Anti‐melanogenic properties of velutin and its analogs?

Article abstract of DOI:10.3390/molecules26103033

Velutin, one of the flavones contained in natural plants, has various beneficial activities, such as skin whitening, as well as anti‐inflammatory, anti‐allergic, antioxidant, and antimicrobial activities. However, the relationship between the structure of velutin and its anti‐melanogenesis activity is not yet investigated. In this study, we obtained 12 velutin derivatives substituted at C5, C7, C3′, and C4′ of the flavone backbone with hydrogen, hydroxyl, and methoxy functionalities by chemical synthesis, to perform SAR analysis of velutin structural analogues. The SAR study revealed that the substitution of functional groups at C5, C7, C3′, and C4′ of the flavone backbone affects biological activities related to melanin synthesis. The coexistence of hydroxyl and methoxy at the C5 and C7 position is essential for inhibiting tyrosinase activity. However, 1,2‐diol compounds substituted at C3′ and C4′ of flavone backbone induce apoptosis of melanoma cells. Further, substitution at C3′ and C4′ with methoxy or hydrogen is essential for inhibiting melanogenesis. Thus, this study would be helpful for the development of natural‐derived functional materials to regulate melanin synthesis.

Full text of DOI:10.3390/molecules26103033

molecules
Article
Anti-Melanogenic Properties of Velutin and Its Analogs †
Se-Hui Jung 1,‡ , Hee-Young Heo 1,‡, Jung-Won Choe 1, Jaehyun Kim 1 and Kooyeon Lee 1,2,
*
1
Department of Bio-Health Technology, Division of Biomedical Convergence, College of Biomedical Science,
Kangwon National University, Chuncheon 24341, Korea; 96-shjung@hanmail.net (S.-H.J.);
gmldud940315@naver.com (H.-Y.H.); cjh9531@naver.com (J.-W.C.); 1342113@naver.com (J.K.)
Research Institute, K-medichem Co., Ltd., Chuncheon 24341, Korea
Correspondence: lky@kangwon.ac.kr; Tel.: +82-33-250-6477
2
*
†
‡
Dedicated to Prof. Phil-Ho Lee on his 60th Birthday.
These authors contributed equally to this work.
Abstract: Velutin, one of the flavones contained in natural plants, has various beneficial activities,
such as skin whitening, as well as anti-inflammatory, anti-allergic, antioxidant, and antimicrobial
activities. However, the relationship between the structure of velutin and its anti-melanogenesis
activity is not yet investigated. In this study, we obtained 12 velutin derivatives substituted at C5, C7,
0
0
C3 , and C4 of the flavone backbone with hydrogen, hydroxyl, and methoxy functionalities by chem-
ical synthesis, to perform SAR analysis of velutin structural analogues. The SAR study revealed that
0
0
the substitution of functional groups at C5, C7, C3 , and C4 of the flavone backbone affects biological
activities related to melanin synthesis. The coexistence of hydroxyl and methoxy at the C5 and C7
position is essential for inhibiting tyrosinase activity. However, 1,2-diol compounds substituted at
C30 and C40 of flavone backbone induce apoptosis of melanoma cells. Further, substitution at C30
0
and C4 with methoxy or hydrogen is essential for inhibiting melanogenesis. Thus, this study would
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
be helpful for the development of natural-derived functional materials to regulate melanin synthesis.
Citation: Jung, S.-H.; Heo, H.-Y.;
Choe, J.-W.; Kim, J.; Lee, K.
Keywords: velutin derivatives; melanin synthesis; tyrosinase activity; SAR study
Anti-Melanogenic Properties of
Velutin and Its Analogs . Molecules
2021, 26, 3033. https://doi.org/
10.3390/molecules26103033
1. Introduction
Flavones, a type of flavonoid consisting of two phenyl rings and a benzopyran func-
tionality, are widely present in natural plants, including vegetables and fruits [1]. In many
compounds, the flavone scaffolds are considered important core structures that act on
various targets molecules via simple modifications. In particular, flavone derivatives with
metabolic or synthetic modification are known to have anti-inflammatory, antiestrogenic,
Academic Editor: H. P.
Vasantha Rupasinghe
Received: 19 April 2021
Accepted: 14 May 2021
Published: 19 May 2021
anti-allergic, antioxidant, antimicrobial, antitumor, and anti-proliferation activities [2–4].
Furthermore, simple substitution of the flavone backbone with functional groups like
hydroxyl and methoxy, results in very different biological activities. Due to the broad bio-
logical activities of flavones, their structure–activity relationship (SAR) attracted interest in
the field of medicinal chemistry, which helped to discover some lead compounds to allevi-
ate numerous diseases. For example, the previous SAR study revealed that, as the number
of hydroxyl groups increased, the radical scavenging effect of flavones increased [5].
Melanin is a natural pigment that is involved in the determination of skin, eye, and
hair color, and has an important role in the protection of skin from damage by harmful light,
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such as ultraviolet rays [
synthesis is responsible for pigmentary disorders that include albinism, vitiligo, melasma,
freckles, and lentigo [ 10]. Melanin is synthesized in melanocytes, which are specialized
cells located at the basal layer of the epidermis, through multistep catalytic reactions of
enzymes such as tyrosinase, tyrosinase-related protein 1, and tyrosinase-related protein
2 [9,11]. Tyrosinase plays a key role in the two rate-limiting reactions of melanin synthesis,
by the hydroxylation of L-tyrosine to L-3,4-dihydroxyphenylalanine (L-DOPA), and the
oxidation of L-DOPA to dopaquinone [12]. Thus, various regulators of the tyrosinase
6–8]. Despite its beneficial effects, abnormal regulation of melanin
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© 2021 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
9
,
Molecules 2021, 26, 3033. https://doi.org/10.3390/molecules26103033
https://www.mdpi.com/journal/molecules

Molecules 2021, 26, 3033
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activity were studied for treating pigmentary disorders [13]. Whitening reagents based
on tyrosinase inhibition, such as hydroquinone, azelaic acid, arbutin, and kojic acid, were
reported to prevent skin hyperpigmentation [11,14]. However, due to their cellular toxicity,
low efficacy, and low stability in the presence of oxygen and water, whitening ingredients
from natural products are considered in the cosmetic research and development field to be
an alternative strategy to prevent hyperpigmentation.
Velutin belongs to a class of flavonoids that have hydroxyl, methoxy, methoxy, and
hydroxyl groups at C5, C7, C30, and C40 of the flavone backbone, respectively (Figure 1).
Velutin is found in the pulp of acai fruit, and is known to exhibit anti-inflammatory
and antioxidant activities [15
of homoflavoyadorinin B in mistletoe extract has an inhibitory effect on tyrosinase and
melanogenesis [ ]. However, the relationship between the structural modification of velutin
,16]. Furthermore, velutin obtained by the deglycosylation
8
and its anti-tyrosinase and anti-melanogenesis activities are not yet investigated.
Figure 1. The chemical structure of flavones.
In this study, we prepared 12 velutin derivatives by substitution at C5, C7, C30, and
C40 of the flavone backbone with hydrogen, hydroxyl, and methoxy functionalities, to
perform SAR analysis of the velutin structural analogues. All 12 derivatives were evaluated
for their inhibitory activity against tyrosinase activity and melanogenesis. We found that
the coexistence of the methoxy group at the C7 position and hydroxyl groups at the C5 and
C40 positions in the flavone skeleton was essential to have inhibitory activity. In addition,
the 1,2-diol form with hydroxyl groups at the C30and C40 positions of the flavone induced
apoptosis in the melanoma cells. Thus, these findings would be helpful for investigating
the effect of flavone derivatives on the biosynthesis of melanin, and for developing new
chemical drugs to suppress melanin synthesis.
2. Results and Discussion
2.1. Chemical Synthesis of Velutin Derivatives
In order to understand the relationship between the structural modification of the
velutin backbone and its biological activities, eleven velutin derivatives (V1–V12, ex-
cept V11) were chemically synthesized from 2-hydroxy acetophenone derivatives and
aromatic aldehyde derivatives, via five-step conventional methods [17]. Three velutin
derivatives, V1 (velutin), V5, and V8, were synthesized via a seven-step reaction, as shown
in Scheme 1. First, to synthesize 1-(2-(benzyloxy)-6-hydroxy-4-methoxyphenyl)ethan-1-
one (V1e), bis-MOM-protected acetophenone (V1b) was synthesized from commercially
available 20,40,60-trihydroxyacetophenone (V1a), subjected to deprotection reaction, after
synthesis of benzylated acetophenone (V1c), and selective substitution of the methoxy
group at the C4 position of 1-(2-(benzyloxy)-4,6-dihydroxyphenyl)ethan-1-one (V1d) was
performed. Then, V1, V5, and V8 derivatives were synthesized by the subsequent aldol
condensation, oxidative cyclization, and reduction from the modified acetophenone (V1e).

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Scheme 1. Synthesis of V1, V5, and V8 derivatives.
V2 and V3 were synthesized via subsequent reactions, as shown in Scheme 2; O-
alkylation at R2 and R1 of the commercially available two dihydroxyacetophnone com-
pounds, aldol condensation, oxidative cyclization, and deprotection.
Scheme 2. Synthesis of V2 and V3 derivatives.
Four derivatives, including V4, V6, V9, and V10 were synthesized via subsequent
reactions, as shown in Scheme 3; O-alkylation at C4 and C6 of the commercially available

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0
0
0
2 ,4 ,6 -trihydroxyacetophnone compound (V1a), aldol condensation, oxidative cyclization,
and deprotection.
Scheme 3. Synthesis of V4, V6, V9, and V10 derivatives.
The V7 derivative was synthesized via subsequent reactions, as shown in Scheme 4;
0
0
0
benzyl protection at C4 and C6 of the commercially available 2 ,4 ,6 -trihydroxyacetophnone
compound (V1a), aldol condensation, oxidative cyclization, and reduction.
Scheme 4. Synthesis of V4, V6, V7, V9, and V10 derivatives.
V12 derivative was synthesized via a two-step reaction, as previously reported [18]; al-
0
dol condensation of two commercially available 2 -hydroxyacetophnone and benzaldehyde,
and oxidative cyclization.

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Table 1 summarizes the substitution group and the overall yield of the synthesized
velutin derivatives.
Table 1. Substitution group and overall yield of the synthesized velutin derivatives.
Compound
R1
R2
R3
R4
Total Yield (%)
V1
V2
V3
V4
V5
V6
V7
V8
V9
OH
H
OMe
OMe
H
OMe
OMe
OMe
OH
OMe
OMe
OMe
OH
OMe
OMe
OMe
H
OMe
OMe
OMe
OH
OH
OH
H
OH
H
OH
OH
OH
OMe
OMe
OH
H
38
18
16
17
20
49
22
13
20
52
-
OH
OH
OH
OMe
OH
OH
OH
OMe
OH
H
OMe
OMe
OH
V10
V11
V12
H
H
78
2.2. Characterization of Synthetic Velutin Derivatives
2.2.1. Synthesis of Velutin (V1)
1
(
V1): H NMR (400 Mhz, DMSO-d6)
δ
(12.97 (s, 1H), 10.01 (br, s, 1H), (7.61–7.59) (m,
2H), (6.96–6.94) (m, 2H), 6.80 (d, J = 1.52 Hz, 1H), 6.37 (d, J = 2.08 Hz, 1H), 3.91 (s, 3H), 3.88
(s, 3H)).
2.2.2. Synthesis of 2-(4-Hydroxy-3-methoxyphenyl)-7-methoxy-4H-chromen-4-one (V2)
1
(
V2): H NMR (400 Mhz, DMSO-d6)
δ
(9.88 (s, 1H), 7.92 (d, J = 8.84 Hz, 1H), (7.61–7.58)
(m, 2H), 7.32 (d, J = 2.36 Hz, 1H), 7.05 (dd, J1 = 8.80 Hz, J2 = 2.40 Hz, 1H), (6.95–6.93) (m,
1H), 6.89 (s, 1H), 3.92 (s, 3H), 3.91 (s, 3H)).
2.2.3. Synthesis of 5-Hydroxy-2-(4-hydroxy-3-methoxyphenyl)-4H-chromen-4-one (V3)
(
V3): 1H NMR (400 Mhz, DMSO-d6)
δ (12.85 (s, 1H), (7.69–7.62) (m, 3H), 7.21 (d,
J = 8.36 Hz, 1H), 7.07 (s, 1H), 6.96 (d, J = 8.16 Hz, 1H), 6.81 (d, J = 8.16 Hz, 1H), 3.91 (s, 3H)).
2.2.4. Synthesis of Genkwanin (V4)
1
(
V4): H NMR (400 Mhz, DMSO-d6)
δ
(12.96 (s, 1H), (7.97–7.95) (m, 2H), (6.95–6.92)
(m, 2H), 6.85 (s, 1H), 6.77 (d, J = 2.24 Hz, 1H), 6.38 (d, J = 2.28 Hz, 1H), 3.87 (s, 3H)).
2.2.5. Synthesis of 5-Hydroxy-7-methoxy-2-(3-methoxyphenyl)-4H-chromen-4-one (V5)
1
(
V5): H NMR (400 Mhz, CDCl3)
δ
(12.71 (s, 1H), (7.45–7.39) (m, 3H), (7.10–7.07) (m,
1H), 6.65 (s, 1H), 6.50 (d, J = 2.28 Hz, 1H), 6.38 (d, J = 2.24 Hz, 1H), 3.90 (s, 3H), 3.89 (s, 3H)).
2.2.6. Synthesis of 2-(4-Hydroxy-3-methoxyphenyl)-5,7-dimethoxy-4H-chromen-4-one (V6
)
1
(
V6): H NMR (400 Mhz, DMSO-d6)
δ
(7.53–7.52) (m, 2H), (6.93–6.86) (m, 1H), 6.86 (d,
J = 2.24 Hz, 1H), 6.49 (d, J = 2.20 Hz, 1H), 3.90 (s, 3H), 3.89 (s, 3H), 3.83 (s, 3H)).
2.2.7. Synthesis of Chrysoeriol (V7)
(
V7): 1H NMR (400 Mhz, DMSO-d6)
J = 8.92 Hz, 1H), 6.88 (s, 1H), 6.47 (d, J = 1.87 Hz, 1H), 6.16 (d, J = 1.91 Hz, 1H), 3.89 (s, 3H)).
δ (12.97 (s, 1H), (7.57–7.55) (m, 1H), 6.93 (d,

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2.2.8. Synthesis of 2-(3,4-Dihydroxyphenyl)-5-hydroxy-7-methoxy-4H-chromen-4-one (V8
)
1
(
V8): H NMR (400 Mhz, DMSO-d6)
δ
(12.99 (br, s, 1H), (7.47–7.44) (m, 2H), 6.90 (d,
J = 8.21 Hz, 1H), (6.74–6.73) (m, 2H), 6.38 (d, J = 2.24 Hz, 1H), 3.88 (s, 3H)).
2.2.9. Synthesis of 2-(3,4-Dimethoxyphenyl)-5-hydroxy-7-methoxy-4H-chromen-4-one (V9
)
1
(
V9): H NMR (400 Mhz, CDCl3)
δ
(12.80 (s, 1H), 7.53 (dd, J1 = 8.48 Hz, J2 = 2.12 Hz,
1H), 7.34 (d, J = 2.08 Hz, 1H), 6.98 (d, J = 8.54 Hz, 1H), 6.50 (d, J = 2.24 Hz, 1H), 6.37 (d,
J = 2.22 Hz, 1H), 3.98 (s, 3H), 3.97 (s, 3H), 3.89 (s, 3H)).
2.2.10. Synthesis of 2-(3,4-Dimethoxyphenyl)-5,7-dimethoxy-4H-chromen-4-one (V10)
1
(
V10): H NMR (400 Mhz, DMSO-d6)
δ
(7.64 (dd, J1 = 8.48 Hz, J2 = 2.16 Hz, 1H), 7.53
(d, J = 2.12, 1H), 7.11 (d, J = 8.62, 1H), 6.87 (d, J = 2.30, 1H), 6.77 (s, 1H), 6.50 (d, J = 2.30,
1H), 3.91 (s, 3H), 3.88 (s, 3H), 3.85 (s, 3H), 3.83 (s, 3H)).
2.2.11. 2-Phenylchromen-4-one (V12)
(
V12): 1H NMR (400 Mhz, CDCl3)
δ (8.24 (dd, 1H, J1 = 1.66 Hz, J2 = 7.94 Hz), (7.96–7.93)
(m, 2H), (7.73–7.69) (m, 1H), (7.59–7.57) (m, 1H), (7.56–7.53) (m, 3H), 7.45–7.41 (m, 1H), 6.84
(s, 1H)).
2.3. Effect of Velutin Derivatives on ABTS Radical Scavenging Activity
The ABTS radical scavenging activity of the twelve velutin derivatives or arbutin
(positive control) was then evaluated in the concentration range 50–200 µM, and Table 2
lists their half-maximal effective concentration (EC50) values. Most derivatives, except
V10, showed dose-dependent radical scavenging activity (data not shown), with EC50
values ranging (5.47–100.13) µM (Table 2). In particular, V8 and V11 (luteolin) compounds
with a 1,2-diol-type structure, showed a better radical scavenging activity than the other
derivatives. In contrast, the V10 or V12 compounds substituted with methoxy or hydro-
gen group at C5, C7, C30, and C40 of the backbone, had no significant effect on radical
scavenging activity. These results indicated that the presence of hydroxyl group on the
velutin backbone was critical for its radical scavenging ac0tivity. Furthermore, increase in
0
the number of methoxy functionality at C5, C7, C3 , and C4 of the backbone lowered ABTS
radical scavenging activity.
Table 2. Inhibitory effect of velutin derivatives on ABTS radical scavenging and mushroom tyrosi-
nase activities.
EC50 in ABTS Radical
Scavenging Activity (µM)
IC50 in Mushroom
Tyrosinase Activity (µM)
Compound
V1
V2
V3
V4
V5
V6
V7
V8
V9
23.13
21.85
23.33
55.73
96.57
23.73
22.07
5.47
100.13
N.D.
6.13
N.D.
12.67
910.1
N.D.
N.D.
203.5
N.D.
N.D.
N.D.
202.3
715.2
N.D.
36.7
V10
V11
V12
Arbutin
N.D.
376.0
N.D.: Not determined.

Molecules 2021, 26, 3033
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2.4. Effect of Velutin Derivatives on Mushroom Tyrosinase Inhibitory Activity
To further understand the effect of the electrostatic potential of the derivatives on
their inhibitory activities against tyrosinase, the inhibitory effect of the twelve velutin
derivatives on mushroom tyrosinase was evaluated. The five velutin derivatives V1
V8 V9, and V11 had potential inhibitory activity against mushroom tyrosinase, with IC50
values ranging 37–910 M, but the other seven velutin derivatives V2 V3 V5 V6 V9,
, V4,
,
µ
,
,
,
,
V10, and V12 had no significant effect (Table 2). These results indicated that the number of
hydroxyl groups at C5, C7, C30, and C40 of the flavone backbone was an important factor
for their inhibitory activity against tyrosinase. In particular, the V11 compound showed the
lowest IC50 value in the twelve derivatives, while V10 and V12 had no significant effect on
tyrosinase activity, suggesting that the existence of hydroxyl group, rather than methoxy or
hydrogen group, at C5, C7, C30, and C40 of the flavone backbone was more important for
its anti-tyrosinase effect. Furthermore, hydroxyl and methoxy functionalities at the R1 and
R2 positions might play an important role in the inhibition of tyrosinase.
2.5. Cytotoxicity of Velutin Derivatives in Melanoma Cells
Cytotoxicity is a concern for any potential compound, and thus after treating the
cells with derivatives, we quantitatively measured the cytotoxicity of the eleven velutin
derivatives that had the potential to inhibit melanogenesis, except for V12, which had
no potential to inhibit melanogenesis through MTT assay. Nine derivatives, excluding
V8
(data not shown). However, the treatment with V8
concentration-dependent manner, with death rates of 71.8, 24.0, and 69.7%, respectively,
,
V10, and V11, had no significant effect on cell viability at concentrations up to 20
µM
,
V10, and V11, induced cell death in a
at 20 µM (Figure 2). This result indicates that the 1,2-diol form with hydroxyl groups at
the C30 and C40 positions of the flavone induced apoptosis in melanoma cells, which was
consistent with the previous report explaining the cytotoxicity of flavonoid derivatives in
1,2-diol form, such as catechin and epicatechin [19].
Figure 2. Cytotoxicity of velutin derivatives in the B16F10 melanoma cells. B16F10 cells were treated
with 0.1% DMSO as vehicle, or with 20 µM of each derivative, for 24 h. Cell viability was determined
by the MTT assay, as described in the Experimental Section. Data are expressed as the mean
three independent experiments (** p < 0.005, *** p < 0.001 compared to the control cells).
±
SD of
2.6. Effect of Velutin Derivatives on Melanogenesis in Melanoma Cells
Next, the effects of the nine velutin derivatives excluding V8 and V11, which have
cytotoxicity, on melanogenesis stimulated by -melanocyte stimulating hormone ( –MSH)
in melanoma cells were determined. Treatment with –MSH stimulated a 1.9-fold increase
of the melanin content of the melanoma cells (Figure 3A). We found that –MSH-stimulated
melanogenesis was completely inhibited by V1 and V2 (p < 0.001), and partially inhib-
α
α
α
α

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ited by V3 (50.7%, p < 0.005), V4 (69.1%, p < 0.001), and V5 (39.3%, p < 0.005), while
α
–MSH-stimulated melanogenesis was not significantly changed by V7
, V9, and V10
(Figure 3A). Surprisingly, the V6 derivative significantly increased melanin synthesis by
3.0-fold (p < 0.001). These results suggest that the velutin derivatives with similar struc-
tures but different substitution groups, have different biological activity on melanogenesis.
Further, the inhibitory activities of the nine velutin derivatives on
tracellular tyrosinase activation were determined, since this enzyme plays a key role
in melanogenesis. Treatment with –MSH induced an approximately 1.7-fold activa-
α–MSH-mediated in-
α
tion of tyrosinase in B16F10 cells (Figure 3B). Four derivatives V1 (87%, p < 0.001), V3
(97%, p < 0.001), V4 (100%, p < 0.001), and V5 (80%, p < 0.001) reversed tyrosinase activa-
tion mediated by
α
–MSH, whereas four other derivatives V2
,
V7
,
V9, and V10 did not
(
Figure 3B). These results indicate that the four derivatives V1
,
V3, V4, and V5 inhibited
α
–MSH-induced melanin synthesis, by inhibiting the intracellular tyrosinase activity in
B16F10 cells.
Figure 3. Effect of velutin derivatives on intracellular melanin synthesis in B16F10 melanoma cells. B16F10 cells were
treated with 0.1% DMSO as vehicle, or with 10 M of each derivative, for 48 h. ( ) Melanin content in the B16F10 cells
was determined using a photometric method, as described in the Experimental Section. ( ) Cellular tyrosinase activity
was determined by measuring dopachrome generated by the enzymatic reaction of cell lysates with L-DOPA. Data are
µ
A
B
expressed as the mean
B16F10 cells).
±
SD of three independent experiments (** p < 0.005, *** p < 0.001 as compared to α-MSH-stimulated
2.7. In Silico Molecular Docking Simulation of Enzyme Inhibition
To predict the interaction between velutin derivatives and tyrosinase, an in silico
molecular docking study was performed using the Maestro software. The molecular dock-
ing study revealed that V9 and V10 derivatives, highly substituted with methoxy groups,
did not bind to the enzyme (Table 3); this result was consistent with our results obtained
from the in vitro experiment. Further, most of the flavone derivatives, except V9 and V10
interact with tyrosinase with binding energies ranging from
4.983 to −5.677 kcal/mol
Table 3). The two derivatives, V1 and V4, showed inhibitory activity against tyrosinase ac-
tivity in melanoma cells, with docking scores of 5.043 and 5.136 kcal/mol, respectively.
They were stabilized by stacking with Phe 264 (A and C ring) and His 259 (B ring),
and –cation interaction with Arg 268 (A ring), as shown in Figure 4. Two derivatives
that were highly substituted with hydroxyl groups, V7 and V11, formed hydrogen bonds
with enzyme, and showed relatively high docking scores of 5.174 and 5.677 kcal/mol,
,
−
(
−
−
π–π
π
−
−
respectively. This result was consistent with our results, showing relatively low IC50 values
against tyrosinase activity obtained from in vitro experiment. However, V7 had no effect
on melanogenesis and tyrosinase activity in melanoma cells, while V11 showed cytotoxicity
in melanoma cells.

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Table 3. Binding sites and docking scores of velutin derivatives in tyrosinase.
Key Interaction
Docking Score
Ligand
(Kcal/mol)
H Bond
π-π Stacking
π-Cation
Phe 264 (A, C ring)
His 259 (B ring)
Phe 264 (A, C ring)
His 259 (B ring)
Phe 264 (B ring)
His 263 (A ring)
V1
V2
V3
−5.043
Arg 268 (A ring)
Arg 268 (A ring)
Arg 268 (A ring)
−5.093
−5.223
Phe 264 (A, C ring)
His 259, His 85 (B ring)
V4
V5
V6
−5.136
−5.321
−4.983
Arg 268 (A ring)
Arg 268 (A, C ring)
Arg 268 (A ring)
Phe 264 (A, C ring)
His 259 (B ring)
His 85 (A ring)
His 244 (B, C ring)
Phe 264 (C ring)
His 263 (B ring)
V7
−5.174
−5.004
No pose
No pose
Glu 322 (C’-4)
Met 280 (C-7)
V8
Arg 268 (A ring)
V9
V10
His 263 (A ring)
Phe 264 (B ring)
V11
−5.677
Figure 4. Molecular docking of tyrosinase binding with the V1 and V4 derivatives (V1: red sticks
and V4: blue sticks).
3. Experimental Section
3.1. Chemistry
Commercially available reagents were used without additional purification. All reac-
tion mixtures were magnetically stirred, and were monitored by thin-layer chromatography,
using silica gel pre-coated glass plates visualized with UV light, and then developed using
either iodine or a solution of anisaldehyde. Flash column chromatography was carried out
1
using silica gel ((230–400) mesh). H NMR (400 Mhz) and spectra were recorded by NMR
spectrometry. Deuterated chloroform was used as the solvent and chemical shift values (δ)
were reported in parts per million, relative to the residual signals of this solvent [δ 7.26 for
1H (chloroform-d), δ 2.50 for 1H (dimethyl sulfoxide-d)].
3.2. Materials
Reagents such as 2,20-azinobis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) and
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), -melanocyte stimu-
α

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lating hormone (a–MSH), and enzymes, such as mushroom tyrosinase, were obtained from
Sigma–Aldrich (St. Louis, MO, USA). Potassium persulfate (K2S2O8), pyrocatechol violet,
and 3,4-dihydroxy-L-phenylalanine (L-DOPA) were purchased from Alfa Aesar (Haverhill,
MA, USA).
3.3. Determination of ABTS Free Radical Scavenging Activity
In order to evaluate the antioxidant activity of the velutin derivatives, the ABTS radical
scavenging activity was determined, as previously described [20]. To generate the ABTS
radical, 10 mL of 7 mM ABTS was mixed with 176 µL of 140 mM potassium peroxydisulfate
in dH2O, and incubated in the dark at room temperature (RT) for 16 h, prior to use. The
ABTS radical solution was diluted with absolute methanol, to obtain an absorbance of near
0.7 at 734 nm. Aliquots of 100
to 200 M were added to 100
µ
L of each derivative in the indicated concentration range 2
L of the diluted ABTS radical solution, and incubated for
µ
µ
10 min in the dark at RT. The absorbance was then measured at 732 nm using a SpectraMax
M5 Multi-Mode microplate reader (Molecular Devices, Sunnyvale, CA, USA). The ABTS
radical scavenging activity was calculated as follows:
ABTS radical scavenging activity (%) = 1 − (Asample/Acontrol) × 100
(1)
3.4. In Vitro Mushroom Tyrosinase Inhibition
The inhibitory effect of velutin derivatives on tyrosinase activity was assessed by the
amount of dopachrome synthesized from the catalytic reaction of tyrosinase [ 21]. In brief,
50 L of velutin derivatives in the concentration range of 0.01–1 mM was mixed with 50
8,
µ
µL
of 50 U/mL mushroom tyrosinase, in 50 mM phosphate buffered saline (PBS; 8.1 mM
Na2HPO4, 1.2 mM KH2PO4, pH 6.8, 2.7 mM KCl, 138 mM NaCl) in a 96-well plate, and
incubated for 30 min at RT. Then, 100 µL of 0.4 mM L-tyrosine was added to each well,
followed by incubation for an additional 10 min at 37 ◦C. The absorbance of the resulting
solution was measured at 475 nm, using a SpectraMax M5 Multi-Mode microplate reader.
3.5. Cell Culture
B16F10 murine melanoma cells were cultured in DMEM medium (Gibco, Gaithersburg,
USA) supplemented with 10% heat-inactivated fetal bovine serum (Gibco), 100 units/mL
penicillin, and 100 µg/mL streptomycin (Gibco), at 37 ◦C under humidified 5% CO2.
3.6. Cell Viability
The viability of B16F10 cells was determined using an MTT assay, as previously
described [22]. In brief, B16F10 cells were seeded in 24-well plates at a density of 1
×
104
cells per well. After 24 h, the cells were treated with the indicated concentrations of velutin
derivatives for 48 h. The cells were then incubated with MTT solution for 4 h, and the
reduced formazan crystals were dissolved in DMSO. The resulting solution was transferred
to 96-well plates, and the absorbance was measured at 540 nm, using a SpectraMax M5
Multi-Mode microplate reader.
3.7. Melanin Content Determination
The melanin content was determined as previously described, with some modifica-
tions [23]. The melanoma cells were cultured in a 6-well plate for 24 h. They were treated
with the indicated concentrations of velutin derivatives for a further 48 h, in the presence
of 100 nM α–MSH. After washing twice with chilled Dulbecco’s phosphate buffered saline
supplemented with calcium chloride and magnesium chloride (D-PBS, Gibco), the resulting
cells were detached by incubation with trypsin–EDTA solution. After centrifugation at
1000 rpm for 3 min,◦the cell pellet was dissolved in 150
µL of 1 M NaOH containing 10%
DMSO for 1 h at 60 C. The melanin content was determined by the absorbance at 405 nm,
using the microplate reader.

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3.8. Determination of Cellular Tryosinase Activity in Melanoma Cells
Tyrosinase activity in B16 cells was examined on the basis of the amount of dopachrome
produced from the catalytic reaction of intracellular tyrosinase [24]. In brief, the melanoma
cells were cultured in a 6-well plate for 24 h, followed by treatment with different con-
centrations of velutin derivatives for a further 48 h, in the presence of 100 nM of
α–
MSH. After washing twice with ice-cold D-PBS, the cells were lysed in 200 L of radio-
µ
immunoprecipitation assay (RIPA) buffer (Sigma-Aldrich), containing protease and phos-
phatase inhibitors. After centrifugation of the cell lysate collected from each well at
15,000× g for 15 min, 100
µL of supernatant was mixed with 100 µL of 1 mM L-DOPA in
PBS (pH 6.8), followed by incubation for 30 min at 37 ◦C. The absorbance of dopachrome
was measured at 475 nm using the microplate reader. Data were normalized with protein
concentration, determined by bicinchoninic acid assay.
3.9. Molecular Modeling
The crystal structure of mushroom tyrosinase for molecular modeling was an Agaricus
bisporus tyrosinase (PDB dose: 2Y9X) obtained from the Protein Data Bank (PDB). The
enzyme was prepared by using the protein wizard preparation workflow embedded in
the Maestro program (Maestro, version 11.9.011, Schrödinger, LLC, New York, NY, USA,
2019). Water, and all the other molecules present in the pdb files, were removed. Molecular
docking was performed using the Induced Fit Docking (IFD) protocol (Schrödinger Suite
2019 Induced Fit Docking protocol), as previously reported [25].
3.10. Statistical Analysis
All data in this study were expressed as the mean
±
standard deviation (SD) from
three independent experiments. Statistical analyses were performed using the Graph-Pad
Prism 8.0 (GraphPad Software Inc., La Jolla, CA, USA). The differences between the mean
values of the control and the exposed groups were analyzed using one-way analysis of
variance (ANOVA). A value of p < 0.05 was considered to be statistically significant.
4. Conclusions
In this study, we obtained twelve velutin derivatives through chemical synthesis,
and comparatively analyzed the relationship between the chemical structure of these
derivatives and their activity related to melanin synthesis. The SAR study revealed that
the substitution of functional groups at C5, C7, C30, and C40 of the flavone backbone
affected the biological activities related to melanin synthesis (Figure 5). The coexistence of
hydroxyl and methoxy at R1 and R2, respectively, was essential for inhibiting tyrosinase
activity. However, the 1,2-diol compounds substituted at R3 and R4 induced apoptosis
of melanoma cells. Further, substitution at R3 and R4 with methoxy or hydrogen was
essential for inhibiting melanogenesis. This study would be helpful for the development of
natural-derived functional materials, to regulate melanin synthesis.
Figure 5. Velutin derivatives along with their structure–activity relationship for the inhibition of melanogenesis.

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Author Contributions: Conceptualization, S.-H.J. and K.L.; methodology, S.-H.J.; software, J.K.;
validation, S.-H.J. and K.L.; formal analysis, S.-H.J. and K.L.; investigation, H.-Y.H. and J.-W.C.;
resources, K.L.; data curation, S.-H.J. and H.-Y.H.; writing—original draft preparation, S.-H.J. and H.-
Y.H.; writing—review and editing, K.L.; visualization, S.-H.J. and H.-Y.H.; supervision, K.L.; project
administration, K.L.; funding acquisition, K.L. All authors have read and agreed to the published
version of the manuscript.
Funding: This work was supported by a 2017 Research Grant from Kangwon National University
(No. 520170418).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: The data presented in this study are available on request from the
corresponding author.
Conflicts of Interest: The authors declare that they have no conflict of interest.
Sample Availability: Samples of the all compounds are available from the authors.
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