Structure, synthesis, and biological activity of a C-20 bisacetylenic alcohol from a marine sponge Callyspongia sp.

Article abstract of DOI:10.1021/np400297p

An optically inactive C-20 bisacetylenic alcohol, (4E,16E)-icosa-4,16- diene-1,19-diyne-3,18-diol, was isolated from a marine sponge Callyspongia sp. as a result of screening of antilymphangiogenic agents from marine invertebrates. An optical resolution using chiral-phase HPLC gave each enantiomer, (-)-1 and (+)-2. Because the natural and synthetic enantiomers 1 and 2 showed different biological properties, we investigated the structure-activity relationships of bisacetylenic alcohols using 11 synthetic derivatives, and it is clarified that the essential structural unit for antiproliferative activity is the "1-yn-3-ol" on both termini and that there is a minimum chain length that connects the "1-yn-3-ol" moieties.

Full text of DOI:10.1021/np400297p

Article
pubs.acs.org/jnp
Structure, Synthesis, and Biological Activity of a C‑20 Bisacetylenic
Alcohol from a Marine Sponge Callyspongia sp.
Takayuki Shirouzu,† Kousuke Watari,‡ Mayumi Ono,‡ Keiichi Koizumi,§ Ikuo Saiki,§ Chiaki Tanaka,†
Rob W. M. van Soest,⊥ and Tomofumi Miyamoto*,†
‡
†Department of Natural Products Chemistry and Department of Pharmaceutical Oncology, Graduate School of Pharmaceutical
Sciences, Kyushu University, Maidashi 3-1-1, Higashi-ku, Fukuoka 812-8582, Japan
§Division of Pathogenic Biochemistry, Institute of Natural Medicine, Toyama University, 2630 Sugitani, Toyama 930-0194, Japan
⊥Department of Marine Zoology, Naturalis Biodiversity Center, P.O. Box 9517, 2300 RA Leiden, The Netherlands
S
* Supporting Information
ABSTRACT: An optically inactive C-20 bisacetylenic alcohol,
(4E,16E)-icosa-4,16-diene-1,19-diyne-3,18-diol, was isolated from a
marine sponge Callyspongia sp. as a result of screening of
antilymphangiogenic agents from marine invertebrates. An optical
resolution using chiral-phase HPLC gave each enantiomer, (−)-1 and
(+)-2. Because the natural and synthetic enantiomers 1 and 2 showed different biological properties, we investigated the
structure−activity relationships of bisacetylenic alcohols using 11 synthetic derivatives, and it is clarified that the essential
structural unit for antiproliferative activity is the “1-yn-3-ol” on both termini and that there is a minimum chain length that
connects the “1-yn-3-ol” moieties.
ecently it has become clear that the interaction between
cancer cells in a primary tumor and the surrounding
for antilymphangiogenic activity. Among them, the extract of a
Callyspongia sp. marine sponge showed a selective inhibitory
effect on TR-LE proliferation. This extract was submitted to
bioassay-guided separation. The Et2O-soluble part of the EtOH
extract of this sponge was separated by a Sephadex LH-20
column (CHCl3) to give an active compound. As the specific
rotation of this active compound was zero, and two
diastereomeric Mosher’s esters [(S)-MTPA-1 and (S)-MTPA-
2] were obtained from this active compound, an optical
resolution using chiral-phase HPLC was applied to give a new
bisacetylenic alcohol (1) and its enantiomer (2).
R
microenvironment plays an important role in tumor pro-
gression, dissemination, and metastasis.1 The lymphatic system
provides the major route for tumor dissemination to regional
lymph nodes, which serve as a clinical indicator of tumor
metastasis. Metastasis of tumor cells is the primary reason for
cancer deaths, and with few exceptions all cancers can
metastasize.2 Primary tumors and cells in the tumor micro-
environment have been found to secrete growth factors that can
induce the formation of new lymphatic vessels called
lymphangiogenesis.3 Blocking lymphangiogenesis may be an
effective way to reduce regional lymph node dissemination and
to improve patient survival rates. In the course of our
continuing research on new antilymphangiogenic agents from
natural resources,4 we isolated an optically inactive bisacetylenic
alcohol that showed an inhibitory effect on the proliferation of
temperature-sensitive rat lymphatic endothelial (TR-LE) cells5
using bioassay-guided separation. Polyacetylenes might be
ubiquitous sponge metabolites, and more than 300 poly-
acetylenes have been isolated from marine sponges of the
genera Petrosia, Xestospongia, Strongylophora, etc. Furthermore,
polyacetylenes have exhibited diverse biological activities, such
as antitumor, antimicrobial, and antiviral activities.6 Because of
the wide spectrum of biological activities of polyacetylenes,
total syntheses of them have been achieved by several groups.7
In this paper, we will describe the isolation, structure, synthesis,
and biological activities of acetylenic alcohols.
Bisacetylenic alcohol (1) was obtained as a white, amorphous
solid, and the specific rotation was found to be −30.
Spectroscopic analysis was done using the racemic compound.
The IR spectrum showed absorption bands due to hydroxy
(3462 cm−1) and alkyne (2358 cm−1) moieties. High-resolution
electrospray ionization time-of flight mass spectrometry
1
(HRESITOFMS) gave a molecular formula of C20H30O2. H
and 13C NMR and HSQC spectroscopic data indicated the
presence of five aliphatic methylenes, one oxygenated methine,
one terminal alkyne, and one disubstituted E-olefin. Consider-
ing all of the spectroscopic information together, 1 was
indicated to be a symmetrical structure. The COSY and HMBC
spectra revealed the correlation from the terminal acetylene,
secondary alcohol, and E-olefin (4-en-1-yn-3-ol unit). This unit
bound to the alkyl chain at the end of the molecule as shown in
Figure 1. The gross structure of 1 was identical with the
bisacetylenic alcohol (2) reported by Braekman et al.8 By
RESULTS AND DISCUSSION
EtOH extracts prepared from 93 specimens of marine
invertebrates collected at Iriomote, Okinawa, were screened
■
Received: April 14, 2013
© XXXX American Chemical Society and
American Society of Pharmacognosy
A
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Journal of Natural Products
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Figure 1. COSY and HMBC correlations for 1.
Scheme 1. Synthesis of Bisacetylenic Alcohols 1, 2, and 3
1
means of the specific rotation and a comparison of the H
NMR chemical shifts of the (S)-MTPA esters with the
corresponding derivatives of Braekman’s acetylenic alcohol,
the structure of 1 was deduced to be (−)-(3R,4E,16E,18R)-
icosa-4,16-diene-1,19-diyne-3,18-diol, and 2 was
(+)-(3S,4E,16E,18S)-icosa-4,16-diene-1,19-diyne-3,18-diol.
The inhibitory effects of 1 and 2 on the proliferation of TR-
LE and HeLa cells were examined. Interestingly, the inhibitory
effect of 1 was higher than that of 2, with the IC50 values of 1
for both cell lines being nearly one-fourth those of 2. To
confirm the unique biological activities of these bisacetylenic
alcohols, each enantiomer (1 and 2) and the meso isomer (3)
were synthesized by reference to the synthesis of acetylenic
alcohols reported by Gung et al.9
Commercially available dodecane-1,12-diol was converted to
dodecanedial (4) by PCC oxidation with a 66% yield. Wittig
reaction of 4 with Ph3PCHCHO gave (E)-hexadecadienedial
(5) with a 53% yield. Then, 5 was reacted with
ethynylmagnesium bromide, providing the isometric mixture
of 1, 2, and 3 with an 89% yield, as shown in Scheme 1.
Enzymatic resolution by Lipase AK and vinyl acetate was
applied to the isometric mixture of 1, 2, and 3,10 which gave
diacetate 6, monoacetate 7, and optically active (3R,18R)-
bisacetylenic alcohol 1. (3S,18S)-Bisacetylenic alcohol 2 and
meso isomer 3 were prepared from the corresponding acetates,
Table 1. IC50 Values (μM) of Compounds 1−8 and 14−16
for Inhibition of TR-LE and HeLa Proliferation
IC50 (μM)
compound
TR-LE
0.11
HeLa
a
1 (−)-natural
1 (−)-synthetic
2 (+)-natural
2 (+)-synthetic
3 (meso)
nt
0.35 ( 0.13)
0.47
5.3 ( 1.1)
nt
1.5 ( 0.29)
1.3 ( 0.16)
1.3
18 ( 5.0)
5.0 ( 2.2)
6 (diAc of 2)
7 (monoAc of 3)
8 (diAc of 1)
14 (C-16)
9.5
1
respectively. The H NMR spectra of natural and synthetic
bisacetylenic alcohol 1 were identical.
0.28
2.7
0.11
9.8
To elucidate the structure−activity relationship of the
bisacetylenic alcohol, nine related derivatives, namely, the
diacetate of 1 (8), the dibenzoate of 1 (9), the mono- and
dioxo derivatives (10, 11), monoacetylenic derivatives (E)-
undec-4-en-1-yn-3-ol (12) and (E)-undec-4-en-1-yn-3-one
(13), the shortened (C-16) and elongated (C-24) derivatives,
hexadeca-4,12-diene-1,15-diyne-3,14-diol (14) and tetracosa-
4,20-diene-1,23-diyne-3,22-diol (15), and the tetrahydro
derivative, icosa-1,19-diyne-3,18-diol (16), were prepared.
The inhibitory effects of the synthetic derivatives on the
proliferation of TR-LE and HeLa cells were examined. As
shown in Table 1, the IC50 values of the synthetic enantiomers
1 and 2 showed relatively weak activity compared with the
9.4
nt
15 (C-24)
0.37
nt
16 (tetrahydro C-20)
mix. 1 (C-20)
paclitaxel
0.17
nt
0.60
nt
0.018
0.0017
0.0043
vincristine
0.080
a
nt: not tested.
natural products. However, both natural and synthetic products
showed selective inhibition of TR-LE cell proliferation. The
inhibitory effect of the (−)-(3R,18R) alcohol (1; IC50 0.35 μM)
was four times higher than that of the (+)-(3S,18S) alcohol (2;
B
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Figure 2. Effects of bisacetylenic alcohols (1, 2) on capillary-like tube formation of TR-LE cells. TR-LE cells were seeded on Matrigel-precoated 96-
well plates, in the presence of 1 and 2. Morphological changes of TR-LE cells were observed and photographed with a phase-contrast microscope
(40×).
Figure 3. FACS data of cell cycle parameters of TR-LE and HeLa cells after treatment with 1. (A) Representative cell cycle distribution of TR-LE
cells in the presence and absence of 0.3 μM 1 and (B) HeLa cells in the presence and absence of 5.0 μM 1 for the indicated time. Cells were
harvested, fixed, and stained with propidium iodide, respectively. Then 20 000 stained cells were subjected to FACScaliber analysis to determine the
distribution of cells throughout the sub G1, G0/G1, S, and G2/M phases.
IC50 1.5 μM), and the activity of the meso alcohol (3; IC50 1.3
μM) was nearly identical with that of 2. The activities of acetyl
derivatives 6, 7, and 8 were slightly increased compared with
the corresponding alcohols (2, 3, 1); however, the benzoyl
derivative 9 of 1 did not show any activity at a concentration of
33 μM. The dioxo derivative 11 was not active, while the
mono-oxo derivative 10 showed a weak inhibitory effect at a
concentration of 10 μM (viability was 78% of control). The
monoacetylenic alcohol (12) and ketone (13) did not show
any activity at 100 μM. Interestingly, the activity of the
shortened alkyl chain derivative (14; IC50 9.4 μM) was
decreased, but the elongated derivative 15 (IC50 0.37 μM)
and the tetrahydro derivative 16 (IC50 0.17 μM) showed
increased activity when compared with the corresponding
alcohol mixture of 1, 2, and 3 (IC50 0.60 μM). The above data
suggest that the essential structural unit for activity is the “1-yn-
3-ol” on both termini and that there is a minimum alkyl chain
length that connects the two “1-yn-3-ol” moieties.
To further investigate the effects of the bisacetylenic alcohols
on lymphangiogenesis, the inhibitory effect on capillary-like
tube formation of TR-LE cells was examined. The bisacetylenic
alcohols 1 and 2 inhibited tube formation, resulting in
shortened capillary-like tubes, as shown in Figure 2. However,
this activity was judged to be irrelevant, because the IC50 values
of 1 and 2 were estimated to be 10.3 and 8.7 μM, and these
concentrations are appreciably higher than the IC50 values for
inhibition of TR-LE proliferation.
Next, the effect of bisacetylenic alcohol 1 on TR-LE and
HeLa cell cycle parameters was examined using flow cytometric
analysis. As shown in Figure 3, the percentage of G2/M phase
cells increased slightly, while the percentage of S phase cells did
not change. This indicated that the inhibitory effect of
bisacetylenic alcohol 1 on the proliferation of TR-LE cells is
not due to cell cycle arrest.
In summary, an optically inactive C-20 bisacetylenic alcohol
was isolated from a Callyspongia sp. marine sponge. A chiral-
ph ase s eparation provided the new alcohol
(−)-(3R,4E,16E,18R)-icosa-4,16-diene-1,19-diyne-3,18-diol (1)
together with the known (+)-enantiomer (2). Biological studies
of the acetylenic alcohols and their synthetic derivatives
revealed the “1-yn-3-ol” on both ends is the essential structural
unit responsible for the activity. Jung et al. reported that the
polyacetylenic alcohol dideoxypetrosynol A, which was isolated
from a Petrosia sp. marine sponge inhibited DNA cleavage by
topoisomerase I and showed cytotoxicity against several human
tumor cell lines.11 We prepared a fluorescent derivative of 1 and
analyzed its intracellular localization. We found that the
fluorescent signal was not localized in the nucleus (unpublished
data). From the results of this study, we propose a mechanism
of action involving the stabilization of protein dimers in the
cytoplasm rather than a DNA duplex. Further studies on the
chemical biology of these bisacetylenic alcohols to identify the
target molecule are in progress.
C
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1
(S)-MTPA-2: white powder; H NMR (CDCl3, 600 MHz) δ 1.23
(12H, brs, H-8-H-13), 1.34 (4H, m, H-7 and H-14), 2.03 (4H, q, J =
7.0, H-6 and H-15), 2.60 (2H, d, J = 1.0, H-1 and H-20), 3.57 (6H, s,
−OCH3), 5.48 (2H, dd, J = 7.0, 15.5, H-4 and H-17), 5.94 (2H, dt, J =
8.0, 15.5, H-5 and H-16), 6.01 (2H, d, J = 6.5, H-3 and H-18), 7.34
(6H, m, phenyl), 7.51 (4H, d, J = 6.5, phenyl).
EXPERIMENTAL SECTION
■
General Experimental Procedures. Optical rotations were
measured with a Jasco DIP-370 digital polarimeter at 24 °C. IR
spectra were measured with a Jasco FT/IR-410 spectrophotometer.
NMR spectra were recorded on a Varian INOVA 600 or 400 operating
1
at 600 MHz (400 MHz) for H and 150 MHz (100 MHz) for 13C in
(−)-(3R,4E,16E,18R)-Icosa-4,16-diene-1,19-diyne-3,18-diol (1):
CDCl3. 1H and 13C NMR chemical shifts are reported in ppm (δ) and
referenced to the solvent signal (CDCl3: δH = 7.24, δC = 77.2).
HRESITOFMS spectra were measured with a Bruker micrOTOF mass
spectrometer. Chromatographic separations were carried out using
Sephadex LH-20 (Amersham Biosciences) and silica gel (Merck silica
gel 60 F254). Chiral-phase HPLC (Chiralcel OD, 250 × 4.6 mm i.d.,
Daicel) was carried out with a Jasco PU-980 Intelligent HPLC pump
and 875-UV detector. Thin-layer chromatography (TLC) analyses
white, amorphous powder; [α]24 −30.0 (c 0.13, MeOH); IR
D
(CHCl3) νmax 3432(br), 2947, 2358(s), 1624, 1310 cm−1; H NMR
1
(CDCl3, 600 MHz) δ 1.25 (12H, brs, H-8−H-13), 1.38 (4H, quint, H-
7 and H-14), 2.05 (4H, q, J = 7.0, H-6 and H-15), 2.54 (2H, d, J = 2.0,
H-1 and H-20), 4.82 (2H, d, J = 6.0, H-3 and H-18), 5.61 (2H, dd, J =
6.0,15.5, H-4 and H-17), 5.90 (2H, dt, J = 8.0, 15.5, H-5 and H-16);
HRESITOFMS m/z 325.2091 [M + Na]+ (calcd for C20H30NaO2,
325.2138).
were carried out using Merck precoated TLC plates, silica gel 60 F254
or RP-8 F254s
,
(+)-(3S,4E,16E,18S)-iIcosa-4,16-diene-1,19-diyne-3,18-diol (2):
white, amorphous powder; [α]24 +24.5 (c 0.10, MeOH), lit.8
.
D
[α]20 +26 (c 1, MeOH); HRESITOFMS m/z 325.2135 [M + Na]+
Collection, Extraction, and Isolation. Callyspongia sp. (wet
weight 93.45 g) was collected by hand at a depth of 10 m off the shore
of Iriomote Island, Okinawa Prefecture, Japan, in June 2009. The
sponge is a mottled blue and light blue-gray lobate mass growing
underneath and among the branches of stony corals. It is an unusual
Callyspongia (Callyspongiidae) because it is soft, and the skeleton is
largely unispicular, mimicking sponges of the genera Chalinula or
Haliclona. The lobes are mostly blind-ending with uncommon
depressed oscules of approximately 3 mm diameter between the
lobes. Rarely lobes may have an apical oscule. At the surface the
skeleton is arranged in rather irregular polygonal meshes of
approximately 200−300 μm in diameter made by aligned spicules
and subdivided by single spicules. The choanosomal skeleton is
likewise largely unispicular, without clearly developed multispicular
tracts. Spongin is present only at the nodes. Spicules are straight or
slightly curved oxeas of 70−95 × 1.2−3 μm. This combination of
characters is not found in the literature, precluding further
identification to species level. A voucher fragment is deposited in
the collections of the Naturalis Biodiversity Center at Leiden, under
reg. no. ZMA Por. 22115. The sample was stored at −20 °C for one
week before use. The sponge was homogenized and extracted with
EtOH (2 × 0.6 L) and filtrated. The extract was evaporated in vacuo,
and the resulting aqueous suspension was diluted with H2O (0.5 L)
and extracted with Et2O (3 × 0.5 L) and n-BuOH (3 × 0.5 L). The
organic layers were evaporated to give an Et2O extract (225.4 mg) and
an n-BuOH extract (3.61 g). The active Et2O extract was subjected to
Sephadex LH-20 column chromatography with CHCl3 to give nine
fractions, and fraction 6 (13.2 mg) was active and spectroscopically
pure. Part of fraction 6 (6.4 mg) was applied to chiral-phase HPLC
(Chiralcel OD) with 2-propanol/n-hexane (7:93) to give 1 (1.9 mg)
and 2 (1.5 mg).
D
(calcd for C20H30NaO2, 325.2138).
Synthesis of a Mixture of 1, 2, and 3. To a solution of
dodecanediol (1.01 g, 5.0 mmol, TCI Co .Ltd.) in CH2Cl2 (40 mL)
stirring at rt under argon were added PCC (pyridinium chlorochro-
mate) (3.25 g, 15 mmol, Sigma-Aldrich) and MS4A (5.0 g. After
stirring for 1 h, the reaction mixture was filtered through a pad of silica.
The filtrate was evaporated and purified by silica gel column
chromatography (n-hexane/EtOAc, 10:1) to give dodecanedial (4,
657.7 mg, 3.32 mmol, 66.4%). To a dry 100 mL flask charged with
(triphenylphosphoranyldiene)acetaldehyde (3.51 g, 11.5 mmol, TCI
Co. Ltd.) was added a CH2Cl2 (40 mL) solution of 4 (501.1 mg, 2.5
mmol). The reaction mixture was refluxed at 110 °C for 12 h under
argon, then filtered. The filtrate was evaporated and purified by silica
gel (n-hexane/EtOAc, 10:1) and RP-8 (85% MeOH/H2O) column
chromatography to give (2E,14E)-hexadecadienedial (5, 343.9 mg,
1.38 mmol, 53.2%). To a solution of 5 (259.3 mg, 1.04 mmol) in THF
(10 mL) stirring at 0 °C under argon was added 0.5 M
ethynylmagnesium bromide in THF (10.4 mL, 5.2 mmol, Sigma-
Aldrich) dropwise over 10 min. After stirring for 30 min at rt, the
reaction was quenched with 5% HCl, then extracted with Et2O. The
Et2O layer was washed with saturated NaHCO3 and NaCl solution.
The Et2O layer was dried (Na2SO4), filtered, and concentrated. The
crude product was purified by silica gel column chromatography (n-
hexane/EtOAc, 4:1) to yield a mixture of 1, 2, and 3 (278.9 mg, 0.924
mmol, 88.8%).
1,12-Dodecanedial (4): colorless oil; IR (CHCl3) νmax 2930, 2857,
1
1719(s), 1410 cm−1; H NMR (CDCl3, 400 MHz) δ 1.27 (12H, br),
1.60 (4H, quint, J = 7.2 Hz), 2.39 (4H, dt, J = 2.0, 7.0 Hz), 9.74 (2H, t,
J = 2.0 Hz); 13C NMR (CDCl3, 100 MHz) δ 22.0 (CH2), 29.1 (CH2),
29.7 (CH2), 43.9 (CH2), 202.8 (C); HRESITOFMS m/z 221.1524
[M + Na]+ (calcd for C12H22NaO2, 221.1512).
(2E,14E)-Hexadeca-2,14-diene-1,16-dial (5): yellow oil; IR
(CHCl3) νmax 2930, 2856, 1685(s) cm−1; 1H NMR (CDCl3, 400
MHz) δ 1.26 (12H, br), 1.48 (4H, quint, J = 7.0 Hz), 2.31 (4H, dt, J =
6.5, 7.0 Hz), 6.09 (2H, dd, J = 8.0, 15.6 Hz), 6.82 (2H, dt, J = 6.5, 15.5
Hz), 9.48 (2H, d, J = 8.0 Hz); 13C NMR (CDCl3, 100 MHz) δ 27.8
(CH2), 29.1 (CH2), 29.3 (CH2), 29.4 (CH2), 32.7 (CH2), 133.0
(CH), 158.9 (CH), 194.1 (C); HRESITOFMS m/z 273.1832 [M +
Na]+ (calcd for C16H26NaO2, 273.1825).
(4E,16E)-Icosa-4,16-diene-1,19-diyne-3,18-diol (mixture of 1, 2,
and 3): white powder; IR (CHCl3) νmax 3462(br), 2941, 2358(s),
2856, 1678(w), 1616(m), 1312(m) cm−1; 1H NMR (CDCl3, 400
MHz) δ 1.25 (12H, br), 1.37 (4H, m), 2.05 (4H, dt, J = 6.5, 7.6 Hz),
2.54 (2H, d, J = 2.0 Hz), 4.82 (2H, brs), 5.59 (2H, dd, J = 6.0, 15.2
Hz), 5.90 (2H, dt, J = 6.5, 15.2 Hz); 13C NMR (CDCl3, 100 MHz) δ
28.8 (CH2), 29.1 (CH2), 29.4 (CH2), 29.5 (CH2), 31.9 (CH2), 62.8
(CH), 73.9 (CH), 83.4 (C), 128.4 (CH), 134.6 (CH); HRESI-
TOFMS m/z 325.2163 [M + Na]+ (calcd for C20H30NaO2, 325.2138).
Enzymatic Resolution of 1, 2, and 3. A flask was charged with
lipase AK Amano (60 mg, Wako Pure Chemicals), MS4A (60 mg),
vinyl acetate (0.1 mL, 1.16 mmol, Wako), and the mixture of 1, 2, and
3 (30.0 mg, 0.099 mmol) in CH2Cl2 (4 mL). The mixture was stirred
Fraction 6: white amorphous powder; [α]25D 0 (c 0.6, MeOH); 13
C
NMR (CDCl3, 600 MHz) δ 28.8 (CH2, C-7 and C-14), 29.1 (CH2, C-
8 and C-13), 29.4 (CH2, C-9 and C-12), 29.5 (CH2, C-10 and C-11),
31.9 (CH2, C-6 and C-15), 62.8 (CH, C-3 and C-18), 73.9 (CH, C-1
and C-20), 83.4 (C, C-2 and C-19), 128.4 (CH, C-4 and C-17), 134.6
(CH, C-5 and C-16).
Preparation of the (S)-MTPA Esters of Fraction 6. To a
solution of fraction 6 (1.9 mg, 6.3 μmol) in CH2Cl2 (1 mL) stirring at
room temperature (rt) were added (S)-MTPA (16.3 mg, 66.3 μmol)
and DCC (14.7 mg, 71.2 μmol). After stirring for 1 h, the reaction was
terminated with H2O and extracted with EtOAc. The concentrated
EtOAc layer was subjected to reversed-phase HPLC (Cosmosil
5C18AR-II) with 90% MeOH/H2O to give two MTPA esters, (S)-
MTPA-1 (1.1 mg) and (S)-MTPA-2 (0.9 mg), respectively.
1
(S)-MTPA-1: white powder; H NMR (CDCl3, 600 MHz) δ 1.24
(12H, brs, H-8-H-13), 1.37 (4H, m, H-7 and H-14), 2.06 (4H, q, J =
7.0, H-6 and H-15), 2.56 (2H, d, J = 2.0, H-1 and H-20), 3.53 (6H, s,
−OCH3), 5.58 (2H, dd, J = 7.0, 15.5, H-4 and H-17), 5.99 (2H, d, J =
5.5, H-3 and H-18), 6.05 (2H, dt, J = 8.0, 15.5, H-5 and H-16), 7.38
(6H, m, phenyl), 7.51 (4H, d, J = 6.5, phenyl); HRESITOFMS m/z
757.2959 [M + Na]+ (calcd for C40H44F6NaO6, 757.2934).
D
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Journal of Natural Products
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at rt for 96 h. The reaction was monitored by TLC. When the amount
of diacetate was about the same as that of the mixture, the reaction was
terminated. The reaction mixture was filtered, then separated by silica
gel chromatography (n-hexane/EtOAc, 9:1 → 4:1 → 2:1) to yield the
diacetate (6, 9.4 mg, 0.024 mmol, 24.2%), monoacetate (7, 11.3 mg,
0.033 mmol, 33.3%), and (−)-diol (1, 3.6 mg, 0.012 mmol, 12.1%).
The monoacetate (7) or diacetate (6) was dissolved in EtOH (2 mL)
and treated with K2CO3 (5 mg) at rt for 4 h. The reaction mixtures
were diluted with H2O, then extracted with Et2O. Et2O layers were
evaporated and purified by silica gel (n-hexane/EtOAc, 3:1) to yield
(+)-diol 2 (3.2 mg, 0.011 mmol, 45.8% from 6) and meso-3 (9.8 mg,
0.032 mmol, 97.0% from 7), respectively.
Syntheses of 10 and 11. To a solution of the mixture of 1, 2, and 3
(50.0 mg, 0.17 mmol) in CH2Cl2 (4 mL) stirring at rt under argon
were added PCC (108 mg, 0.50 mmol) and MS4A (250 mg). After
stirring for 1 h, the reaction mixture was filtrated through a pad of
silica. The filtrate was evaporated and purified by silica gel column
chromatography (n-hexane/EtOAc, 5:1) to give mono-oxo derivative
10 (8.3 mg, 0.028 mmol, 16.7%) and dioxo derivative 11 (6.8 mg,
0.023 mmol, 13.7%).
(4E,16E)-18-Hydroxyicosa-4,16-diene-1,19-diyn-3-one (10): yel-
low powder; UV (n-hexane) λmax 247.0 nm; IR (CHCl3) νmax
3019(s), 2930(s), 2360(s), 1685(s) cm−1; 1H NMR (CDCl3, 400
MHz) δ 1.25 (12H, brs), 1.37 (2H, m),1.48 (2H, m), 2.04 (4H, dt, J =
6.5, 14.0 Hz), 2.28 (2H, dd, J = 6.5, 13.5 Hz), 2.54 (1H, d, J = 2.5 Hz),
3.19 (1H, s), 4.81 (1H, d, J = 5.5 Hz), 5.59 (1H, dd, J = 6.5, 15.5 Hz),
5.89 (1H, dt, J = 6.5, 15.5 Hz), 6.16 (1H, d, J = 15.5 Hz), 7.22 (1H, dt,
J = 6.5, 15.5 Hz); 13C NMR (CDCl3, 100 MHz) δ 27.8, 28.8, 29.1,
29.3, 29.5, 31.9, 32.7, 62.8, 73.9, 78.8, 128.4, 131.9, 134.5, 155.8, 178.3;
HRESITOFMS m/z 323.1982 [M + Na]+ (calcd for C20H28NaO2,
323.1982).
Diacetate 6: colorless oil; [α]24 +27.5 (c 0.49, CHCl3); IR
D
1
(CHCl3) νmax 2928(s), 2359(s), 1733(s), 1540(w), 1237 cm−1; H
NMR (CDCl3, 400 MHz) δ 1.24 (12H, br), 1.37 (4H, m), 2.05 (4H,
m), 2.07 (6H, s), 2.53 (2H, d, J = 2.0 Hz), 4.82 (2H, brs), 5.52 (2H,
dd, J = 6.5, 15.5 Hz), 5.80 (2H, d, J = 6.5 Hz), 5.99 (2H, dt, J = 6.5,
15.5 Hz); 13C NMR (CDCl3, 100 MHz) δ 21.0, 28.6, 29.1, 29.4, 32.0,
64.0, 74.6, 79.9, 124.3, 137.2, 169.6; HRESITOFMS m/z 409.2343 [M
+ Na]+ (calcd for C24H34NaO4, 409.2349).
(4E,16E)-Icosa-4,16-diene-1,19-diyne-3,18-dione (11): yellow
powder; UV (n-hexane) λmax 242.8 nm; IR (CHCl3) νmax 2930(s),
Monoacetate 7: colorless oil; [α]24 +11.0 (c 0.52, CHCl3). IR
D
(CHCl3) νmax 3306(s), 2929(s), 2358(s), 1733(s), 1237 cm−1; 1H
NMR (CDCl3, 400 MHz) δ 1.25 (12H, br), 1.37 (4H, m), 2.05 (4H,
m), 2.07 (3H, s), 2.53 (2H, d, J = 2.0 Hz), 4.81 (1H, brs), 5.51 (1H,
dd, J = 6.5, 15.5 Hz), 5.59 (1H, dd, J = 5.5, 15.5 Hz), 5.81 (1H, d, J =
5.5 Hz), 5.89 (1H, dt, J = 7.0, 15.5 Hz), 5.99 (1H, dt, J = 7.0, 15.5 Hz);
13C NMR (CDCl3, 100 MHz) δ 21.4, 28.9, 29.1, 29.4, 29.5, 29.7, 29.8,
32.2, 32.3, 63.1, 64.4, 74.2, 74.9, 80.2, 83.7, 124.6, 128.7, 134.,8, 137.5,
170.0; HRESITOFMS m/z 367.2242 [M + Na]+ (calcd for
C22H32NaO3, 367.2244).
1
2360(s),1719(s) cm−1; H NMR (CDCl3, 400 MHz) δ 1.26 (12H,
brs), 1.49 (4H, m), 2.28 (4H, dt, J = 6.5, 13.5 Hz), 3.19 (2H, s), 6.16
(2H, d, J = 16.5 Hz), 7.22 (2H, dt, J = 6.5, 16.5 Hz); 13C NMR
(CDCl3, 100 MHz) δ 27.8, 29.1, 29.3, 29.4, 32.6, 79.8, 131.9, 155.7,
178.1; HRESITOFMS m/z 321.1827 [M + Na]+ (calcd for
C20H26NaO2, 321.1825).
Syntheses of 12 and 13. To a solution of 0.5 M ethynylmagnesium
bromide in THF (2.5 mL, 2.5 mmol) stirring at 0 °C under argon was
added dropwise (E)-non-2-enal (70.0 mg, 2.5 mmol, TCI Co. Ltd.)
over 10 min. After stirring for 1 h at rt, the reaction was quenched with
5% HCl, then extracted with Et2O. The Et2O layer was washed with
saturated NaHCO3 and NaCl solution. The Et2O layer was dried
(MgSO4), filtered, and concentrated. The crude product was purified
with silica gel column chromatography (petroleum ether/Et2O, 5:1) to
yield 12 (254.9 mg, 1.5 mmol, 75.0%). To a solution of 12 (344.7 mg,
2.1 mmol) in CH2Cl2 (20 mL) stirring at rt under argon were added
PCC (1.08 g, 5.0 mmol), NaOAc (300 mg), and MS4A (15 g). After
stirring for 3 h, the reaction mixture was filtrated through a pad of
silica. The filtrate was evaporated and purified by silica gel column
chromatography (petroleum ether/Et2O, 10:1) to yield 13 (129.3 mg,
0.79 mmol, 37.6%).
(E)-Undec-4-en-1-yn-3-ol (12): colorless oil; IR (CHCl3) νmax
3306(s), 2929(s), 2353(s) cm−1; 1H NMR (CDCl3, 600 MHz) δ
0.88 (3H, t, J = 6.9 Hz), 1.30 (6H, brs), 1.40 (2H, m), 2.07 (2H, q, J =
6.5 Hz), 2.56 (1H, d, J = 2.5 Hz), 4.83 (1H, brs), 5.61 (1H, dd, J = 6.0,
15.5 Hz), 5.91 (1H, dt, J = 6.0, 15.5 Hz); 13C NMR (CDCl3, 100
MHz) δ 14.0, 22.5, 28.8, 28.8, 31.6, 31.9, 62.7, 73.9, 83.4, 128.4, 134.5;
HRESITOFMS m/z 189.1255 [M + Na]+ (calcd for C11H18NaO,
189.1250).
(-)-1 (synthetic): white powder; [α]25 −30.4 (c 0.24, MeOH);
D
HRESITOFMS m/z 325.2152 [M + Na]+ (calcd for C20H30NaO2,
325.2138).
(+)-2 (synthetic): white powder; [α]25 +29.6 (c 0.21, MeOH);
D
HRESITOFMS m/z 325.2137 [M + Na]+ (calcd for C20H30NaO2,
325.2138.
meso-3 (synthetic): white powder; [α]25 0 (c 0.67, MeOH);
D
HRESITOFMS m/z 325.2152 [M + Na]+ (calcd for C20H30NaO2,
325.2138).
Syntheses of Related Acetylenes: Syntheses of 8 and 9. To a
solution of (−)-1 (12.5 mg, 0.041 mmol) in pyridine (0.5 mL) stirring
at 75 °C was added Ac2O (0.5 mL). After stirring for 1 h, the reaction
mixture was poured into H2O, then extracted with Et2O. The organic
layer was dried with Na2SO4 and purified with silica gel
chromatography (n-hexane/EtOAc, 3:1) to yield diacetate 8 (10.6
mg, 0.027 mmol, 66.3%). To a solution of (−)-1 (14.0 mg, 0.046
mmol) in CH2Cl2 (5 mL) and pyridine (1 mL) stirring at rt was added
benzoyl chloride (0.4 mL). After stirring for 1 h, the reaction mixture
was poured into H2O, then extracted with Et2O. The organic layer was
washed with 5% HCl, saturated NaHCO3, and brine then dried with
Na2SO4. The concentrated organic layer was purified with RP-18 (90%
MeOH/H2O) to yield dibenzoate 9 (12.7 mg, 0.025 mmol, 54.1%).
(E)-Undec-4-en-1-yn-3-one (13): colorless oil; IR (CHCl3) νmax
2928(s), 2353(s), 1735(s) cm−1; 1H NMR (CDCl3, 400 MHz) δ 0.89
(3H, t, J = 7.1 Hz), 1.30 (6H, brs), 1.51 (2H, quint, J = 7.5 Hz), 2.31
(2H, q, J = 7.0 Hz), 3.22 (1H, s), 6.18 (1H, d, J = 15.5 Hz), 7.25 (1H,
dt, J = 7.0, 15.5 Hz); 13C NMR (CDCl3, 100 MHz) δ 14.0, 22.5, 27.8,
28.8, 31.5, 32.6, 78.8, 79.8, 131.9, 155.8, 177.8; HRESITOFMS m/z
187.1099 [M + Na]+ (calcd for C11H16NaO, 187.1093).
Diacetate 8: colorless oil; [α]25 −27.0 (c 0.71, CHCl3); IR
D
1
(CHCl3) νmax 2911(s), 2399(m), 1742(s), 1522(w), 1237 cm−1; H
NMR (CDCl3, 400 MHz) δ 1.24 (12H, br), 1.36 (4H, m), 2.06 (4H,
m), 2.07 (6H, s), 2.53 (2H, d, J = 2.0 Hz), 4.82 (2H, brs), 5.52 (2H,
dd, J = 6.5, 15.5 Hz), 5.80 (2H, d, J = 6.5 Hz), 5.99 (2H, dt, J = 6.5,
15.5 Hz); 13C NMR (CDCl3, 100 MHz) δ 20.7, 28.3, 28.8, 29.1, 29.2,
31.6, 63.7, 74.3, 118.7, 124.0, 136.8, 169.3; HRESITOFMS m/z
409.2336 [M + Na]+ (calcd for C24H34NaO4, 409.2345).
Syntheses of 14 and 15. Syntheses of 14 and 15 were achieved in a
manner similar to synthesis of the mixture of 1, 2, and 3. Octanediol
(365.0 mg, 2.5 mmol, TCI Co.Ltd.) for 14 or hexadecanediol (258.0
mg, 1.0 mmol) for 15 was used as starting material, and final
separation by silica gel chromatography yielded 14 (5.3 mg, 0.021
mmol, 0.84%) and 15 (40.3 mg, 0.13 mmol, 13.0%), respectively.
(4E,12E)-Hexadeca-4,12-diene-1,15-diyne-3,14-diol (14): white
powder; IR (CHCl3) νmax 3306(s), 3019(s), 2930(m), 2360(s),
Dibenzoate 9: colorless oil; [α]25 −22.3 (c 1.11, CHCl3); IR
D
(CHCl3) νmax 3307(s), 3019(s), 2929(s), 2360(s), 1717(s), 1540(w),
1262 cm−1; 1H NMR (CDCl3, 400 MHz) δ 1.24 (12H, br), 1.39 (4H,
m), 2.08 (4H, q, J = 7.0 Hz), 2.57 (2H, s), 5.64 (2H, ddd, J = 1.0, 6.5,
15.5 Hz), 6.06 (2H, m), 6.07 (2H, m), 7.42 (4H, t, J = 7.5 Hz), 7.55
(2H, t, J = 7.5 Hz), 8.05 (4H, d, J = 7.0 Hz); 13C NMR (CDCl3, 100
MHz) δ 28.9, 29.4, 29.7, 29.8, 32.3, 64.9, 75.1, 80.3, 124.7, 128.7,
130.2, 133.5, 137.6, 165.6; HRESITOFMS m/z 533.2702 [M + Na]+
(calcd for C34H38NaO4, 533.2662).
1
1652(w), 1540(w) cm−1; H NMR (CDCl3, 400 MHz) δ 1.29 (4H,
m), 1.38 (4H, m), 2.05 (4H, dt, J = 6.5, 7.5 Hz), 2.54 (2H, d, J = 2.5
Hz), 4.81 (2H, d, J = 5.0 Hz), 5.59 (2H, dd, J = 6.0, 15.5 Hz), 5.89
(2H, dt, J = 6.5, 15.5 Hz); 13C NMR (CDCl3, 100 MHz) δ 28.9, 29.1,
E
dx.doi.org/10.1021/np400297p | J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
32.0, 63.0, 74.1, 83.6, 128.7, 134.6; HRESITOFMS m/z 269.1543 [M
+ Na]+ (calcd for C16H22NaO2, 269.1512).
(4E,20E)-Tetracosa-4,20-diene-1,23-diyne-3,22-diol (15): white
ASSOCIATED CONTENT
■
S
* Supporting Information
1H and 13C NMR spectra of natural and synthetic bisacetylenic
alcohol (1) are available free of charge via the Internet at
http://pubs.acs.org.
powder; IR (CHCl3) νmax 3324(s), 3032(s), 2932(m), 2360(s)
1
cm−1; H NMR (CDCl3, 400 MHz) δ 1.23 (12H, br), 1.37 (4H,
br), 2.05 (4H, d, J = 6.5 Hz), 2.54 (2H, d, J = 1.0 Hz), 4.81 (2H, d, J =
4.5 Hz), 5.59 (2H, dd, J = 6.0, 15.5 Hz), 5.89 (2H, dt, J = 6.5, 15.5
Hz); 13C NMR (CDCl3, 100 MHz) δ 28.9, 29.1, 32.0, 63.0, 74.1, 83.6,
128.7, 134.6; HRESITOFMS m/z 381.2775 [M + Na]+ (calcd for
C24H38NaO2, 381.2764).
Synthesis of 16. To a solution of hexadecanediol (194.5 mg, 0.75
mmol, TCI Co. Ltd.) in CH2Cl2 (25 mL) stirring at rt under argon
were added PCC (488 mg, 2.25 mmol), NaOAc (150 mg), and MS4A
(0.5 g). After stirring for 4 h, the reaction mixture was filtered through
a pad of silica. The filtrate (185 mg) was dissolved in THF (10 mL);
then 0.5 M ethynylmagnesium bromide (10.4 mL, 5.2 mmol) was
added dropwise at 0 °C and stirred for 2 h at rt. The reaction mixture
was quenched with 5% HCl, then extracted with Et2O. The Et2O layer
was washed with a saturated NaHCO3 and NaCl solution. The Et2O
layer was dried (Na2SO4), filtered, and concentrated. The crude
product was purified by silica gel column chromatography (n-hexane/
EtOAc, 4:1) to yield 16 (29.6 mg, 0.097 mmol, 12.9%).
AUTHOR INFORMATION
Corresponding Author
*Tel: +81-92-642-6636. Fax: +81-92-642-6636. E-mail:
miyamoto@phar.kyushu-u.ac.jp.
■
Notes
The authors declare no competing financial interest.
REFERENCES
■
(1) Spano, D.; Zollo, M. Clin. Exp. Metastasis 2012, 29, 381−395.
(2) Peper, M. S. Clin. Cancer Res. 2001, 7, 462−468.
(3) Onimal, M.; Yonemitsu, Y. Front. Biosci. 2011, 3, 216−225.
(4) Jeong, D.; Watari, K.; Shirouzu, T.; Ono, M.; Koizumi, K.; Saiki,
I.; Kim, Y.-C.; Tanaka, C.; Higuchi, R.; Miyamoto, T. Biol. Pharm. Bull.
2013, 36, 152−157.
(5) Matsuo, M.; Koizumi, K.; Yamada, S.; Tomi, M.; Takahashi, R.;
Ueda, M.; Terasaki, T.; Obinata, M.; Hosoya, K.; Ohtani, O.; Saiki, I.
Cell Tissue Res. 2006, 326, 749−758.
Icosa-1,19-diyne-3,18-diol (16): white powder; IR (CHCl3) νmax
3306(s), 2927(m), 2360(s) cm−1; 1H NMR (CDCl3, 400 MHz) δ 1.24
(20H, brs), 1.44 (4H, m), 1.68 (4H, m), 2.43 (2H, s), 4.34 (2H, m);
13C NMR (CDCl3, 100 MHz) δ 24.7, 28.9, 29.2, 29.3, 37.3, 62.0, 72.5,
84.7; HRESITOFMS m/z 329.2439 [M + Na]+ (calcd for
C20H34NaO2, 329.2451).
(6) Jung, H. J.; Im, S. K.; Bae, H. B. Biomaterials from Aquatic and
Terrestrial Organisms; Fingerman, M.; Nagabhushanam, R., Eds.;
Science Publishers: NH, 2006; Chapter 12, pp 451−511.
(7) Gung, B. W. C. R. Chim. 2009, 12, 489−505.
Cell Culture and Cell Proliferation Assay. TR-LE cells were
donated from the library of Prof. I. Saiki and maintained on type I
collagen-coated cell culture dishes (Iwaki) in HuMedia-EB2 and
HuMedia-EG (Kurabo Industries Ltd.) supplemented with 10% fetal
bovine serum (FBS) at a permissive temperature (33 °C). Human
malignant epithelial cells (HeLa) were cultured in Eagle’s minimum
essential medium (EMEM) supplemented with 10% FBS kept in an
incubator at 37 °C in humidified air containing 5% CO2. FBS was
purchased from Nichirei Bioscience Inc. TR-LE cells (8 × 103 cells/
well) were seeded in Dulbecco’s modified Eagle’s medium (DMEM)
containing 10% FBS in type I collagen-coated 96-well cell culture
plates (Nippi Inc.). Cells were allowed to adhere for 3 h and then
grown with compounds at a range of final concentrations between 0.1
and 100 μM for an additional 48 h. Cell viability was determined by a
Cell-Titer 96 aqueous non-radioactive cell proliferation (MTS) assay
(Promega) according to the manufacturer’s protocol. HeLa cells (3 ×
103 cells/well) were seeded in EMEM containing 10% FBS in 96-well
plates and incubated for 24 h and subsequently grown with
compounds for an additional 48 h, and then the cell proliferation
assay was performed. In all experiments, the final concentration of
vehicle (DMSO) did not exceed 0.5% (v/v). Paclitaxel and vincristine
(Wako Pure Chemicals) were used for positive controls.
(8) Braekman, J. C.; Daloze, D.; Devijver, C.; Dubut, D.; van Soest,
R. W. M. J. Nat. Prod. 2003, 66, 871−872.
(9) (a) Gung, B. W.; Omollo, A. O. J. Org. Chem. 2008, 73, 1067−
1070. (b) Gang, B. W.; Dickson, H.; Shockley, S. Tetrahedron Lett.
2001, 42, 4761−4763.
(10) (a) Burgess, K.; Jennings, L. D. J. Am. Chem. Soc. 1991, 113,
6129−6139. (b) Burgess, K.; Jennings, L. D. J. Am. Chem. Soc. 1990,
112, 7434−7436.
(11) Kim, D.-K.; Lee, M.-Y.; Lee, H. S.; Lee, D. S.; Lee, J.-R.; Lee, B.-
J.; Jung, J. H. Cancer Lett. 2002, 185, 95−101.
Tube Formation Assay. Subconfluent TR-LE cells were harvested
with trypsin-EDTA and centrifuged at 1000 rpm for 5 min. A cell
suspension (2 × 104 cells/well) was prepared in 40 μL of DMEM
supplemented with 10% FBS in Matrigel-precoated 96-well plates
(Becton Dickinson Labware). After 5 h incubation at 37 °C, cells were
fixed by using glutaraldehyde and stained with hematoxylin.
Morphological changes were photographed at 40× magnification
with a phase-contrast microscope. The length of the capillary-like tube
structures was measured by hand. We used three photographs per well
and measured the length of about 10 tube structures.
Cell Cycle Analysis. TR-LE or HeLa cells were treated with 0.3 or
5.0 μM 1, respectively, and after 6 and 24 h incubation, cells were
harvested and stained with propidium iodide using the Cycle Test Plus
DNA Reagent kit (BD Biosciences) according to the manufacturer’s
recommendations. Cell distribution according to cell cycle phase was
determined by measuring the DNA content using a BD FACSCalibur
flow cytometer employing Cell Quest software. The percentage of cells
in the G0/G1, S, and G2/M phases was determined using Modifit LT
software (Verity Software House Inc.).
Statistical Analysis. Results are given as means SD.
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dx.doi.org/10.1021/np400297p | J. Nat. Prod. XXXX, XXX, XXX−XXX