Analogues of N-hydroxycinnamoylphenalkylamides as inhibitors of human melanocyte-tyrosinase

Article abstract of DOI:10.1016/j.bmcl.2006.01.022

Melanin play a major role in human skin protection and their biosynthesis is vital. Due to their color, they contribute to the skin pigmentation. Tyrosinase is a key enzyme involved in the first stage of melanin synthesis, catalyzing the transformation of tyrosine to l-dopaquinone. The aim of the present study was to study molecules able to inhibit melanin synthesis through inhibition of tyrosinase and their potential use in treating pigmentation-related disorders. We targeted amides obtained from coupling p-hydroxycinnamic acid derivatives with phenylalkylamines. The biological activity was evaluated on human melanocytes by an assay which measures tyrosine-catalyzed l-Dopa oxidation. The most active amides were: trans-N-caffeoyltyramine, N-dihydrocaffeoyltyramine, and trans-N-dihydro-p- hydroxycinnamoyltyramine which induce complete inhibition at 0.1 mM. At the latter concentration, kojic acid, which was used as the reference inhibitor, was inactive.

Full text of DOI:10.1016/j.bmcl.2006.01.022

Bioorganic & Medicinal Chemistry Letters 16 (2006) 2252–2255
Analogues of N-hydroxycinnamoylphenalkylamides as inhibitors
of human melanocyte-tyrosinase
a
b
b
a
´
Sabrina Okombi, Delphine Rival, Sebastien Bonnet, Anne-Marie Mariotte,
b
a,
*
`
Eric Perrier and Ahcene Boumendjel
aDe´partement de Pharmacochimie Mole´culaire, UMR-CNRS 5063, Faculte´ de Pharmacie de Grenoble,
5 avenue de Verdun, BP 138, 38243 Meylan, France
bEngelhard-Lyon, 32 rue St Jean de Dieu, 69007 Lyon, France
Received 19 December 2005; revised 3 January 2006; accepted 6 January 2006
Available online 26 January 2006
Abstract—Melanin play a major role in human skin protection and their biosynthesis is vital. Due to their color, they contribute to
the skin pigmentation. Tyrosinase is a key enzyme involved in the first stage of melanin synthesis, catalyzing the transformation of
tyrosine to L-dopaquinone. The aim of the present study was to study molecules able to inhibit melanin synthesis through inhibition
of tyrosinase and their potential use in treating pigmentation-related disorders. We targeted amides obtained from coupling p-hy-
droxycinnamic acid derivatives with phenylalkylamines. The biological activity was evaluated on human melanocytes by an assay
which measures tyrosine-catalyzed L-Dopa oxidation. The most active amides were: trans-N-caffeoyltyramine, N-dihydrocaffeoyltyr-
amine, and trans-N-dihydro-p-hydroxycinnamoyltyramine which induce complete inhibition at 0.1 mM. At the latter concentration,
kojic acid, which was used as the reference inhibitor, was inactive.
ꢀ 2006 Elsevier Ltd. All rights reserved.
Skin hyperpigmentation can be dependent on either an
increased number of melanocytes or activity of melano-
genic enzymes, such as tyrosinase (EC 1.14.18.1).1,2 The
latter plays a critical role in the biosynthesis of melanin
and it is considered to be the key enzyme in coloring of
skin, hair, eyes, and food browning.3 Tyrosinase, is a
copper-containing enzyme, widely present in mammals,
plants and fungi and accepts many phenols and cate-
chols as substrates.4,5 In mammals, it is involved in the
transformation of L-tyrosine to dopaquinone which oc-
curs through two steps: hydroxylation of L-tyrosine to
L-3,4-dihydroxyphenylalanine (L-DOPA), then oxida-
tion of the latter to an ortho-quinone (dopaquinone).
Dopaquinone is further transformed through several
reactions to yield brown to black melanin which is
responsible for colour of mammals’ skin.6
increase melanogenesis such as tyrosinase activators
may protect human skin from UV irradiation damage.11
In this regard, several natural and synthetic compounds
acting as tyrosinase inhibitors were reported but only
few of them are used as skin-whitening agents due to
safety concerns. For example, arbutin and kojic acid
were among the most popular before serious side effects
came to limit their human use.12–15
As recently reviewed by Briganti et al.,16 it should be
highlighted that targeting tyrosinase inhibitors/activa-
tors to treat melanogenesis disorders is one of many pos-
sible approaches, due to the complex biochemical
reaction involved in the melanin synthesis. Moreover,
physical therapies such as lasers are now being devel-
oped to overcome whitening agents’ ineffectiveness.16
Inhibitors/activators of tyrosinase have become increas-
ingly important in medicinal and cosmetic products. For
example, tyrosinase inhibitors are used in depigmenta-
tion drugs and cosmetics,7–10 whereas compounds that
Effects of wine phenolic compounds such as caffeic acid,
ferulic acid, and p-coumaric acid have been reported to
act as tyrosinase inhibitors.17 Amides derived from cou-
pling the latter acids with tyramine or dopamine have
been isolated from plants or synthesized and have been
studied in different therapeutic areas such as anticancer
agents,18,19 antioxidants,20–23 and as selective NMDA
receptor antagonists.24 In a recent study, Roh et al. have
Keywords: Melanin; Tyrosinase inhibitors; N-Hydroxycinnamic acid
analogues; Phenylalkylamines; Amides.
*
Corresponding author. Tel.: +33 4 76 04 10 06; fax: +33 4 76 04 10
07; e-mail: Ahcene.Boumendjel@ujf-grenoble.fr
0960-894X/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.01.022

S. Okombi et al. / Bioorg. Med. Chem. Lett. 16 (2006) 2252–2255
2253
O
HO
OH
R2
R2
R3
R1
O
( )n
N
H
R1
N
H
( )n
HO
n = 1, 2
R1, R2, R3 = H, OH, OMe
n = 1, 2
R1, R2 = H, OH, OMe
Figure 1. Structures of targeted compounds.
reported melanogenesis-inhibitory effects of the naturally
occurring N-feruloylserotonin and N-(p-coumaroyl)sero-
tonin, isolated from Safflower (Carthamustinctorius L.).25
ed by measuring the transformation rate of L-Dopa
(tyrosinase substrate) to L-dopaquinone. The latter can
be trapped by MBTH (3-methyl-2-benzothiazolinone
hydrazone), a chromophore easily quantifiable at
490 nm.26,27 A compound able to modify tyrosinase
activity will be accompanied with a decrease in absor-
bance at 490 nm compared to the negative test (without
molecule) (Table 1).27,28
In continuing our program aimed to search for polyphe-
nol-derived compounds acting as skin protectors,26 we
investigated amides derived from coupling of: caffeic
acid, ferulic acid, p-hydroxycinnamic acid, and deriva-
tives with phenylethylamines (Fig. 1). The synthesized
compounds were studied for their effect as inhibitors
of human melanocyte-tyrosinase and their potential as
melanogenesis suppressors.
First, the compounds were assessed for their cytotoxicity
by measuring melanocyte viability at different concen-
trations in the range of 10–100 lM (results not shown)
and ruled out compounds which alter the cell viability.
At the highest concentration (100 lM), kojic acid, which
was used as a reference inhibitor, was inactive and there-
fore it was evaluated at 700 lM.
The amides disclosed in Figure 1 were obtained by cou-
pling of a carboxylic acid (1) with a phenylalkylamine
(2) in DMF in the presence of triethylamine and
BOP (benzotriazol-1-yloxy-tris-(dimethylamino)phos-
phonium hexafluorophosphate) as a coupling agent
(Scheme 1).
From the structure–activity point of view, we notice that
a higher activity was achieved with amides obtained by
coupling tyramine (4-hydroxyphenylethylamine) with
caffeic acid, dihydrocaffeic acid, and dihydro-p-hydroxy-
cinnamic acid (compounds 4, 15, and 17). Evaluation of
free tyramine, dopamine, caffeic acid, ferulic acid, and
dihydro-p-hydroxycinnamic acid shows weak inhibiting
activity, indicating the importance of the amide linkage.
Another structural feature is that the amide function
should be separated from each phenyl group by two car-
bons (compound 13 vs 15). The presence of a catechol
entity and preferably at the acid moiety seems to be
important, because dopamine derivatives are less
active than compounds derived from tyramine.
In the literature, there are two main methods for eval-
uating tyrosinase inhibitors. The first and the most
frequently used is an in vitro assay which uses the
commercially available mushroom tyrosinase as a
model. The second is a cellular test and measures
the tyrosinase activity, either by looking at the intra-
cellular accumulation of melanin or by determining
the transformation rate of a tyrosinase substrate.
Although, the in vitro test is easy to handle, provides
accurate results and allows us to work on the enzymo-
logical aspects, however, it has the drawback that the
results cannot be exploited for developing products for
human use.
As mentioned above, one of the reactions catalyzed by
the tyrosinase is the oxidation of L-Dopa to ortho-
dopaquinone by using molecular oxygen and it is
obvious that antioxidants may inhibit the oxidation
step, without interacting with the tyrosinase. The com-
pounds reported in this study were evaluated at
100 lM for their free radical scavenging activity by
using the DPPH method.29 It revealed that com-
pounds 5, 7, and 9 showed the highest free radical
scavenging activity (ꢀ60% of free radical scavenging
activity) and the most active inhibitor (compound 4)
shows 40% of free radical scavenging. These results
indicate that in our case there is no evidence for direct
correlation between free radical scavenging activity
and tyrosinase inhibition. The latter assumption is
confirmed by the weak inhibition of p-hydroxycinnam-
ic acid and derivatives (caffeic and ferulic acids) which
are known for their strong antioxidant activity.
Because our ultimate goal was to bring the active com-
pounds to human use, we preferred to relay on the cel-
lular test using human melanocytes obtained from
healthy individuals. The inhibitory potency was evaluat-
R1
OH
R2
R3
( )n
X
H2N
+
O
HO
2
1
x = CH2; -(CH2)2-; -CH=CH-
n = 1, 2
R3
R2
H
N
R1
a
X
( )n
O
HO
16-76%
3
Finally, because our ultimate goal is to bring active
compounds to human use, we evaluated the tolerance
Scheme 1. Reagents and condition: (a) BOP, Et3N, DMF, 20 h.

2254
S. Okombi et al. / Bioorg. Med. Chem. Lett. 16 (2006) 2252–2255
Table 1. Inhibition data
Compound
R1
R2
R3
n
% inhibition at 100 lM27
O
R1
R2
R3
( ) n
N
H
HO
1
2
3
4
5
6
7
8
9
10
OH
H
H
H
H
H
2
2
2
2
2
1
1
2
2
2
nd
OMe
OMe
OH
H
19
49
9
4
2
9
OH
OH
OH
OH
OH
OMe
OH
OMe
100
67
OH
OH
OMe
OH
OH
OMe
OMe
OMe
OH
40 11
nd
nd
62
OMe
OMe
2
5
OMe
25
HO
R1
R3
R2
O
( )n
N
H
11
12
OMe
OMe
OH
H
OH
OH
OH
OH
OH
OH
OH
1
1
1
2
2
2
2
16
0
6
9
7
7
2
5
2
2
1
2
3
2
5
OH
H
13
14
34
28
94
42
96
4
OMe
OH
H
15
16
H
OMe
H
OH
H
17
p-OHÆcinnamic acid
Caffeic acid
Ferulic acid
Tyramine
Dopamine
10
12
23
10
20
Kojic acid (evaluated at 700 lM)
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tuent pattern and the chain length which separate phen-
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properties of these compounds. The inhibition data
combined with the free radical scavenging activity indi-
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/
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