Pretrichodermamides D–F from a Marine Algicolous Fungus Penicillium sp. KMM 4672

Three new epidithiodiketopiperazines pretrichodermamides D–F (1–3), together with the known N-methylpretrichodermamide B (4) and pretrichodermamide С (5), were isolated from the lipophilic extract of the marine algae-derived fungus Penicillium sp. KMM 4672. The structures of compounds 1–5 were determined based on spectroscopic methods. The absolute configuration of pretrichodermamide D (1) was established by a combination of modified Mosher′s method, NOESY data, and biogenetic considerations. N-Methylpretrichodermamide B (5) showed strong cytotoxicity against 22Rv1 human prostate cancer cells resistant to androgen receptor targeted therapies.


Introduction
Epithiodiketopiperazines with 1,2-oxazadecaline moiety are rare in nature.To date, only eleven such compounds have been reported [1][2][3][4].Structural differences in compounds of this class consist in N-methylation, C-4-C-5-epoxidation [5,6] and substituent at C-5.In addition, first mono-and trithioderivatives have recently been reported [3,4].The fungi of genus Trichoderma were producers of most of these alkaloids.However, pretrichodermamide A has also been reported to be synthesized by Aspergillus sp.[7], while N-methylated compounds methylgliovirin (the first described compound from this class) [5], N-methylpretrichodermamide B (adametizine A) [8,9], and pretrichodermamide C (adametizine B) [8,9] have only been isolated from Penicillium species.Many oxazadecaline thiodiketopiperazines have been reported and patented as antibiotics, and chloroderivatives have shown cytotoxic activity against murine lymphoma and Jurkat cells with IC 50 of 2-5 µM [2,[8][9][10].During our ongoing search for structurally novel and bioactive metabolites from marine-derived fungi, we investigated the fungus Penicillium sp.KMM 4672 isolated from Vietnamese brown alga Padina sp.A chemical study resulted in the isolation and identification of three new 1,2-oxazadecaline epidithiodiketopiperazines pretrichodermamides D-F (1-3), together with the known pretrichodermamide C (4) and N-methylpretrichodermamide B (5) (Figure 1).Herein, The molecular formula of compound 1 was determined to be C21H24N2O9S2 from a HRESIMS peak at m/z 511.0857 [M − H] − and was in accordance with 13 C NMR data.A thorough analysis of the 1 H and 13 C NMR data (Table 1) of 1 with DEPT and HSQC techniques revealed the presence of two methoxyls, one N-methyl, one methylene, four sp 2 -methines, and five sp 3 -methines together with two sp 3 -quaternary carbons.The remaining functionalities, corresponding to the carbon signals at δC 165.4 (C), 164.2 (C), 153.0 (C), 147.6 (C), 135.9 (C), and 116.3 (С), suggested the presence of two amide carbonyl carbons, three oxygenated, and one C-substituted sp 2 -carbons.

Results
The EtOAc extract of the culture of the fungus was suspended in H 2 O-EtOH (4:1) and successively partitioned with hexane, EtOAc, and n-BuOH.The EtOAc portion was subjected to column chromatography over silica gel and by HPLC to yield individual compounds 1-5 as white powders.
The molecular formula of compound 3 was determined as C 21 H 24 N 2 O 9 S 2 (the same as 1 and 2) on the basis of HRESIMS and 13   Besides the new pretrichodermamides D-F (1-3), the known pretrichodermamide C (4) and N-methylpretrichodermamide B (5) were also isolated from this fungus.For the first time these compounds were found in Egyptian hyper saline lake fungus Penicillium sp.[8] and were later isolated from sponge-derived Penicillium adametzioides and published as new adametizines A and B, respectively.The absolute stereochemistry for adametizines were determined based on X-ray and ECD data [9].The structures of 4 and 5 were established on the basis of 1D and 2D NMR data and high resolution ESIMS analysis (Supplementary data S20-S26).The absolute structures of compounds 4 and 5 were determined the same as for adametizines B and A, respectively, based on identity of their ECD spectra.In a next step, we investigated the effects of compounds 1-5 on viability and the apoptosis induction of human prostate cancer cells.It should be noted that, in a recently published study, N-methylpretrichodermamide B did not show any cytotoxic effect against a number of different cancer cells up to 10 µM [9].MTT assays revealed N-methylpretrichodermamide B (5) to be highly cytotoxic in 22Rv1, PC-3, and LNCaP cells with IC 50 0.51, 5.11, and 1.76 µM, respectively, while revealed IC 50 s of 0.013, 0.015, and 0.004 µM were determined for docetaxel (positive control).Remarkably, 5 induces apoptosis in human prostate cancer 22Rv1 cells (31.3% ˘8.2% apoptosis after treatment with 1 µM for 48 h), which are highly resistant to androgen receptor (AR)-targeted therapies due to a loss of the ligand-binding domain of the AR receptor [12].Compounds 1-4 did not exhibit cytotoxic activity against human prostate cancer cells at concentrations up to 100 µM.No significant effect on cell cycle progression was observed for any of the compounds at concentrations up to 100 µM.22Rv1 cells are known to be resistant to the hormone therapy due to the presence of androgen receptor splice variant AR-V7, while LNCaP cells bearing w/t AR are sensitive to the hormone deprivation [12].Remarkably, 5 was mostly active in AR-V7-positive 22Rv1 cells with IC 50 at nanomolar concentrations (MTT test).In addition, the effect of compounds 1-5 was tested on non-malignant murine cells (splenocytes and erythrocytes).N-methylpretrichodermamide B (5) did not show hemolitic activity up to 100 µM and was cytotoxic for splenocytes only at high doses (ED 50 62.1 µM).

Fungal Strain
The strain was isolated from brown algae Padina sp.(South China Sea, Vietnam) by the plating method using malt extract agar and identified on the basis of morphological and molecular features.For DNA extraction, the culture was grown on malt extract agar under 25 ˝C for 7 d.DNA extraction was performed by HiPurATM Plant DNA Isolation kit (CTAB Method) (HiMedia Laboratories Pvt. Ltd., Mumbai, India) according to the manufacturer 1 s instructions.Fragments containing the ITS regions were amplified using primers ITS1 and ITS4 [13].Amplification of the partial calmodulin gene was performed using Cmd5 and Cmd6 primers [14].The newly obtained sequences were checked visually and compared to available sequences of GenBank by using BLAST-n.According to BLAST analysis of the ITS1-5.8S-ITS2and partial calmodulin datasets, the strain Penicillium sp.KMM 4672 is related to P. citrinum-group and displays the most similarity with P. steckii (99% and 97%, respectively).The sequences were deposited in GenBank nucleotide sequence database under KU 695807 and KU 695808.The strain was deposited in the Collection of Marine Microorganisms under the code KMM 4672.

Cultivation of Fungus
The fungus was grown stationary at 22 ˝C for three weeks in 60 ˆ500 mL Erlenmeyer flasks, each containing 60 g of the solid nutrient medium of the following composition: rice (20.0 g), yeast extract (20.0 mg), KH 2 PO 4 (10 mg), and natural sea water (40 mL).

Extraction and Isolation
The fungal mycelia with the medium were extracted for 24 h with 12 L of EtOAc.Evaporation of the solvent under reduced pressure yielded a brown oil (9.2 g), to which 250 mL of H 2 O-EtOH (4:1) was added, and the combination was thoroughly mixed to yield a suspension.It was extracted successively with hexane (150 mL ˆ2), EtOAc (150 mL ˆ2) and n-BuOH (150 mL ˆ2).The EtOAc fraction was concentrated in vacuo to give a residue (6.0 g), which was separated on a silica gel column (30 ˆ3cm) eluted with a hexane-EtOAc gradient (1:0-0:1).

Preparation of (S)-MTPA and (R)-MTPA Esters of Pretrichodermamide D (1)
4-dimethylaminopyridine (a few crystals) and (R)-MTPA-Cl (5 µL) was added to a solution of the pretrichodermamide D (1.8 mg) in pyridine at room temperature and stirred for 4 h.After evaporation of the solvent, the residue was passed through a silica gel column (20% EtOAc-hexane) to afford the (S)-MTPA ester (1.0 mg).The (R)-MTPA ester (1.2 mg) was prepared in a similar manner using (S)-MTPA-Cl.

Cell Culture
The human prostate cancer cells lines 22Rv1, PC-3, and LNCaP were purchased from ATCC.Cell lines were cultured according to the manufacturers instructions in 10% FBS/RPMI media (Invitrogen) with (for LNCaP) or without (for 22Rv1 and PC-3) 1 mM sodium pyruvate (Invitrogen).Cells were continuously kept in culture for a maximum of 3 months and were routinely inspected microscopically for stable phenotype and regularly checked for contamination with mycoplasma.Cell line authentication was performed by DSMZ (Braunschweig, Germany) using highly polymorphic short tandem repeat loci [15].

Cell Cycle and Apoptosis Induction Analysis
The cell cycle distribution was analyzed by flow cytometry using PI staining as described before with slight modifications [18].In brief, cells were pre-incubated overnight in 6-well plates (2 ˆ10 5 cells/well in 2 mL/well).The medium was changed to fresh medium containing different concentrations of the substances.After 48 h of treatment, cells were harvested with a trypsin-EDTA solution, fixed with 70% EtOH, stained, and analyzed by FACS.The results were quantitatively analyzed using Cell Quest Pro software (Version 5.2.1.,BD Bioscience, Bedford, MA, USA).Cells appeared at sub-G1 peak were assumed as apoptotic.

Hemolytic Activity
The hemolytic activity was evaluated using CD-I mouse erythrocytes as previously described [19,20].

Conclusions
Three new epidithiodiketopiperazines, named pretrichodermamides D-F (1-3) were isolated from the lipophilic extract of marine algae-derived fungus Penicillium sp.KMM 4672.Each new compound contains rare 1,2-oxazadecaline moieties [1].Compounds 1 and 2 are the first isomers at oxazadecaline moiety among the related alkaloids.N-methylpretrichodermamide B (5), highly cytotoxic in 22Rv1 human prostate cancer cells, is resistant to androgen receptor-targeted therapies.At the same time, N-methylpretrichodermamide B was found to be cytotoxic for non-malignant cells (splenocytes and erythrocytes) only at high doses (ED50 62.1 and >100 µM).Therefore, this compound may be a promising candidate for the therapy of human drug-resistant prostate cancer.