molecules Article Methylated Xanthones from the Rootlets of Metaxya rostrata Display Cytotoxic Activity in Colorectal Cancer Cells Eva Mittermair 1,2,*, Hanspeter Kählig 3, Ammar Tahir 1 , Stefanie Rindler 1, Xenia Hudec 2, Hemma Schueffl 2, Petra Heffeter 2, Brigitte Marian 2 and Liselotte Krenn 1 1 Department of Pharmacognosy, University of Vienna, Althanstraße 14, 1090 Vienna, Austria; [email protected] (A.T.); [email protected] (S.R.); [email protected] (L.K.) 2 Institute of Cancer Research, Department of Medicine I, and Comprehensive Cancer Center, Medical University Vienna, Borschkegasse 8a, 1090 Vienna, Austria; [email protected] (X.H.); hemma.schueffl@meduniwien.ac.at (H.S.); petra.heff[email protected] (P.H.); [email protected] (B.M.) 3 Department of Organic Chemistry, University of Vienna, Währingerstraße 38, 1090 Vienna, Austria; [email protected] * Correspondence: [email protected] Academic Editor: Natalizia Miceli Received: 15 September 2020; Accepted: 25 September 2020; Published: 28 September 2020 Abstract: AbstractThe tree fern Metaxya rostrata (Kunth) C. Presl is common in the rainforests of Central and South America, where suspensions of the dried rhizome are traditionally used to treat intestinal diseases. Two compounds from this plant, 2-deprenyl-rheediaxanthone B (XB) and 2-deprenyl-7-hydroxy-rheediaxanthone B (OH-XB), have been shown to be biologically highly active against colorectal cancer (CRC) cells in previous studies. The current investigation resulted in the isolation of the previously undescribed methylated xanthones 2-deprenyl-6-O-methyl-7-hydroxy- rheediaxanthone B, 2-deprenyl-5-O-methyl-7-methoxy-rheediaxanthone B, 2-deprenyl-5-O-methyl- 7-hydroxy-rheediaxanthone B and 2-deprenyl-7-methoxy-rheediaxanthone B. All compounds were isolated by column chromatography, structures were elucidated by one- and two-dimensional NMR-experiments and the identities of the compounds were confirmed by LC-HRMS. In logarithmically growing SW480 CRC cell cultures, cytotoxicity by neutral red uptake and MTT assays as well as caspase activation was analyzed. Cellular targets were examined by Western blot, and topoisomerase I (topo I) inhibition potential was tested. Comparing the structure-activity relationship with XB and OH-XB, the monomethylated derivatives showed qualitatively similar effects/mechanisms to their nonmethylated analogues, while dimethylation almost abolished the activity. Inhibition of topo I was dependent on the presence of an unmethylated 7-OH group. Keywords: Metaxya rostrata; metaxyaceae; 2-deprenyl-6-O-methyl-7-hydroxy-rheediaxanthone B; 2-deprenyl-5-O-methyl-7-methoxy-rheediaxanthone B; 2-deprenyl-5-O-methyl-7-hydroxy-rhee- diaxanthone B; 2-deprenyl-7-methoxy-rheediaxanthone B 1. Introduction Metaxya rostrata (Kunth) C. Presl was first described in 1836, and is the only species of the genus Metaxya that has been investigated phytochemically to date. The tree fern is widespread in lowland Amazonian forests, from Mexico to Panama, Peru, Bolivia and northern Brazil [1,2]. Especially in Costa Rica, its rhizome is used in ethnic medicine to treat intestinal diseases [3]. Considering the interest in natural compounds that mostly originates from traditional use and documentation, numerous Molecules 2020, 25, 4449; doi:10.3390/molecules25194449 www.mdpi.com/journal/molecules Molecules 2020, 25, 4449 2 of 11 Molecules 2020, 25, x FOR PEER REVIEW 2 of 11 valuable anticancer drugs have ultimately been obtained from plants [4–7]. Prenylated xanthones have gainednumerous a lot ofvaluable interest anticancer during the drugs last decade,have ultimate as theyly arebeen well obtained known from for plants their cytotoxic [4–7]. Prenylated effects on malignantxanthones cells. have The gained best studieda lot of amonginterest theseduring are th thee last mangostins decade, as thatthey induce are well cell known cycle arrestfor their and activecytotoxic cell death effects in on a broad malignant range cell ofs. tumor The best cells studied [8–10]. among After establishingthese are the cytotoxicmangostins activity that induce of fractions cell fromcycle an arrest aqueous and and active a methanolic cell death in extract a broad of rangeM. rostrata of tumoragainst cells colorectal[8–10]. After cancer establishing (CRC) cellscytotoxic [3], an activity-guidedactivity of fractions study from led to an the aqueous isolation and ofa methanolic structurally extract related of 2-deprenyl-rheediaxanthoneM. rostrata against colorectal cancer B (XB) and(CRC) 2-deprenyl-7-hydroxy-rheediaxanthone cells [3], an activity-guided study Bled (OH-XB). to the isolation These studies of structurally identified related the two 2-deprenyl- compounds as biologicallyrheediaxanthone highly B active, (XB) blockingand 2-deprenyl-7-hydro cell cycle progressionxy-rheediaxanthone and inducing B cell (OH-XB). death in CRCThese cells studies [3,11 ]. Moreidentified recently, the our two findings compounds suggested as biologically that both highly substances active, also blocking interact cell with cycle the progression cellular targets and forkheadinducing box cell protein death M1 in CRC (FoxM1) cells and[3,11]. topoisomerase More recently, I (topo our findings I) [12]. Thus, suggested we observed that both that substances XB was a strongalso inducerinteract ofwith FoxM1 the cellular degradation, targets whileforkhead OH-XB box hadprotein a stronger M1 (FoxM1) impact and on topoisomerase topo I activity. I (topo Based I) on these[12]. results, Thus, thewe focusobserved of the that current XB was study a strong was inducer the isolation of FoxM1 of further degradation, closely while related OH-XB xanthones had a in orderstronger to explore impact the on e ffitopocacy I andactivity. cellular Based targets on thes ine SW480 results, CRC the cellsfocus inof orderthe current to establish study awas detailed the structure-activityisolation of further relationship. closely related xanthones in order to explore the efficacy and cellular targets in SW480 CRC cells in order to establish a detailed structure-activity relationship. 2. Results and Discussion 2. Results and Discussion A detailed investigation of a CH2Cl2 extract of the dried rootlets from M. rostrata resulted in the isolationA detailed of investigation three xanthones of a CH (1,2Cl2,2 3extract), and of of the an dried additional rootlets compound from M. rostrata (4), which resulted was in the only isolation of three xanthones (1, 2, 3), and of an additional compound (4), which was only accessible accessible as a mixture (M) together with 1 (Figure1). They are all new, natural compounds and as a mixture (M) together with 1 (Figure 1). They are all new, natural compounds and were identified were identified by chromatographic, spectrometric and spectroscopic methods. This led to the by chromatographic, spectrometric and spectroscopic methods. This led to the elucidation of 2- elucidation of 2-deprenyl-6-O-methyl-7-hydroxy-rheediaxanthone B (1) as well as 2-deprenyl-5-O- deprenyl-6-O-methyl-7-hydroxy-rheediaxanthone B (1) as well as 2-deprenyl-5-O-methyl-7- methyl-7-methoxy-rheediaxanthone B (2). Compound 3 was identified as 2-deprenyl-5-O-methyl-7- methoxy-rheediaxanthone B (2). Compound 3 was identified as 2-deprenyl-5-O-methyl-7-hydroxy- hydroxy-rheediaxanthonerheediaxanthone B. In the B. Inmixture, the mixture, together together with with1, compound1, compound 4 was4 wasidentified identified as 2-deprenyl-7- as 2-deprenyl- 1 7-methoxy-rheediaxanthonemethoxy-rheediaxanthone B at a ratio ratio of of 1:0.3, 1:0.3, according according to to the the integration integration in inthe the 1H-NMR.H-NMR. FigureFigure 1. 1.Chemical Chemical structures structures ofof thethe identifiedidentified xanthones. 1 13 InIn general, general, the theH- 1H- and and 13C-NMRC-NMR spectraspectra are very similar to to published published data data for for 2-deprenyl- 2-deprenyl- 1 rheediaxanthonerheediaxanthone B [3B]. [3]. For For the the prenyl prenyl part part in in all all substances, substances, the theH-NMR 1H-NMR spectra spectra show show two two singletssinglets at 1.33at and 1.33 1.62and ppm1.62 ppm (two (two CH 3CH), and3), and one one doublet doublet at at 1.41 1.41 ppm ppm (CH (CH33)) coupledcoupled to to a a quartet quartet at at 4.54 4.54 ppm ppm (CH),(CH), all all within within a a shift shift range range ofof ±0.0150.015 ppm. ppm. In Inthe the aromatic aromatic region, region, proton proton 2 is detected 2 is detected around around 6.13 ± 6.13ppm ppm (±0.014 ( 0.014 ppm). ppm). The The second second aromatic aromatic signal signal for forH-8 H-8 is in is inthe the range range from from 7.08 7.08 to to7.36 7.36 ppm, ppm, ± influencedinfluenced by by the the di differentfferent methylation methylation patterns. As As only only these these two two aromatic aromatic protons protons are present, are present, the thesecond second aromatic aromatic ring ring has has to to carry carry three three phenolic phenolic groups, groups, i.e., i.e., either either one one being being methylated methylated or two or twoin in compoundcompound 22.. These These methyl methyl groups groups can can be beseen seen at around at around 3.95 3.95 ppm. ppm. In the In 13 theC-NMR13C-NMR spectra, spectra, the thesignals signals for for the the prenyl prenyl part part and and the the connected connected arom aromaticatic ring ring match match very very closely. closely. To To elucidate elucidate the the substitution pattern, HMBC and NOESY spectra were recorded. In the HMBC spectra, the methyl Molecules 2020, 25, 4449 3 of 11 substitution pattern, HMBC and NOESY spectra were recorded. In the HMBC spectra, the methyl protons give a three-bond correlation to the quaternary aromatic carbon, where the methoxy group is located. Methoxy substitution at position 6 or 7 shows a correlation to this carbon starting from H-8, either via three or two bonds, respectively. On the other hand, a methoxy group at position 7 gives a correlation in the NOESY spectrum to H-8, and in the case of a substitution at C-5, a minute cross peak can be seen in methyl groups 12 and 13 (Tables1–3).