(Z)-2-(Benzo[d]thiazol-2-ylamino)-5-(substituted benzylidene) thiazol-4(5H)-one derivatives as novel tyrosinase inhibitors

Article abstract of DOI:10.1248/bpb.b15-00300

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, on

Full text of DOI:10.1248/bpb.b15-00300

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Vol. 38, No. 8
Biol. Pharm. Bull. 38, 1227–1233 (2015)
1227
Regular Article
(Z)-2-(Benzo[d]thiazol-2-ylamino)-5-(substituted benzylidene)thiazol-
4(5H)-one Derivatives as Novel Tyrosinase Inhibitors
Kyeong Ha Kang,a,# Bonggi Lee,a,# Sujin Son,a,b Hwi Young Yun,a,b Kyoung Mi Moon,a
Hyoung Oh Jeong,a Dae Hyun Kim,a,b Eun Kyeong Lee,a Yeon Ja Choi,a Do Hyun Kim,a,b
,a,b
,a,b
Pusoon Chun,c Hyung Ryong Moon,* and Hae Young Chung*
b
a College of Pharmacy, Pusan National University; Busan 609–735, Republic of Korea: and ꢀMolecularꢀInflammationꢀ
Research Centor for Ageing Intervention (MRCA), Pusan National University; Busan 609–735, Republic of
c
Korea: and College of Pharmacy, Inje University; Gimhae, Gyeongnam 621–749, Republic of Korea.
Received April 2, 2015; accepted May 18, 2015
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 tyrosi-
nase 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 hydro-
phobic 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 ty-
rosinase inhibitor for prevention and treatment of pigmentation disorders.
Key words skin; melanogenesis; tyrosinase inhibitor
The skin is a protective barrier against UV irradiation. Me-
lanogenesis 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 pho-
toaging.1) In addition, melanin accumulation is able to increase
MATERIALS AND METHODS
Synthesis of Compounds (Chart 1)
N-(Benzo[d]thiazol-2-yl)-2-chloroacetamide (14)
To a stirred solution of 2-aminobenzothiazole (6.0g,
pigmentation disorders such as melasma, freckles, and solar 39.95mmol) in acetone (80mL) was added a solution of chlo-
lentigo.2,3) Therefore, various compounds that prevent mela- roacetyl chloride (6.35mL, 79.84mmol) in acetone (20mL)
nogenesis have been screened for cosmetics and preventing at 0°C and the reaction mixture was refluxed for 3h. After
pigmentation disorders.
cooling, the volatile solvent was removed under reduced pres-
Melanogenesis is the process by which melanin is secreted sure to give a residue, which was neutralized using aqueous
by melanocytes located in the basal layer of the epidermis. sodium bicarbonate solution to pH 7. The precipitates gener-
Tyrosinase, a copper containing metalloenzyme, is one of ated were filtered and washed with water to give 14 (8.40g,
the enzymes responsible for skin pigmentation in mammals.4) 92.8%) as a solid.
Tyrosinase catalyses two rate-limiting steps in melanogenesis:
1H-NMR (400MHz, dimethyl sulfoxide (DMSO)-d6) δ:
the hydroxylation of tyrosine to 3,4-dihydroxyphenylalanine 7.97 (d, 1H, J=7.6Hz), 7.73 (d, 1H, J=8.4Hz), 7.41 (t, 1H,
(DOPA) and the oxidation of DOPA to DOPAquinone. There- J=8.0Hz), 7.29 (t, 1H, J=8.0Hz), 4.43 (s, 2H); 13C-NMR
fore, tyrosinase predominantly regulates melanin production5) (100MHz, DMSO-d6) δ: 166.6, 158.2, 149.1, 132.1, 126.9,
and tyrosinase inhibitors are widely used for cosmetics and 124.5, 122.5, 121.4, 43.2.
treatments for pigmentation disorders compared to other
melanogenesis inhibitors. However, only a few tyrosinase in-
2-(Benzo[d]thiazol-2-ylamino)thiazol-4(5H)-one (15)
A suspension of 14 (4.0g, 17.65mmol) and ammonium
hibitors are currently used in cosmetics and medical products thiocyanate (2.7g, 35.47mmol) in ethyl alcohol (40mL) was
because of their cytotoxicity and lack of selectivity and stabil- refluxed for 4h. After cooling, the reaction mixture was fil-
ity.6–8)
tered and washed with cold EtOH to give 15 (3.01g, 68.4%)
Based on the structure of tyrosinase substrates L-tyrosin as a solid.
and L-DOPA, we synthesized a series of (Z)-2-(benzo[d]-
1H-NMR (400MHz, DMSO-d6) δ: 12.27 (s, 1H), 7.93 (d,
thiazol-2-ylamino)-5-(substituted benzylidene)thiazol-4(5H)- 1H, J=8.0Hz), 7.76 (d, 1H, J=8.0Hz), 7.42 (t, 1H, J=8.0Hz),
one derivatives to screen for novel tyrosinase inhibitors. Here, 7.30 (t, 1H, J=8.0Hz), 4.03 (s, 2H); 13C-NMR (100MHz,
our data show that MHY2081 is the strongest tyrosinase in- DMSO-d6) δ: 175.0, 169.6, 166.8, 151.5, 133.7, 127.0, 124.9,
hibitor among 13 synthesized compounds.
122.6, 122.0, 35.9.
General Procedure of (Z)-2-(Benzo[d]thiazol-2-ylamino)-5-
(substituted benzylidene)thiazol-4(5H)-one Analogs (Com-
# These authors contributed equally to this work.
*To whom correspondence should be addressed. e-mail: mhr108@pusan.ac.kr; hyjung@pusan.ac.kr
© 2015 The Pharmaceutical Society of Japan

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Reagents and conditions: (a) acetone, reflux, 3h, 92.8%; (b) NH4SCN, EtOH, reflux, 4h, 68.4%; (c) NaOAc, AcOH, reflux, 10–35h for 1–2 and 4–13; piperidine, EtOH,
reflux, 10h for 16; (d) 2N-HCl, 1,4-dioxane, DMF, rt, overnight and then 50°C, 2d for 3 (MHY2081).
Chart 1. Synthetic Scheme for Compounds 1–13
pounds 1–2 and 4–13)
151.5, 150.2, 148.7, 134.3, 133.8, 127.2, 125.4, 125.1, 122.8,
A suspension of 15 (80mg, 0.32mmol), a variety of substi- 122.3, 120.7, 116.9, 115.1, 56.2; HR-MS-ESI m/z C18H12N3O3S2
tuted benzaldehyde (1.1eq.), and NaOAc (3.0eq.) in acetic acid (M−H)− Calcd 382.0320, obsd 382.0333.
(1.0mL) was refluxed for 10–35h. After cooling, water was
(Z)-2-(Benzo[d]thiazol-2-ylamino)-5-(3-ethoxy-4-hydroxy-
added and the precipitates generated were filtered and washed bbenzylidene)thiazol-4(5H)-one (5)
1
with water or MeOH/H2O (8:1–1:2) to give compounds 1–2
and 4–13 as solids.
Reaction time, 14h; yield, 95.1%; yellow solid; H-NMR
(400MHz, DMSO-d6) δ: 9.91 (s, 1H, OH), 7.93 (d, 1H,
(Z)-2-(Benzo[d]thiazol-2-ylamino)-5-(4-hydroxybenzyl- J=8.0Hz, 4′-H), 7.79 (d, 1H, J=7.6Hz, 7′-H), 7.64 (s, 1H,
idene)thiazol-4(5H)-one (1) vinylic H), 7.44 (t, 1H, J=7.6Hz, 5′-H), 7.30 (t, 1H, J=7.6Hz,
1
Reaction time, 10h; yield, 66.5%; yellow solid; H-NMR 6′-H), 7.22 (s, 1H, 2″-H), 7.13 (d, 1H, J=8.0Hz, 6″-H), 6.94
(400MHz, DMSO-d6) δ: 12.76 (br s, 1H, NH), 10.35 (s, 1H, (d, 1H, J=8.0Hz, 5″-H), 4.10 (q, 2H, J=6.8Hz, OCH2), 1.38
OH), 7.96 (d, 1H, J=7.6Hz, 4′-H), 7.91 (d, 1H, J=8.4Hz, 7′-H), (t, 3H, J=6.8Hz, CH2CH3); 13C-NMR (100MHz, DMSO-d6)
7.66 (s, 1H, vinylic H), 7.53 (d, 2H, J=7.6Hz, 2″-H, 6″-H), 7.46 δ: 169.0, 167.9, 159.8, 151.5, 150.4, 147.7, 134.3, 133.8, 127.1,
(t, 1H, J=8.0Hz, 5′-H), 7.33 (t, 1H, J=7.6Hz, 6′-H), 6.93 (d, 125.3, 125.0, 122.7, 122.1, 120.6, 117.0, 116.0, 64.5, 15.3; HR-
2H, J=7.6Hz, 3″-H, 5″-H); 13C-NMR (100MHz, DMSO-d6) MS-ESI m/z C19H14N3O3S2 (M−H)− Calcd 396.0477, obsd
δ: 169.0, 167.9, 160.7, 159.6, 151.5, 134.0, 133.8, 133.4, 127.1, 396.0490.
125.1, 124.8, 122.7, 122.5, 120.3, 117.1; high resolution (HR)-
MS-electrospray ionization (ESI) m/z C17H10N3O2S2 (M−H)− oxybenzylidene)thiazol-4(5H)-one (6)
Calcd 352.0214, obsd 352.0226. Reaction time, 15h; yield, 98.7%; yellow solid; H-NMR
(Z)-2-(Benzo[d]thiazol-2-ylamino)-5-(3,4-dihydroxy- (400MHz, DMSO-d6) δ: 12.77 (brs, 1H, NH), 9.58 (s, 1H,
benzylidene)thiazol-4(5H)-one (2) OH), 7.96 (d, 1H, J=7.6Hz, 4′-H), 7.89 (d, 1H, J=8.0Hz,
(Z)-2-(Benzo[d]thiazol-2-ylamino)-5-(3-hydroxy-4-meth-
1
Reaction time, 22h; yield, 89.4%; greenish yellow solid; 7′-H), 7.60 (s, 1H, vinylic H), 7.46 (t, 1H, J=8.0Hz, 5′-H), 7.33
1H-NMR (400MHz, DMSO-d6) δ: 12.74 (brs, 1H, NH), 9.89 (t, 1H, J=7.6Hz, 6′-H), 7.14 (d, 1H, J=8.4Hz, 6″-H), 7.11 (s,
(brs, 1H, OH), 9.56 (brs, 1H, OH), 7.92 (d, 1H, J=7.6Hz, 1H, 2″-H), 7.07 (d, 1H, J=8.4Hz, 5″-H), 3.81 (s, 3H, OCH3);
4′-H), 7.91 (d, 1H, J=7.6Hz, 7′-H), 7.57 (s, 1H, vinylic H), 13C-NMR (100MHz, DMSO-d6) δ: 169.0, 167.9, 159.6, 151.5,
7.44 (t, 1H, J=7.6Hz, 5′-H), 7.30 (t, 1H, J=7.6Hz, 6′-H), 150.8, 147.6, 134.0, 133.9, 127.2, 126.6, 125.1, 124.6, 122.7,
7.11 (s, 1H, 2″-H), 7.02 (d, 1H, J=7.2Hz, 6″-H), 6.89 (d, 1H, 122.4, 121.3, 116.8, 113.1, 56.4; HR-MS-ESI m/z C18H12N3O3S2
J=7.2Hz, 5″-H); 13C-NMR (100MHz, DMSO-d6) δ: 169.0, (M−H)− Calcd 382.0320, obsd 382.0334.
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 idene)thiazol-4(5H)-one (7)
C17H10N3O3S2 (M−H)− Calcd 368.0164, obsd 368.0169.
Reaction time, 14h; yield, 82.9%; yellow solid; H-NMR
(Z)-2-(Benzo[d]thiazol-2-ylamino)-5-(4-hydroxy-3-methoxy- (400MHz, DMSO-d6) δ: 7.94 (d, 1H, J=7.6Hz, 4′-H), 7.89
benzylidene)thiazol-4(5H)-one (4) (d, 1H, J=7.6Hz, 7′-H), 7.68 (s, 1H, vinylic H), 7.61 (d, 2H,
(Z)-2-(Benzo[d]thiazol-2-ylamino)-5-(4-methoxybenzyl-
1
1
Reaction time, 17h; yield, 73.6%; brown solid; H-NMR J=8.0Hz, 2″-H, 6″-H), 7.45 (t, 1H, J=7.6Hz, 5′-H), 7.32 (t, 1H,
(400MHz, DMSO-d6) δ: 12.47 (brs, 1H, NH), 10.00 (s, 1H, J=7.6Hz, 6′-H), 7.09 (d, 2H, J=8.0Hz, 3″-H, 5″-H), 3.81 (s,
OH), 7.96 (d, 1H, J=7.6Hz, 4′-H), 7.84 (d, 1H, J=8.0Hz, 3H, OCH3); 13C-NMR (100MHz, DMSO-d6) δ: 168.9, 167.9,
7′-H), 7.67 (s, 1H, vinylic H), 7.46 (t, 1H, J=8.0Hz, 5′-H), 161.7, 159.4, 151.5, 133.9, 133.4, 133.0, 127.1, 126.4, 125.1,
7.33 (t, 1H, J=8.0Hz, 6′-H), 7.27 (s, 1H, 2″-H), 7.16 (d, 1H, 122.7, 122.4, 121.6, 115.6, 56.1; HR-MS-ESI m/z C18H12N3O2S2
J=8.0Hz, 6″-H), 6.95 (d, 1H, J=8.4Hz, 5″-H), 3.85 (s, 3H, (M−H)− Calcd 366.0371, obsd 366.0383.
OCH3); 13C-NMR (100MHz, DMSO-d6) δ: 169.0, 167.9, 159.8,
(Z)-2-(Benzo[d]thiazol-2-ylamino)-5-(3,4-dimethoxybenzyl-

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idene)thiazol-4(5H)-one (8)
vinylic H), 7.46 (t, 1H, J=8.0Hz, 5′-H), 7.33 (t, 1H, J=8.0Hz,
1
Reaction time, 15h; yield, 85.5%; yellow solid; H-NMR 6′-H); 13C-NMR (100MHz, DMSO-d6) δ: 168.9, 167.5, 158.9,
(400MHz, DMSO-d6) δ: 7.95 (d, 1H, J=7.6Hz, 4′-H), 7.81 153.3, 151.3, 134.7, 133.9, 130.7, 128.3, 127.3, 125.2, 124.3,
(d, 1H, J=8.0Hz, 7′-H), 7.69 (s, 1H, vinylic H), 7.45 (t, 1H, 122.8, 122.2, 113.0; HR-MS-ESI m/z C17H8Br2N3O2S2 (M−H)−
J=8.0Hz, 5′-H), 7.32 (t, 1H, J=8.0Hz, 6′-H), 7.27–7.25 (m, Calcd 507.8425, obsd 507.8438.
2H, 2″-H, 6″-H), 7.13 (d, 1H, J=8.0Hz, 5″-H), 3.83 (s, 3H,
OCH3), 3.80 (s, 3H, OCH3); 13C-NMR (100MHz, DMSO-d6) 5-(2,4-dihydroxybenzylidene)thiazol-4(5H)-one (3, MHY2081)
δ: 169.0, 167.8, 159.6, 151.5, 151.5, 149.6, 133.8, 133.8, 127.2, A solution of 15 (130mg, 0.52mmol) and 2-hydroxy-4-(tetra-
Synthetic Procedure of (Z)-2-(Benzo[d]thiazol-2-ylamino)-
126.6, 125.1, 124.7, 122.8, 122.2, 122.0, 114.2, 112.8, 56.4, hydro-2H-pyran-2-yloxy)benzaldehyde (127.5mg, 0.57mmol)
56.1; HR-MS-ESI m/z C19H14N3O3S2 (M−H)− Calcd 396.0477, in EtOH (2.5mL) was refluxed in the presence of piperidine
obsd 396.0490.
(Z)-2-(Benzo[d]thiazol-2-ylamino)-5-(2,4-dimethoxybenzyl- and the precipitates was filtered and washed with H2O/MeOH
idene)thiazol-4(5H)-one (9) (1/1) to give 16 (140mg) as a crude solid, which was used for
(0.02mL, 0.20mmol) for 10h. After cooling, water was added
1
Reaction time, 17h; yield, 92.2%; yellow solid; H-NMR the next step without further purification. To a solution of 16
(400MHz, DMSO-d6) δ: 7.93 (d, 1H, J=7.6Hz, 4′-H), 7.87–7.81 (140mg) in 1,4-dioxane (0.7mL) and DMF (0.5mL) was added
(m, 2H, vinylic H, 7′-H), 7.44 (d, 1H, J=8.8Hz, 6″-H), 7.43 (t, 2N-HCl solution (0.7mL) at room temperature and the reaction
1H, J=7.2Hz, 5′-H), 7.30 (t, 1H, J=7.6Hz, 6′-H), 6.70 (dd, 1H, mixture was stirred at the same temperature overnight and
J=2.0, 8.8Hz, 5″-H), 6.63 (d, 1H, J=2.0Hz, 3″-H), 3.86 (s, 3H, heated at 50°C for 2d. After cooling, water was added and the
OCH3), 3.80 (s, 3H, OCH3); 13C-NMR (100MHz, DMSO-d6) precipitates generated were filtered and washed with a small
δ: 169.0, 168.0, 163.8, 160.5, 159.9, 151.6, 133.8, 131.3, 128.1, amount of MeOH to give 3 (MHY2081, 100.3mg, 52.1% from
127.1, 125.0, 122.7, 122.3, 121.1, 115.2, 107.2, 99.3, 56.6, 56.3; 15) as a solid.
1
HR-MS-ESI m/z C19H14N3O3S2 (M−H)− Calcd 396.0477, obsd
Yellow solid; H-NMR (500MHz, DMSO-d6) δ: 12.60 (br
s, 1H, NH), 10.50 (brs, 1H, 2″-OH), 10.23 (brs, 1H, 4″-OH),
396.0490.
(Z)-2-(Benzo[d]thiazol-2-ylamino)-5-(3,4,5-trimethoxy- 7.98 (s, 1H, vinylic H), 7.92 (d, 1H, J=7.5Hz, 4′-H), 7.84
benzylidene)thiazol-4(5H)-one (10) (brd, 1H, J=7.5Hz, 7′-H), 7.43 (t, 1H, J=7.5Hz, 5′-H), 7.35
1
Reaction time, 17h; yield, 98.2%; yellow solid; H-NMR (d, 1H, J=8.5Hz, 6″-H), 7.30 (t, 1H, J=7.5Hz, 6′-H), 6.47 (d,
(400MHz, DMSO-d6) δ: 12.03 (brs, 1H, NH), 7.96 (d, 1H, 1H, J=8.5Hz, 5″-H), 6.45 (s, 1H, 3″-H); 13C-NMR (100MHz,
J=8.0Hz, 4′-H), 7.75 (d, 1H, J=8.0Hz, 7′-H), 7.69 (s, 1H, DMSO-d6) δ: 169.0, 168.2, 163.0, 162.5, 160.2, 151.6, 133.8,
vinylic H), 7.46 (t, 1H, J=7.6Hz, 5′-H), 7.33 (t, 1H, J=7.6Hz, 131.1, 129.2, 127.0, 124.9, 122.6, 122.3, 118.2, 112.7, 109.1,
6′-H), 7.01 (s, 2H, 2″-H, 6″-H), 3.86 (s, 6H, 2×OCH3), 3.72 103.3; HR-MS-ESI m/z C17H10N3O3S2 (M−H)− Calcd 368.0164,
(s, 3H, 4″-OCH3); 13C-NMR (100MHz, DMSO-d6) δ: 169.0, obsd 368.0179 (Supplementary Fig. 2).
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 cause of automated docking capability, AutoDock4.2 was
C20H16N3O4S2 (M−H)− Calcd 426.0582, obsd 426.0591.
used for the in silico protein–ligand docking simulation. The
(Z)-2-(Benzo[d]thiazol-2-ylamino)-5-(4-hydroxy-3,5- 3D structure of tyrosinase was used in the crystal structure
dimethoxybenzylidene)thiazol-4(5H)-one (11) of Agaricus bisporus (PDB ID: 2Y9X). As a docking pocket,
Docking Simulation of Tyrosinase and MHY2081 Be-
1
Reaction time, 15h; yield, 74.0%; yellow solid; H-NMR predefined binding site of tyrosine was used. Docking simula-
(400MHz, DMSO-d6) δ: 12.74 (brs, 1H, NH), 9.39 (s, 1H, tions were performed between tyrosinase and MHY2081 or
OH), 7.95 (d, 1H, J=8.0Hz, 4′-H), 7.75 (d, 1H, J=7.6Hz, kojic acid. To prepare compounds for docking simulation, (1)
7′-H), 7.67 (s, 1H, vinylic H), 7.45 (t, 1H, J=7.6Hz, 5′-H), 7.32 2D structures were converted into 3D structures, (2) charges
(t, 1H, J=7.6Hz, 6′-H), 7.00 (s, 2H, 2″-H, 6″-H), 3.85 (s, 6H, were calculated, and (3) hydrogen atoms were added using
2×OCH3); 13C-NMR (100MHz, DMSO-d6) δ: 169.1, 167.8, the ChemOffice program (http://www.cambridgesoft.com).
159.9, 151.4, 148.9, 139.3, 134.5, 133.8, 127.3, 125.1, 124.2, The prediction of possible hydrogen bonding residues between
122.8, 122.1, 121.2, 109.0, 56.7; HR-MS-ESI m/z C19H14N3O4S2 compounds and tyrosinase and generation of pharmacophores
(M−H)− Calcd 412.0426, obsd 412.0438.
were analyzed with LigandScout 3.0 program.
Tyrosinase Activity Assay Using Mushroom Tyrosinase
Tyrosinase activity was measured using commercially avail-
(Z)-2-(Benzo[d]thiazol-2-ylamino)-5-(3-bromo-4-hydroxy-
benzylidene)thiazol-4(5H)-one (12)
1
Reaction time, 16h; yield, 87.7%; brown solid; H-NMR able mushroom tyrosinase. Twenty microliters of mushroom
(400MHz, DMSO-d6) δ: 11.17 (brs, 1H, OH), 7.92 (d, 1H, tyrosinase (1000U) were added to a 96-well microplate (Nunc,
J=7.2Hz, 4′-H), 7.85–7.80 (m, 2H, 7′-H, 2″-H), 7.61 (s, 1H, Denmark) in 200µL assay buffer containing 1mM L-tyrosine
vinylic H), 7.49 (d, 1H, J=8.0Hz, 6″-H), 7.43 (t, 1H, J=7.2Hz, solution and 50mM phosphate buffer (pH 6.5). Various con-
5′-H), 7.30 (t, 1H, J=7.2Hz, 6′-H), 7.10 (d, 1H, J=8.0Hz, centrations of MHY2081 or kojic acid were added to the
5″-H); 13C-NMR (100MHz, DMSO-d6) δ: 168.9, 167.7, 159.3, microplate. After incubating the plate at 25°C for 30min,
156.9, 151.4, 136.3, 133.8, 132.3, 131.4, 127.1, 126.6, 125.0, the dopachrome production was measured using a microplate
122.7, 122.3, 117.6, 110.9; HR-MS-ESI m/z C17H9BrN3O2S2 reader (Berthold, Bad Wildbad, Germany) at 492nm. Based
(M−H)− Calcd 429.9320, obsd 429.9323.
(Z)-2-(Benzo[d]thiazol-2-ylamino)-5-(3,5-dibromo-4- were obtained to calculate IC50 values, the concentration at
hydroxybenzylidene)thiazol-4(5H)-one (13) which the Y-axis equaled to 50% inhibition of tyrosinase ac-
Reaction time, 15h; yield, 93.0%; yellow solid; H-NMR tivity.
on this experiment, log–linear curves with their equations
1
(400MHz, DMSO-d6) δ: 7.96 (d, 1H, J=8.0Hz, 4′-H), 7.83 (s,
Kinetic Analysis of Tyrosinase Inhibition by MHY2081
2H, 2″-H, 6″-H), 7.78 (brd, 1H, J=7.6Hz, 7′-H), 7.61 (s, 1H, Reaction mixture was prepared in a 96 well plate, in which

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20µL of L-tyrosine as a tyrosinase substrate, 10µL of an aque- chased from the Korea Cell Line Band. These cells were
ous mushroom tyrosinase solution (1000U), and 50mM potas- cultured in Dulbecco’s modified Eagle’s medium (DMEM)
sium phosphate buffer (pH 6.5) were included. The initial rate supplemented with 5% fetal bovine serum (FBS) and 1% peni-
of dopachrome formation from the reaction mixture was de- cillin streptomycin at 37°C in a humidified 95% air/5% CO2
termined using a microplate reader (the increase in absorbance atmosphere. For experiments, the cells were seeded in 6-well
at 492nm per min). The inhibitory kinetics of MHY2081 to plates and MHY2081, kojic acid, or α-melanocyte-stimulating
tyrosinase was analyzed using Lineweaver–Burk plots. The hormone (α-MSH) was treated for 48h when the cells were
Michaelis constant (Km) and maximal velocity (Vmax) of ty- grown to 70–80%.
rosinase activity were determined by using Lineweaver–Burk
plots with various concentrations of L-tyrosine substrate.
Cell Viability Assay 3-[4,5-Dimethylthiazol-2-yl]-3,5-di-
phenyltetrazolium bromide (MTT) assay was performed to
Cell Culture B16F10 murine melanoma cells were pur- examine cytototoxic effect of MHY2081. B16F10 melanoma
Fig. 1. The Inhibitory Effects of MHY2081 on Tyrosinase and Melanin Accumulation
(a) B16F10 melanoma cells were treated with various concentrations of MHY2081 for 48h 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 (1µM) for 48h 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 (1µM) for 48h. 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.
Table 1. Substitution Pattern of the Substituted (Z)-2-(Benzo[d]thiazol-2-ylamino)-5-(substituted benzylidene)thiazol-4(5H)-one Derivatives
Compound
MHY
R2
R3
R4
R5
Tyrosinase inhibition (%)
1
2
1887
1886
2081
2079
1885
1880
1883
1881
1888
1942
1884
1879
1882
H
H
H
OH
H
OH
OH
H
H
8.756 0.56
6.580 5.03
96.82 2.85
NI
3
OH
H
OH
H
4
OMe
OEt
OH
H
OH
H
5
H
OH
H
7.983 3.46
5.771 2.64
NI
6
H
OMe
OMe
OMe
OMe
OMe
OH
H
7
H
H
8
H
OMe
H
H
NI
9
OMe
H
H
NI
10
11
12
13
OMe
OMe
Br
OMe
OMe
Br
Br
NI
H
33.17 2.23
3.591 1.14
3.591 1.14
H
OH
H
Br
OH
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.