3. Results and Discussion
Bipolarisorokin A (
1) was isolated as colorless crystals. Its molecular formula of C
15H
24O
3 was determined on the basis of the HR-ESIMS data (measured at
m/z 251.16624 [M–H]
−, calculated for C
15H
23O
3− 251.16527), corresponding to four degrees of unsaturation. The
1H and
13C NMR spectra, in association with the HSQC spectrum, revealed two methyls, four methenes, seven methines, and two quaternary carbons (
Table 1). Of them, signals at
δC 66.9 (t, C-11), 69.6 (d, C-14), and 74.9 (d, C-15) were identified as the oxygenated methylene and methines. Two olefinic carbons at
δC 156.8 (s, C-2) and 103.5 (t, C-12) corresponded to a double bond, which suggested that
1 possessed a tricyclic system. Considering the 15 carbons in
1, as well as those isolates from the same source, compound
1 was suggested to be a tricyclic sesquiterpenoid. In the
1H–
1H COSY spectrum, a fragment was revealed, as shown with bold lines in
Figure 2. The HMBC correlations from to
δH 4.94 (H, s, H-12a) and 4.62 (H, s, H-12b), to
δC 156.8 (s, C-2), 54.3 (d, C-1) and 43.0 (s, C-3), established the connections between C-12, C-2, and C-1. Further analyses of
1H–
1H COSY, as well as HMBC correlations from
δH 0.92 (3H, d,
J = 6.8 Hz, H-10) to
δC 37.6 (d, C-6), 40.5 (d, C-9) and 66.9 (t, C-11), indicated a hydroxy group at C-11. In addition, the connections of C-8/C-3, C-3/C-4, C-3/C-2, and C-3/C-13 were deduced from HMBC correlations from
δH 1.05 (3H, s, H-8) to
δC 43.0 (s, C-3), 39.9 (t, C-4), 156.8 (s, C-2), and 58.2 (d, C-13). Moreover, the proton of an oxygenated methine at
δH 4.02 (H, d,
J = 5.9 Hz, H-14) showed key correlations to C-13, C-3, and
δC 42.2 (d, C-7), which indicated that
δC 69.6 (d, C-14) should be placed at C-13. The above 2D NMR data analysis suggested that compound
1 possessed a sativene type sesquiterpene backbone. A ROESY experiment was carried out to establish the relative configuration of
1 (
Figure 3). The key correlations of H-13/H-8, H-13/H-6, H-8/H-14, and H-7/H-13 suggested that H-6, H-7, H-8, and H-13 were
β oriented, while the correlation of H-1/H-9 indicated that H-1 and H-9 were
α-oriented. Because of the rigid structure and the ROESY correlation of H-8/H-14, both H-14 and H-15 were assigned as an
α orientation [
31]. Finally, the single-crystal X-ray diffraction not only confirmed the planar structure, as elucidated above, but also established the absolute configuration of
1 (Flack parameter = −0.10(7), CCDC: 2124305;
Figure 4).
The molecular formula of bipolarisorokin B (
2) was determined to be C
15H
24O
3 from the HRESIMS data (measured at
m/z 275.16166 [M+Na]
+, calculated for C
15H
24O
3Na
+ 275.16177). Close similarities were observed in the 1D NMR data (
Table 1) of compound
1. However, signals for a methyl (
δH 0.94, d,
J = 6.9 Hz, H-11;
δC 16.4, C-11) and an oxygenated quaternary carbon (
δC 73.7, C-6) in
2 was suggested to replace the oxymethylene (
δH 3.64, overlap, H-11;
δC 66.9, C-11) and the methine (
δH 1.65, m, H-6;
δC 37.6, C-6) in
1. These observations indicated that the hydroxy group at C-10 in
1 migrated to C-6 in
2. The observed
1H−
1H COSY cross-peak of H-10 (
δH 0.88, 3H, d,
J = 6.9 Hz) and H-9 (
δH 1.57, 1H, m), and H-9/H-11, along with the HMBC correlations from H-10 to C-6, C-9, and C-11 confirmed the above deduction (
Figure 2). Furthermore, ROESY correlations of H-13/H-8, H-8/H-14, H-7/H-13, and H-1/H-9 revealed that compounds
2 and
1 shared the same relative configuration. In consideration of its biosynthetic origin, the absolute configuration of compound
2 was identified the same as that of
1.
Bipolarisorokin C (
3) was obtained as colorless needles. Its molecular formula of C
15H
24O
3 was determined on the basis of the HR-ESIMS data (measured at
m/z 253.17971 [M+H]
+, calculated for C
15H
25O
3+ 253.17982), corresponding to four degrees of unsaturation. The
1H NMR data (
Table 1) showed characteristic signals, including three methyls at
δH 0.78 (3H, d,
J = 6.4 Hz, H-10), 1.06 (3H, d,
J = 6.4 Hz, H-11), and 2.13 (3H, s, H-12), and the proton of an aldehyde group at
δH 10.02 (H, s, H-15). The
1H and
13C NMR data, in association with the HSQC data, revealed three methyls, four methenes, five methines, and three nonprotonated carbons (
Table 1). Preliminary analyses on the 1D NMR data revealed that
3 was likely to be a seco-sativene type sesquiterpenoid. Detailed analyses of the 2D NMR data indicated that the majority of the data of
3 was the same as those of helminthosporol [
32], except for a hydroxy group at C-8 (t,
δC 64.6) in
3, which was confirmed by the HMBC correlations from
δH 3.63 (H, d,
J = 11.6 Hz, H-8a) and 3.71 (H, d,
J = 11.6 Hz, H-8b) to
δC 58.0 (s, C-3), 29.6 (t, C-4), 167.0 (s, C-2), and 60.8 (d, C-13) (
Figure 2). A ROESY experiment was carried out to establish the relative configuration of
3 (
Figure 3). The cross peaks of H-13/H-8a, H-13/H-4b, H-4b/H-6, and H-7/H-14b were observed, which indicated that H-6, H-7, H-8, and H-13 were
β oriented. Furthermore, single crystal X-ray diffraction established the relative configuration (Flack parameter = −0.40(17), CCDC: 2124306;
Figure 4), and the absolute configuration of
3 was determined by ECD calculations, as shown in
Figure 5.
Bipolarisorokin D (
4) was isolated as a colorless oil. The molecular formula was determined to be C
15H
24O
3 according to the HRESIMS spectra (measured at
m/z 275.16153 [M+Na]
+, calculated for C
15H
24NaO
3+ 275.16177). Compound
4 had the same molecular formula and NMR spectral patterns to that of
3 (
Table 2). The key difference was an oxygenated quaternary carbon (
δC 73.5, s) in
4 instead of the methine in
3 (
δC 46.4, d). The HMBC correlations from H-4a (
δH 1.38, m), H-5b (
δH 1.61, m), H-7 (
δH 3.16, br s), H-10 (
δH 1.02, d,
J = 6.6 Hz), and H-11 (
δH 0.80, d,
J = 6.6 Hz) to
δC 73.5 established the quaternary carbon to be C-6. In addition, a methyl (s,
δH 1.07, H-8;
δC 18.7, C-8) in
4 replaced the oxygenated methylene (
δC 64.6) of C-8 in
3, which was verified by HMBC correlations from H-8 (
δH 1.07, s) to C-2 (
δC 170.4, s), C-3 (
δC 52.0, s), C-4 (
δC 32.4, t), and C-13 (
δC 55.3, d). Detailed analyses of 2D NMR (HSQC, HMBC,
1H-
1H COSY and ROESY) data confirmed that the other fragments of
4 were the same as those of
3.
Bipolarisorokin E (
5) was obtained as a colorless oil. Its molecular formula C
14H
22O
2 was characterized according to HRESIMS (measured at
m/z 221.15529 [M–H]
-, calculated for C
14H
21O
2− 221.15470), implying four degrees of unsaturation. The general features of its NMR data closely resembled that of
3 (
Table 2). Detailed analyses of 1D and 2D NMR data revealed the differences. At first, the loss of the aldehyde group at C-1 was revealed by the chemical shift of C-1 at
δC 124.2, along with the data from
1H–
1H COSY and HMBC spectra as shown in
Figure 2. Second, the hydroxy migrated from C-8 to C-12 (
δC 59.8, t) as identified by the HMBC correlation from
δH 4.06 (2H, m, H-12) to
δC 124.2 (d, C-1), 147.2 (s, C-2), and 47.7 (s, C-3). Third, one double bond between C-9 and C-10 was built by HMBC correlations from
δH 4.69 (2H, d,
J = 5.1 Hz, H-10) to
δC 22.7 (q, C-11) and 45.2 (d, C-6). The other parts of
5 were elucidated as the same as those of
3 by a detailed analysis of 2D NMR data.
Bipolarisorokin F (
6) was purified as white powder, and its molecular formula C
14H
24O
2 was determinded according to HRESIMS (measured at
m/z 225.18506 [M+H]
+, calculated for C
14H
25O
2+ 225.18491). Analyses of the 1D and 2D NMR data (
Table 2) suggested that
6 showed structural similarities to
3. The distinction between the two compounds was that the
α,
β-unsaturated aldehyde group (
δC 140.0, C-1;
δC 167.0, C-2;
δC 190.0, C-15) in
3 was replaced by a carbonyl (
δC 212.0, C-1) and a methylene group (
δC 50.7, C-2) in
6. It was supported by HMBC correlations from
δH 2.70 (H, br s, H-7), 0.96 (3H, q,
J = 7.2 Hz, H-12), and 1.72 (H, dd,
J = 7.9, 5.0 Hz, H-13) to
δC 212.0 (s, C-1), 50.7(d, C-2), and the COSY cross-peak of
δH 2.10 (1H, m, H-2) and H-12. The hydroxymethyl group (C-8) in
3 was replaced by a methyl group at C-8 (
δC 22.1, q) in
6, as well as the HMBC correlations from
δH 1.09 (3H, s, H-8) to C-2,
δC 41.8 (s, C-3),
δC 36.1 (t, C-4), and
δC 54.9 (d, C-13). The key ROESY cross-peak (
Figure 3) of H-2/Ha-14 (H, dd,
J = 10.7, 5.0 Hz,
δH 3.85) suggested that H-2 was
β oriented. Other ROESY data revealed the same patterns to
3. Finally, regarding the same origin of
6 and
3, the absolute configuration of
6 was identified to be the same as that of
3, as depicted.
The molecular formula of bipolarisorokin G (
7) was assigned as C
18H
26O
2 based on its HRESIMS spectrum (measured at
m/z 275.20059 [M+H]
+, calculated for C
18H
27O
2+, 275.20056), which contained three more carbon atoms than
3. The interpretation of the
1H and
13C NMR data of
7 (
Table 3) indicated the same structure skeleton to that of
3. Analyses of 2D NMR spectra revealed modifications in
7 (
Figure 2). HMBC correlations from
δH 0.97 (3H, s, H-8) to
δC 165.3 (s, C-2), 52.6 (s, C-3), 33.7 (t, C-4), and 63.6 (d, C-13) suggested that a hydroxy group was missing in
7. In addition, an
α,
β-unsaturated ketone group was identified by the HMBC correlations from
δH 6.55 (H, dd,
J = 15.9, 9.6 Hz, H-14), 6.08 (H, d,
J = 15.9 Hz, H-16), and 2.20 (3H, s, H-18) to
δC 198.6 (s, C-17). In the
1H−
1H COSY spectrum, correlations from H-14 to
δH 2.22 (H, d,
J = 9.6 Hz, H-13) and H-16 indicated that the
α,
β-unsaturated carboxyl moiety was located at C-13. Finally, the absolute configuration of
7 can be fully resolved by the ECD calculation, as shown in
Figure 5.
Bipolarisorokin H (
8) was obtained as a colorless oil. Its molecular formula, C
17H
24O
3, was inferred from the pseudomolecular ion peak at m/z 277.17984 [M+H]
+ in the HRESIMS (calculated for C
17H
25O
3+ 277.17982). The NMR data of
8 (
Table 3) resembled that of
7, except for the presence of a carboxyl (
δC 171.1, C-17) in
8 instead of a carbonyl (
δC 198.6, C-17) in
7, as well as the loss of a methyl group. This was supported by HMBC correlations from
δH 6.80 (H, dd,
J = 15.4, 9.9 Hz, H-14) and 5.81 (H, d,
J = 15.5 Hz, H-16) to
δC 171.1 (s, C-17). Detailed analyses of 2D NMR data suggested that the other data were the same as those of
7.
Bipolarisorokin I (
9) was isolated as colorless crystals. Its molecular formula was identified as C
15H
24O
3 by HRESIMS (measured at
m/z 251.16621 [M–H]
−, calculated for C
21H
23O
3− 251.16527). All the spectroscopic data indicated similar patterns to those of longifolene [
33]. Detailed analyses of 1D and 2D NMR data revealed the differences. Signals at
δC 67.0 (d, C-5), 70.5 (d, C-14), and 74.9 (d, C-15) were identified as the oxygenated methines. Therefore, three hydroxyls were suggested to be placed at C-5, C-14, and C-15, respectively, which were identified by the HMBC and
1H–
1H COSY correlations, as shown in
Figure 2. Comprehensive analyses of other data suggested that the other parts of
9 were the same as those of longifolene. The relative configuration of
9 was revealed by a ROESY experiment, as shown in
Figure 3. The ROESY correlations of Me-10/H-13, H-13/H-5, Me-8/H-13, Me-10/H-9, and Me-10/H-5 indicated these groups were cofacial (assigned as
β orientation). In addition, the Me-11/H-1 interaction suggested that H-1 should be
α oriented. Moreover, the coupling constant between H-14 and H-15 (
J14,15 = 6.2 Hz), as well as the ROESY correlations of Me-8/H-14 and Me-8/H-15, suggested that H-14 and H-15 were
α oriented. Finally, the single-crystal X-ray diffraction not only confirmed the planar structure but also established the absolute configuration of
9 (Flack parameter =0.01(3), CCDC: 2124307;
Figure 4).
Bipolarithone A (
10) was isolated as a yellow oil, and its molecular formula was determined to be C
17H
16O
8 by HRESIMS (measured at
m/z 349.09143 [M+H]
+, calculated for C
17H
17O
8+ 349.09179). The NMR data (
Table 4) of
10 were similar to those of the dechlorinated methyl ester (
16) isolated in this study [
34]. The major difference was that
10 exhibited a dihydrofuran ring rather than a furan ring. HMBC correlations from H-8 (H, d,
J = 3.9 Hz,
δH 5.64) to C-8a (
δC 114.7, s), C-7 (
δC 170.0, s), C-9 (
δC 178.3, s), and C-10a (
δC 167.7, s), together with H-5 (H, ddd,
J = 6.6, 4.4, 3.9 Hz,
δH 5.73) to C-10a, C-8a, C-6 (
δC 37.7, t), and C-2′ (
δC 169.5, s), supported the above assignment. The relative configuration of
10 was identified by the analysis of its ROESY data. The ROESY correlation between H-8 and H-5 indicated that H-8 had the same orientation as H-5 (assigned as an
α orientation). The calculated ECD of
10 established the configuration of
10, as shown in
Figure 5. Therefore, the structure of
10 was characterized as depicted.
Bipolarithone B (
11) was isolated as a yellow oil. The HRESIMS spectrum of
11 suggested a molecular formula of C
17H
16O
8 (measured at
m/z 349.09157 [M+H]
+, calculated for C
17H
17O
8+ 349.09179), the same as that of
10. The planar structure of
11 was elucidated to be the same as that of
10 by the analysis of its 1D and 2D NMR data. The main difference was suggested as its stereochemistry at C-8 (
δC 79.8, d). Analyses of the
1H NMR information showed that the coupling constants of H-8, H-5, and H-6 were significantly different from those of
11, as shown in the
Table 4. Furthermore, the ROESY correlation of H-8 (
δH 5.63, 1H, d,
J = 1.7 Hz)/H-5 (
δH 5.62, 1H, ddd,
J = 8.4, 3.8, 1.7 Hz) was not observed in
11. These data suggested that
11 was an epimer of
10. The ECD calculation for
11 was performed, and the results of
11 matched well with the experimental ECD curve (
Figure 5). Hence, the absolute configuration of
11 can be fully assigned, as shown.
Bipolarithone C (
12) was assigned a molecular formula of C
30H
36O
9 based on its HRESIMS data (measured at
m/z 541.24310 [M+H]
+, calculated for C
30H
37O
9+ 541.24321). The NMR data of
12 were very similar to those of bipolenin I (
14) (
Table 5), a novel sesquiterpenoid-xanthone adduct isolated from the fungus
Bipolaris eleusines [
35]. The significant differences were that there was an absence of an aldehyde group and two olefinic carbons, as well as the presence of an additional methine and carbonyl, in
12. These data suggested that the
α,
β-unsaturated aldehyde moiety disappeared in
12. This assignment was confirmed by the HMBC correlations of
δH 2.16 (H, m, H-2), 1.29 (H, m, H-6), 2.56 (1H, br s, H-7), 0.95 (3H, d,
J = 7.2 Hz, C-12), and 1.90 (H, m, H-13) to
δC 50.6 (d, C-2) and 221.6 (s, C-1). The ROESY spectrum displayed similar patterns to those of
14. Furthermore, a cross peak between H-2 and H-14a (
δH 4.05, 1H, dd,
J = 11.3, 5.1 Hz) confirmed the relative configuration of C-2, as shown. The absolute configuration of
12 was elucidated by the quantum chemistry calculations. At first, the ECD calculations were conducted on the four possible conformers (
12a–d), using time-dependent density functional theory (TDDFT) at the B3LYP/6-311G (d) level in methanol with the PCM model. The overall calculated ECD spectrum of each configuration was then generated according to the Boltzmann weighting of the conformers. As a result, the calculated ECD curves of
12a and
12d matched well with the experimental data (
Figure 5). To determine its final structure, the theoretical NMR calculations and DP4+ probabilities were employed. The
13C NMR chemical shifts of
12a and
12d were calculated at the mPW1PW91/6-311+G (d,p) level in the gas phase. According to the DP4+ probability analyses,
12a was assigned with 100% probability (see data in the
Supporting Information). Structurally, compound
12 comprised of a seco-sativene sesquiterpenoid unit and a xanthone unit, whose absolute configurations were in accord with compound
6 and compound
10, respectively. Therefore, the structure of
12 was established as depicted.
Bipolarithone D (
13) had the same molecular formula (C
30H
36O
9) as that of
12, according to their HRESIMS spectra (measured at
m/z 541.24316 [M + H]
+, calculated for C
30H
37O
9+ 541.24321). The NMR resonances for
13 (
Table 5) resembled those of
12, except that the resonances of C-6′ (
ΔδC + 1.5), H-6′a (
ΔδH + 0.08), and H-6′b (
ΔδH + 0.15) were shifted downfield, while the data H-5′ (
ΔδH − 0.08) were shifted upfield. A detailed comparison of the 1D and 2D NMR data of
13 with that of
12 indicated that the two compounds possessed the same planar structure. The main difference was the stereochemistry at C-8′. A key ROESY correlation of H-5′/H-8′ could be detected in
12 but not in
13. In addition, the coupling constants of H-8′ in
13 (
J = 1.8 Hz) were different from that in
12 (
J = 3.9 Hz). All the data suggested that compound
13 was a C-8′ epimer of
12. Finally, the absolute configuration of
13 was confirmed by ECD calculations (
Figure 5).
Five known compounds were determined as bipolenins I and J (
14 and
15), dechlorinated methyl ester (
16), drechslerines A (
17), and (+)-secolongifolene diol (
18) by the comparison of their spectral data with that reported in the literature [
32,
34,
35]. In this study, the absolute configurations of compounds
17 and
18 were confirmed by single crystal X-ray diffractions (
Figure 6), which could support the absolute configurations of
1–
9,
12, and
13 as depicted in the text, since they were obtained from the same source.
All compounds (
1–
18) were evaluated for their anti-Psa activity. As a result, compounds
10 and
15 showed significant inhibitory activity, with MICs of 64 and 16 μg/mL, respectively, while compounds
7,
11,
13, and
16 showed moderate activity, with MICs of 128 μg/mL (
Table 6).
In addition, our previous study on chemicals from
B. eleusines suggested that sativene-xanthone adducts have promising inhibitory activity against plant pathogenic microorganisms [
35]. Therefore, all compounds were evaluated for their inhibitory activity against four plant pathogenic microorganisms, including
P.
infestane,
A.
solani,
R.
solani, and
F.
oxysporum. As a result, many compounds showed certain inhibitory activity, as given in
Table 6.
A brief structure–activity relationship analysis suggested that the aldehyde-containing sativene sesquiterpenoids were more active than the others, while the xanthones or their derivatives showed better inhibitory activities than sativene sesquiterpenoids.