TLN-05220, TLN-05223, new Echinosporamicin-type antibiotics, and proposed revision of the structure of bravomicins

The Journal of Antibiotics (2009) 62, 565–570 & 2009 Japan Antibiotics Research Association All rights reserved 0021-8820/09 $32.00 www.nature.com/ja ORIGINAL ARTICLE TLN-05220, TLN-05223, new Echinosporamicin-type antibiotics, and proposed revision of the structure of bravomicins* Arjun H Banskota1, Mustapha Aouidate2, Dan Sørensen3, Ashraf Ibrahim2, Mahmood Piraee4, Emmanuel Zazopoulos2, Anne-Marie Alarco2, Henriette Gourdeau2, Christophe Mellon2, Chris M Farnet2, Pierre Falardeau2 and James B McAlpine2 The deposited strain of the hazimicin producer, Micromonospora echinospora ssp. challisensis NRRL 12255 has considerable biosynthetic capabilities as revealed by genome scanning. Among these is a locus containing both type I and type II PKS genes. The presumed products of this locus, TLN-05220 (1) and TLN-05223 (2), bear a core backbone composed of six fused rings starting with a 2-pyridone moiety. The structures were confirmed by conventional spectral analyses including MS, and 1D and 2D NMR experiments. Comparison of both the 1H and 13C NMR data of the newly isolated compound with those of echinosporamicin and bravomicin A led us to propose a revision of the structure of the latter to include a 2-pyridone instead of the pyran originally postulated. Both compounds (1 and 2) possessed strong antibacterial activity against a series of grampositive pathogens including several strains of methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococci (VRE), and cytotoxic activities against several human tumor cell lines. The TLN compounds are the first of this group with reported anticancer activity. The Journal of Antibiotics (2009) 62, 565–570; doi:10.1038/ja.2009.77; published online 14 August 2009 Keywords: antibacterial; anticancer; bravomicin; Micromonospora echinospora; polycyclic aromatic; TLN-05220; TLN-05223 INTRODUCTION The genomics of secondary metabolite biosynthesis recently evolved to the point in which analysis of the genome of an organism can define its biosynthetic capabilities for secondary metabolites. A genome scanning technique, requiring minimal amount of sequencing, has been developed in our laboratories, and used with our DECIPHER technology to analyze the genomes of bacteria for their secondary metabolite biosynthetic genes, greatly reducing the amount of sequencing required to define this capability.1–4 This approach not only ascertains the biosynthetic potential of a producing organism, but it provides the scientist with a handle to detect, isolate and structurally define a specific metabolite. We have shown this approach in the isolation and structural determination of an antifungal agent, ECO02301 from Streptomyces aizunensis5 and three 5-alkenyl-3,3(2H)furanones from two different Streptomyces species.6 This genome scanning approach, which was applied to define the biosynthetic capabilities of Micromonospora echinospora ssp. challisensis NRRL 12255 (this strain was deposited (as SCC 1411) to support US Patent 4,440,751),7 revealed 13 secondary metabolite biosynthetic loci. In our experience, this is an unusually high number to find in a Micro- monospora species and led to the prioritization of this strain for investigation. A preliminary DECIPHERIT analysis annotated these loci as encoding a terpenoid, five mixed PKS/NRPS, four NRPS and three PKS biosynthetic systems. In this article, we describe the use of the genome scanning technique8,9 to identify and isolate new polycyclic aromatic antibiotics (TLN-05220, 1 and TLN-05223, 2) from this strain, the former was previously referred to as ECO-3396.1,2 Both compounds (1 and 2) possessed strong antibacterial activity against broadly resistant, gram-positive pathogens, and cytotoxic activities against several human tumor cell lines. RESULTS AND DISCUSSION M. echinospora ssp. challisensis NRRL 12255 was obtained from the National Center for Agricultural Utilization Research (NCAUR) in Peoria, IL, USA and was grown in shaken flasks in a dozen different fermentation media designed for the production of secondary metabolites. The whole cultures were then extracted by equal volumes of MeOH and the resultant aqueous/MeOH extracts were subjected to HPLC/MS/UV analyses. A number of these extracts contained a compound with UV absorption lmax at 248 and 508 nm, as might 1Institute for Marine Biosciences, National Research Council, Halifax, Nova Scotia, Canada; 2Thallion Pharmaceuticals, Alexander-Fleming, Montréal, Quebec, Canada; 3Merck-Frosst Canada, Trans Canada Highway, Kirkland, Quebec, Canada and 4Biotica Technology, Chesterford Research Park, Cambridge, UK Correspondence: Dr JB McAlpine, 730 Benson Lane, Green Oaks, IL 60048, USA. E-mail: *Part IV in a series on Genomic Analysis for the Discovery of Novel Secondary Metabolites. TLN-05220 was previously referred to as ECO-3396. Received 30 April 2009; revised 9 July 2009; accepted 22 July 2009; published online 14 August 2009 TLN-05220, TLN-05223 and revised structure of bravomicins AH Banskota et al 566 be expected for a type II polyketide. This compound had MS peaks at m/z 738.1 (in the positive mode) and 736.1 (in the negative mode). Larger scale (20500 ml) fermentations of M. echinospora ssp. challisensis NRRL 12255 were carried out in the two media, which gave the best yields of this metabolite. On harvest, the EtOAc extract gave TLN05220 (1) and TLN-05223 (2) (Figure 1) by Sephadex LH-20 (SigmaAldrich Canada, Oakville, ON, Canada) column chromatography followed by reversed phase HPLC. TLN-05220 (1) was isolated as a red amorphous solid with molecular formula C30H31N3O13 calculated from the MS data (m/z 738.15 (M+H)+ and 736.10 (MH)). The 1H NMR spectrum (Table 1) of 1 displayed five aromatic protons including two sets of ortho-coupling protons, one methoxy, two methine, three methylene and three C-methyl groups. The carbon NMR spectrum (Table 2) on the other hand had 38 carbon signals including 23 quaternary carbons in aromatic and carbonyl region (d 111.4–187.1), indicating the presence of a complex aromatic system. The UV absorption of TLN05220 (1) at lmax 508 nm further suggested that the rest of the molecule should be constructed by an extended conjugated system. In-depth analyses of the gCOSY together with heteronuclear single quantum coherence (gHSQC) and heteronuclear multiple bond coherence (gHMBC) spectra led to the identification of the piperazinone and 1-methyl propyl groups as two partial structures. Additional sets of proton and carbon signals of the piperazinone moiety were also observed in the NMR spectra and are ascribed to the presence of slowly interchangeable rotamers. The presence of five carbonyl carbons (d 187.1 (2), 183.3, 181.9 and 159.0), two sets of ortho-coupled aromatic protons (d 8.63 and 8.39; 7.47 and 7.44) and a singlet aromatic proton signal (d 6.73) strongly suggested the presence of a polycyclic aromatic with a core backbone composed of six fused rings, similar to the albofung (...truncated)