Design and discovery of tyrosinase inhibitors based on a coumarin scaffold

Article abstract of DOI:10.1039/c5ra14465e

In this manuscript we report the synthesis, pharmacological evaluation and docking studies of a selected series of 3-aryl and 3-heteroarylcoumarins with the aim of finding structural features for the tyrosinase inhibitory activity. The synthesized compounds were evaluated as mushroom tyrosinase inhibitors. Compound 12b showed the lowest IC₅₀(0.19 μM) of the series, being approximately 100 times more active than kojic acid, used as a reference compound. The kinetic studies of tyrosinase inhibition revealed that 12b acts as a competitive inhibitor of mushroom tyrosinase with l-DOPA as the substrate. Furthermore, the absence of cytotoxicity in B16F10 melanoma cells was determined for this compound. The antioxidant profile of all the derivatives was evaluated by measuring radical scavenging capacity (ABTS and DPPH assays). Docking experiments were carried out on mushroom tyrosinase structures to better understand the structure-activity relationships.

Full text of DOI:10.1039/c5ra14465e

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ARTICLE
Design and discovery of tyrosinase inhibitors based on the
coumarin scaffold
M. J. Matos,a,b,* C. Varela,c S. Vilar,b,d G. Hripcsak,d F. Borges,a L. Santana,b E. Uriarte,b A. Fais,e A. Di
Received 00th January 20xx,
Accepted 00th January 20xx
Petrillo,e F. Pintuse and B. Erae
DOI: 10.1039/x0xx00000x
Corresponding Author * Tel: +351 220 402 653; e-mail: mariacmatos@gmail.com
www.rsc.org/
In this manuscript we report the synthesis, pharmacological evaluation and docking studies of a selected series of 3-aryl
and 3-heteroarylcoumarins with the aim of finding structural features for the tyrosinase inhibitory activity. The
synthesized compounds were evaluated as mushroom tyrosinase inhibitors. Compound 12b showed the lowest IC50 (0.19
μM) of the series, being approximately 100 times more active than kojic acid, used as reference compound. The kinetic
studies of tyrosinase inhibition revealed that 12b acts as a competitive inhibitor of mushroom tyrosinase with L-DOPA as
the substrate. Furthermore, it was determined the absence of cytotoxicity in B16F10 melanoma cells for this compound.
The antioxidant profile of all the derivatives was evaluated by measuring radical scavenging capacity (ABTS and DPPH
assays). Docking experiments were carried out on mushroom tyrosinase structure to better understand the structure–
activity
relationships.
Introduction
pathways studies.8
Tyrosinase (EC 1.14.18.1) is a dinuclear copper-containing Tyrosinase inhibitors have a huge importance in medicine,
multifunctional enzyme widely distributed in nature.1 cosmetics and agriculture.9,10 A great amount of research
Tyrosinase oxidizes phenols and diphenols using a catalytic performed in this area is related with the tyrosinase inhibitory
mechanism that depends on the presence of copper at its activity of plant extracts, not isolated compounds.11 Although
active site.2 This enzyme catalyzes both initial reactions in many tyrosinase inhibitors are available, they have
melanogenesis: the hydroxylation of tyrosine to form L-DOPA demonstrated only mild efficacy and safety concerns.
and the subsequent oxidation of L-DOPA into dopaquinone.3 Therefore, from the current arsenal of compounds, none, as of
Tyrosinase is mainly involved in the synthesis of melanin, and yet, have reached the potency and safety requirements
other polyphenolic compounds, in the skin and hair as well as needed to enter clinical trials.12 This has led to a safer, more
neuromelanin in the brain.4 In fact, tyrosinase inhibitors have potent and efficient discovery of novel tyrosinase inhibitors.13
been used as depigmenting agents for the treatment or The emergence of new in vitro and in vivo tests will finally
prevention of hyperpigmentation disorders.5 Also, tyrosinase is allow the arrival of new compounds with more potency and
involved in the process to maintain the appearance, flavour, potential interest in this field.
texture and nutritional value of many fresh-cut products.6 The A large number naturally occurring compounds has been
enzyme extracted from the mushroom Agaricus bisporus (A. reported as moderate to potent inhibitors of tyrosinase.14,15
bisporus) has high homology with the mammalian one.7 So, it is Among them, phenolic compounds, in particular polyphenols,
suited as a model for melanogenesis and tyrosinase bio- were reported to have many human health benefits, including
tyrosinase inhibitory activity.16,17 Many flavonoid derivatives
have been revealed to be the strongest inhibitors of
a CIQUP/Department of Chemistry and Biochemistry, Faculty of Sciences, University of
tyrosinase.18 In recent studies, some coumarins proved to be
Porto, 4169-007 Porto, Portugal
mushroom tyrosinase inhibitors.19,20,21 In the work of
b Department of Organic Chemistry, Faculty of Pharmacy, University of Santiago de
Masamoto et al., esculetin (IC50 = 43 μM) and umbelliferone
(IC50 = 420 μM) (Figure 1A) proved to be some of the best
tyrosinase inhibitors, exhibiting esculetin the strongest
inhibitory activity of the entire series.22 Liu et al. reported that
2-(1-(coumarin-3-yl)ethylidene)hydrazinecarbothioamide
(Figure 1A) exhibited the most potent tyrosinase inhibitory
activity of the studied series, with IC50 value of 3.44 μM.20
Compostela, 15782 Santiago de Compostela, Spain
c CNC-IBILI, Pharmaceutical Chemistry Group, Faculty of Pharmacy, University of
Coimbra, 3000-548 Coimbra, Portugal
d Department of Biomedical Informatics, Columbia University Medical Center, New
York, NY 10032, USA
e Department of Life Science and Environment, University of Cagliari, 09042
Monserrato, Cagliari, Italy
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Recently, and in contrast with the Masamoto’s findings, Sollai inhibitors bearing the coumarin scaffold, the study of
View Article Online
DOI: 10.1039/C5RA14465E
et al. have shown that esculetin is considered a tyrosinase structure–activity relationships based on theoretical
substrate rather than an inhibitor, whereas umbelliferone calculations, mechanisms and kinetic of inhibition and the
seems to be an inhibitor of the mentioned oxidase.23 Structural effect of the inhibitors on the viability of mice melanoma cells
modifications on the coumarin scaffold have become a (B16F10).
powerful tool on tyrosinase inhibitors drug discovery.24 One of
the best strategies was the introduction of hydroxyl groups in Experimental
different positions of the coumarin core (Figure 1A).21,25,26 In Materials and methods
addition, 4-(6-hydroxy-2-naphthyl)-1,3-bezendiol (Figure 1B) Chemistry
proved to be a potent tyrosinase inhibitor (IC50 = 0.07 μM).27 Starting materials and reagents were obtained from
Finally, kojic acid (used as reference compound) is a flavonoid commercial suppliers (Sigma-Aldrich) and were used without
structurally related to the 3-arylcoumarin.27 This compound is further purification. Melting points (mp) are uncorrected and
also tyrosinase inhibitor (IC50 = 38.24 μM).27
were determined with a Reichert Kofler thermopan or in
1
These previous studies proved that both simple capillary tubes in a Büchi 510 apparatus. H NMR (300 MHz)
hydroxycoumarins and multi-hydroxylated 3-arylcoumarins and 13C NMR (75.4 MHz) spectra were recorder with a Bruker
were interesting scaffolds to further modulate the tyrosinase AMX spectrometer using DMSO-d6 as solvent. Chemical shifts
inhibitory activity. Further, a modification of phenyl rings by (δ) are expressed in parts per million (ppm) using TMS as an
other heterocyclic rings (i.e. thiophenyl rings) is a common internal standard. Coupling constants J are expressed in Hertz
approach in Medicinal Chemistry (Figure 1C).
(Hz). Spin multiplicities are given as s (singlet), d (doublet), dd
(doublet of doublets) and m (multiplet). Mass spectrometry
was carried out with a Hewlett-Packard 5988A spectrometer.
Elemental analyses were performed by a Perkin-Elmer 240B
microanalyzer and are within ±0.4% of calculated values in all
cases. The analytical results are ≥ 98% purity for all
compounds. Flash chromatography (FC) was performed on
silica gel (Merck 60, 230-400 mesh); analytical TLC was
performed on precoated silica gel plates (Merck 60 F254).
Organic solutions were dried over anhydrous sodium sulfate.
Concentration and evaporation of the solvent after reaction or
extraction was carried out on a rotary evaporator (Büchi
Rotavapor) operating under reduced pressure.
A
H
N
HO
HO
NH2
N
S
HO
O
O
O
O
O
O
Umbelliferone
2-(1-(Coumarin-3-yl)ethylidene)hydrazinecarbothioamide
Esculetin
(IC50 = 420 µM)
(IC50 = 3.44!µM)
(IC50 = 43 µM)
OH
OH
OH
Br
HO
OH
O
O
O
O
OH
OH
6,8-Dihydroxy-3-(3,4,5-trihydroxyphenyl)coumarin
6-Bromo-8-hydroxy-3-(4-hydroxyphenyl)coumarin
(IC50 = 0.27!mM)
(IC50 = 0.215!mM)
NH2
O
HO
O
1
Mass, H and 13C NMR spectra of the studied compounds are
3-Amino-7-hydroxycoumarin
presented in Supplementary Information.
(IC50 = 50 µM)
B
HO
OH
OH
C
O
General procedure for the synthesis of 3-phenylcoumarin and
3-thiophenylcoumarin (1 and 7).
N,N’-Dicyclohexylcarbodiimide (11.46 mmol) was added to a
solution of ortho-hydroxybenzaldehyde (7.34 mmol) and
phenyl/thiophenylacetic acid (9.18 mmol) in dimethyl sulfoxide
S
HO
HO
4-(6-Hydroxy-2-naphthyl)-
1,3-bezendiol (IC50 = 0.07 µM)
HO
O
O
O
Kojic acid
Studied scaffolds
(IC50 = 17.90 µM)
Figure 1. A – Reference inhibitors and previously described (15 mL). The mixture was heated at 110 °C for 24 h. Then, ice
compounds bearing the coumarin scaffold; B – 4-(6-Hydroxy-2- (100 mL) and acetic acid (10 mL) were added to the reaction
naphthyl)-1,3-bezendiol and Kojic acid; C – Scaffolds studied in mixture. After keeping it at room temperature for 2 h, the
the current work.
mixture was extracted with ether (3 x 25 mL). The organic
layers were combined and washed with sodium bicarbonate
Recent evidences have shown the importance of the solution (50 mL, 5%) and water (20 mL). Subsequently, the
antioxidant activity displayed by compounds with other solvent was evaporated under vacuum and the dry residue
pharmacological activities, such as tyrosinase inhibition.28,29 In was purified by flash chromatography (hexane/ethyl acetate
addition, tyrosinase inhibitors could have broad applications in 9:1), to give the desired 3-phenylcoumarin or 3-
therapeutics. As the ideal drug candidate has not been thiophenylcoumarin [1 (yield 79%)30 or 7 (yield 83%)31].32
attained, an intensive search for new and innovative
tyrosinase inhibitors is still needed. In this context, and in an General procedure for the synthesis of acetoxy-3-
attempt to develop novel tyrosinase inhibitors, in the current aryl/heteroarylcoumarins (2a-6a and 13a-15a/8a-12a).
work we described the synthesis, biological evaluation Compound 2a-6a and 13a-15a/8a-12a were synthesized under
(tyrosinase inhibitory activity, antioxidant activity and anhydrous conditions, using material previously dried at 60 °C
cytotoxicity in melanoma cells) and docking calculations of the for at least 12 h and at 300 °C during few minutes immediately
selected 3-aryl and 3-heteroarylcoumarins. This paper before use. A solution containing anhydrous CH3CO2K (2.94
emphasizes the rationale behind the discovery of tyrosinase mmol), the corresponding phenylacetic acid (for compounds
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2a-6a and 13a-15a) or thiophenylacetic acid (for compounds Anal. Elem. Calc. for C15H10O5: C, 66.67; H, 3.73;. Found: C,
View Article Online
DOI: 10.1039/C5RA14465E
8a-12a)
(1.67
mmol)
and
the
corresponding 66.65; H, 3.70.
hydroxysalicylaldehyde (1.67 mmol), in Ac2O (1.2 mL), was
refluxed for 16 h. The reaction mixture was cooled, neutralized 5,7-Dihydroxy-3-(3’,4’-dihydroxyphenyl)coumarin (14b). Yield
1
with 10% aqueous NaHCO3, and extracted with EtOAc (3 x 30 88%. Mp: 336-7 °C. H NMR (300 MHz, DMSO-d6) δ: 6.18-6.32
mL). The organic layers were combined, washed with distilled (m, 2H, H-6, H-8), 6.74 (d, 2H, J = 8.2, H-6’), 6.94 (dd, 1H, J =
water, dried (anhydrous Na2SO4), and evaporated under 8.2, J = 2.1, H-5’), 7.10 (d, 1H, J = 2.1, H-2’), 7.90 (s, 1H, H-4),
reduced pressure. The product was purified by recrystallization 9.12 (s, 2H, OH), 10.31 (s, 1H, OH), 10.64 (s, 1H, OH). 13C NMR
in EtOH and dried, to afford the desired compound.
(75.4 MHz, DMSO-d6) δ: 93.7, 98.4, 102.5, 115.5, 115.7, 119.4,
119.5, 199.5, 119.9, 126.6, 134.0, 144.9, 145.6, 155.9, 161.6.
General procedure for the synthesis of hydroxy-3- MS m/z: 286 ([M+1]+, 10). Anal. Elem. Calc. for C15H10O6: C,
aryl/heteroarylcoumarins (2b-6b and 13b-15b/8b-12b).
62.94; H, 3.52. Found: C, 62.97; H, 3.52.
Compounds 2b-6b33,34,35,36,37 and 13b-15b/8b-15b were
obtained by hydrolysis of their acetoxylated counterparts 2a- 3-(4’-Bromophenyl)-5,7-dihydroxycoumarin (15b). Yield 90%.
1
6a and 13a-15a/8a-12a, respectively.38,39 In general, the Mp: 288-9 °C. H NMR (300 MHz, DMSO-d6) δ: 6.21-6.29 (m,
appropriate acetoxylated coumarin, mixed with 2 N aqueous 2H, H-6, H-8), 7.54-7.63 (m, 4H, H-2’, H-3’, H-5’, H-6’), 8.09 (s,
HCl and MeOH, was refluxed during 3 h. The resulting reaction 1H, H-4), 10.43 (s, 1H, OH), 10.75 (s, 1H, OH). 13C NMR (75.4
mixture was cooled in an ice-bath and the reaction product, MHz, DMSO-d6) δ: 99.2, 103.8, 107.8, 123.7, 126.4, 135.6,
obtained as solid, was filtered, washed with cold distilled 136.5, 140.1, 141.6, 161.3, 161.8, 165.5, 167.8. MS m/z: 333
water, and dried under vacuum, to afford the desired ([M+1]+, 100). Anal. Elem. Calc. for C15H9BrO4: C, 54.08; H,
compound.
2.72. Found: C, 54.04; H, 2.70.
6-Hydroxy-3-(3-thiophenyl)coumarin (8b). Yield 92%. Mp: Pharmacology
1
263-4 °C. H NMR (300 MHz, DMSO-d6) δ: 7.01 (d, 1H, J = 2.8, Tyrosinase inhibition assay
H-5), 7.06 (dd, 1H, J = 9.0, J = 2.8, H-7), 7.27 (d, 1H, J = 8.8, H- Tyrosinase inhibition assay was carried out using the following
8), 7.65 (dd, 1H, J = 5.0, J = 2.9, H-5’), 7.71 (dd, 1H, J = 5.2, J = protocol. Briefly, in a 96-well plate, 80 μL of phosphate buffer
1.3, H-4’), 8.23 (dd, 1H, J = 2.9, J = 1.2, H-2’), 8.42 (s, 1H, H-4), (0.5 M, pH 6.5), 60 μL of mushroom tyrosinase (240 UmL-1), 20
9.78 (s, 1H, OH). 13C NMR (75.4 MHz, DMSO-d6) δ: 112.4, μL of inhibitor dissolved in DMSO at the desired
116.9, 119.8, 120.0, 121.5, 125.7, 126.4, 127.1, 134.9, 137.4, concentrations or DMSO (control), were mixed. The assay
138.5, 145.9, 154.0. MS m/z: 245 ([M+1]+, 29). Anal. Elem. mixture was then incubated at 37 °C for 10 min. Finally, 40 µL
Calc. for C17H12O6S: C, 63.92; H, 3.30. Found: C, 63.90; H, 3.27.
of 2.5 mM L-DOPA in phosphate buffer were added and
immediately monitored (t = 0) at 492 nm for dopachrome
8-Hydroxy-3-(3-thiophenyl)coumarin (10b). Yield 88%. Mp: formation in the reaction mixture.
208-9 °C. 1H NMR (300 MHz, DMSO-d6) δ: 7.04-7.17 (m, 3H, H- Kojic acid was used as a positive control. Each measurement
5’, H-6, H-7), 7.62-7.71 (m, 2H, H-2’, H-4’), 8.24 (dd, 1H, J = 7.5, was made at least in triplicate. The percentage of inhibition of
J = 1.6, H-5), 8.43 (s, 1H, H-4), 10.25 (s, 1H, OH). 13C NMR (75.4 tyrosinase activity was calculated as inhibition (%) = (A-
MHz, DMSO-d6) δ: 117.9, 118.0, 118.5, 118.6, 124.6, 124.8, B)/A×100, where
A represents the difference in the
125.7, 126.4, 127.0, 139.0, 142.9, 144.5, 153.1. MS m/z: 245 absorbance of control sample between an incubation time of
([M+1]+, 26). Anal. Elem. Calc. for C13H8O3S: C, 63.92; H, 3.30. 0.5 and 1.0 min, and B represents the difference in absorbance
Found: C, 63.91; H, 3.28.
of the test sample between an incubation time of 0.5 and 1.0
min. The IC50 value, a concentration giving 50% inhibition of
5,7-Dihydroxy-3-(3-thiophenyl)coumarin (12b). Yield 92%. tyrosinase activity, was determinate by interpolation of dose-
Mp: 315-6 °C. 1H NMR (300 MHz, DMSO-d6) δ: 6.23 (d, 1H, J = response curves.
2.0, H-8), 6.29 (d, 1H, J = 2.0, H-6), 7.51-7.63 (m, 2H, H-2’, H- The mode of inhibition on the enzyme was performed using
5’), 8.00-8.12 (m, 1H, H-4’), 8.27 (s, 1H, H-4), 10.44 (s, 1H, OH), the Lineweaver-Burk plot. Different concentrations of L-DOPA
10.77 (s, 1H, OH). 13C NMR (75.4 MHz, DMSO-d6) δ: 93.8, 98.5, (0.3, 0.5, 0.7 and 1.0 mM) were used for the assay.
102.2, 105.4, 123.7, 126.2, 126.7, 134.0, 135.4, 140.7, 155.4,
156.3, 162.1. MS m/z: 261 ([M+1]+, 20). Anal. Elem. Calc. for Determination of copper chelation
C13H8O4S: C, 59.99; H, 3.10. Found: C, 60.00; H, 3.12.
The copper chelating capacity of the compound 12b and 15b
was determined by the UV/Vis spectra according to the
5,7-Dihydroxy-3-(3’-hydroxyphenyl)coumarin (13b). Yield protocol described by Kubo et al.40 The compounds (0.05 mM)
1
93%. Mp: 276-7 °C. H NMR (300 MHz, DMSO-d6) δ: 6.21-6.27 were mixed with different concentration of CuSO4 (0.025-0.5
(m, 2H, H-6, H-8), 6.71-6.79 (m, 1H, H-4’), 7.02-7.23 (m, 3H, H- mM) and, after incubation at 25 °C for 10 min, absorption
2’, H-5’, H-6’), 8.00 (s, 1H, H-4), 9.51 (s, 1H, OH), 10.38 (s, 1H, spectra from 200 to 800 nm was recorded.
OH), 10.75 (s, 1H, OH). 13C NMR (75.4 MHz, DMSO-d6) δ: 93.8, For determination of the ability of compound 12b and 15b to
102.4, 113.2, 115.2, 119.8, 120.3, 120.9, 129.4, 131.2, 136.7, chelate copper in the enzyme, the mixture of 1.8 mL of 0.1 M
138.0, 156.3, 157.2, 162.2, 172.1. MS m/z: 270 ([M+1]+, 100). phosphate buffer (pH 6.8), 1 mL of water, 0.1 mL of the sample
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solution (0.05 mM) and 0.1 mL of the aqueous solution of the Molecular Docking
Journal Name
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DOI: 10.1039/C5RA14465E
mushroom tyrosinase (50 units), was incubated at 25 °C for 30 Docking calculations were performed with Glide from the
min, and then UV-Vis spectra were recorded.
Schrödinger package.44
Ligands and protein preparation: Ligands were pre-processed
Antioxidant activity
with the LigPrep module44 that generated tautomers, different
Total free radical-scavenging capacity of the molecules protonation states (pH = 7.0 ± 2.0) and optimized the
presenting hydroxyl groups on their structure was determined molecular structures with OPLS_2005 force field. Crystallized
by ABTS·+ and DPPH· scavenging methods, using trolox as proteins selected to run the calculations were 2Y9W (PDB
antioxidant standard.
code)45 and 2Y9X,45 extracted from the organism A. bisporus. A
water molecule or hydroxyl ion is present in the 2Y9W protein
pocket coordinated with Cu2+. The hydroxyl ion is maintained
ABTS radical scavenging activity
The ABTS·+ method is based on the capacity of an antioxidant in the pocket for the docking calculations. On the other hand,
to scavenge the free ABTS·+ and was performed as previously the crystal structure 2Y9X represents the deoxy form of the
reported.41 ABTS·+ reagent was produced by reacting 7 mM enzyme. The Protein Preparation Workflow44 was used to treat
ABTS with 2.45 mM potassium persulfate (final concentration) the proteins. As an example, some steps described in the
in aqueous solution. The mixture was kept in the dark, at room workflow included: addition and optimization of hydrogens
temperature, for 24 h. The concentration of the blue-green and the hydrogen bonding network, addition of cap termini or
ABTS·+ solution was adjusted to an absorbance of 0.700 ± 0.02 optimization of protonation states of some residues (His, Asp
(mean ± SD) at 734 nm.
and Glu), among others.
The samples of the compounds (10 μL) were added to ABTS·+ Molecular docking calculation: In the first step, we calculated a
solution and incubated in the dark at room temperature for 1 receptor grid centered in the pocket, with a van der Waals
min. Afterwards the decrease in A734 was calculated and radii scaling factor of 1.0 and a partial atomic charge cut-off of
referred to the trolox standard curve. The activity was 0.25. In the second step, we docked the compounds to the
expressed as concentration of sample necessary to give a 50% tyrosinase crystal structure 2Y9W and 2Y9X44 using Glide SP
reduction in the original absorbance (EC50).
(standard precision) and XP (extra-precision). The final binding
mode described in the manuscript was selected taking into
account the best-ranked scoring functions.
DPPH radical scavenging activity
The DPPH· scavenging activity of the compounds was analyzed
according to the procedure previously described.42 Each Results and discussion
compound, in DMSO solution (20 μL), was added to a mixture Chemistry
of 100 mM acetate buffer (pH 6.5, 630 μL) and 0.3 mM DPPH Compounds 1, 7 and 2b-6b and 8b-12b were efficiently
in ethanol (350 μL) in a cuvette, and left at room temperature, synthesized according to the synthetic strategy outlined in
in the dark, for 15 min. The absorbance of the resulting Scheme 1. Compound 1 and 7 are obtained by a Perkin
solutions was measured at 515 nm. Scavenging capacity of reaction, starting from the commercially available ortho-
each sample was compared to that of DMSO (0% radical hydroxybenzaldehyde and the phenyl/2-(thiophen-3-yl)acetic
scavenging) and trolox (positive control, 100% radical acid, using N,Nʹ-dicyclohexylcarbodiimide (DCC) as dehydrating
scavenging). The results were expressed as concentration of agent, and DMSO as solvent, at 110 °C.32 The synthetic
the compounds capable of scavenging 50% of free radicals methodology to obtain 2b-6b and 8b-12b occurs in two
(EC50).
different steps. The first one is a Perkin-Oglialoro condensation
of different commercially available hydroxybenzaldehydes and
aryl/heteroarylacetic acids, using potassium acetate (CH3CO2K)
Cell viability
Murine melanoma B16F10 cells (CRL-6475) were purchased in acetic anhydride (Ac2O), under reflux, for 16 h, to obtain the
from the American Type Culture Collection (ATCC, Manassas, acetoxy-3-aryl/heteroarylcoumarins (2a-6a and 8a-12a).
VA, USA). The cells were cultured in Dulbecco’s Modified Eagle Acetylation of the hydroxyl groups and pyrone ring closure
Medium (DMEM) containing 10% fetal bovine serum (FBS, occurred simultaneously. The second step is a hydrolysis of the
Gibco, NY, USA), and 1% penicillin/streptomycin at 37 °C in a obtained acetoxy derivatives, in the presence of aqueous HCl
humidified atmosphere with 5% CO2.
solution and MeOH, under reflux, for 3 h, to achieve the
Cell viability was detected by the colorimetric 3-(4,5- hydroxyl substituted 3-aryl/heteroarylcoumarins (2b-6b and
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) 8b-12b).46 Structures of the synthesized compounds were
assay.43 Briefly, cells were seeded in a 96-well plate (104 established on the basis of their spectral data (see
cells/well) and exposed to compound 12b or 15b at different Experimental Section).
concentrations (5-100 μM). After 48 h of incubation, cells were
labelled with MTT solution for 3 h at 37 °C. The resulting violet
formazan precipitate was dissolved in isopropanol and the
absorbance of each well was determined at 590 nm, using a
microplate reader with a 630 nm reference.
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a)
a)
In vitro tyrosinase inhibition
View Article Online
DOI: 10.1039/C5RA14465E
O
COOH
O
O
O
The tyrosinase inhibitory activity of all the synthetized
compounds was evaluated in vitro by the measurement of the
enzymatic activity of tyrosinase enzyme extracted from the
mushroom species A. bisporus. The compounds were screened
for o-diphenolase inhibitory activity of tyrosinase, using L-
DOPA as substrate. The IC50 values of all compounds and
reference inhibitor, kojic acid, are summarized in Table 1.
H
1
S
S
OH
COOH
O
7
O
b)
c)
H3C
O
HO
COOH
O
O
O
O
2a = 6-OCOCH3
3a = 7-OCOCH3
4a = 8-OCOCH3
5a = 7,8-OCOCH3
6a = 5,7-OCOCH3
2b = 6-OH
3b = 7-OH
4b = 8-OH
5b = 7,8-OH
6b = 5,7-OH
O
H
HO
Table 1. IC50 values of 3-aryl and 3-heteroarylcoumarins in
mushroom tyrosinase activity.
OH
S
S
S
O
Compounds
1
IC50 (μM)a
b)
c)
H3C
O
HO
COOH
O
O
O
O
> 1000
8a = 6-OCOCH3
9a = 7-OCOCH3
8b = 6-OH
9b = 7-OH
10a = 8-OCOCH3
11a = 7,8-OCOCH3
12a = 5,7-OCOCH3
10b = 8-OH
11b = 7,8-OH
12b = 5,7-OH
2b
3b25
4b26
5b
300.0 ± 25.95
> 1000
> 1000
Scheme 1. Synthetic methodologies. Reagents and conditions:
a) DCC, DMSO, 110 °C, 24 h; b) CH3CO2K, Ac2O, reflux, 16 h; c)
HCl, MeOH, reflux, 3 h.
> 1000
6b
408.37 ± 19.66
> 1000
After the biological studies, and the excellent inhibitory and
antioxidant profile of compounds 6b and 12b, a selected series
with structural variability at position 3, maintaining the
phenolic hydroxyls present in the coumarin skeleton
(compounds 13b-15b) were efficiently synthesized according
to the synthetic strategy outlined in Scheme 2. As described
before, the synthetic methodology occurs in two different
steps: i) Perkin-Oglialoro condensation of different
commercially available hydroxybenzaldehydes and arylacetic
acids to obtain the acetoxy-3-arylcoumarins (13a-15a) ii) and
hydrolysis of the obtained acetoxy derivatives to achieve the
hydroxyl substituted 3-aryl/heteroarylcoumarins (13b-15b).
Structures of the synthesized compounds were established on
the basis of their spectral data (see Experimental Section).
7
8b
> 1000
9b
821.65 ± 137.45
> 1000
10b
11b
12b
13b
14b
15b
Kojic acidb
508.75 ± 4.83
0.19 ± 0.016
727.05 ± 3.05
376.02 ± 0.008
1.05 ± 0.056
17.90 ± 0.98
CH3
R2
R1
R2
R1
R1
OH
O
O
O
OH
R2
a Values were shown as Mean ± SE (n = 3)
b Positive control
H
a)
O
b)
HO
OH
H3
C
O
O
O
HO
O
O
COOH
13b: R1 = OH ; R2 = H
14b: R1 = R2 = OH
R1 = H, OH
R2 = H, OH, Br
13a: R1 = OCOCH3 ; R2 = H
14a: R1 = R2 = OCOCH3
15a: R1 = H ; R2 = Br
15b: R1 = H ; R2 = Br
As shown in Table 1, from the studied series, compounds 2b,
6b, 9b and 11b-15b displayed inhibitory activity. Compounds
12b (IC50 = 0.19 μM) and 15b (IC50 = 1.05 μM) were better
tyrosinase inhibitors than the reference compound, kojic acid
(IC50 = 17.9 μM). In fact, compound 12b proved to be
approximately 100 times more active than kojic acid.
Scheme 2. Synthetic methodologies. Reagents and conditions:
a) CH3CO2K, Ac2O, reflux, 16 h; b) HCl, MeOH, reflux, 3 h.
Compounds
1 and 7, the studied scaffolds without
Pharmacology
substituents, proved to be inactive at the highest tested
concentration. Compounds 3b and 4b were already described
for their tyrosinase activity in previous papers.25,26 They were
included in this manuscript due to their interest to complete
the structure-activity relationship study.
This journal is © The Royal Society of Chemistry 20xx
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In general, the most relevant structural feature for the activity increasing the concentration of 15b resulted in a family of
View Article Online
DOI: 10.1039/C5RA14465E
of both series of compounds (3-phenyl and 3-thiophenyl straight lines with different slope and y-intercepts, which
derivatives), is the presence of two hydroxyl groups at intersected in the second quadrant. This behaviour
positions 5 and 7 of the coumarin moiety, as all these demonstrated that compound 15b can bind not only with the
compounds (6b and 12b-15b) were active against the studied free enzyme, but also with the enzyme-substrate complex, and
target.
their equilibrium constants are different. The equilibrium
The mode of inhibition of the enzyme was determined by constants for inhibitor binding with the free enzyme (KI) and
Lineweaver-Burk plot analysis, as shown in Figures 2 and 3. with the enzyme-substrate complex (KIS) were obtained from
Plots of the initial rates of tyrosinase activity in the presence of the slope or the vertical intercept versus inhibitor
increasing concentrations of substrate yielded a family of concentration, respectively (data presented in Supplementary
straight lines with different slopes but they intersected one Information). The values of KI and KIS of compound 15b were
another on the Y-axis, which indicates that compound 12b is a determined to be 0.89 and 5.38 μM, respectively.
competitive inhibitor (Figure 2a). The inhibition constant (Ki)
for the inhibitor binding with the free enzyme (E) was obtained
from the secondary plot as 0.22 μM (Figure 2b).
20
15
a)
10
80
5
60
0
-1
0
1
2
3
40
20
0
-5
1/[L-DOPA], µM-1
Figure 3. Lineweaver–Burk plots for the inhibition of
compound 15b on mushroom tyrosinase for catalysis of L-
DOPA. The concentrations of inhibitor were 0 (◊), 0.5 (□), 1.0
(●), and 1.5 (○) μM.
0
1
2
3
4
-1
µ
1/[L-DOPA],
M
Tyrosinase inhibitors might exert their effect by chelating
copper ion in the active site of the enzyme. In order to
determine whether the inhibitory activity of the compounds
12b and 15b are dependent on the ability to chelate the
copper ions, tests with copper sulfate or mushroom tyrosinase
were carried out. A solution of the compound 12b or 15b (50
μM) in phosphate buffer was scanned with and without copper
sulfate (up to 500 μM) or tyrosinase (50 units), and the spectra
obtained were compared. UV-Vis spectra showed no
significant change by adding Cu2+ or by incubation with the
enzyme. Thus, no interaction between compounds and copper
was observed.
30
20
10
0
b)
-0,4
0
0,4
0,8
1,2
Antioxidant assays
[I], µM
Radical scavengers can protect the biosynthesis and food
browning process. They are also involved in non-enzymatic
oxidation, as well as its oxidation process. Therefore, in the
present study, antioxidant activity of the hydroxylated
compounds was evaluated by measuring radical scavenging
capacity (ABTS and DPPH assays). The results are summarized
in Table 2.
Figure 2. a) Lineweaver–Burk plots for the inhibition of
compound 12b on mushroom tyrosinase for catalysis of L-
DOPA. The concentrations of inhibitor were 0 (◊), 0.25 (○), 0.5
(▪) and 1.0 (●) μM. b) The secondary plot of slope (Km/Vmax
)
versus concentration of compound 12b, to determine the
inhibition constant (Ki).
Table 2. EC50 values of selected 3-aryl and 3-
heteroarylcoumarins (2b-15b).
In the presence of 15b, the Lineweaver–Burk plots (Figure 3)
showed that this compound was a mixed-type inhibitor since
6 | J. Name., 2012, 00, 1-3
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showed EC50 values ranged from 7.08 to 11.59 μM, being
View Article Online
DOI: 10.1039/C5RA14465E
EC50 (μM)a
DPPH
177.7 ± 1.85 161.81 ± 2.57
> 300 > 300
63.44 ± 1.33 223.07 ± 5.38
compounds 6b, 12b, 13b and 14b in the same range (EC50
7.08, 8.40, 8.62 and 5.46 μM, respectively) than trolox (EC50
5.28 μM).
=
=
Compound
ABTS
Taking into account these results, and the structural
characteristics of the compounds, it could be observed that
the presence of two hydroxyl groups in the coumarin scaffold,
either in the positions 5/7 or in the positions 7/8, seems to be
important for the desired antioxidant activity.
2b
3b
4b
5b
Effect of the compound 12b and 15b on the viability of
B16F10 melanoma cells
11.59 ± 0.39
7.08 ± 0.035
> 600
11.39 ± 0.24
6.30 ± 3.82
> 300
The cytotoxicity of compound 12b, which showed the
strongest mushroom tyrosinase inhibition (IC50 = 0.19 μM),
was carried out to determine the safety of this molecule
(Figure 4a). Cells were treated with different concentration of
compound (5–100 μM) for 48 h and were examined using MTT
test. The results indicate that this inhibitor exhibited no
considerable cytotoxic effect in B16F10 melanoma cells at the
concentration in which inhibits the tyrosinase activity. The
same protocol was applied for compound 15b, which proved
to be a very good tyrosinase inhibitor (IC50 = 1.05 μM). A
similar result was obtained: the compound proved to be non-
cytotoxic to the cells under the experimental conditions
(Figure 4b).
6b
8b
9b
> 600
> 300
10b
11b
12b
13b
14b
15b
Troloxb
> 600
> 300
10.15 ± 1.20
8.40 ± 0.28
8.62 ± 0.18
5.46 ± 0.85
20.77 ± 0.42
5.28 ± 0.15
7.71 ± 0.09
9.42 ± 0.31
15.54 ± 0.19
11.64 ± 1.00
18.65 ± 0.85
28.27 ± 0.45
100
80
60
40
20
0
a)
a The EC50 value is the concentration of antioxidant required to quench
50% of the radicals in the reaction mixture under the experimental
conditions. Each EC50 value is the mean ± SEM from three experiments
(n = 3).
0
20
40
60
80
100
b Positive control
[12b], µM
As shown in Table 2, compounds that incorporate a single
hydroxyl group on their structure (compounds 2b-4b and 8b-
10b) did not present effective radical and radical cation
scavenging activity, measured in both ABTS and DPPH
methods. In particular, compounds 3b and 8b-10b proved to
be inactive at the highest tested concentration in both assays.
The best compounds, due to their capacity to scavenging both
radicals (ABTS·+ and DPPH·) proved to be compounds 5b, 6b
and 11b-14b. Regarding the DPPH· scavenging capacity, these
compounds exhibited higher activity than trolox, with EC50
values ranged from 6.30 to 15.54 μM. Among the different
tested coumarins, compound 6b (EC50 = 6.30 μM) exhibited the
most potent DPPH· scavenging activity, being this compound
about 5 times more potent than standard (trolox, EC50 = 28.27
μM). Regarding the ABTS·+ assay, the highlighted compounds
100
80
60
40
20
0
b)
0
20
40
60
80
100
[15b], µM
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Figure 4. The effect of inhibitors on B16F10 cell viability. a)
compound 12b; b) compound 15b. Cells were treated with
View Article Online
DOI: 10.1039/C5RA14465E
different concentrations of compound (5-100 μM) and studied Figure 5. a) Comparison of the co-crystallized ligand tropolone
by MTT assay. Data are expressed as a percentage of the (pink color) and the binding mode calculated with the
control.
molecular docking (green color) in the tyrosinase crystal
structure (PDB: 2Y9X); b) Hypothetical binding mode
calculated with docking for compound 11b inside the 2Y9W
Docking calculations
Some compounds shown in Scheme 1 were studied with tyrosinase. Hydrogen bonds are represented in yellow dashed
molecular docking techniques to understand the possible key lines.
interactions responsible for the protein-ligand binding. We
used the crystal structure 2Y9W (PDB code)45 in which the Compounds with tyrosinase activity, such as compounds 6b,
crystallized tyrosinase belongs to the mushroom A. bisporus, 9b, 11b and 12b showed a similar binding mode inside the
the same organism used in our experimental in vitro tyrosinase active site of the tyrosinase. The compounds orientated the
inhibition. No ligand is present in the active site of the coumarin ring towards the bottom of the cavity close to the
crystallized enzyme. The protein structure contains a water catalytic site and the 3-aryl or heteroaryl ring towards the
molecule or hydroxyl ion coordinated with the copper ions. protein surface. The hydroxyl group at position
7 of
We considered the oxy form of the enzyme as a suitable compounds 6b, 9b, 11b and 12b established a hydrogen bond
template for docking calculations. Moreover, an additional with the amide moiety of residue Asn260 (see Figure 5 and 6).
docking using the structure 2Y9X (PDB code),45 representing Compound 11b also established a hydrogen bond with the
the deoxy state of the enzyme, was performed to evaluate the same residue Asn260 using the hydroxyl group at position 8
stability of the results. The ligands were docked to the protein (Figure 5b). Moreover, compound 6b and 12b established an
through Glide in the Schrodinger package.44 With the aim of additional hydrogen bond with the residue Met280 using the
validating our docking protocol, we measured the root mean hydroxyl group at position 5 (see Figure 6). This second
square deviation (RMSD) between the atomic coordinates of stabilizing interaction could be responsible for the higher
the co-crystallized and the theoretical pose of the tropolone activity shown by compound 6b compared to the other
inhibitor present in the 2Y9X. The crystal structure (2Y9X) is a compounds in the series with 3-phenyl substitutions. Similar
tetramer with tropolone bound to different subunits with a argument can be made to explain the improved activity shown
slight different conformation depending on the subunit. by compound 12b regarding the series with 3-heteroayl
Docking simulations reproduced the best X-ray crystal substituents. On the other hand, compounds with heteroaryl
parameters with a RMSD of 1.24 and 2.30 Å using the protein moieties showed a better tyrosinase activity than compounds
structures 2Y9W and 2Y9X respectively (see Figure 5a with the with 3-phenyl substituents. Increased polarity in the 3-aryl
co-crystallized tropolone in the 2Y9X structure). In the case of substitution that points out towards the enzyme surface seems
2Y9W the protein was superimposed to 2Y9X and the RMSD to have an important effect in the enzyme affinity. The poses
between the co-crystallized tropolone in 2Y9X and the yielded by the calculations showed that the ligands orientated
theoretical pose determined in 2Y9W was measured.
the 3-substitution towards a polar area defined by the Arg268,
a charged residue with hydrophilic characteristics.
We also performed an alternative docking with the other
available tyrosinase crystal structure from A. bisporus (PDB ID:
2Y9X).45 The main difference in the crystallized active site is
the presence of the inhibitor tropolone and the lack of a water
molecule or hydroxyl between the copper ions. Molecular
docking using the 2Y9X protein yielded similar binding modes
for the compounds.
To better study the interaction profile of compound 12b, we
calculated the contribution of the residues to the interaction
with the ligand. Figure 6d shows the interaction score for the
residues placed in a distance of 4 Å from the ligand (score is
measured as the sum of Coulomb, van der Waals and
hydrogen bonding contributions). The residue that contributes
the most is the Asn260. The compound established Coulomb
interactions mainly with residues Asn260, Gly281 and Met280.
Van der Waals contributions are important in residues such as
His263, Phe264, Val283 and Arg268. Our docking results are in
accordance with the experimental data that showed that the
ligand 12b is a competitive inhibitor, but no interaction with
the copper ions was detected.
8 | J. Name., 2012, 00, 1-3
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coumarins, confirming that these leads could be effectively
View Article Online
DOI: 10.1039/C5RA14465E
optimized in candidates for the treatment of disorders related
to the melanin biosynthesis, such as hyperpigmentation skin
diseases. These findings have encouraged us to continue the
effort towards the optimization of the pharmacological profile
of these coumarins.
Acknowledgements
This work was partially supported by University of Santiago de
Compostela, University of Cagliari, Portuguese Foundation for
Science and Technology (FCT) of Portugal and QREN (FCUP-
CIQ-UP-NORTE-07-0124-FEDER-000065) project, European
Social Fund (ESF), Angeles Alvariño program from Xunta de
Galicia (Plan I2C), Galician Plan of research, innovation and
growth 2011-2015 (Plan I2C - ED481B 2014/086-0) and FCT,
POPH and QREN (SFRH/BPD/95345/2013).
Figure 6. Pose extracted from the docking for compound 6b
(panel a) and 12b (panel b) in the tyrosinase crystal structure
(PDB: 2Y9W). Hydrogen bonds are represented in yellow color.
Panel c) shows the perspective of the whole tyrosinase with
the inhibitor 12b docked to the protein. Panel d) represents
the residue interaction contribution (Coulomb, van der Waals
and hydrogen bonding scores) between the protein (residues
selected in a distance of 4 Å from the ligand) and compound
12b.
Notes and references
Conclusions
This study showed that some of the synthesized derivatives
displayed inhibitory activity against mushroom tyrosinase. The
two most active compounds (12b and 15b) presented
tyrosinase inhibitory activity in the low-micromolar range (IC50
= 0.19 and 1.05 μM, respectively), better than kojic acid (IC50
=
17.90 μM), used as reference compound. The presence of two
hydroxyl groups at positions 5/7 of the coumarin scaffold
improved the inhibitory activity compared to the other
synthesized derivatives and the reference compound. The
mode of inhibition of the enzyme was determined by
Lineweaver-Burk plot analysis, and indicated that compound
12b is a competitive inhibitor and compound 15b is a mixed-
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