Bioactive Bioxanthracene and Cyclodepsipeptides from the Entomopathogenic Fungus Blackwellomyces roseostromatus BCC56290

In the course of our ongoing research targeting the identification of potential biocontrol agents from entomopathogenic fungi (EPF), we explored a solid-state rice fungal extract of Blackwellomyces roseostromatus BCC56290 derived from infected lepidopteran larvae. Chemical and biological prospections afforded four unprecedentedly reported natural products differentiated into a dimeric naphthopyran bioxanthracene ES-242 derivative (1) and three cyclodepsipeptides (2–4) in addition to two known cyclodepsipeptides, cardinalisamides B (5) and C (6). Chemical structures of the isolated compounds were elucidated through comprehensive 1D/2D NMR and HR-ESI-MS data together with comparisons to the reported literature. The absolute configuration of the isolated cyclodepsipeptides was determined using Marfey’s method. All isolated compounds were assessed for their antimicrobial, cytotoxic, and nematicidal activities with some compounds revealing significant activities.


Introduction
Entomopathogenic fungi constitute a subgroup of soil-dwelling trophic fungi, infecting insects, majorly classified under the order Hypocreales, class Sordariomycetes, phylum Ascomycota [1].The Hypocrealean Entomopathogenic Fungi (HEPF) comprise several genera including Beauveria, Cordyceps, and Isaria (family Cordycipitaceae) in addition to Metarhizium (family Clavicipitaceae) [2].Species of Beauveria and Metarhizium serve as the most commercially applied biocontrol agents (BCAs) [3].In addition, the HEPF revealed an immanent capacity to produce secondary metabolites (SMs) of diverse structural and pharmacological features including some marketed pharmaceuticals and/or agrochemicals [2,4,5].The diversity in the HEPF parvome of SMs, ranging from molecules below 200 Da up to cyclic polypeptide (>1200 Da) [1], was reflected in a broad range of bioactivities mitigating the pathologic stresses affecting the hepatic, cardiovascular, immune, and nervous systems [2,4,5].Among the other genera of the family Cordycipitaceae is the genus Blackwellomyces whose species were reported to infect the larvae of coleopteran and lepidopteran insects in particular B. calendulinus and B. minutus [6][7][8].To the best of our knowledge, few SMs were reported from the genus Blackwellomyces cardinalis including the antitrypanosomal cyclohexadepsipeptides, cardinalisamides A-C [9], and oosporein, a bibenzoquinone derivative with potent antimicrobial, antifungal and insecticidal activities [10] whose biosynthetic gene cluster was recently described [11].
Along the course of our ongoing research targeting anti-infective secondary metabolites from HEPF, we came across the fungal strain Blackwellomyces roseostromatus BCC56290 derived from a soil-buried lepidopteran larva in Thailand.The current study reports the chemical and biological characterization of fungal secondary metabolites purified from its solid-state rice culture extract.
Compound 4 was obtained as a yellowish-brown amorphous solid with its molecular formula established as C38H53N5O7 indicating fifteen degrees of unsaturation based on its HR-ESI-MS that revealed a protonated molecular ion peak at m/z 692.4023 [M + H] + (calculated 692.4018) and a sodium adduct at m/z 714.3836 [M + Na] + (calculated 714.3837).The 1D ( 1 H/ 13 C) NMR spectral data of 4 (Table 3, Figure S27) exhibited the characteristic features of a peptide including the presence of three exchangeable amide NH signals (δH 7.42-7.92)together with two N-methyl moieties at δH 3.12 (δC 37.6) and 3.16 (δC 36.2, Figures 3 and S21) revealed comparable characteristic features for 2 apart from the emergence of proton signals ascribed to a second Trp residue such as a deshielded pyrrole NH proton at δ H 10.69 (d, J = 2.4 Hz) that revealed a key cross-peak to an olefinic proton at δ H 6.81 (d, J = 2.4 Hz, H-10 ′′ ) in addition to the presence of a second spin system among four olefinic protons at δ H 7.47 (H-5 ′′ )/6.95 (H-6 ′′ )/7.04 (H-7 ′′ )/7.32 (H-8 ′′ ).By comparing the 1D ( 1 H and 13 C) NMR data and the key HMBC correlations of 2 and 3 (Table 2, Figures 3, S14 and S22), compound 3 was found to have a second Trp residue replacing Ala in 2 and hence has the amino acid sequence as HMDA-Trp 1 -Trp 2 -Phe.As for 2, the absolute configurations of amino acid residues were determined to be 3S,4S-HMDA-L-Trp 1 -L-Trp 2 -L-Phe based on the common biosynthetic origin of beauveriolides [20], and the results of Marefy's method (Figures S45-S46).Based on the aforementioned results, compound 3 was recognized as a previously undescribed cyclic tetradepsipeptide and was named beauveriolide U.  3, Figure S27) exhibited the characteristic features of a peptide including the presence of three exchangeable amide NH signals (δ H 7.42-7.92)together with two N-methyl moieties at δ H 3.12 (δ C 37.6) and 3.16 (δ C 36.5) in addition to five amino acid α-proton signals (δ H 3.47-4.93),and four diastereotopic β-methylene groups (δ H 3.19/3.34,2.90/3.15,1.57/1.64,1.47/1.53).The 1 H-1 H COSY spectrum of 4 (Figure 3) revealed two comparable spin systems each extending over a set of three aromatic protons signals at δ H 7.15/7.27/7.18 and at δ H 7.28/7.20/7.21 with each having a total integration index of five suggesting the presence of two monosubstituted aromatic rings.In addition, the 1 H-1 H COSY spectrum of 4 (Figure 3) revealed two pairs of comparable spin systems, one pair represents two leucine residues by spin systems extending from α-proton signals (δ H 4.93/4.81)to two diastereotopic methylene groups (δ H 1.57/1.64or δ H 1.47/1.53)then to two methine protons (δ H 1.58/1.45)and ending either by pair of two doublet methyl groups (δ H 0.84-0.89).The second pair of comparable spin systems revealed clear COSY cross-peaks between two overlapping α-proton signals at δ H 3.48 to two doublet methyl groups at δ H 1.32/1.19with each having a coupling constant of 7.0 Hz.Based on the obtained results and by searching the reported literature, compound 4 was found to be structurally related to cardinalisamide C (6) [9], a symmetric cyclohexadepsipeptide isolated from Blackwellomyces cardinalis (aka C. cardinalis) NBRC 103832, an entomopathogenic fungus derived from an infected lepidopteran larva.A careful comparison of the 1 H/ 13 C NMR and HR-ESI-MS spectral data of 4 and cardinalisamide C (6) revealed that compound 4 lacks symmetry in 6 due to the replacement of one oxygen atom by nitrogen resulting from having one phenyllactic acid (Pla) and one Phe residue in 4 instead of having two Pla residues in 6.This structural difference led to the asymmetry of 4. The amino acid sequence in 4 was determined by acquiring its HMBC spectrum (Figures 3 and S30) that revealed key correlations from an oxygenated methine proton at δ H 5.05 (dd, J = 10.5, 3.9 Hz, H-2) to two carbonyl carbon atoms at δ C 168.0 (C-1) and 170.0 (C-10): from an α-proton at δ H 4.93 (dt, J = 9.5, 7.0 Hz, H-15) to C-10 and C14 (δ C 171.2); from H-21 and H-30 to C-20 (δ C 169.8) and C-29 (δ C 170.2); and from H-34 to C-29 and C-33 (δ C 171.8).Accordingly, compound 4 was found to be a cyclohexadepsipeptide composed of Pla-NMe-Ala 1 -Leu 1 -Phe-NMe-Ala 2 -Leu 2 .According to the results of Marfey's analysis (Figures S46-S48) and based on the taxonomic proximity with common biosynthetic origin, the absolute configurations of the amino acid residues in 4 were deduced to be all in Lconfiguration.In conclusion, compound 4 was determined to be a previously undescribed cyclohexadepsipeptide and it was given a trivial name cardinalisamide D.   S34 and S40).A literature search of 5/6 and by comparing their 1 H/ 13 C NMR spectra to the reported literature [9], they were identified as cardinalisamides B and C, respectively.

Biological Assays
In our search for novel biological natural products with anti-infective activity, several bioassays were performed.All the isolated compounds 1-6 were assessed for their cytotoxic, antimicrobial, and nematicidal activity against a panel of different cell lines, Gram-positive/negative bacterial, fungal pathogens, and Caenorhabditis elegans, respectively.In cytotoxicity (MTT) assay, the obtained results (Table 4) revealed that among the tested compounds, only cardinalisamides B (5) and C ( 6) revealed significant pancytotoxic activities against almost all tested human cancer cell lines with IC 50 values between 2.2 and 13.9 µM in spite of being relatively non-toxic against normal fibroblast (L929) cell line which gives a positive indication for their safety.The antimicrobial activity assay was conducted against a panel of twelve different bacterial and fungal pathogens (Table S1), however, none of the tested compounds proved to be active.According to the reported literature, related ES-242 derivatives revealed moderate to potent antimalarial activity against Plasmodium falciparum (K1, multidrug-resistant strain) at IC 50 values between 3.3 and 12 µM [15].Herein, the nematicidal activity assay results (Figure 4, Table S2) revealed that only ES-242-9 (1) and cardinalisamide C (6) exhibited significant effects against C. elegans with corrected mortality rates of 51.1 and 53.6% at 100 µg/mL, respectively.Being neither cytotoxic nor antimicrobial in the conducted assays, ES-242-9 (1) could be a suitable candidate for further assessment to develop a nematicidal agent.In the literature, cardinalisamides B ( 5) and (6) were first reported to have an almost equipotent in vitro antitrypanosomal activity against Trypanosoma brucei, with IC 50 values of 12.8 and 12.5 µM, respectively [9].
The two previously undescribed beauveriolides T (2) and U (3) revealed no activity in any of the conducted assays.These cyclodepsipeptides belong to the class of beauveriolides, which are abundantly biosynthesized by HEPF [17][18][19].Beauveriolides were first isolated from the insect pathogenic fungus Beauveria bassiana in 1977 [17].Although various related derivatives have been reported to exhibit pharmacological properties, such as calmodulin (CaM) inhibition [23], and protective effects on HEI-OC1 cells [24], as well as stimulating glucose uptake in cultured rat L6 myoblasts [24], to the best of our knowledge, none has shown antimicrobial or nematicidal activities.Corrected mortality rate of compounds 1-6 (10 µg mL −1 , 50 µg mL −1 , and 100 µg mL −1 ) against C. elegans after 18 h of treatment.A solution of ivermectin (1 µg mL −1 ) was used as a positive control.The data were corrected using Schneider-Orelli's formula based on the negative control as methanol and are shown as the mean ± SD (n ≥ 3).The blue dash line characterizes the IC50.

Fermentation, Extraction and Isolation
We evaluated the antiviral activities of Blackwellomyces species such as B. aurantiacus, B. calendulinus, B. minutus, and B. roseostromatus, against SARS-CoV-2 and CHIKV infections including the inhibitory effect on 3CLpro activity, cytotoxicity of the extracts to Vero E6, Huh7, and HEK293 cells used in the assays and found that B. roseostromatus BCC56290 extract inhibited CHIKV infection.We therefore chose this fungus due to our previous findings against the virus.Fermentation of the selected strain B. roseostromatus BCC56290 was grown on solid-state rice media (fermentations completed in 10 × 1000 mL Erlenmeyer flasks containing 180 g of rice in 180 mL distilled water) and inoculated with 10 pieces of fully grown 7 mm mycelial plugs.The rice cultures were incubated on static conditions in white light/dark cycles in the laboratory, under room temperature, until full growth of the mycelia was achieved (31 days from inoculation).Thereafter, the cultures were soaked overnight in acetone (500 mL) and extracted thrice under sonication.The solvent was evaporated to yield an aqueous phase (400 mL) that was extracted thrice with ethyl acetate in a ratio of 1:1 (v/v).The organic phase was filtered through anhydrous sodium sulfate and evaporated on a rotary evaporator to dryness.The extracts were transferred into vials and dried under nitrogen and thereafter their weights were determined.
The crude extract (4.43 g) was dissolved in 3.0 mL of MeOH and loaded on silica gel by trituration using mortar and pestle and then left to dry overnight.The loaded air-dried extract was applied on the top of a column dry-packed with silica gel.A vacuum liquid chromatography (VLC) procedure was initiated by applying a gradient elution starting with 100% n-hexane, n-hexane/EtOAc (7:3, 1:1, and 3:7) and 100% EtOAc.Then, the gradient was switched to 100% DCM, DCM/MeOH (9:1, 8:2, 1:1) and ended by washing with 100% MeOH (v/v) affording ten fractions (F1-F10).

Derivatization with Marfey's Reagent and Elucidation of Amino Acid Configurations
Determination of amino acid stereochemistry of beauveriolide U (3) and cardinalisamide D (4) was conducted using Marfey's method, following the experimental procedure outlined by Viehrig et al. [25].For the hydrolysis, 500 µL of 6 N HCl was added to 0.5 mg of the compound and incubated at 90 • C for 18 h.The resulting hydrolysate was evaporated under vacuum conditions and suspended in 200 µL of Milli-Q water.Subsequently, 20 µL of 1 M NaHCO 3 and 100 µL of acetone containing 1% derivatization agent Nα-(2,4-dinitro-5-fluorophenyl)-l-alaninamide (FDAA) were added.The mixture was incubated at 40 • C for 40 min and evaporated to dryness under vacuum.Finally, the residual product was diluted in 1 mL MeOH and analyzed using an HPLC system connected to an amaZon speed ESI-MS as described before.The L -or D -configuration of the amino acids was determined by comparing the observed retention times with those of authentic amino acids subjected to the same derivatization procedure.The retention times (in minutes) of the FDAA-derivatized amino acids were as follows: alanine ( L-: 5.61, D-: 6.40), tryptophan ( L-: 7.82, D-: 8.41; leucine ( L-: 8.07, D-: 9.0), and phenylalanine ( L-: 8.01, D-: 8.81).

Nematicidal Activity
The nematicidal activity assay was conducted following a previously described procedure with slight modifications [28,29].C. elegans was cultured on nematode growth medium (NGM) containing 3 g NaCl, 20 g agar, 2.5 g peptone, 1 mL 1 M CaCl 2 , 25 mL of 1 M (pH 6.0) KPO 4 , 1 mL 1 M MgSO 4 , and 1 mL cholesterol (5 mg/mL in ethanol) per liter of medium.Plates were coated with Escherichia coli strain OP50, which served as the food source for nematodes.Synchronization of the nematode population was performed using a high egg density plate (~120 h).The plate was rinsed three times with 4 mL of 0.9% NaCl solution and then transferred to a 15 mL falcon tube.The nematode suspension was centrifuged at 1000 rpm for 3 min, and the supernatant was discarded.This procedure was repeated until a clear nematode suspension was obtained.After the last washing step, 2 mL of the suspension was mixed with 5 mL of bleaching solution (1 mL sodium hypochlorite solution, 0.5 mL 5 M NaOH, and 3.5 mL Milli-Q water).The solution was gently shaken for approximately 5 min to break down the nematode tissue, and monitored under the microscope.The reaction was stopped by adding 7 mL 0.9% NaCl solution when traces of adults were still visible.Subsequently, the suspension was centrifuged at 2500 rpm for 2 min and washed 3 times as previously described.After the final washing step, the supernatant was removed, and the volume was adjusted to 7 mL with 0.9% NaCl solution.The nematode egg suspension was incubated at 23 • C on a rotary shaker at 80 rpm for 18 h.The hatched nematodes were transferred to a fresh NGM plate supplemented with E.coli OP50.After 50-70 h, the J4 and adult C. elegans were washed from the plate as mentioned above.The nematode concentration was determined and diluted to approximately 1000 nematodes/mL.
Pure compounds were tested at concentrations of 10, 50, and 100 µg/mL in 48-well microtiter plates.The compounds, dissolved in MeOH, were added to the well plate and subsequently dried under nitrogen.After complete evaporation of the solvent, 300 µL of the nematode suspension (1000 nematodes/mL) was added to the compounds.Each treatment was replicated three times.MeOH was used as the negative control while 1 µg/mL Ivermectin served as the positive control.Nematodes were monitored 15 min after inoculation, and the plates were incubated at 24 • C and 150 rpm for 18 h.After incubation, both alive and dead nematodes were counted in three replicates under a stereomicroscope, and the mortality rate was calculated.Erect and non-moving nematodes were considered dead.A compound was deemed active if it resulted in mortality rates of at least 50% at a concentration of 100 µg/mL (lethal dose, 50%).The observed percentage of dead nematodes was corrected by considering the natural mortality observed in the negative control, using the Schneider-Orelli formula [30].

Figure 3 .
Figure 3. Key 1 H-1 H COSY and HMBC correlations of 2-4.Compound 3 was purified as a yellowish-white amorphous solid.Its HR-ESI-MS revealed a protonated molecular ion peak at m/z 704.3806 [M + H] + (calculated 704.3806 for C 42 H 50 N 5 O 5 + ) and a sodium adduct at m/z 726.3627 [M + Na] + (calculated 726.3626 for C 42 H 49 N 5 NaO 5 + ).Therefore, the molecular formula of 3 was determined as C 42 H 49 N 5 O 5 indicating twenty-one degrees of unsaturation exceeding those in 2 by six degrees.The 1 H NMR and the 1 H-1 H COSY spectral data of 3 in DMSO-d 6 (Table2, Figures3 and S21) revealed comparable characteristic features for 2 apart from the emergence of proton signals ascribed to a second Trp residue such as a deshielded pyrrole NH proton at δ H 10.69 (d, J = 2.4 Hz) that revealed a key cross-peak to an olefinic proton at δ H 6.81 (d, J = 2.4 Hz, H-10 ′′ ) in addition to the presence of a second spin system among four olefinic protons at δ H 7.47 (H-5 ′′ )/6.95 (H-6 ′′ )/7.04 (H-7 ′′ )/7.32 (H-8 ′′ ).By comparing the 1D ( 1 H and 13 C) NMR data and the key HMBC correlations of 2 and 3 (Table2, Figures3, S14 and S22), compound 3 was found to have a second Trp residue replacing Ala in 2 and hence has the amino acid sequence as HMDA-Trp 1 -Trp 2 -Phe.As for 2, the absolute configurations of amino acid residues were determined to be 3S,4S-HMDA-L-Trp 1 -L-Trp 2 -L-Phe based on the common biosynthetic origin of beauveriolides[20], and the results of Marefy's method (FiguresS45-S46).Based on the aforementioned results, compound 3 was recognized as a previously undescribed cyclic tetradepsipeptide and was named beauveriolide U.Compound 4 was obtained as a yellowish-brown amorphous solid with its molecular formula established as C 38 H 53 N 5 O 7 indicating fifteen degrees of unsaturation based on its HR-ESI-MS that revealed a protonated molecular ion peak at m/z 692.4023 [M + H] + (calculated 692.4018) and a sodium adduct at m/z 714.3836 [M + Na] + (calculated 714.3837).The 1D ( 1 H/ 13 C) NMR spectral data of 4 (Table3, FigureS27) exhibited the characteristic features of a peptide including the presence of three exchangeable amide NH signals (δ H 7.42-7.92)together with two N-methyl moieties at δ H 3.12 (δ C 37.6) and 3.16 (δ C 36.5) in addition to five amino acid α-proton signals (δ H 3.47-4.93),and four diastereotopic β-methylene groups (δ H 3.19/3.34,2.90/3.15,1.57/1.64,1.47/1.53).The 1 H-1 H COSY spectrum of 4 (Figure3) revealed two comparable spin systems each extending over a set

Compound 4
was obtained as a yellowish-brown amorphous solid with its molecular formula established as C 38 H 53 N 5 O 7 indicating fifteen degrees of unsaturation based on its HR-ESI-MS that revealed a protonated molecular ion peak at m/z 692.4023 [M + H] + (calculated 692.4018) and a sodium adduct at m/z 714.3836 [M + Na] + (calculated 714.3837).The 1D ( 1 H/ 13 C) NMR spectral data of 4 (Table

Figure 4 .
Figure 4. Bioassay of compounds 1-6 against Caenorhabditis elegans.Corrected mortality rate of compounds 1-6 (10 µg mL −1 , 50 µg mL −1 , and 100 µg mL −1 ) against C. elegans after 18 h of treatment.A solution of ivermectin (1 µg mL −1 ) was used as a positive control.The data were corrected using Schneider-Orelli's formula based on the negative control as methanol and are shown as the mean ± SD (n ≥ 3).The blue dash line characterizes the IC50.

Figure 4 .
Figure 4. Bioassay of compounds 1-6 against Caenorhabditis elegans.Corrected mortality rate of compounds 1-6 (10 µg mL −1 , 50 µg mL −1 , and 100 µg mL −1 ) against C. elegans after 18 h of treatment.A solution of ivermectin (1 µg mL −1 ) was used as a positive control.The data were corrected using Schneider-Orelli's formula based on the negative control as methanol and are shown as the mean ± SD (n ≥ 3).The blue dash line characterizes the IC 50 .
C , a,c Type δ H b Multi (J [Hz]) Pos.δ C , a,c Type δ H b Multi (J [Hz]) Measured in DMSO-d 6 a at 125 MHz/ b at 500 MHz.c Assignment confirmed by HMBC and HSQC spectra.

Table 2 .
Cont.Measured in DMSO-d 6 a at 125 MHz/ b at 500 MHz.c Assignment confirmed by HMBC and HSQC spectra.
Measured in DMSO-d6 a at 125 MHz/ b at 500 MHz.c Assignment confirmed by HMBC and HSQC spectra.

Table 3 .
Cont.MHz/ b at 500 MHz.c Assignment confirmed by HMBC and HSQC spectra.Compounds 5 and 6 were isolated similarly as yellowish-brown amorphous solids with their molecular formulas determined as C 37 H 50 N 4 O 8 and C 38 H 52 N 4 O 8 through their HR-ESI-MS spectra (Figures