Searching for indole derivatives as potential mushroom tyrosinase inhibitors

Article abstract of DOI:10.3109/14756366.2015.1029470

Tyrosinase is a copper-containing enzyme widely distributed in nature, involved in the biosynthesis of melanin whose role is to protect the skin from ultraviolet damage. A great interest has been shown on the melanin involvement in malignant melanoma and

Full text of DOI:10.3109/14756366.2015.1029470

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ISSN: 1475-6366 (print), 1475-6374 (electronic)
J Enzyme Inhib Med Chem, Early Online: 1–6
2015 Informa UK Ltd. DOI: 10.3109/14756366.2015.1029470
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RESEARCH ARTICLE
Searching for indole derivatives as potential mushroom tyrosinase
inhibitors
1
1
1
1
Stefania Ferro1, Giovanna Certo1,2, Laura De Luca , Maria Paola Germano , Antonio Rapisarda , and Rosaria Gitto
`
1
2
Dipartimento di Scienze del Farmaco e dei Prodotti per la Salute, Universita di Messina, Messina, Italy and Fondazione Prof. Antonio Imbesi,
`
Messina, Italy
Abstract
Keywords
Tyrosinase is a copper-containing enzyme widely distributed in nature, involved in the
biosynthesis of melanin whose role is to protect the skin from ultraviolet damage. A great
interest has been shown on the melanin involvement in malignant melanoma and other
carcinogenetic processes. These phenomena have encouraged the research of tyrosinase
inhibitors useful in therapeutic field as well as in foods and cosmetics to prevent browning. The
idea was to screen our ‘‘in house’’ database to select suitable lead compounds for the discovery
of potential drug-inhibiting enzyme. The obtained biological results demonstrated that
compounds containing 4-fluorobenzyl moiety at N ꢀ 1 position of indole system showed the
best activity. In addition, the role of the portion linked to the carbonyl group at C ꢀ 3 was
discussed. A Lineweaver–Burk kinetic analysis of the most active indoles, CHI 1043 and
derivative 4, showed a mixed-type inhibition in the presence of L-3,4-dihydroxyphenylalanine
(L-DOPA) as substrate.
Indoles, kinetic analysis, synthesis, tyrosinase
History
Received 19 February 2015
Revised 9 March 2015
Accepted 10 March 2015
Published online 31 March 2015
Introduction
classified into four types, namely, competitive, uncompetitive,
non-competitive and mixed4,8–13
.
Tyrosinase (EC1.14.18.1), also known as polyphenoloxidase, is a
copper-containing enzyme that catalyses two distinct reactions
of melanin synthesis: the hydroxylation of tyrosine by mono-
phenolase action (monophenolase activity) and the oxidation of
L-3,4-dihydroxyphenylalanine (L-DOPA) to o-dopaquinone
(diphenolase activity)1. Furthermore, in humans dopaquinone is
converted by a series of complex reactions involving cyclization
and oxidative polymerizations which finally results in the
formation of melanin2,3. The production of abnormal pigmenta-
tion is a serious aesthetic problem in human beings4. Exogenous
causes, particularly ultraviolet light exposure, are a common
factor in pigmentary abnormalities such as melasma, solar
lentigines and ephelides. In addition, tyrosinase is responsible
for the undesired enzymatic browning of fruits and vegetables5.
Therefore, tyrosinase inhibitors have become increasingly import-
ant in food industry as well as in the medicinal and cosmetic
products6,7. The architecture of catalytic site of tyrosinase consists
of two copper ions bound to six histidines. Three types of
tyrosinase (met-, oxy- and deoxy-tyrosinase), exhibiting different
binuclear copper structures of the active site, are involved in the
formation of melanin pigments. Based on the interaction between
the inhibitor and the enzyme, tyrosinase inhibitors can be
In previous articles14–18, we reported the biological activity of
numerous indole diketo acid derivatives as potent Integrase Strand
Transfer Inhibitors (INSTIs) (Figure 1) in the HIV-1 life cycle.
The mechanism of their antiviral action was due to the ability to
coordinate, into the catalytic site, the magnesium ions required for
the integration process, thus blocking the IN function.
On these bases, the idea was to screen our ‘‘in house database’’
to select suitable leads for the future discovery of potential
enzyme tyrosinase inhibitors.
The main goal was the identification of molecular structures
able to efficiently block the two copper cofactors of enzyme,
similarly to what observed in the case of chelating effect of indole
diketo acid derivatives on Mg ions.
Our research group is from many years engaged in the study of
the indole nucleus, representing one of the most important
structural motifs in the drug discovery, and usually described as
‘‘privileged scaffolds’’. As reported in literature, indole deriva-
tives have the unique property of mimicking the structure of
peptides and to bind reversibly to enzymes, providing in this way
the opportunities to discover novel drugs acting on several
biological targets with different mode of action19–23
.
In the light of these considerations and considering the
perspectives on how the indole scaffolds might be exploited in the
future, herein, we report the synthesis and the biological activity
of some compounds containing 4-methoxy-1H-indole scaffold as
potential tyrosinase inhibitors. For the most promising inhibitors,
the Lineweaver–Burk kinetic analysis was performed in order to
Address for correspondence: Stefania Ferro, Dipartimento di Scienze del
`
Farmaco e dei Prodotti per la Salute, Universita di Messina, Viale
Annunziata, I-98168 Messina, Italy. E-mail: sferro@unime.it

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S. Ferro et al.
J Enzyme Inhib Med Chem, Early Online: 1–6
clarify if their mechanism of inhibition is competitive, uncom- General procedures for the synthesis of indoles CHI 1043
petitive, non-competitive or mixed.
and 1–7
Derivatives CHI 1043 and 1–7 were prepared following the
synthetic procedures previously reported by us and all the spectral
Material and methods
data are in accordance with the literature14,24
.
Chemistry
All the commercially available reagents and solvents were used
without any further purification. The microwave-assisted reac-
tions were carried out in a CEM Focused Microwave Synthesis
System, Model Discover, working at the power necessary for
refluxing under atmospheric conditions. Melting points were
determined on a BUCHI Melting Point B-545 apparatus and are
uncorrected. Elemental analyses (C, H and N) were carried out on
a Carlo Erba Model 1106 Elemental Analyzer and the results are
within 0.4% of the theoretical values. Merck silica gel 60 F254
plates were used for analytical TLC; column chromatography was
performed on Merck silica gel 60 (230–400 mesh) and Flash
Tyrosinase inhibition assay
Tyrosinase inhibition was assayed according to the method
of Masamoto25 with minor modifications26. Briefly, aliquots
(0.05 ml) of sample at various concentrations (125–500 mM) were
mixed with 0.5 ml of L-DOPA solution (1.25 mM), 0.9 ml of
sodium acetate buffer solution (0.05 M, pH 6.8) and preincubated
at 25 ꢁC for 10 min. Then, 0.05 ml of an aqueous solution of
mushroom tyrosinase (333 U/ml) was added last to the mixture.
The reaction mixture was immediately monitored for the forma-
tion of dopachrome by measuring the linear increase in
adsorbance (Abs) at 475 nm.
1
Chromatography (FC) on a Biotage SP1 EXP. H-NMR spectra
The extent of inhibition by the addition of samples is expressed
as inhibition percentage and calculated as follows:
were recorded in CDCl3 with TMS as internal standard or
[D6]DMSO on a Varian Gemini-300 spectrometer. Chemical
shifts were expressed in d (ppm) and coupling constants (J) in
hertz (Hz). All the exchangeable protons were confirmed by
addition of D2O. Mushroom tyrosinase, L-DOPA, Kojic acid,
dimethyl sulfoxide (DMSO) and sodium acetate were purchased
from Sigma–Aldrich (St. Louis, MO).
Inhibition% ¼ f½ðA ꢀ BÞ ꢀ ðC ꢀ DÞꢂ=A ꢀ Bg=100
where A: Abs acetate buffer and enzyme; B: Abs acetate buffer;
C: Abs acetate buffer, test sample and enzyme; D: Abs acetate
buffer and test sample
The concentrations leading to 50% activity lost (IC50) were
also calculated by interpolation of the dose-response curves. Kojic
acid [5-hydroxy-2 -(hydroxymethyl)-4H-pyran-4-one], a fungal
secondary metabolite used as skin-whitening agent, was employed
as a positive standard (8–35 mM).
Kinetic analysis
The inhibition kinetics of the most active compounds on the
tyrosinase were studied using Lineweaver–Burk plots27. For the
test, the reaction mixture consisted of four different concentra-
tions of L-DOPA (0.6–5 mM) as substrate and mushroom
tyrosinase in acetate buffer (0.05 M, pH 6.8). Test samples at
various concentrations (125–500 mM) were added to the reaction
mixture.
Figure 1. Chemical structure of INSTIs.
Scheme 1. Reagents and conditions: (i) POCl3, CH3CON(CH3)2, RT, 12 h; (ii) benzyl bromide or chloride, NaH, DMF, mw: 10 min at continuous
temperature (50 ꢁC), 100 W; (iii) diethyl oxalate, dry CH3ONa, THF, two separate steps under the same conditions mw: 2 min, at continuous
temperature (50 ꢁC), 250 W and (iv) 2 N NaOH, MeOH, RT, 1.5 h.

DOI: 10.3109/14756366.2015.1029470
Indole derivatives as potential mushroom tyrosinase inhibitors
3
Statistical analysis
tyrosinase inhibition with an IC50 value of 312 mM; on the
contrary the presence of the same substituent in a different
position (1) and in higher number (2) afforded less active
compounds.
With the aim of improving our knowledge about the enzym-
atic activity of this class of potential indole tyrosinase inhibitors
(ITIs), new 4-methoxy-1H-indoles were designed and
synthesized.
All the experiments were performed at least 3 times in order to
ensure the reproducibility of the results. Values are given as
means SEM and they were analysed statistically by means of
analysis of variance (ANOVA) followed by Student’s t-test. Value
of p40.05 are regarded as significant.
Results and discussion
Particularly, we focused our attention on compounds in which
Our previous studies led to the discovery of potent INSTIs the diketo acid portion has been replaced with other functional-
belonging to the class of diketo acid derivatives14–17
ities capable of performing the chelating effect on the metal ions
.
Following the synthetic procedure reported by us into the catalytic site of enzyme (3, 4 and 5) maintaining in two of
(Scheme 1)16, 4-methoxy-substituted indole (a) was converted them (4 and 5) the typical 4-fluoro substitution (Figure 3).
into the corresponding 3-acetyl derivative (b) under Vilsmeier–
Haack conditions, using phosphoryl chloride and an excess of synthetic chemistry24 that is reported in Schemes 2 and 3.
N,N-dimethylacetamide. Treatment of commercially available 4-methoxy-1H-indole
Derivatives 3–5 were prepared according to a standard
The obtained intermediate was N-alkylated by reaction with with oxalyl chloride at 0 ꢁC produced 2 -(1H-3-indolyl)-2-
the suitable benzyl bromide under microwave (MW) irradiation oxoacetyl chloride as an inseparable intermediate which gave
conditions, thus reducing reaction time, usual thermal reagents the compound 3 after the reaction with the appropriate arylamine.
degradation and increasing the yields.
To prepare the N-alkylated indole 4, the starting material firstly
Reaction of benzyl derivatives (c) with diethyl oxalate under was treated with 4-fluorobenzyl bromide and a catalytic amount
the same conditions provided key diketoester intermediates (d) of potassium t-butoxide and then, the obtained intermediate b,
which were converted into the corresponding diketoacids (CHI was converted into the desired compound by reaction with the
1043, 1 and 2) by hydrolysis in basic medium.
For the initial screening, we focused on the compounds that is
arylamine (Scheme 2).
Scheme 3 shows the pathway to synthesize derivative 5. By
depicted in Figure 2 (CHI 1043, 1 and 2) containing one or two 3-acylation of 4-methoxy-1H-indole (a) with 2,3-dimethoxyben-
fluorine atoms on the benzyl moiety, taking into account the role zoyl chloride (c) that had been obtained from the corresponding
of the ‘‘privileged indole scaffolds’’ drug discovery and the acid (b) and thionyl chloride, the 4-methoxy-1H-indol-3-yl (2,3-
results of a previous article by Liu et al.28 which demonstrated the dimethoxyphenyl)methanone (d) was prepared. It was then
influence of halogenated substituents on the pharmacokinetic alkylated by treatment with 4-fluorobenzyl bromide using a
effects.
catalytic amount of potassium t-butoxide. Finally, the desired
The inhibitory effects of these derivatives were investigated on indole derivative 5 was obtained by reaction of N-alkylated
the diphenolase activity of mushroom tyrosinase using L-DOPA intermediate (d) with BBr3 in CH2Cl2.
as substrate and compared with kojic acid (Figure 2), which is a
widely used skin-whitening material in cosmetic industry.
Results of the above-reported preliminary screening (Table 1) synthetic pathway employed for obtaining the tyrosinase inhibitor
In addition, on the basis of a molecular simplification approach
we also tested some fragments (6 and 7) isolated during the
showed that the diketo acid portion was not crucial for the activity CHI 104314 (Figure 3).
and confirmed the pivotal role of halogen substitution.
The obtained results (Table 1) showed that derivatives 4 and 7
Particularly, only the 4-fluoro-substituted CHI 1043 showed produced inhibitory effects on mushroom tyrosinase activity with
IC50 values of 224 and 372 mM, respectively, while compounds 3,
5 and 6 did not affect enzyme activity.
It was recently reported that the fluoro derivatives showed
higher activity than chloro and bromo analogues and better
pharmacobiological properties. In particular, it was observed that
the position of the substituent plays an important role influencing
the biological activity10. Accordingly, the results of tyrosinase
inhibition assay demonstrated that compounds 3 and 6, in
which the 4-fluorobenzyl moiety was removed, were completely
inactive.
Table 1. Inhibitory activity against mushroom
tyrosinase of indole derivatives.
Compound
IC50
Kojic acid
CHI1043
17.76 0.18 mM*
312 1.92 mM*
NIa mM
1
2
3
4
5
6
7
4400
NIa
224 1.23 mM*
NIa
NIa
372 1.87 mM
aNo obvious inhibition at 500 mM (520%).
*p50.05.
Figure 2. Chemical structure of Kojic acid, CHI 1043 and its analogs.

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S. Ferro et al.
J Enzyme Inhib Med Chem, Early Online: 1–6
Figure 3. New potential indole tyrosinase
inhibitors (ITIs).
Scheme 2. Reagents and conditions: (i) oxalyl
chloride, dry Et2O, N2, 0 ꢁC, 3 h; (ii)
NH2-Het, dry THF, N2, rt, 1.5 h and
(iii) 4-fluorobenzyl bromide, t-BuOK,
THF, rt, 2 h.
Scheme 3. Reagents and conditions: (i) thi-
onyl chloride, D, 2 h; (ii) Et2AlCl, CH2Cl2,
0 ꢁC, 2 h; (iii) 4-fluorobenzyl bromide,
t-BuOK, THF, rt, 2 h and (iv) BBr3 1 M sol.
in CH2Cl2, CH2Cl2, rt, 16 h.

DOI: 10.3109/14756366.2015.1029470
Indole derivatives as potential mushroom tyrosinase inhibitors
5
Figure 4. Lineweaver–Burk plots for the
inhibition of tyrosinase respect to L-DOPA
as substrate in the presence of CHI 1043
(a) and 4 (b).
On the contrary, derivative 7 structurally related to derivative 6 Conclusions
but characterized by the 4-fluorobenzyl substitution showed anti
tyrosinase activity.
In summary, novel indole derivatives have been designed,
synthesized and tested as tyrosinase inhibitors. The biological
results confirmed the positive influence of the 4-fluorophenyl
moiety as substituent at N ꢀ 1 position of indole nucleus and
suggested the key role of the substituent at C ꢀ 3 position of the
same scaffold on tyrosinase inhibition.
Among the 4-fluoro-substituted derivatives, only compound 5
was completely inactive thus suggesting that not only the
substituent at N ꢀ 1 position of the indole system but also the
portion linked to the carbonyl group at C ꢀ 3 plays a key role in
the tyrosinase inhibition.
Finally, the better biological activity of derivative 4 suggested
that an improvement of enzymatic inhibition can be achieved by
modulating the coordinating properties, also according to the
specificity of the catalytic site of tyrosinase.
Declaration of interest
The authors declare no conflict of interest.
For this reason, molecular modelling studies are in progress to
shed new important information useful in the further development
of this new class of compounds as potential tyrosinase inhibitors.
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