Structure-activity studies on the spiroketal moiety of a simplified analogue of debromoaplysiatoxin with antiproliferative activity

Article abstract of DOI:10.1021/jm300566h

Aplog-1, a simplified analogue of tumor-promoting debromoaplysiatoxin, is antiproliferative but not tumor-promoting. Our recent study has suggested that local hydrophobicity around the spiroketal moiety is a crucial determinant for antiproliferative activity. To further clarify the structural features relevant to the activity, we synthesized two methyl derivatives of aplog-1, where a methyl group was installed at position 4 or 10 of the spiroketal moiety. 10-Methyl-aplog-1 (5) bound to the C1B domains of novel PKCs (δ, η, and θ) with subnanomolar K_i values, approximately 10-20 times stronger than aplog-1, and markedly inhibited the growth of many human cancer cell lines, while 4-methyl-aplog-1 (4) had levels of activity similar to those of aplog-1. Interestingly, 5 showed little tumor-promoting activity unlike the tumor promoter debromoaplysiatoxin. These results suggest that 5 is a potent PKC ligand without tumor-promoting activity and could be a therapeutic lead for the treatment of cancer, like bryostatins.

Full text of DOI:10.1021/jm300566h

Article
pubs.acs.org/jmc
Structure−Activity Studies on the Spiroketal Moiety of a Simplified
Analogue of Debromoaplysiatoxin with Antiproliferative Activity
Masayuki Kikumori,† Ryo C. Yanagita,†,‡ Harukuni Tokuda,§ Nobutaka Suzuki,§ Hiroshi Nagai,∥
Kiyotake Suenaga,⊥ and Kazuhiro Irie*,†
†Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan
‡Department of Applied Biological Science, Faculty of Agriculture, Kagawa University, Kagawa 761-0795, Japan
§Department of Complementary and Alternative Medicine, Clinical R&D, Graduate School of Medical Science, Kanazawa University,
Kanazawa 920-8640, Japan
∥Department of Ocean Sciences, Tokyo University of Marine Science and Technology, Tokyo 108-8477, Japan
⊥Faculty of Science and Technology, Keio University, Yokohama 223-8522, Japan
S
* Supporting Information
ABSTRACT: Aplog-1, a simplified analogue of tumor-promoting
debromoaplysiatoxin, is antiproliferative but not tumor-promoting. Our
recent study has suggested that local hydrophobicity around the
spiroketal moiety is a crucial determinant for antiproliferative activity.
To further clarify the structural features relevant to the activity, we
synthesized two methyl derivatives of aplog-1, where a methyl group was
installed at position 4 or 10 of the spiroketal moiety. 10-Methyl-aplog-1
(5) bound to the C1B domains of novel PKCs (δ, η, and θ) with
subnanomolar Ki values, approximately 10−20 times stronger than aplog-
1, and markedly inhibited the growth of many human cancer cell lines,
while 4-methyl-aplog-1 (4) had levels of activity similar to those of aplog-
1. Interestingly, 5 showed little tumor-promoting activity unlike the
tumor promoter debromoaplysiatoxin. These results suggest that 5 is a potent PKC ligand without tumor-promoting activity and
could be a therapeutic lead for the treatment of cancer, like bryostatins.
Bryostatin 1 (bryo-1)15 and its congeners16 isolated from the
INTRODUCTION
Tumor promoters are chemicals that enhance tumor formation
■
marine bryozoan Bugula neritina are fascinating PKC activators
without tumor-promoting activity.17 Bryo-1 binds to PKCs with
when applied repeatedly after the initial administration of a
subnanomolar Ki values.18 Since bryo-1 showed significant
small amount of carcinogen.1 The effects of tumor promoters
growth-inhibitory activity against various cancer cell lines, more
are mediated mainly by protein kinase C (PKC), a family of
than 30 clinical trials in phases I and II have been performed
serine/threonine kinases that play an important role in cellular
against a wide range of tumor types.6,8,16 Although many of
signal transduction associated with cell proliferation, differ-
them gave disappointing results for bryo-1 on its own and in
entiation, and apoptosis.2−4 Potent tumor promoters such as
combination with other anticancer drugs,19−24 further inves-
12-O-tetradecanoylphorbol 13-acetate (TPA), teleocidin B-4,
tigations are in progress.
In addition to its possible role as an anticancer drug, bryo-1
and aplysiatoxin (ATX) activate conventional and novel PKC
isozymes (PKCs) (Figure 1).2,5 Conventional PKCs (α, βI, βII,
has therapeutic potential for AD and AIDS where activation of
PKCs plays a critical role.11−14 However, its limited availability
γ) are calcium-dependent, while novel PKCs (δ, ε, η, θ) do not
require calcium for their activation. Since PKCs are involved in
numerous signal transduction pathways, they are attractive
targets in the treatment of cancer6−9 and other intractable
diseases such as diabetes,10 Alzheimer’s disease (AD),11−13 and
acquired immune deficiency syndrome (AIDS).14 In fact, TPA
and artificial PKC activators with a benzolactam skeleton
reduced levels of amyloid β, a causative protein for AD.11,12
TPA is also reported to activate latent human immunodefi-
ciency virus (HIV) and thus represents a possible adjuvant for
antivirus therapy.14 However, its therapeutic use is strictly
limited because of adverse effects as a tumor promoter.
from natural sources (18 g of bryo-1 from 13 tons of B.
neritina)25 and difficulty in total synthesis26−28 have hampered
further studies on its mode of action and structural
optimization as a therapeutic agent. Recently, excellent practical
methods for producing bryo-1 and its congeners have been
developed by the groups of Wender, Keck, Hale, Trost, and
Krische.29 Wender and colleagues developed simplified
analogues of bryo-1 showing more potent antiproliferative
Received: April 21, 2012
Published: May 24, 2012
© 2012 American Chemical Society
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binding affinity for PKCδ, which plays a tumor suppressor role
and is involved in apoptosis.40−44 The results clearly showed
that the dimethyl groups at position 6 are indispensable to
these activities but that the hydroxyl group at position 18 is not
necessary.40,41 Introduction of the dimethyl groups at position
12 did not change the PKC-binding but increased the
antiproliferative activities.43 Of note, the more hydrophobic
12,12-dimethyl-aplog-1 (3) did not show any tumor-promoting
activity in vitro or in vivo.43,44 These results suggest that
hydrophobicity around the spiroketal moiety of aplog-1 would
enhance antiproliferative activities but not tumor-promoting
activity.
To further investigate the structure−activity relationship
between the antiproliferative and tumor-promoting effects of
aplog-1 and to find more effective analogues, two methyl
derivatives of aplog-1 (4, 5, Figure 2), in which methyl groups
were installed in the spiroketal moiety at positions 4 and 10,
were synthesized because these substituents would be
responsible for strong binding of ATX to PKCδ. In this article,
we describe their antiproliferative activities, tumor-promoting
activity in vitro and in vivo, and PKC binding.
Figure 1. Structure of naturally occurring tumor promoters and
bryo‑1.
RESULTS AND DISCUSSION
effects than bryo-1.30,31 Keck and colleagues identified the
structural factors responsible for the unique biological activities
of bryo-1.32,33 Trost and colleagues established a practical route
for producing bryo-16 as a common intermediate of various
bryostatins.34,35 More recently, Wender, Keck, Hale, and
Krische have reported the total synthesis of bryo-9, bryo-1,
and bryo-7.36−39
In contrast, we identified more synthetically accessible
compounds with bryo-1-like activities (aplogs) as another
way to address the supply problem.40−44 Aplog-1 (Figure 2),40
■
Synthesis of 4 and 5. The 4-methyl derivative of aplog-1
(4) was synthesized from a previously reported epoxide (6)40
(Scheme 1). Coupling of 6 with a Kishi’s dithiane (7)46 was
achieved by the protocol of Ide and Nakata,47 and methanolysis
of the tetrahydropyranyl (THP) ether and tert-butyldimethyl-
silyl (TBS) ether generated a triol (8). O-Isopropylidenation
and oxidation of the primary alcohol provided an aldehyde.
Since attempts to install an allyl group streoselectively into the
aldehyde employing Keck’s48 or Maruoka’s49 method gave no
desired product, probably because of the steric hindrance at the
aldehyde group, a Grignard reaction was employed to generate
a homoallyl alcohol (9) as a 1:1 diastereomeric mixture.
Detachment of the isopropylidene acetal and subsequent
hydrolytic cleavage of the dithiane moiety provided three
spiroketals (10, 11, and a diastereomer) in a ratio of
approximately 1:2:3. The configuration of 10 was determined
by nuclear Overhauser enhancement (NOE) experiments
(Supporting Information). NOEs were observed between
OH-10 and H-2, H-3, or H-12, supporting a desired
conformation. Compound 11 proved to be an undesired
compound with double axial oxygen atoms. An attempt to
convert 11 into 10 employing an acid-catalyzed equilibrium
reaction40 (PPTS, 0.1 equiv, MeOH/DCM = 1:1) gave a
disappointing result. In a protic solvent such as MeOH, the
undesired spiroketal (11) with double anomeric effects could
be much more stable than the desired one (10) with a single
anomeric effect. However, only 10 could form an intra-
molecular hydrogen bond between the hydroxyl group at
position 10 and oxygen of the spiroketal. Since formation of the
desired spiroketal (10) might be hampered by the intermo-
lecular hydrogen bond between the hydroxyl group at position
10 and the protic solvent, an aprotic solvent (MeCN/DCM =
1:1) along with a stronger acid (( )-10-camphorsulfonic acid,
CSA) were employed. This condition led to a 1:1 mixture of 10
and 11. No conversion occurred in DCM even if CSA was used
as an acid catalyst. Use of PPTS in a mixed solvent (MeCN/
DCM = 1:1) also resulted in no conversion. These results
indicate both the acidity of the catalyst and permittivity of the
solvent to be important in the equilibrium reaction.
Figure 2. Structure of the simplified analogues of debromoaplysiatox-
in.
a simple and less hydrophobic analogue of ATX,45 is a new
PKC ligand with little tumor-promoting activity and showed
growth-inhibitory activities against several cancer cell lines with
potency comparable to that of bryo-1. In subsequent efforts to
identify the structural features relevant to the unique biological
profile of aplog-1, we prepared several derivatives altered at
positions 6, 12, and 18 (1−3, Figure 2) and evaluated their
antiproliferative activities against 39 human cancer cell lines and
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a
a
Scheme 1. Synthesis of 4
Scheme 2. Synthesis of 5
a
(a) (1) 7, n-BuLi, THF; (2) PPTS, MeOH; 83% in two steps; (b)
2,2-dimethoxypropane, CSA, 82%; (c) TPAP, NMO, 4 Å molecular
sieves, DCM, 80%; (d) allyl-MgBr, THF, 83%; (e) p-TsOH·H2O,
MeCN, THF, H2O, 95%; (f) Hg(ClO4)2·6H2O, DCM, MeCN, H2O;
10, 11%; 11, 23%; (g) CSA, MeCN, DCM; 10, 41%; 11, 40%; (h) 12,
TCB-Cl, Et3N, DMAP, toluene, 94%; (i) DDQ, H2O, DCM, 94%; (j)
TESCl, imidazole, THF, 89%; (k) KMnO4, NaIO4, t-BuOH, pH 7
buffer, 59%; (l) HF·pyridine, pyridine, THF, quant; (m) TCB-Cl,
Et3N, DMAP, toluene, 74%; (n) 20% Pd(OH)2/C, H2, EtOAc, 76%.
a
(a) trans-2-butene, KO-t-Bu, n-BuLi, (−)-DIP-OMe, BF3·Et2O, THF,
88%; (b) (Boc)2O, NaHMDS, THF, 87%; (c) (1) IBr, toluene, DCM;
(2) K2CO3, MeOH; 53% in two steps; (d) NaOMe, MeOH, 73%; (e)
MPMCl, NaH, DMF, THF, 97%; (f) 19, n-BuLi, THF, 96%; (g)
DDQ, 4 Å molecular sieves, DCM, 87%; (h) PPTS, MeOH, DCM,
82%; (i) TPAP, NMO, 4 Å molecular sieves, DCM, 68%; (j) TiCl4,
Ti(O-i-Pr)4, Ag2O, (S)-BINOL, allyl-SnBu3, 75%; (k) p-TsOH·H2O,
MeCN, THF, H2O, 41%; (l) Hg(ClO4)2·6H2O, DCM, MeCN, H2O,
62%; (m) 12, TCB-Cl, Et3N, DMAP, toluene, 89%; (n) DDQ, H2O,
DCM, 94%; (o) TESCl, imidazole, THF, 95%; (p) KMnO4, NaIO4, t-
BuOH, pH 7 buffer, 69%; (q) HF·pyridine, pyridine, THF, quant; (r)
TCB-Cl, Et3N, DMAP, toluene, 75%; (s) 10% Pd/C, H2, EtOH, 78%.
Yamaguchi’s esterification50 of 11 with a known carboxylic
acid (12),40 followed by switching of the protecting group from
a 4-methoxyphenylmethyl (MPM) group to a triethylsilyl
(TES) group, and subsequent oxidative cleavage of the olefin
group provided a carboxylic acid (14). Deprotection of the TES
group, lactonization, and removal of the two benzyl groups gave
4 in 14 steps from 6 with an overall yield of 2.4%.
Coupling of 18 with a dithiane (19) provided 20 in high yield
(96%).
Synthesis of the 10-methyl derivative of aplog-1 (5) started
with Brown’s diastereoselective crotylation51 of a known
aldehyde (15)40 (Scheme 2) to give a homoallyl alcohol (16)
with 92% ee (Supporting Information). Stereoselective
iodocarbonate cyclization52 of the homoallyl alcohol generated
a cyclic carbonate, which was converted to an epoxide (17) by
methanolysis. Because of steric hindrance by the methyl group,
the methyl carbonate moiety in 17 was a little susceptible to
methanolysis with K2CO3 at room temperature. To overcome
this situation, 17 was subjected to a coupling reaction with 19
according to the same procedure used in the synthesis of 3
reported previously.43 However, the desired reaction at the
epoxide site did not occur possibly because of the instability of
the carbonate under the coupling conditions. Hence, we
cleaved the methyl carbonate under alkaline conditions. A 2 h
treatment of 17 with NaOMe in MeOH at 50 °C gave an
alcohol, without formation of a five-membered ether as a
byproduct, which was converted to an MPM ether (18).
Oxidative acetal formation and deprotection of the TBS
ether provided a primary alcohol, the oxidation of which,
followed by Maruoka’s asymmetric allylation,49 produced a
homoallyl alcohol (21). Because of the high stability of the p-
methoxybenzylidene acetal moiety, acid hydrolysis of 21 gave a
triol in moderate yield (41%). Unlike the synthesis of 4,
hydrolysis of the dithiane moiety of the triol with Hg-
(ClO4)2·6H2O produced the desired isomer 22 as a main
product (62%) because of the 1,3-diaxal interaction between
the hydrogen atom at position 9 and the methyl group at
position 11 in the undesired isomer. Subsequent construction
of the diolide ring was achieved by procedures similar to those
used for the synthesis of 4. Compound 5 was obtained in 20
steps from 15 with an overall yield of 0.85%.
Antiproliferative Activities against 39 Human Cancer
Cell Lines. Antiproliferative activities of 4 and 5 were initially
evaluated using a panel of 39 human cancer cell lines as
described previously.53 The growth inhibitory activity was
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expressed as a GI50 (M), the concentration required to inhibit
cell growth by 50% compared to an untreated control
(Experimental Section). The average of the log GI50 values
for all 39 human cancer cell lines was expressed as a MG-MID.
Since the MG-MID of aplog-1 was −4.97, the cell lines with
log GI50 values less than −5.00 except for SNB-78 are listed in
Table 1 (the rest of the data are provided in the Supporting
induction (<10%) even at 1 μM, like aplog-1 (Figure 3),
suggesting that both methyl groups at positions 4 and 10 are
not crucial for tumor-promoting activity.
Table 1. Growth Inhibition of Debromoaplysiatoxin (DAT),
Aplog-1, 4, and 5 against Several Human Cancer Cell Lines
log GI50 (log M)
a
cancer type
breast
cell line
HBC-4
DAT
aplog-1
4
5
−6.47
−6.03
−4.80
−6.09
−6.46
−5.94
−5.69
−6.44
−6.33
−5.61
−4.72
−5.43
−5.60
−5.32
−5.74
−5.55
−6.56
−5.81
−4.76
−5.39
−5.90
−5.13
−5.38
−5.22
−7.48
−6.90
−6.05
−6.47
−7.07
−6.01
−6.21
−6.24
MDA-MB-231
SNB-78
CNS
colon
lung
HCC2998
NCI-H460
A549
Figure 3. EBV-EA production induced by TPA, debromoaplysiatoxin
(DAT), aplog-1, 4, and 5. 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 sodium n-butyrate. The final concentration of DMSO was
0.4%. Cell viability exceeded 60% in all experiments except for that
with TPA at 10−6 M (>50%). Error bars represent standard error of
the mean (n = 3).
melanoma
stomach
LOX-IMVI
St-4
a
Cited from ref 40.
Information). The MG-MIDs of 4-methyl-aplog-1 (4) and 10-
methyl-aplog-1 (5) were −5.03 and −5.24, respectively.
Interestingly, tumor-promoting debromoaplysiatoxin (DAT)
also showed antiproliferative activity; the MG-MID was −5.22.
As shown in Table 1, the growth inhibitory activities of
aplog-1 and 4-methyl-aplog-1 (4) were similar. On the other
hand, 10-methyl-aplog-1 (5) exhibited a 5−20 times stronger
inhibitory effect on the growth of the cancer cells listed in
Table 1 than aplog-1. Moreover, the antiproliferative activities
of 5 were greater than those of DAT except for St-4. To
examine the relationship between the MG-MID and molecular
hydrophobicity, log P values of these compounds were
evaluated by a method using HPLC54 (Table 2). The
Since 5 exhibited more potent antiproliferative activities than
4 and aplog-1, the in vivo tumor-promoting activity was
evaluated by a two-stage carcinogenesis experiment on mouse
skin (Figure 4). The skin on the back of ICR mice was treated
with a single dose of 390 nmol of 7,12-dimethylbenz[a]-
Table 2. Values of log P of Debromoaplysiatoxin (DAT),
Aplog-1, 4, and 5, Determined by HPLC54
DAT
4.4
aplog-1
3.3
4
5
log P
3.7
3.6
hydrophobicity of 4 and 5 was similar and slightly higher
than that of aplog-1. As expected, DAT was the most
hydrophobic of these compounds. These results indicate that
the antiproliferative activities of aplogs do not simply depend
on molecular hydrophobicity and that the local hydrophobicity
around position 10 would play an important role in enhancing
antiproliferative activities.
Tumor-Promoting Activity. The most likely adverse effect
of aplog-1 and its derivatives (4 and 5) would be tumor
promotion because these compounds possess the skeleton of
tumor-promoting ATX and DAT. Thus, we evaluated the
tumor-promoting activities of 4 and 5 by testing induction of
Epstein−Barr virus early antigen (EBV-EA).55,56 EBVs are
under the strict control of the host human B lymphoblastoid
Raji cells. They are activated when treated with chemicals such
as tumor promoters and produce early antigen, which can be
detected by an indirect immunofluorescence technique. The
EBV-EA-inducing activities are expressed as the percentage of
EA-positive cells. Compared to DAT that showed significant
EA induction like TPA, both 4 and 5 exhibited only weak EA
Figure 4. Tumor-promoting activity of TPA, debromoaplysiatoxin
(DAT), and 5. The back of each male 6-week-old ICR mice was
shaved with surgical clippers. From a week after initiation by a single
application of 390 nmol of DMBA in 0.1 mL acetone, 8.5 nmol of 5 in
0.1 mL of acetone was applied twice a week from week 1 to week 20
○
( ). The control group was treated with DMBA and 1.7 nmol TPA
□
●
( ) or DAT ( ). Each group consisted of 10 mice. (a) Tumor-
bearing mice (%). The differences between TPA and DAT at weeks 8,
9, 11, and 12 were statistically significant (p < 0.05, Fisher’s exact test).
(b) Number of papillomas per mouse. The differences between TPA
and DAT at weeks 9−20 were statistically significant (p < 0.05,
Welch’s t-test).
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anthracene (DMBA) and from 1 week later, with 8.5 nmol of 5
(5-fold excess of TPA and DAT) twice a week. Compound 5
did not induce any tumors at week 20. In a control experiment
using TPA (1.7 nmol twice a week), the first tumor appeared in
week 6 and the proportion of tumor-bearing mice reached
100% at week 11. The number of papillomas/mice was 8.0 in
week 20. Moreover, DAT (1.7 nmol twice a week) also
generated tumors, albeit weakly compared with TPA. DAT’s
weak tumor-promoting activity coincided with that reported
previously.57
According to Suganuma and colleagues, oscillatoxin A, which
lacks DAT’s methyl group at position 29, exhibited tumor-
promoting activity in vitro, suggesting that this methyl group is
not important for tumor-promoting activity.57 Nakamura and
colleagues reported that 3-deoxy-DAT and DAT are equipotent
as PKC activators.58 However, the tumor-promoting activity of
3-deoxy-DAT remains unknown. Taken together, the hemi-
acetal hydroxy group at position 3 and/or the methoxy group at
position 15 in DAT would be responsible for the tumor-
promoting activity.
novel PKC-C1B domains was relatively poor. On the other
hand, 5 displayed stronger affinity, 1 order of magnitude, for all
PKC C1 peptides than aplog-1; the Ki of 5 for PKCδ-C1B was
comparable to that of DAT and bryo-1 (Ki = 0.60 nM),65 and 5
showed some selectivity for novel PKC-C1B domains like
aplog-1. These results suggest that introduction of a methyl
group into position 4 could enhance the affinity for
conventional PKCs rather than for novel PKCs and that
introduction of a methyl group into position 10 would enhance
the affinity for both conventional and novel PKCs. These
results may also imply binding for conventional PKCs to be
linked to tumor-promoting activity rather than antiproliferative
activities. In fact, activation of PKCα was suggested to be
involved in cancer cell growth,66,67 and PKCδ to play a tumor
suppressor role and to be involved in apoptosis.68,69
Since a docking model for ATX and the C1B domain of
PKCδ has not yet been proposed, it is not easy to explain the
affinity of 5 for PKCδ. However, overlapping of the
pharmacophores of ATX and TPA was reported,70 which
indicated that the spiroketal moiety of ATX occupies positions
in space similar to the C-12 residues of TPA. The C-12 region
of phorbol esters was located on a hydrophobic surface of the
crystal structure of the PKCδ C1B domain71 and would be
involved in hydrophobic interaction with the C1B domain and
phosphatidylserine in cellular membranes.72 Moreover, methyl
groups at positions 4 and 10 do not seem to affect significantly
the conformation of the spiroketal moiety and the macro-
lactone ring, since each spiroketal intermediate (10, 22)
showed an NOE pattern similar to that of aplog-1 (Supporting
Information),40 and 1H NMR coupling constants of the diolide
moiety of 4 and 5 were also similar to those of aplog-1.40 Taken
together, the methyl groups at positions 4 and 10 of ATX might
be involved in hydrophobic interaction, analogous to TPA, and
the methyl group at position 10 might interact specifically with
the hydrophobic residues of novel PKCs and/or have a role in
orienting properly the adjacent side chain at position 11.
Binding Affinity for PKCs. The activation of PKCs is likely
to be responsible for the antiproliferative and tumor-promoting
activities of ATX-related compounds. We evaluated the affinity
of DAT and the methyl derivatives of aplog-1 (4, 5) for C1
domains (C1A and C1B) of PKCs (Table 3). The C1A
Table 3. Values of Ki for the Inhibition of [3H]PDBu
Binding by Debromoaplysiatoxin (DAT), Aplog-1, 4, and 5
Ki (nM)
a
b
a
a
PKC C1 peptide
DAT
aplog-1
4
5
α-C1A
β-C1A
γ-C1A
δ-C1B
ε-C1B
η-C1B
θ-C1B
0.26(0.08)
0.17(0.02)
0.38(0.06)
0.20(0.01)
0.63(0.08)
0.11(0.02)
0.11(0.03)
63
89
39
7.4
25
4.4
4.0
15(2)
4.7(0.7)
15(2)
12(0.5)
12(2)
5.5(0.4)
3.3(0.3)
17(2)
0.46(0.06)
2.0(0.2)
5.9(0.5)
2.9(0.2)
0.45(0.05)
0.54(0.08)
CONCLUSION
■
a
Values in parentheses represent the standard deviation from at least
Our previous studies indicated that the spiroketal moiety of
aplog-1 was deeply involved in PKC binding and antiprolifer-
ative activities against several cancer cell lines. To further study
the structure−activity relationship, we synthesized 4-methyl-
and 10-methyl-aplog-1 (4, 5). To our delight, the introduction
of a methyl group at position 10 enhanced the affinity for PKCδ
but not tumor-promoting activity in vitro as well as in vivo.
Compound 5 had the strongest antiproliferative effects against
cancer cell lines of any aplog synthesized to date (aplog-1, 1−
4). The compound could thus be a new drug lead for cancer,
like bryo-1. It is remarkable that the antiproliferative activities
of ATX-related compounds did not simply depend on
molecular hydrophobicity but correlated with the specific
interaction with the C1 domain of PKCs. PKC ligands that
bind more strongly and more selectively to PKCδ might
become a more pertinent lead in the development of drugs for
treating cancer.
b
three separate experiments. Cited from ref 40.
peptides were used as conventional PKC surrogates, and the
C1B peptides were employed as novel PKC surrogates, since
these domains are the main binding sites of tumor
promoters.59−62 Our accumulated data suggest that the binding
assay using PKC C1 peptides is a reliable system for evaluating
binding affinities of tumor promoters for PKC isozymes. The
binding constants of the C1 peptides for tumor promoters such
as phorbol 12,13-dibutyrate (PDBu)59 and (−)-indolactam-V63
(Supporting Information) were almost equivalent to those of
corresponding whole PKC isozymes obtained by the well
established procedure of Sharkey and Blumberg.64 Thus,
assessment of the binding affinities and selectivity for PKC
isozymes can be realized by a simple procedure using PKC C1
peptides. Recently, we reported that tumor promoters like
PDBu and ATX bound potently to the C1 domains of both
conventional and novel PKCs, while antiproliferative com-
pounds such as aplog-1 and bryo-1 exhibited some selectivity
for novel PKCs other than PKCε, that is, PKCδ, PKCη, and
PKCθ.44 The affinity of DAT for the PKC C1 domains was
quite similar to that of ATX as reported previously.41
DAT still retained, albeit weak, tumor-promoting activity in
vivo but inhibited the growth of cancer cells more potently than
aplog-1 (Table 1). On the other hand, 5 showed little tumor-
promoting activity and exhibited stronger antiproliferative
activities than DAT. These observations suggest that DAT is
a “master key” with tumor-promoting and antiproliferative
activities whereas its simplified analogue 5 is a “special key”
with only antiproliferative activities. Such simplification is quite
Although the affinity of 4 for PKCδ-C1B was 2 times
stronger than that of aplog-1, the binding selectivity of 4 for
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filtrate was concentrated in vacuo, and the residue was purified by
column chromatography (silica gel, 10% → 30% EtOAc/hexane) to
afford an acetonide (1.16 g, 1.93 mmol, 82%) as a clear oil. To a
solution of the acetonide (1.16 g, 1.93 mmol), NMO (350 mg, 2.90
mmol, 1.5 equiv), and 4 Å molecular sieves (966 mg) in DCM (16
mL) was added a suspension of tetrapropylammonium perruthenate
(34 mg, 0.097 mmol, 0.05 equiv) in DCM (2 mL). After 15 min of
stirring at room temperature, the reaction mixture was absorbed in a
small amount of silica gel. The resulting gel was directly loaded onto a
silica gel column and eluted with 10% EtOAc/hexane. Concentration
of the fractions gave an aldehyde (922 mg, 1.54 mmol, 80%) as a clear
oil. To a solution of the aldehyde (384 mg, 0.643 mmol) in THF was
added 1 M solution of allyl-MgBr in Et2O (965 μL) at −78 °C under
an Ar atmosphere. After 30 min of stirring at the same temperature,
the reaction mixture was warmed to room temperature. The reaction
mixture was quenched with saturated aqueous NH4Cl (10 mL). After
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, 10% →
20% EtOAc/hexane) to afford 9 (340 mg, 0.532 mmol, 83%) as a clear
important in developing new medicinal leads from natural
products. Further studies are necessary to identify the structural
factors of DAT involved in the tumor-promoting activity.
EXPERIMENTAL SECTION
■
General Remarks. The following spectroscopic and analytical
instruments were used: for UV, UV-2200A (Shimadzu, Kyoto, Japan);
digital polarimeter, DIP-1000 (Jasco, Tokyo, Japan); 1H and 13C
NMR, Avance I 400 and Avance III 500 (reference TMS, Bruker,
Germany); HPLC, model 600E with a model 2487 UV detector
(Waters, Tokyo, Japan); HR-FAB-MS, 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 Industries, Osaka, Japan) and YMC A60-350/
250 gel (ODS, Yamamura Chemical Laboratory, Kyoto, Japan) were
used for column chromatography. [3H]PDBu (22.1 Ci/mmol) was
purchased from Perkin-Elmer Life Sciences Research Products
(Boston, MA, U.S.). The PKC C1 peptides were synthesized as
reported previously.73 All other chemicals and reagents were
purchased from chemical companies and used without further
purification. The purity of the final compounds was greater than
95% as judged by HPLC (for details, see Supporting Information).
Synthesis of 8. To a solution of 7 (1.88 g, 5.68 mmol, 2 equiv) in
dry THF (27.5 mL) was added 1.6 M n-BuLi in hexane (3.9 mL) at
room temperature under an Ar atmosphere. After 45 min of stirring at
room temperature, the mixture was cooled to 4 °C. A solution of 6
(1.25 g, 2.85 mmol) in dry THF (15 mL) was then added, and the
reaction mixture was stirred for 2 h at 4 °C. The reaction was
quenched with saturated aqueous NH4Cl (25 mL). The mixture was
poured into EtOAc (50 mL) and H2O (50 mL). After the organic layer
was separated, 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 (silica gel, 5% → 10% EtOAc/hexane) to
afford a coupling product (2.03 g) as a clear oil. To a solution of the
coupling product (2.03 g) in MeOH (60 mL) was added PPTS (1.34
g, 5.34 mmol, 2 equiv) at room temperature. After 17 h of stirring, the
reaction mixture was concentrated in vacuo. The residue was poured
into EtOAc (20 mL) and H2O (20 mL). After the organic layer was
separated, 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 (silica gel, 40% → 70% EtOAc/hexane) to
afford 8 (1.32 g, 2.36 mmol, 88% in two steps) as a clear oil. 1H NMR
(400 MHz, 297.8 K, CDCl3, 0.023 M) δ 1.00 (3H, d, J = 6.7 Hz), 1.15
(3H, s), 1.18 (3H, s), 1.35−1.72 (11H, m), 1.90−1.97 (3H, m), 2.17
(1H, dd, J = 15.5, 8.9 Hz), 2.60 (2H, t, J = 7.7 Hz), 2.81−3.00 (4H,
m), 3.40 (1H, dd, J = 10.4, 6.6 Hz), 3.46 (1H, dd, J = 10.4, 6.0 Hz),
3.87 (1H, m), 4.52 (1H, m), 5.05 (2H, s), 6.78−6.82 (3H, m), 7.19
(1H, t, J = 7.8 Hz), 7.30−7.45 (5H, m) ppm. 13C NMR (100 MHz,
298.6 K, CDCl3, 0.035 M) δ 19.80, 22.49, 22.91, 25.15, 27.33, 27.52,
31.43, 32.11, 36.00, 37.60, 39.48, 44.36, 45.04, 46.31, 63.24, 69.35,
69.91, 71.22, 71.72, 77.22, 111.79, 115.15, 121.16, 127.54 (2C),
127.89, 128.55 (2C), 129.21, 137.20, 144.46, 158.85 ppm. HR-FAB-
MS (matrix, m-nitrobenzyl alcohol): m/z = 561.3074 ([MH]+, calcd
1
oil and a 1:1 diastereomeric mixture. H NMR (400 MHz, 297.8 K,
CDCl3, 0.087 M) δ 0.97 (3H, d, J = 5.4 Hz), 0.99 (3H, d, J = 5.4 Hz),
1.14 (3H, s), 1.15 (3H, s), 1.17 (3H, s), 1.18 (3H, s), 1.22−1.67 (22H,
m), 1.37 (6H, s), 1.42 (6H, s), 1.75−2.39 (14H, m), 2.59 (4H, t, J =
7.7 Hz), 2.65−2.73 (4H, m), 2.80−2.87 (2H, m), 3.02−3.10 (2H, m),
3.45 (1H, m), 3.52 (1H, m), 3.81 (2H, br s), 4.22 (1H, br s), 4.25
(1H, br s), 5.05 (4H, s), 5.11−5.17 (4H, m), 5.78−5.90 (2H, m),
6.77−6.81 (6H, m), 7.19 (2H, t, J = 7.8 Hz), 7.30−7.45 (10H, m)
ppm. 13C NMR (100 MHz, 298.3 K, CDCl3, 0.087 M) δ 17.12 (2C),
18.55 (2C), 19.65 (2C), 22.58 (2C), 23.18 (2C), 24.67, 24.78, 26.96,
27.04, 30.34 (2C), 31.31 (2C), 33.95 (2C), 34.44 (2C), 35.89 (2C),
36.31 (2C), 37.89 (2C), 38.66 (2C), 39.07 (2C), 43.59 (2C), 44.31,
44.44, 63.64, 63.68, 68.04, 68.08, 69.15 (2C), 69.91 (2C), 75.51, 75.61,
98.57 (2C), 111.73 (2C), 115.20 (2C), 117.79, 118.01, 121.13 (2C),
127.51 (4C), 127.91 (2C), 128.56 (4C), 129.21 (2C), 135.66, 135.68,
137.15 (2C), 144.38 (2C), 158.86 (2C) ppm. HR-FAB-MS (matrix,
m-nitrobenzyl alcohol): m/z = 663.3511 ([M + Na]+, calcd for
C38H56O4S2Na 663.3518).
Synthesis of 10. To a solution of 9 (640 mg, 1.00 mmol) in
MeCN (17 mL) and THF (1.7 mL) were added H2O (1.7 mL) and p-
TsOH·H2O (571 mg, 3.00 mmol, 3.0 equiv) at room temperature.
The mixture was stirred at room temperature for 2 h, and then the
reaction was quenched with saturated aqueous 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, 30% → 50% EtOAc/hexane) to afford a
triol (568 mg, 0.947 mmol, 95%) as a clear oil. To a solution of the
triol (568 mg, 0.947 mmol) in MeCN (1.26 mL), DCM (1.26 mL),
and H2O (1.26 mL) was added Hg(ClO4)2·6H2O (387 mg, 0.762
mmol, 2.0 equiv) at 4 °C. After 1 h of stirring at the same temperature,
the reaction mixture was poured into EtOAc (30 mL) and saturated
aqueous Na2S2O3 (30 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
chromatography (5% → 10% → 20% EtOAc/hexane) to afford a
mixture containing 10 (105 mg) and 11 (43.0 mg, 0.087 mmol, 23%).
The former was further purified by HPLC (column, YMC-Pack ODS-
A AA12S05-1520WT; solvent, 85% MeCN/H2O; flow rate, 8.0 mL/
min; pressure, 900 psi; UV detector, 254 nm; retention time, 50 min)
for C32H49O4S2 561.3072). [α]24.7 +9.2° (c 0.34, CHCl3).
D
Synthesis of 9. To a solution of 8 (1.32 g, 2.36 mmol) in DCM
(20 mL) were added 2,2-dimethoxypropane (2.89 mL, 23.6 mmol, 10
equiv) and ( )-10-camphorsulfonic acid (110 mg, 0.472 mmol, 0.2
equiv) at room temperature. The mixture was stirred at room
temperature for 30 min, and the reaction was quenched with saturated
aqueous NaHCO3 (20 mL). The organic layer was separated, and the
aqueous layer was extracted with DCM (20 mL × 2). The combined
organic layers were washed with brine, dried over Na2SO4, and
concentrated in vacuo. The residue was dissolved in DCM (20 mL),
and silica gel (2.0 g) was added. After the mixture was stirred at room
temperature for 30 min, the silica gel was removed by filtration. The
1
to afford 10 (20.5 mg, 0.042 mmol, 11%). Compound 10: H NMR
(400 MHz, 297.8 K, CDCl3, 0.069 M) δ 0.82 (3H, d, J = 6.5 Hz), 0.84
(3H, s), 0.98 (3H, s), 1.25−1.73 (11H, m), 1.51 (1H, dd, J = 14.4, 3.4
Hz), 2.16 (1H, dt, J = 13.9, 8.6 Hz), 2.28 (1H, ddd, J = 14.4, 2.7, 1.9
Hz), 2.42 (1H, m), 2.59 (2H, t, J = 7.7 Hz), 3.36 (1H, ddd, J = 10.3,
8.6, 2.6 Hz), 3.63 (1H, d, J = 11.5 Hz, OH), 4.06 (1H, m), 4.14 (1H,
m), 5.05 (2H, s), 5.12−5.16 (2H, m), 5.90 (1H, m), 6.78−6.83 (3H,
m), 7.19 (1H, t, J = 7.8 Hz), 7.30−7.45 (5H, m) ppm. 13C NMR (100
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MHz, 298.6 K, CDCl3, 0.069 M) δ 17.78, 22.01, 24.90, 25.91, 27.98,
30.78, 31.15, 35.73, 36.02, 37.80, 38.13, 38.35, 44.10, 63.69, 65.78,
69.89, 78.42, 102.21, 111.71, 115.15, 118.34, 121.15, 127.51 (2C),
127.87, 128.53 (2C), 129.16, 134.87, 137.22, 144.57, 158.85 ppm. HR-
FAB-MS (matrix, m-nitrobenzyl alcohol): m/z = 493.3288 ([MH]+,
3.0 equiv) and chlorotriethylsilane (60 μL, 0.359 mmol, 1.8 equiv) at
room temperature. After the mixture was stirred for 1 h at room
temperature, 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, 2.5% → 5%) to afford a silyl ether (145
mg, 0.182 mmol, 89%) as a clear oil. To a suspension of NaIO4 (103
mg, 0.482 mmol, 8.0 equiv) in pH 7.2 phosphate buffer (4.8 mL) was
added KMnO4 (9.5 mg, 0.0603 mmol, 1.0 equiv) in one portion. After
10 min of stirring at room temperature under an Ar atmosphere, the
mixture was added to a solution of the silyl ether (48.1 mg, 0.0603
mmol) in t-BuOH (4.8 mL). The reaction mixture was stirred at room
temperature for 17 h, and the reaction was quenched with Na2S2O3
(28.9 mg). The resulting mixture was poured into EtOAc (20 mL) and
water (20 mL). The organic layer was separated, and the aqueous layer
was extracted with EtOAc (20 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% → 20% EtOAc/hexane containing 0.1% AcOH) to afford 14
calcd for C32H45O4 493.3318). [α]10.3 +40.6° (c 0.42, CHCl3).
D
1
Compound 11: H NMR (400 MHz, 297.9 K, CDCl3, 0.153 M) δ
0.76 (3H, d, J = 5.9 Hz), 0.85 (3H, s), 0.96 (3H, s), 1.03 (1H, m),
1.22−1.67 (11H, m), 1.95 (1H, m), 2.07 (2H, m), 2.29 (1H, m), 2.59
(2H, t, J = 7.6 Hz), 3.42 (1H, m), 3.88 (1H, m), 4.21 (1H, m), 4.97−
5.03 (2H, m), 5.04 (2H, s), 5.87 (1H, m), 6.78−6.82 (3H, m), 7.19
(1H, t, J = 7.8 Hz), 7.30−7.45 (5H, m) ppm. 13C NMR (100 MHz,
298.8 K, CDCl3, 0.153 M) δ 17.86, 23.69, 25.58, 26.11, 30.62, 31.24,
35.54, 35.99, 36.29, 37.52, 37.59, 37.82, 42.87, 62.43, 69.90, 71.64,
74.56, 101.88, 111.76, 115.19, 115.90, 121.15, 127.51 (2C), 127.89,
128.55 (2C), 129.21, 136.06, 137.18, 144.35, 158.85 ppm. HR-FAB-
MS (matrix, m-nitrobenzyl alcohol): m/z = 493.3303 ([MH]+, calcd
for C32H45O4 493.3318). [α]11.7 +6.4° (c 0.58, CHCl3).
D
To a solution of 11 (21 mg, 0.043 mmol) in MeCN (0.6 mL) and
DCM (0.6 mL) was added ( )-10-camphorsulfonic acid (1.0 mg, 4.3
μmol, 0.1 equiv) at room temperature. After 1 h of stirring at room
temperature, the reaction mixture was quenched with saturated
aqueous NaHCO3 (3 mL). The mixture was extracted with EtOAc (3
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, 5% → 10% EtOAc/hexane) to
afford 10 (8.7 mg, 0.018 mmol, 41%) and 11 (8.5 mg, 0.017 mmol,
40%).
1
(29.2 mg, 0.0358 mmol, 59%) as a clear oil. H NMR (500 MHz,
295.3 K, CDCl3, 0.025 M) δ 0.58 (6H, q, J = 7.9 Hz), 0.82 (3H, d, J =
6.6 Hz), 0.88 (3H, s), 0.91 (9H, t, J = 7.9 Hz), 1.01 (3H, s), 1.20−1.30
(2H, m), 1.31−1.80 (9H, m), 1.71 (1H, dd, J = 15.3, 4.4 Hz), 2.18
(1H, br d, J = 15.3 Hz), 2.48−2.64 (5H, m), 2.67 (1H, dd, J = 15.2, 3.6
Hz), 3.38 (1H, dd, J = 9.5, 6.6 Hz), 3.49 (1H, dd, J = 9.5, 5.0 Hz), 3.50
(1H, m), 4.10 (1H, m), 4.31 (1H, m), 4.51 (1H, d, J = 12.1 Hz), 4.54
(1H, d, J = 12.1 Hz), 5.05 (2H, s), 5.10−5.11 (1H, m), 6.78−6.83
(3H, m), 7.18 (1H, t, J = 7.8 Hz), 7.27−7.45 (10H, m) ppm. 13C
NMR (125 MHz, 295.8 K, CDCl3, 0.025 M) δ 4.88 (3C), 6.77 (3C),
17.52, 22.04, 24.96, 25.93, 26.61, 30.84, 31.19, 34.45, 35.56, 35.99,
38.18, 38.82, 40.60, 43.65, 65.03, 67.58, 68.49, 69.96, 73.44, 74.07,
75.55, 101.58, 111.88, 115.19, 121.22, 127.54 (2C), 127.76, 127.81
(2C), 127.86, 128.41 (2C), 128.54 (2C), 129.20, 137.29, 137.91,
144.45, 158.90, 171.57, 171.65 ppm. HR-FAB-MS (matrix, m-
nitrobenzyl alcohol): m/z = 839.4557 ([M + Na]+, calcd for
Synthesis of 13. To a solution of 12 (110 mg, 0.335 mmol, 1.36
equiv) and Et3N (53 μL, 0.379 mmol, 1.53 equiv) in toluene (2.5 mL)
was added 2,4,6-trichlorobenzoyl chloride (43.0 μL, 0.275 mmol, 1.7
equiv) at room temperature. After 2 h of stirring at room temperature,
a supernatant of the resulting suspension was added to a solution of 10
(122 mg, 0.247 mmol) and DMAP (57 mg, 0.379 mmol, 1.89 equiv)
in toluene (4.5 mL) at 50 °C. The resulting mixture was stirred at the
same temperature for 1 h and then poured into water (10 mL). The
mixture was extracted with EtOAc (20 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, 5% → 10% EtOAc/hexane) to afford 13
(186 mg, 0.231 mmol, 94%) as a clear oil. 1H NMR (400 MHz, 297.3
K, CDCl3, 0.032 M) δ 0.74 (3H, d, J = 6.5 Hz), 0.84 (3H, s), 0.97 (3H,
s), 1.17 (1H, t, J = 13.5 Hz), 1.28 (1H, dd, J = 13.5, 4.7 Hz), 1.32−
1.47 (5H, m), 1.53−1.69 (5H, m), 2.15−2.24 (2H, m), 2.36 (1H, m),
2.56 (2H, t, J = 7.7 Hz), 2.60 (2H, m), 3.05 (1H, m), 3.50 (1H, dd, J =
10.0, 3.5 Hz), 3.58 (1H, dd, J = 10.0, 5.3 Hz), 3.78 (3H, s), 4.08 (1H,
m), 4.20 (1H, m), 4.52 (1H, d, J = 11.2 Hz), 4.53 (2H, s), 4.59 (1H, d,
J = 11.2 Hz), 4.98−5.03 (2H, m), 5.04 (2H, s), 5.10 (1H, m), 6.00
(1H, m), 6.77−6.86 (5H, m), 7.18 (1H, t, J = 7.8 Hz), 7.21−7.45
(12H, m) ppm. 13C NMR (100 MHz, 297.7 K, CDCl3, 0.032 M) δ
17.39, 21.77, 24.90, 25.68, 26.10, 29.93, 31.11, 34.81, 35.53, 36.09,
37.92, 38.07, 38.18, 44.28, 55.25, 63.90, 68.31, 69.87, 71.88, 72.13,
73.33, 74.81, 77.22, 99.73, 111.72, 113.73 (2C), 115.10, 116.24,
121.16, 127.53 (2C), 127.58 (2C), 127.62, 127.86, 128.38 (2C),
128.53 (2C), 129.14, 129.36 (2C), 130.60, 135.44, 137.25, 138.17,
144.67, 158.83, 159.19, 171.61 ppm. HR-FAB-MS (matrix, m-
nitrobenzyl alcohol): m/z = 827.4532 ([M + Na]+, calcd for
C48H68O9SiNa 839.4531). [α]28.6 +16.5° (c 1.00, CHCl3).
D
Synthesis of 4. To a solution of 14 (29.2 mg, 0.0358 mmol) in
THF (3.0 mL) was added a freshly prepared solution of buffered
HF·pyridine (825 μL, prepared by adding 75 μL of HF·pyridine to 150
μL of pyridine in 600 μL of THF) at 4 °C. The reaction mixture was
stirred for 6 h at the same temperature, then diluted with water (5 mL)
and warmed to room temperature. The mixture was extracted with
EtOAc (10 mL × 2) and CHCl3 (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, 20% → 50% EtOAc/hexane containing
0.1% AcOH) to afford a seco-acid (28.9 mg, quantitatively) as a clear
oil. To a solution of the seco-acid (28.9 mg, 0.0358 mol) and Et3N
(149 μL, 1.074 mmol, 30 equiv) in toluene (3.8 mL) was added 2,4,6-
trichlorobenzoyl chloride (102 μL, 0.716 mmol, 20 equiv) at room
temperature. The mixture was stirred at room temperature for 3 h and
then diluted with toluene (20 mL). The supernatant of the mixture
was added dropwise to a solution of DMAP (218 mg, 1.79 mmol, 50
equiv) in toluene (42 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 an additional 1 h of stirring at room
temperature, saturated aqueous NaHCO3 (30 mL) was added and the
resulting biphasic mixture was poured into EtOAc (30 mL). The
organic layer was separated and the aqueous layer 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, 5% → 10%
EtOAc/hexane) to afford the lactone (18.1 mg, 0.0265 mmol, 74% in
two steps) as a clear oil. To 20% Pd(OH)2/C (3.3 mg) in a flask was
added a solution of the lactone (5.0 mg, 7.3 μmol) in EtOAc (0.5 mL)
at room temperature. The mixture was vigorously stirred under an H2
atmosphere at room temperature for 20 min. 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/
C51H64O8Na 827.4499). [α]26.5 +11.3° (c 1.17, CHCl3).
D
Synthesis of 14. To a vigorously stirred solution of 13 (179 mg,
0.223 mmol) in DCM (12 mL) and H2O (2.0 mL) was added DDQ
(104 mg, 0.445 mmol, 2.0 equiv) at room temperature, causing a
brown color to appear. After 1 h of stirring at room temperature, the
color gradually changed to yellow-orange with precipitation of a solid.
The mixture was poured into saturated aqueous NaHCO3 (30 mL)
and extracted with DCM (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, 10% → 30% EtOAc/hexane) to afford an alcohol (143 mg, 0.209
mmol, 94%) as a clear oil. To a solution of the alcohol (139 mg, 0.203
mmol) in THF (1.6 mL) were added imidazole (41 mg, 0.609 mmol,
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CHCl3/hexane = 7.5:12.5:80; flow rate, 3.0 mL/min; pressure, 600
psi; UV detector, 254 nm; retention time, 16.0 min) to afford 4 (2.8
mg, 5.56 μmol, 76%) as a clear oil. H NMR (500 MHz, 295.1 K,
quenched with a 1:1 mixture of 10% aqueous Na2S2O3 and saturated
aqueous NaHCO3 (20 mL). The mixture was extracted with EtOAc
(20 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% → 20% →
40% EtOAc/hexane) to afford an iodocarbonate, which was dissolved
in MeOH (10 mL). To this solution was added K2CO3 (853 mg, 6.18
mmol) at room temperature. After 45 min of stirring, the reaction
mixture was poured into H2O (40 mL) and EtOAc (40 mL). The
organic layer was separated, and the aqueous layer was extracted with
EtOAc (40 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, 15%
EtOAc/hexane; ODS, 70% → 80% MeOH/H2O) to afford 17 (437
mg, 1.10 mmol, 53% in two steps) as a clear oil. 1H NMR (500 MHz,
295.1 K, CDCl3, 0.054 M) δ 0.98 (3H, d, J = 7.1 Hz), 1.40 (2H, m),
1.55−1.75 (5H, m), 2.43 (1H, dd, J = 5.0, 2.7 Hz), 2.59 (2H, t, J = 7.8
Hz), 2.72 (1H, dd, J = 5.0, 4.1 Hz), 2.92 (1H, ddd, J = 7.7, 4.1, 2.7
Hz), 3.79 (3H, s), 4.81 (1H, m), 5.05 (2H, s), 6.77−6.81 (3H, m),
7.18 (1H, t, J = 7.8 Hz), 7.32−7.45 (5H, m) ppm. 13C NMR (125
MHz, 295.9 K, CDCl3, 0.054 M) δ 12.53, 25.08, 31.05, 31.56, 35.79,
40.07, 44.84, 52.97, 54.74, 69.95, 80.89, 111.97, 115.15, 121.15, 127.52
(2C), 127.89, 128.56 (2C), 129.24, 137.26, 144.13, 155.91, 158.93
ppm. HR-FAB-MS (matrix, m-nitrobenzyl alcohol): m/z = 399.2189
1
CDCl3, 0.011 M) δ 0.81 (3H, d, J = 6.5 Hz, H-22), 0.85 (3H, s, H-24),
0.99 (3H, s, H-23), 1.24 (1H, t, J = 12.7 Hz, H-5a), 1.32 (1H, dd, J =
13.8, 4.4 Hz, H-5b), 1.34−1.44 (3H, m, H-10a, H-12a, H-13a), 1.44−
1.56 (2H, m, H-12b, H-13b), 1.56−1.74 (4H, m, H-4, H-10b, H2-14),
1.65 (1H, dd, J = 15.6, 4.2 Hz, H-8a), 2.35 (1H, dd, J = 12.9, 10.9 Hz,
H-2a), 2.39 (1H, t, J = 5.9 Hz, OH), 2.44 (1H, d, J = 15.6 Hz, H-8b),
2.57 (2H, t, J = 7.6 Hz, H-15), 2.74 (1H, dd, J = 16.9, 3.3 Hz, H-26a),
2.78 (1H, dd, J = 12.9, 2.8 Hz, H-2b), 2.83 (1H, dd, J = 16.9, 11.5 Hz,
H-26b), 3.48 (1H, td, J = 10.6, 2.8 Hz, H-3), 3.76 (2H, m, H-28), 4.22
(1H, m, H-11), 5.20 (2H, m, H-9, H-27), 6.38 (1H, br s, PhOH), 6.67
(1H, dd, J = 8.0, 2.4 Hz, H-19), 6.73 (1H, d, J = 7.9 Hz, H-21), 6.83
(1H, s, H-17), 7.13 (1H, t, J = 7.8 Hz, H-20) ppm. 13C NMR (125
MHz, 296.1 K, CDCl3, 0.011 M) δ 17.38 (C-22), 22.03 (C-23), 24.11
(C-13), 25.18 (C-8), 25.89 (C-24), 29.58 (C-14), 31.54 (C-4), 34.49
(C-12), 34.91 (C-10), 35.07 (C-15), 37.01 (C-26), 38.14 (C-6), 40.76
(C-2), 43.97 (C-5), 63.21 (C-11), 64.25 (C-28), 68.67 (C-9), 72.28
(C-27), 76.64 (C-3), 100.29 (C-7), 112.61 (C-19), 115.09 (C-21),
120.73 (C-17), 129.36 (C-20), 144.58 (C-16), 156.17 (C-18), 169.40
(C-25), 173.11 (C-1) ppm. HR-FAB-MS (matrix, m-nitrobenzyl
alcohol): m/z = 505.2811 ([MH]+, calcd for C28H41O8 505.2801).
[α]30.0 +38.9° (c 0.28, CHCl3).
D
Synthesis of 16. To a solution of KO-t-Bu (2.34 g, 20.90 mmol,
2.02 equiv) and trans-2-butene (excess) in dry THF (50 mL) was
added 1.6 M n-BuLi in hexane (11.8 mL, 18.83 mmol, 1.82 equiv) at
−78 °C under an Ar atmosphere. The resulting yellow mixture was
stirred at −40 °C for 15 min. The reaction mixture was recooled to
−78 °C, and a solution of (−)-B-methoxydiisopinocampheylborane
(5.95 g, 18.83 mmol, 1.82 equiv) in dry THF (10 mL) was added. The
resulting colorless reaction mixture was stirred at −78 °C for 30 min.
BF3·Et2O (3.18 mL, 25.05 mmol, 2.42 equiv) was added rapidly
followed immediately by a solution of 15 (2.79 g, 10.35 mmol) in 10
mL of dry THF. After 2 h of stirring at −78 °C, the reaction mixture
was warmed to room temperature. The reaction was then quenched by
addition of 1 M aqueous NaOH (36 mL) followed by 30% aqueous
H2O2 (8 mL). The resulting mixture was stirred for 14 h at room
temperature. The organic layer was separated, and the aqueous layer
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 (ODS,
70% → 90% MeOH/H2O) to afford 16 (2.95 g, 9.12 mmol, 88%) as a
clear oil [92% ee by chiral HPLC analysis (column, CHIRALCEL OJ-
RH; solvent, 90% MeOH/H2O; flow rate, 0.6 mL/min; pressure, 600
([MH]+, calcd for C24H31O5 399.2171). [α]16.0 +4.3° (c 1.29,
D
CHCl3).
Synthesis of 18. To a solution of 17 (1.02 g, 2.55 mmol) was
added a solution of 28% NaOMe in MeOH (0.54 mL) at room
temperature. After 2 h of stirring at 50 °C, saturated aqueous NH4Cl
(10 mL) was added and the resulting mixture was poured into EtOAc
(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 (silica gel,
15% → 30% EtOAc/hexane) to afford an alcohol (630 mg, 1.85 mmol,
73%) as a clear oil. To a suspended solution of the alcohol (12.7 mg,
37.4 μmol) and NaH (3.0 mg, 60% in mineral oil, 74.7 μmol) in
THF−DMF (3 mL, 3:1) was added p-methoxybenzyl chloride (9.0 μL,
66.1 μmol) at room temperature. After 55 h of stirring, the mixture
was quenched with 5% aqueous H3PO4 (5.0 mL). The solution was
extracted with 1:1 mixture of EtOAc and hexane (5 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, 3% → 7% EtOAc/hexane) to
1
afford 18 (16.7 mg, 36.3 μmol, 97%) as a clear oil. H NMR (500
1
psi; UV detector, 254 nm; retention time, 29.6 min)]. H NMR (500
MHz, 295.1 K, CDCl3, 0.070 M) δ 0.93 (3H, d, J = 7.1 Hz), 1.40 (1H,
m), 1.46−1.69 (6H, m), 2.47 (1H, dd, J = 5.0, 2.7 Hz), 2.58 (2H, t, J =
7.7 Hz), 2.72 (1H, dd, J = 5.0, 4.1 Hz), 2.90 (1H, m), 3.43 (1H, m),
3.78 (3H, s), 4.41 (1H, d, J = 11.1 Hz), 4.44 (1H, d, J = 11.1 Hz), 5.04
(2H, s), 6.77−6.82 (3H, m), 6.86 (2H, m), 7.19 (1H, t, J = 7.8 Hz),
7.24 (2H, m), 7.30−7.44 (5H, m) ppm. 13C NMR (125 MHz, 295.3
K, CDCl3, 0.070 M) δ 11.55, 25.54, 30.69, 31.44, 36.00, 38.97, 45.39,
53.62, 55.28, 69.95, 71.48, 80.89, 111.87, 113.77 (2C), 115.19, 121.20,
127.52 (2C), 127.88, 128.55 (2C), 129.21, 129.34 (2C), 131.04,
137.26, 144.42, 158.92, 159.14 ppm. HR-FAB-MS (matrix, m-
nitrobenzyl alcohol): m/z = 460.2619 ([M]+, calcd for C30H36O4
MHz, 295.1 K, CDCl3, 0.035 M) δ 1.02 (3H, d, J = 6.9 Hz), 1.40 (2H,
m), 1.52 (1H, d, J = 4.2 Hz), 1.51−1.55 (2H, m), 1.63 (2H, m), 2.19
(1H, m), 2.60 (2H, m), 3.38 (1H, br s), 5.05 (2H, s), 5.08−5.13 (2H,
m), 5.71−5.78 (1H, m), 6.78−6.82 (3H, m), 7.19 (1H, t, J = 7.7 Hz),
7.30−7.45 (5H, m) ppm. 13C NMR (125 MHz, 295.7 K, CDCl3, 0.035
M) δ 16.30, 25.48, 31.39, 34.13, 36.00, 44.17, 69.96, 74.62, 111.87,
115.22, 116.28, 121.17, 127.51 (2C), 127.88, 128.55 (2C), 129.22,
137.26, 140.33, 144.39, 158.92 ppm. HR-FAB-MS (matrix, m-
nitrobenzyl alcohol): m/z = 324.2090 ([M]+, calcd for C22H28O2
324.2089). [α]13.4 +1.0° (c 0.58, CHCl3).
D
Synthesis of 17. To a solution of 16 (1.13 g, 3.49 mmol) in THF
(10 mL) was added 1 M sodium bis(trimethylsilyl)amide in THF
(4.53 mL, 4.53 mmol, 1.3 equiv) dropwise at 4 °C. After 30 min of
stirring, (Boc)2O (1.15 g, 5.28 mmol, 1.5 equiv) was added, and the
reaction mixture was stirred for 15 min at room temperature. The
reaction was quenched with brine (10 mL) and water (3 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% → 2% EtOAc/hexane) to afford a
carbonate (1.28 g, 3.02 mmol, 87%) as a clear oil. To a solution of the
carbonate (1.28 g, 3.02 mmol) in toluene (15 mL) was added IBr
(2.32 g, 11.2 mmol, 1.5 equiv) in DCM (11 mL) at −78 °C. After the
mixture was stirred for 2 h the same temperature, the reaction was
460.2614). [α]17.5 +12.3° (c 1.03, CHCl3).
D
Synthesis of 20. To a solution of 19 (653 mg, 1.96 mmol, 2
equiv) in dry THF (12 mL) was added 1.6 M n-BuLi in hexane (1.35
mL) at room temperature under an Ar atmosphere. After 45 min of
stirring at room temperature, the mixture was cooled to 4 °C. A
solution of 18 (450 mg, 0.978 mmol) in dry THF (15 mL) was then
added, and the reaction mixture was stirred for 18 h at 4 °C. The
reaction was quenched with saturated aqueous NH4Cl (20 mL). The
mixture was poured into EtOAc (30 mL) and H2O (30 mL). After the
organic layer was separated, the aqueous layer was extracted with
EtOAc (40 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, 3% → 7%
EtOAc/hexane) to afford 20 (743 mg, 0.934 mmol, 96%) as a clear oil.
5621
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1H NMR (500 MHz, 295.0 K, CDCl3, 0.019 M) δ 0.05 (6H, s), 0.89
(3H, d, J = 6.3 Hz), 0.89 (9H, s), 1.11 (6H, br s), 1.39−1.63 (10H,
m), 1.87−2.01 (3H, m), 2.07 (1H, dd, J = 15.4, 9.2 Hz), 2.17 (1H, d, J
= 15.4 Hz), 2.58 (2H, m), 2.81−2.97 (4H, m), 3.59 (2H, t, J = 6.4
Hz), 3.68 (1H, m), 3.78 (3H, s), 4.22 (1H, t, J = 8.2 Hz), 4.27 (1H, s),
4.36 (1H, d, J = 10.9 Hz), 4.47 (1H, d, J = 10.9 Hz), 5.04 (2H, s),
6.77−6.80 (3H, m), 6.84 (2H, m), 7.18 (1H, t, J = 7.8 Hz), 7.25 (2H,
m), 7.30−7.44 (5H, m) ppm. 13C NMR (125 MHz, 295.3 K, CDCl3,
0.019 M) δ −5.21 (2C), 10.82, 18.35, 22.34 (2C, br), 22.85, 25.45,
26.01 (3C), 27.18, 27.47, 28.37, 29.38, 31.54, 32.80, 36.08, 41.43,
42.42, 45.75, 55.29, 63.71, 63.87, 69.95, 70.89, 71.21, 79.24, 111.80,
113.71 (2C), 115.20, 121.22, 127.54 (2C), 127.87, 128.54 (2C),
129.17, 129.28 (2C), 131.47, 137.28, 144.63, 158.91, 159.02 ppm. HR-
FAB-MS (matrix, m-nitrobenzyl alcohol): m/z = 817.4328 ([M +
(2H, m), 5.44 (1H, s), 5.77 (1H, m), 6.79−6.83 (3H, m), 6.86 (2H,
m), 7.19 (1H, t, J = 7.8 Hz), 7.30−7.44 (7H, m) ppm. 13C NMR (125
MHz, 296.3 K, CDCl3, 0.024 M) δ 13.21, 22.42 (br), 23.08 (br),
24.38, 24.90, 26.63, 27.52, 31.36, 32.10, 32.56 (br), 32.94, 36.00, 38.84,
41.52, 41.93, 44.45, 55.26, 63.83, 69.96, 71.47, 80.32, 81.96, 99.59,
111.88, 113.44 (2C), 115.19, 117.98, 121.20, 127.32 (2C), 127.52
(2C), 127.89, 128.56 (2C), 129.22, 131.65, 134.93, 137.26, 144.49,
158.93, 159.66 ppm. HR-FAB-MS (matrix, m-nitrobenzyl alcohol): m/
z = 719.3813 ([MH]+, calcd for C43H59O5S2 719.3804). [α]19.2D −0.3°
(c 0.37, CHCl3).
Synthesis of 22. To a solution of 21 (118 mg, 0.164 mmol) in
MeCN (1 mL) and THF (1 mL) were added H2O (1 mL) and p-
TsOH·H2O (94 mg, 0.493 mmol, 3.0 equiv) at room temperature.
The mixture was stirred at 50 °C for 5 h, and then the reaction was
quenched with saturated aqueous NaHCO3 (5 mL). The mixture was
extracted with EtOAc (10 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, 7% → 15%
→ 60% EtOAc/hexane) to afford a recovered substrate (65 mg, 0.091
mmol, 55%) and a triol (40 mg, 0.067 mmol, 41%) as a clear oil. To a
solution of the triol (65.7 mg, 0.109 mmol) in MeCN (0.36 mL),
DCM (0.36 mL), and H2O (0.36 mL) was added Hg(ClO4)2·6H2O
(111 mg, 0.219 mmol, 2.0 equiv) at 4 °C. After 30 min of stirring at
the same temperature, the reaction mixture was poured into EtOAc (5
mL) and saturated aqueous Na2S2O3 (15 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 (3% → 8% EtOAc/hexane) to afford the
desired spiroketal 22 (33.4 mg, 0.0679 mmol, 62%) as a clear oil. Since
it was difficult to purify the undesired spiroketal, spectral data were not
Na]+, calcd for C46H70O5S2SiNa 817.4332). [α]18.0 +7.3° (c 0.94,
D
CHCl3).
Synthesis of 21. To a suspension of 20 (386 mg, 0.486 mmol)
and 4 Å molecular sieves (1.0 g) in DCM (6 mL) was added DDQ
(137 mg, 0.583 mmol, 1.2 equiv) at room temperature, causing a green
color to appear. After 1 h of stirring at room temperature under an Ar
atmosphere, the color gradually changed to brown with precipitation
of a solid. The mixture was poured into saturated aqueous NaHCO3 (6
mL), H2O (20 mL), and EtOAc (20 mL). After the organic layer was
separated, the aqueous layer was extracted with EtOAc (20 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, 5% EtOAc/hexane) to afford an
acetal (333 mg, 0.420 mmol, 87%) as a clear oil. To a solution of the
acetal (322 mg, 0.407 mmol) in DCM (4.3 mL) and MeOH (4.3 mL)
was added PPTS (204 mg, 0.813 mmol, 2 equiv) at room temperature.
After the mixture was stirred for 1.5 h, the reaction was quenched with
saturated aqueous NaHCO3 (10 mL). The resulting mixture was
extracted with EtOAc (20 mL × 3), and 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, 15% → 30% EtOAc/hexane) to afford an alcohol (226 mg, 0.333
mmol, 82%) as a clear oil. To a solution of the alcohol (213 mg, 0.314
mmol), NMO (55 mg, 0.471 mmol, 1.5 equiv), and 4 Å molecular
sieves (157 mg) in DCM (0.63 mL) was added tetrapropylammonium
perruthenate (5.5 mg, 0.097 mmol, 0.05 equiv). After 20 min of
stirring at room temperature, the reaction mixture was diluted with
DCM (2 mL) and absorbed in a small amount of silica gel. The
resulting gel was directly loaded onto a silica gel column and eluted
with 10% EtOAc/hexane. Concentration of the fractions gave an
aldehyde (144 mg, 0.213 mmol, 68%) as a clear oil. Then 1 M TiCl4 in
DCM (31 μL, 31 μmol, 0.15 equiv) was diluted with DCM (0.5 mL)
and cooled to 4 °C. To the TiCl4 solution was added Ti(O-i-Pr)4 (28
μL, 93 μmol, 0.45 equiv) at 0 °C. The mixture was warmed to room
temperature and stirred for 1 h. Ag2O (14 mg, 62 μmol, 0.3 equiv) was
added in one portion, and stirring was continued with exclusion of
direct light for 5 h. (S)-1,1′-Bi-2-naphthol (35 mg, 0.124 mmol, 0.6
equiv) was then added in one portion. After a further 2 h of stirring,
the mixture was diluted with DCM (1.0 mL) to afford a stock solution
(about 40 mM) of Ti catalyst. To the aldehyde (140 mg, 0.207 mmol)
was added the supernatant (1.03 mL) of the above stock solution at
−15 °C. After 15 min of stirring at the same temperature, allyl-SnBu3
(128 μL, 0.414 mmol, 2 equiv) was added. The resulting reaction
mixture was kept in a cold room for 63 h without stirring. The reaction
was quenched with saturated aqueous NaHCO3 (3 mL), and the
mixture was poured into EtOAc (10 mL) and H2O (5 mL). The
organic layer was separated, and the aqueous layer 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, 10% →
20% EtOAc/hexane) to afford 21 (112 mg, 0.156 mmol, 75%) as a
clear oil. 1H NMR (500 MHz, 295.8 K, CDCl3, 0.024 M) δ 0.93 (3H,
d, J = 6.6 Hz), 1.17 (3H, br s), 1.19 (3H, br s), 1.35−2.06 (15H, m),
2.23 (1H, m), 2.38 (1H, dd, J = 16.2, 7.2 Hz), 2.48 (1H, d, J = 16.2
Hz), 2.60 (2H, m), 2.67−2.79 (2H, m), 2.83−2.93 (2H, m), 3.44−
3.49 (2H, m), 3.78 (3H, s), 3.92 (1H, m), 5.04 (2H, s), 5.08−5.12
1
measured. H NMR (500 MHz, 295.1 K, CDCl3, 0.024 M) δ 0.89
(3H, s), 0.90 (3H, d, J = 7.0 Hz), 0.95 (3H, s), 1.34−1.70 (12H, m),
2.26−2.30 (3H, m), 2.60 (2H, m), 3.43 (1H, d, J = 11.1 Hz, OH), 3.73
(1H, m), 3.80 (2H, m), 5.05 (2H, s), 5.07−5.12 (2H, m), 5.79 (1H,
m), 6.78−6.84 (3H, m), 7.19 (1H, t, J = 7.8 Hz), 7.30−7.45 (5H, m)
ppm. 13C NMR (125 MHz, 297.5 K, CDCl3, 0.024 M) δ 14.03, 22.60,
24.42, 25.53, 26.30, 30.52, 31.33, 32.59, 33.37, 36.07, 36.77, 38.81,
40.93, 68.81, 69.95, 70.34, 72.43, 102.48, 111.76, 115.23, 117.85,
121.20, 127.50 (2C), 127.87, 128.55 (2C), 129.17, 134.78, 137.29,
144.67, 158.02 ppm. HR-FAB-MS (matrix, m-nitrobenzyl alcohol): m/
z = 493.3296 ([MH]+, calcd for C32H45O4 493.3318. [α]19.5D +63.2° (c
0.60, CHCl3).
Synthesis of 23. Compound 22 (41.5 mg, 0.0843 mmol) was
treated in a manner similar to that described for the synthesis of 13 to
give 23 (60.0 mg, 0.0746 mmol, 89%). 1H NMR (500 MHz, 295.7 K,
CDCl3, 0.017 M) δ 0.74 (3H, d, J = 6.9 Hz), 0.85 (3H, s), 0.95 (3H,
s), 1.31−1.61 (10H, m), 1.66 (1H, dd, J = 15.0, 3.9 Hz), 1.67 (1H, m),
2.22 (1H, m), 2.30 (1H, m), 2.33 (1H, dd, J = 15.0, 3.2 Hz), 2.57−
2.61 (3H, m), 2.64 (1H, dd, J = 15.8, 7.7 Hz), 3.38 (1H, m), 3.50 (1H,
dd, J = 10.0, 5.2 Hz), 3.57 (1H, dd, J = 10.0, 5.3 Hz), 3.78 (2H, s),
3.98 (1H, m), 4.12 (1H, m), 4.53 (2H, s), 4.54 (1H, d, J = 11.0 Hz),
4.58 (1H, d, J = 11.0 Hz), 4.95−5.02 (3H, m), 5.04 (2H, s), 5.79 (1H,
m), 6.78−6.85 (5H, m), 7.18 (1H, t, J = 7.8 Hz), 7.21−7.25 (3H, m),
7.27−7.47 (10H, m) ppm. 13C NMR (125 MHz, 296.1 K, CDCl3,
0.017 M) δ 13.06, 21.70, 24.27, 25.57, 26.36, 27.52, 31.21, 32.45,
34.16, 36.14, 36.88, 37.05, 38.10, 41.04, 55.28, 68.62, 69.94, 71.50,
71.97, 72.00, 72.26, 73.36, 74.82, 99.76, 111.76, 113.73 (2C), 115.20,
116.67, 121.21, 127.52 (2C), 127.62 (2C), 127.86, 128.11, 128.37
(2C), 128.54 (2C), 129.14 (1C), 129.41 (2C), 130.66, 135.07, 137.31,
138.20, 144.76, 158.90, 159.21, 171.90 ppm. HR-FAB-MS (matrix, m-
nitrobenzyl alcohol): m/z = 804.4644 ([M]+, calcd for C51H64O8
804.4601). [α]29.0 +33.1° (c 0.69, CHCl3).
D
Synthesis of 24. Compound 23 (60.0 mg, 0.0746 mmol) was
treated in a manner similar to that described for the synthesis of 14 to
1
give 24 (35.5 mg, 0.0435 mmol, 62% in three steps). H NMR (500
MHz, 295.0 K, CDCl3, 0.037 M) δ 0.59 (6H, q, J = 7.9 Hz), 0.80 (3H,
d, J = 6.9 Hz), 0.87 (3H, s), 0.92 (9H, t, J = 7.9 Hz), 0.95 (3H, s),
1.33−1.72 (11H, m), 1.70 (1H, dd, J = 15.3, 3.9 Hz), 2.23 (1H, dd, J =
15.3, 3.0 Hz), 2.51 (1H, dd, J = 15.8, 7.4 Hz), 2.53−2.61 (4H, m), 2.63
5622
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(1H, dd, J = 15.8, 5.0 Hz), 3.39 (1H, dd, J = 9.6, 6.7 Hz), 3.48 (1H, dd,
J = 9.6, 5.1 Hz), 3.78−3.87 (2H, m), 4.35 (1H, m), 4.50 (1H, d, J =
11.9 Hz), 4.54 (1H, d, J = 11.9 Hz), 5.04 (1H, m), 5.05 (2H, s), 6.78−
6.88 (3H, m), 7.19 (1H, t, J = 7.8 Hz), 7.27−7.47 (10H, m) ppm. 13C
NMR (125 MHz, 295.3 K, CDCl3, 0.037 M) δ 4.91 (3C), 6.80 (3C),
13.08, 22.03, 24.39, 25.49, 26.40, 28.02, 31.28, 32.53, 33.19, 36.03,
36.84, 37.03, 40.42, 40.90, 67.90, 69.05, 69.75, 69.96, 70.93, 73.42,
74.27, 101.47, 111.87, 115.21, 121.24, 127.53 (2C), 127.72, 127.79
(2C), 127.86, 128.40 (2C), 128.54 (2C), 129.20, 137.31, 138.07,
144.51, 158.91, 171.67, 171.87 ppm. HR-FAB-MS (matrix, m-
nitrobenzyl alcohol): m/z = 817.4714 ([MH]+, calcd for C48H69O9Si
(T) was measured at 525 nm along with that for the test well at time 0
(T0). Cell growth inhibition (% growth) by each concentration of drug
(10−8, 10−7, 10−6, 10−5, and 10−4 M) was calculated as 100[(T − T0)/
(C − T0)] using the average of duplicate points. By processing of these
values, each GI50 value, defined as 100[(T − T0)/(C − T0)] = 50, was
determined.
EBV-EA Induction Test. Human B-lymphoblastoid Raji cells (5 ×
105/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 compound for the induction test. Each test
compound was added as 2 μL of a DMSO solution (5, 50, and 500 μM
stock solution) along with 2 μL 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 immunofluorescence 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.55,56 In each assay, at least 500 cells were counted
and the proportion of the EA-positive cells was recorded. Cell viability
817.4711). [α]29.4 +48.5° (c 0.40, CHCl3).
D
Synthesis of 5. Compound 24 (35.5 mg, 0.0435 mmol) was
treated in a manner similar to that described for the synthesis of 4 to
give a seco-acid (33.4 mg, quantitatively). To a solution of the seco-acid
(30.5 mg, 0.0434 mol) and Et3N (18 μL, 0.130 mmol, 3 equiv) in
toluene (4 mL) was added 2,4,6-trichlorobenzoyl chloride (9.4 μL,
0.0652 mmol, 1.5 equiv) at room temperature. The mixture was stirred
at room temperature for 4 h and then diluted with toluene (27 mL).
The supernatant of the mixture was added dropwise to a solution of
DMAP (79.5 mg, 0.652 mmol, 15 equiv) in toluene (47 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 an additional
1 h of stirring at room temperature, H2O (30 mL) was added and the
resulting biphasic mixture was poured into EtOAc (30 mL). The
organic layer was separated, and the aqueous layer 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, 5% → 15%
EtOAc/hexane) to afford a lactone (22.3 mg, 0.0326 mmol, 75% in
two steps) as a clear oil. To 10% Pd/C (5 mg) in a flask was added a
solution of the lactone (10.0 mg, 14.6 μmol) in EtOH (0.7 mL) at
room temperature. The mixture was vigorously stirred under an 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/hexane = 6:14:80; flow rate, 3.0 mL/min; pressure, 500 psi;
UV detector, 254 nm; retention time, 20.0 min) to afford 5 (5.7 mg,
exceeded 60% in all experiments except for that with TPA at 10−6
(>50%).
M
Two-Stage Carcinogenesis Experiment. The back of each male
6-week-old ICR mice was shaved with surgical clippers. From a week
after initiation by a single application of 390 nmol of DMBA in 0.1 mL
of acetone, 1.7 nmol of TPA or DAT in 0.1 mL of acetone or 8.5 nmol
of 5 in 0.1 mL of acetone was applied twice a week from week 1 to
week 20. Each group consisted of 10 mice.
Inhibition of Specific Binding of [3H]PDBu to PKC C1
Peptides. The binding of [3H]PDBu to the PKCδ C1 peptides was
evaluated by the procedure of Sharkey and Blumberg64 with
modifications as reported previously59 using 50 mM Tris-maleate
buffer (pH 7.4 at 4 °C), 10−20 nM PKCδ C1 peptides, 20 nM
[3H]PDBu (22.1 Ci/mmol, Perkin-Elmer Life Sciences), 50 μg/mL
1,2-dioleoyl-sn-glycero-3-phospho-L-serine (Sigma), 3 mg/mL bovine
γ-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, the IC50, which
was calculated with PriProbit 1.63 software.76 The inhibition constant,
Ki, was calculated by the method of Sharkey and Blumberg.64 Although
we used each PKC C1 peptide in the range of 10−20 nM, the
concentration of the properly folded peptide was estimated to be
about 3 nM on the basis of Bmax values of Scatchard analyses reported
previously.59 Therefore, the concentration of free PDBu will not
markedly vary over the dose−response curve.
1
11.3 μmol, 78%) as a clear oil. H NMR (500 MHz, 294.9 K, CDCl3,
0.009 M) δ 0.77 (3H, d, J = 7.0 Hz, H-24), 0.87 (3H, s, H-23), 0.96
(3H, s, H-22), 1.27−1.37 (2H, m, H-5a, H-12a), 1.37−1.48 (3H, m,
H2-4, H-13a), 1.48−1.61 (5H, m, H-5b, H-10, H-12b, H-13b, H-14a),
1.73 (1H, dd, J = 15.5, 4.1 Hz, H-8a), 1.75 (1H, m, H-14b), 2.31 (1H,
br s, OH), 2.43 (1H, dd, J = 13.3, 10.8 Hz, H-2a), 2.52 (1H, dd, J =
15.5, 2.3 Hz, H-8b), 2.55 (2H, m, H-15), 2.58 (1H, dd, J = 13.3, 2.6
Hz, H-2b), 2.79 (1H, dd, J = 16.8, 4.5 Hz, H-26a), 2.84 (1H, dd, J =
16.8, 10.1 Hz, H-26b), 3.81 (2H, m, H-28), 3.92 (1H, tt, J = 11.0, 2.9
Hz, H-3), 3.98 (1H, td, J = 10.3, 2.1 Hz, H-11), 5.03 (1H, dd, J = 6.1,
3.4 Hz, H-9), 5.18 (1H, m, H-27), 6.35 (1H, br s, PhOH), 6.6 (1H,
dd, J = 8.0, 2.5 Hz, H-19), 6.73 (1H, d, J = 7.6 Hz, H-21), 6.87 (1H, t,
J = 1.9 Hz, H-17), 7.13 (1H, t, J = 7.8 Hz, H-20) ppm. 13C NMR (125
MHz, 295.2 K, CDCl3, 0.009 M) δ 13.11 (C-24), 21.35 (C-22), 24.00
(C-13), 25.97 (C-23), 26.51 (C-8), 27.27 (C-4), 29.18 (C-14), 31.58
(C-12), 34.57 (C-5), 35.29 (C-15), 36.75 (C-26), 36.98 (C-6), 37.57
(C-10), 42.84 (C-2), 64.09 (C-28), 67.22 (C-11), 70.64 (C-3), 72.43
(C-27), 72.93 (C-9), 100.01 (C-7), 112.55 (C-19), 115.00 (C-17),
120.66 (C-21), 129.31 (C-20), 144.81 (C-16), 156.26 (C-18), 169.71
(C-25), 172.87 (C-1) ppm. HR-FAB-MS (matrix, m-nitrobenzyl
alcohol): m/z = 505.2828 ([MH]+, calcd for C28H41O8 505.2801).
ASSOCIATED CONTENT
* Supporting Information
■
S
Chiral HPLC analysis of 16; HPLC analysis of 4 and 5; NMR
spectra of 4, 5, 10, and 22; growth inhibitory activity of DAT,
aplog-1, 4, and 5 against 39 human cancer cell lines; Kd values
of the specific [3H]PDBu binding for whole PKC isozymes; Ki
values for inhibition of the specific [3H]PDBu binding for PKC
C1 peptides and whole PKC isozymes by (−)-indolactam-V.
This material is available free of charge via the Internet at
http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Phone: +81-75-753-6281. Fax: +81-75-753-6284. E-mail:
irie@kais.kyoto-u.ac.jp.
■
[α]29.8 +76.2° (c 0.28, CHCl3).
D
Measurements of Cell Growth Inhibition. A panel of 39 human
cancer cell lines established by Yamori and colleagues53 according to
the NCI method with modifications was employed, and cell growth
inhibitory activity was measured as reported previously.74 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) parameter was calculated as reported
previously.75 Absorbance for the control well (C) and the test well
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
This work was supported by a Grant-in-Aid for Scientific
Research on Innovative Areas “Chemical Biology of Natural
■
5623
dx.doi.org/10.1021/jm300566h | J. Med. Chem. 2012, 55, 5614−5626

Journal of Medicinal Chemistry
Article
(16) Hale, K. J.; Hummersone, M. G.; Manaviazar, S.; Frigerio, M.
The Chemistry and Biology of the Bryostatin Antitumour Macrolides.
Nat. Prod. Rep. 2002, 19, 413−453.
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Mol. Pharmacol. 1994, 46, 374−379.
Products” (Grant No. 23102011 to K.I.) from The Ministry of
Education, Culture, Sports, Science and Technology, Japan. We
also thank the Screening Committee for Anticancer Drugs
supported by a Grant-in-Aid for Scientific Research on
Innovative Areas “Scientific Support 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.
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ABBREVIATIONS USED
■
TPA, 12-O-tetradecanoylphorbol 13-acetate; ATX, aplysiatoxin;
bryo-1, bryostatin 1; DAT, debromoaplysiatoxin; MG-MID,
mean graph midpoint; EBV-EA, Epstein−Barr virus early
antigen; PDBu, phorbol 12,13-dibutyrate
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