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<front>
<journal-meta>
<journal-id journal-id-type="publisher-id">Front. Mar. Sci.</journal-id>
<journal-title>Frontiers in Marine Science</journal-title>
<abbrev-journal-title abbrev-type="pubmed">Front. Mar. Sci.</abbrev-journal-title>
<issn pub-type="epub">2296-7745</issn>
<publisher>
<publisher-name>Frontiers Media S.A.</publisher-name>
</publisher>
</journal-meta>
<article-meta>
<article-id pub-id-type="doi">10.3389/fmars.2024.1386175</article-id>
<article-categories>
<subj-group subj-group-type="heading">
<subject>Marine Science</subject>
<subj-group>
<subject>Original Research</subject>
</subj-group>
</subj-group>
</article-categories>
<title-group>
<article-title>Discovery of new eremophilanes from the marine-derived fungus <italic>Emericellopsis maritima</italic> BC17 by culture conditions changes: evaluation of cytotoxic and antimicrobial activities</article-title>
</title-group>
<contrib-group>
<contrib contrib-type="author">
<name>
<surname>Viru&#xe9;s-Segovia</surname>
<given-names>Jorge R.</given-names>
</name>
<xref ref-type="aff" rid="aff1">
<sup>1</sup>
</xref>
<xref ref-type="aff" rid="aff2">
<sup>2</sup>
</xref>
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</contrib>
<contrib contrib-type="author">
<name>
<surname>Pinedo</surname>
<given-names>Cristina</given-names>
</name>
<xref ref-type="aff" rid="aff1">
<sup>1</sup>
</xref>
<xref ref-type="aff" rid="aff2">
<sup>2</sup>
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<contrib contrib-type="author">
<name>
<surname>Zorrilla</surname>
<given-names>David</given-names>
</name>
<xref ref-type="aff" rid="aff3">
<sup>3</sup>
</xref>
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<contrib contrib-type="author">
<name>
<surname>S&#xe1;nchez-M&#xe1;rquez</surname>
<given-names>Jes&#xfa;s</given-names>
</name>
<xref ref-type="aff" rid="aff3">
<sup>3</sup>
</xref>
<uri xlink:href="https://loop.frontiersin.org/people/1667581"/>
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<contrib contrib-type="author">
<name>
<surname>S&#xe1;nchez</surname>
<given-names>Pilar</given-names>
</name>
<xref ref-type="aff" rid="aff4">
<sup>4</sup>
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<contrib contrib-type="author">
<name>
<surname>Ramos</surname>
<given-names>Mar&#xed;a C.</given-names>
</name>
<xref ref-type="aff" rid="aff4">
<sup>4</sup>
</xref>
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<contrib contrib-type="author">
<name>
<surname>de la Cruz</surname>
<given-names>Mercedes</given-names>
</name>
<xref ref-type="aff" rid="aff4">
<sup>4</sup>
</xref>
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</contrib>
<contrib contrib-type="author" corresp="yes">
<name>
<surname>Aleu</surname>
<given-names>Josefina</given-names>
</name>
<xref ref-type="aff" rid="aff1">
<sup>1</sup>
</xref>
<xref ref-type="aff" rid="aff2">
<sup>2</sup>
</xref>
<xref ref-type="author-notes" rid="fn001">
<sup>*</sup>
</xref>
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</contrib>
<contrib contrib-type="author" corresp="yes">
<name>
<surname>Dur&#xe1;n-Patr&#xf3;n</surname>
<given-names>Rosa</given-names>
</name>
<xref ref-type="aff" rid="aff1">
<sup>1</sup>
</xref>
<xref ref-type="aff" rid="aff2">
<sup>2</sup>
</xref>
<xref ref-type="author-notes" rid="fn001">
<sup>*</sup>
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</contrib-group>
<aff id="aff1">
<sup>1</sup>
<institution>Departamento de Qu&#xed;mica Org&#xe1;nica, Facultad de Ciencias, Universidad de C&#xe1;diz</institution>, <addr-line>C&#xe1;diz</addr-line>, <country>Spain</country>
</aff>
<aff id="aff2">
<sup>2</sup>
<institution>Instituto de Investigaci&#xf3;n en Biomol&#xe9;culas (INBIO), Universidad de C&#xe1;diz</institution>, <addr-line>C&#xe1;diz</addr-line>, <country>Spain</country>
</aff>
<aff id="aff3">
<sup>3</sup>
<institution>Departamento de Qu&#xed;mica F&#xed;sica, Facultad de Ciencias, Universidad de C&#xe1;diz</institution>, <addr-line>C&#xe1;diz</addr-line>, <country>Spain</country>
</aff>
<aff id="aff4">
<sup>4</sup>
<institution>Centro de Excelencia en Investigaci&#xf3;n de Medicamentos Innovadores en Andaluc&#xed;a, Fundaci&#xf3;n MEDINA</institution>, <addr-line>Granada</addr-line>, <country>Spain</country>
</aff>
<author-notes>
<fn fn-type="edited-by">
<p>Edited by: Susana P. Gaud&#xea;ncio, New University of Lisbon, Portugal</p>
</fn>
<fn fn-type="edited-by">
<p>Reviewed by: Hui Cui, Guangzhou University of Chinese Medicine, China</p>
<p>Chang-Wei Li, Beijing Institute of Pharmacology &amp; Toxicology, China</p>
</fn>
<fn fn-type="corresp" id="fn001">
<p>*Correspondence: Josefina Aleu, <email xlink:href="mailto:josefina.aleu@uca.es">josefina.aleu@uca.es</email>; Rosa Dur&#xe1;n-Patr&#xf3;n, <email xlink:href="mailto:rosa.duran@uca.es">rosa.duran@uca.es</email>
</p>
</fn>
</author-notes>
<pub-date pub-type="epub">
<day>10</day>
<month>05</month>
<year>2024</year>
</pub-date>
<pub-date pub-type="collection">
<year>2024</year>
</pub-date>
<volume>11</volume>
<elocation-id>1386175</elocation-id>
<history>
<date date-type="received">
<day>14</day>
<month>02</month>
<year>2024</year>
</date>
<date date-type="accepted">
<day>22</day>
<month>04</month>
<year>2024</year>
</date>
</history>
<permissions>
<copyright-statement>Copyright &#xa9; 2024 Viru&#xe9;s-Segovia, Pinedo, Zorrilla, S&#xe1;nchez-M&#xe1;rquez, S&#xe1;nchez, Ramos, de la Cruz, Aleu and Dur&#xe1;n-Patr&#xf3;n</copyright-statement>
<copyright-year>2024</copyright-year>
<copyright-holder>Viru&#xe9;s-Segovia, Pinedo, Zorrilla, S&#xe1;nchez-M&#xe1;rquez, S&#xe1;nchez, Ramos, de la Cruz, Aleu and Dur&#xe1;n-Patr&#xf3;n</copyright-holder>
<license xlink:href="http://creativecommons.org/licenses/by/4.0/">
<p>This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.</p>
</license>
</permissions>
<abstract>
<p>In our previous studies, the marine-derived fungus <italic>Emericellopsis maritima</italic> BC17 was found to produce new eremophilane-type sesquiterpenoids on solid media. In order to explore its potential to produce more metabolites, <italic>E. maritima</italic> BC17 was subjected to a one strain-many compounds (OSMAC) analysis leading to the discovery of three new eremophilanes (<bold>1</bold>-<bold>3</bold>) and fourteen known derivatives (<bold>4</bold>-<bold>17</bold>) in the liquid media Czapek Dox and PDB. Their structures were established by extensive analyses of the 1D and 2D NMR, and HRESIMS data, as well as ECD data for the assignment of their absolute configurations. Antitumoral and antimicrobial activities of the isolated metabolites <bold>1</bold>, <bold>3</bold>, <bold>11</bold>, and <bold>15</bold> were investigated. PR toxin 3-deacetyl (<bold>15</bold>) exhibited cytotoxic activity against HepG2, MCF-7, A549, A2058 and Mia PaCa-2 human cancer cell lines with IC<sub>50</sub> values ranging from 2.5 to 14.7 &#xb5;M. In addition, <bold>15</bold> exhibited selective activity against methicillin-sensitive <italic>Staphylococcus aureus</italic> ATCC29213 at the highest concentration tested of 128 &#xb5;g/mL.</p>
</abstract>
<kwd-group>
<kwd>eremophilane</kwd>
<kwd>
<italic>Emericellopsis maritima</italic>
</kwd>
<kwd>marine-derived fungus</kwd>
<kwd>OSMAC approach</kwd>
<kwd>antimicrobial</kwd>
<kwd>antitumoral</kwd>
</kwd-group>
<counts>
<fig-count count="4"/>
<table-count count="3"/>
<equation-count count="0"/>
<ref-count count="54"/>
<page-count count="13"/>
<word-count count="9787"/>
</counts>
<custom-meta-wrap>
<custom-meta>
<meta-name>section-in-acceptance</meta-name>
<meta-value>Marine Biotechnology and Bioproducts</meta-value>
</custom-meta>
</custom-meta-wrap>
</article-meta>
</front>
<body>
<sec id="s1" sec-type="intro">
<label>1</label>
<title>Introduction</title>
<p>Marine fungi strains can be found across diverse habitats holding a notable ecological importance due to their ability to produce a wide range of metabolites (<xref ref-type="bibr" rid="B22">Grossart et&#xa0;al., 2019</xref>; <xref ref-type="bibr" rid="B24">Hafez Ghoran et&#xa0;al., 2022</xref>), including peptides, terpenes, lactones, steroids, alkaloids and polyketides (<xref ref-type="bibr" rid="B26">Jin et&#xa0;al., 2016</xref>).</p>
<p>In recent decades, the interest in marine fungi as chemical producers of bioactive molecules has increased significantly due to the isolation of new compounds with potential pharmaceutical applications (<xref ref-type="bibr" rid="B50">Xu et&#xa0;al., 2015</xref>; <xref ref-type="bibr" rid="B26">Jin et&#xa0;al., 2016</xref>; <xref ref-type="bibr" rid="B36">Pang et&#xa0;al., 2016</xref>; <xref ref-type="bibr" rid="B27">Khalifa et&#xa0;al., 2019</xref>; <xref ref-type="bibr" rid="B46">Wang et&#xa0;al., 2022</xref>), playing a crucial role in discovering and developing new drugs (<xref ref-type="bibr" rid="B1">Agrawal et&#xa0;al., 2023</xref>).</p>
<p>Given the increasing need to find and develop new bioactive molecules to combat global health problems such as microbial resistance and the rising incidence of cancer (<xref ref-type="bibr" rid="B15">Christaki et&#xa0;al., 2020</xref>; <xref ref-type="bibr" rid="B42">Sung et&#xa0;al., 2021</xref>), marine-derived fungi have proven to be a promising source of novel bioactive metabolites. Additionally, cytotoxic and antibacterial activities are the most commonly reported bioactivities of natural products from these fungi (<xref ref-type="bibr" rid="B26">Jin et&#xa0;al., 2016</xref>). In fact, halimide, a potent cytotoxic diketopiperazine isolated from the marine-derived fungus <italic>Aspergillus ustus</italic>, has led to the synthesis of plinabulin (NPI-2358), which is currently undergoing phase II/III clinical trials for cancer treatment (<xref ref-type="bibr" rid="B7">Blayney et&#xa0;al., 2020</xref>, <xref ref-type="bibr" rid="B6">2022</xref>).</p>
<p>Traditional methodologies for the chemical exploration of new bioactive natural products are currently considered inefficient due to recurrent rediscovery of known compounds or the limited yields obtained under laboratory conditions. In this sense, one of the most widely used strategies to exploit the chemical diversity produced by microorganisms involves the manipulation of culture conditions, such as variation of medium composition, temperature, and pH. This approach is known as the &#x201c;one strain many compounds&#x201d; (OSMAC) approach (<xref ref-type="bibr" rid="B8">Bode et&#xa0;al., 2002</xref>). OSMAC strategy, which attempts to activate silenced biosynthetic pathways, has already been successfully employed in marine-derived fungal strains leading to the isolation of a wide range of compounds with interesting antimicrobial and cytotoxic activities (<xref ref-type="bibr" rid="B37">Pinedo-Rivilla et&#xa0;al., 2022</xref>).</p>
<p>
<italic>Emericellopsis</italic>, a genus of <italic>Ascomycota</italic> fungi within the order <italic>Hypocreales</italic>, has a wide environmental amplitude and universal distribution (<xref ref-type="bibr" rid="B23">Grum-Grzhimaylo et&#xa0;al., 2013</xref>). According to the different phylogeny and ecology, it has been classified into two distinct groups, specifically marine and terrestrial clades (<xref ref-type="bibr" rid="B54">Zuccaro et&#xa0;al., 2004</xref>).</p>    <p>
<italic>Emericellopsis</italic> spp. are ubiquitous in marine environments and can survive extreme conditions, including temperature, pressure, salinity, and pH (<xref ref-type="bibr" rid="B23">Grum-Grzhimaylo et&#xa0;al., 2013</xref>). These fungi have also been isolated from different marine macroalgae and sponges (<xref ref-type="bibr" rid="B48">Wiese et&#xa0;al., 2011</xref>; <xref ref-type="bibr" rid="B21">Gon&#xe7;alves et&#xa0;al., 2020</xref>) and have previously been studied for their ability to produce peptides with antifungal (<xref ref-type="bibr" rid="B28">Kuvarina et&#xa0;al., 2021</xref>), antitumoral (<xref ref-type="bibr" rid="B38">Rogozhin and Sadykova, 2019</xref>), and antibiotic activities (<xref ref-type="bibr" rid="B25">Inostroza et&#xa0;al., 2018</xref>; <xref ref-type="bibr" rid="B2">Agrawal and Saha, 2022</xref>).</p>
<p>Specifically, <italic>E. maritima</italic> has previously been isolated from Atlantic sponges and the deep-sea (<xref ref-type="bibr" rid="B3">Batista-Garc&#xed;a et&#xa0;al., 2017</xref>; <xref ref-type="bibr" rid="B10">Bovio et&#xa0;al., 2018</xref>). However, no secondary metabolites from this species had been published, making its study an important starting point as a potential source of bioactive compounds.</p>
<p>The strain <italic>E. maritima</italic> BC17 was isolated from sediments collected along an intertidal gradient of the inner Bay of C&#xe1;diz, Spain (<xref ref-type="bibr" rid="B44">Viru&#xe9;s-Segovia et&#xa0;al., 2023</xref>). Four new eremophilane-type sesquiterpenes, together with thirteen known derivatives were found in the culture broth of this fungus on different solid media (<xref ref-type="bibr" rid="B44">Viru&#xe9;s-Segovia et&#xa0;al., 2023</xref>).</p>
<p>Eremophilanes have been reported to exhibit a wide variety of bioactivities such as phytotoxins, antimicrobials, protein inhibitors, immunomodulators and cytotoxins. Indeed, these sesquiterpenes have previously been isolated from marine fungi such as <italic>Penicillium copticola</italic> (<xref ref-type="bibr" rid="B52">Zhang et&#xa0;al., 2022</xref>), <italic>Xylaria</italic> sp. BL321 (<xref ref-type="bibr" rid="B39">Song et&#xa0;al., 2012</xref>), <italic>Penicillium</italic> sp. PR19N-1 (<xref ref-type="bibr" rid="B49">Wu et&#xa0;al., 2013</xref>; <xref ref-type="bibr" rid="B31">Lin et&#xa0;al., 2014</xref>) or <italic>Aspergillus</italic> sp. SCSIOW2 (<xref ref-type="bibr" rid="B45">Wang et&#xa0;al., 2016</xref>), among others, showing an interesting diversity of bioactive compounds (<xref ref-type="bibr" rid="B18">Ebel, 2010</xref>).</p>
<p>With the aim of continuing the study of its chemical potential, in this work <italic>E. maritima</italic> BC17 was further investigated using liquid culture media following the OSMAC strategy. Consequently, three previously undescribed eremophilanes (<bold>1</bold>-<bold>3</bold>) and fourteen known derivatives (<bold>4</bold>-<bold>17</bold>) were isolated using the liquid media Czapek Dox and potato dextrose broth (PDB), both under surface culture and shaking conditions. Additionally, the antimicrobial and cytotoxic activities of compounds <bold>1</bold>, <bold>3</bold>, <bold>11</bold>, and <bold>15</bold> are reported.</p>
</sec>
<sec id="s2" sec-type="materials|methods">
<label>2</label>
<title>Materials and methods</title>
<sec id="s2_1">
<label>2.1</label>
<title>General experiment procedures</title>
<p>Melting points were measured with a Reichert-Jung Kofler block. ECD spectra were recorded on a JASCO J-1500 CD spectrometer. Optical rotations were determined with a JASCO P-2000 polarimeter. IR spectra were recorded on a PerkinElmer Spectrum BX FT-IR spectrophotometer and reported as wavenumber (cm<sup>&#x2014;1</sup>). TLC was performed on Merck Kiesegel 60 &#xc5; F<sub>254</sub>, 0.25 mm thick. Silica gel 60 PF<sub>254</sub> (60-100 mesh, VWR) was used for column chromatography. HPLC was performed with a Hitachi/Merck L-6270 apparatus equipped with an UV-VIS detector (L 4250) and a differential refractometer detector (RI-7490). LiChroCART LiChrospher Si 60 (5 &#xb5;m, 250 mm &#xd7; 4 mm), LiChroCART LiChrospher Si 60 (10 &#xb5;m, 250 mm &#xd7; 10 mm), and ACE 5 SIL (5 &#xb5;m, 250 mm &#xd7; 4.6 mm id) columns were used for isolation experiments. <sup>1</sup>H and <sup>13</sup>C NMR measurements were recorded on Bruker 400, 500, and 700 MHz spectrometers with SiMe<sub>4</sub> as the internal reference. Chemical shifts are expressed in ppm (<italic>&#x3b4;</italic>), referenced to CDCl<sub>3</sub> (Eurisotop, Saint-Aubiu, France, <italic>&#x3b4;</italic>
<sub>H</sub> 7.25, <italic>&#x3b4;</italic>
<sub>C</sub> 77.0) and CD<sub>3</sub>OD (Eurisotop, Saint-Aubiu, France, <italic>&#x3b4;</italic>
<sub>H</sub> 3.30, <italic>&#x3b4;</italic>
<sub>C</sub> 49.0). COSY, HSQC, HMBC, and NOESY experiments were performed using standard Bruker pulse sequence. NMR assignments were made using a combination of 1D and 2D techniques. High-Resolution Mass Spectroscopy (HRMS) was performed either with a double-focusing magnetic sector mass spectrometer in a QTOF mass spectrometer in the positive-ion ESI mode.</p>
</sec>
<sec id="s2_2">
<label>2.2</label>
<title>Fungal material</title>
<p>The strain used in this work, <italic>E. maritima</italic> BC17, was isolated from sediment samples collected along an intertidal gradient of Bay of C&#xe1;diz, Spain (<xref ref-type="bibr" rid="B44">Viru&#xe9;s-Segovia et&#xa0;al., 2023</xref>). This culture was deposited at the University of C&#xe1;diz, Mycological Herbarium Collection (UCA). Conidial stock suspensions were maintained viable in 80% glycerol at -40&#xb0;C.</p>
</sec>
<sec id="s2_3">
<label>2.3</label>
<title>Culture and extraction conditions</title>
<p>
<italic>E. maritima</italic> BC17 strain was grown in Petri dishes with potato dextrose agar (PDA, Condalab, Madrid, Spain) medium for one week at 25&#xb0;C under white light (day light lamp).</p>
<p>For surface cultures, 26 Roux bottles containing 150 mL of PDB (Condalab, Madrid, Spain) or Czapek-Dox medium (50 g glucose, 1 g yeast extract, 5 g K<sub>2</sub>HPO<sub>4</sub>, 2 g NaNO<sub>3</sub>, 0.5 g MgSO<sub>4</sub>&#xb7;7H<sub>2</sub>O, 0.01 g FeSO<sub>4</sub>&#xb7;7H<sub>2</sub>O, 1L of water and pH adjusted to 6.5-7.0) were inoculated with 5 mycelium plugs (9 mm) from a 7 days-old culture. The bottles were incubated at 25&#xb0;C under white light for 14 and 28 days.</p>
<p>For shaken cultures, the strain <italic>E. maritima</italic> BC17 was fermented in 1.4 L of Czapek-Dox or 3.2 L of PDB liquid media (200 mL per flask) for 14 days at 25&#xb0;C and 120 rpm under continuous white light. Each Erlenmeyer flask was inoculated with 5 PDA discs (9 mm) from a 7 days-old culture. Fermentation continued at 25&#xb0;C under continuous white light for 14 or 28 days.</p>
<p>Once the fermentation was finished, the mycelium was filtered. The broth was extracted three times with ethyl acetate and the organic extract dried over dry Na<sub>2</sub>SO<sub>4</sub>. The solvent was then evaporated and the residue chromatographed, first on a silica gel column and then by HPLC with an increasing gradient of ethyl acetate to <italic>n</italic>-hexane.</p>
</sec>
<sec id="s2_4">
<label>2.4</label>
<title>Isolation and characterization of metabolites</title>
<sec id="s2_4_1">
<label>2.4.1</label>
<title>Surface culture fermentation</title>
<p>Chromatography of the extract fermented in Czapek Dox medium for 14 days (746 mg) gave, in addition to the known compounds 2-phenylethanol (22.0 mg), tyrosol (10.0 mg), <bold>4</bold> (3.5 mg), <bold>5</bold> (3.8 mg), <bold>6</bold> (2.0 mg), <bold>7</bold> (1.0 mg), and <bold>8</bold> (49.1 mg), the new metabolites <bold>1</bold> (1.7 mg), <bold>2</bold> (0.8 mg), and <bold>3</bold> (1.0 mg). Purification of the extract fermented for 28 days (316 mg) afforded the known compounds 2-phenylethanol (31.0 mg), tyrosol (32.0 mg), <bold>4</bold> (10.9 mg), <bold>5</bold> (10.6 mg), <bold>6</bold> (2.9 mg), <bold>8</bold> (1.8 mg), <bold>9</bold> (1.0 mg), and <bold>10</bold> (1.5 mg), and the new metabolites <bold>2</bold> (1.2 mg), and <bold>3</bold> (2.0 mg) (<xref ref-type="fig" rid="f1"><bold>Figure 1</bold></xref>).</p>
<fig id="f1" position="float">
<label>Figure&#xa0;1</label>
<caption>
<p>Eremophilanes (<bold>1</bold>-<bold>17</bold>) isolated from the strain <italic>Emericellopsis maritima</italic> BC17 cultivated in liquid culture media.</p>
</caption>
<graphic mimetype="image" mime-subtype="tiff" xlink:href="fmars-11-1386175-g001.tif"/>
</fig>
<p>3-Deacetyl guignarderemophilane A (<bold>1</bold>): purified through semipreparative HPLC (<italic>n</italic>-hexane: EtOAc : Acetone 55:45:5, flow 2.0 mL/min, <italic>t</italic>
<sub>R</sub> = 31 min). White solid; mp 64.7&#xb0;C; <inline-formula>
<mml:math display="inline" id="im1">
<mml:mrow>
<mml:msubsup>
<mml:mrow>
<mml:mo stretchy="false">[</mml:mo>
<mml:mi>a</mml:mi>
<mml:mo stretchy="false">]</mml:mo>
</mml:mrow>
<mml:mtext>D</mml:mtext>
<mml:mrow>
<mml:mn>21</mml:mn>
</mml:mrow>
</mml:msubsup>
</mml:mrow>
</mml:math>
</inline-formula> -33.7&#xb0; (<italic>c</italic> 0.17, MeOH); ECD (MeOH) <italic>&#x3bb;</italic> (&#x394;<italic>&#x3f5;</italic>) 214 (5.74), 256 (-2.71), 288 (-2.03) nm; IR <italic>&#x3bd;</italic>
<sub>max</sub> (cm<sup>-1</sup>) 3447, 2932, 1637, 1448, 655; HRMS(ESI<sup>+</sup>) calcd. for C<sub>12</sub>H<sub>16</sub>O<sub>4</sub>Na [M+Na]<sup>+</sup> 247.0946, found 247.0933; <sup>1</sup>H and <sup>13</sup>C NMR data in <xref ref-type="table" rid="T1">
<bold>Table&#xa0;1</bold>
</xref>; gCOSY (selected correlations) H-1<italic>&#x3b1;</italic> &#x2192; H-1<italic>&#x3b2;</italic>, H-2<italic>&#x3b1;</italic>; H-1<italic>&#x3b2;</italic> &#x2192; H-1<italic>&#x3b1;</italic>, H-2<italic>&#x3b1;</italic>, H-9; H-2<italic>&#x3b1;</italic> &#x2192; H-1<italic>&#x3b2;</italic>, H-1<italic>&#x3b1;</italic>, H-3<italic>&#x3b1;</italic>; H-3<italic>&#x3b1;</italic> &#x2192; H-2<italic>&#x3b1;</italic>, H-4<italic>&#x3b1;</italic>; H-4<italic>&#x3b1;</italic> &#x2192; H-3<italic>&#x3b1;</italic>, Me-15<italic>&#x3b2;</italic>; H-9 &#x2192; H-1<italic>&#x3b2;</italic>; Me-15<italic>&#x3b2;</italic> &#x2192; H-4<italic>&#x3b1;</italic> (<xref ref-type="supplementary-material" rid="SM1"><bold>Supplementary Figure S3</bold></xref>); gHMBC (selected correlations) H-1<italic>&#x3b1;</italic> &#x2192; C-3, C-5, C-9, C-10; H-1<italic>&#x3b2;</italic> &#x2192; C-2, C-9, C-10; H-4<italic>&#x3b1;</italic> &#x2192; C-5, C-14, C-15; H-6 &#x2192; C-4, C-7, C-8, C-10, C-14; H-9 &#x2192; C-1, C-5, C-7; Me-14<italic>&#x3b2;</italic> &#x2192; C-4, C-5, C-6, C-10; Me-15<italic>&#x3b2;</italic> &#x2192; C-3, C-4, C-5 (<xref ref-type="supplementary-material" rid="SM1"><bold>Supplementary Figure S5</bold></xref>).</p>
<table-wrap id="T1" position="float">
<label>Table&#xa0;1</label>
<caption>
<p>
<sup>1</sup>H and <sup>13</sup>C NMR spectroscopic data for compounds <bold>1</bold>-<bold>3</bold>.</p>
</caption>
<table frame="hsides">
<thead>
<tr>
<th valign="middle" align="center"/>
<th valign="middle" colspan="2" align="center">1</th>
<th valign="middle" colspan="2" align="center">2</th>
<th valign="middle" colspan="2" align="center">3</th>
</tr>
</thead>
<tbody>
<tr>
<th valign="middle" align="center">Position</th>
<th valign="middle" align="center">
<italic>&#x3b4;</italic>
<sub>H</sub>, Mult (<italic>J</italic> in Hz)<sup>a</sup>
</th>
<th valign="middle" align="center">
<italic>&#x3b4;</italic>
<sub>C</sub>, Type<sup>b</sup>
</th>
<th valign="middle" align="center">
<italic>&#x3b4;</italic>
<sub>H</sub>, Mult (<italic>J</italic> in Hz)<sup>c</sup>
</th>
<th valign="middle" align="center">
<italic>&#x3b4;</italic>
<sub>C</sub>, Type<sup>d</sup>
</th>
<th valign="middle" align="center">
<italic>&#x3b4;</italic>
<sub>H</sub>, Mult (<italic>J</italic> in Hz)<sup>e</sup>
</th>
<th valign="middle" align="center">
<italic>&#x3b4;</italic>
<sub>C</sub>, Type<sup>d</sup>
</th>
</tr>
<tr>
<td valign="middle" align="center">1<italic>&#x3b1;</italic>
</td>
<td valign="middle" align="center">2.45, dd (12.0, 4.5)</td>
<td valign="middle" rowspan="2" align="center">37.5, CH<sub>2</sub>
</td>
<td valign="middle" align="center">2.16, m</td>
<td valign="middle" rowspan="2" align="center">28.0, CH<sub>2</sub>
</td>
<td valign="middle" align="center">2.55, m</td>
<td valign="middle" rowspan="2" align="center">29.0, CH<sub>2</sub>
</td>
</tr>
<tr>
<td valign="middle" align="center">1<italic>&#x3b2;</italic>
</td>
<td valign="middle" align="center">2.90, td (12.0, 1.5)</td>
<td valign="middle" align="center">2.90, m</td>
<td valign="middle" align="center">2.87, dddd (14.7, 11.3, 7.6, 1.8)</td>
</tr>
<tr>
<td valign="middle" align="center">2<italic>&#x3b1;</italic>
</td>
<td valign="middle" align="center">3.53, ddd (12.0, 4.5, 3.2)</td>
<td valign="middle" rowspan="2" align="center">74.6, CH</td>
<td valign="middle" align="center">1.67, m</td>
<td valign="middle" rowspan="2" align="center">37.2, CH<sub>2</sub>
</td>
<td valign="middle" align="center">2.19, ddt (14.2, 4.7, 2.3)</td>
<td valign="middle" rowspan="2" align="center">37.4, CH<sub>2</sub>
</td>
</tr>
<tr>
<td valign="middle" align="center">2<italic>&#x3b2;</italic>
</td>
<td valign="middle" align="center">&#x2013;</td>
<td valign="middle" align="center">2.16, m</td>
<td valign="middle" align="center">2.50, m</td>
</tr>
<tr>
<td valign="middle" align="center">3<italic>&#x3b1;</italic>
</td>
<td valign="middle" align="center">3.70, td (3.2, 3.0)</td>
<td valign="middle" rowspan="2" align="center">74.1, CH</td>
<td valign="middle" align="center">3.97, brs</td>
<td valign="middle" rowspan="2" align="center">70.8, CH</td>
<td valign="middle" align="center">&#x2013;</td>
<td valign="middle" rowspan="2" align="center">212.1, C</td>
</tr>
<tr>
<td valign="middle" align="center">3<italic>&#x3b2;</italic>
</td>
<td valign="middle" align="center">&#x2013;</td>
<td valign="middle" align="center">&#x2013;</td>
<td valign="middle" align="center">&#x2013;</td>
</tr>
<tr>
<td valign="middle" align="center">4</td>
<td valign="middle" align="center">1.41, qd (7.0, 3.0)</td>
<td valign="middle" align="center">43.4, CH</td>
<td valign="middle" align="center">1.75, qd (7.1, 3.3)</td>
<td valign="middle" align="center">43.7, CH</td>
<td valign="middle" align="center">2.60, qd (7.0, 3.3)</td>
<td valign="top" align="center">53.6, CH</td>
</tr>
<tr>
<td valign="middle" align="center">5</td>
<td valign="middle" align="center">&#x2013;</td>
<td valign="middle" align="center">45.0, C</td>
<td valign="middle" align="center">&#x2013;</td>
<td valign="middle" align="center">42.2, C</td>
<td valign="middle" align="center">&#x2013;</td>
<td valign="middle" align="center">42.2, C</td>
</tr>
<tr>
<td valign="middle" align="center">6<italic>&#x3b1;</italic>
</td>
<td valign="middle" align="center">6.30, s</td>
<td valign="middle" rowspan="2" align="center">127.4, CH</td>
<td valign="middle" align="center">2.08, d (15.0)</td>
<td valign="middle" rowspan="2" align="center">38.1, CH<sub>2</sub>
</td>
<td valign="middle" align="center">2.34, d (15.0)</td>
<td valign="middle" rowspan="2" align="center">37.5, CH<sub>2</sub>
</td>
</tr>
<tr>
<td valign="middle" align="center">6<italic>&#x3b2;</italic>
</td>
<td valign="middle" align="center">&#x2013;</td>
<td valign="middle" align="center">1.99, d (15.0)</td>
<td valign="middle" align="center">2.49, d (15.0)</td>
</tr>
<tr>
<td valign="middle" align="center">7</td>
<td valign="middle" align="center">&#x2013;</td>
<td valign="middle" align="center">147.9, C</td>
<td valign="middle" align="center">&#x2013;</td>
<td valign="middle" align="center">65.6, C</td>
<td valign="middle" align="center">&#x2013;</td>
<td valign="middle" align="center">127.0, C</td>
</tr>
<tr>
<td valign="middle" align="center">8</td>
<td valign="middle" align="center">&#x2013;</td>
<td valign="middle" align="center">183.9, C</td>
<td valign="middle" align="center">&#x2013;</td>
<td valign="middle" align="center">195.2, C</td>
<td valign="middle" align="center">&#x2013;</td>
<td valign="middle" align="center">190.5, C</td>
</tr>
<tr>
<td valign="bottom" align="center">9</td>
<td valign="middle" align="center">6.17, d (1.5)</td>
<td valign="middle" align="center">124.4, CH</td>
<td valign="middle" align="center">5.92, d (1.3)</td>
<td valign="middle" align="center">123.6, CH</td>
<td valign="middle" align="center">5.90, d (1.8)</td>
<td valign="middle" align="center">128.2, CH</td>
</tr>
<tr>
<td valign="bottom" align="center">10</td>
<td valign="bottom" align="center">-</td>
<td valign="middle" align="center">171.1, C</td>
<td valign="middle" align="center">-</td>
<td valign="middle" align="center">172.4, C</td>
<td valign="middle" align="center">-</td>
<td valign="middle" align="center">162.6, C</td>
</tr>
<tr>
<td valign="bottom" align="center">11</td>
<td valign="middle" align="center">&#x2013;</td>
<td valign="middle" align="center">&#x2013;</td>
<td valign="middle" align="center">&#x2013;</td>
<td valign="middle" align="center">64.7, C</td>
<td valign="middle" align="center">&#x2013;</td>
<td valign="middle" align="center">146.1, C</td>
</tr>
<tr>
<td valign="bottom" align="center">12</td>
<td valign="middle" align="center">&#x2013;</td>
<td valign="middle" align="center">&#x2013;</td>
<td valign="middle" align="center">1.41, s</td>
<td valign="middle" align="center">21.3, CH<sub>3</sub>
</td>
<td valign="middle" align="center">1.85, d (1.4)</td>
<td valign="middle" align="center">23.1, CH<sub>3</sub>
</td>
</tr>
<tr>
<td valign="bottom" align="center">13</td>
<td valign="middle" align="center">&#x2013;</td>
<td valign="middle" align="center">&#x2013;</td>
<td valign="middle" align="center">1.30, s</td>
<td valign="middle" align="center">19.1, CH<sub>3</sub>
</td>
<td valign="middle" align="center">2.15, d (2.0)</td>
<td valign="middle" align="center">22.8, CH<sub>3</sub>
</td>
</tr>
<tr>
<td valign="bottom" align="center">14</td>
<td valign="middle" align="center">1.35, s</td>
<td valign="middle" align="center">22.0, CH<sub>3</sub>
</td>
<td valign="middle" align="center">1.36, s</td>
<td valign="middle" align="center">24.5, CH<sub>3</sub>
</td>
<td valign="middle" align="center">1.20, s</td>
<td valign="middle" align="center">25.1, CH<sub>3</sub>
</td>
</tr>
<tr>
<td valign="bottom" align="center">15</td>
<td valign="middle" align="center">1.28, d (7.0)</td>
<td valign="middle" align="center">13.2, CH<sub>3</sub>
</td>
<td valign="middle" align="center">1.15, d (7.1)</td>
<td valign="middle" align="center">12.6, CH<sub>3</sub>
</td>
<td valign="middle" align="center">1.10, d (7.0)</td>
<td valign="middle" align="center">11.0, CH<sub>3</sub>
</td>
</tr>
</tbody>
</table>
<table-wrap-foot>
<fn>
<p>
<sup>a</sup>500 MHz, CD<sub>3</sub>OD; <sup>b</sup>125 MHz, CD<sub>3</sub>OD; <sup>c</sup>400 MHz, CDCl<sub>3</sub>; <sup>d</sup>125 MHz, CDCl<sub>3</sub>; <sup>e</sup>500 MHz, CDCl<sub>3</sub>. </p>
</fn>
</table-wrap-foot>
</table-wrap>
<p>(3<italic>S</italic>,4<italic>R</italic>,5<italic>R</italic>,7<italic>R</italic>)-3-Hydroxy-7(11)-epoxyeremophil-9-en-8-one (<bold>2</bold>): purified through semipreparative HPLC (<italic>n</italic>-hexane: EtOAc : Acetone 65:30:5, flow 2.5 mL/min, <italic>t</italic>
<sub>R</sub> = 51 min). Colourless oil. <inline-formula>
<mml:math display="inline" id="im2">
<mml:mrow>
<mml:msubsup>
<mml:mrow>
<mml:mo stretchy="false">[</mml:mo>
<mml:mi>a</mml:mi>
<mml:mo stretchy="false">]</mml:mo>
</mml:mrow>
<mml:mtext>D</mml:mtext>
<mml:mrow>
<mml:mn>21</mml:mn>
</mml:mrow>
</mml:msubsup>
</mml:mrow>
</mml:math>
</inline-formula> -25.4&#xb0; (<italic>c</italic> 1.3, CHCl<sub>3</sub>); ECD (MeOH) <italic>&#x3bb;</italic> (&#x394;<italic>&#x3f5;</italic>) 205 (0.76), 220 (-3.12), 268 (-2.56), 331 (2.31) nm; IR <italic>&#x3bd;</italic>
<sub>max</sub> (cm<sup>-1</sup>) 3496, 2850, 1674; HRMS(ESI<sup>+</sup>) calcd. for C<sub>15</sub>H<sub>22</sub>O<sub>3</sub>Na [M+Na]<sup>+</sup> 273.1467, found 273.1446; <sup>1</sup>H and <sup>13</sup>C NMR data in <xref ref-type="table" rid="T1">
<bold>Table&#xa0;1</bold>
</xref>; gCOSY (selected correlations) H-1<italic>&#x3b2;</italic> &#x2192; H-1<italic>&#x3b1;</italic>, H-2<italic>&#x3b1;</italic>, H-2<italic>&#x3b2;</italic>, H-9; H-2<italic>&#x3b1;</italic> &#x2192; H-1<italic>&#x3b2;</italic>, H-1<italic>&#x3b1;</italic>, H-2<italic>&#x3b2;</italic>, H-3<italic>&#x3b1;</italic>; H-3<italic>&#x3b1;</italic> &#x2192; H-2<italic>&#x3b1;</italic>, H-4<italic>&#x3b1;</italic>; H-4<italic>&#x3b1;</italic> &#x2192; Me-15<italic>&#x3b2;</italic>; H-9 &#x2192; H-1<italic>&#x3b2;</italic>; Me-15<italic>&#x3b2;</italic> &#x2192; H-4<italic>&#x3b1;</italic> (<xref ref-type="supplementary-material" rid="SM1"><bold>Supplementary Figure S10</bold></xref>); gHMBC (selected correlations) H-1<italic>&#x3b2;</italic> &#x2192; C-9, C-10; H<sub>2</sub>-6 &#x2192; C-4, C-5, C-7, C-10, C-11, C-14; H-9 &#x2192; C-1, C-5, C-7; Me-12 &#x2192; C-7, C-11, C-13; Me-13 &#x2192; C-7, C-11, C-12; Me-14<italic>&#x3b2;</italic> &#x2192; C-4, C-5, C-6, C-10; Me-15<italic>&#x3b2;</italic> &#x2192; C-3, C-4, C-5 (<xref ref-type="supplementary-material" rid="SM1"><bold>Supplementary Figure S12</bold></xref>).</p>
<p>4-<italic>Epi</italic>isopetasone (<bold>3</bold>): purified through semipreparative HPLC (<italic>n</italic>-hexane:EtOAc 80:20, flow 2.5 mL/min, <italic>t</italic>
<sub>R</sub> = 83 min). Colourless oil. <inline-formula>
<mml:math display="inline" id="im3">
<mml:mrow>
<mml:msubsup>
<mml:mrow>
<mml:mo stretchy="false">[</mml:mo>
<mml:mi>a</mml:mi>
<mml:mo stretchy="false">]</mml:mo>
</mml:mrow>
<mml:mtext>D</mml:mtext>
<mml:mrow>
<mml:mn>21</mml:mn>
</mml:mrow>
</mml:msubsup>
</mml:mrow>
</mml:math>
</inline-formula> +83.3&#xb0; (<italic>c</italic> 0.3, CHCl<sub>3</sub>); ECD (MeOH) <italic>&#x3bb;</italic> (&#x394;<italic>&#x3f5;</italic>) 213 (2.74), 246 (6.13), 282 (-2.80) nm; IR <italic>&#x3bd;</italic>
<sub>max</sub> (cm<sup>-1</sup>) 2924, 1717, 1660, 1458; HRMS(ESI<sup>+</sup>) calcd. for C<sub>15</sub>H<sub>21</sub>O<sub>2</sub> [M+H]<sup>+</sup> 233.1542, found 233.1543; <sup>1</sup>H and <sup>13</sup>C NMR data in <xref ref-type="table" rid="T1">
<bold>Table&#xa0;1</bold>
</xref>; gCOSY (selected correlations) H-1&#x3b2; &#x2192; H-1<italic>&#x3b1;</italic>, H-2, H-9; H-2 &#x2192; H-1<italic>&#x3b2;</italic>, H-1<italic>&#x3b1;</italic>; H-4<italic>&#x3b2;</italic> &#x2192; Me-15<italic>&#x3b1;</italic>; H-9 &#x2192; H-1<italic>&#x3b2;</italic>; Me-15<italic>&#x3b1;</italic> &#x2192; H-4<italic>&#x3b2;</italic> (<xref ref-type="supplementary-material" rid="SM1"><bold>Supplementary Figure S17</bold></xref>); gHMBC (selected correlations) H-1<italic>&#x3b2;</italic> &#x2192; C-2, C-9, C-10; H-4<italic>&#x3b2;</italic> &#x2192; C-5, C-14, C-15; H-9 &#x2192; C-1, C-5, C-7; Me-12 &#x2192; C-7, C-11, C-13; Me-13 &#x2192; C-7, C-11, C-12; Me-14<italic>&#x3b2;</italic> &#x2192; C-4, C-5, C-6, C-10; Me-15<italic>&#x3b1;</italic> &#x2192; C-3, C-4, C-5 (<xref ref-type="supplementary-material" rid="SM1"><bold>Supplementary Figure S19</bold></xref>).</p>
<p>Isopetasone (<bold>4</bold>): purified through semipreparative HPLC (<italic>n</italic>-hexane:EtOAc 85:15, flow 3.0 mL/min, t<sub>R</sub> = 62 min). <inline-formula>
<mml:math display="inline" id="im4">
<mml:mrow>
<mml:msubsup>
<mml:mrow>
<mml:mo stretchy="false">[</mml:mo>
<mml:mi>a</mml:mi>
<mml:mo stretchy="false">]</mml:mo>
</mml:mrow>
<mml:mtext>D</mml:mtext>
<mml:mrow>
<mml:mn>26</mml:mn>
</mml:mrow>
</mml:msubsup>
</mml:mrow>
</mml:math>
</inline-formula> +31&#xb0; (c 0.06, CHCl<sub>3</sub>). <sup>1</sup>H NMR (500 MHz, CDCl<sub>3</sub>) <italic>&#x3b4;</italic> 5.92 (dd, 1H, <italic>J</italic> = 1.8, 0.9 Hz, H-9), 2.82 (d, 1H, <italic>J</italic> = 13.9, H-6<italic>&#x3b2;</italic>), 2.79 &#x2013; 2.68 (m, 2H, H-1), 2.60 &#x2013; 2.48 (m, 2H, H-2), 2.57 (q, 1H, <italic>J</italic> = 6.6 Hz, H-4), 2.45 (br d, 1H, <italic>J</italic> = 13.9 Hz, H-6<italic>&#x3b1;</italic>), 2.11 (d, 3H, <italic>J</italic> = 1.9 Hz, H-12)<sup>a</sup>, 1.86 (d, 3H, <italic>J</italic> = 1.2 Hz, H-13)<sup>a</sup>, 1.08 (d, 3H, <italic>J</italic> = 6.7 Hz, H-15), 0.95 (s, 3H, H-14). <sup>13</sup>C NMR (125 MHz, CDCl<sub>3</sub>) <italic>&#x3b4;</italic> 210.2 (C-3, C), 191.0 (C-8, C), 162.8 (C-10, C), 144.6 (C-11, C), 127.7 (C-9, CH), 126.7 (C-7, C), 52.6 (C-4, CH), 44.6 (C-5, C), 40.7 (C-6, CH<sub>2</sub>), 39.8 (C-2, CH<sub>2</sub>), 31.1 (C-1, CH<sub>2</sub>), 22.7 (C-13, CH<sub>3</sub>)<sup>b</sup>, 22.3 (C-12, CH<sub>3</sub>)<sup>b</sup>, 18.5 (C-14, CH<sub>3</sub>), 7.5 (C-15, CH<sub>3</sub>). <sup>a,b</sup> Interchangeable assignments.</p>
<p>(+)-3-<italic>Epi</italic>isopetasol (<bold>5</bold>): purified through semipreparative HPLC (<italic>n</italic>-hexane: EtOAc : Acetone 80:20, flow 2.5 mL/min, t<sub>R</sub> = 86 min). <inline-formula>
<mml:math display="inline" id="im5">
<mml:mrow>
<mml:msubsup>
<mml:mrow>
<mml:mo stretchy="false">[</mml:mo>
<mml:mi>a</mml:mi>
<mml:mo stretchy="false">]</mml:mo>
</mml:mrow>
<mml:mtext>D</mml:mtext>
<mml:mrow>
<mml:mn>21</mml:mn>
</mml:mrow>
</mml:msubsup>
</mml:mrow>
</mml:math>
</inline-formula> +110&#xb0; (c 0.11, CHCl<sub>3</sub>). <sup>1</sup>H NMR (500 MHz, CDCl<sub>3</sub>) <italic>&#x3b4;</italic> 5.77 (d, <italic>J</italic> = 1.9 Hz, 1H, H-9), 3.93 (q, <italic>J</italic> = 2.8 Hz, 1H, H-3<italic>&#x3b1;</italic>), 2.87 (d, <italic>J</italic> = 13.0 Hz, 1H, H-6<italic>&#x3b2;</italic>), 2.76 (tdd, <italic>J</italic> = 14.4, 5.1, 1.9 Hz, 1H, H-1<italic>&#x3b2;</italic>), 2.12 (ddd, <italic>J</italic> = 14.4, 4.4, 2.6 Hz, 1H, H-1<italic>&#x3b1;</italic>), 2.09 (s, 3H, H-12)<sup>c</sup>, 2.08 (br d, 1H, <italic>J</italic> = 13.0 Hz, H-6<italic>&#x3b1;</italic>), 2.00 (ddt, <italic>J</italic> = 14.0, 5.1, 2.6 Hz, 1H, H-2<italic>&#x3b2;</italic>), 1.83 (d, <italic>J</italic> = 1.0 Hz, 3H, H-13)<sup>c</sup>, 1.69 (tdd, <italic>J</italic> = 14.0, 4.4, 2.8 Hz 1H, H-2<italic>&#x3b1;</italic>), 1.55 (qd, <italic>J</italic> = 7.3, 2.8 Hz, 1H, H-4), 1.15 (s, 3H, H-14),1.12 (d, <italic>J</italic> = 7.3 Hz, 3H, H-15). <sup>13</sup>C NMR (125 MHz, CDCl<sub>3</sub>) <italic>&#x3b4;</italic> 192.2 (C-8, C), 168.3 (C-10, C), 143.1 (C-11, C), 127.9 (C-7, C), 126.1 (C-9, CH), 71.3 (C-3, CH), 45.4 (C-4, CH), 41.9 (C-6, CH<sub>2</sub>), 41.4 (C-5, C), 33.7 (C-2, CH<sub>2</sub>), 26.7 (C-1, CH<sub>2</sub>), 22.6 (C-12, CH<sub>3</sub>)<sup>d</sup>, 22.1 (C-13, CH<sub>3</sub>)<sup>d</sup>, 18.9 (C-14, CH<sub>3</sub>), 12.1 (C-15, CH<sub>3</sub>). <sup>c,d</sup> Interchangeable assignments.</p>
<p>1<italic>&#x3b1;</italic>-Hydroxydehydrofukinone (<bold>6</bold>): purified through semipreparative HPLC (<italic>n</italic>-hexane:EtOAc 72:28, flow 3.0 mL/min, t<sub>R</sub> = 39 min). <inline-formula>
<mml:math display="inline" id="im6">
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<mml:mn>22</mml:mn>
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</inline-formula> +47&#xb0; (c 0.05, CHCl<sub>3</sub>). <sup>1</sup>H NMR (500 MHz, CDCl<sub>3</sub>) <italic>&#x3b4;</italic> 5.84 (s, 1H, H-9), 4.33 (t, <italic>J</italic> = 3.0 Hz, 1H, H-1<italic>&#x3b2;</italic>), 2.90 (d, <italic>J</italic> = 14.2 Hz, 1H, H-6<italic>&#x3b2;</italic>), 2.12 (br d, <italic>J</italic> = 14.2 Hz, H-6<italic>&#x3b1;</italic>), 2.11 (s, 3H, H-12)<sup>e</sup>, 1.99 (dq, <italic>J</italic> = 13.9, 3.0 Hz, 1H, H-2<italic>&#x3b1;</italic>), 1.85 (s, 3H, H-13)<sup>e</sup>, 1.83 (qd, <italic>J</italic> = 13.4, 3.3 Hz, 1H, H-3<italic>&#x3b1;</italic>), 1.67 (tt, <italic>J</italic> = 13.9, 3.5 Hz, 1H, H-2<italic>&#x3b2;</italic>), 1.48 (dqd, <italic>J</italic> = 13.4, 6.9, 3.3 Hz, 1H, H-4), 1.39 (dq, <italic>J</italic> = 13.4, 3.5 Hz, 1H, H-3<italic>&#x3b2;</italic>), 1.14 (s, 3H, H-14), 0.98 (d, <italic>J</italic> = 6.8 Hz, 3H, H-15). <sup>13</sup>C NMR (125 MHz, CDCl<sub>3</sub>) <italic>&#x3b4;</italic> 192.4 (C-8, C), 165.6 (C-10, C), 144.3 (C-11, C), 129.1 (C-9, CH), 128.1 (C-7, C), 72.7 (C-1, CH), 42.3 (C-6, CH<sub>2</sub>), 42.3 (C-4, CH), 41.0 (C-5, C), 32.8 (C-2, CH<sub>2</sub>), 24.8 (C-3, CH<sub>2</sub>), 22.8 (C-12, CH<sub>3</sub>)<sup>f</sup>, 22.4 (C-13, CH<sub>3</sub>)<sup>f</sup>, 18.1 (C-14, CH<sub>3</sub>), 15.5 (C-15, CH<sub>3</sub>). <sup>e,f</sup> Interchangeable assignments.</p>
<p>Eremofortin A alcohol (<bold>7</bold>): purified through semipreparative HPLC (<italic>n</italic>-hexane:EtOAc 60:40, flow 3.0 mL/min, t<sub>R</sub> = 31 min). <inline-formula>
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<mml:mn>23</mml:mn>
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</inline-formula> +71&#xb0; (c 0.06, CHCl<sub>3</sub>). <sup>1</sup>H NMR (600 MHz, CDCl<sub>3</sub>) <italic>&#x3b4;</italic> 6.39 (s, 1H, H-9), 4.11 (dt, <italic>J</italic> = 9.9, 5.1 Hz, 1H, H-3), 3.87 (dd, <italic>J</italic> = 5.1, 3.6 Hz, 1H, H-2), 3.76 (d, <italic>J</italic> = 3.6 Hz, 1H, H-1), 2.09 (dd, <italic>J</italic> = 14.7, 1.0 Hz, 1H, H-6<italic>&#x3b1;</italic>), 1.92 (d, <italic>J</italic> = 14.7 Hz, 1H, H-6<italic>&#x3b2;</italic>), 1.54 (m, 1H, H-4), 1.52 (s, 3H, H-13), 1.38 (s, 3H, H-12), 1.28 (s, 3H, H-14), 1.11 (d, <italic>J</italic> = 7.1 Hz, 3H, H-15). <sup>13</sup>C NMR (150 MHz, CDCl<sub>3</sub>) <italic>&#x3b4;</italic> 194.0 (C-8, C), 160.5 (C-10, C), 131.9 (C-9, CH), 67.7 (C-3, CH), 64.6 (C-11, C), 62.3 (C-7, C), 58.0 (C-1, CH), 58.0 (C-2, CH), 44.0 (C-4, CH), 42.1 (C-6, CH<sub>2</sub>), 37.1 (C-5, C), 23.4 (C-14, CH<sub>3</sub>), 21.5 (C-12, CH<sub>3</sub>), 19.4 (C-13, CH<sub>3</sub>), 10.6 (C-15, CH<sub>3</sub>).</p>
<p>(+)-Aristolochene (<bold>8</bold>): purified through silica gel column (<italic>n</italic>-hexane:EtOAc 100:0). <inline-formula>
<mml:math display="inline" id="im8">
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<mml:mrow>
<mml:mn>24</mml:mn>
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</inline-formula> +47&#xb0; (c 0.23, CHCl<sub>3</sub>). <sup>1</sup>H NMR (500 MHz, CDCl<sub>3</sub>) <italic>&#x3b4;</italic> 5.31 (dt, <italic>J</italic> = 5.6, 2.0 Hz, 1H, H-9), 4.71 (m, 2H, H-13), 2.21 (m, 1H, H-7<italic>&#x3b2;</italic>), 2.13 (m, 1H, H-2<italic>&#x3b1;</italic>)<sup>g</sup>, 2.05 &#x2013; 1.97 (m, 2H, H-2<italic>&#x3b2;</italic>, H-8<italic>&#x3b1;</italic>)<sup>g,i,</sup> 1.87 (dddd, J = 16.9, 11.6, 3.4, 2.0 Hz, 1H, H-8<italic>&#x3b2;</italic>)<sup>i</sup>, 1.77 (dt, <italic>J</italic> = 12.7, 2.3 Hz, 1H, H-6<italic>&#x3b1;</italic>)<sup>h</sup>, 1.74 (t, <italic>J</italic> = 1.1 Hz, 3H, H-12), 1.73 (m, 1H, H-3<italic>&#x3b2;</italic>)<sup>j</sup>, 1.42 (m, 1H, H-1<italic>&#x3b1;</italic>)<sup>k</sup>, 1.35 (td, <italic>J</italic> = 12.2, 11.7, 2.8 Hz, 1H, H-1<italic>&#x3b2;</italic>)<sup>k</sup>, 1.31 &#x2013; 1.22 (m, 2H, H-3<italic>&#x3b1;</italic>, H-4)<sup>j</sup>, 1.16 (td, <italic>J</italic> = 12.7, 0.9 Hz, 1H, H-6<italic>&#x3b2;</italic>)<sup>h</sup>, 0.97 (s, 3H, H-14), 0.84 (d, J = 6.6 Hz, 3H, H-15). <sup>13</sup>C NMR (125 MHz, CDCl<sub>3</sub>) <italic>&#x3b4;</italic> 150.6 (C-11, C), 144.4 (C-10, C), 118.8 (C-9, CH), 108.3 (C-13, CH<sub>2</sub>), 44.2 (C-4, CH), 43.3 (C-6, CH<sub>2</sub>), 38.7 (C-5, C), 37.8 (C-7, CH), 32.6 (C-2, CH<sub>2</sub>), 31.3 (C-8, CH<sub>2</sub>), 31.1 (C-1, CH<sub>2</sub>), 27.8 (C-3, CH<sub>2</sub>), 20.8 (C-12, CH<sub>3</sub>), 18.1 (C-14, CH<sub>3</sub>), 15.7 (C-15, CH<sub>3</sub>). <sup>g&#x2013;k</sup> Interchangeable assignments.</p>
<p>7-Hydroxy-4a,5-dimethyl-3-prop-1-en-2-yl-3,4,5,6,7,8-hexahydronaphthalen-2-one (<bold>9</bold>): purified through analytical HPLC (<italic>n</italic>-hexane:EtOAc 60:40, flow 1.0 mL/min, t<sub>R</sub> = 32 min). <inline-formula>
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</inline-formula> +90&#xb0; (c 0.12, CHCl<sub>3</sub>). <sup>1</sup>H NMR (500 MHz, CDCl<sub>3</sub>) <italic>&#x3b4;</italic> 5.81 (d, <italic>J</italic> = 2.0 Hz, 1H, H-9), 4.97 (p, <italic>J</italic> = 1.5 Hz, 1H, H-13), 4.81 (dq, <italic>J</italic> = 1.8, 0.8 Hz, 1H, H-13&#x2019;), 4.20 (br s, 1H, H-2<italic>&#x3b2;</italic>), 3.13 (dd, <italic>J</italic> = 13.8, 5.1 Hz, 1H, H-7), 2.59 (ddd, <italic>J</italic> = 15.2, 3.4, 2.1 Hz, 1H, H-1<italic>&#x3b2;</italic>), 2.37 (m, 1H, H-1<italic>&#x3b1;</italic>), 2.05 &#x2013; 1.89 (m, 3H, H-4, H-6), 1.73 (dd, <italic>J</italic> = 1.5, 0.8 Hz, 3H, H-12), 1.69 (ddd, <italic>J</italic> = 7.9, 3.0, 1.7, 2H, H-3), 1.16 (s, 3H, H-14). 0.92 (d, <italic>J</italic> = 6.9 Hz, 3H, H-15). <sup>13</sup>C NMR (125 MHz, CDCl<sub>3</sub>) <italic>&#x3b4;</italic> 198.2 (C-8, C), 166.2 (C-10, C), 143.6 (C-11, C), 127.0 (C-9, CH), 114.2 (C-13, CH<sub>2</sub>), 66.8 (C-2, CH), 51.0 (C-7, CH), 41.2 (C-6, CH<sub>2</sub>), 40.0 (C-1, CH<sub>2</sub>), 39.3 (C-5, C), 37.1 (C-3, CH<sub>2</sub>), 36.3 (C-4, CH), 20.0 (C-12, CH<sub>3</sub>), 15.4 (C-14, CH<sub>3</sub>), 14.7 (C-15, CH<sub>3</sub>).</p>
<p>Warburgiadione (<bold>10</bold>): purified through analytical HPLC (<italic>n</italic>-hexane:acetone 95:5, flow 1.0 mL/min, tR = 32 min. <sup>1</sup>H NMR (500 MHz, CDCl<sub>3</sub>) <italic>&#x3b4;</italic> 6.99 (dt, <italic>J</italic> = 9.8, 0.7 Hz, 1H, H-1), 6.21 (dd, <italic>J</italic> = 9.8, 0.7 Hz, 1H, H-2), 6.07 (t, <italic>J</italic> = 0.7 Hz, 1H, H-9), 2.99 (d, <italic>J</italic> = 13.9 Hz, 1H, H-6<italic>&#x3b2;</italic>), 2.61 (q, <italic>J</italic> = 6.8 Hz, 1H, H-4), 2.38 (br d, <italic>J</italic> = 13.9 Hz, 1H, H-6<italic>&#x3b1;</italic>), 2.18 (d, <italic>J</italic> = 2.2 Hz, 3H, H-12)<sup>l</sup>, 1.92 (d, <italic>J</italic> = 1.5 Hz, 3H, H-13)<sup>l</sup>, 1.17 (d, <italic>J</italic> = 6.8 Hz, 3H, H-15), 0.98 (s, 3H, H-14). <sup>13</sup>C NMR (125 MHz, CDCl<sub>3</sub>) <italic>&#x3b4;</italic> 200.0 (C-3, C), 190.6 (C-8, C), 156.6 (C-10, C), 148.4 (C-11, C), 142.0 (C-1, CH), 131.8 (C-2, CH), 131.6 (C-9, CH), 126.3 (C-7, C), 51.4 (C-4, CH), 41.4 (C-5, C), 40.0 (C-6, CH<sub>2</sub>), 23.4 (C-12, CH<sub>3</sub>)<sup>m</sup>, 23.0 (C-13, CH<sub>3</sub>)<sup>m</sup>, 18.1 (C-14, CH<sub>3</sub>), 7.1 (C-15, CH<sub>3</sub>). <sup>l,m</sup> Interchangeable assignments.</p>
<p>Purification of the extracts fermented in PDB medium for 14 days (331 mg) and 28 days (261 mg) only afforded tyrosol (8 mg and 18.0 mg for 14 and 28 days, respectively) and 2-phenylethanol (88 mg for 14 days and 80.0 mg for 28 days).</p>
</sec>
<sec id="s2_4_2">
<label>2.4.2</label>
<title>Shaken culture fermentation</title>
<p>The resulting extract (388 mg) of the Czapek Dox fermentation was chromatographed and subsequently purified employing HPLC to yield the known compounds <bold>4</bold> (2.8 mg), <bold>5</bold> (4.0 mg), <bold>6</bold> (4.0 mg), <bold>7</bold> (0.6 mg), <bold>9</bold> (10.0 mg), <bold>10</bold> (2.0 mg), <bold>11</bold> (1.2 mg), <bold>12</bold> (1.6 mg), <bold>13</bold> (6.0 mg), <bold>14</bold> (0.5 mg), and <bold>15</bold> (9.2 mg) (<xref ref-type="fig" rid="f1"><bold>Figure 1</bold></xref>).</p>
<p>Chromatography and purification of the extract fermented in PDB medium (168 mg) yielded the known compounds <bold>4</bold> (8.0 mg), <bold>9</bold> (0.2 mg), <bold>16</bold> (0.5 mg), and <bold>17</bold> (1.0 mg) (<xref ref-type="fig" rid="f1"><bold>Figure 1</bold></xref>).</p>
<p>(1<italic>R</italic>,7<italic>S</italic>,8a<italic>R</italic>)-1,8a-Dimethyl-7-(prop-1-en-2-yl)-1,7,8,8a-tetrahydronaphthalene-2,6-dione (<bold>11</bold>): purified through analytical HPLC (<italic>n</italic>-hexane:acetone 95:5, flow 1.0 mL/min, t<sub>R</sub> = 39 min). <inline-formula>
<mml:math display="inline" id="im10">
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</inline-formula> +117&#xb0; (c 0.38, CHCl<sub>3</sub>). <sup>1</sup>H NMR (500 MHz, CDCl<sub>3</sub>) <italic>&#x3b4;</italic> 7.03 (d, <italic>J</italic> = 9.8 Hz, 1H, H-1), 6.26 (d, <italic>J</italic> = 9.8 Hz, 1H, H-2), 6.09 (s, 1H, H-9), 5.07 (p, <italic>J</italic> = 1.5 Hz, 1H, H-13), 4.91 (dt, <italic>J</italic> = 1.5, 0.8 Hz, 1H, H-13&#x2019;), 3.24 (dd, <italic>J</italic> = 13.5, 5.0 Hz, 1H, H-7), 2.63 (q, <italic>J</italic> = 6.8 Hz, 1H, H-4), 2.16 (d, <italic>J</italic> = 13.5 Hz, 1H, H-6), 2.09 (dd, <italic>J</italic> = 13.5, 5.0 Hz, 1H, H-6&#x2019;), 1.78 (d, <italic>J</italic> = 1.5, 3H, H-12), 1.25 (s, 3H, H-14), 1.18 (d, <italic>J</italic> = 6.8 Hz, 3H, H-15). <sup>13</sup>C NMR (125 MHz, CDCl<sub>3</sub>) <italic>&#x3b4;</italic> 199.7 (C-3, C), 198.3 (C-8, C), 158.7 (C-10, C), 142.6 (C-11, C), 142.0 (C-1, CH), 132.2 (C-2, CH), 129.0 (C-9, CH), 115.3 (C-13, CH<sub>2</sub>), 52.2 (C-4, CH), 50.9 (C-7, CH), 40.4 (C-5, C), 40.3 (C-6, CH<sub>2</sub>), 19.9 (C-12, CH<sub>3</sub>), 18.6 (C-14, CH<sub>3</sub>), 7.0 (C-15, CH<sub>3</sub>).</p>
<p>Eremofortine B (<bold>12</bold>): purified through analytical HPLC (<italic>n</italic>-hexane: EtOAc 55:45, flow 1.0 mL/min, t<sub>R</sub> = 22 min). <inline-formula>
<mml:math display="inline" id="im11">
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</inline-formula> +115&#xb0; (c 0.09, CHCl<sub>3</sub>). <sup>1</sup>H NMR (400 MHz, CDCl<sub>3</sub>) <italic>&#x3b4;</italic> 6.20 (s, 1H, H-9), 5.00 (br s, 1H, H-13), 4.84 (br s, 1H, H-13&#x2019;), 4.12 (dt, <italic>J</italic> = 9.7, 5.0 Hz, 1H, H-3), 3.85 (dd, <italic>J</italic> = 5.0, 3.7 Hz, 1H, H-2), 3.69 (d, <italic>J</italic> = 3.7 Hz, 1H, H-1), 3.26 (dd, <italic>J</italic> = 14.1, 4.6 Hz, 1H, H-7), 1.90 (dd, <italic>J</italic> = 12.9, 4.6 Hz, 1H, H-6), 1.83 &#x2013; 1.74 (m, 1H, H-6&#x2019;), 1.72 (s, 3H, H-12), 1.51 (m, 1H, H-4), 1.35 (s, 3H, H-14), 1.09 (d, <italic>J</italic> = 7.1 Hz, 3H, H-15).</p>
<p>3<italic>&#x3b2;</italic>-hydroxy-7<italic>&#x3b2;</italic>-eremophil-1(2),9(10),11(12)-trien-8-one (<bold>13</bold>): purified through analytical HPLC (<italic>n</italic>-hexane:EtOAc 70:30, flow 1.0 mL/min, t<sub>R</sub> = 22 min). <inline-formula>
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</mml:mrow>
</mml:math>
</inline-formula> +377&#xb0; (c 0.25, CHCl<sub>3</sub>). <sup>1</sup>H NMR (400 MHz, CDCl<sub>3</sub>) <italic>&#x3b4;</italic> 6.31 &#x2013; 6.23 (m, 2H, H-1, H-2), 5.79 (s, 1H, H-9), 4.98 (p, <italic>J</italic> = 1.5 Hz, 1H, H-13), 4.86 (dt, <italic>J</italic> = 1.8, 0.8 Hz, 1H, H-13&#x2019;), 4.18 (q, <italic>J</italic> = 4.6 Hz, 1H, H-3), 3.24 (dd, <italic>J</italic> = 13.9, 4.9 Hz, 1H, H-7), 2.01 (dd, <italic>J</italic> = 12.9, 4.9 Hz, 1H, H-6)<sup>n</sup>, 1.85 (dd, <italic>J</italic> = 13.9, 12.9 Hz, 1H, H-6&#x2019;)<sup>n</sup>, 1.76 (td, <italic>J</italic> = 7.2, 4.6 Hz, 1H, H-4), 1.72 (dd, <italic>J</italic> = 1.5, 0.8 Hz, 3H, H-12), 1.32 (s, 3H, H-14), 1.15 (d, <italic>J</italic> = 7.2 Hz, 3H, H-15). <sup>13</sup>C NMR (100 MHz, CDCl<sub>3</sub>) <italic>&#x3b4;</italic> 199.0 (C-8, C), 161.6 (C-10, CH), 143.7 (C-11, C), 136.1 (C-2, CH), 129.3 (C-1, CH), 125.5 (C-9, CH), 114.7 (C-13, CH<sub>3</sub>), 68.2 (C-3, CH), 51.4 (C-7, CH), 42.0 (C-4, CH), 40.4 (C-6, CH<sub>2</sub>), 36.2 (C-5, C), 19.8 (C-12, CH<sub>3</sub>), 18.9 (C-14, CH<sub>3</sub>), 10.4 (C-15, CH<sub>3</sub>). <sup>n</sup> Interchangeable assignments.</p>
<p>PR toxin (<bold>14</bold>): purified through analytical HPLC (<italic>n</italic>-hexane:EtOAc 60:40, flow 1.0 mL/min, t<sub>R</sub> = 29 min). <inline-formula>
<mml:math display="inline" id="im13">
<mml:mrow>
<mml:msubsup>
<mml:mrow>
<mml:mo stretchy="false">[</mml:mo>
<mml:mi>a</mml:mi>
<mml:mo stretchy="false">]</mml:mo>
</mml:mrow>
<mml:mtext>D</mml:mtext>
<mml:mrow>
<mml:mn>24</mml:mn>
</mml:mrow>
</mml:msubsup>
</mml:mrow>
</mml:math>
</inline-formula> +239&#xb0; (c 0.10, CHCl<sub>3</sub>). <sup>1</sup>H NMR (500 MHz, CDCl<sub>3</sub>) <italic>&#x3b4;</italic> 9.70 (s, 1H, H-12), 6.41 (s, 1H, H-9), 5.13 (t, <italic>J</italic> = 5.1 Hz, 1H, H-3), 3.95 (dd, <italic>J</italic> = 5.1, 3.5 Hz, 1H, H-2), 3.63 (d, <italic>J</italic> = 3.5 Hz, 1H, H-1), 2.15 (s, 3H, H-17), 2.14 (d, <italic>J</italic> = 14.2 Hz, 1H, H-6<italic>&#x3b1;</italic>), 1.82 (d, <italic>J</italic> = 14.2 Hz, 1H, H-6<italic>&#x3b2;</italic>), 1.77 (qd, <italic>J</italic> = 7.1, 5.1 Hz, 1H, H-4), 1.47 (s, 3H, H-13), 1.43 (s, 3H, H-14), 1.01 (d, <italic>J</italic> = 7.1 Hz, 3H, H-15). <sup>13</sup>C NMR (125 MHz, CDCl<sub>3</sub>) <italic>&#x3b4;</italic> 198.6 (C-12, C), 191.6 (C-8, C), 170.6 (C-16, C), 164.5 (C-10, C), 129.9 (C-9, CH), 69.8 (C-3, CH), 67.4 (C-11, C), 67.3 (C-7, C), 55.9 (C-1, CH), 55.5 (C-2, CH), 42.8 (C-4, CH), 41.6 (C-6, CH<sub>2</sub>), 38.1 (C-5, C), 21.9 (C-14, CH<sub>3</sub>), 20.7 (C-17, CH<sub>3</sub>), 13.7 (C-13, CH<sub>3</sub>), 10.1 (C-15, CH<sub>3</sub>).</p>
<p>PR toxin 3-deacetyl (<bold>15</bold>): purified through analytical HPLC (<italic>n</italic>-hexane:EtOAc 60:40, flow 1.0 mL/min, t<sub>R</sub> = 9 min). <inline-formula>
<mml:math display="inline" id="im14">
<mml:mrow>
<mml:msubsup>
<mml:mrow>
<mml:mo stretchy="false">[</mml:mo>
<mml:mi>a</mml:mi>
<mml:mo stretchy="false">]</mml:mo>
</mml:mrow>
<mml:mtext>D</mml:mtext>
<mml:mrow>
<mml:mn>25</mml:mn>
</mml:mrow>
</mml:msubsup>
</mml:mrow>
</mml:math>
</inline-formula> -142.7&#xb0; (c 0.70, CHCl<sub>3</sub>). <sup>1</sup>H NMR (400 MHz, CDCl<sub>3</sub>) <italic>&#x3b4;</italic> 9.70 (s, 1H, H-12), 6.42 (s, 1H, H-9), 4.13 (dt, <italic>J</italic> = 9.8, 5.1 Hz, 1H, H-3), 3.91 (dd, <italic>J</italic> = 5.1, 3.6 Hz, 1H, H-2), 3.76 (d, <italic>J</italic> = 3.6 Hz, 1H, H-1), 2.10 (dd, <italic>J</italic> = 14.4, 1.1 Hz, 1H, H-6<italic>&#x3b1;</italic>), 1.85 (d, <italic>J</italic> = 14.4 Hz, 1H, H-6<italic>&#x3b2;</italic>), 1.58 &#x2013; 1.50 (m, 1H, H-4), 1.47 (s, 3H, H-13), 1.42 (d, <italic>J</italic> = 1.0 Hz, 3H, H-14), 1.12 (d, <italic>J</italic> = 7.1 Hz, 3H, H-15).</p>
<p>(3<italic>S</italic>)-Acetoxy-7(11)-epoxyeremophil-9-en-8-one (<bold>16</bold>): purified through semipreparative HPLC (<italic>n</italic>-hexane:EtOAc 60:40, flow 3.0 mL/min, t<sub>R</sub> = 42 min). <inline-formula>
<mml:math display="inline" id="im15">
<mml:mrow>
<mml:msubsup>
<mml:mrow>
<mml:mo stretchy="false">[</mml:mo>
<mml:mi>a</mml:mi>
<mml:mo stretchy="false">]</mml:mo>
</mml:mrow>
<mml:mtext>D</mml:mtext>
<mml:mrow>
<mml:mn>23</mml:mn>
</mml:mrow>
</mml:msubsup>
</mml:mrow>
</mml:math>
</inline-formula> +72&#xb0; (c 0.51, CHCl<sub>3</sub>). <sup>1</sup>H NMR (600 MHz, CDCl<sub>3</sub>) <italic>&#x3b4;</italic> 5.94 (s, 1H, H-9), 5.12 (q, <italic>J</italic> = 3.4 Hz, 1H, H-3), 2.73 (td, <italic>J</italic> = 13.5, 4.7 Hz, 1H, H-1<italic>&#x3b2;</italic>), 2.20 (m, 1H, H-1<italic>&#x3b1;</italic>), 2.19 (ddt, <italic>J</italic> = 14.2, 4.7, 2.3 Hz, 1H, H-2<italic>&#x3b2;</italic>), 2.12 (s, 3H, H-17), 2.10 (d, <italic>J</italic> = 15.0 Hz, 1H, H-6)<sup>p</sup>, 2.04 (d, <italic>J</italic> = 15.0 Hz, 1H, H-6&#x2019;)<sup>p</sup>, 1.89 (qd, <italic>J</italic> = 7.0, 3.4 Hz, 1H, H-4), 1.67 (tt, <italic>J</italic> = 14.2, 3.8 Hz, 1H, H-2<italic>&#x3b1;</italic>), 1.41 (s, 3H, H-12), 1.32 (s, 3H, H-14), 1.31 (s, 3H, H-13), 1.03 (d, <italic>J</italic> = 7.0 Hz, 3H, H-15). <sup>13</sup>C NMR (150 MHz, CDCl<sub>3</sub>) <italic>&#x3b4;</italic> 195.0 (C-8, C), 170.7 (C-10, C), 170.3 (C-16, C), 124.1 (C-9, CH), 72.5 (C-3, CH), 65.4 (C-7, C), 64.8 (C-11, C), 42.7 (C-4, CH), 41.9 (C-5, C), 38.0 (C-6, CH<sub>2</sub>), 33.9 (C-2, CH<sub>2</sub>), 28.3 (C-1, CH<sub>2</sub>), 23.8 (C-14, CH<sub>3</sub>), 21.3 (C-12, CH<sub>3</sub>), 21.3 (C-17, CH<sub>3</sub>), 19.2 (C-13, CH<sub>3</sub>), 12.2 (C-15, CH<sub>3</sub>). <sup>p</sup> Interchangeable assignments.</p>
<p>Eremofortine A (<bold>17</bold>): purified through semipreparative HPLC (<italic>n</italic>-hexane:EtOAc 60:40, flow 1.0 mL/min, t<sub>R</sub> = 54 min). <inline-formula>
<mml:math display="inline" id="im16">
<mml:mrow>
<mml:msubsup>
<mml:mrow>
<mml:mo stretchy="false">[</mml:mo>
<mml:mi>a</mml:mi>
<mml:mo stretchy="false">]</mml:mo>
</mml:mrow>
<mml:mtext>D</mml:mtext>
<mml:mrow>
<mml:mn>23</mml:mn>
</mml:mrow>
</mml:msubsup>
</mml:mrow>
</mml:math>
</inline-formula> +137&#xb0; (c 0.07, CHCl<sub>3</sub>). <sup>1</sup>H NMR (500 MHz, CDCl<sub>3</sub>) &#x3b4; 6.38 (s, 1H, H-9), 5.13 (t, <italic>J</italic> = 5.0 Hz, 1H, H-3), 3.89 (dd, <italic>J</italic> = 5.0, 3.5 Hz, 1H, H-2), 3.63 (d, <italic>J</italic> = 3.5 Hz, 1H, H-1), 2.14 (s, 3H, H-17), 2.12 (d, <italic>J</italic> = 14.7 Hz, 1H, H-6<italic>&#x3b1;</italic>), 1.92 (d, <italic>J</italic> = 14.7 Hz, 1H, H-6<italic>&#x3b2;</italic>), 1.77 (m, 1H, H-4), 1.52 (s, 3H, H-13), 1.39 (s, 3H, H-12), 1.30 (s, 3H, H-14), 1.00 (d, <italic>J</italic> = 7.1 Hz, 3H, H-15). <sup>13</sup>C NMR (125 MHz, CDCl<sub>3</sub>) <italic>&#x3b4;</italic> 193.9 (C-8, C), 170.7 (C-16, C), 160.7 (C-10, C), 131.4 (C-9, CH), 69.9 (C-3, CH), 64.6 (C-11, C), 62.4 (C-7, C), 55.9 (C-1, CH), 55.1 (C-2, CH), 42.0 (C-4, CH), 41.8 (C-6, CH<sub>2</sub>), 37.2 (C-5, C), 22.9 (C-14, CH<sub>3</sub>), 21.6 (C-12, CH<sub>3</sub>), 20.7 (C-17, CH<sub>3</sub>), 19.5 (C-13, CH<sub>3</sub>), 10.4 (C-15, CH<sub>3</sub>).</p>
</sec>
</sec>
<sec id="s2_5">
<label>2.5</label>
<title>Acetylation of compound 15</title>
<p>Compound <bold>15</bold> (0.97 mg, 0.0035 mmol) was treated with a small crystal of <italic>p</italic>-toluenesulfonic acid in 2 mL of acetic anhydride. The reaction was stirred for 24 h at room temperature, neutralized with H<sub>2</sub>O and Na<sub>2</sub>CO<sub>3</sub> and extracted with ethyl acetate. The organic layer was dried over dry Na<sub>2</sub>SO<sub>4</sub> and the solvent eliminated by distillation under reduced pressure. Finally, the product was purified using an analytical normal-phase HPLC column (LiChrospher Si 60, 250 x 4 mm, 5 &#xb5;m) applying an isocratic method (<italic>n</italic>-hexane:EtOAc 70:30, 1 mL/min) to yield <bold>14</bold> (0.25 mg, 0.0008 mmol, 22% yield, t<sub>R</sub> 31 min).</p>
</sec>
<sec id="s2_6">
<label>2.6</label>
<title>Computational details of EDC calculations</title>
<p>The molecular structure analysis of compounds <bold>1</bold>-<bold>3</bold> employed the semiempirical PM6 method (<xref ref-type="bibr" rid="B40">Stewart, 2007</xref>). Quantum mechanical computations were carried out using the Gaussian 16 package (<xref ref-type="bibr" rid="B20">Frisch et&#xa0;al., 2016</xref>). A comprehensive geometric optimization was conducted with Density Functional Theory (DFT) employing B3LYP functionals (<xref ref-type="bibr" rid="B29">Lee et&#xa0;al., 1988</xref>; <xref ref-type="bibr" rid="B5">Becke, 1993</xref>) and the 6&#x2212;311+G(2d,p) basis set. Following this, calculations were executed to determine energies, oscillator strengths, and rotational strengths for the initial 20 electronic excitations, employing the TD-DFT methodology (<xref ref-type="bibr" rid="B4">Bauernschmitt and Ahlrichs, 1996</xref>; <xref ref-type="bibr" rid="B14">Casida et&#xa0;al., 1998</xref>). The solvent effect (methanol) was considered in the calculations using the Polarizable Continuum Model (PCM) with the Implicit Solvation Energy (IEF) approach (<xref ref-type="bibr" rid="B12">Canc&#xe8;s et&#xa0;al., 1997</xref>; <xref ref-type="bibr" rid="B33">Mennucci et&#xa0;al., 1997</xref>; <xref ref-type="bibr" rid="B43">Tomasi et&#xa0;al., 2005</xref>). To replicate the ECD spectrum of the conformer, a Gaussian function was utilized with a half-bandwidth of 0.33 eV.</p>
</sec>
<sec id="s2_7">
<label>2.7</label>
<title>
<italic>In vitro</italic> antimicrobial assays</title>
<p>The antimicrobial activities of compounds <bold>1</bold>, <bold>3</bold>, <bold>11</bold>, and <bold>15</bold> were evaluated against six bacterial and two fungal human pathogens. Antibacterial susceptibility of the compounds was tested against <italic>Acinetobacter baumannii</italic> ATCC19606, <italic>Escherichia coli</italic> ATCC25922, <italic>Pseudomonas aeruginosa</italic> PAO-1, <italic>Klebsiella pneumoniae</italic> ATCC700603, and methicillin-resistant <italic>Staphylococcus aureus</italic> MB5393, and sensitive <italic>S. aureus</italic> ATCC29213, while antifungal activity was tested against <italic>Aspergillus fumigatus</italic> ATCC46645 and <italic>Candida albicans</italic> ATCC64124 following previously described methodologies (<xref ref-type="bibr" rid="B53">Zhang et&#xa0;al., 2013</xref>). Briefly, each compound was serially diluted in 100% DMSO with a dilution factor of 2 to provide 10 final assay concentrations in all antimicrobial assays. Rifampicin, aztreonam, vancomycin, gentamicin and amphotericin were used as positive controls, as shown in <xref ref-type="table" rid="T2">
<bold>Table&#xa0;2</bold>
</xref>. The MIC was defined as the lowest concentration of the compound that inhibited &#x2265;90% of the growth of a microorganism after overnight incubation. Genedata Screener software, version 18.0.4-Standard (Genedata, Inc., Basel, Switzerland) was used to process and analyze the data and to calculate the RZ&#x2019; factor, which predicts the robustness of an assay (<xref ref-type="bibr" rid="B53">Zhang et&#xa0;al., 2013</xref>). In all experiments performed in this work, the RZ&#x2019; factor obtained was between 0.89 and 0.98.</p>
<table-wrap id="T2" position="float">
<label>Table&#xa0;2</label>
<caption>
<p>Minimum inhibitory concentration (MIC) of compounds <bold>1</bold>, <bold>3</bold>, <bold>11</bold>, and <bold>15</bold>, and the corresponding positive controls, against eight human pathogens.</p>
</caption>
<table frame="hsides">
<thead>
<tr>
<th valign="middle" colspan="9" align="center">MIC (<italic>&#xb5;</italic>g/mL)</th>
</tr>
</thead>
<tbody>
<tr>
<th valign="middle" align="center">Compound</th>
<th valign="middle" align="center">
<italic>A. fumigatus</italic> ATCC46645</th>
<th valign="middle" align="center">
<italic>C. albicans</italic> ATCC64124</th>
<th valign="middle" align="center">
<italic>K.</italic> <break/><italic>pneumonia</italic> ATCC700603</th>
<th valign="middle" align="center">
<italic>E. coli</italic> ATCC25922</th>
<th valign="middle" align="center">MSSA ATCC29213</th>
<th valign="middle" align="center">MRSA MB5393</th>
<th valign="middle" align="center">
<italic>A. baumannii</italic> ATCC19606</th>
<th valign="middle" align="center">
<italic>P. aeruginosa</italic> PAO-1</th>
</tr>
<tr>
<td valign="middle" align="center">
<bold>1</bold>
</td>
<td valign="middle" align="center">&gt;32</td>
<td valign="middle" align="center">&gt;32</td>
<td valign="middle" align="center">&gt;32</td>
<td valign="middle" align="center">&gt;32</td>
<td valign="middle" align="center">&gt;32</td>
<td valign="middle" align="center">&gt;32</td>
<td valign="middle" align="center">&gt;32</td>
<td valign="middle" align="center">&gt;32</td>
</tr>
<tr>
<td valign="middle" align="center">
<bold>3</bold>
</td>
<td valign="middle" align="center">&gt;128</td>
<td valign="middle" align="center">&gt;128</td>
<td valign="middle" align="center">&gt;128</td>
<td valign="middle" align="center">&gt;128</td>
<td valign="middle" align="center">&gt;128</td>
<td valign="middle" align="center">&gt;128</td>
<td valign="middle" align="center">&gt;128</td>
<td valign="middle" align="center">&gt;128</td>
</tr>
<tr>
<td valign="middle" align="center">
<bold>11</bold>
</td>
<td valign="middle" align="center">&gt;24</td>
<td valign="middle" align="center">&gt;24</td>
<td valign="middle" align="center">&gt;24</td>
<td valign="middle" align="center">&gt;24</td>
<td valign="middle" align="center">&gt;24</td>
<td valign="middle" align="center">&gt;24</td>
<td valign="middle" align="center">&gt;24</td>
<td valign="middle" align="center">&gt;24</td>
</tr>
<tr>
<td valign="middle" align="center">
<bold>15</bold>
</td>
<td valign="middle" align="center">&gt;128</td>
<td valign="middle" align="center">&gt;128</td>
<td valign="middle" align="center">&gt;128</td>
<td valign="middle" align="center">&gt;128</td>
<td valign="middle" align="center">128</td>
<td valign="middle" align="center">&gt;128</td>
<td valign="middle" align="center">&gt;128</td>
<td valign="middle" align="center">&gt;128</td>
</tr>
<tr>
<td valign="middle" align="center">
<bold>Rifampicin</bold>
</td>
<td valign="middle" align="center"/>
<td valign="middle" align="center"/>
<td valign="middle" align="center"/>
<td valign="middle" align="center"/>
<td valign="middle" align="center"/>
<td valign="middle" align="center"/>
<td valign="middle" align="center">2-4</td>
<td valign="middle" align="center"/>
</tr>
<tr>
<td valign="middle" align="center">
<bold>Aztreonam</bold>
</td>
<td valign="middle" align="center"/>
<td valign="middle" align="center"/>
<td valign="middle" align="center"/>
<td valign="middle" align="center">0.25-0.50</td>
<td valign="middle" align="center"/>
<td valign="middle" align="center"/>
<td valign="middle" align="center"/>
<td valign="middle" align="center">1.25-2.50</td>
</tr>
<tr>
<td valign="middle" align="center">
<bold>Vancomycin</bold>
</td>
<td valign="middle" align="center"/>
<td valign="middle" align="center"/>
<td valign="middle" align="center"/>
<td valign="middle" align="center"/>
<td valign="middle" align="center">0.5-1.0</td>
<td valign="middle" align="center">1-2</td>
<td valign="middle" align="center"/>
<td valign="middle" align="center"/>
</tr>
<tr>
<td valign="middle" align="center">
<bold>Gentamicin</bold>
</td>
<td valign="middle" align="center"/>
<td valign="middle" align="center"/>
<td valign="middle" align="center">4-8</td>
<td valign="middle" align="center"/>
<td valign="middle" align="center"/>
<td valign="middle" align="center"/>
<td valign="middle" align="center"/>
<td valign="middle" align="center"/>
</tr>
<tr>
<td valign="middle" align="center">
<bold>Amphotericin</bold>
</td>
<td valign="middle" align="center">2</td>
<td valign="middle" align="center">1-2</td>
<td valign="middle" align="center"/>
<td valign="middle" align="center"/>
<td valign="middle" align="center"/>
<td valign="middle" align="center"/>
<td valign="middle" align="center"/>
<td valign="middle" align="center"/>
</tr>
</tbody>
</table>
</table-wrap>
</sec>
<sec id="s2_8">
<label>2.8</label>
<title>
<italic>In vitro</italic> antitumoral assays</title>
<p>The tumor human cell lines used were liver HepG2 (ATCC HB-8065), breast MCF-7 (ATCC HTB-22), lung A549 (ATCC CCL-185), skin A2058 (ATCC CRL-3601) and pancreas Mia PaCa-2 (ATCC CRL-1420). Purified compounds were dissolved in DMSO 100% at 9 mM (compound <bold>1</bold>), 34.4 mM (compound <bold>3</bold>), 6.4 mM (compound <bold>11</bold>), and at 28.8 mM (compound <bold>15</bold>). The compounds were assayed for cytotoxicity using MTT test. The MTT test is a colorimetric assay for measuring the activity of enzymes that reduce 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, a yellow tetrazole, to formazan dyes, giving a purple color. Briefly, after 24 h, seeded cells were treated with compounds at a maximum dilution of 1/200 from the stocks in 10 points of 2-fold dilution per triplicate, for 72 h. MMS (Sigma Aldrich, St. Louis, Missouri, United States), at 4 mM, was used as a positive control of cell death, DMSO 0.5% as a negative control and doxorubicin (Sigma-Aldrich) was included as a known chemotherapeutic agent. After the incubation period, the plates containing treated cells were washed with 200 &#xb5;L of phosphate-buffered saline (PBS) 1X (137 mM NaCl, 2.7 mM KCl, 10 mM Na<italic>
<sub>2</sub>
</italic>HPO<sub>4</sub>, 1.8 mM KH<sub>2</sub>PO<sub>4</sub>). Then, MTT dye (Thiazolyl blue tetrazolium bromide, ACROS Organics BV, Geel, Belgium) was added at 0.5 mg/mL and plates were incubated for 2 h. The supernatant was then removed and 100 &#xb5;L of DMSO 100% were added to each well in order to dissolve the resulting formazan precipitates. Finally, absorbance was measured at 570 nm with an EnVision Multilabel Plate Reader (PerkinElmer, Waltham, USA). Data obtained was analyzed using Genedata Screener Software inhibitory curves fit to a Hill Equation model to calculate IC<sub>50</sub> and the confidence intervals at 95%.</p>
</sec>
</sec>
<sec id="s3" sec-type="results">
<label>3</label>
<title>Results and discussion</title>
<p>The fungus <italic>E. maritima</italic> BC17 was cultivated in the liquid culture media Czapek-Dox and PDB, and incubated for 14 or 28 days in surface and shaken conditions, as described in Materials and Methods. After extraction and purification by HPLC, in addition to fourteen known compounds (<bold>4</bold>-<bold>17</bold>), three new eremophilanes (<bold>1</bold>-<bold>3</bold>) were isolated and characterized. In particular, the undescribed eremophilanes <bold>1</bold>-<bold>3</bold> were only isolated from Czapek-Dox fermentation broth, which showed the highest chemical diversity.</p>
<p>The molecular formula for compound <bold>1</bold> was established as C<sub>12</sub>H<sub>16</sub>O<sub>4</sub>, deduced from the sodiated molecular ion [M+Na]<sup>+</sup> (<italic>m/z</italic> 247.0933 [M+Na]<sup>+</sup>, calculated for C<sub>12</sub>H<sub>16</sub>O<sub>4</sub>Na 247.0946) observed in its HRESIMS (<xref ref-type="supplementary-material" rid="SM1">
<bold>Supplementary Figure S7</bold>
</xref>). Its IR spectrum showed absorption bands characteristic for hydroxy (3447 cm<sup>&#x2212;1</sup>) and enone carbonyl groups (1637 cm<sup>&#x2212;1</sup>). The <sup>1</sup>H NMR data (<xref ref-type="table" rid="T1">
<bold>Table&#xa0;1</bold>
</xref>) indicated the presence of two methyl signals at <italic>&#x3b4;</italic>
<sub>H</sub> 1.28 (d, <italic>J</italic> = 7.0 Hz) and 1.35 (s), and two olefinic signals at <italic>&#x3b4;</italic>
<sub>H</sub> 6.17 (d, <italic>J</italic> = 1.5 Hz) and 6.30 (s). The <sup>13</sup>C NMR data (<xref ref-type="table" rid="T1">
<bold>Table&#xa0;1</bold>
</xref>) displayed twelve signals including one carbonyl (<italic>&#x3b4;</italic>
<sub>C</sub> 183.9), four olefinic carbons (<italic>&#x3b4;</italic>
<sub>C</sub> 124.4, 127.4, 147.9, 171.1), two methyls (<italic>&#x3b4;</italic>
<sub>C</sub> 13.2, 22.0), one methylene (<italic>&#x3b4;</italic>
<sub>C</sub> 37.5), other three methines, two of which were oxygenated (<italic>&#x3b4;</italic>
<sub>C</sub> 43.4, 74.1, 74.6), and another quaternary carbon (<italic>&#x3b4;</italic>
<sub>C</sub> 45.0).</p>
<p>Two double bonds and one carbonyl group accounted for three of five degrees of unsaturation, indicating the presence of two rings in the structure. The two-ring system was then established based on <sup>1</sup>H&#x2212;<sup>1</sup>H COSY (<xref ref-type="supplementary-material" rid="SM1">
<bold>Supplementary Figure S3</bold>
</xref>), HSQC (<xref ref-type="supplementary-material" rid="SM1">
<bold>Supplementary Figure S4</bold>
</xref>), and HMBC (<xref ref-type="supplementary-material" rid="SM1">
<bold>Supplementary Figure S5</bold>
</xref>) correlations (<xref ref-type="fig" rid="f2">
<bold>Figure&#xa0;2</bold>
</xref>). Inspection of the <sup>1</sup>H&#x2212;<sup>1</sup>H COSY and HSQC data led to the assignment of a C(1)H<sub>2</sub>&#x2212;C(2)H&#x2212;C(3)H&#x2212;C(4)H&#x2212;C(15)H<sub>3</sub> unit. The assigned spin system, together with HMBC correlations from H<sub>2</sub>-1 to C-3/C-5/C-9/C-10, from H-4 to C-14, and from H-15 to C-3/C-4/C-5, permitted the complete elucidation of ring A. HMBC correlations from H-6 to C-7/C-8/C-10/C-4/C-14 and from H-9 to C-1/C-5/C-7 allowed the elucidation of ring B. Thus, compound 1 was established as a <italic>nor</italic>-eremophilane sesquiterpene. Its <sup>1</sup>H and <sup>13</sup>C NMR spectra (<xref ref-type="supplementary-material" rid="SM1">
<bold>Supplementary Figures S1, S2</bold>
</xref>) were very similar to those of compound guignarderemophilane A (<bold>18</bold>), previously reported as a metabolite from the endophytic fungus <italic>Guignardia mangiferae</italic> (<xref ref-type="bibr" rid="B32">Liu et&#xa0;al., 2015</xref>), except for the absence of the acetyl group signals at C-3.</p>
<fig id="f2" position="float">
<label>Figure&#xa0;2</label>
<caption>
<p>Key COSY (red bond) and HMBC (blue arrows) correlations for compounds <bold>1</bold>-<bold>3</bold>.</p>
</caption>
<graphic mimetype="image" mime-subtype="tiff" xlink:href="fmars-11-1386175-g002.tif"/>
</fig>
<p>The relative configuration of <bold>1</bold> was determined by analysis of 1D NOESY (<xref ref-type="supplementary-material" rid="SM1">
<bold>Supplementary Figures S6a&#x2013;g</bold>
</xref>) correlations and the splitting patterns of related protons (<xref ref-type="fig" rid="f3">
<bold>Figure&#xa0;3</bold>
</xref>). The NOESY correlations between H-2, H-3 and H-4 revealed that they showed the same orientation. The NOE effect between the equatorial Me-15 and H-6 revealed that C5&#x2212;C6 was equatorial and that Me-14 was axial. The correlations between Me-14 and Me-15 suggested they were in the <italic>syn</italic> conformation. These correlations permitted us to propose the relative configuration of <bold>1</bold> as shown in <xref ref-type="fig" rid="f3">
<bold>Figure&#xa0;3</bold>
</xref>.</p>
<fig id="f3" position="float">
<label>Figure&#xa0;3</label>
<caption>
<p>Selected NOESY correlations exhibited by compounds <bold>1</bold>-<bold>3</bold>.</p>
</caption>
<graphic mimetype="image" mime-subtype="tiff" xlink:href="fmars-11-1386175-g003.tif"/>
</fig>
<p>Absolute stereochemistry of compound <bold>1</bold> was established by comparison of the experimental electronic circular dichroism (ECD) spectrum with the one obtained from quantum mechanical time-dependent density functional theory (TD-DFT) calculations for the (2<italic>S</italic>,3<italic>R</italic>,4<italic>R</italic>,5<italic>S)</italic> stereoisomer in the 200-400 nm region (<xref ref-type="fig" rid="f4">
<bold>Figure&#xa0;4</bold>
</xref>) (<xref ref-type="bibr" rid="B30">Li et&#xa0;al., 2010</xref>). Moreover, compound <bold>1</bold> showed an ECD spectrum similar to that of the previously reported acetylated compound guignarderemophilane A (<bold>18</bold>) (<xref ref-type="bibr" rid="B32">Liu et&#xa0;al., 2015</xref>). Based on these data, the absolute configuration at C-2, -3, -4 and -5 was determined to be <italic>S</italic>, <italic>R</italic>, <italic>R</italic>, and <italic>S</italic>, respectively. Therefore, the structure of <bold>1</bold> was characterized and named as 3-deacetyl guignarderemophilane A.</p>
<fig id="f4" position="float">
<label>Figure&#xa0;4</label>
<caption>
<p>Experimental and calculated ECD spectra for compounds <bold>1</bold>-<bold>3</bold>. Calculations were performed with the conformers shown in <xref ref-type="fig" rid="f3">
<bold>Figure&#xa0;3</bold>
</xref>.</p>
</caption>
<graphic mimetype="image" mime-subtype="tiff" xlink:href="fmars-11-1386175-g004.tif"/>
</fig>
<p>Compound <bold>2</bold> was obtained as a colourless oil with the molecular formula C<sub>15</sub>H<sub>22</sub>O<sub>3</sub>, as determined by the HRESIMS peak at <italic>m/z</italic> 273.1446 [M+Na]<sup>+</sup> (<xref ref-type="supplementary-material" rid="SM1">
<bold>Supplementary Figure S14</bold>
</xref>), indicating five degrees of unsaturation. Its IR spectrum showed absorptions corresponding to a hydroxyl group (3496 cm<sup>&#x2212;1</sup>) and an <italic>&#x3b1;</italic>,<italic>&#x3b2;</italic>-unsaturated keto group (1674 cm<sup>&#x2212;1</sup>). The <sup>13</sup>C NMR data (<xref ref-type="table" rid="T1">
<bold>Table&#xa0;1</bold>
</xref>) displayed fifteen carbon resonances, some of which could be assigned to one keto group (<italic>&#x3b4;</italic>
<sub>C</sub> 195.2), a C=C double bond (<italic>&#x3b4;</italic>
<sub>C</sub> 123.6, 172.4), one 1,2-epoxy group (<italic>&#x3b4;</italic>
<sub>C</sub> 64.7, 65.6), one oxygenated sp<sup>3</sup> carbon (<italic>&#x3b4;</italic>
<sub>C</sub> 70.8), and four methyl groups in the high-field region (<italic>&#x3b4;</italic>
<sub>C</sub> 12.6, 19.1, 21.3, 24.5). These functionalities account for three degrees of unsaturation, thus hinting the bicyclic nature of <bold>2</bold>. Analysis of the <sup>1</sup>H NMR data (<xref ref-type="table" rid="T1">
<bold>Table&#xa0;1</bold>
</xref>) revealed the presence of four methyl groups, including three singlets (<italic>&#x3b4;</italic>
<sub>H</sub> 1.30, 1.36, 1.41) and one doublet (<italic>&#x3b4;</italic>
<sub>H</sub> 1.15, <italic>J</italic>=7.1 Hz) in the high-field region. These data were characteristic of an eremophilane-type sesquiterpene, i.e., a <italic>cis</italic>-decaline ring with an isopropyl group (C-11/Me-12/Me-13) and two methyl groups, one connected to a methine group (CH-4) and the other one connected to a quaternary carbon (C-5), resulting in one doublet (Me-15) and one singlet (Me-14) signals (<xref ref-type="supplementary-material" rid="SM1"><bold>Supplementary Figure S11</bold></xref>). In addition, the signals for one olefinic proton (<italic>&#x3b4;</italic>
<sub>H</sub> 5.92) and one oxymethine proton (<italic>&#x3b4;</italic>
<sub>H</sub> 3.97) could be seen in the <sup>1</sup>H NMR spectrum (<xref ref-type="supplementary-material" rid="SM1">
<bold>Supplementary Figure S8</bold>
</xref>), which in combination with the signals observed in the <sup>13</sup>C NMR spectrum (<xref ref-type="supplementary-material" rid="SM1">
<bold>Supplementary Figure S9</bold>
</xref>) suggested the occurrence of one trisubstituted double bond, and one oxygenated methine group. The positions of these functional groups in the eremophilane skeleton were clarified by analysis of the HMBC spectrum (<xref ref-type="supplementary-material" rid="SM1">
<bold>Supplementary Figure S12</bold>
</xref>). The presence of an epoxide moiety with <sup>13</sup>C peaks at C-7 (<italic>&#x3b4;</italic>
<sub>C</sub> 65.6) and C-11 (<italic>&#x3b4;</italic>
<sub>C</sub> 64.7) was confirmed by HMBC correlations from Me-13 (<italic>&#x3b4;</italic>
<sub>H</sub> 1.30) to C-7, C-11 and C-12 (<italic>&#x3b4;</italic>
<sub>C</sub> 21.3), from H<sub>2</sub>-6 (<italic>&#x3b4;</italic>
<sub>H</sub> 2.08 and 1.99) to C-7, C-10 (<italic>&#x3b4;</italic>
<sub>C</sub> 172.4), C-11, and C-14 (<italic>&#x3b4;</italic>
<sub>C</sub> 24.5), and from Me-12 (<italic>&#x3b4;</italic>
<sub>H</sub> 1.41) to C-7, C-11, and C-13 (<italic>&#x3b4;</italic>
<sub>C</sub> 19.1) (<xref ref-type="fig" rid="f2">
<bold>Figure&#xa0;2</bold>
</xref>). Correlation between Me-15 (<italic>&#x3b4;</italic>
<sub>H</sub> 1.15, d) and the oxygenated methine (<italic>&#x3b4;</italic>
<sub>C</sub> 70.8) placed the hydroxyl substituent at C-3 (<xref ref-type="fig" rid="f2">
<bold>Figure&#xa0;2</bold>
</xref>). The <sup>1</sup>H and <sup>13</sup>C NMR spectra of <bold>2</bold> (<xref ref-type="supplementary-material" rid="SM1">
<bold>Supplementary Figures S8, S9</bold>
</xref>) were very similar to those of compound 3-acetoxy-7(11)-epoxyeremophil-9-en-8-one (<bold>16</bold>), previously reported by our research&#x2019;s group as an <italic>E. maritima</italic> BC17 metabolite from solid culture media (<xref ref-type="bibr" rid="B44">Viru&#xe9;s-Segovia et&#xa0;al., 2023</xref>), except for the absence of the acetyl group signals at C-3.</p>
<p>The relative configuration of <bold>2</bold> was determined by analysis of 1D NOESY correlations (<xref ref-type="supplementary-material" rid="SM1">
<bold>Supplementary Figures S13a&#x2013;f</bold>
</xref>). The H-1 (<italic>&#x3b4;</italic>
<sub>H</sub> 2.90) and Me-14 protons showed strong NOE correlations with each other, indicating that H-1<italic>&#x3b2;</italic> and Me-14 were in axial orientations on the same face of ring A. The NOE correlations between H-3 and H-1 (<italic>&#x3b4;</italic>
<sub>H</sub> 2.16) and H-4 confirmed that these protons were on the opposite side from H-1<italic>&#x3b2;</italic>/Me-14. NOE correlations between Me-14 and Me-15 indicated both methyls were on the same face. The <italic>&#x3b2;</italic>-orientation of the epoxide at C-7 and C-11 in <bold>2</bold> was evidenced by the NOE effect from Me-12 to H-6<italic>&#x3b1;</italic> (<italic>&#x3b4;</italic>
<sub>H</sub> 2.08) (<xref ref-type="fig" rid="f3">
<bold>Figure&#xa0;3</bold>
</xref>).</p>
<p>The ECD spectrum of <bold>2</bold> was very similar to that obtained for the previously reported compound <bold>16</bold> (<xref ref-type="bibr" rid="B44">Viru&#xe9;s-Segovia et&#xa0;al., 2023</xref>). In addition, the ECD curve for the (3<italic>S</italic>,4<italic>R</italic>,5<italic>R</italic>,7<italic>R</italic>) stereoisomer, calculated with the TD-DFT theoretical method, matched well with the experimental ECD spectrum of <bold>2</bold> (<xref ref-type="fig" rid="f4">
<bold>Figure&#xa0;4</bold>
</xref>). As a result, its absolute stereochemistry was assigned as (3<italic>S</italic>,4<italic>R</italic>,5<italic>R</italic>,7<italic>R</italic>)-3-hydroxy-7(11)-epoxyeremophil-9-en-8-one (<bold>2</bold>).</p>
<p>Compound <bold>3</bold> had a molecular formula of C<sub>15</sub>H<sub>20</sub>O<sub>2</sub>, as deduced from HRESIMS (<xref ref-type="supplementary-material" rid="SM1">
<bold>Supplementary Figure S21</bold>
</xref>), which was consistent with six degrees of unsaturation. The <sup>13</sup>C NMR data (<xref ref-type="table" rid="T1">
<bold>Table&#xa0;1</bold>
</xref>) displayed fifteen signals, including two carbonyl groups (<italic>&#x3b4;</italic>
<sub>C</sub> 190.5, 212.1), four olefinic carbons (<italic>&#x3b4;</italic>
<sub>C</sub> 127.0, 128.2, 146.1, 162.6), four methyls (<italic>&#x3b4;</italic>
<sub>C</sub> 11.0, 22.8, 23.1, 25.1), three methylenes (<italic>&#x3b4;</italic>
<sub>C</sub> 29.0, 37.4, 37.5), another methine (<italic>&#x3b4;</italic>
<sub>C</sub> 53.6), and an additional quaternary carbon (<italic>&#x3b4;</italic>
<sub>C</sub> 42.2). The <sup>1</sup>H NMR data (<xref ref-type="table" rid="T1">
<bold>Table&#xa0;1</bold>
</xref>) showed four methyl groups, including one singlet (<italic>&#x3b4;</italic>
<sub>H</sub> 1.20) and three doublets (<italic>&#x3b4;</italic>
<sub>H</sub> 1.10, <italic>J</italic>=7.0 Hz; 1.85, <italic>J</italic>=1.4 Hz; 2.15, <italic>J</italic>=2.0 Hz) in the high-field region.</p>
<p>NMR data (<xref ref-type="table" rid="T1">
<bold>Table&#xa0;1</bold>
</xref>) of <bold>3</bold> revealed a great similarity with those of isopetasone (<bold>4</bold>) (<xref ref-type="bibr" rid="B11">Brooks and Draffan, 1969</xref>; <xref ref-type="bibr" rid="B51">Yamakawa et&#xa0;al., 1974</xref>). However, the <sup>1</sup>H and <sup>13</sup>C NMR spectra (<xref ref-type="supplementary-material" rid="SM1">
<bold>Supplementary Figures S15, S16</bold>
</xref>) of both compounds differed significantly in the shift of the signals corresponding to H-1<italic>&#x3b2;</italic> (<italic>&#x3b4;</italic>
<sub>H</sub> 2.87 in <bold>3</bold> and 2.72 in <bold>4</bold>), H-6<italic>&#x3b1;</italic> (<italic>&#x3b4;</italic>
<sub>H</sub> 2.49 in <bold>3</bold> and 2.82 in <bold>4</bold>), Me-14 (<italic>&#x3b4;</italic>
<sub>C/H</sub> 25.1/1.20 in <bold>3</bold> and 18.5/0.96 in <bold>4</bold>), and Me-15 (<italic>&#x3b4;</italic>
<sub>C</sub> 11.0 in <bold>3</bold> and 7.4 in <bold>4</bold>). This suggested a possible difference in the stereochemistry of these methyls. The NOESY correlation between H-1 (<italic>&#x3b4;</italic>
<sub>H</sub> 2.87) and Me-14 (<italic>&#x3b4;</italic>
<sub>H</sub> 1.20) (<xref ref-type="supplementary-material" rid="SM1">
<bold>Supplementary Figures S20a, d</bold>
</xref>) revealed that this methyl group was axial, while the correlation from Me-14 to H-4 (<italic>&#x3b4;</italic>
<sub>H</sub> 2.60) (<xref ref-type="supplementary-material" rid="SM1">
<bold>Supplementary Figure S20d</bold>
</xref>) positioned the latter to the equatorial site, indicating that Me-14 and H-4 shared the same <italic>&#x3b2;</italic>-configuration (<xref ref-type="fig" rid="f3">
<bold>Figure&#xa0;3</bold>
</xref>), in contrast to all eremophilanes described in this work. Hence, compound <bold>3</bold> was elucidated to be 4-<italic>epi</italic>isopetasone.</p>
<p>This stereochemistry was confirmed by comparison of the predicted ECD spectrum from TD-DFT calculations with the experimental one of compound <bold>3</bold>. The calculated spectrum reproduced well the experimental data (<xref ref-type="fig" rid="f4">
<bold>Figure&#xa0;4</bold>
</xref>), confirming the 4<italic>S</italic> and 5<italic>R</italic> absolute configuration of <bold>3</bold>.</p>
<p>In view of these results, it is noteworthy that the new metabolites could only be isolated under surface culture conditions and with Czapek-Dox medium. In particular, compound <bold>3</bold> belongs to a different stereochemical series than all eremophilanes previously isolated from the <italic>E. maritima</italic> BC17 strain. This indicates that the conditional changes are useful for accessing cryptic metabolites that would not be obtained under standard laboratory conditions.</p>
<p>The other isolated compounds <bold>4</bold>-<bold>17</bold> were identified by comparing their spectroscopic data with the literature: isopetasone (<bold>4</bold>) (<xref ref-type="bibr" rid="B11">Brooks and Draffan, 1969</xref>; <xref ref-type="bibr" rid="B51">Yamakawa et&#xa0;al., 1974</xref>) (<xref ref-type="supplementary-material" rid="SM1">
<bold>Supplementary Figures S22, S23</bold>
</xref>), (+)-3-<italic>epi</italic>isopetasol (<bold>5</bold>) (<xref ref-type="bibr" rid="B41">Sumarah et&#xa0;al., 2010</xref>) (<xref ref-type="supplementary-material" rid="SM1">
<bold>Supplementary Figures S24, S25</bold>
</xref>), 1<italic>&#x3b1;</italic>-hydroxydehydrofukinone (<bold>6</bold>) (<xref ref-type="bibr" rid="B9">Bohlmann and Knoll, 1979</xref>) (<xref ref-type="supplementary-material" rid="SM1">
<bold>Supplementary Figures S26, S27</bold>
</xref>), eremofortin A alcohol (<bold>7</bold>) (<xref ref-type="bibr" rid="B35">Moreau et&#xa0;al., 1977</xref>) (<xref ref-type="supplementary-material" rid="SM1">
<bold>Supplementary Figures S28, S29</bold>
</xref>), (+)-aristolochene (<bold>8</bold>) (<xref ref-type="bibr" rid="B17">Demyttenaere et&#xa0;al., 2002</xref>) (<xref ref-type="supplementary-material" rid="SM1">
<bold>Supplementary Figures S30, S31</bold>
</xref>), 7-hydroxy-4a,5-dimethyl-3-prop-1-en-2-yl-3,4,5,6,7,8-hexahydronaphthalen-2-one (<bold>9</bold>) (<xref ref-type="bibr" rid="B16">Daengrot et&#xa0;al., 2015</xref>) (<xref ref-type="supplementary-material" rid="SM1">
<bold>Supplementary Figures S32, S33</bold>
</xref>), warburgiadione (<bold>10</bold>) (<xref ref-type="bibr" rid="B11">Brooks and Draffan, 1969</xref>) (<xref ref-type="supplementary-material" rid="SM1">
<bold>Supplementary Figures S34, S35</bold>
</xref>), (1<italic>R</italic>,7<italic>S</italic>,8a<italic>R</italic>)-1,8a-dimethyl-7-(prop-1-en-2-yl)-1,7,8,8a-tetrahydronaphthalene-2,6-dione (<bold>11</bold>) (<xref ref-type="bibr" rid="B19">Fan, 2016</xref>) (<xref ref-type="supplementary-material" rid="SM1">
<bold>Supplementary Figures S36, S37</bold>
</xref>), eremofortine B (<bold>12</bold>) (<xref ref-type="bibr" rid="B34">Moreau et&#xa0;al., 1980</xref>) (<xref ref-type="supplementary-material" rid="SM1">
<bold>Supplementary Figure S38</bold>
</xref>), 3<italic>&#x3b2;</italic>-hydroxy-7<italic>&#x3b2;</italic>-eremophil-1(2),9(10),11(12)-trien-8-one (<bold>13</bold>) (<xref ref-type="bibr" rid="B31">Lin et&#xa0;al., 2014</xref>) (<xref ref-type="supplementary-material" rid="SM1">
<bold>Supplementary Figure S39, S40</bold>
</xref>), PR toxin (<bold>14</bold>) (<xref ref-type="bibr" rid="B34">Moreau et&#xa0;al., 1980</xref>) (<xref ref-type="supplementary-material" rid="SM1">
<bold>Supplementary Figures S41, S42</bold>
</xref>), PR toxin 3-deacetyl (<bold>15</bold>) (<xref ref-type="bibr" rid="B47">Wei et&#xa0;al., 1975</xref>; <xref ref-type="bibr" rid="B13">Capasso et&#xa0;al., 1986</xref>) (<xref ref-type="supplementary-material" rid="SM1">
<bold>Supplementary Figure S43</bold>
</xref>), (3<italic>S</italic>)-acetoxy-7(11)-epoxyeremophil-9-en-8-one (<bold>16</bold>) (<xref ref-type="bibr" rid="B44">Viru&#xe9;s-Segovia et&#xa0;al., 2023</xref>) (<xref ref-type="supplementary-material" rid="SM1">
<bold>Supplementary Figures S44, S45</bold>
</xref>), and eremofortine A (<bold>17</bold>) (<xref ref-type="bibr" rid="B34">Moreau et&#xa0;al., 1980</xref>) (<xref ref-type="supplementary-material" rid="SM1">
<bold>Supplementary Figures S46, S47</bold>
</xref>). Compounds <bold>11</bold>, <bold>12</bold>, and <bold>15</bold> are reported for the first time as fungal metabolites from <italic>E. maritima</italic> BC17 in this work, indicating the usefulness of the OSMAC strategy to increase the chemical diversity of the strain under study.</p>
<p>PR toxin 3-deacetyl (<bold>15</bold>) was previously chemically synthesised from PR toxin (<bold>14</bold>) (<xref ref-type="bibr" rid="B47">Wei et&#xa0;al., 1975</xref>). In order to confirm the configuration at C-3 of <bold>15</bold>, this compound was acetylated with acetic anhydride and <italic>p</italic>-toluenesulfonic acid to give a product whose spectroscopic data coincided with those found in literature for PR toxin (<bold>14</bold>) (<xref ref-type="bibr" rid="B34">Moreau et&#xa0;al., 1980</xref>). This has allowed us to confirm the absolute configuration of compound <bold>15</bold> and report it for the first time as a fungal metabolite.</p>
<p>The undescribed isolated metabolites <bold>1</bold> and <bold>3</bold> and the known compounds <bold>11</bold> and <bold>15</bold> were evaluated for antimicrobial and cytotoxic activities. They were tested in triplicates against a panel of 8 human pathogens, including both Gram-negative and Gram-positive bacteria (<italic>E. coli</italic> ATCC25922, <italic>P. aeruginosa</italic> PAO-1, <italic>A. baumannii</italic> ATCC19606, <italic>K. pneumoniae</italic> ATCC700603, methicillin-resistant <italic>S. aureus</italic> MB5393, and sensitive <italic>S. aureus</italic> ATCC29213), yeast (<italic>C. albicans</italic> ATCC64124) and fungal (<italic>A. fumigatus</italic> ATCC46645) strains. No antimicrobial activity was detected for these metabolites against the human pathogenic strains assayed, except for compound <bold>15</bold> that exhibited selective but moderate activity against methicillin-sensitive <italic>S. aureus</italic> ATCC29213 at the highest concentration tested of 128 &#xb5;g/mL (<xref ref-type="table" rid="T2">
<bold>Table&#xa0;2</bold>
</xref>).</p>
<p>Antitumoral activity of these compounds (<bold>1</bold>, <bold>3</bold>, <bold>11</bold>, and <bold>15</bold>) was also tested against liver (HepG2, ATCC HB-8065), breast (MCF-7, ATCC HTB-22), lung (A549, ATCC CCL-185), skin (A2058, ATCC CRL-3601), and pancreas (Mia PaCa-2, ATCC CRL-1420) human cancer cells using MTT test. Methyl methanesulfonate (MMS) 4 mM was used as positive control of cell death and doxorubicin was included as known chemotherapeutic agent.</p>
<p>In previous studies, <italic>in vitro</italic> tests of compound <bold>11</bold>, isolated from dry root of <italic>Valeriana jatamansi</italic>, indicated that it played a restraining effect to human brain malignant glioblastoma U251 cell by inhibiting cell proliferation and inducing cell apoptosis (<xref ref-type="bibr" rid="B19">Fan, 2016</xref>). However, in this work we did not detect antiproliferative activity of this metabolite, nor of compounds <bold>1</bold> and <bold>3</bold>, against the human cancer cells assayed. Among the eremophilanes tested, only compound <bold>15</bold> was active against all of the tested cell lines with IC<sub>50</sub> values ranging from 2.5 to 14.7 &#xb5;M (<xref ref-type="table" rid="T3">
<bold>Table&#xa0;3</bold>
</xref>). In a previous study, we reported that the PR toxin (<bold>14</bold>) inhibited the same human cancer cells tested with IC<sub>50</sub> values in the range of 3.75-33.44 &#xb5;g/mL (<xref ref-type="bibr" rid="B44">Viru&#xe9;s-Segovia et&#xa0;al., 2023</xref>). These results confirm that the aldehyde group at C-12 present in both compounds, <bold>14</bold> and <bold>15</bold>, is directly related to their biological activity.</p>
<table-wrap id="T3" position="float">
<label>Table&#xa0;3</label>
<caption>
<p>Half-maximal inhibitory concentration (IC<sub>50</sub>) of compounds <bold>1</bold>, <bold>3</bold>, <bold>11</bold>, and <bold>15</bold> of cell viability in five tumor cell lines.</p>
</caption>
<table frame="hsides">
<thead>
<tr>
<th valign="middle" colspan="6" align="center">IC<sub>50</sub> (<italic>&#xb5;</italic>M)</th>
</tr>
</thead>
<tbody>
<tr>
<th valign="middle" align="center">Compound</th>
<th valign="middle" align="center">HepG2</th>
<th valign="middle" align="center">MCF7</th>
<th valign="middle" align="center">A549</th>
<th valign="middle" align="center">A2058</th>
<th valign="middle" align="center">Mia PaCa-2</th>
</tr>
<tr>
<td valign="middle" align="center">
<bold>1</bold>
</td>
<td valign="middle" align="center">&gt;45</td>
<td valign="middle" align="center">&gt;45</td>
<td valign="middle" align="center">&gt;45</td>
<td valign="middle" align="center">&gt;45</td>
<td valign="middle" align="center">&gt;45</td>
</tr>
<tr>
<td valign="middle" align="center">
<bold>3</bold>
</td>
<td valign="middle" align="center">&gt;172</td>
<td valign="middle" align="center">&gt;172</td>
<td valign="middle" align="center">&gt;172</td>
<td valign="middle" align="center">&gt;172</td>
<td valign="middle" align="center">&gt;172</td>
</tr>
<tr>
<td valign="middle" align="center">
<bold>11</bold>
</td>
<td valign="middle" align="center">&gt;33</td>
<td valign="middle" align="center">&gt;33</td>
<td valign="middle" align="center">&gt;33</td>
<td valign="middle" align="center">&gt;33</td>
<td valign="middle" align="center">&gt;33</td>
</tr>
<tr>
<td valign="middle" align="center">
<bold>15</bold>
</td>
<td valign="middle" align="center">7.6 (6.8-7.9)</td>
<td valign="middle" align="center">4.7 (4.0-5.4)</td>
<td valign="middle" align="center">14.7 (12.6-17.3)</td>
<td valign="middle" align="center">12.2 (10.8-14.0)</td>
<td valign="middle" align="center">2.5 (2.2-2.9)</td>
</tr>
<tr>
<td valign="middle" align="center">Doxorubicin</td>
<td valign="middle" align="center">0.21 (0.19-0.23)</td>
<td valign="middle" align="center">0.21 (0.16-0.28)</td>
<td valign="middle" align="center">0.90 (0.70-1.00)</td>
<td valign="middle" align="center">0.10 (0.08-0.13)</td>
<td valign="middle" align="center">0.43 (0.36-0.50)</td>
</tr>
</tbody>
</table>
<table-wrap-foot>
<fn>
<p>Confidence intervals at 95% are shown in brackets as calculated with the Genedata<sup>&#xa9;</sup> Screener software, version 18.0.4-Standard.</p>
</fn>
</table-wrap-foot>
</table-wrap>
</sec>
<sec id="s4" sec-type="conclusions">
<label>4</label>
<title>Conclusions</title>
<p>In this research, three previously undescribed eremophilanes (<bold>1</bold>-<bold>3</bold>) and fourteen known eremophilane derivatives (<bold>4</bold>-<bold>17</bold>) were isolated and identified from the fungal strain <italic>E. maritima</italic> BC17, grown in both Czapek Dox and PDB liquid culture media, in order to increase its chemical diversity. Notably, the new eremophilanes <bold>1</bold>-<bold>3</bold> were only isolated from the Czapek-Dox medium, which showed the highest chemical diversity. Compound 3 belongs to a different stereochemical series than all eremophilanes previously isolated from the strain under study (<xref ref-type="bibr" rid="B44">Viru&#xe9;s-Segovia et&#xa0;al., 2023</xref>). These results confirm that the OSMAC approach is a simple, rapid and effective strategy to increase the chemical diversity of marine natural products by activating silent gene clusters.</p>
<p>In addition, compounds <bold>1</bold>, <bold>3</bold>, <bold>11</bold> and <bold>15</bold> were tested for antimicrobial and cytotoxic activity. Compound <bold>15</bold> exhibited cytotoxic activity against HepG2, MCF-7, A549, A2058 and Mia PaCa-2 human cancer cell lines with IC<sub>50</sub> values ranging from 2.5 to 14.7 <italic>&#xb5;</italic>M. The aldehyde group at C-12 was identified as responsible for its biological activity, as observed in compound <bold>14</bold> (<xref ref-type="bibr" rid="B44">Viru&#xe9;s-Segovia et&#xa0;al., 2023</xref>).</p>
</sec>
<sec id="s5" sec-type="data-availability">
<title>Data availability statement</title>
<p>The original contributions presented in the study are included in the article/<xref ref-type="supplementary-material" rid="SM1">
<bold>Supplementary Material</bold>
</xref>. Further inquiries can be directed to the corresponding authors.</p>
</sec>
<sec id="s6" sec-type="ethics-statement">
<title>Ethics statement</title>
<p>Ethical approval was not required for the studies on humans in accordance with the local legislation and institutional requirements because only commercially available established cell lines were used.</p>
</sec>
<sec id="s7" sec-type="author-contributions">
<title>Author contributions</title>
<p>JRV-S: Writing &#x2013; review &amp; editing, Investigation. CP: Writing &#x2013; review &amp; editing, Methodology, Investigation. DZ: Writing &#x2013; review &amp; editing, Data curation. JS-M: Writing &#x2013; review &amp; editing, Data curation. PS: Writing &#x2013; review &amp; editing, Data curation. MCR: Writing &#x2013; review &amp; editing, Data curation. MdlC: Writing &#x2013; review &amp; editing, Data curation. JA: Writing &#x2013; review &amp; editing, Writing &#x2013; original draft, Supervision, Project administration, Funding acquisition, Conceptualization. RD-P: Writing &#x2013; review &amp; editing, Supervision, Methodology, Funding acquisition, Conceptualization.</p>
</sec>
</body>
<back>
<sec id="s8" sec-type="funding-information">
<title>Funding</title>
<p>The author(s) declare financial support was received for the research, authorship, and/or publication of this article. This work was co-financed by the 2014-2020 ERDF Operational Programme and by the Department of Economy, Knowledge, Business and University of the Regional Government of Andalusia. Project reference: FEDER-UCA18-105749.</p>
</sec>
<ack>
<title>Acknowledgments</title>
<p>J.R. V-S thanks the Spanish Ministry of Universities for a grant of the National Programme FPU 2022.</p>
</ack>
<sec id="s9" sec-type="COI-statement">
<title>Conflict of interest</title>
<p>The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.</p>
</sec>
<sec id="s10" sec-type="disclaimer">
<title>Publisher&#x2019;s note</title>
<p>All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.</p>
</sec>
<sec id="s11" sec-type="supplementary-material">
<title>Supplementary material</title>
<p>The Supplementary Material for this article can be found online at: <ext-link ext-link-type="uri" xlink:href="https://www.frontiersin.org/articles/10.3389/fmars.2024.1386175/full#supplementary-material">https://www.frontiersin.org/articles/10.3389/fmars.2024.1386175/full#supplementary-material</ext-link></p>
<supplementary-material xlink:href="DataSheet_1.pdf" id="SM1" mimetype="application/pdf"/>
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