Furanones and Anthranilic Acid Derivatives from the Endophytic Fungus Dendrothyrium variisporum

Extracts from an endophytic fungus isolated from the roots of the Algerian plant Globularia alypum showed prominent antimicrobial activity in a screening for novel antibiotics. The producer organism was identified as Dendrothyrium variisporum by means of morphological studies and molecular phylogenetic methods. Studies on the secondary metabolite production of this strain in various culture media revealed that the major components from shake flasks were massarilactones D (1) and H (2) as well as two new furanone derivatives for which we propose the trivial names (5S)-cis-gregatin B (3) and graminin D (4). Scale-up of the fermentation in a 10 L bioreactor yielded massarilactone D and several further metabolites. Among those were three new anthranilic acid derivatives (5–7), two known anthranilic acid analogues (8 and 9) and three cyclopeptides (10–12). Their structures were elucidated on the basis of extensive spectroscopic analysis (1D- and 2D-NMR), high-resolution mass spectrometry (HRESIMS), and the application of the modified Mosher’s method. The isolated metabolites were tested for antimicrobial and cytotoxic activities against various bacteria, fungi, and two mammalian cell lines. The new Metabolite 5 and Compound 9 exhibited antimicrobial activity while Compound 9 showed cytotoxicity activity against KB3.1 cells.


Introduction
Fungi are a rich source of secondary metabolites that may serve as leads for the development of badly needed novel antibiotics and anticancer agents.In particular, certain ecological groups of fungi like the endophytes have recently been proven to yield a plethora of novel metabolites exhibiting a variety of biological activities [1].During the course of our studies on endophytic strains derived from plants collected in Algeria, we have encountered various interesting organisms that produced new chemical entities, such as the preussilides from Preussia similis [2].The current study deals with the evaluation of the taxonomy and intensive studies on the secondary metabolite production of another interesting strain Dendrothyrium variisporum, a coniothyrium-like fungus that was isolated during the same campaign from Globularia alypum Linn.(Plantaginaceae).This is the first report describing the secondary metabolites profile of the genus Dendrothyrium Verkley, Göker & Stielow, which was described recently as a new coelomycete genus belonging to the family Montagnulaceae [3].

Results and Discussion
Dendrothyrium variisporum was shown to produce in flasks mainly the polyketide massarilactone D (1) and as well as massarilactone H (2) and two new minor furanone derivatives 3 and 4.However, HPLC-MS analysis of the crude extracts revealed the presence of numerous minor constituents that could not be isolated in sufficient quantities to allow for their complete structure elucidation.To increase the amount of biologically active products, a fully controlled bioreactor experiment on a 10 L scale was conducted.A small amount of the culture medium was collected after each 24 h, extracted and analyzed using HPLCDAD-MS for the estimation of metabolite production.After 8 days, the free glucose was completely depleted as determined by commercial glucose test strips, and the cultures were harvested as previously described [4].From the resulting extracts, Metabolites 5−12 were isolated in addition to massarilactone D (Figure 1).describing the secondary metabolites profile of the genus Dendrothyrium Verkley, Göker & Stielow, which was described recently as a new coelomycete genus belonging to the family Montagnulaceae [3].
From spectroscopic analysis and by comparison with literature data, Compounds 1 and 2 where identified to massarilactone D [13] and massarilactone H [14], respectively.Ethyl anthranilate 8 was previously detected by GC-MS as one of the aroma-active compounds in Pinot Noir wines [15], while 2-phenylethyl anthranilate ( 9) is a fragrance ingredient present in the essential oils from the leaves of   2).Comparison of the 1 H-and 13 C-NMR/DEPT data of 6 with those of 5 indicated that the structures of these two compounds are very similar, except the absence of the hydroxyl group at C-3 in 6 and the substitution of the phenylethyl group by the benzyl moiety.This was confirmed by a comprehensive analysis of the 2D-NMR data, particularly 1 H-1 H COSY, HSQC, and HMBC spectra.The HMBC correlation depicted between the benzylic proton at δ H 5.31 (H-7 ) and the carbon at δ C 169.3 (C-7) further confirmed the structure (Figure 5).Consequently, Compound 6 was elucidated as phenylmethyl anthranilate.Although it was previously reported as a synthetic compound with a fish anesthetic effect [12], this is the first report on its isolation from the natural source to the best of our knowledge.Furthermore, its NMR data have not yet been reported.5).The remaining proton signals were those of two methylenes at δ H 1.94 (t, J = 7.0 Hz, H-2 ) and 4.41 (t, J = 7.0 Hz, H-1 ) and a singlet integrating for six protons at δ H 1.28 (s).The 13 C-NMR and DEPT spectra of this part revealed signals for two symmetrical methyl groups at δ C 29.8 (C-5 and C-6 ), an oxygenated quaternary carbon at δ C 70.6 (C-3 ), two methylenes including an oxygen-bearing one at δ C 62.4 (C-1 ) (Table 2).The HMBC correlation from H-4 /H-5 (δ H 1.28) to carbons C-3 (δ C 70.6) and C-2 (δ C 43.0), then from H-1 (δ H 4.41) to C-3 (δ C 70.6) and C-2 (δ C 43.0) confirmed the residue to be 3-hydroxy-3-methylbutyl (Figure 5).Furthermore, the HMBC correlation observed between the oxymethylene protons at δ H 4.41 (H-1 ) and the anthranilic acid carbonyl proved the structure of 7 to be the previously undescribed 3-hydroxy-3-methylbutyl anthranilate.
From spectroscopic analysis and by comparison with literature data, Compounds 1 and 2 where identified to massarilactone D [13] and massarilactone H [14], respectively.Ethyl anthranilate 8 was previously detected by GC-MS as one of the aroma-active compounds in Pinot Noir wines [15], while 2-phenylethyl anthranilate ( 9) is a fragrance ingredient present in the essential oils from the leaves of Cinnamomum zeylanicum Blume collected in India [16].As far as we know, their 1 Hand 13 C-NMR data are reported in the present work for the first time (Table S2).The 1 H-and 13 C-NMR data of Compounds 10-12 were in agreement with those reported in the literature for cyclo-(L-pro-L-isoleu) [17], cyclo-(L-pro-L-leu) [17,18] and cyclo-(L-pro-L-phe) [18], respectively.Since the crude extracts showed prominent antimicrobial activity, the isolated metabolites were screened against various bacteria and fungi.The minimum inhibitory concentration (MIC) values showed that only the new Metabolite 5 and Compound 9 were active, whereas the remaining compounds were inactive against the organisms tested (Table 3).Compound 5 showed the strongest activity against Bacillus subtilis and Micrococcus luteus with MIC values of 8.33 and 16.66 µg/mL, respectively, while the MIC value of Compound 9 against Mucor hiemalis (16.66 µg/mL) was the same as that of nystatin used as positive control.The two active metabolites are anthranilic acid derivatives with a phenylethyl core.Since Metabolite 6, which contains a phenylmethyl group instead of a phenylethyl residue, was not active, it was concluded that the phenylethyl moiety in Compounds 5 and 9 is essential for their antimicrobial activity.Furthermore, the ability of some of the isolated compounds to inhibit the proliferation of two mammalian cell lines including HeLa cells KB3.1 and mouse fibroblasts L929 was examined.Only 2-phenylethyl anthranilate (9) showed moderate cytotoxic activity against HeLa cells KB3.1 with an IC 50 value of 8.8 µg/mL (Table 4).

Fungal Material
The endophytic fungus Dendrothyrium variisporum was isolated from fresh healthy roots of Globularia alypum Linn.(Plantaginaceae).The plant samples were collected in June 2015 from Ain Touta, Batna 05000 (Algeria).The isolation of the endophyte was achieved following a previously reported method [19,20].Genomic DNA was extracted from fungal colonies growing on YMG using the EZ-10 Spin Column Genomic DNA Miniprep kit (Bio Basic Canada Inc., Markham, Ontario, Canada) following the manufacturer's protocol.The internal transcribed spacer (ITS) regions including the 3′-end of 18S rRNA gene, 5.8S rRNA gene, and the 5′-end of the 28S rRNA gene were

Fungal Material
The endophytic fungus Dendrothyrium variisporum was isolated from fresh healthy roots of Globularia alypum Linn.(Plantaginaceae).The plant samples were collected in June 2015 from Ain Touta, Batna 05000 (Algeria).The isolation of the endophyte was achieved following a previously reported method [19,20].Genomic DNA was extracted from fungal colonies growing on YMG using the EZ-10 Spin Column Genomic DNA Miniprep kit (Bio Basic Canada Inc., Markham, Ontario, Canada) following the manufacturer's protocol.The internal transcribed spacer (ITS) regions including the 3 -end of 18S rRNA gene, 5.8S rRNA gene, and the 5 -end of the 28S rRNA gene were amplified using ITS1F/ITS4 primer [21].The ITS sequence of the endophytic fungus was deposited with GenBank under the accession number MG018984.The producer organism was identified as Dendrothyrium variisporum on the basis of ITS sequencing and morphology (see Supporting Information).

Fermentation and Extraction
For fermentation in flasks, the submerged culture were raised in 30 × 500 mL Erlenmeyer flasks, each containing 200 mL of YMG medium: 1.0% malt extract, 0.4% glucose, 0.4% yeast extract, pH 6.3 [20].The flasks were inoculated with five mycelial plugs from actively growing yeast-malt-glucose-agar (YMG) plates and incubated at 23 • C under constant shaking at 140 rpm on a rotary shaker for 12 days.After separation from fungal mycelia by vacuum filtration, the supernatant was treated with 2% (v/v) of Amberlite XAD-16 resin.The latter was extracted with acetone by sonication at 40 • C for 30 min.The acetone extract was evaporated in vacuo, and the residual aqueous solution was re-extracted with EtOAc.The ethyl acetate extract was dried on Na 2 SO 4 and concentrated by evaporation to yield 1.4 g of crude extract.
For the scale up, a seed culture of the strain with a total volume of 1000 mL was prepared in YMG medium incubated at 23 • C and 140 rpm for 5 days and homogenized with a Heidolph Silent Crusher.A 15 L in-situ autoclavable bioreactor (bbi Germany) was prepared with 10 L of YMG medium and inoculated with 1000 mL of the seed culture.The temperature was set at 23 • C, agitation with a Rushton-impeller was set to 150 rpm, and the aeration rate was set to 1.5 L/min (0.15 vvm) and remained constant during fermentation.The culture was harvested after 8 days as the glucose was depleted, and a stagnation of secondary metabolite production was observed by analytical HPLC.The mycelium was separated from the culture fluid (supernatant) by centrifugation in a Dupont Instrument (Sorvall RC-5B Refrigerated Superspeed Centrifuge) followed by vacuum filtration and was extracted three times with acetone, then with methanol in an ultrasonic bath at 40 • C for 30 min.The acetone and methanol extracts were filtered, combined and evaporated to yield an aqueous phase, which was further extracted with 3 × 500 mL EtOAc in a separating funnel.The ethyl acetate fraction was dried over anhydrous Na 2 SO 4 , filtered, and concentrated under vacuum to yield 373.7 mg of oily mycelial crude extract.The supernatant was treated with 2% adsorber resin Amberlite XAD-16 resin over 2 h at room temperature.The XAD was separated by filtration and extracted three times with acetone, then with methanol in an ultrasonic bath at 40 • C for 30 min.The extract was evaporated to yield an aqueous phase, which was further extracted with ethyl acetate (3 × 500 mL).After drying over anhydrous Na 2 SO 4 , the ethyl acetate fraction was concentrated under vacuum to yield 508.2 mg of crude extract.

Isolation of Compounds 1-12
The crude supernatant extract from the fermentation in flasks (858.7 mg) was subjected to flash chromatography (GRACE Reveleris X2 flash system) with silica gel (40 g) as stationary phase.The column was eluted with the mixture of CH 2 Cl 2 (solvent A) and acetone (solvent B); gradient: 100% A in 5 min, 0-100% B in 35 min, and finally 100% B for 5 min; flow rate: 40 mL/min; UV detection: 254, 280, and 380 nm.Three fractions were collected: Fraction 1 (32.4 mg, t R = 8.5−10.1 min), Fraction 2 (32.0 mg, t R = 11.5−13.5 min) and Fraction 3 (708.7 mg, t R = 14.5−17.1 min), which was pure massarilactone D 1. Fraction 1 was further purified by preparative HPLC using a gradient of 25-60% solvent B in 40 min, 60-100% B for 5 min, and 100% B for 5 min.The fractions were combined according to UV absorption at 220, 280, and 325 nm and concurrent HPLC-MS analyses.Compound 3 (0.4 mg) was eluted at t R = 12.80 min, and Compound 4 (1.8 mg) was eluted at t R = 9.30 min.Fraction 2 was purified using the same preparative HPLC conditions as for Fraction 1 to yield Compound 2 (9.1 mg) at t R = 5.88 min.

3. 5 .
Preparation of the S-and R-MTPA Esters of 4 R-(-)-MTPA-Cl (4 µL) was added to a stirred solution of 4 (0.2 mg) and dry pyridine (5 µL) in dry CDCl 3 (100 µL) at room temperature.After 1 h, the reaction mixture was diluted by the addition of 100 µL of CDCl 3 .The produced S-MTPA ester of 4 was submitted to NMR spectroscopy.In an entirely