Sulfonyl chloride

Antioxidant capacity was measured by the ABTS (2,2-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid) assay previously optimised for cereal samples [9 ] with results expressed as ascorbic acid (AA) equivalents/L. Anti-hypertensive capacity was evaluated by the angiotensin converting enzyme (ACE) inhibitory test [10 (link)] with minor modifications. ACE enzymatic activity was assayed by monitoring the amount of hippuric acid derived from the hydrolysis of the substrate hippuryl-histidyl-leucine in the presence of the sample. Pyridine and benzene sulfonyl chloride (40% and 20%, respectively, on the final assay volume) were added and the absorbance of the developed yellow coloured solution was measured at 410 nm. The percent inhibition curves were plotted using a minimum of five increasing concentrations for each sample and the IC50 value was calculated. Anti-tyrosinase activity was assessed by an optimised tyrosinase inhibition assay [11 (link)]. The kinetic of brown colour formation was evaluated (490 nm absorbance measurement) in a reaction containing 10 U of tyrosinase and 2 mM L-DOPA in the presence of the sample. The results were expressed as kojic acid (KA, a well-known tyrosinase inhibitor) equivalents/L by means of a dose-response calibration curve (between 1 and 10 μg of KA). A bioluminescent cell-based assay for anti-inflammatory activity was performed using human embryonic kidney HEK293 cells (ATCC, American Type Culture Collection, Manassas, VA, USA) routinely grown in Dulbecco Modified Essential Medium (DMEM high glucose 4.5 g/L, GE Healthcare, Milan, Italy), supplemented with 10% (v/v) fetal bovine serum, 2 mM L-glutamine, 50 U/μL penicillin, and 50 μg/mL streptomycin. The day before transfection, HEK293T cells were plated in a 24-well plate at a density of 8 x 10⁴ per well. Cells were co-transfected with plasmid pGL4.32[luc2P/NF-κB-RE/Hygro] containing five copies of the NF-κB response element (NF-κB-RE) driving transcription of the luc2P reporter gene (Promega, Madison, WI, USA), and plasmid pmcherryPRET9 expressing the fluorescent protein mcherry-C1 (Clontech, Mountain View, CA, USA) and a red thermostable P. pyralis luciferase mutant [12 (link)], obtained by standard molecular biology procedures. Co-transfections were performed by using FuGENE®HD according to the manufacturer's instructions and incubated at 37°C with 5% CO₂ for 24 h. Forty-eight hours post-transfection, cells were co-incubated for 20 hours with 500 μL of fresh medium containing sample (1:20 dilution) and 20 ng/mL TNFα. After incubation at 37°C, cells were detached with trypsin-EDTA 1X in PBS, resuspended in 100 μL PBS 0.1 M pH 7.5 and then transferred to black 96-well microplates. Fluorescence (FL) and bioluminescence (BL) measurements were performed with a Varioskan™ Flash Multimode Reader. The FL signal was obtained exciting samples at 570 nm and acquiring the signal at 610 nm, while the BL signal was acquired with band pass green and red filters after injection of the substrate BrightGlo [13 ]. Statistical analysis was performed by using one-way Anova with p < 0.05 accepted as significant. Basal activation of NF-κB or activation with 20 ng/mL TNFα were used to calculate fold of induction of treated cells vs control cells. Cytotoxicity and irritation tests were performed on Sterlab Reconstructed Human Epidermis (RHE). For cytotoxicity evaluation, human tissues were placed in a 24-well plate with medium and exposed topically to pure samples for 24 hours at 37°C. After washing with PBS, a cell viability test was performed. For irritation evaluation, human tissues were placed in a 24-well plate with medium and each tested substance was topically applied for 42 min at room temperature. Exposure to the substance was followed by rinsing with PBS and mechanical drying. RHEs were transferred to fresh medium and incubated at 37°C for 42 additional hours. Then a cell viability test was performed. For each test the cell viability was assessed by incubating the tissues for 3 hours with 0.3 mL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) solution (0.5 mg/mL). Formazan crystals were extracted for 2 hours at room temperature using 1.5 mL isopropanol and quantified by spectrometry at 550 nm absorbance. SDS 0.1% (w/v) and PBS were used as positive and negative controls, respectively. For each treated tissue, the cell viability was expressed as percentage of the mean negative control tissues. A cell viability above 50% indicated the not toxicity or the not irritancy potential of the tested substance.

Materials were of analytical grade; Sodium alginate was purchased from Fisher scientific Co. (UK), viscosity 1%at 25 ^oC: 5–40 cps. p-Toluene sulfonyl chloride, anhydrous lithium chloride (LiCl), N, n-dimethyl acetamide (DMA), triethyl amine (TEA), and carrageenan from Sigma-Aldrich Co. The following products: ethanol (99%), calcium chloride (CaCl₂), sodium hydroxide (NaOH), and hydrochloric acid (HCl) from ELNASER Co. (Egypt).

For all target compounds, the final synthetic procedure was an esterification between the substituted benzene sulfonyl chloride and A-1, B-3 or C-4 respectively. And the reaction condition was completely same. Here, compound A series was taken for example to illustrate the reaction procedure. Intermediate A-1 (1.7 mmol) and triethylamine (5.1 mmol) were added and dissolved in 20 mL of dichloromethane in a clean 50 mL three-necked flask at room temperature. To the flask, different substituted benzene sulfonyl chloride (2.1 mmol) was added accordingly. The mixture was stirred overnight till the reaction was completed (monitored by TLC). The reaction was quenched with 20 mL water. Then ethyl acetate was added to extract and concentrate to get the crude solid, which was purified by the column chromatography (dichloromethane: methanol = 50:1, v/v) and obtained the title compounds A₁-A₁₈ with a yield in the range of 70-96%. For title compound B₁-B_20, the yield ranged 74-90% and C₁-C₂₀ 68-95%.