Silvaticusin A (1), which was obtained as colorless crystals, had the molecular formula C20H30O6 as inferred from the (+)-HRESIMS ion at m/z 389.1936 [M + Na]+ (calcd 389.1535) and supported by 13C NMR data. The IR spectrum exhibited characteristic absorption bands indicative of hydroxy (3391 cm−1), and olefinic (1633 cm−1) functionalities. Its 1H NMR spectrum showed resonances of two methyl groups at δH 1.05 (s) and 1.15 (s), four oxygenated methines at δH 4.21 (d, J = 5.3 Hz), 4.32 (t, J = 8.2 Hz), 5.34 (br s), and 5.72 (br s), and two olefinic protons at δH 5.36 (d, J = 1.9 Hz) and 5.74 (d, J = 1.9 Hz) (Table 1). Its 13C NMR and DEPT spectra featured twenty carbon resonances, including two methyl groups, six methylenes (one olefinic, one oxygenated), seven methines (four oxygenated), five non-protonated carbons (three quaternary) (Table 2). Comprehensive analysis of the 1D and 2D NMR data of 1 manifested that its structure resembled that of the 7,20-epoxy-ent-kauranoid, enmenol [15]. The H2-1/H2-2/H2-3, H-5/H-6, and H-9/H2-11/H-12/H-13/H-14 correlations in the 1H − 1H COSY spectrum, in tandem with the H2-1/C-20, H-6/C-4, H-6/C-7, H-6/C-8 correlations in the HMBC spectrum (Fig. 2) indicated that silvaticusin A (1) possesses a hydroxyl group at the C-12 position, differing from the C-1 hydroxylation in enmenol, while the rest of their structures were consistent.

1H − 1H COSY (bold), selected HMBC (arrow), and key ROESY (dashed arrow) correlations of silvaticusins A–C (1–3)

As for the configurational determination of 1, assigning H-5 as β-oriented, the 3JH-5/H-6 (5.3 Hz) indicated that H-6 was α-oriented. The H-9/H-5β, H-12/H-9, H3-18/H-5β, H-20b/H3-19, and H-14/H-20a correlations in the ROESY spectrum indicated that H-9, H-12, HO-14 adopted β orientation, while C-20 adopted α orientation. But the orientation of HO-15 could not be conclusively assigned. Fortunately, well-formed crystals of compound 1 were successfully procured using methanol and subsequently underwent X-ray crystallographic analysis. The refined Cu Kα data yielded a Flack parameter of –0.13(15), which enabled the determination of its absolute configuration as 5R, 6S, 7R, 9R, 10R, 12S, 13R, 14R, and 15R (Fig. 3). Thus, the structure of silvaticusin A (1) was defined as 6β,7β,12α,14β,15β-pentahydroxy-7,20-epoxy-ent-kaur-16-ene.

Crystallographic structure of silvaticusin A (1)

Silvaticusin B (2), which was obtained as a white amorphous powder, had the molecular formula C20H28O5 as established by the ( +)-HRESIMS ion at m/z 349.2010 [M + H]+ (calcd 349.2010). The 1H and 13C NMR data of compound 2 (Tables 1 and 2) were similar to those of 1. The 1H − 1H COSY spectrum exhibited correlations of H2-1/H2-2/H2-3, H-5/H2-6, H-9/H2-11/H-12, and H-13/H-14 (Fig. 2). And the HMBC spectrum displayed correlations from H-5 to C-6, C-7, C-10, and C-19, from H-13 to C-8, C-12, C-14, C-15, C-16 and C-17. These findings demonstrated that compound 2 features a carbonyl at C-15 instead of a hydroxy group as in 1, and a methylene at C-6 instead of the oxygenated methine in 1. Analysis of the ROESY spectrum of 2 uncovered the β orientations of H-12, HO-14. Thus, the structure of silvaticusin B (2) was assigned as 7β,12α,14β-trihydroxy-7,20-epoxy-ent-kaur-16-en-15-one.

Silvaticusin C (3) was isolated as a white amorphous powder. The molecular formula C20H28O5 was deduced from HREIMS and 13C NMR data. A thorough examination of the 1H and 13C NMR data for compound 3 revealed that its structure bore a close resemblance to that of the C-20 non-oxygenated-ent-kaurane diterpenoid macroclyxin C [16], sharing an identical molecular weight of 348 Daltons. The 1H − 1H COSY spectrum, with key correlations of H2-1/H2-2/H-3, H-5/H2-6/H-7, and H-9/H2-11/H2-12/H-13/H-14, and the HMBC spectrum (Fig. 2), showing correlations from CHO-18 to C-3, C-4, and C-19, and from H-13 to C-12 and C-15, have pinpointed a hydroxyl group at C-12 in silvaticusin C (3). This differs from macroclyxin C's hydroxyl group at C-3, with the rest of their structures remains congruent. As for the stereochemistry of 3, appointing H-5 as β-oriented, the H-3/H-5β, H-7/H-5β, H-9/H-5β, CHO-18/H-5β, H3-20/H3-19, H-14/H3-20 correlations in the ROESY spectrum indicated the β orientation of H-3, H-7, CHO-18, and HO-14. Therefore, the structure of silvaticusin C (3) was assigned as 3α,7α,14β-trihydroxy-ent-kaur-16-en-18-al-15-one.

Silvaticusin D (4) was isolated as a white amorphous powder. The molecular formula of C42H55O14 was inferred from the (+)-HRESIMS ion at m/z 789.3458 [M + Na]+ (calcd 789.3457) and 13C NMR data. Detailed analysis of the 1H and 13C NMR data of 4 suggested that it is a diterpene dimer composed of two 6,7-seco-6,20-epoxy-ent-kauranoids analogous to the structures of epinodosin [17, 18] and enmein-3-acetate [19], respectively. This deduction was further supported by H-1/H2-2/H2-3, and H-9/H-11/H2-12/H-13/H-14, H-1′/H2-2′/H-3′, and H-9′/H2-11′/H2-12′/H-13′/H-14′ correlations in the 1H − 1H COSY spectrum, as well as HMBC correlations from H-6 to C-4, C-5, C-10, C-20, from H-11 to C-8, C-10, C-13, from H-6′ to C-4′, C-10′, C-20′, from H-3′ to C-1′, C-2′, C-4′, C-5′, -OAc. The lack of NMR signals for the typical terminal double bonds at C-16/C-17, a feature usually presents in ent-kauranoids, in both monomers of compound 4, implies that these double bonds are involved in the dimerization. This, combined with the one remaining degree of unsaturation and key 1H − 1H COSY correlation between H-17 and H-17′, along with HMBC correlations of H-13/C-16, H2-17/C-13, H-13′/C-16′, and H2-17′/C-13′, supported a head-to-head [2 + 2] cycloaddition mechanism for dimerization. Accordingly, the planar structure of compound 4 is established as illustrated in Fig. 4.

1H − 1H COSY (bold), selected HMBC (arrow), and key ROESY (dashed arrow) correlations of silvaticusin D (4)

The stereochemistry of most chiral centers was determined using ROESY correlations and coupling constants, except for C-3′, where overlapping NMR signals obscured its configuration determination. As for the two new chiral centers (C-16 and C-16′) formed during dimerization, correlations of H-13/H-17a, H-13/H-17′a, and H-13′/H-17′b in the ROESY spectrum suggested the α orientation of C-17 methylene, and the β orientation of C-17′. Then, the two possible epimers, (1S*, 5R*, 6R*, 8S*, 9S*, 10S*, 11S*, 13S*, 16R*, 1′S*, 3′S*, 5′R*, 6′R*, 8′S*, 9′S*, 10′S*, 13′R*, 16′R*)-4 (4a) and (1S*, 5R*, 6R*, 8S*, 9S*, 10S*, 11S*, 13S*, 16R*, 1′S*, 3′R*, 5′R*, 6′R*, 8′S*, 9'S*, 10′S*, 13′R*, 16′R*)-4 (4b) were subjected to NMR computation at the mPW1PW91-SCRF/6–31 + G(d,p)//B3LYP-D3BJ/6-31G(d) level of theory. In the subsequent DP4 + analysis [20] comparing isomers 4a and 4b, 4a exhibited a conclusive probability of 100% (Figure S46), thereby substantiating its designation as the correct structure. Then, TDDFT ECD calculation of (1S, 5R, 6R, 8S, 9S, 10S, 11S, 13S, 16R, 1′S, 3′S, 5′R, 6′R, 8′S, 9′S, 10′S, 13′R, 16′R)-4 (4A) were carried out at the PBE0-SCRF/6–311 + G(2d,p) and CAM-B3LYP-SCRF/6–311 + G(2d,p) level of theory, respectively, and the calculated curves matched the experimental curve (Fig. 5), thereby determining the absolute configuration of compound 4 as 1S, 5R, 6R, 8S, 9S, 10S, 11S, 13S, 16R, 1′S, 3′S, 5′R, 6′R, 8′S, 9′S, 10′S, 13′R, and 16′R (Table 3).

Experimental ECD spectrum of silvaticusin D (4) (black). Calculated ECD spectra of (1S, 5R, 6R, 8S, 9S, 10S, 11S, 13S, 16R, 1′S, 3′S, 5′R, 6′R, 8′S, 9′S, 10′S, 13′R, 16′R)-4 (4A) at PBE0-SCRF/6–311 + G(2d,p) (red) and CAM-B3LYP-SCRF/6–311 + G(2d,p) (blue, shift =  + 15 nm) level of theory

Previous research on diterpenoids from the Isodon genus has shown that the exo-methylene cyclopentanone D-ring system is crucial for their antitumor and anti-inflammatory effects, with C-14 hydroxylation usually enhancing these effects [21]. In this research, compounds 2 and 3, which possess this D-ring system, were evaluated for cytotoxicity against HL-60, A-549, SMMC-7721, MDA-MB-231, and SW-480 cell lines using the MTS assay [22], with cisplatin and paclitaxel as positive controls. Compounds 2 and 3 exhibited significant cytotoxic activity, with IC50 values ranging from 1.27 ± 0.08 to 7.52 ± 0.33 μM for all five cell lines (Table 4).