Polyketide-Derived Secondary Metabolites from a Dothideomycetes Fungus, Pseudopalawania siamensis gen. et sp. nov., (Muyocopronales) with Antimicrobial and Cytotoxic Activities.

Pseudopalawania siamensis gen. et sp. nov., from northern Thailand, is introduced based on multi-gene analyses and morphological comparison. An isolate was fermented in yeast malt culture broth and explored for its secondary metabolite production. Chromatographic purification of the crude ethyl acetate (broth) extract yielded four tetrahydroxanthones comprised of a new heterodimeric bistetrahydroxanthone, pseudopalawanone (1), two known dimeric derivatives, 4,4'-secalonic acid D (2) and penicillixanthone A (3), the corresponding monomeric tetrahydroxanthone paecilin B (4), and the known benzophenone, cephalanone F (5). Compounds 1-3 showed potent inhibitory activity against Gram-positive bacteria. Compounds 2 and 3 were inhibitory against Bacillus subtilis with minimum inhibitory concentrations (MIC) of 1.0 and 4.2 μg/mL, respectively. Only compound 2 showed activity against Mycobacterium smegmatis. In addition, the dimeric compounds 1-3 also showed moderate cytotoxic effects on HeLa and mouse fibroblast cell lines, which makes them less attractive as candidates for development of selectively acting antibiotics.


Introduction
Fungi are potentially known as a promising source of bioactive compounds for drug discovery [1]. Mushrooms and other Basidiomycota, in particular, are widely used in traditional Chinese medicines and have been shown to provide beneficial activities against cancer and other ailments [2,3], but even the microfungi have various other potential benefits [4]. Dothideomycetes (Ascomycota) is a large and diverse class comprising of mostly microfungi. New species are constantly being discovered from this group and could be promising sources of novel bioactive compounds [5][6][7]. A few contemporary studies in Thailand have been focusing on saprobic fungi in Dothideomycetes as a source for finding novel bioactive compounds. For example, a novel Thai Dothideomycete, Pseudobambusicola thailandica, Biomolecules 2020, 10, 569 3 of 23 TEF1: partial translation elongation factor 1-α gene; and RPB2: partial RNA polymerase II second largest subunit gene sequence data from representative closest relatives to our strains were selected following Hongsanan et al. [20], Crous et al. [21], Hernández-Restrepo et al. [22], and Mapook et al. [23,24], to confirm the phylogenetic placement of our new strains. The phylogenetic analysis based on maximum likelihood (ML) and Bayesian inference (BI) were following the methodology as described in Mapook et al. [23,24]. The sequences used for analyses with accession numbers are given in Table 1. Phylogram generated from ML analysis was drawn using FigTree v. 1.4.2 [25] and edited by Microsoft Office PowerPoint 2013. The new nucleotide sequence data are deposited in GenBank.

General Information of Chromatography and Spectral Methods
Specific optical rotations ([α] D ) were measured using a Perkin-Elmer (Überlingen, Germany) 241 polarimeter in a 100 × 2 mm cell at 22 • C. ECD spectra were recorded on a J-815 spectropolarimeter (JASCO, Pfungstadt, Germany). UV spectra were obtained on a Shimadzu (Duisburg, Germany) UV-Vis spectrophotometer UV-2450 with 1 cm quartz cells. IR spectra were measured with a Nicolet Spectrum 100 FT-IR spectrometer (Perkin-Elmer, Waltham, MA, USA). Nuclear magnetic resonance (NMR) spectra were recorded on a Bruker 700 MHz Avance III spectrometer with a 5 mm TXI cryoprobe ( 1 H 700 MHz, 13 C 175 MHz) and a Bruker 500 MHz Avance III spectrometer with a BBFO (plus) SmartProbe ( 1 H 500 MHz, 13 C 125 MHz). In all cases, spectra were acquired at 25 • C (unless otherwise specified) in solvents as specified in the text, with referencing to residual 1 H or 13 C signals in the deuterated solvents (CDCl 3 or MeOH-d 4 ). HPLC-DAD/MS analysis was conducted using an amaZon Speed ETD ion trap mass spectrometer (Bruker Daltonics, Bremen, Germany). HR-ESI mass spectra was measured using an Agilent 1200 series HPLC-UV system (column 2.1 × 50 mm, 1.7 µm, C18 Waters Acquity UPLC BEH) combined with an maXis (Bruker) ESI-TOF-MS instrument The mobile phase was composed of H 2 O + 0.1% formic acid (solvent A) and acetonitrile + 0.1% formic acid (solvent B), with the following gradient: 5% solvent B for 0.5 min with a flow rate of 0.6 mL/min, increasing to 100% solvent B in 19.5 min and then maintaining 100% solvent B for 5 min. UV/Vis detection at 200-600 nm. Chemicals and solvents were obtained from AppliChem GmbH, Avantor Performance Materials, Carl Roth GmbH & Co. KG (Karlsruhe, Germany) and Merck KGaA (Darmstadt, Germany) in analytical and HPLC grade.

Fermentation and Extraction
Five mycelial plugs from actively growing colonies on malt extract agar (MEA) media (malt extract 20 g/L, D-glucose 20 g/L, peptone 6 g/L, pH 6.3) were cut using a sterile cork borer (0.7 × 0.7 cm 2 ) and placed into a sterilized 500 mL Erlenmeyer flask containing 200 mL of liquid yeast malt (YM) medium (malt extract 10 g/L, D-glucose 4 g/L, yeast extract 4 g/L, pH 6.3). These seed cultures were incubated on a rotary shaker (140 rpm) at 23 • C in the dark for nine days. Ten milliliters of the seed culture were added into 25 × 500 mL sterile Erlenmeyer flasks with 200 mL of YM medium and incubated on a rotary shaker for 14 days. The extraction was conducted 3 days after glucose depletion as monitored by the glucose strip test using Bayer Harnzuckerstreifen, (Bayer, Leverkusen, Germany). Fungal mycelium and supernatant were separated by using vacuum filtration. The supernatant was mixed with 3% Amberlite XAD-16N adsorber resin (Sigma-Aldrich, Deisenhofen, Germany) and stirred for 1 h and filtrated to remove the culture broth. The XAD resin was eluted three times with an equal volume of ethyl acetate. The mycelia were extracted twice with an equal volume of acetone in an ultrasonic bath for 30 min and the combined extracts were passed through a filter, then dissolved in water/ethyl acetate. The aqueous phase (lower) was discarded while the organic phase (upper) was filtered through anhydrous sodium sulfate (Na 2 SO 4 ) for water removal and then evaporated to dryness. This procedure yielded 1580 mg mycelial crude extract and 769 mg of supernatant crude extract. The mycelial extract contained mainly fatty acids and ergosterol derivatives and showed only weak bioactivity. It was therefore not further processed. The supernatant extract contained the majority of the active components and was therefore subjected to preparative isolation of its active ingredients.

Isolation of Compounds 1-5
The supernatant crude extract was dissolved in methanol and initially fractionated on preparative HPLC manufactured by Gilson (Middleton, Wi, USA), comprised of a GX-271 Liquid Handler, a 172 DAD, a 305 and 306 pump, with 50SC Piston Pump Head. A Phenomenex (Torrance, Ca., USA) Gemini 10u C 18 110Å column (250 × 21.20 mm, 10 µm) was used as a stationary phase. The mobile phase was composed of deionised water (Milli-Q, Millipore, Schwalbach, Germany) with 0.05% of trifluoroacetic acid (TFA) as a solvent A and acetonitrile (ACN) HPLC grade with 0.05% TFA as a solvent B. The fractionation proceeded with the following gradient: linear gradient of 10% solvent B for 5 min with a flow rate of 35 mL/min, followed by 10% to 100 % solvent B for 30 min, and 100% solvent B for 10 min. The UV detection was carried out at 210, 254 and 350 nm. Final five compounds were purified from initially 16 fractions (Figure 1). Compound 1 (pseudopalawanone; 5.51 mg) eluted at t R = 7.8 min from fraction 12, compound 2 (4,4 -secalonic acid D; 5.48 mg) eluted at t R = 10.5 min from fraction 15, compound 4 (paecilin B; 1.08 mg) eluted at t R = 6.9 min from fraction 4, and compound 5 (cephalanone F; 1.52 mg) eluted at t R = 3.0 min from fraction 3, while compound 3 (penicillixanthone A; 0.86 mg) eluted at t R = 11.3 min was resulted from the purification of fraction 16 (4.12 mg) on a VarioPrep Nucleodur 100-10 C 18 ec column (150 × 40 mm, 7 µm; Macherey-Nagel, Düren, Germany) using the following gradients: linear gradient of 30% solvent B for 5 min with a flow rate of 15 mL/min, followed by 30% to 100 % solvent B for 20 min, and 100% solvent B for 10 min.

Antimicrobial Activity and Cytotoxicity Assays
Minimum inhibitory concentrations (MIC) of compounds 1-5 were determined against various fungal and bacterial strains by using a 96-well serial dilution technique according to previously described procedures [92,93]. The tested organisms with results are given in Tables 3 and 4. Gentamicin, kanamycin, nystatin, and oxytetracycline were used as positive controls against tested organisms. In vitro cytotoxicity (IC 50 ) of compounds 1-5 were determined using the MTT assay according to previously described procedures [26,27] against the mouse fibroblast cell line (L929) and the human HeLa (KB-3-1) cell line. Epothilone B and methanol were used as positive and negative control, respectively.

Phylogenetic Analysis
The combined dataset of LSU, SSU, RPB2, ITS and TEF sequence data including our new strains were analyzed by maximum likelihood (ML) and Bayesian analyses. The combined sequence alignment is comprised of 155 taxa (6131 characters with gaps), which include representative strains from Lecanoromycetes as outgroup taxa. A best scoring RAxML tree with a final likelihood value of -91,669.392085 is presented in Figure 2  Mycobank number: MB834934. Etymology: The generic epithet refers to the similarity to Palawania. Saprobic on dead rachis of Arecaceae. Sexual morph: Ascomata superficial, solitary or scattered, sub-carbonaceous to carbonaceous, appearing as circular, flattened, dark brown to black spots, covering the host, without a subiculum, with a poorly developed basal layer and an irregular margin. Ostioles central. Peridium comprising dark brown or black to reddish-brown cells of textura epidermoidea to textura angularis. Hamathecium cylindrical to filiform, septate, hyaline, branching pseudoparaphyses. Asci eight-spored, bitunicate, fissitunicate, cylindric-clavate, straight or slightly curved, with an ocular chamber observed clearly when immature. Ascospores overlapping, 2-3-seriate, broadly fusiform to inequilateral, pointed ends, hyaline, 1-septate, constricted at the septum, guttulate when immature, surrounded by hyaline and thin layers of gelatinous sheath, observed clearly when mounted in Indian ink. Asexual morph: Undetermined.

Pseudopalawania siamensis Mapook and K.D. Hyde, sp. nov.
Mycobank number: MB834935; Figure 3 Etymology: Named after the country from where the fungus was collected, using the former name of Siam.
Culture characteristics: Ascospores germinating on MEA within 24 hrs. at room temperature and germ tubes produced from the apex. Colonies on MEA circular, slightly raised, filamentous, mycelium white at the surface and initially creamy-white to pale brown in reverse, becoming dark brown from the centre of the colony with creamy-white at the margin. Notes: Pseudopalawania is similar to Palawania in its superficial and flattened ascomata, with hyaline, 1-septate ascospores, but differs in its peridium wall patterns, shape of asci (cylindric-clavate vs. inequilateral to ovoid) with an ocular chamber and shape of ascospores (broadly fusiform to inequilateral vs. oblong to broadly fusiform) with a thin layer of gelatinous sheath. The gelatinous sheath in Palawania is thicker [24]. Pseudopalawania is also similar to Muyocopron in its superficial, flattened ascomata with similar peridium wall patterns, and asci with an ocular chamber; but differs in its sub-carbonaceous to carbonaceous ascomata, shape of asci and ascospores with surrounded by hyaline gelatinous sheath, 1-septate, while Muyocopron have coriaceous ascomata, aseptate ascospores with granular appearance and without gelatinous sheath [23]. In addition, the genus was compared with genera in Microthyriaceae of which no DNA sequence data are available, but the holotype specimens were re-examined in previous studies with morphological descriptions and illustrations [94][95][96][97][98][99], and neither of them matched our new fungus. Therefore, we introduce Pseudopalawania as a new genus with a new species P. siamensis from Thailand. The fungus is placed in Muyocopronaceae (Muyocopronales) with evidence from morphology and phylogeny. compared with genera in Microthyriaceae of which no DNA sequence data are available, but the holotype specimens were re-examined in previous studies with morphological descriptions and illustrations [94][95][96][97][98][99], and neither of them matched our new fungus. Therefore, we introduce Pseudopalawania as a new genus with a new species P. siamensis from Thailand. The fungus is placed in Muyocopronaceae (Muyocopronales) with evidence from morphology and phylogeny.     Pseudopalawanone (1) was obtained as optically active, pale yellow gum. The IR spectrum showed the presence of hydroxyl groups (3387 cm −1 ), carbonyl functionalities (1787, 1741 cm −1 ) and aromatic residues (1648, 1622 cm −1 ) while the UV spectrum was indicative of absorptions due to chromanone units [102,104]. The molecular formula C31H28O15, indicating eighteen double bond equivalents, was established by HR-ESIMS based on its protonated pseudomolecular ion peak ([M + H] + ) at m/z 641.1492. Observation of two sets of signals in the NMR spectra ( Figure S1 and S2) and careful comparison of the 1 H and 13 C NMR spectroscopic data of 1 ( Table 2) with those of 2-4 immediately revealed 1 to be an asymmetric dimer of an unfamiliar highly oxygenated tetrahydroxanthone subunit and 7-deoxyblennolide D [102]. Thus, the gross structure of the latter fragment along with its connection to 7-deoxyblennolide D was established through analysis of 1D and 2D NMR spectroscopic data and will be the subject of the following discussions. The 13 C and HSQC-DEPT edited spectra ( Figure S3) showed the presence of fifteen resonances comprised of a ketone (δC 194.9), a carboxyl group of an ester functionality (δC 176.6), a hemiacetal carbon (δC 108.9), four quaternary aromatic carbons (δC 106.8, 117.6, 158.3, 160.1), two aromatic methine carbons (δC 108.3, 143.8), two aliphatic quaternary carbons (δC 73.6, 84.7), two methine carbons (δC 30.4, 74.1), a methylene carbon (δC 33.8) and a methyl group (δC 14.9). The 1 H and COSY NMR spectrum ( Figure  S4) revealed two ortho-coupled aromatic protons ( 3 J = 8.6 Hz) for H-3 (δH 7.82) and H-4 (δH 6.77), and a seven-proton spin system comprised of H-5 (δH 4.44) -H-6 (δH 2.23) (H3-11) (δH 1.20) -H2-7 (δH 2.12, 2.36). A C-2 substituted 1-hydroxychromanone unit was elucidated on the basis of HMBC correlations of chelated 1-OH (δH 11.35) with C-1 (δC 160.1), C-2 (δC 117.6) and C-9a (δC 106.8) and of H-4 (δH 6.77) with C-2 and C-4a (δC 158.3). The remaining portion of the molecule was constructed through HMBC correlations of H-6 (δH 2.13) and H-11 (δH 1.20) with C-8 (δC 108.9), of H-5 (δH 4.44) with C-8a (δC 73.6), C-10a (δC 84.7) and C-12 (δC 176.6), and of H2-7 (δH 2.12, 2.36) with C-8 and C-8a. The chemical shifts assigned for C-8 and C-12 were ascribed to hemiacetal and γ-lactone moieties, respectively, by using a combination of 2D NMR experiments ( Figure 5). The lactone ester was plausibly attached to C-8 forming a γ-hydroxylactone subunit of a [3.2.1] bicyclic structure. The remaining 17 mass units was attributed to a hydroxyl group attached to the carbon (C-8a) of the chromanone substructure. This unusual tetrahydroxanthone motif could putatively originate presumably from α hydroxylation of the keto form of blennolide A, followed by nucleophilic attack  of the hydrolyzed C-12 methyl ester ( Figure 6). The relative configurations of C-5 and C-6 were readily established to be similar with blennolide A by the coupling constant ( 3 J5,6 = 4.0 Hz) and the Pseudopalawanone (1) was obtained as optically active, pale yellow gum. The IR spectrum showed the presence of hydroxyl groups (3387 cm −1 ), carbonyl functionalities (1787, 1741 cm −1 ) and aromatic residues (1648, 1622 cm −1 ) while the UV spectrum was indicative of absorptions due to chromanone units [102,104]. The molecular formula C 31 H 28 O 15 , indicating eighteen double bond equivalents, was established by HR-ESIMS based on its protonated pseudomolecular ion peak ([M + H] + ) at m/z 641.1492. Observation of two sets of signals in the NMR spectra ( Figures S1 and S2) and careful comparison of the 1 H and 13 C NMR spectroscopic data of 1 ( Table 2) with those of 2-4 immediately revealed 1 to be an asymmetric dimer of an unfamiliar highly oxygenated tetrahydroxanthone subunit and 7-deoxyblennolide D [102]. Thus, the gross structure of the latter fragment along with its connection to 7-deoxyblennolide D was established through analysis of 1D and 2D NMR spectroscopic data and will be the subject of the following discussions. The 13 C and HSQC-DEPT edited spectra ( Figure S3) showed the presence of fifteen resonances comprised of a ketone (δ C 194.9), a carboxyl group of an ester functionality (δ C 176.6), a hemiacetal carbon (δ C 108.9), four quaternary aromatic carbons (δ C 106.8, 117.6, 158.3, 160.1), two aromatic methine carbons (δ C 108.3, 143.8), two aliphatic quaternary carbons (δ C 73.6, 84.7), two methine carbons (δ C 30.4, 74.1), a methylene carbon (δ C 33.8) and a methyl group (δ C 14.9). The 1 H and COSY NMR spectrum ( Figure S4) revealed two ortho-coupled aromatic protons ( 3 J = 8.6 Hz) for H-3 (δ H 7.82) and H-4 (δ H 6.77), and a seven-proton spin system comprised of H-5 (δ H 4.44) -H-6 (δ H 2.23) (H 3 -11) (δ H 1.20) -H 2 -7 (δ H 2.12, 2.36). A C-2 substituted 1-hydroxychromanone unit was elucidated on the basis of HMBC correlations of chelated 1-OH (δ H 11.35) with C-1 (δ C 160.1), C-2 (δ C 117.6) and C-9a (δ C 106.8) and of H-4 (δ H 6.77) with C-2 and C-4a (δ C 158.3). The remaining portion of the molecule was constructed through HMBC correlations of H-6 (δ H 2.13) and H-11 (δ H 1.20) with C-8 (δ C 108.9), of H-5 (δ H 4.44) with C-8a (δ C 73.6), C-10a (δ C 84.7) and C-12 (δ C 176.6), and of H 2 -7 (δ H 2.12, 2.36) with C-8 and C-8a. The chemical shifts assigned for C-8 and C-12 were ascribed to hemiacetal and γ-lactone moieties, respectively, by using a combination of 2D NMR experiments ( Figure 5). The lactone ester was plausibly attached to C-8 forming a γ-hydroxylactone subunit of a [3.2.1] bicyclic structure. The remaining 17 mass units was attributed to a hydroxyl group attached to the −carbon (C-8a) of the chromanone substructure. This unusual tetrahydroxanthone motif could putatively originate presumably from α hydroxylation of the keto form of blennolide A, followed by nucleophilic attack of the hydrolyzed C-12 methyl ester ( Figure 6). The relative configurations of C-5 and C-6 were readily established to be similar with blennolide A by the coupling constant ( 3 J 5,6 = 4.0 Hz) and the chemical shifts as 5S*, 6S* while that of C-10a was assigned R* based on the observed positive n-π* CD transition at around 331 nm [104]. The chirality of C-8a cannot be established using available methods due to its remoteness to most protons in the molecule.

Structure Elucidation of the New Compound
The axial configuration of C-2/C-4′ was assigned as P based on the CD spectrum of 1 which showed a positive first Cotton effect (225 nm, De = −6.41) and a negative second cotton effect (250 nm, De = +3.15). Thus, compound 1 was given the trivial name pseudopalawanone. To establish unambiguously its relative and absolute configurations especially C-8a in the blennolide A substructure and C-10a' in the 7-deoxyblennolide D substructure, we suggest additional experiments such as asymmetric total synthesis, derivatization with heavy atom/s followed by single crystal x-ray diffraction and/or further ECD-TDDFT measurements and calculations.

Biological Activity of Compounds 1-5
The polyketides 1-5 were evaluated for their antimicrobial activity against selected microorganisms (Table 3) and cytotoxicity against two mammalian cell lines, HeLa cells KB3.1 and mouse fibroblast cell line L929 (Table 4). The starting concentration for antimicrobial assay and cytotoxicity assay were 66.7 and 300 μg/mL, respectively and the substances were dissolved in MeOH (1 mg/mL). MeOH was used as the negative control and showed no activity against the tested organisms and mammalian cell lines. Results were expressed as MIC or minimum inhibitory concentration (μg/mL) and IC50 or half maximal inhibitory concentration (μM) (Tables 3 and 4). The The axial configuration of C-2/C-4′ was assigned as P based on the CD spectrum of 1 which showed a positive first Cotton effect (225 nm, De = −6.41) and a negative second cotton effect (250 nm, De = +3.15). Thus, compound 1 was given the trivial name pseudopalawanone. To establish unambiguously its relative and absolute configurations especially C-8a in the blennolide A substructure and C-10a' in the 7-deoxyblennolide D substructure, we suggest additional experiments such as asymmetric total synthesis, derivatization with heavy atom/s followed by single crystal x-ray diffraction and/or further ECD-TDDFT measurements and calculations.

Biological Activity of Compounds 1-5
The polyketides 1-5 were evaluated for their antimicrobial activity against selected microorganisms (Table 3) and cytotoxicity against two mammalian cell lines, HeLa cells KB3.1 and mouse fibroblast cell line L929 (Table 4). The starting concentration for antimicrobial assay and cytotoxicity assay were 66.7 and 300 μg/mL, respectively and the substances were dissolved in MeOH (1 mg/mL). MeOH was used as the negative control and showed no activity against the tested organisms and mammalian cell lines. Results were expressed as MIC or minimum inhibitory concentration (μg/mL) and IC50 or half maximal inhibitory concentration (μM) (Tables 3 and 4). The The linkage between the chromanone subunit and the ©−lactone in the 7-deoxyblennolide D monomer was indicated by the HMBC correlation of H-5 (δ H 4.38) with C-10a (δ C 84.8) and C-12 (δ C 168.5). The C-5 S* and C-6 S* relative configurations in the lactone moiety were established by coupling constant analysis ( 3 J 5,6 = 2.5 Hz) depicting a pseudodiaxial orientation for H-5 /H-6 and the NOE (Figures S6 and S7) noted between H-5 and H-8a'a (δ H 3.14), H-8a b (δ H 2.98) and H-6 (δ H 2.65), and that of H-6 and H 3 -13 (δ H 3.80) [102]. The spatial arrangements in ring C were similar to 7-deoxyblennolide D corroborated by NOE correlations between H-5 , H 3 -11 (δ H 1.16) and H-7 b (δ H 1.99). Finally, the relative configuration of C-10a may be tentatively assigned as S* on the basis of negative π*-π* transitions below 330 nm and positive n-π* transitions at 346 nm in the ECD spectrum ( Figure S9) of 1 [104]. The overall relative configuration of the blennolide-type tetrahydroxanthone substructure is 5S*, 6S*, and 10aS* thus, structurally similar to 7-deoxyblennolide D.
The planar structure of 1 was established by connecting the two monomers through the linkage of C-2 (δ C = 117.6) of the oxidized secalonic acid subunit and C-4 (δ C 114.0) of 7-deoxyblennolide D evidenced by the diagnostic HMBC correlations of H-3 (δ H 7.82) to C-4 and H-3 (δ H 7.54) to C-2. The axial configuration of C-2/C-4 was assigned as P based on the CD spectrum of 1 which showed a positive first Cotton effect (225 nm, De = −6.41) and a negative second cotton effect (250 nm, De = +3.15). Thus, compound 1 was given the trivial name pseudopalawanone. To establish unambiguously its relative and absolute configurations especially C-8a in the blennolide A substructure and C-10a' in the 7-deoxyblennolide D substructure, we suggest additional experiments such as asymmetric total synthesis, derivatization with heavy atom/s followed by single crystal x-ray diffraction and/or further ECD-TDDFT measurements and calculations.

Biological Activity of Compounds 1-5
The polyketides 1-5 were evaluated for their antimicrobial activity against selected microorganisms (Table 3) and cytotoxicity against two mammalian cell lines, HeLa cells KB3.1 and mouse fibroblast cell line L929 (Table 4). The starting concentration for antimicrobial assay and cytotoxicity assay were 66.7 and 300 µg/mL, respectively and the substances were dissolved in MeOH (1 mg/mL). MeOH was used as the negative control and showed no activity against the tested organisms and mammalian cell lines. Results were expressed as MIC or minimum inhibitory concentration (µg/mL) and IC 50 or half maximal inhibitory concentration (µM) (Tables 3 and 4). The known compounds 4 and 5 showed neither antimicrobial nor cytotoxic activities. The dimeric tetrahydroxanthone 4,4 -secalonic acid D (2) showed inhibition against the pathogenic fungus Candida albicans while penicillixanthone A (3) inhibited Mucor hiemalis with activities comparable to the positive drug control nystatin. Prominent activities were observed for compounds 2 and 3 against Bacillus subtilis with MIC values of 1.0 and 4.2 µg/mL, respectively. Compound 2 also showed inhibitory activity against all Gram-positive bacteria (Bacillus subtilis, Micrococcus luteus, Mycobacterium smegmatis, and Staphylococcus aureus), while compounds 1 and 3 also showed inhibitory activity against the Gram-positive bacterium, Mycobacterium smegmatis. In general, only the dimeric tetrahydroxanthones 1-3 exhibited activity against fungi and bacteria with the secalonic acid-bearing derivatives 2 and 3 exhibiting better antimicrobial profile. However, the dimeric compounds 1-3 also showed moderate cytotoxic activities against two mammalian cell lines (Table 4). These inhibitory concentrations for cytotoxic activities are given traditionally in molar concentrations, but if they are calculated in µg/mL, the IC 50 values would be equivalent to a range of 2-25 µg/mL (i.e., the same or only slightly higher activity range as compared to the MIC). This observation precludes the potential use of these metabolites as candidates for the development of antibiotics, because their selectivity indices are far too low. In addition, the fact that they are broadly active against both, prokaryotic and eukaryotic test organisms suggests that they may address multiple targets and are therefore less suitable for development of any drug.  The in vitro cytotoxicity test of polyketides 1-5 was conducted against two mammalian cell lines, with epothilone B as positive control. Starting concentration for cytotoxicity assay was 66 µg/mL, substances were dissolved in MeOH (1 mg/mL). MeOH was used as negative control and showed no activity against the tested mammalian cell lines. Results were expressed as IC 50 : half maximal inhibitory concentration (µM). (-): no inhibition.
Some information on these and chemically related compounds is even available from the literature. Compound 2 (4,4 -secalonic acid D; 4,4 -SAD) is a regioisomeric structure to SAD with 2,2 -biarylic connectivity, belonging to the secalonic acid family. This compound class has long been known to have non-selective antimicrobial and other biological activities [100][101][102][103][104][105][106]. The compound 4,4 -SAD (2) itself was recently reported to have low toxicity with "potent" antitumor activity against several cancer cell lines through cell proliferation inhibition and apoptosis induction [100]. However, when compared to the precursor for a marketed drug, epothilone, which we used as a positive control in our standard cytotoxicity assays (Table 4), the activities of all the metabolites from Pseudopalawania siamensis are much weaker. Promising candidate compounds for anticancer therapy should have at least activities in the 100 nM range such assays. Penicillixanthone A (3) was also already shown to possess moderate antibacterial activity against four tested bacterial strains (M. luteus, Pseudoalteromonas nigrifaciens, E. coli and B. subtilis [100], and its moderate cytotoxic effects on MDA-MB-435 human melanoma cells and SW620 human colorectal adenocarcinoma cell lines had been previously reported [101]. Furthermore, compound 3 was previously isolated from the marine-derived fungus Aspergillus fumigatus, and was reported to exhibit anti-HIV-1 activities by inhibiting CCR5-tropic HIV-1 and CXCR4-tropic HIV-1 infection [103]. These data also point toward non-selective effects of this metabolite in biological systems.

Conclusions
The current study showed that new genera and species of tropical fungi can still yield numerous new and interesting secondary metabolites. Even though the preliminary characterization of the metabolites 1-5 indicates that they act non-selectively in biological systems, their further evaluation could result in the discovery of additional, more specific biological effects. In any case, it is worthwhile to further explore tropical fungi whose cultures result from taxonomic and biodiversity studies for the production of secondary metabolites and other potentially beneficial properties [107].