Neoantimycins A and B, Two Unusual Benzamido Nine-Membered Dilactones from Marine-Derived Streptomyces antibioticus H12-15

An actinomycete strain (H12-15) isolated from a sea sediment in a mangrove district was identified as Streptomyces antibioticus on the basis of 16S rDNA gene sequence analysis as well as the investigation of its morphological, physiological, and biochemical characteristics. Two novel benzamido nonacyclic dilactones, namely neoantimycins A (1) and B (2), together with the known antimycins A1ab (3a,b), A2a (4), and A9 (5), were isolated from the culture broth of this strain. Compounds 1 and 2 are the first natural modified ATNs with an unusual benzamide unit. The structures of these new compounds, including their absolute configuration, were established on the basis of HRMS, NMR spectroscopic data, and quantum chemical ECD calculations. Their cytotoxicities against human breast adenocarcinoma cell line MCF-7, the human glioblastoma cell line SF-268, and the human lung cancer cell line NCI-H460 were also tested. All compounds exhibited mild cytotoxic activity. However, Compounds 1 and 2 showed no activity against C. albicans at the test concentration of 1 mg/mL via paper disc diffusion, while the known antimycins showed obvious antifungal activity.

During our ongoing research on bioactive secondary metabolites from Streptomyces [17][18][19], two novel nonacyclic dilactone fused with benzamide, neoantimycins A and B (1,2), and three known ANTs (3)(4)(5) were isolated from Streptomyces antibioticus H12-15 from a sea sediment collected at a mangrove district of South China Sea. Interestingly, neoantimycins A and B are the first natural modified ATNs with an unusual benzamide unit. In this paper, we describe the structure determination of two new compounds, as well as the isolation and bioactivity assay of these isolated compounds from the ethanol extract of a fermented broth of strain H12-15.
In addition, three biogenetically related compounds, antimycins A1ab (3a/b), A2a (4) [16], and A9 (5) [12], were identified by HR-TOF-MS and NMR data analyses and by comparison with other data in the literature. Unfortunately, the structural similarities of the inseparable mixtures, Compounds  , and a methyl triplet at δ 0.86 (J = 6.8 Hz, Me-6"). Comparison of its 1 H-NMR spectroscopic data of 2 with those of 1 indicated that the major difference between them was the side chain, that is, an isopropyl group with two methyl doublets at  Figure 2B). Interpretation of the COSY, HSQC, and HMBC data helped to construct the planar structure of 2, and the assignments of all proton and carbon resonances are provided in Table 1. NOE correlations between H-3 and H-4/H-8, H-7, and H-9 were observed, so the conformation of C-1~C-9 can better achieve the steric requirements. Above all, the CD spectra of Compounds 2 and 4 in methanol revealed the same Cotton effects, indicating that there is no configurational difference between these two compounds ( Figure 4). Therefore, Compound 2 was established as (3R,4S,7R,8R,9S)-3-(benzoylamino)-7-hexyl-4,9-dimethyl-1,5-dioxo-2,6-dioxonan-8-yl-isopropyl ester and named as neoantimycin B.
In addition, three biogenetically related compounds, antimycins A 1ab (3a/b), A 2a (4) [16], and A 9 (5) [12], were identified by HR-TOF-MS and NMR data analyses and by comparison with other data in the literature. Unfortunately, the structural similarities of the inseparable mixtures, Compounds 3a/b, bearing a closely related acyl group, have made their separation via HPLC purification difficult. Based on analysis of the NMR spectra, there were two different acyl groups owing to their discrete 1 H and 13 C signals, although most of the 1 H and 13 C signals about the basic skeleton of the dilactone and benzyl rings of the two components were completely overlapped [17].

Antifungal Activity
The isolated metabolites 3-5 exhibited an obvious effect against Candida albicans, while the two new compounds 1-2 showed no bioactivity against the test strain. The antifungal activity of the extract eventually formed the known antimycins.

Cytotoxic Assay
The cytotoxicity of Compounds 1-5 against the human breast adenocarcinoma cell line MCF-7, the human glioblastoma cell line SF-268, and the human lung cancer cell line NCI-H460 has been investigated by the MTT assay. Cis-dichlorodiamine platinum was used as the positive control.
According to the result of the cytotoxicity assay (Table 2), it is worth noting that the dilactone ring may be involved directly in the interaction of antimycin A with its site of action [21]. Compounds 1 and 2 showed moderate growth inhibitory activities toward the SF-268 cell line with IC 50 values of 33.6 µg/mL (68.7 µM) and 41.6 µg/mL (87.6 µM), respectively. The other compounds containing 3-aminosalicylic acid were much more potent against the three cell lines tested, especially SF-268 with IC 50 values lower than 1.6 µg/mL.

General Materials
Melting points were recorded on an X-5 micro-MP apparatus (Huayan Corporation, Shanghai, China). The optical rotations were measured with a JASCO digital polarimeter (JASCO Corporation, Tokyo, Japan) using a thermostable optical glass cell (1 dm path length and c in g/100 mL). UV spectra were taken on a JASCO V-550 UV/VIS spectrometer (JASCO Corporation, Tokyo, Japan). IR spectra were carried out with a Nicolet Impact 410-FTIR instrument (Thermo, San Jose, CA, USA) in KBr pellets. All NMR spectra were obtained on Bruker AV-300 and AV-600 spectrometer (Bruker Instrument, Inc., Zurich, Switzerland) using the residual signals (CHCl 3 : δ H 7.26/δ c 77.5) as the internal standard. HR-ESI-MS was achieved on an Agilent 6210 LC/MS TOF mass spectrometer (Agilent Technologies, Santa Clara, CA, USA). HPLC was performed on an Agilent 1200 HPLC system equipped with a diode array detector, using a Column A (Ultimate XB-C18, 5 µm, 4.6 mm × 250 mm, Welch, Potamac, MA, USA) for analysis and a Column B (Ultimate XB-C18, 5 µm, 10 mm × 250 mm, Welch, Potamac, MA, USA) for semi-preparative purification. Open column chromatography (CC) was performed on silica gel (200-300 mesh, Haiyang Chemical Group Corporation, Qingdao, China) and Sephadex LH-20 (25-100 mm, Pharmacia, Uppsala, Sweden). HSGF254 silica gel TLC plates (0.2 mm thickness, 200 mm × 200 mm, Qingdao Marine Chemical, Qingdao, China) were used as a routine analysis of fractions. 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 actinomycete strain H12-15 was isolated in 2002 from a marine sediment sample collected at the Taishan mangrove site, Guangdong province, China, and kept in a test tube with sandy soil and stored at about 4 • C in a refrigerator in the Guangdong Key Laboratory of New Technique for Plant Protection, Institute of Plant Protection, Guangdong Academy of Agricultural Sciences, Guangzhou, China, before use. Strain H12-15 was activated on a Gauserime synthetic agar medium at 28 • C. Studies on its morphological, physiological, and biochemical characteristics indicated that this strain belonged to the genus Streptomyces. It was further identified as a S. antibioticus strain based on analysis of its 16S rDNA gene sequence (see supplementary materials), which showed similarity with the sequence of S. antibioticus (GenBank accession NR043348).

Computational Details for ECD of Compound 1
The initial 3D structures of (3R,4S,7R,8R,9S,2 S)-1 and (3R,4S,7R,8R,9S,2 R)-1 were generated with Chem3D version 15.0. For each molecule, the initial 3D structure was minimized with Gasteiger-Huckel force field implemented in SYBYL version 8.0 with default parameters before its conformation space was sampled. Conformer databases were also generated using SYBYL version 8.0, with an energy window for acceptable conformers (ewindow) of 5 kcal mol −1 above the ground state using the modified version of the MMFF94 force field mentioned above, an RMSD cutoff (rmsd) of 0.1 Å. The low energy conformers accounting for more than 5% Boltzmann distribution were further optimized successively in the gas phase by a semi-empirical method and the Hartree-Fork (HF) method at the 6-31G (d) level in Gaussian 09 program package, which was reoptimized and analyzed for frequency using the density functional theory (DFT) at the B3LYP/6-31G (d, p) level, and this was also performed in the methanol, which resulted in another epimer. Solvent effects were taken into account by using the polarizable continuum model (PCM). The conformers were calculated electronic circular dichroism (ECD) by the time-dependent density functional theory (TD-DFT) method at the PBE1PBE/6-31G (2d, p) level with the PCM model in a methanol solution. The overall calculated ECD curves were generated by the Boltzmann weighting of their selected low-energy conformers. These steps were performed with software SpecDis 1.53 with σ = 0.2 eV at 25 nm shift [22][23][24].
3.6. Biological Activities 3.6.1. Antifungal Activity Antifungal assays were performed by a paper disc diffusion assay with Candida albicans as an indicator. The test samples were prepared in MeOH solutions at a concentration (1 mg/mL), and 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 in the agar Martin medium (composed of agar 18 g, glucose 20 g, peptone 5 g, yeast extract 4 g, K 2 HPO 4 ·7H 2 O 0.63 g, MgSO 4 ·7H 2 O 1.8 g, H 2 O 1 L) for 24 h at 37 • C.

Cytotoxic 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 • C in a humidified atmosphere of 5% CO 2 . The cytotoxicity of isolated compounds against three cancer cell lines was evaluated with an MTT assay with cis-dichlorodiamine platinum as a positive control. Seven concentrations (1.563, 3.125, 6.25, 12.5, 25, 50 and 100 µg/mL) of Compounds 1-5 were set for the test. Briefly, the cells were seeded at 8 × 10 4 C/mL in 96-well plates overnight. When cells fusion reached 80% of the bottom, five compounds and cis-dichlorodiamine platinum at different concentrations were added and cultured for 48 h. Subsequently, MTT dye (dissolved in deionized water) was added to the 96-well plates. Following this, the 96-well plates were incubated at 37 • C 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% (IC 50 ) were calculated using Origin 8 software (OriginLab, Northampton, MA, USA).

Conclusions
Two new modified ATNs along with three known ones (3)(4)(5) were isolated from the culture broth of Sterptomyces sp. H12-15, which was isolated from a mangrove district and identified as Streptomyces antibioticus on the basis of 16S rDNA gene sequence analysis. Absolute structures of neoantimycins A (1) and B (2) were elucidated on the basis of extensive analysis of spectroscopic data, especially quantum chemical ECD calculations. The cytotoxicities of all compounds were tested by the MTT method. Neoantimycins A (1) and B (2) indicated moderate cytotoxic activity against human glioblastoma cell line SF-268 with IC 50 values of 33.6 µg/mL (68.7 µM) and 41.6 µg/mL (87.6 µM).
Supplementary Materials: Supplementary materials are available online. IR, UV, CD, HR-TOF-MS, and NMR spectra of Compounds 1 and 2 as well as other supporting data.