Chromenone Derivatives as Monoamine Oxidase Inhibitors from Marine-Derived MAR4 Clade Streptomyces sp. CNQ-031

Three compounds were isolated from marine-derived Streptomyces sp. CNQ-031, and their inhibitory activities against monoamine oxidases (MAOs), acetylcholinesterase (AChE), butyrylcholinesterase (BChE), and β-secretase (BACE-1) were evaluated. Compound 1 (5,7-dihydroxy-2-isopropyl-4H-chromen-4-one) was a potent and selective inhibitor of MAO-A, with a 50% inhibitory concentration (IC50) of 2.70 μM and a selectivity index (SI) of 10.0 versus MAO-B. Compound 2 [5,7-dihydroxy-2-(1-methylpropyl)-4H-chromen-4-one] was a potent and low-selective inhibitor of MAO-B, with an IC50 of 3.42 μM and an SI value of 2.02 versus MAO-A. Compound 3 (1-methoxyphenazine) did not inhibit MAO-A or MAO-B. All three compounds showed little inhibitory activity against AChE, BChE, and BACE-1. The Ki value of compound 1 for MAO-A was 0.94 ± 0.28 μM, and the Ki values of compound 2 for MAO-A and MAO-B were 3.57 ± 0.60 and 1.89 ± 0.014 μM, respectively, with competitive inhibition. The 1-methylpropyl group in compound 2 increased the MAO-B inhibitory activity compared with the isopropyl group in compound 1. Inhibition of MAO-A and MAO-B by compounds 1 and 2 was recovered by dialysis experiments. These results suggest that compounds 1 and 2 are reversible, competitive inhibitors of MAOs and can be considered potential therapies for neurological disorders such as depression and Alzheimer’s disease.


Introduction
hydroxy-2-methyl-chroman-4-one (HMC) from the endogenous lichen fungus Daldinia fissa [19]. Marine natural MAO inhibitors in particular have recently been reviewed [20]. In this study, we isolated three compounds from a marine-derived Streptomyces sp., identified their structures and investigated their MAO inhibitory activities, as well as the AChE, BChE, and BACE-1 inhibitory activities, for their possible multitarget inhibitions.

Structural Analysis
Optical rotations were acquired using a Kruss Optronic P-8000 polarimeter with a 5-cm cell. The ultraviolet (UV) spectra were measured with a V-730 UV-visible spectrophotometer (Jasco, USA) using a path length of 1 cm. The infrared spectra were recorded on a Varian Scimitar Series in CHCl 3 . The nuclear magnetic resonance (NMR) spectra were acquired at 300 MHz for 1 H in CD 3 OD and DMSO-d 6 using a solvent signal as an internal reference (δ H 3.31 and δ H 2.50 for the respective solvents). Mass data were obtained on an Agilent Technologies 6120 quadrupole. Electrospray ionization mass spectroscopy (ESIMS) data were collected using an Agilent Technologies 6120 quadrupole mass spectrometer (Santa Clara, CA) coupled with an Agilent Technologies 1260 series HPLC with a reversed-phase column (Phenomenex Luna C-18(2) (100 Å, 50 mm × 4.6 mm, 5 μm) at a flow rate of 1.0 ml/min. The fractions were purified by a Waters 616 quaternary HPLC pump and a Waters 996 photodiode array detector using a Phenomenex Luna C-18(2) (250 mm × 10 mm, 5 μm) reversed HPLC column. HRMS analysis was conducted with a JEOL JMS-AX505WA mass spectrometer.

Bacterial Culture and Isolation of Compounds
The marine-derived actinomycetes strain CNQ-031 was isolated from sediment sampled off the coast of California. It was identified as belonging to the Streptomyces sp. MAR4 clade on the basis of 16S rRNA gene sequence analysis. Strain CNQ-031 was cultured in 40 L of a 2.5-L ultra-yield flask containing 1 L of SYP SW medium (10 g/l of soluble starch, 2 g/l of yeast, 4 g/l of peptone, 10 g/l of CaCO 3 , 20 g/l of KBr, 8 g/l of Fe 2 (SO 4 ) 3 × 4(H 2 O) dissolved in 750 ml natural seawater and 250 ml of distilled water) at 25°C with shaking at 150 rpm for 7 days. The culture medium was extracted with EtOAc (40 L overall), and the EtOAc-soluble fraction was concentrated in vacuo to yield 5.84 g of crude extract. The entire crude extract was fractionated by C-18 open column chromatography with a step gradient from 20% to 100% MeOH in distilled water to obtain nine fractions. The third fraction was subjected to reversed-phase HPLC with 39% aqueous acetonitrile (Phenomenex Luna C-18(2) (100 Å, 250 mm × 100 mm, 2.0 ml/min, 5 μm) to obtain compound 1 (19.8 mg). The fourth fraction was chromatographed and eluted with 50% acetonitrile to isolate compound 3 (6.0 mg), and the fifth fraction was purified with 47% acetonitrile to provide compound 2 (11.4 mg).
MAO-A and MAO-B inhibitory activities were determined using a continuous spectrophotometric method as described previously [23,24]. The K m values of MAO-A for kynuramine and of MAO-B for benzylamine were 0.024 and 0.14 mM, respectively. The concentrations of kynuramine (0.06 mM) and benzylamine (0.3 mM) used were 1.5 and 1.8 times the respective K m values. AChE activities were assayed as described by Ellman et al. [25], with slight modification [26,27]. The inhibitory activities of AChE and BChE were measured after the enzyme was preincubated with inhibitors for 15 min and before adding DTNB and the substrate (ATCI and BTCI, respectively). Reactions were performed using approximately 0.2 U/ml of AChE and BChE in the presence of 0.5 mM DTNB and 0.5 mM substrate (ATCI and BChE) in 0.5-ml reaction mixtures; reactions were continuously monitored for 10 min at 412 nm. Reaction rates were expressed as changes in absorbance per minute [28]. BACE-1 assays were performed using the BACE-1 kit at 320 and 405 nm for excitation and emission wavelengths, respectively, and a fluorescence spectrometer (FS-2, Scinco, Korea) after reaction for 2 h at 37 o C with 7methoxycoumarin-4-acetyl-[Asn670,Leu671]-amyloid β/A4 protein fragment 667-676-(2,4-dinitrophenyl)Lys-Arg-Arg amide trifluoroacetate as a substrate [29].

Analysis of Inhibitory Activities of the Isolated Compounds
Inhibition of MAOs, AChE, BChE, and BACE-1 by the three compounds was investigated at a concentration of 10 μM, and then IC 50 values of the compounds and the reference inhibitors were determined. Kinetic parameters, inhibition types, and kinetic of inhibition (K i ) values of potent MAO-A and MAO-B inhibitors (i.e., compound 1 for MAO-A; compound 2 for MAO-A and MAO-B) were determined, as previously described [26]. The K i values were measured at five different substrate concentrations for each MAO isoform (0.0075, 0.015, 0.03, 0.06, and 0.12 mM for MAO-A; 0.03, 0.06, 0.15, 0.3, and 0.6 mM for MAO-B) and in the absence or presence of each inhibitor at concentrations of approximately 0.5, 1.0, and 2.0 times their IC 50 values [21]. Inhibitory patterns and K i values were determined using Lineweaver-Burk plots and secondary plots of their slopes.

Reversibility Tests of Compounds 1 and 2
The reversibilities of MAO inhibition by compounds 1 and 2 and by the reference compounds (toloxatone and clorgyline for MAO-A; lazabemide and pargyline for MAO-B) at twice the IC 50 concentrations were investigated by dialysis as previously described [22]. After the compounds or reference inhibitors were preincubated with MAOs for 30 min, residual activities for undialyzed and dialyzed samples were measured. The relative values for undialyzed (A U ) and dialyzed (A D ) activities were then calculated, and the reversibilities were determined by comparing the A U and A D values of the inhibitors with those of the references.

Isolation and Identification of the Compounds
According to the procedure, three compounds were isolated, and their properties were as follows: . Compound 3 was identified as 1-methoxyphenazine according to a comparison of the 1 H and 13 C NMR data with previously reported data [31]. The structures of compounds 1, 2, and 3 are shown in Fig. 1.

Reversibilities of Compounds 1 and 2
In reversibility experiments by dialysis, inhibition of MAO-A by compounds 1 and 2 was substantially recovered, from 35.5% (A U ) to 90.9% (A D ) and from 35.6% to 79.4%, respectively ( Fig. 2A). These values were similar to those observed for the reversible inhibitor toloxatone (33.6% to 84.2%). Little recovery was observed for the irreversible inhibitor clorgyline (36.3% to 39.7%) ( Fig. 2A). Inhibition of MAO-B by compound 2 was recovered from 34.8% (A U ) to 76.7% (A D ), and these recovery values were similar to those observed for the reversible inhibitor lazabemide (30.5% to 78.7%), whereas little recovery was observed for the irreversible inhibitor pargyline (35.4% to 39.5%) (Fig. 2B). These results indicated that compound 1 was a reversible inhibitor of MAO-A and that compound 2 was a reversible inhibitor of MAO-A and MAO-B.

Kinetics of Compounds 1 and 2
Modes of MAO-A inhibition by compound 1 and modes of MAO-A and MAO-B inhibition by compound 2 were investigated using Lineweaver-Burk plots. Plots of MAO-A inhibition by compound 1 were linear and intersected the y-axis (Fig. 3A), indicating that compound 1 is a competitive inhibitor of MAO-A. Secondary plots of the slopes of the Lineweaver-Burk plots against the inhibitor concentrations showed a Ki value of 0.94 ± 0.28 μM for compound 1 (Fig. 3B). Similarly, compound 2 was a competitive inhibitor of MAO-A and MAO-B (Figs. 3C and 3E); its Ki values were 3.57 ± 0.60 and 1.89 ± 0.014 μM, respectively (Figs. 3D and 3F).
In this study, three compounds-two chromenones (compounds 1 and 2) and one phenazine (compound 3)were isolated from marine-derived Streptomyces sp. CNQ-031. Compound 1 potently and selectively inhibited MAO-A, and compound 2 inhibited MAO-B and next MAO-A with low selectivity. Compound 3 showed little inhibitory activity against MAO-A and MAO-B. All three compounds weakly inhibited AChE, BChE, and BACE-1. Compound 1 was a reversible competitive inhibitor of MAO-A, and compound 2 was a reversible competitive inhibitor of MAO-A and MAO-B; thus, both compound 1 and compound 2 are effective MAO inhibitors from a microbial source.