Gerardiins A–L and Structurally Related Phenanthrenes from the Halophyte Plant Juncus gerardii and Their Cytotoxicity against Triple-Negative Breast Cancer Cells

Species in the Juncaceae accumulate different types of secondary metabolites, among them phenanthrenes and 9,10-dihydrophenanthrenes in substantial amounts. These compounds have chemotaxonomic significance and also possess interesting pharmacological activities. The present study has focused on the isolation, structure determination, and pharmacological investigation of phenanthrenes from Juncus gerardii. Twenty-six compounds, including 23 phenanthrenes, have been isolated from a methanol extract of this plant. Twelve compounds, the phenanthrenes gerardiins A–L (1–12), were obtained as new natural products. Eleven phenanthrenes [effusol (13), dehydroeffusol (14), effususin A (15), compressin A, 7-hydroxy-2-methoxy-1-methyl-5-vinyl-9,10-dihydrophenanthrene, juncusol, 2-hydroxy-7-hydroxymethyl-1-methyl-5-vinyl-9,10-dihydrophenanthrene, 2,7-dihydroxy-5-formyl-1-methyl-9,10-dihydrophenanthrene, effususol A, 2,7-dihydroxy-5-hydroxymethyl-1-methyl-9,10-dihydrophenanthrene, and jinflexin C], 1-O-p-coumaroyl-3-O-feruloyl-glycerol, and the flavones apigenin and luteolin were isolated for the first time from this plant. The cytotoxicity of the 23 isolated phenanthrenes in both mouse (4T1) and human (MDA-MB-231) triple-negative breast cancer cells and in a nontumor (D3, human cerebral microvascular endothelial) cell line was tested using an MTT viability assay. The results obtained showed that the dimeric compounds gerardiins I (9), J (10), K (11), and L (12), derived biogenetically from effusol and dehydroeffusol, were cytotoxic to both tumor and nontumor cell lines, while the monomeric compounds exerted no or very low cytotoxicity. Impedance measurements were consistent with the results of the MTT assays performed.

T he plant family Juncaceae is an abundant source of phenanthrene derivatives. These compounds can be found in almost all parts (e.g., roots, medulla, leaves) of the plants belonging to this family. 1 In terms of biosynthesis, phenanthrenes and dihydrophenanthrenes are classified as stilbenoids, with 9,10-dihydrophenanthrenes being formed from bibenzyls by an oxidative coupling reaction. These compounds are thus products of the phenylpropanoid metabolism in combination with polyketide formation. Phenanthrenes are synthesized by stilbene synthase from cinnamic acids through stilbene precursors. 2 The biosynthesis of these compounds can be enhanced by different stress conditions, such as fungal infection or wounds or by increases in the salt concentration of the soil. 3 Phenanthrenes occurring in Juncaceae species have chemotaxonomic significance, as several of them contain a vinyl group in the molecule. To date, almost 100 monomers and dimers, substituted with methyl, hydroxy, vinyl, methoxy, oxymethylene, methoxyethyl, ethoxyethyl, and formyl groups, have been isolated from different Juncus and Luzula species. 1 Phenanthrenes possess a wide range of biological activities, including antimicrobial, antiproliferative, anti-inflammatory, anxiolytic, and spasmolytic effects. 4 In a continuation of our work aiming at the isolation of biologically active compounds from Juncaceae species, the halophyte plant Juncus gerardii Loisel. was investigated. Halophytes are specialized plants able to survive and thrive in saline soils. Apart from their physiological adaptation, improved biochemical strategies such as improved antioxidant capacity and transporters determine the tolerance against oxidative stress caused by high salinity conditions. 5 Moreover, these antioxidant systems, including enzymes and bioactive unambiguously showed that it is attached to C-12. NOE correlations of H-4/H-5, H-6/H 2 -12, H 2 -12/H-14b, H 2 -9/H-13, and H 2 -10/H 3 -11 supported the above findings and afforded the structure of gerardiin B (2), as shown.
Compound 5 (gerardiin E) was obtained as a colorless, amorphous solid. Its molecular formula was determined as C 17  . The presence of four aromatic methines (two meta-and two ortho-coupled), two saturated methylenes, and one methyl group in the 1 H NMR spectrum revealed that 5 is a 1,2,5,7-tetrasubstituted 9,10dihydrophenanthrene derivative (Table 2). However, the lack of any characteristic resonances of a vinyl moiety, and the    6 Hz in 6 vs 6.71 and 7.13, each 1H, d, J = 8.4 Hz in 5) clearly suggested that the sugar unit is attached to the skeleton at C-2 (δ C 153.9) ( Table  2). The position of the β-D-glucose was substantiated by HMBC correlations of C-2 (δ C 153.9) with H-4, H 3 -11, and the anomeric H-1′ and by the NOE cross-peak between H-3/ H-1′, and the structure of 6 was proposed as shown.
Gerardiin G (7) was shown to be a structural isomer of 6 by the sodium adduct HRESIMS ion at m/z 455.1675 [M + Na] + (calcd C 23 H 28 O 8 Na, 455.1682). As in the case of gerardiin D, the position of the β-D-glucose at C-7 (δ C 155.5) was determined with the aid of diagnostic HMBC and NOE crosspeaks, leading to the structure of 7 as shown.
Compound 11 (gerardiin K) was obtained as an amorphous solid. According to its protonated molecular ion peak seen at m/z 505.2375 [M + H] + (calcd C 34 H 33 O 4 , 505.2379) in the HRESIMS, the molecular formula of C 34 H 32 O 4 was assigned to this compound. The JMOD spectrum displayed 34 signals, which suggested that compound 11 is also a phenanthrene dimer ( Table 5). The subunits were identified based on their 1D NMR data as effusol (15). The HMBC cross-peak of C-7 (δ C 156.9) with H-12′ (δ H 5.80), and a strong NOE from H-6 to H-12′ revealed that the monomers are linked through an ether bond formed between the OH-7 group of one effusol monomer and the vinyl side chain of the other effusol molecule. The specific optical rotation of 11 was recorded as zero. By HPLC investigation on chiral stationary phase, only one peak was observed. Accordingly, the structure determined for gerardiin K (11) is as shown.
Gerardiin A (1) and gerardiin B (2) are substituted with a methoxymethylene group at C-1 (1) or C-7 (2). The structure of compound 1 is very similar to that of effusol, with the only difference being the presence of a methoxy group at C-11. Gerardiins C (3) and D (4) are glycosides of effusol, substituted with a D-glucose unit at C-2 (3) or C-7 (4), respectively. Gerardiins F (6) and G (7) are also substituted with a D-glucose moiety. Gerardiin E (5) contains a hydroxyethyl group at C-5, instead of a vinyl group. The only difference between gerardiin H (8) and juncunol, isolated previously from other Juncus species (J. acutus, J. ef f usus, J. roemerianus, J. subulatus), 10,17−19 is the presence of an unsaturated B ring in the former phenanthrene.
Phenanthrenoid dimers represent a rare class of secondary metabolites; to date, less than 20 have been reported from species in the plant family Juncaceae. In gerardiins I (9) and J (10), the two effusol monomers are connected through their vinyl groups. Gerardiin K (11) is composed of two effusol monomers that are joined through an ether bond, while in gerardiin L (12) an effusol and a dehydroeffusol unit are attached via a C−C linkage formed between C-8−C-8′. The individual monomers [effusol (13) and dehydroeffusol (14)] were also isolated from the plant.
In order to gain insight into the biological effects of the isolated phenanthrenes, 4T1 mouse breast cancer cells were treated with the isolated compounds, and changes in the viability and impedance were assessed, which reflects proliferation, degree of adhesion, spreading, and viability of the cells. At a concentration of 20 μM, compounds 1−8 (gerardiin A−H) had no cytotoxic effects on 4T1 cells, as assessed by an MTT assay ( Figure S73A, Supporting Information) or impedance measurements ( Figure S73B, Supporting Information).
In contrast to this, the viability of 4T1 cells was reduced significantly in a concentration-dependent manner in response to compounds 9−12 (gerardiins I−L) ( Figure S74, Supporting Information). The effect of these phenanthrenes was comparable to that of doxorubicin, which was applied as a positive control to measure cytotoxicity. Since all these compounds are dimers of effusol (13) (compounds 9−11) or of effusol and dehydroeffusol (14) (compound 12), the cytotoxic effects of the monomers and dimers in both mouse and human tumor cells and in a nontumor cell line (D3) were compared. Besides the aforementioned phenanthrenes, effususin A (15) was also included in this study, since it is also a dimer of effusol ( Figure S74, Supporting Information).
The present results show unequivocally that the dimeric compounds 9−12 and 15 comprising effusol (13) and dehydroeffusol (14) monomers are cytotoxic to both tumor and nontumor cell lines, while the monomers (13,14) alone displayed no or very low cytotoxicity ( Figure S74, Supporting Information). Among the diphenanthrenes tested, effususin A (15) exerted the lowest cytotoxicity, while gerardiins I−L (9− 12) proved to be the most active. Indeed, moderate toxicity of effususin A (15) in A2780 human ovarian cancer cells was reported by Buś et al. 8 Impedance measurements were in line with the results of the MTT assay, indicating a concentrationdependent toxicity of the dimers ( Figure S76, Supporting Information).
IC 50 values of both 9 and 10 were below 10 μM in the two tested tumor cell lines (Table 6). In the case of compound 11,  Considering the already known isolated phenanthrenes, only juncusol (in MTT and impedance assays) and jinflexin C (in impedance measurements) displayed moderate cytotoxicity, while compressin A, 7-hydroxy-2-methoxy-1-methyl-5-vinyl-9,10-dihydrophenanthrene, 2-hydroxy-7-hydroxymethylene-1methyl-5-vinyl-9,10-dihydrophenanthrene, 2,7-dihydroxy-5formyl-1-methyl-9,10-dihydrophenanthrene, effususol A, and 2,7-dihydroxy-5-hydroxymethyl-1-methyl-9,10-dihydrophenanthrene were not cytotoxic in 4T1 cells at the concentration of 20 μM ( Figure S75, Supporting Information). The present results are in agreement with previous findings that demonstrated the antiproliferative activity of juncusol against HeLa cervical cancer cells. 20 ■ EXPERIMENTAL SECTION General Experimental Procedures. Optical rotations were determined in MeOH at ambient temperature using a PerkinElmer 341 polarimeter. NMR spectra were recorded in MeOD and DMSOd 6 on a Bruker Avance DRX 500 spectrometer at 500 MHz ( 1 H) and 125 MHz ( 13 C). The signals of the deuterated solvents were taken as references. The chemical shift values (δ) were given in ppm, and coupling constants (J) are in Hz. Two-dimensional (2D) experiments were performed with standard Bruker software. In the COSY, HSQC, and HMBC experiments, gradient-enhanced versions were used. The HRMS were acquired on a Thermo Scientific Q-Exactive Plus Orbitrap mass spectrometer equipped with ESI ion source in the positive ionization mode. The resolution was over 1 ppm. The data were acquired and processed with MassLynx software.
The fraction obtained from the polyamide column with MeOH− H 2 O 2:1 (3 g) was subjected to VLC on silica gel with a gradient system of cyclohexane−EtOAc−MeOH [from 95:5:0 to 1:1:1 (200 mL/eluent), and finally with MeOH; the volumes of the collected fractions were 50 mL], to yield six major fractions (D/1−6). The fractions were combined according to their TLC patterns. Fraction Memorial Institute) 1640 medium supplemented with 5% fetal bovine serum (FBS) (both from Thermo Fisher Scientific, Waltham, MA, USA). MDA-MB-231 (human triple-negative breast cancer cells, abbreviated as MDA) were cultured in DMEM (Dulbecco's modified Eagle's medium, Thermo Fisher Scientific) + 5% FBS. D3 (hCMEC/ D3 human cerebral microvascular endothelial cells) were kept in rat tail collagen-coated dishes in EBM-2 (endothelial basal medium-2, Lonza, Basel, Switzerland) complemented with 2% FBS and an EGM-2MV kit (Lonza). Viability and impedance measurements were performed in the log growth phase of tumor cells and in the stationary phase of endothelial cells.
For the MTT (3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide) viability assay, cells were plated in 96-well plates (Corning, Corning, NY, USA). The seeding density of the cells was 5000/well (for 4T1 or MDA cells) or 25 000/well (for D3 cells). After 48 h, half of the medium was replaced with serum-free medium, containing the test compounds in a final concentration of 10 or 20 μM. Control wells received solvent (DMSO) in up to a 0.2% concentration. After 48 h, MTT reagent (Sigma-Aldrich, St. Louis, MO, USA) was added to the cells in a final concentration of 2.5 mg/ mL. After incubation at 37°C for 30 min, acidified isopropanol solution was added to each well. Absorbance was measured at 595 nm with a FLUOstar OPTIMA microplate reader (BMG LABTECH, Offenburg, Germany). Doxorubicin was used as a positive control, at a concentration of 10 μM.
For impedance measurements, cells were plated in 96-well E-plates having microelectrodes integrated on the bottom (ACEA Biosciences, San Diego, CA, USA) and allowed to attach onto the electrode surface for 48 h. Afterward, cells were treated with the test compounds as described above. Electrical impedance was recorded every 30 min for 48 h using an xCELLigence real-time cell analysis (RTCA) instrument (ACEA Biosciences). Cell index was automatically calculated by the software of the instrument.
For determining IC 50 values, nine-step, 2-fold serial dilutions of the test compounds were applied, starting from 100 μM. Cells were treated for 48 h, and viability was measured with the MTT assay, as described above. Half-maximum inhibitory concentrations (IC 50 ) were calculated via nonlinear dose−response curve fitting by the log(inhibitor) vs response (variable slope) model of GraphPad Prism 5.01 (GraphPad Software, San Diego, CA, USA) by using automatic outlier elimination at Q = 1.0.