Kojyl cinnamate ester derivatives promote adiponectin production during adipogenesis in human adipose tissue-derived mesenchymal stem cells

Article abstract of DOI:10.1016/j.bmcl.2014.03.034

The subcutaneous fat tissue mass gradually decreases with age, and its regulation is a strategy to develop anti-aging compounds to ameliorate the photo-aging of human skin. The adipogenesis of human adipose tissue-mesenchymal stem cells (hAT-MSCs) can be

Full text of DOI:10.1016/j.bmcl.2014.03.034

Bioorganic & Medicinal Chemistry Letters 24 (2014) 2141–2145
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Kojyl cinnamate ester derivatives promote adiponectin production
during adipogenesis in human adipose tissue-derived mesenchymal
stem cells
a
b
c
c
a
a
Ho Sik Rho , Soo Hyun Hong , Jongho Park , Hyo-Il Jung , Young-Ho Park , John Hwan Lee ,
a,
⇑
b,
⇑
Song Seok Shin , Minsoo Noh
a
Skin Research Institute, AmorePacific Corporation R&D Center, Yongin, Gyeonggi-do 446-729, Republic of Korea
Natural Products Research Institute, College of Pharmacy, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul 151-742, Republic of Korea
School of Mechanical Engineering, Yonsei University, Seoul, Republic of Korea
b
c
a r t i c l e i n f o
a b s t r a c t
Article history:
The subcutaneous fat tissue mass gradually decreases with age, and its regulation is a strategy to develop
anti-aging compounds to ameliorate the photo-aging of human skin. The adipogenesis of human adipose
tissue-mesenchymal stem cells (hAT-MSCs) can be used as a model to discover novel anti-aging com-
pounds. Cinnamomum cassia methanol extracts were identified as adipogenesis-promoting agents by nat-
ural product library screening. Cinnamates, the major chemical components of Cinnamomum cassia
extracts, promoted adipogenesis in hAT-MSCs. We synthesized kojyl cinnamate ester derivatives to
improve the pharmacological activity of cinnamates. Structure–activity studies of kojyl cinnamate deriv-
Received 6 October 2013
Revised 23 February 2014
Accepted 13 March 2014
Available online 21 March 2014
Keywords:
Cinnamic acid
Kojyl cinnamate ester
Adipogenesis
Human mesenchymal stem cells
atives showed that both the
a,b-unsaturated carbonyl ester group and the kojic acid moiety play core
roles in promoting adiponectin production during adipogenesis in hAT-MSCs. We conclude that kojyl cin-
namate ester derivatives provide novel pharmacophores that can regulate adipogenesis in hAT-MSCs.
Ó 2014 Elsevier Ltd. All rights reserved.
1
Subcutaneous fat tissue gradually thins with age. This is asso-
ciated with the increased risk of skin injury, a deteriorated skin
function to regulate body temperature, and impaired dermal elas-
Approximately ten percent of hMSCs in cell culture have an adipo-
cyte phenotype. When sulfonylurea-type antidiabetic drugs like
glibenclamide and peroxisome proliferator-activated receptor
c
2
,3
ticity. Human mesenchymal stem cells (hMSCs) exist in virtually
all human mesenchymal tissues and can differentiate into adipo-
cytes. Therefore, chemical compounds that promote adipocyte
differentiation in MSCs resident in subcutaneous fat tissue may
counteract the aging-related functional decreases. Human adipose
tissue-mesenchymal stem cells (hAT-MSCs) can be used as a model
system to study adipocyte differentiation in fat tissue.4 In murine
pre-adipocytes like 3T3-L1 cells, treatment with an adipogenic
cocktail consisting of insulin, dexamethasone, and 3-isobutyl-1-
methylxanthine (IBMX) (the IDX condition), can induce nearly all
(PPAR ) agonists like troglitazone are added to hMSCs cultured
c
in the IDX condition, the fraction of differentiated adipocytes is
increased. Chemical compounds with the potential to promote adi-
pocyte differentiation in hAT-MSCs can be evaluated by testing
them in combination with the IDX adipogenic cocktail in cell cul-
4
6
,8
ture. To discover novel pharmacophores capable of regulating
the thickness of subcutaneous fat tissue, we have screened natural
product libraries using hAT-MSCs as an adipocyte differentiation
model. Adiponectin can be measured to quantitatively evaluate
9
the level of adipocyte differentiation in hAT-MSCs. A preliminary
5
,6
pre-adipocytes to acquire differentiated adipocyte phenotypes.
However, the IDX condition cannot induce the adipocyte differen-
tiation of all the hMSCs tested in two dimensional cell cultures.
screening showed that ethanol extracts of Cinnamomum cassia
(Ramulus Cinnamomi and Cortex Cinnamomi) promoted adipocyte
6
,7
10
differentiation (Fig. 1A). The major chemical components of Cin-
namomum cassia include cinnamaldehyde, cinnamic acid, and
1
1
eugenol (Fig. 1B). After determining the highest non-cytotoxic
concentrations for cinnamaldehyde, cinnamic acid, and eugenol
on hAT-MSCs (Fig. 1C–E), we evaluated the effects of the major
chemical components in Cinnamomum cassia on adipocyte differ-
entiation. The results (Fig. 1F) showed that supplementation with
Abbreviations: hAT-MSCs, human adipose tissue-derived mesenchymal stem
cells; PPAR , peroxisome proliferator-activated receptor gamma.
Corresponding authors. Tel.: +82 31 280 5915; fax: +82 31 281 8391 (S.S.S.);
c
⇑
tel.: +82 2 880 2481 (M.N.).
E-mail addresses: ssshin@amorepacific.com (S.S. Shin), minsoo@alum.mit.edu
(
M. Noh).
http://dx.doi.org/10.1016/j.bmcl.2014.03.034
960-894X/Ó 2014 Elsevier Ltd. All rights reserved.
0

2
142
H. S. Rho et al. / Bioorg. Med. Chem. Lett. 24 (2014) 2141–2145
A
B
O
H
4
3
2
1
00
00
00
00
0
O
O
H
H 3 CO
H O
C
D
E
1
1
50
00
150
100
50
150
100
50
5
0
0
0
0
F
6
4
2
00
00
00
0
Figure 1. Effects of Cinnamomum cassia extracts on adipogenesis in hAT-MSCs. hAT-MSCs were grown under IDX conditions and/or co-treated with 30 lg/ml of ethanol
extracts of Cinnamomi Ramulus and Cinnamomi Cortex (A). After the induction of adipogenesis, the media were replaced every two or three days. On the 12th day in culture,
cell culture supernatants were harvested. ELISA was performed to measure the levels of adiponectin accumulated in the supernatants during the 48 h after the last medium
exchange. Cytotoxicity of the major chemical components, cinnamaldehdye, cinnamic acid, and eugenol, on hAT-MSCs was determined using 4-3-[4-lodophenyl]-2-4(4-
nitrophenyl)-2H-5-tetrazolio-1,3-benzene disulfonate (WST, Roche Molecular Biochemical, Indianapolis, IN, USA). The absorbance of the samples (A450) was determined, and
absolute optical density expressed as a percentage of the control value (B–E). The effects of cinnamaldehdye, cinnamic acid, and eugenol on adiponectin production in hAT-
⁄
⁄⁄
MSCs during adipogenesis were evaluated (F). Values represent mean ± SD (n = 3). P 6 0.05 and P 6 0.01.
cinnamaldehyde (30
l
M) and cinnamic acid (100
l
M) in the IDX
acid derivatives have in vivo anti-aging activity in human skin,
we intuitively expected that compound 4b may counteract the
functional regression of aged human skin. In human clinical stud-
ies, we demonstrated that compound 4b increased procollagen
synthesis and decreased matrix metalloproteinase-1 (MMP-1) in
photo-aged human skin in vivo. Because compound 4b also
contains a cinnamate moiety, we evaluated whether, like cinna-
maldehyde and cinnamic acid, it could affect adipogenesis in
condition significantly increased adiponectin production (by 70%
and 46%, respectively), compared with that in the IDX control. In
contrast, eugenol did not promote adipogenesis in hAT-MSCs
(Fig. 1F). Therefore, cinnamaldehyde and cinnamic acid may con-
1
2
tribute to the effect of the Cinnamomum cassia methanol extract
in promoting adipogenesis in hAT-MSCs.
Previously, we synthesized the 3,4-methylenedioxy cinnamate
derivative of kojic acid (4b, Seletinoid G, Scheme 1) as a new
hAT-MSCs. In a preliminary screen, we found 100 lM of 4b
1
2
anti-aging compound. Kojic acid is produced from carbohydrate
promoted adiponectin production in hAT-MSCs (data not shown).
Therefore, to study the structure activity relationship of kojyl
cinnamate derivatives of 4b in promoting adiponectin biosynthetic
activity in hAT-MSCs, we synthesized additional cinnamate
1
3
sources in an aerobic process by a variety of microorganisms. It
exerts various biological effects-such as an inhibition of tyrosinase,
chelation of metal ions, free radical scavenging, and prevention of
15
photo-damage due to its
c
-pyranone structure containing an enolic
Compound 4b was designed based on the
assumption that the -pyranone ring of kojic acid would mimic
the carboxylic acid moiety in retinoid structures. Because retinoic
derivatives (4a and 4c) and modified compounds (4d–4f). The
synthetic pathways are shown in Scheme 1. Cinnamate derivatives
(4a–4c), a benzoate derivative (4e), and a hydrocinnamate deriva-
tive (4d) were synthesized by the condensation of kojyl chloride 2
1
4
hydroxyl group.
c

H. S. Rho et al. / Bioorg. Med. Chem. Lett. 24 (2014) 2141–2145
2143
O
c
O
R1
R2
O
O
Cl
O
OMe
OMe
O
1
2
3
4f : R , R = -OCH O-
2
b
O
O
O
O
a
c
HO
Cl
R1
O
O
OH
OH
R2
OH
O
O
1
2
1
, Kojic acid
2
4a : R , R = H
4
4
1
2
b : R , R = -OCH O-
2
1
2
c : R , R = -OCH CH O-
2
2
O
c
R1
R2
O
O
O
OH
O
R1
R2
O
O
O
1
2
4
d : R , R = -OCH O-
2
OH
1
2
4
e : R , R = -OCH O-
2
2 2 3
Scheme 1. Reaction and conditions: (a) SOCl , DMF, rt; (b) dimethylsulfate, K CO , acetone, reflux; (c) potassium salts of acids, DMF, 100–120 °C.
with the potassium salts of acids.15 The 5-methoxy-protected com-
pound (4f) was synthesized by the condensation of 5-methoxy ko-
jyl chloride (3) with the potassium salt of 3,4-methylenedioxy
cinnamic acid. Compared with the IDX condition, the addition
of 4a, 4b, or 4c to the IDX adipogenic cocktail promoted adiponec-
tin production in hAT-MSCs by 280%, 420%, or 370%, respectively.
Kojyl cinnamate ester derivatives 4a, 4b, and 4c, showed more po-
tent activities than cinnamic acid (Table 1 and Fig. 2). Treatment
with the benzoate derivative 4e increased adiponectin production
by 111%, making it almost as potent as cinnamic acid. A hydrocin-
namate derivative 4d was inactive. The inactive compound 4d has
concentration 50 (EC50) values for glibenclamide and troglitazone
were 3.49 and 0.32 M (Fig. 3A). In parallel experiments, the EC50-
values for 4a, 4b, and 4c were 60.5, 40.3, and 45.5 M, respectively.
The increase in adiponectin levels in culture supernatants during
adipogenesis in hAT-MSCs by the addition of cinnamaldehyde
and cinnamic acid were far below the half-maximal adiponectin le-
vel in the dose-response curve (Table 1). Taken together, both the
l
l
1
5
a,b-unsaturated carbonyl ester structure and the kojic acid moiety
play roles in the pharmacological effects of kojyl cinnamate ester
derivatives in promoting adiponectin production during adipogen-
esis in hAT-MSCs.
the reduced form of the
carbonyl ester of the active compound 4b, suggesting that the
,b-unsaturated carbonyl ester structure plays an important role
in promoting adiponectin production by the hAT-MSCs. In addi-
tion, 4e, the compound lacking the ,b-unsaturated double bond
a
,b-unsaturated double bond next to the
The kojyl ester compound of cinnamaldehyde, 4b, was initially
identified as an anti-aging compound because it increased dermal
1
2
a
collagen levels in photo-aged human skin. Although it is clini-
cally effective, the pharmacological mechanism of action of the
compound 4b has not been fully explained. Adiponectin is known
to stimulate the production of extracellular matrix components
such as type I collagen and hyaluronic acid in dermal fibroblasts.16
Because 4b significantly promoted adipocyte differentiation in
hAT-MSCs (Fig. 3), it may increase adipocyte differentiation in tis-
sue-resident hMSCs to affect the mass of subcutaneous fat tissue in
human skin. Therefore, these results suggest that the pharmaco-
logical mechanisms of the 4b compound associated with the in-
creased ECM production in human skin are indirectly mediated
by paracrine effects mediated by subcutaneous fat tissue-derived
adiponectin on dermal fibroblasts in human skin.
a
moiety adjacent to the carbonyl ester, was less potent than com-
pounds with this moiety 4a, 4b and 4c. This supports the core role
of the
a,b-unsaturated carbonyl ester structure in promoting adi-
pogenesis in hAT-MSCs. Importantly, 4f, in which the kojic acid
moiety of compound 4b is 5-methoxy substituted, had no effect
on adipogenesis (Table 1). Because a chemical modification of the
kojic acid moiety in 4b resulted in the loss of pharmacological
activity, the kojic acid moiety of kojyl cinnamate ester derivatives
played an essential role in the promotion of adipogenesis in hAT-
MSCs.
Next, we tested the concentration-dependent effect of 4a, 4b,
and 4c (Fig. 3A), and compared their potencies with those of gli-
benclamide and troglitazone in promoting adiponectin production
during hAT-MSC adipogenesis (Fig. 3B). In dose-response analyses
for glibenclamide or troglitazone, adiponectin in culture superna-
tants reached maximal levels at higher drug concentrations. From
the maximal level in the dose-response curves, the effective
Additionally, the up-regulation of adiponectin production is
also associated with an increase in insulin sensitivity.6 In this
regard, kojyl cinnamate ester derivatives provide novel pharmaco-
phores for developing anti-diabetic drugs. However, the adipogen-
esis model using hMSCs, one example of a phenotypic cell-based
assay for measuring insulin sensitivity, does not provide direct
information about the molecular targets. As both sulfonylurea-type
–8

2
144
H. S. Rho et al. / Bioorg. Med. Chem. Lett. 24 (2014) 2141–2145
Table 1
Effects of cinnamate-kojic acid ester derivatives on adiponectin production during adipogenesis in hAT-MSCs
*
*
Compound
Adiponectin (pg/ml)
Cell viability (CV%, mean ± SD)
Mean ± SD, n = 3
CV50 (lM)
CV90 (lM)
Vehicle control
Glibenclamide 30
32 ± 11
30 ± 5
lM
93 ± 11
56 ± 11
36 ± 14
24 ± 2
79 ± 2
504 ± 32
61 ± 21
82 ± 5
72 ± 18
87 ± 29
95 ± 22
85 ± 32
Troglitazone 10
Cinnamic acid 60
Kojic acid 400
l
M
24 ± 13
26 ± 17
34 ± 3
29 ± 4
27 ± 17
32 ± 8
29 ± 10
35 ± 3
26 ± 15
302 ± 50
1832 ± 110#
lM
202 ± 46
1345 ± 292
151 ± 17
181 ± 10
161 ± 10
197 ± 23
192 ± 29
181 ± 27
l
M
4a 60
4b 60
4c 60
4d 60
4e 60
4f 60
l
l
M
M
lM
l
M
lM
lM
IDX control
IDX + glibenclamide 30
IDX + troglitazone 10
IDX + cinnamic acid 60
IDX + kojic acid 400
l
M
M
#
l
2005 ± 193
501 ± 62
*
l
M
l
M
312 ± 103
IDX + 4a 60
IDX + 4b 60
IDX + 4c 60
IDX + 4d 60
IDX + 4e 60
IDX + 4f 60
l
l
M
M
1068 ± 176#
#
1435 ± 113
lM
1300 ± 140#
364 ± 16
l
M
*
lM
614 ± 77
463 ± 36
*
lM
#
*
Denotes p <0.01 for statistical comparison between the IDX control and the IDX + compound.
Denotes p <0.05.
*
*
Cell viability (CV) was determined using the WST method in hAT-MSCs as described in Figure 1. CV50 and CV90 are the concentration of 50% and 90% of cells are viable
compared to control, respectively.
A 2
000
500
000
*
*
*
*
*
*
1
1
**
*
5
00
0
hAT-MSC control
IDX control
O
O
O
S
O
B
S
O
O
O
OCH
3
O
N
H
N
H
O
O
O
O
N
H
O
O
N
H
O
H
H
O
Cl
IDX+Kojic acid (400 M)
IDX+Cinnamic acid (60 M)
2
1
1
000
500
000
** **
*
** **
*
*
*
Maximum effect
*
*
Glibenclamide
Troglitazone
Compound 4b
ED50
*
*
*
*
5
00
IDX control
0
0.1
1
10 100
IDX + 4b 30
M
IDX+Glibenclamide30
Maximum effect)
M
IDX + Compound ( M)
(
Figure 3. Concentration-dependent effects of kojyl cinnamate ester derivatives on
adiponectin production during adipogenesis in hAT-MSCs. After the induction of
adipogenesis, the media were replaced every two or three days. On the 12th day in
culture, cell culture supernatants were harvested. ELISA was performed to measure
the levels of adiponectin accumulated during the 48 h after the last medium change
Figure 2. Phenotypic changes induced in hAT-MSCs by the kojyl cinnamate ester
derivative 4b. Twelve days after adipogenic stimulation with IDX, lipid droplets in
adipocytes were stained with Oil Red O (ORO). The differentiated adipocytes in hAT-
MSC cultures were photographed using an Olympus IX71 inverted phase-
microscope.
(
A). CA denotes cinnamic acid. Glibenclamide and troglitazone were used as
positive controls to plot the concentration dependence curve for 4b (B). Values
⁄
⁄⁄
represent mean ± SD (n = 3). P 6 0.05 and P 6 0.01.
antidiabetic drugs and PPAR
hMSCs,
c
agonists promote adipogenesis in
it is possible that kojyl cinnamate ester derivatives can
regulate the same molecular targets. When we evaluated whether
6
–8
kojyl cinnamate ester derivatives transactivate PPARc
,6 no signifi-
cant effect on PPARc activity was detected (data not shown).

H. S. Rho et al. / Bioorg. Med. Chem. Lett. 24 (2014) 2141–2145
2145
Therefore, kojyl cinnamate ester derivatives may affect other
molecular targets to regulate adipogenesis. Further studies
examining hAT-MSCs as a model will aim to synthesize novel
compounds with an improved activity to stimulate adiponectin
production during adipogenesis, and to identify the direct
molecular targets of kojyl cinnamate ester derivatives.
(Invitrogen, Carlsbad, CA). To induce adipocyte differentiation, the growth
medium was replaced by DMEM containing a high concentration of glucose
(
4.5 g/L) and supplemented with 10% FBS, Glutamax™, 10 lg/mL insulin, 1 lM
dexamethasone, and 0.5 mM 3-isobutyl-1-methylxanthine (IBMX). Ethanol
extracts of Cinnamomum cassia were obtained from Korea Plant Extract Bank
(http://extract.pdrc.re.kr/). Cinnamaldehyde, cinnamic acid, and eugenol were
purchased from Sigma–Aldrich Chemical Company (St. Louis, MO). Adipocyte
differentiation was assessed using an oil red O (ORO) stain as an indicator of
intracellular lipid accumulation. After hBM-MSCs had differentiated to
adipocytes, cells were rinsed twice with phosphate-buffered saline (PBS),
fixed with 10% formalin in PBS (pH 7.4) for 1 h, and then washed with 60%
isopropanol, before being allowed to dry completely. hBM-MSCs were stained
with 0.2% ORO reagent for 10 min at room temperature, and then washed with
In conclusion, kojyl cinnamate ester derivatives promoted adi-
pogenesis in hAT-MSCs and increased adiponectin production.
Both the
a,b-unsaturated carbonyl ester structure and the kojic
acid moiety are core structural features important in the pharma-
cological activity. These results suggest that the clinically proven
anti-aging activity of kojyl cinnamate ester derivatives in human
skin may be mediated by up-regulation of adiponectin synthesis
in hAT-MSCs residing in subcutaneous fat tissue. In addition, kojyl
cinnamate ester compounds may exert anti-diabetic effects by
increasing insulin sensitivity, as shown using hAT-MSCs in an
adipogenesis model. Therefore, kojyl cinnamate ester derivatives
provide novel pharmacophores that may play anti-aging roles in
photo-aged human skin as well as anti-diabetic roles by improving
insulin sensitivity.
2
H O four times. Following a 10 min elution of each hBM-MSCs sample with
100% isopropanol, absorbance was measured at 500 nm using
a
spectrophotometer. To visualize the nucleus, hBM-MSCs were counterstained
with hematoxylin reagent for 2 min and then washed twice with H O. The level
2
of adipocyte differentiation was observed and counted using an inverted
phase-microscope. For quantitative determination of adiponectin in cell
culture supernatants, Quantikine™ immunoassay kits were used (R&D
Systems, Minneapolis, MN, US.
10. The methanol extracts for 200 medicinal plants traditionally used in China and
Korea were obtained from the Plant Extract Bank of the Korea Research
Institute of Bioscience and Biotechnology (http://extract.kribb.re.kr/extract/
f.htm). Ten
lg/ml of a methanol extract was treated in the adipogenesis-
induced hAT-MSCs for a preliminary screening to discover candidate plant
extracts to promote adiponectin production.
1
1
1
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potassium salt of 3,4-methylenedioxycinnamic acid (6.88 g, 30.0 mmol) with
,4-methylenedioxycinnamic acid (4.32 g, 22.5 mmol). The reaction mixture
1
2
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4
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.
.
.
.
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1
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2
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.
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6
7
8
.
.
148.0, 146.0, 145.7, 139.8, 128.2, 125.3, 114.6, 112.5, 108.4, 106.7, 101.6, 61.2.
+
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