Synthesis and Biological Evaluation of New 2-Azetidinones with Sulfonamide Structures

New series of N-(arylidene)hydrazinoacetyl sulfonamides 4a1–6, 4b1–6 and N-(4-aryl-3-chloro-2-oxoazetidin-1-yl)aminoacetyl sulfonamides 5a1–6, 5b1–6 were synthesized. The structures of the new derivatives was confirmed using spectral methods (FT-IR, 1H-NMR, 13C-NMR). The antibacterial activities of these compounds against Gram positive (Staphyloccoccus aureus ATCC 6583, Staphyloccoccus epidermidis ATCC 12228, Enterococcus faecalis ATCC 25912) and Gram negative (Klebsiella pneumoniae CIP 53153, Proteus vulgaris CIP 104989, Citrobacter freundii CIP 5732, Enterobacter cloacae CIP 103475, Escherichia coli ATCC 25922, Pseudomonas aeruginosa CIP 82118) bacterial strains were evaluated using the broth micro-dilution method. Compound 4a2 displayed the highest antibacterial activity, especially against Staphyloccoccus epidermidis, Enterococcus faecalis and Pseudomonas aeruginosa. The antioxidant potential of the synthesized compounds was also investigated according to ferric reducing power, total antioxidant activity and DPPH radical scavenging assays. All tested compounds showed excellent antioxidant activity in comparison with sulfadiazine and sulfisoxazole which were used as parent sulfonamides. Moreover, some of them showed an antioxidant activity comparable with that of ascorbic acid. In general, the compounds designed based on a sulfadiazine skeleton (compounds 4a1–6, 5a1–6) are more active than those obtained from sulfisoxazole (compounds 4b1–6, 5b1–6), and the N-(arylidene)hydrazinoacetyl sulfonamide derivatives 4a1–6, 4b1–6 are more active than their azetidionone analogues 5a1–6, 5b1–6.


Introduction
The 2-azetidinone skeleton, otherwise known as the β-lactam ring, has been recognized as a useful building block in the synthesis of biologically important compounds. Azetindin-2-one derivatives display interesting biological activities such as antifungal, antimicrobial [1][2][3][4], antitubercular [5,6], analgesic, anti-inflammatory [7,8], chymase inhibitory [9], antitumoral [10][11][12], antiviral, antidiabetic and cholesterol absorption inhibitory properties [13]. The activity of famous antibiotic classes such as the penicillins, cephalosporins, carumonam, aztreonam, thienamicine, nocardicins and carbapenems is attributed to the presence of an 2-azetidinone ring [2]. Unfortunately, the most widely used of them exert selective pressure on bacteria and permit the proliferation of resistant organisms. Several synthetic and semi-synthetic β-lactam antibiotics were developed due to the growing resistance of bacteria towards the classical β-lactam antibiotics and the need for drugs with a more specific antibacterial activity [1]. The biological activity of sulfonamides is also well documented. They have be found to be useful in a variety of applications, including antibacterial, antifungal, antitumor agents, diuretics, carbonic anhydrase inhibitors, hypoglycemic agents, thyroid inhibitors, anticonvulsants and protease inhibitors [14,15]. Among antibacterial sulfonamides, sulfadiazine and its silver and cerium salts have an important place. They are widely used as topical agents for the management of burns where they prevent infections and promote rapid healing with minimal scarring [15].
Wounds are physical injuries that result in an opening of the skin. Proper healing of wounds is essential for the restoration of disrupted anatomical continuity and disturbed functional status of the skin [16]. Normal healing of wounds is a dynamic process following three phases: inflammation, granulation (tissue formation) and re-epithelization (tissue remodeling), which overlap in time [17]. It was proven that reactive oxygen species (ROS) and bacterial infections are deleterious to the wound healing process due to their harmful effects on cells and tissues [18]. ROS are produced in high amounts at wound sites as a defense mechanism against invading bacteria. At the same time, the process of wound healing may be hampered by the presence of free radicals, which can damage the cells surrounding the wound, or by microbial infection [19] and recent data has proved the beneficial effects of antioxidants in the wound healing process [20][21][22]. In the present study, we are reporting the design, synthesis and biological evaluation of some new 2-azetidinone derivatives of sulfadiazine and sulfisoxazole with potential use in wound healing processes.

Ferric Reducing Power
The measurement of reducing power defines an important aspect of the antioxidant activity of the compounds. In this assay, the presence of a reducing agent in the sample results in reducing of the ferric/ferricyanide complex to its ferrous (Fe 2+ ) form. The amount of Fe 2+ is then quantitatively monitored by measuring the intensity of Perl's Prussian blue colour complex at 695 nm [25]. The results expressed as EC 50 values (mg/mL) are presented in Tables 3 and 4. The small value of the EC 50 indicates a higher ferric reducing power. Table 3. Ferric reducing power (EC 50 mg/mL) of the sulfadiazine derivatives 4a 1-6 , 5a 1-6 .
The chemical modulation of the parent sulfonamides improve their ferric reducing power and all tested compounds are more active than sulfadiazine and sulfisoxazole, respectively, but they are less active than ascorbic acid (AA) at the same concentration.

Total Antioxidant Activity
The total antioxidant activity was determined using phophomolybdenum blue complex with a maximum absorption at 695 nm [26]. The data presented in Tables 5 and 6 show that the tested compounds are more active than sulfadiazine and sulfisoxazole, respectively, and moreover, the sulfadiazine derivatives are more active than sulfisoxazole compounds. Table 5. Total antioxidant activity (EC 50 mg/mL) of the sulfadiazine derivatives 4a 1-6 , 5a 1-6 .

General Procedures
Melting points were measured using a Buchi Melting Point B-540 apparatus and are uncorrected. The FT-IR spectra were recorded on an ABB Bomen MB3000 spectrometer, over a 500-4000 cm −1 range, after 32 scans at a resolution of 4 cm −1 . The spectra processing was carried out with Horizon MB TM FTIR Software. The 1 H-NMR (300 MHz) and 13 C-NMR (75 MHz) spectra were obtained on a Bruker Avance ARX-300 spectrometer using tetramethylsilane as internal standard and DMSO-d 6 as solvent. The chemical shifts are shown in  values (ppm). The progress of the reaction was monitored on TLC, using pre-coated Kieselgel 60 F254 plates (Merck) and the compounds were visualized by UV light. Sulfonamides 4a 1-6 ; 4b 1-6 To a solution of hydrazinoacetyl sulfonamide derivatives (10 mmol) in ethanol 50% (200 mL), glacial acetic acid (0.5 mL) and the appropriate aldehyde (10 mmol) were added. The mixture was heated under reflux for 8 h, and then it was cooled at room temperature. The solid was filtered off, dried and recrystallized from isopropyl alcohol.             5a 1-6 ; 5b 1-6 To a solution of N-(arylidene)hydrazinoacetyl sulfonamides 4a 1-6 ; 4b 1-6 (2 mmol) in anhydrous 1,4-dioxane (50 mL), chloracetyl chloride (3 mmol) and triethylamine (2 mmol) were added dropwise at 0-5 °C. The mixture of reaction was stirred at room temperature for 3 h and the solid (triethylamine hydrochloride) was removed. The solution was heated under reflux for 5 h and then the solvent was evaporated under reduced pressure. The solid product was washed with water (20 mL), filtered off, dried and recrystallized from absolute ethanol. The progress of the reaction was monitored by silica gel coated TLC plates.

Antibacterial Assay
In order to evaluate the antibacterial activity of the synthesized compounds 4a 1-6 , 4b 1-6 , 5a 1-6 and 5b 1-6 , a panel of three Gram positive (Staphyloccoccus aureus ATCC 6583, Staphyloccoccus epidermidis ATCC 12228, Enterococcus faecalis ATCC 25912) and six Gram negative (Klebsiella pneumoniae CIP 53153, Proteus vulgaris CIP 104989, Citrobacter freundii CIP 5732, Enterobacter cloacae CIP 103475, Escherichia coli ATCC 25922, Pseudomonas aeruginosa CIP 82118) bacterial strains were used. Minimum inhibitory concentrations (MICs) were assessed according to the guidelines of EUCAST Def. 3.1 [24]. Briefly, stock solutions were prepared by solving the substances mentioned above (200 mg) in dimethyl sulfoxide (DMSO, 19.5 mL). Using these solutions, series of two-fold dilutions were subsequently obtained. In a 9 cm diameter Petri dish, one milliliter of each dilution was mixed thoroughly with Mueller-Hinton agar (19 mL), sterilized by autoclaving and cooled to 50 °C. After this, the concentrations of the substances inside the medium were 512, 256, 128, 64, 32, 16, 8, 4, 2, and 1 μg/mL respectively. A blank plate (control of growth) was also prepared by mixing DMSO (1 mL) with molten agar (19 mL). From each bacterial strain, a 0.5 McFarland suspension was prepared in 0.85% saline solution and after that, the inoculum was standardized in order to assure 10 4 colony-forming units (CFU) per spot (5 μL). All inoculated plates were incubated for 18 h at 36 °C. The MIC was interpreted as the lowest concentration of the substance that completely inhibits the growth of bacteria in the spot area. Each determination was performed in triplicate in order to accurately confirm the MIC values.

Statistical Analysis
All assays (antimicrobial and antioxidant) were carried out in triplicate. Data were analysed by an analysis of variance (ANOVA) (p < 0.05) and were expressed as means ± SD. The total antioxidant antivity (EC 50 values) were calculated by linear interpolation between values above and below 50% activity.

Conclusions
In this study new N-(arylidene)hydrazinoacetyl and new 2-azetidionone derivatives have been designed and synthesized starting from sulfadiazine and sulfizoxazole. The structures of all new compounds were proved using spectral methods. The compounds were evaluated for their antimicrobial and antioxidant activity. Although their antimicrobial potential was reduced, they shown excellent antioxidant properties; for some of them the potential is comparable with the antioxidant activity of ascorbic acid. These results support the antioxidant potential of the synthesized compounds and their applications in several disease mediated by reactive oxygen species (ROS) including the healing of the wounds.