The details of the instruments and equipments can be found in the Supplementary Material .

The strain Penicillium sp. LA032 was isolated from the rhizospheric soil of a mangrove ecosystem collected from Beilun Estuary, Fangchenggang, Guangxi, People’s Republic of China. The fungus LA032 was further identified as Penicillium sp. (GenBank Accession No. OP804236), based on a basic local alignment search tool (BLAST) search. The strain was deposited in the laboratory of the Institute of Microbiology, Chinese Academy of Sciences, Beijing.

The strain was firstly cultured on a potato dextrose agar (PDA) medium at 28°C for 7 days. Then, some pieces of agar plugs were cultured on media (1% malt extract, 0.4% yeast extract, and 0.4% glucose) in 500 ml Fernbach flasks at 28°C on a rotary shaker (200 rpm) for 4 days to obtain the seed culture. Finally, the spore inoculums were incubated on 50 × 500 ml Fernbach flasks that contained 100 g of rice and 100 ml of distilled water at room temperature for 21 days.

The fermented products were extracted repeatedly with EtOAc (4 × 6.0 L), which were concentrated to afford 48.0 g of crude residue. The extract was subjected to silica gel column chromatography (CC) eluting with petroleum ether/EtOAc gradient elution to afford eight subfractions Fr.1−Fr.8. Fr.4 (2.1 g) eluted with 30% EtOAc was subjected to silica gel CC with CH₂Cl₂/MeOH to afford Fr.4.1−Fr.4.8. Fr.4.1 (165.3 mg) was further purified by octadecylsilanized (ODS) CC (20%−100% MeOH/H₂O) to yield six subfractions Fr.4.1.1−Fr.4.1.6. Fr.4.1.5 (70.3 mg) eluted with 70% MeOH/H₂O was further purified by reversed-phase (RP) HPLC (57% CH₃CN/H₂O for 60 min; 2.0 ml/min) to afford compound 6 (1.8 mg, t _R 54.0 min). Fr.5 (560.0 mg) eluted with 50% EtOAc was separated by ODS CC using MeOH/H₂O gradient elution to obtain five subfractions Fr.5.1−Fr.5.5. Fr.5.2 (19.0 mg) eluted with 40% MeOH was further purified by RP-HPLC (51% MeOH/H₂O for 50 min; 2.0 ml/min) to get compounds 9 (3.6 mg, t _R 28.5 min) and 8 (1.8 mg, t _R 43.0 min). Fr.7 (630.0 mg) eluted with 80% EtOAc was separated by ODS CC (30%−100% MeOH/H₂O) to afford five subfractions Fr.7.1−Fr.7.5. Fr.7.2 (45.0 mg) was further purified by RP-HPLC (57% MeOH/H₂O for 40 min; 2.0 ml/min) to get compounds 3 (4.8 mg, t _R 26.8 min), 4 (3.8 mg, t _R 28.0 min), and 5 (3.4 mg, t _R 30.2 min). Fr. 7.3 (1.01 g) was subjected to chromatography using a Sephadex LH-20 column (CH₂Cl₂/MeOH, 1:1) to yield six subfractions Fr.7.3.1−Fr.7.3.6. Fr.7.3.3 (30.4 mg) was further purified by RP-HPLC (47% CH₃CN/H₂O for 30 min; 2.0 ml/min) to get compounds 2 (3.1 mg, t _R 21.6 min) and 1 (2.0 mg, t _R 25.0 min). Fr.8 (860.0 mg) eluted with 90% EtOAc was purified by ODS CC eluting with MeOH/H₂O gradient elution to afford seven subfractions Fr.8.1–Fr.8.7. Fr.8.6 (20.6 mg) eluted with 65% MeOH was further purified by RP-HPLC (70% MeOH/H₂O for 30 min; 2.0 ml/min) to get compound 7 (3.1 mg, t _R 28.0 min).

Penicilamide A ( 1 ): white amorphous powder; [ α ] D 25 +53.3 (c 0.06, MeOH); UV (MeOH) λ _max (log ϵ) 230 (2.12), 273 (1.56) nm; ECD (c 0.82 × 10⁻³ M, MeOH) λ _max (Δϵ) 226 (+6.15), 256 (+1.66), 282 (−0.41) nm; IR (neat) ν _max 3,343, 2,964, 1,735, 1,636, 1,429, 1,362, and 1,123 cm⁻¹; ¹H and ¹³C nuclear magnetic resonance (NMR) data, see Table 1 ; high-resolution electrospray ionisation mass spectrometry (HRESIMS) m/z 362.1508 [M + H]⁺ (calcd for C₂₁H₂₀N₃O₃, 362.1505).

Penicilamide B ( 2 ): white amorphous powder; [ α ] D 25 −140.9 (c 0.10, MeOH); UV (MeOH) λ _max (log ϵ) 246 (2.60), 333 (1.93) nm; ECD (c 0.81 × 10⁻³ M, MeOH) λ _max (Δϵ) 234 (+52.3), 266 (−23.7), 341 (−11.3) nm; IR (neat) ν _max 3,358, 2,973, 1,667, 1,420, and 1,243 cm⁻¹; ¹H and ¹³C NMR data, see Table 1 ; HRESIMS m/z 368.1970 [M + H]⁺ (calcd for C₂₁H₂₆N₃O₃, 368.1969).

Penicinone A ( 6 ): white amorphous powder; [ α ] D 25 −20.0 (c 0.10, MeOH); UV (MeOH) λ _max (log ϵ) 245 (3.50) nm; ECD (c 1.01 × 10⁻³ M, MeOH) λ _max (Δϵ) 246 (+31.0), 285 (−17.9) nm; IR (neat) ν _max 3,399, 2,955, 2,927, 1,765, 1,644, 1,456, and 1,034 cm⁻¹; ¹H and ¹³C NMR data, see Table 2 ; HRESIMS m/z 295.1546 [M − H]⁻ (calcd for C₁₆H₂₃O₅, 295.1545).

Penicinone B ( 7 ): yellow oil; UV (MeOH) λ _max (log ϵ) 224 (1.56), 274 (2.05) nm; IR (neat) ν _max 3,436, 2,949, 1,735, 1,610, 1,438, and 1,219 cm⁻¹; ¹H and ¹³C NMR data, see Table 2 ; HRESIMS m/z 297.1332 [M + H]⁺ (calcd for C₁₅H₂₁O₆, 297.1333).

Penicinone C ( 8 ): colorless oil; [ α ] D 25 −10.0 (c 0.10, CHCl₃); UV (MeOH) λ _max (log ϵ) 217 (2.69), 276 (0.56) nm; ECD (c 1.00 × 10⁻³ M, MeOH) λ _max (Δϵ) 220 (+1.88) nm; IR (neat) ν _max 3,370, 2,958, 1,731, 1,614, 1,516, and 1,225 cm⁻¹; ¹H and ¹³C NMR data, see Table 2 ; HRESIMS m/z 309.1104 [M + Na]⁺ (calcd for C₁₇H₁₈O₄Na, 309.1103).

The ligand-compound 6 (0.2 mg) was added into a dry solution of Mo₂(OAc)₄ (0.7 mg) in dimethyl sulfoxide (DMSO) (400 μl) and was subjected to CD measurements. Firstly, the ECD spectrum was recorded immediately after mixing. Subsequently, its evolution was monitored every 10 min until stationary. Finally, the inherent ECD was subtracted. The diagnostic band observed at ca. 312 nm in the induced ECD spectrum was correlated to the absolute configuration of the 7,8-diol moiety on the basis of Snatzke’s regulations (Frelek et al., 1997).

Compound 8 (0.3 mg) was dissolved in a dry solution of the CH₂Cl₂ (500 μl). Then, 0.6 mg of [Rh₂(OCOCF₃)₄] was added. Subsequently, the first induced ECD spectrum was recorded at once. The following spectrum was measured every 5 min for four times. The correlation between the absolute configuration of C-8 secondary alcohol in 8 and the sign of the E band at 350 nm was inferred using the bulkiness rule (Gerards and Snatzke, 1990; Frelek and Szczepek, 1999).

The ECD calculations were performed as described previously (Zhang et al., 2022). A conformational search was performed using Maestro 10.2 (Ji and Xu, 2021). The conformers were then optimized using the density functional theory method at the B3LYP/6-311G(2d,p) level by the software package Gaussian 09 (Frisch et al., 2013). The 60 lowest energy conformations were calculated by the ECD calculations using the time-dependent density functional theory (TDDFT) methodology at the CAM-B3LYP/6-311G(d,p) level.

Cytotoxic assays were conducted as previously described (Liu et al., 2015). The detailed procedure is described in the Supplementary Material .

The fungus Penicillium sp. LA032 was fermented on rice solid culture and extracted three times with EtOAc. The EtOAc extract (48.0 g) was fractionated by silica gel vacuum liquid chromatography (VLC), Sephadex LH-20 CC, and semipreparative HPLC to afford five new compounds (1, 2 and 6−8) and four known compounds.

Penicilamide A (1) was obtained as white amorphous powder. The molecular formula of 1 was determined as C₂₁H₁₉N₃O₃ by the HRESIMS molecular ion peak at m/z 362.1508 [M + H]⁺ (calcd for C₂₁H₂₀N₃O₃, 362.1505), indicating 14 degrees of unsaturation. The IR spectrum showed absorption bands at 3,343, 2,964, 1,735, 1,636, and 1,429 cm⁻¹, indicating the presence of amino, hydroxy, carbonyl, and aromatic functional groups. The ¹H NMR spectrum ( Table 1 and Figure S1 ) exhibited one exchangeable proton (δ _H 10.03), nine olefinic or aromatic protons (δ _H 7.53, 7.30, 7.14, 6.93, 6.92, 6.75, 6.31, 6.14, and 5.97), one methylene (δ _H 3.99 and 3.54), and two singlet methyls (δ _H 1.57 and 1.48). The ¹³C NMR spectrum ( Figure S2 ) and heteronuclear single quantum coherence (HSQC) spectrum ( Figure S3 ) showed the presence of 21 carbon resonances, including two methyls, one methylene, nine olefinic or aromatic methines, and nine quaternary carbons (two sp³ quaternary carbons at δ _C 38.4 and 73.8; five sp² carbons at δ _C 103.3, 126.3, 128.9, 136.0, and 142.8; and two amide carbonyls at δ _C 165.1 and 156.9). Taking the above NMR data and molecular formula into consideration, an aromatic system, four double bonds and two amide carbonyl functionalities accounted for 10 indices of hydrogen deficiency, suggesting that 1 was a pentacyclic compound. The disubstituted indole moiety (rings A and B) in 1 was assigned by the heteronuclear multiple bond correlations (HMBC) correlations ( Figures 2 and S5 ) from H-4 to C-3 (δ _C 103.3), C-6 (δ _C 121.7), C-8 (δ _C 136.0), and C-9 (δ _C 128.9) and from H-7 to C-5 (δ _C 119.7), C-8, and C-9, together with the ¹H-¹H COSY cross-peaks ( Figures 2 and S4 ) of H-4/H-5/H-6/H-7. The characteristic NMR data of two amide carbonyls (C-12 and C-18: δ _C 165.1 and 156.9, respectively) and the HMBC cross-peaks from H-16 to C-17 (δ _C 126.3) and C-18 and from H-14 to C-17, along with the ¹H-¹H COSY cross-peaks of H-14/H-15/H-16, permitted the completion of the diketopiperazine ring and the unsaturated proline moiety (rings D and E). The other HMBC correlations from H₂-10 to C-2 (δ _C 142.8), C-3, C-9, C-11 (δ _C 89.0) and C-12, from H-20 to C-18 and C-22 (δ _C 38.4), from H-21 to C-2, and from H₃-23/H₃-24 to C-2, C-21 (δ _C 141.9), and C-22, combined with the ¹H-¹H COSY cross-peak of H-20/H-21, safely expounded that rings B and D were cyclized at C-20 and a nitrogen atom to form an azocane subunit (ring C), completing the unusual 6/5/8/6/5 pentacyclic ring system. The chemical shift value for C-11 (δ _C 89.0) indicated that the carbon C-11 was substituted by the hydroxyl group, which was also supported by the molecular formula of 1. Then, the structure of 1 was finally determined.

The absolute configuration for compound 1 was determined unequivocally by the comparison of the experimental and calculated ECD curves. The ECD spectra of 11R-(1a) and 11S-(1b) were performed using the time-dependent density functional theory (TD-DFT) at the CAM-B3LYP/6-311G(d,p) level. The calculated ECD spectrum of 11R-(1a) matched well with the experimental one of 1 ( Figure 3 ), which allowed the assignment of the absolute configuration of 1 as 11R.

Penicilamide B (2) had a molecular formula of C₂₁H₂₅N₃O₃ as deduced from the HRESIMS and ¹³C NMR data, with 11 degrees of unsaturation, which is 16 mass units higher than that of deoxybrevianamide E (3) (Song et al., 2012). The ¹H, ¹³C, and HSQC spectroscopic data ( Table 1 and Figures S7–S9 ) suggested the presence of two methyls, four methylenes (one sp² methylene), two sp³ methines, six olefinic/aromatic methines, and seven quaternary carbons including two amide carbonyls (δ _C 164.3 and 165.7). The ¹H NMR and ¹³C NMR data ( Table 1 ) of 2 were similar to those of the coisolated compound 3, with slight differences in the chemical shift values of C-2, C-3, C-9, C-10, C-11, and C-20, suggesting their structural similarity. A further analysis of their NMR data indicated that the olefin C-2/C-3 in 3 was replaced by the olefin C-3/C-10 in 2 and an extra hydroxy group was located at C-2 in 2, as supported by the HMBC correlations ( Figures 2 and S11) from 1-NH (δ _H 6.26), H-21, H₃-23, and H₃-24 to the sp³ quaternary carbon C-2 (δ _C 98.4) and C-20, from H-4 (δ _H 7.31) to the olefinic quaternary carbon C-3 (δ _C 149.0), from H-10 (δ _H 6.38) to C-2, C-3, C-9, C-11, and C-12 and from H-11 (δ _H 5.26) to C-3 and the olefinic methine carbon C-10 (δ _C 121.1), together with the ¹H-¹H COSY cross-peak ( Figures 2 and S10 ) of H-10/H-11. In the nuclear overhauser effect spectroscopy (NOESY) spectrum ( Figures 4 and S12 ), the NOESY correlation of H-4/H-10 suggested the olefin C-3/C-10 as Z geometry. In addition, the cross-peaks of H-11 with H-17 and H₃-24 suggested that these protons were cofacial, implying the existence of two possible configurations 2R,11S,17S-(2a) and 2S,11R,17R-(2b). Lastly, the absolute configuration of 2 was also established by ECD calculations. The experimental ECD curve was consistent with the calculated one of 2R,11S,17S-(2a), indicating the 2R,11S,17S configuration of 2. Finally, compound 2 was established as a PIDA featuring a rare 3-methyleneindolin-2-ol moiety.

Penicinone A (6), obtained as white amorphous powder, had a molecular formula of C₁₆H₂₄O₅ as deduced from the negative HRESIMS peak at m/z 295.1546 [M − H]⁻ (calcd for C₁₆H₂₃O₅, 295.1545) and the NMR data ( Table 2 ), implying five indices of hydrogen deficiency. The analysis of the ¹H, ¹³C, and HSQC spectra ( Table 2 and Figures S13–S15 ) showed the presence of two methyls, six sp³ methylenes, one sp² methylene (δ _C/H 91.7/4.02, 5.04), two oxygenated methines (δ _C/H 89.0/4.31 and 66.9/4.20), one sp³ quaternary carbon (δ _C 72.2), three sp² quaternary carbons, and one carbonyl (δ _C 167.5). The NMR data of 6 were almost similar to those of isoaigialone B (Silva et al., 2017) except that one oxygenated methine and one methyl group were absent, while the exocyclic double bond (δ _C/H 91.7/4.99, 5.04; δ _C 149.5) signals were observed in 6. The HMBC correlations ( Figures 2 and S17 ) from H₂-10 (δ _H 4.99, 5.04) to C-3 (δ _C 149.5) and C-4 (δ _C 163.7) further confirmed the existence of an exocyclic double bond at C-3 and C-10 positions of 6. A comprehensive analysis of the ¹H-¹H COSY ( Figures 2 and S16 ) and HMBC correlations corroborated the planar structure of compound 6 with the furo[3,4-b]pyran-5-one ring system as shown. The NOESY correlations ( Figures 4 and S18 ) of H₃-11 with H-6 and H-8 suggested that these protons were on the same side of the pyran ring. Furthermore, Snatzke’s method was employed to address the absolute configuration of the 7,8-diol unit. After mixing 6 and dimolybdenum tetraacetate [Mo₂(OAc)₄] in DMSO, ECD data were measured between 250 and 450 nm. The inherent ECD was subtracted. The positive Cotton effect (CE) at 312 nm ( Figure 5 ) in the induced spectrum of 6 indicated a 7R,8R configuration of the 7,8-diol (Frelek et al., 1997; Di Bari et al., 2001). Thus, the absolute configuration of 6 was established as 6R,7R,8R.

Penicinone B (7) was obtained as yellow oil. Its molecular formula was deduced as C₁₅H₂₀O₆ (six degrees of unsaturation) by the analysis of its HRESIMS and 1D NMR data ( Table 2 ). The interpretation of its IR spectrum indicated the presences of hydroxy (3436 cm⁻¹) and α,β-unsaturated lactone carbonyl (1735 cm⁻¹) functional groups. The analysis of the ¹H, ¹³C and HSQC NMR data ( Table 2 and Figures S19–S21 ) of 7 revealed the presence of five methylenes (one oxygenated), two methoxy groups (δ _C/H 60.4/4.02 and 51.6/3.61), six olefinic carbons (three protonated), and two ester carbonyl carbons (δ _C 167.5 and 174.1), accounting for five degrees of unsaturation. Therefore, the remaining one unsaturation indicated the presence of a monocyclic ring system in 7. The characteristic signals of the α,β-unsaturated lactone subunit corresponding to a lactone carbonyl carbon (δ _C 167.5), a quaternary carbon (δ _C 141.0) and two olefinic carbons (δ _C 172.3 and 91.9, respectively), as well as the HMBC correlations ( Figures 2 and S23 ) from H-2 to C-1, C-3 and C-4 and from H₃-15 to C-3, permitted the completion of the 2(5H)-furanone moiety with the methoxy carbon C-15 attached at C-3. The ¹H-¹H COSY cross-peaks ( Figures 2 and S22 ) of H-6/H-7/H₂-8/H₂-9/H₂-10/H₂-11, combined with the HMBC cross-peaks from H₂-10, H₂-11 and H₃-13 to C-12 (δ _C 174.1), established the C-6–C-13 subunit. Furthermore, the HMBC correlations from H-6 to C-4 and C-14, from H-7 to C-5, and from H₂-14 to C-4, C-5 and C-6 demonstrated that both C-6 and C-14 were directly attached to the C-4/C-5 double bond at C-5. The remaining hydroxyl group was substituted at C-14, which are deduced by the chemical shift of C-14 (δ _C 55.6) and the molecular formula of 7. The large coupling constant of the olefinic protons of H-6 and H-7 (15.6 Hz) suggested the olefin C-6/C-7 as E geometry. The NOESY correlation H₃-15 to H-2 and the lack of NOESY cross-peak between H₃-15 and H₂-14 indicated the Z-configuration of the C-4/C-5 double bond based on the previous literature (Phainuphong et al., 2017). Therefore, the structure of 7 was established as depicted.

Penicinone C (8) was isolated as colorless oil. The molecular formula of 8 was established as C₁₇H₁₈O₄ from the positive HRESIMS data (m/z 309.1104 [M + Na]⁺, calcd 309.1103). The ¹H and ¹³C NMR data ( Table 2 ) of 8 were close to those of 4′-hydroxyphenylethyl 4,8(R)-dihydroxyphenylpropionate (Sun et al., 2021), except that the hydroxy group at C-4 disappeared in 8. This observation was supported by the HMBC correlations ( Figures 2 and S29 ) from H₂-7 (δ _H 2.87, 3.01) to C-1 (δ _C 138.6), C-2 (δ _C 130.5), C-6 (δ _C 130.5) and C-9 (δ _C 174.5) and from H-2 (δ _H 7.18) and H-6 (δ _H 7.18) to C-4 (δ _C 127.3), as well as the ¹H-¹H COSY cross-peaks ( Figures 2 and S28 ) of H-3/H-4/H-5. The absolute configuration of C-8 in 8 was determined by the circular dichroism data of the in situ formed [Rh₂(OCOCF₃)₄] complex, with the inherent contribution subtracted (Gerards and Snatzke, 1990; Frelek and Szczepek, 1999). The Rh₂(OCOCF₃)₄-induced ECD spectrum of 8 showed positive CE at 350 nm ( Figure 6 ), suggesting the 8S configuration (Gerards and Snatzke, 1990; Frelek and Szczepek, 1999). Therefore, the absolute configuration of compound 8 was established as 8S.

The structures of four known compounds, deoxybrevianamide E (3) (Song et al., 2012), brevianamide V (4) (Song et al., 2012), 12,13-dehydroprolyl-2-(1,1-dimethylallyltryptophyl)diketopiperazine (5) (Steyn, 1973) and 4-hydroxyphenethyl 2-(4-hydroxyphenyl)acetate (9) (Wang et al., 2009) ( Figure 1 ), were assigned by the interpretation of their spectroscopic data with those reported in the literature.

To the best of our knowledge, the PIDAs featuring the 6/5/8/6/5 pentacyclic moiety are rare in nature, with only 10 compounds being reported (Steyn, 1973; Quang et al., 2013; Zhuravleva et al., 2021). Moreover, the hydroxy group is usually located at the C-3, C-16, or C-17 position in these metabolites (Quang et al., 2013; Zhuravleva et al., 2021). Penicilamide A (1) represents the first example of PIDAs featuring the unique 6/5/8/6/5 pentacyclic skeleton with an α-hydroxy group at the C-11 position. Meanwhile, penicilamide B (2) is a new PIDA with a 3-methyleneindolin-2-ol moiety. Biogenetically, PIDAs 1−5 could be derived from the condensation of _L-tryptophan with _L-proline. The biosynthetic intermediate I was firstly produced, followed by reverse C2 prenylation to afford the intermediate 3. Starting from compound 3, compounds 1, 2, 4, and 5 could be generated via oxidation, dehydrogenation, and cyclization reactions. The plausible biosynthetic pathways were proposed as shown in Scheme 1 . In addition, compound 6 was established as a rare furo[3,4-b]pyran-5-one skeleton with an n-heptyl moiety. Meanwhile, compounds 7 and 8 are γ-butenolide and phenolic acid derivatives, respectively.

Compounds 1−7 were tested for their cytotoxic activity against B16 (murine melanoma cells), MCF-7 (human breast adenocarcinoma cells), and HepG2 (human hepatocellular carcinoma cells) at 50.0 μM. Compound 6 displayed potent cytotoxicity against B16, MCF-7, and HepG2 cell lines at 50.0 µM with inhibitory ratios of 82.7%, 75.1%, and 95.9%, respectively ( Figure 7 ). Furthermore, compound 6 showed more potent cytotoxicity against HepG2 cells (IC₅₀ value of 3.09 ± 0.03 µM) than that of the positive-control cisplatin (IC₅₀ value of 8.71 ± 0.28 μM). Compound 6 also showed cytotoxicity against MCF-7 and B16 cells, with IC₅₀ values of 30.01 ± 0.52 and 27.91 ± 0.17 μM, respectively, whereas the positive-control cisplatin showed IC₅₀ values of 4.22 ± 0.46 and 20.39 ± 3.25 μM, respectively. Other compounds did not show obviously inhibitory effects on the cell lines tested at 50.0 μM ( Table 3 ).