New bioactive compounds from the marine-derived actinomycete Nocardiopsis lucentensis sp. ASMR2

2.1 Morphological, cultural, and phenotypic characteristics

Cultures of strain ASMR2 grew well on certain International Streptomyces Project (ISP) media used, but light growth was observed on ISP4. The color of the aerial mycelium is mostly pale yellow to pale brown; white on ISP3 and Czapeck’s Dox agar, whereas the reverse sides of cultures were yellow on ISP3 and ISP4 and brown on ISP5. Yellow pigments have been observed on starch-nitrate agar and Czapeck’s Dox agar (Table 1). Based on the microscopic examinations (Fig. 1), spores of the strain ASMR2 are elongated and smooth. Physiologically, the strain ASMR2 is a weak H₂S producer and utilizes d-glucose, d-xylose, d-mannose, l-arabinose, and sucrose for growth (Table 2).

Fig. 1:

Spore chain structure of strain ASMR2 grown on ISP4 medium (50% seawater, 10 days, 37°C). (a) Light microscopy of sporulating mycelium (1200×) showing Rectiflexibilis; (b) transmission electron micrography of strain showing spore chain with smooth surface (bar=5 μm).

For taxonomic classification of strain ASMR2, its internal transcribed spacer (ITS) sequence was sequenced was amplified by applying polymerase chain reaction (PCR), and then sequenced and analyzed in a BLAST-based approach. Bioinformatic analysis of the 895-bp-long 16S rDNA sequence, amplified from a single colony of strain ASMR2, confirmed the highly closed relation of the isolate ASMR2 to N. lucentensis (99%). The results were supported by a neighbor-joining-based phylogenetic tree (Fig. 2). However, by comparing the characteristics of strain ASMR2 with those found in the literature, and the description manuals of actinomycetes [13], [14], [15], [16], [17], [18], [19], it has been confirmed that strain ASMR2 differed from the strain types of both phylogenetic neighbors and other closely similar Nocardiopsis species. The strain ASMR2 has been recorded in GenBank database with accession no. KX581217.

Fig. 2:

Phylogenetic tree of strain ASMR2 based on 16S rDNA gene sequences, showing its close relationship to Nocardiopsis species. The tree was constructed using the neighbor-joining method.

2.2 Fermentation and structure elucidation

The N. lucentensis sp. ASMR2 was cultivated on modified solid rice medium containing malt extract (1%) and yeast extract (0.4%). The methanol extract of the strain exhibited strong activity against the yeast Candida albicans (14 mm), and low to moderate activity (9–11 mm) against the yeast Saccharomyces cerevisiae, Gram-positive Bacillus subtilis, and Staphylococcus aureus (Table 6). In the chemical screening monitored by thin layer chromatography (TLC), the bacterial extract exhibited numerous bands in a wide polarity range: some of them showed a group of ultraviolet (UV)-absorbing compounds that were detected as brown, orange, pink, violet, and blue bands by spraying with anisaldehyde/sulfuric acid; others were UV-nonabsorbing, showing violet-blue staining with anisaldehyde/sulfuric acid.

Separation of metabolites produced by the strain using a series of chromatographic techniques afforded the novel butenolide derivative 3′-hydroxy-N-(2-oxo-2,5-dihydrofuran-4-yl)propanamide (1a), and the naturally new 4-methoxy-2H-isoquinolin-1-one (2). Furthermore, the bacterial extract afforded additional eight known compounds: indole-3-carboxylic acid [20], indole-3-acetic acid [20], indole-3-acetic acid methyl ester [20], furan-2,5-dimethanol [20], tyrosol [20], glycerol linoleate [21], cyclo-(Tyr, Pro) [20], and adenosine [20]. Their structures were confirmed by comparison of the spectroscopic data and chromatographic properties with literature. The physico-chemical properties of 3′-hydroxy-N-(2-oxo-2,5-dihydrofuran-4-yl) propanamide (1a) and 4-methoxy-2H-isoquinolin-1-one (2) are listed in Table 3.

2.3 3′-Hydroxy-N-(2-oxo-2,5-dihydro-furan-4-yl)propionamide

Compound 1a was obtained as a middle-polar colorless semisolid showing UV absorbance on TLC and orange staining on spraying with anisaldehyde/sulfuric acid. The compound exhibited a UV absorbing peak at λ_max=252 nm, referring to its aromatic/conjugated olefinic nature. Two quasimolecular ion peaks were revealed in ESI-MS-positive mode, confirming the molecular weight of 1a as 171 Da. EI-HRMS of 1a delivered the molecular formula C₇H₉NO₄, bearing four double bond equivalents (DBE) (Table 3).

The NMR spectra (Table 4) of 1a exhibited two spin systems: one triplet 1H signal (J~1.4 Hz) in the olefinic region at δ=5.70 ppm (δ_C=94.6 ppm), which exhibited a weak H,H-COSY correlation (Fig. 2) with a 2H triplet signal (J~1.4 Hz) originating from an oxygenated methylene group visible at δ=5.15 ppm (δ_C=69.8 ppm). The second spin system consisted of two vicinal triplet (J~6.0 Hz) methylene signals at δ=3.88 ppm (δ_C=57.1 ppm) and δ=2.61 ppm (δ_C=39.0 ppm) as deduced by H,H-COSY connectivity, which are corresponding to an ethanediyl group flanked by oxygen and sp² carbon systems, respectively. Based on the ¹³C NMR/HMQC spectra, compound 1a displayed seven carbon signals, as matched with the molecular formula, which have been classified into two quaternary carbons of ester/amide carbonyls (δ=175.8, 171.4 ppm), an oxygenated (or amide) quaternary sp² carbon (δ=161.3 ppm), an olefinic methine carbon (δ=94.6 ppm, δ_H=5.70 ppm), and three methylene sp³ carbons (δ=69.8, 57.1, 39.0 ppm). Accordingly, the structure of compound 1a contains at least one ring system. A search in different databases [AntiBase [3], Dictionary of Natural Products [22], and Scifinder (https://scifinder.cas.org/scifinder)] considering the NMR results and the molecular formula of compound 1a confirmed its structural novelty.

Consequently, an intensive study of the NMR data based on HMBC and H,H-COSY correlations was carried out (Fig. 3): based on the HMBC connectivities, the olefinic proton (H-3) at δ=5.70 ppm exhibited three HMBC correlations at the ester/amide-carbonyl (C-2, δ=175.8 ppm, ²J), oxygenated/amide-bounded carbon at δ=161.3 ppm (C-4, ²J), and the oxygenated methylene carbon at δ=69.8 ppm (C-5, ³J). Proton signals of the last methylene group (δ=5.15 ppm) displayed three alternative correlations at C-2 (δ=175.8 ppm, ³J), C-3 (δ=94.6 ppm, ³J), and C-4 (δ=161.3 ppm, ²J), suggesting one of two five-membered ring systems: (a) 3-amino-butyro-lactone or (b) 3-hydroxy-butyrolactame. The proton signals of the ethanediyl group (CH₂-2′, and CH₂-3′) system showed ²J and ³J correlations, respectively, at the respite ester/amide carbonyl group (C-1′, δ=171.4 ppm), constructing 3-hydroxy-propionic acid ester or 3-hydroxy-propanamide. Based on previous studies of HMBC, H,H-COSY correlations (Fig. 3), and the molecular formula of 1, two alternative structures have been considered: 3′-hydroxy-N-(2-oxo-2,5-dihydrofuran-4-yl)propanamide (1a) and 2-oxo-2,5-dihydro-1H-pyrrol-4-yl-3′-hydroxypropionate (1b). A search in different databases confirmed the novelty of both structure formulas. On the basis of the NMR chemical shifts of the methylene group CH₂-5 (δ_H=5.15 ppm, δ_C=69.8 ppm), it must be included in a lactone five-membered ring 1a but not in a lactam moiety 1b. In the last structure 1b, the methylene group (CH₂-5) is shifting mostly at δ_H=3.63 ppm (δ_C=45.2 ppm), which differs extremely from our NMR results, excluding such structural probability. Accordingly, 3′-hydroxy-N-(2-oxo-2,5-dihydrofuran-4-yl) propanamide (1a) is the sole reasonable structure in agreement with the chromatographic property and the NMR data discussed above.

Fig. 3:

H,H-COSY (↔, ) and HMBC (→) correlations of 3′-hydroxy-N-(2-oxo-2,5-dihydrofuran-4-yl) propanamide (1a).

Butenolide derivatives are a group of important compounds containing a unique carbon skeleton of 2(5H) and 2(3H) furanones, consisting of unsaturated γ-lactone, which are also known as butenolactones/crotonolactones. They are widely present in many natural products [23], [24], for example, goniobutenolides A and B [23], [25], [26], digitoxigenin, lignans (e.g. podophyllotoxin), ascorbic acid, and antibiotics like patulin [27] and fissohamione [28]. Butenolides exhibit a variety of biological properties and have been considered as potential antitumor agents, fungicides, bactericides, insecticides, antibiotics, and anti-inflammatory and allergy inhibitors [23]. γ-Butyrolactones (GBL) are known to be neuropharmacologically active and exhibit anticonvulsant activity as well [23], [29].

2.4 4-Methoxy-2H-isoquinolin-1-one

As a middle-polar yellow solid, compound 2 was obtained displaying an UV absorbance (λ=254 nm) and blue fluorescence (λ=365 nm) during TLC. Nevertheless, it displayed no color staining with anisaldehyde/sulfuric acid. The UV spectrum of 2 displayed a strong absorbing peak at λ_max=260 nm, referring to its aromatic nature. The molecular weight of 2 was proved as 175 Da according to electrospray ionization mass spectrometry, with a corresponding molecular formula of C₁₀H₉NO₂ based on HR-EIMS, containing seven double bond equivalents.

The ¹H NMR spectrum (Table 5) of 2 displayed five aromatic signals with each 1H, four among them are attributed to 1,2-disubstituted aromatic residue (J~8.3 Hz), which appeared at δ=8.30 (dd), 8.16 (dd), 7.60 (td), and 7.54 (td) ppm. This conclusion was further confirmed by H,H COSY (Fig. 4). The remaining 1H aromatic signal was visible as a singlet (δ=7.19 ppm). In the aliphatic region, a 3H singlet of an aromatic-bounded methyl ether signal was displayed at δ=3.97 ppm. According to the ¹³C NMR/HMQC spectra (Table 5), compound 2 displayed 10 carbon signals, which are identical with the revealed molecular formula. They were classified into four sp² quaternary carbons, two among them are oxygenated (δ=154.9, 147.6 ppm), five are aromatic methines (δ=127.9–101.5 ppm), and one is an aromatic-attached methyl ether (δ=54.6 ppm). Based on its chromatographic properties, the number of unsaturation degrees (7DBE), as well as on the downfield ¹HNMR shifting of the 1,2-disubstituted aromatic residue spectroscopic data, an indole structural nature of 2 must be excluded.

Fig. 4:

H,H-COSY (↔) and HMBC (→) correlations of 4-methoxy-2H-isoquinolin-1-one (2).

A consequent intensive study of compound 2 on the bases of HMBC and H,H-COSY correlations was done (Fig. 4). Based on the HMBC connectivities, the dd proton signal at δ=8.30 ppm (H-8) exhibited five correlations, among them a vital correlation at δ=154.9 ppm (C-1), confirming their location in preposition to each other. The last carbon (C-1, δ=154.9 ppm) is typically for an amide carbonyl present in the aromatized ring system. With respect to the H-8, the para-positioned (H-5) proton signal (δ=8.16 ppm) displayed five HMBC correlations including one ³J coupling with the second oxygenated carbon C-4 (δ=147.6 ppm), confirming their existence in preposition to each other as well. The last carbon (δ=147.6 ppm, C-4) displayed a ³J HMBC cross-section with the methyl ether signal (δ=3.97 ppm) proving their direct attachment. The methine carbon signal at δ=101.5 ppm (C-3, δ_H=7.19 ppm) is flanked by two resonance electron-donating hetero atoms (i.e. O and N). The corresponding singlet proton signal (δ_H=7.19 ppm) of C-3 showed ²J and ³J correlations versus C-4 and C-1, respectively (the remaining HMBC correlation of compound 2 is shown in Fig. 4), confirming our suggestion mentioned previously. In agreement with the chromatographic property and the NMR data discussed previously, structure 2 is deduced as 4-methoxy-2H-isoquinolin-1-one. Synthetically, compound 2 is known [30]; however, we have reported it herein for the first time as a new natural product with full assignment as well.

Isoquinolinone and isoquinolinequinone derivatives are commonly known as anticancer agents [31], [32], [33]. Particularly, isoquinolinone derivatives are anti-breast cancer agents targeting estrogen receptor alpha (ERα) and VEGF receptor 2 (VEGFR-2) through inhibition of ERα and VEGFR-2 simultaneously [34]. Further investigation also showed that they possessed good antiproliferation effects against MCF-7 breast cancer cells and potential antiangiogenesis effects in vivo. These kinds of compounds may provide a new and potential route to develop effective drugs for breast cancer [34], [35], [36].

2.5 Biological activities

Antimicrobial activity testing for the crude extract of the marine actinomycetes N. lucentensis sp. ASMR2 was carried out together with the two new compounds 1a and 2 against a set of microorganisms using the agar diffusion technique. Filtrate and cell extracts of strain ASMR2 showed similarity in their low to moderate activity against Gram-positive bacteria (8–12 mm). Remarkably, the microbial extract from bacteria cultivated on rice medium showed pronounced activity against the yeast C. albicans (14 mm), and low to moderate activity (9–11 mm) against the yeast S. cerevisiae, Gram-positive B. subtilis, and S. aureus. However, the compounds 1a and 2 exhibited no activity (Table 6). An in vitro cytotoxicity assay for the strain extracts and the produced compounds (1a, 2) was studied against the human cervix carcinoma cell line (KB-3-1) and its multidrug-resistant subclone (KB-V1), compared with cryptophycin-52 as reference sample, showing no cytotoxicity as well.

3.1 General experimental procedure

NMR spectra (¹H NMR, ¹³C NMR, DEPT, COSY, HMQC, and HMBC) were measured on Bruker Avance DRX 500 and DRX 600 MHz (Bruker BioSpin, Billerica, MA, USA) spectrometers using standard pulse sequences and referenced to residual solvent signals. EI-HRMS was determined using GCT Premier Spectrometer (Waters, Milford, MA, USA). The UV and visible (UV/Vis) spectra were measured on Spectro UV/Vis Double Beam PC8 scanning auto Cell UVD-3200 (Labomed, Los Angeles, CA, USA), Labomed, Inc. Column chromatography was carried out on silica gel 60 (0.040–0.063 mm, Merck, Darmstadt, Germany) and Sephadex LH-20 (GE Healthcare, Little Chalfont, UK) as the stationary phases. Preparative TLC (0.5 mm thick) and analytical TLC were performed with precoated Merck silica gel 60 PF_254+366. R_f values and visualization of chromatograms was carried out under UV light (λ=254 and 366 nm) and further by spraying with anisaldehyde/sulfuric acid followed by heating.

3.2 Fermentation A (culture broth medium), work up and isolation

1 mL of prepared seed culture of strain ASMR2 was used to inoculate 18 1-L Erlenmeyer flasks containing 300 mL of ISP2 medium (50% seawater). The flasks were incubated in a shaking incubator (100 rpm) at 30°C for 7 days. The culture was harvested and centrifuged (4500 rpm at 5°C) to separate cells. The medium was extracted by ethyl acetate and the cells were extracted with acetone. Acetone and water were evaporated in vacuo. Finally, the residue was re-extracted with ethyl acetate. Chromatographic purification of the afforded bacterial extract (0.9 g) was carried out using silica gel column (100×2 cm), and eluted with petroleum ether-dichloromethane-methanol gradient (0.5 L petroleum ether, 0.5 L petroleum ether-CH₂Cl₂ [1:1], 0.5 L CH₂Cl₂, 0.5 L CH₂Cl₂-MeOH [97:2], 1 L CH₂Cl₂-MeOH [95:5], 0.5 L CH₂Cl₂-MeOH [93:7], 1 L CH₂Cl₂-MeOH [90:10], 0.5 L CH₂Cl₂-MeOH [80:20], 0.5 L CH₂Cl₂-MeOH [50:50]; 0.5 L MeOH). According to TLC monitoring, three fractions were obtained: FI (0.32 g), FII (0.28 g), and FIII (0.3 g). Further column chromatography of fraction III by Sephadex LH-20 (CH₂Cl₂/40% MeOH) and silica gel afforded furan-2,5-dimethanol (14 mg).

3.3 Fermentation B (rice medium), work up and isolation

100 mL of ISP2 medium was inoculated with a spore suspension of strain ASMR2 and cultivated at 28°C for 3 days as seed culture. A total of 5 mL of this seed culture was used to inoculate 1-L Erlenmeyer flasks (12 flasks) containing modified rice medium, consisting of 100 g of commercial rice; 150 mL of distilled water/seawater (1:1) supplemented with 0.4% yeast extract, and 1% malt extract. The flasks were incubated for 14 days at 37°C. After harvesting, 0.5 L methanol was added to each flask, and then applied to vigorous shaking for 5 min. The obtained aqueous methanol extract was then separated from rice by filtration under vacuum. After filtration, the water/methanol fraction was evaporated to remove methanol using a rotary evaporator (Heidolph). After complete evaporation of methanol, the water phase was re-extracted by ethyl acetate. The obtained ethyl acetate extracts were finally in vacuo concentrated to dryness and then applied to further work up.

The crude extract (6.0 g) was separated by column chromatography on silica gel (100×10 cm) with a cyclohexane-CH₂Cl₂-MeOH gradient (0.5 L cyclohexane, 0.5 L cyclohexane-CH₂Cl₂ [1:1], 0.5 L CH₂Cl₂, 0.5 L CH₂Cl₂-MeOH [97:2], 1 L CH₂Cl₂-MeOH [95:5], 0.5 L CH₂Cl₂-MeOH [93:7], 1 L CH₂Cl₂-MeOH [90:10], 0.5 L CH₂Cl₂-MeOH [80:20], 0.5 L CH₂Cl₂-MeOH [50:50]; 0.5 L MeOH). According to TLC monitoring, six fractions were obtained: FI (3 g), FII (0.54 g), FIII (0.1 g), FIV (0.47 g), FV (0.32 g), and FVI (1.57 g). Further column chromatography of fraction III on Sephadex LH-20 (CH₂Cl₂-40% MeOH) afforded indole-3 acetic acid methyl ester as a colorless solid (1 mg). An application of fraction IV to silica gel (column 100×2 cm) and elution with a cyclohexane-CH₂Cl₂-MeOH gradient resulted in five subfractions: FIVa (0.1 g), FIVb (0.07 g), FIVc (0.03 g), FIVd (0.19 g), and FIVe (0.08 g). Purification of subfraction FIVa on silica gel column and elution with DCM-MeOH gradient delivered indole-3 acetic acid as a colorless solid (10 mg). In a similar way, subfraction FIVb afforded indole-3-carboxylic acid (7 mg) as a colorless solid. Subfraction FIVc yielded, in the same way, tyrosol (5 mg) as a colorless solid. Purification of subfraction FIVd using Sephadex LH-20 (MeOH) resulted in the isolation of glyceryl monolinoleate as a colorless oil (54 mg). Fraction V was subjected to Sephadex LH-20 (MeOH) giving 4-methoxy-2H-isoquinolin-1-one (2, 12 mg) as a faint yellow solid, in addition to cyclo-(Tyro, Pro) (3 mg) and 3′-hydroxy-N-(2-oxo-2,5-dihydrofuran-4-yl)propanamide (1a, 5 mg) as colorless solids. Finally, the polar fraction FVI (1.57 g) was purified using silica gel column eluted with DCM-MeOH gradient followed by Sephadex LH-20 (MeOH) to afford adenosine (10 mg) as a colorless solid.

3.4 Antimicrobial assay using agar diffusion test

Antimicrobial activity testing of the crude extract of the marine isolate ASMR2 was carried out together with the two new compounds (1a, 2) against a set of microorganisms using the agar diffusion technique. Paper-disk diffusion assay [42], with some modifications, was followed to measure the antimicrobial activity. A total of 20 mL of medium seeded with test organism were poured into 9 cm sterile Petri dishes. After solidification, the paper disks (6 mm diameter) containing the extract or the purified compounds with a concentration of 10 μL of 25 mg mL⁻¹ of stock solution (i.e. 250 μg per disk) were placed on inoculated agar plates and the loaded substances were allowed to diffuse into the bacterial lawn at 4°C for 2 h. Afterward, the plates were incubated for 24 h at 30°C. Both bacteria and yeasts were grown on nutrient agar medium containing 3 g L⁻¹ beef extract, 10 g L⁻¹ peptone, and 20 g L⁻¹ agar. The pH value was adjusted to 7.2 using NaOH. Fungal strains were grown on potato dextrose agar medium containing 4 g L⁻¹ potato extract, 20 g L⁻¹ dextrose, 15 g L⁻¹ agar no. 1 (pH 6). After incubation, the diameters of the inhibition zones were measured against a wide panel of test microorganisms comprising Gram-positive bacteria (B. subtilis, S. aureus), Gram negative bacteria (Pseudomonas aeruginosa ATCC 27853), yeasts (C. albicans ATCC 10231 and S. cerevisiae ATCC 9080), and the fungus Aspergillus niger.

3.5 Cytotoxicity assays

The KB-3-1 and KB-VI cells were cultivated as a monolayer in Dulbecco’s modified eagle medium with glucose (4.5 g L⁻¹), l-glutamine, sodium pyruvate, and phenol red supplemented with 10% (KB-3-1) fetal bovine serum. The cells were maintained at 37°C and 5.3% CO₂-humidified air. On the day before the test, the cells (70% confluence) were detached with trypsin-ethylenediamine tetra-acetic acid solution (0.05% and 0.02%, respectively, in Dulbecco’s phosphate-buffered saline) and placed in sterile 96-well plates at a density of 10,000 cells in 100 μL medium per well. The dilution series of the compounds were prepared from stock solutions in DMSO of concentrations of 100 mM, 50 mM, or 25 mM. The stock solutions were diluted with culture medium (10% fetal bovine serum) down to the picomolar range. The dilution prepared from the stock solution was added to the wells. Each concentration was tested in six replicates. Dilution series were prepared by pipetting liquid from well to well. The control contained the same concentration of DMSO as the first dilution. After incubation for 72 h at 37°C and 5.3% CO₂-humidified air, 30 μL of an aqueous resazurin solution (175 μM) was added to each well. The cells were incubated under the same conditions for 5 h. Subsequently, the fluorescence was measured. The excitation was affected at a wavelength of 530 nm, whereas the emission was recorded at a wavelength of 588 nm. The IC₅₀ values were calculated as a sigmoidal dose-response curve using GraphPad Prism 4.03 (GraphPad Software, San Diego, USA). The IC₅₀ values equal the drug concentrations, in which vitality is 50% [43], [44].