Streptonaphthyridine A, a new naphthyridine analogue with antiproliferative activity against human glioma cells from mariana trench-associated actinomycete Streptomyces sp. SY2111

Abstract A new naphthyridine analogue, named streptonaphthyridine A (1), together with eight previously reported compounds (2–9), were isolated from a Mariana Trench sediment-associated actinomycete Streptomyces sp. SY2111. Planar structure of streptonaphthyridine A was established by analyses of its HRESIMS data and extensive NMR spectra and its absolute configuration was determined by a combination of single crystal X-ray diffraction analysis and optical rotation calculations. Streptonaphthyridine A (1) had antiproliferative activity against human glioma U87MG and U251 cells with IC50 values of 7.9 ± 1.3 and 13.4 ± 2.7 µM, respectively, and the known compound monomethylsulochrin (7) showed more potent activity with IC50 values of 0.6 ± 0.1 µM for U87MG cells and 0.1 ± 0.0 µM for U251 cells. Graphical Abstract


Introduction
Gliomas are the most common and high mortal brain tumors (Kamran et al. 2016;Ostrom et al. 2020) and usually locate at many important brain function areas that makes surgical excision very difficult. Thus, chemotherapy has played a more important role in the treatment and prevention of gliomas. However, only a few drugs have been approved for the treatment of gliomas so far, including temozolomide, carmustine, lomustine, and procarbazine, which are DNA cytotoxic alkylating agents with limited efficacy and serious toxicity and side-effects (Patil et al. 2013;Mittal et al. 2015). Therefore, there is an urgent need to discover lead compounds for the development of novel antiglioma drugs.
Deep sea-derived natural products are important sources for the discovery of novel bioactive compounds. It was reported that about 75% of deep-sea natural products possess biological activity, about 40% were drug-like, and 2/3 were within Known Drug Space (KDS) (Pilkington 2019). A typical example is the marine obligate Salinospora actinomycetes, which were found in tropical and subtropical marine sediments at the depth of up to 1100 m (Prieto-Dav o et al. 2013;Russo et al. 2015). The secondary metabolites reported to date from the Salinospora actinomycetes are predominantly new, including salinosporamide A, a second-generation proteasome inhibitor isolated from S. tropica strain CNB-392 (Jensen et al. 2015), which was termed as marizomib under investigation for the treatment of malignant glioma and relapsedrefractory multiple myeloma (Di et al. 2016;Badros et al. 2017).
During the course of our ongoing project for the discovery of novel bioactive agents from Mariana Trench-associated microorganisms (Kaleem et al. 2020;Qin et al. 2020;Yi et al. 2020), a strain SY2111 was isolated from a sediment sample collected from the Mariana Trench at depth of 11,000 m. An extract prepared from the culture of this isolated hadal strain SY2111 in rice medium showed activity in inhibiting the proliferation of human glioma U87MG and U251 cells with inhibitory rates of 94.7 ± 2.7 and 86.6 ± 4.4%, respectively. Chemical investigation on this active extract resulted in the isolation of nine compounds (1-9), including one new naphthyridine analogue with antiglioma activity, named streptonaphthyridine A (1) (Figure 1). Herein, we describe the isolation, structure elucidation, and bioactive evaluation of these isolated marine natural products.

Results and discussion
The strain SY2111 ( Figure S1, Supplementary data) was identified as Streptomyces sp. SY2111 based on its 16S rDNA sequence analytic result ( Figure S2), which showed ! 99.18% similarity to those of five strains of Streptomyces (Table S1). An EtOAc extract prepared from the culture of the strain SY2111 in rice medium was separated by repeated column chromatography, followed by HPLC purification to give compounds 1 À 9.
The HRESIMS spectrum of 1 showed ions at m/z 191.0818 [M þ H] þ and 213.0637 [M þ Na] þ , corresponding to a molecular formulate C 10 H 10 N 2 O 2 with seven degrees of unsaturation. Analyses of the 13 C and HMQC spectra indicated the presence of eight olefinic carbons (d C 161.8, 152.8, 150.7, 149.7, 143.2, 119.7, 119.4, and 97.6), one methine carbon (d C 65.2), and one methyl carbon (d C 23.3). In the 1 H NMR spectrum, a pair of double peaks at d H 8.64 and 7.55 with a small coupling constant (J ¼ 5.6 Hz) suggested that the two aromatic protons were located at a nitrogen-contained ring, which was determined as pyridine (ring B) based on HMBC correlations ( Figure S3) of H-5 (d H 7.55) with C-4 (C, d C 143.2), C-6 (CH, d C 150.7), and C-8 (C, d C 119.7), H-6 (d H 8.64) with C-4, C-5 (C, d C 119.4), and C-7 (CH, d C 149.7), and H-7 (d H 9.24, s) with C-4, C-6, and C-8. The fact of two nitrogen atoms in the molecular formulate suggested that structure of 1 had a second nitrogen-contained ring. As depicted in Figure S3, HMBC spectrum showed correlations of H-3 (d H 6.55, s) with C-2 (C, d C 152.8), C-4, and C-8, H-9 (d H 4.56, q, J ¼ 6.4 Hz) with C-2, C-3 (CH, d C 97.6), and C-10 (CH 3 , d C 23.3), and H-10 (d H 1.37, d, J ¼ 6.4 Hz) with C-2 and C-9 (CH, d C 65.2). These HMBC correlations and the presence of two oxygen atoms in the molecular formulate indicated that the second nitrogen-contained ring (B) was also a pyridine substituted with a ¼ O or -OH group at C-1 and a 1-hydroxyethyl at C-2. Finally, HMBC correlations of H-3 with C-5, H-5 with C-3, and H-7 with C-1 (C, d C 161.8) and C-8 established the fused pattern of rings A and B. So far, the planar structure of 1 has been determined as 1 or 1a (Figure 1). The structure of 1 was finally confirmed by its crystal structure ( Figure S3) from single crystal X-ray diffraction. However, the Cu Ka data of X-ray diffraction may not be good enough for the assignment of the absolute configuration because the Flack parameter (-0.2(2)) was a little big, most likely due to crystal quality. Several attempts to develop high quality crystals were unsuccessful. Alternately, optical rotation (OR) calculations (Haghdani et al. 2016;Ren et al. 2019) were used to confirm the 9 R absolute configuration, the only one chiral center. The results (Tables S7 and S9) showed a positive OR value (þ37.67) for 9 R and a negative OR value (-37.66) for 9S. Accordingly, a 9 R configuration was assigned for 1 because of its positive OR value (þ12). Thus, the results of determining the 9 R configuration by using single crystal Xray diffraction analysis and OR calculations are the same. Based on the foregoing evidences, compound 1 was elucidated as a new naphthyridine analogue, named streptonaphthyridine A.

Conclusions
A new naphthyridine analogue streptonaphthyridine A was discovered from a hadal actinomycete Streptomyces sp. SY2111, which was isolated from a sediment sample obtained from the Mariana Trench at a depth of 11,000 m. Structure of streptonaphthyridine A was elucidated based on the HRESIMS data and extensive NMR spectroscopic analyses and its absolute configuration was assigned by a combination of single crystal X-ray diffraction analysis and optical rotation calculations. Streptonaphthyridine A showed potent antiproliferative activity against human glioma U87MG and U251 cells.

Disclosure statement
No potential conflict of interest was reported by the authors.

Funding
This study was supported by the National Key R&D Program of China (No. 2018YFC0310600), the National Natural Science Foundation of China (No. 81773587), and the HPC Center of Zhejiang University (Zhoushan Campus).