Tyrosinase inhibition and anti-melanin generation effect of cinnamamide analogues

Article abstract of DOI:10.1016/j.bioorg.2019.03.001

Abnormal melanogenesis results in excessive production of melanin, leading to pigmentation disorders. As a key and rate-limiting enzyme for melanogenesis, tyrosinase has been considered an important target for developing therapeutic agents of pigment disorders. Despite having an (E)-β-phenyl-α,β-unsaturated carbonyl scaffold, which plays an important role in the potent inhibition of tyrosinase activity, cinnamic acids have not attracted attention as potential tyrosinase inhibitors, due to their low tyrosinase inhibitory activity and relatively high hydrophilicity. Given that cinnamic acids’ structure intrinsically features this (E)-scaffold and following our experience that minute changes in the chemical structure can powerfully affect tyrosinase activity, twenty less hydrophilic cinnamamide derivatives were designed as potential tyrosinase inhibitors and synthesised using a Horner-Wadsworth-Emmons reaction. Four of these cinnmamides (4, 9, 14, and 19) exhibited much stronger mushroom tyrosinase inhibition (over 90% inhibition) at 25 μM compared to kojic acid (20.57% inhibition); crucially, all four have a 2,4-dihydroxy group on the β-phenyl ring of the scaffold. A docking simulation using tyrosinase indicated that the four cinnamamides exceeded the binding affinity of kojic acid, and bound more strongly to the active site of tyrosinase. Based on the strength of their tyrosinase inhibition, these four cinnamamides were further evaluated in B16F10 melanoma cells. All four cinnamamides, without cytotoxicity, exhibited higher tyrosinase inhibitory activity (67.33 – 79.67% inhibition) at 25 μM than kojic acid (38.11% inhibition), with the following increasing inhibitory order: morpholino (9) = cyclopentylamino (14) < cyclohexylamino (19) < N-methylpiperazino (4) cinnamamides. Analysis of tyrosinase activity and melanin content in B16F10 cells showed that the four cinnamamides dose-dependently inhibited both cellular tyrosinase activity and melanin content and that their inhibitory activity at 25 μM was much better than that of kojic acid. The results of melanin content analysis well matched those of the cellular tyrosinase activity analysis, indicating that tyrosinase inhibition by the four cinnamamides is a major factor in the reduction of melanin production. These results imply that these four cinnamamides with a 2,4-dihydroxyphenyl group can act as excellent anti-melanogenic agents in the treatment of pigmentation disorders.

Full text of DOI:10.1016/j.bioorg.2019.03.001

Accepted Manuscript
Tyrosinase inhibition and anti-melanin generation effect of cinnamamide ana-
logues
Sultan Ullah, Chaeun Park, Muhammad Ikram, Dongwan Kang, Sanggwon Lee,
Jungho Yang, Yujin Park, Sik Yoon, Pusoon Chun, Hyung Ryong Moon
PII:
S0045-2068(18)30890-3
DOI:
https://doi.org/10.1016/j.bioorg.2019.03.001
YBIOO 2827
Reference:
Bioorganic Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
16 August 2018
1 February 2019
2 March 2019
Please cite this article as: S. Ullah, C. Park, M. Ikram, D. Kang, S. Lee, J. Yang, Y. Park, S. Yoon, P. Chun, H.
Ryong Moon, Tyrosinase inhibition and anti-melanin generation effect of cinnamamide analogues, Bioorganic
Chemistry (2019), doi: https://doi.org/10.1016/j.bioorg.2019.03.001
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Tyrosinase inhibition and anti-melanin generation effect of cinnamamide analogues
Sultan Ullaha, Chaeun Parka, Muhammad Ikramb,c, Dongwan Kanga, Sanggwon Leea, Jungho
Yanga, Yujin Parka, Sik Yoonb, Pusoon Chund, Hyung Ryong Moona,*
aLaboratory of Medicinal Chemistry, College of Pharmacy, Pusan National University, Busan
46241, South Korea
bDepartment of Anatomy, Pusan National University School of Medicine, 49 Busandaehak-ro,
Mulgeum-eup, Yangsan-si, Gyeongsangnam-do, 50612, South Korea.
cDepartment of Pharmacy, Comsats University Islamabad, Abbottabad Campus, Abbottabad
22060, Pakistan
dCollege of Pharmacy and Inje Institute of Pharmaceutical Sciences and Research, Inje
University, Gimhae, Gyeongnam 50834, South Korea
*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;
Tel.: +82 51 510 2815; Fax: +82 51 513 6754.

Abstract
Abnormal melanogenesis results in excessive production of melanin, leading to
pigmentation disorders. As a key and rate-limiting enzyme for melanogenesis, tyrosinase has
been considered an important target for developing therapeutic agents of pigment disorders.
Despite having an (E)--phenyl-,-unsaturated carbonyl scaffold, which plays an important
role in the potent inhibition of tyrosinase activity, cinnamic acids have not attracted attention
as potential tyrosinase inhibitors, due to their low tyrosinase inhibitory activity and relatively
high hydrophilicity. Given that cinnamic acids’ structure intrinsically features this (E)-
scaffold and following our experience that minute changes in the chemical structure can
powerfully affect tyrosinase activity, twenty less hydrophilic cinnamamide derivatives were
designed as potential tyrosinase inhibitors and synthesised using a Horner-Wadsworth-
Emmons reaction. Four of these cinnmamides (4, 9, 14, and 19) exhibited much stronger
mushroom tyrosinase inhibition (over 90% inhibition) at 25 µM compared to kojic acid
(20.57% inhibition); crucially, all four have a 2,4-dihydroxy group on the -phenyl ring of
the scaffold. A docking simulation using tyrosinase indicated that the four cinnamamides
exceeded the binding affinity of kojic acid, and bound more strongly to the active site of
tyrosinase. Based on the strength of their tyrosinase inhibition, these four cinnamamides were
further evaluated in B16F10 melanoma cells. All four cinnamamides, without cytotoxicity,
exhibited higher tyrosinase inhibitory activity (67.33 – 79.67% inhibition) at 25 M than
kojic acid (38.11% inhibition), with the following increasing inhibitory order: morpholino (9)
= cyclopentylamino (14) < cyclohexylamino (19) < N-methylpiperazino (4) cinnamamides.
Analysis of tyrosinase activity and melanin content in B16F10 cells showed that the four
cinnamamides dose-dependently inhibited both cellular tyrosinase activity and melanin
content and that their inhibitory activity at 25 M was much better than that of kojic acid.
The results of melanin content analysis well matched those of the cellular tyrosinase activity

analysis, indicating that tyrosinase inhibition by the four cinnamamides is a major factor in
the reduction of melanin production. These results imply that these four cinnamamides with a
2,4-dihydroxyphenyl group can act as excellent anti-melanogenic agents in the treatment of
pigmentation disorders.
Key words: cinnamamide, tyrosinase inhibitor, melanin, anti-melanogenic, docking, B10F16
melanoma cells.

1. Introduction
A complicated process of melanogenesis causes skin pigmentation. Melanin is
produced in the melanosomes of melanocytes and transferred to keratinocytes in the
epidermis [1]. UV initiates melanogenesis, which is a human defence mechanism to produce
melanin for protecting skin cells from harmful UV rays [2]. However, abnormal accumulation
of melanin causes skin disorders including Riehl melanosis, melasma, and senile lentigo [3,
4]. To decrease the abnormal accumulation of melanin, numerous approaches have been
attempted involving the inhibition of tyrosinase, the suppression of melanin transfer from
melanocytes to keratinocytes, and the intervention of cell signalling for melanogenesis [5-9].
Although many different chemicals including natural and synthetic substances showed
meaningful anti-melanogenic effects in cell-based assays [10-18], most of them caused
several side effects, in in vivo animal models, or humans, such as an insufficient potency,
carcinogenic property, permanent depigmentation, and dermatitis [19-23]. Even though
hydroquinone and kojic acid are used as whitening agents in limited concentration in a few
countries, these skin-lightening agents are prohibited in most countries due to the risks of
undesirable side effects such as possible carcinogenicity in thyroid [24], nephrotoxicity [25],
genotoxicity [26] and cytotoxicity to melanocytes [27]. Arbutin is a -D-glucopyranoside of
hydroquinone, which is extracted from the bearberry plant in the genus Arctostaphylos. Due
to its fewer side effects, arbutin is more widely used as a whitening agent than hydroquinone,
and its anti-melanogenic effect is known to be due to the inhibition of tyrosinase activity.
Arbutin is hydrolyzed to D-glucose and hydroquinone by the main skin microflora
(Staphylococcus epidermidis and Staphylococcus aureus) [28], enzyme, such as -
o
glycosidase, and temperature (10% decomposition at 20 C for 5 days) [29]. Therefore, the
need to discover novel skin-lightening agents persists.

Tyrosinase is a metalloenzyme carrying two copper atoms at its active site and acts as a
multifunctional enzyme involving hydroxylase and oxidase[30]. During melanogenesis,
tyrosinase controls two rate-limiting steps: the transformation of L-tyrosine to L-dopa (as a
hydroxylase), and the conversion of dopa to dopaquinone (as an oxidase) [31]. Therefore,
tyrosinase has been considered a critical target for the treatment of skin disorders associated
with abnormal pigmentation. Among several approaches to suppress excessive and abnormal
melanogenesis, tyrosinase inhibition studies have been the most studied [10, 11, 14, 32].
Over the past several years, we have synthesized a variety of compounds with an
exomethylene type of -phenyl-,-unsaturated carbonyl scaffold to discover new whitening
agents (Figure 1). Compounds with the scaffold have been verified to exhibit good tyrosinase
inhibition by in vitro and/or in vivo assays [16, 18, 33-37]. In this study, we focussed on the
amide compounds connected to the -phenyl-,-unsaturated carbonyl scaffold to find novel
tyrosinase inhibitors, as shown in Figure 1. Cinnamic acid, a naturally occurring compound,
can be a handy template for tyrosinase inhibitors due to the presence of this scaffold in its
structure. Although caffeic acid, 3,4-dihydroxycinnamic acid, was found to have no
tyrosinase inhibitory activity, chlorogenic acid [38, 39], the quinic acid ester of caffeic acid
[39], showed mild inhibitory activity, compared with kojic acid. According to our previous
cumulative structure-activity relationship data, [35, 36, 40-42] derivatives with a hydroxyl
group on the -phenyl ring of the scaffold generally showed high tyrosinase inhibitory
activity, and the number and position of the hydroxyl groups are closely related to the degree
of tyrosinase inhibition, depending on the overall structure of the molecules. Therefore,
changing the number and position of the hydroxyl groups in cinnamic acids may greatly
increase the inhibitory activity of tyrosinase. However, it is difficult for cinnamic acids to be
absorbed by the melonocytes present in the epidermal basal layer due to the high polarity of
the carboxylic acid functionality. An amide functionality has even less polarity than a

carboxylic acid functionality, and in general, shows more druggable property than an ester
functionality in terms of biostability. On the basis of these findings, we designed the amide
derivatives of cinnamic acid with at least one hydroxyl group in our search for potent
tyrosinase inhibitors (Figure 1). As part of our continuous efforts to find new tyrosinase
inhibitors with a cinnamic acid scaffold, we synthesized a series of the amide derivatives of
cinnamic acid and evaluated their tyrosinase inhibition using mushroom tyrosinase and the
suppression of both tyrosinase activity and melanin production in murine cell systems.
Figure 1. An exomethylene type of -phenyl-,-unsaturated carbonyl scaffold and the
design of cinnamamide derivatives 1 – 20.
2. Results and Discussion
2.1. Chemistry
For the synthesis of various amide analogues of cinnamic acid, amines such as
primary cycloalkylamines and secondary cyclic amines were used in the condensation
reaction with cinnamic acid. As shown in Scheme 1, first of all, five benzaldehydes (3-
hydroxy-4-methoxybenzaldehyde
dihydroxybenzaldehyde 23,
21,
4-hydroxybenzaldehyde
24, and
22,
3,4-
2,4-dihydroxybenzaldehyde
4-hydroxy-3-
methoxybenzaldehyde 25) with at least one hydroxyl group were reacted with benzyl
bromide in the presence of K2CO3, giving the corresponding benzyl ether compounds 26 – 30
in yields of 95 – 97%, which were converted into the corresponding ethyl cinnamates 31 – 35
in yields of 95 – 98% by treatment with triethyl phosphonoacetate in the presence of a

catalytic amount of DBU (1,8-diazabicyclo[5.4.0]undec-7-ene) and 1.0 equiv. K2CO3. The
configuration of the double bond newly generated through a Horner-Wadsworth-Emmons
olefination reaction was determined after hydrolysis of the cinnamates. Treatment with 1N-
NaOH produced the corresponding cinnamic acid derivatives 36 – 40 quantitatively. The
configuration of the double bond of the cinnamic acids 36 – 40 was assigned as an (E)-form,
based on the coupling constant of the vinylic protons’ peaks. All five cinnamic acids had the
coupling constant with 16.0 Hz, implying that the cinnamic acids have the (E)-configuration.
Cinnamic acids 36 – 40 obtained after hydrolysis were coupled with amines involving
primary amines (cyclopentylamine and cyclohexylamine) and secondary amines (4-
methylpiperazine and morpholine) with the help of isobutyl chloroformate in the presence of
N-methylmorpholine to give the corresponding cinnamamides 41 – 60 in yields of 78 – 94%.
Debenzylation of 41 – 60 under acidic conditions using acetic acid and conc-HCl under
reflux produced the desired cinnamamide derivatives 1 – 3, 5 – 8, 10 – 13, 15 – 18, and 20 in
yields of 61 – 76%, but compounds 4, 9, 14, and 19 with a 2,4-dihydroxyl substituent were
not obtained. Debenzylation of compounds, 44, 49, 54, and 59, with a 2,4-dihydroxyl
substituent under these acidic conditions provided a mixture of compounds with high polarity.
Therefore, milder acidic conditions were needed to prevent the formation of undesired by-
products. BBr3 was tried as an alternative acidic reagent for a debenzylation reaction and,
surprisingly, produced the corresponding debenzylated cinnamamides 4, 9, 14, and 19 in
good yields (62 – 70%). The structures of the twenty final cinnamamides 1 – 20 were
confirmed by NMR and mass spectroscopy.

Scheme 1. Synthesis of cinnamamide derivatives 1 – 20. Reagents and conditions: a) BnBr,
K2CO3, CH3CN, reflux, 24 h; b) (EtO)2P(=O)CH2CO2Et, K2CO3, DBU (cat. amount), DCM,
o
DMF, rt for 21, 23 and 25, 70 C for 22, and 24, 24 – 36 h; c) 1M-NaOH aqueous solution,
1,4-dioxane, rt, 48 h; d) i-BuO2CCl, N-methylmorpholine, THF, rt, 30 min, and then, 4-
methylpiperazine, morpholine, cyclopentylamine, or cyclohexylamine, rt, 24 h; and e) c-HCl,
AcOH, reflux, 40 min for 1 – 3, 5 – 8, 10 – 13, 15 – 18, and 20, or 4.0 – 8.0 eq. BBr3, DCM,-
40 0C, 30 min for 4, 9, 14, and 19.
2.2. Mushroom tyrosinase inhibitory effect of cinnamamide derivatives 1 – 20.
The inhibitory effect of the twenty synthesized cinnamamides 1 – 20 on mushroom
tyrosinase was evaluated at 25 M using kojic acid, a well-known tyrosinase inhibitor, as a
positive control. As shown in Table 1, all compounds (2, 3, 7, 8, 12, 13, 17 and 18) having
either a 4-hydroxyl or 3,4-dihydroxyl group on the -phenyl ring did not show any inhibitory
effect regardless of the amide type. Compounds (1, 6, 11, and 16) with a 3-hydroxy-4-
methoxyl group on the -phenyl ring exhibited stronger inhibitory activities (from
31.12±7.93 to 60.15±3.51% inhibition) against mushroom tyrosinase than kojic acid
(20.57±2.02% inhibition). On the other hand, compounds (5, 10, 15, and 20) with a 4-
hydroxy-3-methoxyl group on the -phenyl ring showed similar or more potent inhibitory
activity (from 19.33±1.36 to 68.04±8.25% inhibition) than kojic acid, except for compound
15 (no inhibition). Regardless of the amide type, four of the cinnamamides (4, 9, 14, and 19)
with a 2,4-dihydroxyphenyl group revealed much better inhibitory activity than kojic acid.
All four of these cinnamamides showed an inhibitory activity exceeding 90% with an IC50
values of 0.14±0.00 M for 4, 0.06±0.01 M for 9, 0.12±0.00 M for 14, and 0.16±0.01 M
for 19, respectively, while kojic acid showed an IC50 value of 30.64±1.27 M. Notably,
although a 4-hydroxyl group alone on the -phenyl ring of cinnamamides did not show

tyrosinase inhibitory activity, the insertion of an additional hydroxyl group into the 2-position
of the -phenyl ring dramatically enhanced the inhibition (2 vs. 4, 7 vs. 9, 12 vs. 14, and 17 vs.
19). However, the insertion of an additional hydroxyl group into the 3-position did not
enhance the tyrosinase inhibition. On the other hand, the insertion of a methoxyl group into
the 3-position increased (compounds 5, 10, and 20) or did not influence (compound 15) the
tyrosinase inhibition, depending on their molecular structures. Compounds 1, 6, 11, and 16
have a hydroxyl group at the 3-position of the -phenyl ring and a methoxyl group at the 4-
position of the -phenyl ring, whereas compounds 5, 10, 15, and 20 have the same
substituents in the reverse position. Overall, compounds with a 3-hydroxy-4-methoxy group
on the -phenyl ring exerted stronger inhibitory activities than compounds with a 4-hydroxy-
3-methoxy group (1 vs. 5, 6 vs. 10, and 11 vs. 15). These results suggest that the number and
type of the substituents on the -phenyl ring of cinnamamide derivatives greatly affect the
inhibitory activity of tyrosinase.
Because of showing the highest inhibitory activity against mushroom tyrosinase, the four
cinnamamide derivatives with a 2,4-dihydroxyphenyl group (4, 9, 14, and 19) were used in
the following studies involving docking simulation and murine cell-based experiments.
Table 1. Substitution patterns, and tyrosinase-inhibitory activities of the synthesized
cinnamamide derivatives 1 – 20 and kojic acid

Compounds
R1
H
R2
OH
R3
OMe
Tyrosinase inhibition (%)a
40.22±4.67
1
H
H
H
OH
H
OH
OH
OH
OH
OMe
OH
OH
OH
OH
OMe
OH
OH
OH
OH
OMe
OH
OH
OH
OH
NI
NI
2
3
OH
H
94.39±0.25
19.33±1.36
31.12±7.93
NI
4
OMe
OH
H
5
H
6
H
7
H
OH
H
NI
8
OH
H
95.74±0.12
21.28±3.45
60.15±3.51
NI
9
OMe
OH
H
10
H
11
H
12
H
OH
H
NI
13
OH
H
94.11±1.38
NI
14
OMe
OH
H
15
H
45.61±16.34
NI
16
H
17
H
OH
H
NI
18
19
OH
H
93.78±1.42
68.04±8.25
20.57±2.02
OMe
20
Kojic acid
Tyrosinase inhibition was assayed at 25 M using L-tyrosine as a substrate. Results are
presented as mean ± SEMs.

2.3. Docking studies of cinnamamide derivatives 4, 9, 14, and 19 and kojic acid with
tyrosinase
To examine whether the four cinnamamide derivatives bind to the active site of
tyrosinase and inhibit the enzyme as the result of the binding, in silico docking simulation
was carried out using AutoDock Vina 1.1.2 software (The Scripps Research Institute). For
tyrosinase ligand preparation, 2D structures of the four cinnamamides were drawn using
ChemDraw Ultra 12.0 software (CambridgeSoft Corporation) and converted into the
corresponding 3D structures using the energy minimization tool of Chem3D Pro 12.0
software (CambridgeSoft Corporation). The 3D X-ray crystal structure of Agaricus bisporus
tyrosinase was downloaded from the Protein Data Bank (protein ID: 2Y9X) for docking
simulation with the four cinnamamide derivatives.

Figure 2. Docking simulation of the four cinnamamides 4, 9, 14, and 19 and kojic acid with
tyrosinase and pharmacophore analysis: (a, and b) pharmacophore results for 4, 9, 14, and 19
and kojic acid obtained using LigandScout 4.1.0 showing hydrogen-bonding (green arrow),
- stacking (violet arrow) and hydrophobic (yellow) interactions, (c) docking simulation
results between the four cinnamamide derivatives (4, 9, 14, and 19), and mushroom
tyrosinase, and (d) docking scores between tyrosinase and 4, 9, 14, 19 or kojic acid.

Compared to the binding affinity (-5.7 kcal/mol) of kojic acid, all four cinnamamide
derivatives showed high binding affinities (-6.7 ~ -7.3 kcal/mol), as indicated in Figure 2.
According to the pharmacophore analysis results of kojic acid using LigandScout 4.1.0
software, the 4-pyranone ring of kojic acid interacts with the imidazole ring of His263 amino
acid residue through - stacking and the primary hydroxyl group produces two hydrogen
bonds with two amino acid residues, His259 and His263. All four cinnamamide derivatives
commonly have a 2,4-dihydroxyl substituent on the phenyl ring. Notably, the 2-hydroxyl
groups on the phenyl ring of cinnamamides 4 and 9 belonging to cyclic tertiary amides make
a hydrogen bond with Met280 amino acid residue, whereas the 2-hydroxyl groups on the
phenyl ring of cinnamamides 14 and 19 belonging to cycloalkyl secondary amides make a
hydrogen bond with Asn260 amino acid residue. Further, the 4-hydroxy groups on the phenyl
ring of 4 and 9 do not participate in hydrogen bonding, while those of 14 and 19 create
hydrogen bonds with the same amino acid, Met280. Also, cinnamamide 4 shows same
hydrophobic interactions with two amino acid residues (Val283, and Ala286) as cinnamamide
9, and cinnamamide 14 shows the same hydrophobic interactions (Phe264, Val283, and
Ala286) as cinnamamide 19. All four cinnamamides commonly form a hydrophobic
interaction with two amino acid residues (Val283 and Ala286). These docking simulation
results suggest that the cinnamamide analogues bind to the active site of tyrosinase and
consequently inhibit tyrosinase.
2.4. Cell viability of cinnamamide derivatives 4, 9, 14 and 19 in B16F10 melanoma cells
We performed a WST-8 assay to confirm whether cinnamamides 4, 9, 14 and 19 are
cytotoxic or not. For this purpose, B16F10 melanoma cells were cultured and treated with
four different concentrations (0, 5, 10, and 25 µM) of the test cinnamamides. After an
incubation of 24 h under a humidified atmosphere, the optical densities were measured on a

microplate reader.
According to Figure 3, no significant cytotoxic effect was found on the melanoma cells at
concentrations up to 25 µM, showing that cinnamamides 4, 9, 14 and 19 were safe in murine
melanoma B16F10 cells. Therefore, all cell-based assays involving cellular tyrosinase and
melanin content were carried out at concentrations below 25 µM.

Figure 3. Cell viability assay of four cinnamamide derivatives 4, 9, 14 and 19 in B16F10
melanoma cells. Cinnamamides were treated at concentrations of 5, 10, and 25 μM.
Viabilities are expressed in % to control, and bars represent standard errors.

2.5. Tyrosinase inhibitory activity of cinnamamide derivatives 4, 9, 14 and 19 in α-MSH-
stimulated B16F10 melanoma cells
To determine the tyrosinase inhibitory effect of the four cinnamamide derivatives 4,
9, 14 and 19, α-MSH-stimulated B16F10 melanoma cells were cultured and treated with four
different concentrations of test compounds (0, 5, 10, and 25 μM), kojic acid (25 μM ) and
arbutin (400 μM). After an incubation of 24 h, the tyrosinase inhibitory effect of compounds
on B16F10 cells was determined by measuring the optical densities of the compounds.
According to the results displayed in Figure 4, cinnamamide 4 showed the greatest
tyrosinase inhibitory effect among all four cinnamamides, kojic acid and arbutin. The
inhibition of tyrosinase activity at 25 μM by the four cinnamamides increased in the order of
morpholino (9) = cyclopentylamino (14) < cyclohexylamino (19) < N-methylpiperazino (4),
with 67.33%, 67.67%, 75.89% and 79.67% inhibitions, respectively. Considering that the
cinnamamides did not show cytotoxicity at concentrations below 25 μM, as shown in Figure
3, the inhibition of tyrosinase activity was attributed to direct tyrosinase inhibition by the
cinnamamides, rather than cytotoxicity. Docking simulation results depicted in Figure 2 also
support that the cinnamamides directly inhibit tyrosinase. All four cinnamamide derivatives
having a common 2,4-dihydroxyl substituent on the phenyl ring of the cinnamamide
exhibited more potent tyrosinase inhibitory activity at 25 μM than kojic acid (38.11%, at 25
μM) and arbutin (49.56%, at 400 μM), two natural tyrosinase inhibitors. Also, the tyrosinase
activity was inhibited by the cinnamamide derivatives in a dose-dependent manner. These
results indicate that all four cinnamamide derivatives have adequate lipophilicity to penetrate
well into the cell membrane.

Tyrosinase activity
19
14
9
4
100
50
0
**
***
***
***
***
***
***
***
***
***
***
***
l
n
d
i
M
M
M
M
M
M
M
M
M
M
M
M
o
i
t
r
µ
µ
µ
µ
µ
µ
µ
µ
µ
µ
µ
µ
c
t
u
A
n
5
5
5
5
0
5
0
5
0
5
0
5
b
r
o
1
2
1
2
1
2
1
2
c
i
C
A
j
o
K
Concentration
Figure 4. Tyrosinase inhibitory activity of cinnamamide derivatives 4, 9, 14 and 19 in α-
MSH-stimulated B16F10 melanoma cells that were co-treated with the four cinnamamides 4,
9, 14, and 19 (5, 10, and 25 µM), kojic acid (25 µM), or arbutin (400 µM). The asterisks
represent the significant difference between the columns. **, p<0.01; and ***, p<0.001. The
bars represent standard errors.
2.6. Melanin production inhibitory effect of cinnamamide derivatives 4, 9, 14 and 19 in α-
MSH-stimulated B16F10 melanoma cells
To observe the melanin production inhibitory effect of the cinnamamide derivatives,
melanin content assay was carried out. For this purpose, B16F10 melanoma cells were
stimulated by α-MSH and co-treated with cinnamamides 4, 9, 14 and 19 in four different
concentrations (0, 5, 10, and 25 µM), or kojic acid (25 µM). After an incubation of 24 h, the
optical densities were measured to evaluate the melanin production inhibitory effect of the
four cinnamamides.
According to the results shown in Figure 5, the four cinnamamide derivatives greatly
decreased the melanin content in α-MSH-stimulated B16F10 melanoma cells at 25 µM, as

compared to kojic acid (35.43% inhibition). The inhibition of melanin production by the four
cinnamamides increased in the order of cyclopentylamino (14) < morpholino (9) <
cyclohexylamino (19) < N-methylpiperazino (4), with 58.20%, 62.03%, 74.00% and 75.57%
inhibitions, respectively. Also, all four cinnamamides decreased melanin content significantly
and dose-dependently. The similarity between the inhibition of tyrosinase activity (Figure 4)
and the suppression of melanogenesis (Figure 5) implies that inhibition of melanin production
is due to inhibition of tyrosinase activity by cinnamamides.
Melanin content
19
4
9
14
100
50
0
***
***
***
***
***
***
***
***
***
***
***
***
***
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d
i
M
M
M
µ
M
M
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r
µ
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µ
µ
µ
µ
µ
µ
µ
c
t
A
n
5
5
5
5
0
5
0
5
0
5
0
5
1
2
1
2
1
2
1
2
o
c
i
C
j
o
k
Concentration
Figure 5. Melanin production inhibitory effect of cinnamamide derivatives 4, 9, 14 and 19 in
α-MSH-stimulated B16F10 melanoma cells. B16F10 cells were stimulated by α-MSH and
co-treated with the four cinnamamides 4, 9, 14, and 19 (5, 10, and 25 µM), or kojic acid (25
µM). The asterisks represent the significant difference between the columns. ***, p<0.001.
The bars represent standard errors.
2.7. DPPH radical scavenging activity of cinnamamide derivatives 1 – 20
DPPH radical scavenging assay was performed to evaluate the antioxidant activities
of cinnamamide derivatives 1 - 20. In the final concentration of 1 mM, L-ascorbic acid, and

cinnamamides 1 – 20 were added to the DPPH methanol solution and the DPPH radical
scavenging activity was determined after 30 min (Table 2).
According to the results shown in Table 2, most of the cinnamamides showed
moderate to potent DPPH radical scavenging activity. The DPPH radical scavenging activity
is closely related to the structure of the phenyl ring of the cinnamamides, rather than the
structure of the amine moiety of the cinnamamides. Cinnamamides (2, 7, 12, and 17) with a
4-hydroxyl group on the phenyl ring seldom exhibited DPPH radical scavenging activity and
cinnamamides (1, 4, 6, 9, 11, 14, 16, and 19) with either a 3-hydroxy-4-methoxyl or 2,4-
dihydroxyl group exhibited weak to mild scavenging activity. High DPPH radical scavenging
activity was observed in cinnamamides (3, 5, 8, 10, 13, 15, 18, and 20) with a 3,4-dihydroxyl,
or 4-hydroxy-3-methoxyl group. Their DPPH radical scavenging activity (76.40 ~ 85.57%)
was comparable to that of L-ascorbic acid (84.64%).
Melanogenesis may result from reactive oxygen species (ROS) which are one of the
factors that activate tyrosinase. Considering that cinnamamide derivatives 4, 9, 14 and 19
with a 2,4-dihydroxyphenyl group showed low to moderate DPPH radical scavenging activity,
the strong anti-melanogenic effect of these cinnamamides may be attributable mainly to
direct tyrosinase inhibition, rather than tyrosinase deactivation by antioxidant activity.
Table 2. DPPH radical scavenging activities of the synthesized cinnamamide derivatives 1 –
20 and kojic
acid

Compound
DPPH radical
scavenging activity (%)
3.64±0.83
Compound
DPPH radical
scavenging activity (%)
36.69±0.87
19.83±2.05
85.57±0.18
46.52±1.97
83.71±0.29
39.25±4.96
NA
1
2
3
4
5
6
7
8
11
12
13
14
15
16
17
18
19
20
1.45±1.27
83.38±0.37
7.18±0.79
76.40±0.54
21.15±0.51
NA
83.40±0.56
35.38±0.89
82.44±0.26
84.64±0.32
81.53±0.36
36.94±0.93
78.59±0.23
9
10
L-ascorbic acid
Radical scavenging activities were determined 30 min after the addition of cinnamamide
derivatives, or L-ascorbic acid to a final concentration of 1.0 mM DPPH solution in methanol.
Three independent experiments were performed. Results are presented as the means±SDs of
three experiments.
4. Conclusion
Cinnamic acid has an (E)--phenyl-,-unsaturated carbonyl scaffold, which is an
essential structural moiety for excellent tyrosinase inhibition, but is relatively hydrophilic and
difficult to be absorbed on the skin. Conversion of the carboxylic acid functional group in
cinnamic acid into an amido group can increase the lipophilicity. Thus, twenty of the
cinnamamides were synthesized as tyrosinase inhibitors using a Horner-Wadsworth-Emmons
olefination. Of the twenty cinnamamides, four (4, 9, 14, and 19) showed a high inhibitory
effect on mushroom tyrosinase in excess of 90%. Docking simulation of the cinnamamides
and tyrosinase indicated that the four cinnamamides bind more tightly to the active site of
tyrosinase than kojic acid. Inhibition of tyrosinase activity and melanin content in B16F10
melanoma cells revealed that the four cinnamamides inhibited both cellular tyrosinase
activity and melanin content dose-dependently and more strongly than kojic acid, which was
used as a positive control. Similar inhibition patterns were observed in both cellular
tyrosinase activity and melanin content, suggesting that the reduction of melanin production

by cinnamamides results from tyrosinase inhibition. Notably, all compounds 4, 9, 14, and 19
which exhibited great anti-melanogenesis had a common 2,4-dihydroxyphenyl group.
Comparing the anti-melanogenic effect of the four cinnamamides to that of kojic acid
demonstrates the promising potential of the four cinnamamides as anti-whitening therapeutic
agents.
4. Experimental section
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. The melting
points of synthesized compounds were measured in capillary tubes on a Stuart™ melting
point apparatus SMP3 (Bibby Scientific, Staffordshire, UK) and are uncorrected. All
anhydrous solvents were distilled over CaH and Na/benzophenone. High resolution mass
spectroscopy data were obtained on an Agilent Accurate Mass Q-TOF (quadruple-time of
flight) liquid chromatography mass spectrometer (Agilent, Santa Clara, CA, USA) in ESI
negative mode while low-resolution mass data were also obtained in ESI negative mode on
an Expression CMS spectrometer (Advion Ithaca, NY, USA). Infrared spectroscopy data
were obtained with a PerkinElmer Frontier FT-IR/FIR Spectrometer (PerkinElmer, Waltham,
MA, USA) in ATR (Attenuated total reflection) mode using pure powder. Nuclear magnetic
resonance (NMR) spectra were recorded on a Varian Unity INOVA 400 spectrometer or a
1
Varian Unity AS500 spectrometer (Agilent Technologies, Santa Clara, CA, USA) for H
NMR (400 MHz and 500 MHz) and for 13C NMR (100 MHz). DMSO-d6, CDCl3, and
CDCl3+CD3OD were used as an NMR solvent for NMR samples. The chemical shift and

coupling constant (J) values were measured in parts per million (ppm) and hertz (Hz),
respectively. The abbreviations used in the analysis of 1H NMR data are s (singlet), brs (broad
singlet), d (doublet), brd (broad doublet), dd (doublet of doublets), t (triplet), tt (triplet of
triplets), brt (broad triplet), q (quartet), quint (quintet), m (multiplet), and brm (broad
multiplet).
4.1.1. General procedure for the synthesis of compounds 26 – 30 [43].
Benzaldehyde 21 – 25 (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. Volatiles were evaporated on completion of the reaction and
the resultant residues were partitioned between dichloromethane and water. The organic layer
was dried with anhydrous MgSO4 and evaporated in vacuo to provide the products 26 - 30 as
a white solid in yields of 95 – 97%. Spectroscopic data can be found in SI.
4.1.2. General procedure for the synthesis of cinnamic acids 36 – 40 through cinnamates
31 – 35 [44].
To a stirred solution of compounds 26 – 30 (2.00 g), triethyl phosphonoacetate (1.1
equiv.), and potassium carbonate (2.0 equiv.) in dichloromethane/N,N-dimethylformamide
(15 mL, 2:1) was added
a
catalytic amount of DBU (0.03equiv., 1,8-
diazabicyclo[5.4.0]undec-7-ene). The reaction mixture was stirred at room temperature
(compounds 26, 28, and 30) or 70 0C (compounds 27, and 29) for 24 – 36 h. After removing
volatiles, ice water was added to the reaction mixture which was then stirred at room
temperature for 30 min. After the generated precipitates were filtered, the filter cake was
washed with excess water, and dried to provide ethyl cinnamates 31 – 35 as a white or grey
solid in yields of 95 – 98%. The cinnamates 31 – 35 were used in the next step without
characterization.

To a stirred solution of cinnamates 31 – 35 (2.57 to 4.11 g) in 1,4-dioxane (20 mL)
was added 1N NaOH aqueous solution (8.0 – 16.0 equiv.) and the reaction mixture was
stirred at room temperature for 48 h. On completion of the reaction, the reaction mixture was
then acidified until pH 2 by 2N HCl. The reaction mixture was stirred at room temperature
for 30 min to give precipitates. The filter cake obtained from filtration was washed with
excess water, and dried to give cinnamic acids 36 – 40 as a white or grey solid in yields of
100%. Spectroscopic data can be found in SI.
4.1.3. General procedures for the preparation of compounds 41 – 60 [45].
To a solution of cinnamic acids 36 – 40 (100 mg), and isobutyl chloroformate (2.0 equiv.) in
anhydrous THF (5 mL) was added N-methylmorpholine (2.5 equiv.). After the reaction
mixture was stirred at room temperature for 30 min, appropriate amines (4-methylpiperazine,
morpholine, cyclopentylamine, and cyclohexylamine: 2.0 equiv.) were added and the reaction
mixture was then stirred at room temperature for 24 h. On completion of the reaction, the
reaction mixture was partitioned between ethyl acetate and water. The organic layer was
washed with brine, dried with MgSO4 and filtered. The filtrate was removed under reduced
pressure. The resultant residue was purified by silica gel column chromatography using
dichloromethane and methanol (30 – 70: 1) to afford compounds 41 – 60 as a white or yellow
solid in yields of 78 – 94%. Spectroscopic data of known compounds can be found in SI.
4.1.3.1.
(E)-3-(3-(Benzyloxy)-4-methoxyphenyl)-1-(4-methylpiperazin-1-yl)prop-2-en-1-
oneone (41). Yellowish white solid; 90% yield; 1H NMR (500 MHz, CDCl3) δ 7.55 (d, 1 H, J
= 15.5 Hz, 3-vinylic H), 7.44 (d, 2 H, J = 7.5 Hz, 2′-H, 6′-H), 7.37 (t, 2 H, J = 7.5 Hz, 3′-H,
5′-H), 7.30 (t, 1 H, J = 7.5 Hz, 4′-H), 7.10 (dd, 1 H, J = 8.5, 2.0 Hz, 6-H), 7.13 (d, 1 H, J =
2.0 Hz, 2-H), 6.86 (d, 1 H, J = 8.5 Hz, 5-H), 6.61 (d, 1 H, J = 15.5 Hz, 2-vinylic H), 5.17 (s, 2
H, benzylic H), 3.90 (s, 3 H, OCH3), 3.76 (brm, 2 H), 3.67 (brm, 2 H), 2.48 (brt, 4 H, J = 5.0

Hz, 3′′-H, 5′′-H), 2.36 (s, 3 H, NCH3); 13C NMR (100 MHz, CDCl3) δ 165.8, 151.4, 148.4,
142.9, 137.1, 128.8, 128.2, 128.2, 127.5, 122.4, 114.9, 113.2, 111.7, 71.5, 56.2, 54.8, 46.1,
43.4.
4.1.3.2. (E)-3-(3,4-Bis(benzyloxy)phenyl)-1-(4-methylpiperazin-1-yl)prop-2-en-1-one (43).
Yellowish white solid; 81% yield; 1H NMR (500 MHz, CDCl3) δ 7.55 (d, 1 H, J = 15.0 Hz, 3-
vinylic H), 7.45 – 7.29 (m, 10 H, 2ⅹPh), 7.09 (s, 1 H, 2-H), 7.06 (d, 1 H, J = 8.5 Hz, 6-H),
6.90 (d, 1 H, J = 8.5 Hz, 5-H), 6.61 (d, 1 H, J = 15.0 Hz, 2-vinylic H), 5.18 (s, 2 H, benzylic
H), 5.18 (s, 2 H, benzylic H), 3.78 (brm, 4 H, 2′′-H, 6′′-H), 2.61 (brm, 4 H, 3′′-H, 5′′-H), 2.44
13
(s, 3 H, NCH3); C NMR (100 MHz, CDCl3) δ 166.8, 150.8, 149.1, 143.3, 137.2, 137.0,
128.8, 128.8, 128.1, 127.4, 127.3, 122.5, 114.9, 114.5, 114.3, 71.7, 71.2, 54.6, 45.9, 43.8.
4.1.3.3. (E)-3-(2,4-Bis(benzyloxy)phenyl)-1-(4-methylpiperazin-1-yl)prop-2-en-1-oneone (44).
Yellowish white solid; 78% yield; 1H NMR (500 MHz, CDCl3) δ 7.71 (d, 1 H, J = 15.0 Hz, 3-
vinylic H), 7.47 – 7.33 (m, 11 H, J = 7.5 Hz, 6-H, 2ⅹPh), 6.98 (d, 1 H, J = 15.0 Hz, 2-vinylic
H), 6.45 (d, 1 H, J = 2.0 Hz, 3-H), 6.59 (dd, 1 H, J = 9.0, 2.0 Hz, 5-H), 5.08 (s, 2 H, benzylic
H), 5.04 (s, 2 H, benzylic H), 3.80 (brm, 2 H), 3.35 (brm, 2 H), 2.56 (brm, 4 H, 3′′-H, 5′′-H),
2.41 (s, 3 H, NCH3); 13C NMR (100 MHz, CDCl3) δ 166.7, 161.2, 159.4, 139.9, 136.6, 136.4,
133.2, 128.9, 128.9, 128.6, 128.5, 128.4, 127.7, 117.8, 116.0, 106.4, 100.6, 70.8, 70.4, 54.5,
45.5, 42.9.
4.1.3.4. (E)-3-(4-(Benzyloxy)-3-methoxyphenyl)-1-(4-methylpiperazin-1-yl)prop-2-en-1-one
1
(45). Yellow solid; 82% yield; H NMR (500 MHz, CDCl3) δ 7.60 (d, 1 H, J = 15.5 Hz, 3-
vinylic H), 7.42 (d, 2 H, J = 7.5 Hz, 2′-H, 6′-H), 7.36 (t, 2 H, J = 7.5 Hz, 3′-H, 5′-H), 7.30 (t,
1 H, J = 7.5 Hz, 4′-H), 7.04 (d, 1 H, J = 1.5 Hz, 2-H), 7.03 (dd, 1 H, J = 9.0, 1.5 Hz, 6-H),
6.85 (d, 1 H, J = 9.0 Hz, 5-H), 6.70 (d, 1 H, J = 15.5 Hz, 2-vinylic H), 5.17 (s, 2 H, benzylic
H), 3.92 (s, 3 H, OCH3), 3.77 (brm, 2 H), 3.72 (brm, 2 H), 2.50 (brt, 4 H, J = 5.0 Hz, 3′′-H,

5′′-H), 2.36 (s, 3 H, NCH3); 13C NMR (100 MHz, CDCl3) δ 165.8, 155.7, 149.8, 143.1, 136.9,
128.8, 128.8, 128.2, 127.4, 121.8, 115.0, 113.7, 110.6, 71.0, 56.3, 54.8, 46.1, 43.6.
4.1.3.5. (E)-3-(3-(Benzyloxy)-4-methoxyphenyl)-1-morpholinoprop-2-en-1-one (46). White
solid; 91% yield; 1H NMR (500 MHz, CDCl3) δ 7.58 (d, 1 H, J = 15.5 Hz, 3-vinylic H), 7.44
(d, 2 H, J = 7.5 Hz, 2′-H, 6′-H), 7.36 (t, 2 H, J = 7.5 Hz, 3′-H, 5′-H), 7.30 (t, 1 H, J = 7.5 Hz,
4′-H), 7.10 (d, 1 H, J = 8.5 Hz, 6-H) 7.05 (s, 1 H, 2-H), 6.87 (d, 1 H, J = 8.5 Hz, 5-H), 6.58 (d,
1 H, J = 15.5 Hz, 2-vinylic H), 5.16 (s, 2 H, benzylic H), 3.90 (s, 3 H, OCH3), 3.72 – 3.63 (m,
8 H, morpholine-H); 13C NMR (100 MHz, CDCl3) δ 166.0, 151.6, 148.4, 143.3, 137.1, 128.8,
128.2, 128.2, 127.5, 122.5, 114.4, 113.3, 111.8, 71.6, 67.1, 56.2, 44.1.
4.1.3.6. (E)-3-(3,4-Bis(benzyloxy)phenyl)-1-morpholinoprop-2-en-1-one (48). White solid; 94%
yield; 1H NMR (500 MHz, CDCl3) δ 7.58 (d, 1 H, J = 15.0 Hz, 3-vinylic H), 7.46 – 7.29 (m,
10 H, 2ⅹPh), 7.09 (s, 1 H, 2-H), 7.07 (d, 1 H, J = 8.5 Hz, 6-H), 6.90 (d, 1 H, J = 8.5, 5-H),
6.60 (d, 1 H, J = 15.0 Hz, 2-vinylic H), 5.19 (s, 2 H, benzylic H), 5.18 (s, 2 H, benzylic H),
13
3.72 – 3.66 (m, 8 H, morpholine-H); C NMR (100 MHz, CDCl3) δ 165.9, 150.8, 149.1,
143.2, 137.2, 137.0, 128.8, 128.8, 128.7, 128.1, 127.5, 127.4, 122.5, 114.7, 114.6, 114.4, 71.8,
71.2, 67.1, 44.3.
4.1.3.7. (E)-3-(2,4-Bis(benzyloxy)phenyl)-1-morpholinoprop-2-en-1-one (49). White solid; 90%
yield; 1H NMR (500 MHz, CDCl3) δ 7.74 (d, 1 H, J = 15.5 Hz, 3-vinylic H), 7.47 – 7.33 (m,
11 H, 6-H, 2ⅹPh), 6.99 (d, 1 H, J = 15.5 Hz, 2-vinylic H), 6.65 (d, 1 H, J = 1.5 Hz, 3′-H),
6.60 (dd, 1 H, J = 8.0, 1.5 Hz, 5′-H), 5.09 (s, 2 H, benzylic H), 5.04 (s, 2 H, benzylic H), 3.57
– 3.33 (brm, 8 H, morpholine-H); 13C NMR (100 MHz, CDCl3) δ 167.0, 161.3, 159.5, 140.3,
136.6, 136.4, 133.6, 128.9, 128.9, 128.7, 128.6, 128.4, 127.7, 117.8, 115.8, 106.4, 100.6, 70.8,
70.5, 67.0, 44.7.
4.1.3.8. (E)-3-(3-(Benzyloxy)-4-methoxyphenyl)-N-cyclopentylacrylamide (51). Yellowish

white solid; 83% yield; 1H NMR (500 MHz, CDCl3) δ 7.51 (d, 1 H, J = 15.5 Hz, 3-vinylic H),
7.43 (d, 2 H, J = 7.5 Hz, 2′-H, 6′-H), 7.36 (t, 2 H, J = 7.5 Hz, 3′-H, 5′-H), 7.30 (t, 1 H, J = 7.5
Hz, 4′-H), 7.08 (d, 1 H, J = 8.0 Hz, 6-H), 7.04 (s, 1 H, 2-H), 6.85 (d, 1 H, J = 8.0 Hz, 5-H),
6.22 (d, 1 H, J = 15.5 Hz, 2-vinylic H), 5.83 (brs, 1 H, NH), 5.13 (s, 2 H, benzylic H), 4.33
(brm, 1 H, 1′′-H), 3.89 (s, 3 H, OCH3), 2.03 (m, 2 H), 1.70 (m, 2 H), 1.62 (m, 2 H), 1.45 (m, 2
13
H); C NMR (100 MHz, CDCl3) δ 166.1, 151.4, 148.4, 141.1, 136.9, 128.8, 128.2, 127.9,
127.6, 122.4, 118.6, 112.9, 111.8, 71.3, 56.2, 51.7, 33.4, 24.0.
4.1.3.9. (E)-3-(3,4-Bis(benzyloxy)phenyl)-N-cyclopentylacrylamide (53). Yellowish solid; 81%
yield; 1H NMR (500 MHz, CDCl3) δ 7.51 (d, 1 H, J = 16.0 Hz, 3-vinylic H), 7.44 – 7.29 (m,
10 H, 2ⅹPh), 7.09 (d, 1 H, J = 2.0 Hz, 2-H), 7.03 (dd, 1 H, J = 8.5, 2.0 Hz, 6-H), 6.88 (d, 1 H,
J = 8.5 Hz, 5-H), 6.23 (d, 1 H, J = 16.0 Hz, 2-vinylic H), 5.84 (brs, 1 H, NH), 5.16 (s, 2 H,
benzylic H), 5.15 (s, 2 H, benzylic H), 4.32 (brm, 1 H, 1′′-H), 2.03 (m, 2 H), 1.70 (m, 2 H),
13
1.62 (m, 2 H), 1.45 (m, 2 H); C NMR (500 MHz, CDCl3) δ 166.1, 150.7, 149.1, 141.0,
137.1, 137.0, 128.7, 128.7, 128.5, 128.1, 127.5, 127.4, 122.5, 118.9, 114.6, 114.1, 71.5, 71.2,
51.7, 33.4, 24.0.
4.1.3.10. (E)-3-(2,4-Bis(benzyloxy)phenyl)-N-cyclopentylacrylamide (54). Yellow solid; 78%
yield; 1H NMR (500 MHz, CDCl3) δ 7.82 (d, 1 H, J = 16.0 Hz, 3-vinylic H), 7.43 – 7.31 (m,
11 H, 6-H, 2ⅹPh), 6.58 – 6.54 (m, 2 H, 3-H, 5-H), 6.43 (d, 1 H, J = 16.0 Hz, 2-vinylic H),
5.53 (brd, 1 H, J = 4.5 Hz, NH), 5.10 (s, 2 H, benzylic H), 5.02 (s, 2 H, benzylic H), 4.32 (m,
1 H, 1′′-H), 2.02 (m, 2 H), 1.71 – 1.59 (m, 4 H), 1.42 (m, 2 H); 13C NMR (100 MHz, CDCl3)
δ 166.7, 161.2, 158.8, 136.7, 136.7, 136.2, 130.7, 128.9, 128.8, 128.4, 128.2, 127.7, 127.5,
119.9, 117.8, 106.6, 101.0, 70.6, 70.4, 51.5, 33.4, 23.9.
4.1.3.11. (E)-3-(3-(Benzyloxy)-4-methoxyphenyl)-N-cyclohexylacrylamide (56). Yellow solid;
86% yield; 1H NMR (500 MHz, CDCl3) δ 7.51 (d, 1 H, J = 16.0 Hz, 3-vinylic H), 7.43 (d, 2

H, J = 7.5 Hz, 2′-H, 6′-H), 7.36 (t, 2 H, J = 7.5 Hz, 3′-H, 5′-H), 7.30 (t, 1 H, J = 7.0 Hz, 4′-H),
7.08 (d, 1 H, J = 8.0 Hz, 6-H), 7.05 (s, 1 H, 2-H), 6.84 (d, 1 H, J = 8.0 Hz, 5-H), 6.22 (d, 1 H,
J = 16.0 Hz, 2-vinylic H), 5.75 (brs, 1 H, NH), 5.12 (s, 2 H, benzylic H), 3.88 (m, 4 H, 1′′-H,
OCH3), 1.96 (m, 2 H, 2′′-Ha, 6′′-Ha), 1.72 (m, 2 H, 2′′-Hb, 6′′-Hb), 1.63 (m, 1 H, 4′′-Ha),
13
1.40 (m, 2 H, 3′′-Ha, 5′′-Ha), 1.19 (m, 3 H, 3′′-Hb, 4′′-Hb, 5′′-Hb); C NMR (100 MHz,
CDCl3) δ 165.6, 151.3, 148.4, 141.0, 136.9, 128.8, 128.2, 127.9, 127.6, 122.4, 118.8, 112.7,
111.7, 71.2, 56.2, 48.8, 33.4, 25.7, 25.1.
4.1.3.12. (E)-3-(3,4-Bis(benzyloxy)phenyl)-N-cyclohexylacrylamide (58). Yellowish solid; 80%
yield; 1H NMR (500 MHz, CDCl3) δ 7.51 (d, 1 H, J = 15.5 Hz, 3-vinylic H), 7.45 – 7.29 (m,
10 H, 2ⅹPh), 7.09 (s, 1 H, 2-H), 7.03 (d, 1 H, J = 8.0 Hz, 6-H), 6.88 (d, 1 H, J = 8.0 Hz, 5-H),
6.22 (d, 1 H, J = 15.5 Hz, 2-vinylic H), 5.69 (brs, 1 H, NH), 5.17 (s, 2 H, benzylic H), 5.15 (s,
2 H, benzylic H), 3.89 (brm, 1 H, 1′′-H), 1.97 (m, 2 H, 2′′-Ha, 6′′-Ha), 1.72 (m, 2 H, 2′′-Hb,
6′′-Hb), 1.63 (m, 1 H, 4′′-Ha), 1.40 (m, 2 H, 3′′-Ha, 5′′-Ha), 1.19 (m, 3 H, 3′′-Hb, 4′′-Hb, 5′′-
13
Hb); C NMR (500 MHz, CDCl3) δ 165.6, 150.7, 149.0, 141.1, 137.1, 137.0, 128.7, 128.7,
128.5, 128.1, 127.5, 127.4, 127.3, 122.5, 118.9, 114.5, 113.9, 71.5, 71.1, 48.8, 33.4, 25.7,
25.1.
4.1.3.13. (E)-3-(2,4-Bis(benzyloxy)phenyl)-N-cyclohexylacrylamide (59). Yellowish white
solid; 79% yield; 1H NMR (500 MHz, CDCl3) δ 7.83 (d, 1 H, J = 16.0 Hz, 3-vinylic H), 7.43
– 7.31 (m, 11 H, 6-H, 2ⅹPh), 6.58 – 6.54 (m, 2 H, 3-H, 5-H), 6.43 (d, 1 H, J = 16.0 Hz, 2-
vinylic H), 5.43 (brs, 1 H, NH), 5.10 (s, 2 H, benzylic H), 5.03 (s, 2 H, benzylic H), 3.89 (brm,
1 H, 1′′-H), 1.96 (m, 2 H, 2′′-Ha, 6′′-Ha), 1.71 (m, 2 H, 2′′-Hb, 6′′-Hb), 1.62 (m, 1 H, 4′′-Ha),
13
1.40 (m, 2 H, 3′′-Ha, 5′′-Ha), 1.17 (m, 3 H, 3′′-Hb, 4′′-Hb, 5′′-Hb); C NMR (100 MHz,
CDCl3) δ 166.1, 161.2, 158.8, 136.7, 136.6, 136.3, 130.7, 128.9, 128.4, 128.2, 127.8, 127.5,
119.9, 117.8, 106.6, 100.9, 70.6, 70.4, 48.5, 33.4, 25.8, 25.1.

4.1.4. General procedure for the preparation of cinnamamide derivatives 1 – 3, 5 – 8, 10 – 13,
15 – 18 and 20.
Compounds 41 – 43, 45 – 48, 50 – 53, 55 – 58 and 60 (80-123 mg) were added in a
25 mL round-bottom flask along with concentrated HCl (1.0 mL) and acetic acid (1.0 mL) in
1:1 ratio and the reaction mixture was refluxed for 40 min. On completion of the reaction, the
mixture was neutralised by 2N NaOH and partitioned between dichloromethane and water.
The organic layer was dried with anhydrous MgSO4, filtered, and evaporated in vacuo. The
resulting residue was purified by silica gel column chromatography using dichloromethane
and methanol (10 – 30: 1) as the eluent to give cinnamamide derivatives 1 – 3, 5 – 8, 10 – 13,
15 – 18, and 20 as a white or grey solid in yields of 61 – 76%. Spectroscopic data of known
compounds can be found in SI.
4.1.4.1. (E)-3-(3-Hydroxy-4-methoxyphenyl)-1-(4-methylpiperazin-1-yl)prop-2-en-1-one (1).
White solid (hygroscopic); 68% yield; IR (ATR, cm-1): ν 3343, 2930, 1641, 1584; 1H NMR
(500 MHz, DMSO-d6) δ 9.02 (s, 1 H, OH), 7.32 (d, 1 H, J = 15.5 Hz, 3-H), 7.13 (d, 1 H, J =
2.0 Hz, 2′-H), 7.07 (dd, 1 H, J = 8.0, 2.0 Hz, 6′-H), 6.97 (d, 1 H, J = 15.5 Hz, 2-H), 6.90 (d, 1
H, J = 8.0 Hz, 5′-H), 3.78 (s, 3 H, OCH3), 3.57 (brm, 2 H), 3.52 (brm, 2 H), 2.27 (brm, 4 H,
13
3′′-H, 5′′-H), 2.17 (s, 3 H, NCH3); C NMR (100 MHz, DMSO-d6) δ 165.5, 150.0, 147.1,
143.1, 128.6, 121.4, 115.5, 115.1, 112.5, 56.3, 54.2, 53.7, 44.2; LRMAS (ESI-) m/z 275 (M-
H)-; HRMS (ESI+) m/z C15H21N2O3 (M+H)+ calcd 277.1547, obsd 277.1541, m/z
C15H20N2NaO3 (M+Na)+ calcd 299.1366, obsd 299.1356.
4.1.4.2. (E)-N-Cyclopentyl-3-(3-hydroxy-4-methoxyphenyl)acrylamide (11). Yellowish solid;
mp: 148.1 – 149.4 oC; 65% yield; IR (ATR, cm-1): ν 3347, 3094, 2942, 1651, 1585, 1534; 1H
NMR (500 MHz, CDCl3 + a few drops ofCD3OD) δ 7.45 (d, 1 H, J = 15.5 Hz, 3-H), 7.06 (d,
1 H, J = 1.5 Hz, 2′-H), 6.93 (dd, 1 H, J = 8.5, 1.5 Hz, 6′-H), 6.76 (d, 1 H, J = 8.5 Hz, 5′-H),