Synthesis of N-hydroxycinnamoyl amide derivatives and evaluation of their anti-oxidative and anti-tyrosinase activities

Article abstract of DOI:10.1016/j.bmc.2019.05.031

Twelve N-hydroxycinnamoyl amino acid amide ethyl esters (CAES) were synthesized by using L-amino acid ethyl ester hydrochloride and corresponding cinnamic acid (ferulic acid, acetylferulic acid and caffeic acid) as raw materials in the presence of a catal

Full text of DOI:10.1016/j.bmc.2019.05.031

Accepted Manuscript
Synthesis of N-hydroxycinnamoyl amide derivatives and evaluation of their an-
ti-oxidative and anti-tyrosinase activities
Dong Wang, Jun Zhu, Jin-Rong Xu, Dan-Dan Ji
PII:
S0968-0896(19)30392-X
https://doi.org/10.1016/j.bmc.2019.05.031
BMC 14918
DOI:
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
8 March 2019
15 May 2019
19 May 2019
Please cite this article as: Wang, D., Zhu, J., Xu, J-R., Ji, D-D., Synthesis of N-hydroxycinnamoyl amide derivatives
and evaluation of their anti-oxidative and anti-tyrosinase activities, Bioorganic & Medicinal Chemistry (2019), doi:
https://doi.org/10.1016/j.bmc.2019.05.031
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Synthesis of N-hydroxycinnamoyl amide derivatives and evaluation of
their anti-oxidative and anti-tyrosinase activities
a
a,b,
a
a
Dong Wang , Jun Zhu * Jin-Rong Xu , Dan-Dan Ji
a
School of Science, Beijing Technology and Business University, Beijing 100048, China
Key Laboratory of Cosmeyic, China National Light Industry, Beijing 100048, China
Corresponding author: E-mail address: zhujun@btbu.edu.cn (J. Zhu)
b
*
Abstract
Twelve N-hydroxycinnamoyl amino acid amide ethyl esters (CAES) were synthesized by using L-amino
acid ethyl ester hydrochloride and corresponding cinnamic acid (ferulic acid, acetylferulic acid and
caffeic acid) as raw materials in the presence of a catalytic amount of 1-(3-dimethylaminopropyl)-3-
ethylcarbodiimide-hydrochloride (EDC) and 1-hydroxybenzotriene (HOBt). The 1,1-diphenyl-2-
picrylhydrazyl (DPPH) free radical scavenging activities of CAES were evaluated. The anti-tyrosinase
activities of N-feruloyl amino acid ethyl esters and the hydroxyl (OH·) free radical scavenging activities
of N-caffeoyl amino acid ethyl esters were also examined. DPPH free radical scavenging activity was
shown in all CAES, of which N-caffeoyl amino acid ethyl esters demonstrated higher radical scavenging
activity than N-feruloyl amide derivatives, and (E) -N-(caffeic acid)-L-glycinate ethyl ester (c ) had the
5
strongest ability to scavenge free radicals with an IC50 value of 18.6 µM.. The acetylferuloyl amino acid
esters exhibited the highest tyrosinase inhibition activity among the tested amides.
Keywords: N-hydroxycinnamoyl amino acid amide ethyl esters;L-amino acid ethyl ester
hydrochloride;Radical scavenging; Anti-tyrosinase activit

1
. Introduction
The pigmentation of plants and animals is usually triggered by enzymatic browning, mainly caused by
1
tyrosainase catalysis. The pigmentation of melanin appears in food, causing the undesirable reduction
of nutritional quality and consumer acceptance, and its commercial value may also be affected. The
browning degree of fruits and vegetables is mainly influenced by the content of phenols and the activity
2
of tyrosinase. Moreover, pigmentation-related disorders of human might be caused by melanin, such as
3
melasma and postinflammatory melanoderma. Tyrosinase is the main rate-limiting enzyme in melanin
synthesis. In addition, experimental evidence suggested that the excess free radicals in the body are one
4
of the important factors that exacerbate melanin production. Free radicals are produced continuously
during normal physiological events, which not only lead to the accumulation of lipid peroxides, but also
exacerbate cellular injury and aging process.5-7 However, antioxidant defense mechanisms reduce the
production of free radicals. Therefore, exploration for high-efficiency tyrosinase inhibitors and anti-
oxidants is important for food preservation and medicinal whitening products.
8
Caffeic acids (CA) and Ferulic acids (FA) as hydroxycinnamic acid derivatives, are widely
distributed in fruits, vegetables and drinks.9-12 Natural occurring CA and FA are very unstable in
13
structure, they are usually presented as conjugated (esters and amides) forms. Hydroxycinnamamide is
14
one important kind of hydroxycinnamic acid derivatives and is also widely distributed in plants. The
plant hydroxycinnamamides, exhibited a wide variety of biological activities, including anti-
tyrosinase,8,15 anti-viral, anti-oxidative,16-18 anti-inflammatory19-21 and insulin secretion stimulating
activities in vitro.22
16
Interestingly, hydroxycinnamic acid amide derivatives formed by the hydroxycinnamic acid with
amino acids had a significantly stronger antioxidant activity than hydroxycinnamic acid, and the

irritation were significantly reduced.5,23 Moreover, a large number of experimental datas indicated that
tyrosinase inhibitors have an enhanced inhibitory effect on tyrosinase as their hydrocarbon chain length
increases.1,24
Inspired by the above studies, twelve N-hydroxycinnamoyl amino acid amide ethyl esters (CAES)
(their structure was shown in Table 1) were synthesized by using L-amino acid ethyl ester
hydrochloride and cinnamic acid (ferulic acid, acetylferulic acid and caffeic acid) as raw materials and
the structure-activity relationship (antioxidative and anti-tyrosinase activity) was evaluated between N-
hydroxycinnamic acid and L-amino acid ethyl ester. In this study, the anti-oxidative activity of CAES
was evaluated by DPPH free radical scavenging rate. In addition, the anti-tyrosinase of N-feruloyl
amides and the hydroxyl free radical scavenging activities of N-caffeoyl amides were studied.
Table 1
Synthesized N-hydroxycinnamoyl amino acid amides ethyl esters (CAES)
O
R3
O
R1
R1
EDC/HOBt
DMF
O
N
OH
H
O
R2
R2
d1～d3
a1～a3, b1～b3, c1～c6
Compound
R
1
R
2
R
3
Yields, %
48.4
a
a
a
1
2
3
OCH
OCH
OCH
OCH
3
3
3
3
OH
OH
CH
3
CH(CH
3
)
2
50.6
OH
CH
2
CH(CH
3
)
2
55.8
b
1
OOCCH
3
CH
3
54.8

b
b
2
3
OCH
OCH
OH
3
3
OOCCH
OOCCH
OH
3
CH(CH
CH CH(CH
CH
CH(CH
3
)
2
52.7
54.8
73
3
2
3
)
2
c
1
c
2
c
3
c
4
c
5
c
6
3
OH
OH
3
)
2
79
OH
OH
CH
2
CH(CH
3
)
2
82
OH
OH
CH(CH
3
)CH
2
CH
3
86
OH
OH
H
60
OH
OH
CH
2
C
6
H
6
75
CA(d
1
)
OH
OH
—
—
—
—
FA(d
2
)
OCH
OCH
3
3
OH
—
d
3
OOCCH
3
71.4
2
2
2
. Results and discussion
.1 Biological evaluation
.1.1. DPPH radical scavenging activity
DPPH had an unpaired valence electron at one atom of nitrogen bridge, when combined with a
hydrogen-donating antioxidant, the power of free radical scavenging of an antioxidant can be
25
determined by measuring of a color change at 517 nm. The inhibitory effects of the synthesized amides
see Table 1. for structures) against stable free DPPH radical (IC50) were shown in Table 2. Ferulic
acid, caffeic acid and ascorbic acid were tested as controls. Their DPPH radical scavenging activity
(

among the tested target products decreased in the following order: c
5
> c
2
> CA (d
1
) > c
3
> c
1
> c > FA
6
(d
2
) > c > a > a > a > Vitamin C > b > d , b , b . All CAES exhibited inhibitory effects on free
4
1
3
2
1
3
2
3
DPPH radicals. As shown in Table 2. N-caffeoyl amino acid ethyl esters demonstrated higher radical
scavenging activity than N-feruloyl amide derivatives. This results suggests that the introduction of
(hydroxyl) groups significantly increases the scavenging free radical activity of these compounds.
Furthermore, the scavenging effect of the same parent molecule demonstrated the following sequence:
ferulic acid (FA) > feruloyl amino acid ethyl esters > acetylferuloyl amino acid ethyl esters, with
feruloyl amino acid esters exhibited higher scavenging activity than acetylferuloyl amino acid esters
with phenolic hydroxyl groups. As for the IC50 values, N-caffeoyl amide derivatives showed the order of
c
5
> c
ascorbic acid in DPPH-scavenging activity. Among all N-caffeoyl amino acid ethyl esters, (E) -N-
caffeic acid)-L-glycinate ethyl ester (c ) showed the strongest ability to scavenge free radicals, with an
2
> CA (d
1
). It is noticeable that N-caffeoyl amino acid ethyl esters are more effective than
(
5
IC50 of 18.6 µM. Correspondingly, for the series N-feruloyl amides derivatives had no enhancing effect
on DPPH inhibition in the DPPH-scavenging test. The activity of different CAES correlates with their
alkyl chains.
Table 2
DPPH radical scavenging activity
Compound
IC （µM）
Compounds
IC （µM）
5
0
50
d
3
>500
c
1
29.7

a
a
a
1
2
3
46.4
59.6
56.1
482
c
c
c
c
c
2
3
4
5
6
19.6
28.6
43.5
18.6
33.2
b
b
b
1
2
3
>500
>500
35.7
Vitamin C
CA(d
80.9
27.6
FA(d
2
)
1
)
Note: Ferulic acid, caffeic acid and Vitamin C were tested as positive controls. Each experiment was
measured for 3 times. The IC50 value was defined as the concentration of the antioxidant required when
reaching a 50% inhibition of DPPH free radicals. The IC50 value was determined by interpolation,from
linear regression analysis.
2
.1.2. Hydroxyl radical scavenging activity of N-caffeoyl amino acid ethyl esters
In this study, N-caffeoyl amino acid ethyl esters demonstrated higher DPPH radical scavenging
activity than N-feruloyl amides derivatives. Oxidation is one of the important causes of food
6
deterioration. H
2
O
2
as a nonradical that belongs to the group of ROS. can cause serious damage to
biomolecules via the formation of OH·. Hydroxyl radicals are commonly formed in vivo and
continuously produced during normal physiologic events. Hydroxyl radicals usually cause damage to
lipids, proteins, and nucleic acids.26
The effect of N-caffeoyl amino acid ethyl esters in hydroxyl radical-scavenging activity was measured
in vitro and the obtained results (IC50) were shown in Table 3. The scavenging activity of N-caffeoyl
amino acid esters on OH· radicals decreased in the following order: c
1
> CA (d
1
) > c
6
> c
5
> c
2
> c
3
> c
4

>
Vitamin C. It is noticable that N-caffeoyl amino acid ethyl esters showed higher anti-oxidative effect
than ascorbic acid (a positive control). Fan reported that the antioxidative activity of (E) -N-(caffeic
2
4
acid)-L-phenylalaninate ethyl ester is lower than the free CA. The present work found that (E) -N-
(
caffeic acid) -L-alanine ethyl ester (c
1
) demonstrated higher hydroxyl radical scavenging activity than
(
E) -N-(caffeic acid)-L-phenylalaninate ethyl ester (c
6
) and the free CA (d ).
1
Table 3
Hydroxyl radical-scavenging activity
Compounds
IC （mM）
Compounds
IC （mM）
5
0
50
c
1
c
2
c
3
c
4
0.214
c
5
6
0.340
0.292
0.287
1.616
0.348
0.373
0.411
c
CA(d
1
)
Vitamin C
Note: Vitamin C were tested as positive controls. Each experiment was measured for 3 times. IC50
sample concentration in mM at which the hydroxyl radical inhibition rate reaches 50%). The IC50 value
was determined by interpolation, from linear regression analysis.
(
2
.1.3. Anti-tyrosinase activity
Gómezcordovés reported that caffeic acid and ferulic acid in wine showed inhibition of tyrosinase
2
7
activity. (E) -N-(feruloyl acid)-L-phenylalaninate ethyl ester (IC50 < 0.18 μM) demonstrated higher
inhibition of tyrosinase activity than (E) -N-(caffeic acid)-L-phenylalaninate ethyl ester (IC50 > 29
1
5
μM). Considering feruloyl amino acid esters and their acetylferuloyl amino acid esters derivatives are
potential inhibitors, the effects of the tested target products in suppressing mushroom tyrosinase activity

was measured and the obtained results were summarized in Fig.1. The inhibitory rate decreased in the
following order: b > b > b > a > FA (d ) > a > d > a . It was found that acetylferuloyl amino acid
amide and feruloyl amino acid amide, (E) -ethyl 2-(3-(4-acetoxy-3-methoxyphenyl)acrylamido) -4-
methylpentanoate (b ) showed the most significant inhibitory effects agaist mushroom tyrosinase than
3
2
1
1
2
3
3
2
3
ferulic acid (p < 0.05). Furthermore, the anti-tyrosinase activity of N-feruloyl amides derivatives had the
following sequence: acetylferuloyl amino acid esters > feruloyl amino acid esters and ferulic acid (FA).
The preliminary results of the structure-activity relationship were as follows: 1) the tyrosinase inhibition
activity of acetylferuloyl amino acid esters was more effective than feruloyl amino acid amides, which
mainly derive from the acetyl groups of the molecular structure; 2) The anti-tyrosinase activity of
different feruloyl amino acid esters and their acetylferuloyl amino acid ester derivatives was found to
correlate with the alkyl chains in structure.
50
40
30
20
10
0
*
*
*
a1
a2
a3
b1
b2
b3 ferulic acid d3
N-feruloyl amides
Fig.1.
). The concentrations of the
Anti-tyrosinase activity of different CAES derivatives (a
1
- a3,
b
1
- b
3
and d
3

CAES derivatives are 1 mg/L. The values were presented as mean ± confidence interval (n=3). * P <
0
.05 (Compared with the ferulic acid group, calculated at level of significance 0.05).
3
. Conclusions
Twelve CAES have been synthesized and their efficacy as anti-oxidant was measured by acavenger
activity against DPPH radicals. In addition, the anti-tyrosinase of N-feruloyl amides and the OH·
scavenging-activities of N-caffeoyl amides were studied.
The present study has shown that N-caffeoyl amino acid ethyl esters exhibited the highest antioxidant
activity in scavenging both DPPH· and OH· radicals when compared with Vitamin C (a famous
antioxidant). The radical-scavenging activity of amides mainly depended on the phenolic hydroxyl
groups, carboxyl groups and alkyl chain of the molecular structure. On the other side, acetylferuloyl
amino acid esters exhibited the highest tyrosinase inhibition activity among the tested amides. Further
investigations are required to elucidate the relationship between the activity and alkyl chain structure.
All the data suggested that N-caffeoyl amino acid ethyl esters might serve as new potent anti-oxidative
and acetylferuloyl amino acid esters might serve as new whitening agents used in food or cosmetics
industry.
4
4
. Experimental section
.1. Chemistry
Ferulic acid, Caffeic acids were purchased from Macklin Biochemical (Shanghai) and DPPH was
purchased from TCI (Shanghai). Mushroom tyrosinase and L-ascorbic acid was purchased from Beijing
Huabo Station Bioanalysis Technology Co., Ltd., China. All other chemical reagents were in analytical
grade.

The NMR spectra of the synthesized compounds were acquired on a Bruker AV 300 MHz
1
spectrometers, operating at 300 MHz for H (the synthesized compounds were dissolved in CDCl
3
or
DMSO-d
DPPH and anti-tyrosinase assays were measure by ELISA (Tecan, infinite M200 PRO, Swiss).
.2. Preparation of acetylferulic acid (d
Ferulic acid (3.88 g, 0.02 mol) was dissolved in acetic anhydride (10 mL) with the addition of
anhydrous pyridine (10 drops). After that, the resultant reaction mixture was heating back for 7 h at 120
. The mixture was cooled at 25 ℃ with the addition of water (40 mL), and then the reaction mixture
6
); The model of UPLC-MS was ACQUITY UPLC/Xevo G2. The reduction in absorbance of
4
3
)
℃
was stirred for 2 h until the appearance of white precipitate at 25 ℃. Finally, the white precipitate was
filtered, and the residual paste was collected and purified by EtOH.
4
.3. Preparation of CAES
Feruloyl amino acid ethyl esters a1-3, b1-3 were synthesized from ferulic acid and different amino acid
ethyl esters using 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide-hydrochloride (EDC) /1-
hydroxybenzotriene (HOBt) method with some modifications.28
Ferulic acid (1.07 g, 5.5 mmol) and HOBt (0.74 g, 5.5 mmol) and EDC（0.99 g, 5.5 mmol） were
dissolved in (11 mL) N,N-dimethylformamide (DMF), the reaction mixture was cooled at 0 ℃ and
reacted for 10 min. Then, N-methyl morpholine (0.76 mL) and hydrochloride of HCl•NH
COOR (C-protected amino acid) (5.5 mmol) in 8.5 ml CH Cl were added and well mixed. The reaction
was carried out at 0 ℃ for 1 h, followed by stirring at room temperature for 20 h. When the reaction was
completed, the reaction solution was poured into (100 mL) 5% NaHCO and extracted with ethyl acetate
2 × 75 mL). Then, the organic phase was washed with water (2 × 50 mL) and brine (2 × 50 mL) before
dring with anhydrous Na SO for 24 h. After recovering the solvent under reduced pressure, the residue
2
-CH(R)-
2
2
3
(
2
4

was purified by silica gel column chromatography (Ethyl acetate/Petroleum ether (2:1)) to give a
1
-a .
3
The preparation of acetylferuloyl amino acid ethyl esters b -b was the same as feruloyl amino acid
1
3
esters’ except for the use acetylferulic acid as reaction material instead of ferulic acid. EDC/HOBt
was applied.5
coupling method for the synthesis of caffeoyl amino acid ethyl esters c
1
-c
6
(
E) -3-(4-acetoxy-3-methoxyphenyl) acrylic acid (d ): yield 71.4%, white powder, melting point:
3
1
1
6
93-195 ℃; H NMR (300 MHz, CDCl
3
): 7.74 (d, J = 15.6 Hz, 1H, CH=C), 7.06-7.17 (m, 3H, Ar-H),
), 2.33 (s, 3H, OCOCH ); ESI-MS (m/z): 259.0591
.40 (d, J = 15.6 Hz, 1H, C=CH), 3.88 (s, 3H, OCH
3
3
([M+Na]+).
(
E) -N-(feruloyl) -L-alanine ethyl ester (a
1H NMR (300 MHz, CDCl
): 7.53 (d, J = 15.6 Hz, 1H, CH=C), 7.53 (dd, J = 8.1, 1.8 Hz, 1H, Ar-H),
.96 (d, J = 1.8 Hz, 1H, Ar-H), 6.88 (d, J = 8.1 Hz, 1H, Ar-H), 6.36 (d, J = 7.2 Hz, 1H, -NH), 6.30 (d, J
15.6 Hz, 1H, C=CH), 6.10 (s, 1H, -OH), 4.72 (m, 1H, -CH), 4.22 (q, J = 7.2 Hz, 2H, -CH ), 3.89 (s,
H, -OCH ), 1.46 (d, J = 7.2 Hz, 3H, -CH ), 1.29 (t, J = 7.2 Hz, 3H, -CH ); ESI-MS (m/z): 294.1344
[M+H]+).
1
): yield 48.4%, white powder, melting point: 110-112 ℃;
3
6
=
2
3
3
3
3
(
1
(
E) -N-(feruloyl) -L-valine ethyl ester (a
NMR (300 MHz, CDCl ): 7.55 (d, J = 15.6 Hz, 1H, CH=C), 7.04 (dd, J = 8.4, 1.8 Hz, 1H, Ar-H), 6.99
d, J = 1.8 Hz, 1H, Ar-H), 6.91 (d, J = 8.4 Hz, 1H, Ar-H), 6.34 (d, J = 15.6 Hz, 1H, C=CH), 6.24 (d, J =
2
): yield 50.6%, white powder, melting point: 178-180 ℃; H
3
(
8
2
9
.7 Hz, 1H, -NH), 6.13 (s, 1H, -OH), 4.72 (dd, J = 8.7, 4.8 Hz, 1H, -CH), 4.23 (dddd, J = 21.6, 14.4, 7.2,
.4 Hz, 2H, -CH ), 3.90 (s, 3H, -OCH ), 2.23 (m, 1H, -CH), 1.30 (t, J = 7.2 Hz, 3H, -CH ), 0.97 (dd, J =
.9, 6.9 Hz, 6H, >CH +CH ); ESI-MS (m/z): 322.1662 ([M+H]+).
2
3
3
3
3
(E) -N-(feruloyl) -L-leucine ethyl ester (a
3
): yield 55.8%, white powder, melting point: 180-182 ℃;
1H NMR (300 MHz, CDCl
): 7.53 (d, J = 15.6 Hz, 1H, CH=C), 7.02 (dd, J = 8.1, 1.8 Hz, 1H, Ar-H),
3

6
.96 (d, J = 1.8 Hz, 1H, Ar-H), 6.88 (d, J = 8.1 Hz, 1H, Ar-H), 6.27 (d, J = 15.6 Hz, 1H, C=CH), 6.15
d, J = 8.4 Hz, 1H, -NH), 5.97 (s, 1H, -OH), 4.78 (m, 1H, -CH), 4.21 (q, J = 7.2 Hz, 2H, -CH ), 3.90 (s,
), 0.96 (t, J = 6.6 Hz, 6H,
(
2
3
H, -OCH
CH +CH
3
), 1.56-1.72 (m, 3H, >CH
2
+CH), 1.29 (t, J = 7.2 Hz, 3H, -CH
3
>
3
3
); ESI-MS (m/z): 336.1815 ([M+H]+).
(
E) -ethyl 2-(3-(4-acetoxy-3-methoxyphenyl) acrylamido) propanoate (b ): yield 54.8%, white solid,
1
1
melting point: 80-82 ℃; H NMR (300 MHz, CDCl
3
): 7.57 (d, J = 15.6 Hz, 1H, CH=C), 7.11 (dd, J =
8
.1, 1.8 Hz, 1H, Ar-H), 7.06 (d, J = 1.8 Hz, 1H, Ar-H), 7.02 (d, J = 8.1 Hz, 1H, Ar-H), 6.38 (d, J = 15.6
Hz, 1H, C=CH), 6.34 (d, J = 7.2 Hz, 1H, -NH), 4.72 (m, 1H, -CH), 4.23 (q, J = 7.2 Hz, 2H, -CH ), 3.85
s, 3H, -OCH ), 2.32 (s, 3H, -OCOCH ), 1.47 (d, J = 6.9 Hz, -CH ), 1.30 (t, 3H, J = 7.2 Hz, -CH ); ESI-
MS (m/z): 336.1456 ([M+H]+).
2
(
3
3
3
3
(
E) -ethyl 2-(3-(4-acetoxy-3-methoxyphenyl)acrylamido) -3-methylbutanoate (b ): yield 52.7%, white
2
1
solid, melting point: 100-102 ℃; H NMR (300 MHz, CDCl
3
): 7.57 (d, J = 15.6 Hz, 1H, CH=C), 7.09
(dd, J = 8.1, 1.8 Hz, 1H, Ar-H), 7.07 (d, J = 1.8 Hz, 1H, Ar-H), 7.02 (d, J = 8.1 Hz, 1H, Ar-H), 6.41 (d,
J = 15.6 Hz, 1H, C=CH), 6.25 (d, J = 9 Hz, 1H, -NH), 4.70 (dd, J = 9, 4.8 Hz, 1H, -CH), 4.22 (dddd, J =
2
1
1.3, 14.4, 7.2, 1.8 Hz, 2H, -CH
2
), 3.85 (s, 3H, -OCH
3
), 2.31 (s, 3H, -OCOCH
3
), 2.24 (m, 1H, -CH),
.30 (t, 3H, J = 7.2 Hz, -CH ), 0.97 (dd, J = 9.9, 6.9 Hz, 6H, >CH
3
3
+CH ); ESI-MS (m/z): 364.1770
3
([M+H]+).
(
E) -ethyl 2-(3-(4-acetoxy-3-methoxyphenyl)acrylamido) -4-methylpentanoate (b ): yield 54.8%,
3
1
yellow solid, melting point: 102-104 ℃; H NMR (300 MHz, CDCl
3
): 7.51 (d, J = 15.6 Hz, 1H, CH=C),
6
.97-7.03 (m, 3H, Ar-H), 6.43 (d, J = 8.4 Hz, 1H, -NH), 6.34 (d, J = 15.6 Hz, 1H, CH=C), 4.76 (m, 1H,
CH), 4.20 (q, J = 7.2 Hz, 2H, -CH ), 3.82 (s, 3H, -OCH ), 2.30 (s, 3H, -OCOCH ), 1.55-1.70 (m, 3H,
+CH), 1.28 (t, 3H, J = 7.2 Hz, -CH ), 0.95 (t, J = 6.3 Hz, 6H, >CH +CH ); ESI-MS (m/z):
-
2
3
3
>
CH
2
3
3
3
3
78.1923 ([M+H]+).

(
E) -N-(caffeic acid) -L-isoleucine ethyl ester (c
H NMR (300 MHz, DMSO-d ): 9.36 (s, 1H, OH), 9.12 (s, 1H, OH), 8.19 (d, J = 8.2 Hz, 1H, NH),
.23 (d, J = 15.7 Hz, 1H, HC=C), 6.93 (d, J = 1.7 Hz, 1H, Ar-H), 6.83 (d, J = 8.2 Hz, 1H, Ar-H), 6.73
d, J = 8.1 Hz, 1H, Ar-H), 6.51 (d, J = 15.7 Hz, 1H, C=CH), 4.35-4.25 (m, 1H, CH), 4.10 (dd, J = 7.0,
.1 Hz, 2H, OCH ), 1.88-1.73 (m, 1H, CH), 1.51-1.04 (m, 5H, CH +CH ), 0.92-0.80 (m, 6H,
CH +CH ); ESI-MS (m/z): 322.0833[M+H]+).
4
): yield 86%, yellow solid, melting point: 120-124 ℃
; 1
6
7
(
5
2
2
3
3
3
(
E) -N-(caffeic acid)-L-phenylalaninate ethyl ester (c ): yield 75%, yellow solid, melting point: 164-
6
1
1
66 ℃; H NMR (300 MHz, DMSO-d
6
): 9.37 (s, 1H, OH）, 9.13 (s, 1H, OH), 8.42 (d, J = 10.2 Hz, 1H,
NH), 7.23 (m, J = 15.9 Hz, 7.9 Hz, 5H, HC=C, Ar-H), 6.92 (d, J = 1.6 Hz, 1H, Ar-H), 6.81 (s, 1H, Ar-
H), 6.73 (d, J = 8.2 Hz, 1H, Ar-H), 6.37 (d, J = 15.7 Hz, 1H, C=CH), 4.57-4.49 (m, 1H, CH), 3.09-2.81
(
m, 2H, OCH
2
), 1.10 (t, J = 7.1 Hz, 3H, CH ); ESI+-MS (m/z): 356.0579 [M+H]+).
3
Detailed characterization data of the compounds (E) -N-(caffeic acid) -L-alanine ethyl ester (c1)，(E)
N-(caffeic acid) -L-valine ethyl ester (c2)，(E) -N-(caffeic acid) -L-leucine ethyl ester (c ) and (E) -N-
caffeic acid)-L-glycinate ethyl ester (c
) were showned in our previous paper.23,29
.4. DPPH free radical scavenging activity
The free radical scavenging activity of the CAES was determined by the DPPH , and the activity was
-
(
3
5
4
3
0
measured according to a previous literature, with minor modifications. Testing samples were dissolved
in absolute ethanol and formulated into different concentrations. Then, 1 mL of the sample solution was
mixed with 1 mL of DPPH (0.2 mM) in ethanol with gentle shaking, the mixture was incubated in the
dark for 30 min, and the absorbance at 517 nm was measured against blank control. 1 mL of absolute
ethanol was used instead of the sample as the blank control. The positive control was L-ascorbic acid.
Each experiment was measured for 3 times. For each sample ,an IC50 values was calculated using a
dose-response curve. The IC50 value was defined as the concentration of the antioxidant required when

reaching an inhibition rate of 50%. Sample identified with a lower IC50 indicates higher anti-oxidative
activity.31
4
.5. The hydroxyl free radical-scavenging activity test
The hydroxyl free radical-scavenging activity of caffeoyl amino acid ethyl esters and L-ascorbic acid
1
5
was determined according to the reported method with some modifications. Briefly, 1mL of ethanolic
solution of phenanthroline (0.75 mM), phosphate buffer (0.2 M, pH 7.4), sample solution (dissolved in
DMSO) and aqueous solution of 0.75 mM FeSO
4
was mixed in a test tube with shaking. 1 mL of 0.01%
H
2
O
2
solution (30% H were mixed with deionised water) were added to the tube. Then the reaction
2
O
2
mixed solution was incubated at 37 ℃ in a water bath for 1h, and the absorbance value of the mixture
was measured at 536 nm in a spectrophotometer. Each experiment was measured for 3 times. The
inhibition percentage of hydroxyl radical of the samples was expressed as IC50 (sample concentration in
mM at which the hydroxyl radical inhibition rate reaches 50%). The inhibition percentage of OH· was
determined using the following equation:
Hydroxyl radical inhibition rate (%) = [(C-B) / (A-B)] × 100
where A is the absorbance of the control, B is the initial concentration of the blank and C is the
absorbance in the presence of sample
4
.6. Assay of anti-tyrosinase activity
Anti-tyrosinase activity was determined according to the modified method using L-tyrosine as
8
substrate. Mushroom tyrosinase (25 KU/mg In a 3 ml reaction mixture containing L-ascorbic acid (pH
6
.5, 25 ° C), ΔA265 changes to 0.001 per unit of enzyme activity unit per minute). Briefly, the
compounds (1 g/L) (a and ferulic acid) were dissolved in dimethyl sulfoxide (DMSO).
1
- a3,
b
1
- b3 ,
d
3
HCl (0.1 M) and Phosphate buffer (0.2 M, pH 6.8) were dissolved in deionized water. The type and
amount of reagents were shown in Table 4.

Table 4
The type and amount of reagents
tube PB（mL） L-tyrosine（mL） sample（mL）
DMSO（mL）
MT（mL）
A
B
C
D
1.35
1.65
1.35
1.65
1.00
1.00
1.00
1.00
-
0.35
0.30
-
0.35
-
0.30
-
0.35
0.35
-
-
Note: Phosphate buffer (PB) (65 mL, 0.2 M, pH 6.8); Tyrosine (0.3 mL, 0.507 g/L) was dissolved in
HCl (35 mL, 0.1 M) and phosphate buffer (65 mL, 0.2 M, pH 6.8); Inhibitor (0.350 mL, 1 g/L) was
dissolved in DMSO; Mushroom tyrosinase (MT) (0.300 mL, 200 U/mL) dissolved in phosphate buffer
(0.2 M, pH 6.8). The reference solution was obtained by replacing the inhibitor with DMSO (0.350 mL).
The reaction solution was incubated after adding mushroom tyrosinase solution to the mixture. Then,
the mixture was incubated at 37 ℃ for 30 min. The absorbance was measured at 475 nm. The
inhibition percentage of mushroom tyrosinase activity was determined by using the following formula:
%
Tyrosinase activity inhibition rate = {[(A-B)-(C-D)]/(A-B)}×100%
Each experiment was measured for 3 times. Ferulic acid was used as positive control.
.7. Statistical analysis
4
The IC50 values of DPPH and OH· free radical scavenging assay were obtained by regression
analysis. The other data were presented as mean ± standard deviations and analyzed by SPSS software.
The set of data was considered to have a significant difference (p < 0.05).

Acknowledgements
This work was financially supported by Outstanding Teachers’ Reword of Beijing Colleges and
Universities, China (No. 19005753005).
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[
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2

Graphical Abstract
Synthesis of N-hydroxycinnamoyl amide derivatives and evaluation of their anti-oxidative and
anti-tyrosinase activities
Jun Zhu*, Dong Wang, etal
School of Science, Beijing Technology and Business University, Beijing 100048, China

Research Highlights
(
(
(
(
i) Four new cinnamoyl amides with amino acid ester moiety were designed and synthesized.
ii) The free radical-scavenging and anti-tyrosinase activities of the compounds were evaluated.
iii) The antioxidant activity of compounds was due to the phenolic hydroxyl groups.
iv) The tyrosinase inhibition activity of compouds mainly derive from the acetyl groups.
2