Synthesis and evaluation of (+)-decursin derivatives as inhibitors of the Wnt/β-catenin pathway

Article abstract of DOI:10.1016/j.bmcl.2016.06.029

We synthesized (+)-decursin derivatives substituted with cinnamoyl- and phenyl propionyl groups originating from (+)-CGK062 and screened them using a cell-based assay to detect relative luciferase reporter activity. Of this series, compound 8b, in which a

Full text of DOI:10.1016/j.bmcl.2016.06.029

Bioorganic & Medicinal Chemistry Letters xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and evaluation of (+)-decursin derivatives as inhibitors
of the Wnt/b-catenin pathway
Jee-Hyun Lee a,y, Min-Ah Kim a,y, Seoyoung Park b,y, Soo-Hyun Cho a, Eunju Yun a, Yu-Seok O a, Jiseon Kim a,
Ja-Il Goo d, Mi-Young Yun c, Yongseok Choi d, Sangtaek Oh b, , Gyu-Yong Song a,
⇑
⇑
a College of Pharmacy, Chungnam National University, Daejeon 305-764, Republic of Korea
b Department of Bio and Fermentation Convergence Technology, Kookmin University, Seoul 136-702, Republic of Korea
c Department of Beauty Science, Kwangju Women’s University, Kwangju 506-713, Republic of Korea
d College of Life Sciences and Biotechnology, Korea University, Seoul 136-701, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
We synthesized (+)-decursin derivatives substituted with cinnamoyl- and phenyl propionyl groups orig-
inating from (+)-CGK062 and screened them using a cell-based assay to detect relative luciferase reporter
activity. Of this series, compound 8b, in which a 3-acetoxy cinnamoyl group was introduced, most
potently inhibited (97.0%) the Wnt/b-catenin pathway. Specifically, compound 8b dose-dependently
inhibited Wnt3a-induced expression of the b-catenin response transcription (CRT) and increased b-cate-
nin degradation in HEK293 reporter cells. Furthermore, compound 8b suppressed expression of the
downstream b-catenin target genes cyclin D1 and c-myc and suppressed PC3 cell growth in a concentra-
tion-dependent manner.
Received 4 November 2015
Revised 7 June 2016
Accepted 10 June 2016
Available online xxxx
Keywords:
Cinnamoyl decursin
Phenylpropionyl decursin
Wnt/b-catenin pathway
Prostate cancer
Ó 2016 Published by Elsevier Ltd.
Protein degradation
The Wnt/b-catenin signaling pathway is associated with
embryonic development and regulates cell proliferation and
differentiation. Abnormal activity of this pathway and increased
b-catenin-dependent transcription induces tumor development
including colon and prostate cancer.1–3
In this study, we synthesized (+)-decursin derivatives with var-
ious cinnamoyl and phenyl propionyl groups using (+)-CGK062 as
the lead compound. These compounds were screened using a cell-
based assay to detect relative luciferase reporter activity.
Semi-synthesis of the (+)-decursin derivatives is outlined in
Scheme 1. (+)-Decursinol (4) was the starting material obtained
by hydrolyzing an ethanol extract of Angelica gigas root with
alkaline solvent.8 Esterification by EDC-mediated coupling
provided non-substituted cinnamoyl-, halo-substituted cinnamoyl-,
nitro-substituted cinnamoyl- and methoxy-cinnamoyl-decursin
derivatives 5a–d and 6a–i. Demethylation of (OCH3)n-cinnamoyl
decursin derivatives 6a–d and 6h was accomplished using 1 M
BBr3 solution, leading to 7a–e. The (OH)n-cinnamoyl decursin
derivatives 3 (CGK062) and 7a–e were acetylated using acetyl
chloride to obtain good yields of the (OAc)n-cinnamoyl decursin
derivatives 8a–f.9–16 We synthesized (+)-decursin derivatives by
introducing various substituted phenyl propionyl groups. The
reaction conditions for these compounds were identical to the
conditions used for compounds 6–8.
6,7-Dihydroxycoumarin (1, Fig. 1), also called esculetin, was
recently identified as a potential Wnt/b-catenin pathway inhibitor.
This compound suppresses growth of human colon cancer cells by
disrupting formation of the b-catenin-Tcf complex.4 We also found
that (+)-decursin (2, Fig. 1) and (+)-CGK062 (3, Fig. 1), which have a
coumarin-ring skeleton similar to esculetin, inhibit Wnt/b-catenin
pathway activity.5–7 In particular, (+)-CGK062, in which a 3,4-dihy-
droxy cinnamoyl group was introduced, potently inhibits (99.5%)
the Wnt/b-catenin pathway and dose-dependently antagonizes
Wnt3a-induced b-catenin response transcription (CRT). In addi-
tion, 100 mg/kg (+)-CGK062 inhibits PC3 tumor growth and
50 mg/kg (+)-CGK062 inhibits >80% of subcutaneously established
PC3 xenograft tumor growth in athymic nude mice. This compound
induces no observable signs of toxicity in mice.
We used a cell-based screening system to identify decursin
derivatives that inhibit the Wnt/b-catenin pathway. HEK293 repor-
ter cells were stably transfected with TOPFlash and the hFz-1
expression plasmid. TOPFlash reporter activity was monitored
⇑
Corresponding authors. Tel.: +82 2 910 5732; fax: +82 2 910 5739 (S.O.); tel.:
+82 42 821 5923; fax: +82 42 823 6566 (G.-Y.S.).
E-mail addresses: ohsa@kookmin.ac.kr (S. Oh), gysong@cnu.ac.kr (G.-Y. Song).
These authors contributed equally to this work.
y
using a microplate reader after adding Wnt3a-conditioned
http://dx.doi.org/10.1016/j.bmcl.2016.06.029
0960-894X/Ó 2016 Published by Elsevier Ltd.
Please cite this article in press as: Lee, J.-H.; et al. Bioorg. Med. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.bmcl.2016.06.029

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J.-H. Lee et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
Table 1
O
O
O
O
O
OH
OH
O
Inhibitory percentage of the Wnt/b-catenin pathway for (+)-decursin derivatives,
introduced cinnamoyl group
O
Compd No.
R1
% of inhibitiona
1. esculetin
2. (+)-decursin
IC50= 175.9uM
2
3
(+)-Decursin
(+)-CGK062
–H
–3-F
–3-Br
79.4
99.5
81.1
93.6
84.8
40.1
34.3
88.3
87.5
54.8
57.8
52.8
65.3
42.3
46.9
59.6
94.2
93.3
78.1
95.7
75.3
95.9
97.0
79.6
89.7
94.0
67.4
O
O
O
O
5a
5b
5c
5d
5e
6a
6b
6c
6d
6e
6f
OH
OH
O
–4-Br
–4-NO2
3. (+)-CGK-062
IC50= 12.3uM
–2-OCH3
–3-OCH3
–4-OCH3
–2,3-(OCH3)2
–2,4-(OCH3)2
–2,5-(OCH3)2
–3,4-(OCH3)2
–3,4,5-(OCH3)3
–2,4,5-(OCH3)3
–2-OH
–3-OH
–4-OH
–2,3-(OH)2
–3,4,5-(OH)3
–2-OAc
Figure 1. Inhibitors of the Wnt/b-catenin pathway.
medium (Wnt3a-CM) to each decursin derivative.17–22 The controls
were assayed in the presence or absence of Wnt3a-CM.
6g
6h
6i
Results are shown in Tables 1 and 2. The (+)-decursin deriva-
tives with a cinnamoyl group were more potent inhibitors of the
Wnt/b-catenin pathway than (+)-decursin derivatives with a phe-
nyl propionyl group. This result indicates that the double bond
between the ester and phenyl groups is very important for Wnt/
b-catenin pathway inhibitory activity. The position of the cin-
namoyl group substituent on the decursin derivative was a signif-
icant factor for inhibiting the Wnt/b-catenin pathway. Ortho- or
meta-substituted cinnamoyl decursin derivatives 5b, 5c, 6a, 6b,
7a, 7b, 7d, 8a, 8b, 8d, 8e, and 11d were potent inhibitors of the
Wnt/b-catenin pathway.
7a
7b
7c
7d
7e
8a
8b
8c
8d
8e
8f
–3-OAc
–4-OAc
–2,3-(OAc)2
–3,4-(OAc)2
–3,4,5-(OAc)3
a
These compounds were tested in concentration of 20 lM.
In particular, (+)-decursin derivative 8b, with a 3-acetoxy cin-
namoyl group, exhibited strong inhibitory activity, similar to that
of CGK062 which had two phenolic OH group. To our best knowl-
edge, because the proportion of poly phenolic groups of CGK-062
excreted in the free state was very low due to conjugation phenol
groups with glucuronic acid and sulfuric acid in vivo, compound 8b
with acetyl group instead of phenolic groups had the advantage
compared to CGK-062. (+)-Decursin derivatives with a (OH)n-cin-
namoyl group (3, 7a–e) or a (OAc)n-cinnamoyl (8a–f) group
strongly inhibited the Wnt/b-catenin pathway.
We further characterized the effects of compound 8b, 3-(3-ace-
toxy-phenyl)-acrylic acid, and 8,8-dimethyl-2-oxo-6,7-dihydro-
2H,8H-pyrano[3,2-g]chromen-7yl-ester on the Wnt/b-catenin
pathway. Treating HEK293 reporter cells with varying concentra-
tions of compound 8b dose-dependently decreased CRT induced
Table 2
Inhibitory percentage of the Wnt/b-catenin pathway for (+)-decursin derivatives,
introduced phenyl propionyl group
Compd No.
R2
% of inhibitiona
2
3
9
(+)-Decursin
(+)-CGK062
–H
79.4
99.5
10.6
50.4
49.9
41.5
13.5
18.8
19.9
25.1
10.0
42.0
32.8
81.4
68.0
23.1
27.5
12.0
77.7
46.4
10a
10b
10c
10d
10e
10f
10g
11a
11b
11c
11d
11e
12a
12b
12c
12d
12e
–2-OCH3
–3-OCH3
–4-OCH3
–2,3-(OCH3)2
–2,4-(OCH3)2
–2,5-(OCH3)2
–3,4-(OCH3)2
–2-OH
–3-OH
–4-OH
–2,3-(OH)2
–3,4-(OH)2
–2-OAc
by Wnt3a (IC50 = 9.85 lM) (Fig. 2A). A western blot analysis with
an anti-b-catenin antibody was performed to examine whether
compound 8b affected intracellular b-catenin levels in HEK293
cells, as CRT is regulated by intracellular b-catenin.23 b-Catenin
level increased consistently upon treatment with Wnt3a-CM,
which was downregulated by compound 8b (Fig. 2B), consistent
with its inhibitory effect on Wnt3a-stimulated CRT, and suggesting
–3-OAc
–4-OAc
–2,3-(OAc)2
–3,4-(OAc)2
a
These compounds were tested in concentration of 20 lM.
that compound 8b attenuates the Wnt/b-catenin pathway by low-
ering b-catenin protein levels.24
The Wnt/b-catenin pathway is activated by aberrant upregula-
tion of intracellular b-catenin in PC3 prostate cancer cells. Thus,
we carried out a western blot analysis to determine cytoplasmic
b-catenin levels in compound 8b-treated PC3 cells.25 As shown in
Figure 3A, compound 8b dose-dependently downregulated cyto-
plasmic b-catenin. Next, we examined its effect on the expression
of b-catenin-dependent genes in PC3 cells. After incubating with
increasing concentrations of compound 8b, expression of cyclin
D1 and c-myc, established b-catenin target genes, was quantified
by western blot.24,26 As expected, we found a significant reduction
in cyclin D1 and c-myc protein levels (Fig. 3B).
Scheme 1. Reagents and conditions: (a) cinnamic acid or phenyl propionic acid,
EDC, 4-DMAP, dry MC, (b) 1 M BBr3 solution, dry MC, (c) acetyl chloride, pyridine,
dry MC.
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J.-H. Lee et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
3
Figure 2. (A) Dose-dependent inhibition of the b-catenin transcription response. HEK293 reporter cells were incubated with indicated concentrations of compound 8b in the
presence of Wnt3a-CM. After 15 h, luciferase activities were determined and reported as relative light unit (RLU) normalized to cell titer. The results are the average of three
experiments, and the bars indicate standard deviations. (B) Downregulation of the b-catenin protein by compound 8b. Cytosolic proteins were prepared from HEK293
reporter cells treated with either vehicle (DMSO) or indicated concentrations of compound 8b in the presence of Wnt3a-CM for 15 h and then subjected to western blotting
with b-catenin antibody. The blots were reprobed with anti-actin antibody as fraction controls.
Figure 3. Effects of compound 8b on prostate cancer cells. (A) Cytosolic fractions were prepared from PC3 cells treated with vehicle (DMSO) or compound 8b for western
blotting with b-catenin antibody. (B) PC3 cells were incubated with the vehicle (DMSO) or compound 8b for 15 h and then cell extracts were prepared for western blotting
with anti-cyclin D1 and anti-myc antibodies. The blots were reprobed with anti-actin antibody as fraction controls.
D1, we hypothesized that compound 8b would suppress prostate
cancer cell growth. To explore this hypothesis, androgen-indepen-
dent PC3 cells were incubated with varying concentrations of
compound 8b, and cell proliferation was measured using the Cell-
Titer-Glo assay. As presented in Figure 4, compound 8b efficiently
inhibited PC3 cell proliferation with an IC50 value 8.97 l
M.31
In summary, our results show that the double bond included in
the cinnamoyl group of decursin derivatives is necessary for inhi-
bition of the Wnt/b-catenin pathway. Substituting the functional
group in the ortho- or meta-position on the benzene ring of the cin-
namoyl group was most effective. Compound 8b was the most
potent at inhibiting Wnt/b-catenin pathway activity. Compound
8b dose-dependently inhibited CRT induced by Wnt3a
(IC50 = 9.85 lM) in HEK293 reporter cells and decreased intracel-
lular levels of b-catenin protein in the cytosol of PC3 prostate can-
cer cells. In addition, compound 8b promoted b-catenin
degradation and suppressed the expression of cyclin D1 and c-
myc, downstream b-catenin target genes. Compound 8b may lead
to a candidate anti-tumor agent for prostate cancer.
Figure 4. The effect of compound 8b on prostate cancer cell growth. Cells were
incubated, in the indicated concentrations of compound 8b, for 24 h in 96-well
plates. Cell viability was examined using the CellTiter-Glo assay (Promega). The
results are the average of three experiments, and the bars indicate standard
deviations.
Acknowledgments
This study was supported by Basic Science Research Program
Previous studies have demonstrated that specifically inhibiting
the Wnt/b-catenin pathway by expressing Frzb/secreted Frizzled-
related protein, which is a secreted Wnt antagonist, suppresses
growth of androgen-dependent and -independent prostate can-
cer.27–29 In addition, cyclin D1 regulates growth factor-induced
G1 phase cell cycle progression.30 Because compound 8b inhibited
both the Wnt/b-catenin pathway and downstream targets of cyclin
(NRF-2013R1A1A2062764,
2015R1A2A2A01004599)
through
the National Research Foundation of Korea (NRF) funded by the
Ministry of Science, Education and Technology and by the
Priority Research Centers Program through the National Research
Foundation of Korea (NRF) funded by the Ministry of Education,
Science and Technology (2009-0093815).
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J.-H. Lee et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
described previously. In brief, Wnt3a-secreting
Dulbecco’s modified Eagle’s medium with 10% fetal bovine serum for 4 days.
The medium was harvested and sterilized using 0.22- filter. Fresh
L cells were cultured in
Supplementary data
a
lm
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2016.06.
029.
medium was added, the cells were cultured for another 3 days, and the
medium was collected and combined with the previous medium. Transfection
was performed with Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA)
according to the manufacturer’s instructions. The luciferase assay was
performed using a Dual Luciferase Assay kit (Promega, Madison, WI, USA).
21. Plasmid constructs and transfection. The pTOPFlash reporter plasmid was
obtained from Upstate Biotechnology (Lake Placid NY, USA). The pCMV-RL
and pSV-FL plasmids were purchased from Promega. Transfection was
performed with Lipofectamine 2000 (Invitrogen) according to the
manufacturer’s instructions.
22. Screening for a Wnt/b-catenin signaling inhibitor. HEK293 reporter (TOPFlash)
cells were inoculated into 96-well plates at 15,000 cells per well in duplicate
and grown for 24 h. Next, Wnt3a-CM was added, and the compounds were
added to the wells. After 15 h, the plates were assayed for firefly luciferase
activity and cell viability.
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Please cite this article in press as: Lee, J.-H.; et al. Bioorg. Med. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.bmcl.2016.06.029