Synthesis and structure-activity relationships of phenylpropanoid amides of serotonin on tyrosinase inhibition

Article abstract of DOI:10.1016/j.bmcl.2011.02.028

In this manuscript, we synthesized a series of phenylpropanoid amide of serotonin 1-9, analyzed their structural importance for two biologic activities (antioxidant activity and tyrosinase inhibitory activity). While the serotonin moiety and the amide linkage of serotonin derivatives affect antioxidant activity strongly, the serotonin moiety, the amide linkage and the cinnamic acid moiety affect tyrosinase inhibitory activity. Among tested compounds, compound 4 which contains cathechol moiety exhibited the most antioxidant activity (EC₅₀ = 19.4 ± 2.0 μM), and compound 6 exhibited significant tyrosinase inhibitory activity (IC₅₀ = 5.4 ± 3.6 μM). Our data suggests that a useful clue for the design and development of new tyrosinase inhibitors.

Full text of DOI:10.1016/j.bmcl.2011.02.028

Bioorganic & Medicinal Chemistry Letters 21 (2011) 1983–1986
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and structure–activity relationships of phenylpropanoid amides
of serotonin on tyrosinase inhibition
⇑
Toshiyuki Takahashi, Mitsuo Miyazawa
Department of Applied Chemistry, Faculty of Science and Engineering, Kinki University, 3-4-1, Kowakae, Higashiosaka-shi, Osaka 577-8502, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
In this manuscript, we synthesized a series of phenylpropanoid amide of serotonin 1–9, analyzed their
structural importance for two biologic activities (antioxidant activity and tyrosinase inhibitory activity).
While the serotonin moiety and the amide linkage of serotonin derivatives affect antioxidant activity
strongly, the serotonin moiety, the amide linkage and the cinnamic acid moiety affect tyrosinase inhib-
itory activity. Among tested compounds, compound 4 which contains cathechol moiety exhibited the
Received 26 November 2010
Revised 31 January 2011
Accepted 9 February 2011
Available online 13 February 2011
most antioxidant activity (EC50 = 19.4 2.0 lM), and compound 6 exhibited significant tyrosinase inhib-
Keywords:
itory activity (IC50 = 5.4 3.6
lM). Our data suggests that a useful clue for the design and development of
Tyrosinase inhibitor
Structure–activity relationships
Phenylpropanoid amide
Serotonin
new tyrosinase inhibitors.
Ó 2011 Elsevier Ltd. All rights reserved.
Tyrosinase (EC 1.14.18.1) is known to be a key enzyme for
melanin biosynthesis and is responsible for melanization in ani-
mals and browning in plants.1 Melanin plays a crucial protective
role against skin phytocarnogenesis, however, the production of
abnormal melanin pigmentation is serious esthetic problem in hu-
man beings. In the food industry, tyrosinase is responsible for
enzymatic browning reactions in damaged fruit during post-har-
vest handling and processing. The browning reaction produces
undesirable changes in color, flavor, and nutritive value of the
product. Therefore, the development of tyrosinase inhibitors that
can be used as preservatives for fresh food or as skin-whitening
agents have been pursued. In particular, there is a concerned effort
to research for naturally occurring tyrosinase inhibitors from
plants, because plants constitute a rich source of bioactive chemi-
cals and many of them are free from harmful adverse effects.2,3
Several hydroxycinnamic acid derivatives have been found to pos-
sessed strong antioxidant activities as radical scavengers their
antioxidant activity being strongly related to their structural fea-
tures and the presence of a hydroxyl function in the aromatic
structure4, cinnamic acid derivatives have been reported to act as
tyrosinase inhibitors.5–7 In a recent study, the tyrosinase inhibitory
effects of amides derived from coupling of caffeic acid, ferulic acid
and derivatives with phenylalkylamines (tyramine, dopamine)
have been reported.8,9
industrial additives or health agents10,11, in addition, applications
of antioxidants as preservative in food industry12 and skin-protec-
tive ingredients in cosmetics have also received considerable
attention.13
Serotonin derivatives were identified as the major unique phe-
nolic compound of safflower seeds, there are members of a family
of indole hydroxycinnamic acid amides.14–17 These compounds
have a number of biologic effect, such as, antioxidative activity18
,
anti-tumor activity19, fibroblasts growth promoting activity20, in
particular, melanogenesis-inhibitory effects21 of N-(p-coumaroyl
serotonin) and N-feruloyl serotonin and tyrosinase inhibition of
N-caffeoyl serotonin22 have been reported. Though these com-
pounds were important substance as a new class of potent tyrosi-
nase inhibitors, to our knowledge, there is no report on the
correlation between tyrosinase inhibition and structures of seroto-
nin derivatives in detail. Therefore, we further investigated the
structure–activity relationship of serotonin derivatives 1–9 on
the inhibition of tyrosinase23 and anti oxidant activity24 In this
study, we synthesized a series of serotonin derivatives 1–925,26
and analyzed their structural importance for two biologic activi-
ties. The synthetic pathways are shown Scheme 1.
The antioxidative activity of serotonin derivatives 1–9 and cin-
namic acid derivatives 1a–9a (Fig. 1) was investigated and com-
pared with that of dibutylhydroxytoluene (BHT). The results are
shown in Table 1. All serotonin derivatives 1–9 showed a more ac-
tive DPPH radical scavenging than BHT (EC50 = 87.5 2.6 lM). In
particular, compound 4 which contains cathechol moiety exhibited
On the other hand, antioxidants, used to prevent or inhibit the
natural phenomena of oxidation, have a broad application in di-
verse industrial fields as they have a huge importance either as
the most potent DPPH radical scavenging activity (EC50 = 19.4
2.0
lM). On the other hand, among cinnamic acid derivatives 1a–
⇑
Corresponding author. Tel.: +81 6 6721 2332; fax: +81 6 6727 2024.
E-mail address: miyazawa@kindai.ac.jp (M. Miyazawa).
8a, compound 4a which contains cathecohol moiety showed the
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.02.028

1984
T. Takahashi, M. Miyazawa / Bioorg. Med. Chem. Lett. 21 (2011) 1983–1986
EDC, HOBT,
Et3N,
CH2Cl2, DMF,
rt, 20h
H
N
O
NH
R1
R2
COOH
H2N
R1
R2
N
+
H
yeild 75-96%
R3
OH
R3
OH
R1, R2, R3 =H, OH, OMe
1a-3a, 5a-8a
1-3, 5-8
allyl bromide,
malonic acid,
CHO piperidin, pyridine,
reflux, 2h
K2CO3, NaI,
COOH
CHO
DMF, 60oC, 2h
yeild 98%
O
O
HO
O
yeild 83%
OH
O
EDC, HOBT, Et3N,
CH2Cl2, DMF, rt, 20h
yeild 96%
Pd(PPh3)4,
morpholine, THF,
rt, 12h
NH
NH
O
O
N
H
N
H
yeild 83%
HO
O
OH
OH
OH
O
4
NH
NH
O
O
pyridine, DMF,
rt, 24h
HOOC
N
H
O
H2N
+
yeild 62%
O
OH
OH
9
Scheme 1. Synthesis of serotonin derivatives 1–9.
R1
R2
R3
R1
R2
R3
Compound
Compound
1NH
O
8
COOH
7'
R1
R2
7
3
R1
5'
N
9
1'
10
8'
H
4
R2
R3
3'
R3
OH
H
H
H
H
H
H
H
H
H
H
H
H
H
H
1
2
3
1a
2a
3a
OH
OH
OMe
OMe
H
OH
OH
H
OH
OH
4
4a
H
H
H
OH OMe
OMe OH
OMe OMe
H
H
H
OH OMe
OMe OH
OMe OMe
5
6
7
5a
6a
7a
8
8a
OMe OH OMe
OMe OH OMe
NH
O
HOOC
N
H
OH
Compound 9 (bufobutanoic acid)
Figure 1. Structure of serotonin derivatives and cinnamic acid derivatives.
antioxidant activity (EC50 = 136.5 3.5
l
M). Whereas, all other cin-
radical scavenging activity, to the contrary, compound 9 exhibited
antioxidant activity (EC50 = 38.3 1.8 M). In antioxidant assays,
the amide linkage and the serotonin moiety of serotonin deriva-
namic acid derivatives (1a–3a, 5a–8a) showed low antioxidant
activities. These results indicate that the serotonin moiety affect
the antioxidant activity. Furthermore, serotonin did not exhibit
l
tives are essential for the antioxidant activity. The cinnamic moiety

T. Takahashi, M. Miyazawa / Bioorg. Med. Chem. Lett. 21 (2011) 1983–1986
1985
Table 1
Table 2
Effects of serotonin derivatives and cinnamic acid derivatives against DPPH radical
Inhibitory effects of serotonin derivatives and cinnamic acid derivatives against
tyrosinase
a
a
Compound
EC50
(l
M)
Compound
EC50 (lM)
a
a
Compound
IC50
(l
M)
Compound
IC50
(lM)
1
2
3
4
5
6
7
8
22.2 2.3
24.7 1.9
34.2 2.0
19.4 2.0
22.8 2.6
35.1 2.2
28.1 2.5
30.7 2.0
38.3 1.8
>250
1a
2a
3a
4a
5a
6a
7a
8a
BHT
>250
>250
>250
136.5 3.5
>250
>250
>250
>250
87.5 2.6
1
2
3
4
5
6
7
8
40.4 3.7
8.8 3.7
23.8 3.7
278.1 4.6
8.0 3.8
5.4 3.6
321.8 3.7
>350
1a
2a
3a
4a
5a
6a
7a
8a
>350
121.3 4.0
>350
>350
>350
115.2 4.1
>350
>350
9
Serotonin
9
>350
>350
Kojic acid
Arbutin
51.4 3.6
201.2 2.3
Serotonin
a
Values were determined from logarithmic concentration–effect curves and are
a
the means of three experiments.
Values were determined from logarithmic concentration–effect curves and are
the means of three experiments.
of serotonin derivatives did not affect antioxidant activity strongly.
Therefore, we considered that the serotonin moiety and the amide
linkage of serotonin derivatives have a great influence on the anti-
oxidant activity.
After evaluating the antioxidant activity, we investigated the
tyrosinase inhibitory activity of serotonin derivatives. The inhibi-
tory activity of compounds 1, 2, 3, 5 and 6 was more potent than
kojic acid (IC50 = 51.4 3.6
tives, compound 627 showed the most inhibitory activity (IC50
5.4 3.6 M), whereas, compounds 4, 7, 8, and serotonin
exhibited low inhibitions (Fig. 2 and Table 2).
lM). Among tested serotonin deriva-
=
l
9
Inhibitory mechanism of compound 6 on mushroom tyrosinase
was analyzed by a Lineweaver–Burk plot as shown in Fig. 3. Three
lines representing the uninhibited enzyme and different concentra-
tions of compound 6 intersected on the horizontal axis. Therefore,
this compound is regarded as a non-competitive inhibitor of
mushroom tyrosinase. The inhibitory activity of tyrosinase was
found to be similar to antioxidant activity, that is to say, inhibitory
activities of serotonin derivatives were more potent than cinnamic
acid derivatives. To identify the structural importance of serotonin
derivatives, we evaluated compound 9 and serotonin. These com-
pounds exhibited decreased inhibitory activity, thus, we assumed
that the serotonin moiety and the cinnamic acid moiety of serotonin
derivatives are essential for tyrosinase inhibitory activity. From
the structural–activity point of view, compound 2 and 3 which
have OH or OMe group at 40 position exhibited a increase in tyros-
inase inhibitory activity relative to compound 1. In the case of
Figure 3. Lineweaver–Burk plot of compound 6 on tyrosinase. Concentrations of 6
were 0 mM (square), 2.5 mM (diamond), 10 mM (circle), respectively.
compound 2, 30 position replacement with OH group (compound
4) decreases the inhibitory activity very much, to the contrary, in
the case of compound 3, 30 position replacement with OH group
(compound 6) increased the activity. When the meta-hydroxy
function of compound 2 was substituted by a methoxy group
(compound 5), the activity was similar inhibitory potency to com-
pound 2, on the other hand, in the case of compound 3, compound
7 showed a significant loss in inhibitory activity. Furthermore, the
introduction of another methoxy group in an ortho-position to a
hydroxy group (compound 8) led to less in inhibitory activity rela-
tive to compound 5. Compound 9 exhibited a low inhibitory activ-
ity, thus, the cinnamic acid moiety of serotonin derivatives is
critical factor for tyrosinase inhibitory activity. Among cinnamic
acid derivatives, compounds 2a and 6a exhibited inhibitory po-
tency with IC50 values of 121.3 and 115.2 lM respectively. On
the other hand, these serotonin derivatives (2 and 6) also exhibited
inhibitory potency, therefore, we considered that the cinnamic
moiety of serotonin derivatives also affect tyrosinase inhibitory
activities. These results indicate the cinnamic moiety, the seroto-
nin moiety and the amide linkage of serotonin derivatives are
essential for the tyrosinase inhibitory activity.
In conclusion, we synthesized a series of serotonin derivatives
(1–9). Compound 4 (N-caffeoyl serotonin) showed the most potent
activity in DPPH assay (EC50 = 19.4
lM) and compound 6 showed
significant tyrosinase inhibitory activity (IC50 = 5.4
l
M). In the
two biologic activity (antioxidant activity and tyrosinase inhibitory
activity), while, the serotonin moiety and the amide linkage of
serotonin derivatives affect the antioxidant activity strongly, the
serotonin moiety, the amide linkage and the cinnamic acid moiety
of serotonin derivatives also affect tyrosinase inhibitory activity.
These results indicate that there is no evidence for direct correla-
Figure 2. Dose-dependent inhibitory effects of compound 6 (diamond shape) on
tyrosinase activity. Tyrosinase activity was measured using
substrate.
L-tyrosine as the

1986
T. Takahashi, M. Miyazawa / Bioorg. Med. Chem. Lett. 21 (2011) 1983–1986
20. Takii, T.; Hayashi, M.; Hiroma, H.; Chiba, T.; Kawashima, S.; Zhang, H. L.;
Nagatsu, A.; Sakakibara, J.; Onozaki, K. J. Biochem. 1999, 125, 910.
21. Roh, J. S.; Han, Y. J.; Kim, J. H.; Hwong, J. K. Biol. Pharm. Bull. 2004, 27, 1976.
22. Yamazaki, Y.; Kawano, Y.; Yamanaka, A.; Maruyama, S. Bioorg. Med. Chem. Lett.
2009, 19, 4178.
tion between free radical scavenging activity and tyrosinase inhibi-
tion, however, suggest that serotonin derivatives may serve as the
designed development of novel tyrosinase inhibitors.
23. Method of tyrosinase assay: The tyrosinase assay was performed by using
tyrosine as the substrate. 140 L of 0.1 M phosphate buffer (pH 7.0), 36 L of
1.5 mM -tyrosine and 13 L of sample solution were added to each well of a
96-well plate and then incubated at 37 °C for 10 min. Then 16 L of mushroom
L-
Supplementary data
l
l
L
l
l
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.02.028.
tyrosinase (500 unit/ml, 0.1 M phosphate buffer at pH 7.0) was added, and the
assay mixture was incubated at 37 °C for 25 min. Before and after incubation,
the amount of dopachrome produced in the reaction mixture was measured at
492 nm in a microplate reader (Corona Electric Co. Ltd.). Arbutin and kojic acid
were used as a positive control. The extent of tyrosinase inhibition by the
addition of the different compounds was calculated and expressed as the
percentage necessary for 50% inhibition concentration (IC50). The percentage of
tyrosinase activity was calculated as follows: Tyrosinase activity (%) = [(CꢀD)/
(AꢀB)] ꢁ 100, where A is the absorbance at 492 nm without test sample, B is
References and notes
1. Perez-Gilabert, M.; Garcia-Carmona, F. Biochem. Biophys. Res. Commun. 2001,
285, 257.
2. Kubo, I.; Yokokawa, Y.; Kinst-Hori, I. J. Nat. Prod. 1995, 58, 739.
3. Lee, G. C.; Lee, C. Y. Food Chem. 1997, 60, 231.
the absorbance at 492 nm without test sample and substrate,
C is the
4. Bran-Williams, W.; Cuvelier, ME.; Berset, C. Lebensm-Wiss-Technol. 1995, 28, 25.
5. Gomez-Cordové, C.; Bartolomé, B.; Vieira, W.; Virador, V. M. J. Agric. Food. Chem.
2001, 49, 1620.
absorbance at 492 nm with test sample, D is the absorbance at 492 nm with
test sample, but without substrate.
24. The scavenging activity for the DPPH radical was performed according to the
6. Masamoto, Y.; Murata, Y.; Baba, K.; Shimoishi, Y.; Tada, M.; Takahata, K. Biol.
Pharm. Bull. 2004, 27, 422.
follow method. An amount of 500
was mixed in a cuvette with 500
l
L of a 0.5 mM methanoic DPPH solution
l
L of cinnamic acid derivatives at different
7. Khan, K. M.; Maharvi, G. M.; Abbaskhan, A.; Hayat, S.; Khan, M. T. H.;
Makhmoor, T.; Choudhary, M. I.; Shaheen, F.; Atta-ur-Rahman. Helv. Chim. Acta
2003, 86, 457.
concentration levels. These cuvettes were shaken vigorously. The cuvettes
were allowed to stand at 27 °C for 30 min, the absorbance was measured at
517 nm using
a U-1500 spectrophotometer. The percentage of radical
8. Okombi, S.; Kival, D.; Bonnet, S.; Moriotte, A. M.; Pervier, E.; Boumendzel, A.
Bioorg. Med. Chem. Lett. 2006, 16, 2252.
9. Mellay-Hamon, V. L.; Criton, M. Biol. Pharm. Bull. 2009, 32, 301.
10. Aruoma, O. I.; Halliwell, B. Free Radical Food Addit. 1991, 121.
11. Halliwell, B.; Murcia, M. A.; Chirico, S.; Aruoma, O. I. Crit. Rev. Food Sci. Nutr.
1995, 35, 7.
scavenging activity was calculated using the equation. Radical scavenging
activity (%) = (Control ODꢀSample OD/Control OD) ꢁ 100. All tests were
performed in triplicate. BHT was used as
investigation of radical scavenging activity.
a reference standard for the
25. Koyama, N.; Kuribayashi, K.; Seki, T.; Kobayashi, K.; Furuhata, Y.; Suzuki, K.;
Arisaka, H.; Nakano, T.; Amino, Y.; Ishii, K. J. Agric. Food. Chem. 2006, 54, 4970.
26. Somei, M.; Yamada, F.; Kurauchi, T.; Nagahama, Y.; Hasegawa, M.; Yamada, K.;
Teranishi, S.; Sato, H.; Kaneko, C. Chem. Pharm. Bull. 2001, 49, 87.
12. Winkler, C.; Frick, B.; Schroecksnadelm, K.; Schennach, H.; Fuchs, D. Food Chem.
Toxicol. 2006, 44, 2003.
13. Lupo, M. P. Clin. Dermatol. 2001, 19, 467.
14. Sakamura, S.; Terayama, Y.; Kawakatsu, S.; Ichihara, A.; Saito, H. Agric. Biol.
Chem. 1978, 42, 1805.
15. Sato, H.; Kawagishi, H.; Nishimura, T.; Yoneyama, S.; Yoshimoto, Y.; Sakamura,
S. Agric. Biol. Chem. 1985, 49, 2969.
16. Park, J. B. Phytomedicine 2008, 15, 1093.
17. Kang, K.; Park, S.; Kim, Y. S.; Lee, S.; Back, K. Appl. Microbiol. Biotechnol. 2009, 83,
27.
18. Zhang, H. L.; Nagatsu, A.; Watanabe, T.; Sakakibara, J.; Okuyama, H. Chem.
Pharm. Bull. 1997, 45, 1910.
19. Nagatsu, A.; Zhang, H. L.; Mizukami, H.; Okuyama, H.; Sakakibara, J.; Tokuda,
H.; Nishino, H. Nat. Prod. Lett. 2000, 14, 153.
27. N-Isoferuloyl serotonin (6): This compound was obtained with a yield of 77.0%;
mp: 99–103 °C; IR (KBr): 3390, 1652, 1584, 1509 cmꢀ1 1H NMR (400 MHz,
;
DMSO-d6) d 10.47 (1H, d, J = 2.0 Hz, H-1), 9.16 (1H, s, 30-OH) 8.59 (1H, s, 5-OH),
8.10 (1H, t, J = 6.0 Hz, –CONH), 7.27 (1H, d, J = 15.6 Hz, H-70), 7.10 (1H, d,
J = 8.0 Hz, H-7), 7.04 (1H, d, J = 2.0 Hz, H-2), 6.97 (1H, d, J = 1.2 Hz, H-20), 6.95
(1H, dd, J = 8.4 and 1.2 Hz, H-60), 6.92 (1H, d, J = 8.4 Hz, H-50), 6.86 (1H, d,
J = 1.6 Hz, H-4), 6.58 (1H, dd, J = 8.0 and 1.6 Hz, H-6), 6.39 (1H, d, J = 15.6 Hz, H-
80), 3.78 (3H, s, 40-OMe), 3.41 (2H, dt, J = 6.0 and 7.2 Hz, H-11), 2.77 (2H, t,
13
J = 7.2 Hz, H-10). C NMR (100 MHz, DMSO-d6) d 165.1 (C-90), 150.2 (C-5),
149.1 (C-40), 146.7 (C-30), 138.5 (C-70), 130.8 (C-8), 127.9 (C-9), 127.8 (C-10),
123.1 (C-2), 120.1 (C-60), 119.7 (C-80), 113.3 (C-50), 112.1 (C-7), 111.6 (C-6),
111.3 (C-3), 110.8 (C-20), 102.4 (C-4), 55.6 (40-OMe), 39.4 (C-11), 25.4 (C-10).