Biakamides A-D, Unique Polyketides from a Marine Sponge, Act as Selective Growth Inhibitors of Tumor Cells Adapted to Nutrient Starvation

Article abstract of DOI:10.1021/acs.joc.6b02948

Biakamides A-D, novel unusually unique polyketides, were isolated from an Indonesian marine sponge (Petrosaspongia sp.) with a constructed bioassay using PANC-1 human pancreatic cancer cells. Through detailed analyses of the one- and two-dimensional NMR spectra of biakamides, planar chemical structures possessing a terminal thiazole, two N-methyl amides, a chloromethylene, and a substituted butyryl moiety were obtained. After elucidation of the configuration of the secondary alcohol moiety in biakamides A and B, the absolute stereostructures of the two secondary methyl groups in biakamides A-D were determined by the asymmetric total syntheses of all possible stereoisomers from the optically pure monoprotected 2,4-dimethyl-1,5-diol. Biakamides A-D showed selective antiproliferative activities against PANC-1 cells cultured under glucose-deficient conditions in a concentration-dependent manner. The primary mode of action of biakamides was found to be inhibition of complex I in the mitochondrial electron transport chain.

Full text of DOI:10.1021/acs.joc.6b02948

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Article
Biakamides A–D, unique polyketides from a marine sponge, act as
selective growth inhibitors of tumor cells adapted to nutrient starvation
Naoyuki Kotoku, Ryosuke Ishida, Hirokazu Matsumoto, Masayoshi Arai,
Kazunari Toda, Andi Setiawan, Osamu Muraoka, and Motomasa Kobayashi
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.6b02948 • Publication Date (Web): 16 Jan 2017
Downloaded from http://pubs.acs.org on January 19, 2017
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Biakamides A–D, unique polyketides from a marine sponge, act as selective growth
inhibitors of tumor cells adapted to nutrient starvation
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a
a
Naoyuki Kotoku a,*,†, Ryosuke Ishida a,†, Hirokazu Matsumoto , Masayoshi Arai ,
Kazunari Toda a, Andi Setiawan b, Osamu Muraoka c, and Motomasa Kobayashi a,*
a
Graduate School of Pharmaceutical Sciences, Osaka University, Yamadaꢀoka 1ꢀ6,
Suita, Osaka 565ꢀ0871, Japan
b
Department of Chemistry, Faculty of Science, Lampung University, Jl. Prof. Dr.
Sumantri Brodjonegoro No. 1, Bandar Lampung 35145, Indonesia
c
School of Pharmacy, Kindai University, 3ꢀ4ꢀ1 Kowakae, Higashiꢀosaka, Osaka
577ꢀ8502, Japan
† These authors contributed equally to this work.
* Corresponding authors. Tel: +81ꢀ6ꢀ6879ꢀ8215; Fax: +81ꢀ6ꢀ6879ꢀ8219
Eꢀmail: kotoku@phs.osakaꢀu.ac.jp; kobayasi@phs.osakaꢀu.ac.jp
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Abstract
Biakamides A–D, novel unusually unique polyketides, were isolated from an Indonesian
marine sponge (Petrosaspongia sp.) with a constructed bioassay using PANCꢀ1 human
pancreatic cancer cells. Through detailed analyses of the oneꢀ and twoꢀdimensional
nuclear magnetic resonance (NMR) spectra of biakamides, planar chemical structures
possessing a terminal thiazole, two Nꢀmethyl amides, a chloromethylene, and a
substituted butyryl moiety were obtained. After elucidation of the configuration of the
secondary alcohol moiety in biakamides A and B, the absolute stereostructures of the
two secondary methyl groups in biakamides A–D were determined by the asymmetric
total syntheses of all possible stereoisomers from the optically pure monoprotected
2,4ꢀdimethylꢀ1,5ꢀdiol. Biakamides A–D showed selective antiproliferative activities
against PANCꢀ1 cells cultured under glucoseꢀdeficient conditions in
a
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concentrationꢀdependent manner. The primary mode of action of biakamides was found
to be inhibition of complex I in the mitochondrial electron transport chain.
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Keywords: biakamides; polyketide; marine natural product; structure elucidation; total
synthesis; glucoseꢀdeficient condition; mitochondrial electron transport chain
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Introduction
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The microenvironment surrounding tumors is an important target for anticancer
drug discovery. Because of the disordered vascular network, solid tumors have large
areas exhibiting hypoxia and/or nutrient starvation.1 Cancer cells are known to acquire
tolerance toward such severe conditions, allowing them to survive. Furthermore, these
cancer cells also acquire resistance to cancer chemotherapy and irradiation and
aggravate the pathology of cancer by promoting tumor growth and metastasis.2
Therefore, compounds that exhibit selective growth inhibitory activity against tumor
cells adapted to the tumor microenvironment may be novel, promising anticancer drugs
and important chemical tools for identification of new molecule(s) responsible for the
adaptation of tumor cells to hypoxia and/or nutrient starvation.
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Marine natural products have been shown to be a rich and promising source of
drug candidates, particularly in the field of anticancer drug discovery.3,4 During our
assessment of bioactive substances from marine organisms, we have focused on
searching for compounds targeting the tumor microenvironment. From our studies, we
have identified several active compounds, such as furospinosulinꢀ15 and dictyoceratin6,
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showing hypoxiaꢀselective growth inhibitory activity against cancer cells. Furthermore,
we have also conducted synthetic studies and target analyses of the identified
compounds to validate their potential as anticancer drugs.7ꢀ11
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In contrast, some reports have described the search for selective growth inhibitors
against cancer cells adapted to nutrient starvation conditions. According to the soꢀcalled
“antiꢀausterity” strategy,12,13 several active compounds, including kigamycin D and
arctigenin, have been identified from microbial secondary metabolites or traditional
herbal medicines.14ꢀ17 However, the adaptation system of cancer cells to nutrient
deprivation has not been fully elucidated. Recently, we also established a screening
system to search for substances that selectively inhibit the growth of PANCꢀ1 human
pancreatic cancer cells cultured under glucoseꢀdeficient conditions, and we isolated a
new
3ꢀalkylpyridine
alkaloid
(Nꢀmethylniphatyne
A)
and
3,4,5ꢀtribromoꢀ2ꢀ(2’,4’ꢀdibromophenoxy)ꢀphenol as active substances through
bioassayꢀguided separation.18,19 Further screening of the extract library of marine
organisms led us to isolate unique polyketides named biakamides A–D (1–4) from an
Indonesian marine sponge (Petrosaspongia sp.).
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Here, we report the isolation, structure elucidation, total synthesis, and biological
evaluation of biakamides A–D (Figure 1).
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Figure 1. Chemical Structures of Biakamides A (1), B (2), C (3) and D (4).
Results and Discussion
Isolation and structure elucidation of biakamides A–D
The MeOH extract (240 g) of the marine sponge Petrosaspongia sp. (05A01),
collected in Biak, Indonesia in 2005, showed selective growth inhibitory activity against
PANCꢀ1 cells cultured in glucoseꢀdeficient medium. The MeOH extract was partitioned
into an H2O and AcOEt mixture, and the AcOEt soluble portion was further partitioned
into an nꢀhexane and 90% methanol mixture. Using the bioassay, the active 90%
methanol soluble portion (34 g) was fractionated by successive silica gel column
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chromatography and reversedꢀphase highꢀperformance liquid chromatography (HPLC)
to yield four active constituents, designated biakamides A (1, 2.8 mg), B (2, 1.4 mg), C
(3, 6 mg), and D (4, 6 mg).
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The matrixꢀassisted laser desorption ionization timeꢀofꢀflight (MALDIꢀTOF) mass
spectrometry (MS) results for biakamide A (1) showed pseudomolecular ion peaks at
m/z 534 and 536 [M+Na]+ with a ratio of 3:1. These data suggested that there may be a
chlorine atom within this molecule. The molecular formula of 1 was determined to be
C26H42O3N3SCl by highꢀresolution (HR) MALDIꢀTOF MS, indicating that this
1
compound possessed seven double bond equivalents. Many signals in the H and 13C
NMR spectra of 1 were anomalously broadened, irrespective of solvent, and some
others were observed as doubled signals (Figure 2). Brief analyses of the
twoꢀdimensional (2D)ꢀnuclear magnetic resonance (NMR) spectra indicated that two
Nꢀmethylamide moieties were contained in the molecule, implying the presence of
distinguishable cis/trans rotamers. Thus, structure elucidation of 1 was executed using
the NMR signals of the respective conformers, as described below (Table 1).
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Figure 2. 1H and 13C NMR Spectra of Biakamide A (1).
Table 1. NMR chemical shifts of biakamide A (1) in CDCl3.
Major conformer
Minor conformer a
Pos.
Pos.
δ C
δ H
δ
δ H
C
1
2
173.7 (br)
128.9
138.4 (br)
29.8
1
2
3
5.42 (br s)
2.56 (m)
3
4
4
5
44.2
1.26 (br), 1.13 (br)
1.41 (br)
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6
30.6
6
7
34.0
1.42 (br), 1.18 (br)
2.11 (m), 2.20 (m)
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8
27.6
8
27.5
141.5
31.8
26.1
49.0
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142.0
32.1
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2.05 (m)
1.63 (m)
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25.2
1.68 (m)
3.24 (m)
47.3
3.34 (t, 7.5 Hz)
1.88 (s)
14.2 (br)
20.0
0.95 (br s)
0.89 (br s)
5.79 (s)
19.8
112.4
48.5 (br)
167.0 (br)
113.0
5.80 (s)
4.88 (s)
52.6 (br)
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OH
119.9 (br)
142.3 (br)
172.6
7.33 (br)
7.72 (br)
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26
OH
172.4
40.6
41.2
2.44 (m), 2.30 (m)
4.19 (m)
2.46 (m), 2.28 (m)
1.217 (d, 6.3 Hz)
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64.1
64.3
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22.2
1.22 (d, 6.3 Hz)
3.04 (br s)
22.3
37.0 (br)
35.2
33.3 (br)
33.1
2.95 (s)
4.59 (d, 4.3 Hz) b
2.92 (s)
4.61 (d, 4.3 Hz) b
a Only distinguishable signals were indicated. b Observed in DMSOꢀd6.
Analyses of the 13C NMR and heteronuclear single quantum coherence (HSQC)
spectra of biakamide A (1) revealed that this molecule consisted of six methyls (four
aliphatic and two nitrogenꢀsubstituted), eight methylenes (six aliphatic and two
heteroatomꢀsubstituted), seven methines (two aliphatic, one oxymethine, and four
olefinic), and five quaternary carbons (two olefinic and three possible carbonyls).
Detailed analyses of some 2DꢀNMR spectra (correlation spectroscopy [COSY], HSQC,
and heteronuclear multiple bond correlation [HMBC]) of 1 demonstrated the presence
of seven partial structures, A to G (Figure 3). The characteristic 2ꢀsubstituted thiazole
structure (partial structure A) has been found in some marineꢀderived metabolites, such
as barbamide or dysideathiazole.20,21 The chloromethylene moiety in the partial
structure E was deduced from the HMBC correlations between Hꢀ16/Cꢀ8, Cꢀ9, and
Cꢀ10 and from a comparison of the chemical shifts of the sp2 carbons with those of
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malyngamides and jamaicamides.22,23 Furthermore, the HMBC between Hꢀ13 and Cꢀ1,
together with an exceptionally upfieldꢀshifted vinylic proton24 (Hꢀ3, δH 5.42), indicated
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that the two partial structures B and C were connected to form a
Nꢀmethylated
α,βꢀunsaturated amide. The HMBCs between Hꢀ22, Hꢀ26/Cꢀ21, and Hꢀ26/Cꢀ12 led us
to deduce the connectivities of another terminal (structures E–G). Some pairs of the
distinguishable NMR signals corresponding to this region were observed, probably
because of the presence of the two conformers (ratio of approximately 3:2) for the
N
ꢀmethylamide group at Cꢀ21. The nuclear Overhauser effect spectroscopy (NOESY)
correlation between Hꢀ22 and Hꢀ26 was observed in the spectrum of the major
transꢀconformer of this
Nꢀmethyl amide moiety, whereas the Hꢀ22/Hꢀ12 NOESY
correlation was observed in the minor cisꢀconformer.
In contrast, no correlation signals around the partial structure D were observed in
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the HMBC and HꢀH COSY spectra of biakamide A (1). Most 1H and C NMR signals
corresponding to the left half of the molecule were exceptionally broadened, probably
because of the presence of a large energy barrier of bond rotation around the
N
ꢀmethylamide group. Therefore, we anticipated that an elevated temperature would
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accelerate bond rotation to yield averaged and sharpened NMR signals. As expected, the
1H NMR spectrum of 1 measured at 50°C in DMSOꢀd6 gave somewhat sharpened
signals. Under this condition, the magnetization relay (from Hꢀ15 to Hꢀ4, Hꢀ5, Hꢀ7, and
Hꢀ8) was observed in the total correlation spectroscopy (TOCSY) results. Furthermore,
the HMBCs between Hꢀ15/Cꢀ5 and Cꢀ7 were observed under these conditions,
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providing evidence for the connectivities between the partial structures C, D, and E.
2
The geometries of the two olefins (∆ and ∆9(16)) were determined as
E from the
NOESY correlations among Hꢀ13/Hꢀ4, Hꢀ16/Hꢀ10, and Hꢀ11. Finally, the absolute
configuration of the secondary alcohol moiety at Cꢀ23 was found to be
S by applying
the modified Mosher method25 (Figure 4, Supporting Information). In this way, most of
the chemical structure of biakamide A (1), except for configurations of the two
secondary methyls at Cꢀ4 and Cꢀ6, could be determined as depicted in Figure 4.
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Figure 3. Partial Structures and 2D NMR correlations of Biakamide A (1).
Figure 4. Selected NOEs and Modified Mosher Analysis of Biakamide A (1).
From the HRꢀMALDIꢀTOF MS analysis, the molecular formula of biakamide B
(2) was found to be the same as that of biakamide A (1). The oneꢀdimensional (1D) and
2D NMR analyses revealed that biakamide B (2) possessed almost the same planar
structure as 1, except for the geometry of the chloromethylene moiety, in which the
NOESY correlations between Hꢀ16/Hꢀ7 and Hꢀ8 were observed to define the geometry
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of the ∆9(16) olefin as
Z (Figure 5).
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The molecular formula of biakamide C (3) was determined to be C27H42O3N3SCl,
by HRꢀMALDIꢀTOF MS. Initial attempts to determine the structure of 3 revealed that
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1
many signals in the HꢀNMR spectrum of 3 were further broadened compared with
those of 1. Additionally, compound 3 was unstable and gradually changed to another
compound during prolonged NMR measurement in CDCl3 solvent. Considering the acid
lability of 3, the CDCl3 that passed through basic alumina just before preparation of the
NMR sample was used to suppress the decomposition of 3. Then, the chemical structure
of 3 could be deduced by detailed analysis of the 2D NMR spectra, which was similar to
that of 1, except that a methoxy group (δH 3.60) and an olefinic proton (δH 5.15) were
observed in the terminal acyl moiety. Through analysis of HMBCs (Hꢀ22/Cꢀ21 and
Cꢀ23, Hꢀ24/Cꢀ22 and Cꢀ23, and Hꢀ27/Cꢀ23), as depicted in Figure 5, biakamide C (3)
was found to possess a 3ꢀmethoxyꢀ2ꢀbutenoyl moiety at its terminal. Some reported
metabolites, such as ajudazole,26 also had the same functionality. Furthermore, the
geometry of the ∆22 olefin in 3 was determined as
E from nuclear Overhauser effect
(NOE) experiments. Additionally, biakamide D (4) was assigned as the geometrical
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counterpart of biakamide C (3) at the ∆9(16) chloromethylene moiety (Figure 5).
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Figure 5. Planar Structures and Selected NOEs of Biakamides B (2), C (3) and D (4).
Total syntheses of biakamides A– D (1–4)
Finally, we aimed to determine the relative/absolute stereostructures of the two
secondary methyl groups at Cꢀ4 and Cꢀ6 for biakamides A–D (1–4) . As described
above, the 1H NMR signals at the Cꢀ4–Cꢀ7 positions in biakamides were too broadened,
1
and we could not apply empirical methods27, 28 to analyze the differences in H NMR
chemical shifts. Accordingly, we decided to carry out the total synthesis of 1–4 in order
to determine the absolute stereostructures of these moieties and to generate sufficient
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amounts of the compounds for further biological analyses.
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An outline of our synthetic plan is depicted in Figure 6. To obtain all possible
stereoꢀisomers of biakamides A–D (1–4), we planned to use the monoprotected
2,4ꢀdimethylꢀ1,5ꢀpentanediol as a chiral synthon, which could be obtained in an
optically pure form through the known enzymatic desymmetrization and kinetic
resolution of the synꢀisomer and antiꢀisomer, respectively.29 The central polyketide
skeleton II could be prepared through the CoreyꢀSeebach coupling reaction 30 between
the 1,3ꢀdithiane III and the nitrogenꢀcontained alkyl halide, and the following
successive introduction of the unsaturated amide and chloromethylene moieties was
expected to provide the common precursor I of biakamides. The final acylation using
the corresponding carboxylic acid would afford biakamides A–D (1–4).
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Amide
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N
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O
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Cl
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N
*
*
N
S
NH
O
Amide
formation
I
Cl
Wittig
reaction
Corey-Seebach
reaction
*
*
RO
O
Boc
N
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S
Wittig
reaction
II
*
*
RO
S
S
III
RO
RO
OH
OH
RO
RO
OH
OH
available by enzymatic desymmetrization / resolution
Figure 6. Retrosynthesis of Biakamides.
Initially, the syntheses of biakamides C (3) and D (4) were carried out as shown in
Scheme 1 because the structure determination process for 3 and 4 was expected to be
simpler. The (R, R)ꢀ2,4ꢀdimethylꢀ1,5ꢀpentanediol ((–)-5) was prepared by the known
enzymatic optical resolution method29 using lipase AK. According to a recent report,31
monoprotection of the two hydroxyl groups of (–)-5 as tertꢀbutyldiphenylsilyl (TBDPS)
ether was achieved in acceptable yield. Subsequent oneꢀpot TEMPO oxidation/Wittig
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reaction32 proceeded smoothly to give an unsaturated ester (7). The double bond of 7
was reduced by treatment with magnesium in MeOH, and the subsequent hydrolysis of
the resulting ester (8) provided a carboxylic acid (9). Then, 9 was converted into the
corresponding Weinreb amide (10), which was reduced with DIBAL to give an
aldehyde (11). Treatment of 11 with 1,3ꢀpropanedithiol and a catalytic amount of
iodine33 gave a 1,3ꢀdithiane (12), the designated precursor III of the central fragment.
As expected, the CoreyꢀSeebach coupling reaction between a carbanion generated by
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the n
ꢀBuLi treatment of 12 and an alkyl iodide (21)34, followed by TBAF treatment,
afforded the desired compound (13) in good yield. TPAP oxidation of the liberated
primary alcohol of 13 gave an aldehyde (14), and the subsequent Wittig homologation
with a stabilized ylide yielded an α,βꢀunsaturated ester (15). The concomitant Zꢀisomer
was separated by SiO2 column chromatography. Subsequent hydrolysis of the ester
moiety with NaOH or KOH in aqueous methanol or 1,4ꢀdioxane was found to be very
sluggish, whereas TMSOK treatment35 resulted in decomposition. Gratifyingly,
hydrolysis with LiOH proceeded efficiently to provide a carboxylic acid (16) in good
yield. Epimerization at Cꢀ4, the γꢀposition of the unsaturated ester moiety, did not occur
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during hydrolysis. Subsequent condensation with a secondary amine (22)36 yielded an
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N
ꢀmethylamide (17). We found that almost all the signals in the H NMR spectrum of
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the amide 17 broadened exceptionally, whereas the normal and sharp signals were
1
observed in the H NMR spectrum of the acid 16. These data clearly indicated that the
presence of the unsaturated
Nꢀmethylamide moiety interrupted free bond rotation.
Iodine treatment of 17 under basic conditions resulted in clean removal of 1,3ꢀdithiane
to give a ketone (18), and subsequent introduction of chloromethylene moiety was
quantitatively proceeded to give 19 through the Wittig reaction using
(chloromethyl)triphenylphosphonium chloride (E/Z = 3:2). Finally, removal of the Boc
moiety by TFA treatment and subsequent condensation with
a
known
E
ꢀ3ꢀmethoxyꢀ2ꢀbutenoic acid (23)37 afforded (4
R, 6S)ꢀbiakamides C (3) and D (4).
These two compounds were separated by reversedꢀphase HPLC under the established
conditions.
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Scheme 1. Total Synthesis of (4R,6S)ꢀBiakamides C (3) and D (4). Reagents and
Conditions: (a) TBDPSCl, Et3N, CH3CN/Hexane, 50%; (b) TEMPO, PhI(OAc)2,
CH2Cl2; (c) Ph3P=CHCO2Me, toluene, 100 °C, 82% (2 steps); (d) Mg, MeOH,
quant.; (e) LiOH, MeOH, 96%; (f) MeONHMe·HCl, EDCI·HCl, HOBt, Et3N,
CH2Cl2; (g) DIBAL, THF, 0 °C, 49% (2 steps); (h) 1,3ꢀpropanedithiol, I2, CHCl3,
90%; (i) 21, nꢀBuLi, THF; (j) TBAF, THF, 61% (2 steps); (k) TPAP, NMO, MS4A,
CH2Cl2, 79%; (l) Ph3P=C(Me)CO2Et, toluene, 100 °C, quant.; (m) LiOH,
THF/MeOH/H2O, 77%; (n) 22, EDCI·HCl, HOBt, Et3N, CH2Cl2, 92%; (o) I2,
NaHCO3, aq. CH3CN, 0 °C, 47%; (p) (chloromethyl)triphenylphosphonium chloride,
LHMDS, THF, 99%; (q) TFA, CH2Cl2; (r) 23, EDCI·HCl, HOBt, Et3N, CH2Cl2,
78%; (s) HPLC separation.
According to the synthetic route described above, (4R, 6R)ꢀbiakamides C (3’) and
D (4’) were synthesized starting from (2S, 4R)ꢀ2,4ꢀdimethylꢀ1,5ꢀpentanediol 5ꢀTBDPS
ether (6’) (Scheme 2).38 Each reaction proceeded without problem, resulting in
1
comparable yields. A comparison of the HꢀNMR spectra of these compounds revealed
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that the 1H signals of the natural biakamide C (3) showed good accordance with those of
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the (4R, 6S)ꢀisomer, whereas some discrepancies were observed with those of the (4R,
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6R)ꢀisomer, typically at Hꢀ6 and Hꢀ7 signals (Figure 7). Furthermore, the specific
rotation ([α]D = –17.2) of the (4R, 6S)ꢀisomer matched well with that ([α]D = –18.0) of
the natural biakamide C (3). Similar results were obtained in the case of biakamide D
(4) (data not shown). Therefore, the absolute stereostructures both of biakamides C (3)
and D (4) were unambiguously determined to be (4R, 6S), as shown in Figure 1.
Scheme 2. Synthesis of (4R,6R)ꢀBiakamides C (3’) and D (4’).
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Figure 7. Comparison of 1H NMR Spectra of Natural biakamide C (3) and Synthetic
(4R,6S)ꢀ3 or (4R,6R)ꢀ3’.
Next, the synthesis of biakamides A (1) and B (2) was also investigated. The 23S
configuration for these two compounds was determined as described above. However, a
comparison of the NMR spectra and specific rotations between the four synthetic
stereoisomers [(4R, 6S)ꢀ, (4S, 6R)ꢀ, (4S, 6S)ꢀ, and (4R, 6R)ꢀ] and the natural biakamides
A (1) and B (2) is needed to finalize this stereochemical analysis.
In the same manner as described above, the (4S, 6R)ꢀ and (4S, 6S)ꢀisomers of the
amine 20 were synthesized starting from the (2S, 4S)ꢀ or (2R, 4S)ꢀcongeners of the
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2,4ꢀdimethylꢀ1,5ꢀpentanediol 5ꢀTBDPS ether, respectively. Condensation between
commercially available (S)ꢀ3ꢀhydroxybutyric acid and the respective isomer of the
amine 20 proceeded without problem, and subsequent HPLC separation afforded all
possible stereoisomers of biakamides A (1) and B (2) (Scheme 3).
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Scheme 3. Synthesis of All Possible Isomers of Biakamides A (1) and B (2).
Next, we compared the NMR spectra and specific rotations of the four
diastereomers with those of the natural biakamide A (1) (Figure 8). The potential (4S,
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6S) and (4R, 6R) configurations were easily excluded by considering the obvious
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differences in the chemical shifts of the HꢀNMR signals at Hꢀ6 and Hꢀ7 between the
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1
isomers and the natural biakamide A (1) (Figure 8A). In contrast, the HꢀNMR spectra
of the (4S, 6R)ꢀ and (4R, 6S)ꢀisomers were found to similar to each other throughout the
molecule. Fortunately, only the multiplicity of the signals at Hꢀ12 of the (4S, 6R)ꢀisomer
was different from that of the (4R, 6S)ꢀisomer and the natural biakamide A (1) (Figure
8B). In addition, the specific rotation of the natural biakamide A (1) ([α]D = +6.3) was
similar to that of the (4R, 6S)ꢀisomer ([α]D = +1.2), whereas a large value ([α]D = +33.4)
was observed in the case of the (4S, 6R)ꢀisomer.
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Figure 8. Comparison of 1H NMR Spectra of Natural biakamide A (1) and Synthetic
four isomers of 1.
Furthermore, crucial evidence was obtained from a comparison of the CD spectra
of these compounds. Thus, the CD spectrum of the (4S, 6R)ꢀisomer showed a positive
maximum at 210 nm derived from the α,βꢀunsaturated amide moiety, whereas both the
(4R, 6S)ꢀisomer and natural biakamide A (1) showed a negative maximum (Figure 9).
Similar data were obtained in the case of biakamide B (2) (data not shown). Thus, the
absolute stereochemistry both of biakamides A (1) and B (2) was found to be (4R, 6S),
which was similar to those of biakamides C (3) and D (4).
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Figure 9. Comparison of CD Spectra of Natural biakamide A (1) and Synthetic
(4R,6S)ꢀ1 or (4S,6R)ꢀ1.
Bioactivity of biakamides A–D (1–4)
Biakamides A–D (1–4) exhibited selective antiproliferative activity against PANCꢀ1
cells cultured under glucoseꢀdeficient conditions, in a concentrationꢀdependent manner
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(Table 2). Among the four compounds, biakamides C (3) and D (4) showed more potent
activity, with a halfꢀmaximal inhibitory concentration (IC50) of 0.5 µM. Additionally,
when used at a concentration of less than 30 µM, these biakamides showed almost no
toxicity against PANCꢀ1 cells cultured in normal medium containing 25 mM glucose.
These data indicated that these biakamides may be promising anticancer drugs and that
the anticancer effects of these compounds were realized by the targeting of cancer cells
adapted to a glucoseꢀdeficient microenvironment.
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Table 2. Growth inhibition of biakamides A (1) – D (4)
against PANCꢀ1 cells.
1
2
3
4
IC50
1.0
S.I.
ꢀ
IC50
4.0
S.I.
ꢀ
IC50
0.5
S.I.
ꢀ
IC50
0.5
S.I.
ꢀ
Glucose
(–)
Glucose
(+)
>100
>100 >100 >25
50
100
35
70
IC50 = µM; S.I = selective index: IC50 in Glucose (+) medium /
IC50 in Glucose (–) medium.
Next, we performed a mechanistic analysis of biakamides. Recent studies have
revealed that cancer cells adapt to nutrient starvation by activating the phosphoinositol
3ꢀkinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling pathway and
the unfolded protein response (UPR), such as induction of glucoseꢀregulated protein 78
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(GRP78). Moreover, inhibitors of mitochondrial function or GRP78 are known to
exhibit selective growthꢀinhibitory activity against cancer cells cultured under
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glucoseꢀdeprived
conditions.39,40
Indeed,
we
recently
revealed
that
3,4,5ꢀtribromoꢀ2ꢀ(2',4'ꢀdibromophenoxy)ꢀphenols exhibit antiproliferative activity
against PANCꢀ1 cells cultured under glucoseꢀstarved conditions through weak
inhibition of Akt phosphorylation and GRP78 expression. We clarified that the
antiproliferative activity of this compound may be mediated by inhibition of complex II
in the mitochondrial electron transport chain.19 We then analyzed the effect of
biakamides on these signaling pathways or mitochondrial function using western
blotting and Mito Check Complex Activity Assays (Cayman Chemical).
Western blot analysis revealed that Akt phosphorylation and GRP78 expression
were induced in PANCꢀ1 cells cultured in glucoseꢀdeficient medium in comparison with
those cultured in general glucose medium (Figure 10, lanes 1 and 2). Additionally,
biakamide C (3) weakly inhibited Akt phosphorylation and GRP78 expression in
PANCꢀ1 cells cultured in glucoseꢀdeficient medium (lanes 5 and 6). A similar
phenomenon was observed for antimycin A, a known antiꢀausterity agent that inhibits
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complex III in the mitochondrial electron transport chain (lanes 3 and 4).
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Furthermore, we found that biakamide C (3) selectively inhibited complex I in the
mitochondrial electron transport chain, with an IC50 of 0.45 µM. This inhibitory
concentration was similar to the IC50 of the antiproliferative activity of 3 against
PANCꢀ1 cells cultured under glucoseꢀdeficient conditions, as shown in Table 3. We
also found that rotenone, a known inhibitor of complex I, also exhibited selective
growth inhibitory activity against PANCꢀ1 cells cultured under glucoseꢀdeficient
conditions and showed inhibitory properties similar to those observed for Akt and
GRP78 (Figure S1, Supporting Information). These results strongly implied that
biakamide C (3) showed selective growth inhibitory activity against cancer cells
adapted to glucoseꢀdeficient conditions through inhibition of complex I in the
mitochondrial electron transport chain.
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Figure 10. Effect of biakamide C (3) on the Akt signaling and induction of GRP78 by
western blot analysis. The PANCꢀ1 cells cultured in the Glucose Deficient Medium
(Glucose (—)) or General Glucose Medium (Glucose (+)) were treated with biakamide
C (3) or antimycin A. Cell lysate was resolved by using SDSꢀPAGE and detected with
antibodies against the indicated proteins.
Table 3. Effect of biakamide C (3) on the mitochondrial electron transfer chain.
IC50 value (µM)
complex I
0.45
complex II
>100
complex II/III
complex IV
>100
complex V
37
biakamide C (3)
>100
positive control a
0.13
18
0.003
7.7
0.36
a Compounds used as positive control are rotenone, thenoyltrifluoroacetone,
antimycin A, KCN and oligomycin for complex I, II, III, IV and V, respectively.
Conclusion
Based on the constructed bioassay, we isolated four novel polyketides, designated
biakamides A–D (1–4), from the Indonesian marine sponge (Petrosaspongia sp.), as
selective growth inhibitors against the PANCꢀ1 human pancreatic cancer cells cultured
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