*3.4. Extraction and Isolation*

Freeze dried animal material (29.6 g) was extracted with MeOH (3 × 400 mL) and a butanol:CH2Cl2:H2O (150:50:100 mL) partition performed. The aqueous phase was further partitioned with BuOH:CH2Cl2 (150:50 mL) and the organic phase added to the first organic fraction. The organic fraction (16.8 g) was then subjected to reversed phase C18 flash vacuum chromatography (RP-C18, 25%, 50%, 75%, 100% MeOH in H2O and 1:1 MeOH:CH2Cl2). Activity was observed for the first four fractions. A portion of the 25% MeOH fraction (3.44 g of 10.27 g) was pre-absorbed onto C18, packed into a cartridge, and further separated by preparative C18 HPLC (52 mL/min, isocratic elution at 15% CH3CN:H2O for 3 min followed by gradient elution from 15% CH3CN:H2O to 100% CH3CN:H2O over 50 min and an isocratic elution at 100% CH3CN for 30 min through a 250 × 41.1 mm Varian Dynamax Microsorb 60-8 C18 column), fractions were collected every 30 s (*n* = 176) to yield (in order of elution) (4*R*\*,5*R*\*,9*S*\*,10*R*\*,11*Z*)-4-methoxy-9-((dimethylamino)-methyl)-12,15-epoxy-11(13)-endecahydronaphthalen-16-ol (**1**, fr 19 and 20 combined, 18.3 mg, 0.06% dry wt of extract), lobatrientriol [7] (fr 71, 24.6 mg, 0.08% dry wt of extract), loba-8,10,13(15)-triene-16,17,18-triol (fr

81, 21.1 mg, 0.07% dry wt of extract), 14,17-epoxyloba-8,10,13(15)-trien-18-ol-18-acetate [26] (fr, 83 and 84 combined, 72.8 mg, 0.07% dry wt of extract), lobatrienolide [7] (fr 87, 24.9 mg, 0.08% dry wt of extract), (1*E*,3*E*,7*E*)-11,12-epoxycembratrien-15-ol [8] (fr 89, 30.9 mg, 0.10% dry wt of extract) and 14,18-epoxyloba-8,10,13(15)-trien-17-ol [26] (fr 109, 123.7 mg, 0.42% dry wt of extract). Fractions 100 to 102 were combined (108.4 mg) and further purified by C18 analytical HPLC (1 mL/min, gradient elution from 5% CH3CN:H2O to 100% CH3CN over 18 min, followed by 6 min with 100% CH3CN through 250 × 4.6 mm, 5μ Phenomenex Luna (2) C18 column and fractions collected every 30 s) to yield the known compounds (17*R*)-loba-8,10,13(15)-triene-17,18-diol [27] (fr 34, 6.2 mg, 0.02% dry wt of extract) and sarcophytol-B [24] (fr 36, 18.3 mg, 0.06% dry wt of extract) and the new compound (1*R\**,2*R\**,4*S\**,15*E*)-loba-8,10,13(14),15(16)-tetraen-17,18-diol-17-acetate (**2**, fr 33, 2.3 mg, 0.008% dry wt of extract). The known compounds had identical physical and spectroscopic properties to those previously published [7,8,24,26,27].

3.4.1. (4*R*\*,5*R*\*,9*S*\*,10*R*\*,11*Z*)-4-Methoxy-9-((dimethylamino)-methyl)-12,15-epoxy-11(13)-endecahydronaphthalen-16-ol (**1**)

Pale yellow oil. [α] 24D +72◦ (CH3OH; *c* 0.67); UV (PDA) λmax nm: 195, 208; IR νmax cm−1: 3388, 2969, 2935, 1645, 1468, 1384, 1161, 1080; 1H (300 MHz, CD3OD) and 13C (75 MHz, CD3OD) NMR data Table 1; ESI-FTMS *m/z* [M + H]+ 394.3316 (calcd for C24H44O3N 394.3303), [M + Na]<sup>+</sup> 416.3128 (calcd for C24H43O3NNa 416.3135).

3.4.2. (1*R*\*,2*R*\*,4*S*\*,15*E*)-Loba-8,10,13(14),15(16)-tetraen-17,18-diol-17-acetate (**2**)

Colourless oil. [α] 24D −9.5◦ (CH3OH; *<sup>c</sup>* 0.23); UV (PDA) <sup>λ</sup>max nm: 203, 227; IR <sup>ν</sup>max cm−1: 3408, 2963, 2926, 1734, 1635, 1455, 1372, 1234, 1024, 904; 1H (300 MHz, CD3OD) and 13C (75 MHz, CD3OD) NMR data Table 2; ESI-FTMS *m/z* [M + Na]+ 369.2396 (calcd for C22H34O3Na 369.2400).
