(3β)-3,13-Dihydroxykaur-16-en-2-one (16) is white powder. HRESIMS shows that the molecular formula is C20H30O3 (m/z 319.2265 [M + H]+, calcd 319.2268), and the degree of unsaturation is 6. UV spectra indicated carbon–carbon double bond (λmax = 210 nm), IR spectra indicated carbonyl (σmin = 1712 cm−1) and hydroxyl (σmin = 3424 cm−1). There were 3 methyl signals in 1H-NMR of 16 at δH 0.70 (br s, 3H, H-18), 1.19 (s, 3H, H-19), and 0.97 (s, 3H, H-20). In addition, two alkene hydrogen signals at δH 4.85 (m, H-17), 5.00 (m, H-17), and one methine bearing to hydroxyl at δH 3.87 (br s, H-3) were found. In the 13C-NMR of 16, the signal at δC 210.7 (C-2) suggested the presence of carbonyl. The DEPT spectrum showed 3 primary carbons, 8 secondary carbons, 3 tertiary carbons, and 6 quaternary carbons, including a quaternary carbon connected to a hydroxyl at δC 80.0 (C-13). The methyl remote coupling signal of HMBC can confirm that the δH 0.70 (br s, CH3-18), 1.19 (s, CH3-19) connecting to δC 45.3 (C-4) then to 82.8 (C-3) and 54.4 (C-5), and δH 0.97 (s, CH3-20) connecting to δC 45.2 (C-10) then to 53.2 (C-1), 54.4 (C-5) and 54.0 (C-9). The coupling from δH 3.87 (br s, H-3), 2.03; 2.64 (m; dd, H-1) and δC 210.7 (C-2) confirmed the position of carbonyl at C-2. In the HMBC spectrum, correlations could be observed from δH 4.85; 5.00 (m; m, H-17) at the end side of the alkene to δC 155.3 (C-16), and then to the quaternary carbon 80.0 (C-13) and methylene 47.1 (C-15). Moreover, the HMBC correlations showed that there is an extra D ring in the closure of C-8, 13, 14, 15, and 16, which proves that this is a tetracyclic diterpene (Fig. 2A). 16 is very similar to the structure of the known compound excoecarin K [5]. Excoecarin K is an enantio-kaurane tetracyclic diterpene, indicating that 16 should also be a tetracyclic diterpene of the kaurane skeleton. The difference between 16 and excoecarin K is that C-13 of 16 has a higher chemical shift (δC 43.7 to 80.0) because 13-H is replaced by 13-OH. In NOESY, there are related signals of δH 0.97 (s, CH3-20), 0.70 (br s, CH3-18), and 2.04 (m, H-14β), which can indicate that C-14 on the C ring is tilted in the same direction as CH3-20, CH3-18; δH 1.19 (s, CH3-19), there are also correlation signals between 1.49 (m, H-5), 1.29 (br s, H-9), 3.87 (br s, H-3), and 2.12 (br d, H-15α), indicating that the D ring is tilted in the direction opposite to that of C-14, that is, the configurations of C-15, C-16 and CH3-19, H-5 and H-9 are consistent, that is, CH3-20, CH3-17, CH3-18, 3-OH, 13-OH are β configurations, H-5, H-9, C-15, and C-16 are in α configuration (Fig. 2B). The data determined by ECD showed that the absolute configuration of the compound is 3R, 5R, 8S, 9S, 10R, and 13R (Fig. 2C).

Structural elucidation of compounds 16, 17. A COSY and key HMBC signals. B NOESY correlations and relative configurations. C ECD spectral of 16, 17 versus calculated values

By comparison the 13C-NMR signals of 17 and 16, the difference between them leis in A ring. The C-3 position changed from a hydroxyl connected methine to a carbonyl, which shifted from δC 82.8 to δC 201.0. In addition, the -CH2CH(OH)- moiety at C-1, 2 position of 16 with signals at δC 53.2, 210.7 changes to a hydroxyl connected alkene double bone (δC 128.4 and 144.3) to form an α,β-unsaturated ketone structure. on the aids of HSQC, COSY and HMBC, the planar structure could be established as shown in Fig. 2A. The comparison of NOE correlations (Fig. 2B), optical rotation, ECD spectrum and calculated value showed that the absolute configuration of 17 is consistent with 16 (Fig. 2C).

Other isolated compounds were identified from their NMR and MS data, as well as by comparing these data with reported values, and they were: hinokiresinol (1) [6], methoxynyasol (2) [7], sugiresinol (3) [8], bis (4-hydroxyphenyl) buta-1,3-diene (4) [9], 2,2'-oxybis(1,4)-di-tertbutylbenzene (5) [10], balanophonin (6) [11], todolactol C (7) [12], (1R,3aR,4S,9aR)-1,7-dimethoxy-4-(4'-hydroxy-3'-methoxyphenyl)-1,3,3a,4,9,9a-hexahydronaphtho[2,3-c]furan-6-ol (8) [12], α-conidendrin (9) [13], (7'R)-hydroxymatairesanol (10) [14], pinoresinol (11) [15], epipinoresinol (12) [16], p-hydroxybenzaldehyde (13), 4-(3-furanyl)phenol (14), and cycloeucalenol (15) [17].