Synthesis and biological evaluation of the 12,12-dimethyl derivative of aplog-1, an anti-proliferative analog of tumor-promoting aplysiatoxin

Article abstract of DOI:10.1271/bbb.110130

Aplog-1 is a unique analog of tumor-promoting aplysiatoxin that inhibits tumor-promotion by phorbol diesters and proliferation of tumor cells. While the structural features relevant to the biological activities of Aplog-1 remain to be identified, recent studies by us have suggested that local hydrophobicity around the spiroketal moiety of Aplog-1 is a crucial determinant of its anti-proliferative activity. This hypothesis led us to design 12,12-dimethyl-Aplog-1 (3), in which a hydrophobic geminal dimethyl group is installed proximal to the spiroketal moiety to improve biological potency. As expected, 3 was more effective than Aplog-1 in inhibiting cancer cell growth and binding to protein kinase Cδ, a putative receptor responsible for the biological response of Aplog-1. Moreover, an induction test on Epstein-Barr virus early antigen demonstrated 3 to be a better anti-tumor promoter than Aplog-1. These results indicate that 3 is a superior derivative of Aplog-1, and thus a more promising lead for anti-cancer drugs.

Full text of DOI:10.1271/bbb.110130

Biosci. Biotechnol. Biochem., 75 (6), 1167–1173, 2011
Synthesis and Biological Evaluation of the 12,12-Dimethyl Derivative of Aplog-1,
an Anti-Proliferative Analog of Tumor-Promoting Aplysiatoxin
Yu NAKAGAWA,1;2 Masayuki KIKUMORI,1 Ryo C. YANAGITA,1;3 Akira MURAKAMI,1
y
1;
Harukuni TOKUDA,4 Hiroshi NAGAI,5 and Kazuhiro IRIE
1Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University,
Kyoto 606-8502, Japan
2Synthetic Cellular Chemistry Laboratory, Advanced Science Institute,
RIKEN (The Institute of Physical and Chemical Research), Wako 351-0198, Japan
3Department of Applied Biological Science, Faculty of Agriculture, Kagawa University,
Kagawa 761-0195, Japan
4Graduate School of Medical Science, Kanazawa University, Kanazawa 920-8640, Japan
5Tokyo University of Marine Science and Technology, Minato-ku, Tokyo 108-8477, Japan
Received February 16, 2011; Accepted February 28, 2011; Online Publication, June 13, 2011
[doi:10.1271/bbb.110130]
Aplog-1 is a unique analog of tumor-promoting
aplysiatoxin that inhibits tumor-promotion by phorbol
diesters and proliferation of tumor cells. While the
structural features relevant to the biological activities of
Aplog-1 remain to be identified, recent studies by us
have suggested that local hydrophobicity around the
spiroketal moiety of Aplog-1 is a crucial determinant of
its anti-proliferative activity. This hypothesis led us to
design 12,12-dimethyl-Aplog-1 (3), in which a hydro-
phobic geminal dimethyl group is installed proximal to
the spiroketal moiety to improve biological potency. As
expected, 3 was more effective than Aplog-1 in inhibit-
ing cancer cell growth and binding to protein kinase Cꢀ,
a putative receptor responsible for the biological
response of Aplog-1. Moreover, an induction test on
Epstein-Barr virus early antigen demonstrated 3 to be a
better anti-tumor promoter than Aplog-1. These results
indicate that 3 is a superior derivative of Aplog-1, and
thus a more promising lead for anti-cancer drugs.
PKC, discrepancies exist in ATX-related compounds.
An example is debromo-ATX, which retains affinity for
PKC but shows reduced tumor-promoting activity as
compared to ATX.8,9) This observation has been inter-
preted to result from decreased hydrophobicity due to
the loss of a bromine atom, suggesting that the tumor-
promoting activity of ATX is highly dependent on
hydrophobicity while its binding to PKC is not.
On the basis of this finding, we recently developed a
simple and less hydrophobic analog of ATX (Aplog-1,10)
Fig. 2) as a new PKC activator with much reduced
tumor-promoting activity, like bryostatin 1 (Bryo-1,
Fig. 1), a potent PKC activator without tumor-promot-
ing activity isolated from the marine bryozoan Bugula
neritina.11–13) Aplog-1 inhibited tumor-promotion by
TPA and proliferation of human cancer cell lines despite
retaining the skeleton of tumor-promoting ATX. In
subsequent efforts to identify the structural features
relevant to the unique biological profile of Aplog-1, we
synthesized 18-deoxy derivatives of Aplog-1 (1, 2).10,14)
Biological evaluation of them revealed the importance
of the geminal dimethyl group at position 6 for anti-
proliferative activity and PKC binding of Aplog-1. This
finding suggests that the spiroketal moiety of Aplog-1 is
involved in the hydrophobic interaction with PKC,
suggesting the possibility of enhancing the biological
potency of Aplog-1 by increasing local hydrophobicity
around the spiroketal moiety. This led us to design
12,12-dimethyl-Aplog-1 (3, Fig. 2), in which a hydro-
phobic geminal dimethyl group is installed proximal to
the spiroketal moiety. In this paper, we report the
synthesis, PKC binding, anti-proliferative activity, and
tumor-promoting activity of 3, which proved to be
superior to Aplog-1 in three biological assays, suggest-
ing that it is a promising lead for anti-cancer drugs.
Key words: Aplog-1; aplysiatoxin; bryostatin; protein
kinase C; tumor promoter
Aplysiatoxin (ATX, Fig. 1) is a polyacetate-type
tumor promoter isolated from the digestive gland of
the sea hare Stylocheilus longicauda.1) ATX exhibited
tumor-promoting activity in vivo comparable to other
naturally-occurring tumor promoters, such as 12-O-
tetradecanoylphorbol 13-acetate (TPA) and teleocidin
B-4, in the two-stage mouse skin carcinogenesis
system.2) The mode of action of these tumor promoters
has been proposed to arise from activation of protein
kinase C (PKC).3,4) PKC is a family of serine/threonine
kinases that play crucial roles in cellular signal trans-
duction, including proliferation, differentiation, and
apoptosis.5,6) ATX, TPA, and teleocidin B-4 bind
competitively to the regulatory domain of PKC despite
the differences in their chemical structures (Fig. 1).7)
While the tumor-promoting activity of phorbol diesters
and teleocidins correlates well with their affinity for
Results and Discussion
12,12-Dimethyl-Aplog-1 (3) was synthesized from
known bromide 4,15) as shown in Scheme 1. Substitution
y
To whom correspondence should be addressed. Tel: +81-75-753-6281; Fax: +81-75-753-6284; E-mail: irie@kais.kyoto-u.ac.jp

1168
Y. NAKAGAWA et al.
HO
OAc
O
H3CO2C
O
O
O
H
N
H
OCO(CH2)12CH3
O
O
N
OH
OAc
OH HO
O
O
O
OMe
Br
H
OH
O
O
O
H
H
H
O
OH
N
H
OH
CO2CH3
O
OH
O
OH
OH
HO
TPA
Teleocidin B-4
Bryo-1
ATX
Fig. 1. Structures of Aplysiatoxin (ATX), 12-O-Tetradecanoylphorbol 13-acetate (TPA), Teleocidin B-4, and Bryostatin 1 (Bryo-1).
acid 14,10) followed by switching of the protecting group
R
R
H
H
6
from the 4-methoxyphenylmethyl (MPM) group to a
triethylsilyl (TES) group, provided mono-ester 12. After
oxidative cleavage of the olefin group and deprotection
of the TES group, lactonization was performed by the
Yamaguchi method.20) Final deprotection of the two
benzyl groups provided target compound 3 in 22 steps,
with an overall yield of 2.4%.
Compound 3 was initially evaluated for its ability to
bind PKCꢀ. Several lines of evidence indicate that PKCꢀ
is an important isozyme responsible for the anti-cancer
actions of Bryo-1.21,22,24) We found previously that
Aplog-1 displayed binding and activation profiles for
PKCꢀ similar to those of Bryo-1, and that its anti-
proliferative activity was closely correlated with its
affinity for PKCꢀ.10,14) The affinity of 3 for PKCꢀ was
O
O
12
O
O
H
O
H
O
O
O
O
O
H
O
H
O
OH
X 18
OH
HO
Aplog-1 : R = Me, X = OH
12,12-Dimethyl-Aplog-1 (3)
1
2
: R = X = H
: R = Me, X = H
Fig. 2. Structures of Aplog-1, Its 18-Deoxy-derivatives (1, 2), and
12,12-Dimethyl-Aplog-1 (3).
with ethyl isobutyrate and conversion of the resulting
ester to an aldehyde provided 5. Asymmetric Brown’s
allylation16) of 5 produced an (S)-homoallyl alcohol with
an ee of 67% (Supplemental Fig. 1; see Biosci.
Biotechnol. Biochem. Web site). Attempts to improve
the enantioselectivity of the homoallyl alcohol employ-
ing Keck’s17) and Maruoka’s18) allylations resulted in no
reaction, probably due to steric crowding at the aldehyde
group. Stereoselective iodocarbonate cyclization19) of
the homoallyl alcohol generated a cyclic carbonate,
which was converted to epoxide 6 by methanolysis. Due
to steric hindrance by the geminal dimethyl group, the
methyl carbonate of 6 was less susceptible to meth-
anolysis with the use of K2CO3 in MeOH at room
temperature. Attempts to cleave the methyl carbonate by
methanolysis under reflux conditions or by hydrolysis
using aqueous NaOH in MeOH resulted in the decom-
position of 6. Hence, 6 was used for the coupling
reaction with dithiane 13,10) though the carbonate group
is generally sensitive to strong basic conditions. As
expected, the coupling reaction produced several prod-
ucts derived from side reactions at the carbonate site.
Since most of these by-products were anticipated to
contain ester or carbonate moieties, reduction with
LiAlH4 was performed to convert them into a single
diol. After O-isopropylidenation of the diol followed by
desilylation, 7 was successfully obtained in 69% yield
in 4 steps from 6. Oxidation of the alcohol of 7,
followed by Maruoka’s asymmetric allylation18) and
acid hydrolysis of the isopropylidene acetal, produced
an inseparable mixture of homoallyl alcohol 8 and its
diastereomer. Hydrolysis of the dithiane moiety of the
measured in
a competition binding assay with
[3H]phorbol 12,13-dibutyrate (PDBu).25) Compound 3
exhibited strong affinity for PKCꢀ (Ki ¼ 5:9 nM), about
2.5 times higher than that of Aplog-1 (Ki ¼ 15 nM). This
increase as compared to Aplog-1 indicates that as
expected, the geminal dimethyl group at position 12 of 3
makes a positive contribution to hydrophobic interaction
with PKCꢀ.
Encouraged by this result, we evaluated the anti-
proliferative activities of 3 against a panel of 39 human
cancer cell lines, as described previously.26) Growth
inhibitory activity was expressed as the concentration
required to inhibit cell growth by 50% as compared to an
untreated control, GI50 (M). The results for the cell lines,
in which Aplog-1 showed significant activities with GI50
values of less than 10ꢀ5 M, are listed in Table 1 (the rest
are provided in Supplemental Table 1). As expected
based on the results for PKCꢀ binding, 3 was more
potent than Aplog-1 in most cancer cell lines. The mean-
graph midpoint (MG-MID) values for Aplog-1 and 3
were ꢀ4:98 and ꢀ5:12 respectively. This correlation
between PKCꢀ binding and anti-proliferative activities
of 3 further confirms that PKCꢀ is the receptor
responsible for the anti-proliferative activity of Aplog-
related compounds.
The most critical point in developing derivatives of
Aplog-1 is to confirm that the structural modifications
can be made without increasing tumor-promoting
activity, since Aplog-1 possesses the skeleton of
tumor-promoting ATX. Especially in the case of 3,
attachment of the geminal dimethyl group resulted in
increased hydrophobicity, which is prone to enhance
the tumor-promoting activities of ATX-related com-
pounds.8,9) Hence, we evaluated the tumor-promoting
and its inhibiting activities of 3 by testing to determine
.
diastereomeric mixture with Hg(ClO4)2 6H2O produced
three spiroketals (9, 10, 11), whose configurations were
determined by nuclear Overhauser enhancement (NOE),
as shown in Supplemental Fig. 2. Yamaguchi esterifi-
cation20) of desired spiroketal 9 with known carboxylic

Superior Anti-Proliferative Analog of Aplysiatoxin
1169
O
O
Br
a
b
OHC
MeO
O
OBn
+ Enantiomer
OBn
S
OBn
4
5
6
OH
S
S
S
OH
c
d
e
HO
O
HO
O
OBn
OBn
7
8
+ Enantiomer
+ Diastereomer
OH
O
O
O
O
O
O
+
+
OH
BnO
OH
BnO
11
9
10
OBn
O
O
O
O
g
f
TESO
OBn
O
O
O
O
H
O
H
O
BnO
OH
HO
3
12
S
BnO
CO2H
OMPM
TIPSO
S
13
14
Scheme 1. Synthesis of 3.
.
(a) (1) Ethyl isobutyrate, LDA, THF, HMPA; (2) LiBH4, THF; (3) SO3 Pyr, NEt3, DMSO, CH2Cl2; 88% in 3 steps. (b) (1) AllylMgBr,
(ꢀ)-Ipc2BOMe, THF, 67% ee; (2) (Boc)2O, NaHMDS, THF; (3) IBr, CH2Cl2, Toluene; (4) K2CO3, MeOH; 72% in 4 steps. (c) (1) 13, n-BuLi,
.
THF; (2) LiAlH4, THF; (3) 2,2-Dimethoxypropane, CSA, CH2Cl2; (4) TBAF, THF; 69% in 4 steps. (d) (1) SO3 Pyr, NEt3, DMSO, CH2Cl2;
(2) TiCl4, Ti(OiPr)4, (S)-BINOL, Ag2O, AllylSnBu3, CH2Cl2; (3) TsOH H2O, H2O, CH3CN, THF; 63% in 3 steps. (e) Hg(ClO4)2 6H2O,
.
.
MeCN, CH2Cl2, H2O; 42% for 9, 22% for 10, 18% for 11. (f) (1) 14, TCBCl, NEt3, DMAP, Toluene; (2) DDQ, H2O, CH2Cl2; (3) TESCl,
.
Imidazole, THF; 50% in 3 steps. (g) (1) KMnO4, NaIO4, t-BuOH, pH 7 buffer; (2) HF Pyr, Pyridine, THF; (3) TCBCl, NEt3, DMAP, Toluene;
(4) H2, 10% Pd-C, MeOH, CHCl3, 41% in 4 steps
Table 1. Log GI50 Values for Aplog-1 and 3 against a Subset of 39
Human Cancer Cell Lines
activity, Aplog-1 has been found to exhibit weak EA-
induction by itself (Fig. 3) and suppress markedly the
production of EA induced by TPA (Fig. 4).10) Only
log GI50 (log M)
Cancer type
Cell line
HBC-4
weak EA-induction (9.0% and 7.1% at 10ꢀ6 and 10ꢀ7
M
Aplog-1a
3
respectively) and significant suppression of TPA-in-
duced EA production (5.0% and 7.2% at 10ꢀ6 and
10ꢀ7 M respectively) were observed, suggesting that 3 is
a better anti-tumor promoter than Aplog-1. Although our
previous study using 2 found no correlation between
inhibition of TPA-induced EA production and suppres-
sion of proliferation of tumor cells,14) introduction of the
geminal dimethyl group into position 12 of Aplog-1 was
found to enhance both activities.
Breast
Breast
CNS
ꢀ6:33
ꢀ5:61
ꢀ5:06
ꢀ5:43
ꢀ5:60
ꢀ5:32
ꢀ5:74
ꢀ5:55
ꢀ5:33
ꢀ6:67
ꢀ5:92
ꢀ5:32
ꢀ6:06
ꢀ6:05
ꢀ5:51
ꢀ6:06
ꢀ6:20
ꢀ5:33
MDA-MB-231
SF-295
Colon
Lung
Lung
HCC2998
NCI-H460
A549
Melanoma
Stomach
Stomach
LOX-IMVI
St-4
MKN45
In conclusion, 12,12-dimethyl-Aplog-1 (3) was de-
veloped as a superior derivative of Aplog-1. Three
biological assays demonstrated that 3 was more effective
than Aplog-1 in binding to PKCꢀ, inhibiting cancer cell
growth and suppressing TPA-induced EA production.
These results collectively indicate that 3 is a more
promising lead for anti-cancer drugs, and that the
introduction of a hydrophobic group at pertinent
positions of Aplog-1 can enhance anti-proliferative
activity without increasing tumor-promoting activity.
aCited from reference 11.
whether Epstein-Barr virus early antigen (EBV-EA)
would be induced in Raji cells.27) EBV is activated by
treatment with tumor promoters to produce EA, which
can be detected by an indirect immunofluorescence
technique.28,29) EBV-EA-inducing activity is expressed
as the percentage of EA-positive cells (Figs. 3 and 4).
Compared to ATX which has significant EA-inducing

1170
Y. NAKAGAWA et al.
ꢀ78 ꢂC for 5 min, the mixture was warmed to 0 ꢂC, and stirring was
continued for 10 min. The resulting lithium diisopropylamide (LDA)
solution was recooled to ꢀ78 ꢂC, and ethyl isobutyrate (2.9 mL,
21.8 mmol, 2.0 equiv.) was slowly added. The mixture was stirred at
ꢀ78 ꢂC for 20 min, and then a solution of 412) (3.30 g, 10.9 mmol) and
hexamethylphosphoramide (HMPA, 2.0 mL) in THF (5 mL) was
added. The reaction mixture was stirred at ꢀ78 ꢂC for 4 h, and stirring
was continued in a cold room for 15 h. The reaction was quenched
with saturated aq. NH4Cl (50 mL), and the mixture was extracted with
EtOAc (50 mL ꢁ 3). The combined organic layers were washed with
brine, dried over Na2SO4, filtered, and concentrated in vacuo. The
residue was purified by column chromatography (silica gel, 1% ! 3%
EtOAc/hexane) to afford the crude ester (4.54 g). To a solution of the
ester (4.54 g) in THF (24 mL) was added LiBH4 (650 mg, 29.8 mmol)
at rt. The reaction mixture was stirred at rt for 18 h, and the reaction
was quenched with 0.5 N HCl (20 mL). The resulting mixture was
extracted with EtOAc (100 mL ꢁ 3). The combined organic layers
were washed with brine (100 mL), dried over Na2SO4, filtered, and
concentrated in vacuo. The residue was purified by column chroma-
tography (silica gel, 5% ! 10% EtOAc/hexane) to afford the alcohol
(3.74 g). To a solution of the alcohol (3.74 g) and NEt3 (7.0 mL,
50.4 mmol) in CH2Cl2 (150 mL) were added DMSO (27 mL,
30
20
10
0
Concentration (M) 10-7 10-6
10-7 10-6
ATX
10-7 10-6
Aplog-1
10-7 10-6
of compound
TPA
3
Fig. 3. EBV-EA Production Induced by TPA, ATX, Aplog-1, and 3.
Percentages of EA-positive cells are shown. Sodium n-butyrate
(4 mM) was added to all samples to enhance the sensitivity of the
Raji cells. Only 0.1% of the cells were positive for EA at 4 mM of
sodium n-butyrate. The final concentration of DMSO was 0.4%. Cell
viability exceeded 50% in all experiments, except for ATX at 10ꢀ6
(40%). Error bars represent standard error of the mean (n ¼ 3).
M
.
378 mmol) and SO3 pyridine (6.0 g, 37.8 mmol) at rt. The mixture
20
15
10
5
was stirred at rt for 18 h, and the reaction was quenched with saturated
aq. NH4Cl (100 mL). The organic layer was separated and the aqueous
layer was extracted with CH2Cl2 (100 mL ꢁ 2). The combined
organic layers were dried over Na2SO4 and concentrated in vacuo.
The residue was purified by column chromatography (silica gel, 3%
EtOAc/hexane) to afford 5 (2.85 g, 9.63 mmol, 88% in 3 steps) as a
clear oil. 1H NMR: (400 MHz, CDCl3, 0.067 M) ꢀ 1.02 (6H, s), 1.47–
1.56 (4H, m), 2.57 (2H, t, J ¼ 7:1 Hz), 5.05 (2H, s), 6.76–6.82 (3H,
m), 7.19 (1H, dt, J ¼ 7:4, 0.7 Hz), 7.32–7.45 (5H, m), 9.42 (1H, s)
ppm. 13C NMR: (100 MHz, CDCl3, 0.067 M) ꢀ 21.30 (2C), 25.93,
36.35, 36.73, 45.75, 69.93, 112.02, 115.17, 121.06, 127.51 (2C),
127.92, 128.57 (2C), 129.34, 137.13, 143.61, 158.90, 206.26 ppm.
HR-EIMS: m=z ([Mþ]): Calcd. for C20H24O2: 296.1776, Found:
296.1764.
0
Concentration (M) 10-7 10-6
10-7 10-6
Aplog-1
10-7 10-6
of compound
ATX
3
Fig. 4. Inhibition of TPA-Induced EBV-EA Production by ATX,
Aplog-1, and 3.
Synthesis of 6. To a solution of (ꢀ)-B-methoxydiisopinocampheyl-
borane (Ipc2BOMe, 3.29 g, 10.4 mmol, 2.2 equiv.) in THF (15 mL)
was added 1 M allylMgBr in ether (9.5 mL, 9.46 mmol, 2.0 equiv.) at
0 ꢂC. After stirring at rt for 1 h, the mixture was cooled to ꢀ78 ꢂC.
Aldehyde 5 (1.40 g, 4.73 mmol) in THF (15 mL) was added dropwise
at ꢀ85 ꢂC (dry ice/ether). After stirring at the same temperature for
2 h, the reaction was quenched with MeOH (5 mL), saturated aq.
NaHCO3 (7 mL), and 30% H2O2 (7 mL). The resulting mixture was
stirred at rt for 18 h, and then extracted with EtOAc (50 mL ꢁ 3). The
combined organic layers were dried over Na2SO4, filtered, and
concentrated in vacuo. The residue was purified by column chroma-
tography (silica gel, 1% ! 3% EtOAc/hexane; ODS, 70% ! 80%
MeOH) to afford the homoallyl alcohol (1.75 g, 67% ee, which was
determined by Mosher analysis as shown in Supplemental Fig. 1). To
a solution of the homoallyl alcohol (1.75 g) in THF (13 mL) was
added 1 M sodium hexamethyldisilazane (NaHMDS) in THF (6.7 mL,
6.7 mmol) dropwise at 0 ꢂC. After stirring for 30 min, (Boc)2O (1.47 g,
6.73 mmol) was added, and the reaction mixture was stirred for 15 min
at rt. The reaction was quenched with brine (25 mL) and water
(10 mL), and the mixture was extracted with EtOAc (50 mL ꢁ 3). The
combined organic layers were washed with brine, dried over Na2SO4,
filtered, and concentrated in vacuo. The residue was purified by
column chromatography (silica gel, 0:5% ! 1% EtOAc/hexane) to
afford the carbonate (2.31 g). To a solution of the carbonate (2.31 g) in
toluene (25 mL) was added IBr (1.21 g, 5.83 mmol) in CH2Cl2
(7.8 mL) at ꢀ78 ꢂC. After stirring for 2.5 h at ꢀ78 ꢂC, the reaction
was quenched with a 1:1 mixture of 10% Na2S2O3 and saturated aq.
NaHCO3 (80 mL). The mixture was extracted with EtOAc
(100 mL ꢁ 3). The combined organic layers were washed with brine,
dried over Na2SO4, filtered, and concentrated in vacuo. The residue
was purified by column chromatography (silica gel, 10% ! 15%
EtOAc/hexane) to afford the iodo carbonate (2.28 g). To a solution of
the iodo carbonate (2.28 g) in anhydrous MeOH (25 mL) was added
K2CO3 (1.84 g, 13.4 mmol). After stirring for 3 h at rt, the reaction
was quenched with H2O (60 mL). The mixture was extracted with
Percentages of EA-positive cells in the presence of TPA
(10ꢀ7:5 M) are shown. At 10ꢀ7:5 M of TPA, 30 ꢃ 0:6% of cells were
positive for EA. Sodium n-butyrate (4 mM) was added to all samples
to enhance the sensitivity of the Raji cells. Only 0.1% of the cells
were positive for EA at 4 mM of sodium n-butyrate. The final
concentration of DMSO was 0.4%. Cell viability exceeded 50% in
all experiments, except for ATX at 10ꢀ6 M (40%). Error bars
represent standard error of the mean (n ¼ 3).
Efforts to identify additional superior derivatives of
Aplog-1 are underway.
Experimental
General remarks. The following spectroscopic and analytical
instruments were used: UV, UV-2200A (Shimadzu, Kyoto, Japan);
Digital Polarimeter, DIP-1000 (Jasco, Tokyo, Japan); 1H NMR,
AVANCE I 400 and AVANCE III 500 (ref. TMS, 296 K, Bruker,
Germany); HPLC, Model 600E with a Model 2487 UV detector
(Waters, Tokyo, Japan); (HR) EI-MS, JMS-600H (JEOL, Tokyo,
Japan). HPLC was carried out on a YMC packed A-023 (silica gel,
10 mm i.d. ꢁ 250 mm) column (Yamamura Chemical Laboratory,
Kyoto, Japan). Wako gel C-200 (silica gel, Wako Pure Chemical
Industries, Osaka, Japan) and YMC A60-350/250 gel (ODS,
Yamamura Chemical Laboratory, Kyoto, Japan) were used for column
chromatography. [3H]PDBu (16.3 Ci/mmol) was purchased from
PerkinElmer Life Sciences Research Products (Boston, MA, USA).
All other chemicals and reagents were purchased from chemical
companies, and were used without further purification.
Synthesis of 5. To a solution of diisopropylamine (3.4 mL,
26.2 mmol, 2.4 equiv.) in THF (30 mL) was added 1.6 M n-BuLi in
hexane (15 mL, 24.0 mmol, 2.2 equiv.) at ꢀ78 ꢂC. After stirring at

Superior Anti-Proliferative Analog of Aplysiatoxin
1171
EtOAc (100 mL ꢁ 3). The combined organic layers were washed with
brine, dried over Na2SO4, filtered, and concentrated in vacuo. The
residue was purified by column chromatography (silica gel, 10% !
12:5% EtOAc/hexane) to afford 6 (1.40 g, 3.40 mmol, 72% in 4 steps)
as a clear oil. 1H NMR: (400 MHz, CDCl3, 0.049 M) ꢀ 0.88 (3H, s),
0.90 (3H, s), 1.24–1.39 (2H, m), 1.52–1.76 (4H, m), 2.42 (1H, dd,
J ¼ 4:9, 2.7 Hz), 2.54 (2H, m), 2.73 (1H, dd, J ¼ 4:9, 4.1 Hz), 2.97
(1H, m), 3.79 (3H, s), 4.79 (1H, dd, J ¼ 8:3, 4.5 Hz), 5.03 (2H, s),
6.76–6.82 (3H, m), 7.19 (1H, m), 7.30–7.45 (5H, m) ppm. 13C NMR:
(100 MHz, CDCl3, 0.049 M) ꢀ 22.84, 23.05, 25.46, 33.05, 36.65, 37.05,
38.16, 46.33, 50.31, 54.83, 69.91, 82.79, 111.89, 115.12, 121.08,
127.52 (2C), 127.91, 128.56 (2C), 129.28, 137.16, 144.01, 156.06,
158.88 ppm. HR-EIMS: m=z ([Mþ]): Calcd. for C25H32O5: 412.2250,
Found: 412.2272.
hexane) to afford the aldehyde (336 mg). One M TiCl4 in CH2Cl2
(78 mL, 78 mmol) was diluted with CH2Cl2 (1.25 mL) and cooled to
0 ꢂC. To the TiCl4 solution was added Ti(Oi-Pr)4 (70 mL, 234 mmol) at
0 ꢂC. The mixture was warmed to rt and stirred for 1 h. Ag2O (36 mg,
156 mmol) was added in one portion, and stirring was continued with
exclusion of direct light for 5 h. (S)-1,10-bi(2-naphthol) (BINOL,
89 mg, 312 mmol) was then added in one portion. After stirring for a
further 2 h, the mixture was diluted with CH2Cl2 (1.25 mL) to afford
the stock solution (about 40 mM) of Ti catalyst. To the aldehyde
(320 mg) was added the supernatant (2.6 mL) of the above stock
solution at ꢀ15 ꢂC. After stirring at the same temperature for 15 min,
allyl-SnBu3 (324 mL, 1.05 mmol) was added. The resulting reaction
mixture was stirred in a cold room for 20 h. The reaction was quenched
with saturated aq. NaHCO3 (10 mL), and poured into EtOAc (30 mL)
and H2O (10 mL). The organic layer was separated, and the aqueous
layer was extracted with EtOAc (30 mL ꢁ 2). The combined organic
layers were washed with brine, dried over Na2SO4, filtered, and
concentrated in vacuo. The residue was purified by column chroma-
tography (silica gel, 5% ! 7:5% EtOAc/hexane) to afford the homo-
allyl alcohol (340 mg). To a solution of the homo-allyl alcohol
(340 mg) in CH3CN (9.0 mL) and THF (0.9 mL) were added H2O
Synthesis of 7. To a solution of 1310) (805 mg, 2.14 mmol, 2.0
equiv.) in THF (8.4 mL) was added 1.6 M n-BuLi in hexane (1.3 mL,
2.14 mmol, 2.0 equiv.) at rt. After stirring for 10 min at rt, the mixture
was cooled to ꢀ78 ꢂC. A solution of 6 (439 mg, 1.07 mmol) in THF
(4.2 mL) was then added, and the reaction mixture was stirred for 1.5 h
at ꢀ78 ꢂC. The reaction was quenched with saturated aq. NH4Cl
(10 mL). The mixture was poured into EtOAc (50 mL) and H2O
(30 mL). After the organic layer was separated, the aqueous layer was
extracted with EtOAc (50 mL ꢁ 2). The combined organic layers were
washed with brine, dried over Na2SO4, filtered, and concentrated in
vacuo to give the complex mixture, which was carried forward without
purification. To a solution of the mixture in THF (13 mL) was added
LiAlH4 (101 mg, 2.66 mmol) at rt. The resulting mixture was heated at
reflux for 2 h, and then the reaction was quenched with 1 N NaOH
(10 mL). The mixture was extracted with EtOAc (30 mL ꢁ 3). The
combined organic layers were washed with brine, dried over Na2SO4,
filtered, and concentrated in vacuo. The residue was purified by column
chromatography (silica gel, 2:5% ! 5% ! 8% EtOAc/hexane) to
afford the diol (550 mg). To a solution of the diol (550 mg) and 2,2-
dimethoxypropane (0.92 mL, 7.53 mmol) in CH2Cl2 (10 mL) was
added (1R)-(ꢀ)-10-camphorsulfonic acid (CSA, 35 mg, 0.15 mmol) at
rt. The mixture was stirred at rt for 45 min, and the reaction was
quenched with saturated aq. NaHCO4 (10 mL). The mixture was
poured into CHCl3 (10 mL) and the organic layer was separated. The
aqueous layer was extracted with CHCl3 (10 mL ꢁ 2). The combined
organic layers were washed dried over Na2SO4, filtered, and
concentrated in vacuo. The residue was dissolved in THF (8.0 mL),
and tetra-n-butylammonium fluoride (TBAF, 475 mg, 1.51 mmol) was
added at rt. The mixture was stirred at rt for 1 h and then poured into
EtOAc (10 mL) and 5% KHSO4 (10 mL), and the organic layer was
separated. The aqueous layer was extracted with EtOAc (10 mL ꢁ 2).
The combined organic layers were washed with brine, dried over
Na2SO4, filtered, and concentrated in vacuo. The residue was purified
by column chromatography (silica gel, 8% ! 30% EtOAc/hexane) to
afford 7 (454 mg, 0.739 mmol, 69% in 4 steps) as a clear oil. 1H NMR:
(500 MHz, CDCl3, 0.007 M) ꢀ 0.82 (3H, s), 0.84 (3H, s), 1.13 (6H, s),
1.18–1.44 (6H, m), 1.33 (3H, s), 1.38 (3H, s), 1.51–1.82 (5H, m), 1.98
(1H, m), 2.22 (1H, dd, J ¼ 16:2, 2.2 Hz), 2.34 (1H, dd, J ¼ 16:2,
5.3 Hz), 2.54 (2H, m), 2.68 (2H, m), 2.84 (1H, m), 3.09 (1H, m), 3.52
(1H, dd, J ¼ 11:4, 2.3 Hz), 3.63 (2H, m), 4.19 (1H, m), 5.05 (2H, s),
6.78–6.82 (3H, m), 7.20 (1H, t, J ¼ 7:8 Hz), 7.31–7.45 (5H, m) ppm.
13C NMR: (125 MHz, CDCl3, 0.007 M) ꢀ 19.52, 22.50, 22.69, 22.78,
22.86, 24.83, 25.69, 26.95, 27.05, 28.30, 30.29, 32.56, 32.98, 36.12,
36.98, 38.48, 43.57, 43.96, 63.54, 63.85, 68.45, 69.98, 75.11, 98.41,
111.75, 115.28, 121.11, 127.52 (2C), 127.92, 128.57 (2C), 129.22,
137.24, 144.66, 158.91 ppm. HR-FABMS: m=z (½M þ Hꢄþ): Calcd. for
C36H55O4S2: 615.3542, Found: 615.3527.
.
(0.9 mL) and TsOH H2O (299 mg, 1.57 mmol) at rt. The mixture was
stirred at rt for 2 h, and then the reaction was quenched with saturated
aq. NaHCO3 (20 mL). The mixture was extracted with EtOAc
(30 mL ꢁ 3). The combined organic layers were washed with brine,
dried over Na2SO4, and concentrated in vacuo. The residue was
purified by column chromatography (silica gel, 10% ! 40% EtOAc/
hexane) to afford an inseparable mixture of 8 and its diastereomer
(274 mg, 0.446 mmol, 63% in 3 steps) as a clear oil. 1H NMR:
(400 MHz, CDCl3, 0.049 M) ꢀ 0.85 (3H, s), 0.87 (3H, s), 1.12 (6H, s),
1.24 (1H, m), 1.41–1.69 (9H, m), 1.79 (1H, m), 1.95 (2H, m), 2.12–
2.23 (2H, m), 2.33 (1H, m), 2.56 (2H, m), 2.81–3.00 (4H, m), 3.61 (2H,
m), 4.47 (1H, m), 5.05 (2H, s), 5.15 (2H, m), 5.82 (1H, m), 6.78–6.84
(3H, m), 7.19 (1H, t, J ¼ 7:8 Hz), 7.30–7.46 (5H, m) ppm. 13C NMR:
(100 MHz, CDCl3, 0.049 M) ꢀ 22.46, 22.74, 23.06, 23.32, 25.98, 27.43,
27.64, 32.01, 32.77, 37.14, 37.20, 38.69, 38.76, 42.22, 45.27, 45.74,
45.83, 63.28, 70.05, 71.35, 71.85, 78.39, 111.90, 115.28, 118.51,
121.25, 127.67 (2C), 128.02, 128.68 (2C), 129.34, 134.77, 137.34,
144.83, 158.97 ppm. HR-FABMS: m=z (½M þ Hꢄþ): Calcd. for
C36H55O4S2: 615.3542, Found: 615.3564.
Synthesis of 9. To a solution of a mixture of 8 and its diastereomer
(255 mg, 0.415 mmol) in MeCN (1.4 mL), CH2Cl2 (1.4 mL), and H2O
.
(1.4 mL) was added Hg(ClO4)2 6H2O (422 mg, 0.83 mmol, 2.0 equiv.)
at 0 ꢂC. After stirring at the same temperature for 2 h, the reaction
mixture was poured into EtOAc (20 mL) and saturated aq. Na2S2O3
(20 mL). The organic layer was separated, and the aqueous layer was
extracted with EtOAc (20 mL ꢁ 2). The combined organic layers were
washed with brine, dried over Na2SO4, filtered, and concentrated
in vacuo. The residue was purified by column chromatography
(2:5% ! 4% ! 10% EtOAc/hexane) to afford
9
(88.4 mg,
0.175 mmol, 42%), 10 (45.9 mg, 0.091 mmol, 22%), and 11 (38.1 mg,
0.075 mmol, 18%). Compound 9: 1H NMR: (400 MHz, CDCl3,
0.079 M) ꢀ 0.82 (3H, s), 0.86 (3H, s), 0.87 (3H, s), 0.96 (3H, s), 1.16
(1H, dd, J ¼ 12:6, 9.0 Hz), 1.36–1.76 (10H, m), 2.24 (3H, m), 2.54
(2H, m), 3.74 (1H, m), 3.78 (1H, d, J ¼ 11:3 Hz, OH), 3.93 (1H, dd,
J ¼ 12:4, 2.2 Hz), 4.14 (1H, m), 5.01–5.08 (2H, m), 5.05 (2H, s), 5.79
(1H, m), 6.77–6.85 (3H, m), 7.19 (1H, t, J ¼ 7:8 Hz), 7.30–7.45 (5H,
m) ppm. 13C NMR: (100 MHz, CDCl3, 0.079 M) ꢀ 21.80, 23.17, 23.32,
25.46, 25.88, 26.98, 28.27, 31.74, 34.17, 36.16, 37.05, 37.18, 39.47,
41.20, 65.95, 68.40, 69.88, 72.30, 102.59, 111.59, 115.26, 118.17,
121.15, 127.50 (2C), 127.85, 128.53 (2C), 129.14, 134.47, 137.25,
144.84, 158.85 ppm. HR-FABMS: m=z (½M þ Hꢄþ): Calcd. for
5:6
Synthesis of 8. To a solution of 7 (454 mg, 0.739 mmol) and NEt3
(0.52 mL, 3.70 mmol, 5.0 equiv.) in CH2Cl2 (8.0 mL) were added
C
33H47O4: 507.3474, Found: 507.3434. ½ꢁꢄ
þ42ꢂ (c 0.34, CHCl3).
D
Compound 10: 1H NMR: (400 MHz, CDCl3, 0.079 M) ꢀ 0.84 (3H, s),
0.86 (3H, s), 0.89 (3H, s), 0.97 (3H, s), 1.14 (1H, m), 1.24–1.66 (6H,
m), 1.90 (1H, m), 2.16 (2H, m), 1.99–2.19 (4H, m), 2.55 (2H, m), 3.53
(1H, dd, J ¼ 12:4, 1.8 Hz), 3.93 (1H, m), 4.27 (1H, m), 4.96–5.04 (2H,
m), 5.05 (2H, s), 5.78 (1H, m), 6.78–6.82 (3H, m), 7.18 (1H, t,
J ¼ 7:8 Hz), 7.30–7.45 (5H, m) ppm. 13C NMR: (100 MHz, CDCl3,
0.079 M) ꢀ 22.94, 23.27, 23.34, 25.63, 25.69, 27.71, 32.71, 33.47,
33.82, 36.72, 36.74, 36.97, 38.79, 40.60, 63.09, 68.62, 69.90, 75.60,
101.61, 111.78, 115.15, 115.91, 121.19, 127.51 (2C), 127.89, 128.55
.
DMSO (2.0 mL) and SO3 pyridine (470 mg, 2.96 mmol, 4.0 equiv.) at
rt. The mixture was stirred at rt for 4 h, and the reaction was quenched
with saturated aq. NH4Cl (10 mL). The resulting mixture was poured
into EtOAc (50 mL) and saturated aq. NH4Cl (20 mL). The organic
layer was separated and the aqueous layer was extracted with EtOAc
(50 mL ꢁ 2). The combined organic layers were washed with brine,
dried over Na2SO4, and concentrated in vacuo. The residue was
purified by column chromatography (silica gel, 2:5% ! 5% EtOAc/

1172
Y. NAKAGAWA et al.
(2C), 129.19, 135.88, 137.20, 144.54, 158.85 ppm. HR-FABMS: m=z
6:1
acid (16.1 mg) in THF (1.6 mL) was added a freshly prepared solution
(½M þ Hꢄþ): Calcd. for C33H47O4: 507.3474, Found: 507.3455. ½ꢁꢄ
of buffered HF pyridine (75 mL HF pyridine, 150 mL pyridine, 600 mL
THF) at 0 ꢂC. The reaction mixture was stirred for 15 h in a cold room,
then diluted with water (2 mL) and warmed to rt. The mixture was
extracted with EtOAc (10 mL ꢁ 2) and CHCl3 (10 mL ꢁ 2). The
combined organic layers were dried over Na2SO4, filtered, and
concentrated in vacuo. The residue was purified by column chromatog-
raphy (silica gel, 10% ! 20% EtOAc/hexane containing 0.5% AcOH)
to afford the seco-acid (13.6 mg). To a solution of the seco-acid
(13.6 mg) in toluene (2 mL) were added Et3N (79 mL, 570 mmol) and
TCBCl (55 mL, 380 mmol) at rt. The mixture was stirred at rt for 3 h, and
then diluted with toluene (11 mL). The supernatant of the mixture was
added dropwise to a solution of DMAP (116 mg, 950 mmol) in toluene
(21 mL) over 5 h. The anhydride flask was rinsed twice with toluene
(2 mL) (each rinse was added in one portion to the reaction mixture).
After stirring at rt for an additional 1 h, saturated aq. NaHCO3 (10 mL)
was added, and the resulting biphasic mixture was poured into EtOAc
(20 mL). The organic layer was separated, and the aqueous layer was
extracted with EtOAc (10 mL ꢁ 2). The combined organic layers were
washed with brine, dried over Na2SO4, filtered, and concentrated
in vacuo. The residue was purified by column chromatography (silica
gel, 2:5% ! 5% ! 7:5% EtOAc/hexane) to afford the lactone
(10.9 mg). To 10% Pd-C in a double-neck flask was added a solution
of the lactone (10.9 mg) in MeOH (0.4 mL) and CHCl3 (0.1 mL) at rt.
The mixture was vigorously stirred under a H2 atmosphere at rt for 5 h.
The mixture was filtered, and the filtrate was concentrated in vacuo. The
residue was purified by HPLC (column A-023; solvent i-PrOH: CHCl3:
hexane ¼ 5:15:80; flow rate 3.0 mL/min; pressure 500 psi; UV detector
254 nm; retention time 16.2 min) to afford 3 (6.9 mg, 13.4 mmol, 41% in
4 steps) as a clear oil. 1H NMR: (400 MHz, CDCl3, 0.015 M) ꢀ 0.82 (3H,
s), 0.88 (3H, s), 0.89 (3H, s), 0.96 (3H, s), 1.14 (1H, dt, J ¼ 12:9,
3.9 Hz), 1.31–1.66 (9H, m), 1.90 (1H, m), 2.41 (1H, dd, J ¼ 13:6,
10.8 Hz), 2.46–2.63 (4H, m), 2.67 (1H, t, J ¼ 5:8 Hz, OH), 2.80 (2H,
m), 3.80 (2H, m), 3.94 (1H, m), 4.10 (1H, dd, J ¼ 11:3, 2.6 Hz), 5.15
(1H, m), 5.24 (1H, m), 6.68 (1H, dd, J ¼ 8:0, 2.3 Hz), 6.72 (1H, d,
J ¼ 7:6 Hz), 6.76 (1H, s, Ph-OH), 6.95 (1H, br.s), 7.13 (1H, t,
J ¼ 7:8 Hz) ppm. 13C NMR: (100 MHz, CDCl3, 0.015 M) ꢀ 21.32,
23.01, 23.34, 23.48, 25.03, 25.96, 27.16, 28.75, 34.33, 35.42, 35.88,
37.13, 37.29, 40.12, 42.83, 64.18, 67.33, 69.13, 70.56, 73.12, 100.65,
112.44, 114.12, 120.58, 129.20, 144.61, 156.58, 169.45, 173.33 ppm.
.
.
D
ꢀ15ꢂ (c 0.30, CHCl3). Compound 11: 1H NMR: (400 MHz, CDCl3,
0.079 M) ꢀ 0.87 (3H, s), 0.89 (3H, s), 0.92 (3H, s), 0.96 (3H, s), 1.15
(1H, m), 1.32–1.77 (10H, m), 1.91 (1H, dt, J ¼ 13:3, 4.5 Hz), 2.16 (2H,
m), 2.56 (2H, m), 3.51 (1H, dd, J ¼ 12:1, 2.1 Hz), 3.65 (1H, m), 4.09
(1H, m), 4.20 (1H, d, J ¼ 10:2 Hz, OH), 5.05 (2H, s), 5.07–5.12 (2H,
m), 5.78 (1H, m), 6.78–6.82 (3H, m), 7.19 (1H, t, J ¼ 7:8 Hz), 7.30–
7.45 (5H, m) ppm. 13C NMR: (100 MHz, CDCl3, 0.079 M) ꢀ 22.61,
22.96, 23.48, 25.57, 25.65, 27.62, 31.34, 32.69, 33.58, 36.12, 36.67,
36.98, 38.47, 40.69, 65.23, 69.23, 69.91, 70.06, 102.86, 111.74,
115.20, 117.64, 121.14, 127.51 (2C), 127.90, 128.56 (2C), 129.12,
135.08, 137.20, 144.41, 158.87 ppm. HR-FABMS: m=z (½M þ Hꢄþ):
5:1
Calcd. for C33H47O4: 507.3474, Found: 507.3460. ½ꢁꢄ
(c 0.31, CHCl3).
ꢀ17ꢂ
D
Synthesis of 12. To a solution of 1410) (31.9 mg, 0.097 mmol, 1.5
equiv.) and Et3N (15.1 mL, 0.109 mmol, 1.7 equiv.) in toluene (700 mL)
was added 2,4,6-trichlorobenzoyl chloride (TCBCl, 17.1 mL, 0.109
mmol, 1.7 equiv.) at rt. After stirring at rt for 2 h, a supernatant of the
resulting suspension was added to a solution of 9 (32.6 mg, 0.0644
mmol) and 4-(dimethylamino)pyridine (DMAP, 16.5 mg, 0.135 mmol,
2.1 equiv.) in toluene (700 mL) at rt. The resulting mixture was stirred
at 50 ꢂC for 2 h, and then poured into water (10 mL). The mixture was
extracted with EtOAc (10 mL ꢁ 3). The combined organic layers were
washed with brine, dried over Na2SO4, filtered, and concentrated
in vacuo. The residue was purified by column chromatography (silica
gel, 2:5% ! 4% ! 5% EtOAc/hexane) to afford the ester (34.5 mg).
To a vigorously stirred solution of the ester (34.5 mg) in CH2Cl2
(2.2 mL) and H2O (0.4 mL) was added 2,3-dichloro-5,6-dicyano-p-
benzoquinone (DDQ, 19.2 mg, 0.084 mmol) at rt, causing a brown
color to appear. After stirring at rt for 1 h, the color gradually changed
to yellow-orange with precipitation of a solid. The mixture was poured
into saturated aq. NaHCO3 (10 mL) and extracted with CH2Cl2
(10 mL ꢁ 3). The combined organic layers were dried over Na2SO4,
filtered, and concentrated in vacuo. The residue was purified by column
chromatography (silica gel, 5% ! 30% EtOAc/hexane) to afford the
alcohol (26.8 mg). To a solution of the alcohol (26.8 mg) in THF were
added imidazole (7.8 mg, 0.115 mmol) and chlorotriethyl silane
(TESCl, 7.7 mL, 0.046 mmol) at rt. After stirring at rt for 4 h, the
reaction was quenched with brine (5 mL). The resulting mixture was
extracted with EtOAc (10 mL ꢁ 3). The combined organic layers were
dried over Na2SO4, filtered, and concentrated in vacuo. The residue
was purified by column chromatography (silica gel, 1% EtOAc/
hexane) to afford 12 (26.3 mg, 0.0324 mmol, 50% in 3 steps) as a clear
oil. 1H NMR: (500 MHz, CDCl3, 0.032 M) ꢀ 0.58 (6H, q, J ¼ 8:0 Hz),
0.78 (3H, s), 0.82 (3H, s), 0.85 (3H, s), 0.92 (9H, t, J ¼ 8:0 Hz), 0.96
(3H, s), 1.08–1.14 (1H, m), 1.29–1.38 (2H, m), 1.40–1.63 (7H, m),
1.73–1.79 (1H, m), 2.17–2.28 (3H, m), 2.46–2.60 (4H, m), 3.35–3.40
(2H, m), 3.45 (1H, dd, J ¼ 9:5, 5.3 Hz), 4.03 (1H, dd, J ¼ 11:3,
3.1 Hz), 4.30–4.35 (1H, m), 4.48–4.53 (2H, m), 4.92–4.99 (2H, m),
5.04 (2H, s), 5.11–5.12 (1H, m), 5.76–5.84 (1H, m), 6.77–6.84 (3H,
m), 7.18 (1H, dd, J ¼ 7:8, 7.8 Hz), 7.26–7.44 (10H, m) ppm.
13C NMR: (125 MHz, CDCl3, 0.032 M) ꢀ 4.91 (3C), 6.79 (3C),
21.28, 23.04, 23.05, 25.31, 25.74, 26.41, 26.69, 28.83, 34.64, 36.03,
37.13, 37.21, 39.47, 40.73, 41.11, 68.41, 68.53, 68.64, 69.93, 71.47,
73.33, 74.26, 100.02, 111.66, 115.28, 116.60, 121.21, 127.51 (2C),
127.55, 127.59 (2C), 127.83, 128.31 (2C), 128.52 (2C), 129.10,
134.98, 137.34, 138.26, 144.92, 158.88, 171.58 ppm. HR-FABMS: m=z
HR-FABMS: m=z (½M þ Hꢄþ): Calcd. for C29H43O8: 519.2958, Found:
25:4
519.2960. ½ꢁꢄ
þ29ꢂ (c 0.35, CHCl3).
D
Inhibition of specific binding of [3H]PDBu to PKCꢀ. The binding of
[3H]PDBu to PKCꢀ was evaluated by the procedure of Sharkey and
Blumberg,25) with 50 mM Tris–maleate buffer (pH 7.4 at 4 ꢂC), 12.5 nM
of PKCꢀ (Invitrogen), 10 nM [3H]PDBu, 50 mg/mL of 1,2-dioleoyl-sn-
glycero-3-phospho-L-serine (Sigma), 3 mg/mL of bovine ꢂ-globulin
(Sigma), and various concentrations of inhibitors. Binding affinity was
evaluated based on the concentration required to cause 50% inhibition
of the specific binding of [3H]PDBu, IC50, which was calculated with
PriProbit 1.63 software. The inhibition constant, Ki, was calculated by
the method of Sharkey and Blumberg.25)
Measurements of cell growth inhibition. The panel of 39 human
cancer cell lines established by Yamori26) and coworkers by the NCI
method was employed with modifications, and cell growth inhibition
was measured as reported previously.28) In brief, the cells were plated
in 96-well plates in RPMI 1640 medium supplemented with 5% fetal
bovine serum, and were allowed to attach overnight. They were
incubated with test compounds for 48 h. Cell growth was estimated
(½M þ Hꢄþ): Calcd. for C50H73O7Si: 813.5126, Found: 813.5118.
9:3
½ꢁꢄ
þ22ꢂ (c 1.3, CHCl3).
D
by sulforhodamine B assay. The 50% growth inhibition (GI50
)
parameter was calculated as reported previously.29–31) Absorbance for
the control well (C) and the test well (T) was measured at 525 nm along
with that for the test well at time 0 (T0). GI50 was calculated as
100 ꢁ ½ðT ꢀ T0Þ=ðC ꢀ T0Þꢄ ¼ 50.
Synthesis of 3. To a suspension of NaIO4 (55.5 mg, 0.259 mmol, 8.0
equiv.) in pH 7.2 phosphate buffer (2.6 mL) was added KMnO4
(5.1 mg, 0.0324 mmol, 1.0 equiv.) in one portion. After stirring at rt for
10 min under an Ar atmosphere, the mixture was added to a solution of
12 (26.3 mg, 0.0324 mmol) in t-BuOH (2.6 mL). The reaction mixture
was stirred at rt for 1 h, and the reaction was quenched with Na2S2O3
(15.4 mg). The resulting mixture was extracted with EtOAc (3 mL ꢁ 5).
The combined organic layers were dried over Na2SO4, filtered, and
concentrated in vacuo. The residue was purified by column chromatog-
raphy (silica gel, 5% ! 10% EtOAc/hexane containing 0.5% AcOH)
to afford the carboxylic acid (16.1 mg). To a solution of the carboxylic
EBV-EA induction test. Human B-lymphoblastoid Raji cells
a 5% CO2
(5 ꢁ 105 cells/mL) were incubated at 37 ꢂC under
atmosphere in 1 mL of RPMI 1640 medium (supplemented with 10%
fetal bovine serum) with 4 mM sodium n-butyrate (a synergist) and
100 nM of test compound in the presence and the absence of 32 nM
TPA. TPA was added as 2 mL of a DMSO solution. Since the various

Superior Anti-Proliferative Analog of Aplysiatoxin
1173
test compounds were added as 2 mL of DMSO solutions of the various
20 mM stock solutions, the final DMSO concentration was 0.4%. After
incubation for 48 h, smears were made from the cell suspension, and
the EBV-EA-expressing cells were stained by a conventional indirect
immunofluorescence technique with an NPC patient’s serum (a gift
from Kobe University) and FITC-labeled anti-human IgG (Dako,
Glostrup, Denmark), as reported previously.28,29) In each assay, at least
500 cells were counted and the proportion of EA-positive cells was
recorded. The test compounds (Aplog-1, bryo-1 from Sigma, and
ATX,32) 3) did not induce cell death during the experiment at 100 nM.
Cell viability exceeded 50% in each experiment.
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Acknowledgments
This work was supported partly by The Naito
Foundation and The Uehara Memorial Foundation (to
K.I.), and by a Grant-in-aid for Scientific Research (A)
(no. 21248015 to K.I.) and a Grant-in-aid for JSPS
Fellows (no. 20ꢅ4135 to R.C.Y.) from The Ministry of
Education, Culture, Sports, Science, and Technology of
Japan. Finally, we thank the Screening Committee for
Anticancer Drugs, supported by a Grant-in-aid for
Scientific Research on Priority Area ‘‘Cancer’’ from
The Ministry of Education, Culture, Sports, Science, and
Technology of Japan.
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