Discovery of benzylidenebenzofuran-3(2H)-one (aurones) as inhibitors of tyrosinase derived from human melanocytes

Article abstract of DOI:10.1021/jm050715i

Tyrosinase is a copper-dependent enzyme which converts L- tyrosine to dopaquinone and is involved in different biological processes such as melanogenesis and skin hyperpigmentation. The purpose of this study was to investigate naturally occurring aurones (Z-benzylidenebenzofuran-3(2H)-one) and analogues as human tyrosinase inhibitors. Several aurones bearing hydroxyl groups on A-ring and different substituents on B-ring were synthesized and evaluated as inhibitors of human melanocyte-tyrosinase by an assay which measures tyrosinase-catalyzed L-Dopa oxidation. We found that unsubstituted aurones were weak inhibitors; however, derivatives with two or three hydroxyl groups preferably at 4,6 and 4′ positions are able to induce significant tyrosinase inhibition. The most potent aurone was found to be the naturally occurring 4,6,4′-trihydroxyaurone which induces 75% inhibition at 0.1 mM concentration and is highly effective when compared to kojic acid, one of the best tyrosinase inhibitors known so far (the latter is completely inactive at such concentrations). Active aurones are devoid of toxic effects as shown by in vivo studies.

Full text of DOI:10.1021/jm050715i

J. Med. Chem. 2006, 49, 329-333
329
Discovery of Benzylidenebenzofuran-3(2H)-one (Aurones) as Inhibitors of Tyrosinase Derived
from Human Melanocytes
Sabrina Okombi,† Delphine Rival,‡ Se´bastien Bonnet,‡ Anne-Marie Mariotte,† Eric Perrier,‡ and Ahce`ne Boumendjel*,†
De´partement de Pharmacochimie Mole´culaire, UMR-CNRS 5063, Faculte´ de Pharmacie de Grenoble, 5 aVenue de Verdun,
BP 138, 38243 Meylan, France, and Engelhard-Lyon, 32 rue St Jean de Dieu, 69007 Lyon, France
ReceiVed July 26, 2005
Tyrosinase is a copper-dependent enzyme which converts L- tyrosine to dopaquinone and is involved in
different biological processes such as melanogenesis and skin hyperpigmentation. The purpose of this study
was to investigate naturally occurring aurones (Z-benzylidenebenzofuran-3(2H)-one) and analogues as human
tyrosinase inhibitors. Several aurones bearing hydroxyl groups on A-ring and different substituents on B-ring
were synthesized and evaluated as inhibitors of human melanocyte-tyrosinase by an assay which measures
tyrosinase-catalyzed L-Dopa oxidation. We found that unsubstituted aurones were weak inhibitors; however,
derivatives with two or three hydroxyl groups preferably at 4,6 and 4′ positions are able to induce significant
tyrosinase inhibition. The most potent aurone was found to be the naturally occurring 4,6,4′-trihydroxyaurone
which induces 75% inhibition at 0.1 mM concentration and is highly effective when compared to kojic
acid, one of the best tyrosinase inhibitors known so far (the latter is completely inactive at such concentrations).
Active aurones are devoid of toxic effects as shown by in vivo studies.
Introduction
Tyrosinase, is a copper-containing enzyme, widely present
in mammals, plants, and fungi and accepts many phenols and
catechols as substrates.1,2 In mammals, it is implied in the
transformation of L-tyrosine to dopaquinone which occurs
through two steps: hydroxylation of L-tyrosine to L-3,4-
dihydroxyphenylalanine (L-DOPA) and then oxidation of the
latter to an o-quinone (dopaquinone). Dopaquinone is further
transformed through several reactions to yield brown to black
melanin which is responsible for color of mammals’ skin.3,4
Melanin pigments are also found in the mammalian brain where
tyrosinase plays a key role in the synthesis of neuromelanin
and is linked to dopamine neurotoxicity.5 In the food industry,
Figure 1.
tyrosinase is a target enzyme in controlling the stability of fruits
and vegetables.6,7
zylidenebenzofuran-3(2H)-one (aurones) (Figure 1) as this
subclass of flavonoids have never been studied as tyrosinase
inhibitors.
The tyrosinase-mediated transformation of tyrosine to melanin
is of great importance since melanin has many key functions
such light absorption and scattering. Compounds inhibiting
tyrosinase are desired as therapeutics in treatment of some
dermatological disorders and as cosmetic agents, in relation to
hyperpigmentation. In cosmetology, tyrosinase inhibitors are
used as skin whitening agents. In this regard, the well-known
hydroquinone-O-â-glucopyranoside (arbutin)8-10 and kojic acid11
are considered as reference compounds (Figure 1). Due to
undesirable side effects, their use as depigmenting agents is
being compromised,12 and arbutin use is now prohibited in
several countries. Tropolone, a heptacyclic ketone has been
reported as a powerful inhibitor.13 Benzaldehyde derivatives and
especially hydroxylated analogues have been extensively in-
vestigated.14 Recently, Ley and Bertram have described ben-
zaldoximes as tyrosinase inhibitors.15 Finally, flavonoids and
their biosynthetic precursors have been reported as tyrosinase
inhibitors.14,16-17
Aurones, (2-benzylidenebenzofuran-3(2H)-ones) which are
structurally isomeric of flavones, are met in vegetables, espe-
cially in flowers18 where they are responsible for the gold-yellow
color. Their occurrence in marine organisms has been recently
reported.19 They also have been described as phytoalexins, used
by plants as defense agents against various infections.20-22
Compared to flavones, the medicinal literature on aurones is in
its infancy with trends and design just now emerging.23,24
Like all subclasses of flavonoids, aurones are mostly found
in a hydroxylated form and especially at C-4, C-6, and C-4′ (in
flavones, these positions correspond to C-5, C-7, and C-4′). On
the basis of these remarks, we chose to target aurones possessing
hydroxy groups at C-4 and/or at C-6 and bearing different
substituents on B-ring, hoping to obtain highly active derivatives
and to establish a structure-activity relationship (Figure 2).
A comment is deserved on the biological evaluation of
tyrosinase inhibitors. Although, the mushroom tyrosinase is a
commercially available enzyme and is frequently used for
biological evaluation, it is obvious that this model is not
sufficient for evaluating molecules destined for human use.14
In this study, we have chosen to work on human melanocytes
which express tyrosinase.
In our continuing program aimed to search for natural
products acting as skin protectors, we investigated ben-
* Corresponding author. Tel: (33) 4 76 04 10 06. Fax: (33) 4 76 04 10
07. E-mail: Ahcene.Boumendjel@ujf-grenoble.fr.
† Faculte´ de Pharmacie de Grenoble.
‡ Engelhard-Lyon.
10.1021/jm050715i CCC: $33.50 © 2006 American Chemical Society
Published on Web 11/23/2005

330 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 1
Okombi et al.
Table 1. Tyrosinase Inhibition Results
Figure 2.
Scheme 1a
inhibition %
at 0.1 mM
IC50
(µM)
compd
R1
R2
R3
R4
OH
OH
H
OH
Et
H
7a
7b
8a
8b
8c
10a
10b
10c
10d
10e
10f
10g
10h
10i
10j
H
OH
H
H
H
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
H
H
H
39 ( 6
71 ( 7
0 ( 23
69 ( 3
0 ( 29
3 ( 3
75 ( 8
nd
0 ( 34
4 ( 9
11 ( 4
5 ( 8
5 ( 3
0 ( 14
4 ( 4
H
H
H
H
H
H
H
Et
OH
H
H
H
H
H
31.7 ( 2.6
38.4 ( 2.6
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
Et
H
38.0 ( 2.9
H
OMe
OEt
n-Pr
t-Bu
3′-OMe,
4′-OH
apigenin (5,7,4′-trihydroxyflavone)
kojic acid (evaluated at 7 mM)
0 ( 2
20 ( 5
Percent Inhibition of Tyrosinase
from Different Melanocyte Sources
source of melanocytes (skin color)
concentration of 10b
(IC50 ) 38 µM)
white
dark
black
0.1 mM
0.01 mM
75 ( 5
38 ( 10
67 ( 3
32 ( 3
66 ( 3
39 ( 5
a (a) (Me)3N(Br3)Ph, THF, 6 h; (b) KOH, MeOH/H2O, 60 °C; (c) MEM-
Cl, (i-Pr)2NEt, DMF (R ) MEM); (d) benzaldehyde derivative, KOH,
MeOH/H2O, 60 °C; (e) HCl (1 M in Et2O), 60 °C, 2 h.
zone), easily quantifiable at 490 nm.28 Therefore, the use of a
compound able to modify tyrosinase activity is accompanied
with a decrease in absorbance at 490 nm compared to the
negative test (without molecule).28
Chemistry. Aurones disclosed in this study were synthesized
by means of an aldol condensation of the appropriate benzo-
furanone with the selected benzaldehyde derivative.25,26 The
synthesis required the preparation of benzofuranones 1, 2, and
3 (Scheme 1) which are needed for the synthesis of 4-hy-
droxyaurones, 6-hydroxyaurones, and 4,6-dihydroxyaurones,
respectively. The synthesis of 2 and 3 has been already
reported.25 The synthesis of 4-hydroxybenzofuranone 1 was
accomplished starting from 2′,6′-diacetyloxyacetophenone 4.
The latter was brominated with trimethylphenylammonium
tribromide to yield bromoacetophenone 5.27 Subsequent base
hydrolysis and cyclization afforded the desired compound 1.
Prior to aldol condensation, a protection step of 3 was needed,
otherwise poor yields and purification problems were faced. The
protection of the hydroxyl groups with a methoxyethyloxy-
methyl (MEM) group affords benzofuranone 6. Having 1, 2,
and 6 in hand, the final step was a condensation with
benzaldehyde derivatives in the presence of an excess of KOH
in H2O/MeOH to provide aurones 7, 8, and 9. Aurones 9 were
submitted to a deprotection step by using molar HCl in diethyl
ether for removing MEM groups and affording dihydroxyau-
rones 10.
Results and Discussion
The strategy adopted in this study was to submit all
synthesized compounds to a screening test aimed to evaluate
the cytotoxic effect. This step is needed prior measuring
tyrosinase inhibition because it allows us to rule out cytotoxic
compounds. Cytotoxicity was assessed by measuring cell
viability after incubation for 24 h with a molecule at 0.1 mM.
Compounds which do not alter cell viability (viability remains
higher than 75%) were retained for inhibitory study. In this
study, our reference inhibitor was kojic acid, which was used
at its effective concentration (7 mM).
A preliminary screening on a set of representative compounds
ruled out any tyrosinase inhibition with aurones without
hydroxyl groups on A or/and B-rings (results not shown).
With regards to hydroxylated aurones, it emerged that the
presence of hydroxy groups at 4,4′ (compound 7b), 6,4′
(compound 8b), or 4,6,4′ (compound 10b) positions was vital
for good tyrosinase inhibitory potencies. Interestingly, deletion
of the hydroxyl group on the B-ring (10a) led to complete loss
of activity. However, maintaining the 4′-hydroxy group and
removing those on the A-ring led to aurone (7a) which induces
39% of inhibition. With regard to these results, it emerged that
a hydroxyl group at the B-ring plays a more vital role than those
on the A-ring. Attempts to identify other important substituents
on the B-ring was unsuccessful as indicated by weak inhibitory
activity shown in Table 1. The crucial importance of C-4′
Biology. As indicated in the Introduction, we decided to use
a cellular assay which measure the tyrosinase activity of human
melanocytes obtained from healthy individuals. The inhibitory
potencies against tyrosinase were evaluated by the transforma-
tion of L-Dopa to L-dopaquinone. The latter can be trapped by
a chromophore MBTH (3-methyl-2-benzothiazolinone hydra-

Inhibitors of Tyrosinase
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 1 331
obtained on a Spectrometer Varian MAT 311. Elemental analysis
were performed by the analytical Department of CNRS, Vernaison,
France. Thin-layer chromatography (TLC) was carried out using
Machery-Nagel Polygram SIL/UV plates (thickness 0.20 mm).
Chromatography was carried out using Merck silica gel 60, 200-
400 mesh. Chemicals and reagents were obtained either from
Aldrich or Acros companies.
The synthesis of 4,6-dihydroxybenzofuran-3(2H)-one (1) and
6-hydroxybenzofuran-3(2H)-one (2) have already been reported
elsewhere.25,26
hydroxyl group was checked by protection of the latter as a
methoxy group, which led to inactive aurone 10f. The same
observation was made when either or both hydroxyls at C-4
and C-6 were methylated (data not shown). Interestingly, when
hydroxyl groups at C-4 and C-4′ are maintained and 6-OH is
methylated (data not shown), the inhibitory activity is completely
lost, indicating probably that steric hindrance in this region is
not tolerated. It is worth noting that antityrosinase activity was
dramatically decreased when the most active aurone 10b was
substituted at C-3′ (10j). This may indicate an unfavorable
sterical hindrance at this region of molecule. To confirm that
the C-4′ is the most suitable position for B-ring hydroxylation,
we prepared and tested 4,6,2′-trihydroxyaurone 10e which led
to this order of potency: 4′-position . 2′-position. It is
important to mention that the tyrosinase inhibitory activity is
specific to aurones among flavonoids subclasses. For example,
apigenin (5,7,4′-trihydroxyflavone), which is the structurally
closest flavone (Figure 1) to aurone 10b, was inactive at 0.1
mM.
On the basis of these results, we selected compounds 7, 8b,
and 10b and evaluated IC50 and found them (Table 1) very much
stronger than arbutin (IC50, 8.4 mM)29 and kojic acid (IC50, 280
µM ).11 As outlined before, this comparison is not highly
significant since arbutin and kojic acid are evaluated on
mushroom tyrosinase.
To investigate the correlation between melanocyte source
(skin color) and inhibitory activity, we selected aurone 10b and
tested it on melanocytes obtained from three individuals with
white, dark, and black skin (Table 1). The inhibitory activity
evaluated at two different concentrations (0.1 and 0.01 mM)
showed no significant difference in inhibitory activity. Because
our ultimate goal is to bring active aurones to human use, we
evaluated its tolerance in animals and found that it is devoid of
any toxicity after oral administration to rats at 5 g/kg dose. In
rabbits, topical and eye application of aurone 10b did not show
any significant irritation.
In the literature, it is assumed that most inhibitors act by
complexing the two copper atoms which are present in the active
site of the enzyme.30 Although, we do not have evidence
regarding the mechanism of action of aurones, it is possible
that aurones hydroxylated at the 4-position such as 7 and 10b
may act by the same mechanism. It is well established that
5-hydroxyflavones are good metal-complexing compounds, due
to the proximity of 4-carbonyl group to the 5-hydroxyl. This
hypothesis is challenged by 8b. Indeed, the latter, which lacks
the 4-hydroxy group and therefore its complexation capability
and should be considerably weakened, is still as active as 7
and 10b. Considering these data, we propose that copper
complexation is not the only mechanism of action but that the
aurones may bind to an another essential domain of tyrosinase.
In conclusion, we report for the first time the inhibition of
human tyrosinase activity by aurones. The natural aspect of these
molecules combined with absence of toxicity make them highly
attractive. Currently, we are developing 4,6,4′-trihydroxyaurone
as an active agent for treatment of hyperpigmentation-related
anomalies. Further investigations are underway in our laboratory
to clarify the possible mechanism of action.
4-Hydroxybenzofuran-3(2H)one (1). A solution of 2,6-diac-
etoxyacetophenone 4 (1 g, 4.23 mmol) was dissolved in anhy-
drous THF, trimethylphenylammonium tribromide (1.2 equiv) was
added under stirring, and the resulting mixture was stirred 6 h at
room temperature. The reaction was quenched with water and
extracted with ethyl acetate (2 × 10 mL). The ethyl acetate was
dried over MgSO4 and removed by evaporation to provide 5 as an
amber oil which was used without purification. 1H NMR (CDCl3):
δ 7.43 (t, 1H, J ) 8.4 Hz, H4); 7.05 (d, 2H, J ) 8.4 Hz, H3, H5);
4.28 (s, 2H, CH2Br); 2.24 (s, 6H, 2 × Ac). The crude material was
dissolved in MeOH (10 mL) and treated with 0.5 mL of KOH (50%
in H2O). After refluxing for 2 h, the methanol was evaporated and
the mixture was partitioned between HCl (1 N) and ethyl acetate.
The ethyl acetate was separated, washed with water, brine, and
concentrated, and the crude compound was purified by chroma-
tography column eluted with ethyl acetate:hexane to provide 1
having spectral and physical data in accordance with literature.29,30
1H NMR (CDCl3): δ 7.42 (t, 1H, J ) 8.4 Hz, H6); 6.55 (d, 1H, J
) 8.4 Hz, H7); 6.42 (d, 1H, J ) 8.4 Hz, H5); 4.58 (s, 2H, H2).
4,6-Di-MEM-benzofuran-3(2H)-one (6). A solution of 3 (3 g,
18 mmol) and N,N-diisopropylethylamine (2 equiv) in anhydrous
DMF is cooled to 0 °C, and then 2-methoxyethoxymethyl chloride
(MEM-Cl) was added dropwise (2 equiv). The resulting mixture
was stirred at room temperature for 20 min. The reaction mixture
was diluted with a large amount of water and extracted with ethyl
acetate. The organic layer was dried over MgSO4 and evaporated
to dryness to yield crude 6 which was used without purification.
1H NMR (CDCl3): δ 6.31 (m, 2H, H5, H7); 5.28 (s, 2H, OCH2O);
5.19 (s, 2H, OCH2O); 4.48 (s, 2H, H2); 3.76 (m, 2H, CH2O); 3.71
(m, 2H, CH2O); 3.46 (m, 4H, 2 × OCH2); 3.28 (s, 3H, OCH3);
3.27 (s, 3H, OCH3).
Condensation of benzaldehyde derivatives with benzofuran-
3-ones 1, 2 or 6, general procedure: To a solution of 1, 2, 6, or
benzofuran-3(2H)-one (commercially available) in methanol (10
mL/mmol) was added an aqueous solution of potassium hydroxide
(50%, 1.5 mL/mmol) followed by the addition of benzaldehyde
derivative (1.5 equiv). The solution was heated at 60 °C for 1 h,
and then methanol was evaporated. The residue was diluted in water,
and the resulting mixture was extracted with ethyl acetate. The
organic layer was dried over MgSO4 and filtered, and solvent was
removed by evaporation to yield crude products. Pure aurones 7
and 8 were obtained by purification on column chromatography
(silica gel) eluted with EtOAc:cyclohexane (1:2). In the case of
aurones 9, a deprotection step was performed prior purification:
to a methanolic solution (10 mL/mmol) of crude 9 was added a
molar solution of HCl in Et2O (5 mL/mmol). The reaction mixture
was stirred at 60 °C for 2 h and cooled to room temperature, water
(40 mL) was added, and the mixture was extracted with CH2Cl2.
The organic layer was washed with water and brine, dried over
MgSO4, and purified by column chromatography using cyclohex-
ane:ethyl acetate 6:4 to provide pure aurones 10 as orange or yellow
powders, and no further recrystallization was needed.
(Z)-2-(4-Hydroxybenzylidene)benzofuran-3(2H)-one (7a): 23%,
1
yellow powder, mp 256-258 °C; H NMR (acetone-d6): δ 7.86
Experimental Section
(d, 2H, J ) 6.8 Hz, H2′, H6′); 7.71 (m, 2H, H-4, H6); 7.42 (d, 1H,
J ) 7.6 Hz, H7); 7.24 (t, 1H, J ) 7.6 Hz, H5); 6.93 (d, 2H, J ) 6.8
Hz, H3′, H5′); 6.76 (s, 1H, dCH). MS (EI) m/z 238 [M]+•; Anal.
(C15H10O3) C, H.
Chemistry. Melting point were measured on a Fisher micromelt-
ing point apparatus and are uncorrected. 1H and 13 C NMR spectra
were recorded on Bruker Avance 400 operating at 400 and 100
MHz for 1H and 13C, respectively. Chemical shifts are reported in
ppm, in reference to TMS. Mass spectra were obtained on a JEOL
HX-110 spectrometer and high-resolution mass spectra were
(Z)-2-(4-Hydroxybenzylidene)-4-hydroxybenzofuran-3(2H)-
one (7b): 35%; orange powder; mp 275-277 °C; 1H NMR
(acetone-d6): δ 7.81 (d, 2H, J ) 8.8 Hz, H2′, H6′); 7.50 (t, 1H, J )

332 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 1
Okombi et al.
8 Hz, H6); 6.91 (d, 2H, J ) 8.8 Hz, H3′, H5′); 6,81 (d, 1H, J ) 8.4
Hz, H7); 6.66 (s, 1H, dCH); 6.59 (d, 1H, J ) 8.4 Hz, H5). MS
(EI) m/z 254 [M]+; Anal. (C15H10O3) C, H.
(Z)-2-Benzylidene-6-hydroxybenzofuran-3(2H)-one (8a): 17%;
yellow powder; mp 262 °C; 1H NMR (acetone-d6): δ 7.91 (d, 2H,
J ) 8.4 Hz, C6H5); 7.58 (d, 1H, J ) 8.4 Hz, H4); 7.42 (m, 3H,
C6H5); 6.80 (d, 1H, J ) 2 Hz, H7); 6.74 (dd, J ) 2 Hz, J ) 8.4 Hz,
H5); 6.66 (s, 1H, dCH). MS (EI) m/z 238 [M]+; Anal. (C15H10O3)
C, H.
H7); 6.09 (d, 1H, J ) 2 Hz, H5); 2.59 (t, 2H, J ) 7.2 Hz, Ph-CH2);
1.60 (m, 2H, CH2); 0.91 (t, 3H, 7.6 Hz, CH3). MS (EI) m/z 296
[M]+; Anal. (C18H16O4‚0.5H2O) C, H.
(Z)-2-(4-tert-Butylbenzylidene)-4,6-dihydroxybenzofuran-3(2H)-
1
one (10i): 93%; yellow powder; mp 218 °C; H NMR (acetone-
d6): δ 7.82 (d, 2H, J ) 8.8 Hz, H2′, H6′); 7.46 (d, 2H, J ) 8.8 Hz,
H3′, H5′); 6.56 (s, 1H, dCH); 6.29 (br s, 1H, H7); 6.10 (br s, 1H,
H5); 1.27 (s, 9H, t-Bu). MS (EI): m/z 296 [M]+; Anal. (C19H18O4)
C, H.
(Z)-2-(4-Hydroxybenzylidene)-6-hydroxybenzofuran-3(2H)-
one (8b): 92%; yellow powder; mp 286-288 °C; 1H NMR (DMSO-
d6): δ 10.1 (br s, 1H, OH); 7.73 (d, 2H, J ) 8.8 Hz, H2′, H6′); 7.52
(d, 1H, J ) 8.8 Hz, H4); 6.81 (d, 2H, J ) 8.8 Hz, H3′, H5′); 6.71
(d, 1H, J ) 1.6 Hz, H7); 6.65 (s, 1H, dCH); 6.63 (dd, 1H, J ) 1.6
Hz, J ) 8.8 Hz, H5). MS (DCI: NH3 + isobutane) m/z 255 [MH]+;
Anal. (C15H10O4) C, H.
(Z)-4,6-Dihydroxy-2-(4-hydroxy-3-methoxybenzylidene)ben-
zofuran-3(2H)-one (10j): 17%; yellow powder; mp 262 °C; H
1
NMR (acetone-d6): δ 7.50 (d, 1H, J ) 2 Hz, H2′); 7.42 (dd, 1H, J
) 2 Hz, J ) 8.4 Hz, H6′); 6.87 (d, 1H, J ) 8.4 Hz, H5′); 6.54 (s,
1H, dCH); 6.29 (d, 1H, J ) 2 Hz, H7), 6.08 (d, 1H, J ) 2 Hz,
H5); 3.86 (OCH3). MS (EI) m/z 300 [M]+; Anal. (C16H12O6‚0.5H2O)
C, H.
(Z)-2-(4-Ethylbenzylidene)-6-hydroxybenzofuran-3(2H)-one
(8c): 60%; yellow powder; mp 228 °C; H NMR (acetone-d6): δ
Tyrosinase Inhibition. Human melanocytes were obtained from
an abdominal plastic surgery. Melanocytes were seeded in 24 well
plates (80000 cells per well) and were cultivated until confluence.
Samples for evaluation were applied in cell culture medium during
24 h at 37 °C. After this time, cell culture medium was taken off
and human tyrosinase was extracted from melanocytes by thermal
choc lysis. After centrifugation (to eliminate cell membrane),
supernatants were incubated with MBTH and L-Dopa during 30
min and then analyzed by spectrophotometry at 490 nm. Inhibition
of tyrosinase activity was expressed in percentage of inhibition,
compared with negative control (medium without tested molecule).
A protein assay was performed on each extracellular medium to
correlate the enzymatic activity to a protein content (BCA assay).
Inhibition was calculated using the following formula: inhibition
(%) ) 100 - [(ODsample/protein content sample)/(ODnegative
control/protein content negative control)] × 100.
1
7.83 (d, 2H, J ) 8 Hz, H2′, H6′); 7.56 (d, 1H, J ) 8.4 Hz, H4); 7.28
(d, 2H, J ) 8 Hz, H3′, H5′); 6.78 (d, 1H, J ) 2 Hz, H7); 6.72 (dd,
1H, J ) 2 Hz, J ) 8.4 Hz, H5); 6.65 (s, 1H, dCH-); 2.63 (q, 2H,
CH2); 1.19 (t, J ) 7.8 Hz, CH3). MS (EI) m/z 266 [M]+; HRMS.
Calcd for C17H14O3: 266.09429. Found: 266.0919. Anal. (C17H14O3)
C, H.
(Z)-Benzylidene-4,6-dihydroxybenzofuran-3(2H)-one (10a):
1
40%; yellow powder; mp 252 °C; H NMR (acetone-d6): δ 6.09
(d, 1H, J ) 1.6 Hz, H7); 6.30 (d, 1H, J ) 1.6 Hz, H5); 6.57 (s, 1H,
dCH-); 7.35 (m, 1H, -C6H5); 7.42 (m, 2H, -C6H5); 7.87 (2H,
-C6H5). MS (EI) m/z [M]+; Anal. (C15H10O4) C, H.
(Z)-4,6-Dihydroxy-2-(4-hydroxybenzylidene)benzofuran-3(2H)-
one (10b): 31%; yellow powder; 295 °C (decomposition); yellow
1
powder; mp 295 °C; H NMR (acetone-d6): δ 7.82 (d, 2H, J )
7.8 Hz, H3′, H5′); 6.94 (d, 2H, J ) 7.8 Hz, H2′, H6′); 6.59 (s, 1H,
dCH); 6.33 (d, 1H, J ) 1.7 Hz, H7); 6.12 (d, 1H, J ) H5). MS
(DCI) m/z 270 [MH]+; Anal. (C15H15O5) C, H.
Cytotoxic Assay. Cellular viability was assessed using a “pNPP
assay as described by Yang et al.31 The pNPP (p-nitrophenyl
phosphate) was used for measuring AP (alkaline phosphatase)
activity. In the presence of AP, pNPP is hydrolyzed rapidly to
p-nitrophenol and inorganic phosphate. The released p-nitrophenol
(yellow) was measured at 405 nm. The absorbance at 405 nm is
directly proportional to the cell viability.
(Z)-4,6-Dihydroxy-2-(4-ethylbenzylidene)benzofuran-3(2H)-
1
one (10c): 65%; yellow powder; mp 236 °C; H NMR (acetone-
d6): δ 7.80 (d, 2H, J ) 8.4 Hz, H2′, H6′); 7.27 (d, 2H, J ) 8.4 Hz,
H3′, H5′); 6.56 (s, 1H, dCH-); 6.29 (d, 1H, J ) 1.6 Hz, H7); 6.09
(d, 1H, J ) 1.6 Hz, H5); 2.62 (q, 2H, J ) 7.6 Hz, CH2); 1.20 (t,
3H, J ) 7.2 Hz, CH3). MS (DCI) m/z 283 [MH]+; Anal. (C17H17O4)
C, H.
(Z)-4,6-Dihydroxy-2-(2-ethylbenzylidene)benzofuran-3(2H)-
one (10d): 84%; yellow powder; mp 208-210 °C; 1H NMR
(acetone-d6): δ 8.09 (m, 1H, H4′); 7.25 (m, 3H, H3′, H5′, H6′); 6.79
(s, 1H, dCH-); 6.26 (d, 1H, J ) 2 Hz, H7); 6.09 (d, 1H, J ) 2 Hz,
H5); 2.80 (q, 2H, J ) 7.6 Hz, CH2); 1.22 (t, 3H, J ) 7.6 Hz, CH3).
MS (EI) m/z 282 [MH]+; Anal. (C17H14O4‚H2O) C, H.
(Z)-4,6-Dihydroxy-2-(2-hydroxybenzylidene)benzofuran-3(2H)-
one (10e): 61%; yellow powder; mp 262-264 °C; 1H NMR
(acetone-d6): δ 8.12 (d, 1H, J ) 7.2 Hz, H6′); 7.17 (m, 1H, H3′);
7.12 (s, 1H, dCH-); 6.90 (m, 2H, H4′+H5′); 6.29 (d, 1H, J ) 2
Hz, H5); 6.08 (d, 1H, J ) 2 Hz, H7). MS (DCI) m/z 270 [MH]+•;
Anal. (C15H10O5) C, H.
(Z)-4,6-Dihydroxy-2-(4-methoxybenzylidene)benzofuran-3(2H)-
one (10f): 20%; yellow powder; mp 208-210 °C; 1H NMR
(acetone-d6): δ 7.83 (d, 2H, J ) 8.8 Hz, H2′, H6′); 6.97 (d, 2H, J
) 8.8 Hz, H3′, H5′); 6.55 (s, 1H, dCH); 6.28 (d, 1H, J ) 1.6 Hz,
H7); 6.07 (d, 1H, J ) 1.6 Hz, H5); 3.80 (s, 3H, OCH3). MS (EI)
m/z 284 [M]+•; Anal. (C16H12O5) C, H.
(Z)-4,6-Dihydroxy-2-(4-ethyloxybenzylidene)benzofuran-3(2H)-
one (10g): 29%; yellow powder; mp 215-217 °C; 1H NMR
(acetone-d6): δ 7.82 (d, 2H, J ) 6.8 Hz, H2′, H6′); 6.95 (d, 2H, J
) 6.8 Hz, H3′, H5′); 6.55 (s, 1H, dCH); 6.28 (d, 1H, J ) 1.6 Hz,
H5); 6.08 (d, 1H, J ) 1.6 Hz, H7); 4.05 (q, 2H, J ) 7.2 Hz, CH2);
1.32 (t, 3H, J ) 7.2 Hz, CH3). MS (EI) m/z 298 [M]+; Anal.
(C17H14O5‚H2O) C, H.
Melanocytes were seeded in 96 well plates with 50000 cells/
cm2, and cells were cultivated until confluence. Molecules were
added at different concentrations (10-4 M and 10-5 M) to the cell
culture medium at 37 °C in the presence of CO2 (5%). After 24 h,
the culture cell medium was taken off, a solution of p-nitrophenyl
phosphate in sodium acetate buffer (pH 8) was added for 2 h, and
the reaction was stopped by adding NaOH (1 N). The absorbance
at 405 nm was measured with a multiplate reader, and results were
expressed in percentage of viability compared to negative control
(without molecule).
In Vivo Toxicity. The toxicological studies were realized on
compound 10b incorporated at 10% in a xanthane gel.
Oral Toxicity in Rats. Acute oral toxicity test in the rat acute
toxic class method, OECD Guideline 423 (12-17-2001). The aim
of the study was to assess qualitatively and quantitatively the toxic
effects and the delay of appearance after single oral administration
of a predefined dose of 5 g/kg of body weight, of test preparation
as such, in six female rats, using a stepwise procedure. Similarly,
six control rats (six females) received 2000 mg/kg of distilled water.
The animals were daily observed for 14 days after administration,
and the signs of toxicity (mortality, etc.) were noted. This test
provided results allowing the test preparation to be classified
according to the Globally Harmonized System (GHS) for the
classification of chemicals which cause acute toxicity (OECD 1998).
In this study, no mortality at the dose of 2 g/kg was observed.
Therefore, the compounds were classified in the hazard category 5
or unclassified with a LD50 higher than 2 g/kg.
Primary Eye Irritation and Acute Dermal Irritation/Corro-
sion Test in the Rabbit. After single application of 0.1 mL (eye)
or 0.5 mL (skin) of test preparation in three rabbits, the ocular
reactions (redness, chemosis of conjunctiva, discharge, iris and
cornea lesions) and the skin reactions (erythema and edema) were
(Z)-4,6-Dihydroxy-2-(4-propylbenzylidene)benzofuran-3(2H)-
one (10h): 89%; yellow powder; mp 232-234 °C; 1H NMR
(acetone-d6): δ 7.87 (d, 2H, J ) 8.4 Hz, H2′, H6′); 7.25 (d, 2H, J
) 8.4 Hz, H3′, H5′); 6.56 (s, 1H, dCH); 6.29 (d, 1H, J ) 2 Hz,

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no alteration nor irritation occurred, the compounds were not
classified among the preparation irritant to the eyes or to the skin
in accordance with the criteria defined in the European Communities
decree of 04-20-1994.
Supporting Information Available: Elemental analysis and
high-resolution mass spectrometry (HRMS). These materials are
available free of charge via the Internet at http://pubs.acs.org.
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