Role of the phenolic hydroxyl group in the biological activities of simplified analogue of aplysiatoxin with antiproliferative activity

Article abstract of DOI:10.1016/j.bmcl.2010.08.051

The 18-deoxy derivative (3) of a simplified analogue (1) of aplysiatoxin with antiproliferative activity was synthesized to examine the role of the phenolic hydroxyl group at position 18 in the biological activities of 1. Compound 3 as well as 1 showed significant affinity for protein kinase Cδ (PKCδ), and the antiproliferative activity of 3 was slightly higher than that of 1. However, the anti-tumor-promoting activity of 3 was less than that of 1 in vitro, suggesting that the phenolic hydroxyl group of 1 is necessary for the anti-tumor-promoting activity but not for the binding of PKCδ and antiproliferative activity. Moreover, PKC isozyme selectivity of 3 was similar to that of 1, suggesting non-PKC receptors for these compounds to play some roles in the anti-tumor-promoting activity of 1.

Full text of DOI:10.1016/j.bmcl.2010.08.051

Bioorganic & Medicinal Chemistry Letters 20 (2010) 6064–6066
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Role of the phenolic hydroxyl group in the biological activities of
simplified analogue of aplysiatoxin with antiproliferative activity
Ryo C. Yanagita a, Hiroaki Kamachi a, Keisuke Tanaka a, Akira Murakami a, Yu Nakagawa b,
Harukuni Tokuda c, Hiroshi Nagai d, Kazuhiro Irie a,
⇑
a Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan
b Synthetic Cellular Chemistry Laboratory, Advanced Science Institute, RIKEN (The Institute of Physical and Chemical Research), Wako 351-0198, Japan
c Graduate School of Medical Science, Kanazawa University, Kanazawa 920-8640, Japan
d Tokyo University of Marine Science and Technology, Minato-ku, 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:
Received 3 July 2010
Revised 10 August 2010
Accepted 11 August 2010
Available online 13 August 2010
The 18-deoxy derivative (3) of a simplified analogue (1) of aplysiatoxin with antiproliferative activity was
synthesized to examine the role of the phenolic hydroxyl group at position 18 in the biological activities
of 1. Compound 3 as well as 1 showed significant affinity for protein kinase Cd (PKCd), and the antipro-
liferative activity of 3 was slightly higher than that of 1. However, the anti-tumor-promoting activity of 3
was less than that of 1 in vitro, suggesting that the phenolic hydroxyl group of 1 is necessary for the anti-
tumor-promoting activity but not for the binding of PKCd and antiproliferative activity. Moreover, PKC
isozyme selectivity of 3 was similar to that of 1, suggesting non-PKC receptors for these compounds to
play some roles in the anti-tumor-promoting activity of 1.
Keywords:
Protein kinase C
Tumor promoter
Bryostatin
Ó 2010 Elsevier Ltd. All rights reserved.
Antiproliferative
Epstein-Barr virus
Aplysiatoxin
Phorbol ester
Protein kinase C (PKC) isozymes are serine/threonine kinases
involved in cell proliferation, differentiation, and apoptosis.1,2 The
PKC family comprises at least eleven members of three distinct
tetradecanoylphorobol 13-acetate (TPA) showed strong tumor-
promoting activity,5 less hydrophobic derivatives such as 12-
deoxyphorbol 13-phenylacetate,12 prostratin (12-deoxyphorbol
13-acetate),13 phorbol 12,13-diacetate,14 and PDBu15 showed
anti-tumor-promoting activity in vivo. Moreover, loss of the bro-
mine atom of aplysiatoxin did not affect the affinity for PKC but
reduced the tumor-promoting activity of ATX.16 Based on these
findings, we recently developed simplified analogues of ATX
(1 and 2; Fig. 1) with less hydrophobicity and stereogenic centers,
showing bryo-1-like activity.17 Compound 1 was produced in only
22 steps, three times less than is needed to produce bryo-1, and
showed antiproliferative effects against many human cancer cell
lines comparable to bryo-1. Moreover, 1 showed very weak tu-
mor-promoting activity, but significant anti-tumor-promoting
activity, in vitro. However, its affinity for the C1B domain of PKCd
(d-C1B) and ability to activate the PKCd isozyme were one order
of magnitude lower than those of bryo-1.
In that study, we also suggested the geminal dimethyl group at
position 6 and/or the phenolic hydroxyl group at position 18 of 1 to
be important both for the binding of PKCd and for the antiprolifer-
ative activity.17 However, the exact role of the phenolic hydroxyl
group in these activities remains unknown. In this communication,
we report the synthesis and biological activities of the 18-deoxy
analogue (3) of 1.
types: conventional (
a, bI, bII, and c), novel (d, e, g, and h), and
atypical (k/ and
i
1
) PKCs.3 Naturally-occurring PKC activators such
as 12-O-tetradecanoylphorbol 13-acetate (TPA),4 teleocidin B-4,5
and aplysiatoxin6 (ATX; Fig. 1) bind to tandem C1 domains (C1A
and C1B) in the regulatory region of conventional and novel PKCs.
While these activators exhibit strong tumor-promoting activity,5
bryostatin 17 (bryo-1; Fig. 1), a PKC activator isolated from the
marine bryozoan Bugula neritina, shows significant anticancer
and anti-tumor-promoting activities possibly through PKCd-
dependent mechanisms.8–10 Bryo-1 has also attracted attention
as a therapeutic lead for the treatment of central nervous system
(CNS) diseases such as Alzheimer’s disease and depression.11
Despite its fascinating activities, however, bryo-1’s low availability
from natural sources and structural complexity have hampered its
optimization for therapeutic use.
Several studies have indicated that the hydrophobicity of PKC
activators is a critical determinant for tumor-promoting activity.
For example, while hydrophobic phorbol esters such as 12-O-
⇑
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 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.08.051

R. C. Yanagita et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6064–6066
6065
HO
O
OAc
R1
MeO2C
R1
O
O
O
H
H
6
O
O
O
O
1: R1 = Me, R2 = OH
2: R1 = R2 = H
HO
O
OMe
Br
OH
H
OH
O
O
O
O
O
O
O
H
O
H
O
3: R1 = Me, R2 = H
OH
OH
R2
18
OH
HO
O
CO2Me
Aplysiatoxin (ATX)
Bryostatin 1 (bryo-1)
Figure 1. Structure of bryostatin 1 (bryo-1), aplysiatoxin (ATX), and the simplified analogues of ATX (1–3).
Compound 3 was synthesized from 1 with a transfer-hydroge-
nation method (Scheme 1). Selective triflation of the phenolic
hydroxyl group followed by transfer-hydrogenation using palla-
dium(II) acetate, formic acid, and triethylamine gave 3.18 The affin-
ity of 3 for the C1 domains of PKCd was evaluated by inhibiting the
specific binding of [3H]phorbol 12,13-dibutyrate (PDBu) to the syn-
thetic PKCd C1 peptides (d-C1A, d-C1B) as described previously.19
The affinity of 3 for these peptides was almost equal to that of 1
(Table 1) and was significantly greater than that of 2, suggesting
the geminal dimethyl group at position 6, not the phenolic hydro-
xyl group at position 18 of 1, to play an important role in the bind-
ing to the C1 domains. It is worth noting that the removal of the
hydrophilic phenolic hydroxyl group from the side chain of 1 did
not affect the binding of PKCd. This indicates that the side chain
of these ATX analogues (1 and 3) is not a pharmacophore equiva-
lent to the ester side chains of phorbol esters such as TPA that in-
sert into phospholipid membranes. Since the introduction of
oxygen or hydrophilic groups into the ester side chains of phorbol
esters abolished the binding to PKC, the structure–activity data on
1 and 3 are not consistent with the pharmacophore model pro-
posed previously based on computational methods.20 However,
the hydrophobicity of the PKC activators are important for the
binding to PKC. The alkyl chain and the hydrophobic part of the
benzene ring of the side chain of the analogues could contribute
as the hydrophobic moiety such as the terpene moiety of teleocidin
B-4 without a straight alkyl chain.
One important biological property of 1 is its antiproliferative
activity. We evaluated the antiproliferative activity of 3 against a
panel of 39 human cancer cell lines as described previously.21 The
growth inhibitory activity is expressed as the concentration
required to inhibit cell growth by 50% compared to an untreated
control [GI50 (M)]. The results for the cell lines in which 1 and 3
showed GI50 values greater than each full panel mean-graph
midpoint (MG-MID) are listed in Table 2; the MG-MID values of 1
and 3 were À4.98 and À5.09, respectively. The rest are provided as
Supplementary data. The antiproliferative activities of 3 were
equivalent to or higher than those of 1 except for the LOX-IMVI
Table 1
Ki values for the inhibition of [3H]PDBu’s binding by 1–3 and ATX
PKC C1 peptides
Ki (nM)
Kd (nM)
PDBua
1
2
3
ATX
a
-C1A
b-C1A
-C1A
63 (5)b
89 (5)
39 (3)
140c
2400 (100) 120 (10) 0.40 (0.05) 1.1
3500 (400) 140 (20) 0.45 (0.08) 1.3
c
1600 (200) 80 (2)
0.63 (0.14) 1.5
130 (30) 12c
51.9
9.8 (0.5) 0.41c
d-C1A
d-C1B
6800c
170c
7.4c
0.53
0.81
e-C1B
25 (2)
4.4 (0.2) 180 (20)
4.0 (0.5) 170 (20)
820 (80)
37 (5)
12 (2)
1.3 (0.2)
0.36 (0.02) 0.45
g
-C1B
h-C1B
8.1 (1.0) 0.16 (0.03) 0.72
a
b
c
Cited from Ref. 28.
Standard deviation from triplicate experiments.
Cited from Ref. 17.
Table 2
log GI50 values for 1 and 3 against a subset of 39 human cancer cell lines
Cancer type
Cell line
log GI50 (log M)
1a
3
Bryo-1b
Breast
Breast
Breast
CNS
Colon
Lung
HBC-4
BSY-1
À6.33
À4.87
À5.61
À5.06
À5.43
À5.60
À5.32
À5.74
À5.55
À5.33
À4.96
À6.28
À5.17
À5.67
À5.14
À5.53
À5.83
À5.49
À5.17
À6.05
À6.09
À5.26
NRc
NR
MDA-MB-231
SF-295
HCC2998
NCI-H460
A549
LOX-IMVI
St-4
MKN45
PC-3
À5.20
À5.20
À5.30
À5.60
À5.20
NR
NR
NR
À5.30
Lung
Melanoma
Stomach
Stomach
Prostate
a
Cited from Ref. 17. Antiproliferative assays of 1 and 3 were carried out simul-
taneously by the same method.
b
Cited from Ref. 22. Antiproliferative assay of bryo-1 could not carried out since
the supply of bryo-1 is extremely limited.
c
Not reported.
melanoma cell lines. Notably, the GI50 values of 3 against St-4 and
MKN45 stomach cancer cells were below 10À6 M. Since the affinity
of 3 for PKCd was almost equal to that of 1, the slight increase in
the antiproliferative activity of 3 might be due to the increase in
hydrophobicity associated with the ability of the molecule to
permeate membranes.
As mentioned above, the hydrophobicity of the PKC activators is
a critical determinant for tumor-promoting activity. The tumor-
promoting/anti-tumor-promoting activity of 3 was estimated by
the Epstein-Barr virus early antigen (EBV-EA) induction test using
Raji cell (EBV genome-carrying lymphoblastoid cell) as described
previously.23
H
H
O
O
O
O
a, b
O
O
O
O
O
O
H
O
H
O
OH
HO
OH
1
3
As shown in Figure 2, the ability to induce EA of each compound
Scheme 1. Synthesis of 3. Reagents and conditions: (a) N-phenyl triflimide, Et3N,
CH2Cl2 (91%); (b) Pd(OAc)2, Ph3P, Et3N, HCO2H, DMF (25%).
at 10À7 M was measured since potent tumor promoters such as

6066
R. C. Yanagita et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6064–6066
Acknowledgments
This work was partly supported by The Naito Foundation and
The Uehara Memorial Foundation (K.I.), and by a Grant-in-aid for
Scientific Research (A) (No.21248015) (K.I.) and a Grant-in-aid for
JSPS Fellows (No. 20Á4135) (R.C.Y.) from The Ministry of Education,
Culture, Sports, Science and Technology, Japan. Finally, we thank
the Screening Committee of Anticancer Drugs supported by a
Grant-in-aid for Scientific Research on the Priority Area ‘Cancer’
from The Ministry of Education, Culture, Sports, Science and Tech-
nology, Japan.
Supplementary data
Figure 2. EBV-EA-inducing ability of TPA, ATX, bryo-1, 1, and 3. Percentages of
EA-positive cells are shown. Sodium n-butyrate (4 mM) was added to all samples to
enhance the sensitivity of Raji cells. Only 0.1% EA-induction was demonstrated at
4 mM. The final concentration of dimethyl sulfoxide (DMSO) was 0.4%. Cell viability
exceeded 60% in each experiment except for ATX (50%). Error bars represent
standard errors of the mean (n = 3).
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2010.08.051.
References and notes
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Not only PKCd but also other PKC isozymes (PKCs) play important
physiological roles, for example, Raji cells express PKCa, bII, and d
isozymes.26,27 To investigate the mode of action of the ATX ana-
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activators.19,28 The affinities were, thus, measured by the C1A pep-
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results are summarized in Table 1. While ATX showed little selectiv-
ity between conventional and novel PKCs like PDBu,28 affinities of
1–3 for novel PKCs were about ten times higher than those for
conventional PKC. Given that the affinity and the selectivity for
PKC isozymes of 3 were comparable to those of 1, these results also
suggest an unknown receptor of 1 other than PKCs to be responsible
for the anti-tumor-promoting ability of 1.
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s), 1.34–1.69 (13H, m), 2.21 (1H, t, J = 6.0 Hz), 2.40 (1H, dd, J = 13.1, 10.8 Hz),
2.49 (1H, d, J = 15.6 Hz), 2.53 (1H, dd, J = 13.1, 2.7 Hz), 2.62 (2H, t, J = 7.8 Hz),
2.70 (1H, dd, J = 16.7, 3.4 Hz), 2.80 (1H, dd, J = 16.7, 11.5 Hz), 3.70 (1H, quintet,
J = 5.7 Hz), 3.77 (1H, ddd, J = 12.0, 5.7, 4.0 Hz), 3.88 (1H, m), 4.17 (1H, m), 5.18
(1H, m), 5.20 (1H, m), 7.15–7.26 (3H, m), 7.27–7.29 (2H, m) ppm; 13C NMR
(125 MHz, CDCl3, 0.022 M, 297 K) d 21.19, 24.77, 25.13, 25.93, 27.25, 31.28,
34.54, 34.72, 35.52, 35.93, 36.87, 36.98, 42.69, 63.72, 64.43, 68.77, 70.57, 71.74,
100.20, 125.52, 128.19 (2C), 128.44 (2C), 142.91, 169.60, 171.51 ppm; HR-FAB-
MS m/z 475.2700 ([M+H]+). Calcd for C27H39O7 475.2696; [
CHCl3, 22.7 °C).
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a] +41 (c 0.218,
D
In summary, we synthesized the 18-deoxy derivative (3) of a sim-
plified analogue of ATX (1) to investigate the effect of the phenolic
hydroxyl group at position 18 on the affinity for PKCd, antiprolifera-
tive activity in 39 human cancer cell lines, and tumor-promoting/
anti-tumor-promoting activity in vitro. Although 1 and 3 were al-
most the same in terms of binding to PKC isozymes including PKCd,
PKC isozyme selectivity, and antiproliferative activity, they differed
significantly in the EBV-EA induction test with 3 showing less anti-
tumor-promoting activity than 1. The present results indicate that
the phenolic hydroxyl group at position 18 is not involved in the
antiproliferative activity of 1 in vitro. This means that the phenolic
hydroxyl group would be available as a tunable site to develop supe-
rior analogues and molecular probes for analyzing the mechanism of
the antiproliferative and anti-tumor-promoting activity of 1.