Sporochartines A–E, A New Family of Natural Products from the Marine Fungus Hypoxylon monticulosum Isolated from a Sphaerocladina Sponge

Four new sporochartines B–E were isolated from the marine fungus Hypoxylon monticulosum CLL-205, isolated from a sponge belonging to the Sphaerocladina order and collected in Tahiti coast. Sporochartine A ( 1 ), the ﬁrst representative of this family was previously isolated from the same fungus. The structures of sporochartines B–E were elucidated using 1D and 2D NMR, HRMS and IR data. Their conﬁgurations were established according to ROE correlations and comparison with the absolute conﬁguration of sporochartine A ( 1 ) previously obtained from X-ray analysis. Sporochartines A–D ( 2–4 ) may be derived from endo Diels-Alderase type catalysis and sporochartine E ( 5 ) from an exo Diels-Alderase catalysis. The spatial conformation of sporochartines drastically inﬂuences the results of the cytotoxic bioassay against HCT-116, PC-3, and MCF-7 human cancer cell lines. the on the proliferation of MCF-7 (breast cancer The the ranged to were and the absorbance was (490 to measure the inhibition of cell proliferation compared to untreated cells. IC 50 determination experiments were performed in separate duplicate experiments.

Sporothriolide belongs to the furofurandione family of natural compounds first published in 1994 (Krohn et al., 1994). This compound exhibits antifungal activity and benefits from substantial synthetic efforts (Sharma and Krishnnudu, 1995;Yu et al., 2001;Fernandes and Ingle, 2009;Ishihara et al., 2014). The name sporothriolide is related to Sporothrix sp. Hektoen and Perkins (strain 700), from which this compound was first isolated. Sporothrix genus belongs to a different ascomycete family, ophiostomataceae. The first report on sporothriolide in 1994 detailed both the structure and bioactivity of this product (Krohn et al., 1994). It shows that Sporothrix produces sporothriolide, dihydrosporothriolide, as well as various sporothriolide analogs with different side-chain length (canadensolide, discosiolide, avenaciolide, ethiosolide). The authors reported also the antifungal/herbicidal activities of these compounds (Krohn et al., 1994) (Figure 1).
Twenty years later, sporothriolide and dihydrosporothriolide were isolated from Hypoxylon monticulosum together with three monocyclic acid precursors: sporothric acid, isosporothric acid and dihydroisosporothric acid (Figure 1) . More recently, sporothriolide was isolated from Nodulisporium sp., an anamorph of Hypoxylon, and the herbicidal activity was confirmed (Cao et al., 2016).
In our previous contribution, we added to the scarce sporothriolide family two new compounds, deoxysporothric FIGURE 1 | Top: Sporothriolide and analogs reported in 1994 (Krohn et al., 1994). Middle: Monocyclic precursors reported in 2014 . Bottom: Deoxysporothric acid and sporochartine A reported in our previous work (Leman-Loubière et al., 2017). acid and a new complex architecture sporochartine A, combining sporothriolide and trienylfuranol A moieties. The trienylfuranol A was recently isolated from a different Hypoxylon submoniticulosum (Burgess et al., 2017).
In the present work we report four new sporochartines B to E. Their structures were elucidated using 1D and 2D NMR, HRMS, IR and comparison with sporochartine A data, for which the absolute configuration was previously established by X-Ray analysis. A Diels-Alderase type reaction is probably involved in the biosynthesis of the five isolated sporochartines, as discussed below.
The human cancer cell-lines cytotoxicity bioassay shows that the conformation of sporochartines has an impact on the biological activity.

RESULTS AND DISCUSSION
According to our previous work, sporochartine A (1) was obtained after 5 days cultivation of H. monticulosum CLL-205 in PDB broth (Leman-Loubière et al., 2017). By extending the cultivation of the same microorganism by a further 4 days, the ethyl actetate extract gives the HPLC chromatogram presented in Figure 2.
Sporochartines B-D were isolated as white powders by flash chromatography followed by semi-preparative HPLC. They had similar [M+H] + HRESIMS molecular weights, molecular formula C 24 H 34 O 6 and IR spectra compared with sporochartine A (1) ( Table 2) (Leman-Loubière et al., 2017). The 1 H and 13 C NMR spectra of sporochartines B-D were similar to those of compound 1 (Tables 1, 2). Optical rotations [α] D 25 , IR bands and HRESIMS are reported in Table 2. COSY and HMBC spectra, confirmed that sporochartines A-D had the same connectivities supporting similar planar scaffold (Figure 3). In addition, the common coupling constant of 15.4-15.6 Hz between H-18 and H-19 confirmed that the double bond C-18/C-19 is in E configuration.
Based on the previously reported absolute configuration of sporochartine A (1) and ROE correlations, we deduced the absolute configuration of sporochartines B-D (2-4) (Figure 4).
The common ROE correlations between H-2 and H-5 and between H-5 and H-6 requiring a cis orientation of these three protons was found in the sporochartine A-D. Therefore, the stereochemistry of the sporothriolide moiety was identical. Moreover, based on ROE correlations between H-20 and H-21 and between H-21 and H-23, the strerochemistry of the tetrahydrofurane moiety is also a common feature in sporochartines A-D.
For sporochartine B (2) (Figure 4), we did not observe ROE correlations between H-17 and H-2 and between H-17 and H-14b as in sporochartine A (1), while a new correlation is observed between H-17 and H-13. This data suggests that the carbon C-17 have opposite stereochemistry compared to 1 supporting a 3S,17R configuration of 2 (instead of 3S,17S in 1).
For sporochartine C (3) (Figure 4), the H-17/H-2 and H-17/H-14b correlations observed in sporochartine A (1) are absent. In addition, we observed a correlation between H-17 and H-13 and H-2 and H-14a in compound 3. Based on this data, we suggest that compound 3 has a 3R,17S configuration.
Sporochartine D (4) (Figure 4) conserved the correlations between H-17 and H-2 and between H-17 and H-14b reported for sporochartine A (1). Furthermore, the correlation between H-17 and H-13 is absent in both 4 and 1. In 4 we have an additional correlation between H-13 and H-2, absent in 1. These observations support the conclusion that 4 is the 3R,17R isomer of 1.
A new compound referred as sporochartine E (5) was also isolated as a white powder. Compound 5 has the same molecular formula C 24 H 34 O 6 as compound 1, deduced from HRESIMS m/z [M+H] + 419.2433. Here again we have eight degrees of unsaturation accounting for two γ-lactones, two double bonds, one six-membered cycle moiety and one tetrahydrofurane moiety.  Compound 5 has a terminal methylene group (at δ C 119.6, δ H 5.27 and δ H 5.21) while the tetrahydrofuran moiety connected to C-19 in 1 is connected to C-14 in 5.
Based on COSY correlations (Figure 5), the sporothriolide moiety was the same in compound 5 as in 1. correlations between H-20 and C-14 and C-15 allowed us to connect this tetrahydrofurane moiety to the sp 3 methine C-14.
The absolute configuration of sporochartine E (5) was suggested using ROE correlations compared to the absolute configuration of sporochartine A (1) (Figure 6).
ROE correlations between H-2 and H-5/H-6 in the sporothriolide moiety and the ROE correlation between H-20 and H-21 and between H-23 and H-21 in the tetrahydrofuran moiety indicated a similar to that in 1.
Sporochartine E (5), showed a correlation between H-17 and H-2, like in compounds 1 and 4. H-2 also exhibited a correlation with H-13b but not with H-14. This suggests that C-3 and C-17 has the same relative configuration than 4. For C-14, we observed ROE correlations between H-14 and H-13a, H-13a, and H-17 and H-14 and H-24, suggesting a 3R, 14S, 17S configuration for compound 5.
Based on the structure of sporothriolide and the recently reported trienylfuranol A isolated from H. submoniticulosum, we suggested a hypothetic biosynthetic pathway of sporochartines, involving a "spiro" Diels-Alderase reaction as shown in The cytotxicity of sporochartines was evaluated on three human cancer cell lines, HCT-116 (human colon carcinoma), PC-3 (prostate cancer cell lines) and MCF-7 (breast cancer cell line). The results presented in Table 3 are highly contrasting but nevertheless clearly indicate the impact of sporochartine conformation on the bioassay results.
Our future efforts will focus on the cytotoxic profile, biosynthesis and synthesis of sporochartines. The cytotoxicity profile reveals a non-cytotoxic sporochartine A (1), a large spectrum cytotoxic sporochartine C (3) and more cell line specific  sporochartines B (2), D (4), and E (5). This finding merits future investigation on the mechanisms of action of these new scaffolds of cytotoxic compounds.
The biosynthesis of sporochartines, and the biosynthesis of its two moieties, sporothriolide and trienylfuranol A are still unknown. This opens new and promising opportunities for the discovery of novel biosynthetic microbial clusters. Finally, having in hand hundreds of milligrams of sporothriolide, the hemi-synthesis of sporochartines is currently in progress based on a final Diels-Alder connection. The selectivity of the chemical catalysis and the proportion of different isomers will be compared to the microbial counterpart. According to our expertise in biocatalysis-based chemodiversification of natural compounds (Adelin et al., 2011;Martins et al., 2015), sporothriolide will be submitted to a FIGURE 7 | Hypothetic biosynthesis of sporochartines through Diels-Alderase type reaction between sporothtriolide and trienylfuranol A.

General Experimental Procedures
Optical rotations [α] D were measured using an Anton Paar MCP-300 polarimeter. IR spectra were obtained using a Perkin Elmer BX FT-IR spectrometer. NMR experiments were performed using a Bruker Avance 500 MHz in CDCl 3 at room temperature. High-resolution mass spectra were obtained on a Waters LCT Premier XE spectrometer equipped with an ESI-TOF (electrospray-time of flight) by direct infusion of the purified compounds. Preparative HPLC was performed using Waters modules consisting of an autosampler 717, a pump 600, a photodiode array detector 2996 and an evaporative light-scattering detector, ELSD 2420. Prepacked silica gel Redisep columns were used for flash chromatography using a Combiflash-Companion chromatogram (Serlabo, France).
All other chemicals and solvents were purchased from SDS (France).

Animal Material
The

Cytotoxicity Assays
A tetrazolium dye [3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium-bromide; MTT]-based colorimetric assay was used to measure the inhibition on the proliferation of various human tumor cell lines HCT-116 (human colon carcinoma), PC-3 (prostate cancer cell lines) and MCF-7 (breast cancer cell line). The tested compounds were formulated in DMSO and added to the cells such that the final DMSO concentration ranged from 1 to 3%. Cells were grown in D-MEM medium supplemented with 10% fetal calf serum (Invitrogen), in the presence of penicillin, streptomycin, and fungizone, and plated in 96-well microplates. After 24 h of growth, cells were treated with target compounds from 100 µM to 10 nM. After 72 h, MTS reagent (Promega) was added, and the absorbance was monitored (490 nm) to measure the inhibition of cell proliferation compared to untreated cells. IC 50 determination experiments were performed in separate duplicate experiments.

ASSOCIATED CONTENT
Detailed 1D and 2DNMR, MS and IR spectra of sporochartines are available free of charge via the Internet at http://pubs.acs.org.

AUTHOR CONTRIBUTIONS
CL-L: microbiologie chemistry; GL: chemistry; CD: invetebrate investigation; JO: head of the team and science manager.