Carascynol A, a hybrid of caryophyllane-type terpenoid and a C₆ unit degraded by polyprenylated acylphloroglucinols from Hypericum ascyron

1 Introduction

Colorectal cancer (CRC) ranks as the second most lethal cancer and the third most prevalent malignant tumor worldwide [1]. Colon cancer, one of three types of CRC, accounts for the highest percentage of incidence and mortality rate [1, 2]. For cancer patients, surgery and chemotherapy are usually the first choices. Current chemotherapy includes single-agent therapy, mainly fluoropyrimidine (5-FU)-based, and multiple-agent regimens containing one or several drugs [3]. Due to chemical and biological diversity, natural products have always been a major source for pharmacotherapy, especially for cancer diseases [4].

Polycyclic polyprenylated acylphloroglucinols (PPAP), a special class of structurally diverse and biologically broad natural products, are rich in the plants of Hypericum. As one of the most widely distributed Hypericum species in China, Hypericum ascyron is a medicinal herb used in the treatment of abscesses and wounds [5]. Our previous studies on this plant have led to the characterization of some seco- and nor-PPAPs with anti-cancer activities [6, 7]. As a part of our systematic search for novel and bioactive natural PPAPs from Hypericum plants, further investigation on H. ascyron obtained an unprecedented hybrid condensed by a caryophyllane-type sesquiterpenoid and an uncommon C₆ unit (Fig. 1). It is proposed that the C₆ unit was derived from polyprenylated acylphloroglucinols by a cascade of ring-contracting isomerization, addition, and degradation reactions. Herein, the isolation, structure elucidation, plausible biosynthetic pathways, and biological evaluation of compound 1 were elaborated in this paper.

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2 Result and discussion

2.1 Structure elucidation

Carascynol A (1) was obtained as colorless needles crystals. Its molecular formula C₂₁H₃₄O₄ was established by its ¹³C NMR and HRESIMS data (m/z 373.2351, [M + Na]⁺, calcd for 373.2355), corresponding to 5 indices of hydrogen deficiency. The IR absorptions implied the presence of hydroxy (3430 cm⁻¹), carbonyl (1712 cm⁻¹), and terminal double-bond (3086, 1643, and 908 cm⁻¹) groups. Its ¹H NMR spectrum exhibited two olefinic protons (δ_H 4.82 and 4.96) and five methyl singlets (δ_H 0.96, 1.00, 1.01, 1.21, and 1.40). The ¹³C NMR and DEPT data presented a total of 21 carbon signals, including one non-conjugated carbonyl (δ_C 207.4), four quaternary carbons (including one unsaturated hydrocarbon at δ_C 151.7 and two oxygenated ones at δ_C 88.0 and 83.9), four methines (including an oxygenated one at δ_C 71.1), seven methylenes (including an olefinic one at δ_C 110.5), and five methylenes (Table 1).

The correlations from a gem-dimethyl at δ_H 1.00 (Me-12) and 0.96 (Me-13) to C-1 (δ_C 57.9)/C-10 (δ_C 36.3)/C-11 (δ_C 34.6), from H₂-10 (δ_H 1.75 and 1.60) to C-9 (δ_C 41.3)/C-11, and from H-9 (δ_H 2.42) to C-1/C-10 in the HMBC spectrum, combined with the correlations of H-1 (δ_H 1.66)/H-9/H₂-10 in the ¹H-¹H COSY spectrum, suggested the presence of a cyclobutane, which is characteristic for caryophyllene with 4/9-fused ring nucleus. Another nine-membered ring was established by the ¹H-¹H COSY correlations of H₂-2 (δ_H 1.43 and 1.64)/H₂-3 (δ_H 1.44 and 1.88) and H-5 (δ_H 2.79)/H₂-6 (δ_H 1.59 and 1.99)/H₂-7 (δ_H 1.93 and 2.10), together with the HMBC correlations from H₂-2 to C-1, from H₂-14 (δ_H 4.82 and 4.96) to C-1/C-7 (δ_C 36.2)/C-8 (δ_C 151.7)/C-9 (δ_C 41.3), and from Me-15 (δ_H 1.21) to C-3 (δ_C 39.6)/C-4 (δ_C 88.0)/C-5 (δ_C 52.3) (Fig. 2a).

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Besides the existence of a caryophyllane-type sesquiterpenoid monomer, the remaining 6 carbons were connected by the correlations of H-5 to C-2′ (δ_C 207.4), H₂-3′ (δ_H 2.51 and 2.55) to C-2′/C-4′ (δ_C 71.1), and Me-6′/7′ (δ_H 1.40 and 1.01) to C-4′/C-5′ (δ_C 83.9) in the HMBC spectrum. Additionally, a changeable proton and three additional oxygen atoms were unassigned in the molecular formula of 1. Except for three unsaturated degrees attributed to the caryophyllane unit and one deficiency due to the carbonyl group, there should be one unsaturated degree left, which indicated that the C₆ unit should be involved in further cyclization. We assumed that a peroxide bridge should lie between C-4 and C-5′ according to their apparently downfield chemical shifts at δ_C 88.0 and 83.9, respectively, while a hydroxyl group was located at C-4′ (δ_C 71.1). The single crystals of 1 [Flack parameter = − 0.09(10), CCDC 2212335] were obtained in methanol and subjected to an X-ray diffraction experiment with Cu Kα radiation. The XRD result confirmed the planar structure (Fig. 2b).

In the ROESY spectrum (Fig. 2a), the cross-peaks of H-7b (δ_H 1.93)/H-5, H-5/H-4′ (δ_H 4.64), and H-7a (δ_H 2.10)/H-9 suggested that H-5 and H-4′ placed in the same orientation, while H-9 adopted the opposite orientation and was assigned as α. This deduction was consistent with the XRD result (Fig. 2b). Consequently, the absolute configuration of 1 was determined as 1R, 4R, 5R, 9S, 4′R.

Polycyclic polyprenylated acylphloroglucinols (PPAP) possess highly oxygenated acylphloroglucinol-derived cores decorated with isoprenyl or geranyl side chains. Biosynthetically, prenylation of the acylphloroglucinols core moiety affords monocyclic polyprenylated acylphloroglucinols (MPAPs), which may be further cyclized to PPAP-type metabolites with diverse carbon skeletons [8-13]. In this study, we reckon that prenylation of acyphloroglucinols could also obtain polyprenylated acyphloroglucinols metabolites such as α-acids, which then naturally isomerized as its ring-contracted isomer (iso-α-acids) [14, 15]. Subsequently, iso-α-acids underwent an addition reaction with caryophyllene to form an intermediate i, which was oxidized to create a peroxide ii. Then, ii performed a degradation to produce iii and a humulinic acid [16]. Finally, the epoxidation of iii generated iv, which was cyclized to afford 1 (Scheme 1).

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2.2 Biological activity

The cytotoxicity of compound 1 against three human colon cancer cell lines (HCT116, SW480, and LoVo) was evaluated with fluorouracil (5-FU) as the positive control. As shown in Table 2, compound 1 showed the strongest activity against LoVo with IC₅₀ values 12.30 ± 0.19 µM, while exhibited weaker cytotoxicity to SW480 and HCT116 cell lines (IC₅₀ 18.33 ± 1.68 and 24.57 ± 3.09 µM). Compound 1 did not significantly alter the viability of PBMCs, suggesting its selective cytotoxicities on colon cancer cells.

3 Conclusion

Actually, the hybridization of acylphloroglucinols core and β-caryophyllene unit has been described previously [9, 17]. However, it is the first time that the condensation of a sesquiterpenoids unit with an unusual C₆ polyprenylated acylphloroglucinols degraded moiety in Hypericum species has been reported. Biogenetically, β-caryophyllene and polyprenylated acylphloroglucinols conjugated through a nucleophilic addition to produce a key intermediate, which upon multistep degradation, oxidation, and cyclization afforded 1. Besides, our study revealed that compound 1 exhibited cytotoxicities on LoVo, SW480, and HCT116, with IC₅₀ values in the range of 12.30–24.57 µM. To sum up, the present study may provide a new perspective for the structural and biological explorations of the terpenoid and PPAP hybrids.

Notes

Acknowledgements

This work was financially supported by the NSFC-Joint Foundation of Yunnan Province (Grant No. U1902213) the Second Tibetan Plateau Scientific Expedition and Research (STEP) program (Grant No. 2019QZKK0502-0303), Natural Science Foundation of Yunnan (Grant No. 2019FA003), CAS Interdisciplinary Innovation Team of CAS "Light of West China" Program, and the Fund of State Key Laboratory of Phytochemistry and Plant Resources in West China (Grant No. E0230211Z1).

Author contributions

All authors conceived and designed the study and experiments. HYL conducted the experiments, analyzed the results, and wrote the manuscript. LXR proposed the biosynthetic pathways. XG designed the project and revised the manuscript. All authors read and approved the final manuscript.

Declarations

Competing interests

The authors declare that there are no competing interests associated with this work.