Antimycic Acid and its Acetyl Derivative from Deep-sea-derived Alcanivorax sp. SHA4 with Neuroprotective Properties

Abstract

Chemical investigation of secondary metabolites of the deep-sea-derived Alcanivorax sp. SHA4 identified a new compound 1 which was antimycic acid (2)'s acetyl derivative, and 11 known compounds (2-12). Their structures were elucidated by extensive nuclear magnetic resonance and mass spectrometry spectroscopic analyses, and the absolute configuration of compound 1 was determined by Marfey's method. Bioactivity assays indicated that compounds 1 and 2 exhibited significant neuroprotective properties against glutamate-induced PC12 cell death in 0.02-0.31 μM.

Introduction

Previous studies have shown that the ocean can provide marine drugs with health benefits including antidiabetic effects, ¹ blood pressure modulation, ² osteoporosis recovery, ³ and antituberculosis agents. ⁴ Salinosporamide A (marizomib) isolated from deep-sea bacterium is a powerful proteasome inhibitor, ⁵ and the Food and Drug Administration has approved the orphan drug marizomib for the treatment of malignant glioma. Alcanivorax is an important alkane-degrading bacterium in the marine environments, which provides an optimal benefit to the petroleum pollution bioremediation. ⁶ The attention to Alcanivorax has been focused on the mechanism and related enzymes of alkane-degrading, ⁷ but pharmacological studies of its metabolites have not been reported. As part of our research to find pharmacologically active natural products from secondary metabolites of the deep-sea-derived Alcanivorax sp. SHA4, we obtained antimycic acid (2) and its new acetyl derivative (1), together with 10 other known compounds (3-12) (Figure 1). This paper describes the isolations and structural determinations of the above 12 compounds and neuroprotective activities of antimycic acid and its acetyl derivative.

Figure 1.

Structures of compounds 1-12 from Alcanivorax sp. SHA4.

Results and Discussion

Compound 1 was isolated as pale yellow-green powder, with high-resolution electrospray ionization mass spectrometry (HRESIMS) analysis at m/z 297.1086 [M + H]⁺ showing a molecular formula of C₁₃H₁₆N₂O₆ and corresponding to 7 degrees of unsaturation. The signals suggested a characteristic 1,2,3-trisubstituted aromatic ring at δ_H 7.90 (1H, d, J = 7.6 Hz), 6.89 (1H, t, J = 7.6 Hz), 7.76 (1H, d, J = 7.6 Hz), and δ_C 115.7, 151.4, 127.6, 126.5, 118.1, and 123.7, and 1 threonine residue at δ_H 4.41 (1H, br s), 4.22 (1H, qd, J = 6.4, 3.9 Hz), 1.14 (3H, d, J = 6.4 Hz) and δ_C 171.8, 58.6, 66.3, and 20.6 ⁸ in ¹H and ¹³C nuclear magnetic resonance (NMR) spectra (Table 1) of 1. Based on HRESIMS analysis at m/z 255.0977 [M + H]⁺, compound 2 possessed a molecular formula of C₁₁H₁₄N₂O₅, corresponding to 6 degrees of unsaturation. By comparing detailed spectroscopic analysis (Supplemental Figures S10 and S11) with reported data, ⁹ compound 2 was identified as antimycic acid. The difference between 1 and 2 can be observed in an additional acetyl group at δ_H 2.11 (3H, s) and δ_C 169.5, 27.5 in the spectra of 1. The 1H detected heteronuclear multiple bond correlation (HMBC) correlations (Figure 2, Supplemental Figure S5) of 3-NH (δ_H 9.50) with C-1″ (δ_C 169.4), C-2 (δ_C 151.4), C-3 (δ_C 127.6) were observed, suggesting the acetyl group linked to the C-3 amino group.

Figure 2.

Key 2-dimensional nuclear magnetic resonance (2D NMR) correlations of compound 1.

Table 1.

¹H (600 MHz) and ¹³C (150 MHz) NMR Spectral Data of Compounds 1 and 2.

No.	1 (in DMSO-d6)		2 (in methanol-d4)
	δ C	δH (J in Hz)	δ C	δH (J in Hz)
1	115.7		115.8
2	151.4		153.4
3	127.6		129.7
4	126.5	7.90, d (7.6)	124.5	7.23, dd (8.0, 1.3)
5	118.1	6.89, t (7.6)	119.9	6.91, t (8.0)
6	123.7	7.76, d (7.6)	122.5	7.58, dd (8.0, 1.3)
1-CO	168.9		171.8
3-NH		9.50, s
1′	171.8		173.5
2'	58.6	4.41, brs	59.2	4.71, d (3.1)
3'	66.3	4.22, qd (6.4, 3.9)	68.4	4.56, qd (6.4, 3.1)
4'	20.6	1.14, d (6.4)	20.6	1.27, d (6.4)
2'-NH		8.73, brs
1″	169.4
2"	23.6	2.11, s

Abbreviations: NMR, nuclear magnetic resonance; DMSO-d₆, dimethylsulfoxide-d₆.

The absolute configuration of the threonine residue of compound 1 was determined by Marfey's method. Firstly, compound 1 was hydrolyzed with hydrochloric acid and then reacted with Marfey's reagent (1-fluoro-2,4-dinitrophenyl-5-L-alanine amide) to obtain Marfey's derivative. ¹⁰ Then by their injection into a high-performance liquid chromatography (HPLC) apparatus, compared it with authentic amino acid derivatives. The chromatography result showed that the amino acid was in the L-configuration (Supplemental Figure S9). Therefore, the absolute configuration of compound 1 was established as shown in Figure 1.

The remaining 10 known compounds were assigned as 2′-O-methyluridine (3), ¹¹ hypoxanthine riboside (4), ¹² 2-acetaminobenzoic acid (5), ¹³ 2-aminobenzoic acid (6), ¹⁴ 4-acetaminobenzoic acid (7), ¹⁵ 4-aminobenzoic acid (8), ¹⁶ tryptophol (9), ¹⁷ IAA (10), ¹⁸ 6-hydroxyisatin (11), ¹⁹ and 7-hydroxy-2H-1,4-benzothiazin-3(4H)-one (12), ²⁰ respectively, by a comparative analysis of their spectral data with the literature (Figure 1 and Supplemental material).

The structures of compounds 3-12 are relatively simple and their bioactivities have been well understood, so we did not test their bioactivities. Against HCT-116, SW620, and PC-3 human cancer cell lines, compounds 1 and 2 were evaluated for their cytotoxicity at 50 μM, but both compounds showed no potent inhibitory activities in the bioassay. The adrenal pheochromocytoma (PC12) cell line has high consistency with the characteristics of dopaminergic neurons and is relatively easy to culture, having been widely used as a model for neural differentiation. ²¹ Then their neuroprotective activities were tested in glutamate-induced PC12 cells, as shown in Figure 3, both compounds exhibited potent protective effects at 0.02-0.31 μM. Compared to antimycic acid (2), its acetyl derivative (1) has a lower optimal treatment concentration of 0.04 μM. It may be because acetylation improves the physical and chemical properties of small molecules, which is beneficial to permeation.

Figure 3.

Results of neuroprotective activities tested in glutamate-induced PC12 cells. (A) Effects of acantimycic acid (1) with various concentrations on glutamate-induced PC12 cell viability for 24 h. (B) Effects of antimycic acid (2) with various concentrations on glutamate-induced PC12 cell viability for 24 h. Survival rates were measured by the SRB assay. Each value represents the mean ± SD, n = 3, Z factor = 0.67. Δp < .05 versus control group; ∗p < .05 versus glutamate group.

Conclusion

Alcanivorax mainly plays an important role in the biodegradation of natural petroleum, and we also speculate that there may be some pharmacologically active natural products in its secondary metabolites. In the study, we obtained antimycic acid (2) and its new acetyl derivative (1), together with 10 other known compounds from deep-sea-derived Alcanivorax sp. SHA4. Compounds 1 and 2 exhibited potent protective effects in 0.02-0.31 μM, and maybe it is because of its acetylation, compound 2 has a lower optimal treatment concentration of 0.04 μM. Therefore, the structure–activity relationship and molecular mechanism of neuroprotection of antimycic acid derivatives need to be further explored.

Experimental

General

Marfey's reagent was purchased from J&K Scientific. Digital polarimeter JASCO P-1010 was used to measure optical rotation. Ultraviolet (UV) spectra were recorded on a SHIMAZU UV-1800 spectrophotometer. Infrared (IR) spectra were acquired on a Bruker Vector 22 spectrophotometer. NMR data were collected using a Bruker AVANCE III 600/400 NMR spectrometer. HRESIMS were measured using an Agilent 6230 TOF LC-MS. GF254 silica gel plates and Silica gel (300-400 mesh) were purchased from Qingdao Haiyang Chemical Company and were used for thin-layer chromatography experiments and column chromatography. Sephadex LH20 was purchased from Amersham Pharmacia Biotech. The LC3000 liquid chromatograph (Beijing Tong Heng Innovation Technology Co., China) with an Agilent XRs C-18 (5 μm, 9.4 × 250 mm) column was used for semipreparative HPLC.

Alcanivorax Material

The bacterial strain was isolated from muddy deep-sea sediments (supplied by the Second Institute of Oceanography, Hangzhou, China) that were collected from the western pacific at a depth of 5180 m. The strain SHA4 was identified as a member of the genus Alcanivorax sp., using the analysis of the 16S rDNA by TaKaRa, which was identical with Alcanivorax xenomutans strain P40 (accession no. CP012331.1). Alcanivorax sp. SHA4 was deposited at −80 °C in the Ocean College, Zhejiang University, China.

Fermentation

The spores of Alcanivorax sp. SHA4 growing on Gause's agar medium (soluble starch 20 g, agar 20 g, sea salt 25 g, KNO₃ 1 g, MgSO₄·7H₂O 0.5 g, K₂HPO₄ 0.5 g, NaCl 0.5 g, and FeSO₄·7H₂O 0.01 g, made up to 1 L with distilled water, pH = 7.2-7.4) were transferred to 80 bottles of 500 mL Erlenmeyer flasks with 250 mL of Gause's liquid medium while shaking at 28 °C and 180 r/min for 7 days. Selected 3 bottles after 3 days of culture to produce seed broth, and then 5 mL of seed broth was transferred to 120 bottles of 500 mL Erlenmeyer flasks with solid rice medium (rice 40 g and 35‰ salt water 60 mL) while statically culturing at room temperature for 60 days at which time the pH value stabilized at around 6.5. All the medium were sterilized.

Extraction and Isolation

The above 2 kinds of fermented cultures were extracted, respectively, with EtOAc 3 times and then evaporated to dryness to provide organic extracts. The extract (4.55 g) from the solid rice medium was separated on a silica gel chromatography (182 g) using a light CH₂Cl₂/MeOH gradient system (1:0, 50:1, 30:1, 10:1, 5:1, 1:1, and 0:1, v/v) to obtain 7 fractions (A-G). With fraction F (652 mg), 6 subfractions (F-1 to F-6) were obtained by Sephadex LH20 column (CH₂Cl₂/MeOH, 1:1). Fraction F-5 (30 mg) was purified by preparative HPLC (5%-45% MeOH/H₂O with 0.05% trifluoroacetic acid [TFA], 20 mL/min, 60 min) to afford 1 (2.2 mg, t_R = 52 min), 2 (3.8 mg, t_R = 50 min) and 3 (5.5 mg, t_R = 22 min). Sephadex LH20 column (MeOH) was used again to separate fraction F-4 (24 mg) to afford 4 (7.3 mg). With fraction D (212 mg), 5 subfractions (D-1 to D-5) were obtained by Sephadex LH20 column (MeOH). Preparative HPLC (10%-50% MeCN/H₂O, 15 mL/min, 60 min) was used to purify fraction D-2 (23 mg) to afford 5 (3.6 mg, t_R = 32 min), with preparative HPLC (70%-100% MeOH/H₂O with 0.05% TFA, 15 mL/min, 60 min) for fraction D-3 (68 mg) to afford 6 (4.2 mg, t_R = 24 min), 7 (3.1 mg, t_R = 29 min) and 8 (2.8 mg, t_R = 35 min). Fraction A (865mg) was dissolved with petroleum ether and extracted 3 times with 90% MeOH/H₂O, and the resulting MeOH/H₂O-soluble portions were dried in vacuum to give an extract (34 mg). Then the extract was submitted to preparative HPLC (20%-60% MeOH/H₂O with 0.05% TFA, 20 mL/min, 60 min) to afford 9 (2.2 mg, t_R = 25 min) and 10 (2.4 mg, t_R = 59 min). With the extract (0.52 g) from Gause's liquid medium, 3 fractions (H-J) were obtained by Sephadex LH20 column (CH₂Cl₂/MeOH, 1:1). By preparative HPLC (20%-60% MeOH/H₂O, 20 mL/min, 40 min), fraction J (128 mg) yielded 11 (3.2 mg, t_R = 16 min) and 12 (2.4 mg, t_R = 38 min).

Compound 1: Pale yellow-green powder; $[\alpha {]}_D^{25}$  + 90 (c 0.02, MeOH); UV (MeOH) λ_max: 222 (3.3), 315 (2.4) nm; IR v_max: 2924, 1540, 1376, and 1255 cm⁻¹; HRESIMS m/z 297.1086 [M + H]⁺ (calcd for C₁₃H₁₇N₂O₆, 297.1081); ¹H and ¹³C NMR, Table 1.

Cell Culture and Treatment

The rat pheochromocytoma (PC12) cell line was obtained from the Cell Bank of the Shanghai Institute of Life Science (Chinese Academy of Sciences). An RPMI-1640 containing 5% fetal bovine serum (FBS) and 10% horse serum (HS) (v/v) (Gibco) was used to incubate PC12 cells at 37 °C and 5% CO₂. PC12 cells were sown (10 000 cells/well) in 96-well plates. After having been cultured overnight, a fresh medium containing various concentrations of test compounds was given to the cells, and 2 h later, stimulated with 15 mM glutamate (final concentration). Meanwhile, the control group was supplemented with a pure culture medium without compounds and glutamate. ²² After 24 h incubation, the sulforhodamine B (SRB) assay ²³ was used for the evaluation of neuroprotection of the compounds. The absorbance at 515 nm was determined by a Tecan Spark 10M Microplate Reader. All the tests were repeated 3 times.

Supplemental Material

sj-docx-1-npx-10.1177_1934578X221110661 - Supplemental material for Antimycic Acid and its Acetyl Derivative from Deep-sea-derived Alcanivorax sp. SHA4 with Neuroprotective Properties

Supplemental material, sj-docx-1-npx-10.1177_1934578X221110661 for Antimycic Acid and its Acetyl Derivative from Deep-sea-derived Alcanivorax sp. SHA4 with Neuroprotective Properties by Shuang Chen, Da-shan Zhang and Jin-hui Wang in Natural Product Communications