Synthesis and Bioevaluation of 4,5,6,7-Tetrahydrobenzo[d]isoxazole Derivatives as Melanogenesis Inhibitors

Experimental

General Procedure for Synthesis of 3a-b. To a solution of 4,5,6,7-tetrahydrobenzo[d] isoxazole-3-carboxylic acids 2a-b (1.0 equiv.), oxalyl chloride (3.0 equiv.) in dichloromethane, was added DMF (0.01 equiv.). The mixture was stirred for 2 h at room temperature. The reaction mixture was concentrated in rotary evaporator, dried in vacuo. The crude product was used next step without further purification

General Procedure for Synthesis of 4a-j. To a solution of acid chloride 3a-b (1.0 equiv.), triethylamine (3.0 equiv.) in dry THF, was added arylpiperazines (1.2 equiv.) in dry THF dropwise. The mixture was stirred for 1 h at room temperature. The reaction mixture was diluted with EtOAc, washed with water and brine, dried over anhydrous MgSO4, and concentrated in vacuo. The crude product was purified by flash column chromatography on silica gel (EtOAC: hexanes = 1:1-1:5) to yield the desired products (4a-j).

Compound 4a : 1H NMR (300 MHz, CDCl3 ) δ 6.82 (m, 4H), 3.90 (m, 4H), 3.11 (s, 4H), 3.02 (m, 2H), 2.59 (m, 1H), 2.35 (m, 1H), 2.11 (m, 1H), 1.34 (m, 2H), 0.95 (s, 9H).

Compound 4b : 1H NMR (600 MHz, CDCl3 ) δ 6.90-6.93 (m, 2H), 6.84-6.87 (m, 2H), 3.87-4.01 (m, 4H), 3.77 (s, 3H), 3.10-3.18 (m, 4H), 2.96-3.06 (m, 2H), 2.53-2.61 (m, 1H), 2.30-2.37 (m, 1H), 2.00-2.13 (m, 1H), 1.28-1.42 (m, 2H), 0.96 (s, 9H).

Compound 4c : 1H NMR (600 MHz, CDCl 3 ) δ 7.01-7.06 (m, 1H), 6.87-6.94 (m, 3H), 3.91-4.02 (m, 4H), 3.89 (s, 3H), 3.09-3.17 (m, 4H), 2.96-3.06 (m, 2H), 2.53-2.61 (m, 1H), 2.31-2.38 (m, 1H), 2.06-2.13 (m, 1H), 1.27-1.42 (m, 2H), 0.96 (s, 9H).

Compound 4d : 1H NMR (300 MHz, CDCl 3 ) δ 7.52 (d, J = 8.6 Hz, 2H), 6.95 (d, J = 8.6 Hz, 2H), 3.92 (m, 4H), 3.37 (s, 4H), 3.03 (m, 2H), 2.56 (m, 1H), 2.35 (m, 1H), 2.13 (m, 1H), 1.34 (m, 2H), 0.96 (s, 9H).

Compound 4e : 1H NMR (300 MHz, CDCl 3 ) δ 6.94 (m, 4H), 3.93 (m, 4H), 3.17 (s, 4H), 3.02 (m, 2H), 2.59 (m, 1H), 2.35 (m, 1H), 2.11 (m, 1H), 1.37 (m, 2H), 0.97 (s, 9H).

Compound 4f : 1H NMR (600 MHz, CDCl 3 ) δ 6.80-6.84 (m, 2H), 6.73-6.76 (m, 2H), 3.82-3.92 (m, 4H), 3.07 (s, 4H), 2.68-2.77 (m, 4H), 2.17 (s, 1H), 1.69-1.82 (m, 4H).

Compound 4g : 1H NMR (300 MHz, CDCl 3 ) δ 6.81-6.95 (m, 4H), 3.92 (s, 4H), 3.78 (s, 3H), 3.18 (s, 4H), 2.69-2.84 (m, 4H), 1.69-1.87 (m, 4H).

Compound 4h : 1H-NMR (300 MHz, CDCl3)δ 7.00-7.09 (m, 1H), 6.86-6.95 (m, 3H), 3.86-4.02 (m, 4H), 3.89 (s, 3H), 3.13 (s, 4H), 2.71-2.83 (m, 4H), 1.69-1.88 (m, 4H).

Compound 4i : 1H NMR (300 MHz, CDCl 3) δ 7.52 (d, J = 8.5 Hz, 2H), 6.95 (d, J = 8.5 Hz, 2H), 3.95 (m, 4H), 3.37 (m, 4H), 2.79 (m, 4H), 1.80 (m, 4H).

Compound 4j : 1H NMR (300 MHz, CDCl3) δ 6.99 (m, 2H), 6.90 (m, 2H), 3.93 (m, 4H), 3.17 (m, 4H), 1.06 (m, 4H), 1.79 (m, 4H).

Compound 5 : A mixture of 4f (20 mg, 0.061 mmol), EtI (19 mg, 0.122 mmol) and K2CO3 (84 mg, 0.61 mmol) in DMF (1 mL) was stirred overnight at 80 °C. the solids were filtered off and the filtrate was evaporated to give a residue. The residue was purified by flash column chromatography on silica gel (EtOAc:hexanes = 1:3) to afford 16 mg of compound 5 (75%) as a yellowish solid. 1H NMR (300 MHz, CDCl3) δ 6.87 (m, 4H), 3.98 (m, 6H), 3.13 (s, 4H), 2.77 (m, 4H), 1.79 (m, 4H), 1.39 (t, J = 7.0 Hz, 3H).

Measurement of Melanin Content . Extracellular melanin release was measured as described previously.10 Briefly, melanin biosynthesis inhibitory activity of synthesized compounds was evaluated for α-melanocyte-stimulating hormone (α-MSH) induced melanogenesis in mouse melanoma cell line B16F10. The melanoma cells were treated with compounds (1 µM, 10 µM) in the presence of α-MSH (1 µM) and incubated for 3 days. Absorbance was measured at 400 nm to quantify the melanin content in culture media and was normalized by the number of viable cells. The cells were then counted using a hemocytometer. Melanin production was expressed as the percentage of α-MSH-treated controls.