Synthesis and biological evaluation of caffeic acid derivatives as potent inhibitors of α-MSH-stimulated melanogenesis

Article abstract of DOI:10.1016/j.bmcl.2017.06.011

We have disclosed our effort to develop caffeic acid derivatives as potent and non-toxic inhibitors of α-MSH-stimulated melanogenesis to treat pigmentation disorders and skin medication including a cosmetic skin-whitening agent. The SAR studies revealed t

Full text of DOI:10.1016/j.bmcl.2017.06.011

Accepted Manuscript
Synthesis and Biological Evaluation of Caffeic Acid Derivatives as Potent In-
hibitors of α-MSH-stimulated Melanogenesis
Hyeju Jo, Minho Choi, Jaeuk Sim, Mayavan Viji, Siyuan Li, Young Hee Lee,
Youngsoo Kim, Seung-Yong Seo, Yuanyuan Zhou, Kiho Lee, Wun-Jae Kim,
Jin Tae Hong, Heesoon Lee, Jae-Kyung Jung
PII:
S0960-894X(17)30613-3
DOI:
http://dx.doi.org/10.1016/j.bmcl.2017.06.011
BMCL 25045
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
12 March 2017
22 May 2017
2 June 2017
Please cite this article as: Jo, H., Choi, M., Sim, J., Viji, M., Li, S., Lee, Y.H., Kim, Y., Seo, S-Y., Zhou, Y., Lee,
K., Kim, W-J., Hong, J.T., Lee, H., Jung, J-K., Synthesis and Biological Evaluation of Caffeic Acid Derivatives as
Potent Inhibitors of α-MSH-stimulated Melanogenesis, Bioorganic & Medicinal Chemistry Letters (2017), doi:
http://dx.doi.org/10.1016/j.bmcl.2017.06.011
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Synthesis and Biological Evaluation of
Caffeic Acid Derivatives as Potent Inhibitors
of α-MSH-stimulated Melanogenesis
Hyeju Joa, Minho Choia, Jaeuk Sima, Mayavan Vijia, Siyuan Lia, Young Hee Leea, Youngsoo Kima, Seung-Yong
Seob,*, Yuanyuan Zhouc, Kiho Leec, Wun-Jae Kimd, Jin Tae Hong, Heesoon Leea, Jae-Kyung Junga,*

Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com
Synthesis and Biological Evaluation of Caffeic Acid Derivatives as Potent
Inhibitors of α-MSH-stimulated Melanogenesis
Hyeju Joa, †, Minho Choia, †, Jaeuk Sima, Mayavan Vijia, Siyuan Lia, Young Hee Leea, Youngsoo Kima,
Seung-Yong Seob,*, Yuanyuan Zhouc, Kiho Leec, Wun-Jae Kimd, Jin Tae Honga, Heesoon Leea, Jae-Kyung
Junga,∗
aCollege of Pharmacy and Medicinal Research Center (MRC), Chungbuk National University, Cheongju 28160, Republic of Korea
bCollege of Pharmacy and Gachon Institute of Pharmaceutical Sciences, Gachon University, Incheon 21936, Republic of Korea
cCollege of Pharmacy, Korea University, Sejong 30019, Republic of Korea
dCollege of Medicines, Chungbuk National University, Cheongju 28644, Republic of Korea
ARTICLE INFO
ABSTRACT
Article history:
Received
We have disclosed our effort to develop caffeic acid derivatives as potent and non-toxic
inhibitors of α-MSH-stimulated melanogenesis to treat pigmentation disorders and skin
medication including a cosmetic skin-whitening agent. The SAR studies revealed that
cyclohexyl ester and secondary amide derivatives of caffeic acid showed significant
inhibitory activities.
Revised
Accepted
Available online
Keywords:
Caffeic acid
Melanogenesis
α-MSH
2014 Elsevier Ltd. All rights reserved.
caffeamide
skin whitening
Melanogenesis is a complex and multistep biosynthetic
process that results in the production of melanin pigment, which
plays a protecting role against harmful effects of ultra violet
(UV) induced skin damages.1-3 Upon exposure to UV radiation,
alpha-melanocyte stimulating hormone (α-MSH) is produced and
it stimulates melanocortin 1 receptor that results in the activation
of adenylyl cyclase. The production of cAMP affects the pathway
of cAMP-responsive element-binding protein (CREB), MAP
kinase, and PI3K, finally resulting in the formation of melanin.
cAMP leads to the phosphorylation of CREB transcription
factors, which induces microphthalmia-associated transcription
factor (MITF). MITF is the core controller in the expression of
melanogenesis, which regulates the melanogenesis enzymes
tyrosinase (TYR), catalyzing the rate-limiting step in
melanogenesis. This key enzyme catalyses the hydroxylation of
tyrosine into L-dopa and the subsequent oxidation to generate
dopaquinone, which leads to the synthesis of pheomelanin and
eumelanin; the basic melanin pigments which are responsible of
Fig. 1. Structures of caffeic acid-related derivatives to inhibit α-MSH-
induced melanogenesis.
However, excessive accumulations of epidermal pigmentation
can cause various hyperpigmentation disorders, such as,
melasma, age spots, and sites of actinic damage and reducing the
synthesis of melanin is important in cosmetic whitening.5-8
Therefore, it is very important to control the related enzyme in
human skin and color.4
———
† These authors contributed equally to this work
∗ Corresponding author. Tel.: +82 32 820 4949; fax: +82 32 820 4829. E-mail: syseo@gachon.ac.kr (S.-Y. Seo)
∗ Corresponding author. Tel.: +82-43-261-2635; fax: +82-43-268-2732. E-mail: orgjkjung@chungbuk.ac.kr (J.-K. Jung)

treating pigmentation disorders as well as in the development of
skin medication including a cosmetic skin-whitening agent. This
has led to the discovery of several tyrosinase inhibitors such as
kojic acid, arbutin, and various herbal extracts.9-19
DMF. Cycloalkylamines, phenylamines, benzylamines were
selected for the secondary amides, whereas Boc-piperazine and
piperidine were chosen for tertiary amide synthesis. Free amino
group was obtained by treating Boc-piperazyl amide 9a with 4 M
HCl to afford 9b.
Hydroxycinnamic acids are class of phenolic acid derivatives,
which are widely found in various plants with numerous
biological activities; widely utilized in food, medicine, and
cosmetics industries.20 Of them caffeic acid occurs in foods
mainly as an ester with L-quinic acid called chlorogenic acid (5-
caffeoylquinic acid, CGA). CGA and caffeic acid are potent anti-
oxidants in vitro/in vivo which present anti-inflammatory and
immune-modulatory effects. Also, they might inhibit the
formation of mutagenic and carcinogenic N-nitroso compounds
and inhibit DNA damage. Recently, there are many efforts in
which caffeic acid derivatives were found to inhibit α-MSH-
induced melanogenesis (Figure 1).21-23 Thus ester and amide
derivatives of caffeic acid, isolated from plants or synthesized
examined with respect to their abilities to control human
melanogenesis.24,25 Ha group reported that caffeic acid phenethyl
ester (CAPE), found in various plants and propolis, has an anti-
melanogenic activity in α-MSH-stimulated B16-F10 melanoma
cells by suppression of melanogenic enzyme expression.26 In
contrast to the CAPE analogs, phenethyl alcohol, caffeic acid,
and phenethyl cinnamate were not inhibitors of α-MSH-
stimulated melanogenesis. On the other hands, Kim group
recently discovered that 4-hydroxy-3-methoxycinnamaldehyde
(4H3MC) inhibits α-MSH-induced melanin production in B16
mouse melanoma cells and primary human melanocytes, whereas
4H3MC analogs such as cinnamaldehyde, cinnamic acid, 4-
hydroxycinnamic acid, 3-methoxycinnamic acid, 4-hydroxy-3-
methoxycinnamic acid and 3-(4-hydroxyphenyl)propionic acid
have the lower inhibitory activity of α-MSH-induced melanin
production than 4H3MC.27
O
O
HO
HO
RO
RO
a
OH
caffeic acid
O
3
4
(R = Ac)
(R = H)
b
c
O
O
HO
HO
c
N
HO
HO
R
N
X
H
5
6
7a
8a
9a
9b
10
(R = cyclopentyl)
(R = cyclohexyl)
(R = Ph)
(R = Bn)
(X = NBoc)
(X = NH)
(X = CH2)
d
Scheme 2. Synthesis of caffeate and caffeamide derivatives. Reagents and
conditions: (a). i) Ac2O, Py, DMAP, rt, 12 h, 42%; ii) SOCl2, benzene, 50 ºC,
3 h; iii) DMAP, cyclohexanol, DCM, rt, 12 h, 20%; (b). 2% Na2CO3, MeOH,
rt, 4 h, 50%; (c). HOBt, EDCI, DMF, amine, DIPEA, rt, 12 h; (d) 4 M HCl,
1,4-dioxane, rt, 3 h, 85%.
Synthetic analogs of CGA and caffeic acid derivatives were
evaluated for their inhibitory potential on the production of
melanin in α-MSH-activated B16 melanoma cells in Tables 1~3.
Arbutin and kojic acid were used as positive controls. This assay
was to determine the inhibitory activity of melanin formation in
B16 melanoma cells in the presence of α-MSH (100 nM)
throughout three-day incubation. The amount of melanin released
into the culture media was determined by measuring sample
absorbance at 405 nm against a synthetic melanin standard.28-33
Both the saturated compound 1 and methyl ester 2 of CGA are
inactive even with 40 µM.22
In this report, we have disclosed our effort to develop potent
and non-toxic inhibitors of α-MSH-stimulated melanogenesis
through the ester and amide derivatives of caffeic acid. Thus we
first chose to synthesize two chlorogenic acid derivatives; the
saturated compound 1 was prepared from chlorogenic acid by
catalytic hydrogenation using 10% Pd/C, and methyl ester 2 by
the treatment of (trimethylsilyl)diazomethane, repectively
(Scheme 1).
Table 1. The inhibitory activity of caffeic acid analogs
a
Compound
IC50 (µM)
400 9.50
57 1.0
> 40
cell viability % (µM)
NDd
clogPc
Arbutinb
Kojic acidb
ND
1
2
ND
> 40
ND
3
0.20 0.05
7.0 1.0
30 2.0
2.8 0.10
7.6 0.85
8.0 1.0
5.8 0.26
> 40
24 (2.5)
30 (10)
0 (40)
2.91
2.96
1.87
2.29
2.4
4
5
6
23 (10)
21 (10)
23 (10)
31 (20)
31 (20)
0 (40)
7a
8a
9a
9b
10
2.47
1.46
0.35
1.71
Scheme 1. Synthesis of dihydrochlorogenic acid and methyl ester analogs.
Reagents and conditions: (a). 10% Pd/C, H2, EtOH, 12 h, rt, 95%; (b).
TMSCHN2, MeOH, rt, 12 h, 70%.
24 2.0
a Data are taken as a mean from 3 independent experiments; b Arbutin and
kojic acid were used as the positive control; c clogP value was calculated by
chemdraw ver. 13; d ND: not determined.
We turned our attention to the synthesis of various derivatives
of caffeic acid such as esters, secondary and tertiary amides as
depicted in Scheme 2. In order to prepare cyclohexyl ester,
sequences of reactions were performed. Acetylation of the caffeic
acid using Ac2O, followed by the treatment of thionyl chloride in
benzene at 50 ºC and finally esterification using DMAP and
cyclohexanol afforded the compound 3. Deacetylation of 3 using
2% sodium carbonate in methanol afforded the cyclohexyl ester
of caffeic acid 4. Caffeamides were prepared from caffeic acid
with appropriate amines by using HOBt, EDCI, and DIPEA in
However, cyclohexyl ester 3 of caffeic acid with diacetate on
catechol moiety exhibited potent inhibitory activity with IC50 of
0.2 µM, whereas reduced inhibitory activity (7 µM) was observed
in the case of deacetylated ester 4. Of secondary amides (5, 6, 7a,
and 8a), the cyclohexyl amide 6 exhibited more potent, than the
cyclopentyl amide 5. The inhibitory activity of tertiary amides

8d
8e
8f
H
CF3
H
H
H
CH3
H
3.7 0.10
20 (10), 22-30 (20)
having piperidine and piperazine is lower than that of secondary
amides. Ring size (5 vs 6) and secondary amide are critical to
have the promising activity.
1.8 0.26 22-28 (5), 33-49 (10)
CF3
H
H
1.4 0.36
1.6 0.40
7.0 2.6
6.2 1.7
6.5 0.60
6.0 1.6
1.8 0.26
>40
24 (5), 29-47 (10)
26 (5), 39-48 (10)
20 (20), 35-46 (40)
20 (20), 37-54 (40)
0 (40)
Next, the effect of substitution on phenyl and benzyl amide
derivatives which are expected to have the improved activity and
drug-like properties. Aryl and benzyl amides having substituents
on phenyl ring were prepared like as the synthesis of amides such
as 7a and 8a in Scheme 2. Caffeamides (7a~7g) containing
substituted-phenyl as well as 3-methyl-4-(4-bromophenyl)-5-
pyrazolyl caffeamide (11) as a heteroaromatic analog were
evaluated for their inhibitory effect on the melanin production in
α-MSH-activated B16 melanoma cells (Table 2); all the tested
compounds except phenyl amide 7a and p-isoproylphenyl amide
7d, inhibited the melanin production with the IC50 values ranging
from 0.7 to 2.1 µM. The cell viability of them was measured
using the MTT assay, even though the tested compounds had no
significant cytotoxic activity; some of these derivatives exhibited
the cytotoxicity with higher concentration than IC50 value of
inhibitory effect on the melanin production. Interestingly,
irrespective of electronic and steric effect, substituent on phenyl
ring is beneficial, displays higher activity than unsubstituted
phenyl amide 7a.
8g
8h
8i
H
CF3
H
OCH3
H
H
H
OCH3
H
8j
Cl
H
8k
8l
H
Cl
H
H
22 (10), 59-62 (20)
23 (5), 39-53 (10)
0 (40)
H
Cl
8m
H
H
SO2NH2
a Data are taken as a mean from 3 independent experiments.
In summary, a series of novel caffeate and caffeamide analogs
were synthesized and evaluated inhibitory activity screening for
the production of melanin in α-MSH-activated B16 melanoma
cells identified potent and non-toxic agents such as 3, 7c, 7g, 11,
8g and 8l. SAR studies revealed that cyclohexyl ester and amide
showed potent inhibitory effect and some substituents on phenyl
group of secondary amides are very crucial. Tertiary amides are
showing weaker inhibitory activity than secondary amides. Our
ongoing efforts are to improve biological activities and drug-like
properties. These findings provide important information of the
structural features that influence the biological activities within
this class of compounds, and offer new possibilities for further
explorations in analog design. Based on SAR studies, further
study for mode of action of these caffeates and caffeamide is
under progress.
Table 2. The inhibitory effect of aryl amide analogs
compound
R1
R2
R3
H
IC50 (µM)a
cell viability % (µM)
21 (10)
Acknowledgement
7a
7b
7c
7d
7e
7f
H
H
H
H
H
H
H
-
H
CF3
H
7.6 0.85
1.4 0.47
1.4 0.45
3.4 0.058
1.2 0.46
1.9 0.21
1.4 0.17
1.0 0.15
This work was supported by the National Research
Foundation of Korea grant funded by the Korea Government
(MSIP) (No. MRC, 2008-0062275), the International Science and
Business Belt Program through the Ministry of Science, ICT and
Future Planning (2016K000297), and the research grant of
Chungbuk National University in 2015.
H
22-36 (2.5), 60-82 (5)
56-58 (5), 77-90 (10)
15 (5), 82 (10)
CF3
iPr
n-Bu
H
H
H
33-49 (2.5), 68-96 (5)
24-35 (5), 48 (10)
30-35 (5), 51-62 (10)
29-56 (2.5), 68-93 (5)
NO2
H
7g
11
NO2
-
-
Supplementary Material
a Data are taken as a mean from 3 independent experiments.
Supplementary data associated with this article can be found,
in the online version, at doi:
Next, we have evaluated the inhibitory activity of the
synthesized benzyl caffeamide analogues (8a~8m) on the
melanin production in α-MSH-activated B16 melanoma cells
(Table 3). Interestingly, electron-withdrawing group such as CF3
on phenyl moiety exhibited more potent effects than electron-
donating group except 8j, 8k, and 8m. Later, cell viability of
them was measured by using the MTT assay; all the compounds
had no significant cytotoxic activity.
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