Isolation of C11 Cyclopentenones from Two Didemnid Species, Lissoclinum sp. and Diplosoma sp.

A series of new C11 cyclopentenones 1–7 was isolated, together with four known metabolites 9/10, 12 and 13, from the extract of the didemnid ascidian Lissoclinum sp. The other didemnid ascidian Diplosoma sp. contained didemnenones 1, 2 and 5, and five known metabolites 8–12. The structures of 1–7 were elucidated by spectroscopic analyses. Cytotoxicity of the isolated compounds was evaluated against three human cancer cell lines (HCT116, A431 and A549).


Introduction
It has been amply demonstrated that ascidians are a prolific source of novel bioactive secondary metabolites [1−4]. Ascidians belonging to the family Didemnidae, e.g., Lissoclinum spp. and OPEN ACCESS Diplosoma spp., harbor obligate cyanobacterial symbionts of the genus Prochloron [5−7], and have yielded structurally unique and pharmacologically interesting compounds such as patellazoles, varacin, virenamides, haterumalide and haterumaimides [8−15]. A series of C 11 compounds having the distinctive exo-allylidene-lactone named didemnenone was isolated from didemnid ascidians, Trididemnum cyanophorum (didemnenones A and B) and Didemnum voeltzkowi (didemnenones C and D) [16]. They showed a wide range of biological activities, including cytotoxicity against leukemia cells as well as antimicrobial and antifungal activities [16−18]. Their structures were determined based on an X-ray investigation of the methylacetal of didemnenone A and from synthetic results [16−18]. As described previously, as part of our ongoing research aiming at the isolation of biologically active metabolites from marine organisms living in the tidal zone, we have isolated several C 11 compounds, dinemnenone congeners 14−17 [19−22] and pentylphenols 18 and 19 [22] from ascidian Diplosoma spp. (Figure 1). Recently, we examined the constituents of ascidians, Lissoclinum sp. collected on the coast of Tarama island and Diplosoma sp. from dead corals of Hateruma island. From the Lissoclinum sp. we identified the new metabolites 1−7, along with the known metabolites, didemnenones A (9) and B (10) as an inseparable mixture, a methylacetal of didemnenone B (12) [16], and inosine (13) (Figure 2). The Diplosoma sp. contained didemnenones 1, 2 and 5 along with five known metabolites 8−12 ( Figure 2) [16,23−34]. In this report, we describe the isolation, structure elucidation and bioactivity of these metabolites, and we also discuss the biosynthesis of didemnenones and the related compounds.
Analysis of 1 by NMR (Tables 1 and 2) 3.51 (dd) and δ C 56.6 (t); δ H 4.64 (dd), 4.36 (dd)] and an oxygenated quaternary carbon [δ C 80.5 (s)] in 1. Degrees of unsaturation for these partial structures amount to four. Thus, 1 must be monocyclic to account for the five degrees of unsaturation required by the molecular formula. The connectivity of the aforementioned partial structures was established from the HMBC correlations of H 2 -1/C-2, H-3/C-6, H-4/C-5, H-4/C-6, H-8/C-11, H 3 -10/C-7, H 3 -10/C-8, H 3 -10/C-9, H 2 -11/C-6 and H 2 -11/C-7, as shown in Figure 3, to describe the entire carbon framework of 1. Geometric configuration of two olefins in 1 at C-6/C-7 and C-8/C-9 were assigned to be E by NOEDS experiments (Figure 4), in which irradiation of H-9 caused enhancement of H-11 and irradiation of H-1 resulted in enhancement of the H-3 and OH-11 proton signals. Therefore, the planar structure of 1 was established as a class of didemnenone as shown in 1.      (Tables 1 and 2) of 4 resembled those of 3, except for the presence of a proton signal at δ H 3.31 (s) and a carbon signal at δ C 54.9 (q) in 4. Geometric configuration of the double bond at C-7/C-8 in 4 was assigned to be E by NOEDS experiments, in which irradiation of H-6 caused enhancement of H-9 and irradiation of H-8 caused enhancement of H-11 and H-10. Therefore, the planar structure of 4 was elucidated to be a methylacetal of 3. The NOE between a hydroxyl proton (OH-2) and H-6 also indicated the ring junction to have the cis-geometry. The NOEs; OH-2/ H-1b; H-1a /H-11 allowed the assignment of the H-11 as β. We cannot affirm that 4 is a natural product, because it is conceivable it arises from 3 in the isolation process.
The molecular formula of 5 was determined to be C 11 (Table 2) and the IR absorption band at ν max 1717 cm −1 further supported the presence of the carbonyl groups.
The 1 H-and 13 C-NMR (Tables 1 and 2) and 2D NMR spectral data of 5 are similar to those of γ-lactone didemnenone [16], except for the HMBC correlation observed between an oxymethylene proton H 2 -1 at δ H 4.13 and a carbonyl carbon C-11 at δ C 166.9. Geometric configuration of the double bond at C-7/C-8 in 5 was assigned to be E by NOEDS experiments, in which irradiation of H-6 caused enhancement of H-9. Therefore, the planar structure of 5 was concluded to be a class of didemnenone, as depicted in 5.The NOE between a hydroxyl proton (OH-2) and H-6 allowed the ring junction to be assigned as cis.
Analysis of the 13 (Table 2) and the IR absorption band at ν max 1720 cm −1 further supported the presence of the carbonyl group. Extensive analysis of 1 H-and 13 C-NMR data (Tables 1 and 2  The structure of 7 was elucidated to be a dimeric didemnenone composed of 1 and 3, from the molecular formula, the NMR data and the HMBC correlations of H-11/C-11' and H 2 -11'/C-11. Geometric configuration of three olefins in 7 at C-7/C-8, C-6'/C-7' and C-8'/C-9' was assigned to be E by NOEDS experiments, in which irradiation of H-6 caused enhancement of H-9, irradiation of H-8 caused enhancement of H-11 and H-10, irradiation of H-9' caused enhancement of H-11' and irradiation of H-3' resulted in enhancement of the H-1' proton signal. Therefore, the structure of 7 was established as a didemnenone dimer as shown in 7. We could not determine the C-2/C-6 ring junction stereochemistry owing to decomposition of 7.  The structure of marine metabolite 8 was determined to be 4-amino-7-(5'-deoxy-β-Dxylofuranosyl) -5-iodopyrrolo [2,3-d]pyrimidine by 1D and 2D NMR spectra for 8 and 23, and by CD spectra of compounds 8, 24 and 25 ( Figure 5), as previously described [23]. The absolute stereochemistry of the new compounds was tentatively deduced to be as depicted in 1−7 based on the assumption that there is a similar biogenetic relationship between these compounds and (+)-didemnenone A. (+)Didemnenones 9−12 and inosine (13) were unambiguously identified by comparison of their spectral data with those described in the literature [16].
Compounds 1−13 were tested in vitro for their cytotoxic activities against the HCT116, A431 and A549 cancer cell lines (Table 4). Compounds 1, 2 and 8 were significantly cytotoxic against the HCT116, A431 and A549 cancer cell lines, and compounds 3, 4, 7, 9/10 and 12 were significantly cytotoxic against two cell lines, HCT116 and A431. In contrast to 12 (a β-anomer), its isomer 11 (an α-anomer) was not cytotoxic against any of the three cell lines. Among the isolated compounds tested, the iodinated nucleoside 8 showed the strongest cytotoxic activity against the HCT116, A431 and A549 cancer cell lines, with IC 50 values of 1.8, 3.1 and 3.5 μg/mL, respectively. To date, a variety of C 11 compounds have been isolated from ascidians (compounds 9 and 10), cyanobacteria (compounds 20 and 21) and a sponge (compound 22) (Figures 1 and 2) [16,35,36]. Compounds 16 and 17 have been isolated from the ascidian Diplosoma virens and a sponge Ulosa sp. (Figure 2) [19,20]. Isolation of a series of the C 11 compounds including compounds 18 and 19 ( Figure 2) from unrelated marine organisms supports the potential microbial origin of these compounds. From this viewpoint, we assume that the ascidian Diplosoma sp. might not be the actual producer of the C 11 compounds, but suggest a possible microorganism source such as Prochloron spp. We conducted, therefore, the following experiments. The Prochloron spp., which are obligatory symbionts of ascidians, were separated from the body of the ascidians Lissoclinum sp. and Diplosoma spp. by squeezing through the plankton net. 1 H-NMR spectra of the acetone extracts of the separated Prochloron spp. showed the presence of the same peaks as present in those of didemnenones. This confirms our assumption that Prochloron spp. are the actual producers of didemnenones.
Most C 11 compounds are derived from polyketides (six acetates−C 1 ) or polyketides (five acetates + C 1 ) [37]. Pentylphenols such as 18 and 19, and some compounds which have a carbon skeleton of 5-methyldecane are known to be derived from the former with a loss of CO 2 from C 12 parent (six acetates). Some C 11 metabolites are ascertained to be derived from a polyketide precursor (five acetates + C 1 ) which has a carbon skeleton of 4-methyldecane [37]. We found that didemnenonerelated compounds 1−7, 9−12, 14−17 and 20−22 have a common carbon skeleton of 4-methyldecane from a consideration of the carbon skeleton of these compounds. Consequently, We propose that these compounds should be derived from the polyketides (five acetates + C 1 ) via cyclization between C-9/C-5, between C-11/C-7 or between C-10/C-5 (Scheme 1).

Scheme 1.
Plausible biosynthesis of the carbon skeletons for didemnenones and related compounds.

General experimental procedures
Optical rotations were measured on either a JASCO P-1020 or JASCO DIP-1000 polarimeter. Ultraviolet-visible spectra were obtained in methanol on a JASCO V-550 spectrophotometer. Infrared spectra were recorded as dry films on either JASCO FT/IR-300 or Spectrum 2000 Explorer (PERKIN ELMER). CD spectra were recorded on a JASCO J-720W Circular Dichroism Spectrometer. 1

Animal material
The colonial brown ascidian was collected by hand at the tidal zone of Tarama island, Okinawa, Japan, and the colonial green ascidian was collected by hand from the coast of Hateruma island, Okinawa, Japan. The ascidians were stored at −15 °C until extraction. The brown ascidian and the green ascidian were identified as Lissoclinum sp. and Diplosoma sp., respectively, by Euichi Hirose, University of the Ryukyus, Japan. The voucher specimens were deposited at the University of the Ryukyus (Specimen no. URKU-801 for the brown ascidian and URKU-802).

Acetal 23
To a solution of iodinated nucleoside 8, (10.0 mg, 26.6 μmol) in 2,2-dimethoxypropane (1 mL) and acetone (2 mL) was added a catalytic amount of camphorsulfonic acid. The mixture was stirred at rt for 24 h and at 45 °C for 4 h. The reaction mixture was diluted with ether, washed with saturated aqueous Na 2 CO 3 and brine. The organic phase was dried (MgSO 4 ) and concentrated in vacuo. The residual oil was purified by preparative TLC [CHCl 3 -MeOH (3:0.2)] to give the acetal as colorless oil (23, 3.0 mg, 25%). HR-FABMS and 1 H NMR data for compound 23 were described in the earlier paper [23].