Total synthesis and evaluation of Wnt signal inhibition of melleumin A and B, and their derivatives

Article abstract of DOI:10.1039/c0ob00352b

The total synthesis of melleumin A (1), a novel cyclic depsipeptide isolated from the myxomycete Physarum melleum, and 3-epi-melleumin A (6) was achieved. Melleumin A-like compounds were also designed and synthesized; analysis of these melleumin A-like co

Full text of DOI:10.1039/c0ob00352b

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www.rsc.org/obc | Organic & Biomolecular Chemistry
Total synthesis and evaluation of Wnt signal inhibition of melleumin A and B,
and their derivatives†
Midori A. Arai,* Shuwa Hanazawa, Yujiro Uchino, Xiaofan Li and Masami Ishibashi*
Received 30th June 2010, Accepted 16th August 2010
DOI: 10.1039/c0ob00352b
The total synthesis of melleumin A (1), a novel cyclic depsipeptide isolated from the myxomycete
Physarum melleum, and 3-epi-melleumin A (6) was achieved. Melleumin A-like compounds were also
designed and synthesized; analysis of these melleumin A-like compounds showed moderate Wnt signal
inhibition. Comparison of the inhibition activity of melleumin B and its three epimers, melleumin A,
3-epi-melleumin A and three melleumin A-like compounds led to further investigation of the structural
conformation of the active molecules. The scaffold of melleumin is a potential target in the search for
“peptide-like” Wnt signaling inhibitors.
Introduction
Isolation of new natural products and the synthesis of small
molecules based on their scaffolds is of chemical relevance to living
cells and organs, implicated in the binding to proteins, absorption,
distribution, metabolism, and excretion.1 Recently, we have been
attempting natural product isolation by guided bioactivity, such
as Wnt signal inhibition,2 Hh signal inhibition,3 and TRAIL
resistance overcoming activity.4 Wnt signaling shows aberrant
activation in many cancer cells, especially in colon cancers.5 The
aberrant activation of this signal is caused by mutation or loss
of function in b-catenin, T-cell factor/lymphoid enhancer factor
(TCF/LEF), and glycogen synthase kinase 3b (GSK3b) and other
things. Small molecules that act on certain molecular components
in the Wnt signal pathway would be potential candidates for
treating cancer. Several Wnt signal inhibitors have been previously
reported.6 However, there are not many effective Wnt signal
Fig. 1 Structure of melleumin A, B and derivatives.
inhibitors. We have also reported natural products or their
bond8 and found moderate Wnt inhibition in 4-epi-melleumin B
derivatives as Wnt signal inhibitors, such as bisindole alkaloid
cis-dihydroarcyriarubin C,2a melleumin derivatives,2b,c and new
and 4-epi-deoxymelleumin A, but not in melleumin A (1) and 4-
natural products eleutherinoside B–E.2d
epi-melleumin A. In this report, we describe the total synthesis of
Novel peptide lactone, melleumin A (1), and its seco acid methyl
ester, melleumin B (2) were discovered by our group in 2005 from
cultured plasmodium of the myxomycete Physarum melleum (Fig.
1).7 Stereochemistry of 1 and 2 at the chiral centers was established
as 3S, 10S, and 11R-configurations. The absolute stereochemistry
of the C-4 position was determined as S by total synthesis of
melleumin B (2).2b Because the quantity of isolated 1 and 2
was very small, we decided to synthesize those compounds to
evaluate biological activity. After synthesizing 2 and its related
isomers, we found Wnt signal inhibitory activities in the isomers
of 2 using a cell-based assay. The 10R-epimer (3), 3R-epimer (4)
and (3R, 10R)-epimer (5) of 2 showed moderate activity.2b,c Luo
et al. reported the first total synthesis of 1, 4R-epimer and other
derivatives by intramolecular lactamization at the N5–C6 amide
melleumin A (1) and its 3R-epimer (6). In order to analyze Wnt
inhibitory activities on cyclic compounds related to 1, melleumin
A-like compounds that do not contain a lactone subunit that
is easily cleaved in cells were designed and synthesized. These
compounds showed moderate Wnt inhibitory activities.
Results and discussion
Total synthesis of mellumin A (1) and 3-epi-melleumin A (6)
Melleumin A (1) and B (2) consist of four residues: p-
methoxybenzoic acid (pMBz), L-threonine (L-Thr), glycine (Gly),
and an unusual amino acid, a tyrosine-attached acetic acid (TyrA).
Because compound 2 is a seco acid methyl ester of 1, we attempted
intramolecular lactone formation of the seco acid of 1 that was
obtained by hydrolysis of synthesized 2. However, several condi-
tions prevented cyclization.9 Therefore, we changed strategies to
synthesize 1 by N8–C9 amide bond formation as a cyclization
step (Scheme 1). Compound 7 could be prepared from dipep-
tide 8 (TyrA-Gly unit) and N-p-methoxybenzoyl-L-threonine
Graduate School of Pharmaceutical Sciences, Chiba University, 1-33 Yayoi-
cho, Inage-ku, Chiba, 263-8522, Japan. E-mail: marai@p.chiba-u.ac.jp;
Fax: +81-43-290-2913; Tel: +81-43-290-2914
† Electronic supplementary information (ESI) available: NMR spectra and
crystal data. For ESI and crystallographic data in CIF or other electronic
format see DOI: 10.1039/c0ob00352b
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Scheme 1 Retrosynthetic approach to melleumin A (1).
Table 1 Streoselective synthesis of TyrA unit
and tert-butyl ester of 18 were deprotected to give compound
7. Macrolactamization of 7 was investigated for various coupling
reagents. HBTU, TBTU, PyBOP, and DEPBT were unsuccessful,
but FDPP and HATU provided 20 in 28% and 24% yield (2
steps), respectively. The benzyl group was finally removed by
1
hydrogenolysis to give melleumin A (1). The H and 13C NMR
spectral data of the synthetic material were identical to those of the
natural product.1 The optical rotation of synthetic melleumin A
(1) showed the same sense as the natural product {[a]1D8 +16 (c 0.97,
MeOH); [a]1D8 +27 (c 0.15, MeOH) lit.7}. X-Ray crystallographic
analysis of synthetic melleumin A (1) was successful as shown in
Fig. 2.11 This result strongly confirmed the absolute structure of
melleumin A (1). Starting from (3R,4S)-b-hydroxy ester 15, 3-epi-
melleumin A (6) was also synthesized as shown in Scheme 2. To
Entry Reagent (eq.)
Solvent T/◦C Time/h Ratioa 9 : 15 Yield (%)
1
2
K-selectride (2.4) THF
NaBH4 (1.5)
0
9
1
93 : 7
9 : 91
69
94
EtOH -78
a The ratio was determined by HPLC.
12 (pMBz-L-Thr unit). Dipeptide 8 could be obtained from
(3S,4S)-b-hydroxy ester 9 and Boc-glycine (10).
To obtain (3S,4S)-TyrA unit 9 stereoselectively, the reduction
of b-ketoester 14 was carried out (Table 1).2c b-Ketoester 14 was
synthesized from N-Boc-O-phenylmethyl-L-tyrosine. As shown in
Table 1, desired (3S,4S)-b-hydroxy ester 9 was obtained in 69%
yield with a ratio of 9 : 15 = 93 : 7, with K-selectride. When NaBH4
was used, (3R,4S)-TyrA unit 15 was predominately obtained as
expected, which was used for the synthesis of 3-epi-melleumin A
(6).
After deprotection of the Boc group, coupling with N-Boc-
glycine gave 16 in good yield (Scheme 2). Compound 16 was
hydrolyzed by lithium hydroxide to give a desired carboxylic
acid that was used in the next step without further purification.
Compound 12 (pMBz-L-Thr unit), synthesized from L-threonine
tert-butyl ester hydrochloride and p-methoxybenzoyl chloride, and
the aforementioned carboxylic acid were coupled using DCC
to give compound 18 in 50% yield in 2 steps. EDC gave com-
parable yield with DCC, but 2-methyl-6-nitrobenzoic anhydride
(MNBA)10 reagent provided 18 in low yield. The Boc group
Fig. 2 X-Ray structure of melleumin A (1).
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Scheme 2 Synthesis of melleumin A (1) and 3-epi-melleumin A (6). Reagents and conditions: (a) TFA or HCl; (b) Boc-Gly, EDC, HOBt, DMAP, 80%
(16 (3S), 2 steps from 9), 61% (17 (3R), 2 steps from 15); (c) LiOH, 0 ◦C; (d) 12, DCC, DMAP, 50% (18 (3S), 2 steps from 16), 36% (19 (3R), 2 steps from
17); (e) TFA; (f) FDPP, 28% (20 (3S), 2 steps from 18), 22% (21 (3R), 2 steps from 19); (g) H2, 10% Pd/C, 97% (1), 77% (6).
ensure the absolute configuration at C-10 of 6, the hydrolysate of 6
with 6 N HCl was analyzed by chiral TLC with authentic samples:
L-threonine (10S) and D-allo-threonine (10R). The Rf value of
hydrolysate was identical to that of L-threonine.12
of melleumin A, have moderate Wnt inhibitory activity, greater
flexibility would be important to fit the target molecule (larger
ring). The lactone moiety was removed for compound stability in
cells. At this time, a 13-membered ring was chosen instead of the
12-membered ring of melleumin A.
Because the allyl ether moiety at C-3 is used for cyclization,
compound 15 (3R) was used to maintain the stereochemistry
of 1; substitution on the ring at C-3 and C-4 was predicted
as trans. Synthesis of melleumin A-like compounds is shown
in Scheme 3. Allylation of 15 by Pd catalyst gave compound
22 in 96% yield. Deprotection of the Boc group followed by
coupling with glycine gave 23 in good yield. Coupling reaction
with N-Boc-L-allylglycine13 provided compound 24 in 83% yield
in 2 steps. Ring-closing metathesis (RCM) reaction was selected
for construction of the ring system.14 RCM by second genera-
tion Grubbs’ catalyst proceeded smoothly to give cyclized 13-
membered ring 25 in 98% yield in 20 min. When Grubbs’ catalyst
( bis(tricyclohexylphosphine)benzylidine ruthenium(IV) dichlo-
ride) was used, 25 was obtained in 67% yield under reflux condi-
tions in CH2Cl2 for 24 h. The yield with second generation Grubbs’
catalyst at 80 ◦C in toluene decreased to 82%. The Boc group of
25 was removed, then two acyl chlorides gave each derivative (26,
27). Structural elucidation of 26 was performed by H–H COSY,
HMBC and HMQC (see the ESI†). Compound 26 exhibited the
same substitution as melleumin A (1), a p-methoxybenzoyl unit.
Compound 27 has a greater electron withdrawing acyl unit, a 2,4,6-
trichlorobenzoyl unit. We also synthesized compound 28 from 26
by hydrogenation (Scheme 4).
Synthesis of mellumin A-like compounds
Next, we examined the biological activity of melleumin A (1)
and 3-epi-melleumin A (6). Unfortunately, melleumin A (1) had
weak Wnt signal inhibitory activity and 3-epi-melleumin A (6)
had no inhibitory activity. However, we had previously explored
and found that melleumin B derivatives became Wnt signal
inhibitors.2b,c Therefore, we thought it possible to add Wnt in-
hibition to melleumin A-like compounds. The successful synthesis
and results of moderate inhibition by 4-epi-deoxymelleumin A
reported by Luo et al. also encouraged us to attempt the synthesis
of melleumin A-like compounds. We designed melleumin A-like
compounds as shown in Fig. 3. Although the target molecule of
active melleumins is unknown at this stage, most melleumins show
moderate or weak Wnt inhibition. Therefore we hypothesized
that two amides in the ring and aryl groups could be important
as a common structure (Fig. 3, same moieties as melleumin A).
Because melleumin B derivatives, which are open chain analogues
Wnt signal inhibitory activity
Next we examined Wnt signal inhibitory activity of the synthe-
sized compounds using a luciferase reporter gene assay. Wnt
signaling activates gene transcription with a complex between
Fig. 3 Design of melleumin A-like compounds.
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Melleumin A (1) showed weak inhibitory activity. We evaluated
three newly synthesized melleumin A-like compounds (26–28).
Interestingly, all compounds showed moderate inhibitory activi-
ties. Of them, compound 26, exhibited the strongest activity in a
dose-dependent manner with high cell viability. Comparing the
activities of 26 and 28, the Bn unit and C–C double bond seemed
to be critical. From these results, modification of the melleumin A
scaffold is effective to construct cyclic peptides like Wnt inhibitors.
Fig. 5 shows the comparison of Wnt signal inhibitory activities
of melleumin A, B, their derivatives and melleumin A-like
compounds (50 mM). Natural products, melleumin A (1) and
melleumin B (2), reduced Wnt transcriptional activity to 74% and
64%, respectively. Of them, 10-epi-melleumin B (3) seemed to be
the most active (46%). Regarding cyclic peptide type (melleumin
A type) compounds, 3-epi-melleumin A (6) lost activity; however,
melleumin A-like compound (26) showed 20% more inhibition
(54%) compared with 1 (74%).
To evaluate the structural differences between melleumin A (1)
and melleumin A-like compounds, DFT calculation of 26, the
most active of the three melleumin A-like compounds, was carried
out (Fig. 6). The DFT calculation at the level of B3LYP/6-31G*
suggested a slight difference in the three-dimensional orientation
of two amide units in the ring. The two amide torsion angle (f) was
changed to 86.7◦ (26) from 112.4◦ (1). The distances of N1–N2 and
Scheme 3 Synthesis of melleumin A-like compounds. Reagents and
conditions: (a) allyl ethyl carbonate, Pd2(dba)3, dppb, THF, 65 ◦C, 96%;
(b) TFA; (c) Fmoc-glycine, PyBOP, HOBt, DIPEA, 61% (2 steps); (d)
piperidine; (e) N-Boc-L-allyglycine, PyBOP, HOBt, iPr2NEt, 83% (2 steps);
(f) 2nd Grubbs’ cat. (20 mol%), CH2Cl2, reflux, 98%; (g) TFA; (h)
p-methoxy benzoyl chloride, (2,4,6-trichlorobenzoyl chloride for 27), TEA,
54% (26), 53% (27) (2 steps).
˚
˚
˚
N1–N3 were changed to 3.35 A (26) from 2.93 A (1), and 3.64 A
˚
(26) from 3.71 A (1), respectively. Since two amides in the ring and
aryl groups are common structures in bioactive melleumins, Wnt
inhibition of 26 might be due to this conformational change in the
structure.
Conclusion
The total synthesis of melleumin A (1), 3-epi-melleumin A (6)
and X-ray crystal analysis of synthesized compound 1 were
achieved. Moreover, we designed and synthesized melleumin
A-like compounds (26–28) to seek a possible Wnt signaling
inhibitor. We succeeded to add the Wnt inhibitory activity of a
melleumin A-like compound, though natural product 1 showed
weak activity. Since few small molecules are known as Wnt signal
inhibitors and their clinical use has received great attention, we
believe that peptide-based inhibitors will be worth synthesizing to
evaluate their potential. Synthesis of melleumin A-like compounds
and application using solid-phase reactions to construct small
molecule libraries based on melleumin A-like compounds are in
progress.
Scheme 4 Synthesis of melleumin A-like compounds.
b-catenin and TCF/LEF, a DNA-binding protein. Super TOP-
Flash,15 a reporter plasmid with multiple TCF-binding sites
(CCTTTGATC), was stably transfected into 293 cells. Super FOP-
Flash has eight mutated TCF-binding sites (CCTTTGGCC);
therefore, a selective inhibitor would not affect transcription
in Super FOP-Flash-transfected cells. We tested Super FOP-
Flash activity on all samples. Because it is also possible that
a decrease of luciferase reporter activity in this assay may be
due to cytotoxicity of the test samples, we also examined the
cytotoxicity of samples using fluorometric microculture cytotoxi-
city assay.16 Therefore, compounds exhibiting both low luciferase
reporter activity and high cell viability are to be considered
“potent” compounds. Results are shown in Fig. 4, along with cell
viability.
Experimental
General
Optical rotations were measured with a JASCO P-1020 polarime-
ter. IR spectra were measured on ATR on a JASCO FT-IR 230
spectrophotometer. NMR spectra were recorded on JEOL A 400
and A 500 spectrometers with deuterated solvents, the chemical
shift of which was used as an internal standard. FABMS was
measured on a JEOL JMS-AX500 and HR-FABMS using a JEOL
HX-110A spectrometer. HREIMS was measured on a JEOL JMS-
GC Mate and HR-ESIMS using an Exactive spectrometer.
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Fig. 4 Wnt signal inhibitory activities of melleumin A (1) and melleumin A-like compounds (26, 27, 28). Fold activation of Super TOP-Flash (solid
columns) and cell viability (solid curves). STF/293 cells (a 293 human embryonic kidney cell line stably transfected with Super Top-Flash, 3 ¥ 104) were
split into 96-well plates and 24 h later cells were treated with 15 mM LiCl and testing samples (DMSO solution). Super FOP-Flash activities at each
concentration were not affected (data not shown). N = 3, Bars = sd.
Fig. 5 Comparison of Wnt signal inhibitory activities of melleumin A,
B, their derivatives and melleumin A-like compounds at 50 mM. Fold
activation of Super TOP-Flash (solid columns). STF/293 cells (3 ¥ 104)
were split into 96-well plates and 24 h later cells were treated with 15 mM
LiCl and testing samples (DMSO solution). N = 3, Bars = sd. The activity
of the untreated cells was defined as 100%.
Fig. 6 Structural comparison of 26 (without Bn) and 1. Hydrogen atoms
have been omitted for clarity.
(3S,4S)-Ethyl-5-(4-(benzyloxy)phenyl)-4-(2-(tert-butoxycarbo-
nylamino)acetamido)-3-hydroxypentanoate (16). A solution of 9
(940.8 mg, 2.12 mmol) in CH2Cl2 (10.5 mL) was treated with
TFA (10.5 mL) under argon atmosphere. The reaction mixture
stirred for 30 min. The solvent was removed in vacuo, the residue
was used without further purification in the following reaction.
A solution of N-Boc-glycine (757 mg, 2.55 mmol), EDC·HCl
(488 mg, 2.55 mmol), HOBt·H2O (344 mg, 2.55 mmol) and
DMAP (311 mg, 2.55 mmol) in CH2Cl2 (11.0 mL) was stirred
concentrated in vacuo. The residue was chromatographed on silica
gel (hexane–ethyl acetate = 1/1) to afford 16 (850 mg, 80%, 2 steps
from 9) as a pale yellow powder. 1H NMR (400 MHz, CDCl3) d
1.23 (t, J = 7.1 Hz, 3H), 1.46 (s, 9H), 2.33 (dd, J = 2.4, 17.1 Hz,
1H), 2.51 (dd, J = 17.1, 10.5 Hz, 1H), 2.83 (dd, J = 13.7, 7.0 Hz,
1H), 2.88 (dd, J = 13.7, 8.7 Hz, 1H), 3.60 (br s, 1H), 3.73 (dd, J =
16.8, 5.7 Hz, 1H), 3.78 (dd, J = 16.8, 6.2 Hz, 1H), 3.99–4.16 (m,
4H), 5.03 (s, 2H), 5.06 (br s, 1H), 6.46 (d, J = 9.0 Hz, 1H), 6.91
(d, J = 8.7 Hz, 2H), 7.16 (d, J = 8.7 Hz, 2H) and 7.29–7.44 (m,
5H); 13C NMR (100 MHz, CDCl3) d 14.0, 28.2, 37.1, 38.5, 44.2,
54.0, 60.7, 66.8, 69.8, 80.1, 114.7, 127.4, 127.8, 128.4, 130.0, 130.2,
136.9, 156.0, 137.4, 169.5, and 173.2; HRMS (FAB) [M+H]+: calcd
◦
for 10 min at 0 C under argon atmosphere. To the mixture was
added a solution of the deprotected material obtained above in
CH2Cl2 (11.0 mL). After stirring overnight at RT, the reaction
mixture was diluted with ethyl acetate, washed successively with
water, saturated aqueous NaHCO3, brine, dried over Na2SO4, and
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for C27H37N2O7 501.2601, found 501.2642; IR (ATR); 3321, 2979,
2926, 1712, 1654, 1509, 1239, 1161, 1022, 735 and 696 cm-1; [a]D22
-39.8 (c 3.0, CHCl3).
N -((2R,3S,9S,10S)-9-(4-(Benzyloxy)benzyl)-10-hydroxy-2-
methyl-4,7,12-trioxo-1-oxa-5,8-diazacyclododecan-3-yl)-4-meth-
oxybenzamide (20). A solution of 18 (136 mg, 0.178 mmol) in
◦
CH2Cl2 (3.6 mL) was treated with TFA (0.89 mL) at 0 C under
(2S,3R)-3-Hydroxy-2-(4-methoxybenzamido)butanoic tert-butyl
ester (12). A solution of L-threonine-tert-butyl ester hydrochlo-
ride (334 mg, 1.58 mmol) and triethylamine (0.55 mL, 3.94 mmol)
in CH2Cl2 (6.3 mL) was treated with 4-methoxybenzoyl chloride
(0.24 mL, 1.73 mmol) at 0 ◦C under argon atmosphere. After
stirring overnight at RT, the reaction mixture was quenched
with H2O and extracted with CH2Cl2. The organic layer was
dried over Na2SO4, and concentrated in vacuo. The residue was
chromatographed on silica gel (hexane–ethyl acetate = 2/1) to
argon atmosphere. After stirring for 4 h, the solvent was removed
in vacuo, the residue was used without further purification in
i
the following reaction. To a solution of the residue and Pr2NEt
(0.15 mL, 0.89 mmol) in CH2Cl2 (89 mL) was added FDPP
(136 mg, 0.356 mmol) at 0 ◦C. After stirring overnight at RT,
the reaction mixture was concentrated in vacuo. The residue was
chromatographed on silica gel (hexane–ethyl acetate = 1/1 to 0/1)
1
to afford 20 (24 mg, 23%) as a white solid. H NMR (400 MHz,
DMSO-d6) d 1.21 (3H, d, J = 6.4 Hz), 2.36 (1H, t, J = 11.6 Hz),
2.57 (1H, dd, J = 12.5, 5.0 Hz), 2.64 (1H, dd, J = 14.2, 11.6 Hz),
2.91 (2H, br d, J = 12.6 Hz), 3.44–3.49 (1H, m), 3.72–3.78 (1H,
m), 3.81 (3H, s), 4.11–4.18 (1H, m), 4.98–5.03 (1H, m), 5.04 (2H,
s), 5.47 (1H, d, J = 4.4 Hz), 5.61–5.65 (1H, m), 6.23 (1H, d, J =
10.1 Hz), 6.90 (2H, d, J = 8.7 Hz), 7.00 (2H, d, J = 9.0 Hz), 7.10
(2H, d, J = 8.7 Hz), 7.29–7.44 (5H, m), 7.91 (2H, d, J = 9.0 Hz),
8.09 (1H, br d, J = 8.8 Hz) and 8.47 (1H, br t, J = 5.7 Hz);
13C NMR (100 MHz, CDCl3/CD3OD = 4/1) d 16.1, 29.4, 31.6,
36.9, 44.6, 55.0, 55.1, 55.9, 68.6, 69.8, 72.3, 113.6 (2C), 114.5
(2C), 125.1, 127.2n, 127.7, 128.3 (2C), 129.0 (2C), 129.6 (2C),
130.4, 136.8, 157.1, 162.6, 167.9, 169.8, 170.4 and 172.0; HRMS
(FAB) [M+H]+: calcd for C32H36N3O8 590.2502, found 590.2518;
IR (ATR); 3319, 2970, 2926, 1737, 1651, 1509, 1366, 1250, 1076,
1027, 844, 735 and 696 cm-1; [a]D21 +19.9 (c 0.25, CHCl3–MeOH =
1/1).
1
afford 12 (485 mg, 99%) as a pale yellow amorphous solid. H
NMR (400 MHz, CDCl3) d 1.24 (d, J = 5.3 Hz, 2H), 1.48 (9H,
s), 3.80 (3H, s), 3.88 (1H, br s), 4.37 (1H, br d, J = 4.4 Hz), 4.66
(1H, dd, J = 8.8, 1.2 Hz), 6.85 (2H, d, J = 8.6 Hz), 7.20 (1H, d,
J = 8.7 Hz) and 7.80 (2H, d, J = 8.8 Hz); 13C NMR (100 MHz,
CDCl3) d 20.1, 28.0, 55.3, 58.2, 68.6, 82.4, 113.6, 126.0, 129.0,
162.3, 167.4 and 170.3; IR (ATR) 3394, 2980, 2935, 1733, 1630,
1606, 1537, 1255, 1086, 839 and 764 cm-1; HRMS (FAB) [M+H]+:
calcd for C16H24NO5 310.1654, found 310.1656. IR (ATR); 3394,
2980, 2935, 1733, 1629, 1606, 1606, 1537, 1506, 1349, 1255, 1159,
1123, 1085, 1011, 838 and 764 cm-1; [a]D20 +32.2 (c 3.0, CHCl3).
(3S,4S)-((2R,3S)-4-tert-Butoxy-3-(4-methoxybenzamido)-4-
oxobutan-2-yl)-5-(4-(benzyloxy)phenyl)-4-(2-(tert-butoxycarbony-
lamino)acetamido)-3-hydroxypentanoate (18). To a solution of 16
(412 mg, 0.823 mmol) in solvent (THF–H2O = 1/1) was added
LiOH·H2O (86 mg, 2.06 mmol) at 0 ◦C, and the mixture was
stirred for 2.5 h. The mixture was acidified with 2 N HCl, and
then extracted with ethyl acetate. The organic layer was dried over
Na2SO4 and concentrated in vacuo, the residue was used without
further purification in the following reaction. The residue was
dissolved in CH2Cl2 (3.9 mL). To this solution DCC (131 mg,
0.633 mmol), DMAP (28 mg, 0.231 mmol) and a solution of
12 (310 mg, 1.00 mmol) in CH2Cl2 (3.9 mL) were added. After
stirring for 48 h at RT, the reaction mixture was quenched with
10% aqueous KHSO4 and extracted with CH2Cl2. The organic
layer was washed with saturated aqueous NaHCO3, water and
brine. The organic layer was dried over Na2SO4, and concentrated
in vacuo. The residue was chromatographed on silica gel (hexane–
ethyl acetate = 2/1 to 1/1) to afford 18 (257 mg, 44%) as a colorless
Melleumin A (1). To a solution of 20 (18 mg, 31 mmol)
in solvent (EtOAc–EtOH = 1/2) was added 25% palladium
on carbon (4 mg). The reaction mixture was stirred for 24 h
under hydrogen and then filtered through Celite and concentrated
in vacuo. The crude product was purified by preparative TLC (silica
gel, MeOH–CHCl3 = 1/10), to give melleumin A (15 mg, 97%) as a
white solid. For X-ray analysis, the compound was further purified
by HPLC (Develosil C30, 25% CH3CN, 2.5 ml min-1, 32 min). 1H
NMR (400 MHz, DMSO-d6) d 1.20 (3H, d, J = 6.2 Hz), 2.35 (1H,
t, J = 11.7 Hz), 2.53–2.61 (2H, m), 2.85 (1H, dd, J = 14.2, 11.9 Hz),
3.37–3.49 (3H, m), 3.71–3.77 (1H, m), 3.81 (3H, s), 4.08–4.13 (1H,
m), 4.99 (1H, dd, J = 9.0, 3.5 Hz), 5.45 (1H, br s), 5.52 (1H, qd,
J = 6.5, 3.7 Hz), 6.26 (1H, d, J = 9.9 Hz), 6.63 (2H, d, J = 8.4 Hz),
6.96 (2H, d, J = 8.4 Hz), 7.00 (2H, d, J = 9.0 Hz), 7.91 (2H, d, J =
9.0 Hz) 8.09 (1H, br d, J = 9.0 Hz) and 8.63 (1H, brs); 13C NMR
(125 MHz, CDCl3) d 16.1, 30.8, 38.7, 44.4, 54.9, 55.1, 69.5, 71.7,
113.4, 114.9, 126.0, 129.4, 129.5, 155.4, 161.8, 166.6, 169.1, 169.2
and 170.7; HRMS (FAB) [M+H]+: calcd for C25H30N3O8 500.2033,
found 500.2030; IR (ATR); 3369, 3031, 2943, 1739, 1635, 1507,
1438, 1365, 1229, 1217 and 1109 cm-1; [a]D18 +16 (c 0.97, MeOH).
1
amorphous solid. H NMR (400 MHz, CDCl3) d 1.29 (d, J =
6.4 Hz, 3H), 1.43 (s, 9H), 1.44 (s, 9H), 2.34 (dd, J = 15.6, 3.0 Hz.
1H), 2.48 (dd, J = 15.6, 9.9 Hz, 1H), 2.81 (dd, J = 13.4, 6.8 Hz,
1H), 2.87 (dd, J = 13.4, 8.4 Hz, 1H), 3.65 (br s, 1H), 3.78 (d, J =
5.7 Hz, 2H), 3.85 (s, 3H), 3.97 (br d, J = 9.9 Hz, 1H), 4.12 (q, J =
8.4 Hz, 2H), 4.88 (dd, J = 9.0, 2.6 Hz, 1H), 5.02 (s, 2H), 5.18 (br s,
1H), 5.52 (qd, J = 6.4, 2.6 Hz, 1H), 6.47 (d, J = 9.5 Hz, 1H), 6.87
(d, J = 8.6 Hz, 2H), 6.96 (d, J = 8.5 Hz, 2H), 7.13 (d, J = 8.5 Hz,
2H), 7.30–7.44 (m, 5H), 7.86 (d, J = 8.6 Hz, 2H); 13C NMR (100
MHz, CDCl3) d 17.1, 27.8 (3C), 28.2 (3C), 37.3, 39.5, 54.1, 55.4,
56.2, 67.1, 6.9, 71.3, 80.3, 83.1, 113.8 (2C), 114.9 (2C), 125.8, 127.5
(2C), 127.9, 128.5 (2C), 129.2 (2C), 129.8, 130.3 (2C), 136.9, 157.5,
162.5, 167.2, 169.4, 169.5 and 171.7; HRMS (FAB) [M+H]+: calcd
for C41H54N3O11 764.3758, found 764.3794; IR (ATR); 3309, 2970,
2935, 1737, 1653, 1507, 1231, 1155, 1026, 844 and 696 cm-1; [a]D21
+9.9 (c 3.0, CHCl3).
(3R,4S)-Ethyl-5-(4-(benzyloxy)phenyl)-4-(2-(tert-butoxycar-
bonylamino)acetamido)-3-hydroxypentanoate (17). 1H NMR
(500 MHz, CDCl3) d 1.25 (3H, t, J = 7.1 Hz), 1.43 (9H, s), 2.46–
2.55 (2H, m), 2.78 (1H, dd, J = 14.3, 8.9 Hz), 2.90 (1H, dd, J =
14.3, 4.6 Hz), 3.62 (1H, dd, J = 16.3, 5.6 Hz), 3.67 (1H, br dd,
J = 16.3, 6.0 Hz), 3.92 (1H, br d, J = 4.0 Hz), 4.03–4.07 (1H, m),
4.12–4.18 (1H, m),3.78 (1H, q, J = 7.1 Hz), 5.03 (2H, s), 5.16 (1H,
br t, J = 5.7 Hz), 6.46 (1H, d, J = 8.9 Hz), 6.89 (2H, d, J = 8.5 Hz),
7.11 (2H, d, J = 8.5 Hz) and 7.29–7.43 (5H, m); 13C NMR (125
MHz, CDCl3) d 14.1, 28.2 (3C), 34.3, 38.1, 44.3, 54.2, 60.8, 69.6,
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69.9, 80.2, 114.8 (2C), 127.4 (2C), 127.9, 128.5 (2C), 129.5, 130.2
(2C), 136.9, 156.1, 157.4, 169.6 and 172.7; HRMS (FAB) (M)+:
calcd for C27H36N2O7 500.2522, found 500.2497; IR (ATR); 3342,
3281, 1732, 1685, 1654, 1541, 1508, 1365, 1218, 1165, 1018 and
696 cm-1; [a]1D9 -7.7 (c 3.0, CHCl3).
(ATR); 3291, 2923, 2851, 1732, 1717, 1637, 1516, 1506, 1256, 1177
and 1055 cm-1; [a]2D5 +42.3 (c 1.0, EtOH).
(3R,4S)-Ethyl-3-(allyloxy)-5-(4-(benzyloxy)phenyl)-4-(tert-but-
oxycarbonylamino)-pentanoate (22). To a solution of 15 (254 mg,
0.574 mmol), Pd2(dba)3 (13 mg, 14 mmol) and dppb (25 mg,
0.57 mmol) in THF (2.3 mL) was added allyl ethyl carbonate
(0.45 mL, 3.44 mmol) under argon atmosphere. The mixture was
heated to 65 ◦C and stirred for 4 h and then concentrated in vacuo.
The residue was chromatographed on silica gel (hexane–diethyl
ether = 5/1 to 1/1) to afford 22 (258 mg, 96%) as a white solid. 1H
NMR (400 MHz, CDCl3) d 1.26 (t, 3H, J = 7.1 Hz), 1.46 (s, 9H),
2.53 (dd, 1H, J = 5.5, 15.5,Hz), 2.57 (dd, 1H, J = 6.5, 15.5 Hz),
2.59–2.69 (m, 1H), 2.91 (dd, 1H, J = 4.4, 14.2 Hz), 3.85–3.92 (m,
2H), 4.02 (ddt, 1H, J = 12.4, 6.0, 1.4 Hz), 4.07–4.18 (3H, m), 4.41
(1H, br s), 5.03 (2H, s), 5.15 (1H, dq, J = 10.3, 1.5 Hz), 5.26 (1H,
dq, J = 17.2, 1.7 Hz), 5.85–5.94 (1H, m), 6.88 (2H, d, J = 8.8 Hz),
7.09 (2H, d, J = 8.8 Hz), 7.29–7.42 (5H, m); 13C NMR (100 MHz,
CDCl3) d 13.9, 28.0, 35.6, 37.0, 54.6, 60.1, 69.8, 71.0, 77.6, 78.7,
114.7, 116.3, 127.0, 127.4, 128.1, 129.9, 130.3, 134.6, 137.1, 155.0,
157.3 and 171.1; HRMS (EI) [M]+: calcd for C28H37NO6 483.2621,
found 483.2618; IR (ATR); 3345, 2989, 1726, 1683, 1529, 1509,
1237, 1158 and 1004 cm-1; [a]D19 -20.5 (c 3.0, CHCl3).
(3R,4S)-((2R,3R)-4-tert-Butoxy-3-(4-methoxybenzamido)-4-
oxobutan-2-yl)-5-(4-(benzyloxy)phenyl)-4-(2-(tert-butoxy-
carbonylamino)acetamido)-3-hydroxypentanoate (19).
1H NMR (400 MHz, CDCl3) d 1.34 (3H, d, J = 6.6 Hz), 1.43
(9H, s), 1.45 (9H, s), 2.51 (2H, d, J = 6.5 Hz), 2.79 (1H, dd, J =
14.2, 8.7 Hz), 2.88 (1H, dd, J = 14.2, 4.6 Hz), 3.65 (2H, br d, J =
5.9 Hz), 3.72 (1H, br t, J = 4.9 Hz), 3.84 (3H, s), 3.97 (1H, br
d, J = 9.9 Hz), 4.01–4.07 (1H, m), 4.12–4.20 (1H, m), 4.91 (1H,
dd, J = 9.0, 2.9 Hz), 5.02 (2H, s), 5.55 (1H, qd, J = 6.6, 2.9 Hz),
6.15–6.24 (1H, m), 6.15–6.24 (1H, m), 6.85–6.89 (1H, m), 6.89
(2H, d, J = 8.7 Hz), 6.95 (2H, d, J = 8.9 Hz), 7.09 (2H, d, J =
8.9 Hz), 7.30–7.44 (5H, m), 7.84 (2H, d, J = 8.7 Hz); 13C NMR
(100 MHz, CDCl3) d 17.1, 27.9 (3C), 28.2 (3C), 34.3, 38.6, 54.1,
55.4, 56.3, 69.4, 69.9, 71.5, 80.4, 83.1, 113.8 (2C), 114.9 (2C), 125.9,
127.5 (2C), 127.9, 128.6 (2C), 129.2 (2C), 130.2 (2C), 136.9, 157.6,
162.5, 167.2, 169.3, 169.6 and 170.9; HRMS (FAB) [M+H]+: calcd
for C41H54N3O11 764.3758, found 763.3755.; IR (ATR); 2979, 1733,
1652, 1507, 1367, 1247, 1155, 1028 and 751 cm-1; [a]D19 +36.6 (c
3.0, CHCl3).
(3R,4S)-Ethyl-3-allyloxy-4-(2-(9-fluorenylmethoxycarbony-
lamino)acetamide)-5-(4-(benzyloxy)phenyl)pentanoate (23). To a
solution of 22 (242 mg, 0.50 mmol) in CH2Cl2 (2.5 mL) was treated
with TFA (2.5 mL) at 0 ◦C under argon atmosphere. After stirring
for 1 h at RT, the solvent was removed in vacuo. The residue
was used without further purification in the following reaction. A
solution of Fmoc-glycine (223 mg, 0.750 mmol), PyBOP (390 mg,
0.75 mmol), HOBt·H2O (101 mg, 0.75 mmol) and DIEA (0.26 mL,
1.50 mmol) in CH2Cl2 (2.5 mL) was stirred for 10 min at 0 ◦C
under argon atmosphere. To this mixture was added a solution
of the deprotected material obtained above in CH2Cl2 (2.5 mL).
After stirring overnight, the reaction mixture was quenched with
5% aqueous KHSO4 and extracted with CH2Cl2. The organic
layer was washed with 5% aqueous NaHCO3, water and brine,
dried over Na2SO4, and concentrated in vacuo. The residue was
chromatographed on silica gel (hexane–EtOAc = 2/1 to1/1) to
afford 23 (321 mg, 0.483 mmol, 61%, 2 steps from 22) as a white
solid. 1H NMR (400 MHz, CDCl3) d 1.24 (t, J = 7.1 Hz, 3H), 2.53
(dd, J = 5.4, 15.6 Hz, 1H), 2.60 (dd, J = 7.1, 15.6 Hz, 1H), 2.68
(dd, J = 9.5, 14.2 Hz, 1H), 2.91 (dd, J = 4.9, 14.2 Hz, 1H), 3.70
(d, J = 5.6 Hz, 2H), 3.86–3.93 (m, 1H), 4.00 (dd, J = 6.1, 13.0 Hz,
1H), 4.07–4.41 (m, 7H), 4.97 (s, 2H), 5.15 (dd, J = 10.3, 1.5 Hz,
1H), 5.25 (dd, J = 17.3, 1.2 Hz, 1H), 5.27 (br s, 1H), 5.82–5.92 (m,
1H), 6.14 (br t, J = 9.0 Hz, 1H), 6.84 (d, J = 8.6 Hz, 2H), 7.04
(d, J = 8.6 Hz, 2H), 7.27–7.42 (m, 9H), 7.58 (br d, J = 7.1 Hz,
2H), and 7.75 (d, J = 7.6 Hz, 2H); 13C NMR 100 MHz (CDCl3) d
14.1, 35.0, 37.0, 44.4, 47.0, 53.0, 60.8, 67.1, 69.8, 71.3, 114.7, 117.1,
120.0, 125.0, 127.0, 127.4, 127.7, 127.8, 128.4, 129.7, 130.0, 134.4,
136.9, 141.2, 141,6, 156.4, 157.3, 168.4, and 171.4; HRMS (FAB)
[M+Na]+: calcd for C40H42N2O7Na 685.2890, found 685.2912; IR
(ATR); 3307, 3272, 1726, 1696, 1652, 1539, 1509, 1232 and 731
cm-1; [a]1D9 -25.2 (c 3.0, CHCl3).
N -((2R,3R,9S,10R)-9-(4-(Benzyloxy)benzyl)-10-hydroxy-2-
methyl-4,7,12-trioxo-1-oxa-5,8-diazacyclododecan-3-yl)-4-meth-
oxybenzamide (21). 1H NMR (400 MHz, DMSO-d6) d 1.16 (3H,
d, J = 6.3 Hz), 2.35–2.48 (2H, m), 2.63 (1H, dd, J = 13.7, 8.6 Hz),
2.94 (1H, dd, J = 13.7, 3.2 Hz), 3.25 (1H, dd, J = 13.2, 3.4 Hz),
3.72 (1H, qd, J = 9.2, 3.4 Hz), 3.80 (3H, s), 3.81–4.05 (2H, m),
4.83 (1H, dd, J = 9.3, 3.6 Hz), 5.03 (2H, s), 5.33 (1H, d, J =
6.6 Hz), 5.56 (1H, qd, J = 6.4, 3.8 Hz), 6.78 (1H, d, J = 9.8 Hz),
6.88 (2H, d, J = 8.8 Hz), 7.00 (2H, d, J = 9.0 Hz), 7.08 (2H, d,
J = 8.8 Hz), 7.29–7.44 (5H, m), 7.89 (2H, d, J = 9.0 Hz) and
8.18–8.23 (2H, m); 13C NMR (100 MHz, CDCl3) d 15.9, 36.1,
38.6, 40.9, 44.1, 55.4, 56.2, 56.4, 69.1, 69.4, 70.2, 113.4 (2C), 114.2
(2C), 126.0, 127.6 (2C), 127.7, 128.3 (2C), 129.6 (2C), 130.3 (2C),
130.7, 137.2, 156.6, 161.8, 166.6, 169.0, 169.3 and 170.8; HRMS
(FAB) [M+H]+: calcd for C32H36N3O8 590.2502, found 590.2468;
IR (ATR); 3314, 2926, 1720, 1641, 1606, 1509, 1455, 1254, 1176
and 1021 cm-1; [a]D21 +53.3 (c 0.25, CHCl3–MeOH = 1/1).
3-epi-Melleumin A (6). 1H NMR (500 MHz, DMSO-d6) d 1.15
(d, J = 6.5 Hz, 3H, H-12), 2.36 (dd, J = 9.8, 15.1 Hz, 1H, H-2),
2.45 (d, J = 15.1 Hz, 1H, H-2), 2.57 (dd, J = 8.5, 13.6 Hz, 1H,
H-1¢¢), 2.87 (dd, J = 3.2, 13.6 Hz, 1H, H-1¢¢), 3.25 (dd, J = 4.0,
13.7 Hz, 1H, H-7), 3.67 (m, 1H, H-4), 3.80 (s, 3H, OCH3), 3.82
(m, 2H, H-3, H-7), 4.83 (dd, J = 3.7, 9.2 Hz, 1H, H-10), 5.34 (d,
J = 6.4 Hz, 1H, CHOH), 5.54 (dq, J = 3.7, 6.5 Hz, 1H, H-11),
6.61 (d, J = 8.4 Hz, 2H, H-4¢¢ and H-6¢¢), 6.75 (d, J = 9.5 Hz,
1H, H-5), 6.94 (d, J = 8.4 Hz, 2H, H-3¢¢ and H-5¢¢), 6.99 (d, J =
8.9 Hz, 2H, H-5¢ and H-7¢), 7.90 (d, J = 8.9 Hz, 2H, H-4¢ and H-8¢),
8.25 (m, 2H, H-8, H-1¢) and 9.11 (br s, 1H, C6H4OH); 13C NMR
(125 MHz, DMSO-d6) d 15.6, 36.0, 40.9, 44.1, 55.4, 56.2, 56.5,
69.3, 70.3, 113.4 (2C), 114.8 (2C), 126.0, 128.5, 129.7 (2C), 130.2
(2C), 155.4, 161.8, 166.6, 169.0, 169.3 and 170.9; HRMS (FAB)
[M+Na]+: calcd for C25H29N3O8Na 522.1852, found 522.1849; IR
(3R,4S)-Ethyl-3-(allyloxy)-4-(2-((S)-2-(tert-butoxycarbony-
lamino)pent-4-enamido)acetamido)-5-(4-(benzyloxy)phenyl)pen-
tanoate (24). A solution of 23 (441 mg, 0.665 mmol) in DMF
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(6.7 mL) was treated with piperidine (0.67 mL, 6.7 mmol) for
1 h. The residue after evaporation was used without further
purification in the next reaction. A solution of N-Boc-L-allyglycine
(147 mg, 0.682 mmol), PyBOP (519 mg, 1.00 mmol), HOBt·H2O
(135 mg, 1.00 mmol) and DIPEA (0.35 mL, 1.50 mmol) in DMF
(1.5 mL) was stirred for 20 min at 0 ◦C under argon atmosphere.
To this solution was added a solution of the deprotected material
obtained above in DMF (3.3 mL). After stirring overnight at RT,
the reaction mixture was quenched with 5% aqueous KHSO4 and
extracted with dichloromethane. The organic layer was washed
with 5% aqueous NaHCO3, water and brine, dried over Na2SO4
and concentrated in vacuo. The residue was chromatographed on
silica gel (hexane–EtOAc = 3/1 to 1/2) to afford 24 (352 mg,
with 5% aqueous NaHCO3, and extracted with CH2Cl2. The
organic layer was washed with water and brine, dried over Na2SO4
and concentrated in vacuo. The crude product was purified by
preparative TLC (silica gel, MeOH–CHCl3 = 1/10), to give 26
(8 mg, 54%) as a white solid. 1H NMR (500 MHz, CDCl3–
CD3OD = 4/1) d 1.24 (t, J = 7.0 Hz, 3H), 2.37 (dd, J = 5.4,
15.6 Hz, 1H), 2.54 (dd, J = 7.6, 15.6 Hz, 1H), 2.61–2.69 (m, 2H),
2.78–2.85 (m, 1H), 2.88 (dd, J = 5.9, 14.0 Hz, 1H), 3.81 (s, 3H),
3.87 (dd, J = 6.2, 13.1 Hz, 1H), 3.92 (ddd, J = 1.5, 5.4, 7.6 Hz,
1H), 4.02 (dd, J = 6.3, 13.5 Hz, 1H), 4.12 (q, J = 7.0 Hz, 2H),
4.17 (dd, J = 6.2, 13.1 Hz, 1H), 4.20–4.26 (m, 1H), 4.81–4.86 (m,
1H), 5.01 (s, 2H), 5.85 (dt, J = 6.3, 15.6 Hz, 1H), 5.92 (dt, J = 6.2,
15.6 Hz, 1H), 6.63 (br d, J = 9.2 Hz, 1H), 6.87 (d, J = 8.6 Hz,
2H), 6.95 (d, J = 8.7 Hz, 2H), 6.95 (d, J = 7.3 Hz, 2H), 7.14 (d,
J = 8.5 Hz, 2H), 7.29–7.43 (m, 5H), and 7.72 (d, J = 8.7 Hz, 2H);
13C NMR 125 MHz (CDCl3) d 14.2, 34.2, 35.5, 38.4, 43.8, 52.8,
53.0, 55.4, 60.8, 69.8, 69.9, 77.6, 113.9 (2C), 114.6 (2C), 125.3,
127.5 (2C), 127.9, 128.5 (2C), 129.0 (2C), 130.2, 130.4 (2C), 130.5,
131.2, 137.1, 157.3, 162.7, 167.0, 167.3, 171.0, and 171.6; HRMS
(FAB) [M+H]+: calcd for C33H38N3O7 644.2941, found 644.2971;
IR (ATR) 3281, 2923, 1746, 1661, 1540, 1507,1253, 1177, 1025
and 695 cm-1; [a]2D4 -22 (c 0.2, DMSO).
1
0.552 mmol, 83%, 2 steps from 23) as a colorless oil. H NMR
(400 MHz, CDCl3) d 1.24 (t, J = 7.1 Hz, 3H), 1.42 (s, 9H),
2.37–2.60 (m, 4H), 2.67 (dd, J = 9.3, 14.1 Hz, 1H), 2.87 (dd,
J = 4.7, 14.1 Hz, 1H), 3.71 (dd, J = 4.6, 16.5 Hz, 1H), 3.86–4.09
(m, 4H), 4.13 (q, J = 7.1 Hz, 2H), 4.15–4.22 (m, 1H), 4.33–4.39
(m, 1H), 4.96 (s, 2H), 5.66–5.91 (m, 2H), 6.86 (d, J = 8.7 Hz, 2H),
7.08 (d, J = 8.7 Hz, 2H) and 7.29–7.40 (m, 5H); 13C NMR 100
MHz (CDCl3) d 13.9, 28.0 (3C), 35.3, 36.5, 36.6, 42.8, 52.7, 53.8,
60.4, 69.5, 71.0, 76.9, 79.7, 114.5 (2C), 116.7, 118.7, 127.2 (2C),
127.6, 128.2 (2C), 129.8, 130.0 (2C), 132.8, 134.4, 139.8, 155.3,
157.1, 168.2, 171.5, and 171.9; HRMS (FAB) [M+Na]+: calcd for
C35H47N3O8Na 660.3261, found 660.3248; IR (ATR) 3299, 2979,
1645, 1509, 1366, 1240, 1164, 1020, 920, 737 and 695 cm-1; [a]D19
+4.9 (c 3.0, MeOH).
Ethyl-2-((2R,3S,9R,E)-3-(4-(benzyloxy)benzyl)-5,8-dioxo-9-
(2,4,6-trichlorobenzamido)-1-oxa-4,7-diazacyclotridec-11-en-2-
yl)acetate (27). 1H NMR (500 MHz, CDCl3–CD3OD = 4/1) d
1.24 (t, J = 7.0 Hz, 3H), 2.37 (dd, J = 5.4, 15.6 Hz, 1H), 2.54 (dd,
J = 7.6, 15.6 Hz, 1H), 2.61–2.69 (m, 2H), 2.78–2.85 (m, 1H), 2.88
(dd, J = 5.9, 14.0 Hz, 1H), 3.81 (s, 3H), 3.87 (dd, J = 6.2, 13.1 Hz,
1H), 3.92 (ddd, J = 1.5, 5.4, 7.6 Hz, 1H), 4.02 (dd, J = 6.3, 13.5 Hz,
1H), 4.12 (q, J = 7.0 Hz, 2H), 4.17 (dd, J = 6.2, 13.1 Hz, 1H), 4.20–
4.26 (m, 1H), 4.81–4.86 (m, 1H), 5.01 (s, 2H), 5.85 (dt, J = 6.3,
15.6 Hz, 1H), 5.92 (dt, J = 6.2, 15.6 Hz, 1H), 6.63 (br d, J = 9.2 Hz,
1H), 6.87 (d, J = 8.6 Hz, 2H), 6.95 (d, J = 8.7 Hz, 2H), 6.95 (d,
J = 7.3 Hz, 2H), 7.14 (d, J = 8.5 Hz, 2H), 7.29–7.43 (m, 5H), and
7.72 (d, J = 8.7 Hz, 2H); 13C NMR 125 MHz (CDCl3) d 14.2,
34.2, 35.5, 38.4, 43.8, 52.8, 53.0, 55.4, 60.8, 69.8, 69.9, 77.6, 113.9
(2C), 114.6 (2C), 125.3, 127.5 (2C), 127.9, 128.5 (2C), 129.0 (2C),
130.2, 130.4 (2C), 130.5, 131.2, 137.1, 157.3, 162.7, 167.0, 167.3,
171.0, and 171.6; HRMS (FAB) [M+H]+: calcd for C35H37Cl3N3O7
716.1697, found 716.1681; IR (ATR) 3281, 2923, 1746, 1661, 1540,
1507,1253, 1177, 1025 and 695 cm-1; [a]D24 -22 (c 0.2, DMSO).
Ethyl-2-((2R,3S,9R,E)-3-(4-(benzyloxy)benzyl)-9-(tert-butoxy-
carbonylamino)-5,8-dioxo-1-oxa-4,7-diazacyclotridec-11-en-2-yl)-
acetate (25). To a solution of 24 (32 mg, 50 mmol) in CH2Cl2
(25 mL) was added Grubbs’ catalyst (second-generation) (8.4 mg,
10 mmol). The mixture was heated under reflux conditions for 20
min and concentrated in vacuo. Purification of this residue by flash
chromatography (hexane–EtOAc = 1/1 to 0/1) afforded 25 (30 mg,
98%) as a white powder. 1H NMR (400 MHz, DMSO-d6) d 1.15
(t, J = 7.1 Hz, 3H), 1.36 (s, 9H), 2.30–2.47 (m, 4H), 2.59 (dd, J =
7.6, 15.8 Hz, 1H), 2.76 (dd, J = 4.4, 14.1 Hz, 1H), 3.29 (dd, J =
4.6, 16.3 Hz, 1H), 3.62–3.76 (m, 3H), 4.00–4.15 (m, 5H), 5.02 (s,
2H), 5.72 (dt, J = 6.6, 15.4 Hz, 1H), 5.80 (dt, J = 6.4, 15.4 Hz,
1H), 6.75 (br d, J = 9.3 Hz, 1H), 6.86 (d, J = 8.5 Hz, 2H), 7.11 (d,
J = 8.5 Hz, 2H), 7.18 (d, J = 7.3 Hz, 1H) and 7.29–7.43 (m, 5H);
13C NMR 100 MHz (CDCl3) d 14.0, 28.2 (3C), 33.4, 34.6, 37.4,
43.0, 52.0, 53.1, 60.1, 68.7, 69.1, 76.7, 78.2, 114.3 (2C), 127.7 (2C),
127.8, 128.4 (2C), 129.5, 130.1 (2C), 131.0, 131.1, 137.2, 154.9,
156.7, 167.5, 170.7, and 171.5; HRMS (FAB) [M+Na]+: calcd for
C33H43N3O8Na 632.2948, found 632.2924; IR (ATR) 3325, 2926,
1732, 1655, 1514, 1245, 1170, 1018, 733 and 695 cm-1; [a]D19 -40 (c
0.1, CHCl3).
Ethyl-2-((2R,3S,9R)-3-(4-hydroxybenzyl)-9-(4-methoxybenza-
mido)-5,8-dioxo-1-oxa-4,7-diazacyclotridecan-2-yl)acetate (28).
To a solution of 26 (5 mg, 8 mmol) in solvent (THF–EtOH = 2/1)
was added 25% palladium on carbon (1 mg). The reaction mixture
was stirred for 7 h under hydrogen atomosphere and then filtered
through Celite and concentrated in vacuo. The crude product was
purified by preparative TLC (silica gel, MeOH–CHCl3 = 1/9), to
give 28 (3 mg, 6 mmol, 70%) as a white solid. 1H NMR (400 MHz,
CDCl3/CD3OD = 4/1) d 1.29 (t, J = 7.1 Hz, 3H), 1.49–1.92 (m,
6H), 2.49 (dd, J = 6.6, 15.3 Hz, 1H), 2.62 (dd, J = 10.0, 14.2 Hz,
1H), 2.69 (dd, J = 6.6, 15.3 Hz, 1H), 2.89 (dd, J = 4.9, 14.2 Hz,
1H), 3.23 (t, J = 11.7 Hz, 1H), 3.46 (d, J = 16.5 Hz, 1H), 3.66–3.85
(m, 2H), 3.87 (s, 3H), 3.92 (d, J = 16.5 Hz, 1H), 4.16 (q, J =
7.1 Hz, 2H), 4.21–4.29 (m, 1H), 4.47 (dd, J = 3.1, 9.9 Hz, 1H),
6.73 (d, J = 8.6 Hz, 2H), 6.94 (d, J = 8.9 Hz, 2H), 7.04 (d, J =
8.6 Hz, 2H), and 7.79 (d, J = 8.9 Hz, 2H); 13C NMR 125 MHz
(CDCl3) d 14.2, 20.9, 28.5, 31.8, 33.6, 37.2, 43.5, 53.0, 54.2, 55.6,
Ethyl-2-((2R,3S,9R,E)-3-(4-(benzyloxy)benzyl)-9-(4-methoxy-
benzamido)-5,8-dioxo-1-oxa-4,7-diazacyclotridec-11-en-2-yl)acet-
ate (26). A solution of 25 (13 mg, 21.6 mmol) in CH2Cl2 (1.0 mL)
was treated with TFA (1.0 mL) at 0 ◦C under argon atmosphere.
After stirring for 30 min, the solvent was removed in vacuo. The
residue after evaporation was used without further purification
in the next reaction. To a solution of the deprotected material
and triethylamine (8 mL, 108 mmol) in CH2Cl2 (2.2 mL) was
added p-methoxybenzoyl chloride (6 mL, 43 mmol) at 0 ◦C.
After stirring overnight at RT, the reaction mixture was quenched
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61.3, 68.1, 78.7, 114.0 (2C), 115.3 (2C), 125.8, 129.0 (2C), 129.3,
130.4 (2C), 155.5, 162.8, 167.5, 168.6, 171.5 and 173.7; HRMS
(FAB) [M+H]+: calcd for C29H38N3O8 556.2659, found 556.2608;
IR (ATR) 3394, 3290, 2920, 1731, 1651, 1540, 1506,1458, 1258,
1183, 1110, 1026 and 846 cm-1; [a]1D9 -16.9 (c 0.5, MeOH).
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Cell cultures. STF/293 cells were a generous gift from Prof.
Jeremy Nathans (Johns Hopkins Medical School). STF/293 and
293T cells were cultured in Dulbecco’s modified eagle medium
(Wako) with 10% FBS. Cultures were maintained in a humidified
incubator at 37 ◦C in 5% CO2/95% air.
Super TOP-Flash reporter assay. STF/293 cells (a 293 human
embryonic kidney cell line stably transfected with Super Top-
Flash, 3 ¥ 104) were split into 96-well plates and 24 h later cells were
treated with 15 mM LiCl and testing samples (DMSO solution)
with a medium containing FBS. After incubation for 24 h, cells
were lysed with CCLR (cell culture lysis reagent; 20 mL/well,
Promega) and luciferase activities were measured with a Luciferase
Assay System (Promega). We checked this system worked correct
by using quercetin as a standard positive compound. Assays were
performed in triplicate.
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9 The reaction did not proceed in conditions; EDC, HOBT, CH2Cl2;
EDC, DMAP, CH2Cl2, reflux; 2,4,6-trichlorobenzoyl chloride, Et3N,
DMAP, toluene, rt or 50 ◦C; MNBA, DMAP, CH2Cl2, rt; MNBA,
DMAP, Et3N, CH2Cl2, rt.
Super FOP-Flash reporter assay. 293T cells (1 ¥ 105) were split
into 24-well plates and were transfected 24 h later with 1 mg/well
Super Fop-Flash reporter plasmid and 0.05 mg of pRL-CMV plas-
mid (Promega, USA) for normalization using Lipofectamine2000
(Invitrogen; 2.5 mL/well). After 3 h transfection, compounds were
added with a medium containing FBS. Of note, 293T cells were
treated with compounds in a FBS-containing medium combined
with 15 mM of LiCl. Cells incubated for 24 h were lysed in Passive
lysis buffer (Promega, 50 mL/well) and luciferase activity was
measured with a Dual-Glo Luciferase Assay System (Promega).
Assay of cell viability. 293T cells (6 ¥ 103) were split into
96-well plates and incubated for 24 h. Cells were treated with
compounds and incubated for 24 h. They were treated with
fluorescein diacetate (Wako) in PBS buffer (10 mg mL-1), and
after 1 h of incubation, fluorescence was detected. Assays were
performed at least in triplicate.
10 I. Shiina, M. Kubota, H. Oshiumi and M. Hashizume, J. Org. Chem.,
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˚
11 Crystal data: Monoclinic; space group P21; a = 10.9532(17) A, b =
3
˚
˚
˚
7.9598(13) A, c = 17.259(3) A; V = 1494.4(4) A , Z = 2; Mo Ka radiation
(298 K); R = 0.0693, Rw = 0.1875.
12 Merk, HPTLC plate CHIR Art. 14101; CH3CN–CH3OH–H2O
(4/1/1); Rf value was compared with that of L-threonine (Rf 0.45),
D-allo-threonine (Rf 0.40). Example of determination of components
of cyclic depsipeptide by hydrolysis; see O. Ohno, Y. Ileda, R. Sawa, M.
Igarashi, N. Kinoshita, Y. Suzuki, K. Miyake and K. Umezawa, Chem.
Biol., 2004, 11, 1059–1070.
Acknowledgements
We thank Prof. Jeremy Nathans (John Hopkins Medical School)
for kindly providing STF cells (293 cell line stably transfected
with Super TOP-Flash by Prof. R.T. Moon) and Prof. Randall
T. Moon (University of Washington) for the Super FOP-Flash
reporter plasmid. This work was supported by a Grant-in-Aid for
Scientific Research from the Japan Society for the Promotion of
Science (JSPS).
13 H. M. E. Duggan, P. B. Hitchcock and D. W. Young, Org. Biomol.
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14 For recent reviews on RCM see: (a) A. Fu¨rstner, Top. Catal., 1997, 4,
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15 Q. Xu, Y. Wang, A. Dabdoub, P. M. Smallwood, J. Williams, C. Woods,
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16 R. Larsson, J. Kristensen, C. Sandberg and P. Nygren, Int. J. Cancer,
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©