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

Ya-Li Hu, Xing-Ren Li and Gang Xu*

Abstract:Carascynol A, an unprecedented 4/9/8 ring system hybrid with a peroxide bridge, was characterized from Hypericum ascyron. The architecture contains a caryophyllane-type moiety and a C6 unit derived from polyprenylated acylphloroglucinols. Its structure and absolute configuration were determined by comprehensive spectroscopic and X-ray diffraction data. Biologically, compound 1 inhibited cell proliferation in LoVo, SW480, and HCT116 cell lines(IC50 = 12.30–24.57 μM).

Keywords: Hypericum ascyron, Caryophyllane, Polyprenylated acylphloroglucinols, Cytotoxicity, Colon cancer

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].

Fig. 1 Structure of carascynol A (1)

Polycyclic polyprenylated acylphloroglucinols (PPAP),a special class of structurally diverse and biologically broad natural products, are rich in the plants ofHypericum. As one of the most widely distributedHypericumspecies in China,Hypericum ascyronis a medicinal herb used in the treatment of abscesses and wounds [5]. Our previous studies on this plant have led to the characterization of someseco- andnor-PPAPs with anti-cancer activities [6, 7]. As a part of our systematic search for novel and bioactive natural PPAPs fromHypericumplants, further investigation onH. ascyronobtained an unprecedented hybrid condensed by a caryophyllanetype sesquiterpenoid and an uncommon C6unit (Fig. 1).It is proposed that the C6unit was derived from polyprenylated acylphloroglucinols by a cascade of ringcontracting isomerization, addition, and degradation reactions. Herein, the isolation, structure elucidation,plausible biosynthetic pathways, and biological evaluation of compound 1 were elaborated in this paper.

2 Result and discussion

2.1 Structure elucidation

Carascynol A (1) was obtained as colorless needles crystals. Its molecular formula C21H34O4was established by its13C 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. Its1H NMR spectrum exhibited two olefinic protons (δH4.82 and 4.96) and five methyl singlets (δH0.96, 1.00, 1.01, 1.21, and 1.40). The13C NMR and DEPT data presented a total of 21 carbon signals,including one non-conjugated carbonyl (δC207.4), four quaternary carbons (including one unsaturated hydrocarbon atδC151.7 and two oxygenated ones atδC88.0 and 83.9), four methines (including an oxygenated one atδC71.1), seven methylenes (including an olefinic one atδC110.5), and five methylenes (Table 1).

The correlations from a gem-dimethyl atδH1.00 (Me-12) and 0.96 (Me-13) to C-1 (δC57.9)/C-10 (δC36.3)/C-11 (δC34.6), from H2-10 (δH1.75 and 1.60) to C-9 (δC41.3)/C-11, and from H-9 (δH2.42) to C-1/C-10 in the HMBC spectrum, combined with the correlations of H-1 (δH1.66)/H-9/H2-10 in the1H-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 the1H-1H COSY correlations of H2-2 (δH1.43 and 1.64)/H2-3(δH1.44 and 1.88) and H-5 (δH2.79)/H2-6 (δH1.59 and 1.99)/H2-7 (δH1.93 and 2.10), together with the HMBC correlations from H2-2 to C-1, from H2-14 (δH4.82 and 4.96) to C-1/C-7 (δC36.2)/C-8 (δC151.7)/C-9 (δC41.3),and from Me-15 (δH1.21) to C-3 (δC39.6)/C-4 (δC88.0)/C-5 (δC52.3) (Fig. 2a).

Table 1 13C (150 MHz) and 1H (600 MHz) NMR spectroscopic data of compound 1 in CDCl3

Besides the existence of a caryophyllane-type sesquiterpenoid monomer, the remaining 6 carbons were connected by the correlations of H-5 to C-2′ (δC207.4), H2-3′(δH2.51 and 2.55) to C-2′/C-4′ (δC71.1), and Me-6′/7′ (δH1.40 and 1.01) to C-4′/C-5′ (δC83.9) 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 C6unit 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 atδC88.0 and 83.9, respectively, while a hydroxyl group was located at C-4′ (δC71.1). 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 Kαradiation. The XRD result confirmed the planar structure (Fig. 2b).

Fig. 2 Key 2D NMR correlations (a) and ORTEP drawing (b) of 1

In the ROESY spectrum (Fig. 2a), the cross-peaks of H-7b (δH1.93)/H-5, H-5/H-4′ (δH4.64), and H-7a (δH2.10)/H-9 suggested that H-5 and H-4′ placed in the same orientation, while H-9 adopted the opposite orientation and was assigned asα. 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 asα-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).

Scheme 1 Plausible biosynthetic pathway to compound 1

Table 2 Cytotoxicity of compound 1

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 IC50values 12.30 ± 0.19 μM, while exhibited weaker cytotoxicity to SW480 and HCT116 cell lines (IC5018.33 ± 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 andβ-caryophyllene unit has been described previously [9, 17]. However, it is the first time that the condensation of a sesquiterpenoids unit with an unusual C6polyprenylated acylphloroglucinols degraded moiety inHypericumspecies 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 IC50values 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.

Supplementary Information

The online version contains supplementary material available at https:// doi.org/ 10. 1007/ s13659- 022- 00362-z.

Additional file 1.The details of isolation and biological experimental procedures, physical and crystal data, and original NMR and MS spectra.

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.

Author details

1State Key Laboratory of Phytochemistry and Plant Resources in West China,Kunming Institute of Botany, Chinese Academy of Sciences, and Yunnan Key Laboratory of Natural Medicinal Chemistry, Kunming 650201, Yunnan,People’s Republic of China.2University of Chinese Academy of Sciences,Beijing 100049, People’s Republic of China.

Received: 16 October 2022 Accepted: 25 October 2022