The S. fulvorobeus was obtained from fresh feces of E. maximus collected in the Xishuangbanna National Nature Reserve. The fermentation broth of S. fulvorobeus was clarified with a centrifuge to collected 150 L of culture supernatant. The clarified supernatant was extracted with ethyl acetate and the extract was isolated by repeated column chromatography over silica gel, Sephadex LH-20, and ODS to afford thirty-six sequiterpenoids (Fig. 1).

Chemical structures of compounds 1–36 from S. fulvorobeus

Compound 1 was isolated as a colorless oil with the molecular formula C15H24O3 based on HRESIMS and 13C NMR data. The IR spectrum of 1 showed characteristic absorption bands for hydroxy groups (3382 cm−1) and carbonyl (1699 cm−1). The 1H NMR (Table 1) of 1 displayed the presence of an oxygenated methylene (δH 3.14, 3.11), two singlet methyls (δH 1.01, 0.88), and other aliphatic residues at δH 0.98–2.82. The 13C NMR data (Table 2) of 1 showed 15 carbons signals, including one carbonyl (δC 216.9), two oxygen bearing carbons (δC 71.0, 68.1), two quaternary carbons (δC 46.5, 38.4), six methylenes (δC 49.6, 38.6, 36.9, 34.7, 30.3, 26.4), two methines (δC 43.5, 39.9), and two methyls (δC 31.1, 17.7). Analysis of the 1D and 2D NMR data indicated that 1 was consistent with a caryolane-type sesquiterpenoid and exhibited similarity to the reported bacaryolane A [19], except for the presence of an additional hydroxymethyl (δC 71.0, δH 3.14, 3.11) and the absence of a methyl. The HMBC correlations from H-13 (δH 3.14, 3.11) to C-3 (δC 30.3) and C-14 (δC 17.7), from H-3β (δH 1.78) to C-13 (δC 71.0), and from H-14 (δH 0.88) to C-13 (δC 71.0) established the hydroxymethyl was located at C-4. Moreover, the HMBC correlations from H-10 (δH 2.82, 2.17), H-11 (δH 1.84, 1.70), H-12 (δH 1.89), and H3-15 (δH 1.01) to C-9 (δC 216.9) confirmed that the carbonyl was located at C-9 (Fig. 2A). Caryolanes derived from plants and bacteria possessed varied stereochemical structures as they were biosynthesized by different cyclization from the humulyl cation [19]. The carbon skeleton of caryolanes in the current study was defined the same as those from bacteria such as bacaryolanes A-C [19]. The NOESY correlations found between H-13 (δH 3.14, 3.11) and H-5 (δH 1.82), between H3-14 (δH 0.88) and H-2 (δH 1.59) established that H-2 and H3-14 were β-orientated, whereas H-5 and H2-13 were α-orientated. In addition, NOE correlations between H3-15 (δH 1.01) and H-10α (δH 2.82), between H-10β (δH 2.17) and H-2 (δH 1.59) further confirmed the structure (Fig. 3). The absolute configuration 1S,2R,4S,5S,8R were deduced from comparison of experimental and calculated ECD spectra of 1 (Fig. 4A). According to the literature, the proton signals of the oxymethylene protons in the (S)- and (R)-α-methoxy-α-trifluoromethylphenylacetyl (MTPA) esters of primary alcohol showed a unique split pattern [24]. 1 was treated with (R)-MTPA-Cl and (S)-MTPA-Cl to afford the (S)- or (R)-MTPA ester derivatives 1a and 1b, respectively. The signals of oxymethylene protons at C-13 for the (S)-MTPA ester 1a appeared at δlow 4.26 and δhigh 4.24 (Δδ 0.02), while those for the (R)-MTPA ester 1b were observed as two separated doublet signals at δlow 4.30 and δhigh 4.22 (Δδ 0.08). The (R)-MTPA esters of primary alcohol analogue possessing 4S-configuration had relatively larger Δδ (δlow–δhigh) values. Therefore, the structure of 1 was elucidated as (1S,2R,4S,5S,8R)-9-oxocaryolane-1,13-diol.

Main HMBC and COSY correlations of compounds 1–4, 13–15, 17, 18, 22, 23, 25–28 (A). proposed biosynthetic pathway of compounds 17, 25, 27 (B)

Main NOE correlations of compounds 1–4, 13–15, 17, 18, 22, 23, 25–28

Experimental ECD spectra and calculated ECD spectra of compounds 1 (A), 13–15 (B−D), 22 (E), 23 (F), 28 (G)

Compound 2 had the molecular formula C15H26O2 by its HRESIMS and 13C NMR data. Its 1H and 13C NMR data revealed that 2 was a caryolane type sesquiterpenoid and related to the known compound caryolan-1-ol [25]. The distinct difference was that a methyl of caryolan-1-ol was replaced by a hydroxymethyl (δC 65.3, δH 3.48, 3.40) in 2. The HMBC correlations between H-14 (δH 3.48, 3.40) and C-3 (δC 30.6) and C-13 (δC 26.6), between H-3 (δH 1.61, 1.40), H-13 (δH 0.98) and C-14 (δC 65.3) determined the hydroxymethyl at C-4 (Fig. 2A). NOE correlations between H-5 (δH 1.85) and 1-OH (δH 3.94), H3-13 (δH 0.98), between H-2 (δH 2.22) and H-14 (δH 3.48, 3.40), H-7β (δH 1.50), between H-7α (δH 1.00) and H3-15 (δH 0.81) indicated that H-2, H-14 were β-orientation whereas 1-OH, H-5, H3-13, H3-15 were α-orientation (Fig. 3). The absolute configuration of C-4 was determined by Mosher’s method. Treatment of 2 with (R)-MTPA-Cl or (S)-MTPA-Cl obtained the S or R Mosher’s esters 2a and 2b. The signals of oxymethylene protons at C-14 for the (S)-MTPA ester 2a showed two separated doublet signals at δlow 4.69 and δhigh 4.55 (Δδ 0.14), while those for the (R)-MTPA ester 2b were presented at δH 4.63 as a broad singlet peak. These findings suggested the R-configuration of C-4 in 2 by comparing the Δδ values of oxymethylene protons with those of 4S-configuration analogue 1. Thus, 2 was deduced to be (1S,2R,4R,5S,8R)-caryolane-1,14-diol as the same biosynthesis pathway from bacteria.

Compound 3 afforded a molecular formula of C15H26O3 on the basis of HRESIMS and 13C NMR data. Analysis of its 1H and 13C NMR data (Tables 1, 2) indicated that 3 has a close structural relationship to 2. The only difference was that one methylene was absent and one oxygenated methine was present (δC 77.1, δH 3.10) in 3. The HMBC correlations from H-10 (δH 1.63, 1.58), H-12 (δH 1.46, 0.89), H3-15 (δH 0.82) to C-9 (δC 77.1) as well as 1H–1H COSY correlations between H-9 (δH 3.10) and H-10 (δH 1.63, 1.58) confirmed a hydroxy at C-9 (Fig. 2A). The NOE cross peaks observed between H3-13 (δH 0.98) and H-5 (δH 1.89), between H3-15 (δH 0.82) and H-9 (δH 3.10), between H-9 (δH 3.10) and H-11α (δH 1.30), and between H-2 (δH 2.27) and H-14 (δH 3.49, 3.42) and H-11β (δH 1.43) indicated that H-2, H-14, and 9-OH were β-oriented, while H-5, H-9, H3-13, and H3-15 were α-oriented (Fig. 3). The (S)-MTPA ester (3a) or (R)-MTPA ester (3b) were obtained by the same derivative process as those of 1 and 2. Both 9-OH and 14-OH in compound 3 were esterified by MTPA-Cl according to NMR data. Unfortunately, it was impossible to assign the absolute configuration of C-4 as the Δδ values of two separated peaks of H2-14 for 3a and 3b were very approximate (Δδ = 0.12 for 3a and Δδ = 0.10 for 3b) being influenced by two MTPA groups. As 3 presented almost identical 13C NMR data of C-4, C-13, and C-14 with those of compound 2, the R-configuration of C-4 could be determined. The configuration of C-9 could be deduced by comparing the chemical shift of H3-15 in (S)- and (R)-MTPA esters. The signal of H3-15 for (S)-MTPA ester 3a was observed at lower field (δH 0.99) compared to the signal for (R)-MTPA ester 3b (δH 0.88), which exhibited the absolute configuration of C-9 to be S. Thus, the structure of 3 was confirmed as (1S,2R,4R,5S,8R,9S)-caryolane-1,9,14-triol.

The molecular formula of 4 was deduced as C15H26O3 according to its HRESIMS and 13C NMR data. The 1H NMR and 13C NMR data (Tables 1, 2) indicated that 4 was similar to bacaryolane C (6) [19], also isolated in the current study. The obvious alteration was that a methylene in 6 was replaced by an oxygenated methine (δC 66.1, δH 3.68) in 4. The HMBC correlations from H-9 (δH 1.57, 0.88), H-11 (δH 1.74, 1.05) to C-10 (δC 66.1) confirmed that a hydroxy was located at C-10. The COSY correlations from H-10 (δH 3.68) to H-9 (δH 1.57, 0.88), H-11 (δH 1.74, 1.05), and 10-OH (δH 4.37) further supported the above inference (Fig. 2A). The NOESY cross peaks from H-2 (δH 1.86) to H-6 (δH 3.51) and H-10 (δH 3.68), from H-10 (δH 3.68) to H-7β (δH 1.51) indicated that H-2, H-6, and H-10 were on the same side. Correspondingly, the NOESY correlations from H3-15 (δH 0.95) to H-7α (δH 1.15), from H-7α (δH 1.15) to H-5 (δH 1.61) suggested that H-5, H3-15 were on the opposite side (Fig. 3). Consequently, 4 was deduced to be caryolane-1,6α,10α-triol.

Compound 13 was isolated as a colorless oil with a molecular formula C15H26O3 by HRESIMS and 13C NMR data. Its IR spectrum revealed the presence of hydroxy groups (3315 cm−1) and double bonds (1668 cm−1). The 1H NMR data (Table 3) showed three olefinic hydrogens (δH 5.41, 4.90, 4.65), two oxygenated methines (δH 4.19, 3.80), three singlet methyls (δH 1.53, 1.14, 0.93), one doublet methyl (δH 0.97), and other aliphatic hydrogens (δH 1.37–2.41). The 13C NMR data (Table 2) of 13 displayed 15 carbon signals, including four olefinic carbons (δC 139.7, 136.1, 132.9, 121.7), three oxygen-bearing carbons (δC 73.4, 67.6, 63.8), two methines (δC 61.8, 32.3), two methylenes (δC 52.1, 40.5), and four methyls (δC 30.8, 24.5, 18.4, 16.1), as determined in an HSQC experiment. The 1H and 13C NMR data of 13 were very similar to those of 1(10)E,5E-germacradiene-2α,11-diol (16) [26], a germacrane-type sesquiterpenoid. The major differences were the disappearance of a methylene in 16 and the existence of an oxygen-bearing methine (δC 67.6, δH 3.80) in 13. The 1H–1H COSY correlations from H-8 (δH 3.80) to H-7 (δH 2.28) and H-9 (δH 2.27, 2.25) as well as the HMBC correlations between H-7 (δH 2.28), H-9 (δH 2.27, 2.25) and C-8 (δC 67.6) indicated that a hydroxy was connected with C-8 (Fig. 2A). Thus, the planar structure of compound 13 was established. The NOE interactions observed between H-2 (δH 4.19) and H3-15 (δH 0.97), between H-6 (δH 4.65) and H-8 (δH 3.80), H3-15 (δH 0.97), between H3-13 (δH 1.14) and H-6 (δH 4.65), H-8 (δH 3.80) revealed that H-2, H-8, and H3-15 were on the same side, while 2-OH, 8-OH, H-4, and H-7 were located on the opposite side (Fig. 3). Furthermore, the coupling constants of H-2 (δH 4.19, td, J = 10.2, 4.2 Hz) suggested that the 2-OH presented in the equatorial position. In addition, NOE interactions observed from H3-14 (δH 1.53) to H-2 (δH 4.19) and H-8 (δH 3.80), from H-1 (δH 4.90) to H-5 (δH 5.41) as well as the large coupling constant of H-5/H-6 elucidated the E configurations of C-1/C-10 and C-5/C-6 double bonds. The experimental ECD spectrum of 13 had a consistent trend with its corresponding calculated ECD curve which determined the 2S,4S,7S,8S-configurations (Fig. 4B). Consequently, 13 was established as (2S,4S,7S,8S)-1(10)E,5E-germacradiene-2,8,11-triol.

The 1H and 13C NMR data (Tables 2, 3) as well as a molecular formula of C16H28O2 exhibited that 14 bore a close resemblance to the known compound 16 [26], except for the presence of an additional methoxy (δC 54.5, δH 3.06). The HMBC correlation between methoxy (δH 3.06) and C-2 (δC 73.8) determined that the methoxy was located at C-2 (Fig. 2A). In NOESY spectrum, the key cross peaks from H-2 (δH 3.90) to H3-15 (δH 1.02), from H-5 (δH 5.35) to H-4 (δH 2.41) and H-7 (δH 2.10) indicated that H-2 and H3-15 were β-orientation, while H-4 and H-7 were α-orientation (Fig. 3). Moreover, the double bonds at C-5/C-6, C-1/C-10 were elucidated as E geometry based on the NOE correlations from H3-14 (δH 1.57) to H-2 (δH 3.90) and H-6 (δH 4.84), from H-1 (δH 4.80) to H-5 (δH 5.35) as well as the large coupling constant of H-5/H-6 (J = 15.6 Hz). The absolute configurations of C-2, C-4, and C-7 were elucidated as 2S,4S,7R based on comparing the experimental and calculated ECD spectra (Fig. 4C). Therefore, the structure of 14 was confirmed as (2S,4S,7R)-1(10)E,5E-germacradiene-2,11-diol 2-methyl ether.

Compound 15 was assigned a molecular formula of C17H28O5 based on its HRESIMS and 13C NMR data. The characteristic 1H and 13C NMR data (Tables 2, 3) of 15 suggested that it was a germacrane-type sesquiterpenoid and had a close structural relationship to 13, except for the different oxygenated position and an additional acetyl (δC 169.9, 21.5). HMBC correlations from H-3 (δH 1.76, 1.48), H-6 (δH 4.72) and H3-15 (δH 1.15) to C-4 (δC 70.5) confirmed that the extra hydroxy was located at C-4. HMBC correlations from H-2 (δH 5.21) to C-16 (δC 169.9) as well as 1H–1H COSY correlations from H-2 (δH 5.21) to H-1 (δH 4.82) and H-3 (δH 1.76, 1.48) indicated that the acetyl was located at C-2. (Fig. 2A). 15 presented the same relative configuration as those of 13 and 14 on the basis of NOE correlations between H-2 (δH 5.21) and H3-15 (δH 1.15), H3-14 (δH 1.61), between H3-14 (δH 1.61) and H-8 (δH 3.87), between H3-13 (δH 1.14) and H-6 (δH 4.72), H-8 (δH 3.87) (Fig. 3). Besides, the geometry at C-5/C-6 double bonds was assigned as E according to the large coupling constants of H-5/H-6 (J = 15.6 Hz). The (+) Cotton effect at 200 nm (+ 29.25) and (−) Cotton effect at 231 nm (− 9.42) of 15 detected by CD spectrum was consistent with those of the calculated ECD curve of 2S,4R,7S,8S-15 (Fig. 4D). Thus, 15 was identified as (2S,4R,7S,8S)-1(10)E,5E-germacradiene-2,4,8,11-tetraol 2-acetate.

The HRESIMS and 13C NMR data determined the molecular formula of 17 as C15H26O3. The NMR data (Tables 3, 4) suggested 17 was a 6,11-epoxyisodaucane type sesquiterpenoid and related to the known compound 6,11-epoxyisodaucane [27]. The difference was that two oxygenated methines replaced two methylenes in 17. 1H–1H COSY correlations from H-3 (δH 4.17) to H-4 (δH 2.30) and H-2 (δH 1.82, 1.53), from H-9 (δH 3.51) to H-10 (δH 1.62, 1.48) and H-8 (δH 1.82, 1.04) determined the two hydroxys at C-3 and C-9, respectively. The HMBC correlations between H3-15 (δH 1.16) and C-1 (δC 38.9), C-2 (δC 51.9), C-5 (δC 61.6), and C-10 (δC 51.7), between H-6 (δH 3.13) and C-1 (δC 38.9), C-8 (δC 47.7), between H-5 (δH 1.94) and C-2 (δC 51.9), C-3 (δC 73.0), C-10 (δC 51.7), and C-15 (δC 31.3), between H-12 (δH 1.17) and C-4 (δC 64.2), C-11 (δC 80.0) confirmed the planar structure (Fig. 2A). The absolute configuration of 6,11-epoxyisodaucane was determined by total synthesis method [27]. The similar coupling constants of H-5 (δH 1.94, t, J = 9.5 Hz), H-6 (δH 3.13, t, J = 9.9 Hz) indicated that 17 possessed the same 4α-H, 5α-H, 6β-H, 7β-methyl configuratinos as those of synthesized 6,11-epoxyisodaucane. The NOE interactions from H-5 (δH 1.94) to H-9 (δH 3.51), H-7 (δH 1.42), from H3-14 (δH 0.87) to H-6 (δH 3.13), from H-6 (δH 3.13) to H-3 (δH 4.17), from H3-15 (δH 1.16) to H-9 (δH 3.51) determined the configurations as in Fig. 3.

Analysis of 1H NMR, 13C NMR (Tables 3, 4), and HRESIMS data of 18 indicated it was a sesquiterpenoid and was related to the reported ganodermanol L (19) [28]. The only difference was one more oxygenated methine (δC 67.0, δH 3.38) in 18 replaced a methylene in 19. The HMBC correlations between 8-OH (δH 4.56) and C-7 (δC 59.1), C-8 (δC 67.0), and C-9 (δC 52.8) determined the C-8 position of extra hydroxy. Moreover, COSY correlations from H-8 (δH 3.38) to H-7 (δH 1.38), H-9 (δH 1.77, 1.40), and 8-OH (δH 4.56) further supported the conclusion (Fig. 2A). NOE cross peaks observed between H-6 (δH 1.16) and H-2 (δH 3.67), H-8 (δH 3.38), H3-15 (δH 0.84), between H-5 (δH 3.46) and H-1 (δH 1.22) demonstrated that H-2, H-6, H-8, and H3-15 were located on the same side, whereas H-1, H-5, and H-7 were located on the opposite side (Fig. 3). There were potential inaccuracies of NOE correlations due to the overlapped signals of H-6 (δH 1.16) and H3-14 (δH 1.17). The configuration of C-10 was then confirmed through comparing the 13C NMR data of C-1, C-2, C-10, and C-14 with those of ganodermanol L (19) [28], which was elucidated the structure by X-ray crystallographic analyses. In addition, the peak shapes and coupling constants of H-1 (δH 1.22, dd, J = 12.5, 9.7 Hz), H-2 (δH 3.67, brt J = 9.6 Hz), and H-5 (δH 3.46, dd, J = 10.2, 4.8 Hz) further confirmed the relative configuration.

The 1H and 13C NMR data (Tables 4, 5) as well as molecular formula, C15H26O3, of 22 showed it was a cadinene-type sesquiterpenoid and related to 15-hydroxy-α-cadinol (21) [29], except an additional oxygenated methine (δC 70.1, δH 3.82) in 22 instead of a methylene in 21. 1H–1H COSY correlations from H-2 (δH 3.82) to H-3 (δH 2.20, 1.89) and H-1 (δH 1.30) as well as HMBC correlations from H-1 (δH 1.30), H-3 (δH 2.20, 1.89) to C-2 (δC 70.1) revealed that a hydroxy was located at C-2 (Fig. 2A). The large coupling constants of H-1 (δH 1.30, brt, J = 10.6 Hz), H-6 (δH 1.73, brt, J = 10.6 Hz) indicated a trans fusion of the bicyclic system. NOE correlations from H-6 (δH 1.73) to H-2 (δH 3.82), H3-14 (δH 1.18), and H3-12 (δH 0.73) indicated that the H-2, H-6, and H3-14 were β-orientated, whereas H-1, H-7, 2-OH, and 10-OH were α-orientated (Fig. 3). Comparison of the experimental and calculated ECD spectra of 22 revealed the absolute configuration as 1S,2S,6R,7S,10R (Fig. 4E). Thus, compound 22 was elucidated as (1S,2S,6R,7S,10R)-cadinane-2,10,15-triol.

The molecular formula of 23 was determined to be C15H26O3 according to HRESIMS and 13C NMR data. Analysis of its 1H and 13C NMR data (Tables 4, 5) indicated that 23 possessed a resemble structural relationship to 3β-hydroxyepicubenol [13], except for one oxygenated methine (δC 71.3, δH 3.00) instead of a methylene in 23. 1H–1H COSY correlations from H-9 (δH 3.00) to H-8 (δH 1.67, 0.91) and H-10 (δH 1.23) supported that an additional hydroxy was located at C-9. This conclusion was confirmed by HMBC correlations between H-8 (δH 1.67, 0.91), H-10 (δH 1.23) and C-9 (δC 71.3) (Fig. 2A). The relative configuration of 23 was established from detailed analysis of NOE correlations. NOE interactions from H-9 (δH 3.00) to H-7 (δH 1.16) and H3-14 (δH 0.96), from H-6 (δH 1.56) to H-10 (δH 1.23) revealed that H-7, H-9, and H3-14 were α-orientation, while H-6 and H-10 were β-orientation (Fig. 3). Although there was no obvious NOE correlation to demonstrate the configuration of 1-OH and 3-OH, comparing the 13C NMR data of C-1 (δC 74.6), C-2 (δC 34.3), C-3 (δC 68.0), C-4 (δC 136.4), C-5 (δC 124.2), and C-6 (δC 47.4) of 23 with those of the analogues indicated that 23 possessed the same configurations of C-1 and C-3 as 3β-hydroxyepicubenol [13] and muurol-4-ene-1β,3β,10β-triol [30]. The absolute configurations of the chiral carbons were determined to be 1R,3S,6R,7S,9S,10R according to the experimental ECD spectrum of 23 closely matched the calculated ECD curve (Fig. 4F).

Compound 25 had a molecular formula of C15H28O3 from its HRESIMS and 13C NMR data. The characteristic 1H and 13C NMR data (Tables 4, 5) of 25 demonstrated an oplopanane sequiterpenoid and related to the known oplopanane-4,10α-diol [31]. The difference was a 2-hydroxypropan-2-yl (δC 72.4, 31.9, 24.5, δH 1.09, s, δH 1.02, s) in 25 at C-7 replaced an isopropyl, which was confirmed by HMBC correlations between H3-12 (δH 1.02) and C-7 (δC 55.5), C-11 (δC 72.4), between H3-13 (δH 1.09) and C-7 (δC 55.5), C-11 (δC 72.4). The large coupling constants of H-5 (δH 2.00, brt, J = 9.0 Hz), H-6 (δH 1.37, td, J = 10.9, 8.0 Hz), and H-7 (δH 1.18, ddd, J = 13.3, 10.5, 3.0 Hz) indicated the both trans configurations of H-5/H-6, H-6/H-7. In addition, the trans configuration of H-1/H-6 also could be deduced from the peak shape and coupling constants of H-6 (δH 1.37, td, J = 10.9, 8.0 Hz). The relative configuration of 25 was further determined by NOE correlations observed from H-5 (δH 2.00) to H-7 (δH 1.18) and H-1 (δH 1.26), from H-6 (δH 1.37) to H3-14 (δH 0.98) and H-4 (δH 4.23) (Fig. 3). Furthermore, comparing the 13C NMR data of C-14 (δC 20.6) with that of oplopanone (δC 20.3) or 10-epi-oplopanone (δC 28.2) further confirmed above inference [31, 32]. Thus, the structure of 25 was identified as 4,10α,11-oplopananetriol and the configuration of C-4 undetermined as the less amount.

The molecular formula of compound 26 was determined as C15H28O3 based on HRESIMS and 13C NMR data. The 1H NMR data (Table 5) presented one oxygenated methine (δH 3.50), three singlet methyls (δH 1.22, 1.16, 1.05), one doublet methyl (δH 1.04), and three active hydrogens (δH 4.26, 4.24, 3.95). The 13C NMR data (Table 4) of 26 displayed 15 carbon signals, which were assigned to three oxygen-bearing carbons (δC 78.8, 72.9, 67.1), four methines (δC 52.0, 36.3, 23.8, 23.1), four methylenes (δC 39.4, 37.2, 27.6, 26.2), and four methyls (δC 30.6, 26.5, 26.4, 24.2) with the aid of HSQC experiment. The obviously upfield of H-6 (δH 0.13, dt, J = 10.3, 3.4 Hz) and H-7 (δH 0.57, ddd, J = 10.0, 8.0, 3.5 Hz) hinted 26 was a pallenane and related to 3β,4β-dihydroxypallenone [33, 34]. The C5/C3 bicyclic skeleton of 26 was established by 1H–1H COSY correlations between H-1/H-2/H-3, between H-1/H-6, H-1/H-5, and H-5/H-6. Furthermore, the 1H–1H COSY correlations from H-6/H-7/H-8/H-9/H-10/H3-14 and H-10/10-OH revealed a 4-hydroxypentyl were connected with C-6. HMBC correlations from H-12 (δH 1.05) to C-7 (δC 52.0), C-11 (δC 72.9), from H-13 (δH 1.16) to C-7 (δC 52.0), C-11 (δC 72.9) elucidated a 2-hydroxypropan-2-yl was connected with C-7. Moreover, HMBC correlations from H-3 (δH 1.34) to C-4 (δC 78.8), C-5 (δC 36.3), from H-6 (δH 0.13) to C-2 (δC 26.2) and C-4 (δC 78.8), from H-7 (δH 0.57) to C-6 (δC 23.1), C-8 (δC 27.6), C-9 (δC 39.4), and C-11 (δC 72.9), from H3-15 (δH 1.22) to C-3 (δC 37.2), C-4 (δC 78.8), C-5 (δC 36.3) further confirmed the planar structure (Fig. 2A). Comparing the 1H NMR data of H-5 (δH 1.13, dd, J = 6.0, 3.6 Hz), H-6 (δH 0.13, dt, J = 10.3, 3.4 Hz), and H-7 (δH 0.57, ddd, J = 10.0, 8.0, 3.5 Hz) with those of 3β,4β-dihydroxypallenone [33, 34] indicated 26 possessed the same H-1/H-5 cis, H-1/H-6 trans, H-5/H-6 trans configurations. This conclusion was confirmed by NOE correlations observed from H-7 (δH 0.57) to H-1 (δH 1.04) and H-5 (δH 1.13) (Fig. 3). The methyl at C-4 was determined as β deduced from NOE correlations observed from H-6 (δH 0.13) to H3-15 (δH 1.22), from H-5 (δH 1.13) to 4-OH (δH 4.24). As there were potential inaccuracies of NOE correlations as the overlapped signals of H-1 and H3-14. 1D and 2D NMR spectra of 26 were remeasured in pyridine-d5 to get the distinct signals of H-1, H-5, H-6 and analysis of NOE correlations (in pyridine-d5) further confirmed the configurations (Table S1, Additional file 1). Due to the configuration of methyl at C-4 differed from the congeners, 26 was named as 4-epi-pallenane-4α,10,11-triol.

The characteristic 1H and 13C NMR data (Tables 4, 5) of 27 showed it was also a pallenane sesquiterpenoid. The NMR data of 27 were similar to those of 26, except for the presence of one methine (δC 35.0) instead of one oxygenated quaternary carbon as well as a doublet methyl replaced a singlet methyl. 1H–1H COSY correlations from H-4 (δH 2.15) to H3-15 (δH 0.99) combining with HMBC correlations between H3-15 (δH 0.99) and C-3 (δC 30.2), C-4 (δC 35.0), C-5 (δC 30.1) confirmed the methyl was located at C-4. (Fig. 2A). The similar 1H NMR data of H-5 (δH 1.12, dt, J = 6.3, 3.5 Hz), H-6 (δH 0.27, dt, J = 10.3, 3.3 Hz), and H-7 (δH 0.56, ddd, J = 10.6, 7.8, 3.5 Hz) with those of 26 suggested they possessed the identical configurations. In NOESY spectrum, the key cross peaks from H-6 (δH 0.27) to H3-15 (δH 0.99), from H-4 (δH 2.15) to H-5 (δH 1.12), from H-7 (δH 0.56) to H-1 (δH 0.97) and H-5 (δH 1.12) further indicated that H-1, H-4, H-5, and H-7 were α-oriented, while H-6, and H3-15 were β-oriented (Fig. 3). Finally, the structure of compound 27 was confirmed. Pallenane is a kind of rarely reported sesquiterpene with a distinctive C5/C3 bicyclic skeleton. Till now, only two pallenane congeners (3β,4β-dihydroxypallenone and 3β-acetoxy-4β-hydroxypallenone) were found from the plant Pallenis spinosa [33, 34]. The current compounds 26 and 27 were firstly obtained from streptomycete. According to the literature [33, 34], the isodaucane (17), oplopanane (25), and pallenane (26, 27) skeleton metabolites were derived from the pinacol-type cadinane glycols through Wagner rearrangement in organisms (Fig. 2B). The 2-hydroxypropan-2-yl groups at C-7 in 26 and 27 were suggested as β-orientation according to the biogenetic way.

The characteristic 1H and 13C NMR signals implied that 28 was an eudesmane-type sesquiterpene and related to eudesmane-1β,6α,11-triol (29) [10]. The alterations were one more oxygenated methine replaced a methylene and a double bond replaced two methines. The HMBC correlations from H-4 (δH 2.39), H-7 (δH 2.02) to C-5 (δC 145.9) and H-4 (δH 2.39), H-7 (δH 2.02) to C-6 (δC 126.6) indicated double bond was located at C-5 and C-6, from 3-OH (δH 4.50) to C-2 (δC 35.6) and C-3 (δC 68.7) demonstrated an extra hydroxy was connected with C-3 (Fig. 2A). The relative configuration was determined by analysis of coupling constants and NOE correlations. The large coupling constants of H-1 (δH 3.03, dt, J = 11.6, 4.8 Hz), H-3 (δH 3.52, m) and H-7 (δH 2.02, ddd, J = 11.0, 6.2, 2.0 Hz) suggested that H-1, H-3, H-7 were axial orientation. While the peak shape and coupling constants of H-4 (δH 2.39, quint, J = 7.0 Hz) suggested H-4 was equatorial orientation. NOE interactions from H-3 (δH 3.52) to H-4 (δH 2.39), H-1 (δH 3.03), from H-6 (δH 5.53) to H-4 (δH 2.39), H-7 (δH 2.02), from H3-14 (δH 0.95) to 1-OH (δH 4.45) demonstrated that H-1, H-3, H-4, and H-7 were α-oriented, while 1-OH, 3-OH, H3-14, and H3-15 were β-oriented (Fig. 3). The configurations of chiral carbons of 28 were identified as 1R,3S,4R,7R,10R by comparing the experimental ECD spectrum of 28 with calculated ECD spectrum (Fig.