Saccharobisindole, Neoasterric Methyl Ester, and 7-Chloro-4(1H)-quinolone: Three New Compounds Isolated from the Marine Bacterium Saccharomonospora sp.

Analysis of the chemical components from the culture broth of the marine bacterium Saccharomonospora sp. CNQ-490 has yielded three novel compounds: saccharobisindole (1), neoasterric methyl ester (2), and 7-chloro-4(1H)-quinolone (3), in addition to acremonidine E (4), pinselin (5), penicitrinon A (6), and penicitrinon E (7). The chemical structures of the three novel compounds were elucidated by the interpretation of 1D, 2D nuclear magnetic resonance (NMR), and high-resolution mass spectrometry (HRMS) data. Compound 2 generated weak inhibition activity against Bacillus subtilis KCTC2441 and Staphylococcus aureus KCTC1927 at concentrations of 32 μg/mL and 64 μg/mL, respectively, whereas compounds 1 and 3 did not have any observable effects. In addition, compound 2 displayed weak anti-quorum sensing (QS) effects against S. aureus KCTC1927 and Micrococcus luteus SCO560.


Introduction
Actinomycetes, a group of aerobic and anaerobic Gram-positive mycelial bacteria, are known to produce a variety of bioactive secondary metabolites. In fact, more than 70% of the currently used antibiotics were originally isolated from Streptomyces, the largest genus of actinomycetes [1]. Rare actinomycetes have also been regarded as potential sources for the discovery of bioactive compounds, including antibiotics [1]. In the past five years, 31% of new bioactive compounds were isolated from rare actinomycete strains, although the Streptomyces genus continues to dominate this field, contributing 65% of the reported bioactive compounds [2].
The genus Saccharomonospora, a rare actinomycete, was first described in 1971 [3,4] and only a few secondary metabolites have been isolated from this genus [5,6]. The strain CNQ-490, Saccharomonospora sp., was isolated from a sediment sample of La Jolla Submarine

Results
Saccharobisindole (1) was obtained as a pale, yellow oil. The molecular formula this compound (C26H28N2O3) was deduced from high-resolution fast atom bombardme mass spectrometry (HRFABMS) coupled with the detection of an ion at m/z 417.21 [M+H] + (calcd for C26H29N2O3, 417.2178) indicating 14 degrees of unsaturation. The infr red (IR) spectrum of this compound indicated the presence of a hydroxy group at 34 cm −1 , a carbonyl group at 1676 cm −1 , and a double bond at 1638 cm −1 .
Compound 3 has been reported as a synthesized product [22] but this is the first re port from a natural source (i.e., a bacterium). Compound 3 was approved as an antitumo drug for the treatment of late mammary cancer and non-small cell lung cancer by the 7-Chloro-4(1H)-quinolone (3) was isolated as a white amorphous powder and its chemical formula was assigned as C 9 H 6 35 ClNO based on the observation of a protonated molecular ion peak at m/z 180.0218 [M+H] + (calcd for C 9 H 6 35 ClNO, 180.0216) in the HRFAB mass spectrum. The 1 H NMR spectrum of 3 displayed two deshielded olefinic protons H-2 (δ H 8.01, 1H, d, J = 7.3 Hz) and H-3 (δ H 6.37, 1H, d, J = 7.3 Hz), and a 1,3,4-trisubstituted benzene ring with protons at H-5 (δ H 8.24, 1H, d, J = 8.8 Hz) and H-6 (δ H 7.42, 1H, dd, J = 8.8, 1.9 Hz), and H-8 (δ H 7.63, 1H, d, J = 1.9 Hz). The 13 C NMR spectrum exhibited nine carbons C-2 (δ C 142.2), C-3 (δ C 110.3), C-4 (δ C 179.8), C-4a (δ C 125.1), C-5 (δ C 128.3), C-6 (δ C 126.1), C-7 (δ C 142.2), C-8 (δ C 118.9), C-8a (δ C 139.8). A quinolone moiety was identified based on the interpretation of COSY and HMBC spectroscopic data. The COSY correlation of H-2 (δ H 8.01) with H-3 (δ H 6.37) and long-range HMBC correlations from H-2 to C-4 (δ C 179.8) indicated the presence of an α,β-unsaturated carbonyl group. Furthermore, three-bond HMBC correlations from H-2 to C-8a and from H-3 to C-4a, along with the carbon chemical shifts of C-2 (δ C 142.2) and C-8a (δ C 142.2), and correlations from H-6 to C-4a (δ C 125.1) and C-8 (δ C 118.9), and from H-5 to C-4, C-7 (δ C 142.2) and C-8a (δ C 139.8) allowed for the construction of the quinolone moiety. The carbon chemical shift at δ C 142.2 and the isotope ratio (3:1) of two pseudomolecular ion peaks [M + H] + and [M + H + 2] + in the LR-ESI-MS spectroscopic data ( Figure S19), allowed the attachment of a chlorine atom at C-7. Compound 3 was assigned as 7-chloro-4(1H)-quinolone (Figure 4). The structural assignment of 3 was completed by comparing the NMR data with the data from the literature [22]. Compound 3 has been reported as a synthesized product [22] but this is the first report from a natural source (i.e., a bacterium). Compound 3 was approved as an antitumor drug for the treatment of late mammary cancer and non-small cell lung cancer by the SFDA (State Food and Drug Administration of China) due to its capacity to damage DNA and block DNA synthesis in tumor cells [23]. Furthermore, 3 was found to exhibit weak inhibitory activity on cysteine protease [24].
In addition to 1-3, four known natural products were isolated and identified as acremonidin E (4), pinselin (5), penicitrinone A (6), and penicitrinone E (7) by comparing their NMR and MS spectroscopic data with those of previously reported compounds. Interestingly, the known compounds were originally isolated from fungi and had never been reported to be produced by actinomycetes. Acremonidine E (4), penicitrinon A (6), and penicitrinon E (7) were isolated from Penicillium sp. [25][26][27], whereas pinselin (5) was previously identified in the endophytic fungus Phomopsis sp. [28], the marine fungus Scopuariopsis sp. [29], and the soil fungus Penicillium sp. [30]. In this study, a largescale regrowth of the strain CNQ-490 allowed us to identify the minor compounds (1-7) produced by this strain as well as to rediscover the major compounds such as lodopyridones A-C, sacchromonopyrones A-C, and saccharoquinoline [7][8][9][10]. These data suggest that strain CNQ-490 has very good potential in producing biosynthetically-diverse secondary metabolites.

General Experimental Procedures
UV spectra were recorded in MeOH on a Scinco UVS2100 spectrophotometer. IR spectra were collected using a Nicolet iS10 FT-IR spectrometer (Thermo Scientific Inc., Waltham, MA, USA). NMR spectra were obtained using an Agilent NMR spectrometer (Agilent, Santa Clara, CA, USA, at 400 for 1 H and at 100 MHz for 13 C) and a Bruker NMR spectrometer (Bruker, Middlesex, MA, USA, at 300 for 1 H and and 75 MHz for 13 C) using the signals of the residual solvent as internal references (δ H 2.50 ppm and δ C 39.5 ppm for dimethyl sulfoxide-d 6 (DMSO-d 6 ) and δ H 4.87 and 3.31 ppm and δ C 49.1 ppm for deuterated methanol (CD 3 OD). Low-resolution LC/MS measurements were performed using the Agilent Technologies 1260 quadrupole and Waters Micromass ZQ LC/MS system using a reversed-phase column (Phenomenex Luna C18 (2) 100 Å, 50 mm × 4.6 mm, 5 µm) at a flow rate of 1.0 mL/min at the National Research Facilities and Equipment Center (NanoBioEnergy Materials Center) at Ewha Womans University. Open column chromatography was performed using silica (40-63 µm, Merck silica gel 60) eluting with a gradient solvent of dichloromethane (CH 2 Cl 2 ) and methanol (MeOH). The fractions were purified via semi-preparative HPLC using a Waters 996 Photodiode Array Detected HPLC coupled with a reversed-phase Phenomenex Luna C18 (2) (100 Å, 250 nm × 10 mm, 5µm) column at a 2.0 mL/min flow rate. High-resolution mass spectra were recorded on a JMS-700 mass spectrometer (JEOL Ltd., Tokyo, Japan) at Seoul National University.

Strain Isolation and Fermentation
Strain CNQ-490 is an actinomycete that was isolated from a marine sediment sample obtained at a depth of 45 m from a submarine canyon in La Jolla, CA. Strain CNQ-490 was assigned to the genus Saccharomonospora based on 16S ribosomal DNA gene sequence similarity analyses and BLAST searches (GeneBank accession number EU214929). A total of 80 L of CNQ-490 was cultured in 20 × 2.5 L Ultra-Yield flasks (Thomson Scientific, Oceanside, CA, USA) each containing 1 L of the medium (10 g/L soluble starch, 2 g/L yeast, 4 g/L peptone, 10 g/L CaCO 3 , 20 g/L KBr, 8 g/L Fe 2 (SO 4 ) 3 ·4H 2 O dissolved in 750 mL natural seawater and 250 mL of distilled water) at 27 • C with constant shaking at 150 rpm. After 7 days of cultivation, the broth was extracted with ethyl acetate (EtOAc, 80 L overall) and the EtOAc-soluble fraction was dried in vacuo to obtain 6.5 g of organic extract.

Purification
The organic extract of CNQ-490 was fractionated by reversed-phase C-18 flash vacuum chromatography eluting with a step gradient from 0 to 100% methanol in water, which resulted in 11 fractions. Both fractions 3 and 4 (eluted with 9% and 10% MeOH in water, respectively) were further purified by HPLC (Phenomenex 100 Å, 250 nm × 10 mm, 5 µm, UV = 285 nm), with 60% of acetonitrile in H 2 O at a flow rate of 2.0 mL/min to yield 1 (1.2 mg, t R 33.7 min), 45% of acetonitrile in H 2 O at a flow rate 2.0 mL/min to yield 2 (4.1 mg,

Detection of Anti-Quorum Sensing Activity
The 96-well plate method was employed to detect the anti-quorum sensing activity of different compounds against six bacterial strains (C. marina JEA023, M. luteus SCO560, S. aureus KCTC1927, P. aeruginosa SNC165, P. fluorescens SNA239, and A. tumefaciens SND195). S. aureus KCTC1927 and A. tumefaciens SND195 were cultured in tryptic soy broth (TSB), whereas P. aeruginosa SNC165 and P. fluorescens SND204 were cultured in King's broth (KB). C. marina JEA023 and M. luteus SCO560 were cultured in marine broth maintaining a 0.5 McFarland standard turbidity (1.0 × 10 8 CFU/mL). Dimethyl sulfoxide (100% DMSO) was used as a negative control, and kanamycin and rifampin (10 mg/mL each) were used as a positive control. Next, 50 µL of inoculum was inoculated into each well and 10 mg/mL of compound 2 and 4-7 dissolved in DMSO was diluted with marine broth media to produce concentrations ranging from 0.25 to 256 µg/mL. The mixtures of compounds and broth were dispensed into each inoculated well. The 96-well plates were then incubated for 16-18 h at 37 • C depending on the bacterial strains.

Conformational Analysis and ECD Spectrum Calculations
A conformational analysis of saccharobisindole (1) was carried out by MacroModel with the Merck molecular force field (gas phase), a 10 kJ/mol upper energy limit, and a 0.001 kJ (mol Å) −1 convergence threshold on the rms gradient to minimize computational complexity and expense. The possible enantiomers of 1 were selected based on NOESY NMR data and the energy-minimized enantiomer structures were generated by Avogadro 1.2.0. Energy minimization of the two structures was performed by Turbomole X 4.3.2. The calculated ECD spectra corresponding to two enantiomer models were calculated using DFT at the functional B3LYP/DFT level and the def-SV(P) basis set. The ECD spectra were simulated by overlapping each transition, where σ is the width of the band at 1/e height. ∆Ei is the excitation energies and Ri is rotatory strengths for transition i. In this calculation, the σ value was at 0.10 eV. The observed ECD spectrum of compound 1 showed positive cotton effect around 291 nm. Comparing the calculated spectra of the two enantiomers with the measured ECD spectrum, the absolute configurations of compound 1 were deduced as R, S.

Conclusions
The identification of the chemical components produced by strain CNQ-490 has led to the discovery of the new compounds saccharobisindole (1), neoasterric methyl ester (2), and 7-chloro-4(1H)-quinolone (3), in addition to four known compounds. Compound 1 is an isatin derivative and 2 contains two substituted benzene methyl esters with an ether linkage. Compound 3 possesses a chloroquinolone moiety and our study was the first to isolate this compound from a natural source. Additionally, our study was the first to isolate compounds 4-7, reported as fungal metabolites, from a bacterial strain. Compound 2 exhibited antibacterial activity against B. subtilis KCTC 1021 and S. aureus KCTC 1927, as well as a weak QS inhibitory activity against S. aureus KCTC 1927.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.

Conflicts of Interest:
The authors declare no conflict of interest.