3878) and its derivatives Chem. Pharm. Bull, 1982, 30, 3580-3600.] with different aromatic aldehydes in glacial acetic acid and using catalytic amount of sodium acetate. Alkylation of 6a-6d with 1 in DMF in presence of K₂CO₃ at rt afforded the target thiazolidin-2,4-dione derivatives 7a-7d in satisfied yields (Scheme 5).

Target compounds 4a-4d and 7a-7d can potentially exist in either E or Z geometrical isomeric forms at the 5-exocyclic double bond. The Z configuration was postulated due to the deshielded methine proton signals by adjacent carbonyl group for both 4a-4d (δ 7.83-8.18) and 7a-7d (δ 7.90-8.02) in the ¹H NMR spectra. These higher chemical shifts were in coordination with the Z configuration in comparison with the E configuration which expected to have lower chemical shifts [39Bruno, G; Costantino, L; Curinga, C; Maccari, R; Monforte, F; Nicolo`, F; Ottana`, R; Vigorita, M Synthesis and aldose reductase inhibitory activity of 5-arylidene-2,4-thiazolidinediones Bioorg. Med. Chem, 2002, 10, 1077-1084., 40Vicini, P; Geronikaki, A; Anastasia, K; Incertia, M; Zania, F Synthesis and antimicrobial activity of novel 2-thiazolylimino-5-arylidene-4-thiazolidinones Bioorg. Med. Chem, 2006, 14, 3859-3864.
[http://dx.doi.org/10.1016/j.bmc.2006.01.043] ]. All the newly synthesized compounds were characterized by physical analytical and spectral data and found to be in agreement with predicted values.

The antibacterial activity of the synthesized compounds was determined by using the cup-diffusion technique [35Wayne, P Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically, 5^th ed., Approved standard M7- A5. National Committee for Clinical Laboratory Standards, vol. 20, 2000.] against Staphylococcus aureus and Bacillus subtilis as examples for Gram-positive bacteria, Escherichia coli and Pseudomonas aeruginosa as examples for Gram-negative bacteria and Candida albicans and Candida tropicalis as example for fungi. The antibacterial assay was carried out using NCCLS (National Committee for Clinical Laboratory Standards) broth dilution method. Compounds that exhibited significant inhibition zones were selected for determination of the minimum inhibitory concentrations (MICs). The minimum inhibitory concentrations (MICs) were determined using 96-well microtitre plates [36Pelczar, M J; Chan, E C S; Krieg, N R Microbiology Concepts and Applications, 6th ed; McGraw-Hill Inc: New York, 1999. , 37Collee, J G; Fraser, A G; Marmion, B P; Simmons, A Mackie and McCartney In: Practical medical microbiology, 14th ed; Churchill Livingstone: London, 1996. ].

The results were compared with Ampicillin and Gentamicin for bacteria and Clotrimazole for fungi. The data showed no antifungal activity towards Candida albicans and Candida tropicalis. Whereas, the synthesized compounds exhibited activity against Bacillus subtilis (7a=Ampicillin > 4a=7d) more than Staphylococcus aureus (Ampicillin > 7d > 7a > 4a) as Gram-positive bacteria and weak antibacterial activity was observed against Gram-negative bacteria.

The anti-inflammatory activity of the synthesized compounds was carried out using carrageenan-induced rat paw edema model [38Winter, C A; Risley, E A; Nuss, G W Carrageenin-induced edema in hind paw of the rat as an assay for antiiflammatory drugs Proc. Soc. Exp. Biol. Med, 1962, 111, 544-547.
[http://dx.doi.org/10.3181/00379727-111-27849] ]. The results revealed that compounds of thiazolidin-2,4-dione series (7a-7d) exhibited greater anti-inflammatory activity than the 2-iminothiazolidin-4-one series (4a-4d). The best anti-inflammatory activity was observed with compound 7b. The anti-inflammatory activities of other thiazolidin-2,4-dione derivatives were found in the following order (7c>7a>7d). For compounds of iminothiazolidin-4-one series (4a-4d), their anti-inflammatory activities were found to be relatively low and were in the following order (4a>4b>4c>4d).