Bioorganic & Medicinal Chemistry 24 (2016) 5960–5968

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Bioorganic & Medicinal Chemistry

journal homepage: www.elsevier.com/locate/bmc

Synthesis and biological evaluation of furocoumarin derivatives on

melanin synthesis in murine B16 cells for the treatment of vitiligo
Chao Niu a,b, Guang Xian Pang a,b, Gen Li a,b, Jun Dou a,b, Li Fei Nie a,b, Helimay Himit a,b, Madina Kabas a,b,
Haji Akber Aisa a,b,⇑
a
The Key Laboratory of Plant Resources and Chemistry of Arid Zone, Chinese Academy of Sciences, Urumqi 830011, China
b
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

a r t i c l e i n f o a b s t r a c t

Article history: Furocoumarins, isolated from Psoralen corylifolia L., were found to be the most effective drug in the treat-
Received 18 July 2016 ment of vitiligo nowadays. Twenty-five furocoumarin derivatives were thus designed and synthesized in
Revised 20 September 2016 order to improve the melanogenesis in B16 cells for the first time. Among them, twenty-three compounds
Accepted 22 September 2016
were more potent than the positive control (8-MOP), the commonly used drug for vitiligo in clinic.
Available online 23 September 2016
Noticeably, compounds 6m (350.5%) and 6p (313.1%) based on the scaffold of 6k (2H-benzofuro[2,3-h]
chromen-2-one) were nearly 3-fold stronger than 8-MOP (114.50%). The in vitro melanin synthesis eval-
Keywords:
uation of these structurally diverse analogues had also led to an outline of structure–activity relationship.
Furocoumarin
Synthesis
Ó 2016 Elsevier Ltd. All rights reserved.
Melanin
Vitiligo
SAR

1. Introduction
Vitiligo is a condition characterized by the loss of pigment cells
in the epidermis.1 It can involve any part of the body where mela-
nocytes resided and caused both functional and physiological
abnormalities in the affected skin. So far, several theories had been
put forward to explain the pathogenesis of vitiligo.2 It was believed
that the disease was mainly resulted from destruction of the mel-
anocyte and obstruction of the melanin synthesis.3,4
The melanin biosynthesis proceed in melanocytes, which
responded to a wide variety of intrinsic and extrinsic factors pro-
duced by the environment or neighboring cells in the skin, includ-
ing UV, melanocyte stimulating hormone (MSH), agouti signal
protein (ASP), endothelin 1 (ET1), dickkopf-1 (DKK1), many kinds
of growth factors and cytokines.5,6 This process was catalyzed by
three melanocyte-specific enzymes: tyrosinase, tyrosinase-related
protein 1 (TRP-1) and tyrosinase-related protein 2 (TRP-2).7
Since thousand years ago, the plant specie Psoralen corylifolia L.
(Fig. 1)8 was used for repigmentation of vitiligo with natural sun-
light in India, Egypt and other oriental countries.9,10 Similarly,
the extract of Psoralen corylifolia L. seeds was one of the most
⇑ Corresponding author.
E-mail address: haji@ms.xjb.ac.cn (H.A. Aisa).
popular Uygur medicines used for vitiligo and initially recorded
in ‘Yao Yong Zong Ku’ around 300 years ago.11–13
In 1930s, 8-methoxypsoralen (8-MOP) and 5-methoxypsoralen
(5-MOP) were isolated from the Psoralen corylifolia L.14,15 Later,
other psoralens, such as 4,5,8-trimethylpsoralen (TMP) was syn-
thesized as well (Fig. 2). Continuous researches proved that these
compounds showing strong photosensitivity,16 which may be used
for the treatment of vitiligo with subsequent exposure to long-
waved ultraviolet radiation.17,18 Although PUVA(psoralen+UVA)19
was accompanied with some undesired side effects, such as gene
mutation, skin phototoxicity and risk of skin cancer,20–22 the ther-
apy is still the most successful one for the disease today.
The precise mechanism of PUVA in the treatment of vitiligo is
not clear today. However, it is probably beneficial via a variety of
mechanisms: (1) PUVA therapy may elicit the release of a certain
melanocyte stimulating growth factor that was capable of stimu-
lating melanocyte proliferation.23 (2) PUVA stimulated hypertro-
phy, proliferation and enzymatic activity of the melanocytes
resided in the outer root sheath of hair follicles, as well as melano-
cytes located at the margins of vitiliginous lesions. Repigmentation
was therefore the result of the migration of these stimulated mel-
anocytes into the depigmented skin area.24,25 (3) It was also argued
that the PUVA-induced repigmentation was, at least in part,
immunologically mediated. Investigators found that the so called
‘vitiligo associated melanocyte antigens’ and antimelanocyte

http://dx.doi.org/10.1016/j.bmc.2016.09.056
0968-0896/Ó 2016 Elsevier Ltd. All rights reserved.
 C. Niu et al. / Bioorg. Med. Chem. 24 (2016) 5960–5968
Figure 1. The plant of the Psoralen corylifolia L.8
antibodies decreased after a course of PUVA therapy.26,27 Thus
exploration of the mechanism of the furocoumarin compounds
used for vitiligo remains to be a challenge task.
Furocoumarin compounds are very versatile for physiologically
activity, such as antiinflammatory,28 antihyperplasia,29,30 antimi-
crobial and anticancer.31,32 Nonetheless, few furocoumarin deriva-
tives possessed anti-vitiligo activity were reported. Our group has
been dedicated on the drug development of the vitiligo for many
years.33–37 In the continuing developing a better medication for
the vitiligo, nine angular furocoumarins (angelicins: 5a–5i) and
sixteen linear furocoumarins (psoralens: 6a–6p) were prepared
and submitted to the activity assay of melanogenesis in murine
B16 cells, and the SAR was summarized as well. Some structural
modification were further performed on 4-methyl of 6k (com-
pound with a best activity) to search for better candidate com-
pounds for the vitiligo.
2. Result and discussion
2.1. Synthesis
The synthetic route of the target compounds was described in
Scheme 1. The intermediate 1 (7-hydroxycoumarin or 4-methy-
lumbelliferone) prepared from resorcine via Pechmann reac-
tion,38,39 was converted to 2 and 3 by Williamson reaction
refluxing with different alkylation reagents (chloroacetone, 3-
chloro-2-butanone, chloroethanol and 2-bromocyclohexanone) in
the presence of anhydrous K2CO3. The desired two sets of com-
pounds (angelicins: 5a–5f and psoralens: 6a–6h) were produced
in one step by refluxing intermediate 2 in 4% KOH ethanolic solu-
tion for several hours.40 Among them, 6a–6f were isomers of 5a–5f
respectively, and yielded about 7 times as much as 5a–5f.
The intermediate 3 was further oxidized to aldehyde 4 at
78 °C by an optimized Swern oxidation in excellent yields. Inter-
molecular cycloaddition of the intermediate 4 yielded the target
compounds 5g–5h and 6i–6j in the presence of a catalytic amount
of NaOH. It is notable that a mixed solvent (Vwater:V1,4-dioxane = 1:1)
was applied to get a homogeneous solution of aldehyde 4 for the
easy cycloaddition.
O
O O O effect to the activity than none, mono- or bis- substituents in above
O O O O
O positions. The orders of their activation on melanin synthesis was
TMP
8-MOP 5-MOP
Figure 2. The structure of the psoralens for vitiligo.
5961
Compounds 5f or 6f was aromatized in toluene with
2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) and catalyst
Pd/C to give the corresponding 5i or 6k.41
Selenium dioxide was applied in selective oxidation of com-
pound 6k to produce 4-carbaldehyde 6l, and condensation of 6l
with arylamine in 95% ethanol gave 6m;41 Compound 6l was
reduced with NaBH4 to achieve alcohol 6n,42 which was further
esterified to give 6o and 6p respectively.38,43
The oxidation of intermediate 3 to 4 (Scheme 2) was the crucial
step for the preparation of compounds 5g–5h and 6i–6j, which
bearing no substituent group on furan ring. A number of oxidant
systems, such as PCC, DMSO–trifluoroacetic anhydride (TFAA),
and DMSO–(COCl)2 (Swern oxidation)44 were tried to improve
the yield of 4 (Table 1). The oxidant PCC gave a mixture of 4a
and a byproduct 7a (Table 1, Entry 1) in low yield at room temper-
ature. By using a smaller amount of PCC and silica as supported
reagent, still afforded a mixture with the slightly improved selec-
tivity. Fortunately, with the application of DMSO-based oxidation
system as alternatives, 3 was smoothly transformed to 4 in a good
yield, especially under DMSO–(COCl)2, aldehyde 4 were obtained
in almost quantitative yield.
The 4% KOH ethanolic solution was used as we did above in the
cyclization of intermediate 2 to catalyze the similar reaction from 4
to compounds 5g–5h and 6i–6j. Unfortunately, no products were
observed, thus an alternative treatment of 4 in NaOH aqueous solu-
tion successfully gave, after acidification, a mixture with average
overall yields 80–90%, in which psoralens 6i–6j were predominant
with few amounts of isomer angelicins 5g–5h.45 We wished point
out that the yield of psoralen in mixture were much higher than
angelicin when the cyclization occurred under basic condition.
The proposed mechanism of the reaction was elucidated in
Scheme 3. The aldehyde first underwent lactone ring opening to
generate the intermediate dianion which in turn gave rise to a
nucleophilic addition–elimination reaction on the aldehyde carbon
atom with furan ring formation. Subsequent acidification lead to
lactone ring closure with formation of the target compounds.
Furthermore, PPA (polyphosphoric acid) was adopted to
improve the yield of the angelicins as solvent and catalyst at
120 °C in cyclization reaction.46 Interestingly, psoralens and angel-
icins were obtained in a mixture with a reverse proportion com-
pared with reactions catalyzed by bases. However, PPA was
viscous and the method required very high temperature, resulting
in dark tarry materials, which was difficult to treat.
As shown in Figure 3, the crystal of the compound 6b used for
X-ray diffraction analysis were obtained by slow evaporation of
acetone–ethanol (1:3) mixed solution at room temperature (CCDC
No. 1476860).
2.2. In vitro melanin synthesis evaluation
All these compounds were screened for their activity on mela-
nin synthesis in murine B16 cells, with a known method.47 Accord-
ing to the result (Fig. 4), most tested compounds (5a–5i, 6a–6k, in
grey) exhibited a stronger activity than the positive control
(8-MOP) except 5f and 6j. Among them, psoralen derivatives were
more potent than angelicin derivatives, such as 6d and 6f com-
pared with their corresponding isomers 5d and 5f.
The most important factor in efficacy of the psoralen
compounds was the numbers of the substituent groups.
Tri-substituents on 4,6,7-positions displayed more advantageous
O O
6k > 6f > 6d > 6a–6c, 6i–6j, which suggested that unsaturated
aromatic ring may contribute more than saturated and small
groups (–CH3) on 6 and 7-position.
5962 C. Niu et al. / Bioorg. Med. Chem. 24 (2016) 5960–5968

R1 R1
R3 R1
R1

i ii O O O iii O O O R2
O O O
HO OH O
HO O O R2 R3
R2
1 R3
2 5 6
1a R1 =H 2a R1 =R2 =H, R3 =CH 3 5a R 1=R 2=H, R3 =CH 3 6a R1 =R2 =H, R3 =CH 3
1b R1 =CH 3 2b R 1=H, R 2=R 3=CH 3 5b R1 =H, R2 =R3 =CH 3 6b R1 =H, R 2 =R 3=CH3
2c R1 =R3 =CH 3, R2 =H 5c R 1=R 3=CH3 , R2 =H 6c R1 =R3 =CH 3, R2 =H
2d R1 =R 2 =R 3 =CH3 5d R1 =R2 =R3 =CH 3 6d R1 =R 2 =R 3 =CH3
2e R1 =H, R2 ,R 3 =(CH 2 )4 5e R 1=H, R 2,R3 =(CH2 )4 6e R1 =H, R2 ,R 3 =(CH 2 )4
iv 2f R 1=CH 3 , R2 ,R3 =(CH2 )4 5f R 1=CH3 , R 2,R 3=(CH2 )4 6f R 1=CH 3 , R2 ,R3 =(CH2 )4
2g R1 =R 2 =H, R 3=Ph 6g R1 =R 2 =H, R 3=Ph
2h R1 =CH 3, R2 =H, R 3 =Ph 6h R1 =CH 3, R2 =H, R 3 =Ph

R1 R1
R1 R1
v vi
O O O O O O
O O O O O O
O

OH 3 H 4 5g R 1=H 6i R1 =H
5h R 1=CH3 6j R 1 =CH 3
3a R1 =H 4a R1 =H
3b R1 =CH 3 4b R1 =CH 3

O O O
vii or
5f or 6f
O O O

5i 6k

OH
CHO O

viii x O
6k xii
O O O O O O O O O
6l 6n 6p

ix xi

N O

O O O O O O
6m 6o

Scheme 1. Synthetic route for the furocoumarin derivatives. Reagents and conditions: (i) for 1a: malic acid, H2SO4, 120 °C; for 1b: ethyl acetoacetate, H2SO4, 60 °C; (ii) for 2a,
2c: chloroacetone, K2CO3, acetone, reflux; for 2b, 2d: 3-chloro-2-butanone, K2CO3, acetone, reflux; for 2e–2f: 2-bromocyclohexanone, K2CO3, acetone, reflux; for 2g–2h: 2-
bromoacetophenone, K2CO3, acetone, reflux; (iii) 4% KOH ethanolic solution, reflux; (iv) chloroethanol, K2CO3, acetone, reflux; (v) 78 °C, oxalyl chloride, DMSO,
triethylamine, DCM; (vi) 1 M NaOH aqueous solution, 1,4-dioxane (vii) Pd–C, DDQ, toluene, reflux; (viii) SeO2, xylene, reflux; (ix) o-toluidine, 95% ethanol, reflux; (x) NaBH4,
ethanol, rt; (xi) acetic anhydride, DMAP, pyridine, rt; (xii) 4-methylbenzoic acid, DCC, DMAP, THF, rt.

R1 R1 Table 1
Oxidation from 3 to 4 under different oxidant systems

oxidation Entry Substrates Oxidation systems Product Yielda (%)

O O O O O
O 1 3a PCC b
4a 30
O 3a: R1=H 4a:R1=H
3b: R1=CH 3 4b:R1=CH3 7a 41
OH H 2 3a PCC–Silicab 4a 35
3 4 7a 29
3 3a DMSO–TFAA 4a 56
4 3b DMSO–TFAA 4b 52
5 3a DMSO–(COCl)2 4a 98
O 6 3b DMSO–(COCl)2 4b 97
O O O O O O
O a
Isolated yields.
b
7a (byproduct) Reactions were carried out in CH2Cl2 at 25 °C (1.5 equiv PCC), monitored by TLC
until the starting material was fully reacted.
Scheme 2. Oxidation from 3 to 4.
 C. Niu et al. / Bioorg. Med. Chem. 24 (2016) 5960–5968 5963

R1 R1
H H
OH
H H COO
O O O O O
O O

-H 2O -H 2O
R1 R1

O COO COO
O O
O

H H
R1 R1

O O O O O O

Scheme 3. Possible mechanism of the cyclization reaction under alkaline condition.

interesting that Schiff base 6m and benzoate 6p which both bear-

ing a benzene ring, exhibited the strongest activate effect with the
value of 350.5% and 313.1%, which were nearly 3-fold potent than
the positive control 8-MOP (8-MOP with the value of 114.50%).
Surprisingly, the introduction of hydroxyl to 6n failed to promote
the melanogenesis, probably because of its strong hydrophilicity.
It was apparent that more structure optimization should be pro-
ceed on these active molecules to discover the leading compounds
for the treatment of vitiligo.

3. Conclusion

Figure 3. Chemical structure of the crystal of 6b.

The 4-methyl substituted psoralen 6d, 6f and 6h stimulated
melanogenesis highly than 6b, 6e and 6g respectively, which bear-
ing no substituent on 4-position, indicating that the substituent on
4-position would play a significant role in activity.
Based on these results, five C-4 modified analogues of most
potent compound 6k were synthesized. As showed in Figure 4(in
black), the activity of these analogues 6l, 6m, 6n, 6p were poten-
tially increased as compared with the lead compound 6k. It was
In summary, a series of furocoumarin derivatives had been pre-
pared via total synthesis or structural modification. Most of com-
pounds exhibited better activities on melanin synthesis in vitro
than positive control (8-MOP) and the SAR was summarized. It
was noteworthy that the activity of psoralen derivatives 6m
(350.5%) and 6p (313.1%) based on the scaffold of 6k were nearly
3-fold stronger than 8-MOP (114.50%) on melanin synthesis in
murine B16 cells. These two compounds were specific and promis-
ing candidates against vitiligo, further studies on action mecha-
nism of them and animal experiment on vitiligo transgenic
mouse is under way.

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 was treated
with 50 lM of different furocoumarin derivatives for 48 h. After that, melanin content was measured directly. The data are the mean ± SD (bars) of three experiments in
duplicate.
5964 C. Niu et al. / Bioorg. Med. Chem. 24 (2016) 5960–5968
4. Experimental section
4.1. Chemistry
Reagents and solvents were purchased from Sigma, and used
without further purification. Thin-layer chromatography (TLC)
was carried out on glass plates coated with silica gel (Qingdao
Haiyang Chemical Co., 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 the NMR spectra were recorded with a Vari-
an400 MHz NMR spectrometer in CDCl3 or Acetone-d6, using TMS
as an internal standard. High-resolution mass spectra (HRMS) were
recorded on AB SCIEX QSTAR Elite quadrupole time-of-flight mass
spectrometry. The IR data were recorded on a Thermo Fisher Scien-
tific Nilolet 6700 FT-IR infrared spectrometer (KBr).
4.1.1. General procedure of preparation of intermediate 1
4.1.1.1. 7-Hydroxy-2H-chromen-2-one (1a). A mixture of
resorcinol (1.4 g, 12.7 mmol), 2-hydroxysuccinic acid (1.9 g,
14.0 mmol) and concentrated sulfuric acid (5 mL) was stirred at
room temperature for 0.5 h. The above mixture was heated to
120 °C and stirred for another 4 h. After cooling to room tempera-
ture, ice water was added to the mixture and stirred for 0.5 h. The
reaction mixture was filtered and dried to afford compound as
white solid. Yield 71%, mp 234–236 °C.
4.1.1.2. 7-Hydroxy-4-methyl-2H-chromen-2-one (1b). 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 recrystal-
lized from methanol to give compound as white needle crystals.
Yield 92%, mp 202–204 °C.
4.1.2. General procedure of preparation of intermediate 2 and 3
A mixture of 1 (5.0 mmol) with chloroethanol or suitable a-
haloketone (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 sil-
ica gel chromatography with petroleum ether/ethyl acetate or
chloroform to give intermediate 2 and 3.
4.1.2.1. 7-(2-Oxopropoxy)-2H-chromen-2-one (2a). Yield
89%, white solid mp 165–167 °C; 1H NMR (400 MHz, Acetone-d6)
d 7.90 (d, J = 9.5 Hz, 1H), 7.59 (d, J = 8.6 Hz, 1H), 6.94 (dd, J = 8.6,
2.4 Hz, 1H), 6.85 (d, J = 2.4 Hz, 1H), 6.23 (d, J = 9.5 Hz, 1H), 4.95
(s, 2H), 2.25 (s, 3H).
4.1.2.2. 4-Methyl-7-(2-oxopropoxy)-2H-chromen-2-one (2c).
Yield 91%, white solid, mp 97–98 °C; 1H NMR (400 MHz, Ace-
tone-d6) d 7.67 (d, J = 8.8 Hz, 1H), 6.94 (dd, J = 8.8, 2.5 Hz, 1H),
6.82 (d, J = 2.5 Hz, 1H), 6.13 (d, J = 1.0 Hz, 1H), 4.95 (s, 2H), 2.43
(d, J = 1.1 Hz, 4H), 2.25 (s, 3H).
4.1.2.3. 4-Methyl-7-((2-oxocyclohexyl)oxy)-2H-chromen-2-one
(2f). Yield 93%, white solid, mp 158–159 °C; 1H NMR
(400 MHz, Acetone-d6) d 7.63 (d, J = 8.8 Hz, 1H), 6.89 (dd, J = 8.8,
2.5 Hz, 1H), 6.76 (d, J = 2.5 Hz, 1H), 6.11 (s, 1H), 5.15 (dd, J = 10.6,
6.2 Hz, 1H), 2.82–2.64 (m, 2H), 2.43 (s, 2H), 2.03–1.84 (m, 2H),
1.77–1.64 (m, 2H).
4.1.2.4. 7-(2-Oxo-2-phenylethoxy)-2H-chromen-2-one (2g).
Yield 80%, light yellow solid, mp 205–207 °C; 1H NMR (400 MHz,
CDCl3) d 7.99 (dd, J = 8.2, 1.0 Hz, 2H), 7.68–7.61 (m, 2H), 7.53 (t,
J = 7.7 Hz, 2H), 7.39 (d, J = 8.6 Hz, 1H), 6.92 (dd, J = 8.6, 2.5 Hz,
1H), 6.80 (d, J = 2.4 Hz, 1H), 6.26 (d, J = 9.5 Hz, 1H), 5.38 (s, 2H).
4.1.2.5. 4-Methyl-7-(2-oxo-2-phenylethoxy)-2H-chromen-2-one
(2h). Yield 83%, light yellow solid, mp 162–164 °C; 1H NMR
(400 MHz, CDCl3) d 7.98 (dd, J = 8.2, 1.0 Hz, 2H), 7.64 (t,
J = 7.4 Hz, 1H), 7.51 (dd, J = 12.3, 5.4 Hz, 3H), 6.94 (dd, J = 8.8,
2.6 Hz, 1H), 6.79 (d, J = 2.5 Hz, 1H), 6.13 (d, J = 1.1 Hz, 1H), 5.38
(s, 2H), 2.38 (d, J = 1.1 Hz, 3H).
4.1.2.6. 7-(2-Hydroxyethoxy)-2H-chromen-2-one (3a). Yield
96%, white solid, mp 92–93 °C; 1H NMR (400 MHz, CDCl3) d 7.85 (d,
J = 9.3 Hz, 1H), 7.56 (d, J = 8.6 Hz, 1H), 6.94–6.85 (m, 2H), 6.17 (d,
J = 9.3 Hz, 1H), 4.15 (t, J = 8.5 Hz, 2H), 4.05 (m, 2H).
4.1.2.7. 7-(2-Hydroxyethoxy)-4-methyl-2H-chromen-2-one (3b).
Yield 95%, white solid, mp 108–110 °C; 1H NMR (400 MHz, CDCl3) d
7.51 (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).
4.1.3. General procedure of preparation of intermediate 4
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 solution of alcohols 3 (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 65 °C and allowed to warm up
to room temperature. The reaction mixture was diluted with CH2-
Cl2 and filtered through a pad of silica gel. Removal of the solvent
under vacuum afforded the aldehydes 4.
4.1.3.1. 7-(2-Oxoethoxy)- 2H-chromen-2-one (4a). Yield
94%, white solid, mp 123–124 °C; 1H NMR (400 MHz, CDCl3) d
9.90 (s, 1H), 7.88 (d, J = 9.2 Hz, 1H), 7.57 (d, J = 8.4 Hz, 1H), 6.92
(dd, J = 8.4, 2.5 Hz, 1H), 6.80 (d, J = 2.5 Hz, 1H), 6.20 (d, J = 9.2 Hz,
1H), 4.70 (s, 2H).
4.1.3.2. 7-(2-Oxoethoxy)-4-methyl-2H-chromen-2-one (4b).
Yield 97%, white solid, mp 140–142 °C; 1H NMR (400 MHz, CDCl3)
d 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, 1H), 6.15 (d, J = 1.0 Hz, 1H), 4.68 (s, 2H), 2.39 (d,
J = 0.9 Hz, 3H).
4.1.4. General procedure of preparation of angelicins 5a–5f and
psoralens 6a–6h
To an ethanolic solution (500 mL) of intermediate 2 (10 mmol)
was added a 4% ethanol potassium hydroxide solution (70 mL) and
the mixture was refluxed for 4 h. After cooling, the solution was
acidified with 1 M hydrochloric acid and extracted with ethyl acet-
ate three times. The organic phase was dried over night and evap-
orated under reduced pressure. The resulting residue was purified
by silica gel chromatography with petroleum ether/ethyl acetate to
give a pair of isomer.
4.1.4.1. 9-Methyl-2H-furo[2,3-h]chromen-2-one (5a). Yield
10%, white solid, mp 132–134 °C; 1H NMR (400 MHz, CDCl3) d
7.80 (d, J = 9.5 Hz, 1H), 7.43 (s, 1H), 7.37 (d, J = 8.6 Hz, 1H), 7.33
(d, J = 8.5 Hz, 1H), 6.37 (d, J = 9.6 Hz, 1H), 2.53 (s, 3H). 13C NMR
(101 MHz, CDCl3) d 161.02 (s), 158.02 (s), 149.90 (s), 144.76 (s),
 C. Niu et al. / Bioorg. Med. Chem. 24 (2016) 5960–5968
142.39 (s), 123.83 (s), 117.58 (s), 116.08 (s), 113.87 (s), 113.48 (s),
108.98 (s), 9.77 (s); IR (KBr) v: 2924, 1386, 1508, 1261, 1104,
1050 cm 1; HRMS (ESI) calcd for C12H9O3 [M+H]+ 201.0552, found
201.0561.
4.1.4.2. 6-Methyl-2H-furo[3,2-g]chromen-2-one (6a). Yield
70%, white solid, mp 179–181 °C; 1H NMR (400 MHz, CDCl3) d
7.78 (d, J = 9.6 Hz, 1H), 7.55 (s, 1H), 7.44 (s, 1H), 7.36 (s, 1H), 6.34
(d, J = 9.6 Hz, 1H), 2.25 (s, 3H). 13C NMR (101 MHz, CDCl3) d
161.22 (s), 156.80 (s), 152.11 (s), 144.26 (s), 143.26 (s), 126.77
(s), 118.24 (s), 115.62 (s), 114.98 (s), 114.41 (s), 99.78 (s), 7.93
(s); IR (KBr) v: 2922, 1714, 1577, 1385, 1208, 1133, 1065,
871 cm 1; HRMS (ESI) calcd for C12H9O3 [M+H]+ 201.0552, found
201.0539.
4.1.4.3. 8,9-Dimethyl-2H-furo[2,3-h]chromen-2-one (5b).
Yield 11%, white solid, mp 126–128 °C; 1H NMR (400 MHz, CDCl3)
d 7.77 (d, J = 9.6 Hz, 1H), 7.28 (d, J = 8.4 Hz, 1H), 7.24 (d, J = 8.4 Hz,
1H), 6.34 (d, J = 9.5 Hz, 1H), 2.46 (s, 3H), 2.40 (s, 3H). 13C NMR
(101 MHz, CDCl3) d 161.03 (s), 156.32 (s), 151.76 (s), 148.91 (s),
130.87(s), 128.80 (s), 122.47 (s), 118.60 (s), 113.38 (s), 109.93 (s),
108.06 (s), 11.65 (s), 9.60 (s); IR (KBr) v: 2925, 1735, 1611, 1125,
831 cm 1; HRMS (ESI) calcd for C13H11O3 [M+H]+ 215.0708, found
215.0715.
4.1.4.4. 6,7-Dimethyl-2H-furo[3,2-g]chromen-2-one (6b).
Yield 75%, white solid, mp 156–158 °C; 1H NMR (400 MHz, CDCl3)
d 7.77 (d, J = 9.6 Hz, 1H), 7.41 (s, 1H), 7.29 (s, 1H), 6.32 (d, J = 9.5 Hz,
1H), 2.39 (s, 3H), 2.16 (s, 3H). 13C NMR (101 MHz, CDCl3) d 161.33
(s), 155.51 (s), 152.80 (s), 151.59 (s), 144.28 (s), 128.22 (s), 116.86
(s), 114.59 (s), 113.97 (s), 109.48 (s), 98.99 (s), 11.93 (s), 7.86 (s); IR
(KBr) v: 2922, 1710, 1581, 1389, 1142, 923, 874 cm 1; HRMS (ESI)
calcd for C13H11O3 [M+H]+ 215.0708, found 215.0692.
4.1.4.5. 4,9-Dimethyl-2H-furo[2,3-h]chromen-2-one (5c).
Yield 13%, white solid, mp 151–153 °C; 1H NMR (400 MHz, CDCl3)
d 7.48 (d, J = 8.7 Hz, 1H), 7.42 (s, 1H), 7.37 (d, J = 8.7 Hz, 1H), 6.26 (s,
1H), 2.53 (s, 3H), 2.49 (s, 3H). 13C NMR (101 MHz, CDCl3) d 160.88
(s), 157.78 (s), 153.58 (s), 149.25 (s), 142.14 (s), 120.34 (s), 117.40
(s), 116.17 (s), 114.36 (s), 112.53 (s), 108.41 (s), 19.54 (s), 9.71 (s);
IR (KBr) v: 2924, 1715, 1608, 1369, 1096, 1060, 845 cm 1; HRMS
(ESI) calcd for C13H11O3 [M+H]+ 215.0708, found 215.0697.
4.1.4.6. 4,6-Dimethyl-2H-furo[3,2-g]chromen-2-one (6c).
Yield 71%, white solid, mp 239–241 °C; 1H NMR (400 MHz, CDCl3)
d 7.67 (s, 1H), 7.46 (s, 1H), 7.40 (s, 1H), 6.25 (s, 1H), 2.52 (s, 3H),
2.29 (s, 3H). 13C NMR (101 MHz, CDCl3) d 161.21 (s), 156.63 (s),
152.77 (s), 151.62 (s), 143.11 (s), 126.39 (s), 116.08 (s), 115.64
(s), 114.74 (s), 113.23 (s), 99.77 (s), 19.25 (s), 7.90 (s); IR (KBr) v:
2923, 1706, 1576, 1388, 1139, 1038, 873 cm 1; HRMS (ESI) calcd
for C13H11O3 [M+H]+ 215.0708, found 215.0720.
4.1.4.7. 4,8,9-Trimethyl-2H-furo[2,3-h]chromen-2-one (5d).
Yield 12%, white solid, mp 145–146 °C; 1H NMR (400 MHz, CDCl3)
d 7.38 (d, J = 8.7 Hz, 1H), 7.28 (d, J = 8.7 Hz, 1H), 6.22 (s, 1H), 2.47
(d, J = 0.9 Hz, 3H), 2.45 (s, 3H), 2.40 (s, 3H). 13C NMR (101 MHz,
CDCl3) d 161.06 (s), 156.21 (s), 153.67 (s), 151.59 (s), 148.39 (s),
119.08 (s), 118.55 (s), 114.31 (s), 112.30 (s), 110.13 (s), 107.64
(s), 19.48 (s), 11.65 (s), 9.66 (s); IR (KBr) v: 2927, 1718, 1614,
1389, 1081, 866 cm 1; HRMS (ESI) calcd for C14H13O3 [M+H]+
229.0865, found 229.0873.
4.1.4.8. 4,6,7-Trimethyl-2H-furo[3,2-g]chromen-2-one (6d).
Yield 73%, white solid, mp 182–184 °C; 1H NMR (400 MHz, CDCl3)
d 7.51 (s, 1H), 7.30 (s, 1H), 6.22 (s, 1H), 2.50 (s, 3H), 2.39 (s, 3H),
2.18 (s, 3H). 13C NMR (101 MHz, CDCl3) d 161.38 (s), 155.39 (s),
5965
152.91 (s), 152.71 (s), 151.13 (s), 127.92 (s), 115.72 (s), 113.45
(s), 112.88 (s), 109.59 (s), 99.00 (s), 19.22 (s), 11.94 (s), 7.90 (s);
IR (KBr) v: 2926, 1726, 1577, 1390, 1277, 1140, 830 cm 1; HRMS
(ESI) calcd for C14H13O3 [M+H]+ 229.0865, found 229.0851.
4.1.4.9. 8,9,10,11-Tetrahydro-2H-benzofuro[2,3-h]chromen-2-
one (5e). Yield 11%, white solid, mp 209–210 °C; 1H NMR
(400 MHz, CDCl3) d 7.76 (d, J = 9.6 Hz, 1H), 7.30 (d, J = 8.4 Hz, 1H),
7.23 (d, J = 8.4 Hz, 1H), 6.33 (d, J = 9.5 Hz, 1H), 3.03–2.95 (m, 2H),
2.73–2.79 (m, 2H), 2.00–1.92 (m, 2H), 1.92–1.83 (m, 2H). 13C
NMR (101 MHz, CDCl3) d 159.99 (s), 155.72 (s), 154.12 (s), 147.64
(s), 143.55 (s), 121.25 (s), 116.49(s), 112.56 (s), 112.36 (s), 111.61
(s), 107.29 (s), 22.41 (s), 21.70 (s), 21.56(s), 20.82 (s); IR (KBr) v:
2936, 1716, 1612, 1453, 1274, 1110, 842 cm 1; HRMS (ESI) calcd
for C15H13O3 [M+H]+ 241.0865, found 241.0881.
4.1.4.10. 6,7,8,9-Tetrahydro-2H-benzofuro[3,2-g]chromen-2-
one (6e). Yield 74%, white solid, mp 200–202 °C; 1H NMR
(400 MHz, CDCl3) d 7.76 (d, J = 9.6 Hz, 1H), 7.41 (s, 1H), 7.32 (s,
1H), 6.32 (d, J = 9.5 Hz, 1H), 2.69–2.77 (m, 2H), 2.57–2.65 (m,
2H), 1.99–1.90 (m, 2H), 1.90–1.81 (m, 2H). 13C NMR (101 MHz,
CDCl3) d 161.30 (s), 156.35 (s), 156.03 (s), 151.44 (s), 144.26 (s),
126.58 (s), 116.57 (s), 114.60 (s), 113.98 (s), 112.55 (s), 99.34 (s),
23.37 (s), 22.63 (s), 22.37 (s), 20.27 (s); IR (KBr) v: 2933, 1716,
1575, 1391, 1136, 972 cm 1; HRMS (ESI) calcd for C15H13O3 [M
+H]+ 241.0865, found 241.0848.
4.1.4.11. 4-Methyl-8,9,10,11-tetrahydro-2H-benzofuro[2,3-h]
chromen-2-one (5f). Yield 17%, white solid, mp 160–
162 °C; 1H NMR (400 MHz, CDCl3) d 7.39 (d, J = 8.7 Hz, 1H), 7.32
(d, J = 8.6 Hz, 1H), 6.23 (s, 1H), 3.05–2.98 (m, 2H), 2.81–2.74 (m,
2H), 2.48 (s, 3H), 2.00–1.92 (m, 2H), 1.92–1.85 (m, 2H). 13C NMR
(101 MHz, CDCl3) d 160.98(s), 156.60(s), 154.93 (s), 130.80 (s),
128.79 (s), 118.83 (s), 114.36(s), 112.81 (s), 112.41 (s), 107.85 (s),
23.41 (s), 22.64 (s), 21.85 (s), 19.33 (s). IR (KBr) v: 2945, 1723,
1607, 1442, 1274, 1070, 872 cm 1; HRMS (ESI) calcd for C16H15O3
[M+H]+ 255.1021, found 255.1005.
4.1.4.12. 4-Methyl-6,7,8,9-tetrahydro-2H-benzofuro[3,2-g]chro-
men-2-one (6f). Yield 72%, white solid, mp 185–187 °C; 1H
NMR (400 MHz, CDCl3) d 7.53 (s, 1H), 7.34 (s, 1H), 6.22 (s, 1H),
2.71–2.79 (m, 2H), 2.68–2.61 (m, 2H), 2.49 (s, 3H), 2.00–1.92 (m,
2H), 1.92–1.84 (m, 2H). 13C NMR (101 MHz, CDCl3) d 161.39 (s),
156.30 (s), 155.94 (s), 152.92 (s), 151.03 (s), 126.34 (s), 115.77
(s), 113.21 (s), 112.94 (s), 112.70 (s), 99.42 (s), 23.42 (s), 22.70
(s), 22.45 (s), 20.36 (s), 19.23 (s); IR (KBr) v: 2938, 1716, 1570,
1388, 1194, 1131, 871 cm 1; HRMS (ESI) calcd for C16H15O3 [M
+H]+ 255.1021, found 255.1033.
4.1.4.13. 6-Phenyl-2H-furo[3,2-g]chromen-2-one (6g).
Yield 95%, light yellow solid, mp 103–105 °C; 1H NMR (400 MHz,
CDCl3) d 7.89 (s, 1H), 7.84 (s, 1H), 7.82 (d, J = 9.6 Hz, 1H), 7.60–
7.66 (m, 2H), 7.48–7.55 (m, J = 8.3, 6.7 Hz, 3H), 7.42 (td, J = 7.4,
1.1 Hz, 1H), 6.40 (d, J = 9.6 Hz, 1H). 13C NMR (101 MHz, CDCl3) d
161.04 (s), 157.34 (s), 152.33 (s), 144.18 (s), 143.00 (s), 131.06
(s), 129.33 (s), 128.21 (s), 127.64 (s), 124.35 (s), 122.30 (s),
119.40 (s), 115.73 (s), 114.97 (s), 100.34 (s); IR (KBr) v: 2919,
1735, 1577, 1389, 1155, 1110, 872 cm 1; HRMS (ESI) calcd for
C17H11O3 [M+H]+ 263.0708, found 263.0716.
4.1.4.14. 4-Methyl-6-phenyl-2H-furo[3,2-g]chromen-2-one (6h).
Yield 97%, light yellow solid, mp 171–173 °C; 1H NMR (400 MHz,
CDCl3) d 7.98 (s, 1H), 7.83 (s, 1H), 7.64 (dd, J = 8.2, 1.1 Hz, 2H),
7.50–7.56 (m, 3H), 7.44 (td, J = 7.4, 1.1 Hz, 1H), 6.29 (s, 1H), 2.52
(s, 3H). 13C NMR (101 MHz, CDCl3) d 160.99 (s), 157.10 (s),
152.64 (s), 151.80 (s), 142.88 (s), 131.06 (s), 129.25 (s), 128.06
5966 C. Niu et al. / Bioorg. Med. Chem. 24 (2016) 5960–5968
(s), 127.58 (s), 123.96 (s), 122.32 (s), 116.80 (s), 115.77 (s), 113.64
(s), 100.23 (s), 19.26 (s); IR (KBr) v: 2925, 1735, 1611, 1447, 1280,
1125, 1063, 831 cm 1; HRMS (ESI) calcd for C18H13O3 [M+H]+
277.0865, found 277.0853.
4.1.5. General procedure of preparation of angelicins 5g–5h and
psoralens 6i–6j
A solution of aldehydes 4 (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 left overnight. The resultant mixture
was extracted with ethyl acetate three 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 petroleum ether/ethyl
acetate to afford a pair of isomer.
4.1.5.1. 2H-Furo[2,3-h]chromen-2-one (5g). Yield 10%,
white solid, mp 149–151 °C; 1H NMR (400 MHz, CDCl3) d 7.81 (d,
J = 9.6 Hz, 1H), 7.70 (d, J = 2.2 Hz, 1H), 7.44 (dd, J = 8.5, 0.8 Hz,
1H), 7.38 (d, J = 8.5 Hz, 1H), 7.14 (dd, J = 2.2, 0.8 Hz, 1H), 6.40 (d,
J = 9.6 Hz, 1H). 13C NMR (101 MHz, CDCl3) d 160.96 (s), 156.02
(s), 145.39 (s), 144.63 (s), 124.55 (s), 123.96 (s), 114.28 (s),
108.95 (s), 104.26 (s), 103.98 (s); IR (KBr) v: 2923, 1709, 1616,
1272, 1123, 1054, 832 cm 1; HRMS (ESI) calcd for C11H7O3 [M
+H]+ 187.0395, found 187.0411.
4.1.5.2. 4-Methyl-2H-furo[2,3-h]chromen-2-one (5h). Yield
9%, white solid, mp 119–120 °C; 1H NMR (400 MHz, CDCl3) d 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, J = 2.1 Hz, 1H), 6.28 (s, 1H), 2.51 (s, 3H). 13C NMR
(101 MHz, CDCl3) d 160.89 (s), 157.25 (s), 153.58 (s), 145.76 (s),
120.48 (s), 117.00 (s), 112.86 (s), 110.02 (s), 108.41 (s), 104.37
(s), 19.44 (s); IR (KBr) v: 2919, 1718, 1617, 1264, 1065,
761 cm 1; HRMS (ESI) calcd for C12H9O3 [M+H]+ 201.0552, found
201.0559.
4.1.5.3. 2H-Furo[3,2-g]chromen-2-one (6i). Yield 76%, white
solid, mp 155–156 °C; 1H NMR (400 MHz, CDCl3) d 7.80 (d,
J = 9.6 Hz, 1H), 7.71–7.67 (m, 2H), 7.48 (s, 1H), 6.83 (d, J = 2.2,
1H), 6.38 (d, J = 9.6 Hz, 1H). 13C NMR (101 MHz, CDCl3) d 161.15
(s), 156.55 (s), 152.17 (s), 147.04 (s), 144.20 (s), 125.02 (s),
119.97 (s), 115.55 (s), 114.80 (s), 106.51 (s), 100.01 (s). IR (KBr)
v: 2920, 1719, 1617, 1389, 1126, 1022 cm 1; HRMS (ESI) calcd
for C11H7O3 [M+H]+ 187.0395, found 187.0377.
4.1.5.4. 4-Methyl-2H-furo[3,2-g]chromen-2-one (6j). Yield
80%, white solid, mp 160–162 °C; 1H NMR (400 MHz, CDCl3) d
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, 1H), 2.50 (s, 3H). 13C NMR (101 MHz, CDCl3)
d 161.24 (s), 156.43 (s), 152.78 (s), 151.75 (s), 146.96 (s), 124.77 (s),
116.70 (s), 113.62 (s), 106.67 (s), 100.00 (s), 19.30 (s); IR (KBr) v:
2921, 1723, 1630, 1385, 1137, 1031, 928 cm 1; HRMS (ESI) calcd
for C12H9O3 [M+H]+ 201.0552, found 201.0540.
4.1.6. General procedure of preparation of 5i and 6k
A mixture of 5f or 6f (10 mmol), 2,3-dichloro-5,6-dicyano-1,4-
benzoquinone (DDQ, 25 mmol) and a catalytic amount of Pd/C in
anhydrous toluene (500 ml) was refluxed for 6 h. After cooling,
the solid was filtered off and the solvent evaporated under reduced
pressure. The residue was purified by column chromatography and
crystallized from MeOH to give 5i or 6k.
4.1.6.1. 4-Methyl-2H-benzofuro[3,2-g]chromen-2-one
(5i). Yield 86%, white solid, mp 233–235 °C; 1H NMR
(400 MHz, CDCl3) d 8.13 (s, 1H), 7.98 (d, J = 7.6 Hz, 1H), 7.59 (d,
J = 8.2 Hz, 1H), 7.53–7.48 (m, 2H), 7.39–7.43 (m, 1H), 6.31 (s, 1H),
2.58 (s, 3H). 13C NMR (101 MHz, CDCl3) d 161.08 (s), 158.05 (s),
153.53 (s), 152.78 (s), 129.28 (s), 128.01 (s), 123.66 (s), 121.79
(s), 120.75 (s), 116.23 (s), 113.57 (s), 112.07 (s), 100.38 (s), 19.39
(s); IR (KBr) v: 2922, 1720, 1605, 1435, 1390, 1213, 1135,
1025 cm 1; HRMS (ESI) calcd for C16H11O3 [M+H]+ 251.0708, found
251.0689.
4.1.6.2. 4-Methyl-2H-benzofuro[2,3-h]chromen-2-one (6k).
Yield 82%, white solid, mp 228–230 °C; 1H NMR (400 MHz, CDCl3)
d 8.40 (d, J = 7.6 Hz, 1H), 7.64 (d, J = 8.7 Hz, 1H), 7.59 (d, J = 8.2 Hz,
1H), 7.52 (t, J = 7.8 Hz, 1H), 7.40–7.49 (m, 2H), 6.29 (s, 1H), 2.50 (s,
3H). 13C NMR (101 MHz, CDCl3) d 160.65 (s), 158.42 (s), 156.27 (s),
153.34 (s), 149.61 (s), 127.89 (s), 123.93 (s), 123.73 (s), 123.33 (s),
121.98 (s), 115.12 (s), 113.07 (s), 111.62 (s), 108.40 (s), 19.48 (s); IR
(KBr) v: 2922, 1728, 1609, 1431, 1384, 1214, 1070, 849 cm 1;
HRMS (ESI) calcd for C16H11O3 [M+H]+ 251.0708, found 251.0717.
4.1.7. Preparation of 2H-benzofuro[3,2-g]chromen-4-carbalde-
hyde (6l)
Powdered SeO2 (30 mmol) was added to a solution of 6k
(20 mmol) in 20 ml of hot dry xylene and the mixture were
refluxed for 12 h with vigorous stirring under the nitrogen. The
reaction mixture was filtered to remove black Se, and the deep
orange filtrate was allowed to stand overnight. Almost pure crys-
tals of 6l could be separated from the solution. Yield 63%, yellow
solid, mp 254–256 °C; 1H NMR (400 MHz, CDCl3) d 10.18 (s, 1H),
9.23 (s, 1H), 8.06 (dd, J = 7.7, 0.6 Hz, 1H), 7.63–7.57 (m, 2H),
7.56–7.50 (m, 1H), 7.43 (t, J = 7.6 Hz, 1H), 6.90 (s, 1H). 13C NMR
(101 MHz, CDCl3) d 190.05 (s), 161.30 (s), 157.20 (s), 156. 30(s),
153.20 (s), 141.70 (s), 130.10 (s), 127.20 (s), 125.10 (s), 122.06(s),
121.04(s), 120.10 (s), 118.50 (s), 112.10 (s), 108.90 (s), 100.30 (s);
IR (KBr) v: 3060, 2864, 2730, 1645, 1453, 1385, 1256, 1055,
860 cm 1; HRMS (ESI) calcd for C16H9O4 [M+H]+ 265.0501, found
265.0523.
4.1.8. Preparation of 4-((o-tolylimino)methyl)-2H-benzofuro
[3,2-g]chromen-2-one (6m)
A mixture of 6l (5 mmol) and o-Toluidine (15 mmol) in 95%
ethanol (25 ml) was refluxed for 24 h. After cooling, the precipitate
was collected, washed with 95% ethanol to give 6m. Yield 73%, yel-
low solid, mp 268–270 °C; 1H NMR (400 MHz, CDCl3) d 9.71 (s, 1H),
8.64 (s, 1H), 7.97 (d, J = 7.6 Hz, 1H), 7.63–7.57 (m, 2H), 7.51 (td,
J = 7.6 Hz, J = 1.2 Hz, 1H), 7.43–7.28 (m, 4H), 7.11 (d, J = 7.6 Hz,
1H), 6.80 (s, 1H), 2.56 (s, 3H). 13C NMR (101 MHz, CDCl3) d 161.8
(s), 158.2 (s), 157.2 (s), 154.5 (s), 152.0 (s), 148.3 (s), 147.6 (s),
138.2 (s), 131.0 (s), 129.8 (s), 128.7 (s), 125.1 (s), 124.2 (s), 123.0
(s), 122.6 (s), 122.3 (s), 121.9 (s), 120.3 (s), 119.5 (s), 118.0 (s),
113.2 (s), 101.1 (s), 87.3 (s), 23.1 (s); IR (KBr) v: 3059, 2914,
2710, 1650, 1503, 1427, 1270, 1140, 735 cm 1; HRMS (ESI) calcd
for C25H16NO3 [M+H]+ 354.1130, found 354.1141.
4.1.9. Preparation of 4-(hydroxymethyl)-2H-benzofuro[3,2-g]
chromen-2-one (6n)
Compound 6l was dissolved in ethanol (130 mL), sodium boro-
hydride (380 mg, 10.0 mmol) was added, and the solution was stir-
red for 2 h at room temperature. Thereafter the suspension was
carefully hydrolyzed with 1 M HCl (20 mL), diluted with H2O and
extracted three times with CH2Cl2. The organic phase was washed
with brine, dried over Na2SO4 and evaporated under reduced pres-
sure. The residue was purified by flash chromatography on silica
 C. Niu et al. / Bioorg. Med. Chem. 24 (2016) 5960–5968
gel eluted with Vchloroform:Vmethanol = 30:1 to afford alcohol 6n.
Yield 90%, white solid, mp 209–211 °C; 1H NMR (400 MHz, Ace-
tone-d6) d 8.48 (s, 1H), 8.19–8.15 (m, 1H), 7.68 (d, J = 8.2 Hz, 1H),
7.61 (s, 1H), 7.59–7.53 (m, 1H), 7.44 (td, J = 7.6, 0.9 Hz, 1H), 6.58
(t, J = 1.5 Hz, 1H), 5.08 (d, J = 1.6 Hz, 2H). 13C NMR (101 MHz, Ace-
tone-d6) d 156.85 (s), 138.87 (s), 132.60 (s), 129.10 (s), 127.93 (s),
126.95 (s), 123.52 (s), 121.37 (s), 120.65 (s), 115.03 (s), 111.91
(s), 110.62 (s), 109.99 (s), 105.41 (s), 100.49 (s), 61.24 (s); IR
(KBr) v: 3443, 2930, 1700, 1628, 1157, 1074, 1033, 860,
775 cm 1; HRMS (ESI) calcd for C16H11O4 [M+H]+ 267.0657, found
267.0643.
4.1.10. Preparation of (2-oxo-2H-benzofuro[3,2-g]chromen-4-
yl)methyl acetate) (6o)
A mixture of 6n (0.09 mmol), 4-dimethylaminopyridine DMAP
(1 mg) and one drop of acetic anhydride in 2 mL of anhydrous pyr-
idine was stirred at room temperature overnight under nitrogen
atmosphere, 5 mL of icy water was then added. The reaction mix-
ture was extracted with ethyl acetate three times, and the organic
layer was washed with brine and dried over anhydrous Na2SO4.
After removal of solvent, the crude product was purified by flash
column chromatography over silica gel eluted with Vpetroleum ether/
Vethyl acetate = 3:1 to give acetate 6o. Yield 91%, white solid, mp 190–
192 °C; 1H NMR (400 MHz, CDCl3) d 8.05 (s, 1H), 8.01–7.96 (m,
1H), 7.61 (d, J = 8.3 Hz, 1H), 7.56 (s, 1H), 7.55–7.50 (m, 1H), 7.42
(td, J = 7.7, 0.8 Hz, 1H), 6.53 (t, J = 1.2 Hz, 1H), 5.46 (d, J = 1.3 Hz,
2H), 2.26 (s, 3H). 13C NMR (101 MHz, CDCl3) d 170.35 (s), 160.60
(s), 153.65 (s), 149.30 (s), 128.29 (s), 123.83 (s), 122.11 (s),
120.88 (s), 115.04 (s), 113.32 (s), 112.15 (s), 111.61 (s), 100.88
(s), 61.51 (s), 20.93 (s). IR (KBr) v: 3082, 1725, 1630, 1278, 1160,
1077, 898, 765, 550 cm 1; HRMS (ESI) calcd for C18H13O5 [M+H]+
309.0763, found 309.0775.
4.1.11. Preparation of (2-oxo-2H-benzofuro[3,2-g]chromen-4-
yl)methyl 4-methylbenzoate (6p)
1,3-Dicyclohexylcarbodiimide (DCC, 4 mmol) was added to a
solution of 6n (4 mmol), 4-methylbenzoic acid (5 mmol) and
DMAP (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 sil-
ica gel eluted with Vpetroleum ether:Vethyl acetate = 3:1 to give benzoate
6p. Yield 94%, white solid, mp 233–234 °C; 1H NMR (400 MHz,
CDCl3) d 8.14 (s, 1H), 8.04 (d, J = 8.2 Hz, 2H), 7.99 (d, J = 7.6 Hz,
1H), 7.61 (d, J = 8.2 Hz, 1H), 7.58 (s, 1H), 7.56–7.50 (m, 1H), 7.42
(td, J = 7.6, 0.6 Hz, 1H), 7.31 (d, J = 8.1 Hz, 2H), 6.65 (s, 1H), 5.69
(d, J = 1.3 Hz, 2H), 2.45 (s, 3H). 13C NMR (101 MHz, CDCl3) d
165.98 (s), 160.66 (s), 158.18 (s), 157.39 (s), 153.69 (s), 149.69
(s), 144.90 (s), 130.06 (s), 129.59 (s), 128.28 (s), 126.36 (s),
123.85 (s), 123.11 (s), 122.16 (s), 120.91 (s), 115.12 (s), 113.41
(s), 112.14 (s), 111.57 (s), 100.89 (s), 61.81 (s), 21.92 (s); IR (KBr)
v: 3054, 1745, 1680, 1610, 1433, 1262, 1141, 1077, 900, 845,
564 cm 1; HRMS (ESI) calcd for C24H17O5[M+H]+ 385.1076, found
385.1059.
4.2. Biological activity
4.2.1. Cell culture
Murine B16 melanoma cell lines (B16F10) were obtained from
CAS (Chinese Academy of Sciences, China). The B16F10 cells were
grown in DMEM medium (GIBICO, USA) supplemented with 10%
heat-inactivated fetal bovine serum (GIBICO, USA), 100 U/mL peni-
cillin and 100 mg/mL streptomycin (GIBCO, USA) in a humidified
atmosphere with 5% CO2 at 37 °C.
5967
4.2.2. Melanin contents assay
Exponentially growing cells were seeded into 6-well plates at a
concentration of 5  105 cells per well. After 24 h incubation at
37 °C, the culture medium was removed and replaced with fresh
medium containing the candidate compounds in different concen-
trations. The cells were incubated for another 48 h, washed with
ice cold PBS, followed by lysis with RIPA buffer for 40 min on ice,
and the lysates were centrifuged at 10,000g for 20 min. Super-
natants containing protein were subject to the protein assay and
the pellets with intracellular melanin were solubilized in 200 ll
of 1 M NaOH for 2 h at 60 °C. Melanin amount was determined
spectrophotometrically at 405 nm by a multi-plate reader. The
melanin amount was calculated by normalizing the total melanin
values with protein content (abs melanin/lg protein).
Acknowledgements
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).
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