Two new mohangic acid derivatives from the deep-sea bacteria Alcanivorax dieselolei BC-5

Abstract Mohangic acids are a class of p-aminoacetophenonic acids that contain a conjugated triene or diene moiety. Herein, this paper reports two new mohangic acids E and F (1-2) together with a known compound mohangic acid A (3), which were isolated from the deep-sea sediment-derived bacteria Alcanivorax dieselolei BC-5. The structures of 1 and 2 were established by HRESIMS, 1 D and 2 D NMR, and IR spectroscopy. Graphical Abstract


Introduction
Natural products have been central to drug discovery and organic chemistry, but research on natural products in the pharmaceutical industry has declined since 1990s due to multiple challenges (Albarano et al. 2020) (Shinde et al. 2019).In recent years, advances in analytical technology and other scientific developments have led to a revived interests in natural products (Atanasov et al. 2021).Marine organisms under extreme growth conditions are considered to be largely unexplored natural resources, with great potential to produce novel and unique natural products (Li and Lou 2018) (Khalifa et al. 2019).
As part of the efforts to discover novel natural products from marine derived organisms, bacteria BC5 was isolated from deep-sea sediment.Subsequent chemical investigation led to the identification of two novel p-aminoacetophenonic acid derivatives mohangic acids F-G (1-2), along with a known compound mohangic acid A (3) (Bae et al. 2016) (Figure 1).The group of mohangic acid analogues has been reported only about five compounds so far which were extracted from marine mudflat-derived Streptomyces sp.(Bae et al. 2016).Herein, we report the isolation, structure elucidation and biological activity of mohangic acids F-G.

Results and discussion
Mohangic acid F (1), a yellowish oil with a molecular formula of C 24 H 33 NO 5 , was determined on the basis of HRESIMS (high resolution electrospray ionization mass spectrometry) data (m/z 416.2431 [M þ H] þ , calcd 416.2431), deduced nine degrees of unsaturation.The 1 H NMR and HMQC spectra of 1 indicated the presence of three methyls (d H 0.91, 0.89, 1.05), two methylenes (d H 3.02, 2.95, 1.33, 1.24), three methines (d H 1.72, 1.63, 2.50), two oxygenated methines (d H 4.05, 3.17), six olefinic protons (d H 5.83, 7.30, 6.31, 6.62, 6.22, 5.99) and two overlapped aromatic protons (d H 7.81, 6.72) (Table S1).Furthermore, the 13 C NMR spectrum of 1 showed one ketone carbonyl at d C 200.2 (C-15), one carboxylic acid carbon at d C 170.7 (C-1), two quaternary carbons at d C 128.1 (C-1 0 ) and d C 154.0 (C-4 0 ).The presence of a p-substituted benzene ring was deduced based on the typical p-substituted aromatic ring protons, d H 7.81 (H-2 0 / 6 0 ) and d H 6.72 (H-3 0 /5 0 ), as well as COSY correlations between H-2 0 and H-3 0 and HMBC correlations from H-2 0 to C-1 0 .Further analysis of the COSY spectra revealed the connectivity from C-2 to C-14 which contained a triene moiety from C-2 to C-7 (2E, 4E, 6E based on the trans 1 H-1 H coupling constants, J ¼ 15.0 Hz).The connectivity of moieties was determined based on HMBC correlations from H-2 0 /6 0 and H-14 to C-15, and from H-2 to C-1.The location of three methyl groups were also determined according to COSY correlations (H-16/H-8, H-17/H-10, and H-18/H-12).A primary amine group deduced from the molecular formula was assigned at C-4 0 (d C 154.0) which was consistent with the observed chemical shifts of C-1 0 to C-6 0 .Therefore, the planar structure of 1 was established and it was an analog of known compound mohangic acid A. The absolute configuration of the mohangic acid A was determined by J-based configuration analysis, 13 C NMR analysis and chemical derivatization which had been reported before (Bae et al. 2016) (Jacqueline et al. 1998).The similar 1 H and 13 C NMR data between 1 and mohangic acid A indicated that 1 possessed the same relative configuration as mohangic acid A.
Mohangic acid G (2) was obtained as yellow gum with a molecular formula of C 22 H 31 NO 5 , as determined via HRESIMS ions at m/z 390.2272 [M þ H] þ (calcd 390.2275) and 1 D NMR data (Table S1).The 1 H and 13 C NMR data for 2 were similar to those of 1, differing only by the absence of two olefinic carbons, which was confirmed by the HMQC and 1 H-1 H coupling constants of H-2 (d H 5.79, d, J ¼ 15.3 Hz), H-3(d H 7.26, dd, J ¼ 15.2 Hz, 10.5 Hz), H-4(d H 6.28, dd, J ¼ 15.2 Hz, 10.5 Hz) and H-5(d H 6.19, dd, J ¼ 15.3 Hz, 8.5 Hz).Analysis of 1 D and 2 D NMR spectra of 2 indicated the connectivity from C-2 to C-12 which contains a diene moiety from C-2 to C-5.Also, a p-substituted benzene ring was connected to C-13.Comparing the 1 H and 13 C NMR data of compounds 1 and 2 showed that the absolute configuration of 2 was same as 1.
Compounds 1 and 2 were evaluated for cytotoxic activity against PC-3 and HCT-116 cancer cell lines.However, they exhibited no cytotoxicity (IC 50 >10 lM).In addition, these compounds were also evaluated for anti-inflammatory activity, unfortunately, they showed no positive effect (IC 50 >10 lM).

General experimental procedures
Optical rotations and UV were obtained by using a JASCO DIP-370 digital polarimeter and a Horiba SEPA-300 polarimeter, respectively.IR spectra were recorded on a Bruker Vector 22 spectrophotometer. 1 D and 2 D NMR spectra were measured on a JEOL 600 spectrometer using TMS as an internal standard.HRESIMS data was recorded on an Agilent 1260 HPLC-6230 TOF tandem spectrometer.The crude extract was separated by ODS gel (75-150 lm; Mitsubishi Chemical Corporation, Japan), silica gel (100-200 or 300-400 meshes, Qingdao Haiyang Chemical Company, Qingdao, China), and Sephadex LH-20 (Amersham Pharmacia Biotech, Sweden) column chromatography (CC), and further purified with preparative HPLC (Beijing Chuangxintongheng LC3000 system equipped with a Agilent Pursuit C-18 column (10 lm, 21.2 Â 250 mm).Fractions were monitored by TLC under UV light, and spots were visualized by heating silica gel plates sprayed with 10% H 2 SO 4 in EtOH.

Actinomycic material
Strain Alcanivorax dieselolei BC-5 was isolated from deep sea sediments of South China sea by using a standard dilution plating method.The strain was purified in Gause's solid medium (20 g of soluble starch, 1 g of KNO 3 , 0.5 g of K 2 HPO 4 , 0.5 g of MgSO 4 Á7H 2 O, 0.01 g of FeSO 4 Á7H 2 O, 20 g of agar and 25 g of sea salt dissolved in 1 L of H 2 O, pH ¼ 7.2-7.4).BC-5 was identified using 16S rDNA sequence analysis by TaKaRa (Dalian, China), and its DNA sequence using BLAST (nucleotide sequence comparison) was compared to the GenBank database.The 16S rDNA sequence of strain BC-5 has been deposited in GenBank (accession number MN901214.1).BC-5 is preserved at the Laboratory of Institute of Marine Biology and Pharmacology, Ocean College, Zhejiang University.

Extraction and isolation
Colonies of the strain BC-5 growing on Gause's agar plate were inoculated into a 500 mL Erlenmeyer flask, containing 250 mL of Gause's liquid medium, and then incubated at 28 C for 3 days on a rotary shaker (180 rpm) to produce seed broth.The seed broth (8 mL) was then transferred to a 500 mL Erlenmeyer flask, each containing 250 mL of Gause's liquid medium.After incubation in cultivation for 7 days on a rotary shaker (180 rpm) at 28 C, a total of 100 Erlenmeyer flasks were prepared for this study.
The whole fermented culture was extracted with ethyl acetate at room temperature for three times.The resulting EtOAc part was dried in vacuo, and the crude extract was then separated by Sephadex LH-20 chromatography (MeOH) to produce 7 fractions (A-G) after TLC analysis.Fraction C was further purified by preparative HPLC applying a 20-100% MeCN/H 2 O for 40 min at a flow rate of 10 mL/min, to yield 1 (2.9 mg, t R 24.2 min) and 2 (1.8 mg, t R 25.5 min).

Cytotoxic assay
Compounds 1 and 2 were tested for cytotoxic activity against PC-3 and HCT-116 cancer cell lines, assayed by the sulforhodamine B (SRB) method (Belal 2015).Detailed procedures were previously described (Zhou et al. 2018).

Anti-inflammatory assay
Anti-inflammatory activity of compounds 1 and 2 were evaluated by measuring inhibitory effects on the NO production in LPS-induced RAW 264.7 macrophage cells (Yeon et al. 2015).RAW 264.7 cells were firstly cultured in DMEM supplemented with 10% FBS, and then seeded for 24 h in a 96-well plates at a concentration of 1.5 Â 10 5 cells/ well.Finally, the cells were stimulated with 1 lg/ml of LPS in the presence of 100 lM compounds or L-NMMA (positive control).After incubation at 37 C for 24 h, 100 lL of cell-free supernatant was mixed with 100 lL of Griess reagent (Beyotime, S0021) to determine nitrite production.Abosorbance was measured at 540 nm against a calibration curve with sodium nitrite standards.

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