The dried aerial parts of the plant were defatted and extracted with methanol (MeOH). This solvent was evaporated and the residue was extracted with EtOAc, then extracted with BuOH. The BuOH extract was separated by column chromatography on silica gel. Radial chromatography and HPLC yielded twenty pure compounds (Fig. 1). Their structures were elucidated using 1D and 2D nuclear magnetic resonance (NMR) and high-resolution electrospray ionisation mass spectrometry (HRESIMS) data. Their absolute configurations cannot be confirmed from these data but are assumed on the basis of precedent for related compounds.9,25,26
Compound 1. HRESIMS showed pseudomolecular ions at m/z 457 [M + K]+, m/z 441.1509 [M + Na]+ (calc 441.1525) and m/z 419.1690 [M + H]+ (calc 419.1706), for the formula C22H26O8. An ion was also observed at m/z 401 [M + H - H2O]+, indicating a hydroxy group. Negative pseudomolecular ions were detected at m/z 463.1605 [M + formate]− (calc 463.1605) and m/z 453.1316 [M + 35Cl]− (calc 453.1316). The 13C NMR spectrum (Table S1, Supplementary Information (SI)) showed 22 discrete resonances: 2 ⋅ CH3, 6 ⋅ CH2, 7 ⋅ CH, 7 ⋅ Cq. The core structure was shown to be a decalin and related to the neoclerodane diterpenoids.9,15,16,25 The infra-red (IR) spectrum showed an OH (3536 cm− 1) and one γ-lactone carbonyl peak (1761 cm− 1).
In the upper part of the structure of 1 (Figure 1), the aromatic furan was characterised by 1H NMR signals at δ 6.47 (H-14), δ 7.54 (H-15) and δ 7.61 (H-16) (Table S1, SI). A 1H-1H correlation spectrum (COSY) cross-peak linked δ 6.47 (H-14) and δ 7.54 (H-15). Heteronuclear single quantum (HSQC) correlation linked the 1H signals to 13C signals at δ 109.1 (C-14), δ 145.6 (C-15) and δ 141.80 (C-16); the signal for C-13 (δ 126.3) was identified by a strong 3-bond heteronuclear multi-bond (HMBC) correlation to H-15 and weaker 2-bond correlations to H-14 and H-16. These signals match well with those observed previously for the furans in fatimanol B, fatimanol D and fatimanol E.15 The spiro-lactone was identified through the chemical shift of H12 (δ 5.46) (cf. corresponding signals in fatimanol B (δ 5.51) and fatimanol D (δ 5.46)1. HMBC tied this proton signal to each of the furan 13C signals. COSY correlation linked this H-12 signal to the doublet signal at δ 2.49 (2 H) and a 2-bond HMBC correlation confirmed that C-11 resonated at δ 41. Although the two H-11 protons are formally diastereotopic, they are coincident for fatimanol D15 and for 1 (δ 2.49). Curiously, no 3-bond HMBC cross-peaks were seen linking H-12 to the lactone carbonyl (20-C, δ 179.27) or the spiro-carbon (9-C, δ 49.06), although examination of the MM2-minimised conformation indicated that the corresponding dihedral angles (H-12)-(C-12)-(O)-(C-9) and (H-12)-(C-12)-(C-11)-(C-9) are very close to 90°, the coupling constant minimum in the Karplus relationship. This model also suggested a rigid trans-decalin conformation for the lower part of 1. This conformational and configurational assignment was supported by H-10 resonating as a broad doublet at δ 1.89 with 3J = 11.2 Hz corresponding to a trans-diaxial coupling with H-1ax. A nuclear Overhauser effect correlation spectroscopy (NOESY) experiment ((CD3)2SO solvent) showed a cross-peak between H-18endo (δ 3.69 (δ 3.87 in CD3OD)) and H-11 (δ 2.49); this demonstrated that C-18 (δ 58.3 in CD3OD) is axial. H-3 was identified by its chemical shift (δ 4.25) and by HMBC correlations to C-1 (δ 29) and C-4 (δ 87.1). The corresponding C-3 (δ 71.4) correlated by HSQC to H-3 and by HMBC to both 1-H (δ 1.29 and δ 1.60) and to both 2-H (δ 1.60 and δ 2.20). H-3 had a trans-diaxial coupling with H-2ax (3J = 10.9 Hz) and was thus axial, making 3-OH equatorial and confirming the conformation of ring A as chair. A strong NOESY correlation between 3-H and H-19exo (δ 3.91) showed that CH2-19 was close in space to H-3 and also axial. With H-10 and CH2-19 both axial, the decalin must be trans-fused. CH3-17 resonated as expected as a doublet at δ 1.08 (1H) and δ 16.1 (13C), linked by a HSQC cross-peak. Strong 3-bond HMBC cross-peaks from H3-17 to C-9 (δ 49.1) and to C-7 (δ 35.2) and from C17 to H-7ax (δ 2.27) confirmed the location of this methyl group. The 1H signal for H-7ax was a dd (2J(H7ax)−(H-7eq) = 14.1 Hz, 2J(H-7ax)−(H-8) = 12.9 Hz), showing that H-8 is axial and, therefore, CH317 is equatorial. The NOESY experiment ((CD3)2SO) showed a cross-peak between H3-20 and the H-11 resonance at δ 2.36 (δ 2.49 in CD3OD), which is only possible if CH3-17 is equatorial.
The orthoacetate unit of 1 was more challenging to identify. The methyl protons gave a singlet at δ 1.43, which is inappropriate for an acetate ester, with the 13CH3 signal at δ 23.9. 2-Bond HMBC linked this CH3 to the orthoester carbon signal at δ 107.7 / 107.8, which is inappropriate for an ester carbonyl. Thus this 2-carbon unit was not a conventional acetate ester, which was consistent with no loss of 60 Da (HOAc) in the MS fragmentation. The 1H NMR spectrum in (CD3)2SO showed only one OH resonance (HO-3, δ 5.23, with COSY and NOESY correlations with H-3); thus the oxygens in the lower part of the structure must be ethers. H-18endo (δ 3.87) and H-18exo both formed HMBC cross-peaks with the 13C signal(s) at δ 107.7 / 107.8. Thus one of these signals must have been due to the orthoester carbon (four bonds from H2-18) and the other due to acetal carbon C-5 (three bonds from H2-18). This confirmed the ring-closure of the (C-6)ࣧ(C-5)ࣧ(C-4)ࣧ(C-18)ࣧ(O) tetrahydrofuran. C-4 (δ 87.1), C-18 (δ 58.3) and C-19 (δ 74.6) all carry an oxygen as shown by their 13C chemical shifts. These data are consistent with the orthoacetate structure shown in Fig. 1 for 1 (fatimanol F, a novel compound). Analogue 21, which contains the acetal and orthoacetate structures but only differs from 1 in that it lacks HO-3, was reported27 as a product of the high-temperature pyrolysis of 19-acetylgnaphalin.
Compound 2. HRESIMS showed a pseudomolecular ion at m/z 421.1855 [M + H]+ (calc 421.1862) for the formula C22H28O8. Fragment ions were seen at m/z 379 [M + H - ketene]+ and m/z 361 [M + H - AcOH]+, showing an acetate ester. Twenty-two discrete 13C NMR signals were observed (2 ⋅ CH3, 6 ⋅ CH2, 8 ⋅ CH, 6 ⋅ Cq (ester and ketone). IR confirmed these carbonyls, with bands at 1712 cm− 1 and 1796 cm− 1, respectively. A hydroxy group absorbed at 3478 cm− 1.
The NMR data (Table S1, SI) showed that 2 had a neoclerodane structure. The aromatic furan was shown by 1H NMR signals at δ 6.41 (H-14), δ 7.43 (H-15) and δ 7.42 (H-16), with HSQC correlations to C-14 (δ 108.8), C-15 (δ 143.8) and C-16 (δ 139.6), respectively. The C-13 signal (δ 124.8) was identified by HMBC correlations with H-14, H-15, and H16. C-13, C-14, and C-16 showed HMBC cross-peaks with a double doublet (dd) aliphatic proton signal at δ 5.25, which was assigned as H-12. HSQC correlated this signal with C-12 (δ 71.2). The corresponding signal for H-12 in the lactone 1 is downfield at δ 5.46, whereas H-12 in the alcohol 3 resonates upfield at δ 4.85. These comparisons suggest that the electron-density at H-12 in 2 is intermediate between that in the lactone 1 and the alcohol 3 and is consistent with the hemiacetal / lactol structure in 2. A strong 3-bond HMBC cross-peak linked C-12 with the hemiacetal H-20 singlet at δ 5.54. HSQC identified C-20 (δ 99.7). The five-membered lactol was completed by identification of both H-11 signals (δ 1.93 dd, δ 2.34 dd) by HBMC correlations with C20, location of C-12 (δ 44.2) by HSQC correlation with H-12, and characterisation of the quaternary spiro carbon C-9 (δ 53.6) by HMBC correlations with H-12 and H-20. COSY linked both H-11 and H-12. In the lower part, the hydroxy group was located at C-3 through the chemical shifts of H-3 (δ 4.11) and C-3 (δ 66.4). H-3 had been identified by HMBC correlation to C-5 (δ 63.6) and C-3 had been identified by HMBC correlation to H-1 (δ 2.22 and δ 2.64) and H-2 (δ 1.36 and δ 2.22). Observation of two geminally coupled doublets (H-18) at δ 2.81 and δ 3.14 (2J = 5.3 Hz) revealed the spiro-oxirane. This was shown to be at C-4 by HMBC from these H-18 protons to C-5 (δ 63.6) and from H-3 to C-18 (δ 43.7). C-6 was a ketone, as shown by its chemical shift (δ 206.2) and by HMBC cross-peaks to both H-7 signals (δ 2.28 and δ 2.72) and a 4-bond HMBC correlation with CH3-17. Examination of a model of 2 suggested that ring B was in a flattened-chair conformation, such that the dihedral angles (C6)ࣧ(C5)ࣧ(C-10)ࣧ(H-10) and (C-6)ࣧ(C-7)ࣧ(C-8)ࣧ(H-8) were close to 90°, explaining the lack of these 3-bond HMBC interactions. The acetoxymethyl (AcOCH2-) group was defined by CH32’ (δH 2.07, δC 21.1) and the ester carbonyl C-1’ (δ 171.2). HMBC cross-peaks between C1’ and the geminally coupled doublets for H2-19 (δ 4.72 and δ 4.83) confirmed the AcOCH2-; HMBC cross-peaks from these two protons to the ketone (C-6), to quaternary C-5 and to the spiro-oxirane carbon (C-4) demostrated that this unit was located at C-5. Rings A and B were shown to be in chair conformations by examining relevant 3J coupling constants. For example, the H-7ax signal (δ 2.72) was a broad triplet with 3J = 2J = 14.6 Hz, indicating diaxial and geminal couplings, respectively. H-7eq (δ 2.28) only showed axial-equatorial and geminal couplings. Thus Me-17 is equatorial and H-8 is axial and the chair conformation is confirmed. The boat conformation would have shown only eq-eq and eq-ax couplings for 3J(H−7)ࣧ(H−8).
The NMR spectra showed, in addition to the peaks for this major compound, a full set of peaks for a minor component, with similar chemical shifts and multiplicities. This minor set of peaks integrated for ca. 10% of the major compound present. As the sample gave only one pure peak on HPLC, we ascribed these peaks to a minor diastereoisomer in slow equilibrium with the major diastereoisomer. These are likely to be epimers at the lactol hemiacetal C-20. This assignment was supported by the largest differences in chemical shift between the epimers being for H-3 (Δδ 0.1 ppm), H-14 (Δδ 0.05 ppm), and H2-19 (Δδ 0.2), as these four protons are close in space to the epimeric C-20. We assign the structure 2 (Fig. 1) to this novel compound, fatimanone B.
Compound 3. Negative-ion HRESIMS showed ions at m/z 407.1690 [M - H]− (calc 407.1706) for the formula C21H28O8. Positive-ion HRESIMS revealed a pseudomolecular ion at m/z 409 [M + H]+ and a fragment ion at m/z 379.1747 [M + H – H2C = O]+ (calc 379.1757). Twenty-one discrete 13C NMR signals were observed: 2 ⋅ CH3, 6 ⋅ CH2, 7 ⋅ CH, 6 ⋅ Cq, including a carbonyl (δ 172.5). IR showed bands for OH (3513 cm− 1) and one carbonyl (1716 cm− 1).
The NMR data (Table S1, SI) indicated that 3 was a neoclerodane. The upper part was an aromatic furan, with 1H NMR signals (Table S1, SI) at δ 6.42 (H-14), δ 7.39 (H-16) and δ 7.40 (H-15). The furan 13C NMR signals were at δ 108.4 (C-14), δ 130.1 (C-13), δ 138.6 (C-16) and δ 143.9 (C-15), with appropriate HSQC and HMBC connectivities. The chemical shift of H-12 (δ 4.85) showed that it was not part of a lactone or lactol system, confirmed by the lack of a HMBC cross-peak to the signal for carbonyl C-20 (δ 172.5). The identity of H-12 signal was confirmed by HMBC cross-peaks to C-13, C-14, and C-16. Further HMBC cross-peaks were seen from H-12 to C-11 (δ 35.8 or δ 40.0) and to C-9 (δ 48.5), linking this upper side-chain to the main decalin. The configuration at C-12 could not be established. The bridging lactone was established by HMBC cross-peaks between carbonyl C-20 and H219 (δ 4.45 and δ 4.68). The identity of H219 had been confirmed by each signal being a doublet with only geminal coupling 2J = 12.5 Hz) and by HMBC cross-peaks to the C-6 (δ 110.0) and to C-4 (δ 84.1). C-19 (δ 66.4) was identified by HSQC cross-peaks to H2-19 and by strong HMBC to H-10 (δ 2.24–2.48 m). The signal at δ 72.8 was assigned to C-3 on the basis of HMBC cross-peaks to both H-2 (δ 1.4 and δ 2.1) and to one of the H-1 signals (δ 2.35). H-3 (δ 3.87) was characterised by a HSQC cross-peak to C-3, COSY cross-peaks to both H-2 and HMBC cross-peaks to C-1 (δ 21.9), C-2 (δ 30.3) and C-4 (δ 84.1). H-3 is axial, as shown by the large 3Jax−ax (11.6 Hz) to H-2ax. The H-1 signal at δ 1.29 is a quartet (J = 13.2 Hz); thus this signal is for H-1ax. NOESY correlations from H-3 to one H-19 (δ 4.45) and to H-1ax showed that these are on the same face of the decalin and thus that the decalin is trans-fused and that ring A is in the chair conformation. The conformation of ring B is less clear, owing to overlap of 1H NMR signals but MM2-minimisation suggests that it may be a flattened boat. The fused tetrahydrofuran and the acetal were identified as follows. Acetal carbon C-6 was characterised by its chemical shift (δ 110.0) and by HMBC cross-peaks to both H-19. Further HMBC correlations were seen to the doublets at δH 3.90 and δH 4.45, showing that they were due to H2-18 and demonstrating the closure of the tetrahydrofuran ring. The acetal at C-6 was identified by observation of an OMe group (δH 3.40, δC 48.9), linked by HMBC to C-6. These data characterise the structure of 3, fatimanol G, as shown in Fig. 1. This structure is identical to that of teulepicephin 22, with the exception of the acetal (hemiacetal in 14)15; the spectroscopic features are very similar, suggesting a similar conformation.
Compound 4. HRESIMS showed pseudomolecular ion peaks at m/z 383.1113 [M + Na]+ (calc 383.1107) and m/z 384.1148, [M + Na]+ (calc for 12C1813CH20NaO7, 384.1140), appropriate to the formula C19H20O7. Low-intensity pseudomolecular ions were also observed at m/z 361 [M + H]+ and m/z 399 [M + K]+. The 13C NMR spectrum contained signals for 19 discrete carbons: 1 ⋅ CH3, 4 ⋅ CH2, 7 ⋅ CH, 7 ⋅ Cq (including two carbonyls). The IR indicated hydroxy groups (3614 cm1) and two carbonyls (1715, 1701 cm− 1).
The 1H NMR spectrum (Table S2, SI) contained two very similar sets of signals, in ca. 1:1 ratio, suggesting diastereoisomers which interconverted slowly on the 1H NMR timescale. This may indicate a cyclic hemiacetal (cf. 2). Detailed assignment of the signals was challenging, as many overlapped between the two stereoisomers. Taken together, the NMR data showed that 4 had a neoclerodane core. As for 1–3, the upper part was an aromatic furan, with 1H NMR signals at δ 6.49 (H-14), δ 7.55 (H-15) and δ 7.61 (H-16). The signals for H-15 and H-16 were distinguished by a NOESY cross-peak from the former to H-14. The 13C NMR signals for this ring were at δ 124.7 (C-13), δ 107.9 (C-14), δ 139.6 (C-15) and δ 144.3 (C-16), which were identified by appropriate HSQC and HMBC. The spiro-lactone was initially identified by the chemical shift of H-12 (δ 5.59), corresponding to a benzylic ester. This signal showed an HSQC cross-peak to C-12 (δ 72.3) and HMBC cross-peaks to C-13, C-14, C-16, and C-11 (δ 40.2, weak 2-bond cross-peak). HSQC then identified the dd signal at δ 2.73 as being due to one H-11 and the two signals at δ 2.56 and δ 2.57 (both dd, with each integrating for 0.5 H) as due to the other H11. The 13C signal at δ 176.4 was shown to be due to the lactone carbonyl C-20 by HMBC cross-peaks to H-11 (δ 2.73), H-8 (δ 2.2), and H-10 (δ 2.85). H-3 (δ 4.41) and C-3 (δ 60.5) had the expected downfield chemical shifts arising from the OH. COSY then identified H-2 at δ 1.66 and δ 2.10, with HSQC showing C-2 (δ 21.6). Further COSY cross-peaks then showed the resonances for H-1 at δ 1.64 and δ 2.3. The lower fused butenolide became evident through HMBC cross-peaks from H-10 to the alkene Cq peaks for C-4 (δ 128.5) and C-5 (δ 163.0). These were distinguished from each other by their chemical shifts and by observation of a HMBC cross-peak from C-4 to H-2 (δ 2.01). The carbonyl C-18 signal was at δ 170.1. The lactone was completed by C-6 (δ 102.4), which shows HMBC correlations with both H-7 signals, H-8 and H-10. Interestingly, there is also a weak 4-bond HMBC cross-peak between H-17 (the methyl group) and C-6. The extensive overlap of many of the 1H NMR signals for the two diastereoisomers precluded detailed assignments of the conformations of the decalins. We assign structure 4 (Fig. 1) to this novel compound, fatimanol H.
Compound 5. HRESIMS showed pseudomolecular ion peaks at m/z 425.1197 [M + Na]+ (calc 425.1212) and m/z 403.1378 [M + H]+ (calc 403.1393), corresponding to the formula C21H22O8. The spectrum also contained a peak at m/z 827.2509 [2 M + Na]+ (calc 827.2527). The 13C NMR spectrum contained signals for twenty-one discrete carbons: 2 ⋅ CH3, 4 ⋅ CH2, 7 ⋅ CH, 8 ⋅ Cq. The IR showed absorbances for a hydroxy group (3618 cm− 1) and three carbonyls (1763, 1715, 1701 cm− 1). The NMR spectra (Table S2, SI) were very similar to those for 4, with the exception of additional methyl signals at δH 2.05 / δC 20.9, an additional carbonyl signal at δC 172.1, and a marked downfield change in the chemical shifts of H-3 (δ 5.59, Δδ 1.18 ppm) and C-3 (δ 64.65, Δδ 4.16 ppm). These indicate that 5 is the 3-O-acetate ester of 4. The 1H NMR spectrum contained only one set of signals, showing that one of the possible hemiacetal diastereoisomers had significantly lower energy than the other but it was not possible to determine which from the spectroscopic data. The methyl protons (H-2’, δ 2.05) of the acetate showed a strong HMBC to the corresponding ester carbonyl (C-1’, δ 170). C-1’ also showed a cross-peak to H-3, confirming the location of the ester. We assign structure 5 (Fig. 1) to this novel compound, fatimanol I.
Compound 6 was also closely related to 4. Pseudomolecular ions were seen in HRMS at m/z 397.1248 [M + Na]+ (calc 397.1263) and m/z 375.1429 [M + H]+ (calc 375.1444), consistent with the formula C20H22O7. A 13C isotopomer peak was observed at m/z 398.1282 (calc 398.1297). The 13C NMR spectrum showed discrete signals for 2 ⋅ CH3, 4 ⋅ CH2, 7 ⋅ CH and 7 ⋅ Cq. The 1H and 13C NMR spectra (Table S2, SI) (with 2D spectra) were very similar to those for 4, with the addition of signals for a methoxy group (δH 3.19, δC 51.0). The protons of this group showed a strong HMBC cross-peak to the 13C signal for C-6 (δ 150.8), identifying the methoxy group as being part of an acetal at this position. These data demonstrate that 6 (fatimanol J, Fig. 1) has the novel structure shown.
Compound 7. HRESIMS contained a pseudomolecular ion at m/z 425.1459, corresponding to [M - H]− (calc 425.1526) for the formula C20H26O10. The 13C NMR spectrum showed discrete resonances for twenty carbons: 1 ⋅ CH3, 6 ⋅ CH2, 6 ⋅ CH, 7 ⋅ Cq. The IR contained absorbances for two carbonyls (1769 cm− 1, 1737 cm− 1) and OH (3433 cm− 1). The NMR data (Table S2, SI) suggested a neoclerodane core. The upper part contained a hydroxyfuranone ring, as in fatimanol A15. This cyclic acetal was represented by H-14 (δ 7.17), which showed an HSQC cross-peak to C14 (δ 146.6); the downfield shifts of these signals were due to the enone system. From H-14, 2-bond HMBC cross-peaks were seen to C-13 (δ 108.1) and to C-15 (δ 99.0), and 3-bond cross-peaks were evident to the carbonyl C-16 (δ 172.2) and (weakly owing to the adverse dihedral angle) to C-12 (δ 64.0). H-14 did not show a COSY cross-peak to the hemiacetal proton H-15 (δ 5.88), again owing to the small coupling constant consequent to a dihedral angle approaching 90°. HSQC identified H-12 as part of a complex multiplet at δ ca. 4.6. A 2-bond HMBC from H-12 then led to identification of C-11 (δ 41.3), from which HSQC showed the two signals for H-11 (δ 1.6, δ 2.25). From the downfield H-11 signal, it was then possible to use HMBC to identify the signals for C-9 (δ 50.2), C-10 (δ 43.9) and the saturated lactone carbonyl C-20 (δ 174.6). This lactone was confirmed by HMBC cross-peaks from C-20 to H-19 (δ 4.58, δ 4.64), and the latter were linked on to C-4 (δ 85.6), C-5 (δ 49.7) and C-6 (δ 108.2). A 2-bond HMBC correlation from C-4 identified H-3 (δ 3.83). The latter then gave an HMBC cross-peak to the methylene C-18 (δ 75.6) with H-18 (δ 3.97 d (J = 10.4 Hz), δ 4.35 d (J = 10.4)). The downfield H-19 also gave a 3-bond HMBC with C-10, completing the lower lactone ring. H-3 resonated as a dd (J = 11.6, 5.9 Hz), the larger of the two coupling constants indicating that this proton is axial. Completing the features of the lower part of the structure, H-18 (δ 4.35) showed a 3-bond HMBC with the hemiacetal carbon C-6. We assign the novel structure 7 (Fig. 1) to this compound, fatimanol K.
Compound 8. HRESIMS showed a pseudomolecular ion at m/z 519.2212 [M + Na]+ (calc 519.2206) for the formula C25H36O10. The 13C NMR spectrum showed twenty-five discrete resonances: 4 ⋅ CH3, 8 ⋅ CH2, 6 ⋅ CH, and 7 ⋅ Cq. Bands for OH (3615 cm1) and three ester / lactone carbonyls (1744, 1734, 1695 cm− 1) were seen in the IR spectrum. Combined interpretation of the NMR spectra (Table S3, SI) showed that 8 was a neoclerodane. The upper ring was a furan-2-one. The acetal proton H-15 resonated as a doublet at δ 5.84, with a small coupling constant (2 Hz) to H-14 (δ 7.02) confirmed by COSY. HSQC then identified C-15 (δ 104.6) and C-14 (δ 144.8). The latter was appropriate for the βcarbon of an enone. HMBC linked H-15 to C-13 (δ 138.9) and the lactone carbonyl C-16 (δ 173.3). H-14 and C-16 also showed a cross-peak in HMBC. The IR band at 1695 cm− 1 was assigned to this α,β-unsaturated ester. The methoxy protons (δ 3.52) showed a HMBC cross-peak to C-15, demonstrating the location of the OMe. HSQC identified the methoxy carbon signal at δ 57.3. HMBC from C-13 to both H-12 (δ 2.15 and δ 2.28) and to both H11 (δ 1.4 and δ 2.25) confirmed the attachment of the methoxyfuranone ring at C-12. C-11 (δ 34.6) and C-12 (δ 19.3) were identified by HSQC. A further HMBC cross-peak from H-11 (δ 1.4) to the 13C signal at δ 66.5 (CH2) showed that the latter was due to C-20. HSQC confirmed the geminally coupled doublets (δ 3.97 and δ 4.03) (J = 12.0 Hz) as due to the two H-20. These were linked by HMBC to the carbonyl 13C signal at δ 172.6 and thence to the acetate protons at δ 2.06. Both H-20 signals had 3-bond HMBC correlations with C-10 (δ 47) and C-20 had HMBC correlation with the signal with H-8 (δ 1.7). H-10 (δ 1.7) and C-8 (δ 35.2 or 35.1) were then identified by HSQC. The C-Me group (H-17) resonated as a doublet at high field (δ 0.97), with C-17 at δ 16.5. HMBC cross-peaks from H3C-20 linked to both H-7 (δ 1.7 and δ 1.8), from which C-7 (δ 30.8) could be identified by HSQC. A weak 4-bond HMBC cross-peak was observed between H-17 and C-6 (δ 74.3), as in 2 and 4. The chemical shift of this carbon and of the attendant H-6 (δ 3.78) suggested the presence of an oxygen substituent. H-6 had 3-bond HMBC correlations with C-4 (δ 66.9) and with C-19 (δ 63.7). HSQC identified the geminally coupled H-19 protons (δ 4.43 and δ 4.65). HMBC from these latter protons confirmed the lower acetate ester carbonyl (δ 172.7), linked on to the methyl proton signal at δ 2.06, co-incident with the other acetate 1H NMR signal. Weak 2-bond HMBC cross-peaks from C-4 identified the oxirane protons H-18 at δ 3.01 and δ 3.21, while a stronger 2-bond cross-peak showed H-3 (δ 4.00), with its corresponding C-3 (δ 66.5).
The relative stereochemical configurations were largely determined by use of NOESY. An MM2-minimised structure suggested that the decalin would have both rings in chair conformations. Strong NOESY cross-peaks showed that H-17 and H-20 were cis to each other on the lower face. Similarly, H2-19 were shown to be on the lower face by strong NOESY cross-peaks to H-20, with these methylenes being diaxial on ring B. Further strong NOESY cross-peaks from both H-19 to H-3 confirmed the latter as axial down on ring A and a cross-peak to H-1 (δ 2.1) also suggested that this was on the lower face. On the upper face, the downfield H-18 signal (δ 3.21) gave a cross-peak to H-6 (δ 3.78), which also allowed differentiation of the two oxirane proton signals. The relative configuration at C-15 could not be determined. These data show the structure of 8 (Fig. 1), fatimanol L.
Compound 9. HRESIMS showed pseudomolecular ion peaks at m/z 535.2189 [M + Na]+ (calc 535.2155) and m/z 513.2350 [M + H]+ (calc 531.2336), corresponding to the formula C25H36O11. The 13C NMR spectrum complied, with twenty-five discrete signals: 4 ⋅ CH3, 7 ⋅ CH2, 7 ⋅ CH, 7 ⋅ Cq. The IR showed OH (3546 cm− 1) and three carbonyls (1764, 1752, 1708 cm1). The NMR spectra (Table S3, SI) showed considerable similarity to those for 8, except in the C-11 / C-12 region and the upper methoxyfuran. The structure of the trans-decalin and the lower appendages were identical to those of 8. HMBC from C-11 (δ 38.0) identified H-12 (δ 4.56); the downfield chemical shift of this peak indicated that a hydroxy group. HSQC identified C-12 (δ 63.8). H-12 was also linked by COSY cross-peaks to both H-11 (δ 1.75 and δ 1.80). A strong 3-bond HMBC from H-12 showed the signal at δ 144.8 to be due to C-14, with HSQC identifying H14 (δ 7.13). Appropriate HMBC and HSQC correlations then identified C-13 (δ 144.2), C-15 (δ 104.2), H-15 (δ 5.88) and C-16 (δ 171.7). The methoxy group protons resonated as two singlets (δ 3.54 and δ 3.55), each integrating for 1.5 H, suggesting that 9 was a mixture of epimers at C-15. The corresponding methoxy 13C signal was linked by HMBC to H-15. NOESY also linked together the upper part of the structure. Strong cross-peaks were seen linking H-15 with the methoxy group and with H-14. Furthermore, H-14 was linked by NOESY with H-12. Addressing the relative configurations of 9 (except for the mixture of epimers at C-15), strong NOESY cross-peaks were seen from H-8 (δ 1.94) to H-6 (δ 3.78), to H-10 (δ 2.22) and to H-12, showing that all of these are on the upper face of the bicycle. Noesy also linked H-6 with one H-18 (δ 3.17), confirming that the latter is on the upper face and differentiating the two H-18 signals. On the lower face, one H-19 (δ 4.42) formed a NOESY cross-peak with H-3 (δ 4.00) and the other H-19 (δ 4.71) was close in space with H-20 (δ 3.95). The 1H signals for H-2 were differentiated by a NOESY cross-peak from H-10 to H-2ax (δ 2.10). The relative configuration at C-12 could not be determined. The structure of this novel compound is thus 9 (Fig. 1), fatimanol M.
Compound 10. HRESIMS showed a pseudomolecular ion at m/z 425.1452 [M - H]− (calc 425.1448), corresponding to the formula C20H26O10. Smaller pseudomolecular ions were seen at m/z 426.1485 [M - H]− (calc 426.1481) and m/z 427.1520 [M - H]− (calc 427.1515) for 13C1 and 13C2 isotopomers, respectively. In positive-ion mode, the HRESIMS contained the pseudomolecular ion at m/z 427.1596 [M + H]+ (calc 427.1604), in addition to a very abundant ion at m/z 409 [M + H – H2O]+ showing an aliphatic alcohol. Twenty discrete 13C NMR peaks were evident: 1 ⋅ CH3, 7 ⋅ CH2, 5 ⋅ CH, 7 ⋅ Cq. Three Cq were carbonyls, with two coincident 13C NMR signals at δ 174.5 and a singleton at δ 173.1 and IR bands at 1795, 1790, and 1689 cm− 1. The higher-frequency C = O absorptions suggested that they were likely to be a cyclic anhydride. As with the other examples, the combined NMR data (Table S3, SI) suggested the neoclerodane skeleton for 10. However, the upper five-membered ring was unusual, in that it was a succinic anhydride. Carbonyl C-15 resonated at δ 174.5 and C-16 appeared at δ 173.1. The latter showed a 3-bond HMBC correlation with one H-14 (δ 2.04). COSY then identified the other H-14 (δ 1.42) and H-13 (δ 3.1), with HSQC then establishing C-14 (δ 33) and C-13 (δ 49.7). COSY linked H-13 to H-12 (δ 4.76) and HSQC identified C-12 (δ 65.8). A 2-bond HMBC from the latter led to assignment of one H-11 (δ 1.89 dd), which was shown by HSQC to be attached to the same carbon (C-11, δ 34.2) as the signal at δH 2.43 for the other H-11. An additional 2-bond HMBC led to identification of C-9 (δ 36.2). In the lower part, a 3-bond HMBC was observed from the methyl H-17 (δ 0.94) to C-7 (δ 41.2). A 3-bond HMBC from H-8 to the hemiacetal carbon (C-6, δ 108.1) confirmed its location. A further HMBC from C-6 to one H-18 (δ 4.34) showed the closure of the lower tetrahydrofuran ring / hemiacetal. An MM2-minimised model explained the absence of a HMBC cross-peak from the other H-18 to C-6, in that the dihedral angle is ca. 90°. H-18 (δ 4.34) also gave an HMBC with C-3 (δ 73.3), which carries an oxygen. HSQC then identified H-3 (δ 3.83) as a dd with J = 9.4, 4.6 Hz. The larger coupling constant implies a trans-diaxial coupling; thus H-3 is axial. HMBC correlation from H-3 identified C2 (δ 32.8). A 2-bond HMBC linked H-3 with C-4. The (C-19)—O―(C-20) ester bridge was also confirmed by HMBC, in that both H-19 (δ 4.59 and δ 4.65) showed cross-peaks with C-5 (δ 85.6), C-6 (δ 50.2) and C-20 (δ 174.5). C-19 was identified by HSQC at δ 68.5, consistent with the ester. Thus the structure of 10 (fatimanol N) is as in Fig. 1.
Compound 11. Instability under MS conditions precluded obtaining useful mass spectra. The 13C NMR showed discrete peaks for twenty-one carbons: 2 × CH3, 6 × CH2, 8 × CH, 5 × Cq. The IR contained a band for OH (3414 cm1) but no carbonyls were evident. The NMR data (Table S3, SI) showed a neoclerodane structure. The 3-substituted aromatic furan was shown by the downfield resonances of the ring-H. H-14 resonated at δ 6.41, while H-15 and H-16 were co-incident at δ 7.43. HMBC correlated H-14 with C-13 (δ 126.7), C-15 (δ 143.6) and C-16 (δ 139.3). Similarly, H-15 was correlated with C-16 and H-16 was correlated with C-14 (δ 108.6) and with C-15. HMBC was also useful in linking C-13, C-13, and C-16 with H-12 (δ 5.10 m) and HSQC identified C-12 at δ 71.6. A COSY cross-peak from H-12 to the multiplet signal at δ 1.85 identified the latter as one H-11 and HSQC led to C-11 (δ 40.5) and thence to the other H-11 (δ 2.24). C-20 was an acetal carbon, as shown by its chemical shift (δ 101.2), and HSQC identified H-20 as the singlet at δ 5.08. H-20 gave HMBC cross-peaks to C-8 (δ 35.2) and C-9 (δ 44.6), and to C-12, demonstrating the ether linkage. HMBC from C-8 to both H-11, completed this ring. The C-Me group (C-17, H-17) was readily identified as the origin of the most upfield NMR signals (δC 16.3, δH 1.02). From here, a 2-bond HMBC cross-peak identified H-8 (δ 1.85), confirmed by a COSY cross-peak from H-17. Three-bond HMBC interactions also identified both H-7 (δ 1.65 t and δ 2.36 dd). The multiplicities of these indicated that the former was axial and the latter was equatorial. C-7 (δ 36.1) was located by HSQC. Acetal C-6 (δ 110.9) was confirmed by 3-bond HMBC with both H-19 (δ 3.94 and δ 4.15, geminally coupled). Both H-19 also gave HMBC cross-peaks to C-20, confirming the ether bridge between C-19 (δ 58.5) and C-20. The closure of the lower tetrahydrofuran ring was demonstrated by HMBC from C-6 to both H-18 (δ 3.89, δ 4.42). Moving clockwise around the decalin, HMBC from C-18 (δ 75.1) identified H-3 (δ 3.89), the large 3J of which indicated a trans-diaxial relationship with one H-2 (δ 1.41). Further HMBC, HSQC, and COSY analysis established the assignments of H-1 (δ 1.95 and δ 1.99), C-1 (δ 23.2), H-2 (δ 1.41 and δ 2.15), C-2 (δ 30.3) and C-3 (δ 73.2). Thus 11 has the novel polycyclic structure shown in Figure 1, with many fused rings and bridges. Fortunately, these fusions and bridges make the structure fairly rigid and it was straightforward to assign the relative stereochemical configurations by use of coupling constants (Karplus relationship) and NOESY. Figure 2 shows the key NOESY interactions used in this assignment. Particularly useful was the NOE interaction between the methoxy protons and H-18exo (δ 3.89), which confirms that the methoxy is on the lower face of the trans-decalin. The name fatimanol O is assigned to the novel compound 11 (Figure 1).
Compound 12. HRESIMS gave pseudomolecular ions at m/z 443.1706 [M + Na]+ (calc 443.1682) and m/z 421.1854 [M + H]+ (calc 421.1862), corresponding to the formula C22H28O8. The 13C NMR spectrum showed 22 discrete signals: 2 ⋅ CH3, 6 ⋅ CH2, 8 ⋅ CH, 6 ⋅ Cq. The NMR data (Table S4, SI) showed that 12 was a neoclerodane. In the upper furan, C-13 resonated at δ 126.7 and gave HMBC cross-peaks to H-14 (δ 6.46), H-15 (δ 7.53) and H-16 (δ 7.59). The corresponding 13C resonated at δ 109.2 (C-14), δ 145.6 (C-15) and δ 141.4 (C-16). C15 and C-16 were differentiated by a HMBC correlation between the latter and H-12 (δ 5.48). HSQC identified C-12 (δ 73.5). Three-bond HMBC from C-13 also identified both H-11 (δ 2.38 and δ 2.51), from which C-11 was shown to be at δ 43.6. The downfield H-11 signal also showed HMBC cross-peaks to C-9 (δ 52.0), C-10 (δ 51.8) and carbonyl C-20 (δ 178.9). The methyl group gave the most upfield signals (C-17 δ 16.9, H-17 δ 0.99). Strong HMBC cross-peaks identified C-8 (δ 38.65) and C-7 (δ 33.5), whereas a weak cross-peak suggested the peak at δ 74.7 to be due to C6. H-8 resonated at δ 1.78 and H-7 at δ 1.56 and δ 2.22. The chemical shifts of C-6 and of H-6 (δ 4.99) were consistent with an ester oxygen being attached thereto and HMBC from H-6 to the carbonyl signal at δ 171.6 confirmed that this appendage was an acetoxy group (AcO-) (MeCO2 δC 21.3 δH 2.04). H-6 also gave a strong HMBC 2-bond to C-5 (δ 48.0) and a cross-peak to C-19 (δ 61.4). The diastereotopic H-19 protons gave well-separated doublet signals at δ 3.90 and δ 4.62. C-19 gave a strong 3-bond HMBC to H-10 (δ 1.89). The upfield H-19 gave a 2-bond HMBC with quaternary C-4 (δ 52.8). The relatively upfield chemical shift of C-4 was consistent with the strained oxirane and C-18 (a methylene) was also identified by its chemical shift at δ 42.5. HSQC then linked it to the doublets for H-18 (δ 2.72, δ 2.80). C-18 gave a moderate HMBC to H-3 (δ 4.28), from which COSY and HSQC identified the signals for C-3 (δ 68.7), C-2 (δ 34.1), H-2 (δ 1.45, δ 2.22), C-1 (δ 23.1) and H-1 (δ 1.78, δ 2.12). Completing ring A, the upfield H-1 gave a strong COSY cross-peak with H-10. The structure of 12 is less rigid than 11 but it was still possible to assign its relative configurations by NOESY (Fig. 2). These data confirm the structure of 12, fatimanol P (Fig. 1).
Compound 13. HRESIMS showed a pseudomolecular ion at m/z 405.1880 [M + Na]+ (calc 405.1889), corresponding to the formula C20H30O7. Other ions were observed at m/z 787.3875 [2 M + Na]+ (calc 787.3881). Fragment ions were observed at m/z 729.3844 [2 M + Na - C2H2O2]+ (calc 729.3826), m/z 711 [2 M + Na - C2H2O2 - H2O]+, m/z 693 [2 M + Na - C2H2O2 − 2 × H2O]+, m/z 347.1850 [M + Na – C2H2O2]+ (calc 347.1834), m/z 329.1744 [M + Na - C2H2O2 - H2O]+ (calc 329.1729), and m/z 311.1639 [M + Na – C2H2O2 − 2 × H2O]+ (calc 311.1623), indicating the presence of at least two hydroxy groups. In the negative-ion HRESIMS, confirmatory peaks were seen at m/z 427.1970 [M + formate]− (calc 427.1968), m/z 417.1689 [M + 35Cl]− (calc 417.1680), and m/z 381.1920 [M – H]− (calc 381.1913). The 13C NMR spectrum contained signals for twenty carbons: 1 ⋅ CH3, 7 ⋅ CH2, 8 ⋅ CH, and 4 ⋅ Cq, although two signals were co-incident at δ 143.5.
The NMR data (Table S4, SI) suggested that 13 had a structure broadly similar to those of the neoclerodanes but with important differences. The upper ring was a furan, with 1H NMR signals at δ 6.48 (H-14), δ 7.56 (H-15) and δ 7.50 (H-16). The signals for H-15 and H-16 were differentiated through COSY from H-15 to H14 and by HMBC from H-16 to C-12 (δ 62.1). HSQC then linked these to the 13C peaks at δ 109.7 (C-14) and δ 143.5 (C-15, C-16). HMBC from H-14, H-15, and H16 then identified the 13C signal at δ 132.4 as being due to C-13. HMBC from C13, C-14 and C-16 to the 1H signal at δ 4.68, along with HSQC from C-12, identified this multiplet signal as H-12. This chemical shift suggested that this proton was benzylic and it gave COSY cross-peaks to 12-OH (δ 5.02, doublet) and both 11-H (δ 1.60, δ 2.01, both dd). HSQC from the latter then gave 11-C (δ 41.7). Linkage of this upper side-chain to the core bicycle was demonstrated by a HMBC correlation from H-12 to C-9 (δ 40.4, Cq) and by HMBC from both H-11 to C-8 (δ 35.6) and to C-10 (δ 51.5). The 3-bond HMBC cross-peak between H-11 (δ 1.60) and C-8 was weak, owing to the dihedral angle (H-11)—(C-11)—(C-9)—(C-8) being close to 90°. H-8 (δ 1.47) also gave a HMBC cross-peak with C-11. The other side-chain at C-9 was a hydroxymethyl (HOCH2-) unit, with C-20 (δ 53.3) giving weak HMBC with both H-11. The geminal protons H-20 (δ 3.21 and δ 3.32) gave strong COSY cross-peaks with each other and with HO-20 (δ 4.46). The methyl group was at C-8, as shown by a COSY cross-peak from H-8 to the doublet signal at δ 0.71 (H-17), from which HSQC identified the 13C peak at δ 16.3 as C-17. Moving clockwise around the cyclohexane B-ring, a strong 2-bond HMBC from C-8 identified Hax-7 at δ 1.21 (q, J = 11 Hz). HSQC revealed C-7 (δ 37.9) and thence Heq-7 (δ 1.47). Both H-7 gave strong 2-bond HMBC correlations with C-6 (δ 69.4), from which HSQC showed the signal at δ 3.36 as being H-6. The COSY cross-peak from Hax-7 to H-6 was strong, whereas that from Heq-7 to H-6 was much weaker, suggesting that the dihedral angle (Heq-7)—(C-7)—(C-6)—(H-6) was ca. 90° and thus that H-6 was axial. COSY linked H-6 to HO-6 (δ 4.46). Three-bond HMBC correlations from both H-7 located quaternary C-5 (δ 59.8). Ring B was completed by observation of a 2-bond HMBC cross-peak from C-5 to H-10 (δ 2.07).
Establishing ring A was more challenging. A 2-bond HMBC cross-peak from H-10 identified C-1 at δ 24.6, from which HSQC showed that the H-1 signals were at δ 1.87 and δ 1.98. A strong 3-bond HMBC linked C-10 with one H-2 signal (δ 1.87), whereas the cross-peak to the other H-2 (δ 1.27) was weaker. A 3-bond HMBC cross-peak was also seen linking H-1 (δ 1.27) to C-5; the 3-bond path between these two nuclei cannot pass through C-1 and C-10, thus ring A must be five-membered. Quaternary C-3 (δ 57.7) was identified through a 2-bond HMBC with H-2 (δ 1.27) and a 3-bond correlation with H-10, completing the cyclopentane. Three-bond HMBC from both H-2 showed the methylene C-4 (δ 62.5) as being attached at C-3 and the H-4 protons were observed as dd at δ 3.27 and δ 3.66. These H-4 signals were linked by COSY to each other and to HO-4 (δ 4.46). A 2-bond HMBC correlation between H-4 (δ 3.27) and C-3 confirmed the attachment of the HOCH2-. A 2-bond HMBC from C-3 and a 3-bond cross-peak from H-2 (δ 1.27) revealed the other substituent at quaternary C-3 by identifying C-18 (δ 102.0). The hemiacetal was confirmed by a COSY from H-18 (δ 4.76) to HO-18 (δ 6.26). HMBC from C-5, C-6 and C-10 to the doublet signals at δ 3.80 and δ 3.87 identified both H-19. C-19 (δ 67.3) was shown to be a methylene by HSQC and 135DEPT. The chemical shifts of C-19 and both H-19 suggested the attachment of an oxygen but this was not an OH (no COSY cross-peak). However, HMBC linked C-19 with H-18 and C-18 with both H-19. The only structure consistent with these connectivities is the lactol / cyclic acetal shown. It was not possible to obtain a good NOESY spectrum, so the relative configuration shown in Chart 1 is speculative, except where suggested by 3JH−H coupling constants. The NMR spectra contained a second (smaller) set of peaks, which we ascribe to the presence of a minor diastereoisomer in slow equilibrium, probably the epimer at the acetal C-18. Thus 13 (fatimanol Q) has the novel structure shown in Fig. 1.
Compound 14. HRESIMS showed a pseudomolecular ion m/z 447.1985 [M + Na]+ (calc 447.1995), consistent with the molecular formula C22H32O8. Additional ions were seen at m/z 871.4083 [2 M + Na]+ (calc 871.4091) and m/z 425.2167 [M + H]+ (calc 425.2175) in positive-ion mode, and 469.2075 [M + formate]− (calc 469.2074), and 459.1795 [M + 35Cl]− (calc 459.1786) in negative-ion mode. The 13C NMR spectrum (in (CD3)2SO) contained discrete signals for twenty-two carbon atoms: 2 ⋅ CH3, 7 ⋅ CH2, 8 ⋅ CH, 5 ⋅ Cq.
Combined analysis of the NMR data ((CD3)2SO, Table S4, SI) showed that 14 was a conventional neoclerodane. The upper ring was a furan, with 1H signals for H-14 (δ 6.42), H-15 (δ 7.56) and H-16 (δ 7.49) and 13C signals for C-13 (δ 132.8), C-14 (δ 109.6), C-15 (δ 143.6) and C-16 (δ 138.6) all duly linked by HSQC and HMBC. Strong 3-bond HMBC cross-peaks were evident from H-12 (δ 4.59) to C-14 and C-16. HSQC linked this proton to C-12 (δ 61.7), which also gave HMBC cross-peaks to H-14 and H-16. A 2-bond HMBC from H-12 identified C-11 (δ 39.7), from which both H-11 (δ 1.67 and δ 1.86) could be identified by HSQC. HO-12 (δ 4.90) was located through a 3-bond HMBC from C-11. A 3-bond HMBC from H-12 identified quaternary C-9 (δ 43.3), while a similar correlation from H-11 (δ 1.67) confirmed C-10 (δ 46.8) and thence H-10 (δ 1.96). From here, C-20 (δ 63.0) was located by HMBC to H-10; the H-20 protons were approximately co-incident at δ 3.28, with HMBC cross-peaks to C-9 (weak, 2-bond), C-10, and C-11. Two-bond HMBC linked C-10 to both H-1 (δ 1.75 and δ 2.00), from which C-1 (δ 21.5) was identified by HSQC. The upfield H-1 signal gave a weak 2-bond HMBC with C-2 (δ 34.5), whereas the downfield H-1 signal correlated strongly with C-3 (δ 65.0). The signal at δ 1.17 was a double quartet (J = 4, 11 Hz), indicating that this was due to H-2ax, while H-2eq resonated as a narrow multiplet at δ 1.91. Thus the coupling to H-3 (δ 3.80) shows that this proton is axial / down) and HO-3 is equatorial / up. Furthermore, both H-3 and H-10 are axial, showing that ring A is in the chair conformation. A 2-bond HMBC correlation from H-3 identified C-4 (δ 70.0) and a 3-bond correlation revealed C-18 (δ 43.0). The H-18 protons resonated as doublets at δ 2.83 and δ 3.06; the chemical shifts suggested the spiro-oxirane ring. Strong HMBC cross-peaks from these protons were observed to C-4 and C-5 (δ 45.47). An AcOCH2- group was present at C-5, as demonstrated by HMBC from both H-19 (δ 4.40, δ 4.57) to C-4 and C-5. These protons also correlated with the ester carbonyl MeCO2-19 (δ 170.8), with the adjacent methyl group (MeCO2-19) resonating at δH 2.01 / δC 21.5. Three-bond HMBC cross-peaks were also seen from both H-19 to C-6 (δ 73.3) and HSQC then located the H-6 signal at δ 3.62 (brd, J = ca. 11 Hz). The H-6 signal was better resolved when the 1H NMR spectrum was obtained on a solution in CDCl3, which showed it as δ 3.70 (dd, J = 10.0, 6.0 Hz). The larger axial-axial coupling shows that H-6 is axial and up on the trans-decalin. Both H-7 (δ 1.39, δ 1.52) were located both by HSQC with C-7 and by HMBC with C-6, whence C-7 (δ 34.7) was revealed by HSQC. H-7 also formed HMBC correlations with the methyl C-17 (δ 17.0), from which H-17 was identified at δ 0.82. Completing ring B, H-17 gave an HMBC cross-peak with C-9. These spectroscopic interpretations were aided, in part, by 1H, COSY and HSQC spectra of a solution in CDCl3, which were better resolved, although paucity of sample precluded identification of the quaternary carbons (Table S4). Examination of the coupling constants allowed confirmation of the trans-decalin structure and the relative configurations of most of the substituents, although the relative configuration at C-12 could not be established. We assign the novel structure shown in Fig. 1 to 14, fatimanol R.
Compound 15. The HRESIMS contained a pseudomolecular ion at m/z 379.1748 [M + H]+ (calc 379.1757) which showed the formula C20H26O7. Other ions were observed at m/z 779.3248 [2 M + Na]+ (calc 779.3255), m/z 401.1567 [M + Na]+ (calc 401.1576), m/z 361.1642 [M + H - H2O]+ (calc 361.1651), 343.1537 [M + H − 2 × H2O]+ (calc 343.1546), and m/z 325.1341 [M + H − 3 × H2O]+ (calc 325.1440). The latter three fragment ions indicated the presence of three hydroxy groups. Negative ions were present at m/z 423.1656 [M + formate]− (calc 423.1655), and m/z 377.1605 [M - H]− (calc 377.1600). The 13C NMR spectrum (in (CD3)2SO) contained individual signals for twenty carbon atoms: 1 ⋅ CH3, 6 ⋅ CH2, 8 ⋅ CH, 5 ⋅ Cq. The NMR data (Table S5, SI) for 15 indicated a conventional neoclerodane. The upper furan showed 1H signals for H-14 (δ 6.49), H-15 (δ 7.74) and H-16 (δ 7.78). HSQC linked these to C-14 (δ 109.1), C-15 (δ 145.0) and C-16 (δ 141.1), respectively. The C-13 13C NMR signal was identified at δ 125.6 by HMBC. Moving south, 3-bond HMBC cross-peaks from C-14 and C-16 identified H-12 as a triplet at δ 5.43. The HMBC from C-16 to H-12 and the NOESY interaction between H-16 and H-12 distinguished H-16 from H15. The chemical shift of H-12 strongly suggested that it carried a lactone oxygen, confirmed by HMBC from H-12 to the signal at δ 177.0 (C-20). HSQC linked H-12 to C-12 (δ 71.4). A 2-bond HMBC linked H-12 to C-11 (δ 41.2), with HSQC from this signal the H-11 protons (δ 2.25, δ 2.45). Both H-11 gave HMBC correlations to the lactone carbonyl C-20, further confirming the ring. Both H-11 also gave HMBC cross-peaks to the quaternary carbon signal at δ 51.2, which was shown to be C-9, where the spiro-lactone joins the decalin. Working clockwise around the lower decalin, C-19 gave a strong HMBC to the methyl H-17 protons (δ 0.91), from which C-17 (δ 16.9) was identified. A 2-bond HMBC from H-17 located C-8 (δ 37.3) and a 3-bond correlation located the methylene C-7 (δ 34.9), HSQC then showed H-8 (δ 1.62) and both H-7 (δ 1.48 (H-7eq), δ 1.96 (H-7ax)). C-7 showed a 2-bond HMBC with H-6 (δ 3.67); this chemical shift indicates an attached alcohol. C-6 (δ 72.5) was shown by HMBC with H-7eq and HSQC with H-6. The signal for 6-OH was broad but gave HMBC correlation with C-6. The signal at δ 46.4 was due to C-5. HMBC interactions then tied C-5 to the attached CH2OH group (H-19 δ 3.69, δ 4.36; HO-19 δ 4.07) and HSQC identified C-19 (δ 59.3). Both H-19 gave strong 3-bond HMBC cross-peaks to quaternary C-4 (δ 68.8), which was shown to be part of a spiro-oxirane. In the oxirane, H-18left resonated as a doublet at δ 2.69 and H-18right as a doublet at δ 2.89. These diastereotopic protons were distinguished by NOESY correlations from H-18left to HO-3 (δ 4.66) and one H-2 (δ 1.30) and from H-18right to H-19 (δ 3.69), H-6 and HO-6. Three-bond HMBC from both H-18 identified C-3 (δ 64.3), from which H-3 (δ 4.19) was shown by HSQC. HMBC from C-3 identified its HO-3 as a doublet at δ 4.66. The ring was completed by appropriate HMBC and HSQC cross-peaks identifying C-2 (δ 34.1), H-2 (δ 1.30, δ 2.05), C-1 (δ 22.0) and H-1 (δ 1.59, δ 1.96). Finally, there was a strong HMBC linking H-2 (δ 1.30) with C-10 (δ 51.2), closing the ring. H-10 resonated at δ 1.67.
The relative configurations were largely demonstrated by NOESY spectroscopy, with some consideration of 1H J values. Firstly, a strong NOESY interaction was seen between H-16 and H-17 and a weaker one between H-14 and H-17. This demonstrates that the furan and the methyl group are on the same face of the lactone and that the configuration at C-12 is S. The signal for H-7 (δ 1.48) is a broad doublet, thus this proton is equatorial up as the only large coupling constant would be 2Jgem to H-7ax (δ 1.96). The methyl (C-17, H-17) is equatorial down, as for the vast majority of neoclerodanes. H-7ax makes a strong NOESY interaction with H-19 (δ 4.36), consistent with C-19 being axial down. HO-6 is located on the lower face of the decalin, as H-6 experiences a strong NOESY correlation with H-7eq on the upper face. Running the spectrum in DMSO also allowed a NOESY interaction between HO-6 and H-19 (δ 4.36), confirming the orientation of HO-6. The configuration at C-4 of the spiro-oxirane was determined. H-1 (δ 1.59) is axial and down, as it has three large coupling constants (trans-diaxial to H-10 and H-2 (δ 1.30) and geminal to H-1eq (δ 1.96)). Therefore, H-2 (δ 1.30) is axial up. H-18left (δ 2.69) makes strong NOESY contacts with H-2ax and with HO-4 (upper face), which shows that the CH2 of the oxirane is on the upper face, as in 2, 8, 9, and 12. Finally, H-10 is on the upper face, as it shows a trans-diaxial coupling to H-1ax. Thus the bicycle is a trans-decalin. These spectroscopic assignments were aided partly by 1H, COSY, and HSQC spectra obtained of a solution in CDCl3, which were better resolved, although shortage of sample precluded identification of the quaternary carbons (Table S5). Minor differences in chemical shift were seen for H-2eq, H-3, H-14, H-15, H-18, and H-19, probably reflecting minor changes in hydrogen-bonding and consequent minor changes in conformation. The COSY spectrum confirmed the H-H connectivities within the molecule. We assign the novel structure shown in Fig. 1 to 15, fatimanol S.
Compound 16. The HRESIMS contained a pseudomolecular ion at m/z 379.1748 [M + H]+ (calc 379.1757), consistent with the formula C20H26O7. Ions were also seen at m/z 779.3250 [2 M + Na]+ (calc 779.3255), m/z 401.1568 [M + Na]+ (calc 401.1576), 361.1643 [M + H - H2O]+ (calc 361.1651), and m/z 325.1432 [M + H − 3 × H2O]+ (calc 325.1440), indicating three hydroxy groups. The 13C NMR spectrum (in (CD3)2SO) contained signals for twenty carbon atoms: 1 ⋅ CH3, 6 ⋅ CH2, 8 ⋅ CH, and 5 ⋅ Cq. Therefore, 16 is a closely structurally related isomer of 15. Analysis of the COSY, HSQC, HMBC and some NOESY connectivities showed an identical network to 15, strongly suggesting that they were stereoisomers. The NMR signals (in (CD3)2SO, Table S5, SI) for the lower part of the structure were very similar: position-2 (δH 1.20, δH 2.00, δC 34.1), position-3 (δH 4.16, δC 64.5), position-4 (δC 68.8), position-5 (δC 46.2) and position-6 (δH 3.65, δC 72.5). However, slightly more significant differences in chemical shift were observed for the upper and right parts of the decalin, at position-1 (δH 1.43, δH 1.68, δC 21.5), position-7 (δH 1.42, δH 1.74, δC 35.6), position-8 (δH 1.74, δC 40.0), and position-10 (δH 1.50, δC 49.2). The signal for C-9 (δ 51.5) was identical in 16 and 15. Moving into the upper spiro-lactone, the differences were again observed for position-11 (δH 2.33, δH 2.41, δC 42.6) but less significant for position-12 (δH 5.42, δC 71.3) and the lactone carbonyl C-20 (δ 176.8). Small differences were also seen for the furan: position-13 (δC 125.8), position-14 (δH 6.51, δC 109.3), position-15 (δH 7.71, δC 145.0) and position-16 (δH 7.77, δC 140.7). The larger differences were in the upper part of the decalin and in the lactone, which suggested that the stereochemical difference between 16 and 15 was at C-9 or at C-12. A detailed study of NOESY data in that area was undertaken. Firstly, the equatorial methyl H-17 gave a strong NOESY cross-peak to H-12, suggesting that these were on the same face of the γ-lactone. Secondly, H-12 gave a strong NOESY correlation with one H-11 (δ 2.41) but only weakly with the other H-11 (δ 2.33). Since the furan protons H-14 and H-16 both formed strong NOESY cross-peaks with H-11 (δ 2.33), the furan and this upfield H-11 must be on the same face of the γ-lactone and this face must be opposite to that carrying H-12. These data are consistent with 16 having the opposite configuration at C-12 from 15. Other NOESY interactions, COSY cross-peaks and 1H-1H coupling constants in the decalin were consistent with trans-configuration. The spectroscopic assignments were aided, in part, by 1H, COSY, and HSQC spectra of a solution in CDCl3, which were better resolved, although shortage of sample precluded identification of the quaternary carbons and NOESY data could not be obtained. We assign the novel structure shown in Fig. 1 to 16, fatimanol T.
Compound 17. The HRESIMS contained a pseudomolecular ion at m/z 503.1886 [M + Na]+ (calc 503.1893), confirming the formula C24H32O10. Other positive ions at m/z 983.3881 [2 M + Na]+ (calc 983.3889), m/z 481 [M + H]+, m/z 463.1961 [M + H - H2O]+ (calc 463.1968), m/z 445.1856 [M + H − 2 × H2O]+ (calc 445.1862), m/z 421 [M + H - AcOH]+, m/z 403.1750 [M + H - H2O - AcOH]+ (calc 403.1757), and m/z 361.1644 [M + H − 2 × AcOH]+ (calc 361.1651) confirmed the formula and indicated at least two hydroxy groups and at least two acetate esters. Negative-mode ions confirming the formula were present at m/z 525.1974 [M + formate]− (calc 525.1972), m/z 515.1693 [M + 35Cl]− (calc 515.1684), and m/z 479.1923 [M - H]− (calc 479.1917). The 13C NMR spectrum (in (CD3)2SO) contained individual signals for twenty-four carbon atoms: 3 ⋅ CH3, 6 ⋅ CH2, 8 ⋅ CH, 7 ⋅ Cq. Compound 17 was a conventional neoclerodane. As for 15, the upper part of the structure was a furan linked to a γ-lactone. H-14, H-15, and H-16 resonated at δ 6.51, δ 7.71 and δ 7.79, respectively, with the carbon signals at δ 109.1 (C-14), δ 144.9 (C-15) and δ 141.2 (C-16) (Table S6, SI). C-13 (δ 125.7) was identified through HMBC interactions with H-15 and H-16. HMBC from H-16 also revealed C-12 (δ 74.1), from which H-12 (δ 5.45) was shown by HSQC. COSY then linked H-12 to both H-11 (δ 2.32 and δ 2.45) and thence by HSQC to C-11 (δ 43.1). The chemical shift of H-12 confirmed the lactone and 3-bond HMBC from both H-12 identified the lactone carbonyl C-20 at δ 177.1. The lactone was tied to the decalin through HMBC from the upfield H-11 to C-8 (δ 37.7). H-8 resonated at δ 1.70 and this signal correlated in HMBC with the methyl group (H-17 δ 0.96, C-17 δ 16.8). Strong HMBC cross-peaks linked C-17 to both H-7 (δ 1.54, δ 1.87) and thence by HSQC to C-7 (δ 36.1). HMBC from H-8 and both H-7 to the signal at δ 72.8 identified the latter as C-6, with the chemical shift indicating an oxygen. H-6 (δ 4.20) gave HMBC correlations to the quaternary C-5 (δ 48.2) and C-4 (δ 76.9). The downfield chemical shift of C-4 indicated an oxygen but contraindicated a spiro-oxirane. H-19 of the pendant methylene resonated as a pair of geminally coupled doublets at δ 4.77 and δ 4.86, which were linked by HMBC to C-5 and C-4. The chemical shifts of H-19 suggested an AcO- group and this was confirmed by HMBC to the ester carbonyl 19-MeCO2, which linked onwards to the acetate methyl (δH 2.00, δC 169.9). H-18 / C-18 is a pendant HOCH2- group, with 2-bond HMBC from both H-18 (δ 3.73 and δ 4.04) to C-4 and 3-bond cross-peaks to C-5. Both H-18 resonated as dd, with 2J = 10 Hz and smaller couplings to HO-18 (δ 5.06). This spin-set was confirmed by COSY. A 3-bond HMBC identified C-3 (δ 71.4). H-3 (δ 5.45) had a HMBC cross-peak with a carbonyl at δ 170.4, demonstrating that the oxygen at C-3 was acetylated. The methylenes at position-2 and position-1 were also identified by application of HMBC, (C-2 δ 36, H-2 δ 1.80, δ 2.45; C-1 δ 22.6, H-1 δ 1.95, δ 2.0). The closure of the decalin system was confirmed by a weak HMBC from C-2 to H-10 (δ 1.80) and a strong peak from C-4 to H-10. The above spectroscopic assignments were aided by 1H, COSY, HSQC, and HMBC spectra of a solution in CDCl3, which were better resolved, although shortage of sample precluded identification some quaternary carbons. The relative configurations corresponded to those of most of the neoclerodanes, particularly 15, as demonstrated largely by 1H NMR coupling constants. The structure shown in Fig. 1 was assigned to 17, fatimanol U.
Compound 18. The HRESIMS contained ions at m/z 803.3255 [2 M + Na]+ (calc 803.3255) and m/z 413.1568 [M + Na]+ (calc 413.1576) confirming the formula C21H26O7. An ion was also seen at m/z 371 [M + H]+. The 13C NMR spectrum (in (CD3)2SO) contained discrete signals for twenty-one carbon atoms: 3 ⋅ CH3, 4 ⋅ CH2, 8 ⋅ CH, 6 ⋅ Cq. Combined analysis of the NMR data (Table S6, SI) allowed assignment of all the signals and confirmed that the overall structure was similar to a conventional neoclerodane. However, C-6 was quaternary but not a carbonyl (cf. 2). Two methoxy groups were also evident. The upper part of the structure comprised a furan, with the usual chemical shifts (position-13: δC 125.4; position-14: δH 6.52, δC 109.2; position-15: δH 7.72, δC 145.0; position-16: δH 7.82, δC 141.2). HMBC cross-peaks from C-13 linked this to H-12 (δ 5.48) and to one H-11 (δ 2.42). The other H-11 (δ 2.57) was identified by COSY cross-peaks to H-12 and to its geminal partner H-11. HSQC then located C-12 (δ 71.6) and C-11 (δ 40). The carbonyl (C-20) of the γ-lactone resonated at δ 176.6 and showed an HMBC cross-peak to the downfield H-11. It also gave an HMBC with H-10 (δ 2.45) and both H-11 gave HMBC correlations with C-9 (δ 53.0), confirming the attachment of the spiro-lactone. In the lower bicycle, H-10 also correlated in HMBC with C-8 (δ 35.8), from which H-8 (δ 2.01) and the methyl doublet H-17 (δ 0.92) were identified. COSY from H-8 led to one H-7 (δ 1.87) and thence to its geminal partner H-7 (δ 2.27). HSQC identified C-7 (δ 39.6). Two-bond HMBC linked both H-7 to C-6 (δ 109.16). This chemical shift implied that C-6 was an acetal or hemiacetal carbon, confirmed by a 3-bond HMBC with the upfield methoxy group MeO-6 (δ 2.97). Both H-7 also showed HMBC correlations to C-5 (δ 137.4), an alkene carbon. Thus C-19 is missing from the usual neoclerodane structure. H-10 is present, thus the other alkene carbon is C-4 (δ 139.5). HMBC from C-4 located HO-3 (δ 5.06), from which H-3 was shown (COSY) to be at δ 4.06 and thence C-3 was identified at δ 51.5 by HSQC. COSY joined H-3 to both H-2 (δ 1.39, δ 2.01) and HMBC from HO-4 identified C-2 (δ 33.0). CH2-1 resonated at δH 1.25, δH 2.27, and δC 23.7. In the bottom dihydrofuran, HMBC from C-3 and C-5 identified H-18 (δ 6.02) and HSQC confirmed C-18 (δ 107.0), where the chemical shifts were consistent with an acetal. One arm of this acetal was the oxygen linking through to C-6 and the other was a methoxy (MeO-18) (δH 3.15, δC 51.5), as shown by HMBC to H-18 and C-18.
Turning to the configuration of 18, the 1H chemical shift of MeO-6 was unusually low (δ 2.97). Examination of a molecular model showed that this methyl group, if on the lower face, would be held in the anisotropic shielding zone of the C-20 carbonyl; thus it is likely to be located on the lower face of the fused tricycle. The configuration at the other acetal C-18 could not be determined, although it was clear that only one diastereoisomer was present. A 1H NMR spectrum of 18 was also obtained in CDCl3, which was consistent with the structure determined above. The novel structure shown in Fig. 1 was assigned to 18, fatimanol V.
Compound 19. Instability under MS conditions precluded obtaining useful mass spectra. The 13C NMR spectrum (in (CD3)2SO) (Table S7, SI) contained twenty-two discrete 13C signals: 3 ⋅ CH3, 8 ⋅ CH2, 5 ⋅ CH, 6 ⋅ Cq. The combined spectra showed it to be a modified neoclerodane. The upper ring was a furanone, with the H-16 protons resonating accidentally equivalently as a singlet at δ 4.86, with C-16 at δ 73.53. These chemical shifts suggested a γ-lactone. Three-bond HMBC cross-peaks from H-16 identified lactone carbonyl C-15 (δ 173.4) and C-14 (δ 114.0), with H-15 (δ 5.96) identified by HSQC. A 2-bond HMBC identified C-13 (δ 174.4). Initially, it was unexpected that the resonance for C-13 (an alkene) would be downfield of that for C-15 (a carbonyl) but comparison with the chemical shifts29 for 4-ethyl-5H-furan-2-one gave precedent. Moreover, the assignment was shown to be correct by HMBC from H-12 (δ 2.25) to C-13. HSQC then identified the other H-12 (δ 2.15) and C-12 (δ 21.9). HMBC from both H-12 led to C-11 (δ 34.38), confirmed by interactions of C-12 with both H-11 (δ 1.50, δ 1.98). C-9 resonated at δ 38.0 and was linked by HMBC to the two upfield methyl signals, δ 0.78 for H-17 and δ 0.65 for H-20. A 3-bond HMBC from H-20 led to C-10 (δ 46.3) and thence H-10 (δ 1.38). C-10 formed a HMBC cross-peak to H-8 (δ 1.24), from which C-8 (δ 4.1) was identified by HSQC. Confirmation was supplied by HMBC from C-8 to C-17 and C-20. A weak 4-bond HMBC from H-17 to C-6 (δ 72.7) identified the latter. H-6 resonated at δ 3.63, indicating the OH. HMBC from C-6 to δH 1.43 led to identification of one H-7, from which the other H-7 (δ 1.50) and C-7 (δ 34.41) were correlated by HSQC. C-6 also gave an HMBC cross-peak with H-10, closing the lower-right carbocycle, while a cross-peak from the upfield H-7 located C-5 (δ 45.2). C-5 was shown to carry C-19 by HMBC to both H-19 (δ 4.34, δ 4.49, geminally coupled doublets). Both H-19 linked on to the carbonyl at δ 170.8, demonstrating that C-19 carries an AcO- group. C-5 also gave HMBC cross-peaks to both H-18 (δ 2.84, δ 3.09). HSQC identified C-18 (δ 43.0) and 2-bond HMBC from both H-18 located the signal for C-4 at δ 69.6. A weaker HMBC interaction between C-4 and HO-3 (δ 7.76) linked on to C-3 (δ 64.5) and thence H-3 (δ 3.82). Finally, the remaining methylenes were identified through further HMBC correlations (H-2: δ 1.24, δ 1.55, C-2 δ 34.42; H-1: δ 1.50, δ 1.66, C-1 δ 20.0). The structure shown in Fig. 1 was assigned to 19, fatimanol W.
Compound 20. Instability under MS conditions precluded obtaining useful mass spectra. The 13C NMR spectrum (in (CD3)2SO) (Table S7, SI) contained twenty discrete 13C signals: 2 ⋅ CH3, 8 ⋅ CH2, 5 ⋅ CH, and 5 ⋅ Cq. The combined NMR spectra showed it to be very similar to 19, with similar HMBC and HSQC connectivities. Only the points of significant difference are discussed here. This compound lacked an acetate ester. Relative to 19, the signals for H-19 and C-19 have moved markedly upfield (δH 3.78, δH 3.98, δC 60.3). This was consistent with the C-19 substituent being HO-. This was confirmed by observation of the HO-19 signal as a dd at δ 4.12, with HMBC to C-5 and C-19. Changes in chemical shift were also seen for the oxirane H-18 (δ 2.70, δ 2.89) and C-18 (δ 40.5), and for C-5 (δ 46.9), reflecting changes in steric effects in that region and the absence of through-space effects from an ester carbonyl. Thus 20 (fatimanol X) was the desacetyl analogue of 19, with the structure shown in Fig. 1.
The occurrence of orthoesters in plant natural products was reviewed by Liao et al. in 2008.28 Orthoacetates have been reported in only a limited number of frameworks, principally the daphnane diterpenoids, phragmalin limonoids, bufadienolide and ergostanoid steroids. Several of these diterpene orthoacetates have potent biological activities, including systemic toxicity. In each case, the three oxygen atoms of the orthoacetate unit were situated appositely in space for formation of the orthoester and the rigidity of the framework of the diterpene contributed to the stability of this usually highly acid-labile functionality. We propose the mechanism shown in Figure 3 for formation of the orthoacetate in 1. Proposed intermediate 23 is 19-acetylteulepicin, reported by Savona et al.16 to be a secondary metabolite in T. buxifolium and the formal oxidation product of 2 at the lactol. It is therefore feasible that 23 is the true biosynthetic precursor of 1 in T. yemense. The carbonyl oxygen of the acetate attacks the adjacent electrophilic ketone carbonyl from the lower face, generating intermediate 24. Here the alkoxide anion is held close to the CH2 of the electrophilic oxirane and attacks it, opening the strained ring and forming a new bond. Finally, the new alkoxide in 30 is perfectly placed for attack as the electrophilic carbon of the acetate to form the orthoester 1. Bruno et al.30 observed a related cyclisation from chemical pyrolysis of fruticolone (a constituent of T. fruticans) at 200°C and a chemical acid-catalysed epoxyester-orthoester rearrangement has been reported31 in a synthesis of petuniasterone D.
Neoclerodanes with the A-ring contracted to a cyclopentane, as in 13, have been reported previously as plant natural products but with more complex substitution patterns. Examples with a cyclobutene fused to the cyclopentane were reported in the 1980s.32,33 Ring-contracted neoclerodanes were later identified34-37 from Pteronia, Conyza, and Microglossa species. In these neoclerodanes, there is extensive transannular bridging. Fatimanol Q (13) is the simplest ring-contracted neoclerodane identified to date. Bohlmann’s group suggested that their ring-contracted compounds had arisen from rearrangements involving migration of C-2 to bond with C-4. Mechanisms proposed include protonation of a hydroxy group at C-4 to initiate the rearrangement,34 trapping the aldehyde formed from C-3 with a hydroxy group at C-1034,35 and formation of a C-1 = C-10 double bond.34,36,37 In each case, the C-10 position is oxidised. However, in the present case, C10 is not oxidised with either an oxygen function or an alkene. We propose the mechanism in Figure 4 for the biosynthetic ring-contraction. In this mechanism, the rearrangement is triggered by protonation and ring-opening of the strained oxirane. The leaving group oxygen is on the lower face of the ring and thus almost antiperiplanar to the C-3—C-2 bond. The oxirane-opening and the migration of C-2 are probably concerted, given this conformation. C-2 will approach C-4 from the opposite side to the leaving group, resulting in stereochemical inversion at C-4. This places C-18 on the upper face and the newly generated carbocation (C-3) on the lower face, where it can readily form a hemiacetal with HO-19. This hemiacetal closes the second 5-membered ring such that the two 5-membered rings are cis-fused, an energetically favoured arrangement.
Acid anhydrides are relatively uncommon in natural products, owing to their potential for electrophilic reactivity and hydrolysis. However, we have firmly identified 10 as having a succinic anhydride moiety as the upper ring; this is the first such neoclerodane to be reported. Only one natural product containing a simple (non-fused) succinic anhydride, tubogenic anhydride A, has previously been isolated, from Aspergillus tubingensis.38
Compounds 2,3,7-9,11,12,14-17,19,20 contain the conventional neoclerodane carbon framework with various differences in oxidation level, acetylation, bridging rings and ring-opening at the C-4/C-18 oxirane, whereas 4-6,18 lack C-19. The C-12–C-20 lactol in 2 has precedent in gnaphalin (isolated from T. gnaphalodes),39 although the latter lacks HO-3. The C-19–C-20 bridging lactone of 3,7,10 is present in teulepicephin15 and many other neoclerodanes. Compound 11 contains a related bridging acetal, giving a rigid polycyclic structure. This polycycle is also present in teucrin P1 (also from T. gnaphalodes),40 teupyrenone (from T. pyrenaicum),41 and teupolin III,42 although the latter do not have the additional lower fused tetrahydrofuran to stiffen the structure further. The upper hydroxyfuranone hemiacetal in 7 is also present in the neoclerodane salvidivin (from Salvia divinorum),43 whereas the methoxyfuranone acetal moiety in 8,9 has precedent in the labdane 15-methoxyvelutine C (from Marrubium thessalum).44 Most neoclerodanes have the 12-S configuration, so the identification of 16 as a 12-R neoclerodane is noteworthy. Gács-Baitz et al.42 used NOE NMR spectroscopy to determine configuration at C-12 in neoclerodanes featuring the upper aromatic furan and the spiro-lactone but did not have an exact epimeric pair for their study; 15 and 16 are exact epimers which facilitated their stereochemical identification. The lower hydroxyfuranone hemiacetal of 4,5 and the corresponding methoxyfuranone acetal feature of 6 have scant precedent, in teucvisin C45 and cracroson B,46 respectively, while the dimethoxydihydrofuran diacetal of 18 is completely novel in the series. The ajugamarins and related neoclerodanes have the upper furanone unit of 19 and 20 but all known ajugamarins are oxygenated at C-12.47
Compounds 1–12 were evaluated for their ability to enhance the glucose-triggered secretion of insulin by freshly isolated murine pancreatic islets, using our previous assay.48 In negative-control islets, insulin secretion was 9.1 ± 0.3 ng islet− 1 h− 1, triggered by glucose (16.7 mM) (Fig. 5). This release was increased 2.2-fold by the standard drug tolbutamide (20.2 ± 1.3 ng islet− 1 h1). The tested compounds showed a range of activities. Compounds 1,2,12 showed little or no effect on the secretion of insulin. Compounds 3–6 and 11 increased the glucose-triggered release of insulin by approximately the same extent as the positive control tolbutamide. Encouragingly, 7–10 showed strong enhancement of insulin secretion, by 3–4, although these are not as potent as the coumarins cluteolin D and clueolin J (from Clutia lanceolata).49