Dearomatized Isoprenylated Acylphloroglucinol Derivatives with Potential Antitumor Activities from Hypericum henryi

A series of dearomatized isoprenylated acylphloroglucinols derivatives, hyperhenols A–E (1–5), as well as seven known analogues (6–12), were characterized from Hypericum henryi. Their structures were determined by combination of NMR, ECD spectroscopy, and X-ray diffraction analysis. Compounds 1 and 6–8 were tested to exhibit potential antitumor properties, of which 6 and 7 inhibited cell growth through inducing apoptosis and cell cycle arrest. In addition, these compounds could induce autophagy and PINK1/Parkin-mediated mitophagy in cancer cell lines, as well as suppress lung cancer A549 cells metastasis in vitro. Electronic supplementary material The online version of this article (10.1007/s13659-019-00229-w) contains supplementary material, which is available to authorized users.


Introduction
Natural phloroglucinol derivatives are widely distributed in Myrtaceae, Guttiferae, Euphorbiaceae, Aspidiaceae families as well as appeared in marine and microbial sources [1]. In which prenylated acylphloroglucinols are a special kind of hybrid natural products originated from a polyketide combined isoprenylation biosynthetic pathways, and were mainly reported from the plants of genus Hypericum and Garcinia in the family Guttiferae [2][3][4]. With their wide range of biological profiles and diverse molecular architectures exemplified by hyperforin [5], hypersubone B [6], hyperuralone A [7] and chinesins Ι/ΙΙ [8], prenylated acylphloroglucinol derivatives have attracted great interest of chemists and pharmaceutists.
As a traditional folk medicine in China, Hypericum henryi has been used to treat hepatitis [9]. Previous investigations on this plant have reported structurally diverse polycyclic polyprenylated acylphloroglucinols (PPAPs) such as hyphenrones A-X [10][11][12]. As a part of systematic search on bioactive acylphloroglucinol derivatives, five new dearomatized isoprenylated acylphloroglucinols (DIAPs) derivatives, hyperhenols A-E (1-5) together with seven known analogues (6)(7)(8)(9)(10)(11)(12) were isolated from H. henryi (Fig. 1). In the bioactive study, compounds 1 and 6-8 were found to exhibit promising cytotoxic activities against three human cancer cell lines in vitro. And the further studies indicated compounds 6 and 7 could trigger autophagy, PINK1/Parkinmediated mitophagy in cancer cell lines, and also suppress lung cancer A549 cells metastasis targeting Akt and cofilin signaling pathways. In addition, 6 and 7 also displayed significant anti-proliferation activities by inducing apoptosis and cell cycle arrest. Herein, the isolation, structure elucidation, and bioactivities evaluation of these compounds were reported.
The relative configuration of monoterpenoid moiety was determined by detailed interpretation of the ROESY spectrum. The NOE correlations of H-5′/H-1′ and H-1′/Me-7′ indicated that H-5′, H-1′ and Me-7′ were on the same side. However, due to the rotation of carbon-carbon single bond (C-3/C-1′) between DIAP core and monoterpenoid, determination of the configuration of C-5 is still challenging. For instance, an analogue of 1 (hyperhenone E, 8), has also been reported with the configuration of C-5 undefined [12]. In this study, hyperhenone E, as well as its crystals, was fortunately obtained, which unambiguously determined absolute configurations of 8 as 5S, 1′R, 2′R, 5′S (Fig. 3). Furthermore, the absolute configurations of C-5, C-1′, C-2′ and C-5′ in 1 were also determined to be the same with those of 8 via their well-matched ECD curves (Fig. 4).
Hyperhenol B (2) was obtained as yellow oil. A molecular formula of C 33 H 42 O 5 , was deduced by its 13 [12]. Comparative analyses of their NMR data revealed that the isopropyl in hyperhenone F was replaced by a phenyl, which was supported by the HMBC correlations from H-9/H-13 (δ H 7.43) to C-7 (δ C 195.5) and C-8 (δ C 139.2) in 2 (Fig. 2). Because of the rigidity of the bicyclic skeleton, cyclohexane moiety tended to form boat conformation. Hence, the ROESY cor-  .2843), implying 9 indices of hydrogen deficiency. The characteristic information for a DIAPs core was clearly observed in the 13 C NMR spectra (δ C 106.5, C-1; δ C 189.2, C-2; δ C 103.3, C-3; δ C 170.6 C-4; δ C 52.4 C-5; δ C 196.4 C-6). A comparison of the 1D NMR data of 4 with those of chinesin Ι suggested that they shared closely similar plane structures [8]. The molecular formulas (C 27 H 40 O 4 for chinesin Ι; C 27 H 38 O 4 for 4) revealed that 4 possessed one more degree of unsaturation [8], which could derived by the loss of H 2 O between hydroxyls of monoterpenoid and DIAPs core of chinesin Ι to afford 4. The ether linkage of C-4 and C-3′ was evidenced by indices of hydrogen deficiency, the downfield chemical shift of C-3′ (δ C 86.8) and the ROESY correlation of Me-7′/Me-17. The relative configurations of C-2′, C-3′, and C-6′ were elucidated by key ROESY correlations of Me-7′/H-2′, Me-7′/H-6′, and H-2′/H-6′. Unfortunately, the configuration of C-5 also could not be determined since the absence of sufficient evidence.
Hyperhenol E (5)   In the searching for their anticancer properties, compounds 1 and 6-8 were found to effectively inhibit cell growth in HeLa, A549, and MDAMB-231 cell lines (Table 3). Of which 6 and 7 could significant inhibit cancer cells growth with the IC 50 up to 0.07 and 0.09 μM, respectively. Both the two compounds could also obviously increase mitochondrial fission and further activated the caspase-3, caspase-9, and increased PARP cleavage in HeLa cells (Fig. 6a, c). Treatment with 6 and 7 also increased the percentage of cells in G0/G1 phase and decreased in G2/M phase (Fig. 6b). Moreover, western blot results indicated that these two compounds efficiently suppressed the expression of cyclin D1 and Cdk 6 in HeLa cells, suggesting 6 and 7 induced cell cycle arrest. (Fig. 6c). Taken together, these results demonstrated that these compounds inhibited cell growth through inducing apoptosis and cell cycle arrest.
Autophagy is widely implicated in human diseases, offering a potential target for drug discovery [15]. Then, the effects of 6 and 7 on autophagy were assessed. GFP-LC3 puncta were significantly increased upon these compounds treatment (Fig. 7a). Western blot analysis showed that 6 and 7 inhibited autophagy, as assessed by the increased expression of LC3 II and P62 (Fig. 7b). Similar to CCCP (mitophagy inducer) treatment, 6 and 7 also increased the YFP-Parkin puncta formation (Fig. 7c). These data suggested that the compounds could induce PINK1/Parkinmediated mitophagy. In addition, the antimetastasis effects  of these compounds were also studied. As shown in Fig. 8, wound healing and migration assay suggested 6 and 7 could efficiently suppress cell metastasis consistent with sorafenib (SFB) treated, which also decreased the expression of vimentin, p-AKT and cofilin (Fig. 8). Together, these results indicated that these isolates could suppress lung cancer A549 cells metastasis in vitro and may affect tumor metastasis targeted by Akt and cofilin signaling pathways. In summary, five new and seven known DIAPs derivatives were isolated from H. henryi. Structurally, these compounds were characterized by a dearomatized isoprenylated acylphloroglucinol core combined a functionalized cyclohexane or cyclopentane skeleton. It is worthy to note that several isolates exhibited significant cytotoxic activities in vitro. In addition, they also possess inducing autophagy, mitophagy, and anti-metastasis activities, which provided sufficient information on the potential application of these compounds on future drug development. Therefore, the finding of these DIAPs derivatives with potential antitumor properties may provide a new clue for the discovery of antitumor lead compounds, which should attract great interest from pharmacological and total synthetic communities.

General Experimental Procedures
Optical rotations were measured on a Jasco P-1020 polarimeter. UV spectra were detected on a Shmadzu UV-2401PC spectrometer. IR spectra were determined on a Bruker FT-IR Tensor-27 infrared spectrophotometer with KBr disks. All 1D and 2D NMR spectra were recorded on Bruker DRX-600 spectrometers using TMS as an internal standard. Unless

Plant Material
The plants of Hypericum henryi were collected in Dongchuan prefecture (Yunnan Province, People's Republic of China) in September 2018. The plant was identified by ZHANG Yong-Zeng. A voucher specimen (No. 2018H01) was deposited in Kunming Institute of Botany.

Cell Culture
HeLa cells, GFP-LC3 HeLa cells, YFP-Parkin HeLa cells and A549 cells were maintained in DMEM (Gibco, 1 3 D11527) supplemented with 10% fetal bovine serum, FBS (HyClone, SV30160.03) and 100 U/mL penicillin-streptomycin (Gibco/Invitrogen, 15,140-122) in a humidified atmosphere containing 5% CO 2 at 37 °C. 50 The cells were seeded in a 96-well tissue culture plate at a predetermined density in 100 μL of complete medium, attached overnight, and then treated with a series of concentrations of compound for 72 h. At the end of the incubation period, 10 μL MTT solution was added into each well of a 96-well plate for 4 h at 37℃. After the medium was removed, 100 μL DMSO was added to dissolve the purple crystals. After shaking for 5 min, the optical densities at 490 nm were measured using a Microplate Reader.

MitoTracker Red Staining
HeLa cells were seeded on coverslips and treated with compounds 6 and 7 for 48 h. We then removed the media from the dish and added staining solution containing MitoTracker red (100 nM) incubation 30 min at 37 °C. The cells were fixed with 4% PFA in PBS for 15 min and observed using a fluorescence microscope.

Flow Cytometry Analysis
HeLa cells were treated with various concentrations of 7 and 8 for 48 h. Subsequently, the cells were harvested, washed with PBS and fixed with 70% alcohol at 4 °C overnight. Then cells were washed with PBS and stained with 20 μg/mL PI/RNase staining buffer for 30 min and analyzed using FACSCalibur flow cytometer (Becton Dickinson, USA).

Immunofluorescence Microscopy
The GFP-LC3 or YFP-Parkin HeLa cells were treated with compounds for the indicated time point, and then the cells were fixed with 4% PFA in PBS for 15 min at room temperature. The cells were observed under a fluorescence microscope (Olympus, IX83).

Wound Healing Assay
Wound healing was used to evaluate cell motility as our previous study [16]. Briefly, A549 cells were seeded into a 24-well culture plate. When the cells grew to 90% confluence, then a scratch was gently created through the cell monolayer by sterile 10 μL pipette tips and loose cells were washed away. The cell migration was observed and imaged under an IX83 microscope for each condition and timepoint (0, 48 h). (Olympus, Tokyo, Japan).

Cell Migration Assay
Cell migration assay were performed as described previously [17]. In brief, cell migration was estimated using transwell chambers (Millicell, Germany) with a pore size of 8 μM. For the migration assay, 4.5 × 104 A549 cells resuspended in 100 μL serum-free medium were seeded in the upper chamber with serum-containing medium in the lower chamber of 24-well transwell plates (BD Biosciences, San Jose, CA). After 24 h, the experiment was terminated by wiping the cells from the wells with a cotton swab and fixed and stained with 0.05% crystal violet for 20 min, scored under a light microscope in five random fields.

Western Blotting Analysis
Cells were harvested and lysed in a lysis buffer (62.5 mM Tris at pH 6.8, 20% glycerol, 2% SDS, phosphatase inhibitor), proteins were separated on SDS polyacrylamide gels and transferred to PVDF membranes (Millipore, Billerica, MA, USA). The membranes were blocked with 5% nonfat milk, and immunoblotted with primary antibodies at 4 °C overnight. After washed three times with TBST, membranes were incubated for 1 h with appropriate secondary antibodies at room temperature. otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/4.0/.