New Antiproliferative Compounds against Glioma Cells from the Marine-Sourced Fungus Penicillium sp. ZZ1750

Seven novel compounds, namely peniresorcinosides A–E (1–5), penidifarnesylin A (6), and penipyridinone A (7), together with the 11 known ones 8–17, were isolated from a culture of the marine-associated fungus Penicillium sp. ZZ1750 in rice medium. The structures of the new compounds were established based on their high-resolution electrospray ionization mass spectroscopy (HRESIMS) data, extensive nuclear magnetic resonance (NMR) spectroscopic analyses, chemical degradation, Mosher’s method, 13C-NMR calculations, electronic circular dichroism (ECD) calculations, and single crystal X-ray diffraction. Peniresorcinosides A (1) and B (2) are rare glycosylated alkylresorcinols and exhibited potent antiglioma activity, with IC50 values of 4.0 and 5.6 µM for U87MG cells and 14.1 and 9.8 µM for U251 cells, respectively.


Introduction
Gliomas are one of the most common types of primary brain tumors and despite advances in cancer therapy, have remained particularly challenging to treat [1]. Gliomas usually locate at many important brain function areas, which makes surgical resection very difficult, therefore, chemotherapy has played a more important role in the treatment and prevention of glioma. However, most of the current anti-glioma drugs, such as temozolomide (the first line chemotherapeutic agent for glioma), carmustine, lomustine, and procarbazine, are DNA cytotoxic alkylating agents with drug resistance, serious toxicity, and other side-effects [2,3]. Although antibodies (such as bevacizumab) and molecularly targeted anticancer drugs are constantly being evaluated for the treatment of glioma, their overall curative effect is poor [4]. Therefore, the discovery and development of new anti-glioma drugs with unique mechanisms of action is an urgent need.
Marine fungi are important resources for the discovery of novel bioactive natural products and drug lead compounds [5][6][7][8][9][10][11]. Among them, Penicillium species fungi have been proved to be one of the most novel bioactive compound producers [9][10][11]. For example, 390 new compounds with different structural types were identified from the marine-derived Penicillium fungi during 1991-2014, of which 58% showed some form of antitumor, antiviral, antibacterial, and anti-inflammatory activity [9]. It was also reported that 188 secondary metabolites with diverse bioactivities were isolated from marine Penicillium fungi from 2015 to 2020 [11].
In recent years, we have carried out research on the discovery of natural products from marine microorganisms with antiproliferative activity against human glioma cells. These studies have resulted in the isolation and identification of a number of novel compounds with potent antiglioma activity, including the polycyclic anthraquinones N-acetyl-Ndemethylmayamycin and streptoanthraquinone A from Streptomyces sp. 182SMLY [12], the cyclodepsipeptide streptodepsipeptides P11A and P11B from Streptomyces sp. P11-23B [13], bagremycin C from Streptomyces sp. Q22 [14], pyrrospirone G and penicipyrroether A from Penicillium sp. ZZ380 [15,16], 1-hydroxymethyl-8-hydroxyanthraquinone-3-carboxylic acid from Streptomyces sp. ZZ406 [17], streptoglutarimide H from Streptomyces sp. ZZ741 [18], and the compounds marinacarbolines G and M and caerulomycin N from Actinoalloteichus sp. ZZ1866 [19]. The antiglioma activity of some compounds were related to the downregulation of several important glioma glycolytic enzymes [13,17].
As a part of our ongoing research program to discover novel natural antiglioma products from marine microorganisms, a Penicillium sp. ZZ1750 fungus was isolated from a marine mud sample collected from the Arabian Sea, close to Karachi, Pakistan. An ethyl acetate (EtOAc) extract prepared from a scale-up culture of the strain ZZ1750 in rice medium showed antiproliferative activity against human glioma U87MG and U251 cells with inhibition rates of 79.68% and 78.27%, respectively. Chemical investigation of this active extract resulted in the isolation of 17 secondary metabolites, including seven new compounds (Figure 1), namely the peniresorcinosides A-E (compounds 1-5), penidifarnesylin A (6), and penipyridinone A (7). Herein, we report the details of the isolation, structure elucidation and antiglioma activity evaluation of all isolated compounds. pounds with potent antiglioma activity, including the polycyclic anthraquinones N-acetyl-N-demethylmayamycin and streptoanthraquinone A from Streptomyces sp. 182SMLY [12], the cyclodepsipeptide streptodepsipeptides P11A and P11B from Streptomyces sp. P11-23B [13], bagremycin C from Streptomyces sp. Q22 [14], pyrrospirone G and penicipyrroether A from Penicillium sp. ZZ380 [15,16], 1-hydroxymethyl-8-hydroxyanthraquinone-3-carboxylic acid from Streptomyces sp. ZZ406 [17], streptoglutarimide H from Streptomyces sp. ZZ741 [18], and the compounds marinacarbolines G and M and caerulomycin N from Actinoalloteichus sp. ZZ1866 [19]. The antiglioma activity of some compounds were related to the downregulation of several important glioma glycolytic enzymes [13,17]. As a part of our ongoing research program to discover novel natural antiglioma products from marine microorganisms, a Penicillium sp. ZZ1750 fungus was isolated from a marine mud sample collected from the Arabian Sea, close to Karachi, Pakistan. An ethyl acetate (EtOAc) extract prepared from a scale-up culture of the strain ZZ1750 in rice medium showed antiproliferative activity against human glioma U87MG and U251 cells with inhibition rates of 79.68% and 78.27%, respectively. Chemical investigation of this active extract resulted in the isolation of 17 secondary metabolites, including seven new compounds (Figure 1), namely the peniresorcinosides A-E (compounds 1-5), penidifarnesylin A (6), and penipyridinone A (7). Herein, we report the details of the isolation, structure elucidation and antiglioma activity evaluation of all isolated compounds.

Results and Discussion
The marine-sourced strain ZZ1750 ( Figure S1, Supplementary Materials) was identified as Penicillium sp. ZZ1750 based on its internal transcribed spacer (ITS) rDNA sequence (563 bp, Figure S2), which was a 100% match to those of six other Penicillium strains (Table S1). An EtOAc extract prepared from a large-scale culture of the strain ZZ1750 in rich medium was separated by column chromatography, followed by high performance liquid chromatography (HPLC) purification, to afford compounds 1-17.
Compound 1 was obtained as a yellowish oil with an optical rotation value of + 76 and a UV absorption at 232 nm. Its molecular formula C30H48O8 was deduced from the HRESIMS (high resolution electrospray ionization mass spectroscopy) ion peak at m/z 559.3248 [M + Na] + (calcd. for C30H48NaO8 + , 559.3247) as well as its 13 C-NMR data. Analyses of its 1 H, 13 C, distortionless enhancement by polarization transfer (DEPT), and heteronuclear multiple quantum correlation (HMQC) spectra showed the presence of ten olefinic carbons, six oxymethines, one oxymethylene, one methine, ten methylenes, and two methyls (Table 1). A double peak signal at δH 6.02 (2H, d, J = 2.0 Hz, H-2 and H-6) showed HMQC correlation with a carbon signal at δC 106.3 (C-2 and C-6) and also showed HMBC

Results and Discussion
The marine-sourced strain ZZ1750 ( Figure S1, Supplementary Materials) was identified as Penicillium sp. ZZ1750 based on its internal transcribed spacer (ITS) rDNA sequence (563 bp, Figure S2), which was a 100% match to those of six other Penicillium strains (Table S1). An EtOAc extract prepared from a large-scale culture of the strain ZZ1750 in rich medium was separated by column chromatography, followed by high performance liquid chromatography (HPLC) purification, to afford compounds 1-17.
Compound 2 was also obtained as a yellowish oil with a similar optical rotation value and UV absorption to those of 1. The molecular formula C 32 H 50 O 9 of 2 was deduced from its HRESIMS ion peak at m/z 601.3346 [M + Na] + , 42 mass units higher than that of 1, corresponding to a -C 2 H 2 O group. The IR spectrum of 2 showed an absorption at 1724 cm −1 , indicating the presence of a carbonyl group. Detailed comparison of the 13 C-and 1 H-NMR spectra of 2 and 1 indicated that their chemical shifts bore a very close resemblance, except for two additional 13 C-NMR signals (δ C 170.2 and 20.6) and one extra 1 H-NMR signal (δ H 1.98, 3H, s) for an acetyl observed in the NMR spectra of 2. The HMBC correlations of H 2 -30 (δ H 4.25, dd, J = 11.7, 1.9 Hz; 3.95, dd, J = 11.7, 7.3) with C-1 (δ C 170.2) and H 3 -2 (δ H 1.98) with C-1 demonstrated the acetyl was at the C-30 position. The difference between the 13 C-NMR chemical shifts for C-30 (∆ +3.2 ppm) between 2 and 1 also supported the location of this acetyl group. Compound 2 was thus elucidated as a new glycosylated alkylresorcinol analogue, named peniresorcinoside B. The 13 C-and 1 H-NMR data (Table 1) were fully assigned based on the HMQC, COSY, and HMBC correlations ( Figure 2). Compound 3 was obtained as a yellowish oil. Its HRESIMS spectrum showed ion peaks at m/z 775.5719 [M + H] + and 797.5543 [M + Na] + , corresponding to a molecular formula C 46 H 78 O 9 , which was 16 carbons, 30 protons, and one oxygen more than that of 1. Careful analyses of the 13 C-and 1 H-NMR spectra of 3 and 1 demonstrated that 3 and 1 shared the same glycosylated alkylresorcinol structural backbone. Compared to the NMR signals of 1, compound 3 showed additional NMR signals for a carbonyl (δ C 172.7), a methyl (δ C 13.9; δ H 0.84, 3H, t, J = 7.0 Hz), and some methylenes, whose NMR signals were high overlapped. Based on these characteristic NMR signals, in consideration of the presence of additional 16 carbons and one oxygen in 3, it was deduced that compound 3 had a structural unit of sixteen-carbon saturated acid (palmitic acid). The HMBC correlations of H 2 -30 (δ H 4.26, 1H, d, J = 10.8; 3.95, 1H, dd, J = 10.8, 7.3 Hz) with C-1 (δ C 172.7) established the linkage of this sixteen-carbon unit. Therefore, compound 3 was elucidated as a new glycosylated alkylresorcinol analogue, and named peniresorcinoside C. Its 13 Cand 1 H-NMR data ( Table 2) were assigned based on the HMQC, COSY, and HMBC correlations ( Figure S4).  30 protons, and one oxygen more than that of 1. Analyses of the 13 C-and 1 H-NMR spectra of 4 and 1 led to the conclusion that 4 also had the same glycosylated alkylresorcinol structural unit of 1. Compared to the NMR signals of 1, compound 4 exhibited additional NMR signals for a carbonyl (δ C 172.7), two pairs of double bonds (δ C 129.7, 129.6, 127.8, 127.7; δ H 5.28, 2H, m, 5.31, 2H, m), a methyl (δ C 13.9; δ H 0.84, 3H, t, J = 7.3 Hz), and some signal-overlapped methylenes. These characteristic NMR signals, together with the fact of additional 18 carbons and one oxygen in 4 mentioned above, suggested that compound 4 had an eighteen-carbon unsaturated fatty acid structural unit. Alkaline hydrolysis of 4 gave peniresorcinoside A (1) as determined by co-HPLC analysis ( Figure S5) with the authentic sample and 9(Z),12(Z)-octadecadienoic acid (linoleic acid, 4a) based on its NMR data (Table S9) and the comparison with the reference data [24,25] as well as co-HPLC analysis ( Figure S6) with standard linoleic acid. The HMBC correlations of H 2 -30 (δ H 4.27, 1H, d, J = 11.2; 3.95, 1H, dd, J = 11.2, 7.5 Hz) with C-1 (δ C 172.7) demonstrated this linoleic acid unit at C-30 position. The structure of 4 was thus identified as a new glycosylated alkylresorcinol analogue, named peniresorcinoside D. Its 13 C-and 1 H-NMR data ( Table 2) were assigned based on the HMQC, COSY, and HMBC correlations ( Figure S4).
The molecular formula C 48 H 80 O 9 of 5 was determined based on its HRESIMS ions at m/z 823.5694 [M + Na] + , two mass units higher than that of 4. Detailed analyses of the 13 C-and 1 H-NMR spectra of 5 and 4 indicated that the structures of both compounds had the same glycosylated alkylresorcinol backbone with a difference in the number of double bonds of the eighteen-carbon unsaturated fatty acid unit. The 13 C-and 1 H-NMR spectra of 5 only exhibited a pair of double bonds (δ C 129.7, 129.6; δ H 5.29, 2H, m), instead of the two pairs of double bonds in 4.
Compound 6 was obtained as orthorhombic crystals from a mixture solvent of EtOAc and CHCl 3 (1:1) and its HRESIMS ion peak at m/z 497.3604 [M + Na] + , corresponding to a molecular formula C 30 H 50 O 4 . However, only 15 carbon signals were observed in the 13 C NMR spectrum, suggesting its symmetrical structure. The 15 carbon signals were assigned for six olefin carbons, two oxymethine carbons, three methylene carbons, and four methyl carbons (Table 3) based on its HMQC spectrum. The half planar structure (C 1 -C 15 ) of 6 was established as a farnesyl derivative by further analyses of its COSY and HMBC correlations (Figure 4). A HMBC correlation of H 2 -12 (δ H 1.92, 2H, m) with C-12 (δ C 27.9, CH 2 ), in consideration of its molecular formula, suggested the whole planer structure of 6, which was further confirmed by its crystal structure ( Figure 5) obtained from X-ray diffraction analysis. However, the data from the X-ray diffraction was not good enough for the assignment of the absolute configuration of 6 because of a poor Flack parameter of 0.3 (3). Therefore, a computational method was applied to assign the absolute configuration of 6 by comparing its experimental electronic circular dichroism (ECD) spectrum with the calculated ECD spectra. The X-ray CIF profile of 6 (5R,8R,5 R,8 R-6) and its enantiomer (5S,8S,5 S,8 S-6) were initially optimized at B3LYP/6-31g (d, p) level in MeOH. The theoretical calculations of ECD were conducted in MeOH using Timedependent Density functional theory (TD-DFT) at the B3LYP/6-311+g (d, p) level. The results ( Figure 6) showed that the experimental ECD spectrum of 6 was agreement with the calculated ECD curve of the model molecule 5R,8R,5 R,8 R-6, indicating a 5R,8R,5 R,8 Rconfiguration for 6. Based on the foregoing evidence, the structure of 6 was determined to correspond to a new dimeric farnesene derivative, named penidifarnesylin A. Its 13 C and 1 H NMR data (Table 3) were assigned according to the HMQC, COSY, and HMBC correlations (Figure 4).         13 C-and HMQC NMR spectra demonstrated the presence of two carbonyls, ten olefin carbons, eight olefin protons, three oxymethines, one oxymethylene, one methoxy, one methine, three methylenes, and four methyls ( Table 4). The two carbonyls and ten olefin carbons (five pairs of double bonds) accounted for seven out of the nine degrees of unsaturation required by the molecular formula, suggesting that 7 had a structure with two rings. Two COSY spin systems of H2-1/H-2(H3-15)/H-3/H-4/H-5 and H-7/H-8/H-9/H-10/H-11/H2-12/H2-13/H3-14 and the HMBC correlations as described in Figure 7 determined that the first cycle-related partial structure (7a, Figure 7) was a tetrahydropyran derivative. Similarly, the second     13 C-and HMQC NMR spectra demonstrated the presence of two carbonyls, ten olefin carbons, eight olefin protons, three oxymethines, one oxymethylene, one methoxy, one methine, three methylenes, and four methyls ( Table 4). The two carbonyls and ten olefin carbons (five pairs of double bonds) accounted for seven out of the nine degrees of unsaturation required by the molecular formula, suggesting that 7 had a structure with two rings. Two COSY spin systems of H2-1/H-2(H3-15)/H-3/H-4/H-5 and H-7/H-8/H-9/H-10/H-11/H2-12/H2-13/H3-14 and the HMBC correlations as described in Figure 7 determined that the first cycle-related partial structure (7a, Figure 7) was a tetrahydropyran derivative. Similarly, the second   13 C-and HMQC NMR spectra demonstrated the presence of two carbonyls, ten olefin carbons, eight olefin protons, three oxymethines, one oxymethylene, one methoxy, one methine, three methylenes, and four methyls ( Table 4). The two carbonyls and ten olefin carbons (five pairs of double bonds) accounted for seven out of the nine degrees of unsaturation required by the molecular formula, suggesting that 7 had a structure with two rings. Two COSY spin systems of H 2 -1/H-2(H 3 -15)/H-3/H-4/H-5 and H-7/H-8/H-9/H-10/H- Figure 7 determined that the first cycle-related partial structure (7a, Figure 7) was a tetrahydropyran derivative. Similarly, the second cycle-related partial structure (7b) was established as a pyridinone derivative by the COSY correlation of H-23 (δ H 5   . These assigned configurations for 7 were the same as those of restrictinol (7c) [28,29]. Based on the forgoing evidences, the structure of 7 was elucidated as a new restrictinol analogue with a unique pyridinone functionality, named penipyridinone A. Its 13 C-and 1 H-NMR data (Table 4) were assigned based on the HMQC, COSY, HMBC, and NOESY correlations (Figure 7).
All isolated compounds 1-17 were tested for their antiproliferative activity against human glioma U87MG and U251 cells by using the Sulforhodamine B (SRB) assay [42]. Doxorubicin (DOX, an anticancer drug) was used as a positive control. The results (Table 5) showed that peniresorcinosides A (1) and B (2) had potent antiglioma activity with IC 50 values of 4.0 and 5.6 µM for U87MG cells and 14.1 and 9.8 µM for U251 cells, respectively. Penidifarnesylin A (6) also showed antiproliferative activity with IC 50 values of 5.9 µM against U87MG cells and 27.6 µM against U251 cells. However, peniresorcinosides C-E (3-5) with a long-chain fatty acid unit only showed antiproliferative activity against U87MG cells with IC 50 values of 53.0, 19.4, and 22.1 µM, respectively. Other tested compounds were inactive at a concentration of 50 µM.
Marine-derived Penicillium fungi are still important sources for the discovery of novel bioactive natural products. The current study described the isolation and structural elucidation of 17 metabolites, including new glycosylated alkylresorcinols of peniresorcinosides A-E (1-5), penidifarnesylin A (6), and penipyridinone A (7) as well as some known indole alkaloids with complicated structures, from the marine fungus Penicillium sp. ZZ1750 cultured in rice medium.
Alkylresorcinols are resorcinol units alkylated with a long odd-numbered carbon chain and had high levels (over 500 µg/g) in wheat, rye, and triticale [43]. It has been reported that some of alkylresorcinols exhibited antimicrobial, antiparasitic, and cytotoxic activities [44]. Resorcinosides A and B [45], recently isolated from the marine fungus Penicillium janthinellum, are the first reported alkylresorcinol derivatives containing a glucose moiety linked to a hydroxy group of the alkyl side chain. Peniresorcinosides A-E (1)(2)(3)(4)(5) are second example of this type of rare glycosylated alkylresorcinols. Peniresorcinosides C-E (3-5) have more complicated structures, with a long-chain fatty acid moiety attached to the C-6 position of the glucosyl unit. Evaluation of the antiglioma activity showed that peniresorcinosides A (1) and B (2) had potent antiproliferative activity against both glioma U87MG and U251 cells, while peniresorcinosides C-E (3-5) only exhibited moderate antiglioma activity against U87MG cells. The cyclotryprostatin [34,46], fumiquinazoline [35,[46][47][48], and verruculogen [31,34] indole alkaloids were most frequently isolated from Aspergillus and Penicillium fungi. Some of them have reported antibacterial, antifungal, cytotoxic, and antidiabetic activities [48,49]. In this study, seven such known indole alkaloids (compounds 9-15) were obtained from the metabolites produced by the marine-sourced Penicillium fungus ZZ1750. Fumiquinazoline C (13) was previously reported to have moderate cytotoxicity against murine lymphocytic leukemia P388 cells [47]. However, none of these isolated indole alkaloids 9-15 were active against human glioma U87MG and U251 cells at a concentration of 50 µM.

Isolation and Taxonomic Identification of Strain ZZ1750
The strain ZZ1750 was isolated from a sample of marine mud, which was collected from the Arabian Sea near Karachi, Sindh, Pakistan in January 2019. Briefly, the mud sample was air dried at 28 • C for 7 days and the dried sample (1.0 g) was diluted with sterile water to make up the dilutions of 10 −2 , 10 −3 , and 10 −4 g/mL. Each dilution (200 µL) was transferred on the surface of ten different solid media of B, BS, PDA, PDAS, E, ES, ISP-2, ISP-2S, ISP-4, and ISP-4S on Petri dishes and then incubated at 28 • C for 14 days. The single colony of ZZ1750 was picked from the 10 −2 g/mL suspension in PDA medium and then transferred to another PDA medium in dish. After growth for another 7 days at 28 • C, the single pure colony (ZZ1750) that grew well was transferred onto PDA slant medium and stored at 4 • C for further study.
The ITS rDNA sequence analysis of strain ZZ1750 was conducted by Legenomics (Hangzhou Lizhen Biotechnology Co., Ltd., Hangzhou, China). The ITS rDNA sequence was compared to those in the GenBank database using the nucleotide Basic Local Alignment Search Tool (BLAST). The ITS rDNA sequence of strain ZZ1750 has been deposited in GenBank (accession number: MT159428). The strain Penicillium sp. ZZ1750 was preserved at the Laboratory of Institute of Marine Biology and Pharmacology, Ocean College, Zhoushan campus, Zhejiang University, Zhoushan, China.

Mass Culture of Strain ZZ1750
The colony of strain ZZ1750 from the PDA slant medium was inoculated into a 500 mL Erlenmeyer flask, which contained 250 mL potato dextrose broth (PDB) medium and then incubated for 3 days in a shaker (180 rpm, 28 • C) to produce the seed broth ( Figure S1). The seed broth (5 mL) was transferred into rice medium (40 g rice, 60 mL 3.5% sea salt solution) in 500 mL Erlenmeyer flask and then all these flasks were incubated at 28 • C for 30 days in a static state. For this study, a total of 210 cultured flasks were prepared.

Extraction and Isolation of Compounds 1-17
The culture of strain ZZ1750 in rice medium in each flask was extracted with EtOAc (250 mL) three times. The combined EtOAc extract was dried in vacuo to give an extract (100 g). This extract was fractionated on a column (160 × 10 cm) of silica gel (1600 g) eluting with a mixture of cyclohexane and EtOAc in different ratios (10:1, 5:1, 2:1, 1:1, 1:2, each 1000 mL) to give four fractions of Frs. A-D based on the results of TLC analyses.
Fr. A was separated by using an Agilent 1260 HPLC system equipped with an Agilent Zorbax SB-C 18 column (250 × 9. Finally, Fr. D was separated by using the Zorbax SB-C 18 (8)  were used in all calculations. The final R 1 was 0.0877 (I > 2σ (I)) and wR 2 was 0.2366 (all data). The crystal data and structure refinement parameters of penidifarnesylin A (6) were also reported in Table S6. Crystallographic data of penidifarnesylin A (6) has been deposited at the Cambridge Crystallographic Data Centre (CCDC Number: 1976942

Enzymatic Hydrolysis of Peniresorcinoside A (1)
Peniresorcinoside A (1, 3.0 mg) was equilibrated at 37 • C for 5 min in a solution of water (2.0 mL) and 0.1 M phosphate buffer (pH = 7.0, 2.0 mL) and then 0.5 mL of α-glucosidase solution (0.2 M potassium phosphate solution containing 1 mM EDTA and 0.05% Tween-20, pH 7.0) was added. The mixture was incubated at 37 • C for 1 h and then 2.0 mL of 0.2 M Na 2 CO 3 solution were added to terminate the enzymatic reaction. The enzymatic product was extracted with EtOAc (each 5 mL) three times to give an EtOAc extract and a water solution. The EtOAc extract was separated on an Agilent Zorbax SB-C18 column (250 × 9.4 mm, 5 µm; mobile phase: MeOH/H 2 O, 85/15; flow rate: 1.0 mL/min, UV detection: 210 nm) to furnish 1a (1.8 mg, t R 37.4 min). The aqueous solution was dried under reduced pressure to afford a residue which was first treated with 10 mg hydroxylamine hydrochloride in 2 mL pyridine at 90 • C for 30 min in a water bath and then mixed with 2 mL acetic anhydride at 90 • C for 1 h in a water bath. Finally, the reaction products were dried in vacuo and dissolved in 2 mL chloroform for GC analysis. The aldonitrile acetate of sugar in 1 was identified as the aldonitrile acetate of D-glucose (t R 7.20 min) ( Figure S3) by GC analysis with aldonitrile acetates of D-glucose (t R 7.20 min), L-glucose (t R 7.28 min), D-galactose (t R 7.40 min), and L-galactose (t R 7.45 min) as references. Compound

13 C-and 1 H-NMR Calculations
Monte Carlo conformational searches were carried out by means of the Spartan's 10 software using Merck Molecular Force Field (MMFF). The conformers with Boltzmannpopulation of over 5% for NMR calculations were initially optimized at B3LYP/6-31g (d, p) level in MeOH. Gauge-independent atomic orbital (GIAO) calculations of 13 C and 1 H NMR chemical shifts were accomplished by density functional theory (DFT) at the mPWLPW91-SCRF (DMSO)/6-311+g (d, p) level with the PCM solvent continuum model in Gaussian 09 software. The calculated NMR data of the lowest energy conformers for model molecules 20R,21S-1a, 20S,21S-1a, 20R,21R-1a, and 20S,21R-1a were averaged according to the Boltzmann distribution theory and their relative Gibbs free energy. The 13 C-NMR and 1 H-NMR chemical shifts for TMS were calculated by the same protocol as reported in the reference [23] and the experimental and calculated data of the isomeric compounds were analyzed by the improved probability DP4 + method [23]. A significant higher DP4 + probability score of the model molecules suggested the correctness of its configuration.

ECD Calculations
The X-ray CIF profile of 5S,8S,5 S,8 S-6 was initially optimized at B3LYP/6-31g (d, p) level in MeOH. The theoretical calculation of ECD was conducted in MeOH using Timedependent Density functional theory (TD-DFT) at the B3LYP/6-311+g (d, p) level. Under the same conditions, the enantiomer 5R,8R,5 R,8 R-6 was also calculated. Rotatory strengths for a total of 30 excited states were calculated. ECD spectra were generated using the program SpecDis 1.6 (University of Würzburg, Würzburg, Germany) and GraphPad Prism 5 (University of California San Diego, San Diego, CA, USA) from dipole-length rotational strengths by applying Gaussian band shapes with sigma = 0.2 eV.

Sulforhodamine B (SRB) Assay
Human glioma U87MG and U251 cells were cultured in Minimum Essential Medium (MEM, Gibco, Thermo Fisher Scientific Inc., Waltham, MA, USA) and Dulbecco's Modified Eagle Medium (DMEM, Gibco) with 10% FBS, respectively. All cells were incubated at 37 • C in a humidified incubator with 5% CO 2 incubator. Cells from the third repeated culture were used for experiments. The SRB assay as describe in previous publication [42] was used to evaluate the antiproliferative activity of all isolated compounds 1-17 against human glioma U87MG and C251 cells. Doxorubicin (DOX) was used as a positive control. Briefly, glioma cells in logarithmic growth (4 × 10 3 cells/well) were plated in a 96-well plate, treated with different concentrations of each tested compound after 24 h of cells adhesion, and then incubated for 72 h. After that, the treated cells were fixed with 50 µL of 50% cold TCA (trichloroacetic acid) solution at 4 • C for 1 h, washed with distilled water five times, and then dried at 37 • C in a drying oven. The dried cells were stained with 50 µL of 0.4% SRB for 15 min, rinsed with 1% glacial acetic acid solution five times, then dried at 37 • C. Finnally, the dried dye was dissolved in 100 µL of 10 mM Tris buffer and the optical density (OD) value measured at 515 nm on a microplate reader (BioTech, Winooski, VT, USA). The cell viability (%) was calculated from the formula of T OD /C OD × 100% (T OD : OD value of tested compound; C OD : OD value of negative control) and IC 50 value was obtained based on the cell viability (%) by logistic calculation using SPSS software.

Conclusions
Chemical investigation of the metabolites produced by the marine-sourced fungus Penicillium sp. ZZ1750 cultured in rice medium resulted in the characterization of seven new compounds: peniresorcinosides A-E (1-5), penidifarnesylin A (6), and penipyridinone A (7), which enrich the structural diversity of the metabolites of marine Penicillium fungi. Peniresorcinosides A (1) and C (2) are rare glycosylated alkylresorcinols and had potent antiproliferative activity against both human glioma U87MG and U251 cells and might be the main components responsible for the antiglioma activity of the crude extract prepared the culture of strain ZZ1750 in rice medium.
Author Contributions: K.Y. and S.K. conducted the isolation and culture of stain ZZ1750, the isolation and structural elucidation of compounds, and the bioactive assay; B.W. and Z.Z. designed and supervised the experiments and wrote the manuscript. All authors have read and agreed to the published version of the manuscript.