New streptophenazines from marine Streptomyces sp. 182SMLY

Abstract Six phenazines including three new ones were isolated from the culture of a marine actinomycete Streptomyces sp. 182SMLY. Based on the analyses of NMR, HRESIMS, optical rotation value, and CD data, the structures of these isolated compounds were determined as new phenazines of (-)-streptophenazines M–O and known phenazines of 1-carbomethoxyphenazine and (-)-streptophenazines A and B. (-)-Streptophenazine B showed activity in suppressing the growth of methicillin-resistant Staphylococcus aureus with MIC value of 4.2 μg/mL.


Introduction
Phenazine natural products consist of two benzene rings linked through two nitrogen atoms and are primarily isolated from Pseudomonas and Streptomyces strains from soil or marine habitats. Phenazines from Pseudomonas are mostly simple hydroxyl-and carboxylsubstituted structures, while phenazines isolated from Streptomyces usually have more complex structures with one or more C-substituents on the phenazine backbone. Some of phenazines had antibiotic, antitumor, antimalaria, and antiparasitic activities (Laursen & Nielsen 2004).
Streptophenazines are 1,6-disubstituted phenazines with a long alkyl chain at C-6 position. This type of phenazines (streptophenazines A-H) was first isolated from the culture of sponge-derived Streptomyces sp. strain HB202 (Mitova et al. 2008). However, the stereochemistry of the side chain of these streptophenazines was not determined therein. The total synthesis and the absolute configuration of streptophenazines A, B, E, and g were investigated, resulting in the structural revisions of streptophenazines A, B, E, and g to (-)-streptophenazines A, B, E, and g (Yang et al. 2011;Yang et al. 2012). In the structures of (-)-streptophenazines A, B, E, and g, the alkyl chain at C-6 had a hydroxyl at C-1′ and a carbomethoxyl at C-2′, instead of the hydroxyl at C-2′ and the carbomethoxy at C-1′ in the structures of streptophenazines A, B, E, and g ( Figure S1). Recently, more new streptophenazine derivatives such as streptophenazines I-L were further obtained from Streptomyces strain HB202 (Kunz et al. 2014) and Streptomyces sp. BCC21835 (Bunbamrung et al. 2014).
Actinomycetes from the genus Streptomyces are still the important resources of novel bioactive natural products (Yang et al. 2014;Zhang et al. 2014;Chen et al. 2015;Yang et al. 2015;Hu et al. 2016;Luo et al. 2016;Ou et al. 2016). Marine actinomycete Streptomyces sp. 182SMLY isolated from a sediment sample was recently reported to produce two new anthraquinones of N-acetyl-N-demethylmayamycin and streptoanthraquinone A with potent activity against the proliferation of different glioma cells (Liang et al. 2016). Further chemical investigation on the culture of this marine strain 182SMLY resulted in the isolation of six phenazines including three new streptophenazine analogues. In this study, we report the isolation and structural elucidation of these phenazines and their activities in inhibiting the proliferation of glioma cells and the growth of methicillin-resistant Staphylococcus aureus ATCC 43300 and Escherichia coli ATCC 25922.

Results and discussion
The isolated marine actinomycete 182SMLY was scaled up in the gause's liquid medium to 60 L. The culture broth was extracted by EtOAc and the EtOAc extract was fractionated by ODS column chromatography, following by HPLC purification, to yield compounds 1-6 ( Figure 1).
Compound 3 displayed [M + H] + ion at m/z 425.2072 and [M + Na] + ion at m/z 447.1894, 14 mass units lower than that of 1. Detailed 1 H NMR spectroscopic comparison of compounds 1 and 3 concluded that the major difference of the 1 H NMR signals for these two phenazines was the absence of a signal at δ H 3.68 (3H, s) for the methoxyl C-10′. This difference, in combination with the HRESIMS data of 3, suggested that compound 3 is an analogue of 1 but had no methoxyl at C-10′. Just like (-)-streptophenazines A (4), B (6), and M (1), the absolute configuration of 3 were also assigned as 1′S, 2′R based on its large 3 J 1′-2′ coupling constant (8.0 Hz), negative optical rotation value, and negative Cotton effect at 251 nm. Therefore, the structure of 3 was elucidated as (-)-streptophenazine N, a new analogue of streptophenazines.
Compounds 5 had the same molecular formula C 24 H 28 N 2 O 5 as that of (-)-streptophenazine N (3) based on their HRESIMS data, but different retention times from HPLC analysis. Detailed 1 H NMR spectroscopic analysis indicated that the structures of phenazines 5 and 3 were different in the terminal substitute of the long alkyl chain. A triplet signal at δ H 0.84 (3H, t, J = 6.9 Hz) observed in the 1 H NMR spectrum of 5 suggested that the long alkyl chain of 5 had a methyl as the terminal group, instead of an isopropyl in 3. The 1′S,2′R absolute configurations of 5 were also deduced from its large 3 J 1′-2′ coupling constant (8.1 Hz), negative optical rotation value, and negative Cotton effect at 251 nm. The structure of 5 was thus identified as (-)-streptophenazine O, a new member of streptophenazine family.
The isolated phenazines 1-6 were tested for their activity in inhibiting the proliferation of four glioma cell lines of C6, U87-Mg, SHg-44, and U251 by Sulforhodamine B (SRB) assay (Liang et al. 2016). Doxorubicin (DOX, one of the chemotherapeutic drugs) (Tacara et al. 2013) was used as a positive control. Unfortunately, none of these phenazines showed activity against the tested glioma cells.
Phenazines 1-6 were also assayed for their activity against the growth of methicillin-resistant S. aureus and E. coli using micro broth dilution method (Ye et al. 2015). Norfloxacin was used as a positive control. The result indicated that only (-)-Streptophenazine B (6) exhibited activity against the growth of methicillin-resistant S. aureus with MIC 4.2 μg/mL. The positive control norfloxacin inhibited the growth of both S. aureus and E. coli with MIC values of 20.0 and 10.0 μg/mL, respectively.

General experimental procedures
IR spectra were recorded on an AVATAR 370 FT-IR spectrometer (Thermo Nicolet). Optical rotations were measured on a JASCO DIP-370 digital polarimeter. CD spectra were recorded on a JASCO J 715 spectropolarimeter. NMR spectra were acquired on a Bruker 500 spectrometer and chemical shifts were expressed in δ (ppm). HRESIMS data were acquired on an Agilent 6230 TOF LC/MS spectrometer. Octadecyl-functionalized silica gel (ODS, Cosmosil 75C 18 Prep, Nacalai Tesque Inc., Kyoto, Japan) was used for column chromatography. HPLC purification was performed on an Agilent 1260

Experimental materials
A total of 60.0 L culture broth of strain Streptomyces sp. 182SMLY was prepared according to the previously reported method (Liang et al. 2016).

SRB assay
SRB assay (Yu et al. 2014) was used to evaluate the activity of tested compounds against the proliferation of glioma C6, U87-Mg, U251, and SHg-44 cells. Doxorubicin (DOX) was used as a positive control.

Antimicrobial assay
The micro broth dilution method as described in the previous study (Ye et al. 2015) was applied to determine the antibacterial activity of compounds 1-6 against methicillin-resistant S. aureus ATCC 43300 and E. coli ATCC 25922. Norfloxacin, a broad-spectrum antibiotic against both gram-positive and gram-negative bacteria, was used as a positive control.

Conclusion
More than 6000 phenazines have been reported during the past century (Laursen & Nielsen 2004). Streptophenazines are 1,6-disubstituted phenazines with a long alkyl chain at C-6 position. So far, only 12 streptophenazine-type phenazines have been identified. In this study, three new streptophenazine-type phenazines, named as (-)-streptophenazines M-O (1, 3, 5), together with three known phenazines of 1-carbomethoxyphenazine (2) and (-)-streptophenazines A (4) and B (6), were isolated from the culture of marine actinomycete Streptomyces sp. 182SMLY. The planar structures of these streptophenazines were determined by the analyses of NMR and HRESIMS data, while the absolute configurations of these streptophenazine-type phenazines can be assigned based on their 3 J 1′-2′ coupling constant, optical rotation value, and Cotton effect at around 251 nm. (-)-Streptophenazine B exhibited potent activity against the growth of methicillin-resistant S. aureus.