Okamurene A (1) was obtained as a colorless oil and its molecular formula was established by HRESIMS to be C₁₅H₂₁BrO, corresponding to five degrees of unsaturation. The ¹H 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 ¹³C 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 (¹H–¹H COSY, HSQC, and HMBC). The ¹H–¹H COSY experiment established the connectivity for a –CH₂–CH₂–CH– unit (C-8 through C-10, Figure 2). The C-10 methine of this unit was connected to CH₃-12 and CH₃-13 via the oxygenated quaternary carbon C-11 (δ_C 75.2) as evidenced by the observed HMBC correlations from the methyl protons H₃-12 and H₃-13 to C-10 and C-11, while the C-8 methylene was linked to the CH₃-14 via the oxygenated quaternary carbon C-7 (δ_C 74.6) as supported by the observed HMBC correlation from the methyl protons H₃-14 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 H₃-13 with both H-10 and H₃-14 placed the methyl groups CH₃-13 and CH₃-14 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 ¹H and ¹³C 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 ¹H and ¹³C NMR data as well as ¹H–¹H 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 H₃-12 in the NOESY spectrum indicated an equatorial face of CH₃-12, while the NOE correlation from H₃-13 to H₃-14 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 ¹H 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.

The molecular formula of compounds 3 and 4 were determined to be C₁₅H₂₂O (five degrees of unsaturation) on the basis of HRESIMS data. Examination of the ¹H and ¹³C NMR data (Table 2) revealed that they resembled 9S- and/or 9R-2-bromo-3-chloro-6,9-epoxybisabola-7(14),10-diene [11], except for the presence of signals for a trisubstituted vinyl group at C-2 and, accordingly, the lack of the resonances due to a brominated methine at C-2 and a chlorinated quaternary carbon at C-3 [11]. The chemical shifts for the vinyl carbons at δ_C 119.1/119.0 (C-2) and 133.4/133.7 (C-3) as well as for one of the neighboring methylene groups C-4 (δ_C 27.7/28.0) in the ¹³C NMR spectrum of 3 and 4 were very similar to those reported for 8-bromochamigra-1,11(12)-dien-9-ol (with C-2 at δ_C 119.4, C-3 at δ_C 132.9, and C-4 at δ_C 27.5) [12], and these data strongly supported the presence of the trisubstituted vinyl group at C-2 in 3/4. These data indicated that compounds 3 and 4 were the dehalogenated derivatives corresponding to 9S- and/or 9R-2-bromo-3-chloro-6,9-epoxybisabola-7(14),10-diene [11]. The ¹H–¹H COSY and HMBC correlations (Figure 2) further verified the planar structures of 3/4 to be 6,9-epoxybisabola-2,7(14),10-triene. Assignment of the relative configuration at C-6 by NOESY experiment is not applicable for compounds 3 and 4 since there is no proton around C-6 in the tetrahydrofuran ring. However, the C-6 relative configuration was tentatively assigned to be the same as that of 9S- and/or 9R-2-bromo-3-chloro-6,9-epoxybisabola-7(14),10-diene based on the similar NMR data around the chiral center, as well as on biogenetic consideration [11].

Okamurene E (5), a colorless oil, was shown to have the molecular formula of C₁₅H₂₃BrO by the interpretation of HRESIMS data. The IR absorption at 3401 cm⁻¹ exhibited the presence of a hydroxyl group. The ¹H 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 ¹³C 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 ¹H–¹H 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 C₁₂H₁₈Br₂O₃ 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 ¹H and ¹³C 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 ¹H–¹H 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 CH₂-2, 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.

In addition to the six new compounds, the other nine sesquiterpenes including isobromocuparene [14], 7-hydroxylaurene [15], laurene [16], filiformin [17], debromofiliformin [18], 6-bromo-filiformin [19], deoxyprepacifenol [20], 2-bromo-3-chloro-2,7-epoxy-9-chamigren-8α-ol [11], and 2,10-dibromo-3-chloro-7-chamigren-9-ol [21], together with four C₁₅-acetogenins including 3E, 12Z-laurediol [22], neolaurallene [23], E-stereoisomer of neoisoprelaurefucin [24], and 3Z-laurentin [25], were all identified by comparison of their spectral data with those previously reported.

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 LD₅₀ 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).

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 ¹H and ¹³C, respectively, on a Bruker Advance 500 MHz NMR spectrometer in CDCl₃ 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).

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.

The dried and powdered alga L. okamurai (3.8 kg) was extracted with a mixture of CHCl₃ and MeOH (1:1, v/v). The concentrated extracts were partitioned between H₂O 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).

Okamurene A (1): Colorless oil; [α]¹⁸_D +2.3 (c 0.11, MeOH); UV (MeOH) λ_max (log ε) 221 (3.56) nm; IR (KBr) ν_max 3065, 2964, 2857, 1514, 1479, and 1205 cm⁻¹; ¹H and ¹³C NMR data, see Table 1; HRESIMS m/z 297.0748 [M + H]⁺ (calcd for C₁₅H₂₂⁷⁹BrO, 297.0854).

Okamurene B (2): Colorless oil; [α]¹⁸_D +3.6 (c 0.06, MeOH); UV (MeOH) λ_max (log ε) 221 (3.66) nm; IR (KBr) ν_max 3068, 2964, 2857, 1514, 1477, and 1208 cm⁻¹; ¹H and ¹³C NMR data, see Table 1; HRESIMS m/z 319.0726 [M + Na]⁺ (calcd for C₁₅H₂₁BrONa, 319.0673).

Okamurenes C (3) and D (4): Colorless oil; IR (KBr) ν_max 3096, 2924, 2854, 1637, 1457, and 1024 cm⁻¹; ¹H and ¹³C NMR data, see Table 2; HRESIMS m/z 219.1757 [M + H]⁺ (calcd for C₁₅H₂₃O, 219.1749).

Okamurene E (5): Colorless oil; [α]¹⁸_D +7.6 (c 0.09, MeOH); IR (KBr) ν_max 3401, 2971, 2928, 1549, 1447, 1367 and 1121 cm⁻¹; ¹H and ¹³C NMR data, see Table 2; HRESIMS m/z 281.0846 [M − H₂O + H]⁺ (calcd for C₁₅H₂₂⁷⁹Br, 281.0905), and 283.0860 [M − H₂O + H]⁺ (calcd for C₁₅H₂₂⁸¹Br, 283.0884).

Okamuragenin (6): Colorless oil; [α]¹⁸_D +11.2 (c 0.18, MeOH); IR (KBr) ν_max 3060, 2926, 2854, 2762, 1728, 1421, and 1134 cm⁻¹; ¹H and ¹³C NMR data, see Table 2; HRESIMS m/z 385.9926 [M + NH₄]⁺ (calcd for C₁₂H₂₂N⁷⁹Br₂O₃, 385.9966), 387.9986 [M + NH₄]⁺ (calcd for C₁₂H₂₂N⁷⁹Br⁸¹BrO₃, 387.9946).

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