Potent inhibitory effects of N-aryl S-alkylthiocarbamate derivatives on the dopa oxidase activity of mushroom tyrosinase

Article abstract of DOI:10.1248/cpb.53.747

This study reports the potent inhibitory effect of N-aryl S-alkylthiocarbamate derivatives on mushroom tyrosinase (MT) activity. N-Aryl S-alkylthiocarbamate derivatives were found to exhibit a potent inhibitory effect on the dopa (3,4-dihydroxyphenylalanine) oxidase activity of mushroom tyrosinase. Most of the N-aryl S-alkylthiocarbamate derivatives (compounds from A to J) exhibited higher inhibitory effects than kojic acid (IC₅₀=318 μm), a well known tyrosinase inhibitor. Tyrosinase was the most inhibited by S-phenetyl N-phenylthiocarbamate (compound E, IC₅₀=7.25 μM), and this inhibition was 44 times stronger than that of kojic acid. Compound E exhibited 95.0% of inhibition at 100 μM. A kinetic study of MT inhibition by compound E using the Lineweaver-Burk plots analysis was performed. And the kinetics profiles observed suggest that compound E competitively inhibits MT.

Full text of DOI:10.1248/cpb.53.747

July 2005
Chem. Pharm. Bull. 53(7) 747—749 (2005)
747
Potent Inhibitory Effects of N-Aryl S-Alkylthiocarbamate Derivatives on
the Dopa Oxidase Activity of Mushroom Tyrosinase
Kun Ho LEE,a Mamoru KOKETSU,b Sang Yoon CHOI,c Kang Jin LEE,a Pyeongjae LEE,d
e
,a
*
Hideharu ISHIHARA, and Sun Yeou KIM
a Graduate School of East-West Medical Science, Kyung Hee University; Yongin 449–701, Korea: b Division of
c
Instrumental Analysis, Life Science Research Laboratory, Gifu University; Gifu 501–1193, Japan: Korea Food Research
Institute; Songnam 463–746, Korea: d Rural Development Administration, National Institute of Agricultural Science and
Technology; Suwon 441–857, Korea: and e Department of Chemistry, Faculty of Engineering, Gifu University; Gifu
501–1193, Japan. Received November 9, 2004; accepted April 8, 2005; published online April 18, 2005
This study reports the potent inhibitory effect of N-aryl S-alkylthiocarbamate derivatives on mushroom ty-
rosinase (MT) activity. N-Aryl S-alkylthiocarbamate derivatives were found to exhibit a potent inhibitory effect
on the dopa (3,4-dihydroxyphenylalanine) oxidase activity of mushroom tyrosinase. Most of the N-aryl S-alkyl-
thiocarbamate derivatives (compounds from A to J) exhibited higher inhibitory effects than kojic acid
(IC50ꢀ318 mm), a well known tyrosinase inhibitor. Tyrosinase was the most inhibited by S-phenetyl N-phenylthio-
carbamate (compound E, IC50ꢀ7.25 mM), and this inhibition was 44 times stronger than that of kojic acid. Com-
pound E exhibited 95.0% of inhibition at 100 mM. A kinetic study of MT inhibition by compound E using the
Lineweaver–Burk plots analysis was performed. And the kinetics profiles observed suggest that compound E
competitively inhibits MT.
Key words N-aryl S-alkylthiocarbamate; mushroom tyrosinase; diethyldithiocarbamate
aryl S-alkylthiocarbamate derivatives under mild conditions in high yields.
Briefly, the synthesis of S-ethyl N-phenylthiocarbamate (A) was conducted
as follows; Phenyl isocyanate (0.22 ml, 2.0 mmol) was added to a THF solu-
tion (10 ml) of LiAlHSH (1.0 mmol). The reaction mixture was stirred at
room temperature for 1 h, and ethyl iodide (0.06 ml, 1.0 mmol) was added,
Tyrosinase is melanogenic copper-containing enzyme that
catalyzes the transformation of tyrosine to dopaquinone.1,2)
This enzyme is responsible for melanization in plants and an-
imals, which leads to the undesirable browning of farm prod-
ucts and the coloring of an animal’s skin, eyes, inner ear, and and stirred at room temperature for 3 h. The mixture was then extracted with
hair.3,4) Numerous tyrosinase inhibitors, such as kojic acid
dichloromethane, washed with distilled water, and the organic layer was
dried over sodium sulfate and evaporated to dryness. The residue was puri-
fied by flash chromatography on silica gel with dichloromethane : hexane
(1 : 2) to give:
and oxyresveratrol, have been developed to remove of unde-
sirable pigment.5—7) In this study, we examined the inhibitory
effects of diethyldithiocarbamate derivatives on mushroom
tyrosinase. Diethyldithiocarbamate (DETC) has been re-
ported to act as a nitric oxide synthase inhibitor and as a xan-
thine oxidase inhibitor.8,9) In particular, DETC has also been
reported to potently inhibit tyrosinase,10,11) which lead us to
investigate the inhibitory effects of diethyldithiocarbamate
derivatives on tyrosinase. To identify more potent tyrosinase
inhibitors, several N-aryl S-alkylthiocarbamate derivatives
were synthesized by reacting isocyanates with LiAlHSH and
then with alkyl halides. Thiocarbamates have been used as
key intermediates for the synthesis of thioureas12) and of
isothiocyanates,13) and are important moieties in pesticides
components.14,15) In this work, we investigated the struc-
ture–activity-relationships (SARs) of synthetic N-aryl S-
alkylthiocarbamate derivatives on tyrosinase inhibitory activ-
ity and on these inhibition patterns.
Compound A; S-Ethyl N-Phenylthiocarbamate (Yield 71%): White crys-
tals; mp 63.9—65.1 °C; IR (KBr) 1651, 3281 cmꢁ1 1H-NMR (CDCl3); d
;
1.33 (3H, t, Jꢂ7.2 Hz, CH3), 2.98 (2H, q, Jꢂ7.2 Hz, CH2), 7.09 (1H, t,
Jꢂ7.2 Hz, Ar), 7.21 (1H, br s, NH), 7.30 (2H, t, Jꢂ7.2 Hz, Ar), 7.41 (2H, d,
Jꢂ7.2 Hz, Ar); 13C-NMR (CDCl3); d 15.5, 24.7, 119.7, 124.4, 129.1 137.6
(Ar), 165.8; MS (CI): m/zꢂ182 [Mꢃꢃ1].
Compound B; S-Propyl N-Phenylthiocarbamate (Yield 66%): White crys-
tals; mp 73.2—75.6 °C; IR (KBr) 1653, 3280 cmꢁ1 1H-NMR (CDCl3); d
;
1.00 (3H, t, Jꢂ7.6 Hz, CH3), 1.67 (2H, m, CH2), 2.95 (2H, t, Jꢂ7.6 Hz,
CH2), 7.02 (1H, br s, NH), 7.10 (1H, t, Jꢂ7.6 Hz, Ar), 7.31 (2H, t,
Jꢂ7.6 Hz, Ar), 7.41 (2H, d, Jꢂ7.6 Hz, Ar); 13C-NMR (CDCl3); d 13.2, 23.6,
32.2, 119.7—137.7 (Ar), 165.9; MS (CI): m/zꢂ196 [Mꢃꢃ1].
Compound C; S-Isopentyl N-Phenylthiocarbamate (Yield 50%): White
1
crystals; mp 66.1—69.3 °C; IR (KBr) 1655, 3290 cmꢁ1; H-NMR (CDCl3);
d 0.92 (6H, t, Jꢂ6.8 Hz, CH3), 1.54 (2H, q, CH2), 1.68 (1H, m, CH), 2.97
(2H, t, Jꢂ8.0 Hz, CH2), 7.09 (1H, t, Jꢂ7.2 Hz, Ar), 7.19 (1H, br s, NH),
7.30 (2H, t, Jꢂ7.2 Hz, Ar), 7.41 (2H, d, Jꢂ7.2 Hz, Ar); 13C-NMR (CDCl3);
d 22.1, 27.4, 28.3, 30.8, 39.1, 119.6—137.7 (Ar), 165.9; MS (CI): m/zꢂ224
[Mꢃꢃ1].
Compound D; S-Benzyl N-Phenylthiocarbamate (Yield 55%): White pow-
Experimental
der; mp 92.1—94.2 °C; IR (KBr) 1653, 3250 cmꢁ1 1H-NMR (CDCl3); d
;
General Methods Melting points were determined using a Yanagimoto
4.22 (2H, s, CH2), 7.08—7.40 (10H, m, Ar), 7.10 (1H, br s, NH); 13C-NMR
micromelting point apparatus. IR spectra were obtained using a Perkin- (CDCl3); d 34.4, 119.8—137.8 (Ar), 165.1; MS (CI): m/zꢂ244 [Mꢃꢃ1].
Elmer 1600 spectrometer, and 1H- and 13C-NMR spectra were recorded on a
JEOL-JNM-a 400 (400 MHz) spectrometer. Mass spectra were obtained
using a Shimadzu 9020-DF mass spectrometer, and UV spectra using a Mo-
lecular Devices E09090 microplate reader.
Compound E; S-Phenetyl N-Phenylthiocarbamate (Yield 44%): White
crystals; mp 108.1—110.3 °C; IR (KBr) 1652, 3382 cmꢁ1 1H-NMR
;
(CDCl3); d 2.96 (2H, t, Jꢂ6.8 Hz, CH2), 3.21 (2H, t, Jꢂ7.7 Hz, CH2),
7.08—7.40 (10H, m, Ar), 7.43 (1H, br s, NH); 13C-NMR (CDCl3); d 31.5,
36.6, 119.7—139.9 (Ar), 165.5; MS (CI): m/zꢂ258 [Mꢃꢃ1].
Materials Mushroom tyrosinase, L-dopa (3-(3,4-dihydroxyphenyl)-L-
alanine), Kojic acid (5-hydroxy-2-(hydroxymethyl)-4H-pyran-4-one) and
Compound F; S-3-Phenyl-propyl N-Phenylthiocarbamate (Yield 36%):
DETC (diethyldithiocarbamate) were purchased from Aldrich Chemical, White crystals; mp 78.3—79.8 °C; IR (KBr) 1652, 3386 cmꢁ1
;
1H-NMR
Inc. (U.S.A.). Oxyresveratrol (3,5,2ꢀ,4ꢀ-tetrahydroxy stilbene) was synthe- (CDCl3); d 1.99 (2H, quint, Jꢂ7.2 Hz, CH2), 2.73 (2H, t, Jꢂ7.2 Hz, CH2),
sized in our previous work.16) Solvents for organic synthesis were redistilled. 2.99 (2H, t, Jꢂ7.2 Hz, CH2), 7.08—7.41 (10H, m, Ar), 7.17 (1H, br s, NH);
All other chemicals and solvents were of analytical grade and used without
13C-NMR (CDCl3); d 29.7, 31.8, 34.7, 119.6—141.5 (Ar), 165.7; MS (CI):
further purification. N-Aryl S-alkylthiocarbamate derivatives were prepared m/zꢂ272 [Mꢃꢃ1]
according to the previously reported method.17,18) This method give the N-
∗ To whom correspondence should be addressed. e-mail: sunnykim@khu.ac.kr
Compound G; S-Methyl N-(4-Methylphenyl)thiocarbamate (Yield 59%):
© 2005 Pharmaceutical Society of Japan

748
Vol. 53, No. 7
Yellow crystals; mp 102.1—103.5 °C; IR (KBr) 1654, 3242 cmꢁ1; H-NMR
(CDCl3); d 2.30 (3H, s, CH3), 2.39 (3H, s, CH3), 7.10 (2H, d, Jꢂ8.4 Hz, Ar),
7.27 (1H, br s, NH), 7.28 (2H, t, Jꢂ8.4 Hz, Ar); 13C-NMR (CDCl3); d 12.5,
20.7, 119.9—135.0, (Ar), 166.3; MS (CI): m/zꢂ182 [Mꢃꢃ1].
1
Table 1. Inhibitory Effects of N-Aryl S-Alkylthiocarbamate Derivatives,
DETC, Oxyresveratrol and Kojic Acid on Mushroom Tyrosinase
Compound H; S-Butyl N-(4-Methylphenyl)thiocarbamate (Yield 46%):
White crystals; mp 73.9—75.1 °C; IR (KBr) 1651, 3297 cmꢁ1 1H-NMR
;
(CDCl3); d 0.91 (3H, t, Jꢂ7.6 Hz, CH3), 1.40 (2H, m, Jꢂ7.6 Hz, CH2), 1.62
(2H, quint, Jꢂ7.6 Hz, CH2), 2.29 (3H, s, CH3), 2.95 (2H, t, Jꢂ7.6 Hz, CH2),
7.08 (2H, d, Jꢂ8.4 Hz, Ar), 7.23 (1H, br s, NH), 7.28 (2H, t, Jꢂ8.4 Hz, Ar);
13C-NMR (CDCl3); d 13.5, 20.7, 21.8, 29.9, 32.3, 119.9—135.1 (Ar), 166.0;
MS (CI): m/zꢂ224 [Mꢃꢃ1].
Substituent
b)
Inhibition at
IC50
Compounds
100 mM (%)a)
(mM)
R1
R2
CH2CH3
(CH2)2CH3
(CH2)2CH(CH3)2
CH2C6H5
(CH2)2C6H5
(CH2)3C6H5
CH3
(CH2)3CH3
(CH2)2CH(CH3)2
CH2CH3
Compound I; S-Isopentyl N-(4-Methylphenyl)thiocarbamate (Yield 52%):
White crystals; mp 33.1—35.6 °C; IR (KBr) 1657, 3318 cmꢁ1 1H-NMR
;
A
B
C
D
E
F
G
H
H
H
H
H
H
H
CH3
CH3
CH3
Cl
3.4ꢄ5.9
36.8ꢄ12.5 ꢅ100
ꢅ100
(CDCl3); d 0.92 (6H, t, Jꢂ6.8 Hz, CH3), 1.53 (2H, q, CH2), 1.67 (1H, m,
CH), 2.30 (3H, s, CH3), 2.96 (2H, t, Jꢂ7.2 Hz, CH2), 7.06 (1H, br s, NH),
7.10 (2H, d, Jꢂ8.4 Hz, Ar), 7.28 (2H, t, Jꢂ8.4 Hz, Ar); 13C-NMR (CDCl3);
d 20.8, 22.1, 27.4, 28.3, 30.8, 39.1, 120.0—135.1 (Ar), 164.2; MS (CI):
m/zꢂ238 [Mꢃꢃ1].
82.2ꢄ7.3
93.2ꢄ4.9
95.0ꢄ4.0
59.9ꢄ7.6
74.3ꢄ1.7
55.1ꢄ7.3
70.9ꢄ5.2
20.2ꢄ4.5
13.5ꢄ3.5
61.9ꢄ0.5
73.4ꢄ1.8
19.0ꢄ2.6
38.1
18.4
7.3
46.6
48.5
87.5
47.4
Compound J; S-Ethyl N-(4-Chlorophenyl)thiocarbamate (Yield 47%):
White crystals; mp 95.1—96.2 °C; IR (KBr) 1651, 3271 cmꢁ1 1H-NMR
;
(CDCl3); d 1.33 (3H, t, Jꢂ7.6 Hz, CH3), 2.98 (2H, q, Jꢂ7.6 Hz, CH2), 7.13
(1H, br s, NH), 7.26 (2H, t, Jꢂ8.4 Hz, Ar), 7.36 (2H, t, Jꢂ8.4 Hz, Ar); 13C-
NMR (CDCl3); d 15.4, 24.7, 120.8—136.2 (Ar), 166.0; MS (CI): m/zꢂ216
[Mꢃꢃ1].
I
J
K
ꢅ100
ꢅ100
61.2
53.7
318.0
DETC
Oxyresveratrol
Kojic acid
—
—
—
Compound K was synthesized by following method; to a mixture of ben-
zoic acid (300 mg, 2.5 mM) and trichloro acetonitrile (490 ml, 4.9 mM) in
CH2Cl2 (7 ml), Ph3P (1.3 g, 4.9 mM) in CH2Cl2 (5 ml) was added under nitro-
gen at room temperature. After stirring for 4 h, the reaction mixture was
treated with aniline (225 ml, 5 mM) and mixture was stirring for 12 h.
The reaction mixture was poured into water and extracted with ethlyac-
etate. The extract was washed with brine, and the organic layer was dried
over sodium sulfate and evaporated to dryness. The residue was purified by
flash chromatography on silica gel with dichloromethane : methanol (60 : 1)
to give:
a) Each value represents the meanꢄS.E. of three experiments. b) 50% inhibitory
concentration (IC50).
Compound K; N-Phenyl-benzamide (Yield 40%): White crystals; 1H-
NMR (CDCl3); d 7.08 (1H, m), 7.30 (2H, m), 7.38—7.51 (4H, m), 7.56
(1H, t, Jꢂ1.2 Hz, Ar), 7.59 (1H, t, Jꢂ1.2 Hz, Ar), 7.79 (1H, t, Jꢂ1.5 Hz,
Ar), 7.81 (1H, t, Jꢂ1.5 Hz, Ar); 13C-NMR (CDCl3); d 120.2, 124.6, 127.0,
128.8, 129.1, 131.8, 135.0, 137.9, 165.7; MS (CI): m/zꢂ197 [Mꢃꢃ1].
Assay of Tyrosinase Activity The test compounds were dissolved in
methanol at various concentrations (500 mM, 250 mM, 50 mM, 5 mM). 120 ml of
L-dopa (8 mM, dissolved in 67 mM phosphate buffer, pH 6.8) and 40 ml of
each N-aryl S-alkylthiocarbamate compound solution was added to a 96-well
microplate, and 40 ml of mushroom tyrosinase (125 U) was added. After in-
cubation at 37 °C for 20 min, the amount of dopachrome in the reaction mix-
ture was determined. Based on the optical density at 490 nm, the inhibitory
activity was expressed as a concentration, i.e., the concentration required to
inhibit the enzyme activity by 50% (IC50). Kojic acid was used as a positive
control. The pattern of inhibition of the test compound was determined by
Lineweaver–Burk’s plot at various L-dopa concentrations.
Fig. 1. Inhibitory Effects of Compound E, DETC, and Kojic Acid on
Mushroom Tyrosinase at Several Concentrations
Each value represents the meanꢄstandard error of experiments performed in tripli-
cate.
Statistical Analysis Data are presented as the meansꢄS.E. of three in-
dependent experiments. Different treatments were compared using the Stu-
dent’s t-test.
Results and Discussion
Inhibitory Effects of Compounds on Tyrosinase Activ-
ity Tyrosinase inhibitory effects by the N-aryl S-alkylthio-
carbamate derivatives are presented in Table 1. The majority
of these N-aryl S-alkylthiocarbamate compounds inhibited
tyrosinase more strongly than kojic acid. Compound E had
the highest inhibitory effects with an IC50 of 7.25 mM, and the
level of inhibition increased dose dependently over the con-
centration range 1—100 mM. At 50 mM, the inhibitory effect
of compound E exceeded 90% (Fig. 1).
Fig. 2. Lineweaver–Burk Plots of Mushroom Tyrosinase Activity Changes
Caused by Compound E
10 mM (rectangle), 5 mM (diamond) and blank (triangle).
SARs of N-Aryl S-Alkylthiocarbamate Derivatives
Compounds D, E and F with aromatic ring containing sub-
stituents at R2 showed higher tyrosinase inhibition than the
other N-aryl S-alkylthiocarbamate derivatives or DETC.
Among these compounds, compound E (R2ꢂphenetyl) had
the greatest potency versus compound D (R2ꢂS-benzyl) or
compound F (R2ꢂS-3-phenyl-propyl). However, introduction
of the aliphatic chain at R2 (compounds A, B, C) reduced in-
hibitory activity. This tendency was confirmed by comparing
the inhibitory abilities of compounds A and D, with those of

July 2005
749
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chemical skeleton.
In addition, the presence of sulfur may play a very impor-
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compound K including no sulfur atom, the inhibitory activity
of the compound was very low.
Inhibition Pattern of Compound E Kinetic analysis
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at several concentrations, but the Km value reduced with in-
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fied as competitive inhibitor of mushroom tyrosinase. As
competitive inhibitor, kojic acid is well known.
In this study, compound E exhibited 44-fold higher tyrosi-
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tyrosinase inhibitor. The present study identified a useful
candidate for potent tyrosinase inhibitor. Furthermore, our
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Acknowledgements This work was supported by the grants from the
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