Sargassopenillines A–G, 6,6-Spiroketals from the Alga-Derived Fungi Penicillium thomii and Penicillium lividum

Seven new 6,6-spiroketals, sargassopenillines A–G (1–7) were isolated from the alga-derived fungi Penicillium thomii KMM 4645 and Penicillium lividum KMM 4663. The structures of these metabolites were determined by HR-MS and 1D and 2D NMR. The absolute configurations of compounds 1, 5 and 6 were assigned by the modified Mosher’s method and by CD data. Sargassopenilline C (3) inhibited the transcriptional activity of the oncogenic nuclear factor AP-1 with an IC50 value of 15 µM.


Introduction
Marine fungi isolated from the surface of marine algae have received great attention as a prolific source of chemically diverse bioactive metabolites [1,2].As a part of our ongoing search for structurally novel and bioactive metabolites from marine-derived fungi, we have previously isolated ten new OPEN ACCESS austalide meroterpenoids from the strains of Penicillium thomii KMM 4645 and Penicillium lividum KMM 4663 associated with the marine brown alga Sargassum miyabei [3].Further investigation of metabolites of these fungal strains has now led to the isolation of seven new 6,6-spiroketals, sargassopenillines A-G.We report herein the isolation and structure determination of compounds 1-7 (Figure 1) and their biological assay results.

Structure Elucidation
The fungi were cultured for 21 days on specially modified rice medium [4].The EtOAc extracts of the mycelia were purified by a combination of silica gel column chromatography and reversed-phase HPLC to yield compounds 1 and 5 from the P. thomii and 2-7 from the P. lividum as amorphous solids.
Compound 6 exhibited a nearly identical CD spectrum in the high-energy region to that of sargassopenilline E (5), which allowed us to determine the 7R configuration of 6. Esterification of 6 with (R)-and (S)-MTPA chloride occurred at the C-6 hydroxy group to give the (S)-and (R) MTPA esters 6a and 6b, respectively.The observed chemical shift differences Δδ(δS − δR) (Figure 2) revealed the 6R configuration.The NOE correlation H-12/H-1b (H 4.05) indicated a β-orientation for the 14-methyl group and suggested the configuration of C-3 to be S.The small coupling constants of the H-9 signal at δ 5.02 (1H, t, 2.9) and biogenetic relationship between sargassopenilline A and 6 suggested an α-orientation for the 9-acetoxy group.Unfortunately, the correlations observed in the NOESY spectrum could not unequivocally establish the relative configuration at C-4. Compound 6 was named sargassopenilline F.
The effect of compounds 1-3 and 7 on the basal AP-1-dependent transcriptional activity was also studied using JB6 Cl41 cells stably expressing a luciferase reporter gene controlled by an AP-1-DNA binding sequence [9][10][11][12].We found that compound 3 is able to inhibit the transcriptional activity of the oncogenic nuclear factor AP-1 with IC50 value of 15 µM after 12 h of treatment.
The sargassopenillines 1, 2, 4-7 were assayed for their cytotoxic activity against CD-1 mouse splenocytes and membranolytic activity to erythrocytes up to 100 µM.Sargassopenilline E (5) exhibited cytotoxicity against splenocytes with a IC50 value 38 µM.The effects of the compounds 1, 2 and 4-7 on the functional activity of CD-1 murine peritoneal macrophages were also studied.It was shown that sargassopenillines D and F at a non-toxic concentration (10 µM) inhibit the adhesion of macrophages (30%-40% of inhibition).
In addition, compounds 1 and 5 showed radical scavenging activity against DPPH with IC50 values of 100 and 50 µM, respectively, while others were inactive.

General Experimental
Optical rotations were measured on a Perkin-Elmer 343 polarimeter.UV spectra were recorded on a Shimadzu UV-1601PC spectrometer in MeOH.CD spectra were measured with a Chirascan-Plus CD Spectrometer (Leatherhead, IR spectra were determined on a Bruker OPUS Vector-22 infrared spectrophotometer in CHCl3. 1 H and 13 C NMR spectra were recorded in CDCl3, MeOH-d4 and pyridine-d5 on a Bruker Avance-500 and Avance III-700 spectrometers operating at 500.13 MHz and 125.77MHz and 700.13 and 176.04 MHz, respectively, using TMS as an internal standard.HRESIMS spectra were measured on an Agilent 6510 Q-TOF LC mass spectrometer. Low-pressure liquid column chromatography was performed using Si gel L (40/100 μm, Sorbpolimer, Russia).Glass plates (4.5 × 6.0 cm) precoated with Si gel (5-17 μm, Sorbfil) were used for thin layer chromatography.Preparative HPLC was carried out on a Beckman-Altex chromatograph, using a Supelco Discovery C-18 (5 μm, 4.6 × 250 mm) column with an RIDK-122 refractometer.
The energy-minimized conformations for 1, 5 and 7 have been determined using crystallographic data (CCDC 940798) for the structure of peniciketal A [13] by the MM2 force field calculation method using ChemBioDraw Ultra 12.0, CambridgeSoft Corporation (Cambridge, MA, USA).

Fungal Material and Fermentation
The strains of the fungi Penicillium lividum and Penicillium thomii were isolated from superficial mycobiota of the brown alga Sargassum miyabei (Lazurnaya Bay, the Sea of Japan) and were identified on the basis of morphological evaluation by Natalya N. Kirichuk from the G.B. Elyakov Pacific Institute of Bioorganic Chemistry (PIBOC).Strains are stored at the Collection of Marine Microorganisms, PIBOC, Vladivostok, Russia with the codes KMM 4663 and KMM 4645, respectively.The fungi were grown stationary at 22 C for 21 days in 20 Erlenmeyer flasks (500 mL) (for each strain), each flask containing 20 g of rice, 20 mg of yeast extract, 10 mg of KH2PO4, and 40 mL of natural sea water (Marine Experimental Station of G.B. Elyakov Pacific Institute of Bioorganic Chemistry, Troitsa (Trinity) Bay, Sea of Japan).

Extraction
At the end of the incubation period, the mycelia and medium were homogenized and extracted with EtOAc (2 L).The extract of each fungus was concentrated to dryness.The residue was dissolved in 20% MeOH-H2O (1 L) and was extracted with n-hexane (0.2 L × 3) and EtOAc (0.2 L × 3).After evaporation of the EtOAc layer, the residual materials (1.5 g, P. thomii and 1.3 g, P. lividum) were passed over silica columns (4 × 20 cm), which were eluted first with n-hexane (1 L) followed by a step gradient from 5% to 100% EtOAc in n-hexane (total volume 7 L).Fractions of 200 mL were collected and combined on the basis of TLC (Si gel, toluene-isopropanol 6:1, v/v).

Preparation of (S)-MTPA and (R)-MTPA Esters of 6
4-Dimethylaminopyridine (a few crystals) and (R)-MTPACl (20 μL) were added to a solution of the 6 (3.0 mg) in pyridine and stirred at room temperature (25 °C) for 24 h.After evaporation of the solvent, the residue was passed through a silica gel column (15% EtOAc-hexane) to generate the (S)-MTPA ester (6a).The (R)-MTPA ester (6b) was prepared in a similar manner using (S)-MTPACl.The spectra of compounds 1-7 are all given in the Supplementary Information.

Cytotoxicity Assay
The effect of the compounds on the cells viability was evaluated using the MTS test, which is based on the reduction of MTS into its formazan product by alive cells [14][15][16].Cytotoxicity towards CD-I mouse splenocytes was determined according to Freshney [17].Hemolytic activity towards CD-I mouse erythrocytes was determined as previously described [18].

Determination of the Effects of Compounds on the Basal Transcriptional Activity of AP-1
The effects of the compounds on the basal transcriptional activities of AP-1 were evaluated using the JB6 Cl41 cell line stably expressing a luciferase reporter gene controlled by an AP-1-DNA binding sequence [9].The experiments were performed as previously reported [16] with slight modifications.

Figure 3 .
Figure 3. Chem3D representation of the minimum conformation of 1, 5 and 7 showed observed NOE correlations.