**2. Results and Discussion**

The 13C NMR and ESI-FTMS of **1** established its molecular formula to be C24H43O3N, requiring four degrees of unsaturation. The 1H and 13C NMR spectral data of **1** (Table 1) showed the molecule to contain a trisubstituted double-bond (δ<sup>C</sup> 142.5, s, C-11; 117.8, d, C-13; δ<sup>H</sup> 5.59, d, 5.2, H-13) as the only multiple bond within the molecule and accounted for one of the degrees of unsaturation. This information, in combination with the molecular formula, showed the molecule to be tricyclic.

**Table 1.** 1H and 13C NMR data (300 MHz and 75 MHz, CD3OD) for (4*R*\*,5*R*\*,9*S*\*,10*R*\*,11*Z*)-4-methoxy-9-((dimethylamino)-methyl)-12,15-epoxy-11(13)-en-decahydronaphthalen -16-ol (**1**).


From the 1H-1H COSY spectrum of **1** spin systems from Hb-1 (δ<sup>H</sup> 1.24, m) to H2-3 (δ<sup>H</sup> 1.85, m; 1.60, m) via Ha/b-2 (δ<sup>H</sup> 1.59, m; 1.45, m) and from H-5 (δ<sup>H</sup> 1.49, m) to H2-22 (δ<sup>H</sup> 3.25, dd, 3.2, 11.0; 2.93, dd, 11.0, 13.1) via H2-6 (δ<sup>H</sup> 1.82, m), H-7 (δ<sup>H</sup> 1.86 m), H2-8 (δ<sup>H</sup> 1.85, m; 1.52 m) and H-9 (δ<sup>H</sup> 1.52, m) could be discerned. This information together with cross-peaks in the HMBC spectrum from Hb-1 and H-5 to C-19 (δ<sup>C</sup> 15.2, q), from H-20 (δ<sup>H</sup> 1.08, s) to C-3, C-4 and C-5, and from H-7 to the olefinic C-11 (δ<sup>C</sup> 142.5, s), showed the presence of a substituted bicyclic ring system.

The 1H-NMR spectrum of **1** displayed singlet resonances of a methoxy (-OCH3) at δ<sup>H</sup> 3.16 and an *N*,*N*-dimethyl substituted tertiary amine (-N(CH3)2) at δ<sup>H</sup> 2.90. The HMBC correlation from δ<sup>H</sup> 3.16 (s) to δ<sup>C</sup> 76.8 (s, C-4) located the methoxy at C-4 while correlations from both H-23/24 to and δ<sup>C</sup> 60.5 (C-22) located the tertiary amine at C-22.

Further analysis of the 1H-1H COSY indicated the presence of a spin system from δ<sup>H</sup> 5.59 (d, 5.2, H-13) to δ<sup>H</sup> 3.25 (m, H-15) via δ<sup>H</sup> 2.12 and 2.00 (m, H2-14). HMBC correlations from H-13 to C-7 located the C-11 side chain at C-7 of the bicyclic ring system. Based on HMBC correlations from H3-17/H3-18 to δ<sup>C</sup> 72.7 (C-16), the two methyl groups with resonances at δ<sup>H</sup> 1.17 (H-17/18) were connected to a tertiary carbon bearing an OH, forming a propan-2-ol-2-yl moiety [28]. The data so far accounted for three of the four oxygens, the double-bond, two of the rings, and the nitrogen, leaving one oxygen and one ring unassigned. An ether linkage, forming the third ring, was deduced between C-12 and C-15 based on the HMBC correlation from δ<sup>H</sup> 4.16 (brs, H-12) to δ<sup>C</sup> 81.9 (C-15), and was further supported by a C-O-C stretch at 1080 cm−<sup>1</sup> in the IR spectrum of **1**. Hence the planar structure of **1**, a diterpene, is best described as (4*R*\*,5*R*\*,9*S*\*,10*R*\*,11*Z*)-4-methoxy-9-((dimethylamino)-methyl)-12,15-epoxy-11(13)-endecahydronaphthalen-16-ol (Scheme 1).

**Scheme 1.** Structures of (4*R*\*,5*R*\*,9*S*\*,10*R*\*,11*Z*)-4-methoxy-9-((dimethylamino)-methyl)-12,15-epoxy-11(13)-en -decahydronaphthalen-16-ol (**1**), and (1*R\**,2*R\**,4*S\**,15*E*)-loba-8,10,13(14),15(16)-tetraen-17,18-diol-17-acetate (**2**).

The nOe data of **1** showed correlations between H3-19 (δ<sup>H</sup> 0.89, s) and Hb-1, Hb-2, H3-20 and Hb-22. These cross peaks revealed the two fused six-membered rings to have a trans-ring junction, CH3-19 and CH3-20 to be axial and therefore on the same side of **1**, and the side-chain at C-9 to be on the same side as C-19 (Figure 1). Furthermore, nOe correlations were observed between H3-21 and H-5 indicating they were on the same side of the molecule as each other but the opposite side of C-19 (Figure 1). The configurations at C-7 and C-15 remain unresolved. Based on the above findings, the relative configurations of chiral carbons C-10, C-5, C-9 and C-4 of **1** were assigned as 4*R*\*, 5*R*\*, 9*S*\* and 10*R*\* (Scheme 1).

**Figure 1.** Diagnositic nOe correlations for partial structure of **1**.

The 13C NMR and ESI-FTMS of **2** established its molecular formula as C20H34O3, indicating the molecule to have four degrees of unsaturation. The 1H and 13C NMR spectral data of **2** (Table 2) showed it contained a vinyl moiety (δ<sup>C</sup> 151.6, d, C-8; 110.4, t, C-9; δ<sup>H</sup> 5.87, dd, 10.8, 17.6 Hz, H-8; δ<sup>H</sup> 4.93, dd, 1.2, 17.6 Hz, Ha-9; δ<sup>H</sup> 4.90, dd, 1.2, 10.8 Hz, Hb-9), an isopropenyl group (δ<sup>C</sup> 148.9, s, C-10; 112.7, t, C-11, 25.3, q, C-12; δ<sup>H</sup> 4.81, brt, 1.5 Hz, Ha-11; 4.59, brs, Hb-11; 1.71, brs, H3-12) and a tertiary methyl group (δ<sup>C</sup> 17.1, q, C-7; δ<sup>H</sup> 1.03, s, H3-7), characteristic of the 3-isopropenyl-4-methyl-4-vinylcyclohexane-1-yl moiety found in lobane-type diterpenoids [7,25–27]. This partial structure was confirmed by analysis of the COSY, HMQC, and HMBC NMR spectral data of **2**. The relative configuration about C-1 and C-2 in **2** was found to be the same as in elemol and several other lobanes, as established by comparison of the 1H and 13C NMR data for each molecule at centres C-1, C-2, C-6, C-7, C-8, C-9, C-10, C-11 and C-12 [25,27]. Also evident from this data was an exo-methylene (δ*<sup>C</sup>* 151.9, s, C-13; 114.3, t, C-14; δ<sup>H</sup> 5.06, s, H2-14), an endo-disubstituted double-bond (δ*<sup>C</sup>* 137.4, d,C-15; 124.4, d, C-16; δ<sup>H</sup> 6.26, d, 16.0, H-15; 5.78, dd, 7.6, 16.0, H-16), a carbonyl group in the form of an acetate (δ<sup>C</sup> 172.1, s, O-CO-CH3; 21.1, q; δ<sup>H</sup> 2.09, s, O-CO-CH3), a CH bearing the acetate (δ<sup>C</sup> 82.2, d, C-17; δ<sup>H</sup> 5.14, brd, 7.6, H-17), and a propan-2-ol-2-yl moiety the same as that found in **1**. These assignments were corroborated by the IR data with terminal vinyl C-H stretches at 3079 and 3012 cm−1, a carbonyl ester band at 1734 cm−1, and an alcohol OH stretch at 3084 cm−1. This data accounted for all of the remaining unsaturation within the molecule as well as the previously unaccounted for C10H15O3. From the HMBC data of **2** (Table 2), it was evident that C-17 bonded to both C-15 and C-18, as well as the oxygen of the acetyl function. Further, HMBC correlations between H-14 and the carbons C-4, C-13 and C-15, confirmed the side-chain to be attached at C-4 and that the two double-bonds were conjugated, an observation supported by the UV maxima of **2** at 227 nm. With the planar structure of **2** deduced, the double-bond geometry and stereochemistry required resolution. The magnitude of the coupling constant between H-15 and H-16 (*J* = 16.0 Hz), showed Δ<sup>15</sup> to have *E* geometry. The relative configurations at C-1 and C-2 were confirmed to be the same as in the known lobane loba-8,10,13(15)-triene-16,17,18-triol [25] on the basis of comparable 13C NMR chemical shift for the same centres. The relative configurations at C-1, C-2 and C-4 were assigned based on NOESY NMR correlations from H-4 to H-2, H2-5, Ha-6, H-14, H-15, H-16, H3-19, H3-20 and O-CO-CH3, and from H-12 to H-2, Ha-6, H-7, H-8, Ha-9, H2-11, H3-19 and confirmed them to be 1*R\**, 2*R\** and 4*S\**, as shown for **2** [6,7,25–27]. The configuration at C-17 remains unresolved. Compound **2** is thus best described as (1*R\**,2*R\**,4*S\**,15*E*)-loba-8,10,13(14),15(16)-tetraen-17,18-diol-17-acetate.


**Table 2.** 1H and 13C NMR data (300 MHz and 75 MHz, CD3OD) for (1*R\**,2*R\**,4*S\**,15*E*)-loba-8,10,13(14),15(16)-tetraen-17,18-diol-17-acetate (**2**).

Complete 1D and 2D NMR data for the known cembranes: sarcophytol-B and (1*E*,3*E*,7*E*)-11,12-epoxycembratrien-15-ol, and the six known lobanes: loba-8,10,13(15)-triene-16,17,18-triol, 14,18-epoxyloba-8,10,13(15)-trien-17-ol, lobatrientriol, lobatrienolide, 14,17-epoxyloba-8,10,13(15)-trien-18-ol-18-acetate and (17*R*)-loba-8,10,13(15)-trien-17,18-diol, are provided for the first time (Supplementary Information). Raju *et al.* reported that loba-8,10,13(15)-triene-16,17,18-triol was the product of long-term, cold storage of the natural product 17,18-epoxyloba-8,10,13(15)-trien-16-ol in CDC13 [25]. Closer inspection of the FTMS and 13C NMR of the fresh extract in CD3OD showed the presence of only the triol in our study.

The cytotoxic activities of compounds **1** and **2**, and of the known compounds loba-8,10,13(15)-triene-16,17,18-triol, 14,17-epoxyloba-8,10,13(15)-trien-18-ol-18-acetate, lobatrienolide, (1*E*,3*E*,7*E*)-11,12-epoxycembratrien-15-ol and sarcophytol-B towards a panel of human tumour cell lines are given in Table 3. With the exception of **1** (GI50s all over 70 μM), all compounds showed good

activity with GI50s in the range 6.8–18.5 μM. From these data there appears to be no obvious SAR. The four lobanes (including **2**) and the two cembranes all had approximately the same overall activities against the human tumour cell lines SF-268, MCF-7 and H460, with no selectivity.

**Table 3.** Cytotoxicity data [GI50 (μM)] for compounds **1**, **2** and the known compounds loba-8,10,13(15)-triene-16,17,18-triol, 14,17-epoxyloba-8,10,13(15)-trien-18-ol-18-acetate, lobatrienolide, (1*E*,3*E*,7*E*)-11,12-epoxycembratrien-15-ol and sarcophytol-B against the human tumour cell lines SF-268, MCF-7 and H460.


<sup>a</sup> SF-268 Central nervous system-glioblastoma cells; <sup>b</sup> MCF-7 Breast-pleural effusion adenocarcinoma cells; <sup>c</sup> H460 Lung-large cell carcinoma cells; NT = Not tested.
