Prenylated Indolediketopiperazine Peroxides and Related Homologues from the Marine Sediment-Derived Fungus Penicillium brefeldianum SD-273

Three new indolediketopiperazine peroxides, namely, 24-hydroxyverruculogen (1), 26-hydroxyverruculogen (2), and 13-O-prenyl-26-hydroxyverruculogen (3), along with four known homologues (4–7), were isolated and identified from the culture extract of the marine sediment-derived fungus Penicillium brefeldianum SD-273. Their structures were determined based on the extensive spectroscopic analysis and compound 1 was confirmed by X-ray crystallographic analysis. The absolute configuration of compounds 1–3 was determined using chiral HPLC analysis of their acidic hydrolysates. Each of the isolated compounds was evaluated for antibacterial and cytotoxic activity as well as brine shrimp (Artemia salina) lethality.


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Some of these compounds showed tremor-producing, cytotoxic, antibacterial, and brine shrimp lethal activity [3][4][5][6][7]. In our continuing investigation aimed to explore new and bioactive secondary metabolites from marine-derived fungi [9][10][11][12][13][14][15], we recently focused on a fungal strain Penicillium brefeldianum SD-273 that was isolated from the sediment samples collected from the estuary of the Pearl River in the South China Sea. The EtOAc extract of the fermentation broth showed moderate brine shrimp (Artemia salina) lethality in the preliminary assays. Further investigation of the culture extracts of this fungus resulted in the isolation of three new indolediketopiperazine peroxides (1)(2)(3) and four known homologues (4-7) (Figure 1). The structure of these compounds was determined by spectroscopic analysis and compound 1 was confirmed by X-ray crystallographic experiment. The absolute configuration of compounds 1-3 was determined by chiral HPLC analysis of their acidic hydrolysates. It should be noted that, based on our 1D and 2D NMR data, the original assignment of the carbon signals for the two methyls in the isoprenyl moiety of verruculogen (4) [4] should be interchanged. This paper describes the isolation, structure determination, and bioactivity evaluation of the isolated compounds.

Structure Elucidation of the New Compounds 1-3
The cultured broth and mycelia of the fungus were exhaustively extracted with EtOAc and MeOH, respectively. Since the HPLC and TLC profiles of the two extracts were nearly identical, they were combined before further separation. The combined extract was fractionated by a combination of column chromatography (CC) on Si gel, reversed-phase Si gel C18, and Sephadex LH-20 as well as by semi-preparative HPLC and preparative thin layer chromatography (pTLC), to yield seven indolediketopiperazine derivatives (1-7) (Figure 1).
Compound 1 was obtained as colorless crystals. The HRESIMS experiment established its molecular formula C 27 H 33 N 3 O 8 . Inspection of the 1 H-, 13 C-, and DEPT NMR data of 1 revealed the presence of four methyls (with one O-methyl group), five sp 3 methylenes (with one oxygenated), four sp 3 and four sp 2 methines, and ten quaternary carbons (with two oxygenated sp 3 and six sp 2 carbons and two ester/amide carbons), as well as three exchangeable protons ( Table 1). The 1 H-and 13 C-NMR data assignments matched well with those of the corresponding signals for verruculogen (4), a tremorgenic mycotoxin peroxide isolated from peanuts-derived fungus Penicillium verruculosum [4,5], except for the presence of the C-24 hydroxy group, which was consistent with the difference in the molecular formula. This difference was supported by the fact that the NMR signals for one of the two methyls in the prenyl moiety of 4 [4,5] were replaced by the downfield oxygenated CH 2 signals at δ H 3.80/δ C 65.2 (CH 2 -24) in the NMR spectra of 1 ( Table 1). The HMBC correlations from H-22 to C-24 and C-25, from H 2 -24 to C-22 and C-23, and from H 3 -25 to C-22, C-23, and C-24 verified the above deduction ( Figure 2). The observed NOE correlations from H-26α to H-3 and H-6 and from H-3 to H-22 indicated that these protons located on the same face of the molecule (Figure 3). In addition, NOE correlation from H-22 to the proton of 24-OH established the E-geometry for the double bond at C-22. However, the relative configuration for 12-OH and 13-OH was not assigned since no diagnostic NOE correlations could be detected.  To unambiguously assign the structure, a single X-ray diffraction study was performed. However, compound 1 was difficult to get suitable crystal for X-ray analysis and after many attempts, X-ray quality crystals were obtained by slow evaporation of a solution of 1 in CHCl 3 -MeOH (1:1). The X-ray diffraction crystallographic analysis confirmed the structure and relative configuration for 1 (Figure 4).
The absolute configuration of 1 was deduced after chiral HPLC analysis of the degradation products of the acidic hydrolysate. The HPLC profile of the hydrolysate was compared with authentic standard to establish the configuration of the amino acid-derived unit as L-proline ( Figure 5), corresponding to the S-configuration for C-6. The absolute configuration of compound 1 was therefore assigned to be 3S, 12R, 13S, and 21R. The structure for compound 1 was finally determined and it was named as 24-hydroxyverruculogen. Compound 2 was isolated as a pale amorphous solid. Its molecular formula was demonstrated as C 27 H 33 N 3 O 8 by HRESIMS data. Analysis of its 1 H and 13 C NMR spectral data ( Table 1) with those of verruculogen (4) indicated that both compounds shared the same indolediketopiperazine skeleton [4,5]. The main difference between the two compounds was the methylene carbon resonating at δ C 45.3 (C-26, this carbon was numbered as C-19 in the reference 4) in the 13 C NMR spectrum of 4 [4] being replaced by an oxygenated methine (δ C 80.0, C-26) in that of 2. This difference was verified by the 1 H-1 H COSY correlations from oxymethine proton H-26 to H-3 (δ H 6.13, d, J = 8.8 Hz) and to the exchangeable proton signal at δ H 5.52 (br s, 26-OH). The observed HMBC correlations from H-3, H 3 -28, and H 3 -29 to C-26 ( Figure 2) supported this deduction. Other 1 H-1 H COSY and HMBC correlations confirmed the structure of 2 ( Figure 2). However, the remaining two exchangeable protons resonating at δ H 5.26 and 6.32, which belonging to 12-OH and 13-OH, could not be unambiguously ascribed since they didn't show any correlations in the 2D NMR spectra.
The relative configuration of compound 2 was determined by NOESY experiment, proton coupling constant, and NMR data comparison. The trans-relationship between H-3 and H-26 was deduced on the basis of large coupling constant (J 3,26 = 8.8 Hz) and by the fact that there is no NOE correlation could be detected among them in the NOESY spectrum. The NOE correlations from the proton of 26-OH to the protons of 12-OH and 13-OH implied the same direction of these OH groups, while the observed NOE correlation from H-3 to the olefinic H-22 indicated the same orientation of H-3 and CH-22 ( Figure 2). Unfortunately, the NOESY data for compound 2 proved to be insufficient to solve the relative configuration at C-6. However, based on the fact of the very similar NMR data of H-6 to H-9 and of C-6 to C-9 (Table 1) in 1 and 2, as well as from the biogenetic consideration, the configuration of H-6 was assigned as α-orientation, the same as that of compound 1.
The absolute configuration of 2 was also determined by amino acid analysis of its acidic hydrolysate ( Figure 5), which assigned S-configuration for C-6. The other chiral centers in 2 were therefore deduced as 3R, 12R, 13S, 21R, and 26S. The structure for compound 2 was thus determined and it was named as 26-hydroxyverruculogen.

Biological Activities of the Isolated Compounds
The isolated compounds 1-7 were examined for antibacterial, cytotoxicity, and brine shrimp lethality. None of them showed potent antibacterial activity against two bacteria (Escherichia coli and Staphyloccocus aureus), or cytotoxicity against eight tumor cell lines (B16, HuH-7, SW-1990, Hela, Du145, H460, MCF-7, and SGC-7901). However, compound 3 showed potent lethality against brine shrimp (Artemia salina), with LD 50 value of 9.44 μΜ, while the positive control colchicine had the LD 50 value of 99.0 μΜ, and the activity might be related to the prenyl substitution at 13-OH.

General
The melting point was determined with an SGW X-4 micro-melting-point apparatus. The optical rotations were measured on an Optical Activity A-55 polarimeter. UV data were obtained on a Lengguang Gold S54 spectrophotometer. The 1 H, 13 C, and 2D NMR spectroscopic data were recorded on Bruker Advance 500 spectrometer. Mass spectra were measured on a VG Autospec 3000 mass spectrometer. HPLC analysis was performed using a Dionex HPLC System equipped with a P680 pump, an ASI-100 automated sample injector, a TCC-100 column oven, a UV-DAD 340U detector, and a Dionex Acclaim ODS column (4.6 × 250 mm, 5 μm). Semi-preparative HPLC was operated using a Dionex UltiMate U3000 system using an Agilent Prep RP-18 column (21.

Fungal Material
The fungus Penicillium brefeldianum SD-273 was isolated from a sediment sample collected from the estuary of the Pearl River in South China Sea at a depth of 100 m, in October 2010. The fungal strain grew fast on potato dextrose agar plate and the pale yellow mycelia with few spores could be observed in about 3 days at 28 °C. Fungal identification was carried out using a molecular biological protocol by DNA amplification and sequencing of the ITS region as well as by calmodulin (cmd) sequencing as described previously [16]. The sequence data derived from the fungal strain have been submitted to and deposited at GenBank under accession no. JQ306332 (ITS) and KJ160447 (cmd). A BLAST search result showed that the ITS rDNA sequence was same (100%) to the sequence of Eupenicillium brefeldianum B37 (GenBank accession no. EF488446. It should be noted that the genus Eupenicilium is not used anymore and was re-defined to belong in Penicillium [17]), while the calmodulin sequence of the strain SD-273 was similar (99%) to that of Eupenicillium brefeldianum AS3.6689 (accession no. AY678593). The strain is preserved at China General Microbiological Culture Collection Center, CGMCC (Culture Collection Number CGMCC 7.160).

Fermentation
For chemical investigation, the fungal strain was statically cultivated in liquid potato-dextrose broth medium (1000 mL seawater, 20 g glucose, 5 g peptone, 3 g yeast extract, pH 6.5-7.0, liquid medium/flask = 300 mL) in 1 L Erlenmeyer flasks for 30 days at room temperature.

X-ray Crystallographic Analysis of Compound 1
All crystallographic data [18] were collected on a Bruker Smart-1000 CCD diffractometer equipped with graphite-monochromatic Mo Kα radiation (λ = 0.71073 Å) at 293(2) K. The data were corrected for absorption by using the program SADABS [19]. The structure was solved by direct methods with the SHELXTL software package [20]. All non-hydrogen atoms were refined anisotropically. The H atoms were located by geometrical calculations, and their positions and thermal parameters were fixed during the structure refinement. The structure was refined by full-matrix least-squares techniques [21].
Crystal data for 1: