Alkaloids and Sesquiterpenes from the South China Sea Gorgonian Echinogorgia pseudossapo

Five zoanthoxanthin alkaloids (1–5) and four sesquiterpenes (6–9) were isolated from the South China Sea gorgonian Echinogorgia pseudossapo. Their structures were determined on the bases of extensive spectroscopic analyses, including 1D and 2D NMR data. Among them, pseudozoanthoxanthins III and IV (1–2), 8-hydroxy-6β-methoxy-14- oxooplop-6,12-olide (6) and 3β-methoxyguaian-10(14)-en-2β-ol (7) were new, 1 and 3 showed mild anti-HSV-1 activity, and 7 showed significant antilarval activity towards Balanus amphitrite larvae.

The suggestion was supported by 1 H-1 H COSY and HMBC spectra ( Figure 3). The presence of five membered ring substructure was concluded from the 1  with C-4 (δ C 44.5), indicated that one methoxy group and one methyl were connected with C-3 and C-4, respectively.
The relative configuration of 7 was determined by a NOESY experiment ( Figure 3) and comparison with that of guaia-1(10),11-diene and guaia-9,11-diene [13]. Considering the bulky isopropyl group to keep a pseudo equatorial position and being β-oriented, H-7 had to be α-oriented. In vitro antiviral activity of 1-4 against HSV-1 was evaluated using plaque reduction assay. First, the completely non-toxic concentration (CC 0 ) of 1-4 and positive control ACV on Vero cells were tested to be 270.3, 523.6, 185.2, 195.3, >7500 μM by MTT assays, respectively, then for further antivirus studies, the concentrations of tested compounds were kept below their CC 0 values. The antiviral assays displayed that 1-4 exhibited anti-HSV-1 activity with EC 50 (50% effective concentration required to inhibit virus-induced cytopathicity 50%) values of 108.1, 471.2, 70.4, 117.2, 6.08 μM, respectively. The results suggested that the side chain at the nitrogen N(3) in 1-4 could affect their antiviral activity. Although 1 and 3 showed mild anti-HSV-1 activity, their activities were far lower than that of the positive control ACV.
Compound 7 was evaluated for its antilarval activity against B. amphitrite and B. neritina larvae. The results showed that 7 had significant antilarval activity towards B. amphitrite larvae with EC 50 value of 17.2 μg/mL (68.2 μM), and showed 50% inhibition towards the settlement of B. neritina larvae at concentration of 25 μg/mL. The EC 50 value of 7 is lower than the standard requirement of an EC 50 of 25 μg/mL established by the US Navy program as an efficacy level for natural antifoulants, indicating that 7 is a potential natural antifouling agent.

General
Optical rotations were measured with a Horiba SEAP-300 spectropolarimeter. UV spectra were measured with a Shimadzu double-beam 210A spectrophotometer in MeOH solution. 1 H, 13 C NMR and 2D NMR spectra were recorded on a Bruker AV-500 MHz NMR spectrometer with TMS as internal standard. MS spectral data were obtained on an LCQDECA XP HPLC/MSn spectrometer for ESIMS.

Animal Material
The South China Sea gorgonian coral E. pseudossapo

Viruses and Cells
HSV-1 (15577) strain and Vero cells were obtained from American Type Culture Collection. Cytotoxicity assay and cytopathic effect reduction assay were undertaken with the reported methods [14]. ACV was used as the positive control.

Larval Settlement Bioassays
Antilarval activity of the compounds was evaluated in settlement inhibition assays with laboratory-reared Balanus amphitrite and Bugula neritina larvae. The procedures were the same as previously reported [15].