Synthesis, determination of stereochemistry, and evaluation of new bisindole alkaloids from the myxomycete Arcyria ferruginea: An approach for Wnt signal inhibitor

Article abstract of DOI:10.1016/j.bmcl.2007.05.033

To determine the stereochemistry of dihydroarcyriarubin C (1), new bisindole alkaloid isolated from the myxomycete Arcyria ferruginea, cis- (2) and trans-dihydroarcyriarubin C (3) were synthesized. Comparison of their NMR characteristics allowed the trans

Full text of DOI:10.1016/j.bmcl.2007.05.033

Bioorganic & Medicinal Chemistry Letters 17 (2007) 4254–4257
Synthesis, determination of stereochemistry, and evaluation of new
bisindole alkaloids from the myxomycete Arcyria ferruginea:
An approach for Wnt signal inhibitor
Kouken Kaniwa, Midori A. Arai, Xiaofan Li and Masami Ishibashi*
Graduate School of Pharmaceutical Science, Chiba University, Chiba 263-8522, Japan
Received 26 April 2007; revised 8 May 2007; accepted 10 May 2007
Available online 16 May 2007
Abstract—To determine the stereochemistry of dihydroarcyriarubin C (1), new bisindole alkaloid isolated from the myxomycete
Arcyria ferruginea, cis- (2) and trans-dihydroarcyriarubin C (3) were synthesized. Comparison of their NMR characteristics allowed
the trans stereochemistry of the natural product to be confirmed. Moreover, the Wnt signal inhibitory activities of 2 and 3 were
compared with that of arcyriaflavin C (4), which is a natural product containing a bond between C-2 and C-20. The cis-dihydroar-
cyriarubin C (2) showed moderate inhibition of Wnt signal transcription, which suggests that bisindole frameworks might be useful
as small-molecule Wnt signal inhibitors.
Ó 2007 Elsevier Ltd. All rights reserved.
Bisindole alkaloids have received considerable attention
because they have potential for biological activity. Our
recent interest in myxomycetes, an unusual group of
primitive organisms, has led us to isolate new natural
compounds;1–3 for instance, we have recently isolated
bon in methanol afforded an 82% yield of the meso
succimide (10). The dl isomer (11) was readily prepared
by reduction with magnesium in refluxing methanol.6a–d
To clarify the configuration, the cis-isomer (10) was
treated with magnesium in refluxing methanol to give
a
new bisindole alkaloid, dihydroarcyriarubin
C
1
the thermally stable trans-isomer (11). The H NMR
(Fig. 1: 1), together with arcyriaflavin C (4),3 from the
myxomycete Arcyria ferruginea.4 However, we had not
determined its stereochemistry and bioactivity. Here,
we describe the synthesis of two stereoisomers of 1,
cis- (2) and trans-dihydroarcyriarubin C (3), and use
them to determine its stereochemistry. Moreover, we
found the Wnt signal inhibitory activity of cis-dihyd-
roarcyriarubin C (2) and arcyriaflavin C (4).
peak patterns were similar for natural product 1 and
the trans-isomer (11) strongly suggesting that 1 has a
trans configuration.7
Several methods for deprotecting the isomers were
examined, but the corresponding bisindole products
From a number of possible synthetic approaches to the
bisindole compounds, we first attempted a coupling
between the appropriate indolyl-glyoxlate and indolyl-
acetamide (Scheme 1).5 Reduction of 6-methoxyindolyl
glyoxylamide (5) with NaBH4 followed by dehydroxyla-
tion under TMSCl, NaI conditions, gave methyl indolyl-
acetamide (7). Methyl indolyl-glyoxlate (8), prepared
from 6-methoxy indole, reacted with 7 to give bis-indo-
lylmaleimide (9). Hydrogenation with palladium on car-
H
H
N
10
N
O
O
O
O
9
8
8'
4
4'
3a
5
3
2
HO 6
OH
HO
OH
OH
1'N
H
1
7a
N
H
N
N
7
7'
H
H
4
1
H
N
H
N
O
H
O
H
O
H
O
H
HO
HO
OH
N
N
N
N
H
H
H
H
Keywords: Arcyria ferruginea; Bisindole alkaloid; Wnt signal inhibitor.
*
3
2
Corresponding author. Tel./fax: +81 43 290 2913; e-mail: mish@
p.chiba-u.ac.jp
Figure 1. Structures of compounds 1–4.
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.05.033

K. Kaniwa et al. / Bioorg. Med. Chem. Lett. 17 (2007) 4254–4257
4255
O
NH2
O
HO
NH2
O
NH2
O
a
b
MeO
MeO
MeO
N
H
N
H
N
H
5
6
7
H
H
N
N
O
O
O
O
O
OMe
O
H
H
d
c
7
+
MeO
MeO
OMe
MeO
OMe
N
N
N
H
N
N
H
H
H
H
8
9
10
f
H
N
e
O
O
H
H
MeO
OMe
N
N
H
H
11
Scheme 1. Reagents and conditions: (a) NaBH4, MeOH, 4 h, 61%; (b) TMSCl, NaI, CH3CN, 0 °C, 5 h, 35%; (c) KOtBu, THF, 0 °C to rt, 50%; (d)
cat. Pd/C, H2, MeOH, 48 h, 82%; (e) Mg, MeOH, reflux, 10 h, 56%; (f) Mg, MeOH, reflux, 2 h, 57%.
were not obtained. From the desire to change the pro-
tective group and to improve the yield of the coupling
of 7 and 8, the synthetic route was changed to use the
reaction of 2,3-dibromo-N-methylmaleimide and 4-ben-
zyloxyindole. The compound 12 was synthesized in four
steps following the procedure of Ohkubo et al.8 The
conditions for conversion of 12 to the maleic anhydride
13 were examined (Table 1). Compound 12 is quite
insoluble in H2O, so no reaction took place under aque-
ous conditions (run 1); using an organic solvent (diox-
ane, ethanol) allowed synthesis of 13 in good yield
under reflux conditions (run 3, 4).
(3) are shown in Table 2. The chemical shifts of 3 were
identical to those of 1; the cis- and trans-isomers were
obviously different. Thus, the stereochemistry of 1 was
shown to be trans.9
With synthetic cis- and trans-dihydroarcyriarubin C in
hand, we evaluated their biological activities.10 Arcyria-
flavin C (4), which has a C–C bond between C-2 and C-
20, was also synthesized.6b,8 The Wnt signal transduction
pathway has important functions in vertebrate develop-
ment, and its deregulation is believed to occur early in
human colorectal cancer.11,12 Wnt target genes have
recently been found to be highly expressed in colon can-
cers; some of the genes are likely to contribute to cancer
formation.13 Small-molecule inhibitors of this pathway
are desired because it is believed their clinical use would
suppress cancer cell growth. Several potent small-mole-
cule inhibitors have been reported;14a–4d however, the
development of therapies to specifically target the Wnt
pathway in cancer cells is still in its infancy.
After ammonolysis of 13, compound 14 was reduced to the
cis-isomer (2) using Pd/C under H2 atmosphere (Scheme 2).
Treatment of 14 with Mg gave compound 15 in 84% yield,
and then 3 was obtained by reduction. Product 3, produced
by epimerization of 2 under basic conditions, proved to be
the thermally stable trans-isomer.
NMR data from the natural isolated dihydroarcyriaru-
bin C (1) and the synthesized cis- (2) and trans-isomers
We examined the Wnt signal inhibitory activity of our
synthesized compounds using the luciferase reporter
gene assay. Wnt signaling activates gene transcription
by forming a complex between DNA-binding proteins
of the Tcf/LEF family and b-catenin. SuperTOP-Flash,
a b-catenin-responsive reporter plasmid with multiple
TCF-binding sites (CCTTTGATC), was activated in
cells.15,16 SuperFOP-Flash has eight mutated TCF-bind-
ing sites (CCTTTGGCC), so a selective inhibitor would
not show enhanced transcription in SuperFOP-Flash-
transfected cells; thus, it provides a negative control
for the assay and the TOP/FOP-Flash reporter activa-
tion ratio provides a measure of selective Wnt signal
inhibition.
Table 1.
Me
N
O
O
O
O
O
KOH (1.0 eq)
conditions
BnO
OBn
BnO
OBn
N
N
N
N
H
H
H
H
12
13
Runa Solvent (v/v)
Temperature Time Yield (%)
(h)
(°C)
1
2
3
4
H2O
Dioxane/H2O (5:1) rt
Reflux
10
96
24
4.5
No reaction
0
The results are shown in Figure 2, along with cell viabil-
ity in order that a decrease in cell number should not
resemble inhibition. The cis-isomer (2) exhibited moder-
ate dose-dependent Wnt signal inhibition, whereas the
Dioxane/H2O (5:1) Reflux
Reflux
84
89
EtOH/H2O (5:1)
a All reactions were carried out using 2 M aq KOH.

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K. Kaniwa et al. / Bioorg. Med. Chem. Lett. 17 (2007) 4254–4257
H
N
inhibit a b-catenin–TCF protein interaction. A protein
level mechanistic study is in progress.
O
O
b
a
2
13
In summary, the configuration of the new bisindole
compound dihydroarcyriarubin C (1), which had previ-
ously been isolated by our group, was determined to be
trans by comparison of its NMR data with that of its
synthesized cis- (2) and trans-isomers (3). The cis-dihyd-
roarcyriarubin C (2) showed moderate inhibition of Wnt
signal transcription. Thus, we suggest that a bisindole
framework would be included as a candidate for
small-molecule Wnt signal inhibitor.
BnO
OBn
N
N
H
H
14
e or f
c
H
N
O
H
O
H
d
3
BnO
OBn
N
N
H
H
15
Acknowledgments
Scheme 2. Reagents and conditions: (a) NH4OAc, 140 °C, 3 h, 97%;
(b) cat. Pd/C, H2, EtOH, 12 h, 95%; (c) Mg, MeOH, reflux, 6 h, 84%;
(d) cat. Pd/C, H2, EtOH, 5 h, 98%; (e) Mg, MeOH, reflux, 7 h, 81%; (f)
DBU, THF, rt to reflux, 15 h, 93%.
We appreciate prof. Jeremy Nathans (John Hopkins
Medical School) for kindly offering STF cells (293 cell
line stably transfected with a SuperTOP-Flash by prof.
R.T. Moon) and prof. Randall T. Moon (University
of Washington) for the SuperFOP-Flash reporter plas-
mid. This work was supported by a Grant-in-Aid for
Young Scientists (B) from the Ministry of Education,
Culture, Sports, Science and Technology, Japan (No.
18790012 to M.A.A.), and in part by Grants-in-Aid
for Scientific Research on Priority Areas (No.
18032020 to M.I.). M.A.A. also gratefully acknowledges
Uehara Memorial Foundation for financial support.
trans-isomer (3) had no effect. Interestingly, arcyriafla-
vin C (4) also showed dose-dependent inhibition
between 12.5 and 50 lM, although the Wnt signal acti-
vation effect seems to occur at low concentration. There
is no reported potent inhibitor with bisindole frame-
work, we believe that these results are interesting for a
search of Wnt signal inhibitor from many known or
new indole compounds. A large number of bisindole
compounds have been reported as kinase inhibitors.
We also reported kinase inhibitory activities of bisindole
alkaloids from a myxomycete.2 Wnt signal pathway
includes several kinases such as GSK3b which
phosphorylates b-catenin for degradation. Because these
kinase inhibition may lead to activation of b-catenin–
TCF transcription, these bisindole compounds might
Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.bmcl.
2007.05.033.
Table 2. 1H and 13C NMR data of compounds 1–3 (in acetone-d6)
Dihydroarcyriarubin C (1)
2 (cis)
3 (trans)
a
dc
dH (Hz)
dH (Hz)
dc
dH (Hz)
dc
1,10
9.88 br s
7.15 d (2.2)
9.48 br s
6.60 d (2.0)
9.88 br s
7.16 d (2.4)
2,20
122.3
111.9
121.0
120.1
110.2
154.7
97.7
123.4
109.7
122.2
120.0
109.8
154.0
97.3
122.6
112.0
121.0
120.1
110.4
139.1
97.7
3,30
3a,3a0
4,40
7.25 d (8.5)
6.61 dd (2.2, 8.5)
7.28 d (8.8)
7.26 d (8.4)
5,50
6.49 dd (2.0, 8.8)
6.76 d (2.0)
4.90 s
6.61 dd (2.4, 8.4)
6.85 d (2.4)
4.45 s
6,60
7,70
6.85 d (2.2)
4.44 s
7,7a0
8,80
139.1
48.8
178.5
138.0
46.3
179.2
139.1
48.9
178.3
9,90
6,60-OH
10-NH
7.91 br s
9.51 br s
7.73 s
10.28 br s
7.91 br s
10.29 br s
a The 13C NMR chemical shifts of natural dihydroarcyriarubin C (1) were reinvestigated and revised as shown here.
Figure 2. Wnt signal inhibitory activity. (a) cis-dihydroarcyriarubin C (2); (b) trans-dihydroarcyriarubin C (3); (c) arcyriaflavin C (4).

K. Kaniwa et al. / Bioorg. Med. Chem. Lett. 17 (2007) 4254–4257
4257
9. Natural product (1) showed no particular CD curve,3
indicating that natural product (1) was also dl.
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