Anthenosides LâU, Steroidal Glycosides with ... - ACS Publications

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Anthenosides L−U, Steroidal Glycosides with Unusual Structural Features from the Starfish Anthenea aspera Timofey V. Malyarenko,† Sofiya D. Kharchenko,† Alla A. Kicha,† Natalia V. Ivanchina,† Pavel S. Dmitrenok,† Ekaterina A. Chingizova,† Evgeny A. Pislyagin,† Evgeny V. Evtushenko,† Tatyana I. Antokhina,‡ Chau Van Minh,§ and Valentin A. Stonik*,†,⊥ †

G.B. Elyakov Pacific Institute of Bioorganic Chemistry, Far Eastern Branch of Russian Academy of Sciences, Pr. 100-let Vladivostoku 159, 690022 Vladivostok, Russian Federation ‡ Severtsov Institute of Ecology and Evolution, Russian Academy of Sciences, Leninskii Pr. 33, 119071 Moscow, Russian Federation § Institute of Marine Biochemistry, Vietnam Academy of Science and Technology, 18 Hoang Quoc Viet, Caugiay, Hanoi, Viet Nam ⊥ Far Eastern Federal University, Sukhanova Str. 8, 690000 Vladivostok, Russian Federation S Supporting Information *

ABSTRACT: Ten new polyhydroxysteroidal glycosides, anthenosides L−U (1−10), with rare positions of carbohydrate fragment attachments, were isolated from the starfish Anthenea aspera. The structures of 1−10 were established by NMR and ESIMS techniques as well as by chemical transformations. The unoxidized Δ22-24-nor-cholestane (1), (24S)-Δ22-24methylcholestane (2−5), and Δ22-cholestane (7) side chains of the steroidal aglycons, 3-O-methyl-β-D-galactofuranosyl residue (2, 8), and 5α-cholest-8(14)-ene-3α,7β,16α-trihydroxysteroidal nucleus (9, 10) have not been found previously in starfish polar steroidal compounds. The mixture of glycosides 9 and 10 showed hemolytic activity with an EC50 = 8 μM. Compound 4 at a dose of 10 μM exhibited a potential immunomodulatory action, decreasing by 24% the intracellular ROS content in RAW 264.7 murine macrophages, induced by pro-inflammatory endotoxic lipopolysaccharide from E. coli.

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attachment positions of carbohydrate fragments at C-16 or together at C-16 and C-7 of the aglycons, Δ 8(14) 3β,4β,6β,7β,16α-pentahydroxy or Δ8(14)-3β(α),6β,7β,16α-tetrahydroxy substitutions, and unoxygenated side chains in aglycons. Herein, we report the results of the our studies on steroidal glycosides from the ethanolic extract of the tropical starfish Anthenea aspera (order Valvatida, family Oreasteridae), collected at Tu Long Bay near Khuan Lan Island in the South China Sea. As a result, we have established the structures of 10 new polyhydroxysteroidal glycosides, anthenosides L−U (1−10), having some structural similarity with glycosides from the starfish A. chinensis.2,3 In addition, we have studied hemolytic and cytotoxic effects of the glycosides against

olar steroidal metabolites from starfish (phylum Echinodermata, class Asteroidea) are characterized by a remarkable diversity and include polyhydroxysteroids and related mono-, di-, and triglycosides as well as steroidal oligoglycosides named asterosaponins.1 The polar steroids have been reported to show a wide spectrum of biological activities, such as hemolytic, cytotoxic, antibiofouling, neuritogenic, antiinflammatory, antitumor, and cancer preventive effects.1 Generally, polyhydroxysteroidal glycosides from starfish have oxygenated steroidal aglycons with the number of hydroxy groups ranging from three to nine, and one, two, and rarely three monosaccharide residues attached to C-3 in the steroidal nucleus and/or to C-24, C-26, C-28, and C-29 in the side chain. In most cases monosaccharides of the polyhydroxysteroidal glycosides are presented by β-D-xylopyranosyl and α-Larabinofuranosyl residues or their methylated or sulfonated derivatives. In contrast with a great majority of these secondary metabolites, anthenosides A−K, recently isolated from the starfish Anthenea chinensis,2,3 have atypical structure fragments: © XXXX American Chemical Society and American Society of Pharmacognosy

Received: July 18, 2016

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DOI: 10.1021/acs.jnatprod.6b00667 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

Δ8(14)-steroid moiety of 1. The key ROESY cross-peaks H-5/ Hα-1, H-9; H3-19/Hβ-2, Hβ-4, Hβ-11; H3-18/Hβ-11; H-6/ Hα-4; H-7/H-5, Hα-15; and H-16/H3-18 showed the common 5α-H/9α-H/10β-CH3/13β-CH3 configurations of the steroid nucleus and the 6β,7β,16α-configurations of oxygenated substituents in 1 (Figure 1). The NMR spectra of the aglycon side chain indicated the existence of three secondary methyls, CH3-21 [δH 1.08 d (J = 7.1); δC 23.8], CH3-26 [δH 0.97 d (J = 6.7); δC 23.1], and CH3-27 [δH 0.97 d (J = 6.8); δC 23.2], and a 22(23) double bond [δH 5.72 ddd (J = 15.3, 8.7, 1.2), 5.30 dd (J = 15.3, 6.6); δC 133.8, 137.3)]. The proton sequence from H-17 to H-27, correlated with the corresponding carbon atoms of the side chain of 1, was assigned using the COSY and HSQC experiments (Tables 1 and 3). The HMBC correlations H3-21/ C-17, C-20, C-22; H-22/C-25; H-23/C-20, C-25, C-26, C-27; H-25/C-22, C-23, C-26, C-27; H3-26/C-23, C-25, C-27; and H3-27/C-23, C-25, C-26 and ROESY correlations H-23/H-20, H3-26, H3-27 supported the total structure of the unoxygenated Δ22-24-nor-cholestane side chain (Figure 1). The 20Rconfiguration was assumed on the basis of ROESY correlations of H3-18/H-20, H3-21 and H3-21/Hβ-12, H-22/H-16 as well as the chemical shift of H3-21 at δH 1.08 (δH 1.10 for 20R-Δ22 and δH 1.00 for 20S-Δ22 steroids4). The trans configuration of the 22(23)-double bond followed from J22,23 = 15.3 Hz. The 1H NMR spectrum of 1 exhibited one resonance signal in the deshielded region due to an anomeric proton of the monosaccharide unit at δH 4.31, correlated in the HSQC experiment with a carbon signal at δC 102.5. The NMR spectroscopic data indicated the presence of an O-methylhexose unit in compound 1. The chemical shifts and coupling constants of H-1′−H-6′ of a monosaccharide unit were determined by the irradiation of the anomeric H atom in the 1D TOCSY experiment. The coupling constant (7.7 Hz) of the anomeric H atom corresponded to the β-configuration of the glycosidic bond. The C and H signals and the corresponding coupling constants of the monosaccharide unit coincided well with those of a terminal 3-O-methyl-β-glucopyranosyl residue.5 The attachment of the 3-O-methyl-β-glucopyranosyl residue to the steroidal aglycon at C-16 was determined by the HMBC and ROESY spectra, where the HMBC cross-peak between H1′ of the sugar and C-16 of the aglycon was observed, and the ROESY cross-peak between H-1′ of the sugar and H-16 of the aglycon was observed (Figure 1). The D-configuration of the 3O-methyl-β-glucopyranose unit was proposed by analogy with co-occurring glycoside 6, in which it was firmly established (Experimental Section). On the basis of these results, the structure of anthenoside L was determined as (20R,22E)-16-O(3-O-methyl-β-D-glucopyranosyl)-24-nor-5α-cholesta-8(14),22(23)-diene-3α,6β,7β,16α-tetraol (1). The molecular formula of compound 2 was determined to be C35H58O9 by (+)HRESIMS. The comparison of the molecular masses of 1 and 2 showed that the difference between 1 and 2 is 28 amu. The 1H and 13C NMR data of 2 differed from those of 1 only by the signals of the steroidal side chain and monosaccharide residue. The NMR data for the side chain indicated the existence of four secondary methyls, CH3-21 [δH 1.09 d (J = 7.1); δC 24.7], CH3-26 [δH 0.88 d (J = 6.8); δC 20.6], CH3-27 [δH 0.85 d (J = 6.7); δC 20.1], and CH3-28 [δH 0.95 d (J = 6.9); δC 18.5], and the 22(23) double bond [δH 5.62 dd (J = 15.4, 9.2), 5.27 dd (J = 15.4, 8.5); δC 135.6, 135.3]. The proton sequence from H-17 to H-28, correlated with the corresponding carbon atoms of the side chain of 2, was assigned using the COSY and HSQC experiments. The HMBC

mouse erythrocytes, splenocytes, and cancer cells and immunomodulatory activities on macrophages.

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RESULTS AND DISCUSSION The concentrated ethanolic extract of the starfish A. aspera was subjected to sequential separation by chromatography on columns with Polychrom-1 and Si gel followed by HPLC on semipreparative Diasorb-130-C16T and analytical Discovery C18 columns to yield 10 new pure glycosides 1−10, named anthenosides L−U.

The molecular formula of compound 1 was determined to be C33H54O9 from the [M + Na]+ sodiated adduct ion peak at m/z 617.3662 in the (+)HRESIMS spectrum. The 1H, 13C, and DEPT NMR spectroscopic data belonging to the tetracyclic moiety of the aglycon of 1 showed the resonances of protons and carbons of two angular methyls, CH3-18 and CH3-19 (δH 0.91 s, 0.84 s; δC 20.5, 15.4), an 8(14) double bond (δC 128.2, 147.2), three oxygenated methines, CH-3 (δH 4.08 m; δC 67.4), CH-6 [δH 3.54 t (J = 2.7); δC 77.2], CH-7 [δH 4.27 d (J = 2.7); δC 72.0], and one oxygenated CH-16 group bearing a monosaccharide residue [δH 4.62 td (J = 9.0, 5.1); δC 78.9]. The chemical shifts and coupling constants of the CH-3, CH-6, CH-7, and CH-16 signals and the width of the multiplet of H-3 of about 10 Hz characteristic of a 3α-OH (vs more then 30 Hz for 3β-OH) were similar to the corresponding signals in the NMR spectra of anthenosides E and G3 and were indicative of a 3α,6β,7β,16α-tetrahydroxysteroidal nucleus glycosylated at C16 position in 1 (Tables 1 and 3). The COSY and HSQC correlations attributable to the steroidal nucleus revealed the corresponding sequences of protons at C-1 to C-7, C-9 to C-12 through C-11, and C-15 to C-17. The HMBC cross-peaks H-6/ C-8, C-10; H-7/C-9, C-14; H-9/C-8; H-15/C-8, C-13, C-14; H-17/C-13, C-18; H3-18/C-12, C-13, C-14, C-17; and H3-19/ C-1, C-5, C-9, C-10 confirmed the overall structure of the B



Article

Table 1. 1H NMR Spectroscopic Data (δ, J in Hz, CD3OD) for Anthenosides L−P (1−5)a position 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 29 1′ 2′ 3′ 4′ 5′ 6′ OCH3

1 1.54, 1.30, 1.62, 4.08, 1.97, 1.38, 2.15, 3.54, 4.27,

m m m m td (13.5, 2.7) br d (13.5) m t (2.7) d (2.7)

2 1.53, 1.31, 1.62, 4.08, 1.97, 1.38, 2.16, 3.53, 4.25,

m m m m td (13.7, 2.9) m m t (2.8) d (2.8)

3 1.53, 1.37, 1.62, 4.09, 1.97, 1.37, 2.15, 3.53, 4.25,

m m m m m m m m d (2.6)

m m m br s td (13.5, 2.9) br d (13.5) dt (13.5, 2.3) t (2.3) d (2.3)

5 1.53, 1.29, 1.62, 4.08, 1.97, 1.36, 2.12, 3.61, 4.19,

m m m m m m dt (13.7, 2.9) t (2.9) d (2.9)

2.25, m

2.25, m

2.25, m

2.25, m

2.26, m

1.65, 1.53, 1.83, 1.26,

m m dt (12.1, 3.2) m

1.66, 1.53, 1.79, 1.20,

m m dt (12.2, 3.5) m

1.67, 1.52, 1.80, 1.20,

m m dt (12.5, 3.3) m

1.66, 1.52, 1.83, 1.24,

m m dt (12.4, 3.3) m

1.65, 1.53, 1.79, 1.22,

m m m m

2.86, 2.48, 4.62, 1.51, 0.91, 0.84, 2.38, 1.08, 5.72, 5.30,

ddd (17.0, 9.0, 3.1) ddd (17.0, 5.1, 2.0) td (9.0, 5.1) dd (9.0, 4.2) s s m d (7.1) ddd (15.3, 8.7, 1.2) dd (15.3, 6.6)

2.94, 2.39, 4.51, 1.48, 0.89, 0.83, 2.40, 1.09, 5.62, 5.27, 1.96, 1.50, 0.88, 0.85, 0.95,

ddd (16.7, 8.8, 3.0) m ddd (10.0, 8.8, 5.9) dd (10.0, 2.2) s s m d (7.1) dd (15.4, 9.2) dd (15.4, 8.5) m m d (6.8) d (6.7) d (6.9)

2.95, 2.41, 4.53, 1.50, 0.89, 0.83, 2.42, 1.11, 5.66, 5.27, 1.98, 1.50, 0.88, 0.86, 0.96,

ddd (11.8, 8.9, 3.3) m m m s s m d (7.2) dd (15.5, 9.0) dd (15.5, 8.3) m m d (6.6) d (6.7) d (6.7)

2.87, 2.48, 4.64, 1.51, 0.92, 0.84, 2.40, 1.09, 5.76, 5.26, 1.97, 1.51, 0.87, 0.85, 0.95,

ddd (17.1, 8.6, 3.0) ddd (17.1, 5.1, 1.8) td (8.6, 5.1) m s s m d (7.1) dd (15.4, 8.9) dd (15.4, 8.0) m m d (6.8) d (6.8) d (6.8)

2.90, 2.56, 4.53, 1.50, 0.90, 0.84, 2.43, 1.13, 5.66, 5.27, 1.99, 1.50, 0.88, 0.86, 0.96,

ddd (17.0, 8.7, 2.8) ddd (17.0, 5.9, 2.3) m m s s m d (7.2) dd (15.5, 9.4) dd (15.5, 8.2) m m d (6.8) d (6.8) d (6.8)

2.26, m 0.97, d (6.7) 0.97, d (6.8)

3-OMe-β-D-Glcp 4.31, d (7.7) 3.21, dd (9.0, 7.7) 3.08, t (9.0) 3.37, dd (9.8, 9.0) 3.27, ddd (9.8, 5.6, 2.7) 3.88, dd (11.6, 2.7) 3.70, dd (11.6, 5.6) 3.62, s

3-OMe-β-D-Galf 4.98, br s 4.01, dd (2.5, 1.2) 3.76, dd (5.5, 2.5) 4.08, dd (5.5, 3.0) 3.75, m 3.66, br d (6.5) 3.65, br d (5.3) 3.41, s

6-OMe-β-D-Galf 4.95, br d (2.2) 3.91, dd (4.4, 2.2) 4.07, dd (6.7, 4.4) 3.96, dd (6.7, 2.3) 3.87, m 3.53, m 3.39, s

1″ 2″ 3″ 4″ 5″ 6″ OCH3 a

4 1.54, 1.31, 1.62, 4.09, 1.97, 1.38, 2.16, 3.54, 4.27,

3-OMe-β-D-Glcp 4.32, d (7.8) 3.21, dd (9.0, 7.8) 3.08, t (9.0) 3.38, m 3.27, ddd (9.8. 5.4, 2.6) 3.88, dd (11.5, 2.6) 3.72, dd (11.5, 5.4) 3.63, s

6-OMe-β-D-Galf 4.97, br d (2.2) 3.98, dd (4.4, 2.2) 4.06, dd (6.8, 4.4) 3.96, dd (6.8, 2.4) 3.87, br d (2.4) 3.54, m 3.39, s 6-OMe-β-D-Galf 4.98, br d (1.9) 3.91, dd (3.8, 1.9) 3.95, dd (6.2, 3.8) 3.87, dd (6.2, 4.0) 3.84, dd (5.9, 4.0) 3.53, d (5.9) 3.39, s

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

for the 24R-configuration in the 13C NMR spectra of mycalosides A and K having similar side chains6,7). The trans configuration of the 22(23)-double bond followed from J22,23 = 15.4 Hz. Thus, this structural fragment of glycoside 2 was determined to be a (20R,22E,24S)-24-methylcholestane side chain.

and ROESY correlations supported the total structure of the unoxidized Δ22-24-methylcholestane side chain (Tables 1 and 3). The 20R-configuration was assumed in the same manner as for glycoside 1 on the basis of ROESY correlations of H3-18/H20, H3-21 and H-22/H-16 and the chemical shift of H3-21 at δH 1.09.4 The S-configuration at C-24 was suggested based on the chemical shift of C-28 at δC 18.5 (δC 18.1 for the 24S- and 17.4 C



Article

Table 2. 1H NMR Spectroscopic Data (δ, J in Hz, CD3OD) for the Anthenosides Q−U (6−10) position 1 2 3 4

5 6

7 8 9 10 11 12

13 14 15

6 1.53, m 1.31, m 1.62, m 4.08, m 1.95, td (13.7, 2.8) 1.37, m

mixture of 9 and 10 m m m t (2.6) m

2.12, dt (13.6, 2.7) 3.62, t (2.7)

2.16, m

7

4.27, br d (2.8)

4.23, d (2.6)

4.22, d (2.7)

2.27, m

2.26, m

2.26, m

2.25, m

4′

3.31, m

3.38, m

1.64, m 1.53, m 1.87, dt (12.7, 3.3) 1.31, m

1.65, m 1.53, m 1.81, dt (12.2, 3.2) 1.25, m

1.65, m 1.54, m 1.80, dt (12.6, 3.3) 1.24, m

1.65, m 1.46, m 1.79, dt (12.4, 3.4) 1.24, m

5′

3.26, m

6′

3.88, dd (11.7, 2.5) 3.65, dd (11.7, 5.8) 3.62, s 6-OMe-β-DGalf 5.05, br d (2.2) 3.90, dd (3.0, 2.2) 3.94, dd (5.9, 3.0) 3.92, dd (5.9, 3.1) 3.82, ddd (7.0, 4.8, 3.1) 3.53, d (4.8)

3.27, ddd (10.0, 5.4, 2.6) 3.88, dd (11.7, 2.6) 3.72, dd (11.7, 5.4) 3.63, s 6-OMe-β-DGalf 5.02, br d (2.1) 3.90, m

dd 3.0) (14.0,

1.53, 1.39, 1.62, 3.98, 1.46,

6

1.56, dt (14.0, 2.6) 1.24, m 4.39, t (2.6)

(14.0,

1.53, m 1.29, m 1.61, m 4.08, m 1.96, td (13.5, 2.6) 1.37, m

position

2.13, dt (13.4, 2.8) 3.64, t (2.8)

24 25 26 27 28

23

1.41, m 1.46, m 1.17, m

2.86, ddd (17.5, 9.0, 3.2) 2.64, ddd (17.5, 5.0, 2.1) 4.62, td (9.0, 5.0) 1.53, m 0.91, s 0.84, s 2.41, m 1.11, d (6.9) 5.79, dd (15.3, 9.0) 5.34, dd (15.3, 7.3)

1.52, 1.18, 1.54, 0.88, 0.88,

m m m d (6.6) d (6.6)

1.91, m

2.78, ddd (17.0, 8.4, 3.2) 2.55, ddd (17.0, 5.0, 2.5) 4.46, m

3-OMe-β-DGlcp 4.33, d (7.9) 3.23, dd (9.2, 7.9) 3.09, t (9.2)

6-OMe-β-D-Galf

3′

3-OMe-β-DGlcp 4.32, d (7.7) 3.21, dd (9.0, 7.7) 3.09, t (9.0)

1.42, m 2.22, m

1.27, m 1.48, m

1.92, m

1.08, m

1″ 2″ 3″

1.46, m 0.90, 0.67, 1.59, 1.04, 1.62,

m d (6.9) d (6.9) br s br d (1.3)

29

1′ 2′

4″

s s m d (6.9) m

5″ 6″

4.99, br d (1.5) 3.92, dd (3.7, 1.5) 3.95, dd (6.0, 3.7) 3.87, dd (6.0, 3.7) 3.84, dd (5.8, 3.7) 3.53, d (5.8)

3.39, s 3-OMe-β-D-Galf

3.83, ddd (7.1, 4.6, 2.0) 3.52, m

4.99, br s 4.07, dd (3.0, 1.7) 3.73, dd (6.0, 3.0) 3.98, dd (6.0, 3.0) 3.69, dd (6.0, 3.0) 3.59, d (6.0)

3.38, s

3.40, s

3.94, dd (6.0, 3.7) 3.91, m

3.52, d (7.0) OCH3

3.38, s

mixture of 9 and 10 1.04, m

2.26, 1.04, 1.04, 4.74, 4.71,

1.36, m

2.88, ddd (17.0, 9.0, 2.8) 2.58, ddd (17.0, 6.0, 2.1) 4.45, td (9.0, 6.0) 1.45, dd (9.0, 4.0) 0.92, s 0.85, s 1.65, m 1.04, d (6.9) 1.78, m

8

1.59, m 0.88, d (6.7) 0.89, d (6.7)

OCH3

18 19 20 21 22

17

8

1.53, m 1.30, m 1.61, m 4.07, m 1.96, td 3.0) 1.37, br (14.0, 2.12, dt 3.0) 3.62, m

2.84, ddd (17.2, 8.7, 2.9) 2.68, ddd (17.2, 4.3, 1.6) 4.54, ddd (10.0, 8.7, 4.3) 1.50, dd (8.7, 6.5) 0.94, s 0.85, s 1.69, m 1.01, d (6.8) 1.67, m

16

7

a

1.74, m 0.86, d (6.8) 0.85, d (6.7) 1.36, m 1.20, m 0.89, t (7.5) β-D-Galf 4.95, br d (2.3) 3.95, dd (4.6, 2.3) 4.03, dd (7.0, 4.6) 3.88, dd (7.0, 2.8) 3.73, ddd (7.6, 4.6, 2.8) 3.64, dd (11.3, 7.6) 3.61, dd (11.3, 4.6) 6-OMe-β-DGalf 4.99, br d (2.0) 3.93, dd (3.8, 2.0) 3.94, m 3.89, dd (5.9, 3.8) 3.83, ddd (7.3, 4.5, 3.8) 3.53, dd (10.1, 4.5) 3.50, dd (10.1, 7.3) 3.38, s

a

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

The NMR spectroscopic data indicated the presence of an Omethylhexose unit in compound 2. The 1H NMR spectrum of 2 exhibited one resonance in the deshielded region due to an anomeric proton of a monosaccharide unit at δH 4.98, correlated in the HSQC experiment with a carbon signal at δC 108.3 as well as one resonance due to O-methyl protons of the monosaccharide unit at δH 3.41, correlated in the HSQC experiment with a carbon signal at δC 58.1. The chemical shifts and coupling constants of H-1′−H-6′ and chemical shifts of C1′−C-6′ of an O-methylhexose unit were determined by COSY and HSQC experiments (Tables 1 and 3). The HMBC correlations H-1′/C-3′, C-4′; H-3′/C-2′, O-CH3; H-4′/C-3′; H-5′/C-6′; H-6′/C-4′, C-5′; and O-CH3 /C-3′ and ROESY correlations H-3′/H-5′, O-CH3 and H-4′/H-6′ supported the total structure of the 3-O-methylgalactofuranosyl residue. The chemical shift of the anomeric C atom at δC 108.3 corresponded to the β-configuration of the glycosidic bond (δC 109.2 for β-O-methylgalactofuranose and δC 103.1 for α-Omethylgalactofuranose8). Acid hydrolysis of glycoside 2 with 2 M trifluoroacetic acid (TFA) was carried out to ascertain the

stereochemical series of its monosaccharide unit. Alcoholysis of the obtained monosaccharide by (R)-(−)-2-octanol followed by acetylation, GC analysis, and comparison with the corresponding derivatives of standard monosaccharides allowed us to establish the D-configuration of the 3-O-methyl-β-galactofuranose. The attachment of the 3-O-methyl-β-galactofuranosyl residue to the steroidal aglycon was confirmed by the HMBC and ROESY spectra, where the HMBC cross-peak between H1′ of the sugar and C-16 of the aglycon was observed, and the ROESY cross-peak between H-1′ of the sugar and H-16 of the aglycon was observed. On the basis of the above-mentioned data, the structure of anthenoside M was elucidated to be 2. The molecular formula of compound 3 was determined to be C35H58O9 from the [M + Na]+ sodiated adduct ion peak at m/z 645.3970 in the (+)HRESIMS spectrum. The fragment ion peak at m/z 469 [(M + Na) − C7H12O5]+ in the (+)ESIMS/ MS spectrum from the precursor ion at m/z 645 [M + Na]+ showed the presence of an O-methylhexose unit in 3. The thorough comparison of the 1H and 13C NMR data of compound 2 with those of 3 revealed that they differed from D



Article

Table 3. 13C NMR Spectroscopic Data (δ, CD3OD) for Anthenosides L−U (1−10) position 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 29

1′ 2′ 3′ 4′ 5′ 6′ OCH3

1 34.6, CH2 29.5, CH2 67.4, CH 33.4, CH2 37.7, CH 77.2, CH 72.0, CH 128.2, C 45.8, CH 38.6, C 19.5, CH2 37.4, CH2 45.1, C 147.2, C 34.0, CH2 78.9, CH 63.0, CH 20.5, CH3 15.4, CH3 37.3, CH 23.8, CH3 133.8, CH 137.3, CH 32.3, CH 23.1, CH3 23.2, CH3

3-OMe-β-DGlcp 102.5, CH 75.1, CH 88.0, CH 71.7, CH 77.8, CH 63.2, CH2 61.0, CH3

2

3

4

5

6

7

8

34.5, CH2 29.5, CH2 67.4, CH 33.4, CH2 37.8, CH 77.3, CH 72.1, CH 128.4, C 45.8, CH 38.7, C 19.3, CH2 36.8, CH2 44.9, C 146.7, C 33.6, CH2 76.9, CH 62.6, CH 20.7, CH3 15.3, CH3 37.1, CH 24.7, CH3 135.6, CH 135.3, CH 44.5, CH 34.6, CH 20.6, CH3 20.1, CH3 18.5, CH3




34.6, CH2 29.6, CH2 67.5, CH 33.3, CH2 37.9, CH 75.5, CH 78.7, CH 126.6, C 45.7, CH 38.9, C 19.6, CH2 37.5, CH2 45.4, C 148.1, C 34.7, CH2 80.2, CH 62.7, CH 19.8, CH3 15.5, CH3 34.0, CH 20.9, CH3 35.6, CH2 25.8, CH2 40.7, CH2 29.2, CH 23.3, CH3 23.1, CH3

34.6, CH2 29.7, CH2 67.5, CH 33.3, CH2 38.0, CH 75.2, CH 78.6, CH 126.7, C 46.0, CH 38.9, C 19.5, CH2 37.2, CH2 45.4, C 147.6, C 34.5, CH2 79.6, CH 62.8, CH 20.2, CH3 15.4, CH3 37.3, CH 24.0, CH3 138.1, CH 128.8, CH 43.2, CH2 29.9, CH 22.9, CH3 22.9, CH3

34.5, CH2 29.7, CH2 67.5, CH 33.1, CH2 38.0, CH 75.2, CH 78.3, CH 127.0, C 45.9, CH 38.8, C 19.5, CH2 37.2, CH2 44.9, C 147.2, C 33.3, CH2 76.9, CH 62.8, CH 20.1, CH3 15.4, CH3 32.8, CH 21.4, CH3 33.5, CH2 33.8, CH2 157.7, C 34.8, CH 22.5, CH3 22.3, CH3 107.2, CH2

3-OMe-β-DGalf 108.3, CH 80.8, CH 88.8, CH 84.5, CH 73.2, CH 65.2, CH2 58.1, CH3

6-OMe-β-DGalf 107.9, CH 83.7, CH 78.2, CH 84.6, CH 70.0, CH 76.0, CH2 59.4, CH3

3-OMe-β-DGlcp 102.7, CH 75.2, CH 88.0, CH 71.6, CH 77.7, CH 63.2, CH2 60.9, CH3

6-OMe-β-DGalf 108.1, CH 83.7, CH 78.3, CH 84.5, CH 70.0, CH 76.0, CH2 59.3, CH3 6-OMe-β-DGalf 108.5, CH 83.4, CH 78.7, CH 85.0, CH 70.7, CH 75.5, CH2 59.4, CH3

3-OMe-β-DGlcp 102.8, CH 75.1, CH 87.8, CH 71.7, CH 77.8, CH 63.4, CH2 61.0, CH3 6-OMe-β-DGalf 108.5, CH 83.4, CH 78.6, CH 84.9, CH 70.8, CH 75.5, CH2 59.4, CH3

3-OMe-β-DGlcp 103.1, CH 75.2, CH 87.9, CH 71.6, CH 77.7, CH 63.2, CH2 61.0, CH3 6-OMe-β-DGalf 108.5, CH 83.3, CH 78.8, CH 85.1, CH 70.8, CH 75.5, CH2 59.4, CH3

6-OMe-β-DGalf 108.5, CH 83.4, CH 78.8, CH 85.0, CH 70.8, CH 75.6, CH2 59.3, CH3 3-OMe-β-DGalf 108.2, CH 81.2, CH 88.6, CH 84.0, CH 73.1, CH 65.2, CH2 58.2, CH3

1″ 2″ 3″ 4″ 5″ 6″ OCH3

each other only in the position of an O-methyl group in the monosaccharide unit (Tables 1 and 3). The carbon and proton atom signals and the corresponding coupling constants of the monosaccharide unit coincided well with those of a terminal 6O-methyl-β-galactofuranosyl residue.3 The chemical shift of the anomeric C atom at δC 107.9 corresponded to the βconfiguration of the glycosidic bond.8 The D-series of the 6O-methyl-β-galactofuranosyl unit was proposed by analogy with co-occurring glycoside 6. The attachment of this monosaccharide unit to C-16 of the steroidal aglycon was determined by the HMBC spectrum, where the cross-peak between H-1′ of 6-OMe-Gal f and C-16 of the aglycon was observed.

mixture of 9 and 10 32.7, CH2 29.6, CH2 67.2, CH 36.2, CH2 33.1, CH 34.2, CH2 73.8, CH 128.9, C 46.3, CH 38.6, C 19.8, CH2 37.0, CH2 44.8, C 144.8, C 33.6, CH2 77.4, CH 63.0, CH 20.3, CH3 11.6, CH3 33.3, CH 21.7 21.5, CH3 33.1, CH2 29.8, CH2 47.2 47.0, CH 30.2 30.5, CH 20.0 20.1, CH3 19.3 19.4, CH3 24.0 24.2, CH2 12.7 12.4, CH3 β-D-Galf 107.6, CH 83.6, CH 78.4, CH 84.6, CH 72.5, CH 65.4, CH2 6-OMe-β-D-Galf 107.4, CH 83.5, CH 78.8, CH 84.9, CH 70.9, CH 75.4, CH2 59.3, CH3

Accordingly, the structure of anthenoside N was determined as 3. The molecular formula of compound 4 was determined to be C35H58O9 by (+)HRESIMS. The fragment ion peak at m/z 217 [C7H14O6 + Na]+ in the (+)ESIMS/MS spectrum from the precursor ion at m/z 645 [M + Na]+ showed the presence of an O-methylhexose unit in compound 4. A thorough comparison of the 1H and 13C NMR data of compound 3 with those of 4 exhibited that they differed from each other only in the Omethylhexose unit. Analysis of the 1H and 13C NMR spectra of 4 and 1 clearly revealed that compound 4 had the same 3-Omethyl-β-D-glucopyranosyl residue. The attachment of the 3-OE



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

showed that they differed from each other only in the steroidal side chain and one monosaccharide unit. A detailed analysis of the carbon and proton NMR signals and the corresponding coupling constants for the side chain of 6 testified that compound 6 contained the (20R)-cholestane side chain as in the previously known anthenoside B3 (Tables 2 and 3). The MS data and the presence of two anomeric proton signals at δH 4.33 and 5.02 and the corresponding carbon signals at δC 103.1 and 108.5 revealed the existence of two O-methylhexose units in compound 6. A comparison of the NMR data of 6 with those of 1 and 5 clearly showed that glycoside 6 contained the same 3-O-methyl-β-glucopyranosyl and 6-O-methyl-β-galactofuranosyl residues, respectively (Tables 2 and 3). Glycoside 6 was hydrolyzed with 2 M TFA and treated in the same manner as glycoside 2 to ascertain a stereochemical series of its monosaccharide units. The D-configurations of 3-O-methyl-βglucopyranose and 6-O-methyl-β-galactofuranose were determined on the basis of the GC retention times of the corresponding acetylated octylglycoside derivatives. The attachments of these residues to the steroidal aglycon were determined by the HMBC and ROESY spectra, where the HMBC cross-peaks between H-1′ of 3-OMe-Glcp and C-16 of the aglycon and H-1″ of 6-OMe-Galf and C-7 of the aglycon were observed, and the ROESY cross-peaks between H-1′ of 3OMe-Glcp and H-16 of the aglycon and H-1″ of 6-OMe-Galf and H-7 of the aglycon were observed. Accordingly, the structure of anthenoside Q was elucidated as 6.

methyl-β-D-glucopyranosyl residue to the steroidal aglycon was determined by the HMBC and ROESY spectra, where the HMBC cross-peak between H-1′ of the sugar and C-16 of the aglycon was observed, and the ROESY cross-peak between H1′ of the sugar and H-16 of the aglycon was observed (Tables 1 and 3). Thereby, the structure of anthenoside O was proved to be 4. The molecular formula of compound 5 was determined to be C42H70O14 from the [M + Na]+ sodiated adduct ion peak at m/ z 821.4663 in the (+)HRESIMS spectrum. The MS data and two anomeric proton signals at δH 4.97 and 4.98 along with the corresponding carbon signals at δC 108.5 and 108.2 showed the presence of two O-methylhexose units in 5. A comparison of the NMR and MS data of 5 with those of 3 and previously known anthenosides J and K3 clearly showed that glycoside 5 contained two 6-O-methyl-β-galactofuranosyl residues (Tables 1 and 3). The attachments of the 6-O-methyl-β-galactofuranosyl residues to the steroidal aglycon were determined by the HMBC and ROESY spectra, where the HMBC cross-peaks between H-1′ of 6-OMe-Galf and C-16 of the aglycon and H-1″ of 6-OMe-Galf and C-7 of the aglycon were observed, and the ROESY cross-peaks between H-1′ of 6-OMe-Galf and H-16 of the aglycon and H-1″ of 6-OMe-Galf and H-7 of the aglycon were observed. Thus, the structure of anthenoside P was established as 5. The molecular formula of compound 6 was determined to be C41H70O14 by (+)HRESIMS. The thorough comparison of the 1 H and 13C NMR and MS data of compound 6 with those of 5 F



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tuted steroidal nucleus. The NMR data of the side chain indicated the existence of three secondary methyls, CH3-21 [δH 1.04 d (J = 6.9); δC 21.7, 21.5], CH3-26 [δH 0.86 d (J = 6.8); δC 20.0, 20.1], and CH3-27 [δH 0.85 d (J = 6.7); δC 19.3, 19.4], and one primary CH3-29 [δH 0.89 t (J = 7.5); δC 12.7, 12.4]. The complexity of the methyl region of the 13C NMR data clearly showed the presence of two epimeric glycosides with 24R and 24S ethyl groups. The proton and carbon resonances of the side chains, established by 2D NMR experiments, were similar to those of the side chains in the previously known anthenosides H and I.3 The chemical shift of C-29 of the 24S epimer was more deshielded than that in the 24R epimer, while the chemical shifts of C-25, C-26, C-27, and C-28 of the 24S epimer were more shielded than those in the 24R epimer (Table 3).3 Therefore, the structures of the aglycon parts of 9 and 10 were defined as (20R,24S)- and (20R,24R)-24-ethyl-5αcholest-8(14)-ene-3α,7β,16α-triols, respectively. Evaluation of the intensities of C-29 resonances revealed a ratio of 4:1 for the 9 + 10 mixture, showing the predominance of the 24S epimer (9). The 1H NMR spectrum of mixture 9 and 10 showed two resonances at δH 4.99 and 4.95 in the deshielded region belonging to anomeric H atoms of the monosaccharide units that correlated in the HSQC spectrum with C atom signals at δC 107.4 and 107.6, respectively. The fragment ion peaks at m/ z 613 [(M + Na) − C7H14O6]+, 411 [(M + Na) − C7H14O6 − (C6H12O6 + Na)]+, 217 [C7H14O6 + Na]+, and 203 [C6H12O6 + Na]+ in the (+)ESIMS/MS spectrum from the precursor ion at m/z 807 [M + Na]+ and fragment ion peaks at m/z 603 [(M − H) − C6H12O6]−, 589 [(M − H) − C7H14O6]−, 193 [C7H13O6]−, and 179 [C6H11O6]− in the (−)ESIMS/MS spectrum from the precursor ion at m/z 783 [M − H]− showed the presence of O-methylhexose and hexose units in 9 and 10. Applying 1D TOCSY, COSY, HSQC, HMBC, and ROESY experiments led to the assignment of all carbon and proton resonances of the carbohydrate moieties in the NMR spectra of the 9 + 10 mixture (Tables 2 and 3). The carbon and proton signals and the corresponding coupling constants of the monosaccharide units agreed well with those of the terminal 6-O-methyl-β-galactofuranosyl residue in the NMR spectra of 3 and a β-galactofuranosyl residue.9 The D-series of both monosaccharide units were expected by analogy with cooccurring glycosides 2 and 6. The attachments of the 6-Omethyl-β-D-galactofuranosyl and β-D-galactofuranosyl units to the steroidal aglycon were determined by the HMBC and ROESY spectra, where the HMBC cross-peaks between H-1′ of Galf and C-16 of the aglycon and H-1″ of 6-OMe-Galf and C-7 of the aglycon were observed, and the ROESY cross-peaks between H-1′ of Galf and H-16 of the aglycon and H-1″ of 6OMe-Galf and H-7 of the aglycon were observed. On the basis of these results, the structures of anthenosides T (9) and U (10) were determined as (20R,24S)-7-O-(6-O-methyl-β-Dgalactofuranosyl)-16-O-(β-D-galactofuranosyl)-24-ethyl-5α-cholest-8(14)-ene-3α,7β,16α-triol and (20R,24R)-7-O-(6-O-methyl-β-D-galactofuranosyl)-16-O-(β-D-galactofuranosyl)-24-ethyl5α-cholest-8(14)-ene-3α,7β,16α-triol, respectively. The new anthenosides L−U (1−10) from A. aspera have structural similarities to the previously mentioned anthenosides A−K from A. chinensis. These substances belong to the new series of atypical steroidal glycosides of starfish. Anthenosides L−S (1−8) as well as anthenosides A−K have a range of shared structural features: a Δ8(14)-3α,6β,7β,16α-tetrahydroxysteroidal nucleus, unoxidized steroidal side chains, a 6-O-methylgalacto-

The molecular formula of compound 7 was determined to be C41H68O14 from the [M + Na]+ sodiated adduct ion peak at m/ z 807.4496 in the (+)HRESIMS spectrum. The analysis of 1H and 13C NMR data of glycosides 7 and 6 indicated that 7 differed from 6 only in the presence of an additional double bond in the steroidal side chain in accordance with the molecular mass difference of 2 amu between 7 and 6 [δH 5.79 dd (J = 15.3, 9.0), 5.34 dd (J = 15.3, 7.3); δC 138.1, 128.8]. The trans configuration of the 22(23)-double bond followed from J22,23 = 15.3 Hz. Thus, the aglycon side chain of glycoside 7 was determined to be (20R,22E)-cholest-22(23)-ene type, and the structure of anthenoside R was elucidated as 7. The molecular formula of compound 8 was determined to be C42H70O14 by (+)HRESIMS. The 1H and 13C NMR data of 8 differed from those of 5 only in signals of the steroidal side chain and the monosaccharide residue at C-7. The NMR data attributable to the side chain indicated the existence of three secondary methyls, CH3-21 [δH 1.04 d (J = 6.9); δC 21.4], CH326 [δH 1.04 d (J = 6.9); δC 22.5], and CH3-27 [δH 1.04 d (J = 6.9); δC 22.3], and a 24(28) double bond [δH 4.74 br s, 4.71 br d (J = 1.3); δC 107.2, 157.7] (Tables 1 and 3). These resonances were similar to those of the Δ24(28)-24-methylcholestane side chain of known anthenosides F and G.3 The MS data and the presence of signals of two anomeric protons at δH 4.99 and the corresponding carbon signals at δC 108.1 and 108.5 showed the existence of two O-methylhexose units in 8. A comparison of the NMR data of 8 with those of 2 and 5 clearly indicated that glycoside 8 contained the same 3-Omethyl-β-D-galactofuranosyl and 6-O-methyl-β-D-galactofuranosyl residues, respectively. The attachments of the 3-O-methyl-βD-galactofuranosyl and 6-O-methyl-β-D-galactofuranosyl residues to the steroidal aglycon were determined by the HMBC and ROESY spectra, where the HMBC cross-peaks between H1′ of 6-OMe-Galf and C-16 of the aglycon and H-1″ of 3-OMeGalf and C-7 of the aglycon were observed, and the ROESY cross-peaks between H-1′ of 6-OMe-Galf and H-16 of the aglycon and H-1″ of 3-OMe-Galf and H-7 of the aglycon were observed. Hence, the structure of anthenoside S was established as 8. Compounds 9 and 10 were not separated by repeated reversed-phase HPLC. They have the same molecular formula, C42H72O13, determined by (+)HRESIMS. The 1H, 13C, and DEPT NMR spectra of these compounds showed the resonances of protons and carbons of two angular methyls, CH3-18 and CH3-19 (δH 0.90 s, 0.67 s; δC 20.3, 11.6), the 8(14) double bond (δC 128.9, 144.8), one oxygenated methine, CH-3 [δH 3.98 t (J = 2.6); δC 67.2], and two methines, CH-7 [δH 4.39 t (J = 2.6); δC 73.8] and CH-16 (δH 4.46 m; δC 77.4], bearing O-monosaccharide residues. The COSY, HSQC, HMBC, and ROESY experiments led to the assignment of all proton and carbon resonances in the NMR spectra of the mixture of 9 and 10 (Tables 2 and 3). Most of the signals in the NMR spectra of the mixture, attributable to the steroidal nucleus, were similar to those of 1 with the exception of some resonances belonging to the steroid A- and B-rings. The signals of H3-19 in the mixture of 9 and 10 were shielded from δH 0.84 to 0.67 in comparison with H3-19 of 1. The CH-6 (δH 3.54) in 1 was replaced by a CH2-6 (δH 1.56; 1.34) in the spectrum of 9 and 10, indicating the absence of one hydroxy group at C-6 (δC 34.2) in 9 and 10. A detailed analysis of the carbon and proton signals and the corresponding coupling constants in the NMR spectra of the aglycon part of 9 and 10 indicated that these compounds contained the Δ8(14)-3α,7β,16α-trihydroxy-substiG



Article

20 m by hand using scuba at Tu Long Bay near Khuan Lan Island in the South China Sea during the 34th scientific cruise of the research vessel Akademik Oparin in May 2007. Species identification was carried out by one of the authors (T.I.A.). A voucher specimen [no. 034-142] is on deposit at the marine specimen collection of the G.B. Elyakov Pacific Institute of Bioorganic Chemistry of the FEB RAS, Vladivostok, Russia. Extraction and Isolation. The fresh animals of A. aspera (1.3 kg, crude weight) were chopped into small pieces and extracted three times with EtOH. The H2O/EtOH layer was evaporated, and the residue was dissolved in H2O (1.0 L). The H2O-soluble material was passed through a Polychrom-1 column (7 × 26.5 cm), eluted with distilled H2O (4.0 L) until a negative chloride ion reaction was obtained, and then eluted with EtOH (3.5 L). The combined EtOH eluate was evaporated to give a reddish residue (7.0 g). This material was chromatographed over a Si gel column (6 × 18.5 cm) using CHCl3/EtOH (stepwise gradient, 6:1−EtOH, v/v) to yield eight fractions, 1−8, which were then analyzed by TLC in the eluent system n-BuOH/EtOH/H2O (4:1:2, v/v/v). Fractions 1−6 mainly contained the polyhydroxysteroidal glycosides and admixtures of pigments and concomitant lipids. HPLC separation of fraction 3 (42 mg) on a Diasorb-130-C16T column (2.5 mL/min) with EtOH/H2O (75:25, v/ v) as an eluent system yielded pure 1 (3.0 mg, tR 25.3 min) and subfractions 3.7 (5.0 mg), 3.8 (2.0 mg), and 3.9 (3.5 mg), which were additionally submitted to a purification on a Discovery C18 analytical column (1.0 mL/min) with EtOH/H2O (70:30, v/v) as an eluent system to give pure 2 (3.5 mg, tR 19.7 min), 4 (1.5 mg, tR 20.8 min), and 3 (1.0 mg, tR 22.1 min). HPLC separation of fractions 2 (113 mg) and 4 (183 mg) on a Diasorb-130-C16T column (2.5 mL/min) with EtOH/H2O (75:25, v/v) as an eluent system and further separation on a Discovery C18 analytical column (1.0 mL/min) with EtOH/H2O (70:30, v/v) as an eluent system yielded pure 5 (4.0 mg, tR 20.9 min), 6 (2.5 mg, tR 21.5 min), 7 (6.5 mg, tR 18.6 min), and 8 (2.5 mg, tR 19.1 min) and a mixture of 9 and 10 (1.0 mg, tR 28.2 min). Anthenoside L (1): amorphous, white powder, [α]20D −24 (c 0.1, MeOH); IR (CDCl3) νmax 3421, 3020, 2856, 1602, 1261, 1216, 1034 cm−1; 1H and 13C NMR data, Tables 1, 3; HRESIMS m/z 617.3662 [M + Na]+ (calcd for C33H54O9Na, 617.3660), m/z 629.3460 [M + Cl]− (calcd for C33H54ClO9, 629.3440). Anthenoside M (2): amorphous, white powder, [α]20D −16 (c 0.1, MeOH); 1H and 13C NMR data, Tables 1, 3; HRESIMS m/z 645.3973 [M + Na]+ (calcd for C35H58O9Na, 645.3977), m/z 657.3775 [M + Cl]− (calcd for C35H58ClO9, 657.3773). Anthenoside N (3): amorphous, white powder, [α]20D −11 (c 0.1, MeOH); 1H and 13C NMR data, Tables 1, 3; HRESIMS m/z 645.3970 [M + Na]+ (calcd for C35H58O9Na, 645.3973), m/z 657.3770 [M + Cl]− (calcd for C35H58ClO9, 657.3775); ESIMS/MS of the [M + Na]+ ion at m/z 645: 627 [(M + Na) − H2O]+, 469 [(M + Na) − C7H12O5]+, 217 [C7H14O6 + Na]+. Anthenoside O (4): amorphous, white powder, [α]20D −22 (c 0.1, MeOH); IR (CDCl3) νmax 3414, 3020, 2856, 1602, 1261, 1216, 1032 cm−1; 1H and 13C NMR data, Tables 1, 3; HRESIMS m/z 645.3974 [M + Na]+ (calcd for C35H58O9Na, 645.3973), HRESIMS m/z 657.3776 [M + Cl]− (calcd for C35H58ClO9, 657.3775); ESIMS/MS of the [M + Na]+ ion at m/z 645: 627 [(M + Na) − H2O]+, 217 [C7H14O6 + Na]+. Anthenoside P (5): amorphous, white powder, [α]20D −31 (c 0.2, MeOH); IR (CDCl3) νmax 3415, 3020, 2856, 1602, 1261, 1216, 1033 cm−1; 1H and 13C NMR data, Tables 1, 3; HRESIMS m/z 821.4663 [M + Na]+ (calcd for C42H70O14Na, 821.4658), m/z 833.4461 [M + Cl]− (calcd for C44H70ClO14, 833.4460); ESIMS/MS of the [M + Na]+ ion at m/z 821: 645 [(M + Na) − C7H12O5]+, 627 [(M + Na) − C7H14O6]+, 217 [C7H14O6 + Na]+; ESIMS/MS of the [M − H]− ion at m/z 797: 603 [(M − H) − C7H14O6]−, 193 [C7H13O6]−. Anthenoside Q (6): amorphous, white powder, [α]20D −89 (c 0.2, MeOH); IR (CDCl3) νmax 3393, 3020, 2873, 2857, 1602, 1261, 1216, 1074 cm−1; 1H and 13C NMR data, Tables 2, 3; HRESIMS m/z 809.4658 [M + Na]+ (calcd for C41H70O14Na, 809.4656), m/z 821.4460 [M + Cl]− (calcd for C41H70ClO14, 821.4463); ESIMS/MS

furanoside residue, and attachment positions of the carbohydrate moiety at C-16 or together at C-16 and C-7 of the steroidal nucleus. At the same time the newly isolated anthenosides have some structural peculiarities, which were not found in starfish polar steroidal compounds previously, for example, a new type of steroidal nucleus (Δ8(14)-3α,7β,16αtrihydroxysteroidal nucleus) in 9 and 10, unoxidized side chains, such as the Δ22-24-nor-cholestane side chain in 1, the (24S)-Δ22-24-methylcholestane side chain in 2−5, and the Δ22cholestane side chain in 7, and the 3-O-methyl-β-D-galactofuranosyl residue in 2 and 8. It is of interest that all of the glycosides that we isolated have the 3α-hydroxy configuration and unoxidized side chains in the aglycon moieties, rare features in starfish polar steroids. Thus, the discovery of anthenosides L−U (1−10) showed that this series of unusual polyhydroxysteroidal glycosides is characteristic of at least two starfish species of the genus Anthenea. The anthenosides, therefore, may be considered as chemotaxonomic markers of this genus. We have studied the cytotoxicities and potential immunomodulatory properties of the new polyhydroxysteroidal glycosides using different murine cell cultures. According to the obtained data, none of the studied compounds were cytotoxic. The hemolytic activities for the studied substances also were not significant. Only the mixture of glycosides of 9 and 10 showed marked hemolytic activity, with EC50 = 8 μM, compared with other glycosides (Table S1, Supporting Information). In vitro cytotoxicities of compounds 1−10 against RAW 264.7 murine macrophage cells were evaluated by the MTT assay. None of the studied compounds exhibited cytotoxic effects. At a dose of 10 μM, the studied polyhydroxysteroidal glycosides did not show any increase in reactive oxygen species (ROS) formation in the RAW 264.7 cells (data not shown). However, the glycoside 4 showed a reduction in ROS levels when the cells were co-stimulated with the pro-inflammatory endotoxin lipopolysaccharide (LPS) from E. coli, which induces a relatively high ROS level in macrophages. Actually, glycoside 4 at a concentration of 10 μM decreased the ROS content in macrophages by 24% (p = 0.0045) (Figure S64, Supporting Information).

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were determined on a PerkinElmer 343 polarimeter. IR spectra were determined on a Bruker OPUS Vector-22 infrared spectrophotometer in CDCl3. The 1H 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 tetramethylsilane used as an internal standard. 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; Diasorb-130-C16T (11 μm, 250 × 16 mm) (Biochemmack) and Discovery C18 analytical (5 μm, 250 × 4 mm) (Supelco) columns were used. GC analysis was performed on an Agilent 6850 Series chromatograph equipped with HP-5 MS capillary column (30 m × 0.25 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 the 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 thin-layer chromatography. Animal Material. Specimens of Anthenea aspera Döderlein, 1915 (order Valvatida, family Oreasteridae) were collected at a depth of 3− H



Article

of the [M + Na]+ ion at m/z 809: 615 [(M + Na) − C7H14O6]+, 217 [C7H14O6 + Na]+. Anthenoside R (7): amorphous, white powder, [α]20D −52 (c 0.1, MeOH); 1H and 13C NMR data, Tables 2, 3; HRESIMS m/z 807.4496 [M + Na]+ (calcd for C41H68O14Na, 807.4501), m/z 819.4299 [M + Cl]− (calcd for C41H68ClO14, 819.4303); ESIMS/MS of the [M + Na]+ ion at m/z 807: 613 [(M + Na) − C7H14O6]+, 217 [C7H14O6 + Na]+. Anthenoside S (8): amorphous, white powder, [α]20D −31 (c 0.1, MeOH); 1H and 13C NMR data, Tables 2, 3; HRESIMS m/z 821.4652 [M + Na]+ (calcd for C42H70O14Na, 821.4658), m/z 833.4462 [M + Cl]− (calcd for C44H70ClO14, 833.4460); ESIMS/MS of the [M + Na]+ ion at m/z 821: 627 [(M + Na) − C7H14O6]+, 217 [C7H14O6 + Na]+; ESIMS/MS of the [M − H]− ion at m/z 797: 603 [(M − H) − C7H14O6]−, 193 [C7H13O6]−. Mixture of anthenosides T and U (9 and 10): amorphous, white powder, [α]20D −60 (c 0.1, MeOH); 1H and 13C NMR data, Tables 2, 3; HRESIMS m/z 807.4859 [M + Na]+ (calcd for C42H72O13Na, 807.4865), m/z 819.4665 [M + Cl]− (calcd for C42H72ClO13, 819.4667); ESIMS/MS of the [M + Na]+ ion at m/z 807: 613 [(M + Na) − C7H14O6]+, 411 [(M + Na) − C7H14O6 − (C6H11O6 + Na)]+, 217 [C7H14O6 + Na]+, 203 [C6H12O6 + Na]+; ESIMS/MS of the [M − H]− ion at m/z 783: 603 [(M − H) − C6H12O6]−, 589 [(M − H) − C7H14O6]−, 193 [C7H13O6]−, 179 [C6H11O6]−. Acid Hydrolysis and Determination of Absolute Configurations of Monosaccharides. Determination of absolute configurations of monosaccharides was carried out according to the procedure of Leontein et al.10 The acid hydrolysis of 2 (1.5 mg) was carried out in a solution of 2 M TFA (1 mL) in a sealed vial on a glycerol bath at 100 °C for 2 h. The H2O layer was washed with CHCl3 (3 × 1.0 mL) and concentrated in vacuo. One drop of concentrated TFA and 0.5 mL of (R)-(−)-2-octanol (Aldrich) were added to the sugar, 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 the monosaccharide were detected in the hydrolysate of 2: 3-O-Me-D-galactose (tR 26.39, 26.61, 27.34, and 27.58 min). The acid hydrolysis of 6 (2.0 mg) was carried out in the same manner as described for 2, and the obtained acetylated 2-octylglycosides were analyzed by GC. The following peaks were detected in the hydrolysate of 6: 3-O-Me-D-glucose (tR 26.74, 27.84, 27.95, and 28.22 min) and 6-O-Me-D-galactose (tR 26.02, 26.82, and 26.94 min). The samples of 3-O-methyl-D-glucose, 3-O-methyl-D-galactose, and 6-O-methyl-D-galactose were synthesized as described previously.11,12 The retention times of the authentic samples were as follows: 3-OMe-D-galactose (tR 26.41, 26.63, 27.32, and 27.56 min), 3-O-Me-Dglucose (tR 26.77, 27.90, 28.01, and 28.27 min), 6-O-Me-D-galactose (tR 26.02, 26.81, and 26.94 min), 3-O-Me-L-galactose (tR 26.62, 26.88, 27.12, and 27.32 min), 3-O-Me-L-glucose (tR 27.15, 27.49, 27.57, and 28.54 min), and 6-O-Me-L-galactose (tR 26.27, 26.34, and 26.42 min). Cell Cultures. Erythrocytes were isolated from CD-1 mouse blood and washed three times with PBS (pH 7.4) using centrifugation (450g, for 5 min), and the residue of erythrocytes was resuspended in PBS (pH 7.4) to a final optical density of 1.5 at 700 nm and kept on ice. The museum tetraploid strain of mouse Ehrlich ascite carcinoma was provided by the N.N. Blokhin Russian Oncology Center (Russian Academy of Medical Sciences, Moscow, Russia). The cells of Ehrlich carcinoma were inoculated into the peritoneal cavity of 18 to 20 g albino CD-1 mice (male and female). Cells for experimentation were collected 7−10 days after inoculation, washed two times by centrifugation (450g for 5 min) in PBS (pH 7.4), and then resuspended in RPMI-1640 culture medium without serum. The final cell concentration in the media was usually (1−2) × 106 cells/ mL. The total fraction of mouse lymphocytes (splenocytes) was isolated from the spleen of BALB/C line mice. The spleen was homogenized in

PBS solution (pH 7.4). The cell suspension was filtered through nylon voile (280 mesh). The suspension of splenocytes was washed three times with PBS (pH 7.4), centrifuged (450g for 5 min) to remove debris, and resuspended in PBS by the final cell concentration (2−5) × 106 cells/mL. The RAW 264.7 murine macrophage cell line was obtained from the American Type Culture Collection. The cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum at 37 °C under a humidified 5% CO2 atmosphere in an incubator (MCO-18AIC, Sanyo). Cell Viability Assay. The cell viability assay was carried out according to the standard MTT method13 against the mouse Ehrlich carcinoma cell line, splenocyte cells, and RAW 264.7 murine macrophages. Hemolytic Activity. For the hemolytic assay 20 μL of solution of the tested substance, in different concentrations, was mixed with 180 μL of erythrocyte suspension and incubated at 37 °C for 1 h in a 96well U-bottom shape microplate. After that the residual cells were sedimented by centrifugation, aliquots of supernatant (150 μL) were transferred to the wells of 96-well flat bottom microplates, and the hemoglobin concentration in the supernatant was evaluated spectroscopically at λex = 570 nm with a Multiskan FC (Thermo Scientific) plate reader. The results were expressed as percent of hemolysis compared to control. Immunomodulatory Activity. For immunomodulatory activity RAW 264.7 murine macrophages were plated into 96-well microplates and incubated at 37 °C with 5% CO2 for 2 h. After adhesion, cells were incubated with the test compounds (10 μM) for 24 h. To study ROS formation, 20 μL of 2,7-dichlorodihydrofluorescein diacetate solution (Molecular Probes, final concentration 10 μM) was added to each well, and the microplate was incubated for an additional 10 min at 37 °C. In each experiment tested compounds were co-incubated with LPS from E. coli serotype 055:B5 (Sigma, 1.0 μg/mL). Fluorescent intensity 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. The results are expressed as the mean ± standard deviation. A Student’s t test was used to evaluate the data with the significance level of p < 0.05. The means and standard deviations for each treatment were calculated and plotted using SigmaPlot 3.02 software (Jandel Scientific).

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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.6b00667. 1 H, 13C, COSY, HSQC, HMBC, ROESY, and HRESIMS spectra of compounds 1−10, Figure S64 (Influence of anthenosides L−U (1−10) upon ROS level in RAW 264.7 murine macrophages, co-incubated with LPS from E. coli), and Table S1 (Hemolytic and cytotoxic activity of anthenosides L−U (1−10)) (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Tel: +7(423)2312360. Fax: +7(423)2314050. E-mail: [email protected]. ORCID

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 No. 14-04I



Article

00341-a from the RFBR. The bioassay study was supported by Grant No. 14-25-00037 from the RSF (Russian Science Foundation).

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REFERENCES

(1) (a) Minale, L.; Riccio, R.; Zollo, F. Fortschr. Chem. Org. Naturst. 1993, 62, 75−308. (b) Stonik, V. A. Russ. Chem. Rev. 2001, 70, 673− 715. (c) Iorizzi, M.; De Marino, S.; Zollo, F. Curr. Org. Chem. 2001, 5, 951−973. (d) Stonik, V. A.; Ivanchina, N. V.; Kicha, A. A. Nat. Prod. Commun. 2008, 3, 1587−1610. (e) Ivanchina, N. V.; Kicha, A. A.; Stonik, V. A. Steroids 2011, 76, 425−454. (f) Dong, G.; Xu, T. H.; Yang, B.; Lin, X. P.; Zhou, X. F.; Yang, X. W.; Liu, Y. H. Chem. Biodiversity 2011, 8, 740−791. (2) Ma, N.; Tang, H. F.; Qiu, F.; Lin, H. W.; Tian, X. R.; Zhang, W. Chin. Chem. Lett. 2009, 20, 1231−1234. (3) Ma, N.; Tang, H. F.; Qiu, F.; Lin, H. W.; Tian, X. R.; Yao, M. N. J. Nat. Prod. 2010, 73, 590−597. (4) Vanderah, D. J.; Djerassi, C. J. Org. Chem. 1978, 43, 1442−1448. (5) Kicha, A. A.; Kalinovsky, A. I.; Stonik, V. A. Khim. Prir. Soedin. 1990, 2, 218−221. (6) Kalinovsky, A. I.; Antonov, A. S.; Afiyatullov, S. S.; Dimitrenok, P. S.; Evtuschenko, E. V.; Stonik, V. A. Tetrahedron Lett. 2002, 43, 523− 525. (7) Afiyatullov, S. S.; Antonov, A. S.; Kalinovsky, A. I.; Dmitrenok, P. S. Nat. Prod. Commun. 2008, 3, 1581−1586. (8) Shashkov, A. S.; Chizhov, O. V. Russ. J. Bioorg. Chem. 1976, 2, 437−496. (9) Iorizzi, M.; De Marino, S.; Minale, L.; Zollo, F.; Le Bert, V.; Roussakis, C. Tetrahedron 1996, 52, 10997−11012. (10) Leontein, K.; Lindberg, B.; Lönngren, J. Carbohydr. Res. 1978, 62, 359−362. (11) Evtushenko, E. V.; Ovodov, Y. S. Khim. Prir. Soedin. 1982, 1, 21−23. (12) Evtushenko, E. V.; Plisova, E. Y.; Ovodov, Y. S. Bioorg. Khim. 1986, 12, 1366−1371. (13) Carmichael, J.; DeGraff, W. G.; Gazdar, A. F.; Minna, J. D.; Mitchell, J. B. Cancer Res. 1987, 47, 943−946.

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