In our continuous research for new compounds from the fungi [14,15,16], Penicillium chrysogenum wis54-1255 was selected for further chemical investigation. To further expand its secondary metabolites, this strain was cultured in different media employing the OSMAC approach. Then HPLC fingerprints analysis showed that the metabolic profile of rice media exhibited the higher abundance and diversity of compounds (Figure S1). Subsequent fractionation of the rice fermentation extract led to the isolation of one new indole diketopiperazine penichrysogenone A (1), and two new natural products 2 and 3 first reported from a natural source, together with 10 known compounds (4-13) (Fig. 1). The isolation, structural elucidation, and bioactivity are reported herein.

Penichrysogenone A (1) was isolated as a yellow oil. Its molecular formula, CHNO, was deduced from the HRESIMS data, which displayed a [M + H] ion peak at m/z 326.1866 (calculated for CHNO m/z 326.1863), with ten degrees of unsaturation. Infrared spectroscopy reveals absorption bands at 3271, 1725, 1675, and 1455 cm, indicative of the presence of amino, hydroxyl, carbonyl, and aromatic ring functionalities. The H NMR data of 1 (Table 1) exhibited the presence of two exchangeable protons (δ 10.99 and 8.60), four aromatic protons (δ 7.42, 7.31, 7.07, and 6.98), two methylenes (δ 3.23, 2.71, and 1.53), four methines (δ 5.58, 4.28, 3.97, and 1.71), and three methyls (δ 1.43, 0.83, and 0.80). The C NMR data and HSQC (Table 1) spectra showed 19 carbon resonances, including three methyls, two methylenes, four sp methines, four aromatic methines, four sp quaternary carbons, and two amide carbonyls (δ 166.2 and 167.8). These data accounted for all the H and C NMR resonances, suggesting a tetracyclic structure of 1. The planar structure of 1 was further determined by interpretation of its 2D NMR data. HMBC correlations (Fig. 2) from H-13 to C-11, C-12 and C-17, from H-16 to C-11, C-14 and C-17, together with the H-H COSY (Fig. 2) correlations of H-13/H-14/H-15/H-16 constructed the disubstituted indole ring (rings A and B, Fig. 2). Other HMBC cross-peaks from H-6 and H-9 to C-5 and C-8, from NH-7 to C-5 and C-9, and from H-19 to C-5, from H₃-21/H₃-22 to C-19/C-20, from H₃-21 to C-22, and from H₂-20 to C-6 and C-19, together with H-H COSY (Fig. 2) correlations of H-6/H-19/H-20/H-21(22), as well as the characteristic NMR data of two amide carbonyls C-5 and C-8 (δ 166.2 and 167.8) indicated the presence of diketopiperazine moiety (ring D) with isobutyl located at C-6 position. Furthermore, key HMBC correlations from H-3 to C-2, C-5, C-9 and C-11, and from H-10 to C-2, C-8, C-9 and C-11, combined with H-H COSY (Fig. 2) correlations of H-3/H-18 and H-9/H-10 permitted the construction of ring C, thereby completing the 6/5/6/6 tetracyclic pyrazino[1',2':1,6]pyrido[3,4-b]indole-1,4-dione skeleton. Therefore, the planar structure of 1 was established as shown.

NOESY correlation of H-9 with H-18 suggested their co-facial orientation, estalishing relative configuration of C-3 and C-9 in 1. However, the relative configuration at C-6 in 1 remained unassigned due to lack of definitive NOESY correlation. Consequently, the absolute configuration of 1 was determined by comparing its experimental ECD spectrum with the calculated ECD spectra generated by the time-dependent density functional theory (TDDFT) for four possible enantiomers (3R,6S,9S)-1 (1a), (3S,6R,9R)-1 (1b), (3R,6R,9S)-1 (1c) and (3S,6S,9R)-1 (1d). The experimental spectrum of compound 1 matched well with the calculated spectrum for the (3R,6S,9S)-1 (1a) (Fig. 3), indicating the 3R,6S,9S absolute configuration of 1.

Structurally, penichrysogenone A (1) features the same 6/5/6/6 tetracyclic pyrazino[1',2':1,6]pyrido[3,4-b]indole-1,4-dione core skeleton as fumitremorgin C [17]. However, compound 1 is characterized by a distinct substitution pattern, with a methyl group at C-3 instead of a prenyl group and an isobutyl group at C-6 in place of the pyrrolidine moiety. Biogenetically, compound 1 is proposed to derived from tryptophan and leucine, with the initial formation of a diketopiperazine (DKP) skeleton, followed by acetylation at N-4 position. A subsequent Pictet-Spengler reaction then leads to form the tetrahydropyridine ring (ring C), thereby constructing the 6/5/6/6 tetracyclic pyrazino[1',2':1,6]pyrido[3,4-b]indole-1,4-dione core skeleton.

Furthermore, two new natural products reported here for the first time from a natural source 4-(N-benzoyl-N-hydroxyamino)-butyric acid (2) [18], 2-acetamido-N-benzyl-2-(ethylamino)acetamide (3) [19], and ten known products 4-hydroxymandelamide (4) [20], pestalotiopamide E (5) [21], fuscoatramide (6) [22], cyclo (Ile-Leu) (7) [23], (E)-ferulic acid (8) [24], 4-hydroxybenzaldehyde (9) [25], 4-hydroxyphenylacetic acid (10) [26], 3β-hydroxy-5β,8β-epidioxyergosta-6,9,22-triene (11) [27], 5α,8α-epidioxy-(22E,24 R)-23-methylergosta-6,22-dien-3β-ol (12) [28] and β-sitosterol (13) [29] were determined by comparison of their NMR data with those reported in the literature.

Compound 1 was not evaluated activity due to sample limits. The antimicrobial activities of compounds 2-13 were evaluated against a panel of pathogenic bacteria, including Methicillin-resistance Staphylococcus aureus 8325-4 (MRSA), Bacillus paranthracis, Xanthomonas campestris pv. campestris BLY013 (Xcc) and Pseudomonas solanacearum BXL01, using vancomycin (Van) as the positive control (Table 2). For P. solanacearum and Xcc, chlortetracycline was also used as an additional positive control, showing MIC values of >64 µg/mL and 2 µg/mL. Compound 4 showed antibacterial activity against P. solanacearum (MIC = 64 μg/mL), with vancomycin as the positive control (MIC > 64 μg/mL). In addition, the isolated compounds 2-13 were also evaluated antifungal activity against Candida albicans SC5314 and C. auris TY. Compound 10 exhibited inhibitory activity against C. auris TY, with MIC value of 32 μg/mL, while the positive control fluconazole showed MIC value of 2 μg/mL. The antioxidant activities of compounds 2-13 were further evaluated using the ABTS radical scavenging assay (Table 3). Compounds 2 and 10 displayed significant radical scavenging activity, with IC values of 2.36 ± 0.11 and 2.45 ± 0.14 μg/mL, respectively, which are superior to the positive control ascorbic acid (5.11 ± 0.08 μg/mL).