Paspalines C–D and Paxillines B–D: New Indole Diterpenoids from Penicillium brefeldianum WZW-F-69

Five new indole diterpenoids named paspaline C–D (1–2) and paxilline B–D (3–5), as well as eleven known analogues (6–16), were identified from fungus Penicillium brefeldianum strain WZW-F-69, which was isolated from an abalone aquaculture base in Fujian province, China. Their structures were elucidated mainly through 1D- and 2D-NMR spectra analysis and ECD comparison. Compound 1 has a 6/5/5/6/6/8 hexacyclic ring system bearing 2,2-dimethyl-1,3-dioxocane, which is rare in natural products. Compound 2 has an unusual open F-ring structure. The cytotoxic activities against 10 cancer cell lines and antimicrobial activities against model bacteria and fungi of all compounds were assayed. No compound showed antimicrobial activity, but at a concentration of 1 μM, compounds 1 and 6 exhibited the highest inhibition rates of 71.2% and 83.4% against JeKo-1 cells and U2OS cells, respectively.


Introduction
Indole diterpenoids are a family of secondary metabolites featuring a common core structure comprising an indole derived from indole-3-glycerol phosphate, and a cyclic diterpene skeleton derived from geranylgeranyl diphosphate. A select group of ascomycetous fungi, including aspergillus, penicillium, and zygomycetous fungi, is the main producer [1,2]. They were known initially for tremorgenic and neurotoxic activities partly due to their inhibition of potassium ion channels in the nervous systems [3,4], but in recent years, more diverse bioactivities, such as acyl-CoA:cholesterol acyltransferase (ACAT) inhibitors [5], M phase cell cycle inhibitors [6], and in vitro anti-proliferative, anti-insetan activities [7], etc., were discovered, which indicated their potency as drug lead compounds. Among the known indole diterpenoids, paspaline-type and paxiline-type congeners are the largest classes with the most multitudinous structures [8]. Paspline (6) is the simplest member of complex indole diterpenoids, and paxilline is the proposed precursor of terpendoles and other indole ditrpenoids, such as lotirems and janthitrems [9].
During our searching for bioactive natural compounds from marine fungi, one fungus, Penicillium brefeldianum, strain WZW-F-69, was isolated from an aquaculture base of abalone at Fujian province, China. From the culture, 16 indole diterpenoids, most of which belonged to the paspaline and paxiline types, were identified; thereinto, 5 were elucidated for the first time ( Figure 1). Their cytotoxic and antimicrobial activities were tested, some of them showed to be biologically active. Herein, the structural elucidation and bioassay assay are described.
of them showed to be biologically active. Herein, the structural elucidation and bioassay assay are described.

Structure Elucidation of the New Compounds
The ethyl acetate extract of the fermentation broth of WZW-F-69 was chromatographed on preparative HPLC columns, Sephadex LH-20 to give compounds 1-16.
The H-H COSY from H-20 to H-23, as well as the HMBC correlations ( Figure 2) from H-1 to C-2/C-18/C-19/C-24, hinted at the presence of a 2,3-disubstituted indole residue (ring A and B). The indole molecule had six degrees of unsaturation; the remaining four degrees of unsaturation indicated that compound 1 has a hexacyclic skeleton. Further analysis of the HMBC correlation from H3-25 to C-2/C-3/C-4/C-16 and from H-17 to C-2/C-3/C-16/C-18 revealed that ring C was ortho-fused with ring B at C-2 and C-18. Ring D and ring E were established mainly by the COSY correlations from H-13 to H-15 through H-14, and HMBC correlations from H3-26 to C-3/C-4/ C-5/C-13, as well as H3-30 to C-7/C-11/C-12/C-13. A sub-structure featuring a 6/5/5/6/6-fused system (ring A-E) search resulted in many hits, most of which were indole diterpenoids, mainly the derivatives of paspaline (6), which was co-isolated with 1. Detailed comparison of the NMR data between 1 and 6 showed that compound 1 had an additional 2-oxypropyl unit. The observed HMBC correlations from H-9 to C-31 and from H-32 to C-7 established a 2,2-dimethyl-1,3dioxocane ring (ring F), so the planar structure of compound 1 was solved. It had a 6/5/5/6/6/8 hexacyclic system.

Structure Elucidation of the New Compounds
The ethyl acetate extract of the fermentation broth of WZW-F-69 was chromatographed on preparative HPLC columns, Sephadex LH-20 to give compounds 1-16.
The relative configuration of 2 was established by analysis of the NOE spectrum (  . Comparison of the 1 H-and 13 C-NMR spectra of 1 and 2 showed two similar sets of data, the main differences being C-7 and C-31. For compound 1, C-31 (δ106.5 ppm) was an aldehyde acetal carbon, and H-7 was a typical oxygenous chemical shift (δ3.59 ppm), while in compound 2, C-31 (δ170.7 ppm) was a carbonyl carbon, and the chemical shift of H-7 (δ4.80 ppm) shifted downfield obviously. Detailed analysis of 2D-NMR spectra ( Figure 2) revealed that compound 2 had an identical 6/5/5/6/6 system, but had no ring F; an acetoxyl group and a 3, 4-dihydroxy-4-methylpentyl group were attached at C-7 and C-12 with ring E, respectively, so its planar structure was established.
The relative configuration of 2 was established by analysis of the NOE spectrum ( Figure 3). The α orientations of H-7/H-13/H 3 -25 and β orientations of H-16/H 3 -26/H 3 -30 were the same as those in compound 1. The H-7/H-9 correlation helped in confirming the configuration of C-9 as S*.
The ECD experiment was used to determine the absolute configuration (AC) of compounds 1 and 2 ( Figure 4). A negative cotton curve at 230-250 nm corresponds to the AC of 3S, 4S, 13R and 16S of indole diterpenoids with the 6/5/5/6/6 skeleton of ring A-E as the π→π* transition of the indole ring [10]. Compounds 1, 2 and 6 had the same negative at about 230 nm ( Figure 4) and positive at about 260 nm. Based on the result and biosynthetic considerations, the AC of compounds 1 and 2 was established as 3S, 4S, 7S, 9S, 12S, 13R, and 16S. This was confirmed by single crystal X-ray crystallographic analysis of compound 6 ( Figure S69).    Table 2), except for the position of the isopentenyl group. Detailed analysis of 2D-NMR spectra (Table S4) revealed that for compound 4, the isopentenyl is at C-21, so the planar structure of 4 is 21-prenylated -27-O-acetyl paxilline.
The relative configurations of 3 and 4 can be inferred through NOE spectra analysis. Two compounds had the same NOE correlations from H-16 to H 3 -26, from H-7 to H-9, and H 3 -25. Their ECD spectra are highly consistent with those of compound 7 ( Figure S63), indicating that compounds 3 and 4 have the absolute configuration of 3S, 4R, 7S, 9R, 13S, and 16S.
The relative structure of compound 5 was determined by NOE through connection from H-9 to H-13, H 3 -30 and H 3 -25, which indicated the same orientation of these atoms (Table S5). Compound 5 showed a negative cotton effect at 200-250 nm in the ECD spectrum, that corresponded to that of paxilline. Therefore, the absolute configuration of compound 5 was assigned as 3S, 4S, 7R, 9R, 12R, 16S.

Bioactivities of Compounds
Compounds 1-16 were evaluated for their antimicrobial and cytotoxic activities with the cell lines and microbial strains in our lab. All tested compounds showed no activities against Escherichia coli CMCC44103, Staphlococcus aures CMCC26003, Aspergillus niger ACCC3005, and Candida albicans AS2.538 by the disk diffusion method at 10 mg/mL, nor cytotoxic activity at 1 μM against MDA-MB-231 (human breast cancer cells), A375 (human melanoma cells), BGC-823 (human pancreatic cancer cells), SKGT4 and Kyle 450 (two human esophagus cancer cell) lines. However, compounds 1 and 6 exhibited moderate inhibitory activity against HepG-2 (human hepatoma carcinoma cells), U2OS (human bond on ring F; compounds 3, 4, 8 and 9 are the 3rd group which have one isopentene group on the indole ring; and compounds 12, 15 and 16 have two isopentyl substituent groups on the indole ring. The biogenetic pathway of the five new compounds is proposed in Figure 6. Emindole SB is likely a biosynthetic precursor of 6 and other advanced indole diterpenoids.

Bioactivities of Compounds
Compounds 1-16 were evaluated for their antimicrobial and cytotoxic activities with the cell lines and microbial strains in our lab. All tested compounds showed no activities against Escherichia coli CMCC44103, Staphlococcus aures CMCC26003, Aspergillus niger ACCC3005, and Candida albicans AS2.538 by the disk diffusion method at 10 mg/mL, nor cytotoxic activity at 1 μM against MDA-MB-231 (human breast cancer cells), A375 (human melanoma cells), BGC-823 (human pancreatic cancer cells), SKGT4 and Kyle 450 (two human esophagus cancer cell) lines. However, compounds 1 and 6 exhibited moderate inhibitory activity against HepG-2 (human hepatoma carcinoma cells), U2OS (human

Bioactivities of Compounds
Compounds 1-16 were evaluated for their antimicrobial and cytotoxic activities with the cell lines and microbial strains in our lab. All tested compounds showed no activities against Escherichia coli CMCC44103, Staphlococcus aures CMCC26003, Aspergillus niger ACCC3005, and Candida albicans AS2.538 by the disk diffusion method at 10 mg/mL, nor cytotoxic activity at 1 µM against MDA-MB-231 (human breast cancer cells), A375 (human melanoma cells), BGC-823 (human pancreatic cancer cells), SKGT4 and Kyle 450 (two human esophagus cancer cell) lines. However, compounds 1 and 6 exhibited moderate inhibitory activity against HepG-2 (human hepatoma carcinoma cells), U2OS (human osteosarcoma cells), MCF-7 (human breast cancer cells), Jeko-1 (human mantle cell lymphoma cells) and HL-60 (human acute promyelocytic leukemia cells). Their average inhibition rates were in the range of 50% to 80% at a concentration of 1 µM (Table 3). Our result is in line with the literature [3,22] that paspline-and paxilline-type indole diterpenoids seldom have anti-microbial activity, while some of them have cytotoxic activity, which differs from different cancer cell lines.

Fungal Material
The fungal strain WZW-F-69 was isolated from soil near an abalone aquaculture base of Fujian province, China (N 23 • 47 727 , E 117 • 29 079 ), in August 2015. After being purified, the strain was identified with the ITS gene sequence (Sequence S1) to be Penicillium brefeldianum (100% consistent with Genebank KM243921.1). The strain was preserved in our lab at −80 • C.

Fermentation and Extraction
For chemical investigation, the fungal strain WZW-F-69 was cultivated in 100 mL liquid seawater potato-dextrose medium (including extract of potato 200 g, glucose 20 g, sea salt 30 g, water 1 L), 220 rpm, at 28 • C for 5-7 days to prepare the seed culture. Then, 5 mL of the seed solution was inoculated on solid rice culture media (150 g rice in 150 mL water) in a 2.5 L stainless steel box. A total of 3 kg of the rice media was incubated at 28 • C for 20 days. The rice culture media were collected and soaked in ethyl acetate (EA) overnight. After the solvent was removed in vacuum, it yielded a crude extract (20 g). The crude extract was redissolved in MeOH: petroleum ether (PE, 1:1, v/v) three times. The combined MeOH extract (10 g) was grouped by the RP-18 silica gel column mid pressure chromatography (28 × 4 cm, 170 g), and the elute solution changed from 100% H 2 O to 100% MeOH in 2 h, 30 mL/min.

Antimicrobial Activity
The antimicrobial activity was evaluated with the disk diffusion methodology [23]. Four indicator organisms (Staphococcus aureus CMCC26003, Escherichia coli CMCC44103, Aspergillus niger ACCC3005, Canidida albicans AS2.538) were used. The spores at 1×10 6 /mL were poured onto culture media on a Petri dish (ϕ = 9 cm): PDA medium for fungal indicators, and Luria-Bertain (LB) medium for bacterial indicators. The sterilized filter paper (ϕ = 5 cm) with 5 µL of compound solution at 10 mg/mL was put on the surface of the culture medium; 24-48 h later, the inhibition diameters were measured. Amphotricin B and gentamicin at 1 mg/mL were used as positive controls for the fungal and bacterial indicators, respectively.

Cytotoxic Activity
A375, MDA-MB-231, HepG2, BGC823, U2OS and MCF-7 cell lines were grown in DMEM medium. SKGT4, Kyse450, JeKo-1 and HL60 cells were grown in RPMI1640 medium. Two media were supplemented with 10% FBS and 1% penicillin or streptomycin. Then, 198 µL of the medium with about 5000 cancer cells was plated in each well of a 96-well plate. Then, 2 µL of the test compound at 10 mM in DMSO was added in triplicate. Forty-eight hours later, the cell viability was determined using CellTiter 96 ® AQ ueous Assay Reagents according to the instruction manual at 490 nm. Data were normalized to the control group (DMSO).

Conclusions
In summary, 5 new indole diterpenoids (1-5) and 11 known congeners (6-16) were identified from sea-mud-derived fungal Penicillium Brefeldianum WZW-F-69. Even though the main structural elements resemble the reported indole diterpenes, paspaline C (1) has a rare 2,2-dimethyl-1,3-dioxocane ring F, while paspaline D (2) has an unusual open ring F structure. Paxilline B (3) and paxilline C (4) are isopentenyl ring A derivatives of paxilline, while paxilline D (5) has an aldehyde acetyl carbon (C-7), which makes them chemically unique. Although the AC of new compounds was established mainly by the comparison of ECD spectra with known compounds 6 and 7, the consistence with known compounds, such as 6-hydroxylpaspslinine [3], 4a-demethylpaspaline-4a-carboxylic acid [7], whose AC was confirmed with experimental and calculated ECD, supported our assignment. Compounds 1 and 6 exhibited cytotoxic activity against several cancer cell lines. In summary, the identification of diverse structural compounds from WZW-F-69 illustrates the huge potential of finding more new bioactive lead compounds from marine fungi for future research.

Conflicts of Interest:
The authors declare no conflict of interest.