Structure, Synthesis, and Biological Activity of a C-20 Bisacetylenic

Jul 15, 2013 - 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 Cal...
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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.

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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)-MTPA2] 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). 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 (HRESITOFMS) gave a molecular formula of C20H30O2. 1H 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. Considering 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

ecently it has become clear that the interaction between cancer cells in a primary tumor and the surrounding microenvironment plays an important role in tumor progression, 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 microenvironment 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 polyacetylenes 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.

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RESULTS AND DISCUSSION EtOH extracts prepared from 93 specimens of marine invertebrates collected at Iriomote, Okinawa, were screened © XXXX American Chemical Society and American Society of Pharmacognosy

Received: April 14, 2013

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Figure 1. COSY and HMBC correlations for 1.

Scheme 1. Synthesis of Bisacetylenic Alcohols 1, 2, and 3

means of the specific rotation and a comparison of the 1H 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-4,1 6-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 TRLE 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, respectively. The 1H NMR spectra of natural and synthetic bisacetylenic alcohol 1 were identical. 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 tetracosa4,20-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

Table 1. IC50 Values (μM) of Compounds 1−8 and 14−16 for Inhibition of TR-LE and HeLa Proliferation IC50 (μM)

a

compound

TR-LE

HeLa

1 (−)-natural 1 (−)-synthetic 2 (+)-natural 2 (+)-synthetic 3 (meso) 6 (diAc of 2) 7 (monoAc of 3) 8 (diAc of 1) 14 (C-16) 15 (C-24) 16 (tetrahydro C-20) mix. 1 (C-20) paclitaxel vincristine

0.11 0.35 (±0.13) 0.47 1.5 (±0.29) 1.3 (±0.16) 1.3 0.28 0.11 9.4 0.37 0.17 0.60 0.018 0.080

nta 5.3 (±1.1) nt 18 (±5.0) 5.0 (±2.2) 9.5 2.7 9.8 nt nt nt nt 0.0017 0.0043

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 96well 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-yn3-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 chiralphase separation 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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(S)-MTPA-2: white powder; 1H 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). (−)-(3R,4E,16E,18R)-Icosa-4,16-diene-1,19-diyne-3,18-diol (1): white, amorphous powder; [α]24D −30.0 (c 0.13, MeOH); IR (CHCl3) νmax 3432(br), 2947, 2358(s), 1624, 1310 cm−1; 1H NMR (CDCl3, 600 MHz) δ 1.25 (12H, brs, H-8−H-13), 1.38 (4H, quint, H7 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). (+)-(3S,4E,16E,18S)-iIcosa-4,16-diene-1,19-diyne-3,18-diol (2): white, amorphous powder; [α]24D +24.5 (c 0.10, MeOH), lit.8 [α]20D +26 (c 1, MeOH); HRESITOFMS m/z 325.2135 [M + Na]+ (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 chlorochromate) (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, SigmaAldrich) 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 (nhexane/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, 1719(s), 1410 cm−1; 1H 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); HRESITOFMS 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

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 at 600 MHz (400 MHz) for 1H and 150 MHz (100 MHz) for 13C in 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 were carried out using Merck precoated TLC plates, silica gel 60 F254, or RP-8 F254s. 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). Fraction 6: white amorphous powder; [α]25D 0 (c 0.6, MeOH); 13C NMR (CDCl3, 600 MHz) δ 28.8 (CH2, C-7 and C-14), 29.1 (CH2, C8 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. (S)-MTPA-1: white powder; 1H 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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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): yellow 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). (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), 2360(s),1719(s) cm−1; 1H 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). (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). 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), 1652(w), 1540(w) cm−1; 1H 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,

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. Diacetate 6: colorless oil; [α]24D +27.5 (c 0.49, CHCl3); IR (CHCl3) νmax 2928(s), 2359(s), 1733(s), 1540(w), 1237 cm−1; 1H 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). Monoacetate 7: colorless oil; [α]24D +11.0 (c 0.52, CHCl3). IR (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); 13 C 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 (synthetic): white powder; [α]25D −30.4 (c 0.24, MeOH); HRESITOFMS m/z 325.2152 [M + Na]+ (calcd for C20H30NaO2, 325.2138). (+)-2 (synthetic): white powder; [α]25D +29.6 (c 0.21, MeOH); HRESITOFMS m/z 325.2137 [M + Na]+ (calcd for C20H30NaO2, 325.2138. meso-3 (synthetic): white powder; [α]25D 0 (c 0.67, MeOH); 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 Na 2 SO 4 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%). Diacetate 8: colorless oil; [α]25D −27.0 (c 0.71, CHCl3); IR (CHCl3) νmax 2911(s), 2399(m), 1742(s), 1522(w), 1237 cm−1; 1H 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). Dibenzoate 9: colorless oil; [α]25D −22.3 (c 1.11, CHCl3); IR (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). E

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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 powder; IR (CHCl3) νmax 3324(s), 3032(s), 2932(m), 2360(s) cm−1; 1H 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%). 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); 13 C 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). 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. 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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ASSOCIATED CONTENT

S Supporting Information *

1 H and 13C NMR spectra of natural and synthetic bisacetylenic alcohol (1) are available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel: +81-92-642-6636. Fax: +81-92-642-6636. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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REFERENCES

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dx.doi.org/10.1021/np400297p | J. Nat. Prod. XXXX, XXX, XXX−XXX