Anti-tyrosinase, antioxidant, and antibacterial activities of novel 5-hydroxy-4-acetyl-2,3-dihydronaphtho[1,2- b ]furans

Article abstract of DOI:10.1016/j.ejmech.2014.09.025

Novel 5-hydroxy-4-acetyl-2,3-dihydronaphtho[1,2-b]furans (7a-k) were synthesized using ceric ammonium nitrate (CAN)-catalyzed formal [3 + 2] cycloaddition. Synthesized compounds were evaluated for their tyrosinase inhibitory, antioxidant, and antibacterial activities. A modified spectrophotometric method using l-DOPA as substrate was used to determine tyrosinase inhibitory activities, and a 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay was used to evaluate antioxidant properties. Antibacterial activities against gram-negative Escherichia coli (KCTC-1924) and gram-positive Staphylococcus aureus (KCTC-1916) were evaluated using the disc diffusion technique. Of the synthesized compounds, 7b with a 4-acetyl and an electron-enriched dihydronaphthofuran ring showed the highest tyrosinase-inhibition activity (IC₅₀ Combining double low line 8.91 μg/mL), which was comparable with that of standard kojic acid (IC₅₀ Combining double low line 10.16 μg/mL), potent antioxidant activity (IC₅₀ Combining double low line 3.33 μg/mL), which was comparable with that of BHT (IC₅₀ Combining double low line 34.67 μg/mL), and excellent antibacterial activities (MICs: 0.50 μg/mL against E. coli and S. aureus strains). A mechanistic analysis of 7b demonstrated that its tyrosinase inhibitory activity was reversible and competitive. Compounds 7c and 7d showed potent antioxidant activities (IC₅₀: 6.30 and 5.01 μg/mL), and compound 7d also exhibited potent inhibitory activity against E. coli with a MIC of 0.5 μg/mL. Furthermore, compounds 7a, 7e, 7f, and 7i showed potent antibacterial activities against S. aureus with MICs of 0.5 μg/mL, which was comparable to that of ampicillin (MIC Combining double low line 0.5 μg/mL).

Full text of DOI:10.1016/j.ejmech.2014.09.025

European Journal of Medicinal Chemistry 86 (2014) 605e612
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Anti-tyrosinase, antioxidant, and antibacterial activities of novel 5-
hydroxy-4-acetyl-2,3-dihydronaphtho[1,2-b]furans
,
Likai Xia a, Akber Idhayadhulla a, Yong Rok Lee a *, Young-Jung Wee b, Sung Hong Kim c
a School of Chemical Engineering, Yeungnam University, Gyeongsan 712-749, Republic of Korea
b Department of Food Science and Technology, Yeungnam University, Gyeongsan 712-749, Republic of Korea
c Analysis Research Division, Daegu Center, Korea Basic Science Institute, Daegu 702-701, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
Novel 5-hydroxy-4-acetyl-2,3-dihydronaphtho[1,2-b]furans (7aek) were synthesized using ceric
ammonium nitrate (CAN)-catalyzed formal [3 þ 2] cycloaddition. Synthesized compounds were evalu-
ated for their tyrosinase inhibitory, antioxidant, and antibacterial activities. A modified spectrophoto-
Received 22 June 2014
Received in revised form
5 September 2014
Accepted 8 September 2014
Available online 9 September 2014
metric method using L-DOPA as substrate was used to determine tyrosinase inhibitory activities, and a
1,1-diphenyl-2-picrylhydrazyl (DPPH) assay was used to evaluate antioxidant properties. Antibacterial
activities against gram-negative Escherichia coli (KCTC-1924) and gram-positive Staphylococcus aureus
(KCTC-1916) were evaluated using the disc diffusion technique. Of the synthesized compounds, 7b with a
4-acetyl and an electron-enriched dihydronaphthofuran ring showed the highest tyrosinase-inhibition
Keywords:
Dihydronaphtho[1,2-b]furans
CAN
Formal [3 þ 2] cycloaddition
Anti-tyrosinase
Antioxidant
activity (IC50 ¼ 8.91
mL), potent antioxidant activity (IC50 ¼ 3.33
(IC50 ¼ 34.67 g/mL), and excellent antibacterial activities (MICs: 0.50
strains). A mechanistic analysis of 7b demonstrated that its tyrosinase inhibitory activity was reversible
m
g/mL), which was comparable with that of standard kojic acid (IC50 ¼ 10.16
g/mL), which was comparable with that of BHT
g/mL against E. coli and S. aureus
mg/
m
m
m
Antibacterial
and competitive. Compounds 7c and 7d showed potent antioxidant activities (IC50: 6.30 and 5.01
and compound 7d also exhibited potent inhibitory activity against E. coli with a MIC of 0.5
m
m
g/mL),
g/mL.
Furthermore, compounds 7a, 7e, 7f, and 7i showed potent antibacterial activities against S. aureus with
MICs of 0.5 g/mL).
m
g/mL, which was comparable to that of ampicillin (MIC ¼ 0.5
m
© 2014 Elsevier Masson SAS. All rights reserved.
1. Introduction
tyrosinase has also been linked to the neurodegenerations associ-
ated with Parkinson's disease and other degenerative diseases [9].
In addition, tyrosinase is known to be involved in the insect molting
process and the adhesion of marine organisms to substrates
[10e13]. In fruits and vegetables, enzymatic browning results
nutritional and commercial losses [14e17]. Thus, the development
of effective tyrosinase inhibitors has become an issue of importance
in the agricultural, medicinal, and cosmetic industries. Numerous
natural and synthetic tyrosinase inhibitors have been reported in
the literature [18e26], but a few, such as, kojic acid and arbutin, are
used practically as therapeutic agents or components of cosmetic
products. As a result, researchers are trying to identify novel
tyrosinase inhibitors with higher activities and lower side effects.
Nevertheless, it has been reported that antioxidants of natural or
synthetic origin could be of great importance as therapeutic agents
for the treatment of several diseases caused by oxidative stress [27].
Furthermore, the use of antioxidants as preservatives by the food
industry and as skin-protectives in cosmetics has also attracted
much interest [28].
Tyrosinase is
a copper-containing enzyme that is widely
distributed in microorganisms, plants, and animals [1,2], and is
involved in two distinct reactions required for melanin synthesis,
that is, the hydroxylation of monophenols and the conversion of o-
diphenols to corresponding o-quinones [3e6]. o-Quinones are
highly active compounds that can polymerize spontaneously to
form high molecular weight melanin pigments via a series of re-
actions with amino acids and proteins [1,2]. Melanin is one of the
most important natural biopolymers responsible for pigmentation
and the color patterns of mammalian skin, and protects skin from
solar radiation [7]. However, the accumulation of epidermal
pigmentation can cause some dermatological disorders associated
with melasma, freckles, and senile lentigines [8]. Recently, melanin
pigments were found in the mammalian brain, and interestingly,
* Corresponding author.
E-mail address: yrlee@yu.ac.kr (Y.R. Lee).
http://dx.doi.org/10.1016/j.ejmech.2014.09.025
0223-5234/© 2014 Elsevier Masson SAS. All rights reserved.

606
L. Xia et al. / European Journal of Medicinal Chemistry 86 (2014) 605e612
The naphthofuran structural motif is frequently found in bio-
methine proton at
dihydronaphthofuran ring and three methyl protons at
d
5.83 (1H, d, J ¼ 5.7 Hz) ppm attributed to a
2.58 ppm
logically active synthetic compounds and natural products [29e33].
For example, furomollugin and rubicordifolin were isolated from
Rubia cordifolia and found to possess significant anticancer prop-
erties [34e38]. In the research field, naphthofuran containing
molecules have been widely studied, for example, as hepatocyte
d
as a singlet due to the functional acetyl group. Furthermore,
treatment of 4 with 2-methoxypropene gave compound 7e in 38%
yield, whereas treatment with ethyl 1-propenyl ether (as a 30:70
mixture of cis and trans-isomers) provided both cis-7f and trans-7g
as racemates in 46 and 42% yield, respectively. The stereochemistry
of the cis-7f (J ¼ 6.3 Hz) and trans-7g (J ¼ ~0 Hz) was confirmed by
the coupling constants between vicinal protons on the dihy-
drofuran ring. Reaction of 4 with 2,3-dihydrofuran or 3,4-dihydro-
2H-pyran in the presence of 5 mol% of CAN for 0.5 h gave cis-7h and
nuclear factor 4 alpha (HNF4
choline receptor (nAChR) agonists [40], inhibitory kappa-
(IKK- ) inhibitors [40], and nuclear transcription factor kappa-
(NF-k ) inhibitors [41]. Naphthofuran derivatives have also been
a) inhibitors [39], nicotinic acetyl-
b
kinase
b
b
b
reported to possess diverse biological activities, including anti-
diabetic [42], anti-inflammatory [43], antimicrobial [44], and
analgesic activities [45]. Recently, we have reported that mollugin
and furomollugin, which possess a naphtho[1,2-b]pyran or naphtho
[1,2-b]furan skeleton bearing a hydroxyl and an ester group on the
ring, show potent antioxidant and antibacterial activities (Fig. 1)
[46,47]. To the best of our knowledge, the tyrosinase inhibitory
activities of naphthofuran derivatives have not been previously
investigated. Because mollugin and furomollugin did not show
potent anti-tyrosinase activities, we have designed other new
compounds bearing an acyl group on the naphtha[1,2-b]furan ring.
Thus, during our continued studies on the synthesis and biological
evaluations of new naphtho[1,2-b]furan derivatives, we prepared
various 5-hydroxy-4-acetyl-2,3-dihydronaphtho[1,2-b]furan de-
rivatives and evaluated their anti-tyrosinase, antioxidant, and
antibacterial activities in vitro.
cis-7i in 82 and 94% yield, respectively, whereas the use of
a-
methylstyrene or 4-methoxystyrene resulted in 7j and 7k in 72 and
78% yield, respectively. The stereochemistry of the cis-7h and cis-7i
was confirmed by comparing with the chemical shifts and the
vicinal coupling constants of the related reported cis-compounds
[54].
2.2. Biological activity
2.2.1. Anti-tyrosinase activity
To evaluate tyrosinase inhibitory activities, the synthesized 5-
hydroxy-4-acetyl-2,3-dihydronaphtho[1,2-b]furans were sub-
jected to a tyrosinase inhibition assay using 3,4-dihydroxy-
L-
phenylalanine ( -DOPA) as substrate, as described previously by
L
Bradford with slight modification [55]. Kojic acid, a well-known
tyrosinase inhibitor, which is heavily used as one of the basic skin
whitening ingredients in Asian countries, was selected as a refer-
ence. The results of studies conducted on the inhibitory effects of
2. Results and discussion
the
5-hydroxy-4-acetyl-2,3-dihydronaphtho[1,2-b]furans
on
2.1. Chemistry
diphenolase are summarized in Table 1. IC50 values were deter-
mined from logarithmic concentrationeinhibition curves and re-
sults are presented as the means of three independent
experiments. Compound 7b bearing a 2-n-propyloxy substituent
showed better inhibitory activity (IC50 ¼ 8.91
compounds or kojic acid (IC50 ¼ 10.16 g/mL). Compounds 7a, 7c
and 7d exhibited good mushroom tyrosinase inhibition with IC50
values ranging from 59.56 to 83.17 g/mL. In particular, compounds
7e, cis-7f, 7h, 7j and 7k poorly inhibited tyrosinase (IC50 > 100 g/
mL). Compound cis-7i had greater inhibitory activity than cis-7h,
probably due to the stereoelectronic constraint. Interestingly, trans-
7g demonstrated much greater inhibitory activity than the cis-7f
isomer.
5-Hydroxy-4-acetyl-2,3-dihydronaphtho[1,2-b]furans (7aek)
were synthesized as shown in Scheme 1 [48]. First, the diac-
etylation of 1 with acetyl chloride (2.1 equiv.) in the presence of
N,N-diisopropylethylamine (DIPEA, 3.0 equiv.) and 4-(dimethyla-
mino)pyridine (DMAP, 0.05 equiv.) in CH2Cl2 at room temperature
for 3 h gave naphthalene-1,4-diyl diacetate (2) in 95% yield. Fries
rearrangement of 2 in boron trifluoride complex afforded 1-(4-
ethoxy-1-hydroxynaphthalen-2-yl)ethan-1-one (3) in 88% yield
[49]. Compound 3 was then oxidized to the corresponding naph-
thoquinone 4 (90% yield) using ceric ammonium nitrate (CAN,
2.0 equiv.) [50e52]. As another method, the photo FriedeleCrafts
acylation of 1,4-naphthoquinone (5) followed by silver(I) oxide-
mediated oxidation provided compound 4 in 36% yield [53]. Next,
further reactions of 4 with vinyl ethers, such as, ethyl vinyl ether, n-
propyl vinyl ether, i-butyl vinyl ether, and n-butyl vinyl ether, in the
presence of 5 mol% of ceric ammonium nitrate (CAN) in acetonitrile
at room temperature for 20 min provided the desired products
7aed as racemates in 81, 79, 70, and 78% yield, respectively.
Compounds 7aed were easily separated by column chromatog-
raphy and identified by spectroscopic analyses. For example,
compound 7b showed a strong ketone carbonyl absorption peak at
1722 cmꢀ1 in its IR spectra, and its 1H NMR spectrum displayed a
mg/mL) than the other
m
m
m
2.2.1.1. Inhibitory mechanism. The kinetic behavior of the most
active compound 7b was studied with respect to the oxidation of L-
DOPA by mushroom tyrosinase at different concentrations. Initial
reaction rates were determined by monitoring dopachrome for-
mation at 475 nm. As shown in Fig. 2, LineweavereBurk plots of 1/V
versus 1/[S] resulted in a family of straight lines with the same
intercept on the vertical axis. In this figure, the abscissa 1/[
is the reciprocal of the -DOPA concentrations, whereas the ordi-
nate 1/V is the reciprocal of the reaction rate, which reflects the
reciprocal of tyrosinase activity. The plots obtained indicated that
L-DOPA]
L
compound 7b (Ki ¼ 11.2
mM at 25 mM) is a competitive inhibitor and
that its inhibitory activity decreases with increasing substrate
concentration. Compound 7b has a 2-hydroxyketone unit and kojic
acid contains an
a-hydroxyketone group, which suggest that
inhibitory mechanism of 7b and kojic acid is similar (Scheme 2)
[56].
2.2.2. Antioxidant activity
The synthesized 5-hydroxy-4-acetyl-2,3-dihydronaphtho[1,2-b]
furans (7aek) were also evaluated with respect to their antioxidant
activities using 1,1-diphenyl-2-picrylhydrazyl (DPPH), a stable free
Fig. 1. Structures of mollugin, furomollugin, and naphtho[1,2-b]furans.

L. Xia et al. / European Journal of Medicinal Chemistry 86 (2014) 605e612
607
Scheme 1. Synthetic route of 5-hydroxy-4-acetyl-2,3-dihydronaphtho[1,2-b]furan derivatives. Reagents and conditions: (a) acetyl chloride (2.1 equiv), DIPEA (3.0 equiv), DMAP
(0.2 equiv), CH2Cl2, RT, 3 h, 96%; (b) BF3$Et2O (5 mL), reflux, 4 h, 90%; (c) CAN (2.0 equiv), acetonitrile, RT, 30 min, 90%; (d) acetaldehyde (5.0 equiv), h , degased benzene, RT, 48 h,
45%; (e) Ag2O (3.3 equiv), MgSO4 (2.0 equiv), Et2O, RT, 6 h, 80%; (f) olefin (2.0 equiv), catalyst CAN (0.05 equiv), acetonitrile, RT, 20 min, (7aek, 38e96%).
n
radical species that is often used to study the free radical scav-
enging activities of antioxidants. Initially, the stabilities of DPPH
methanol solutions containing compounds 7aek were examined
by storing them in the dark at 25 ꢁC and measured absorbances at
regular intervals (Fig. 3). Absorbances decreased rapidly (within
10 min) in the presence of compounds 7aee, and less rapidly for
compounds 7fek (within 15 min) to the lower detection limit.
Thus, in subsequent studies, a minimum incubation time of 15-min
was required for determining the DPPH radical scavenging activ-
ities of 5-hydroxy-4-acetyl-2,3-dihydronaphtho[1,2-b]furans.
Next, the scavenging activities of the synthesized 5-hydroxy-4-
acetyl-2,3-dihydronaphtho[1,2-b]furans (7aek) were examined,
and the majority were found to be potent DPPH scavengers
(Table 2). More specifically, 7b, 7c, and 7d potently scavenged DPPH
growths. MIC (Minimal inhibitory concentration) was defined at
lowest concentration that completely inhibited visible bacterial
growth, and ampicillin was used as reference standard. All tested
compounds exhibited moderate to high inhibitory activity
(Table 3), and most of them showed potent activities against Gram-
positive and Gram-negative strains. Of the compounds tested,
compounds 7b and 7d exhibited greatest inhibitory activity against
E. coli (MIC ¼ 0.5
cillin (MIC: 1 g/mL). Compounds 7a, 7b, 7e, 7f, and 7i had inhib-
itory activities similar to that of standard ampicillin against
mg/mL for both) and greater activity than ampi-
m
S. aureus (all MICs: 0.5 mg/mL).
3. Conclusions
(IC50: 3.33, 6.30, and 5.01
showed moderate activity (IC50: 25.11, 31.47
(IC50: 34.67 g/mL). The antioxidant mechanism of the 5-hydroxy-
m
g/mL, respectively), whereas 7a, and 7e
We synthesized eleven acetyl-5-hydroxy-naphtho[1,2-b]furan
derivatives (7aek) and evaluated their anti-tyrosinase, antioxidant,
and antibacterial activities. Compound 7b significantly inhibited
mg/mL) versus BHT
m
4-acetyl-2,3-dihydronaphtho[1,2-b]furans could be explained
based on its chemical structures and comparisons with Vitamin E
[56]. For example, compound 7b, which bears a 2-hydroxyketone,
can donate an H atom from its phenol group to DPPHꢂ to form the
resonance-stabilized free-radical intermediate 8 (Scheme 3). The
existence of an aromatic residue on the oxygen atom and a dihy-
dronaphthofuran ring would stabilize the phenoxy radical formed,
and thus, enhance antioxidant activity. Furthermore, intermediate
8 could react with a second DPPHꢂ to form an inactive anion, which
on cleavage by protonation would give naphthalene-1,4-dione 9
[57].
tyrosinase (IC50 ¼ 8.91
m
g/mL) and potently reduced the free
g/mL). In terms of in vitro antibacterial
radical, DPPH (IC50 ¼ 3.33
m
activities, compounds 7b and 7d were most active against E. coli,
and compounds 7a, 7b, 7e, 7f, and 7i were most active against
S. aureus. These results suggest that 5-hydroxy-4-acetyl-2,3-
dihydronaphtho[1,2-b]furan derivatives are of biological impor-
tance and could serve as a starting point for the development of
new anti-tyrosinase, antioxidant, and antibacterial agents.
4. Experimental section
4.1. Chemistry
2.2.3. Antibacterial activity
The antibacterial activities of compounds (7aek) were screened
using the disk diffusion method. Experiments were performed
using test Gram-negative and Gram-positive organisms, namely,
Escherichia coli (E. coli, KCTC-1924) and Staphylococcus aureus
(S. aureus, KCTC-1916), respectively. The results obtained revealed
that the majority of the synthesized compounds inhibited bacterial
4.1.1. General methods
All chemicals were purchased from SigmaeAldrich, Fluka, or
Tokyo Chemical Industries (TCI), and used without further purifi-
cation. Solvents were dried and distilled prior to use. All synthetic
experiments were carried out in a N2 atmosphere. Merck precoated
silica gel plates (Art. 5554) with a fluorescent indicator were used

608
L. Xia et al. / European Journal of Medicinal Chemistry 86 (2014) 605e612
Table 1
4.1.2. Synthesis of naphthalene-1,4-diyl diacetate (2)
The mushroom tyrosinase inhibitory activities of compounds (7aek) and standard
To a solution of 1,4-dihydroxynaphthalene (1, 16.02 g, 0.10 mol)
in CH2Cl2 (150 mL) was added N,N-diisopropylethylamine (DIPEA,
52.3 mL, 0.30 mol) and 4-(dimethylamino)pyridine (DMAP, 2.44 g,
0.02 mol) at room temperature. The reaction mixture was stirred at
room temperature for 30 min, and then, acetyl chloride (14.9 mL,
0.21 mol) was added incrementally by syringe. Reaction progress
was monitored by TLC. After all the starting material was
consumed, the reaction mixture was quenched with water
(100 mL) and the aqueous solution was extracted with CH2Cl2
(100 mL ꢃ 3). Combined organic extracts were washed with water,
dried (MgSO4), and evaporated in vacuo. Flash chromatography on
silica gel using hexane/ethyl acetate (4:1) as eluant afforded 2
(23.4 g, 96%) as a yellow solid: mp 126e127 ꢁC [58]; 1H NMR
kojic acid.
Compound
Concentration (
25
m
g/mL)a
50
IC50 (m
g/mL)a
100
7a
7b
7c
7d
7e
7f
7g
7h
18.75 0.65
53.41 0.94
38.06 0.78
32.95 0.46
12.50 0.96
28.41 0.65
46.21 0.49
36.93 0.96
48.83 0.92
20.45 0.65
3.043 0.85
55.69 0.21
27.84 0.24
64.20 0.98
44.31 0.45
38.63 0.65
19.10 0.74
34.65 0.16
57.98 0.49
40.34 0.74
50.0 0.56
23.86 0.47
9.09 0.69
68.18 0.32
58.52 0.95
89.47 0.48
59.09 0.11
59.28 0.98
25.56 0.78
45.45 0.65
68.06 0.96
47.72 0.14
57.95 0.98
32.95 0.58
11.93 0.36
85.79 0.13
83.17 0.12
8.91 0.24
59.56 0.95
69.82 0.72
>100
>100
30.61 0.91
>100
7i
7j
50.00 0.82
>100
>100
7k
Kojic acid
(300 MHz, CDCl3)
d
7.82e7.79 (m, 2H), 7.49e7.46 (m, 2H), 7.18 (brs,
169.35, 144.22, 127.53,
10.16 0.16
2H), 2.38 (s, 6H); 13C NMR (75 MHz, CDCl3)
d
a
The IC50 values shown are the means SEs of three different experiments.
126.93, 121.52, 117.60, 20.95; IR (KBr): 3082, 2937, 1755, 1431, 1370,
1205, 1153, 1056, 1010, 902, 765, 602 cmꢀ1; HRMS m/z (Mþ) calcd.
for C14H12O4: 244.0736, found 244.0736.
4.1.3. Synthesis of 1-(4-ethoxy-1-hydroxynaphthalen-2-yl)
ethanone (3)
This synthesis was conducted as described by Wigal et al. [49].
Briefly, boron trifluoride diethyl etherate (80.0 mL) and 2 (22.0 g,
90 mmol) were placed in a 250-mL round-bottom flask equipped
with a condenser and a stirrer bar and refluxed for 4 h. The
resulting solution was allowed to cool to room temperature, poured
into water, and extracted with dichloromethane. The organic phase
was washed with water and dried over anhydrous MgSO4. The
solvent was removed under reduced pressure, and the crude
product obtained was purified by flash chromatography on silica
gel using hexane/ethyl acetate (4:1) to afford 3 (18.7 g, 90%) as a
yellow solid; mp 113e114 ꢁC; 1H NMR (300 MHz, CDCl3)
d 13.72 (s,
1H), 8.41 (1H, d, J ¼ 8.1 Hz), 8.19 (1H, d, J ¼ 8.1 Hz), 7.64 (1H, dd,
J1 ¼ J2 ¼ 8.1 Hz), 7.55 (1H, dd, J1 ¼ J2 ¼ 8.1 Hz), 6.77 (s, 1H), 4.11 (q,
J ¼ 6.9 Hz, 2H), 2.63 (s, 3H), 1.53 (t, J ¼ 6.9 Hz, 3H); 13C NMR
(75 MHz, CDCl3)
d 203.74, 157.20, 146.63, 130.42, 129.62, 126.50,
125.82, 124.26, 121.96, 111.99, 101.83, 64.06, 27.03, 14.84; IR (KBr):
3013, 2981, 2878,1627, 1576,1456,1383,1325,1240,1103, 1027, 979,
861, 814, 773 cmꢀ1; HRMS m/z (Mþ) calcd. for C14H14O3: 230.0943,
found 230.0944.
Fig. 2. LineweavereBurk plot for the inhibition of the diphenolase activity of tyrosi-
nase by compound 7b. Concentrations of compound 7b for curves were 0
and 87.38 M, respectively.
mM, 3.50 mM,
m
4.1.4. Synthesis of 2-acetylnaphthalene-1,4-dione (4)
A solution of ammonium cerium(IV) nitrate (CAN, 87.7 g,
0.16 mol) in water (150 mL) was added to an acetonitrile solution
(80 mL) of 3 (18.4 g, 80 mmol). The mixture was stirred for 50 min
and then poured into water-CH2C12. The aqueous phase obtained
was further extracted with CH2Cl2, and the combined organic phase
7a
0.7
7b
7c
7d
7e
7f
0.6
0.5
0.4
0.3
0.2
0.1
0.0
Scheme 2. The reversible competitive binding of 7b to the binuclear active site of
tyrosinase.
7g
7h
7i
for analytical TLC. Flash column chromatography (FC) was per-
formed using silica gel 7734 (Merck). 1H and 13C NMR spectra were
recorded on a Bruker Model ARX spectrometer (at 300 and 75 MHz,
respectively) using TMS as an internal standard in deuterated
chloroform or dimethylsulfoxide. IR spectra were recorded on a
BioRad FTS 3000 spectrophotometer. High-resolution mass spectra
(HR-MS) were recorded on a JEOL JMS-600 mass spectrometer at
the Korean Basic Science Institute.
7j
7k
BHT
0
5
10
15
20
25
30
Time (min)
Fig. 3. The temporal effect of compounds 7aek and BHT (100 ug/mL) on the absor-
bance of DPPH solution.

L. Xia et al. / European Journal of Medicinal Chemistry 86 (2014) 605e612
609
Table 2
The in vitro DPPH radical scavenging activities of compounds (7aek).
Compound
Concentration (
1.0
m
g/mL)a
IC50 (m
g/mL)a
25
50
100
7a
7b
7c
7d
7e
7f
7g
7h
7i
21.57 0.16
35.32 0.45
26.94 0.19
25.05 0.39
e
45.02 0.69
70.23 0.56
61.15 0.69
76.02 0.16
37.26 0.97
26.94 0.64
30.56 0.28
28.31 0.46
27.68 0.61
26.67 0.26
25.05 0.16
40.77 0.46
81.36 0.26
89.74 0.46
81.90 0.55
85.83 0.49
75.47 0.86
41.47 0.13
30.77 0.31
27.40 0.15
26.94 0.29
30.63 0.84
26.03 0.16
65.62 0.49
88.94 0.88
91.03 0.43
90.94 0.63
88.98 0.24
80.26 0.64
63.68 0.99
36.06 0.54
28.94 0.64
33.36 0.96
59.89 0.14
28.63 0.32
76.47 0.62
25.11 0.26
3.33 0.16
6.30 0.32
5.01 0.21
31.47 0.45
62.51 0.55
>100
>100
>100
77.62 0.22
>100
34.67 0.69
e
10.22 0.19
5.23 0.26
8.94 0.29
16.73 0.19
5.10 0.24
e
7j
7k
BHT
a
Values represent mean SE of three different reactions.
was washed with water then brine, and dried over Na2SO4. Evap-
oration the solvent yielded 4 (14.4 g, 90%), which was pure enough
for use in subsequent experiments without further purification. The
spectral data of 4 were in accord with literature values [53,59,60].
Compound 4 can also be prepared by the photoacylation of 1,4-
naphthoquinone with acetaldehyde followed by Ag2O mediated
oxidation, as previously described [53].
J ¼ 8.1 Hz, 1H), 7.61 (dd, J ¼ 8.1, 7.2 Hz, 1H), 7.47 (dd, J ¼ 8.1, 7.2 Hz,
1H), 5.82 (d, J ¼ 6.6 Hz, 1H), 3.76e3.71 (m, 1H), 3.68e3.61 (m, 1H),
3.41e3.35 (m, 2H), 2.59 (s, 3H), 1.92e1.81 (m, 1H), 0.90 (t, J ¼ 6.9 Hz,
6H); 13C NMR (75 MHz, CDCl3)
d 203.55, 158.86, 145.67, 129.93,
125.77, 125.22, 124.98, 124.92, 121.19, 113.49, 111.28, 105.57, 75.40,
41.22, 30.70, 28.44, 19.35, 19.25; IR (neat): 3064, 2958, 1713, 1631,
1452, 1391, 1282, 1110, 1025, 941, 887, 766 cmꢀ1; HRMS (EIþ): m/z:
calcd for C18H20O4: 300.1362, found 300.1360.
4.1.5. General procedure for the preparation of 5-hydroxy-4-acetyl-
2,3-dihydronaphtho[1,2-b]furan derivatives 7aek
4.1.5.4. 1-(2-Butoxy-5-hydroxy-2,3-dihydronaphtho[1,2-b]furan-4-
To
a
solution of methyl 1,4-dihydroxynaphthalene-2-
yl)ethanone (7d). Yellow liquid; yield 234 mg, 78%; 1H NMR
carboxylate (9, 218 mg, 1.0 mmol) and the corresponding olefin
(2.0 mmol) in MeCN (5.0 mL) was added cerium (IV) ammonium
nitrate (CAN, 5.0 mol%) at room temperature. The reaction was
monitored by TLC. After all starting material had been consumed,
water (10 mL) was added and the solution was extracted with ethyl
acetate (10 mL ꢃ 3). Evaporation of the solvent and purification by
silica gel column chromatography using hexane-ethyl acetate
(10:1) as eluant gave the desired products [48].
(300 MHz, CDCl3)
d
14.20, (s, 1H), 8.39 (d, J ¼ 8.4 Hz, 1H), 7.87 (d,
J ¼ 8.1 Hz, 1H), 7.60 (dd, J ¼ 8.1, 7.2 Hz, 1H), 7.46 (dd, J ¼ 8.4, 7.2 Hz,
1H), 5.82 (d, J ¼ 6.6 Hz, 1H), 3.98e3.91 (m, 1H), 3.66e3.60 (m, 2H),
3.39e3.33 (m, 1H), 2.57 (s, 3H), 1.61e1.50 (m, 2H), 1.42e1.30 (m,
2H), 0.90 (t, J ¼ 7.2 Hz, 3H); 13C NMR (75 MHz, CDCl3)
d 203.52,
158.83, 145.60, 129.90, 125.74, 125.17, 124.94, 124.87, 121.14, 113.43,
111.19, 105.34, 68.37, 41.20, 31.61, 30.66, 19.17, 13.76; IR (neat): 2977,
2928, 1733, 1623, 1450, 1393, 1248, 1112, 1004, 887, 846, 766,
640 cmꢀ1; HRMS (EIþ): m/z: calcd for C18H20O4: 300.1362, found
300.1360.
4.1.5.1. 1-(2-Ethoxy-5-hydroxy-2,3-dihydronaphtho[1,2-b]furan-4-
yl)ethanone (7a). Yellow liquid; yield 220 mg, 81%; 1H NMR
(300 MHz, CDCl3)
d
14.24, (s, 1H), 8.41 (d, J ¼ 8.4 Hz, 1H), 7.89 (d,
J ¼ 8.1 Hz, 1H), 7.62 (dd, J ¼ 8.1, 7.2 Hz, 1H), 7.48 (dd, J ¼ 7.8, 7.5 Hz,
1H), 5.86 (d, J ¼ 5.7 Hz, 1H), 4.07e3.97 (m, 1H), 3.73e3.64 (m, 2H),
3.40 (d, J ¼ 16.5 Hz,1H), 2.60 (s, 3H),1.25 (t, J ¼ 7.2 Hz, 3H); 13C NMR
4.1.5.5. 1-(5-Hydroxy-2-methoxy-2-methyl-2,3-dihydronaphtho[1,2-
b]furan-4-yl)ethanone (7e). Yellow solid; yield 103 mg, 38%; mp
124e125 ꢁC; 1H NMR (300 MHz, CDCl3)
d 14.23, (s, 1H), 8.42 (d,
(75 MHz, CDCl3)
d 203.57, 158.96, 145.65, 129.98, 125.84, 125.25,
J ¼ 8.4 Hz, 1H), 7.91 (d, J ¼ 8.1 Hz, 1H), 7.62 (dd, J ¼ 7.8, 7.2 Hz, 1H),
7.49 (dd, J ¼ 7.8, 7.2 Hz, 1H), 3.60 (d, J ¼ 16.8 Hz, 1H), 3.50 (d,
J ¼ 17.1 Hz, 1H), 3.35 (s, 3H), 2.62 (s, 3H), 1.78 (s, 3H); 13C NMR
125.02, 124.94, 121.18, 113.41, 111.24, 105.17, 64.09, 41.32, 30.74,
15.17; IR (neat): 2971, 1710, 1665, 1597, 1445, 1347, 1228, 1161, 1150,
1027, 944, 876, 764 cmꢀ1; HRMS (EIþ): m/z: calcd for C16H16O4:
272.1049, found 272.1050.
(75 MHz, CDCl3)
d 203.53, 158.92, 145.90, 130.04, 125.93, 125.36,
125.05, 124.68, 121.25, 113.84, 111.15, 110.76, 49.74, 45.44, 30.74,
24.27; IR (KBr): 2962, 1713, 1623, 1451, 1387, 1312, 1233, 1056, 849,
768, 642 cmꢀ1; HRMS (EIþ): m/z: calcd for C16H16O4: 272.1049,
found 272.1046.
4.1.5.2. 1-(5-Hydroxy-2-propoxy-2,3-dihydronaphtho[1,2-b]furan-4-
yl)ethanone (7b). Yellow liquid; yield 226 mg, 79%; 1H NMR
(300 MHz, CDCl3)
d
14.22, (s, 1H), 8.39 (d, J ¼ 8.1 Hz, 1H), 7.88 (d,
J ¼ 8.1 Hz, 1H), 7.61 (t, J ¼ 7.5 Hz, 1H), 7.47 (dd, J ¼ 7.8, 7.5 Hz, 1H),
5.83 (d, J ¼ 5.7 Hz, 1H), 3.94e3.87 (m, 1H), 3.69e3.35 (m, 3H), 2.58
(s, 3H), 1.68e1.52 (m, 2H), 0.90 (t, J ¼ 6.9 Hz, 3H); 13C NMR (75 MHz,
4.1.5.6. 1-(cis-2-Ethoxy-5-hydroxy-3-methyl-2,3-dihydronaphtho
[1,2-b]furan-4-yl)ethanone (7f). Yellow liquid; yield 132 mg, 46%;
mp 166e167 ꢁC; 1H NMR (300 MHz, CDCl3)
d 14.15 (s, 1H), 8.40 (d,
CDCl3)
d 203.56, 158.95, 145.70, 129.97, 125.82, 125.28, 125.02,
124.97, 121.20, 113.45, 111.29, 105.40, 70.35, 41.30, 30.72, 22.89,
10.52; IR (neat): 2962, 2874,1722, 1623,1452,1392,1281, 1111,1004,
888, 765 cmꢀ1; HRMS (EIþ): m/z: calcd for C17H18O4: 286.1205,
found 286.1209.
J ¼ 8.4 Hz, 1H), 7.87 (d, J ¼ 8.1 Hz, 1H), 7.66e7.45 (m, 2H), 5.88 (d,
J ¼ 6.3 Hz, 1H), 4.17e4.09 (m, 1H), 3.86e3.69 (m, 2H), 2.68 (s, 3H),
1.36 (t, J ¼ 7.2 Hz, 3H), 1.23 (t, J ¼ 6.9 Hz, 3H); 13C NMR (75 MHz,
CDCl3)
d 203.42, 158.77, 146.62, 129.92, 126.04, 125.41, 124.97,
124.91, 121.49, 119.90, 110.96, 108.76, 66.42, 42.81, 30.07, 15.23,
14.48; IR (neat): 1629, 1451, 1387, 1320, 1240, 1157, 1090, 980, 918,
871, 770 cmꢀ1; HRMS (EIþ): m/z: calcd for C17H18O4: 286.1205,
found 286.1209.
4.1.5.3. 1-(5-Hydroxy-2-isobutoxy-2,3-dihydronaphtho[1,2-b]furan-
4-yl)ethanone (7c). Yellow liquid; yield 210 mg, 70%; 1H NMR
(300 MHz, CDCl3)
d
14.22, (s, 1H), 8.40 (d, J ¼ 8.4 Hz, 1H), 7.88 (d,

610
L. Xia et al. / European Journal of Medicinal Chemistry 86 (2014) 605e612
Scheme 3. Oxidation of 7b with DPPH.
Table 3
4.1.5.10. 1-(5-Hydroxy-2-methyl-2-phenyl-2,3-dihydronaphtho[1,2-
b]furan-4-yl)ethanone (7j). Yellow solid; yield 229 mg, 72%; mp
119e120 ꢁC; 1H NMR (300 MHz, CDCl3)
14.23, (s, 1H), 8.43 (d,
Minimal inhibitory concentrations (
m
g/mL) of compounds (7aek).
d
Compound
E. coli
S. aureus
J ¼ 8.1 Hz, 1H), 8.05 (d, J ¼ 7.2 Hz, 1H), 7.67 (t, J ¼ 6.9 Hz, 1H),
7.56e7.49 (m, 3H), 3.35 (dd, J ¼ 7.5, 6.9 Hz, 2H), 7.28 (t, J ¼ 6.9 Hz,
1H), 3.78 (s, 2H), 2.59 (s, 3H), 1.88 (s, 3H); 13C NMR (75 MHz, CDCl3)
7a
7b
7c
7d
7e
7f
7g
7h
16
0.5
ND
0.5
16
32
ND
2
0.5
0.5
32
8
0.5
0.5
ND
8
d
203.57, 158.78, 146.96, 146.47, 130.01, 128.50, 127.23, 125.92,
125.06, 124.97, 124.37, 121.45, 113.65, 111.54, 102.07, 88.41, 49.69,
30.81, 29.40; IR (KBr): 3067, 2977, 2926, 1663, 1450, 1383, 1288,
1157, 1102, 902, 833, 769, 725 cmꢀ1; HRMS (EIþ): m/z: calcd for
7i
7g
32
2
ND
1
0.5
32
32
0.5
C
21H18O3: 318.1256, found 318.1252.
7k
Ampicillin
4.1.5.11. 1-(5-Hydroxy-2-(4-methoxyphenyl)-2,3-dihydronaphtho
[1,2-b]furan-4-yl)ethanone (7k). Yellow solid; yield 261 mg, 78%;
ND: Not determined.
mp 138e139 ꢁC; 1H NMR (300 MHz, CDCl3)
d 14.26, (s, 1H), 8.44 (d,
J ¼ 8.4 Hz, 1H), 7.92 (d, J ¼ 8.1 Hz, 1H), 7.61 (dd, J ¼ 8.4, 6.9 Hz, 1H),
7.50 (dd, J ¼ 8.4, 6.9 Hz, 1H), 7.37 (d, J ¼ 9.0 Hz, 2H), 6.91 (d,
J ¼ 8.7 Hz, 2H), 5.83 (dd, J ¼ 9.6, 8.4 Hz, 1H), 3.95 (dd, J ¼ 15.6,
9.6 Hz, 1H), 3.52 (dd, J ¼ 15.6, 8.1 Hz, 1H), 3.80 (s, 3H), 2.58 (s, 3H);
4.1.5.7. 1-(trans-2-Ethoxy-5-hydroxy-3-methyl-2,3-dihydronaphtho
[1,2-b]furan-4-yl)ethanone (7g). Yellow liquid; yield 120 mg, 42%;
1H NMR (300 MHz, CDCl3)
d
14.31, (s, 1H), 8.41 (d, J ¼ 8.4 Hz, 1H),
13C NMR (75 MHz, CDCl3)
d 203.85, 159.79, 159.00, 147.47, 134.30,
7.91 (d, J ¼ 8.1 Hz, 1H), 7.61 (dd, J ¼ 8.1, 6.9 Hz, 1H), 7.47 (dd, J ¼ 8.1,
7.2 Hz, 1H), 5.39 (brs, 1H), 4.04e3.93 (m, 1H), 3.74e3.66 (m, 2H),
2.71 (s, 3H), 1.29 (d, J ¼ 7.2 Hz, 3H), 1.22 (t, J ¼ 7.2 Hz, 3H); 13C NMR
130.21, 127.38, 126.16, 125.57, 125.21, 124.91, 121.67, 114.34, 114.30,
111.59, 83.60, 55.53, 43.35, 31.02; IR (KBr): 3057, 2931, 2840, 1630,
1513, 1450, 1389, 1254, 1174, 1086, 1031, 886, 834, 765 cmꢀ1; HRMS
(EIþ): m/z: calcd for C21H18O4: 334.1205, found 334.1202.
(75 MHz, CDCl3)
d 203.43, 159.34, 144.87, 129.98, 126.56, 126.00,
125.89, 125.03, 121.19, 119.69, 111.09, 110.75, 63.98, 46.42, 30.00,
19.15, 15.14; IR (neat): 2973, 2929, 2877, 1716, 1622, 1449, 1389,
1287, 1238, 1105, 1045, 979, 915, 880, 769 cmꢀ1; HRMS (EIþ): m/z:
calcd for C17H18O4: 286.1205, found 286.1206.
4.2. Biological activity
4.2.1. Anti-tyrosinase activity
Tyrosinase inhibitory activities were measured spectrophoto-
metrically according to a modified method [55] using Mushroom
tyrosinase (EC 1.14.18.1, lyophilized powder, ꢄ1000 unit/mg solid,
4.1.5.8. cis-1-(5-Hydroxy-6b,7,8,9a-tetrahydrofuro[2,3-b]naphtho
[2,1-d]furan-6-yl)ethanone (7h). Yellow solid; yield 221 mg, 82%;
mp 148e149 ꢁC; 1H NMR (300 MHz, CDCl3)
d 14.23, (s, 1H), 8.40 (d,
Sigma Chemical Co.) and L-DOPA as substrate. Briefly, a reaction
mixture (3.0 mL of final volume) containing 1.5 mM L-DOPA,
J ¼ 8.4 Hz, 1H), 7.90 (d, J ¼ 8.1 Hz, 1H), 7.63 (dd, J ¼ 8.1, 7.2 Hz, 1H),
7.51 (dd, J ¼ 8.1, 7.2 Hz, 1H), 6.50 (d, J ¼ 6.0 Hz, 1H), 4.36 (t,
J ¼ 8.7 Hz, 1H), 4.11 (t, J ¼ 8.4 Hz, 1H), 3.70e3.61 (m, 1H), 2.70 (s,
3H), 2.50e2.36 (m, 1H), 2.01e1.96 (m, 1H); 13C NMR (75 MHz,
0.1 mM sodium phosphate buffer (pH 6.5), 12.428 U of mushroom
tyrosinase, and the test sample was incubated at 30 ꢁC for 2 min.
The formation of dopachrome was determined spectrophotomet-
rically by monitoring absorption at 475 nm using a Shimadzu UV-
160A spectrophotometer. Kojic acid was used as a positive con-
trol. Percentage tyrosinase inhibition was calculated using the
following equation:
CDCl3)
d 202.96, 159.56, 146.49, 130.30, 126.40, 125.62, 124.94,
124.12, 121.79, 115.25, 110.78, 110.44, 66.39, 49.63, 34.35, 30.41; IR
(KBr): 3059, 2982, 2882, 1616, 1448, 1394, 1310, 1243, 1154, 1088,
984, 884, 771 cmꢀ1; HRMS (EIþ): m/z: calcd for C16H14O4: 270.0892,
found 270.0888.
Tyrosinase inhibitory activityð%Þ
¼ ½ðA ꢀ BÞ ꢀ ðC ꢀ DÞꢅ=ðA ꢀ BÞ ꢃ 100
4.1.5.9. cis-1-(5-Hydroxy-7,8,9,10a-tetrahydro-6bH-naphtho
Where A is the absorbance of the blank solution after incubation, B
is the absorbance of the blank solution before incubation, C is the
absorbance of a sample solution after incubation, and D is the
absorbance of the sample solution before incubation.
[2',1':4,5]furo[2,3-b]pyran-6-yl)ethanone (7i). Yellow solid; yield
267 mg, 94%; mp 100e101 ꢁC; 1H NMR (300 MHz, CDCl3)
d 14.20, (s,
1H), 8.39 (d, J ¼ 8.4 Hz, 1H), 7.88 (d, J ¼ 8.4 Hz, 1H), 7.60 (dd, J ¼ 8.4,
6.9 Hz, 1H), 7.46 (dd, J ¼ 8.4, 6.9 Hz, 1H), 5.83 (d, J ¼ 6.6 Hz, 1H),
3.94e3.86 (m, 1H), 3.67e3.50 (m, 1H), 3.40e3.34 (m, 1H), 2.58 (s,
3H), 1.66e1.51 (m, 4H); 13C NMR (75 MHz, CDCl3)
d
203.19, 159.16,
4.2.2. Antioxidant activity by DPPH assay
144.49, 130.15, 126.21, 125.46, 125.01, 121.59, 120.11, 113.44, 110.68,
104.41, 65.44, 40.95, 29.88, 27.00, 22.65; IR (KBr): 2940, 1725, 1626,
1449, 1388, 1276, 1130, 1075, 975, 869, 768 cmꢀ1; HRMS (EIþ): m/z:
calcd for C17H16O4: 284.1049, found 284.1048.
Methanolic solutions of DPPH exhibit a strong purple color and
strong absorption at 517 nm, and this is reduced in the presence of
an antioxidant [61,62]. Thus, there is an inverse relationship be-
tween DPPH concentration and anti-radical activity. Various

L. Xia et al. / European Journal of Medicinal Chemistry 86 (2014) 605e612
611
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~
ꢀ
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Acknowledgments
This research was supported by the Nano Material Technology
Development Program through the Korean National Research
Foundation (NRF) funded by the Korean Ministry of Education,
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