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Aug 26, 2013 - Institute of Chemistry, Far East Branch of the Russian Academy of Sciences, Prospect 100-let Vladivostoku 159, Vladivostok 690022,. Rus...
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Isolation, Structures, and Biological Activities of Triterpenoids from a Penares sp. Marine Sponge Sophia A. Kolesnikova,† Ekaterina G. Lyakhova,† Anatoly I. Kalinovsky,† Michail A. Pushilin,‡ Shamil Sh. Afiyatullov,† Ekaterina A. Yurchenko,† Sergey A. Dyshlovoy,† Chau V. Minh,§ and Valentin A. Stonik*,† †

G. B. Elyakov Pacific Institute of Bioorganic Chemistry, Far-Eastern Branch of the Russian Academy of Sciences, Prospect 100-let Vladivostoku 159, Vladivostok 690022, Russian Federation ‡ Institute of Chemistry, Far East Branch of the Russian Academy of Sciences, Prospect 100-let Vladivostoku 159, Vladivostok 690022, Russian Federation § Institute of Marine Biochemistry, Vietnam Academy of Science and Technology, 18 Hoang Quoc Viet, Caugiay, Hanoi, Vietnam S Supporting Information *

ABSTRACT: Six new triterpenoids (1−6) and the previously known penasterone, acetylpenasterol, and ergosta-4,24(28)-dien3-one were isolated from a Penares sp. sponge collected from Vietnamese waters. Structures of the obtained compounds were established by extensive 1D and 2D NMR spectroscopy and mass spectrometry. Configurations of the triterpene epoxy lactones (1− 4) were determined on the basis of NOESY and CD data and calculation of spin coupling constants and confirmed by X-ray crystallographic analysis of compound 2. The isolated triterpenoid 6 was cytotoxic against human leukemia HL-60 cells (IC50 = 9.7 μM).

M

histamine release from rat peritoneal mast cells induced by antiIgE, were later isolated from the sponge Penares incrustans.13 A Penares sp. sponge was collected in Vietnamese waters during the 30th cruise of R/V Akademik Oparin in January 2005. The sponge differs in the morphology of one type of spicule (calthrops-like megascleres instead of dichotrienes) from earlier described species of the genus Penares. This motivated us to study the major components of the alcoholsoluble materials in continuation of our previous studies on marine natural products from sponges.14 An initial examination of the chemistry of this Penares specimen led to the discovery of carbazole-derived and indolophenanthridine alkaloids.15 Herein, we report the structures and biological activities of six new triterpenoids (1−6) and three earlier known compounds isolated by us from a Penares sp. sponge.

arine sponges are well known to be a rich source of new natural compounds with unusual structures and diverse biological properties of medical interest.1 Among them, sponges belonging to the genus Penares contain different secondary metabolites such as the bromoindole alkaloid penaresin with sarcoplasmic-reticulum Ca2+-inducing properties,2 sphingosine-derived azetidine alkaloids with potent actomyosin ATPase activity3 and inhibitory properties against protein kinase C,4 the penaramide bis-amides, which inhibit binding of ω-conotoxin GVIA to N-type Ca2+ channels,5 30and 31-membered macrolides that inhibit α-glucosidase,6 the ancorinoside tetramic acid glycosides, possessing inhibitory activities against type 1 membrane matrix metalloproteinase,7 the isoquinoline alkaloid schulzeines with alpha-glycosidase inhibitory properties,8 iminopentitols and methylpipecolates acylated by sulfated fatty acids, which also show alphaglycosidase inhibitory properties,9,10 and the cytotoxic longchain sphingoid base penasins.11 A group of natural products of particular interest from Penares spp. includes unusual triterpenoids with a carboxy group attached to C-14. Such triterpenoids are very rare in nature and demonstrate interesting bioactivities. Penasterol, an antileukemic triterpenoid, was found from an Okinawan Penares sp. in 1988 as the first natural compound belonging to this group.12 Penasterone and acetylpenasterol, potent inhibitors of © 2013 American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION Nine metabolites including six new triterpenoids (1−6) have been isolated from the major nonpolar fraction of the ethanolic extract of a Penares sp. sponge by LH-20 and silica gel column chromatography followed by HPLC. The structure elucidation of the new metabolites 1−6 and identification of the previously known ergosta-4,24(28)-diene-3-one,16,17 penasterone, and Received: June 13, 2013 Published: August 26, 2013 1746

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acetylpenasterol13 was made using 1D and 2D NMR techniques accompanied by mass spectrometry, IR, CD spectroscopy, and X-ray diffraction analysis of compound 2. Compound 1 has the molecular formula C30H44O4 based on its HREIMS and 13C NMR spectroscopic data (Table 1). There were six methyl singlets at δH 1.02 (Me-18), 1.06 (Me-29), 1.10 (Me-28), 1.19 (Me-19), 1.59 (Me-26), and 1.68 (Me-27) and one doublet at δH 0.89 (Me-21, J = 6.6 Hz) in the 1H NMR spectrum of 1, suggesting its triterpene nature. The 13C NMR and DEPT spectra of this compound indicated 30 carbon signals, including seven methyls, five methines, nine methylenes, and nine quaternary carbons. The side chain with a 24(25)-double bond (δC 131.2, 124.7 and δH 5.06, tt, J = 7.1, 1.4 Hz) in the structure of 1 was identical to that of lanosterol,18 as was confirmed by the HMBC correlations from Me-26 and Me-27 to C-24 and C-25, from Me-21 to C-17, CTable 1. 13C NMR (176.04 MHz) and 1H NMR (500.13 MHz) Spectroscopic Data (CDCl3) for Compounds 1 and 2 1 pos.

δC

δH (J in Hz)

1

31.1

2

33.7

α: 2.14, ddd (13.7, 13.7, 5.0) β: 1.79, ddd (13.7, 5.0, 5.0) α: 2.34, ddd (14.9, 4.5, 4.5) β: 2.60, ddd (14.9, 13.9, 5.5)

3 4 5 6

214.6 46.8 43.1 21.8

7 8 9 10 11

53.6 64.6 85.5 37.1 22.8

12

34.0

13 14 15

46.8 59.3 20.2

16

27.5

17 18 19 20 21 22

52.2 13.9 16.3 34.4 18.1 35.6

23

24.7

24 25 26 27 28 29 30

124.7 131.2 17.6 25.7 25.7 22.4 176.8

a

2 2, 2, 1, 1,

3, 3, 3, 3,

6, 7, 7, 6,

HMBC

δC

δH (J in Hz)

5,b 9, 10, 19 5, 9,b 10, 19 4, 10 10

31.9

α: 2.07, m β: 1.87, m α: 2.34, ddd (14.6, 4.7, 3.0) β: 2.43, ddd (14.6, 14.6, 5.8)

2, 2, 1, 1,

2.16, dq (12.6, 6.4) 1.76, ddd (12.6, 12.6, 4.6) α: 2.11, ddd (15.8, 6.5, 4.6) β: 1.63, dd (15.8, 12.6) 3.07, d (6.4)

3, 5, 6, 28 4, 6, 9, 10, 19, 28 7, 8, 10 4,b 7, 8 5, 6, 8, 14b

α: 2.01, m β: 2.09, m α: 1.60, m β: 2.03, m

8, 9, 12, 13 10, 12, 13 11 9, 11, 14, 18

α: 1.54, m β: 1.24, ddd (12.5, 12.5, 5.4) a: 1.36, m b: 2.15, m 1.68, m 0.99, s 1.03, s 1.42, m 0.86, d (6.5) 1.07, m 1.41, m 1.85, m 2.02, m 5.06, tt (7.1; 1.4)

13, 14, 16, 30 8, 14, 16, 30 14, 17, 20b 13, 14, 17 13, 16,b 18, 20 12, 13, 14, 17 1, 5, 9, 10 13,b 17, 21,b 22 17, 20, 22 20, 21, 23, 24 20, 21, 23,b 24 22, 24, 25 24,b 25b 23, 26, 27

1.59, s 1.68, s 1.03, d (6.5)

24, 25, 27 24, 25, 26 3, 4, 5

1.89 m α: 1.92, m β: 1.88, m 3.07, d (6.4)

4, 5, 5, 5,

7, 10, 19, 28, 29 10, 19, 28, 29 10, 19, 28, 29 8,b 14b

α: 1.95, m β: 2.07, m α: 1.58, m β: 2.02, m

5,b 8, 9, 12, 13 5b, 9, 12, 13, 14 11, 13, 14, 17,b 18 9, 11, 13, 14, 18

α: 1.53, m β: 1.25, ddd (12.5, 12.5, 5.3) a: 1.37, m b: 2.15, m 1.66, m 1.02, s 1.19, s 1.41, m 0.89, d (6.6) 1.05, m 1.40, m 1.84, m 2.02, m 5.06, tt (7.1, 1.4)

13, 14, 16, 17,b 30 8, 14, 16, 17,b 30 14, 15, 17, 20 14, 15, 17, 20 13, 16,b 18, 20, 22 12, 13, 14, 17 1, 5, 9, 10 13, 16, 17, 21, 22, 23 17, 20, 22 20, 23, 24b 16, 17, 20, 21, 23, 24 22, 24, 25 22,b 24,b 25b 22, 23, 26, 27

1.59, 1.68, 1.10, 1.06,

24, 25, 27 24, 25, 26 3, 4, 5, 29 3, 4, 5, 28

s s s s

36.5 210.6 44.8 42.2 25.4 52.8 64.9 84.6 36.7 23.0 34.0 46.8 59.3 20.3 27.5 52.2 13.9 14.4 34.4 18.1 35.7 24.7 124.7 131.2 17.7 25.7 11.6

c

HMBC 10, 19 3, 5, 10 3, 4, 10, 28b 3, 10

176.8

a

Assignments were made with the aid of the COSY and HSQC spectra. bCross-peaks of low intensity. cMultiplicity and J for H-4, H-5, and H-6α,β were determined using 1D-TOCSY data. 1747

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20, and C-22, and from H-24 to C-22, C-23, Me-26, and Me27. The IR spectrum of 1 exhibited absorption bands at 1707 and 1768 cm−1 characteristic of ketone and γ-lactone functionalities. Therefore, the signal at δC 214.6 was assigned to the oxo functionality that was placed at C-3 according to the HMBC cross-peaks H2-2, H-5, Me-28, and Me-29/C-3. An ester-type carbonyl group signal at δC 176.8 (C-30) and two carbon signals at δC 85.5 (C-9) and 59.3 (C-14) confirmed the presence of a γ-lactone in the structure of 1. The 30,9connectivity of this functional group was established by the HMBC correlations Me-18/C-14; H2-15/C-30; H2-11/C-9; H11α/C-8; and Me-19/C-9. The 30,9α-olide with S configuration at C-14 in compound 1 seemed to be more probable due to biogenetic evidence, as only 14α-carboxy-triterpenes or related aglycones in the corresponding glycosides have previously been isolated from sponges.1,19 In accordance with the HSQC data, the doublet signal at δH 3.07 (J = 6.4 Hz) in the 1H NMR spectrum of 1 was due to the proton linked to the δC 53.6 carbon with a one-bond 1H−13C coupling constant 1JCH = 178.1 Hz, characteristic of epoxide carbons.20,21 Together with the quaternary carbon signal at δC 64.6 the mentioned signals were assigned to a C-7/C-8 epoxide. This position of an epoxide was confirmed by the long-range correlations H-7/C-5, C-6, C-8, C-14; H2-6/C-7, C-8; and H5/C-6, C-7, C-8. A doublet character for the H-7 signal can be explained by the close to zero value of one of the 1H−1H coupling constants (3JH6−H7). The above data suggested the 3-oxo-7β,8β-epoxy-5αlanost-24-en-30,9α-olide or the 3-oxo-7α,8α-epoxy-5α-lanost24-en-30,9α-olide structure and relative configuration for compound 1. To resolve the configuration of the epoxide group, the strain-energy-minimized conformations for both possible structures were analyzed. It was shown that a dihedral angle near 90° and the corresponding zero coupling constant for vicinal protons H-6β and H-7 are realized for the 7β,8βepoxide. Moreover, this presumption was in a good agreement with observed NOE correlations H-7/H2-15. The configurations of 1 and related epoxides 2−4 will be discussed further during the description of the structure elucidation of 2. The molecular formula of compound 2 was determined by HREIMS as C29H42O4. The 13C and 1H NMR signals of compound 2 and HMBC (Table 1) and COSY data showed that its chemical structure is closely related to that of 1, but 2 contains only one methyl group at C-4 (Me-28, δH 1.03, d, J = 6.5 Hz, δC 11.6; CH-4 δC 44.8, δH 2.16, dq, J = 12.7; 6.4 Hz). A crystal of 2 was obtained by slow evaporation of its solution in MeOH, and X-ray diffraction analysis (Supporting Information) established the 29-nor-3-oxo-7β,8β-epoxy-5αlanost-24-en-30,9α-olide structure of 2 (Figure 1). To distinguish between the two alternative enantiomeric structures, the CD spectrum of 2 was recorded. It showed a positive Cotton effect at 290 nm (Δε 3.48). The octant rule predicts a positive Cotton effect for the 3-oxo-4α-methyl-5α-configuration in 2 and a negative effect for the enantiomeric 3-oxo-4β-methyl5β-configuration. Moreover, comparison with CD spectra of known 3-oxo-4α-methyl-5α-cholestane and cycloartane derivatives,22 which also demonstrate positive Cotton effects at around 300 nm, indicated that 2 has the 4S, 5S, 7S, 8S, 9S, 10S, 13R, 14S, 17R, 20R absolute configuration. It was also an indirect confirmation of the analogous spatial structure of 1. The revealed configurations of the epoxide and γ-lactone in 1 and 2 were in good agreement with the multiplicities and

Figure 1. ORTEP drawing of the compound 2 asymmetric unit containing three independent molecules.

coupling constants for H-5 (δH 1.76, ddd, J = 12.6, 12.6, 4.6 Hz), H2-6 (Hα: δH 2.11, ddd, J = 15.8, 6.5, 4.6 Hz, and Hβ: δH 1.63, dd, J = 15.8, 12.6 Hz), and H-7 (δH 3.07, d, J = 6.4 Hz) of 2 that were measured by a 1D TOCSY experiment and calculated using an empirical generalization of the classical Karplus equation.23 The same character of multiplicity for the H-7 signal and coincidence of the long-range COSY correlations CH3-19/H-5 and CH3-18/H-12α, H-17 as well as observed NOE correlations CH3-19/H-2β, H-6β; CH3-18/ H-11β, H-20; and H-7/H2-15 for both compounds confirmed the same configurations in 1 and 2. Compound 3, isolated as a minor component, showed an ion peak with m/z 512.3476 [M]+ in the HREIMS experiment that indicated its molecular formula as C32H48O5. The EIMS fragmentation peaks at m/z 467 [M − COOH]+ and m/z 453 [M − OAc]+ along with IR bands at 1766 and 1727 cm−1 indicated the presence of a γ-lactone function and an acetate group in 3. Examination of the 1H and 13C NMR data (Table 2) revealed that 3 differed from 1 only in an acetoxy group instead of a carbonyl at C-3. Indeed, the 1H NMR spectrum contained an additional CH3 singlet at δH 2.04 and an axial H-3 signal at δH 4.51 (dd, J = 11.6, 4.2 Hz). The 13C NMR spectrum of 3 showed an additional ester carbonyl at δC 170.6 and the signal of an oxygen-bearing carbon at δC 79.7 instead of a carbonyl signal at δC 214.6 in the spectrum of 1. All of the signals of atoms belonging to ring A as well as Me-19 (δH 1.03, δC 16.4), Me-28 (δH 0.90, δC 28.1), and Me-29 (δH 0.90, δC 1748

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Table 2. 13C NMR (125.7 MHz) and 1H NMR (500.13 MHz) Spectroscopic Data (CDCl3) for Compounds 3 and 4 3 pos.a 1

30.3

2

22.8

3 4 5 6

79.7 37.3c 42.0 20.8

7 8 9 10 11

53.8 64.4 85.7 37.4c 22.7

12

34.1

13 14 15 16

46.8 59.4 20.2 27.5

17 18 19 20 21 22

52.2 13.9 16.4 34.5 18.1 35.7

23

24.7

24 25 26 27 28 29 30 Ac a

δC

124.8 131.2 17.7 25.7 28.1 16.7 177.0 170.6 (C) 21.2 (CH3)

4

δC (J in Hz)

HMBC

α: 1.92, m β: 1.55, m α:1.69, m β: 1.56, m 4.51, dd (11.6, 4.2)

4, 28, 29, 170.6 (C)

1.53 m α: 1.96, m β: 1.77, t (14.6) 3.02, d (6.4)

3, 4, 4, 5,

α: 1.90, m β: 2.01, m α: 1.58, m β: 1.99, m

8, 9, 13

10, 19 10, 19

δC

δC (J in Hz)

HMBC

30.5

α: 1.83, ddd (14.0, 14.0, 3.8) β: 1.54, m α: 1.63, m β: 1.57, m 3.26, dd (11.4, 4.3)

2, 3, 10, 19 2, 3, 5, 10 1, 3,b 4,b 10 3, 4b 1,b 2,b 4,b 28, 29

1.44, dd (13.5, 4.1) α: 1.97, m β: 1.76, br t (14.7) 3.02, d (6.5)

4, 5, 5, 5,

α: 1.90, m β: 2.03, m α: 1.56, m β: 1.99, m

8, 9, 11, 13 12, 13 11, 13,b 14, 18 9, 11, 13,b 14, 18

α: 1.54, m β: 1.25, ddd (12.5, 12.5, 5.3) a: 1.36, m b: 2.15, m 1.67, m 0.99, s 1.01, s 1.41, m 0.88, d (6.6) 1.07, m 1.41, m 1.85, m 2.03, m 5.06, tt (7.1; 1.4)

13, 14, 16, 17, 30 8, 14, 16, 17, 30 14,b 15, 17, 20 13, 14, 15, 17 12, 13,b 18 12, 13, 14, 17 1, 5, 9, 10 17, 21 17, 20, 22 17,b 20, 21, 23, 24 24 22, 24, 25 24,b 25b 23, 26, 27

1.59, 1.68, 1.01, 0.83,

24, 25, 27 24, 25, 26 3, 4, 5, 29 3, 4, 5, 28

26.4

4, 10, 19 10 5, 7, 10 6, 8b

78.0 38.4 41.9 20.9 53.9 64.4 85.9 37.5 22.7 34.2

14 46.8 59.5 20.2

α: 1.54, m β: 1.24, ddd (12.5; 12.5, 5.3) a: 1.37, m b: 2.15, m 1.68, m 0.99, s 1.03, s 1.42, m 0.88, d (6.6) 1.08, m 1.41, m 1.85, m 2.01, m 5.06, tt (7.1, 1.4)

24, 25b

24.8

26, 27

1.59, 1.67, 0.90, 0.90,

23, 24, 25, 27 23, 24, 25, 26 3, 4, 5, 29 3, 4, 5, 28

124.8 131.2 17.7 25.7 28.1 15.6 177.3

s s s s

2.04, s

8, 14, 30 27.6 13, 14 12, 20, 21 12, 13, 14, 17 1, 5, 9, 10 21 17, 20

52.3 13.9 16.3 34.5 18.1 35.7

s s s s

6, 7, 7, 6,

7, 10, 19 10 8, 10b 8, 14b

2, 3, 9,b 10, 19 2, 3, 5, 10

Ac (170.6)

Assignments were made with the aid of COSY and HSQC spectra. bCross-peaks of low intensity. cValues can be interchanged.

NOESY cross-peaks allowed us to assign the 3β-hydroxy-7β,8βepoxy-5α-lanost-24-en-30,9α-olide structure for 4. Compound 5 (29-nor-penasterone) was isolated as a pale yellow, amorphous solid together with the previously known penasterone, and its structure was determined by NMR (Table 3), EIMS, and comparison with previously published data.13 Compound 5 has the C29H44O3 molecular formula on the basis of the peak with m/z 440.3305 [M]+ in the HREIMS spectrum. The 13C and 1H NMR spectra were very similar to those of penasterone, but the signals of the atoms belonging to ring A as well as Me-19 (δH 1.23, s; δC 17.7) were shifted. Moreover, there were a new H-4 signal (δH 2.31, dq, J = 13.0, 6.6 Hz) and a Me-28 doublet (δH 1.02, J = 6.6 Hz) in the 1H NMR spectrum of 5 instead of two methyl singlets [Me-28 (δH 1.07) and Me-29 (δH 1.09)] observed for penasterone. The equatorial position of Me-28 was established from the NOE correlation

16.7) in 3 were slightly shifted, while all other chemical shifts were identical to those of 1. Furthermore, 2D NMR experiments including HSQC, HMBC, COSY, and NOESY confirmed the 3β-acetoxy-7β,8β-epoxy-5α-lanost-24-en-30,9αolide structure of 3. According to its NMR characteristics and HREIMS data (m/ z 470.3381 [M]+, C30H46O4), compound 4 seemed to be the deacetyl derivative of 3. It was in good agreement with its IR spectrum, which had absorption bands of hydroxy and γ-lactone groups at 3617 and 1764 cm−1, respectively. The presence of a 3β-hydroxy in the structure of 4 was deduced from 1H NMR chemical shift and coupling constants of axial H-3 (δH 3.26, dd, J = 11.4, 4.3 Hz) along with the EIMS fragmentation peak with m/z 452 [M − H2O]+. The similarity in chemical shifts of the corresponding protons and carbon atoms in the NMR spectra of 3 and 4 (Table 2) as well as their similar HMBC and key 1749

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Table 3. 13C NMR (176.04 MHz) and 1H NMR (500.13 MHz) Spectroscopic Data (CDCl3) for Compounds 5 and 6 5

a

6

pos.a

δC

δH (J in Hz)

1

36.7

2

37.8

α: 2.08, m β: 1.64, m α: 2.37, ddd (14.9; 5.1; 2.4) β: 2.47, ddd (14.3; 14.3, 6.6)

2, 2, 1, 1,

3, 5, 10, 19 10, 19 3, 4, 10 3

3 4 5 6

213.2 44.9 49.1 22.0 26.7

3, 4, 5, 5, 6 6,

5, 6, 28 6, 7,b 10, 19 7, 8, 10 7,b 10

7

2.31, dq (13.0; 6.6) 1.46, m α: 1.78, m β: 1.45, m α: 2.03, m β: 2.09, m

8 9 10 11

128.5 138.6 37.1 23.1

12

31.4

13 14 15

47.3 62.8 27.7

16

29.2

17 18 19 20 21 22

50.8 17.6 17.7 35.8 18.4 36.0

23

24.9

24 25 26 27 28 29 30

125.0 131.0 17.6 25.7 11.4

α: 2.25, m β: 2.16, m α: 2.14, m β: 1.76, m

δC

HMBC

30.0 26.9 78.3 38.4 46.2 21.8 120.2

δH (J in Hz)

HMBC

α: 1.91, m β: 1.61, m α: 1.68, m β: 1.62, m 3.29, dd (11.4, 4.5) 1.26, 2.01, 2.01, 5.59,

m m m dd (7.0, 3.0)

8,b 9b 140.1 88.9 38.4 25.9

8, 9, 12b 8, 9, 12b 11, 13, 14,b 18 9, 11, 13, 14, 18

34.3

α: 1.56, m β: 2.06, m α: 2.13, m β: 1.40, m 1.53, m 0.81, s 1.23, s 1.43, m 0.93, d (6.4) 1.05, m 1.42, m 1.84, m 2.03, m 5.08, tt (7.1, 1.4)

14, 16 13, 14, 16, 17 15, 17 15,b 20 12,b 13, 16, 18, 20 12, 13, 14, 17 1, 5, 9, 10 17, 21, 22b 17, 20, 22 20, 21, 23, 24 20, 21, 23,b 24 20, 22, 24, 25 20, 24,b 25b 22, 23, 26, 27

1.59, s 1.67, s 1.02, d (6.6)

24, 25, 27 24, 25, 26 3, 4, 5

181.4

46.0 65.2 22.1 27.9 52.5 14.2 17.2 35.3 18.4 35.9 24.9 124.9 131.1 17.7 25.7 28.9 16.5 179.0

α: 1.87, m β: 1.78, ddd (13.9, 12.0, 5.6) α: 1.55, m β: 1.90, m

a: 1.69, m b: 1.71, m a: 1.41, m b: 2.20, m 1.74, m 0.82, s 1.03, s 1.40, m 0.89, d (6.5) 1.09, m 1.43, m 1.86, m 2.04, m 5.07, tt (7.1; 1.4) 1.60, 1.68, 1.02, 0.92,

s s s s

30

12, 13, 14, 17 1, 5, 9, 10 20, 22

24, 25, 27 24, 25, 26 3, 4, 5, 29 3, 4, 5, 28

Assignments were made with the aid of COSY and HSQC spectra. bCross-peaks of low intensity.

The complete assignments of the spectroscopic data were made with the aid of a combination of COSY, HSQC, HMBC, and NOESY experiments. The double bond was located at the 7(8) position based on COSY correlations H-7/H2-6 and H-7/H-5 as well as the NOESY cross-peak H-7/H2-15. As a result, the 3β-hydroxy-5α-lanosta-7,24-dien-30,9α-olide structure was proposed for 6. Penasterol and its derivatives are also known as aglycones of glycosides from sponges belonging to the genus Erylus.19 Compounds 1−4 are structurally related to the aglycone of eryloside U from Erylus gof frilleri.24 According to the new experimental data reported above, we propose a revision of the orientation of the 7α,8α-epoxide group in the previously published eryloside U to 7β,8β. This change follows from the same multiplicities and similar coupling constant values for H26 and H-7 in our compounds 1−4 and eryloside U, for which such spectroscopic data as H-6α, δH 1.93, ddd (J = 15.0, 6.6, 4.0

between H-4 and Me-19 and from the large vicinal coupling constant (J = 13.0 Hz) of trans-diaxial protons H-4 and H-5. Of the free triterpenoids containing a 14-carboxy group, only penasterol,12 penasterone, and acetylpenasterone13 have been found in Penares spp. sponges. The isolated compound 5 is the first representative of nor-triterpenoids of this series. Compound 6 was obtained as a minor component of the sponge extract and had an HREIMS peak at m/z 454.3459 [M]+ that suggested a C30H46O3 empirical formula. Detailed analysis of the 1H and 13C NMR data (Table 3) indicated the presence of a lanosterol-type side chain in 6. Other signals were attributed to four quaternary methyl groups, Me-18 (δH 0.82, s, δC 14.2), Me-19 (δH 1.03, s, δC 17.2), Me-28 (δH 1.02, s, δC 28.9), and Me-29 (δH 0.92, s, δC 16.5); a 3β-hydroxy group (δH 3.29, dd, J = 11.4, 4.5 Hz, δC 78.3); one γ-lactone (C-14, δC 65.1; C-9, δC 88.9, C-30, δC 179.0); and one trisubstituted double bond (δH 5.59, dd, J = 7.0, 3.0 Hz, δC 120.2 and 140.0). 1750

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Hz); H-6β, δH 1.72, t (J = 14.6 Hz); and H-7, δH 3.13, br d (J = 6.4 Hz) were reported.24 Therefore, compounds 1−4 and eryloside U represent a series of free triterpenoids and a triterpene glycoside with the unique combination of a 30,9αlactone and a 7β,8β-epoxide in their structures. Minor compound 6 has a polycyclic core identical to that in the aglycone of eryloside T24 and could be considered as a biosynthetic precursor of more oxygenated compounds 1−4 and the aglycones of some triterpene glycosides from sponges. We measured the cytotoxicities of all of the isolated triterpenoids against several tumor cell lines, including Ehrlich ascites carcinoma, human promyelocytic leukemia HL-60, and human cervical carcinoma HeLa, as well as normal mouse epithelial cells JB6 Cl41. Metabolite 6 was active against human leukemia HL-60 cells with an IC50 = 9.7 μM. This observation correlates with the previously reported data for the structurally related antileukemic triterpenoid penasterol.12

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well as pure compound 5 (41.6 mg, 0.037%), penasterone (72.9 mg, 0.066%), and acetylpenasterol (6.8 mg, 0.006%). Compounds 4 (8.2 mg, 0.007%) and 6 (2.0 mg, 0.002%) were further isolated and purified using a normal-phase HPLC column in n-hexane−EtOAc (4:1). 3-Oxo-7β,8β-epoxy-5α-lanost-24-en-30,9α-olide (1): 19.7 mg; pale yellow oil; [α]27 D −45.8 (c 0.65, CHCl3); IR (CHCl3) νmax 1768, 1707 cm−1; 1H and 13C NMR data (CDCl3), see Table 1; EIMS m/z 468 [M]+ (21), 423 (24), 407 (20), 384 (31), 339 (28), 311 (37), 269 (12), 201 (15), 173 (24), 105 (38), 81 (44), 69 (100), 55 (79), 41 (98); HREIMS m/z 468.3259 [M]+ (calcd for C30H44O4, 468.3240). 29-Nor-3-oxo-7β,8β-epoxy-5α-lanost-24-en-30,9α-olide (2): 12.2 mg; colorless crystals (MeOH); mp 143−145 °C; [α]27 D −43.6 (c 0.55, CHCl3); UV (EtOH) λmax (log ε) 273 (2.76), 204 (3.84); CD (c 2.8 × 10−4 M, EtOH) λmax (Δε) 208 (2.37), 222 (−15.42), 290 (3.48) nm; IR (CHCl3) νmax 1770, 1710 cm−1; 1H and 13C NMR data (CDCl3), see Table 1; EIMS m/z 454 [M]+ (35),409 (25), 370 (36), 325 (30), 302 (44), 287 (57), 233 (26), 209 (25), 149 (39), 109 (100), 69 (95), 55 (79), 41 (93); HREIMS m/z 454.3104 [M]+ (calcd for C29H42O4, 454.3083). 3β-Acetoxy-7β,8β-epoxy-5α-lanost-24-en-30,9α-olide (3): 2.3 mg; white, amorphous powder; [α]27 D −29.6 (c 0.23, CHCl3); IR (CHCl3) νmax 1766, 1727 cm−1; 1H and 13C NMR data (CDCl3), see Table 2; EIMS m/z 512 [M]+ (17), 467 (19), 453 (12), 428 (20), 355 (27), 339 (14), 295 (14), 253 (10), 173 (23), 147 (22), 121 (30), 107 (34), 69 (100), 55 (68), 41 (86); HREIMS m/z 512.3476 [M]+ (calcd for C32H48O5, 512.3502). 3β-Hydroxy-7β,8β-epoxy-5α-lanost-24-en-30,9α-olide (4): 8.2 mg; colorless crystals (EtOH); mp 180−183 °C; [α]27 D −12.6 (c 0.82, CHCl3); IR (CHCl3) νmax 3617, 1765 cm−1; 1H and 13C NMR data (CDCl3), see Table 2; EIMS m/z 470 [M]+ (32), 452 (11), 425 (34), 409 (17), 386 (40), 341 (30), 313 (45), 295 (16), 271 (15), 195 (11), 161 (26), 109 (29), 95 (38), 81 (37), 69 (100), 55 (56), 41 (72); HREIMS m/z 470.3381 [M]+ (calcd for C30H46O4, 470.3396). 29-Nor-penasterone (5): 41.6 mg; pale yellow, amorphous solid; [α]27 D −24.2 (c 0.83, CHCl3); UV (EtOH) λmax (log ε) 204 (3.88); CD (c 2.8 × 10−4 M, EtOH) λmax (Δε) 207 (35.59), 224 (−33.81), 234 (−31.40), 289 (4.73) nm; IR (CHCl3) νmax 1703 cm−1; 1H and 13C NMR data (CDCl3), see Table 3; EIMS m/z 440 [M]+ (22), 395 (100), 327 (9), 243 (45), 231 (22), 159 (11), 109 (40), 69 (62), 55 (35), 41 (37); HREIMS m/z 440.3305 [M]+ (calcd for C29H44O3, 440.3291). 3β-Hydroxy-5α-lanosta-7,24-dien-30,9α-olide (6): 2.0 mg; color1 13 less oil; [α]27 D −10 (c 0.06, CHCl3); H and C NMR data (CDCl3), + see Table 3; EIMS m/z 454 [M] (53), 439 (7), 409 (33), 377 (10), 328 (14), 299 (24), 246 (28), 232 (30), 158 (27), 109 (46), 81 (49), 69 (100), 55 (90), 41 (89); HREIMS m/z 454.3459 [M]+ (calcd for C30H46O3, 454.3447). X-ray Crystallography. The colorless crystal of 2 was grown from MeOH. Crystal data: C29H42O4; Mr 454.63; T = 140(1) K; wavelength 0.71073 Å; crystal system monoclinic; space group P21; unit cell dimensions a = 20.9983(6) Å, b = 7.8332(2) Å, c = 23.1219(9) Å, β = 93.695(1)°; volume 3795.3(2) Å3; Z = 6; Dcalc = 1.194 Mg/m3; absorption coefficient 0.077 mm−1; F(000) = 1488; crystal size 0.03 × 0.22 × 0.26 mm; reflections collected 16 663; independent reflections 7292 [R(int) 0.028]. Complete crystallographic data of 2 including atomic coordinates, bond lengths and angles, thermal parameters, and additional experimental details have been deposited with the Cambridge Crystallographic Data Center (CCDC 934294). Copies of the data can be obtained, free of charge, on application to the Director, CCDC, 12 Union Road, Cambridge CB2 1EZ, UK (fax: +44(0)1223-336033 or e-mail: [email protected]). Cytotoxicity Assays. The cell lines HL-60 and HeLa were purchased from the ATCC collection. Both cell lines were cultured at 37 °C and 5% CO2 in RPMI medium containing 10% FBS (HyClone), 2 mM L-glutamine, and 1% penicillin−streptomycin. The normal mouse epithelial JB6 Cl41 cell line was provided by Dr. Zigang Dong (Hormel Institute). It was cultured in a monolayer at 37 °C and 5% CO2 in MEM, containing 5% FBS, 2 mM L-glutamine, and 1% penicillin−streptomycin. The effects of the compounds on the HL-60,

EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured using a Perkin-Elmer 343 polarimeter. The CD spectra were recorded on a Jasco J-500A spectropolarimeter. IR spectra were recorded on a Bruker Vector 22 IR spectrometer. 1H NMR (700.13 MHz, 500.13 MHz) and 13C NMR (176.04 MHz, 125.75 MHz) spectra were recorded in CDCl3 on Avance-III 700 and DRX-500 Bruker spectrometers. The 1H and 13C NMR chemical shifts were referenced to the solvent peak for CDCl3 at δH 7.26 and δC 77.0. EIMS and HREIMS spectra were obtained on an AMD-604 S (AMD Intectra, 70 eV). X-ray crystallographic analysis was carried out on a Bruker Kappa APEX II diffractometer with graphite-monochromated Mo Kα radiation (λ = 0.71073 Å). APEX2 software was used in data collection, cell refinement, and data reduction processes (Bruker APEX2, Bruker AXS Inc., 2005). The structure was solved by direct methods and refined by a full-matrix least-squares method using the SHELXTL program. Low-pressure column liquid chromatography was performed using Sephadex LH-20 (Sigma Chemical Co.) and silica gel (KSK, Russia, 0.05−0.16 mm). HPLC was performed using an Agilent Series 1100 Instrument equipped with a RID-G1362A differential refractometer and ULTRASPHERA Si (5 μ, 250 × 10 mm), YMCPack ODS-A (250 × 10 mm), and YMC-Pack ODS-A (5 μ, 250 × 4.6 mm) columns. Yields are based on dry weight of the sponge. Animal Material. The specimens of Penares sp. were collected by dredging at a depth of 95 m (16°07′ N, 114°47′ E) during the 30th cruise of R/V Akademik Oparin in Vietnam in January 2005, as previously described.15 A voucher specimen (PIBOC O30-271) has been deposited in the collection of marine invertebrates of the G. B. Elyakov Pacific Institute of Bioorganic Chemistry (Vladivostok, Russian Federation) and identified by taxonomist Vladimir B. Krasokhin of the same institute. Extraction and Isolation. The fresh collection of the sponge Penares sp. was frozen and kept at −20 °C. The sponge specimen (wet weight 400 g) was cut and extracted with EtOH. The extract was concentrated in vacuo to an aqueous residue, which was further extracted with n-hexane (3 × 250 mL). The organic layers were combined and evaporated in vacuo to give a dark brown oil (2.8 g), which was fractionated using LH-20 column chromatography (CHCl3−EtOH, 1:1) into four fractions. 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, stepwise gradient, CHCl3− EtOH). The first subfraction eluted with 100% CHCl3 was separated by normal-phase HPLC in n-hexane−EtOAc (5:1) to obtain 36.1 mg (0.032%) of ergosta-4,24(28)-diene-3-one, 19.7 mg (0.009%) of 1, 12.2 mg (0.011%) of 2, and 7.7 mg of a fraction containing 3 as a major component. Compound 3 (2.3 mg; 0.002%) was further purified using an analytical reversed-phase HPLC column (95% EtOH). The next subfraction that eluted with 100% CHCl3 was chromatographed using a semipreparative reversed-phase HPLC column (95% EtOH) to yield 26 mg of a mixture containing compounds 4 and 6 as 1751

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HeLa, and JB6 Cl41 cell viability were evaluated using the MTS test.25 The experiment was performed as previously described27 with slight modifications (Supporting Information). Murine Ehrlich ascites carcinoma cells was obtained from AllRussian Cancer Center of RAMS (Moscow, Russian Federation). The cells of Ehrlich ascites carcinoma were separated from ascites that were collected on day 7 after inoculation into CD-1 line mice. The cells were washed of ascites three times and then resuspended in RPMI1640 medium containing gentamicin 8 μg/mL (BioloT). The cytotoxicities of the compounds against these cells were measured by the MTT method26 (Supporting Information). The results of both experiments are represented as IC50 values of the substances against corresponding cells. Cisplatin (cisdiamminedichloroplatinum(II), 1 mg/mL in dH2O, NeoCorp) was used as a reference substance, demonstrating IC50 values of 0.25 μM (HL-60), 4.1 μM (HeLa), 18.2 μM (JB6 Cl41), and 6.8 μM (Ehrlich ascites carcinoma).

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(10) Nakao, Y.; Maki, T.; Matsunaga, S.; van Soest, R. W. M.; Fusetani, N. J. Nat. Prod. 2004, 67, 1346−1350. (11) Ando, H.; Ueoka, R.; Okada, S.; Fujita, T.; Iwashita, T.; Imai, T.; Yokoyama, T.; Matsumoto, Y.; van Soest, R. W. M.; Matsunaga, S. J. Nat. Prod. 2010, 73, 1947−1950. (12) Cheng, J. F.; Kobayashi, J.; Nakamura, H.; Ohizumi, Y.; Hirata, Y.; Sasaki, T. J. Chem. Soc., Perkin Trans. 1 1988, 8, 2403−2406. (13) Shoji, N.; Umeyama, A.; Motoki, S.; Arihara, S.; Ishida, T.; Nomoto, K.; Kobayashi, J.; Takei, M. J. Nat. Prod. 1992, 11, 1682− 1685. (14) Makarieva, T. N.; Tabakmaher, K. M.; Guzii, A. G.; Denisenko, V. A.; Dmitrenok, P. S.; Shubina, L. K.; Kuzmich, A. S.; Lee, H. S.; Stonik, V. A. J. Nat. Prod. 2011, 74, 1952−1958. (15) Lyakhova, E. G.; Kolesnikova, S. A.; Kalinovsky, A. I.; Afiyatullov, Sh. Sh.; Dyshlovoy, S. A.; Krasokhin, V. B.; Minh, Ch. V.; Stonik, V. A. Tetrahedron Lett. 2012, 53, 6119−6122. (16) Sheikh, Y. M.; Djerassi, C. Tetrahedron 1974, 30, 4095−4103. (17) Guella, G.; Mancini, I.; Pietra, F. Comp. Biochem. Physiol. 1988, 90B, 113−115. (18) Knight, S. A. Org. Magn. Reson. 1974, 6, 603−611. (19) Ivanchina, N. V.; Kicha, A. A.; Stonik, V. A. Steroids 2011, 76, 425−454. (20) Seto, H.; Furihata, K.; Otake, N.; Itoh, Y.; Takahashi, S.; Haneishi, T.; Ohuchi, M. Tetrahedron Lett. 1984, 25, 337−340. (21) Uhrin, D.; Batta, G.; Hruby, V. J.; Barlow, P. N.; Kövér, K. E. J. Magn. Reson. 1998, 130, 155−161. (22) Djerassi, C.; Halpern, O.; Halpern, O.; Riniker, B. J. Am. Chem. Soc. 1958, 80, 4001−4015. (23) Haasnoot, C. A. G.; de Leeuw, F. A. A. M.; de Leeuw, H. P. M.; Altona, C. Org. Magn. Reson. 1981, 15, 43−52. (24) Afiyatullov, Sh. Sh.; Kalinovsky, A. I.; Antonov, A. S.; Ponomarenko, L. P.; Dmitrenok, P. S.; Aminin, D. L.; Krasokhin, V. B.; Nosova, V. M.; Kisin, A. V. J. Nat. Prod. 2007, 70, 1871−1877. (25) Barltrop, J. A.; Owen, T. C.; Cory, A. H.; Cory, J. G. Bioorg. Med. Chem. Lett. 1991, 1, 611−614. (26) Carmichael, J.; DeGraff, W. G.; Gazdar, A. F.; Minna, J. D.; Mitchell, J. B. Cancer Res. 1987, 47, 936−942. (27) Dyshlovoy, S. A.; Naeth, I.; Venz, S.; Preukschas, M.; Sievert, H.; Jacobsen, C.; Shubina, L. K.; Gesell Salazar, M.; Scharf, C.; Walther, R.; Krepstakies, M.; Priyadarshini, P.; Hauber, J.; Fedorov, S. N.; Bokemeyer, C.; Stonik, V. A.; Balabanov, S.; Honecker, F. J. Proteome Res. 2012, 11, 2316−2330.

ASSOCIATED CONTENT

S Supporting Information *

1D and 2D NMR spectra of all new compounds; details of the bioassay experiments; photograph of a Penares sp. (PIBOC O30-271) marine sponge; CIF file with the X-ray crystallographic data for 2. These materials are available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel/Fax: +7-432-231-1168. Fax: +7-432-231-4050. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The research described in this publication was supported by Grant NSS 546.2012.4 from the President of Russian Federation, Grant 12-04-93009-Viet from RFBR, the Program of Presidium of RAS “Molecular and Cell Biology”, and FEB RAS Grant 13-III-B-05-009. We thank taxonomist V. B. Krasokhin (G. B. Elyakov Pacific Institute of Bioorganic Chemistry, Vladivostok, Russian Federation) for identification of the animal material and Dr. Z. Dong (Hormel Institute, University of Minnesota, MN, USA) for providing the JB6 cell lines.

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REFERENCES

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