Expanding the chemical diversity of an endophytic fungus Bulgaria inquinans, an ascomycete associated with mistletoe, through an OSMAC approach

An endophytic fungus Bulgaria inquinans (isolate MSp3-1), isolated from mistletoe (Viscum album), was subjected to fermentation on solid Czapek medium. Chromatographic workup of the crude EtOAc extract yielded five new natural products (1–5). Subsequent application of the “One Strain, MAny Compounds” (OSMAC) strategy on this strain by the addition of a mixture of salts (MgSO4, NaNO3 and NaCl) to solid Czapek medium induced the accumulation of nine additional new secondary metabolites (6–13, 16), with most of them (8, 10–12) not detectable in cultures lacking the salt mixture. The structures of the new compounds were established on the basis of the 1D/2D NMR and HRESIMS data. The TDDFT-ECD method was applied to determine the absolute configurations of the new compounds 1, 4 and 6 as well as of the previously reported bulgarialactone B (14), for which the absolute configuration was unknown so far. The modified Mosher's method was performed to assign the absolute configurations of 12 and 13. TDDFT-ECD analysis also allowed determining the absolute configuration of (+)-epicocconone, which had an enantiomeric absolute configuration in the tricyclic moiety compared to that of bulgarialactone B (14). All the isolated metabolites were evaluated for their cytotoxic activity. Compound 2 was found to possess strong cytotoxic activity against the murine lymphoma cell line L5178Y with an IC50 value of 1.8 μM, while the remaining metabolites were shown to be inactive.


Introduction
Fungi are well-known producers of novel drug leads, as exem-plied by the fascinating discovery of beta-lactam antibiotics, cyclosporin A, caspofungin, lovastatin and ngolimod. 1-3 In particular, endophytic fungi, which live asymptomatically within plant tissues, have been recognized for their capability to produce therapeutically interesting natural products. 4 Remarkable examples include the antimycotic natural products cryptocandin 5 and cryptocin, 6 the insecticidal compound nodulisporic acid A, 7 the mitochondrial toxin phomoxanthone A 8,9 as well as the immunosuppressant diterpene pyrones subglutinols A and B, 10 among others. Interestingly, isolation of the important anticancer agents, paclitaxel from Taxomyces andreanae, an endophyte of Taxus brevifolia, 11 and camptothecin produced by Entrophospora infrequens, a fungus associated with Nothapodytes foetida, 12 as well as the identication of lycopodine-type alkaloids recently detected in an UV-irradiated strain of Paraboeremia, a fungal endophyte of Lycopodium serratum var. longipetiolatum, 13 highlights the special importance of endophytes as a reservoir of metabolites previously known only from the host plant. 14,15 Gene clusters involved in the biosynthesis of fungal secondary metabolites oen remain silent under standard laboratory culture conditions, leading to a frequent rediscovery of known metabolites. 16,17 To overcome this problem, several strategies for enhancing the biosynthetic potential of fungi can be applied. One of them is the OSMAC (One Strain, MAny Compounds) approach, 18 a powerful experimental method used to enhance the chemical diversity of microorganisms using the selective modication of the fermentation parameters, such as the media type and composition, the physical parameters (pH value, temperature, aeration conditions), the addition of enzyme inducers/inhibitors and chemical elicitors. [16][17][18][19] Even a difference in water quality in preparing the culture media, such as exchanging tap water for distilled water, was reported to inuence the pattern of the main metabolites of Paraphaeosphaeria quadriseptata, due to the presence of traces of metal ions (Cu 2+ , Cd 2+ and Cr 3+ ) in tap water. 20 Successful application of the OSMAC approach on the fungal endophyte, Dothideomycete sp. CRI7 by changing the medium type (PDB vs. Czapek malt medium) as well as by using different nutrient sources (potato for PDB medium and malt extract for Czapek malt medium) resulted in distinct secondary metabolite production of this fungus. 21,22 Interestingly, a series of studies have reported the isolation of halogenated natural products from fungal cultures grown on media to which different halide salts had been added, thus highlighting the fungal capability to utilize different halogen sources when present in the media. [23][24][25][26] In addition, supplementing media with trace elements, e.g. the addition of CuCl 2 to cultures of Pestalotiopsis sp. Z233 or the addition of ZnSO 4 to cultures of Aspergillus clavatus, induced the production of new sesquiterpenes possessing tyrosinase inhibitory activity, 27 and the production of a new metabolite, clavatustide C, 28 respectively.
As part of our ongoing studies aimed at inuencing the biosynthetic capacity of endophytic fungi utilizing the OSMAC approach, we investigated the metabolic proles of Bulgaria inquinans (isolate MSp3-1), an ascomycete fungus isolated from sprouts of common mistletoe (Viscum album). Previous chemical investigations of this fungus resulted in the isolation of azaphilone pigments, namely bulgarialactones A-D, 29,30 of which bulgarialactones A and B exhibited antimicrobial, cytotoxic and nematocidal activities. 29 Moreover, quinones containing a benzouoranthenequinone nucleus, bulgarhodin and bulgarein 31 as well as cytotoxic anthraquinone dimers, bulgareones A and B, 32 were isolated from this fungus.
In the present study, we report the isolation and structure elucidation of 14 new natural products, including ve new natural products (1-5) derived from B. inquinans cultured on solid Czapek medium and nine new compounds (6)(7)(8)(9)(10)(11)(12)(13)16) isolated from this fungus when grown on solid Czapek medium with the addition of a salt mixture (MgSO 4 , NaNO 3 and NaCl). Furthermore, determination of the absolute congurations of the new compounds and of the known derivative, bulgarialactone B (14), is described herein. The cytotoxicity assay results of the isolated natural products are briey discussed.

Results and discussion
Chemical investigation of B. inquinans grown on solid Czapek medium resulted in the isolation of ve new natural products, namely butyrolactones (1-3, 5) and a new metabolite bearing a diol moiety (4) together with known compounds, such as the azaphilone pigment bulgarialactone B (14) 29 as well as phenylbutyrolactone IIa (15) 33,34 and xenofuranone B (17) 35 (Fig. 1). Aer this fermentation, we studied the chemical proles of the fungus when grown in the presence of different salts that had been added to solid Czapek medium. B. inquinans was cultured on solid Czapek medium following the addition of either NaCl, NaBr, NaI, NaNO 3 , or (NH 4 ) 2 SO 4 (3.5 g of each) or following the addition of salt mixtures: (a) MgSO 4 $7H 2 O, NaNO 3 and NaCl (2.5 g each), (b) FeSO 4 $7H 2 O, NaNO 3 and NaCl (2.5 g each) or (c) ZnSO 4 $7H 2 O, NaNO 3 and NaCl (2.5 g each), as described in the Experimental section. B. inquinans failed to grow on media containing either (NH 4 ) 2 SO 4 , or mixtures (b) and (c). No changes in the chromatographic proles were observed following the addition of either NaCl, NaBr, NaI or NaNO 3 to solid Czapek medium when compared to chromatograms of the fungus grown on the medium without salts. However, the presence of a mixture of MgSO 4 , NaNO 3 and NaCl in the medium resulted in a signicant change in the metabolite prole of B. inquinans, as indicated by HPLC-DAD analysis (Fig. S1, ESI †). Chromatographic workup of this fungal extract led to the isolation of nine new secondary metabolites (6)(7)(8)(9)(10)(11)(12)(13)16), including a butyrolactone derivative 6, two unusual 1,3-oxazine containing natural products (7 and 8), ve new a-pyrones (9-13) and (À)-(S)-avipesin B (16), together with the known compound bulgarialactone D (18) 30 (Fig. 1). Compounds 9, 13 and 18 were subsequently also detected (albeit in minor amounts) in HPLC chromatograms of the fungus following cultivation on solid Czapek medium without the addition of salts. The OSMAC approach nevertheless enhanced their production, enabling isolation and structural characterization of these compounds. Moreover, the accumulation of compounds 8 and 10-12 was only induced in the presence of the salt mixture, whereas these latter compounds were not detected in fungal cultures lacking salts.
Compound 1 was obtained as a yellow solid. Its UV spectrum displayed a maximum absorption at 288 nm, which was characteristic for butyrolactone-type metabolites. 36 -9), implying the presence of a mono-substituted benzene ring. Moreover, the 13 C NMR data of 1 (Table 1) displayed signals of a carbonyl carbon at d C 170.3 (C-2), two sp 2 carbons at d C 140.6 (C-3) and 131.2 (C-4), and one oxygenated sp 3 carbon at d C 85.2 (C-5), attributed to a conjugated ve-membered lactone ring. The HMBC correlations observed from H-7/H-11 to C-4 and from H 3 -12 to C-4, C-5 and C-1 0 (Fig. 2) provided the connections of the aromatic ring and of the methyl group to the butyrolactone ring as well as the attachment of the carboxylic acid group to C-5.
In order to elucidate the absolute conguration of 1, the TDDFT-ECD protocol was performed on the arbitrarily chosen (S) stereoisomer. B3LYP/6-31+G(d,p) and CAM-B3LYP/TZVP PCM/MeCN reoptimization of the initial 13 MMF conformers resulted in three and eight low-energy conformers over a 1% Boltzmann-population, respectively. ECD spectra computed at various levels for both sets of conformers effectively reproduced the experimental ECD spectrum of 1 (Fig. 3), allowing the elucidation of the absolute conguration as (S). Accordingly, the structure of 1 was established as a new natural product, for which the name bulgariline A is proposed (Fig. 1).
The molecular formulae of 2 and 3 were assigned as C 17 H 14 O 4 and C 18 H 16 O 4 , respectively, based on their prominent pseudomolecular ion peaks in the HRESIMS spectra. The 1 H and 13 C NMR data of 2 (Table 1) were almost identical to those of phenylbutyrolactone IIa (15), except for the replacement of the carboxylic acid functionality with a hydroxyl group, as indicated by the absence of a carbonyl signal in the 13 C NMR spectrum, as well as a 28 amu difference in the molecular weight of 2 compared with that of phenylbutyrolactone IIa (15). The NMR data of 3 (Table 1) were in good agreement with those of 2, apart from the presence of a methoxy signal at d H 3.24 (d C 50.9). Accordingly, a methoxy substituent was assigned to C-5 instead of a hydroxyl group as in 2, based on the evident HMBC correlation from 5-OMe to C-5. Thus, 2 and 3 were elucidated as new butyrolactone derivatives and were named as bulgarilines B and C, respectively. The zero value of their specic rotations indicated that 2 and 3 were obtained as racemic mixtures.
The molecular formula of 4 was determined as C 15 H 16 O 3 based on its HRESIMS data, implying 8 degrees of unsaturation. Investigation of its 1 H NMR data (Table 2) revealed typical signals of phenyl and p-hydroxy phenyl moieties in the structure   , which permitted the assignment of a 1,2-propanediol moiety. The HMBC correlations from H 2 -7 to C-3, C-4, C-5, C-8 and C-9 and in turn from H-9 to C-10, C-11 and C-15 established the connectivity of the partial structures. The relative conguration of 4 was deduced to be erythro, based on the large coupling constant value between the two vicinal methines at positions 8 and 9 (5.8 Hz), while a smaller value (J ¼ 2.5 Hz) is suggested for threo, as previously reported for related vicinal diols. 37,38 Hence, the structure of 4 was elucidated and the trivial name bulgariol is suggested for this compound. DFT reoptimization of the initial 41 MMFF conformers of (8R,9S)-4 resulted in 15 and 22 low-energy conformers over a 1% Boltzmann-population. Despite the exibility of the molecule and the substantially different ECD spectra of the individual low-energy conformers, the Boltzmann-averaged ECD spectra computed at various levels for both sets of conformers gave moderate to good mirror-image agreement with the experimental ECD spectrum (Fig. 4). Furthermore, the sign of the highest-wavelength ECD transition was the same for all the conformers over a 1.2% Boltzmann-population, allowing elucidation of the absolute conguration as (8S,9R).
Compound 5 had the molecular formula C 17 H 12 O 3 , requiring 12 degrees of unsaturation. The 1 H and 13 C NMR data of 5 (Table 1) were similar to those of 2 and 3. However, an additional olenic signal appeared at d H 5.97 (H-12), which provided HMBC correlations to C-4, C-5, C-13, C-14/C-18, along with a long-range correlation to C-2. Thus, the position of the double bond was deduced at D 5 (12) , contributing to one additional degree of unsaturation of 5 compared to 2 and 3. The geometry of the exocyclic double bond C-5/C-12 in 5 was determined based on the NOESY spectrum. The NOE correlations observed between H-12 with H-7/H-11 implied the close proximity of H-12 to the aromatic ring, thus conrming the Z conguration of the double bond. 39,40 Compound 5, (5Z)-3-hydroxy-4-phenyl-5-(phenylmethylene)-2(5H)-furanone, was reported previously only as a synthetic product. 41 In the present study, we report 5 for the rst time as a natural product and provide its complete NMR data ( Table 1).
The molecular ion of 6 was 14 amu larger than that of 1, as indicated by the HRESIMS spectrum, corresponding to the molecular formula C 13 H 12 O 5 . The 1 H and 13 C NMR data of 6 (Table 1) were in perfect agreement with those of 1, except for the additional methoxy group signal at d H 3.76/d C 53.9, which showed the respective HMBC correlation to C-1 0 , hinting at a substitution of the carboxylic acid group with a carbomethoxy attached to C-5 in the structure of 6. Finally, 6 was elucidated as a methyl ester of bulgariline A to which the trivial name bulgariline D is given.   The absolute conguration of 6 was concluded to be the same as that of 1 on the basis of biogenetic considerations, similar positive values of their specic rotation, [a] 20 D +16 for 1 and [a] 25 D +26 for 6, as well as the identical ECD spectra of 1 and 6. Furthermore, TDDFT-ECD calculations performed on the arbitrarily chosen (R) enantiomer gave mirror-image agreement (Fig. S106, ESI †), verifying the (S) absolute conguration of 6. It is interesting to compare the ECD spectra of 1 and 6 with those of the related compounds chaetobutenolide C, WF-3681 and WF-3681 methyl ester described by some of us in 2017 and where the carboxylic acid/ester group is attached through two carbons to the central unit. 36 The positive transition at ca. 260-280 nm, which is stronger in two recent cases, and the negative one around 220 nm, belonging to the homochiral derivatives, becomes more complex in 1 and 6, but they are still in accordance with the two characteristic bands. Moreover, 6 was proven to be a true natural product, by incubating 1.0 mg of 1 in 1.0 mL MeOH containing 0.1% formic acid at room temperature for 1 week, which did not result in methylation, as indicated by HPLC analysis.
Nitrogen-containing metabolites (7 and (Table 3) revealed the resonances of four aromatic protons, three sets of methylenes and one methoxy group. The COSY correlations of the aromatic protons from H-9 until H-12 suggested a 1,2-disubstituted aromatic ring for 7, as corroborated by HMBC correlations from H-9 to C-11 and C-13 as well as from H-12 to C-8 and C-10. The last spin system observed in the COSY spectrum between d H 2.60/2.67 (H 2 -3) and 2.30/2.54 (H 2 -4) afforded HMBC correlations from H 2 -3 to C-2 and C-5 and from H 2 -4 to C-5 and C-1 0 , respectively. Additionally, the HMBC correlation from 1 0 -OMe to C-1 0 established the substructure of an oxoproline residue bearing a 5-carbomethoxy group (Fig. 2). Further HMBC correlations from the methylene at d H 4.96/5.10 (H 2 -7) to C-5, C-8, C-9 and C-13 established the linkage between these two substructures, which accounted for the last degree of unsaturation in 7. The 1 H and 13 C NMR data of 8 were in a good agreement with those of 7, apart from the absence of an OMe signal, which is in accordance with the 14 amu difference in the molecular weight of 8 compared to 7, thus indicating a carboxylic acid group attached at C-5, instead of a carbomethoxy function. Accordingly, 7 and 8 possess unusual heterocyclic structures containing a 1,3-oxazine nucleus, which rarely occurs in natural products. A few examples of natural 1,3-oxazines include the antimycobacterial oxazinin A, isolated from the Eurotiomycetes strain 110162, 42 as well as salinazinones A and B from the bacterial strain Streptomyces sp. KMF-004. 43 The latter two compounds have been also reported from the marine-derived bacterium Streptomyces spinoverrucosus as spinoxazines A and B. 44 However, compared to the aforementioned natural products, the structures of 7 and 8 lack a keto function in the 1,3oxazine core, which is mostly attributed to synthetic compounds. 45,46 The trivial names bulgarixines A (7) and B (8) were assigned to these compounds. Considering the possibility that 7 might arise as an artifact of its non-methoxylated analogue (8) during the isolation process, an experiment was carried out by incubating 0.5 mg of 8 in 0.5 mL MeOH containing 0.1% formic acid for 1 week at room temperature. HPLC analysis showed that no methylated product was present in the chromatogram, thus indicating that 7 is a natural product. The baseline ECD curves of both 7 and 8 indicated that both of them were isolated as a racemate.
Compound 9 was afforded as a brown solid. Its molecular formula was determined as C 17 H 28 O 5 on the basis of its HRE-SIMS spectrum, requiring 4 degrees of unsaturation. Its UV  -5). COSY correlations observed from H-1 0 to H 3 -9 0 allowed the assignment of a 1-nonanol side-chain (Fig. 2). The HMBC correlations from H-5 to C-3, C-4 and C-6, as well as from H 2 -1 00 to C-2, C-3 and C-4, conrmed the presence of a conjugated a-pyrone nucleus in the structure of 9. Additionally, the position of two methoxy groups (4-OMe and 1 00 -OMe) as substituents of the a-pyrone ring was deduced based on their respective HMBC correlations. Finally, the presence of HMBC correlations from H-5 to C-1 0 and in turn from H-1 0 to C-5 and C-6, indicated the connection of the 1-nonanol side-chain with the a-pyrone nucleus at C-6 ( Fig. 2). The structure of 9 is closely related to dothideopyrone B, which was isolated from the endophyte Dothideomycete sp. LRUB20, 49 and with dothideopyrone F, a recently reported a-pyrone from the endolichenic fungus Dothideomycetes sp. EL003334. 47 However, 9 features two extra methylene protons in the saturated aliphatic chain in comparison with dothideopyrone B, 49 and an additional OMe at C-1 00 in comparison with dothideopyrone F. 47 Therefore, compound 9 was elucidated as a new a-pyrone derivative, for which the trivial name bulgariapyrone A is suggested. Compounds 10-12 exhibited similar UV spectra as 9, indicative of pyrone derivatives (see ESI †). Their molecular formulae were determined as C 18 H 28 O 7 , C 19 H 30 O 7 and C 17 H 28 O 6 , respectively, as indicated by their HRESIMS spectra. The 1 H and 13 C NMR data of 10-12 (Table 4) were similar to those of 9 and indicated that 10-12 retained the same a-pyrone core structure as 9, differing from the latter only in the nature of their aliphatic side-chains. The molecular weight of 10 was 44 amu larger than that of 9, which together with an additional methoxy signal at d H 3.65/d C 51.9 and an additional carbonyl resonating at d C 176.0 (C-9 0 ), implied that the aliphatic chain of 10 is terminated by a carbomethoxy group instead of a methyl group. This deduction was corroborated by the observed HMBC correlation from H 2 -7 0 /H 2 -8 0 to C-9 0 , in addition to a further degree of unsaturation of 10 in comparison with 9. The 1 H NMR data of 11 (Table 4) displayed an additional oxygenated methylene at d H 4.05 (H 2 -9 0 ) and a signal of a downeld shied methyl group at d H 2.02 (H 3 -12 0 ), which along with the observed HMBC correlations from H 2 -9 0 to C-7 0 , C-8 0 and C-11 0 , as well as from H 3 -12 0 to C-11, hinted at the presence of an acetoxy group at the terminus of the aliphatic side-chain in 11. The molecular weight  of compound 12 was 16 amu larger than that of 9. Moreover, the 1 H NMR data of 12 (Table 4) displayed an extra oxygenated methylene (d H 3.53) split into a triplet, which revealed a COSY correlation to d H 1.52 (H-8 0 ), suggesting the presence of a hydroxyl group at C-9 0 . Accordingly, compounds 10-12 were established as new a-pyrone analogues, for which the trivial names bulgariapyrones B-D are proposed. In order to assign the absolute congurations of 9-12, the modied Mosher's reaction was carried out, and 12 was chosen as a model compound for this reaction. Both primary and secondary alcohol groups in 12 were converted to either (S)-or (R)-MTPA esters. Based on the calculated values of Dd (S)-(R) of MTPA esters, the absolute conguration at C-1 0 was established as (S), which is in agreement with the literature data of previously described a-pyrone derivatives related to 12 (Fig. 5). 47 Similar specic optical rotation values of 9-11 suggested that these a-pyrones share the same (S) absolute conguration at C-1 0 .
The molecular formula of 13 was determined as C 33 H 52 O 11 , based on the prominent pseudomolecular ion peak at m/z 625.3587 [M + H] + in the HRESIMS spectrum, corresponding to 8 degrees of unsaturation. Analysis of the molecular formula and of the 1D/2D NMR data of 13 (Table 4, ESI †) suggested it contained two substructures similar to bulgariapyrone D (12). However, the NMR data of 13 revealed additional signals of an isolated methylene at d H 4.34/d C 62.3 (H 2 -1 00000 ) and one set of methylene as a triplet at d H 3.46/d C 71.6 (H 2 -9 0 ), suggesting an asymmetry of the molecule. The HMBC correlations observed from H 2 -1 00000 to C-2 000 , C-3 000 , C-4 000 and C-9 0 , and in turn from H 2 -9 0 to C-7 0 , C-8 0 and C-1 00000 , together with the NOESY correlation H 2 -9 0 /H 2 -1 00000 allowed connecting these two monomeric units via an ether bridge between C-1 00000 and C-9 0 , suggesting that 13 could be formed through a condensation reaction between two molecules of 12. Thus, the planar structure of 13 was elucidated as shown and the name bulgariapyrone E is proposed. Notably, only two related naturally occurring symmetrical a-pyrone dimers have been reported so far, multiforisin D, previously isolated from Gelasinospora multiforis, 50 and dothideopyrone D, a metabolite from the endophytic fungus Dothideomycete sp. LRUB20, 49 which emphasize the rare nature of these dimeric compounds. Moreover, compound 13 seems to be a product of a "head to tail" condensation, where the side chain of one monomeric unit is connected through an ether bond to the C-1 00000 of the a-pyrone nucleus of the other monomeric unit, which creates an asymmetry in the structure of 13 and makes it structurally different from known symmetrical C2-type bispyrone dimers. As the condensation of primary alcohols might take place under acidic conditions, an experiment was carried out by incubating 0.2 mg of 12 in 0.2 mL MeOH containing different concentrations of formic acid (0.1%, 1%, 5% and 10%), at room temperature for 1 week, followed by HPLC analysis. The HPLC chromatograms showed no formation of compound 13, which indicated that 13 is a true natural product, and not an artifact arising during the isolation procedure.
Following the same Mosher's reaction protocol for compound 13 as for 12, the (S)-or (R)-MTPA ester products were obtained (see the Experimental section). The MALDI-MS spectra of the reaction products indicated that all the primary and secondary alcohol groups in 13 reacted with the reagent (m/z 1295 [M + Na] + ). Thus, the absolute conguration of 13 was unequivocally assigned as (S) for both stereocenters at C-1 0 and C-1 0000 , which is in accordance with biosynthetic considerations and the reported conguration of the structurally related homodimer dothideopyrone D. 49 Compound 14 was obtained as a dark red solid. Its molecular formula was determined as C 26 H 28 O 7 , on the basis of a prominent pseudomolecular ion peak at m/z 453.1910 [M + H] + in the HRESIMS spectrum, requiring 13 degrees of unsaturation. Detailed analysis of the 1D and 2D NMR spectra showed that 14 is a known compound, bulgarialactone B, 29 rst isolated from the same fungus more than two decades ago. However, in this rst paper, the absolute conguration was not determined for bulgarialactone B and the relative conguration was elucidated on the basis of the weak NOESY correlation between H-3 and H 3 -27. Further reports on bulgarialactone B focused on the Hsp90 inhibitory activity and were not engaged in the elucidation of the absolute conguration. 30,51 The SciFinder database indicates the absolute conguration for the closely related compound epicocconone, which lacks a chirality centre in the unsaturated side-chain and exhibits potent uorescent properties, but the original paper 52 reported only the relative conguration of the two chirality centres as elucidated by the comparison of the HF/6-31G(d,p) optimized low-energy geometries of the high-temperature molecular dynamics trajectories with the experimental NMR data. Subsequent papers did not address the elucidation of the absolute conguration of epicocconone.
Bulgarialactone B (14) had positive Cotton effects (CEs) at 437 and 269 nm and negative ones at 315 and 232 nm. In order to determine the absolute conguration of bulgarialactone B, the solution TDDFT-ECD method was applied on the arbitrarily chosen (3S,11S,23R) and (3S,11S,23S) stereoisomers. 53,54 Although the core part was expected to govern mostly the ECD spectrum, the C-23 chirality centre located in the allylic position of the conjugated p system of the side-chain was also supposed to have at least a minor contribution, which would have allowed distinguishing the C-23 epimers. A MMFF (Merck Molecular Force Field) conformational search of the two epimers resulted in 549 and 541 conformer clusters in a 21 kJ mol À1 energy window, respectively, indicating high conformational exibility. These geometries were reoptimized at the CAM-B3LYP/TZVP PCM/MeCN level. Despite the large number of low-energy conformers, the Boltzmann-averaged ECD spectra of both epimers computed at four different levels (B3LYP/TZVP, BH&HLYP/TZVP, CAM-B3LYP/TZVP and PBE0/TZVP, all with PCM for MeCN) gave consistently moderate to good agreement with the experimental ECD spectrum (Fig. 6 and 7). Interestingly, the difference between the computed ECD spectra of the epimers was rather small, which suggested that the inuence of C-23 on the overall ECD is marginal. Accordingly, the absolute conguration of the core part could be elucidated as (3S,11S), while the C-23 chirality centre remained unassigned. By analyzing the individual ECD spectra of the low-energy computed conformers, it turned out that the features of the ECD spectra were inuenced by the helicity of the dihydropyrane ring and the orientation of the conjugating side-chain, the latter of which was also described by Syzgantseva et al. to inuence the UV characteristics. 55 The experimental ECD spectra of 14 and that of (+)-epicocconone reported by Bell and Karuso 52 have a mirror image relationship, suggesting that (+)-epicocconone showing an intense negative CE above 400 nm has an (3R,11R) absolute conguration in Epicoccum nigrum, 52 which is an example of the chiral switching of the tricyclic skeleton. 56 It is also clear that the absolute conguration presented in the SciFinder database for epicocconone 57 is incorrect and it should specify only the relative conguration.
The planar structure of compound 16 was shown to be identical to the previously described avipesin B, isolated from the fungal endophyte Aspergillus avipes AIL8, 58 which has been previously reported as a synthetic compound. 59,60 In a recent study, it was isolated from an engineered strain of A. nidulans. 61 However, 16 possesses an opposite sign of the specic optical rotation, [a] 25 D À72 (MeOH), compared to that of the reported value for avipesin B: [a] 25 D +133 (acetone). 58 Therefore, in this study, we assigned 16 as (À)-(S)-avipesin B.
The structures of the remaining known compounds isolated from B. inquinans were established as phenylbutyrolactone IIa (15), 33,34,59 xenofuranone B (17) 35,67 and bulgarialactone D (18), 30 based on their spectroscopic data as well as on a comparison with the literature.
All the isolated compounds were evaluated for cytotoxicity towards the murine lymphoma cell line L5178Y. However, only 2 exhibited pronounced activity against the L5178Y cell line, with an IC 50 value of 1.8 mM, which was stronger than that of the positive control kahalalide F (IC 50 4.3 mM). The presence of a hydroxy group attached to C-5 in the 2-furanone ring in the structure of 2 was apparently important for its cytotoxic activity as no activity was found for compounds 3, phenylbutyrolactone IIa (15), 16 and xenofuranone B (17). Furthermore, a closely related derivative, 4-O-demethylisobutyrolactone II, which bears an additional hydroxy group on both aromatic rings at the positions 9 and 16 (tyrosine-derived residues) compared to 2, was shown to be inactive against the L5178Y cell line, 62 thus suggesting that the phenyl substituents were required for its cytotoxicity. The remaining compounds showed no signicant cytotoxic properties. Interestingly, related a-pyrones were previously reported to be nontoxic or exhibited only moderate cytotoxicity against several cancer cell lines. 49 A number of apyrone analogues, however, revealed promising biological activities as antibacterial agents, 63 HIV-1 protease inhibitors, 64 tyrosinase inhibitors 65 or inhibitors of nitric oxide production, 47 making these metabolites attractive scaffolds for synthetic chemical studies. In our study, bulgarialactone B (1) revealed no cytotoxicity against the tested L5178Y cell line, albeit in a previous study it was reported to possess antitumor activity against an ascitic ovarian carcinoma xenogra. 30 Butyrolactone analogues similar to the derivatives isolated in this study were previously described from Aspergillus species, 58,62,66-68 Here we report B. inquinans as a source of this type of metabolites for the rst time. Moreover, the results obtained upon application of the OSMAC approach employing a mixture of salts (MgSO 4 , NaNO 3 and NaCl) highlighted the usefulness of this strategy not only for diversifying secondary metabolites produced by this strain, but also to afford rare natural product scaffolds, as exemplied by the isolation of 1,3oxazine derivatives (7 and 8) and of an unusual a-pyrone dimer 13. Another set of OSMAC experiments was performed to investigate which salt mainly contributes to the changes in the metabolic prole of B. inquinans, by adding separately MgSO 4 -$7H 2 O (2.5 and 3.5 g), and a mixture of NaNO 3 and NaCl (2.5 g each) to Czapek medium with and without MgSO 4 . The fungus failed to grow in the presence of the mixture of NaNO 3 and NaCl, when MgSO 4 was completely excluded from Czapek medium, suggesting that Mg 2+ ions are apparently critical for fungal growth. However, the addition of MgSO 4 alone did not result in any changes in the HPLC chromatogram in comparison with the chromatogram of the fungus grown on solid Czapek medium, suggesting that the fungus was only metabolically affected by adding the mixture of these three salts simultaneously. Interestingly, OSMAC studies with the marinederived fungus, Ascotricha sp. ZJ-M-5, involving MgCl 2 in Czapek Dox broth medium, previously reported that Mg 2+ ions inuenced the secondary metabolites prole of this fungus. 69 With respect to the ndings in this study, the compounds obtained from the OSMAC experiments in the presence of Mg 2+ were mostly methoxylated derivatives. Thus, it may be speculated that Mg 2+ ions under certain conditions might trigger Oalkylation reactions.

Fungal material and fermentation
The fungus Bulgaria inquinans (isolate MSp3-1) was isolated from a healthy sprout of mistletoe (Viscum album), 70 collected in January 2017 at Jülich, Germany. Fungal identication was carried out according to a standard molecular biology protocol, 70 followed by a BlastN search in the NCBI database. The sequence was submitted to the GenBank (accession no. MK246763). The fungal strain is kept in one of the author's laboratory (P. P.).
The fungus was cultivated on solid Czapek medium, which was prepared by autoclaving 200 mL of liquid Czapek medium with the addition of 5.0 g of bacto agar in a 1 L Erlenmeyer ask. The composition of liquid Czapek medium was 10.0 g dextrose, 20.0 g mannitol, 20.0 g maltose, 3.0 g yeast extract, 1.0 g corn steep liquor, 0.5 g tryptophan, 0.5 g K 2 HPO 4 $3H 2 O, 0.3 g MgSO 4 $7H 2 O and 1 L of distilled water (pH value of the medium adjusted between 7.2-7.8). The fermentation was performed in 15 asks at room temperature, under static conditions for 27 days.
OSMAC experiments were carried out by growing the fungus on solid Czapek medium containing either 3.5 g NaCl, 3.5 g NaBr, 3.5 g NaI, 3.5 g NaNO 3 , 3.5 g (NH 4 ) 2 SO 4 or mixtures of (a) MgSO 4 $7H 2 O, NaNO 3 and NaCl (2.5 g of each), (b) FeSO 4 $7H 2 O, NaNO 3 and NaCl (2.5 g of each), or (c) ZnSO 4 $7H 2 O, NaNO 3 and NaCl (2.5 g of each), added to each 1 L ask followed by extraction when the asks were completely overgrown by the fungus. Based on the chromatographic proles obtained from these experiments, a large-scale fermentation of B. inquinans was carried out by adding a mixture of MgSO 4 $7H 2 O, NaNO 3 and NaCl (2.5 g of each) to solid Czapek medium. The fungus was grown under static conditions for 33 days followed by extraction.