Pseudonocardians A–C, New Diazaanthraquinone Derivatives from a Deap-Sea Actinomycete Pseudonocardia sp. SCSIO 01299

Pseudonocardians A–C (2–4), three new diazaanthraquinone derivatives, along with a previously synthesized compound deoxynyboquinone (1), were produced by the strain SCSIO 01299, a marine actinomycete member of the genus Pseudonocardia, isolated from deep-sea sediment of the South China Sea. The structures of compounds 1–4 were determined by mass spectrometry and NMR experiments (1H, 13C, HSQC, and HMBC). The structure of compound 1, which was obtained for the first time from a natural source, was confirmed by X-ray analysis. Compounds 1–3 exhibited potent cytotoxic activities against three tumor cell lines of SF-268, MCF-7 and NCI-H460 with IC50 values between 0.01 and 0.21 μm, and also showed antibacterial activities on Staphylococcus aureus ATCC 29213, Enterococcus faecalis ATCC 29212 and Bacillus thuringensis SCSIO BT01, with MIC values of 1–4 μg mL−1.


Introduction
Marine microorganisms, especially marine actinomycetes, are continuing to be rich sources of bioactive metabolites, with 273 new compounds reported from marine microbes in 2009 [1]. Natural products derived from marine actinomycetes displayed a wide range of bioactivities, such as antitumor [2,3], antiinfective [4], and antimalarial [5]. In recent years, South China Sea has been emerging as a potentially abundant source of novel species/genera of marine actinomycetes [6][7][8][9][10]. Some new bioactive compounds, such as marinactinones A-C [10], lobophorins E and F [11], were reported from marine actinomycetes isolated from the South China Sea.
In our continuous search for bioactive secondary metabolites from South China Sea-derived actinomycetes, crude extracts of the strain SCSIO 01299 were found to exhibit significant cytotoxic and antibacterial activities. Upon large fermentation of the strain SCSIO 01299, four products (1)(2)(3)(4) were isolated and the main product 1 showed UV absorptions at 276, 354, 461 nm, characteristic of a highly conjugated chromophore related to anthraquinone compounds [12]. Herein we report the preliminary characterization of the strain SCSIO 01299 as a member of the genus Pseudonocardia by 16S rRNA gene sequence analysis, the isolation and structural elucidation of four diazaanthraquinone derivatives including a previously synthesized compound deoxynyboquinone (DNQ, 1) and three new analogues, pseudonocardians A-C (2)(3)(4). In addition, antibacterial and cytotoxic activities for compounds 1-4 were investigated.

Taxonomy of the Producing Strain
The strain SCSIO 01299 was isolated from deep-sea sediment (−3258 m) of the South China Sea, and was preliminarily identified as an actinomycetal species based on morphology observation. The 16S rRNA gene of the strain SCSIO 01299 was PCR amplified, sequenced and submitted to GenBank (accession number is JN204514). BLAST results showed that the new isolate had the highest similarity (98%) with Pseudonocardia autotrophica IMSNU 20050 T [13]. The phylogenetic tree generated by a neighbor-joining method clearly revealed the evolutionary relationship of the strain SCSIO 01299 to a group of Pseudonocardia species (Figure 1). Thus, this strain was designated Pseudonocardia sp. SCSIO 01299.

Figure 1.
Phylogenetic dendrogram of the strain SCSIO 01299 and its closest relatives reconstructed by the neighbor-joining method based on 16S rRNA gene sequences.  Table 1). The structure of ring A in 1 was constructed based on HMBC correlations of the methyl H 3 -15 to C-2/C-11, the methyl H 3 -16 to C-3/C-4/C-12, and H-3 to C-2/C-12/C-16. The structure of ring B was deduced from HMBC correlations of H-7 to C-8/C-13/C-17 and H 3 -17 to C-6/C-7/C-13. These two moieties were then connected through two carbonyls [δ C 182.4 and 176.9]. Finally, 1 was unambiguously identified to be deoxynyboquinone (DNQ, Figures 2 and 3), a chemically synthesized compound [14,15], by X-ray crystallographic analysis. Compound 2, designated pseudonocardian A, was isolated as a white solid. The molecular formula of compound 2 was established as C 18 Figure 4A), C-19 was supposed to be connected to N-9. The assumption was confirmed by the downfield shift of C-19 at δ C 97.6. After careful analysis of HMBC correlations, the planar structure of 2 was established ( Figure 2). In order to assign the relative configuration of 2, a NOESY experiment was carried out in DMSO-d 6 . NOESY correlation of OH-10 to H 3 -20 was found ( Figure 4B), indicating the two hydroxyls (OH-10 and OH-19) were on the opposite sides. This was consistent with the mimic configuration using the MM2 minimum energy calculation by ChemBio3D Ultra 11.0 ( Figure 4B). Therefore, the relative configuration of compound 2 was shown in Figure 2.    (Table 1). This substitution was confirmed by HMBC correlations of H-21 to C-19/C-20 and of H-20 to C-18/C-19/C-21. Therefore, the structure of 3 was established as shown in Figure 2.

Structural Elucidation
Compound 4, designated pseudonocardian C, was isolated as a red brown powder. The molecular formula of 4 was established as C 21  And the HMBC correlation from H-1 to C-10 showed that the glucose was located at C-10. On these bases of these cumulative evidences, the structure of 4 was established as shown in Figure 2.  (Table 2). DNQ (1) showed a slightly better activity than pseudonocardians A (2) and B (3) against NCI-H460 (Table 2). Pseudonocardian C (4) showed no antibacterial activity, while preserved certain in vitro cytotoxic activities comparable to those of the control compound cisplatin. However, its cytotoxicities were largely reduced when compared to 1-3 (Table 2).
In this study, we reported the isolation of DNQ (1) for the first time from a natural source, the marine actinomycete Pseudonocardia sp. SCSIO 01299. In addition, we confirmed the structure of DNQ (1) by X-ray crystallographic analysis for the first time. More interestingly, we isolated 3 novel DNQ (1) analogues, pseudonocardians A-C (2-4), from the same strain. Consistent with previous report [15], DNQ (1) exhibited potent in vitro cytotoxic activities against three tumor cell lines of SF-268, MCF-7 and NCI-H460, with IC 50 values of 22, 15, and 80 nM, respectively. Pesudonocardians A (2) and B (3) had almost the same in vitro cytotoxic activities as those of DNQ (1), with the added benefit of being ~10-fold more aqueously soluble than 1. Pseudonocardian C (4), although being >300-fold less potent than 1-3, showed comparable cytotoxicity to the positive control cisplatin ( Table 2). Unlike 1-3, pseudonocardian C (4) displayed no observable antibacterial activities, probably due to the glucosylation at C-10, which resembled the inactivation of macrolide antibiotics by glycosylation as a resistance mechanism [31].

Microbiological Material
The strain SCSIO 01299 was isolated from a sediment sample (E 120°0.975′, N 19°0.664′) at the depth of 3258 m collected from an open voyage to the South China Sea in August 2007, and was deposited in the type culture collection of Center for Marine Microbiology, Research Network of Applied Microbiology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou, China. Genomic DNA isolation, PCR amplification of 16S rRNA gene, sequence alignment, and phylogenetic tree construction of the strain SCSIO 01299 were performed as described previously [6]. A single colony of SCSIO 01299 was inoculated into 50 mL seed medium (soybean meal 0.5%, soluble starch 1.5%, peptone bacteriological 1.5%, glycerol 1.5%, CaCO 3 0.2%, sea salt 3%, pH 7.4, adjusted before sterilization) in 250 mL Erlenmeyer flasks, and was cultured on a rotary shaker at 200 r.p.m. and 28 °C for 2 days. 10% inoculums were transferred into 50 mL production medium (the same as the seed medium) in 250 mL Erlenmeyer flasks, and were subsequently incubated on a rotary shaker at 200 r.p.m., 28 °C for 5 days.

Conclusions
We have found that deep-sea actinomycete Pseudonocardia sp. SCSIO 01299 is a natural producer of the promising anticancer drug candidate deoxynyboquinone (1) and three diazaanthraquinone derivatives pseudonocardians A-C (2-4). In comparison with 1, pseudonocardians A (2) and B (3) showed the same anticancer potency but had enhanced aqueous solubility. These findings once again highlighted the potential of marine actinomycetes for novel drug discovery.