Cytochalasins from an Australian Marine Sediment-Derived

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Cytochalasins from an Australian Marine Sediment-Derived Phomopsis sp. (CMB-M0042F): Acid-Mediated Intramolecular Cycloadditions Enhance Chemical Diversity Zhuo Shang,† Ritesh Raju,‡ Angela A. Salim, Zeinab G. Khalil, and Robert J. Capon* Institute for Molecular Bioscience, The University of Queensland, St. Lucia, Queensland 4072, Australia S Supporting Information *

ABSTRACT: Chemical analysis of an Australian coastal marine sediment-derived fungus, Phomopsis sp. (CMBM0042F), yielded the known cytochalasins J (1) and H (2), together with five new analogues, cytochalasins J1−J3 (3−5) and H1 and H2 (6 and 7). Structures of 1−7 were assigned on the basis of detailed spectroscopic analysis, chemical interconversion, and biosynthetic and mechanistic considerations. Of note, 1 and 2 proved to be highly sensitive to acidmediated transformation, with 1 affording 3−5 and 2 affording 6 and 7. Whereas 1, 2, 4, and 5 were detected as natural products in crude culture extracts, 3, 6, and 7 were designated as acid-mediated handling artifacts. We propose novel stereoand regiospecific intramolecular cycloadditions, under tight functional group control, that facilitate selective conversion of 1 and 2 to the rare 5/6/6/7/5- and 5/6/5/8-fused heterocycles 5 and 7, respectively. Knowledge of acid sensitivity within the cytochalasin family provides a valuable cautionary lesson that has the potential to inform our analysis of past and future investigations into this structure class and inspire novel biomimetic transformations leading to new chemical diversity.

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INTRODUCTION Cytochalasins are a diverse group of polyketide synthasenonribosomal peptide synthetase (PKS-NRPS)-derived fungal metabolites characterized by a perhydroisoindolone moiety, typically fused to a macrocyclic ring (i.e., a carbocycle, lactone, or cyclic carbonate). With over 100 examples reported from many genera, including Phomopsis, Hyposylon, Xylaria, Phoma, Penicillium, Aspergillus, Chaetomium, and Zygosporium, selected cytochalasins have been attributed to a range of biological properties.1 As part of our ongoing studies into the chemistry of Australian marine-derived fungi, we examined a Phomopsis sp. (CMB-M0042F) isolated from marine sediment collected near Shorncliffe, Queensland, Australia. These investigations led to discovery of the known cytochalasins J (1) and H (2), together with five new congeners, cytochalasins J1−J3 (3−5) and H1 and H2 (6 and 7). This report describes the isolation and structure elucidation of 1−7 (Figure 1), as well as an account of their chemical and biological properties. Particular attention is paid to acid-mediated stereo- and regiospecific intramolecular cycloadditions that yield novel fused carbocyclic and heterocyclic cytochalasins.

Australia. Cultivation trials carried out on one such marinederived fungus, Phomopsis sp. (CMB-M0042F) (Figure S1), established its potential for producing secondary metabolites. Cultivation on nonsaline and saline PYG agar, as well as saline rice solid media and saline M1 agar, revealed significant and overlapping levels of production of an array of related secondary metabolites (Figure S2), encouraging more detailed examination. An EtOAc extract of a 25 days scaled up saline rice solid media cultivation of CMB-M0042F was concentrated in vacuo to yield a crude extract that was subjected to sequential solvent trituration to deliver hexane, CH2Cl2, and MeOH soluble materials. Following HPLC-DAD profiling, the CH2Cl2 soluble material was prioritized for fractionation by reversed-phase SPE and HPLC, affording 1−5. In a parallel study, an EtOAc extract of a 25 days scaled up saline M1 agar cultivation was concentrated in vacuo and subjected to sequential solvent trituration and reversed-phase HPLC fractionation to afford 2, 6, and 7. The structure elucidation of 1−7 was achieved by detailed spectroscopic analysis, chemical interconversion, and biosynthetic considerations, as outlined below. Spectroscopic analysis readily identified the major metabolites as cytochalasin J (1) (Table S1) and its acetate, cytochalasin H (2) (Table S2). Cytochalasin H (2) was first

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RESULTS AND DISCUSSION During investigations into the chemistry of Australian microbes, we assembled a library of bacterial and fungal isolates from coastal sediment and sand samples, collected near Brisbane, © 2017 American Chemical Society

Received: July 19, 2017 Published: August 23, 2017 9704

DOI: 10.1021/acs.joc.7b01793 J. Org. Chem. 2017, 82, 9704−9709

Article

The Journal of Organic Chemistry

whereas absolute configurations were assigned by analysis of ROESY data and chemical interconversion with 1 (see below). HRESI(+)MS analysis of 5 afforded a sodium adduct ion attributed to a molecular formula (C28H35NO3, Δmmu −0.5) isomeric with 4. Comparison of NMR (CDCl3) data for 5 (Table S5) with those for 4 revealed the absence of resonances for Δ13 and Δ19 moieties, and the appearance of resonances for three methines (H-14, δH 3.69, C-14, δC 88.2; H-13, δH 1.83, C13, δC 45.2; H-19, δH 2.34, C-19, δC 35.5) and one methylene (H2-20, δH 2.63, 1.95; C-20, δC 34.7). Detailed analysis of 2D NMR data (Figure 3) permitted assignment of a planar

Figure 1. Structures of 1−7.

Figure 3. Diagnostic NMR (CDCl3) correlations for 5.

reported in 1976 from a Phomopsis sp. isolated from weevil damaged pecan kernels,2 and its structure was determined by Xray analysis3 and total synthesis.4 Cytochalasin J (1) was first reported in 19815 as deacetylcytochalasin H from the same Phomopsis sp. Despite the scientific literature being rich with structurally diverse cytochalasins, our preliminary analyses suggested the co-metabolites 3−7 were new cytochalasins. HRESI(+)MS analysis of 3 and 4 afforded sodium adduct ions attributed to molecular formulas C29H39NO4 (Δmmu +0.1) and C28H35NO3 (Δmmu −0.5), consistent with Omethylated and dehydrated analogues of 1. Comparison of the NMR (CDCl3) data for 3 (Table S3) with those for 1 revealed differences attributed to the addition of a methoxy moiety (OCH 3 , δ H 3.22; OCH 3 ,δ C 50.8), with the 18-OMe regiochemistry evident from HMBC correlations (Figure 2).

structure strongly suggestive of an intramolecular cycloaddition product of 4, incorporating a fused heterocycle from 7-OH to C-14 and carbocycle from C-13 to C-19. Diagnostic 2D NMR ROESY correlations (Figure 3) positioned H-3, H3-11, H-7, H13, and H-21 on the α-face and H-4, H-5, H-8, H-14, H-16, and H-19 on the β-face of 5, whereas the absolute configuration was assigned by chemical interconversion with 1 (see below). Although 4 and 5 were readily detected as minor metabolites in saline rice cultivations (Figure S2), the absence of 3 in any cultivation extracts suggested the possibility of handling artifacts. To test this hypothesis, an aliquot of 1 (20 μg) was exposed to 1% TFA/MeOH (50 μL) at room temperature for 20 h, with HPLC-DAD analysis detecting quantitative conversion to 3−5 (Figure 4 inset). Similar treatment of 1 with 50% TFA/MeOH or 1% TFA/H2 O resulted in quantitative conversion to 4 and 5, whereas exposure to 10% TFA/H2O yielded exclusively 5 (Figure S3). Interestingly, 1 was stable to prolonged storage in MeOH. Collectively, these observations prompted a mechanistic explanation (Figure 4) in which mild acid (i.e., 1% TFA) causes the C-18 3°-OH in 1 to undergo dehydration to an intermediate C-18 3°-carbocation, which is in turn quenched by SN1 addition of either H2O or MeOH to yield 1 or 3, respectively. As the SN1 addition of H2O to 1 demonstrates reversibility, the failure to detect 18-epi-1 confirms that quenching of the 3°-carbocation is stereospecific (i.e., β-facial). In a parallel mechanism, we propose exposure to acid causes the C-18 3°-OH in 1 to undergo a nonreversible E2 elimination to yield 4. Significantly, Δ13 and Δ19 moieties in 4 are ideally positioned to undergo an acid-mediated intramolecular cycloaddition with concomitant quenching by 7-OH to yield 5 (Figure 4). This cycloaddition reaction is particularly intriguing, as it provides a rapid and efficient route to a rare, stereorich, cytochalasin scaffold. It also confirms structure assignments to 3−5, including absolute configurations. HRESI(+)MS analysis of 6 afforded a sodium adduct ion attributed to a molecular formula (C31H41NO5, Δmmu −0.5) consistent with an O-methylated analogue of 2. As with 1 and 3, comparison of the NMR (CDCl3) data for 6 (Table S6) with those for 2 revealed a new methoxy moiety (OCH3, δH 3.15; OCH3, δC 50.9), with the C-18 regiochemistry evident from

Figure 2. Diagnostic 2D NMR (CDCl3) correlations for 3 and 4.

Comparison of the NMR (CDCl3) data for 4 (Table S4) with those for 1 revealed addition of a Δ17 moiety, evidenced by resonances for an sp2 methine (H-17, δH 5.26, C-17, δC 136.1), an sp2 quaternary carbon (C-18, δC 132.6), and an olefinic methyl (H3-23, δH 1.83, C-23, δC 21.1), with regiochemistry confirmed by COSY correlations between H-17 and H-16 and HMBC correlations from H3-23 to C-17, C-18, and C-19 (Figure 2). A Z Δ17 configuration was evident from ROESY correlations between H-17 and H3-23, whereas 1H NMR couplings confirmed E configurations about Δ13 (J 15.8 Hz) and Δ19 (J 16.6 Hz). Planar structures for 3 and 4 were also validated by an array of 2D NMR correlations (Figure 2), 9705

DOI: 10.1021/acs.joc.7b01793 J. Org. Chem. 2017, 82, 9704−9709

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The Journal of Organic Chemistry

Figure 4. Proposed mechanism for acid-mediated conversion of 1 to 3−5. Inset: HPLC-DAD (210 nm) chromatograms of (i) cytochalasin J (1), (ii) 1 in 1% TFA in MeOH for 20 h, and (iii) 1 in 1% TFA in H2O for 20 h.

attributed to a Δ18 bearing a olefinic methyl (C-18, δC 133.7; H-19, δH 5.64, C-19, δC 127.4; H3-23, δH 1.75, C-23, δC 27.0), as well as a methoxy (OCH3, δH 3.34, OCH3, δC 54.5) and two methines (H-13, δH 3.00, C-13, δC 50.7; H-20, δH 2.65, C-20, δC 52.2). Detailed analysis of the 2D NMR data (Figure 5) permitted assembly of a planar structure strongly suggestive of an intramolecular cycloaddition product of 2, incorporating a fused carbocycle from C-13 to C-20. Diagnostic 2D NMR ROESY correlations (Figure 5) positioned H-3, H3-11, H-7, H13, and H-21 on the α-face and H-4, H-5, H-8, H-14, H-16, and H-19 on the β-face of 7, whereas the absolute configuration was assigned by chemical interconversion with 2 (see below). A failure to detect either 6 or 7 in any cultivation extracts suggested these may be handling artifacts (as was observed for 3). In support of this hypothesis, aliquots of 2 exposed to 1% TFA/MeOH were observed to undergo partial (∼20%) conversion to 6 (Figure S4), whereas samples exposed to 10 and 50% TFA/MeOH experienced either ∼50% conversion to 6 with traces of 7 or increased conversion to 7, respectively (Figure 6 inset). A possible mechanistic explanation parallels that noted above for 1 (Figure 4) and involves 2 undergoing elimination of H2O to generate an intermediate C-18 3°carbocation, which can be quenched by stereospecific (i.e., βfacial) addition of either H2O or MeOH to yield 2 or 6, respectively (Figure 6). In a parallel process, an alternate resonance structure with a C-20 2°-carbocation can undergo concomitant intramolecular cyclization and quenching by MeOH to afford 7 (Figure 6). These transformations confirm

HMBC correlations (Figure 5). Additional 2D NMR correlations permitted assembly of the planar structure (Figure

Figure 5. Diagnostic NMR (CDCl3) correlations for 6 and 7.

5). Excellent comparisons between the NMR data for 6 and 2 (Tables S2 and S6), together with biosynthetic considerations, suggested a common absolute configuration, subsequently confirmed by the acid-mediated conversion of 2 into 6 (see below). HRESI(+)MS analysis of 7 afforded a sodium adduct ion attributed to a molecular formula (C31H41NO5, Δmmu −0.5) isomeric with 6. Comparison of NMR (CDCl3) data for 7 (Table S7) with those for 6 revealed the absence of resonances for Δ13 and Δ19 moieties and the appearance of resonances

Figure 6. Proposed mechanism for acid-mediated conversion of 2 to 6 and 7. Inset: HPLC-DAD (210 nm) chromatograms of (i) cytochalasin H (2), (ii) 2 in 10% TFA in MeOH for 20 h, and (iii) 2 in 50% TFA in MeOH for 20 h. 9706

DOI: 10.1021/acs.joc.7b01793 J. Org. Chem. 2017, 82, 9704−9709

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The Journal of Organic Chemistry Table 1. Cytotoxicity Data (IC50, μM) for 1−7

structure assignments for 6 and 7, including absolute configurations. The observations outlined above (Figures 4 and 6) reveal a remarkable level of stereo- and regiochemical control over acidmediated intramolecular cycloaddition within the cytochalasin J/H framework. For example, under TFA/MeOH conditions the 21-OH-substituted cytochalasin J (1) undergoes transformation to the 5/6/6/5/7-fused 5, whereas the 21-OAcsubstituted cytochalasin H (2) yields the 5/6/5/8-fused 7. We reason that under these conditions, differing functionality at C21 perturbs the conformation and subsequent reactivity of the 11-membered carbocycle common to 1 and 2, as illustrated in Figures 4 and 6. Interestingly, the fused scaffolds present in 5 and 7 are exceptionally rare, with the only known examples being phomopchalasins A (8) and B (9) (Figure 7), first reported

cell linea

1

2

3

4

5

6

7

SW620 NCI-H460 HepG2 HEK

2.3 3.2 0.4 0.5

0.1 0.4 0.2 0.02

1.7 2.9 0.3 0.1

12.8 >30 0.2 0.9

3.7 12.9 0.3 0.1

1.2 1.5 0.3 0.03

6.1 4.5 0.3 0.2

a

SW620, colon adenocarcinoma; NCI-H460, lung carcinoma; HepG2, hepatocellular carcinoma; HEK, human embryonic kidney.

these transformations. These mechanisms, which reveal the stereo- and regiospecific intramolecular cycloaddition of 1 to 5/ 6/6/5/7-fused 5 and 2 to 5/6/5/8-fused 7, call into question the natural product status of the recently reported Phomopsis metabolites 8 and 9. Knowledge of the acid sensitivity of 1 and 2 provides a cautionary lesson that will inform future investigations into cytochalasin natural products. Likewise, knowledge of acid-mediated intramolecular cycloadditions within the cytochalasin family has the potential to inspire novel biomimetic syntheses, enabling access to new cytochalasins.

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EXPERIMENTAL SECTION

General Experimental Procedures. Specific optical rotations ([α]D) were measured on a polarimeter in a 100 × 2 mm cell at 22 °C. UV−visible spectra were obtained on a UV−visible spectrophotometer with 1 cm quartz cells. Circular dichroism (CD) spectra were recorded on a spectropolarimeter in 1 mm quartz cells at room temperature with scanning speed of 50 nm/min and 0.5 nm step scan over the wavelength from 200 to 600 nm. Nuclear magnetic resonance (NMR) spectra were acquired on a 600 MHz spectrometer with either a 5 mm PASEL 1 H/D- 1 3 C Z-gradient probe or 5 mm CPTCI 1 H/19F-13C/15N/DZ-gradient cryoprobe. In all cases, spectra were acquired at 25 °C (unless otherwise specified) in solvents as specified in the text, with referencing to residual 1H or 13C signals in the deuterated solvents. Electrospray ionization mass spectrometry (ESIMS) experiments were carried out on an LC/MSD (quadrupole) instrument in both positive and negative modes. HPLC-QTOF data were acquired on Q-TOF LC/MS system. High-resolution ESIMS spectra were obtained on TOF mass spectrometer by direct injection in MeCN at 3 μL/min using sodium formate clusters as an internal calibrant. Fungal Isolation and DNA Taxonomy. The fungus Phomopsis sp. (CMB-M0042F) was isolated in 2008 from a marine sediment sample collected at Shorncliffe, Queensland. The fresh sediment sample (∼5 g) was air-dried in a laminar flow hood for 24 h, after which serial dilutions were applied to M1 agar plates (1% soluble starch, 0.4% yeast extract, 0.2% peptone, 1.5% agar, and 0.0005% rifampicin), and the sealed plates were incubated at 26.5 °C for 4 weeks. A pure culture of fungus CMB-M0042F obtained by singlecolony serial transfer on M1 agar plates was cryopreserved at −80 °C in 15% aqueous glycerol. The fungus grew slowly and formed light brown colonies after 10 days of cultivation on M1 agar plate and rice solid media. A BLAST search of the amplified ITS sequence (see Supporting Information) (GenBank accession no. KU641387) revealed 99% homology with other members of the genus Phomopsis sp. Analytical Cultivation and Chemical Profiling. Analytical cultivations of Phomopsis sp. (CMB-M0042F) were generated on distilled water-based PYG agar (2% glucose, 1% peptone, 0.5% yeast extract, and 1.5% agar), saline PYG agar (2% glucose, 1% peptone, 0.5% yeast extract, 1.5% agar, and 3.5% sea salt), saline M1 agar (1% soluble starch, 0.4% yeast extract, 0.2% peptone, 1.5% agar, and 3.5% sea salt), and saline rice solid media (70 g rice, 0.3% peptone, 0.3% yeast extract, 0.1% monosodium glutamate, and 3.5% sea salt prepared in 100 mL of distilled water). All agar plates were incubated at 26.5 °C for 25 days, after which they were extracted with EtOAc, and the

Figure 7. Structures of 8 and 9.

in 2016 as co-metabolites with cytochalasin J (1) from an endophytic fungus, Phomopsis sp. shj2.6 Of note, the reported purification of 8 and 9 included silica column chromatography of the crude extract with CHCl3/acetone, followed by “repeated chromatography over silica gel”. As both silica chromatography and CHCl3 are potentially acidic, it is possible that 8 and 9 are acid-mediated intramolecular cycloaddition artifacts of 1. Evidence of a possible mechanism for transforming 1 to 9 can be found in a recent report on the polyene polyketide natural product heronamides; heronamide C undergoes selective air oxidation to a monoepoxide that in turn activates intramolecular cycloadditions to yield heronamides A and B.7 Although we cannot replicate the exact isolation conditions reported for 8 and 9, in our hands, exposure of 1 to CHCl3/ acetone, without and with addition of a trace of HCl, resulted in ∼50% and quantitative conversion to a range of products, respectively (Figure S5). Significantly, the dominant product encountered under both conditions possessed a molecular formula in common with 9 (Figure S6). Whether 8 and/or 9 are natural products or artifacts, or indeed both, remains unresolved at this time, but the level of uncertainty highlights the need for caution when dealing with acid-labile natural products. Given the mycotoxin reputation of cytochalasins, the cytotoxicity of 1−7 was quantified against a panel of human cell lines. These results confirmed 1 and 2 as potent cytotoxins, with 3−7 exhibiting very significant, albeit variable and lower, levels of cytotoxicity (Table 1).

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CONCLUSION In summary, this report provides an account of the isolation and structure elucidation of two known (1 and 2) and five new (3−7) cytochalasins from an Australian marine sedimentderived fungus, Phomopsis sp. (CMB-M0042F). It also documents the acid-mediated transformation of 1 to 3−5 and 2 to 6 and 7 and provides plausible mechanisms to account for 9707

DOI: 10.1021/acs.joc.7b01793 J. Org. Chem. 2017, 82, 9704−9709

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ESI(−)MS m/z 478 [M + FA − H]−; HRMS (ESI-TOF) m/z [M + Na]+ calcd for C28H35NO3Na 456.2509; found 456.2514. Cytochalasin H1 (6): White powder; [α]D22 +71.2 (c 0.17, MeOH); NMR (600 MHz, CDCl3) see Table S6; ESI(+)MS m/z 530 [M + Na]+, ESI(−)MS m/z 506 [M − H]−; HRMS (ESI-TOF) m/z [M + Na]+ calcd for C31H41NO5Na 530.2877; found 530.2882. Cytochalasin H2 (7): White powder; [α]D22 +9.1 (c 0.14, MeOH); CD (MeOH) λmax (Δε) 205 (−12.3) nm; NMR (600 MHz, CDCl3) see Table S7; ESI(+)MS m/z 530 [M + Na]+, 490 [M − H2O + H]+; HRMS (ESI-TOF) m/z [M + Na]+ calcd for C31H41NO5Na 530.2877; found 530.2882. Acid-Mediated Transformation of 1 and 2. Samples of 1 (20 μg) and 2 (20 μg) were treated for 20 h with (i) 1, 10, and 50% TFA in MeOH (50 μL) at rt or (ii) 1, 10, and 50% TFA in H2O (50 μL) at rt, after which the reaction mixtures were concentrated to dryness under dry N2 and redissolved in MeOH (20 μL) prior to HPLC-DADESI(±)MS analysis (Agilent Zorbax SB-C8 column, 150 × 4.6 mm column, 5 μm, 1 mL/min gradient elution from 90% H2O/MeCN to 100% MeCN over 15 min, with constant 0.05% formic acid modifier). Authentic standards of 1−7 were analyzed by the same HPLC method. Results of analyses are shown in Figures S3 and S4. Sample of 1 (20 μg) was treated for 15 h with (i) 1:1 CHCl3/ acetone (100 μL) at rt or (ii) 1:1 CHCl3/acetone (100 μL) with traces of HCl at rt, after which the reaction mixtures were concentrated to dryness under dry N2 and redissolved in MeCN (20 μL) prior to HPLC-DAD-ESI(±)MS analysis (Agilent Zorbax SB-C8 column, 150 × 4.6 mm column, 5 μm, 1 mL/min gradient elution from 90% H2O/ MeCN to 100% MeCN over 15 min, with constant 0.05% formic acid modifier). The result is shown in Figure S5. The reaction mixtures were also analyzed with HPLC-QTOF to generate an accurate molecular formula for the peak with 450 mass unit (see Figure S6). Cytotoxicity Assay of 1−7. The MTT assay was performed using adherent cell lines SW620, NCIH-460, HepG2, and HEK293. SW620 and NCIH-460 were cultured in RPMI medium 1640, and HepG2 and HEK293 were cultured in DMEM as adherent monolayer in flasks supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 100 units/mL penicillin, and 100 μg/mL streptomycin in a humidified 37 °C incubator supplied with 5% CO2. Briefly, cells were harvested with trypsin and dispensed into 96-well microtiter assay plates at 2000 cells/well for all cell lines and then incubated for 18 h at 37 °C with 5% CO2 (to allow cells to attach). Testing compounds were dissolved in 5% DMSO in PBS (v/v) and aliquots (10 μL) tested over a series of final concentrations ranging from 10 nM to 30 μM. Control wells were treated with 5% aqueous DMSO. After 68 h incubation at 37 °C with 5% CO2, an aliquot (10 μL) of MTT in PBS (5 mg/mL) was added to each well (final concentration of 0.5 mg/mL), and the microtiter plates were incubated for a further 4 h at 37 °C with 5% CO2. After this final incubation, the medium was aspirated and precipitated formazan crystals dissolved in DMSO (100 μL/well). The absorbance of each well was measured at 580 nm with a PowerWave XS microplate reader from Bio-Tek Instruments Inc. (Vinooski, VT). IC50 values were calculated using Prism 5.0 (GraphPad Software Inc., La Jolla, CA), as the concentration of analyte required for 50% inhibition of cancer cell growth (compared to negative controls). Vinblastine was used as a positive control (20 μg/mL in H2O). All experiments were performed in duplicate.

organic phase was dried in vacuo to yield crude extracts. Analytes of crude extracts (5 mg/mL in MeOH) were subjected to HPLC-DADESI(±)MS analysis (Agilent Zorbax SB-C8 column, 150 × 4.6 mm, 5 μm, 1 mL/min gradient elution from 90% H2O/MeCN to 100% MeCN over 15 min, with constant 0.05% formic acid modifier). Based on biomass production and chemical diversity, saline rice solid media and saline M1 agar were chosen for scale-up fermentation of Phomopsis sp. (CMB-M0042F). Production, Isolation, and Structure Characterization of 1− 7. A 1 L Erlenmeyer flask containing sterile rice solid medium inoculated with the fungal strain Phomopsis sp. (CMB-M0042F) was incubated at 26.5 °C for 25 days. The fungal mycelia as well as the rice media were exhaustively extracted with EtOAc and concentrated in vacuo to afford a combined EtOAc extract (1073.0 mg), which was sequentially triturated to yield hexane (260.0 mg), CH2Cl2 (573.3 mg), and MeOH (175.5 mg) solubles. Following HPLC-DAD chemical profiling, the CH2Cl2 solubles were subjected to C18 SPE fractionation (10% stepwise gradient elution from 80 to 20% H2O/ MeOH) to yield seven fractions. The 50% H2O/MeOH fraction (33.1 mg) was subjected to preparative HPLC (Phenomenex Luna C8 column, 250 × 21.2 mm, 10 μm, 20 mL/min gradient elution from 70 to 40% H2O/MeCN over 20 min with a constant 0.01% TFA modifier) to yield cytochalasin H (2) (tR = 15.1 min; 10.6 mg, 1.0%). The combined remaining eluant was freeze-dried (9.1 mg) and further fractionated by HPLC (Agilent Zorbax SB-C3 column, 250 × 9.4 mm, 5 μm, 3 mL/min gradient elution from 65 to 45% H2O/MeCN over 20 min) to afford cytochalasin J (1) (tR = 8.8 min; 6.7 mg, 0.6%). The 60% H2O/MeOH fraction (47.0 mg) was subjected to preparative HPLC (Phenomenex Luna C8 column, 250 × 21.2 mm, 10 μm, 20 mL/min gradient elution from 75 to 35% H2O/MeCN over 20 min with a constant 0.01% TFA modifier) to yield cytochalasin J1 (3) (tR = 12.0 min; 1.1 mg, 0.1%), cytochalasin J2 (4) (tR = 15.2 min; 1.4 mg, 0.1%), and cytochalasin J3 (5) (tR = 16.4 min; 1.1 mg, 0.1%). A set of saline M1 agar plates (x15) inoculated with the fungal strain Phomopsis sp. (CMB-M0042F) were incubated at 26.5 °C for 25 days. After cultivation, the recovered agar was extracted with EtOAc (500 mL) for 48 h, and the organic phase was concentrated in vacuo to yield a crude extract (85.7 mg), which was sequentially triturated to yield hexane (25.9 mg), CH2Cl2 (30.3 mg), and MeOH (12.8 mg) solubles. The CH2Cl2 fraction (30.3 mg) was subsequently subjected to C18 SPE fractionation (MeOH/H2O) and semipreparative HPLC (Zorbax C8 column, 250 × 9.4 mm, 5 μm, 4 mL/min gradient elution 90% H2O/MeCN to 100% MeCN over 30 min) to yield cytochalasin H (2) (tR = 23.6 min, 2.5 mg, 2.9%), cytochalasin H1 (6) (tR = 26.0 min, 2.2 mg, 2.6%), and cytochalasin H2 (7) (tR = 26.3 min, 2.7 mg, 3.2%). Note: percent yields for 1−7 are calculated as weight-to-weight estimate against the crude extract. Cytochalasin J (1): White powder; [α]D22 +23.6 (c 0.06, MeOH); NMR (600 MHz, CDCl3) see Table S1; ESI(+)MS m/z 452 [M + H]+, 434 [M − H2O + H]+, ESI(−)MS m/z 496 [M + FA − H]−; HRMS (ESI-TOF) m/z [M + Na]+ calcd for C28H37NO4Na 474.2615; found 474.2620. Cytochalasin H (2): White powder; [α]D22 +58.6 (c 0.05, MeOH); NMR (600 MHz, CDCl3) see Table S2; ESI(+)MS m/z 476 [M − H2O + H]+, 516 [M + Na]+, ESI(−)MS m/z 492 [M − H]−; HRMS (ESI-TOF) m/z [M + Na]+ calcd for C30H39NO5Na 516.2720; found 516.2725. Cytochalasin J1 (3): White powder; [α]D22 +38.9 (c 0.07, MeOH); NMR (600 MHz, CDCl3) see Table S3; ESI(+)MS m/z 466 [M + H]+, 488 [M + Na]+, ESI(−)MS m/z 510 [M + FA − H]−; HRMS (ESI-TOF) m/z [M + Na]+ calcd for C29H39NO4Na 488.2771; found 488.2770. Cytochalasin J2 (4): White powder; [α]D22 +18.5 (c 0.04, MeOH); NMR (600 MHz, CDCl3) see Table S4; ESI(+)MS m/z 434 [M + H]+, 456 [M + Na]+, 416 [M − H2O + H]+, ESI(−)MS m/z 478 [M + FA − H] −; HRMS (ESI-TOF) m/z [M + Na]+ calcd for C28H35NO3Na 456.2509; found 456.2514. Cytochalasin J3 (5): White powder; [α]D22 −19.4 (c 0.05, MeOH); CD (MeOH) λmax (Δε) 206 (−15.8) nm; NMR (600 MHz, CDCl3) see Table S5; ESI(+)MS m/z 434 [M + H]+, 416 [M − H2O + H]+,

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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.joc.7b01793. Fungal taxonomy, HPLC chromatograms and acidmediated conversion studies, tabulated 1D and 2D NMR data and spectra for 1−7 (PDF) 9708

DOI: 10.1021/acs.joc.7b01793 J. Org. Chem. 2017, 82, 9704−9709

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: +61-7-3346-2979. Fax: +617-3346-2090. ORCID

Robert J. Capon: 0000-0002-8341-7754 Present Addresses

† Centre for Marine Biotechnology and Biomedicine, Scripps Institution of Oceanography, University of California, San Diego, La Jolla, California 92093, United States. ‡ School of Medicine, Western Sydney University, Locked Bag 1797, Penrith, NSW 2751, Australia.

Author Contributions

R.R. isolated CMB-M0042F and performed preliminary studies on 2 and 6−7. Z.S. completed cultivation trials, isolated and identified 1−7, confirmed acid-mediated relationships, and assembled the SI. A.A.S. investigated the transformation of 1 to 9 and reviewed all spectroscopic data. Z.G.K. completed all bioassays. R.J.C. supervised all the above, proposed mechanisms, and wrote the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS R.R. thanks A.M. Piggott for NMR advice. Z.S. acknowledges the provision of a University of Queensland, International Postgraduate Research Scholarship (IPRS), and a Centennial Scholarship. This research was funded in part by the Institute for Molecular Bioscience, the University of Queensland and the Australian Research Council (DP120100183).

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REFERENCES

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DOI: 10.1021/acs.joc.7b01793 J. Org. Chem. 2017, 82, 9704−9709