Antimicrobial acylphloroglucinol derivatives from linarioides ()

S. Crockett1, O. Kunert2, M. Jacob3, R. Bauer1 and P. Nedialkov4

1Department of Pharmacognosy, Karl-Franzens-University Graz, 8010 Graz, Austria 2Department of Pharmaceutical Chemistry, Karl-Franzens-University Graz, 8010 Graz, Austria 3Natural Center for Natural Products Research, University of Mississippi, University, MS 38677, U.S.A. 4Department of Pharmacognosy, Faculty of Pharmacy, Medical University of Sofia, 1000 Sofia, Bulgaria

Abstract: Hypericum linarioides Bosse (Fig. 1, taxonomic section Taeniocarpium) has one center of distribution in western and southern Transcaucasia, adjacent parts of Russia and northern Iran and Turkey, and a second center in southern Serbia, northern Greece and western Bulgaria. Naphthodianthrones, simple anthrones, flavonoids, flavonol glycosides, caffeic acid derivatives and the xanthone-C-glycoside mangiferin have been previously reported from this species [1, 2]. TLC and HPLC screening of an extract of this species collected in western Bulgaria revealed the presence of a rich diversity of acylphloroglucinol derivatives. Subsequently, bioassay-guided fractionation of the dichloromethane extract of dried, ground aerial parts of

H. linarioides was performed, testing extracts against a range of bacterial and fungal pathogens. Upon Photo: P. Nedialkov observation of promising activity against Candida glabrata (IC50=16.91-21.86 μg/mL), Cryptococcus neoformans (IC50=2.48-8.43 μg/mL), Staphylococcus aureus (IC50=2.98-24.90 μg/mL) and MRSA (IC50=2.72- Figure 1: Habit (left) and flower 18.99 μg/mL), extracts were further fractionated, resulting in the isolation of several structurally related bud (above) of Hypericum acylphloroglucinol derivatives. Extracts were analyzed by chromatographic means (TLC, OC, HPLC) and linarioides growing in Bulgaria. structure elucidation performed using data from NMR and MS. This work resulted in the isolation of 3- Note reddish-black marginal geranyl-1-(2′-methylbutanoyl)phloroglucinol (1) and 3-geranyl-1-(2′-methylpropanoyl)phloroglucinol (2) as glands on the petals, indicative of naphthodianthrone content. main constituents, as well as several minor derivatives. 1 has been previously reported from H. Photo: P. Nedialkov empetrifolium (sect. Coridium) [3] and 2 has been previously reported from H. empetrifolium, H. jovis (sect. Coridium) [4] and H. styphelioides (sect. Brathys) [5]. Implications of this chemical convergence are „Euhypericum“: discussed. „Hirtella,“ „Taeniocarpium,“ Coridium, Origanifolia, Drosocarpium, Crossophyllum, Thasia, „Adenosepalum,“ Elodes, Elodeoida, Monanthema, Olympia, „Oligostema,“ Campylopus, Concinna, Figure 2: Isolation scheme and „Humifusoideum,“ Sampsonia, Hypericum, „Graveolentia“ H. linarioides Dried Aerial Material structures of compounds isolated (360 g) from H. linarioides.

O 1. DCM extraction (10 L, percolation) Webbia, „Adenosepalum“ „Arthrophyllum“ O O

38.2 g Extract O CH3

CH3

1. CC on Diaion HP-20, MeOH:H2O gradient (70:30 to 100:0)

2. Merge fractions on basis of TLC similarities O „Brathys s.l.“: Brathys, Trigynobrathys A B C D E F G H I J K O O CH3 Photo: P. Nedialkov Inodora, Triadenioides O

Heterophylla, Adenotrias CH3 Umbraculoides 2.08 g Ext. 1.50 g Ext. „Myriandra-Ascyreia s.l.“: 1. VLC, hexane:EtOAc gradient 3.45 g Ext. Frs. 15-20 Frs. 21-25 Campylosporus, Psorophytum, O 2. Fractions merged on basis of TLC similarities „Takasagoya,“ „Ascyreia,“ 3. Preparative HPLC of selected LC fractions, Myriandra, Androsaemum O O ACN:H2O gradient (70:30 to 100:0) UV280 CH3 Roscyna O

C A 1. Preparative HPLC, ACN:H O isocratic (98:2) „Humifusoideum“ NMR and MS evaluation of 2 B 2. Peaks with UV nm absorption collected purified compounds 280 Outgroup D Figure 3: Schematic illustration of phylogenetic relationships among taxonomic sections in the E Frs. A-B Frs. A-H Hypericum, based on a cladistic analysis of morphological characters, modified from [7], indicating sections from which compounds containing an isobutanoyl-phloroglucinol moiety (blue text), isopropanoyl- Compounds 1 - 2 Compounds 1 - 2 Compound 3 phloroglucinol moiety (red text), both of these (pink text), or both plus the O-geranyl-substituted phloroglucinol (green text) have been isolated. Section names in quotation marks are polyphyletic in the Major Phloroglucinol Derivatives Minor Phloroglucinol Derivative tree. The outgroup taxa included representatives from the genera Lianthus, Santomasia, Thornea, Vismia, and Harungana, Triadenum and Cratoxylum. CH3 CH3

CH CH OH CH CH O CH OH 3 3 3 3 3 Results and Discussion:

CH3 CH3

HO OH HO OH This work resulted in the isolation of 3-geranyl-1-(2′-

H3C HO OH H3C O O methylbutanoyl)-phloroglucinol (1) and 3-geranyl-1-(2′-methylpropanoyl)- H3C CH3 CH3 O phloroglucinol (2) as the major phloroglucinol derivatives present in the 3-geranyl-1-(2′-methylbutanoyl)- 3-geranyl-1-(2′-methylpropanoyl)- CH3 aerial material of H. linarioides (sect. Taeniocarpium), as well as 4-O- phloroglucinol (1) phloroglucinol (2) 4-O-geranyl-1-(2′-methylpropanoyl)- geranyl-1-(2′-methylpropanoyl)-phloroglucinol (3) as a minor constituent phloroglucinol (3) (Fig. 2). Column fractions containing (1) and (2) demonstrated bioactivity Materials and Methods: against Candida glabrata (IC =1.44-4.80 μg/mL), Cryptococcus neoformans Aerial material of Hypericum linarioides Bosse were collected near Petrohan, Bulgaria. A voucher specimen (SOM-Co-1196) has been 50 deposited at the Herbarium of Botanical Institute-Sofia (Bulgarian Academy of Sciences). Aerial material was air-dried at room temperature, in (IC50= 0.62-3.08 μg/mL), Staphylococcus aureus (IC50=2.58-3.78 μg/mL) the dark, to a moisture content of > 2% (dry weight = ca. 360 g). Percolation with 10 L DCM yielded ca. 38.2 g waxy greenish semi-solid extract. This extract was mixed with Diaion HP-20 resin (20 g) and CC was conducted on a Diaion HP-20 column (4.7 X 76 cm), eluting with and MRSA (IC50=1.39-3.32 μg/mL). (1), (2) and (3) have been previously 70-90% aqueous MeOH (2 L per gradient step), then 95% aq. MeOH (6 L). All solvents used for isolation and TLC were ACS grade and all reported as major constituents of the lipophilic extract of the flowering solvents used during HPLC isolation/analysis were HPLC grade (Roth Inc., Germany). TLC precoated silica gel 60 F254 (Merck, Germany); detection by spraying with vanillin/H SO , followed by heating. Instrumentation: Analytical HPLC -- Agilent 1100 Separations Module (Agilent 2 4 and/or fruiting material from other species in the taxonomic sections Technologies, USA) using a Luna Phenomenex C18 column (5 μm, 4 X 150 mm; Phenomenex), coupled to diode array UV detector with flow rate of 750 μl/min. Preparative HPLC -- Varian R PrepStar Model SD-1 with Dynamax R Solvent Delivery System Model SD-1 (Varian Inc., Brathys, Coridium and, most recently, Adenosepalum (Crockett et al., in USA) using LichroSpher 100-C18 column (10 μm, 25 X 250 mm, Varian), coupled to Dynamax R Absorbance Detector Model UV-1 (detection at 280 nm) with flow rate of 18 ml/min. HPLC–DAD/ESI–MS (neg.) was performed on Thermo Finnigan Surveyor liquid chromatography preparation). In a cladistic analysis of the genus using morphological instrument with Thermo Quest Surveyor photodiode array detector, autosampler, and MS pump, and Thermo Finnigan LCQ-XP mass detector equipped with an electrospray ionization (ESI) source run by Xcaliber software (Thermo Fisher Scientific, USA). Mass spectra were detected characters, these sections were placed in different clades (both basal and and recorded in scan range of m/z 100–1000, using transfer capillary temperature of 350˚C, spray voltage of 5.00 kV and sheath gas flow of 70 units. NMR Spectra: 1H-, 13C-, and 2D-NMR experiments (HSQC, HMBC, DQFCOSY) performed on Varian Unitylnova-400 and -600 MHz derived) within Hypericum [7], suggesting that the biosynthetic pathways spectrometers; experimental parameters as in Seebacher et al. (6); δ in ppm, J in Hz. Compounds dissolved in chloroform- d1, with leading to these secondary metabolites have been conserved within the tetramethylsilane (TMS) as an internal standard. genus (Fig. 3). The considerable energetic investment of the toward Acknowledgements: Funding for S. Crockett was provided by a grant from the Austrian Science Foundation (FWF, Project T345). Sincere thanks to N. Nürk for fruitful discussions regarding the phylogeny of Hypericum. This work was partially supported by the NIH, NIAID, Division of AIDS, Grant No. AI 27094 (antifungal producing these compounds also indicates that they may play an ecological testing) and the USDA Agricultural Research Service Specific Cooperative Agreement No. 58-6408-2-0009 (antibacterial testing). role as attractant (e.g. UV absorbing, scent) or protectant (e.g. anti- References : 1. Smelcerović et al. (2006) Phytochemistry 67: 171-77. 2. Ayan and Çirak (2008) Pharmaceutical Biology 46: 288-91. 3. Crockett et al. (2008) Phytochemical Letters 1: 37-43. 4. Athanas et al. (2004) Journal of Natural Products 67: 973-77. 5. Gamiotea-Turro et al. (2004) Journal of Natural Products 67: 869-71. 6. microbial) molecules, as has been demonstrated for related compounds [8]. Seebacher, W. et al. (2003) Magn. Reson. Chem. 41:636–638. 7. Nürk and Blattner (2010) Taxon 59, in press. 8. Gronquist et al. (2001) PNAS, 98: 13745-13750 .