New Deoxyisoaustamide Derivatives from the Coral-Derived Fungus Penicillium dimorphosporum KMM 4689

Seven new deoxyisoaustamide derivatives (1–7) together with known compounds (8–10) were isolated from the coral-derived fungus Penicillium dimorphosporum KMM 4689. Their structures were established using spectroscopic methods, X-ray diffraction analysis and by comparison with related known compounds. The absolute configurations of some alkaloids were determined based on CD and NOESY data as well as biogenetic considerations. The cytotoxic and neuroprotective activities of some of the isolated compounds were examined and structure-activity relationships were pointed out. New deoxyisoaustamides 4–6 at concentration of 1 µM revealed a statistical increase of PQ(paraquat)-treated Neuro-2a cell viability by 30–39%.

In our search for fungal secondary metabolites possessing novel chemical structures and/or biological activity, we have investigated the strain Penicillium dimorphosporum KMM 4689.
Fungi of the genus Penicillium have a special position in nature and human life. The marine environment is no exception. These fungi were found in the extremely salty

Results and Discussion
The fungus was cultured for 21 days on solid rice medium. The EtOAc extract of the mycelium was purified by a combination of a Si gel and an ODS-A column chromatography, and a reversed phase HPLC to yield compounds 1-10 ( Figure 1).
The  The configuration of the chiral center C-11 in 1 as S was established based on obvious biogenetic relationships with (+)-deoxyisoaustamide (8), the optical rotation value of which was in full agreement with the literature data [2,9]. Thus, the absolute configuration of 1 was established as 11S,16S,17S. Compound 1 was named 16α-hydroxy-17β-methoxy-deoxydihydroisoaustamide.
The   The planar structures of the compounds 5 and 6 were found by extensive NMR spectroscopy ( 1 H, 13 C, DEPT, HSQC and HMBC) (Tables 1 and 2 and Figures S50-S63) to be the same as those of 4. The general features of 1 H and 13 C NMR spectra of 5 and 6 closely resembled those of 2 and 3, respectively.
The absolute configurations of the chiral centers in 2 were defined based on NOESY ( Figure S34 The planar structures of the compounds 5 and 6 were found by extensive NMR spectroscopy ( 1 H, 13 C, DEPT, HSQC and HMBC) (Tables 1 and 2 and Figures S50-S63) to be the same as those of 4. The general features of 1 H and 13 C NMR spectra of 5 and 6 closely resembled those of 2 and 3, respectively.
Of note, we have tried to determine the absolute configuration of the asymmetric centers at C-16 for compounds 1, 2, 4 and 5 using the modified Mosher's method. Esterification of compounds with (S)-MTPA chloride at the C-16 hydroxy groups produced (R)-MTPA esters. However, the attempts to obtain (S)-MTPA esters from (R)-MTPA-Cl were unsuccessful.
We further examined potential protective effects of the isolated compounds 1-10 against the acute toxicity of paraquat (PQ) in murine neuroblastoma Neuro-2a cells, which is a well-established model for neuroprotective activity studies [16]. In our experiments, treatment of Neuro-2a cells with 500 µM of PQ induced a decrease of cell viability by 51.8% ( Figure 5). Whereas co-treatment with 1 µM of 4 and 6 could increase a viability of PQ-treated cells by 38.6% and 30.3%, respectively. Compound 5 increased a viability of the cells by 36.5% and 39.4% at concentrations at 1 µM and 10 µM, respectively. At the same time, the investigated compounds were non-cytotoxic to Neuro-2a cells, as well as to human prostate epithelial PNT-2 cells (compounds 1, 2, 4, 8-10, Figure S71), used as non-cancer human cell lines to demonstrate the lack of cytotoxic activity to human cells (IC 50 s > 100 µM, data not shown). This is the first report on neuroprotective activity of deoxyisoaustamides. Earlier, it was reported that (+)-deoxyisoaustamide (8) and deoxydihydroisoaustamide (9) were studied in glutamate and t-BHP-induced cytotoxicity assays [3]. In addition, inhibitory effects of the metabolites on nitrite production of LPS-stimulated RAW264.7 and BV2 cells were evaluated. These compounds showed no significant effects on cytoprotection This is the first report on neuroprotective activity of deoxyisoaustamides. Earlier, it was reported that (+)-deoxyisoaustamide (8) and deoxydihydroisoaustamide (9) were studied in glutamate and t-BHP-induced cytotoxicity assays [3]. In addition, inhibitory effects of the metabolites on nitrite production of LPS-stimulated RAW264.7 and BV2 cells were evaluated. These compounds showed no significant effects on cytoprotection or nitrite inhibition [3]. In our experiments, new deoxyisoaustamides 4-6 revealed a statistical increase of PQ-treated Neuro-2a cell viability.
The analyses of structure-activity relationships suggest a key role of both hydroxy groups at C-16 and C-17 for the neuroprotective activity of investigated deoxyisoaustamide alkaloids. Indeed, the presence of the mentioned feature in structures 4-6 correlates with a higher activity of the compounds.

General Experimental Procedures
Optical rotations were measured on a Perkin-Elmer 343 polarimeter (Perkin Elmer, Waltham, MA, USA). UV spectra were recorded on a Shimadzu UV-1601PC spectrometer (Shimadzu Corporation, Kyoto, Japan) in methanol. CD spectra were measured with a Chirascan-Plus CD spectrometer (Leatherhead, UK) in methanol. NMR spectra were recorded in CD 3 OD and DMSO-d 6 , on a Bruker DPX-500 (Bruker BioSpin GmbH, Rheinstetten, Germany) and a Bruker DRX-700 (Bruker BioSpin GmbH, Rheinstetten, Germany) spectrometer, using TMS as an internal standard. HRESIMS spectra were measured on a Maxis impact mass spectrometer (Bruker Daltonics GmbH, Rheinstetten, Germany). Microscopic examination and photography of fungal cultures were performed with Olympus CX41 microscope fitted with (equipped with) an Olympus SC30 digital camera. Detailed examination of ornamentation of the fungal conidia was performed by scanning electron microscopy (SEM) EVO 40.

Fungal Strain
Soft coral samples were collected using a Van Veen bottom grab at various points in the South China Sea. The samples were collected in individual sterile plastic bags and stored frozen (−18 • C) before use. Isolation of fungal colonies was made by the plating methods using Tubaki agar. Isolation of pure cultures was made by transferring of inoculums to the slant wort agar. Macroscopical characters were studied on the agar media Czapek yeast extract agar (CYA), yeast extract agar (YES) and malt extract agar (MEA). Preparation of these media is detailed by Frisvad and Samson (2004). The strains were inoculated at three points on 9-cm Petri dishes and incubated for 7 d at 25 • C in darkness. In addition, inoculated CYA plates were incubated for 7 d at 37 • C according to the recommendations of Pitt. Color names are from Ridgway. To determine the degree of halotolerance, the strains were grown on MEA supplemented with 5, 10, 15 and 20% NaCl at 25 • C for 7 days.
The cultures used for the molecular studies were grown on malt extract agar under 25 • C for 7 d. DNA extraction was performed by DNA kit (DNA-TechnologyLtd., Moscow, Russia) according to the manufacturer's instructions. Fragments containing the ITS regions were amplified using primers ITS1 and ITS4 [20].Newly generated sequences were compared to the available sequences of the National Center for Biotechnology Informatic (NCBI) by using BLAST. BLAST search results indicated that the sequence was 99% identical with the sequenceof Penicillium dimorphosporum strain CBS 456.70(GenBank accession numberMH859796.1). The sequence was deposited in GenBank nucleotide sequence database under MW325972 code.

Cultivation of Fungus
The fungus was cultured at 22 • C for three weeks in 60 × 500 mL Erlenmeyer flasks, each containing rice (20.0 g), yeast extract (20.0 mg), KH 2 PO 4 (10 mg) and natural sea water from the Marine Experimental Station of PIBOC, Troitsa (Trinity) Bay, Sea of Japan (40 mL).

Extraction and Isolation
At the end of the incubation period, the mycelia and medium were homogenized and extracted with EtOAc (1 L). The obtained extract was concentrated to dryness. The residue (2.8 g) was dissolved in H 2 O−EtOH (4:1)(100 mL) and was extracted with n-hexane (0.2 L × 3) and EtOAc (0.2 L × 3). After evaporation of the EtOAc layer, the residual material (1.5 g) was passed over a silica column (3 × 14 cm), which was eluted first with n-hexane (200 mL) followed by a step gradient from 5% to 50% EtOAc in n-hexane (total volume 20 L). Fractions of 250 mL were collected and combined on the basis of TLC (Si gel, toluene-isopropanol 6:1 and 3:1, v/v).

MTT Cell Viability Assay
The Neuro-2a cells (1 × 10 4 cells/well) or PNT-2 cells (0.6 × 10 4 cells/well)were seeded in 96-well plate, incubated overnight. Then, the culture medium was exchanged to the fresh corresponding medium containing the different concentrations of the investigated compounds and the cells were further incubated for an additional 24 h. After that, cell viability was determined by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) method according to the manufacturer's instructions (Sigma-Aldrich, St. Louis, MO, USA). Absorbance of the converted formazan was measured using a Multiskan FC microplate photometer (Thermo Scientific, Waltham, MA, USA) at λ = 570 nm with background subtraction at λ = 630-690 nm. The results were presented as percentages of control data.

Paraquat-Induced Neurotoxicity
The Neuro-2a cells (1 × 10 4 cells/well of 96-well plate) were pretreated with the studied compounds at concentrations of 1 and 10 µM for 1 h, and then 500 µM of PQ (Sigma-Aldrich, St. Louis, MO, USA) was added (500 µM, final concentration) to the neuroblastoma cells. Cells incubated without PQ and compounds, and with PQ alone, were used as positive and negative controls, respectively. The viability of cells was measured after 24 h using MTT method. The results were presented as percentages of positive control data.
All data were obtained in three independent replicates and calculated values were expressed as mean ± SEM. Student's t-test was performed using SigmaPlot 14.0 (Systat Software Inc., San Jose, CA, USA) to determine statistical significance.

Conclusions
Seven new deoxyisoaustamidederivatives (1-7) together with known compounds (8)(9)(10) were isolated from the fungus Penicillium dimorphosporum KMM 4689 associated with unidentified marine soft coral. The absolute configurations of compounds 1-3, 5-7 were determined using combined CD and NOESY data as well as biogenetic considerations. All new compounds were found to have an indoloazocine tricyclic subunit which is rare in natural alkaloids. New deoxyisoaustamides 4-6 at concentration of 1 µM revealed a statistical increase of PQ-treated Neuro-2a cell viability by 30-39%.