HETEROCYCLES

Short Paper | Regular issue | Vol. 83, No. 9, 2011, pp. 2149-2155
Received, 9th May, 2011, Accepted, 1st July, 2011, Published online, 11th July, 2011.
DOI: 10.3987/COM-11-12251

■ Cytotoxic Eunicellin-Type Diterpenes from the Soft Coral Litophyton viscudium

Tetsuo Iwagawa,* Daiki Kusatsu, Keiko Tsuha, Toshiyuki Hamada, Hiroaki Okamura, Tatsuhiko Furukawa, Shin-ichi Akiyama, Matsumi Doe, Yoshiki Morimoto, Fumihito Iwase, and Kaoru Takemura

Department of Oncology, Graduate School of Medical and Dental Sciences, Kagoshima University, 8-35-1 Sakuragaoka Kagoshima 890-8544, Japan

Abstract

Five new eunicellin-type diterpenes were isolated from the soft coral Litophyton viscudium. The structures of the compounds were elucidated mainly on the basis of extensive spectroscopic analysis. Compounds exhibited cytotoxic activity against HL-60 with IC50 values of 4.2–50 μM.

The soft coral Litophyton species is known to produce a sesquiterpene,1 cembranes,1 eunicellin-based diterpenes,2-5 and sterols6 possessing biological activities, such as antiproliferative activity against the cell lines L-929 and K-562, cytotoxicity against HeLa cells,1 and hemolytic,2 molluscicidal,3 repellent,4 insect growth inhibitory,5 brine shrimp lethality,6 and antileukemic7 activities. As a continuation of our survey on bioactive compounds from marine organisms,8 the CH2Cl2-soluble portion (23 g) of a MeOH extract of the soft coral L. viscudium (5.3 kg, wet wt.), collected in the area of Otsuki Town area, Kochi Prefecture, Japan, showed moderate cytotoxic activity (IC50 = 6.9 µg/mL) against the proliferation of human promyelocytic leukemia cells (HL-60). Further investigation of this CH2Cl2 fraction has led to the isolation of five new eunicellin-type diterpenes: (1 (3.7 mg), 2 (2.0 mg), 3 (1.8 mg), 4 (3.7 mg), and 5 (11.7 mg)) and a known compound, litophynin F (6)5(a) (7.3 mg). The isolates exhibited moderate cytotoxic activity against HL-60 cells. In this paper, we describe the isolation and structure elucidation of these new compounds.
Compound 1 was isolated as an amorphous powder and had a molecular formula of C24H36O5, which was determined by HRFABMS [m/z 405.2629, (M + H)+, calcd m/z (405.2641)]. The IR spectrum showed absorptions characteristic of a hydroxy group at 3426 cm-1, an ester carbonyl at 1732 cm-1, and a conjugated carbonyl at 1688 cm-1. The molecular formula implied seven degrees of unsaturation and the presence of one carbonyl, one ester carbonyl, and four olefinic carbons in the 13C NMR spectrum (Table 1) indicated that compound 1 had a tricyclic structure. Resonances due to i-propyl protons at C-14, methyl protons at C-3, and two terminal methylene protons in the 1H NMR spectrum (Table 1) were observed along with signals due to two oxygenated methine carbons at δC 90.7 and 80.1 that were correlated to the signals δ 3.77 (s, H-2) and 4.47 (m, H-9), respectively, in an HMQC experiment. This suggested that compound 1 was a eunicellin-based diterpenoid, which is commonly found as a chemical constituent of the Litophyton genus.2-6 The 1H NMR spectrum further indicated the presence of another oxygenated methine proton (δ 4.45, br s, W1/2 = 8.4 Hz) and an n-butyroxyl group at C-3 (δ 0.95, 1.63, 2.20). The gross structure was elucidated on the basis of 1H­–1H COSY and HMBC experiments (Figure 1). HMBC correlations of both H2-5 (δ 2.47, 2.73) and H2-16 (δ 5.23, 5.39) to C-6 (δ 206.0) and C-7 (δ 147.6) indicated that the carbonyl group was located at C-6. Another terminal methylene and the hydroxy group were determined to be attached to C-11 and C-12, respectively, on the basis of the correlations of H-10 (δ 3.06), H-12 (δ 4.45) and the methylene protons (δ 4.87, 5.12) to C-11 (δ 146.2). Correlations of Me-15 (δ 1.53) to C-2 (δ 90.7), C-3 (δ 84.1), and C-4 (δ 31.9) indicated that C-3 was attached to C-2 and C-4, and the chemical shift of C-3 suggested the presence of the n-butyroxyl group at C-3.
The relative stereochemistry was determined on the basis of NOESY analysis (Figure 2). NOESY correlation of H-1 to H-10, Me-19, and Me-20; H-10 to H-16b (δ 5.23) and H-17b (δ 4.87); H-16b to H-17b, and H-12 to H-17a (δ 5.12) suggested that H-1, H-10, H-12, and the i-propyl moiety were on the same face (β) of the ring. The configuration of H-12 was also indicative of a pseudo-equatorial orientation (β) based on the half-height width, W1/2 = 8.4 Hz, of this proton. H-2 showed NOE’s to H-9, H-14, and Me-15, and H-9 gave an NOE to H-14, confirming α-orientations for H-2, H-9, H-14, and Me-15. Thus, compound 1 has the relative structure corresponding to the 6-oxo derivative of litophynin H.
Absorptions of compound 2, C26H38O6, in the IR spectrum indicated the presence of an ester carbonyl at 1736 cm-1 and a conjugated carbonyl at 1692 cm-1. The 1H NMR spectrum was similar to that of 1, except for resonances due to additional acetyl protons (δ 2.00, 3H, s). The position of the acetyl group was determined to be located at C-12 based on the downfield shift of H-12 (δ 5.48, 1H, m) by 1.03 ppm when compared with that of 1. The β-configuration of H-12 was supported by the

observation of NOE’s between H-12 and H-10. Therefore, compound 2 is the 12-O-acetyl analogue of 1.
Compound 3, C24H36O5, was isomeric with 1, and the presence of a hydroxy group, an ester carbonyl, and a conjugated carbonyl was observed in the IR spectrum similar to the case of 1. The NMR spectra were essentially similar to those of 1; however, the chemical shifts corresponding to C-8 (δ 79.9) and C-12 (δ 31.3) in the 13C NMR spectrum were shifted downfield by 43.0 ppm and upfield by 40.2 ppm, respectively when compared with those of 1. This suggested that the hydroxy group was attached to C-8. The configuration was inferred to be α-oriented from NOE correlations of H-8 (δ 4.26) to H-16b (δ 5.44) and H-17b (δ 4.68), the latter of which was also correlated to H-10. The relative configurations of the remaining chiral centers in 3 were deduced from the similarity of their coupling patterns and chemical shifts in the NMR spectra and NOE correlations to those of 1. Thus, compound 3 has the relative structure corresponding to the keto derivative of litophynol A.2

The IR spectrum of compound 4, C24H38O5, indicated the presence of hydroxy groups at 3366 cm-1, an ester carbonyl at 1732 cm-1, and olefins at 1645 cm-1. However, unlike that observed for 1–3, absorption due to a conjugated carbonyl was not observed. A resonance due to H-6 (δ 5.07, 1H, dd, J = 11.4, 3.4 Hz) in the 1H NMR spectrum suggested that the carbonyl group in 3 was reduced to a hydroxy group in 4. The remaining resonances were similar to those of 3. The configuration of the hydroxy group was found to be the α-oriented based on an NOE correlation between H-6 and H-10. Therefore, the relative structure of 4 is as same as that of the 6-epi litophynol A.2
The molecular formula of compound 5, C25H42O5, indicated that the MW of 5 was 16 mass units higher than 4. The presence of a hydroxy group at 3495 cm-1 and an ester carbonyl at 1736 cm-1 was indicated by the IR spectrum. The 1H NMR spectrum was essentially similar to that of 4, except for the presence of signals extra side chains on the 10-membered ring. Thus, unique resonances due to an additional methoxy group at δ 3.34 (3H, s) and a methyl group at δ 1.11 (3H, s) bearing an oxygenated carbon were observed instead of the terminal methylene protons in 4. The positions of the methoxy and the hydroxy groups were confirmed to be C-6 and C-7, respectively, by interpretation of HMBC correlations between the methoxy protons and C-6 (δ 90.7) and between the methyl protons and C-7 (δ 76.0). The relative configuration of chiral carbons on the five-membered ring and six-membered ring was determined by the similarity of the NOESY data for 4 compared with those of 3 and 5. Regarding the configurations of C-3, C-6, and C-7, NOESY correlations of Me-15 to H-2 and H-6 indicated a β-orientation of the methoxy group, while an α-orientation of the hydroxy group was suggested from NOESY correlations between the methoxy protons and H-5exo (δ 1.58, 1H, dd, 15.5, 8.9 Hz) and between Me-16 and H-5endo (δ 1.34, 1H, m), as shown in Figure 3. This meant that C-3, C-6, and C-7 had relative configurations as shown in Figure 5. Therefore, the relative structure of 5 is identical with that of the 6-methyl ether of litophynol B.2 However, compound 5 may well be an artifact of the methanol extraction method used, because a hydroxy substituent at C-7 that could form hydrogen bond with the 6-hydroxy would facilitate nucleophilic attack at the 6-position by methanol.
The cytotoxicity of compounds 1–5 and litophynin F (6) in human promyelocytic leukemia cells was determined by an MTT assay. Compounds 1–6 exhibited activity with the following IC50 values: 1 (20 µM), 2 (20 µM), 3 (5.7 µM), 4 (4.2 µM), 5 (50 µM), and 6 (18 µM). Compounds 1 and 2, possessing a hydroxy group or acetoxy group at C-12 exhibited moderate cytotoxic activity with an IC50 = 20 µM, while compounds 3 having an additional hydroxy group at C-8 and its reduced compound 4 showed significant cytotoxic activity with an IC50 = 4.2–5.7 µM. The C-6 methoxy and C-7 hydroxy groups apparently reduced the toxicity of compound 5. Compound 6 with the absence of a hydroxy group at C-8 and the presence of a β-hydroxy group at C-6 displaying less cytoxocic activity with an IC50 = 18 µM than that of 4.

EXPERIMENTAL
Optical rotations were measured at 25 ˚C on a JASCO DIP-370S polarimeter. UV spectra were measured using a Hitachi U-2001 double-beam spectrophotometer. IR spectra were recorded on a MASCO FT/IR 5300. NMR spectra were recorded with a Bruker AVANCE 600 MHz NMR instrument using CDCl3 as a solvent. Chemical shifts are given on a δ (ppm) scale (1H, 7.26 ppm; 13C, 77.0 ppm as the internal standard). MS spectra were obtained with a JEOL JMS XD-303 instrument. VCC separation was performed with silica gel 60H (Merck, 90% < 45 µm). Column chromatography was carried out on silica gel 60 (Merck, 70—230 µm). Silica gel 60F (Merck, 0.25 mm thick) was used for TLC. HPLC was performed using a Waters 501 HPLC pump with a Shodex UV-41 detector. A Develosil ODS-UG-5 C18 column (10 mm× 250 mm) was used for HPLC.
Soft Coral Material. The soft coral Litophyton viscudium (collection number 253) was collected in the Otsuki Town area, Kochi Prefecture, Japan (32°44’10”N, 132°43’56”E), on June 15th 2004 and was identified by Dr. F. Iwase. A voucher specimen has been deposited at Faculty of Science, Kagoshima University (voucher specimen: 253).
Extraction and Isolation. The organism (wet weight: 5.3 kg, dry weight: 748 g) was chopped into small pieces and extracted with MeOH several times. The dried MeOH extract was suspended in H2O and extracted with CH2Cl2. The CH2Cl2 extract (23 g) was subjected to vacuum column chromatography on silica gel. Fractions of 300 mL were collected as follows: 1—3 (CH2Cl2/­n-hexane, 4:1), 4—6 (CH2Cl2), 7—11 (MeOH/CH2Cl2, 1:49), 12—18 (MeOH/CH2Cl2, 1:19), 19—21 (MeOH/CH2Cl2, 1:9), 22—25 (MeOH/CH2Cl2, 1:4), 26—28 (MeOH). Fractions 15—18 (5.4 g) were subjected to silica gel column chromatography with n-hexane/CH2Cl2 (4:1) and then MeOH/CH2Cl2 of increasing polarity. Fractions eluted with MeOH/CH2Cl2 (1:49 to 1:4) were chromatographed on silica gel successively with acetone/CH2Cl2 (3:97 to 1:9) and then MeOH/CH2Cl2 (1:49) followed by reversed-phase HPLC with CH3OH/H2O (7:3 to 3:2) to yield compounds (2) (2.0 mg) and (3) (1.8 mg). Compounds (4) (3.7 mg), (5) (11.7 mg), and (6) (7.3 mg) were isolated from the fractions eluted with acetone/CH2Cl2 (1:49 to 1:9) followed by reversed-phase HPLC using MeCN/H2O (3:2 to 1:1). Fractions eluted slowly with acetone/CH2Cl2 (1:9) were subjected to reversed-phase HPLC with MeCN/H2O (1:1) afforded compound (1) (3.7 mg).
Compound (1): amorphous powder; [α]25D -22.4 (c 0.11, MeOH); TLC Rf 0.41 (3:97, MeOH/CH2Cl2); UV (MeOH) λmax (log ε) 216 (3.41) nm; IR (NaCl) νmax 3426, 1732, 1688 cm-1; 1H NMR and 13C NMR, see Table 1; HRFABMS m/z 405.2629 [M + H]+ (calcd for C24H37O5, 405.2641).
Compound (2): amorphous powder; [α]25D -9.6 (c 0.06, MeOH); TLC Rf 0.36 (3:17, EtOAc/n-hexane); UV (MeOH) λmax (log ε) 216 (3.54) nm; IR (NaCl) νmax 1736, 1692 cm-1; 1H NMR and 13C NMR, see Table 1; HRFABMS m/z 447.2745 [M + H]+ (calcd for C26H39O6, 447.274 7).
Compound (3): amorphous powder; [α]25D -5.0 (c 0.06, MeOH); TLC Rf 0.25 (3:17, EtOAc/n-hexane); UV (MeOH) λmax (log ε) 215 (3.58) nm; IR (NaCl) νmax 3451, 1736, 1686 cm-1; 1H NMR and 13C NMR, see Table 1; HRFABMS m/z 405.2647 [M + H]+ (calcd for C24H37O5, 405.2641).
Compound (4): amorphous powder; [α]25D +25.0 (c 0.06, MeOH); TLC Rf 0.29 (3:97, MeOH/CH2Cl2); IR (NaCl) νmax 3366, 1732, 1645 cm-1; 1H NMR and 13C NMR, see Table 1; HRFABMS m/z 407.2764 [M + H]+ (calcd for C24H39O5, 407.2797).
Compound (5): amorphous powder; [α]25D +33.4 (c 0.35, MeOH); TLC Rf 0.55 (3:97, MeOH/CH2Cl2); IR (NaCl) νmax 3495, 1736, 1645 cm-1; 1H NMR and 13C NMR, see Table 1; HRFABMS m/z 445.2917 [M + Na]+ (calcd for C25H42O5Na, 445.2920), m/z 421.2958 [M – H]+ (calcd for C25H41O5, 421.2954).
Litophynin F (6): amorphous powder; [α]25D +12.4 (c 0.28, MeOH); TLC Rf 0.28 (1:99, MeOH/CH2Cl2); νmax 3614, 1734, 1644 cm-1; HRFABMS m/z 391.2845 [M + H]+ (calcd for C24H39O4, 391.2848).
Cell lines. Human leukemia HL-60 cells were maintained in RPMI1640 containing 10% fetal calf serum, 2 mM glutamine and the antibiotic/antimycotic solution at 37 °C in a 5 % CO2 humidified atmosphere.

ACKNOWLEDGEMENT
This study was supported in part by the Kagoshima Science Scholarship Foundation and JSPS via Grants-in-Aid for Scientific Research (No. 22510236).

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