Tetrachlorodibenzodioxin

Example 1

Each of the peptides having amino acid sequences of SEQ ID NOS: 1, 2, and 3 mixed with a coating buffer (20 mM sodium phosphate, pH 9.6) at a concentration of 1.8 mM was seeded on a plate for an enzyme-linked immunosorbent assay (ELISA) and cultured at 4° C. overnight. Subsequently, the peptide was washed with phosphate buffered saline with Tween-20 (PBST) and blocked with 3% of bovine serum albumin (BSA) for 2 hours at room temperature. After washing with PBST, 2 μM of 2,3,7,8-tetrachlorodibenzo-p-dioxin (hereinafter, referred to as TCDD) was added to each well and cultured at room temperature for 2 hours. Subsequently, after washing with PBST, treatment with anti-TCDD antibody conjugated with fluorescein isothiocyanate (FITC) was conducted at a ratio of antibody:PBST=1:100 and the resultant was cultured for 2 hours at room temperature. Then, after washing with PBST, an excitation 488 nm/emission 520 nm value was measured using a fluorescence meter, and the results are shown in FIGS. 1A to 1C, and Table 2.

As shown in FIGS. 1A to 1C and Table 2, it was confirmed that the peptide consisting of an amino acid sequence of SEQ ID NO: 1, 2, or 3 directly binds to TCDD.

Example 2

HaCaT cells, human keratinocyte cells, were seeded on a 6-well plate at a density of 3×10⁵ cells/well and cultured overnight. Subsequently, 10 nM of TCDD and 50 μM of the peptide consisting of an amino acid sequence of SEQ ID NO: 1, 2, or 3 were added to the culture medium. After 30 minutes of reaction, the cells were treated for 1 hour and collected to obtain nuclei and cytoplasmic proteins separated from each other. Westin blotting was performed using an aryl hydrocarbon receptor (AhR) antibody (Santa Cruz Biotechnology, U.S.A.) to identify activated nuclear translocation of AhR, and the results are shown in FIGS. 2A to 2F and Table 3.

As shown in FIGS. 2A to 2F and Table 3, it was confirmed that the peptide consisting of an amino acid sequence of SEQ ID NO: 1, 2, or 3 inhibits nuclear translocation of AhR by TCDD.

Example 174

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Step 1.

tert-Butyl 6-(8-methoxy-7-quinolyl)spiro[4H-1,3-benzodioxine-2,4′-piperidine]-1′-carboxylate. tert-Butyl 6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)spiro[4H-1,3-benzo-dioxine-2,4′-piperidine]-1′-carboxylate (0.80 g, 1.9 mmol), 7-bromo-8-methoxy-quinoline (0.45 g, 1.9 mmol), palladium(II) acetate (0.024 g, 0.11 mmol) and triphenylphosphine (0.10 g, 0.38 mmol) in 1,4-dioxane (30 mL), DMF (50 mL) was added aq. Na₂CO₃ (0.5 M) (6.0 mL, 3.0 mmol). The mixture was vacuum degassed then heated at 85° C. overnight. The mixture was diluted with EtOAc (200 mL) and water (100 mL) and extracted. The aqueous extract was washed with EtOAc (50 mL) and the combined organics were dried over Na₂SO₄, filtered and concentrated. The residue was dissolved in DCM, applied to a silica gel loading cartridge (5 g) and purified on silica gel (80 g, 0-40% EtOAc:hexanes) to afford tert-butyl 6-(8-methoxy-7-quinolyl)spiro[4H-1,3-benzodioxine-2,4′-piperidine]-1′-carboxylate (0.38 g, 0.82 mmol, 43% Yield). LCMS m/z=463.

Step 2.

6-(8-Methoxy-7-quinolyl)spiro[4H-1,3-benzodioxine-2,4′-piperidine]. A mixture of tert-butyl 6-(8-methoxy-7-quinolyl)spiro[4H-1,3-benzodioxine-2,4′-piperidine]-1′-carboxylate (0.38 g, 0.82 mmol) and TFA (0.5 mL, 7 mmol) in DCM (10 mL) was stirred at RT for 24 h, then was diluted with DCM (20 mL) and NaOH (1M, 24 mL). The layers were separated and the aqueous phase was further extracted with DCM (2×20 mL). The combined organics were filtered through a phase separator, then dried over Na₂SO₄, filtered, and concentrated in vacuo to give a white foam. A small amount (50 mg) was purified by preparative HPLC to afford 6-(8-methoxy-7-quinolyl)spiro[4H-1,3-benzodioxine-2,4′-piperidine] TFA salt (20 mg). Analysis: LCMS m/z=363; ¹H NMR (400 MHz, DMSO-d₆) δ: 9.02 (dd, J=4.4, 1.6 Hz, 1H), 8.71 (br s, 2H), 8.57 (d, J=7.5 Hz, 1H), 7.87 (d, J=8.5 Hz, 1H), 7.71-7.64 (m, 2H), 7.54 (dd, J=8.5, 2.3 Hz, 1H), 7.43 (d, J=2.0 Hz, 1H), 7.03 (d, J=8.5 Hz, 1H), 5.00 (s, 2H), 3.88 (s, 3H), 3.29-3.16 (m, 4H), 2.19-2.06 (m, 4H). The remainder was used in the next step without further purification.

Step 3.

6-(8-Methoxy-7-quinolyl)spiro[4H-1,3-benzodioxine-2,4′-piperidine]-1′-carboxamide. A mixture of 6-(8-methoxy-7-quinolyl)spiro[4H-1,3-benzodioxine-2,4′-piperidine] (0.198 g, 0.546 mmol), trimethylsilyl isocyanate (0.30 mL, 1.9 mmol), DIPEA (0.50 mL, 2.9 mmol), and DCM (10.0 mL) was stirred overnight. The solution was concentrated and the resulting material was diluted with DCM an put on a 5 g preload silica gel. The material was purified on silica gel chromatography (24 g, 0-10% EtOAc:hexanes) to afford 6-(8-methoxy-7-quinolyl)spiro[4H-1,3-benzodioxine-2,4′-piperidine]-1′-carboxamide (0.183 g, 0.451 mmol, 83%) as an off-white solid. Analysis: LCMS m/z=406 (M+1); ¹H NMR (400 MHz, DMSO-d₆) δ 8.95 (dd, J=4.0, 1.8 Hz, 1H), 8.38 (dd, J=8.3, 1.8 Hz, 1H), 7.77 (d, J=8.5 Hz, 1H), 7.61-7.47 (m, 3H), 7.36 (d, J=2.3 Hz, 1H), 6.97 (d, J=8.5 Hz, 1H), 6.04 (s, 2H), 4.95 (s, 2H), 3.94 (s, 3H), 3.52-3.37 (m, 4H), 1.91-1.77 (m, 3H), 1.88-1.77 (m, 1H).