Synthesis of cinnamic amide derivatives and their anti-melanogenic effect in α-MSH-stimulated B16F10 melanoma cells

Article abstract of DOI:10.1016/j.ejmech.2018.10.025

Of the three enzymes that regulate the biosynthesis of melanin, tyrosinase and its related proteins TYRP-1 and TYRP-2, tyrosinase is the most important because of its ability to limit the rate of melanin production in melanocytes. For treating skin pigmentation disorders caused by an excess of melanin, the inhibition of tyrosinase enzyme is by far the most established strategy. Cinnamic acid is a safe natural product with an (E)-β-phenyl-α,β-unsaturated carbonyl motif that we have previously shown to play an important role in high tyrosinase inhibition. Since cinnamic acid is relatively hydrophilic, which hinders its absorption on the skin, fifteen less hydrophilic cinnamic amide derivatives (1–15) were designed as safe and more potent tyrosinase inhibitors and were synthesized through a Horner-Wadsworth-Emmons reaction. The use of conc-HCl and acetic acid for debenzylation of the O-benzyl-protected cinnamic amides 40–54 produced the following three results. 1) Cinnamic amides 43, 48, and 53 with a 2,4-dibenzyloxyphenyl group, irrespective of the amine type of the amides, produced complex compounds with high polarity. 2) Cinnamic amides 40–42, 44, 50–52, and 54 with a benzylamino, or diethylamino group produced the desired debenzylated cinnamic amides 1–3, 5, 10–13, and 15. 3) Cinnamic amides 45–47, and 49 with an anilino moiety provided 3,4-dihydroquinolinones 16–19 through intramolecular Michael addition of the anilide group. Notably, the use of BBr₃ as an alternative debenzylating agent for debenzylation of cinnamic amides 45–49 with the anilino moiety provided our desired cinnamic amides 6–10 without inducing the intramolecular Michael addition. Debenzylation of cinnamic amides 43, 48, and 53 with a 2,4-dibenzyloxyphenyl group was also successfully accomplished using BBr₃ to give 4, 9, and 14. Among the nine compounds that inhibited mushroom tyrosinase more potently at 25 μM than kojic acid, four cinnamic amides 4, 5, 9, and 14 showed 3-fold greater tyrosinase inhibitory activity than kojic acid. The docking simulation using tyrosinase indicated that these four cinnamic amides (?6.2 to ?7.9 kcal/mol) bind to the active site of tyrosinase with stronger binding affinity than kojic acid (?5.7 kcal/mol). All four cinnamic amides inhibited melanogenesis and tyrosinase activity more potently than kojic acid in α-MSH-stimulated B16F10 melanoma cells in a dose-dependent manner without cytotoxicity. The strong correlation between tyrosinase activity and melanin content suggests that the anti-melanogenic effect of cinnamic amides is due to tyrosinase inhibitory activity. Considering that the cinnamic amides 4, 9, and 14, which exhibited strong inhibition on mushroom tyrosinase and potent anti-melanogenic effect in B16F10 cells, commonly have a 2,4-dihydroxyphenyl substituent, the 2,4-dihydroxyphenyl substituent appears to be essential for high anti-melanogenesis. These results support the potential of these four cinnamic amides as novel and potent tyrosinase inhibitors for use as therapeutic agents with safe skin-lightening efficiency.

Full text of DOI:10.1016/j.ejmech.2018.10.025

Accepted Manuscript
Synthesis of cinnamic amide derivatives and their anti-melanogenic effect in α-MSH-
stimulated B16F10 melanoma cells
Sultan Ullah, Dongwan Kang, Sanggwon Lee, Muhammad Ikram, Chaeun Park, Yujin
Park, Sik Yoon, Pusoon Chun, Hyung Ryong Moon
PII:
S0223-5234(18)30887-0
10.1016/j.ejmech.2018.10.025
EJMECH 10811
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 16 August 2018
Revised Date: 8 October 2018
Accepted Date: 10 October 2018
Please cite this article as: S. Ullah, D. Kang, S. Lee, M. Ikram, C. Park, Y. Park, S. Yoon, P. Chun, H.R.
Moon, Synthesis of cinnamic amide derivatives and their anti-melanogenic effect in α-MSH-stimulated
B16F10 melanoma cells, European Journal of Medicinal Chemistry (2018), doi: https://doi.org/10.1016/
j.ejmech.2018.10.025.
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ACCEPTED MANUSCRIPT
Graphical abstract

ACCEPTED MANUSCRIPT
Synthesis of cinnamic amide derivatives and their anti-melanogenic effect in α-MSH-
stimulated B16F10 melanoma cells
a,
a,
a,
§
b,c
a
§
§
Sultan Ullah , Dongwan Kang , Sanggwon Lee , Muhammad Ikram , Chaeun Park ,
a
c
d
a,
Yujin Park , Sik Yoon , Pusoon Chun , Hyung Ryong Moon *
a
Laboratory of Medicinal Chemistry, College of Pharmacy, Pusan National University, Busan
4
6241, South Korea
b
Department of Pharmacy, Comsats University Islamabad, Abbottabad Campus, Abbottabad
2
2060, Pakistan
c
Department of Anatomy, Pusan National University School of Medicine, 49 Busandaehak-ro,
Mulgeum-eup, Yangsan-si, Gyeongsangnam-do, 50612, South Korea.
d
College of Pharmacy and Inje Institute of Pharmaceutical Sciences and Research, Inje
University, Gimhae, Gyeongnam 50834, South Korea
§These authors (U. Sultan, D. Kang, and S. Lee) equally contributed to this work.
*
Author to whom correspondence should be addressed.
Hyung Ryong Moon, Laboratory of Medicinal Chemistry, College of Pharmacy, Pusan
National University, Busan 46241, Republic of Korea. E-mail address: mhr108@pusan.ac.kr;
1

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Tel.: +82 51 510 2815; Fax: +82 51 513 6754.
Abstract
Of the three enzymes that regulate the biosynthesis of melanin, tyrosinase and its related
proteins TYRP-1 and TYRP-2, tyrosinase is the most important because of its ability to limit
the rate of melanin production in melanocytes. For treating skin pigmentation disorders
caused by an excess of melanin, the inhibition of tyrosinase enzyme is by far the most
established strategy. Cinnamic acid is a safe natural product with an (E)-β-phenyl-α,β-
unsaturated carbonyl motif that we have previously shown to play an important role in high
tyrosinase inhibition. Since cinnamic acid is relatively hydrophilic, which hinders its
absorption on the skin, fifteen less hydrophilic cinnamic amide derivatives (1 – 15) were
designed as safe and more potent tyrosinase inhibitors and were synthesised through a
Horner-Wadsworth-Emmons reaction. The use of conc-HCl and acetic acid for debenzylation
of the O-benzyl-protected cinnamic amides 40 – 54 produced the following three results. 1)
Cinnamic amides 43, 48, and 53 with a 2,4-dibenzyloxyphenyl group, irrespective of the
amine type of the amides, produced complex compounds with high polarity. 2) Cinnamic
amides 40 – 42, 44, 50 – 52, and 54 with a benzylamino, or diethylamino group produced the
desired debenzylated cinnamic amides 1 – 3, 5, 10 – 13, and 15. 3) Cinnamic amides 45 – 47,
and 49 with an anilino moiety provided 3,4-dihydroquinolinones 16 – 19 through
intramolecular Michael addition of the anilide group. Notably, the use of BBr as an
3
alternative debenzylating agent for debenzylation of cinnamic amides 45 – 49 with the
anilino moiety provided our desired cinnamic amides 6 – 10 without inducing the
intramolecular Michael addition. Debenzylation of cinnamic amides 43, 48, and 53 with a
2
,4-dibenzyloxyphenyl group was also successfully accomplished using BBr to give 4, 9,
3
2

ACCEPTED MANUSCRIPT
and 14. Among the nine compounds that inhibited mushroom tyrosinase more potently at 25
µM than kojic acid, four cinnamic amides 4, 5, 9, and 14 showed 3-fold greater tyrosinase
inhibitory activity than kojic acid. The docking simulation using tyrosinase indicated that
these four cinnamic amides (-6.2 – -7.9 kcal/mol) bind to the active site of tyrosinase with
stronger binding affinity than kojic acid (-5.7 kcal/mol). All four cinnamic amides inhibited
melanogenesis and tyrosinase activity more potently than kojic acid in α-MSH-stimulated
B16F10 melanoma cells in a dose-dependent manner without cytotoxicity. The strong
correlation between tyrosinase activity and melanin content suggests that the anti-
melanogenic effect of cinnamic amides is due to tyrosinase inhibitory activity. Considering
that the cinnamic amides 4, 9, and 14, which exhibited strong inhibition on mushroom
tyrosinase and potent anti-melanogenic effect in B16F10 cells, commonly have a 2,4-
dihydroxyphenyl substituent, the 2,4-dihydroxyphenyl substituent appears to be essential for
high anti-melanogenesis. These results support the potential of these four cinnamic amides as
novel and potent tyrosinase inhibitors for use as therapeutic agents with safe skin-lightening
efficiency.
Key words: cinnamic amide, anti-melanogenic effect, tyrosinase inhibitor, melanin content,
docking, B10F16 melanoma cells.
3

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1
. Introduction
Melanin is the primary cellular component responsible for eye, hair, and skin colour in
humans [1,2] and is biosynthesized in the melanosomes of melanocytes starting from
L
-
tyrosine through complicated chemico-enzymatic processes [3,4]. Mainly three enzymes are
involved in the regulation of melanogenesis: tyrosinase and two tyrosinase-related proteins,
TYRP1 and TYRP2 [5-8]. Among these three enzymes, tyrosinase (EC 1.14.18.1) is the
pivotal enzyme that controls the rate of melanin production, and as the rate-limiting enzyme
for melanin biosynthesis is responsible for the conversion of
dopa using monophenolase and diphenolase activities [1,9,10]. Depending on the presence of
thiol materials such as glutathione and -cysteine, the reactive dopaquinone undergoes two
L-tyrosine to dopaquinone via L-
L
pathways [5,9]. In the presence of thiol materials, the dopaquinone reacts with thiols via
Michael addition and is finally converted to eumelanin (pigments responsible for a yellow-
red colour) after a series of multi-reactions [8]. On the other hand, in the absence of thiol
materials, the dopaquinone is finally transformed to eumelanin (pigments responsible for a
brown-black colour) by several reactions [11-15]. The proportion and amount of pheomelanin
and eumelanin mainly determine the phenotype of human skin colour [16-19]. Melanin is
widely distributed in organisms ranging from bacteria, plants, animals, and humans[19].
Melanin not only plays a positive role in protecting skin cells from UV radiation but also has
negative functions involved in hyperpigmentation diseases and browning of fruits and
vegetables.
In addition to melanin biosynthesis, tyrosinase is also involved in the defensive and
developmental functions of the pests [20,21]. Excessive dopaquinone produced by the
4

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catalysis of tyrosinase has been reported to cause the neurodegeneration associated with
Parkinson’s disease in the brain [22-26]. Therefore, suitable new tyrosinase inhibitors might
be applied to pesticides with a novel mode of action and medications associated with
neuronal damages. The abnormal excessive melanin accumulation in a specific region may
cause melasma, freckles, age spots and melanoma, a malignant tumour of melanocytes and
aesthetic problems [27-34].
Despite the few clinically used whitening agents, a crucial need remains for novel agents to
combat some unmet needs, such as side effects of potential carcinogenicity, and low clinical
efficacy demonstrated by currently available tyrosinase inhibitors [32,35-38]. Cinnamic acid
is a natural, safe substance extracted from balsams or cinnamon oil and is mainly used as a
source for fragrances and pharmaceuticals [39]. As part of our ongoing efforts to discover
safer and more potent tyrosinase inhibitors, cinnamic amide derivatives that can be prepared
by coupling of cinnamic acid with arylamine, arylalkylamine or noncyclic secondary amine,
were designed and synthesized. These cinnamic amide derivatives were expected to show
strong tyrosinase inhibitory activity due to the presence of the (E)-β-phenyl-α,β-unsaturated
carbonyl motif. Several of our previous studies have demonstrated that the motif plays an
essential role in tyrosinase inhibitory activity [40-49]. The synthesized cinnamic acid
analogues were evaluated for their inhibitory activity against mushroom tyrosinase and the
cinnamic amides with good mushroom tyrosinase activity were further studied in cell-based
experiments and docking simulation.
2
. Results and Discussion
.1. Chemistry
2
To examine the effect of the amine moiety of cinnamic amide derivatives on tyrosinase
inhibition, cinnamic amides having a benzylamino (as a representative of an arylalkylamino
5

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group), anilino (as a representative of an arylamino group), or diethylamino (as a
representative of noncyclic secondary amino group) group were designed as our desired
compounds. Based on our finding that at least one hydroxyl group on the β–phenyl ring of
the (E)-β-phenyl-α,β-unsaturated carbonyl motif is required to exhibit high tyrosinase
inhibitory activity, cinnamic amide analogues with a substituent, such as 3-hydroxy-4-
methoxy, 4-hydroxy, 3,4-dihydroxy, 2,4-dihydroxy, and 4-hydroxy-3-methoxy on the phenyl
ring were designed. Five commercially available benzaldehydes were used as starting
materials (Scheme 1): isovanillin (20), 4-hydroxybenzaldehyde (21), 3,4-
dihydroxybenzaldehyde (22), 2,4-dihydroxybenzaldehyde (23) and vanillin (24). Reaction of
these five benzaldehydes with benzyl bromide in the presence of K CO gave the
2
3
corresponding benzyl ether products 25 – 29, which, in turn, were subjected to a Horner-
Wadsworth-Emmons reaction using triethyl phosphonoacetate, K CO (2.0 equiv.) and DBU
2
3
(1,8-diazabicyclo[5.4.0]undec-7-ene, a catalytic amount) to afford the corresponding ethyl
cinnamates 30 – 34. After saponification under basic conditions using 1.0M-NaOH aqueous
solution, in order to activate the carboxylic functionality the resultant carboxylic acids 35 –
3
9 were converted to the corresponding mixed anhydrides by treatment with isobutyl
chloroformate in the presence of morpholine as a base. Reaction of the mixed anhydrides
with benzylamine, aniline and diethylamine produced compounds 40 – 54, and the benzyl
o
ethers of 40 – 54 were removed under acidic conditions using BBr at -40 C, or conc-HCl,
3
and acetic acid under reflux.
In case of compounds 40 – 44 with a benzylamino group, and 50 – 54 with a diethylamino
group, the corresponding debenzylated cinnamic amides 1 – 3, 5, 11 – 13, and 15 were
produced during the debenzylation reaction using conc-HCl, and acetic acid, except for
cinnamic amides 4, and 14 with a 2,4-dihydroxy substituent. Notably, conversion to acidic
6

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conditions using BBr successfully induced the formation of cinnamic amides 4, and 14 with
3
a 2,4-dihydroxy substituent. Compounds 45 – 49 having an anilino group also were treated
with conc-HCl and acetic acid to obtain the desired cinnamic amides 6 – 10. However, we
could not obtain our desired cinnamic amide derivatives 6 – 10. Instead, 3,4-
dihydrocarbostyril compounds, i.e., 3,4-dihydroquinolinones 16 – 19 were obtained from
compounds 45 – 47 and 49, respectively, via a cyclization reaction along with debenzylation.
On the other hand, reaction of compound 48 under the same acidic conditions afforded only
complex compounds with high polarity. A plausible mechanism for the formation of 3,4-
dihydroquinolinone derivatives is depicted in Scheme 2. Protonation of the carbonyl group by
HCl/acetic acid may activate the Michael acceptor, an α,β-unsaturated amide and induce a
Michael addition of the anilino group to give compound A, which may be spontaneously
tautomerised into more stable 3,4-dihydroquinolinone derivatives. Among the four obtained
3
,4-dihydroquinolinone derivatives 16 – 19, two (17 and 18) were unknown compounds and
although the remaining two (16 and 19) are known compounds, these compounds were
prepared via a different synthetic method: a reaction of monoanilides of malonic acid with
benzaldehydes in trifluoroacetic acid under reflux. Many studies [50-53] have revealed
intramolecular cyclization of the anilides of cinnamic acid in the presence of a variety of
Bronsted-Lowry acids or Lewis acids: polyphosphoric, hydrobromic, hydroiodic, sulfuric,
trifluoromethanesulfonic, and trifluoroacetic acids, and AlCl . To the best of our knowledge,
3
this study is the first to use HCl/acetic acid for converting cinnamic acid anilides into 3,4-
dihydroquinolinone derivatives. A milder acidic reagent and conditions to avoid
intramolecular cyclization were required for the desired cinnamic amides 6 – 10. For this
purpose, BBr was chosen because the Lewis acid is a common reagent for debenzylation of
3
phenolic benzyl ether and because a debenzylation reaction can be generally conducted at
7

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o
o
0
low temperatures (-78 C ~ 0 C). Surprisingly, treatment of 45 – 49 with BBr at -40 C
3
provided our desired cinnamic amide analogues 6 – 10 in moderate yields. These results
indicate that the intramolecular cyclization of the cinnamic acid anilides requires more
vigorous acidic conditions than those for debenzylation. The structures of the fifteen
1
13
cinnamic amides 1 – 15 and four dihydroquinolinones 16 – 19 were identified by H and C
nuclear magnetic resonance (NMR) and mass spectroscopy. The double bond geometry of
cinnamic amides 1 – 15 was assigned as an (E)-configuration on the basis of J values (> 15.0
1
Hz) of the vinylic protons in the H NMR spectra.
8

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Scheme 1. Synthetic scheme of cinnamic amide derivatives 1 – 19. Reagents and
conditions: a) BnBr, K CO , CH CN, reflux, 24 h; b) (EtO) P(=O)CH CO Et, K CO , DBU
2
3
3
2
2
2
2
3
o
(
cat. amount), DCM, DMF, rt for 20, 22, and 24, or 70 C for 21, and 23, 24 – 36 h; c) 1N-
NaOH aqueous solution, 1,4-dioxane, rt, 48 h; d) i-BuO CCl, N-methylmorpholine, THF, rt,
2
3
0 min, and then, benzylamine or aniline and diethylamine, rt, 24 h; and e) c-HCl, AcOH,
0
reflux, 40 min; f) 4 – 8 equiv. BBr DCM, -40 C, 30 min.
3
,
Scheme 2. A plausible mechanism for the formation of 3,4-dihydroquinolinone derivatives.
2.2.Mushroom tyrosinase activity
The inhibitory activity of cinnamic amide derivatives 1 – 19 against mushroom tyrosinase
was investigated at 25 µM. Kojic acid was used as a positive control, as typically done in
evaluation of tyrosinase inhibitors. As depicted in Table 1, among the 15 cinnamic amides,
cinnamic amides 2, 7, and 12 with the 4-hydroxyl group on the β-phenyl ring of the (E)-β-
phenyl-α,β-unsaturated carbonyl motif did not exhibit tyrosinase inhibitory activity and
9

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cinnamic amides 3, 8, and 13 with a catechol group showed no inhibition or weak inhibition
against tyrosinase activity. Cinnamic amide derivatives 1 (31.52±2.85% inhibition), 6
(29.68±12.09% inhibition), and 11 (20.66±4.79% inhibition) with a 3-hydroxy-4-methoxy
group on the β-phenyl ring exerted similar or slightly stronger tyrosinase inhibition than kojic
acid (21.39±5.37% inhibition). On the other hand, cinnamic amides 5, 10, and 15, which have
hydroxyl and methoxyl substituents at opposite positions to cinnamic amides 1, 6, and 11,
showed 2- to 3-fold greater tyrosinase inhibitory effects (59.05±13.94% to 82.88±3.01%
inhibitions) than cinnamic amides 1, 6, and 11 with a 3-hydroxy-4-methoxy group. The
highest inhibitory activities were observed from cinnamic amides 4, 9, and 14 with a
resorcinol moiety (85.37±0.54% to 96.20±1.44% inhibition). These results suggest that the
degree of tyrosinase inhibition is closely dependent on the chemical structure of the β-phenyl
ring of cinnamic amides. Regardless of the amide species, the tyrosinase inhibition of the
cinnamic amides decreased in the order of 2,4-dihydroxy < 4-hydroxy-3-methoxy < 3-
hydroxy-4-methoxy < 4-hydroxy, 2,3-dihydroxy.
One methylene reduction in the amine residue of cinnamic amides 1 – 5 generally resulted in
a reduction in the inhibitory activity as seen in the tyrosinase inhibition of cinnamic amides 6
–
4
10. In particular, the phenomenon was clearly found in the cinnamic amide (5 vs. 10) with a
-hydroxy-3-methoxyphenyl ring. One exception was found in the cinnamic amide (4 vs. 9)
with a 2,4-dihydroxyphenyl group, and the latter increased tyrosinase inhibition from 82.88%
to 93.70%.
While the cinnamic amides 3, 8, and 13 with a 3,4-dihydroxyphenyl substituent showed very
low or no tyrosinase inhibition, the cinnamic amides 4, 9, and 14 with a 2,4-dihydroxyphenyl
substituent showed high inhibition. Presumably, this result means that the former structurally
1
0

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similar to
L-dopa can be rapidly consumed as a substrate for tyrosinase, whereas the latter
structurally less similar to
for tyrosinase.
L-dopa can act as an inhibitor of tyrosinase rather than a substrate
Dihydroquinolinones 16 – 19 obtained through intramolecular cyclization were also
evaluated for their tyrosinase inhibitory effects. Only dihydroquinolinone 16 with a 3-
hydroxy-4-methoxyphenyl substituent showed similar inhibitory activity to kojic acid,
whereas the remaining three dihydroquinolinones 17 – 19 did not show tyrosinase inhibition.
Taken together, cinnamic amides having a structural characteristic of the (E)-β-phenyl-α,β-
unsaturated carbonyl motif exhibited very potent tyrosinase inhibition, whereas
dihydroquinolinones showed no or only moderate tyrosinase inhibition, indicating that the
motif plays an essential role in tyrosinase inhibition.
Finally, four cinnamic amides 4, 9, and 14 with a 2,4-dihydroxyphenyl group and 5 with a 4-
hydroxy-3-methoxyphenyl group, were selected for further investigation because they
exhibited the most potent tyrosinase inhibitory activities.
1
1

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Table 1. Substitution patterns, and tyrosinase-inhibitory activities of the synthesized cinnamic amide derivatives 1 – 19, and kojic acid
1
2
3
1
2
3
Compound
R
R
R
Tyrosinase
inhibition (%)
31.52±2.85
NI
Compound
R
R
R
Tyrosinase
inhibition (%)
20.66±4.79
NI
a
a
1
2
3
4
5
6
7
8
9
H
H
OH
H
OMe
OH
OH
OH
OH
OMe
OH
OH
OH
OH
11
H
H
OH
H
OMe
OH
OH
OH
OH
OMe
OH
OH
OH
12
H
OH
H
10.02±3.55
85.37±0.54
82.88±3.01
29.68±12.09
NI
13
H
OH
H
NI
OH
H
14
OH
H
96.20±1.44
61.93±12.34
25.94±2.87
NI
OMe
OH
H
15
OMe
OH
H
H
16
H
H
17
18
H
H
OH
H
NI
H
OH
OMe
NI
OH
H
93.70±1.48
59.05±13.94
19
H
NI
1
0
OMe
Kojic acid
21.39±5.37
a
Tyrosinase inhibition was assayed at 25 µM using L-tyrosine as substrate. The results are presented as mean ± SEMs.
1
2

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2.3. Types of inhibition of cinnamic amide derivatives 9 and 14 on mushroom tyrosinase
Since cinnamic amide derivatives 9 and 14 exhibited the most potent tyrosinase inhibition
with the IC values of 25.6 ± 2.0 nM and 11.2 ± 3.0 nM, respectively, we have determined
5
0
their inhibitory mode of action by using Lineweaver-Burk double reciprocal plots. The
kinetics of the enzyme were shown in Figure 1. The results showed that the plots of 1/V
versus 1/[S] gave straight lines with different slopes depending on concentrations of the
inhibitor, and the lines intersected on the vertical axis. The Lineweaver-Burk plot showed that
Vmax is the same regardless of concentration of the inhibitor, and KM increases with
increasing concentration of the inhibitor. This behavior indicates that cinnamic amide
derivatives 9 and 14 inhibit the enzyme tyrosinase in a competitive manner.
Figure 1. Types of inhibition of cinnamic amide derivatives 9 and 14 against mushroom
tyrosinase. Inhibition types were determined using Lineweaver–Burk plots. Results are mean
1
/V values, where V is the increase in the absorbance per minute at different
L-tyrosine
concentrations. The modified Michaelis–Menten equation was utilized: 1/Vmax = 1/K (1 +
M
[
S]/K ), where V: reaction velocity, S: L-tyrosine concentration, K : Michaelis-Menten
i M
constant, K : inhibition constant. All experiments were performed in triplicate.
i
1
3

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2.4. Docking studies
To determine whether the four selected cinnamic amides are able to bind directly to the
active site of tyrosinase, AutoDock Vina 1.1.2 software developed by The Scripps Research
Institute was utilised for docking simulation. 3D-structures of the four tyrosinase ligands 4, 5,
9
, and 14 were prepared through the energy minimization of the 2D-structures using Chem3D
Pro 12.0 software (CambridgeSoft Corporation). The 3D structure of tyrosinase for docking
simulation was obtained from Agaricus bisporus tyrosinase (Protein Data Bank ID: 2Y9X).
Although the correlation between the degree of inhibition of mushroom tyrosinase and the
binding affinities of the four ligands was not perfect, all four cinnamic amides showed higher
binding affinities (-6.2 ~ -7.9 kcal/mol) than kojic acid (-5.7 kcal/mol), which was used as a
reference control (Figure 2d). LigandScout 3.1.2 software showed interactions between the
amino acid residues of tyrosinase and ligands. Kojic acid interacts with two amino acid
residues (His259 and His263) of tyrosinase as shown in Figure 2b. The branched hydroxyl
group of kojic acid creates two hydrogen bonds with both amino acids and the ring of kojic
acid interacts with His263 through π-π stacking. The four tested ligands also generate various
interactions such as hydrophobic interactions, hydrogen bonding, and π-π stacking, as shown
in Figure 2a and 1c. Cinnamic amides 4 and 9 create two hydrogen bonds while cinnamic
amides 5 and 14 generate only one hydrogen bond. Cinnamic amide 14 also forms π-π
stacking between the β-phenyl ring and His263, which is the same amino acid as the π-π
stacking interaction of kojic acid. Interestingly, Met280 creates hydrogen bonds with the
hydroxyl group of ligands at different positions, depending on the simulation ligands. Met280
hydrogen bonds with the 4-hydroxyl group on the β-phenyl ring in cinnamic amides 4, 5, and
9
, whereas it hydrogen bonds with the 2-hydroxyl group on the β-phenyl ring in cinnamic
1
4

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amide 14. Notably, although most hydroxyl substituents form hydrogen bonds, the 4-
hydroxyl group in cinnamic amide 14 does not form a hydrogen bond with the amino acids of
tyrosinase. Val283 forms hydrophobic interactions with the β-phenyl ring of all four ligands,
and Phe264 interacts with both phenyl rings of 4, 5, and 9 via hydrophobic interactions.
Although the phenyl ring of 14 does not interact with Phe264, one N-ethyl group interacts
with Phe264 via hydrophobic interaction. In addition to Phe264, the phenyl ring of the amino
moiety of the amides 4, 5, and 9 forms additional hydrophobic interactions with two amino
acids Thr261 and Met257 (compound 4), one amino acid Met257 (compound 5), and two
amino acids Val248 and Met257 (compound 9). It is speculated that various hydrophobic
interactions and hydrogen bonds between tyrosinase and the ligands may result in stronger
binding affinities than kojic acid. Summarising these results, these cinnamic amide ligands
have the potential to bind to the active site of tyrosinase and inhibit tyrosinase.
1
5

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Figure 2. Docking simulation of the cinnamic amide derivatives 4, 5, 9, and 14 and of kojic
acid with tyrosinase and pharmacophore analysis. (a, and b) Pharmacophore results for 4, 5, 9,
1
4 and kojic acid obtained from LigandScout 4.1.0 indicate possible hydrogen-bonding
(green arrow), π-π stacking (violet arrow) and hydrophobic (yellow) interactions between the
ligands tested and the amino acid residues of tyrosinase. (c) Docking simulation result
between cinnamic amides 4, 5, 9, and 14 and mushroom tyrosinase. (d) Docking scores
between tyrosinase and 4, 5, 9, 14 or kojic acid.
1
6

ACCEPTED MANUSCRIPT
2.5. Cell viability of cinnamic amide derivatives 4, 5, 9 and 14 in B16F10 melanoma cells
Cell viabilities were measured by performing WST-8 assay. Treatment of the four
cinnamic amide derivatives at four concentrations (0, 5, 10, and 25 µM) in B16F10
melanoma cells, gave no significant cytotoxicity after an incubation of 24 h.
According to Figure 3, negligible cytotoxicity was noted for cinnamic amides 4, 5, 9 and 14
at concentrations below 25 µM as compared to the control. On the basis of these findings,
further evaluation of the four cinnamic amide derivatives in α-MSH-stimulated B16F10
melanoma cells was carried out at concentrations up to 25 µM.
1
7

ACCEPTED MANUSCRIPT
Figure 3. Cell viability assay of cinnamic amide derivatives 4, 5, 9 and 14 in B16F10
melanoma cells. Test cinnamic amides were treated at the concentrations of 5, 10, and 25 µM.
Viabilities are expressed in % to control, and the bars represent standard errors.
2.6. Tyrosinase inhibitory activity of cinnamic amide derivatives 4, 5, 9 and 14 in α-MSH-
stimulated B16F10 melanoma cells
The tyrosinase inhibition effect of the four cinnamic amides was assessed by using
B16F10 melanoma cells stimulated by α-MSH (melanin-stimulating hormone). Once the
tyrosinase activity level had been increased by the α-MSH treatment, the cells were treated
with four concentrations (0, 5, 10, and 25 µM) of the four cinnamic amides or with two
natural tyrosinase inhibitors used as positive controls: kojic acid (25 µM) or arbutin (400 µM).
After an incubation of 24 h, the tyrosinase inhibition effect of the cinnamic amides was
observed in B16F10 cells by measuring the optical densities spectrophotometrically.
As shown in Figure 4, all four cinnamic amide derivatives revealed an impressive inhibition
of tyrosinase activity, superior to that of kojic acid and arbutin in α-MSH-stimulated B16F10
melanoma cells at 25 µM concentration. Among the four cinnamic amide, 5 (79.17%
inhibition) with a 4-hydroxy-3-methoxyphenyl group and 14 (73.69% inhibition) with a 2,4-
dihydroxyphenyl group showed the highest tyrosinase inhibition at 25 µM as compared to
kojic acid (39.97% inhibition) and arbutin (53.17% inhibition). The other two cinnamic
amides 4 (63.11% inhibition) and 9 (60.33% inhibition) also outperformed tyrosinase
inhibition at 25 µM, which is superior to kojic acid and arbutin. All cinnamic amides dose-
dependently inhibited the activity of tyrosinase. As in our previous report that the 2,4-
dihydroxyphenyl group plays an important role in the inhibition of tyrosinase,[42,43,54-56]
all three cinnamic amides 4, 9, and 14 with a 2,4-dihydroxyphenyl group showed potent
inhibition of tyrosinase activity in α-MSH-stimulated B16F10 melanoma cells as well as the
1
8

ACCEPTED MANUSCRIPT
inhibition of mushroom tyrosinase shown in Table 1. Interestingly, cinnamic amides 4 and 5
at a concentration of 10 µM inhibited the activity of tyrosinase more potently than kojic acid
at 25 µM.
Tyrosinase activity
4
5
9
14
100
*
**
*
**
***
*
**
*
**
***
*
**
*
**
50
*
**
*
**
***
*
**
0
Concentration
Figure 4. Tyrosinase inhibition activity of cinnamic amide derivatives 4, 5, 9 and 14 in α-
MSH-stimulated B16F10 melanoma cells that were co-treated with cinnamic amides 4, 5, 9,
and 14, kojic acid (25 µM), or arbutin (400 µM). The asterisks represent the significance
difference between the columns: ***, p<0.001. The bars represent standard errors.
2.7. Melanin content inhibition effect of cinnamic amide derivatives 4, 5, 9 and 14 in α-
MSH-stimulated B16F10 melanoma cells
To evaluate the melanin content in B16F10 melanoma cells, first, the cells were
stimulated by α-MSH and then co-treated with the four cinnamic amides in four
concentrations (0, 5, 10, and 25 µM), or kojic acid (25 µM). After an incubation of 24 h,
optical densities were measured to evaluate the melanin production inhibitory effect of the
cinnamic amides.
1
9

ACCEPTED MANUSCRIPT
The results indicated in Figure 5 revealed that all four cinnamic amide derivatives
vigorously and potently decreased the melanin content in α-MSH-stimulated B16F10
melanoma cells as compared to kojic acid at 25 µM concentration. The greatest decreases in
melanin content were observed for cinnamic amides 5 (80.43% inhibition) and 14 (78.07%
inhibition), followed by 4 (65.84% inhibition) and 9 (63.97% inhibition), as compared to
kojic acid (44.26% inhibition). Interestingly, all four cinnamic amides at a concentration of
1
0 µM reduced the melanin content in α-MSH-stimulated B16F10 melanoma cells to an
extent similar to or greater than kojic acid. Of the four cinnamic amides, 5 with a 4-hydroxy-
-methoxyphenyl group exhibited the greatest reduction of melanin content at all tested
3
concentrations. All four cinnamic amides inhibited melanin production in B16F10 melanoma
cells in a concentration-dependent manner. The pattern of decrease in melanin content was
similar to the inhibition pattern of tyrosinase as shown in Figure 4, suggesting a strong
correlation between inhibition of tyrosinase and decreased melanin content.
Melanin content
4
5
9
14
100
**
**
***
***
***
*
**
***
***
***
50
***
*
**
***
*
**
0
Concentration
Figure 5. Melanin production inhibitory effect of cinnamic amide derivatives 4, 5, 9 and 14
2
0

ACCEPTED MANUSCRIPT
in α-MSH-stimulated B16F10 melanoma cells that were co-treated with cinnamic amides 4, 5,
9
, and 14, or kojic acid (25 µM). The asterisks represent the significant difference between
the columns: **, p<0.01; and ***, p<0.001. The bars represent standard errors.
2.8. Antioxidant activities of cinnamic amide derivatives 1 – 19 in DPPH radical
The antioxidant activity of cinnamic amide derivatives 1 – 19 was evaluated by adding
1
.0 mM of cinnamic amides, or L-ascorbic acid to a DPPH methanol solution. The decrease
in absorbance was measured with a spectrophotometer at 517 nm, indicating the scavenging
capacity of the DPPH radical by cinnamic amides. The results are presented in Table 2.
Overall, cinnamic amides 8 (81.88% inhibition), and 13 (84.55% inhibition) with a 3,4-
dihydroxyphenyl group, cinnamic amides 10 (82.58% inhibition), and 15 (78.94% inhibition)
with a 4-hydroxy-3-methoxyphenyl group, and dihydroquinolinone 19 (83.05% inhibition)
with a 4-hydroxy-3-methoxyphenyl group showed high DPPH scavenging activities
comparable to L-ascorbic acid (84.64% inhibition) used as a positive control. The DPPH
radical scavenging activity of cinnamic amides having a 4-hydroxyphenyl group was very
susceptible to their amino moiety and showed very weak to strong DPPH radical scavenging
activity (7 and 12 vs. 2) depending on their structure. The four cinnamic amides 4, 5, 9, and
1
4 that exhibited strong inhibition of tyrosinase activity and suppression of melanin
production in cellular experiments exerted moderate to strong DPPH radical scavenging
activity (46.04 ~ 80.60% inhibition). Since ROS and RNS can induce melanogenesis, it is
speculated that the DPPH radical scavenging effect of cinnamic amides contributes in part to
the anti-melanogenesis effect.
Table 2. DPPH radical scavenging activities of the synthesized cinnamic amide derivatives 1
–
19, and kojic acid
2
1

ACCEPTED MANUSCRIPT
Compound
DPPH radical
scavenging activity (%)
Compound
DPPH radical
scavenging activity (%)
24.33±0.32
3.87±0.47
1
2
3
4
5
6
7
8
9
NA
11
12
13
14
15
16
17
18
19
81.57±0.48
46.67±0.99
80.60±0.22
49.19±1.21
53.27±1.52
16.55±1.06
81.88±0.07
46.07±0.99
82.58±0.04
84.55±0.97
55.31±0.62
78.94±0.51
NA
81.80±1.19
69.21±0.22
83.05±0.63
84.64±0.32
10
L
-ascorbic acid
Radical scavenging activities were determined 30 min after the addition of cinnamic a
mides to DPPH in methanol to a final concentration of 1.0 mM. Three independent e
xperiments were performed. NA means not active. The results are presented as the m
eans±SDs of three experiments.
Conclusion
In summary, fifteen cinnamic amide derivatives 1 – 15 having an (E)-β-phenyl-α,β-
unsaturated carbonyl motif were synthesized as potential tyrosinase inhibitors, via
debenzylation of the O-benzyl-protected cinnamic amides 40 – 54 using conc-HCl/acetic acid,
or BBr . Four 3,4-dihydroquinolinones 16 – 19 were also synthesized from cinnamic amides
3
4
5 – 47, and 49 with an anilino moiety during debenzylation using conc-HCl and acetic acid,
via an intramolecular Michael addition of the anilide group. These nineteen compounds 1 –
2
2

ACCEPTED MANUSCRIPT
1
9 were evaluated for mushroom tyrosinase inhibition activity. Nine compounds exhibited
greater inhibition at 25 µM than kojic acid, indicating that the motif closely affects tyrosinase
inhibition. Especially, four of the cinnamic amides (4, 5, 9, and 14) showed tyrosinase
inhibitory activity three times greater than that of kojic acid. The docking simulation using
AutoDock Vina exhibited that these four cinnamic amides (-6.2 – -7.9 kcal/mol) have greater
binding affinities to the active site of tyrosinase than kojic acid (-5.7 kcal/mol). Cell-based
experiments on B16F10 melanoma cells demonstrated that these four cinnamic amides are
not cytotoxic and exhibit more potent anti-melanogenic effect than kojic acid through the
inhibition of cellular tyrosinase activity. Notably, a 2,4-dihydroxyphenyl substituent appears
to play a major role in the great inhibition of tyrosinase, taking into account that the four
cinnamic amides commonly have the substituent. These results imply that the (E)-β-phenyl-
α,β-unsaturated carbonyl motif plays a key role in tyrosinase inhibition and that cinnamic
amide derivatives having the motif are inherently promising candidates for safe and potent
tyrosinase inhibitors.
3
. Materials and Methods
4
.1. General methods
All the chemicals and reagents were obtained commercially and used without further
purification. Thin layer chromatography (TLC) and column chromatography were conducted
on Merck precoated 60F245 plates and MP Silica 40-63, 60 Å, respectively. All anhydrous
solvents were distilled over CaH and Na/benzophenone. High resolution mass spectroscopy
data were obtained on an Agilent accurate Mass quadruple time of flight (Q-TOF) liquid
2
3

ACCEPTED MANUSCRIPT
chromatography (LC) mass spectrometer (Agilent, Santa Clara, CA, USA) in electrospray
ionization (ESI) negative mode while low-resolution mass data were obtained in ESI positive
mode on an Expression CMS spectrometer (Advion Ithaca, NY, USA). NMR spectra were
recorded on a Varian Unity INOVA 400 spectrometer, or a Varian Unity AS500 spectrometer
1
(
Agilent technologies, Santa Clara, CA, USA) for 400 MHz or 500 MHz H NMR and 100
1
3
MHz C NMR. CDCl , DMSO-d , and CDCl +CD OD were used as an NMR solvent for
3
6
3
3
NMR samples. All chemical shifts were measured in parts per million (ppm) versus residual
solvent or deuterated peaks (δH 7.24 and δC 77.0 for CDCl3, δH 2.50 and δC 39.7 for DMSO-
d6). Coupling constant (J) values were measured in hertz (Hz). The following abbreviations
1
are used for H NMR: s (singlet), brs (broad singlet), d (doublet), brd (broad doublet), dd
(
doublet of doublets), t (triplet), brt (broad triplet), td (triplet of doublets), q (quartet), brq
broad quartet), and m (multiplet).
(
4
.1.1. General procedure for the synthesis of compounds 25 – 29 [57].
Benzaldehydes 20 – 24 (5.00 g), benzyl bromide (1.0 equiv.), potassium carbonate
(1.0 equiv.) and acetonitrile (50 mL) were added to a 250 mL round-bottom flask and the
reaction mixture was refluxed for 24 h. Acetonitrile was evaporated on completion of the
reaction and the residues were partitioned between dichloromethane and water. The
dichloromethane layer was dried with anhydrous MgSO and evaporated in vacuo to give
4
benzyl-protected benzaldehydes 25 - 29 as a white or grey solid in yields of 95 – 97%.
1
4
.1.1.1 3-(Benzyloxy)-4-methoxybenzaldehyde (25). White solid, 97% yield. H NMR (500
MHz, CDCl ) δ 9.82 (s, 1 H, CHO), 7.47 (dd, 1 H, J = 8.0, 2.0 Hz, 6-H), 7.47 – 7.44 (m, 3 H,
3
2
1
-H, 2′-H, 6′-H), 7.38 (t, 2 H, J = 7.5 Hz, 3′-H, 5′-H), 7.32 (t, 1 H, J = 7.5 Hz, 4′-H), 7.00 (d,
13
H, J = 8.0 Hz, 5-H), 5.19 (s, 2 H, benzylic H), 3.96 (s, 3 H, OCH ); C NMR (100 MHz,
3
2
4

ACCEPTED MANUSCRIPT
CDCl ) δ 191.0, 155.2, 148.9, 136.5, 130.2, 128.8, 128.3, 127.7, 127.1, 111.6, 111.0, 71.1,
3
5
6.4.
.1.1.2. 4-(Benzyloxy)benzaldehyde (26). White solid, 97% yield. H NMR (500 MHz, CDCl )
1
4
3
δ 9.89 (s, 1 H, CHO), 7.84 (d, 2 H, J = 8.5 Hz, 2-H, 6-H), 7.44 (d, 2 H, J = 8.0 Hz, 2′-H, 6′-
H), 7.41 (t, 2 H, J = 8.0 Hz, 3′-H, 5′-H), 7.35 (t, 1 H, J = 7.5 Hz, 4′-H), 7.08 (d, 2 H, J = 8.5
1
3
Hz, 3-H, 5-H), 5.15 (s, 2 H, benzylic H); C NMR (100 MHz, CDCl ) δ 191.0, 163.9, 136.1,
3
1
32.2, 130.3, 128.9, 128.5, 127.7, 115.3, 70.5.
.1.1.3. 3,4-Bis(benzyloxy)benzaldehyde (27). White solid, 96% yield. H NMR (500 MHz,
1
4
CDCl ) δ 9.81 (s, 1 H, CHO), 7.49 (d, 1 H, J = 2.0 Hz, 2-H), 7.48 – 7.36 (m, 9 H, 6-H, 2′-H,
3
3
8
′-H, 5′-H, 6′-H, 2′′-H, 3′′-H, 5′′-H, 6′′-H), 7.34 – 7.30 (m, 2 H, 4′-H, 4′′-H), 7.02 (d, 1 H, J =
1
3
.0 Hz, 5-H), 5.26 (s, 2 H, benzylic H), 5.22 (s, 2 H, benzylic H); C NMR (100 MHz,
CDCl ) δ 191.0, 154.4, 149.4, 136.7, 136.4, 130.5, 128.9, 128.8, 128.3, 128.2, 127.5, 127.2,
3
1
26.9, 113.3, 112.5, 71.2, 71.0.
.1.1.4. 2,4-Bis(benzyloxy)benzaldehyde (28). Grey solid, 95 % yield. H NMR (500 MHz,
1
4
CDCl ) δ 10.39 (s, 1 H, CHO), 7.84 (d, 1 H, J = 9.0 Hz, 6-H), 7.45 – 7.38 (m, 8 H, 2′-H, 3′-H,
3
5
′-H, 6′-H, 2′′-H, 3′′-H, 5′′-H, 6′′-H), 7.37 – 7.34 (m, 2 H, 4′-H, 4′′-H), 6.64 (dd, 1 H, J = 9.0,
.0 Hz, 5-H), 6.60 (d, 1 H, J = 2.0 Hz, 3-H), 5.14 (s, 2 H, benzylic H), 5.11 (s, 2 H, benzylic
2
1
3
H); C NMR (100 MHz, CDCl ) δ 188.5, 165.4, 162.9, 136.1, 136.1, 130.7, 129.0, 128.9,
3
1
28.6, 128.5, 127.8, 127.5, 119.7, 107.2, 100.3, 70.6, 70.4.
.1.1.5. 4-(Benzyloxy)-3-methoxybenzaldehyde (29). White solid, 97% yield. H NMR (500
1
4
MHz, CDCl ) δ 9.84 (s, 1 H, CHO), 7.45 – 7.42 (m, 3 H, 2-H, 2′-H, 6′-H), 7.40 – 7.37 (m, 3
3
H, 6-H, 3′-H, 5′-H), 7.33 (t, 1 H, J = 7.5 Hz, 4′-H), 6.99 (d, 1 H, J = 8.0 Hz, 5-H), 5.25 (s, 2 H,
13
benzylic H), 3.95 (s, 3 H, OCH ); C NMR (100 MHz, CDCl ) δ 191.1, 153.8, 150.3, 136.2,
3
3
2
5

ACCEPTED MANUSCRIPT
1
30.5, 128.9, 128.4, 127.4, 126.8, 112.6, 109.5, 71.1, 56.3.
4
.1.2. General procedure for the synthesis of cinnamic acid derivatives 35 – 39 via ethyl
cinnamates 30 – 34.
To a stirred solution of compounds 25 – 29 (2.00 g), triethyl phosphonoacetate (1.1
equiv.), and potassium carbonate (2.0 equiv.) in dichloromethane/N,N-dimethylformamide
(2:1, 15 mL) was added a catalytic amount of DBU (0.03 equiv.). The reaction mixture was
0
stirred at room temperature (compounds 25, 27, and 29) or 70 C (26, and 28) for 24 – 36 h.
After removal of dichloromethane by evaporation, ice water was added to the reaction
mixture which was then stirred for 30 min to give precipitates. After filtration, the filter cake
was washed with plenty of water, and dried to produce ethyl cinnamates 30 – 34 as a white or
grey solid in yields of 95 – 98%. The ethyl cinnamates were used directly in the next reaction
without characterization.
Ethyl cinnamates 30 – 34 (2.57 to 4.11 g) were added to a 100 mL round-bottom flask
and 1,4-dioxane (20 mL) and 1N-NaOH aqueous solution (8.0 – 16.0 equiv.) were added
subsequently. The reaction mixture was stirred at room temperature for 48 h. After
completion of the reaction, the reaction mixture was acidified until pH 2 using 2N-HCl
aqueous solution. The reaction mixture was stirred at room temperature for 30 min and then
the resulting solid was filtered. The filter cake was washed with a plenty of water, and dried
to give the cinnamic acid derivatives 35 – 39 as a white or grey solid in yields of 100%.
1
4
.1.2.1. (E)-3-(3-(Benzyloxy)-4-methoxyphenyl)acrylic acid (35). White solid, 100% yield. H
NMR (500 MHz, DMSO-d ) δ 12.19 (brs, 1 H, COOH), 7.49 (d, 1 H, J = 16.0 Hz, 3-vinylic
6
H), 7.45 (d, 2 H, J = 7.5 Hz, 2′-H, 6′-H), 7.41 (d, 1 H, J = 2.0 Hz, 2´-H), 7.38 (t, 2 H, J = 8.0
2
6

ACCEPTED MANUSCRIPT
Hz, 3′-H, 5′-H), 7.32 (t, 1 H, J = 7.5 Hz, 4′-H), 7.20 (dd, 1 H, J = 8.0, 2.0 Hz, 6´-H), 6.98 (d,
1
H, J = 8.0 Hz, 5´-H), 6.40 (d, 1 H, J = 16.0 Hz, 2-vinylic H), 5.12 (s, 2 H, benzylic H), 3.78
1
3
(
s, 3 H, OCH ); C NMR (100 MHz, DMSO-d ) δ 168.5, 151.7, 148.6, 144.7, 137.6, 129.1,
3
6
1
28.6, 128.5, 127.6, 123.7, 117.4, 112.5, 112.4, 70.5, 56.3.
.1.2.2. (E)-3-(4-(Benzyloxy)phenyl)acrylic acid (36). White solid, 100% yield. H NMR
1
4
(
500 MHz, DMSO-d ) δ 12.25 (brs, 1 H, COOH), 7.61 (d, 2 H, J = 9.0 Hz, 2-H, 6-H), 7.52 (d,
6
1
3
=
H, J = 16.0 Hz, 3-vinylic H), 7.43 (d, 2 H, J = 7.5 Hz, 2′-H, 6′-H), 7.38 (t, 2 H, J = 7.5 Hz,
′-H, 5′-H), 7.31 (t, 1 H, J = 7.5 Hz, 4′-H), 7.02 (d, 2 H, J = 8.0 Hz, 3-H, 5-H), 6.36 (d, 1 H, J
1
3
16.0 Hz, 2-vinylic H), 5.14 (s, 2 H, benzylic H); C NMR (100 MHz, DMSO-d ) δ 168.5,
6
1
60.6, 144.3, 137.4, 130.6, 129.1, 128.6, 128.4, 127.7, 117.4, 115.8, 70.0.
.1.2.3. (E)-3-(3,4-Bis(benzyloxy)phenyl)acrylic acid (37). White solid, 100% yield. H NMR
500 MHz, DMSO-d ) δ 12.22 (brs, 1 H, COOH), 7.47 (d, 1 H, J = 16.0 Hz, 3-vinylic H),
1
4
(
6
7
2
2
2
1
.46 -7.41 (m, 5 H, 2-H, 2′-H, 6′-H, 2′′-H, 6′′-H), 7.37 (t, 2 H, J = 7.5 Hz, 3′-H, 5′-H), 7.36 (t,
H, J = 7.5 Hz, 3′′-H, 5′′-H), 7.30 (t, 2 H, J = 7.5 Hz, 4′-H, 4′′-H), 7.17 (dd, 1 H, J = 8.5,
.0 Hz, 6-H), 7.05 (d, 1 H, J = 8.5 Hz, 5-H), 6.40 (d, 1 H, J = 16.0 Hz, 2-vinylic H), 5.18 (s,
1
3
H, benzylic H), 5.16 (s, 2 H, benzylic H); C NMR (100 MHz, DMSO-d ) δ 168.5, 150.7,
6
48.9, 144.6, 137.8, 137.6, 129.1, 129.1, 128.5, 128.5, 128.3, 128.1, 123.5, 117.7, 117.5,
114.5, 113.5, 70.6, 70.5.
1
4
.1.2.4. (E)-3-(2,4-Bis(benzyloxy)phenyl)acrylic acid (38). Grey solid, 100% yield. H NMR
(
(
(
500 MHz, DMSO-d ) δ 12.10 (s, 1 H, COOH), 7.77 (d, 1 H, J = 16.0 Hz, 3-vinylic H), 7.62
6
d, 1 H, J = 9.0 Hz, 6-H), 7.45 – 7.31 (m, 10 H, 2ⅹPh), 6.81 (d, 1 H, J = 2.0 Hz, 3-H), 6.65
dd, 1 H, J = 9.0, 2.0 Hz, 5-H), 7.38 (d, 1 H, J = 16.0 Hz, 2-vinylic H), 5.19 (s, 2 H, benzylic
1
3
H), 5.14 (s, 2 H, benzylic H); C NMR (100 MHz, DMSO-d ) δ 168.8, 162.1, 158.8, 139.3,
6
2
7

ACCEPTED MANUSCRIPT
1
7
37.3, 137.3, 130.6, 129.2, 129.1, 128.7, 128.6, 128.5, 128.4, 117.3, 116.6, 107.8, 101.2, 70.5,
0.2.
1
4
.1.2.5. (E)-3-(4-(Benzyloxy)-3-methoxyphenyl)acrylic acid (39). White solid, 100% yield. H
NMR (500 MHz, DMSO-d ) δ 12.17 (brs, 1 H, COOH), 7.50 (d, 1 H, J = 16.0 Hz, 3-vinylic
6
H), 7.42 (d, 2 H, J = 7.5 Hz, 2′-H, 6′-H), 7.38 (t, 2 H, J = 7.5 Hz, 3′-H, 5′-H), 7.32 (t, 1 H, J =
7
.5 Hz, 4′-H), 7.32 (d, 1 H, J = 2.0 Hz, 2-H), 7.16 (dd, 1 H, J = 8.0, 2.0 Hz, 6-H), 7.04 (d, 1 H,
J = 8.0 Hz, 5-H), 6.43 (d, 1 H, J = 16.0 Hz, 2-vinylic H), 5.11 (s, 2 H, benzylic H), 3.80 (s, 3
1
3
H, OCH ); C NMR (100 MHz, DMSO-d ) δ 168.5, 150.3, 149.9, 144.7, 137.4, 129.1, 128.6,
3
6
1
28.5, 128.0, 123.1, 117.5, 113.7, 111.2, 70.4, 56.3.
4
.1.4. General procedure for the preparation of compounds 40 – 54 [58].
Cinnamic acid derivatives 35 – 39 (100 mg), isobutyl chloroformate (2.00 equiv.), N-
methyl morpholine (2.5 equiv.), and anhydrous THF (5 mL) were added to a 25 mL round-
0
bottom flask and stirred at 25 C for 30 min. Then benzylamine, aniline or diethylamine (2.00
equiv.) was added to the reaction flask which was then stirred at room temperature for 24 h.
After completion of the reaction, the reaction mixture was partitioned between ethyl acetate
and H O. The ethyl acetate layer was washed with brine, dried with MgSO and evaporated
2
4
under reduced pressure. The resultant residue was purified by column chromatography using
methylene chloride and methanol (30 – 70:1) as the eluent. The products 40 – 54 were
obtained as a white or yellow solid in yields of 75 – 96%.
4
.1.3.1. (E)-N-Benzyl-3-(3-(benzyloxy)-4-methoxyphenyl)acrylamide (40). Yellowish white
1
solid, 91% yield. H NMR (500 MHz, CDCl ) δ 7.55 (d, 1 H, J = 15.5 Hz, 3-vinylic H), 7.42
3
(d, 2 H, J = 7.5 Hz, 2′′-H, 6′′-H), 7.36 (t, 2 H, J = 7.5 Hz, 3′-H, 5′-H), 7.34 – 7.28 (m, 6 H, 4′-
H, Ph), 7.08 (dd, 1 H, J = 8.5, 1.5 Hz, 6-H) 7.03 (d, 1 H, J = 1.5 Hz, 2-H), 6.85 (d, 1 H, J =
2
8