0six g, 0.13 mmol) and was obtained as a white solid (0.03 g, 0.11 mmol, 81 %). 1 H NMR (500 MHz, [D6]DMSO): ?= (s, 1H), 9.63 (br s, 1H), 9.27 (br s, 1H), 8.42 (s, 1H), 8.04 (d, J=8.0 Hz, 1H), 7.65 (d, J=7.7 Hz, 1H), 7.46 (t, J=7.9 Hz, 1H), 7.41 (s, 1H), 7.38 (dd, J=8.2, 1.9 Hz, 1H), 6.83 (d, J=8.2 Hz, 1H), 4.32 (q, J=7.1 Hz, 2H), 1.33 (t, J=7.1 Hz, 3H); 13 C NMR (126 MHz, [D6]DMSO): ?=, , , , , , , , , , , , , , , ; HRMS (ESI + ) [M+H] + CsixteenH165 calculated Da, found: m/z.
Ethyl 3-(3,5-dihydroxy-benzamido)benzoate (6): Compound 6 was synthesized according to the general procedure using 23 (0.09 g, 0.18 mmol) and was obtained as a grey solid (0.05 g, 0.15 mmol, 86 %). 1 H NMR (500 MHz, [D6]DMSO): ?= (s, 1H), 9.57 (s, 2H), 8.44 (t, J=1.9 Hz, 1H), 8.04 (ddd, J=8.2, 2.2, 1.0 Hz, 1H), 7.67 (dt, J=7.7, 1.3 Hz, 1H), 7.47 (t, J=8.0 Hz, 1H), 6.80 (d, J=2.2 Hz, 2H), 6.43 (t, J=2.2 Hz, 1H), 4.33 (q, J=7.1 Hz, 2H), 1.33 (t, J=7.1 Hz, 3H). 13 C NMR (126 MHz, [D6]DMSO): ?=, , , , , , , , , , , , , ; Anal. calcd for C16H155 (301.3): C, ; H, 5.02; N, 4.65; found: C, ; H, 5.02; N, 4.66 %; HRMS (ESI + ) [M+H] + C16H165 calculated Da, found: m/z.
Zero
Preparation of 1-(2,4-dihydroxyphenyl)-2-(3,4-dimethoxyphenyl)ethan-1-one (5): To a solution of 2-(3,4-dimethoxyphenyl)acetic acid (1.57 g, 8.00 mmol, 1 equiv) in boron trifluoride diethyl etherate ( mL, mmol, 7 equiv) at 0 °C resorcinol (1.32 g, 12 mmol, 1.5 equiv) was added. The mixture was heated for 5 h at 110 °C and then cooled to 0 °C. Cold water (200 mL) was added, the resulting precipitate collected by filtration, washed with water and recrystallized from EtOH to afford 5 as a white solid (0.44 g, 1.52 mmol, 19 %). 1 H NMR (400 MHz, [D6]DMSO): ?= (s, 1H), (s, 1H), 7.95 (d, J=8.9 Hz, 1H), 6.90 (d, J=1.9 Hz, 1H), 6.87 (d, J=8.2 Hz, 1H), 6.79 (dd, J=8.2, 2.0 Hz, 1H), 6.39 (dd, J=8.8, 2.4 Hz, 1H), 6.25 (d, J=2.4 Hz, 1H), 4.19 (s, 2H), 3.72 (s, 3H), 3.71 (s, 3H); 13 C NMR (101 MHz, [D6]DMSO): ?=, , , , , , , , , , , , , , , ; HRMS (ESI + ) [M+H] + C16H17O5 calculated Da, found: m/z.
Physiological validation
Cell culture: Human embryonic kidney (HEK)-Blue hTLR2 cells (InvivoGen, Touluse, France) were used from passage 5 to 20. Stimulation of the cells with TLR2 agonists leads to production and secretion of NF-?B-inducible embryonic alkaline phosphatase (SEAP). The enzyme transforms the substrate QUANTI-Blue (InvivoGen) into a blue dye that was detected by optical density (OD) at 640 nm as previously described. 11a, 12 THP-1 cells (DSMZ, Braunschweig, Germany) from passage 10 to 15 were cultured as previously reported. 13 For the generation of THP-1 macrophages, THP-1 monocytes were first incubated with 25 ng/mL Phorbol 12-myristate 13-acetate (PMA, Sigma-Aldrich) for 48 h and afterwards rested for 24 h. The cell lines were cultured at 37 °C in a humidified atmosphere of 5 % CO2 and 95 % air and were regularly tested negative for mycoplasma contamination (VenorGeM Classic Mycoplasma PCR detection kit, Minerva Biolabs, Berlin, Germany).
TLR2 ligands: The synthetic lipopeptides Pam3CSK4 and Pam2CSK4, lipopolysaccharide from Escherichia coli O111:B4 (LPS-EB), flagellin from Salmonella typhimurium (FLA-ST), the thiazoloquinoline compound CL075 and class B CpG oligonucleotide ODN2006 were purchased from Invivogen and the TLR2 antagonist CU-CPT22 was obtained from Sigma-Aldrich.
Cell pleasure: HEK-Bluish hTLR2 structure (4?10 4 structure/well) and you may THP-step one macrophages (2?10 5 structure/well) was in fact cultured into the 96-really dishes and you will 24-really dishes (TPP, Trasadingen, Switzerland). Immediately following structure have been washed having phosphate-buffered saline (PBS, Sigma-Aldrich), cell stimulation is done in OptiMEM (ThermoFisher Medical). The newest tested ingredients and you may CU-CPT22 (fifty mM) had been demolished when you look at the DMSO. Latest DMSO levels during the telephone culture was indeed below 0.dos % (v/v). Vehicle controls displayed no significant difference so you can nonstimulated control (research perhaps not revealed). For co-pleasure out-of TLR2 agonists and you can tested modulators, muscle was basically preincubated towards the taimi ne demek substances or CU-CPT22 for just one h and you can afterwards stimulated towards the agonists.
