Selective synthesis of 7-O-substituted luteolin derivatives and their melanonenesis and proliferation inhibitory activity in B16 melanoma cells

Article abstract of DOI:10.1016/j.bmcl.2018.05.051

In our previous study, the isolation of ugonin J, K, and L, which are luteolin derivatives, from the roots of Helminthostachys zeylanica and their identification as potent melanogenesis inhibitors, was described. The structure activity relationship (SAR) investigation in that study revealed that the catechol moiety in the B-ring of the flavone skeleton of ugonin K was important for its melanogenesis inhibitory activity, and the presence of the low polarity substituents at the C-7 position enhanced this activity. In order to further investigate the SAR of the C-7-substituent in the luteolin derivatives, different groups were selectively introduced at the C-7 position of luteolin after borax protection of the catechol hydroxyl group and the C-5 hydroxyl group. NMR and MS analysis of the borax protected derivatives revealed that the borax protects not only hydroxyl groups of catechol on the B ring but also the 5-hydroxyl group on the A ring. Eight luteolin derivatives were synthesized and evaluated for melanogenesis inhibitory effect in B16 melanoma cells. Two bulky groups and six alkoxyl groups were introduced at the C-7 position. The resulting luteolin derivatives showed improved melanogenesis and cell proliferation inhibitory activities. From among these derivatives, 7-O-hexylluteolin (7) showed the highest activity and inhibited the melanogenesis to 14% at 6.25 μM. The present study also revealed that the length of the carbon chain rather than the bulky substituent was more important for the melanogenesis inhibitory activity.

Full text of DOI:10.1016/j.bmcl.2018.05.051

Accepted Manuscript
Selective synthesis of 7-O-substituted luteolin derivatives and their melanone-
nesis and proliferation inhibitory activity in B16 melanoma cells
Kosei Yamauchi, Akari Fujieda, Tohru Mitsunaga
PII:
S0960-894X(18)30463-3
https://doi.org/10.1016/j.bmcl.2018.05.051
BMCL 25868
DOI:
Reference:
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
2 May 2018
25 May 2018
26 May 2018
Please cite this article as: Yamauchi, K., Fujieda, A., Mitsunaga, T., Selective synthesis of 7-O-substituted luteolin
derivatives and their melanonenesis and proliferation inhibitory activity in B16 melanoma cells, Bioorganic &
Medicinal Chemistry Letters (2018), doi: https://doi.org/10.1016/j.bmcl.2018.05.051
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

1
Selective synthesis of 7-O-substituted luteolin derivatives and their
melanonenesis and proliferation inhibitory activity in B16 melanoma
cells
Kosei Yamauchi, Akari Fujieda, Tohru Mitsunaga*
Faculty of Applied Biological Sciences, Gifu University, 1-1 Yanagido 501-1193, Gifu,
Japan
*
Corresponding author.
Prof. Dr. Tohru Mitsunaga,
Faculty of Applied Biological Sciences, Gifu University, 1-1 Yanagido 501-1193, Gifu,
Japan
Tel.: +81 58 293 2920; Fax: +81 58 293 2920
E-mail: mitunaga@gifu-u.ac.jp

2
ABSTRACT
In our previous study, the isolation of ugonin J, K, and L, which are luteolin derivatives,
from the roots of Helminthostachys zeylanica and their identification as potent
melanogenesis inhibitors, was described. The structure activity relationship (SAR)
investigation in that study revealed that the catechol moiety in the B-ring of the flavone
skeleton of ugonin K was important for its melanogenesis inhibitory activity, and the
presence of the low polarity substituents at the C-7 position enhanced this activity. In
order to further investigate the SAR of the C-7-substituent in the luteolin derivatives,
different groups were selectively introduced at the C-7 position of luteolin after borax
protection of the catechol hydroxyl group and the C-5 hydroxyl group. NMR and MS
analysis of the borax protected derivatives revealed that the borax protects not only
hydroxyl groups of catechol on the B ring but also the 5-hydroxyl group on the A ring.
Eight luteolin derivatives were synthesized and evaluated for melanogenesis inhibitory
effect in B16 melanoma cells. Two bulky groups and six alkoxyl groups were
introduced at the C-7 position. The resulting luteolin derivatives showed improved
melanogenesis and cell proliferation inhibitory activities. From among these derivatives,
7
-O-hexylluteolin (7) showed the highest activity and inhibited the melanogenesis to
4 % at 6.25 μM. The present study also revealed that the length of the carbon chain
1

3
rather than the bulky substituent was more important for the melanogenesis inhibitory
activity.
Keywords: B16 melanoma cells; Luteolin derivatives; Borax; Protecting group
Melanoma, a malignant skin tumor caused by ultraviolet (UV) damage to
melanocytes, that are pigment-producing cells distributed on the surface of the skin, is
1
–3
responsible for 75% of deaths due to skin cancer . . Melanoma metastasizes to the
4
,5
lungs, lymph nodes, and bones. Numerous studies have reported anti-metastasis
agents for melanoma. Rho kinase inhibitors such as AT13148 and CCT129254
6
dramatically inhibit invasion of melanoma. . Galangin suppresses proliferation and
metastasis of B16 melanoma by inhibiting focal adhesion kinase (FAK), a cytoplasmic
7
tyrosine kinase involved in proliferation, adhesion, and invasion of the cancer cells.
Melanoma are also known to biosynthesize melanin, a pigment found in human hair
and skin, which plays an important role in protecting skin from the damage caused by
8
–11
ultraviolet rays.
A reduction in the rate of melanogenesis is associated with aging
and stress that causes gray hair and leukoderma, while an excess production of melanin
leads to mottling or sunlight-induced skin darkening. A number of food ingredients and

4
herbal medicines have been evaluated for their effectiveness in modulating
melanogenesis for maintaining the health and cosmetic appearance of the human body.
1
2–18
Flavonoids have also been identified as melanogenesis modulators. For instance,
1
9
inulavosin, a pentamethylflavan, first isolated from Inula nervosa
melanogenesis by a lysosomal degradation of tyrosinase in B16 melanoma cells.
Apigenin (4′,5,7-trihydroxyflavone) and its derivatives induce melanogenesis in B16
inhibits
2
0
1
8
melanoma cells. . Quercetin (3,5,7,3′,4′-pentahydroxyflavone), a representative
2
1–23
flavonoid present in a wide variety of foods and medicinal plants,
has been
investigated as a melanogenesis modulator, an antioxidant, and as an anti-cancer agent.
2
4,25
Quercetin derivatives exhibit extracellular melanogenesis stimulatory activity in
2
6–30
B16 melanoma cells and in human three dimensional skin model.
In contrast,
luteolin (5,7,3′,4′-tetrahydroxyflavone) and its derivatives, ugonins J and K, potently
3
1,32
inhibit extracellular melanogenesis.
Ugonin A-L were isolated from Helminthostachys zeylanica, which belongs to the
3
3–35
Ophioglossaceae family.
They have shown high bioactivity against a number of
3
6
diseases. Ugonin K has been reported to inhibit osteoclast differentiation. Ugonin J, K,
and L showed antioxidant activity that was more potent than trolox, which was used as a

5
3
5
positive control. . Ugonin K has also been reported to protect against hydrogen
3
7
peroxide induced osteoblast cell damage. In our previous study, the SAR of ugonin J
3
2
and K, as melanogenesis inhibitors in B16 melanoma cells, was investigated. That
study established that the catechol and the flavone skeletons play an essential role in the
melanogenesis inhibitory activity, and that the substituent on ring A of luteolin enhances
this activity. Likewise, the 7-methoxyl group on the A ring of ugonin K stimulated this
effect. However, the detailed role of the C-7 substituent on anti-melanogenesis activity
of luteolin derivatives is still unknown. Luteolin has also been reported to act as a
3
8–40
potent anticancer agent by stimulating the apoptosis pathway of tumor cells.
However, the role of the substituent groups of the luteolin derivatives on its anticancer
activity has not been investigated as yet. In this study, eight luteolin derivatives (Fig. 1)
having different substituents were synthesized and their anti-proliferation and
melanogenesis inhibitory effects were evaluated, using B16 melanoma cells, in order to
understand the role of the substituents on melanogenesis and proliferation.

6
Compounds
luteolin R= H
5
2
’
’
OH
OH
6
’
2
3
4
5
R= CH3-
8
1
4’
3’
1
’
O
8a
4
O
R
2
7
R= CH3CH2-
6
3
a
4
5
R= CH3CH2CH2-
R= CH3CH2CH2CH2-
OH
O
OH
B
O
O
6
7
8
R= CH3CH2CH2CH2CH2-
HO
O
O
R= CH3CH2CH2CH2CH2CH2-
12CH3
1
1
O
9
R= H C 10
3
B
1
1
2
1
1
9
10
OH
9
R=
1
3
14
1
5
Fig.1. Structures of synthesized luteolin derivatives
Selective protection of the hydroxyl groups of flavonoids has been reported so far
because of the high chemical reactivity of the flavonoid derivatives. Recently
dichlorodiphenylmethane was used to protect the hydroxyl groups on the B ring of the
4
1,42
flavonoids.
However, since this reaction required a high temperature of around
1
80°C, it was difficult to protect the 5-hydroxyl group. Consequently, borax (sodium
tetraborate) protecting method available to react even under the room temperature was
selected to protect the catechol hydroxyl groups as well as the 5-hydroxyl group. The
borax protected luteolin was identified using NMR and MS analysis. The changes in the
chemical shifts of borax-protected luteolin were shown in figure 2. The NMR signals

7
for H-2’ and H-5’ were shifted upfield by 0.49 ppm and 0.43 ppm respectively.
Furthermore, the protons on A and C rings also showed a comparable upfield shift. In
contrast, the NMR signals showed a downfield shift by 7.3 ppm and 7.1 ppm for C-3’
and C-4’ respectively, due to the borax protection of luteolin. The signal for C-5 also
showed a dramatic downfield shift from 162.0 ppm to 175.0 ppm, in protected luteolin.
These changes suggested that borax can interact with the 5-hydroxyl group and the
4
-carbonyl group on the A ring as well as the hydroxyl groups on the B ring. HMBC
+
correlations (Fig. 3) and a m/z value of 361.9009 for [M+Na] also supported the
attachment of the borax to the luteolin molecule. All these data supported the structure
of the luteolin-borax complex as shown in figure 1 and established borax as a new
protecting agent for flavonoids.

8
OH
B
O
6
.87
1
53.6
46.3
5
6
.96
.41
7
.36
157.3
150.2
1
H
104.4
113.9
163.8
O
H
107.6
116.5
9
6.0
1
1
61.6
64.7
94.4 158.6
157.8
HO
O
O
164.4
1
1
20.0
22.0
1
1
17.4
19.5
H 6.42
6.85
1
1
01.3
03.4
H7.13
7.37
9
9.9
04.2
1
9
01.9
9.4
1
180.4
182.2
5
.73H
H
6.29
6.63
1
75.0
1
62.0
6
.15
O
B
OH
Fig.2. Chemical shift of 13C and H-NMR of borax-protected luteolin and luteolin (italic)
1
Underline: high magnetic shift comparing to luteolin, Outline: low magnetic shift.
Fig.3. Key HMBC correlations of borax-protected luteolin
7
-O-selective methylation and ethylation were performed in water. The routes for
synthesis of 7-O-methylluteolin (2) or 7-O-ethylluteolin (3) are shown in Scheme 1.

9
HPLC analysis of the reaction mixture showed the presence of almost exclusively the
desired product (data not shown), suggesting that the reaction was highly selective.
However, the longer hydrocarbon chain containing groups did not react under these
conditions. Consequently, we tried to obtain the borax protected luteolin powder after
freeze drying, and then the substitution reactions using the powder were conducted in
DMF replacing water. The exclusive formation of the desired products was confirmed
by the presence of just one prominent peak in the HPLC analysis. As a result,
7
-O-propylluteolin (4), 7-O-butylluteolin (5), 7-O-pentylluteolin (6), 7-O-hexylluteolin
7), 7-O-(1-methyl)propylluteolin (8), and 7-O-methylcyclohexylluteolin (9) were
obtained (Scheme 2) in a favorable yields of 70.3, 65.0, 77.0, 75.6, 46.0, and 72.5%
(
1
13
respectively. The structures of the reactants were identified using H-NMR, C-NMR,
COSY, HMQC, HMBC, UV and TOFMS. The binding position of the substituent group
was confirmed through HMBC correlation between C-7 and the methylene protons
directly connected to the oxygen atom in each of the substituents.

10
OH
O
O
B
OH
OH
HO
O
O
HO
O
O
Borax (Na B O ・ 10H O)
2
2
4
7
in water
room temperature
3
0 min
O
OH
B
OH
OH
O
O
B
OH
OH
(
C H ) SO or (CH )SO 9 eq
2
5
2
4
3
4
O
O
O
R
O
O
in water
.5 M NaOHaq 9 eq
R
0
6 M HClaq
room temperature
O
6
-16 h
B
OH
O
OH
R= CH
3
or C
2
H
5
R= CH
3
or C2H5
Scheme 1. Synthesis route of compounds 2 and 3
OH
O
B
OH
O
HO
O
O
Borax (Na B O ・ 10H O)
2
OH
2
in water
4
7
HO
O
O
Freeze dry
room temperature
3
0 min
OH
O
B
OH
O
OH
O
B
OH
OH
R-Br 18 eq
in DMF
K CO 9 eq
O
O
O
R
O
O
O
6
M HClaq
R
2
3
room temperature
2-16 h
O
B
1
OH
OH
Scheme 2. Synthesis route of compounds 4-9

11
Measurement of cellular melanin content and proliferation was done according to a
27
previously described method (shown in supplementary data) . Intra and extracellular
melanogenesis activity of B16 melanoma cells, treated with luteolin derivatives, were
determined as shown in Table 1. Luteolin decreased the extracellular and intracellular
melanogenesis to 15% and 66% respectively at a concentration of 25 μM. The cell
proliferation was also decreased to the intracellular melanogenesis level, at this
concentration. The decrease in the extracellular melanogenesis level was lower than the
decrease in cell proliferation. This result suggested that luteolin did not show any
intracellular melanogenesis inhibitory activity, but only extracellular melanogenesis
inhibitory activity. Compound 2 decreased extracellular melanin to 51% at 12.5 μM and
thus was more potent than luteolin. Compounds 3-6 inhibited the extracellular
melanogenesis even more than 2, at 12.5 μM. Compound 7 reduced extracellular
melanin to under 20% even at 6.25 μM. The concentration of extracellular melanin was
decreased to 7.4 μg/mL (μg/well) by compound 7 at 6.25 μM comparing to the control
cells (99.6 μg/mL) (shown in Supplementary data). These results suggest that the length
of hydrocarbon substituent plays a central role in this effect. In order to investigate the
influence of bulkiness of the substituent, compound 8 and 9 were synthesized (Fig. 1).

12
The inhibition was of the same magnitude as that due to the compounds 3 - 6, indicating
that the bulkiness of substituent at position 7 was not so important for the
melanogenesis inhibitory effect as the length of the linear hydrocarbon substituent.
Luteolin acts as an anticancer agent by stimulating the apoptosis in cancer cells. The
anticancer property of luteolin is manifested in the form of inhibition of the proliferation,
3
8
metastasis and angiogenesis of the cancer cells . Luteolin inhibited proliferation to
4% at 50 μM as shown in Table 1. Compounds 4-9 increased the proliferation
inhibitory activity. Luteolin induces apoptosis pathway by activating p53 and c-Jun
3
4
3
N-terminal kinase (JNK).
JNK is also related to the melanogenesis via down
4
4,45
regulation of MITF, which is the transcriptional factor of melanogenic enzymes.
The 7-O- substituent group attached with luteolin may be stimulating the activation of
the JNK that induces apoptosis and suppression of melanogenesis in B16 melanoma
cells. The determinations of these protein expressions in the cells treated with the
luteolin derivatives were needed to elucidate the mechanism of the activity. These
results suggested that the 7-O-substituted luteolin derivatives have the potency as an
anticancer agent as well as a whitening agent.
Ugonin K showed strong extracellular melanogenesis inhibitory activity,
3
2
comparable to the activity of the synthesized luteolin derivatives in this study but

13
without inhibiting the cell proliferation. Then the bulky and low polarity substituent
groups at position 6 of the ugonin may be involved in reducing the proliferation
inhibitory activity. A detailed SAR study, using 6 substituted luteolin derivatives, is
needed to clarify the mechanism of luteolin derivatives on the inhibitory effects.

14
Table 1.
Intra- and extracellular melanogenesis activities and cell
proliferation effect of luteolin derivatives in B16 melanoma cells
Compounds
and concentration
μM)
luteolin
name
Cell
proliferation
(%)
Intracellular
melanogeneis
activity (%)
Extracellular melanogenesis
activity (%)
(
5
2
1
6
2
5
2
1
6
3
5
2
1
6
4
5
2
1
6
5
0.0
5.0
2.5
.25
34±2.6**
68±1.3**
110±1.7
106±1.8
55±1.0**
66±0.4**
118±2.4**
122±1.8**
16±0.2**
15±0.1**
109±0.6**
114±0.8**
0.0
5.0
2.5
.25
41±0.5**
55±2.1**
85±2.1
50±1.1**
50±3.8**
87±1.1**
108±4.4
16±0.2**
14±0.2**
51±10.9**
107±2.6
98±1.1
0.0
5.0
2.5
.25
68±1.9**
73±0.3**
59±3.7**
83±1.5**
70±8.6
65±0.4**
73±1.1**
98±2.2
15±0.2**
15±0.8**
14±0.1**
127±2.1**
0.0
5.0
2.5
.25
32±0.8**
46±1.1**
55±1.1**
70±1.7**
54±0.8**
56±0.4**
66±0.1**
92±2.7*
15±0.4**
14±0.2**
12±0.0**
123±3.8**
5
2
1
6
6
5
2
1
6
7
5
2
1
6
8
5
2
1
6
9
5
2
1
6
0.0
5.0
2.5
.25
52±1.0**
51±0.8**
50±0.9**
69±2.1**
56±0.4**
56±0.4**
61±0.1**
85±1.0**
13±0.1**
13±0.1**
14±0.4**
117±2.1**
0.0
5.0
2.5
.25
47±2.7**
44±5.1**
43±1.9**
66±0.4**
67±1.7**
69±0.6**
69±1.9**
88±1.6*
13±0.2**
13±0.1**
13±0.2**
84±4.5**
0.0
5.0
2.5
.25
50±4.0**
53±2.4**
44±0.5**
40±1.4**
67±4.3**
70±5.9**
67±2.0**
65±0.9**
13±0.2**
13±0.3**
13±0.4**
14±0.5**
0.0
5.0
2.5
.25
6±0.5**
24±0.9**
50±2.1**
74±4.1**
54±0.2**
63±0.4**
70±1.0**
93±2.6*
21±0.0**
18±0.2**
18±0.3**
78±7.3**
0.0
5.0
2.5
.25
33±1.0**
30±1.6**
51±3.9**
77±4.0**
85±1.4**
72±1.1**
84±3.1**
101±0.5
14±0.2**
14±0.4**
36±0.8**
117±2.8**
Arbutin
30.0
7
105±6.5
89±3.7*
45±2.4**
Data are expressed as means ± S. D. against the ratio of melanogenesis activity and cell proliferation. * p≤0.05 and
* p≤0.01 compared with respective control values. n=3.
*

15
Acknowledgements
This work was supported by a Grant-in-Aid for Start-up Research Activity from Japan
Society for the Promotion of Science (JSPS).
References
1
.
Lawrence MS, Stojanov P, Polak P, et al. Mutational heterogeneity in cancer and
the search for new cancer-associated genes. Nature. 2013;499(7457):214-218.
doi:10.1038/nature12213.
2
3
4
.
.
.
Schadendorf D, Fisher DE, Garbe C, et al. Melanoma. Nat Rev Dis Prim.
2
015;1:15003. doi:10.1038/nrdp.2015.3.
Schadendorf D, Hauschild A. Melanoma in 2013: Melanoma—the run of success
continues. Nat Rev Clin Oncol 2014 112. January 2014.
De Angelis R, Sant M, Coleman MP, et al. Cancer survival in Europe 1999–2007
by country and age: results of EUROCARE-5—a population-based study. Lancet
Oncol. 2014;15(1):23-34. doi:10.1016/S1470-2045(13)70546-1.
5
.
Livingstone E, Windemuth-Kieselbach C, Eigentler TK, et al. A first prospective
population-based analysis investigating the actual practice of melanoma

16
diagnosis, treatment and follow-up. Eur J Cancer. 2011;47(13):1977-1989.
doi:10.1016/j.ejca.2011.04.029.
6
7
8
.
.
.
Sadok A, McCarthy A, Caldwell J, et al. Rho Kinase Inhibitors Block Melanoma
Cell Migration and Inhibit Metastasis. Cancer Res. 2015;75(11):2272-2284.
doi:10.1158/0008-5472.CAN-14-2156.
Zhang W, Tang B, Huang Q, Hua Z. Galangin inhibits tumor growth and
metastasis of B16F10 melanoma. J Cell Biochem. 2013;114(1):152-161.
doi:10.1002/jcb.24312.
Sánchez-Ferrer Á, Neptuno Rodríguez-López J, García-Cánovas F,
García-Carmona F. Tyrosinase: a comprehensive review of its mechanism.
Biochim Biophys Acta (BBA)/Protein Struct Mol. 1995;1247(1):1-11.
doi:10.1016/0167-4838(94)00204-T.
9
1
.
Cooksey CJ, Garratt PJ, Land EJ, Ramsden CA, Riley PA. Tyrosinase kinetics:
failure of the auto-activation mechanism of monohydric phenol oxidation by
rapid formation of a quinomethane intermediate. Biochem J. 1998;333:685-691.
0. Pérez-Sánchez A, Barrajón-Catalán E, Herranz-López M, Castillo J, Micol V.
Lemon balm extract (Melissa officinalis, L.) promotes melanogenesis and
prevents UVB-induced oxidative stress and DNA damage in a skin cell model. J

17
Dermatol Sci. 2016;84(2):169-177. doi:10.1016/J.JDERMSCI.2016.08.004.
1
1
1. Wang H, Pan Y, Tang X, Huang Z. Isolation and characterization of melanin
from Osmanthus fragrans’ seeds. LWT - Food Sci Technol. 2006;39(5):496-502.
doi:10.1016/j.lwt.2005.04.001.
2. Tsuji-Naito K, Hatani T, Okada T, Tehara T. Modulating effects of a novel
skin-lightening agent, α-lipoic acid derivative, on melanin production by the
formation of DOPA conjugate products. Bioorganic Med Chem.
2
007;15(5):1967-1975. doi:10.1016/j.bmc.2006.12.042.
1
1
3. Yamauchi K, Mitsunaga T, Batubara I. Isolation, Identification and Tyrosinase
Inhibitory Activities of the Extractives from Allamanda cathartica. Nat Resour.
2
011;2(3):167-172. doi:10.4236/nr.2011.23022.
4. Chiang HM, Chien YC, Wu CH, et al. Hydroalcoholic extract of Rhodiola rosea
L. (Crassulaceae) and its hydrolysate inhibit melanogenesis in B16F0 cells by
regulating the CREB/MITF/tyrosinase pathway. Food Chem Toxicol.
2
014;65:129-139. doi:10.1016/j.fct.2013.12.032.
1
5. Jang JY, Kim HN, Kim YR, et al. Partially purified components of Nardostachys
chinensis suppress melanin synthesis through ERK and Akt signaling pathway
with cAMP down-regulation in B16F10 cells. J Ethnopharmacol.

18
2
011;137(3):1207-1214. doi:10.1016/j.jep.2011.07.047.
1
1
6. Batubara I, Darusman LK, Mitsunaga T, Rahminiwati M, Djauhari E. Potency of
Indonesian medicinal plants as tyrosinase inhibitor and antioxidant agent. J Biol
Sci. 2010;10(2):138-144. doi:10.3923/jbs.2010.138.144.
7. Tangke Arung E, Matsubara E, Wijaya Kusuma I, Sukaton E, Shimizu K, Kondo
R. Inhibitory components from the buds of clove (Syzygium aromaticum) on
melanin formation in B16 melanoma cells. Fitoterapia. 2011;82(2):198-202.
doi:10.1016/j.fitote.2010.09.008.
1
1
2
2
8. Ye Y, Chou GX, Wang H, Chu JH, Yu ZL. Flavonoids, apigenin and icariin exert
potent melanogenic activities in murine B16 melanoma cells. Phytomedicine.
2
010;18(1):32-35. doi:10.1016/j.phymed.2010.06.004.
9. Yoshida T, Mori K, He G. Inulavosin, a New Thymol Dimer with Piscicidal
Activity from Inula nervosa. Heterocycles. 1995;41(9):1923.
doi:10.3987/COM-95-7148.
0. Fujita H, Motokawa T, Katagiri T, et al. Inulavosin, a melanogenesis inhibitor,
leads to mistargeting of tyrosinase to lysosomes and accelerates its degradation. J
Invest Dermatol. 2009;129(6):1489-1499. doi:10.1038/jid.2008.376.
1. Hertog MGL, Hollman PCH, Hertog MGL, Katan MB. Content of Potentially

19
Anticarcinogenic Flavonoids of 28 Vegetables and 9 Fruits Commonly
Consumed in the Netherlands. J Agric Food Chem. 1992;40(12):2379-2383.
doi:10.1021/jf00024a011.
2
2
2
2. Manach C, Scalbert A, Morand C, Rémésy C, Jiménez L. Polyphenols: Food
sources and bioavailability. Am J Clin Nutr. 2004;79(5):727-747.
doi:10.1038/nature05488.
3. Hollman PCH, Katan MB. Dietary flavonoids: Intake, health effects and
bioavailability. Food Chem Toxicol. 1999;37(9-10):937-942.
doi:10.1016/S0278-6915(99)00079-4.
4. Gibellini L, Pinti M, Nasi M, et al. Interfering with ROS metabolism in cancer
cells: The potential role of quercetin. Cancers (Basel). 2010;2(2):1288-1311.
doi:10.3390/cancers2021288.
2
2
5. Chen QX, Kubo I. Kinetics of mushroom tyrosinase inhibition by quercetin. J
Agric Food Chem. 2002;50(14):4108-4112. doi:10.1021/jf011378z.
6. Yamauchi K, Mitsunaga T, Batubara I. Novel quercetin glucosides from
Helminthostachys zeylanica root and acceleratory activity of melanin
biosynthesis. J Nat Med. 2013;67(2):369-374. doi:10.1007/s11418-012-0672-9.
2
7. Yamauchi K, Mitsunaga T, Inagaki M, Suzuki T. Synthesized quercetin

20
derivatives stimulate melanogenesis in B16 melanoma cells by influencing the
expression of melanin biosynthesis proteins MITF and p38 MAPK. Bioorganic
Med Chem. 2014;22(13):3331-3340. doi:10.1016/j.bmc.2014.04.053.
2
2
8. Yamauchi K, Mitsunaga T, Batubara I. Synthesis of quercetin glycosides and
their melanogenesis stimulatory activity in B16 melanoma cells. Bioorganic Med
Chem. 2014;22(3):937-944. doi:10.1016/j.bmc.2013.12.062.
9. Yamauchi K, Mitsunaga T, Inagaki M, Suzuki T. Quercetin derivatives regulate
melanosome transportation via EPI64 inhibition and elongate the cell shape of
B16 melanoma cells. Biomed Pharmacother. 2015;70(C):206-212.
doi:10.1016/j.biopha.2015.01.031.
3
3
3
0. Yamauchi K, Mitsunaga T. Methylquercetins stimulate melanin biosynthesis in a
three-dimensional skin model. J Nat Med. 2018;72(2):563-569.
doi:10.1007/s11418-018-1175-0.
1. Choi MY, Song HS, Hur HS, Sim SS. Whitening activity of luteolin related to
the inhibition of cAMP pathway in α-MSH-stimulated B16 melanoma cells. Arch
Pharm Res. 2008;31(9):1166-1171. doi:10.1007/s12272-001-1284-4.
2. Yamauchi K, Mitsunaga T, Itakura Y, Batubara I. Extracellular melanogenesis
inhibitory activity and the structure-activity relationships of ugonins from

21
Helminthostachys zeylanica roots. Fitoterapia. 2015;104:69-74.
doi:10.1016/j.fitote.2015.05.006.
3
3
3
3
3
3
3. Murakami T, Hagiwara M, Tanaka K, Chen C-M. Chemische Untersuchungen
uber die Inhaltsstoffe von Helminthostachys zeylanica (L) HOOK. I. Chem
Pharm Bull (Tokyo). 1973;21(8):1849-1851. doi:10.1248/cpb.21.1849.
4. Murakami T, Hagiwara M, Tanaka K, Chen C-M. Chemische Untersuchungen
uber die Inhaltsstoffe von Helminthostachys zeylanica (L) HOOK. II. Chem
Pharm Bull (Tokyo). 1973;21(8):1851-1852. doi:10.1248/cpb.21.1851.
5. Huang Y-L, Yeh P-Y, Shen C-C, Chen C-C. Antioxidant flavonoids from the
rhizomes of Helminthostachys zeylanica. Phytochemistry. 2003;64(7):1277-1283.
http://www.ncbi.nlm.nih.gov/pubmed/14599526. Accessed March 22, 2018.
6. Huang Y-L, Shen C-C, Shen Y-C, Chiou W-F, Chen C-C. Anti-inflammatory
and Antiosteoporosis Flavonoids from the Rhizomes of Helminthostachys
zeylanica. J Nat Prod. 2017;80(2):246-253. doi:10.1021/acs.jnatprod.5b01164.
7. Huang Y-L, Lee C-H, Liao J-F, Liu Y-W, Chiou W-F. Protective effects of
ugonin K on hydrogen peroxide-induced osteoblast cell damage. J Funct Foods.
2
015;15:487-496. doi:10.1016/J.JFF.2015.03.056.
8. Lin Y, Shi R, Wang X, Shen H-M. Luteolin, a flavonoid with potential for cancer

22
prevention and therapy. Curr Cancer Drug Targets. 2008;8(7):634-646.
http://www.ncbi.nlm.nih.gov/pubmed/18991571. Accessed March 26, 2018.
9. Jiang Z-Q, Li M-H, Qin Y-M, Jiang H-Y, Zhang X, Wu M-H. Luteolin Inhibits
3
Tumorigenesis and Induces Apoptosis of Non-Small Cell Lung Cancer Cells via
Regulation of MicroRNA-34a-5p. Int J Mol Sci. 2018;19(2).
doi:10.3390/ijms19020447.
4
4
4
0. Han K, Meng W, Zhang J-J, et al. Luteolin inhibited proliferation and induced
apoptosis of prostate cancer cells through miR-301. Onco Targets Ther.
2
016;9:3085. doi:10.2147/OTT.S102862.
1. Li N-G, Shi Z-H, Tang Y-P, Yang J-P, Duan J-A. An efficient partial synthesis of
’-O-methylquercetin via regioselective protection and alkylation of quercetin.
4
Beilstein J Org Chem. 2009;5:60. doi:10.3762/bjoc.5.60.
2. Bouktaib M, Lebrun S, Atmani A, Rolando C. Hemisynthesis of all the
O-monomethylated analogues of quercetin including the major metabolites,
through selective protection of phenolic functions. Tetrahedron.
2
002;58(50):10001-10009. doi:10.1016/S0040-4020(02)01306-6.
4
3. Shi R, Huang Q, Zhu X, et al. Luteolin sensitizes the anticancer effect of cisplatin
via c-Jun NH2-terminal kinase-mediated p53 phosphorylation and stabilization.

23
Mol Cancer Ther. 2007;6(4):1338-1347. doi:10.1158/1535-7163.MCT-06-0638.
4. Kim D-S, Jeong Y-M, Park I-K, et al. A new 2-imino-1,3-thiazoline derivative,
KHG22394, inhibits melanin synthesis in mouse B16 melanoma cells. Biol
Pharm Bull. 2007;30(1):180-183.
4
http://www.ncbi.nlm.nih.gov/pubmed/17202683. Accessed March 27, 2018.
5. Bu J, Ma P-C, Chen Z-Q, et al. Inhibition of MITF and Tyrosinase by
Paeonol-Stimulated JNK/SAPK to Reduction of Phosphorylated CREB. Am J
Chin Med. 2008;36(2):245-263. doi:10.1142/S0192415X08005758.
4

24
OH
O
Borax protection
7-selective introduction of substituent group
O
B
OH
HO
O
7
O
7
O
O
R
OH
O
O
OH
B
OH
Anti-melanogenesis and anticancer activity

25
・
・
・
・
7-substituted luteolin derivatives were selectively synthesized by borax protection.
7-substituent group stimulated the melanogenesis inhibitory activity.
7-substituent group stimulated the proliferation inhibitory activity in melanoma cells.
The length of substituent group is involved in the melanogenesis inhibitory activity.