Synthesis and biological activities of simplified aplysiatoxin analogs focused on the CH/π interaction

Article abstract of DOI:10.1016/j.bmcl.2020.127657

Debromoaplysiatoxin (DAT) is a potent protein kinase C (PKC) activator with tumor-promoting and pro-inflammatory activities. Irie and colleagues have found that 10-methyl-aplog-1 (1), a simplified analog of DAT, has strong anti-proliferative activity against several cancer cell lines with few adverse effects. Therefore, 1 is a potential lead compound for cancer therapy. We synthesized a new derivative 2 which has a naphthalene ring at the side chain terminal position instead of a benzene ring, to increase CH/π interactions with Pro-241 of the PKCδ-C1B domain. Based on the synthetic route of 1, 2 was convergently synthesized in 26 linear steps from 6-hydroxy-1-naphthoic acid with an overall yield of 0.18%. Although the anti-proliferative activity of 2 was more potent than that of 1, the binding potency of 2 to the PKCδ-C1B domain did not exceed that of 1. Molecular dynamics simulation indicated the capability of 2 to simultaneously form hydrogen bonds and CH/π interactions with the PKCδ-C1B domain. Focusing on the hydrogen bonds, their geometry in the binding modes involving the CH/π interactions seemed to be sub-optimal, which may explain the slightly lower affinity of 2 compared to 1. This study could be of help in optimizing such interactions and synthesizing a promising lead cancer compound.

Full text of DOI:10.1016/j.bmcl.2020.127657

Journal Pre-proofs
Synthesis and biological activities of simplified aplysiatoxin analogs focused
on the CH/π interaction
Takumi Kobayashi, Ryo C. Yanagita, Kazuhiro Irie
PII:
DOI:
Reference:
S0960-894X(20)30768-X
https://doi.org/10.1016/j.bmcl.2020.127657
BMCL 127657
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Bioorganic & Medicinal Chemistry Letters
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22 October 2020
25 October 2020
Please cite this article as: Kobayashi, T., Yanagita, R.C., Irie, K., Synthesis and biological activities of simplified
aplysiatoxin analogs focused on the CH/π interaction, Bioorganic & Medicinal Chemistry Letters (2020), doi:
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Synthesis and biological activities of simplified aplysiatoxin analogs focused on the
CH/ interaction
Takumi Kobayashia, Ryo C. Yanagitab, and Kazuhiro Iriea,∗
aDivision of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto
University, Kyoto 606-8502, Japan
bDepartment of Applied Biological Science, Faculty of Agriculture, Kagawa University,
Kagawa 761-0795, Japan
Keywords: anti-proliferative activity, aplysiatoxin, CH/ interaction,
debromoaplysiatoxin, protein kinase C, tumor promotion.
*Corresponding author.
Tel.: +81-75-753-6281; Fax: +81-75-753-6284; e-mail: irie.kazuhiro.2z@kyoto-u.ac.jp
1

Abstract
Debromoaplysiatoxin (DAT) is a potent protein kinase C (PKC) activator with
tumor-promoting and pro-inflammatory activities. Irie and colleagues have found that 10-
methyl-aplog-1 (1), a simplified analog of DAT, has strong anti-proliferative activity
against several cancer cell lines with few adverse effects. Therefore, 1 is a potential lead
compound for cancer therapy. We synthesized a new derivative 2 which has a naphthalene
ring at the side chain terminal position instead of a benzene ring, to increase CH/
interactions with Pro-241 of the PKC-C1B domain. Based on the synthetic route of 1, 2
was convergently synthesized in 26 linear steps from 6-hydroxy-1-naphthoic acid with an
overall yield of 0.18%. Although the anti-proliferative activity of 2 was more potent than
that of 1, the binding potency of 2 to the PKC-C1B domain did not exceed that of 1.
Molecular dynamics simulation indicated the capability of 2 to simultaneously form
hydrogen bonds and CH/π interactions with the PKC-C1B domain. Focusing on the
hydrogen bonds, their geometry in the binding modes involving the CH/π interactions
seemed to be sub-optimal, which may explain the slightly lower affinity of 2 compared
to 1. This study could be of help in optimizing such interactions and synthesizing a
promising lead cancer compound.
2

Protein kinase C (PKC) is a family of serine/threonine kinases that plays critical
roles in cell proliferation, differentiation, and apotosis.1,2 Recent studies have suggested
that PKC could suppress tumor growth in many human cancers.3 Therefore, PKC
activation is considered to be an effective strategy in cancer treatment.4 Naturally
occurring PKC activators, such as 12-O-tetradecanoylphorbol 13-acetate (TPA),
teleocidin B-4, and aplysiatoxins (Fig. 1) bind to the C1 domains (C1A and C1B) of
conventional PKC (, I, II, ) and novel PKC (, , , ) isozymes, and show anti-
proliferative and proapoptotic activities against several cancer cell lines.5–7 However, the
application of natural PKC ligands (Fig. 1) to medicinal uses is strictly limited as they
exhibit serious adverse effects such as tumor-promoting and pro-inflammatory activities.8
H
CH3(CH2)12OCO
OCOCH3
H
N
N
OH
O
H
N
O
H
N
OH
H
O
HO
H
N
H
HO
BzO
O
HO
N
H
HO
OH
HO
TPA
Ingenol 3-benzoate
Teleocidin B-4
(–)-Indolactam-V
Fig. 1. Structures of naturally occurring protein kinase C (PKC) activators.
Bryostatin-1 (bryo-1) is an exceptionally potent PKC ligand with little tumor-
promoting activity.9,10 Effective synthetic routes of bryo-1 have been established11,12 and
its simplified analogs have been developed.13,14 In addition, Irie and colleagues developed
10-methyl-aplog-1 (1), a simplified analog of debromoaplysiatoxin (DAT),15 as a new
anti-cancer seed.16,17 Compound 1 showed significant anti-proliferative activity against
several human cancer cell lines, but did not exhibit tumor-promoting and pro-
inflammatory activities, unlike DAT and TPA.17 Subsequent structure–activity studies
revealed that the phenolic hydroxy group and the aromatic ring in the side chain of 1
increased its affinity for the PKC-C1B (δ-C1B) domain through a hydrogen bond or
3

CH/ interaction18 with Met-239 or Pro-241 of the δ-C1B domain, resulting in potent
anti-proliferative activity against several cancer cell lines.19 Moreover, the presence of
the phenolic hydroxy group and an aromatic ring was found to suppress adverse effects,
such as pro-inflammatory and tumor-promoting activities.19
To develop more promising aplogs as anti-cancer seeds, we focused on the CH/
interaction of the phenolic moiety of 1 with Pro-241 of the -C1B domain. Irie and
colleagues reported the involvement of the indole ring of (–)-indolactam-V in the CH/
interaction with Pro-241 of the -C1B domain,20 and developed more potent and/or PKC
isozyme-selective ligands based on this interaction.21–23 Our recent study also suggested
that the analogs of 1 with different side-chain linker lengths could retain hydrogen
bonding involving the phenolic hydroxy group.24 Based on this research, we synthesized
a new derivative 2, which has a naphthalene ring in the side chain of 1 instead of a benzene
ring (Fig. 2), and evaluated its affinity for the C1 domains of PKC and PKC, anti-
proliferative activity against 39 human cancer cell lines, and pro-inflammatory activity
on the ears of mice.
H
H
H
4
3
O
O
O
O
O
O
12
O
O
O
OH
O
OMe
10
15
O
O
O
O
O
O
O
O
30
OH
HO
OH
HO
OH
RO
1)
10-Methyl-aplog-1 (
DAT
2
R = H :
3
R = Bn :
Fig. 2. Structures of 10-Methyl-aplog-1 (1) and its derivatives (2 and 3) along with DAT.
The synthesis of 2 was carried out by referring to the synthetic scheme for 125 with
appropriate modifications to the synthesis of the side chain. Scheme 1 shows the
retrosynthetic analysis of 2. The dissection of 2 at two ester linkages gave subunits A and
4

B. Fragment A provided fragments C and D, which were named as an epoxide unit and a
dithiane unit, respectively.
H
H
O
O
O
HO2C
O
O
O
O
O
HO
OMPM
CO2H
OH
RO
RO
OBn
2
R = H :
A
3
R = Bn :
B
O
OTES
S
S
OTIPS
+
RO
C
D
Scheme 1. Retrosynthetic analyses of 2 and 3.
The synthesis of 2 (Schemes 2 and 3) commenced with the methyl esterification of
6-hydroxy-1-naphthoic acid, which was followed by protection of the phenol group as a
benzyl ether, followed by reduction and bromination26 to provide bromide 7. SN2
substitution of 7 with diethyl malonate gave a diester 8, which was decarboxylated to
produce the ester 9. The repetition of this two-carbon homologation sequence gave the
ester 13. Ester 13 was partially reduced to aldehyde 14, which was immediately used for
asymmetric Brown crotylation27 to furnish the homoallyl alcohol 15 (> 90% de). The
vanadium-catalyzed epoxidation28 of 15 generated the desired epoxide 16 as the main
product (98% ee). At this step, 16 was separated from any undesirables by medium
pressure silica gel column chromatography. The hydroxy group was protected as a
triethylsilyl (TES) ether to give epoxide 17 with an overall yield of 5.2% from 6-hydroxy-
1-naphtoic acid.
5

R
CO2H
CO2Me
a
c
HO
RO
BnO
6-hydroxy-1-naphthoic Acid
4: R = H
6: R = OH
b
d
5:
7:
R = Br
R = Bn
CO2Et
R
CO2Et
R
R
e
g
i
BnO
BnO
BnO
8:
9:
10:
11:
12:
13:
R = CO2Et
R = H
R = OH
R = Br
R = CO2Et
R = H
h
f
j
OHC
O
OH
OR
k
l
m
BnO
BnO
BnO
14
15
16:
17:
R = H
n
R = TES
Scheme 2. Synthesis of the epoxide unit C. (a) SOCl2, MeOH, 87%; (b) BnBr, K2CO3, acetone, 98%; (c)
LiAlH4, THF, 97%; (d ) CBr4, PPh3, CH2Cl2, 83%; (e) diethylmalonate, NaH, DMF, 76%; (f) LiCl, H2O,
DMSO, 50%; (g) LiAlH4, THF, 97%; (h) CBr4, PPh3, CH2Cl2, 93%; (i) diethylmalonate, NaH, DMF, 92%;
(j) LiCl, H2O, DMSO, 78%; (k) DIBAL-H, toluene, 93%; (l) trans-2-butene, t-BuOK, n-BuLi, (–)-
Ipc2BOMe, THF, BF3·Et2O, 76%; (m) VO(acac)2, TBHP, CH2Cl2, 52%; (n) TESCl, imidazole, THF, 83%.
The coupling of 17 with the known dithiane unit D16 provided 18 according to the
protocol of Ide and Nakata.29 After deprotection of TES and triisopropylsilyl (TIPS)
groups of 18, secondary hydroxy groups were protected as an isopropylidene acetal to
give the primary alcohol 20. Following Parikh–Doering oxidation30 of 20, Maruoka’s
asymmetric allylation31 yielded the homoallylic alcohol 22 (> 95% de) as a mixture with
(S)-BINOL. Acid hydrolysis of 22 gave triol 23. The use of Selectfluor®,32 an
electrophilic fluorinating reagent, achieved the cleavage of 1,3-dithiane and subsequent
6

spiroketalization. The desired spiroketal 24, and the undesired one 25, as well as its
diastereomer, were obtained at a ratio of 2.2:1:1.2, and were separated by HPLC
purification. The presence of the diastereomer might be partly due to the inadequate
separation of 16 from the undesirable stereoisomers. Treatment of 25 with pyridinium p-
toluenesulfonate (PPTS) produced a 3:2 mixture of 24 and 25.
Condensation of 24 with the known carboxylic acid B16 was achieved using
Yamaguchi’s method.33 After detachment of the p-methoxyphenylmethyl (MPM) group
of 25 under neutral conditions by employing a phosphate buffer (pH 7.2) to prevent
isomerization, oxidative cleavage of the olefin group produced the carboxylic acid 27.
Yamaguchi’s macrolactonization of the seco-acid 27 was performed at 2 mM to
efficiently produce lactone 28 without formation of the corresponding dimer. Finally,
catalytic hydrogenolysis of 28 gave the target compound 2 and its intermediate 3 with a
benzyl group in the side chain. The additional benzyl group of 3 might increase the CH/π
interaction with the -C1B domain. Compound 2 was obtained in 26 steps from 6-
hydroxy-1-naphthoic acid with an overall yield of 0.18%.
The analogs (2 and 3) were initially evaluated for their anti-proliferative activities in
growth inhibition tests against 39 human cancer cell lines established by Yamori et al.34
Growth inhibitory activity was expressed as GI50 (M), the concentration required to
inhibit cell growth by 50% compared to an untreated control. The average log GI50 value
for all 39 human cancer cells was expressed as MG-MID (mean-graph midpoint value).
Nine cell lines with log GI50 values less than –6.00 when treated with 2 are listed in Table
1. The average log GI50 values of the 9 cancer cell lines treated with 2 and 3 were smaller
than that treated with 1, suggesting that the anti-proliferative activities of 2 and 3 were
more potent than those of 1. The increase in anti-proliferative activities might be ascribed
to the increased hydrophobicity of the molecules; Clog P values of 1–3 are 3.4, 4.0, and
4.5, respectively. Previous studies on side-chain-modified aplogs suggested that the
optimal hydrophobicity range of aplogs in terms of anti-proliferative activity is 4.0–4.5,35
7

and that the presence of the phenolic hydroxy group suppresses tumor-promoting-related
activities.19,35 Those results were consistent with the trends for 1–3. Increased
hydrophobicity might enhance the cellular uptake of 2 and 3 compared with 1. The
involvement of the CH/ interaction will be discussed later in this paper.
TIPSO
O
S
S
OTES
OH OTES
BnO
a
b
BnO
17
18
HO
R
S
S
S
S
OH OH
BnO
O
O
c
e
BnO
19
20: R = CH2OH
21: R = CHO
d
OH
H
O
O
O
S
S
O
OH
OR OR
BnO
g
+
OH
BnO
BnO
24
22: R, R = C(CH3)2
23: R = H
25
f
h
H
H
O
O
O
HO2C
O
O
i
k
l
RO
O
OH O
O
OBn
BnO
OBn
BnO
26: R = MPM
27: R = H
28
j
H
H
O
O
O
O
O
m
O
O
O
O
O
O
O
OBn
BnO
OH
RO
29
R = H : 2
R = Bn : 3
8

Scheme 3. Syntheses of 2 and 3. (a) D, n-BuLi, THF, 78%; (b) TsOH·H2O, CH3CN, THF, H2O, CHCl3,
99%; (c) 2,2-dimethoxypropane, (+)-CSA, CH2Cl2, 87%; (d) SO3·Pyr, Et3N, DMSO, CH2Cl2, 90%; (e)
allyl-SnBu3, Ti(OiPr)4, TiCl4, Ag2O, (S)-BINOL, CH2Cl2; (f) TsOH·H2O, CH3CN, THF, H2O, 59% in
two steps; (g) Selectfluor®, CH3CN, H2O, 31%; (h) PPTS, CH2Cl2, CH3CN, 51%; (i) B, TCBCl, Et3N,
DMAP, toluene, quant.; (j) DDQ, pH 7.2 phosphate buffer, CH2Cl2, 82%; (k) KMnO4, NaIO4, pH 7.2
phosphate buffer, 53%; (l) TCBCl, Et3N, DMAP, toluene, 85%; (m) H2, 10% Pd/C, EtOH; 2, 80%; 3,
62%.
Table 1. Log GI50 values of growth inhibition of several human cancer cell lines for DAT and its
analogs.
log GI50 (M)
Cancer type
Breast
Cell line
HBC-4
DAT17
–6.47
117
2
3
–7.48
–7.04
–7.20
–6.27
MDA-MB-231
–6.03
–6.90
–6.05
CNS
SF-295
HCC2998
NCI-H460
A549
–5.53
–6.09
–6.46
–5.94
–5.69
–6.44
–4.98
–5.96
–5.22
–4.98
–6.47
–7.07
–6.01
–6.21
–6.24
–4.97
–6.25
–5.24
–6.02
–6.30
–6.79
–6.12
–6.35
–6.62
–6.32
–6.40
–5.40
–6.06
–6.40
–6.94
–6.16
–6.74
–7.28
–6.71
–6.64
–5.19
Colon
Lung
Melanoma
Stomach
LOX-IMVI
St-4
MKN45
Average of 9 cell lines
MG-MIDa of 39 cell lines
aMean-graph midpoint value.
9

PKCδ is related to the growth inhibition and apoptosis induced by phorbol esters.36–
39 Recently, Hanaki et al. reported that the ability to activate PKCδ and PKC could be
closely correlated to the anti-proliferative activity against aplog-sensitive cancer cell
lines.40 Thus, we evaluated the affinity of 2 and 3 for these isozymes following the
procedure of Sharkey and Blumberg.41 Since it was difficult to obtain reproducible results
using whole PKC isozymes prepared by molecular biology techniques, synthetic PKCδ-
C1B (-C1B) and PKC-C1A (-C1A) peptides were employed for the binding
assay.42,43 Although each PKC isozyme has two C1A and C1B domains, the main binding
sites of phorbol esters for PKC and PKC were identified as C1B and C1A,
respectively.42–44 Contrary to expectations, the binding affinity of 2 and 3 to -C1B did
not exceed that of 1 as the affinity of 2 (Ki = 0.98 nM) and 3 (Ki = 0.74 nM) for δ-C1B
was weaker than that of 1 (Ki = 0.46 nM),17 indicating that the CH/ interaction between
2/3 and -C1B was not enhanced (Table 2). Alternatively, the hydrogen bonding of
phenolic hydroxy group in 2 with PKC-C1B might not be optimal compared with that
of 1. A similar tendency was observed in their binding to -C1A.
Table 2. Ki values for the inhibition of [3H]PDBu binding by 1, 2, and 3.
Ki (nM)
PKC C1 peptides
-C1A
1 17)
4.7
0.46
2
3
6.4 (1.2)a
0.98 (0.08)
7.6 (1.2)
0.74 (0.01)
-C1B
aStandard deviation from at least two separate experiments.
To predict the binding mode of 2 with -C1B, we performed a molecular dynamics
(MD) simulation of -C1B in complex with 2 in a phospholipid membrane environment
as previously described.24 Figure 3A shows four snapshots from 100 ns of trajectory from
10

the MD simulation with each having a representative binding mode involving hydrogen
bonding with the carbonyl group of Met-239. In three out of four binding modes (t = 4,
12, and 28 ns), 2 formed hydrogen bonds with Met-239 and CH/π interactions with Pro-
241. Importantly, in the binding mode of t = 28 ns, there existed three CH/π interactions
between the naphthalene ring in 2 and Pro-241 (Figure 3B, right). These simulation results
indicate the ability of 2 to simultaneously form hydrogen bonds and CH/π interactions
with -C1B. Focusing on the hydrogen bonds, their geometry in the binding modes
involving the CH/π interactions seems not to be optimal, which may explain the slightly
lower affinity of 2 compared to 1, despite the possible existence of additional interactions
with the protein. These results suggested that there is room for structural optimization of
2. The introduction of the β-methyl group at position 12 and a substituent at position 14
into 2 is a possible strategy to restrict the orientation of the side chain. In fact, Kikumori
et al. reported that the introduction of the -methyl group at position 12 in 1 increased its
anti-proliferative activities without increasing its tumor-promoting and pro-inflammatory
activities.45 Another approach is to change the phenolic group into bioisosteres such as an
indole or a benzimidazole ring to adjust hydrophobicity and hydrogen bond donor ability.
11

A
B
C
1
Fig. 3. MD simulation of the PKCδ-C1B (δ-C1B) domain in complex with 2 in a phospholipid membrane
environment. A: snapshots of 100 ns of MD trajectory. B: Close-ups of the interaction between Pro-241
and the naphthalene ring in 2. C: Representative binding mode of 1 with δ-C1B.24 Compound 2 is drawn
as a stick model. δ-C1B is depicted in cartoon form, and amino acid residues that could form hydrogen
bonds or be involved in CH/π interactions with the aromatic ring of each compound are represented in the
stick model colored green (carbon), red (oxygen), blue (nitrogen), yellow (sulfur), and white (hydrogen).
Yellow and orange dashed lines represent hydrogen bonds and CH/π interactions, respectively.
12

Fig. 4. Change in the relative weight of each of the ears of mice 24 hr after the application of TPA, 2, or
3 (nmol). Error bars show the standard error of three samples. ****, p < 0.0001; ***, 0.0001 ≤ p < 0.001;
**, 0.001 ≤ p < 0.01 versus the vehicle (EtOH) group (Dunnett’s test).
The most likely adverse effects of ATX analogs are tumor promotion and
inflammation. Previous studies indicated that chronic inflammation was closely related
to tumor promotion.46 Therefore, we evaluated the pro-inflammatory activities of 2 and 3
on the ears of mice.47
Ears of each ICR mouse (female) were treated with each compound and EtOH for
24 h. Pro-inflammatory activity was measured as an increase in the relative weight of
each ear after treatment with each compound. Although 2 as well as 1 induced slight
inflammation at a dose of 170 nmol, 3 exhibited significant pro-inflammatory activity
even at a dose of 17 nmol (Fig. 4). Since the hydrophobicity of a molecule was one of the
factors that enhanced tumor-promoting activities of phorbol esters,36 the higher pro-
inflammatory activity of 3 likely results from its higher hydrophobicity and lack of a
hydroxy group which could form hydrogen bonds with -C1B as Hanaki et al. proposed.19
13

In summary, we synthesized a new aplog derivative 2 with a naphthalene ring in the
side chain, to enhance the CH/ interaction with Met-239 or Pro-241 of the PKC-C1B
domain. Although the anti-proliferative activity of 2 was more potent than that of 1,
possibly due to an increase of molecular hydrophobicity, the ability of 2 to bind to -C1B
did not exceed that of 1. The MD simulation indicated the ability of 2 to simultaneously
form hydrogen bonds and CH/π interactions with -C1B. However, the spatial
arrangement of the naphthalene ring might not be optimal. Further investigation to
optimize such interactions and anti-proliferative activities is currently underway in our
laboratory.
Declaration of Competing Interest
The authors declare no conflicts of interest associated with this manuscript.
Acknowledgements
This work was supported by JSPS KAKENHI (grant number 17H06405) to K. I. and
R. C. Y. The authors thank the Molecular Profiling Committee, Grant-in-Aid for
Scientific Research on Innovative Areas “Advanced Animal Model Support (AdAMS)”
from the Ministry of Education, Culture, Sports, Science and Technology, Japan (JSPS
KAKENHI Grant Number JP 16H06276). They also thank Ms. Akiko Fujihashi in the
Joint Usage/Research Center (JURC) at the Institute for Chemical Research, Kyoto
University, Japan, for mass measurements with the JEOL JMS-MS700V spectrometer.
Supplementary data
Supplementary data associated with this article can be found, in the online version,
at http://dx.doi.org./10.1016/j.bmcl.
14

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was approved by the above-mentioned committee (approval number R2-58).
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