Hypulatone C (1) was obtained as yellow gum. Its molecular formula C41H60O5 with 12 degrees of unsaturation was established by analysis of 13C NMR (Table 1) and HRESIMS data (m/z 671.4082, [M + K]+, cacld for 671.4072). The FTIR spectrum displayed absorption bands due to carbonyl (1719 and 1699 cm−1) functionalities. The 1H NMR spectrum (Table 1) exhibited the signals of an isopropyl (δH 3.07, sept; 1.16, d; 1.09, d; J = 6.9 Hz), four olefinic protons (δH 4.51–5.01), and nine singlet methyls (δH 0.64–1.67). Analysis of its 13C and DEPT-NMR data revealed a total of 41 carbon resonances, including a shielded sp2 carbon at δC 115.3 (C-1) and three deshielded carbons at δC 178.7 (C-6), 199.3 (C-2), and 203.2 (C-7), which were indicative of the presence of an enol-β-triketo system. The above signals, in combination with a nonconjugated ketone at δC 207.4 (C-4) and two quaternary carbons at δC 57.4 (C-3) and 62.6 (C-5), suggested that compound 1 should possess a dearomatized acylphloroglucinol core. This assumption was further confirmed by the HMBC correlations from a singlet methyl at δH 1.31 (Me-16) to C-2, C-3, and C-4, and from δH 3.29 and 2.28 (H2-17) to C-4, and C-5, and C-6. An isoprenyl group (δC 39.5, C-11; 118.6, C-12; 135.3, C-13; 26.0, C-14; 17.4, C-15) linked to C-3 was deduced by the correlation of δH 4.82 (H-12)/δH 2.67 and 2.31 (H2-11) in the 1H–1H COSY spectrum, together with the correlations of both δH 1.58 (Me-14) and 1.53 (Me-15) with C-11 and C-12, of H2-11 with C-2, C-3, and C-4 in the HMBC spectrum. Moreover, C-17 was proved to be the head of a geranyl group (δC 40.3, C-17; 116.6, C-18; 141.4, C-19; 17.6, C-20; 39.9, C-21; 26.3, C-22; 123.9, C-23; 131.5, C-24; 25.7, C-25; 17.7, C-26) by the HMBC correlations from δH 1.62 (Me-20) to C-18, C-19, and C-21, from both δH 1.67 (Me-25) and 1.59 (Me-26) to C-23 and C-24, coupled with the proton spin systems of H2-17/δH 4.74 (H-18) and δH 1.97 (H2-21)/δH 2.03 (H2-22)/δH 5.02 (H-23) in the 1H–1H COSY spectrum. The HMBC correlations of both doublet methyls at δH 1.16 and 1.09 with C-7 assigned the location of the isopropyl group (δC 39.8, C-8; 18.5, C-9; 18.1, C-10) (Fig. 3).

1H–1H COSY and HMBC correlations of 1–4

Besides the aforementioned 26 carbon signals in the 13C and DEPT NMR spectra of 1, the remaining 15 resonances assignable to three nonprotonated carbons (δC 135.5, C-8′; 92.4, C-1′; and 33.8, C-4′), three methines (δC 123.6, C-9′; 64.2, C-3′; and 59.4, C-2′), six methylenes, and three methyls indicated a humulane-type sesquiterpenoid moiety. This deduction was further confirmed by the correlations of 2.63 (H-2′)/δH 2.85 (H-3′), and δH 1.24 (H-5′)/δH 1.41 (H-6′)/δH 1.76 (H-7′), and δH 5.13 (H-9′)/δH 2.41 (H-10′)/δH 1.71 (H-11′) in the 1H–1H COSY spectrum, along with the HMBC correlations from δH 2.31 (H-12′) to δC 92.4 (C-1′), 59.4 (C-2′), and 40.3 (C-11′), from both singlet methyls at δH 0.64 (Me-14′) and 1.00 (Me-13′) to δC 64.2 (C-3′), 33.8 (C-4′), and 39.3 (C-5′), and from singlet methyl at δH 1.64 (H-15′) to δC 39.5 (C-7′), 135.5 (C-8′), and 123.6 (C-9′) (Fig. 3).

The linkage of C-5/C-12′ was deduced by the HMBC correlations from H2-12′ to C-4, C-5, and C-6, which combined the acylphloroglucinol and sesquiterpenoid moieties. The formation of the 2′,3′-epoxide and deduced furan ring were indicated by the indices of hydrogen deficiency along with the special chemical shifts of C-1′ (δC 92.4), C-2′ (δC 59.4), C-3′ (δC 64.2), and C-6 (δC 178.7). So far, the planar structure of 1 was elucidated as shown (Fig. 1).

Comparison of the structure of 1 with that of hyperkouytin C (6) [31] indicated that the benzoyl and prenyl groups in 6 were replaced by an isobutyryl and a methyl in 1, respectively. The 13C chemical shifts of carbons around C-1′, C-2′ C-3′, and C-5 chiral centers are very close to those of 6, which suggests that the relative configurations of C-1′, C-2′ C-3′, and C-5 are identical to those of 6. Furthermore, the chemical shift of H-17a (δH 3.29) was 1.01 ppm downfield of H-17b (δH 2.28), indicating that compound 1 had a close O2′/H-17a contact and the geranyl group was located at the same side of C-2′. This deduction was confirmed by a computationally optimized model (Fig. 4) and the crystallographic data of hypulatone B and hyperkouytins A and B [14, 31]. These evidences further confirmed the relative configurations four chiral carbons mentioned above. Finally, the NOESY correlation between Me-16 and H-18 established the configuration of C-3, thus assigning the relative configuration of 1 as 3S*, 5S*, 1'R*, 2'R*, 3'S* (Fig. 4). Considering that all the meroterpenoids of this type were reported in enantiomeric pairs [14, 31], the optical rotation of 1 ([α]D =  + 19) suggested it might be scalemic mixtures. Nevertheless, the lack of sufficient sample quantities precluded the further chiral separation.

Configuration optimized molecular model of 1. yellow arrows, key NOE correlation; yellow dashed line, close O2′/H-17a contact

Hypulatone D (2) was assigned the molecular formula C26H38O5 by analysis of its 13C NMR (Table 2) and HREIMS data (m/z 429.2643 [M − H]−). Comparing the 1H and 13C NMR data of 2 to those of hyperpatulone E [15] indicates that they are structurally similar. The sec-butyl group in hyperpatulone E is replaced by an isopropyl (δH 1.21, d; 1.13, d; 3.51, sept.; J = 6.8 Hz) in 2, as evidenced by the HMBC correlations from Me-25 (δH 1.21) and Me-26 (δH 1.13) to δC 206.7 (C-23). It is worth noting that one could barely determine the relative configuration of spirocyclic PPAPs characterized by six chiral centers, like 2, unless one used a combination of 1H–1H coupling constants, conformational analysis, and NOE correlations. Firstly, in the 1H spectrum of 2 measured in CDCl3 (Table S1), the 3J coupling constant of H-14b (δH 1.23, t, J = 13.0 Hz) was 13.0 Hz. So, the corresponding six-membered ring adopted chair conformations (Fig. 5), and H-14a (δH 2.00, brd, J = 13.0 Hz) and Me-15 were equatorial while H-14b and H-13 (δH 1.25) were axial. Secondly, the NOE contacts of Me-15 with H-11eq (δH 1.86) and of H-11ax (δH 1.11) with H-8 (δH 1.57) indicated the trans configuration of the octahydro-indene moiety, as well as the relationship between H-12 and H-13. Thirdly, due to the constraints of the spirocyclic framework, the phloroglucinol ring is perpendicular to the octahydro-indene moiety, which itself lies nearly coplanar with the plane formed by the two C-6 substituents. The NOE correlations of H-14a/Me-22 (δH 1.43), H-18 (δH 4.70)/H-7b (δH 1.79), and H-7b/Me-16 (δH 1.28) measured in CD3OD defined the relative configurations of C-6 and C-9. Thus, the structure of 2 was determined as shown and named as hypulatone D (Fig. 1).

Configuration optimized molecular model of 2. Pink arrows, coupling constants; yellow arrows, NOE correlations

The molecular formula of hypulatone E (3) was determined as C26H40O6 by analysis of its 13C NMR (Table 2) and HRESIMS data (m/z 447.2745 [M–H]−). The 1H and 13C NMR data of 3 resembled those of hyperhenone E (15) [32]. Instead of two olefinic carbons in hyperhenone E, an oxygen-bearing quaternary carbon at δC 73.2 (C-13) and a methyl at δC 27.2 (Me-14) appeared in 3, suggesting that 3 could be derived from hyperhenone E by adding water across the Δ13,14 double bond of the latter. This suggestion was further supported by the correlations of both Me-14 (δH 1.15) and Me-15 (δH 1.14) with C-13 and C-12 (δC 50.8). The NOE contacts of H-7 (3.18) with H-12 (1.48) and Me-16 (1.16) indicated that the relative configurations of C-7, C-9, and C-12 were identical to those of hyperhenone E. Furthermore, the well matched ECD curves of 3 and hyperhenone E (15) suggested that their absolute configuration of C-5 was identical [32,33,34]. Considering that compounds 3 and 15 were co-isolated and the absolute configuration of 15 was determined by X-ray diffraction data [33], the absolute configuration of 3 could be defined as 5R, 7R, 9R, 12S (Fig. 1).

The molecular formula of hypulatone F (4) was determined to be C35H44O5 from its HRESIMS and 13C NMR data (Table 2). On the basis of analysis of its 1D and 2D NMR data, compound 4 was assigned to possess the same backbone as hypseudohenrin F [35]. The structural novelty of 4 involved the presence of a hemiacetal hydroxyl (δH 3.62, OH-23) rather than a methoxy group, which was confirmed by the 1H–1H COSY correlations of H-22a (δH 2.59) with H-23 (δH 6.05), in combination with the HMBC correlations from H-22a to C-5 (δC 58.4) and C-6 (δC 40.2) and C-9 (δC 204.7), and from H-23 to C-4 (δC 171.8) (Fig. 3). The 2D NMR data showed that the other structural features of 4 were identical to those of hypseudohenrin F.

Twenty-four known compounds were identified as hypulatone A (5) [14], ( +)-hyperkouytin C (6) [31], hypulatone B (7) [14], ( −)-hyperkouytin D (8) [31], tomoeone A (9) [36], tomoeone B (10) [36], chipericumin D (11) [37], chipericumin E (12) [38], hypercohone G (13) [39], spirohypatone A (14) [13], hyperhenone E (15) [32], bellumone I (16) [40], hyphenrone J (17) [23], hyphenrone K (18) [23], hyphenol J (19) [34], uralione E (20) [41], hookerione K (21) [42], attenuatumione D (22) [43], sampsonione H (23) [44], hypersampsone D (24) [45], sampsonione D (25) [44], sampsonione C (26) [44], hypersampsone I (27) [46], and hypersampsonone G (28) [47], by comparison of their spectroscopic and physical data with those of related literature (Fig. 2).

All the isolates (compounds 1–28) were tested for their cytotoxic activities on Huh-7 and Panc-1 cell lines by CCK-8 assay. Sorafenib and paclitaxel were used as the positive control. As shown in Table 3, compounds 6, 8, and 15 showed moderate inhibitory activity against two human cancer cell lines with IC50 values in the range of 9.7–19.2 µM.

In summary, four previously undescribed PAPs, hypulatones C–F (1–4), together with twenty-four known analogues, were isolated from the fruit of Hypericum patulum and structurally characterized. Compounds 6, 8, and 15 showed moderate inhibitory activity against two human cancer cell lines with IC50 values in the range of 9.7–19.2 µM. Our findings enriched the structural diversity of natural PAPs, and also provided a useful method for configurational assignments of spirocyclic PPAPs that bear six chiral centers.