Secondary metabolites from marine-derived Streptomyces antibioticus strain H74-21

Abstract A new secondary metabolite, (2S,3R)-l-threonine, N-[3-(formylamino)-2-hydroxybenzoyl]-ethyl ester (streptomyceamide C, 1), together with four known compounds 1, 4-dimethyl-3-isopropyl-2,5-piperidinedione (2), cyclo-((S)-Pro-8- hydroxy-(R)-Ile (3), cyclo-((S)-Pro-(R)-Leu (4), and seco-((S)-Pro-(R)-Val) (5), were isolated from the EtOH extract of the fermented mycelium of the marine-derived streptomycete strain H74-21, which was isolated from sea sediment in a mangrove site. The structure of the new compound was established on the basis of its spectroscopic data, including 1D and 2D NMR, HR-TOF-MS. Their antifungal activities against Candida albicans and cytotoxicities against human breast adenocarcinoma cell line MCF-7, human glioblastoma cell line SF-268 and human lung cancer cell line NCI-H460 were tested. Compounds 1 only displayed cytotoxicity against human breast adenocarcinoma cell line MCF-7 with the IC50 value of 27.0 μg/mL. However, compounds 1–5 do not show antifungal activities at the test concentration of 1 mg/mL, and 2–5 have no cytotoxicities at the test concentration of 50 μg/mL.


Introduction
Microbial secondary metabolites are one of the main sources of bioactive natural products, and are also one of the main sources of drugs or leads (Yang et al. 2009;Amedei & D'Elios 2012). It is estimated that around 60% of all known antibiotics are derived from secondary metabolites produced by filamentous actinomycete bacteria, most notably Streptomyces species (Javed et al. 2011;Sun et al. 2014;Deng & Lu 2016). Marine environment has characteristic features of high salinity, high pressure, low temperature, low nutrition and less lightness, so marine-derived micro-organisms have a diversity of species, and their particularities are different from those of terrestrial micro-organisms. Marine-derived Streptomyces are a large group of marine micro-organisms. It has an outstanding performance in the production of bioactive substances (Chen et al. 2015;Sivaperumal et al. 2015;Yang et al. 2015) and drug discovery. Previous chemical studies of marine Streptomyces antibioticus strain reported the isolation of a number of bioactive natural products such as indanomycin-related antibiotics, simocyclinones, saphenamycin and actinomycins (Garkavenko & Dukhovnaya 1975;Adrian et al. 1988;Schimana et al. 2001;Lian & Zhang, 2013). Some of these antibiotics exhibited antibacterial activities, others are of antifungal, or antiviral activities. We obtained an antifungal marine-derived Streptomyces H74-21 by activity screening, and we want to isolate some new antifungal compounds from H74-21. In this paper, we describe the isolation and structural elucidation of a new secondary metabolite, (2S,3R)l-threonine, N-[3-(formylamino)-2-hydroxybenzoyl]-ethyl ester (1), together with four known compounds 1,4-dimethyl-3-isopropyl-2,5-piperidinedione (2) (Luo et al. 2016), cyclo-((S)-Pro-8-hydroxy-(R)-Ile (3), cyclo-((S)-Pro-(R)-Leu (4), and seco-((S)-Pro-(R)-Val) (5) (Renner & Cheng 1999)

Characterisation of the compounds
Compound 1 was isolated as yellowish oil. , which requires 7° of unsaturation. The 13 C NMR and DEPT NMR spectra of 1 showed 14 signals: two CH 3 , one CH 2 , six CH, and five quaternary C-atoms. The 1 H NMR data (see Table 1s) of 1 exhibited aromatic-ring resonances at δ H 8.30 (1H, d, J = 7.9 Hz, H-4), 7.64 (1H, d, J = 7.9 Hz, H-6), and 6.91 (1H, t, J = 7.9 Hz, H-5), indicating three H-atoms successively in ortho-positions. The HMBC experiment of 1 showed correlations between the proton signal at δ H 8.30 (1H, d, J = 7.9 Hz, H-4) and the carbon signals at δ C 152.6 (C-2), 128.2 (C-3) and 123.5 (C-6), between those protons at δ H 6.91 (1H, t, J = 7.8 Hz, H-5) and the carbons at δ C 152.6 (C-2), 128.2 (C-3) and 115.7 (C-1), and between the signals at δ H 7.64 (1H, d, J = 7.9 Hz, H-6) and δ C 171.9 (C-7), 152.6 (C-2), 126.4 (C-4) and 115.7 (C-1), respectively. These further confirmed the mutual ortho arrangement of the aromatic H-atoms, in accord with a 2-oxygenated, 3-substituted benzoic acid (Zhao et al. 2006). The 3-substituent at the aromatic ring was speculated to be an NH group based on the molecular formula and the chemical shift for δ C 128.2 (C-3). Further, the long-range correlation between the formyl H-atom at δ H 8.37 (1H, s, H-12) and C-atom at δ C 128.2 (C-3) confirmed that this H-atom was linked with the 3-NH group on the aromatic ring through an amide function (formamide). In addition, the 1 H, 1 H-COSY spectrum of 1 showed that H-8 (1H, d, J = 6.9 Hz) was correlated with H-13 (1H, m), and H-13 (1H, m) with H 3 -14 (3H, d, J = 7.3 Hz), giving a CH-CHCH 3 moiety. And H 2 -10 (2H, q, J = 7.1 Hz) was correlated with H 3 -11 (3H, t, J = 7.1 Hz), giving a CH 2 CH 3 group. Moreover, another amide group was identified by correlations between the CH proton at δ H 4.71 (1H, d, J = 6.9 Hz, H-8) and carbonyl carbon at δ C 171.9 (C-7). An ester group was identified by correlations between the oxygenated CH 2 protons resonance at δ H 4.21 (H 2 -10) and carbon at δ C 172.1 (C-9), between the oxygenated methine proton at δ H 4.41 (H-13) and carbonyl carbon at δ C 172.1 (C-9), respectively. The relative configuration of 1 was established by a NOESY experiment: although NOE correlation between H-8 and H-13 was not observed in methanol, there was NOE correlation between H-8 and 13-OH in DMSO-d 6 . Moreover, the coupling constant value of H-8 with H-13 was 7.2 Hz in DMSO-d 6 . We calculated and compared the configurations between C-8 and C-13 with the lowest energy of compound 1 in ChemDraw, and believed that threo is a rationale one for the relative configuration between H-8 and H-13 with the consideration of the J value and dihedral angle ( Figure 12s). Moreover, weak proton correlation between H-8 and H-13 is typical pattern of l-threonine. From these data, compound 1 was determined to be (2S,3R)-l-threonine,N-[3-(formylamino)-2-hydroxybenzoyl]-ethyl ester, and named as streptomyceamide C. Structurally, streptomyceamide C is same as the partial structure of antimycins, several known ones of which we have isolated from the same cultural material now and other strain in literature (Xu et al. 2011). Thus, we reasonably assume that streptomyceamide C is the precursor of antimycins with biosynthesis consideration (Seipke & Hutchings 2013).
Compound 2 was early from degradation of peptides (Mauger 1971) or synthesised (Mauger et al. 1972), and the structure was deduced using elemental analysis and GC-MS method. We report for the first time the isolation of 2 from the marine-derived Streptomyces H74-21 as a natural product and the determination of their structures on the basis of whole spectroscopic data.

Biological activity
The compounds 1-5 do not show antifungal activities against Candida albicans at the test concentration of 1 mg/mL with the paper disc diffusion method, though the crude extract displayed a moderate activity against C. albicans. The antifungal activity of the extract may come from the known antimycins (see Table 3s), which we have isolated from the extract. The cytotoxicity of these five compounds against human breast adenocarcinoma cell line MCF-7, human glioblastoma cell line SF-268, and human lung cancer cell line NCI-H460 had also been investigated using an MTT assay. Only compound 1 displayed cytotoxicity against human breast adenocarcinoma cell line MCF-7 with the IC 50 value of 27.0 μg/mL (see Table  4s).

General experimental procedures
Optical rotations were obtained using a JASCO P-1020 automatic digital polarimeter (JASCO Corporation, Tokyo, Japan). uV spectra were taken on a JASCO V-550 uV/VIS spectrometer (JASCO Corporation). IR spectra were recorded on a Fourier Transform IR-480 plus infrared spectrometer (JASCO Corporation). 1D and 2D NMR spectra were recorded in CD 3 OD or CDCl 3 using Bruker AV-300 spectrometer (Bruker Instrument, Inc, Zurich, Switzerland) and in DMSO-d 6 using Bruker AV-600 spectrometer (Bruker Instrument) with tetramethylsilane (TMS) as the internal standard, and the chemical shifts were expressed in δ values (ppm). HR-ESI-MS data were obtained from an Agilent 6210 LC/MS TOF mass spectrometer (Agilent Technologies, Santa Clara, CA, uSA). LR-MS were obtained from an LCQ Advantage Max mass spectrometer (Thermo Finnigan, San Diego, CA, uSA). Reversed phase HPLC was performed on a column XB-C18 (5 μm, 4.6 mm × 250 mm, Welch Materials, Inc) for analysis and ultimate TM XB-C18 (5 μm, 10 mm × 250 mm, Welch Materials, Inc, Shanghai, China) for the semi-preparation of sample purification. Open column chromatography was performed on silica gel (300-400 mesh, Qingdao, Haiyang Chemical Group Corporation, Qingdao, China) and ODS (50 mm, YMC, Tokyo, Japan). Thin-layer chromatography was performed on precoated aluminium silica gel plate (aluminium gel HSAF254, 1 mm, Yantan, China). The spraying reagent used for TLC was 10% H 2 SO 4 in EtOH. All the other reagents and solvents were purchased from Tianjin Damao Chemical Company (Tianjin, China).

Strain isolation and identification
The Streptomycete H74-21 was isolated from the sea sediment about 1-2 m depth at Yamen Bridge district of XinHui, GuangDong, China. The temperature of sea water at the site for collecting sediment sample is about 29 °C and pH are about 8.2. H74-21was kept in a sandy soil tube and stored at about 4 °C refrigerator before use in the Guangdong Key Laboratory of New Technique for Plant Protection, Institute of Plant Protection, Guangdong Academy of Agricultural Sciences, Guangzhou, China. Strain H74-21 was activated on Gauserime synthetic agar medium at 28 °C. It was identified to be a S. antibioticus strain based on its 16S rDNA (see Figure 24s), as well as its morphological, cultural, and physiological and biochemical characteristics (Fu et al. 2016).

Antifungal activity
Antifungal assays were assessed with a paper disc diffusion assay. The test samples were prepared in MeOH solutions and six concentrations (31.25, 62.5, 125, 250, 500 and 1000 μg/mL) were set for the test, the filter paper discs (6 mm) were soaked in 10 μL of the solutions. Then, the discs were placed on inoculated agar plates after evaporating the solvent and incubated for 24 h at 37 ºC for Candida albicans. The mycelium extract of Streptomyces anandii, the corresponding compounds 1-5 and two known antimycins were examined.

Cytotoxicity assay
Three cancer cell lines MCF-7, SF-268, and NCI-H460 were obtained from School of Medicine, Jinan university (Guangzhou, China), and were cultured in RPMI 1640 medium (Life Technologies, Grand Island, NY, uSA) supplemented with 10% fetal bovine serum (FBS, Gibco, Carlesbad, CA, uSA), 100 u/mL penicillin and 100 μg/mL streptomycin (Invitrogen, Carlsbad, CA, uSA). Cells were cultured at 37 ºC in a humidified atmosphere of 5% CO 2 . The cytotoxicity of isolated compounds against three cancer cell lines was evaluated by an MTT assay with cis-dichlorodiamine platinum as a positive control. Seven concentrations (1.56, 3.13, 6.25, 12.5, 25, 50 and 100 μg/mL) of compounds 1-5 in DMSO were set for the test. Briefly, the cells were seeded at 8 × 10 4 /mL in 96-well plates overnight. When cells fusion reached 80% of the bottom, five compounds and cis-dichlorodiamine platinumat different concentrations were added, and cultured for 48 h. Subsequently, MTT dye (dissolved in deionised water) was added to the 96-well plates. Following this, the 96-well plates were incubated at 37 ºC for another 4 h. The liquid supernatant was removed softly and DMSO was added (100 μL per well). The absorbance at 570 nm was monitored using a microplate reader (Bio-Rad, Hercules, CA, uSA). The concentrations required to inhibit cell growth by 50% (IC50) were calculated using Origin 8 software (OriginLab, Northampton, MA, uSA).

Disclosure statement
No potential conflict of interest was reported by the authors.