Cytotoxic phenazine and antiallergic phenoxazine alkaloids from an arctic Nocardiopsis dassonvillei SCSIO 502F

Microbes well-adapted to the Arctic Ocean are promising for producing novel compounds, due to their fancy strategies for adaptation and being under-investigated. Two new phenazine alkaloids (1 and 2) and one new phenoxazine (3) were isolated from Nocardiopsis dassonvillei 502F, a strain originally isolated from Arctic deep-sea sediments. AntiSMASH analysis of the genome of Nocardiopsis dassonvillei 502F revealed the presence of 16 putative biosynthetic gene clusters (BGCs), including a phenazine BGC. Most of the isolated compounds were evaluated for their antibacterial, antiallergic, and cytotoxic activities. Among them, compounds 4 and 5 exhibited potent in vitro cytotoxic activities against osteosarcoma cell line 143B with IC50 values 0.16 and 20.0 μM, respectively. Besides, the results of antiallergic activities of compounds 6–8 exhibited inhibitory activities with IC50 values of 10.88 ± 3.05, 38.88 ± 3.29, and 2.44 ± 0.17 μg/mL, respectively (IC50 91.6 μM for the positive control loratadine). Graphical Abstract Supplementary Information The online version contains supplementary material available at 10.1007/s13659-023-00408-w.


Introduction
Phenazines and phenoxazines consist of a large group of nitrogen-containing heterocyclic compounds that differ in their chemical and physical properties depending on the type and location of the functional groups present [1,2].More than 100 different phenazine structural derivatives have been found in nature.Microbes inhabiting the extreme environments in the polar regions are attracting increasing attention in ecology and biotechnology.The polar regions are generally characterized by permanently low temperature, high pressure, high salinity, and extreme gradients of nutrients and light etc.To survive and propagate in such harsh environments, microbes have developed various strategies including producing novel bioactive compounds [3].Therefore, polar microorganisms are promising sources for novel natural products or chemical scaffolds with pharmaceutically relevant biological activity, considering their diversity and novelty.Previous studies have shown great potential of polar microorganisms in producing natural products with various bioactivities, including antimicrobial [4], anticancer [5], and immunosuppressive activities etc. [6] (as summarized in [7] and [8]).More recently, genome-based analyses have revealed biosynthetic potential of microbes from the polar regions, especially rare Actinobacteria groups (i.e., non-Streptomyces Actinobacteria) [9][10][11].Much greater biosynthetic potential has been indicated by genomic data mining than previous investigation based merely on traditional isolation.
The chemical shift of C-3 (δ C 147.3) indicated oxygenation at this site.Besides, the exchangeable proton 3-OH (δ H 10.26) in turn possessed HMBC correlations to C-2, C-3, and C-4 (δ C 103.4) confirmed the -OH group to be located at C-3.The oxymethylene H 2 -14 (δ H 4.28) demonstrated a HMBC correlation to C-13 (δ C 171.1), allowing for a hydroxyacetyl moiety to be constructed.This part has no further relevance in any 2D NMR experiments, 2-NH 9.32, s 3-OH 10.26, s Fig. 2 The key 1 H-1 H COSY, HMBC, and ROESY correlations of 1-3 suggesting that it is attached to the molecule by heteroatoms.Due to the presence of 2-NH and 3-OH, there was only one position for the hydroxyacetyl fragment located at 10-N, which was also consistent with the degrees of unsaturation and molecular formula.Furthermore, this deduction was supported by the ROESY correlations of H 2 -14 (δ H 4.28) with H-1 (δ H 7.88) and H-9 (δ H 7.58) (Fig. 2).Consequently, the structure of 3 was determined to be N-(3-hydroxy-10-(2-hydroxyacetyl)-10H-phenoxazin-2-yl)acetamide.Phenazine and phenoxazine-based alkaloids are a large group of structurally unique natural products containing a tricyclic core consist of two nitrogen atoms or nitrogen and oxygen atoms, respectively.Since 1859, when Fordos reported the isolation of the blue pigment known as pyocyanin (5-N-methylphenazine-1-one), more than 100 different natural phenazines have been identified.Although phenoxazinone-type alkaloids are also widely distributed in nature, alkaloids with benzoxazine nuclei are relatively rare compared to phenazines.Furthermore, phenoxazinone-type compound 3 containing carbon substitution at N-10 is the third reported in nature.Naturally occurring phenazine and phenoxazine-based alkaloids have received much attention due to their interesting biological activities, including anti-bacterial, anti-cancer, anti-parasitic.
AntiSMASH analysis of the genome of Nocardiopsis dassonvillei 502F revealed the presence of 16 putative biosynthetic gene clusters (BGCs) (Additional file 1: Table S1).The BGCs contained quite diverse types, including phenazine, ectoine, PKS, NRPS, and small RPS etc.Most of the remaining BGCs shared quite low identities with known clusters, indicating their potential in producing new compounds.A putative phenazine biosynthetic gene cluster was predicted in the genome, containing the core biosynthetic genes (phzA/B, phzD, phzE, phzF, phzG and phzNO1), possible modifying genes (phzS, phzX, phzNO1, phzO and a VOC family proteinencoding gene), transporter and transcription regulation genes (Fig. 3).The core biosynthetic genes were responsible for conversion of the precursor chorismate to phenazine-1-carboxylic acid (PCA) or phenazine-1,6-dicarboxylic acid (PDC).PCA or PDC are two precursors that can be modified further to create more complex phenazine derivatives, resulting in a serial of phenazines such as compounds 1, 2, 4, and 5.

Fig. 3
Fig. 3 Putative biosynthetic gene cluster of phenazines predicted in the genome of strain 502F

Table 1 1
H and 13 C NMR Data for 1-3 (700, 175 MHz, TMS, δ ppm) a Data were recorded in CD 3 OD.b Data were recorded in DMSO-d 6 .*Not observed