Piltunines A–F from the Marine-Derived Fungus Penicillium piltunense KMM 4668

Six new carotane sesquiterpenoids piltunines A–F (1–6) together with known compounds (7–9) were isolated from the marine-derived fungus Penicillium piltunense KMM 4668. Their structures were established using spectroscopic methods. The absolute configurations of 1–7 were determined based on circular dichroism (CD) and nuclear Overhauser spectroscopy (NOESY) data as well as biogenetic considerations. The cytotoxic activity of some of the isolated compounds and their effects on regulation of reactive oxygen species (ROS) and nitric oxide (NO) production in lipopolysaccharide-stimulated macrophages were examined.


Introduction
Species of the genus Penicillium can grow on many different substrates and they are the most widespread fungi on our planet. These fungi are commonly isolated from terrestrial substrates and are also known as permanent components of marine ecosystems. Penicillium species are frequently found associated with sea grasses, algae, vertebrates, and invertebrates. Among microscopic fungi, divaricate Penicillium species are one of the most difficult to identify. This group comprises species with asymmetric, strongly divaricate penicillia bearing terminal and subterminal clusters of monoverticillate structures. Morphological, physiological and molecular analyses of the Penicillium spp. isolates of mycobiota in the northeastern part of the Sakhalin Island shelf (near the Piltun Bay, baymouth) [1,2] revealed that the strain Penicillium piltunense KMM 4668 isolated from subaqueous soilswas not assignable to any previously known species. Phylogenetic analysis showed that the strain was more closely related to members of the Penicillium canescens group including eight species: P. canescens, P. antarcticum, P. atrovenetum, P. coralligerum, P. novae-zeelandiae, P. jensenii, P. murcianum, and P. janczewski and was described as a new species [3].
The HRESIMS of 2 showed the quasimolecular ion at m/z 323.1500 [M -H] -. These data, coupled with 13 C NMR spectral data (DEPT), established the molecular formula of 2 as C 17 H 24 O 6 . The general features of 1 H and 13 C NMR spectra (Table 1; Table 2; Table S2; Figures S33-S39) of 2 showed a close similarity of the carbon chemical shifts to the ones for piltunine A (1), with the exception of the C-10, C-11 and C-14 carbon signals. The molecular mass difference of 42 mass units between 1 and 2 and HMBC correlations from H 3 -17 (δ H 2.03) to C-14 (δ C 69.4) and C-16 (δ C 173.6) indicate the presence of an acetoxy group at C-14 in 2.
The absolute configurations of the chiral centers in 2 were elucidated as 2R,3R,6S,7S based on NOESY data ( Figure S39, Table S2) and biogenetic considerations. Compound 2 was named piltunine B.
The absolute configurations of the chiral centers in penigrisacid D (7)were defined based on NOESY (Figure 4, Figure S73) correlations H-6 (δ H  (δH2.26) to C-10 and C-11 elucidated the structures of a five-membered ring and indicated the 9-en-11-one position for the trisubstituted enonechromophore in 5. The absolute configurations of the chiral centers in 5 were elucidated based on a NOESY experiment ( Figure S60) and biogenetic considerations as for piltunine D (4). Compound 5 was named piltunine E.
The absolute configurations of the chiral centers in penigrisacid D (7)were defined based on NOESY (Figure 4, Figure S73  We have investigated the effect of the isolated compounds1, 1a, 2, 7-9 on the viability of human drug-resistant prostate cancer 22Rv1 cell as well as on human prostate non-cancer PNT-2 cells using an MTT assay (Table 3). We have also determined a selectivity index (SI) at for all the tested substances (Table 3). Compounds showing high SI values are more active in cancer cells in comparison with non-cancer cells lines. Thus, 8 had the highest SI value among the tested substances, which makes it a promising candidate for the further synthetic modifications in order to optimize its anticancer activity and selectivity. Interestingly, 8 has been also identified to be the most cytotoxic in cancer cells in comparison with the other substances (Table 3). Note, 22Rv1 cells are We have investigated the effect of the isolated compounds 1, 1a, 2, 7-9 on the viability of human drug-resistant prostate cancer 22Rv1 cell as well as on human prostate non-cancer PNT-2 cells using an MTT assay (Table 3). We have also determined a selectivity index (SI) at for all the tested substances (Table 3). Compounds showing high SI values are more active in cancer cells in comparison with non-cancer cells lines. Thus, 8 had the highest SI value among the tested substances, which makes it a promising candidate for the further synthetic modifications in order to optimize its anticancer activity and selectivity. Interestingly, 8 has been also identified to be the most cytotoxic in cancer cells in comparison with the other substances (Table 3). Note, 22Rv1 cells are known to be resistant to the hormone therapy due to the expression of androgen receptor splice variant AR-V7 [15]. Thus, the compounds which are active inAR-V7-positive 22Rv1 cells are of high clinical interest.
Additionally, we have investigated the effects of the isolated compounds 1, 2, 3 and 5-9 on reactive oxygen species (ROS) and nitric oxide (NO) production in murine macrophages following lipopolysaccharide (LPS) stimulation. NO, which is produced in large quantities by inducible nitric oxide synthase (iNOS), is known to be responsible for vasodilation and hypotension observed during septic shock and inflammation.
Compounds 5 and 9 induced a significant down-regulation of ROS production ( Figure 5). LPS from E. coli, an inflammatory agent, was used as a positive control in our study. Table 3. Cytotoxic activity of the investigated compounds. Cells were treated for 72 h and the viability was measured using MTT assay. IC 50 s were calculated using GraphPad Prism software. Additionally, the viability of the cells treated with 100 µM of the compounds is presented and the selectivity index (SI) was calculated as [viability of   Table 3. Cytotoxic activity of the investigated compounds. Cells were treated for 72 h and the viability was measured using MTT assay. IC50s were calculated using GraphPad Prism software. Additionally, the viability of the cells treated with 100 µM of the compounds is presented and the selectivity index (SI) was calculated as [viability of  Additionally, we have investigated the effects of the isolated compounds 1, 2, 3 and 5-9 on reactive oxygen species (ROS) and nitric oxide (NO) production in murine macrophages following lipopolysaccharide (LPS) stimulation. NO, which is produced in large quantities by inducible nitric oxide synthase (iNOS), is known to be responsible for vasodilation and hypotension observed during septic shock and inflammation.
Compounds 5 and 9 induced a significant down-regulation of ROS production ( Figure 5). LPS from E. coli, an inflammatory agent, was used as a positive control in our study. Compound 9 suppressed NO production in LPS-stimulated macrophages at non-toxic concentration of 1 µM. NO level in these cells was decreased by 24.1 ± 0.7% (p < 0.05, Student's t-test) in comparison with LPS-treated control (data not shown). Therefore, compound 9 may be a promising candidate for the therapy of inflammatory diseases accompanying overproduction of NO. Compound 9 suppressed NO production in LPS-stimulated macrophages at non-toxic concentration of 1 µM. NO level in these cells was decreased by 24.1 ± 0.7% (p < 0.05, Student's t-test) in comparison with LPS-treated control (data not shown). Therefore, compound 9 may be a promising candidate for the therapy of inflammatory diseases accompanying overproduction of NO.

Fungal Strain
The strain of Penicillium piltunense KMM 4668 was isolated from marine subaqueous soil (Piltun Bay, Sea of Okhotsk, the northeastern shelf of the Sakhalin Island, Russia). The fungus was identified according to a molecular biology protocol by DNA amplification and sequencing of the ITS region (MycoBank accession number is MB818671) and morphological and physiological studies. The strain is stored at the Collection of Marine Microorganisms (KMM) of G.B. Elyakov Pacific Institute of Bioorganic Chemistry (Vladivostok, Russia).

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 (4.8 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 (2.5 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).
The n-hexane-EtOAcfraction (70:30, 70 mg) was separated on a Gel ODS-A column (1.5 × 8 cm) eluting with  and then was purified on an YMC ODS-A column eluting with  to yield 3 (3 mg).

In Vitro MTT-Based Drug Sensitivity Assay
The in vitro cytotoxicity of individual substances was evaluated using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, which was performed as previously described [16]. The vehicle (DMSO) treated cells were used as a negative control. Treatment time was 72 h.

Reactive Oxygen Species (ROS) Level Analysis in Lipopolysaccharide (LPS)-Treated Cells
The suspension of macrophages on 96-well plates (2 × 10 4 cells/well) were washed withphosphate-buffered saline (PBS) and treated with 180 µL/well of the tested compounds (10 µM) for 1 h and 20 µL/well LPS from E. coli serotype 055:B5 (Sigma-Aldrich, MA, USA, 1.0 µg/mL), which were both dissolved in PBS and cultured at 37 • C in a CO 2 -incubator for one hour. For the ROS levels measurement, 200 µL of 2,7-dichlorodihydrofluorescein diacetate (DCF-DA, Sigma, final concentration 10 µM) fresh solution was added to each well, and the plates were incubated for 30 min at 37 • C. The intensity of DCF-DA fluorescence was measured at λex 485 n/λem 518 nm using the plate reader PHERAstar FS (BMG Labtech, Offenburg, Germany) [17].

Reactive Nitrogen Species (RNS) Level Analysis in LPS-Treated Cells
The suspension of macrophages on 96-well plates (2 × 10 4 cells/well) were washed with the PBS and treated with 180 µL/well of the tested compounds (10 µM) for 1 h and 20 µL/well LPS from E. coli serotype 055:B5 (Sigma, 1.0 µg/mL), which were both dissolved in PBS and cultured at 37 • C in a CO 2 -incubator for one hour. For the RNS levels measurement, 200 µL Diaminofluorescein-FM diacetate (DAF FM-DA, Sigma, final concentration 10 µM) fresh solution was added to each well, and the plates were incubated for 40 min at 37 • C, then replace with fresh PBS, and then incubated for an additional 30 min to allow complete de-esterification of the intracellular diacetates. The intensity of DAF FM-DA fluorescence was measured at λex 485 n/λem 520 nm using the plate reader PHERAstar FS (BMG Labtech, Offenburg, Germany).

Peritoneal Macrophage Isolation
Mice BALB/c were sacrificed by cervical dislocation. Peritoneal macrophages were isolated using standard procedures. For this purpose, 3 ml of PBS (pH 7.4) was injected into the peritoneal cavity and the body intensively palpated for 1-2 min. Then the peritoneal fluid was aspirated with a syringe. Mouse peritoneal macrophage suspension was applied to a 96-well plate left at 37 • C in an incubator for 2 h to facilitate attachment of peritoneal macrophages to the plate. Then a cell monolayer was triply flushed with PBS (pH 7.4) for deleting attendant lymphocytes, fibroblasts and erythrocytes and cells were used for further analysis.
All animal experiments were conducted in compliance with all rules and international recommendations of the European Convention for the Protection of Vertebrate Animals used for experimental and other scientific purposes. All procedures were approved by the Animal Ethics Committee at the G. B. Elyakov Pacific Institute of Bioorganic Chemistry, Far Eastern Branch of the Russian Academy of Sciences (Vladivostok, Russia), according to the Laboratory Animal Welfare guidelines.

Statistical Analysis
Average value, standard error, standard deviation and p-values in all experiments were calculated and plotted on the chart using SigmaPlot 3.02 (Jandel Scientific, San Rafael, CA, USA) or GraphPad Prism software v. 5.01 (GraphPad Prism software Inc., La Jolla, CA, USA). Statistical difference was evaluated by the t-test, and results were considered as statistically significant at p < 0.05.

Conclusions
Six carotane sesquiterpenoids piltunines A-F (1-6) together with known compounds (7)(8)(9) were isolated from the fungus Penicillium piltunense KMM 4668 obtained from marine subaqueous soils. The absolute configurations of 1-7 were determined using combined CD and NOESY dataas well as biogenetic considerations. Piltunine C (3) was established as a 13-hydroxy derivative of aspterric acid. Piltunines E (5) and F (6) contain the 9-en- 11-one and 6-en-11-one enone chromophoresin structure of molecules. Piltunine A (1) is reported here for first time as a natural product. Some of the isolated compounds exhibited cytotoxic activity in human drug-resistant prostate cancer cells as well as being able to inhibitROS and NO production in LPS-stimulated macrophages.

Funding:
The study was supported by Russian Science Foundation (grant No 19-74-10014).