Synthesis and biological evaluation of polyhydroxy benzophenone as mushroom tyrosinase inhibitors

Abstract

A series of polyhydroxy benzophenone were synthesized and evaluated as mushroom tyrosinase inhibitors. The results demonstrated that most of the target compounds had remarkable inhibitory activities on mushroom tyrosinase. Among all these compounds, 2,3,4,3′,4′,5′-hexahydroxy-diphenylketone 10 was found to be the most potent tyrosinase inhibitor with IC₅₀ value of 1.4 μM. In addition, the inhibition kinetics analyzed by Lineweaver–Burk plots revealed that such compounds were competitive inhibitors. These results suggested that such compounds might be utilized for the development of new candidate for treatment of dermatological disorders.

Keywords::

• Polyhydroxy benzophenone
• tyrosinase
• inhibitors

Notes

¹The synthesis of compounds 1–18: To the mixture of appropriate phenol (50 mmol) and polyhydroxybenzoic acid (55.6 mmol), anhydrous zinc chloride (27.2 g) and phosphorus oxychloride (25 mL) were added. The reaction mixture was refluxed for 2.5 h in the water bath of 70°C with stirring. Then add the mixture to appropriate ice and cool to 4°C for 24 h. The precipitate solid was filtered, washed with 3% sodium bicarbonate twice, and purified by recrystallization from boiling water to afford compounds 1–18 (Figure 1). The synthetic route of compounds 1–18 is shown in Scheme 1. Compound 10: 2,3,4,3′,4′,5′-hexahydroxydiphenylketone (10). Yield: 90%; yellow powders; mp 272–273°C; ¹H NMR (dimethyl sulfoxide-d6, 300 MHz) δ (ppm): 6.4 (1H, d, J = 6.9 Hz, ArH), 6.6 (2H, s, ArH), 7.0 (1H, d, J = 4.2 Hz, ArH), 8.6 (1H, s, OH-R₃), 8.9 (1H, s, OH-R₂), 9.3 (2H, s, OH-R₆, OH-R₈), 10.0 (1H, s, OH-R₇), 12.1 (1H, s, OH-R₁). ¹³C NMR(dimethyl sulfoxide-d6, 300 MHz) δ: ¹³C NMR(dimethyl sulfoxide-d6, 300 MHz) δ: 198.60(C_carbonyl), 175.94 (C_phenyl-4), 152.43 (C_phenyl-3′), 145.96 (C_phenyl-3,5), 138.27 (C_phenyl-4′), 133.45 (C_phenyl-2′), 128.61 (C_phenyl-5′), 125.31 (C_phenyl-6′), 113.76 (C_phenyl-1′), 109.64 (C_phenyl-2,6), 107.83 (C_phenyl-1).

²Tyrosinase Inhibition Assay: In brief, a 10 μL sample was added to an assay mixture containing with 10 μL tyrosinase solution (0.5 mg/mL) and 900 μL phosphate buffer (pH 6.8) to a total volume assay mixture of 920 μL, for 20 min at 25°C. Then 80 μL of l-dopa (1.50 mg/mL) was added to the reaction mixture and the enzyme reaction was monitored by measuring the change in absorbance at 475 nm of the l-dopa for 1 min. IC₅₀ value, a concentration giving 50% inhibition of tyrosinase activity, was determined by interpolation of the dose–response curves. Here, kojic acid (IC₅₀ = 19.0 μmol/L) was used as the reference inhibitors.

³Kinetic Assay of Tyrosinase Inhibition: Various concentrations of l-dopa (0.1–1.2 mM) as the substrate, 40 μL of mushroom tyrosinase (1 units/mL), and 50 mM potassium phosphate buffer (pH 6.8) were added to test tube in a total volume assay mixture of 1000 μL. The initial rate of dopa chrome formation in the reaction mixture was determined by the increase of absorbance at 475 nm/min (ΔOD₄₇₅/min). Michaelis constant (K_m) and maximal velocity (V_max) of the tyrosinase activity were determined by the Lineweaver–Burk plot.