Jugiones A–D: Antibacterial Xanthone–Anthraquinone Heterodimers from Australian Soil-Derived Penicillium shearii CMB-STF067

The Australian roadside soil-derived fungus Penicillium shearii CMB-STF067 was prioritized for chemical investigation based on an SDA cultivation extract exhibiting both antibacterial properties and natural products with unprecedented molecular formulae (GNPS). Subsequent miniaturized 24-well plate cultivation profiling (MATRIX) identified red rice as optimal for the production of the target chemistry, with scaled-up cultivation, extraction and fractionation yielding four new xanthone–anthraquinone heterodimers, jugiones A–D (1–4), whose structures were assigned by detailed spectroscopic analysis and biosynthetic considerations. Of note, where 1–2 and 4 were active against the Gram-positive bacteria vancomycin-resistant Enterococcus faecalis (IC50 2.6–3.9 μM) and multiple-drug-resistant clinical isolates of Staphylococcus aureus (IC50 1.8–6.4 μM), and inactive against the Gram-negative bacteria Escherichia coli (IC50 > 30 μM), the closely related analog 3 exhibited no antibacterial properties (IC50 > 30 μM). Furthermore, where 1 was cytotoxic to human carcinoma (IC50 9.0–9.8 μM) and fungal (IC50 4.1 μM) cells, 2 and 4 displayed no such cytotoxicity (IC50 > 30 μM), revealing an informative structure activity relationship (SAR). We also extended the SAR study to other known compounds of this heterodimer class, which showed that the modification of ring G can reduce or eliminate the cytotoxicity while retaining the antibacterial activity.


Introduction
Soil microbes have been the source of many modern antibiotics in clinical use, including β-lactams, streptomycins, aminoglycosides and tetracyclines.With a global increase in multidrug-resistant pathogens challenging healthcare across the world, the need for new and more effective antibiotics is both urgent and compelling.As part of our ongoing search for new microbial natural products with antibiotic properties, we screened extracts obtained from agar plate cultivations of ×139 bacterial (on ISP2 medium) and ×254 fungal (on SDA or PDA media) isolates obtained from roadside soils collected in New South Wales (NSW), Australia, against Gram-positive and Gram-negative bacterial pathogens.This survey drew our attention to the extract prepared from an SDA cultivation of Penicillium shearii CMB-STF067, which exhibited promising Gram-positive antibacterial properties.Importantly, a Global Natural Products Social (GNPS) [1] molecular network comparison of this extract against an in-house library of ~2000 microbial extracts confirmed that CMB-STF067 was unique in producing a suite of metabolites with unprecedented molecular formulae (i.e., not attributable to any reported microbial natural product).
Capitalizing on the knowledge that the transcriptional status of natural-product biosynthetic gene clusters can be influenced by culture conditions (i.e., media composition, temperature, aeration. . . ) [2][3][4], we employed a miniaturized 24-well plate approach to cultivation profiling known in lab as the MATRIX [5].When integrated with UPLC-DAD and GNPS molecular networking, MATRIX analyses can be particularly effective and have proved pivotal to our prior discovery and reporting of many new natural-product structure classes (e.g., noonindoles [6], chrysosporazines [7], talarolides [8] and terreusides [9]).In this study, we employed both the standard MATRIX [5] and newly developed grain/pulse and cereal MATRIX [9] variations to probe the metabolite production capabilities of CMB-STF067.
This report provides an account of an optimized, scaled-up fermentation and chemical fractionation of CMB-STF067 to yield four new xanthone-anthraquinone heterodimers, jugiones A-D (1)(2)(3)(4).The jugiones belong to a class of rare fungal metabolites and are the first examples of xanthone-anthraquinone heterodimers to be reported from the genus Penicillium.In addition to structure elucidation by detailed spectroscopic analysis, we carried out a structure activity relationship (SAR) assessment of the jugiones against vancomycinresistant Enterococcus faecalis and multiple-drug-resistant isolates of Staphylococcus aureus, and against human colon and lung carcinoma and fungal cells.What follows is an account of these investigations.

Results and Discussion
MATRIX methodology comparing CMB-STF067 metabolite production in ×11 standard media compositions (Table S1) under solid phase (2.0 mL agar) as well as shaken and static broth (1.5 mL) conditions was further extended to include ×23 grain/pulse (Table S2) (grain MATRIX) and ×11 cereal (Table S3) (cereal MATRIX)-based solid media compositions, inclusive of uninoculated media controls.Subsequent in situ solvent (EtOAc) extraction followed by UPLC-DAD (Figures S5, S7 and S9) and UPLC-QTOF-MS/MS (GNPS) (Figures S6, S8 and S10) chemical profiling identified red rice as the preferred production media, with scale-up cultivation followed by solvent extraction and trituration, and gel and reversed-phase chromatography (Scheme S1) yielding the target chemistry, jugiones A-D (1-4) (Figure 1).temperature, aeration…) [2][3][4], we employed a miniaturized 24-well plate approach to cultivation profiling known in lab as the MATRIX [5].When integrated with UPLC-DAD and GNPS molecular networking, MATRIX analyses can be particularly effective and have proved pivotal to our prior discovery and reporting of many new natural-product structure classes (e.g., noonindoles [6], chrysosporazines [7], talarolides [8] and terreusides [9]).In this study, we employed both the standard MATRIX [5] and newly developed grain/pulse and cereal MATRIX [9] variations to probe the metabolite production capabilities of CMB-STF067.This report provides an account of an optimized, scaled-up fermentation and chemical fractionation of CMB-STF067 to yield four new xanthone-anthraquinone heterodimers, jugiones A-D (1)(2)(3)(4).The jugiones belong to a class of rare fungal metabolites and are the first examples of xanthone-anthraquinone heterodimers to be reported from the genus Penicillium.In addition to structure elucidation by detailed spectroscopic analysis, we carried out a structure activity relationship (SAR) assessment of the jugiones against vancomycin-resistant Enterococcus faecalis and multiple-drug-resistant isolates of Staphylococcus aureus, and against human colon and lung carcinoma and fungal cells.What follows is an account of these investigations.

Results and Discussion
MATRIX methodology comparing CMB-STF067 metabolite production in ×11 standard media compositions (Table S1) under solid phase (2.0 mL agar) as well as shaken and static broth (1.5 mL) conditions was further extended to include ×23 grain/pulse (Table S2) (grain MATRIX) and ×11 cereal (Table S3) (cereal MATRIX)-based solid media compositions, inclusive of uninoculated media controls.Subsequent in situ solvent (EtOAc) extraction followed by UPLC-DAD (Figures S5, S7 and S9) and UPLC-QTOF-MS/MS (GNPS) (Figures S6, S8 and S10) chemical profiling identified red rice as the preferred production media, with scale-up cultivation followed by solvent extraction and trituration, and gel and reversed-phase chromatography (Scheme S1) yielding the target chemistry, jugiones A-D (1-4) (Figure 1).HRESI(+)MS measurement established a molecular formula for 1 (C 39 H 34 O 13 , ∆mmu +2.7) requiring ×23 double bond equivalents (DBEs), while analysis of the 1D and 2D NMR (CDCl 3 ) (Tables 1, 2 and S4, Figures S11-S16) data allowed assembly of four sub-structures A to D (Figure 2) accounting for ×21 DBE and necessitating two additional ring systems.Sub-structure A was identified as a conjugated triene octanoate with an all E configuration evident from diagnostic J values, while sub-structure B was attributed to a disubstituted phenol.An HMBC correlation from H-10 to C-1', and from H-2 to C-7, allowed assembly of the consolidated sub-structure A-B.By contrast, sub-structures C and D featured more complex carbocycles, with an HMBC correlation from H-15 to C-10, and a ROESY correlation between H-15 and H-10, establishing a C-9 to C-10 linkage supportive of the consolidated sub-structure A-B-C.With the full suite of sub-structures A to D accounting for an oxygen atom in excess of the molecular formula necessitating a C-18 to C-24 ether bridge, the remaining disconnections and requirement for two additional rings required C-7 and C-22 ketone bridges to form rings B and F, to arrive at the complete planar structure for 1 as indicated (Figure 1).In this regard, 1 shares a planar carbo/heterocyclic core framework (rings A to G) in common with JBIR-99 (5) first reported in 2010 [10] from an Okinawan marine spongederived fungus, Tritirachium sp.SpB081112MEf2, and subsequently re-isolated in 2021 [11] from the Indian Ocean marine seawater-derived fungus, Meyerozyma guilliermondii Y39, and assigned a relative configuration based on X-ray crystallographic analysis.Interestingly, a 2016 [12] re-isolation of 5 together with its isomer engyodontochone B (6) from a Croatian marine sponge-derived fungus, Engyodontium album LF069, employed ROESY and ECD correlations to assign absolute configurations to both 5 and 6 (Figure 3).Also noteworthy are the biosynthetically related xanthoquinodins B10 (7) and B11 (8), first reported in 2020 [13] from a Nepalese soil-derived fungus, Jugulospora vestita CBS 135.91, and assigned structures and absolute configurations based on spectroscopic and ECD analysis (Figure 3).Careful consideration of the spectroscopic data for 5-7, which collectively encompass the full array of C-24/C-25 stereoisomers, reveals useful empirical rules.For example, 5 (24R,25R) and 7 (24S,25S), which feature α and β facial cis disposed 24-CO 2 Me and 25-OH moieties, respectively, are characterized by comparable NMR (CDCl 3 ) chemical shifts for C-25 (δ C 72.0 and 71.7, respectively), which differ from those of the alternate trans disposed isomers 6 (24S,25R) and 8 (24R,25S) (δ C 66.9 and 66.9, respectively).Furthermore, the 24R isomers 5 and 8 possess comparable experimental ECD spectra (Figure S18), which differ from the equally comparable experimental ECD spectra reported for the 24S isomers 6 and 7 (Figure S19).Drawing on these NMR and ECD observations, as 1 possesses a 13 C NMR (CDCl 3 ) chemical shift for C-25 (δ C 68.9) in common with 6 and 8, and an ECD spectra (Figure S20) in common with 5 and 8, we propose that 1 shares a 10S,9R,12S,24R,25S configuration in common with 8. Finally, a ROESY correlation between 24-CO 2 CH 3 and H b -26 in 1, together with a J 26b,27 of 10.2 Hz, is consistent with a large H b -26/H-27 dihedral angle and 27S configuration.This hypothesis was further validated by the consideration of predicted dihedral angles and J values about ring G in energy-minimized models of 1 and its unnatural 27R epimer (Figure S21).Based on all of the above, the structure inclusive of absolute configurations for jugione A (1) was assigned as shown.
lation between H-15 and H-10, establishing a C-9 to C-10 linkage supportive of the consolidated sub-structure A-B-C.With the full suite of sub-structures A to D accounting for an oxygen atom in excess of the molecular formula necessitating a C-18 to C-24 ether bridge, the remaining disconnections and requirement for two additional rings required C-7 and C-22 ketone bridges to form rings B and F, to arrive at the complete planar structure for 1 as indicated (Figure 1).common with JBIR-99 (5) first reported in 2010 [10] from an Okinawan marine spongederived fungus, Tritirachium sp.SpB081112MEf2, and subsequently re-isolated in 2021 [11] from the Indian Ocean marine seawater-derived fungus, Meyerozyma guilliermondii Y39, and assigned a relative configuration based on X-ray crystallographic analysis.Interestingly, a 2016 [12] re-isolation of 5 together with its isomer engyodontochone B (6) from a Croatian marine sponge-derived fungus, Engyodontium album LF069, employed ROESY and ECD correlations to assign absolute configurations to both 5 and 6 (Figure 3).Also noteworthy are the biosynthetically related xanthoquinodins B10 (7) and B11 (8), first reported in 2020 [13] from a Nepalese soil-derived fungus, Jugulospora vestita CBS 135.91, and assigned structures and absolute configurations based on spectroscopic and ECD analysis (Figure 3).Careful consideration of the spectroscopic data for 5-7, which collectively encompass the full array of C-24/C-25 stereoisomers, reveals useful empirical rules.For example, 5 (24R,25R) and 7 (24S,25S), which feature α and β facial cis disposed 24-CO2Me and 25-OH moieties, respectively, are characterized by comparable NMR (CDCl3) chemical shifts for C-25 (δC 72.0 and 71.7, respectively), which differ from those of the alternate trans disposed isomers 6 (24S,25R) and 8 (24R,25S) (δC 66.9 and 66.9, respectively).Furthermore, the 24R isomers 5 and 8 possess comparable experimental ECD spectra (Figure S18), which differ from the equally comparable experimental ECD spectra reported for the 24S isomers 6 and 7 (Figure S19).Drawing on these NMR and ECD observations, as 1 possesses a 13 C NMR (CDCl3) chemical shift for C-25 (δC 68.9) in common with 6 and 8, and an ECD spectra (Figure S20) in common with 5 and 8, we propose that 1 shares a 10S,9R,12S,24R,25S configuration in common with 8. Finally, a ROESY correlation between 24-CO2CH3 and Hb-26 in 1, together with a J26b,27 of 10.2 Hz, is consistent with a large Hb-26/H-27 dihedral angle and 27S configuration.This hypothesis was further validated by the consideration of predicted dihedral angles and J values about ring G in energy-minimized models of 1 and its unnatural 27R epimer (Figure S21).Based on all of the above, the structure inclusive of absolute configurations for jugione A (1) was assigned as shown.HRESI(+)MS measurement established a molecular formula for 2 (C 39 H 34 O 13 , ∆mmu +1.4) isomeric with 1.Comparison of the 1D and 2D NMR (CDCl 3 ) data for 2 (Tables 1, 2 and S6, Figure 4 and Figures S22-S27) with 1 allowed the key differences to be attributed to a reverse Claisen condensation-like opening of ring G (Figure 5) with concomitant replacement of the sp 2 quaternary C-23 in 1 (δ C 99.8) with a diastereotopic H 2 -23/C-23 methylene in 2 (δ H 3.18/3.02,AB q , J 17.0 Hz; δ C 38.2) and associated formation of a sidechain butyrolactone moiety, as evident from deshielding of H-25 in 2 compared to 1 (∆δ H +0.37).On the basis of the spectroscopic similarities between 1 and 2 including near-identical ECD spectra (Figure S44) and biogenetic considerations, the structure inclusive of absolute configuration for jugione B (2) was assigned as shown.
Alert to the possibility that chemically reactive natural products can form artifacts during handling and storage [14], careful analysis of fresh CMB-STF067 extracts prior to chemical fractionation detected 1-4 and confirmed their status as natural products.Supportive of this, no chemical stability issues were observed during the handling and storage of pure samples of 1-4.
The antibacterial activity against drug-sensitive Staphylococcus aureus ATCC 25923 that prompted our initial interest in CMB-STF067 was shown to be due to 1-2 and 4 (IC 50 1.8 to 6.6 µM), with the closely related carboxylic acid 3 being inactive (IC 50 >30 µM) (Table 3 and Figure S45).Of note, this antibacterial activity extended to both drug-sensitive and resistant Enterococcus faecalis strains (IC 50 0.5 to 1.8 µM and 2.6 to 3.9 µM, respectively) and multiple-drug-resistant strains of S. aureus (IC 50 1.8 to 6.4 µM) (Table 3).Notwithstanding, one of the challenges associated with discovering new natural product antibiotics is the need for selectivity, particularly in favor of pathogens over host (human) cells.To address this challenge, we assessed the ability of 1-4 to inhibit the growth of eukaryote cells, namely human colon and lung carcinoma and fungal cells.Significantly, while 1 was cytotoxic to carcinoma (IC 50 9-10 µM) and fungal (IC 50 4.1 µM) cells, the γ-lactoneand seco-ring G analogs 2 and 4, respectively, displayed no such cytotoxicity (IC 50 >30 mM) and were selectively effective against Gram-positive bacteria (Table 3 and Figure S45).Similar SARs have been observed for JBIR-97/98 and engyodontochones A and C (heterodimer scaffold i) and JBIR-99 and engyodontochones B, E and F (heterodimer scaffold ii) (Figure S48), where metabolites inclusive of ring G exhibit antibacterial, antifungal and cytotoxic properties, while those featuring a seco-ring G (including the γ-lactone) are antibacterial against Gram-positive bacteria but do not exhibit cytotoxicity towards eukaryote cells [12].It has also been reported that xanthoquinodins A6-A8 (heterodimer scaffold iii) and xanthoquinodins B11, B14 and B15 (heterodimer scaffold iv) (Figure S48) with an intact ring G are antibacterial and cytotoxic to eukaryotic cells (fungi and human), while seco-ring G analogs (included the γ-lactone) are antibacterial but with reduced cytotoxicity to eukaryotic cells [13,21].

Collection and Isolation of CMB-STF067
The fungus CMB-STF067 was isolated from roadside soil collected in 2019 near Jugiong, NSW, Australia.The soil sample was transported to the laboratory in a sealed container at room temperature, after which, a portion (1 g) in sterile water (10 mL) was heated for 30 min at 55 • C, with an aliquot (100 µL) serially diluted and applied to SDA and ISP-2 agar plates supplemented with both cycloheximide (100 µg/mL) and rifampicin (5 µg/mL).The plates were sealed with parafilm and incubated at 29 • C with periodic inspection over 4 weeks.The fungus CMB-STF067 (Figure S1) was manually recovered by colony picking from the SDA plate, and after recultivation on SDA medium, the pure isolate was cryopreserved at −80 • C in 15% aqueous glycerol.

Taxonomic Identification of CMB-STF067
Genomic DNA was extracted from the mycelia of an SD static broth culture of CMB-STF067 using the DNeasy Plant Mini Kit (QIAGEN) as per the manufacturer's protocol.The 18S rRNA genes were amplified by PCR using the universal primers ITS 1 (5 ′ -TCCGTAGGTGAACCTGCGG-3 ′ ) and ITS 4 (5 ′ TCCTCCGCTTATTGATATGC-3 ′ ) purchased from Sigma-Aldrich (Merck, Darmstadt, Germany).The PCR mixture (50 µL) contained genomic DNA (2 µL, 20-40 ng), EmeraldAmpn GT PCR Master Mix (2× Premix, 25 µL), primer (0.2 µM, each) and H 2 O (up to 50 µL).PCR was performed using the following conditions: initial denaturation at 95 • C for 2 min, 40 cycles in series of 95 • C for 20 s (denaturation), 56 • C for 20 s (annealing) and 72 • C for 30 s (extension), followed by one cycle at 72 • C for 5 min.The PCR products were purified with a PCR purification kit (QIAGEN).Amplification products were examined by agarose gel electrophoresis.The DNA sequencing was performed by the Australian Genome Research Facility (AGRF) at The University of Queensland.A GenBank BLAST analysis (NCBI database) (Figure S2) on the resulting ITS gene sequence (accession number OR730993) and following a phylogenetic analysis revealed 100.0%identity with the fungal strain Penicillium shearii (Figure S3).

UPLC-DAD Profiling of MATRIX Extracts
Aliquots (1 µL, 5 mg/mL MeOH) of all MATRIX extracts prepared as noted above were analyzed by UPLC-DAD using the method outlined in the general experimental procedure (Figures S5, S7 and S9).

GNPS Molecular Network Profiling of MATRIX Extracts
Aliquots (1 µL, 0.05 mg/mL MeOH) of MATRIX extracts prepared as noted above were subjected to UPLC-QTOF analysis using the method outlined in the general experimental procedure.The resulting MS/MS Mass Hunter data files (.d) were converted to .mzXMLfile format using MS convert software version 3.0 [29] prior to being uploaded to the GNPS platform (gnps.ucsd.edu,accessed on 24 October 2023) [1] through FTP approaches (FileZilla).A molecular network analysis was performed using an online workflow at GNPS, setting the minimum cluster size at 2, cosine score 0.5 and the minimum number of fragments at 6.The spectral networks were imported into Cytoscape (version 3.9.1)[30] and visualized using a ball-stick layout where nodes represented parent masses and edge thickness corresponded to cosine score (Figures S4, S6, S8 and S10).

Scaled-Up Cultivation and Chemical Fractionation of CMB-STF067
The UPLC and GNPS profiling as outlined above prompted the optimized scaled-up cultivation of CMB-STF067 on red rice.To this end, cells recovered from a 7-day inhibitory mold agar (IMA, Table S1) cultivation of CMB-STF067 were used to inoculate flasks (2 × 2 L) containing red rice media (140 g, 200 mL distilled water), which were subsequently incubated at 27 • C for 21 days.After incubation, the red rice cultures were extracted with EtOAc (4 × 500 mL), and the organic phase was concentrated in vacuo at 40 • C to yield an extract (2.37 g) which was subjected to sequential trituration followed by concentration under N 2 at 40 • C to yield an n-hexane (201 mg) and a combined CH 2 Cl 2 and MeOH (1.80 g) soluble extract.The latter was subjected to gel chromatography (Sephadex LH-20, 2.5 × 85 cm, gravity isocratic elution with 50% CH 2 Cl 2 /MeOH) to yield fractions which were combined based on analytical HPLC-DAD-MS analysis.

Antibacterial Assay
LB agar plates inoculated with the bacterial isolate to be tested were incubated at 37 • C for 24 h, after which, several colonies were transferred to fresh sterile LB broth which was incubated at 37 • C for 24 h, and following the measurement of optical density, the cell density was adjusted to 5 × 10 5 CFU/mL.Analytes (1-4 and controls) were dissolved in DMSO and diluted with H 2 O to afford stock solutions (600 µM, 20% DMSO) which were serially diluted with 20% DMSO to yield analyte concentrations ranging from 600 to 0.2 µM.An aliquot (10 µL) of each analyte dilution was transferred to a 96-well microtiter plate along with freshly prepared bacterium broth (190 µL) to final concentrations of 30-0.01 µM in 1% DMSO.The resulting assay plates were incubated at 37 • C for 18 h and the optical density of each well was measured spectrophotometrically at 600 nm using the POLARstar Omega plate reader (BMG LABTECH, Offenburg, Germany).Antibacterial screening was carried out against Gram-positive Staphylococcus aureus ATCC 25923, clinical isolates of daptomycin-resistant Staphylococcus aureus 587701692:1, methicillin-resistant Staphylococcus aureus AUS-RBWH-MRSA-01/02, vancomycin-resistant Enterococcus AUS-RBWH-VRE-01, Enterococcus faecalis ACM 5184 and Gram-negative Escherichia coli ATCC11775.The positive control was rifampicin (10 µM in 1% DMSO) and the negative control was 1% DMSO in culture broth, together with extracts prepared from LB broth medium without bacterial inoculation.Each analysis was repeated two times and the data represented graphically, and IC 50 and MIC values were calculated using GraphPad Prism version 10.0.1 (Figure S45).

Antifungal Assay
SD agar plates inoculated with Candida albicans ATCC 10231 were incubated at 27 • C for 48 h, after which, several colonies were transferred to fresh sterile SD broth (4 mL) which was incubated at 27 • C for 48 and, following the measurement of optical density, the cell density was adjusted to 5 × 10 5 CFU/mL.An aliquot (10 µL) of analytes as prepared above for antibacterial assays was transferred to a 96-well microtiter plate and freshly prepared fungal broth (190 µL) was added to each well to give final concentrations of 30-0.01 µM in 1% DMSO.The resulting assay plates were incubated at 27 • C for 48 h and the optical density of each well was measured spectrophotometrically at 600 nm using the POLARstar Omega plate reader (BMG LABTECH, Offenburg, Germany).The positive control was amphotericin (10 µM in 1% DMSO) and the negative control was 1% DMSO, together with extracts prepared from SD broth without fungal inoculation.Each analysis was repeated two times and the data represented graphically, and IC 50 and MIC values were calculated using GraphPad Prism version 10.0.1 (Figure S45).

Cytotoxic Assay
Aliquots (3000 cells/well in 190 µL of Roswell Park Memorial Institute medium supplemented with 10% fetal bovine serum) of human colorectal (SW620) and lung (NCI-H460) carcinoma cells were transferred to 96-well plates and incubated at 37 • C in 5% CO 2 for 3 days.An aliquot (10 µL) of analytes as prepared above for antibacterial assays was transferred to a 96-well microtiter plate and incubated again for 24 h, after which, an aliquot (10 µL) of a solution of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) in phosphate-buffered saline (5 mg/mL) was added to each well which were again incubated for 4 h.The media were then carefully removed (pipette) and the residue dissolved in DMSO (100 µL) by shaking at 50 rpm for 2 min.Finally, the absorbance of each well was measured spectrophotometrically at 600 nm using the POLARstar Omega plate reader (BMG LABTECH, Offenburg, Germany).The positive control was sodium dodecyl sulfate (SDS) and the negative control was 1% DMSO.Each analysis was repeated two times and the data represented graphically, and IC 50 and MIC values were calculated using GraphPad Prism version 10.0.1 (Figure S45).

Conclusions
The jugiones are rare xanthone-anthraquinone heterodimers, which were only produced by one Penicillium strain (class Eurotiomycetes) in our in-house soil-derived microbe collection (~2000 isolates).Such heterodimers have only previously been reported from fungi belonging to the Sordariomycetes class isolated from various substrates (marine sponges, soil, insects and plants).An SAR assessment of jugiones A-D determined that 1 was antibacterial against vancomycin-resistant Enterococcus faecalis and multiple-drugresistant isolates of Staphylococcus aureus and was cytotoxic to human carcinoma cells, while 2 and 4 retained antibacterial properties but were not cytotoxic, and 3 was neither antibacterial nor cytotoxic.
While most xanthone-anthraquinone heterodimers are cytotoxic towards eukaryotic cells, our investigations into the jugiones reveal that ring G modification can reduce cytotoxicity while retaining the antibacterial activity-as evidenced by jugiones B and D which exhibit antibacterial activity against both drug-susceptible and resistant Grampositive bacteria but display no cytotoxicity against eukaryotic cells.Given the increase in multidrug-resistant bacteria in the community, jugiones and other related heterodimers could be viewed as antibacterial scaffolds with potential.

Figure 2 .
Figure 2. Selected NMR (CDCl3) correlations and J values for jugione A (1), with individual substructures A to D highlighted.