Introduction

Gliding bacteria are the Gram-negative, motile bacteria which use gliding motility instead of flagella. The most studied group of the gliding bacteria are terrestrial Myxobacteria which yielded more than 500 novel chemical structures exhibiting highly interesting biological activities such as antimicrobial and cytotoxic activities. Regardless of the high biological and chemical diversities, only a few reports of gliding bacteria isolated from marine environment have been known so far. However, the number of reports on newly isolated taxa of marine gliding bacteria has been increasing during the past few years i.e. Olleya, Aureispira, Mariniflexile, Fulvivirga, Rapidithrix, Perexilibacter, Limibacter, Cellulophaga, Winogradskyella, Ekhidna, Salinimicrobium, Corallibacter, Tenacibaculum, Sunxiuqinia, Catalinimonas and Muricauda (Nichols et al. 2005; Hosoya et al. 2006; Nedashkovskaya et al. 2006, 2007, 2009, 2010; Srisukchayakul et al. 2007; Yoon et al. 2007, 2008; Kahng et al. 2009; Alain et al. 2010; Kim et al. 2012; Piñeiro-Vidal et al. 2012; Takai et al. 2012; Choi et al. 2013; Kim et al. 2013).

Interestingly, the study of secondary metabolites produced by marine gliding bacteria is still limited. Fudou et al. (2001) firstly reported the isolation of haliangicin from Haliangium luteum which showed antifungal activity against Pythium ultimum. Spyere et al. (2003) isolated the neoverrucosane diterpenoids from Saprospira grandis when cultivated in RL 1 medium in seawater but no biological activity of these compounds were reported. Iizuka et al. (2006) reported the isolation of miuraenamides from a novel Paraliomyxa miuraensis with antifungal activity against Phytophthora capsici. Our group recently reported the novel metabolites from Rapidithrix thailandica including marinoquinoline A (Kanjana-opas et al. 2006) and other pyrrole derivatives (Sangnoi et al. 2008). It is worth mentioning that marinoquinoline A is the first report of natural product obtained from the marine gliding bacterium R. thailandica. The compound exhibited a strong inhibiting effect against acetylcholinesterase (AChE) with an IC50 of 4.90 ± 0.9 μM without cytotoxicity against the human cancer cells i.e. MCF-7, HeLa, HT-29 and KB (Sangnoi et al. 2008). Recently, Oku et al. (2008) isolated polyketide-peptide metabolites, ariakemicins, from an un-identified species of Rapidithrix which showed antibacterial activity against Staphylococcus aureus. However, the compounds were not stable to maintain their antibacterial activities.

In this study, we report the isolation of additionally novel metabolite of R. thailandica (TISTR 1741) by using an antibacterial bioassay guided fractionation and spectroscopic methods.

Materials and methods

Microorganisms

Rapidithrix thailandica strain TISTR 1741 (AB265183) was isolated from the biofilm on a shell sample collected from the Yong Ling beach, Trang province, Thailand. The biofilm sample was rinsed with sterile seawater and inoculated on to a modified seawater glutamate agar medium (SWG) [l-glutamic acid monosodium salt, 1.0 g; NH4NO3, 0.01 g; K2HPO4, 0.01 g; agar 15 g and seawater, 1/L] (Hosoya et al. 2006) and the pure culture was obtained by a subculturing technique followed by a single cell isolation with a micromanipulator. The isolate was maintained on modified SAP2 agar medium [tryptone, 1 g; yeast extract, 1 g; agar, 15 g; sea water, 1/L] (Reichenbach 1991) at 25 °C. The identification of the isolate was completed by 16S rRNA gene sequence and chemotaxonomic analyses described in Srisukchayakul et al. (2007).

Fermentation of TISTR 1741 isolate

The TISTR 1741 isolate was cultivated into 40 × 250-ml Erlenmeyer flasks each containing 100 ml of modified VY/2 medium [Baker’s yeast paste, 5 g; seawater, 1/L] and 2 g of amberlite XAD-16 resins (Spyere et al. 2003) at 25 °C, 200 rpm for 7 days. The resins were separated by a filtration and rinsed twice with deionized water in order to remove salt and remaining culture medium. The resins were combined and left to dry at room temperature for 10 min before soaked twice with 2/l of methanol for 6 h at room temperature. Crude extract was obtained after filtering the resins and evaporating of the solvent phase with a rotary evaporator.

Isolation of amino phenyl pyrrolidone derivative (1)

The crude extract obtained from 4-L fermentation (0.9 g) was subjected to a size exclusion chromatography using Sephadex LH-20 as a stationary phase and 100 % methanol as a mobile phase yielded seven fractions. After the antibacterial assay, an active fraction (fraction 3; 61.5 mg) was further separated and purified by a reversed phase C-18 HPLC (Phenomenex, 250 × 10 mm, 10 μm, 30:70 MeOH in H2O, 4.5 ml/min, 210 nm) to yield compound 1 (Fig. 1) (1.2 mg, tR = 15 min).

Fig. 1
figure 1

Structures of new amino phenyl pyrrolidone derivative [3-(2-amino-phenyl)-5-methoxy-1,5-dihydro-pyrrol-2-one] (1), 3-(2′-aminophenyl)-pyrrole (2) and pistaciamide (3)

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Physicochemical properties

UV spectra were recorded on a Hewlett Peckard® 8452A diode array spectrophotometer (France). IR spectra were recorded on a Jasco® IR-810 infrared spectrophotometer (Japan). Mass spectra, both low and high resolutions, were operated on an MAT 95 XL mass spectrometer (Germany). NMR spectra were recorded on an FT-NMR, Varian Unity® Inova 500 spectrometer (Germany) at 500 MHz for 1H. The NMR chemical shifts for 1H and 13C, respectively, were referenced to the solvent peaks of δ H 7.24 and δ C 77.0 ppm for CDCl3 solution.

Antimicrobial activities

Compound 1 was tested for antimicrobial activities against nine microorganisms including methicillin-resistant S. aureus (MRSA) ATCC 43300, vancomycin-resistant Enterococcus faecalis (VRE) ATCC 51299, S. aureus TISTR 517, E. faecalis TISTR 459, Bacillus subtilis ATCC 6633, Salmonella typhi (clinical strain), Salmonella sonei, Pseudomonas aeruginosa ATCC 27853 and Candida albicans ATCC 10231. The antimicrobial assay was performed by a colorimetric microdilution broth technique using RPMI1640 medium and AlamarBlue as an indicator as described elsewhere (Boonsri et al. 2006). In brief, the tested microorganisms were prepared as suspension in RPMI1640 medium and mixed with AlamarBlue indicator at the final concentration of 1 %. The cell suspension was then transferred into a 96-well microtiter plate (100 μl/well except for first row which contained 190 μl/well). A 10 μl of the tested compound dissolved in DMSO at a final concentration of 50 μg/ml was added to each well of the first row and mixed well with a micropipette. Half of the mixtures in the first row were then transferred to the next well in the second row to perform a half-fold dilution. The dilution process was repeated until the extracts were diluted 128 times in the last row. The excess 100 μl of the mixture in the last row was discarded. The microtiter plates were incubated at 37 °C for 8–12 h and 24 h for antibacterial and antifungal assays, respectively. The antimicrobial activity was determined as the minimal inhibitory concentration (MIC) value which was the least concentration of the extract that could inhibit the growth of tested microorganisms.

Results

Isolation of amino phenyl pyrrolidone derivative (1) and its physicochemical properties

The methanolic-extract (0.9 g) of R. thailandica TISTR 1741 yielded 1.2 mg of 1 after consecutive chromatography on Sephadex LH-20 and preparative RP C-18 HPLC, respectively. The molecular formula of 1 was proposed as C11H12O2N2, according to the pseudomolecular signal at m/z 205.1 [M + H] + which was confirmed by the pseudomolecular signal at m/z 205.0970 [M + H] + (calc for C11H12O2N2 205.0977) in ESIMS spectrum. The other properties are summarized in Table 1. The 13C NMR spectrum exhibited the signals of four sp2 methines (δ 117.1, C-3′; 130.5, C-4′; 118.7, C-5′ and 130.6, C-6′) and two quarternary carbons (δ 116.9, C-1′; and 145.4, C-2′). Among these, six belonged to a phenyl moiety. From the 1H NMR spectrum (500 MHz, CDCl3) (Table 2), series of resonances assigned to an o-disubstituted phenyl moiety were observed at δ 6.73 (d; J = 8.0 Hz; H-3′), 7.18 (dd; J = 7.0, 8.0 Hz; H-4′), 6.78 (dd; J = 7.0, 7.5 Hz; H-5′) and 7.28 (m; H-6′). The evidence of primary amino group was confirmed from the IR absorption band at ν max 3,300 cm−1 and from the exchangeable proton resonance at δ 4.36 (br s). A primary amino group was proposed to attach to C-2′ (δ 145.4) of a phenyl ring moiety due to the low-fielded chemical shift. Another moiety, 13C NMR spectrum demonstrated the signal at δ 171.7 (C-2) indicated the amide carbonyl group. This was clearly confirmed from the IR absorption band at ν max 1,700 cm−1 and the broad signal at δ 6.22 (br s) indicating the presence of carbonyl and an exchangeable proton, respectively. The remaining resonances of one sp2 methine (δ 6.96 s; H-4), one oxygenated methine (δ 5.59 s; H-5) and an O-methoxyl group (δ 3.35 s; H-6) were put together and established the presence of pyrrolidone moiety which clearly confirmed by HMBC correlations from H-4 (δ 6.96) to C-2 (δ 171.7) and C-5 (δ 84.0), from H-5 (δ 5.59) to C-4 (δ 140.9) and C-6 (δ 52.3) and from H-6 (δ 3.35) to C-5 (δ 84.0). Connection of phenyl and pyrrolidone moieties was observed from the correlation between H-6′ (δ 7.28) and C-3 (δ 140.5). Compound 1 was therefore proposed as a new amino phenyl pyrrolidone derivative [3-(2-Amino-phenyl)-5-methoxy-1,5-dihydro-pyrrol-2-one].

Table 1 Physico-chemical properties of 3-(2-amino-phenyl)-5-methoxy-1,5-dihydro-pyrrol-2-one
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Table 2 1H and 13C NMR spectral data of 3-(2-Amino-phenyl)-5-methoxy-1,5-dihydro-pyrrol-2-one (500 MHz for 1H; CDCl3)
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Biological activities

Antimicrobial assay of 1 against a panel of microbial strains including five Gram-positive bacteria (MRSA, VRE, B. subtilis, E. faecalis and S. aureus), three Gram-negative bacteria (P. aeruginosa, S. sonei and S. typhi) and yeast (C. albicans) showed selective inhibitory activity against VRE (MIC 5.97 mM) followed by S. aureus (MIC 12.19 mM) as shown in Table 3. However, compound 1 showed no an inhibitory activity against neither Gram-negative bacteria nor yeast used in this study.

Table 3 Antimicrobial spectrum of the 3-(2-amino-phenyl)-5-methoxy-1,5-dihydro-pyrrol-2-one
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Discussion

Compound 1 isolating from the marine gliding bacterium R. thailandica TISTR 1741 showed a similar structure to 3-(2′-aminophenyl)-pyrrole (2) which was isolated from another strain of R. thailandica as previously reported by our group (Sangnoi et al. 2008). In addition, compound 1 exhibited a relative structure with pistaciamide (3) which was isolated from plant, Pistacia chinensis (Lui et al. 2008). Sangnoi et al. (2008) reported the acetylcholinesterase (AChE), which had regulated the brain’s cognitive functions, inhibitory activity and cytotoxicity of 2. It was inactive in the AChE inhibition and in the cytotoxicity bioassays (enzyme inhibition <30 % at 0.1 g/l, > 80 % cell viability at 20 g/l, respectively). Compound 2 showed no antimicrobial activities (data not shown) whereas no biological activities of 3 were reported. Unfortunately, we could not determine the AChE inhibition and the cytotoxic activity of 1 due to the scantiness of the compound available. The AChE inhibitory activities of 1 and 3 may be similar to that of 2 because of the structural similarity. However, the pyrrolidone derivatives belong to a racetam family which is known to have important value in pharmaceutical industry (Lui et al. 2008). A limited number of racetam (pyrrolidone derivatives) have been developed for clinical use as nootropic drugs that mean enhancement of learning and memory (Shorvon 2001). Most drugs in the pyrrolidone (racetam) class stimulate acetylcholine production and turnover. The examples of drugs in this class that go to a clinical development are such as aniracetam, fasoracetam and nefiracetam were used as anti-Alzheimer’s disease agents (Fujita et al. 2002; Shorvon 2001). Piracetam, oxiracetam, nebracetam and pramiracetam were used for cognition enhancer whereas brivaracetam, seletracetam and levetiracetam were used for antiepilepsy (Kenda et al. 2004; Matagne et al. 2009; Shorvon 2001). The further investigation on biological activities like racetam family of 1 should also be considered.