Synthesis and biological activity of hydroxy substituted phenyl-benzo[d]thiazole analogues for antityrosinase activity in B16 cells

Article abstract of DOI:10.1016/j.bmcl.2011.02.064

In this study, we synthesized hydroxy and/or alkoxy substituted phenyl-benzo[d]thiazole derivatives using substituted benzaldehydes and 2-aminothiophenol in MeOH. The structures of these compounds were established by ¹H and ¹³C NMR a

Full text of DOI:10.1016/j.bmcl.2011.02.064

Bioorganic & Medicinal Chemistry Letters 21 (2011) 2445–2449
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and biological activity of hydroxy substituted
phenyl-benzo[d]thiazole analogues for antityrosinase activity in B16 cells
Young Mi Ha a, Ji Young Park b, Yun Jung Park b, Daeui Park a, Yeon Ja Choi a, Ji Min Kim a, Eun Kyeong Lee a,c
,
a,
Yu Kyeong Han a, Jin-Ah Kim b, Ji Yeon Lee b, Hyung Ryong Moon b, , Hae Young Chung
⇑
⇑
a Molecular Inflammation Research Center for Aging Intervention (MRCA), College of Pharmacy, Pusan National University, Kumjeong-Gu, Busan 609-735, Republic of Korea
b Laboratory of Medicinal Chemistry, College of Pharmacy, Pusan National University, Busan 609-735, Republic of Korea
c Research Center, Dongnam Institute of Radiological and Medical Sciences, Busan 619-953, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
In this study, we synthesized hydroxy and/or alkoxy substituted phenyl-benzo[d]thiazole derivatives
using substituted benzaldehydes and 2-aminothiophenol in MeOH. The structures of these compounds
were established by 1H and 13C NMR and mass spectral analyzes. All synthesized compounds were eval-
uated for their mushroom tyrosinase inhibition activity. Out the 12 generated compounds, 2a and 2d
Received 3 December 2010
Revised 11 February 2011
Accepted 15 February 2011
Available online 23 February 2011
exhibited much higher tyrosinase inhibition activity (45.36–73.07% and 49.94–94.17% at 0.01–20
respectively) than kojic acid (9.29–50.80% at 1.25–20 M), a positive control.
lM,
l
Keywords:
The cytotoxicity of 2a and 2d was evaluated using B16 cells and the compounds were found to be non-
toxic. Compounds 2a and 2d were also demonstrated to be potent mushroom tyrosinase inhibitors, dis-
Melanin biosynthesis
Tyrosinase inhibitor
Hyperpigmentation
Inflammation
playing IC50 values of 1.14 0.48 and 0.01 0.0002
lM, respectively, compared with kojic acid, which has
an IC50 value of 18.45 0.17 M. We also predicted the tertiary structure of tyrosinase, simulated the
l
docking with compounds 2a and 2d and confirmed that the compounds strongly interact with mushroom
tyrosinase residues. Kinetic plots showed that 2a and 2d are competitive tyrosinase inhibitors. Substitu-
tions with a hydroxy group at R3 or both R3 and R1 of the phenyl ring indicated that these groups play a
major role in the high binding affinity to tyrosinase. We further found that compounds 2a and 2d inhibit
melanin production and tyrosinase activity in B16 cells. These results may assist in the development of
new potent tyrosinase inhibitors against hyperpigmentation.
Docking analysis
Ó 2011 Elsevier Ltd. All rights reserved.
Tyrosinase, a copper containing metalloenzyme, is one of the
enzymes responsible for skin pigmentation in mammals and is
the rate-determining enzyme that catalyzing the first two steps
similar to N-phenylthiourea. Additionally, because the nitrogen
atom of the substituted phenyl-benzo[d]thiazole derivatives can
in the melanin-biosynthesis pathway: 30-hydroxylation of
L
-tyro-
-3,4-dihydroxyphenylalanine) and oxidation of
-DOPA to DOPA quinone (Scheme 1).1 Most melanin-biosynthesis
inhibitors are phenol or catechol analogues, which are structurally
similar to tyrosinase substrate, tyrosine or
-DOPA.2 However,
sine to L-DOPA (L
HO
L
O
O
Tyrosinase
HO
HO
L
O
O
N-phenylthiourea (Fig. 1) has been reported to inhibit a catechol
oxidase enzyme that belonging to the tyrosinase type-3 copper
protein group because its sulfur atom is bound to both copper ions
of the enzyme in the active site.3,4 On the basis of these findings,
we considered that it might be interesting to synthesize hydroxyl
and/or alkoxy substituted phenyl-benzo[d]thiazole derivatives
(Fig. 1), in which the sulfur atom of the benzothiazole ring can
act as a chelator of copper ions in the active site of tyrosinase,
NH3
NH3
DOPA
Tyrosine
Tyrosinase
O
O
Melanin
O
O
NH3
DOPA quinone
⇑
Corresponding authors. Tel.: +82 51 510 2814; fax: +82 51 518 2821 (H.Y.C.);
tel.: +82 51 510 2815; fax: +82 51 513 6754 (H.R.M.).
E-mail addresses: mhr108@pusan.ac.kr (H.R. Moon), hyjung@pusan.ac.kr (H.Y.
Chung).
Scheme 1. Pathway for melanin biosynthesis.
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.02.064

2446
Y. M. Ha et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2445–2449
Table 1
R2
R1
H
N
Substitution pattern and mushroom tyrosinase inhibition of the synthesized substi-
tuted-phenyl-benzo[d]thiazole analogues 2a–2l
NH2
S
N
R3
R2
R1
S
S
N
R4
R3
N
-Phenylthiourea
2a - 2m
R4
Figure 1. Chemical structures of N-phenylthiourea and substituted phenyl-
benzo[d] thiazole analogues (2a–2l).
Compound
R1
R2
R3
R4
Tyrosinase inhibitiona (%)
2a
2b
2c
2d
2e
2f
2g
2h
2i
2j
2k
2l
H
OH
H
OH
H
H
H
H
OMe
H
H
H
H
H
H
OH
OH
OMe
OEt
OH
H
OMe
OH
OH
OMe
OH
OH
OMe
OMe
OH
H
H
H
H
H
H
H
H
H
H
OMe
OMe
73.07 0.27
ꢀ3.91 0.18
33.02 0.16
94.17 2.29
36.10 0.33
35.04 0.61
49.76 0.30
18.42 0.20
31.52 0.18
42.48 0.65
38.49 0.20
36.32 0.23
50.80 0.57
form a quaternary ammonium ion at physiological pH, similar to
tyrosine and DOPA, the nitrogen atom might act as a positive cen-
ter capable of interacting with anionic or partially anionic groups
of amino acid residues existing in the tyrosinase active site. There-
fore, hydroxy and/or alkoxy substituted phenyl-benzo[d]thiazole
derivatives might serve as potent competitive tyrosinase inhibi-
tors. The target compounds, substituted phenyl-benzo[d]thiazole
analogues,5–7 were prepared by stirring substituted benzaldehydes
and 2-aminothiophenol in MeOH (Scheme 2). The structures of the
synthesized compounds were determined by 1H and 13C NMR and
mass spectral analyzes.
H
OMe
OMe
OMe
H
OMe
Kojic acid
a
Tyrosinase inhibition was measured using L-tyrosine as the substrate at 20 lM.
Values represent the means S.E. of three experiments.
The inhibitory activities of the synthesized compounds were
examined using mushroom tyrosinase as described previously
with minor modification.8 The inhibition of mushroom tyrosinase
using the synthesized compounds is summarized in Table 1.
Our results showed that 20,40-dihydroxyphenyl-benzo[d]thiazole
(2d) exhibited the most potent inhibitory activity against
Table 2
Inhibitory effects of compounds kojic acid, 2a, and 2d on mushroom tyrosinase
activity
Concna
(lM)
Tyrosinase inhibition (%)
IC50
(lM)
b
mushroom tyrosinase, with 94.17% inhibition at 20 l
M. 40-
Compound
Kojic acid
Hydroxyphenyl-benzo[d]thiazole (2a) and 40-hydroxy-30-methoxy-
phenyl-benzo[d]thiazole (2g) lagged behind 2d in decreasing order
of inhibitory potency, with 73.07% and 49.76% inhibition at 20 lM,
respectively. Mushroom tyrosinase inhibition with compounds 2a
and 2d was more potent than that obtained with a reference tyros-
inase inhibitor, kojic acid, which exhibited 50.80% inhibition at a
0.31
1.25
5
ꢀ0.98 0.49
9.29 0.37
30.75 0.80
50.80 0.57
18.45 0.17
20
2a
2d
0.01
0.08
0.31
1.25
5
45.36 0.38
46.18 0.47
47.97 0.40
54.67 1.75
57.87 0.71
73.07 0.27
1.14 0.48
concentration of 20 lM. Compounds 2c, 2e, 2f, and 2i–2l exhibited
moderate mushroom tyrosinase inhibition, and the remaining
compounds, 2b and 2h, showed no or low inhibitory activity
against mushroom tyrosinase. It is notable that the benzo[d]thia-
zole analogue, 2d, with a 20,40-dihydroxyphenyl scaffold, has a
more potent inhibitory effect against mushroom tyrosinase than
2e with a catechol (30,40-dihydroxyphenyl) scaffold, even though
compound 2e with catechol is structurally more similar to DOPA,
one of substrates for tyrosinase (Scheme 1). This indicates that a
20,40-dihydroxyphenyl scaffold can exhibit more potent inhibitory
activity against tyrosinase than a catechol scaffold, depending on
the remaining template (e.g., benzo[d]thiazole) and excluding the
phenyl scaffold.
20
0.01
0.08
0.31
1.25
5
49.94 0.07
70.75 0.27
80.88 0.48
85.60 2.31
87.73 0.53
94.17 2.29
0.01 0.0002
20
a
Values represent the means S.E. of three experiments.
50% inhibitory concentration (IC50).
b
was used as a reference compound. We predicted the tertiary
structure of tyrosinase and simulated docking to compounds 2a
and 2d. Using DOCK6, we searched for tyrosinase residues that
may bind to 2a and 2d. We found that the most important ex-
pected binding residues interacting with compounds 2a were
VAL 93, PRO 96, THR 97, TRP 239, LEU 297, TRP 300, SER 301,
TRP 319, PRO 323, ASP 324, GLY 328, and LYS 329, and 2d were
VAL 93, PRO 96, THR 97, TRP 239, LEU 297, SER 301, ASP 324,
GLY 328, LYS 329, and THR 350, according to DOCK6 (Fig. 2). Res-
idues were located within 3 Å of the ligand. Docking simulation
provided support for the slope–parabolic mixed-type inhibition
observed, as this type of inhibition is generally observed when
there are multiple-inhibitor binding sites. Docking results showed
that compound 2a (ꢀ30.33 kcalꢁmolꢀ1) combines with mushroom
tyrosinase more strongly than compound 2d (ꢀ27.37 kcalꢁmolꢀ1).
To explore the mechanism of inhibition, we examined the ki-
netic behaviors of tyrosinase activity in the presence of compounds
2a and 2d. The data are shown in Figure 3. The mode of enzyme
In this study, we investigated in greater detail the bioactivities
of compounds 2a and 2d, which exhibit more potent activity than
kojic acid. As shown in Table 2, kojic acid, 2a, and 2d were found to
inhibit mushroom tyrosinase activity in a concentration-depen-
dent manner. The data showed that compounds 2a
(IC50 = 1.14
lM) and 2d (IC50 = 0.01 lM) are more potent inhibitors
of mushroom tyrosinase than kojic acid (IC50 = 18.45
l
M), which
R1
O
R2
R4
R1
R2
R3
HS
S
N
H
R3
+
H2N
R4
1a - 1l
2-aminothiophenol
2a - 2l
Scheme 2. Synthesis of the target compounds, substituted phenyl-benzo[d]thiazole
analogues, 2a–2l. Reagents and conditions: MeOH, RT, 5–96 h, 15.2–82.6%.

Y. M. Ha et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2445–2449
2447
Z < 3
Z < 3
#0 VAL 93
#0 PRO 96
#0 THR 97
#0 TRP 239
#0 LEU 297
#0 TRP 300
#0 SER 301
#0 TRP 319
#0 PRO 323
#0 ASP 324
#0 GLY 328
#0 LYS 329
#0 VAL 93
#0 PRO 96
#0 THR 97
#0 TRP 239
#0 LEU 297
#0 SER 301
#0 ASP 324
#0 GLY 328
#0 LYS 329
#0 THR 350
Figure 2. Computational structure prediction for mushroom tyrosinase and docking simulation with compounds 2a and 2d. Predicted 3D structure of mushroom tyrosinase.
The boxes indicate compound 2a or 2d binding sites with tyrosinase residues. The yellow area indicates the tyrosinse–ligand interactions (Z <3).
Compound 2a
A
2000
1500
20 µM
1000
1.25 µM
500
0
0 µM
-2
-1
0
1
2
3
4
5
1/[L-tyrosine], mM-1
- 500
Compound 2d
600
500
400
300
200
100
0
B
20 µM
1.25 µM
0 µM
-2
-1
0
1
2
3
4
5
1/[L-tyrosine], mM-1
- 100
Figure 3. Lineweaver–Burk plot of mushroom tyrosinase. Data were obtained as mean values of 1/V, the inverse of the absorbance increase at a wavelength of 492 nm per
min ( -tyrosine as a substrate. Enzyme inhibitors are indicated as follows: 1.25 M (square),
A492 minꢀ1), of three independent tests with different concentrations of
20.0 M (diamond), and no of compound 2a or 2d (triangle).
D
L
l
l

2448
Y. M. Ha et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2445–2449
**
the value of Vmax, suggesting that both compounds 2a and 2d are
***
***
***
***
***
***
100
80
60
40
20
0
***
competitive inhibitors of mushroom tyrosinase. Thus, compounds
2a and 2d might exert enzymatic inhibitory activities by binding
to the copper active site of mushroom tyrosinase.
To evaluate the cytotoxic effects of compounds 2a and 2d, we
used the murine B16F10 melanoma cell line (B16 cells). The effects
of compounds 2a and 2d on cell viability are presented in Figure 4.
***
Compound 2a
Compound 2d
At doses of 0.2, 1.0, 2.5, 5.0, and 10.0 lM of compounds 2a or 2d for
48 h, cell viability was 99.70%, 98.92%, 97.63%, 96.46%, and 95.77%
for compound 2a, and 96.28%, 94.92%, 94.70%, 94.57%, and 94.41%
for compound 2d, respectively, compared with a control. Thus, nei-
ther compound 2a nor 2d is cytotoxic to B16 cells in the concentra-
Control
0.2
1
2.5
5
10
Concentration (μM)
Figure 4. Effect of compounds 2a and 2d on B16 cell viability. Cells were treated
tion range of 0.2–10 lM.
with varying doses of compound 2a or 2d (0–10 lM) and examined using an MTT
assay. Data are expressed as a percentage of the control. ⁄⁄p <0.01 and ⁄⁄⁄p <0.001,
To assess the inhibitory effects of compounds 2a and 2d on
melanogenesis, we quantified the melanin content of B16 cells
treated with compounds 2a and 2d (Fig. 5). The melanin content
of B16 cells after treatment with compound 2a in the presence of
compared with the untreated control.
inhibition was determined by Lineweaver–Burk plot analysis, in
which the inhibitory kinetics of 2a exhibited Ki values of
100 nM
dose-dependently, showing 201.43% at 0.2
1.0 M, 128.97% at 2.5 M, 104.05% at 5.0
10.0
a-melanocyte-stimulating hormone (a-MSH) decreased
5.67 ꢂ 10ꢀ7 M and 1.70 ꢂ 10ꢀ6 M at 1.25 and 20
lM, respectively,
lM, 187.61% at
whereas those of 2d exhibited Ki values of 6.73 ꢂ 10ꢀ7 M and
l
l
l
l
M, and 97.14% at
7.70 ꢂ 10ꢀ6 M at 1.25 and 20
lM of 2d. Mushroom tyrosinase
M of 2a (Fig. 5A), compared with the 100 nM a-MSH-only-
activity in the presence of compound 2a at 1.25 and 20 lM, respec-
treated group (242.34%) and the control group (100%). Similarly,
cells treated with compound 2d, exhibited melanin contents of
tively. Mushroom tyrosinase activity in the presence of compound
2a at 1.25 and 20 lM exhibited Km values of 1.02 and 5.65 mM,
223.81% at 0.2
140.97% at 5.0
with 100 nM -MSH-only-treated group (240.13%) and the control
l
M, 216.91% at 1.0
lM, 189.85% at 2.5
lM,
respectively, and the same Vmax value of 1.54 ꢂ 10ꢀ2. In the pres-
ence of compound 2d, mushroom tyrosinase activity at 1.25 and
lM, and 130.66% at 10.0
l
M (Fig. 5B), compared
a
20 lM exhibited Km values of 1.16 and 1.46 mM, respectively,
group (100%). On the basis of the cytotoxicity results (Fig. 4),
inhibition of melanogenesis appears to be due neither to cytotoxic
effects nor to cell growth inhibition by compounds 2a and 2d. The
suppression of melanogenesis by compounds 2a and 2d could be
and the same Vmax value of 1.37 ꢂ 10ꢀ2. Accompanying the in-
crease in L-tyrosine concentration there was a dose-dependent in-
crease in the Km value of mushroom tyrosinase without a change in
Compound 2a
A
***
***
Tyrosinase activity (%)
Melanin contents (%)
***
***
***
***
***
***
***
***
***
0
50
100
150
200
250
300
% of control
Compound 2d
B
***
***
Tyrosinase activity (%)
Melanin contents (%)
***
***
***
***
***
***
**
***
***
***
0
50
100
150
200
250
300
% of control
Figure 5. Inhibitory effect of compound 2a (A) or 2d (B) after treatment with 100 nM
mean S.E. of three experiments. Data are expressed as a percentage of the control. ⁄⁄p <0.01 and ⁄⁄⁄p <0.001, compared with the 100 nM
compound 2a or 2d on tyrosinase activity: In the presence of 100 nM -MSH, B16 cells were treated with varying doses of compound 2a or 2d (0.2–10.0
are expressed as the percentage of control and each column represents the mean S.E. of three determinations. ⁄⁄⁄p <0.001, compared with the 100 nM
a
-MSH in B16 cells. Melanin contents were measured at 405 nm. Values represent the
-MSH-treated group. Effect of
M) for 24 h. Results
-MSH-treated group.
a
a
l
a

Y. M. Ha et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2445–2449
2449
attributed to their inhibition of murine-derived tyrosinase.
Although compound 2a (IC50 = 1.14 M) is a less potent inhibitor
of mushroom tyrosinase than compound 2d (IC50 = 0.01 M), as
shown in Table 2, 2a is a more potent inhibitor of melanogenesis
than 2d in B16 cells.
To analyze the mechanisms by which compounds 2a and 2d in-
hibit melanin biosynthesis, we examined the inhibitory effects of
these compounds on cellular tyrosinase activity using murine-de-
rived tyrosinase. After incubation for 24 h with compound 2a or
2d, mushroom-derived tyrosinase activities were 242.99% at
bit more potent inhibitory activities against tyrosinase than the
catechol scaffold, which depends on the remaining template
excluding the phenyl scaffold. Both 2a and 2d are promising candi-
dates for the development of safe pharmacological or cosmetic
agents that have potent inhibitory effects against tyrosinase activ-
ity and melanin biosynthesis.
l
l
Acknowledgments
This work was supported by a National Research Foundation of
Korea (NRF) grant funded by the Korea government (MEST) (No.
2009-0083538) and by the Basic Science Research Program
through the National Research Foundation of Korea (NRF) funded
by the Ministry of Education, Science and Technology (No. 2010-
0012038). We thank the Aging Tissue Bank for providing research
materials for the study.
0.2
and 95.17% at 10.0
100 nM -MSH-only-treated group (244.46%) and the control
group (100%), and were 242.63% at 0.2 M, 200.52% at 1.0 M,
110.84% at 2.5 M, 107.73% at 5.0 M, and 99.59% at 10.0 M of
2d (Fig. 5B), compared with the 100 nM -MSH-only-treated group
l
M, 133.27% at 1.0
lM, 98.78% at 2.5 lM, 97.66% at 5.0 lM,
l
M of 2a (Fig. 5A), compared with the
a
l
l
l
l
l
a
(245.72%) and the control group (100%). Interestingly, contrary to
results with mushroom tyrosinase, compound 2a showed more po-
tent inhibitory activity against murine-derived tyrosinase and
more potent inhibition of melanogenesis in B16 cells than 2d.
In summary, among substituted phenyl-benzo[d]thiazole ana-
logues, compounds 2a and 2d were found to be approximately
16-fold and 1845-fold, respectively, more potent than kojic acid,
which is often used as a positive reference compound. Docking
simulation with 2a and 2d confirmed that they bind strongly to
mushroom tyrosinase. Both 2a and 2d were identified as compet-
itive inhibitors of mushroom tyrosinase in a kinetic study. The lack
of 2a and 2d cytotoxicity indicates that the suppression of melano-
genesis by compound 2a or 2d can be attributed to the inhibition of
murine tyrosinase. These data suggest that both 2a and 2d have
strong de-pigmenting activities without discernible cytotoxicity
in a B16 cell system. These results provide the strongest evidence
to date that the 20,40-dihydroxyphenyl and 40-hydroxyphenyl scaf-
folds among substituted phenyl-benzo[d]thiazole analogues exhi-
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.02.064.
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