Thiodiketopiperazines with two spirocyclic centers extracted from Botryosphaeria mamane, an endophytic fungus isolated from Bixa orellana L.

Phytochemistry 158 (2019) 142–148

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Thiodiketopiperazines with two spirocyclic centers extracted from Botryosphaeria mamane, an endophytic fungus isolated from Bixa orellana L.

T

Fatima Barakata, Marieke Vansteelandta,∗∗, Asih Triastutia, Patricia Jargeatb, Denis Jacqueminc, Jérôme Gratonc, Kember Mejiad, Billy Cabanillasd, Laure Vendiere, Jean-Luc Stiglianie, Mohamed Haddada, Nicolas Fabrea,∗ a

UMR 152 Pharma Dev, Université de Toulouse, IRD, UPS, France Laboratoire Evolution et Diversité Biologique UMR 5174, Université de Toulouse, CNRS, IRD, UPS, France c Laboratoire CEISAM, UMR CNRS n° 6230, University of Nantes, 2, rue de la Houssinière, 44322 Nantes, Cedex 2, France d Instituto de Investigaciones de la Amazonía Peruana, Avenida Abelardo Quiñonez Km. 4.5, Iquitos, Peru e Laboratoire de Chimie de Coordination du CNRS, Centre National de la Recherche Scientifique, 205 route de Narbonne, BP 44099, 31077, Toulouse Cedex 4, France b

A R T I C LE I N FO

A B S T R A C T

Keywords: Botryosphaeria mamane D.E. Gardner (Botryosphaeriaceae) Bixa orellana L. (Bixaceae) Thiodiketopiperazine Cytotoxic activity

Three thiodiketopiperazines, botryosulfuranols A-C (1–3) were isolated from the endophytic fungus Botryosphaeria mamane. The three compounds present sulfur atoms on α- and β-positions of phenylalanine derived residues and unprecedented two spirocyclic centers at C-4 and C-2′. Their planar structures were determined by spectroscopic analysis and absolute configurations were achieved by X-ray diffraction analysis and ECD and NMR chemical shifts calculations. Botryosulfuranol A (1) was the most cytotoxic compound against four cancer cell lines (HT-29, HepG2, Caco-2, HeLa) and two healthy cell lines (IEC6, Vero) highlighting the importance of an electrophilic center for cell growth inhibition.

1. Introduction Endophytic fungi have been recognized as a source of numerous structurally unique and biologically active natural specialised metabolites (Gunatilaka, 2006; Zhang et al., 2006). Thiodiketopiperazines (TDKPs) alkaloids are an important class of fungal compounds divided into nearly twenty distinct families and characterized by a sulfurbridged (or reduced) six-membered diketopiperazine core (Jiang and Guo, 2011; Welch and Williams, 2014). Biogenetically, TDKPs are derived from at least one phenylalanine, tyrosine and/or tryptophan (Welch and Williams, 2014). In most compounds reported previously (over a hundred), the disulfide functionality is attached in α-positions of the amino acids residues (C-2 and C-2′). In contrast, both α- and βdisulfide positions were very rarely described (Liu et al., 2015). Also, very few compounds have been reported to bear one spiro[furan-pyrazino] feature in their structure (Meng et al., 2016; Niu et al., 2017; Zhu et al., 2017). TDKPs display broad spectra of biological activities such as antibacterial (Seephonkai et al., 2006; Zheng et al., 2006), antiviral (Curtis et al., 1977; Nagarajan et al., 1968), antifungal (Kajula et al., 2016; Seya et al., 1986), immunosuppressive (Fujimoto et al., 2004;

∗

Mullbacher et al., 1986), phytotoxic (Elliott et al., 2007; Pedras et al., 1989; Pedras and Biesenthal, 2001; Wang et al., 2017) and antitumoral properties which were particularly investigated (Chen et al., 2009; Vigushin et al., 2004). It has been demonstrated that the sulfide functionalities (disulfide or polysulfide) play an essential role in the bioactivities and an opening of the bridge can lead to a reduction or even inhibition in the effect depending on the activities measured (Jiang and Guo, 2011; Zhu et al., 2017). In the course of our ongoing search for bioactive compounds from endophytic fungi, mycochemical investigation was performed on a strain of Botryosphaeria mamane D.E. Gardner (= Cophinforma mamane (D.E. Gardner) A. J. L. Phillips & A. Alves) (Botryosphaeriaceae). This species of filamentous fungus was first described by Donald E. Gardner in 1997 as a pathogenic fungus associated with witches'-brooms on Sophora chrysophylla, an endemic forest tree in Hawai (Gardner, 1997). This fungus has been then described as endophyte of the Amazonian plant Carapa guianensis Aublet (Ferreira et al., 2015) and of Garcinia mangostana (Pongcharoen et al., 2007). Very few chemical studies have been conducted to date, with only 7 compounds isolated from an endophytic strain of Garcinia mangostana: a new dihydrobenzofuran derivative (botryomaman) and

Corresponding author. Corresponding author. E-mail address: [email protected] (N. Fabre).

∗∗

https://doi.org/10.1016/j.phytochem.2018.11.007 Received 30 July 2018; Received in revised form 8 November 2018; Accepted 9 November 2018 0031-9422/ © 2018 Elsevier Ltd. All rights reserved.

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those of a thiodiketopiperazine derived from two phenylalanines represented by an aromatic ring on one hand and by a cyclohexenone on the other hand (Meng et al., 2014, 2016; Zhu et al., 2017). Investigating the east part of the molecule, key HMBC (Fig. 2 & Fig. S4) cross peaks between the protons of NMe and two quaternary carbons including the carbonyl C-1 and more specifically the sp3 hybrid carbon C-2′ resonance at 97.8 ppm, allow the construction of a spiro[furan-pyrazino] functionality and leans to a phenyl system. This rare spirocyclic skeleton located between an aromatic ring and the diketopiperazine core has recently been described in TDKPs family with δ values resonances ranged from 92 to 101 ppm for the diagnostic C-2′ (Liu et al., 2015; Meng et al., 2016). A methylthio substituent can be easily connected at C-3′ according to HMBC cross peaks observed between SMe and C-3′ and H-3′ with C-1′ (Fig. 2 & Fig. S4). Exploring the west part of 1, key HMBC couplings observed between the protons of the cyclohexenone moiety (methylene protons 5 and 6 and olefinic H-8) with quaternary carbon resonating at 87.8 ppm (C-4) suggest the arrangement of another spiro[isoxazolidino-cyclohexenone] scaffold, the isoxazolidine cycle being linked to the diketopiperazine core. The second methylthio substituent together with a hydroxyl group are located at C-2 and C-3, respectively, according to HMBC cross peaks observed between the nonexchangeable hydroxyl proton at 4.73 ppm (br s) with C-3 and thiomethyl protons at 2.42 ppm (s) with C-2 (Fig. 2 & Fig. S4). The planar structure of 1 was depicted in Fig. 1. The relative configuration can be deduced from NOESY experiments (Fig. S6). The NOE correlations of H-5/H-3/SMe-2/H-3′/H-5′ in 1 (Fig. 3) show clearly that these protons are located on the same side of the plane formed by the diketopiperazine core and suggest the relative configurations of C-4, C-3, C-2 and C-3′. The relative configuration of the spiro carbon C-2′ remains unsolved due to the lack of diagnostic correlation. Fortunately, obtaining compound 1 as a single crystal led to confirm its planar structure and allowed the determination of its unambiguous absolute configuration (2R,3R,4R,2′R,3′S) by analysis of the single-crystal X-ray diffraction data (Fig. 4). Botryosulfuranol B (2) was obtained as a white powder and assigned the molecular formula C21H24N2O6S2 according to the positive HRESIMS protonated ion [M+H]+ at m/z 465.1131 (calcd for C21H25N2O6S2 465.1149, Δ 3.7 ppm), thus possessing two additional protons compared to 1. Both the IR and UV spectra are superimposable to those of 1. In addition, the NMR data (Table 1) of these compounds are also similar except for two olefinic signals at δH 6.99/6.12 ppm and δC 149.9/129.7 ppm (CH-7/CH-8), respectively in 1 that are replaced by two methylene carbons signals at δH7 1.81/1.94, δH8 2.55/2.87 and δC 26.7/42.1 (CH2-7/CH2-8) in 2. These observations are further confirmed by the relevant HMBC and COSY cross peaks (Fig. 2, Figs. S12 and S13). Only 2 key NOESY cross peaks are observed between H-3 and SMe-2 and between SMe-2 and H-3′ without correlation between SMe-2 and SMe-3′ (Fig. 3 & Fig. S14). However, the presence of two spirocyclic centers implies a perpendicular relative position of cycles between them as depicted in the ORTEP drawing for 1 and in Fig. 3. This makes an interpretation of NOESY data quite hazardous but four plausible relative configurations (see Fig. 5) were considered. Therefore, the computation of electronic circular dichroism (ECD) data using timedependent density functional theory (TDDFT) was used to draw conclusions regarding the configuration of compound 2. To assess the quality of the level of theory selected for the modelling studies and having the unambiguous absolute configuration of very similar structure, the ECD spectra of 1 was first computed and superimposed over its experimental one (Fig. S25). As it can be seen, the general experimental trends were nicely reproduced with two positive maxima around 240 and 280 nm and a strong negative band at smaller wavelength. The ECD spectra of the four plausible configurations of 2: i) 2R,3R,4R,2′R,3′R; ii) 2R,3R,4R,2′R,3′S; iii) 2S,3S,4S,2′S,3′R and iv) 2S,3S,4S,2′S,3′S, determined at the same level of theory are depicted in Fig. 5. The X-ray diffraction structure of 1 served as starting point for these simulations and Fig. S26 gave the lowest energy conformers for

Fig. 1. Chemical structures of compounds 1–3.

six known compounds: 2,4-dimethoxy-6-pentylphenol, an alkylquinone (primin), isocoumarins (R-(-)-mellein, cis-4-hydroxymellein and trans-4hydroxymellein) and 4,5-dihydroxy-2-hexenoic acid (Pongcharoen et al., 2007). In our study, the B. mamane strain E224 has been isolated from the fresh leaves of Bixa orellana (Bixaceae), a native plant from South America, commonly known as annatto, roucou, achiote or lipstick tree (Shahid-ul-Islam et al., 2016). Seeds of this plant are mainly used topically by native people to improve the beauty of body and lips. It is also used as a dye in textile and food industry (Shahid-ul-Islam et al., 2016). Bioguided fractionation of EtOAc of B. mamane E224 led to the isolation of three undescribed TDKPs. Botryosulfuranols A (1), B (2) and C (3) present an unprecedented skeleton possessing two spirocyclic centers at C-4 and C-2′ (Fig. 1). We describe here the isolation, structure determination including stereochemical assignments and cytotoxic activities of isolated compounds. The structure of 1 was confirmed by single crystal X-ray diffraction analysis.

2. Results and discussion 2.1. Mycochemical investigation Botryosulfuranol A (1) was isolated as colourless crystals. Its molecular formula was established as C21H22N2O6S2 based on the positive HRESIMS m/z 463.0974 [M+H]+ (calcd for C21H23N2O6S2 463.0992, Δ 3.9 ppm), implying twelve degrees of unsaturation. The presence of two sulfur atoms in the structure was confirmed by the isotopic pattern, including a M+2/M ratio of 9% (Fig. S7). The IR spectrum of 1 (Fig. S8) showed diagnostic absorption bands at 3331, 1714, 1666 and 1594 cm−1, corresponding to the hydroxyl group, α,β-unsaturated ketone and two amide stretching bands, respectively. A UV spectrum with a λmax of 279 nm suggested an aromatic ring in the structure of 1. Inspection of the 13C NMR (Table 1 and Fig. S2) and HSQC spectroscopic data (Fig. S3) of botryosulfuranol A showed twenty-one carbon resonances revealing the presence of three methyls (two sulfurated and one nitrogenated), two methylenes, eight methines (including six aromatic/olefinic (C-7 and C-8) and two sulfur/oxygenated sp3) and eight quaternary carbons (with three sp3 hybrid carbons and five sp2 including two amide carbonyls). Investigation of 1H NMR (Table 1) and HSQC spectra (Fig. S3) confirmed the presence of two SMe (singlets at 2.20 and 2.42 ppm) and one N-Me (2.98 ppm), two cyclic methylenes (AB systems between 2.47 and 2.68 ppm for CH2-5 and 6), four aromatic protons (H-5′ to H-8′) between 6.96 and 7.29 ppm allowing an ortho-substituted phenyl ring (two doublets and two triplets) and two olefinic protons suggesting an enone moiety (signals at 6.99 and 6.12 ppm with a coupling constant of 10.1 Hz for H-7 and H-8, respectively). Two methine singlets linked to heteroatoms at 4.89 and 4.97 ppm as well as an exchangeable OH group exist also. At this stage, all NMR signals are in good agreement with 143

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Table 1 1 H and 13C NMR data for compounds 1–3. no.

1 (in CDCl3) δC , type a

2 (in CDCl3) δH (mult, J, Hz)

δC , type

4.89, s

162.7 C 76.2 C 84.4 CH 92.4 C 37.4 CH2

b

1 2 3 4 5

163.2 C 72.6 C 86.4 CH 87.8 C 31.2 CH2

6

24.3 CH2

7

149.9 CH

Ha 2.47, m Hb 2.56, m Ha 2.62, m Hb 2.68, m 6.99, dd (10.1, 4.1)

8

129.7 CH

6.12, dt (10.1, 2.0)

9 2-SMe 1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′ 9′ N-Me 3′-SMe 3-OH

192.4 C 14.1 CH3 159.9 C 97.8 C 57.5 CH 125.8 C 124.9 CH 122.6 CH 130.3 CH 110.2 CH 156.9 C 30.8 CH3 16.9 CH3

a b

2.42, s

4.97, s 7.29, 7.01, 7.25, 6.96,

d (7.6) td (7.6, 0.9) td (7.7, 0.9) d (8.0)

2.98, s 2.20, s 4.73, br s

a

3 (in CD3OD) δH (mult, J, Hz)

δCa, type

δHb (mult, J, Hz)

4.87, d (2.1)

165.6 C 74.8 C 86.8 CH 86.1 C 30.8 CH2

4.69, s

b

Ha 2.29, m Hb 2.43, m Ha 1.81, m Hb 2.05, m Ha 1.81, m Hb 1.94, m Ha 2.55, m Hb 2.87, m

22.7 CH2 26.7 CH2 42.1 CH2 205.2 C 14.1 CH3 161.4 Cq 97.9 C 58.3 CH 126.2 C 124.7 CH 122.6 CH 130.3 CH 110.0 CH 156.8 C 30.8 CH3 17.7 CH3

24.6 CH2

2.37, m

154.3 CH

Ha 2.65, m Hb 2.70, m 7.22, dt (10.0, 4.25)

129.9 CH

6.05, dt (10.0, 2.0)

195.5 C 2.40, s

4.96, s 7.30, 7.01, 7.24, 6.91,

d (7.6) td (7.6, 0.9) td (7.6, 1.0) d (7.9)

2.86, s 2.32, s 4.48, d (2.2)

159.7 C 97.5 C 57.2 CH 122.7 C 125.9 CH 125.0 CH 132.7 CH 111.8 CH 158.3 C 27.5 CH3

5.57, t (1.2) 7.32, 7.14, 7.43, 7.15,

dtd (7.5, 1.1, 0.5) td (7.5, 1.0) td (7.5, 1.0) ddd (7.5, 1.0, 0.5)

2.97, s

Measured at 125 MHz. Measured at 500 MHz. δ in ppm.

Fig. 2. Key COSY (bold lines) and HMBC (arrows) correlations of 1, 2 and 3.

Fig. 4. ORTEP drawing of compound 1 based on single X-ray crystallographic analysis.

configuration iv). As expected, ECD spectra of the enantiomers all-R (i) and all-S (iv) are perfect mirror images of each other. The experimental spectrum of 2 shows a positive band at ca. 280 nm, a negative band at ca. 250 nm and a more intense positive band at ca. 225 nm. Therefore, configurations i) and ii) can be straightforwardly discarded. Concerning the two last configurations, it seems reasonable to state that iv) better matches experiment as iii) leads an additional negative band at ca. 265 nm, unseen experimentally. Further simulations (conformational search and ECD obtained through Boltzmann averaging) are available

Fig. 3. Key NOESY correlations of compounds 1 and 2. 144

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Fig. 5. Experimental and computed ECD spectra for compound 2 using four “NMR-compatible" configurations.

Fig. 6. Experimental and computed ECD spectra for compound 3 for 16 plausible configurations.

in supplementary information (Fig. S27) and confirm the proposed all-S absolute configuration for 2. According to HRESIMS data, compound 3 was assigned the molecular formula C19H16N2O6S2 (m/z 433.0527, [M+H]+ Δ 0.92 ppm). A decrease of 30.0447 mass units for 3 when compared to 1 can be noted, thus corresponding to C2H6 (Δ 8 ppm). The UV and IR spectra were very similar to those of 1 and 2. In addition, the 1H and 13C-NMR data (Table 1) of 3 are superimposable to those of compound 1 except for the two SMe signals that are lacking in the NMR spectra of 3. The same is true for 2D-NMR data HMBC and COSY (Fig. 2, Figs. S20 and S21). Particularly, as observed in the HMBC spectrum of 1, the HMBC spectrum of 3 displayed the same key cross peaks for both H-3 (coupling with C-2, C-4, C-5 and C-9) and H-3′ (coupling with C-2′, C-4′, C-5′ and C-9′) thus confirming the presence of the two spirocyclic centers. According to these facts, it can be deducted a planar structure for 3 identical to that of 1 with only one difference, the absence of the two methyl groups beared by sulfur atoms thus suggesting a sulfur-bridged thiodiketopiperazine skeleton. Close examination of the NOESY spectrum of 3 (Fig. S22) does not show any diagnostic correlation allowing relative positions of various substituents beared by asymmetric carbon atoms since the observed correlations are the same as those visible on the COSY spectrum. Experimental ECD spectrum of 3 was compared to the sixteen theoretical spectra compatible with NMR data, and sterically feasible. Indeed, the disulfide bridge connecting the C-2 and C-3′ carbons involves certain configuration constraints due to the C-2′ spiro carbon which places the furan and diketopiperazine cycles perpendicular to each other as depicted in the ORTEP drawing of 1 (Fig. 4). This fact implies that configurations at C-2 and C-2′ for 3 must be the same in order to position the C-2 and C-3′ carbons (bearing the sulfur atoms) on the same side with respect to the diketopiperazine ring because the disulfide bond cannot cross the plane of this ring. Fig. 6 shows the sixteen ECD spectra over 32 possibilities corresponding to the sixteen possible diastereoisomers (8 for 2S,2′S and 8 for 2R,2′R) due to the above-mentioned steric constraints. When compared to experimental CD of 3 (Fig. 6), no doubt remains concerning the R configurations at C2 and C-2′ regarding the prominent negative Cotton effect near 250 nm for the 8 2R,2′R ECD traces (solid lines). Concerning the remaining 8 possibilities, no clear solution seems to emerge even if the simulated spectrum of the 2R,3R,4R,2′R,3′S derivative (red line), seems to be the most similar to the experimental spectrum due to a slight negative Cotton effect close to 220 nm. In order to confirm this assumption, Gauge-Independent Atomic Orbital (GIAO) NMR chemical shifts calculations supported by the advanced statistical method DP4 (Smith and

Goodman, 2010) was performed on the 8 possibilities let unresolved after ECD calculations. In this approach, the differences between ab initio computed 13C and 1H NMR scaled chemical shifts and experimental data are considered. Then, the probability of the error in each chemical shift of each configuration is computed using Student t-test. The product of these probabilities divided by the sum of the probabilities gives the DP4 probability. In recent years, the Smith and Goodman DP4 method was widely used to confirm absolute configurations of natural products (Cooper et al., 2018; Novaes et al., 2015; Smith and Goodman, 2010). Therefore, the DP4 probability applied on the 8 possible isomers of 3, unambiguously confirmed the 2R,3R,4R,2′R,3′S configuration as the correct diastereoisomer with high confidence level. Indeed, histograms displaying the differences between scaled and experimental for 13C (Fig. S28) and 1H (Fig. S29) chemical shifts led to calculate probabilities of 96.99, 99.13 and 99.97% for 13C-, 1 H- and combined 1H-13C-NMR chemical shifts, respectively. Therefore, Botryosulfuranol C (3) was identified as the disulfur-bridged derivative of 1 with the same absolute configuration.

2.2. Cytotoxic effects against cancerous and non-cancerous cell lines Compounds 1–3 were evaluated for cell-growth inhibition of four cancer cell lines (human hepatocellular carcinoma HepG2, human colon adenocarcinoma HT29, human colorectal adenocarcinoma Caco2 and human cervical adenocarcinoma HeLa) and two healthy cell lines (monkey kidney epithelial Vero and rat small intestine epithelial IEC6). Doxorubicin was used as positive control. Table 2 (and Fig. S30) clearly shows that, first at all, none of the compounds or positive control has a selective action against cancer cells when compared to healthy lines. Table 2 GI50a values (μM) of compounds 1–3.

HepG2 HT29 Caco-2 HeLa IEC6 Vero

b

145

1

2

8.0 ± 2.4 11.4 ± 1.2 23.0 ± 0.8 18.2 ± 0.0 23.5 ± 0.6 9.3 ± 2.3

63.2 ± 56.1 ± > 100 61.2 ± 49.9 ± 64.7 ±

a GI50 is the concentration Dox = doxorubicin.

15.2 4.7 4.9 3.6 17.4

that

3

Doxb

15.9 ± 3.7 31.1 ± 2.4 > 100 115.7 ± 0.0 41.5 ± 36 65.4 ± 11.9

0.50 0.22 1.98 0.97 0.64 1.60

inhibited

50%

of

cell

± ± ± ± ± ±

0.03 0.02 0.17 0.08 0.11 0.23

growth.

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removed by filtration on regenerated-cellulose 0.45 μm filters (Whatman). All EtOAc soln. were combined and evaporated to dryness under reduced pressure to afford a brown crude extract (6 g). The EtOAC crude extract (6 g) was subjected to MPLC (Kieselgel 60, 15–40 μm) eluted with a gradient of CH2Cl2/MeOH (100:0-0:100 v/v) to give 40 fractions (F1 to F40). The pooled F14/15 (344 mg) was applied to a silica gel CC (Kieselgel 60, 40–63 μm) eluted with a gradient of CH2Cl2/MeOH (100:0-0:100 v/v) to give 8 fractions (F14/15.1 to F14/15.8). Fraction 16 (300 mg) was subjected to semi-prep. HPLC (eluted by a 0.1% formic acid MeCN/0.1% formic acid in water gradient 65:35-10:90 v/v) to obtain compounds 1 (9.0 mg, Rt = 14.5 min) and 2 (6.8 mg, Rt = 16.5 min). F14/15.6 (39 mg) was subjected to semiprep. HPLC (eluted by a 0.1% formic acid MeCN/0.1% formic acid in water gradient 65:35-5:95 v/v) to afford compound 3 (5.4 mg, Rt = 18.2 min). Botryosulfuranol A (1): colorless crystals; mp 168-172 °C with degradation; [α]20 D +76.0 (c 2.5 g/L, MeOH); UV (MeOH) λmax (log ε) 203 (4.58), 229 (4.2) nm, 279 (3.54), 286 (3.53) nm; CD {MeOH, λ [nm] (Δε), c = 0.025 × 10−3 M}: 280 (+3.45), 240 (+3.79) nm; IR νmax 3331,1714, 1666, 1594 cm−1; 1H and 13C NMR data, see Table 1; HRESIMS m/z 463.0974 [M + H]+ (calcd for C21H23N2O6S2, 463.0992). Botryosulfuranol B (2): white amorphous powder; [α]20 D +13.7 (c 1.83 g/L, MeOH); UV (MeOH) λmax (log ε) 203 (4.64), 215 (4.34), 279 (3.57) nm, 286 (3.55) nm; CD {MeOH, λ [nm] (Δε), c = 0.025 × 10−3 M}: 280 (+2.14), 250 (−1.21), 225 (+4.54) nm; IR νmax 3417,1730, 1670, 1594 cm−1; 1H and 13C NMR data, see Table 1; HRESIMS m/z 465.1131 [M + H]+ (calcd for C21H25N2O6S2, 465.1149). Botryosulfuranol C (3): white amorphous powder; [α]20 D +34.9 (c 3.85 g/L, MeOH); UV (MeOH) λmax (log ε) 203 (4.82), 220 (4.5) nm, 278 (3.67), 284 (3.62) nm; ECD {MeOH, λ [nm] (Δε), c = 0.1 × 10−3 M}: 297 (+0.50), 281 (−1.02), 249 (−5.56) nm; IR νmax 3408,1736, 1685, 1595 cm−1; 1H and 13C NMR data, see Table 1; HRESIMS m/z 433.0527 [M + H]+ (calcd for C19H17N2O6S2, 433.0523). Crystal data of 1: C21H22N2O6S2, Mr = 462.75, Triclinic space group, P 1, unit cell dimensions a = 9.6618(6) Å, b = 12.0903(8) Å, c = 18.3402(13) Å, V = 2091.3(2) Å 3, α = 99.974(3), β = 97.503(3),γ = 90.087(3), Z = 4, d calcd = 1.469 mg/m3, crystal dimensions 0.16 × 0.03 × 0.02 mm, μ = 0.297 mm −1, F(000) = 968. 79820 reflections were measured, 15358 independent reflections were applied. The final refinement gave R 1 = 0.049 and wR 2 = 0.112 [I > 2σ (I)]. The absolute structure parameter (Flack parameter) was 0.05 (3). Crystallographic data for compound 1 has been deposited at the Cambridge Crystallographic Data Center, with deposition N° 1851532.

Generally, the cytotoxicities of 1–3 are weak to moderate but botryosulfuranol A (1) is always the most active whatever the cell line tested here, thus revealing the importance of α,β-unsaturated ketone and an open sulfur bridge depending on the tested cell line. This observation is particularly marked for the Caco-2 cells which proved to be the most resistant of the four cancerous lines used in this study. 3. Conclusions In conclusion, botryosulfuranols A-C (1–3) with an unprecedent spiro[isoxazolidino-cyclohexenone] scaffold were isolated from Botryosphaeria mamane E224 strain, an endophytic fungus isolated from the leaves of Bixa orellana. Preliminary in vitro cytotoxic evaluation highlights the importance of electrophilic center for inhibition of cancer cells growth. 4. Experimental section 4.1. General experimental procedures Melting points (mp) were determined with an Electrothermal IA 9200 melting-point apparatus. Optical rotations were measured on a JASCO P-2000 polarimeter. UV and ECD spectra were recorded on a JASCO J-815 spectropolarimeter. IR spectra were taken on a Bruker Perkin Elmer FT-IR/FIR spectrometer.1D and 2D NMR spectra were recorded on a Bruker AVANCE 500 NMR spectrometer operating at 500 MHz for 1H and 125 MHz for 13C, using TMS as internal standard and chemical shifts were recorded as δ-values. Mass spectra were obtained on a Thermo Scientific LTQ Orbitrap XL mass spectrometer. Semipreparative HPLC was performed on Phenomenex EVO C18 column (250 × 10 mm, 5 μm, 3 mL/min). Medium-pressure liquid chromatography (MPLC) was performed on BUCHI Pump Module C601(BUCHI Labortechnik AG, Flawil, Switzerland). Column chromatography (CC) was performed with silica gel (40–63 μm, Merck KGaA, Darmstadt, Germany). 4.2. Fungal material The Botryosphaeria mamane D.E. Gardner E224 (Botryosphaeriaceae) strain was isolated as an endophytic fungus from the fresh leaves of Bixa orellana L. (Bixaceae), collected in Peru in November 2013 (end of the dry season) in Iquitos (national reserve of Allpahuayo Mishana, Amazonian rainforest, GPS coordinates: 3°58′02.3 S, 73°25′03.9 W). Leaves surface was sterilized with 70% EtOH for 2 min followed by treatment with 4% sodium hypochlorite (2 min) then sterilized with 70% EtOH (2 min) and finally washed with sterile distilled water. Sterile leaves were placed on a Petri dish and left under the laminar air flow (LAF) to dryness. The tissues were then cut into 5 mm pieces and deposited on Malt Extract Agar (MEA) plates containing chloramphenicol (100 μg/mL). The fungus was identified based on the ITS sequence (GenBank accession number: MG457709). The voucher specimen is deposited in UMR152 laboratory at −80 °C under the number E224. Working stocks were prepared on Malt Extract Agar cryotubes stored at 4 °C.

4.4. Computational details for ECD and GIAO NMR chemical shifts calculations All DFT and TD-DFT calculations were performed using the Gaussian16.A03 program (Frisch et al., 2016). The calculations of the ECD spectra consisted in three successive steps. First, we optimized the ground-state geometries of the anticipated enantiomers at the B3LYP/6311G(d,p) level (Becke, 1993). These calculations used the so-called tight optimization threshold, as well as improved DFT integration grids (the ultrafine grid was selected) and SCF convergence thresholds (10−10 au). The bulk solvent effects (here methanol) were accounted for using the Polarizable Continuum Model (PCM) (Tomasi et al., 2005) and selecting the Gaussian16 default settings to build the cavity. In a second stage, the vibrational frequencies have been evaluated at the same level of theory to confirm the nature of the obtained structures (absence of imaginary frequency). In a third step, TD-DFT calculations were performed at the B3LYP/6-311 + G(2d,p) level in order to model the ECD spectra of the envisaged diastereoisomers. These calculations included

4.3. Fermentation, extraction and isolation Agar plugs of B. mamane E224 were used to inoculate 250 × 250 mL Erlenmeyer flasks, each containing 50 mL of MEA culture medium (prepared with 20 g malt extract, 20 g glucose, 20 g agar, 1 g peptone, 0.005 g CuSO4, 5H2O, 0.01 g ZnSO4, 7H2O per L of distilled water). Flask cultures were incubated under static conditions and natural light at 27 °C for 14 days. After cultivation, each fungal culture (biomass and medium) was extracted twice with 100 mL of EtOAc at room temp. in shaking water bath for 1 h, followed by 30 min sonication in an ultrasonic bath. Organic phases were dehydrated on MgSO4. Spores were 146

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the 25 lowest single excited-states and used the PCM model in its linearresponse non-equilibrium form, which is suited for modelling absorption and ECD spectra (Jacquemin and Adamo, 2015). The presented spectra have been convoluted, from the individual vertical contributions, using a Gaussian with of half-width at half-minimum of 0.2 eV. Concerning the determination of DP4 probabilities, the following protocol was performed: each of the eight isomers was first subjected to a quenched molecular dynamics conformational search at high temperature (1000 K) using the MMFF94 force-field as implemented in TINKER software tools. For each configuration, the resulting geometries were minimized and all conformers within 10.0 kcal mol−1 of the lowest energy were subjected to further re-optimization at the B3LYP/6-31G** level of theory, in the gas phase. The magnetic shielding constants (σ) were computed using the gauge-including atomic orbitals (GIAO) method (Barone et al., 2002; Schreckenbach and Ziegler, 1995), with the B3LYP/6-31G** level of theory. The resulting shielding tensors were averaged using the Boltzmann distribution. The chemical shifts were calculated from TMS as reference standard. The systematic errors were removed by scaling according to δscaled = (δcalc – Intercept)/Slope, where Intercept and Slope, result from a linear regression calculation on a plot of δcalc against δexp (Barone et al., 2002). The DP4 probabilities were then computed as originally described by Smith and Goodman (2010)

We thank the Faculty of Pharmacy of Lebanese University for financial support. Strain Botryosphaeria mamane E224 was obtained with the appropriate legal permissions (authorization n°010-2017SERNANP-RNAM-JEF) and with a revenue-sharing agreement in place. This work used the computational resources of the CCIPL and CALMI installed in Nantes and in Toulouse, respectively. The ‘Institut de Chimie de Toulouse’ (ICT) is greatly acknowledged for NMR, CD, and XRay crystallography facilities. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.phytochem.2018.11.007. References Barone, G., Gomez-Paloma, L., Duca, D., Silvestri, A., Riccio, R., Bifulco, G., 2002. Structure validation of natural products by quantum-mechanical GIAO calculations of 13C NMR chemical shifts. Chem. Eur. J. 8, 3233–3239. https://doi.org/10.1002/ 1521-3765(20020715)8:14<3233::AID-CHEM3233>3.0.CO;2-0. https://doi.org/. Becke, Axel D., 1993. Density-functional thermochemistry. III. The role of exact exchange. J. Chem. Phys. 98, 5648–5652. https://doi.org/10.1063/1.464913. Chen, Y., Guo, H., Du, Z., Liu, X.-Z., Che, Y., Ye, X., 2009. Ecology-based screen identifies new metabolites from a cordyceps-colonizing fungus as cancer cell proliferation inhibitors and apoptosis inducers: gliocladicillins as antitumor agents. Cell Prolif 42, 838–847. https://doi.org/10.1111/j.1365 2184.2009.00636.x. Cooper, J.K., Li, K., Aubé, J., Coppage, D.A., Konopelski, J.P., 2018. Application of the DP4 probability method to flexible cyclic peptides with multiple independent stereocenters: the true structure of cyclocinamide A. Org. Lett. 20, 4314–4317. https:// doi.org/DOI:10.1021/acs.orglett.8b01756. Curtis, P.J., Greatbanks, D., Hesp, B., Cameron, A.F., Freer, A.A., 1977. Sirodesmins A, B, C, and G, antiviral epipolythiopiperazine-2,5-diones of fungal origin: X-Ray analysis of sirodesmin A diacetate. J. Chem. Soc. 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4.5. Cytotoxic assay Caco-2 (Homo sapiens colon colorectal adenocarcinoma), HT29 (Homo sapiens small intestine normal), IEC6 (rattus norvegicus small intestine normal), HeLa (Homo sapiens cervix adenocarcinoma), Vero (Cercopithecus aethiops kidney normal) and HepG2 (Homo sapiens liver hepatocellular carcinoma) cell lines were purchased from the American Type Culture Collection (ATCC® HTB-37™, ATCC® HTB-38™, ATCC® CRL-1592™, ATCC®-CCL-2™, ATCC®-CCL-81™ and ATCC®-HB-8065™, respectively, USA). Caco-2, HT-29 and IEC6 cells were cultured at 37 °C, 5% CO2, in Dulbecco's Modified Eagle's Medium (DMEM) High glucose supplemented with 5% foetal calf serum, and penicillin/streptomycin (100 U/mL) and supplemented with 10% NEAA 1X for Caco-2 cells. HeLa cells were cultured at 37 °C, 5% CO2, in Modified Eagle's Medium (MEM) supplemented with 5% foetal calf serum, and penicillin/streptomycin (100 U/mL). Vero cells were cultured at 37 °C, 5% CO2, in Dulbecco's modified Eagle's medium (DMEM) supplemented with 5% foetal calf serum, 100 U/mL penicillin, 100 μg/mL streptomycin and 10% NEAA 1X for. HepG2 cells were cultured at 37 °C, 5% CO2 in Roswell Park Memorial Institute (RPMI) 1640 medium (Gibco®, Life Technologies, USA), supplemented with 10% fetal bovine serum, and penicillin/streptomycin (100 U/mL). For the anti-proliferative assays, cells were cultured in the same conditions but without antibiotics. The medium was renewed twice a week. Caco-2, HT29, IEC6, HeLa cells under exponential growth were seeded in 96-well plates at a density of 105 cells per well. Vero and HepG2 cells under exponential growth were seeded in 96-well plates at a density of 104 cells per well. After overnight growth, cells were treated with various conc. of compounds for 72 h. The conc. tested ranged from 1.95 to 250 μg/mL. Then, the cells were washed with phosphate-buffered saline (Gibco®, Life Technologies, USA), and incubated with 3-(4, 5-dimethylthiazol-2-yl)2,5-diphenyl tetrazolium bromide (MTT) (Sigma Aldrich, USA) at a conc. of 0.5 mg/mL for 4 h. Next, DMSO (100 μL/well) was added into each well and the plates were read at 570 nm using a microplate reader (EON, BioTek, USA). The cell growth and inhibition rate were calculated and GI50 values determined using Graph Pad Prism version 7 software (Graph Pad, USA). For each set of experiments, a positive control (Doxorubicine) was used to induce 100% cell death. All experiments were performed in triplicate (Mahlo et al., 2013).

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