Evaluation of anti-pigmentary effect of synthetic sulfonylamino chalcone

Article abstract of DOI:10.1016/j.ejmech.2010.01.049

The 4′-(p-toluenesulfonylamino)-4-hydroxychalcone (TSAHC), which bears inhibitory chemotypes for both α-glucosidase and tyrosinase, was evaluated for tyrosinase activity and depigmenting ability relative to compounds designed to only target tyrosianse act

Full text of DOI:10.1016/j.ejmech.2010.01.049

European Journal of Medicinal Chemistry 45 (2010) 2010–2017
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Evaluation of anti-pigmentary effect of synthetic sulfonylamino chalcone
Woo Duck Seo b, Young Bae Ryu c, Marcus J. Curtis-Long d, Chan Woo Lee e, Hyung Won Ryu a,
,
Ki Chang Jang b, Ki Hun Park a
*
a Division of Applied Life Science (BK21 program), EB-NCRC, Institute of Agriculture & Life Science, Graduate School of Gyeongsang National University, Jinju 660-701,
Republic of Korea
b Department of Functional Crop, NICS, RDA, Miryang 627-803, Republic of Korea
c Bioindustry Research Center, KRIBB, Jeongeup 580-185, Republic of Korea
d 12 New Road, Nafferton, Driffield, East Yorkshire YO25 4JP, UK
e R&D Center, Amore-Pacific Corporation, 314-1,Bora-dong, Kiheung-gu, Yongin-si 449-720, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
The 40-(p-toluenesulfonylamino)-4-hydroxychalcone (TSAHC), which bears inhibitory chemotypes for
Article history:
Received 28 September 2009
Received in revised form
14 January 2010
Accepted 20 January 2010
Available online 28 January 2010
both a-glucosidase and tyrosinase, was evaluated for tyrosinase activity and depigmenting ability rela-
tive to compounds designed to only target tyrosianse activity. TSAHC emerged to be a competitive
reversible inhibitor of mushroom tyrosinase. More importantly, it was also able to return the melanin
content of a-melanocyte stimulated by a-MSH to base levels unlike other inhibitors that only targeted
tyrosinase. The Western blot for expression levels of proteins involved in melanogenesis showed that
TSAHC significantly decreased three main tyrosinase related protein in melanin biosynthesis, tyrosinase,
TRP-1 and TRP-2.
Keywords:
Chalcone
Depigmentation
Sulfonylamino chalcone
Tyrosinase
Ó 2010 Elsevier Masson SAS. All rights reserved.
1. Introduction
(monophenolase) and then to dopaquinone (diphenolase). Tyrosi-
nase is thus a perfect target in many ways because it is produced
Excessive/uneven pigmentation is the root cause of numerous
skin problems including age spots, melasma and chloasma. These
maladies can be initiated by ultraviolet light, chronic inflammation
only by melanocytic cells and to reach its fully active form it
requires processing (glycosylation) in the ER and Golgi, after which
time it is trafficked to specialized organelles, melamosomes [3].
Thus numerous highly tyrosinase specific processes can be targeted
to effect inhibition of melanin production. In principle, tyrosinase
activity can be attenuated by regulating the transcription of
tyrosinase mRNA, tyrosinase related protein-1 (TRP-1), tyrosinase
related protein-2 (TRP-2) [4] and its maturation via asparagine-
liked oligosaccharide processing [5–7]. However, arguably the most
direct route is to inhibit tryosinase (EC 1.14.18.1).
as well as abnormal levels of
a-melanocyte stimulating hormone
(a
-MSH) which results in overproduction of melanin [1,2]. It is thus
not unsurprising that many efforts have been devoted to screening
both recognized and putative depigmenting agents in a bid to
develop drugs to attenuate hyperpigmentation. Most of these
screens have in some way targeted tyrosinase, which has been
considered to be an effective approach to treat a variety of hyper-
pigmentary disorders [3]. Tyrosinase in Homo sapiens is
a membrane-bound glycoprotein with an active site containing two
copper ions. This enzyme catalyzes two different processes
involving the oxidation of tyrosine, first to L-DOPA
Numerous reports have focused on the inhibition of tyrosinase
as the sole route to depigmentation of the epidermal layer [8,9].
Kojic acid is a representative tyrosinase inhibitor with an IC50 of
16 mM against mushroom tyrosinase. Its activity is ascribed to
copper chelation. However, despite the wide knowledge garnered
to date concerning depigmentation and the numerous ways to
bring it about, we noticed that there has so far been no effort to
target multiple proteins in the pathway to melanogenis in one
inhibitor. For instance, one of the most widely employed strategies
to effect a decrease in cellular tyrosinase activity which does not
involve direct tyrosinase inhibition is the inhibition of enzymes
involved in tyrosinase N-glycosylation [10]. This reduces tyrosinase
Abbreviations: IC50, the inhibitor concentration leading to 50 % activity loss;
Ki, inhibition constant; TRP-1, tyrosinase related protein-1; TRP-2, tyrosinase
related protein-2;
toluenesulfonylamino)-4-hydroxychalcone; NMR, nuclear magnetic resonance;
TM4SF5, four-transmembrane L6 family member 5.
a-MSH, a
-melanocyte stimulating hormone; TSAHC, 40-(p-
* Corresponding author. Tel.: þ82 55 751 5472; fax.:þ82 55 757 0178.
E-mail address: khpark@gsnu.ac.kr (K.H. Park).
0223-5234/$ – see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.01.049

W.D. Seo et al. / European Journal of Medicinal Chemistry 45 (2010) 2010–2017
2011
O
O
O
O
S
O
HO
OH
OH
R
N
H
OH
O
1
S N
OH
For tyrosinase
O
H
O
3: R = CH3
5: R = OH
7: R = NH2
4: R = H
6: R = NO2
8: R = F
For glycosidase
H2N
2
Fig. 1. Different chemotypes within TSAHC and their presumed targets.
Fig. 2. Chemical structures of chalcone derivatives (1–8).
procesing and lowers tyrosinase activity because the tyrosinase can
not reach its full maturity, and hence remains inactive. It thus
occurred to us that a substance which can inhibit both tyrosinase
and glycosidase may be a more promising depimenting agent.
Chalcones are natural products of widespread occurrence in
plants. They have been reported to exhibit a wide range of phar-
maceutical effects including antioncogenetic, anti-inflammatory,
anti-ulcerative, antimalarial, antiviral, antifungal, and antibacterial
activities [11]. Recently, they were highlighted as a new class of
tyrosinase inhibitor in several publications. Nerya Ohad et al.
reported that an OH group at the 4-position in B-ring of the chal-
cone is the major factor affecting inhibitory potency. This is
presumably because these species have a phenol chemotype which
the enzyme’s native substrate, tyrosine, also contains [12]. In
a previous communication, we showed that sulfonylamino cha-
reduce pigmentation, which is the principal objective of this study.
Furthermore, sulfonylamino chalcones bearing an unsubstituted
phenyl unit on the ring B were not effective tyrosinase inhibitors in
vitro, which is consistent with the general belief that a 4-hydroxy
phenyl moiety on ring B of the chalcone is required for tyrosinase
inhibitory activity.
2. Chemistry
Sulfonylamino chalcone derivatives were easily obtained
through the Claisen–Schmidt condensation of hydrox-
ybenzaldehyde and derivertized acetophenones using an acidic
catalyst [13]. The 40-(p-toluenesulfonylamino)-4-hydroxychalcone
(TSAHC) was prepared from hydroxybenzaldehyde and N-sulfony-
lamino acetophenone using a catalytic amount of H2SO4 in MeOH
(Scheme 1).
cones are potent
values. Our structure activity relationship (SAR) studies unearthed
some important requirements for -glucosidase inhibitory activity
a-glucosidase inhibitors with nanomolar IC50
a
(Fig. 1) [13,14]. In subsequent work, amino chalcones were able to
reduce the tumorigenic proliferation induced by the trans-
membrane four L6 family member 5 (TM4SF5), the function of
which is deeply related to N-glycosylation [15].
When all the above results were considered, we hypothesized that
sulfonylamino hydroxy-chalcones (hybrids of tyrosinase inhibiting
hydroxychalcones and glucosidase inhibiting sulfonamide chalcones)
may have formidable antimelanogenic activity as they can inhibit
both processing of tyrosinase as well as tyrosinase catalytic activity.
Herein, we report that 40-(p-toluenesulfonylamino)-4-hydrox-
ychalcone (TSAHC) is an excellent depigmentaryagent in a number of
cell based assays through inhibition of tyrosinase’s catalytic function
and tyrosinase related proteins such as TRP-1 and TRP-2.
3. Results and discussion
The aim of the present work is to evaluate the anti-pigmentary
effects of the bifunctional inhibitor TSAHC (3), which is adorned
with functionalities for controlling both glycosidase and tyrosinase
activities (Fig. 1). To allow us to draw meaningful comparisons we
compare the efficacy of TSAHC with structurally similar inhibitors
which only target tyrosinase. In our previous study, TSAHC
inhibited
a-glucosidase with 0.58 mM Ki value [13]. Molecular
docking simulations revealed that sulfonylamino chalcones bind to
the active site in a similar fashion to known inhibitors like acarbose
and voglibose [14]. The sulfonylamino group plays the critical role
in inhibitor/protein interaction. For example His111 and His348
In these studies, we chose to focus our efforts on inhibitors
bearing a para-hydroxy substituent in the B ring of the sulfonyla-
mino chalcone. This is because it is well known that the corre-
sponding catechol derived species are highly cytotoxic, being
rapidly oxidized to ortho-quinones by tyrosinase [16]. As such they
are not viable start points for in vivo screening of compounds which
within a-glucosidase can interact with the SO2 function and indeed
both these residues are crucial for an efficient enzyme/substrate
binding interaction [14]. As shown in Fig. 2, the sulfonylamino
chalcones (3–8) in this study also have a 4-hydroxychalcone moiety
that was suggested to be instrumental in tyrosinase inhibiton by
Nerya Ohad et al. [12].
O
O
(ii)
1, R1 = NH2
2, R1 = OH
OH
R1
R1
O
O
O
(i)
(ii)
O
O
S N
O
R1
S N
R1
OH
H2N
H
O
H
3, R1 = CH3
6, R1 = NO2
7, R1 = NH2
8, R1 = F
4, R1 = H
5, R1 = OH
Scheme 1. Synthesis of chalcone derivatives: (i) p-toluenesulfonyl chloride, pyridine, CH2Cl2, r.t, (ii) 4-Hydroxybenzaldhyde, H2SO4, MeOH, reflux.

2012
W.D. Seo et al. / European Journal of Medicinal Chemistry 45 (2010) 2010–2017
Table 1
In order to investigate and evaluate the activity of TSAHC against
Tyrosinase inhibitory activity and melanin formation of chalcone derivatives (1–8).
Compound Tyrosinase (enzyme) Mealnin formation (cell)
M) IC50 M)
tyrosinase, we examined its tyrosinase inhibitory activity and
compared it to all chalcone derivatives (1–8) as shown Fig 3A. Table 1
shows that all compounds exhibited a significant degree of mono-
IC50
(m
M) Inhibition type Ki (
m
(m
phenolase (L-tyrosine substrate to dopaquinone) inhibition, although
1
2
3
4
5
6
7
8
4.8
8.3
Competitive
Competitive
Competitive
Competitive
Competitive
Competitive
Competitive
Competitive
–
2.9
3.9
48.7
28.1
10.4
24.2
32.1
sulfonylamino chalcones showed slightly lower inhibitory activities
compared to the parent chalcones (1, 2). The sulfonylamino chalcones
inhibit tyrosinase dose-dependently with a similar pattern to the
parent chalcones (1, 2) (Fig. 3A). The inhibition of mushroom tyrosi-
nase by TSAHC is illustrated in Fig. 3B representatively. Plots of the
initial velocity versus enzyme concentration in the presence of
different concentrations of TSAHC gave a family of straight lines, all of
which passed through the origin. Increasing the inhibitor concen-
tration resulted in a lowering of the line gradients, indicating that
TSAHC was a reversible inhibitor. The mode of inhibition was
analyzed by Lineweaver–Burk plots (Fig. 3C), which showed that
TSAHC behaved as a competitive inhibitor because increasing
concentration of substrate resulted in a family of lines which declined
with a common intercept on the y-axis. The inhibition kinetics were
illustrated by Dixonplots, which were obtained by plotting 1/V versus
[I] with varying concentrations of substrate. Dixon plots give a family
of straight lines passing through the same point at the second quad-
rant, giving the inhibition constant (ꢀKi) (Fig. 3D). Most importantly,
the plots of initial rate vs enzyme concentration (Fig. 3B) for TSAHC all
consisted of straight lines passing through the origin, which strongly
suggest that TSAHC is a competitive inhibitor for the monophenolase
23.3
22.5
58.8
15.2
30.7
29.1
16.3
12.6
13.3
25.2
11.4
16.7
14.9
–
–
21.2
19.8
>50
Kojic acid
activity of tyrosinase. Through a similar analysis, all synthetic chal-
cones (1–8) have respective inhibition constants of 2.9, 3.9,12.6,13.3,
25.2, 11.4, 16.7, and 14.9 mM (Table 1).
The above data show that the chalcone skeleton exhibits
optimal mushroom tyrosinase inhibition when sterically unen-
cumbering groups are present on the A ring: only inhibitors 1 and 2
have IC50 and Ki values in the single digit micromolar range,
whereas all inhibitors bearing the sulfonyl group had IC50 and Ki
values around 3–8 fold higher. As all inhibitors studied were
competitive, it is likely that there are unfavorable steric interactions
between the bulky (tetrahedral) sulfonyl function and the active
site. The crystal structure of inhibitor 3 shows that the aryl
Fig. 3. (A) Effect of chalcone derivatives (1–3) on the oxidation of L-tyrosine by mushroom tyrosinase. (B) Relationship between the catalytic activity of tyrosinase and concen-
trations of compound 3. Concentrations of compound 3 for lines from top to bottom were 0, 10, 15, 20, and 25 M, respectively. (C) Lineweaver–Burk plots for the inhibition of the
monophenoase activity of mushroom tyrosinase by compound 3. (D) Dixon plots for the inhibition of the monophenoase activity of mushroom tyrosinase by compound 3 in the
presence of different concentrations of L-tyrosine (90, 180, and 360 M, respectively).
m
m

W.D. Seo et al. / European Journal of Medicinal Chemistry 45 (2010) 2010–2017
2013
appendage within the sulfonamide moiety is orientated perpen-
A
100
80
60
40
20
0
*
dicular to the ring system, which may further impede inhibitor
binding (Fig. 4) [17]. The complex but relatively minor variation in
IC50s seen by varying the 4-substituent on the phenyl unit within
the sulfonamide shows steric effects are much less important at
this position.
Having characterized these inhibitors in vitro, we progressed to
investigate their effects in vivo using cultured cells as well as in an
animal model. It is well known that the hormone a-MSH upregu-
*
*
**
**
*
lates tyrosinase and tyrosinase related protein production through
the activation of a transcription factor, MITF. Therefore, we exam-
3
ined the effect of target compounds (1–3) on
tyrosinase expression levels in B16 melanoma cells. In this assay,
B16 melanoma cells were incubated with 20 M compounds (1–3)
in presence of 10 ng/ml of -MSH for 2 days. Cells under the same
conditions but without added inhibitors were used as a control and
the cells without any additives ( -MSH or inhibitor) were used as
a
-MSH-controlled
2
1
Compounds
m
B
a
a
a baseline reference. As shown in Fig. 5, exposure of the cells to
MSH more than doubled the melanin content of the cells. However,
upon treatment with compounds 1–3 the pigmentation and
melanin content of the MSH stimulated cells were both reduced. As
may have been expected based upon its double inhibitor motif,
TSAHC was the best inhibitor and was able to return the melanin
α
-
-
+
-
+
+
+
+
-MSH
Compounds (20 μM)
2
KA
1
3
0.5
0.4
0.3
0.2
0.1
0
pigment content to the levels seen in cells without a-MSH treat-
*
ment. Most pleasingly, this compound also emerged to have very
low cytotoxicity. An important point of note is that hydroxy-4-
hydroxychalcone 1, which showed the best inhibitory activity
*
*
*
(IC50 ¼ 4.8
mM) against mushroom tyrosinase in vitro, was not
particularly effective at decreasing cell pigmentation. This result
can be ascribed in part to the fact that the structure/regulation of
mushroom tyrosinase differs significantly in several respects from
mammalian tyrosinase: compounds that are active against mush-
room tyrosinase do not show comparable results with mammalian
tyrosinase [18]. In addition inhibitors 1 and 2 do not have any
glucosidase ability and hence may be expected to be less potent
than the hybrid inhibitors in the in vivo assays. For instance, 4-
amino-40-hydroxychalcone 2 showed a potent inhibitory effect on
tyrosinase in vitro, but its efficacy against melanogenesis
α
-MSH
Compounds (20 μM)
+
-
+
+
+
+
-
-
1
2
KA
3
Fig. 5. (A) Effect of compounds 1–3 on the growth of B16 melanoma cells. (Black,
M; dark gray, 10 M; light gray, 20 M; white, 30 M). Cytotoxicity was measured
0
m
m
m
m
by MTT assay of chalcone compounds (1–3) and control (DMSO) treated B16 melanoma
cells after 48 h at the indicated concentrations. (B) Effect of chalcone compounds on
melanin content in B16 melanoma cells. Cells (5 ꢁ106 cells per well) were incubated
separately with 20
the presence of 10 ng mLꢀ1
a percentage of
mM of the indicated concentration of three chalcone compounds in
(IC50 ¼ 48.7
in vivo. In a similar vein, the control inhibitor, kojic acid, was not
able to reduce melanogenesis to the base level at 20 M. The most
mM) was significantly less than TSAHC (IC50 ¼ 10.4
mM)
a
-MSH for 48 h; KA, Kojic acid. Results are showed as
a
-MSH induced control and presented as mean ꢂ SEM of three inde-
m
pendent experiments. The data shown are representative of combined mean of three
important aspect of these assays is that the effects of the three
chalcones studied are not due to their cytostatic activities because
concentrations of inhibitors were chosen to be well below
independent experiments. *P < 0.05.
Fig. 4. ORTEP view of TSAHC (CCDC no. ¼ 703762).

2014
W.D. Seo et al. / European Journal of Medicinal Chemistry 45 (2010) 2010–2017
A
-MSH
α
-
-
+
-
+
+
+
μ
Compound 3 ( M)
20
10
30
0.5
0.4
0.3
0.2
0.1
0
*
**
**
Fig. 7. Effect of compound 3 on a-MSH-induced increase of microphthalmia-associ-
ated transcription factor (MITF), tyrosinase, tyrosinase related protein 1 (TRP-1) and
TRP-2 expression analyzed by western blot (average of three results).
**
+
+
+
+
-
Repeating the experiments above using differing concentrations of
the inhibitor showed that the effects of the latter were dose
dependent (Fig. 6). Furthermore, the survival of cells grown in the
-MSH
Compound 3 ( M)
-
-
α
μ
20
10
30
B
presence of TSAHC varied as follows: 92% 10
78% at 30 M. These detailed results give quantitative backing to
our claim that TSAHC inhibits melanin formation through a non-
cytotoxic/cytostatic pathway. Thus at 20 M concentration this
inhibitor is able to regulate the melanin concentrations of hyper-
stimulated cells to normal levels, while the vast majority of cells
remain viable.
The above results spurred us on to investigate the effects of
TSHAC at the biochemical level. Three enzymes are known to be
involved in melanin biosynthesis in mammals, namely tyrosinase,
tyrosinase related protein-1 (TRP-1) and tyrosinase related protein-
2 (TRP-2) [4]. Therefore, we investigated the dose dependent effects
that TSAHC exerted on the expression levels of tyrosinase and
tyrosinase related proteins by western blot analysis in MSH-treated
B16 melanoma cells. As anticipated, cells treated with MSH alone
(in the absence of inhibitor) showed significant upregulation of
MITF (transcription factor) which in turn led to increased TRP-1 and
2 and tyrosinase. However, the expression levels of MITF and
tyrosinase related proteins were significantly reduced by a 2-day
mM, 84% at 20 mM, and
*
100
m
80
60
40
20
0
*
m
**
**
0
5
10
20
μ
30
Concentration of compound 3( M)
Fig. 6. Effect of various concentrations of compound 3 on cellular melanin content and
celluar tyrosinase activity in B16 melanoma cells. (A) Effect of various concentrations of
compound 3 on melanin content in B16 melanoma cells. (B) Cells (5 ꢁ106 cell per well)
were treated with the indicated concentration of compound 3 in the presence of
10 ng mLꢀ1
a-MSH for 48 h. Cellular tyrosinase activity was determined as described in
material and methods. The data shown are representative of combined mean of three
independent experiments. *P < 0.05; **P < 0.02.
treatment of TSAHC at 20 mM concentration (Fig. 7). These results
concentrations leading to significant cell growth inhibition. The
effects of the inhibitors on growth were assessed using a calori-
metric MTT-based assay as described in the experimental section.
We progressed to analyze the action of TSAHC more thoroughly.
indicate that TSAHC exerts its strong depigmenting activity at least
partially by downregulating expression levels of the various
proteins required for tyrosinase processing/transcription. As we
Fig. 8. (A) Photographs of the dorsal skin of guinea pig. Representative photographs of UV-irradiatted dorsal skin of guinea pig treated with 0.3% of TSAHC. (B) Degree of
pigmentation. The degree of pigmentation (DL value) before and daily topical application of vehicle (6), non-treated (-), 2% of hydroquinone (;), 1% of kojic acid (B), and 0.3% of
TSAHC (C) was measured.

W.D. Seo et al. / European Journal of Medicinal Chemistry 45 (2010) 2010–2017
2015
specifically designed TSAHC to exhibit dual inhibition characteris-
tics this relatively complex behavior is not unexpected.
dd, J1 ¼6.83, J2 ¼ 2.0 Hz), 7.52 (1H, d, J ¼ 15.6 Hz), 7.53 (2H, d,
J ¼ 8.9 Hz), 7.70 (1H, d, J ¼ 15.5 Hz), 7.98 (2H, dd, J1 ¼6.8,
TheskinwhiteningeffectofTSAHCwasnextexaminedusingaUV-
induced hyperpigmentation model in brown guinea pigs. These
animals are excellent models because they have functional melano-
cytes in their epidemis, which respond well to several stimuli
including UV light. TSAHC was topically applied twice daily for 4
weeks to the dorsal skin of brown guinea pigs which had been tanned
by irradiation once a week for three consecutive weeks. Hydroqui-
none (2%) and kojic acid (1%) were used as positive controls. A visible
decrease in hyperpigmentation was observed 4 weeks after treat-
ment with TSAHC, hydroquinone, kojic acid, and vehicle group. The
J2 ¼ 2.0 Hz); 13C NMR (75 MHz; MeOD)
d 115.1, 115.6, 118.3, 126.5,
129.9, 130.3, 130.9, 144.4, 159.9, 162.3 and 189.8; EIMS m/z 240
[Mþ]; HREIMS m/z 240.0788 [Mþ] (calculated for C15H12O3,
240.0786).
5.2.2. 40-Amino-4-hydroxychalcone (2)
m.p. 79–80 ꢃC; IR (KBr) : 3510, 1665 cmꢀ1 1H NMR (300 MHz;
;
MeOD)
d
6.44 (2H, dd, J1 ¼6.9, J2 ¼ 1.9 Hz), 6.85 (2H, dd, J1 ¼6.9,
J2 ¼1.8 Hz), 7.55 (3H, m), 7.68 (1H, d, J ¼ 15.5 Hz), 7.91 (2H, dd,
J1 ¼7.9, J2 ¼ 2.0 Hz); 13C NMR (75 MHz; MeOD)
d 113.1, 115.5, 118.5,
change in pigmentation (
etertoquantifythedegreeofpigmentationreductioninducedbyeach
compound. As shown in Fig. 8, the L values of 0.3% TSAHC, 1% kojic
DL) values were measured using a colorim-
126.5, 126.8, 130.1, 131.0, 143.4, 153.8, 159.7 and 189.1; EIMS m/z 239
[Mþ]; HREIMS m/z 239.0948 [Mþ] (calculated for C15H13NO2,
239.0946).
D
acid, and 2% hydroquinone were obtained as 1.06, 1.49, and 2.18,
respectively. Fig. 8 indicates that the skin returned to its original color
after the TSAHC treatment without apparent side-effect.
5.2.3. 40-(p-Toluenesulfonamide)-4-hydroxychalcone (3)
m.p. 105–107 ꢃC; IR (KBr): 3520, 1675 cmꢀ1; 1H NMR (300 MHz;
MeOD)
d
2.25 (3H, s), 6.82 (2H, d, J ¼ 8.6 Hz), 7.23 (4H, m), 7.42 (1H, d,
4. Conclusion
J ¼ 15.5 Hz), 7.51 (2H, d, J ¼ 8.6 Hz), 7.66 (1H, d, J ¼ 15.5 Hz), 7.73 (2H,
d, J ¼ 8.3 Hz), 7.90 (2H, dd, J1 ¼8.7, J2 ¼ 2.0 Hz); 13C NMR (75 MHz;
In conclusion, we have rigorously investigated a range of
inhibitors designed to be able to target both tyrosinase and glyco-
sidase simultaneously. The 40-(p-toluenesulfonylamino)-4-
hydroxychalcone (TSAHC) bearing inhibitory chemotypes of both
MeOD) d 20.1, 115.6, 118.0, 118.5, 126.4, 126.9, 129.4, 129.8, 130.5,
133.5, 136.6, 142.4, 144.1, 145.2, 160.2 and 189.7; EIMS m/z 393 [Mþ];
HREIMS m/z 393.1033 [Mþ] (calculated for C22H19NO4S, 393.1035).
a
-glucosidase and tyrosinase, showed potent inhibitory activities
5.2.4. 40-(Benzensulfonamide)-4-hydroxychalcone (4)
against mushroom tyrosinase in vitro as well strong depigmenting
ability against B16 melanoma cells and UV-induced hyperpig-
mentation in brown guinea pigs skin. Our data suggest that TSAHC
downregulated tyrosinase expression as well as a number of pro-
cessing enzymes and transcription factors (TRP1, TRP2, and MITF).
Our work has thus gone a long way to showing that the strategy of
designing hybrid inhibitors targeting different aspects of tyrosinase
maturation and activity can be used to treat skin pigmentation.
m.p. 206–208 ꢃC; IR (KBr): 3535,1655 cmꢀ1; 1H NMR (300 MHz;
MeOD)
d
6.84 (2H, d, J ¼ 8.6 Hz), 7.27 (2H, d, J ¼ 8.7 Hz), 7.55 (6H,
m), 7.71 (1H, d, J ¼ 15.5 Hz), 7.88 (2H, d, J ¼ 8.6 Hz), 7.96 (2H, d,
J ¼ 8.7 Hz); 13C NMR (75 MHz; MeOD)
d 115.5, 117.9, 118.5, 126.3,
126.8, 128.4, 129.7, 130.5, 132.8, 133.6, 139.7, 142.3, 145.2, 160.3 and
189.6; EIMS m/z 379 [Mþ]; HREIMS m/z 379.0873 [Mþ] (calculated
for C21H17NO4S, 379.0878).
5.2.5. 40-(4-Hydroxylbenzensulfonamide)-4-hydroxychalcone (5)
5. Experimental and methods
m.p. 102–103 ꢃC; IR (KBr): 3525, 1670 cmꢀ1; 1H NMR (300 MHz;
Acetone-d6)
d
6.94 (3H, m), 7.37 (1H, d, J ¼ 8.7 Hz), 7.67 (3H, m), 7.77
5.1. General
(2H, d, J ¼ 8.8 Hz), 8.04 (2H, m), 8.70 (3H, s); 13C NMR (75 MHz;
Acetone-d6)
d 115.6, 115.8, 118.4, 118.5, 126.8, 129.6, 128.9, 130.3,
All reactions were monitored by thin layer chromatography (TLC)
using commercially available glass-backed plates. Column chroma-
tography was carried out using 230–400 mesh silica gel. The final
solution before evaporation was washed with brine and dried over
anhydrous Na2SO4. Melting points were measured on a Thomas
Scientific Capillary Point Apparatus and are uncorrected. Infrared
spectra (IR) were recorded on a Bruker IFS 66. 1H and 13C NMR data
were obtained on a Bruker AM 300 (1H NMR at 300 MHz and 13C
NMR at 75 MHz) spectrometer in either MeOD or acetone-d6; EIMS
and HREIMS data were collected on a JEOL JMS-700 spectrometer.
130.7, 133.7, 142.4, 144.0, 159.9, 161.5 and 187.6; EIMS m/z 395 [Mþ];
HREIMS m/z 395.0830 [Mþ] (calculated for C21H17NO5S, 395.0827).
5.2.6. 40-(4-Nitrobenzensulfonamide)-4-hydroxychalcone (6)
m.p. 136–138 ꢃC; IR (KBr): 3513, 1656 cmꢀ1; 1H NMR (300 MHz;
Acetone-d6)
d
6.92 (2H, d, J ¼ 8.6 Hz), 7.42 (2H, d, J ¼ 8.7 Hz), 7.68
(4H, m), 8.08 (2H, d, J ¼ 8.7 Hz), 8.17 (2H, d, J ¼ 8.9 Hz) and 8.41 (2H,
d, J ¼ 8.9 Hz); 13C NMR (75 MHz; Acetone-d6)
d 116.3, 118.7, 119.3,
125.3, 126.3, 128.8, 130.5, 131.4, 134.2, 141.7, 144.6, 145.1, 150.5, 160.6
and 187.9; EIMS m/z 424 [Mþ]; HREIMS m/z 424.0728 [Mþ]
(calculated for C21H16N2O6S, 424.0729).
5.2. General method for synthesis of chalcone and sulfonylamino
chalcone derivatives
5.2.7. 40-(4-Aminobenzensulfonamide)-4-hydroxychalcone (7)
m.p. 216–217 ꢃC; IR (KBr): 3526, 1663 cmꢀ1; 1H NMR (300 MHz;
A solution of the required para-substituted acetophenone
(0.1 mol) and 4-hydroxybenzaldehyde (0.12 mol) in MeOH with
a catalytic amount of H2SO4 was refluxed for 1 day after which time
it was neutralized with 15 % NaOH (50 mL). The organic layer was
extracted with EtOAc, and dried over anhydrous Na2SO4, and
evaporated in vacuo. The residue was purified by column chro-
matography eluted with hexane/acetone to give pure chalcone
derivatives.
Acetone-d6)
d
6.70 (2H, d, J ¼ 8.7), 6.93 (2H, d, J ¼ 8.6 Hz), 7.37 (2H,
d, J ¼ 8.7 Hz), 7.61 (2H, d, J ¼ 8.8 Hz), 7.70 (4H, m) and 8.06 (2H, d,
J ¼ 8.7 Hz); 13C NMR (75 MHz; Acetone-d6)
d 113.1, 115.9, 118.2,
118.6,125.7,126.9,129.2,129.8,130.6,133.4,142.9,143.9,153.1,159.9
and 187.6; EIMS m/z 394 [Mþ]; HREIMS m/z 394.0985 [Mþ]
(calculated for C21H18N2O4S, 394.0987).
5.2.8. 40-(4-Fluorobenzensulfonamide)-4-hydroxychalcone (8)
m.p. 179–180 ꢃC; IR (KBr): 3544, 1671 cmꢀ1; 1H NMR (300 MHz;
5.2.1. 40,4-Dihydroxychalcone (1)
MeOD)
d
6.84 (2H, d, J ¼ 8.6 Hz), 7.26 (4H, m), 7.55 (3H, m), 7.72 (1H,
m.p. 205 ꢀ 206 ꢃC; IR (KBr) 3440, 1687 cmꢀ1
;
1H NMR
d, J ¼ 15.5 Hz) and 7.94 (4H, m); 13C NMR (75 MHz; MeOD)
d 115.5,
(300 MHz; MeOD)
d
6.85 (2H, dd, J1 ¼8.64, J2 ¼ 2.0 Hz), 6.90 (2HHH,
115.8, 116.1, 117.9, 118.7, 126.3, 129.7, 129.9, 130.5, 133.8, 135.8, 142.1,

2016
W.D. Seo et al. / European Journal of Medicinal Chemistry 45 (2010) 2010–2017
145.2, 160.3 and 189.6; EIMS m/z 397 [Mþ]; HREIMS m/z 397.0782
pretreated with the indicated concentrations of compound,
were treated with -MSH for 48 h. Then the media was aspi-
[Mþ] (calculated for C21H16FNO4S, 397.0784).
a
rated and the cells were washed twice with PBS. Cells were
harvested from each well in a 20 mM Tris – 0.2 % Triton X-100
buffer. Cells were pelleted by centrifugation (10,000 g) and the
melanin was dissolved by treatment with 1 N NaOH for 15 min
5.2.9. X-ray crystal structure analysis of 3
C22H19NO4S, M ¼ 393.44, monoclinic, space group, P21,
a ¼ 11.0821(7) Å, b ¼ 13.1497(8) Å, c ¼ 13.5886(8) Å,
a
¼ 90ꢃ,
b
¼ 102.2520(10)ꢃ,
g
¼ 90ꢃ, V ¼ 1935.1(2) Å3, Z ¼ 4, T ¼ 446(2) K,
at 80 ꢃC. Then, 200
mL crude cell extracts were transferred into
D ¼ 1.350 mg/m3,
m
¼ 0.196 mmꢀ1
,
F(000) ¼ 824, R1 ¼0.0561,
96-well plates. Relative melanin concentration was measured at
400 nm with microplate reader (Molecular Devices, Sunnyvale,
CA, USA).
Rw ¼ 0.1280 for 4195[R(int) ¼ 0.0916] independent reflections [17].
5.3. Mushroom tyrosinase assay
5.7. Measurement of cell-free tyrosinase activity
The mushroom tyrosinase (EC 1.14.18.1) (Sigma Chemical Co.)
was used for in vitro bioassays as described previously with some
modifications [19]. In this experiment, L-tyrosine was used as
a substrate. In a spectrophotometric experiment, enzyme activity
was monitored by dopachrome formation at 475 nm with a UV-Vis
spectrophotometer (Spectro UV-Vis Double beam; UVD-3500, Lab-
omed, Inc.) at 30 ꢃC. All samples were first dissolved in EtOH at
The tyrosinase activity was measured by the method of Akao
et al. [22] with a slight modification. Briefly, B16 melanoma cells,
pretreated with the indicated concentrations of chalcones for 3 h,
were treated for 48 h with
a
-MSH (10 ng mLꢀ1). The cells were
washed twice with ice-cold phosphate-buffered saline (PBS) and
extracted and then centrifuged at 10,000 g for 10 min. Samples of
cell extract supernatant were incubated in duplicate for 1 h at 37 ꢃC
10 mM and used for the experiment with dilution. First, 200
mL of
sodium phosphate buffer (pH 7.4) containing 0.1% L-DOPA. Dop-
achrome formation was monitored by measuring absorbance at
470 nm.
a 2.7 mM
with 2687
L
-tyrosine (Km ¼ 180
m
M) aqueous solution was mixed
m
L of 0.25 M phosphate buffer (pH 6.8). Then, 100
mL of
the sample solution and 13 mL of the same phosphate buffered
solution of mushroom tyrosinase (144 units) were added in this
order to the mixture. Each assay was conducted in triplicate. The
inhibitor concentration leading to 50% activity loss (IC50) was
obtained by fitting experimental data to the logistic curve by Eq. (1):
5.8. Western blotting
In order to measure cellular levels of MITF, tyrosinase, TRP-1
and TRP-2, B16 cells, pretreated with the indicated concentrations
of chalcones for 3 h, were treated with
a
-MSH (10 ng mLꢀ1) for
Activity ð%Þ ¼ 100½1=ð1 þ ð½Iꢄ=IC50ÞÞꢄ
(1)
48 h. Cells were washed with PBS and scraped in lysis buffer
[40 mM Tris–HCl (pH 8.0), 120 mM NaCl, 0.1 % Nonidet P-40, and
protease inhibitors]. They were then centrifuged at 14,000 g for
15 min at 4 ꢃC and supernatants (total cell lysates) were collected
and aliquoted. These lysates were either used on the day of
preparation or were immediately stored at ꢀ78 ꢃC until required.
5.4. Cell culture and chemicals
The B16 murine melanoma cells were grown in Dulbecco’s
modified minimum essential medium (DMEM) (Invitrogen, NY,
USA) supplemented with 10 % heat-inactivated fetal bovine serum
(Invitrogen, CA, USA),
pure, CA, USA)], and antibiotics [penicillin (50 unit/mL) and strep-
tomycin (50
g/mL)] (Gibco, Invitrogen, CA, USA)] at 37 ꢃC in a 5%
CO2 humidified atmosphere. MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolidium bromide (MTT), -MSH and kojic acid were
L-glutamine (2.5 mM), HEPES [40 mM (Bio-
For western blot analysis, 30 mg of lysate was resolved on 12.5%
and 16% polyacrylamide gels and then transferred to nitrocellulose
membranes. Membranes were blocked with 5% fat-free milk in
PBS (pH 7.4) for 30 min at room temperature and then incubated
with appropriate mono- and polyclonal primary antibodies in
blocking buffer for 2 h to overnight at 4 ꢃC. After washing,
membranes were incubated with anti-mouse or anti-rabbit horse
radish peroxidase conjugated secondary antibodies for 1 h at room
temperature, and then subjected to ECL solution and visualized
using Hyperfilm (Amersham Biosciences). Densitometric analysis
of Western blots was performed with the use of the Quantity one
software system (Bio-Rad XRS).
m
a
obtained from Sigma. Anti-tyrosinase, anti-MITF, anti-TRP-1 and
anti-TRP-2 were purchased from SantaCruz (USA, CA). TSAHC
treatment began 24 h after plating, and cells were harvested after 2
days of incubation.
5.5. Cell viability assay
After treatment with kojic acid or one of the three compounds,
cells were assayed for growth activity using a MTT 3-((4,5-dime-
thylthiazol-2-yl)-2,5-diphenyltetrazolidium bromide, Sigma, USA)-
based colorimetric method, as previously described [20]. Briefly,
Cells were seeded at densities of 5000 cells/well in 96-well culture
plates. Cells were treated with chalcone compounds for 72 h. After
treatment with these compounds, the attached cells were incu-
bated with 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl tetrazolium
bromide (MTT, 0.5 mg/mL, 1 h) and subsequently solubilized in
DMSO. The absorbency at 550 nm was then measured using
a microplate reader. The GI20 is the concentration of agent that
reduced the cell viability by 20% under the experimental
conditions.
5.9. UV-induced hyperpigmentation in brown guinea pigs
UV-induced hyperpigmentation was induced on the backs of
brownish guinea pigs weighing approximately 500 g (Jungang
Animal Co) using a modification of the methods reported by
Hideya et al. and Imokawa et al. [23,24]. The guinea pigs were
anesthetized with pentobarbital (30 mg/kg), and separate areas
(1 cm2) on the back of each animal were exposed to UV irradi-
ation (Waldmann UV 800, Herbert Waldman GmbH, Philis TL/12
lamp emitting 280–305 nm). The total energy dose of UV was
500 mJ/cm2 per exposure period. Each animal was exposed to
UV radiation once a week for three consecutive weeks. The
DL
value was calculated using the value (brightness index)
measured with the colorimeter (CR-300; Minolta, Osaka, Japan)
as follows:
L
5.6. Measurement of melanin content
The melanin content was measured using the method
reported by Park et al. [21] with a slight modification. The cells,
D
L ¼ L ðat each day measureÞ ꢀ L ðat day 0Þ
(2)

W.D. Seo et al. / European Journal of Medicinal Chemistry 45 (2010) 2010–2017
2017
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