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New cytotoxic polyacetylene amides from the Egyptian marine sponge Siphonochalina siphonella
Fitoterapia 142 (2020) 104511
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New cytotoxic polyacetylene amides from the Egyptian marine sponge Siphonochalina siphonella
T
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Dae-Won Kia, Ahmed H. El-Desokya,b, , Takeshi Kodamaa, Chin Piow Wonga, ⁎ Mohamed Abdel Ghanic, Ahmed Atef El-Beihd, Mineyuki Mizuguchie, Hiroyuki Moritaa, a
Institute of Natural Medicine, University of Toyama, 2630-Sugitani, Toyama 930-0194, Japan Pharmaceutical and Drug Industries Research Division, Pharmacognosy Department, National Research Centre, P.O. 12622, 33 El Bohouth St. (Former El Tahrir St.), Dokki, Giza, Egypt c Red Sea Marine Parks, Egyptian Environmental Affairs Agency (EEAA), Egypt d Chemistry of Natural & Microbial Products Department, National Research Centre, P.O. 12622, 33 El Bohouth Street (Former El Tahrir Street), Dokki, Giza, Egypt e Faculty of Pharmaceutical Sciences, University of Toyama, 2630-Sugitani, Toyama 930-0194, Japan b
A R T I C LE I N FO
A B S T R A C T
Keywords: Siphonochalina siphonella Polyacetylene amides Siphonellamide Cytotoxicity
Four new polyacetylene amides, siphonellamides A-D (1–4), and one new fatty amide, siphonellamide E (5), together with a known indole fatty amide (6) and callyspongamide A (7), were isolated from the Red Sea marine sponge Siphonochalina siphonella. The structures of 1–5 were elucidated by extensive analyses of their 1D- and 2D-NMR spectra and MS. The isolated compounds were assessed for their cytotoxicity against HeLa, MCF-7, and A549 cancer cell lines. Compounds 1 and 2 exhibited cytotoxic activities with IC50 values ranging from 9.4 to 34.1 μM, while 5 was only cytotoxic to HeLa cells, with an IC50 value of 78.4 μM. Compound 7 showed moderate cytotoxicity against all tested cell lines.
1. Introduction Although open chain polyacetylenes are common natural products in marine sponges, with abundant occurrence in species of the families Petrosiidae [1–5], Callyspongiidae [6–9], Chalinidae [10], Halichondriidae [11], and Niphatidae [12,13], polyacetylene amides are rare metabolites in marine sponges. To date, only the cytotoxic polyacetylenic amide, callyspongamide A, has been reported as an example of this class in marine sponges [9]. Meanwhile, alkamides are common metabolites in plants and bacteria [14–16] and have several biological properties, such as cytotoxicity [14], immunomodulatory [14], and anti-inflammatory [14] activities. The isolation of hermitamides structurally similar to callyspongamide A, from the marine cyanobacteria Lyngbya majuscule, suggested the contribution of the microbial biosynthetic machinery in the biosyntheses of these compounds [17]. Siphonochalina siphonella is a Red Sea endemic sponge belonging to the Callyspongiidae family [18]. It contains clusters of several violet tubes up to 60 cm in size, ending in a central oval oscula with a 26–40 mm diameter [18]. Previous studies of this marine sponge have revealed the presence of triterpenoids [19–21], brominated oxindole alkaloids [22], sterols [23], and polyacetylenes [6], with anti-osteoclastogenesis [20], cytotoxic [19,21], antibacterial [22], and anti-
⁎
inflammatory [23] activities. We have also isolated three new polyacetylene alcohols, named siphonellanols A-C, with cytotoxicity against human breast MCF-7, human lung A549, and human cervical HeLa cancer cell lines, from the methanol extract of S. siphonella collected in Hurghada, Egypt [24]. In our continuous studies of natural products, we herein report the isolation and structure elucidations of three new diacetylenic amides (1–3), one new monoacetylenic amide (4), and one new fatty amide (5), designated as siphonellamides A-E, respectively, together with a known alkamide, N-[2-(1H-indol-3-yl)ethyl]hexadecanamide (6) [25] and callyspongamide A (7) [9], as well as their cytotoxic activities against the HeLa, MCF-7, and A549 cell lines. 2. Experimental section 2.1. General experimental procedures Infrared spectra were measured with KBr pellets on a JASCO FT/IR460 Plus spectrometer (Japan). UV-spectra were obtained with a NANODROP 2000C spectrophotometer (Thermo Fisher Scientific, USA). NMR spectra were recorded at 500 or 400 MHz on JEOL ECA500II or ECA400II spectrometers (Japan). 1He1H COSY and 1He1H
Corresponding authors at: Institute of Natural Medicine, University of Toyama, 2630-Sugitani, Toyama 930-0194, Japan. E-mail addresses: [email protected] (A.H. El-Desoky), [email protected] (H. Morita).
https://doi.org/10.1016/j.fitote.2020.104511 Received 9 January 2020; Received in revised form 12 February 2020; Accepted 12 February 2020 Available online 13 February 2020 0367-326X/ © 2020 Elsevier B.V. All rights reserved.
Fitoterapia 142 (2020) 104511
D.-W. Ki, et al.
TOCSY spectra were recorded on a Bruker Avance III 800 spectrometer. Chemical shift values are expressed in δ (ppm) downfield from TMS, as an internal standard. HR-ESI-MS and ESI-MS/MS data were obtained from the positive mode on SHIMADZU LC-MS-IT-TOF (Japan) and Agilent Technologies 6420 Triple Quad LC/MS spectrometers (ESI voltage: +3.0 kV), respectively. Open column chromatography was performed with normal phase silica gel (silica gel 60 N, spherical, neutral, 40–50 μM (Kanto Chemical, Japan) and reversed phase silica gel (Cosmosil 75C18-OPN) (Nacalai Tesque, Japan). TLC was performed on silica gel GF254 pre-coated (Merck) plates, with detection by visualization with a UV lamp at 254 and 365 nm, followed by spraying with a p-anisaldehyde stain solution (Nacalai Tesque, Japan) and heating to 120 °C. HPLC was performed with an Agilent Technologies 1260 quaternary pump with a HEWLETT PACKARD 1100 detector, and an OSAKA SODA C18 CAPCELL PAK (10 mm I.D × 250 mm) column was used for the reversed-phase HPLC column chromatography.
2927, 2865, 1731, 1645, 1545, 1448, 1232, 1106, 752 cm−1; UV (MeOH) λmax (log ε) 290 (3.2), 228 (4.0), 220 (4.0); 1H NMR (500 MHz): see Table 1. 13C NMR was deduced from HMQC and HMBC: see Table 2; HR-ESI-MS (+): m/z 441.2881 [M + Na]+ (calcd. for C28H38N2ONa, 441.2882); ESI-MS/MS (daughter ion, CID 35 eV) m/z 441 [M + Na]+ (100), 163 [M-C16H19N2O]+ (10), 144 [MC18H28NO]+ (38). Siphonellamide E (5): yellowish oil. IR (KBr) vmax 3406, 2925, 1642, 1542, 1457, 1094, 744 cm−1; UV (MeOH) λmax (log ε) 282 (3.6), 226 (4.2), 219 (4.2), 215 (4.1); 1H (400 MHz) and 13C (100 MHz) NMR, see Tables 1 and 2; HR-ESI-MS (+): m/z 419.3025 [M + Na]+ (calcd. for C26H40N2ONa, 419.3038); ESI-MS/MS (daughter ion, CID 40 eV) m/z 419 [M + Na]+ (60), 159 [M-C16H29O]+ (60), 144 [M-C16H30NO]+ (100).
2.2. Sponge material
The cytotoxic activities of the isolated compounds were evaluated human cervical HeLa, human breast MCF-7, and human lung A549 cancer cell lines using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay (MTT; Nacalai Tesque, Japan) [26], according to the published procedure. α-Minimum essential medium with L-glutamine and phenol red (α-MEM; Wako, Japan) was used to culture the cancer cell lines [27–30]. All media were supplemented with 10% fetal bovine serum (FBS; Sigma, USA) and 1% antibiotic antimycotic solution (Sigma, USA). For the MCF-7 cancer cells, the growth medium was supplemented with 1% 0.1 mM non-essential amino acids (NEAA; Gibco, USA) and 1% 1 mM sodium pyruvate (Gibco, USA). Each cancer cell line was seeded in 96-well plates (1 × 104 cells per well) and incubated in the respective medium at 37 °C, under a 5% CO2 and 95% air atmosphere, for 24 h. After the cells were washed with phosphate-buffered saline (PBS), different concentrations of the tested samples (10, 50, and 100 μM) were added. After a 72 h incubation, the cells were washed with PBS, and 100 μL of medium containing MTT solution (5 mg/mL) was added to each well and incubated for 3 h. Subsequently, the absorbance of each well was measured at a 570 nm wavelength. 5-Fluorouracil (Wako, Japan) was used as the positive control.
2.4. Cell culture and cytotoxicity assay
The marine sponge S. siphonella was collected in January 2018 from the reefs southwest of Magawish Island, Hurghada, Egypt, at a depth of 7 m, and immediately soaked in MeOH. A voucher specimen was deposited with the Red Sea Marine Parks, Hurghada, Egypt. 2.3. Extraction and isolation The chloroform-soluble fraction of S. siphonella was prepared as reported previously [24]. The chloroform fraction was subjected to normal phase silica gel open column chromatography, eluted with nhexane/EtOAc (10:0, 9.5:0.5, 9:1, 7:3, 5:5, 3:7, 0:10) and EtOAc/MeOH (5:5, 0:10) with increasing polarities as in our previous report, and fraction 17 (83.2 mg) obtained from n-hexane/EtOAc (3:7) was subjected to reverse-phase open column chromatography, eluted with MeCN/H2O (3:1) to give 1 (4.3 mg) and 7 (1.2 mg). Fraction 18 (35.4 mg) obtained from EtOAc was subjected to reverse phase open column chromatography, using MeCN/H2O (3:1) as the elutant to afford 5 (4.8 mg) and 6 (1.2 mg). Fraction 19 (105.1 mg), obtained from EtOAc, was separated by reverse-phase semi-preparative HPLC equipped OSAKA SODA C18 CAPCELL PAK (10 mm I.D × 250 mm) column, with gradient elution by MeCN/H2O (3:2, 1–60 min), MeCN/ H2O (7:3, 60–120 min), and MeCN (120–150 min) at a flow rate of 2 mL/min, monitored at 254 nm, to afford 2 (1.5 mg, tR 90 min), 3 (0.6 mg, tR 83 min), and 4 (0.7 mg, tR 93 min). Siphonellamide A (1): yellowish oil; IR (KBr) vmax 3434, 3287, 2926, 2854, 2352, 1648, 1539, 1457, 755, 664 cm−1; UV (MeOH) λmax (log ε) 228 (3.9), 218 (4.0), 215 (4.0), 211 (4.0); 1H (400 MHz) and 13C (100 MHz) NMR, see Tables 1 and 2. HR-ESI-MS (+) m/z 414.2759 [M + Na]+ (calcd. for C27H37NONa: 414.2773). ESI-MS/MS (daughter ion, CID 30 eV) m/z 414 [M + Na]+ (100), 200 [M-C14H23]+ (20), 121 [M + H-C19H27O]+ (18), 105 [M-C19H28NO]+ (18). Siphonellamide B (2): yellowish oil. IR (KBr) vmax 3422, 3276, 2926, 2860, 2356, 1728, 1642, 1548, 1457, 1227, 1109, 752 cm−1; UV (MeOH) λmax (log ε) 290 (3.3), 227 (4.0), 218 (4.0); 1H (400 MHz) and 13 C (100 MHz) NMR, see Tables 1 and 2; HR-ESI-MS (+) m/z 453.2865 [M + Na]+ (calcd. for C29H38N2ONa, 453.2882); ESI-MS/MS (daughter ion, CID 15 eV) m/z: 431 [M + H]+ (100), 187 [M-C18H27]+ (10), 160 [M + H-C19H27O]+ (11), 144 [M-C19H28NO]+ (82). Siphonellamide C (3): yellowish oil. IR (KBr) vmax 3404, 3299, 2926, 2860, 2357, 1734, 1646, 1548, 1454, 1229, 1109, 752 cm−1; UV (MeOH) λmax (log ε) 290 (3.3), 227 (4.0), 219 (4.0); 1H NMR (500 MHz): see Table 1. 13C NMR was deduced from HMQC and HMBC: see Table 2; HR-ESI-MS (+): m/z 451.2712 [M + Na]+ (calcd. for C29H36N2ONa, 451.2725); ESI-MS/MS (daughter ion, CID 45 eV) m/z: 429 [M + H]+ (20), 144 [M-C19H26NO]+ (100), 130 [M-C20H28NO]+ (15), 116 [M-C21H30NO]+ (10), 105 [M-C21H27N2O]+ (15). Siphonellamide D (4): yellowish oil. IR (KBr) vmax 3414, 3299,
2.5. Ozonolysis A solution of 5 (0.2 mg) in dichloromethane (1.0 mL) was treated with O3 at −78 °C for 15 min, and then triphenylphosphine (0.5 mg) was added to the reaction mixture at −78 °C for 3 h. The product was warmed to room temperature and evaporated in vacuo, and the resultant solution was subjected to ESI-MS and ESI-MS/MS (CID 30 eV) in the positive mode without further purification. 3. Results and discussion In our previous study, we reported that the methanol extract from S. siphonella collected in Egypt showed cytotoxic activities, with IC50 values of 25.5, 13.0, and 27.5 μg/mL, against the HeLa, MCF-7, and A549 cancer cells lines, respectively [24]. Further investigation of the cytotoxic compounds in the chloroform-soluble fraction from the methanol extract led to the isolation of siphonellamides A-E (1–5), together with two known compounds, N-[2-(1H-indol-3-yl)ethyl]hexadecanamide (6) and callyspongamide (7) (Fig. 1). The chemical structures of 6 and 7 were identified by comparisons of their experimental data with those reported in the literature [9,25]. Compound 1 was obtained as yellowish oil. The HR-ESI-MS displayed a pseudo-molecular ion peak at m/z 414.2759 [M + Na]+ (calcd. for C27H37NONa, 414.2773) and the molecular formula of 1 was established as C27H37NO, in conjugation with the NMR data (Tables 1 and 2). The UV spectrum exhibited maximum absorptions at 228, 218, 215, and 211 nm characteristic for polyacetylenic amide [9]. The IR 2
Fitoterapia 142 (2020) 104511
D.-W. Ki, et al.
Table 1 1 H NMR spectroscopic data for siphonellamides A-E (1–5) (δ in ppm and J values in (Hz) in parentheses). Position 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 1′ 2′ 3′ 4′, 8′ 5′, 7′ 6′ -NH 1′′ 2′′ 1′-NH 2′ 3′ 3'a 4′ 5′ 6′ 7′ 7'a
1a
2a
3b
4b
5a
2.15 (m) 2.29 (m)
2.11 (m) 2.13⁎ (m)
2.14 (m) 2.32 (m)
2.13 (m) 1.47 (m) 1.30⁎ (m) 1.30⁎ (m) 1.25⁎ (m) 1.25⁎ (m) 1.30⁎ (m) 1.30⁎ (m) 1.49 (m) 2.32 (m) 5.99 (dt, 10.9, 7.5) 5.43 (d, 10.9)
2.13⁎ (m) 1.48 (m) 1.30⁎ (m) 1.30⁎ (m) 1.30⁎ (m) 1.30⁎ (m) 1.30⁎ (m) 1.30⁎ (m) 1.46 (m) 2.33 (m) 6.00 (dt, 10.7, 7.0) 5.44 (d, 10.7)
2.34 1.53 1.54 1.39 1.40 2.27 5.82 5.42 1.73 2.31 5.99 5.44
2.10 (m) 2.02 (m) 5.34 (dt, 10.8, 6.4) 5.33 (dt, 10.8, 6.4) 2.00 (m) 1.41 (m) 1.30⁎ (m) 1.30⁎ (m) 1.26⁎ (m) 1.26⁎ (m) 1.30⁎ (m) 1.30⁎ (m) 2.32 (m) 5.99 (dt, 10.9, 7.5) 5.44 (d, 10.9)
2.09 (m) 1.57 (m) 1.25⁎ (m) 1.25⁎ (m) 1.25⁎ (m) 2.01⁎ (m) 5.35⁎ (m) 5.35⁎ (m) 2.01⁎ (m) 1.25⁎ (m) 1.25⁎ (m) 1.25⁎ (m) 1.25⁎ (m) 1.25⁎ (m) 0.87 (t, 6.9)
3.07 (d, 2.1) 3.53 (td, 6.9, 6.0) 2.82 (t, 6.9)
3.07 (d, 2.3)
3.07 (d, 2.3)
5.51 3.61 2.99 8.08 7.05
(br s) (td, 6.7, 6.0) (t, 6.7) (br s) (s)
5.50 3.63 2.99 8.04 7.05
(br s) (td, 6.7, 6.0) (t, 6.7) (br s) (s)
5.47 3.62 2.98 8.02 7.05
(br s) (td, 6.7, 6.0) (t, 6.7) (br s) (s)
5.50 3.62 2.99 8.10 7.04
(br s) (td, 6.7, 6.0) (t, 6.7) (br s) (s)
7.39 7.14 7.22 7.62
(dd, 8.1, 1.0) (ddd, 8.1, 7.0, 1.0) (ddd, 7.9, 7.0, 1.0) (d, 7.9)
7.39 7.14 7.22 7.62
(dd, 8.0, 1.0) (ddd, 8.0, 7.0, 1.0) (ddd, 8.0, 7.0, 1.0) (dd, 8.0, 1.0)
7.39 7.14 7.23 7.62
(dd, 8.1, 1.0) (ddd, 8.1, 7.0, 1.0) (ddd, 8.0, 7.0, 1.0) (dd, 8.0, 1.0)
7.39 7.13 7.21 7.62
(dd, 8.1, 1.0) (ddd, 8.1, 7.0, 1.0) (ddd, 8.1, 7.0 1.0) (d, 8.1)
(m) (m) (m) (m) (m) (m) (dt, 10.8, 7.4) (br d, 10.8) (m) (m) (dt, 10.8, 7.0) (d, 10.8)
3.07 (d, 2.3)
7.20 (dd, 7.6, 1.4) 7.32 (t, 7.6) 7.23 (tt, 7.6, 1.4)
a 1
H NMR at 400 MHz measured in CDCl3. H NMR at 500 MHz measured in CDCl3. Overlapping resonances within the same column.
b 1 ⁎
spectrum showed absorption bands at 3434 cm−1 (NeH), 3287 cm−1 (C^H), 2926 cm−1, 2854 cm−1 (CeH), 2352 cm−1 (C^C), and 1648 cm−1 (C=C). The 1H NMR spectrum of 1 (Table 1) showed five aromatic proton signals at δH 7.32 (t, J = 7.6 Hz, H-5′ and H-7′), 7.23 (tt, J = 7.6, 1.4 Hz, H-6′), and 7.20 (dd, J = 7.6, 1.4 Hz, H-4′ and H-8′), two cis-oriented olefinic proton signals at δH 5.99 (dt, J = 10.9, 7.5 Hz, H-16) and 5.43 (d, J = 10.9 Hz, H-17), a terminal acetylene proton signal at δH 3.07 (d, J = 2.1 Hz, H-19), and 14 methylenes, which accounted for the remaining protons in the molecule. The 13C NMR (Table 2) and HMQC spectra displayed signals assignable to a carbonyl carbon at δC 171.5 (C-1), six aromatic carbons including a quaternary carbon at δC 138.9, five aromatic carbons at δC 128.8 (C-4′ and C-8′), 128.6 (C-5′ and C-7′), and 126.5 (C-6′), two mono-substituted olefinic carbons at δC 146.2 (C-16) and 107.9 (C-17), four acetylenic carbons including three quaternary carbons at δC 81.2 (C-5), 80.7 (C-18), and 79.5 (C-4), and a terminal acetylenic carbon at δC 81.2 (C-19), together with 14 methylene carbons. The NMR data of 1 were similar to those of callyspongamide A (7) isolated from Callyspongia fistularis [9] as well as in this study, except for the presence of two more methylenes. Further elucidation of the chemical structure of 1 was achieved by 2D NMR spectroscopic and ESI-MS/MS analyses (Figs. 2 and 3). The spin systems [C(4′)H-C(5′)H-C(6′)H-C(7′)H-C(8′)H and C(1′)H2-C(2′)H2] in the 1 He1H COSY spectrum and the cross peaks from H-5′ to C-3′, from H-7′ to C-3′, from H2–2′ to C-1′/C-4′/C-8′, and from H2–1′ to C-3′ in the HMBC spectrum suggested the presence of an ethyl benzene moiety in 1, which was further confirmed by the observation of an ion fragment at m/z 105 [M-C19H28NO]+ obtained from the ESI-MS/MS spectrum of
the pseudo-molecular ion at m/z 414 [M + Na]+ of 1 (Fig. 3). The spin system [C(6)H2-C(7)H2] in the 1He1H COSY spectrum and the HMBC correlations from H2–1′ to C-1, from H2–3 to C-1, from H2–2 to C-4, and from H2–7 to C-5, as well as the HOHAHA correlation of H2–3/H2–7, established a hept-4-ynamide moiety. Furthermore, one of the key HMBC correlations from H2–1′ to C-1 allowed us to connect both moieties at C-1 and C-1′ via an amide bond. In contrast, the 1He1H COSY linear spin system [C(14)H2-C(15)H2-C(16)H-C(17)H] and the HOHAHA long range correlation between H-17 and H-19 revealed the presence of a (3Z)-pent-3-en-1-yne moiety. Meanwhile, the ESI-MS/MS ion fragments at m/z 121 [M + H-C19H27O]+ and 200 [M-C14H23]+ suggested that the remaining 6 methylene groups were located between C-7 and C-14 (Figs. 2 and 3). The structure of 1 was thus assigned as (16Z)-N-(2-phenylethyl)nonadec-16-en-4,18-diynamide, and was named siphonellamide A. Compound 2 was obtained as yellowish oil. The molecular formula of 2 was determined as C29H38N2O, indicating twelve degrees of unsaturation, based on the pseudo-molecular ion peak [M+ Na]+ (m/z 453.2865) observed in the HR-ESI-MS, in conjunction with the NMR data (Tables 1 and 2). The UV spectrum exhibited maximum absorptions at 290, 227, and 218 nm characteristic for polyacetylenic amide bearing indole moiety [9,17]. The IR spectrum showed absorption bands at 3422 cm−1 (NeH), 3276 cm−1 (C^H), 2926 cm−1, 2860 cm−1 (CeH), 2356 cm−1 (C^C), 1728 cm−1 (C=O), and 1642 cm−1 (C=C). The 1H and 13C NMR data, in conjunction with the HMQC experiment, revealed the signals ascribable to the (16Z)-Nethylnonadec-16-en-4,18-diynamide, as in the case of 1, as well as 3
Fitoterapia 142 (2020) 104511
D.-W. Ki, et al.
Table 2 13 C NMR spectroscopic data for siphonellamides A-E (1–5) (δ in ppm). Position
1a
2a
3b
4b
5a
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 1′ 2′ 3′ 4′, 8′ 5′, 7′ 6′ -NH 1′′ 2′′ -NH 2′ 3′ 3'a 4′ 5′ 6′ 7′ 7'a
171.5 36.2 18.6 79.5 81.2 18.5 28.7 28.8 29.3 29.0 28.8 28.6 29.1 29.1 30.2 146.2 107.9 80.7 81.2 40.5 35.7 138.9 128.8 128.6 126.5
172.7 36.3 18.5 79.6 81.2 18.7 28.7 29.1⁎ 29.3 28.6 29.1⁎ 29.0⁎ 29.0⁎ 28.8 30.2 146.3 107.9 80.6 81.2
173.3 36.0 18.8 79.0 82.8 19.2 28.6 29.6 29.8⁎ 29.8⁎ 30.6 143.2 136.0 25.0 30.6 146.3 110.0 80.7 81.3
173.6 36.8 27.1 130.6⁎ 130.6⁎ 27.1 31.8 29.3⁎ 29.3⁎ 29.7⁎ 29.7⁎ 29.3⁎ 29.3⁎ 30.1 146.0 108.3 80.7 81.3
173.2 36.9 25.3 29.0 29.2 29.3 27.2⁎ 129.8 130.0 27.2⁎ 29.6⁎ 29.6⁎ 27.2⁎ 31.8 22.6 14.1
39.6 25.4
40.4 25.9
40.7 25.4
39.6 25.3
122.3 113.1 127.2 111.2 119.5 122.0 118.7 136.6
122.6 113.3 127.3 111.5 119.1 122.4 118.8 136.5
122.4 113.2 127.7 111.1 119.7 122.9 118.8 136.6
122.2 113.1 127.3 111.2 119.5 122.0 118.7 136.4
a 13
C NMR at 100 MHz measured in CDCl3. C NMR determined from HMQC and HMBC. Overlapping resonances within the same column.
b 13 ⁎
typical signals corresponding to the indole ring [δH 7.05 (s)/δC 122.3 (C-2′), 7.39 (dd, J = 8.1, 1.0 Hz)/δC 111.2 (C-4′), 7.14 (ddd, J = 8.1, 7.0, 1.0 Hz)/δC 119.5 (C-5′), 7.22 (ddd, J = 7.9, 7.0, 1.0, Hz)/δC 122.0 (C-6′), δH 7.62 (d, J = 7.9 Hz)/δC 118. 7 (C-7′), δC 136.6 (C-7'a), 127.2 (C-3'a), 113.1 (C-3′)] (Tables 1 and 2), suggesting that 2 was a structural analogue of 1 with an indole ring instead of the phenyl group of 1. The 1He1H COSY correlation between C(4′)H and C(7′)H and the HMBC correlations from H-7′/H-6′ to C-7'a, from H-5′/H-4′ to C-3'a, and H-2′ to C-3'a/C-7'a confirmed the presence of the indole moiety in 2. The COSY cross peak of NH/H2–1′′/H2–2′′ and the HMBC correlations from H2–2′′ to C-1′′/C-2′/C-3′/C-3'a further verified the attachment of the indole ring at C-2′′. Comprehensively considering the spectroscopic data, including the 1D- and 2D-NMR results shown in Fig. 2 and the ESIMS/MS analysis measured on the pseudo-molecular ion at m/z 431 [M + H]+ shown in Fig. 3, compound 2 was determined to be (16Z)-N[2-(1H-indol-3-yl)ethyl]nonadec-16-en-4,18-diynamide, and was given the trivial name siphonellamide B. Compound 3 was obtained as yellowish oil. The HR-ESI-MS of 3 showed a pseudo-molecular ion peak at m/z 451.2712 [M + Na]+ (calcd. for C29H36N2ONa, 451.2725), and the molecular formula of 3 was determined to be C29H36N2O, indicating an additional degree of unsaturation as compared with that of 2. The IR and UV absorption spectra of 3 showed similar patterns to those of 2. The 1H NMR data and carbon signals assigned by HMQC and the HMBC cross peaks (Tables 1 and 2) exhibited similar features to those of 2, with the appearance of an additional cis-oriented olefinic group [δH 5.82 (dt, J = 10.8, 7.4 Hz)/δC 143.2 and δH 5.42 (br d, J = 10.8 Hz)/δC 136.0] and the
Fig. 1. Structures of compounds 1–7 from S. siphonella.
disappearance of two methylene protons, suggesting that 3 is the monodehydro analogue of 2. A part of the 1He1H COSY cross peaks starting from H2–10 to H-13 and the HOHAHA long range correlation of H2–10/ H-16, as well as the ESI-MS/MS fragment peak at m/z 105 [MC21H27N2O]+, confirmed the presence of a Δ12-double bond with the cis-configuration in 3 (Figs. 2 and 3). Conversely, the structure of 3 was determined as (12Z,16Z)-N-[2-(1H-indol-3-yl)ethyl]nonadec-12,16dien-4,18-diynamide, and named siphonellamide C. Compound 4 was obtained as yellowish oil. The IR and UV data as well as the NMR spectra of 4 (Tables 1 and 2) showed similar structural features to those of 3, whereas the HR-ESI-MS of 4 indicated a molecular formula of C28H38N2O requiring eleven degrees of unsaturation, which is less than that of 3 by one carbon unit and two degrees of unsaturation. The 1H NMR analysis in conjunction with the HMQC and HMBC spectra of 4 (Tables 1 and 2 and Fig. 2) revealed the existence of an indole ring, a terminal acetylene [δH 3.07 (d, J = 2.3 Hz)/δC 81.3 (C-18), δC 80.7 (C-17)], an amide carbonyl [δC 173.6 (C-1)], two cisoriented olefins [δH 5.33 (dt, J = 10.8, 6.4 Hz)/δC 130.6 (C-5), 5.34 (dt, J = 10.8, 6.4 Hz)/δC 130.6 (C-4), 5.44 (d, J = 10.9 Hz)/δC 108.3 (C16), 5.99 (dt, J = 10.9, 7.5 Hz)/δC 146.0 (C-15)], and 13 methylenes. Furthermore, the HMBC correlations from H-1′′ to C-1/C-3′ and from H2′′ to C-1′′/C-3′, in conjunction with the 1He1H COSY correlations of NH-C(1′′)H2-C(2′′)H2 and C(14)H2-C(15)H-C(16)H and the HOHAHA 4
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Fig. 2. Key HMBC (arrows), 1He1H COSY (bold lines), and HOHAHA (dotted lines) correlations of 1–5.
spectrum were similar to those of compounds 2–4, except for the lack of the absorption bands corresponding to the acetylene unit. The NMR data of 5 (Tables 1 and 2) showed close structural resemblance to the known indole fatty amide (6) isolated from Pseudomonas spp. [25], as well as in this study. The only difference between 5 and 6 was the presence of an olefin [δH 5.35 (m)/δC 130.0 (C-9), 5.35 (m)/δC 129.8(C8)] in 5. The geometry of the olefin was assigned as the cis-configuration, based on the chemical shifts of 27.2 ppm for C-7 and C-10 [31,32]. Furthermore, after the ozonolysis of 5 followed by the triphenylphosphine treatment, the ESI-MS analysis of the derived products revealed the appearance of the pseudo-molecular ion at m/z 301 [M + H]+, consistent with an N-[2-(1H-indol-3-yl)ethyl]-8-oxooctanamide (Fig. S34), which clarified the location of the olefin functionality at C-8 and C-9. In light of all the spectroscopic data (Figs. 2 and 3), the structure of 5 was confirmed to be (8Z)-N-[2-(1H-indol-3-yl)ethyl]hexadec-8-enamide and named siphonellamide E. The cytotoxic activities of 1, 2, and 5–7 were evaluated against the three human cancer cell lines (HeLa, MCF-7 and A549) (Table 3), while 3 and 4 were excluded from the evaluation in this study due to their low
long range correlation of H-16/H-18, confirmed that the terminal structures in 4 consisted of N-2-(1H-indol-3-yl)ethyl amide and (3Z)pent-3-en-1-yne moieties, as in the case of 3. Based on the presence of the aforementioned moieties and the molecular formula of 4, including its degrees of unsaturation, 4 should lack an internal acetylene unit as compared with 3, suggesting that 4 was a monoacetylenic amide analogue of 3. However, the structural fragment of C(2)H2-C(3)H2-C(4)HC(5)H-C(6)H2 in the 1He1H COSY spectrum and the cross peaks of H2–2 to C-1 and of H2–7 to C-5 in the HMBC spectrum indicated that one of the cis-oriented olefins was located at C-4 in 4, in a different manner from 3. The ESI-MS/MS fragments of 4 at m/z 163 [M-C16H19N2O]+ and m/z 144 [M-C18H28NO]+ also indicated that the seven methylene groups (C-7 to C-13) were located between C-6 and C-14. Taken together, 4 was determined to be (4Z,15Z)-N-[2-(1H-indol-3-yl)ethyl]octadec-4,15-dien-17-ynamide and named siphonellamide D. Compound 5 was obtained as yellowish oil. Its HR-ESI-MS analysis, in conjugation with the NMR data, displayed a pseudo-molecular ion peak at m/z 419.3025 [M + Na]+ (calcd. for C26H40N2ONa, 419.3038) attributable to C26H40N2O (Tables 1 and 2). The UV spectrum and IR 5
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Fig. 3. ESI-MS/MS fragmentation patterns of 1–5. 6
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Table 3 Cytotoxic activities of 1, 2, and 5–7 against three human cancer cell lines. Samples
1 2 5 6 7 5-FUa a
[3] E.J. Mejia, L.B. Magranet, N.J. De Voogd, K. Tendyke, D. Qiu, Y.Y. Shen, Z. Zhou, P. Crews, Structures and cytotoxic evaluation of new and known acyclic Ene-Ynes from an American Samoa Petrosia sp, Sponge, J. Nat. Prod. 76 (2013) 425–432. [4] M. Yang, L.F. Liang, L.G. Yao, H.L. Liu, Y.W. Guo, A new brominated polyacetylene from Chinese marine sponge Xestospongia testudinaria, J. Asian Nat. Prod. Res. 21 (2019) 573–578. [5] S.E. Ayyad, D.F. Katoua, W.M. Alarif, T.R. Sobahi, M.M. Aly, L.A. Shaala, M.A. Ghandourah, Two new polyacetylene derivatives from the Red Sea sponge Xestospongia sp, Z. Naturforsch. 70c (2015) 297–303. [6] S.E. Ayyad, R. Angawy, W.M. Alarif, E.A. Saqer, F.A. Badria, Cytotoxic polyacetylenes from the Red Sea sponge Siphonochalina siphonella, Z. Naturforsch. 69c (2014) 117–123. [7] C.W. Chiu, H.J. Su, M.C. Lu, W.H. Wang, J.H. Sheu, J.H. Su, Cytotoxic polyacetylenes from a Formosan marine sponge Callyspongia sp, Bull. Chem. Soc. Jpn. 87 (2014) 1231–1234. [8] Y. Nakao, T. Uehara, S. Matsunaga, N. Fusetani, R.W. van Soest, Callyspongynic acid, a polyacetylenic acid which inhibits alpha-glucosidase, from the marine sponge Callyspongia truncata, J. Nat. Prod. 65 (2002) 922–924. [9] D.T. Youssef, R.W. van Soest, N. Fusetani, Callyspongamide A, a new cytotoxic polyacetylenic amide from the Red Sea sponge Callyspongia fistularis, J. Nat. Prod. 66 (2003) 861–862. [10] M.J. Ortega, E. Zubia, J.L. Carbello, J. Salva, Fulvinol, a new long-chain diacetylenic metabolite from the sponge Reniera fulva, J. Nat. Prod. 59 (1996) 1069–1071. [11] J.R. Dai, Y.F. Hallock, J.H. Cardellina, G.N. Gray, M.R. Boyd, Triangulynes A−H and triangulynic acid, new cytotoxic polyacetylenes from the marine sponge Pellina triangulate, J. Nat. Prod. 59 (1996) 860–865. [12] J. Wang, L.Y. Liu, L. Liu, K.X. Zhan, W.H. Jiao, H.W. Lin, Pellynols M−O, cytotoxic polyacetylenic alcohols from a Niphates sp. marine sponge, Tetrahedron 74 (2018) 3701–3706. [13] Y.F. Hallock, J.H. Cardellina, M.S. Balaschak, M.R. Alexander, T.R. Prather, R.H. Shoemaker, M.R. Boyd, Antitumor activity and stereochemistry of acetylenic alcohols from the sponge Cribrochalina vasculum, J. Nat. Prod. 58 (1995) 1801–1807. [14] D.A. Konovalov, Polyacetylene compounds of plants of the Asteraceae family, Pharm. Chem. J. 48 (2014) 613–631. [15] A. Proschak, K. Schultz, J. Herrmann, A.J. Dowling, A.O. Brachmann, R. FfrenchConstant, R. Müller, H.B. Bode, Cytotoxic fatty acid amides from Xenorhabdus, ChemBioChem. 12 (2011) 2011–2015. [16] G. Chianese, C. Sirignano, Y. Shokoohinia, Z. Mohammadi, L. Bazvandi, F. Jafari, F. Jalilian, TRPA1 modulating C14 polyacetylenes from the Iranian endemic plant Echinophora platyloba, Molecules 23 (2018) E1750. [17] L.T. Tan, T. Okino, W.H. Gerwick, Hermitamides A and B, toxic malyngamide-type natural products from the marine cyanobacterium Lyngbya majuscule, J. Nat. Prod. 63 (2000) 952–955. [18] J.N.A. Hooper, R.W. van Soest, SYSTEMA PORIFERA: A Guide to the Classification of Sponges, Springer US, New York, 2002, pp. 849–851. [19] S.M. Al-Massarani, A.A. El-Gamal, M.S. Al-Said, S.S. Al-Lihaibi, O.A. Basoudan, In vitro cytotoxic, antibacterial and antiviral activities of triterpenes from the Red Sea sponge, Siphonochalina siphonella, Trop. J. Pharm. Res. 14 (2015) 33–40. [20] A.A. El-Beih, A.H. El-Desoky, M.A. Al-Hammady, A.I. Elshamy, M.F. Hegazy, H. Kato, S. Tsukamoto, New inhibitors of RANKL-induced osteoclastogenesis from the marine sponge Siphonochalina siphonella, Fitoterapia 128 (2018) 43–49. [21] R.F. Angawi, E. Saqer, A. Abdel-Lateff, F.A. Badria, S.E. Ayyad, Cytotoxic neviotane triterpene-type from the Red Sea sponge Siphonochalina siphonella, Pharmacogn. Mag. 10 (2014) S334–S341. [22] S.S. El-Hawary, A.M. Sayed, R. Mohammed, H.M. Hassan, M.E. Rateb, E. Amin, T.A. Mohammed, M. El-Mesery, A. Bin Muhsinah, A. Alsayari, H. Wajant, M.A. Anany, U.R. Abdelmohsen, Bioactive brominated oxindole alkaloids from the Red Sea sponge Callyspongia siphonella, Mar. Drugs 17 (2019) 465. [23] D.T. Youssef, A.K. Ibrahim, S.I. Khalifa, M.K. Mesbah, A.M. Mayer, R.W. van Soest, New anti-inflammatory sterols from the Red Sea sponges Scalarispongia aqabaensis and Callyspongia siphonella, Nat. Prod. Commun. 5 (2010) 27–31. [24] D.W. Ki, A.H. El-Desoky, C.P. Wong, M. Abdel-Ghani, A.A. El-Beih, M. Mizuguchi, H. Morita, New cytotoxic polyacetylene alcohols from the Egyptian marine sponge Siphonochalina siphonella, J. Nat. Med. (2019), https://doi.org/10.1007/s11418019-01377-6. [25] F. Grundmann, V. Dill, A. Dowling, A. Thanwisai, E. Bode, N. Chantratita, R. Ffrench-Constant, H.B. Bode, Identification and isolation of insecticidal oxazoles from Pseudomonas spp. Beilstein J. Org. Chem. 8 (2012) 749–752. [26] T. Mosmann, Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays, J. Immunol. Methods 65 (1983) 55–63. [27] C.P. Wong, Prema, A.E. Nugroho, M.D. Awouafack, Y.Y. Win, N.N. Win, H. Ngwe, H. Morita, H. Morita, Two new quassinoids and other constituents from Picrasma javanica wood, and their biological activities, J. Nat. Med. 73 (2019) 589–596. [28] H.M. Nguyen, T. Ito, N.N. Win, H.Q. Vo, H.T. Nguyen, H. Morita, A new sterol from the Vietnamese marine sponge Xestospongia testudinaria and its biological activities, Nat. Prod. Res. 33 (2019) 1175–1181. [29] H.M. Nguyen, T. Ito, S. Kurimoto, M. Ogawa, N.N. Win, V.Q. Hung, H.T. Nguyen, T. Kubota, J. Kobayashi, H. Morita, New merosesquiterpenes from a Vietnamese marine sponge of Spongia sp. and their biological activities, Bioorg. Med. Chem. Lett. 27 (2017) 3043–3047. [30] T. Ito, H.M. Nguyen, N.N. Win, H.Q. Vo, H.T. Nguyen, H. Morita, Three new sesquiterpene aminoquinones from a Vietnamese Spongia sp. and their biological activities, J. Nat. Med. 72 (2018) 298–303. [31] Y. Hitora, K. Takada, S. Okada, Y. Ise, S. Matsunaga, (−)-Duryne and its homologues, cytotoxic acetylenes from a marine sponge Petrosia sp. J. Nat. Prod. 74 (2011) 1262–1267. [32] E. Breitmaier, W. Voelter, In Carbon-13 NMR Spectroscopy, Weinheim, VCH Verlagsgesellschaft, 1987, pp. 192–194.
IC50 (μM) HeLa
MCF-7
A549
9.4 17.4 78.4 > 100 25.5 28.4
18.0 34.1 > 100 > 100 19.4 34.7
24.2 25.9 > 100 > 100 30.0 14.6
5-Fluorouracil: positive control.
amounts. Compound 1 possessed strong cytotoxic activity against the HeLa cancer cell line, with an IC50 value of 9.4 μM. Compound 1 also showed moderate cytotoxic activities against MCF-7 and A549, with IC50 values of 18.0 and 24.2 μM, respectively. Compound 2 had moderate cytotoxic activities against the HeLa, MCF-7, and A549 cancer cell lines, with IC50 values of 17.4, 34.1, and 25.9 μM, respectively. In contrast, 5 exhibited weak activity against the HeLa cancer cell line, and lacked activity against MCF-7 and A549. A similar case was also observed in the assay of the known compound 7, which exhibited moderate activity against the HeLa cell line as previously reported [9]. Our assay also indicated that 7 possessed moderate activities against the MCF-7 and A549 cell lines, with IC50 values of 19.4 and 30.0 μM, respectively. However, 6 lacked cytotoxic activity against the three cancer cell lines. In conclusion, four new acetylene amides, siphonellamides A-D (1–4), and a new indole fatty amide, siphonellamide E (5), were isolated from S. siphonella, together with the related known amides 6 and 7. The cytotoxic assay suggested that the acetylene functionality might be crucial for their cytotoxic activities. As mentioned above, only callyspongamide A has been reported from the same sponge so far [9]. Further isolation of N-acylated tryptamides or phenethylamides from the marine sponge S. siphonella (Syn. Callyspongia siphonella) may provide a new chemotaxonomic incentive to these sponges. On the other hand, the biosynthetic origin of the polyacetylene amides still remains unclear. However, these compounds may produce by cyanobacteria in S. siphonella, as previous report, where hermitamides, the N-acyltryptamide type of compounds, have been isolated from cyanobacteria L. majuscule [17]. Declaration of Competing Interest The authors declare that they have no conflicts of interest. Acknowledgements This work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology, Japan (JSPS KAKENHI Grants JP17H02203 and 19H04649) and the TWAS-COMSTECH Research Grant Award_17-056 RG/PHA/AF/AC_C, TWAS/UNESCO, 2018. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.fitote.2020.104511. References [1] C. Okamoto, Y. Nakao, T. Fujita, T. Iwashita, R.W. van Soest, N. Fusetani, S. Matsunaga, Cytotoxic C47-polyacetylene carboxylic acids from a marine sponge Petrosia sp, J. Nat. Prod. 70 (2007) 1816–1819. [2] J.S. Kim, Y.J. Lim, K.S. Im, J.H. Jung, C.J. Shim, C.O. Lee, J. Hong, H. Lee, Cytotoxic polyacetylenes from the marine sponge Petrosia sp, J. Nat. Prod. 62 (1999) 554–559.
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New cytotoxic polyacetylene amides from the Egyptian marine sponge Siphonochalina siphonella
T
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Dae-Won Kia, Ahmed H. El-Desokya,b, , Takeshi Kodamaa, Chin Piow Wonga, ⁎ Mohamed Abdel Ghanic, Ahmed Atef El-Beihd, Mineyuki Mizuguchie, Hiroyuki Moritaa, a
Institute of Natural Medicine, University of Toyama, 2630-Sugitani, Toyama 930-0194, Japan Pharmaceutical and Drug Industries Research Division, Pharmacognosy Department, National Research Centre, P.O. 12622, 33 El Bohouth St. (Former El Tahrir St.), Dokki, Giza, Egypt c Red Sea Marine Parks, Egyptian Environmental Affairs Agency (EEAA), Egypt d Chemistry of Natural & Microbial Products Department, National Research Centre, P.O. 12622, 33 El Bohouth Street (Former El Tahrir Street), Dokki, Giza, Egypt e Faculty of Pharmaceutical Sciences, University of Toyama, 2630-Sugitani, Toyama 930-0194, Japan b
A R T I C LE I N FO
A B S T R A C T
Keywords: Siphonochalina siphonella Polyacetylene amides Siphonellamide Cytotoxicity
Four new polyacetylene amides, siphonellamides A-D (1–4), and one new fatty amide, siphonellamide E (5), together with a known indole fatty amide (6) and callyspongamide A (7), were isolated from the Red Sea marine sponge Siphonochalina siphonella. The structures of 1–5 were elucidated by extensive analyses of their 1D- and 2D-NMR spectra and MS. The isolated compounds were assessed for their cytotoxicity against HeLa, MCF-7, and A549 cancer cell lines. Compounds 1 and 2 exhibited cytotoxic activities with IC50 values ranging from 9.4 to 34.1 μM, while 5 was only cytotoxic to HeLa cells, with an IC50 value of 78.4 μM. Compound 7 showed moderate cytotoxicity against all tested cell lines.
1. Introduction Although open chain polyacetylenes are common natural products in marine sponges, with abundant occurrence in species of the families Petrosiidae [1–5], Callyspongiidae [6–9], Chalinidae [10], Halichondriidae [11], and Niphatidae [12,13], polyacetylene amides are rare metabolites in marine sponges. To date, only the cytotoxic polyacetylenic amide, callyspongamide A, has been reported as an example of this class in marine sponges [9]. Meanwhile, alkamides are common metabolites in plants and bacteria [14–16] and have several biological properties, such as cytotoxicity [14], immunomodulatory [14], and anti-inflammatory [14] activities. The isolation of hermitamides structurally similar to callyspongamide A, from the marine cyanobacteria Lyngbya majuscule, suggested the contribution of the microbial biosynthetic machinery in the biosyntheses of these compounds [17]. Siphonochalina siphonella is a Red Sea endemic sponge belonging to the Callyspongiidae family [18]. It contains clusters of several violet tubes up to 60 cm in size, ending in a central oval oscula with a 26–40 mm diameter [18]. Previous studies of this marine sponge have revealed the presence of triterpenoids [19–21], brominated oxindole alkaloids [22], sterols [23], and polyacetylenes [6], with anti-osteoclastogenesis [20], cytotoxic [19,21], antibacterial [22], and anti-
⁎
inflammatory [23] activities. We have also isolated three new polyacetylene alcohols, named siphonellanols A-C, with cytotoxicity against human breast MCF-7, human lung A549, and human cervical HeLa cancer cell lines, from the methanol extract of S. siphonella collected in Hurghada, Egypt [24]. In our continuous studies of natural products, we herein report the isolation and structure elucidations of three new diacetylenic amides (1–3), one new monoacetylenic amide (4), and one new fatty amide (5), designated as siphonellamides A-E, respectively, together with a known alkamide, N-[2-(1H-indol-3-yl)ethyl]hexadecanamide (6) [25] and callyspongamide A (7) [9], as well as their cytotoxic activities against the HeLa, MCF-7, and A549 cell lines. 2. Experimental section 2.1. General experimental procedures Infrared spectra were measured with KBr pellets on a JASCO FT/IR460 Plus spectrometer (Japan). UV-spectra were obtained with a NANODROP 2000C spectrophotometer (Thermo Fisher Scientific, USA). NMR spectra were recorded at 500 or 400 MHz on JEOL ECA500II or ECA400II spectrometers (Japan). 1He1H COSY and 1He1H
Corresponding authors at: Institute of Natural Medicine, University of Toyama, 2630-Sugitani, Toyama 930-0194, Japan. E-mail addresses: [email protected] (A.H. El-Desoky), [email protected] (H. Morita).
https://doi.org/10.1016/j.fitote.2020.104511 Received 9 January 2020; Received in revised form 12 February 2020; Accepted 12 February 2020 Available online 13 February 2020 0367-326X/ © 2020 Elsevier B.V. All rights reserved.
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TOCSY spectra were recorded on a Bruker Avance III 800 spectrometer. Chemical shift values are expressed in δ (ppm) downfield from TMS, as an internal standard. HR-ESI-MS and ESI-MS/MS data were obtained from the positive mode on SHIMADZU LC-MS-IT-TOF (Japan) and Agilent Technologies 6420 Triple Quad LC/MS spectrometers (ESI voltage: +3.0 kV), respectively. Open column chromatography was performed with normal phase silica gel (silica gel 60 N, spherical, neutral, 40–50 μM (Kanto Chemical, Japan) and reversed phase silica gel (Cosmosil 75C18-OPN) (Nacalai Tesque, Japan). TLC was performed on silica gel GF254 pre-coated (Merck) plates, with detection by visualization with a UV lamp at 254 and 365 nm, followed by spraying with a p-anisaldehyde stain solution (Nacalai Tesque, Japan) and heating to 120 °C. HPLC was performed with an Agilent Technologies 1260 quaternary pump with a HEWLETT PACKARD 1100 detector, and an OSAKA SODA C18 CAPCELL PAK (10 mm I.D × 250 mm) column was used for the reversed-phase HPLC column chromatography.
2927, 2865, 1731, 1645, 1545, 1448, 1232, 1106, 752 cm−1; UV (MeOH) λmax (log ε) 290 (3.2), 228 (4.0), 220 (4.0); 1H NMR (500 MHz): see Table 1. 13C NMR was deduced from HMQC and HMBC: see Table 2; HR-ESI-MS (+): m/z 441.2881 [M + Na]+ (calcd. for C28H38N2ONa, 441.2882); ESI-MS/MS (daughter ion, CID 35 eV) m/z 441 [M + Na]+ (100), 163 [M-C16H19N2O]+ (10), 144 [MC18H28NO]+ (38). Siphonellamide E (5): yellowish oil. IR (KBr) vmax 3406, 2925, 1642, 1542, 1457, 1094, 744 cm−1; UV (MeOH) λmax (log ε) 282 (3.6), 226 (4.2), 219 (4.2), 215 (4.1); 1H (400 MHz) and 13C (100 MHz) NMR, see Tables 1 and 2; HR-ESI-MS (+): m/z 419.3025 [M + Na]+ (calcd. for C26H40N2ONa, 419.3038); ESI-MS/MS (daughter ion, CID 40 eV) m/z 419 [M + Na]+ (60), 159 [M-C16H29O]+ (60), 144 [M-C16H30NO]+ (100).
2.2. Sponge material
The cytotoxic activities of the isolated compounds were evaluated human cervical HeLa, human breast MCF-7, and human lung A549 cancer cell lines using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay (MTT; Nacalai Tesque, Japan) [26], according to the published procedure. α-Minimum essential medium with L-glutamine and phenol red (α-MEM; Wako, Japan) was used to culture the cancer cell lines [27–30]. All media were supplemented with 10% fetal bovine serum (FBS; Sigma, USA) and 1% antibiotic antimycotic solution (Sigma, USA). For the MCF-7 cancer cells, the growth medium was supplemented with 1% 0.1 mM non-essential amino acids (NEAA; Gibco, USA) and 1% 1 mM sodium pyruvate (Gibco, USA). Each cancer cell line was seeded in 96-well plates (1 × 104 cells per well) and incubated in the respective medium at 37 °C, under a 5% CO2 and 95% air atmosphere, for 24 h. After the cells were washed with phosphate-buffered saline (PBS), different concentrations of the tested samples (10, 50, and 100 μM) were added. After a 72 h incubation, the cells were washed with PBS, and 100 μL of medium containing MTT solution (5 mg/mL) was added to each well and incubated for 3 h. Subsequently, the absorbance of each well was measured at a 570 nm wavelength. 5-Fluorouracil (Wako, Japan) was used as the positive control.
2.4. Cell culture and cytotoxicity assay
The marine sponge S. siphonella was collected in January 2018 from the reefs southwest of Magawish Island, Hurghada, Egypt, at a depth of 7 m, and immediately soaked in MeOH. A voucher specimen was deposited with the Red Sea Marine Parks, Hurghada, Egypt. 2.3. Extraction and isolation The chloroform-soluble fraction of S. siphonella was prepared as reported previously [24]. The chloroform fraction was subjected to normal phase silica gel open column chromatography, eluted with nhexane/EtOAc (10:0, 9.5:0.5, 9:1, 7:3, 5:5, 3:7, 0:10) and EtOAc/MeOH (5:5, 0:10) with increasing polarities as in our previous report, and fraction 17 (83.2 mg) obtained from n-hexane/EtOAc (3:7) was subjected to reverse-phase open column chromatography, eluted with MeCN/H2O (3:1) to give 1 (4.3 mg) and 7 (1.2 mg). Fraction 18 (35.4 mg) obtained from EtOAc was subjected to reverse phase open column chromatography, using MeCN/H2O (3:1) as the elutant to afford 5 (4.8 mg) and 6 (1.2 mg). Fraction 19 (105.1 mg), obtained from EtOAc, was separated by reverse-phase semi-preparative HPLC equipped OSAKA SODA C18 CAPCELL PAK (10 mm I.D × 250 mm) column, with gradient elution by MeCN/H2O (3:2, 1–60 min), MeCN/ H2O (7:3, 60–120 min), and MeCN (120–150 min) at a flow rate of 2 mL/min, monitored at 254 nm, to afford 2 (1.5 mg, tR 90 min), 3 (0.6 mg, tR 83 min), and 4 (0.7 mg, tR 93 min). Siphonellamide A (1): yellowish oil; IR (KBr) vmax 3434, 3287, 2926, 2854, 2352, 1648, 1539, 1457, 755, 664 cm−1; UV (MeOH) λmax (log ε) 228 (3.9), 218 (4.0), 215 (4.0), 211 (4.0); 1H (400 MHz) and 13C (100 MHz) NMR, see Tables 1 and 2. HR-ESI-MS (+) m/z 414.2759 [M + Na]+ (calcd. for C27H37NONa: 414.2773). ESI-MS/MS (daughter ion, CID 30 eV) m/z 414 [M + Na]+ (100), 200 [M-C14H23]+ (20), 121 [M + H-C19H27O]+ (18), 105 [M-C19H28NO]+ (18). Siphonellamide B (2): yellowish oil. IR (KBr) vmax 3422, 3276, 2926, 2860, 2356, 1728, 1642, 1548, 1457, 1227, 1109, 752 cm−1; UV (MeOH) λmax (log ε) 290 (3.3), 227 (4.0), 218 (4.0); 1H (400 MHz) and 13 C (100 MHz) NMR, see Tables 1 and 2; HR-ESI-MS (+) m/z 453.2865 [M + Na]+ (calcd. for C29H38N2ONa, 453.2882); ESI-MS/MS (daughter ion, CID 15 eV) m/z: 431 [M + H]+ (100), 187 [M-C18H27]+ (10), 160 [M + H-C19H27O]+ (11), 144 [M-C19H28NO]+ (82). Siphonellamide C (3): yellowish oil. IR (KBr) vmax 3404, 3299, 2926, 2860, 2357, 1734, 1646, 1548, 1454, 1229, 1109, 752 cm−1; UV (MeOH) λmax (log ε) 290 (3.3), 227 (4.0), 219 (4.0); 1H NMR (500 MHz): see Table 1. 13C NMR was deduced from HMQC and HMBC: see Table 2; HR-ESI-MS (+): m/z 451.2712 [M + Na]+ (calcd. for C29H36N2ONa, 451.2725); ESI-MS/MS (daughter ion, CID 45 eV) m/z: 429 [M + H]+ (20), 144 [M-C19H26NO]+ (100), 130 [M-C20H28NO]+ (15), 116 [M-C21H30NO]+ (10), 105 [M-C21H27N2O]+ (15). Siphonellamide D (4): yellowish oil. IR (KBr) vmax 3414, 3299,
2.5. Ozonolysis A solution of 5 (0.2 mg) in dichloromethane (1.0 mL) was treated with O3 at −78 °C for 15 min, and then triphenylphosphine (0.5 mg) was added to the reaction mixture at −78 °C for 3 h. The product was warmed to room temperature and evaporated in vacuo, and the resultant solution was subjected to ESI-MS and ESI-MS/MS (CID 30 eV) in the positive mode without further purification. 3. Results and discussion In our previous study, we reported that the methanol extract from S. siphonella collected in Egypt showed cytotoxic activities, with IC50 values of 25.5, 13.0, and 27.5 μg/mL, against the HeLa, MCF-7, and A549 cancer cells lines, respectively [24]. Further investigation of the cytotoxic compounds in the chloroform-soluble fraction from the methanol extract led to the isolation of siphonellamides A-E (1–5), together with two known compounds, N-[2-(1H-indol-3-yl)ethyl]hexadecanamide (6) and callyspongamide (7) (Fig. 1). The chemical structures of 6 and 7 were identified by comparisons of their experimental data with those reported in the literature [9,25]. Compound 1 was obtained as yellowish oil. The HR-ESI-MS displayed a pseudo-molecular ion peak at m/z 414.2759 [M + Na]+ (calcd. for C27H37NONa, 414.2773) and the molecular formula of 1 was established as C27H37NO, in conjugation with the NMR data (Tables 1 and 2). The UV spectrum exhibited maximum absorptions at 228, 218, 215, and 211 nm characteristic for polyacetylenic amide [9]. The IR 2
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Table 1 1 H NMR spectroscopic data for siphonellamides A-E (1–5) (δ in ppm and J values in (Hz) in parentheses). Position 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 1′ 2′ 3′ 4′, 8′ 5′, 7′ 6′ -NH 1′′ 2′′ 1′-NH 2′ 3′ 3'a 4′ 5′ 6′ 7′ 7'a
1a
2a
3b
4b
5a
2.15 (m) 2.29 (m)
2.11 (m) 2.13⁎ (m)
2.14 (m) 2.32 (m)
2.13 (m) 1.47 (m) 1.30⁎ (m) 1.30⁎ (m) 1.25⁎ (m) 1.25⁎ (m) 1.30⁎ (m) 1.30⁎ (m) 1.49 (m) 2.32 (m) 5.99 (dt, 10.9, 7.5) 5.43 (d, 10.9)
2.13⁎ (m) 1.48 (m) 1.30⁎ (m) 1.30⁎ (m) 1.30⁎ (m) 1.30⁎ (m) 1.30⁎ (m) 1.30⁎ (m) 1.46 (m) 2.33 (m) 6.00 (dt, 10.7, 7.0) 5.44 (d, 10.7)
2.34 1.53 1.54 1.39 1.40 2.27 5.82 5.42 1.73 2.31 5.99 5.44
2.10 (m) 2.02 (m) 5.34 (dt, 10.8, 6.4) 5.33 (dt, 10.8, 6.4) 2.00 (m) 1.41 (m) 1.30⁎ (m) 1.30⁎ (m) 1.26⁎ (m) 1.26⁎ (m) 1.30⁎ (m) 1.30⁎ (m) 2.32 (m) 5.99 (dt, 10.9, 7.5) 5.44 (d, 10.9)
2.09 (m) 1.57 (m) 1.25⁎ (m) 1.25⁎ (m) 1.25⁎ (m) 2.01⁎ (m) 5.35⁎ (m) 5.35⁎ (m) 2.01⁎ (m) 1.25⁎ (m) 1.25⁎ (m) 1.25⁎ (m) 1.25⁎ (m) 1.25⁎ (m) 0.87 (t, 6.9)
3.07 (d, 2.1) 3.53 (td, 6.9, 6.0) 2.82 (t, 6.9)
3.07 (d, 2.3)
3.07 (d, 2.3)
5.51 3.61 2.99 8.08 7.05
(br s) (td, 6.7, 6.0) (t, 6.7) (br s) (s)
5.50 3.63 2.99 8.04 7.05
(br s) (td, 6.7, 6.0) (t, 6.7) (br s) (s)
5.47 3.62 2.98 8.02 7.05
(br s) (td, 6.7, 6.0) (t, 6.7) (br s) (s)
5.50 3.62 2.99 8.10 7.04
(br s) (td, 6.7, 6.0) (t, 6.7) (br s) (s)
7.39 7.14 7.22 7.62
(dd, 8.1, 1.0) (ddd, 8.1, 7.0, 1.0) (ddd, 7.9, 7.0, 1.0) (d, 7.9)
7.39 7.14 7.22 7.62
(dd, 8.0, 1.0) (ddd, 8.0, 7.0, 1.0) (ddd, 8.0, 7.0, 1.0) (dd, 8.0, 1.0)
7.39 7.14 7.23 7.62
(dd, 8.1, 1.0) (ddd, 8.1, 7.0, 1.0) (ddd, 8.0, 7.0, 1.0) (dd, 8.0, 1.0)
7.39 7.13 7.21 7.62
(dd, 8.1, 1.0) (ddd, 8.1, 7.0, 1.0) (ddd, 8.1, 7.0 1.0) (d, 8.1)
(m) (m) (m) (m) (m) (m) (dt, 10.8, 7.4) (br d, 10.8) (m) (m) (dt, 10.8, 7.0) (d, 10.8)
3.07 (d, 2.3)
7.20 (dd, 7.6, 1.4) 7.32 (t, 7.6) 7.23 (tt, 7.6, 1.4)
a 1
H NMR at 400 MHz measured in CDCl3. H NMR at 500 MHz measured in CDCl3. Overlapping resonances within the same column.
b 1 ⁎
spectrum showed absorption bands at 3434 cm−1 (NeH), 3287 cm−1 (C^H), 2926 cm−1, 2854 cm−1 (CeH), 2352 cm−1 (C^C), and 1648 cm−1 (C=C). The 1H NMR spectrum of 1 (Table 1) showed five aromatic proton signals at δH 7.32 (t, J = 7.6 Hz, H-5′ and H-7′), 7.23 (tt, J = 7.6, 1.4 Hz, H-6′), and 7.20 (dd, J = 7.6, 1.4 Hz, H-4′ and H-8′), two cis-oriented olefinic proton signals at δH 5.99 (dt, J = 10.9, 7.5 Hz, H-16) and 5.43 (d, J = 10.9 Hz, H-17), a terminal acetylene proton signal at δH 3.07 (d, J = 2.1 Hz, H-19), and 14 methylenes, which accounted for the remaining protons in the molecule. The 13C NMR (Table 2) and HMQC spectra displayed signals assignable to a carbonyl carbon at δC 171.5 (C-1), six aromatic carbons including a quaternary carbon at δC 138.9, five aromatic carbons at δC 128.8 (C-4′ and C-8′), 128.6 (C-5′ and C-7′), and 126.5 (C-6′), two mono-substituted olefinic carbons at δC 146.2 (C-16) and 107.9 (C-17), four acetylenic carbons including three quaternary carbons at δC 81.2 (C-5), 80.7 (C-18), and 79.5 (C-4), and a terminal acetylenic carbon at δC 81.2 (C-19), together with 14 methylene carbons. The NMR data of 1 were similar to those of callyspongamide A (7) isolated from Callyspongia fistularis [9] as well as in this study, except for the presence of two more methylenes. Further elucidation of the chemical structure of 1 was achieved by 2D NMR spectroscopic and ESI-MS/MS analyses (Figs. 2 and 3). The spin systems [C(4′)H-C(5′)H-C(6′)H-C(7′)H-C(8′)H and C(1′)H2-C(2′)H2] in the 1 He1H COSY spectrum and the cross peaks from H-5′ to C-3′, from H-7′ to C-3′, from H2–2′ to C-1′/C-4′/C-8′, and from H2–1′ to C-3′ in the HMBC spectrum suggested the presence of an ethyl benzene moiety in 1, which was further confirmed by the observation of an ion fragment at m/z 105 [M-C19H28NO]+ obtained from the ESI-MS/MS spectrum of
the pseudo-molecular ion at m/z 414 [M + Na]+ of 1 (Fig. 3). The spin system [C(6)H2-C(7)H2] in the 1He1H COSY spectrum and the HMBC correlations from H2–1′ to C-1, from H2–3 to C-1, from H2–2 to C-4, and from H2–7 to C-5, as well as the HOHAHA correlation of H2–3/H2–7, established a hept-4-ynamide moiety. Furthermore, one of the key HMBC correlations from H2–1′ to C-1 allowed us to connect both moieties at C-1 and C-1′ via an amide bond. In contrast, the 1He1H COSY linear spin system [C(14)H2-C(15)H2-C(16)H-C(17)H] and the HOHAHA long range correlation between H-17 and H-19 revealed the presence of a (3Z)-pent-3-en-1-yne moiety. Meanwhile, the ESI-MS/MS ion fragments at m/z 121 [M + H-C19H27O]+ and 200 [M-C14H23]+ suggested that the remaining 6 methylene groups were located between C-7 and C-14 (Figs. 2 and 3). The structure of 1 was thus assigned as (16Z)-N-(2-phenylethyl)nonadec-16-en-4,18-diynamide, and was named siphonellamide A. Compound 2 was obtained as yellowish oil. The molecular formula of 2 was determined as C29H38N2O, indicating twelve degrees of unsaturation, based on the pseudo-molecular ion peak [M+ Na]+ (m/z 453.2865) observed in the HR-ESI-MS, in conjunction with the NMR data (Tables 1 and 2). The UV spectrum exhibited maximum absorptions at 290, 227, and 218 nm characteristic for polyacetylenic amide bearing indole moiety [9,17]. The IR spectrum showed absorption bands at 3422 cm−1 (NeH), 3276 cm−1 (C^H), 2926 cm−1, 2860 cm−1 (CeH), 2356 cm−1 (C^C), 1728 cm−1 (C=O), and 1642 cm−1 (C=C). The 1H and 13C NMR data, in conjunction with the HMQC experiment, revealed the signals ascribable to the (16Z)-Nethylnonadec-16-en-4,18-diynamide, as in the case of 1, as well as 3
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Table 2 13 C NMR spectroscopic data for siphonellamides A-E (1–5) (δ in ppm). Position
1a
2a
3b
4b
5a
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 1′ 2′ 3′ 4′, 8′ 5′, 7′ 6′ -NH 1′′ 2′′ -NH 2′ 3′ 3'a 4′ 5′ 6′ 7′ 7'a
171.5 36.2 18.6 79.5 81.2 18.5 28.7 28.8 29.3 29.0 28.8 28.6 29.1 29.1 30.2 146.2 107.9 80.7 81.2 40.5 35.7 138.9 128.8 128.6 126.5
172.7 36.3 18.5 79.6 81.2 18.7 28.7 29.1⁎ 29.3 28.6 29.1⁎ 29.0⁎ 29.0⁎ 28.8 30.2 146.3 107.9 80.6 81.2
173.3 36.0 18.8 79.0 82.8 19.2 28.6 29.6 29.8⁎ 29.8⁎ 30.6 143.2 136.0 25.0 30.6 146.3 110.0 80.7 81.3
173.6 36.8 27.1 130.6⁎ 130.6⁎ 27.1 31.8 29.3⁎ 29.3⁎ 29.7⁎ 29.7⁎ 29.3⁎ 29.3⁎ 30.1 146.0 108.3 80.7 81.3
173.2 36.9 25.3 29.0 29.2 29.3 27.2⁎ 129.8 130.0 27.2⁎ 29.6⁎ 29.6⁎ 27.2⁎ 31.8 22.6 14.1
39.6 25.4
40.4 25.9
40.7 25.4
39.6 25.3
122.3 113.1 127.2 111.2 119.5 122.0 118.7 136.6
122.6 113.3 127.3 111.5 119.1 122.4 118.8 136.5
122.4 113.2 127.7 111.1 119.7 122.9 118.8 136.6
122.2 113.1 127.3 111.2 119.5 122.0 118.7 136.4
a 13
C NMR at 100 MHz measured in CDCl3. C NMR determined from HMQC and HMBC. Overlapping resonances within the same column.
b 13 ⁎
typical signals corresponding to the indole ring [δH 7.05 (s)/δC 122.3 (C-2′), 7.39 (dd, J = 8.1, 1.0 Hz)/δC 111.2 (C-4′), 7.14 (ddd, J = 8.1, 7.0, 1.0 Hz)/δC 119.5 (C-5′), 7.22 (ddd, J = 7.9, 7.0, 1.0, Hz)/δC 122.0 (C-6′), δH 7.62 (d, J = 7.9 Hz)/δC 118. 7 (C-7′), δC 136.6 (C-7'a), 127.2 (C-3'a), 113.1 (C-3′)] (Tables 1 and 2), suggesting that 2 was a structural analogue of 1 with an indole ring instead of the phenyl group of 1. The 1He1H COSY correlation between C(4′)H and C(7′)H and the HMBC correlations from H-7′/H-6′ to C-7'a, from H-5′/H-4′ to C-3'a, and H-2′ to C-3'a/C-7'a confirmed the presence of the indole moiety in 2. The COSY cross peak of NH/H2–1′′/H2–2′′ and the HMBC correlations from H2–2′′ to C-1′′/C-2′/C-3′/C-3'a further verified the attachment of the indole ring at C-2′′. Comprehensively considering the spectroscopic data, including the 1D- and 2D-NMR results shown in Fig. 2 and the ESIMS/MS analysis measured on the pseudo-molecular ion at m/z 431 [M + H]+ shown in Fig. 3, compound 2 was determined to be (16Z)-N[2-(1H-indol-3-yl)ethyl]nonadec-16-en-4,18-diynamide, and was given the trivial name siphonellamide B. Compound 3 was obtained as yellowish oil. The HR-ESI-MS of 3 showed a pseudo-molecular ion peak at m/z 451.2712 [M + Na]+ (calcd. for C29H36N2ONa, 451.2725), and the molecular formula of 3 was determined to be C29H36N2O, indicating an additional degree of unsaturation as compared with that of 2. The IR and UV absorption spectra of 3 showed similar patterns to those of 2. The 1H NMR data and carbon signals assigned by HMQC and the HMBC cross peaks (Tables 1 and 2) exhibited similar features to those of 2, with the appearance of an additional cis-oriented olefinic group [δH 5.82 (dt, J = 10.8, 7.4 Hz)/δC 143.2 and δH 5.42 (br d, J = 10.8 Hz)/δC 136.0] and the
Fig. 1. Structures of compounds 1–7 from S. siphonella.
disappearance of two methylene protons, suggesting that 3 is the monodehydro analogue of 2. A part of the 1He1H COSY cross peaks starting from H2–10 to H-13 and the HOHAHA long range correlation of H2–10/ H-16, as well as the ESI-MS/MS fragment peak at m/z 105 [MC21H27N2O]+, confirmed the presence of a Δ12-double bond with the cis-configuration in 3 (Figs. 2 and 3). Conversely, the structure of 3 was determined as (12Z,16Z)-N-[2-(1H-indol-3-yl)ethyl]nonadec-12,16dien-4,18-diynamide, and named siphonellamide C. Compound 4 was obtained as yellowish oil. The IR and UV data as well as the NMR spectra of 4 (Tables 1 and 2) showed similar structural features to those of 3, whereas the HR-ESI-MS of 4 indicated a molecular formula of C28H38N2O requiring eleven degrees of unsaturation, which is less than that of 3 by one carbon unit and two degrees of unsaturation. The 1H NMR analysis in conjunction with the HMQC and HMBC spectra of 4 (Tables 1 and 2 and Fig. 2) revealed the existence of an indole ring, a terminal acetylene [δH 3.07 (d, J = 2.3 Hz)/δC 81.3 (C-18), δC 80.7 (C-17)], an amide carbonyl [δC 173.6 (C-1)], two cisoriented olefins [δH 5.33 (dt, J = 10.8, 6.4 Hz)/δC 130.6 (C-5), 5.34 (dt, J = 10.8, 6.4 Hz)/δC 130.6 (C-4), 5.44 (d, J = 10.9 Hz)/δC 108.3 (C16), 5.99 (dt, J = 10.9, 7.5 Hz)/δC 146.0 (C-15)], and 13 methylenes. Furthermore, the HMBC correlations from H-1′′ to C-1/C-3′ and from H2′′ to C-1′′/C-3′, in conjunction with the 1He1H COSY correlations of NH-C(1′′)H2-C(2′′)H2 and C(14)H2-C(15)H-C(16)H and the HOHAHA 4
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Fig. 2. Key HMBC (arrows), 1He1H COSY (bold lines), and HOHAHA (dotted lines) correlations of 1–5.
spectrum were similar to those of compounds 2–4, except for the lack of the absorption bands corresponding to the acetylene unit. The NMR data of 5 (Tables 1 and 2) showed close structural resemblance to the known indole fatty amide (6) isolated from Pseudomonas spp. [25], as well as in this study. The only difference between 5 and 6 was the presence of an olefin [δH 5.35 (m)/δC 130.0 (C-9), 5.35 (m)/δC 129.8(C8)] in 5. The geometry of the olefin was assigned as the cis-configuration, based on the chemical shifts of 27.2 ppm for C-7 and C-10 [31,32]. Furthermore, after the ozonolysis of 5 followed by the triphenylphosphine treatment, the ESI-MS analysis of the derived products revealed the appearance of the pseudo-molecular ion at m/z 301 [M + H]+, consistent with an N-[2-(1H-indol-3-yl)ethyl]-8-oxooctanamide (Fig. S34), which clarified the location of the olefin functionality at C-8 and C-9. In light of all the spectroscopic data (Figs. 2 and 3), the structure of 5 was confirmed to be (8Z)-N-[2-(1H-indol-3-yl)ethyl]hexadec-8-enamide and named siphonellamide E. The cytotoxic activities of 1, 2, and 5–7 were evaluated against the three human cancer cell lines (HeLa, MCF-7 and A549) (Table 3), while 3 and 4 were excluded from the evaluation in this study due to their low
long range correlation of H-16/H-18, confirmed that the terminal structures in 4 consisted of N-2-(1H-indol-3-yl)ethyl amide and (3Z)pent-3-en-1-yne moieties, as in the case of 3. Based on the presence of the aforementioned moieties and the molecular formula of 4, including its degrees of unsaturation, 4 should lack an internal acetylene unit as compared with 3, suggesting that 4 was a monoacetylenic amide analogue of 3. However, the structural fragment of C(2)H2-C(3)H2-C(4)HC(5)H-C(6)H2 in the 1He1H COSY spectrum and the cross peaks of H2–2 to C-1 and of H2–7 to C-5 in the HMBC spectrum indicated that one of the cis-oriented olefins was located at C-4 in 4, in a different manner from 3. The ESI-MS/MS fragments of 4 at m/z 163 [M-C16H19N2O]+ and m/z 144 [M-C18H28NO]+ also indicated that the seven methylene groups (C-7 to C-13) were located between C-6 and C-14. Taken together, 4 was determined to be (4Z,15Z)-N-[2-(1H-indol-3-yl)ethyl]octadec-4,15-dien-17-ynamide and named siphonellamide D. Compound 5 was obtained as yellowish oil. Its HR-ESI-MS analysis, in conjugation with the NMR data, displayed a pseudo-molecular ion peak at m/z 419.3025 [M + Na]+ (calcd. for C26H40N2ONa, 419.3038) attributable to C26H40N2O (Tables 1 and 2). The UV spectrum and IR 5
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Fig. 3. ESI-MS/MS fragmentation patterns of 1–5. 6
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Table 3 Cytotoxic activities of 1, 2, and 5–7 against three human cancer cell lines. Samples
1 2 5 6 7 5-FUa a
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IC50 (μM) HeLa
MCF-7
A549
9.4 17.4 78.4 > 100 25.5 28.4
18.0 34.1 > 100 > 100 19.4 34.7
24.2 25.9 > 100 > 100 30.0 14.6
5-Fluorouracil: positive control.
amounts. Compound 1 possessed strong cytotoxic activity against the HeLa cancer cell line, with an IC50 value of 9.4 μM. Compound 1 also showed moderate cytotoxic activities against MCF-7 and A549, with IC50 values of 18.0 and 24.2 μM, respectively. Compound 2 had moderate cytotoxic activities against the HeLa, MCF-7, and A549 cancer cell lines, with IC50 values of 17.4, 34.1, and 25.9 μM, respectively. In contrast, 5 exhibited weak activity against the HeLa cancer cell line, and lacked activity against MCF-7 and A549. A similar case was also observed in the assay of the known compound 7, which exhibited moderate activity against the HeLa cell line as previously reported [9]. Our assay also indicated that 7 possessed moderate activities against the MCF-7 and A549 cell lines, with IC50 values of 19.4 and 30.0 μM, respectively. However, 6 lacked cytotoxic activity against the three cancer cell lines. In conclusion, four new acetylene amides, siphonellamides A-D (1–4), and a new indole fatty amide, siphonellamide E (5), were isolated from S. siphonella, together with the related known amides 6 and 7. The cytotoxic assay suggested that the acetylene functionality might be crucial for their cytotoxic activities. As mentioned above, only callyspongamide A has been reported from the same sponge so far [9]. Further isolation of N-acylated tryptamides or phenethylamides from the marine sponge S. siphonella (Syn. Callyspongia siphonella) may provide a new chemotaxonomic incentive to these sponges. On the other hand, the biosynthetic origin of the polyacetylene amides still remains unclear. However, these compounds may produce by cyanobacteria in S. siphonella, as previous report, where hermitamides, the N-acyltryptamide type of compounds, have been isolated from cyanobacteria L. majuscule [17]. Declaration of Competing Interest The authors declare that they have no conflicts of interest. Acknowledgements This work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology, Japan (JSPS KAKENHI Grants JP17H02203 and 19H04649) and the TWAS-COMSTECH Research Grant Award_17-056 RG/PHA/AF/AC_C, TWAS/UNESCO, 2018. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.fitote.2020.104511. References [1] C. Okamoto, Y. Nakao, T. Fujita, T. Iwashita, R.W. van Soest, N. Fusetani, S. Matsunaga, Cytotoxic C47-polyacetylene carboxylic acids from a marine sponge Petrosia sp, J. Nat. Prod. 70 (2007) 1816–1819. [2] J.S. Kim, Y.J. Lim, K.S. Im, J.H. Jung, C.J. Shim, C.O. Lee, J. Hong, H. Lee, Cytotoxic polyacetylenes from the marine sponge Petrosia sp, J. Nat. Prod. 62 (1999) 554–559.
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