Synthesis and biological activities of the amide derivative of aplog-1, a simplified analog of aplysiatoxin with anti-proliferative and cytotoxic activities

Article abstract of DOI:10.1080/09168451.2014.1002452

Aplog-1 is a simplified analog of the tumorpromoting aplysiatoxin with anti-proliferative and cytotoxic activities against several cancer cell lines. Our recent findings have suggested that protein kinase Cδ (PKCδ) could be one of the target proteins of a

Full text of DOI:10.1080/09168451.2014.1002452

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Synthesis and biological activities of the amide
derivative of aplog-1, a simplified analog of
aplysiatoxin with anti-proliferative and cytotoxic
activities
Yusuke Hanakia, Ryo C. Yanagitaab, Takahiro Sugaharac, Misako Aidac, Harukuni Tokudad,
Nobutaka Suzukid & Kazuhiro Iriea
a Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto
University, Kyoto, Japan
b Faculty of Agriculture, Department of Applied Biological Science, Kagawa University,
Kagawa, Japan
c Center for Quantum Life Sciences, Department of Chemistry, Graduate School of
Science, Hiroshima University, Higashi-Hiroshima, Japan
Click for updates
d Department of Complementary and Alternative Medicine, Clinical R & D, Graduate
School of Medical Science, Kanazawa University, Kanazawa, Japan
Published online: 23 Jan 2015.
To cite this article: Yusuke Hanaki, Ryo C. Yanagita, Takahiro Sugahara, Misako Aida, Harukuni Tokuda, Nobutaka
Suzuki & Kazuhiro Irie (2015): Synthesis and biological activities of the amide derivative of aplog-1, a simplified analog
of aplysiatoxin with anti-proliferative and cytotoxic activities, Bioscience, Biotechnology, and Biochemistry, DOI:
10.1080/09168451.2014.1002452
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Bioscience, Biotechnology, and Biochemistry, 2015
Synthesis and biological activities of the amide derivative of aplog-1, a
simplified analog of aplysiatoxin with anti-proliferative and cytotoxic activities
Yusuke Hanaki1, Ryo C. Yanagita1,2, Takahiro Sugahara3, Misako Aida3, Harukuni Tokuda4,
Nobutaka Suzuki4 and Kazuhiro Irie1,*
1Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Kyoto, Japan;
3
2Faculty of Agriculture, Department of Applied Biological Science, Kagawa University, Kagawa, Japan; Center for
Quantum Life Sciences, Department of Chemistry, Graduate School of Science, Hiroshima University, Higashi-
4
Hiroshima, Japan; Department of Complementary and Alternative Medicine, Clinical R & D, Graduate School of
Medical Science, Kanazawa University, Kanazawa, Japan
Received November 12, 2014; accepted December 11, 2014
http://dx.doi.org/10.1080/09168451.2014.1002452
Aplog-1 is a simplified analog of the tumor-
promoting aplysiatoxin with anti-proliferative and
cytotoxic activities against several cancer cell lines.
Our recent findings have suggested that protein
kinase Cδ (PKCδ) could be one of the target pro-
teins of aplog-1. In this study, we synthesized amide-
aplog-1 (3), in which the C-1 ester group was
replaced with an amide group, to improve chemical
stability in vivo. Unfortunately, 3 exhibited seventy-
fold weaker binding affinity to the C1B domain
of PKCδ than that of aplog-1, and negligible anti-
proliferative and cytotoxic activities even at 10−4 M.
A conformational analysis and density functional
theory calculations indicated that the stable confor-
mation of 3 differed from that of aplog-1. Since
27-methyl and 27-methoxy derivatives (1, 2) without
the ability to bind to PKC isozymes exhibited
marked anti-proliferative and cytotoxic activities at
10−4 M, 3 may be an inactive control to identify the
target proteins of aplogs.
Bryostatin-1 (bryo-1)9) which was isolated from the
marine bryozoan Bugula neritina, is a unique PKC
activator that does not exhibit tumor-promoting or
inflammatory activity. Bryo-1 has been reported to have
significant anti-cancer and anti-proliferative activities,
and these have been attributed to activation of the
PKCδ isozyme,10–12) which plays a tumor suppressor
role and is involved in apoptosis.13–15) Bryo-1 is also
expected to become a therapeutic candidate for AD16)
and AIDS.17) Despite its potential as a new medicinal
lead, further studies on its mode of action and struc-
tural optimization have been hampered due to its lim-
ited availability from natural sources and synthetic
complexity. A functional oriented synthesis of the sim-
plified analogs of bryo-1 was recently conducted
address these issues.18,19)
As an alternative approach, we developed aplog-1, a
simplified analog of ATX.20) Aplog-1, supplied in only
27 steps via standard reactions, was not tumor-promoting
or inflammatory, but was anti-proliferative even though
it has the skeleton of tumor-promoting ATX. The anti-
proliferative activity of aplog-1 against several cancer
cell lines was previously shown to be similar to that of
bryo-1.20) Furthermore, aplog-1 behaved in a similar
manner to bryo-1 rather than TPA for the translocation
of GFP-tagged PKCδ using CHO-K1 cells; aplog-1 as
well as bryo-1 translocated GFP-tagged PKCδ to the
nuclear membrane and perinuclear region rather than to
the plasma membrane, unlike TPA.20) To examine the
contribution of PKCδ to the anti-proliferative activity of
aplog-1, we recently synthesized 27-methyl and 27-
methoxy derivatives (1, 2) that lacked the ability to bind
to PKCδ and evaluated their anti-proliferative activities
against 39 human cancer cell lines.21) Compounds 1 and
2 only exhibited weak anti-proliferative activities against
all the human cancer cell lines tested,21) suggesting that
the activation of PKCδ was involved in growth inhibi-
tory activities against several cancer cell lines that were
at least sensitive to aplog-1.
Key words: aplysiatoxin; anti-proliferative; protein
kinase C; tumor promoter
Aplysiatoxin (ATX) is a potent tumor promoter that
has been isolated from the digestive gland of the sea
hare Stylocheilus longicauda.1) ATX strongly binds to
and activates protein kinase C (PKC) isozymes, as well
as 12-O-tetradecanoylpholbol 13-acetate (TPA) and
teleocidin B-4.2,3) Since PKC is a family of serine/thre-
onine kinases that play pivotal roles in cellular signal
transduction including proliferation, differentiation, and
apoptosis,4–6) tumor promoters may become therapeutic
agents for intractable diseases such as cancer, Alzheimer’s
disease (AD), and acquired immune deficiency syn-
drome (AIDS). However, difficulties are associated
with their application to therapeutic uses due to their
potent tumor-promoting and inflammatory activities.7,8)
*Corresponding author. Email: irie@kais.kyoto-u.ac.jp
© 2015 Japan Society for Bioscience, Biotechnology, and Agrochemistry

2
Y. Hanaki et al.
The next step is to evaluate and improve the
Yamaguchi’s esterification29) of 6 with 5 provided 7.
Oxidative cleavage of the olefin group, followed by
esterification with N-hydroxysuccinimide, gave an acti-
vated ester. Immediately after deprotection of the benzyl
(Bn) and carbobenzyloxy (Cbz) groups by catalytic
hydrogenation, lactamization occurred to give 3 in a
single step. The yield (23% in two steps) was not
good because the Bn group resisted the catalytic
hydrogenation.
We initially evaluated the ability of 3 to bind to PKCδ
using a synthetic PKCδ-C1B peptide (δ-C1B),30) which
is a major binding site and plays a predominant role in
the activation of PKCδ by PKC ligands such as bryo-1,
ATX, and TPA. The concentration required to cause
50% inhibition (IC50) of [3H]phorbol 12,13-dibutyrate
(PDBu) was measured using a competitive binding
assay.31,32) Affinity for δ-C1B was expressed as a Ki
value calculated from the IC50 value of 3 and the Kd
value of [3H]PDBu, as reported by Sharkey and Blum-
berg.31) Table 1 lists the Ki value of 3 for δ–C1B along
with those of aplog-1 and its 27-methyl and 27-methoxy
derivatives (1 and 2). As previously reported,21,33) the
stereochemistry at position 27 was critical for PKC bind-
ing, and the free hydroxyl group at position 27 was
indispensable. Although the amide analog 3 fulfilled
these structural requirements at position 27 for PKCδ
binding, the affinity of 3 (Ki = 520 nM) was approxi-
mately two orders of magnitude weaker than that of
aplog-1 (Ki = 7.4 nM).
Regarding PKC activators such as 1,2-diacyl-sn-glyc-
erol (DAG) and indolactam-V (Fig. 1), the replacement
of an ester group with an amide group and vice versa
significantly affected their abilities to bind to PKC iso-
zymes by changing their conformation. The replace-
ment of either the sn-1 or sn-2 ester group of DAG, an
endogenous second messenger, with an amide group
markedly reduced its ability to activate PKC iso-
zymes.34) The conformationally constrained analogs of
DAG (DAG-lactones) with similar modifications devel-
oped by Marquez and colleagues also showed approxi-
mately ninety-fold to two hundred-fold lower binding
affinities for PKCα than those of their ester
counterparts.35) On the other hand, amide-to-ester
anti-proliferative activity of aplog-1 in vivo. When a
bioactive compound is applied to in vivo studies, its
chemical stability is critical to the drug efficacy. Com-
pounds with ester groups are generally susceptible to
hydrolysis by esterases in vivo. For example, epothilone
B22) showed cytotoxicity by inhibiting the depolymer-
ization of microtubules,23) but its efficacy was limited
in vivo due to its ester group. Ixabepilone is a deriva-
tive of epothilone B, in which the ester group is
replaced with an amide group. Ixabepilone was
approved as an anti-breast cancer agent because of its
potent cytotoxicity in vivo.24,25) Aplog-1 has two ester
linkages at C-1 and C-24 in the macrolactone ring. We
previously reported that benzolactams, the simplified
analogs of teleocidins, bound to PKC isozymes more
strongly than their lactone counterparts, benzolac-
tones,26,27) which prompted us to develop a new deriv-
ative of aplog-1 with a more stable amide linkage. We
herein described the synthesis of amide-aplog-1 (3), in
which the ester group at C-1 of aplog-1 was replaced
with an amide group, along with several of its biologi-
cal activities such as PKCδ binding, in vitro tumor-
promoting, anti-proliferative, and cytotoxic activities.
We chose to replace the C-1 ester group because this
site and benzolactones (Fig. 1) share a common hydro-
philic substructure, –C(=O)–O–CHR–CH2OH, with an
inversed stereochemistry, and the amide proton of 3
was expected to be at a spatial position similar to that
of the hydrogen atom in the hemiacetal 3-OH group of
ATX.
Results and discussion
The synthesis of 3 was accomplished in a convergent
approach from a spiroketal (6)20) and carboxylic acid
(5), as shown in Scheme 1. Compound 4 was prepared
from Z-D-Asp(OtBu)-OH as reported previously,28) and
the subsequent removal of the t-butyl ester with trifluoro-
acetic acid (TFA) afforded the carboxylic acid (5). The
spiroketal 6 was synthesized from m-hydroxycinnamic
acid in a similar manner to the synthesis of aplog-1.20)
HO
OAc
MeO2C
O
O
H
H
N
H
N
O
O
3
O
2
O
N
OH
N
OH
HO
OH
OH
O
OMe
Br
O
O
O
O
O
O
O
OH
N
H
N
H
OH
HO
O
CO2Me
Aplysiatoxin (ATX)
Indolactam-V
Bryostatin 1
Teleocidin B-4
22
23
5
O
4
H
O
H
12
6
7
3
O
3
13
14
2
11
2
X
N
O
O
O
Benzolactam-V8s : X = NH
Benzolactone-V8s: X = O
1
H
O
1
H
10
8
15
16
9
O
O
NH
O
OH
R1
17
18
21
20
27
26
24
26
24
O
O
27
25
25
R
R2
HO
OH
HO
19
: R1 = H, R2 = OH
: R1 = Me, R2 = OH
: R1 = H, R2 = OMe
Aplog-1
3
1
2
Fig. 1. Structures of bryostatin-1, aplysiatoxin, its simplified analogs (1–3), teleocidin-B4, indolactam-V, and its 8-membered analogs.

Biological Activities of the Amide Derivative of Aplog-1
3
CbzHN
HO2C
CbzHN
BnO
CbzHN
BnO
ref. 28
a
CO2tBu
CO2tBu
CO2H
3 steps
5
4
H
H
O
H
O
O
O
O
O
O
CO2H
ref. 17
20 steps
H
b
c-e
CbzHN
OBn
O
O
NH
O
OH
BnO
O
OH
BnO
OH
HO
6
7
3
Scheme 1. (a) TFA, dichloromethane (95%); (b) 5, 2,4,6-trichlorobenzoyl chloride, Et3 N, DMAP, toluene (80%); (c) KMnO4, NaIO4, t-BuOH,
pH 7 buffer (74%); (d) N-Hydroxysuccinimide, DCC, MeCN; (e) 20% Pd(OH)2-C, MeOH (23% in two steps).
Table 1. Ki values for the inhibition of [3H]PDBu-binding by
aplog-1, 1, 2, and 3.
(DFT) calculations to estimate the relative stabilities of
possible conformers.
A set of possible conformations of the macrolactone
core structure of 3 was generated by the simulated
Ki (nM)
Aplog-1a
7.4
1b
2b
3
annealing method, and we chose three possible con-
formers: the global-minimum with a trans-amide, an
ATX-like conformation with a trans-amide, and a
conformation with a cis-amide because the active con-
formation of indolactam compounds is known to be a
cis-amide form.37,38) Side chains at C-11 were attached
to them and dihedral angles in the side chain were
manually rotated to search for an energetically stable
orientation, in which the 18-OH group is involved in
intramolecular hydrogen bonding as suggested by its
δ–C1B peptide
>2,500
>2,500
520 (19)c
aCited from Ref. 20.
bCited from Ref. 21.
cValues in parentheses represent the standard deviation from triplicate
experiments.
modification in the indolactam-V analogs gave opposite
results. A lactone analog of the nine-membered indo-
lactam-V, the core structure of teleocidins, showed a
different conformational preference and was completely
inactive,36) while a lactone analog of the eight-mem-
bered benzolactam-V8 took a ring conformation similar
to that of benzolactam-V8 with cis-amide, and exhib-
ited twenty to ninety-fold lower affinity for PKC C1
domains than the corresponding benzolactam-V8 ana-
log.27) Therefore, both ester and amide analogs could
bind to PKC isozymes if it adopted an appropriate
conformation.
The strong binding ability of ATX to PKC isozymes
was attributed to the rigid conformation of the macro-
cyclic ring, which included a hydrophilic pharmaco-
phore,33) and an NMR analysis of aplog-120) indicated
that its preferred macrocyclic ring conformation was
similar to that of 3-deoxy-debromo-ATX (e.g.,
J2,3 = 10.8 and 2.8 Hz for aplog-1; 11 and 3 Hz for 3-
deoxy-debromo-ATX).33) In the case of 3, an NOE cor-
relation between NH-26 and H-11 was observed in a
2D NOESY NMR experiment in CDCl3 (Supplemental
Fig. 2), suggesting that the conformation of 3 differed
to those of ATX33) and aplog-1, in which the distance
between these atoms could be more than 4 Å. Since
ATX and aplogs were involved in the hydrophobic
environment when bound to PKCδ-C1B in the presence
of phosphatidylserine, the conformation of 3 in CDCl3
would reflect a conformation in a ternary complex of 3,
PKCδ-C1B, and phosphatidylserine membrane. In order
to clarify the effects of conformational changes in 3 on
decreases in PKCδ binding, we performed a conforma-
tional search followed by density functional theory
1
sharp H NMR signal in CDCl3. The candidate struc-
tures were pre-optimized using the molecular mechan-
ics method with the MMFF94s force field, and the
final DFT geometry optimizations were then performed
at the ωB97X-D/6–31G* level of theory.39) Fig. 2
shows the resulting three possible conformers of 3
(A-C) and their relative ωB97X-D/6–31G* energies.
As described above, conformers A and B had a
trans-amide bond, while C had a cis one. Conformer B
resembles a stable conformation of ATX.33) In con-
formers A and B, NH-26 and H-11 were spatially close
(2.06 Å in A, 2.61 Å in B), which is consistent with
the NOE correlation. Furthermore, the sharp and down-
field-shifted 1H NMR signal of NH-26 (7.45 ppm) in
CDCl3 could be explained by intramolecular hydrogen
bonding between N–H and an oxygen atom in these
conformers. DFT calculations showed that conformer A
had the lowest energy, and differences in energies
between A-B and A-C were 1.365 and 13.259 kcal -
mol−1, respectively. These results suggested that 3
existed as conformer A in CDCl3. Since conformer B
resembled but significantly diverged from the stable
conformation of ATX due to the rotation of amide
plane by 45º (based on N–C–1–C–2–C–3 dihedral
angle), the difference in energies between conformer A
and the ATX-like active conformation would be more
than 1.365 kcal mol−1. Thus, a seventy-fold decrease in
the binding ability of 3 from aplog-1, which is
equivalent to a 2.34 kcal mol−1 change in free energy,
could be ascribable mainly to the change in the
conformational preference.

4
Y. Hanaki et al.
trans-amide form A
trans-amide form B (ATX-like)
0.000 kcal mol−1
1.365 kcal mol−1
cis-amide form C
13.259 kcal mol−1
Fig. 2. Cross-eyed stereo views of possible conformations of 3 (A–C) and their relative energies at the ωB97X-D/6–31G* level.
Note: Dashed lines represent hydrogen bonding.
We then evaluated the tumor-promoting activity of 3
in vitro by testing the induction of Epstein-Barr virus
early antigen (EBV-EA) production.40,41) EBV is acti-
vated by treating cells with tumor promoters such as
TPA in order to produce EA, which can be detected by
an indirect immunofluorescence technique. Aplog-1 and
its C-27 derivatives (1 and 2) induced EA production
more weakly than the potent tumor promoter TPA. The
ability of 3 to induce EA production was weaker than
those of 1 and 2 without the ability to bind to PKC iso-
zymes21,33) (Fig. 3). Although EA production is consid-
ered to be related to the activation of PKC isozymes,42)
this result suggested that some part of the induction of
EA caused by aplog-1 and its derivatives could be
attributed to other mechanisms.
examples, because aplog-1 showed stronger growth
inhibitory activity against these cell lines. Similar
results were observed in MDA-MB-231, SNB-78,
HCC2998, A549, LOX-IMVI, and St-4 cell lines
(Supplemental Table 1). Cell growth was estimated by
the sulforhodamine B assay and expressed as a percent-
age of the control without 3. As expected from the
weak binding affinity for PKCδ, 3 hardly inhibited their
growth at low concentrations (<10−5 M) as well as 1
and 2. Furthermore, 3 exhibited weak cytotoxicity even
at 10−4 M, whereas aplog-1, 1, and 2 induced cell
death regardless of their binding affinities for PKCδ at
the same concentration, 10−4 M. This result suggests
that other targets may be involved in the cytotoxicity
of aplogs at 10−4 M.
The anti-proliferative activity of 3 was evaluated
with a panel of 39 human cancer cell lines established
by Yamori and colleagues44,45) The results for HBC-4
and NCI-H460 were shown in Fig. 4 as typical
In summary, we synthesized a new amide derivative
of aplog-1 (3) in order to improve the stability of ap-
log-1 against esterases and pH changes in vivo.
Although the very weak binding of 3 to the C1B
domain of PKCδ could be detected, 3 showed weak
anti-proliferative activity against the 39 cancer cell
lines examined, even at 10−4 M (Supplemental Table 1),
and hardly induced the production of EBV-EA. In con-
trast, aplog derivatives without binding affinity to
PKCδ and its isozymes (1, 2)21) retained these activities
at 10−4 M. These results suggest that some of the anti-
proliferative and cytotoxic activities of aplogs were
attributed to unidentified targets other than PKC iso-
zymes. A conformational analysis and DFT calculations
indicated that the stable conformation of 3 differed
from that of aplog-1. This conformational change could
prevent 3 from binding to PKCδ and other target pro-
teins responsible for anti-proliferative activity and
EBV-EA induction. Given that conformational changes
in PKC ligands could affect not only biological activi-
ties, but also cellular targets, these effects must be
taken into account to successfully derivatize the
skeletons of aplogs in the future.
30
20
10
0
–8 –7 –6 –8 –7 –6 –8 –7 –6–8 –7 –6 –8 –7 –6
aplog-1a
TPA
1b
2b
3
Concentration of compounds (log M)
Fig. 3. EBV-EA production induced by TPA, aplog-1, 1, 2, and 3.
Notes: The percentages of EA-positive cells are shown. Sodium n-
butyrate (4 mM) was added to all samples to enhance the sensitivity
of Raji cells. Only 0.1% of cells were positive for EA at 4 mM
sodium n-butyrate. The final concentration of DMSO was 0.4%. Cell
viability exceeded 60%. Error bars show the standard error of the
mean (n = 3). aCited from Ref. 43. bCited from Ref. 21.
We recently developed aplog-based molecular probes
and are currently attempting to identify its target pro-
teins other than PKC isozymes. As described above,

Biological Activities of the Amide Derivative of Aplog-1
HBC-4 (Breast) NCI-H460 (Lung)
5
100
50
0
100
50
0
aplog-1
1
2
3
21%
9%
50
50
–59%
–71%
–78%
–83%
–81%
–88%
100
100
–8
–7
–6
–5
–4
–8
–7
–6
–5
–4
Concentration (log M)
Concentration (log M)
Fig. 4. Effects of aplog-1, 1, 2, and 3 on the growth of human cancer cell lines: HBC-4 (breast) and NCI-H460 (non-small cell lung).
Note: Cell growth was expressed as a percentage of the control (media only). The results were presented as the average of duplicate points.
27-methyl and 27-methoxy derivatives (1, 2) did not
exhibit the ability to bind to PKC isozymes, but had
marked anti-proliferative and cytotoxic activities at
10−4 M, indicating that 1 and 2 were not suitable as
inactive controls for the target analysis. Since 3 exhib-
ited weak binding affinity for PKC, but little cytotoxic
activity even at 10−4 M, it would be suitable as an
inactive control for the identification of targets other
than PKC isozymes.
2,4,6-trichlorobenzoyl chloride (28.0 μL, 179 μmol,
1.7 equiv.) was added to a solution of 5 (51.1 mg,
149 μmol, 1.4 equiv.) and Et3 N (24.9 μL, 179 μmol,
1.7 equiv.) in toluene (1.1 mL) at room temperature.
After 3 h of stirring at room temperature, the superna-
tant of the suspension was added to a solution of 620)
(50.0 mg, 105 μmol) and DMAP (27.0 mg, 221 μmol,
2.1 equiv.) in toluene (1.1 mL) at room temperature.
The mixture was stirred at 50 °C for 2 h and then
poured into H2O (5.0 mL). The mixture was extracted
with EtOAc (5 mL x 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 7 (67.8 mg, 84.4 μmol, 80%)
Experimental
The following spectroscopic and analytical instru-
ments were used: digital polarimeter, DIP-1000 (Jasco,
Tokyo, Japan); 1H and 13C NMR, Avance III 400,
Avance III 500, and Avance II 800 (reference TMS,
Bruker, Germany); HPLC, model 600E with a model
2487 UV detector (Waters, Tokyo, Japan); and HR-
FAB-MS, JMS-600H (JEOL, Tokyo, Japan), and JMS-
700 (JEOL, Tokyo, Japan). HPLC was carried out on a
YMC-packed ODS-A AA12S05–2510WT (Yamamura
Chemical Laboratory, Kyoto, Japan). Wakogel® C-200
(silica gel, Wako Pure Chemical Laboratory, Osaka,
Japan) was used for column chromatography. [3H]
PDBu (18.7 Ci/mmol) was custom-synthesized by Per-
kin-Elmer Life Sciences Research Products (Boston,
MA). The PKCδ C1B peptide was synthesized as
reported previously.30) All other chemicals and reagents
were purchased from chemical companies and used
without further purification.
1
as a clear oil. H NMR (500 MHz, CDCl3, 0.0027 M)
ppm: δ 0.86 (3H, s), 0.95 (3H, s), 1.33–1.49 (9H, m),
1.57–1.66 (4H, m), 2.19 (1H, br. d, J = 15.5 Hz), 2.26
(1H, m), 2.35 (1H, m), 2.57 (2H, t, J = 7.8 Hz), 2.61
(1H, dd, J = 15.8, 8.0 Hz), 2.69 (1H, dd, J = 5.8 Hz),
3.49–3.60 (4H, m), 4.16–4.21 (2H, m), 4.47–4.52 (2H,
m), 4.95–5.02 (2H, m), 5.04 (2H, s), 5.06–5.09 (3H,
m), 5.56 (1H, br. d, J = 8.8 Hz), 5.80 (1H, m), 6.78–
6.82 (3H, m), 7.18 (1H, t, J = 7.8 Hz), 7.27–7.45 (14H,
m); 13C NMR (125 MHz, CDCl3, 0.0027 M) ppm: δ
22.0, 24.8, 25.3, 26.1, 27.2, 31.3, 33.7, 34.5, 35.6,
36.0, 36.5, 36.8, 40.9, 48.0, 63.9, 66.7, 68.4, 70.0,
71.1, 71.7, 73.3, 100.1, 111.8, 115.2, 116.6, 121.2,
127.5 (2C), 127.6 (2C), 127.8, 127.9 (2C), 128.1,
128.1, 128.4 (2C), 128.5 (2C), 128.6 (2C), 129.2,
135.4, 136.6, 137.3, 137.9, 144.6, 155.8, 158.9, 171.3;
HR-FAB-MS (matrix, m-nitrobenzyl alcohol) m/z:
826.4324 ([M + Na]+ Calcd. for C50H61NO8Na
826.4295) [α]D + 19.7º (c = 0.14, CHCl3, 28.9 °C).
KMnO4 (13.1 mg, 83.2 μmol, 1 equiv.) was added to
a suspension of NaIO4 (143 mg, 0.666 mmol, 8 equiv.)
in pH 7.2 phosphate buffer (6.9 mL) in one portion.
After 5 min of stirring at room temperature under an
Ar atmosphere, the mixture was added to a solution of
7 (66.8 mg, 83.2 μmol) in t-BuOH (6.9 mL). The reac-
tion mixture was stirred at room temperature for 1 h,
and the reaction was quenched with Na2S2O3
(39.5 mg). The organic layer was separated, and the
aqueous layer was extracted with EtOAc (20 mL × 3).
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, 20% EtOAc/hexane containing 1% AcOH)
Synthesis of 3.
Compound 4 was prepared as
reported previously.28) [α]D + 13.2º (c = 0.44, MeOH,
10.9 °C). TFA (0.9 mL) was added to a solution of 4
(35.8 mg, 89.7 μmol) in dichloromethane (0.9 mL) at
0 °C. After 4 h of stirring at room temperature, the
reaction mixture was concentrated in vacuo to afford 5
(29.3 mg, 85.4 μmol, 95%) as a clear oil. 1H NMR
(500 MHz, CDCl3, 0.015 M) ppm: δ 2.70 (2H, d,
J = 5.9 Hz), 3.55–3.59 (2H, m), 4.23 (1H, br. s), 4.50
(2H, s), 5.09 (2H, s), 5.41 (1H, br. d, J = 8.1 Hz),
7.27–7.37 (10H, m); 13C NMR (125 MHz, CDCl3,
0.015 M) ppm: δ 35.8, 47.8, 66.9, 70.8, 73.4, 127.7
(2C), 127.9 (2C), 128.1, 128.2, 128.5 (2C), 128.6 (2C),
136.4, 137.7, 155.9, 174.1; HR-EI-MS m/z: 343.1427
([M]+ Calcd. for C19H21NO5 343.1420) [α]D + 16.1º
(c = 0.15, CHCl3, 10.3 °C).

6
Y. Hanaki et al.
to afford a carboxylic acid (50.8 mg, 61.9 μmol, 74%)
as a clear oil.
concentration required to inhibit the specific binding of
[3H]PDBu by 50%, the IC50, which was calculated by
a computer program with a probit procedure.46) The
inhibition constant, Ki, was calculated by the method
of Sharkey and Blumberg.31)
N,N′-dicyclohexylcarbodiimide (7.3 mg, 35.4 μmol,
1.5 equiv.) in MeCN (0.20 mL) was added to a solution
of the carboxylic acid (19.4 mg, 23.6 μmol) and N-
hydroxysuccinimide (5.4 mg, 47.2 μmol, 2 equiv.) in
MeCN (0.20 mL) at 0 °C. The reaction mixture was
stirred at 0 °C for 10 h, then concentrated in vacuo.
The residue was purified by column chromatography
(silica gel, 5% → 10% → 20% → 30% EtOAc/hexane)
to afford a crude activated ester (28.1 mg). A solution
of the activated ester (26.7 mg) in MeOH (1.0 mL) was
added to 20% Pd(OH)2-C (wet support, Aldrich)
(7.8 mg) in a flask at room temperature. The mixture
was vigorously stirred under a H2 atmosphere at room
temperature for 4.5 h. The mixture was filtered, and the
filtrate was concentrated in vacuo. The residue was dis-
solved in MeOH (0.6 mL), and again added to 20% Pd
(OH)2-C (wet support, Aldrich) (5.9 mg) in a flask at
room temperature. The mixture was vigorously stirred
under a H2 atmosphere at room temperature for 2.5 h.
The mixture was filtered, and the filtrate was concen-
trated in vacuo. This procedure was repeated two times
and a total of 38.5 mg (46.9 μmol) of the carboxylic
acid was reacted. These residues were purified by
HPLC (column, YMC-Pack ODS-A AA12S05–
2510WT; solvent MeOH/H2O = 75:25, flow rate
3.0 mL/min; pressure, 2100 psi; UV detector 254 nm;
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. 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 the serum of an NPC patient’s (a gift from Kobe
University, Japan) and FITC-labeled anti-human IgG
(DAKO, Glostrup, Denmark) as reported previously.41)
At least 500 cells were counted in each assay and the
proportion of EA-positive cells was recorded. Cell via-
bility exceeded 60% in all experiments.
Measurements of cell growth inhibition.
A panel
of 39 human cancer cell lines established by Yamori
and colleagues44) according to the NCI method with
modifications was employed, and cell growth inhibitory
activity was measured as reported previously.45) In
brief, cells were plated on 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.
Absorbance for the control well (C) and test well (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 the 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.
retention time, 20.1 min) to afford
3
(5.2 mg,
10.6 μmol, 23%) as a clear oil. 1H NMR (400 MHz,
CDCl3, 0.0036 M) ppm: δ 0.96 (3H, s, H3–23), 1.00
(3H, s, H3–22), 1.38–1.59 (9H, m, H2–4, H2–5, H-10a,
H2–12, H2–13), 1.64 (2H, m, H2–14), 1.71 (1H, dd,
J = 15.5, 3.7 Hz, H-8α), 1.79 (1H, m, H-10b), 2.28
(1H, dd, J = 15.1, 1.1 Hz, H-2a), 2.46 (1H, m, H-8β),
2.55–2.65 (4H, m, H-2b, H2–15, H-25a), 2.95 (1H, dd,
J = 16.5, 10.7 Hz, H-25b), 3.70–3.87 (4H, m, H-3, H-
26, H2–27), 4.09 (1H, m, H-11), 4.45 (1H, dd, J = 7.6,
5.0 Hz, OH), 5.21 (1H, m, H-9), 6.27 (1H, s, Ph-OH),
6.65–6.74 (3H, m, H-17, H-19, H-21), 7.13 (1H, t,
J = 7.8 Hz, H-20), 7.45 (1H, br. d, J = 4.7 Hz, NH);
13C NMR (125 MHz, CDCl3, 0.0029 M) ppm: δ 21.5
(C-22), 24.2 (C-13), 25.6 (C-8), 25.9 (C-23), 27.8 (C-
4), 30.2 (C-14), 34.2 (C-5 or 10 or 12), 34.4 (C-5 or
10 or 12), 34.7 (C-5 or 10 or 12), 35.3 (C-15), 36.3
(C-25), 37.3 (C-6), 43.8 (C-2), 51.6 (C-26), 63.9 (C-
11), 64.5 (C-27), 68.5 (C-9), 71.3 (C-3), 101.3 (C-7),
112.8 (C-19), 115.1 (C-17), 120.6 (C-21), 129.4 (C-
20), 144.4 (C-16), 156.3 (C-18), 170.3 (C-24), 173.4
(C-1); HR-FAB-MS (matrix, m-nitrobenzyl alcohol) m/
z: 490.2812 ([M + H]+ Calcd. for C27H40NO7
490.2805) [α]D + 38.9º (c = 0.074, CHCl3, 9.6 °C).
Conformational search of 3. The generation of a
conformer library of 3 by a simulated annealing
method under a vacuum was performed using the
GROMACS program47) (version 4.6.5) with a general
AMBER force field.48) The side chain at C11 of 3 was
replaced with a methyl group. All bonds were con-
strained using the LINCS algorithm. The annealing
temperature was initially set to 1,500 K in order to sur-
pass the cis-trans isomerization barrier in this system
and the temperature was kept constant for 1 ps. The
temperature was linearly dropped to 100 K over 1 ps
and then to 0 K over 1 ps, and kept at the same tem-
perature for 1 ps. This 5-ps cycle was repeated 200
times to give the conformer library.
Inhibition of specific binding of [3H]PDBu to the
PKCδ-C1B peptide. The binding of [3H]PDBu to the
δ-C1B peptide was evaluated by the procedure of
Sharkey and Blumberg31) with modifications as
reported previously32) using 50 mM Tris-maleate buffer
(pH 7.4 at 4 °C), 13.8 nM δ-C1B peptide, 20 nM [3H]
PDBu (18.7 Ci/mmol), 50 μg/mL 1,2-dioleoyl-sn-glyce-
ro-3-phospho-L-serine sodium salt (Sigma), 3 mg/mL
bovine γ-globulin, and various concentrations of an
inhibitor. Binding affinity was evaluated based on the
Three possible conformers were selected from this
library: the global-minimum with trans-amide,
ATX-like conformation with trans-amide, and
a
conformation with cis-amide. The side chains at C-11
were attached to them, and dihedral angles in the side

Biological Activities of the Amide Derivative of Aplog-1
7
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Supplemental material
The supplemental material for this paper is available
at http://dx.doi.org/10.1080/09168451.2014.1002452.
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Acknowledgments
We thank the Screening Committee for Anticancer
Drugs supported by a Grant-in-Aid for Scientific
Research on Innovative Areas (“Scientific Support Pro-
grams for Cancer Research”) from the Ministry of Edu-
cation, Culture, Sports, Science and Technology, Japan.
Mass measurements were 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. The DFT calcula-
tions were carried out at the Research Center for Com-
putational Science, Okazaki National Research
Institutes. The NOESY spectrum of 3 was measured by
Dr Ken-ichi Akagi at the National Institute of Biomedi-
cal Innovation (Osaka).
[17] Gustafson KR, Cardellina JH, McMahon JB, Gulakowski RJ,
Ishitoya J, Szallasi Z, Lewin NE, Blumberg PM, Weislow OS. A
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synthesis, step economy, and drug design. Acc. Chem. Res.
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Funding
This work was partly supported by the Ministry of Educa-
tion, Culture, Sports, Science and Technology, Japan under a
Grant-in-Aid for Scientific Research on Innovative Areas
“Chemical Biology of Natural Products” [number 23102011]
(to KI and RCY), [number 26102733] (to MA).
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Blumberg PM, Keck GE. Synthesis of a des -B-ring bryostatin
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H, Saito N, Nagai H, Irie K. A simple analogue of tumor-
promoting aplysiatoxin is an antineoplastic agent rather than a
ORCID
Kazuhiro Irie
http://orcid.org/0000-0001-7109-8568
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