Steroids 96 (2015) 37–43

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Further study on Penares sp. from Vietnamese waters: Minor lanostane and nor-lanostane triterpenes Ekaterina G. Lyakhova a, Sophia A. Kolesnikova a, Anatoly I. Kalinovsky a, Pavel S. Dmitrenok a, Nguyen H. Nam b, Valentin A. Stonik a,⇑ a b

G.B. Elyakov Pacific Institute of Bioorganic Chemistry, Far East Branch of the Russian Academy of Sciences, Pr. 100-let Vladivostoku 159, 690022 Vladivostok, Russia Institute of Marine Biochemistry, Vietnam Academy of Science and Technology, 18 Hoang Quoc Viet, Caugiay, Hanoi, Viet Nam

a r t i c l e

i n f o

Article history: Received 17 March 2014 Received in revised form 3 December 2014 Accepted 12 January 2015 Available online 29 January 2015 Keywords: Trimethylsteroids Dimethylsteroids Marine sponge Penares Triterpenoids Biochemical parallelism

a b s t r a c t Eight new oxidized lanostane and nor-lanostane derivatives (1–8) along with the previously known penasterol (9) and 24-ethylcholesta-4,24(28)-dien-3-one (10) were isolated from a sponge Penares sp. collected from the Vietnamese waters. Structures of these minor compounds were elucidated by the detailed NMR spectroscopic and mass-spectrometric analyses and by comparison with earlier reported spectroscopic data. A hypothetic scheme of metabolism of the lanostane derivatives in sponges belonging to Penares and Erylus genera was proposed and discussed. Ó 2015 Elsevier Inc. All rights reserved.

1. Introduction Marine organisms are a prolific source of novel natural products, including steroids and their biosynthetic precursors such as tri-, di- and monomethylsterols with unique structures and biological activities of interest. Actually, as it is well known, marine sponges often produce unusual sterols and related metabolites, especially oxidized polar derivatives [1]. The steroidal and related compounds isolated from sponges form, as a rule, very complex mixtures of highly functionalized metabolites, many of which have no terrestrial counterpart and are bioactive [2]. From the sponges belonging to the genus Penares, diverse indole and azetidine alkaloids [3] along with sphingolipids, [4] proline containing macrolides, [5] and triterpenes [6–8] were earlier isolated. Triterpenoids, their demethylated derivatives and thereof derived steroids from Penares spp. represent a particular group of natural products, some of which are of physiological relevance as antileukemic agents [6] and inhibitors of histamine release from rat peritoneal mast cells [7]. Structurally, the peculiarity of these compounds consists in the presence of a rare in nature carboxy group, attached to C-14 or 30,9-lactone fragment in lanostane nucleus. In continuation of our studies on the natural products of ⇑ Corresponding author. Tel.: +7 4232 312360; fax: +7 4232 314050. E-mail address: [email protected] (V.A. Stonik). http://dx.doi.org/10.1016/j.steroids.2015.01.009 0039-128X/Ó 2015 Elsevier Inc. All rights reserved.

the Penares sp. from Vietnamese waters [8,9] and attempts to obtain the more detailed picture of biosynthetic transformations of lanostane triterpenoids in this animal, herein we describe the isolation and structure elucidation of new minor polar constituents from its ethanol extract. 2. Experimental 2.1. General methods Optical rotations were measured using a Perkin-Elmer 343 polarimeter. Melting points were determined using a hot stage melting point apparatus model Leica GALEN III equipped with microscope. 1H NMR (700.13 MHz, 500.13 MHz) and 13C NMR (176.04 MHz, 125.75 MHz) spectra were recorded on Avance III 700 and DRX-500 Bruker spectrometers, using TMS as an internal standard. HRESI mass spectra and ESIMS/MS spectra were recorded on an Agilent 6510 Q-TOF LC/MS mass spectrometer; samples were dissolved in MeOH (c 0.01 mg/mL). HPLC separations were carried out on an Agilent 1100 Series chromatograph equipped with a differential refractometer. YMC-Pack ODS-A (250  10 mm), YMCPack ODS-A (5 l, 250  4.6 mm) and ULTRASPHERA Si (5 l, 250  10 mm) columns were used. Low pressure column liquid chromatography was performed using Sephadex LH-20 (Sigma Chemical Co.) and Si gel KSK (50–160 lm, Sorbpolimer, Krasnodar, Russia). Sorbfil Si gel plates (5–17 lm, Sorbpolimer, Krasnodar,

38

E.G. Lyakhova et al. / Steroids 96 (2015) 37–43

Russia) were used for thin-layer chromatography. Yields are based on dry weight of the sponge. 2.2. Animal material The specimens of Penares sp. were collected in Vietnamese waters in January 2005, as previously described [9]. A voucher specimen (PIBOC O30-271) has been deposited at the collection of marine invertebrates of the G.B. Elyakov Pacific Institute of Bioorganic Chemistry (Vladivostok, Russian Federation). 2.3. Extraction and isolation The sponge specimen (wet weight 400 g) was finely chopped and extracted with EtOH. The ethanol soluble materials (2.8 g) were fractionated using LH-20 column chromatography (CHCl3–EtOH, 1:1) into four fractions. A half of the second fraction (1.3 g of yellow oil; 2.6 g total) was further separated by silica gel column chromatography (12  3 cm, step-wise gradient CHCl3– EtOH) to yield four subfractions (A–D). From the subfraction A, a number of triterpenoids were isolated and described previously [8]. In the present study, we also isolated 24-ethylcholesta-4,24(28)-dien-3-one (10) ½a27 D + 48.2 (c 0.28, CHCl3) lit ½a26 D + 52.0 (c 0.24, CHCl3) [10] (analytical reversed phase HPLC, 100% EtOH) (2.8 mg; 0.002%) as a minor compound in addition to ergosta-4,24(28)-diene-3-one [11,12] (36.1 mg; 0.032%). Subfraction B, eluted with 100% CHCl3, was chromatographed using a semi-preparative reversed phase HPLC column (95% EtOH)

to give a series of previously published major components of sponge extract [8] and the sum (57.3 mg) of minor compounds. The latter was further separated using a semi-preparative reversed phase HPLC column (85% EtOH) to give subsubfractions B1 and B2. The subsubfraction B1 was purified by preparative HPLC (75% CH3CN) to yield compounds 1 (0.5 mg; 0.0005%) and 2 (0.8 mg; 0.0007%). The subsubfraction B2 was chromatographed on a preparative HPLC column (75% CH3CN) to obtain two fractions (B2a and B2b) and epimers 3 (1.4 mg; 0.0013%) and 4 (1.1 mg; 0.0010%) as individual compounds. Further purification of B2a on an analytical normal phase HPLC column (n-hexane–EtOAc, 3:1) led to the isolation of triterpenoids 5 (0.7 mg; 0.0006%) and 7 (0.1 mg; 0.0001%). The fraction B2b contained compound 6 (0.3 mg; 0.0003%), which was purified by analytical normal phase HPLC (n-hexane–EtOAc, 4:1). The subfraction C, eluted with CHCl3–EtOH (20:1), was rechromatographed using a semi-preparative (90% EtOH) and analytical (85% EtOH) reversed phase HPLC columns to yield penasterol (9), mp 193–202 °C, lit [6] 197–201 °C (3.0 mg; 0.0027%) and compound 8 (0.3 mg; 0.0003%), the latter that was further purified using analytical normal phase HPLC column (CHCl3–EtOH, 40:1) and analytical reversed phase HPLC column (75% CH3CN). 2.3.1. 29-Nor-24(R),25-dihydroxypenasterone (1) White amorphous powder; 1H and 13C NMR data (CDCl3) see Tables 1 and 3; ms/ms () ESI m/z: 473 [MH], 429 [MCOOH], 427, 413, 411, 383, 369, 341; HRESI m/z 473.3272 [MH] (calcd. for C29H45O5, 473.3272).

Table 1 H NMR data of compounds 1–4 (d in ppm, J in Hz, CDCl3).

1

Position

1

2

3

4

1

2.10, m 1.62, m a: 2.38, m b: 2.48, ddd (15.0; 5.1; 2.4)

a: 2.08, m

a: 2.03, m

a: 2.03, m

b: 1.88, m a: 2.34, dt (14.5; 3.8) b: 2.43, td (14.5; 5.8)

b: 1.70, m a: 2.60, ddd (15.8; 11.1; 7.3) b: 2.43, ddd (15.8; 6.9; 3.7)

a: 2.59, m

1.65, 1.63, 1.65, 2.05, 2.17,

m m m m m

1.64, 1.62, 1.67, 2.05, 2.17,

m m m m m

m m m m

2.17, 2.17, 1.78, 2.14,

m m m m

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 OOH

b: 1.70, m b: 2.44, ddd (15.9; 6.8; 3.6)

2.31, 1.45, 1.45, 1.80, 2.05, 2.17,

m m m m m m

2.16, m 1.76, m a: 2.11, m b: 1.63, dd (15.6; 13.1) 3.01, d (6.7)

2.25, 2.19, 1.80, 2.12,

m m m m

a: 2.02, m b: 2.09, m a: 1.60, m b: 2.02, m

2.17, 2.17, 1.78, 2.12,

1.61, 2.06, 1.46, 2.18, 1.62, 0.83, 1.24, 1.47, 0.94, 1.05, 1.77, 1.13, 1.57, 3.30,

m m m m m s s m d (6.5) m m m m dd (10.0, 2.0)

a: 1.55, m b: 1.28, m a: 1.39, m b: 2.18, m 1.69, m 1.04, s 1.20, s 1.56, m 0.91, d (6.6) 1.88, m 2.16, m 5.65, ddd (15.7; 8.8; 5.4)

a: 1.61, m b: 2.07, m 1.47, m 2.17, m 1.61, m 0.81, s 1.16, s 1.47, m 0.94, d (6.6) 1.02, m 1.77, m 1.14, m 1.57, m 3.28, dd (10.0; 1.8)

1.62, 2.08, 1.44, 2.18, 1.62, 0.81, 1.16, 1.47, 0.94, 1.28, 1.50, 1.38, 1.38, 3.33,

m m m m m s s m d (6.6) m m m m dd (6.8, 5.6)

1.16, 1.21, 1.08, 1.09,

1.16, 1.21, 1.08, 1.10,

s s s s

1.16, s 1.21, s 1.05, d (6.5)

5.56, d (15.9) 1.31, s 1.35, s 1.03, d (5.9) 7.61, br d

s s s s

39

E.G. Lyakhova et al. / Steroids 96 (2015) 37–43 Table 2 H NMR data of compounds 5–8 (d in ppm, J in Hz, CDCl3).

1

Position

5

6

7

8

1

a: 2.14, td (13.7; 5.0) b: 1.79, dt (13.7; 5.0) a: 2.59, td (14.3; 4.5) b: 2.35, dt (15.0; 4.6)

a: 2.14, td (13.7; 5.0)

a: 2.08, m

a: 1.83, m

b: 1.79, dt (13.7; 5.0) a: 2.59, ddd (14.9; 13.8; 5.4) b: 2.35, ddd (15.8; 6.9; 3.7)

b: 1.87, ddd (13.5; 5.9; 3.0) a: 2.34, ddd (14.4; 4.5; 3.0) b: 2.43, td (14.5; 5.9)

a: 1.63, m

1.90, 1.90, 1.90, 3.07,

1.90, 1.90, 1.90, 3.07,

2.16, m 1.77, td (12.2; 4.6) a: 2.11, m b: 1.63, m 3.01, d (6.8)

a: 1.97, m

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 OEt

m m m d (6.0)

m m m d (6.0)

b: 1.55, m b: 1.57, m 3.26, dd (11.4, 4.3) 1.44, dd (13.5, 4.0) b: 1.77, br t (14.7) 3.02, d (6.8)

a: 1.96, m b: 2.08, m a: 1.60, m b: 2.02, m

a: 1.96, m

a: 2.03, m

a: 1.90, m

b: 2.08, m a: 1.60, m b: 2.02, m

b: 2.10, m a: 1.63, m b: 2.03, m

a: 1.56, m

a: 1.55, m b: 1.26, m a: 1.39, m b: 2.14, m 1.66, m 1.02, s 1.19, s 1.44, m 0.89, d (6.5) 0.96, m 1.48, m 1.40, m 1.64, m 4.00, t (6.6)

a: 1.57, m b: 1.27, m a: 1.38, m b: 2.17, m 1.68, m 1.02, s 1.19, s 1.45, m 0.89, d (6.6) 1.12, m 1.40, m 1.44, m 1.62, m 4.01, brt (6.2)

a: 1.55, m

a: 1.53, m

b: 1.27, td (12.5; 5.4) a: 1.44, m b: 2.17, m 1.69, m 1.03, s 1.21, s 1.47, m 0.90, d (6.5) 1.06, m 1.82, m 1.18, m 1.68, m 3.42, ddd (10.0; 5.5; 1.8)

b: 1.25, m a: 1.44, m b: 2.17, m 1.68, m 1.00, s 1.01, s 1.46, m 0.89, d (6.6) 1.02, m 1.81, m 1.13, m 1.48, m 3.34, dd (9.9, 1.8)

4.83, 4.91, 1.71, 1.10, 1.06,

4.83, 4.92, 1.71, 1.10, 1.06,

1.54, s

1.08 s

1.60, s 1.04, d (5.9)

1.13, 1.01, 0.83, 3.42, 1.16,

quintet (1.6) br s s s s

quintet (1.6) br s s s s

OH

b: 2.03, m b: 1.99, m

s s s q (3.5) t (7.0)

2.05, br s

2.3.2. 29-Nor-25-hydroperoxy-3-oxo-7b,8b-epoxy-5a-lanost-23(E)en-30,9a-olide (2) Colorless thin crystal needles; mp 103–105 °C; 1H and 13C NMR data (CDCl3) see Tables 1 and 3; ms/ms (+) ESI m/z: 509 [M+Na]+, 491, 477, 461, 451, 437, 393; HRESI m/z 509.2882 [M+Na]+ (calcd. for C29H42O6Na, 509.2874).

2.3.6. 24(S)-Hydroxy-3-oxo-7b,8b-epoxy-5a-lanost-25-en-30,9aolide (6) 1 13 Colorless oil; ½a28 C NMR data D 71.3 (c 0.07, CHCl3); H and (CDCl3) see Tables 2 and 3; ms/ms (+) ESI m/z: 502 [M+NH4]+, 484 [M]+, 467, 449, 421, 405, 357, 339, 311; HRESI m/z 507.3072 [M+Na]+ (calcd. for C30H44O5Na, 507.3081).

2.3.3. 24(R),25-Dihydroxypenasterone (3) 1 White amorphous powder; ½a27 D 14.3 (c 0.14, CHCl3); H and 13 C NMR data (CDCl3) see Tables 1 and 3; ms/ms () ESI m/z: 487 [MH], 443 [MCOOH], 441, 427, 397, 383, 355; HRESI m/z 487.3428 [MH] (calcd. for C30H47O5, 487.3429).

2.3.7. 29-Nor-24n-hydroxy-25-chloro-3-oxo-7b,8b-epoxy-5a-lanost30,9a-olide (7) White amorphous powder; 1H and 13C NMR data (CDCl3) see Tables 2 and 3; ms/ms (+) ESI m/z: 529/531 (100/37) [M+Na]+, 493 [MHCl+Na]+; HRESI m/z 529.2681 [M+Na]+ (calcd. for C29H3543 ClO5Na, 529.2691).

2.3.4. 24(S),25-Dihydroxypenasterone (4) 1 13 White amorphous powder; ½a27 C D 7.2 (c 0.11, CHCl3); H and NMR data (CDCl3) see Tables 1 and 3; ms/ms () ESI m/z: 487 [MH], 443 [MCOOH], 441, 427, 397, 355; HRESI m/z 487.3429 [MH] (calcd. for C30H47O5, 487.3429).

2.3.8. 3b, 24-Dihydroxy-7b,8b-epoxy-5a-lanost-24-en-30,9a-olide (8) Colorless oil; 1H and 13C NMR data (CDCl3) see Tables 2 and 3; ms/ms (+) ESI m/z: 550 [M+NH4]+, 487 [M-OEt]+, 469, 451, 441, 423, 405, 395, 359, 341, 313; HRESI m/z 555.3658 [M+Na]+ (calcd. for C32H52O6Na, 555.3656).

2.3.5. 24(R)-Hydroxy-3-oxo-7b,8b-epoxy-5a-lanost-25-en-30,9aolide (5) 1 13 Colorless oil; ½a25 C NMR data D 63.3 (c 0.03, CHCl3); H and (CDCl3) see Tables 2 and 3; ms/ms (+) ESI m/z: 507 [M+Na]+, 489, 463, 447, 421, 381, 353, 321; HRESI m/z 507.3079 [M+Na]+ (calcd. for C30H44O5Na, 507.3081).

3. Results and discussion As a continuation of our previous studies on triterpenes from the Vietnamese marine sponge Penares sp. [8] herein we describe the isolation and structural elucidation of a series of minor compounds from the nonpolar fraction of ethanolic extract.

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E.G. Lyakhova et al. / Steroids 96 (2015) 37–43

stereochemistry of the both 1–8 and 9, 10 were elucidated using NMR spectroscopy, ESI-MS and by comparison of the obtained spectral data with those of the previously isolated compounds. The molecular formula of 1 was determined by HRESI-MS using ion peak at m/z 473.3272 [MH] and 13C NMR data (Table 3) as C29H45O5. Analysis of NMR spectra established that 1 has the same tetracyclic core as in the previously isolated 29-nor-penasterone [8]. Actually, 1H NMR spectra (Table 1) showed four methyl singlets at d 0.83 (Me-18), 1.16 (Me-26), 1.21 (Me-27) and 1.24 (Me-19) and two doublets at d 0.94 (Me-21; J = 6.5 Hz) and 1.05 (Me-28; J = 6.5 Hz) as well as the characteristic signals at d 2.38 (H-2, m) and 2.48 (H-2, ddd J = 15.0, 5.1, 2.4 Hz). Other signals in NMR spectra of 1, attributed to the side chain atoms, were differing from those of 29-nor-penasterone. There were downfield shifted singlets of Me-26 (dH 1.16, s) and Me-27 (dH 1.21, s) and an additional methine signal at dH 3.30 (dd, J = 10.0, 2.0 Hz) as well as signals of two oxygen-bearing carbons at d 73.2 (C-25) and d 79.5 (C-24) instead of double bond signals in the spectrum of 29-nor-penasterone. Positions of hydroxy groups at C-24 and C-25 were confirmed by the long-range correlations: Me-27/C-24, C-25, C-26; Me-26/C-24, C-25, C-27 and Me-21/C-17, C-20, C-22. The R configuration of C-24 was proposed on the basis of comparison of the 1H and 13C NMR spectral data of 1 with those of compounds with analogous side chains [13,14]. Compound 2 has a molecular formula C29H42O6 as determined by HRESI-MS with an ion peak at m/z 509.2882 [M+Na]+ and 13C NMR spectroscopic data. The 13C and 1H NMR signals of compound 2, HMBC and COSY data showed that its chemical structure is closely related to that of recently described 29-nor-3-oxo-7b, 8b-epoxy-5a-lanost-24-en-30,9a-olide [8], but 2 contains 23(E) double bond (dH 5.65, ddd, J = 15.7, 8.8, 5.4 Hz and 5.56, d, J = 15.9 Hz; dC 129.1 and 135.6) and an additional hydroperoxy group at C-25 (Me-26, dH 1.31, s; dC 24.8, Me-27, dH 1.35, s; dC 23.7; C-25 dC 81.9). The hydroperoxy functionality was confirmed by the presence of a broad singlet at dH 7.61 (OOH) [15] along with fragmentary ion peak at m/z 477 [M+NaO2]+ in ESI MS. Two isomeric penasterone-derived diols 3 and 4 were isolated as individual compounds using reversed-phase HPLC in 75% CH3 CN. Their molecular formulae C30H47O5 are identical for the both

Table 3 C NMR data of compounds 1–8 (d in ppm, CDCl3).

13

Position

1

2

3

4

5

6

7

8

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 30 OEt

36.7 37.8 212.7 45.0 49.3 22.0 26.7 129.3 139.0 37.2 23.1 31.5 47.2 62.9 27.9 29.2 50.6 17.4 17.7 36.2 18.6 33.3 28.5 79.5 73.2 23.3 26.6 11.5

31.9 36.5 210.5 44.8 42.2 25.4 52.9 64.9 84.8 36.8 23.0 33.9 46.6 59.3 20.3 27.4 51.4 14.0 14.5 34.8 18.3 38.6 129.1 135.6 81.9 24.8 23.7 11.6 177.0

30.5 26.4 217.1 47.3 50.8 19.4 27.5 129.3 139.6 37.5 22.3 31.5 47.2 62.9 28.0 29.3 50.8 17.6 18.9 35.6 18.4 32.8 28.3 78.7 73.2 23.3 26.6 21.3 26.3 176.6

31.1 33.7 214.6 46.8 43.2 21.8 53.7 64.6 85.5 37.1 22.9 34.1 46.8 59.3 20.2 27.6 52.2 13.9 16.3 34.7 18.2 31.4 31.4 76.6 147.3 111.5 17.2 25.8 22.4 176.8

31.1 33.7 214.6 46.8 43.2 21.8 53.7 64.6 85.5 37.1 22.9 34.1 46.8 59.3 20.2 27.6 52.2 13.9 16.3 34.6 18.2 31.2 31.4 76.1 147.5 111.0 17.6 25.8 22.4 176.8

31.9 36.5 210.5 44.8 42.2 25.4 52.8 64.9 84.6 36.8 23.1 34.0 46.8 59.3 20.3 27.5 52.2 14.0 14.5 35.0 18.3 32.8 28.4 79.8 76.6 26.9 29.4 11.6

176.6

35.7 34.4 217.2 47.4 50.8 19.4 27.5 129.2 139.5 37.4 22.3 31.5 47.2 62.9 28.0 29.2 50.7 17.5 18.9 36.3 18.6 33.3 28.6 79.5 73.3 23.2 26.5 21.3 26.2 177.1

30.5 26.4 78.0 38.4 41.9 20.9 53.9 64.4 85.9 37.5 22.7 34.2 46.8 59.5 20.2 27.5 52.3 13.9 16.3 35.0 18.3 33.2 28.1 77.6 77.4 19.3 21.5 28.1 15.6 177.3 56.4 (CH2) 16.1 (CH3)

176.8

Using different chromatographic procedures, eight new unusual triterpenoids and 29-nor-triterpenoids (1–8) (Fig. 1) were isolated together with previously known penasterol (9) [6] and 24-ethylcholesta-4,24(28)-dien-3-one (10) [10] from the EtOH extract of Penares sp., collected from the Vietnamese waters. Structures and

21 18

20

19

1

OH

O 26

H

17

OH

O

OH

14

OH

H

H

H OH

OOH

OH

27 10

O

4

H

COOH

H

30

H

O

H

COOH

O

2

3 O

OH

O

COOH

O

1

28

O

O

O

OH

O

H

4 OH

O

H

H Cl

H

O

H

O

O

H

O 5

6

O

O

O 7

OH

O

H

H

H

OEt H

H

O

COOH

HO

HO 8

O 9

Fig. 1. Structures of compounds 1–10.

10

41

E.G. Lyakhova et al. / Steroids 96 (2015) 37–43

compounds, as it was deduced by HRESI-MS and 13C NMR data. All the spectroscopic data of 3 and 4 showed that their structures include the polycyclic core of the known Penares sp. metabolite penasterone [7] and side chains identical (3) or similar (4) to that of compound 1. According to the observed difference of CH2-22, CH2-23, CH-24 protons and carbons shifts in the NMR spectra (Tables 1 and 3) the compound 4 should be an epimer of 3 at C-24. In particular, the most significant difference was in chemical shifts and multiplicities of CH-24 atoms for 3 (dH 3.28, dd, J = 10.0, 1.8; dC 79.5) and 4 (dH 3.33, dd J = 6.8, 5.6; dC 78.7). The chemical shifts of the corresponding signals for other epimeric compounds with the same side chains were earlier reported [13]. According to the comparison with the reported data, 3 and 4 should be assigned as having 24R and 24S configurations, correspondingly. Compound 5 was obtained as a colorless oil. Its molecular formula was assigned as C30H44O5 on the basis of the positive ion peak at 507.3079 [M+Na]+ in HRESI MS together with 13C NMR data (Table 3). The 1H (Table 2) and 13C NMR data of 5 were very close to those of 3-oxo-7b,8b-epoxy-5a-lanost-24-en-30,9a-olide [8] except for the signals of the side chain. There were an additional CH–O signals at dH 4.00 (t, J = 6.6 Hz) and dC 76.6 and the signals of 25(26) double bond (dH 4.83, quin, J = 1.6 Hz; 4.91, br s; dC 111.5, 147.3) with methyl singlet at dH 1.71; dC 17.2 instead of two methyl singlets at dH 1.59; dC 17.2 and 1.68; dC 25.7 in 3-oxo-7b,8b-epoxy-5a-lanost-24-en-30,9a-olide. All these findings were confirmed by COSY cross-peaks for H3-27/H2-26;

H-24/H2-23, H-26 and suggested metabolite 5 to have the 24-hydroxy-3-oxo-7b,8b-epoxy-5a-lanost-25-en-30,9a-olide structure, while the stereochemistry at C-24 asymmetric atom was still unclear. We made an attempt to disclose this question with the aid of modified Mosher’s method [16]. Obtained MTPA derivatives of 5 showed irregular sign distributions for DdH (=dH (R)-ester  dH (S)-ester) along with oversized 0.3 ppm DdH value for H-24 (see Supplementary data). Unfortunately, this result demonstrated that Mosher’s methodology cannot be correctly used for the absolute configuration assignment of these compounds [17]. The configuration of 5 will be discussed further during the description of the structure elucidation of 6. Compound 6, colorless oil, had the same molecular formula C30H44O5 as 5. The 1H and 13C NMR spectra (Tables 2 and 3) of 6 were almost the same as those of 5, but there were visible differences in the chemical shifts of C-24 (5 dC 76.6; 6 dC 76.1), C-25 (5 dC 147.3; 6 dC 147.5), C-26 (5 dC 111.5; 6 dC 111.0) and C-27 (5 dC 17.2; 6 dC 17.6). Hence, compound 6 may considered to be an epimer of 5 at C-24. Thoroughly comparison of the NMR characteristics of the isolated metabolites with the reported data for C-24 epimeric cycloartanes with the same type of side chain [13] allowed us to suggest the 24R and 24S stereochemistry of 5 and 6, correspondingly. Compound 7 has a molecular formula C29H43ClO5 determined by positive-ion HRESI-MS and 13C NMR data. The presence of a chlorine atom was confirmed by the characteristic ratio of

Compounds 5-8 Vietnamese Penares sp. OH

Side chain oxidation O

Squalene

O

O Vietnamese Penares sp.

HO

COOH

Carbohydrate chains O

R

Erylus formosus

Side chain oxidation

R=H or CH3

7,8-Epoxodation O

Lanosterol

HO

O COOH

Carbohydrate chains O HO

Vietnamese Penares sp.

Glycosylation

E r ylus spp. 30,9-Lactonization Side chain alkylatin and oxidation

30,9-Lactonization

O

COOH HO Acetylation COOH

Penasterol

P enar es spp.

AcO Oxidation Demethylation

Penares incrustans

Decarboxylation Demethylation Alkylation in side chain Oxidation Carbohydrate chains O R

OH

O

Er ylus goffrileri

7,8-Epoxidation O COOH O

Side chain oxidation

O

R

R=H or CH 3 Compounds 1, 3, 4 P enar es incrustans, R=H or CH3 Vietnamese Penares sp. Vietnamese Penares sp. Vietnamese Penar es sp.

OH

O

Carbohydrate chains O

Scheme 1. Hypothetical biogenetic correlation of lanostane triterpenoids in Penares and Erylus sponges.

O

Er y lus goffrileri

42

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intensities of ion peaks with m/z 529 [M+Na]+ and 531 [M+2+Na]+ and by the presence of a fragmentation ion peak at 493 [MHCl+Na]+ in the ESI MS spectrum. According to the 1H and 13 C NMR spectra metabolite 7 contains in its structure the same type of triterpenoid nucleus as compound 2. Due to the results of the HMBC, HSQC and COSY experiments the rest of the observed NMR shifts were attributed to the side chain atoms including one CH–O group at C-24 (dH 3.42, ddd, J = 10.0, 5.5, 1.8 Hz; dC 79.8), Me-21 (0.90, d, J = 6.5 Hz; dC 18.3) and two downfield shifted methyls Me-26 (dH 1.54, s; dC 26.9) and Me-27 (dH 1.60, s; dC 29.4) bonded with chlorine-bearing carbon C-25 (dC 76.6). Therefore, the structure of the new compound 7 was established as 29-nor-24n-hydroxy-25-chloro-3-oxo-7b,8b-epoxy-5a-lanost30,9a-olide. Compound 8 was isolated as a colorless oil. Its molecular formula C32H52O6 was deduced by HRESI peak with m/z 555.3658 [M+Na]+. A number of NMR signals of this compounds including those of four quaternary methyl groups: Me-18 (dH 1.00, s; dC 13.9), Me-19 (dH 1.01, s; dC 16.3), Me-28 (dH 1.01, s; dC 28.1) and Me-29 (dH 0.83, s; dC 15.6); 3b-hydroxy group (dH 3.26, dd, J = 11.4, 4.3 Hz; dC 78.0); one c-lactone (C-14, dC 59.5; C-9, dC 85.9; C-30, dC 177.3) and one 7b,8b epoxide (dH 3.02, d, J = 6.8 Hz; dC 53.9 and 64.4) showed 8 to have previously described polycyclic core of 3b-hydroxy-7b,8b-epoxy-5a-lanost-24-en30,9a-olide [8]. The side chain of compound 8 seemed to be the ethylated one of compounds 1, 3 or 4. The presence of ethoxy group was detected from chemical shifts and multiplicities of signals at dH 3.42, q, J = 3.5 Hz (CH2CH3) and 1.16, t, J = 7.0 Hz (CH2CH3). The C-25 position of this group led from the HMBC correlations from Me-26, Me-27 to C-25 and from CH2CH3 to C-25. We suggest that compounds 7 and 8 are rather artificial products, derived from the corresponding 24(25)-epoxide at reaction with hydrochloride and ethanol, respectively. Thus, application of different chromatographic procedures and modern spectral methods gave a series of new metabolites with established structures, in majority lanostane or nor-lanostane derivatives from the studied sponge. The isolated compounds bear either two methyl groups or a-methyl at C-4 and either a free carboxyl group at C-14 or this group, included into 30,9-c-lactone. Structures of numerous metabolites, isolated at all studies on this group of sponges suggest that Penares spp. use lanosterol as a common biosynthetic precursor of above-mentioned compounds. Isolation by us of major [8] and minor metabolites from the Vietnamese Penares sp. allowed us to complement the total picture of biosynthetic transformations of lanosterol in the sponges belonging to this genus. The oxidation of methyl group at C-14 to carboxyl in lanosterol is probably the first stage of such transformations, which gives penasterol in the majority of these species (Scheme 1). Further transformations of the latter in Penares spp. include oxidation at C-3 with the formation of 3-keto-derivatives or sometimes acetylation (with formation of acetylpenasterol in Penares incrustans) followed by oxidative removal of b-methyl group at C-4, producing 29-nor-penasterone (the Vietnamese P. sp.). Simultaneously, alkylation in side chains takes a place in many species as well as further demethylation and decarboxylation, that lead to sterol ketones such as ergosta-4,24-diene-3-one and 24-etylcholesta-4,24(28)-dien-3-one. In the Vietnamese Penares sp., 30,9-lactonization followed by 7,8-epoxidation and oxidative transformations in side chain take a place. It is of special interest that there is a biochemical parallelism between Penares spp. and Erylus spp. In fact, the both these sponge groups contain penasterol derivatives in free (Penares) or glycosylated (Erylus) forms [18]. Moreover, the characteristic features of aglycone biosynthesis such as 30,9-lactonization followed by 7,8epoxidation and oxidation in side chain in some Erylus sponges

[19] are the same as of triterpenoids from the Vietnamese Penares sp., studied by us. This biochemical parallelism confirms the recent conclusion, that the subfamily Erylinae, combining genera Penares, Erylus and some others, may be discriminated within the family Geodiidae on the basis of molecular biological and morphological studies [20,21]. Acknowledgements The research described in this publication was supported by Grant 148.2014.4 from the President of Russian Federation, Grants 14-03-31318-mol_a and 14-04-93008-Viet_a from RFBR. We thank taxonomist V.B. Krasokhin (G.B. Elyakov Pacific Institute of Bioorganic Chemistry, Vladivostok, Russian Federation) for identification of the animal material.

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E.G. Lyakhova et al. / Steroids 96 (2015) 37–43 [19] Afiyatullov ShSh, Kalinovsky AI, Antonov AS, Ponomarenko LP, Dmitrenok PS, Aminin DL, Krasokhin VB, Nosova VM, Kisin AV. Isolation and structures of erylosides from the Carribean sponge Erylus goffrilleri. J Nat Prod 2007;70:1871–7. [20] Cárdenas P, Rapp HT, Schander C, Tendal OS. Molecular taxonomy and phylogeny of the Geodiidae (Porifera, Demospongiae, Astrophorida)

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Further study on Penares sp. from Vietnamese waters: minor lanostane and nor-lanostane triterpenes.

Eight new oxidized lanostane and nor-lanostane derivatives (1-8) along with the previously known penasterol (9) and 24-ethylcholesta-4,24(28)-dien-3-o...
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