A new curvularin glycoside and its cytotoxic and antibacterial analogues from marine actinomycete Pseudonocardia sp. HS7

Five curvularin macrolides (1–5) were isolated from the cultured broth of marine actinomycete Pseudonocardia sp. HS7 that was obtained from the cloacal aperture of sea cucumber Holothuria moebii. The structures of these isolates were characterized as (11S,15R)-11-hydroxycurvularin (1), (11R,15R)-11-hydroxycurvularin (2), curvularin-7-O-α-D-glucopyranoside (3), trans-dehydrocurvularin (4) and curvularin (5) based on their NMR and HRESIMS data as well as chemical degradation. Compound 3 is a new macrolide with a rare α-D-glucopyranose substituent. Compounds 1–4, 5a and 5c (the acyl products of 5), suppressed the proliferation of all six tested cancer cell lines and 4 is the most active compound with IC50 values ranging from 0.59 to 3.39 μM. The 11-hydroxycurvularins 1 and 2 also showed antibacterial activity inhibiting the growth of Escherichia coli.


Introduction
Gliomas are one of the most challenging cancers to treat and account for 80% of all malignant brain tumours. Chemotherapy is an important adjunctive therapy for treating gliomas and temozolomide (TMZ) is the only drug that has been independently used for the treatment of gliomas. The efficacy of the currently used anticancer drugs including TMZ is limited (Chamberlain 2010;Patil et al. 2013). There is therefore a need to discover lead compounds for the development of novel anti-glioma drugs. Natural products are highly significant sources of new drug leads (Newman & Cragg 2012).
During the course of our ongoing programme for the discovery of novel anti-glioma agents from natural sources (Xin et al. 2012;Ye et al. 2014;Yu, Ye, Chen et al. 2014;Yu et al. 2015;Yu, Ye, Xin et al. 2014), the cultured broth of marine actinomycete strain HS7 isolated from the cloacal aperture of sea cucumber Holothuria moebii was found to be active against the proliferation of glioma cells. The strain HS7 was identified by 16S rDNA sequence analysis and its top16S rDNA sequence was 100% sequence match to Pseudonocardia antitumoralis strain SCSIO 01299. This actinomycete strain SCSIO 01299 was recently isolated as a new species from deep-sea sediment collected from the northern South China (Tian et al. 2013). Previously chemical investigation indicated that strain SCSIO 01299 produced deoxynyboquinone and three new diazaanthraquinone derivatives of pseudonocardians A, B and C. These diazaanthraquinones were shown to have potent cytotoxic and antibacterial activities (Li et al. 2011).
In this study, chromatographic separation of an active EtOAc extract prepared from the cultured actinomycete strain HS7 afforded five curvularin analogues (1 -5, Figure 1). Three acyl products (5a-5c) of curvularin (5) were also prepared for bioactivity assay. Compound 3 is a new curvularin macrolide glycoside with a rare a-D-glucopyranose substituent and 5a is a new synthetic compound. We herein report the isolation and structural elucidation of these compounds and their activities inhibiting the proliferation of cancer cells and the growth of Staphylococcus aureus and Escherichia coli.

Results and discussion
Marine actinomycete strain HS7 was isolated from the cloacal aperture of sea cucumber H. moebii. The taxonomic identity of this isolated actinomycete was determined by 16S rDNA sequence analysis. The top sequence of HS7 was 100.0% sequence similarity to P. antitumoralis strain SCSIO 01299 (accession number: NR_109460.1) and 99% sequence similarity to other nine Pseudonocardia sp. strains Table S1) in the GenBank database. Therefore, the taxonomy of actinomycete HS7 was proposed to be Pseudonocardia sp. HS-7.
The compounds (1 -5, 5a-5c) were also assayed for their activity against S. aureus and E. coli. The antibacterial activity of compounds was initially tested at 100 mg/mL by spot method (Supplementary material). Only compounds 1 and 2 showed activity inhibiting the growth of E. coli. None of tested compounds was active against S. aureus. The MIC (minimum inhibitory concentration) and MBC (minimum bactericidal concentration) of the active compounds 1 and 2 were further determined by micro broth dilution method (Supplementary material) and nor oxacin was used as positive control. The results indicated that both 1 and 2 had activity against E. coli with an MIC value of 20 mg/mL and an MBC value of 30 mg/mL. The positive control drug nor oxacin had activity against E. coli with MIC 1.2 mg/mL.
Curvularin macrolides were previously found in fungi mainly from genus Curvularia (Greve et al. 2008) and Penicillium (Meng et al. 2013). This type of macrolides was reported to be cytotoxic towards tumour cells (He et al. 2004;Greve et al. 2008;Meng et al. 2013) and also inhibit the growth of fungal and bacterial organisms (Dai et al. 2010). In the current study, several curvularin macrolides including a new curvularin macrolide glycoside (3) were isolated from marine actinomycete strain HS7. This new macrolide and other isolates were shown to be active against the proliferation of different cancer cell lines, further confirming the antitumour property of this type of macrolides. (11S,15R)-11-hydroxycurvularin (1) and (11R,15R)-11hydroxycurvularin (2) were found to have antibacterial activity inhibiting the growth E. coli. To the best of our knowledge, this type of curvularin macrolides is isolated from a bacterial source for the first time.

Experimental
Experimental section was supplied as online Supplementary material.