Microbial Transformation of Licochalcones

Microbial transformation of licochalcones B (1), C (2), D (3), and H (4) using the filamentous fungi Aspergillus niger and Mucor hiemalis was investigated. Fungal transformation of the licochalcones followed by chromatographic separations led to the isolation of ten new compounds 5–14, including one hydrogenated, three dihydroxylated, three expoxidized, and three glucosylated metabolites. Their structures were elucidated by combined analyses of UV, IR, MS, NMR, and CD spectroscopic data. Absolute configurations of the 2″,3″-diols in the three dihydroxylated metabolites were determined by ECD experiments according to the Snatzke’s method. The trans-cis isomerization was observed for the metabolites 7, 11, 13, and 14 as evidenced by the analysis of their 1H-NMR spectra and HPLC chromatograms. This could be useful in better understanding of the trans-cis isomerization mechanism of retrochalcones. The fungal transformation described herein also provides an effective method to expand the structural diversity of retrochalcones for further biological studies.


Introduction
Licorice is one of the oldest and most commonly used herbal medicines in the world [1]. The name 'licorice' is derived from the dried roots of Glycyrrhiza species (Leguminosae family) and is the common name for these plants [2]. A variety of pharmacological activities have been described for licorice, and these bioactivities are attributed to the chemical constituents of licorice [3]. To date, more than 600 compounds have been isolated from licorice, and the major active constituents are saponins and flavonoids [4]. Among them, a series of retrochalcones, licochalcones A, B, C, D, E, G and the closely related compound echinatin, were identified from the roots of licorice [5]. Structurally, these retrochalcones belong to an unusual phenolic compound family and are distinguished from ordinary chalcones by the lack of a hydroxyl group at the C-2 and C-6 positions [6]. Several biological mechanism studies have proved that these compounds exhibited diverse biological effects such as anti-oxidant [7], antibacterial [8], anti-inflammatory [9,10], anticancer [11][12][13][14], osteogenesis [15], anti-hepatotoxic, antidiabetic and anti-allergic effects [16]. Moreover, the novel synthetic licochalcone F, a regioisomer of licochalcone E, showed in vivo anti-inflammatory and glucose intolerance effects without side effects [17]. Another synthetic licochalcone H, a regioisomer of licochalcone C, was found to exhibit anti-cancer activity by promoting apoptosis in human esophageal squamous cell carcinoma cells [18]. Even though promising biological studies of these small molecules were investigated, it is still essential to explore and generate more novel candidates for possible therapeutic uses [19].
Microbial transformation by filamentous fungi can be used as a feasible alternative to the conventional chemical methods when searching for new derivatives with potential biological properties [20]. These fungi could improve the regio-and stereo-selectivity of some chemical reactions [21]. The application of microbes for biotransformation of chalcones led to the formation of many novel derivatives by means of cyclization, hydroxylation, reduction, and dehydrogenation Metabolite 5 was obtained as a yellow amorphous powder. Its molecular formula was determined as C 16 H 16 O 5 by the positive [M + Na] + peak at m/z 311.0896 (calcd for C 16 O 16 O 5 Na, 311.0895) on the basis of its HRESIMS spectrum, which indicated that it was a dihydrogenerated derivative of 1. In the Molecules 2020, 25, 60 3 of 13 1 H-NMR spectrum of 5, the trans-olefinic proton signals of H-α and H-β were replaced by two new methylene proton signals at δ H 3.15 (2H, t, J = 7.4 Hz) and 2.87 (2H, t, J = 7.4 Hz). Consistently, 13 C-NMR spectrum of 5 showed the absence of low field olefinic carbon signals and the presence of two new high field carbon signals at δ C 39.2 and 25.1. Location of the hydrogenation was further determined to be at C-α and C-β based on the correlations of H-α (δ H 3. 15) and H-β (δ H 2.87) with C=O (δ C 200.0) in the HMBC spectrum of 5. Thus, coupound 5 was established to be α,β-dihydrolicochalcone B.
Microbial transformation of licochalcone C (2) by A. niger for three days produced metabolites 6 and 7. They were obtained as yellow amorphous powders. Metabolite 6 gave a molecular formula of C 21 H 24 O 6 from its HRESIMS, indicating that two oxygen atoms and two hydrogen atoms were inserted into the substrate 2. UV spectrum of 6 showed characteristic absorption maxima at 207 and 352 nm, indicating that the chalcone skeleton remained intact. The 1 H-NMR spectrum of 6 showed two doublet proton signals at δ H 8.00 and 7.63 (each d, J = 15.6 Hz), which are characteristic of the E-configuration of the α,β-double bond in the chalcone derivatives. In comparison with the 1 H-NMR spectrum of 2, two proton signals H-1" (δ H 3.45, 2H, d, J = 6.9 Hz) and H-2" (δ H 5.23, m, 1H) on the prenyl moiety disappeared, whereas three new proton signals δ H 2.69 (1H, dd, J = 14.2, 10.3 Hz), 3.12 (1H, dd, J = 14.2, 2.0 Hz) and 3.65 (1H, dd, J = 10.3, 2.0 Hz) were observed in 6. It was suggested that two hydroxyl groups were newly introduced at C-2" and C-3". Dihydroxylation was also supported by 13 C-NMR based on the presence of two new oxygen-bearing 13 C signals at δ C 72.6 (C-3") and 78.9 (C-2") instead of two olefinic carbon signals [24]. In HMBC spectrum, the proton signals at δ H 2.69 (H-1"b) and 3.12 (H-1"a) were correlated with the carbon signal at δ C 120.9 (C-3), and the proton signal at δ H 3.65 (H-2") was correlated with the carbon signal at δ C 72.6 (C-3"). Absolute configuration of the 2",3"-diols in 6 was determined using the ECD spectrum obtained by the Snatzke's method [25][26][27]. Based on the empirical rule, induced CD (ICD) curve of the Mo-complex of 6 showed the negative Cotton effect at around 310 nm, indicating that the O-C-C-O dihedral angle in the favored conformation was negative for 6 ( Figure 1). Therefore, the absolute configuration at C-2" of 6 was assigned R. Based on these data, the structure of 6 was elucidated as (2"R)-2",3"-dihydroxylicochalcone C. Metabolite 5 was obtained as a yellow amorphous powder. Its molecular formula was determined as C16H16O5 by the positive [M + Na] + peak at m/z 311.0896 (calcd for C16O16O5Na, 311.0895) on the basis of its HRESIMS spectrum, which indicated that it was a dihydrogenerated derivative of 1. In the 1 H-NMR spectrum of 5, the trans-olefinic proton signals of H-α and H-β were replaced by two new methylene proton signals at δH 3.15 (2H, t, J = 7.4 Hz) and 2.87 (2H, t, J = 7.4 Hz). Consistently, 13 C-NMR spectrum of 5 showed the absence of low field olefinic carbon signals and the presence of two new high field carbon signals at δC 39.2 and 25.1. Location of the hydrogenation was further determined to be at C-α and C-β based on the correlations of H-α (δH 3.15) and H-β (δH 2.87) with C=O (δC 200.0) in the HMBC spectrum of 5. Thus, coupound 5 was established to be α,βdihydrolicochalcone B.
Microbial transformation of licochalcone C (2) by A. niger for three days produced metabolites 6 and 7. They were obtained as yellow amorphous powders. Metabolite 6 gave a molecular formula of C21H24O6 from its HRESIMS, indicating that two oxygen atoms and two hydrogen atoms were inserted into the substrate 2. UV spectrum of 6 showed characteristic absorption maxima at 207 and 352 nm, indicating that the chalcone skeleton remained intact. The 1 H-NMR spectrum of 6 showed two doublet proton signals at δH 8.00 and 7.63 (each d, J = 15.6 Hz), which are characteristic of the Econfiguration of the α,β-double bond in the chalcone derivatives. In comparison with the 1 H-NMR spectrum of 2, two proton signals H-1'' (δH 3.45, 2H, d, J = 6.9 Hz) and H-2" (δH 5.23, m, 1H) on the prenyl moiety disappeared, whereas three new proton signals δH 2.69 (1H, dd, J = 14.2, 10.3 Hz), 3.12 (1H, dd, J = 14.2, 2.0 Hz) and 3.65 (1H, dd, J = 10.3, 2.0 Hz) were observed in 6. It was suggested that two hydroxyl groups were newly introduced at C-2'' and C-3''. Dihydroxylation was also supported by 13 C-NMR based on the presence of two new oxygen-bearing 13 C signals at δC 72.6 (C-3'') and 78.9 (C-2'') instead of two olefinic carbon signals [24]. In HMBC spectrum, the proton signals at δH 2.69 (H-1''b) and 3.12 (H-1''a) were correlated with the carbon signal at δC 120.9 (C-3), and the proton signal at δH 3.65 (H-2'') was correlated with the carbon signal at δC 72.6 (C-3''). Absolute configuration of the 2'',3''-diols in 6 was determined using the ECD spectrum obtained by the Snatzke's method [25][26][27]. Based on the empirical rule, induced CD (ICD) curve of the Mo-complex of 6 showed the negative Cotton effect at around 310 nm, indicating that the O-C-C-O dihedral angle in the favored conformation was negative for 6 ( Figure 1). Therefore, the absolute configuration at C-2'' of 6 was assigned R. Based on these data, the structure of 6 was elucidated as (2''R)-2'',3''dihydroxylicochalcone C. The molecular formula of the metabolite 7 was revealed as C21H22O5 based on the HRESIMS ion peak at 377.1376 (calcd for C21O22O5Na, 377.1365), indicating the addition of one oxygen atom compared to the substrate 2. Interestingly, the HPLC chromatogram of the purified 7 showed two peaks at tR of 22.66 and 26.06 min. The corresponding UV λmax of the two peaks were 240, 357 nm and 293 nm, respectively. Moreover, the 1 H-NMR peak pattern of 7 showed two sets of resonance signals in a ratio of approximately 1:0.3. They were clearly assignable to the suggested major (E)-and minor (Z)-isomers by observing the two sets of H-α and H-β proton signals including those at δH 7.64 (1H, d, J = 15.6 Hz) and 8.01 (1H, d, J = 15.6 Hz) in one set, and those at δH 6.55 (1H, d, J = 12.8 Hz) and 7.11 (1H, d, J = 12.8 Hz) in another. Assignment of the major trans isomer was confirmed by the presence Location of the epoxidation of 7 was confirmed to be at C-2" and C-3" according to the HMBC correlation from H-1"a and H-1"b to the additional quaternary carbon signal at δ c 77.3 (C-3"). Minor cis-form showed the same correlation patterns comparing with those of trans set signals in the 1D and 2D NMR spectra. Moreover, the assignments of the proton signals revealed general up-field shifts in the cis isomer as compared to those of the trans isomer in the 1 H-NMR spectrum. Therefore, the structure of 7 was confirmed as 2", 3"-expoxylicochalcone C.
Microbial transformation of licochalcone D (3) by A. niger for three days produced the metabolites 8 and 9. Compounds 8 and 9 were obtained as yellow amorphous powders. The molecular formula of 8 (C 21 H 24 O 7 ) was compatible with the insertion of two oxygen and two hydrogen atoms to the substrate 3, which was supported by the analysis of its HRESIMS data (m/z 389.1603 [M + H] + , calcd for C 21  The locations of dihydroxylation were confirmed to be at C-2" and C-3" by the HMBC correlations from H-4" and H-5" to C-2" and C-3", and from H-1" to C-2" and C-3". The absolute configuration at C-2" was determined by comparison of their ICD spectra using the Snatzke's method. The Mo-complex of compound 8 presented a negative Cotton effect at around 310 nm ( Figure S35), indicating the R configuration at C-2". Therefore, metabolite 8 was identified as (2"R)-2",3"-dihydroxylicochalcone D. , suggesting that the differences between 8 and 9 were at C-2" and C-3". In the HMBC spectrum, the H-1"a and H-1"b were coupled with the oxygen-bearing carbon signals at δ C 68.6 (C-2") indicating the epoxidation of the double bond in the prneyl group. Moreover, the proton signals H-4" (δ H 1.36) and H-5" (δ H 1.31) showed correlations with C-2" and C-3". Thus, metabolite 9 was identified as 2",3"-expoxylicochalcone D.
Microbial transformation of licochalcone H (4) by A. niger for three days produced metabolites 10 and 11. The molecular formula of metabolite 10, C 21  7 Hz) appeared in the 1 H NMR spectrum. Similar changes were also observed in the 13 C-NMR spectrum, including two oxygenated carbon signals at δ C 78.5 and δ C 72.5. This suggested that 10 was a dihydroxylated derivative of 4. Locations of the two hydroxyl groups were confirmed to be at C-2" and C-3" by observation of HMBC correlations from H-4"/5" (δ H 1.26) to C-2" (δ C 78.5) and C-3" (δ C 72.5). On the basis of the ICD spectroscopic analysis, the absolute configuration at C-2" was confirmed as R for the compound 10 ( Figure S36). Therefore, structure of the compound 10 was elucidated as (2"R)-2",3"-dihydroxylicochalcone H.
The molecular formula of the metabolite 11 was determined as C 21  , it was revealed that 11 exists as a mixture of (Z)-and (E)-isomers. The integrals of (Z)-form and (E)-form (approximate ratio 0.3:1) indicated that the (E)-form was the major one. The NMR spectral data of the major trans-form showed three oxygenated proton signals at δ H 3.67 (1H, dd, J = 7.5, 5.2 Hz, H-2"), 2.92 (1H, dd, J = 16.2, 5.2 Hz, H-1"a) and 2.60 (1H, dd, J = 16.2, 7.5 Hz, H-1"b) coupled with the signals at δ c 68.4 (C-2") and 30.6 (C-1") on the basis of the combined analyses of 1 H-NMR, 13 C-NMR and HSQC of 11, which indicated the epoxidation of the double bond between C-2" and C-3" of 4. Further, location of the epoxidation was deduced to be between C-2" and C-3" based on the HMBC correlations from H-1"a and H-1"b to the carbon signals at δ c 68.4 (C-2") and 113.3 (C-5). Assignment of the minor cis-form was made possible by observing the same correlation patterns as those of trans isomer signals in the 1D and 2D NMR spectra. Therefore, the structure of 11 was confirmed as 2",3"-expoxylicochalcone H (11). Further, location of the epoxidation was deduced to be between C-2'' and C-3'' based on the HMBC correlations from H-1''a and H-1''b to the carbon signals at δc 68.4 (C-2'') and 113.3 (C-5). Assignment of the minor cis-form was made possible by observing the same correlation patterns as those of trans isomer signals in the 1D and 2D NMR spectra. Therefore, the structure of 11 was confirmed as 2'',3''expoxylicochalcone H (11). The other corresponding proton signals were assigned by the correlations in the spectrum of HSQC. Anomeric proton at δH 4.74 revealed that the glycoside linkage was formed between the anomeric hydroxyl group of the sugar moiety and the hydroxyl group of the C-3 (δH 138.7) of the aglycone based on their HMBC correlation. Acid hydrolysis of 12 afforded a D-glucose and the aglycone 1 which was identified by TLC and HPLC. All these evidences indicated that this sugar moiety was a β-D-glucopyranose. Therefore, the structure of 12 was assigned as licochalcone B 3-O-β-D-glucopyranoside.

Microbial Transformation of
Microbial transformation of licochalcone C (2) by M. hiemalis for three days produced the metabolite 13. Compound 13 was isolated as a yellow amorphous powder, with a molecular formula The other corresponding proton signals were assigned by the correlations in the spectrum of HSQC. Anomeric proton at δ H 4.74 revealed that the glycoside linkage was formed between the anomeric hydroxyl group of the sugar moiety and the hydroxyl group of the C-3 (δ H 138.7) of the aglycone based on their HMBC correlation. Acid hydrolysis of 12 afforded a d-glucose and the aglycone 1 which was identified by TLC and HPLC. All these evidences indicated that this sugar moiety was a β-d-glucopyranose. Therefore, the structure of 12 was assigned as licochalcone B 3-O-β-d-glucopyranoside.
Microbial transformation of licochalcone C (2) by M. hiemalis for three days produced the metabolite 13. Compound 13 was isolated as a yellow amorphous powder, with a molecular formula of C 27 H 32 O 9 , which was determined by HRESIMS at m/z 501.2123 [M + H] + (calcd for C 27 H 33 O 9 , 501.2125). In the comparison of its 1 H-and 13 C-NMR spectra with those of the substrate 2, compound 13 displayed NMR resonance signals for the presence of a sugar moiety, showing an anomeric proton signal at δ H 5.02 (1H, d, J = 7.2 Hz). Complete assignments of the 1 H-and 13 C-NMR signals of the sugar moiety were accomplished by HMBC and HSQC experiments. HMBC correlation from the anomeric proton signal at δ H 5.02 (H-1"') to the carbon signal at δ C 158.6 (C-4) confirmed the assignment of glycosylation at C-4. In the sugar analysis, d-glucopyranose was confirmed using TLC after acid hydrolysis of 13. All these indicated the presence of a β-d-glucopyranose unit. The aglycone moiety of the 1 H-and 13  Generally, trans-chalcones are thermodynamically more stable than their corresponding cis isomers and most chalcones are thus isolated in trans form [28]. However, during the structure elucidation of the metabolites, the trans-cis isomerizations were observed by the analyses of the NMR spectra and HPLC chromatograms of metabolites 7, 11, 13, and 14 (Schemes 1 and 2). The (Z)-form observed in the structures of these metabolites might be considered as artefacts formed during incubation, sample processing, or isolation procedure. There might be interconversion between the (E)-form and its corresponding (Z)-form because it was detected again after separation of the peaks of corresponding (E)-and (Z)-forms. Recently, two dihydrobenzofuran congeners of licochalcone A isoated from the roots of Glycyrrhiza inflata were found to rapidly isomerize and yield trans and cis isomers when solutions were exposed to sunlight [29]. Additionally, one metabolite of licochalcone A produced by human liver microsomes was observed as a mixture of transand cis-forms [30]. Based on these evidences, it could be deduced that some substituents introduced in the B ring of the retrochalcones may affect the stability of trans-form. Therefore, further studies will be necessary to evaluate the mechanism of trans-cis isomerization. Also the thermodynamics of retrochalcone isomerization should be considered in biological evaluation

Conclusions
Fungal transformation of the four licochalcones 1-4 by A. niger and M. hiemalis resulted in the formation of ten different metabolites 5-14, including hydrogenated, dihydroxylated, expoxidized and glucosylated metabolites. Of them, the metabolites 5, 6, 8, 9, 10, and 12 were confirmed to be in (E)-form; whereas the metabolites 7, 11, 13, and 14 were confirmed as a mixture of (E)-and (Z)-isomers which were present in the integral ratios ranging from 1:0.3 to 1: 0.4 as evidenced by their 1 H-NMR spectra. Although further investigations such as thermodynamic stability sutdies on these isomers may be needed, it was concluded that 7, 11, 13, and 14 are present as interconverting mixtures of the major (E)-and minor (Z)-isomers.
Prenyl groups could affect the generation of the metabolites produced by A. niger and M. hiemalis (Figure 2). When prenyl groups were introduced in different positions of the retrochalcone backbone, it was observed that dihydroxylation or expoxidation of the corresponding substrates was preferentially performed on the prenyl side chains by A. niger. The position of glucosylation by M. hiemalis was preferred to be at the hydroxyl groups nearest to the prenyl groups. It was deduced that the flexibility of the prenyl substituents might have an effect on the regio-selectivity during the microbial transformation.
isomers which were present in the integral ratios ranging from 1:0.3 to 1: 0.4 as evidenced by their 1 H-NMR spectra. Although further investigations such as thermodynamic stability sutdies on these isomers may be needed, it was concluded that 7, 11, 13, and 14 are present as interconverting mixtures of the major (E)-and minor (Z)-isomers.
Prenyl groups could affect the generation of the metabolites produced by A. niger and M. hiemalis (Figure 2). When prenyl groups were introduced in different positions of the retrochalcone backbone, it was observed that dihydroxylation or expoxidation of the corresponding substrates was preferentially performed on the prenyl side chains by A. niger. The position of glucosylation by M. hiemalis was preferred to be at the hydroxyl groups nearest to the prenyl groups. It was deduced that the flexibility of the prenyl substituents might have an effect on the regio-selectivity during the microbial transformation. Microbial transformation is regarded as an efficient tool for the structural modification of bioactive natural and synthetic compounds [31]. Microbial transforamtion of the licochalcones can provide alternative approach to prepare the targeted licochaclcone derivatives under mild conditions by utilizing microbial system as biocatalysts.

General Experimental Procedures
Optical rotations were recorded with a 343 Plus polarimeter (Perkin Elmer, Waltham, MA, USA). UV spectra were recorded on a V-530 spectrophotometer (Jasco, Tokyo, Japan). IR spectra were obtained on a Jasco FT/IR 300-E spectrometer, and CD spectra were recorded on a Jasco J-815 CD spectrometer. NMR experiments were recorded using an Avance III 400 spectrometer (Bruker, Billerica, MA, USA) with TMS as the internal standard. HRESIMS were determined on Waters Synapt G2 QTOF (Waters, Milford, MA, USA). TLC was carried out on Merck silica gel F254-precoated glass plates and RP-18 F254s plates. Chromatography was performed on a Waters 1525 Binary HPLC pump connected to a 996 Photodiode Array (PDA) detector using Isco Allsphere ODS-2 (10 μm, 10 × 250 mm) and Nova-Pak C18 (4 μm, 3.9 × 150 mm) columns.

Preparation of Substrates
Licochalcones B (1) and D (3) were synthesized through acid-mediated Claisen-Schmidt condensation using 4-hydroxyacetophenone as a starting material [32]. And starting from 2,4dihydroxybenzaldehyde, licochalcone C (2) and its regio-isomer licochalcone H (4) were synthesized by acid-mediated Claisen-Schmidt condensation [24]. The 1 H-NMR data of licochlacones B (1), C (2), D (3), and H (4) agreed with data in the literature data [24,32]. The purity of each substrate was determined to be above 95% by HPLC analysis.  Microbial transformation is regarded as an efficient tool for the structural modification of bioactive natural and synthetic compounds [31]. Microbial transforamtion of the licochalcones can provide alternative approach to prepare the targeted licochaclcone derivatives under mild conditions by utilizing microbial system as biocatalysts.

General Experimental Procedures
Optical rotations were recorded with a 343 Plus polarimeter (Perkin Elmer, Waltham, MA, USA). UV spectra were recorded on a V-530 spectrophotometer (Jasco, Tokyo, Japan). IR spectra were obtained on a Jasco FT/IR 300-E spectrometer, and CD spectra were recorded on a Jasco J-815 CD spectrometer. NMR experiments were recorded using an Avance III 400 spectrometer (Bruker, Billerica, MA, USA) with TMS as the internal standard. HRESIMS were determined on Waters Synapt G2 QTOF (Waters, Milford, MA, USA). TLC was carried out on Merck silica gel F 254 -precoated glass plates and RP-18 F 254 s plates. Chromatography was performed on a Waters 1525 Binary HPLC pump connected to a 996 Photodiode Array (PDA) detector using Isco Allsphere ODS-2 (10 µm, 10 × 250 mm) and Nova-Pak C18 (4 µm, 3.9 × 150 mm) columns.

Procedure for Microbial Transformation
Microbial metabolism studies were carried out according to the standard two-stage procedure [21]. Briefly, the actively growing microbial cultures were inoculated in 250 mL Erlenmeyer flasks containing 50 mL of a suitable medium, and incubated with gentle agitation (200 rpm) at 25 • C in a temperature-controlled shaking incubator. The DMSO solutions (20 mg/mL, 100 µL) of the substrates (1, 2, 3 or 4) were added to each flask 24 h after inoculation, and further incubated at the same conditions for another 5 days. Sampling and TLC monitoring were performed at an interval of 24 h. UV (254 and 365 nm) and anisaldehyde-sulfuric acid reagent were used for identification of metabolites. Culture controls were carried out as a result of enzymatic activity, but not a consequence of degradation or non-metabolic changes.
Similarly, the preparative-scale fermentations were carried out in 500 mL flasks containing 150 mL of culture medium, and the selected microorganisms were pre-cultured under the culture conditions mentioned above for 24 h to obtain sufficient amounts for biotransformation. After that, 50 mg of 1, 80 mg of 2, 35 mg of 3 or 80 mg of 4 dissolved in DMSO (20 mg/mL) were used for preparative-scale fermentations. The other procedures and culture conditions were same as those of the screening experiments.

Extraction and Isolation of Metabolites
After incubation, the cultures of 1, 2, 3, or 4 were extracted with equal volume of EtOAc two times, and the organic layer was collected and concentrated. The EtOAc extract of 1 incubated with A. niger was subjected to reversed-phased HPLC with a gradient solvent system of 35% MeOH to 45% MeOH to afford 5 (5.8 mg, t R = 10.92 min) at a flow rate of 1.0 mL/min. The EtOAc extract of 1 incubated with M. hiemalis was subjected to reversed-phased HPLC with an isocratic solvent system of 34% MeOH to afford 12 (4.0 mg, t R = 8.79 min) at a flow rate of 1.0 mL/min. The EtOAc extract of 2 incubated with A. niger was subjected to reversed-phased HPLC with a gradient solvent system of 53% MeOH to 69% MeOH to afford 6 (4.6 mg, t R = 22.17 min) and 7 (5.2 mg, t R = 30.46 min) at a flow rate of 2.0 mL/min. The EtOAc extract of 2 incubated with M. hiemalis was subjected to reversed-phased HPLC with a gradient solvent system of 65% MeOH to 80% MeOH to afford 13 (5.0 mg, t R = 12.94 min) at a flow rate of 2.0 mL/min. The EtOAc extract of 3 with incubated with A. niger was subjected to reversed-phased HPLC with a gradient solvent system of 58% MeOH to 65% MeOH to afford 8 (2.5 mg, t R = 12.62 min) and 9 (3.2 mg, t R = 20.08 min) at a flow rate of 2.0 mL/min. The EtOAc extract of 4 incubated with A. niger was subjected to reversed-phased HPLC with a gradient solvent system of 60% MeOH to 70% MeOH to afford 10 (4.3 mg, t R = 14.40 min) and 11 (5.5 mg, t R = 20.48 min) at a flow rate of 2.0 mL/min. The EtOAc extract of 4 incubated with M. hiemalis was subjected to reversed-phased HPLC with a gradient solvent system of 60% MeOH to 75% MeOH to afford 14 (5.0 mg, t R = 16.51 min) at a flow rate of 2.0 mL/min.