Structure-activity studies on the side chain of a simplified analog of aplysiatoxin (aplog-1) with anti-proliferative activity

Article abstract of DOI:10.1016/j.bmc.2013.03.013

We have recently developed a simplified analog of aplysiatoxin (aplog-1) as an activator of protein kinase C (PKC) with anti-proliferative activity like bryostain 1. To identify sites in aplog-1 that could be readily modified to optimize therapeutic performance and to develop a molecular probe for examining the analog's mode of action, substituent effects on the phenol ring were systematically examined. Whereas hydrophilic acetamido derivatives were less active than aplog-1 in inhibiting cancer cell growth and binding to PKCδ, introduction of hydrophobic bromine and iodine atoms enhanced both biological activities. The anti-proliferative activity was found to correlate closely with molecular hydrophobicity, and maximal activity was observed at a log P value of 4.0-4.5. On the other hand, an induction test with Epstein-Barr virus early antigen demonstrated that these derivatives have less tumor-promoting activity in vitro than aplog-1 regardless of the hydrophobicity of their substituents. These results would facilitate rapid preparation of molecular probes to examine the mechanism of the unique biological activities of aplog-1.

Full text of DOI:10.1016/j.bmc.2013.03.013

Bioorganic & Medicinal Chemistry 21 (2013) 2695–2702
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Bioorganic & Medicinal Chemistry
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Structure–activity studies on the side chain of a simplified analog
of aplysiatoxin (aplog-1) with anti-proliferative activity
Hiroaki Kamachi a, Keisuke Tanaka a, Ryo C. Yanagita a,b, Akira Murakami a, Kazuma Murakami a,
Harukuni Tokuda c, Nobutaka Suzuki c, Yu Nakagawa a,d, 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 Synthetic Cellular Chemistry Laboratory, RIKEN Advanced Science Institute, Saitama 351-0198, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
We have recently developed a simplified analog of aplysiatoxin (aplog-1) as an activator of protein kinase
C (PKC) with anti-proliferative activity like bryostain 1. To identify sites in aplog-1 that could be readily
modified to optimize therapeutic performance and to develop a molecular probe for examining the ana-
log’s mode of action, substituent effects on the phenol ring were systematically examined. Whereas
hydrophilic acetamido derivatives were less active than aplog-1 in inhibiting cancer cell growth and
binding to PKCd, introduction of hydrophobic bromine and iodine atoms enhanced both biological activ-
ities. The anti-proliferative activity was found to correlate closely with molecular hydrophobicity, and
maximal activity was observed at a logP value of 4.0–4.5. On the other hand, an induction test with
Epstein–Barr virus early antigen demonstrated that these derivatives have less tumor-promoting activity
in vitro than aplog-1 regardless of the hydrophobicity of their substituents. These results would facilitate
rapid preparation of molecular probes to examine the mechanism of the unique biological activities of
aplog-1.
Received 30 January 2013
Revised 13 March 2013
Accepted 14 March 2013
Available online 25 March 2013
Keywords:
Anti-proliferative activity
Aplysiatoxin
aplog-1
Protein kinase C
Structure–activity relationship
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
bryo-1 is a difficulty in supply both by isolation from natural
sources (only 1 g from 10 tons of B. neritina)16 and organic synthe-
Protein kinase C (PKC), a family of serine/threonine kinases, are
a component of intracellular signaling pathways that cause differ-
entiation, proliferation, and apoptosis.1,2 Regulation of PKC activity
has thus been recognized as an important therapeutic strategy for
various cancers.3,4 Several PKC inhibitors against the ATP-binding
domain were shown to be anti-proliferative for some cancer
cells.5,6 In contrast, there exist some PKC activators that show
anti-proliferative activity, representative of which is bryostatin 1
(bryo-1), a marine natural product isolated from the bryozoan
Bugula neritina.7,8 Although most exogenous PKC activators such
as phorbol esters, teleocidins, and aplysiatoxins are strong tumor
promoters,9 bryo-1 possesses little tumor-promoting activity.10
PKCd, one of the PKC isozymes, plays a tumor suppressor role
and is involved in apoptosis.11,12 The unique biological activities
of bryo-1 could be ascribable in part to the ability to bind to and
activate PKCd.13 bryo-1 is under clinical trials for the treatment
of several cancers.3,8 Although some of these trials have been
disappointing, bryo-1 is also expected to be promising as a thera-
peutic agent for Alzheimer’s disease14 and AIDS.15 A drawback of
sis,17–19 though more efficient methods of producing bryo-1-re-
lated compounds have recently been reported.20–23 Simplification
of bryo-1 is a promising approach to solving this problem.24,25
Our approach to this issue is to focus on a skeleton other than
bryo-1. As described in previous papers,26–28 we identified a sim-
plified analog of the tumor-promoting aplysiatoxin (ATX)29 that
behaved like bryo-1. This analog, named aplog-1 (Fig. 1), was
anti-proliferative for many cancer cell lines but exhibited little tu-
mor-promoting activity. The next step is to optimize the anti-pro-
liferative activity of aplog-1. Introduction of a methyl group on the
spiroketal moiety at position 10 drastically increased the anti-pro-
liferative activity and the ability to bind to the C1B domain of PKCd,
whereas this derivative (10-methyl-aplog-1) showed little tumor-
promoting activity both in vitro and in vivo.30
Another method to increase the anti-proliferative activity of
aplog-1 is to modify the phenolic side chain. Previous computer-
aided analyses indicated that the phenolic side chain of ATX is in-
volved not in the specific interaction with PKC but in non-specific
hydrophobic interaction with membranous lipids.31,32 Precise
structure–activity studies on the side chain would afford the opti-
mal hydrophobicity for anti-proliferative activity and be connected
to the development of molecular probes for the identification of
⇑
Corresponding author. Tel.: +81 75 753 6281; fax: +81 75 753 6284.
E-mail address: irie@kais.kyoto-u.ac.jp (K. Irie).
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.03.013

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H. Kamachi et al. / Bioorg. Med. Chem. 21 (2013) 2695–2702
Figure 1. Structure of bryostatin 1 (bryo-1), aplysiatoxin (ATX), debromoaplysiatoxin (DAT), aplog-1, and 10-Me-aplog-1.
receptors other than PKC isozymes, since the unique biological
activities of aplog-1 could not be explained only by PKC.26–28 In
fact, a molecular target of bryo-1 other than PKC has recently been
reported.33
This paper deals with a comprehensive structure–activity study
on the side chain of aplog-1 in binding to PKCd C1 domains,
anti-proliferative activity against 39 cancer cell lines, and tumor-
promoting activity. The data clearly showed that optimal hydro-
phobicity exists, and that the steric requirements at this moiety
are low.
These derivatives were selected based on a lowering or raising of
hydrophobicity, an increase in steric hindrance around the
aromatic ring, and the role of the phenolic hydroxyl group. We
attempted at first the amination of aplog-1 to lower the hydropho-
bicity but failed because of catalytic poisoning at the debenzylation
step. Subsequent acetylation followed by debenzylation gave
acetamido derivatives. In brief, nitration of 1 with tetrabutylam-
monium nitrate and trifluoroacetic anhydride provided dibenzyl-
21-NO2-aplog-1 and dibenzyl-19-NO2-aplog-1. 18-Crown-6 was
added to increase the selectivity for the para position of a phenoxy
ether.34 Each nitro compound was selectively reduced to the corre-
sponding aniline derivative, followed by acetylation. Finally, two
benzyl groups in each derivative were deprotected to give acetam-
ido-aplog-1 (2, 3).
2. Results and discussion
2.1. Synthesis of aplog-1 derivatives at the phenol side chain
Bromination of the phenol moiety of aplog-1 was performed
with the solid tribromide reagent.35 Mono-, di-, and tri-substitu-
tions were controlled by the amount of the tribromide reagent
A series of side chain derivatives of aplog-1 was synthesized
from dibenzyl-aplog-1 (1, Bn2-aplog-1) or aplog-126 (Scheme 1).
Scheme 1. Synthesis of 2–10.

H. Kamachi et al. / Bioorg. Med. Chem. 21 (2013) 2695–2702
2697
Table 1
Our group reported that aplog-1 behaved like bryo-1 in the trans-
location of PKCd in CHO-K1 cells.26 Thus, the aplog-1 derivatives
synthesized in this study were subjected to evaluations of their
affinity for synthetic C1 domains of PKCd42 by inhibition of the
specific binding of [3H]phorbol 12,13-dibutylate (PDBu) in the
logP values for aplog-1, 10-Me-aplog-1, 2–10, DAT,
and ATX determined by the HPLC method38,39
Compounds
logP
aplog-1
3.3
3.6
2.3
2.7
4.0
5.0
5.5
4.3
4.2
4.4
4.8
4.4
5.4
10-Me-aplog-1a
presence of phosphatidylserine as reported previously.43,44
A
2
3
4
5
6
7
8
9
binding assay using whole PKCd reflects binding to both the
C1A and C1B domains, and its instability diminishes accuracy
and reproducibility. This is why we use synthetic C1A and C1B
peptides (d-C1A and d-C1B) that are stable and inexpensive. As
reported previously, binding constants of some PKC ligands in
the inhibition of specific [3H]PDBu-binding were almost equal
to those of whole PKCd.30
Table 2 summarizes Ki values of all aplog-1 derivatives. All
derivatives except for 2 and 3 with logP values of less than 3.0
bound strongly to d-C1B. Although these derivatives bound to d-
C1B more strongly than to d-C1A, they showed substantial bind-
ing to d-C1A with Ki values of about 100 nM. Hydrophobic halo-
gen derivatives (4–8) exhibited slightly higher binding than
aplog-1. Lack of the phenolic hydroxyl group hardly changed
the affinity for the C1 peptides, indicating the hydroxyl group
not to be critical to the binding.45 It is remarkable that the highly
bulky and hydrophobic tribromide 6 did not lose binding ability,
suggesting that the aromatic side chain of aplog-1 is not close to
the binding pocked of the C1 domain. This speculation is sup-
ported by computer-aided molecular modeling by several
groups.31,32
10
DAT
ATX
a
Cited from Ref. 30.
added. Iodination of aplog-1 gave 7 and 8 in one step. Selective
methylation of the phenolic hydroxyl group was achieved by
less-explosive TMS-diazomethane36 to give 18-OMe-aplog-1 (9).
2.2. Measurement of the partition coefficient (logP) of the
aplog-1 derivatives
As expressed in ‘Lipinski’s rule of five’,37 the partition coefficient
(logP) between 1-octanol and water is an important parameter for
drug design. The hydrophobic side chain of tumor promoters plays
a critical role in tumor promotion.9 To analyze the relationship be-
tween biological activity and molecular hydrophobicity, a logP va-
lue for each derivative was estimated by the HPLC method
recommended by OECD.38,39 The retention time of each derivative
on a reverse-phase column could be correlated to logP using
appropriate reference compounds with known logP values.
Table 1 summarizes the logP values along with those of debro-
moaplysiatoxin (DAT) and ATX as references. Regardless of the ab-
sence of a bromine atom, DAT as well as ATX behaved as a tumor
promoter in vivo.30,40 Although all compounds contained many
oxygen atoms, they showed comparatively high values, over 3.0,
with the exception of 2 and 3. This might be ascribable to the mac-
rolactone structure where many oxygen atoms are oriented inside
the ring, avoiding interaction with the solvent. Compounds 5, 6, 9,
and 10 showed a hydrophobicity equal to or greater than that of
the tumor promoter DAT.9 In contrast, the acetamido derivatives
2 and 3 were more hydrophilic than aplog-1, especially the p-
substituted derivative 2, which was one order of magnitude more
hydrophilic than aplog-1. The hydrophobicity of aplog-1 (3.3) was
almost equal to that of bryo-1 (2.9) reported by Bignami et al.41
2.4. Anti-proliferative activity of the aplog-1 derivatives
The anti-proliferative activity of the aplog-1 derivatives was
evaluated by growth inhibition against a panel of 39 human cancer
cell lines established by Yamori et al. as described previously.46 The
concentration required to inhibit cell growth by 50% compared to
an untreated control was expressed as the GI50 (M). Representative
cancer cell lines whose log GI50 was less than MG-MID are shown
in Table 3. MG-MID is defined as the average of the log GI50 values
of all 39 human cancer cell lines. For calculation of the MG-MID of
2, the log GI50 was taken as ꢀ4.00 in the cell lines that showed a
value of more than ꢀ4.00 (Supplementary data). The profile of
anti-proliferative activities against 39 cancer cell lines of aplogs
synthesized in this study was similar to that of the parent com-
pound, DAT and 10-methyl-aplog1 (Table 3).30
Hydrophilic acetamido substitution (2, 3) weakened the anti-
proliferative activity as observed in the PKCd binding assay; the
activity of the most hydrophilic acetamide (2) decreased markedly.
In contrast, mono-substitution with a hydrophobic halogen atom
enhanced the activity of aplog-1. The bromide (4) was found to
be slightly superior to the iodides (7, 8). However, introduction
of additional bromine atoms (5, 6) attenuated the activity of 4. Lack
of the phenolic hydroxyl group little influenced the activity as ob-
served in 9 and 10. The results for the anti-proliferative activity
correlated well with those for the binding affinity for the PKCd
C1 peptides.
2.3. Ability of the aplog-1 derivatives to bind to the C1 domains
of PKCd
As mentioned above, translocation and activation of PKCd is
considered critical to the unique biological activities of bryo-1.13
Table 2
Ki values for inhibition of specific binding of [3H]PDBu by aplog-1, 10-Me-aplog-1, DAT, and 2–10
PKCd C1 peptide
Ki (nM)
Kd (nM)
aplog-1a
10-Me-aplog-1
DAT
2
3
4
5
6
7
8
9
10c
PDBud
d-C1A
d-C1B
140
7.4
22
9.7
8700
690
290
36
66
3.5
93
4.1
79
6.9
82
1.3
110
4.6
330
9.7
130
9.8
52
0.53
0.46b
0.20b
a
b
c
Cited from Ref. 26.
Cited from Ref. 30.
Cited from Ref. 45.
Cited from Ref. 44.
d

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H. Kamachi et al. / Bioorg. Med. Chem. 21 (2013) 2695–2702
Table 3
log GI50 values for aplog-1, 10-Me-aplog-1, DAT, and 2–10 against 39 human cancer cell lines
Cancer type
Cell line
Log GI50 (M)
aplog-1a
10-Me-aplog-1b
DATb
2
3
4
5
6
7
8
9
10c
Breast
Breast
CNS
Colon
Lung
Lung
Melanoma
Stomach
Stomach
Prostate
MG-MIDd
HBC-4
MDA-MB-231
SF-295
HCC2998
NCI-H460
A549
LOX-IMVI
St-4
ꢀ6.33
ꢀ5.61
ꢀ5.06
ꢀ5.43
ꢀ5.60
ꢀ5.32
ꢀ5.74
ꢀ5.55
ꢀ5.33
ꢀ4.96
ꢀ4.98
ꢀ7.48
ꢀ6.90
ꢀ4.98
ꢀ6.47
ꢀ7.07
ꢀ6.01
ꢀ6.21
ꢀ6.24
ꢀ4.97
ꢀ4.94
ꢀ5.24
ꢀ6.47
ꢀ6.03
ꢀ5.53
ꢀ6.09
ꢀ6.46
ꢀ5.94
ꢀ5.69
ꢀ6.44
ꢀ4.98
ꢀ4.98
ꢀ5.22
>ꢀ4.00
>ꢀ4.00
ꢀ4.22
ꢀ5.61
ꢀ4.78
ꢀ4.88
ꢀ4.86
ꢀ4.69
ꢀ4.82
ꢀ4.89
ꢀ5.67
ꢀ4.54
ꢀ4.68
ꢀ4.68
ꢀ7.01
ꢀ6.33
ꢀ5.26
ꢀ6.10
ꢀ5.78
ꢀ5.30
ꢀ6.10
ꢀ6.02
ꢀ5.74
ꢀ5.53
ꢀ5.20
ꢀ6.65
ꢀ5.53
ꢀ5.18
ꢀ6.05
ꢀ5.91
ꢀ5.31
ꢀ5.55
ꢀ5.89
ꢀ5.43
ꢀ5.41
ꢀ5.17
ꢀ6.06
ꢀ4.92
ꢀ4.88
ꢀ5.12
ꢀ5.49
ꢀ4.90
ꢀ4.93
ꢀ5.51
ꢀ4.94
ꢀ4.88
ꢀ4.83
ꢀ6.53
ꢀ5.80
ꢀ5.39
ꢀ5.79
ꢀ5.22
ꢀ5.48
ꢀ5.54
ꢀ6.03
ꢀ4.93
ꢀ5.51
ꢀ5.18
ꢀ6.80
ꢀ5.81
ꢀ5.20
ꢀ6.05
ꢀ4.98
ꢀ5.39
ꢀ5.70
ꢀ6.37
ꢀ4.92
ꢀ5.33
ꢀ5.19
ꢀ5.77
ꢀ5.18
ꢀ4.99
ꢀ5.10
ꢀ5.59
ꢀ5.31
ꢀ4.96
ꢀ5.60
ꢀ5.02
ꢀ4.92
ꢀ4.95
ꢀ6.28
ꢀ5.67
ꢀ5.14
ꢀ5.53
ꢀ5.83
ꢀ5.49
ꢀ5.17
ꢀ6.05
ꢀ6.09
ꢀ5.26
ꢀ5.09
ꢀ4.14
>ꢀ4.00
>ꢀ4.00
ꢀ4.13
>ꢀ4.00
>ꢀ4.00
>ꢀ4.00
ꢀ4.04
MKN45
PC-3
a
b
c
Cited from Ref. 26.
Cited from Ref. 30.
Cited from Ref. 45.
d
Average of 39 cancer cell lines. The list is shown in the Supplementary data.
Figure 2. Correlation between anti-proliferative activity and logP. ‘R’ means the macrolide skeleton and alkyl side chain of aplog-1.
introduced into the spiroketal moiety at position 10.30 Since the
logP of 10-methyl-aplog-1 is 3.6,30 introduction of a bromine atom
into the side chain would slightly enhance the anti-proliferative
activity. 10-Methyl-aplog-1 could not be incorporated in the regres-
sion analysis since the effect of modification by substituents on the
phenyl ring is not similar to that of modification by the methyl
group at position 10. The methyl group increased more potently
the ability to bind PKCd C1 domains and anti-proliferative activities
against several cancer cell lines compared with the bromine and io-
dine atoms at the phenyl ring as shown in Table 3.
2.5. Relationship between the anti-proliferative activity and
logP of the aplog-1 derivatives
Figure 2 shows a plot of –MG-MID against the logP of each
aplog-1 derivative. The plot could be approximated to a quadratic
curve relevant to logP. The least squares regression equation is
shown below:
2
ꢀMG-MID ¼ ꢀ0:25 ðꢁ0:10Þ ðlogPÞ þ 2:2 ðꢁ0:8Þ ðlogPÞ
þ 0:50 ðꢁ1:5Þ
ðn ¼ 10; s ¼ 0:10; r ¼ 0:95Þ
where n is the number of compounds, s is the standard deviation,
and r is the correlation coefficient. Figures in parentheses are 95%
confidence limits. These results indicate that the aplog-1 skeleton
shows maximum anti-proliferative activity at a logP value of 4.0–
4.5. A logP value greater than 4.5 decreased the activity possibly be-
cause of trapping by the cellular membrane. A logP value less than
3.0 also decreased the activity as observed in 2 and 3, suggesting
that these ligands could not sufficiently penetrate the cellular mem-
brane. These results clearly indicate that the hydrophobicity of 4, 7
and 8 is optimal, and that modification of the side chain did not
drastically enhance the anti-proliferative activity of aplog-1. Re-
cently, we have identified 10-methyl-aplog-1 with more potent
anti-proliferative activity than aplog-1, where a methyl group was
2.6. Tumor-promoting activity of the aplog-1 derivatives
The most critical point in developing derivatives of aplog-1 is to
confirm that the structural modifications do not increase tumor-
promoting activity in vivo. Since tumor promoters such as ATX
and DAT9 have higher hydrophobicity (logP >4.4), hydrophobic
halogen substitutions at the side chain of aplog-1 might get back
tumor-promoting activity. Induction of the Epstein–Barr virus
early antigen (EBV-EA) is one of the most reliable assays for the
evaluation of tumor-promoting activity in vitro.47,48 EBV is acti-
vated by tumor promoters to produce early antigen (EA), that is de-
tected by an indirect immunofluorescence technique.49

H. Kamachi et al. / Bioorg. Med. Chem. 21 (2013) 2695–2702
2699
Figure 3. EBV-EA-inducing ability of TPA, DAT,30 aplog-1,45 2–9, and 10.45 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. Error bars represent standard errors of the mean (n = 3).
As shown in Figure 3, a strong tumor promoter, 12-O-tetradeca-
noylphorbol 13-acetate (TPA), produced 29.4% EA-positive cells at
10ꢀ7 M. Tumor promoters ATX and DAT also generated 20–25%
EA-positive cells at this concentration as reported.26,30 On the other
The present results also indicated that the skeleton of aplog-1 is
qualitatively different from that of DAT and ATX with tumor-pro-
moting activity. The aplog-1 derivatives with a logP greater than
that of DAT (4.4) did not show any tumor-promoting activity.
These results support our recent conclusion that the hemiacetal
hydroxyl group at position 3 and/or the methoxy group at the ben-
zyl position of the phenolic side chain play an important role in tu-
mor promotion.30 DAT and ATX behave as a master key with both
tumor-promoting and anti-proliferative activities, whereas aplog-1
behaves as a special key with anti-proliferative activity. The next
step is to identify molecular targets of aplog-1 other than PKCd
using molecular probes derived from aplog-1 based on the present
structure–activity relationship. The involvement of PKCd in the
anti-proliferative activity of the cell lines sensitive to aplog-1 (Ta-
ble 3) should also be confirmed using siRNA.
hand, aplog-126 and its deoxy as well as methoxy analogs (9, 1045
)
showed weaker EA induction even at 10ꢀ6 M. The hydrophilic acet-
amido derivatives (2, 3) were almost inactive as expected. How-
ever, hydrophobic halogen substitution caused little EA induction
either. Moreover, 5–10 with logP values similar to or higher than
DAT hardly exhibited any EA induction even at 10ꢀ6 M (Table 1
and Fig. 3). This is an unexpected result, indicating the EBV-EA
induction by tumor promoters ATX and DAT to be mainly caused
by their macrolactone structure rather than their molecular
hydrophobicity.
We have recently reported that aplog-1 and its derivatives
(12,12-dimethyl-aplog-1 and 10-methyl-aplog-1) did not show
any tumor-promoting activity in a two-stage carcinogenesis exper-
iment using ICR mice even when applied at a fivefold excess
compared with DAT and TPA.28,30,50 Therefore, we examined the
tumor-promoting activity of 4 with more anti-proliferative activity
and less ability to induce EBV-EA using 10 male ICR mice. From a
week after initiation by a single application of 390 nmol of 7,12-
dimethylbenz[a]anthracene (DMBA), 8.5 nmol of 4 was applied
twice a week from week 1 to 20. The control group was treated
with DMBA and 1.7 nmol TPA twice a week. TPA-treated mice be-
gan to develop tumors in week 6 and all had tumors at week 11. On
the other hand, mice given 4 did not develop tumors even at week
20. This result is consistent with that of the EBV-EA induction test,
and supports the development of anti-proliferative compounds
using the skeleton of aplog-1.
4. Experimental
4.1. General remarks
The following spectroscopic and analytical instruments were
used: Digital Polarimeter, DIP-1000 and P-2200 (Jasco, Tokyo, Ja-
pan); 1H, 13C NMR, AVANCE 400 and AVANCE III 500 (Bruker, Ger-
many, TMS as an internal reference); HPLC, model 600E with a
model 2487 UV Detector (Waters, Tokyo, Japan); HR-FAB-MS,
JMS-600H and JMS-700 (JEOL, Tokyo, Japan). Reactions were mon-
itored by TLC (TLC Silica gel 60 F254, Merck). HPLC was carried out
on YMC packed ODS-A AA12S05-1520WT (SH-342-5), AM-323,
AQ-311, and A-023 (Yamamura Chemical Laboratory, Kyoto, Ja-
pan). Wakogel™ C-200 (silica gel, Wako Pure Chemical Industries,
Osaka, Japan) was used for column chromatography. [3H]PDBu
(19.6 Ci/mmol) was purchased from Perkin–Elmer Life Sciences Re-
search Products (Boston, MA, USA). The PKCd C1 peptides were
synthesized as reported previously.42 Bn2-aplog-1 (1) and aplog-1
were also synthesized as reported.26 All other chemicals and re-
agents were purchased commercially and used without further
purification.
3. Conclusions
Our group developed aplog-1 as a simplified analog of ATX that
showed anti-proliferative activity without tumor-promoting activ-
ity like bryo-1.26 To optimize its anti-proliferative activity, the phe-
nolic side chain was modified systematically. As shown in Figure 2,
the change in anti-proliferative activity against 39 cancer cell lines
was dominated by the change in the logP values of the aplog-1
derivatives. Although the optimal logP proved to be 4.0–4.5, perti-
nent modification at the macrolactone ring was found to be indis-
pensable to the development of highly potent analogs of aplog-1.
These attempts continue in our laboratory as exemplified by 10-
methyl-aplog-1.30
4.2. Synthesis of the aplog-1 derivatives
4.2.1. Synthesis of 21-NHAc-aplog-1 (2) and 19-NHAc-aplog-1
(3)
To a solution of Bn2-aplog-1 (1) (11.6 mg, 17.3
(1.2 mL) and trifluoroacetic anhydride (350
l
mol) in CH2Cl2
l
L) were added

2700
H. Kamachi et al. / Bioorg. Med. Chem. 21 (2013) 2695–2702
18-Crown-6 (48.0 mg, 181
l
mol, 10 equiv) and tetrabutylammo-
calcd for C29H41NO9Na, 570.2679).19-NO2-Bn2-aplog-1 (9.0 mg,
12.6 mol) was treated in a manner similar to that described for
the synthesis of 2 to give 19-NHAc-Bn2-aplog-1 (6.0 mg, 7.6 mol,
60% in three steps) as a clear oil. To a solution of 19-NHAc-Bn2-
aplog-1 (6.0 mg, 7.6 mol) in EtOH (200 L) was added a suspen-
sion of Pd/C (4.0 mg) in EtOH (300 L) at rt. The mixture was
stirred at rt under a H2 atmosphere for 4 h, and then filtered. The
filtrate was concentrated in vacuo, and purified by HPLC (column:
SH-342-5; solvent: 70% MeOH/H2O; flow rate 8.0 mL/min; pres-
sure: 2000 psi; UV detector: 254 nm; retention time: 21.8 min)
nium nitrate (8.2 mg, 27.0 mmol, 1.6 equiv). After 1 h of stirring
at rt, the reaction was quenched with saturated aq NaHCO3
(2 mL). The organic layer was separated, and the aqueous layer
was extracted with CH2Cl2 (2 mL ꢂ 2). The combined organic layers
were washed with brine, dried over Na2SO4, filtered, and concen-
trated in vacuo. The residue was purified by HPLC (column:
YMC-Pack ODS-A, solvent: 90% MeOH/H2O; flow rate 8.0 mL/min;
pressure: 1600 psi; UV detector: 254 nm) to afford 21-NO2-Bn2-
l
l
l
l
l
aplog-1 (5.8 mg, 8.1 lmol, 47%, retention time: 22 min) and 19-
NO2-Bn2-aplog-1 (1.5 mg, 2.1 mmol, 12%, retention time: 26 min)
as clear oils.
to afford 19-NHAc-aplog-1 (3) (3.3 mg, 6.0
lmol, 78%) as a clear
oil. [a]
+42.6° (c 0.165, EtOH, 25.3 °C). 1H NMR (400 MHz, 297 K,
D
To a solution of 21-NO2-Bn2-aplog-1 (19 mg, 26.6
NiCl2–6H2O (13.9 mg, 58.5 mol, 2.2 equiv) in MeOH (4 mL) and
THF (2 mL) at 4 °C was added NaBH4 (5.6 mg, 148 mol, 5.6 equiv).
l
mol) and
CDCl3, 0.012 M): d 0.87 (3H, s), 0.97 (3H, s), 1.36–1.67 (13H, m),
2.23 (3H, s), 2.42 (1H, dd, J = 13.0, 10.8 Hz), 2.47–2.57 (5H, m),
2.77 (2H, m), 3.75 (2H, m), 3.89 (1H, m), 4.18 (1H, m), 5.20 (2H,
m), 6.69 (1H, dd, J = 8.1, 1.8 Hz), 6.88 (1H, d, J = 1.7 Hz), 7.16 (1H,
d, J = 8.1 Hz), 7.57 (1H, br s, Ph-OH), 8.60 (1H, br s, NH) ppm. 13C
NMR (100 MHz, 298 K, CDCl3, 0.012 M): d 21.17, 23.93, 24.33,
25.14, 25.92, 27.23, 30.19, 34.51, 34.78, 34.81, 34.90, 36.89,
37.04, 42.75, 63.26, 64.30, 68.82, 70.59, 72.19, 100.34, 118.38,
120.64, 121.48, 123.35, 141.70, 147.83, 169.54, 169.77,
172.23 ppm. HR-FAB-MS m/z: 548.2878 (MH+, calcd for
C29H42NO9, 548.2860).
l
l
After 10 min of stirring at the same temperature, the solvent was
removed in vacuo, and CHCl3 (10 mL) and H2O (2.5 mL) were
added. The mixture was filtered through Celite, and the Celite
pad was washed with CHCl3 (100 mL). The combined organic lay-
ers were concentrated in vacuo to approximately 10 mL, and the
organic layer was washed with saturated aq NaHCO3 (10 mL).
The organic layer was separated, and the aqueous layer was ex-
tracted with CHCl3 (10 mL ꢂ 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, 10–50% EtOAc in hexane) to afford 21-NH2-Bn2-aplog-1
4.2.2. Synthesis of 21-Br-aplog-1 (4)
To a solution of aplog-1 (3.8 mg, 7.8
was added a solution of benzyltrimethylammonium tribromide
(3.0 mg, 7.8 mol, 1.0 equiv) and CaCO3 (3.8 mg, 38 mol,
lmol) in MeOH (600 lL)
(15.0 mg, 21.9
(15.0 mg, 21.9
l
l
mol, 82%). To a solution of 21-NH2-Bn2-aplog-1
mol) and NEt3 (6.1 L, 43.8 mol, 2.0 equiv) in
L) was added acetyl chloride (6.1 L, 88.7 mol,
l
l
l
l
CH2Cl2 (400
l
l
l
4.9 equiv) in CH2Cl2 (1.3 mL). The mixture was stirred at rt for
3 h, and then filtered. The filtrate was concentrated in vacuo. The
residue was purified by HPLC (column: SH-342-5; solvent: 80%
MeOH/H2O; flow rate 8.0 mL/min; pressure: 1700 psi; UV detector:
254 nm; retention time: 24 min) to afford 21-Br-aplog-1 (4)
4.1 equiv) at rt. After 4 h of stirring at the same temperature, the
reaction was quenched with saturated aq NaHCO3 (1 mL). The
resulting mixture was poured into CHCl3 and water. After the or-
ganic layer was separated, the aqueous layer was extracted with
CHCl3 (5 mL ꢂ 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, 30–
50% EtOAc in hexane) to afford 21-NHAc-Bn2-aplog-1 (8.0 mg,
(3.0 mg, 5.3 lmol, 68%) as a clear oil. [a] +38.4° (c 0.114, EtOH,
D
15.1 °C). 1H NMR (400 MHz, 298 K, CDCl3, 0.011 M): d 0.88 (3H,
s), 0.96 (3H, s), 1.34–1.79 (13H, m), 2.25 (1H, br s, OH), 2.41 (1H,
dd, J = 13.3, 10.8 Hz), 2.51 (1H, d, J = 15.7 Hz), 2.62 (3H, m), 2.80
(2H, m), 3.79 (2H, m), 3.94 (1H, tt, J = 10.7, 3.1 Hz), 4.30 (1H, m),
5.17–5.23 (2H, m), 6.60 (1H, dd, J = 8.6, 3.0 Hz), 6.94 (1H, d,
J = 3.0 Hz), 6.98 (1H, br s, Ph-OH), 7.35 (1H, d, J = 8.6 Hz) ppm.
13C NMR (100 MHz, 298 K, CDCl3, 0.011 M): d 21.18, 23.85, 25.20,
25.91, 27.18, 27.23, 33.97, 34.47, 34.92, 35.00, 36.96, 36.98,
42.89, 62.31, 64.02, 68.88, 70.68, 72.74, 100.50, 114.34, 114.61,
116.52, 133.20, 142.87, 155.87, 169.37, 173.53 ppm. HR-FAB-MS
m/z: 569.1766 (MH+, calcd for C27H38O8Br, 569.1750).
11.0
1 (8.0 mg, 11.0
of Pd/C (4.2 mg) in EtOH (300
l
mol, 50%) as clear oil. To a solution of 21-NHAc-Bn2-aplog-
mol) in EtOH (200 L) was added a suspension
L) at rt. The mixture was stirred
l
l
l
at rt under H2 atmosphere for 4 h, and then filtered. The filtrate
was concentrated in vacuo, and purified by HPLC (column: SH-
342-5; solvent: 65% MeOH/H2O; flow rate 8.0 mL/min; pressure:
2100 psi; UV detector: 254 nm; retention time: 13.9 min) to afford
21-NHAc-aplog-1 (2) (5.4 mg, 9.9
7:3 mixture of conformational isomers. [
l
mol, 90%) as a clear oil and a
D +53.4° (c 0.270, EtOH,
a]
23.5 °C). 1H NMR (400 MHz, 297 K, CDCl3, 0.018 M) major con-
former: d 0.85 (3H, s), 0.86 (3H, s), 1.31–1.69 (13H, m), 2.32–2.45
(3H, m), 2.49–2.63 (2H, m), 2.71–2.89 (2H, m), 3.76 (2H, m), 3.89
(1H, m), 4.26 (1H, m), 5.19 (2H, m), 6.64 (1H, dd, J = 8.6, 2.8 Hz),
6.76 (1H, br s, Ph-OH), 6.79 (1H, d, J = 2.8 Hz), 7.24 (1H, d,
J = 8.6 Hz), 7.30 (1H, br s, NH) ppm; minor conformer: d 0.86 (3H,
s), 0.93 (3H, s), 1.31–1.69 (13H, m), 1.82 (3H, s), 2.19 (3H, s),
2.32–2.45 (3H, m), 2.49–2.63 (2H, m), 2.71–2.89 (2H, m), 3.66
(1H, dd, J = 11.6, 5.5 Hz), 3.76 (1H, m), 3.89 (1H, m), 4.26 (1H, m),
5.19 (1H, m), 5.27 (1H, m), 6.68 (1H, dd, J = 8.5, 2.8 Hz), 6.76 (1H,
br s, Ph-OH), 6.86 (1H, d, J = 2.8 Hz), 6.99 (1H, d, J = 8.8 Hz), 7.54
(1H, br s, NH) ppm. 13C NMR (100 MHz, 298 K, CDCl3, 0.018 M) ma-
jor conformer: d 21.07, 23.71, 24.11, 25.20, 25.86, 27.19, 30.82,
33.83, 34.38, 34.46, 35.02, 36.90, 37.00, 42.91, 62.22, 63.89,
68.85, 70.61, 72.30, 100.39, 113.37, 116.02, 127.17, 127.60,
138.68, 154.90, 169.47, 169.60, 172.98 ppm; minor conformer: d
21.10, 23.71, 24.36, 25.24, 25.86, 27.19, 30.96, 33.83, 34.38,
34.52, 35.20, 36.90, 37.00, 42.91, 61.74, 63.89, 68.75, 70.61,
72.30, 100.39, 113.67, 116.32, 128.16, 130.03, 138.68, 156.10,
169.47, 172.70, 172.98 ppm. HR-FAB-MS m/z: 570.2690 ([M+Na]+,
4.2.3. Synthesis of 19,21-dibromo-aplog-1 (5)
To a solution of aplog-1 (6.0 mg, 12.2
11.0 mol, 0.9 equiv) in MeOH (60 L) and CH2Cl2 (140
added benzyltrimethylammonium tribromide (9.5 mg, 24.4
2.0 equiv) at rt. After 1.5 h of stirring at the same temperature, sat-
urated aq Na2S2O3 (330 L) and H2O (660 L) were added to the
l
mol) and CaCO3 (1.1 mg,
L) was
mol,
l
l
l
l
l
l
mixture. The resulting mixture was extracted with EtOAc
(2 mL ꢂ 4). The combined organic layers were dried over Na2SO4,
and concentrated in vacuo. The residue was purified by column
chromatography (silica gel, 30 to 35% EtOAc in hexane) to afford
the mixture of Br2-aplog-1 (5) and Br3-aplog-1 (6). The mixture
was purified by HPLC (column: AM-323; solvent: 85% MeOH/
H2O; flow rate 3.0 mL/min; pressure: 1800 psi; UV detector:
254 nm; retention time: 24 min) to afford 19,21-dibromo-aplog-1
(5) (4.6 mg, 7.1
l
mol, 58%) as a clear oil. [
a]
+41.0° (c 0.807,
D
CHCl3, 15.5 °C). 1H NMR (500 MHz, 295 K, CDCl3, 0.014 M): d 0.87
(3H, s), 0.95 (3H, s), 1.34–1.76 (13H, m), 2.24 (1H, t, J = 5.6 Hz,
OH), 2.41 (1H, dd, J = 13.2, 10.9 Hz), 2.50 (1H, d, J = 15.6 Hz),
2.56–2.66 (3H, m), 2.80 (2H, m), 3.78 (2H, m), 3.92 (1H, m), 4.25
(1H, m), 5.20 (2H, m), 7.02 (1H, s), 7.03 (1H, br s, Ph-OH), 7.61

H. Kamachi et al. / Bioorg. Med. Chem. 21 (2013) 2695–2702
2701
(1H, s) ppm. 13C NMR (125 MHz, 298 K, CDCl3, 0.014 M): d 21.26,
24.12, 25.23, 25.91, 27.23, 27.93, 34.47, 34.53, 35.02, 36.96,
37.01, 42.86, 62.73, 64.11, 68.83, 70.69, 72.39, 77.60, 100.45,
107.29, 114.60, 117.04, 135.04, 142.89, 152.28, 169.43,
172.95 ppm. HR-FAB-MS m/z: 647.0849 (MH+, calcd for
139.75, 146.16, 156.95, 169.40, 173.50 ppm. HR-FAB-MS m/z:
617.1611 (MH+, calcd for C27H38O8I, 617.1611).
4.2.6. Synthesis of 18-OMe-aplog-1 (9)
To a solution of aplog-1 (1.5 mg, 3.1
CHCl3 (250 L) was added 2 M TMS-diazomethane in hexane
(20 L, 40 mol, 13 equiv) at rt. After 35 h of stirring at the same
temperature, the reaction was quenched with acetic acid (20 L).
lmol) in MeOH (50 lL) and
C27H37O8Br2, 647.0855).
l
l
l
l
4.2.4. Synthesis of 17,19,21-tribromo-aplog-1 (6)
To a solution of aplog-1 (4.3 mg, 8.76 mol) and CaCO3 (1.3 mg,
13.1 mol, 1.5 equiv) in MeOH (44 L) and CH2Cl2 (100 L) was
added benzyltrimethylammonium tribromide (10.9 mg, 28.0 mol,
The mixture was concentrated in vacuo, and purified by HPLC (col-
umn: A-023; solvent: iPrOH:CHCl3:hexane = 5:15:80; flow rate
3.0 mL/min; pressure: 600 psi; UV detector: 254 nm; retention
l
l
l
l
l
time: 14.5 min) to afford 18-OMe-aplog-1 (9) (1.0 mg, 2.0
lmol,
3.2 equiv) at rt. After 1.5 h of stirring at the same temperature, sat-
urated aq Na2S2O3 was added to the mixture. The resulting mixture
was extracted with EtOAc (2 mL ꢂ 4). The combined organic layers
were dried over Na2SO4, and concentrated in vacuo. The residue
was purified by column chromatography (silica gel, 35% EtOAc in
64%) as a clear oil. [
a
]
D
+55.2° (c 0.519, EtOH, 24.9 °C). 1H NMR
(500 MHz, 295 K, CDCl3, 0.014 M): d 0.86 (3H, s), 0.98 (3H, s),
1.34–1.68 (13H, m), 2.25 (1H, t, J = 5.7 Hz, OH), 2.40 (1H, dd,
J = 12.9, 10.9 Hz), 2.49 (1H, m), 2.53 (1H, dd, J = 12.9, 2.8 Hz), 2.59
(2H, t, J = 7.8 Hz), 2.71 (1H, dd, J = 16.6, 3.3 Hz), 2.80 (1H, dd,
J = 16.6, 11.3 Hz), 3.71 (1H, m), 3.77 (1H, m), 3.80 (3H, s), 3.88
(1H, m), 4.17 (1H, m), 5.18–5.23 (2H, m), 6.72 (1H, dd, J = 8.2,
2.5 Hz), 6.76 (1H, d, J = 2.3 Hz), 6.80 (1H, d, J = 7.6 Hz), 7.19 (1H,
t, J = 7.9 Hz) ppm. 13C NMR (125 MHz, 296 K, CDCl3, 0.014 M): d
21.20, 24.81, 25.19, 25.93, 27.29, 31.13, 34.59, 34.77, 35.51,
36.02, 36.89, 37.02, 42.01, 55.15, 63.72, 64.44, 68.79, 70.58,
71.72, 100.24, 110.87, 114.27, 120.69, 129.12, 144.61, 159.61,
169.61, 171.43 ppm. HR-FAB-MS m/z: 505.2813 (MH+, calcd for
hexane) to afford 17,19,21-tribromo-aplog-1 (6) (5.9 mg, 8.1
lmol,
92%) as a clear oil. [
a]
D
+25.7° (c 0.569, CHCl3, 17.5 °C). 1H NMR
(500 MHz, 295 K, CDCl3, 0.015 M): d 0.87 (3H, s), 0.99 (3H, s),
1.34–1.67 (13H, m), 2.41 (1H, dd, J = 12.9, 10.9 Hz), 2.55 (2H, m),
2.71 (1H, dd, J = 16.6, 3.3 Hz), 2.80 (1H, dd, J = 16.6, 11.3 Hz), 2.92
(2H, t, J = 7.5 Hz), 3.71 (1H, dd, J = 11.9, 5.3 Hz), 3.78 (1H, dd,
J = 11.9, 3.9 Hz), 3.89 (1H, m), 4.17 (1H, m), 5.19–5.23 (2H, m),
7.67 (1H, s) ppm. 13C NMR (125 MHz, 298 K, CDCl3, 0.015 M): d
21.23, 25.20, 25.95, 27.30, 27.88, 34.60, 34.84, 35.36, 36.92,
37.02, 37.36, 42.70, 63.74, 64.47, 68.76, 70.61, 71.79, 77.60,
100.26, 107.01, 113.00, 114.79, 134.55, 141.85, 149.09, 169.58,
171.48 ppm. HR-FAB-MS m/z: 724.9947 (MH+, calcd for
C28H41O8, 505.2801).
4.3. Inhibition of specific binding of [3H]PDBu to PKCd C1
peptides
C27H36O8Br3, 724.9960).
The binding of [3H]PDBu to the PKCd C1 peptides was evaluated
by the procedure of Sharkey and Blumberg43 with modifications as
reported previously44 with 50 mM Tris–maleate buffer (pH 7.4 at
4 °C), 14 or 40 nM PKCd C1 peptide, 20 nM [3H]PDBu (19.6 Ci/
4.2.5. Synthesis of 19-I-aplog-1 (7) and 21-I-aplog-1 (8)
To a solution of aplog-1 (17.9 mg, 36.5 mol) in MeOH (350
and CH2Cl2 (600 L) was added benzyltrimethylammonium
dichloroiodate (15.9 mg, 45.7 mol, 1.3 equiv) and CaCO3
(11.3 mg, 113 mol, 3.1 equiv). The mixture was stirred at rt for
l
lL)
l
l
mmol), 50
l
g/mL 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (Sig-
l
ma), 3 mg/mL bovine
c-globulin (Sigma), and various concentra-
4 h, and then filtered. The filtrate was concentrated in vacuo. The
residue was purified by HPLC (column: SH-342-5; solvent: 75%
MeOH/H2O; flow rate 8.0 mL/min; pressure: 1800 psi; UV detector:
254 nm; retention time: 36 min) to afford the mixture of 19-I-
aplog-1 (7) and 21-I-aplog-1 (8). The mixture was purified by HPLC
(column: A-023; solvent: iPrOH:CHCl3:hexane = 5:15:80; flow rate
3.0 mL/min; pressure: 600 psi; UV detector: 254 nm) to afford 7
tions of inhibitors. Binding affinity was evaluated on the basis of
the concentration required to cause 50% inhibition of the specific
binding of [3H]PDBu, IC50, which was calculated with Microsoft Ex-
cel. The inhibition constant, Ki, was calculated by the method of
Sharkey and Blumberg.43 Although we used each PKC C1 peptide
at 14 or 40 nM, the concentration of the properly folded peptide
was estimated to be about 3–4 nM on the basis of Bmax values of
Scatchard analyses reported previously.44 Therefore, the concen-
tration of free PDBu would not have drastically varied over the
dose–response curve.
(6.6 mg, 10.7
7.6 mol, 21%; retention time: 17 min) as clear oils. 21-I-aplog-1
(7): [
+37.7° (c 0.333, EtOH, 26.5 °C). 1H NMR (400 MHz,
lmol, 29%; retention time: 13 min) and 8 (4.7 mg,
l
a
]
D
297 K, CDCl3, 0.019 M): d 0.86 (3H, s), 0.95 (3H, s), 1.34–1.69
(13H, m), 2.34 (1H, t, J = 5.8 Hz, OH), 2.42 (1H, dd, J = 13.1,
10.8 Hz), 2.47–2.59 (4H, m), 2.79 (2H, m), 3.78 (2H, m), 3.91 (1H,
tt, J = 3.0, 10.8 Hz), 4.21 (1H, m), 5.16–5.22 (2H, m), 6.51 (1H, dd,
J = 8.0, 2.0 Hz), 6.71 (1H, br s, Ph-OH), 6.92 (1H, d, J = 1.9 Hz),
7.54 (1H, d, J = 8.0 Hz) ppm. 13C NMR (100 MHz, 298 K, CDCl3,
0.019 M): d 21.14, 24.00, 25.16, 25.91, 27.21, 29.57, 34.50, 34.50,
34.75, 34.89, 36.91, 37.02, 42.79, 62.85, 64.12, 68.78, 70.64,
72.27, 80.77, 100.35, 115.04, 122.80, 138.13, 145.44, 155.15,
169.52, 172.67 ppm. HR-FAB-MS m/z: 617.1611 (MH+, calcd for
4.4. Anti-proliferative activity against a panel of 39 human
cancer cell lines
A panel of 39 human cancer cell lines established by Yamori
et al.46 according to the NCI method with modifications was em-
ployed, and cell growth inhibitory activity was measured as re-
ported previously.51 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
C
27H38O8I, 617.1611). 21-I-aplog-1 (8): [a] +25.9° (c 0.235, EtOH,
D
25.8 °C). 1H NMR (400 MHz, 297 K, CDCl3, 0.015 M): d 0.88 (3H, s),
0.96 (3H, s), 1.34–1.76 (13H, m), 2.31 (1H, t, J = 5.9 Hz, OH), 2.41
(1H, dd, J = 13.3, 10.8 Hz), 2.51 (1H, d, J = 15.6 Hz), 2.55–2.64 (3H,
m), 2.81 (2H, m), 3.79 (2H, m), 3.94 (1H, m), 4.31 (1H, m), 5.17–
5.23 (2H, m), 6.48 (1H, dd, J = 8.5, 3.0 Hz), 6.94 (1H, d, J = 2.9 Hz),
7.11 (1H, s, Ph-OH), 7.62 (1H, d, J = 8.5 Hz) ppm. 13C NMR d
(100 MHz, 298 K, CDCl3, 0.015 M): d 21.17, 23.79, 25.20, 25.91,
27.18, 27.64, 33.98, 34.47, 34.99, 36.96, 36.96, 39.84, 42.88,
62.29, 63.97, 68.87, 70.69, 72.68, 88.68, 100.50, 115.26, 116.14,
the sulforhodamine B assay. The 50% growth inhibition (GI50
)
parameter was calculated as reported previously.52 Absorbance
for the control well (C) and the test well (T) were measured at
525 nm along with that for the test well at time 0 (T0). Cell growth
inhibition (% growth) by each concentration of compound (10ꢀ8
,
10ꢀ7, 10ꢀ6, 10ꢀ5 and 10ꢀ4 M) was calculated as 100[(T ꢀ T0)/
(C ꢀ T0)] using the average of duplicate points. By processing these
values, each GI50 value, defined as 100[(T ꢀ T0)/(C ꢀ T0)] = 50, was
determined.

2702
H. Kamachi et al. / Bioorg. Med. Chem. 21 (2013) 2695–2702
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4.5. Determination of logP of the aplog-1 derivatives using
HPLC
8. Hale, K. J.; Hummersone, M. G.; Manaviazar, S.; Frigerio, M. Nat. Prod. Rep. 2002,
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The HPLC method used to determine logP values of the aplog-1
derivatives was from the ‘OECD GUIDELINE FOR TESTING OF
CHEMICALS’.39 In brief, 35% or 75% MeCN/H2O was used for the
mobile phase on
a
reverse-phase column AQ-311 (100 ꢂ
6.0 mm). For preparing a calibration curve, two sets of six com-
pounds, 2-butanone (logP: 0.3), 4-acetylpyridine (0.5), acetanilide
(1.0), phenol (1.5), acetophenone (1.7) and anisole (2.1), or phenol
(1.5), anisole (2.1), 1-naphthol (2.7), cumene (3.7), n-butylbenzene
(4.6) and triphenylamine (5.7), were used as reference compounds.
Dead time was measured using unretained formamide. To confirm
the accuracy of this measurement, logP values of 2 and 3 were also
measured by the conventional 1-octanol/water shake-flask method
(OECD Test Guideline 107). The results gave quite similar logP
values.
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4.6. EBV-EA induction test
Human B-lymphoblastoid Raji cells (5 ꢂ 105/mL) were incu-
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medium (supplemented with 10% fetal bovine serum) with 4 mM
sodium n-butyrate (a synergist) and 10ꢀ7 or 10ꢀ6 M of each test
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l
L of a DMSO
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NPC patient’s serum (a gift from Kobe University) and FITC-labeled
anti-human IgG (DAKO, Glostrup, Denmark) as reported previ-
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portion of the EA-positive cells was recorded. Cell viability
exceeded 50% in most cases as shown in Supplementary data.
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Acknowledgements
This work was supported by a Grant-in-aid for Scientific Re-
search on Innovative Areas ‘Chemical Biology of Natural Products’
(Grant No. 23102011 to K.I.) from The Ministry of Education, Cul-
ture, Sports, Science and Technology, Japan. 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 Re-
search, Kyoto University. Finally, we thank the Screening Commit-
tee of Anticancer Drugs supported by Grant-in-aid for Scientific
Research on Innovative Areas, Scientific Support Programs for Can-
cer Research, from The Ministry of Education, Culture, Sports, Sci-
ence and Technology, Japan.
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Supplementary data
Supplementary data (these data include a full version of the re-
sults of the growth inhibition assay against 39 human cancer cell
lines and the EBV-EA induction test) associated with this article
can be found, in the online version, at http://dx.doi.org/10.1016/
j.bmc.2013.03.013.
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