A new diketopiperazine derivative from a deep sea-derived Streptomyces sp. SCSIO 04496

A new diketopiperazine (DKP) derivative, (6R,3Z)-3-benzylidene-6-isobutyl-1-methyl piperazine-2,5-dione (1), as well as five known DKPs 2–6 was isolated from a deep sea-derived Streptomyces sp. SCSIO 04496. The structure of 1 was elucidated using a combination of 1D and 2D NMR, HR-ESI-MS and chiral-phase HPLC techniques. Compounds 1–6 did not show cytotoxic activity at a concentration of 100 μM in bioactivity assay.


Introduction
Diketopiperazine is a small family among natural products. Nowadays, Diketopiperazine derivatives (DKPs) have received much attention because of their significant bioactivities including cytotoxicity , antibacterial activity (Graz et al. 1999) and antiviral activity (Wang et al. 2013). Natural DKPs have provided important inspiration for drug discovery (Borthwick 2012). For example, the vascular disrupting agent NPI-2358 (Plinabulin) is a synthetic analogue of natural diketopiperazine NPI-2350, which was isolated from Aspergillus sp. (Nicholson et al. 2006). NPI-2358 is now in phase II clinical trial as an anticancer agent (Mita et al. 2010). Marine-derived microorganisms have been looked upon as potential sources of bioactive compounds due to the unique and extreme environment characterised by high pressure, low temperature, lack of light and variable salinity and oxygen concentration (Blunt et al. 2013). As part of our ongoing research program to discover bioactive metabolites with novel structures from the South China Sea-derived actinomycetes, we have reported an antimicrobial and cytotoxic polythiazole cyclopeptide marthiapeptide A from Marinactinospora thermotolerans SCSIO 00652 (Zhou et al. 2012), cytotoxic angucyclines grincamycins B-F from Streptomyces lusitanus SCSIO LR32 . Recently, an actinomycete strain was isolated from a marine sediment sample collected in the South China Sea. This strain was subsequently identified as Streptomyces sp. SCSIO 04496 on the basis of 16S rDNA sequence analysis. Chemical investigation of the culture of this strain resulted in the isolation and identification of six DKPs, including a new DKP 1 and five known DKPs 2-6 ( Figure 1 and Figure S1). We reported herein the isolation, structure elucidation of the new compound 1, as well as the full assignment of the NMR data for the known compound 2.
Compounds 1 -6 were evaluated for their cytotoxic activities against a panel of five tumour cells, including SF-268, MCF-7, NCI-H460, HepG-2 and LX-2 cells using the Sulforhodamine B (SRB) method . None of these compounds showed significant anti-proliferation activity at a concentration of 100 mM.

General experimental procedures
Materials for column chromatography were silica gel (100 -200 mesh; Jiangyou Silica Gel Development, Inc.). Thin layer chromatography (TLC) was conducted with precoated glass plates (0.1 -0.2 mm; silica gel GF254, 10 -40 nm). Semi-preparative HPLC were performed with L-2000 system (Hitachi) using a YMC-Pack ODS-A column (250 £ 10 mm, 5 mm). The analytical chiral-packed column (MCIGEL CRS10W, 4.6 £ 50 mm), and a 1260 infinity HPLC system (Agilent) were used for the chirality analysis. Low and high resolution mass spectral data were obtained on amazon SL ion trap mass spectrometer and MaXis quadrupole-TOF mass spectrometer (Bruker), respectively. Optical rotations were recorded with a MCP 300 polarimeter (Anton Paar). NMR spectra were recorded on an Aonvance 500 spectrometer (Bruker) at 500 MHz for 1 H and 125 MHz for 13 C.

Strain material
Strain SCSIO 04496 was isolated from a sediment sample collected from the South China Sea (E 12080.250 0 and N 20822.971 0 ) at a depth of 3536 m using isolation media plates containing Gauze's medium No. 1 (soluble starch 2.0%, KNO 3 0.1%, K 2 HPO 4 0.05%, MgSO 4 ·7H 2 O 0.05%, FeSO 4 ·7H 2 O 0.01%, pH 7.4) after incubation at 288C for 2 weeks. This strain was deposited in the type culture collection of the Center for Marine Microbiology, Research Network of Applied Microbiology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou, China.
The almost full 16S rDNA gene sequence (1422bp) of the strain SCSIO 04496 was submitted to GenBank under the accession number KM077017, which showed the highest similarity (98.2%) with that of Streptomyces flocculus NBRC 13041T (AB184272). The phylogenetic tree generated by a neighbour-joining method clearly revealed the evolutionary relationship of the strain SCSIO 04496 with a group of Streptomyces species ( Figure S2). On the basis of the 16S rDNA gene sequence analysis, the strain SCSIO 04496 was designated as a species of Streptomyces. Pure culture in dish and scanning electron micrographs of spore chains and aerial mycelia of strain SCSIO 04496 see Figure S3.

Acid hydrolysis
Compound 1 was dissolved in 6 N HCl (1 mL) and heated at 1108C for 18 h. After cooling to room temperature, the solvent was removed under reduced pressure, and the standard amino acids were prepared according to the published method ). The dried hydrolysate was dissolved in 100 mL of 2 mM CuSO 4 -H 2 O solution. Ten microlitres of this sample were then analysed by HPLC with a chiral column (MCIGEL CRS10W) using 2 mM CuSO 4 -H 2 O solution as the mobile phase at a flow rate of 0.5 mL/min with UV detection at 254 nm. The retention times of the N-Me-D-Leu and N-Me-L-Leu were 14.8 and 22.5 min, respectively. Thus, the N-Me-Leu residue in compound 1 was assigned as N-Me-D-Leu (14.7 min) ( Figure S10).