Dimeric cinnamoylamide analogues for regulation of tyrosinase activity in melanoma cells: A role of diamide-link chain length

Article abstract of DOI:10.1016/j.bmc.2018.10.036

Dimeric cynnamoyl analogues (DCAs) with depigmenting activity have been developed. In this study, a role of diamide linkage chain length of DCAs as a tyrosinase inhibitor was investigated on tyrosinase inhibitory activity, antioxidative activity, hydropho

Full text of DOI:10.1016/j.bmc.2018.10.036

Bioorganic&MedicinalChemistryxxx(xxxx)xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Dimeric cinnamoylamide analogues for regulation of tyrosinase activity in
melanoma cells: A role of diamide-link chain length
Ji hoon Ha, Soo Nam Park⁎
Department of Fine Chemistry, Cosmetic R&D Center, Seoul National University of Science and Technology, 232 Gongneungro, Nowon-gu, Seoul 01811, Republic of Korea
A R T I C L E I N F O
A B S T R A C T
Keywords:
Dimeric cynnamoyl analogues (DCAs) with depigmenting activity have been developed. In this study, a role of
diamide linkage chain length of DCAs as a tyrosinase inhibitor was investigated on tyrosinase inhibitory activity,
antioxidative activity, hydrophobicity and anti-melanogenesis as well as structural characteristics and dipole
moments based on density functional theory. DCAs with different diamide-link chain lengths (n = 2, 3, and 4)
and various functional groups (m-coumaroyl, p-coumaroyl, isoferuloyl and feruloyl groups) were synthesized.
DCAs with a diamide-link chain length of three indicated high inhibitory effect of melanin production on α-
melanocyte stimulating hormone (α-MSH) stimulated B16F1 cells. Approach of p-hydroxyl group of DCAs to
active site of tyrosinase, an important melanogenic enzyme, is interfered by addition of m-methoxy group. In
structural modeling based on density functional theory, DCAs with a diamide-link chain length of three showed
folded shapes, and they had lower dipole moment than with a diamide-link chain length of two or four. Thus, for
the enhancement of the depigmenting activities of dimeric compounds, the diamide-link chain length is im-
portant. Our results provide an important index for the design of dimeric compounds with physiological ac-
tivities.
Dimeric compound
Cinnamoyl analogues
Depigementing activity
Diamide-link chain length
Density functional theory
1. Introduction
formation and pigmentation. In particular, the increase of reactive
oxygen species (ROS) stimulates the keratinocytes of the epidermis to
The skin color of a person depends on the distribution of blood
vessels, thickness of stratum corneum, and various components such as
melanin, carotene, and hemoglobin.1,2 Melanin, which is responsible
for the dark color of the skin, is a lipophilic natural polymer. Melanin is
produced by a defense mechanism to protect the skin against ultraviolet
radiation. However, excessive production and accumulation of melanin
causes aesthetic problems, such as freckles and melasma, by hy-
perpigmentation in melanocytes.3–6
promote melanin production in melanocytes through the secretion of
compounds such as NO and α-melanocyte stimulating hormone (α-
MSH).11 In addition, free iron released after cell damage produces ROS
such as the hydroxyl radical (%OH) through the Fenton reaction with
hydrogen peroxide, which promotes oxidation of the melanin precursor
and melanin production.12 Therefore, antioxidative activity is very
important for inhibiting melanogenesis as well as the inhibitory activity
of tyrosinase.
Melanin is biosynthesized in melanocytes distributed in the basal
layer of the skin epidermis, then transferred to the keratinocytes.6–8
Copper-containing tyrosinase is a major enzyme involved in melanin
synthesis. Tyrosinase is an important rate-controlling step in melanin
biosynthesis through the catalysis of two distinct reactions: hydro-
xylation of L-tyrosinase to L-DOPA, and the subsequent oxidation of L-
DOPA to DOPA quinone.9,10
L-Ascorbic acid, p-coumaric acid, and ferulic acid are known to in-
hibit the activity of tyrosinase through antioxidative activity and
blocking the active sites of tyrosinase.13–15,16
melanin production by supplying hydrogen toLo-AxisdciozrebdicDaOcPidAdqeucirneoansees,
resulting in the restoration of DOPA quinone to DOPA.17
Strong inhibitors of tyrosinase such as hydroquinone, kojic acid, and
arbutin, act as competitive inhibitors that bind to the active site of
tyrosinase owing to their similar structure to tyrosine.18,19 However,
rhododendrol, known as a potential tyrosinase inhibitor, causes an
Ultraviolet rays damage the skin both directly and indirectly, re-
sulting in the acceleration of skin aging characteristics such as wrinkle
Abbreviations: DCAs, dimeric cinnamoylamide analogues; α-MSH, α-melanocyte stimulating hormone; ROS, reactive oxygen species; DFP, N,N′-diferuloyl-pu-
trescine; DPPH radical, 1,1-diphenyl-2 picrylhydrazyl radical
⁎ Corresponding author: Department of Fine Chemistry, Cosmetic R&D Center, Cosmetic Industry Coupled Collaboration Center, Seoul National University of
Science and Technology, 232 Gongneung-ro, Nowon-gu, Seoul 01811, Republic of Korea.
E-mail address: snpark@seoultech.ac.kr (S.N. Park).
https://doi.org/10.1016/j.bmc.2018.10.036
Received 22 August 2018; Received in revised form 20 October 2018; Accepted 29 October 2018
0968-0896/©2018ElsevierLtd.Allrightsreserved.
Pleasecitethisarticleas:Ha,J.h.,Bioorganic&MedicinalChemistry,https://doi.org/10.1016/j.bmc.2018.10.036

J.h. Ha, S.N. Park
Bioorganic&MedicinalChemistryxxx(xxxx)xxx–xxx
acute problem on the skin.20,21 Therefore, development of a safe
whitening material is required.22
8.12(2H, s), 9.85(2H, s).13C NMR (DMSO‑d6): 166.1, 159.3, 139.2,
129.7, 126.3, 119.0, 116.2, 39.1.
Dimeric cinnamoylamide analogues (DCAs) as depigmenting agents
were isolated from Sophora japonica L. and corn bran23,24 and syn-
thesized to enhance depigmenting activity. In particular, DCAs with
different cinnamoyl derivatives and a diaminoethyl group.25 Kim
et al.26 synthesized DCAs containing hydroxyl groups at various posi-
tions and different diamide-link chain lengths. However, the role of
diamide-link chain lengths on DCAs with hydroxymethoxyl group was
not reported for depigmenting activity.
N,N'-di-p-coumaroyl-1,3-diaminopropane (5): White power, Yield
79.9%, λmax (310 nm), HRMS (EI+): m/z: calcd for C21H22N2O4:
367.16, 1H NMR(400 MHz, DMSO‑d6): 1.63(2H, t, J = 7.0),
3.21–3.18(4H, m, J = 7.0), 6.41(2H, d, J = 15.4), 6.79–6.78(4H, m,
J = 2.1), 7.33(2H, d, J = 16.1), 7.40–7.38(2H, m, J = 5.6), 9.84(2H, s).
13C NMR (DMSO‑d6): 164.8, 158.2, 138.0, 128.6, 125.6, 125.3, 118.0,
115.1, 35.9, 28.8.
N,N'-di-p-coumaroyl-1,4-diaminobutane (6): White power, Yield
60.9%, λmax (309 nm), HRMS (EI+): m/z: calcd for C22H24N2O4:
381.17, 1H NMR(400 MHz, DMSO‑d6): 1.47(4H, s), 3.02(4H, t, J = 6.0),
6.42(2H, d, 15.6), 6.79(4H, d, J = 8.4), 7.34(2H, d, J = 16), 7.39(4H,
d, J = 8.8), 8.01(2H, t, J = 5.6) 9.83(2H, s).13C NMR (DMSO‑d6):
165.8, 148.7, 148.3, 139.3, 126.9, 121.3, 119.5, 116.1, 111.1, 55.9,
38.8, 27.3.
In this study, DCAs with cinnamoyl derivatives (feruloyl, iso-
feruloyl, p-coumaroyl or m-coumaroyl) and different diamide-link
chain lengths (n = 2, 3, or 4) were synthesized and evaluated on tyr-
osinase and melanogenesis inhibitory effect. Our results provide useful
information about structure-activity relationship of DCAs for de-
pigmenting activities.
N,N'-diisoferuloyl-1,2-diaminoethane (7): White power, Yield 64.2%,
λmax (320 nm), HRMS (EI+): m/z: calcd for C22H24N2O6: 413.16, 1H
NMR(400 MHz, DMSO‑d6): 3.28(4H, d), 3.79(6H, s), 6.40(2H, d,
J = 16.0), 6.99–6.92(6H, m, J = 8.8), 7.13(2H, d, J = 15.6), 8.18(2H,
s), 9.20(2H, s). 13C NMR(DMSO‑d6): 165.9, 149.6, 139.3, 128.2, 120.8,
119.9, 113.7, 112.5, 55.0.
2. Experimental section
2.1. Chemistry
2.1.1. Instruments and chemicals
The UV-VIS spectrophotometer used in this study was a Varian
(Australia) Cary 50, a Spectronic 20D system (Milton Roy Co., USA) was
used to measure the cellular protective effect. A Berthold (Germany) 6-
channel LB9505 LT system was used for the chemiluminescence assay,
and the nuclear magnetic resonance (NMR) spectrometer used here was
a 400 MHz NMR system (Varian Inc., USA). Na2HPO4, NaH2PO4,
ethylenediaminetetraacetic acid (EDTA), H2O2, luminol and heparin
were purchased from Sigma Chemical Co. (St. Louis, MO). FeCl3·6H2O
was bought from Junsei Chemical Co. (Japan). Ethanol (EtOH), me-
thanol (MeOH), ethyl acetate (EtOAc), and acetonitrile were purchased
from as the highest purity and HPLC grade from Sigma Chemical Co.
(St. Louis, MO). Dulbecco’s modified Eagle’s medium (DMEM), fetal
bovine serum (FBS), and penicillin-streptomycin, and bovine serum
albumin (BSA) were obtained from PAA Co. (Pasching, Austria).
N,N'-diisoferuloyl-1,3-diaminopropane (8): White power, Yield
66.5%, λmax (322 nm), HRMS (EI+): m/z: calcd for C23H26N2O6:
427.18, 1H NMR(400 MHz, DMSO‑d6): 1.68–1.64(2H, m, J = 6.8),
3.23–3.19(4H, m, J = 6.4), 6.44(2H, d, J = 15.6), 7.01(6H, m,
J = 1.6), 7.30(2H, d, J = 16), 8.14(2H, s), 8.14(2H, s), 9.25(2H, s). 13
C
NMR(DMSO‑d6): 164.7, 148.6, 146.1, 138.1, 127.0, 119.7, 118.9,
112.7, 111.4, 54.9, 36.0, 35.9, 28.8
N,N'-diisoferuloyl-1,4-diaminobutane (9): White power, Yield 66.1%,
λmax (321 nm), HRMS (EI+): m/z: calcd for C24H28N2O6: 441.19, 1H
NMR(400 MHz, DMSO‑d6): 1.47(4H, s), 3.19(4H, d, J = 5.2), 3.79(6H,
s), 6.40(2H, d, J = 12.4), 6.91(2H, d, J = 12.4), 6.98(6H, m, J = 2.0),
7.28(2H, d, J = 15.6), 8.03(2H, t, J = 5.6), 9.20(2H, s). 13C NMR
(DMSO‑d6): 165.6, 149.6, 147.1, 139.0, 128.3, 120.7, 113.7, 112.5,
56.0, 38.8, 27.3.
N,N'-diferuloyl-1,2-diaminoethane (10): White power. Yield 64.5%,
λmax (321 nm), HRMS (EI+): m/z: calcd for C22H24N2O6: 413.16, 1H
NMR(400 MHz, DMSO‑d6): 3.28(4H, t, J = 2.0), 3.80(6H, s), 6.46(2H,
d, J = 15.6), 6.80(2H, d, J = 8.0), 7.00(2H, t, J = 1.6), 7.13(2H, d,
J = 1.6), 7.36(2H, d, J = 15.6), 8.09(2H, s), 9.46(2H, s). 13C NMR
(DMSO‑d6): 166.1, 148.7, 148.3, 139.6, 126.8, 122.0, 119.3, 116.1,
111.2, 55.9
2.1.2. Physicochemical characteristics of dimeric cinnamoylamide
analogues
N,N'-di-m-coumaroyl-1,2-diaminoethane (1): White power, Yield
51.8%, λmax (278 nm), HRMS (EI+): m/z: calcd for C20H20N2O4:
353.14, 1H NMR(400 MHz, DMSO‑d6): 3.28(4H, d), 6.55(2H, d,
J = 15.4), 6.79–6.77(2H, m, J = 2), 6.93(2H, s), 6.97–6.93(2H, m,
J = 6.6), 7.22–7.19(2H, t, J = 7.7), 7.53(2H, d, J = 16.1), 8.26(2H, t,
J = 5.6), 9.59(2H, s). 13C NMR (DMSO‑d6): 164.7, 157.1, 138.4, 135.6,
129.3, 121.3, 118.1, 116.1, 113.1, 24.5.
N,N'-diferuloyl-1,3-diaminopropane (11): White power. Yield 66.5%,
λmax (320 nm), HRMS (EI+): m/z: calcd for C23H26N2O6: 427.18, 1H
NMR(400 MHz, DMSO‑d6): 1.65(2H,t, J = 6.8), 3.21(4H, m, J = 6.8),
6.47(2H, d, J = 16), 6.80(2H, d, J = 8.40), 7.00–6.98(2H, dd, J = 1.6),
7.13(2H, d, J = 1.6), 7.34(2H, d, J = 15.6), 8.01(2H, t, J = 5.6),
9.44(2H, s). 13C NMR (DMSO‑d6):164.7, 148.6, 146.1, 138.1, 127.0,
119.7, 118.9, 112.7, 111.4, 54.9, 36.0, 35.9, 28.8
N,N'-di-m-coumaroyl-1,3-diaminopropane (2): White power, Yield
75.7%, λmax (280 nm), HRMS (EI+): m/z: calcd for C21H22N2O4:
366.16, 1H NMR(400 MHz, DMSO‑d6): 1.69–1.66(2H, m, J = 7.0),
3.24–3.21(2H, d, J = 7.0), 6.56–6.54(2H, d, J = 15.4), 6.79–6.76(2H,
m, J = 1.4), 6.94(2H, t, J = 2.1), 6.99(2H, d, J = 7.7), 7.21(2H, t,
J = 7.7), 7.34(2H, d, J = 15.4, 8.16(2H, t, J = 5.6), 6.99(2H, d,
J = 7.7), 7.21(2H, t, J = 7.7), 7.34(2H, d, J = 15.4), 8.16(2H, t,
J = 5.6), 9.59(2H, s). 13C NMR (DMSO‑d6): 164.4, 157.0, 138.2, 135.6,
129.3, 121.4, 118.1, 116.0, 113.0, 36.6, 28.7.
N,N'-diferuloyl-1,4-diaminobutane (12): White power. Yield 66.5%,
λmax (328 nm), HRMS (EI+): m/z: calcd for C24H28N2O6: 441.19, 1H
NMR(400 MHz, DMSO‑d6): 1.47(4H, s), 3.18(4H, d, J = 4.8), 3.80(6H,
s), 6.47(2H, d, J = 15.6), 6.80(2H, d, J = 8.4), 6.99(2H, t, J = 2.0),
7.12(1H, d, J = 1.60), 7.34(2H, d, J = 18.4), 8.01(2H. t, J = 5.6),
9.45(2H, s). 13C NMR (DMSO‑d6): 165.7, 148.6, 148.2, 139.3, 126.9,
121.9, 119.5, 116.1, 111.1, 55.9, 38.8, 27.3.
N,N'-di-m-coumaroyl-1,4-diaminobutane (3): White power, Yield
61.4%, λmax (278 nm), HRMS (EI+): m/z: calcd for C22H24N2O4:
381.17, 1H NMR(400 MHz, DMSO‑d6): 1.49(4H, s), 3.20(4H, d,
J = 5.20), 6.54(2H, d, J = 9.6), 6.79–6.76(2H, dd, J = 2.0), 6.92(2H,
s), 6.98(2H, d, J = 7.6), 7.18(2H, t, J = 7.6), 7.34(2H, d, J = 15.6),
8.14(2H, t, J = 5.2), 9.59(2H, s). 13C NMR (DMSO‑d6): 164.8, 157.7,
157.6, 138.6, 129.8, 121.9, 118.4, 116.6, 113.6, 38.3, 26.7.
N,N'-di-p-coumaroyl-1,2-diaminoethane (4): White power, Yield
59.2%, λmax (310 nm), HRMS (EI+): m/z: calcd for C20H20N2O4:
353.14, 1H NMR(400 MHz, DMSO‑d6): 3.27(4H, d), 6.42(2H, s),
6.80(4H, d, J = 8.40), 7.36(2H, d, J = 15.6), 7.40(4H, d, J = 8.8),
2.2. Biological activity
2.2.1. Cell culture
B16F1 cells were gained from Korean Cell Line Bank (Seoul,
Korea)22 and were cultured in Dulbecco’s modified Eagle’s medium
(DMEM; Capricorn, Germany) supplemented with 10% fetal bovine
serum (FBS; Capricorn, Germany) and 1% penicillin/streptomycin in a
humidified atmosphere containing 5% CO2 at 37 °C.
2

J.h. Ha, S.N. Park
Bioorganic&MedicinalChemistryxxx(xxxx)xxx–xxx
2.2.2. Measurement of cell viability
Table 1
Substitution pattern of DCAs (1–12).
Cell viability assay was performed using 3-(4,5-cimethylthiazol-2-
yl)-2,5-diphenyl tetrazolium bromide (MTT) by measuring the reduc-
tion value of MTT to formazan. B16F1 cells were seeded at a density of
1 × 104 cells/well in a 96-well plates. Then, cells were treated in-
dicated concentration of compounds. After incubation for 72 h, the
medium was removed and washed, and MTT solution (200 μL) was
added to each well. After cells were treated for 1 h, the media is re-
moved and added DMSO for solubilization of formazan. Then, absor-
bance was measured using a microplate reader (TECAN, Salzburg,
Austria) at 570 nm.
Compound
Formula
Diamide-link chain length (n)
R1
R2
2.2.3. Measurement of melanin content
1
C20H20N2O4
C21H22N2O4
C22H24N2O4
C20H20N2O4
C21H22N2O4
C22H24N2O4
C22H24N2O6
C23H26N2O6
C24H28N2O6
C22H24N2O6
C23H26N2O6
C24H28N2O6
2
3
4
2
3
4
2
3
4
2
3
4
OH
OH
OH
H
H
B16F1 cells were seeded at 1 × 105 cells/well in 6-well culture
plates.27 Then, cells were treated with indicated concentration of
MAHDP for 72 h. B16F1 cells were stimulated with 200 nM α-MSH
together. To analyze intracellular melanin content, cells were washed
twice with PBS and dissolved in 1 N NaOH solution containing 10%
DMSO at 80 °C for 2 h. The cell lysates was transferred to a new 96 well
plate and determined by estimating the absorbance at 405 nm using a
microplate reader.
2
H
3
H
4
OH
OH
OH
OCH3
OCH3
OCH3
OH
OH
OH
5
H
6
H
7
OH
OH
OH
OCH3
OCH3
OCH3
8
9
10
11
12
2.2.4. Intracellular TYR inhibitory activity
To obtain intracellular TYR, B16F1 cells were harvested and lysed
by stripper with RIPA buffer (cell lysis buffer). The cell lysates cen-
trifuged at 13,000 rpm for 30 min at 4 °C and supernatant was collected.
The protein concentrations in lysates were evaluated using the BCA
protein assay kit (Pierce, Biotechnology, Rockford, IL, USA). Then, the
lysate containing the same amount of protein (60 μg) was placed in a
96-well plate and treated with L-DOPA (10 mM), 0.1 M phosphate
buffer (pH 6.5), sample solution or arbutin for 30 min at 37 °C. The
absorbance was measured at 490 nm using a microplate reader.
diaminopropane (2,5,8,11) or 1,4-diaminobutane (3,6,9,12) was used
for the synthesis of DCAs as depicted (Table 1.). Representatively, the
synthetic pathway of N,N'-diferuloyl-1,2-diaminoethane (10) is dis-
played (Scheme 1). Ferulic acid (a) added to the solution including
acetic anhydride and dimethylaminopyridine (DMAP) in triethylamine
(Et3N), and then acetylferulic acid (b) was obtained. The compound b
was refluxed in thionyl chloride (SOCl2), while reacting to ethylene-
diamine (636 mg, 25.4 mmol) to produce N,N'-acetylferuloyl-1,2-dia-
minoethane. Finally, N,N'-diferuloyl-1,2-diaminoethane (10) was ob-
tained after hydrolysis as shown in Scheme 1.
2.3. DPPH radical scavenging activity
Free radical scavenging activity was conducted by 1,1-diphenyl-2-
picryl hydrazyl (DPPH), which is used because it has a stable free ra-
dical. Various concentrations of 0.3 mL the samples were mixed with
0.3 mL 0.2 mM DPPH and 0.3 mL 100% EtOH and incubated for 10 min.
The levels of free radicals was measured by absorbance at 517 nm (λmax
of DPPH).24 Results were expressed as concentration of sample neces-
sary to give a 50% reduction in the original absorbance (EC50). The free
radical scavenging activity was calculated using the following Eq. (1):
3.2. Mushroom tyrosinase inhibitory activity
Tyrosinase inhibitory activity of DCAs is shown in Table 2, and the
activity was compared to that of arbutin as positive controls.
The results indicated that the evaluated compounds present dif-
ferent profiles of mushroom tyrosinase inhibitory activity. Tyrosinase
inhibitory activity of DCAs without hydroxy groups has been also
evaluated but they did not exert any activity (data not shown).
In the experimental results, compound 3 and 10 did not exert tyr-
osinase inhibitory activity. Among the tested compounds, p-coumaroyl
DCAs (4–6) indicated to be strong inhibitors, with IC50 values of 4.6,
3.4 and 6.5 μM, respectively and they showed approximately 2.3, 3.1
and 1.6 times higher active than the standard inhibitor
(IC50 = 10.4 μM). In particular, p-coumaroyl DCAs showed more 16
times higher inhibitory activity than m-coumaroyl DCAs (1–2) and
isoferuloyl DCAs (7–9) at the same amide-link chain length. These ef-
fects can be explained that p-hydroxybenzyl group acts as a strong
competitive inhibitor because its structure is similar to the binding site
of tyrosine.29 In addition, m-methoxy group interferes with approaches
of p-hydroxyl group to the active site of tyrosinase through steric hin-
drance.30 Interestingly, DCAs showed high tyrosinase inhibitory activ-
ities when the diamide chain length was three (2, 5, 8 and 11). These
results suggest that a methoxy group and the diamide-link chain lengths
in DCAs may affect the tyrosinase inhibitory activity directly.
AExperiment - ABlank
Radicalscavenging% = 1-
× 100
AControl
(1)
2.4. Determination of chelating activity
To evaluate the ability of DCAs to chelate copper, the UV–visible
spectra (220–400 nm) of DCAs and
Cu-complexed counterparts were measured by the modified
method.28 The mixture, consisting of 50 μL of DCAs (50 μM) ethanol
solution and 0.95 mL of the aqueous solution of CuSO4 (10 μM) or water
(control) was incubated at 25 °C for 10 min before UV-Vis spectral
analysis. All tests and analyses were carried out in triplicate and dis-
played representative spectra.
3. Results and discussion
3.1. Chemistry
3.3. Antimelanogenic effects in B16F1 melanoma cells
The synthesis of DCAs with m-coumaric acid (1–3), p-coumaric acid
(4–6), isoferuloyl acid (7–9), feruloyl acid (10–12) was performed by
Samkyung costech Co. In addition, 1,2-diaminoethane (1,4,7,10), 1,3-
3.3.1. Effect on cell viability
To evaluate the potential cytotoxicity of DCAs on melanoma cells,
the cells were treated with different concentration of each DCAs (100,
3

J.h. Ha, S.N. Park
Bioorganic&MedicinalChemistryxxx(xxxx)xxx–xxx
Scheme 1. Reagents and reaction conditions: (a) acetic anhydride, Et3N, DMAP (b) 1,2-diaminoethane, SOCl2, Et3N, MC (c) KOH, H2O.
Table 2
Table 3
Half maximal inhibitory concentration (IC50) va-
lues of DCAs against mushroom tyrosinase activity.
Effects of DCAs on tyrosinase inhibitory activity and melanin production in
B16F1 cells.
Compound
IC50 (μM)a
Compound
IC50 (μM)a
Tyrosinase inhibitory activity
Melanin production
> 150
1
95.3
55.2
> 150
4.6
1.2
2
0.5
1
> 150
3
2
40.2
1.2
95.3
0.8
4
0.04
0.06
0.03
1.1
3
> 150
> 150
4.6
> 150
> 150
45.6
5
3.4
4
6
6.5
5
0.1
0.4
7
84.2
55.3
80.2
> 150
98.3
108.4
10.4
6
> 150
> 150
46.7
> 150
> 150
111.3
> 150
> 150
122.8
> 150
148.3
8
0.1
7
9
1.6
8
1.5
2.5
10
9
> 150
> 150
65.2
11
2.1
10
12
2.5
11
1.9
3.3
3.1
1.4
2.1
Arbutinb
0.11
12
88.4
Arbutinb
87.5
a
Data represent the mean ( standard devia-
tion, SD) of three independent experiments.
a
Data represent the mean ( standard deviation, SD) of three independent
b
experiments.
Positive control.
Positive control.
b
150 and 200 μM) for 72 h and were examined using MTT assay. Results
indicated that compounds 1–12 had no considerable cytotoxic effect in
B16F1 melanoma cells at the concentration in which tyrosinase activity
was inhibited (Fig. 1). Cell viabilities of compounds 1, 3 and 6 slightly
decreased at 200 μM. Therefore, the following experiments were per-
formed using up to 150 μM of DCAs.
suppressed (IC50) (Table 3). Compounds 1, 3, 4, 6, 7, 9 and 10 did not
exert any effect (> 150 μM) on both intracellular tyrosinase activity
and melanin production on α-MSH stimulated B16F1 cells. On the other
hand, compounds 2, 5, 8, 11 reduced melanin production, as well as
intracellular tyrosinase activity. Although compounds 1, 4, 6, 7, 9 and
12 indicated inhibitory effects on mushroom tyrosinase, they didn’t
exert inhibitory effect on both tyrosinase activity and melanin pro-
duction on α-MSH stimulated B16F1 cells. This may be due to the
cellular mechanisms, not reflected in the mushroom tyrosinase
screening assays.25 Interestingly, compounds 2, 5, 8 and 11 with a
3.3.2. Effect on intracellular tyrosinase activity and melanin production
To investigate the inhibitory effect of DCAs on melanogenesis,
B16F1 cells were treated with DCAs and α-MSH (200 nM), and eval-
uated for the concentration at which 50% of melanogenesis is
Fig. 1. Cell viability of DCAs on B16F1 cells at 100 μM (dark blue), 150 μM (cobalt blue), 200 μM (light blue). Data are presented as the mean
independent experiments. *p < 0.01 compared with non-treated cells (NC).
SD of three
4

J.h. Ha, S.N. Park
Bioorganic&MedicinalChemistryxxx(xxxx)xxx–xxx
Fig. 2. Copper ions chelating ability of DCAs. The representative UV–vis spectra of compound 1–12 at 50 μM was displayed (solid line). Spectrum recorded again
with the addition of 10 μM CuSO4 (dotted line).
diamide-link chain length of three possessed higher inhibitory effect of
tyrosinase activity and melanin production than DCAs with a diamide-
link chain length of two or four. They showed more anti-melanogenesis
activity than arbutin as compared with the control. Thus, the position of
methoxy groups and/or hydroxyl groups in DCAs, as well as the dia-
mide-link chain length, are important for forming the optimal structure
to bind to the tyrosinase active site.
increase in the UV–Vis spectra of compounds with Cu (II) addition and
the compounds indicated tyrosinase inhibitory activities.27 These re-
sults suggest that melanogenesis of DCAs might be dependent on their
tyrosinase inhibitory activity through copper-chelating properties.
3.5. Antioxidative activity
Free radical scavenging activity of DCAs was performed using
DPPH∙ assay and L-ascorbic acid and arbutin were used as the positive
controls.31,32 Antioxidative activity of DCAs without hydroxy groups
was also evaluated but they did not exert any activity (data not shown).
Half maximal effective concentration (EC50) values for the assay were
indicated in Table 4.
3.4. Effect on intracellular tyrosinase activity and melanin production
Tyrosinase inhibitory effects of DCAs and arbutin might be due to
binding with copper as the active site of tyrosinase. The copper-che-
lating properties of DCAs were evaluated by spectrophotometric ana-
lyses.
As a results, m-coumaroyl (1–3) and p-coumaroyl DCAs (4–6) did
not exert antioxidative activities in the assay. Regarding DPPH∙ assay,
isoferuloyl (7–9) and feruloy DCAs (10–12) showed the best EC50 va-
lues in the same range (EC50 = 2.21, 2.31, 5.20, 0.09, 0.15 and
0.83 μM, respectively) as L-ascorbic acid (EC50 = 0.18 μM) and arbutin
(EC50 = 1.71 μM). In particular, feruloyl DCAs (10–12) showed free
radical scavenging activity 5–40 times higher than isoferuloyl DCAs
(7–9) in the same rage. These effects can be explained due to the
As shown in Fig. 2, the max absorbance indicated widely from 280
to 320 nm for compound 1–6 with a hydroxyl group (Fig. 2, solid line).
The max absorbance of compound 7–12 with hydroxyl group and
methoxy group indicated two λmax between 280 and 330 nm. Com-
pound 2, 5, 8, 11, 12 showed increasing absorbance (Fig. 2, dote line)
with addition of Cu (II). Interestingly, they possessed inhibitory activity
on intracellular tyrosinase of B16F1 cells. There are previous studies on
5

J.h. Ha, S.N. Park
Table 4
Bioorganic&MedicinalChemistryxxx(xxxx)xxx–xxx
Table 5
Free radical scavenging activities (EC50) of DCAs.
Dipole moments and partition coefficient (Log P) of DCAs.
a
Compound
EC50 (mM)a
Compound
Dipole moment
log Pb
1
> 5
> 5
> 5
> 5
> 5
> 5
2.21
2.31
4.20
0.09
0.15
0.83
0.18
1.7
1
7.348
5.975
6.914
5.543
5.248
5.279
8.189
6.199
7.618
6.747
6.335
7.054
3.594
2.01
2.12
2.57
2.01
2.12
2.57
1.76
1.86
2.32
1.76
1.86
2.32
−0.58
2
2
3
3
4
4
5
5
6
6
7
0.25
0.14
0.22
0.001
0.01
0.02
0.01
7
8
8
9
9
10
10
11
11
12
12
L-Ascorbic acidb
Arbutinb
Arbutinb
0.07
a
b
Debye.
a
Data represent the mean ( standard deviation, SD)
of three independent experiments.
Log P (Log[octanol/water] partition coefficient) values were calculated
using Chemdraw version 7.0.
b
Positive control.
presence of a methoxy group on their structures. The methoxy group as
an electron-donating group enhances stabilization of the electrons
produced after the transfer of hydrogen from the hydroxyl group to the
free radical as DPPH.33 Especially, m-methoxy group in ferulic moiety
can stabilize more than p-methoxy group in isoferulic moiety.34 Inter-
estingly, compounds 7, 8, 10 and 11 with a diamide-link chain length
of two or three were higher anti-oxidative activities than compounds 9
and 12 with a diamide-link chain length of four. Their high activities
are presumed that their diamide-link chain length is important to be
stabilized after electron donation.35 These results suggest that addition
and position of methoxy groups as well as length of diamide-link chain
in DCAs are important to enhance antioxidative activity. Although
feruloy DCAs indicated high free radical scavenging activity, they did
not affect anti-melanogenesis activity.
3.6. Structure, dipole moment, log P and cell uptake
In order to study the effects of the structure and dipole moment of
the DCAs in water phase, we optimized the structures of the DCAs using
a Gaussian 09 W software package at the DFT level of theory (B3LYP
Fig. 3. DFT-optimized structures of the DCAs (1–12). Calculations were conducted using a Gaussian 09 W. All structure optimizations were performed by molecular-
mechanics computations and were further refined by DFT computation (B3LYP at the 6-31G* level).
6

J.h. Ha, S.N. Park
Bioorganic&MedicinalChemistryxxx(xxxx)xxx–xxx
Fig. 4. Cell uptake level (μmol/mg protein) of DCAs on B16F1 cells. The cells Data are presented as the mean
SD of three independent experiments.
hybrid functional) together with a 6-31G*(d,p) basis set in a water
phase.
4. Conclusion
Compounds 2, 5, 8 and 11, which are containing a diamide-link
chain length of three, displayed folded structures with their functional
groups pointed in the same direction (Fig. 3). The structure of them
shows both hydroxyl groups are close to each other than that of DCAs
with a diamide-link chain length of two or four, which display slightly
bent shapes (1, 3, 4, 6, 7, 9, 10 and 12).
Dimeric compounds are widely developed to enhance the biological
properties such as antioxidative and tyrosinase inhibitory activities.
However, structure–activity relationship of dimeric compounds with
regard to inhibitory effects on tyrosinase are not well known. In the
present paper, DCAs (1–12) were synthesized to investigate the effect of
functional groups and the diamide-link chain length in dimeric com-
pounds. In detail, we adjusted two factors of the DCAs: 1) the position
of the hydroxy and methoxy groups in the cinnamoyl group, 2) the
diamide-link chain lengths (n = 2, 3, and 4) between the functional
groups in the DCAs. In melanin production in α-MSH induced B16F1
cells, DCAs with the diamide-link chain length was three (2, 5, 8 and
11) only showed inhibitory effects. The compounds displayed folded
structure and low dipole moments of DCAs with the same functional
group. These characteristics of them are optimized to inhibit tyrosinase
activity through copper-chelation as an active cite of tyrosinase. The
results suggest that the diamide linkage chain length of DCAs is im-
portant to block the active site of tyrosinase, decreasing in melanin
production effectively.
In the dipole moment evaluation (Table 5), the calculated dipole
moment of these compounds (2: 5.975 Debye, 5: 5.248 Debye, 8: 6.199
Debye, 11: 6.335 Debye) showed a linear relationship for higher in-
hibitory activity with decreasing dipole moment (for inhibitory activity,
5 > 2 > 8 > 11). Their diamide-link chin length is three. Their va-
lues of dipole moment were the lowest of DCAs with the same func-
tional group and a diamide-link chain lengths of two or four. This re-
lation is elucidated that the decrease in the dipole moment of DCAs
increases tyrosinase inhibitory activity for possibility of binding to
copper ions in the active cite of tyrosinase.
To compare the hydrophobic characteristics of DCAs, we calculated
partition coefficient (Log P) values using Chemdraw program (Table 5).
The hydrophobic characteristics were increased as the increase in dia-
mide-link chain lengths and addition of a methoxy group. DCAs (1–12)
indicated higher 1.5 of log P value, which increase in accessibility to the
active site of tyroisnase and penetration into melanocytes. On the other
hand, arbutin indicated very low log P value (-0.58) decreasing these
abilities for depigmenting activity.
Recently, it has been reported that arbutin, 4-hexylresorcinol, and
hydroquinone can be used as substrates for tyrosinase in the presence of
hydrogen peroxide, ascorbic acid, or 4-tert-butylcatechol.36–38 It has
also been reported that the product of the reaction between hydro-
quinone and tyrosinase is toxic. Although the compounds we synthe-
sized were not cytotoxic, the compounds also have phenolic groups
similar to hydroquinone. Therefore, these compounds are likely to be
converted by tyrosinase in the presence of hydrogen peroxide and
various reducing agents, which may result in cytotoxicity. These issues
will be thoroughly reviewed before future clinical studies on whitening
effects.
In the cell uptake evaluation (Fig. 4) of DCAs, compounds 3 and 6
showed the highest levels of cellular uptake and the lowest Log P values
among the compounds having hydroxyl groups (–OH). Their cell uptake
showed a correlation with log P indicating physical properties. However,
the presence of –OH groups at the binding positions, R1 and R2, did not
affect the cellular uptake of these compounds. Compounds 8 and 11 had
a relatively low dipole moment and a diamide connection length of 3,
and they showed the highest cellular uptake ratios within the group of
compounds having both methoxy and hydroxyl groups (7–12).33 Al-
though the groups had low log P values, which was probably due to their
highly hydrophilic nature, they showed higher cellular uptake ratios than
the compounds with hydroxyl groups alone. These results suggest that
the affinity between the methoxy group and the cell membrane leads to
an increase in cellular uptake of DCAs. Arbutin, as a control, had a very
low cellular uptake ratio due to its hydrophilic structure
In conclusion, these results provide a useful insight into the devel-
opment of new dimeric compounds for various physiological active
agents in the future. To understand the structure–activity relationship
of dimeric compounds in the biological systems further detailed studies
are required.
References
1. Slominski A, Tobin DJ, Shibahara S, Wortsman J. Melanin pigmentation in mam-
malian skin and its hormonal regulation. Physiol Rev. 2004;84:1155–1228.
2. Olivares C, Solano F. New insights into the active site structure and catalytic me-
chanism of tyrosinase and its related proteins. Pigm Cell Melan Res. 2009;22:750–760.
3. Costin GE, Hearing VJ. Human skin pigmentation: melanocytes modulate skin color
in response to stress. FASEB J. 2007;21:976–994.
Taken together, anti-melanogenic effects of DCAs are enhanced
when their diamide-link chain length are three involving a folded
shape, a low dipole moment, and appropriate hydrophobicity (> 1.5)
and folded structure increase in accessibility to the active site of
tyrosinase. Therefore, our results provide useful information for the
design of dimeric compounds with antioxidant and whitening activ-
ities.
4. Parvez S, Kang M, Chung HS, et al. Survey and mechanism of skin depigmenting and
lightening agents. Phytother Res. 2006;20:921–934.
5. Paramonov BA, Turkovskii II IL, Potokin NA, Chebotarev Yurlova VY.
Photoprotective activity of melanin preparations in human skin exposed to UV ir-
radiation: dependence on previous photoexposure. Bull Exp Biol Med.
7

J.h. Ha, S.N. Park
Bioorganic&MedicinalChemistryxxx(xxxx)xxx–xxx
2002;134:366–369.
2018.
6. Shin EM, Zhou HY, Xu GH, Lee SH, Merfort I, Kim YS. Anti-inflammatory activity of
hispidol A 25-methyl ether, a triterpenoid isolated from Ponciri Immaturus Fructus.
Eur J Pharmacol. 2010;627:318–324.
23. Lo YH, Lin RD, Lin YP, Liu YL, Lee MH. Active constituents from Sophora japonica
exhibiting cellular tyrosinase inhibition in human epidermal melanocytes. J
Ethnopharmacol. 2009;124:625–629.
7. Yaar M, Wu C, Park HY, Panova I, Schutz G, Gilchrest BA. Bone morphogenetic
protein-4, a novel modulator of melanogenesis. J Biol Chem. 2006;281:25307–25314.
8. Kim M, Shin S, Lee JA, Park D, Lee J, Jung E. Inhibition of melanogenesis by
Gaillardia aristata flower extract. BMC Compl Altern Med. 2015;15:449.
9. Park HY, Kosmadaki M, Yaar M, Gilchrest BA. Cellular mechanisms regulating human
melanogenesis. Cell Mol Life Sci. 2009;66:1493–1506.
24. Choi SW, Lee SK, Kim EO, et al. Antioxidant and antimelanogenic activities of
polyamine conjugates from corn bran and related hydroxycinnamic acids. J Agric
Food Chem. 2007;55:3920–3925.
25. Criton M, Le Mellay-Hamon V. Dimeric cinnamoylamide derivatives as inhibitors of
melanogenesis. Biol Pharm Bull. 2011;34:420–425.
26. Kim MK, Youk HS, Yoo JJ, et al. Synthesis of dimeric cinnamoylamide derivatives
and evaluation of their depigmenting activities. Bull Korean Chem Soc.
2014;35:3085–3088.
10. Won YK, Lin CB, Seiberg M, et al. Galvanic zinc-copper microparticles inhibit mel-
anogenesis via multiple pigmentary pathways. Arch Dermatol Res. 2014;306:27–35.
11. Romero-Graillet C, Aberdam E, Clement M, Ortonne JP, Ballotti R. Nitric oxide
produced by ultraviolet-irradiated keratinocytes stimulates melanogenesis. J Clin
Invest. 1997;99:635–642.
27. Ha JH, Park SN. Mechanism underlying inhibitory effect of six dicaffeoylquinic acid
isomers on melanogenesis and the computational molecular modeling studies. Bioorg
Med Chem. 2018;26:4201–4208.
12. Novellino L, Napolitano A, Prota G. 5,6-Dihydroxyindoles in the fenton reaction: a
model study of the role of melanin precursors in oxidative stress and hy-
perpigmentary processes. Chem Res Toxicol. 1999;12:985–992.
28. Maisuthisakul P, Gordon MH. Antioxidant and tyrosinase inhibitory activity of
mango seed kernel by product. Food Chem. 2009;117:332–341.
29. Briganti S, Camera E, Picardo M. Chemical and instrumental approaches to treat
hyperpigmentation. Pigment Cell Res. 2003;16:101–110.
13. Ebanks JP, Wickett RR, Boissy RE. Mechanisms regulating skin pigmentation: the rise
and fall of complexion coloration. Int J Mol Sci. 2009;10:4066–4087.
14. Ito S, Ojika M, Yamashita T, Wakamatsu K. Tyrosinase-catalyzed oxidation of rho-
dodendrol produces 2-methylchromane-6,7-dione, the putative ultimate toxic me-
tabolite: implications for melanocyte toxicity. Pigm Cell Melan Res. 2014;27:744–753.
15. Song K, An SM, Kim M, Koh JS, Boo YC. Comparison of the antimelanogenic effects of
p-coumaric acid and its methyl ester and their skin permeabilities. J Dermatol Sci.
2011;63:17–22.
30. Selinheimo E, NiEidhin D, Steffensen C, et al. Comparison of the characteristics of
fungal and plant tyrosinases. J Biotechnol. 2007;130:471–480.
31. Hachinohe M, Matsumoto H. Involvement of reactive oxygen species generated from
melanin synthesis pathway in phytotoxicty of L-DOPA. J Chem Ecol.
2005;31:237–246.
32. Park SN, Kim MJ, Ha JH, et al. Protective effects of TES trioleate, an inhibitor of
phospholipase A2, on reactive oxygen species and UVA-induced cell damage. J
Photochem Photobiol B. 2016;164:30–35.
16. Shirota S, Miyazaki K, Aiyama R, Ichioka M, Yokokura T. Tyrosinase inhibitors from
crude drugs. Biol Pharm Bull. 1994;17:266–269.
33. Hwang JP, Ha JH, Kim MK, Park SN. Synthesis and antioxidative activities of N, N-
diferuloyl-putrescine (DFP) and its derivatives. Appl Chem Eng. 2015;26:29–34.
34. Karamac M, Koleva L, Kancheva VD, Amarowicz R. The structure-antioxidant activity
relationship of ferulates. Molecules. 2017;22.
17. Mishima S, Inoh Y, Narita Y, et al. Identification of caffeoylquinic acid derivatives
from Brazilian propolis as constituents involved in induction of granulocytic differ-
entiation of HL-60 cells. Bioorg Med Chem. 2005;13:5814–5818.
18. Ortonne JP, Passeron T. Melanin pigmentary disorders: treatment update. Dermatol
Clin. 2005;23:209–226.
35. Musa EM, Ali EO. Density functional theory (DFT) B3LYP study of antioxidant ac-
tivity of 6-(2,2'-bithiophen-5yl) pyridine-3-carboximidamide, new model bichalco-
phenes derivatives. J Appl Indust Sci. 2015;3:126–132.
19. Curto EV, Kwong C, Hermersdorfer H, et al. Inhibitors of mammalian melanocyte
tyrosinase: in vitro comparisons of alkyl esters of gentisic acid with other putative
inhibitors. Biochem Pharmacol. 1999;57:663–672.
36. Garcia-Jimenez A, Teruel-Puche JA, Berna J, Rodriguez-Lopez JN, Tudela J, Garcia-
Canovas F. Action of tyrosinase on alpha and beta-arbutin: a kinetic study. PLoS ONE.
2017;12.
20. Shin HJ, Oh CT, Kwon TR, et al. A novel adamantyl benzylbenzamide derivative,
AP736, inhibits melanogenesis in B16F10 mouse melanoma cells via glycogen syn-
thase kinase 3beta phosphorylation. Int J Mol Med. 2015;36:1353–1360.
21. Park SH, Kim DS, Kim WG, et al. Terrein: a new melanogenesis inhibitor and its
mechanism. Cell Mol Life Sci. 2004;61:2878–2885.
37. Ortiz-Ruiz CV, Berna J, Rodriguez-Lopez JN, Tomas V, Garcia-Canovas FJ.
Tyrosinase-catalyzed hydroxylation of 4-hexylresorcinol, an antibrowning and de-
pigmenting agent: a kinetic study. Agric Food Chem. 2015;12:7032–7040.
38. Garcia-Molina M, Munoz Munoz JL, Martinez-Ortiz F, et al. Tyrosinase-catalyzed
hydroxylation of hydroquinone, a depigmenting agent, to hydroxyhydroquinone: a
kinetic study. Bioorg Med Chem. 2014;22:3360–3369.
22. Ha JH, Jeong YJ, Xuan SH, Lee JY, Park J, Park SN. Methyl-2-acetylamino-3-(4-
hydroxyl-3,5-dimethoxybenzoylthio)propanoate suppresses melanogenesis through
ERK signaling pathway mediated MITF proteasomal degradation. J Dermatol Sci.
8