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Cite This: J. Nat. Prod. 2018, 81, 1517−1526

Banksialactones and Banksiamarins: Isochromanones and Isocoumarins from an Australian Fungus, Aspergillus banksianus Nirmal K. Chaudhary,† John I. Pitt,‡ Ernest Lacey,†,§ Andrew Crombie,§ Daniel Vuong,§ Andrew M. Piggott,† and Peter Karuso*,† †

Department of Molecular Sciences, Macquarie University, Sydney, NSW 2109, Australia Commonwealth Scientific and Industrial Research Organisation, North Ryde, NSW 2113, Australia § Microbial Screening Technologies Pty. Ltd., Smithfield, NSW 2164, Australia Downloaded via STEPHEN F AUSTIN STATE UNIV on July 27, 2018 at 06:56:12 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

‡

S Supporting Information *

ABSTRACT: Chemical investigation of an Australian fungus, Aspergillus banksianus, led to the isolation of the major metabolite banksialactone A (1), eight new isochromanones, banksialactones B−I (2−9), two new isocoumarins, banksiamarins A and B (10 and 11), and the reported compounds, clearanol I (12), dothideomynone A (13), questin (14), and endocrocin (15). The structures of 1−11 were established by NMR spectroscopic data analysis, and the absolute configurations were determined from optical rotations and ECD spectra in conjunction with TD-DFT calculations. The secondary metabolite profile of A. banksianus is unusual, with the 11 most abundant metabolites belonging to a single isochromanone class. Conjugation of 1 with endocrocin, 5-methylorsellinic acid, 3,5-dimethylorsellinic acid, mercaptolactic acid, and an unknown methylthio source gave rise to five unprecedented biosynthetic hybrids, 5−9. The isolated compounds were tested for cytotoxicity, antibacterial, and antifungal activities, with hybrid metabolites 7−9 displaying weak cytotoxic and antibiotic activities.

S

Continuing our investigations into the chemical diversity of Australian Aspergillus species, herein we report the identification and secondary metabolite profile of a new species, A. banksianus. A total of 15 polyketide-derived natural products, including the major metabolite banksialactone A (1), eight new isochromanones, banksialactones B−I (2−9), two new isocoumarins, banksiamarins A and B (10 and 11), and the previously reported compounds clearanol I (12), dothideomynone A (13), questin (14), and endocrocin (15), were isolated from A. banksianus.

oil microorganisms are a rich source of secondary metabolites with potentially useful applications as drugs, agrichemicals, and cellular probes. Aspergilli are the most prolific producers of fungal secondary metabolites in the family Trichocomaceae, with over 2000 secondary metabolites reported to date from the 129 formally described Aspergillus species.1 Despite intense efforts focused on the discovery of novel chemical diversity in Aspergillus, there are no signs that productivity is on the wane. Recent characterization of the total genomes of over 40 Aspergillus species2 demonstrates that secondary metabolite gene clusters frequently outnumber the metabolites identified from each respective species.3 Importantly, the secondary metabolite profile of each Aspergillus species provides a unique fingerprint that can act as a specific chemotaxonomic descriptor of that species.4 In the exploration of previously described and novel Aspergillus species, Australian strains have proven to be rich sources of new chemical scaffolds. For example, chemical investigation of new Australian species, A. hancockii and A. kumbius, revealed a unique family of piperazines, hancockiamides A−F,3 and novel bis-indolyl benzenoids, kumbicins A−D, repectively.5 Other notable examples from Australian strains of Aspergillus include aspergillazines A−E from A. unilateralis,6 aspergillicins A−E from a marine-derived strain of A. carneus,7 and cottoquinazoline A and cotteslosins A and B from A. versicolor.8 © 2018 American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION A. banksianus sp. nov. was isolated from a soil sample collected at Collaroy, New South Wales, Australia, in 2004. On agar, A. banksianus formed small green colonies bearing a superficial morphological resemblance to Penicillium species, but detailed examination indicated that the strain was a novel Aspergillus species related to A. viridinutans in the clade associated with the sexual genus Neosartorya. A formal taxonomic description of A. banksianus will be published elsewhere in due course. Preparative cultivation of A. banksianus on rice resulted in a luxuriant growth, with fungus covering, and almost entirely Received: September 26, 2017 Published: June 19, 2018 1517

DOI: 10.1021/acs.jnatprod.7b00816 J. Nat. Prod. 2018, 81, 1517−1526

Journal of Natural Products

Article

Chart 1

Figure 1. UV and ECD spectra of banksialactone A (1) compared to calculated spectra. Solid lines are the experimental data with dashed gray lines for the calculated spectra (TD-DFT-D3//BP86/TZVPP) with COSMO solvent simulation (MeOH) blue-shifted by 24 nm. (A) UV spectrum of 1 (MeOH). (B) ECD spectrum of 1 (solid line) and the calculated ECD spectrum of the (3S,4R)/(3R,4R) mixture of the isomer at 5.8:1 as per the NMR spectrum (Figure S1, Supporting Information) in methanol-d4. (C) ECD spectrum of 1 (solid line) and the calculated ECD spectrum for a 5.8:1 mixture of (3R,4S)/(3S,4S)-1 in methanol.

consuming, the grain after 14 days at 24 °C. The pooled mycelial mass was extracted into acetone, reduced to an aqueous concentrate, and then partitioned against ethyl acetate to recover the organic metabolites. The crude extract was defatted and fractionated on a Sephadex LH-20 column, to yield four fractions. Fractions 2−4 contained small molecules (TLC) and were purified by reversed-phase HPLC to yield 15 compounds in quantities sufficient for full spectroscopic characterization. The UV spectra of 1−8, 12, and 13 (Figures S14−S21, S25−S26; Supporting Information) were very similar, with maxima at ca. 210, 270, and 310 nm in a ratio of 3:2:1, respectively, indicative of an isochromanone chromophore.9 The IR spectra of the metabolites were also

very similar, with characteristic carbonyl absorptions at ca. 1650 cm−1 and hydroxy absorptions at ca. 3300 cm−1. Banksialactone A (1), the most abundant metabolite, was obtained as a colorless oil. LC-MS analysis revealed a single peak with a mass of 269.1 ([M + H]+), which together with 13 C NMR spectroscopy (13 carbons) suggested a molecular formula of C13H16O6. Despite showing a single peak by HPLC, the 1H NMR of 1 revealed two sets of closely related signals with a ratio of 5.8:1 (Figure S1, Supporting Information) The presence of one aromatic methoxy group (δC 56.0; δH 3.84), one singlet aromatic methyl (δC 9.8; δH 2.00), and one singlet aromatic proton (δC 97.0; δH 6.45) in the major isomer, in conjunction with the UV spectrum, suggested an isochromanone skeleton. The remaining signals in the 1H NMR 1518



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Table 1. 1H (600 MHz) and 13C (150 MHz) NMR Data for Banksialactones B−D (2−4) in DMSO-d6 banksialactone B (2) position

δC, type

1 3 4 4a 5 6 7 8 8a 9 10 11 12 13 4-OH 6-OH 8-OH 9-OH

167.7, C 68.8, CH 88.4, C 152.0, C 110.2, C 163.6, C 98.9, CH 156.3, C 103.8, C 17.1, CH3 21.1, CH3 11.1, CH3 56.1, CH3

δH, mult (J in Hz) 4.06, q (6.3)

6.48, s

0.73, d (6.3) 1.60, s 2.05, s 3.81, s

banksialactone C (3) δC, type 167.8, C 68.7, CH 88.1, C 153.0, C 108.9, C 162.6, C 102.2, CH 155.8, C 103.1, C 17.1, CH3 21.1, CH3 11.0, CH3

δH, mult (J in Hz) 4.05, q (6.3)

6.40, s

0.71, d (6.3) 1.58, s 2.02, s

a

a

10.31, br

10.39, s 10.09, br

banksialactone D (4) δC, type 167.0, C 68.7, CH 88.2, C 152.8, C 111.0, C 164.0, C 95.6, CH 157.7, C 104.8, C 17.1, CH3 21.1, CH3 11.1, CH3 56.4, CH3 55.8, CH3

δH, mult (J in Hz) 4.07, q (6.3)

6.68, s

0.71, d (6.3) 1.61, s 2.09, s 3.93, s 3.90, s 5.13, br

a

Not observed or not assignable.

Figure 2. UV and ECD spectra of banksialactone B (2) compared to calculated spectra. Solid lines are the experimental data with dashed gray lines for the calculated spectra (TD-DFT-D3//BP86/TZVPP) with COSMO solvent simulation (MeOH) blue-shifted by 38 nm. (A) UV spectrum of 1 (MeOH) compared to the calculated spectrum of 2. All isomers showed an identical calculated UV spectrum. (B) ECD spectrum of 2 (solid line) and the calculated ECD spectrum of (3S,4S)-banksialactone B. (C) Calculated ECD spectra of (3R,4R)-fusaraisochromanone (dotted line) and (3S,4R)-fusaraisochromanone (dashed line) that fit the published ECD data for fusaraisochromanone.32

spectrum were a doublet methyl (δC 16.3; δH 1.06) coupled to a benzylic methine (δC 35.0; δH 3.28) and a CH2OH group (δC 63.6; δH 3.64, 3.72) (see Table S1, Supporting Information, for full spectroscopic data). A search of the literature revealed structure 1, which has closely matching NMR data.10,11 Although 1 has been previously reported from the ascomycete Allantophomopsis lycopodina10 and subsequently from the fungi Leptosphaeria sp.11 and Paraphoma radicina,12 it has not been assigned a trivial name. As 1 is the most abundant and the key metabolite in A. banksianus, acting as the putative biosynthetic precursor to a diverse range of isochromanone conjugates (5− 9), for convenience we have assigned the trivial name banksialactone A. The observed specific rotation of 1 (+79) is in agreement, in terms of sign and general magnitude, with literature values of +55,12 +61,11 and +106.10 Given that the compound exists as a mixture of diastereomers resulting from rapid opening and closing of the hemiketal, which would be influenced by solvent, temperature, and possibly concentration, such variations in optical rotation are not unexpected. The

absolute configuration of 1 at C-4 was determined computationally. First, the four possible stereoisomers of 1 were energyminimized (molecular mechanics, MMFF94x) and subjected to a conformational search (low-mode molecular dynamics, MMFF94x). The lowest energy geometry was further optimized using density functional theory (DFT//BP86/ TZVPP-COSMO), and the UV spectrum was calculated using time-dependent DFT (TD-DFT//BP86/TZVPP), which gave a good match to the experimental data (Figure 1A). The four possible stereoisomers of 1 gave very similar calculated UV spectra at this level of theory. The electronic circular dichroism (ECD) spectrum of each isomer was also calculated, with a 5.8:1 ratio (as per the NMR spectrum in methanol-d4) of 3R,4S/3S,4S, giving a good match for the experimental ECD (Figure 1C). In contrast, a 5.8:1 ratio of 3S,4R/3R,4R did not fit the experimental data (Figure 1B). Thus, the major isomer of 1 is (3R,4S)-banksialactone A. The very similar ECD spectra of 5−9 and 13 (Figures S33−S39, 1519



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C12H14O5 by HRMS (Δmmu −0.4). The 1H and 13C NMR data of 3 were almost identical to those of 2, with the only significant differences being the absence of signals for an Omethyl group and the presence of an additional exchangeable proton at δH 10.39 (Table 1). This suggested that 3 is the 6-Odesmethyl analogue of 2. Banksialactone D (4) was also isolated as an optically active, colorless oil. The molecular formula of 4 was determined as C14H18O5 by HRMS (Δmmu −0.4). The difference in the molecular formula of CH2 from 2 to 4, together with similar 1 H NMR (Table 1) spectra, suggested the presence of an additional O-methyl group (δC 55.8; δH 3.90, s). HMBC correlations (Table S4 and Figure S3, Supporting Information) from the additional methoxy protons to C-8 confirmed that 4 is the 8-O-methyl analogue of 2. Banksialactone E (5) was isolated as a pale yellow solid. Although LC-MS revealed a single chromatographic peak and molecular mass, the 1H NMR spectrum of 5 clearly indicated a mixture of two sets of signals (3.75:1) reminiscent of 1 (Figure S4, Supporting Information). A molecular formula of C16H20O8S was assigned by HRMS (Δmmu −0.8). The presence of sulfur was also suggested by the intensity of the [M + 2] ion in the mass spectrum (∼4.5% of the monoisotopic peak). The NMR spectra of 5 were similar to those of 1 (Table 2), with a notable difference being the presence of an additional −CH2−CH− spin system as determined by COSY. Methylene protons (H2-11) from this spin system showed HMBC correlation to C-9 (Figure S5, Supporting Information), and the presence of a sulfur bridge between these carbons was suggested based on their chemical shifts of 9-CH2 (δC 38.9; δH 3.13, 3.19) and 11-CH2 (δC 36.6; δH 2.88, 2.98). The methine (C-12) of this spin system was an oxymethine (δC 70.4), and both H-12 and H2-11 protons showed HMBC correlations to a carbonyl carbon (δC 174.0) (Table S5, Supporting Information). These observations suggested the presence of a 3-mercaptolactate group attached to C-9 of 1 through its sulfhydryl group, thus allowing the structure of 5 to be assigned as indicated. The 3mercaptolactate motif presumably arises from the oxidative deamination of cysteine. Natural products incorporating a 3mercaptolactate moiety have been reported in the literature but are rather uncommon.14−22 Banksialactone F (6) was isolated as a pale yellow solid. Although LC-MS revealed a single chromatographic peak and single molecular mass, the 1H NMR spectrum clearly indicated a mixture of four stereoisomers in the ratio of 1:4.1:6.7:14.3 due to the ring opening−closing equilibrium at C-3 and mixed chirality at the sulfur atom (Figure S6, Supporting Information). The ratios indicate that the configuration on the sulfur atom must be partially racemic. A molecular formula of C14H18O6S was established by HRMS (Δmmu −0.8). The 1 H NMR spectrum of the major isomer showed resonances for a singlet aromatic/olefinic methine (δH 6.47), two methyls (δH 1.14, d and δH 1.98, s), one methoxy (δH 3.85, s), and one methine (δH 3.43), whereas the 13C NMR spectrum included resonances for an ester carbonyl carbon (δC 168.1) and hemiketal carbon (δC 104.2) similar to 1, together with some additional signals in the 1H and 13C NMR spectrum (Table 2). A methyl (H3-11) showed HMBC correlation to C-9, and the presence of a sulfinyl bridge between these carbons was assigned based primarily on the chemical shifts of adjacent atoms: 9-CH2 (δC 59.3; δH 3.27, 3.57) and 11-CH3 (δC 39.3; δH 2.72) (Table S6, Supporting Information). In addition, a

Supporting Information) suggested the same absolute configurations at C-3 and C-4 for all the compounds. Banksialactone B (2) was isolated as a colorless oil, and its UV spectrum was characteristic of a 2,4-dihydroxybenzoyl chromophore, with peak maxima at 215, 255, and 297 nm. The molecular formula of 2 was determined as C13H16O5 by highresolution mass spectrometry (HRMS; Δmmu −0.2). The IR spectrum (Figure S56; Supporting Information) indicated the presence of carbonyls (νmax 1611, 1721 cm−1) and hydroxy groups (νmax 3368 cm−1). Unlike 1, a hemiketal carbon (C-3) was not present in the 13C NMR spectrum of 2 (Table 1). Rather, the presence of a tertiary alcohol was indicated by a 13 C NMR resonance at 88.4 ppm, while the 13C NMR resonance for C-3 was observed around 68.8 ppm. The observed 1H and 13C NMR chemical shifts of the aromatic part of the molecule are almost identical to 1, and a strong ROESY correlation between H3-11 and 6-OMe confirmed an ortho regiochemistry. These data suggested that 2 could be fusaraisochromanone, which has recently been isolated from a Fusarium sp.13 However, the observed optical rotation of 3, [α]24D −17 (c 1.01, MeOH), did not match with the reported value for fusaraisochromanone, [α]25D +21 (c 1.01, MeOH). The observed ECD spectrum of 2 showed Cotton effects at 214 (−10.65) and 260 (+3.92) nm, whereas the ECD spectrum reported in the literature for fusaraisochromanone is completely different, having Cotton effects at 227 (−13.2), 242 (+7.26), 262 (−4.95), and 280 (+10.22) nm. TD-DFT calculations were able to reproduce the UV spectrum of 2, albeit red-shifted by 38 nm (Figure 2A). All four possible stereoisomers of 2 had similar calculated UV spectra. The calculated ECD spectrum for (3S,4S)-banksialactone B matched most closely with the experimental data, although (3R,4S)-banksialactone B could not be discounted (Figure 2B). Fortunately, the absolute configuration of C-3 could be assigned relative to (S)-C-4 with the help of ROESY data and the DFT-minimized structure for the two configurations at C-3 (Figure 3). As there was a strong ROESY correlation between

Figure 3. DFT-minimized (DFT-D3//BP86/TZVPP-COSMO; MeOH) structures. (A) (3S,4S)-2 methyl groups are trans-diaxial and should not show an NOE to each other, but an NOE between H3 and H3-10 (red arrow) should be apparent. In contrast, (B) (3R,4S)-2 should not show an NOE between H-3 and H3-10.

H-3 and H3-10 (Figure S2 and Table S2, Supporting Information), C-3 was assigned an S-configuration. ECD calculations for the other two stereoisomers (Figure 2C) indicated a reasonable match with the published ECD spectrum for fusaraisochromanone.13 It was not possible to determine unequivocally whether 2 is the enantiomer or diastereomer of fusaraisochromanone, but considering the nearly opposite specific rotation, the enantiomer is most likely. Furthermore, the very similar optical rotations and ECD spectra (Figures S30−S32, Supporting Information) for 2−4 suggested all possess the same absolute configurations, namely, 3S,4S. Banksialactone C (3) was isolated as an optically active, colorless oil. The molecular formula of 3 was determined as 1520



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Table 2. 1H (600 MHz) and 13C (150 MHz) NMR Data for Banksialactone E (5) and Banksialactone F (6) in DMSO-d6 banksialactone E (5) major isomer position

δC, type

1 3 4

168.7, C 105.7, C 35.8, CH

4a 5 6 7 8 8a 9

143.3, C 114.5, C 163.9, C 97.1, CH 161.9, C 99.4, C 38.9b, CH2

11

36.6, CH2

12

70.4, CH

13 14 15 16 3-OH 8-OH

174.0, C 16.4, CH3 9.9, CH3 56.0, CH3

δH, mult (J in Hz)

δC, type

3.37, q (7.1)

168.7, C 106.7, C 34.8, CH

6.46, s

3.13, d (13.8) 3.19, d (13.8) 2.88, dd (13.8, 6.9) 2.98, dd (13.8, 4.7) 4.21, dd (6.9, 4.7) 1.05, d (7.1) 2.00, s 3.84, s 7.40, br 11.21, s

banksialactone F (6)

minor isomer

142.6, C 114.6, C 163.9, C 97.6, CH 162.1, C 98.6, C 40.0,b CH2

37.0, CH2

70.4, CH 173.9, C 14.6, CH3 9.8, CH3 56.1, CH3

isomer I

δH, mult (J in Hz)

δC, type

3.63, q (7.1)

168.1, C 104.2, C 37.7, CH

6.49, s

2.69 d, (14.2) 3.02 d, (14.2) 2.72, dd (13.8, 5.1) 2.73, dd (13.8, 6.6) 4.03, dd (6.6, 5.1) 1.08, d (7.1) 2.03, s 3.84, s 7.72, br 11.22, s

isomer II

δH, mult (J in Hz)

143.3, C 114.7, C 164.2, C 97.3, CH 162.0, C 99.3, C 59.3, CH2

39.3,b CH3

17.3, CH3 10.0, CH3 56.2, CH3

δC, type 168.5, C 104.2, C 36.8, CH

3.43, q (7.1)

6.47, s

3.27, d (13.7) 3.57, d (13.7) 2.72, s

1.14, d (7.1) 1.98, s 3.85, s

142.7, C 115.1, C 164.0, C 97.6, CH 162.0, C 98.4, C 60.4, CH2

38.8,b CH3

14.1, CH3 9.5, CH3 56.0, CH3

7.87, br 11.21, s

δH, mult (J in Hz)

3.65, q (7.1)

6.49, s

3.02, d (13.4) 3.12, d (13.4) 2.51,b s

1.10, d (7.1) 1.99, s 3.85, s

8.22, br 11.23, s

isomer III δC, type 168.0, C 104.0, C 37.9, CH 42.8, C 14.7, C 64.0, C 97.2, CH 161.9, C 99.2, C 60.3, CH2

40.0,b CH3

16.9, CH3 9.9, CH3 56.0, CH3

isomer IV

δH, mult (J in Hz)

3.37, q (7.1)

6.47, s

3.25, d (14.1) 3.61, d (14.1) 2.75, s

δC, type 168.2, C 104.8, C 36.2, CH 142.3, C 114.7, C 164.2, C 97.2, CH 162.0, C 99.4, C 61.2, CH2

39.5,b CH3

1.09, d (7.1) 2.00, s 3.85, s

10.0, CH3 56.1, CH3

a

a

11.24, s

16.9, CH3

δH, mult (J in Hz)

3.62, q (7.1)

6.49, s

2.96, d (14.3) 3.26, d (14.3) 2.55, s

1.12, d (7.1) 2.02, s 3.85, s

a

11.26, s

a

Not observed or not assignable. bMasked by DMSO-d6 peak

strong band at 1022 cm−1 in the IR spectrum (Figure S60, Supporting Information) suggested the presence of a sulfoxide. Banksialactone G (7) was isolated as an off-white solid. The 1 H NMR spectrum clearly indicated a mixture of two stereoisomers in a ratio of 4.8:1 (Figure S7, Supporting Information) reminiscent of 1. A molecular formula of C22H24O9 was assigned by HRMS (Δmmu −0.1). The 1H NMR spectrum showed resonances typical of the other isochromanones: a singlet aromatic/olefinic methine (δH 6.48), two methyls (δH 1.11 and 2.00), a methoxy singlet (δH 3.85), one oxymethylene (δH 4.35, 4.60), and a methine quartet (δH 3.35), in addition to an ester carbonyl carbon (δC 168.3) and a hemiketal carbon (δC 103.0), characteristic of 1. However, the 1H and 13C NMR data (Table 3) revealed additional signals for two methyls, two hydroxy groups, one aromatic/olefinic proton, one carbonyl, and six sp2-hybridized carbons, suggesting that the molecule had an additional benzoate fragment connected to the isochromanone. The chemical shifts of the six sp2-hybridized carbons and the presence of only one aromatic proton (δH 6.32) were consistent with a pentasubstituted benzene ring. The chemical shift of the protonated aromatic carbon C-3′ (δC 100.0) suggested it was flanked by two hydroxy groups. HMBC correlations (Table S7, Supporting Information) arising from the additional two methyl singlets (δH 1.97 and 2.16), two exchangeable hydroxy signals (δH 9.74 and 9.66), and one aromatic/olefinic singlet (δH 6.32) established the remaining portion as 5′-methylorsellinate. The point of attachment was determined by a strong correlation from H2-9 to C-11 (Figure S8 and Table S7, Supporting Information), suggesting 7 as the structure.

Banksialactone H (8) was isolated as an off-white solid. The H NMR spectrum again revealed a mixture of two stereoisomers in a ratio of 3.8:1 arising from the epimerizable center at C-3 (Figure S9, Supporting Information). A molecular formula of C23H26O9 was assigned by HRMS (Δmmu −0.5). The 1H and 13C NMR data were similar to 7 (Table 3) except for an additional methyl singlet (δH 2.04, δC 9.3) in place of the aromatic singlet (δH 6.32) found in methyl orsellinate, consistent with a corresponding increase in molecular weight by 14 amu compared to 7. The carbon chemical shifts of the three non-isochromanone methyls indicated three different electronic environments, with the first methyl (δC 9.3) being adjacent to two hydroxy groups, the second methyl (δC 12.2) adjacent to a single hydroxy group, and the third methyl (δC 17.9) group not adjacent to any hydroxy groups. These data suggested banksialactone H was 9(6-O-methyldothideomynone)-3′,5′-dimethylorsellinate (8). This was further confirmed by key 2D NMR correlations such as H2-9 to C-11 in the HMBC spectrum (Table S8, Supporting Information). Although orsellinic acid and its analogues have been reported from a number of fungi,23,24 7 and 8 are esters of 1 with 5-methylorsellinic acid and 3,5dimethylorsellinic acid, respectively, and are the first reported isochromanone conjugates of orsellinic acids. Banksialactone I (9) was isolated as an orange-yellow solid. A molecular formula of C29H24O12 was assigned by HRMS (Δmmu −0.3). The UV spectrum did not resemble 1−8, but the 1H and 13C NMR spectra did contain all the characteristic resonances of an isochromanone (cf. 8). For example, the 1H NMR spectrum showed a singlet aromatic/olefinic methine (δH 6.48), two methyls (δH 1.14, d and δH 1.99, s), a methoxy 1

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Table 3. 1H (600 MHz) and 13C (150 MHz) NMR Data for Two Major Isomers of Banksialactone G−I (7−9) in DMSO-d6 banksialactone G (7) major isomer δH, mult (J in Hz)

banksialactone H (8)

minor isomer δH, mult (J in Hz)

position

δC, type

1 3 4

168.0, C 103.0, C 35.4, CH

4a 5 6 7 8 8a 9

143.1, C 114.6, C 164.1, C 97.2, CH 162.0, C 99.3, C 65.0, CH2

11 12

168.3, C 16.4, CH3

13 14 1′ 2′ 3′ 4′ 5′

9.9, CH3 56.0, CH3 111.7, C 154.5, C 100.0, CH 157.5, C 114.0, C

6′ 7′

136.2, C 11.0, CH3

1.97, s

136.4, C 11.0, CH3

1.93, s

8′ 9′ 10′ 11′ 4a′ 10a′ 8a′ 9a′ 3-OH 8-OH 1′-OH 2′-OH 4′-OH 6′-OH 8′-OH

17.1, CH3

2.16, s

17.1, CH3

2.05, s

3.35,a q (7.1)

6.48, s

4.35, d (11.5) 4.60, d (11.5) 1.11, d (7.1) 2.00, s 3.85, s

6.32, s


168.4, C 14.4, CH3 9.9, CH3 56.0, CH3 111.7, C 154.5, C 99.8, CH 157.7, C 113.6, C

3.35,a q (7.1)

6.42, s

4.15, d (11.5) 4.25, d (11.5) 1.10, d (7.1) 1.97, s 3.82, s

6.26, s

major isomer δC, type 167.9, C 102.8, C 35.6, CH 143.0, C 114.7, C 164.1, C 97.2, CH 162.0, C 99.3, C 65.4, CH2

169.9, C 16.4, C 9.9, CH3 56.1, CH3 109.3, C 155.3, C 109.2, C 156.9, C 116.0, C

minor isomer

δH, mult (J in Hz)

3.35,a q (7.1)

6.49, s

4.50, d (11.5) 4.58, d (11.5) 1.12, d (7.1) 2.00, s 3.85, s

banksialactone I (9)


169.6, C 14.8, C 9.7, CH3 55.9, CH3 108.7, C 156.0, C 108.0, C 151.1, C 115.9, C

δH, mult (J in Hz)

3.35,a q (7.1)

6.29, s

4.50, d (11.5) 4.57, d (11.5) 1.11, d (7.1) 1.95, s 3.76, s

134.3, C 9.3, CH3

2.04, s

134.9, C 9.1, CH3

1.99, s

12.2, CH3 17.9, CH3

2.07, s 2.31, s

12.1, CH3 18.0, CH3

2.03, s 2.16, s

7.64, br 11.24, s

7.95, br 11.21, s

7.74, br 11.22, s

8.02, br 11.18, s

9.74, s 9.66, s

9.74, s 9.67, s

10.05, s 8.86, s

10.05, s 8.85, s

major isomer δC, type 167.9, C 102.6, C 35.5, CH 143.0, C 114.7, C 164.2, C 97.3, CH 162.0, C 99.2, C 65.8, CH2

165.1, C 16.4, CH3 9.9, CH3 56.1, CH3 158.2, C 128.0, C 144.7, C 120.7, CH 109.1, CH 165.9, C 108.2, CH 164.6, C 189.5, C 181.1, C 19.8, CH3 135.2, C 133.4, C 109.2, C 114.2, C

minor isomer δH, mult (J in Hz)

3.35,a q (7.0)

6.48, s

4.55, d (11.5) 4.73, d (11.5) 1.14, d (7.1) 1.99, s 3.84, s

7.63, br s 7.17, d (2.4) 6.65, d (2.4)

2.46, s

δC, type 168.2, C 104.8, C 36.2, C 142.3, C 114.7, C 164.2, C 97.2, CH 162.0, C 99.4, C 66.7, CH2

165.1, C 16.9, CH3 9.9, CH3 56.1, CH3 158.2, C 128.0, C 144.7, C 120.7, CH 109.1, CH 165.9, C 108.2, CH 164.6, C 189.5, C 181.1, C 19.8, CH3 135.2, C 133.4, C 109.1, C 114.2, C

δH, mult (J in Hz)

3.35,a q (7.0)

6.47, s

4.30, d (11.5) 4.38, d (11.5) 1.14, d (7.1) 1.99, s 3.80, s

7.59, br s 7.17, d (2.4) 6.64, d (2.4)

2.34, s

7.75, br 11.21, s 11.50, s

7.57, br 11.18, s 11.50, s

12.54, s 11.93, s

12.44, s 11.92, s

a

Masked by water peak.

inseparable stereoisomers in a ratio of 4.6:1 (Figure S10, Supporting Information). The 13C NMR spectrum also revealed the presence of two additional carbonyl carbons at δ C 189.5 and 181.1, consistent with an asymmetric anthraquinone scaffold. The downfield shift of one of the carbonyls (δC 189.5) can be attributed to strong intramolecular hydrogen bonding, suggesting the presence of peri-hydroxy groups. A meta coupling between 5′ and 7′ protons (J = 2.4 Hz), a benzylic coupling between 11′ and 4′ protons (J ≈ 1 Hz), and HMBC correlations (Table S9, Supporting Information) established endocrocin as the anthraquinone fragment. The connectivity between the endocrocin fragment and the isochromanone fragment was established by the

singlet (δH 3.84), an oxymethylene as an AB quartet (δH 4.55 and 4.73), and a methine quartet (δH 3.35) typical of an isochromanone (Table 3). The additional fragment dominated the UV−visible spectrum of the molecule and was consistent with a 1,8-dihydroxyanthraquinone (λmax 441).25 The IR spectrum (Figure S63, Supporting Information) indicated at least three different carbonyl groups with sharp, strong absorption bands at 1652 and 1618 cm−1 indicative of a strongly H-bonded carbonyl such as in a 1-hydroxy-substituted anthraquinone. The mass difference between 9 and 1 (296 amu) is consistent with a trihydroxylated anthraquinone carboxylic acid. Like the other hemiketal isochromanones, the 1H NMR spectrum of 9 revealed a mixture of two 1522



Article

shift of C-8a (δC 99.5) suggested that it was flanked by two carbons bearing oxygen. The presence of a hydrogen-bonded hydroxy group at C-8 implied that C-1 must be a carbonyl carbon, and its chemical shift (δC 165.0) further indicated it was an ester. These observations, together with the HMBC correlations from the two methyls, two hydroxy groups, and the olefinic proton, led to the unambiguous assignment of the structure as 10, an isocoumarin. Moreover, the assigned structure is consistent with the observed UV spectrum as well as 8 degrees of unsaturation as predicted from the molecular formula. Banksiamarin B (11) was isolated as an off-white solid. The UV spectrum was similar to 10, suggesting this was also an isocoumarin. A molecular formula of C13H10O8 was assigned by HRMS (Δmmu −0.3). The 1H and 13C NMR spectra of 11 (Table 4), along with the UV spectrum, were very similar to those of 10. The major differences were an additional carbonyl carbon (δC 166.7) and one less aromatic methyl. HMBC correlations from H-4 to C-5 and C-9 (δC 160.8; Table S11, Supporting Information) suggested the presence of a carboxylic acid at C-3, rather than a methyl ester as in 10. This required a carbomethoxy group at C-7, thus completing the structure of 11. Four previously reported compounds, clearanol I (12),29 dothideomynone A (13),30 questin (14),31,32 and endocrocin (15),33 were also isolated from A. banksianus and were identified by analysis of spectroscopic data (Supporting Information) and comparison with literature values. Compounds 1−14 were tested for in vitro biological activity against the Gram-positive bacterium Bacillus subtilis (ATCC 6633), the Gram-negative bacterium Escherichia coli (ATCC 25922), the fungi Candida albicans (ATCC 10231) and Saccharomyces cerevisiae (ATCC 9763), the protozoan Tritrichomonas fetus (KV-1), and NS-1 mouse myeloma cells (ATCC TIB-18). The simpler banksialactones 1, 2, 4, and 12, together with the related dothideomynone A (13) and the thioether (5) and methylsulfoxide (6), displayed no activity in any of the assays. Weak to moderate activity (Table 5) was

HMBC correlations from H2-9 oxymethylene protons of the banksialactone A fragment to the carbonyl of the endocrocin fragment (Figure S11 and Table S9, Supporting Information), thereby establishing its structure as the isochromanyl ester of endocrocin, 9. Similar to compounds 7 and 8, the major isomer had an ROE between H3-12 and H2-9, while in the minor isomer this was replaced by an ROE between H-4 and H2-9 (Table S9, Supporting Information). Esters of anthraquinones, other than the methyl esters, are very rare in Nature, with paeciloquinone A,26 variecolorquinone,27 and trihydroxy labdanyl anthraquinoate28 being the only other examples. This is the first report of an isochromanyl ester of an anthraquinone. Banksiamarin A (10) was isolated as an off-white solid. The UV spectrum (Figure S23, Supporting Information) was significantly different from those of the isochromanones 1−8 and showed peak maxima at wavelengths of 204, 259 (strong), 320, and 352 nm, with a shoulder at 284 nm. A molecular formula of C13H12O6 was assigned from the HRMS of the [M + Na]+ ion (Δmmu 0.0). The 1H NMR spectrum (Table 4) Table 4. 1H (600 MHz) and 13C (150 MHz) NMR Data for Banksiamarins A and B (10 and 11) in DMSO-d6 banksiamarin A (10) position

δC, type

1 3 4 4a 5 6 7 8 8a 9 10 11 12 13 6-OH 8-OH 9-OH

165.0, C 140.9, C 110.7, CH 131.0, C 113.4, C 161.6, C 113.7, C 158.5, C 99.5, C 160.2, C 52.9, CH3 11.0, CH3 9.1, CH3

δH, mult (J in Hz)

7.63, s

3.88, s 2.27, s 2.14, s 10.05, br 11.28, s

banksiamarin B (11) δC, type 164.9, C 143.7, C 109.3, CH 136.1, C 113.6, C 161.8, C 108.4, C 159.5, C 99.9, C 160.8, C 10.4, CH3 166.7, C 52.7, CH3

δH, mult (J in Hz)

7.57, s

2.24, s

3.87, s 11.49, br 11.75, s

Table 5. Antibacterial (ProTOX), Antifungal (EuTOX), Cytotoxic (CyTOX), and Antiprotozoal (TriTOX) Minimum Inhibitory Concentrations (MICs) of Compounds 1−14

a

a

Not observed or not assignable. compound

indicated two methyls (δH 2.14 and 2.27) as singlets, one methoxy singlet (δH 3.88), one olefinic singlet (δH 7.63), and two exchangeable protons (δH 11.28 and 10.05) consistent with phenols. The 13C NMR chemical shifts of the two methyls C-12 and C-13 (δC 11.0 and 9.1, respectively) suggested that they were flanked by one and two hydroxy groups, respectively, which was supported by HMBC correlations from H3-12 to C6 (δC 161.6) and H3-13 to C-6 and C-8 (δC 158.5) (Table S10, Supporting Information). HMBC correlations from H3-11 methoxy protons and the H-4 olefinic proton to C-9 established the attachment of a methyl ester side chain on the carbon adjacent to the olefinic proton (C-3). A ROESY correlation between H-4 and H 3 -12 and the HMBC correlations from these protons aided in the assignment of their relative positions (Table S10, Supporting Information). The presence of a sharp downfield signal at δH 11.28 in the 1H NMR indicated the presence of a hydrogen-bonded hydroxy group, which was assigned as 8-OH. The upfield 13C NMR

7 8 9 14 Ce

ProTOXa (μg/mL)

EuTOXb (μg/mL)

CyTOXc (μg/mL)

50 25 25

200 50

100 100 100

d

d

d

0.4

0.2

0.8

d

TriTOX (μg/mL) d

50 12.5 12.5 1.6

a

Bacillus subtilis (ATCC 6633), no activity for Escherichia coli (ATCC 25922). bSaccharomyces cerevisiae (ATCC 9763), no activity for Candida albicans (ATCC 10231). cMouse myeloma NS-1 cell line (ATCC TIB-18). dNot active up to 100 μg/mL. ePositive controls: ampicillin; clotrimazole; 5-fluorouracil; mebendazole, respectively.

observed for the “hybrid” metabolites 7−9 across all of the bioassays, suggesting that scavenging for aromatic acids offers a convenient strategy for broadening the bioprofile. Questin (14) displayed a moderate level of selective antiprotozoal activity that deserves further investigation, as this type of activity has not previously been reported for 14. 1523



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CONCLUSION

Article

EXPERIMENTAL SECTION

General Experimental Procedures. Size exclusion chromatography was performed using Sephadex LH-20 (GE Healthcare BioSciences Corp.). Thin-layer chromatography (TLC) was performed using precoated silica gel GF254 plates (Merck, Darmstadt, Germany) with various solvent systems, and spots were visualized with UV light (254 nm). Preparative and semipreparative HPLC separations were performed on a Gilson HPLC system, equipped with a Gilson 215 liquid handler, 819 injection module, 322 pump, 506C system interface, and Gilson single variable wavelength UV−vis 152 detector by reversed-phase chromatography using a gradient of acetonitrile (Sigma-Aldrich, USA) and water containing 0.025% HPLC-grade trifluoroacetic acid (Sigma-Aldrich, USA) unless mentioned otherwise. Phenomenex Hydro-RP (250 × 22.4 mm, 10 μm) and Phenomenex Hydro-RP (250 × 10 mm, 5 μm) columns were used for preparative and semipreparative purifications, respectively. UV− visible readings were taken on an Eppendorf UV−visible spectrophotometer (Biospectrometer Kinetic) in a 1 × 1 cm quartz cell. Optical rotations were recorded in MeOH using a JASCO P-1020 polarimeter (Jasco Corp., Japan) in a 1 cm path length cell. ECD spectra were recorded on a Jasco J-810 spectropolarimeter, model CDF-426S/ 426L Peltier-type ECD/fluorescence simultaneous measurement attachment (Jasco Corp., Japan), in a 1 × 1 cm cell. The IR spectra were recorded on a Thermo Scientific Nicolet iS10 ATR FTIR spectrometer. 1H NMR, 13C NMR, and 2D NMR spectra were recorded in deuterated DMSO using a Bruker AVANCE II 600 MHz NMR spectrometer (Bruker Corp.). ESIMS spectra were recorded using an Agilent 6130 single quadrupole mass spectrometer (Agilent Corp.). HR-ESIMS data were obtained using a Q Exactive Plus hybrid quadrupole-orbitrap mass spectrometer (Thermo Scientific, Bremen, Germany). Biological Material. A. banksianus sp. nov. was collected from under a specimen of Banksia integrifolia in Collaroy, New South Wales, Australia, in 2004 as part of an investigation of the diversity of the fungal family Trichocomaceae in the Sydney basin and was accessioned into the Commonwealth Scientific and Industrial Research Organisation (CSIRO) fungal collection as FRR 6047.36 A full taxonomic description of A. banksianus will be published elsewhere in due course. We have deposited the rRNA sequence of A. banksianus under SUB3902971 in the NIH GenBank. A. banksianus was cultivated for extraction on hydrated rice in Erlenmeyer flasks (100 g; 40 × 250 mL) at 24 °C for 14 days and pooled (2620 g) then extracted with acetone (2 × 3 L) for 2 h on a rotatory platform at 100 rpm. The macerated solid was removed by filtration and the filtrate concentrated to an aqueous concentrate (261 g) by rotatory evaporation. The concentrate was partitioned against ethyl acetate (2 × 2 L), then evaporated to dryness to provide an organic extract (59.3 g). The extract was dissolved in methanol (360 mL), diluted with distilled water (140 mL), and defatted by partition against hexane (2 × 500 mL) to provide a crude extract (10.5 g). Isolation. A portion of the crude extract of A. banksianus (2.7 g) was subjected to size exclusion chromatography on Sephadex LH-20 (methanol/chloroform, 1:1). Eluted fractions were combined based on TLC into four fractions, S1−S4. Fraction S1 was mostly viscous polymeric material and was not further investigated. Fractions S2−S4 were pretreated on a C18 SPE cartridge by washing with 10% methanol/water and eluting with 90% methanol/water, before reversed-phase HPLC purification (acetonitrile/water). Preparative HPLC purification of fraction S2 yielded dothideomynone A (13; 35 mg; tR 12.3 min), clearanol I (12; 2 mg; tR 15.2 min), banksialactone E (5; 2 mg; tR 17.6 min), banksialactone A (1; 72 mg; tR 18.2 min), endocrocin (15; 6 mg; tR 21.6 min), banksiamarin B (11; 1 mg; tR 22.4 min), banksialactone B (2; 2 mg; tR 23.2 min), banksiamarin A (10; 1.5 mg; tR 24.6 min), questin (14; 1 mg; tR 25.5 min), banksialactone G (7; 3 mg; tR 28.8 min), banksialactone H (8; 5 mg; tR 30.2 min), and banksialactone I (9; 3 mg; tR 32.3 min). Preparative HPLC purification of fraction S3 yielded banksialactone F (6; 7 mg; tR 14.5 min), banksialactone C (3; 6 mg; tR 16.0 min), banksialactone E (5; 3 mg; tR 16.2 min), banksialactone B (2; 6 mg; tR 22.8 min), and

Chemical investigation of the novel fungal species A. banksianus revealed that polyketides (isochromanones, isocoumarins, orsellinates, and anthraquinones) dominated the secondary metabolite profile. Isochromanone 1 was the most abundant and the key metabolite in A. banksianus and likely acts as the direct precursor to several other unique natural products (5−9), thereby increasing chemical diversity. These unprecedented conjugates illustrate the use of parallel biosynthetic pathways in Nature to generate hybrid metabolites. Biogenesis of the ester secondary metabolites 7−9 involves two polyketide synthase pathways merging, whereas the S-hybrids 5 and 6 represent an apparent primary metabolite and secondary metabolite fusion. Sumalarins A−C from the fungus Penicillium sumatrense14 and pulcherrimine from the sea urchin Hemicentrotus pulcherrimus incorporate 3mercaptolactate groups.15 Periconiasin H, a metabolite of the endophytic fungus Periconia sp., has an esterified 3mercaptolactate group in which the sulfur is oxidized to a rare sulfoxide group.16 Pandegolide 3, from the fungus Cladosporium herbarum,17 and dichrostachinsäure, an amino acid from the seeds of Dichrostachys glomerata and Neptunia oleracea, also have 3-mercaptolactate groups.18 Similarly, the macrocyclic polyketides cyclothiocurvularins A and B, cyclothiocurvularin B methyl ester, dimethoxymethylcyclothiocurvularin B methyl ester, cyclosulfoxicurvularin, and cyclosulfoxicurvularin methyl ester isolated from the marine fungus Penicillium sp. all contain a 3-hydroxytetrahydrothiophene-3carboxylic acid moiety, which is presumed to arise from the condensation of 3-mercaptopyruvate with 10,11-dehydrocurvularin via a Michael-type addition to the conjugated double bond, followed by cyclization at the α-carbonyl group of mercaptopyruvate.19 The endophytic fungus Paraphaeosphaeria neglecta produces paraphaeosphaeride A, which contains a 4pyranone-γ-lactam-1,4-thiazine moiety that presumably arises by the nucleophilic addition of mercaptolactate to paraphaeosphaeride C.20 Bioconjugation of a 3-mercaptolactate moiety into the enone-bearing fungal metabolite Sch-642305 produced by Phomopsis sp., catalyzed by the fungus Aspergillus niger, has been reported by Adelin et al.22 Pharbitic acid, a gibberellin-related diterpenoid isolated from the immature seeds of Japanese glory (Pharbitis nil), also contains a cyclized 3-mercaptolactate moiety.21 Sulfoxide-bearing natural products are extremely rare, and to our knowledge, periconiasin H,16 dichrostachinsäure,18 cyclosulfoxicurvularin, and cyclosulfoxicurvularin methyl ester19 are the only examples reported to date. Banksialactones F and G are the first reported sulfurcontaining isochromanones. The high proportion of new secondary metabolites compared to previously reported metabolites (10 vs 5) is a further testament to the fact that novel species can yield novel metabolites, even within highly explored genera such as Aspergillus. In addition, further diversity is generated by the epimerization of C-4 in the hemiketal compounds (1, 5−9, and 13) and apparent enantiodivergence of 2 from the known metabolite fusaraisochromanone.34,35 The isolated secondary metabolites did not show any significant activities in the available bioassays, with only the ester hybrids 7−9 exhibiting weak antibiotic, antiprotozoal, and cytotoxic activities. The bioactivities of these secondary metabolites warrant further investigation to fully understand their role in the ecology of A. banksianus. 1524



Article

721, 702, 666, 600 cm−1; NMR (600 MHz, DMSO-d6) see Table 1 and Table S3, Supporting Information; HR-ESI(+)MS m/z 239.0910 [M + H]+ (calcd for C12H15O5+, 239.0914). Banksialactone D (4): colorless oil; [α]24D −23 (c 0.51, MeOH); ECD (MeOH) λmax (Δε) 214 (−15.51), 261 (+4.91), nm; UV (MeOH) λmax (log ε) 222 (4.19), 261 (3.96), 300 (3.71) nm; IR (ATR) νmax 3453, 2981, 2940, 2843, 1720, 1594, 1498, 1466, 1432, 1356, 1321, 1246, 1210, 1178, 1142, 1069, 1050, 1001, 939, 921, 892, 811, 719, 601 cm−1; NMR (600 MHz, DMSO-d6) see Table 1 and Table S4, Supporting Information; HR-ESI(+)MS m/z 267.1223 [M + H]+ (calcd for C14H19O5+, 267.1227). Banksialactone E (5): pale yellow solid; [α]23D +25 (c 1.83, MeOH); ECD (MeOH) λmax (Δε) 226 (−3.36), 238 (+3.54), 251 (+1.19), 271 (+5.63), 312 (−2.61) nm; UV (MeOH) λmax (log ε) 213 (4.32), 270 (4.03), 313 (3.75) nm; IR (ATR) νmax 3200, 2939, 1730(w), 1657, 1617, 1442, 1361, 1291, 1249, 1151, 1023, 954, 798, 702, 598 cm−1; NMR (600 MHz, DMSO-d6) see Table 2 and Table S5, Supporting Information; HR-ESI(+)MS m/z 395.0763 [M + Na]+ (calcd for C16H20O8SNa+, 395.0771). Banksialactone F (6): pale yellow solid; [α]23D +9 (c 2.03, MeOH); ECD (MeOH) λmax (Δε) 228 (−6.7), 240 (+1.16), 249 (−0.47), 272 (+3.80), 312 (−3.58) nm; UV (MeOH) λmax (log ε) 212 (4.37), 270 (4.04), 315 (3.74) nm; IR (ATR) νmax 3132, 2938, 1720(w), 1659, 1616, 1591, 1463, 1440, 1355, 1318, 1291, 1245, 1201, 1150, 1022, 943, 877, 798, 719 cm−1; NMR (600 MHz, DMSO-d6) see Table 2 and Table S6, Supporting Information; HRESI(+)MS m/z 337.0708 [M + Na]+ (calcd for C14H18O6SNa+, 337.0716). Banksialactone G (7): off-white solid; [α]23D +17 (c 0.71, MeOH); ECD (MeOH) λmax (Δε) 210 (−9.21), 222 (−2.71), 227 (−4.99), 237 (+7.00), 257 (−1.12), 277 (+7.25), 321 (−3.99) nm; UV (MeOH) λmax (log ε) 212 (4.28), 268 (4.01), 312 (3.70) nm; IR (ATR) νmax 3382, 2359, 1668, 1617, 1435, 1314, 1292, 1244, 1202, 1139, 1059, 1023, 951, 837, 801, 722, 668, 604 cm−1; NMR (600 MHz, DMSO-d6) see Table 3 and Table S7, Supporting Information; HR-ESI(+)MS m/z 433.1492 [M + H]+ (calcd for C22H25O9+, 433.1493). Banksialactone H (8): off-white solid; [α]23D +9 (c 0.98, MeOH); ECD (MeOH) λmax (Δε) 222 (−2.26), 227 (−3.96), 237 (+5.03), 256 (−1.30), 275 (+4.19), 324 (−2.77) nm; UV (MeOH) λmax (log ε) 216 (4.41), 270 (4.07), 312 (3.81) nm; IR (ATR) νmax 3386, 3147, 2979, 2942, 1777(w), 1650, 1616, 1589, 1435, 1358, 1315, 1289, 1246, 1148, 1127, 1080, 1061, 1022, 984, 931, 871, 829, 794, 781, 762, 729, 702, 670, 634, 599 cm−1; NMR (600 MHz, DMSO-d6) see Table 3 and Table S8, Supporting Information; HR-ESI(+)MS m/z 469.1463 [M + Na]+ (calcd for C23H26NaO9+, 469.1469). Banksialactone I (9): yellow-orange solid; [α]23D +16 (c 0.86, MeOH); ECD (MeOH) λmax (Δε) 225 (−4.79), 239 (+1.02), 270 (+3.41), 315 (−2.53) nm; UV (MeOH) λmax (log ε) 216 (4.29), 266 (4.10), 288 (4.03), and 440 (3.63) nm; IR (ATR) νmax 3191, 1739(w), 1652, 1618, 1441, 1359, 1290, 1239, 1147, 1062, 1024, 995, 932, 876, 797, 761, 703, 582 cm−1; NMR (600 MHz, DMSO-d6) see Table 3 and Table S9, Supporting Information; HR-ESI(+)MS m/z 587.1157 [M + Na]+ (calcd for C29H24O12Na+, 587.1160). Banksiamarin A (10): off-white solid; UV (MeOH) λmax (log ε) 206 (3.76), 259 (4.23), 284sh (3.70), 320 (3.59), and 352 (3.62) nm; IR (ATR) νmax 3349, 1731(w), 1657, 1616, 1442, 1361, 1291, 1151, 1023, 953, 798, 702 cm−1; NMR (600 MHz, DMSO-d6) see Table 4 and Table S10, Supporting Information; HR-ESI(+)MS m/z 287.0526 [M + Na]+ (calcd for C13H12O6Na+, 278.0526). Banksiamarin B (11): off-white solid; UV (MeOH) λmax (log ε) 207 (3.57), 245 (4.19), 277sh (3.21) and 328 (3.08) nm; IR (ATR) νmax 3348, 1674, 1436, 1388, 1352, 1275, 1237, 1139, 994, 800, 721, 667, 633, 603 cm−1; NMR (600 MHz, DMSO-d6) see Table 4 and Table S11, Supporting Information; HR-ESI(−)MS m/z 293.0300 [M − H]− (calcd for C13H9O8−, 293.0303).

banksialactone D (4; 4 mg; 23.4 min). Fraction S4 contained very minor quantities of low molecular weight compounds that were not sufficient for further investigation. Bioassays. Test compounds were dissolved in DMSO to provide 10 mg/mL stock solutions (or 1 mg/mL for compounds of limited quantities). An aliquot of each stock solution was transferred to the first lane of rows B to G in a 96-well microtiter plate and 2-fold serially diluted across the 12 lanes of the plate to provide a 2048-fold concentration gradient. Bioassay medium was added to an aliquot of each test solution to provide a 100-fold dilution into the final bioassay, thus yielding a test range of 100 to 0.05 μg/mL in 1% DMSO. Row A was used as the negative control (no inhibition), and Row H was used as the positive control (uninoculated). CyTOX5 is an indicative bioassay platform for discovery of antitumor actives. NS-1 (ATCC TIB-18) mouse myeloma cells were inoculated in 96-well microtiter plates (190 μL) at 50 000 cells/mL in DMEM (Dulbecco’s modified Eagle’s medium + 10% fetal bovine serum (FBS) + 1% penicillin/streptomycin (10 mL/L, Life Technologies)) and incubated in a 37 °C (5% CO2) incubator. At 48 h, resazurin (250 μg/mL; 10 μL) was added to each well, and the plates were incubated for a further 48 h. MIC end points were determined visually. ProTOX5 is a generic bioassay platform for antibiotic discovery. Bacillus subtilis (ATCC 6633) and Escherichia coli (ATCC 25922) were used as indicative species for Gram-positive and -negative antibacterial activity, respectively. A bacterial suspension (50 mL in a 250 mL flask) was prepared in nutrient broth by cultivation for 24 h at 100−250 rpm, 28 °C. The suspension was diluted to an absorbance of 0.01 absorbance unit per mL, and 10 μL aliquots were added to the wells of a 96-well microtiter plate, which contained the test compounds dispersed in nutrient agar (Amyl) with resazurin (12.5 μg/mL). The plates were incubated at 28 °C for 48 h, during which time the negative control wells change from a blue to light pink color. MIC end points were determined visually. EuTOX5 is a generic bioassay platform for antifungal discovery. The yeasts Candida albicans (ATCC 10231) and Saccharomyces cerevisiae (ATCC 9763) were used as indicative species for antifungal activity. A yeast suspension (50 mL in a 250 mL flask) was prepared in 1% malt extract broth by cultivation for 24 h at 250 rpm, 24 °C. The suspension was diluted to an absorbance of 0.005 and 0.03 absorbance unit per mL for C. albicans and S. cerevisiae, respectively. Aliquots (20 and 30 μL) were applied to the wells of a 96-well microtiter plate, which contained the test compounds dispersed in malt extract agar containing bromocresol green (50 μg/mL). The plates were incubated at 24 °C for 48 h, during which time the negative control wells change from a blue to yellow color. MIC end points were determined visually. TriTOX5 is a bioassay focused on the discovery of inhibitors of the animal protozoan pathogen Tritrichomonas fetus (strain KV-1). T. fetus were inoculated in 96-well microtiter plates (200 μL) at 4 × 104 cells/ mL in T. fetus medium (0.2% tryptone, Oxoid; 0.1% yeast extract, Difco; 0.25% glucose; 0.1% L-cysteine; 0.1% K2HPO4; 0.1% KH2PO4; 0.1% ascorbic acid; 0.01% FeSO4·7H2O; 1% penicillin/streptomycin (10 mL/L), 10% new born calf serum, Life Technologies). The plates were incubated in anaerobic jars (Oxoid AG25) containing an Anaerogen satchel (Oxoid AN25) in a 37 °C (5% CO2) incubator. At 72 h, T. fetus proliferation was counted and % inhibition was graphed to determine the MIC values. Banksialactone B (2): colorless oil; [α]24D −17 (c 1.01, MeOH); ECD (MeOH) λmax (Δε) 214 (−10.65), 260 (+3.92) nm; UV (MeOH) λmax (log ε) 222 (4.19), 260 (3.94), 298 (3.69) nm; IR (ATR) νmax 3368, 2938, 1721, 1611, 1461, 1369, 1314, 1235, 1216, 1199, 1148, 1107, 999, 947, 924, 905, 838, 795, 720, 660, 602, 559 cm−1; NMR (600 MHz, DMSO-d6) see Table 1 and Table S2, Supporting Information; HR-ESI(+)MS m/z 253.1069 [M + H]+ (calcd for C13H17O5+, 253.1071). Banksialactone C (3): colorless oil; [α]24D −20 (c 0.56, MeOH); ECD (MeOH) λmax (Δε) 214 (−8.72), 261 (+2.07) nm; UV (MeOH) λmax (log ε) 215 (4.18), 255 (3.92), 297 (3.67) nm; IR (ATR) νmax 3214, 1713, 1609, 1332, 1246, 1146, 1053, 1023, 847, 1525



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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.7b00816. Tabulated full NMR data, NMR spectra, CD spectra, UV−visible spectra, biological activity data, and details of DFT calculations (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: +61-2-9850-8290. Fax: +61-2-9850-8313. ORCID

Peter Karuso: 0000-0002-0217-6021 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Dr. M. McKay (APAF, Macquarie University) for the acquisition of HRMS data. This research was funded, in part, by the Australian Research Council (DP130103281 to P.K. and A.M.P.; FT130100142 to A.M.P.) and Macquarie University (iMQRES scholarship to N.C.).

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REFERENCES

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