Efficient Synthesis and Antibacterial Evaluation of (±)-Yanglingmycin and Its Analogues

An efficient synthetic route was developed for the large-scale preparation of (±)-Yanglingmycin and its analogues. Three series of derivatives of (±)-Yanglingmycin were synthesized and the structures of all compounds were elucidated by analyses of NMR and ESI-MS spectra data. Moreover, their antibacterial activities against seven species of bacteria were systematically evaluated by the micro-broth dilution method, most of which displayed considerable activity. It was worth noting that compounds 5b, 5c, 5d, 6g, and 7 were found to be the most promising leading candidates, with peak MIC values of 0.98 μg·mL−1 for Bacillus subtilis, which is superior to positive controls (MIC = 3.91 μg·mL−1). The above results might lay the firm foundation for the design and synthesis of novel antibacterial drugs based on (±)-Yanglingmycin.


Introduction
Since the 1940s, antibiotics have been widely used in daily life mainly due to their key role in prevention and control of human, animal, and plant diseases, etc. [1]. Since the effective targets of the antibiotics are still rather limited [2], the abuse of antibiotics resulted in the target sites becoming less sensitive. This context has led to the emergence of antibiotic resistance, for higher doses of antibiotics required to successfully cure certain bacterial infections, with some antibiotics losing their antibiotic activity completely [3,4]. In 2013, biologically-active natural product (´)-Yanglingmycin ( Figure 1) was carefully isolated from the fermentation broth of Streptomyces djakartensis by our group [5]. As a 2-aryl-substituted 4,5-dihydrooxazol derivative, (´)-Yanglingmycin has been found to possess wide-range functionality and exhibited a variety of biological activities useful for applications, such as pharmaceutical drugs, polymeric materials, insecticides, and so on [6,7]. As reported by us recently, both (´)-Yanglingmycin and (+)-Yanglingmycin exhibited satisfying antibacterial activity for Pseudomonas syringae pv. Actinidiae and Ralstonia solanacearum with peak MIC values 7.81 and 15.62 µg¨mL´1, respectively [8]. As far as the antibacterial data of Yanglingmycin was concerned, we postulated that this compound has the potential to act as the lead compound for the development of new antibacterial drugs. Unfortunately, the content of (´)-Yanglingmycin in Streptomyces djakartensis fermentation broth was less than 2 µg¨mL´1, obviously, which could not provide enough pure target compound for our further study. In the last decades, the total synthesis of oxazole compounds has attracted more and more attention of researchers

Chemistry
General unless otherwise noted, all reagents and solvents were purchased from commercial suppliers and used without further purification, which were of analytical reagent (AR) grade. TLC was performed on GF 254 silica gel plates (Qingdao Haiyang Co., Ltd., Qingdao, Shandong, China). Column chromatography was carried out with silica gel (Qingdao Haiyang Co., Ltd.); all compounds were eluted with petroleum ether and ethyl acetate in sequence. Melting point (m.p.) was determined on a Yanagimoto apparatus (uncorrected). 1 H-NMR and 13 C-NMR spectra were performed on a Bruker-Avance-500 spectrometer (Bruker Daltonics Inc., Bremen, Germany) with DMSO-d 6 or CDCl 3 as solvent and SiMe 4 (tetramethylsilane) as the internal standard. MS were recorded on an electrospray ionization (ESI) conditions by using a Thermo LCQ Fleet instrument (Thermo Fisher Scientific Co., Waltham, MA, USA). HR-MS were recorded on an Agilent 1290-6224 instrument (Agilent, Santa Clara, CA, USA).

Synthesis of 4a-4h
The solution of substituted benzonitrile (100 mmol), serinol (54.660 g, 600 mmol), and Na 2 CO 3 (10.599 g, 100 mmol) in anhydrous methanol (100 mL) was heated to reflux for 10 h. After the reaction solution was cooled to room temperature, the solvent was removed under vacuum, the resulting residue was then diluted with anhydrous CH 2 Cl 2 (30 mL). The organic fractions were successively washed with an aqueous saturated solution of NH 4 Cl (30 mLˆ2), brine (30 mLˆ3), and dried over anhydrous Na 2 SO 4 . The solvent was got rid of under vacuum to afford a scarlet residue which was purified by flash column chromatography using petroleum ether/ethyl acetate (8:1, v/v) as the eluent.

Synthesis of 5a-5z
A solution of compound 4a (193 mg, 1.0 mmol), carboxylic acid (1.2 mmol), EDC¨HCl (288 mg, 1.5 mmol), and DMAP (6 mg, 5%) in anhydrous CH 2 Cl 2 (8 mL) is stirred at room temperature for 6 h. Then, the reaction solution was quenched with saturated aqueous NaHCO 3 (50 mL), concentrated under reduced pressure until approximately 30 mL remained. The organic layer was separated with CH 2 Cl 2 (30 mLˆ3) and washed sequentially with water (30 mLˆ2), brine (30 mLˆ3), and dried over anhydrous Na 2 SO 4 . The solvent is concentrated under reduced pressure, and purified by flash column chromatography using petroleum ether/ethyl acetate (10:1, v/v) as the eluent.        173.67, 166.70, 159.94, 133.70, 128.15, 118.71, 116.86, 110.32, 69.10, 65.14, 64.29, 34.14 13 134.10, 133.72, 130.22, 129.45, 128.37, 128.27, 126.86, 118.83, 116.39, 110.38, 69.27, 65.69, 64.54. The structure of 5n was also confirmed by 2D NMR data and can be seen in Table 3 and Figure 2.                 was also confirmed by 2D-NMR data and can be seen in Table 4 and Figure 3.   Table 5 and Figure 4.  Antibacterial activities were evaluated by the micro-broth dilution method in 96-well culture plates using the Mueller-Hinton broth, according to the National Committee for Clinical Laboratory Standards [20,21]. The tested bacteria were incubated in the Mueller-Hinton broth at 30˝C at 190 rpm for 12 h and the spore concentration was diluted to approximately 1ˆ10 5 -1ˆ10 6 CFU/mL with Mueller-Hinton broth. After incubation at 30˝C for 24 h, the MICs were examined. Briefly, bacteria were grown to mid-log phase, diluted with fresh Luria-Bertani culture broth to an optical density of 0.08-0.1 at 600 nm (OD 600 ) and diluted again 1:100. This suspension (50 µL) was added to wells in a 96 well microtiter plate (Sarstedt) and 50 µL of compound dissolved in DMSO-water was added to give a final concentration of from 125 to 0.245 µg¨mL´1 and at most 1‰DMSO by volume. A DMSO negative control and standard antibiotic positive controls (Ampicillin Sodium, 10 µg¨mL´1) were included in each plate. All compounds were tested in triplicate for each concentration. Plates were sealed with parafilm and incubated at 37˝C (Pseudomonas syringae pv. actinidiae and Pseudomonas solanacearum E. F. Smith at 28˝C) for 12-16 h. The OD 600 values for each well were determined with a plate reader (Shimadzu, UV1800, Kyoto, Japan) and the data were standardized to the Luria-Bertani culture broth control wells.

Conclusions
A novel, efficient, synthetic method was developed for the large-scale preparation of (˘)-Yanglingmycin and its analogues. In this way, arylnitriles reacted with serinol under the condition of sodium carbonate in dry methanol to obtain 2-aryl substituent of 4,5-dihydrooxazol analogues with good yields. Series of (˘)-Yanglingmycin derivatives were designed and synthesized as potent antibacterial compounds, among which 5b, 5c, 5d, 6g, and 7 were identified as the most promising candidates with good antibacterial activity, but 4b-h did not possess antibacterial activity at all. This result indicated that the alternation in antibacterial activity of these compounds was dominated by unsaturated bond, electronic interaction, intramolecular hydrogen bond, and stereoscopic effect. Neither electron-drawing nor electron-donating groups could perish the antibacterial activities unless introduction 2-hydroxy to the 2-aryl substituent of 4,5-dihydrooxazol analogues. Further research on the mechanism and toxicology for their bioactivity is ongoing and will be reported in due time.