Angucycline antibiotics and its derivatives from marine-derived actinomycete Streptomyces sp. A6H

Abstract Vineomycin A1 (1) and B2 (2) were isolated from the culture broth of marine actinomycete Streptomyces sp. A6H. Five hydrolysis products were obtained by rational hydrolysis and methanolysis of the fermentation extract. Their structures were characterised as aquayamycin (3), vineomycinone B2 (4), 9-C-D-olivosyltetrangulol (5), 7-O-methylgaltamycinone (6) and vineomycinone B2 methyl ester (7). In addition to these compounds, two ester derivatives, vineolactone A (8) and vineomycinone B2 benzyl ester (9) of compound 4 were generated semisynthetically. Compound 6 is a new analogue of galtamycinone, while compounds 8 and 9 are new members of vineomycins. Cytotoxic activities and antimicrobial activities were determined for all compounds. The results indicate that only compound 1 showed significant activities with IC50 value of 0.34 μM against H1975 and MIC value of 4 μg/mL against Staphylococcus aureus.


Introduction
Marine micro-organisms are increasingly a focus of natural products research due to their unique physiological features enabling them to survive under extremes of temperature, salinity and pressure. Actinomycetes are known for their unprecedented ability to produce novel metabolites with potent bioactivities (Blunt et al. 2013;Kodani et al. 2013;Lian & Zhang 2013;Pu et al. 2013;Rao et al. 2013;Yang XQ et al. 2013;Yang Y et al. 2013;Abdelmohsen et al. 2014;Ai et al. 2014;Awad et al. 2014;Shaaban et al. 2014;Sun et al. 2014;Yang et al. 2014;Zhang et al. 2014;Zhou, Yang, Peng, et al. 2014;Balachandran et al. 2015).
During the course of our screening programme for antitumour antibiotics from marine-derived actinomycetes, we found a strain classified as Streptomyces sp. A6H produced vineomycin A 1 (1) and B 2 (2), which were first isolated by Omura et al. in 1977 from Streptomyces matensis subsp. vineus (Omura et al.1977). These compounds have anthracycline chromophores and sugar moieties, possessing antimicrobial activities against Gram-positive bacteria and antitumour activities against S-180 solid tumour on mice. Vineomycin A 1 was also found to inhibit prolyl hydroxylase (Okazaki et al. 1981). So far, the total synthesis of vineomycinone B 2 methyl ester and vineomycin B 2 has been accomplished due to their important biological activities and novel molecular architecture (Chen et al. 2006;Kusumi et al. 2013).
In the present study, we detected a series of vineomycins by uV spectrum comparison, but failed to separate them because of their extremely similar physicochemical properties as well as sensitivity to both acid and base. Fortuitously, we found the hydrolysates still have significant cytotoxicity. Hence, we rationally designed the hydrolysis and methanolysis of the crude extract and yielded five stable hydrolysis products (3-7, Figure 1). To acquire more bioactive analogues, we therefore generated two ester derivatives (8 and 9) of compound 4 semisynthetically. We report herein on the isolation and structure elucidation of these compounds and their antitumour and antimicrobial activities.

Results and discussion
The actinomycetes were isolated from a sediment sample collected in Taiwan Strait, China. In the course of antitumuor screening, etOAc extract of strain A6H displayed significant cytotoxicity. The taxonomic identity of the strain A6H was determined by 16S rDNA sequence analysis. The top sequence of A6H was 99% sequence similarity to two Streptomyces cellulosae strains NRRL B-2889 and NBRC 13027 (accession numbers: NR_043815.1 and NR_112346.1) and 99% sequence similarity to other nine Streptomyces sp. strains (Table S 1 ) in the GenBank database.
Compound 6 was isolated as a brown solid. Its molecular formula (C 26 H 24 O 8 ) was determined upon analysis of the HReSIMS peak at m/z 465.1543 [M + H] + (calcd for C 26 H 25 O 8 , 465.1549). The uV spectrum of 6 showed absorption peaks at 319, 260, 227 nm, indicating the presence of a highly conjugated system. Analysis of the 1 H and 13 C NMR spectroscopic data (Table S 2 ) for 6 revealed the presence of olivose moiety as the same as other known compounds. The remaining signals consisted of two carbonyls, sixteen aromatic carbons, including five aromatic methines, two hydroxyl groups, a methyl group and a methoxy group. These signals were similar to those of galtamycinone (Takashi et al. 1997). The main difference was that a methoxy group replaced a hydroxyl group at C-7, which was confirmed by NOeSY correlations of 14-CH 3 to both H-6 and H-8. There was also a big different chemical shift of H-6 between galtamycinone and compound 6. In the HMBC spectrum, H-6 showed correlations to C-5, C-7, C-10a and C-11a, which confirmed its position. The structure of compound 6 was thus assigned as 7-O-methylgaltamycinone.
Compound 8  In the 1 H NMR, the signal corresponding to 1-OH of compound 4 was missing, while the signal of 2″-OH was still detectable (Table S 2 ). The HMBC correlations from 3-OH (2″-OH in compound 4) to C-2, C-3 and C-4 confirmed the position of 3-OH in compound 8. Additionally, big different chemical shifts of the aromatic ring between compounds 4 and 8 were observed. Consequently, the esterification reaction took place between -COOH and 1-OH.
Compound 9 was obtained as yellow powder by treating compound 4 with benzyl bromide under KOH (Lee et al. 2006). Its HReSIMS peak at m/z 577.2068 [M + H] + indicated the addition of a molecule of benzyl. The signals of benzyl were also observed in 1 H and 13 C NMR data (Table S 2 ). The addition of benzyl to carboxy group was confirmed by HMBC correlation between benzyl CH 2 (δ H 5.15) and carboxy carbon (δ C 172.6).
The cytotoxic activities were assayed for all compounds against human lung cancer cell line H1975, gastric cancer SGC-7901 and oesophageal cancer eC109 by CCK8 kit. Only compound 1 showed activity inhibiting the proliferation of all three cancer cell lines. IC 50 value of the active compound against HI975 was further determined and the result turned out to be 0.34 μM.
The compounds (1-9, except 2) were also assayed for their antimicrobial activities against Staphylococcus aureus, methicillin-resistant S. aureus, Pseudomonas aeruginosa, Canidia albicans and Fusarium graminearum by micro broth dilution method (Table S 3 ). Only compound 1 had activities against S. aureus and methicillin-resistant S. aureus. None of the tested compounds showed activities against P. aeruginosa, C. albicans and F. graminearum. The MIC values of compound 1 against S. aureus and methicillin-resistant S. aureus both were 4 μg/mL, and the MIC values of positive control vancomycin against S. aureus and methicillin-resistant S.aureus were 2 and 1 μg/mL, respectively.
In the current study, nine angucycline antibiotics including three new ones were obtained, among which compound 1 showed promising antitumour and antimicrobial activities. The main difference in structure between compounds 1 and 2 was the open side chain. We proposed that the exposure of carboxy group resulted in poor solubility and weak bioactivity. Thus, we semisynthetically generated two ester analogues of compound 4, the stable aglycone of compound 2. However, the bioactivities of compounds 8 and 9 turned out to be disappointing. This result in turn demonstrated that the R group was the key point to the bioactivity of compound 1.

Large-scale fermentation and extraction
Spores of strain A6H were inoculated to 18 500-mL erlenmeyer flasks containing 250-mL medium described above and shake cultured for 3 days at 28 °C. Afterwards, 5-mL portions of the culture were inoculated to 500-mL erlenmeyer flasks containing 250 mL of the same medium. A total of 180L fermentation was carried out at 28 °C on a rotary shaker at 180 rpm for 7 days. Then the fermentation broth was combined and filtered. And the filtrate was extracted with etOAc 3 times and dried in vacuo to provide the crude extract (15g).

Methanolysis
The combined mixture (100 mg) was dissolved in 0.1N HCl-MeOH (5 mL) and stirred at 40 °C for 10 h. The reaction mixture was neutralized with saturated NaHCO 3 and evaporated to dryness. The residue was resuspended with 10 mL water, extracted with etOAc (10 mL × 3), washed with water and concentrated to dryness. The products were subjected to silica gel chromatography to yield compound 7 (9 mg) as an orange powder.

Synthesis of 8 from 4
A solution of compound 4 (24.3 mg, 0.05 mmol) and trimethylamine (24 µL, 0.175 mmol) in 2 mL THF and 8 mL CH 2 Cl 2 was added over 6 h to a solution of 2,4,6-trichlorobenoyl chloride (11.8 µL, 0.075 mmol) and DMAP (6.5 mg, 0.055 mmol) in CH2Cl2 (1 mL). The solution was then allowed to stir for 1h after complete addition after which time it was quenched with H 2 O (10 mL). The phases were separated and the aqueous phase was extracted with CH 2 Cl 2 (2 × 10 mL). The organic phases were combined and the solvent was removed via rotary evaporation to yield a brown solid which was purified by preparative HPLC to yield compound 8 (10 mg, 42%) as a yellow powder.

Synthesis of 9 from 4
A solution of compound 4 (4.86 mg, 0.01 mmol) and KOH (1.8 mg, 0.032 mmol) in DMF (2mL) was treated with benzyl bromide (3 µL, 0.025 mmol) and stirred at 40 °C for 4 h. The reaction mixture was diluted with water and then extracted with CH 2 Cl 2 three times. The organic phases were combined, washed with water ten times and dried in vacuo to yield a brown oil which was purified by preparative HPLC to yield compound 9 (3.2 mg, 56%) as a yellow powder.

Antimicrobial assay
Antimicrobial activities were measured against S. aureus ATCC25923, methicillin-resistant S. aureus ATCC43300, P. aeruginosa ATCC27853, C. albicans ATCC10231 and F. graminearum by broth microdilution method. The microbial cultures were prepared by diluting precultured broths to 5 × 105 cfu/mL. The assay was performed in 96-well plates. each compound was tested in dilution series ranging from 64 to 0.0625 μg/mL. The plates were incubated at 35 °C for 24 h. Then the MICs were recorded as the lowest concentrations that completely inhibited bacterial growth, assessed with visual inspection.

Cytotoxicity assay
Human cancer cell lines H1975, SGC-7901 and eC109 were cultured in 1640 medium (Gibco Company) with 10% FBS (Hyclone Company) at 37 °C in a humidified incubator (Thermo Company) with 5% CO 2 . The cells (5 × 103/well) were inoculated in 96-well plates and treated with compounds for 24 h. Then 10 µL of CCK8 (Dojindo Company) was added and reacted for 1-3h. The OD values were measured at 450nm using a Multiskan Spectrum (Plus 384, MD Company).