New Enantiomers of a Nor-Bisabolane Derivative and Two New Phthalides Produced by the Marine-Derived Fungus Penicillium chrysogenum LD-201810

Marine-derived fungi are a treasure house for the discovery of structurally novel secondary metabolites with potential pharmaceutical value. In this study, a pair of new nor-bisabolane derivative enantiomers (±)−1 and two new phthalides (4 and 5), as well as four known metabolites, were isolated from the culture filtrate of the marine algal-derived endophytic fungus Penicillium chrysogenum LD-201810. Their structures were established by detailed interpretation of spectroscopic data (1D/2D NMR and ESI-MS). The optical resolution of compound (±)−1 by chiral HPLC successfully afforded individual enantiomers (+)−1 and (−)−1, and their absolute configurations were determined by TDDFT-ECD calculations. Compound (±)−1 represents the first example of bisabolane analogs with a methylsulfinyl substituent group, which is rare in natural products. All of the isolated compounds 1–7 were evaluated for their cytotoxic activity against A549, BT-549, HeLa, HepG2, MCF-7, and THP-1 cell lines, as well as for antifungal activity against four plant pathogenetic fungi (Alternaria solani, Botrytis cinerea, Fusarium oxysporum, and Valsa mali). Compound 2, a bisabolane-type sesquiterpenoid, was shown to possess excellent activity for control of B. cinerea with half-maximal inhibitory concentration (IC50) of 13.6 μg/mL, whereas the remaining investigated compounds showed either weak or no cytotoxic/antifungal activity in this study.


INTRODUCTION
Marine-derived fungi that inhabit the marine environment possess the unique metabolic pathways to produce a great diversity of bioactive secondary metabolites, which play an important role in agrochemical and pharmaceutical industries Zhang et al., 2020). It is well-known that a large number of new marine natural products have been discovered and reported every year (Carroll et al., 2021). Mining natural products with novel structures and remarkable bioactivities from marine-derived fungi is still a research hotspot.
Filamentous fungi belonging to the genus Penicillum are important and untapped producers of structurally diverse metabolites (Bai et al., 2019). Penicillum from marine environment have gained particular attention, not only due to their unusual chemical skeletons but also their significant bioactivities with pharmaceutical potential . In our continuing study on bioactive metabolites of marine-derived fungi, we investigated Penicillium chrysogenum LD-201810, a marine alga-associated fungus isolated from the marine red alga Grateloupia turuturu (Jiang et al., 2020). Previous solid cultivation of this fungus on rice medium led to the isolation of a new pentaketide derivative, two new hydroxyphenylacetic acid derivatives, as well as the known bisabolane-type sesquiterpenoids and meroterpenoids (Jiang et al., 2020). Motivated by OSMAC (one strain-many compounds) strategy (Zhao et al., 2020), the fungal strain was cultivated on liquid PDB medium. A follow-up examination of this cultivation yielded a pair of new nor-bisabolane derivative enantiomers (±)−1, two previously reported bisabolenes (2 and 3), and two new phthalides (4 and 5) (Figure 1). The aromatic bisabolenes are a rarely found family of sesquiterpenes. Mulholland et al. firstly reported a new trisnor-bisabolane sesquiterpene boivinianin A (Mulholland et al., 2006). Then Li et al. (2015) reported the second occurrence of a new nor-bisabolane derivative, 1-hydroxyboivinianin A. Herein we reported the first example of nor-bisabolane analogs with a methylsulfinyl substituent group, which is rare in natural products. Moreover, the structure elucidation of the new phthalides (4 and 5), as well as the cytotoxicity and antifungal activity of the isolated compounds, are also described.

General Experimental Procedures
The UV and optical rotations data were obtained on a Shimadzu UV-2700 spectrometer (Shimadzu Co., Ltd., Kyoto, Japan) and Jasco P-1020 automatic polarimeter (JASCO, Tokyo, Japan), respectively. 1 H (500 MHz), 13 C (125 MHz), and 2D NMR spectra were measured on an Agilent DD2 spectrometer (Agilent Technologies, Waldbronn, Germany). The mass spectra (ESI-MS) were measured under the positive and negative ion modes by a Waters Xevo G2-XS QTof mass spectrometer (Waters, Milford, MA, United States). Column chromatography was performed on silica gel Qingdao Marine Chemical Inc.,Qingdao,China),Merck,Darmstadt,Germany), and on Sephadex LH-20 (Merck). Preparative TLC plates precoated with silica gel GF 254 were purchased from Qingdao Marine Chemical Industry Company.

Fungal Material
The producing fungal strain P. chrysogenum LD-201810 was isolated from Grateloupia turuturu, a marine red alga which collected in Qingdao coastal zone. The gene sequencing in the ITS region of the rDNA (GenBank no. MT075873) was applied to identify the fungus (Jiang et al., 2020). To identify the phylogenetic location of this fungus, phylogenetic trees were constructed based on the ITS region sequences using maximum likelihood (ML) method, and the bootstrap support was calculated using 1,000 replicates. The fungus has been deposited in School of Life Sciences, Ludong University, Yantai, China.

No.
Compound

Computational Section
The conformational search was performed by the molecular mechanics with MM + method in HyperChem 8.0 software.
Next, the geometries were optimized at B3LYP/6-31G(d) level with Gaussian 09 software to afford the energy-minimized conformers (Frisch et al., 2013). The optimized conformers were subjected to TD-DFT ECD calculations at PBE0/TZVP, CAM-B3LYP/TZVP, and BH&HLYP/TZVP level. The solvent effects (MeCN) were evaluated at the same DFT level with the SCRF/PCM method.

Cytotoxic Assay
Cytotoxicity of compounds 1−7 toward A549, BT-549, HeLa, HepG2, MCF-7, and THP-1 cell lines was tested by the Cell Counting Kit-8 (CCK-8) method . All of the cell lines were purchased from the Chinese Academy of Sciences Committee on Type Culture Collection Cell Bank (Shanghai, China). The six cell lines (3.0 × 10 4 cells per well) were initially inoculated into 96-well plates for 24 h. Subsequently, the cells were exposed to various concentrations of tested compounds (0,5,10,20,40,80, and 100 µg/mL). With the treatment of 24, 48, and 72 h, 10 µL of 5 g/L CCK-8 solution (CCK-8 Cell Proliferation and Cytotoxicity Assay Kit, #CA1210, Solarbio, Beijing, China) was applied to each well and the cells were cultured for 1.5 h at 37 • C. Absorbance data were obtained with a microplate spectrophotometer reader (Multiskan GO, Thermo Fisher Scientific, Waltham, MA, United States) at 490 nm.

Antifungal Assay
The antifungal activities against four phytopathogenic fungi (Alternaria solani, Botrytis cinerea, Fusarium oxysporum, and Valsa mali) were evaluated in 96-well microtiter plates using a modified broth microdilution method  (Shi et al., 2017). Carbendazim was used as a positive control. The tested compounds were added to autoclaved PDA medium to a final concentration of 3.12, 6.25, 12.5, 25, and 50 µg/mL, while 95% ethanol was treated as black control. The blocks (about 5 mm diameter) from four phytopathogenic fungi were cultured in the center of plates at 20 • C. Colony diameters were measured with the cross method. The mycelial growth inhibition rate was calculated as follows, while IC 50 values were obtained by the logarithm method.

Identification of the Producing Strain
To clarify the evolutionary position of the producing strain LD-201810, we performed phylogenetic analysis based on its ITS  Frontiers in Microbiology | www.frontiersin.org sequence, together with those from other Penicillium species. Results indicated that the strain LD-201810 located at the basal position of the whole tree with high confidence (100%, Figure 2). The result demonstrated that Penicillium chrysogenum LD-201810 belongs to the Penicillium genus.

Structural Elucidation
Compound (±)−1 was a white amorphous powder (MeOH), and its molecular formula was determined to be C 13  (d, J = 13.0 Hz, H-12β), and three aromatic protons at δ H 7.33 (d, J = 8.4 Hz, H-3), 6.83 (d, J = 8.4 Hz, H-4), and 6.82 (s, H-6). The 13 C NMR data identified 13 carbon signals that were highly resolved, categorized as two methyls, three methylenes, three sp 2 methines, and five quaternary carbons including three sp 2 , one oxygenated sp 3 , and one carbonyl carbon at δ C 179.6 (C-10). Detailed analysis of the 1D and 2D NMR (Figure 3) spectra of 1 indicated that they were similar to those of 1-hydroxyboivinianin A, a trisnor-bisabolane derivative identified from the culture of a deep-sea sediment-derived fungus Penicillium aculeatum SD-321 (Li et al., 2015). By comparison of the NMR data of 1hydroxyboivinianin A with those of 1, the main differences in 1 were the presence of an additional methylene group at δ C 59.7 (C-12) and a distinctive methyl at δ C 37.4 (C-13). The downfield chemical shifts of 13-CH 3 (δ H/C 2.58/37.4) and 12-CH 2 (δ C  Frontiers in Microbiology | www.frontiersin.org 59.7) were ascribed to that bearing a heteroatom between them. Initially, the common-observed heteroatoms, such as oxygen (1a), nitrogen (1b), and sulfur (1c) atoms, were assumed between C-13 and C-12. However, the predicted 13 C NMR shifts in ChemBioDraw didn't match well with that for measured data (Figure 3) In view of its molecular weight, a remaining S and O atom could be accounted for by inserting the S = O group between C-13 and C-12 to form a methylsulfinyl substituent. The predicted data for 1d were in good agreement with the authentic data. Moreover, Fu et al. reported a series of synthetic compounds with a methylsulfinyl group . The chemical shifts of C-12 and C-13 in 1 were accordant with those of known compounds, which further confirmed the presence of such a rare substituent in natural products. On the basis of the above discussion, the structure of compound 1 was determined as methylsulfinyl-1-hydroxyboivinianin A.
Compound 1 had only one chiral center at C-7. The zero specific rotation value and baseline ECD curve indicated its racemic nature (Meng et al., 2016). Subsequent chiral HPLC analysis of (±)−1 successfully led to the separation of the two individual enantiomers (+)−1 and (−)−1 with a ratio of approximately 1:1, which exhibited opposite optical rotations ( Figure 4A). To determine the absolute configurations of (+)−1 and (−)−1, their ECD spectra were measured in MeOH and simulated by the time-dependent density function theory (TD-DFT) method. The experimental ECD spectrum of (+)−1 showed a positive (+220 nm) Cotton effect, whereas the experimental (−)−1 showed an almost mirror image ECD curve ( Figure 4B). The calculated ECD curves of 7R and 7S matched the experimental ECD curves of (+)−1 and (−)−1, thus the absolute configurations of (+)−1 and (−)−1 were proposed as 7R and 7S, respectively.
Chrysoalide A (4), a white amorphous powder, was shown to possess a molecular formula of C 10 H 10 O 5 by its HR-ESI-MS (m/z 209.0431 [M − H] − , calcd 209.0450). Its UV spectrum showed absorption peaks at 216, 239, and 330 nm, indicating the presence of a conjugated carbonyl chromophore (Phainuphong et al., 2018;Saetang et al., 2021). The 1 H NMR data for 4 ( Table 1) showed signals of one methyl singlet at δ H 1.73 (s, H 3 -8), one methoxy singlet at δ H 2.93 (s, H 3 -9), and a pair of intercoupling aromatic protons at δ H 7.01 (d, J = 8.7 Hz, H-5) and 6.84 (d, J = 8.7 Hz, H-6), which can be easily deduced the presence of a 1,2,3,4-tetrasubstituted benzene group. The 13 C NMR spectrum (Table 1) displayed signals for two methyls at δ C 24.2 (C-8) and 51.0 (C-9), two sp 2 methines at δ C 124.3 (C-5) and 119.7 (C-6), five quaternary carbons, and one ester carbonyl at δ C 166.2 (C-1). Detailed analysis of 2D NMR data established the planar structure of 4 ( Figure 5A). Moreover, the experimental ECD spectrum of 4 displayed a similar shape of curves and Cotton effects to those of the calculated ECD spectrum of the R-configuration (Figure 5B), which established the absolute configuration of C-3 to be R.
Chrysoalide B (5) was also obtained as a white amorphous powder with a molecular formula of C 11 H 12 O 5 as determined by HR-ESI-MS. The molecular weight of 5 was more than that of 4 by 14 units (CH 2 ). The 1D and 2D NMR spectra of 5 (Table 1 and Figure 6A) suggested that it resembled 4 structurally, but possessed an additional methoxy group at δ H/C 3.80/56.3. The extra methoxy group was shown to be linked to C-7, as evidenced from the HMBC correlation from 10-CH 3 to C-7. Compound 5 was elucidated as a methoxy derivative of 4. The absolute configuration of 5 was considered to be identical with that of 4 by its similar ECD curve, which gave a positive Cotton effect at 220 nm. Comparison of the experimental ECD data with those of calculated spectra further proved the above assignment ( Figure 6B).

Bioactivity of the Isolated Compounds
All of the isolated compounds were evaluated for their cytotoxicity against six different types of cancer cell lines, A549 (a human lung adenocarcinoma epithelial cell line), BT-549 (a human breast cancer cell line), HeLa (a human cervix carcinoma cell line), HepG2 (a human liver carcinoma cell line), MCF-7 (a human breast adenocarcinoma cell line), and THP-1 (a human monocytic cell line). However, none of them exhibited obvious inhibitory activity at 20 µg/mL (the highest concentration tested, data were shown in Supplementary Table 1).
Previous studies indicated that bisabolane sesquiterpenoids and phthalides possessed promising antimicrobial activity (Li et al., 2015;Saetang et al., 2021). Marine natural products considered to be new sources of lead molecules with agrochemical significance (Oppong-Danquah et al., 2020). To discover new marine fungal agrochemicals, the isolated compounds were evaluated for antifungal activity against several plant pathogenetic fungi (A. solani, B. cinerea, F. oxysporum, and V. mali). The bisabolane-type sesquiterpenoid 2 was shown to possess excellent activity for control of B. cinerea with an IC 50 value of 13.6 µg/mL (Figure 7) (compared with the positive control carbendazim, with an IC 50 value of 19.2 µg/mL), whereas other compounds showed either weak or no activity (Data were shown in Supplementary Table 2). It should be pointed out that limited amounts of these metabolites were obtained, which prevented us to perform more biological experiments. Further study should be particularly focused on more agricultural activities, such as antifeedant and phytotoxic activities, to fully evaluate their agricultural potentials.

CONCLUSION
Marine-derived fungi have been proven to be prolific producers of secondary metabolites with potent bioactivities. In this study, chemical investigation of a marine algal-derived endophytic fungus P. chrysogenum LD-201810 led to the isolation and identification of a pair of new nor-bisabolane derivative enantiomers (±)−1 and two new phthalides (4 and 5), as well as four known metabolites (2, 3, 6, and 7). Compound (±)−1 represents the first example of bisabolane analogs with a methylsulfinyl substituent group, which is rare in natural products. The aromatic bisabolanes are a rarely found family of sesquiterpenes, and the discovery of (±)−1 added greatly to the diversity of this kind of molecules. The cytotoxic and antifungal activities were evaluated. Hydroxysydonic acid (2), a bisabolanetype sesquiterpenoid, showed strong inhibition against B. cinerea, compared with that of the positive control carbendazim. The results indicated that some marine natural products may be regarded as candidate agents of antifungal agrochemicals.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding authors.