Novel furocoumarin derivatives stimulate melanogenesis in b16 melanoma cells by up-regulation of mitf and tyr family via Akt/GSK3β/β-catenin signaling pathways

Article abstract of DOI:10.3390/ijms19030746

The extracts of Ficuscarica L. and Psoralen corylifolia L. are traditional Uygur medicines for the treatment of vitiligo, and its active ingredients furocoumarins, were are found to be the most effective agents against this skin disorder nowadays. Therefore, a series of novel easter derivatives (8a–8p) of furocoumarin were designed and synthesized based on our previous research to improve this activity in the present study. The synthesized derivatives were biologically evaluated for melanin synthesis in murine B16 cells and the SAR (structure-activity relationship) was summarized. Eight derivatives were more potent than positive control (8-MOP, 8-methoxypsoralan), especially compounds 8n (200%) and 8o (197%), which were nearly 1.5-fold potency when compared with 8-MOP (136%). Furthermore, the signaling pathway by which 8n activates the melanin biosynthesis was defined. Our results showed that it not only elevated the melanin content, but also stimulated the activity of tyrosinasein a concentration-dependent manner. Increasing of phosphorylation of Akt (also named PKB, protein kinase B) and non-activated GSK3β (glycogen synthase kinase 3 beta), which inhibited the degradation of β-catenin were observed through Western blot analysis. The accumulation of β-catenin probably led to the activation of transcription of MITF (microphthalmia-associated transcription factor) and TYR (tyrosinase) family, as well as the subsequent induction of melanin synthesis.

Full text of DOI:10.3390/ijms19030746

International Journal of
Molecular Sciences
Article
Novel Furocoumarin Derivatives Stimulate
Melanogenesis in B16 Melanoma Cells by
Up-Regulation of MITF and TYR Family via
Akt/GSK3β/β-Catenin Signaling Pathways
Chao Niu 1, Li Yin 1,2 and Haji Akber Aisa 1,
*
ID
1
Key Laboratory of Plant Resources and Chemistry of Arid Zone, State Key Laboratory Basis of Xinjiang
Indigenous Medicinal Plants Resource Utilization, Xinjiang Technical Institute of Physics and Chemistry,
Chinese Academy of Sciences, Urumqi 830011, China; niuchao@ms.xjb.ac.cn (C.N.);
jinshiyi2003@163.com (L.Y.)
2
University of Chinese Academy of Sciences, Beijing 101408, China
*
Correspondence: haji@ms.xjb.ac.cn; Tel.: +86-0991-383-5679
Received: 15 January 2018; Accepted: 16 February 2018; Published: 6 March 2018
Abstract: The extracts of Ficuscarica L. and Psoralen corylifolia L. are traditional Uygur medicines for the
treatment of vitiligo, and its active ingredients furocoumarins, were are found to be the most effective
agents against this skin disorder nowadays. Therefore, a series of novel easter derivatives (8a–8p) of
furocoumarin were designed and synthesized based on our previous research to improve this activity
in the present study. The synthesized derivatives were biologically evaluated for melanin synthesis
in murine B16 cells and the SAR (structure-activity relationship) was summarized. Eight derivatives
were more potent than positive control (8-MOP, 8-methoxypsoralan), especially compounds 8n (200%)
and 8o (197%), which were nearly 1.5-fold potency when compared with 8-MOP (136%). Furthermore,
the signaling pathway by which 8n activates the melanin biosynthesis was defined. Our results
showed that it not only elevated the melanin content, but also stimulated the activity of tyrosinasein
a concentration-dependent manner. Increasing of phosphorylation of Akt (also named PKB,
protein kinase B) and non-activated GSK3
β
(glycogen synthase kinase 3 beta), which inhibited the
-catenin
degradation of -catenin were observed through Western blot analysis. The accumulation of
β
β
probably led to the activation of transcription of MITF (microphthalmia-associated transcription
factor) and TYR (tyrosinase) family, as well as the subsequent induction of melanin synthesis.
Keywords: vitiligo; furocoumarin; melanogenesis; SAR; Akt/GSK3β/β-catenin signaling pathways
1. Introduction
Vitiligo also named leukoderma, is an autoimmune disease that results in prominent white
patches of the skin [ ]. It can involve any part of the body where melanocytes resided and cause
1
both functional and physiological abnormalities in the affected skin. Many possible causes of vitiligo
have been proposed including immunologic, genetic, stress, neural mechanism, and biochemical
factors [2]. However, it is believed that the disease is mainly caused by destruction of the melanocyte
and obstruction of the melanin synthesis [3,4].
Melanin, derived from dopaquinone and synthesized in the melanosomes of melanocytes, serves
a number of valuable physiological functions [
cascade, such as tyrosinase, tyrosinase-related protein 1 (TRP-1) and tyrosinase-related protein 2
(TRP-2) [ ]. Among them, tyrosinase is regarded as the rate-limiting enzyme of process, which
5]. The melanin biosynthesis is regulated by enzymatic
6
modulates this process by catalyzing the hydroxylation of tyrosine into 3,4-dihydroxyphenylalanine
(DOPA) and the further oxidation of DOPA into dopaquinone [7,8].
Int. J. Mol. Sci. 2018, 19, 746; doi:10.3390/ijms19030746
www.mdpi.com/journal/ijms

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Several signaling pathways are presented to clarify the specific mechanism controlling melanin
biosynthesis via tyrosinase family, as described in Figure 1 [ 10]. MITF, a master regulator of
9
,
melanogenesis that is involved in these pathways, upregulates the melanogenesis enzymes TYR,
TRP-1 and TRP-2 via binding to the M-box motif in their promoter regions [11]. In addition, MITF
modulates melanocyte function including melanocyte differentiation, pigmentation, proliferation,
and cell survival [12].
Figure 1. Regulation of melanogenesis through different signaling pathways [10].
α-MSH
(
α
-melanocyte-stimulating hormone), SCF (stem cell factor), AC (adenylate cyclase), MC1R
(melanocortin 1 receptor), cAMP (cyclic Adenosine monophosphate), PKA (protein kinase A),
MAPK (mitogen-activated protein kinase), GSK (glycogen synthase kinase), JNK (c-Jun N-terminal
kinase),ERK (extracellular regulated protein kinases), TYR (tyrosinase), TRP1 (Tyrosinase-related
protein 1), TRP2 (Tyrosinase-related protein 2) CREB (cAMP-response element binding protein), CRE
(cAMP response element), MITF (microphthalmia-associated transcription factor), TCF-LEF (T cell
factor/lymphoid-enhancing factor-1).
The extract of Ficuscarica L. and Psoralen corylifolia L. (Figure 2) [13
are popular Uygur medicines that are used for vitiligo in Xinjiang and other Central Asian countries
hundreds of years ago [15 16]. During the last century, several furocoumarins (psoralens), such as
8-methoxypsoralen (8-MOP), 5-methoxypsoralen (5-MOP), and 4,5,8-trimethylpsoralen (TMP) were
isolated from the plants or totally synthesized [17 18]. These compounds were proved to show
strong photosensitivity [19] later, which may be used for the treatment of vitiligo with subsequent
exposure to long-waved ultraviolet radiation [20 21]. Although PUVA (psoralens + UVA) (ultraviolet
radiation A) [22] was accompanied with some undesired side effects [23 25], the therapy is still the
,14] alone or in combination
,
,
,
–
most successful (or less disappointing) one for skin repigmentation today. Unfortunately, the precise
mechanism of PUVA in the treatment of vitiligo is obscure, let al.one the target of these drugs.

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Figure 2. The structures of furocoumarins (psoralens) isolated from the plant Ficuscarica L. and Psoralen
corylifolia L. or synthesized [13]. 5-MOP (5-methoxypsoralan), TMP (trimethylpsoralen).
However, few furocoumarin derivatives and analogues with potential anti-vitiligo activity were
reported in spite of their high efficiency against the disease. Our group had been dedicated on the drug
development of the vitiligo for many years [26–31]. In our previous research, a great augumentation
to the melanin synthesis was observed when aromatic groups were introduced to C-5 position of the
furocoumarin derivative, which suggested that more structural modification should be pursued on
this position to search for novel bioactive molecules on pigmentation that may be developed as better
medication for the vitiligo.
Therefore, sixteen ester derivatives (8a–8p) of furocoumarin were prepared, then submitted to
the activity assay of melanogenesis in B16 cells and the SAR was summarized as well. Furthermore,
the tyrosinase activity and expression of proteins related to melanin biosynthesis were determined by
Western blot analysis in cells treated with the most promising derivative (8n) for understanding the
mechanism underlying the observed effect.
2. Results and Discussion
2.1. Synthesis
The synthetic route of the target compounds was described in Scheme 1. The intermediate
1
(4-methylumbelliferone) prepared from resorcinol via Pechmann reaction [32 33], was converted to
,
compound
Compound
2
by Williamson reaction refluxing with chloroethanol in the presence of anhydrous K2CO3.
◦
2
was further oxidized to aldehyde
3
at
−
78 C by an optimized Swern oxidation in
excellent yields. Intermolecular cycloaddition of the intermediate
3 yielded compound 4a in the
presence of 1M NaOH. It is notable that a mixed solvent (Vwater:V1,4-dioxane = 1:1) was applied to get a
homogeneous solution of aldehyde 3 for the easy cycloaddition.
Scheme 1. Synthetic route for the furocoumarin derivatives (i) ethyl acetoacetate, H2SO4, 60◦C;
(ii) chloroethanol, K2CO3, acetone, reflux; (iii)
−
78◦C, oxalyl chloride, DMSO (dimethyl sulfoxide),
triethylamine, DCM (dichloromethane); (iv) 1M NaOH aqueous solution, 1,4-dioxane; (v) SeO2, xylene,
reflux; and, (vi) NaBH4, ethanol, rt; (vii) pyridine, Ac2O, rt. (vii) DCC (dicyclohexylcarbodiimide),
◦
DMAP (4-dimethylaminopyridine), DCM, 0 C-rt (room temperature).

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Selenium dioxide was applied in selective oxidation of compound 4a to produce aldehyde
Compound was reduced with NaBH4 to achieve alcohol 6 [34,35], which was further esterified to
give 7 and 8a–8p under different conditions, respectively [32,36].
The oxidation of intermediate to was the crucial step in the preparation of compounds 4a
which bearing no substituent group on the furan ring. With the application of DMSO-(COCl)2 oxidation
system, compound was smoothly transformed to aldehyde in almost quantitative yield as we did
in our previous work. In the cyclization of intermediate , two sets of compounds (psoralen: 4a and
5.
5
2
3
,
2
3
3
angelicin: 4b) were produced in one step, which was isomer to each other (Figure 3). The yield of 4a in
the mixture was much higher than angelicin (4b) under basic condition.
Figure 3. Structure of psoralen 4a and angelicin 4b.
2.2. Melanin Synthesis Evaluation of 8a–8p
All of the synthesized compounds were screened for their activity on melanin synthesis in murine
B16 cells, with a known method (Figure 4) [37]. In order to avoid the possibility that inhibition of
melanin synthesis was due to cytotoxicity, we performed CCK-8 assay as well to determine whether
these active ones were cytotoxic to B16 cells. The result showed that the cells treated with compounds
for 48 h caused little cytotoxicity when compared with the control at the dosage of 50 µM (Figure 5).
Figure 4. Stimulation of melanin content of B16 cells by furocoumarin derivatives. N means negative
control; P means positive control (8-MOP); The B16 cells were treated with 50
µM of different
furocoumarin derivatives for 48 h. After that, melanin content was measured directly. Values are
expressed as the mean ± SD of three separate experiments.
According to the result (Figure 4), half of the tested compounds (7, 8a–8d, 8l, 8n–8o) exhibited a
stronger activity than the positive control (8-MOP) with a value from 182% to 200%. For compounds
6
and 7, further esterification of hydroxymethyl with acetic anhydride caused an increase in activity.
The activity of ester derivatives, was which substituted by halogens, decreased dramatically
8j 8p) as compared with 8k, especially for the ones bearing F and I, regardless of the number and
position of halogens on benzene. Compounds with -CH3 (8a 8b) and -OCH3 (8c 8d) on ortho- and para-
(8e– ,
–
–

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position of benzene showed a much higher activity than the halogenated ones, which was consistent
with our previous findings [28]. The interesting thing was that the strong electron-withdrawing group
(EWG), such as -NO2, may be favorable to promote the melanin content, especially for 8n (200%) and
8o (197%), which demonstrated the best activity of these derivatives. It seemed that this effect had no
relationship with the position and number of the -NO2 on benzene.
Figure 5. Effect of furocoumarin derivatives on B16 cells viability. N means negative control; The B16
cells were incubated with 50 µM of different furocoumarin derivatives for 48 h and the cell viability
was assayed by adding CCK-8 (Cell Counting Kit) solution. Values are expressed as the mean
three separate experiments.
±
SD of
Overall, both EDG (electron-donating group) (-CH3, -OCH3) and EWG (-NO2) on benzene
greatly improved the melanin biosynthesis, which indicated that the electrostatic interaction between
derivatives and receptor protein may have little correlation with activity.
2.3. Effect of 8n on B16 Melanoma Cells Viability
The murine melanoma B16 cells were treated with 8n at concentrations of 0–50 µM for 24 h
and examined the cytotoxicity by CCK-8 assay. As shown in Figure 6A(a–d), compound 8n did not
induce any change in cell morphology when compared with untreated cells and showed no increase in
cytotoxicity (Figure 6B). Accordingly, 0–50 µM 8n was applied in the subsequent experiments.
Figure 6.
sulfoxide) as vehicle (
under a microscope. Magnification,
were treated with 50 M 8-MOP as post control and with 8n at concentrations of 0–50
viability was measured by Cell Counting Kit-8 assays. The data are shown as the mean ± SD; n = 3.
(A(a–
d
)) Effects of 8n on cell morphology. B16 cells were treated with 0.1% DMSO (dimethyl
) or with 8n at 1 ( ), 10 ( ) and 50 M ( ) for 48 h. Cell morphology was observed
200. ( ) Effects of 8n on B16 melanoma cell viability. B16 cells
M for 48 h. Cell
a
b
c
B
µ
d
×
µ
µ

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2.4. Effect of 8n on Melanogenesis and Tyrosinase in B16 Melanoma Cells
The melanin production was measured in B16 melanoma cells after 48 h of treatment with
8n at 0–50 M. As shown in Figure 7A, treatment with 8n improved the melanin synthesis in a
dose-dependent manner, and the melanin content increased 63% compared with 8-MOP at 50 M.
(0 M, 100 15.4%; 1 M, 89.7 4.0%; 10 M, 125.9 3.7%; 50 M, 217.6 7.5%; 8-MOP, 50 M,
µ
µ
µ
±
µ
±
µ
±
µ
±
µ
133.5 ± 12.1%). After that, the tyrosinase activity in B16 cells was studied as well and it showed
a similar increasing trend in response to 8n treatment in Figure 7B. Specifically, when compared
with 8-MOP treated cells, treatment with compound at a concentration of 50
approximately 98% activation of intracellular tyrosinase in the B16 melanoma cells.(0
1 µM, 150.7 ± 8.7%; 10 µM, 192.4 ± 6.4%; 50 µM, 293.9 ± 4.3%; 8-MOP, 50 µM, 148.4 ± 12.9%).
µM resulted in an
µ
M, 100 9.2%;
±
Figure 7.
with 0.1% DMSO as vehicle or with 8n at 1, 10, 50
or 24 h to evaluate melanin content and tyrosinase activity, respectively. Each percentage value for
(A
,
B
) Effects of 8n on melanogenesis and tyrosinase in B16 melanoma cells. Cells were treated
µ
M, and 50 M 8-MOP as positive control for 48 h
µ
treated cells is reported relative to that of 0.1% DMSO cells. The data are shown as the mean
n = 3, * p < 0.05, *** p < 0.001 as compared with 0.1% DMSO cells.
±
SD;
2.5. Effects of 8n on TYR Family and MITF Expression Levels in B16 Melanoma Cells
In order to clarify the mechanism that is responsible for the elevation in pigmentation,
the expression levels of TYR family and MITF were examined by Western blot. B16 melanoma
cells were treated with various concentrations of 8n (0–50 µM) for 48 h. As shown in Figure 8,
the tyrosinase, TRP-1, and TRP-2 levels markedly increased following treatment with 8n. As the TYR
family was the downstream genes of MITF, the level of MITF was measured, and MITF expression
exhibited comparative changes. These results suggested that 8n induced melanin synthesis through
the up-regulation of the TYR family and MITF at protein levels.

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Figure 8. Representative western blots illustrating expression of TRPs and MITF. B16 cells were treated
with 8n at 0, 1, 10 and 50 M for 48 h. Tyrosinase, TRP1, TRP2, and MITF protein expression were
detected by Western blotting. Results were normalized against -actin expression; Densitometric
scanning of band intensities obtained from three individual experiments was used to quantify change
µ
β
of protein expression (control value taken as one-fold in each case). The Data are shown as mean
of three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. controls.
±
SD
2.6. Effect of 8n on Akt and Wnt/β-Catenin Signaling Pathways Involved Inmelanogenesis
The Wnt signaling pathway was demonstrated as one of the signaling processes in melanin
synthesis [38 39]. In order to determine the molecular mechanism of the superior pigmented effect
of 8n, Western blot analysis for the changes of -catenin was introduced. As shown in Figure 9A,
the phosphorylation of -catenin at Ser33, decreased with a concomitant increase of total -catenin
induced by 8n. Moreover, -catenin content in the nucleus (the active -catenin) enhanced obviously
,
β
β
β
β
β
after 12 h of 8n treatment as compared with untreated cells, while the content in the cytoplasm did not
change (Figure 9B).

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Figure 9. Effect of 8n on Akt and Wnt/
β
-catenin signaling pathways involved in melanogenesis.
M for 48 h, the expression of Akt, GSK3 and
) B16 cells were treated with 8n of 50 M for 12 h, the
β-catenin in cytoplasm and nucleus were detected by using
(A
) B16 cells were treated with 8n at the 0, 1, 10, and 50
µ
β
β-catenin were measured by Western blot. (
B
µ
expression levels of proteins including
Western blot; Densitometric scanning of band intensities obtained from three individual experiments
was used to quantify change of protein expression (control value taken as one-fold in each case).
The Data are shown as mean
vs. controls.
±
SD of three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001
GSK3
-catenin, which contributes to the degradation of
the level of phosphorylation of GSK3 rose up in B16 cells treated with 8n (Figure 9A). BIO
(6-bromoindirubin-3-oxime) was able to inhibit the phosphorylation of GSK3 and regarded as
β, which is a negative regulator of Wnt signaling, could phosphorylate the residues of
β
β-catenin through proteasome [40]. As expected,
β
β

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a selective GSK3
-catenin content induced by 8n (Figure 10A). Being consistent with this, it also improved the TYR
activity and melanin content in B16 cells in the presence of 8n evidently (Figure 10B,C).
β inhibitor [41]. The graph revealed that the BIO increased the accumulation of
β
Figure 10.
with 8n (50
B16 cells for 2 h before addition of 8n at 50
(
A
µ
)
β
-catenin was detected in B16 cells pre-treated with or without BIO (5
µM) and incubated
M) or not. ( ) Inhibitors (BIO 5 M, Akt inhibitor IV 1 M) were pre-incubated with
B–
E
µ
µ
µM, followed by an additional incubation for 24 h or 48 h
for tyrosinase activity and melanin content, respectively. * p < 0.05compared with control; # p < 0.05,
##
p < 0.01 as compared with 8n stimulation. “-” means pre-treated without 8n or Bio or Akt; “+” means
pre-treated with 8n or Bio or Akt.
Currently, there are numerous reports about the close relationship between PI3K/Akt
(phosphatidylinositide 3-kinases/protein kinase B) signal pathway and GSK3
β [42–46]. Activated
Akt can phosphorylate GSK3 resulting in inactivating GSK3 and inhibit the degradation
β
β

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of
β-catenin [47]. Our data indicated that content of p-Akt and p-GSK3
β
increased in B16
cells in the presence of 8n after 48 h (Figure 9A). However, co-treatment with Akt inhibitor
IV (5-(2-benzothiazolyl)-3-ethyl-2-[2-(methylphenylamino)ethenyl]-1-phenyl-1H-benzimidazolium
iodide, a selective inhibitor of Akt that decreased the phosphorylation level of Akt and inhibited
PI3K/Akt signal pathway [48]) distinctly aborted 8n-mediated tyrosinase activity and melanin content
(Figure 10D,E).
These results suggested that 8n exerts a pigmented effect through the activation Wnt/β-catenin
signaling pathway by regulating Akt signal molecule.
The results of the present study indicated that 50
µM of 8n was more effective than the
melanogenesis agent (8-MOP) since it increased melanin content (Figure 7A) and tyrosinase activity
better than the latter (Figure 7B). In addition, the compound significantly up-regulated the level of
MITF, and its related proteins, including TYR, TRP1, and TRP2, in a dose-dependent manner. Taken
together, these findings suggested that 8n was an effective tyrosinase activator to promote the melanin
production in B16 cells.
According to the literature, the Wnt signalling pathway played a pivotal role in melanogenesis.
By phosphatidylinositol-3-kinase (PI3K)/Akt activation and GSK3
to the M-box of the tyrosinase promoter, thereby up-regulated melanogenic protein expression and
induced melanogenesis [49 51]. Western blot analysis proved 8n could activate the phosphorylation of
Akt and GSK-3 , indicating that it raised MITF transactivation of tyrosinase through GSK-3 , which
β phosphorylation, MITF binded
–
β
β
was similarly as mentioned in previous study [52]. Moreover, it was found that pretreatment with
BIO enhanced 8n-induced melanin production and tyrosinase activation (Figure 10B,C) when the
up-regulation of melanin synthesis by the compound regulated by Akt/GSK3β/β-catenin signaling
was confirmed. However, co-treatment with Akt inhibitor IV significantly reversed these results
(Figure 10D,E). Thus, Melanin production mediated by compound 8n was probably triggered through
both Akt and Wnt pathways, consistent with reports revealing that activation of Akt/GSK3
influenced melanin production in B16 melanoma cells [53].
β/β-catenin
3. Materials and Methods
3.1. Chemistry
Reagents and solvents were purchased from Sigma (Shanghai, China), and used without further
purification. Thin-layer chromatography (TLC) was carried out on glass plates coated with silica gel
(Qingdao Haiyang Chemical Co., Qingdao, China, G60F-254) and visualized by UV light (254 nm).
The products were purified by column chromatography over silica gel (Qingdao Haiyang Chemical
Co., 200–300 mesh). Melting points were determined on a Buchi B-540 apparatus and uncorrected. All
of the NMR (nuclear magnetic resonance) spectra were recorded with a Varian400, 600 MHz NMR
spectrometer in CDCl3, using TMS (tetramethylsilane) as an internal standard. High-resolution mass
spectra (HRMS) were recorded on AB SCIEX QSTAR Elite quadrupole time-of-flight mass spectrometry.
The IR (infrared spectroscopy) data were recorded on a Thermo Fisher Scientific Nilolet 6700 FT-IR
infrared spectrometer (KBr).
3.1.1. Preparation of 4-Methylumbelliferone (1)
To an ice-cold solution of resorcinol (2.0 g, 18.2 mol) in dioxane, conc. H2SO4 (0.5 mL) was added
dropwise under 20 ◦C. After the addition of conc. H2SO4, ethyl acetoacetate (2.8 g, 21.8 mmol) was
◦
added, and the mixture was heated to 60 C for 4 h. Then, the mixture was poured into cold water, and
the precipitate was filtered and dried under reduced pressure. The resulting mixture was recrystallized
from methanol to give compound as white needle crystals. Yield 92%, m.p. 202–204 ◦C.

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3.1.2. Preparation of 7-(2-Hydroxyethoxy)-4-methyl-2H-chromen-2-one (2)
A mixture of 1 (0.88 g, 5.0 mmol) with chloroethanol (0.60 g, 7.5 mmol) and anhydrous K2CO3
(1.4 g, 10 mmol) in acetone (50 mL) was refluxed under stirring for 4 h. After cooling, the reaction
mixture was filtered, and the filtrate was evaporated under reduced pressure. The obtained residue
was purified by silica gel chromatography with petroleumether/ethylacetate or chloroform to give
◦
1
intermediate 3. Yield 95%, white solid, m.p. 108–110 C; H NMR (400 MHz, CDCl3) δ7.51 (d,
J = 9.0 Hz, 1H), 6.91–6.83 (m, 2H),6.15 (d, J = 1.1 Hz, 1H), 4.15 (t, J = 8.7 Hz, 2H), 4.01 (m, 2H), 2.40 (d,
J = 1.1 Hz, 3H).
3.1.3. Preparation of 7-(2-Oxoethoxy)-4-methyl-2H-chromen-2-one (3)
◦
A solution of oxalyl chloride (0.252 g, 2 mmol) in anhydrous CH2Cl2 (5 mL) was cooled to
−
78 C
under a nitrogen atmosphere, and a solution of DMSO (0.156 g, 2 mmol) in CH2Cl2 (2 mL) was added
dropwise (the temperature was kept below
−
65 ◦C), and stirred for 10 min. A solu◦tion of alcohols
2
(0.22 g, 1 mmol) in CH2Cl2 (15 mL) was added dropwise at a temperature below –65 C and stirred for
another 30 min. Triethylamine (0.505 g, 5 mmol) was then added dropwise. The reaction mixture was
stirred for 15 min at a temperature below
reaction mixture was diluted with CH2Cl2 and was filtered through a pad of silica gel. Removal of the
solvent under vacuum afforded the aldehydes
. Yield 97%, white solid, m.p. 140–142 ◦C; 1H NMR
(400 MHz, CDCl3) 9.85 (s, 1H), 7.53 (d, J = 8.8 Hz, 1H), 6.89 (dd, J = 8.8, 2.6 Hz, 1H), 6.78 (d, J = 2.5 Hz,
−
65 ◦C and allowed to warm up to room temperature. The
3
δ
1H), 6.15 (d, J = 1.0 Hz, 1H), 4.68 (s, 2H), 2.39 (d, J = 0.9 Hz, 3H).
3.1.4. Preparation of Compounds 4
A solution of aldehydes
3 (0.5 mmol) in H2O-dioxane (1:1, 3 mL) was added dropwise to a
refluxing aqueous solution of NaOH (1.0 M, 10 mL) under stirring for more than 30 min. The resulted
mixture was stirred at reflux for an additional 4 h and then cooled down to room temperature. The
solution was acidified to pH = 3 with 85% phosphoric acid and was left overnight. The resultant
mixture was extracted with ethylacetatethree times. The organic phase was then washed with brine
and dried over anhydrous Na2SO4. After removal of the solvents, the residue was purified by flash
chromatography on silica gel eluted with Vpetroleumether:Vethylacetate = 1:10 to afford a pair of isomer.
◦
1
5-Methyl-7H-furo[3,2-g]chromen-7-one (4a). Yield 73%, white solid, m.p. 160–162 C; H NMR
(400 MHz, CDCl3) 7.81 (s, 1H), 7.69 (d, J = 2.2 Hz, 1H), 7.47 (s, 1H), 6.85 (d, J = 2.1 Hz, 1H), 6.27 (s,
δ
1H), 2.50 (s, 3H). 13C NMR (101 MHz, CDCl3)
δ 161.24, 156.43, 152.78, 151.75, 146.96, 124.77, 116.70,
113.62, 106.67, 100.00, 19.30; IR (KBr) v: 2921, 1723, 1630, 1385, 1137, 1031, 928 cm−1; HRMS (ESI) calcd
for C12H9O3[M + H]+ 201.0552, found 201.0540.
◦
1
4-Methyl-2H-furo[2,3-h]chromen-2-one (4b). Yield 11%, white solid, m.p. 119–120 C; H NMR
(400 MHz, CDCl3) 7.69 (d, J = 2.2 Hz, 1H), 7.53 (d, J = 8.8 Hz, 1H), 7.45 (d, J = 8.7 Hz, 1H), 7.15 (d,
δ
J = 2.1 Hz, 1H), 6.28 (s, 1H), 2.51 (s, 3H). 13C NMR (101 MHz, CDCl3)
δ 160.89, 157.25, 153.58, 145.76,
120.48, 117.00, 112.86, 110.02, 108.41, 104.37, 19.44; IR (KBr) v: 2919, 1718, 1617, 1264, 1065, 761 cm−1
;
HRMS (ESI) calcd for C12H9O3 [M + H]+ 201.0552, found 201.0559.
3.1.5. Preparation of 7-Oxo-7H-furo[3,2-g]chromene-5-carbaldehyde (5)
Powdered SeO2 (3.33 g, 30 mmol) was added to a solution of 4a (8.0 g, 20 mmol) in 20 mL
of hot dry xylene and the mixture were refluxed for 12 h with vigorous stirring underthenitrogen.
The reaction mixture was filtered to remove black Se, and the deep orange filtrate was allowed to stand
overnight. Almost pure crystals of
5 could be separated from the solution. Yield 63%, yellow solid,
m.p. 178–180 ◦C; 1H NMR (400 MHz, CDCl3)
δ 10.14 (s, 1H), 8.90 (s, 1H), 7.72 (d, J = 2.1 Hz, 1H), 7.54
(s, 1H), 6.89 (d, J = 2.0 Hz, 1H), 6.86 (s, 1H).

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3.1.6. Preparation of 5-(Hydroxymethyl)-7H-furo[3,2-g]chromen-7-one (6)
Compound
5 (4.28g, 20 mmol) was dissolved in ethanol (130 mL), sodium borohydride (380 mg,
10.0 mmol) was added, and the solution was stirred for 2 h at room temperature. Thereafter the
suspension was carefully hydrolyzed with 1M HCl (20 mL), diluted with H2O and extracted three
times with CH2Cl2. The organic phase was washed with brine, dried over Na2SO4, and then evaporated
under reduced pressure. The residue was purified by flash chromatography on silica gel eluted with
◦
1
V
chloroform:Vmethanol = 30:1 to afford alcohol 7. Yield 76%, white solid, m.p. 204–206 C; H NMR
(400 MHz, CDCl3) 1H NMR (600 MHz, CD3OD)
δ 7.95 (s, 1H), 7.87 (d, J = 1.9 Hz, 1H), 7.55 (s, 1H),
6.97 (d, J = 2.0 Hz, 1H), 6.56 (s, 1H), 4.94 (s, 2H). 13C NMR (101 MHz, CD3OD)
δ 163.57, 158.32, 152.82,
148.71, 129.23, 127.39, 117.21, 115.30, 110.23, 107.75, 100.51, 61.09.
3.1.7. Preparation of (7-Oxo-7H-furo[3,2-g]chromen-5-yl)methyl acetate (7)
A mixture of 7 (0.022 g, 0.09 mmol), 4-dimethylaminopyridine DMAP (1 mg), and one drop of
acetic anhydride in 2 mL of anhydrous pyridine was stirred at room temperature overnight under
nitrogen atmosphere, 5 mL of icy water was then added. The reaction mixture was extracted with ethyl
acetate three times, and the organic layer was washed with brine and dried over anhydrous Na2SO4.
After the removal of solvent, the crude product was purified by flash column chromatography over
silica gel eluted with Vpetroleumether/Vethylacetate = 3:1 to give acetate
7. Yield 80%, white solid, m.p.
198–200 ◦C; 1H NMR (400 MHz, CDCl3)
δ
7.82 (s, 1H), 7.71 (d, J = 2.1 Hz, 1H), 7.47 (s, 1H), 6.86 (d,
J = 2.0 Hz, 1H), 6.49 (s, 1H), 5.37 (s, 2H), 2.23 (s, 3H).
3.1.8. General Procedure Of preparation of Esters 8a–8p
1,3-Dicyclohexylcarbodiimide (DCC, 0.82 g, 4 mmol) was added to a solution of 7 (0.86 g, 4 mmol),
different benzoic acid (5 mmol) and DMAP (0.49 g, 4 mmol) in dry CH2Cl2 (25 mL) at 0 ◦C under
nitrogen. After 10 min at 0 ◦C, the mixture was stirred at room temperature for 12 h. After filtration,
the organic filtrate was washed with 1.2 M hydrochloric acid, saturated aqueous sodium hydrogen
carbonate, then dried over sodium sulfate and evaporated under reduced pressure. The residue
was purified by flash chromatography on silica gel eluted with petroleum etherethyl acetate to give
corresponding benzoate 8a–8p.
(7-Oxo-7H-furo[3,2-g]chromen-5-yl)methyl-4-methylbenzoate (8a). Yield 81%, white solid, m.p.
181–182 ◦C; 1H NMR (600 MHz, CDCl3)
δ
8.02 (d, J = 8.1 Hz, 2H), 7.81 (s, 1H), 7.71 (d, J = 2.1 Hz, 1H),
7.53 (s, 1H), 7.29 (d, J = 8.0 Hz, 2H), 6.86 (d, J = 2.1 Hz, 1H), 6.60 (s, 1H), 5.60 (s, 2H), 2.44 (s, 3H). 13
NMR (101 MHz, CDCl3) 165.77, 160.75, 156.37, 151.71, 149.62, 147.14, 144.67, 129.89, 129.40, 126.23,
C
δ
124.94, 115.46, 113.68, 111.57, 106.57, 100.37, 61.65, 21.76; IR (KBr) v: 2925, 1717, 1634, 1576, 1447, 1278,
1264, 1156, 1135, 874, 750 cm−1; HRMS (ESI) calcd for C20H15O5[M + H]+ 335.0914, found 335.0931.
(7-Oxo-7H-furo[3,2-g]chromen-5-yl)methyl-2-methylbenzoate (8b). Yield 84%, white solid, m.p.
169–171 ◦C; 1H NMR (400 MHz, CDCl3)
δ
8.04 (dd, J = 7.8, 0.8 Hz, 1H), 7.83 (s, 1H), 7.72 (d, J = 2.1 Hz
1H), 7.54 (s, 1H), 7.50–7.44 (m, 1H), 7.33–7.28 (m, 2H), 6.87 (d, J = 2.2 Hz, 1H), 6.59 (s, 1H), 5.60 (s,
2H), 2.65 (s, 3H). 13C NMR (101 MHz, CDCl3)
166.28, 160.68, 156.35, 151.71, 149.55, 147.11, 141.04,
,
δ
132.82, 132.00, 130.76, 125.99, 124.91, 115.45, 113.69, 111.74, 106.53, 100.36, 61.61, 21.88; IR (KBr) v: 2926,
1723, 1632, 1577, 1449, 1278, 1263, 1155,1135, 873, 749 cm−1; HRMS (ESI) calcd for C20H15O5[M + H]+
335.0914, found 335.0944.
(7-Oxo-7H-furo[3,2-g]chromen-5-yl)methyl-4-methoxybenzoate (8c). Yield 80%, white solid, m.p.
155–157 ◦C; 1H NMR (400 MHz, CDCl3)
δ
8.09 (d, J = 8.8 Hz, 2H), 7.82 (s, 1H), 7.71 (d, J = 2.1 Hz, 1H),
7.54 (s, 1H), 6.97 (d, J = 8.9 Hz, 2H), 6.86 (d, J = 2.0 Hz, 1H), 6.59 (s, 1H), 5.60 (s, 2H), 3.89 (s, 3H). 13
NMR (101 MHz, CDCl3) 165.56, 160.91, 156.52, 151.87, 149.90, 147.28, 132.12, 129.20, 125.08, 121.44,
C
δ
115.62, 114.11, 113.91, 111.67, 106.72, 100.51, 61.68, 55.69; IR (KBr) v: 2927, 1724, 1634, 1575, 1448, 1277,
1265, 1156, 1134, 872, 748 cm−1; HRMS (ESI) calcd for C20H15O6[M + H]+ 351.0863, found 351.0855.

Int. J. Mol. Sci. 2018, 19, 746
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(7-Oxo-7H-furo[3,2-g]chromen-5-yl)methyl-2-methoxybenzoate (8d). Yield 82%, white solid, m.p.
148–149 ◦C; 1H NMR (400 MHz, CDCl3)
7.92 (dd, J = 7.9, 1.8 Hz, 1H), 7.82 (s, 1H), 7.71 (d, J = 2.2 Hz
1H), 7.57–7.50 (m, 1H), 7.06–7.00 (m, 2H), 6.86 (d, J = 2.2 Hz, 1H), 6.73 (s, 1H), 5.60 (s, 2H), 3.95 (s,
3H). 13C NMR (101 MHz, CDCl3)
165.56, 160.86, 159.54, 156.33, 151.72, 149.65, 147.10, 134.49, 132.20,
δ
,
δ
124.87, 120.35, 118.61, 115.50, 113.73, 112.11, 111.79, 106.57, 100.30, 61.78, 55.94; IR (KBr) v: 2927, 1718,
1633, 1576, 1450, 1279, 1264, 1157, 1136, 873, 760, 750 cm−1; HRMS (ESI) calcd for C20H15O6[M + H]+
351.0863, found 351.0839.
(7-Oxo-7H-furo[3,2-g]chromen-5-yl)methyl-4-chlorobenzoate (8e). Yield 78%, light yellow solid, m.p.
◦
201–202 C; 1H NMR (400 MHz, CDCl3)
δ
8.07 (d, J = 8.6 Hz, 2H), 7.80 (s, 1H), 7.72 (d, J = 2.3 Hz,
1H), 7.55 (s, 1H), 7.48 (d, J = 8.6 Hz, 2H), 6.87 (d, J = 2.1 Hz, 1H), 6.57 (s, 1H), 5.62 (s, 2H). 13C NMR
(101 MHz, CDCl3) 164.89, 160.63, 156.42, 151.72, 149.20, 147.22, 140.40, 131.23, 129.10, 127.41, 125.00,
δ
115.41, 113.58, 111.72, 106.56, 100.46, 62.03; IR (KBr) v: 1717, 1624, 1574, 1541, 1457, 1270, 1243, 1129,
1090, 669 cm−1; HRMS (ESI) calcd for C19H12ClO5[M + H]+ 355.0368, found 355.0390.
(7-Oxo-7H-furo[3,2-g]chromen-5-yl)methyl-2-chlorobenzoate (8f). Yield 75%, light yellow solid, m.p.
◦
183–185 C; 1H NMR (400 MHz, CDCl3)
δ
7.95 (dd, J = 7.6, 1.3 Hz, 1H), 7.82 (s, 1H), 7.72 (d, J = 2.3 Hz
1H), 7.54 (s, 1H), 7.53–7.48 (m, 2H), 7.41–7.35 (m, 1H), 6.87 (d, J = 2.2 Hz, 1H), 6.64 (s, 1H), 5.63 (s,
2H). 13C NMR (101 MHz, CDCl3)
164.92, 160.79, 156.55, 151.89, 149.04, 147.33, 134.37, 133.55, 131.94,
,
δ
131.60, 128.88, 127.01, 125.11, 115.68, 113.74, 112.24, 106.71, 100.56, 62.57; IR (KBr) v: 1734, 1707, 1635,
1577, 1450, 1412, 1240, 1156, 1113, 1028, 746 cm−1; HRMS (ESI) calcd for C19H12ClO5[M + H]+ 355.0368,
found 355.0387.
(7-Oxo-7H-furo[3,2-g]chromen-5-yl)methyl-3,4-dichlorobenzoate (8g). Yield 70%, light yellow solid,
m.p. 198–200 ◦C; 1H NMR (400 MHz, CDCl3)
δ
8.20 (d, J = 2.0 Hz, 1H), 7.95 (dd, J = 8.4, 1.9 Hz, 1H),
7.80 (s, 1H), 7.73 (d, J = 2.3 Hz, 1H), 7.59 (d, J = 8.4 Hz, 1H), 7.55 (s, 1H), 6.87 (d, J = 2.1 Hz, 1H), 6.55 (s,
1H), 5.63 (s, 2H). 13C NMR (101 MHz, CDCl3)
163.97, 160.54, 158.21, 153.31, 147.28, 143.32, 138.63,
δ
136.04, 131.73, 130.90, 128.83, 127.59, 122.64, 120.23, 115.41, 111.86, 106.56, 100.50, 62.34; IR (KBr) v: 1716,
1633, 1620, 1568, 1513, 1447, 1361, 1250, 1089, 748 cm−1; HRMS (ESI) calcd for C19H11Cl2O5[M + H]+
388.9978, found 388.9955.
(7-Oxo-7H-furo[3,2-g]chromen-5-yl)methyl-3,5-dichlorobenzoate (8h). Yield 71%, light yellow solid,
m.p. 223–224 ◦C; 1H NMR (400 MHz, CDCl3)
δ
7.98 (d, J = 1.9 Hz, 2H), 7.79 (s, 1H), 7.73 (d, J = 2.2 Hz
1H), 7.62 (t, J = 1.9 Hz, 1H), 7.55 (s, 1H), 6.87 (d, J = 2.1 Hz, 1H), 6.54 (s, 1H), 5.63 (s, 2H). 13C NMR
(101 MHz, CDCl3) 163.67, 160.63, 156.59, 151.86, 148.84, 147.41, 135.83, 133.77, 131.88, 128.96, 128.32,
,
δ
125.18, 115.52, 112.04, 106.68, 100.63, 62.64; IR (KBr) v: 1718, 1636, 1571, 1451, 1261, 1156, 1130, 870, 748
cm−1; HRMS (ESI) calcd for C19H11Cl2O5[M + H]+ 388.9978, found 388.9967.
(7-Oxo-7H-furo[3,2-g]chromen-5-yl)methyl-4-fluorobenzoate (8i). Yield 83%, off-white solid, m.p.
177–178 ◦C; 1H NMR (400 MHz, CDCl3) δ 8.19–8.12 (m, 2H), 7.81 (s, 1H), 7.72 (d, J = 2.2 Hz, 1H), 7.54
(s, 1H), 7.18 (t, J = 8.6 Hz, 2H), 6.87 (d, J = 2.1 Hz, 1H), 6.57 (s, 1H), 5.62 (s, 2H). 13C NMR (101 MHz,
CDCl3)
δ 164.87, 160.78, 156.54, 151.85, 149.47, 147.35, 146.81, 132.62, 125.13, 116.20, 115.98, 115.56,
111.78, 106.69, 100.56, 62.04; IR (KBr) v: 1716, 1604, 1508, 1456, 1265, 1237, 1154, 849, 743 cm−1; HRMS
(ESI) calcd for C19H12FO5[M + H]+ 339.0663, found 339.0689.
(7-Oxo-7H-furo[3,2-g]chromen-5-yl)methyl-2,4-difluorobenzoate (8j). Yield 77%, off-white solid, m.p.
185–186 ◦C; 1H NMR (400 MHz, CDCl3)
δ
8.08 (td, J = 8.5, 6.5 Hz, 1H), 7.81 (s, 1H), 7.72 (d, J = 2.2 Hz
1H), 7.54 (s, 1H), 7.04–6.90 (m, 2H), 6.87 (d, J = 2.0 Hz, 1H), 6.63 (s, 1H), 5.63 (s 2H). 13C NMR (101 MHz,
CDCl3) 165.18, 164.55, 162.94, 160.79, 156.54, 151.85, 149.00, 147.33, 134.41, 125.10, 115.56, 113.67,
,
δ
112.34, 112.12, 111.97, 106.70, 105.76, 100.56, 62.40; IR (KBr) v: 1718, 1615, 1507, 1452, 1259, 1125, 1090,
852, 747 cm−1; HRMS (ESI) calcd for C19H11F2O5[M + H]+ 357.0569, found 339.0545.
◦
(7-Oxo-7H-furo[3,2-g]chromen-5-yl)methyl-benzoate (8k). Yield 85%, off-white solid, m.p. 213–215 C;
1H NMR (400 MHz, CDCl3)
δ 8.16-8.11 (m, 2H), 7.82 (s, 1H), 7.72 (d, J = 2.2 Hz, 1H), 7.64 (t, J = 7.4 Hz,

Int. J. Mol. Sci. 2018, 19, 746
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1H), 7.56–7.47 (m, 3H), 6.87 (d, J = 2.2 Hz, 1H), 6.61 (s, 1H), 5.63 (s, 2H). 13C NMR (101 MHz, CDCl) δ
165.87 160.85, 156.54, 151.88, 149.61, 147.32, 133.94, 131.04, 130.01, 128.85, 125.11, 115.60, 113.81, 111.79,
106.72, 100.55, 61.96; IR (KBr) v: 1717, 1635, 1506, 1451, 1268, 1158, 1116, 1094, 847, 745 cm−1; HRMS
(ESI) calcd for C19H13O5[M + H]+ 321.0757, found 321.0760.
(7-Oxo-7H-furo[3,2-g]chromen-5-yl)methyl-4-nitrobenzoate (8l). Yield 70%, off-white solid, m.p.
◦
1
201–203 C; H NMR (400 MHz, CDCl3)
δ
8.38–8.26 (m, 5H), 7.81 (s, 1H), 7.73 (d, J = 2.3 Hz, 1H),
164.07,
7.56 (s, 1H), 6.87 (d, J = 2.1 Hz, 1H), 6.57 (s, 1H), 5.68 (s, 2H). 13C NMR (101 MHz, CDCl3)
δ
156.71, 151.89, 149.63, 148.77, 147.48, 135.63, 131.18, 129.03, 126.84, 124.01, 115.49, 114.44, 112.12, 106.68,
100.72, 62.78; IR (KBr) v: 1720, 1624, 1577, 1527, 1451, 1270, 1244, 1089 cm−1; HRMS (ESI) calcd for
C19H12NO7[M + H]+ 366.0608, found 366.0586.
(7-Oxo-7H-furo[3,2-g]chromen-5-yl)methyl-4-(trifluoromethyl)benzoate (8m). Yield 66%, off-white
◦
solid, m.p. 219–220 C; 1H NMR (400 MHz, CDCl3)
δ
8.25 (d, J = 8.0 Hz, 2H), 7.81 (s, 1H), 7.77 (d,
J = 8.3 Hz, 2H), 7.73 (d, J = 2.3 Hz, 1H), 7.56 (s, 1H), 6.87 (d, J = 2.2 Hz, 1H), 6.58 (s, 1H), 5.66 (s, 2H). 13
NMR (101 MHz, CDCl3) 166.84, 160.71, 156.60, 151.87, 149.06, 147.42, 137.47, 130.44, 127.12, 125.94,
C
δ
125.18, 115.53, 112.03, 110.18, 106.70, 100.67, 62.48; IR (KBr) v: 1734, 1718, 1625, 1578, 1559, 1326, 1271,
1131 cm−1; HRMS (ESI) calcd for C20H12F3O5[M + H]+ 389.0631, found 389.0642.
(7-Oxo-7H-furo[3,2-g]chromen-5-yl)methyl-3-nitrobenzoate (8n). Yield 68%, off-white solid, m.p.
202–204 ◦C; 1H NMR (400 MHz, CDCl3)
δ
8.52–8.37 (m, 4H), 7.83 (s, 1H), 7.73 (d, J = 2.2 Hz, 1H), 7.56
(s, 1H), 6.88 (d, J = 2.1 Hz, 1H), 6.56 (s, 1H), 5.69 (s, 2H). 13C NMR (101 MHz, CDCl3)
δ
164.82, 162.22,
156.99, 156.59, 147.46, 142.58, 135.51, 133.05, 130.20, 129.53, 128.34, 125.05, 115.55, 112.20, 109.31, 106.70,
100.71, 62.77; IR (KBr) v: 1733, 1719, 1624, 1576, 1532, 1351, 1270, 1087 cm−1; HRMS (ESI) calcd for
C19H12NO7[M + H]+ 366.0608, found 366.0633.
(7-Oxo-7H-furo[3,2-g]chromen-5-yl)methyl-3,5-dinitrobenzoate (8o). Yield 52%, off-white solid, m.p.
◦
210–211 C; 1H NMR (400 MHz, CDCl3)
δ
9.29 (d, J = 1.9 Hz, 1H), 9.23 (d, J = 2.0 Hz, 1H), 7.83 (s,
1H), 7.74 (d, J = 2.2 Hz, 1H), 7.57 (s, 1H), 6.89 (d, J = 2.1 Hz, 1H), 6.53 (s, 1H), 5.75 (s, 2H). 13C NMR
(101 MHz, CDCl3) 165.43, 161.06, 156.74, 152.15, 148.91, 146.64, 144.84, 135.53, 132.04, 129.76, 128.71,
δ
124.84, 116.13, 112.15, 107.32, 100.31, 62.46; IR (KBr) v: 1718, 1623, 1575, 1540, 1436, 1311, 1243, 1089
cm−1; HRMS (ESI) calcd for C19H11N2O9[M + H]+ 411.0459, found 411.0476.
(7-Oxo-7H-furo[3,2-g]chromen-5-yl)methyl-2-iodobenzoate (8p). Yield 78%, yellow solid, m.p.
186–187 ◦C; H NMR (400 MHz, CDCl3)
δ 8.06 (d, J = 8.0 Hz, 1H), 7.92 (d, J = 7.6 Hz, 1H), 7.82
1
(s, 1H), 7.72 (d, J = 1.9 Hz, 1H), 7.54 (s, 1H), 7.50–7.42 (m, 2H), 6.87 (d, J = 2.0 Hz, 1H), 6.62 (s, 1H),
5.63 (s, 2H). 13C NMR (101 MHz, CDCl3)
δ
160.79, 156.46, 149.03, 147.35, 141.89, 133.56, 131.41, 128.33,
125.13, 123.26, 116.70, 115.69, 113.65, 112.28, 110.17, 106.72, 100.58, 62.59; IR (KBr) v: 1735, 1705, 1636,
1564, 1516, 1441, 1230, 1282, 1149, 1123, 748 cm−1; HRMS (ESI) calcd for C19H12IO5[M + H]+ 446.9724,
found 446.9741.
3.2. Biological Activity
Akt (also named PKB, protein kinase B), p-Akt (Ser308), GSK3
β
, p-GSK3
β
(Ser9), and
β-actin
antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA).
β-catenin, p-β-catenin
(Ser33), antibodies against TYR, TRP1, and TRP2 were bought from Santa Cruz Technology (Dallas, TX,
USA). Anti-MITF antibody was purchased from Millipore (Billerica, MA, USA). Anti-mouse, anti-goat
and anti-rabbit IgG antibodies (horseradish peroxidase conjugated) were purchased from Santa Cruz
Biotechnology (Dallas, TX, USA).
Dimethylsulfoxide (DMSO) was bought from Sigma (St. Louis, MO, USA), [2-(2-methoxy-
4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium] (CCK-8) was purchased from
Trans Gen Biotechnology (Beijing, China). Nuclear and cytoplasmic protein extraction kit was bought
from Beyotime Biotechnology, (Shanghai, China). Akt inhibitor IV was bought from EMD Biosciences,
(La Jolla, CA, USA). BIO was purchased from AMQUAR Biology, (Shanghai, China).

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3.2.1. Cell Cultures
The murine B16 melanoma cell line was purchased from Chinese Academy of Sciences (Beijing,
China). The cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM, Gibco Life
Technologies, Paris, France), supplemented with 10% (v/v) FBS, penicillin G (100 U/mL), and
streptomycin (100 mg/mL) (Gibco-BRL, Grand Island, NY, USA) in 5% CO2 at 37 ◦C.
3.2.2. Cell Morphology and Cell Viability Measurement
Cell morphology was examined under a LEICA DMI8 microscope (LEICA microsystems CMS
GmbH, Wetzlar, Germany). The cell viability was assayed by adding CCK-8 solution. Generally
speaking, The B16 cells were seeded in 96-well plates at a density of 1
allowed to adhere for 24 h. The medium was replaced with medium containing 8n diluted to the
×
104 cells per well and were
appropriate concentrations. The control cells were treated with DMSO at a final concentration of 0.1%.
After 48 h, the culture medium of the cells was discarded, 10 µL of CCK-8 solution was added into
each well and cells were incubated at 37 ◦C for another 2 h. The absorbance was measured at 450 nm
using a Spectra Max M5 (Molecular Devices, San Jose, CA, USA). All of the assays were performed in
triplicate. Absorbance of cells without treatment was regarded as 100% of cell survival. Cell viability
was calculated using the following formula: cell viability (%) = (Asample/Acontrol) × 100%.
3.2.3. Melanin Measurement
B16 cells were seeded at a density of 2
incubation, test samples were added to individual wells, cells were incubated for 48 h, and were
washed twice with ice-cold PBS (phosphate buffered solution). After cells lysed, the harvested cells
×
105 cells/well in a 6-well plate. After overnight
◦
were centrifuged, and the pellet was dissolved by adding 1 N NaOH, followed by incubation at 80 C
for 1 h. Each lysate (150
405 nm by a multi-plate reader. Protein concentration of each sample was determined by BCA Protein
Assay Kit (Biomed, Beijing, China). Intracellular melanin amount was expressed as abs/ g protein
µL) was put in a 96-well microplate, and measured spectro-photometrically at
µ
was shown as a percentage value. The percentage value of the 8n-treated cells was calculated with
respect to the untreated cells.
3.2.4. Tyrosinase Activity Assay
The assay for tyrosinase activity was carried out, as previously described [54], with a slight
modification. B16 cells were seeded in a 6-well plate at a density of 2
×
105 cells per well and allowed
to attach for 24 h. Test samples were then added to individual wells. After a 24 h incubation, cells
were washed with ice-cold PBS twice, lysed with 1% Triton X-100 solution containing 1% sodium
deoxycholate for 30 min at
supernatant. After protein quantification and adjustment, 90
−
80 ◦C, each lysate was centrifuged at 12,000
×
g for 15 min to obtain the
µL of the supernatant was incubated in
duplicate with 10
µ
L of freshly prepared substrate solution (10 mM L-DOPA) in a well of a 96-well
◦
plate. Then the cells were incubated at 37 C in dark for 60 min, the absorbance was measured at
490 nm and the 8n-treated cells was presented as percentage against the untreated cells.
3.2.5. Western Blot Analysis
B16 cells were treated with different concentrations of 8n in a 6-well plate for 48 h. Cells were
then lysed in cold RIPA (radio immunoprecipitation assay) Lysis Buffer (pH 7.4) containing protease
and protease inhibitor cocktail [1 M 4-nitrophenyl phosphate disodium salt hexahydrate (PNPP), 1 M
sodium fluoride (NaF), 10mMphenylmethanesulfonylfluoride (PMSF), 100 mM benzamidine, 100 mM
DL-Dithiothreitol (DTT), 200mM sodium orthovanadate (OV)] for 30 min on ice. The lysates were
◦
centrifuged at 12,000 rpm for 20 min at 4 C before the supernatant was collected. The protein samples
concentration was measured by BCA Protein Assay Kit (Biomed, Beijing, China) and was separated by
10% SDS polyacrylamide gels and transferred onto polyvinylidene fluoride (PVDF) membranes (Merck

Int. J. Mol. Sci. 2018, 19, 746
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Millipore Ltd., Billerica, MA, USA), Membranes were incubated with the primary antibodies at 4 ◦C
overnight, and then incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h at
room temperature. The targeted proteins were detected by ECL western blotting detection reagents
(GE Healthcare, Beijing, China), and were visualized using the ChemiDoc MP Imaging system (Bio-Rad
Laboratories, Inc., Berkeley, CA, USA). All western bolt assay results were performed in triplicate.
3.2.6. Statistical Analysis
All data were expressed as the means
by one-way ANOVA followed by Tukey post hoc test for multiple comparison tests. A p value of < 0.05
±
standard deviations and statistical analysis was performed
was considered a significant difference.
4. Conclusions
In summary, a series of ester furocoumarin derivatives had been prepared via total synthesis
or structural modification. Half of compounds exhibited a better activity on melanin synthesis than
positive control (8-MOP) in B16 melanoma cells. Among them, compounds 8n (200%) and 8o (197%)
were nearly 1.5-fold stronger than 8-MOP (136%).
Western blot analysis showed that 8n promoted phosphorylation of Akt, and thus probably
increased the non-activated GSK3β, which inhibits the phosphorylation of
β-catenin and ceased
its degradation via proteasome. The accumulation of
β-catenin probably leads to the activation
of transcription of MITF, TYR family, and thus the subsequent induction of melanin synthesis.
These results indicated that 8n stimulated melanin biosynthesis by up-regulation of MITF and TYR
family via Akt/GSK3β/β-catenin signaling pathways.
In the view of the widespread application of furocoumarins in vitiligo nowadays, similar structural
modification on C-5 position may be beneficial to explore potential melanogenesis-modulating
candidates. Signaling pathway research would shed light on the molecular function of furocoumarins
in melanogenesis. Further studies in vivo on vitiligo model mice are under way to assess the safety
and efficacy of 8n for clinical use.
Acknowledgments: This work was supported by the Funds for the Xinjiang Key Research and Development
Program (2016B03038-3); West Light Foundation of the Chinese Academy of Science (No. XBBS201403); Natural
Science Foundation of Xinjiang, China (No. 2017D01A76).
Author Contributions: Chao Niu and Li Yin performed experiments. Chao Niu prepared the manuscript.
Haji Akber Aisa designed the study and revised the paper. All authors read and reviewed the manuscript.
Conflicts of Interest: The authors declare no conflict of interest.
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