Spirocyclic lactams and curvulinic acid derivatives from the endophytic fungus Curvularia lunata and their antibacterial and antifungal activities

Journal Pre-proof Spirocyclic lactams and curvulinic acid derivatives from the endophytic fungus Curvularia lunata and their antibacterial and antifungal activities Felipe Hilario, Giulia Polinário, Marcelo Rodrigues do Amorim, Victor de Sousa Batista, Nailton Monteiro do Nascimento Júnior, Angela Regina Araújo, Taís Maria Bauab, Lourdes Campaner dos Santos PII: S0367-326X(19)32187-2 DOI: https://doi.org/10.1016/j.fitote.2019.104466 Reference: FITOTE 104466 To appear in: Fitoterapia Received date: 28 October 2019 Revised date: 19 December 2019 Accepted date: 19 December 2019 Please cite this article as: F. Hilario, G. Polinário, M.R. do Amorim, et al., Spirocyclic lactams and curvulinic acid derivatives from the endophytic fungus Curvularia lunata and their antibacterial and antifungal activities, Fitoterapia (2019), https://doi.org/10.1016/ j.fitote.2019.104466 This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier. 1 Journal Pre-proof Spirocyclic lactams and curvulinic acid derivatives from the endophytic fungus Curvularia lunata and their antibacterial and antifungal activities Felipe Hilarioa, Giulia Polinárioa, Marcelo Rodrigues do Amorimb , Victor de Sousa Batista b , Nailton Monteiro do Nascimento Júniorb , Angela Regina Araújob , Taís Maria Bauaba and Lourdes Campaner dos Santos b,* a São Paulo State University (UNESP), School of Pharmaceutical Sciences, Road Araraquara-Jaú km1, Araraquara 14800-903, Brazil b São Paulo State University (UNESP), Institute of Chemistry, Av. Prof. Francisco Degni n.55, Araraquara 14800 -060, Brazil Abstract -p ro o f Curvularia lunata, isolated from the capitula of Paepalanthus chiquitensis (Eriocaulaceae), was cultured in potato dextrose broth (PDB) medium. The ethyl acetate extract yielded two new spirocyclic -lactams (3 and 4), and five known compounds, namely: triticones E (1) and F (2), 5-O-methylcurvulinic acid (5), curvulinic acid (6) and curvulin (7). Their structures were elucidated by spectroscopic analysis and by the comparison with literature data. Besides, a computational study was used to elucidate the absolute configuration of the C−3' in the compounds (3) and (4). The extract and the compounds (1 and 2), (6) and (7) were assayed against gram-positive and gram-negative bacteria and fluconazole-resistant yeast. The triticones (1) and (2) showed good antibacterial activity for Escherichia coli, with a minimum inhibitory concentration of 62.5 µg/mL. re Keywords: Paepalanthus chiquitensis; Curvularia lunata; spirocyclic lactams; curvulinic acid; antibacterial activity 1. Introduction lP Corresponding author (Santos, L.). Tel.: +55-16-3301-9657; fax: +0-000-000-0000; e-mail: lourdes.campaner@unesp.br Jo ur na Plant-associated fungi are prolific producers of structurally diverse small-molecule natural products with interesting biological activities (Gunatilaka, 2012)Bashyal et al., 2017). Fungal endophytes were isolated from Paepalanthus chiquitensis Herzog (synonym Paepalanthus giganteus Sano (Eriocaulaceae) (Trovó and Sano, 2010). Paepalanthus Mart comprises approximately 400 species that are predominantly found throughout South and Central America with their center of diversity located in the Espinhaço Range in Minas Gerais and Bahia, Brazil (Andrino et al., 2015). From the taxonomic point of view, Paepalanthus is the largest and most complex genus of Eriocaulaceae (Giulietti et al., 2012). Rupestrian grasslands are tropical regions and the natural habitat of Eriocaulaceae; they occur at altitudes above 900 m and as the soil consists predominantly of quartzite and ironstone there is low water retention, resulting in a high diversity of habitats with different environmental conditions (Conceição et al., 2016). Considering the environmental conditions of the rupestrian grasslands in which P. chiquitensis survives, it is interesting to investigate endophytic fungi associated with this plant species in order to obtain unique microorganisms. Furthermore, with the potential to produce active metabolites. During our investigation of plant-associated endophytic fungi, we screened 25 endophytic fungi isolated from the aerial parts of P. chiquitensis for antimicrobial activity against the Gram-positive bacterium Staphylococcus aureus, Gram-negative bacteria Escherichia coli and Salmonella sp., and the fluconazoleresistant yeast Candida albicans (Hilario et al., 2017). One of the endophytic fungi with promising antimicrobial activities was the strain Curvularia lunata, isolated from a healthy capitula of P. chiquitensis (Eriocaulaceae) collected in February 2012 in Serra do Cipó in Minas Gerais State, Brazil. The C. lunata, a known human pathogen (Rižner and Wheeler, 2003), is a member of the group of dematiaceous (brownpigmented) fungi that have melanin, which is responsible for the dark pigmentation of their spores and conidia (Hoffmann et al., 2011). Melanin seems to behave as a virulence factor (Brunskole et al., 2008). Herein, we report the isolation and structure elucidation of seven metabolites from the endophytic fungus C. lunata. Including two new spirocyclic lactams, metabolites (3) and (4). Their absolute configuration at the C−3' was elucidated by the computational study. The antimicrobial activities were assayed against four human pathogens. 2 Journal Pre-proof 2. Results and discussion ro o f The large-scale cultivation in PDB medium of C. lunata, afforded the EtOAc extract used for fractionation by Sephadex LH-20 column chromatography followed of the isolation of the secondary metabolites by high-performance liquid chromatography with diode-array detector (HPLC-DAD) (see experimental section) yielding the triticones E and F (1 and 2), spirocyclic lactams (3 and 4), 5-Omethylcurvulinic acid (5), curvulinic acid (6) and curvulin (7), Figure 1. -p Figure 1. Chemical structures of triticones E (1) and F (2), spirocyclic lactams (3 and 4), curvulinic acid (6) and its derivatives (5) and (7) Jo ur na lP re Compounds 1 and 2 were characterized by spectroscopic methods, such as UV, HRMS, one- and twodimensional 1 H and 13 C NMR experiments (gCOSY, gHSQC, gHMBC, and NOESY) and by comparison with published data. It was confirmed that the compound 1 is triticone E ( Hallock, Yali F., et a., 1993) and the 2 is spirostaphylotrichin R or triticone F (Masi et al., 2014a), the C−6 configuration is the only difference existing between them (Wang et al., 2018). These compounds were isolated as a mixture (2:1), and chemical isolation was not possible by chromatographic methods (Masi et al., 2014a). The reason why it was not possible may be due to the capacity of the epimers to interconvert and the mechanism involved in the interconversion is a retro-aldol type reaction (Sugawara et al., 1988; Hallock et al., 1993). Triticones and spirostaphylotrichins are known to be inseparable compounds by chromatographic methods (Sugawara et al., 1988; Hallock et al., 1993; Rawlinson et al., 2019). The biogenetic relationship of 19 known spirostaphylotrichins resulted in the identification of the artifacts formed during the isolation, among the metabolites identified is the spirostaphylotrichin R (compound 2) formed from spirostaphylotrichin A (Sandmeier and Tamm, 1990). These authors were the first to describe the spirostaphylotrichin R but could not determine the configuration at C‒3. It was determined in 1995 by Abraham et al. being reported the isolation of spirostaphylotrichin R produced by Curvularia pallescens instead of an artifact (Abraham et al., 1995). Likewise, the triticones E (1) and F (2), the compounds 3 and 4 were obtained as an interconverting mixture (2:1). Even though, after the extensive chromatographic work, it was not possible the isomers isolation. The two new spirocyclic -lactams (3) and (4) are interesting triticone-like compounds and possibly may share biogenetic similarities with the spirostaphylotrichins/triticones. Similarly, the spirostaphylotrichin R or triticone F (2) and triticone E (1), the spirocyclic -lactams (3) and (4) the only existing difference is the C‒6 configuration, and also were obtained as an inseparable epimeric mixture. There is evidence reporting which all the chemical class just to be considered as a biosynthetic or intermediate just when the absolute configuration of C‒6 has been characterized (Walser-Volken and Tamm, 1996). Controversially, in recent works, other compounds have been described as natural products even though in the absent absolute configuration (Masi et al., 2014; Almeida et al., 2018; Rawlinson et al., 2019). On this hand, the two new spirocyclic -lactams (3) and (4) are being reported on the manuscript as a new natural product due to their hybrid characteristic of a diastereoisomer and triticone-like compound due to the spirocyclic -lactam core structure. The IR spectrum of the compounds 3 and 4 showed hydroxyl,  -  -unsaturated and amidic carbonyl groups. The UV spectrum showed absorption maxima at 213 and 295 nm typical of an extended conjugated  , -unsaturated carbonyl group (Masi et al., 2014a). 3 Journal Pre-proof The 1 H NMR spectrum of 3 and 4 showed signals in pairs for all the spectrum, the integration indicated the presence of 27 hydrogens, being assigned 18 hydrogen signals to 3 and the remaining signals assigned to the 4, due to its ratio of 3. Besides, the 1 H NMR spectrum of 3 and 4 showed signals attributed respectively to three olefinic protons of each structure at  7.52 and 7.48 (d, J = 12 Hz, H−3); 5.84 and 5.82 (d, J = 12 Hz, H−4); 5.65 and 5.57 (t, J = 7.5 Hz, H−7). The hydrogens H−3 and H−4 are corresponding to one double bond between them on the cyclohexenone ring, indicating that these hydrogens are cis. Two diastereotopic hydrogens of the methylene groups were observed at   2.14 (m, H2 −4'a) and 1.82 (m, H2 −4'b); 1.86 (m, H2 −8a) and 1.79 (m, H2 −8b). Additionally, two methines at   4.11 and 3.91 (t, H−3'); 4.47 and other two oxygenated methine groups at 4.46 (d, J = 6.5 Hz, H−6) were observed. Furthermore, the presence of methoxy groups was observed at   3.86 and 3.84 (s, N−OMe−10). Finally, two methyl groups at   0.96 and 0.95 (t, J = 7.5 Hz, H3 −9). Table 1. 1 H (600 MHz) and 13 Jo ur na lP re -p ro o f C (150 MHz) NMR data for 3 and 4  a,b  a,b Position  C H m (J)c  C H m (J)c 1 59.9 (C) 60.6 (C) 2 130.0 (C) 131.5 (C) 3 142.8 (CH) 7.52 d (12.0) 143.1 (CH) 7.48 d (12.0) 4 124.0 (CH) 5.84 d (12.0) 124.2 (CH) 5.82 d (12.0) 5 198.6 (C) 199.1 (C) 6 73.1 (CH) 4.47 d (6.5) 73.2 (CH) 4.46 d (6.5) 7 141.0 (CH) 5.65 t (7.5) 142.8 (CH) 5.57 t (7.5) 1.86 m 1.86 m 8 22.4 (CH2 ) 22.4 (CH2 ) 1.79 m 1.79 m 9 14.2 (CH3 ) 0.96 t (7.5) 14.3 (CH3 ) 0.95 t (7.5) 2' 152.4 (C) 152.7 (C) 3' 58.8 (CH) 4.11 t (13.2) 58.8 (CH) 3.91 t (13.2) 2.14 m 2.14 m 4' 28.3 (CH2 ) 28.3 (CH2 ) 1.82 m 1.82 m 5' 200.7 (C) 200.8 (C) 6' 130.0 130.1 7' 166.1 (C) 167.6 (C) 10 65.0 (OCH3 ) 3.86 s 65.3 (OCH3 ) 3.84 s 6'-OH 7.92 brs 7.92 brs a b c Chemical shift in  values (ppm) from TMS; recorded in DMSO-d6 ; J values in Hz The 1 H- 1 H gCOSY spectrum showed a correlation between the olefinic hydrogens H−3 and H−4. The spin system observed of H−7 coupled with both protons of the methylene group of H2 −8 as well the methyl group of H3 −9. Besides, it was observed the gCOSY correlation of H−3' coupled with the protons of the methylene group of H2 −4' (Table 1, Figure 2). The 13 C NMR data of 3 and 4 showed a wide variety of signals, existing mostly in pairs for all the spectrum. It was possible to assign 32 carbons, being attributed equal number of carbons to presence of 16 carbons, to each isomer, corresponding to three methine sp2 carbons (C−3, C−4, and C−7), including another two quaternary sp2 carbons (C−2 and C−6') and the sp3 quaternary carbon (C−1), two methylene (C−4' and C−8), one N-methoxy group (C−10), one methyl group (C−9), and carbinolic carbon (C−6). As well as three carbonyl groups (C−5, C−5' and C−7'). The gHMBC spectrum showed correlations from H−3' to C−1 and C−6 evidencing the junction of the cyclohexenone and lactam ring. The 1-propenyl side chain was localized at C−2 by the couplings observed between H−3 and C−1 and confirmed from the correlations of H−3 to C−2 and C−7. These results allowed chemical shifts to be assigned to all the carbons and the corresponding hydrogens (Table 1). The chemical structures were confirmed by the data of the ESIQTOF-HRMS analysis exhibiting an ion at m/z 320.1137 [M+H]+ (calcd. 320.1134) stablished the molecular formula as C 16 H18 NO6 . Journal Pre-proof Figure 2. COSY and HMBC key correlations for 3 and 4 lP re -p ro o f The relative stereochemistry of 3 and 4 was determined by the NOESY correlation (Figure 3) and proton coupling constant analysis. The Z configuration of the C−4/C−7 double bond was established based on the interaction between H−4/H−7. Besides, the H−3/H−4 showed 3 J coupling constant of 12.0 Hz, confirming the Z configuration. The relative configuration of C−6 to 4 was observed by the correlation between H−6 with H−3', while it was absent to 3. Therefore, the relative configuration to 4 is S* while to 3 is R*. na Figure 3. NOESY key correlations for 3 Jo ur The absolute stereochemistry of the C−3' to the compounds 3 and 4 was determined by the computational study. The 1 H and 13 C correlation tables of the structures under evaluation are presented in Tables 2 and 3. Table 2. Calculated proton absolute error (||) and Mean Absolute Error (MAE) for candidate structures 3 and 4 as calculated with B3LYP/6-311+G(2d,p)//M06-2X/6-31+G(d,p) level of theory. 3exp 4exp 1 H Aavg MAE || 0.25 2.77 0.26 2.89 Bavg MAE || 0.41 4.50 0.43 4.68 Table 3. Calculated carbon absolute error (||) and Mean Absolute Error (MAE) for candidate structures 3 and 4 as calculated with B3LYP/6-311+G(2d,p)//M06-2X/6-31+G(d,p) level of theory. 13 3exp 4exp C Aavg MAE || 4.38 70.08 4.43 70.95 Bavg MAE || 4.01 64.19 3.99 63.87 The 1 H MAE analysis presented in Table 2 indicated that the calculated chemical shifts of the diatereoisomeric mixture of 3 and 4 correlate better with the substructures from group A (Figure 3), 5 Journal Pre-proof meaning that the proton from C−3' is directed towards the back of the bicyclic ring system. This is also observed for the absolute error, given that the error for the incorrectly matched pair B is approximately 1.6 times bigger for both cases. This trend is reversed for the correlations made from 13 C chemical shifts, as the MAE and absolute error for the substructures of group B are that 13 C calculated chemical shifts from this level of theory is not useful for a clear distinction between stereoisomers and being so, one can safely assume that the 1 H analysis is more relevant for this study (Wiitala et al., 2007; Willoughby et al., 2014). Thus, the calculated chemical shifts indicate that the C−3' stereocenter has S absolute configuration. The chemical structures of 3 and 4 were characterized respectively as the (1S*,2Z,3Z,3'S,6R*)- and (1S*,2Z,3Z,3'S,6S*)-6,6'-dihydroxy-1'-methoxy-2-propylidene-3',4'-dihydro-2'H-spiro[cyclohexane-1,3'cyclopenta[b]pyrrol]-3-ene-2',5,5'(1'H)-trione.The spirocyclic -lactams 3 and 4 are being reported for the first time in the literature. f Compound (5) was obtained as a pale amorphous solid. The ESI-QTOF-HRMS analysis exhibited an ion at m/z 223.0608 [M – H]– (calcd 223.0606) identifying the molecular formula is C10 H12 O5 . The HRMS spectrum showed fragmentation ions at m/z 179 [M – 44 – H]– , m/z 164 [M – 44 – 15 – H]– and m/z 136 [M – 44 – 15 – 28 – H] – . The fragmentation Figure proposed for compound (5) is shown in SI: S18. The UV spectrum of (5) (max 233, 269 and 300 nm) was characteristic of an aromatic compound. ro o The spectral properties of compound (5) were compared with those published in the literature, it was identified as being the 5-O-methyl curvulinic acid that has been already published as a metabolite produced by Penicillium griseofulvum (Varma et al., 2006). Besides, it has been isolated for the first time from the genus Curvularia. The 5-O-methyl curvulinic (5) is a chemical analogue of the compound (6) known as curvulinic acid. Its H NMR spectrum is similar to (5), with an OH group in position 11 instead of the methoxy group as was confirmed by HRMS data. The ESI-QTOF-HRMS analysis to (6) exhibited an ion at m/z 211.0602 [M+H]+ (calcd. 211.0606) demonstrating the molecular formula as C 10 H10 O 5 . This metabolite is produced by C. lunata,(Liu et al., 2017; Varma et al., 2006) C. ellisii(Coombe et al., 1968), Peyronellaea sp(Ying et al., 2014), and C. siddigui (Kamal et al., 1962). re -p 1 Jo ur na lP Compound (7), ethyl 2-(2-acetyl-3,5-dihydroxyphenyl)acetate or curvulin is a metabolite that has already been isolated from C. lunata (Varma et al., 2006). It was obtained as a pale amorphous solid. The ESIQTOF-HRMS analysis exhibited an ion at m/z 237.0769 [M–H]– (calcd 237.0763) demonstrating the molecular formula as C 12 H14 O The antimicrobial activities and the minimal inhibitory concentrations (MIC) were evaluated to four human pathogenic microorganisms: S. aureus, Salmonella sp., E. coli and C. albicans (Table 3). The literature does not provide a consensus in terms of the MIC obtained with natural products (Webster et al., 2008). Plant extracts with MIC of less than 500 µg/mL can be considered potent inhibitors, MIC between 600 and 1500 µg/mL are moderate inhibitors and MIC above 1600 µg/mL are weak inhibitors. In another work, a MIC equal to or less than 1000 µg/mL was considered satisfactory (Aligiannis et al., 2001). Overall, the EtOAc extract and the compounds tested showed from low to good activity against the four microorganisms tested. The MIC was 1000-62.5 µg/mL against the bacterial strains tested but there was no activity against the fluconazole-resistant yeast C. albicans. The secondary metabolites of 1 and 2 showed MIC of 62.5 µg/mL for E. coli, demonstrating that they are good natural antimicrobial agents against this bacterium. On the other hand, compounds 6 and 7 had MIC higher than the tested sample concentrations. The EtOAc extract had a low antimicrobial activity of 1000 µg/mL against E. coli and S. aureus. In addition, the results of the minimum bactericidal concentrations (MBC) for the EtOAc extract and for the isolated compounds did not show bactericidal activity, thus demonstrating the bacteriostatic behavior of the compounds against all three bacterial strains tested (Table 4). Journal Pre-proof Table 4. Antimicrobial activity of the EtOAc extract and isolated compounds MIC (MBC)a Sample E. coli Salmonella sp. S. aureus Extract 1000 (1000) ‒ 1000 (-) 1-2 62.5 (1000) ‒ 1000 (-) 6 ‒ ‒ ‒ 7 ‒ ‒ ‒ (+)b 7.8 12.5 0.15 c1 (+) NA NA NA (+)c2 NA NA NA a b C1 Values given as μg/mL; ampicillin for bacteria, amphoterecin B and MIC/MBC/MFC > 1000 μg/mL; NA: not applicable; R: resistant. MIC (MFC)a C. albicans ‒ ‒ ‒ ‒ NA 8.0 R C2 fluconazole for yeast; (‒) ro o f The fungi belonging to the Bipolaris genus and Curvularia are morphologically well-known with slight differences in the median cells of the conidia: Bipolaris is characterized by median cells of conidia of similar widths, while those of Curvularia have enlarged dark-colored median cells that are curved.(Manamgoda et al., 2014), (Shoemaker, 1959) re -p The chemical study of the EtOAc extract led to the isolation and identification of seven secondary metabolites produced by the endophytic fungus, C. lunata, isolated from the capitula of P. chiquitensis (Eriocaulaceae). Here, the spirocyclic -lactams (3) and (4) are being described for the first time. The isolated metabolites (1) and (2) showed good antibacterial activity against E. coli, with MIC of 62.5 g/mL. In conclusion, the endophytic fungus C. lunata has proved to be an interesting source of new active compounds against E. coli. lP 3. Experimental Jo ur na 3.1. General Experimental Procedures The 1D and 2D NMR analyses were obtained using Bruker AVANCE III 600 MHz spectrometers with a non-deuterated residual solvent signal as a reference. Chemical shifts (δ) are expressed in ppm. The highresolution mass spectra were recorded on a Q-TOF Bruker MaXis ImpactT M mass spectrometer (HRESIQTOF-MS) and were ionized by Electrospray (ESI) in negative or positive mode (HRESI-QTOF-MS). Analytical HPLC was performed on a Jasco equipped with a PDA detector. The analytical column used was the Phenomenex Luna (2) RP18 (250.0 × 4.6 mm i.d.; 5 m). Semi-preparative HPLC was performed on a Jasco equipped with a MD-2010 PDA detector, using a Phenomenex Luna (2) RP18 column (250 mm × 10 mm i.d.; 10 µm). The HPLC-grade ACN was purchased from JT Baker. HPLC-grade H2 O was prepared with a Milli-Q purification system. The chromatographic column employed Sephadex LH-20 (Pharmacia Biotech, Sweden). TLC analyses were performed using a Sorbent Technologies silica gel 60, spots on the TLC plates were visualized under UV light and after being sprayed with an anisaldehyde − H2 SO4 reagent followed by heating at 130 °C. 3.2. Fungal Isolation and Identification The fungal strain of C. lunata was isolated from healthy capitula of P. chiquitensis and was deposited in the Nuclei of Bioassays, Biosynthesis, and Ecophysiology of Natural Products (NuBBE) fungi collection in Araraquara, Brazil. The plant material was collected in February 2012 in Serra do Cipó, in Minas Gerais State, Brazil (19o 14'58.92"S, 43o 31'04.40"W) and authenticated by Prof. Dr. Paulo Takeo Sano of the Universidade de São Paulo (USP), Brazil. A voucher specimen (3402 SPF) was deposited at the Herbarium of IB-USP. Fungal identification was carried out by sequence analysis (GenBank Accession No. HQ631009.1). 3.3. Fungal Growth and Extraction The endophytic fungus C. lunata was cultured in solid potato dextrose agar for 12 days. The mycelia were removed from the Petri dish and placed into centrifuge tubes containing sterile water. The suspension was vortexed for 30 sec. (3 times) and then filtered through a 100 M nylon cell strainer. This sporehyphae suspension was used to inoculate the fungus in liquid medium PDB. For large-scale cultures, 1.0 mL of spore-hyphae suspension was used to inoculate the fungus into 23 Erlenmeyer flasks (500 mL) each containing 300 mL of liquid PDB medium, incubated at 25 °C in static mode for 28 days. The mycelia were 7 Journal Pre-proof separated from the liquid medium by filtration using filter paper. The filtrate was extracted using EtOAc (3 x 1/3 volume filtrate). The organic layers were combined and washed with H2 O (3 x 1/5 volume filtrate), the remaining water was removed using a drying agent (MgSO 4 ), and the solid was removed by filtration. The solvent was removed under reduced pressure yielding the EtOAc extract used for the isolation of the metabolites by chromatographic techniques. 3.4 Fractionation and Isolation The amount of 1.3 g of EtOAc extract obtained from large-scale culturing of C. lunata was fractionated by Sephadex LH-20 column chromatography (85.0 × 2.5 cm) and eluted with 100% MeOH affording 43 fractions (10.0 mL each), which were analyzed by silica gel TLC eluted with (CHCl3 /MeOH/n-PrOH/H2 O, 5:6:1:4, v/v/v/v, organic phase). The spots were visualized under UV light at 254 nm and anisaldehyde−H2 SO 4 . The fractions (Fr) 18-23 (74 mg) and Fr 24-29 (144 mg) were separated by semipreparative HPLC-PDA. The mobile phase consisted of H2 O (Eluent A) and MeOH (Eluent B), both containing 0.05% TFA. The isocratic mode was used to purify the Fr 18-23 (10% B for 30 min,  295 nm) yielding compounds 1 and 2 (3.1 mg) and 3 and 4 (2.7 mg). The gradient mode was used to purify Fr 24-29 (10-100% B for 30 min, 295 nm) affording compounds 5 (2.0 mg), 6 (1.2 mg) and 7 (5.7 mg). ro o f 3.4.1 Triticones E (1) and F (2). Amorphous solids, 1 H and 13 C NMR data were similar to those previously reported (Yali F Hallock et al., 1993). HRMS: [2M+Na]+ m/z 617.2322, [M+Na]+ m/z 320.1105 (calcd. for C14 H19 NNaO 6 , 320.1110). -p 3.4.2. (1S*,2Z,3Z,3'S,6R*)- and (1S*,2Z,3Z,3'S,6S*)-6,6'-dihydroxy-1'-methoxy-2-propylidene-3',4'dihydro2'H-spiro[cyclohexane-1,3'-cyclopenta[b]pyrrol]-3-ene-2',5,5'(1'H)-trione (3 and 4, respectivelly). Pale amorphous solid, []25 D 18.33 (c 0.06, MeOH). The UV spectrum showed max. at 213 and 295 nm.  max 3355, 2971, 1655, 1620, 1456, 1120 cm-1 . ESI-QTOF-HRMS analysis exhibited an ion at m/z 320.1137 [M+H]+ (calcd. for C 16 H18 NO 6 , 320.1134) re 3.4.3. 2-Acetyl-3-hydroxy-5-methoxyphenylacetic acid (5). Amorphous solid, 1 H and 13 C NMR data were similar to those already reported.17 ESI-QTOF-HRMS m/z 223.0608 (calcd. for C 11 H12 O5 , 223.0606). 3.5 Computacional Methodology na lP 3.4.4. 2-Acetyl-3,5-dihydroxyphenylacetic acid (6). Amorphous solid, 1 H and 13 C NMR data were similar to those already reported.17-21 HRMS: [M+Na]+ m/z 233.0422 (calcd. for C 10 H10 NaO 5 , 233.0426); [M+H]+ m/z 211.0602 (calcd. for C 10 H11 O5 , 211.0606). 3.4.5. Ethyl-2-(2-acetyl-3,5-dihydroxyphenyl)acetate (7). Pale amorphous solid, 1 H and 13 C NMR data were similar to those already reported.(Varma et al., 2006) ESI-QTOF-HRMS m/z 237.0769 (calcd. for C12 H14 O5 , 237.0763). Jo ur In order to elucidate the absolute stereochemistry of the C−3' carbon nucleus, a computational study was undertaken following the guidelines proposed by Willoughby, Jansma and Hoye, with slight modifications Initially, all candidate structures, namely 3a, 3b, 4a, and 4b, were modeled in Discovery Studio Visualizer (v.17.0.2.1076) with all hydrogen atoms shown explicitly (“Dassault Systèmes BIOVIA, Discovery Studio Visualizer, V.17.0.2.1076,” 2017). The letters a and b represent the two possible stereochemical configurations of the C−3', as shown in Figure 4. Figure 4. Chemical structures under computational evaluation. Journal Pre-proof Conformers for all these structures were generated using CSD Mercury conformer generator module. In this step, the software was requested to minimize the input geometry before starting the conformational search. The maximum number of conformations was set to 200, with at most 2 unusual torsions, sampling a maximum of 1.000.000 conformations per molecule and with a minimum rotamer probability of 0.05 %. The generated conformers were inspected to ensure that all logical anticipated rotamers and invertamers were found, as well as to remove redundant structures. This step resulted in 16 conformers for structures 3a and 4a and 20 conformers for structures 3b and 4b. All conformers were then submitted to geometric optimization and frequency calculation in Gaussian 09 using the M06-2X functional with the 6-31+G(d,p) basis set (FRISCH et al., 2013; Petersson et al., 1988; Petersson and Mohammad A, 1991; Zhao and Truhlar, 2008). A finer integration grid was requested during the calculations. DMSO solvation effects were considered via the integral equation formalism polarized continuum model (IEFPCM) and the solute cavities were constructed using default united-atom radii (UA0) (Barone et al., 1997; Mennucci et al., 1997; Mennucci and Tomasi, 2002; Tomasi et al., 1999). The output geometries of the candidate structures were inspected to remove redundant conformations and this step resulted in 12 conformations for structure 3a, 8 for structures 3b, 4a, and 4b. lP re -p ro o f After the removal of redundant geometries, the 1 H and 13 C atomic chemical shielding tensors σ for the optimized conformers were computed at the density functional level using the gauge independent atomic orbital (GIAO) formalism (Ditchfield, 1974; London, 1937; Wolinski et al., 1990). The B3LYP (Becke, 1993; Lee et al., 1988; Stephens et al., 1994; Vosko et al., 1980) functional was used with the 6311+G(2d,p) basis set and a finer integration grid was requested once again. DMSO solvation was addressed through IEFPCM again, but the substrate solvent cavities in this step were modeled by individual atomic radii (Bondi, 1964). The computed NMR shielding tensors were converted into referenced chemical shifts as defined by Equation 1, where σ is the computed NMR shielding tensor and δ is the referenced chemical shift (Lodewyk et al., 2012). Jo ur na The intercept and slope are empirically derived scaling factors that are applicable to the level of theory used in this work and they are independent of the structure under study. The values for these parameters, summarized in Table 5, were obtained from the reference for the method and were created by the approach proposed by Lodewyk, Siebert and Tantillo (Lodewyk et al., 2012). Table 5. Scaling factors for B3LYP/6-311+G(2d,p)//M06-2X/6-31+G(d,p) level of theory Scaling Factors Slope Intercept 1 -1.0767 31.9477 H 13 C -1.0522 181.2412 Next, the Boltzmann weighting factors for each conformer are determined at 25° C by using the free energies obtained from the frequency calculations, resulting in a mole fraction contribution for each conformer, χi. These contributions are then applied to the computed NMR shielding tensors for each nucleus of each individual conformer to give the Boltzmann-weighted average NMR shielding tensors for them. The mole fraction contributions are calculated as defined by Equation 2, where -E*i is the difference in free energy of the ith conformer minus that of the most stable conformer, R is the universal gas constant and T is the temperature in Kelvin. ∑ 9 Journal Pre-proof The experimental ( ) and computed ( ) chemical shifts are then compared to one another through the absolute error (| |)and mean absolute error (MAE) between them, as described in Equations 3 and 4, where N is the number of unique chemical shifts used in the comparison. The shielding tensors for chemically equivalent nuclei are averaged before the MAE analysis, in this case, this was necessary for the methyl hydrogens of C−9 and C−10. The computed chemical shifts for structures labeled A and B were also averaged between them, given that molecules 3 and 4 exist in an enantiomeric mixture. | ∑| | | | ∑| | ro o f | 3.6 Antimicrobial Assay Jo ur na lP re -p The evaluation of the antimicrobial activity and the MIC was determined by the broth microdilution method as described in the M7-A10 reference guidelines(CCLSI. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically, M7-A8.. 8th ed. Wayne ( PA): Clinical and Laboratory Standards Institute; 2009, 2009) of the Clinical & Laboratory Standards Institute (CLSI) for antibacterial assays and M27-A3(CLSI. Reference method for broth dilution antifungal susceptibility testing of yeasts, M27-A3. 3rd ed. Wayne ( PA): Clinical and Laboratory Standards Institute; 2008, 2008) for antifungal assays, with modifications (Duarte et al., 2005). The biological activity was evaluated against E. coli (ATCC 25922), S. aureus (ATCC 25923) and Salmonella sp. (ATCC 19196) and against the fluconazole-resistant yeast C. albicans (ATCC 10231). The bacterial strains were incubated in MullerHinton broth (MHB) for 24 h and C. albicans was incubated in RPMI 1640 for 48 h, at 37o C. The bacteria inoculums were standardized at 1.0 × 10 8 CFU mL-1 (optical density: 0.10−0.15 at 620 nm) and the yeast at 5.0 × 106 CFU mL-1 (optical density: 0.12−0.15 at 530 nm), corresponding to 0.5 McFarland standards. The assays were performed in triplicate in 96-well microplates. For the bacteria, the wells contained 80 μL of MHB with ampicillin being used as a positive control. For the yeast, the wells contained 100 μL of RPMI 1640 with fluconazole and amphotericin B being used as positive controls. The plates were incubated at 37 o C for 24-48 h. The MIC was detected by adding 30 μL of 0.01 % aqueous resazurin solution for the antibacterial activity and 20 μL of 0.5 % aqueous triphenyl tetrazolium chloride (TTC) solution for the antifungal activity. The MIC was defined as the lowest concentration of sample that inhibited visible growth. A portion from each well that showed antimicrobial activity was plated in agar and incubated at 37 °C for 24-48 h to determine the MBC and MFC. The lowest concentration that showed no bacteria or yeast growth in the subcultures was used as the MBC and MFC. Acknowledgments The authors gratefully acknowledge the financial support of Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) which provided a fellowship for F.H. [grant# 2016/05480-6] and a project for L.C.S. [grant#2015/04899-3] and T.M.B. [grant#2013/25432-0]. 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Highlights  Antibacterial activity against Escherichia coli  New spirocyclic- -lactams  Addition of epigenetic modifiers and chemical elicitors and effects on the production and diversification of the metabolites f o l a n J r u o r P e o r p