Biologically Active Dibenzofurans from Pilidiostigma glabrum, an

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Biologically Active Dibenzofurans from Pilidiostigma glabrum, an Endemic Australian Myrtaceae Qingyao Shou,† Linda K. Banbury,† Dane E. Renshaw,† Eleanore H. Lambley,† Htwe Mon,‡ Graham A. Macfarlane,§ Hans J. Griesser,‡ Michael M. Heinrich,†,⊥ and Hans Wohlmuth*,† †

Southern Cross Plant Science, Southern Cross University, PO Box 157, Lismore NSW 2480, Australia Ian Wark Research Institute, University of South Australia, Mawson Lakes SA 5095, Australia § School of Chemistry & Molecular Biosciences, University of Queensland, Brisbane QLD 4072, Australia ⊥ UCL School of Pharmacy, 29-39 Brunswick Square, London WC1N 1AX, United Kingdom ‡

S Supporting Information *

ABSTRACT: In an effort to identify new anti-inflammatory and antibacterial agents with potential application in wound healing, five new dibenzofurans, 1,3,7,9-tetrahydroxy-2,8dimethyl-4,6-di(2-methylbutanoyl)dibenzofuran (1), 1,3,7,9tetrahydroxy-2,8-dimethyl-4-(2-methylbutanoyl)-6-(2methylpropionyl)dibenzofuran (2), 1,3,7,9-tetrahydroxy-2,8dimethyl-4,6-di(2-methylpropionyl)dibenzofuran (3), 1,3,7,9tetrahydroxy-4,6-dimethyl-2-(2-methylbutanoyl)-8-(2methylpropionyl)dibenzofuran (4), and 1,3,7,9-tetrahydroxy4,6-dimethyl-2,8-di(2-methylpropionyl)dibenzofuran (5), were isolated from the leaves of Pilidiostigma glabrum together with one previously described dibenzofuran. Structure elucidation was achieved by way of spectroscopic measurements including 2D-NMR spectroscopy. Compounds with 2,8-acyl substitutions had potent antibacterial activity against several Gram-positive strains (MIC in the low micromolar range), while compounds with 4,6acyl substitutions were less active. All compounds except 3 inhibited the synthesis of nitric oxide in RAW264 macrophages with IC50 values in the low micromolar range. Compounds with 2,8-acyl substitutions also inhibited the synthesis of PGE2 in 3T3 cells, whereas 4,6-acyl-substituted compounds were inactive. None of the compounds inhibited the synthesis of TNF-α in RAW264 cells. The compounds showed variable but modest antioxidant activity in the oxygen radical absorbance capacity assay. These findings highlight that much of the Australian flora remains unexplored and may yet yield many new compounds of interest. Initial clues are provided on structure/activity relationships for this class of bioactives, which may enable the design and synthesis of compounds with higher activity and/or selectivity. espite Australia being home to a rich flora with a high percentage of endemism, few Australian plants have been exploited as sources of modern pharmaceuticals or phytomedicines. Well-known exceptions are the oils of eucalyptus and tea tree, both of which are terpenoid-rich essential oils, distilled from the leaves of Eucalyptus and Melaleuca species, respectively. Both these genera belong to the Myrtaceae, a family richly represented in Australia by around 70 genera and approximately 1400 species.1 The genus Pilidiostigma also belongs to the family Myrtaceae and comprises six Australian species of shrubs or trees growing in rainforest and wet sclerophyll forest in northeastern New South Wales and Queensland, with one of these species extending to Papua New Guinea.2 The genus forms part of the tribe Myrteae within the subfamily Myrtoideae, based on matK phylogeny.3 Only one Pilidiostigma species, P. tropicum L.S.Sm., has previously been investigated for biological activity. Setzer and co-workers reported rhodomyrtoxin B (a dibenzofuran) and ursolic acid-3-p-coumarate with cytotoxic and antibacterial

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© XXXX American Chemical Society and American Society of Pharmacognosy

activity from the bark of this species.4 Rhodomyrtoxin B was first isolated from Rhodomyrtus macrocarpa Benth. in a closely related genus in the tribe Myrteae.5 The leaf essential oils of five Pilidiostigma species have been characterized and found to consist almost entirely of sesquiterpenes.6 As part of our work on identifying natural products with potential application in wound healing, we here report six (including five new) biologically active dibenzofurans from Pilidiostigma glabrum Burret, an endemic Australian species commonly known as plum myrtle. This species is a shrub or small tree to 5 m that grows in rainforests and wet sclerophyll forests along Australia’s east coast, north from approximately 31° S.1

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RESULTS AND DISCUSSION

Five new dibenzofurans (1−5), along with one known such compound (6), were isolated from the leaves of P. glabrum. Received: June 19, 2012

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dx.doi.org/10.1021/np300433r | J. Nat. Prod. XXXX, XXX, XXX−XXX

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Article

molecular formula of C23H26O7. Compared to 1, the 1H NMR spectrum of 2 showed two methine multiplets separately at δH 3.98 and δH 4.08, which indicated the presence of asymmetric substituents of the two benzene rings. A single methyl multiplet at δH 0.95 and one methylene multiplet at δH 1.64, 1.93, along with the presence of three methyls at δH 1.31−1.33, suggested a 2-methylpropionyl moiety of 2 instead of one of the two 2methylbutanoyl moieties found in 1. The JMOD spectrum showed exactly 23 carbons containing six methyls (δC 19.9, 19.6, 18.2, 11.9, 8.3, 8.3), one methylene (δC 26.7), and two methines (δC 39.2, 45.8), which supported the presence of a 2methylpropionyl moiety. Thus, the identity of compound 2 was deduced as 1,3,7,9-tetrahydroxy-2,8-dimethyl-4-(2-methylbutanoyl)-6-(2-methylpropionyl)dibenzofuran. Compound 3, isolated as light yellow, needle-shaped crystals, showed a [M + Na]+ peak at m/z 423.1415, indicating a molecular formula of C22H24O7. The appearance of only 11 signals in the 13C NMR spectrum suggested that 3 has a symmetric structure. The 1H NMR signals indicated the presence of two 2-methylpropionyl moieties with four methyl doublets at δH 1.33 (d, J = 6.7 Hz) and two methine multiplets at δH 4.08. One singlet (6H) was observed at δH 2.25, evidencing two aromatic methyls. The UV absorption at 276 and 309 nm was similar to compound 1, which indicated the two 2-methylpropionyl moieties to be located at C-4 and C-6. Accordingly, the structure of compound 3 was elucidated as 1,3,7,9-tetrahydroxy-2,8-dimethyl-4,6-di(2-methylpropionyl)dibenzofuran. Compound 4 was obtained as yellow, needle-shaped crystals. Its HRESIMS displayed a [M + Na]+ peak at m/z 437.1577, in agreement with the formula C23H26O7. The UV spectrum was similar to that of rhodomyrtoxin C (compound 6), with absorptions at 279, 291, 307, and 380 nm, but different from compounds 1−3 (see Supporting Information), which implied that the acyl groups are attached at C-2 and C-8. The 1H NMR spectrum of compound 4 was very similar to that of compound 2, with a 2-methylpropionyl moiety as well as a 2methylbutanoyl moiety, but the chemical shifts of the two aromatic methyls moved from δH 2.11 and 2.13 to both δH 2.24, indicating a different substitution position from that of compound 2. The chemical shifts of the carbonyl carbons as well as the oxygenated aromatic carbons at δC 213.1, 213.3, 160.8, 159.9, 153.7, and 153.6 supported the assignment of 2,8acyl substitutions and were compared to data of compound 2 at δC 208.3, 208.2, 164.4, 164.3, 154.3, 154.2, 154.1, and 154.1. Thus, compound 4 was established as 1,3,7,9-tetrahydroxy-4,6dimethyl-2-(2-methylbutanoyl)-8-(2-methylpropionyl)dibenzofuran. Compound 5, yellow, needle-shaped crystals, gave an ion peak at m/z 423.1412 [M + Na]+, corresponding to the

Compound 1, obtained as light yellow needle-shaped crystals, showed a [M + Na]+ peak at m/z 451.1723 (calcd 451.1733) by high-resolution electrospray-ionization time-offlight mass spectrometry, corresponding to a molecular formula of C24H28O7. The characteristic UV absorptions at 277 and 310 nm as well as an IR absorption at 1609 cm−1 were similar to ψ rhodomyrtoxin,7 which suggested the dibenzofuran nature of compound 1. The appearance of only 12 signals in the 13C NMR spectrum revealed that 1 has a symmetrical structure. The 1H NMR signals indicated the presence of two 2methylbutanoyl moieties with two methyl triplets at δH 0.95 (t, J = 7.7 Hz), two methyl doublets at δH 1.33 (d, J = 6.7 Hz), two methylene multiplets at δH 1.62, 1.97, and two methine multiplets at δH 3.99. One singlet (6H) was observed at δH 2.25, thus evidencing two aromatic methyls. Two downfield protons at δH 14.27 assignable to the OH chelated to carbonyls of the 2-methylbutanoyl groups were also observed. In addition, the JMOD experiment showed three methyl groups (δC 11.6, 18.2, and 8.0), one methylene (δC 26.5), one methine (δC 45.2), and seven quaternary carbons (δC 102.0, 104.4, 107.5, 152.7, 153.7, 164.0, 207.7), which supported one side of the benzene ring being substituted with a methyl group, a 2methylbutanoyl moiety, and two hydroxy groups. The 2methylbutanoyl moieties were placed unequivocally at C-4 and C-6 according to the different chemical shift of C-1′ (δC 207.7); C-1, 3, 4a (152.7, 164.0, 153.7), compared to those of rhodomyrtoxin B with C-1′ (δC 211.0); C-1, 3, 4a (159.6, 158.8, 152.2).5 The different UV spectrum from rhodomyrtoxin B as well as the presence of only two chelated hydroxy group protons at δH 14.27 also supported 4,6-acyl substitutions of compound 1. The stereochemistry at C-2′, 2″ in the 2methylbutanoyl substituents was assigned tentatively as S because of the positive sign of rotation {[α]25 D = +287 (c 0.61, CHCl3)}.8 Thus, compound 1 was deduced to be 1,3,7,9tetrahydroxy-2,8-dimethyl-4,6-di(2-methylbutanoyl)dibenzofuran. 1H and 13C NMR spectroscopic signals of 1 were assigned on the basis of the JMOD, 1H−1H COSY, HSQC, and HMBC spectra. Compound 2, isolated as light yellow, needle-shaped crystals, showed a [M + Na]+ peak at m/z 437.1577, indicating a

Table 1. Antibacterial Activity of Dibenzofurans from Pilidiostigma glabruma compound microorganism

1

2

3

4

5

6

Staphylococcus aureus ATCC 29213 Staphylococcus aureus ATCC 25923 Staphylococcus epidermidis ATCC 35984 Escherichia coli ATCC 25922 Pseudomonas aeruginosa ATCC 27853

7.2 (14.7) 3.7 (3.7) 3.7 (3.7) NA NT

12.1 (24.1) 6.0 (24.1) 6.0 (6.0) NA NT

26.5 (26.5) 13.2 (26.5) 13.2 (13.2) NA NA

3.9 (3.9) 0.5 (1.9) 1.9 (3.9) NA NT

2.7 (11.4) 2.7 (5.7) 2.7 (22.7) NA NA

7.2 (7.2) 0.9 (0.9) 1.9 (1.9) NA NT

Values shown are MIC (MBC) in μM. NA: not active at maximum concentration tested. NT: not tested. MIC for levofloxacin (positive control) 200

22.3 (18.6−26.7) >200

44.0 (38.8−49.8) >200

9.4 (7.8−11.3) 6.0 (3.1−11.5)

15.3 (12.0−19.5) 4.5 (2.2−9.1)

7.0 (4.4−11.0) 3.4 (1.8−6.4)

3637 ± 629

6281 ± 325

609 ± 75

2576 ± 144

1451 ± 153

1008 ± 222

a

ED50 (95% CI) is shown for cytotoxic activity (n = 3), IC50 (95% CI) values for inhibition of nitric oxide (n = 3) and PGE2 synthesis (n = 3), and antioxidant activity is expressed as micromoles of Trolox equivalent per gram (mean ± SD μmol TE/g, n = 4). 2011 and authenticated by one of the authors (H.W.). A voucher specimen (PHARM110026) has been deposited in the Medicinal Plant Herbarium at Southern Cross University. Extraction and Isolation. The powdered, dried leaves of P. glabrum (300 g) were extracted with 95% ethanol at room temperature. The ethanol extract was suspended in H2O and extracted in successive steps using CHCl3 and EtOAc. The CHCl3 portion was evaporated under reduced pressure to afford a crude extract (25.0 g). The crude CHCl3 extract was subjected to MCI gel (CHP20P) CC, eluted with a gradient of MeOH/H2O (80:20−95:5), to give 10 fractions (A−J). Fraction A was subjected to CC on C18-E (5 × 40 cm) with MeOH/H2O (4:1) to give compound 3 (19 mg). Fraction C (120 mg) was further separated by preparative HPLC [Phenomenex Luna C18 column (150 × 21.20 mm) 5 μm; mobile phase acetonitrile and H2O containing 0.05% TFA (0−5 min: 50% acetonitrile, 5−13 min: 50−95% acetonitrile, 13−20 min: 95% acetonitrile); flow rate 20 mL/min; UV detection at 210 and 280 nm] to give compounds 1 (43 mg) and 2 (51 mg). Fraction H (6.394 g) was subjected to silica gel CC, eluted with a gradient of hexane/EtOAc (4:1−2:1), to give fraction H1 (300.1 mg), which was applied to a C18-E column (5 × 40 cm) with a stepwise gradient of MeOH/H2O (80−95%) to give compounds 4 (73 mg), 5 (17 mg), and 6 (105 mg). 1,3,7,9-Tetrahydroxy-2,8-dimethyl-4,6-di(2-methylbutanoyl)dibenzofuran (1): light yellow, needle-shaped crystals; mp 111−112 °C; [α]25 D +287 (c 0.61, CHCl3); UV (MeOH) λmax (log ε) 277.0 (4.23), 310.0 (4.21) nm; IR (neat) νmax 2964.7, 1609.3 (chelated C O), 1583.7, 1366.1, 1215.1, 1078.6 cm−1; 1H NMR (500 MHz, CDCl3) δ 14.27 (2H, s, OH-3, 7), 3.99 (2H, m, H-2′, 2″), 2.25 (6H, s, CH3-2, 8), 1.97 (2H, m, H-3′a, H-3″a), 1.62 (2H, m, H-3′b, H-3″b), 1.33 (6H, d, J = 6.7 Hz, H-5′, 5″), 0.95 (6H, t, J = 7.7 Hz, H-4′, 4″); 13 C NMR (125 MHz, CDCl3) δ 207.7 (C, C-1′, 1″), 164.0 (C, C-3, 7), 153.7 (C, C-4a, 6a), 152.7 (C, C-1, 9), 107.5 (C, C-2, 8), 104.4 (C, C1a, 9a), 102.0 (C, C-4, 6), 45.2 (CH, C-2′, 2″), 26.5 (CH2, C-3′, 3″), 18.2 (CH3, C-5′, 5″), 11.6 (CH3, C-4′, 4″), 8.0 (CH3, CH3-2, 8); HRESIMS m/z 451.1723 [M + Na]+ (calcd for C24H28NaO7, 451.1733); ESIMS m/z 429.1 [M + H]+. 1,3,7,9-Tetrahydroxy-2,8-dimethyl-4-(2-methylbutanoyl)-6-(2methylpropionyl)dibenzofuran (2): light yellow, needle-shaped crystals; mp 226−227 °C; [α]D25 +110 (c 1.23, CHCl3); UV (MeOH) λmax (log ε) 276.0 (4.31), 309.0 (4.20) nm; IR (neat) νmax 3681.9, 2973.2, 1608.3 (chelated CO), 1209.8, 1055.7, 1033.0 cm−1; 1 H NMR (500 MHz, MeCO-d6) δ 14.22 (1H, s, OH-3), 14.13 (1H, s, OH-7), 4.08 (1H, m, H-2″), 3.98 (1H, m, H-2′), 2.13 or 2.11 (3H, s, CH3-2), 2.11 or 2.13 (3H, s, CH3-8), 1.93 (1H, m, H-3′a), 1.64 (1H, m, H-3′b), 1.33 (3H, overlap, H-5′), 1.31 (6H, overlap, H-3″, 4″), 0.95 (3H, m, H-4′); 13C NMR (125 MHz, MeCO-d6) δ 208.3 (C, C-1′), 208.2 (C, C-1″), 164.4 (C, C-3), 164.3 (C, C-7), 154.3 (C, C-4a), 154.2 (C, C-6a), 154.1 (C, C-1, 9), 108.2 (C, C-8), 108.1 (C, C-2), 105.4 (C, C-1a, 9a), 102.2 (C, C-4), 101.5 (C, C-6), 45.8 (CH, C-2′), 39.2 (CH, C-2″), 19.6 or 19.9 (CH3, C-3″), 19.9 or 19.6 (CH3, C-4″), 26.7 (CH2, C-3′), 18.2 (CH3, C-5′), 11.9 (CH3, C-4′), 8.3 (CH3, CH3-

Table 3. Selectivity Indices for Dibenzofurans from Pilidiostigma glabruma compound inhibition of nitric oxide synthesis (RAW264) inhibition of PGE2 synthesis (3T3)

1

2

3

4

5

6

1.36

4.00

1.60

10.83

3.53

1.17

n/a

n/a

n/a

1.57

3.40

2.06

a

Selectivity index is the ratio between the ED50 value for cytotoxic activity and the IC50 value for the assay of interest.

conventional chemistry principles of the effects of electrondonating and electron-withdrawing substituents and their positions on the reactivity of phenolic −OH groups; acyl groups located at positions 2 and 8 in between two hydroxy groups on either side would be expected to have a greater influence than if located at the more remote 4,6 positions. Unfortunately, compounds 1−6 provide few opportunities for the elaboration of additional related compounds by semisynthesis, and further insight into structure/activity relationships in this class appears to require isolation of further natural compounds. The existing literature data, particularly the potent activity of usnic acid,12 suggest that a better understanding of structure/activity relationships in dibenzofurans may reveal compounds of medicinal utility.

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MATERIALS AND METHODS

General Experimental Procedures. Melting points are uncorrected and were determined with a Stuart SMP3 melting point apparatus (Bibby Scientific Ltd., Stone, Staffordshire, U.K.). Optical rotations were measured on a Polarriz-D DPL polarimeter (Nippon Optical Works Co., Ltd., Tokyo, Japan). UV spectra were measured on a Hewlett-Packard 8453 polarimeter at room temperature. The IR spectra were acquired using a Bruker Vector 33 spectrometer. NMR spectra were acquired on a Bruker AVANCE 500 MHz spectrometer with TMS as the internal standard. High-resolution electrospray ionization (HRESIMS) accurate mass measurements were carried out on a Bruker micrOTOF-Q instrument with a Bruker ESI source. Column chromatography (CC) separations were carried out using silica gel (silica-amorphous, precipitated, 200−425 mesh, SigmaAldrich), Sepra C18-E (50 μm, 65A; Phenomenex Torrance, CA, USA), and MCI gel CHP20P (Supelco, Bellafonte, PA, USA). Preparative HPLC was performed on a Gilson 322 system with a UV/ vis-155 detector and a FC204 fraction collector using a Phenomenex Luna 5 μm (150 × 21.2 mm i.d.) C18 column. Plant Material. The leaves of Pilidiostigma glabrum were collected from a cultivated plant in the Medicinal Plant Garden at Southern Cross University, Lismore, Australia (28°49′ S, 153°18′ E), in July D

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well. Following incubation for 1 h, LPS solution (10 μL/well, 10 μg/ mL) was added and the plate incubated for a further 20 h. Following this incubation, the plate was centrifuged (1500g, 3 min), and 90 μL of the supernatant transferred to a clear flat-bottom assay plate (PerkinElmer, Glen Waverley, VIC, Australia) and assayed immediately for nitrite. Nitrite standards (0−100 μM) were prepared in the medium. Then 90 μL of each standard and cell supernatant were transferred to a flat-bottom microplate (Greiner Bio-One, Frickenhausen, Germany) with 90 μL of Griess reagent (0.1% N-1naphthylethylenediamine dihydrochloride, 1% sulfanilic acid in 5% phosphoric acid) added to each well, followed by incubation (23 °C, 20 min) on an orbital plate shaker. Following incubation the absorbance was read at 550 nm in a Wallac Victor 2 plate reader (Wallac, Turku, Finland). Samples and controls were assayed in triplicate. Standard curves were calculated for nitrite standards, and R2 values determined to verify linearity. Mean and standard deviation were calculated for replicates. The nitric oxide (as measured by nitrite) production in sample wells was calculated as a percentage of the production in solvent control wells. TNF-α Assay. This assay was performed on the same cell supernatants as the nitrite assay (see above). TNF-α was quantified using a Quantikine Mouse TNF-α immunoassay kit (R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s instructions. PGE2 Assay. 3T3 Swiss albino mouse embryonic fibroblast cells (ATCC, Manassas, VA, USA) were grown at 37 °C, 5% CO2 in Dulbecco’s modified Eagle medium containing 5% FBS (Interpath, Heidelberg, VIC, Australia), 5% newborn calf serum, L-glutamine (2 mM), sodium pyruvate (1 mM), penicillin (50 U/mL), and streptomycin (50 μg/mL) (all except FBS from Invitrogen, Mulgrave, VIC, Australia). For the assay, cells were seeded into 96-well cell culture plates (Interpath, Heidelberg, VIC, Australia) at a concentration of 27 × 103 cells/well in the medium as for the maintenance medium but without phenol red. Cells were allowed to attach overnight. Test samples dissolved in DMSO were added to the cells and incubated for 3 h at 37 °C, 5% CO2. The final DMSO concentration was 0.5% in all wells. Calcium ionophore A23187 (0.5 mM, 10 μL/well) (Sigma-Aldrich, St Louis, MO, USA) was added to the wells to stimulate PGE2 production, and the cells were incubated for a further 15 min. Culture plates were centrifuged (1500g, 3 min), and the supernatant was removed and stored at −80 °C until assayed using a prostaglandin E2 EIA kit-monoclonal (Cayman Chemical Company, Ann Arbor, MI, USA) according to the manufacturer’s protocol. The cell culture supernatant was diluted 81-fold in kit assay buffer before determination of PGE2 content. A standard curve (%B/ B0 vs log10 PGE2 concentration) was plotted, and the curve was fitted to a four-parameter logistic equation using GraphPad Prism software. The percentage inhibition of PGE2 production by each sample (assayed in triplicate) was calculated relative to the DMSO control. Selectivity Index. This index was calculated as the ratio between the ED50 value for cytotoxic activity and the IC50 value for the assay of interest.20 ORAC Assay. This assay was carried out in black 96-well fluorescence assay plates (Interpath, Heidelberg, VIC, Australia). All samples were diluted by combining 40 μL of sample in DMSO (20 mg/mL) with 960 μL of phosphate buffer (75 mM, pH 7.4) to give a concentration of 0.8 mg/mL. Further 2-fold serial dilutions were performed in 75 mM phosphate buffer/2% DMSO. Each sample was tested in two concentrations, which gave AUC values within the range of the Trolox standard curve (12.5−100 μM). Epicatechin (SigmaAldrich, St Louis, MO, USA) was used as a positive control. Into each well were added fluorescein solution (10 μL, 5 μM), 20 μL of sample, Trolox standard, epicatechin, or solvent control solution, and last 170 μL of AAPH (2,2′-azobis-2-methylpropanimidamide dihydrochloride) solution (20 mM). Immediately after the addition of the AAPH solution, the assay plates were placed in a Wallac Victor 2 plate reader (Wallac, Turku, Finland), and the fluorescence was recorded at 37 °C every min for 35 min. The fluorescence readings were referenced to solvent blank wells. Final ORAC values were determined using a regression equation for Trolox concentration versus net area under the fluorescein decay curve. Antioxidant activity

2, 8); HRESIMS m/z 437.1577 [M + Na]+ (calcd for C23H26NaO7, 437.1576); ESIMS m/z 415.2 [M + H]+. 1,3,7,9-Tetrahydroxy-2,8-dimethyl-4,6-di(2-methylpropionyl)dibenzofuran (3): light yellow, needle-shaped crystals; mp 248−249 °C; UV (MeOH) λmax (log ε) 276.0 (4.23), 309.0 (4.13) nm; IR (neat) νmax 3455.4, 2964.7, 1609.3 (chelated CO), 1583.8, 1366.1, 1267.2, 1047.8 cm−1; 1H NMR (500 MHz, CDCl3) δ 14.13 (2H, s, OH-3, 7), 4.08 (2H, m, H-2′, 2″), 2.25 (6H, s, CH3-2, 8), 1.33 (12H, d, J = 6.7 Hz, H-3′, 4′, 3″, 4″); 13C NMR (125 MHz, CDCl3) δ 207.7 (C, C-1′, 1″), 164.0 (C, C-3, 7), 153.7 (C, C-4a, 6a), 152.4 (C, C-1, 9), 107.4 (C, C-2, 8), 104.2 (C, C-1a, 9a), 101.4 (C, C-4, 6), 38.7 (CH, C-2′, 2″), 19.5 (CH3, C-3′, 3″, 4′, 4″), 8.0 (CH3, CH3-2, 8); HRESIMS m/z 423.1415 [M + Na]+ (calcd for C22H24NaO7, 423.1420); ESIMS m/z 401.2 [M + H]+. 1,3,7,9-Tetrahydroxy-4,6-dimethyl-2-(2-methylbutanoyl)-8-(2methylpropionyl)dibenzofuran (4): yellow, needle-shaped crystals; mp 203−204 °C; [α]25 D +162 (c 0.74, CHCl3); UV (MeOH) λmax (log ε) 279.0 (4.75), 291.0 (4.68), 307.0 (4.51), 380.0 (3.61) nm; IR (neat) νmax 3681.9, 2972.9, 1615.5 (chelated CO), 1414.8, 1097.8, 1055.6, 1033.1; 1H NMR (500 MHz, MeCO-d6) δ 4.09 (1H, m, H2″), 3.97 (1H, m, H-2′), 2.24 (6H, s, CH3-4, 6), 1.90 (1H, m, H-3′a), 1.45 (1H, m, H-3′b), 1.22 (9H, overlap, H-5′, 3″, 4″), 0.95 (3H, m, H4′); 13C NMR (125 MHz, MeCO-d6) δ 213.3 (C, C-1″), 213.1 (C, C1′), 160.8, 159.9, 153.7, 153.6 (C, C-1, 3, 4a, 6a, 7, 9), 107.4, 106.8, 105.8, 101.4 (C, C-1a, 2, 4, 6, 8, 9a), 47.1 (CH, C-2′), 40.4 (CH, C2″), 27.8 (CH2, C-3′), 19.7 or 19.8 (CH3, C-3″), 19.8 or 19.7 (CH3, C-4″), 17.0 (CH3, C-5′), 12.3 (CH3, C-4′), 8.4 (CH3, CH3-4, 6); HRESIMS m/z 437.1577 [M + Na]+ (calcd for C23H26NaO7, 437.1576); ESIMS m/z 415.2 [M + H]+. 1,3,7,9-Tetrahydroxy-4,6-dimethyl-2,8-di(2-methylpropionyl)dibenzofuran (5): yellow, needle-shaped crystals; mp 183−184 °C; UV (MeOH) λmax (log ε) 278.0 (4.43), 378.0 (3.37) nm; IR (neat) νmax 3403.0, 2961.1, 1617.1 (chelated CO), 1248.0, 1045.2, 926.9; 1 H NMR (500 MHz, CDCl3) δ 4.07 (2H, m, H-2′, 2″), 2.34 (6H, s, CH3-4, 6), 1.26 (12H, d, J = 6.8 Hz, H-3′, 4′, 3″, 4″); 13C NMR (125 MHz, CDCl3) δ 212.7 (C, C-1′, 1″), 159.2, 153.3 (C, C-1, 3, 4a, 6a, 7, 9), 105.9, 105.1, 100.2 (C, C-1a, 2, 4, 6, 8, 9a), 40.0 (CH, C-2′, 2″), 19.5 (CH3, C-3′, 4′, 3″, 4″), 8.1 (CH3, CH3-4, 6); HRESIMS m/z 423.1412 [M + Na]+ (calcd for C22H24NaO7, 423.1420); ESIMS m/z 401.1 [M + H]+. Cytotoxicity Assay. Cytotoxicity in RAW264 murine leukemic monocyte macrophages (ATCC, Manassas, VA, USA) was assayed in 96-well plates using the ATPlite assay kit (PerkinElmer, Glen Waverley, Australia) with chlorambucil (Sigma C0253) as a positive control. Cells were grown in clear 96-well plates. The growth medium consisted of color-free Dulbecco’s modified Eagle’s medium containing 10% (v/v) fetal bovine serum (FBS; Interpath, Heidelberg, Australia), L-glutamine (2 mM), sodium pyruvate (1 mM), penicillin (200 U/ mL), and streptomycin (200 μg/mL) (all from Invitrogen, Mulgrave, Australia). Cells were plated at a concentration of 30 000 cells/well (90 μL of cell suspension/well), test and control compounds dissolved in DMSO at six concentrations and further diluted 20-fold in the medium were added to the cell suspension at 10 μL/well, and the plates were incubated at 37 °C with 5% CO2 for 24 h. Following incubation, cell lysates were assayed for ATP with the ATPlite assay kit as per the manufacturer’s instructions. Briefly, all kit components were equilibrated to room temperature. Mammalian cell lysis solution (50 μL) was added to each well of the cell culture microplate, the plate was shaken on an orbital microplate shaker (500 rpm, 5 min), then substrate solution (50 μL/well) was added, and the plate was further shaken (500 rpm, 5 min). The plate was dark adapted for 10 min, and the luminescence measured on a Wallac 1450 Microbeta luminescence counter (Wallac, Turku, Finland). Half-maximal inhibitory concentration (IC50) values were calculated using GraphPad Prism version 4 (La Jolla, CA, USA). Samples were assayed in triplicate. Nitrite (Griess) Assay. RAW264 cells were cultivated as described above. Cell suspension (120 μL/well, 106 cells/mL) was added to the wells of a 96-well microplate and incubated for 20 h (37 °C, 5% CO2), after which test compounds (dissolved in DMSO and further diluted 20-fold in the medium) were added to the cell suspension at 10 μL/ E

dx.doi.org/10.1021/np300433r | J. Nat. Prod. XXXX, XXX, XXX−XXX

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was based on the mean value for two sample concentrations and expressed in micromoles of Trolox equivalents per gram of sample. All assays were performed in quadruplicate. Antibacterial Assays. Bacterial test strains (Staphylococcus aureus ATCC 29213 and ATCC 25923, S. epidermidis ATCC 35984 (biofilm forming), Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853) collected from stock cultures preserved at −80 °C in the Microbiology Laboratory, University of South Australia, were utilized. All bacterial strains were grown on Columbia blood agar base (Oxoid CM331), supplemented with 2.5% (w/v) defibrinated horse blood. Tryptone soya broth (TSB) with 0.025 g/mL glucose prepared to manufacturer’s specifications was used for the determination of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). Broth microdilution was performed in 96-well round-bottom plates (Sarstedt, Technology Park, South Australia) according to the Clinical Laboratory Standards Institute’s method M7A7 with modification (TSB broth plus glucose in place of MuellerHinton broth).21 Serial 1-in-2 dilutions of the test compounds in TSB containing 2% DMSO were made in the wells, in a total of 50 μL. Bacterial strains were prepared by adding sterile saline to actively growing broth culture to achieve a turbidity equivalent to a standardized reading of 0.5 McFarland unit representing 108 cfu/ mL. These suspensions were diluted 1 in 100 in TSB broth, and 50 μL of inoculum was added to each well, delivering approximately 5 × 105 cfu in a total volume of 100 μL. The microtiter plate was incubated overnight in an incubator at 37 °C. The MIC was determined as the lowest concentration at which no growth was observed in the microdilution wells as detected by the unaided eye. Following determination of the MIC, a 10 μL aliquot from each of the wells at the concentration corresponding to the MIC and those concentrations above were transferred to 190 μL of TSB in a microtiter plate, which was incubated at 37 °C overnight. The MBC was determined as the lowest concentration at which no growth occurred and was confirmed by viable cell count on Columbia agar plates.

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ASSOCIATED CONTENT

S Supporting Information *

NMR and UV spectra of compounds 1−5. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the Wound Management Innovation CRC (established and supported under the Australian Government’s Cooperative Research Centres Program).

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REFERENCES

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dx.doi.org/10.1021/np300433r | J. Nat. Prod. XXXX, XXX, XXX−XXX