Versixanthones A–F, Cytotoxic Xanthone–Chromanone Dimers from

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Versixanthones A−F, Cytotoxic Xanthone−Chromanone Dimers from the Marine-Derived Fungus Aspergillus versicolor HDN1009 Guangwei Wu,†,§ Guihong Yu,†,§ Tibor Kurtán,‡ Attila Mándi,‡ Jixing Peng,† Xiaomei Mo,† Ming Liu,† Hui Li,† Xinhua Sun,† Jing Li,† Tianjiao Zhu,† Qianqun Gu,† and Dehai Li*,† †

Key Laboratory of Marine Drugs, Chinese Ministry of Education, School of Medicine and Pharmacy, Ocean University of China, 5 Yushan Road, Qingdao, Shandong 266003, People’s Republic of China ‡ Department of Organic Chemistry, University of Debrecen, POB 20, 4010 Debrecen, Hungary S Supporting Information *

ABSTRACT: Six unusual xanthone−chromanone dimers, versixanthones A−F (1−6), featuring different formal linkages of tetrahydroxanthone and 2,2-disubstituted chroman-4-one monomers, were isolated from a culture of the mangrove-derived fungus Aspergillus versicolor HDN1009. The absolute configurations of 1−6, representing the central and axial chirality elements or preferred helicities, were established by a combination of X-ray diffraction analysis, chemical conversions, and TDDFT-ECD calculations. The interconversion of different biaryl linkages between 1 and 4 and between 2 and 3 in DMSO by a retro-oxa-Michael mechanism provided insight into the formation of the xanthone−chromanone dimers and supported the assignments of their absolute configurations. Compounds 1− 6 exhibited cytotoxicities against the seven tested cancer cell lines, with the best IC50 value of 0.7 μM. Compound 5 showed further inhibitory activity against topoisomerase I.

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During our search for antitumor leads from the secondary metabolites of fungi isolated from various habitats,8−10 a mangrove-derived Aspergillus versicolor strain, HDN1009, was selected due to significant cytotoxicity (inhibitory rate = 73% at 100 μg/mL concentration against P388 cells) and the characteristic HPLC-UV profile of the EtOAc extract. Guided by the UV characteristics, six rare heterogeneous xanthone− chromanone dimers, versixanthones A−F (1−6), along with a well-known 2,2′-linked symmetrical tetrahydroxanthone dimer, secalonic acid D (SAD, 7), were isolated from a scale-up fermentation. Distinguished from the reported heterodimers, versixanthones A−F have diverse dimeric patterns including the common 2−2′ linkage (4 and 6) and the less common 4−4′ (2), 2−4′ (3), and 4−2′ (1 and 5) linkages. The absolute configurations including both axial and central chirality elements were achieved by the combination of X-ray crystal diffraction analysis, chemical conversions, and TDDFT-ECD calculations. The new compounds 1−6 exhibited cytotoxicities against all seven tested cancer cell lines with low-micromolar IC50 values.

he xanthone monomer and its related chromanone (2,2disubstituted chroman-4-one) monomer are structurally complex polyketides widely distributed in plants, fungi, and lichens.1,2 Dimerized via a biaryl single bond (Figure S1), the heterodimers (xanthone−chromanone) that comprise a tetrahydroxanthone monomer and a chromanone lactone (biogenetically related to the xanthone monomer) are rare, with only six related cases reported including (±)-blennolide G,3,4 gonytolides D and E,5 and blennolids I and J6 (note: the names of blennolids I and J were also used in ref 4, but represented different structures). Due to the multiple chirality centers, different monomeric units, and sometimes additional axial chirality elements, the determination of the relative and absolute configurations of the heterodimeric dimers are a challenge. Although the absolute configurations for the foregoing heterodimers have been established indirectly by ECD calculations of the corresponding monomers combined with biogenetic considerations, the current approaches are ambiguous and lack efficiency, especially when (1) the ECD curves are dominated by the exciton coupled interaction of the two aryl moieties in axially chiral biaryl dimers, (2) they lack dominant conformations to calculate the time-dependent density functional theory (TDDFT)-ECD curves, especially for dimers with flexible moieties,7 and (3) the tetrahydroxanthone or chromanone monomers and their corresponding dimers were often not co-isolated. © XXXX American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION The organic extract was fractionated by repeated silica gel chromatography, Sephadex LH-20 column chromatography, Received: July 21, 2015

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DOI: 10.1021/acs.jnatprod.5b00636 J. Nat. Prod. XXXX, XXX, XXX−XXX

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linkage patterns. For 2, the 4−4′ linkage of two monomers was established by HMBC correlations of H-3 to C-4′ and of H-3′ to C-4 (Figure S3). The two monomers in 3 were connected via a 2−4′ linkage evidenced by HMBC correlations from H-3 to C-4′ and from H-3′ to C-2, together with the correlations between 1-OH and C-2 (Figure S3). Although 4, with a 2−2′ linkage, was proved to possess the same planar structure as blennolides G, the distinct chemical shifts for the stereogenic centers (δC/H, compound 4 vs blennolide G, CH-5′: 82.6/4.81 vs 87.6/4.45; CH-6′: 33.5/2.99 vs 30.0/2.85; CH2-7′: 36.7/ 2.70 vs 36.1/2.91, 2.23) demonstrated that they are diastereoisomers.3,6 The relative configurations in the two monomeric units of 2−4 were proposed to be the same as 1, as indicated by the similar NMR values (Figure 2, Tables 1 and 2) and by consideration of the biogenetic origin. Compounds 5 and 6 share the same molecular formula of C33H34O15. Comparison of the 1H NMR resonances for 5 with those of 1, particularly the signals at H-2/H-3 and H-3′/H-4′, indicated that 5 has the same 4−2′ linkage, supported by the HMBC correlations of H-3 with C-2′, H-3′ with C-4, and 1′OH with C-2′ (Figure S3). The major difference between them was that the γ-butyrolactone moiety in 1 was changed to a linear side chain in 5, confirmed by the COSY and HMBC correlations (Figure 1). Compound 6 was also composed by the same two monomers as 5, but had a 2−2′ linkage, evidenced by the HMBC correlations and the NMR values for the two linked benzene rings, which were similar to those in compound 4 (Figure S3, Tables 1 and 2). Compounds 5 and 6 were likely the methanolysis products of 1 and 4, respectively. The relative configurations of tetrahydroxanthone monomeric units in 5 and 6 were proposed to be the same as those in 1−4 on the basis of NMR data, especially of the NOESY experiment and coupling constants (Figure 2, Tables 1 and 2), as well as biogenetic grounds. The small coupling constants between H-5′ and H-6′ (1.8 and 1.6 Hz, respectively) in 5 and 6 revealed their gauche relationships,13 consistent with those in 1−4. A considerable number of xanthone dimers are axially chiral biaryl natural products, and the axial chirality or preferred biaryl torsional angle (M- or P-helicity) of biaryl natural products plays a crucial role in their pharmacological activities.14 The hindered rotation about the biaryl linkage and thus the possibility of axial chirality depend on the type of linkage and substitution pattern, and it can be tested by the computation of torsional energy scans and energy of the transition states.15,16 The TDDFT-ECD calculations have been proven to be a powerful tool to study the axial chirality of biaryl natural products.14,17,18 However, when a biaryl natural product contains both axial and central chirality elements, the TDDFT-ECD calculations can afford only the assignment of the axial chirality, as the ECD spectrum is dominated by the exciton coupled interaction of the two aryl moieties, which is governed by the sign and value of the biaryl torsional angle.16 The absolute configurations of 1−6, implying the central and axial chirality elements or preferred helicities, were established by a combination of X-ray diffraction analysis, chemical conversions, and TDDFT-ECD calculations. Crystallization was attempted for 1−6 in various solvent systems, but only single crystals were obtained for compound 1 in a mixture of MeOH and H2O. The absolute configuration of 1 was unambiguous determined as (5R,6S,10aR,5′R,6′S,10a′S) by Cu Kα irradiation [Flack parameter = 0.02(5)] (Figure 3). Although only a single solid-state conformer with M-helicity (denoting a negative ωC‑4a,C‑4,C‑2′,C‑1′ torsional angle) could be

reversed-phase HPLC, and semipreparative HPLC to afford the compounds 1−7. Versixanthones A−D (1−4) showed the common molecular formula of C32H30O14, determined by HRESIMS, with 18 degrees of unsaturation. The high similarity of their UV−vis spectra indicated that they share related skeletons. The doubled resonances in the 1D NMR spectra (Tables 1 and 2), especially in the 13C NMR spectra of 1−4, suggested that they are xanthone heterodimers. Compound 1 was obtained as pale yellow crystals. The presence of a tetrahydroxanthone monomeric unit in 1 was deduced by the near-identical 1D NMR data of 1 to those of SAD (7) and blennolide B. 3,11 The 2,2-disubstituted chromanone monomeric unit was assigned by 2D NMR correlations and the similar 1D NMR data to those of a synthetic analogue (Tables 1 and 2 and Figure S2 in the Supporting Information).12 The connection between the chromanone monomeric unit and the lactone moiety was suggested by the HMBC correlation of H-5′ (δH 4.82) with C12′ (δC 169.1) (Figure 1). The planar structure of 1 was constructed by connecting the two monomeric units via the linkage of tetrahydroxanthone C-4 and chromanone C-2′, evidenced by the HMBC correlations of H-3 (δH 7.47) with C2′ (δC 118.0), H-3′ (δH 7.96) with C-4 (δC 115.0), and 1′-OH (δH 11.82) with C-2′ (Figure 1). The relative configuration of the tetrahydroxanthone monomer was readily established to be the same as that of blennolide B by the coupling constant (3JH‑5,H‑6 = 11.5 Hz), the NOESY correlation between H-5 and H3-11 (Figure 2), and the chemical shifts (Tables 1 and 2). The cis configuration of H-5′ and H-6′ in the γ-butyrolactone moiety was supported by comparison of the coupling constant (3JH‑5′,H‑6′ = 7.1 Hz) with that of a synthetic analogue (Figure S2).12 Finally, the structure of 1 was confirmed by X-ray singlecrystal diffraction analysis (Figure 3). Similarly to 1, compounds 2−4 were also constructed from tetrahydroxanthone and chromanone monomers, with different B

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Table 1. 13C NMR Spectroscopic Data for 1−6 (100 MHz, CDCl3, TMS, δ ppm) no.

1

1 2 3 4 4a 5 6 7 8 8a 9 9a 10a 11 12 13 1′ 2′ 3′ 4′ 4a′ 5′ 6′ 7′ 8′ 8a′ 9′ 9a′ 10a′ 11′ 12′ 13′ 14′

162.0, 110.5, 140.8, 115.0, 155.4, 77.3, 29.2, 36.3, 177.4, 101.6, 187.2, 107.7, 84.9, 18.1, 170.1, 53.8, 159.3, 118.0, 141.6, 107.3, 158.5, 82.8, 33.6, 36.7, 174.9, 39.9, 194.2, 107.1, 84.7, 15.0, 169.1, 53.3,

2 C CH CH C C CH CH CH2 C C C C C CH3 C CH3 C C CH CH C CH CH CH2 C CH2 C C C CH3 C CH3

161.4, 109.9, 141.0, 116.6, 156.2, 76.2, 28.8, 36.3, 178.2, 102.0, 187.2, 107.6, 85.4, 18.1, 170.2, 52.8, 161.7, 110.3, 139.6, 116.1, 156.1, 81.9, 33.9, 34.8, 174.4, 39.8, 193.5, 106.9, 85.8, 14.5, 168.4, 53.6,

3 C CH CH C C CH CH CH2 C C C C C CH3 C CH3 C CH CH C C CH CH CH2 C CH2 C C C CH3 C CH3

161.8, 110.3, 141.7, 115.3, 156.0, 77.3, 29.3, 36.4, 177.8, 101.8, 187.3, 107.9, 85.0, 18.1, 170.4, 53.3, 159.1, 108.3, 140.5, 117.5, 158.5, 82.4, 34.0, 35.5, 174.9, 40.0, 194.1, 106.9, 86.0, 14.7, 168.8, 53.8,

4 C C CH CH C CH CH CH2 C C C C C CH3 C CH3 C CH CH C C CH CH CH2 C CH2 C C C CH3 C CH3

159.1, 117.8, 141.3, 107.3, 158.3, 76.9, 29.2, 36.2, 177.6, 101.5, 187.1, 107.4, 84.7, 18.0, 170.2, 53.3, 159.3, 118.0, 140.1, 107.6, 158.3, 82.6, 33.5, 36.7, 174.9, 39.7, 194.1, 106.8, 84.4, 14.9, 169.0, 53.7,

5 C C CH CH C CH CH CH2 C C C C C CH3 C CH3 C C CH CH C CH CH CH2 C CH2 C C C CH3 C CH3

161.9, 110.5, 140.8, 115.2, 155.4, 77.3, 29.2, 36.3, 177.3, 101.6, 187.2, 107.6, 84.9, 18.0, 170.2, 53.5, 159.3, 117.8, 141.3, 107.1, 158.8, 76.2, 30.9, 40.4, 173.3, 40.0, 196.0, 107.2, 86.8, 13.8, 170.6, 53.3, 51.9,

6 C CH CH C C CH CH CH2 C C C C C CH3 C CH3 C C CH CH C CH CH CH2 C CH2 C C C CH3 C CH3 CH3

159.4, 118.0, 140.3, 108.5, 158.4, 77.0, 29.3, 36.3, 177.7, 101.6, 187.2, 107.0, 84.8, 18.1, 170.4, 53.5, 159.2, 117.8, 141.0, 108.1, 158.8, 76.4, 30.9, 40.2, 173.2, 40.1, 196.1, 107.7, 86.9, 13.8, 170.6, 53.4, 51.9,

C C CH CH C CH CH CH2 C C C C C CH3 C CH3 C C CH CH C CH CH CH2 C CH2 C C C CH3 C CH3 CH3

Table 2. 1H NMR Spectroscopic Data of 1−6 (400 MHz, CDCl3, TMS, δ ppm) no. 2 3 4 5 6 7 11 13 1-OH 8-OH 2′ 3′ 4′ 5′ 6′ 7′ 8a′ 11′ 13′ 14′ 1′-OH

1

2

6.60, d (8.8) 7.47, d (8.8)

6.63, d (8.6) 7.46, d (8.6)

6.63, d (8.8) 7.54, d (8.8)

3.80, d (11.5) 2.39, m 2.27, dd (10.4, 19.3); 2.71, dd (6.1, 19.3) 1.10, d (6.6) 3.71, s 11.41, s 13.69, s

3.75, d (10.9) 2.39, m 2.38, dd (13.0, 17.1); 2.68, dd (4.2, 17.1) 1.10, d (5.7) 3.61, s 11.42, s 13.84, s 6.57, d (8.6) 7.30, brs

3.97, d (11.0) 2.45, m 2.33, overlap; 2.75, dd (6.1, 19.3) 1.18, d (6.6) 3.73, s 11.61, s 13.77, s

7.52, d (8.5) 6.63, d (8.5) 3.93 (11.0) 2.42, m 2.75, dd (5.8, 18.8); 2.32, dd (10.6, 18.8) 1.18, d (6.2) 3.73, s 11.94, s 13.78, s

4.71, d (7.8) 2.88, m 1.79, dd (10.3, 17.0); 2.19, dd (8.6, 17.0) 3.06, d (17.0); 3.31, d (17.0) 1.20, d (7.2) 3.79, s

7.66, d (8.7) 6.66, d (8.7) 4.67, d (7.7) 2.90, m 2.30, overlap; 1.96, dd (11.6, 17.0) a: 3.17, d (17.0); β: 3.27, d (17.0) 1.23, d (7.1) 3.78, s

7.46, d (8.5) 6.63, d (8.5) 4.81, d (6.8) 2.99, m 2.70, dd (8.2, 17.2); 2.49, dd (8.2, 17.2) 3.29, d (17.2); 3.20, d (17.2) 1.34, d (7.1) 3.77, s

11.51, s

11.62, s

11.77, s

7.96, d (8.2) 6.63, d (8.2) 4.82, d (7.1) 2.99, m 2.49, dd (7.7, 17.6); 2.71, dd (8.3, 17.6) 3.28, d (17.0); 3.21, d (17.0) 1.35, d (7.1) 3.77 11.82, s

3

4

C

5

6

6.60, d (8.6) 7.47, d (8.6) 3.79, d (11.2) 2.39, m 2.27, dd (10.6, 19.3); 2.70, dd (6.2, 19.3) 1.10, d (6.4) 3.71, s 11.40, s 13.69, s

7.44, d (8.8) 6.62, d (8.8) 3.91, d (11.0) 2.39, m 2.73, dd (6.1, 19.3); 2.31, dd (12.1, 19.3) 1.16, d (6.6) 3.75, s 11.73, s 13.78, s

7.94, d (8.6) 6.61, d (8.6) 4.06, d (1.8) 2.38, m 2.38, overlap; 2.60, dd (9.4, 18.0) 3.28, d (17.1); 3.24, d (17.1) 1.07, d (6.7) 3.76, s 3.68, s 11.92, s

7.48, d (8.8) 6.60, d (8.8) 4.04, d (1.6) 2.38, overlap 2.60, dd (9.4, 18.7); 2.38, overlap 3.27, d (17.0); 3.23, d (17.0) 1.06, d (6.6) 3.72, s 3.69, s 12.03, s

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Figure 1. HMBC and COSY correlations of 1 and 5.

identified in the single crystals, the energy barrier for the rotation about the biaryl bond is not large enough in 1 to enable atropisomers. Thus, the M-helicity of the solid-state conformer most likely also represents the low-energy solution helicity form of 1. While the absolute configuration of the corresponding chirality centers C-5, C-6 and C-5′, C-6′ are identical in the two tetrahydroxanthone monomers of 1, C-10a and C-10a′ have different configurations, which suggested that the C10a′−O bond opened and recyclized with opposite configuration during the transformations of the pyranone ring.1 Versixanthone B (2) has a 4−4′ linkage, which is known to bring about atropisomers as reported for the homodimeric tetrahydroxanthone biaryl phomoxanthone A, having an estimated 37 kcal/mol (about 155 kJ/mol) rotational barrier.19 On the basis of biogenetic considerations and NMR analysis supported by DFT calculations, the relative and absolute configurations of the chirality centers in the tetrahydroxanthone and chromanone monomers of 2−6 were proposed to be the same as those in 1, which were also confirmed by DFT conformational analysis and ECD calculations for compounds 2 (Figure 4). The Boltzmann-weighted ECD spectra of 2 computed with three functionals for both the in vacuo and solvent model conformers reproduced the main features of the experimental ECD curve satisfactorily, allowing the assignment

Figure 3. ORTEP diagram for the single-crystal X-ray geometry of versixanthone A (1).

of the axial chirality as (aR). The observed NOE correlation between the 6-Me group of the tetrahydroxanthone unit and the methyl group of the chromanone C-10a′ methoxycarbonyl confirmed the 10a′S absolute configuration of the chromanone moiety, in accordance with the geometry of the low-energy computed conformers. Thus, the absolute configuration of 2 was finally assigned as (aR,5R,6S,10aR,5′R,6′S,10a′S). The torsional angle scan of 3 (2−4′ linkage) and 4 (2−2′ linkage) showed that the energy barriers for the inversion of helicity are 45 kJ/mol (3) and 20 kJ/mol (4), respectively (Figure S6), which are not large enough (2%) of (aR,5R,6S,10aR,5′R,6′S,10a′S)-2. (b) Experimental ECD spectrum of versixanthone B (2) and Boltzmannweighted BH&HLYP/TZVP-calculated ECD spectrum of (aR,5R,6S,10aR,5′R,6′S,10a′S)-2 with the PCM solvent model for MeCN computed for the B97D/TZVP (PCM/MeCN) conformers. Bars represent the calculated rotational strengths of the lowest energy conformer.

Figure 5. Experimental ECD and calculated spectra of versixanthones C (3) and D (4). (a) Experimental ECD spectrum of 3 and PBE0/ TZVP-calculated ECD spectrum of (5R,6S,10aR,5′R,6′S,10a′S)-3. (b) Experimental ECD spectrum of 4 and BH&HLYP/TZVP-calculated ECD spectrum of (5R,6S,10aR,5′R,6′S,10a′S)-4. The calculated ECD spectra were carried out with the PCM solvent model for MeCN computed for the B97D/TZVP PCM/MeCN conformers. Bars represent the calculated rotational strengths of the lowest energy conformers.

S,10a′S)-3 and (5R,6S,10aR,5′R,6′S,10a′S)-4 absolute configurations proposed by biosynthetic consideration were confirmed by the agreement of the experimental ECD curves and the calculated ones (Figure 5). The ECD curves of 5 were almost identical to those of 1, indicating the same preferred helicity as that of compound 1 (Figure S8, Supporting Information). Similarly to 4, compound 6 also has a 2−2′ linkage, indicating free interconversion of the P- and M-helicity conformers (Figure S8). Although the absolute configurations of the chirality centers in the two monomeric units of 1−6 were proposed to be the same on the grounds of the biogenetic considerations and this was confirmed by the calculated ECD curves, except for 1 and 2, the assignment of the central chirality elements could not be achieved by direct evidence. The tetrahydroxanthone-containing natural products have been demonstrated to be unstable under basic conditions, and they can easily undergo isomerizations arising from ether linkage replacement, such as in the beticolins,21 parnafungins,22 SAD,23 and γ-butyrolactones like the xanthoquinodins, which can easily undergo ring-opening and ring-closure reactions in CHCl3 and MeOH.24 Inspired by the above conversions and our findings that 1−6 were unstable in some solvents such as deuterated DMSO during the measurement of NMR spectra, we assigned the central chirality elements of 3−6 by chemical conversions. All experiments were

monitored by HPLC-UV and by comparison of retention times with pure standard substances. The chemical conversions (Figure S9) revealed the specific interconversions between 1 (4−2′ linkage) and 4 (2−2′ linkage) and between 2 (4−4′ linkage) and 3 (2−4′ linkage) and the irreversible chemical conversions from 1 to 5 in MeOH and from 6 to 4 in CHCl3 (Figure S10). This finding also indicated that the absolute configurations of the two monomeric units in all isolates remained the same, which further confirmed the assignments of the absolute configurations of 1−6. A retro-oxa-Michael addition could be invoked as a key reaction during the interconversion of 1/4 and 2/3, undergoing ring-opening via cleavage of the ether bond at C10a and ring-reclosing with the other phenolic hydroxy group, resulting in a formal change of the biaryl linkage pattern (Figure 6). Biogenetically, the biaryl carbon−carbon linkage itself does not change. The numbering of the ring system changes with the rearrangement, leading to the change in the linkage nomenclature. In light of the potent cytotoxicity of SAD (7), we evaluated the in vitro cytotoxic effects of compounds 1−6 against a panel of cancer cell lines (Tables 3). Compounds 1−3 displayed quite selective potent cytotoxicity against HL-60 and K562, with lowE

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Figure 7. Compound 5 inhibits Topo I-mediated supercoiled DNA relaxation. PBR322 plasmid DNA (0.5 μg) alone (lane 6) or with Topo I in the presence of 0 (lane 5), 5 (lane 4), 10 (lane 3), 20 (lane 2), and 40 μM (lane 1) compound 5. All DNA samples were resolved by electrophoresis on 1% agarose gels and stained with ethidium bromide. Negatively supercoiled pBR322 (SC) and relaxed DNA (RLX) are marked.

anthone unit. The absolute configurations of the axial and central chirality elements were unambiguously established by a combination of X-ray, ECD, and chemical conversions, in particular the solvent-induced retro-oxa-Michael reaction, which provided an alternative approach for assigning the absolute configuration of tetrahydroxanthone-containing dimeric compounds. The conversions also indicated the possibility that some of the xanthone−chromanone dimers might form spontaneously from the naturally occurring dimers during the fermentation or extraction process. Thus, compounds 5 and 6 were likely methanolysis artifacts in the workup procedure of extraction and isolation.

Figure 6. Proposed interconversion mechanism between 1 and 4.

Table 3. Cytotoxic Effects of 1−6 (IC50, μM) compd

HL-60

K562

A549

H1975

803

HO-8910

HCT-116

1 2 3 4 5 6 7 Doxb

2.6 9.9 7.8 3.1 1.6 2.7 1.00 0.02

7.1 NT 18.2 9.1 11.1 6.7 2.0 0.3

>50 >50 >50 12.7 NT 10.6 2.1 0.2

11.2 >50 25.6 >50 2.7 NT NT 0.8

>50 21.6 NT 9.8 2.2 NT 1.5 0.2

10.1 >50 >50 13.9 2.0 20.8 1.5 0.5

NTa NT >50 6.1 NT 0.7 0.9 0.2

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EXPERIMENTAL SECTION

General Experimental Procedures. The melting point was measured using a Yanaco MP-500D micromelting point apparatus and was uncorrected. Specific rotations were obtained on a JASCO P-1020 digital polarimeter. UV spectra were recorded on a Beckman DU 640 spectrophotometer. ECD spectra were measured on a JASCO J-715 spectropolarimeter. IR spectra were taken on a Bruker Tensor-27 spectrophotometer in KBr discs. NMR spectra were recorded on a Bruker-400 spectrometer using TMS as internal standard, and chemical shifts were recorded as δ values. ESIMS utilized a Thermo Scientific LTQ Orbitrap XL mass spectrometer or a Micromass QTOF ULTIMA GLOBAL GAA076 LC mass spectrometer. X-ray diffraction was realized on a Bruker APEX DUO instrument at 133 K using Cu Kα radiation. Semiprepartive HPLC was performed using an ODS column [HPLC (YMC-Pack ODS-A, 10 × 250 mm, 5 μm, 4 mL/min)]. Medium-pressure liquid chromatography (MPLC) was performed on a Bona-Agela CHEETAHTM HP100 (Beijing Agela Technologies Co., Ltd.). Column chromatography (CC) was performed with silica gel (200−300 mesh, Qingdao Marine Chemical Inc.) and Sephadex LH-20 (Amersham Biosciences), respectively. Fungal Material. The fungal strain Aspergillus versicolor HDN1009 was isolated from soil around a mangrove collected in Guangzhou, China, and was identified by the ITS sequence. The sequence data have been submitted to GenBank (accession number: KP765236). The voucher specimen is deposited in our laboratory at −20 °C. Working stocks were prepared on potato dextrose agar slants stored at 4 °C. Fermentation and Extraction. The fungus A. versicolor HDN1009 was cultured under static conditions at 28 °C in 1 L Erlenmeyer flasks containing 300 mL of liquid culture medium, composed of maltose (20.0 g/L), mannitol (20.0 g/L), glucose (10.0 g/L), monosodium glutamate (10.0 g/L), MgSO4·7H2O (0.3 g/L), KH2PO4 (0.5 g/L), yeast extract (3.0 g/L), corn steep liquor (1.0 g/ L), and seawater (Huiquan Bay, Yellow Sea). After 2 weeks of cultivation, 100 L of whole broth was filtered through cheesecloth to separate the supernatant from the mycelia. The former was extracted

a

NT: not test. bDox stands for doxorubicin hydrochloride, which was used as a reference drug.

micromolar IC50 values, while compounds 4−6 exhibited extensive cytotoxicities against all seven cancer lines, with IC50’s ranging from 0.7 to 14.0 μM. Inspired by the fact that SAD (7) inhibited the activity of topoisomerase I,25,26 all of the new compounds 1−6 were investigated on this target. Interestingly, only compound 5 showed activity (Figure 7). In summary, an unusual group of xanthone−chromanone dimers (1−6) with high structural diversity and complexity was isolated from the fermentation of a marine-derived fungus, A. versicolor, which represents a new class of heterodimers containing the same monomer as that of SAD. They contain a common tetrahydroxanthone and a biogenetically related chromanone monomer or its derivatives coupled by a variety of biaryl linkages. Although the biosynthetic relationships between the tetrahydroxanthone and chromanone units and among the different linkages are unclear, the chromanone unit is proposed to be formed chemically from the tetrahydroxanthone unit through a series of intermediates.1 The formation of the different linkages may be explained by the intramolecular oxaMichael recyclization of the freely rotating benzophenone intermediate which can be obtained from the tetrahydroxF

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λmax (Δε) 365 (7.12), 333 (20.1), 228 (−55.85); ESIMS m/z 639.2 [M + H]+. X-ray Crystallographic Analysis of Compound 1. Crystals of 1 were obtained in the mixed solvent of CHCl3−MeOH−H2O, and crystallographic data (excluding structure factors) for 1 (Cu Kα radiation) have been deposited with the Cambridge Crystallographic Data Center as supplementary publication number CCDC 984115. These data can be obtained free of charge from the Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_request/ cif. Crystal Data for 1: orthorhombic, C32H32O15, space group P212121 (no. 19), a = 7.08100(10) Å, b = 13.9644(2) Å, c = 31.0653(6) Å, V = 3071.80(9) Å3, Z = 4, T = 290(2) K, μ(Cu Kα) = 0.971 mm−1, Dcalcd = 1.420 g/mm3, 27 551 reflections measured (6.94 ≤ 2θ ≤ 139.236), 5706 unique (Rint = 0.0227), which were used in all calculations. The final R1 was 0.0347 [I > 2σ(I)] and wR2 was 0.1033 (all data). Flack parameter = 0.02(5). Transformation between Versixanthones A−G (1−6). Compounds 1−6 (0.2 mg) were dissolved in DMSO, MeOH, or CHCl3, respectively, and then these solutions were placed at 30 °C for 3 or 7 days. The experiments were monitored by HPLC-UV and by comparison of retention times with pure compounds [60:40 to 100:0 MeOH−H2O (with 0.2% trifluoroacetic acid)], 30 min, 1 mL/ min. Computational Section. Mixed torsional/low-frequency mode conformational searches were carried out by means of the Macromodel 9.9.223 software using the Merck Molecular Force Field (MMFF) with an implicit solvent model for CHCl3.28 Geometry reoptimizations were carried out at the B3LYP/6-31G(d) level in vacuo and the B97D/TZVP level29,30 with the PCM solvent model for MeCN. TDDFT ECD calculations were run with various functionals (B3LYP, BH&HLYP, PBE0) and the TZVP basis set as implemented in the Gaussian 09 package.31 Torsional scan calculations were carried out at the B3LYP/6-31G(d) level in vacuo. ECD spectra were generated as sums of Gaussians with 2400 and 3000 cm−1 widths at half-height (corresponding to ca. 28 and 35 at 340 nm), using dipolevelocity-computed rotational strength values.32 Boltzmann distributions were estimated from the ZPVE-corrected B3LYP/6-31G(d) energies in the gas-phase calculations and from the B97D/TZVP energies in the solvated ones. The MOLEKEL software package was used for visualization of the results.33 Biological Assay. Cytotoxic activities of 1−7 were evaluated by an MTT method using HL-60, K562, A549, H1975, 803, HEK293, HO8910, and HCT-116 cell lines. The detailed methodology for biological testing has already been described in a previous report.34 Topoisomerase I-Mediated DNA Cleavage Assay. The detailed methodology has already been described in a previous report.35,36

three times with EtOAc, while the latter was extracted three times with acetone and concentrated under reduced pressure to afford an aqueous solution, which was extracted three times with EtOAc. Both EtOAc solutions were combined and concentrated under reduced pressure to give the organic extract (50 g). Isolation. The organic extract was subjected to vacuum liquid chromatography over a silica gel column using a gradient elution with petroleum ether−CH2Cl2−MeOH to give six fractions (fractions 1− 6). Fraction 3 (5.1 g), eluted with 97:3 CH2Cl2−MeOH, was applied on a C-18 ODS column using a stepped gradient elution of MeOH− H2O, yielding seven subfractions (fractions 3.1−3.7). Fraction 3.4, eluted with 75:25 MeOH−H2O, was chromatographed on Sephadex LH-20 with CH2Cl2−MeOH (1:1) and further separated by MPLC (C-18 ODS) using a stepped gradient elution of MeOH−H2O (70:30 to 90:10) to furnish eight subfractions (fractions 3.4.1−3.4.8). Compound 7 (100 mg) was obtained by recrystallization of fraction 3.4.2. Fraction 3.4.5 was chromatographed on Sephadex LH-20 (MeOH) and further purified by semipreparative HPLC (50:50 MeCN−H2O, 3 mL/min) to afford compounds 1 (22.0 mg) and 5 (33.0 mg). Fraction 3.4.4 was fractionated on Sephadex LH-20 (MeOH) and further purified by semipreparative HPLC (45:55 MeOH−H2O, 3 mL/min) to afford compounds 2 (5.8 mg), 3 (14.7 mg), 4 (19.1 mg), and 6 (8.7 mg). Compound 1 was crystallized in a mixture of MeOH−H2O (drops). Versixanthone A (1): pale yellow crystals (a mixed solvent of CHCl3−MeOH−H2O); mp 214−216 °C; [α]20D −69 (c 0.1, CHCl3); UV (MeOH) λmax (log ε) 221 (4.10), 255 (3.96), 333 (3.80) nm; ECD (c 4.08 × 10−4 M, MeOH), λmax (Δε) 373 (−7.5), 333 (35.1), 306 (3.65), 253 (−128.1), 219 (135.5) nm; 1H and 13C NMR data, Tables 1 and 2, respectively; HRESIMS m/z 639.1708 [M + H]+ (calcd for C32H31O14, 639.1708). Versixanthone B (2): pale yellow powder; [α]20D +89 (c 0.1, CHCl3); UV (MeOH) λmax (log ε) 221 (4.11), 248 (3.92), 333 (3.81) nm; ECD (c 1.56 × 10−4 M, MeCN), λmax (Δε) 368sh (1.65), 339 (3.87), 305 (−2.46), 275 (−1.56), 256 (1.63), 227 (−11.33), 204 (−12.79) nm, positive below 193 nm; ECD (c 4.7 × 10−4 M, MeOH), λmax (Δε) 337 (14.8), 307 (−7.3), 278 (−3.6), 255 (9.6), 226 (−41.3), 206 (−45.8) nm; 1H and 13C NMR data, Tables 1 and 2, respectively; HRESIMS m/z 639.1698 [M + H]+ (calcd for C32H31O14, 639.1708). Versixanthone C (3): pale yellow powder; [α]20D +103 (c 0.1, CHCl3); UV (MeOH) λmax (log ε) 221 (4.10), 240 (3.90), 263 (3.93), 331 (3.80) nm; ECD (c 2.0 × 10−4 M, MeCN), λmax (Δε) 370sh (2.11), 335 (7.49), 284 (−1.16), 254 (7.21), 220 (−31.43) nm, positive below 199 nm; ECD (c 6.11 × 10−4 M, MeOH) λmax (Δε) 338 (29.3), 289 (−1.9), 255 (54.3), 219 (−158.3); 1H and 13C NMR data, Tables 1 and 2, respectively; HRESIMS m/z 639.1709 [M + H]+ (calcd for C32H31O14, 639.1708). Versixanthone D (4): pale yellow powder; [α]20D −16 (c 0.1, CHCl3); UV (MeOH) λmax (log ε) 221 (4.08), 263 (3.93), 333 (3.75) nm; ECD (c 1.56 × 10−4 M, MeCN), λmax (Δε) 378sh (1.06), 334 (7.76), 315sh (3.78), 289sh (1.25), 265sh (−7.59), 238 (−12.97), 226sh (−8.35), 207 (13.72) nm, negative below 198 nm; ECD (c 4.08 × 10−4 M, MeOH) λmax (Δε) 331 (20.1), 240 (−42.6), 212 (16.2) nm; 1H and 13C NMR data, Tables 1 and 2, respectively; HRESIMS m/z 639.1699 [M + H]+ (calcd for C32H31O14, 639.1708). Versixanthone E (5): pale yellow powder; [α]20D −60 (c 0.1, CHCl3); UV (MeOH) λmax (log ε) 221 (4.13), 255 (3.92), 333 (3.82) nm; ECD (c 2.69 × 10−4 M, MeOH) λmax (Δε) 332 (27.4), 306 (−2.1), 290 (9.9), 253 (−109.9), 218 (118.2) nm; 1H and 13C NMR data, Tables 1 and 2, respectively; HRESIMS m/z 671.1972 [M + H]+ (calcd for C33H35O15, 671.1970). Versixanthone F (6): pale yellow powder; [α]20D −10 (c 0.1, CHCl3); UV (MeOH) λmax (log ε) 221 (4.03), 261 (3.87), 333 (3.71) nm; ECD (c 5.52 × 10−4 M, MeOH) λmax (Δε) 338 (18.0), 240 (−33.0), 203 (23.7) nm; 1H and 13C NMR data, Tables 1 and 2, respectively; HRESIMS m/z 671.1970 [M + H]+ (calcd for C33H35O15, 671.1970). Secalonic acid D (7): pale yellow powder; [α]20D +55 (c 0.1, CHCl3), [α]20D +61 (c 0.11, CHCl3);27 UV (MeOH) λmax (log ε) 221 (4.01), 255 (3.86), 335 (4.10) nm; ECD (c 2.99 × 10−4 M, MeOH)

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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.5b00636.

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HRESIMS and NMR spectra of compounds 1−7 (PDF) Crystallographic data (CIF)

AUTHOR INFORMATION

Corresponding Author

*Tel: 0086-532-82031619. Fax: 0086-532-82033054. E-mail: [email protected]. Author Contributions §

G. Wu and G. Yu contributed equally.

Notes

The authors declare no competing financial interest. G

DOI: 10.1021/acs.jnatprod.5b00636 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

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(21) Ducrot, P. H. C. R. C. R. Acad. Sci., Ser. IIc: Chim. 2001, 4, 273− 283. (22) Parish, C. A.; Smith, S. K.; Calati, K.; Zink, D.; Wilson, K.; Roemer, T.; Jiang, B.; Xu, D.; Bills, G.; Platas, G.; Peláez, F.; Dáez, M. T.; Tsou, N.; McKeown, A. E.; Ball, R. G.; Powles, M. A.; Lai, Y.; Liberator, P.; Harris, G. J. Am. Chem. Soc. 2008, 130, 7060−7066. (23) Kurobane, I. M. W.; Vining, L. C.; Mcinnes, A. G.; Halifax, N. S. Germany patent DE3002671, 1980. (24) Chen, G.; Chen, Y.; Gao, H.; Shen, L.; Wu, Y.; Li, X.; Li, Y.; Guo, L.; Cen, Y.; Yao, X. J. Nat. Prod. 2013, 76, 702−709. (25) Guo, Z.; She, Z.; Shao, C.; Wen, L.; Liu, F.; Zheng, Z.; Lin, Y. Magn. Reson. Chem. 2007, 45, 777−780. (26) Ren, H. Pharm. Biol. 2011, 49 (8), 796−799. (27) Ren, H.; Tian, L.; Gu, Q. Q.; Zhu, W. M. Arch. Pharmacal Res. 2006, 29 (1), 59−63. (28) MacroModel; Schrödinger, LLC, 2012, http://www.schrodinger. com/MacroModel. (29) Grimme, S. J. Comput. Chem. 2006, 27, 1787−1799. (30) Sun, P.; Xu, D.-X.; Mándi, A.; Kurtán, T.; Li, T. J.; Schulz, B.; Zhang, W. J. Org. Chem. 2013, 78, 7030−7047. (31) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.; Scalmani, G.; Barone, V.; Mennucci, B.; Petersson, G. A.; Nakatsuji, H.; Caricato, M.; Li, X.; Hratchian, H. P.; Izmaylov, A. F.; Bloino, J.; Zheng, G.; Sonnenberg, J. L.; Hada, M.; Ehara, M.; Toyota, K.; Fukuda, R.; Hasegawa, J.; Ishida, M.; Nakajima, T.; Honda, Y.; Kitao, O.; Nakai, H.; Vreven, T.; Montgomery, J. A., Jr.; Peralta, J. E.; Ogliaro, F.; Bearpark, M.; Heyd, J. J.; Brothers, E.; Kudin, K. N.; Staroverov, V. N.; Kobayashi, R.; Normand, J.; Raghavachari, K.; Rendell, A.; Burant, J. C.; Iyengar, S. S.; Tomasi, J.; Cossi, M.; Rega, N.; Millam, J. M.; Klene, M.; Knox, J. E.; Cross, J. B.; Bakken, V.; Adamo, C.; Jaramillo, J.; Gomperts, R.; Stratmann, R. E.; Yazyev, O.; Austin, A. J.; Cammi, R.; Pomelli, C.; Ochterski, J. W.; Martin, R. L.; Morokuma, K.; Zakrzewski, V. G.; Voth, G. A.; Salvador, P.; Dannenberg, J. J.; Dapprich, S.; Daniels, A. D.; Farkas, O.; Foresman, J. B.; Ortiz, J. V.; Cioslowski, J.; Fox, D. J. Gaussian 09, revision B.01; Gaussian, Inc.: Wallingford, CT, 2010. (32) Stephens, P. J.; Harada, N. Chirality 2010, 22, 229−233. (33) Varetto, U. MOLEKEL, v. 5.4; Swiss National Supercomputing Centre: Manno, Switzerland, 2009. (34) Du, L.; Zhu, T. J.; Liu, H. B.; Fang, Y. C.; Zhu, W. M.; Gu, Q. Q. J. Nat. Prod. 2008, 71, 1837−1842. (35) Gao, H.; Liu, W.; Zhu, T.; Mo, X.; Mándi, A.; Kurtán, T.; Li, J.; Ai, J.; Gu, Q.; Li, D. Org. Biomol. Chem. 2012, 10, 9501−9506. (36) Che, Q.; Zhu, T.; Qi, X.; Mándi, A.; Kurtán, T.; Mo, X.; Li, J.; Gu, Q.; Li, D. Org. Lett. 2012, 14 (13), 3438−3441.

ACKNOWLEDGMENTS This work was financially supported by the National Natural Science Foundation of China (21372208), the Shandong Provincial Natural Science Fund for Distinguished Young Scholars (JQ201422), the Program for New Century Excellent Talents in University (NCET-12-0499), the National High Technology Research and Development Program of China (2013AA092901), and NSFC-Shandong Joint Fund for Marine Science Research Centers (U1406402). T.K. thanks the Hungarian National Research Foundation (OTKA K105871) for financial support and the National Information Infrastructure Development Institute (NIIFI 10038) for CPU time.

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REFERENCES

(1) Masters, K.; Bräse, S. Chem. Rev. 2012, 112, 3717−3776. (2) Wezeman, T.; Bräse, S.; Masters, K. Nat. Prod. Rep. 2015, 32, 6− 28. (3) Zhang, W.; Krohn, K.; Ullah, Z.; Flörke, U.; Pescitelli, G.; Di Bari, L.; Antus, S.; Kurtán, T.; Rheinheimer, J.; Draeger, S.; Schulz, B. Chem. - Eur. J. 2008, 14, 4913−4923. (4) Cai, S.; King, J. B.; Du, L.; Powell, D. R.; Cichewicz, R. H. J. Nat. Prod. 2014, 77, 2280−2287. (5) Kikuchi, H.; Isobe, M.; Kurata, S.; Katou, Y.; Oshima, Y. Tetrahedron 2012, 68, 6218−6223. (6) El-Elimat, T.; Figueroa, M.; Raja, H. A.; Graf, T. N.; Swanson, S. M.; Falkinham, J. O., III; Wani, M. C.; Pearce, C. J.; Oberlies, N. H. Eur. J. Org. Chem. 2015, 2015, 109−121. (7) Berova, N.; Polavarapu, P. L.; Nakanishi, K.; Woody, R. W. Comprehensive Chiroptical Spectroscopy: Applications in Stereochemical Analysis of Synthetic Compounds, Natural Products, and Biomolecules, Vol. 2; John Wiley & Sons, Inc.: Hoboken, NJ, USA, 2012. (8) Du, L.; Liu, H.; Fu, W.; Li, D.; Pan, Q.; Zhu, T.; Geng, M.; Gu, Q. J. Med. Chem. 2011, 54, 5796−5810. (9) Li, L.; Li, D.; Luan, Y.; Gu, Q.; Zhu, T. J. Nat. Prod. 2012, 75, 920−927. (10) Du, L.; Ai, J.; Li, D.; Zhu, T.; Wang, Y.; Knauer, M.; Bruhn, T.; Liu, H.; Geng, M.; Gu, Q.; Bringmann, G. Chem. - Eur. J. 2011, 17, 1319−1326. (11) Ren, H.; Tian, L.; Gu, Q.; Zhu, W. Arch. Pharmacal Res. 2006, 29, 59−63. (12) Qin, T.; Johnson, R. P.; Porco, J. A., Jr. J. Am. Chem. Soc. 2011, 133, 1714−1717. (13) Bifulco, G.; Dambruoso, P.; Gomez-Paloma, L.; Riccio, R. Chem. Rev. 2007, 107, 3744−3779. (14) Bara, R.; Zerfass, I.; Aly, A. H.; Goldbach-Gecke, H.; Raghavan, V.; Sass, P.; Mándi, A.; Wray, V.; Polavarapu, P. L.; Pretsch, A.; Lin, W.; Kurtán, T.; Debbab, A.; Brötz-Oesterhelt, H.; Proksch, P. J. Med. Chem. 2013, 56, 3257−3272. (15) Zhang, Q.; Mándi, A.; Li, S.; Chen, Y.; Zhang, W.; Tian, X.; Zhang, H.; Li, H.; Zhang, W.; Zhang, S.; Ju, J.; Kurtán, T.; Zhang, C. Eur. J. Org. Chem. 2012, 2012, 5256−5262. (16) Rönsberg, D.; Debbab, A.; Mándi, A.; Vasylyeva, V.; Böhler, P.; Stork, B.; Engelke, L.; Hamacher, A.; Sawadogo, R.; Diederich, M.; Wray, V.; Lin, W.; Kassack, M. U.; Janiak, C.; Scheu, S.; Wesselborg, S.; Kurtán, T.; Aly, A. H.; Proksch, P. J. Org. Chem. 2013, 78, 12409− 12425. (17) Bringmann, G.; Hinrichs, J.; Henschel, P.; Kraus, J.; Peters, K.; Peters, E. Eur. J. Org. Chem. 2002, 2002, 1096−1106. (18) Debbab, A.; Aly, A. H.; Edrada-Ebel, R.; Wray, V.; Pretsch, A.; Pescitelli, G.; Kurtan, T.; Proksch, P. Eur. J. Org. Chem. 2012, 2012, 1351−1359. (19) Elsässer, B.; Krohn, K.; Flörke, U.; Root, N.; Aust, H.; Draeger, S.; Schulz, B.; Antus, S.; Kurtán, T. Eur. J. Org. Chem. 2005, 2005, 4563−4570. (20) Bringmann, G.; Mortimer, A. J. P.; Keller, P. A.; Gresser, M. J.; Garner, J.; Breuning, M. Angew. Chem., Int. Ed. 2005, 44, 5384−5427. H

DOI: 10.1021/acs.jnatprod.5b00636 J. Nat. Prod. XXXX, XXX, XXX−XXX