Furostane Series Asterosaponins and Other Unusual Steroid

Oct 5, 2017 - Seven new asterosaponins, pentaregulosides A–G (1–7), including two furostane-type steroid oligoglycosides (2, 3), along with four p...
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Article Cite This: J. Nat. Prod. 2017, 80, 2761-2770

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Furostane Series Asterosaponins and Other Unusual Steroid Oligoglycosides from the Tropical Starfish Pentaceraster regulus Alla A. Kicha,*,† Anatoly I. Kalinovsky,† Natalia V. Ivanchina,† Timofey V. Malyarenko,†,‡ Pavel S. Dmitrenok,† Alexandra S. Kuzmich,† Ekaterina V. Sokolova,† and Valentin A. Stonik†,‡ †

G.B. Elyakov Pacific Institute of Bioorganic Chemistry, Far East Branch of the Russian Academy of Sciences, Pr. 100-let Vladivostoku 159, 690022 Vladivostok, Russian Federation ‡ Far Eastern Federal University, Sukhanova Str. 8, 690000 Vladivostok, Russia S Supporting Information *

ABSTRACT: Seven new asterosaponins, pentaregulosides A−G (1−7), including two furostane-type steroid oligoglycosides (2, 3), along with four previously known compounds (8−11) were isolated from the ethanolic extract of the starfish Pentaceraster regulus, collected off the coast of Vietnam. The structures of 1−7 were elucidated by extensive NMR and ESIMS techniques as well as chemical transformations. The aglycons of compounds 1 and 3 have not previously been observed in starfish steroid oligoglycosides, while the aglycons of compounds 2 and 4−6 are very rare for this structural group. Compound 1 exhibited cytotoxic activity with an IC50 value of 6.4 ± 0.3 μM against RAW 264.7 murine macrophages. In contrast, nontoxic asterosaponins 3, 4, and 5 showed a potential immunomodulatory action at a concentration of 5 μM, reducing by 40%, 28%, and 55%, respectively, reactive oxygen species formation in the RAW 264.7 cells, co-stimulated with the pro-inflammatory endotoxic lipopolysaccharide from E. coli.

A

Pacific Oceans from the northern shores of Australia to the southern coast of Japan. Recently we have isolated and structurally elucidated five new polyhydroxysteroid glycosides, including one monoglycoside and four diglycosides, and five previously known related compounds from the ethanolic extract of the starfish P. regulus, collected near the Cham Islands (Vietnam) in the South China Sea.3 However, no polar steroid oligoglycosides (asterosaponins) have been isolated so far from the starfish P. regulus. Herein, we report results of the studies on the asterosaponin fraction from the same species. We describe the structures of seven new oligoglycosides, pentaregulosides A−G (1−7), which were isolated along with the earlier known four asterosaponins (8−11), as well as their cytotoxic and potential immunomodulatory effects on RAW 264.7 murine macrophage cell cultures. This fraction contains furostane-type compounds 2 and 3.

steroidea (sea star or starfish) contain structurally diverse low molecular weight natural products, which have a variety of reported pharmacological properties.1 Steroid glycosides are common secondary metabolites of starfish, resembling steroid saponins of higher plants. These starfish secondary metabolites are polar compounds, including mono-, di-, and triglycosides of polyhydroxysteroids as well as steroid oligoglycosides named asterosaponins, which never coincide with structural fragments of higher plant steroid metabolites. The majority of asterosaponins contain 3-O-sulfonated Δ9(11)3β,6α-dihydroxysteroid aglycons and carbohydrate chains, comprising usually five or six monosaccharide residues, attached at the C-6 position of the aglycon. These steroid glycosides have been reported to exhibit a wide spectrum of biological activities, such as cytotoxic, hemolytic, antiviral, antibacterial, antibiofouling, antifungal, anticancer, and cancer preventive effects and others.1 Glycosides of the furastanol series in higher plants, as a rule, contain an additional five-membered oxygen-containing ring formed by the cycle D and a part of the side chain. Unexpectedly, we have found similar compounds in starfish. Only two less polar steroid epimeric monoglycosides containing a furastanol moiety have been reported from starfish.2 The starfish Pentaceraster regulus Müller & Troschel, 1842 (order Valvatida, family Oreasteridae), also known as the “spotted starfish”, inhabits tropical waters of the Indian and © 2017 American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION The concentrated EtOH extract of P. regulus (3.15 kg, fresh weight) was subjected to sequential separation by chromatography on columns with powdered Teflon and Si gel followed by HPLC on C18 columns to yield seven new asterosaponins, named pentaregulosides A−G (1−7), along with four known asterosaponins, 8−11. The known compounds were identified Received: July 4, 2017 Published: October 5, 2017 2761

DOI: 10.1021/acs.jnatprod.7b00574 J. Nat. Prod. 2017, 80, 2761−2770

Journal of Natural Products

Article

Chart 1

by comparison of their 1H and 13C NMR and MS spectra with those reported previously for asterone analogue of thornasteroside A (8),4 protoreasteroside (9),5 thornasteroside A (10),1a,6 and acanthaglycoside C (11).7 The molecular formula of pentareguloside A (1) was determined to be C58H95O29SNa from the [M − Na]− ion peak at m/z 1287.5683 in the (−)HRESIMS spectrum and the [M + Na]+ sodium adduct ion peak at m/z 1333 in the (+)ESIMS spectrum, where M is the molecular mass of the intact sodium salt. The fragment ion peak at m/z 97 [HSO4]− in the (−)ESIMS/MS spectrum of the ion at m/z 1287 [M − Na]− showed the presence of a sulfate group in 1. The 1H, 13C, and DEPT NMR spectroscopic data attributable to the tetracyclic moiety of the aglycon of 1 (Tables 1 and 2) exhibited the resonances of protons and carbons of two angular methyls CH3-18 and CH3-19 (δH 1.39 s, 0.98 s; δC 14.7, 19.0), a 9(11) double bond (δH 5.26 m; δC 145.8, 116.4), an oxygenated methine CH-3 (δH 4.91 m; δC 77.3) bearing a sulfate group, an oxygenated methine CH-6 [δH 3.80 td (J = 11.0, 4.7); δC 80.5] bearing an O-carbohydrate chain, and an oxygenated methine CH-16 [δH 5.00 m; δC 73.7]. The chemical shifts and coupling constants of the CH3-18, CH3-19, C-9, CH11, CH-3, CH-6, and CH-16 signals were characteristic of a Δ9(11)-3β,6α,16β-trihydroxysteroid nucleus sulfonated at the C3 position and glycosylated at the C-6 position, as seen in two asterosaponins: archasteroside B8 and downeyoside K.9 The proton and carbon signals belonging to the aglycon side chain of 1 revealed the presence of one tertiary CH3 group (δH 1.49 s, δC 23.9), three secondary CH3 groups (δH 0.92 d, 0.98 d, 0, 91 d; δC 19.0, 20.2, 12.8), one oxygenated tertiary C atom (δC 72.3), and an 22,23-epoxy group [δH 3.04 d (J = 2.2), 3.11 dd (J = 2.3, 8.0); δC 65.2, 57.0]. Based on these data, a 20-hydroxy22,23-epoxy-24-methylcholestane side chain has been determined for 1. The COSY and HSQC correlations belonging to the aglycon moiety revealed the corresponding sequences of protons at C-1 to C-8, C-11 to C-12, C-8 to C-17, C-22 to C27, and C-24 to C-28. The HMBC cross-peaks H-11/C-8, C13; H-17/C-22; H3-18/C-12, C-13, C-14, C-17; H3-19/C-1, C5, C-9, C-10; H3-21/C-17, C-20, C-22; H-22/C-23, C-24; H25/C-28; H3-26/C-24, C-25, C-27; H3-27/C-24, C-25, C-26; and H3-28/C-23, C-24, C-25, C-26 supported the total structure of the steroid aglycon of 1. The key ROESY

correlations, such as H3-19/H-2β, H-4β, H-6β, H-8; H3-18/ H-8, H-12β; H-5/H-3α, H-7α; H-14/H-7α, H-12α, H-17; H11/2H-1; and H-15β/H-8 confirmed the 3β,6α,16β configurations of the oxygenated carbons and H-5α/H-8β/10β-CH3/ 13β-CH3/H-14α/H-17α configurations of the steroid nucleus in 1 (Figure 1). The (20R,22R) configurations were assumed on the basis of ROESY correlations of H3-21/H-12β and H-22/ H-16, H-17 as well as the chemical shift of H3-21 at δH 1.49.8 The configurations of stereogenic centers C-22, C-23, and C-24 were expected as (22R,23S,24S) on the basis of similarity of 1H and 13C NMR data of the aglycon side chain of 1 with those of regularoside A, in which the same configurations were determined by comparison with synthesized model compounds with a (22S,23S,24R), (22R,23R,24R), (22S,23S,24S), and (22R,23R,24S)-22,23-epoxy-24-methylcholestane side chain10 and other steroid glycosides having the same configuration of the aglycon side chains.11 The chemical shift of C-24 at δC 41.8 corresponded well to the same signal observed in the 13C NMR spectra of models with 22,23-epoxy-trans-cholestane side chains,10 while in the 13C NMR spectrum of a model with a 22,23-epoxy-cis-cholestane side chain this signal was shielded by 4.5 ppm.12 These data clearly indicated the presence of a 22,23epoxy-trans group and (22R,23S) configurations in 1 respectively. The signals of H-22 at δH 3.04 d (J = 2.2) and H-23 at δH 3.11 dd (J = 8.0, 2.3) were deshielded slightly compared to those of regularoside A probably due to the influence of the 16-hydroxy group, but the multiplicity of these signals and the corresponding coupling constants were close to the same signals of regularoside A,10 which also suggested (22R,23S) configurations of the epoxy group in 1. The chemical shifts of carbons from C-20 to C-28 were in good agreement with those of regularoside A, which also verified the (22R,23S,24S) configurations in 1. The overall structure of the side chain of 1 was confirmed additionally by the key ROESY correlations H-22/H-17, H-24; H-23/H-25, H3-26, H328; and H-24/H3-27. The 1H NMR spectrum of 1 exhibited five resonances in the deshielded region due to anomeric protons at δH 4.81, 4.90, 4.95, 5.05, and 5.26 that correlated in the HSQC experiment with corresponding carbon signals at δC 105.1, 103.7, 102.8, 106.9, and 104.6, respectively. The anomeric proton coupling constants with values of 7.2−7.9 Hz were indicative of a β2762

DOI: 10.1021/acs.jnatprod.7b00574 J. Nat. Prod. 2017, 80, 2761−2770

Journal of Natural Products

Article

Table 1. 1H NMR Spectroscopic Data (δ, J in Hz, C5D5N) for the Steroid Moieties of 1−7a position 1 2 3 4 5 6 7 8 9 10 11 12

m m brd (13.2) brq (13.0) m brd (11.8) m m td (11.0, 4.7) dt (12.0, 4.0) m brt (13.3)

2 1.63, 1.36, 2.80, 1.90, 4.90, 3.46, 1.73, 1.49, 3.85, 2.69, 1.29, 2.24,

m td (13.8, 3.6) m m m brd (12.3) m m td (10.9, 4.8) dt (12.5, 4.6) m m

3 1.62, 1.36, 2.75, 1.84, 4.86, 3.45, 1.70, 1.48, 3.82, 2.72, 1.32, 2.24,

m m m m m brd (11.2) m m m m m m

6

7

1.66, m 1.39, m 2.80, brd (12.8) 1.89, m 4.89, m 3.47, brd (12.8) 1.69, m 1.48, m 3.70, m 2.71, dt (12.8, 4.7) 1.29, m 2.15, m

4 1.65, 1.39, 2.81, 1.89, 4.90, 3.47, 1.69, 1.49, 3.78, 2.67, 1.26, 2.11,

m m brd (12.8) m m brd (12.7) q (12.4) m m dt (4.4, 12.2) m m

5

1.64, m 1.36, m 2.80, m 1.88, m 4.90 m 3.46, brd (11.8) 1.69, m 1.48, m 3.77, m 2.63, dt (12.4, 4.4) 1.23, q (12.9) 2.08, m

1.62, m 1.37, m 2.75, m 1.84, m 4.85, m 3.41, m 1.68, m 1.45, m 3.77, m 2.67, dt (12.3, 4.7) 1.27, m 2.07, m

5.26, m 2.32, dd (16.5, 5.8) 2.04, brd (16.7)

5.24, brd (5.8) 2.45, dd (16.1, 5.0) 2.01, brd (17.3)

5.25, m 2.46, m 2.03, m

5.26, brd (5.8) 2.35, m 2.12, m

5.23, brd (5.7) 2.30, dd (15.4, 5.4) 2.12, brd (15.5)

5.20, brd (5.5) 2.24, dd (16.2, 5.4) 1.98, m

5.25, brt (3.6) 2.15, m

1.16, ddd (12.2, 10.4, 8.2) 2.47, dt (13.3, 7.9) 1.64, td (13.2, 3.9) 5.00, m

1.19, m

1.21, m

1.31, m

1.26, m

1.11, m

1.32, m

2.24, m 1.50, td (13.0, 4.9) 4.57, td (7.6, 5.0)

2.26, m 1.54, m 4.58, td (7.6, 4.9)

17 18 19 20 21 22 23

1.44, d (7.0) 1.39, s 0.98, s

1.91, d (7.7) 1.44, s 1.00, s

1.93, d (7.7) 1.45, s 1.01, s

2.35, 2.08, 1.85, 1.32, 2.35, 1.13, 0.98,

m m m m m s s

2.00, 1.44, 1.76, 1.25, 2.02, 1.03, 0.96,

1.67, 1.19, 1.98, 1.40, 1.96, 1.02, 0.95,

1.76, 1.21, 2.34, 1.62, 2.51, 0.58, 0.94,

1.49, s 3.04, d (2.2) 3.11, dd (8.0, 2.3)

1.67, s 4.41, d (9.1) 4.02, dd (9.2, 4.7)

1.66, s 4.42, d (9.8) 3.95, dd (9.8, 2.8)

1.59, s

2.81, dd (8.3, 6.8)

7.40, d (15.1)

1.24, m

1.92, m

2.04, m

1.63, m

7.56, d (15.1)

25

1.74, m

2.47, m

26 27 28

0.92, d (6.2) 0.98, d (6.8) 0.91, d (6.5)

1.06, d (6.8) 1.08, d (6.8) 1.21, d (7.0)

2.00, m 1.54, m 1.06, t (7.3)

s m m m m m m

1.58, s

24

1.61, 3.75, 2.13, 1.65, 1.83, 1.36, 1.60,

13 14 15 16

a

1 1.68, 1.41, 2.81, 1.89, 4.91, 3.49, 1.70, 1.51, 3.80, 2.71, 1.30, 2.26,

0.91, d (6.6) 0.89, d (6.6)

m m m m m s s

m m m m m s s

m m brq (10.9) m t (9.4) s s

2.08, s

1.59, m 0.89, d (6.4) 0.89, d (6.4)

1.53, s 1.51, s

1.25, d (6.8)

Assignments from 1H 700.13 MHz, 13C 176.04 MHz, DEPT, COSY, HSQC, HMBC (8 Hz), and ROESY (250 ms) data. Recorded at 35 °C.

configuration for all glycosidic bonds. Four methyl doublets at δH 1.55, 1.73, 1.48, and 1.76 in the 1H NMR spectrum of 1 revealed the presence of four 6-deoxyhexsose units. The (−)ESIMS/MS spectrum exhibited fragment ion peaks at m/z 1141 [(M − Na) − C6H10O4]−, 995 [(M − Na) − 2 × C6H10O4]−, 979 [(M − Na) − C6H10O4 − C6H10O5]−, 833 [(M − Na) − 2 × C6H10O4 − C6H10O5]−, 687 [(M − Na) − 3 × C6H10O4 − C6H10O5]−, 541 [(M − Na) − 4 × C6H10O4 − C6H10O5]−, and 523 [(M − Na) − 4 × C6H10O4 − C6H10O5 − H2O]− corresponding to the successive losses of one, two, three, and four 6-deoxyhexose units and one hexose unit. These spectroscopic data showed the presence of five monosaccharide residues, four 6-deoxyhexoses and one hexose, in the carbohydrate moiety of 1. Analysis of the COSY, HSQC, HMBC, H2BC, 1D TOCSY, and ROESY experiments led to the assignment of all of the proton and carbon signals of the oligosaccharide chain of 1 (Table 3). The 1D TOCSY experiments with the irradiation of anomeric protons indicated the resonances of H-1−H-6 of one glucose unit, H-1−H-6 of

three quinovose units, and H-1−H-4 of one fucose unit, whereas the irradiation of the resonance of the corresponding methyl group resulted in a signal for H-5 of the fucose unit. The 1H and 13C NMR spectroscopic data of the oligosaccharide moiety were similar to those of terminal β-D-fucopyranosyl and β-D-quinovopyranosyl residues, and internal 3-O-substituted βD-quinovopyranosyl, 2,4-di-O-substituted β-D-quinovopyranosyl, and 2-O-substituted β-D-glucopyranosyl residues in the earlier reported spectra of the known archasterosides A and B and pectinioside A.8,13 In order to determine the absolute configurations of the monosaccharide residues, acid hydrolysis of glycoside 1 with 2 M trifluoroacetic acid (TFA) was carried out. Alcoholysis of the obtained mixture of sugars with (R)(−)-2-octanol followed by acetylation, GC analysis, and comparison with corresponding derivatives of standard monosaccharides allowed us to establish the presence of Dquinovose, D-fucose, and D-glucose units in the carbohydrate chain of 1. The attachment of the oligosaccharide chain to the steroid aglycon at C-6 and the positions of the interglycosidic 2763

DOI: 10.1021/acs.jnatprod.7b00574 J. Nat. Prod. 2017, 80, 2761−2770

Journal of Natural Products

Article

Table 2. 13C NMR Spectroscopic Data (δ, C5D5N) for the Steroid Moieties of 1−7a

a

position

1

2

3

4

5

6

7

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28

35.8, CH2 29.4, CH2 77.4, CH 30.7, CH2 49.2, CH 80.5, CH 41.4, CH2 34.9, CH 145.8, C 38.2, C 116.4, CH 42.5, CH2 41.6, C 52.0, CH 38.5, CH2 73.7, CH 61.4, CH 14.7, CH3 19.0, CH3 72.3, C 23.9, CH3 65.2, CH 57.0, CH 41.8, CH 31.6, CH 19.0, CH3 20.2, CH3 12.8, CH3

35.9, CH2 29.3, CH2 77.4, CH 30.6, CH2 49.1, CH 79.9, CH 41.4, CH2 34.1, CH 145.6, C 38.3, C 116.6, CH 41.9, CH2 41.3, C 52.9, CH 36.6, CH2 82.1, CH 66.3, CH 14.1, CH3 19.1, CH3 80.5, C 26.3, CH3 82.8, CH 74.0, CH 43.7, CH 27.5, CH 18.6, CH3 22.5, CH3 11.2, CH3

35.8, CH2 29.2, CH2 77.4, CH 30.6, CH2 49.0, CH 80.7, CH 41.5, CH2 34.2, CH 145.4, C 38.2, C 116.7, CH 41.9, CH2 41.3, C 52.9, CH 36.5, CH2 82.1, CH 66.7, CH 14.2, CH3 19.1, CH3 80.3, C 26.2, CH3 82.3, CH 75.1, CH 38.7, CH 23.1, CH2 12.4, CH3 − 16.2, CH3

35.8, CH2 29.3, CH2 77.3, CH 30.6, CH2 49.3, CH 80.3, CH 41.5, CH2 35.2, CH 145.4, C 38.1, C 116.7, CH 42.6, CH2 41.5, C 53.9, CH 22.5, CH2 25.2, CH2 54.9, CH 13.4, CH3 19.1, CH3 76.2, C 21.5, CH3 78.2, CH 29.5, CH2 37.2, CH2 28.4, CH 22.8, CH3 22.5, CH3

35.8, CH2 29.3, CH2 77.3, CH 30.6, CH2 49.3, CH 80.3, CH 41.1, CH2 35.1, CH 145.5, C 38.2, C 116.3, CH 42.1, CH2 42.0, C 53.5, CH 22.6, CH2 25.1, CH2 56.1, CH 13.3, CH3 19.0, CH3 80.6, C 24.8, CH3 215.7, C 34.5, CH2 33.0, CH2 27.6, CH 22.3, CH3 22.3, CH3

35.8, CH2 29.3, CH2 77.3, CH 30.6, CH2 49.3, CH 80.3, CH 41.3, CH2 35.0, CH 145.4, C 38.1, C 116.3, CH 42.0, CH2 42.0, C 53.4, CH 25.0, CH2 22.5, CH2 55.5, CH 19.0, CH3 13.4, CH3 79.6, C 24.3, CH3 203.6, C 119.4, CH 157.0, CH 70.1, CH 29.7, CH3 29.5, CH3

35.7, CH2 29.1, CH2 78.1, CH 30.5, CH2 49.1, CH 80.3, CH 41.5, CH2 35.4, CH 145.8, C 38.2, C 115.9, CH 40.4, CH2 42.3, C 53.4, CH 25.4, CH2 23.0, CH2 63.2, CH 12.9, CH3 19.0, CH3 207.9, C 30.8, CH3

Recorded at 35 °C.

Figure 1. Key COSY, HMBC, and ROESY correlations of compound 1.

linkages were established from long-range correlations in the ROESY and HMBC spectra, where the cross-peaks between H1 of Quip 1 and H-6 (C-6) of the aglycon, H-1 of Quip 2 and H3 (C-3) of Quip 1, H-1 of Glcp and H-4 (C-4) of Quip 2, H-1 of Fucp and H-2 (C-2) of Glcp, and H-1 of Quip 3 and H-2 (C-2)

of Quip 2 were observed. On the basis of the above-mentioned data, the structure of pentareguloside A (1) was elucidated to be sodium (20R,22R,23S,24S)-6α-O-{β-D-fucopyranosyl-(1→ 2)-β-D-glucopyranosyl-(1→4)-[β-D-quinovopyranosyl-(1→2)]β-D-quinovopyranosyl-(1→3)-β-D-quinovopyranosyl}-16β,202764

DOI: 10.1021/acs.jnatprod.7b00574 J. Nat. Prod. 2017, 80, 2761−2770

Journal of Natural Products

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Table 3. 1H and 13C NMR, HMBC, and ROESY Spectroscopic Data for the Oligosaccharide Moieties of 1−7 (δ, J in Hz, C5D5N)a 1−3b δH

position Qui 1 1

4.81, d (7.9)

2

δC

HMBC

4−7c

C6-agl

3.95, t (8.0)

105.1, CH 73.7, CH

3

3.72, t (9.0)

91.2, CH

4 5 6 Qui 2 or Xyl 1

3.52, t (9.0) 3.67, m 1.55, d (5.6)

74.5, CH 71.7, CH 18.1, CH3

C2-Qui 1, C1Qui 2 C3,C5-Qui 1

4.90, d (7.2)

2

4.07, t (8.7)

103.7, CH 81.8, CH

3 4 5

4.10, t (8.9) 3.58, t (8.4) 3.86, m

75.3, CH 86.3, CH 71.2, CH

6 Glc or Qui 2 1

1.73, d (5.7)

18.3, CH3

C4,C5-Qui 2

H4-Qui 2

4.95, d (7.8)

C4-Qui 2

2

4.05, t (8.6)

102.8, CH 84.1, CH

3 4 5 6

4.25, t (8.8) 4.11, t (9.3) 3.95, m 4.20, dd (11.4, 6.3) 4.52, dd (11.6, 2.7)

78.0, 71.1, 78.1, 62.1,

C2,C5-Glc

5.05, d (7.6)

106.9, CH 73.6, CH

C2-Glc

74.9, CH

C2-Fuc

H1,H4,H5-Fuc

72.3, CH 71.8, CH 16.9, CH3

C2,C3-Fuc C1,C4-Fuc C4,C5-Fuc

H3,H5,H6-Fuc H1,H3,H4-Fuc H4-Fuc

104.6, CH 75.5, CH 76.5, CH 75.3, CH 73.3, CH 17.6, CH3

C2-Qui 2

H5-Qui 3, H2Qui 2

Fuc 1 2 3

4.41, dd (9.4, 8.4) 4.05, dd (9.6, 3.2) 3.98, d (3.8) 3.76, q (6.7) 1.48, d (6.3)

4 5 6 Qui 3 1

5.26, d (7.2)

2 3 4 5 6

4.05, 4.05, 4.05, 3.62, 1.76,

m m m m d (6.4)

CH CH CH CH2

C1,C3-Qui 1

C4,C5-Qui 1

C3-Qui 1 C1-Qui 2, C1Qui 3 C2,C4-Qui 2 C3-Qui 2, C1-Glc

C1-Glc, C1-Fuc

C5-Glc

H3,H5-Qui 1, H6agl H4-Qui 1 H1-Qui 1, H1Qui 2 H2,H6-Qui 1 H1-Qui 1 H4-Qui 1

H5-Qui 2, H3Qui 1 H1-Qui 3

4.79, d (7.8) 3.98, dd (8.8, 8.0) 3.80, t (8.9)

δC

HMBC

ROESY

105.0, CH 73.9, CH

C6-agl C1,C3-Qui 1

H3,H5-Qui 1, H6agl H4-Qui 1

90.2, CH

C1-Xyl

H5-Qui 1, H1-Xyl

3.55, t (8.8) 3.66, m 1.55, d (5.5)

74.4, CH C5-Qui 1 71.8, CH 18.2, CH3 C4,C5-Qui 1

H2,H6-Qui 1 H1,H3-Qui 1 H4-Qui 1

5.02, d (7.7)

104.4, CH 81.6, CH

H3,H5-Xyl, H1Qui 1 H1-Qui 3

4.11, t (8.2)

C3-Qui 1 C1,C3-Xyl

4.19, t (8.9) 4.16, m 4.48, dd (11.8, 5.0) 3.78, dd (11.9, 9.9)

75.6, CH C2,C4-Xyl 78.6, CH 64.3, CH2

H1,H5-Xyl H1-Qui 2 H1,H3-Xyl

H3,H5-Glc, H4Qui 2 H4-Glc, H1-Fuc

4.91, d (7.8) 3.94, t (8.6)

101.5, CH 84.9, CH

H3,H5-Qui 2, H4Xyl H4-Qui 2, H1-Fuc

H1,H5-Glc H2,H6-Glc H1,H3-Glc H4-Glc

4.12, 3.63, 3.66, 1.49,

77.3, 75.7, 72.8, 17.9,

H3,H5-Fuc, H2Glc

4.95, d (8.1)

H1-Glc, H6-Qui 2 H1-Qui 2

C1-Fuc

C4,C5-Qui 3

δH

ROESY

4.41, dd (9.6, 7.2) 4.03, dd (9.4, 2.8) 3.97, d (3.8) 3.73, q (6.4) 1.49, d (6.7) 5.31, d (7.9) 4.07, 4.11, 4.04, 3.71, 1.76,

H6-Qui 3 H1-Qui 3 H4-Qui 3

t (9.1) t (8.8) m d (5.9)

brt (8.1) brt (10.3) brt (9.4) m d (6.6)

C4-Xyl C1,C3-Qui 2, C1Fuc

CH CH C5-Qui 2 CH CH3 C4,C5-Qui 2

106.9, CH 73.7, CH

C2-Qui-2

H1-Qui 2 H2,H6-Qui 2 H1-Qui 2 H4-Qui 2

H3,H5-Fuc, H2Qui 2

C1,C3-Fuc

74.8, CH

H1,H5-Fuc

72.4, CH 71.8, CH C4-Fuc 17.0, CH3 C4,C5-Fuc

H5,H6-Fuc H1,H3,H4-Fuc H4-Fuc

104.7, C2-Xyl CH 76.0, CH 76.6, CH 74.8, CH 73.7, CH 17.7, CH3 C4,C5-Qui 3

H2-Xyl, H5-Qui 3

H6-Qui 3 H1-Qui 3 H4-Qui 3

a

Assignments from 1H 700.13 MHz, 13C 176.04 MHz, DEPT, COSY, 1D TOCSY (200 ms), HSQC, HMBC (8 Hz), H2BC, and ROESY (250 ms) data. Recorded at 35 °C. bThe signals are selected from NMR spectra of 1. cThe signals are selected from NMR spectra of 4.

(+)ESIMS spectrum. The molecular weight and the set of fragment ion peaks in the (−)ESIMS/MS spectrum of 2 were substantially the same as compared to those of 1. A thorough analysis of the 1H, 13C, COSY, HSQC, HMBC, 1D TOCSY, and ROESY NMR spectroscopic data of compounds 2 and 1 revealed that the proton and carbon resonances of the

dihydroxy-22,23-epoxy-24-methyl-5α-cholest-9(11)-en-3β-yl sulfate. The molecular formula of pentareguloside B (2) was elucidated to be C58H95O29SNa from the [M − Na]− ion peak at m/z 1287.5680 in the (−)HRESIMS spectrum and the [M + Na]+ sodium adduct ion peak at m/z 1333 in the 2765

DOI: 10.1021/acs.jnatprod.7b00574 J. Nat. Prod. 2017, 80, 2761−2770

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Figure 2. Key COSY, HMBC, and ROESY correlations of compound 2.

C-22, and C-23 and the same but unknown configuration at C24.2 The observed ROESY correlations of the side chain protons, such as H3-21/H-16, H-17, H-23; H-22/H3-18, H-25, H3-28; H-23/H3-26, H3-28; and H3-27/H3-28, confirmed the relative configuration and total structure of the aglycon side chain of 2 (Figure 2). Thus, the structure of pentareguloside B (2) was defined to be sodium (20R,22S,23S)-6α-O-{β-Dfucopyranosyl-(1→2)-β-D-glucopyranosyl-(1→4)-[β-D-quinovopyranosyl-(1→2)]-β-D-quinovopyranosyl-(1→3)-β-D-quinovopyranosyl}-20,23-dihydroxy-16β,22-epoxy-24-methyl-5αcholest-9(11)-en-3β-yl sulfate. It is assumed that the glycoside 1 is a biogenetic precursor of glycoside 2, which formed from 1 by anti attack of the 16-hydroxy group on the 20-hydroxy22,23-epoxy-24-methyl steroid side chain.2 The molecular formula of pentareguloside C (3) was determined to be C57H93O29SNa by (−)HRESIMS, (+)ESIMS, and (−)ESIMS/MS data. The NMR spectroscopic data of 3 obtained due to extensive NMR techniques were shown to be related to those of 2 and differed only in the absence of a CH327 group in the aglycon side chain (Tables 1−3), agreeing with the molecular mass difference of 14 amu between 2 and 3. The replacement of the signal of H-25 with two signals for H2-25 and the replacement of the proton doublet of H3-26 with the proton triplet of H3-26 in the 1H NMR spectrum of 3 in comparison with those of 2 were observed. We presumed the same (20R,22S,23S) configurations of the stereogenic centers in the aglycon side chain of glycoside 3 by analogy with the side chains of glycoside 2 and downeyoside A.2 The similarity of the NMR spectroscopic data of the aglycon side chains of glycosides 2 and 3 and the presence of the cross-peaks H321/H-16, H-17, H-23; H-22/H3-18, H-25; H-23/H3-28; H-24/ H3-26; and H3-28/H-25, H3-26 in the ROESY spectrum of 3 supported the relative configuration and complete structure except the C-24 configuration of the aglycon side chain of 3. Hence, the structure of pentareguloside C was elucidated as 3. The molecular formula of pentareguloside D (4) was elucidated to be C56H93O27SNa by (−)HRESIMS, (+)ESIMS,

oligosaccharide moiety and most of the signals of the steroid nucleus of 2 were identical to those of 1, while the proton and carbon resonances of ring D, i.e., CH2-15, CH-16, CH-17, CH318, and the aglycon side chain of 2 differed from those of 1 (Tables 1−3, Figure 2). The proton and carbon resonances attributable to the aglycon side chain of 2 revealed the presence of one tertiary Me group (δH 1.67 s, δC 26.3), three secondary Me groups (δH 1.06 d, 1.08 d, 1.21 d; δC 18.6, 22.5, 11.2), one oxygenated tertiary C atom (δC 80.5), and two oxygenated CH groups [δH 4.41 d (J = 9.1), 4.02 dd (J = 9.2, 4.7); δC 82.8, 74.0]. The signals of H-16 and C-16 in the NMR spectra of 2 were shifted from δH 5.00 to 4.57 and from δC 73.7 to 82.1 in comparison with those of 1. These facts indicated that the steroid aglycon has an ether ring linking carbon atom C-16 to one of the methine groups of the side chain. Detailed comparison of 13C NMR data of steroid aglycons of glycoside 2 and earlier reported downeyosides A and B from the starfish Henricia downeyae clearly showed that 2 includes the same steroid moiety, namely, 3β,6α,16β,20,23-pentahydroxy-16,22epoxy-24-methyl-5α-cholest-9(11)-ene.2 Downeyosides A and B were defined to be C-24 methyl epimers possessing a glucuronic acid unit at the C-3 position and an O-sulfonate group at the C-6 position of the aglycon. In contrast to them, the NMR spectroscopic data of 2 indicated that an oligosaccharide chain was attached to the C-6 position and an O-sulfonate group was attached to the C-3 position of the steroid aglycon. The relative configuration of the side chain of downeyosides A and B was determined on the basis of NOEDS experiments and analysis of proton coupling constants in combination with molecular dynamics and mechanics calculations as (20R,22S,23S).2 The absolute configuration of C-24 could not be assigned by this method.2 The similarity of the proton coupling constants and the signals of the carbon atoms belonging to the aglycon side chains in the 1H and 13C NMR spectra of compound 2 and one of the two C-24 methyl epimers, i.e., downeyoside A, allowed us to assume the (20R,22S,23S) configurations of the stereogenic centers C-20, 2766

DOI: 10.1021/acs.jnatprod.7b00574 J. Nat. Prod. 2017, 80, 2761−2770

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(1→2)-β-D-quinovopyranosyl-(1→4)-[β-D-quinovopyranosyl(1→2)]-β- D -xylopyranosyl-(1→3)-β- D -quinovopyranosyl}20,22-dihydroxy-5α-cholest-9(11)-en-3β-yl sulfate. On the basis of COSY, HSQC, HMBC, ROESY, and 1D TOCSY experiments for glycosides 4−7, we determined that the oligosaccharide moiety and steroid nucleus of 4 are identical to those of glycosides 5, 6, and 7. The molecular formula of pentareguloside E (5) was determined to be C56H91O27SNa from the [M − Na]− ion peak at m/z 1227.5469 in the (−)HRESIMS spectrum and the [M + Na]+ sodium adduct ion peak at m/z 1273 in the (+)ESIMS spectrum. A thorough comparison of the NMR spectroscopic data of 5 with those of glycoside 4 clearly showed that glycosides 5 and 4 differed from each other only by the resonances of their side chains. The NMR spectroscopic data attributed to the aglycon side chain of 5 displayed signals due to one tertiary CH3 group (δH 1.58 s, δC 24.8), two secondary CH3 groups [δH 0.89 d × 2 (J = 6.4); δC 22.3 × 2], two CH2 groups [δH 2.81 dd (J = 8.3, 6.8); δC 34.5; and δH 1.63 m; δC 33.0], one oxygenated tertiary C atom (δC 80.6), and a 22-oxo group (δC 215.7) (Tables 1 and 2). The COSY and HSQC correlations allowed us to assign the proton sequence from C23 to the end of the side chain. The HMBC correlations H321/C-17, C-20, C-22; H-23/C-22, C-24; H-24/C-23, C-25, C26; H-25/C-24, C-26; H3-26/C-24, C-25, C-27; and H3-27/C24, C-25, C-26 confirmed the total structure of the 20-hydroxy22-oxo-cholestane side chain. The common (20R) configuration was supported by the ROESY correlation of H3-21/H12β. Based on these data, the structure of the aglycon of 5 was deduced as (20R)-6α,20-dihydroxy-22-oxo-5α-cholest-9(11)en-3β-yl sulfate, which was earlier reported for goniopectenoside B.16 Hence, the structure of pentareguloside E was established to be 5. The molecular formula of pentareguloside F (6) was determined to be C56H89O28SNa from the [M − Na]− ion peak at m/z 1241.5260 in the (−)HRESIMS spectrum and the [M + Na]+ sodium adduct ion peak at m/z 1287 in the (+)ESIMS spectrum. Analysis of the NMR spectra of glycosides 6 and 5 clearly indicated that these compounds differed from each other only by the signals of their side chains. The NMR spectroscopic data belonging to the aglycon side chain of 6 revealed resonances due to three tertiary CH3 groups (δH 1.59 s, 1.53 s, 1.51 s; δC 24.3, 29.7, 29.5), two oxygenated tertiary C atoms (δC 79.6, 70.1), a double bond [δH 7.40 d (J = 15.1), 7.56 d (J = 15.1); δC 119.4, 157.0], and a 22-oxo group (δC 203.6) (Tables 1 and 2). Moreover, the signals of a double bond were characteristic of a typical AB pattern and indicated the existence of a trans-α,β-unsaturated carbonyl group. A detailed comparison of the spectroscopic data of compounds 6 and 5 showed a 23,24-double bond and a 25-hydroxy group in the side chain of 6, in agreement with the molecular mass difference of 14 amu between 6 and 5. The HMBC cross-peaks H3-21/C-17, C-20, C-22; H3-26/C-24, C-25, C-27; and H3-27/ C-24, C-25, C-26 and the ROESY cross-peak H3-21/H-12β confirmed the structure of the (20R)-Δ23-20,25-dihydroxy-22oxo-cholestane side chain, which was earlier found in goniopectenoside A.16 So, the structure of pentareguloside F was determined to be as 6. The molecular formula of pentareguloside G (7) was determined to be C50H79O26SNa from the [M − Na]− ion peak at m/z 1127.4580 in the (−)HRESIMS spectrum and the [M + Na]+ sodium adduct ion peak at m/z 1173 in the (+)ESIMS spectrum. A thorough analysis of the proton and

and (−)ESIMS/MS data. The proton and carbon resonances of two angular methyls CH3-18 and CH3-19 (δH 1.13 s, 0.98 s; δC 13.4, 19.1), a 9(11) double bond (δH 5.26 brd (J = 5.8); δC 145.4, 116.7), an oxygenated methine CH-3 (δH 4.89 m; δC 77.3), bearing an O-sulfonate group, an oxygenated methine CH-6 (δH 3.70 m; δC 80.3), bearing an O-carbohydrate chain, characteristic of the steroid nucleus of most of the asterosaponins, and one tertiary methyl CH3-21 (δH 1.61 s, δC 21.5), two secondary methyls CH3-26 and CH3-27 (δH 0.91 d, 0.89 d; δC 22.8, 22.5), an oxygenated tertiary carbon C-20 (δC 76.2), and an oxygenated methine CH-22 (δH 3.75 m; δC 78.2), belonging to the steroid side chain, were observed in the 1 H and 13C NMR spectra of 4. All of the proton and carbon signals associated with the steroid moiety were assigned by COSY, HSQC, HMBC, and ROESY experiments (Tables 1 and 2). These data clearly indicated the 3β,6α,20,22tetrahydroxy-5α-cholest-9(11)-en-3β-yl sulfate as the steroid aglycon in 4, which was previously found in reticulatoside B from the starfish Oreaster reticulatus.14 The configurations of the stereogenic centers C-20 and C-22 in reticulatoside B were established as (20R,22S) by comparison of the proton and carbon chemical shifts of the methyl group CH3-21 with the corresponding reported data of four stereoisomer 5α-cholesta3β,20,22-triol models (δH 1.60 s, δC 21.9 for CH3-21 of 20R,22S-stereoisomer).14,15 So, the same (20R,22S) configurations in the side chain of 4 were elucidated on the basis of the chemical shifts of CH3-21 (δH 1.61 s, δC 21.5) and similarity of the other carbon signals of the steroid side chain in the 13C NMR spectrum of 4 to those reported for reticulatoside B.14,15 The ROESY correlations between H3-21/H3-18, H-12β also confirmed the (20R) configuration of C-20 in the side chain of 4. The examination of the 1D and 2D NMR spectra of glycosides 1 and 4 exhibited that both compounds have related oligosaccharide moieties, differing from each other in the replacement of the internal 2,4-di-O-substituted β-D-quinovopyranosyl residue for the 2,4-di-O-substituted β-D-xylopyranosyl unit and the internal 2-O-substituted β-D-glucopyranosyl residue for the 2-O-substituted β-D-quinovopyranosyl unit (Table 3). The carbon resonances of the oligosaccharide moiety of 4 were consistent with those from the previously reported 13C NMR spectrum of co-occurring protoreasteroside (9).5 The ESIMS/MS data confirmed the sequence of monosaccharide units in the oligosaccharide chain in 4. The (−)ESIMS/MS spectrum indicated a series of fragmentations with the successive losses of one, two, three, and four 6deoxyhexose units and one pentose unit at m/z 1083 [(M − Na) − C6H10O4]−, 937 [(M − Na) − 2 × C6H10O4]−, 791 [(M − Na) − 3 × C6H10O4]−, 659 [(M − Na) − 3 × C6H10O4 − C5H8O4]−, 513 [(M − Na) − 4 × C6H10O4 − C5H8O4]−, and 495 [(M − Na) − 4 × C6H10O4 − C5H8O4 − H2O]−. The Dconfigurations of all monosaccharide residues of 4 (quinovose, fucose, and xylose) were determined by hydrolysis of 4 and analysis of the acetylated 2-octylglycosides in the same manner as described for 1. The ROESY and HMBC experiments allowed us to prove the positions of the glycosidic linkages and the attachment of the carbohydrate chain to the steroid aglycon at C-6. The cross-peaks between H-1 of Qui 1 and H-6 (C-6) of the aglycon, H-1 of Xyl and H-3 (C-3) of Qui 1, H-1 of Qui 2 and H-4 (C-4) of Xyl, H-1 of terminal Fuc and C-2 of Qui 2, and H-1 of terminal Qui 3 and H-2 (C-2) of Xyl were observed. Accordingly, the structure of pentareguloside D (4) was elucidated to be sodium (20R,22S)-6α-O-{β-D-fucopyranosyl2767

DOI: 10.1021/acs.jnatprod.7b00574 J. Nat. Prod. 2017, 80, 2761−2770

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and Discovery HSC18 (10 μm, 250 mm × 10 mm) (Supelco) columns were used. GC analysis was performed on an Agilent 6580 Series chromatograph equipped with an HP-1 MS capillary column (30 m × 0.32 mm) over the temperature range 100−270 °C at 5 °C/min with the carrier gas He (1.7 mL/min); the temperatures of the injector and the detector were 250 and 270 °C, respectively. Low-pressure liquid column chromatography was carried out with Polychrom 1 (powdered Teflon, Biolar) and Si gel KSK (50−160 μm, Sorbpolimer). Sorbfil Si gel plates (4.5 × 6.0 cm, 5−17 μm, Sorbpolimer) were used for thinlayer chromatography. Animal Material. Specimens of Pentaceraster regulus Müller & Troschel, 1842 (order Valvatida, family Oreasteridae) were collected at a depth of 5−20 m using scuba at the Cham Islands (Vietnam) in the South China Sea during the 38th scientific cruise of the research vessel Akademik Oparin in May 2010. Species identification was carried out by Dr. T. I. Antokhina (Severtsov Institute of Ecology and Evolution of RAS, Moscow, Russia). A voucher specimen [no. 038-223] is on deposit at the marine specimen collection of G.B. Elyakov Pacific Institute of Bioorganic Chemistry of FEB RAS, Vladivostok, Russia. Extraction and Isolation. Freshly collected specimens of P. regulus were immediately frozen and stored at −21 °C until used. The frozen animals (3.15 kg) were cut into small pieces and extracted twice with EtOH at room temperature (1.5 L/kg). The extract was concentrated in vacuo, and the residue (100 g) was dissolved in H2O (1.5 L). The H2O-soluble fraction was passed through a Polychrom 1 column (8 × 62 cm) and eluted with distilled H2O until a negative chloride ion reaction was obtained, followed by elution with 50% aqueous EtOH. The combined aqueous EtOH eluate was evaporated to give a brownish material (4.0 g). The resulting total fraction was chromatographed over a Si gel column (7 × 18 cm) using CHCl3/ EtOH (stepwise gradient, 3:1−1:3, v/v), EtOH, and EtOH/H2O (stepwise gradient, 10:1−10:3, v/v) to yield nine main fractions (1− 9), which were then analyzed by TLC in the eluent system n-BuOH/ EtOH/H2O (4:1:2, v/v/v). Fractions 7−9 mainly contained steroid oligoglycosides (asterosaponins). Fractions 7 (59 mg), 8 (106 mg), and 9 (112 mg) were subjected to HPLC (Discovery C18 column, 55% aqueous EtOH, 1.5 mL/min) to yield subfractions 7.1−7.4, 8.1− 8.4, and 9.1. All subfractions were purified by HPLC (Discovery HSC18 column, 63% aqueous MeOH, 2.5 mL/min) to give pure 1 (3.4 mg, tR 14.1 min), 2 (3.4 mg, tR 11.0 min), 3 (1.6 mg, tR 10.3 min), 4 (1.3 mg, tR 8.7 min), 5 (1.1 mg, tR 15.9 min), 6 (1.3 mg, tR 9.7 min), 7 (2.3 mg, tR 9.1 min), 8 (9.0 mg, tR 11.8 min), 9 (1.0 mg, tR 6.6 min), 10 (4.8 mg, tR 7.6 min), and 11 (3.9 mg, tR 11.5 min). Pentareguloside A (1): amorphous, white powder, [α]20D +18 (c 0.1, MeOH); IR (KBr) νmax 3460, 1637, 1245, 1212, 1061 cm−1; 1H and 13C NMR data, Tables 1−3; HRESIMS m/z 1287.5683 [M − Na]− (calcd for C58H95O29S, 1287.5685); ESIMS m/z 1333 [M + Na]+; ESIMS/MS of the [M − Na]− ion at m/z 1287: 1269 [(M − Na) − H2O]−, 1173 [(M − Na) − C7H14O]−, 1141 [(M − Na) − C6H10O4]−, 995 [(M − Na) − 2 × C6H10O4]−, 979 [(M − Na) − C6H10O4 − C6H10O5]−, 833 [(M − Na) − 2 × C6H10O4 − C6H10O5]−, 687 [(M − Na) − 3 × C6H10O4 − C6H10O5]−, 541 [(M − Na) − 4 × C6H10O4 − C6H10O5]−, 523 [(M − Na) − 4 × C6H10O4 − C6H10O5 − H2O]−, 409 [(M − Na) − 4 × C6H10O4 − C6H10O5 − C7H14O − H2O]−, 97 [HSO4]−. Pentareguloside B (2): amorphous, white powder; [α]20D +4 (c 0.3, MeOH); IR (KBr) νmax 3437, 1637, 1246, 1212, 1061 cm−1; 1H and 13 C NMR data, Tables 1−3; HRESIMS m/z 1287.5680 [M − Na]− (calcd for C58H95O29S, 1287.5685); ESIMS m/z 1333 [M + Na]+; ESIMS/MS of the [M−Na]− ion at m/z 1287: 1141 [(M − Na) − C6H10O4]−, 995 [(M − Na) − 2 × C6H10O4]−, 979 [(M − Na) − C6H10O4 − C6H10O5]−, 833 [(M − Na) − 2 × C6H10O4 − C6H10O5]−, 687 [(M − Na) − 3 × C6H10O4 − C6H10O5]−, 541 [(M − Na) − 4 × C6H10O4 − C6H10O5]−, 523 [(M − Na) − 4 × C6H10O4 − C6H10O5 − H2O]−, 97 [HSO4]−. Pentareguloside C (3): amorphous, white powder; [α]20D +8 (c 0.1, MeOH); 1H and 13C NMR data, Tables 1−3; HRESIMS m/z 1273.5519 [M − Na]− (calcd for C57H93O29S, 1273.5529); ESIMS m/ z 1319 [M + Na]+; ESIMS/MS of the [M − Na]− ion at m/z 1273: 1127 [(M − Na) − C6H10O4]−, 981 [(M − Na) − 2 × C6H10O4]−,

carbon resonances in the 1H and 13C NMR spectra of 7 showed that this glycoside has a different side chain compared to glycosides 4−6. The presence of the distinctive proton and carbon resonances of the methyl CH3-21 (δH 2.08 s, δC 30.8) and a 20-oxo group (δC 207.9) indicated that the aglycon of 7 is the known 3β,6α-dihydroxy-5α-pregn-9(11)-en-20-one (asterone).4 All of the proton and carbon resonances of 7 were assigned from the COSY, HSQC, HMBC, and ROESY experiments and confirmed the structure of both the aglycon and the carbohydrate moieties (Tables 1−3). Therefore, the structure of pentareguloside G was established to be 7. We propose the D-configurations of fucose, quinovose, glucose, and xylose units in all co-occurring asterosaponins 2, 3, and 5−7 by analogy with glycosides 1 and 4. Thus, seven new asterosaponins, pentaregulosides A−G (1− 7), were isolated from the tropical starfish P. regulus. The steroid aglycons of asterosaponins 1 and 3, (20R,22R,23S,24S)22,23-epoxy-24-methyl-5α-cholest-9(11)-en-3β,6α,16β,20-tetraol (in 1) and (20R,22S,23S)-16β,22-epoxy-24-methyl-27-nor5α-cholest-9(11)-en-3β,6α,20,23-tetraol (in 3), have not previously been observed in starfish steroid glycosides, while the aglycons of compounds 2 and 4−6 are very rare for this structural group. The steroid moieties of 2 and 3 were determined to possess a 5α-furostane skeleton. Previously, only two epimeric monoglycosides, downeyosides A and B, of the furostane series were isolated from the starfish Henricia downeyae.2 It is known that steroid glycosides with a furostane skeleton are common in terrestrial plants. Pentaregulosides B and C along with downeyosides A and B are rare examples of steroid glycosides of the furostane series found in starfish. We have investigated the cytotoxicities and potential immunomodulatory activity of the isolated asterosaponins 1− 11 using RAW 264.7 murine macrophage cell cultures. In vitro cytotoxicities of the compounds against these cells were evaluated by the MTS assay. Only the asterosaponins 1 and 10 demonstrated cytotoxic activities with IC50 values of 6.4 ± 0.3 and 7.0 ± 0.2 μM, respectively. The other studied compounds showed no marked cytotoxic effects within the applied concentration range (1.25−10.0 μM). At a dose of 5 μM, the studied asterosaponins 1−11 were completely inactive in the induction of reactive oxygen species (ROS) formation in the RAW 264.7 cells compared to untreated cells (data not shown). Nevertheless, the glycosides 3−5 reduced the ROS levels when the cells were co-stimulated with the proinflammatory endotoxin lipopolysaccharide (LPS) from E. coli, which induces the relatively high ROS level in macrophages. In fact, new asterosaponins 3, 4, and 5 at a concentration of 5 μM decreased the ROS content in the RAW 264.7 cells by 40%, 28%, and 55%, respectively (Figure S50, Supporting Information).

■

EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were determined on a PerkinElmer 343 polarimeter. IR spectra were recorded on a Bruker OPUS Vector-22 infrared spectrophotometer. UV spectra were recorded on a UV-1601PC spectrophotometer. The 1 H and 13C NMR spectra were recorded on Bruker Avance III 500 and Bruker Avance III 700 spectrometers at 500.13 and 125.76 MHz and 700.13 and 176.04 MHz, respectively, with C5D5N used as an internal standard (δH 7.21, δC 149.65). The HRESIMS spectra were recorded on an Agilent 6510 Q-TOF LC mass spectrometer; the samples were dissolved in MeOH (c 0.001 mg/mL). HPLC separations were carried out on an Agilent 1100 Series chromatograph equipped with a differential refractometer; Discovery C18 (5 μm, 250 mm × 10 mm) 2768

DOI: 10.1021/acs.jnatprod.7b00574 J. Nat. Prod. 2017, 80, 2761−2770

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819 [(M − Na) − 2 × C6H10O4 − C6H10O5]−, 673 [(M − Na) − 3 × C6H10O4 − C6H10O5]−, 527 [(M − Na) − 4 × C6H10O4 − C6H10O5]−, 509 [(M − Na) − 4 × C6H10O4 − C6H10O5 − H2O]−, 97 [HSO4]−. Pentareguloside D (4): amorphous, white powder; [α]20D +2 (c 0.3, MeOH); 1H and 13C NMR data, Tables 1−3; HRESIMS m/z 1229.5620 [M − Na]− (calcd for C56H93O27S, 1229.5630); ESIMS m/ z 1275 [M + Na]+; ESIMS/MS of the [M − Na]− ion at m/z 1229: 1083 [(M − Na) − C6H10O4]−, 937 [(M − Na) − 2 × C6H10O4]−, 791 [(M − Na) − 3 × C6H10O4]−, 659 [(M − Na) − 3 × C6H10O4 − C5H8O4]−, 513 [(M − Na) − 4 × C6H10O4 − C5H8O4]−, 495 [(M − Na) − 4 × C6H10O4 − C5H8O4 − H2O]−, 97 [HSO4]−. Pentareguloside E (5): amorphous, white powder; [α]20D +6 (c 0.1, MeOH); 1H and 13C NMR data, Tables 1−3; HRESIMS m/z 1227.5469 [M − Na]− (calcd for C56H91O27S, 1227.5474); ESIMS m/ z 1273 [M + Na]+; ESIMS/MS of the [M − Na]− ion at m/z 1227: 1127 [(M − Na) − C6H12O]−, 1081 [(M − Na) − C6H10O4]−, 935 [(M − Na) − 2 × C6H10O4]−, 789 [(M − Na) − 3 × C6H10O4]−, 657 [(M − Na) − 3 × C6H10O4 − C5H8O4]−, 511 [(M − Na) − 4 × C6H10O4 − C5H8O4]−, 493 [(M − Na) − 4 × C6H10O4 − C5H8O4 − H2O]−, 393 [(M − Na) − 4 × C6H10O4 − C5H8O4 − C6H12O − H2O]−, 97 [HSO4]−. Pentareguloside F (6): amorphous, white powder; [α]20D ±0 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 224 (3.85) nm; 1H and 13C NMR data, Tables 1−3; HRESIMS m/z 1241.5260 [M − Na]− (calcd for C56H89O28S, 1241.5267); ESIMS m/z 1287 [M + Na]+; ESIMS/MS of the [M − Na]− ion at m/z 1241: 1127 [(M − Na) − C6H10O2]−, 1095 [(M − Na) − C6H10O4]−, 949 [(M − Na) − 2 × C6H10O4]−, 803 [(M − Na) − 3 × C6H10O4]−, 671 [(M − Na) − 3 × C6H10O4 − C5H8O4]−, 525 [(M − Na) − 4 × C6H10O4 − C5H8O4]−, 507 [(M − Na) − 4 × C6H10O4 − C5H8O4 − H2O]−, 97 [HSO4]−. Pentareguloside G (7): amorphous, white powder; [α]20D +7 (c 0.5, MeOH); 1H and 13C NMR data, Tables 1−3; HRESIMS m/z 1127.4580 [M − Na]− (calcd for C50H79O26S, 1127.4586); ESIMS m/ z 1173 [M + Na]+; ESIMS/MS of the [M − Na]− ion at m/z 1127: 981 [(M − Na) − C6H10O4]−, 835 [(M − Na) − 2 × C6H10O4]−, 689 [(M − Na) − 3 × C6H10O4]−, 557 [(M − Na) − 3 × C6H10O4 − C5H8O4]−, 411 [(M − Na) − 4 × C6H10O4 − C5H8O4]−, 393 [(M − Na) − 4 × C6H10O4 − C5H8O4 − H2O]−, 97 [HSO4]−. Acid Hydrolysis and Determination of Absolute Configurations of Monosaccharides. Determination of the absolute configurations of the monosaccharides was carried out according to the procedure of Leontein et al.17 Acid hydrolysis of 1 (1.3 mg) was carried out in a solution of 2 M TFA (0.5 mL) in a sealed vial on a H2O bath at 100 °C for 2 h. The H2O layer was washed with CHCl3 (3 × 0.5 mL) and concentrated in vacuo. One drop of concentrated TFA and 0.5 mL of (R)-(−)-2-octanol (Aldrich) were added to the sugar mixture, and the sealed vial was heated on a glycerol bath at 130 °C for 6 h. The solution was evaporated in vacuo and treated with a mixture of pyridine/acetic anhydride (1:1, 0.6 mL) for 18 h at room temperature. The acetylated 2-octylglycosides were analyzed by GC using the corresponding authentic samples prepared by the same procedure. The following peaks of four tautomeric forms (two pyranoses and two furanoses) for each monosaccharide were detected in the hydrolysate of 1: D-quinovose (tR 18.90, 19.08, 19.43, and 19.72 min), D-fucose (tR 18.90, 19.43, and 19.80 min), and D-glucose (tR 22.88, 23.49, 23.73, and 24.04 min). Acid hydrolysis of 4 (1.2 mg) and formation of the acetylated 2octylglycosides were carried out in the same manner as described for 1. The following peaks were detected by GC in the hydrolysate of 4: Dquinovose (tR 18.89, 19.09, 19.44, and 19.72 min), D-fucose (tR 18.89, 19.44, and 19.81 min), and D-xylose (tR 19.27, 19.44, and 19.72 min). The retention times of the authentic samples were as follows: Dquinovose (tR 18.89, 19.12, 19.47, and 19.75 min), D-fucose (tR 19.00, 19.51, and 19.88 min), D-glucose (tR 22.94, 23.58, 23.82, and 24.11 min), D-xylose (tR 19.51, 19.64, and 20.03 min), L-quinovose (tR 18.73, 19.20, and 19.77 min), L-fucose (tR 19.20, 19.30, 19.44, and 19.58 min), L-glucose (tR 23.13, 23.38, and 24.12 min), and L-xylose (tR 19.14, 19.67, and 19.95 min).

Cell Culture. The RAW 264.7 murine macrophage cell line was obtained from the ATCC collection. The cells were cultured in DMEM supplemented with 10% fetal bovine serum at 37 °C under a humidified 5% CO2 atmosphere in an incubator (MCO-18AIC, Sanyo). Cytotoxicity Assay (MTS Test). The effect of the compounds on the cells viability was evaluated using the MTS test, which is based on the reduction of MTS into its formazan product by living cells.18 Briefly, cells were cultured for 2 h in 96-well plates (6 × 103 cells per well), 200 μL/well. The medium was then replaced with fresh medium containing substances at various concentrations in a total volume of 200 μL/well, and the cells were incubated for 24 h. Subsequently 20 μL of Cell Titer 96 Aqueous One Solution Reagent was added into each well, and MTS reduction was measured 2 h later spectrophotometrically at 492 and 690 nm as background using μQuant equipment (Bio-Tek Instruments). Results are represented as IC50 values (inhibition concentration 50%) of the substances. Immunomodulatory Activity. For immunomodulatory activity, RAW 264.7 murine macrophages were plated into 96-well plates and incubated at 37 °C with 5% CO2 for 2 h. After adhesion, cells were incubated with the test compounds (5 μM) for 24 h. To study ROS formation, 20 μL of 2,7-dichlorodihydrofluorescein diacetate solution (Sigma-Aldrich, final concentration 10 μM) was added to each well, and the plate was incubated for an additional 10 min at 37 °C with 5% CO2. In each experiment test compounds were co-incubated with (or without) LPS from E. coli serotype 055:B5 (Sigma, 1.0 μg/mL). The intensity of dichlorofluorescein fluorescence was measured at λex/λem 485 nm/518 nm using a PHERAstar FS plate reader (BMG Labtech). Statistical Analysis. All assays were performed at least in triplicate. All data were expressed as mean ± standard deviation. Statistical analysis was done by one-way Anova. Differences were considered to be statistically significant if p < 0.05.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.7b00574. Figures S1−S49: 1H, 13C, COSY, HSQC, HMBC, ROESY, and HRESIMS spectra of compounds 1−7; Figure S50: influence of 1−7 on ROS level in RAW 264.7 murine macrophages, co-incubated with LPS from E. coli; Table 4: GC chromatograms of the acetylated 2octylglycosides of the individual monosaccharides and the hydrolysates of compounds 1 and 4 (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Tel: +7(423)2311168. Fax: +7(423)2314050. E-mail: kicha@ piboc.dvo.ru, [email protected]. ORCID

Alla A. Kicha: 0000-0002-1371-0103 Valentin A. Stonik: 0000-0002-8213-8411 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The work on the isolation and study of the chemical structures of substances was partially supported by Grant Nos. 17-0400034-a and 16-54-540003 Viet_a from the RFBR (Russian Foundation for Basic Research). The mass spectrometric study was supported by Grant No. 16-13-10185 from the RSF (Russian Science Foundation). The authors are grateful to Dr. T. I. Antokhina (Severtsov Institute of Ecology and Evolution 2769

DOI: 10.1021/acs.jnatprod.7b00574 J. Nat. Prod. 2017, 80, 2761−2770

Journal of Natural Products

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of the RAS, Moscow, Russia) for species identification of the starfish.

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DOI: 10.1021/acs.jnatprod.7b00574 J. Nat. Prod. 2017, 80, 2761−2770