Structure-activity studies at position 27 of aplog-1, a simplified analog of debromoaplysiatoxin with anti-proliferative activity

Article abstract of DOI:10.1016/j.tet.2013.02.008

Aplog-1, a simplified analog of debromoaplysiatoxin with anti-proliferative activity, bound to and activated protein kinase Cδ (PKCδ), that is involved in tumor suppression and apoptosis. To examine the contribution of PKCδ to the anti-proliferative activity of aplog-1, we synthesized 27-methyl and 27-methoxy derivatives that differed in the ability to activate PKCδ. Activators of PKCδ like aplog-1 and 27-(R)-Me-aplog-1 strongly inhibited the growth of several cancer cell lines. On the other hand, 27-(S)-Me-aplog-1 and 27-O-Me-aplog-1 without the ability to activate PKCδ, exhibited only weak growth inhibitory activity against these cell lines. These results suggest indirectly that the activation of PKCδ might be involved in the anti-proliferative activity of aplog-1 against aplog-sensitive cancer cell lines.

Full text of DOI:10.1016/j.tet.2013.02.008

Tetrahedron 69 (2013) 7636e7645
Contents lists available at SciVerse ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Structureeactivity studies at position 27 of aplog-1, a simplified
analog of debromoaplysiatoxin with anti-proliferative activity
Yusuke Hanaki a, Masayuki Kikumori a, Sayo Ueno a, Harukuni Tokuda b,
,
Nobutaka Suzuki b, Kazuhiro Irie a
*
a Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan
b Department of Complementary and Alternative Medicine, Clinical R & D, Graduate School of Medical Science, Kanazawa University,
Kanazawa 920-8640, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Aplog-1, a simplified analog of debromoaplysiatoxin with anti-proliferative activity, bound to and acti-
Received 13 December 2012
Received in revised form 29 January 2013
Accepted 1 February 2013
Available online 8 February 2013
vated protein kinase C
contribution of PKC to the anti-proliferative activity of aplog-1, we synthesized 27-methyl and 27-
methoxy derivatives that differed in the ability to activate PKC . Activators of PKC like aplog-1 and
27-(R)-Me-aplog-1 strongly inhibited the growth of several cancer cell lines. On the other hand, 27-(S)-
Me-aplog-1 and 27-O-Me-aplog-1 without the ability to activate PKC , exhibited only weak growth
d (PKCd), that is involved in tumor suppression and apoptosis. To examine the
d
d
d
d
Keywords:
inhibitory activity against these cell lines. These results suggest indirectly that the activation of PKC
might be involved in the anti-proliferative activity of aplog-1 against aplog-sensitive cancer cell lines.
d
Protein kinase C
Tumor-promoter
Aplysiatoxin
Ó 2013 Elsevier Ltd. All rights reserved.
Anti-proliferative
1. Introduction
its limited availability from natural sources (just 18 g of bryo-1 was
isolated from 13 t of the bryozan B. neritina)14 and synthetic
complexity.15e17
Protein kinase C (PKC) is a family of serine/threonine kinases
that play a critical role in cell proliferation, differentiation, and
apoptosis.1,2 PKC isozymes are classified into three groups accord-
ing to structural features and cofactor requirements: conventional
Recently, more practical synthetic methods for bryo-1 and re-
lated compounds have been reported. Keck’s group accomplished
the total synthesis of bryo-1 via a very short route.18 In addition, the
groups of Wender, Hale, Trost, and Krische have reported excellent
total syntheses of bryo-1’s congeners.19 Moreover, Wender and
colleagues have developed several simplified analogs of bryo-1
with stronger anti-proliferative activity.20,21 In contrast, we tried
to develop a more synthetically accessible PKC activator with bryo-
1-like activity as another way to address the problem of supply.
We focused on the skeleton of aplysiatoxin (ATX),22 a potent
tumor-promoter isolated from the digestive gland of a sea hare
Stylocheilus longicauda, since ATX has a macrolactone structure
similar to bryo-1. An interesting feature of ATX is the role of the
bromine atom. Kakunaga and colleagues reported that while
debromoaplysiatoxin (DAT) bound to PKCs with an affinity similar
to ATX, its ability to increase malignant transformation was lower
than that of ATX.23 Suspecting that molecular hydrophobicity is
closely related to the tumor-promoting activity of ATX-related
compounds, we synthesized aplog-1,24 a simplified and less hy-
drophobic analog of DAT. Fortunately, aplog-1 was anti-
proliferative but not tumor-promoting, and its anti-proliferative
activity against several cancer cell lines was comparable to that of
bryo-1.24 Moreover, we found that aplog-1 exhibited an activation
PKCs (
a
,
b
I,
b
II,
g
), novel PKCs (
d, 3 , h, q), and atypical PKCs (z, l/i
).3
Naturally occurring tumor-promoters such as 12-O-tetradecanoyl-
phorbol 13-acetate (TPA), teleocidin B-4, and aplysiatoxin (ATX)
bind to cysteine-rich C1 domains (C1A, C1B) in the regulatory re-
gions of conventional and novel PKCs, and activate them exten-
sively, resulting in tumor formation.3,4 However, bryostatin 1 (bryo-
1)5 isolated from the marine bryozoan Bugula neritina is a fasci-
nating PKC activator without tumor-promoting activity. Bryo-1
exhibits significant anti-cancer and anti-tumor-promoting activi-
ties possibly through the activation of PKCd,
6e8 which is suggested
to be involved in apoptosis and to play a tumor suppressor role.9e11
Bryo-1 has also attracted attention as a therapeutic lead for other
intractable diseases such as Alzheimer’s disease (AD)12 and ac-
quired immune deficiency syndrome (AIDS).13 Although further
studies on its mode of action and structural optimization as
a therapeutic agent are indispensable, they have been hampered by
* Corresponding author. Tel.: þ81 75 753 6281; fax: þ81 75 753 6284; e-mail
address: irie@kais.kyoto-u.ac.jp (K. Irie).
0040-4020/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tet.2013.02.008

Y. Hanaki et al. / Tetrahedron 69 (2013) 7636e7645
7637
profile for PKC
translocated PKC
d
similar to that of bryo-1. Aplog-1 as well as bryo-1
from the cytosol to the nuclear membrane and
d
perinuclear region in Chinese hamster ovary cells, while TPA in-
duced its translocation mainly to the plasma membrane.24
Our recent structureeactivity studies of aplogs indicated that
the hydrophobicity around the spiroketal moiety is critical for both
the ability to bind to PKCd and the anti-proliferative activity against
cancer cell lines.25e27 Among the derivatives tested, 10-methyl-
aplog-1 (1) was one order of magnitude more potent than aplog-1
both in its ability to bind to PKCd and in its anti-proliferative activity
against cancer cell lines. Interestingly, 10-methyl-aplog-1 did not
show any tumor formation in a mouse two-stage carcinogenesis
experiment even when applied at a 5-fold excess compared with
DAT and TPA, which showed strong tumor-promoting activity.27
The next step is to reveal the mechanism of the unique bi-
ological activities of aplogs. Although there existed a correlation
between the anti-proliferative activity against several cancer cell
lines and the ability to bind to PKCd, the affinity of aplog-1 for PKCd
was one order of magnitude lower than that of bryo-1, despite
significant anti-proliferative activity comparable to bryo-1.24 To
clarify the role of PKC
we prepared three derivatives of aplog-1 (2e4) that differ in affinity
for PKC . This paper describes their synthesis and biological activ-
d in the anti-proliferative activity of aplogs,
Fig. 1. Structure of bryo-1, aplysiatoxin analogs, and their simplified analogs.
d
ities, and discusses the involvement of PKC
proliferative activity of aplogs.
d in the anti-
and 4 was similarly achieved (2, 13%; 3, 15%; 4, 29% in seven steps
from 11).
First, we evaluated affinity for PKC
peptide34 and human recombinant PKC
PKC is suggested to be involved in tumor suppression and
apoptosis,9e11 and bryo-1 exhibited activation profiles for PKC
d
using a synthetic PKC
d-C1B
2. Results and discussion
d. As mentioned above,
d
The three derivatives (2e4) of aplog-1 were designed based on
the following consideration. 27-(R)-Me-aplog-1 (2) has a chiral
methyl group at position 27, whose absolute configuration was the
same as that of DAT at position 30. 27-(S)-Me-aplog-1 (3), an epi-
mer of 2 at position 27, and 27-O-Me-aplog-1 (4) were prepared as
inactive controls in PKC-binding since Kishi and Rando’s study on
the pharmacophore of DAT suggested the hydroxyl group at posi-
tion 30 to be indispensable to bind to PKC C1 domains.28,29 They
also revealed that the stereo center at position 30 of DAT is im-
portant for PKC activation, based on the finding that the epimer of
DAT at position 30 showed only marginal activity.28,29 Suganuma
and colleagues showed that oscillatoxin A, which lacks DAT’s
methyl group at position 30, inhibited the specific binding of TPA to
a mouse epidermal particulate fraction, suggesting this methyl
group not to be essential for the activity.30 According to these
studies, 2 should bind to and activate PKC isozymes with a potency
similar to aplog-1, while 3 and 4 should not (Fig. 1).
d
distinct from tumor-promoters like TPA. Although bryo-1 induced
down-regulation of most PKC isozymes similar to TPA, it protected
PKC
translocated PKC
d
from the down-regulation by TPA.6,7 Moreover, bryo-1
d
to nuclear membrane and perinuclear region in
Chinese hamster ovary cells, while TPA induced its translocation to
the plasma membrane.8 We have found that aplog-1 bound more
potently to PKC
d
,
h
and
q
rather than to conventional PKC iso-
similar to
to evaluate the activity of
zymes,26,27 and showed the translocation profile for PKC
d
bryo-1.24 Therefore, we selected PKC
aplogs synthesized in this study.
d
The affinity was measured by a competition binding assay with
[3H]phorbol 12,13-dibutyrate (PDBu) as described previously by
Sharkey and Blumberg.35,36 A binding assay using whole PKC
d re-
flects binding to both the C1A and C1B domains, but instability can
diminish accuracy and reproducibility. Therefore, we performed
simultaneously a more reliable assay using our synthetic d-C1B
The synthesis of 2e4 is outlined in Scheme 1. They were syn-
thesized from a previously reported spiroketal (11)24 and each
carboxylic acid moiety (10aec). Coupling of (S)-glycidol (5) with
1,3-dithiane gave a diol. Transacetalization of the diol with p-ani-
saldehyde dimethyl acetal afforded the cyclic acetal, which was
reductively cleaved by DIBAL; the result was a 3:1 mixture of
regioisomers in which the desired primary alcohol (6) predomi-
nated. Subsequently, oxidation of 6, and a stereoselective Grignard
reaction generated an alcohol (7a) in 97% ee. Protection by a benzyl
group, deprotection of dithioacetal, and oxidation gave a carboxylic
acid (10a). The Mitsunobu reaction31 of 7a was employed to gen-
erate an epimer alcohol (7b) in 92% ee. The synthesis of 10b from 7b
was achieved in a similar manner to the synthesis of 10a. Com-
pound 10c was provided by oxidation of the known aldehyde
(9c).32 Yamaguchi’s esterification33 of 11 with the carboxylic acid
(10a), followed by switching of the protecting group from a 4-
methoxyphenylmethyl (MPM) group to a triethylsilyl (TES) group,
and oxidative cleavage of the olefin group provided a carboxylic
acid (13a). Deprotection of the TES group, lactonization, and
deprotection of the two benzyl groups gave 2. The synthesis of 3
peptide, which is stable and inexpensive. In addition, the main
binding site of naturally occurring PKC ligands was found to be the
C1B domain of PKC
d rather than the C1A domain, and the binding
constants of some PKC ligands in the inhibition of specific [3H]
PDBu-binding were almost equal to those of whole PKCd.
27,37,38 We
also confirmed that the ability of [3H]PDBu to bind to the
d-C1B
peptide was equal to that in our previous reports.36
As shown in Table 1, the affinity of 2 was several times weaker
than that of aplog-1, while 3 and 4 hardly bound to PKC and -C1B
at all. Their abilities to activate PKC were also evaluated by mea-
suring phosphorylation levels with a fluorescence-labeled peptide
substrate (Fig. 2). Both aplog-1 and 2 activated PKC in a concen-
tration-dependent manner, and the data reflected the binding to
PKC . In contrast, 3 and 4 did not activate PKC even at 10 M. Since
d
d
d
d
d
d
m
PKC activation requires higher concentration than PKC-binding as
shown in many PKC ligands,39 these activation profiles correlated
with the results of the binding assay.
Since 2 possesses the skeleton of ATX and DAT, 2 might be
a tumor-promoter. According to Suganuma and colleagues, DAT’s
tumor-promoting activity was slightly stronger than that of

7638
Y. Hanaki et al. / Tetrahedron 69 (2013) 7636e7645
Scheme 1. (a) 1,3-Dithiane, n-BuLi, THF; (b) p-MeO(C6H4)CH(OMe)2, CSA, DMF; (c) DIBAL, DCM (70% in three steps); (d) SO3$pyridine, DMSO, Et3N, DCM (82%); (e) MeMgBr,
MgBr2$Et2O, DCM (83%); (f) BnBr, NaH, THF (a, 37%; b, 49%); (g) MeI, NaHCO3, MeCN, H2O (a, 73%; b, 87%); (h) NaClO2, 2-methyl-2-butene, t-BuOH, satd NaH2PO4 aq (a, 77%; b, 88%;
c, 95%); (i) (1) p-nitrobenzoic acid, diisopropyl azodicarboxylate, PPh3, THF (73%); (2) NaOH, H2O, THF, MeOH (93%); (j) 7aec, 2,4,6-trichlorobenzoyl chloride, Et3N, DMAP, toluene
(a, 97%; b, quant.; c, quant.); (k) DDQ, H2O, DCM (a, 79%; b, 75%; c, 91%); (l) TES-Cl, imidazole, THF (a, 84%; b, quant.; c, 90%); (m) KMnO4, NaIO4, t-BuOH, pH 7 buffer (a, 45%; b, 43%;
c, 66%); (n) HF$pyridine (a, 83%; b, 74%; c, 96%); (o) 2,4,6-trichlorobenzoyl chloride, Et3N, DMAP, toluene (a, 65%; b, 79%; c, 76%); (p) 10% Pd/C, EtOH or MeOH (a, 83%; b, 77%; c, 74%).
Table 1
control of host human B-lymphoblastoid Raji cells, are activated by
treatment with tumor-promoters to produce early antigen (EA),
which can be detected by an in direct immunofluorescence tech-
nique. Compared with the potent EA-inducer TPA, 2e4 induced
Values of Ki for the inhibition of [3H]PDBu-binding by aplog-1, 2, 3, and 4
Ki (nM)
Aplog-1a
3
4
5
d
-C1B peptide
7.4 (1.1)
15 (1.0)
24 (2.6)b
31 (1.9)
>2500
>5000
>2500
>5000
only weak EA production even at 1 mM like aplog-1. This result
Whole PKC
d
suggests that the methyl group at position 27 of 2 does not con-
tribute to the tumor-promoting activity. This methyl group might
enhance the tumor-promoting activity in combination with other
substituents eliminated when DAT was simplified (Fig. 3).
a
Cited from Ref. 24.
b
Values in parentheses represent the standard deviation from triplicate
experiments.
The anti-proliferative activities of 2e4 were evaluated using
a panel of 39 human cancer cell lines established by Yamori and
colleagues.42 The 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 where
oscilatoxin A,30 indicating that the methyl group at position 30
might enhance the activity. We evaluated the tumor-promoting
activities of 2e4 in vitro by testing the induction of EpstaineBarr
virus early antigen (EBV-EA) production.40,41 EBVs under the strict

Y. Hanaki et al. / Tetrahedron 69 (2013) 7636e7645
7639
Table 3
Growth inhibition of aplog-1, 2, 3, and 4 against aplog-insensitive cancer cell lines
Cancer type
Cell line
log GI50 (log M)
Aplog-1a
2
3
4
Breast
Colon
Lung
Ovarian
Stomach
Prostate
MCF-7
HT-29
DMS114
OVCAR-3
MKN74
DU-145
ꢀ4.72
ꢀ4.77
ꢀ4.79
ꢀ4.78
ꢀ4.76
ꢀ4.85
ꢀ4.73
ꢀ4.77
ꢀ4.82
ꢀ4.77
ꢀ4.80
ꢀ4.80
ꢀ4.79
ꢀ4.75
ꢀ4.82
ꢀ4.78
ꢀ4.76
ꢀ4.73
ꢀ4.88
ꢀ4.71
ꢀ4.88
ꢀ4.83
ꢀ4.80
ꢀ4.67
a
Cited from Ref. 24.
correlation between the anti-proliferative activity and ability to
bind to and activate PKC . This might reflect the lower degree of
PKC expression, or higher degree of expression of other PKC iso-
d
Fig. 2. Kinase activity of PKCd stimulated by TPA, aplog-1, 2, 3, and 4. The values were
d
expressed as a percentage relative to the control (EtOH). The final concentration of
EtOH was 2.0%. The results were presented as the average of duplicated points when all
compounds were simultaneously tested. Error bars represent the standard error of the
mean (n¼2). Another experiment gave a similar result.
zymes in these cell lines.
3. Conclusions
In summary, new derivatives (2e4) of aplog-1 at position 27
were prepared and evaluated as to their biological activities in or-
der to confirm the involvement of PKC
activities of aplogs. Both aplog-1 and 2 bound to and activated
PKC , and showed substantial inhibitory activities especially in
several cancer cell lines shown in Table 2. On the other hand, 3 and
4, without the ability to activate PKC , showed only weak growth
inhibition in all 39 cancer cell lines (Supplementary data). From
these results, we speculated that PKC might play a key role in the
d in the anti-proliferative
d
d
d
anti-proliferative activity of aplog-1 at least in the aplog-sensitive
cell lines. This hypothesis should be proved by knock-out of each
PKC isozyme in these cell lines. Moreover, there might exist targets
other than PKC isozymes responsible for the unique biological ac-
tivities of aplogs. Further study is necessary to identify these targets
using beads technology.43
Fig. 3. EBV-EA production induced by TPA, aplog-1, 2, 3, and 4. Percentages of EA-
positive cells are shown. Sodium n-butyrate (4 mM) was added to all samples to en-
hance the sensitivity of Raji cells. Only 0.1% of the cells were positive for EA at 4 mM
sodium n-butyrate. The final concentration of DMSO was 0.4%. Cell viability exceeded
60% in all experiments except for that with TPA at 10ꢀ6 M (>50%). Error bars present
the standard error of the mean (n¼3). aCited from Ref. 27.
4. Experimental section
4.1. General remarks
aplog-1 showed activity with log GI50 values of less than ꢀ5.30, are
listed in Table 2 (the rest of the data are provided in Supplementary
data). Although the growth inhibitory activity of 2 was slightly
weaker than that of aplog-1, 2 showed substantial activity like
aplog-1 especially in the cell lines listed in Table 2. On the other
hand, 3 and 4 exhibited weak activity in all cancer cell lines. These
results indirectly suggest that aplog-1 and its analogs might inhibit
The following spectroscopic and analytical instruments were
used: digital polarimeter, DIP-1000 (Jasco, Tokyo, Japan); 1H and 13
C
NMR, Avance III 400 and Avance III 500 (reference TMS, Brucker,
Germany); HPLC, model 600E with a model 2487 UV detector
(Waters, Tokyo, Japan); and HR-FABMS, JMS-600H (JEOL, Tokyo,
Japan) and JMS-700 (JEOL, Tokyo, Japan). HPLC was carried out on
YMC-packed ODS-A AA12S05-1520WT and SIL SL12S05-2510WT
(Yamamura Chemical Laboratory, Kyoto, Japan) and CHIRALCEL
OJ-RH (Daicel Corporation, Osaka, Japan). Wakogel C-200 (silica gel,
Wako Pure Chemical Laboratory, Osaka, Japan) was used for column
chromatography. [3H]PDBu (22.1 Ci/mmol) was custom synthesized
by PerkineElmer Life Sciences Research Products (Boston, MA). The
the growth of these cancer cell lines through PKCd-dependent
mechanisms. However, it is worth noting that 3 and 4 retained anti-
proliferative activity though it was weak. We consider that the
growth of the cancer cell lines sensitive to aplogs might be partly
inhibited by activation of PKCd, but that other mechanisms could
also exist. In aplog-insensitive cell lines (Table 3), we observed no
human recombinant PKC
(Carlsbad, CA). PKC
d was purchased from Life Technologies
Table 2
d
-C1B peptide was synthesized as reported
Growth inhibition of aplog-1, 2, 3, and 4 against aplog-sensitive cancer cell lines
previously.34 All other chemicals and reagents were purchased
from chemical companies and used without further purification.
The purity of 2e4 was greater than 95% as judged by HPLC.
Cancer type
Breast
Cell line
log GI50 (log M)
Aplog-1a
2
3
4
HBC-4
ꢀ6.33
ꢀ5.61
ꢀ5.43
ꢀ5.60
ꢀ5.32
ꢀ5.74
ꢀ5.55
ꢀ5.33
ꢀ6.15
ꢀ5.03
ꢀ5.08
ꢀ5.66
ꢀ5.09
ꢀ5.18
ꢀ5.17
ꢀ4.99
ꢀ4.78
ꢀ4.80
ꢀ4.79
ꢀ4.78
ꢀ4.80
ꢀ4.76
ꢀ4.71
ꢀ4.77
ꢀ4.71
ꢀ4.50
ꢀ4.70
ꢀ4.70
ꢀ4.76
ꢀ4.80
ꢀ4.70
ꢀ4.75
MDA-MB-231
HCC2998
NCI-H460
A549
LOX-IMVI
St-4
4.2. Synthesis of 27-(R)-Me-aplog-1 (2)
Colon
Lung
To a solution of 1,3-dithiane (2.18 g, 18.2 mmol, 4 equiv) in THF
(50 mL) was added 1.6 M n-BuLi in hexane (11.3 mL, 18.2 mmol,
4 equiv) at 4 ꢁC. After stirring for 1 h at 4 ꢁC, a solution of (S)-gly-
cidol (5) (336 mg, 4.54 mmol) in THF (1 mL) was added at 4 ꢁC. The
reaction mixture was stirred for 2 h at 4 ꢁC, and the reaction was
Melanoma
Stomach
MKN45
a
Cited from Ref. 24.

7640
Y. Hanaki et al. / Tetrahedron 69 (2013) 7636e7645
26.6
quenched with saturated aq NH4Cl (40 mL). The mixture was
extracted with EtOAc (70 mLꢂ3). 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, 60% EtOAc/hexane) to afford a crude diol.
329.1247 ([MþH]þ calcd for C16H25O3S2 329.1245); [
a
]
þ4.7 (c
D
0.30, CHCl3).
To a solution of 60% NaH in oil (23.5 mg, 0.588 mmol, 1.1 equiv)
in THF (5.9 mL) was added a solution of 7a (175 mg, 0.534 mmol) in
THF (0.5 mL). After stirring for 30 min at 0 ꢁC, BnBr (67
mL,
After the diol was dissolved in DMF (12 mL), p-anisaldehyde
dimethyl acetal (1.24 g, 6.81 mmol,1.5 equiv) and (ꢃ)-CSA (70.0 mg,
0.302 mmol, 0.07 equiv) were added to the mixture at room tem-
perature, which was stirred at room temperature for 1.5 h. The
reaction was quenched with saturated aq NaHCO3 (12 mL), and the
mixture was extracted with EtOAc (15 mLꢂ3). The combined or-
ganic layers were washed with brine, dried over Na2SO4, filtered,
and concentrated in vacuo. The residue was purified by column
chromatography (silica gel, 15% EtOAc/hexane) to afford an acetal.
The acetal was dissolved in DCM (20 mL), and 1 M DIBAL in
toluene (9.00 mL, 9.00 mmol, 2 equiv) was added at ꢀ78 ꢁC. The
reaction mixture was stirred at ꢀ78 ꢁC for 1 h, and then 1 M DIBAL
in toluene (4.00 mL, 4.00 mmol, 0.9 equiv) was added at ꢀ78 ꢁC.
After an additional 1 h of stirring at ꢀ78 ꢁC, the reaction was
quenched with MeOH (6 mL). The mixture was warmed to room
temperature, and saturated aq Rochelle salt (20 mL) was added. The
mixture was poured into EtOAc (30 mL). After the organic layer was
separated, the aqueous layer was extracted with EtOAc (30 mLꢂ3).
The combined organic layers were washed with brine, dried over
Na2SO4, filtered, and concentrated in vacuo. The residue was puri-
fied by column chromatography (silica gel, 10%/20% EtOAc/hex-
ane) to afford 6 (991 mg, 3.16 mmol, 70% in three steps) as a clear
oil, in which the two regioisomers existed in a ratio of 3:1. 1H NMR
0.564 mmol, 1.05 equiv) was added at 0 ꢁC. The reaction mixture
was stirred at room temperature for 21.5 h, and the reaction was
quenched with saturated aq NH4Cl (6 mL). After the organic layer
was separated, the aqueous layer was extracted with EtOAc
(10 mLꢂ3). The combined organic layer was washed with brine,
dried over Na2SO4, filtered, and concentrated in vacuo. The residue
was purified by column chromatography (silica gel, 5% EtOAc/
hexane) to afford 8a (82.7 mg, 0.198 mmol, 37%) as a clear oil. 1H
NMR (500 MHz, CDCl3, 0.012 M; ppm):
d
1.16 (3H, d, J¼6.4 Hz),
1.82e1.93 (2H, m), 2.01e2.11 (2H, m), 2.72 (1H, m), 2.79e2.83 (3H,
m), 3.65 (1H, m), 3.81 (3H, s), 3.82 (1H, m), 4.02 (1H, dd, J¼9.9,
4.9 Hz), 4.50e4.60 (4H, m), 6.86e6.89 (2H, m), 7.25e7.36 (7H, m);
13C NMR (125 MHz, CDCl3, 0.012 M; ppm):
d 14.8, 26.1, 29.8, 30.2,
35.7, 44.1, 55.3, 71.1, 72.8, 75.1, 77.0, 113.8 (2C), 127.5, 127.7 (2C),
128.4 (2C), 129.7 (2C), 131.0, 138.8, 159.3; IR (KBr; cmꢀ1): 2898,
1612,1513,1460,1246,1087, 818; HR-FABMS (matrix, glycerol); m/z:
11.4
419.1716 ([MþH]þ calcd for C23H31O3S2 419.1715); [
a]
þ20.4 (c
D
0.50, CHCl3).
To a solution of 8a (78.0 mg, 0.187 mmol) and NaHCO3 (47.0 mg,
0.560 mmol, 3 equiv) in MeCN (1.8 mL) and H2O (0.52 mL) was
added MeI (0.58 mL, 9.31 mmol, 50 equiv) at room temperature.
The reaction mixture was stirred at room temperature for 19 h, then
diluted with H2O (3 mL). The mixture was extracted with EtOAc
(5 mLꢂ3), and the combined organic layer was washed with brine,
dried over Na2SO4, filtered, and concentrated in vacuo. The residue
was purified by column chromatography (silica gel, 15% EtOAc/
hexane) to afford the known aldehyde 9a44 (44.7 mg, 0.136 mmol,
73%) as a clear oil.
(400 MHz, CDCl3, 0.017 M; ppm): d 1.82 (1H, m), 1.84e1.93 (2H, m),
2.06e2.14 (2H, m), 2.78e2.91 (4H, m), 3.52 (1H, m), 3.76 (1H, m),
3.81 (3H, s), 3.83 (1H, m), 4.12 (1H, dd, J¼8.7, 5.8 Hz), 4.54 (1H, d,
J¼11.1 Hz), 4.58 (1H, d, J¼11.1 Hz), 6.88e6.91 (2H, m), 7.28e7.31
(2H, m); 13C NMR (100 MHz, CDCl3, 0.017 M; ppm):
d 25.9, 30.1,
30.4, 37.2, 43.8, 55.3, 64.1, 71.9, 76.0, 113.9 (2C), 129.6 (2C), 130.4,
To a solution of 9a44 (2.6 mg, 7.9
saturated aq NaH2PO4 (44 L) were added 2 M 2-mehyl-2-butene in
THF (22 L, 44.0 mol, 5.6 equiv) and NaClO2 (0.8 mg, 8.8 mol,
mmol) in t-BuOH (44 mL) and
159.4; IR (KBr; cmꢀ1): 3452, 2933, 1645, 1516, 1247, 1040, 821; HR-
m
FABMS (matrix, glycerol) m/z: 315.1085 ([MþH]þ calcd for
m
m
m
28.9
C15H23O3S2 315.1089); [
a]
ꢀ3.5 (c 0.27, CHCl3).
1.1 equiv) at 4 ꢁC. The mixture was stirred at 4 ꢁC for 30 min, and the
reaction was quenched with 5% aq H3PO4 (0.5 mL). The resulting
mixture was extracted with EtOAc (2 mLꢂ3). The combined organic
layers were washed with brine, dried over Na2SO4, filtered, and
concentrated in vacuo. The residue was purified by column chro-
matography (silica gel, 30% EtOAc/hexane containing 0.1% AcOH) to
D
To a solution of 6 (538 mg, 1.71 mmol) in DCM (6.8 mL) and
DMSO (1.8 mL) were added Et3N (1.67 mL, 12.0 mmol, 7 equiv) and
SO3$pyridine (1.09 g, 6.86 mmol, 4 equiv) at 0 ꢁC. After stirring at
room temperature for 2.5 h, the reaction was quenched with sat-
urated aq NH4Cl (10 mL). The organic layer was separated, and the
aqueous layer was extracted with DCM (10 mLꢂ3). The combined
organic layer was washed with brine, dried over Na2SO4, filtered,
and concentrated in vacuo. The residue was purified by column
chromatography (silica gel, 10%/15% EtOAc/hexane) to afford an
aldehyde (438 mg, 1.40 mmol, 82%) as a clear oil.
afford 10a (2.1 mg, 6.1
cedure was repeated several times. 1H NMR (400 MHz, CDCl3,
0.010 M; ppm):
mmol, 77%) as a pale yellow oil. This pro-
d
1.18 (3H, d, J¼6.4 Hz), 2.54 (1H, dd, J¼16.0, 8.4 Hz),
2.72 (1H, dd, J¼16.0, 4.2 Hz), 3.69 (1H, m), 3.79 (3H, s), 3.98 (1H, m),
4.48e4.60 (4H, m), 6.83e6.87 (2H, m), 7.20e7.36 (7H, m); 13C NMR
To a solution of the aldehyde (438 mg, 1.40 mmol) in DCM
(6.5 mL) was added MgBr2$Et2O (433 mg, 1.68 mmol, 1.2 equiv) at
ꢀ78 ꢁC. After stirring for 1 h, 3 M MeMgBr in Et2O (0.70 mL,
2.10 mmol, 1.5 equiv) was added at ꢀ78 ꢁC. The reaction mixture
was stirred at ꢀ78 ꢁC for 1.5 h, and the reaction was quenched with
saturated aq NH4Cl (10 mL). After the organic layer was separated,
the aqueous layer was extracted with DCM (10 mLꢂ3). The com-
bined organic layer was washed with brine, dried over Na2SO4,
filtered, and concentrated in vacuo. The residue was purified by
column chromatography (silica gel, 20% EtOAc/hexane) to give 7a
(381 mg, 1.16 mmol, 83%) as a clear oil. 1H NMR (400 MHz, CDCl3,
(100 MHz, CDCl3, 0.010 M; ppm): d 14.5, 35.1, 55.3, 71.2, 72.6, 74.8,
76.8, 113.8 (2C), 127.7, 127.7 (2C), 128.4 (2C), 129.6 (2C), 130.0, 138.2,
159.4, 175.2; IR (KBr; cmꢀ1): 3452, 2934, 1709, 1645, 1516, 1249,
1084, 822; HR-FABMS (matrix, glycerol) m/z : 345.1714 ([MþH]þ
calcd for C20H25O5 345.1702); [
a
]
25 ꢀ15.2 (c 0.19, CHCl3).
D
To a solution of 10a (28.6 mg, 83.1 mmol, 1.5 equiv) and Et3N
(12.9
mL, 92.7
mmol, 1.7 equiv) in toluene (580
mL) was added 2,4,6-
trichlorobenzoyl chloride (14.5
m
L, 92.7 mol, 1.7 equiv) at room
m
temperature. After 3 h of stirring at room temperature, the super-
natant of the suspension was added to a solution of 11 prepared as
reported previously24 (26.1 mg, 54.6
mmol) and DMAP (14.0 mg,
0.019 M; ppm):
d
1.20 (3H, d, J¼6.4 Hz), 1.87 (1H, m), 1.97 (2H, dd,
115 mmol, 2.1 equiv) in toluene (580 mL) at room temperature. The
J¼7.1, 6.1 Hz), 2.08e2.16 (2H, m), 2.77e2.91 (4H, m), 3.56 (1H, dd,
J¼11.6, 5.8 Hz), 3.74 (1H, m), 3.81 (3H, s), 4.10 (1H, t, J¼7.2 Hz), 4.56
(1H, d, J¼11.0 Hz), 4.67 (1H, d, J¼11.0 Hz), 6.88e6.91 (2H, m),
7.28e7.32 (2H, m); 13C NMR (100 MHz, CDCl3, 0.019 M; ppm):
mixture was stirred at 50 ꢁC for 1.5 h, and then poured into H2O
(2.0 mL). The mixture was extracted with EtOAc (2 mLꢂ3). The
combined organic layers were washed with brine, dried over
Na2SO4, filtered, and concentrated in vacuo. The residue was puri-
fied by column chromatography (silica gel, 2.5%/7.5% EtOAc/
d
19.3, 25.9, 30.2, 30.5, 37.1, 43.9, 55.3, 69.5, 73.4, 80.0, 113.9 (2C),
129.7 (2C),130.5, 159.4; IR (KBr; cmꢀ1): 3451, 2900,1613,1513,1247,
hexane) to afford 12a (42.7 mg, 53.1
NMR (400 MHz, CDCl3, 0.0027 M; ppm):
m
mol, 97%) as a clear oil. 1H
0.87 (3H, s), 0.96 (3H, s),
1067, 820; HR-FABMS (matrix, m-nitrobenzyl alcohol) m/z:
d

Y. Hanaki et al. / Tetrahedron 69 (2013) 7636e7645
7641
1.15 (3H, d, J¼6.4 Hz), 1.23e1.67 (13H, m), 2.19e2.26 (2H, m), 2.34
(1H, m), 2.53 (1H, dd, J¼15.9, 9.0 Hz), 2.57 (2H, t, J¼7.8 Hz), 2.63 (1H,
dd, J¼15.9, 3.7 Hz), 3.46 (1H, m), 3.67 (1H, m), 3.78 (3H, s), 4.07 (1H,
m), 4.21 (1H, m), 4.48e4.57 (4H, m), 4.95e5.04 (2H, m), 5.04 (2H, s),
5.11 (1H, m), 5.83 (1H, m), 6.77e6.85 (5H, m), 7.16e7.45 (13H, m);
a refrigerator for 22 h, then diluted with H2O (4 mL) and warmed at
room temperature. The mixture was extracted with CHCl3
(6 mLꢂ3). 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%/50%
EtOAc/hexane containing 0.1% AcOH) to afford a seco-acid (14.1 mg,
13C NMR (100 MHz, CDCl3, 0.0020 M; ppm):
d 14.6, 21.6, 24.8, 25.6,
26.5, 26.8, 31.3, 34.2, 34.7, 35.6, 35.8, 36.0, 36.9, 41.1, 55.3, 63.9, 68.2,
70.0, 71.1, 71.7, 72.7, 75.0, 77.6, 100.1, 111.8, 113.8 (2C), 115.2, 116.6,
121.2, 127.5, 127.5 (2C), 127.6 (2C), 127.9, 128.3 (2C), 128.6 (2C),
129.2, 129.4 (2C), 130.8, 135.2, 137.3, 138.7, 144.7, 158.9, 159.2, 172.3;
IR (KBr; cmꢀ1): 2935, 1724, 1514, 1449, 1249, 1070; HR-FABMS
20.1
To a solution of the seco-acid (12.7 mg, 18.1
(7.6 L, 54.6 mol, 3 equiv) in toluene (1.7 mL) was added 2,4,6-
trichlorobenzoyl chloride (5.6 L, 39.0 mol, 2.2 equiv) at room
temperature. The mixture was stirred at room temperature for 3 h,
and then diluted with toluene (11 mL). The supernatant of the
mixture was added dropwise to a solution of DMAP (33.3 mg,
mmol, 83%) as a clear oil.
mmol) and Et3N
m
m
m
m
(matrix, m-nitrobenzyl alcohol) m/z: 827.4497 ([MþNa]þ calcd for
28.6
C51H64O8Na 827.4499); [
a
]
D
þ5.2 (c 0.12, CHCl3).
To a vigorously stirred solution of 12a (40.4 mg, 50.2
DCM (2.6 mL) and H2O (0.47 mL) was added DDQ (22.7 mg,
100 mol, 2 equiv) at room temperature. The mixture turned
mmol) in
273 mmol, 15 equiv) in toluene (19 mL) over 2.5 h. The anhydride
flask was rinsed twice with toluene (4.3 mL) (each rinse was
added in one portion to the reaction mixture). After an additional
1 h of stirring at room temperature, H2O (15 mL) was added and
the resulting biphasic mixture was poured into EtOAc (20 mL). The
organic layer was separated, and aqueous layer was extracted with
EtOAc (20 mLꢂ3). 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, 7.5%
m
brown. After stirring at room temperature for 1 h, the color grad-
ually changed to yellow-orange with precipitation. The mixture
was poured into saturated aq NaHCO3 (8 mL), and extracted with
DCM (10 mLꢂ3). 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, 5%/
20% EtOAc/hexane) to afford an alcohol (27.1 mg, 39.6 mmol, 79%)
as a clear oil.
EtOAc/hexane) to afford 14a (8.1 mg, 11.8
m
d
mol, 65%) as a clear oil.
0.86 (3H, s), 0.98 (3H,
1H NMR (500 MHz, CDCl3, 0.011 M; ppm):
To a solution of the alcohol (26.2 mg, 38.3
were added imidazole (7.8 mg, 115 mol, 3 equiv) and TES-Cl
(7.7 L, 45.9 mol, 1.2 equiv) at room temperature. After stirring
at room temperature for 4 h, the reaction was quenched with brine
(2 mL). The resulting mixture was extracted with EtOAc (3 mLꢂ3).
The combined organic layers were washed with brine, dried over
Na2SO4, filtered, and concentrated in vacuo. The residue was puri-
fied by column chromatography (silica gel, 2% EtOAc/hexane) to
m
mol) in THF (380
m
L)
s), 1.14 (3H, d, J¼6.4 Hz), 1.33e1.69 (13H, m), 2.34 (1H, dd, J¼12.5,
10.9 Hz), 2.43 (1H, br d, J¼15.5 Hz), 2.47 (1H, dd, J¼12.5, 2.7 Hz),
2.60 (2H, t, J¼7.8 Hz), 2.70 (1H, dd, J¼16.9, 3.0 Hz), 2.80 (1H, dd,
J¼16.9, 11.6 Hz), 3.82e3.88 (2H, m), 4.16 (1H, m), 4.52 (1H, d,
J¼11.8 Hz), 4.65 (1H, d, J¼11.8 Hz), 5.05 (2H, s), 5.17 (1H, m), 5.25
(1H, m), 6.77e6.85 (3H, m), 7.18 (1H, t, J¼7.8 Hz), 7.27e7.44 (10H,
m
m
m
m); 13C NMR (125 MHz, CDCl3, 0.011 M; ppm):
d 14.2, 21.2, 24.9,
25.4, 25.9, 27.3, 31.2, 34.6, 34.7 34.8, 35.6, 36.0, 36.8, 42.7, 63.7,
68.6, 69.9, 70.5, 70.6, 71.4, 73.8, 100.0, 111.8, 115.2, 121.2, 127.5 (2C),
127.7, 127.7 (2C), 127.8, 128.4 (2C), 128.5 (2C), 129.1, 137.4, 138.4,
144.7, 158.9, 170.1, 170.6; IR (KBr; cmꢀ1): 2937, 1725, 1448, 1264,
afford a silyl ether (25.6 mg, 32.0
To a suspension of NaIO4 (15.6 mg, 72.9
phosphate buffer (750 L) was added KMnO4 (1.4 mg, 8.9
m
mol, 84%) as a clear oil.
mol, 8 equiv) in pH 7.2
mol,
m
m
m
1 equiv) in one portion. After 3 min of vortexing at room temper-
ature under an Ar atmosphere, the mixture was added to a solution
1067; HR-FABMS (matrix, m-nitrobenzyl alcohol) m/z: 685.3716
15.6
([MþH]þ calcd for C42H53O8 685.3740); [
a
]
þ16.4 (c 0.39,
D
of the silyl ether (7.1 mg, 8.9
mmol) in t-BuOH (750
mL). The reaction
CHCl3).
mixture was vortexed at room temperature for 8 min, and the re-
action was quenched with Na2S2O3 (4.3 mg). The resulting mixture
was poured into EtOAc (3 mL) and H2O (5 mL). The organic layer
was separated, and the aqueous layer was extracted with EtOAc
(5 mLꢂ3). This procedure was repeated three times and a total of
To 10% Pd/C (wet support, Degussa type E101 NE/W, Aldrich)
(4.0 mg) in a flask was added a solution of 14a (8.0 mg, 11.7 mol) in
m
EtOH (1.2 mL) at room temperature. The mixture was vigorously
stirred under a H2 atmosphere at room temperature for 2 h. The
mixture was filtered, and the filtrate was concentrated in vacuo.
The residue was purified by HPLC (column, YMC-Pack SIL SL12S05-
2510WT; solvent, i-PrOH/CHCl3/hexane¼5:15:80, flow rate, 3.0 mL/
min; pressure, 420 psi; UV detector, 254 nm; retention time,
25.0 mg (31.3 mmol) of the silyl ether was reacted. 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%/20% EtOAc/hexane containing
17.2 min) to afford 2 (4.9 mg, 9.7
(500 MHz, CDCl3, 0.0040 M; ppm): d
m
mol, 83%) as a clear oil. 1H NMR
0.87 (3H, s, H3-22 or 23), 0.98
0.1% AcOH) to afford 13a (11.6 mg, 14.2
NMR (400 MHz, CDCl3, 0.0025 M; ppm):
m
mol, 45%) as a clear oil. 1H
0.55 (6H, q, J¼7.9 Hz),
d
(3H, s, H3-22 or 23),1.21 (3H, d, J¼6.5 Hz, H3-28), 1.26e1.71 (13H, m,
H2-4, H2-5, H-8a, H2-10, H2-12, H2-13, H2-14), 2.44 (1H, dd, J¼13.0,
10.8 Hz, H-2a), 2.49e2.53 (2H, m, H-8b, OH), 2.56e2.59 (3H, m, H-
2b, H2-15), 2.73 (1H, dd, J¼16.5, 10.3 Hz, H-25a), 2.79 (1H, dd,
J¼16.5, 4.0 Hz, H-25b), 3.90 (1H, m, H-3), 4.01 (1H, m, H-27), 4.25
(1H, m, H-11), 5.08 (1H, ddd, J¼10.3, 5.2, 4.0 Hz, H-26), 5.17 (1H, m,
H-9), 6.35 (1H, br s, PheOH), 6.68 (1H, dd, J¼8.1, 1.9 Hz, H-19), 6.73
(1H, d, J¼7.6 Hz, H-21), 6.83 (1H, t, J¼1.8 Hz, H-17), 7.14 (1H, t,
0.90 (3H, s), 0.90 (9H, t, J¼7.9 Hz), 0.98 (3H, s), 1.15 (3H, d, J¼6.4 Hz),
1.34e1.87 (13H, m), 2.18 (1H, br d, J¼15.9 Hz), 2.44 (1H, dd, J¼15.6,
8.6 Hz), 2.55e2.64 (5H, m), 3.56 (1H, m), 3.90 (1H, m), 4.11 (1H, m),
4.36 (1H, m), 4.50 (1H, d, J¼11.8 Hz), 4.58 (1H, d, J¼11.8 Hz), 5.05
(2H, s), 5.10 (1H, m), 6.78e6.82 (3H, m), 7.18 (1H, t, J¼7.8 Hz),
7.26e7.45 (10H, m); 13C NMR (125 MHz, CDCl3, 0.014 M; ppm):
d 4.9
(3C), 6.8 (3C), 13.3, 21.9, 24.9, 25.6, 26.6, 27.1, 31.3, 33.5, 34.4, 35.6,
36.0, 37.1, 37.1, 41.0, 64.8, 67.5, 69.2, 70.0, 70.2, 70.9, 77.6, 101.6,
111.9, 115.2, 121.2, 127.5 (2C), 127.6, 127.8 (2C), 127.9, 128.4 (2C),
128.6 (2C), 129.2, 137.3, 138.5, 144.5, 158.9, 171.7, 172.3; IR (KBr;
cmꢀ1): 3424, 2928, 1712, 1647, 1449, 1163, 1085; HR-FABMS (matrix,
m-nitrobenzyl alcohol) m/z: 839.4537 ([MþNa]þ calcd for
J¼7.8 Hz, H-20); 13C NMR (125 MHz, CDCl3, 0.0040 M; ppm):
d 18.5
(C-28), 21.2 (C-22 or 23), 24.0 (C-13), 25.3 (C-8), 25.9 (C-22 or 23),
27.4 (C-4), 29.6 (C-14), 34.4 (C-5 or 12), 34.7 (C-5 or 12), 34.9 (C-10
or 15), 35.1 (C-10 or 15), 36.0 (C-25), 36.9 (C-6), 42.8 (C-2), 63.0 (C-
11), 68.5 (C-27), 69.1 (C-9), 70.6 (C-3), 74.5 (C-26), 100.4 (C-7), 112.6
(C-19), 115.1 (C-17), 120.7 (C-21), 129.4 (C-20), 144.6 (C-16), 156.2
(C-18), 170.0 (C-24), 172.5 (C-1); IR (KBr; cmꢀ1): 3439, 2933, 1716,
C48H68O9SiNa 839.4530); [
a
]
28.5 þ23.4 (c 0.10, CHCl3).
D
To a solution of 13a (19.7 mg, 24.1 mmol) in THF (2 mL) was
added a freshly prepared solution of buffered HF$pyridine (500
prepared by adding 75 L of HF$pyridine to 150 L of pyridine in
600
L of THF) at 4 ꢁC. The reaction mixture was stood in
m
L:
1629, 1449, 1291, 1068; HR-FABMS (matrix, glycerol) m/z: 505.2806
24.5
m
m
([MþH]þ calcd for C28H41O8 505.2801); [
a
]
þ44.0 (c 0.085,
D
m
CHCl3).

7642
Y. Hanaki et al. / Tetrahedron 69 (2013) 7636e7645
4.3. Synthesis of 27-(S)-Me-aplog-1 (3)
FABMS (matrix, glycerol) m/z: 345.1708 ([MþH]þ calcd for C20H25O5
345.1702); [
a]
25.3 þ14.5 (c 1.27, CHCl3).
D
To a solution of 7a (381 mg, 1.16 mmol), p-nitrobenzoic acid
(581 mg, 3.48 mmol, 3 equiv), and Ph3P (911 mg, 3.48 mmol,
3 equiv) in THF (6 mL) was added 1.9 M diisopropyl azodicarbox-
ylate in toluene (1.83 mL, 3.48 mmol, 3 equiv) at 0 ꢁC. The reaction
mixture was stirred at room temperature for 21 h, and the reaction
was quenched with saturated aq NaHCO3 (6 mL). After the organic
layer was separated, the aqueous layer was extracted with EtOAc
(10 mLꢂ3). The combined organic layer was washed with brine,
dried over Na2SO4, filtered, and concentrated in vacuo. The residue
was purified by column chromatography (silica gel, 2%/5%/7%/
10% EtOAc/hexane) to afford an ester (403 mg, 0.845 mmol, 73%) as
a clear oil.
To a solution of the ester (435 mg, 0.912 mmol) in THF (4.0 mL),
MeOH (1.8 mL) and H2O (2.4 mL) was added NaOH (91.0 mg,
2.28 mmol, 2.5 equiv) at 0 ꢁC. The reaction mixture was stirred at
room temperature for 12 h, and then diluted with H2O (10 mL). The
mixture was extracted with EtOAc (20 mLꢂ3). The combined or-
ganic layer was washed with brine, dried over Na2SO4, filtered, and
concentrated in vacuo. The residue was purified by column chro-
matography (silica gel, 20% EtOAc/hexane) to afford 7b (279 mg,
0.851 mmol, 93%) as a clear oil. 1H NMR (400 MHz, CDCl3, 0.046 M;
Compounds 1124 and 10 were treated in a manner similar to that
described for the synthesis of 12a to give 12b quantitatively. 1H
NMR (400 MHz, CDCl3, 0.0037 M; ppm): d 0.86 (3H, s), 0.96 (3H, s),
1.22 (3H, d, J¼6.3 Hz), 1.35e1.67 (13H, m), 2.17e2.27 (2H, m), 2.35
(1H, m), 2.56 (2H, t, J¼7.8 Hz), 2.55e2.67 (2H, m), 3.45 (1H, m), 3.60
(1H, m), 3.78 (3H, s), 3.95 (1H, m), 4.21 (1H, m), 4.51e4.62 (4H, m),
4.96e4.99 (2H, m), 5.04 (2H, s), 5.09 (1H, m), 5.82 (1H, m),
6.78e6.85 (5H, m), 7.16e7.45 (13H, m); 13C NMR (100 MHz, CDCl3,
0.0037 M; ppm):
d 16.0, 21.6, 24.8, 25.5, 26.4, 26.7, 31.3, 34.1, 34.6,
35.6, 36.0, 36.9, 37.2, 41.0, 55.3, 63.9, 68.2, 69.9, 71.0, 71.7, 72.6, 76.4,
79.1, 100.0, 111.7, 113.7 (2C), 115.1,116.6, 121.2, 127.4,127.5 (2C),127.5
(2C), 127.9, 128.3 (2C), 128.5 (2C), 129.2, 129.4 (2C), 130.7, 135.2,
137.2, 138.7, 144.6, 158.9, 159.2, 172.1; IR (KBr; cmꢀ1): 2936, 1725,
1515, 1455, 1250, 1068; HR-FABMS (matrix, m-nitrobenzyl alcohol)
28.1
m/z: 827.4497 ([MþNa]þ calcd for C51H64O8Na 827.4499); [
þ11.7 (c 0.12, CHCl3).
a]
D
Compound 12b was treated in a manner similar to that de-
scribed for the synthesis of 13a to give 13b (32% in three steps). 1H
NMR (400 MHz, CDCl3, 0.0042 M; ppm):
d
0.61 (6H, q, J¼7.9 Hz),
0.89 (3H, s), 0.93 (9H, t, J¼7.9 Hz), 0.97 (3H, s), 1.18 (3H, d, J¼6.3 Hz),
1.31e1.75 (13H, m), 2.12 (1H, br d, J¼14.2 Hz), 2.48e2.63 (6H, m),
3.47 (1H, m), 3.91 (1H, m), 4.09 (1H, m), 4.17 (1H, dt, J¼7.8, 4.5 Hz),
4.54 (1H, d, J¼11.9 Hz), 4.62 (1H, d, J¼11.9 Hz), 5.05 (2H, s), 5.08 (1H,
m), 6.78e6.82 (3H, m), 7.18 (1H, t, J¼7.8 Hz), 7.26e7.45 (10H, m); 13C
ppm):
d
1.15 (3H, d, J¼6.5 Hz), 1.83e1.92 (2H, m), 1.98e2.14 (3H, m),
2.73e2.91 (4H, m), 3.66 (1H, dt, J¼9.5, 3.2 Hz), 3.81 (3H, s), 4.03 (1H,
m), 4.13 (1H, dd, J¼10.1 4.4 Hz), 4.53 (1H, d, J¼11.0 Hz), 4.60 (1H, d,
J¼11.0 Hz), 6.88e6.92 (2H, m), 7.28e7.31 (2H, m); 13C NMR
NMR (125 MHz, CDCl3, 0.017 M; ppm): d 5.1 (3C), 6.9 (3C), 15.7, 22.1,
(100 MHz, CDCl3, 0.046 M; ppm):
d
17.7, 26.0, 30.0, 30.5, 34.7, 44.0,
24.9, 25.5, 26.6, 27.3, 31.3, 33.3, 34.4, 35.6, 36.0, 37.0, 39.7, 41.1, 64.8,
67.5, 69.2, 70.0, 71.1, 72.7, 78.6, 101.6, 111.9, 115.2, 121.2, 127.5, 127.5
(2C), 127.6 (2C), 127.9, 128.3 (2C), 128.5 (2C), 129.2, 137.3, 138.7,
144.5, 158.9, 171.9 (2C); IR (KBr; cmꢀ1): 3440, 2939, 1713, 1646,
55.3, 67.6, 72.2, 79.2, 113.9 (2C), 129.6 (2C), 130.5, 159.4; IR (KBr;
cmꢀ1): 3435, 2921, 1626, 1513, 1247, 1065, 817; HR-FABMS (matrix,
m-nitrobenzyl alcohol) m/z: 329.1255 ([MþH]þ calcd for
C16H25O3S2 329.1245); [
a
]
25.1 þ28.9 (c 0.74, CHCl3).
1449, 1158, 1091; HR-FABMS (matrix, m-nitrobenzyl alcohol) m/z:
D
28.4
Compound 7b was treated in a manner similar to that described
839.4537 ([MþNa]þ calcd for C48H68O9SiNa 839.4530); [
þ20.1 (c 0.18, CHCl3).
a]
D
for the synthesis of 8a to give 8b (49%). 1H NMR (500 MHz, CDCl3,
0.0043 M; ppm):
d
1.21 (3H, d, J¼6.4 Hz), 1.85e1.93 (2H, m), 2.03
Compound 13b was treated in a manner similar to that de-
scribed for the synthesis of 14a to give 14b (58% in two steps). 1H
(1H, m), 2.10 (1H, m), 2.72e2.87 (4H, m), 3.64 (1H, m), 3.79 (1H, m),
3.81 (3H, s), 4.14 (1H, dd, J¼10.0, 4.6 Hz), 4.50 (1H, d, J¼11.0 Hz),
4.58 (1H, d, J¼12.0 Hz), 4.62 (1H, d, J¼12.0 Hz), 4.66 (1H, d,
J¼11.0 Hz), 6.86e6.89 (2H, m), 7.25e7.37 (7H, m); 13C NMR
NMR (500 MHz, CDCl3, 0.013 M; ppm):
d 0.86 (3H, s), 0.98 (3H, s),
1.19 (3H, d, J¼6.5 Hz), 1.27e1.67 (13H, m), 2.34 (1H, dd, J¼12.6,
10.9 Hz), 2.43 (1H, dd, J¼12.6, 2.8 Hz), 2.48 (1H, br d, J¼15.5 Hz),
2.59 (2H, t, J¼7.8 Hz), 2.72 (1H, dd, J¼16.8, 3.0 Hz), 2.95 (1H, dd,
J¼16.8, 11.6 Hz), 3.82e3.88 (2H, m), 4.16 (1H, m), 4.52 (1H, d,
J¼11.9 Hz), 4.57 (1H, d, J¼11.9 Hz), 5.03 (1H, m), 5.05 (2H, s), 5.16
(1H, br s), 6.78e6.84 (3H, m), 7.18 (1H, t, J¼7.9 Hz), 7.27e7.45 (10H,
(125 MHz, CDCl3, 0.0043 M; ppm): d 15.7, 26.1, 30.0, 30.4, 37.1, 44.3,
55.3, 71.1, 72.6, 76.7, 78.3, 113.8 (2C), 127.5, 127.6 (2C), 128.3 (2C),
129.6 (2C),131.1,138.9,159.2; IR (KBr; cmꢀ1): 2899,1614,1515,1463,
1246, 1039, 819; HR-FABMS (matrix, glycerol); m/z: 419.1734
([MþH]þ calcd for C23H31O3S2 419.1715); [
a
]
9.8 þ8.8 (c 0.19, CHCl3).
m); 13C NMR (125 MHz, CDCl3, 0.013 M; ppm):
d 16.5, 21.3, 24.8,
D
Compound 8b was treated in a manner similar to that de-
25.3, 25.9, 27.3, 31.2, 34.5, 34.7 (2C), 35.6, 36.0, 36.9, 42.8, 63.7, 68.7,
70.0, 70.6, 71.8, 73.2, 75.2, 100.1, 111.8, 115.2, 121.2, 127.5 (2C), 127.6
(3C), 127.9, 128.3 (2C), 128.5 (2C), 129.1, 137.4, 138.5, 144.7, 158.9,
170.2, 170.6; IR (KBr; cmꢀ1): 2937, 1726, 1447, 1265, 1067; HR-
scribed for the synthesis of 9a to give 9b (87%). 1H NMR (500 MHz,
CDCl3, 0.013 M; ppm):
d
1.23 (3H, d, J¼5.5 Hz), 2.60 (1H, ddd,
J¼16.5, 4.8, 2.1 Hz), 2.74 (1H, ddd, J¼16.5, 7.1, 2.2 Hz), 3.65 (1H, m),
3.80 (3H, s), 3.91 (1H, dt, J¼7.1, 4.8 Hz), 4.52 (1H, d, J¼11.0 Hz), 4.53
(1H, d, J¼11.9 Hz), 4.56 (1H, d, J¼11.0 Hz), 4.62 (1H, d, J¼11.9 Hz),
6.85e6.88 (2H, m), 7.22e7.36 (7H, m), 9.75 (1H, t, J¼2.2 Hz);
FABMS (matrix, m-nitrobenzyl alcohol) m/z: 685.3737 ([MþH]þ
19.7
calcd for C42H53O8 685.3740); [
a]
þ43.4 (c 0.44, CHCl3).
D
Compound 14b was treated in a manner similar to that de-
13C NMR (125 Hz, CDCl3, 0.013 M; ppm):
d 16.2, 45.3, 55.3, 71.2,
scribed for the synthesis of 2 to give 3 (77%). 1H NMR (500 MHz,
72.0, 76.1, 77.6, 113.9 (2C), 127.6, 127.7 (2C), 128.4 (2C), 129.5
CDCl3, 0.0079 M; ppm): d 0.87 (3H, s, H3-22 or 23), 0.97 (3H, s, H3-
(2C), 130.2, 138.4, 159.4, 201.1; IR (KBr; cmꢀ1): 2871, 1723, 1615,
22 or 23), 1.18 (3H, d, J¼6.6 Hz, H3-28), 1.34e1.72 (13H, m, H2-4, H2-
5, H-8a, H2-10, H2-12, H2-13, H2-14), 2.44 (1H, dd, J¼13.2, 10.9 Hz,
H-2a), 2.50e2.54 (2H, m, H-8b, OH), 2.57 (2H, t, J¼7.8 Hz, H2-15),
2.59 (1H, dd, J¼13.2, 2.9 Hz, H-2b), 2.68 (1H, dd, J¼16.5, 3.0 Hz, H-
25a), 2.86 (1H, dd, J¼16.5, 11.5 Hz, H-25b), 3.91 (1H, tt, J¼11.0,
2.9 Hz, H-3), 4.11 (1H, m, H-27), 4.25 (1H, m, H-11), 5.12 (1H, dt,
J¼11.5, 3.0 Hz, H-26), 5.17 (1H, m, H-9), 6.29 (1H, br s, Ph-OH), 6.68
(1H, dd, J¼8.1, 2.0 Hz, H-19), 6.72 (1H, d, J¼7.6 Hz, H-21), 6.82 (1H, t,
J¼1.8 Hz, H-17), 7.13 (1H, t, J¼7.8 Hz, H-20); 13C NMR (125 MHz,
1515, 1463, 1247, 1173, 821; HR-FABMS (matrix, glycerol); m/z:
329.1761 ([MþH]þ calcd for C20H25O4 329.1753); [
a]
þ31.6 (c
10.9
D
0.21, CHCl3).
Compound 9b was treated in a manner similar to that described
for the synthesis of 10a to give 10b (88%). 1H NMR (400 MHz, CDCl3,
0.074 M; ppm):
d
1.23 (3H, d, J¼6.3 Hz), 2.64 (1H, dd, J¼15.9, 5.2 Hz),
2.69 (1H, dd, J¼15.9, 6.8 Hz), 3.64 (1H, m), 3.78 (3H, s), 3.85 (1H, m),
4.54 (1H, d, J¼11.8 Hz), 4.57 (2H, s), 4.62 (1H, d, J¼11.8 Hz),
6.83e6.86 (2H, m), 7.22e7.33 (7H, m); 13C NMR (100 MHz, CDCl3,
CDCl3, 0.0079 M; ppm): d 18.1 (C-28), 21.2 (C-22 or 23), 24.3 (C-13),
0.074 M; ppm):
d
16.1, 36.4, 55.3, 71.2, 72.4, 76.0, 78.6, 113.8 (2C),
25.2 (C-8), 25.9 (C-22 or 23), 27.3 (C-4), 29.6 (C-14), 34.6 (C-5 or 12),
34.6 (C-5 or 12), 34.9 (C-10 or 25), 35.0 (C-10 or 25), 35.2 (C-15),
36.9 (C-6), 42.9 (C-2), 62.9 (C-11), 68.4 (C-27), 69.0 (C-9), 70.6 (C-3),
127.6, 127.7 (2C), 128.4 (2C), 129.6 (2C), 130.1, 138.3, 159.3, 176.7; IR
(KBr; cmꢀ1): 3430, 2929, 1710, 1647, 1518, 1249, 1068, 820; HR-

Y. Hanaki et al. / Tetrahedron 69 (2013) 7636e7645
7643
75.4 (C-26), 100.4 (C-7), 112.6 (C-19), 115.0 (C-17), 120.7 (C-21),
129.4 (C-20), 144.6 (C-16), 156.3 (C-18), 169.9 (C-24), 172.4 (C-1); IR
(KBr; cmꢀ1): 3430, 2937, 1717, 1539, 1455, 1291, 1068; HR-FABMS
(matrix, glycerol) m/z: 505.2799 ([MþH]þ calcd for C28H41O8
(125 MHz, CDCl3, 0.032 M; ppm):
d
4.9 (3C), 6.8 (3C), 22.3, 24.9,
25.5, 26.6, 27.5, 31.3, 33.3, 34.4, 35.7, 36.0, 37.0, 40.4, 41.3, 59.1, 64.6,
67.6, 68.3, 69.3, 70.0, 76.6, 101.4, 111.9, 115.2, 121.2, 127.5 (2C), 127.9,
128.5 (2C), 129.2, 137.3, 144.5, 158.9, 171.5, 172.6; IR (KBr; cmꢀ1):
3439, 2939, 1726, 1646, 1449, 1160, 1066; HR-FABMS (matrix, m-
nitrobenzyl alcohol) m/z: 726.4154 ([M]þ calcd for C41H62O9Si
505.2801); [
a
]
24.7 þ49.8 (c 0.18, CHCl3).
D
4.4. Synthesis of 27-O-Me-aplog-1 (4)
726.4163) [
a
]
28.6 þ16.9 (c 0.18, CHCl3).
D
Compound 13c was treated in a manner similar to that de-
scribed for the synthesis of 2 to give a seco-acid (96%). To a solution
Compound 9c prepared as reported32 was treated in a manner
similar to that described for the synthesis of 10a to give 10c (95%).
of the seco-acid (8.4 mg, 13.7
3 equiv) in toluene (1.3 mL) was added 2,4,6-trichlorobenzoyl
chloride (3.0 L, 20.9 mol, 1.5 equiv) at room temperature. The
mmol) and Et3N (5.7 mL, 41.0 mmol,
1H NMR (400 MHz, CDCl3, 0.21 M; ppm):
d
2.61 (1H, dd, J¼15.9,
7.1 Hz), 2.66 (1H, dd, J¼15.9, 5.6 Hz), 3.37 (3H, s), 3.44 (1H, dd,
J¼10.1, 5.2 Hz), 3.51 (1H, dd, J¼10.1, 5.0 Hz), 3.78 (3H, s), 4.01 (1H,
m), 4.56 (1H, d, J¼11.2 Hz), 4.60 (1H, d, J¼11.2 Hz), 6.84e6.87 (2H,
m), 7.24e7.27 (2H, m); 13C NMR (100 MHz, CDCl3, 0.21 M; ppm):
m
m
mixture was stirred at room temperature for 3.5 h, and then diluted
with toluene (8.5 mL). The supernatant of the mixture was added
dropwise to a solution of DMAP (25.1 mg, 206 mmol, 15 equiv) in
d
37.3, 55.3, 59.3, 72.1, 74.0 (2C), 113.8 (2C), 129.5 (2C), 130.1, 159.3,
toluene (15 mL) over 3 h. The anhydride flask was rinsed twice with
toluene (2.0 mL) (each rinse was added in one portion to the re-
action mixture). After an additional 1 h of stirring at room tem-
perature, H2O (20 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
(20 mLꢂ3). 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, 5%/20%
176.9; IR (KBr; cmꢀ1): 3440, 2929, 1712, 1617, 1514, 1247, 1035, 824;
HR-FABMS (matrix, glycerol) m/z: 225.1230 ([MþH]þ calcd for
C13H19O5 255.1232); [
a]
7.4 þ14.5 (c 0.48, CHCl3).
D
Compounds 1124 and 10c were treated in a manner similar to
that described for the synthesis of 12a to give 12c quantitatively. 1H
NMR (400 MHz, CDCl3, 0.011 M; ppm):
d 0.87 (3H, s), 0.97 (3H, s),
1.33e1.68 (13H, m), 2.20e2.27 (2H, m), 2.35 (1H, m), 2.53e2.63 (4H,
m), 3.35 (3H, s), 3.41 (1H, dd, J¼10.0, 5.0 Hz), 3.45 (1H, m), 3.48 (1H,
dd, J¼10.0, 5.2 Hz), 3.78 (3H, s), 4.04 (1H, m), 4.21 (1H, m), 4.54 (1H,
d, J¼11.2 Hz), 4.59 (1H, d, J¼11.2 Hz), 4.99e5.06 (2H, m), 5.04 (2H,
s), 5.12 (1H, m), 5.84 (1H, m), 6.77e6.87 (5H, m), 7.18 (1H, t,
J¼7.8 Hz), 7.23e7.45 (7H, m); 13C NMR (100 MHz, CDCl3, 0.011 M;
EtOAc/hexane) to afford 14c (6.2 mg, 10.4
m
d
mol, 76%) as a clear oil.
0.86 (3H, s), 0.97 (3H,
1H NMR (500 MHz, CDCl3, 0.021 M; ppm):
s), 1.32e1.68 (13H, m), 2.36 (1H, dd, J¼12.7, 10.0 Hz), 2.45 (1H, br d,
J¼15.6 Hz), 2.52 (1H, dd, J¼12.7, 2.7 Hz), 2.59 (2H, t, J¼7.7 Hz), 2.71
(1H, dd, J¼16.9, 3.1 Hz), 2.86 (1H, dd, J¼16.9, 11.5 Hz), 3.36 (3H, s),
3.52 (2H, m), 3.87 (1H, m), 4.17 (1H, m), 5.05 (2H, s), 5.18e5.22 (2H,
m), 6.78e6.82 (3H, m), 7.19 (1H, t, J¼7.9 Hz), 7.30e7.45 (5H, m); 13C
ppm):
d 21.5, 24.8, 25.5, 26.5, 26.7, 31.3, 34.2, 34.6, 35.6, 36.0, 36.9,
38.0, 41.1, 55.3, 59.2, 63.9, 68.2, 69.9, 71.7, 72.0, 74.4, 74.6, 100.0,
111.7, 113.7 (2C), 115.1, 116.6, 121.2, 127.5 (2C), 127.9, 128.5 (2C),
129.2, 129.4 (2C), 130.6, 135.2, 137.2, 144.6, 158.8, 159.2, 171.6; IR
(KBr): 2935, 1726, 1515, 1454, 1249, 1072; HR-FABMS (matrix, m-
nitrobenzyl alcohol) m/z: 714.4132 ([M]þ calcd for C44H58O8
NMR (125 Hz, CDCl3, 0.021 M; ppm):
d 21.2, 24.8, 25.3, 25.9, 27.3,
31.1, 34.7, 34.8, 35.6, 36.0, 36.9, 37.3, 42.8, 59.4, 63.7, 68.5, 68.9, 70.0,
70.6, 72.9, 100.2, 111.9, 115.2, 121.3, 127.5 (2C), 127.9, 128.5 (2C),
129.1, 137.4, 144.7, 158.9, 170.0, 170.3; IR (KBr; cmꢀ1): 2937, 1726,
1448, 1267, 1065; HR-FABMS (matrix, m-nitrobenzyl alcohol) m/z:
714.4132); [
a]
26.8 þ17.6 (c 0.37, CHCl3).
D
Compound 12c was treated in a manner similar to that de-
scribed for the synthesis of 13a to give an alcohol (91%). To a so-
lution of the alcohol (101 mg, 0.170 mmol) in THF (1.7 mL) were
595.3265 ([MþH]þ calcd for C35H47O8 595.3271) [
a]
10 þ38.3 (c 0.94,
D
CHCl3).
added imidazole (34.7 mg, 0.510 mmol, 3 equiv) and TES-Cl (34
m
L,
To 10% Pd/C (wet support, Degussa type E101 NE/W, Aldrich)
(3.0 mg) in a flask was added a solution of 14c (6.2 mg, 10.4 mol)
0.203 mmol, 1.2 equiv) at room temperature. After stirring at room
temperature for 4 h, the reaction was quenched with brine
(2.5 mL). The resulting mixture was extracted with EtOAc
(4 mLꢂ3). 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%/2% EtOAc/
hexane) to afford a silyl ether (108 mg, 0.153 mmol, 90%) as a clear
oil.
m
in MeOH (0.6 mL) at room temperature. The mixture was vigor-
ously stirred under a H2 atmosphere at room temperature for 1 h.
The mixture was filtered, and the filtrate was concentrated in
vacuo. The residue was purified by HPLC (column, YMC-Pack SIL
SL12S05-2510WT; solvent, i-PrOH/CHCl3/hexane¼1:19:80, flow
rate, 3.0 mL/min; pressure, 500 psi; UV detector, 254 nm; retention
time, 23.3 min) to afford 4 (3.9 mg, 7.7
NMR (500 MHz, CDCl3, 0.0032 M; ppm):
m
mol, 74%) as a clear oil. 1H
0.86 (3H, s, H3-22 or 23),
To a suspension of NaIO4 (41.5 mg, 0.194 mmol, 8 equiv) in pH
d
7.2 phosphate buffer (2 mL) was added KMnO4 (3.8 mg 24.1
1 equiv) in one portion. After 10 min of stirring at room tempera-
ture under an Ar atmosphere, the mixture was added to a solution
m
mol,
0.96 (3H, s, H3-22 or 23), 1.33e1.73 (13H, m, H2-4, H2-5, H-8a, H2-
10, H2-12, H2-13, H2-14), 2.41 (1H, dd, J¼13.2, 10.9 Hz, H-2a), 2.47
(1H, br d, J¼15.6 Hz, H-8b), 2.56 (2H, t, J¼7.8 Hz, H2-15), 2.58 (1H,
dd, J¼10.4, 2.7 Hz, H-2b), 2.75 (1H, dd, J¼16.8, 3.2 Hz, H-25a), 2.89
(1H, dd, J¼16.8, 11.3 Hz, H-25b), 3.38 (3H, s, OMe), 3.56 (2H, d,
J¼4.5 Hz, H2-27), 3.92 (1H, dt, J¼11.0, 2.8 Hz, H-3), 4.27 (1H, m, H-
11), 5.20e5.24 (2H, m, H-9, H-26), 6.39 (1H, s, PheOH), 6.68 (1H,
dd, J¼8.1, 1.7 Hz, H-19), 6.72 (1H, d, J¼7.7 Hz, H-21), 6.86 (1H, br s,
H-17), 7.13 (1H, t, J¼7.8 Hz, H-20); 13C NMR (125 MHz, CDCl3,
of the silyl ether (17.2 mg, 24.3 mmol) in t-BuOH (2 mL). The reaction
mixture was stirred at room temperature for 1 h, and the reaction
was quenched with Na2S2O3 (11.5 mg). The resulting mixture was
poured into EtOAc (10 mL) and water (10 mL). The organic layer was
separated, and the aqueous layers were washed with brine, dried
over Na2SO4, filtered, and concentrated in vacuo. The residue was
purified by column chromatography (silica gel, 5%/10% EtOAc/
0.099 M);
d 21.2 (C-22 or 23), 24.2 (C-13), 25.3 (C-8), 25.9 (C-22 or
hexane containing 0.5% AcOH) to afford 13c (11.6 mg, 16.0
m
d
mol,
0.60
23), 27.3 (C-4), 29.5 (C-14), 34.6 (C-5 or 12), 34.6 (C-5 or 12), 35.0
(C-10 or 15), 35.1 (C-10 or 15), 36.9 (C-6), 37.2 (C-25), 42.9 (C-2),
59.4 (OMe), 62.7 (C-11), 68.6 (C-9), 69.7 (C-26), 70.7 (C-3), 72.7 (C-
27), 100.3 (C-7), 112.6 (C-19), 115.0 (C-17), 120.6 (C-21), 129.3 (C-
20), 144.6 (C-16), 156.4 (C-18), 169.9 (C-24), 171.8 (C-1); IR (KBr;
cmꢀ1): 3431, 2935, 1723, 1597, 1456, 1289, 1067; HR-FABMS (matrix,
66%) as a clear oil. 1H NMR (500 MHz, CDCl3, 0.017 M; ppm):
(6H, q, J¼7.8 Hz), 0.92 (3H, s), 0.93 (9H, t, J¼7.9 Hz), 0.99 (3H, s),
1.36e1.77 (13H, m), 2.23 (1H, br d, J¼15.7 Hz), 2.49 (1H, dd, J¼15.3,
8.4 Hz), 2.54 (1H, dd, J¼15.3, 4.5 Hz), 2.57e2.65 (4H, m), 3.30 (1H,
dd, J¼9.5, 6.4 Hz), 3.36 (3H, s), 3.39 (1H, dd, J¼9.5, 5.1 Hz), 4.01 (1H,
m), 4.13 (1H, m), 4.29 (1H, m), 5.05 (2H, s), 5.13 (1H, m), 6.78e6.83
(3H, m), 7.19 (1H, t, J¼7.8 Hz), 7.30e7.45 (5H, m); 13C NMR
glycerol) m/z: 505.2846 ([MþH]þ calcd for C28H41O8 505.2801);
25.9
[a
]
þ54.0 (c 0.20, CHCl3).
D

7644
Y. Hanaki et al. / Tetrahedron 69 (2013) 7636e7645
4.5. Inhibition of the specific binding of [3H]PDBu to PKC
and -C1B peptide
d
Screening Committee for Anticancer Drugs supported by a Grant-
in-Aid for Scientific Research on Innovative Areas ‘Scientific Sup-
port Programs for Cancer Research’ from The Ministry of Education,
Culture, Sports, Science and Technology, Japan. This study was
partly carried out with the JEOL JMS-700 MS spectrometer in the
joint Usage/Research Center (JURC) at the Institute for Chemical
Research, Kyoto University, Japan.
d
The binding of [3H]PDBu to PKC
d
and
d
-C1B peptide was eval-
uated by the procedure of Sharkey and Blumberg35 with modifi-
cations as reported previously36 using 50 mM tris-maleate buffer
(pH 7.4 at 4 ꢁC), 13.8 nM
PDBu (22.1 Ci/mmol), 50
d
m
-C1B peptide or 3 nM PKC
g/mL 1,2-dioleoyl-sn-glycero-3-phospho-
-serine sodium salt (Sigma), 3 mg/mL bovine g-globulin, and var-
d
, 20 nM [3H]
L
Supplementary data
ious concentrations of an inhibitor. Binding affinity was evaluated
based on the concentration required to cause 50% inhibition of the
specific binding of [3H]PDBu, the IC50, which was calculated by
a computer program with a probit procedure. The inhibition con-
stant, Ki, was calculated by the method of Sharkey and Blumberg.35
Results of HPLC analyses and NMR spectra of 2e4, and growth
inhibitoryactivities of aplog-1, 2, 3, and 4 against 39 human cancer cell
lines. Supplementary data associated with this article can be found in
the online version, at http://dx.doi.org/10.1016/j.tet.2013.02.008.
4.6. Activation of PKC
d
References and notes
The activation of PKC
d
was evaluated with the Pep TagÒ non-
1. Nishizuka, Y. Nature 1984, 308, 693e698.
2. Nishizuka, Y. FASEB J. 1995, 9, 484e496.
radioactive protein kinase assay (Promega, Tokyo, Japan) accord-
ing to the manufacturer’s instructions. After the reaction had been
stopped by heating at 95 ꢁC for 10 min, the samples were loaded on
to a 0.8% agarose gel (Nippon Gene, Toyama, japan), which was then
quantified by densitometry software (NIH ImageJ, Bethesda, MD).
3. Newton, A. C. Chem. Rev. 2001, 101, 2353e2364.
4. Arcoleo, J. P.; Weinstein, I. B. Carcinogenesis 1985, 6, 213e217.
5. Pettit, G. R.; Herald, C. L.; Doubek, D. L.; Herald, D. L.; Arnold, E.; Clardy, J. J. Am.
Chem. Soc. 1982, 104, 6846e6848.
ꢀ
ꢀ
6. Szallasi, Z.; Smith, C. B.; Pettit, G. R.; Blumberg, P. M. J. Biol. Chem. 1994, 269,
2118e2124.
ꢀ
ꢀ
7. Szallasi, Z.; Denning, M. F.; Smith, C. B.; Dlugosz, A. A.; Yuspa, S. H.; Pettit, G. R.;
Blumberg, P. M. Mol. Pharmacol. 1994, 46, 840e850.
8. Wang, Q. M. J.; Bhattacharyya, D.; Garfield, S.; Nacro, K.; Marquez, V. E.;
Blumberg, P. M. J. Biol. Chem. 1999, 274, 37233e37239.
9. Lu, Z. M.; Hornia, A.; Jiang, Y. W.; Zang, Q.; Ohno, S.; Foster, D. A. Mol. Cell. Biol.
1997, 17, 3418e3428.
10. Reddig, P. J.; Dreckschmidt, N. E.; Ahrens, H.; Simsiman, R.; Tseng, C. P.; Zou, J.;
Oberley, T. D.; Verma, A. K. Cancer Res. 1999, 59, 5710e5718.
11. Jackson, D.; Zheng, Y.; Lyo, D.; Shen, Y.; Nakayama, K.; Nakayama, K. I.;
Humphries, M. J.; Reyland, M. E.; Foster, D. A. Oncogene 2005, 24, 3067e3072.
12. Nelson, T. J.; Alkon, D. L. Trends Biochem. Sci. 2009, 34, 136e145.
13. Mehla, R.; Bivalkar-Mehla, S.; Zhang, R.; Handy, I.; Albrecht, H.; Giri, S.;
Nagarkatti, P.; Nagarkatti, M.; Chauhan, A. PLoS One 2010, 5, e11160.
14. Schaufelberger, D. E.; Koleck, M. P.; Beutler, J. A.; Vatakis, A. M.; Alvarado, A. B.;
Andrews, P.; Marzo, L. V.; Muschik, G. M.; Roach, J.; Ross, J. T.; Lebherz, W. B.;
Reeves, M. P.; Eberwein, R. M.; Rodgers, L. L.; Testerman, R. P.; Snader, K. M.;
Forenza, S. J. Nat. Prod. 1991, 54, 1265e1270.
4.7. EBV-EA induction test
Human B-lymphoblastoid Raji cells (5ꢂ105/mL) were incubated
at 37 ꢁC under a 5% CO2 atmosphere in 1 mL of RPMI 1640 medium
(supplemented with 10% fetal bovine serum) with 4 mM sodium n-
butyrate (a synergist) and 10, 100, or 1000 nM of each test com-
pound. Each test compound was added as 2
m
L of a DMSO solution
(5, 50, and 500 M stock solution) along with 2
m
mL of DMSO; the
final DMSO concentration was 0.4%. After 48 h of incubation,
smears were made from the cell suspension, and the EBV-EA-
expressing cells were stained by a conventional indirect immuno-
fluorescence technique with an NPC patient’s serum (a gift from
Kobe University, Japan) and FITC-labeled anti-human IgG (DAKO,
Glostrup, Denmark) as reported previously.40,41 In each assay, at
least 500 cells were counted and the proportion of the EA-positive
cells was recorded. Cell viability exceeded 60% in all experiments
except for that with TPA at 10ꢀ6 M (>50%).
15. Kageyama, M.; Tamura, T.; Nantz, M. H.; Roberts, J. C.; Somfai, P.; Whirtenour, D. C.;
Masamune, S. J. Am. Chem. Soc. 1990, 112, 7407e7408.
16. Evans, D. A.; Carter, P. H.; Carreira, E. M.; Charette, A. B.; Prunet, J. A.; Lautens,
M. J. Am. Chem. Soc. 1999, 121, 7540e7552.
17. Ohmori, K.; Ogawa, Y.; Obitsu, T.; Ishika, Y.; Nishiyama, S.; Yamamura, S. Angew.
Chem., Int. Ed. 2000, 39, 2290e2294.
18. Keck, G. E.; Poudel, Y. B.; Cummins, T. J.; Rudra, A.; Covel, J. A. J. Am. Chem. Soc.
2011, 133, 744e747.
4.8. Measurements of cell growth inhibition
19. Halford, B. Chem. Eng. News October 24 2011, 10e19.
20. Wender, P. A.; Baryza, J. L.; Bennett, C. E.; Bi, C.; Brenner, S. E.; Clarke, M. O.;
Horan, J. C.; Kan, C.; Lacote, E.; Lippa, B.; Nell, P. G.; Turner, T. M. J. Am. Chem.
Soc. 2002, 124, 13648e13649.
21. Wender, P. A.; Baryza, J. L.; Brenner, S. E.; DeChristopher, B. A.; Loy, B. A.; Schrier,
A. J.; Verma, V. A. Proc. Natl. Acad. Sci. U.S.A. 2011, 108, 6721e6726.
22. Kato, Y.; Scheuer, P. J. J. Am. Chem. Soc. 1974, 96, 2245e2246.
23. Shimomura, K.; Mullinix, M. G.; Kakunaga, T.; Fujiki, H.; Sugimura, T. Science
1983, 222, 1242e1244.
A panel of 39 human cancer cell lines established by Yamori and
colleagues42 according to the NCI method with modifications was
employed, and cell growth inhibitory activity was measured as
reported previously.45 In brief, the cells were plated in 96-well
plates in RPMI 1640 medium supplemented with 5% fetal bovine
serum and allowed to attach overnight. The cells were incubated
with each test compound for 48 h. Cell growth was estimated by
the sulforhodamine B assay. The 50% growth inhibition (GI50) pa-
rameter was calculated as reported previously.46 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). Cell growth in-
24. Nakagawa, Y.; Yanagita, R. C.; Hamada, N.; Murakami, A.; Takahashi, H.; Saito, N.;
Nagai, H.; Irie, K. J. Am. Chem. Soc. 2009, 131, 7573e7579.
25. Nakagawa, Y.; Kikumori, M.; Yanagita, R. C.; Murakami, A.; Tokuda, H.; Nagai,
H.; Irie, K. Biosci. Biotechnol. Biochem. 2011, 75, 1167e1173.
26. Irie, K.; Kikumori, M.; Kamachi, H.; Tanaka, K.; Murakami, A.; Yanagita, R. C.;
Tokuda, H.; Suzuki, N.; Nagai, H.; Suenaga, K.; Nakagawa, Y. Pure Appl. Chem.
2012, 84, 1341e1351.
27. Kikumori, M.; Yanagita, R. C.; Tokuda, H.; Suzuki, N.; Nagai, H.; Suenaga, K.; Irie,
hibition (% growth) by each concentration of drug (10ꢀ8, 10ꢀ7, 10ꢀ6
,
K. J. Med. Chem. 2012, 55, 5614e5626.
28. Nakamura, H.; Kishi, Y.; Pajares, M. A.; Rando, R. R. Proc. Natl. Acad. Sci. U.S.A.
1989, 86, 9672e9676.
29. Kishi, Y.; Rando, R. R. Acc. Chem. Res. 1998, 31, 163e172.
30. Suganuma, M.; Fujiki, H.; Tahira, T.; Cheuk, C.; Moore, R. E.; Sugimura, T. Car-
cinogenesis 1984, 5, 315e318.
10ꢀ5, and 10ꢀ4 M) was calculated as 100[(TꢀT0)/(CꢀT0)] using the
average of duplicate points. By processing of these values, each GI50
value, defined as 100[(TꢀT0)/(CꢀT0)]¼50, was determined.
31. Mitsunobu, O. Synthesis 1981, 1e28.
Acknowledgements
32. Nicolaou, K. C.; Fylaktakidou, K. C.; Monenschein, H.; Li, Y. W.; Weyershausen,
B.; Mitchell, H. J.; Wei, H. X.; Guntupalli, P.; Hepworth, D.; Sugita, K. J. Am. Chem.
Soc. 2003, 125, 15433e15442.
This work was supported by a Grant-in-Aid for Scientific Re-
search on Innovative Areas ‘Chemical Biology of Natural Products’
(Grant No. 23102011 to K.I.) from The Ministry of Education, Cul-
ture, Sports, Science and Technology, Japan. We also thank the
33. Inanaga, J.; Hirata, K.; Saeki, H.; Katsuki, T.; Yamaguchi, M. Bull. Chem. Soc. Jpn.
1979, 52, 1989e1993.
34. Irie, K.; Oie, K.; Nakahara, A.; Yanai, Y.; Ohigashi, H.; Wender, P. A.; Fukuda, H.;
Konishi, H.; Kikkawa, U. J. Am. Chem. Soc. 1998, 120, 9159e9167.

Y. Hanaki et al. / Tetrahedron 69 (2013) 7636e7645
7645
35. Sharkey, N. A.; Blumberg, P. M. Cancer Res. 1985, 45, 19e24.
36. Shindo, M.; Irie, K.; Nakahara, A.; Ohigashi, H.; Konishi, H.; Kikkawa, U.; Fukuda,
H.; Wender, P. A. Bioorg. Med. Chem. 2001, 9, 2073e2081.
37. Irie, K.; Nakagawa, Y.; Ohigashi, H. Curr. Pharm. Des. 2004, 10, 1371e1385.
38. Masuda, A.; Irie, K.; Nakagawa, Y.; Ohigashi, H. Biosci. Biotechnol. Biochem. 2002,
66, 1615e1617.
39. Fujiki, H.; Tanaka, Y.; Miyaki, R.; Kikkawa, U.; Nishizuka, Y.; Sugimura, T. Bio-
chem. Biophys. Res. Commun. 1984, 120, 339e343.
40. zur Hausen, H.; Bornkamm, G. W.; Schmidt, R.; Hecker, E. Proc. Natl. Acad. Sci. U.
S.A. 1979, 76, 782e785.
42. Yamori, T.; Matsunaga, A.; Sato, S.; Yamazaki, K.; Komi, A.; Ishizu, K.; Mita, I.;
Edatsugi, H.; Matsuba, Y.; Takezawa, K.; Nakanishi, O.; Kohno, H.; Nakajima, Y.;
Komatsu, H.; Andoh, T.; Tsuruo, T. Cancer Res. 1999, 59, 4042e4049.
43. Sakamoto, S.; Hatakeyama, M.; Ito, T.; Handa, H. Bioorg. Med. Chem. 2012, 20,
1990e2001.
44. Wender, P. A.; Brabander, J. D.; Harran, P. G.; Jimenez, J. M.; Koehler, M. F. T.;
Lippa, B.; Park, C. M.; Shiozaki, M. J. Am. Chem. Soc. 1998, 120, 4534e4535.
45. Monks, A.; Scudiero, D.; Skehan, P.; Shoemaker, R.; Paull, K.; Vistica, D.; Hose, C.;
Langley, J.; Cronise, P.; Vaigro-Wolff, A.; Gray-Goodrich, M.; Cabpbell, H.; Mayo, J.;
Boyd, M. J. Natl. Cancer Inst. 1991, 83, 757e766.
41. Ito, Y.; Yanase, S.; Fujita, J.; Harayama, T.; Takashima, M.; Imanaka, H. Cancer
Lett. 1981, 13, 29e37.
46. Skehan, P.;Storeng, R.;Scudiero, D.;Monks, A.;McMahon,J.;Vistica, D.;Warren,J.T.;
Bokesch, H.; Kenney, S.; Boyd, M. R. J. Natl. Cancer Inst. 1990, 82, 1107e1112.