Apoptosis-inducing effect of epolactaene derivatives on BALL-1 cells

Article abstract of DOI:10.1016/j.bmc.2005.10.057

Epolactaene, a neuritogenic compound in human neuroblastoma SH-SY5Y, induces apoptosis in a human leukemia B-cell line, BALL-1. The apoptosis-inducing activities of 34 epolactaene derivatives, including those of the newly synthesized α-alkyl-α,β-epoxy-γ-l

Full text of DOI:10.1016/j.bmc.2005.10.057

Bioorganic & Medicinal Chemistry 14 (2006) 2151–2161
Apoptosis-inducing effect of epolactaene derivatives
on BALL-1 cells
Kouji Kuramochi,a Rie Matsui,b Yasuaki Matsubara,c Junko Nakai,b Takashi Sunoki,c
Satoshi Arai,b Seigo Nagata,c Yukitoshi Nagahara,b Yoshiyuki Mizushina,d
Masahiko Ikekitaa,b and Susumu Kobayashia,c,
*
aFrontier Research Center for Genome and Drug Discovery, Tokyo University of Science, 2641 Yamazaki,
Noda, Chiba 278-8510, Japan
bDepartment of Applied Biological Science, Faculty of Science and Technology, Tokyo University of Science,
2641 Yamazaki, Noda, Chiba 278-8510, Japan
cFaculty of Pharmaceutical Sciences, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-8510, Japan
dDepartment of Nutritional Science, High Technology Research Center, Kobe-Gakuin University,
Nishi-ku, Kobe, Hyogo 651-2180, Japan
Received 3 October 2005; revised 30 October 2005; accepted 31 October 2005
Available online 18 November 2005
Abstract—Epolactaene, a neuritogenic compound in human neuroblastoma SH-SY5Y, induces apoptosis in a human leukemia
B-cell line, BALL-1. The apoptosis-inducing activities of 34 epolactaene derivatives, including those of the newly synthesized a-al-
kyl-a,b-epoxy-c-lactam derivative and cyclopropane derivatives, were also tested. The structure–activity relationships of the epol-
actaene derivatives as an inducer of apoptosis are described. The a-acyl-a,b-epoxy-c-lactam moiety as well as the hydrophobicity
derived from the long alkyl side chain are both important for activity. Compound 1e displayed the strongest activity among all
the synthesized compounds with an IC50 value of 0.70 lM.
ꢀ 2005 Elsevier Ltd. All rights reserved.
1. Introduction
Epolactaene (1a) is a microbial metabolite that was orig-
inally isolated by Kakeya and Osada et al. from the cul-
tured mycelium of Penicillium sp. BM 1689-P.16
Epolactaene was found to induce neurite outgrowth
and cell cycle arrest at the G1 phase in a human neuro-
blastoma cell line, SH-SY5Y.16–19 The potent biological
properties of epolactaene, along with its structural com-
plexity, have initiated intensive synthetic research ef-
forts.20–24 We are particularly interested in the unique
biological activity of epolactaene and have undertaken
a series of both synthetic and biological studies.25–31
Apoptosis, the process of programmed cell death, pro-
ceeds with several biochemical and cytological features,
including the condensation and fragmentation of nuclei,
apoptotic body formation, and chromosomal DNA
fragmentation into ca. 180 bp oligomers.1–3 Apoptosis
is tightly regulated with the cell cycle by a cascade of ini-
tiator and effector caspsases that are activated sequen-
tially.4–8 Inappropriate control of apoptosis plays a
role in many human diseases.9–11 Abnormal inhibition
of apoptosis is a hallmark of cancer and autoimmune
diseases,12–14 while excessive apoptosis is implicated in
neurodegenerative disorders such as Alzheimerꢀs
disease.9,15
We have previously described the synthesis of
epolactaene and several of its analogues (1–12)
(Figs. 1 and 2).25–27,30 Mizushina et al. reported that
epolactaene 1a and its analogue 1b selectively inhibit-
ed the activities of mammalian DNA polymerases a
(pol a), b (pol b) and human DNA topoisomerase
II (topo II).28 More recently, analogues 1e, 7b, 8b,
and 12a have also been reported to inhibit the activi-
ties of mammalian pol a and pol b.30 Among these
analogues, compound 1e was found to be the most
Keywords: Epolactaene; Apoptosis; Structure–activity relationships;
BALL-1 cells.
*
Corresponding author. Tel./fax: +81
kobayash@rs.noda.tus.ac.jp
4
7121 3671; e-mail:
0968-0896/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.10.057

2152
K. Kuramochi et al. / Bioorg. Med. Chem. 14 (2006) 2151–2161
O
O
O
O
O
O
O
O
R
R
R
R
R
H3C
HO
H3C
HO
O
O
H3C
O
N
H
N
H
H3C
O
O
OH NH2
1 (a-e)
3 (a-d)
4 (a-d)
2a
O
O
O
OH
OH
O
O
O
R
R
R
H3C
HO
H3C
O
O
H3C
O
H3C
O
N
N
H
H3CO
O
OH NH2
C12H25
5 (a-d)
6 (a-d)
7b
8b
CH3
a: R =
c: R =
b: R=
CH3
CH3
CH3 CO2CH3
d: R =
e R =
Figure 1. Structure of epolactaene and its derivatives.
O
O
O
O
H3C
HO
H3C
HO
H3C
RO
O
N
CONHR
OH
O
O
H3C
N
N
H
H
R
10 (a-b)
11 (a-b)
a: R = n-C12H25 b: R = n-C6H13
12 (a-b)
9
Figure 2. Structure of a,b-epoxy-c-lactams (9–12).
potent inhibitor of DNA pols. The same group has
recently reported that some epolactaene derivatives
inhibit TPA (12-O-tetradecanoylphorbol-13-acetate)-
induced inflammation in the mouse inflammatory
test.31 Because epolactaene derivatives possess anti-in-
flammatory activity in vivo, these agents have poten-
tial therapeutic utility.
mode of action, we examined the structure–activity rela-
tionships of these epolactaene derivatives as inducers of
apoptosis.
2. Chemistry
We have previously reported the preparation of 1–6,
based on the bridgehead oxiranyl anion strategy,27 and
the preparation of 7–12.30 Here, we describe the prepa-
ration of a-alkyl-a,b-epoxy-c-lactam and cyclopropane
analogues in order to examine the influence of epoxyke-
tone on the biological activity of the derivatives (Fig. 3).
We are interested in compounds that induce apoptosis
as potential anti-cancer agents, because many cancer
cells exhibit abnormal inhibition of apoptosis.13,32 Thus,
we investigated the effect of epolactaene derivatives on
several cancer cell lines. We found that synthetic ana-
logues 1(a–d) induced apoptosis in a human leukemia
B-cell line, BALL-1, in a dose- and time-dependent
manner.29 The a,b-epoxy-c-lactam at the core of the
molecule and the linear extended alkyl group in the side
chain were both very important for maintaining biolog-
ical activities.
Scheme 1 outlines the preparation of a-alkyl-a,b-epoxy-
c-lactam 15. A solution of 13 in THF was added to a
solution of LDA and dodecyl trifluoromethanesulfonate
in THF at ꢀ78 ꢁC to give the alkylated product, along
with several unknown byproducts. The formation of
O
It has recently been reported that epolactaene binds to
Hsp60 as a Michael acceptor, thereby inhibiting Hsp60
chaperone activity.33,34 However some epolactaene
derivatives, which lack an a,b-unsaturated ketone, also
inhibit growth in cancer cell lines. These results suggest
that alternative protein targets exist. To investigate the
O
H
H3C
R
R
H3C
HO
O
O
N
N
HO
H
H
Figure 3. Structure of newly designed epolactaene derivatives.

K. Kuramochi et al. / Bioorg. Med. Chem. 14 (2006) 2151–2161
2153
O
1) LDA
2) CH3(CH2)11OTf
O
O
HF aq.
CH3CN
CO2CH3
H3C
H3C
O
THF
OTBS
-78 ˚C
13
14
1) NH3 aq., CH3OH
2) TFAA, DMSO, CH2Cl2
then Et3N
O
H3C
HO
O
N
H
3) LiOH, THF-H2O
15
Scheme 1. Synthesis of a-alkyl-a,b-epoxy-c-lactam 15.
byproducts presumably results from both the instability
of dodecyl triflate during silica gel purification and also
the high reactivity of the oxiranyl anion derived from 13.
Decomposition of 13 was observed when dodecyl iodide
was used in this reaction. No significant improvement
was observed when the reaction was carried out at a
lower temperature (ꢀ100 ꢁC). Thus without any purifi-
cation, treatment of the alkylated product with aqueous
HF in CH3CN, desilylation, and lactonization proceed-
ed to give 14. After ammonolysis of 14, the resulting
hydroxyamide was oxidized by Swern oxidation.27 Since
O-trifluoroacetylated 15 was observed, the crude oxi-
dized product was hydrolyzed to give 15.
between H-4 and H-5 in 19 is greater than that (J4–5 =
ꢁ0 Hz) in 20. NOEs between H-6 and methyl group
and NOEs between H-4 and H-5 in 19 were observed.
These results suggest that the cyclopropane ring and
methyl group in 19 exist on the same face. The major
isomer 19 will be derived from trans-epoxide, whereas
the minor 20 will be derived from cis-epoxide. The prod-
uct 20 was non-cyclic a-alkylated malonate, which can
be converted into 19 by treatment with Cs2CO3 in THF.
Hydrolysis of methyl ester in 19 gave a carboxylic acid
22, which was transformed into Weinreb amide 23
(Scheme 3).40 After ammonolysis of 23, the resulting hy-
droxy group was protected as TBS ether to give 25.
Treatment of 25 with n-BuLi in THF gave 26 in 50%
yield (29% of 25 was recovered). Deprotection of the
TBS group with 3HF-Et3N gave hydroxyamide 27 and
lactone 28. Oxidation of 27 with Dess–Martin period-
inane41 afforded 29 as a 1.2:1 tautomeric mixture and
lactone 28. Compound 29 is one of the desired cyclopro-
pane epolactaene analogues. The mechanism for the for-
mation of lactone 28 is unclear, although it was also
observed by treatment of 27 with Dess–Martin period-
inane in the presence of NaHCO3 or pyridine. Thus,
the results suggest that lactone formation does not occur
under acidic conditions. Although 28 was formed, we
We also aimed to synthesize cyclopropane epolactaene
analogues. Construction of the cyclopropane ring was
conducted according to Temnikovaꢀs procedure
(Scheme 2).35 Crotyl alcohol (16) was treated with tert-
butylhydroperoxide (TBHP) and Ti(O-i-Pr)4 in the pres-
ence of MS3A, followed by p-nitrosulfonate (NsCl) and
Et3N in one pot to give 18 in a racemic form.36,37 Treat-
ment of 18 with dimethyl malonate and Cs2CO3 in the
presence of 0.1 equivalent amount of 18-crown-6 affor-
ded 19, 20, and 21.38 The stereochemistry of 19 and 20
1
was determined by H NMR spectra and NOE experi-
ments (Fig. 4).39 The coupling constant (J4–5 = 4.6 Hz)
TBHP
NsCl
O
Et3N
Ti(Oi-Pr)4
O
H3C
ONs
H3C
OH
H3C
OH
CH2Cl2
MS3A
20 to 0 ºC
CH2Cl2
16 (trans : cis =15 : 1)
17
18 (trans : cis = 15 : 1)
20 ºC
O
O
CH2(CO2CH3)2
Cs2CO3
CO2CH3
H
H
O
OCH3
OCH3
O
H3C
+
CO2CH3
H3C
O
H3C
18-crown-6
THF
O
O
19
64%
20
21
trace
34%
O
CO2CH3
CO2CH3
H
H3C
O
Cs2CO3
THF
OCH3
H3C
O
O
57%
19
21
Scheme 2. Synthesis of compound 19.

2154
K. Kuramochi et al. / Bioorg. Med. Chem. 14 (2006) 2151–2161
NOE
CH3O2C
J = ~0 Hz
H
O
O
O
H
J = 4.6 Hz
O
H
H
H3C
H
H
OCH3
OCH3
H
H3C
H
CH3
O
O
O
O
NOE
20
19
19
Figure 4. Determination of the stereochemistry of 19 and 20.
O
O
O
CH3ONHCH3·HCl
BOP, i-Pr2NEt
OCH3
H
H
H
NaOH aq.
OCH3
OH
N
CH2Cl2
86%
CH3
O
CH3OH
99%
H3C
O
H3C
O
O
H3C
O
O
19
22
23
O
O
OCH3
OCH3
H
H
TBSCl, Imid.
n-BuLi
NH3 aq.
N
N
THF, -78 ˚C
50% and 29%
of recovered 25
CH3
DMF
72%
CH3
CH3OH
89% and 10%
of recovered 23
H3C
O
H3C
TBSO
O
OH NH2
NH2
25
24
O
O
O
O
H
H
H
3HF-Et3N
CH2Cl2
+
H3C
TBSO
O
NH2
H3C
O
NH2
H3C
O
HO
28
26
27
23%
77%
O
O
O
Dess-Martin
periodinane
H
H
H3C
H
+
CH2Cl2
H3C
HO
O
NH2
O
N
H3C
O
HO
O
H
27
29
28
32%
68%
O
O
H
NH3
H
H3C
CH3OH
99%
H3C
HO
O
NH2
O
O
28
27
Scheme 3. Synthesis of cyclopropane derivative 29.
found that ammonolysis of 28 in MeOH regenerated the
hydroxyamide 27.
Thus, we attempted to introduce an N-alkyl-N-methyla-
mide moiety into the side chain to act as a bioisostere of
the acyl group (Fig. 5).42 Condensation of 22 and hexa-
decanoic acid with BOP in the presence of i-Pr2NEt gave
30 as a 1.7:1 rotational mixture in 92% yield. After
ammonolysis of 30, the resulting hydroxy group was
oxidized with the Dess–Martin reagent to give 32 as a
1.1:1 rotational mixture. Judging from the 1H NMR
spectrum (singlet at 2.35 ppm) and IR spectrum
(absorption at 1712 cmꢀ1), 32 appears to exist as a ke-
tone. The formation of c-lactam was not observed in
Since we have already established that a long alkyl
group in the side chain is important for the apoptosis-
inducing activity, we attempted to obtain other
cyclopropane analogues. However, the addition of n-
octadecyl lithium or n-octadecyl magnesium bromide
to 25 did not proceed (Scheme 4). When 25 was treated
with excess amount (30–50 equiv) of n-octadecyl lithium
or n-octadecyl magnesium bromide under higher
temperature (from room temperature to reflux), none
of the desired products was obtained and 25 was recov-
ered in 76–86% yield. The lack of reaction is presumably
due to the low reactivity of n-octadecyl lithium and
n-octadecyl magnesium bromide. Although the addition
of 1-heptadec-1-enyl lithium to 25 was investigated,
almost all of the starting material 25 was recovered.
1
the H NMR spectrum (see Scheme 5).
3. Apoptosis-inducing activity in BALL-1 cells
We examined the cell death-inducing action of epolact-
aene derivatives in BALL-1 cells by means of the

K. Kuramochi et al. / Bioorg. Med. Chem. 14 (2006) 2151–2161
2155
O
O
Table 1. Fifty percent inhibitory concentrations (IC50s) acting against
BALL-1 cell viability and the calculated octanol/water partition
coefficients (ClogP) of epolactaene and its derivatives
OCH3
H
H
N
CH3(CH2)17
THF
M
R
CH3
H3C
TBSO
O
NH2
H3C
TBSO
O
NH2
Compound
IC50 (lM)
ClogPb
3.82a
1.65a
5.04a
7.40a
0.70
3.26
>10
M = Li or MgBr
1a
1b
1.85
2.80
0.20
0.29
5.30
1.85
—
25
R = C18H37
1c
1d
Scheme 4. The addition of alkyl metal reagents to Weinreb amide 25.
1e
2a
3(a–d)
4(a–d)
5(a–d)
6(a–d)
7b
O
O
>10
>10
—
—
R'
H
H
N
R
H3C
H3C
CH3
>10
—
O
O
N
N
HO
HO
H
H
6.51
3.50
>10
7.61
3.16
ꢀ1.13
3.69
1.18
—
8b
9
R = C18H37
R' = C16H37
10a
10b
11(a–b)
12(a–b)
15
3.96
8.34
>10
Figure 5. Modification at the side chain of cyclopropane derivative.
MTT assay.29 The assay can indirectly calculate the cell
survival rate by determining the action of a mitochon-
drial dehydrogenase on MTT in metabolically active
cells.43 The 50% growth inhibitory concentrations
(IC50ꢀs) are summarized in Table 1.
>10
>10
—
3.96
0.78
5.28
29
32
>10
>10
a Ref. 29.
b The ClogP values were obtained using CS ChemDraw version 7.0
software (Cambridge Soft, USA).
Compounds1 (a–e), 2a, 7b, 8b, and 10(a–b) induced cell
death in BALL-1 cells at concentrations lower than
10 lM. Each compound induced cell death in a dose-
and time-dependent manner. The formation of DNA
ladder on agarose gels in DNA fragmentation assay
was observed after treatment with 5 lM of each com-
pound for 6 h. These results indicate that these com-
pounds induced apoptosis.29
the apoptosis-inducing effect by increasing the perme-
ability of the compound across the cell membrane.44
Although the N-dodecyl derivative (7b) and O-methyl
derivative (8b) both have greater ClogP values than
1b, their biological activities were slightly lower. Thus,
substitution at the N- or O-atoms appears to influence
the activity.
Because the hydroxyamides 3, 6 and lactones 4, 5 did
not inhibit BALL-1 cells at concentrations lower than
10 lM, the c-lactam moiety appears to be very impor-
tant for activity. There was no difference in the biologi-
cal action of (+)-epolactaene (1a) and (ꢀ)-epolactaene
(2a), signifying that the stereochemistry of the epoxide
does not influence the apoptosis-inducing activity.
Compound 9, which lacks the acyl side chain at the
a-position, had no effect on BALL-1 cells. We reasoned
that compound 9, being very hydrophilic, might not
readily permeate the cell membrane. Intriguingly,
the N-alkyl-substituted compounds 10(a–b) possess
apoptosis-inducing activity, whereas the hydroxyamides
11(a–b) and O-alkyl-substituted compounds 12(a–b) had
no activity at concentrations lower than 10 lM.
Of all the compounds tested in this study, 1e was the
most potent inducer of apoptosis. Interestingly, 1e had
the greatest calculated logP (i.e., octanol/water partition
coefficient) value among 1(a–e), inferring that hydro-
phobicity affects the activity. We reasoned that the
hydrophobic side chain of epolactaene might enhance
Compounds 15 had no effect on BALL-1 cells at concen-
trations lower than 10 lM. Compound 15 lacks the
carbonyl group at the side chain of 1b. The carbonyl
BOP
O
O
CH3(CH2)15NHCH3
H
(CH2)15CH3
H
NH3 aq.
CH3OH
OH
N
i-Pr2NEt
CH2Cl2
92%
CH3
O
H3C
O
H3C
O
O
64% and 32% of
recovered 30
22
30
O
O
Dess-Martin
(CH2)15CH3
(CH2)15CH3
H
H
N
N
periodinane
CH2Cl2
96%
CH3
CH3
H3C
O
H3C
O
O
NH2
32
OH NH2
31
Scheme 5. Synthesis of cyclopropane derivative 32.

2156
K. Kuramochi et al. / Bioorg. Med. Chem. 14 (2006) 2151–2161
group at the side chain affected the activity, because
compound 1b was found to induce apoptosis with an
IC50 value of 1.65 lM. Compounds 29 and 32 did not
have any effect on BALL-1 cells at concentrations lower
than 10 lM, either. Compounds 29 and 32 have a cyclo-
propane ring instead of an epoxide ring. Because the
epoxide derivatives 1(a–e) induced apoptosis at concen-
trations lower than 10 lM, the activity of the cyclopro-
pane derivatives was less potent than those of the
epoxide derivatives. These results indicate that the a-ac-
yl-substituted a,b-epoxy-c-lactam moiety is important
for the apoptosis-inducing activity.
DNA polymerases at the concentrations required to in-
duce apoptosis.28,30,31 Thus, the inhibition of these
DNA-modifying enzymes may play some part in the
apoptosis-inducing effect of epolactaene and its deriva-
tives, but is not the primary mechanism of action. Nagu-
mo et al. have recently reported that epolactaene
covalently binds Hsp60 and inhibits Hsp60 chaperone
activity.33,34 The reports demonstrate that Micheal addi-
tion of Hsp60 through Cys-442 to the a,b-unsaturated
ketone moiety of epolactaene is responsible for the inhi-
bition of chaperone activity. However, some epolactae-
ne derivatives that lack the a,b-unsaturated ketone
also inhibit the growth of cancer cell lines (e.g., com-
pounds 1(b–e), 7b, and 8b). These results suggest that
the apoptosis-inducing effect of epolactaene is not al-
ways explained by the inhibition of Hsp60. In addition
to these results, we have also found that epolactaene
derivative 1b induces apoptosis more effectively in B
lymphocytes than T lymphocytes. Epolactaene deriva-
tives did not induce apoptosis in attached cells, HepG2,
HeLa, and A549.29 These results also suggest that addi-
tional target proteins are involved in the biological ac-
tion of epolactaene.
4. Discussion
Epolactaene (1a) was originally reported to possess a
potent neurite outgrowth activity in a human neuroblas-
toma cell line, SH-SY5Y.16,17 In our precedent paper,
we found that epolactaene and some of its derivatives in-
duced apoptosis in a human leukemia B-cell line, BALL-
1, in a dose- and time-dependent manner.29 In this
study, we describe the structure–activity relationships
of epolactaene derivatives as inducers of apoptosis. We
tested the apoptosis-inducing activity of 34 epolactaene
derivatives, including some newly synthesized deriva-
tives. We found that compounds 1(a–e), 2a, 7b, 8b,
and 10(a–b) induced apoptosis in BALL-1 cells at con-
centrations lower than 10 lM. Compound 1e was the
most potent inducer of apoptosis with an IC50 value of
0.70 lM. We also found that the hydrophobicity of
epolactaene derivatives, which was calculated as ClogP,
affected the apoptosis-inducing activity. We propose
that the side-chain moiety of the molecule may facilitate
permeation across the cell membrane.
In conclusion, we have described the structure–activity
relationships of epolactaene derivatives as inducers of
apoptosis. We have demonstrated that the a-acyl-a,b-
epoxy-c-lactam moiety as well as the length of the side
chain (i.e., the hydrophobicity) are important for the
activity. Based on the structure–activity relationships
of these derivatives, we are currently attempting to iden-
tify the target molecules of epolactaene.
5. Experimental procedures
5.1. General
Our structure–activity relationship studies suggest that
the a-acyl-a,b-epoxy-c-lactam moiety is important for
activity, because neither hydroxy amides (3 and 6),
lactones (4 and 5), a-alkyl-a,b-epoxy-c-lactam (15)
nor cyclopropanes (29 and 32) affected BALL-1 cells
at concentrations lower than 10 lM. Substitution at
the N-or O-position of the a-acyl-a,b-epoxy-c-lactam
(compounds 7b and 8b) caused a slight decrease in
activity. Although neither a,b-epoxy-c-lactam 9 nor
O-substituted analogues 12(a–b) induced apoptosis at
concentrations lower than 10 lM, N-substituted ana-
logues 10(a–b) did show the activity. However, the dif-
ferences between a-acyl-a,b-epoxy-c-lactams 1(a–e)
and 10(a–b) in terms of the mode of action were
not examined in detail.
1H and 13C NMR were recorded on a JEOL JNM-
LD400 or JNM-LD500, or on a BRUKER DXR400
or DRX600. Chemical shifts were reported in d, parts
per million (ppm), relative to TMS as an internal stan-
dard or calibrated using residual undeuterated solvent
as an internal reference. IR spectra were recorded on a
JASCO FT/IR-410 spectrometer. Mass spectra were
obtained on an API QSTAR Pulsar i spectrometer.
Optical rotations were measured on a JASCO P-1030
digital polarimeter. Melting points were determined with
a Yanaco MP-3S melting point apparatus. Column
chromatography was carried out on Fuji Silisia
PSQ100B. Analytical thin-layer chromatography
(TLC) was performed on precoated Merck silica gel 60
F254 plates, and compounds were visualized by UV
illumination (254 nm) or heating 150 ꢁC after spraying
phosphomolybdic acid in ethanol. THF was distilled
from sodium/benzophenone. CH2Cl2 was distilled
from P2O5. DMSO was distilled from CaH2. All other
solvent and reagents were obtained from commercial
sources and used without further purification. Organic
extracts were dried over Na2SO4 or MgSO4, filtered,
and concentrated using a rotary evaporator. Involatile
oils and solids were vacuum-dried.
Mizushina et al. reported that epolactaene and its deriv-
atives inhibit the activity of mammalian pol a, b, and k,
and human Topo II.28,30,31 It is known that inhibition of
these enzymes can result in cell cycle arrest, apoptosis,
and anti-inflammatory activity.45–47 Indeed, Mizushina
et al. have recently reported that the anti-inflammatory
effect of epolactaene might be related to the inhibition
of pol k. In addition, the structure–activity relationships
of epolactaene derivatives as apoptosis-inducers are sim-
ilar to those of DNA polymerase inhibitors. However,
epolactaene derivatives weakly inhibit the activity of

K. Kuramochi et al. / Bioorg. Med. Chem. 14 (2006) 2151–2161
2157
Fetal bovine serum (FBS), kanamycin sulfate, dimethyl
sulfoxide (DMSO) and 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT) were purchased
from Wako Pure Chemical Industries (Tokyo, Japan).
RPMI 1640 was purchased from Nissui (Tokyo, Japan).
2-Mercaptoethanol was purchased from Nakalai Tesque
(Kyoto, Japan).
A solution of the residue and LiOH (2.5 mg, 0.10 mmol)
in THF-H2O (4:1, 2.5 mL) 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 (MgSO4), and concentrated. The resi-
due was purified by silica gel chromatography (1:2
EtOAc/hexane) to afford 15 (10.8 mg, 67% in 2 steps)
as a ca. 17:1 tautomeric mixture, as a white solid.
23
D
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 (64 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).
Mp = 35 ꢁC; ½aꢂ ꢀ12.6 (c 0.10, MeOH); 1H NMR
(600 MHz, CDCl3) d: 6.40 (1H, br s, NH), 3.67 (1H, s,
H-5), 1.96 (1H, m, H-10), 1.87 (1H, m, H-10), 1.73
(1H, br s, OH), 1.54 (3H, s, 4-CH3), 1.42 (1H, m, H-
20), 1.36 (1H, m, H-20), 1.33–1.25 (18H, m, H-30–H-
110), 0.88 (3H, t, J = 6.9 Hz); 13C NMR (150 MHz,
CDCl3) d: 173.2, 83.5, 62.8, 61.7, 31.9, 29.7, 29.7, 29.6,
29.6, 29.5, 29.4, 29.3, 24.7, 24.1, 22.7, 21.9, 14.9; IR
(KBr) cmꢀ1: 3425, 3224, 2918, 2852, 1705, 1469, 1432,
1372, 1161, 951, 779, 717, 626; HRMS, calcd for
C17H31NO3Na (M+Na)+ 320.2201. Found 320.2200.
5.1.1. (1S,4S,5S)-1-Dodecyl-4-methyl-3,6-dioxabicyclo-
[3.1.0]hexan-2-one (14). To a solution of diisopropyl-
amine (0.8 mL, 5.71 mmol) in THF (30 mL) was added
n-BuLi (3.7 mL of a 1.56 M solution in hexane,
5.8 mmol) at 0 ꢁC. The mixture was stirred at 0 ꢁC for
10 min. A solution of dodecyl trifluoromethanesulfonate
(1.2 g, 3.77 mmol) in THF (10 mL) was added to the
mixture at 0 ꢁC and the mixture was cooled to ꢀ78 ꢁC.
A solution of 13 (1.0 g, 3.84 mmol) in THF (40 mL)
was added to the mixture in portions for 20 min. Then
the mixture was quenched by the addition of H2O and
extracted with EtOAc. The extract was washed with
brine, dried (MgSO4), and concentrated.
5.1.3. ( )-2,3-Epoxy-1-butyl p-nitrobenzenesulfonate
(18). To a solution of crotyl alcohol (3.4 g, 47.2 mmol,
E:Z = 15:1) and Ti(Oi-Pr)4 (1.4 mL, 4.72 mmol) in
CH2Cl2 (50 mL) was added TBHP (14 mL of a 3.7 M
solution in CH2Cl2, 51.9 mmol) at ꢀ20 ꢁC in portions
for 30 min. After stirring at ꢀ20 ꢁC for 2 h, P(OCH3)3
(8.9 mL, 75.4 mmol) was added to the mixture. The mix-
ture was warmed to ꢀ10 ꢁC. Then Et3N (9.7 mL,
69.8 mmol), DMAP (0.69 g, 5.7 mmol), followed by a
solution of p-nitrobenzensulfonyl chloride (10.5 g,
47.2 mmol) in CH2Cl2 (70 mL), were added to the mix-
ture. The mixture was filtered through Celite, and the fil-
trate was washed with a 10% solution of tartaric acid in
H2O, satd NaHCO3 aq, brine, dried (Na2SO4), and con-
centrated. The residue was purified by silica gel chroma-
tography (1:5 ! 1:1 EtOAc/hexane) to afford 18 (9.6 g,
75%) as a 15:1 mixture of E and Z isomers, as a yellow
To a solution of the residue in CH3CN (10 mL) was
added a solution of 5% aqueous HF in CH3CN
(10 mL) at rt. After stirring for 3 h, the mixture was
quenched by the addition of a saturated aqueous solu-
tion of NaHCO3 and extracted with EtOAc. The ex-
tract was washed with brine, dried (MgSO4), and
concentrated. The residue was purified by silica gel
chromatography (1:5, EtOAc/hexane) to yield 14
(14.8 mg). 1H NMR (400 MHz, CDCl3) d: 4.59 (1H,
q, J = 6.7 Hz, H-4), 3.79 (1H, s, H-5), 2.09 (1H, m,
H-10), 1.84 (1H, m, H-10), 1.38 (3H, d, J = 6.8 Hz,
4-CH3), 1.38–1.22 (16H, m, H-20–H-110), 0.88 (3H, t,
J = 7.0 Hz, H-120). No further characterization was
attempted on this compound, which was used directly
in the next step.
1
solid. Mp = 83–86 ꢁC; H NMR (500 MHz, CDCl3) d:
8.41 (2H, d, J = 8.5 Hz, Ar), 8.13 (2H, d, J = 8.5 Hz,
Ar), 4.41 (1H, dd, J = 11.4 Hz, 3.20 Hz, H-1), 4.05
(1H, dd, J = 11.4 Hz, 6.3 Hz, H-1), 2.96–2.94 (1H, m,
H-2), 2.90 (1H, qd, J = 5.2 Hz, 1.8 Hz, H-3), 1.32 (3H,
d, J = 5.2 Hz, 3-CH3); IR (CHCl3) cmꢀ1: 3106, 3028,
3001, 1608, 1536, 1380, 1351, 1312, 1291, 1210, 1187,
1096, 1016, 961, 857; HRMS, calcd for C10H12O6NS
(M+H)+ 274.0385. Found 274.0386.
5.1.2. (1R,5R)-1-Dodecyl-4-hydroxy-4-methyl-6-oxa-3-
azabicyclo-[3.1.0]hexan-2-one (15). A solution of 14
(14.6 mg, 0.052 mmol) and NH3 (0.1 mL, a 28% solution
in H2O) in MeOH (2 mL) was stirred at rt for 30 min.
The solvent was removed under reduced pressure.
5.1.4. (1SR,4RS,5RS)-Methyl 2-oxo-3-oxa-bicyclo[3.1.0]-
hexane-1-carboxylate (19). To a solution of Cs2CO3
(6.2 g, 19.0 mmol) and 18-crown-6 (167.1 mg, 0.63 mmol)
in THF (59 mL) was added dimethyl malonate (0.87 mL,
7.6 mmol) at rt. After stirring for 15 min, a solution of
18 (1.7 g, 6.3 mmol) in THF (20 mL) was added to the
mixture in portions for 30 min. The mixture was stirred
for 30 h. Then the mixture was quenched by the
addition of H2O and extracted with EtOAc (3·). The
combined extract was washed with brine, dried (Na2SO4),
and concentrated. The residue was purified by silica
gel chromatography (1:5 ! 1:1 EtOAc/hexane) to afford
19 (690 mg, 64%), 20 (trace), and 21 (433 mg, 34%). 19:
1H NMR (CDCl3, 500 MHz) d: 3.57 (1H, qd, J =
6.3 Hz, 4.6 Hz, H-4), 3.82 (3H, s, COOCH3), 2.73 (1H,
ddd, J = 9.5 Hz, 4.8 Hz, 4.6 Hz, H-5), 1.96 (1H, dd,
To a solution of DMSO (25 lL, 0.35 mmol) in CH2Cl2
(0.5 mL) was added TFAA (25 lL, 0.18 mmol) at
ꢀ78 ꢁC. After 10 min at ꢀ78 ꢁC, a solution of the resi-
due in CH2Cl2 (2 mL)–DMSO (20 lL) was added to
the mixture and the mixture was stirred at ꢀ78 ꢁC for
45 min. Then Et3N (70 lL, 0.50 mmol) was added and
the mixture was stirred at rt for 10 min. The mixture
was quenched by the addition of H2O and extracted
with CH2Cl2. The extract was washed with brine, dried
(MgSO4), and concentrated.

2158
K. Kuramochi et al. / Bioorg. Med. Chem. 14 (2006) 2151–2161
J = 9.5 Hz, 4.6 Hz, H-6), 1.46 (1H, dd, J = 4.8 Hz,
4.6 Hz, H-6), 1.37 (3H, d, J = 6.3 Hz, 4-CH3); 13C
NMR (CDCl3, 100 MHz) d: 171.1, 169.2, 73.6, 56.7,
32.6, 30.9, 17.9, 17.5; IR (CHCl3) cmꢀ1: 3026, 2989,
2956, 1780, 1728, 1441, 1387, 1340, 1320, 1110, 1093,
1051, 1024, 943, 760; HRMS, calcd for C8H10O4 (M)+
170.0579. Found 170.0568.
IR (CHCl3) cmꢀ1: 3030, 3019, 2985, 2948, 2896, 2872,
2825, 1777, 1656, 1528, 1459, 1422, 1391, 1350, 1335,
1290, 1217, 1112, 1092, 1022, 771, 669; HRMS, calcd
for C9H13O4N (M)+ 199.0845. Found 199.0847.
5.1.9. (1RS,2RS,10RS)-N-Methoxy-N-methyl-2-(10-hy-
droxyethyl)cyclopropane-1,1-dicarboxamide (24). To a
solution of 23 (113 mg, 0.57 mmol) in MeOH (2 mL)
was added NH3 (2 mL, 28% solution in H2O), and the
mixture was stirred overnight. Then the solvent was re-
moved, the residue was purified by silica gel chromatog-
raphy (1:50–1:20 EtOH/EtOAc) to yield 24 (109 mg,
89%) as a colorless oil and recovered 23 (11 mg, 10%).
1H NMR (CDCl3, 500 MHz) d: 5.90 (1H, br, NH),
5.65 (1H, br, NH), 3.83 (1H, m, H-10), 3.73 (3H, s,
OCH3), 3.26 (3H, s, NCH3), 1.83 (1H, m, H-2), 1.80
(1H, m, H-3), 1.36 (1H, dd, J = 5.0 Hz, 4.3 Hz, H-3),
1.34 (3H, d, J = 6.4 Hz, H-20); 13C NMR (CDCl3,
125 MHz) d: 170.3, 169.6, 65.1, 61.2, 34.6, 33.4, 32.9,
23.2, 15.6; IR (CHCl3) cmꢀ1: 3685, 3599, 3498, 3413,
3010, 2938, 1766, 1671, 1587, 1458, 1421, 1390, 1335,
1112, 1092, 1048, 1022, 1001, 939; HRMS, calcd for
C9H13O4N (MꢀNH3)+ 199.0845. Found 199.0836.
5.1.5. (1RS,4RS,5SR)-Methyl 2-oxo-3-oxa-bicyclo[3.1.0]-
hexane-1-carboxylate (20). 1H NMR (CDCl3, 500 MHz)
d: 4.45 (1H, q, J = 6.3 Hz, H-4), 3.82 (3H, s, CO2CH3),
2.53 (1H, dd, J = 8.2 Hz, 5.2 Hz, H-5), 2.05 (1H, dt,
J = 8.2 Hz, 5.2 Hz, H-6), 1.42 (1H, dd, J = 5.5 Hz,
5.2 Hz, H-6), 1.37 (1H, d, J = 6.3 Hz, 4-CH3).
5.1.6. Dimethyl 3,4-epoxypentane-1,1-dicarboxylate (21).
1H NMR (CDCl3, 500 MHz) d: 3.77 (6H, s, CO2CH3),
3.57 (1H, dd, J = 5.8 Hz, 3.1 Hz, H-1), 2.79 (1H, m,
H-3), 2.73 (1H, m, H-4), 2.27 (1H, m, H-1), 1.98 (1H,
m, H-1), 1.29 (3H, d, J = 6.8 Hz, H-5); 13C NMR
(CDCl3, 125 Hz) d: 169.2, 169.2, 56.7, 52.6, 52.2, 48.4,
31.2, 17.3; IR (CHCl3) cmꢀ1: 3028, 3007, 2955, 1733,
1438, 1344, 1287, 1159, 1022, 838, 701; HRMS, calcd
for C9H14O5 (M)+ 202.0841. Found 202.0849.
5.1.10.
(1RS,2RS,10RS)-N-Methoxy-N-methyl-2-
5.1.7. (1SR,4RS,5RS)-4-Methyl-2-oxo-3-oxa-bicyclo[3.1.0]-
hexane-1-carboxylic acid (22). To a solution of 19
(144 mg, 0.85 mmol) in methanol (2 mL) was added a
3 M aqueous NaOH solution (1.2 mL, 3.4 mmol) at
0 ꢁC, and the mixture was stirred at 0 ꢁC for 1 h. Then
the solvent was removed, and a 1 M aqueous solution
of HCl was added to the residue. The mixture was
extracted with CHCl3 (10·). The combined organic layer
was dried (Na2SO4) and concentrated. The residue was
purified by silica gel chromatography (1:20 MeOH/
CHCl3) to give 22 (130 mg, 99%) as a white solid.
Mp = 162–164 ꢁC; 1H NMR (CDCl3, 400 MHz) d:
4.92 (1H, qd, J = 6.3 Hz, 4.6 Hz, H-4), 2.99 (1H, ddd,
J = 8.0 Hz, 5.1 Hz, 4.6 Hz, H-5), 1.96 (1H, dd,
J = 8.0 Hz, 4.6 Hz, H-6), 1.63 (1H, dd, J = 5.1 Hz,
4.6 Hz, H-6), 1.45 (1H, d, J = 6.3 Hz, 4-CH3); IR
(CHCl3) cmꢀ1: 3606, 3304, 3027, 2990, 2935, 1772,
1732, 1442, 1400, 1354, 1336, 1130, 1092, 1054, 1019,
944, 907; HRMS, calcd for C7H8O4 (M)+ 156.0423.
Found 156.0424.
{10-(tert-butyldimethylsilyloxy)ethyl}cyclopropane-1,1-
dicarboxamide (25). To a solution of 24 (78 mg,
0.36 mmol) and imidazole (73 mg, 1.1 mmol) in DMF
(5 mL) was added TBSCl (81 mg, 0.54 mmol) at 0 ꢁC.
The mixture was stirred at rt for 3 h. Then the mixture
was quenched by the addition of H2O and extracted
with EtOAc (3·). The combined organic layer was
washed with brine, dried (Na2SO4), and concentrated.
The residue was purified by silica gel chromatography
(1:50 EtOAc/hexane) to yield 25 (86 mg, 72%) as a col-
1
orless oil. H NMR (CDCl3, 500 MHz) d: 3.71 (3H, s,
OCH3), 3.52 (1H, dq, J = 9.2 Hz, 6.1 Hz, H-10), 3.24
(3H, s, NCH3), 1.92 (1H, td, J = 9.2 Hz, 7.6 Hz, H-2),
1.72 (1H, dd, J = 7.6 Hz, 4.8 Hz, H-3), 1.26 (3H, d,
J = 6.1 Hz, H-20), 1.22 (1H, dd, J = 9.2 Hz, 4.8 Hz, H-
3), 0.89 (9H, s, t-Bu), 0.06 (3H, s, SiCH3), 0.05 (3H, s,
SiCH3); 13C NMR (CDCl3, 125 MHz) d: 168.7, 168.7,
67.8, 61.1, 35.1, 33.5, 32.9, 25.8 (3·), 24.0,
18.0,17.7,ꢀ4.4, ꢀ4.6; IR (CHCl3) cmꢀ1: 3512, 3404,
3012, 2957, 2930, 2856, 1686, 1649, 1592, 1471, 1375,
1256, 1117, 1096, 1016, 780; HRMS, calcd for
C15H29O4N2Si (MꢀH)+ 320.1897. Found 329.1895.
5.1.8. (1RS,4RS,5RS)-N-Methoxy-N-methyl-4-methyl-2-
oxo-3-oxa-bicyclo[3.1.0]hexane-1-carboxamide (23). To a
solution of 22 (149 mg, 0.96 mmol), i-Pr2NEt (0.39 mL,
2.3 mmol), and N,O-dimethylhydroxylamine hydrochlo-
ride (186 mg, 1.9 mmol) in CH2Cl2 (5 mL) was added
benzotriazol-1-yloxytris(dimethylamino)phosphonium
hexafluorophosphate (BOP) (634 mg, 1.4 mmol) at 0 ꢁC.
The mixture was stirred at rt for 1 h. Then the mixture
was quenched by the addition of H2O and extracted
with CH2Cl2 (3·). The combined organic layer was
washed with brine, dried (Na2SO4), and concentrated.
The residue was purified by PTLC (2:1 EtOAc/hexane)
5.1.11.
(1SR,2RS,10RS)-2-{100-(tert-butyldimethylsilyl-
oxy)ethyl}-1-pentanoylcyclopropane-1-carboxamide (26).
To a solution of 25 (50 mg, 0.15 mmol) in THF (2 mL)
was added n-BuLi (0.48 mL, 1.6 M solution in hexane,
0.76 mmol) at ꢀ78 ꢁC. The mixture was stirred at
ꢀ78 ꢁC for 5 h. The mixture was quenched by the addi-
tion of saturated NH4Cl aq and extracted with Et2O.
The organic layer was washed with brine, dried
(Na2SO4), and concentrated. The residue was purified
by silica gel chromatography (1:7–2:1 EtOAc/hexane)
to yield 26 (25 mg, 50%) as a colorless oil and recovered
1
to yield 23 (164 mg, 86%) as a colorless oil. H NMR
1
(CDCl3, 400 MHz) d: 4.85 (1H, qd, J = 6.1 Hz, 4.6 Hz,
H-4), 3.73 (3H, s, OCH3), 3.24 (3H, s, NCH3), 2.41
(1H, m, H-5), 1.84 (1H, m, H-6), 1.37 (3H, d,
J = 6.1 Hz, 4-CH3), 1.23 (1H, br t, J = 5.2 Hz, H-6);
25 (14 mg, 29%). H NMR (CDCl3, 500 MHz) d: 8.24
(1H, br s, NH), 5.63 (1H, br s, NH), 3.83 (1H, dq,
J = 8.3 Hz, 6.1 Hz, H-100), 2.26 (2H, t, J = 7.4 Hz, H-
20), 2.08 (1H, dd, J = 8.3 Hz, 4.9 Hz, H-3), 1.76 (1H,

K. Kuramochi et al. / Bioorg. Med. Chem. 14 (2006) 2151–2161
2159
dt, J = 9.2 Hz, 8.3 Hz, H-2), 1.66 (1H, dd, J = 9.2 Hz,
4.9 Hz, H-3), 1.54 (2H, m, H-30), 1.28 (2H, m, H-40),
1.16 (3H, d, J = 6.1 Hz, H-200), 0.89 (3H, s, J = 6.8 Hz,
H-50), 0.89 (9H, s, t-Bu), 0.09 (3H, s, SiCH3), 0.07
(3H, s, SiCH3); 13C NMR (CDCl3, 125 MHz) d: 202.8,
169.3, 67.3, 40.0, 38.6, 36.9, 26.3, 25.9 (3·), 23.8, 22.2,
19.6, 18.1, 13.9,ꢀ4.4, ꢀ4.6; IR (CHCl3) cmꢀ1: 3649,
3487, 3154, 3058, 2996, 2958, 2930, 2857, 1682, 1563,
1465, 1379, 1097, 1005, 903, 836; HRMS, calcd for
C17H33O3NSi (M)+ 327.2230. Found 327.2252.
(2H, m, H-30), 1.57* (2H, m, H-30), 1.53 (1H, m, H-6),
1.50* (1H, m, H-6), 1.52 (3H, s, 4-CH3), 1.32 (2H, m,
H-40), 1.32* (2H, m, H-40), 0.91 (3H, t, J = 7.3 Hz, H-
50), 0.90* (3H, t, J = 7.3 Hz, H-50); IR (CHCl3) cmꢀ1
:
3642, 3568, 3425, 3025, 3007, 2957, 2929, 2858, 1699,
1587, 1458, 1388, 12203, 1124,963, 850; HRMS, calcd
for C11H17NO3 (M+Na)+ 234.1106. Found 234.1123.
5.1.15. (1RS,4RS,5RS)-N-Hexadecyl-N-methyl-4-methyl-
2-oxo-3-oxa-bicylco[3.1.0]hexane-1-carboxamide (30). To
a solution of 22 (84 mg, 0.54 mmol), i-Pr2NEt (0.22 mL,
1.29 mmol), and N-methylhexadecylamine (275 mg,
1.08 mmol) in CH2Cl2 (9 mL) was added benzotriazol-1-
yloxytris(dimethylamino)phosphonium hexafluorophos-
phate (BOP) (357 mg, 0.81 mmol) at 0 ꢁC. The mixture
was stirred at rt for 2 h. Then the mixture was quenched
by the addition of H2O and extracted with CH2Cl2 (3·).
The combined organic layer was washed with brine, dried
(Na2SO4), and concentrated. The residue was purified by
silica gel chromatography (1:1 hexane/EtOAc) to yield 30
(195 mg, 92%) as a 1.7:1 rotational mixture, as a colorless
5.1.12.
(1SR,2RS,10RS)-2-(100-hydroxyethyl)-1-penta-
noylcyclopropane-1-carboxamide (27). To a solution of
26 (24 mg, 73 lmol) in THF (1 mL) was added
3HFÆEt3N (28 mL, 180 lmol). After the mixture was
stirred for 3 days, the solvent was removed. The residue
was purified by silica gel chromatography (1:5 EtOAc/
hexane) to yield 27 (12 mg, 77%) as a colorless oil and
28 (3.3 mg, 23%) as a colorless oil. 27: 1H NMR (CDCl3,
500 MHz) d: 8.19 (1H, br s, NH), 5.78 (1H, br s, NH),
3.92 (1H, quin, J = 6.4 Hz, H-100), 2.29 (2H, m, H-20),
2.14 (1H, dd, J = 7.7 Hz, 5.2 Hz, H-3), 1.81 (1H, ddd,
J = 9.5 Hz, 7.7 Hz, 5.2 Hz, H-2), 1.71 (1H, dd,
J = 9.5 Hz, 5.2 Hz, H-3), 1.55 (2H, m, H-30), 1.33 (2H,
m, H-20), 1.30 (3H, d, 3H, J = 6.4 Hz, H-100), 0.91 (3H,
t, H-50); 13C NMR (CDCl3, 125 MHz) d: 205.5, 164.2,
64.8, 38.5, 37.1, 36.5, 26.5, 23.3, 22.2, 18.4, 13.8; IR
(CHCl3) cmꢀ1: 3649, 3005, 2957, 1682, 1559, 1466,
1382, 1271, 1175, 1096, 993, 822; HRMS, calcd for
C11H16O3 (MꢀNH3)+ 196.1099. Found 196.1095.
1
oil. H NMR (CDCl3, 400 MHz) d: 4.88 (1H, m, H-4),
4.88* (1H, m, H-4), 3.39 (2H, m H-30), 3.39* (2H, m, H-
30), 3.17 (3H, s, NCH3), 2.94 (3H, s, NCH3), 2.63 (1H, m,
H-5), 2.63* (1H, m, H-5), 1.56 (2H, m, H-40), 1.56 (1H,
m, H-6), 1.56* (2H, m, H-40), 1.56* (1H, m, H-6), 1.37
(3H, d, 4-CH3), 1.37* (3H, d, 4-CH3), 1.33 (1H, m, H-6),
1.33* (1H, m, H-6), 1.26 (26H, H-50–H-170), 1.26* (26H,
m, H-50–H-170), 0.88 (3H, t, J = 7.1 Hz, H-180), 0.88*
(3H, t, J = 7.1 Hz, H-180); IR (CHCl3) cmꢀ1: 3028, 2958,
2928, 2854, 1771, 1764, 1718, 1638, 1625, 1494, 1457,
1409, 1385, 1352, 1210, 1110, 1048, 1018, 938; HRMS,
calcd for C24H43NO3 (M)+ 393.3243. Found 393.3242.
5.1.13.
(1SR,4RS,5RS)-4-Methyl-1-pentanoyl-3-oxa-
bicyclo[3.1.0]hexan-2-one (28). 1H NMR (CDCl3,
500 MHz) d: 4.75 (1H, dq, J = 6.1 Hz, 4.9 Hz, H-4),
3.11 (1H, m, H-20), 2.86 (1H, m, H-20), 2.73 (1H, ddd,
J = 7.7 Hz, 5.2 Hz, 4.6 Hz, H-5) 1.92 (1H, dd,
J = 7.7 Hz, 4.6 Hz, H-6), 1.59 (2H, m, H-30), 1.43 (1H,
dd, J = 5.2 Hz, 4.6 Hz, H-6), 1.36 (3H, d, J = 6.1 Hz,
4-CH3), 1.34 (2H, m, H-40), 0.92 (3H, t, J = 7.3 Hz, H-
50); 13C NMR (CDCl3, 100 MHz) d: 203.0, 162.2, 73.8,
41.4, 34.3, 26.8, 25.5, 22.2, 20.7, 17.6, 13.9; IR (CHCl3)
cmꢀ1: 3660, 3591, 3052, 2298, 2957, 1768, 1696, 1552,
1489, 1459, 1302, 1250, 1188, 1112, 1091, 1039, 996,
940, 918, 893, 863, 839; HRMS, calcd for C11H16O3
(M)+ 196.1099. Found 196.1092.
5.1.16. (1RS,2RS)-N-Hexadecyl-N-methyl-2-(10-hydroxy-
ethyl)clopropane-1,1-dicarboxamide (31). A solution of 30
(136 mg, 0.35 mmol) and NH3 (8 mL, 28% solution in
H2O) was stirred at rt overnight. The solvent was
removed, and the residue was purified by silica gel chro-
matography (2:3 EtOAc/hexane and 1:20 EtOH/EtOAc)
to yield 31 (91 mg, 64%) as a 1.7:1 rotational mixture,
1
as a colorless oil, and recovered 30 (44 mg, 32%). H
NMR (CDCl3, 300 MHz) d: 6.19 (1H, br s, NH), 6.19*
(1H, br s, NH), 5.59 (1H, br s, NH), 5.59* (1H, br s,
NH), 3.80 (1H, m, H-10), 3.80* (1H, m, H-10), 3.38
(2H, m, H-300), 3.38* (2H, m, H-300), 3.18 (3H, s,
NCH3), 2.94* (3H, s, NCH3), 1.83* (1H, m, H-3), 1.75
(1H, m, H-3), 1.56 (2H, m, H-400), 1.56 (1H, m, H-3),
1.56* (2H, m, H-400), 1.56* (1H, m, H-3), 1.35 (3H, d,
J = 6.3 Hz, H-20), 1.35* (3H, d, J = 6.3 Hz, H-20), 1.25
(26H, m, H-500-H-1200), 1.25* (26H, m, H-500-H-1200),
0.88 (3H, t, J = 7.2 Hz, H-1800), 0.88* (3H, t, J = 7.2 Hz,
H-1800). No further characterization was attempted
on this compound, which was used directly in the next
step.
5.1.14. (1SR,5RS)-4-Hydroxy-4-Methyl-1-pentanoyl-3-
aza-bicyclo[3.10]hexan-2-one (29). To a solution of 27
(20 mg, 0.12 mmol) in CH2Cl2 (5 mL) was added
Dess–Martin periodinane (149 mg, 0.35 mmol), and
the mixture was stirred at rt for 6 h. The mixture was
quenched by the addition of satd Na2SO3 aq and diluted
with EtOAc. The layers were separated, and the organic
layer was washed with satd NaHCO3 aq, H2O, brine,
dried (Na2SO4), and concentrated. The residue was puri-
fied by silica gel chromatography (1:5–2:1 hexane/
EtOAc) to yield 29 (7.9 mg, 32%) as a 1.2:1 tautomeric
5.1.17. (1RS,2RS)-N-Hexadecyl-N-methyl-2-acetylcyclo-
propane-1,1-dicarboxamide (32). To a solution of 31
(88 mg, 0.21 mmol) in CH2Cl2 (3 mL) was added Dess–
Martin periodinane (271 mg, 0.64 mmol). The mixture
was stirred for 1 h. Then the mixture was quenched by
the addition of satd Na2SO3 aq and diluted with EtOAc.
The layers were separated, and the organic layer was
1
mixture, as a colorless oil, and 28 (15.6 mg, 68%). H
NMR (CDCl3, 400 MHz) d: 5.61 (1H, br s, NH),
5.42* (1H, br s, NH), 3.08 (2H, m, H-20), 2.88* (2H,
m, H-20), 2.56 (1H, m, H-5), 2.51* (1H, m H-5), 1.94
(1H, dd, J = 8.0 Hz, 4.4 Hz, H-6), 1.88* (1H, dd,
J = 8.0 Hz, 4.4 Hz, H-6), 1.66* (3H, s, 4-CH3), 1.57

2160
K. Kuramochi et al. / Bioorg. Med. Chem. 14 (2006) 2151–2161
11. Reed, J. C. Nat. Rev. Drug. Discovery 2002, 1, 111.
12. Rathmell, J. C.; Thompson, C. B. Cell 2002, 109, 97.
13. Reed, J. C. J. Clin. Oncol. 1999, 17, 2941.
14. Reed, C. J. Semin. Hematol. 2000, 37, 9.
15. Robertson, G. S.; Crocker, S. J.; Nicholson, D. W.;
Schulz, J. B. Brain Pathol. 2000, 10, 283.
16. Kakeya, H.; Takahashi, I.; Okada, G.; Isono, K.; Osada,
H. J. Antibiot. 1995, 48, 733.
17. Kakeya, H.; Onozawa, C.; Sato, M.; Arai, K.; Osada, H.
J. Med. Chem. 1997, 40, 391.
18. Watabe, M.; Kakeya, H.; Osada, H. Oncogene 1999, 18,
5211.
19. Watabe, M.; Machida, K.; Osada, H. Cancer Res. 2000,
60, 5214.
20. Hayashi, Y.; Narasaka, K. Chem. Lett. 1998, 313.
21. Hayashi, Y.; Kanayama, J.; Yamaguchi, J.; Shoji, M. J.
Org. Chem. 2002, 67, 9443.
22. Marumoto, S.; Kogen, H.; Naruto, S. J. Org. Chem. 1998,
63, 2068.
washed with satd NaHCO3 aq, H2O, brine, dried
(Na2SO4), and concentrated. The residue was purified
by silica gel chromatography (1:8–1:1 hexane/EtOAc) to
yield 32 (83 mg, 96%) as a 1.1:1 rotational mixture, as a
1
colorless oil. H NMR (CDCl3, 400 MHz) d: 6.25 (1H,
br d, NH), 6.25* (1H, br d, NH), 5.90 (1H, br d, NH),
5.90* (1H, br d, NH), 3.51* (1H, m, H-3), 3.37 (2H, m,
H-300), 3.31* (2H, m, H-300), 3.12 (3H, s, NCH3), 2.91*
(3H, s, NCH3), 2.74 (1H, m, H-2), 2.74* (1H, m, H-2),
2.35 (3H, s, COCH3), 2.35* (3H, s, COCH3), 2.14 (1H,
m, H-3), 1.51 (2H, m, H-400), 1.51* (2H, m, H-400), 1.50*
(1H, m, H-3), 1.41 (1H, dd, J = 8.3 Hz, 4.9 Hz, H-3),
1.25 (26H, m, H-500-H-1700), 1.25* (26H, m, H-500-H-
1700), 0.87 (3H, t, J = 6.8 Hz, H-1800); 13C NMR
(100 MHz, CDCl3) d: 201.9, 201.8, 165.8, 165.7, 48.1,
46.7, 40.2, 39.8, 33.7, 32.8, 32.5, 31.6, 30.2, 29.4, 28.0,
28.0, 27.9, 27.7, 26.2, 25.0, 25.0, 21.0, 16.5, 16.3, 12.4.,
12.4 (rotational mixture); IR (CHCl3) cmꢀ1: 3007, 2927,
2855, 1712, 1692, 1633, 1590, 1489, 1466, 1407, 1386,
1309, 1239, 1176, 1090, 991, 908; HRMS, calcd for
C24H44N2O3 (M)+ 408.3352. Found 408.3350.
23. Marumoto, S.; Kogen, H.; Naruto, S. Tetrahedron 1999,
55, 7129.
24. Marumoto, S.; Kogen, H.; Naruto, S. Tetrahedron 1999,
55, 7145.
25. Kuramochi, K.; Itaya, H.; Nagata, S.; Takao, K.;
Kobayashi, S. Tetrahedron Lett. 1999, 40, 7367.
26. Kuramochi, K.; Nagata, S.; Itaya, H.; Takao, K.;
Kobayashi, S. Tetrahedron Lett. 1999, 40, 7371.
27. Kuramochi, K.; Nagata, S.; Itaya, H.; Matsubara, Y.;
Sunoki, T.; Uchiro, H.; Takao, K.; Kobayashi, S. Tetra-
hedron 2003, 59, 9743.
28. Mizushina, Y.; Kobayashi, S.; Kuramochi, K.; Nagata, S.;
Sugawara, F.; Sakaguchi, K. Biochem. Biophys. Res.
Commun. 2000, 273, 784.
29. Nakai, J.; Kawada, K.; Nagata, S.; Kuramochi, K.;
Uchiro, H.; Kobayashi, S.; Ikekita, M. Biochim. Biophys.
Acta 2002, 1581, 1.
30. Kuramochi, K.; Mizushina, Y.; Nagata, S.; Sugawara, F.;
Sakaguchi, K.; Kobayashi, S. Bioorg. Med. Chem. 2004,
12, 1983.
31. 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.
32. Kemnitzer, W.; Drewe, J.; Jiang, S.; Zhang, H.; Wang,
Y.; Zha, J.; Jia, S.; Herich, J.; Labreque, D.; Storer,
R.; Meerovitch, K.; Bouffard, D.; Rej, R.; Denis, R.;
Blais, C.; Lamothe, S.; Attardo, G.; Gourdeau, H.;
Tseng, B.; Kasibhatla, S.; Cai, S. X. J. Med. Chem.
2004, 47, 6299.
5.2. Detection of cell death by MTT assay
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 epolactaene or its deriv-
atives. After incubation at 37 ꢁC for 24 h, cell viability
was determined by the MTT assay, a method for deter-
mining cell viability by measuring the mitochondrial
dehydrogenase action. In this assay, 11 lL 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
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.
References and notes
33. Nagumo, Y.; Kakeya, H.; Yamaguchi, J.; Uno, T.; Shoji,
M.; Hayashi, Y.; Osada, H. Bioorg. Med. Chem. Lett.
2004, 14, 4425.
34. Nagumo, Y.; Kakeya, H.; Shoji, M.; Hayashi, Y.;
Dhomae, N.; Osada, H. Biochem. J. 2005, 387, 835.
35. Temnikova, T. I.; Semenova, S. N. USSR J. Org. Chem.
Engl. Trans. 1965, 1, 2076.
36. Klunder, J. M.; Onami, T.; Sharpless, K. B. J. Org. Chem.
1989, 54, 1295.
37. Gao, Y.; Hanson, R. M.; Klunder, J. M.; Ko, S. Y.;
Masamune, H.; Sharpless, K. B. J. Am. Chem. Soc. 1987,
109, 5765.
38. Kitaori, K.; Mikami, M.; Furukawa, Y.; Yoshimoto, H.;
Otera, J. Synlett 1988, 499.
39. Meyers, A. I.; Romine, J.; Fleming, S. A. J. Am. Chem.
Soc. 1988, 110, 7245.
40. Nahm, S.; Weinreb, S. M. Tetrahedron Lett. 1981, 22,
3815.
1. Kerr, J. F.; Wyllie, A. H.; Currie, A. R. Br. J. Cancer
1972, 26, 239.
2. Wyllie, A. H.; Kerr, J. F.; Currie, A. R. Int. Rev. Cytol.
1980, 68, 251.
3. Lin, C.-F.; Yan, J.-S.; Chang, C.-Y.; Kuo, S.-C.; Lee, M.-
R.; Huang, L.-J. Bioorg. Med. Chem. 2005, 13, 1537.
4. OꢀDriscoll, L.; Linehan, R.; Clynes, M. Curr. Cancer Drug
Target 2003, 3, 131.
5. Denault, J. B.; Salvesen, G. S. Chem. Rev. 2002, 102, 4489.
6. Thornberry, N. A. Chem. Biol. 1998, 5, R97.
7. Cai, S. X.; Guan, L.; Jia, S.; Wang, Y.; Yang, W.;
Tseng, B.; Drewe, J. Bioorg. Med. Chem. Lett. 2004, 14,
5295.
8. Sun, H.; Nikolovska-Coleska, Z.; Yang, C.-Y.; Xu, L.;
Tomita, Y.; Krajewski, K.; Roller, P. P.; Wang, S. J. Med.
Chem. 2004, 47, 4147.
9. Thomoson, C. B. Science 1995, 267, 1456.
10. Nicholson, D. W. Nature 2000, 407, 810.
41. Dess, D. B.; Martin, J. C. J. Am. Chem. Soc. 1991, 113,
7277.

K. Kuramochi et al. / Bioorg. Med. Chem. 14 (2006) 2151–2161
2161
42. Tanatani, A.; Yamaguchi, K.; Azumaya, I.; Fukutomi, R.;
Shudo,K.;Kagechika,H.J.Am.Chem.Soc.1998,120,6433.
43. Alley, M. C.; Scudiero, D. A.; Monks, A.; Hursey, M. L.;
Czerwinski, M. J.; Fine, D. L.; Abbott, B. J.; Mayo, J. C.;
Schoemaker, R. H.; Boyd, M. R. Cancer Res. 1988, 48, 589.
44. Pouillart, P.; Douillet, O.; Scappini, B.; Villa, P. Eur. J.
Pharmacol. Sci. 1998, 7, 93.
45. Kaufmann, S. H. Cancer Res. 1989, 49, 5870.
46. Mizushina, Y.; Miyazaki, S.; Ohta, K.; Hirota, M.;
Sakaguchi, K. Biochim. Biophys. Acta 2000, 1475, 1.
47. Mizushina, Y.; Hirota, M.; Murakami, C.; Ishidoh, T.;
Kamisuki, S.; Shimazaki, N.; Takemura, M.; Perelescu,
M.; Suzuki, M.; Yoshida, H.; Sugawara, F.; Koiwai, O.;
Sakaguchi, K. Biochem. Pharmacol. 2003, 66, 1935.