2015 Volume 38 Issue 8 Pages 1227-1233
Inhibiting tyrosinase is an important goal to prevent melanin accumulation in skin and thereby to inhibit pigmentation disorders. Therefore, tyrosinase inhibitors are an attractive target in cosmetics and treatments for pigmentation disorders. However, only a few tyrosinase inhibitors are currently available because of their toxic effects to skin or lack of selectivity and stability. Here, we newly synthesized thirteen (Z)-2-(benzo[d]thiazol-2-ylamino)-5-(substituted benzylidene)thiazol-4(5H)-one derivatives and examined their effect on melanogenesis. Of these compounds, MHY2081 had the strongest inhibitory effect on tyrosinase without cytotoxicity in B16F10 melanoma cells. Consistently, melanogenesis was notably decreased by MHY2081 treatment. As an underlying mechanism, docking simulation showed that compared to kojic acid, a well-known competitive tyrosinase inhibitor which forms a hydrogen bond and aromatic interaction with tyrosinase, MHY2081 has stronger affinity with tyrosinase by forming three hydrogen bonds and a hydrophobic interaction with residues of tyrosinase. In parallel with this, Lineweaver–Burk plot analysis showed that MHY2081 is a strong competitive inhibitor of tyrosinase. In conclusion, MHY2081 may be a novel tyrosinase inhibitor for prevention and treatment of pigmentation disorders.
The skin is a protective barrier against UV irradiation. Melanogenesis is an important function for skin to protect itself from damage.1) However, chronic and repeated exposures of UV lead to premature aging of the skin, which is called photoaging.1) In addition, melanin accumulation is able to increase pigmentation disorders such as melasma, freckles, and solar lentigo.2,3) Therefore, various compounds that prevent melanogenesis have been screened for cosmetics and preventing pigmentation disorders.
Melanogenesis is the process by which melanin is secreted by melanocytes located in the basal layer of the epidermis. Tyrosinase, a copper containing metalloenzyme, is one of the enzymes responsible for skin pigmentation in mammals.4) Tyrosinase catalyses two rate-limiting steps in melanogenesis: the hydroxylation of tyrosine to 3,4-dihydroxyphenylalanine (DOPA) and the oxidation of DOPA to DOPAquinone. Therefore, tyrosinase predominantly regulates melanin production5) and tyrosinase inhibitors are widely used for cosmetics and treatments for pigmentation disorders compared to other melanogenesis inhibitors. However, only a few tyrosinase inhibitors are currently used in cosmetics and medical products because of their cytotoxicity and lack of selectivity and stability.6–8)
Based on the structure of tyrosinase substrates L-tyrosin and L-DOPA, we synthesized a series of (Z)-2-(benzo[d]thiazol-2-ylamino)-5-(substituted benzylidene)thiazol-4(5H)-one derivatives to screen for novel tyrosinase inhibitors. Here, our data show that MHY2081 is the strongest tyrosinase inhibitor among 13 synthesized compounds.
To a stirred solution of 2-aminobenzothiazole (6.0 g, 39.95 mmol) in acetone (80 mL) was added a solution of chloroacetyl chloride (6.35 mL, 79.84 mmol) in acetone (20 mL) at 0°C and the reaction mixture was refluxed for 3 h. After cooling, the volatile solvent was removed under reduced pressure to give a residue, which was neutralized using aqueous sodium bicarbonate solution to pH 7. The precipitates generated were filtered and washed with water to give 14 (8.40 g, 92.8%) as a solid.
1H-NMR (400 MHz, dimethyl sulfoxide (DMSO)-d6) δ: 7.97 (d, 1H, J=7.6 Hz), 7.73 (d, 1H, J=8.4 Hz), 7.41 (t, 1H, J=8.0 Hz), 7.29 (t, 1H, J=8.0 Hz), 4.43 (s, 2H); 13C-NMR (100 MHz, DMSO-d6) δ: 166.6, 158.2, 149.1, 132.1, 126.9, 124.5, 122.5, 121.4, 43.2.
2-(Benzo[d]thiazol-2-ylamino)thiazol-4(5H)-one (15)A suspension of 14 (4.0 g, 17.65 mmol) and ammonium thiocyanate (2.7 g, 35.47 mmol) in ethyl alcohol (40 mL) was refluxed for 4 h. After cooling, the reaction mixture was filtered and washed with cold EtOH to give 15 (3.01 g, 68.4%) as a solid.
1H-NMR (400 MHz, DMSO-d6) δ: 12.27 (s, 1H), 7.93 (d, 1H, J=8.0 Hz), 7.76 (d, 1H, J=8.0 Hz), 7.42 (t, 1H, J=8.0 Hz), 7.30 (t, 1H, J=8.0 Hz), 4.03 (s, 2H); 13C-NMR (100 MHz, DMSO-d6) δ: 175.0, 169.6, 166.8, 151.5, 133.7, 127.0, 124.9, 122.6, 122.0, 35.9.
General Procedure of (Z)-2-(Benzo[d]thiazol-2-ylamino)-5-(substituted benzylidene)thiazol-4(5H)-one Analogs (Compounds 1–2 and 4–13)A suspension of 15 (80 mg, 0.32 mmol), a variety of substituted benzaldehyde (1.1 eq.), and NaOAc (3.0 eq.) in acetic acid (1.0 mL) was refluxed for 10–35 h. After cooling, water was added and the precipitates generated were filtered and washed with water or MeOH/H2O (8 : 1–1 : 2) to give compounds 1–2 and 4–13 as solids.
(Z)-2-(Benzo[d]thiazol-2-ylamino)-5-(4-hydroxybenzylidene)thiazol-4(5H)-one (1)Reaction time, 10 h; yield, 66.5%; yellow solid; 1H-NMR (400 MHz, DMSO-d6) δ: 12.76 (br s, 1H, NH), 10.35 (s, 1H, OH), 7.96 (d, 1H, J=7.6 Hz, 4′-H), 7.91 (d, 1H, J=8.4 Hz, 7′-H), 7.66 (s, 1H, vinylic H), 7.53 (d, 2H, J=7.6 Hz, 2″-H, 6″-H), 7.46 (t, 1H, J=8.0 Hz, 5′-H), 7.33 (t, 1H, J=7.6 Hz, 6′-H), 6.93 (d, 2H, J=7.6 Hz, 3″-H, 5″-H); 13C-NMR (100 MHz, DMSO-d6) δ: 169.0, 167.9, 160.7, 159.6, 151.5, 134.0, 133.8, 133.4, 127.1, 125.1, 124.8, 122.7, 122.5, 120.3, 117.1; high resolution (HR)-MS-electrospray ionization (ESI) m/z C17H10N3O2S2 (M−H)− Calcd 352.0214, obsd 352.0226.
(Z)-2-(Benzo[d]thiazol-2-ylamino)-5-(3,4-dihydroxybenzylidene)thiazol-4(5H)-one (2)Reaction time, 22 h; yield, 89.4%; greenish yellow solid; 1H-NMR (400 MHz, DMSO-d6) δ: 12.74 (br s, 1H, NH), 9.89 (br s, 1H, OH), 9.56 (br s, 1H, OH), 7.92 (d, 1H, J=7.6 Hz, 4′-H), 7.91 (d, 1H, J=7.6 Hz, 7′-H), 7.57 (s, 1H, vinylic H), 7.44 (t, 1H, J=7.6 Hz, 5′-H), 7.30 (t, 1H, J=7.6 Hz, 6′-H), 7.11 (s, 1H, 2″-H), 7.02 (d, 1H, J=7.2 Hz, 6″-H), 6.89 (d, 1H, J=7.2 Hz, 5″-H); 13C-NMR (100 MHz, DMSO-d6) δ: 169.0, 168.0, 160.0, 151.6, 149.5, 146.6, 134.5, 133.9, 127.1, 125.3, 125.1, 125.0, 122.7, 122.4, 120.1, 117.3, 117.0; HR-MS-ESI m/z C17H10N3O3S2 (M−H)− Calcd 368.0164, obsd 368.0169.
(Z)-2-(Benzo[d]thiazol-2-ylamino)-5-(4-hydroxy-3-methoxybenzylidene)thiazol-4(5H)-one (4)Reaction time, 17 h; yield, 73.6%; brown solid; 1H-NMR (400 MHz, DMSO-d6) δ: 12.47 (br s, 1H, NH), 10.00 (s, 1H, OH), 7.96 (d, 1H, J=7.6 Hz, 4′-H), 7.84 (d, 1H, J=8.0 Hz, 7′-H), 7.67 (s, 1H, vinylic H), 7.46 (t, 1H, J=8.0 Hz, 5′-H), 7.33 (t, 1H, J=8.0 Hz, 6′-H), 7.27 (s, 1H, 2″-H), 7.16 (d, 1H, J=8.0 Hz, 6″-H), 6.95 (d, 1H, J=8.4 Hz, 5″-H), 3.85 (s, 3H, OCH3); 13C-NMR (100 MHz, DMSO-d6) δ: 169.0, 167.9, 159.8, 151.5, 150.2, 148.7, 134.3, 133.8, 127.2, 125.4, 125.1, 122.8, 122.3, 120.7, 116.9, 115.1, 56.2; HR-MS-ESI m/z C18H12N3O3S2 (M−H)− Calcd 382.0320, obsd 382.0333.
(Z)-2-(Benzo[d]thiazol-2-ylamino)-5-(3-ethoxy-4-hydroxybbenzylidene)thiazol-4(5H)-one (5)Reaction time, 14 h; yield, 95.1%; yellow solid; 1H-NMR (400 MHz, DMSO-d6) δ: 9.91 (s, 1H, OH), 7.93 (d, 1H, J=8.0 Hz, 4′-H), 7.79 (d, 1H, J=7.6 Hz, 7′-H), 7.64 (s, 1H, vinylic H), 7.44 (t, 1H, J=7.6 Hz, 5′-H), 7.30 (t, 1H, J=7.6 Hz, 6′-H), 7.22 (s, 1H, 2″-H), 7.13 (d, 1H, J=8.0 Hz, 6″-H), 6.94 (d, 1H, J=8.0 Hz, 5″-H), 4.10 (q, 2H, J=6.8 Hz, OCH2), 1.38 (t, 3H, J=6.8 Hz, CH2CH3); 13C-NMR (100 MHz, DMSO-d6) δ: 169.0, 167.9, 159.8, 151.5, 150.4, 147.7, 134.3, 133.8, 127.1, 125.3, 125.0, 122.7, 122.1, 120.6, 117.0, 116.0, 64.5, 15.3; HR-MS-ESI m/z C19H14N3O3S2 (M−H)− Calcd 396.0477, obsd 396.0490.
(Z)-2-(Benzo[d]thiazol-2-ylamino)-5-(3-hydroxy-4-methoxybenzylidene)thiazol-4(5H)-one (6)Reaction time, 15 h; yield, 98.7%; yellow solid; 1H-NMR (400 MHz, DMSO-d6) δ: 12.77 (br s, 1H, NH), 9.58 (s, 1H, OH), 7.96 (d, 1H, J=7.6 Hz, 4′-H), 7.89 (d, 1H, J=8.0 Hz, 7′-H), 7.60 (s, 1H, vinylic H), 7.46 (t, 1H, J=8.0 Hz, 5′-H), 7.33 (t, 1H, J=7.6 Hz, 6′-H), 7.14 (d, 1H, J=8.4 Hz, 6″-H), 7.11 (s, 1H, 2″-H), 7.07 (d, 1H, J=8.4 Hz, 5″-H), 3.81 (s, 3H, OCH3); 13C-NMR (100 MHz, DMSO-d6) δ: 169.0, 167.9, 159.6, 151.5, 150.8, 147.6, 134.0, 133.9, 127.2, 126.6, 125.1, 124.6, 122.7, 122.4, 121.3, 116.8, 113.1, 56.4; HR-MS-ESI m/z C18H12N3O3S2 (M−H)− Calcd 382.0320, obsd 382.0334.
(Z)-2-(Benzo[d]thiazol-2-ylamino)-5-(4-methoxybenzylidene)thiazol-4(5H)-one (7)Reaction time, 14 h; yield, 82.9%; yellow solid; 1H-NMR (400 MHz, DMSO-d6) δ: 7.94 (d, 1H, J=7.6 Hz, 4′-H), 7.89 (d, 1H, J=7.6 Hz, 7′-H), 7.68 (s, 1H, vinylic H), 7.61 (d, 2H, J=8.0 Hz, 2″-H, 6″-H), 7.45 (t, 1H, J=7.6 Hz, 5′-H), 7.32 (t, 1H, J=7.6 Hz, 6′-H), 7.09 (d, 2H, J=8.0 Hz, 3″-H, 5″-H), 3.81 (s, 3H, OCH3); 13C-NMR (100 MHz, DMSO-d6) δ: 168.9, 167.9, 161.7, 159.4, 151.5, 133.9, 133.4, 133.0, 127.1, 126.4, 125.1, 122.7, 122.4, 121.6, 115.6, 56.1; HR-MS-ESI m/z C18H12N3O2S2 (M−H)− Calcd 366.0371, obsd 366.0383.
(Z)-2-(Benzo[d]thiazol-2-ylamino)-5-(3,4-dimethoxybenzylidene)thiazol-4(5H)-one (8)Reaction time, 15 h; yield, 85.5%; yellow solid; 1H-NMR (400 MHz, DMSO-d6) δ: 7.95 (d, 1H, J=7.6 Hz, 4′-H), 7.81 (d, 1H, J=8.0 Hz, 7′-H), 7.69 (s, 1H, vinylic H), 7.45 (t, 1H, J=8.0 Hz, 5′-H), 7.32 (t, 1H, J=8.0 Hz, 6′-H), 7.27–7.25 (m, 2H, 2″-H, 6″-H), 7.13 (d, 1H, J=8.0 Hz, 5″-H), 3.83 (s, 3H, OCH3), 3.80 (s, 3H, OCH3); 13C-NMR (100 MHz, DMSO-d6) δ: 169.0, 167.8, 159.6, 151.5, 151.5, 149.6, 133.8, 133.8, 127.2, 126.6, 125.1, 124.7, 122.8, 122.2, 122.0, 114.2, 112.8, 56.4, 56.1; HR-MS-ESI m/z C19H14N3O3S2 (M−H)− Calcd 396.0477, obsd 396.0490.
(Z)-2-(Benzo[d]thiazol-2-ylamino)-5-(2,4-dimethoxybenzylidene)thiazol-4(5H)-one (9)Reaction time, 17 h; yield, 92.2%; yellow solid; 1H-NMR (400 MHz, DMSO-d6) δ: 7.93 (d, 1H, J=7.6 Hz, 4′-H), 7.87–7.81 (m, 2H, vinylic H, 7′-H), 7.44 (d, 1H, J=8.8 Hz, 6″-H), 7.43 (t, 1H, J=7.2 Hz, 5′-H), 7.30 (t, 1H, J=7.6 Hz, 6′-H), 6.70 (dd, 1H, J=2.0, 8.8 Hz, 5″-H), 6.63 (d, 1H, J=2.0 Hz, 3″-H), 3.86 (s, 3H, OCH3), 3.80 (s, 3H, OCH3); 13C-NMR (100 MHz, DMSO-d6) δ: 169.0, 168.0, 163.8, 160.5, 159.9, 151.6, 133.8, 131.3, 128.1, 127.1, 125.0, 122.7, 122.3, 121.1, 115.2, 107.2, 99.3, 56.6, 56.3; HR-MS-ESI m/z C19H14N3O3S2 (M−H)− Calcd 396.0477, obsd 396.0490.
(Z)-2-(Benzo[d]thiazol-2-ylamino)-5-(3,4,5-trimethoxybenzylidene)thiazol-4(5H)-one (10)Reaction time, 17 h; yield, 98.2%; yellow solid; 1H-NMR (400 MHz, DMSO-d6) δ: 12.03 (br s, 1H, NH), 7.96 (d, 1H, J=8.0 Hz, 4′-H), 7.75 (d, 1H, J=8.0 Hz, 7′-H), 7.69 (s, 1H, vinylic H), 7.46 (t, 1H, J=7.6 Hz, 5′-H), 7.33 (t, 1H, J=7.6 Hz, 6′-H), 7.01 (s, 2H, 2″-H, 6″-H), 3.86 (s, 6H, 2×OCH3), 3.72 (s, 3H, 4″-OCH3); 13C-NMR (100 MHz, DMSO-d6) δ: 169.0, 167.7, 159.7, 153.9, 151.3, 140.0, 133.8, 133.7, 129.5, 127.3, 125.1, 124.2, 122.8, 122.2, 108.5, 60.9, 56.6; HR-MS-ESI m/z C20H16N3O4S2 (M−H)− Calcd 426.0582, obsd 426.0591.
(Z)-2-(Benzo[d]thiazol-2-ylamino)-5-(4-hydroxy-3,5-dimethoxybenzylidene)thiazol-4(5H)-one (11)Reaction time, 15 h; yield, 74.0%; yellow solid; 1H-NMR (400 MHz, DMSO-d6) δ: 12.74 (br s, 1H, NH), 9.39 (s, 1H, OH), 7.95 (d, 1H, J=8.0 Hz, 4′-H), 7.75 (d, 1H, J=7.6 Hz, 7′-H), 7.67 (s, 1H, vinylic H), 7.45 (t, 1H, J=7.6 Hz, 5′-H), 7.32 (t, 1H, J=7.6 Hz, 6′-H), 7.00 (s, 2H, 2″-H, 6″-H), 3.85 (s, 6H, 2×OCH3); 13C-NMR (100 MHz, DMSO-d6) δ: 169.1, 167.8, 159.9, 151.4, 148.9, 139.3, 134.5, 133.8, 127.3, 125.1, 124.2, 122.8, 122.1, 121.2, 109.0, 56.7; HR-MS-ESI m/z C19H14N3O4S2 (M−H)− Calcd 412.0426, obsd 412.0438.
(Z)-2-(Benzo[d]thiazol-2-ylamino)-5-(3-bromo-4-hydroxybenzylidene)thiazol-4(5H)-one (12)Reaction time, 16 h; yield, 87.7%; brown solid; 1H-NMR (400 MHz, DMSO-d6) δ: 11.17 (br s, 1H, OH), 7.92 (d, 1H, J=7.2 Hz, 4′-H), 7.85–7.80 (m, 2H, 7′-H, 2″-H), 7.61 (s, 1H, vinylic H), 7.49 (d, 1H, J=8.0 Hz, 6″-H), 7.43 (t, 1H, J=7.2 Hz, 5′-H), 7.30 (t, 1H, J=7.2 Hz, 6′-H), 7.10 (d, 1H, J=8.0 Hz, 5″-H); 13C-NMR (100 MHz, DMSO-d6) δ: 168.9, 167.7, 159.3, 156.9, 151.4, 136.3, 133.8, 132.3, 131.4, 127.1, 126.6, 125.0, 122.7, 122.3, 117.6, 110.9; HR-MS-ESI m/z C17H9BrN3O2S2 (M−H)− Calcd 429.9320, obsd 429.9323.
(Z)-2-(Benzo[d]thiazol-2-ylamino)-5-(3,5-dibromo-4-hydroxybenzylidene)thiazol-4(5H)-one (13)Reaction time, 15 h; yield, 93.0%; yellow solid; 1H-NMR (400 MHz, DMSO-d6) δ: 7.96 (d, 1H, J=8.0 Hz, 4′-H), 7.83 (s, 2H, 2″-H, 6″-H), 7.78 (brd, 1H, J=7.6 Hz, 7′-H), 7.61 (s, 1H, vinylic H), 7.46 (t, 1H, J=8.0 Hz, 5′-H), 7.33 (t, 1H, J=8.0 Hz, 6′-H); 13C-NMR (100 MHz, DMSO-d6) δ: 168.9, 167.5, 158.9, 153.3, 151.3, 134.7, 133.9, 130.7, 128.3, 127.3, 125.2, 124.3, 122.8, 122.2, 113.0; HR-MS-ESI m/z C17H8Br2N3O2S2 (M−H)− Calcd 507.8425, obsd 507.8438.
Synthetic Procedure of (Z)-2-(Benzo[d]thiazol-2-ylamino)-5-(2,4-dihydroxybenzylidene)thiazol-4(5H)-one (3, MHY2081)A solution of 15 (130 mg, 0.52 mmol) and 2-hydroxy-4-(tetrahydro-2H-pyran-2-yloxy)benzaldehyde (127.5 mg, 0.57 mmol) in EtOH (2.5 mL) was refluxed in the presence of piperidine (0.02 mL, 0.20 mmol) for 10 h. After cooling, water was added and the precipitates was filtered and washed with H2O/MeOH (1/1) to give 16 (140 mg) as a crude solid, which was used for the next step without further purification. To a solution of 16 (140 mg) in 1,4-dioxane (0.7 mL) and DMF (0.5 mL) was added 2 N-HCl solution (0.7 mL) at room temperature and the reaction mixture was stirred at the same temperature overnight and heated at 50°C for 2 d. After cooling, water was added and the precipitates generated were filtered and washed with a small amount of MeOH to give 3 (MHY2081, 100.3 mg, 52.1% from 15) as a solid.
Yellow solid; 1H-NMR (500 MHz, DMSO-d6) δ: 12.60 (br s, 1H, NH), 10.50 (br s, 1H, 2″-OH), 10.23 (br s, 1H, 4″-OH), 7.98 (s, 1H, vinylic H), 7.92 (d, 1H, J=7.5 Hz, 4′-H), 7.84 (br d, 1H, J=7.5 Hz, 7′-H), 7.43 (t, 1H, J=7.5 Hz, 5′-H), 7.35 (d, 1H, J=8.5 Hz, 6″-H), 7.30 (t, 1H, J=7.5 Hz, 6′-H), 6.47 (d, 1H, J=8.5 Hz, 5″-H), 6.45 (s, 1H, 3″-H); 13C-NMR (100 MHz, DMSO-d6) δ: 169.0, 168.2, 163.0, 162.5, 160.2, 151.6, 133.8, 131.1, 129.2, 127.0, 124.9, 122.6, 122.3, 118.2, 112.7, 109.1, 103.3; HR-MS-ESI m/z C17H10N3O3S2 (M−H)− Calcd 368.0164, obsd 368.0179 (Supplementary Fig. 2).
Docking Simulation of Tyrosinase and MHY2081Because of automated docking capability, AutoDock4.2 was used for the in silico protein–ligand docking simulation. The 3D structure of tyrosinase was used in the crystal structure of Agaricus bisporus (PDB ID: 2Y9X). As a docking pocket, predefined binding site of tyrosine was used. Docking simulations were performed between tyrosinase and MHY2081 or kojic acid. To prepare compounds for docking simulation, (1) 2D structures were converted into 3D structures, (2) charges were calculated, and (3) hydrogen atoms were added using the ChemOffice program (http://www.cambridgesoft.com). The prediction of possible hydrogen bonding residues between compounds and tyrosinase and generation of pharmacophores were analyzed with LigandScout 3.0 program.
Tyrosinase Activity Assay Using Mushroom TyrosinaseTyrosinase activity was measured using commercially available mushroom tyrosinase. Twenty microliters of mushroom tyrosinase (1000 U) were added to a 96-well microplate (Nunc, Denmark) in 200 µL assay buffer containing 1 mM L-tyrosine solution and 50 mM phosphate buffer (pH 6.5). Various concentrations of MHY2081 or kojic acid were added to the microplate. After incubating the plate at 25°C for 30 min, the dopachrome production was measured using a microplate reader (Berthold, Bad Wildbad, Germany) at 492 nm. Based on this experiment, log–linear curves with their equations were obtained to calculate IC50 values, the concentration at which the Y-axis equaled to 50% inhibition of tyrosinase activity.
Kinetic Analysis of Tyrosinase Inhibition by MHY2081Reaction mixture was prepared in a 96 well plate, in which 20 µL of L-tyrosine as a tyrosinase substrate, 10 µL of an aqueous mushroom tyrosinase solution (1000 U), and 50 mM potassium phosphate buffer (pH 6.5) were included. The initial rate of dopachrome formation from the reaction mixture was determined using a microplate reader (the increase in absorbance at 492 nm per min). The inhibitory kinetics of MHY2081 to tyrosinase was analyzed using Lineweaver–Burk plots. The Michaelis constant (Km) and maximal velocity (Vmax) of tyrosinase activity were determined by using Lineweaver–Burk plots with various concentrations of L-tyrosine substrate.
Cell CultureB16F10 murine melanoma cells were purchased from the Korea Cell Line Band. These cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 5% fetal bovine serum (FBS) and 1% penicillin streptomycin at 37°C in a humidified 95% air/5% CO2 atmosphere. For experiments, the cells were seeded in 6-well plates and MHY2081, kojic acid, or α-melanocyte-stimulating hormone (α-MSH) was treated for 48 h when the cells were grown to 70–80%.
Cell Viability Assay3-[4,5-Dimethylthiazol-2-yl]-3,5-diphenyltetrazolium bromide (MTT) assay was performed to examine cytototoxic effect of MHY2081. B16F10 melanoma cells were seeded into 96-well cell culture plates. After 24 h, the cells were exposed to MHY2081 at various concentrations for 48 h at 37°C under an atmosphere of 5% CO2. MTT reagent (1 mg/mL in phosphate buffered saline) was added to each well and incubated for 2 h. Afterward, the media was removed, and the formazan crystals were dissolved in DMSO. Absorbance was measured at 560 nm. Cell viability was evaluated as the relative absorbance of the control group.
Determination of Melanin Level in B16F10 Melanoma CellsCellular melanin content was measured based on the previously described method with a slight modification.9) B16F10 melanoma cells were seeded in 6-well dishes and allowed to grow to 70–80% confluence. Cells were incubated with kojic acid or MHY2081 for 2 h before α-MSH treatment. After MSH was treated, cells were incubated for 48 h. The cells were washed twice with phosphate buffered saline (PBS), detached by trypsin/ethylenediaminetetraacetic acid (EDTA), and dissolved in 80 µL of 1 N sodium hydroxide (NaOH) solution including 5% DMSO. The samples were incubated at 60°C for 1 h and mixed to solubilize melanin. Melanin concentration was analyzed by measuring absorbance at 405 nm.
Statistical AnalysisAll results are expressed as mean±standard error of the mean (S.E.M.). Treatments were compared using one-way ANOVA followed by Bonferroni test. A p values <0.05 were considered statistically significant.
To screen for effective tyrosinase inhibitors, we synthesized (Z)-2-(benzo[d]thiazol-2-ylamino)-5-(substituted benzylidene)thiazol-4(5H)-one derivatives based on the structure of well-known tyrosinase substrates L-tyrosine and L-DOPA (Table 1, Supplementary Fig. 1). In vitro tyrosinase activity assay using mushroom tyrosinase was performed to screen for the strongest tyrosinase inhibitor among thirteen derivatives. As shown in Table 1, introduction of two methoxy substituent at R3 and R5 positions on the phenyl ring of compound 1 (MHY 1887) increased tyrosinase inhibition 4-fold (33% inhibition) compared to compound 1 (8.6% inhibition) (Table 1). Surprisingly, introduction of a hydroxyl substituent at R2 position on the phenyl ring of compound 1 increased tyrosinase inhibition by 11-fold (97% inhibition, MHY2081) while introduction of a hydroxyl substituent at R3 position did not affect tyrosinase activity (Table 1). Introductions of hydroxyl, methoxy, ethoxy, or bromo substituent at other positions or/and replacement of the 4-hydroxyl group by a methoxy group on the phenyl ring of compound 1 did not affect tyrosinase activity (Table 1). Therefore, we focused on MHY2081 for further analysis. To further characterize the inhibitory effect of MHY2081 on tyrosinase, we examined dose-dependent inhibitory action of MHY2081 and calculated IC50 values. An in vitro tyrosinase activity assay using mushroom tyrosinase showed that MHY2081 efficiently inhibited tyrosinase activity with a dose-dependent manner. In addition, the inhibitory effect was greater than a well-known tyrosinase inhibitor kojic acid based on IC50 values (kojic acid=16.6 µM and MHY2081=1.8 µM) (Table 2), suggesting that MHY2081 efficiently blocks tyrosinase activity.
 | ||||||
|---|---|---|---|---|---|---|
| Compound | MHY | R2 | R3 | R4 | R5 | Tyrosinase inhibition (%) |
| 1 | 1887 | H | H | OH | H | 8.756±0.56 |
| 2 | 1886 | H | OH | OH | H | 6.580±5.03 |
| 3 | 2081 | OH | H | OH | H | 96.82±2.85 |
| 4 | 2079 | H | OMe | OH | H | NI |
| 5 | 1885 | H | OEt | OH | H | 7.983±3.46 |
| 6 | 1880 | H | OH | OMe | H | 5.771±2.64 |
| 7 | 1883 | H | H | OMe | H | NI |
| 8 | 1881 | H | OMe | OMe | H | NI |
| 9 | 1888 | OMe | H | OMe | H | NI |
| 10 | 1942 | H | OMe | OMe | OMe | NI |
| 11 | 1884 | H | OMe | OH | OMe | 33.17±2.23 |
| 12 | 1879 | H | Br | OH | Br | 3.591±1.14 |
| 13 | 1882 | H | Br | OH | Br | 3.591±1.14 |
Tyrosinase inhibition was measured using mushroom tyrosinase and L-tyrosine as the substrate. Results are expressed as percentage of control and each value represents the mean±S.E.M. Repeated experiments showed the similar results. NI represents no inhibition.
| Compound | MHY | Concentration (µM) | Tyrosinase inhibition (%) | IC50 (µM) |
|---|---|---|---|---|
| 3 | 2081 | 0.2 | 24% | 1.80±2.53 |
| 2.5 | 57% | |||
| 10 | 70% | |||
| Kojic acid | 5 | 21% | 16.6±2.3 | |
| 10 | 33% | |||
| 20 | 58% |
IC50 represents 50% inhibitory concentration.
Because some well-known tyrosinase inhibitors including hydroquinone and kojic acids have cytotoxicity, we performed a MTT assay and confirmed that MHY2081 had no cytotoxicity up to 2 µM concentration in B16F10 melanoma cells (Fig. 1a). To further confirm whether MHY2081 inhibits intracellular tyrosinase function, we measured tyrosinase activity in B16F10 melanoma cells. A known tyrosinase activator α-MSH notably increased cellular tyrosinase activity (Fig. 1b). However, MHY2081 treatment significantly decreased cellular tyrosinase activity in α-MSH-treated melanoma cells with a dose-dependent manner (Fig. 1b). Tyrosinase is a key factor for melanin synthesis.1) To examine whether MHY2081-mediated inhibition of tyrosinase affects cellular melanin content, we measured melanin concentration in B16F10 melanoma cells. As expected, α-MSH increased cellular melanin concentration. However, MHY2081 treatment at 0.5 µM markedly reduced melanin content in α-MSH treated cells to the levels comparable to the control group without α-MSH treatment (Fig. 1c). Although MHY2081 at 1 and 2 µM further reduced melanin content, it was not dramatic. Therefore, MHY2081 at 0.5 µM may be enough for decreasing cellular melanin content.
(a) B16F10 melanoma cells were treated with various concentrations of MHY2081 for 48 h and then MTT assay was performed (n=3/group). Data are expressed as the percent of cell viability compared to a non-treated group. (b) Tyrosinase activity in B16F10 cells treated with MHY2081. B16F10 melanoma cells were pre-treated with MHY2081 or kojic acid for 2h and then the cells were treated with α-MSH (1 µM) for 48 h to stimulate melanogenesis (n=4/group). (c) B16F10 melanoma cells were pre-treated with MHY2081 or kojic acid for 2h and the cells were stimulated with α-MSH (1 µM) for 48 h. Melanin concentrations were determined using a microplate reader (n=4/group). The data are expressed as a mean±S.E.M. (n=4). * p<0.05 and ** p<0.01 compared to the α-MSH-treated control group. ## p<0.01 compared to the control group without α-MSH treatment.
Because MHY2081 was synthesized based on the structure of tyrosine with somewhat similarity, we examined whether MHY2081 directly binds to tyrosinase as an inhibitory mechanism using docking simulation. Docking simulation using AutoDock4.2 can calculate binding affinity between MHY2081 and tyrosinase. The structure images of tyrosinase from docking simulation showed that MHY2081 can directly bind to tyrosinase (Figs. 2a, b). Furthermore, binding energy for MHY2081 was greater than kojic acid (Fig. 2b), indicating that the binding affinity of MHY2081 to tyrosinase is greater than that of kojic acid (tyrosinase docking score-kojic acid: −4.2 kcal/mol, MHY2081: −6.1 kcal/mol). To search for the binding residues of tyrosinase that correlated to kojic acid or new compound MHY2081, a LigandScout 3.1 program was applied. While kojic acid forms a hydrogen bond with a MET280 residue of tyrosinase and an aromatic interaction with HIS263, MHY2081 forms three hydrogen bonds with HIS61, HIS263, and ASN260 and a hydrophobic interaction with VAL283 (Figs. 2c, d), which contribute to higher affinity of MHY2081 to tyrosinase.
(a–d) Computational structure prediction for docking simulation between mushroom tyrosinase and MHY2081. (a) Predicted 3D structure of mushroom tyrosinase. The black lines indicate the binding site for MHY2081. (b) Magnified image of the binding site of MHY2081 in tyrosinase and a small table shows predicted binding affinity of MHY2081 and kojic acid with tyrosinase. The binding sites of (c) kojic acid and (d) MHY2081 with tyrosinase are shown.
Based on docking simulation, we hypothesized that MHY2081 may competitively bind to tyrosinase with tyrosine. We analyzed the mode of inhibition of tyrosinase by Lineweaver–Burk plot analysis. As the concentrations of MHY2081 increased, Km values gradually increased while Vmax values were unchanged (Fig. 3), suggesting that MHY2081 is a competitive tyrosinase inhibitor.
Lineweaver–Burk plot of MHY2081 on mushroom tyrosinase. Data were obtained as mean values of 1/V, inverse of the increase of absorbance at a wavelength 492 nm per min, of three independent tests with different concentrations of L-tyrosine as a substrate.
In conclusion, MHY2081 competitively binds to tyrosinase with its substrate and inhibits tyrosinase activity without cytotoxicity, indicating that MHY2081 has an antimelanogenic effect. Therefore, MHY2081 is a novel compound for whitening cosmetics or treating pigmentation disorders.
Reagents and conditions: (a) acetone, reflux, 3 h, 92.8%; (b) NH4SCN, EtOH, reflux, 4 h, 68.4%; (c) NaOAc, AcOH, reflux, 10–35 h for 1–2 and 4–13; piperidine, EtOH, reflux, 10 h for 16; (d) 2 N-HCl, 1,4-dioxane, DMF, rt, overnight and then 50°C, 2 d for 3 (MHY2081).
This work was supported by the National Research Foundation of Korea (NRF) Grant funded by the Korean government (MSIP) (Grant No. 2009-0083538) and Research Program through NRF funded by the Ministry of Education, Science and Technology (Grant No. NRF-2013R1A1A2009949). We thank Aging Tissue Bank for providing materials and research information.
The authors declare no conflict of interest.
The online version of this article contains supplementary materials.
