Optical rotations were measured with a JASCO P-1020 digital polarimeter (Tokyo, Japan). UV spectra were obtained on a Lambda 35 UV/Vis spectrophotometer (Perkin Elmer, Waltham, United States). HRESIMS data were acquired with a scientific LTQ Orbitrap XL spectrometer (Thermo Scientific, Waltham, United States). 1D (500 and 125 MHz for ¹H and ¹³C, respectively) and 2D (HSQC, COSY, and HMBC) NMR spectra were performed by an Agilent DD2 500 MHz spectrometer (Agilent Technologies, Santa Clara, United States). X-ray diffraction data were collected on an Agilent Xcalibur Gemini E diffractometer equipped with Eos charge-coupled device (CCD) detector with graphite monochromated Cu Kα radiation (λ = 1.54178 Å). Column chromatography was undertaken by using various packing materials including silica gel (100-200/200-300 mesh, Qingdao Marine Chemical Factory, Qingdao, China), octadecylsilyl (ODS) reversed-phase gel (30-50 μm, YMC CO., Ltd., Japan), and Sephadex LH-20 (GE Healthcare, United States).

The fungal strain Penicillium sp. YT2019-3321 was previously isolated from the traditional Chinese medicine Lonicera Japonica. The taxonomic identification of this fungus was performed based on a molecular protocol by DNA amplification and sequencing of the internal transcribed spacer (ITS) of the rRNA locus. The ITS sequence showed 99% identical to that of P. oxalicum (GenBank accession no. KY400080.1). A voucher specimen of this fungal strain was stored at –80°C at the Third Affiliated Hospital of Wenzhou Medical University. This fungus was cultured on potato dextrose agar medium (PDA, Solarbio Life Sciences CO., Ltd., Beijing, China) at 28°C for 5 days. Then all of agar plugs were cut into small pieces (0.5 × 0.5 cm²). Each piece was inoculated in a 1 L Erlenmeyer flask containing 250 mL of potato dextrose broth (PDB) medium (Solarbio). A total of 100 flasks were statically fermented at room temperature for 30 days.

The fermentation materials were adequately extracted with EtOAc (3 × 25 L), and the organic solvent was evaporated in vacuum to yield ca. 20 g of crude extracts. The crude extracts were subjected to a silica gel vacuum liquid chromatography column, which was eluted with an increasing gradient of EtOAc/petroleum ether (from 30:1 to 1:1) to afford six fractions (Fr. 1-Fr. 6). Fr. 4 (3.2 g), eluting with EtOAc/petroleum ether 5:1, was further fractionated over an ODS reversed-phase silica gel with a mixed solvent system of MeOH/H₂O (from 10 to 100%, v/v). This afforded a total of eight subfractions (Fr. 4.1-Fr. 4.8). Fr. 4.6 was further purified over an open silica gel column chromatography by using the solvent system CH₂Cl₂ and MeOH with the ratio 20:1 to afford 16 mg of compound 2. Fr. 5 (2.5 g), eluting with EtOAc/petroleum ether 2:1, was applied to ODS silica gel with gradient elution of MeOH/H₂O (from 10 to 100%, v/v) to yield eight subfractions (Fr. 5.1-Fr. 5.8). Compound 1 (10.2 mg) was isolated from a two-step purification process, first from Fr. 5.3 over an open silica gel column chromatography using the solvent system CH₂Cl₂ and MeOH with the ratio 20:1, followed by preparative TLC (CH₂Cl₂/MeOH, 15:1, v/v). Compound 1 was further resolved into the pure enantiomers (+)-1 (4.9 mg, t_R = 9.6 min) and (–)-1 (4.7 mg, t_R = 10.9 min) by chiral HPLC using a (R,R). Whelk-O1 chiral column (10 mm; 4.6 × 250 mm; n-hexane-ethanol eluent 6:4, v/v; 1.0 mL/min). Compound 3 (10.2 mg, t_R 7.7 min) was isolated from Fr. 5.4 by semipreparative HPLC (YMC-pack ODS-A, 5 μm; 10 × 250 mm; 55% MeOH/H₂O; flow rate 2 mL/min). Compound 7 (5.6 mg) was isolated from Fr. 5.5 by preparative TLC (CH₂Cl₂/MeOH/acetic acid, 15:1:0.4, v/v). Compound 6 (11.3 mg) was isolated from Fr. 5.6 by preparative TLC (CH₂Cl₂/MeOH/acetic acid, 20:1:0.4, v/v). Fr. 6 (4.0 g), eluting with EtOAc/petroleum ether 1:1, was fractionated by Sephadex LH-20 column chromatography in MeOH to give subfractions Fr. 6.1-Fr. 6.3. Fr. 6.1 was subjected to semipreparative HPLC (65% MeOH/H₂O) to give compounds 4 (20.2 mg, t_R 6.9 min) and 5 (6.2 mg, t_R 8.8 min), respectively. Finally, compound 8 (4.9 mg) was obtained by preparative TLC (CH₂Cl₂/MeOH, 20:1, v/v) from Fr. 6.3.

(±)-Chrysoalide B (1): white amorphous powder; [α]²⁰_D + 9.6 (c 0.10, MeOH) for (+)-1 and [α]²⁰_D –10.2 (c 0.10, MeOH) for (–)-1; UV (MeOH) λ_max (log ε) 213 (2.16), 239 (1.60), 331 (1.49) nm; ¹H and ¹³C NMR data (measured in DMSO-d₆) (see Table 1); HRESIMS m/z 223.0644 [M - H]^– (calcd for C₁₁H₁₁O₅, 223.0606).

Penicidone E (2): colorless oil; [α]²⁰_D + 13.5 (c 0.10, MeOH); UV (MeOH) λ_max (log ε) 220 (3.88), 254 (3.26), 309 (2.98); ¹H and ¹³C NMR data (measured in DMSO-d₆) (see Table 1); HRESIMS m/z 390.1547 [M + H]⁺ (C₂₀H₂₄NO₇) and 412.1369 [M + Na]⁺ (C₂₀H₂₃NO₇Na).

Suitable crystals of (+)-1 and (–)-1 were obtained by slowly evaporating the solvent mixture of MeOH and H₂O. Single-crystal X-ray diffraction data were obtained on an Agilent Xcalibur Gemini E diffractometer equipped with Eos CCD detector with graphite monochromated Cu Kα radiation (λ = 1.54178 Å). Structures were solved by direct methods using the SHELXTL software package (Sheldrick, 1997a). All non-hydrogen atoms were refined anisotropically. H atoms were located by geometrical calculations, and their positions and thermal parameters were fixed during structure refinement. Structure was refined by full-matrix least-squares techniques (Sheldrick, 1997b).

Crystal data for (+)-1: C₂₂H₂₆O₁₁ (2 C₁₁H₁₂O₅ + H₂O), F.W. = 466.43, monoclinic space group P2₁, unit cell dimensions a = 7.3590 (9) Å, b = 14.4044 (16) Å, c = 10.4189 (12) Å, α = β = γ = 90°, V = 1103.9 (2) ų, Z = 2, d_calcd = 1.403 mg/m³. Crystal size: 0.08 × 0.05 × 0.04 mm³, μ = 0.967 mm^–1, F (000) = 492.0. Reflections collected/unique: 19,174/4,377 [R (int) = 0.0438]. Final indices resulted in R₁ = 0.0424 and wR₂ = 0.1067 [I > 2σ(I)] Flack parameter = 0.13 (6).

Crystal data for (–)-1: C₂₂H₂₆O₁₁ (2 C₁₁H₁₂O₅ + H₂O), F.W. = 466.43, monoclinic space group P2₁, unit cell dimensions a = 7.3638 (2) Å, b = 14.3632 (4) Å, c = 10.4176 (3) Å, α = β = γ = 90°, V = 1101.11 (5) ų, Z = 2, d_calcd = 1.407 mg/m³. Crystal size: 0.12 × 0.07 × 0.04 mm³, μ = 0.970 mm^–1, F (000) = 492.0. Reflections collected/unique: 25,073/4,450 [R (int) = 0.0590]. Final indices resulted in R₁ = 0.0358 and wR₂ = 0.0827 [I > 2σ(I)] Flack parameter = 0.05 (10).

The conformer rotamer ensemble sampling tool (crest) (Pracht et al., 2020) was used to afford candidate conformers for S-2 and DFT calculations were performed with the Gaussian 16 program (Frisch et al., 2016). The conformers within an energy window of 10 kcal/mol were optimized at B3LYP/6-31G (d) level of theory with Grimme’s D3 dispersion correction (“EmpiricalDispersion = GD3” key words in input files). Frequency analysis of all optimized conformations was undertaken at the same level of theory to ensure they were true local minima on the potential energy surface. Then, energies of all optimized conformations were evaluated by M062X/6-311 + G (2d,p) with D3 dispersion correction. Gibbs free energies of each conformers were calculated by adding “Thermal correction to Gibbs Free Energy” obtained by frequency analysis to electronic energies obtained at M062X/6-311 + G (2d,p). Room-temperature (298.15 K) equilibrium populations were calculated according to Boltzmann distribution law. Those conformers accounting for over 2% population were subjected to subsequent calculations. Calculation of optical rotations of different conformers were carried out using the TDDFT method at CAM-B3LYP/6-311 + g (2d,p) level in methanol (λ = 589 nm). Detailed computational data have shown in Supplementary material.

(±)-Chrysoalide B (1) was isolated as white amorphous powder. Its molecular formula, C₁₁H₁₂O₅, was established by HRESIMS at m/z 223.0644 [M-H]^– (calcd for C₁₁H₁₁O₅, 223.0606). Observation of the ¹H NMR data of 1 (Table 1) revealed the presence of three methyl groups including two methoxy groups at δ_H 2.94 (s, H₃-9) and 3.80 (s, H₃-10) as well as two coupled aromatic methines at δ_H 7.13 (d, J = 8.8 Hz, H-5) and 7.05 (d, J = 8.8 Hz, H-6). The ¹³C spectroscopic data of 1 (Table 1) displayed 11 carbon resonances, including an ester carbonyl at δ_C 165.1 (C-1), five quaternary carbons including one oxygenated sp³ at δ_C 106.8 (C-3), two aromatic methines at δ_C 123.5 (C-5) and 115.2 (C-6), and three methyl groups including two methoxy groups at δ_C 50.7 (C-9) and 55.9 (C-10). Considering the functional groups observed for compound 1 as well as the characteristic UV absorption peaks at 213, 239, and 331 nm (Saetang et al., 2021), the presence of an isobenzofuran core framework was deduced. HMBC correlations (Figure 2) from H-5 to C-4a and C-7 as well as from H-6 to C-4 and C-7a deduced the presence of a 1,2,3,4-tetrasubstituted benzene group. Finally, HMBC correlations from H₃-9 to C-3 and from H₃-10 to C-7 led to the location of two methoxy groups at C-3 and C-7, respectively. The planar structure of 1 was thus determined as shown in Figure 1.

Compound 1 possessed the same planar structure as chrysoalide B, a new phthalide produced by the marine-derived fungus Penicillium chrysogenum LD-201810 (Ge et al., 2021). Surprisingly, the specific rotation value of 1 was found to be zero. Considering its baseline ECD curve, compound 1 was existed as racemic enantiomers. Compound 1 was then subjected to chiral HPLC separation, which successfully afforded two individual enantiomers (+)-1 and (–)-1 with a ratio of 1:1. Both (+)-1 and (–)-1 were cultured into suitable single crystals in MeOH/H₂O mixed solution. Single-crystal X-ray diffraction of (+)-1 and (–)-1 (Figure 3) not only confirmed the proposed structure but also the absolute configurations of (+)-1 and (–)-1. It should be pointed out that Ge et al. (2021) reported the new compound chrysoalide B with the optical rotation of + 15.8° and established the absolute configuration of C-3 to be R based on ECD calculations. However, in our study, the absolute configuration of (+)-chrysoalide B (1) was revised as 3S by single-crystal X-ray diffraction.

Penicidone E (2), isolated as colorless oil, was found to possess the molecular formula of C₂₀H₂₃NO₇ on the basis of HRESIMS (m/z 390.1547 [M + H] ⁺ for C₂₀H₂₄NO₇ and 412.1369 [M + Na] ⁺ for C₂₀H₂₃NO₇Na). Overall inspection of the ¹H and ¹³C NMR spectra of 2 (Table 1) indicated that it contained two carbonyls at δ_C 192.4 (C-8) and 175.8 (C-10), one ester carbonyl at δ_C 166.3 (C-1), ten sp²-hybridized carbons which resonated between δ_C 103.2 and 160.0, one methylene at δ_C 39.2 (C-15), one oxygenated sp³ methine at δ_C 75.3 (C-16), and five methyls including four methoxy groups at δ_C 52.5 (1-OMe), 56.0 (4-OMe), 56.5 (6-OMe), and 56.0 (16-OMe). The ¹H and ¹³C NMR spectra of 2 were partially similar to that of penicidone C, a cytotoxic alkaloidal metabolite isolated from an endophytic Penicillium sp. (Ge et al., 2008). However, the resonances at δ_C 125.3 and 134.2 ascribable to the two sp² methine groups in penicidone C were replaced by a methylene (C-15) and an oxygenated sp³ methine (C-16). Moreover, an extra methoxyl signal at δ_H 3.23 and δ_C 56.0 (16-OMe) appeared in the ¹H and ¹³C NMR spectra of 2. The observation could be explained by assuming that 2 was an oxidative derivative of penicidone C at the location of C-15 and C-16. This assumption was reinforced by the HMBC correlations from H₃-17 to C-15 and C-16, from H₂-15 to C-11 and C-12, and from 16-OMe to C-16 (Figure 2). Compound 2 was named as penicidone E. The γ-pyridone nucleus found in 2 is rare in natural products, with only four analogs, penicidones A-D, possessing similar structures (Liu et al., 2015). Calculation of optical rotations of different conformers 16R and 16S were carried out using the TDDFT method at CAM-B3LYP/6-311 + g (2d,p) level in methanol (λ = 589 nm). The calculated optical rotation was –24.4, which was of the opposite sign to the experimental value ([α]²⁰_D + 13.5). Therefore, the absolute configuration of 2 was established as 16R.

In addition to the new compounds 1 and 2, the structures of the remaining six known compounds were established based on their spectroscopic data, as well as by comparison with the literatures. These compounds were identified as 6,7-dihydroxy-3-methoxy-3-methylphthalide (3) (Wang et al., 2013), oxisterigmatocystin C (4) (Cai et al., 2011), penicillide (5) (Jeon and Shim, 2020), a diphenyl ether 6 (Wu et al., 2018), diorcinol L (7) (Li et al., 2018), and 3-[2-(1-hydroxy-1-methyl-ethyl)-6-methyl-2,3-dihydrobenzofuran-4-yloxy]-5-methylphenol (8) (Zhuravleva et al., 2013).

The isolated compounds 1-8 were detected for their cytotoxicity against human pancreatic cancer cell line PATU8988T by using the CCK-8 method (Yuan et al., 2020). The results of CCK-8 showed that only the new compound 2 showed a promising activity (Supplementary Table 6). 2 demonstrated a dose-dependent cytotoxicity against the cells treated for 48 h, with the IC₅₀ value of 11.4 μM (Figure 4A). Cell apoptosis might inhibit tumor cell proliferation. Therefore, compounds which could aggravate tumor cell apoptosis might possess application prospect for the anti-tumor therapy. To further study whether the inhibition of PATU8988T cells proliferation was caused by cell apoptosis, we examined apoptosis-related indicators. After treated with compound 2 at the concentration of 20 μM for 48 h, PATU8988T cell number reduction and cell morphology abnormity occurred in both doxorubicin (dox) and 2-treated groups while the cells in the control group and DMSO group performed normally (Figure 4B), suggesting 2 as well as dox induced PATU8988T cell death. Additionally, Annexin V-FITC/PI assay was used to examine apoptosis percentage by flow cytometry. As shown in Figure 4C, dox significantly up-regulated the proportion of apoptotic cells with a percentage of 81.44% while 2 increased the proportion of apoptotic cells with a percentage of 8.85% in comparison with 0.55% in the control group and 0.74% in DMSO group. In addition, Bcl-2 family members including Bcl-2 and Bax can regulate apoptosis. The result of western blotting demonstrated that both dox and 2 markedly decreased the ratio of Bcl-2/Bax, indicating that 2 might induce pancreatic tumor cell apoptosis (Figure 4D). The above results proved that 2 might kill pancreas tumor cells by inducing the cell apoptosis.