Synthesis and evaluation of isomers of melleumin B from the myxomycete Physarum melleum; an approach for Wnt signal inhibitor trusted in nature

Article abstract of DOI:10.3987/COM-08-S(N)108

Two stereoisomers of melleumin B (1), seco acid methyl ester of melleumin A (2), which are novel peptide compounds isolated from the myxomycete Physarum melleum, were synthesized and evaluated for their inhibition of Wnt signal transcription. The 3R-epimer and (3R, 4S, 10R, 11R)-isomer of 1 showed moderate inhibition of the Wnt signal.

Full text of DOI:10.3987/COM-08-S(N)108

HETEROCYCLES, Vol. 76, No. 2, 2008
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HETEROCYCLES, Vol. 76, No. 2, 2008, pp. 1425 - 1438. © The Japan Institute of Heterocyclic Chemistry
Received, 30th April, 2008, Accepted, 12th June, 2008, Published online, 19th June, 2008. COM-08-S(N)108
SYNTHESIS AND EVALUATION OF ISOMERS OF MELLEUMIN B
FROM THE MYXOMYCETE PHYSARUM MELLEUM; AN APPROACH
FOR WNT SIGNAL INHIBITOR TRUSTED IN NATURE
Midori A. Arai, Yujiro Uchino, Shuwa Hanazawa, Xiaofan Li, Naoki
Kimura, and Masami Ishibashi*
Graduate School of Pharmaceutical Science, Chiba University, Chiba 263-8522,
Japan e-mail address: mish@p.chiba-u.ac.jp
Dedicated to Professor Ryoji Noyori on the occasion of his 70th birthday
Abstract – Two stereoisomers of melleumin B (1), seco acid methyl ester of
melleumin A (2), which are novel peptide compounds isolated from the
myxomycete Physarum melleum, were synthesized and evaluated for their
inhibition of Wnt signal transcription. The 3R-epimer and (3R, 4S, 10R,
11R)-isomer of 1 showed moderate inhibition of the Wnt signal.
INTRODUCTION
Natural products have important meanings in biological systems. Isolation of new natural products and
synthesis of small molecules based on their scaffolds would be of chemical relevance to living cells and
organs, binding to proteins, absorption, distribution, metabolism, and excretion.1 Recently, we focused on
Wnt signaling, the pathway of which shows aberrant activation in many cancer cells, especially in colon
cancers.2 Efforts have been continuing to obtain a seed compound for a medicine that acts on certain
molecular components in the Wnt signal pathway, such as β-catenin, T-cell factor/lymphoid enhancer
factor (TCF/LEF), and glycogen synthase kinase 3 β (GSK3β) etc. Because in many cases there are
mutated components upstream of this signal, it would be better for inhibitors to act on the transactivation
event more downstream in this pathway. We previously reported that cis-dihydroarcyriarubin C, which is
a synthesized small molecule based on bisindole alkaloid isolated from myxomycete by our group,
showed moderate Wnt signal inhibitory activity.3 We also found that a synthesized 10R-epimer of
melleumin B (1), seco acid methyl ester of melleumin A (2), which are novel peptide compounds isolated

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HETEROCYCLES, Vol. 76, No. 2, 2008
by our group from the cultured myxomycete Physarum melleum, showed moderate inhibition of Wnt
signal transcription.4 These compounds consist of four units, p-methoxy benzoic aid (pMBz), L-threonine
(L-Thr), glycine (Gly), and an unusual amino acid, a tyrosine-attached acetic acid (TyrA). Herein, we
report the synthesis of two isomers of melleumin B (1), the 3R-epimer and (3R, 4S, 10R, 11R)-isomer,
and evaluated their inhibition of Wnt signal transcription.
RESULTS AND DISCUSSION
We previously determined the absolute stereochemistry of a novel peptide lactone, melleumin A (2) and
its seco acid methyl ester, melleumin B (1), as shown in Figure 1, by an analytical method and total
synthesis of 1.4 It was revealed that melleumin A (2) has 3S, 4S, 10S, 11R configuration. We synthesized
1 and 10R-epimer (3) to determine the absolute stereochemistry. In a previous report, we found that 3 has
moderate Wnt signal inhibitory activity although a natural product 1 does not have activity. Because few
Wnt signal inhibitors have been reported and peptide-based lead compounds seem to be good candidates
to treat cancers, we planned to synthesize 3R-epimer of 1 (4) and (3R, 4S, 10R, 11R)-isomer of 1 (5) and
evaluate their Wnt signal inhibitory activities.
OMe
6'
5'
4'
7'
8'
3'
5'
8'
2'
MeO
6'
4'
3'
pMBz
HN1'
11R
O
7'
O
O
O
9
12
2'
10S
10S
HN
8
OH HN
9
1'
Gly
O
7
NH
8
L-Thr
O
7
11R
O
1
6
12
O
6
HN
OH
5
NH
3S
MeO
1
1"
2"
5
2
3S
4S
2
4S
3"
2"
O
OH
1"
4"
5"
3"
4"
7"
L-TyrA
7"
6"
OH
(L-tyrosine-attached6a" cetic acid)
5"
OH
melleumin B (1)
melleumin A (2)
10-epi-melleumin B (3)
3-epi-melleumin B (4)
(3R,4S,10R,11R)-melleumin B (5)
Figure 1. Structures of melleumin B (1), melleumin A (2) and synthesized isomers.
To obtain (3R, 4S)-TyrA unit steroselectively, the reduction of β-ketoester 7 was examined. β-Ketoester 7
was synthesized from N-Boc-O-benzyl-L-tyrosine 6 (Scheme 1). As shown in Table 1, although several
typical conditions of chelating control were used, the desired (3R, 4S)-β-hydroxy ester 8 was obtained in

HETEROCYCLES, Vol. 76, No. 2, 2008
1427
NHBoc
NHBoc
HO2C
EtO2C
a
O
6
7
OBn
OBn
Scheme 1. Reagents and Conditions: (a) CDI, THF, rt, overnight, then EtOAc, LDA, -78 °C, 68%.
Table 1. Stereoselective synthesis of 8
NHBoc
NHBoc
EtO2C
EtO2C
reagent
solvent
OH
OH
+
7
OBn
OBn
8
9
reagent (eq.)
Zn(BH4)2 (10)
solvent temp
( C)
time
(h)
5
5
5
5
1
1
1
yield
(%)
76
44
52
90
72
95
7
(%)
13
35
37
0
0
0
0
ND
0
entry
8 : 9a)
73 : 27
78 : 22
82 : 18
57 : 43
87 : 13
80 : 20
91 : 9
- : -
1
2
3
4
EtOH
-78
-78
-78
-78
-78
0
-78
0 to rt
0
Zn(BH4)2 (10), ZnCl2 (1.5) EtOH
NaBH4 (1.5), TiCl4 (1.5) MeOH
NaBH4 (3.0), CeCl3 (1.5) EtOH
5b) BH3-Py (1.5), TiCl4 (1.5) CH2Cl2
6
7
8
9
10
NaBH4 (1.5)
NaBH4 (1.5)
L-Selectride (3.0)
K-Selectride (2.4)
K-Selectride (2.4)
EtOH
EtOH
THF
94
no reaction
69
216
9
9
THF
7 : 93
5 : 95
THF
-78
41
46
a) Diastereoselectivity was determined by HPLC; b) Diastereoselectivity was determined by 1H NMR.
ND: Not determined.
94% yield with a ratio of 8/9 = 91/9, with only NaBH4 (entry 7). 5 When K-Selectride was used, (3S,
4S)-TyrA unit 9 was predominately obtained as expected (entries 9, 10).Compound 8 was protected as the
TBS ether in a one-pot reaction. Compound 8 was treated with TBSOTf for cleavage of the Boc group,
and then 2,6-lutidine was added to give the TBS ether.4 The reaction mixture, containing free amine 10,
was evaporated and the residue was subjected to coupling with Fmoc-Gly to give compound 11 in good
yield (61%, 2 steps from 8). Deprotection of the Fmoc group gave the desired TyrA-Gly unit 12, which
was used in the next reaction without purification. Coupling of the TyrA-Gly unit 12 with L-Thr-pMBz
unit 134 gave tripeptide 14 along with its 10R-epimer 15, which was caused by racemization in the
reaction (67%, 2 steps from 11; 14/15 = 85/5, the ratio was determined by 1H NMR). The structure of 14
was elucidated by H-H COSY, HMQC, and HMBC spectra, after purification by HPLC. Although we

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HETEROCYCLES, Vol. 76, No. 2, 2008
examined several conditions, racemization occurs at the C10 position. Because 10R-isomer was also
interesting to check a Wnt signal inhibition activity, we decided to separate the isomers at the final step.
Deprotection of the TBS and MOM groups on the tripeptide, followed by hydrolysis of the ethyl ester
with LiOH, afforded a mixture of seco acids.
O
O
NHFmoc
c
NH2
NH2
HN
HN
EtO2C
TBSO
EtO2C
TBSO
EtO2C
TBSO
a
b
8
10
11
12
OBn
OBn
OBn
OMe
OMe
OMe
HN
O
HN
O
O
HN
O
O
MOMO CO2H
10
10
13
HN
HN
MOMO
MOMO
d
e - h
+
HN
O
HN
3
O
3
EtO2C
EtO2C
TBSO
TBSO
OBn
OBn
14
15
OMe
OMe
O
OMe
O
OMe
HN
O
O
HN
HN
HN
O
O
10
10
O
10
10
O
HN
HN
HN
HO
HO
HN
HO
HN
HO
i
+
+
O
HN
O
O
HN
O
HN
3
3
3
3
MeO2C
MeO2C
MeO2C
MeO2C
HO
HO
HO
HO
OBn
OBn
16
17
4
5
OH
OH
Scheme 2. Reagents and Conditions: (a) TBSOTf, CH2Cl2, 0°C, 3.5 h then 2,6-lutidine, 0.5 h; (b)
Fmoc-Gly, EDC, HOBt, 4-DMAP, CH2Cl2, rt, overnight, 61% (2 steps from 8); (c) piperidine, CH2Cl2,
0°C, 1 h; (d) EDC, HOOBt, 4-DMAP, CH2Cl2, rt, overnight, 68% (2 steps from 11), 14/15 = 85/15; (e)
TBAF, THF, rt, 3 h, 83%; (f) HCl, EtOH, 60°C, 45 min, 76%; (g) LiOH, THF/H2O = 1/1, 0°C, 45 min; (h)
TMS-diazomethane, MeOH, rt, overnight, 84% in 2 steps, 16/17 = 83/14; (i) H2, 10% Pd/C, EtOH, rt,
overnight, 94%, 4/5 = 83/17.
Conversion to the methyl ester with TMS-diazomethane gave compounds 16 and 17 (84% in 2 steps,
16/17 = 83/14). The structure of 17 was determined by H-H COSY, HMQC, and HMBC spectra, after
purification by HPLC. The benzyl group was finally removed by hydrogenolysis to give a mixture of
3R-epimer of 1 (4) and (3R, 4S, 10R, 11R)-isomer of 1 (5) in 94% yield (4/5 = 83/17, the ratio was
determined by reversed-phase HPLC). Compounds 4 and 5 were separated as a pure form by
1
reversed-phase HPLC. These structures were carefully determined by using H and 13C NMR, H-H

HETEROCYCLES, Vol. 76, No. 2, 2008
1429
COSY, HMQC, and HMBC spectra and comparison of the characteristic peak pattern with those of
synthetic 1 and its 10R-epimer (3).
The Wnt signal transduction pathway has important functions in vertebrate development, and its
deregulation is believed to occur early in human colorectal cancer.2, 6 Wnt target genes have recently been
found to be highly expressed in colon cancers; some of these genes are likely to contribute to cancer
formation.7 Small-molecule inhibitors of this pathway are desired because it is believed that their clinical
use would suppress cancer cell growth. Several potent small-molecule inhibitors have been reported;8
however, the development of therapies to specifically target the Wnt pathway in cancer cells is still in its
infancy.
We examined Wnt signal inhibitory activity of the two synthesized compounds (4 and 5) using a
luciferase reporter gene assay. Wnt signaling activates gene transcription with a complex between
β-catenin and TCF/LEF, which is a DNA-binding protein. SuperTOP-Flash, a reporter plasmid with
multiple TCF-binding sites (CCTTTGATC), was stably transfected in 293 cells.9 SuperFOP-flash has
eight mutated TCF-binding sites (CCTTTGGCC); therefore, a selective inhibitor would not show
enhanced transcription in SuperFOP-Flash-transfected cells; thus, these cells provide a negative control
for the assay and the TOP/FOP-Flash reporter activation ratio provides a measure of the selectivity of
Wnt signal inhibition. It is also possible that a decrease of luciferase reporter activity in this assay is due
to the cytotoxicity of the test samples. We therefore also examined the cytotoxicity of samples using a
fluorimetric microculture cytotoxicity assay (FMCA). Compounds exhibiting both low luciferase reporter
activity and high cell viability are "potent" compounds.
The results are shown in Figure 2, along with the cell viability. We have already found that 10R-epimer
(3) of melleumin B is a moderate inhibitor of Wnt signal transcription, although melleumin B (1) did not
show inhibition. We evaluated two newly synthesized isomers of 1. For control experiments, the activities
of 1 and 3 were also examined at the same time. The 10R-epimer (3) exhibited moderate dose-dependent
SuperTOP-Flash-transfected cell inhibition, as reported previously.4 The 3R-epimer (4) and (3R, 4S, 10R,
11R)-isomer (5) exhibited moderate Wnt signal inhibition in a dose-dependent manner with high cell
viability. These results suggest that small molecules using a template of melleumin B (1) with unnatural
stereocenters of amino acids might be lead compounds as peptide-based Wnt signal inhibitors.
Since few small molecules are known as Wnt signal inhibitors, and their clinical use has received great
attention, we believe that peptide-based inhibitors would be worth synthesizing to evaluate their potential.
Application using solid-phase reactions to construct small molecule libraries based on melleumins A and
B, isolated from the myxomycete Physarum melleum, is in progress.

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HETEROCYCLES, Vol. 76, No. 2, 2008
Figure 2. Wnt signal inhibitory activities of mellumins.
EXPERIMENTAL
3-(tert-Butoxycarbonylamino)-2-oxo-4-(benzyloxy)benzenepentanoic acid ethyl ester (7)
To a THF (24 mL) solution of (S)-N-(tert-butoxycarbonyl)-O-benzyltyrosine 6 (877 mg, 2.36 mmol) was
added CDI (765 mg, 7.05 mmol) at 0 °C, and stirred for 0.5 h at 0 °C. The reaction mixture was allowed
to warm to rt and stirred overnight. In another flask, to a stirred solution of diisopropylamine (1.60 mL,
11.3 mmol) in THF (24 mL) was added a 1.58 M n-hexane solution of n-butyllithium (7.02 mL, 11.1
mmol) at 0 °C under argon atmosphere. After stirring for 30 min, the reaction mixture was cooled to
-78 °C, EtOAc (1.04 mL, 10.6 mmol) was added and the mixture was stirred for 15 min at -78 °C. The
above reaction mixture of 6 was added to this mixture and the resulting mixture was stirred for 2 h at
-78 °C. The mixture was warmed to rt, and acidified with 2 N HCl, then extracted with EtOAc. The
organic layer was washed with brine, dried over Na2SO4, filtered, and concentrated in vacuo. The residue
was chromatographed on silica gel (hexane:EtOAc = 5:1) to afford 7 (710 mg, 68% yield as white solid).
1H NMR (400 MHz, CDCl3) δ 1.27 (t, J = 7.1 Hz, 3H), 1.41 (s, 9H), 2.94 (dd, J = 7.1, 14.1 Hz, 1H), 3.07
(dd, J = 7.1, 14.1 Hz, 1H), 3.44 (d, J = 16.1 Hz, 1H), 3.50 (d, J = 16.1 Hz, 1H), 4.19 (q, J = 7.1 Hz, 2H),
4.52 (q, J = 7.1 Hz, 1H), 5.02 (s, 2H), 5.12 (m, 1H), 6.92 (brd, J = 8.7 Hz, 2H), 7.09 (d, J = 8.7 Hz, 2H)
and 7.29-7.42 (m, 5H); 13C NMR 100 MHz (CDCl3) δ 13.9, 28.1 (3C), 35.9, 46.7, 60.5, 61.3, 69.8, 80.0,
114.9 (2C), 127.3 (2C), 127.8, 128.2, 128.4 (2C), 130.2 (2C), 136.8, 155.1, 157.7, 166.8, and 202.0; IR
(ATR) 3336, 2983, 2926, 1747, 1718, 1686, 1512, 1313, 1242, 1143 and 1013 cm-1; HRMS (EI)
C25H31NO6 [M]+ calcd 441.2151, found 441.2146; [α]2D2 +0.3 (c 1.0, CHCl3).

HETEROCYCLES, Vol. 76, No. 2, 2008
1431
(3R,4S)-5-[4-(Benzyloxy)phenyl]-4-(tert-butoxycarbonylamino)-3-hydroxypentanoic acid ethyl ester
(8), (3S,4S)-5-[4-(benzyloxy)phenyl]-4-(tert-butoxycarbonylamino)-3-hydroxypentanoic acid ethyl
ester (9)
To a stirred solution of 7 (50 mg, 0.11 mmol) in ethanol (0.29 mL) was added sodium tetrahydroborate
(6.2 mg) at -78 °C under argon atmosphere. After 2 h, the reaction mixture was warmed to rt, and
acidified with 2 N HCl, then extracted with EtOAc. The organic layer was dried over Na2SO4, filtered and
concentrated in vacuo. Chlomatography of the residue on silica gel eluting with a gradient (hexane:EtOAc
= 3:1 to 1:1) afforded a mixture of 8 and 9 (47.3 mg, 94%) (8:9 = 91: 9, Diastereoselectivity was
determined by reversed-phase HPLC; PEGASIL-ODS, 4.6 x 250 mm; UV detection at 274 nm; eluent,
60% MeOH; flow rate, 0.4 mL/min; retention time: 8 (19.3 min), 9 (22.1 min)). Recrystallization from
EtOAc and n-hexane gave compound 8 as a pure form.
Spectral data of 8: 1H-NMR (400 MHz, CD3OD) δ 1.26 (t, J = 7.1 Hz, 3H,CH3CH2OCO), 1.31 (s, 9H,
Boc), 2.40 (dd, J = 9.3, 15.3 Hz, 1H, H-2), 2.49 (dd, J = 10.5, 13.9 Hz, 1H, H-5), 2.62 (dd, J = 3.5, 15.3
Hz, 1H, H-2), 3.04 (dd, J = 3.7, 13.9 Hz, 1H, H-5), 3.59-3.64 (m, 1H, H-3), 3.91-3.96 (m, 1H, H-4),
4.09-4.19 (m, 2H, CH3CH2OCO), 5.04 (s, 2H, C6H5CH2O), 6.47 (brd, J = 9.5 Hz, 1H), 6.88 (d, J = 8.5 Hz,
2H, H-3’and H-5’), 7.12 (d, J = 8.5 Hz, 2H, H-2’and H-6’) and 7.29-7.42 (m, 5H, C6H5CH2O); 13C NMR
(100 MHz, CD3OD) δ 14.5, 28.7 (3C), 36.8, 40.5, 58.1, 61.6, 70.9, 71.9, 80.0, 115.7 (2C), 128.4, 128.4
(2C), 129.4 (2C), 131.4 (2C), 132.4, 138.9, 158.0, 158.7 and 173.7; IR (ATR) 3352, 2981, 2926, 1730,
1682, 1511, 1307 and 1233 cm-1; HRMS (FAB) calcd for C25H34NO6 444.2386 [M+H]+, found 444.2388;
[α]2D3 +4.8 (c 1.0, CHCl3).
Spectral data of 9: 1H NMR (400 MHz, CDCl3) δ 1.24 (t, J = 7.0Hz, 3H), 1.39 (s, 9H), 2.37 (dd, J = 2.4,
16.8 Hz, 1H), 2.58 (dd, J = 10.4, 16.8 Hz, 1H), 2.85 (d, J = 7.8 Hz, 2H), 3.54 (s, 1H), 3.67 (q, J = 7.8 Hz,
1H), 3.98 (m, 1H), 4.13 (q, J = 7.0 Hz, 2H), 4.98 (m, 1H), 5.02 (s, 2H), 6.90 (d, J = 8.4 Hz, 2H), 7.16 (d,
J = 8.4 Hz, 2H) and 7.27-7.44 (m, 5H); 13C NMR (125 MHz, CDCl3) δ 14.1, 28.3 (3C), 37.7, 38.6, 55.4,
60.8, 66.9, 70.0, 79.3, 114.8 (2C), 127.4 (2C), 127.9, 128.5 (2C), 130.3 (2C), 130.5, 137.1, 155.8, 157.4,
and 173.5; IR (ATR) 3368, 2978, 1717, 1679, 1509, 1366, 1242 and 1163 cm-1; HRMS (FAB) calcd for
C25H34NO6 444.2386 [M+H]+, found 444.2361; [α]2D3 -12 (c 1.0, CHCl3).
(3R,4S)-4-Amino-3-[(tert-butyldimethylsilyl)oxy]-5-[4-(benzyloxy)phenyl]pentanoic acid ethyl ester
(10): A solution of 8 (585 mg, 1.32 mmol) in CH2Cl2 (13.2 mL) was treated with tert-butyldimethylsilyl
trifluoromethansulfonate (0.91 mL, 3.96 mmol) at 0 °C under argon atmosphere. The mixture was stirred
for 3.5 h at 0 °C, and then 2,6-lutidine (0.54 mL, 4.63 mmol) was added, and the mixture was further
stirred at 0 °C for 0.5 h. After addition of water, the mixture was extracted with EtOAc, dried over
Na2SO4, filtered, and concentrated in vacuo, to afford 10, which was used without further purification in

1432
HETEROCYCLES, Vol. 76, No. 2, 2008
the following reaction.
(3R,4S)-4-[2-(9-Fluorenylmethoxycarbonylamino)acetylamino]-3-[(tert-butyldimethylsilyl)oxy]-5-
[4-(benzyloxy)phenyl]pentanoic acid ethyl ester (11): A solution of Fmoc-glycine (544 mg, 1.83 mmol),
EDC･HCl (351 mg, 1.83 mmol), HOBt･H2O (247 mg, 1.83 mmol) and 4-DMAP (223 mg, 1.83 mmol) in
CH2Cl2 (6.1 mL) were mixed at 0 °C under argon atmosphere. The mixture was stirred for 10 min, and
then a solution of 10 in CH2Cl2 (6.1mL) was added. The reaction mixture was allowed to warm to rt and
stirred overnight. The solvent was evaporated under reduced pressure and the residue was dissolved in
EtOAc. The organic layer was washed with water, saturated aqueous NaHCO3, 10% aqueous citric acid,
water, and brine. The organic layer was dried over Na2SO4, filtered and concentrated in vacuo. The
residue was chromatographed on silica gel (hexane:EtOAc = 3:1) to afford 11 (592 mg, 0.80 mmol, 61%)
as a yellow powder. 1H NMR (400 MHz, CDCl3) δ 0.04 (s, 3H), 0.07 (s, 3H), 0.89 (s, 9H), 1.26 (t, J = 7.1
Hz, 3H), 2.51-2.59 (m, 2H), 2.65 (dd, J = 9.6, 14.3 Hz, 1H), 2.95 (dd, J = 4.7, 14.3 Hz, 1H), 3.64 (dd, J =
5.4, 16.5 Hz, 1H), 3.72 (dd, J = 5.6, 16.5Hz, 1H), 4.08-4.82 (m, 5H), 4.40 (d, J = 6.6 Hz, 2H ), 4.98 (s,
2H), 5.13 (brs, 1H), 5.96 (brd, J = 8.5 Hz, 1H), 6.84 (d, J = 8.5 Hz, 2H), 7.05 (d, J = 8.3 Hz, 2H),
7.27-7.42 (m, 9H), 7.58 (brd, J = 6.6 Hz, 2H,), and 7.76 (d, J = 7.6 Hz, 2H); 13C NMR 100 MHz (CDCl3)
δ -4.9, -4.7, 14.1, 18.0, 25.7 (3C), 34.6, 39.9, 44.4, 47.0, 55.1, 60.7, 67.1, 69.8, 70.6, 114.8 (2C), 120.0
(2C), 125.0 (2C), 127.1 (2C), 127.4 (2C), 127.7, 127.8, 128.5 (4C), 130.0 (2C), 136.9 (2C), 141.2, 141,3,
143.7, 156.3, 157.3, 168.1, and 171.2; IR (ATR) 2952, 2928, 1728, 1664, 1509, 1449, 1240, 1175, 1081,
1025, 831, 737, and 695 cm-1; HRMS (FAB) calcd for C43H53N2O7Si 737.3970 [M+H]+, found 737.3544;
[α]20 -12.6 (c 1.0, CHCl3).
D
(3R,4S)-4-[(2-Aminoacetyl)amino]-3-[(tert-butyldimethylsilyl)oxy]-5-[(4-benzyloxy)phenyl]-
pentanoic acid ethyl ester (12): A solution of 11 (592 mg, 0.80 mmol) in DMF (8.0 mL) was treated
with piperidine (0.8 mL, 8.1 mmol) for 1 h at 0 °C under argon atmosphere. The reaction mixture was
concentrated in vacuo. The mixture was used without further purification in the following reaction.
(3R,4S)-5-(4-Benzyloxyphenyl)-3-[(tert-butyldimethylsilyl)oxy]-4-[N-(4-methoxybenzoyl)-3-methoxy-
methyl-L-threonylglycyl]amino]pentanoic acid ethyl ester (14) and its 10R-epimer (15): A solution
of 12 (287 mg, 1.01 mmol), EDC･HCl (171 mg, 0.894 mmol), HOOBt (146 mg, 0.894 mmol) and
4-DMAP (109 mg, 0.894 mmol) in CH2Cl2 (2.8 mL) were mixed at 0 °C under argon atmosphere. The
mixture was stirred for 30 min, and then 13 in CH2Cl2 (2.8 mL) was added. The reaction mixture was
allowed to warm to rt and stirred overnight. The solvent was evaporated under reduced pressure and the
residue was dissolved in EtOAc. The organic layer was washed with water, saturated aqueous NaHCO3,
10% aqueous citric acid, water, and brine, dried over Na2SO4, filtered and concentrated in vacuo. The
residue was chromatographed on silica gel (hexane:EtOAc = 2:3) to afford a mixture of 14 and 15 (491
mg, 77%, two steps from 12) (14:15 = 85:15, determined by reversed-phase HPLC).

HETEROCYCLES, Vol. 76, No. 2, 2008
1433
Purification of 14: The mixture (14 and 15) was separated by preparative HPLC (Develosil C-30 UG-5,
10 x 250 mm; eluent, 89% MeOH; UV detection at 254 nm; flow rate, 2.5 mL/min). Retention time: 14
(42.0 min), 15 (46.5 min).
1
Spectral data of 14: H NMR (400 MHz, CDCl3) δ 0.04 (s, 3H, Si(CH3)2C(CH3)3), 0.08 (s, 3H,
Si(CH3)2C(CH3)3), 0.89 (s, 9H, Si(CH3)2C(CH3)3), 1.19 (d, J = 6.3 Hz, 3H, H-12), 1.25 (t, J = 7.1 Hz, 3H,
CH3CH2OCO), 2.50-2.60 (m, 2H, H-2), 2.64 (dd, J = 9.6, 14.2 Hz, 1H, H-1’’), 2.92 (dd, J = 4.6, 14.2 Hz,
1H, H-1’’), 3.39 (s, 3H, CH3OCH2-), 3.79 (dd, J = 5.2, 16.8 Hz, 1H, H-7), 3.85 (s, 3H, PhOCH3), 3.90
(dd, J = 6.1, 16.8 Hz, 1H, H-7), 4.06-4.26 (m, 4H, H-3 and H-4 and -COOCH2CH3), 4.34-4.37 (m, 1H,
H-11), 4.60 (dd, J = 4.1, 5.9 Hz, 1H, H-10), 4.74 (d, J = 6.5Hz, 2H, CH3OCH2O-), 4.99 (s, 2H, PhCH2O),
6.36 (brd, J = 8.8 Hz, 1H, H-5), 6.85 (d, J = 8.5 Hz, 2H, H-4’’ and H-6’’), 6.93 (d, J = 9.0 Hz, 2H, H-5’
and H-7’), 6.99 (m, 1H, H-8), 7.04 (d, J = 8.5 Hz, 2H, H-3’’ and H-7’’), 7.20 (brd, J = 5.9 Hz, 1H, H-1’),
7.29-7.52 (m, 5H, C6H5CH2O), and 7.82 (d, J = 8.8 Hz, 2H, H-4’and H-8’); 13C NMR 100 MHz (CDCl3)
δ -4.8, -4.6, 14.1, 16.5, 18.0, 25.8 (3C), 35.2, 39.9, 43.0, 55.4, 55.57, 55.61, 57.9, 60.7, 69.9, 70.7, 73.4,
77.2, 113.8 (2C), 114.8 (2C), 125.6, 127.5 (2C), 127.9, 128.5 (2C), 129.0 (2C), 130.0 (2C), 130.2, 137.1,
157.4, 162.6, 167.0, 167.9, 169.9 and 171.6; IR (ATR) 3270, 2930, 1734, 1629, 1542, 1507, 1463, 1383,
1252, 1176, 1111, 1029, 838, 777, and 696 cm-1 ; HRMS (FAB) calcd for C42H60N3O10Si 794.3970
[M+H]+, found 794.4039; [α]20 -8.2 (c 1.0, CHCl3).
D
Spectral data of 151H NMR (400 MHz, CDCl3) δ 0.00 (s, 3H), 0.06 (s, 3H), 0.86 (s, 9H), 1.22 (t, J = 7.2
Hz, 3H), 1.36 (d, J = 6.4 Hz, 3H), 2.46 (dd, J = 6.0, 15.2 Hz, 2H), 2.56 (dd, J = 5.4, 15.4 Hz, 1H), 2.64
(dd, J = 9.6, 14.0 Hz, 1H), 2.95 (dd,J = 4.5, 14.0 Hz, 1H), 3.39 (s, 3H), 3.81 (dd, J = 3.0, 5.4 Hz, 2H),
3.85 (s, 3H), 4.01-4.18 (m, 5H), 4.54 (dd, J = 5.2, 6.6 Hz, 1H), 4.69 (dd, J = 7.7, 11.0 Hz, 2H), 5.02 (s,
2H), 6.24 (brd, J =8.3 Hz, 1H), 6.58 (brt, J = 5.4 Hz, 1H), 6.88 (d, J = 8.5 Hz, 2H), 6.94 (d, J = 8.8 Hz,
2H), 7.10 (d, J = 8.5 Hz, 2H), 7.29-7.44 (m, 5H) and 7.82 (d, J = 8.8 Hz, 2H).
Removal of TBS protective group.
(3R,4S)-5-(4-Benzyloxyphenyl)-4-[[N-(4-methoxybenzoyl)-3-methoxymethyl-L-threonylglycyl]-
amino]pentanoic acid ethyl ester and its 10R-epimer: To a stirred solution of a mixture of 14 and 15
(109.5 mg, 0.138 mmol) in THF (0.41 mL) was added a 1.0 M THF solution of tetrabutylammonium
fluoride (0.41 mL, 0.41 mmol) at 0 °C under argon atmosphere. After 10 min, the reaction mixture was
allowed to warm to rt and stirred for 5 h. The reaction mixture was quenched with H2O and extracted with
EtOAc. The organic layer was dried over Na2SO4, filtered and concentrated in vacuo. The residue was
chromatographed on silica gel (hexane:EtOAc = 1:5) to afford a mixture of alchols (78.2 mg, 0.115 mmol,
84%), which was used without further purification in the following reaction.
Spectral data of mixture: 1H-NMR (400 MHz, CDCl3) δ 1.19-1.27 (m, 5.5H), 1.34 (d, J = 9.9 Hz, 0.5H),

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HETEROCYCLES, Vol. 76, No. 2, 2008
2.06 (brs, 2H), 2.45-2.57 (m, 2H), 2.70-2.94 (m, 2H), 3.36 (s, 2.5H), 3.38 (s, 0.5H), 3.74-3.81 (m, 2H),
3.83 (s, 3H), 3.84-4.00 (m, 2H), 4.05-4.30 (m, 6H), 4.45 (t, J = 4.9 Hz, 0.2H), 4.49 (t, J = 5.1 Hz, 0.8H),
4.64-4.77 (m, 2H), 4.96 (s, 1.6H), 5.00 (s, 0.4H), 6.80-7.16 (m, 8H), 7.26-7.42 (m, 6H), and 7.78-7.83 (m,
2H).
Removal of MOM protective group.
(3R,4S)-5-(4-Benzyloxyphenyl)-4-[[N-(4-methoxybenzoyl)-L-threonylglycyl]amino]pentanoic acid
ethyl ester and its 10R-epimer: The above mixture (69.3 mg, 0.10 mmol) was dissolved in EtOH (10
mL) containing conc. aq HCl (0.2 mL), and the mixture was heated to 60 °C and stirred for 4 h. The
reaction mixture was cooled to rt, quenched with saturated aqueous NaHCO3 and extracted with EtOAc.
The organic layer was dried over Na2SO4, filtered and concentrated in vacuo. The residue was
chromatographed on silica gel (CHCl3/MeOH = 20/1) to afford a mixture of diols (48.6 mg, 0.08 mmol,
76%), which was used without further purification in the following reaction.
1
Spectral data of mixture: H-NMR (400 MHz, CDCl3) δ 1.16-1.38 (m, 6H), 2.43-2.84 (m, 4H),
3.61-3.69 (m, 1H), 3.78 (s, 3H), 3.92 (dd, J = 6.3, 16.6 Hz, 0.8H), 3.95-4.19 (m, 4.2H), 4.38-4.53 (m, 2H),
4.93-4.99 (m, 2H), 6.80-6.89 (m, 4H), 7.03-7.06 (m, 1.6H), 7.09 (d, J = 8.5 Hz, 0.5H), 7.16 (d, J = 8.5 Hz,
1H), 7.26-7.44 (m, 6H), and 7.69-7.90 (m, 3H).
(3R,4S)-5-(4-Benzyloxyphenyl)-4-[[N-(4-methoxybenzoyl)-L-threonylglycyl]amino]pentanoic acid
methyl ester (16) and its 10R-epimer (17):
A solution of the above mixture of protected tripeptide compounds (48.6 mg, 0.08 mmol) in solvent
(THF/H2O = 1/1, 0.76 mL) was treated with LiOH-H2O (6.4 mg, 0.153 mmol) at 0 °C, and the mixture
was stirred for 40 min. The mixture was acidified with 2 N HCl, and then extracted with EtOAc. The
organic layer was dried over MgSO4, filtered, and concentrated in vacuo to afford a mixture of seco acid
compounds, which was used without further purification in the following reaction. A solution of the
mixture of seco acid compounds in MeOH (2.6 mL) was treated with 10% hexane solution of
TMS-diazomethane (0.58 mL, 0.34 mmol) at rt under argon atmosphere. After stirring overnight, the
reaction mixture was concentrated in vacuo. Then, the residue was chromatographed on silica gel
(CHCl3:MeOH = 20:1) to afford 16 as a mixture with its 10R-epimer 17 (39.5 mg, 0.06 mmol, 84%)
(16:17 = 83:17, determined by reversed-phase HPLC), which was used without further purification in the
following reaction. A solution of the mixture (11.1 mg) in MeOH (0.36 mL) was treated with 10% hexane
solution of TMS-diazomethane (1.8 mL) at rt under argon atmosphere. The mixture was stirred overnight.
The reaction mixture was concentrated in vacuo. Then, the residue was chromatographed on silica gel
(CHCl3:MeOH = 10:1) to afford a mixture of 16 and 17 (8.3 mg, 0.06 mmol, 74%) (16:17 = 83:17, the
ratio was determined by reversed-phase HPLC).

HETEROCYCLES, Vol. 76, No. 2, 2008
1435
Purification of 16: The mixture (16 and 17) was separated by preparative HPLC (Develosil C-30 UG-5;
10 x 250 mm; eluent, 70% MeOH; UV detection at 254 nm; flow rate, 3.0 mL/min). Retention time: 16
(16.2 min), 17 (20.4 min).
Spectral data of 16: 1H NMR (400 MHz, CDCl3) δ 1.23 (d, J = 6.3 Hz, 3H, H-12), 1.87 (brs, 2H, -OH ),
2.51 (d, J = 2.2 Hz, 1H, H-2), 2.52 (s, 1H, H-2), 2.71 (dd, J = 9.0, 14.3 Hz, 1H, H-1’’), 2.83 (dd, J = 4.9,
14.3 Hz, 1H, H-1’’), 3.64 (s, 3H, CH3OCO-), 3.69 (dd, J = 5.2, 6.5 Hz, 1H, H-7), 3.81 (s, 3H,
CH3OC6H4-), 3.93 (dd, J = 6.5, 16.5 Hz, 1H, H-7), 4.11 (brs, 1H, H-3), 4.16-4.21 (m, 1H, H-4), 4.26 (brs,
1H, H-5), 4.42 (brd, J = 3.9 Hz, 1H, H-11), 4.47 (brdd, J = 2.2, 7.1 Hz, 1H, H-10), 4.97 (s, 2H,
C6H5CH2-), 6.83 (d, J = 8.8 Hz, 2H, H-4’’ and H-6’’), 6.88 (d, J = 9.0 Hz, 2H, H-5’ and H-7’), 6.95 (brd,
J = 9.0 Hz, 1H, H-5), 7.05 (d, J = 8.5 Hz, 1H, H-3’’ and H-7’’), 7.24 (m, 1H, H-1’), 7.29-7.52 (m, 5H,
13
C6H5CH2O), 7.54 (brs, 1H, H-8), and 7.80 (d, J = 8.8 Hz, 2H, H-4’ and H-8’); C NMR 100 MHz
(CDCl3) δ 19.2, 34.2, 37.8, 43.1, 52.0, 54.8, 55.4, 58.9, 66.9, 69.8, 69.9, 113.8 (2C), 114.8 (2C), 125.1,
127.5 (2C), 127.9, 128.5 (2C), 129.3 (2C), 129.8, 130.1 (2C), 137.0, 157.4, 162.8, 168.1, 169.4, 172.2
and 173.1; HRMS (FAB) calcd for C33H39N3NaO9 644.2584 [M+Na]+, found 644.2552; IR (ATR) 3296,
2926, 2852, 1730, 1637, 1606, 1540, 1508, 1455, 1439, 1410, 1379, 1298, 1253, 1176, 1111, 1074, 1024,
844, 768, 738, and 696 cm-1; [α]2D3 +4.1 (c 1.0, MeOH).
Spectral data of 17: 1H NMR (400 MHz, CDCl3) δ: 1.32 (d, J = 6.3 Hz, 3H), 1.87 (brs, 2H), 2.46 (d, J =
6.3 Hz, 2H), 2.74 (dd, J = 8.8, 14.4 Hz, 1H), 2.86 (dd, J = 4.6, 14.4 Hz, 1H), 3.63 (s, 3H), 3.76 (dd, J =
5.2, 5.6 Hz, 1H), 3.82 (s, 3H), 3.89 (dd, J = 5.6, 16.6 Hz, 1H), 4.00-4.21 (m, 4H), 4.41 (t, J = 6.7 Hz, 1H),
5.00 (s, 2H), 6.87 (d, J = 8.5 Hz, 2H), 6.89 (d, J = 5.8 Hz, 2H), 7.02 (brd, J = 7.6 Hz, 1H), 7.10 (d, J = 8.6
13
Hz, 2H), 7.29-7.42 (m, 6H), and 7.79 (d, J = 8.8 Hz, 2H); C NMR 100 MHz (CDCl3) δ : 20.2, 34.1,
37.8, 43.1, 52.0, 54.9, 55.4, 59.1, 68.8, 69.9, 70.0, 113.9, 114.8, 125.1, 127.5, 127.9, 128.3, 128.5, 129.2,
130.2, 137.0, 157.4, 162.8, 167.7, 169.2, 171.8 and 173.1; HRMS (FAB) calcd for C33H40N3O9 622.2765
[M+H]+, found 622.2768; IR (ATR) 3296, 2927, 2852, 1733, 1636, 1606, 1541, 1507, 1456, 1438, 1376,
1297, 1252, 1176, 1112, 1065, 1025, 845, 768, 738, and 696 cm-1; [α]2D3 -27.9 (c 0.5, MeOH).
3-epi-Melleumin B (4) and (3R,4S,10R,11R)-melleumin B (5):
A mixture of 16 and 17 (3.8 mg, 6.1 µmol) was dissolved in EtOH (0.1 mL), and then 10% palladium on
carbon (0.4 mg) was added. The reaction mixture was stirred overnight under hydrogen and then filtered
through celite and concentrated in vacuo. The residue was chromatographed on silica gel (CHCl3:MeOH
= 10:1) to afford 4 as a mixture with its 10R-epimer 5 (3.1 mg, 5.8 µmol, 94%) (4:5 = 83:17, The ratio
was determined by reversed-phase HPLC).
Purification of 4: The mixture (4 and 5) was separated by preparative HPLC (Develosil C-30 UG-5; 10 x
250 mm; eluent, 50% MeOH; UV detection at 254 nm; flow rate, 3.0 mL/min). Retention time: 4 (13.5
min), 5 (18.6 min).

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HETEROCYCLES, Vol. 76, No. 2, 2008
Spectral data of 3-epi-melleumin B (4): 1H NMR (400 MHz, CD3OD) δ 1.25 (d, J = 6.3 Hz, 3H, H-12),
2.43 (dd, J = 9.5, 15.5 Hz, 1H, H-2), 2.58 (dd, J = 9.8, 14.2 Hz, 1H, H-1”), 2.68 (dd, J = 2.9, 15.5 Hz, 1H,
H-2), 3.00 (dd, J = 3.5, 14.1 Hz, 1H, H-1”), 3.67 (s, 3H, -COOCH3), 3.69 (d, J = 16.8 Hz, 1H, H-7), 3.81
(d, J = 16.8 Hz, 1H, H-7), 3.85 (s, 3H, -C6H4OCH3), 3.94-4.08 (m, 2H, H-3 and H-4), 4.22-4.28 (m, 1H,
H-11), 4.39 (d, J = 4.6 Hz, 1H, H-10), 6.59-6.65 (m, 2H, H-4’’ and H-6’’), 6.92-6.96 (m, 2H, H-3’’ and
13
H-7’’), 6.99-7.03 (m, 2H, H-5’ and H-7’), and 7.87-7.91 (m, 2H, H-4’ and H-8’); C NMR 100 MHz
(CD3OD) δ 20.2, 36.4, 40.1, 43.6, 52.1, 56.0, 57.1, 61.7, 68.4, 71.5, 114.8 (2C), 116.1 (2C), 126.8, 130.4
(2C), 130.5, 131.2 (2C), 156.8, 164.3, 170.0, 171.4, 173.6 and 174.1; HRMS (FAB) calcd for C26H34N3O9
532.2295 [M+H]+, found: 532.2253; IR (ATR) 3852, 3649, 3291, 2940, 1717, 1635, 1606, 1541, 1492,
1457, 1436, 1254, 1177, 1108, 1026, 844, 757, and 701 cm-1; [α]23 +22.9 (c 0.5, MeOH).
D
1
Spectral data of (3R,4S,10R,11R)-melleumin B (5): H-NMR (400 MHz, CD3OD): δ 1.33 (d, J = 6.1
Hz, 3H, H-12), 2.31-2.38 (m, 1H, H-2), 2.60-2.63 (m, 1H, H-2), 2.60-2.66 (m, 1H, H-1’’), 3.05 (dd, J =
2.7, 14.2 Hz, 1H, H-1”), 3.62 (s, 3H, -COOCH3), 3.71 (d, J = 17.1 Hz, 1H, H-7), 3.80 (d, J = 17.1 Hz, 1H,
H-7), 3.86 (s, 3H, -C6H4OCH3), 3.96-4.10 (m, 2H, H-3 and H-4), 4.12-4.18 (m, 1H, H-11), 4.29 (d, J =
7.6 Hz, 1H, H-10), 6.68 (d, J = 8.5 Hz, 2H, H-4’’ and H-6’’), 7.01 (d, J = 8.9Hz, 2H, H-5’ and H-7’), 7.04
13
(d, J = 8.5 Hz, 2H, H-3’’ and H-7’’), and 7.88 (d, J = 8.9 Hz, 2H, H-4’ and H-8’); C NMR 125 MHz
(CD3OD) δ 20.7, 36.4, 40.0, 43.6, 52.0, 56.0, 57.2, 61.8, 68.7, 71.6, 114.8 (2C), 116.1 (2C), 127.0, 130.60,
130.63 (2C), 131.3 (2C), 156.8, 164.3, 170.1, 171.5, 173.7 and 174.0; HRMS (FAB) calcd for
C26H34N3O9 532.2295 [M+H]+, found 532.2253; IR (ATR) 3820, 3302, 2925, 2852, 1726, 1639, 1607,
1541, 1505, 1441, 1256, 1179, 1111, 1065, 1028, 847, and 770 cm-1; [α]16 +25.0 (c 0.1, MeOH).
D
Luciferase Assays (Wnt signal inhibition assay)
TOPflash reporter assay
STF/293 cells (3x104) were split into 96-well plates and 24 h later cells were treated with 15 mM LiCl
and testing samples (DMSO solution). After incubation for 24 h, cells were lysed with CCLR (cell culture
lysis reagent; 20 µL/well, Promega) and luciferase activities were measured with a Luciferase Assay
System (Promega). We checked this system worked collect by using quercetin as a standard positive
compound. Assays were performed in triplicate.
FOPflash reporter assay
293T cells (1x105) were split into 24-well plates and were transfected 24 h later with 1 µg/well
superFop-flash reporter plasmid using Lipofectamine2000 (Invitrogen; 2.5 µL/well). 3 h after transfection,
cells were treated with 15 mM LiCl and testing samples (DMSO solution). After incubation for 24 h, cells
were lysed with Reporter Lysis buffer (Promega; 50 µL/well) and luciferase activities were measured
with a Dual-Glo Luciferase Assay System (Promega). Assays were performed in triplicate.
Assay of cell viability

HETEROCYCLES, Vol. 76, No. 2, 2008
1437
STF/293 cells (3x104) were split into 96-well plates and incubated for 24 h. Cells were treated with
testing samples (DMSO solution) and incubated for 24 h. Cells were treated with fluorescein diacetate
(Wako) in PBS buffer (3.5 µg/mL), after 1 h incubation, fluorescence were detected. Assays were
performed in triplicate.
ACKNOWLEDGEMENTS
We thank Prof. Jeremy Nathans (John Hopkins Medical School) for kindly providing STF cells (293 cell
line stably transfected with SuperTOP-Flash by Prof. R.T. Moon) and Prof. Randall T. Moon (University
of Washington) for the SuperFOP-Flash reporter plasmid. This work was supported by Grants-in Aid
from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (No. 18032020 and
19310136 to M.I. and No. 18790012 to M.A.A.). M.A.A. also gratefully acknowledges financial support
from the Uehara Memorial Foundation.
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