N-Benzylbenzamides: A new class of potent tyrosinase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2006.02.018

A series of potent inhibitors of tyrosinase and their structure-activity relationships are described. N-Benzylbenzamide derivatives (1-21) with hydroxyl(s) were synthesized and tested for their tyrosinase inhibitory activity. With this series, compound 15 provided a potent tyrosinase inhibition: it effectively inhibited the oxidation of l-DOPA catalyzed by mushroom tyrosinase with an IC₅₀ of 2.2 μM.

Full text of DOI:10.1016/j.bmcl.2006.02.018

Bioorganic & Medicinal Chemistry Letters 16 (2006) 2682–2684
N-Benzylbenzamides: A new class of potent tyrosinase inhibitors
Sung Jin Cho,a Jung Seop Roh,b Won Suck Sun,c Sung Han Kimd and Ki Duk Parke,*
aMarine Biotechnology Laboratory, School of Earth and Environmental Sciences,
Seoul National University, Seoul 151-747, Republic of Korea
bHAITAI Confectionary R&D Center, Seoul, Republic of Korea
cLife Science Division, Life Science R&D Center, SK Chemicals, Suwon, Republic of Korea
dNutrex Technology, 1446-11 Seocho-dong, Seoul 137-864, Republic of Korea
eLaboratory of Cellular Function Modulator, Korea Research Institute of Bioscience and Biotechnology,
Daejeon 305-333, Republic of Korea
Received 25 November 2005; revised 27 January 2006; accepted 8 February 2006
Available online 2 March 2006
Abstract—A series of potent inhibitors of tyrosinase and their structure–activity relationships are described. N-Benzylbenzamide
derivatives (1–21) with hydroxyl(s) were synthesized and tested for their tyrosinase inhibitory activity. With this series, compound
15 provided a potent tyrosinase inhibition: it effectively inhibited the oxidation of L-DOPA catalyzed by mushroom tyrosinase with
an IC50 of 2.2 lM.
Ó 2006 Elsevier Ltd. All rights reserved.
Tyrosinase (E.C. 1.14.18.1), also known as polyphenol
oxidase, is a multifunctional copper-containing enzyme
widely distributed in nature. It is the key enzyme in
the undesirable browning of fruits and vegetables, and
coloring of skin, hair, and eyes in animals.1,2 It catalyzes
two distinct reactions of melanin biosynthesis: the
hydroxylation of monophenols to o-phenols, and con-
version of the o-diphenols to the corresponding o-qui-
nones, which are the initial steps in the pathway.
These quinones are highly reactive and tend to polymer-
ize spontaneously to form brown pigments of high
molecular weight, namely melanin, which determine
the color of mammalian skin and hair.3 This enzymatic
oxidation of L-tyrosine to melanin is of considerable
importance because melanin has many functions, and
alterations in melanin synthesis occur in many disease
states. Therefore, tyrosinase inhibitors have become
increasingly important in the food industry as well as
in medicinal and cosmetic products.4,5
distinct substructures: a 4-substituted resorcinol or cate-
chol moiety. It was suggested that 4-substituted resor-
cinol-type inhibitors bind to the enzyme binuclear
active site.7 The catechol structure may behave as a che-
lator to the copper ions in the tyrosinase. These poly-
phenols generally compete in inhibition with
tyrosinase.6 Recently, a naturally occurring tyrosinase
inhibitor, N-(p-coumaroyl)serotonin, has been de-
scribed, which contains two aromatic rings connected
by an amide bond (Fig. 1).8
In our continuing search for tyrosinase inhibitor, a new
group of compounds, with a similar structure to the ref-
erenced inhibitors, was designed, synthesized, and test-
ed. N-Benzylbenzamides also consist of two aromatic
rings like stilbene separated by three atoms like chalcone
B
A
O
Many tyrosinase inhibitors are polyphenol derivatives of
trans-stilbene or chalcone which have been investigated
intensively.3,6 They are constructed with one of two
Chalcone
OH
t-Stilbene
HO
H
N
Keywords: Tyrosinase inhibitor; N-Benzylbenzamides; 4-Substituted
resorcinol; SAR.
O
NH
N-(p-coumaroyl)serotonin
*
Corresponding author. Tel.: +82 42 860 4564; fax: +82 42 860
2675; e-mail: kdpark@kribb.re.kr
Figure 1. Structure of tyrosinase inhibitors.
0960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.02.018

S. J. Cho et al. / Bioorg. Med. Chem. Lett. 16 (2006) 2682–2684
2683
Table 1. The inhibitory effect of N-benzylbenzamide (1) and its
derivatives (2–21) on mushroom tyrosinase activities
and connected by an amide bond like N-(p-
coumaroyl)serotonin.
R1
B
R2
A
H
N
To evaluate this class of compounds further, a number
of analogues were constructed with various hydroxyl
substituents at both aromatic rings of the molecule.
Our hypothesis was that the position of the hydroxyl(s)
attached to the rings of N-benzylbenzamide must be of
major importance in that activity in the same manner
of stilbene and chalcone. Therefore, changing the posi-
tion of hydroxyl(s) would be very useful to find a potent
tyrosinase inhibitor.
O
a
Compound
R1
H
R2
IC50 (lM)
1
H
H
1990
780
1180
555
17
ndb
>2000
>2000
280
11
2
3,4,5-OH
3,4,5-OH
3,4,5-OH
3,4,5-OH
3,4,5-OH
3,4-OH
3,4-OH
3,4-OH
3,4-OH
3,4-OH
3,5-OH
3,5-OH
3,5-OH
3,5-OH
3,5-OH
2,4-OH
2,4-OH
2,4-OH
2,4-OH
2,4-OH
3
40-OH
4
30,40,50-OH
20,40-OH
30,40-OH
H
5
6
N-Benzylbenzamides were synthesized according to the
details in Scheme 1. Thus, commercially available benzyl
amine, with hydroxyl(s) or methoxy group(s), was first
reacted with an acyl chloride, with methoxy group(s),
to give the hydroxy or methoxy substituted N-benzylb-
enzamide. The methoxy group(s) was then demethylated
with boron tribromide to give the desired final com-
pound in good yields.9
7
8
40-OH
9
10
30,40,50-OH
20,40-OH
30,40-OH
H
11
12
nd
700
710
705
2.2
13
14
40-OH
30,40,50-OH
20,40-OH
30,40-OH
H
15
16
nd
The two aromatic rings in N-benzylbenzamides are not
symmetric, thus the inhibitory effects on tyrosinase were
tested and compared according to the position of hydro-
xy group(s) on both rings A and B.10 The in vitro results
for compounds 1–21 are given in Table 1. Compounds 5,
10, 15, and 20 with resorcinol on ring B (20,40-OH) dem-
onstrated high tyrosinase inhibitory activity. However,
compounds 17–19 and 21 with resorcinol on ring A
(2,4-OH) remarkably decreased inhibitory activity.
Especially, compound 5 exhibited 30 times the activity
than compound 19 which has opposite positions of
hydroxyls on its rings to compound 5. These observa-
tions suggests that 20,40-substituted resorcinol substruc-
ture on ring B plays an important role in determining
their activity. Among compounds 5, 10, 15, and 20,
compound 15 with resorcinols on both rings A and B
(3,5,20,40,-OH) exerted the most potent inhibitory activ-
ity with an IC50 of 2.2 lM.
17
18
1660
1820
550
29
40-OH
19
20
30,40,50-OH
20,40-OH
30,40-OH
21
Kojic acid
nd
16.3
a Values were determined from logarithmic concentration–inhibition
curves (at least eight points) and are given as means of three
experiments.
b Means not determined because of promoting effect which could act as
cofactor like diphenol.
much affected by the catechol subunit on ring A in com-
pounds 9 and 10. It is likely that 3,4-OH of ring A is not
oxidized by tyrosinase as cofactor like another diphenol.
The kinetic behavior of the oxidation of L-DOPA cata-
lyzed by tyrosinase at different concentrations of com-
pound 15 was studied. The inhibition data were
further analyzed by a Dixon plot, which showed that,
the plot of 1/V versus [I] was characterized by straight
lines at different fixed substrate concentrations intersect-
ing in the second quadrant (Fig. 2A). The replot of
slopes represented a straight line not going through
the origin reflecting the linear mixed-type inhibition
(Fig. 2B). In the Lineweaver–Burk plot, straight lines
at different fixed inhibitor concentrations intersected
with a common intersection point in the second quad-
rant (data not shown). The KI value estimated from this
Dixon plot was 1.3 lM as shown in Figure 2A.
In compounds 6, 11, 16, and 21, the catechol subunit on
ring B (30,40-OH) was oxidized by tyrosinase to its o-qui-
none derivatives in addition to the oxidization of origi-
nal substrate, L-DOPA. Tyrosinase inhibitory activity
of those compounds thus could not be determined being
tested because the absorbance at 475 nm in the test was
abnormally increased by those o-quinone derivatives.
Interestingly, tyrosinase inhibitory activity was not so
i
H
N
Cl H2N
n[R]
n[R]
n[R]
n[R]
In similar experiment testing, the tyrosinase effect of
chalcone derivatives substituted with hydroxyl(s), the
inhibitory potency was determined by the position rath-
er than the number of hydroxyl(s).11 In this study as
well, the position of hydroxyl(s) was the major factor
affecting potency. Besides, removal of the hydroxyl or
its substitution with another small group (OMe, NO2,
Cl, and F) significantly reduced the activity toward
mushroom tyrosinase (data not shown). Therefore, the
O
O
R = -OH or -OMe
ii
H
N
n[OH]
n[OH]
O
Scheme 1. Reagents and conditions: (i) TEA, THF, rt, 80–93%; (ii)
BBr3, CH2Cl2, ꢀ20 °C, 65–78%.

2684
S. J. Cho et al. / Bioorg. Med. Chem. Lett. 16 (2006) 2682–2684
Acknowledgment
We thank Nutrex Technology for the financial support.
References and notes
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9. All compounds provided satisfactory spectral data (1H
NMR and LC–MS) and were homogeneous by TLC.
10. The tyrosinase assay was performed by the method of
Masamoto et al. with slight modifications.13 First, 65 ll of
2.5 mM L-DOPA solution, 10 ll of DMSO with or
without a sample, and 105 ll of 0.1 M phosphate buffer
(pH 6.8) were mixed. The mixture was preincubated at
25 °C for 10 min before 20 ll of 1380 U/ml tyrosinase in
aqueous solution was added, and the reaction was
monitored at 475 nm. A control reaction was conducted
with DMSO. The OD values were measured by a UV
spectrophotometer at 475 nm using an ELISA Microplate
Reader (PowerWaveX, Bio-Tek, USA). Kojic acid was
used as a reference.
Figure 2. (A) Dixon plot for the inhibitory effect of compound 15 on
L-DOPA oxidation catalyzed by mushroom tyrosinase. The inhibitor
concentrations used were 0, 1.1, and 2.2 lM. The L-DOPA concen-
trations used were 1 mM (s), 0.5 mM (j), 0.25 mM (m), and
0.125 mM (d). (B) Secondary replot of slopes from Dixon plot versus
l/[L-DOPA]. Values are means of three separate experiments.
presence and the position of hydroxyl(s) proved to be
important for their activity in this class of compounds.
11. Khatib, S.; Nerya, O.; Musa, R.; Shmuel, M.; Tamir, S.;
Vaya, J. Bioorg. Med. Chem. 2005, 13, 433.
12. Spectral data for compound 15. 1H NMR (300 MHz,
DMSO-d6): d 9.85 (s, 1H), 9.79 (s, 1H), 9.26 (s, 2H), 8.00
(br s, 1H), 7.02 (s, 2H), 6.72 (d, 2H, J = 7.6 Hz), 6.47 (s,
1H), 6.17 (d, 2H, J = 7.6 Hz), 6.11 (s, 1H), 4.02 (d, 2H).
MS (EI) m/z: 275 (M+).
In summary, a series of N-benzylbenzamides has been
established as a new class of tyrosinase inhibitors. Struc-
tural feature of resorcinol on aromatic ring B (20,40-OH)
was most important for potent inhibitory activity. Com-
pound 15, N-(2,4-dihydroxybenzyl)-3,5-dihydroxyben-
zamide,12 was found to be eight times more potent
than the positive control, kojic acid.
13. Masamoto, Y.; Ando, H.; Murata, Y.; Shimoishi, Y.;
Tada, M.; Takahata, K. Biosci. Biotechnol. Biochem. 2003,
67, 631.