New halogenated phenylcoumarins as tyrosinase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2011.04.012

With the aim to find out structural features for the tyrosinase inhibitory activity, in the present communication we report the synthesis and pharmacological evaluation of a new series of phenylcoumarin derivatives with different number of hydroxyl or eth

Full text of DOI:10.1016/j.bmcl.2011.04.012

Bioorganic & Medicinal Chemistry Letters 21 (2011) 3342–3345
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
New halogenated phenylcoumarins as tyrosinase inhibitors
Maria João Matos a, , Lourdes Santana a, Eugenio Uriarte a, Giovanna Delogu b, Marcella Corda c,
⇑
Maria Benedetta Fadda c, Benedetta Era c, Antonella Fais c
a Departamento de Química Orgánica, Facultad de Farmacia, Universidad de Santiago de Compostela, 15782 Santiago de Compostela, Spain
b Dipartimento Farmaco Chimico Tecnologico, Facoltà di Farmacia, Università degli Studi di Cagliari, 09124 Cagliari, Italy
c Dipartimento di Scienze della Vita e dell’Ambiente, Università degli Studi di Cagliari 09042 Monserrato, Cagliari, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
With the aim to find out structural features for the tyrosinase inhibitory activity, in the present commu-
nication we report the synthesis and pharmacological evaluation of a new series of phenylcoumarin
derivatives with different number of hydroxyl or ether groups and bromo substituent in the scaffold.
The synthesized compounds 5–12 were evaluated as mushroom tyrosinase inhibitors showing, two of
Received 25 February 2011
Revised 30 March 2011
Accepted 3 April 2011
Available online 9 April 2011
them, lower IC50 than the umbelliferone. Compound 12 (IC50 = 215 lM) is the best tyrosinase inhibitor
of this series.
Keywords:
Ó 2011 Elsevier Ltd. All rights reserved.
Coumarins
Tyrosinase inhibitors
Perkin reaction
Hydrolysis reaction
Tyrosinase (EC 1.14.18.1) is a multifunctional dinuclear copper
centre enzyme widely distributed in nature and mainly involved
in the formation of pigments such as melanins and other polyphe-
nolic compounds.1 Tyrosinase oxidizes phenols and diphenols
using a catalytic mechanism that depends on the presence of cop-
per at its active site.1 In fungi and vertebrates, tyrosinase catalyzes
the two initial steps for the formation of the melanin pigments
(melanogenesis), starting from the tyrosine.2 This enzyme cataly-
ses the conversion of tyrosine to DOPA and the oxidation of the
resultant DOPA.2 So, this enzyme is responsible for the pigmenta-
tion of the skin, eyes and hair.3 In fact, tyrosinase inhibitors have
been used as depigmenting agents for the treatment or prevention
of hyperpigmentation disorders.4 Also, tyrosinase is involved in the
process to maintain the appearance, flavour, texture and nutri-
tional value of many fresh-cut products.5 The enzyme extracted
from the mushroom A. bisporus has high homology with the mam-
malian one. So, it is suited as a model for melanogenesis and tyros-
inase bio-pathways studies.
Resveratrol –3,40,5-trihydroxystilbene—is a natural polypheno-
lic compound present in grapes and red wine.15 It is a phytoalexin
produced by some species in response to an external or internal
damage (such as a fungal infection).15,16 In in vitro, ex vivo and
in vivo experiments, resveratrol has shown important pharmaco-
logical activities including antiinflammatory, antioxidant, antican-
cer and cardioprotective properties, besides inhibitory activity
towards several enzymes.15–19 Therefore, this compound has been
attracting a huge pharmacological interest since the last decade.
In recent studies, some coumarins proved to be mushroom
tyrosinase inhibitors.20,21 In these studies, esculetin and umbellif-
erone exhibited some of the strongest inhibitory activities of the
tested series. Masamoto et al. investigated the structure–activity
relationship of 18 coumarins for their inhibitory activity against
mushroom tyrosinase. They found that esculetin exhibited the
strongest inhibitory activity of those series.20 Recently, and in con-
trast with the Masamoto’s findings, Sollai et al. have shown that
esculetin is considered to be a tyrosinase substrate rather than
an inhibitor, whereas umbelliferone seems to be an inhibitor of
the mentioned oxidase.22 These recent findings revealed that
tyrosinase affinity can be efficiently modulated by appropriate
substitutions in the coumarin moiety. The introduction of hydroxyl
groups in different positions is particularly suitable to these
modifications.20,21 Recently, it has been demonstrated that also
resveratrol and other stilbenes23 have inhibitory effects against
mushroom tyrosinase activity.24 Resveratrol showed stronger
DOPA oxidase inhibitory activity than kojic acid (reference inhibi-
tor).25 In fact, until the last years, polyphenols are the largest
scaffold in tyrosinase inhibition.23,26 Their activity depends on
Coumarins are a large family of compounds, of natural and syn-
thetic origin, which presents different pharmacological activities.6
Structurally they are lactones of the cinnamic acid. Due to their
structural variability, they occupy an important place in the realm
of natural products and synthetic organic chemistry.7 Recent stud-
ies pay special attention to their antioxidative, antiinflamatory,
anticancer and enzymatic inhibition properties.8–14
⇑
Corresponding author.
E-mail address: mariacmatos@gmail.com (M.J. Matos).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.04.012

M. J. Matos et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3342–3345
3343
the presence and position of additional substituents whether a pol-
O
R
O
yphenol may act as an inhibitor.26 So, it was proved that both stil-
bene and coumarin derivatives show interesting inhibitory effects
against tyrosinase.
R2
OH
R2
H
a
OH
O
O
R1
R1
Tyrosinase inhibitors could have broad applications. As the
ideal drug candidate has not been attained, an intensive search
for new and innovative tyrosinase inhibitors is still needed. This ef-
fort has considerably increased in recent years. In this context, and
in an attempt to develop novel tyrosinase inhibitors, we had previ-
ously synthesized and described 3-arylcoumarin derivatives in
which both the coumarin and the resveratrol templates were
present.21
R
5 R = H; R1 = OEt; R2 = H
6 R = H; R1 = OMe; R2 = Br
7 R = OMe; R1 = OEt; R2 = H
3 R1 = OEt; R2 = H
4 R1 = OMe; R2 = Br
1 R = H
2 R = OMe
R = OMe; R1 = OMe; R2 = Br
8
b
Resveratrol ( Fig. 1, A) and umbelliferone (B) are very good
tyrosinase inhibitors.2,20 Umbelliferone-resveratrol hybrid (C) and
3-phenylumbelliferone (D) are less active than the mentioned
compounds (IC50 3.68 mM and >10, respectively). Although, the pre-
viously derivative described by us 6,8,30,40,50-pentahydroxy proved
to be the most active compound of the evaluated 3-arylcoumarin
series, with an IC50 of 0.27 mM.21 This molecule have two hydroxyl
groups under the coumarin ring, being like the coumarin-resveratrol
hybrid (E).
R
R2
O
O
R1
9 R = H; R1 = OH; R2 = H
10 R = H; R1 = OH; R2 = Br
11 R = OH; R1 = OH; R2 = H
1
2
12
R = OH; R = OH; R = Br
Based on these results,21 in the present work we proposed to
continue the 3-phenylcoumarin scaffold study, with different sub-
stitutions like methoxyl, ethoxyl, hydroxyl and/or bromo under the
6, 8 and 40 positions (Scheme 1). We decided to explore the impor-
tance of the number and position of ether and hydroxyl groups lo-
cated in the 3-phenyl ring or in the aromatic ring of the coumarin
and, at the same time, the bromination of the coumarin moiety.
The coumarin derivatives 5–1227 were efficiently synthesized
according to the synthetic protocol outlined in Scheme 1. The 3-
phenylcoumarins 5–8 were prepared starting from the conve-
niently substituted phenylacetic acid 1 or 2 and the appropriate
salicylaldehyde 3 or 4, using dicyclohexylcarbodiimide (DCC) as
dehydrating agent, by Perkin reaction,28–31 in dimethylsulfoxide
(DMSO). These reactions gave 55%, 59%, 61% and 60% yield, respec-
tively. Hydrolysis of the methoxyl/ethoxyl groups, by treatment
with hydriodic acid 57% in acetic acid/acetic anhydride (1:1),32
gave the hydroxyl derivatives 9, 10, 11 and 12 in 64%, 60%, 61%
and 53% yield, respectively. The obtained products were easy to
Scheme 1. Reagents and conditions: (a) DCC, DMSO, 110 °C, 12 h; (b) HI/AcOH/
Ac2O, 0 °C–rt, 3 h.
purify by flash chromatography, using a mixture of hexane/ethyl
acetate, in a proportion 9:1, as eluent.
The tyrosinase inhibitory activity of compounds 5–12 was eval-
uated in vitro by the measurement of the enzymatic activity of
mushroom tyrosinase enzyme extracted from the mushroom spe-
cie A. bisporus.33 Then, the IC50 values for inhibitory effects of the
new compounds were calculated (Table 1).
In the present communication, the effect of the introduction of a
halogen substituent into different hydroxy-3-phenylcoumarins
was proposed. In fact, a higher tyrosinase inhibitory activity,
regarding the non-halogenated compounds, was observed. The
structural change obtained from compound 9 to compound 10
and from 11 to 12, with the bromo atom in the coumarin nucleus,
leads to a significant increase of the tyrosinase activity (from mil-
imolar to micromolar range).
As it is shown in the Table 1, compound 12 is the most active
compound of this series. This compound, with a bromo atom and
two hydroxyl groups in the 3-phenylcoumarin moiety, has an
OH
OH
IC50 in the micromolar range (IC50 = 215 lM). As expected, the
HO
HO
O
O
more hydroxyl groups in the coumarin moiety, the better activity
the compounds have. When compared with compound 10, the
introduction of one hydroxyl group more in compound 12 in-
creases at least 1.5 times the tyrosinase inhibitory activity. Com-
pound 12 shows, in fact, a lightly higher inhibitory activity than
trans-Resveratrol (A)
Umbelliferone (B)
OH
the 6,8-dihydroxy-3-(30,40,50-trihydroxyphenyl)coumarin (IC50
270
M), previously described by us.21 The presence in 6 and 40
=
l
OH
HO
O
O
HO
O
O
Table 1
Inhibitory effect of compounds 5–12 and umbelliferone on mushroom
tyrosinase activity
Umbelliferone-resveratrol
hybrid (C)
3-Phenylumbelliferone (D)
Compounds
IC50 (mM) (L-DOPA 0.5 mM)
5
6
2.07 0.07
>5.0
OH
7
>5.0
HO
8
9
10
11
1.36 0.12
>5.0
0.302 0.002
1.30 0.08
0.215 0.085
0.42a
O
O
OH
Coumarin-resveratrol
hybrid (E)
12
Umbelliferone
a
Obtained from data in Ref.21
Figure 1. Tyrosinase inhibitors with coumarin/resveratrol derivative structures.

3344
M. J. Matos et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3342–3345
3.5
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Figure 2. Lineweaver–Burk plots for inhibition of compound 12 on mushroom
tyrosinase for catalysis of -DOPA. Inhibitor concentrations were 0 (ꢀ), 0.050 mM
(j), 0.100 mM (N), 0.200 mM (d). The inset represents the secondary plot of 1/Vmax
versus concentration of compound 12, to determine the inhibition constant (Ki).
L
17. Orallo, F., Jr. In Resveratrol in Health and Disease; Aggarwal, B. B., Shishodia, S.,
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positions, at the same time, of a bromo and a hydroxyl substituent,
respectively, contributed to an increase of the inhibitory activity.
The experimental results show that the methoxyl and ethoxyl
derivatives (compounds 5–8) do not present any significant activ-
ity against the tested enzyme. These results suggest that the bro-
mination of the hydroxycoumarins, in spite of the bromination of
methoxycoumarins, could be an important step in the synthesis
of novel tyrosinase inhibitors.
The inhibitory mechanism of the compound 12 was determined
using a Lineweaver–Burk double reciprocal plot (Figure ure2). The
data, displayed as a plot of 1/V versus 1/[S], gave three straight lines
with different slopes and a horizontal line that intersected at the
same point. With an increase in compound concentration, the Vmax
value decreased, whereas the Km value was unchanged, suggesting
that this compound is a non-competitive tyrosinase inhibitor. The
inhibition constant of this compound (KI = 0.189 mM) was deter-
mined by plotting the intercept values versus the concentration of
the corresponding compound, as shown in Figure 2.
In conclusion, in the present study it was shown that the syn-
thesized coumarin-resveratrol hybrid compounds have inhibitory
activity against mushroom tyrosinase. Some of them present tyros-
inase inhibitory activity in the micromolar range. The presence of a
bromo atom in position 6 of the hydroxycoumarins improves the
inhibitory activity respect to the other synthesized derivatives.
So, the introduction of a bromo atom improves the pharmacologi-
cal potential of these 3-phenylcoumarins, confirming that this lead
could be effectively optimized in a candidate for the treatment of
some hyperpigmentation skin diseases. These finds have encour-
aged us to continue the efforts towards the optimization of the
pharmacological profile of these 3-phenylcoumarins.
24. Likhitwitayawuid, K. Curr. Sci. 2008, 94, 44.
25. Likhitwitayawuid, K.; Sritularak, B.; De-Eknamkul, W. Planta Med. 2000, 66,
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26. Chang, Te.-S. Int. J. Mol. Sci. 2009, 10, 2440.
27. 8-Ethoxy-3-phenylcoumarin (5). It was obtained with a yield of 55% mp 117–
118 °C. 1H NMR (CDCl3) d (ppm), J (Hz): 1.50 (t, 3H, –CH3, J = 7.0), 4.21 (dd, 2H,
–CH2, J = 14.0 and J = 7.0), 7.09 (t, 2H, H-6, H-7, J = 6.9), 7.21 (t, 1H, H-5, J = 7.8),
7.42–7.48 (m, 3H, H-30, H-40, H-50), 7.72 (dd, 2H, H-20, H-60, J = 7.7 and J = 1.4),
7.79 (s, 1H, H-4). 13C NMR (CDCl3) d (ppm): 14.8, 65.0, 114.5, 119.3, 120.4,
124.3, 128.3, 128.4, 128.5, 128.8, 134.8, 140.1, 143.4, 146.3, 160.2. MS m/z (%):
267 (16), 266 (M+, 83), 239 (16), 238 (20), 212 (14) 211 (20), 181 (15), 153 (25).
Anal. Calcd for C17H14O3: C, 76.68; H 5.30. Found: C, 76.66; H, 5.35.
6-Bromo-8-methoxy-3-phenylcoumarin (6). It was obtained with a yield of 59%
mp 153–154 °C. 1H NMR (CDCl3) d (ppm), J (Hz): 3.97 (s, 3H, –OCH3), 7.16 (d,
1H, H-7, J = 2.0), 7.26 (d, 1H, H-5, J = 2.0), 7.42–7.47 (m, 3H, H-30, H-40, H-50),
7.68–7.78 (m, 3H, H-20, H-60, H-4). 13C NMR (CDCl3) d (ppm): 57.2, 117.0, 117.3,
121.9, 122.1, 129.2, 129.8, 130.3, 134.9, 139.1, 142.9, 148.3, 160.0. MS m/z (%):
332 (99), 331 (30), 330 (M+, 100), 304 (40), 302 (40), 261 (25), 259 (26), 194
(16), 165 (12), 153 (14), 151 (23), 102 (19), 76 (34). Anal. Calcd for C16H11BrO3:
C, 58.03; H, 3.35. Found: C, 58.01; H, 3.30.
8-Ethoxy-3-(40-methoxyphenyl)coumarin (7). It was obtained with a yield of 61%
mp 99–100 °C. 1H NMR (CDCl3) d (ppm), J (Hz): 1.50 (t, 3H, –CH3, J = 7.0), 3.84
(s, 3H, –OCH3), 4.19 (dd, 2H, –CH2, J = 14.0, J = 7.0), 6.84–7.26 (m, 5H, H-30, H-50,
H-5, H-6, H-7), 7.69 (t, 2H, H-20, H-60, J = 7.7), 7.73 (s, 1H, H-4). 13C NMR (CDCl3)
d (ppm): 14.8, 55.4, 64.8, 113.8, 114.0, 119.1, 120.49, 124.2, 127.1, 127.8, 129.7,
129.8, 138.6, 138.7, 146.2, 160.0. MS m/z (%): 297 (35), 296 (M+, 100), 268 (54),
240 (47) 225 (45), 197 (13), 152 (11), 139 (15). Anal. Calcd for C18H16O4: C,
72.96; H, 5.44. Found: C, 72.91; H, 5.39.
6-Bromo-8-methoxy-3-(40-methoxyphenyl)coumarin (8). It was obtained with a
yield of 60% mp 183–184 °C. 1H NMR (CDCl3) d (ppm), J (Hz): 3.85 (s, 3H, –
OCH3), 3.96 (s, 3H, –OCH3), 6.94–6.98 (m, 2H, H-30, H-50), 7.12 (d, 1H, H-7,
J = 1.8), 7.23 (dd, 1H, H-5, J = 2.1 and J = 0.9), 7.63–7.69 (m, 3H, H-4, H-20, H-60).
13C NMR (CDCl3) d (ppm): 55.7, 56.8, 114.2, 116.2, 116.8, 121.5, 126.8, 129.4,
130.2, 130.8, 137.3, 142.2, 147.8, 159.8, 160.6. MS m/z (%): 363 (19), 362 (M+,
100), 361 (19), 334 (24), 332 (23), 319 (33), 317 (34), 291 (11), 289 (11), 182
(18), 167 (17), 139 (21), 91 (11). Anal. Calcd for C17H13BrO4: C, 56.53; H 3.63.
Found: C, 56.55; H, 3.68.
8-Hydroxy-3-phenylcoumarin (9). It was obtained with a yield of 64% mp 199–
200 °C. 1H NMR (CDCl3) d (ppm), J (Hz): 7.14–7.19 (m, 3H, H-5, H-6, H-7), 7.40–
7.49 (m, 3H, H-30, H-40, H-50), 7.70–7.78 (m, 2H, H-20, H-60), 8.19 (s, 1H, H-4),
10.25 (s, 1H, –OH). 13C NMR (CDCl3) d (ppm): 118.0, 118.6, 120.4, 124.6, 126.7,
128.2, 128.5, 134.7, 141.0, 141.7, 144.3, 159.6. MS m/z (%): 239 (16), 238 (M+,
100), 210 (80), 181 (13), 153 (22) 152 (20), 105 (9), 76 (15), 51 (6). Anal. Calcd
for C15H10O3: C, 75.62; H, 4.23. Found: C, 75.57; H, 4.28.
Acknowledgement
Thanks to the Spanish Ministry (PS0900501) and to Xunta da
Galicia (INCITE09E2R203035ES and PGIDIT09CSA030203PR) for
financial support. We are also grateful to the RAS–LR7/2007
(CRP2_133). This work was also supported by funds of Cagliari Uni-
versity (for the biological assays). M.J.M. also thanks the FCT for a
PhD grant (SFRH/BD/61262/2009). The authors are grateful to
Professor Gianni Podda, of Cagliari University, for insightful
suggestions.
6-Bromo-8-hydroxy-3-phenylcoumarin (10). It was obtained with a yield of 60%
mp 163–164 °C. 1H NMR (CDCl3) d (ppm), J (Hz): 7.19 (d, 1H, H-7, J = 2.0), 7.42–
7.51 (m, 4H, H-5, H-30, H-40, H-50), 7.70 (dd, 2H, H-20, H-60, J = 7.6 and J = 1.6), 8.13
(s, 1H, H-4), 10.79 (s, 1H, –OH). 13C NMR (CDCl3) d (ppm): 115.5, 119.9, 120.4,
121.8, 127.9, 128.3, 128.5, 128.8, 134.4, 139.7, 141.1, 145.6, 159.1. MS m/z (%):
References and notes
1. Lerch, K. Life Chem. Rep. 1987, 5, 221.
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M. J. Matos et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3342–3345
3345
318 (99), 317 (17), 316 (M+, 100), 291 (12), 289 (13), 153 (22), 152 (51), 151 (12),
76 (25). Anal. Calcd for C15H9BrO3: C, 56.81; H, 2.86. Found: C, 56.77; H, 2.81.
8-hydroxy-3-(40-hydroxy)phenylcoumarin (11). It was obtained with a yield of
61% mp 237–238 °C. 1H NMR (CDCl3) d (ppm), J (Hz): 6.81-6.85 (m, 2H, H-30, H-
50), 7.03-7.12 (m, 3H, H-5, H-6, H-7), 7.55–7.59 (m, 2H, H-20, H-60), 8.05 (s, 1H, H-
4), 9.80 (s, 1H, –OH), 10.15 (s, 1H, –OH). 13C NMR (CDCl3) d (ppm): 115.3, 117.8,
118.6, 120.9, 124.7, 125.6, 126.8, 130.1, 139.2, 141.6, 144.5, 158.2, 160.1. MS m/z
(%): 255 (18), 254 (M+, 100), 227 (16), 226 (98), 197 (15), 169 (12), 115 (12). Anal.
Calcd for C15H10O4: C, 70.83; H, 3.87. Found: C, 70.80; H, 3.83.
30. Matos, M. J.; Viña, D.; Janeiro, P.; Borges, F.; Santana, L.; Uriarte, E. Bioorg. Med.
Chem. Lett. 2010, 20, 5157.
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33. Pre-incubationwith the enzyme:1/15 M phosphoric acidbuffer solution(pH 6.8,
1.8 mL), an aqueous solution of mushroom tyrosinase (1000 U/mL, Sigma
Chemical Co., 0.1 mL) and DMSO (0.1 mL) with or without the sample. The
6-Bromo-8-hydroxy-3-(40-hydroxyphenyl)coumarin (12). It was obtained with a
yield of 53% mp 249–250 °C. 1H NMR (CDCl3) d (ppm), J (Hz): 6.83 (d, 2H, H-30, H-
50, J = 8.8), 7.14 (t, 1H, H-7, J = 1.5), 7.36 (d, 1H, H-5, J = 2.0), 7.56 (d, 2H, H-20, H-60,
J = 8.5), 8.00 (s, 1H, H-4). 13C NMR (CDCl3) d (ppm): 115.6, 116.0, 119.9, 120.6,
122.5, 125.4, 128.2, 130.4, 138.0, 141.2, 146.0, 158.6, 159.8. MS m/z (%): 334 (99),
333 (45), 332 (M+, 100), 307 (31), 305 (35), 225 (26), 197 (29), 169 (35), 168 (46),
153 (24), 141 (21), 140 (16), 118 (17), 115 (28), 98 (13), 89 (14), 84 (18), 75 (11),
63 (13). Anal. Calcd for C15H9BrO4: C, 54.08; H 2.72. Found: C 54.01, H 2.69.
28. Hans, N.; Singhi, M.; Sharma, V.; Grover, S. K. Indian J. Chem. 1996, 35B, 1159.
29. Perkin, W. H. J. Chem. Soc. 1868, 21, 53.
mixture was incubated at 25 °C for 10 min. Then, a 1.05 mM of L-3,4-
dihydroxyphenylalanine (DOPA) solution (1 mL) was added and the reaction
was monitored at 475 nm, for 5 min. The percent of tyrosinase activity inhibition
was calculated as: inhibition (%) = (AꢀB)/A ꢁ 100, where A represents the
difference in the absorbance of control sample between 0.5 and 1.0 min, and B
represents the difference in absorbance of the test sample between 0.5 and
1.0 min. The IC50 value, a concentration giving 50% inhibition of tyrosinase
activity, was determinate by interpolation of dose–response curves. The
mushroom tyrosinase activity was determinate by spectrophotometric assays
(Varian Cary 50). Umbelliferone was used as a reference tyrosinase inhibitor.