Inhibition of tyrosinase activity by N,N-unsubstituted selenourea derivatives

Article abstract of DOI:10.1248/bpb.28.838

This study investigated inhibitory effects of N,N-unsubstituted selenourea derivatives on tyrosinase activity. Three types of N,N-unsubstituted selenoureas derivatives exhibited inhibitory effect on dopa (3,4-dihydroxyphenylalanine) oxidase activity of mushroom tyrosinase. Compound D at a concentration of 200/M exhibited 55.5% of inhibition on dopa oxidase activity of mushroom tyrosinase. This inhibitory effect was higher than that of kojic acid (39.4%), a well known tyrosinase inhibitor. Moreover, the compound D identified as a noncompetitive inhibitor by Lineweaver-Burk plot analysis. In addition, compound D also inhibited the melanin production in melan-a cells.

Full text of DOI:10.1248/bpb.28.838

838
Biol. Pharm. Bull. 28(5) 838—840 (2005)
Vol. 28, No. 5
Inhibition of Tyrosinase Activity by N,N-Unsubstituted Selenourea
Derivatives
Sang Keun HA,a Mamoru KOKETSU,b Kunho LEE,a Sang Yoon CHOI,c Ji-Ho PARK,a
d
,a
*
Hideharu ISHIHARA, and Sun Yeou KIM
a Graduate School of East-West Medical Science, Kyung Hee University; Suwon 449–701, Korea: b Division of Instrumental
Analysis, Life Science Research Center, Gifu University; Gifu 501–1193, Japan: c Korea food Research Institute; Songnam
463–746, Korea: and d Department of Chemistry, Faculty of Engineering, Gifu University; Gifu 501–1193, Japan.
Received October 2, 2004; accepted March 9, 2005; published online March 14, 2005
This study investigated inhibitory effects of N,N-unsubstituted selenourea derivatives on tyrosinase activity.
Three types of N,N-unsubstituted selenoureas derivatives exhibited inhibitory effect on dopa (3,4-dihydroxy-
phenylalanine) oxidase activity of mushroom tyrosinase. Compound D at a concentration of 200 mM exhibited
55.5% of inhibition on dopa oxidase activity of mushroom tyrosinase. This inhibitory effect was higher than that
of kojic acid (39.4%), a well known tyrosinase inhibitor. Moreover, the compound D identified as a noncompeti-
tive inhibitor by Lineweaver–Burk plot analysis. In addition, compound D also inhibited the melanin production
in melan-a cells.
Key words N,N-unsubstituted selenourea; tyrosinase inhibitor
quently, LiAlHSeH (1 eq)5) was added to the reaction mix-
Melanin formation is the most important determinant of
ture. The reaction mixture was further stirred at 0 °C for 3 h.
After usual workup, N,N-dimethyselenourea (Compound A)
was obtained in a 70% yield as yellow crystals
N,N-Dimethyselenourea: Compound A: mp 172.2—
172.8 °C; IR (KBr) 3366, 3162, 1551 cmꢀ1, 1H-NMR
(CDCl3) d 3.18 (6H, br s, CH2), 7.60 (2H, br s, NH), 13C-
NMR (CDCl3) d 37.9, 45.3, 177.7, 77Se-NMR (CDCl3) d
230.4, MS (CI) m/zꢁ153 [Mꢂꢂ1], HR-MS (EI) Calcd for
C3H8N2Se 151.98522, Found 151.98346.
N,N-Diethyselenourea: Compound B: mp 121.8—
122.7 °C; IR (KBr) 3340, 3176, 1534 cmꢀ1, 1H-NMR
(CDCl3) d 1.27 (6H, t, Jꢁ6.8 Hz, CH3), 3.49 (2H, br s, CH2),
3.97 (2H, br s, CH2), 6.48 (2H, br s, NH), 13C-NMR (CDCl3)
d 12.2, 42.9, 51.1, 176.6, 77Se-NMR (CDCl3) d 209.8, MS
(CI) m/zꢁ181 [Mꢂꢂ1], HR-MS (EI) Calcd for C5H12N2Se
180.0165, Found 180.0147.
1-Selenocarbamoylpyrrolidine: Compound C: mp 215.1—
215.9 °C; IR (KBr) 3292, 3159, 1523 cmꢀ1, 1H-NMR
(DMSO-d6) d 1.80 (2H, m, CH2), 1.98 (2H, m, CH2), 3.26
(2H, t, Jꢁ6.8 Hz, CH2), 3.62 (2H, t, Jꢁ6.8 Hz, CH2), 7.73
(2H, br s, NH), 13C-NMR (DMSO-d6) d 24.4, 25.9, 47.5,
54.2, 173.6, 77Se-NMR (DMSO-d6) d 245.1, MS (CI)
m/zꢁ179 [Mꢂꢂ1], HR-MS (EI) Calcd for C5H10NSe
163.1011, Found 163.1014.
1-Selenocarbamoylpiperidine: Compound D: mp 145.0—
146.2 °C; IR (KBr) 3303, 3169, 1522 cmꢀ1, 1H-NMR
(DMSO-d6) d 1.47 (4H, m, CH2), 1.59 (2H, m, CH2), 3.76
(4H, br s, CH2), 7.75 (2H, br s, NH), 13C-NMR (DMSO-d6) d
23.6, 25.3, 176.0, 77Se-NMR (DMSO-d6) d 218.5, MS (CI)
m/zꢁ193 [Mꢂꢂ1], HR-MS (EI) Calcd for C6H12N2Se
192.0165, Found 192.01499.
Assay of Tyrosinase Activity Tyrosinase activity was
measured by determining its dopa oxidase activity using a
modification of the method reported by Shono et al.6) Test
substances were dissolved in MeOH to 1 mM, 500 mM,
100 mM, or 10 mM. 120 ml of L-dopa (8 mM, dissolved in
67 mM phosphate buffer, pH 6.8) and 40 ml of either the same
buffer or test sample were added to each well of a 96-well
microplate, and then 40 ml of mushroom tyrosinase (125 U)
mammalian skin color. Melanin is secreted by melanocyte
cells, which are distributed in the basal layer of the dermis.1)
One of the roles of melanin is to protect the skin and under-
lying tissues from UV-induced skin injury. However, exces-
sive melanin production in the skin has negative hyperpig-
menting effects such as melasma, freckles, and senile lentig-
ines. The synthesis of melanin starts with the conversion of
the amino acid L-tyrosine to L-dopa (3,4-dihydroxyphenylala-
nine), the subsequent oxidation of L-dopa then yields
dopaquinone. Tyrosinase is the key enzyme in the biosynthe-
sis of melanin, and participates in the oxidation of tyrosine to
L-dopa, and of dopa to dopaquinone.2,3) Therefore, tyrosinase
inhibitors are accepted as important constituents of cosmetics
and as depigmenting agents in cases of hyperpigmentation.4)
In this study, we investigated inhibitory effects of N,N-un-
substituted selenourea derivatives on mushroom tyrosinase
and their depigmenting effect in melan-a cells.
MATERIALS AND METHODS
General Methods Melting points were determined
using a Yanagimoto micromelting point apparatus. IR spectra
were obtained using a Perkin-Elmer 1600 spectrometer, and
1H- and 13C-NMR spectra were recorded on a JEOL-JNM-
a400 (400 MHz) spectrometer. Mass spectra were obtained
using a Shimadzu 9020-DF mass spectrometer, and UV spec-
tra using a Molecular Devices E09090 microplate reader.
Materials Mushroom tyrosinase, L-dopa (3-(3,4-dihy-
droxyphenyl)-L-alanine), and kojic acid (5-hydroxy-2-(hy-
droxymethyl)-4H-pyran-4one) were purchased from Aldrich
Chemical; Inc. (U.S.A.). Solvents used for organic syntheses
were redistilled. All other chemicals and solvents were of an-
alytical grade and used without further purification.
Typical procedure for preparation of N,N-unsubstituted
selenourea derivatives, 1 M HCl (2 eq) in diethyl ether was
added to N,N-dimethylcyanamide (1 eq) of THF solution.
The reaction mixture became milky white suspension from
colorless solution in less than 30 s and then was stirred at
0 °C for 2 h. The compound was formed in situ. Subse-
∗ To whom correspondence should be addressed. e-mail: sunnykim@khu.ac.kr
© 2005 Pharmaceutical Society of Japan

May 2005
839
Table 1. Inhibitory Effects of N,N-Unsubstituted Selenourea Derivatives
and Kojic Acid on Mushroom Tyrosinase.
was added.
The amount of dopachrome in the reaction mixture was
determined after 30 min at 37 °C. Inhibitory activity was ex-
pressed as the concentration that inhibited 50% of tyrosinase
activity (IC50), as determined by optical density at 490 nm
(OD490). Kojic acid was used as a positive control. The test
compound inhibition patterns were determined using
Lineweaver–Burk’s plot at various L-dopa concentrations.
Cell Line and Culture Procedures Melan-a cells were
kindly donated by Dr. B. Lee at the Skin Research Institute at
the Pacific Co., Korea. The cells were cultured in RPMI1640
medium containing 10% FBS and 200 nM phorbol 12-myris-
tate 13-acetate (TPA). Cells were then seeded in a 100 mm
diameter tissue culture dish (5ꢃ105 cells/well). Cells grew to
confluency after 3 to 4 d at 37 °C in 5% CO2, and were
seeded at 105 cells/well in a 24 well plate, and then incubated
for a further 24 h. Each well was renewed with 990 ml of
RPMI1640 medium daily and treated with 10 ml of 10000,
1000 or 100 mM of test sample for 3 d (solvent system: propy-
lene glycol/EtOH/H2Oꢁ5/3/2). And then it was incubated for
1 d in CO2 incubator.
Compounds
Inhibition at 200 mMa) (%) IC50b) (mM)
A
B
C
36.7ꢄ9.8
10.6ꢄ6.1
43.3ꢄ5.3
ꢅ200
ꢅ200
232
D
55.5ꢄ4.9
39.4ꢄ7.9
170
277
Kojic acid
a) Values represent meansꢄS.E. of three experiments. b) 50% inhibitory concentra-
tion (IC50).
Cell Viability Percentages of viable melan-a cells were
determined by staining the cell population with crystal violet.
After removing the medium from each well, the cells were
washed with PBS, and 200 ml of crystal violet (CV 0.1%,
EtOH 10%, PBS 89.9%) was added. The cells were then in-
cubated at room temperature for 5 min and washed twice
with distilled water. After adding 1 ml of EtOH to the cells,
they were shaken at room temperature for 10 min. Crystal vi-
olet absorption was measured at 590 nm.
Fig. 1. Inhibitory Effects of Compound D and of Kojic Acid on Mush-
room Tyrosinase at Several Concentrations
Determination of Melanin Level Melanin contents
were measured using a modification of the methods de-
scribed by Hosoi et al.7) After removing the media from each
wall, it was washed with PBS. 1 ml of 1 M NaOH was then
added to the well to dissolve the melanin. The absorption
maxima of melanin was measured at 400 nm, and the
melanin content per well calculated, and expressed as a per-
centage to the vehicle. Phenylthiourea was used as a positive
control.8,9)
Values represent the meansꢄstandard errors of three independent experiments per-
formed in triplicate.
Statistical Analysis Data are presented as meansꢄS.E.
of three independent experiments. Different treatments were
compared using the Student’s t-test.
Fig. 2. Lineweaver–Burk Plots of Mushroom Tyrosinase Activity Changes
Caused by Compound D
RESULTS
100 mM (rectangle), 50 mM (diamond), and blank (triangle).
Inhibitory Effects of Compounds against Tyrosinase
Activity The tyrosinase inhibitory effects of N,N-unsubsti- creased the Vmax value of tyrosinase in a dose-dependent
tuted selenourea derivatives, compounds A, B, C, and D, and manner but did not change the Km value. Therefore, com-
of kojic acid are detailed in Table 1. Among N,N-unsubsti- pound D was identified as a noncompetitive tyrosinase in-
tuted selenourea derivatives, 1-selenocarbamoylpiperidine hibitor by Lineweaver–Burk plot analysis (Fig. 2).
(compound D) showed the highest inhibitory effect on mush-
Melanin Production and Cell Viability of Melan-a Cells
room tyrosinase. Compound D at 2, 20, 100, and 200 mM in- Melan-a cells are synergistic with the B16 melanoma cell
hibited tyrosinase by 3.8%, 15.1%, 37.0%, and 55.5%, re- line and its sublines, and provide an excellent parallel non-tu-
spectively (Fig. 1). The tyrosinase inhibitory effect of com- morigenic line for studying melanoma malignancy.10) In this
pound D was greater than kojic acid (39.4% at 200 mM); the study, the melan-a cell line was used for testing the melanin
IC50’s of compound D and kojic acid were 170 and 277 mM, production and cell toxicity. Compound D, kojic acid and
respectively.
phenylthiourea were treated in the cultured melan-a cells for
Inhibition Pattern of Mushroom Tyrosinase by Com- 3 d. The testing results, cell viability and melanin contents,
pound D The kinetics behavior of compound D during the were represented at Table 2. As the results, compound D
oxidation of L-DOPA was studied. When various concentra- slightly attenuates the melanin content at above 50 mM (Fig.
tions of L-DOPA was used as a substrate, compound D de- 3). The difference between cell viability and melanin produc-

840
Vol. 28, No. 5
Table 2. Effects of PTU, Kojic Acid and Compound D on Cell Growth and
the Melanin Production in Melan-a Cells
In terms of chemical structure, 5-membered ring piperi-
dine (compound D) had a greater inhibitory effect than 6-
membered ring pyrrolidine (compound C). N,N-Dimethyse-
lenourea (compound A) and N,N-diethyselenourea (com-
pound B) did not show strong inhibitory effects.
In kinetics study, compound D identified as a noncompeti-
tive inhibitor of the mushroom tyrosinase with same Km
value. Therefore, compound D might indicate the enzyme in-
hibitory activity by binding at other sites except cupper ac-
tive site of mushroom tyrosinase.
Concentrations
Melanin
production (%)
Cell viability
(%)
Samples
(mM)
Phenylthiourea
Kojic acid
10
100
10
100
10
76.3ꢄ6.5
33.5ꢄ5.0
98.1ꢄ3.2
96.9ꢄ4.8
95.7ꢄ3.4
49.5ꢄ5.9
102.8ꢄ5.2
99.0ꢄ5.4
97.8ꢄ2.9
95.3ꢄ5.6
103.3ꢄ5.6
60.6ꢄ4.8
Compound D
100
Furthermore, compound D was tested with respect to its
anti-melanogenic effect in cultured melan-a cells at various
concentrations (1, 25, 50, 75, 100 mM). Compound D showed
melanin inhibitory effect in range from 50 to 100 mM (Fig. 3).
Compound D was found to slightly attenuate the melanin
content. Based on these results, we can suggest that com-
pound D may act as a depigmenting agent.
Test sample and medium were renewed daily. The cell viabilities and the melanin
contents of melan-a cells were determined after 3 d. The data shown represents the
meansꢄS.E. of four independent experiments performed in duplicate.
Acknowledgments This work was supported by the
grants from the 2002 Good Health Research and Develop-
ment Project (Ministry of Health and Welfare, Korea, 02-
PJ1-PG1-CH11-0001) and Brain Korea 21 Projects (Ministry
of Education, Korea).
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This study investigated inhibitory effects of N,N-unsubsti-
tuted selenourea derivatives on tyrosinase activity. Among
N,N-unsubstituted selenourea derivatives, 1-selenocarba-
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