1. Introduction
The ocean covers more than 70% of the Earth’s surface and hosts huge biological and chemical diversity. Because marine environmental conditions are quite different from terrestrial ones, natural products from marine organisms have unique structures and biological activities. Marine cyanobacteria, in particular, are known to produce various secondary metabolites and have been recognized as a source of pharmaceutical lead compounds [
1,
2,
3]. For example, bisebromoamide, isolated from
Lyngbya sp., showed potent cytotoxicity against HeLa S
3 cells [
4]. Bisebromoamide inhibited the phosphorylation of extracellular signal-regulated protein kinase (ERK) and was identified as an actin filament stabilizer [
5]. Meanwhile, hoshinolactam was found to possess both a cyclopropane ring and γ-lactam ring, and exhibited potent antitrypanosomal activity without cytotoxicity against human fetal lung fibroblast MRC-5 cells [
6]. The malyngamide series of natural products have been isolated from various marine filamentous cyanobacteria. Malynagmide A, the first compound of this group, was isolated from
Lyngbya majuscule in 1979 [
7]; since then, over 30 malyngamide analogs have been isolated [
8]. As part of our ongoing effort to identify novel bioactive natural products, we have focused on the constituents of marine cyanobacteria and isolated odoamide [
9,
10] and odobromoamide [
11]. We recently discovered three new malyngamides, 6,8-di-
O-acetylmalyngamide 2 (
1), 6-
O-acetylmalyngamide 2 (
2), and
N-demethyl-isomalyngamide I (
3), from the Okinawan cyanobacterium belonging to the genus
Moorea producens. (
Figure 1). Herein, we report the isolation, structure determination, and biological evaluation of these compounds.
3. Experimental Section
3.1. General Experimental Procedures
Chemicals and solvents were the best grade available and used as received from commercial sources. Rat L6 myoblasts were purchased from JCRB Cell Bank (Osaka, Japan). Optical rotations were measured on a JASCO P-1010 polarimeter (JASCO Corporation, Tokyo, Japan). IR spectra were measured on a JASCO FT/IR-6100 spectrometer (JASCO Corporation, Tokyo, Japan). All NMR spectra were recorded on a Bruker AVANCE III 500 NMR spectrometer (500 and 125 MHz for 1H and 13C NMR, respectively, Bruker BioSpin Corporation, Billerica, MA, USA). Chemical shifts were reported as δ values in parts per million (ppm) relative to the residual solvent signals (CD3Cl: δH 7.26, δC 77.0), and coupling constants were in hertz (Hz). ESIMS data were obtained using a Waters Quattro micro API mass spectrometer (Waters Corporation, Milford, MA, USA), HRESIMS data were obtained using a Waters Micromass Q-TOF spectrometer (Waters Corporation, Milford, MA, USA). HPLC was carried out on a JASCO PU-2080 Plus Intelligent HPLC pump (JASCO Corporation, Tokyo, Japan) and a JASCO UV-2075 Plus Intelligent UV/VIS detector (JASCO Corporation, Tokyo, Japan). Absorbance of assay mixture was completed using a BioTek ELx800 absorbance microplate reader (BioTek Instruments Inc., Winooski, VT, USA).
3.2. Collection, Extraction, and Isolation
Samples of the marine cyanobacterium, Moorea producens were collected by hand from the coast of Bise, Okinawa Prefecture, Japan, in April 2016. The cyanobacterium was identified by 16S rRNA sequence analysis. Approximately 30 g (wet weight) of the samples were extracted with MeOH (1.0 L). The extract was filtered, and the filtrate was concentrated. The residue was partitioned between H2O (0.2 L) and EtOAc (0.2 L × 3). The material obtained from the organic layer was further partitioned between 90% aqueous MeOH (0.1 L) and n-hexane (0.1 L × 3). The aqueous MeOH fraction (0.23 g) was separated by column chromatography on ODS (2.0 g) using 60% aqueous MeOH, 80% aqueous MeOH, and MeOH. The fraction (131.4 mg) eluted with 80% aqueous MeOH was subjected to reversed-phase HPLC [Cosmosil 5C18-AR-II (20 mm × 250 mm), 85% MeOH at 5.0 mL/min, and UV detection at 215 nm] to give five fractions (Fractions 1–5). Fraction 1 was subjected to further HPLC [Cosmosil 5C18-AR-II (20 mm × 250 mm), 80% MeOH at 5.0 mL/min, and UV detection at 215 nm] to yield compound 2 (26.8 mg, tR = 34.2 min). Fraction 2 was subjected to further HPLC [Cosmosil 5C18-AR-II (20 mm × 250 mm), 70% MeCN at 5.0 mL/min, and UV detection at 215 nm] to yield compound 3 (12.6 mg, tR = 38.4 min) and two fractions (Fractions 2-1 and 2-2). Fraction 2-2 was further subjected to HPLC [Cosmosil π NAP (20 mm × 250 mm), 65% MeCN at 5.0 mL/min, and UV detection at 215 nm] to yield compound 1 (12.0 mg, tR = 35.1 min).
6,8-Di-O-Acetylmalyngamide 2 (
1): Colorless oil;
+4.8 (
c 1.20, MeOH); IR (neat) 3302, 2929, 2856, 1731, 1651, 1539, 1445, 1373, 1232, 1132, 1096 cm
−1;
1H NMR,
13C NMR and HMBC data, see
Table 1; HRESIMS
m/
z 572.2989 [M + H]
+ (calcd. for C
29H
47ClNO
8 572.2985).
6-O-Acetylmalyngamide 2 (
2): Colorless oil;
+22.9 (
c 2.68, MeOH); IR (neat) 3375, 2929, 2856, 1730, 1650, 1540, 1446, 1375, 1239, 1066 cm
−1;
1H NMR,
13C NMR and HMBC data, see
Table 1; HRESIMS
m/
z 552.2692 [M + Na]
+ (calcd. for C
27H
44ClNO
7Na 552.2699).
N-Demethyl-isomalyngamide I (
3): Colorless oil;
+108.2 (
c 1.12, MeOH); IR (neat) 3313, 2928, 2856, 1714, 1647, 1541, 1456, 1433, 1094 cm
−1;
1H NMR,
13C NMR and HMBC data, see
Table 2; HRESIMS
m/
z 470.2692 [M + H]
+ (calcd. for C
25H
41ClNO
5 470.2668).
3.3. Identification of the Marine Cyanobacterium
Morphological observation was performed using a phase contrast microscopy ECLIPSE Ti-S (Nicon, Tokyo, Japan). The mean cell size and standard deviation of 50 cells were measured. The cell width was observed 36.3 ± 2.0 μm and length was observed 4.0 ± 0.9 μm. The cells were surrounded by thick (2.5–14.5 µm) firm and laminated sheaths. These morphological characters were consistent with description of
Moorea producens [
24]. Therefore, the marine cyanobacterium was identified as
M. producens.
3.4. Preparation of MTPA Esters of 2
Compound 2 (1.1 mg) was reacted with R-MTPACl (10 μL) and DMAP (1.1 mg) in pyridine (50 μL), and the mixture was stirred for 5 h at room temperature. The reaction mixture was concentrated, and the residue was partitioned between EtOAc and 0.1 M NaHCO3 (1:1). The extract obtained from the organic layer was subjected to reversed-phase HPLC [Cosmosil 5C18-AR-II (20 mm × 250 mm), 85% MeCN at 5.0 mL/min, and UV detection at 215 nm] to yield S-MTPA ester (0.8 mg). Using the same procedure as described above, R-MTPA (0.5 mg) ester was obtained from 2 (1.0 mg).
S-MTPA ester: 1H NMR (500 MHz, CDCl3) δ 4.03 (H-4), 5.29 (H-6), 2.41 (H-7a), 2.66 (H-7b), 5.28 (H-8), 1.12 (H-10), 2.15 (6-OAc); ESIMS m/z [M + Na]+ 768.3.
R-MTPA ester: 1H NMR (500 MHz, CDCl3) δ 4.08 (H-4), 5.11 (H-6), 2.40 (H-7a), 2.61 (H-7b), 5.21 (H-8), 1.24 (H-10), 2.13 (6-OAc); ESIMS m/z [M + Na]+ 768.3.
3.5. Preparation of Acetylated Compound 2
Compound 2 (5.0 mg) was reacted with Ac2O (50 μL) in pyridine (50 μL), and the mixture was stirred for 2 h at room temperature. The reaction mixture was subjected to reversed-phase HPLC [Cosmosil 5C18-AR-II (10 mm × 250 mm), 85% MeOH at 4.0 mL/min, and UV detection at 215 nm] to yield acetylated compound of 2 (4.7 mg).
Acetylated compound of 2: +5.5 (c 1.20, MeOH); 1H NMR (500 MHz, CDCl3) δ 6.63 (1H, t, J = 6.0 Hz), 6.29 (1H, d, J = 1.6 Hz), 5.69 (1H, s), 5.47 (1H, m), 5.45 (1H, m), 5.41 (1H, dd, J = 12.9, 6.7 Hz), 5.11 (1H, t, J = 2.8 Hz), 4.27 (1H, s), 4.23 (1H, dd, J = 16.5, 6.7 Hz), 3.95 (1H, ddd, J = 16.5, 5.8, 2.0 Hz), 3.31 (3H, s), 3.17 (1H, m), 2.60 (1H, dt, J = 13.3, 2.6 Hz), 2.32 (1H, m), 2.28 (2H, m), 2.27 (2H, m), 2.19 (3H, s), 2.18 (2H, m), 2.16 (3H, s), 1.42 (2H, m), 1.27 (10H, m), 1.26 (3H, s), 0.87 (3H, t, J = 6.7 Hz); ESIMS m/z [M + H]+ 572.3.
3.6. Base Hydrolysis of Compounds 2 and 3
Compound 2 (7.3 mg) was dissolved in a 5.0 mL solution of 10% KOH in 80% aqueous EtOH and refluxed for 14 h. The reaction mixture was concentrated, and the residue was partitioned between EtOAc and H2O. The organic layer was subjected to reversed-phase HPLC [Cosmosil 5C18-AR-II (10 mm × 250 mm), 80% MeOH with 0.1% TFA at 5.0 mL/min, and UV detection at 215 nm] to yield lyngbic acid (1.6 mg). Using the same procedure as described above, lyngbic acid (4.7 mg) ester was obtained from 3 (13.5 mg).
Lyngbic acid from 2: −10.8 (c 0.16, CHCl3); 1H NMR (500 MHz, CDCl3) δ 5.49 (2H, m), 3.32 (3H, s), 3.15 (1H, m), 2.42 (2H, m), 2.35 (2H, m), 2.19 (2H, m), 1.43 (2H, m), 1.27 (10H, m), 0.88 (3H, t, J = 6.8 Hz).
Lyngbic acid from 3: −19.6 (c 0.47, CHCl3); 1H NMR (500 MHz, CDCl3) δ 5.49 (2H, m), 3.32 (3H, s), 3.15 (1H, m), 2.42 (2H, m), 2.35 (2H, m), 2.19 (2H, m), 1.43 (2H, m), 1.27 (10H, m), 0.88 (3H, t, J = 6.8 Hz).
3.7. Preparation of MTPA Ester of Compound 3
Compound 3 (7.3 mg) was reacted with R-MTPACl (10 μL) and DMAP (0.5 mg) in pyridine (50 μL), and the mixture was stirred for 3 h at room temperature. The reaction mixture was concentrated, and the residue was partitioned between EtOAc and 0.1M NaHCO3 (1:1). The extract obtained from the organic layer was subjected to reversed-phase HPLC [Cosmosil 5C18-AR-II (20 mm × 250 mm), 85% MeOH at 5.0 mL/min, and UV detection at 215 nm] to yield compound 4 (2.3 mg).
Compound
4:
+40.0 (
c 0.23, MeOH);
1H NMR,
13C NMR and HMBC data, see
Table S1; HRESIMS
m/
z 474.2381 [M + Na]
+ (calcd. for C
25H
38ClNO
4Na 474.2382).
3.8. Culture of L6 Myoblasts
L6 myoblasts (5 × 103 cells/well in 96-well plates or 2 × 105 cells/well in 60-mm culture dishes) were maintained in high glucose Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS), streptomycin (100 µg/mL), and penicillin G (100 units/mL) at 37 °C with 5% CO2. After reaching 80% confluence, the cells were cultured in low glucose DMEM containing 2% FBS for 1 week to differentiate into myotubes. The medium was renewed every 2 days.
3.9. Determination of Glucose Uptake
L6 myotubes were incubated in filter-sterilized Krebs-Henseleit buffer (1.2 mM MgSO
4, 1.2 mM KH
2PO
4, 4.7 mK KCl, 119 mM NaCl, 2.5 mM CaCl
2·2H
2O, and 25 mM NaHCO
3, pH 7.4) containing 0.1% bovine serum albumin (BSA), 10 mM HEPES, and 2 mM sodium pyruvate (KHH buffer) for 2 h. The myotubes were then cultured in KHH buffer containing 5 mM glucose with or without compounds
1,
2, and
3 (10–40 µM) for 16 h and without or with compound C (30 µM), an AMPK inhibitor for 6 h. Nepodin [
25] was used as a positive control, and DMSO alone was used as a negative control. The concentrations of glucose remaining in KHH buffer were determined by a commercial assay kit (Glucose CII-Test Wako) and a microplate reader at 490 nm. The amounts of glucose uptake by myotubes were calculated from the differences in glucose concentrations between before and after culture.
3.10. Western Blotting
L6 myotubes were lysed in Blue Loading Buffer for 1 min after washing with ice-cold PBS. The lysates were sonicated for 10 s, boiled at 100 °C for 10 min and centrifuged at 15,000 rpm for 5 min. The protein concentrations of the supernatants were determined by a commercial assay kit (RC DC Protein Assay, Bio-Rad laboratories Inc., Hercules, CA, USA). Equal amounts of protein samples (50 µg/lane) were electrophoresed on 10% Mini-PROTEAN TGX precast gels (Bio-Rad laboratories Inc., Hercules, CA, USA) and transferred to nitrocellulose membranes (Bio-Rad laboratories Inc., Hercules, CA, USA). The membranes were washed with Tris buffered saline (TBS) for 15 min and blocked with 3% nonfat dry milk, or 80% BSA in TBS containing 0.05% Tween 20 (TBST), at room temperature for 1 h. The membranes were then washed with TBST and incubated with anti-AMPK or anti-phosphorylated AMPK primary antibodies (1:1000 or 1:2000 in blocking buffer, respectively) at 4 °C overnight. The membranes were then washed with TBST and incubated with HRP-linked anti-rabbit IgG secondary antibodies (1:2000 in blocking buffer) at room temperature for 1 h. After washing with TBST, immunoreactive bands were detected by a chemiluminescent reagent (Clarity Western ECL Substrate, Bio-Rad laboratories Inc., Hercules, CA, USA) and quantified by densitometry analysis using a ChemiDoc XRS Plus system (Bio-Rad laboratories Inc., Hercules, CA, USA) and Image Lab software (version 5.2, Bio-Rad laboratories Inc., Hercules, CA, USA).