Design, synthesis and bioevaluation of novel umbelliferone analogues as potential mushroom tyrosinase inhibitors

Abstract

A series of umbelliferone analogues were synthesized and their inhibitory effects on the DPPH and mushroom tyrosinase were evaluated. The results showed that some of the synthesized compounds exhibited significant mushroom tyrosinase inhibitory activities. Especially, 2-oxo-2-[(2-oxo-2H-chromen-7-yl)oxy]ethyl-2,4-dihydroxybenzoate (4e) bearing 2,4-dihydroxy substituted phenyl ring exhibited the most potent tyrosinase inhibitory activity with IC₅₀ value 8.96 µM and IC₅₀ value of kojic acid is 16.69. The inhibition mechanism analyzed by Lineweaver–Burk plots revealed that the type of inhibition of compound 4e on tyrosinase was non-competitive. The docking study against tyrosinase enzyme was also performed to determine the binding affinity of the compounds. The compounds 4c and 4e showed the highest binding affinity with active binding site of tyrosinase. The initial structure activity relationships (SARs) analysis suggested that further development of such compounds might be of interest. The statistics of our results endorses that compounds 4c and 4e may serve as a structural template for the design and development of novel tyrosinase inhibitors.

• Antioxidant activity
• kinetic studies
• molecular modeling
• mushroom tyrosinase inhibitors
• synthesis
• umbelliferone analogues

Introduction

Tyrosinase or polyphenol oxidase (EC 1.14.18.1; PPO) is a binuclear copper containing enzyme present in biological systems. Two types of reactions are catalyzed by this enzyme in which molecular oxygen participates: (i) The orthohydroxylation of monophenols to o-diphenols (monophenolase activity) and (ii) the oxidation of o-diphenols to their corresponding o-quinones (diphenolase activity)1. Roughly speaking, mushroom tyrosinases consist of three domains, of which the central domain contains two copper binding sites, called CuA and CuB. The enzyme seems to be almost universally distributed in animals, plants, fungi and bacteria but much is still unknown about its biological function.

Melanins high molecular weight brown-pigments are synthesized by polymerization of quinine because of their high reactivity and react with amino acids and proteins to enhance brown color of the pigment2. First two steps of biosynthesis of melanin are catalyzed by the tyrosinase, the over accumulation of melanin results in hyperpigmentation such as senile lentigo, melasma, freckles and pigmented acne scars is of particular concern to women. The treatment usually involves the use of medicines or medicinal cosmetics containing de-pigmenting agents or skin whitening agents3. Tyrosinase inhibitors are used for treatments of dermatological disorders related to melanin hyperaccumulation and are essential in cosmetics for depigmentation4^,5. For example, age spots and freckle are caused by the accumulation of an excessive level of epidermal pigmentation6.

Previous reports confirmed that tyrosinase was one of the main causes of most fruits and vegetables quality loss during post-harvest handling and processing, leading to faster degradation and shorter shelf life7. Seo et al.8 reviewed the importance of mushroom tyrosinase and defines its biochemical character and inhibition and activation by several inhibitors from natural and synthetic origin. Zhu et al.9 also reported that tyrosinase enzyme has also been linked to Parkinson’s disease. Loizzo et al.10 and Chang11 reviewed the tyrosinase inhibitors from both natural and synthetic origin for their use in food and cosmetic industry. Khan12 also reviewed the molecular design of tyrosinase inhibitors from synthetic origin.

The coumarins are especially abundant in the Umbelliferae and Rutaceae families13 and also found in higher plants where they originate from phenylpropanoid pathway14. Some coumarins from natural sources have also been used as therapeutic agents for humans15 and their antibacterial, antifungal and anticancer activities make these compounds attractive for further derivatization and for screening as therapeutic agents16. Umbelliferone also known as 7-hydroxy coumarin is a natural product widely distributed in many familiar plants Apiaceae, Asteraceae, Hydrangeaceae and Acanthaceae. Coumarin derivatives are of great interest due to their diverse structural features and versatile biological properties, such as anti-inflammatory, antioxidant, vasorelaxant, cytotoxic, anti-HIV, antitubercular and antimicrobial17–22.

Coumarin derivatives have been synthesized and extensively studied for their biological response, but the tyrosinase inhibitor activities of this kind of compounds have hardly ever appeared in the literature. The literature survey revealed that clinically used tyrosinase inhibitors such as kojic acid, arbutine, oxyresveratrol, p-coumaric acid kaempferol, etc., all possess free phenolic hydroxyl moiety Figure 1. The presence of hydroxy substituted phenyl ring is considered to be important in tyrosinase inhibitory activity23^,24. Inspired by these findings, we synthesized the hydroxylated umbelliferone derivatives (4a–g) and (6a–b) in order to explore their inhibition mechanisms to offer a source for the development of new effective tyrosinase inhibitors. The title compounds 4a–g are synthesized by incorporating hydroxy substituted benzoic acids while compounds 6a–b by p-substituted cinnamic acids.

Figure 1. Structures of some mushroom tyrosinase inhibitors.

Experimental

All chemicals used for the synthesis of compounds were purchased from Sigma Chemical Co. (St. Louis, MO) Melting points were determined using a Digimelt MPA 160 (Holland, MI) melting point apparatus and are reported uncorrected. The FT-IR spectra were recorded with Shimadzu FTIR-8400S spectrometer (Kyoto, Japan, υ, cm⁻¹). The ¹H NMR and ¹³C NMR spectra (CDCl₃) and (DMSO-d₆) were recorded using a Bruker 400 MHz spectrometer. Chemical shifts (δ) are reported in ppm downfield from the internal standard tetramethylsilane (TMS). The purity of the compounds was checked by thin layer chromatography (TLC) on silica gel plate using n-hexane and ethyl acetate as mobile phase. The procedure for the synthesis of the desired compounds is depicted in Schemes 1 and 2.

Scheme 1. Synthesis of coumarin analogues (4a–g).

Scheme 2. Synthesis of coumarin analogues (6a–b).

Reagents

Mushroom tyrosinase was purchased from Sigma (St. Louis, MO); l-DOPA and umbelliferone were purchased from Sigma (St. Louis, MO). Stock solutions of the reducing substrates were prepared in phosphate buffer (20 mM, pH 6.8).

Synthesis of umbelliferone chloroacetyl derivative (2)

A mixture of 7-hydroxy coumarin (1) (0.01 mol), triethylamine (0.01 mol) in anhydrous dichloromethane (25 mL) was cooled in an ice salt mixture to 0–5 °C. To this reaction mixture, chloroacetyl chloride (0.01 mol) in dry dichloromethane was added drop wise with constant stirring over a period of 1 h maintaining the temperature constant. The reaction mixture was then stirred at room temperature for further 5 h, washed with 5% HCl, 5% sodium hydroxide and finally with brine solution. The organic layer was dried over anhydrous magnesium sulfate, filtered and the solvent was removed under reduced pressure to afford the corresponding umbelliferone chloroacetyl derivative (2).

2-oxo-2H-chromen-7-yl chloroacetate (2)

White solid; reaction time, 6 h; yield, 85%; melting point, 162–164 °C; R_f 0.64 (n-hexane:ethyl acetate 2:1), FTIR ν_max cm⁻¹: 3079 (sp² C–H), 2973 (sp³ C–H), 1766 (C=O ester), 1728 (C=O, lactone), 1615 (C=C aromatic); ¹H NMR (CDCl₃, δ ppm): 7.73 (d, J = 9.2 Hz, 1H, H-4), 7.54 (d, J = 4.4 Hz, 1H, H-5), 7.19 (d, J = 2.4 Hz, 1H, H-8), 7.13 (dd, J = 2.4, 6.0 Hz, 1H, H-6), 6.26 (d, J = 9.6 Hz, 1H, H-3), 4.36 (s, 2H, –CH₂); ¹³C NMR (CDCl_3, δ ppm): 165.2 (C=O ester), 160.0 (C-2), 154.7 (C-7), 152.5 (C-10), 142.6 (C-4), 128.7 (C-5), 117.8 (C-9), 117.1 (C-6), 116.5 (C-8), 110.1 (C-3), 40.6 (CH₂).

Synthesis of title compounds 4a–g and 6a–b

A mixture of umbelliferone chloroacetyl derivative (2) (0.01 mol), hydroxy substituted benzoic acids (3a–g) (0.01 mol), triethyl amine (0.01 mol), potassium iodide (0.01 mol) in dimethyl formamide (25 mL) was stirred overnight at room temperature (Scheme 1). The reaction mixture was poured into finely crushed ice with stirring and extracted with ethyl acetate (4 × 25 mL). The combined organic layer was washed with 5% HCl, 5% sodium hydroxide and finally with brine solution. The organic layer was dried over anhydrous magnesium sulfate, filtered and the solvent was removed under reduced pressure to afford the final products (4a–g). The title compounds 4a–g were purified by recrystallization in a mixture of hexane and ethyl acetate. The same procedure was used for the preparation of compounds 6a and 6b (Scheme 2).

2-oxo-2-[(2-oxo-2H-chromen-7-yl)oxy]ethyl-3-hydroxybenzoate (4a)

Solid; reaction time, 24 h; yield, 73%; melting point, 184–186 °C; R_f 0.42 (n-hexane:ethyl acetate 2:1), FTIR ν_max cm⁻¹: 3126 (O–H), 3014 (sp² C–H), 2873 (sp³ C–H), 1705 (C=O ester), 1680 (C=O lactone), 1589 (C=C aromatic), 1148 (C–O, ester); ¹H NMR (DMSO-d₆, δ ppm): 7.81 (dd, J = 2.0, 2.1 Hz, 1H, H-2′), 7.80 (m, 2H, H-4′, H-6′), 7.66 (t, J = 4.0 Hz, 1H, H-5′), 7.58 (d, J = 1.2 Hz, 1H, H-8), 7.55 (d, J = 2.8 Hz, 1H, H-4), 7.53 (d, J = 4.8 Hz, 1H, H-5), 6.81 (dd, J = 2.4, 2.0 Hz, 1H, H-6), 6.21 (d, J = 9.2 Hz, 1H, H-3), 5.28 (s, 2H, –CH₂), 2.50 (s, 1H, 3′-OH); ¹³C NMR (DMSO-d_6, δ ppm): 177.7 (C-1″), 161.7 (C=O ester), 160.8 (C-2), 155.9 (C-3′), 153.1 (C-7), 144.9 (C-10), 143.8 (C-4), 139.3 (C-1′), 132.2 (C-5′), 130.1 (C-5), 119.3 (C-6′), 116.5 (C-4′), 113.6 (C-6), 111.8 (C-2′), 111.7 (C-8), 107.5 (C-6′), 102.6 (C-3), 40.2 (CH₂).

2-oxo-2-[(2-oxo-2H-chromen-7-yl)oxy]ethyl-4-hydroxybenzoate (4b)

Solid; reaction time, 24 h; yield, 78%; melting point, 177–179 °C; R_f 0.44 (n-hexane:ethyl acetate 2:1), FTIR ν_max cm⁻¹: 3093 (O–H), 2962 (sp² C–H), 2852 (sp³ C–H), 1772 (C=O ester), 1710 (C=O lactone), 1602 (C=C aromatic), 1159 (C–O, ester); ¹H NMR (DMSO-d₆, δ ppm): 7.92 (dd, J = 9.2, 1.6 Hz, 2H, H-2′, H-6′), 7.52 (d, J = 8.4, 1H, H-4), 6.92 (dd, J = 5.6, 1.2 Hz, 2H, H-3′, H-5′), 6.81 (dd, J = 6.4, 2.0 Hz, 1H, H-6), 6.73 (d, J = 2.0 Hz, 1H, H-8), 6.51 (d, J = 9.6 Hz, 1H, H-5), 6.21 (d, J = 9.6 Hz, 1H, H-3), 5.18 (s, 2H, –CH₂), 2.51 (s, 1H, 4′-OH); ¹³C NMR (DMSO-d_6, δ ppm): 173.4 (C-1″), 162.4 (C=O ester), 160.8 (C-2), 155.9 (C-4′), 149.1 (C-7), 144.9 (C-10), 143.4 (C-4), 135.3 (C-1′), 132.3 (C-3′, C-5′), 130.1 (C-5), 114.4 (C-6), 113.4 (C-8), 111.8 (C-2′, C-6′), 110.5 (C-9), 102.5 (C-3), 40.5 (CH₂).

2-oxo-2-[(2-oxo-2H-chromen-7-yl)oxy]ethyl-3,4-dihydroxybenzoate (4c)

Solid; reaction time, 24 h; yield, 71%; melting point, 226–228–186 °C; R_f 0.39 (n-hexane:ethyl acetate 2:1), FTIR ν_max cm⁻¹: 3126 (O–H), 2868 (sp² C–H), 2827 (sp³ C–H), 1712 (C=O ester), 1681 (C=O lactone), 1567 (C=C aromatic), 1128 (C–O, ester); ¹H NMR (DMSO-d₆, δ ppm): 7.86 (d, J = 9.6 Hz, 1H, H-6′), 7.80 (d, J = 8.6 Hz, 1H, H-2′), 7.48 (d, J = 8.6 Hz, 1H, H-4), 7.39 (d, J = 7.2 Hz, 1H, H-5), 6.88 (d, J = 6.2 Hz, 1H, H-5′), 6.82 (dd, J = 6.4, 2.4 Hz, 1H, H-6), 6.73 (d, J = 2.4 Hz, 1H, H-8), 6.21 (d, J = 9.6 Hz, 1H, H-3), 4.90 (s, 2H, –CH₂), 3.01 (s, 2H, 2′, 4′-OH); ¹³C NMR (DMSO-d_6, δ ppm): 173.6 (C-1″), 162.4 (C=O ester), 161.5 (C-2), 156.2 (C-4′), 150.2 (C-3′), 148.1 (C-7), 144.6 (C-10), 136.8 (C-4), 136.3 (C-1′), 133.2 (C-5′), 129.2 (C-5), 124.3 (C-6′), 113.1 (C-4′), 111.6 (C-6), 110.9 (C-2′), 108.7 (C-8), 105.5 (C-6′), 102.0 (C-3), 49.2 (CH₂).

2-oxo-2-[(2-oxo-2H-chromen-7-yl)oxy]ethyl-3,5-dihydroxybenzoate (4d)

Solid; reaction time, 24 h; yield, 78%; melting point, 221–223 °C; R_f 0.40 (n-hexane:ethyl acetate 2:1), FTIR ν_max cm⁻¹: 3170 (O–H), 3010 (sp² C–H), 2962 (sp³ C–H), 1775 (C=O ester), 1679 (C=O lactone), 1567 (C=C aromatic), 1130 (C–O, ester); ¹H NMR (DMSO-d₆, δ ppm): 7.84 (d, J = 9.6 Hz, 1H, H-4), 7.68 (d, J = 8.4 Hz, 1H, H-5), 7.38 (dd, J = 1.6, 2.0 Hz, 2H, H-2’,6’), 7.03 (dd, J = 1.2, 1.6 Hz, 1H, H-4′), 6.84 (dd, J = 8.0, 2.4 Hz, 1H, H-6), 6.73 (d, J = 2.0 Hz, 1H, H-8), 6.21 (d, J = 9.6 Hz, 1H, H-3), 4.88 (s, 2H, –CH₂), 3.32 (s, 2H, 3′, 5′-OH); ¹³C NMR (DMSO-d_6, δ ppm): 166.3 (C-1″), 162.2 (C=O ester), 161.7 (C-2), 158.5 (C-3′, C-5′), 155.8 (C-7), 144.6 (C-10), 143.5 (C-4), 129.2 (C-1′), 115.3 (C-2′, C-6′), 113.1 (C-5), 111.7 (C-4′), 110.9 (C-6), 109.7 (C-8), 102.0 (C-3), 59.2 (CH₂).

2-oxo-2-[(2-oxo-2H-chromen-7-yl)oxy]ethyl-2,4-dihydroxybenzoate (4e)

Solid; reaction time, 24 h; yield, 64%; melting point, 204–206 °C; R_f 0.45 (n-hexane:ethyl acetate 2:1), FTIR ν_max cm⁻¹: 3125 (O–H), 2924 (sp² C–H), 2852 (sp³ C–H), 1709 (C=O ester), 1677 (C=O lactone), 1603 (C=C aromatic), 1128 (C–O, ester); ¹H NMR (DMSO-d₆, δ ppm): 8.09 (d, J = 9.6 Hz, 1H, H-6′), 7.94 (d, J = 9.6 Hz, 1H, H-4), 7.53 (d, J = 8.4 Hz, 1H, H-5), 7.22 (d, J = 2.4 Hz, 1H, H-3′), 6.80 (dd, J = 6.4, 2.4 Hz, 1H, H-5′), 6.73 (d, J = 2.0 Hz, 1H, H-8), 6.50 (dd, J = 9.6, 2.4 Hz, 1H, H-6), 6.33 (d, J = 2.0 Hz, 1H, H-8), 6.21 (d, J = 9.6 Hz, 1H, H-3), 4.60 (s, 2H, –CH₂), 3.34 (s, 2H, 2′, 4′-OH); ¹³C NMR (DMSO-d_6, δ ppm): 176.5 (C-1″), 161.7 (C=O ester), 160.8 (C-2), 155.9 (C-2′, C-4′), 144.9 (C-7), 143.8 (C-10), 142.3 (C-4), 132.6 (C-1′), 130.9 (C-5), 113.6 (C-6′), 111.8 (C-3′, C-5′), 111.7 (C-6), 107.7 (C-8), 102.6 (C-3), 58.2 (CH₂).

2-oxo-2-[(2-oxo-2H-chromen-7-yl)oxy]ethyl-2,6-dihydroxybenzoate (4f)

Solid; reaction time, 24 h; yield, 74%; melting point, 190–192 °C; R_f 0.46 (n-hexane:ethyl acetate 2:1), FTIR ν_max cm⁻¹: 3103 (O–H), 2912 (sp² C–H), 2853 (sp³ C–H), 1678 (C=O ester), 1601 (C=O lactone), 1566 (C=C aromatic), 1122 (C–O, ester); ¹H NMR (DMSO-d₆, δ ppm): 7.92 (d, J = 9.2 Hz, 1H, H-4), 7.54 (d, J = 8.4 Hz, 1H, H-5), 7.18 (m, 3H, H-3′,4′,5′), 6.85 (dd, J = 9.6, 2.4 Hz, 1H, H-6), 6.79 (d, J = 2.4 Hz, 1H, H-8), 6.20 (d, J = 9.2 Hz, 1H, H-3), 5.20 (s, 2H, –CH₂), 4.09 (s, 2H, 2′,6′-OH); ¹³C NMR (DMSO-d_6, δ ppm): 180.8 (C-1″, 166.4 (C=O ester), 161.3 (C-2), 155.6 (C-2′, C-6′), 150.1 (C-7), 144.9 (C-10), 142.8 (C-4), 136.1 (C-4′),132.4 (C-1′) 130.1 (C-3′, C-5′), 119.5 (C-5), 111.9 (C-6), 111.8 (C-8), 102.5 (C-3), 48.5 (CH₂).

2-oxo-2-[(2-oxo-2H-chromen-7-yl)oxy]ethyl-3,4,5-trihydroxybenzoate (4g)

Solid; reaction time, 24 h; yield, 52%; melting point, 230–232 °C; R_f 0.42 (n-hexane:ethyl acetate 2:1), FTIR ν_max cm⁻¹: 3124 (O–H), 2914 (sp² C–H), 2829 (sp³ C–H), 1705 (C=O ester), 1678 (C=O lactone), 1566 (C=C aromatic), 1129 (C–O, ester); ¹H NMR (DMSO-d₆, δ ppm): 7.87 (d, J = 0.4 Hz, 1H, H-2′, H-6′), 7.85 (d, J = 0.4 Hz, 1H, H-8), 7.48 (d, J = 8.8 Hz, 1H, H-4), 6.82 (dd, J = 6.0, 2.4 Hz, 1H, H-6), 6.73 (d, J = 2.4 Hz, 1H, H-5), 6.21 (d, J = 9.6 Hz, 1H, H-3), 4.90 (s, 2H, –CH₂), 3.90 (s, 1H, –OH); ¹³C NMR (DMSO-d_6, δ ppm): 178.7 (C-1″), 162.3 (C=O ester), 161.7 (C-2), 155.6 (C-3′, 4′,5′), 144.6 (C-7), 141.7 (C-10), 138.3 (C-4), 131.6 (C-1′), 129.2 (C-2′, C-6′), 113.1 (C-5), 111.7 (C-6), 110.9 (C-8), 102.0 (C-3), 48.2 (CH₂).

2-oxo-2-[(2-oxo-2H-chromen-7-yl)oxy]ethyl(2E)-3-(4-hydroxyphenyl)prop-2-enoate (6a)

Solid; reaction time, 24 h; yield, 76%; melting point, 201–203 °C; R_f 0.45 (n-hexane:ethyl acetate 2:1), FTIR ν_max cm⁻¹: 3166 (O–H), 2931 (sp² C–H), 2843 (sp³ C–H), 1678 (C=O ester), 1624 (C=O lactone), 1601 (C=C aliphatic) 1567 (C=C aromatic), 1128 (C–O, ester); ¹H NMR (DMSO-d₆, δ ppm): 8.08 (d, J = 9.6 Hz, 1H, H-4), 7.93 (d, J = 9.2 Hz, 2H, H-2′, H-6′), 7.68 (d, J = 3.2 Hz, 1H, H-5), 7.53 (d, J = 8.4 Hz, 2H, H-3′, H-5′), 6.83 (d, J = 2.4 Hz, 1H, H-8), 6.78 (dd, J = 6.0, 2.4 Hz, 1H, H-6), 6.49 (d, J = 16.0 Hz, 1H, H-1″), 6.31 (d, J = 16.0 Hz, 1H, H-2″), 6.20 (d, J = 9.2 Hz, 1H, H-3), 5.07 (s, 2H, –CH₂), 2.52 (s, 1H, –OH); ¹³C NMR (DMSO-d_6, δ ppm): 162.3 (C-ester), 161.7 (C-3″, C=O), 160.8 (C-2), 155.9 (C-4′), 146.3 (C-7), 144.9 (C-10), 132.1 (C-1′), 131.3 (C-4), 130.1 (C-2′, C-6′), 116.3 (C-5), 115.8 (C-2″), 113.6 (C-3′, C-5′), 111.8 (C-6), 111.7 (C-8), 102.6 (C-3), 36.2 (CH₂).

2-oxo-2-[(2-oxo-2H-chromen-7-yl)oxy]ethyl(2E)-3-(4-chlorophenyl)prop-2-enoate (6b)

Solid; reaction time, 24 h; yield, 84%; melting point, 154–156 °C; R_f 0.47 (n-hexane:ethyl acetate 2:1), FTIR ν_max cm⁻¹: 3083 (sp² C–H), 2867 (sp³ C–H), 1716 (C=O ester), 1692 (C=O lactone), 1615 (C=C aliphatic), 1568 (C=C aromatic), 1123 (C–O, ester); ¹H NMR (DMSO-d₆, δ ppm): 7.77 (d, J = 16.0 Hz, 1H, H-1″), 7.70 (d, J = 9.2 Hz, 1H, H-4), 7.54 (d, J = 6.0 Hz, 1H, H-5), 7.51 (dd, J = 4.0, 2.4 Hz, 2H, H-2′, H-6′), 7.39 (dd, J = 4.8, 1.6 Hz, 2H, H-3′, H-5′), 7.21 (d, J = 2.0 Hz, 1H, H-8), 7.14 (dd, J = 6.0, 2.4 Hz, 1H, H-6), 6.53 (d, J = 16.0 Hz, 1H, H-2″), 6.43 (d, J = 9.6 Hz, 1H, H-3), 5.03 (s, 2H, –CH₂); ¹³C NMR (DMSO-d_6, δ ppm): 165.8 (C=O ester), 160.1 (C-3″, C=O), 154.7 (C-2), 152.4 (C-4′), 145.3 (C-7), 142.6 (C-10), 136.8 (C-1′), 132.5 (C-3′, C-5′), 129.4 (C-2′, C-6′), 129.3 (C-4), 128.6 (C-5), 118.0 (C-2″), 117.0 (C-6), 116.4 (C-8), 110.2 (C-3), 60.7 (CH₂).

Free radical scavenging assay

Radical scavenging activity was determined by modifying method by 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay25^,26. The assay solution consisted of 100 µL of (150 µM) 2,2-diphenyl-1-picrylhydrazyl (DPPH), 20 µL of increasing concentration of test compounds and the volume was adjusted to 200 µL in each well with DMSO. This reaction mixture was then incubated for 30 min at room temperature. Ascorbic acid (Vitamin C) was used as a reference inhibitor. The measurements were carried out by using a micro plate reader (OPTI_Max, tunable) at 517 nm. The reaction rates were compared and the percent inhibition due to the presence of tested inhibitors was calculated. Each concentration was analyzed in three independent experiments. The IC₅₀ values were determined after serial dilution by the Data analysis and graphing software Origin 8.6, 64-bit (OriginLab Corporation, Northampton, MA).

Mushroom tyrosinase inhibition assay

The mushroom tyrosinase was used for in vitro bioassays as described previously with some modifications27^,28. Briefly, 140 μL of phosphate buffer (20 mM, pH 6.8), 20 μL of mushroom tyrosinase (30 U/mL) and 20 μL of the inhibitor solution were placed in the wells of a 96-well micro plate. After pre-incubation for 10 min at room temperature, 20 μL of L-DOPA (3,4-dihydroxyphenylalanine; 0.85 mM) was added and the plate was further incubated at 25 °C for 20 min. Subsequently, the absorbance of dopachrome was measured at 492 nm using a micro plate reader. Kojic acid was used as a reference inhibitor and phosphate buffer was used as negative control. Each concentration was analyzed in three independent experiments. The IC₅₀ values were determined by the data analysis and graphing software Origin 8.6, 64-bit. The percent of inhibition of tyrosinase reaction was calculated as following:

Here, the B and S are the absorbances for the blank and samples.

Kinetic analysis of the inhibition of tyrosinase

A series of experiments were performed to determine the inhibition kinetics by following this method: for example, for each of five different inhibitor concentrations of compound 4e (0.00, 0.007, 0.014, 0.028 and 0.056 mM, respectively), l-DOPA concentration was varied (0.5, 1.0, 1.5 and 2.0 mM). Pre-incubation and measurement time was the same as discussed above in the “Mushroom tyrosinase inhibition assay” section. Maximal initial velocity was determined from initial linear portion of absorbance up to 5 min after addition of mushroom tyrosinase at a 15 s interval. The inhibition type on the enzyme was assayed by Lineweaver–Burk plots of inverse of velocities (1/V) versus inverse of substrate concentration 1/[S] mM⁻¹, and the inhibition constant K_i was determined by the second plots of the apparent K_m/V_max or 1/V_max versus the concentration of compound 4e.

Molecular modeling

The experimental X-ray structure of human tyrosinase has not been determined till now. To overcome this problem, a homology model was made with the crystal structure of mushroom tyrosinase (PDB accession code 2ZWE) taken from the research collaborator for structural bioinformatics (RCSB) Protein Data Bank. The attached ligand and water molecules were removed from the model using the AutoDock Tools 1.5.6 (La Jolla, CA). The charges were stabilized and polar hydrogen molecules were added to the protein structure. The ligand molecules were also prepared for docking using the same program. Among the synthesized coumarin analogues compounds 4a, 4c, 4e and 6a were selected for docking studies. The binding pocket of tyrosinase has already been defined with the residues surrounding the dicopper ions since they are implied to the recognition of the l-DOPA substrate29. The values of additional starting conformations per molecule and maximum number of poses per ligand were both expanded to 40 to increase the accuracy of binding mode. Other parameters were based on the software default setting. Molecular docking was, then, carried out by providing appropriate search space details to AutoDock Vina 1.1.2 (La Jolla, CA). The results were obtained in the form of binding affinities in kcal/mol. The docking process was replicated 10 times on 10 separate computing systems to decrease the biased experimentation. The binding modes were visualized using Discovery Studio 4.0 (San Diego, CA). Both the two- and three-dimensional images were studied to ascertain the interacting molecules in the study.

Results and discussion

Chemistry

The umbelliferone derivatives were synthesized by following the previously described method30 with slight modification shown in Schemes 1 and 2. Chloroacetyl derivative (2) was synthesized by esterification of 7-hydroxyl group of umbelliferrone with chloroacetyl chloride in the presence of anhydrous methylene chloride. The title compounds 4a–g and 6a–b were prepared by simple nucleophilic substitution at 2 with hydroxy substituted benzoic acids (3a–g) and para-substituted cinnamic acids (5a–b), respectively. In the presence of triethylamine as catalyst, the reaction was carried out at room temperature for 24 h. The yields of these compounds were from moderate to good. All of the target compounds possess hydroxyl moieties except compound 6b in which halogen substituent is present to evaluate the role of halogens in tyrosinase inhibition. The structures of all of the synthesized compounds were confirmed by FTIR, ¹H and ¹³C NMR spectroscopic data.

Bioassay for inhibition of tyrosinase activity

The kojic acid is a competitive tyrosinase inhibitor and exhibited a mixed inhibitory effect on diphenolase activity of mushroom tyrosinase31. Therefore, we used kojic acid as the standard inhibitor of tyrosinase for comparison purposes. In this study, the effect of coumarin derivatives (4a–g) and (6a–b) on the inhibition of tyrosinase activity was measured at six different inhibitor concentrations. When l-DOPA solution was incubated with tyrosinase at 25 °C, its color turned to red-brown because of the formation of dopachrome. In the presence of kojic acid and coumarin derivatives, the color intensity of the solution became faint as the result of enzyme activity inhibition. The IC₅₀ values for each of kojic acid and compounds 4a–g and 6a–b are presented in Table 1 which shows that the standard kojic acid is more active than all the synthesized coumarin derivatives except compound 4e.

Table 1. The inhibitory effects of coumarin analogues (4a–g) and (6a–b) on mushroom tyrosinase activity: (substrate: L-DOPA).

Entry	Compounds	Log p*	IC50 µM
1	2	0.96 ± 0.30	362.18 ± 12.1
2	4a	2.54 ± 0.37	173.23 ± 8.5
3	4b	2.51 ± 0.37	179.11 ± 9.2
4	4c	2.34 ± 0.38	118.48 ± 8.5
5	4d	2.25 ± 0.38	233.33 ± 17.3
6	4e	2.57 ± 0.40	8.96 ± 4.3
7	4f	2.94 ± 0.41	198.87 ± 5.7
8	4g	2.91 ± 0.41	258.06 ± 5.1
9	6a	2.29 ± 0.39	123.77 ± 11
10	6b	3.35 ± 0.39	80.20 ± 10.1
11	Kojic acid	–	16.69 ± 2.8
12	Umbelliferone†	–	420

*Log p calculated from chemSketch ACD labs 2012.

†Obtained from data given by Vilar et al32.

From results, it was observed that compounds 4e, 4c and 6b exhibited potent inhibition of mushroom tyrosinase with an IC₅₀ values ranged from 8.96 to 118.48 µM. Especially, compound 4e and 4c possessing 2,4-dihydroxy and 3,4-dihydroxy phenyl scaffold showed more potent inhibitory activities than the other compounds. Rest of the coumarin analogues showed lower or negligible activities against tyrosinase compared to standard kojic acid. The IC₅₀ value of umbelliferone was also mentioned in table taken from literature and it was found that all of umbelliferone analogues have high potential to inhibit the activity of tyrosinase than umbelliferone. Excellent enzyme inhibition was exhibited by compound 4e as it is more potent than the standard kojic acid. From Figure 2, it was observed that compound 4e produced same enzyme inhibition at a lower dose than the reference kojic acid which indicates its higher potency than kojic acid.

Figure 2. Effect of compound 4e (a) and kojic acid (b) on the diphenolase activity against mushroom tyrosinase for the catalysis of L-DOPA at 25 °C.

The following general structural features of SAR can be deduced from compounds 4a–g and 6a–b. Compounds 4a–g possess the hydroxyl substituted benzoic acid moiety while (6a–b) have hydroxyl and chloro-substituted cinnamic acid scaffolds. When activities of these scaffolds possessing different substituents viz. monohydroxy, dihydroxy and chloro compared, then it was observed that coumarin analogues with dihydroxy phenyl rings exhibited more potent inhibitory activities than the monohydroxy phenyl rings. In addition, the activities of coumarin derivatives with 2,4- and 3,4-dihydroxy substituted phenyl have different activities than 3,5-dihydroxy phenyl ring. The presence of 4-hydroxy along with the presence of 2 or 3 hydroxy groups on phenyl ring play very important role to form a more tight interaction with mushroom tyrosinase. We also explore the role of halogen atom for the inhibition of activity of tyrosinase enzyme in this study because compound 6b possess 4-chloro while 6a have 4-hydroxy substituted cinnamic acid scaffold. The compound 6b exhibited less enzyme inhibition than compounds with dihydroxy substituted phenyl rings but it showed higher activity than compound 6a which possess 4-hydoxy substituted cinnamic acid residue. Further investigation to explore the type of interactions of halogen with tyrosinase enzyme is needed. The initial structure–activity relationships (SARs) analysis recommended that additional progress in these structural motifs might be of interest. The Figure 3 depicted the three-dimensional view and highlighted the functionalities involved in the tyrosinase inhibition. Our results propose that compounds 4c and 4e may serve as a structural template for the design and development of novel tyrosinase inhibitors.

Figure 3. Three-dimensional view of compounds 4c and 4e with highlighted functionalities involve in enzyme binding.

Kinetic studies

To understand the inhibitory mechanism of compound 4e on mushroom tyrosinase kinetic studies were conducted. The results were presented in Table 2 and Figure 4. From these kinetic studies, inhibition mechanism of compound 4e was determined to be non-competitive inhibitor of mushroom tyrosinase from Figure 4a. The inhibition constant K_i (0.0215 mM) was determined from Figure 4b. The value of 1/V_max is increased to a new value while that of K_m remains same which indicated that compound 4e simply lowers the concentration of functional enzyme by a non-competitively binding mode at enzyme. Mushroom tyrosinase having central copper containing active binding sites where most of the competitive inhibitors bind. The non-competitive behavior of compound 4e can be verified by the presence of 2,4-dihydroxy substituted phenyl ring a different structural feature as compared to substrate l-DOPA which possess ortho-diphenol scaffold.

Figure 4. (a) Lineweaver–burk plots for the inhibition of compound 4e on mushroom tyrosinase for catalysis of L-DOPA. Inhibitor concentrations were 0, 0.007, 0.014, 0.028 and 0.056 mM. The (b) represents the secondary plot of 1/V_max versus concentration of compound 4e to determine the inhibition constant (K_i).

Table 2. Kinetic analysis of compound 4e.

Entry	Concentration [mM]	1/Vmax	Km	Ki
1	0.000	59 293	0.027	0.0215
2	0.007	118 586	0.027	0.0215
3	0.014	177 879	0.027	0.0215
4	0.028	237 172	0.027	0.0215
5	0.056	296 465	0.027	0.0215

1/V_max is the inverse of reaction velocities. K_m is the Michaelis–Menten constant. K_i is the inhibition constant.

Molecular bocking study

The three-dimensional structure of tyrosinase enables us to gain a better understanding of the tyrosinase inhibition mechanism. Considering compounds 4a, 4c, 4e and 6a having mono- and di-hydroxy substituted phenyl rings, we selected them as representative compounds to study the interaction mode by docking. The docking results were obtained in the form of binding affinities in kcal/mol and average docking values are presented in Table 3. The docking results show that compounds 4c and 4e possess highest binding affinity with the enzyme. The potential ligand–protein interaction of compounds 4a, 4c, 4e and 6a with the active site of tyrosinase (generated using Discovery Studio 4.0 are presented in Figures 5–8), respectively. Both two- and three-dimensional views of the ligand–protein interaction are portrayed in figures.

Figure 5. The potential ligand–protein interaction of compound 4a with the active site of tyrosinase (PDB ID 2ZWE) generated using Discovery Studio 4.0. The (A) and (B) show the three-dimensional docking of the compound in the binding pocket. Dashed lines indicate the interactions between the ligand and the amino acids of the receptor. The (C) shows the two dimensional interaction patterns. The legend inset represents the type of interaction between the ligand atoms and the amino acid residues of the protein.

Figure 6. The potential ligand–protein interaction of compound 4c with the active site of tyrosinase (PDB ID 2ZWE) generated by using Discovery Studio 4.0. The (A) and (B) show the three-dimensional docking of the compound in the binding pocket. Dashed lines indicate the interaction between the ligand and the amino acids of the receptor. The (C) shows the two dimensional interaction patterns. The legend inset represents the type of interaction between the ligand atoms and the amino acid residues of the protein.

Figure 7. The potential ligand–protein interaction of compound 4e with the active site of tyrosinase (PDB ID 2ZWE) generated using Discovery Studio 4.0. The (A) and (B) show the three-dimensional docking of the compound in the binding pocket. Dashed lines indicate the interaction between the ligand and the amino acids of the receptor. The (C) shows the two dimensional interaction patterns. The legend inset represents the type of interaction between the ligand atoms and the amino acid residues of the protein.

Figure 8. The potential ligand–protein interaction of compound 6a with the active site of tyrosinase (PDB ID 2ZWE) generated using Discovery Studio 4.0. The (A) and (B) show the three-dimensional docking of the compound in the binding pocket. Dashed lines indicate the interaction between the ligand and the amino acids of the receptor. The (C) show the two dimensional interaction patterns. The legend inset represents the type of interaction between the ligand atoms and the amino acid residues of the protein.

Table 3. The average binding energy calculated after docking of compounds 4a, 4c, 4e and 6a.

Entry	Compounds	Average binding affinity (kcal/mol)
1	4a	−7.5
2	4c	−7.7
3	4e	−7.6
4	6a	−6.0

In Figure 6, it was found that compound 4c formed π–π stacks between lactonic phenyl ring of the inhibitors and ASP113 of tyrosinase. There were hydrogen bonds between the 3- and 4-position of phenyl ring of (4c) and the ARG140 and GLN116 residue of the active site of tyrosinase. This result further confirmed that hydroxyl groups in the phenyl ring play a crucial role in the tyrosinase inhibitory activity. The lactonic carbonyl in case of (4e) also involved in hydrogen bonding with SER96 and carboxylic carbonyl form hydrogen bonding with GLN116 amino acid residue of the enzyme Figure 7. Both of the phenolic hydroxyls present at 2 and 4 position of phenyl in compound 4e tightly bound by HOH609, HOH646 and HOH695 of the target protein.

DPPH radical scavenging assay

The free radical scavenging activity was determined because most of the tyrosinase inhibitors possess antioxidant activity. The interaction of the synthesized compounds 4a–g and 6a–b with the stable free radical DPPH indicates their radical scavenging ability. The synthesized compounds exhibited no radical scavenging potential at low concentration 20 µM but as the concentration is increased up to 100 µM then compound 4f showed antioxidant activity. Table 4 presented the results and it was found that the interactions of the synthesized coumarin analogues are concentration dependent. All of the synthesized compounds show smaller interactions when compared to the standard ascorbic acid.

Table 4. DPPH free radical scavenging activity of the coumarin analogues (4a–g) and (6a–b).

Entry	Compounds	DPPH free radical Scavenging activity (IC50 ± SEM in µM)
1	2	>1000
2	4a	>1000
3	4b	>1000
4	4c	538.93 ± 17.9
5	4d	>1000
6	4e	>1000
7	4f	87.39 ± 6.4
8	4g	>1000
9	6a	>1000
10	6b	>1000
11	Ascorbic acid	14.82 ± 1.6

Conclusion

In conclusion, we describe the synthesis and tyrosinase inhibitory activity of coumarin derivatives (4a–g) and (6a–b). The title compound (4e) showed the excellent tyrosinase inhibitory activity among all the synthesized compounds. The in silico docking results also support our findings. The kinetic analysis of compound 4e demonstrated that it is non-competitive tyrosinase inhibitor with same value of inhibition constant K_i regardless of the concentration. The position of free hydroxyl group –OH on the side chain phenyl ring of compound 4e plays very significant role in masking tyrosinase activity.

Declaration of interest

This work (Grants No. C0036335) was supported by Business for Cooperative R & D between Industry, Academy, and Research Institute and funded by Korea Small and Medium Business Administration in 2012.