HETEROCYCLES

Communication | Special issue | Vol. 84, No. 1, 2012, pp. 309-314
Received, 2nd June, 2011, Accepted, 27th June, 2011, Published online, 8th July, 2011.
DOI: 10.3987/COM-11-S(P)28

■ Chemistry of Renieramycins. Part 10: Structure of Renieramycin V, a Novel Renieramycin Marine Natural Product Having a Sterol Ether at C-14 Position

Naoki Saito,* Miho Yoshino, Kornvika Charupant, and Khanit Suwanborirux

Meiji College of Pharmacy, 2-522-1 Noshio, Kiyose, Tokyo 204-8588, Japan

Abstract

A new renieramycin-type marine natural product, renieramycin V, was isolated from the low polar fraction obtained from the Thai blue sponge Xestospongia sp., and its structure was elucidated by comparing its spectral data with those of renieramycin O and cholesterol. This is the first example of renieramycin that has a sterol moiety attached to it. It is interesting that renieramycin V does not show any cytotoxicity to several human cancer cell lines.

Renieramycins are isoquinoline marine natural products that are structurally and biologically related to other isoquinoline natural products, such as saframycins and ecteinascidins.1 In our ongoing search for new anticancer metabolites in Thai marine animals, we were able to isolate and elucidate the structures of renieramycins M-O and Q-U from the Thai blue sponge, Xestospongia sp., after stabilization of the sponge homogenized in phosphate buffer solution by the addition of potassium cyanide.2-4 In the course of our chemical studies on isoquinoline marine natural products, we found renieramycin V (2), which is the first example of renieramycin (specifically renieramycin O (1o)) with a sterol moiety attached to it. We present here the structure of 2, which was elucidated by means of spectroscopic analyses, along with its biological data (Figure 1).

New compound 2,5 named renieramycin V,6 was confirmed to have the molecular formula C58H77N3O9 [m/z 959.5659 (M+)] by HRFABMS. All proton and carbon signals of 2 were assigned after extensive NMR measurements using COSY, HMQC, and HMBC techniques. The molecular formula of 2 indicated 22 degrees of unsaturation and the detected resonance attributable to 12 olefinic carbons, five carbonyl groups, and one nitrile carbon in 2 accounted for 13 degrees of unsaturation. This compound was presumed to have nine rings. Comparison of the 13C NMR data of 2 with those of renieramycins O (1o) and R (1r) revealed that the 31 carbon signals of 2, including the characteristic signals of both 10 sp2 and five carbonyl carbons, were almost identical with those of the renieramycin framework (Table 1-1). 1H and 13C NMR spectra led us to speculate that 2 might be 14-O-alkylated renieramycin M with a characteristic 14-H proton signal at δ 4.09.7 The remaining proton and carbon signals could be assigned to a sterol moiety whose formula was presumed to be C27H45. To discuss the NMR spectra of the sterol moiety, the data for 27 carbons, including some characteristic protons, are listed in Table 1-2. The 13C NMR data of the sterol moiety of renieramycin V are quite similar to those of cholesterol8 and the slight differences in the chemical shifts of C-2, C-3, and C-4 carbon signals are thought to be due to the presence of bulky renieramycin at C-3 position. The 1H NMR spectrum of this moiety contained five methyl proton signals [δ 0.69 (3H, s), 0.87 (3H, d, J = 6.7 Hz), 0.87 (3H, d, J = 6.7 Hz), 0.92 (3H, d, J = 6.7 Hz), 1.00 (3H, s)] (Table 1-2). The 1H NMR spectrum also had a characteristic oxygenated methine proton signal at δ 3.52 ppm and the 13C NMR spectrum revealed that the carbon signal corresponding to this appeared at δ 80.9 ppm. Furthermore, a one-proton signal appeared at δ 5.43, which indicated the presence of an olefinic proton at C-6’ owing to the double bond between C-5’ (δ 140.5 ppm) and C-6’ (δ 122.1 ppm) of the sterol moiety.
Evidence of the attachment of the sterol moiety to the renieramycin framework was found in a 3-bond correlation from H-14 at 4.09 to C-3’ at 80.9 in the HMBC spectrum. Thus, the structure of 2 was confirmed to be cholesterol-3-O-renieramycin O.

The cytotoxicity of renieramycin V (2) to three cancer cell lines is shown in Table 2. It was surprising that renieramycin V (2) did not show any cytotoxicity. Nevertheless, we expect that the sterol moiety in renieramycin V, which was introduced by oxidation process, will play a role in protecting the life of the marine organism.

In conclusion, a minor marine natural product having a novel renieramycin structure, renieramycin V, was isolated from the Thai blue sponge, Xestospongia sp., and its structure was elucidated by spectroscopic analysis. To our knowledge, this is the first example of a nontoxic renieramycin derivative, the structure of which consists of cholesterol at C-14 position of renieramycin M.

ACKNOWLEDGEMENTS
This work is supported by a Grant-in Aid (No. 23590019) for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology (MEXT), Japan. This work is also partially supported by the Japan Society for the Promotion of Science (JSPS) Asia-Africa Science Platform Program (2010-2012) and a grant from the High-Tech Research Center Project, MEXT, Japan (S0801043). We are grateful to Dr. Nobuo Shimma (Chugai Pharmaceutical Company Kamakura Research Center) for conducting the cytotoxicity assay.

References

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5. For details of the 2006 recollection (18 kg, wet weight) of the Thai blue sponge Xestospongia sp., see ref. 4. Silica gel chromatography (solvent gradient from hexane to ethyl acetate) provided three primary fractions. The most lipophilic fraction (9.47 g) was purified by silica gel (several times), followed by preparative TLC to give renieramycin V (2: 10.5 mg) along with renieramycin O (1o: 57.0 mg).
6. Renieramycin V (2): amorphous powder, 1H and 13C NMR data, see Table 1: UV λmax (log ε) 269 (4.62), 370 (3.09) nm; EIMS m/z (%) no M+, 575 (3), 398 (46), 386 (78), 368 (30), 314 (100), 300 (45), 299 (30), 275 (31), 273 (37), 272 (30), 271 (63), 255 (62), 243 (35), 220 (32), 213 (40), 161 (31), 159 (39), 147 (31), 145 (43), 133 (34), 107 (42), 105 (33), 95 (34), 93 (30), 81 (37), 55 (47); HRFABMS m/z 959.5659 (M+, calcd for C58H77N3O9, 959.5660): IR (KBr) 3435, 2945, 2868, 2320, 1717, 1655, 1616, 1456, 1375, 1307, 1290, 1261, 1231, 1150, 1080, 1045, 1028, 883 cm-1: [α]D17 －41.6 (c, 0.6, CHCl3); CD (c 0.013 mmol/L, MeOH, 24 oC) －2.6 (353), －1.6 (310), －20.3 (275), －2.0 (246), 1.7 (239), 26.5 (212), 25.9 (210).
7. The absence of coupling between 13-H (δ 3.38 ppm) and 14-H indicated the presence of a dihedral angle measuring approximately 80-90 o.
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