Antioxidant, anti-tyrosinase and anti-melanogenic effects of (E)-2,3-diphenylacrylic acid derivatives

Article abstract of DOI:10.1016/j.bmc.2019.04.020

During our continued search for strong skin whitening agents over the past ten years, we have investigated the efficacies of many tyrosinase inhibitors containing a common (E)-β-phenyl-α,β-unsaturated carbonyl scaffold, which we found to be essential for

Full text of DOI:10.1016/j.bmc.2019.04.020

Accepted Manuscript
Antioxidant, anti-tyrosinase and anti-melanogenic effects of (E)-2,3-dipheny-
lacrylic acid derivatives
Sultan Ullah, Yujin Park, Chaeun Park, Sanggwon Lee, Dongwan Kang, Jungho
Yang, Jinia Akter, Pusoon Chun, Hyung Ryong Moon
PII:
S0968-0896(19)30388-8
https://doi.org/10.1016/j.bmc.2019.04.020
BMC 14874
DOI:
Reference:
Bioorganic & Medicinal Chemistry
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7 March 2019
12 April 2019
13 April 2019
Please cite this article as: Ullah, S., Park, Y., Park, C., Lee, S., Kang, D., Yang, J., Akter, J., Chun, P., Moon, H.R.,
Antioxidant, anti-tyrosinase and anti-melanogenic effects of (E)-2,3-diphenylacrylic acid derivatives, Bioorganic
& Medicinal Chemistry (2019), doi: https://doi.org/10.1016/j.bmc.2019.04.020
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Antioxidant, anti-tyrosinase and anti-melanogenic effects of (E)-2,3-
diphenylacrylic acid derivatives
a,
§
a,
§
a
a
a
a
Sultan Ullah , Yujin Park , Chaeun Park , Sanggwon Lee , Dongwan Kang , Jungho Yang , Jinia
Aktera, Pusoon Chunb, Hyung Ryong Moona,*
aLaboratory of Medicinal Chemistry, College of Pharmacy, Pusan National University, Busan
46241, South Korea
bCollege of Pharmacy and Inje Institute of Pharmaceutical Sciences and Research, Inje University,
Gimhae, Gyeongnam 50834, South Korea
§These authors (U. Sultan, and Y. Park) 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; Tel.: +82 51
510 2815; Fax: +82 51 513 6754.

Abstract
During our continued search for strong skin whitening agents over the past ten years, we have
investigated the efficacies of many tyrosinase inhibitors containing a common (E)-β-phenyl-α,β-
unsaturated carbonyl scaffold, which we found to be essential for the effective inhibition of
mushroom and mammalian tyrosinases. In this study, we explored the tyrosinase inhibitory effects
of 2,3-diphenylacrylic acid (2,3-DPA) derivatives, which also possess the (E)-β-phenyl-α,β-
unsaturated carbonyl motif. We synthesized fourteen (E)-2,3-DPAderivatives 1a – 1n and one (Z)-
2,3-DPA-derivative 1l' using a Perkin reaction with phenylacetic acid and appropriate substituted
benzaldehydes. In our mushroom tyrosinase assay, 1c showed higher tyrosinase inhibitory activity
(76.43±3.53%, IC50 = 20.04±1.91 µM) with than the other 2,3-DPA derivatives or kojic acid
(21.56±2.93%, IC50 = 30.64±1.27 M). Our mushroom tyrosinase inhibitory results were
supported by our docking study, which showed compound 1c (-7.2 kcal/mole) exhibited stronger
binding affinity for mushroom tyrosinase than kojic acid (-5.7 kcal/mole). In B16F10 melanoma
cells (a murine cell-line), 1c showed no cytotoxic effect up to a concentration of 25 μM and
exhibited greater tyrosinase inhibitory activity (68.83%) than kojic acid (49.39%). In these cells,
arbutin (a well-known tyrosinase inhibitor used as the positive control) only inhibited tyrosinase
by 42.67% even at a concentration of 400 μM. Furthermore, at 25 µM, 1c reduced melanin contents
in B16F10 melanoma cells by 24.3% more than kojic acid (62.77% vs. 38.52%). These results
indicate 1c is a promising candidate treatment for pigmentation-related diseases and potential skin
whitening agents.
Key words: 2,3-diphenylacrylic acid, Perkin reaction, tyrosinase, inhibitor, B16F10 melanoma
cells, skin whitening.

1. Introduction
A white skin appearance is viewed as an attractive feature by today’s fashion industry. According
to recent surveys, almost fifteen percent of the world’s population purchase skin whitening
products1. Numbers of users continue to increase, and the world market for skin whitening
products has been predicted to reach $ 23 billion by 2020.2 Many substances influence skin color,
and melanin is the most important substance in skin that determines color. Melanin is a dark
pigment produced by melanocytes in skin by a process called melanogenesis.3,4 Melanocytes are
derived from melanoblasts and reside in the basal skin layer surrounded by keratinocytes5, and
during melanogenesis, melanocytes accumulate melanin in melanosomes, and subsequently, it is
transported to keratinocytes through dendrites.6,7 Melanin protects skin against various noxious
challenges like those posed by UV radiation, toxic chemicals, drugs, and environmental toxicants2.
However, irregular melanin production causes various skin problems such as age spots, melasma,
pigmented acne, lentigo, freckles, post-inflammatory melanoderma and scar-associated
pigmentation.8-11 In addition to melanogenesis disorders, melanin has also been associated with
neurodegenerative diseases such as Parkinson’s, Huntington’s, and Alzheimer’s diseases.12-16
Tyrosinase is the enzyme responsible for the conversion of tyrosine to dopaquinone, which is the
rate-limiting step during melanogenesis.6,7 Dopaquinone is used as the substrate for biosynthesis
of pheomelanin and eumelanin.1,17 Since tyrosinase plays an important role in the production of
melanin, many attempts have been made to develop skin lighting agents based on its inhibition.

2,3-Diphenylacrylic acid (2,3-DPA) derivatives have various biological activities, which include
antiviral18 and anticancer activities19. In addition, the anti-diabetic and anti-angiogenesis activities
of some 2,3-DPA derivatives have been attributed to inhibitions of protein tyrosine phosphatase
enzyme and vascular endothelial growth factor, respectively.20,21 Some 2,3-DPA derivatives have
also been used in sun creams22 and in dry powder inhaler compositions for the treatment of asthma,
chronic obstructive pulmonary disease, and other respiratory diseases.23 Over the past 10 years,
we have reported many tyrosinase inhibitors developed based on considerations of the chemical
structures of L-tyrosine and L-dopa, that is, the natural substrates of tyrosinase.24-29 During these
studies, we found that the (E)-β-phenyl-α,β-unsaturated carbonyl scaffold is essential for the
effective inhibition of mushroom and mammalian tyrosinases. Therefore, as part of our ongoing
efforts to identify new, potent skin whitening agents, we designed (E)-2,3-diphenylacrylic acid
((E)-2,3-DPA) derivatives containing the (E)-β-phenyl-α,β-unsaturated carbonyl scaffold and
synthesized fourteen (E)-2,3-DPA derivatives and one (Z)-2,3-DPA derivative. Based on
measured mushroom tyrosinase inhibitory activities, the most potent (E)-2,3-DPA analog was
assayed for cytotoxicity and cellular tyrosinase inhibitory activity and for the inhibition of
melanogenesis in B16F10 cells.
2. Results and Discussions
2.1. Chemistry
A schematic for the preparation of (E)-2,3-DPA derivatives is shown in Scheme 1. We considered
these derivatives could be produced using the Perkin reaction because this reaction between
benzaldehydes and phenylacetic acids is known to favour production of the (E)-stereoisomer.30,31
Perkin reactions between hydroxy- or methoxy-substituted benzaldehydes and phenylacetic acid
afforded O-acetylated (E)-2,3-DPA derivatives (2a – 2l) and (E)-2,3-DPA derivatives (1m and
1n), respectively. Interestingly, Perkin reaction between 3,5-dibromo-4-hydroxybenzaldehyde and

phenylacetic acid produced O-acetylated (Z)-2,3-DPA derivative 2l′ as well as O-acetylated (E)-
2,3-DPA derivative 2l in a 2:1 ratio (yields of 44% and 20%, respectively). To the best of our
knowledge, this is the first report of the synthesis of a (Z)-2,3-DPA derivative as the major product
of a Perkin reaction. Previously Hadfield et al. synthesized (E)-3-(4-hydroxy-3-methoxyphenyl)-
2-(3,4,5-trimethoxyphenyl)acrylic acid and confirmed its stereochemistry by comparing extinction
coefficients and using the NOESY technique.30 Thus, we confirmed the stereochemistries of
derivatives 2l and 2l′ by comparing the chemical shifts of vinylic protons (7.66 ppm for 2l and
6.89 ppm for 2l′) with the chemical shift of the vinylic proton (7.58 ppm) of the compound
synthesized by Hadfield and co-workers. Conversion of the O-acetylated 2,3-DPA derivatives (2a
– 2l and 2l′) into the desired 2,3-DPA derivatives (1a – 1l, and 1l′) was achieved using 1N-NaOH.
The vinylic protons of all (E)-2,3-DPA derivatives (1a – 1l) appeared in range of 7.53 – 8.15 ppm,
indicating an (E)-geometry, whereas the vinylic proton of 1l′ appeared at 6.70 ppm, which
indicated 2l′ was a (Z)-2,3-DPA derivative. The vinylic protons of compounds 1c and 1h, which
possess a 2-hydroxyl group produced peaks downfield (at 8.00 and 8.15 ppm, respectively) of
those of other (E)-2,3-DPA derivatives (7.53 – 7.90 ppm). This phenomenon has been previously
reported for benzylidene derivatives.25,32,33

Scheme 1. Synthetic scheme for (E)-2,3-DPA derivatives 1a – 1n and (Z)-2,3-DPA derivative
1l′. Reagents and conditions: a) acetic anhydride, Et3N, 90oC, 3 h, 20–73%; b) 1N-NaOH, 1,4-
dioxane, room temperature, overnight, 88 – 99%.

2.2. The mushroom tyrosinase inhibitory activities of (E)-2,3-DPA derivatives 1a – 1n
The mushroom tyrosinase inhibitory activities of (E)-2,3-DPA derivatives 1a – 1n and of the (Z)-
2,3-DPA derivative 1l′ were evaluated at a concentration of 25 µM. Kojic acid (a well-known
tyrosinase inhibitor) was used as a positive control at the same concentration. Tyrosinase inhibitory
activities are summarized in Table 1. Four (E)-2,3-DPA derivatives (1c, 1d, 1h, and 1k) potently
inhibited mushroom tyrosinase. Although all 2,3-DPA derivatives contained the β-phenyl-α,β-
unsaturated carbonyl motif, substitution patterns at the β-phenyl moiety greatly influenced the
tyrosinase inhibition. Compound 1a (12.78±3.86% inhibition) with a 4-hydroxyl group on the β-
phenyl ring exhibited weaker tyrosinase inhibition than kojic acid (21.56±2.93% inhibition), but
1c (76.43±3.53% inhibition, IC50 = 20.04±1.91 M) with an additional hydroxyl group at the 2-
position inhibited tyrosinase more than kojic acid (IC50 = 30.64±1.27 M). On the other hand,
compound 1b (10.84±2.65% inhibition) with an additional hydroxyl group at the 3 position of 1a
did not exhibit any increase in inhibitory activity. Although the introduction of an additional
hydroxyl group at the 3-position did not improve inhibitory activity, tyrosinase inhibition was
enhanced by inserting a methoxyl group at the 3 position of 1a (1d; 56.82±3.14% inhibition).
Notably, insertion of a methyl group at the 3 position of 1a did not increase inhibition (1g;
5.74±3.40% inhibition), and inserting an ethoxyl group at the 3 position (1e; 11.69±2.40%
inhibition) did not increase inhibition. The low inhibition of 1e was probably caused by an increase
in steric hindrance. Interestingly, compound 1f (10.55±1.59% inhibition), which had the same
substituents as 1d, but in the reverse positions had less inhibitory activity. Compound 1k
(43.86±2.63% inhibition) with an additional bromo group at the 3-position of 1a increased
inhibition as was observed for 1d, whereas compounds 1l and 1l′ with additional bromines at the
3 and 5 positions showed no increase in inhibition, regardless of their geometric dispositions. Like

1l and 1l′, compound 1j with additional methoxyl group at positions 3 and 5 of 1a did not increase
tyrosinase inhibitory activity. As was expected, compounds 1m and 1n, which did not possess a
hydroxyl group, showed almost no inhibitory activity. On the other hand, compound 1h
(48.87±2.40% inhibition) with a 2-hydroxyl group on its β-phenyl showed a high level of
tyrosinase inhibitory activity, whereas compound 1i (6.78±2.86% inhibition) with a 3-hydroxy
group on its β-phenyl showed only weak inhibitory activity. The above results indicate that the
(E)-β-phenyl-α,β-unsaturated carbonyl motif is important for tyrosinase inhibition, and that
substituents in appropriate positions on the β-phenyl ring also importantly influence the inhibition
of tyrosinase.

Table 1. Tyrosinase inhibitory activities of (E)-2,3-DPA derivatives 1a – 1n, the (Z)-2,3-DPA
derivative 1l′, and kojic acid
Compound
R1
H
R2
H
R3
OH
OH
OH
OH
OH
OMe
OH
H
R4
H
Tyrosinase inhibition (%)α
12.78±3.86
10.84±2.65
76.43±3.53
56.82±3.14
11.69±2.40
10.55±1.59
5.74±3.40
1a
H
OH
H
H
1b
OH
H
H
1c
OMe
OEt
OH
Me
H
H
1d
H
H
1e
H
H
1f
H
H
1g
OH
H
H
48.87±2.40
6.78±2.86
1h
OH
OMe
Br
H
H
1i
H
OH
OH
OH
OH
OMe
OMe
OMe
H
5.63±2.88
1j
H
43.86±2.63
7.56±1.88
1k
1l
H
Br
Br
Br
H
H
Br
13.20±2.80
5.23±1.55
1l′
H
H
1m
H
OMe
H
3.40±2.67
1n
Kojic acid
21.56±2.93
αTyrosinase inhibitory activities were determined at a concentration of 25 µM. L-Tyrosine was
used as a substrate. Results are presented as mean ± SEMs.

2.3. Binding affinities and interactions between compound 1c or kojic acid and tyrosinase.
AutoDock Vina 1.1.2, a docking software package published by the Scripps Research Institute was
used to determine the binding affinities and binding mode of compound 1c, which exhibited the
greatest inhibition of mushroom tyrosinase, was selected for the docking simulation. By using the
energy minimization tool in Chem3D Pro ver. 12.0 from CambrigeSoft Corporation, we created a
stable 3D structure of ligand 1c; the 3D structure of tyrosinase was obtained from Protein Data
Bank (ID: 2Y9X). As shown in Figure 1d, ligand 1c showed greater binding affinity for tyrosinase
(-7.2 kcal/mole) than kojic acid (-5.7 kcal/mole; the positive control). To investigate binding
interactions between tyrosinase and ligand 1c, we used LigandScout 4.2.1 software. As shown in
Figure 1b, the positive control kojic acid exhibited π-π stacking interaction with His263, and
formed two hydrogen bonds with His259 and His263. On the other hand, compound 1c formed
two hydrogen bonds with Asn260 and Met280, and exhibited hydrophobic interactions between
its two phenyl groups and the two amino acids of tyrosinase at Phe264 and Val283 (Figures 1a and
1c). Accordingly, docking simulation results suggested the inhibitory effect of 1c was due to strong
binding with the active site of tyrosinase.

Figure 1. Binding affinities and interactions between compound 1c or kojic acid and
tyrosinase. (a, and b) Pharmacophore results of compound 1c and kojic acid obtained using
LigandScount 4.3.0 showing possible hydrogen-bonding (green arrows), π-π stacking (violet
arrow), and hydrophobic interactions (yellow) between ligand 1c or kojic acid and tyrosinase
amino acids residues, (c) 3D pharmacophore model of ligand 1c and tyrosinase, and (d) binding
scores between ligand 1c or kojic acid and tyrosinase.
2.4. The cytotoxic effect of compound 1c on B16F10 melanoma cells
Of the 2,3-DPA derivatives synthesized, 1c was chosen for cell viability analysis because it had
the highest tyrosinase inhibitory activity in the mushroom tyrosinase assay. WST-8 assays were
performed using B16F10 murine melanoma cells to check the safety of 1c. B16F10 melanoma
cells were treated with compound 1c at concentrations of 0, 5, 10, or 25 µM in an incubator for 24
h. As shown in Figure 2, the safety profile of 1c was satisfactory; only a negligible reduction in

cell numbers was observed at 25 µM. Thus, 1c was deemed non-cytotoxic at concentrations below
25 µM.
Figure 2. The cytotoxic effect of compound 1c on B16F10 melanoma cells at concentrations of 5,
10, or 25 µM. Cytotoxic effects are expressed as percentages versus non-treated controls. Bars
represent standard errors.
2.5. The tyrosinase inhibitory effect of compound 1c in B16F10 melanoma cells
To evaluate the tyrosinase activity of 1c with 2,4-dihydroxyphenyl further, B10F16 melanoma
cells were stimulated with α-MSH and treated with 1c at concentrations of 0, 5, 10, or 25 μM, kojic
acid (25 μM) or arbutin (400 μM; the positive control). The cells were incubated in a 5% CO2
environment for 24 h. Results are summarized in Figure 3.

As shown in Figure 3, compound 1c more potently inhibited tyrosinase than kojic acid or arbutin.
At 25 µM, 1c and kojic acid inhibited tyrosinase activity by 68.83% and 49.39%, respectively, and
arbutin at even 400 μM only inhibited its activity by 42.67%. Furthermore, compound 1c inhibited
tyrosinase activity in a concentration-dependent manner. These results indicate that the (E)-β-
phenyl-α,β-unsaturated carbonyl motif potently inhibits both mushroom and mammalian
tyrosinase. Notably, these results also showed that the presence of hydroxyl groups at the 2 and 4
positions of the β-phenyl ring importantly contribute to the inhibition of tyrosinase.
Tyrosinase activity
100
***
***
***
***
50
0
***
ol
id
c
M
M
M
tin
u
5 µ
A
b
10 µ
25 µ
Contr
Ar
Kojic
1c
Concentration
Figure 3. The tyrosinase inhibitory effects of compound 1c, kojic acid (25 μM), and arbutin (400
μM) on α-MSH stimulated B16F10 melanoma cells. Asterisks *** indicate significant differences
between columns (p<0.001). Standard errors are represented by bars.
2.6. The effect of compound 1c on the melanin contents of B16F10 melanoma cells
The anti-melanogenic effect of compound 1c was investigated using the B16F10 melanoma cell

line. Initially, cells were activated with α-MSH and treated with compound 1c (0, 5, 10, or 25 μM)
or kojic acid (25 μM) for 24 h. Inhibitory effects on melanin production were quantified by
measuring optical densities.
Treatment with compound 1c at 25 µM significantly and more strongly reduced melanin levels
than kojic acid at the same concentration (by 62.77% and 38.52%, respectively), which suggested
1c had a greater inhibitory effect on tyrosinase (Figure 3). Furthermore, a comparison of Figures
3 and 4 revealed similar patterns of tyrosinase activity and melanin level inhibitions, which also
indicated the antimelanogenic effect of 1c was mainly due to tyrosinase inhibition.
Melanin content
100
**
***
***
50
***
0
l
d
i
M
M
M
o
r
µ
µ
µ
c
t
a
n
5
0
5
1
2
o
c
i
j
C
o
K
1c
Concentration
Figure 4. The effect of compound 1c on the melanin contents of B16F10 melanoma cells
stimulated by α-MSH. B16F10 cells were treated with compound 1c (5, 10, or 25 μM) or kojic
acid (25 μM). Asterisks indicate significant differences between columns: **, p<0.01; and ***,
p<0.001. Standard errors are represented as bars.

2.7. DPPH radical scavenging activities of (E)-2,3-DPA derivatives 1a – 1n, the (Z)-2,3-DPA
derivative 1l′, and of L-ascorbic acid
DPPH radical scavenging assays were performed to assess the radical scavenging effects of the
fifteen 2,3-DPA derivatives (1a – 1n and 1l′). 2,3-DPA derivatives and L-ascorbic acid were added
at a concentration 1 mM to a solution of 0.2mM DPPH in methanol, and 30 min later, radical
scavenging was assessed by measuring percentage absorbances. Results are summarized in Table
2.
According to our previously reported structure-activity data26,27, compounds with a 3,4-dihydroxyl
or 4-hydroxy-3-methoxyl substituent on the β-phenyl ring usually exhibit considerable DPPH
radical scavenging activity. The results obtained for compounds 1b (83.70±0.10 inhibition), which
has a 3,4-dihydroxyl substituent, 1d (81.56±0.23 inhibition), which has a 4-hydroxy-3-methoxyl
substituent, and L-ascorbic acid (84.64±0.32 inhibition; positive control) were similar. Compounds
1g (78.07±0.18 inhibition) and 1j (85.03±1.07 inhibition), which possess a 4-hydroxy-3-methyl
and a 4-hydroxy-3,5-dimethoxyl substituent, respectively, had almost the same potency as L-
ascorbic acid. However, the other 2,3-DPA derivatives exhibited only weak to moderate radical
scavenging activity. Furthermore, it has been well established that reactive oxygen species increase
tyrosinase expression, and thus, positively affect melanin production, which suggests the
antioxidant activities of 2,3-DPA derivatives may partially contribute to the reduction of melanin
levels.

Table 1. DPPH radical scavenging activities of (E)-2,3-DPA derivatives 1a – 1n, the (Z)-2,3-DPA
derivative 1l′, and of L-ascorbic acid.
Compound
DPPH Radical
Compound
DPPH Radical
scavenging activity (%)
scavenging activity (%)
1a
1b
1c
1d
1e
1f
1i
6.04±0.40
83.70±0.10
52.11±0.59
81.56±0.23
43.58±1.50
24.04±0.19
78.07±0.18
4.43±0.33
12.43±2.22
85.03±1.07
19.42±0.36
23.88±0.45
11.56±0.34
5.68±1.22
1j
1k
1l
1l′
1m
1n
1g
1h
10.16±1.15
84.64±0.32
L-ascorbic acid
The radical scavenging effects of the fifteen 2,3-DPA derivatives (1a – 1n and 1l′) and of L-
ascorbic acid were measured at a concentration of 1 mM. Three independent experiments were
performed and results are expressed as means ± standard deviations.

3. Conclusion
Fourteen (E)-2,3-DPA derivatives 1a – 1n and one (Z)-2,3-DPA derivative 1l′ containing the (E)-
β-phenyl-α,β-unsaturated carbonyl motif were synthesized using phenylacetic acid and substituted
benzaldehydes. This is the first report of the production of a (Z)-2,3-DPAderivative 1l′ as the major
product using the Perkin reaction. The tyrosinase inhibitory effects of these fifteen compounds
were evaluated using mushroom tyrosinase. The (E)-2,3-DPA derivative 1c most inhibited
tyrosinase activity (76.43±3.53% inhibition at 25 µM), and this inhibition also exceeded that of
kojic acid (21.56±2.93% inhibition at 25 µM). Our results show tyrosinase inhibition is dependent
on substituent type(s) and location(s) on the β-phenyl ring of the (E)-β-phenyl-α,β-unsaturated
carbonyl motif. Furthermore, our experimental mushroom tyrosinase inhibitory results were
supported by the docking study, according to which compound 1c (-7.2 kcal/mole) exhibited
stronger binding to tyrosinase than kojic acid (-5.7 kcal/mole). B16F10 melanoma cells were used
to investigate the cytotoxicity of derivative 1c, and its effects on cellular tyrosinase activity and
melanin contents. We found 1c was not cytotoxic to B16F10 melanoma at concentrations up to 25
μM, and at a concentration of 25 µM inhibited tyrosinase activity significantly more than kojic
acid (25 µM) and arbutin (400 μM). Furthermore, 1c at the same concentration reduced melanin
contents 24.3% more than kojic acid. These results indicate 1c is a promising candidate for treating
hyperpigmentation-related diseases.

4. Experimental Section
4.1. Chemistry
All reactions were monitored by thin layer chromatography (TLC), which was performed using
Merk 60 F254 glass silica gel plates. All intermediates and final products were purified by extraction,
recrystallization, and MP silica 40–63 (60 Å) column chromatography. Structures were elucidated
using a Varian Unity INOVA 1H NMR and a 13C NMR spectrophotometer (Agilent Technologies,
Santa Clara, CA, USA) using DMSO-d6, CDCl3 and CD3OD as solvents. All chemical shifts were
measured in ppm (parts per million) against residual solvents or deuterated peaks (for DMSO-d6
H 2.50 and C 39.7, and for CDCl3 H 7.24and C 77.0). Coupling constants are presented in Hertz.
The following abbreviations were used in the analysis of NMR data: s (singlet), brs (broad singlet),
d (doublet), dd (doublet of doublets), brd (broad doublet), t (triplet), q (quartet) and m (multiplet).
Mass spectrometers were used to determine the masses of the synthesized 2,3-DPA derivatives.
Low-resolution mass spectra were recorded on an Expression CMS from Advion Ithaca, NY, USA,
and high-resolution mass spectra were recorded using an Agilent Accurate-Mass Q-TOF liquid
chromatography/mass spectrometer from Agilent, Santa Clara, CA, USA.
4.1.1. General procedure for the syntheses of 2a – 2n, and 2l′, 1m and 1n
Substituted benzaldehydes (70 mg) and phenylacetic acid (1.0 equiv.) were added to a 25 mLround
bottom flask and then 1.5 mL of acetic anhydride and 0.7 mL of triethylamine were added
sequentially. This reaction mixture was then stirred at 900C for 3 h. On completion of the reaction,
conc. HCl was added at 00C and the pH was adjusted to 2. The reaction mixture was then
partitioned between ethyl acetate and water, and the organic layer was dried over anhydrous
MgSO4, filtered, and evaporated under reduced pressure. The residue was then purified either by

recrystallization from ethanol or column chromatography using hexane/ethyl acetate gradient
elution at 2–5:1 to give 2,3-DPA derivatives 2a – 2l, 2l′, 1m, and 1n as solids in yields of 20 –
73%.
4.1.1.1. (E)-3-(4-Acetoxyphenyl)-2-phenylacrylic acid (2a). Yellowish white solid, 44% yield.
1H NMR (400 MHz, CDCl3) δ 7.88 (s, 1 H, vinylic H), 7.37 – 7.34 (m, 3 H, 3-H, 4-H, 5-H), 7.22
(d, 2 H, J = 7.6 Hz, 2-H, 6-H), 7.04 (d, 2 H, J = 8.4 Hz, 2′-H, 6′-H), 6.87 (d, 2 H, J = 8.4 Hz, 3′-H,
5′-H), 2.23 (s, 3 H, Ac); 13C NMR (100 MHz, CDCl3) δ 172.7, 169.3, 151.5, 141.4, 135.3, 132.2,
132.1, 129.9, 129.0, 128.9, 128.3, 121.7, 21.3.
1
4.1.1.2. (E)-3-(3,4-Diacetoxyphenyl)-2-phenylacrylic acid (2b). Brown solid, 67% yield. H
NMR (500 MHz, CDCl3) δ 7.86 (s, 1 H, vinylic H), 7.41 – 7.37 (m, 3 H, 3-H, 4-H, 5-H), 7.24 (d,
2 H, J = 7.5 Hz, 2-H, 6-H), 6.99 (d, 1 H, J = 9.0 Hz, 6′-H), 6.93 (d, 1 H, J = 9.0 Hz, 5′-H), 6.87 (s,
1 H, 2′-H), 2.24 (s, 3 H, Ac), 2.21 (s, 3 H, Ac); 13C NMR (100 MHz, CDCl3) δ 172.7, 168.1, 143.0,
141.9, 140.5, 134.8, 133.2, 132.7, 129.8, 129.2, 129.1, 128.5, 126.0, 123.4, 20.8, 20.7.
1
4.1.1.3. (E)-3-(2,4-Diacetoxyphenyl)-2-phenylacrylic acid (2c). Brown solid, 73% yield. H
NMR (500 MHz, CDCl3) δ 7.90 (s, 1 H, vinylic H), 7.35 – 7.32 (m, 3 H, 3-H, 4-H, 5-H), 7.24 (d,
2 H, J = 7.5 Hz, 2-H, 6-H), 6.93 (d, 1 H, J = 1.5 Hz, 3′-H), 6.76 (d, 1 H, J = 8.5 Hz, 5′-H), 6.66 (d,
13
1 H, J = 8.5, 1.5 Hz, 6′-H), 2.37 (s, 3 H, Ac), 2.24 (s, 3 H, Ac); C NMR (100 MHz, CDCl3) δ
172.6, 169.0, 168.8, 151.6, 150.5, 135.2, 134.6, 133.7, 131.2, 130.1, 128.8, 128.4, 125.3, 118.9,
116.2, 21.3, 21.1.
4.1.1.4. (E)-3-(4-Acetoxy-3-methoxyphenyl)-2-phenylacrylic acid (2d). Yellowish white solid,
66% yield. 1H NMR (400 MHz, CDCl3) δ 7.86 (s, 1 H, vinylic H), 7.39 – 7.33 (m, 3 H, 3-H, 4-H,
5-H), 7.24 (d, 2 H, J = 7.6 Hz, 2-H, 6-H), 6.88 (d, 1 H, J = 8.4 Hz, 5′-H), 6.80 (d, 1 H, J = 8.4 Hz,

6′-H), 6.50 (s, 1 H, 2′-H), 3.32 (s, 3 H, OCH3), 2.24 (s, 3 H, Ac); 13C NMR (100 MHz, CDCl3) δ
172.8, 168.9, 150.7, 141.8, 140.9, 135.6, 133.1, 131.6, 130.0, 129.1, 128.3, 125.1, 122.8, 113.9,
55.4, 20.8.
4.1.1.5. (E)-3-(3-Acetoxy-4-ethoxyphenyl)-2-phenylacrylic acid (2e). White solid, 54% yield.
1H NMR (400 MHz, CDCl3) δ 7.85 (s, 1 H, vinylic H), 7.38 (t, 2 H, J = 7.6 Hz, 3-H, 5-H), 7.33 (t,
1 H, J = 7.6 Hz, 4-H), 7.24 (d, 2 H, J = 7.6 Hz, 2-H, 6-H), 6.86 (d, 1 H, J = 8.4 Hz, 5′-H), 6.77 (d,
1 H, J = 8.4, 1.6 Hz, 6′-H), 6.49 (d, 1 H, J = 1.6 Hz, 2′-H), 3.50 (q, 2 H, J = 7.2 Hz, CH2CH3), 2.23
(s, 3 H, Ac), 1.14 (t, 3 H, J = 7.2 Hz, CH2CH3); 13C NMR (100 MHz, CDCl3) δ 172.6, 168.9, 150.0,
141.8, 141.2, 135.6, 133.0, 131.5, 130.0, 129.1, 128.2, 124.9, 122.7, 114.9, 63.9, 20.7, 14.7.
4.1.1.6. (E)-3-(3-Acetoxy-4-methoxyphenyl)-2-phenylacrylic acid (2f). Yellowish white solid,
68% yield. 1H NMR (500 MHz, CDCl3) δ 7.84 (s, 1 H, vinylic H), 7.41 – 7.36 (m, 3 H, 3-H, 4-H,
5-H), 7.24 (d, 2 H, J = 7.5 Hz, 2-H, 6-H), 6.93 (d, 1 H, J = 8.5 Hz, 6′-H), 6.74 (d, 1 H, J = 8.5 Hz,
5′-H), 6.71 (s, 1 H, 2′-H), 3.78 (s, 3 H, OCH3), 2.23 (s, 3 H, Ac); 13C NMR (100 MHz, CDCl3) δ
172.9, 168.9, 152.4, 141.4, 139.4, 135.5, 130.4, 130.1, 129.9, 129.1, 128.2, 127.4, 125.6, 112.0,
56.1, 20.8.
4.1.1.7. (E)-3-(4-Acetoxy-3-methylphenyl)-2-phenylacrylic acid (2g). Yellowish White solid,
47% yield. 1H NMR (400 MHz, CDCl3) δ 7.85 (s, 1 H, vinylic H), 7.36 – 7.33 (m, 3 H, 3-H, 4-H,
5-H), 7.21 (d, 2 H, J = 7.6 Hz, 2-H, 6-H), 6.92 (d, 1 H, J = 2.0 Hz, 2′-H), 6.84 (dd, 1 H, J = 8.4,
2.0 Hz, 6′-H), 6.77 (d, 1 H, J = 8.4 Hz, 5′-H), 2.25 (s, 3 H, CH3), 1.98 (s, 3 H, Ac); 13C NMR (100
MHz, CDCl3) δ 172.9, 169.1, 150.3, 141.6, 135.4, 133.3, 132.3, 131.7, 130.3, 129.9, 129.7, 129.0,
128.3, 122.1, 21.0, 16.3.
1
4.1.1.8. (E)-3-(2-Acetoxyphenyl)-2-phenylacrylic acid (2h). White solid, 51% yield. H NMR

(400 MHz, CDCl3) δ 7.91 (s, 1 H, vinylic H), 7.30 – 7.27 (m, 3 H, 3-H, 4-H, 5-H), 7.23 – 7.19 (m,
3 H, 2-H, 6-H, 5′-H), 7.02 (d, 1 H, J = 8.0 Hz, 6′-H), 6.86 (t, 1 H, J = 8.0 Hz, 4′-H), 6.74 (d, 1 H,
13
J = 8.0 Hz, 3′-H), 2.35 (s, 3 H, Ac); C NMR (100 MHz, CDCl3) δ 172.3, 169.5, 150.0, 136.0,
134.8, 134.0, 130.8, 130.4, 130.2, 128.6, 128.3, 128.0, 125.7, 122.6, 21.1.
4.1.1.9. (E)-3-(3-Acetoxyphenyl)-2-phenylacrylic acid (2i). Off-white solid, 74% yield. 1H NMR
(500 MHz, CDCl3) δ 7.89 (s, 1 H, vinylic H), 7.39 – 7.31 (m, 3 H, 3-H, 4-H, 5-H), 7.24 (d, 2 H, J
= 7.5 Hz, 2-H, 6-H), 7.16 (t, 1 H, J = 7.5 Hz, 5′-H), 6.95 (d, 1 H, J = 7.5 Hz, 4′- or 6′-H), 6.90 (d,
1 H, J = 7.5 Hz, 4′- or 6′-H), 6.77 (s, 1 H, 2′-H), 2.21 (s, 3 H, Ac); 13C NMR (100 MHz, CDCl3) δ
172.4, 169.4, 150.6, 141.1, 136.1, 135.2, 133.1, 129.9, 129.4, 129.0, 128.3, 124.0, 122.8, 21.2.
4.1.1.10. (E)-3-(4-Acetoxy-3,5-dimethoxyphenyl)-2-phenylacrylic acid (2j). Yellowish grey
solid, 47% yield. 1H NMR (500 MHz, DMSO-d6) δ 12.66 (brs, 1 H, COOH), 7.73 (s, 1 H, vinylic
H), 7.42 (t, 2 H, J = 7.5 Hz, 3-H, 5-H), 7.35 (t, 1 H, J = 7.5 Hz, 4-H), 7.20 (d, 2 H, J = 7.5 Hz, 2-
H, 6-H), 6.44 (s, 2 H, 2′-H, 6′-H), 3.41 (s, 6 H, 2ⅹOCH3), 2.17 (s, 3 H, Ac); 13C NMR (100 MHz,
DMSO-d6) δ 168.8, 168.5, 151.8, 139.3, 137.2, 134.1, 133.0, 130.1, 129.3, 129.1, 128.2, 108.0.
56.1, 20.7.
4.1.1.11. (E)-3-(4-Acetoxy-3-bromophenyl)-2-phenylacrylic acid (2k). Yellowish white solid,
36% yield. 1H NMR (500 MHz, CDCl3) δ 7.83 (s, 1 H, vinylic H), 7.42 – 7.37 (m, 3 H, 3-H, 4-H,
5-H), 7.30 (s, 1 H, 2′-H), 7.22 (d, 2 H, J = 7.5 Hz, 2-H, 6-H), 6.98 (d, 1 H, J = 8.0 Hz, 6′-H), 6.91
(d, 1 H, J = 8.0 Hz, 5′-H), 2.31 (s, 3 H, Ac); 13C NMR (100 MHz, CDCl3) δ 172.4, 168.5, 148.9,
139.7, 135.7, 134.7, 134.0, 133.3, 131.1, 129.7, 129.1, 128.6, 123.6, 116.3, 21.0.
4.1.1.12. (E)-3-(4-Acetoxy-3,5-dibromophenyl)-2-phenylacrylic acid (2l). Yellow solid, 20%
yield. 1H NMR (500 MHz, CDCl3) δ 7.66 (s, 1 H, vinylic H), 7.41 – 7.37 (m, 3 H, 3-H, 4-H, 5-H),

7.18 (brd, 2 H, J = 7.5 Hz, 2-H, 6-H), 7.16 (s, 2 H, 2′-H, 6′-H), 2.32 (s, 3 H, CH3); 13C NMR (100
MHz, CDCl3 + 1 drop of CD3OD) δ 169.1, 167.3, 146.3, 136.6, 135.6, 135.4, 134.9, 134.2, 129.5,
129.0, 128.5, 115.3, 20.5.
4.1.1.13. (Z)-3-(4-Acetoxy-3,5-dibromophenyl)-2-phenylacrylic acid (2l′). Brown solid, 44%
yield. 1H NMR (500 MHz, CDCl3) δ 7.65 (s, 2 H, 2′-H, 6′-H), 7.48 (d, 2 H, J = 7.0 Hz, 2-H, 6-H),
7.40 – 7.36 (m, 3 H, 3-H, 4-H, 5-H), 6.87 (s, 1 H, vinylic H), 2.39 (s, 3 H, CH3); 13C NMR (100
MHz, CDCl3) δ 171.4, 167.2, 146.1, 137.2, 136.3, 132.3, 129.1, 129.0, 127.0, 119.6, 117.9, 115.3,
20.6.
4.1.1.14. (E)-3-(4-Methoxyphenyl)-2-phenylacrylic acid (1m). Brownish yellow solid, 21%
yield. 1H NMR (500 MHz, CDCl3) δ 7.90 (s, 1 H, vinylic H), 7.41 (t, 2 H, J = 7.5 Hz, 3-H, 5-H),
7.37 (t, 1 H, J = 7.5 Hz, 4-H), 7.25 (d, 2 H, J = 7.5 Hz, 2-H, 6-H), 7.01 (d, 2 H, J = 9.0 Hz, 2′-H,
6′-H), 6.69 (d, 2 H, J = 9.0 Hz, 3′-H, 5′-H), 3.76 (s, 3 H, OCH3); 13C NMR (100 MHz, CDCl3) δ
173.1, 160.8, 142.3, 136.0, 132.9, 130.0, 129.2, 129.0, 128.1, 127.1, 114.0, 55.4; LRMS (ESI-)
m/z 253 (M-H)-.
4.1.1.15. (E)-3-(3,4-Dimethoxyphenyl)-2-phenylacrylic acid (1n). Yellowish grey, 31% yield.
1H NMR (400 MHz, CDCl3 + 1 drop of CD3OD) δ 7.76 (s, 1 H, vinylic H), 7.35 (d, 2 H, J = 7.6
Hz, 3-H, 5-H), 7.27 (t, 1 H, J = 7.6 Hz, 4-H), 7.21 (d, 2 H, J = 7.6 Hz, 2-H, 6-H), 6.78 (dd, 1 H, J
= 8.4, 2.0 Hz, 6′-H), 6.66 (d, 1 H, J = 8.4 Hz, 5′-H), 6.33 (d, 1 H, J = 2.0 Hz, 2′-H), 3.77 (s, 3 H,
CH3), 3.30 (s, 3 H, CH3); 13C NMR (100 MHz, CDCl3 + 1 drop of CD3OD) δ 170.6, 150.2, 148.3,
141.2, 136.8, 130.1, 129.0, 127.9, 127.5, 125.9, 112.4, 110.6, 55.9, 55.2; LRMS (ESI-) m/z 283
(M-H)-.
4.1.2. General procedure for the syntheses of 2,3-DPA derivatives 1a – 1l and 1l′

Compounds 2a – 2l, and 2l′ (22.1 mg to 112.1 mg) were added to a 25 mL round bottom flask and
then 1,4-dioxane (1 – 3 mL) and aqueous 1N-NaOH (4.0 equiv.) were sequentially added. The
reaction was allowed to proceed at room temperature overnight with stirring. After completion of
the reaction, aqueous 2N-HCl was added to the flask to adjust the pH to 2. The reaction mixture
was then stirred at room temperature for 30 min and solid material was filtered. The filter cake so
obtained was then washed with a large amount of water and dried to give 2,3-DPA derivatives 1a
– 1l and 2l′ in yields of 88 – 99%.
4.1.2.1. (E)-3-(4-Hydroxyphenyl)-2-phenylacrylic acid (1a). Yellowish white solid, 95% yield.
1H NMR (500 MHz, CDCl3 + 1 drop of CD3OD) δ 7.76 (s, 1 H, vinylic H), 7.33 (t, 2 H, J = 7.5
Hz, 3-H, 5-H), 7.29 (t, 1 H, J = 7.5 Hz, 4-H), 7.18 (d, 2 H, J = 7.5 Hz, 2-H, 6-H), 6.85 (d, 2 H, J =
8.5 Hz, 2′-H, 6′-H), 6.54 (d, 2 H, J = 8.5 Hz, 3′-H, 5′-H); 13C NMR (100 MHz, CDCl3 + 1 drop of
CD3OD) δ 170.8, 158.2, 141.4, 136.7, 132.9, 129.9, 129.3, 128.9, 127.8, 126.4, 115.4; LRMS
(ESI-) m/z 239 (M-H)-.
4.1.2.2. (E)-3-(3,4-Dihydroxyphenyl)-2-phenylacrylic acid (1b). Brownish grey solid, 96%
yield. 1H NMR (400 MHz, DMSO-d6) δ 12.34 (brs, 1 H, COOH), 9.43 (s, 1 H, OH), 8.78 (s, 1 H,
OH), 7.53 (s, 1 H, vinylic H), 7.36 – 7.27 (m, 3 H, 3-H, 4-H, 5-H), 7.09 (d, 2 H, J = 7.6 Hz, 2-H,
6-H), 6.48 (d, 1 H, J = 8.0 Hz, 5′- or 6′-H), 6.38 (s, 1 H, 2′-H), 6.34 (d, 1 H, J = 8.0 Hz, 5′- or 6′-
13
H); C NMR (100 MHz, DMSO-d6) δ 169.4, 147.7, 145.4, 140.3, 137.6, 130.2, 130.1, 129.1,
128.0, 126.3, 123.8, 118.4, 115.8; LRMS (ESI-) m/z 255 (M-H)-, 211 (M-H-CO2)-.
4.1.2.3. (E)-3-(2,4-Dihydroxyphenyl)-2-phenylacrylic acid (1c). Yellowish brown solid, 98%
yield. 1H NMR (500 MHz, DMSO-d6) δ 12.19 (brs, 1 H, COOH), 9.91 (s, 1 H, OH), 9.62 (s, 1 H,
OH), 8.00 (s, 1 H, vinylic H), 7.34 (t, 2 H, J = 7.5 Hz, 3-H, 5-H), 7.28 (t, 1 H, J = 7.5 Hz, 4-H),

7.12 (d, 2 H, J = 7.5 Hz, 2-H, 6-H), 6.29 (s, 1 H, 3′-H), 6.26 (d, 1 H, J = 8.5 Hz, 6′-H), 5.80 (d, 1
H, J = 8.5 Hz, 5′-H); 13C NMR (100 MHz, DMSO-d6) δ 169.6, 160.5, 159.2, 138.2, 134.8, 131.1,
130.4, 129.0, 128.2, 127.7, 113.3, 107.4, 102.8; LRMS m/z 211 (M-H-CO2)-; HRMS (ESI+) m/z
C15H13O4 (M+H)+ calcd 257.0808, obsd 257.0801, m/z C15H12NaO4 (M+Na)+ calcd 279.0628,
obsd 279.0620.
4.1.2.4. (E)-3-(4-Hydroxy-3-methoxyphenyl)-2-phenylacrylic acid (1d). Yellow solid, 97%
yield. 1H NMR (500 MHz, DMSO-d6) δ 9.47 (s, 1 H, OH), 7.64 (s, 1 H, vinylic H), 7.40 (t, 2 H, J
= 7.5 Hz, 3-H, 5-H), 7.33 (t, 1 H, J = 7.5 Hz, 4-H), 7.17 (d, 2 H, J = 8.0 Hz, 2-H, 6-H), 6.66 (dd,
1 H, J = 8.0, 2.0 Hz, 6′-H), 6.61 (d, 1 H, J = 8.0 Hz, 5′-H), 6.40 (d, 1 H, J = 2.0 Hz, 2′-H), 3.69 (s,
3 H, OCH3); 13C NMR (100 MHz, DMSO-d6) δ 169.2, 148.7, 147.5, 140.2, 137.8, 130.3, 129.3,
128.0, 126.2, 126.0, 115.8, 113.7, 55.3; LRMS (ESI-) m/z 269 (M-H)-.
4.1.2.5. (E)-3-(3-Ethoxy-4-hydroxyphenyl)-2-phenylacrylic acid (1e). Yellow solid, 88% yield.
1H NMR (500 MHz, CDCl3 + 1 drop of CD3OD ) δ 7.75 (s, 1 H, vinylic 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), 7.22 (d, 2 H, J = 7.5 Hz, 2-H, 6-H), 6.74 (dd, 1 H,
J = 8.0, 1.5 Hz, 6′-H ), 6.70 (d, 1 H, J = 8.0 Hz, 5′-H), 6.32 (d, 1 H, J = 1.5 Hz, 2′-H), 3.52 (q, 2
H, J = 7.5 Hz, CH2CH3), 1.17 (t, 3 H, J = 7.5 Hz, CH2CH3); 13C NMR (100 MHz, CDCl3 + 1 drop
of CD3OD) δ 170.6, 147.4, 145.5, 141.5, 136.9, 130.0, 129.5, 129.0, 127.7, 126.6, 126.4, 114.4,
113.2, 63.9, 14.7; LRMS (ESI-) m/z 283 (M-H)-.
4.1.2.6. (E)-3-(3-Hydroxy-4-methoxyphenyl)-2-phenylacrylic acid (1f). Yellowish white solid,
99% yield. 1H NMR (500 MHz, DMSO-d6) δ 8.87 (s, 1 H, OH), 7.59 (s, 1 H, vinylic H), 7.37 (t,
2 H, J = 7.5 Hz, 3-H, 5-H), 7.33 (t, 1 H, J = 7.5 Hz, 4-H), 7.13 (d, 2 H, J = 7.5 Hz, 2-H, 6-H), 6.72
(d, 1 H, J = 8.5 Hz, 5′-H), 6.50 (dd, 1 H, J = 8.5, 2.0 Hz, 6′-H), 6.42 (d, 1 H, J = 2.0 Hz, 2′-H),
3.69 (s, 3 H, OCH3); 13C NMR (100 MHz, DMSO-d6) δ 169.2, 149.4, 146.5, 139.9, 137.4, 131.1,

130.1, 129.2, 128.1, 127.7, 123.4, 117.9, 112.1, 56.1; LRMS (ESI-) m/z 269 (M-H)-.
4.1.2.7. (E)-3-(4-Hydroxy-3-methylphenyl)-2-phenylacrylic acid (1g). Yellowish white solid,
99% yield. 1H NMR (500 MHz, CDCl3 + 1 drop of CD3OD) δ 7.72 (s, 1 H, vinylic H), 7.31 (t, 2
H, J = 7.5 Hz, 3-H, 5-H), 7.29 (t, 1 H, J = 7.5 Hz, 4-H), 7.16 (d, 2 H, J = 7.5 Hz, 2-H, 6-H), 6.73
(d, 1 H, J = 2.0 Hz, 2′-H), 6.64 (dd, 1 H, J = 8.5, 2.0 Hz, 6′-H), 6.45 (d, 1 H, J = 8.5 Hz, 5′-H),
1.96 (s, 3 H, CH3); 13C NMR (100 MHz, CDCl3 + 1 drop of CD3OD) δ 170.8, 156.5, 141.6, 136.9,
134.3, 130.1, 129.9, 129.0, 128.8, 127.6, 126.2, 124.5, 114.6, 15.9; LRMS (ESI-) m/z 253 (M-H)-.
4.1.2.8. (E)-3-(2-Hydroxyphenyl)-2-phenylacrylic acid (1h). White solid, 89% yield. 1H NMR
(400 MHz, CDCl3 + 1 drop of CD3OD) δ 8.15 (s, 1 H, vinylic H), 7.27 – 7.22 (m, 3 H, 3-H, 4-H,
5-H), 7.18 (d, 2 H, J = 7.6 Hz, 2-H, 6-H,), 6.98 (t, 1 H, J = 8.0 Hz, 5′-H), 6.71 (d, 1 H, J = 8.0 Hz,
6′-H), 6.55 (d, 1 H, J = 8.0 Hz, 2′-H), 6.41 (t, 1 H, J = 8.0 Hz, 3′-H); 13C NMR (100 MHz, CDCl3
+ 1 drop of CD3OD) δ 170.7, 156.3, 136.4, 136.2, 131.8, 130.6, 130.5, 130.2, 128.5, 127.8, 122.1,
119.4, 115.8; LRMS (ESI-) m/z 239 (M-H)-.
1
4.1.2.9. (E)-3-(3-Hydroxyphenyl)-2-phenylacrylic acid (1i). Off-white solid, 99% yield. H
NMR (500 MHz, CDCl3 + 1 drop of CD3OD) δ 7.78 (s, 1 H, vinylic H), 7.34 (t, 2 H, J = 7.5 Hz,
3-H, 5-H), 7.31 (t, 1 H, J = 7.5 Hz, 4-H), 7.21 (d, 2 H, J = 7.5 Hz, 2-H, 6-H), 6.97 (t, 1 H, J = 8.0
Hz, 5′-H), 6.66 (d, 1 H, J = 8.0 Hz, 6′-H), 6.54 (d, 1 H, J = 8.0 Hz, 4′-H), 6.47 (s, 1 H, 2′-H); 13C
NMR (100 MHz CDCl3 + 1 drop of CD3OD) δ 170.6, 156.4, 141.3, 136.3, 136.0, 132.4, 130.0,
129.4, 128.7, 127.8, 122.8, 117.3, 116.7; LRMS (ESI-) m/z 239 (M-H)-.
4.1.2.10. (E)-3-(4-Hydroxy-3,5-dimethoxyphenyl)-2-phenylacrylic acid (1j). Yellowish grey
solid, 96% yield. 1H NMR (500 MHz, CDCl3 + 1 drop of CD3OD) δ 7.75 (s, 1 H, vinylic H), 7.39
(t, 2 H, J = 7.5 Hz, 3-H. 5-H), 7.30 (t, 1 H, J = 7.5 Hz, 4-H), 7.24 (d, 2 H, J = 7.5 Hz, 2-H, 6-H),

6.29 (s, 2 H, 2′-H, 6′-H), 3.53 (s, 6 H, 2ⅹOCH3); 13C NMR (100 MHz, CDCl3 +1 drop of CD3OD)
δ 174.8, 146.8, 141.6, 136.8, 136.4, 130.1, 129.0, 127.9, 125.7, 108.3, 56.0; LRMS (ESI-) m/z 299
(M-H)-.
4.1.2.11. (E)-3-(3-Bromo-4-hydroxyphenyl)-2-phenylacrylic acid (1k). Yellowish white solid,
96% yield. 1H NMR (500 MHz, CDCl3 + 1 drop of CD3OD) δ 7.69 (s, 1 H, vinylic H), 7.35 (t, 2
H, J = 7.5 Hz, 3-H, 5-H), 7.32 (t, 1 H, J = 7.5 Hz, 4-H), 7.17 (d, 2 H, J = 7.5 Hz, 2-H, 6-H), 7.11
(d, 1 H, J = 2.0 Hz, 2′-H), 6.78 (dd, 1 H, J = 8.5, 2.0 Hz, 6′-H), 6.63 (d, 1 H, J = 8.5 Hz, 5′-H); 13C
NMR (100 MHz, CDCl3 + 1 drop of CD3OD) δ 170.3, 154.4, 139.6, 136.1, 135.5, 131.4, 131.1,
129.8, 128.9, 128.1, 128.0, 115.9, 109.9; LRMS (ESI-) m/z 317 (M-H)-, 319 (M+2-H)-.
4.1.2.12. (E)-3-(3,5-Dibromo-4-hydroxyphenyl)-2-phenylacrylic acid (1l). Yellow solid, 95%
yield. 1H NMR (500 MHz, DMSO-d6) δ 12.66 (brs, 1 H, COOH), 10.29 (brs, 1 H, OH), 7.61 (s, 1
H, vinylic H), 7.42 – 7.37 (m, 3 H, 3-H, 4-H, 5-H), 7.17 – 7.13 (m, 4 H, 2-H, 6-H, 2′-H, 6′-H); 13C
NMR (100 MHz, DMSO-d6) δ 168.7, 151.9, 136.9, 136.6, 134.8, 133.6, 129.9, 129.4, 129.4, 128.5,
112.0; LRMS (ESI-) m/z 395 (M-H)-, 397 (M+2-H)-, 399 (M+4-H)-.
4.1.2.13. (Z)-3-(3,5-dibromo-4-hydroxyphenyl)-2-phenylacrylic acid (1l′). Yellowish white
solid, 97% yield. 1H NMR (500 MHz, CDCl3 + 1 drop of CD3OD) δ 7.54 (s, 2 H, 2′-H, 6′-H), 7.41
(d, 2 H, J = 7.5 Hz, 2-H, 6-H), 7.31 (t, 1 H, J = 7.5 Hz, 3-H, 5-H), 7.27 (t, 1 H, J = 7.5 Hz, 4-H),
13
7.26 (t, 1 H, J = 7.5 Hz, 5-H), 6.70 (s, 1 H, vinylic H); C NMR (100 MHz, CDCl3 + 1 drop of
CD3OD) δ 171.4, 150.3, 136.7 136.1, 134.4, 132.1, 128.7, 128.5, 127.1, 126.5, 110.7; LRMS (ESI-)
m/z 395 (M-H)-, 397 (M+2-H)-, 399 (M+4-H)-.
4.2. Docking study of 2,3-DPA derivative 1c and kojic acid
The docking study procedure used was performed as previously described.34 Chem3D Pro 12.0

software was used to create a 3D structure for 1c. AutoDock Vina and chimera software were used
to calculate binding scores between 1c or kojic acid and tyrosinase. The three-dimensional
structure of tyrosinase (PDB ID: 2Y9X, Agaricus Bisporus) was obtained from the Protein Data
Bank, and LigandScout software ver. 4.3.0 was used to generate a pharmacophore model showing
possible interactions between 1c and amino acid residues of tyrosinase.
4.3. Biological Investigation
4.3.1. Mushroom tyrosinase inhibition assay of the fifteen 2,3-DPA derivatives
Mushroom tyrosinase inhibition was assayed as previously described.35 Ten µL of 2,3-DPA
derivatives (1a – 1n and 1l′, 25 µM) and 20 µL of tyrosinase solution (1000 units per mL) were
added to 170 µL solution of substrate solution (14.7 mM of phosphate buffer and 293 µL of L-
tyrosine solution, 1:1, v/v), mixed, and added to 96-well plates, which were then incubated for 30
minutes at 370C. Tyrosinase inhibition percentages were calculated from optical densities
measured at 450 nm using a VersaMax shaking microplate reader. Kojic acid (25 µM) was used as
the control. Three independent experiments were performed. The formula used to calculate %
inhibition was;
%Inhibition = [1-(A/B)ⅹ100]
where A is the absorbance of a 2,3-DPA derivative and B is the absorbance of the non-treated
control.
4.3.2. Cell culture
B16F10 cells (a murine melanoma cell-line) were obtained from the American Type Culture

Collection (ATCC, VS, USA). Cells were cultured in DMEM (Dulbecco’s medium) containing 10%
fetal bovine serum (FBS), 100 µg/mL streptomycin, and 100 IU/ml penicillin in a humidified 5%
CO2 atmosphere at 370C. These cells were subsequently used for cell viability, tyrosinase inhibition,
and melanin content assays.
4.3.3. Determination of the cell viabilities of B16F10 melanoma cells treated with compound 1c
A previously described WST-8 assay was used to assess cell viabilities.36 Cells were incubated in
a 96-well plate at 5x104 cells per well in a humidified 5% CO2 atmosphere for 24 h at 370C.
Compound 1c was added to the cells at concentrations of 0, 5, 10, or 25 µM and cells were re-
incubated for 24 h under the same conditions. Cells were then treated with WST-8 reagents and
incubated for 1 h at 370C. The cell viabilities were determined using the EZ-Cytox assay (EZ-
3000, Daeil Lab Service, Seoul) by reading optical densities at 450 nm. Experiments were repeated
three times.
4.3.4. Tyrosinase inhibition assay in B16F10 melanoma cells
The tyrosinase inhibition assay was performed as previously described with slight changes.37
Briefly, B16F10 cells were inoculated at 5x104 cells per well into 96-well plates and incubated in
a humidified 5% CO2 atmosphere for 24 h at 370C. Cells were then treated with α-MSH (1 µM)
and kojic acid (25 µM) or compound 1c (0, 5, 10, or 25 µM) and re-incubated for 24 h under the
same conditions. Cells were then washed with PBS buffer, lysed with lysis buffer (100 µL
containing 50 mM PBS, 0.1 mM PMSF (5 µL), and Triton X-100 (5 µL)), and frozen at -80 0C for
30 min. Cell lysates were then centrifuged at 12,000 rpm for 30 min at 4 0C, transferred to 96-well
plates in a total volume of 100 µL (80 µL of lysate supernatant and 20 µL of 10 mM L-dopa), and
incubated for 30 min at 370C. Tyrosinase inhibition was quantified by measuring optical densities

at 500 nm using a Tecan, Mannedorf microplate reader. The experiment was performed in
triplicate.
4.3.5. Anti-melanogenic assays performed using B16F10 cells
The anti-melanogenic effect of compound 1c was determined as previously described melanin
assay with minor changes.38 In brief, B16F10 cells were inoculated at 5x104 cells per well in 96-
well plates and incubated in a humidified 5% CO2 atmosphere for 24 h at 370C. Cells were then
treated with α-MSH (1 µM) and kojic acid (25 µM) or compound 1c (0, 5, 10, or 25 µM) and re-
cultured for 24 h under the same conditions. Cells were then cleaned with PBS buffer and melanin
0
contents were dissolved by incubating cells with 1 N NaOH solution (200 µL) at 50 C for 1 h.
Melanin contents were determined by measuring optical densities at 405 nm in 96-well plates using
a Tecan, Mannedorf microplate reader. The experiment was performed in triplicate.
4.3.6. Procedure for anti-oxidant activity determination
A slightly modified version of a previously described DPPH radical scavenging assay was used to
assess anti-oxidant activities.39 Briefly, 20 µL of a solution (in DMSO) of each 2,3-DPA derivative
was mixed with 180 µL of DPPH methanol solution (0.2 mM) in 96-well plates. L-Ascorbic acid
was used as the positive control. Mixture were then incubated for 30 min in the dark, and
antioxidant activities were determined by measuring absorbances at 517 nm using a VersaMax
microplate reader. Experiments were performed in triplicate. The formula used to determine DPPH
radical scavenging activity was;
Radical scavenging activity (%) = [(Ac-As)ⅹ100/Ac]
where Ac is absorbance of the non-treated control and As is the absorbance of a sample containing