Unantimycin A, a new neoantimycin analog isolated from a microbial metabolite fraction library

Abstract

In the course of our screening for structurally unique secondary metabolites from a microbial metabolite fraction library by spectral database search, a new neoantimycin analog, unantimycin A (1) was discovered and isolated with a known neoantimycin analog, SW-163A (2). The structure of 1 was determined based on extensive spectroscopic methods, including NMR and MS. It had a 3-hydroxybenzoic acid moiety instead of a 2-hydroxy-3formylaminobenzoic acid moiety and showed moderate cytotoxicity against several cancer cell lines. Microorganisms, such as actinomycetes and fungi, have a great capacity to produce a wide variety of structurally and biologically interesting secondary metabolites.1 They have been used for pharmaceutical drugs, agrochemicals and/or as leads.2,3 They are also important bioprobes, which are chemical tools to investigate biological functions in chemical biology studies.4,5 To discover and isolate such valuable metabolites, we have developed a methodology constructing a microbial metabolite fraction library combined with an original spectral database, named Natural Products Plot (NPPlot).6,7 The fraction library is composed of fractions, which are semi-purified extracts, prepared by basic chromatographic techniques, such as middle pressure liquid chromatography (MPLC) and HPLC, and some fractions contain an almost pure single metabolite. Each fraction is submitted to diode-array detector (DAD)-LC/MS analysis to collect physicochemical information including UV absorption and mass spectra of a metabolite within the fraction. This information is used to construct an NPPlot, which is a distribution map based on the physicochemical properties of each compound. These are plotted as dots on a 2D display with retention times and m/z values on the x and y axes, respectively. On the basis of this methodology, we have discovered and isolated several new metabolites with unreported structures, such as verticilactam,8 spirotoamides9 and pyrrolizilactone.10 New quinomycin derivatives, RK-1355A and B from Streptomyces sp. RK88-1355 were discovered using NPPlot screening, in which five NPPlots generated from different strains were compared and the distinctive metabolite group was identified as comprising isolated quinomycins.11 In our search for new metabolites by NPPlot screening of RK88-1355, another characteristic distribution was found in the region of retention time around 27min with an m/z value around 650 (Supplementary Figure S1). From this region, a new compound (1) was isolated together with a known neoantimycin analog, SW-163A12 (2) from the related fractions by C18-HPLC. We report herein the isolation, structure and biological activities of 1 (Figure 1). A 30 l of culture broth of Streptomyces sp. RK88-1355 was used to construct the fraction library composed of ~ 400 fractions. The culture condition and construction of the library and NPPlot were described in the previous paper.11 The 34th and 35th fraction of the second MPLC fraction was separated by C18-HPLC with acetonitrile/water (55:45) and (70:30) under isocratic elution to afford enriched fractions of compounds 2 and 1, respectively. The compound 1 rich fraction was further purified by C18-HPLC with isocratic elution of acetonitrile/water (58:42) to afford 1 (25.7mg) as a colorless amorphous solid. The compound 2 rich fraction was purified by the same condition as that of 1 to afford 2 (3.3mg) as a colorless amorphous solid. Compound 1: colorless amorphous solid; [α]589 (c 0.1, CHCl3) − 10.8°; UV (MeOH) λmax (logε) 240sh (3.88), 290 (3.42) nm; IR (ATR) νmax (cm 1) 3370, 2970, 1750, 1720, 1650, 1585, 1520, 1455, 1190; HRESIMS m/z 626.2589 [M+H]+ calcd for C33H40NO11: 626.2601; 1H and 13C NMR data were summarized in Table 1. Compound 2 was found to be identical with SW-163A12 by the comparison of their physicochemical properties including UV, IR, specific rotation, HRMS and NMR (described in Supplementary Information). The molecular formula of 1 was determined to be C33H39NO11 by HRESIMS. The IR absorption spectrum implied the presence of ester (1750 and 1720 cm 1) and amide (1650 and 1520 cm 1) groups. The 1H NMR spectrum (Supplementary Figure S2) suggested that 1 was a compound related to 2, containing six methyl groups and a benzene

DOI: 10.1038/ja.2015.124

Cite this paper

@article{Lim2016UnantimycinAA, title={Unantimycin A, a new neoantimycin analog isolated from a microbial metabolite fraction library}, author={Chung Liang Lim and Toshihiko Nogawa and Akiko Okano and Yushi Futamura and M. Kawatani and Shunji Takahashi and Darah Ibrahim and Hiroyuki Osada}, journal={The Journal of Antibiotics}, year={2016}, volume={69}, pages={456-458} }