Synthesis and biological evaluation of resveratrol derivatives as melanogenesis inhibitors

Article abstract of DOI:10.3390/molecules200916933

Resveratrol (1), a naturally occurring stilbene compound, has been suggested as a potential whitening agent with strong inhibitory activity on melanin synthesis. However, the use of resveratrol in cosmetics has been limited due to its chemical instability and poor bioavailability. Therefore, resveratrol derivatives were prepared to improve bioavailability and anti-melanogenesis activity. Nine resveratrol derivatives including five alkyl ether derivatives with C₂H₅, C₄H₉, C₅H₁₁, C₆H₁₃, and C₈H₁₇ (2a-2e) and four ester derivatives with CH₃, CH=C(CH₃)₂, CH(C₂H₅)C₄H₉, C₇H₁₅ (3a-3d) were newly synthesized and their effect on melanin synthesis were assessed. All the synthetic derivatives efficiently reduced the melanin content in α-MSH stimulated B16F10 melanoma cells. Further investigation showed that the inhibitory effect of 2a on melanin synthesis was achieved not by the inhibition of tyrosinase activity but by the inhibition of melanogenic enzyme expressions such as tyrosinase and tyrosinase-related protein (TRP)-1. Our synthetic resveratrol derivatives have more lipophilic properties than resveratrol by the addition of alkyl or acyl chains to free hydroxyl moiety of resveratrol; thus, they are expected to show better bioavailability in skin application. Therefore, we suggest that our synthetic resveratrol derivatives might be promising candidates for better practical application to skin-whitening cosmetics.

Full text of DOI:10.3390/molecules200916933

Molecules 2015, 20, 16933-16945; doi:10.3390/molecules200916933
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Synthesis and Biological Evaluation of Resveratrol Derivatives
as Melanogenesis Inhibitors
1
2
1
1
1
Qing Liu , CheongTaek Kim , Yang Hee Jo , Seon Beom Kim , Bang Yeon Hwang
1
,
and Mi Kyeong Lee *
1
College of Pharmacy, Chungbuk National University, Cheongju, Chungbuk 28644, Korea;
E-Mails: liuqing7115@hotmail.com (Q.L.); qow0125@naver.com (Y.H.J.);
suntiger85@hanmail.net (S.B.K.); byhwang@chungbuk.ac.kr (B.Y.H.)
2
RNS Inc., Yuseong-Gu, Daejeon 34182, Korea; E-Mail: happilion@rns.co.kr
*
Author to whom correspondence should be addressed; E-Mail: mklee@chungbuk.ac.kr;
Tel.: +82-43-261-2818; Fax: +82-43-268-2732.
Academic Editor: Derek J. McPhee
Received: 15 June 2015 / Accepted: 10 September 2015 / Published: 17 September 2015
Abstract: Resveratrol (1), a naturally occurring stilbene compound, has been suggested as
a potential whitening agent with strong inhibitory activity on melanin synthesis. However,
the use of resveratrol in cosmetics has been limited due to its chemical instability and poor
bioavailability. Therefore, resveratrol derivatives were prepared to improve bioavailability
and anti-melanogenesis activity. Nine resveratrol derivatives including five alkyl ether
derivatives with C
2
H
5
, C
4
H
9
, C
5
H11, C
6
H
13, and C
8
H17 (2a–2e) and four ester derivatives
with CH , CH=C(CH
3
3
)
2
, CH(C
2
H
5
)C
4
H
9
, C H
7
15 (3a–3d) were newly synthesized and their
effect on melanin synthesis were assessed. All the synthetic derivatives efficiently reduced
the melanin content in α-MSH stimulated B16F10 melanoma cells. Further investigation
showed that the inhibitory effect of 2a on melanin synthesis was achieved not by the inhibition
of tyrosinase activity but by the inhibition of melanogenic enzyme expressions such as
tyrosinase and tyrosinase-related protein (TRP)-1. Our synthetic resveratrol derivatives have
more lipophilic properties than resveratrol by the addition of alkyl or acyl chains to free
hydroxyl moiety of resveratrol; thus, they are expected to show better bioavailability in skin
application. Therefore, we suggest that our synthetic resveratrol derivatives might be
promising candidates for better practical application to skin-whitening cosmetics.
Keywords: melanin; resveratrol derivatives; tyrosinase; tyrosinase-related protein (TRP)-1

Molecules 2015, 20
. Introduction
Melanin is produced in melanocytes by melanogenesis and determines skin and hair color. It also
16934
1
plays an important role in skin protection from UV radiation. However, excessive accumulation by
pathological and environmental factors induces pigmentation problems and has become a critical issue
in the cosmetic field [1,2]. Therefore, melanogenesis inhibitors have become important constituents in
cosmetic products for depigmentation [3–5].
Melanogenesis is a complex biosynthetic process regulated by enzymatic and chemical reactions.
Exposure of the skin to UV radiation stimulates keratinocytes to secrete α-melanocyte stimulating hormone
(α-MSH). α-MSH then binds to melanocortin 1 receptor (MC1R) on melanocyte and induces melanogenesis.
Microphthalmia-associated transcriptional factor (MITF) is a key transcriptional factor in melanogenesis
that involved in the regulation of melanogenic enzymes such as tyrosinase, tyrosinase-related protein
(
TRP)-1 and TRP-2. Tyrosinase catalyzes the conversion of L-tyrosine to dopaquinone, the first rate-
limiting step in the melanogenesis. TRP-1 and TRP-2 are also involved in the major steps of melanin
synthesis. Therefore, tyrosinase, TRP-1 and TRP-2 have become important targets in the development
of depigmenting agents, such as skin-whitening cosmetics [6–9].
Resveratrol, 3,4′,5-trihydroxy-trans-stilbene, has diverse biological activities, such as antioxidant,
anti-inflammatory and cardiovascular protective effects [10–12]. Resveratrol also has been reported to
reduce melanin synthesis [13–15]. However, its utilization and development in products are limited due
to the chemical stability, poor solubility, and low bioavailability [16,17]. Therefore, attempts to increase
the bioavailability of resveratrol have been made in many fields. Drug delivery systems using new
formulations, such as encapsulation are suggested to enhance bioavailability [18–20]. The resveratrol
derivatives, such as resveratrol dimers or synthetic derivatives, also have been reported to have better
biological activity with fewer drawbacks [21–25]. Recently, the acetylated derivative of resveratrol has
been synthesized with more efficient anti-melanogenic activity and better stability [26]. For the purpose
of developing anti-melanogenesis inhibitory resveratrol derivatives with better bioavailability, nine
resveratrol derivatives, five alkyl ether derivatives and four alkyl ester derivatives by chemical reaction
and were synthesized in the present study. Effect on melanogenesis and the mode of action were
also investigated.
2
. Results and Discussion
2
.1. Chemistry
The resveratrol ether derivatives were synthesized as shown in Scheme 1. Resveratrol (1) was converted
to the corresponding ether derivatives (2a–2e) in the presence of corresponding alkyl bromides. The
synthesis of resveratrol ester derivatives is shown in Scheme 2. Resveratrol ester derivatives (3a–3d)
were synthesized by the addition of corresponding acyl chlorides. The structures of synthetic derivatives
were confirmed by spectroscopic analysis including NMR, IR and MS analysis (Figure 1).

Molecules 2015, 20
16935
Reagents and conditions: (i) DMF, NaOH, 10 min; (ii) 1-bromoethane, 1-bromobutane, 1-bromopentane,
-bromohexane, or 1-bromooctane, 40 °C, 24 h.
1
Scheme 1. Synthesis of resveratrol ether derivatives 2a–e.
Reagents and conditions: (i) CH
2 2
Cl , 10 °C, 5 min; (ii) TEA, DMAP, 10 °C; (iii) acetyl chloride,
3
,3-dimethylacryloyl chloride, 2-ethylhexanoyl chloride, or octanoyl chloride, 10 °C; 1 h.
Scheme 2. Synthesis of resveratrol ester derivatives 3a–d.
Figure 1. Chemical structure of resveratrol (1) and its derivatives (2a–e and 3a–d).
2.2. Effect on Melanogenesis
2
.2.1. Effect on Melanin Content in B16F10 Melanoma Cells
The effect of resveratrol derivatives on melanogenesis and cell viability was first investigated using
B16F10 melanoma cells. Stimulation of B16F10 melanoma cells with 100 nM α-MSH for 72 h

Molecules 2015, 20
16936
significantly increased the melanin synthesis. Resveratrol derivatives dose-dependently reduced the
melanin content concentration from 5 to 20 µg/mL without any cytotoxicity (Figure 2A,B). Although
the inhibition was slightly increased compared to resveratrol in some derivatives, there was no significant
difference among resveratrol and synthetic derivatives. In addition, all the synthetic derivatives showed
similar inhibition regardless of side chains.
Figure 2. Effects of resveratrol derivatives on (A) melanin content and (B) cell viability in
B16F10 melanoma cells. NC: vehicle treated normal control; PC: α-MSH stimulated positive
control. * p < 0.05 compared with PC group.
2
.2.2. Effect on Tyrosinase Activity
Inhibition of melanin synthesis can be achieved either by inhibiting tyrosinase activity or by reducing
melanogenic enzyme expression [8,9]. Therefore, the effect of resveratrol derivatives on tyrosinase
activity and the expression of melanogenic enzymes were investigated.
Tyrosinase catalyzes the first rate-limiting step in the melanogenesis and plays a pivotal role in
melanin synthesis [6,7]. The effect of resveratrol derivatives on tyrosinase activity was first evaluated
in vitro using mushroom tyrosinase. Although resveratrol effectively inhibited the tyrosinase activity,
both alkyl ether (2a–2e) and ester derivatives (3a–3d) showed little inhibition (Figure 3). These results
suggest that free hydroxyl groups of resveratrol are important for the inhibition of tyrosinase activity,
which is consistent with previous reports [27].

Molecules 2015, 20
16937
Figure 3. Effects of resveratrol derivatives (100 µg/mL) on tyrosinase activity. NC: vehicle
treated normal control. * p < 0.05 compared with NC group.
2
1
.2.3. Effect on Melanin Synthesis in B16F10 Melanoma Cells
Melanin synthesis is also regulated by the expression of melanogenic enzymes. Tyrosinase and TRP-
are key enzymes involved in the major steps of melanin synthesis [8,9]. Therefore, the effect of the
resveratrol derivative 2a on the expressions of tyrosinase and TRP-1 was determined. The expression of
tyrosinase was dramatically reduced by the treatment of compound 2a (Figure 4). Treatment of 2a also
inhibited the expression of TRP-1 expression. These results suggest that 2a efficiently inhibited the
melanogenic enzyme expression.
Figure 4. Effect of resveratrol derivative 2a on the expression of tyrosinase and TRP-1 in
B16F10 melanoma cells. NC: vehicle treated normal control; PC: α-MSH stimulated
positive control.
2
.3. Discussion
Botanical ingredients are good sources of medicine, functional foods and cosmetics. They provide
numerous compounds with diverse skeletons and biological activities. However, their applications are
often limited due to their small amounts, poor bioavailability, etc. Resveratrol is well known for its
potential biological activities. As a cosmetic ingredient, it has antioxidant and melanogenesis inhibitory
activities. However, it has limitations for cosmetic development, such as chemical instability and low
solubility. In addition, the hydroxyl moiety of resveratrol contributes to poor skin absorption. Many
attempts have been made to overcome its limitations, and the synthetic derivatives of resveratrol have
been suggested as effective in increasing stability and bioavailability [26,28–30].

Molecules 2015, 20
16938
In our present study, we synthesized nine resveratrol derivatives, including five ether derivatives (2a–2e)
and four ester derivatives (3a–3d) and then evaluated melanogenesis inhibitory activity. Our present
study showed that all the synthetic ether and ester derivatives of resveratrol inhibited melanin synthesis
in melanoma cells. Further study also showed that resveratrol derivative 2a inhibited melanin synthesis
in melanoma cells by inhibiting the expression of melanogenic enzymes, tyrosinase and TRP-1 (Figures 2
and 4). However, it showed little effect on tyrosinase activity (Figure 3). Taken together, we suggest that
2
a reduced melanin synthesis by the inhibition of melanogenic enzyme expressions rather than direct
inhibition on tyrosinase activity.
Skin is a barrier of our body with lipophilic membrane. Therefore, skin absorption of chemical is
determined by its physicochemical properties. Lipophilicity is one of the important factors that affect
the skin permeation process. Melanocytes and keratinocytes exist in epidermis, the outermost layers of
the skin. Thus, lipophilic compounds penetrate deeper in the skin layers [31]. Our synthetic resveratrol
derivatives have alkyl or acyl chains instead of free hydroxyl moiety of resveratrol. They exhibit more
lipophilic properties than resveratrol, thus these synthetic derivatives are expected to show better
bioavailability in skin application. Consistent with our suggestion, triacetyl resveratrol has been reported
to have anti-melanogenic activity with better stability [26]. Moreover, resveratrol triacetate reduced
hyperpigmented spots in human skin models without any skin irritation [28]. Although the exact
mechanism of our synthetic derivatives needs to be confirmed in further animal study, these derivatives
might be promising candidates for skin-whitening cosmetics with better practical applications.
3. Experimental Section
3.1. General Information
NMR spectra were recorded using a Bruker DRX 500 MHz NMR spectrometer (Bruker, Karlsruhe,
Germany). EI-mass spectra were obtained using VG Autospec Ultima mass spectrometers (Waters,
Milford, MA, USA). Semipreparative HPLC was performed using a Waters HPLC system equipped
with Waters 600 Q-pumps (Waters, Milford, MA, USA), a 996 photodiode array detector Waters,
Milford, MA, USA), and Waters Empower software using a Gemini-NX ODS-column (5 μm, 10 mm ×
150 mm) (Phenominex, Inc., Torrance, CA, USA). Silica gel (70–230 mesh, Merck, Darmstadt, Germany)
was used for open column chromatography (CC). Thin-layer chromatography (TLC) was performed on
a precoated silica gel 60 F254 (0.25 mm, Merck). All other chemicals and reagents were analytical grade.
3
.2. Synthesis of Resveratrol Derivatives
(
E)-1,3-Diethoxy-5-(4-ethoxystyryl)benzene (2a). Resveratrol (1) (15.0 g, 65.7 mmol) was dissolved in
a solution of DMF (150 mL) and NaOH (8.9 g), and stirred for 10 min. Then, 1-bromoethane (25.8 g,
36.6 mmol) was added and the reaction was kept for 24 h at 40 °C. After the removal of DMF by
evaporation under vacuum, the reaction mixture was extracted with toluene (45 mL × 2). The combined
organic layer were dried over anhydrous MgSO and evaporated under vacuum. The crude product was
purified by silica gel column chromatography with n-hexane–EtOAc (95:5) as an eluent to give
2
4
−1
1
compound 2a (15.4 g, 75.0% yield). Compound 2a: IR νmax cm : 1587, 1509, 1247; H-NMR
400 MHz, CDCl ) δ 7.45 (2H, d, J = 8.4 Hz), 7.05 (1H, d, J = 16.4 Hz), 6.91 (2H, J = 8.4 Hz), 6.92
(
3

Molecules 2015, 20
16939
(
1H, d, J = 16.0 Hz), 6.66 (2H, d, J = 2.4 Hz), 6.39 (1H, t, J = 2.0 Hz), 4.08 (6H, m), 1.45 (9H, m);
13C-NMR (100 MHz, CDCl
) δ 160.3, 158.8, 139.6, 129.8, 128.6, 127.8, 126.6, 114.7, 104.9, 100.5,
3
+
6
3.5, 14.9, 14.8; ESI-MS (positive mode) m/z: 313 [M + H] , HRESIMS: m/z 313.1798 (calcd. for
C
20
H
25
O
3
, 313.1798)
.
(
E)-1,3-Dibutoxy-5-(4-butoxystyryl)benzene (2b). Resveratrol (1) (15.0 g, 65.7 mmol) was dissolved in
a solution of DMF (150 mL) and NaOH (8.9 g), and stirred for 10 min. Then, 1-bromobuthane (30.6 g,
23.5 mmol) was added and the reaction was kept for 24 h at 40 °C. After the removal of DMF by
evaporation under vacuum, the reaction mixture was extracted with toluene (45 mL × 2). The combined
organic layer were dried over anhydrous MgSO and evaporated under vacuum. The crude product was
purified by silica gel column chromatography with n-hexane–EtOAc (95:5) as an eluent to give
2
4
−1
1
compound 2b (21.2 g, 81.5% yield). Compound 2b: IR νmax cm : 1589, 1509, 1385, 1294; H-NMR
(
(
(
400 MHz, CDCl
3
) δ 7.45 (2H, d, J = 8.4 Hz), 7.08 (1H, d, J = 16.4 Hz), 6.93 (2H, J = 8.4 Hz), 6.94
1H, d, J = 16.0 Hz), 6.69 (2H, d, J = 2.4 Hz), 6.43 (1H, t, J = 2.0 Hz), 4.02 (6H, m), 1.83 (6H, m), 1.57
13
6H, m), 1.05 (9H, m); C-NMR (100 MHz, CDCl
14.7, 104.9, 100.5, 67.7, 31.5, 31.4, 19.4, 19.3, 13.9; ESI-MS (positive mode) m/z: 397 [M + H]+,
HRESIMS: m/z 435.2296 (calcd. for C26 , 435.2296)
3
) δ 160.5, 159.0, 139.6, 129.8, 128.6, 127.7, 126.6,
1
H36KO
3
.
(
E)-1,3-Bis(pentyloxy)-5-(4-(pentyloxy)styryl)benzene (2c). Resveratrol (1) (50.0 g, 219.1 mmol) was
dissolved in a solution of DMF (200 mL). After adding NaOH (28.0 g) and KI (1.8 g), the reaction
mixture was stirred for 5 min at RT. Then, 1-bromopentane (109.2 g, 223.5 mmol) was added and the
reaction was kept for 12 h at 45 °C. After the removal of DMF by evaporation under vacuum, the reaction
mixture was extracted with toluene (45 mL × 2). The combined organic layer were dried over anhydrous
MgSO
4
and evaporated under vacuum. The crude product was purified by silica gel column chromatography
with n-hexane–EtOAc (95:5) as an eluent to give compound 2b (21.2 g, 81.5% yield). Compound 2c:
−
1
1
IR νmax cm : 1692, 1385, 1344; H-NMR (400 MHz, CDCl
3
) δ 7.47 (2H, d, J = 8.4 Hz), 7.07 (1H, d,
J = 16.4 Hz), 6.93 (2H, J = 8.4 Hz), 6.94 (1H, d, J = 16.0 Hz), 6.68 (2H, d, J = 2.4 Hz), 6.42 (1H, brs),
1
3
4
1
.00 (6H, m), 1.85 (6H, m), 1.48 (12H, m), 0.99 (9H, m); C-NMR (100 MHz, CDCl ) δ 160.5, 159.0,
3
39.6, 129.8, 128.6, 127.7, 126.6, 114.7, 104.9, 100.5, 68.0, 29.1, 29.0, 28.3, 28.2, 22.5, 14.1; ESI-MS
+
(
positive mode) m/z: 439 [M + H] , HRESIMS: m/z 477.2761 (calcd. for C29
H42KO
3
, 477.2765)
.
(E)-1,3-Bis(hexyloxy)-5-(4-(hexyloxy)styryl)benzene (2d). Resveratrol (1) (10.0 g, 43.8 mmol) was dissolved
in a solution of DMF (100 mL) and NaOH (5.96 g), and stirred for 10 min. Then, 1-bromohexane
25.8 g, 236.6 mmol) was added and the reaction was kept for 24 h at 40 °C. After the removal of DMF
by evaporation under vacuum, the reaction mixture was extracted with toluene (45 mL × 2). The combined
organic layer were dried over anhydrous MgSO and evaporated under vacuum. The crude product was
purified by silica gel column chromatography with n-hexane–EtOAc (95:5) as an eluent to give compound
(
4
−
1
1
2
d (16.3 g, 77.3% yield). Compound 2d: IR νmax cm : 1698, 1593, 1457, 1162; H-NMR (400 MHz, CDCl
δ 7.45 (2H, d, J = 8.4 Hz), 7.06 (1H, d, J = 16.4 Hz), 6.91 (2H, J = 8.4 Hz), 6.92 (1H, d, J = 16.0 Hz),
.67 (2H, d, J = 2.4 Hz), 6.42 (1H, t, J = 2.0 Hz), 4.00 (6H, m), 1.82 (6H, m), 1.52 (6H, m), 1.39 (12H,
3
)
6
1
3
m), 0.97 (9H, m); C-NMR (100 MHz, CDCl ) δ 160.5, 158.9, 139.6, 129.8, 128.6, 127.7, 126.6, 114.7,
3

Molecules 2015, 20
16940
1
04.8, 100.5, 68.0, 31.6, 29.4, 29.3, 25.8, 25.7, 22.6, 14.0; ESI-MS (positive mode) m/z: 481 [M + H]+,
HRESIMS: m/z 519.3235 (calcd. for C32 , 519.3235)
H48KO
3
.
(
E)-1,3-Bis(octyloxy)-5-(4-(octyloxy)styryl)benzene (2e). Resveratrol (1) (10.0 g, 43.8 mmol) was dissolved
in a solution of DMF (100 mL) and NaOH (5.9 g), and stirred for 10 min. Then, 1-bromooctane (25.8 g,
36.59 mmol) was added and the reaction was kept for 24 h at 40 °C. After the removal of DMF by
evaporation under vacuum, the reaction mixture was extracted with toluene (45 mL × 2). The combined
organic layer were dried over anhydrous MgSO and evaporated under vacuum. The crude product was
purified by silica gel column chromatography with n-hexane–EtOAc (95:5) as an eluent to give compound
2
4
−1
1
2
e (21.0 g, 84.7% yield). Compound 2e: IR νmax cm : 1694, 1507, 1383, 1298; H-NMR (400 MHz, CDCl
3
)
δ 7.45 (2H, d, J = 8.4 Hz), 7.06 (1H, d, J = 16.4 Hz), 6.91 (2H, J = 8.4 Hz), 6.92 (1H, d, J = 16.0 Hz),
6
0
1
.66 (2H, d, J = 2.4 Hz), 6.40 (1H, t, J = 2.0 Hz), 4.00 (6H, m), 1.83 (6H, m), 1.50 (6H, m), 1.36 (24H, m),
13
.94 (9H, m); C-NMR (100 MHz, CDCl
04.8, 100.5, 68.0, 31.8, 29.4, 29.3, 29.2, 26.1, 26.0, 22.6, 14.1; ESI-MS (positive mode) m/z: 565 [M + H]+,
, 603.4174)
3
) δ 160.5, 158.9, 139.6, 129.8, 128.6, 127.7, 126.5, 114.7,
HRESIMS: m/z 603.4176 (calcd. for C38
H60KO
3
.
(
E)-5-(4-Acetoxystyryl)-1,3-phenylene diacetate (3a). Resveratrol (1) (5.0 g, 21.9 mmol) was dissolved
in CH Cl (40 mL) and kept cool at 10 °C. TEA (7.1 g, 70.1 mmol) and DMAP (0.26 g, 2.19 mmol)
2
2
were added to the reaction mixture at 10 °C. Acetyl chloride (5.3 g, 67.9 mmol) was added carefully and
the reaction was kept for 1 h at 10 °C. After adding distilled water (40 mL) to the reaction mixture, the
CH Cl
2 2
fraction was extracted. The CH
Cl
2 2
layers were dried over anhydrous MgSO and evaporated under
4
vacuum. The crude product was purified by silica gel column chromatography with n-hexane–EtOAc (30:1)
−1
as an eluent to give compound 3a (6.9 g, 88.2% yield). Compound 3a: IR νmax cm : 1759, 1707, 1532,
1
1
6
1
124; H-NMR (500 MHz, CDCl
3
) δ 7.54 (2H, d, J = 8.5 Hz), 7.12 (4H, m), 7.08 (1H, J = 16.0 Hz),
1
3
.98 (1H, d, J = 16.0 Hz), 6.85 (1H, d, J = 2.0 Hz), 2.33 (9H, s); C-NMR (100 MHz, CDCl ) δ 169.3,
3
69.0, 151.3, 150.4, 139.5, 134.4, 129.6, 127.6, 127.2, 121.9, 116.9, 114.4, 21.1; ESI-MS (positive mode)
+
m/z: 377 [M + Na] , HRESIMS: m/z 393.0734 (calcd. for C20
H18KO
6
, 393.0734)
.
(
E)-5-(4-(3-Methylbut-2-enoyloxy)styryl)-1,3-phenylene bis(3-methylbut-2-enoate) (3b). Resveratrol (1)
(
5.0 g, 21.9 mmol) was dissolved in CH Cl (40 mL) and kept cool at 10 °C. TEA (7.3 g, 72.3 mmol)
2
2
and DMAP (0.26 g, 2.2 mmol) were added to the reaction mixture at 10 °C. 3,3-Dimethylacroyl chloride
8.3 g, 70.1 mmol) was added carefully and the reaction was kept for 1 h at 10 °C. After adding distilled
water (50 mL) to reaction mixture, the CH Cl fraction was extracted. The CH Cl layers were dried
over anhydrous MgSO and evaporated under vacuum. The crude product was purified by silica gel
column chromatography with n-hexane–EtOAc (95:5) as an eluent to give compound 3b (6.9 g, 92.5%
(
2
2
2
2
4
−1
1
yield). Compound 3b: IR νmax cm : 1738, 1645, 1379, 1343; H-NMR (400 MHz, CDCl
3
) δ 7.50 (2H,
dd, J = 7.2, 2.0 Hz), 7.11 (4H, m), 7.01 (1H, d, J = 16.4 Hz), 6.86 (1H, t, J = 2.0 Hz), 2.26 (9H, d,
1
3
J = 1.2 Hz), 2.02 (9H, s); C-NMR (100 MHz, CDCl
3
) δ 164.7, 164.4, 160.4, 160.1, 151.4, 150.4, 139.3,
1
34.3, 129.4, 127.5, 127.2, 122.0, 116.8, 115.1, 115.0, 114.8, 27.7, 27.6, 20.6, 20.5; ESI-MS (positive
+
mode) m/z: 497 [M + Na] , HRESIMS: m/z 513.1672 (calcd. for C29
H30KO
6
, 513.1673)
.
(
E)-5-(4-(2-Ethylhexanoyloxy)styryl)-1,3-phenylene bis(2-ethylhexanoate) (3c). Resveratrol (1) (10.0 g,
3.8 mmol) was dissolved in CH Cl (50 mL) and kept to cool at 10 °C. TEA (7.3 g, 72.3 mmol) and
4
2
2

Molecules 2015, 20
16941
DMAP (0.54 g, 4.4 mmol) were added to the reaction mixture. After stirring at 10 °C, 2-ethylnexanoyl
chloride (22.8 g, 140.2 mmol) was added carefully and the reaction was kept for 1 h at 10 °C. After
adding distilled water (50 mL) to reaction mixture, the CH
were dried over anhydrous MgSO and evaporated under vacuum. The crude product was purified by
silica gel column chromatography with n-hexane–EtOAc (50:1) as an eluent to give compound 3c
2
Cl
2
fraction was extracted. The CH
2
Cl
2
layers
4
−1
1
(
25.2 g, 94.8% yield). Compound 3c: IR νmax cm : 1754, 1644, 1376, 1201; H-NMR (500 MHz, CDCl )
3
δ 7.52 (2H, d, J = 8.5 Hz), 7.11 (4H, m), 7.03 (1H, J = 16.0 Hz), 6.98 (1H, d, J = 16.0 Hz), 6.78 (1H, d,
J = 2.0 Hz), 2.56 (3H, m), 1.87 (6H, m), 1.76 (8H, m), 1.07 (9H, m), 0.97 (12H, m); 13C-NMR
(
100 MHz, CDCl
7.4, 47.3, 31.7, 31.6, 29.6, 25.5, 25.4, 22.6, 13.9, 11.9; ESI-MS (positive mode) m/z : 629 [M + Na]+,
HRESIMS: m/z 645.3550 (calcd. for C38 , 645.3551)
3
) δ174.7, 174.4, 151.5, 150.5, 139.4, 134.3, 129.6, 127.6, 127.2, 121.9, 116.8, 114.3,
4
H54KO
6
.
(
E)-5-(4-(Octanoyloxy)styryl)-1,3-phenylene dioctanoate (3d). Resveratrol (1) (5.0 g, 21.9 mmol) was
dissolved in CH Cl (40 mL) and kept cool at 10 °C. TEA (7.1 g, 70.1 mmol) and DMAP (0.26 g, 2.2 mmol)
2
2
were added to the reaction mixture. After stirring at 10 °C, octanoyl chloride (11.0 g, 67.9 mmol) was
added carefully and the reaction was kept for 1 h at 10 °C. After adding distilled water (40 mL) to reaction
mixture, the CH
Cl
2 2
fraction was extracted. The CH
Cl
2 2
layer were dried over anhydrous MgSO and
4
evaporated under vacuum. The crude product was purified by silica gel column chromatography with
n-hexane–EtOAc (30:1) as an eluent to give compound 3d (12.9 g, 96.7% yield). Compound 3d: IR
−1
1
ν
max cm : 1758, 1644, 1367, 1267; H-NMR (500 MHz, CDCl
3
) δ 7.51 (2H, d, J = 8.5 Hz), 7.11 (4H, m),
7
1
1
.08 (1H, J = 16.0 Hz), 6.99 (1H, d, J = 16.0 Hz), 6.82 (1H, d, J = 2.0 Hz), 2.58 (6H, m), 1.79 (6H, m),
1
3
.34-1.42 (24H, m), 0.94 (12H, m); C-NMR (100 MHz, CDCl ) δ 172.2, 171.8, 151.4, 150.5, 139.4,
3
34.3, 129.6, 127.6, 127.2, 121.9, 116.8, 114.4, 34.4, 34.4, 31.6, 29.0, 28.9, 24.9, 24.8, 22.6, 14.0;
+
ESI-MS (positive mode) m/z: 629 [M + Na] , HRESIMS: m/z 645.3550 (calcd. for C38
H54KO
6
, 645.3551)
.
3.3. Evaluation of Anti-Melanogenesis Activity
3
.3.1. Assessment of Tyrosinase Activity
Tyrosinase inhibitory assays were performed using enzyme solution, which was prepared by the
reconstitution of mushroom tyrosinase (Sigma, St. Louis, MO, USA) in 0.1 U/mL phosphate buffer (pH 6.5).
A test sample was mixed with 50 μL enzyme buffer, and incubated for 5 min at 37 °C. Then, 50 μL of
tyrosine solution, which was diluted with phosphate buffer to 1 mM, was added and the enzyme reaction
was allowed to proceed for 20 min at 37 °C. After incubation, the amount of dopachrome formed in the
reaction mixture was determined by measuring the absorbance at 490 nm in an ELISA reader (Bio-Tek
Synergy HT, Winooski, VT, USA).
3
.3.2. Cell Culture
B16F10 mouse melanoma cells were obtained from the American Type Culture Collection (Manassas,
VA, USA). Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with
0% fetal bovine serum (FBS), 100 IU/mL penicillin and 100 μg/mL streptomycin. Cells were maintained
at 37 °C in a humidified atmosphere of 95% air-5% CO
1
2
.

Molecules 2015, 20
.3.3. Measurement of Cellular Melanin Contents
B16F10 cells were stimulated with α-MSH and then treated with resveratrol derivatives at the
16942
3
concentration of 5, 10 and 20 μg/mL for 72 h. After washing with PBS, the cells were harvested and
solubilized the melanin by vortexing in 1N NaOH-10% DMSO at 80 °C. The melanin contents were
measured by absorbance value at 490 nm with synthetic melanin as a standard.
3
.3.4. Cell Viability
B16F10 melanoma were treated with resveratrol derivatives at the concentration of 5, 10 and 20 µg/mL
for 72 h. Cell viability was assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
MTT) assay in an ELISA plate reader.
(
3
.3.5. Western Blot Analysis
Stimulated B16F10 cells were treated with resveratrol derivative 2a for 72 h. Cells were lysed with an
SDS lysis buffer containing a protease inhibitor cocktail. The lysates were centrifuged and the supernatants
were collected. Proteins were separated in an SDS–polyacrylamide gel and transferred to a PVDF
membrane. After blocking in TBST with 5% non-fat dry milk, the membrane was washed and incubated
with the primary antibodies including tyrosinase, TRP-1, or β-actin (Santa Cruz Biotechnology Inc.,
Dallas, TX, USA) at 4 °C overnight. After washing, the membrane was incubated with horseradish
peroxidase-conjugated IgG secondary antibody (Cell Signaling Technology Inc., Danvers, MA, USA).
The membrane was detected by chemiluminescent reaction, and then exposure to X-ray Kodak film. The
proteins of tyrosinase and TRP-1 were normalized by β-actin and the intensities of bands were quantified
using a Scion-Image for Windows program (Informer Technologies, Inc., Shingle Springs, CA, USA).
3
.3.6. Statistical Analysis
Evaluation of statistical significance was determined by one-way ANOVA test with a value of
p < 0.05 considered statistically significant.
4
. Conclusions
Nine resveratrol derivatives including five ether derivatives (2a–2e) and four ester derivatives (3a–3d)
were newly synthesized and evaluated for melanogenesis inhibitory activity. All the synthetic ether and
ester derivatives of resveratrol inhibited melanin synthesis in melanoma cells. Further Western blot
analysis showed that resveratrol derivative 2a inhibited melanin synthesis by the inhibition of melanogenic
enzyme expressions rather than direct tyrosinase activity. Our synthetic resveratrol derivatives have
more lipophilic properties than resveratrol by the addition of alkyl or acyl chains to free hydroxyl moiety
of resveratrol, thus are expected to show better bioavailability in skin application. Taken together, we
suggest that our synthetic resveratrol derivatives might be promising candidates for better practical
application to skin-whitening cosmetics.

Molecules 2015, 20
16943
Acknowledgments
This work was supported by the Ministry of Trade, Industry and Energy (MOTIE) and the Korea
Institute for Advancement of Technology (KIAT) through the Promoting Regional Specialized Industry
(
A006500037) and Medical Research Center program (2008-0062275) of the National Research
Foundation of Korea.
Author Contributions
Qing Liu, Yang Hee Jo and Seon Beom Kim designed the experiments and performed the biological
assay. Cheong Taek Kim executed synthesis and identification of derivatives. Bang Yeon Hwang and
Mi Kyeong Lee analyzed the data and wrote the paper. All authors discussed the results and commented
the manuscript.
Conflicts of Interest
The authors declare no conflicts of interest.
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(