Effects of the methoxy group in the side chain of debromoaplysiatoxin on its tumor-promoting and anti-proliferative activities

Article abstract of DOI:10.1016/j.bmcl.2013.05.096

Debromoaplysiatoxin (DAT) is a tumor promoter isolated from sea hare and exhibits anti-proliferative activity against several cancer cell lines. To clarify key residues that are responsible for its tumor-promoting activity, we focused on the chiral methoxy group in the side chain, whose role had not yet been discussed or examined before. Demethoxy-DAT (8) was derived from DAT and we evaluated its tumor-promoting activity, anti-proliferative activity, and ability to bind to protein kinase C (PKC) isozymes. Compound 8 showed somewhat weaker tumor-promoting activity than that of DAT both in vitro and in vivo, but showed higher anti-proliferative activity against several cancer cell lines. Although the affinity to novel PKC isozymes of 8 was comparable to that of DAT, the affinity to conventional PKC isozymes decreased slightly. These results suggest that the methoxy group of DAT is one of the key residues critical for tumor-promoting activity but not for anti-proliferative activity. Since the methoxy group has little influence on the molecular hydrophobicity, this is the first report showing that structural factors other than hydrophobicity in the side chain of DAT affected its biological activities.

Full text of DOI:10.1016/j.bmcl.2013.05.096

Bioorganic & Medicinal Chemistry Letters 23 (2013) 4319–4323
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Effects of the methoxy group in the side chain
of debromoaplysiatoxin on its tumor-promoting
and anti-proliferative activities
Ryo C. Yanagita a,b, Hiroaki Kamachi a, Masayuki Kikumori a, Harukuni Tokuda c, Nobutaka Suzuki c,
Kiyotake Suenaga d, Hiroshi Nagai e, Kazuhiro Irie a,
⇑
a Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan
b Department of Applied Biological Science, Faculty of Agriculture, Kagawa University, Kagawa 761-0795, Japan
c Department of Complementary and Alternative Medicine, Clinical R&D, Graduate School of Medical Science, Kanazawa University, Kanazawa 920-8640, Japan
d Faculty of Science and Technology, Keio University, Yokohama 223-8522, Japan
e Department of Ocean Sciences, Tokyo University of Marine Science and Technology, Tokyo 108-8477, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Debromoaplysiatoxin (DAT) is a tumor promoter isolated from sea hare and exhibits anti-proliferative
activity against several cancer cell lines. To clarify key residues that are responsible for its tumor-promot-
ing activity, we focused on the chiral methoxy group in the side chain, whose role had not yet been
discussed or examined before. Demethoxy-DAT (8) was derived from DAT and we evaluated its tumor-
promoting activity, anti-proliferative activity, and ability to bind to protein kinase C (PKC) isozymes.
Compound 8 showed somewhat weaker tumor-promoting activity than that of DAT both in vitro and
in vivo, but showed higher anti-proliferative activity against several cancer cell lines. Although the affin-
ity to novel PKC isozymes of 8 was comparable to that of DAT, the affinity to conventional PKC isozymes
decreased slightly. These results suggest that the methoxy group of DAT is one of the key residues critical
for tumor-promoting activity but not for anti-proliferative activity. Since the methoxy group has little
influence on the molecular hydrophobicity, this is the first report showing that structural factors other
than hydrophobicity in the side chain of DAT affected its biological activities.
Received 1 May 2013
Revised 25 May 2013
Accepted 30 May 2013
Available online 7 June 2013
Keywords:
Aplysiatoxin
Debromoaplysiatoxin
Protein kinase C
Tumor promotion
Cancer
Anti-proliferative activity
Ó 2013 Elsevier Ltd. All rights reserved.
Protein kinase C (PKC) is a family of serine/threonine protein ki-
nases consisting of at least 10 isozymes and is involved in many
cellular events such as proliferation, differentiation, and apopto-
sis.1 The PKC family is subdivided into three groups based on their
conducted so far,6,7 for example, phase I trials of TPA for patients
with hematological malignancies, but serious adverse events were
observed in that case.7 Recently, ingenol 3-angelate (ingenol
mebutate), isolated from Euphorbia peplus, was approved by the
US Food and Drug Administration (FDA) for the treatment of actinic
keratosis, a precancerous skin lesion;8 however, the acute inflam-
matory response by the ingenol ester might be a limiting factor
in uses other than topical application.
Bryostatin 1 (bryo-1), isolated from bryozoan Bugula neritina, is
a scarce PKC activator9 displaying little tumor-promoting activity
in vivo10 and a promising anticancer activity that has led to clinical
trials for cancer under the condition of bryo-1 alone or in combina-
tion with other chemotherapeutic agents.10 In spite of its potential
as a new medicinal lead, the isolation yield of bryo-1 from natural
sources is quite low (0.00014%),11 and there is no reliable method
to obtain bryo-1 in large quantities sufficient for further research
and medicinal use. Recently, Wender’s and Keck’s groups have
been developed truncated analogues of bryo-1, which provided
the promising ways to address the problem of supply.12,13
structures and cofactor requirements: conventional PKC (cPKC:
bI, bII, and ), novel PKC (nPKC: d, , and h), and atypical PKC
(aPKC: f and k/
) isozymes.1 Tumor promoters such as 12-O-tetra-
a,
c
e, g
i
decanoylphorbol 13-acetate (TPA), ingenol esters, teleocidins, and
aplysiatoxins (Fig. 1) bind to the tandem C1 domains (C1A and
C1B) in the regulatory domain of cPKCs and nPKCs, and strongly
activate them.1
Since over-expression and/or down-regulation of cPKCs and
nPKCs are observed in many cancers,2 PKC isozymes are expected
to be potential therapeutic targets in cancer.3 Recent studies also
indicated that the modulation of PKC isozymes could be a possible
strategy in the treatment of Alzheimer’s disease4 and acquired
immunodeficiency syndrome (AIDS).5 Since PKC modulators could
be promising reagents for the treatment of cancer, clinical trials of
TPA-type PKC activators for certain types of cancer have been
Aplysiatoxin (ATX) and debromoaplysiatoxin (DAT) are
TPA-type tumor promoters14 isolated from the sea hare Stylocheilus
longicauda15 and marine cyanobacteria belonging to genera
⇑
Corresponding author. Tel.: +81 75 753 6281; fax: +81 75 753 6284.
E-mail address: irie@kais.kyoto-u.ac.jp (K. Irie).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.05.096

4320
R. C. Yanagita et al. / Bioorg. Med. Chem. Lett. 23 (2013) 4319–4323
R
R
4
H
H
H
12
O
O
O
3
12
O
O
O
15
H
O
H
O
OMe
17
OH
O
10
O
O
O
O
O
O
H
O
H
O
H
O
X
30
OH
X
OH
HO
OH
HO
20
4
R = Me, X = OH: Aplog-1 (1)
R = X = H: 2
R = Me, X = H: 3
X = Br, Aplysiatoxin (ATX)
X = H, Debromoaplysiatoxin
(DAT)
R1
H
O
H
4
O
O
H
O
O
H
O
R2
10
O
O
O
O
H
O
H
O
27
OH
HO
OH
HO
R1 = Me, R2 = H: 5
R1 = H, R2 = Me: 6
7
Figure 1. Structure of aplysiatoxin (ATX), debromoaplysiatoxin (DAT), and synthetic analogues of DAT.17–23
Lyngbya, Schizothrix, and Planktothrix (Oscillatoria).16 Among natu-
rally-occurring potent tumor promoters, DAT has relatively low
lipophilicity and weak tumor-promoting activity. In order to ad-
dress the supply problem of bryo-1, we have recently developed
simplified, synthetically accessible, and less lipophilic analogs of
DAT, named ‘aplogs’ (1–7, Fig. 1).17–23 Like bryo-1, aplog-1 (1)
inhibited the growth of several human cancer cell lines and
showed little tumor-promoting activity in vitro and in vivo.17,21
In particular, the ability of 10-methyl-aplog-1 (6) to bind to nPKC
isozymes was comparable to that of DAT, whereas 6 did not show
any tumor-promoting activity in vivo.20
To develop more potent and safer aplogs, we need to know the
key structural components indispensable for the tumor-promoting
activity of ATX and DAT. In aplog-1 (1), a hydroxyl group at posi-
tion 3, methyl groups at positions 4, 10, 12, and 30, a methoxy
group at position 15, and a bromine atom at position 17 were re-
moved from ATX. Kishi and colleagues reported that 3-deoxy-
DAT and DAT are equipotent as PKC activators,24 and Shimomura
and colleagues reported that removal of the bromine atom dimin-
ished the tumor-promoting activity.12,25 Our previous studies have
shown that re-installation of each methyl group at positions 4,20
10,20 12,19 or 2723 did not restore the tumor-promoting activity;
however, the role of the chiral methoxy group of aplysiatoxins re-
mains unclear. In this Letter, we report the preparation and biolog-
ical activities of demethoxy-DAT (8) along with its dehydration
product (9).
chromatography. The spectroscopic data (1H and 13C 1D NMR spec-
tra, and optical rotation in Supplementary data) were in good
agreement with those reported in the literature.26,27 Catalytic
hydrogenation of DAT afforded 8 (42%) along with the dehydrated
derivative 9 (18%) in a single step (Scheme 1). A hemiacetal group
at position 3 of ATX is easily dehydrated under acidic conditions,28
indicating that 9 would be generated from 8 during biological eval-
uations. Thus, the biological activities of 9 as well as 8 were also
evaluated.
We first assessed the in vitro tumor-promoting activity of 8 and
9 by the Epstein–Barr virus early antigen (EBV-EA) induction test
using Raji cells (EBV genome-carrying lymphoblastoid cells) as de-
scribed previously.29 Tumor promoters such as TPA activate the la-
tent EBV genome and induce EA. Figure 2 shows the EBV-EA
inducing ability of DAT, 8, and 9. DAT strongly induced EA at
10À8, 10À7, and 10À6 M (21.1%, 22.9%, and 24.4%, respectively),
while the EA-inducing ability of 8 (14.3%, 17.9%, and 18.6%, respec-
tively) was significantly lower than that of DAT. EA-inducing abil-
ity of 9 was slightly but significantly lower than that of 8.
Evans et al. reported that, in the EBV-EA induction test, non-tu-
mor-promoting diterpene esters such as sapintoxin A were virtu-
ally equipotent to tumor-promoting compounds;30 therefore, the
tumor-promoting activity of 8 in vivo was further evaluated by
the two-stage carcinogenesis test on mouse skin (Fig. 3). The skin
on the back of male ICR mice was treated with a single dose of
390 nmol of 7,12-dimethylbenz[a]anthracene (DMBA) and from
1 week later, with 1.7 or 3.4 nmol of DAT or 8 twice a week. In a
control experiment using TPA (1.7 nmol twice a week), the first tu-
mor appeared in week 6 and the proportion of tumor-bearing mice
reached 100% at week 11 (Fig. 3A). The number of papillomas per
Compound 8 was prepared from DAT, which was isolated from
the extracts of cyanobacteria collected in Ishigaki Island (Okinawa)
and a red alga Gracilaria coronopifolia, which was parasitized by
cyanobacteria, collected in Hawaii26 by a sequence of column
H
H
O
H
O
O
O
O
O
15
H2, Pd(OH)2/C
EtOAc
OMe
OH
O
OH
O
O
O
O
O
O
O
O
H
O
H
O
H
O
OH
HO
OH
HO
OH
HO
DAT
8 (42%)
9 (18%)
Scheme 1. Synthesis of 8 and 9.

R. C. Yanagita et al. / Bioorg. Med. Chem. Lett. 23 (2013) 4319–4323
4321
30
25
20
15
10
5
pair comparisons showed significant (P <0.01) positive correlation.
As mentioned above, DAT and 8 were tumor promoters, while 1 and
DAT
8
9
a
a
a
b
100
b
A
TPA (1.7 nmol)
DAT (3.4 nmol)
c
b
c
80
60
40
20
0
DAT (1.7 nmol)
8 (3.4 nmol)
8 (1.7 nmol)
c
0
-8
-7
-6
10
10
10
Sample concentration (M)
Figure 2. EBV-EA-inducing ability of debromoaplysiatoxin (DAT), 8, and 9.
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. Cell viability exceeded 50% in most
cases, as shown in the Supplementary data. Values are the means standard errors
of the mean (n = 3). Bars marked with different letters in each concentration differ
significantly (P <0.05, Tukey’s HSD test).
5
10
Weeks of promotion
15
20
B
8
TPA (1.7 nmol)
7
6
5
4
3
2
1
0
DAT (3.4 nmol)
DAT (1.7 nmol)
8 (3.4 nmol)
mouse was 7.3 in week 20 (Fig. 3B). In groups treated with
3.4 nmol of DAT and 8, 1.7 nmol of DAT, and 1.7 nmol of 8, the pro-
portion reached up to 90%, 90%, 80%, and 70%, respectively, and
Fisher’s exact test indicated no significant difference between
DAT-treated and 8-treated groups. However, the number of papil-
lomas per mouse in the DAT-treated groups (4.7 for 3.4 nmol and
2.4 for 1.7 nmol at week 20) was higher than in the 8-treated
groups (2.9 for 3.4 nmol and 1.2 for 1.7 nmol at week 20), and sig-
nificant differences were observed in several weeks (Fig. 3B and C).
These results indicate that 8 is a weaker tumor promoter than DAT
and that the methoxy group is important for tumor-promoting
activity. Although the hydrophobicity of tumor promoters gener-
ally correlated with their tumor-promoting ability,31 removal of
8 (1.7 nmol)
5
10
Weeks of promotion
15
20
8
7
6
5
4
3
2
1
0
C
the methoxy group did not increase hydrophobicity (p of methoxy
group = À0.02),32 implying some kind of role of this chiral methoxy
group in tumor-promoting activity.
*
Due to low availability, similar experiments using the dehy-
drate compound 9 were not possible; however, preliminary exper-
iments using 3 mice at a dose of 3.4 nmol did not yield any tumors
after 20 weeks (data not shown), suggesting that 9 could not be a
tumor promoter.
*
+
Since DAT showed anti-proliferative activity against several hu-
man cancer cell lines,20 anti-proliferative activities of 8 and 9 were
evaluated using a panel of 39 human cancer cell lines established
by Yamori et al.33 The growth inhibitory activity was expressed
as a GI50 (M), the concentration required to inhibit cell growth
by 50% compared to an untreated control. The average of the log
GI50 values for all 39 human cancer cell lines was expressed as a
MG-MID. The cell lines with log GI50 values less than À6.00 for
either 8 or 9 are listed in Table 1 (the rest of the data are provided
as Supplementary data). Surprisingly, 8 showed much higher anti-
proliferative activity than DAT, and GI50 values of 8 were at least
one order of magnitude lower than those of DAT against most cell
lines listed in Table 1. Anti-proliferative activity of 9 was lower
than that of 8 in most cell lines, suggesting that 9 could not affect
the anti-proliferative activity of 8 even if 8 degraded into 9 during
the assays.
+
+
+
DAT
8
DAT
8
(3.4 nmol) (3.4 nmol) (1.7 nmol) (1.7 nmol)
Figure 3. Tumor-promoting activity of TPA, debromoaplysiatoxin (DAT), and 8. The
back of each male 6-week-old ICR mouse was shaved with surgical clippers. From a
week after initiation by a single application of 390 nmol DMBA in 0.1 mL acetone,
3.4 or 1.720 nmol DAT or 8 was applied twice a week from week 1 to week 20. The
control group was treated with DMBA and 1.7 nmol TPA. Each group consisted of 10
mice. (A) Tumor-bearing mice (%). There was no significant difference between
DAT-treated and 8-treated groups (Fisher’s exact test). (B) Number of papillomas
per mouse. The differences between DAT- (3.4 nmol) and 8- (3.4 nmol) treated
groups at weeks 11–17, 19, and the differences between DAT- (1.7 mol) and
8- (1.7 nmol) treated groups at weeks 13, 14, 17, 18 were statistically significant
(P <0.05, Welch’s t-test). (C) Box plot of the number of papillomas at week 17.
Horizontal lines across the box represent the median. Plus signs represent the
mean. The box represents the 25th and 75th percentiles. The maximum length of
each whisker is 1.5 times the interquartile range. Outliers are represented by dots.
⁄P <0.05 (Welch’s t-test).
Comparing the cell growth profiles (fingerprints) of agents pro-
vides valuable information on their molecular targets,33 since
agents sharing same molecular targets would show similar profiles.
Table 2 lists Pearson’s correlation coefficient (r) for the GI50 values
of DAT, 1, 6, and 8 for the panel of 39 human cancer cell lines. All

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R. C. Yanagita et al. / Bioorg. Med. Chem. Lett. 23 (2013) 4319–4323
mains (C1A and C1B) of PKCs35 were employed for the binding as-
say. The binding constants of some PKC ligands were almost equal
to those of whole PKC isozymes, as reported previously.20 The C1A
peptides were used as cPKC surrogates, and the C1B peptides were
employed as nPKC surrogates, since these domains are the main
binding sites of tumor promoters.36 While the affinity of 8 for
nPKCs was almost equal to that of DAT, those for cPKCs were about
2–9 times weaker than that of DAT. Affinity of 9 for d-C1B
(Ki = 8.2 nM) was about 25 times weaker than that of 8. Since the
loss of a bromine atom of ATX did not lower the binding affinity
for all PKC isozymes, the weaker tumor-promoting activity of
DAT than ATX may be simply explained by the decrease of hydro-
phobicity (logP of ATX, 5.4; logP of DAT, 4.4).22 In contrast, as
noted above, loss of the methoxy group did not result in the reduc-
tion of hydrophobicity. Thus, weaker tumor-promoting activity of
8 than DAT might be attributed to its relatively lower affinity for
cPKCs, as suggested in the case of 10-methyl-aplog-1 (6).20 Further
studies are needed to understand the precise role of PKC isozymes
in tumor promotion and the anti-proliferative effect by DAT and its
derivatives.
Table 1
Growth inhibition of debromoaplysiatoxin (DAT), 1, 6, 8, and 9 against several human
cancer cell lines
Cancer type
Breast
Cell line
logGI50 (M)
DATa
1b
6a
8
9
HBC-4
MDA-MB-231
HCC2998
À6.47
À6.03
À6.09
À4.94
À6.46
À5.94
À5.69
À6.44
À6.33
À5.61
À5.43
À4.81
À5.60
À5.32
À5.74
À5.55
À7.48
À6.90
À6.47
À6.15
À7.07
À6.01
À6.21
À6.24
<À8.00
À7.31
À7.16
À7.28
À8.00
À6.84
À5.79
À7.89
À6.26
À5.97
À5.73
À4.97
À6.06
À5.68
À6.08
À6.13
Colon
Lung
NCI-H226
NCI-H460
A549
LOX-IMVI
St-4
Melanoma
Stomach
MG-MIDc
À5.22
À4.97
À5.24
À5.61
À5.31
a
b
c
Cited from Ref. 20.
Cited from Ref. 17.
Full panel mean-graph midpoint.
Table 2
In summary, we synthesized the demethoxy derivative (8) of
DAT to investigate the role of the methoxy group in tumor-promot-
ing ability. The EBV-EA induction test using Raji cells and two-
stage carcinogenesis test in mouse skin indicated that 8 is a weaker
tumor promoter than DAT. Furthermore, 8 exhibited stronger anti-
proliferative activity against many human cancer cell lines. It is
quite noteworthy that the structural factors other than simple
hydrophobicity in the side chain of DAT affected its tumor-promot-
ing activity, anti-proliferative activity, and PKC isozyme-selective
binding. While the relationship between tumor-promoting and
anti-proliferative activities of PKC activators remains unclear, the
finding that removal of the methoxy group resulted in an increase
of anti-proliferative activity without the increase of tumor-pro-
moting activity may provide valuable information for the develop-
ment of more potent and safer analogues of DAT. The effect of the
stereochemistry of the methoxy group on tumor-promoting activ-
ity also seems to be interesting.
Pearson’s correlation coefficient (r) matrix for the GI50 values of DAT, 1, 6, and 8 for a
panel of 39 human cancer cell lines
DAT
1
6
8
DAT
1
6
1.000
0.852a
0.815a
0.861a
1.000
0.848a
0.793a
1.000
8
0.909a
1.000
a
Correlation is significant at the 0.01 level (two-tailed).
1.6
ATX
DAT
8
1.4
1.2
1
0.8
0.6
0.4
0.2
Acknowledgments
This work was partly supported by a Grant-in-aid for Scientific
Research on Innovative Areas ‘Chemical Biology of Natural Prod-
ucts’ (Grant No. 23102011 to K.I.) from The Ministry of Education,
Culture, Sports, Science and Technology, Japan. This study was
partly carried out using a JEOL JMS-700 MS spectrometer in the
Joint Usage/Research Center (JURC) at the Institute for Chemical
Research, Kyoto University. Finally, we thank Scientific Support
Programs for Cancer Research supported by a Grant-in-Aid for Sci-
entific Research on Innovative Areas from The Ministry of Educa-
tion, Culture, Sports, Science and Technology, Japan.
0
α-C1A β-C1A γ-C1A δ-C1B ε-C1B η-C1B θ-C1B
Figure 4. Ki values for the inhibition of [3H]PDBu binding by aplysiatoxin (ATX),18
debromoaplysiatoxin (DAT),20 and 8.
6 are not. All these compounds were potently anti-proliferative
against several cancer cell lines as shown in Table 1. Correlation be-
tween 6 and 8 was the highest (r = 0.909) and that between 1 and 8
was the lowest (r = 0.793). No significant difference between tu-
mor-promoting (DAT and 8) and non-tumor-promoting com-
pounds (1 and 6) existed. Although there were significant
differences in the tumor-promoting activity among these com-
pounds, these results clearly suggest that they could share same
molecular targets that are responsible for the anti-proliferative
activity.
To investigate the reason for the difference in these biological
activities between DAT and 8, the ability to bind to conventional
and novel PKC isozymes was evaluated by the procedure of Shar-
key and Blumberg (Fig. 4).34 These isozymes are believed to be
the main cellular targets of DAT.12 Since it was difficult to obtain
stable results using recombinant PKC isozymes, synthetic C1 do-
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2013.05.
096.
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