Sesquiterpene and Acetogenin Derivatives from the Marine Red Alga Laurencia okamurai

In addition to 13 known compounds, four new bisabolane sesquiterpenes, okamurenes A–D (1–4), a new chamigrane derivative, okamurene E (5), and a new C12-acetogenin, okamuragenin (6), were isolated from the marine red alga Laurencia okamurai. The structures of these compounds were determined through detailed spectroscopic analyses. Of these, okamurenes A and B (1 and 2) are the first examples of bromobisabolane sesquiterpenes possessing a phenyl moiety among Laurencia-derived sesquiterpenes, while okamuragenin (6) was the first acetogenin aldehyde possessing a C12-carbon skeleton. Each of the isolated compounds was evaluated for the brine shrimp (Artemia salina) lethal assay and 7-hydroxylaurene displayed potent lethality with LD50 1.8 μM.


Structure Elucidation of the New Compounds
Okamurene A (1) was obtained as a colorless oil and its molecular formula was established by HRESIMS to be C 15 H 21 BrO, corresponding to five degrees of unsaturation. The 1 H NMR spectrum of 1 (Table 1) exhibited resonances for a para-substituted phenyl unit, four methyl groups, and a brominated or oxygenated methine group. There were also four signals for two diastereotopic methylene protons. The 13 C NMR and DEPT spectroscopic data (Table 1) revealed the presence of 15 carbon signals including six aromatic carbons (corresponding to a para-substituted phenyl unit) and nine aliphatic carbons (corresponding to four methyls, two methylenes, one brominated methine, and two oxygenated quaternary carbons). These units accounted for 4 degrees of unsaturation, requiring one additional ring to be present in 1. The structure of the non-phenyl portion of 1 was determined by analysis of 2D NMR data ( 1 H-1 H COSY, HSQC, and HMBC). The 1 H-1 H COSY experiment established the connectivity for a -CH 2 -CH 2 -CH-unit (C-8 through C-10, Figure 2). The C-10 methine of this unit was connected to CH 3 -12 and CH 3 -13 via the oxygenated quaternary carbon C-11 (δ C 75.2) as evidenced by the observed HMBC correlations from the methyl protons H 3 -12 and H 3 -13 to C-10 and C-11, while the C-8 methylene was linked to the CH 3 -14 via the oxygenated quaternary carbon C-7 (δ C 74.6) as supported by the observed HMBC correlation from the methyl protons H 3 -14 to C-8 ( Figure 2). Given the fact that only one oxygen atom existed in the structure, the linkage of C-7/O/C-11 could be constructed, leading to the formation of a tetrahydropyran moiety, which accounted for the remaining degree of unsaturation. Thus, the planar structure of 1 was assigned.  Analysis of the proton coupling constants and NOESY data enabled assignment of the relative configuration of 1. The appearance of the bromomethine proton H-10 as a double doublet, with coupling constants of 7.9 and 4.4 Hz, suggesting the equatorial orientation of H-10 for 1. In the NOESY spectrum, NOE correlations of H 3 -13 with both H-10 and H 3 -14 placed the methyl groups CH 3 -13 and CH 3 -14 on the same face (axial or pseudoaxial) of the tetrahydropyran ring ( Figure 3). On the basis of the above evidence, the structure of 1 was determined, and the trivial name okamurene A was assigned. The 1 H and 13 C NMR spectroscopic data of okamurene B (2), an isomer of 1 as established by HRESIMS data, were very similar to those of 1 except for some chemical shift variations of signals corresponding to the C-8, C-9, and C-12 through C-14 (Table 1). Therefore, compound 2 was presumed to be a stereoisomer of 1. Detailed analysis of the 1 H and 13 C NMR data as well as 1 H-1 H COSY and HMBC correlations supported the conclusion that 2 possesses the same planar structure as 1. However, comparisons of the J-value and NOESY data of 2 with those of 1 revealed a difference in relative configuration at C-10. A trans-diaxial J-value for H ax -10 and H ax -9 (12.1 Hz) indicated an equatorial orientation for the Br-atom at C-10. The NOE correlation from H-10 to H 3 -12 in the NOESY spectrum indicated an equatorial face of CH 3 -12, while the NOE correlation from H 3 -13 to H 3 -14 placed these two methyl groups in axial orientation ( Figure 3). Based on the above data, the structure of compound 2 was identified and it was named okamurene B.
Okamurenes C (3) and D (4) were obtained as a colorless oily mixture in a 2:1 ratio, as indicated by the 1 H NMR spectrum. Attempts to separate the mixture by various CC steps using different solvent systems failed. On the other hand, there is no conjugated system in compounds 3 and 4, making these compounds unsuitable for HPLC separation using the available UV detector. A similar unseparable mixture containing (9S)-and (9R)-2-bromo-3-chloro-6,9-epoxybisabola-7(14),10-diene from L. saitoi was previously described [11]. Most of the NMR signals for compounds 3 and 4 were duplicated or overlapped. By detailed analysis of 1D and 2D NMR data, their structures were determined to be C-9 epimer of 6,9-epoxybisabola-2,7(14),10-triene.
Okamurene E (5), a colorless oil, was shown to have the molecular formula of C 15 H 23 BrO by the interpretation of HRESIMS data. The IR absorption at 3401 cm −1 exhibited the presence of a hydroxyl group. The 1 H NMR spectrum (Table 2) delineated four methyl singlets, one double doublet ascribable to an oxygenated/halogenated methine, and one multiplet and two doublets attributable to three olefinic protons. The 13 C and DEPT NMR spectra (Table 2) displayed four methyls, three methylenes, four methines, and four quaternary carbons. Compared to the reported NMR data for 10-bromo-7α, 8α-expoxychamigr-1-en-3-ol [12], compound 5 exhibited no resonances for the epoxy moiety in the NMR spectra. Instead, it showed additional signals at δ H 5.23 (H-8) and δ C 139.5 (C-7) and 120.8 (C-8) for a trisubstituted vinyl group, which was positioned at C-7 based on the observed HMBC correlations from H-14 to C-6, C-7, and C-8. Further analysis of the 1 H-1 H COSY and HMBC correlations (Figure 2) confirmed the structure of 5 as 10-bromo-1,7-chamigradien-3-ol. The relative configurations at C-3, C-6, and C-10 of 5 were deduced to be same as those of 10-bromo-7α, 8α-expoxychamigr-1-en-3-ol [12] by the NOESY correlation between H-5 and H-10 as well as by their similar NMR data. Okamuragenin (6), isolated as a colorless oil, was assigned the molecular formula C 12 H 18 Br 2 O 3 on the basis of HRESIMS, consistent with three degrees of unsaturation. The IR spectrum exhibited strong absorptions at 2762 and 1728 cm −1 , indicating the existence of an aldehyde group. In accordance with the IR signals, the 1 H and 13 C NMR data ( Table 2) also indicated the presence of an aldehyde group at δ H (9.80, H-1) and δ C 199.3 (CH, C-1). The 1 H-1 H COSY spectrum revealed that the aldehyde group was extended to a straight spin system consisting of six methines, four methylenes, and terminated by a methyl group (Figure 2). Compound 6 was deduced to be bicyclic, since no other unsaturated functionalities were indicated by the NMR data ( Table 2). The connectivity of C-3/O/C-9 was deduced by the correlation from H-3 to C-9 in the HMBC spectrum ( Figure 2). Taking into account the downfield chemical shifts of C-4 (δ C 81.6) and C-6 (δ C 80.9) and the calculated 3 degrees of unsaturation, C-4 and C-6 had to be linked through an oxygen atom. Finally, the two remaining Br-atoms indicated by the molecular formula could only be located at C-7 and C-10 based on the chemical shifts [13]. The relative configuration was determined by NOESY experiment. The same orientation of CH 2 -2, H-4, and H-9 was evidenced by the NOE correlations of H-2 to H-4 and H-9, while H-9 was syn to H-7 based on the NOE correlation between them. The above data established the structure of 6, trivially named okamuragenin.
The isolated compounds were evaluated for the brine shrimp (Artemia salina) lethal activity [26,27]. Among them, 7-hydroxylaurene was found to possess potent lethality with LD 50 1.8 μM, which is more active than that of 7-hydroxylaurene acetate, allolaurinterol acetate, and laurene [12]. Analysis of structure-activity relationship showed that the 7-hydroxyl group in laurene sesquiterpenes may play a key role in the brine shrimp toxicity, and the activity reduced significantly after acetylation. The above data suggested that 7-hydroxylaurene may be a potent chemical defensive agent with cytotoxicity, although the hatchability test was not performed [27]. The other tested compounds only displayed moderate or weak activity (data not shown).

General
IR spectra were measured on a Nicolet NEXUS 470 FT-IR spectrophotometer. Optical rotations were recorded on an Atago Polax-L polarimeter. UV spectra were determined on a Spectrumlab 54 UV-visible spectrophotometer. HRESIMS were run on a VG Autospec 3000 mass spectrometer. 1D and 2D NMR spectra were obtained at 500 and 125 MHz for 1 H and 13 C, respectively, on a Bruker Advance 500 MHz NMR spectrometer in CDCl 3 with TMS as internal standard. Column chromatography (CC) was performed on Si gel (200-300 mesh, Qingdao Haiyang Chemical Co., Qingdao, China) and Sephadex LH-20 (Sigma). TLC was carried out with precoated Si gel plates (GF-254, Qingdao Haiyang Chemical Co., Qingdao, China).

Algal Material
The marine red alga Laurencia okamurai Yamada was collected along Weihai coastline in Shandong Province, China, in May, 2007, and was identified by B.-M. Xia, Institute of Oceanology, Chinese Academy of Sciences (IOCAS). A voucher specimen (HZ0705) has been deposited at the Key Laboratory of Experimental Marine Biology of IOCAS.

Extraction and Isolation
The dried and powdered alga L. okamurai (3.8 kg) was extracted with a mixture of CHCl 3 and MeOH (1:1, v/v). The concentrated extracts were partitioned between H 2 O and EtOAc. The EtOAc-soluble fraction was loaded to Si gel column, eluting with a step gradient of increasing EtOAc (0%-100%) in petroleum ether (PE) to give eight fractions I-VIII. Fraction II eluted with PE/EtOAc 100:1 and was further purified by preparative TLC to afford a mixture of 3 and 4 (5.6 mg). Fraction IV eluted with PE/acetone 100:1 and was further separated by preparative TLC to afford 1 (3.7 mg), 2 (4.7 mg), 6 (13.1 mg). Fraction VI eluted with PE/acetone 30:1 and was further separated by Sephadex LH-20 (MeOH) CC and preparative TLC to afford 5 (10.7 mg).

Brine Shrimp Toxicity
Brine shrimp (Artemia salina) toxicity of crude extract and pure compounds was determined as detailed previously [26,27].