Isolation, Characterization, and Bioactivity Evaluation of 3-((6-Methylpyrazin-2-yl)methyl)-1H-indole, a New Alkaloid from a Deep-Sea-Derived Actinomycete Serinicoccus profundi sp. nov

One new alkaloid, 3-((6-methylpyrazin-2-yl)methyl)-1H-indole (1) was obtained from the deep-sea actinomycete Serinicoccus profundi sp. nov., along with five known compounds (2–6). Their structures were determined on the basis of detailed analysis of the 1D and 2D NMR as well as MS data. The new indole alkaloid displayed weak antimicrobial activity against Staphylococcus aureus ATCC 25923 with an MIC value of 96 μg/mL. It showed no cytotoxicity on a normal human liver cell line (BEL7402) and a human liver tumor cell line (HL-7702).


Introduction
In recent years, many novel genera and species of actinomycetes have been isolated from marine sediments [1][2][3][4]. Subsequently, more and more secondary metabolites have been discovered with novel structures and potent bioactivities [5][6][7][8][9]. This, in turn, attracted more and more attention from scientists discovering novel compounds from marine-derived actinomycetes, especially those from deep-sea sediments.
The genus Serinicoccus belongs to the family Intrasporangiaceae, suborder Micrococcineae. It was first described in 2004 [10]. Up to now only three species were found: Serinicoccus profundi sp. nov., S. marinus gen. nov., and S. chungangensis sp. nov. [10][11][12]. All of them were isolated from marine habitats. The type strain of S. profundi MCCC 1A05965 T was isolated from a deep-sea sediment of the Indian Ocean collected at a depth of 5368 m [12]. Herein, we describe the isolation and structural elucidation of a new indole alkaloid (1) from this actinomycete, together with five known compounds (2-6, Figure 1). Moreover, antimicrobial and cytotoxic activities are reported for compound 1. This is the first report on the secondary metabolites from this genus.

Bioactive Tests
Compound 1 was tested for cytotoxic activity against a normal human liver cell line (HL-7702) and another human liver tumor cell line (BEL7402) at a concentration of 50 μg/mL. However, only weak activities were found with the inhibitory rate of 6.3% on HL-7702 and 1.7% on BEL7402.

General Experimental Procedures
NMR spectra were recorded on a Bruker Avance III 400 MHz NMR spectrometer with TMS as internal standard. ESIMS were measured on a Bruker amazon SL spectrometer, and HRESIMS were measured on a Bruker En Apex ultra 7.0T FT-MS spectrometer. UV spectroscopic data were obtained on a UV-2100 PC spectrophotometer. Materials for CC were silica gel (Huiyou Silical Gel Development Co. Ltd., Yantai, China), Sephadex LH-20 (Amersham Pharmacia Biotech AB, Uppsala, Sweden), and YMC-GEL ODS-A (YMC, USA). Preparative TLC was conducted with glass precoated silica gel GF254 (Yantai, China).

Microbial Source and Culture Conditions
The microorganism MCCC 1A05965 T , identified as Serinicoccus profundi sp. nov., was isolated from a deep-sea sediment of the Indian Ocean, and was deposited in the Marine Culture Collection of China, China General Microbiological Culture Collection Center and German Collection of Microorganisms and Cell Cultures [12].

Extraction and Isolation
After 6 days of cultivation, the broth was centrifuged to separate the mycelia cake from the fermentation liquid. The liquid (10 L) was extracted by ethyl acetate (EtOAc, 10 L) for three times to afford residue A after removal of the solvent. The mycelia cake was extracted three times with acetone (1 L). After concentration under reduced vacuum, the residue was re-extracted with EtOAc (1 L) to give residue B. Based on the results of HPLC and TLC analysis, residues A and B were combined as the crude extract.

Cytotoxic Assay
Compound 1 was tested for cytotoxic activity against a human liver tumor cell line (HL-7702) and a normal human liver cell line (BEL7402) as described previously [16]. Briefly, two cell lines were seeded into a 96-well plate and kept in a humidified atmosphere of 5% CO 2 and 95% air at 37 °C . After 24 h, compound 1 was added and the incubation continued for another 48 h. An MTT solution was then added to evaluate the cell viability by measuring the optical density of the color produced by MTT dye reduction with a microplate reader at 570 nm.

Antibacterial Activity
MIC values for compound 1 were determined against four bacterial strains of Staphylococcus aureus ATCC 25923, Bacillus thuringiensis ATCC 10792, Escherichia coli ATCC 35218, and Bacillus subtilis CMCC63501 as described previously [17].

Conclusions
This is the first report on chemical constituents form Serinicoccus profundi sp. nov., a rare actinomycete from a deep-sea sediment. Six components were isolated and purified including two indole alkaloids (1, 2), two diketopiperazines (3,4), and two phenolics (5, 6). Compound 1 is a new secondary metabolite whose skeleton consists of an indole and a pyrazine. It was tested for cytotoxic and antibacterial activities. However, only a weak antibacterial activity was found on Staphylococcus aureus ATCC 25923 with an MIC value of 96 μg/mL.