Syntheses and applications of fluorescent and biotinylated epolactaene derivatives: Epolactaene and its derivative induce disulfide formation

Article abstract of DOI:10.1016/j.bmc.2008.03.029

Epolactaene, isolated from cultured Penicillium sp. BM 1689-P mycelium, induces neurite outgrowth and arrests the cell cycle of the human neuroblastoma cell line, SH-SY5Y, at the G1 phase. We have found that epolactaene and its derivatives induce apoptosi

Full text of DOI:10.1016/j.bmc.2008.03.029

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 5039–5049
Syntheses and applications of fluorescent and biotinylated
epolactaene derivatives: Epolactaene and its derivative
induce disulfide formation
Kouji Kuramochi,a, Shunsuke Yukizawa,a, Seiki Ikeda,a Takashi Sunoki,b Satoshi Arai,a
Rie Matsui,a Akinori Morita,a Yoshiyuki Mizushina,c Kengo Sakaguchi,a
Fumio Sugawara,a Masahiko Ikekitaa and Susumu Kobayashib,*
aDepartment of Applied Biological Science, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-8510, Japan
bFaculty of Pharmaceutical Sciences, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-8510, Japan
cDepartment of Nutritional Science, Kobe-Gakuin University, Nishi-Ku, Kobe, Hyogo 651-2180, Japan
Received 5 February 2008; revised 10 March 2008; accepted 11 March 2008
Available online 14 March 2008
Abstract—Epolactaene, isolated from cultured Penicillium sp. BM 1689-P mycelium, induces neurite outgrowth and arrests the cell
cycle of the human neuroblastoma cell line, SH-SY5Y, at the G1 phase. We have found that epolactaene and its derivatives induce
apoptosis in the human leukemia B-cell line, BALL-1. In this study, we prepared fluorescent and biotinylated epolactaene deriva-
tives. We characterized the cellular location and the identification of BALL-1 proteins that reacted with these compounds. The
results obtained from the reaction of epolactaene or its derivative with N-acetylcysteine methyl ester indicate that these compounds
induce the disulfide formation and the a-position of the epoxylactam core is the reactive site.
ꢀ 2008 Elsevier Ltd. All rights reserved.
1. Introduction
Concerning the mechanism of action of 1, Nagumo et al.
reported that, in cell lysates, the heat shock protein 60
(Hsp60) is targeted by a biotinylated epolactaene deriv-
ative (bio-ETB), which covalently binds to Cys442 of
Hsp60, thereby inhibiting the protein’s chaperone activ-
ity.7 Furthermore, they suggested that Cys442 reversibly
reacts with the a,b-unsaturated ketone of 1 via a
Michael addition. However, epolactaene derivatives,
such as Epo-C12 (2), which do not have an a,b-unsatu-
rated ketone, still are cytotoxic and apoptosis-induc-
ing.6,7 Thus, the mechanism of action of 1 and its
derivatives is still obscure.
Epolactaene (1; Fig. 1), isolated from cultured Penicil-
lium sp. BM 1689-P mycelium, induces neurite out-
growth and arrests the cell cycle of the human
neuroblastoma cell line, SH-SY5Y, at the G1 phase.1,2
After we accomplished a total synthesis of 1, we further
characterized its biological activities.3,4 We found that 1
and its derivatives inhibited mammalian DNA polymer-
ases and human DNA topoisomerase II in vitro.5 We
also found that they induce apoptosis in the human leu-
kemia B-cell line, BALL-1.6 Our structure–activity stud-
ies indicate that the epoxylactam core defines the
pharmacophore and that the long hydrophobic side-
chain probably interacts with complementary regions
of target proteins.5,6
2. Results and discussion
2.1. Preparation of fluorescent and biotinylated epolact-
aene derivatives
To further catalog the types of proteins targeted by 1,
we synthesized a fluorescent (Flu-Epo-C12) and a biotin-
ylated (Bio-Epo-C12) derivative. Both probes were syn-
thesized using the bridgehead oxiranyl anion strategy
(Scheme 1), as described before.3 By individually
Keywords: Epoxides; Proteins; Antibiotics; Binding proteins; Reaction
mechanism.
*
Corresponding author. Tel./fax: +81 4 7121 3671; e-mail: kobayash@
rs.noda.tus.ac.jp
These authors contributed equally to this work.
0968-0896/$ - see front matter ꢀ 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.03.029

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K. Kuramochi et al. / Bioorg. Med. Chem. 16 (2008) 5039–5049
O
Me
O
O
O
Me
HO
Me
HO
Me
Me CO2Me
O
O
N
H
N
H
Epolactaene (1)
Epo-C12 (2)
O
O
Me
NH
H
HN
H
O
H
N
Me
HO
Me
Me
O
O
O
S
N
H
10
O
bio-ETB
Figure 1. Structure of epolactaene (1), Epo-C12 (2), and bio-ETB. The synthesis of bio-ETB was reported by Nagumo et al.7
from azide 7b. Reduction of 7b with Pd(OH)2 under
O
TMS
O
O
an H2 atmosphere in the presence of biotin N-hydroxy-
succinimide ester gave Bio-Epo-C12. Both Flu-Epo-C12
and Bio-Epo-C12 are cytotoxic for BALL-1 cells (Table
1).
+
R
H
Me
O
3
4
5
OH
2.2. Cellular localization Flu-Epo-C12
O
a
R
To identify wherein cell 1 and its derivatives might accu-
mulate, Flu-Epo-C12 and MitoTracker were introduced
into BALL-1 cells and localized by confocal laser scan-
ning microscopy. MitoTracker is a fluorescent mito-
chondrial marker that binds cysteine thiols.9 As shown
in Figure 2, Flu-Epo-C12 accumulates mainly in the
mitochondria, as most of its fluorescence colocalized
with that of MitoTracker. However, a low level of Flu-
Epo-C12 fluorescence was emitted from the cytoplasm.
Therefore, both mitochondrial and cytoplasmic proteins
bind Flu-Epo-C12.
Me
O
O
O
O
b, c
R
R
CONH2
Me
Me
OH
6
O
O
d
2.3. Identification of binding protein of Bio-Epo-C12
O
N
H
HO
a: R = OBn
b: R = N3
7
Next, Bio-Epo-C12 was used to identify cellular proteins
that might bind to 1 and its derivatives. BALL-1 cell ly-
sates were incubated with Bio-Epo-C12, biotin, or
DMSO, and then exposed to streptavidin agarose beads.
Proteins, isolated by this treatment were separated in
SDS–PAGE gels and then stained with CBB (Fig. 3A
and B). We detected seven bands in the Bio-Epo-C12-
treated sample (lane 1) that were absent in the biotin-
treated sample (lane 2) and the DMSO-treated sample
(lane 3). Therefore, these proteins bound to Bio-Epo-
C12.
Scheme 1. Synthesis of 7. Reagents and conditions: (a) TBAF
(0.1 equiv), MS4A, THF–hexane, then aq HF, MeCN, 5a: 59%, 5b:
54% in 2 steps; (b) TFAA, DMSO, CH2Cl2, then Et3N; (c) NH3,
MeOH, 6a: 76%, 6b: 71% in 2 steps; (d) TFAA, DMSO, CH2Cl2, then
Et3N; LiOH, THF–H2O, 7a: 79%, 7b: 72% in 2 steps.
coupling silylated epoxylactone 3 with aldehydes 4, and
desilylating the products, the aldol products 5 were ob-
tained as ca. 1:1 diastereomeric mixtures. After oxida-
tion of 5 with trifluoroacetic anhydride and DMSO,
ammonolysis of the corresponding ketones gave hydrox-
yamides 6. Finally oxidation of 6 gave the a,b-epoxy-c-
lactam derivatives 7.
We used peptide mass fingerprinting (PMF) to identify
the proteins. Gel protein bands were isolated and the
proteins trypsin-digested in situ (Supplementary infor-
mation). The molecular weights of the tryptic peptides
were measured by MALDI-TOF MS and compared
with those derived theoretically for proteins of the data-
base (NCBInr), using Mascot search program (Matrix
science Inc., USA). The proteins identified are listed in
Table 2 and include Hsp60 and fatty acid synthase
(FAS), which bind epolactaene and cerulenin, respec-
tively (Figs. 1 and 4).7,10
Flu-Epo-C12 was prepared from 7a (Scheme 2). Depro-
tection of the benzyl group gave the corresponding
alcohol. Thermal decomposition of 7-diethylamino-
coumarin-3-carbonyl azide gave the corresponding iso-
cyanate, which reacted with the alcohol, gave the
carbamate Flu-Epo-C12.8 Bio-Epo-C12 was synthesized

K. Kuramochi et al. / Bioorg. Med. Chem. 16 (2008) 5039–5049
5041
O
H
N
O
O
a, b
H3C
HO
7a
O
O
O
O
NEt2
N
H
Flu-Epo-C12
O
NH
H
HN
H
O
c
H
O
N
7b
S
H3C
HO
O
O
N
H
Bio-Epo-C12
Scheme 2. Synthesis of Flu-Epo-C12 and Bio-Epo-C12. Reagents and condition: (a) H2, Pd(OH)2–C, THF, 85%; (b) 7-diethylaminocoumarin-3-
isocyanate, THF–toluene, 50 ꢁC, 86%; (c) H2, Pd(OH)2–C, biotin N-hydroxysuccinimide ester, THF, 88%.
Table 1. Cytotoxicity of epolactaene (1), Epo-C12 (2), Flu-Epo-C12,
and Bio-Epo-C12 against BALL-1
tylhistidine methyl ester, N-acetyllysine methyl ester,
and N-acetylarginine methyl ester did not react with 2.
Compound
IC50 value (lM)
Therefore, Epo-C12 (2) seems to react specifically with
cysteine thiols.
Epolactaene (1)
Epo-C12 (2)
Flu-Epo-C12
Bio-Epo-C12
3.82
1.65
1.56
48.3
Interestingly, treatment of 2 with an excess amount of 8
(2.4 equiv) in a 1:1 MeOH/0.5 M NaHCO3 aq solution
for 24 h gave dodecanoic acid (10) in 36% yield and
disulfide (11) in 64% yield (Scheme 4). Yields were calcu-
lated using the amounts of 2 and 8, respectively. The
proposed reaction mechanism involves the initial forma-
tion of the b-sulfanylketoamide, followed by a slow ret-
ro-Claisen reaction to give 10. Excess 8 then reacts with
methyl N-acetyl-S-(2-amino-2-oxoethyl)cysteinate, the
intermediate of the retro-Claisen reaction, yielding
11.13 Thus, 2 might induce intramolecular or intermolec-
ular disulfide formation between protein cysteines.
Fifty percent inhibitory concentrations (IC50) acting against BALL-1
cell viability are shown.
Nagumo et al. reported that the Cys442 of Hsp60 might
react with the a,b-unsaturated ketone group of 1 via a
Michael addition.7 However, Bio-Epo-C12 does not con-
tain an a,b-unsaturated ketone and yet it still binds
Hsp60, suggesting that, instead, the thiol reacts with
the epoxide of Bio-Epo-C12.
Cerulenin, which has a similar a,b-epoxy-c-lactam moi-
ety, binds and inhibits FAS, causing cytotoxicity and
apoptosis in human cancer cell lines (Fig. 4).10 The crys-
tal structure of the cerulenin/Escherichia coli b-ketoacyl-
acyl carrier protein synthase complex shows that cerule-
nin is covalently attached to the active site cysteine at
the a-position of the a,b-epoxy-c-lactam moiety.11
Therefore, probably 1 and its derivatives also react with
FAS at the a,b-epoxy-c-lactam group.
The reaction of 1 and 8 (1.2 equiv) smoothly produced
the carboxylic acid 12 in 72% yield and the disulfide
11 in 77% yield (Scheme 5). Yields were calculated using
the amounts of 1 and 8, respectively. After the forma-
tion of the b-sulfanylketoamide, the retro-Claisen reac-
tion and disulfide formation immediately proceeded.13b
Thus, the intermediate b-sulfanylketoamide was not iso-
lated in this reaction. Although the reactivity of 1
against 8 is different from that of 2, the formation of
11 and 12 strongly indicates that 1 binds proteins at
the lactam epoxide. The formation of 12 would explain
the fact that, after reaction of Hsp60 with bio-ETB, the
biotin-labeled amount of Hsp60 reduced with time.7b
Furthermore, the formation of disulfide 11 suggests that
1 might smoothly induce intramolecular or intermolecu-
lar disulfide formation between protein cysteines.
2.4. A hypothetical mechanism of action of epolactaene
and its derivatives
Next, to clarify how epolactaene (1) and its derivatives
react with protein cysteines, 1 and Epo-C12 (2) were re-
acted with the model compound, N-acetylcysteine
methyl ester (8).12
Treatment of 2 and 8 (1.2 equiv) in a 1:1 MeOH/0.5 M
NaHCO3 aq solution for 20 min gave 9 in 82% yield
(Scheme 3). The proposed mechanism involves attack
by the thiolate of 8 at the a-position of 2, followed by
a retro-aldol reaction, and then removal of pyruvic alde-
hyde to give 9. No reaction occurred at neutral or acidic
conditions. Other amino acid derivatives such as N-ace-
3. Conclusion
We prepared fluorescent and biotinylated epolactaene
derivatives. We characterized the cellular location and
the identification of BALL-1 proteins that reacted with
these compounds. The results obtained from the reac-
tion of epolactaene (1) or Epo-C12 (2) with N-acetylcys-

5042
K. Kuramochi et al. / Bioorg. Med. Chem. 16 (2008) 5039–5049
Figure 3. Detection of proteins that react with Bio-Epo-C12. BALL-1
cell lysates were incubated with 5 lM Bio-Epo-C12, 5 lM biotin, or
5 lM DMSO, and then exposed to streptavidin agarose beads. The
proteins that were recovered from the beads were separated by
electrophoresis in (A) 6% SDS–PAGE and (B) 12% SDS–PAGE gels
and stained with CBB. Lane 1, Bio-Epo-C12; lane 2, biotin; lane 3,
DMSO; lane 4, BALL-1 cell lysates.
Figure 2. Staining of BALL-1 cells by Flu-Epo-C12 and MitoTracker.
BALL-1 cells were treated with Flu-Epo-C12 and MitoTracker for 1 h
and then washed with PBS. Confocal images of Flu-Epo-C12 (A) and
MitoTracker (B). The superimposed fluorescent images are shown in
(C).
Table 2. The identification of BALL-1 proteins that react with Bio-
Epo-C12
teine methyl ester (8) indicate that the a-position of the
epoxylactam core is the reactive site. Both epolactaene
(1) and Epo-C12 (2) might induce intramolecular or
intermolecular disulfide formation between protein
cysteines.
Band
Protein
Mw (kDa)
Location
1
2
3
4
5
6
Fatty acid synthase
ATP citrate lyase
273
121
95
Cytoplasm
Cytoplasm
Cytoplasm
Cytoplasm
Mitochondria
Mitochondria
Elongation factor 2
Heat shock protein 90b
Heat shock protein 60
Adenine nucleotide
translocator 2
83
61
Additional biological studies concerning the mechanism
of action of 1 and its derivatives are currently underway.
33
7
Peroxiredoxin 1 (Prx1)
22
Cytoplasm
4. Experimental procedure
4.1. General (Chemistry)
The protein gel bands were separated and digested with trypsin. Mw is
the protein’s calculated molecular weight.
All non-aqueous reactions were carried out by using
freshly distilled solvents under argon atmosphere.
THF was distilled from sodium/benzophenone prior to
use. Dichloromethane was distilled from P2O5 prior to
use. N,N-Dimethylformamide and propylene oxide were
distilled from calcium hydride prior to use. All other re-
agents were commercially available and used without
further purification. 7-(Diethylamino)coumarin-3-car-
bonyl azide was purchased from Molecular Probes
(now known as Invitrogen, USA). N-Acetyl-L-cystine
dimethyl ester was obtained from Bachem (Switzerland).
N-Acetyl-L-cysteine (8) was prepared by a reduction of
N-acetyl-L-cystine dimethyl ester (11) with zinc.14
All reactions were monitored by TLC, which was carried
out on Silica Gel 60 F254 plates (Merck, Germany).

K. Kuramochi et al. / Bioorg. Med. Chem. 16 (2008) 5039–5049
5043
600, Avance DRX-400) or a JEOL 400 MHz spectrom-
eter (JNM-LD400), using CDCl3 (with TMS for 1H
NMR and chloroform-d for 13C NMR as the internal
reference) solution, unless otherwise noted. Chemical
shifts were expressed in d (ppm) relative to Me4Si or
residual solvent resonance, and coupling constants (J)
were expressed in Hz.
O
O
N
H
HO
Cerulenin
Figure 4. Structure of cerulenin.
Flash chromatography separations were performed on
PSQ 100B (Fuji Silysia Co., Ltd, Japan).
Melting points were determined with Yanaco MP-3S
and were uncorrected.
1H and 13C NMR spectra were recorded on a Bruker
600 MHz or 400 MHz spectrometer (Avance DRX-
Optical rotations were recorded on a JASCO P-1030
digital polarimeter at room temperature.
O
O
NHAc
+
HS
Me
HO
CO2Me
8 (1.2 eq.)
O
N
H
2
AcNH
HO
CO2Me
AcNH
CO2Me
0.5 M NaHCO3 aq.
retro-aldol
O
S
O
S
O
Me
HO
MeOH
20 min
R
R
Me
HO
O
O
N
H
N
H
O
O
S
O
H2N
H
+
Me
O
AcHN
CO2Me
9
82%
Scheme 3. Treatment of Epo-C12 (2) with 1.2 equiv of N-acetylcysteine methyl ester (8): a plausible mechanism for the formation of 9 (R = C11H23).
NHAc
HS
CO2Me
O
CO2Me
8 (2.4 eq)
O
AcNH
O
Me
HO
S
O
N
H
0.5 M NaHCO3 aq.
2
MeOH
24 hr
O
NH2
9
O
HO
10
36% from 2
OH
NHAc
CO2Me
retro-Claisen reaction
slow
NHAc
CO2Me
HS
MeO2C
S
S
NHAc
8
MeO2C
NH2
S
NHAc
CH3CONH2
–
11
64% from 8
Scheme 4. Treatment of Epo-C12 (2) with excess amount of N-acetylcysteine methyl ester (8): a plausible mechanism for the formation of 10 and 11.
O

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K. Kuramochi et al. / Bioorg. Med. Chem. 16 (2008) 5039–5049
O
Me
O
NHAc
HS
+
Me
HO
CO2Me
8 (1.2 eq)
Me
Me CO2Me
O
N
H
1
CO2Me
AcNH
O
Me
0.5 M NaHCO3 aq.
S
MeOH
20 min
Me
Me CO2Me
O
NH2
O
Me
NHAc
CO2Me
, 8
OH
MeO2C
S
+
HO
S
NHAc
retro-Claisen reaction
Me
Me CO2Me
and disulfide fromation
fast
11
12
72% from 1
77% from 8
Scheme 5. Treatment of epolactaene (1) with 1.2 equiv of N-acetylcysteine methyl ester (8): a plausible mechanism for the formation of 11 and 12.
Infrared spectra (IR) were recorded on a Jasco FT/IR-
410 spectrometer using NaCl (neat) or KBr pellets (so-
lid), and were reported on wavenumbers (cmꢀ1).
(160 mg, 0.55 mmol),15 and MS4A (0.5 g) in THF–hex-
ane (1:1, 6 mL) was added TBAF (100 lL of a 1.0 M
solution in THF, 0.1 mmol), which had been dried with
MS4A for 3 h before use. The mixture was stirred at rt
for 24 h, and then filtered through Celite and washed
with EtOAc. The filtrate was washed with H2O, brine,
dried (Na2SO4), and concentrated. A solution of the res-
idue in CH3CN (1 mL) was added a solution of 5%
aqueous HF in CH3CN (1.5 mL), and the mixture was
stirred at rt for 20 min. The mixture was quenched by
the addition of saturated aqueous NaHCO3 solution
and extracted with EtOAc. The combined extract was
washed with brine, dried (Na2SO4), and concentrated.
The residue was purified by chromatography (hexane/
EtOAc = 3:1 to 1:1) to afford 5a (130 mg, 59%) as a
1:1 diastereomeric mixture as colorless oil. IR (neat)
3444, 3025, 2928, 2855, 1777, 1455, 1339, 1217, 1096,
1071, 1005, 939, 853 cmꢀ1; 1H NMR (600 MHz, CDCl3)
d 7.34 (4H, m), 7.34 (4H, m)*, 7.28 (1H, m), 7.28 (1H,
m)*, 4.66 (1H, m), 4.66 (1H, m)*, 4.50 (2H, s), 4.50
(2H, s)*, 4.23 (1H, dt, J = 8.8 Hz, 3.2 Hz)*, 4.14 (1H,
m), 3.97 (1H, s), 3.97 (1H, s)*, 3.46 (2H, t, J = 6.7 Hz),
3.46 (2H, t, J = 6.7 Hz)*, 2.19 (1H, m)*, 2.10 (1H, br
s), 1.78–1.69 (2H, m), 1.78–1.69 (2H, m)*, 1.63–1.49
(4H, m), 1.63–1.49 (4H, m)*, 1.40 (3H, d,J = 6.8 Hz),
1.40 (3H, d,J = 6.8 Hz)*, 1.38–1.27 (14H, m), 1.38–1.27
(14H, m)*; 13C NMR (100 MHz, CDCl3) d 170.9, 170.
5, 138.6 (·2), 128.2 (·4), 127.5 (·4), 127.4 (·2), 75.34,
75.31, 72.7 (·2), 70.5 (·2), 66.2, 64.7, 62.0, 61.7 (·2),
61.6, 32.4, 32.2, 29.7 (·2), 29.5 (·2), 29.43 (·4), 29.37
(·4), 29.23, 29.16, 26.1 (·2), 25.3, 25.2, 17.8, 17.7 (dia-
stereomeric mixture); HRMS, calcd for C24H36O5Na
([M+Na]+) 427.2454, found 427.2457.
Mass spectra (MS) were obtained on an Applied Biosys-
tems mass spectrometer (APIQSTAR pulsar i) under
conditions as High resolution, using poly (ethylene gly-
col) as internal standard.
4.2. General (Biology)
Fetal bovine serum (FBS) was purchased from Biowest
(Loire Valley, French) Kanamycin sulfate, dimethyl
sulfoxide (DMSO), biotin and 3-(4,5-dimethylthiazol-
2-yl)-2,5-diphenyltetrazolium bromide (MTT) were pur-
chased from Wako Pure Chemical Industries (Tokyo,
Japan). RPMI 1640 was purchased from Nissui (Tokyo,
Japan). 2-Mercaptoethanol was purchased from Nacalai
Tesque (Kyoto, Japan). Protease inhibitor cocktail was
obtained from Sigma–Aldrich (ST. Louis, MO). Strepta-
vidin agarose gel was purchased from GE Healthcare
(London, UK). MitoTracker Red CMXROS dye was
purchased from Molecular Probes (now known as Invit-
rogen, USA).
The confocal images were taken by LSM 5 PASCAL
EXCITER laser scanning confocal microscopy (Zeiss,
Germany).
MALDI-TOF MS measurements were performed using
a Bruker Daltonics Reflex IV MALDI-TOF mass spec-
trometer (Bremen, Germany). Spectra were externally
calibrated with bradykinin, neurotensin, and melittin.
4.3. Preparation of Fluo-Epo-C12 and Bio-Epo-C12
4.3.2. (2S,3S)-3-[(S)-1-Hydroxyethyl]-2-(120-Benzyloxy-
dodecanoyl)oxirane-2-carboxamaide (6a). To a solution
of DMSO (30 lL, 0.42 mmol) in CH2Cl2 (1 mL) was
added TFAA (30 lL, 0.21 mmol) at ꢀ78 ꢁC. After
10 min, a solution of 5a (28 mg, 0.07 mmol) in CH2Cl2
4.3.1. (1R,10RS*,5R)-1-(10-Hydroxy-120-benzyloxydode-
cyl)-4-hydroxy-4-methyl-6-oxa-3-aza-bicyclo[3.1.0]hexan-
2-one (5a). To a solution of 3 (151 mg, 0.81 mmol),3 4a

K. Kuramochi et al. / Bioorg. Med. Chem. 16 (2008) 5039–5049
5045
(1.5 mL) was added to the mixture, and the mixture was
ꢀ78 ꢁC for 30 min. Then Et3N (90 lL, 0.65 mmol) was
added to the mixture, and the mixture was stirred at rt
for 5 min. The mixture was quenched by the addition
of H2O and extracted with CHCl3. The combined ex-
tract was washed with brine, dried (Na2SO4), and
concentrated.
4.3.4. (1R,5R)-1-(120-Hydroxydodecanoyl)-4-hydroxy-4-
methyl-6-oxa-3-aza-bicyclo[3.1.0]hexan-2-one (13).
solution of 7a (67.1 mg, 0.16 mmol) and Pd(OH)2 on
carbon powder (20% Pd, 28.1 mg) in THF (6 mL) was
stirred at room temperature for 9.5 h. The mixture was
filtered through Celite and washed with EtOAc. The fil-
trate was concentrated. The residue was purified by col-
A
umn chromatography (hexane/EtOAc = 1:2) to give 13
24
(44.7 mg, 85%) as white solid. Mp = 112–115 ꢁC; ½aꢂ
To a solution of NH3 (ꢁ0.3 mL) in MeOH (2 mL) was
added a solution of the residue in MeOH (2 mL) at 0 ꢁC,
and the mixture was stirred for 30 min. Then the mixture
was concentrated. The residue was purified by chromatog-
D
ꢀ93.3 (c 0.65, MeOH); IR (KBr) 3521, 3369, 2920,
2852, 1737, 1685, 1469, 1423, 1241, 1160, 1112, 1051,
1
957, 937, 881, 814, 754, 718, 674, 628 cmꢀ1; H NMR
raphy (hexane/EtOAc = 1:1 to 1:2) to afford 6a (22 mg,
(400 MHz, CD3OD) d 4.07 (1H, s), 3.53 (2H, t,
J = 6.7 Hz), 2.60 (2H, t, J = 7.3 Hz), 1.58 (2H, m),
1.51 (2H, m), 1.46 (3H, s),1.30 (14H, br m); 13C NMR
(100 MHz, CD3OD) d 202.2, 170.7, 84.1, 66.9, 63.0,
40.3, 33.7, 30.73, 30.65 (· 2), 30.59, 30.56, 30.5, 30.1,
26.9, 23.8, 21.9; HRMS, calcd for C17H29NO5Na
([M+Na]+) 350.1937, found 350.1919.
22
D
76%) as white solid. Mp = 68–69 ꢁC; ½aꢂ +16.1 (c 1.1,
MeOH); IR (KBr) 3391, 3196, 2919, 2849, 2797, 1717,
1641, 1462, 1395, 1368, 1290, 1210, 1110, 1060, 964, 898,
1
740 cmꢀ1; H NMR (400 MHz, CDCl3) d 7.34 (4H, m),
7.28 (1H, m), 6.84 (1H, br s), 6.35 (1H, br s), 4.50 (2H,
s), 3.65 (1H, m), 3.46 (2H, t, J = 6.6 Hz), 3.16 (1H, d,
J = 7.8 Hz), 2.66 (1H, ddd, J = 18.0 Hz, 8.0 Hz, 6.6 Hz),
2.49 (1H, dt, J = 18.0 Hz, 8.0 Hz, 6.7 Hz), 1.97 (1H, br
s), 1.59 (4H, m), 1.38 (3H, d, J = 6.3 Hz), 1.26 (14H, br
s); 13C NMR (100 MHz, CDCl3) d 203.7, 167.0, 138.6,
128.3 (·2), 127.6 (·2), 127.4, 72.8, 70.5, 65.6, 65.42,
65.39, 38.0, 29.7, 29.5, 29.43, 29.41, 29.33, 29.25, 28.9,
26.1, 23.0, 20.2; HRMS, calcd for C24H37NO5Na
([M+Na]+) 442.2563, found 442.2565.
4.3.5. (1R,5R)-(700-Diethylamino-200-oxo-200H-chromen-
300-yl)carbamic acid 120-(4-hydroxy-4-methyl-2-oxo-6-
oxa-3-aza-bicyclo[3.1.0]hex-1-yl)-120-oxo-dodecyl ester
(Flu-Epo-C12). A solution of 7-(diethylamino)couma-
rin-3-carbonyl azide (15.6 mg, 0.061 mmol) in toluene
(4 mL) was stirred at 100 ꢁC for 10 min. The solution
of the crude isocyanate was added to a solution of 13
(12.0 mg, 0.042 mmol). After the mixture was stirred at
50 ꢁC for 9 h, the mixture was concentrated. The residue
was purified by PTLC (hexane/EtOAc = 1:2) to give
4.3.3. (1R,5R)-1-(120-Benzyloxydodecanoyl)-4-hydroxy-
4-methyl-6-oxa-3-aza-bicyclo[3.1.0]hexan-2-one (7a). To
a solution of DMSO (160 lL, 2.25 mmol) in CH2Cl2
(3 mL) was added TFAA (200 lL, 1.41 mmol) at
ꢀ78 ꢁC and the mixture was stirred at ꢀ78 ꢁC. After
5 min, a solution of 6a (158 mg, 0.38 mmol) in CH2Cl2
(5 mL) was added to the mixture and the mixture was
stirred at ꢀ78 ꢁC for 60 min. Then Et3N (0.5 mL) was
added to the reaction mixture and the mixture was stir-
red at room temperature for 5 min. The mixture was
quenched by the addition of H2O and extracted with
EtOAc. The extract was washed with brine, dried
(Na2SO4), and concentrated. To a solution of the resi-
due in THF–H2O (4:1, 5 mL) was added LiOH (9 mg,
0.38 mmol) at 0 ꢁC. After the mixture was stirred at
0 ꢁC for 10 min, the mixture was quenched by the addi-
tion of H2O and extracted with EtOAc. The extract was
washed with brine, dried (Na2SO4), and concentrated.
The residue was purified by column chromatography
(hexane/EtOAc = 1:1) to give 7a (125 mg, 79%) as a
Fluo-Epo-C12 (21.1 mg, 86%) as yellow solid.
24
D
Mp = 115–116 ꢁC; ½aꢂ ꢀ48.2 (c 0.39, MeOH); IR
(KBr) 3405, 3314, 3020, 2929, 2855, 1740, 1698, 1611,
1523, 1410, 1361, 1305, 1217, 1160, 1131, 1036, 945,
1
757, 667 cmꢀ1; H NMR (400 MHz, CD3OD/CDCl3) d
8.14 (1H, br s), 7.31 (1H, d, J = 8.8 Hz), 6.70 (1H, dd,
J = 8.8 Hz, 2.4 Hz), 6.51 (1H, d, J = 2.4 Hz), 4.17 (2H,
t, J = 6.6 Hz), 4.03 (1H, s), 3.44 (4H, q, J = 7.1 Hz),
2.62 (2H, t, J = 7.3 Hz), 1.72–1.61 (2H, m), 1.61–1.58
(2H, m), 1.48 (3H, m), 1.41–1.31 (14H, br m), 1.21
(6H, t, J=7.1 Hz); 13C NMR (100 MHz, CD3OD/
CDCl3) d 202.1, 170.3, 160.7, 155.5, 153.7, 150.6,
129.3, 110.8, 109.3, 98.0, 83.8, 66.7 (·2), 63.0, 45.4
(·2), 40.3, 30.3 (·2), 30.27, 30.2, 30.1, 29.84, 29.76,
26.7, 23.6, 21.8, 12.8 (·2); HRMS, calcd for
C31H44N3O8Na ([M+H]+) 586.3122, found 586.3112.
4.3.6. 12-Azidododecanal (4b). To a solution of DMSO
(0.36 mL, 5.1 mmol) in CH2Cl2 (3 mL) was added
(COCl)2 (0.22 mL, 2.5 mmol) at ꢀ78 ꢁC. After 10 min,
a solution of 12-azidododecanol (194 mg, 0.85 mmol)16
in CH2Cl2 (3 mL) was added to the mixture, and the
mixture was ꢀ78 ꢁC for 30 min. Then Et3N (0.7 mL,
5.0 mmol) was added to the mixture, and the mixture
was stirred at rt for 15 min. The mixture was quenched
by the addition of H2O and extracted with CHCl3. The
combined extract was washed with brine, dried
(Na2SO4), and concentrated. The residue was purified
by column chromatography (hexane/EtOAc = 4:1) to
yield 4b (198 mg, quant.) as colorless oil. IR (neat)
2928, 2855, 2717, 2096, 1726, 1463, 1410, 1390, 1350,
7.5:1 tautomeric mixture, as colorless needle.
24
D
3463, 3289, 2926, 2853, 1740, 1690, 1496, 1455, 1403,
Mp = 53–55 ꢁC; ½aꢂ ꢀ71.9 (c 1.2, MeOH); IR (KBr)
1363, 1160, 1115, 1028, 945, 736, 698, 651, 621 cmꢀ1
;
1H NMR (600 MHz, CDCl3) d 7.87 (1H, br m), 7.35–
7.28 (4H, br m), 7.26 (1H, m), 5.08 (1H, br s), 4.50
(2H, s), 4.17 (1H, d, J = 1.9 Hz), 3.46 (2H, t,
J = 6.7 Hz), 2.50–2.45 (1H, m), 2.32–2.25 (1H, m), 1.60
(2H, m), 1.55 (3H, s), 1.54–1.50 (1H, m), 1.36–1.32
(2H, m), 1.43–1.38 (1H, m)1.30–1.22 (12H, br m); 13C
NMR (100 MHz, CDCl3) d 202.1, 169.0, 138.7, 128.3
(·2), 127.6 (·2), 127.4, 83.5, 72.8, 70.5, 66.0, 61.3,
38.3, 29.7, 29.5 (·2), 29.4 (·2), 29.3, 28.9, 26.2, 22.6,
21.5; HRMS, calcd for C24H35NO5Na ([M+Na]+)
440.2407, found 440.2400.
1
1260, 893, 723, 671 cmꢀ1; H NMR (600 MHz, CDCl3)
d 9.77 (1H, t, J = 1.8 Hz), 3.26 (2H, t, J = 7.0 Hz), 2.42

5046
K. Kuramochi et al. / Bioorg. Med. Chem. 16 (2008) 5039–5049
(2H, td, J = 7.4 Hz, 1.8 Hz), 1.62 (4H, m), 1.33 (14H,
m); 13C NMR (100 MHz, CDCl3) d 202.5, 51.2, 43.6,
29.2 (· 2), 29.14, 29.10, 28.91, 28.90, 28.6, 26.5, 21.8;
HRMS, calcd for C12H23N3ONa ([M+Na]+) 248.1733,
found 248.1736.
1H NMR (400 MHz, CDCl3) d 6.95 (1H, br s), 6.69
(1H, br s), 3.66 (1H, m), 3.56 (1H, br s), 3.26 (2H, t,
J = 7.0 Hz), 3.17 (1H, d, J = 7.7 Hz), 2.66 (1H, m),
2.49 (1H, m), 1.58 (4H, m), 1.38 (3H, d, J = 6.3 Hz),
1.27 (14H, br s); 13C NMR (100 MHz, CDCl3) d
203.7, 167.1, 65.6, 65.4, 65.1, 51.4, 37.8, 29.35 (· 2),
29.29, 29.2, 29.0, 28.9, 28.7, 26.6, 22.9, 20.2; HRMS,
calcd for C17H30N4O4Na ([M+Na]+) 377.2159, found
377.2166.
4.3.7. (1R,10RS*,5R)-1-(10-Hydroxy-120-azidododecyl)-4-
hydroxy-4-methyl-6-oxa-3-aza-bicyclo[3.1.0]hexan-2-one
(5b). To a solution of 3 (253 mg, 1.36 mmol), 4b
(198 mg, 0.88 mmol), and MS4A (0.6 g) in THF–hexane
(1:1, 10 mL) was added TBAF (130 lL of a 1.0 M solu-
tion in THF, 0.1 mmol), which had been dried with
MS4A for 3 h before use. The mixture was stirred at rt
for 24 h, and then filtered through Celite and washed
with EtOAc. The filtrate was washed with H2O, brine,
dried (Na2SO4), and concentrated. A solution of the res-
idue in CH3CN (2 mL) was added a solution of 5%
aqueous HF in CH3CN (3 mL), and the mixture was
stirred at rt for 20 min. The mixture was quenched by
the addition of saturated aqueous NaHCO3 solution
and extracted with EtOAc. The combined extract was
washed with brine, dried (Na2SO4), and concentrated.
The residue was purified by chromatography (hexane/
EtOAc = 3:1 to 1:1) to afford 5b (160 mg, 54%) as a
1:1 diastereomeric mixture as pale yellow oil. IR (neat)
3477, 2927, 2855, 2096, 1778, 1459, 1381, 1340, 1255,
4.3.9. (1R,5R)-1-(120-Azidododecanoyl)-4-hydroxy-4-
methyl-6-oxa- 3-aza-bicyclo[3.1.0]hexan-2-one (7b). To
a solution of DMSO (400 lL, 5.6 mmol) in CH2Cl2
(5 mL) was added TFAA (520 lL, 3.7 mmol) at
ꢀ78 ꢁC and the mixture was stirred at ꢀ78 ꢁC. After
5 min, a solution of 6b (428 mg, 1.2 mmol) in CH2Cl2–
DMSO (30:1, 3.1 mL) was added to the mixture and
the mixture was stirred at ꢀ78 ꢁC for 60 min. Then
Et3N (1.5 mL) was added to the reaction mixture and
the mixture was stirred at room temperature for 5 min.
The mixture was quenched by the addition of H2O
and extracted with EtOAc. The extract was washed with
brine, dried (Na2SO4), and concentrated. To a solution
of the residue in THF–H2O (4:1, 5 mL) was added LiOH
(30 mg, 1.3 mmol) at 0 ꢁC. After the mixture was stirred
at 0 ꢁC for 10 min, the mixture was quenched by the
addition of H2O and extracted with EtOAc. The extract
was washed with brine, dried (Na2SO4), and concen-
trated. The residue was purified by column chromatog-
1070, 1007, 938, 892, 853, 785, 725, 695 cmꢀ1 1H
;
NMR (400 MHz, CDCl3) d 4.65 (1H, m), 4.65 (1H,
m)*, 4.22 (1H, m), 4.14 (1H, m)*, 3.98 (1H, s), 3.98
(1H, s)*, 3.26 (2H, t, J = 6.9 Hz), 3.26 (2H, t,
J = 6.9 Hz)*, 2.48 (1H, br s)*, 2.36 (1H, br s), 1.78–
1.50 (4H, m), 1.78–1.50 (4H, m)*, 1.41 (3H,
d,J = 6.7 Hz), 1.41 (3H, d,J = 6.7 Hz)*, 1.36–1.28
(14H, br m), 1.38–1.27 (14H, br m)*; 13C NMR
(100 MHz, CDCl3) d 170.9*, 170.5, 75.4*, 75.3, 66.3*,
64.7, 62.1*, 61.73, 61.67, 61.6*, 51.4, 51.4*, 32.3, 32.2*,
29.38, 20.38*, 29.35 (·3), 29.35 (·3)*, 29.3, 29.2*, 29.0,
29.0*, 28.7, 28.7*, 26.6, 26.6*, 25.3*, 25.2, 17.8, 17.7*;
HRMS, calcd for C17H29N3O4Na ([M+Na]+)
326.3050, found 326.2038.
raphy (hexane/EtOAc = 1:1) to give 7b (307 mg, 72%)
24
D
as white solid. Mp = 55–57 ꢁC; ½aꢂ ꢀ88.5 (c 4.5,
MeOH); IR (KBr) 3463, 3264, 3019, 2929, 2855, 2097,
1743, 1690, 1465, 1433, 1402, 1350, 1250, 1217, 1163,
1118, 1052, 948, 887, 758, 667, 651, 623 cmꢀ1 1H
;
NMR (400 MHz, CDCl3) d 8.35 (1H, d, J = 2.4 Hz),
5.20 (1H, br s), 4.22 (1H, d, J = 2.4 Hz), 3.20 (2H, t,
J = 7.0 Hz), 2.41 (1H, m), 2.16 (1H, m), 1.54 (2H, m),
1.54 (3H, s), 1.45 (1H, m), 1.40–1.19 (15H, br m); 13C
NMR (100 MHz, CDCl3) d 202.3, 169.6, 83.7, 66.0,
61.1, 51.4, 37.8, 29.4, 29.4, 29.3, 29.3, 29.1, 28.9, 28.8,
26.6, 22.5, 21.1; HRMS, calcd for C17H28N4O4Na
([M+Na]+) 375.2002, found 375.2038.
4.3.8. (2S,3S)-3-[(S)-1-Hydroxyethyl]-2-(120-azidododeca-
noyl)oxirane-2-carboxamaide (6b). To a solution of
DMSO (0.2 mL, 2.8 mmol) in CH2Cl2 (3 mL) was added
TFAA (0.2 mL, 1.4 mmol) at ꢀ78 ꢁC. After 10 min, a
solution of 5b (160 mg, 0.47 mmol) in CH2Cl2 (3 mL)
was added to the mixture, and the mixture was ꢀ78 ꢁC
for 30 min. Then Et3N (0.7 mL, 5.0 mmol) was added
to the mixture, and the mixture was stirred at rt for
15 min. The mixture was quenched by the addition of
H2O and extracted with CHCl3. The combined extract
was washed with brine, dried (Na2SO4), and
concentrated.
4.3.10. (1R,5R,300R,400S,500S)-500-(200-Oxo-hexahydro-thie-
no[3,4-d]imidazol-400-yl)pentanoc acid [120-(4-hydroxy-4-
methyl-2-oxo-6-oxa-3-aza-bicyclo[3.1.0]hex-1-yl)-120-oxo-
dodecyl]amide (Bio-Epo-C12). A solution of 7b (51 mg,
0.14 mmol), biotin N-hydroxysucimide ester (55 mg,
0.16 mmol) and Pd(OH)2 on carbon powder (20% Pd,
25 mg) in THF–DMF (6:1, 7 mL) was stirred at rt for
9.5 h. Then the mixture was filtered through Celite and
washed with THF. The filtrate was concentrated. The
residue was purified by column chromatography
(CHCl3/MeOH = 9:1) to give Bio-Epo-C12 (70.2 mg,
24
D
To a solution of NH3 (ꢁ0.3 mL) in MeOH (2 mL) was
added a solution of the residue in MeOH (2 mL) at
0 ꢁC, and the mixture was stirred for 30 min. Then the
mixture was concentrated. The residue was purified by
88%) as white solid. Mp = 138–140 ꢁC; ½aꢂ ꢀ16.7 (c
1.45, DMSO); IR (KBr) 3295, 2925, 2853, 1738, 1700,
1642, 1548, 1465, 1424, 1322, 1265, 1206, 1156, 1021,
949, 886, 654 cmꢀ1 1H NMR (400 MHz, CD3OD/
;
chromatography (hexane/EtOAc = 1:1 to 1:2) to afford
6b (119 mg, 71%) as white solid. Mp = 72–74 ꢁC; ½aꢂ
CDCl3) d 4.40 (1H, dd, J = 7.8 Hz, 5.0 Hz), 4.21 (1H,
dd, J = 7.8 Hz, 4.4 Hz), 4.00 (1H, s), 3.11 (1H, m),
3.06 (2H, t, J = 6.9 Hz), 2.84 (1H, dd, J = 12.7 Hz,
5.0 Hz), 2.62 (1H, d, J = 12.7 Hz), 2.52 (2H, t, J = 7.2
Hz), 2.10 (2H, t, J = 7.3 Hz), 1.60–1.68 (2H, m), 1.48–
22
D
+16.0 (c 0.5, MeOH); IR (KBr) 3425, 3342, 3236,
2922, 2853, 2104, 1716, 1669, 1539, 1463, 1403, 1369,
1304, 1263, 1147, 1118, 1065, 973, 924, 898, 760 cmꢀ1
;

K. Kuramochi et al. / Bioorg. Med. Chem. 16 (2008) 5039–5049
5047
1.59 (4H, m), 1.38 (3H, s), 1.31–1.43 (4H, m), 1.22 (14H,
br m); 13C NMR (100 MHz, CD3OD/CDCl3) d 201.0,
174.5, 169.0, 164.5, 82.7, 65.5, 61.9, 61.5, 60.1, 55.6,
39.8, 39.1 (·2), 35.5, 29.22, 29.19, 29.11, 29.05, 29.0
(·2), 28.7, 28.3, 28.0, 26.6, 25.5, 22.4, 20.7; HRMS,
calcd for C27H44N4O6NaS ([M+Na]+) 575.2873, found
575.2868.
1641, 1436, 1383, 1216, 1134, 1057, 1024, 965, 938,
866 cmꢀ1 1H NMR (600 MHz, CDCl3) d 6.94 (2H,
;
m), 6.25 (1H, d, J = 15.6 Hz), 5.96 (1H, br s), 5.72
(1H, dt, J = 15.6 Hz, 6.7 Hz), 3.74 (3H, s), 2.32 (4H,
m), 1.94 (3H, s), 1.73 (3H, dd, J = 7.1 Hz, 0.9 Hz),
1.63 (3H, s); 13C NMR (100 MHz, CDCl3) d 173.1,
167.9, 144.2, 139.7, 137.8, 134.9, 130.5, 128.7, 127.5,
122.8, 51.9, 31.6, 28.9, 15.8, 14.4, 12.1; HRMS, calcd
for C16H22O4Na ([M+Na]+) 301.1410, found 301.1436;
HRMS, calcd for C16H21O4 ([MꢀH]ꢀ) 277.1445, found
277.1455.
4.4. Reaction of Epo-C12 with N-acetylcysteine methyl
ester (8)
4.4.1. Treatment of Epo-C12 (2) with 1.2 equiv of N-
acetylcysteine methyl ester (8). To a solution of 2 (3.1
mg, 10 lmol) and 8 (2.1 mg, 12 lmol)14 in MeOH (0.5
mL) was added a 0.5 M aqueous solution of NaHCO3
(0.5 mL), and the mixture was stirred at rt for 20 min.
The mixture was quenched by the addition of 1 M HCl
(1 mL) and extracted with EtOAc. The extract was
washed with brine, dried (Na2SO4), and concentrated.
The residue was purified by column chromatography
(hexane/EtOAc = 1:1) to give methyl N-acetyl-S-[1-(ami-
4.6. Cell line and culture
BALL-1 cells were purchased from Riken Cell Bank
(Tsukuba, Japan). BALL-1 cells were maintained at
37 ꢁC with 5% CO2 in RPMI 1640 supplemented with
kanamycin sulfate (65 mg/L), 2-mercaptoethanol
(3.5 lL/L), sodium bicarbonate (2 g/L), and heat-inacti-
vated 10% (v/v) FBS. These cells were routinely diluted
with the above medium to the appropriate concentra-
tions (1.0–4.0 · 105 cells/mL).
nocarbonyl)-2-oxotridecanyl]-L-cysteinate (9) (3.4 mg,
22
D
82%) as colorless oil. 9: ½aꢂ +13.3 (c 0.7, CHCl3); IR
(film) 3445, 3321, 3016, 2926, 2854, 1743, 1664, 1586,
1438, 1374, 1308, 1217, 1176, 1128, 1082, 1010, 758,
4.7. Detection of cell death by MTT assay
667 cmꢀ1
;
1H NMR (600 MHz, CDCl3 with 0.03%
BALL-1 cells were plated onto a 96-well plate (Sumito-
mo Bakelite Co., Tokyo, Japan) at a concentration of
2.0 · 104 cells/well and preincubated at 37 ꢁC for 1 h.
The cells were then treated with Flu-Epo-C12 or
Bio-Epo-C12. After incubation at 37 ꢁC for 24 h, cell via-
bility was determined by MTT assay, a method for deter-
mining cell viability by measuring the mitochondrial
dehydrogenase action. In this assay, 11 lL of MTT stock
solution (5 mg/mL in phosphate-buffered saline (PBS))
was added to the cells, and the plate was incubated
37 ꢁC for 1 h. After centrifugation for 5 min at
1500 rpm, the supernatant was discarded, and 100 lL
of DMSO was added to dissolve MTT formazan. Absor-
bance at 570 nm was measured with a microplate reader
(Bio-Rad Model 550, Bio-Rad, Tokyo, Japan), and the
percentage of cell viability was taken as the percentage
absorbance at 570 nm of epolactaene-treated cells and
control.
TFA) d 6.44 (1H, br s), 5.82 (1H, br s), 4.82 (1H, br m),
3.78 (3H, s), 3.10–2.90 (2H, br m), 2.08 (3H, s), 1.62
(2H, m), 1.38–1.26 (20H, br m), 0.88 (3H, t, J =
6.9 Hz); 13C NMR (100 MHz, CDCl3 with 0.03% TFA)
d 187.1, 175.3, 171.0, 170.7, 53.0, 52.5, 39.5, 34.0, 31.9,
29.61, 29.59 (x 2), 29.5, 29.40, 29.36, 29.3, 26.7,
23.0, 22.7, 14.1; HRMS, calcd for C20H36N2O5NaS
([M+Na]+) 439.2237, found 439.2242.
4.4.2. Treatment of Epo-C12 (2) with excess amount of N-
acetylcysteine methyl ester (8). To a solution of 2
(4.7 mg, 15 lmol) and 8 (6.4 mg, 36 lmol) in MeOH
(1.0 mL) was added a 0.5 M aqueous solution of NaH-
CO3 (1.0 mL), and the mixture was stirred at rt for
24 h. The mixture was quenched by the addition of
1 M HCl (1 mL) and extracted with EtOAc. The extract
was washed with brine, dried (Na2SO4), and concen-
trated. The residue was purified by column chromatog-
raphy (hexane/EtOAc = 1:1) to give dodecanoic acid
(10) (1.1 mg, 36%) as white solid and N-acetyl-L-cystine
dimethyl ester (11)17 (4.1 mg, 64%) as white solid.
4.8. Confocal microscopy
To determine the localization of Flu-Epo-C12, BALL-1
cells were seeded in 3.5-cm diameter cell culture dishes.
Cells were treated with 5 lM of Flu-Epo-C12 and
0.1 lM of MitoTracker. The cells were incubated at
37 ꢁC for 1 h and moved to microtubes. The cells were
washed with PBS three times. Live cells were examined
using a LSM 5 laser scanning confocal microscope at
405 nm to assess Flu-Epo-C12 and at 543 nm to assess
MitoTracker.
4.5. Treatment of epolactaene (1) with N-acetylcysteine
methyl ester (8)
4.5.1. (2E,6E,8,E,10E)-10-(Methoxycarbonyl)-2,8-dim-
ethyldodeca-2,6,8,10-tetraenoic acid (12). To a solution
of 1 (9.0 mg, 23 lmol) and 8 (6.0 mg, 34 lmol) in MeOH
(1.0 mL) was a 0.5 M aqueous solution of NaHCO3
(1.0 mL), and the mixture was stirred at rt for 20 min.
The mixture was quenched by the addition of 1 M
HCl (1 mL) and extracted with EtOAc. The extract
was washed with brine, dried (Na2SO4), and concen-
trated. The residue was purified by column chromatog-
raphy (hexane/EtOAc = 1:1) to give 12 (4.6 mg, 72%
from 1) as colorless and 11 (4.6 mg, 77% from 8) as
white solid. 12: IR (film) 3020, 2927, 2855, 1711, 1689,
4.9. Preparation of cell lysates and isolation of binding
proteins of Bio-Epo-C12
BALL-1 cells were washed three times with cold PBS
and then treated with lysis buffer (50 mM Hepes,
150 mM NaCl, 10% glycerol, 1% Triton X-100,
1.5 mM MgCl2, 1 mM EGTA, 1% protease inhibitor

5048
K. Kuramochi et al. / Bioorg. Med. Chem. 16 (2008) 5039–5049
cocktail, pH 7.5). The lysed cells were centrifuged at
14,000g for 15 min at 4 ꢁC, and the supernatant was col-
lected as the cell lysate. The protein concentration of the
lyste was adjusted to be 1 mg/mL by using the Bradford
method.18
1241.277, 1251.199, 1263.333, 1290.171, 1299.382,
1340.374, 1368.443, 1636.501, 1685.728, 713.642,
1741.695, 1916.836, 1968.908, 2116.94, 2233.155,
2265.074, 2368.107, 2472.442, 2668.492.
The following peaks of peptide fragments derived from
band 2 were identical to those theoretically calculated
from the sequence of ATP citrate lyase; [M+H+] =
884.084, 896.187, 920.123, 934.204, 958.179, 1060.213,
1090.246, 1129.298, 1367.583, 1395.521, 1408.485,
1417.539, 1422.556, 1427.58, 1491.634, 1567.835,
1646.917, 1660.797, 1738.018, 1872.086, 1881.021.
The lysate in the lysis buffer (1 mL) was incubated with
5 lM of Bio-Epo-C12, 5 lM of biotin, and 5 lM of vehi-
cle (DMSO), respectively, at 4 ꢁC for 18 h. Streptavidin
agarose beads (100 lL) were added to the lysate, and
incubated at 4 ꢁC for 2 h. After the beads were washed
with the lysis buffer three times, the proteins were eluted
by the addition of 15 lL of sample buffer (284 mM Tris–
HCl, 9% SDS, 27% glycerol, 10% mercaptoethanol,
0.02% bromophenol blue) and 15 lL of lysis buffer, fol-
lowed by heat at 90 ꢁC. The beads were separated by
centrifuging at 14,000g for 5 min at 4 ꢁC, and the super-
natants were collected as the eluted samples. The eluted
samples were analyzed by SDS–PAGE (acrylamide: 12%
and 6% for separating gels, and 4% for stacking gels),
and the proteins were visualized by CBB staining.
The following peaks of peptide fragments derived from
band 3 were identical to those theoretically calculated
from the sequence of translation elongation factor 2
(eEF-2); [M+H+] = 890.175, 922.1, 969.222, 1084.26,
1091.314, 1138.276, 1153.333, 1206.435, 1222.365,
1274.533, 1308.491, 1402.7, 1444.689, 1543.805,
1615.856, 1800.104, 2007.303, 2079.46, 2143.528,
2176.567, 2220.597.
4.10. Identification of the binding proteins by PMF
The following peaks of peptide fragments derived from
band 4 were identical to those theoretically calculated
from the sequence of heat shock protein 90 b; [M+H+] =
886.209, 891.111, 901.219, 951.171, 1141.322, 1194.442,
1236.471, 1249.463, 1311.464, 1348.602, 1416.495,
1513.832, 1527.749, 1783.228, 1809.194, 1848.014,
1911.373, 1988.232, 2177.454, 2256.612, 2374.834.
The visualized bands were sliced and decolorized with
decoloring buffer (50% acetonitrile, 25 mM NH4HCO3).
After the gels were dried by the addition of acetonitrile
(100 lL), the resulting gels were separated and dried in
vacuo. The dried gels were reduced by incubating with
50 lL of reducing buffer (10 mM dithiothreitol, 25 mM
NH4HCO3) at 56 ꢁC for 1 h. After removing the excess
reducing buffer, 100 lL of alkylating buffer (55 mM
iodoacetamide, 25 mM NH4HCO3) was added, and
the mixture was incubated at rt for 45 min. The superna-
tants were removed, and the gels were washed twice by
incubation with 100 lL of 25 mM NH4HCO3, followed
by 100 lL of the buffer (50% acetonitrile, 25 mM
NH4HCO3). The resulting gels were separated and dried
in vacuo. The dried gels were digested by incubation
with 50 lL of digesting solution (50 mM NH4HCO3,
100 lg/mL tripsin) at 0 ꢁC for 30 min. After the excess
digesting solution was removed, the mixture was incu-
bated at 37 ꢁC for 20 h. The peptide fragments were
eluted by incubation with 50 lL of elution solution
(50% aqueous acetonitrile, 1% trifluoroacetic acid) at
0 ꢁC. The extracts were concentrated in vacuo, and dis-
solved in 10 lL of the elution solution. The solution of
the extracts was absorbed on a ZipTip pipette tip (Mil-
lipore Co., MA, USA), and the tip was washed with the
elution solution, and the solution was concentrated. A
mixture of the resulting peptides and DHBA matrix
(20 mg/ml 2,5-dihydroxybenzoic acid in a 2:1 solution
of 0.1% trifluoroacetic acid and acetonitrile) was loaded
on a MALDI-TOF MS target plate. The samples were
applied to MALDI-TOF mass in the positive reflection
mode. Identification of proteins from MALDI-TOF
spectra was achieved by using the Mascot search pro-
gram (Matrix science Inc., USA). Figure S9 shows the
digested peptide derived from seven bands.
The following peaks of peptide fragments derived from
band 5 were identical to those theoretically calculated
from the sequence of heat shock protein 60; [M+H+] =
961.253, 1504.757, 1556.979, 1584.865, 1642.935,
1919.421, 2038.413, 2047.578, 2113.577, 2365.923,
2508.781, 2560.936.
The following peaks of peptide fragments derived from
band 6 were identical to those theoretically calculated
from the sequence of adenine nucleotide translocator 2
(ANT2);
[M+H+] = 902.305,
951.265,
976.308,
1121.336, 1132.392, 1136.409, 1219.351, 1268.361,
1446.446, 1816.563, 1926.7828, 2795.858.
The following peaks of peptide fragments derived from
band 7 were identical to those theoretically calculated
from the sequence of peroxiredoxin 1 (Prx1); [M+H+] =
894.249, 920.305, 980.280, 1107.300, 1164.59, 1196.305,
1211.361, 1263.355, 1359.453, 1622.462, 1750.601,
1778.577, 2125.763, 2405.740, 2752.795.
Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.bmc.
2008.03.029.
References and notes
The following peaks of peptide fragments derived from
band 1 were identical tothose theoreticallycalculated from
the sequence of fatty acid synthase; [M+H+] = 1042.161,
1. Kakeya, H.; Takahashi, I.; Okada, G.; Isono, K.; Osada,
H. J. Antibiot. 1995, 48, 733.

K. Kuramochi et al. / Bioorg. Med. Chem. 16 (2008) 5039–5049
5049
2. Kakeya, H.; Onozawa, C.; Sato, M.; Arai, K.; Osada, H.
J. Med. Chem. 1997, 40, 391.
14010; (b) Schweitzer, D.; Zhu, J.; Jarori, G.; Tanaka, J.;
Higa, T.; Davisson, V. J.; Helquist, P. Bioorg. Med. Chem.
2007, 15, 3208.
3. (a) Kuramochi, K.; Itaya, H.; Nagata, S.; Takao, K.;
Kobayashi, S. Tetrahedron Lett. 1999, 40, 7367; (b)
Kuramochi, K.; Nagata, S.; Itaya, H.; Takao, K.;
Kobayashi, S. Tetrahedron Lett. 1999, 40, 7371; (c)
Kuramochi, K.; Nagata, S.; Itaya, H.; Matsubara, Y.;
Sunoki, T.; Uchiro, H.; Takao, K.; Kobayashi, S. Tetra-
hedron 2003, 59, 9743.
4. Total synthesis of epolactaene (a) Hayashi, Y.; Narasaka,
K. Chem. Lett. 1998, 313; (b) Hayashi, Y.; Kanayama, J.;
Yamaguchi, J.; Shoji, M. J. Org. Chem. 2002, 67, 9443; (c)
Marumoto, S.; Kogen, H.; Naruto, S. J. Org. Chem. 1998,
63, 2068; (d) Marumoto, S.; Kogen, H.; Naruto, S.
Tetrahedron 1999, 55, 7129; (e) Marumoto, S.; Kogen, H.;
Naruto, S. Tetrahedron 1999, 55, 7145; (f) Tan, Z.;
Negishi, E. Org. Lett. 2006, 8, 2783.
5. (a) Mizushina, Y.; Kobayashi, S.; Kuramochi, K.; Nag-
ata, S.; Sugawara, F.; Sakaguchi, K. Biochem. Biophys.
Res. Commun. 2000, 273, 784; (b) Kuramochi, K.;
Mizushina, Y.; Nagata, S.; Sugawara, F.; Sakaguchi, K.;
Kobayashi, S. Bioorg. Med. Chem. 2004, 12, 1983; (c)
Mizushina, Y.; Kuramochi, K.; Ikawa, H.; Kuriyama, I.;
Shimazaki, N.; Takemura, M.; Oshige, M.; Yoshida, H.;
Koiwai, O.; Sugawara, F.; Kobayashi, S.; Sakaguchi, K.
Int. J. Mol. Med. 2005, 15, 785.
´
9. Buckman, J. F.; Hernandez, H.; Kress, G. J.; Votyakova,
T. V.; Pal, S.; Reynolds, I. J. J. Neurosci. Methods 2001,
104(2), 165.
˜
˜
10. (a) Omura, S. Bacteriol. Rev. 1976, 40, 681; (b) Omura, S.
Methods Enzymol. 1981, 72, 520; (c) Pizer, E. S.; Wood, F.
D.; Pasternack, G. R.; Kuhajda, F. P. Cancer Res. 1996,
56, 745; (d) Pizer, E. S.; Jackisch, C.; Wood, F. D.;
Pasternack, G. R.; Davidson, N. E.; Kuhajda, F. P.
Cancer Res. 1996, 56, 2745.
11. Moche, M.; Schneider, G.; Edwards, P.; Dehesh, K.;
Lindqvist, Y. J. Biol. Chem. 1999, 274, 6031.
˜
12. (a) Funabashi, H.; Iwasaki, S.; Okuda, S.; Omura, S.
Tetrahedron Lett. 1983, 24, 173; (b) Funabashi, H.;
˜
Kawaguchi, A.; Tomoda, H.; Omura, S.; Okuda, S.;
Iwasaki, S. J. Biochem. 1989, 105, 751.
13. (a) Magnus, P.; Ladlow, M.; Elliott, J. J. Am. Chem. Soc.
1987, 109, 7929; (b) Kato, E.; Oya, M.; Iso, T.; Iwao, J.-I.
Chem. Pharm. Bull. 1986, 34, 486.
14. (a) Kelleman, A.; Mattern, R.-H.; Pierschbacher, M. D.;
Goodman, M. Biopolymers 2003, 71, 686; (b) Geng, Y.;
Discher, D. E.; Jusynska, J.; Schlaad, H. Angew. Chem.,
Int. Ed. 2006, 45, 7578; (c) Conchillo, A.; Camps, F.;
Messeguer, A. J. Org. Chem. 1990, 55, 1728.
6. (a) Nakai, J.; Kawada, K.; Nagata, S.; Kuramochi, K.;
Uchiro, H.; Kobayashi, S.; Ikekita, M. Biochim. Biophys.
Acta 2002, 1581, 1; (b) Kuramochi, K.; Matsui, R.;
Matsubara, Y.; Nakai, J.; Sunoki, T.; Arai, S.; Nagata, S.;
Nagahara, Y.; Mizushina, Y.; Ikekita, M.; Kobayashi, S.
Bioorg. Med. Chem. 2006, 14, 2151.
7. (a) Nagumo, Y.; Kakeya, H.; Shoji, M.; Hayashi, Y.;
Dohmae, M.; Osada, H. Biochem. J. 2005, 387, 835; (b)
Nagumo, Y.; Kakeya, H.; Yamaguchi, J.; Uno, T.; Shoji,
M.; Hayashi, Y.; Osada, H. Bioorg. Med. Chem. Lett.
2004, 14, 4425.
15. (a) Hamilton, L.; Stevenson, M. H.; Boyd, D. R.;
Brannigan, I. N.; Treacy, A. B.; Hamilton, J. T. G.;
McRoberts, W. C.; Elliott, C. T. J. Chem. Soc., Perkin
Trans.1 1996, 139; (b) Seike, H.; Ghosh, I.; Kishi, Y. Org.
Lett. 2006, 8, 3865.
16. (a) Murozuka, Y.; Kasuya, M. C. Z.; Kobayashi, M.;
Watanabe, Y.; Sato, T.; Hatanaka, K. Chem. Biodiv.
2005, 2, 1063; (b) Sato, T.; Fujita, S.; Kasuya, M. C.
Z.; Hatanaka, K.; Yamagata, T. Chem. Lett. 2004, 33,
580.
`
17. Langlois, B.; Montegre, D.; Roidot, N. J. Fluorine Chem.
1994, 68, 63.
18. Bradford, M. M. Anal. Biochem. 1976, 2, 248.
8. (a) Chatterjee, S. K.; Laffray, J.; Patel, P.; Ravindra, R.;
Qin, Y.; Kuehne, M. E.; Bane, S. L. Biochemistry 2002, 41,