Mangromicin analogs were discovered in a cultured broth of a rare actinomycete, Lechevalieria aerocolonigenes K10-0216.1,2 The mangromicin analogs showed antitrypanosomal and reactive oxygen species (ROS) scavenging bioactivities. Mangromicin analogs have unique structures, including common partial structures, notably a cyclopentadecane skeleton with a tetrahydrofuran unit and a 5,6-dihydro-4-hydroxy-2-pyrone moiety. In a previous report, we described eight new mangromicin analogs, which possessed anti-oxidative properties. At that time, we discovered mangromicin C (1) but were unable to determine its structure, which was clearly different from all the other analogs. We have now found that 1 has a structure which includes a tetrahydropyran ring (Figure 1). In this paper, we report the fermentation, isolation, structural elucidation and biological activity of 1.

Figure 1
figure 1

Relative configurations of mangromicin C (1).

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A loop of spores of the strain K10-0216 was inoculated into 100 ml of the seed medium, consisting of 2.4% starch (Wako Pure Chemical Industries, Osaka, Japan), 0.1% glucose (Wako), 0.3% peptone (Kyokuto Pharmaceutical Industrial, Tokyo, Japan), 0.3% meat extract (Kyokuto), 0.5% yeast extract (Oriental Yeast, Tokyo, Japan) and 0.4% CaCO3 (Wako) (adjusted to pH 7.0 before sterilization) in a 500 ml Erlenmeyer flask. The flask was incubated on a rotary shaker (210 r.p.m.) at 27 °C for 3 days. A 1 ml portion of the seed culture was transferred to 500 ml Erlenmeyer flasks (total 150) containing 100 ml of starch medium, consisting of 2% soluble starch (Wako), 0.5% glycerol (Wako), 1.0% defatted wheat germ (Nisshin Pharma, Tokyo, Japan), 0.3% meat extract, 0.3% dry yeast (JT Foods, Tokyo, Japan) and 0.3% CaCO3 (adjusted to pH 7.0 before sterilization) and fermentation was carried out on a rotary shaker (210 rpm) at 27 °C for eight days.

The cultured broth of K10-0216 strain (15 l) was centrifuged to separate the mycelium and supernatant. The supernatant was extracted three times with ethyl acetate (15 l). The organic layer was concentrated to dryness in vacuo to afford a crude material (6 g). The ethyl acetate extract was subjected to silica gel column chromatography FL100D (60 i.d. × 200 mm, Fuji Silysia, Tokyo, Japan), which was sequentially eluted with 1 l of a mixture of CHCl3-CH3OH (100:0, 100:1, 50:1 and 10:1) in that order. The eluate fraction (100:1) was concentrated to yield 550 mg and applied to an ODS column (40 i.d. × 150 mm; Senshu Scientific, Tokyo, Japan). After washing with 50% MeOH, the fractions containing 1 were eluted with 60% MeOH, followed by concentration in vacuo. The eluate fractions (278 mg) were purified by high performance liquid chromatography on an Inertsil ODS-4 column (14 i.d. × 250 mm, GL Sciences, Tokyo, Japan) with 40% MeOH at 9.3 ml min−1 and subsequently detected under UV light of 254 nm. The yield of 1 was 45.0 mg.

Compound 1 was obtained as a white powder ([α]D25.3=+23.2; c=0.1 in MeOH); UV (MeOH) λmax (ɛ): 251 nm (5595), and its infra-red spectrum showed the characteristic absorptions of hydroxyl and carbonyl groups (νmax, 3430 and 1657 cm−1). The HR-ESIMS of 1 produced the [M+H]+ ion at m/z 395.2413 indicating the molecular formula was C22H34O6 (calculated value for C22H35O6, 395.2434). Since some broadening signals were observed in CD3OD, the 1D and 2D nuclear magnetic resonance (NMR) spectra of 1 were obtained in DMSO-d6. The sharpest NMR signals of 1 were observed at 70°C among various temperatures (see Supplementary Information).

Mangromicin C (1) structure was elucidated by comparison of the 1H and 13C NMR of other mangromicin analogs. The 1H and 13C NMR spectral data of 1 measured in DMSO-d6 at 70 °C are listed in Table 1. The 1H NMR data indicated the presence of three oxygenated sp3 methines, three sp3 methines, containing two methines coupled to methyl groups, seven methylenes, one primary methyl, two secondary methyls and one tertiary methyl. The 13C NMR spectrum showed the resonances of 22 carbons, which were classified into two olefinic carbons, two carbonyl carbons at δc 164.6 and 209.3, one oxygenated sp3 quaternary carbon, three oxygenated sp3 methine carbons, three sp3 methine carbons, seven sp3 methylene carbons and four methyl carbons by heteronuclear-single-quantum coherence spectra.

Table 1 1H and 13C NMR spectral data for mangromicin C (1)
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The 1H-1H COSY indicated the presence of three partial structures (a) C-8/C-12, C-8/C-8-Me and C-12/C-12-Me, (b) C-15/C-16 (c) C-4/C-3′ (Me), as shown in Figure 2. Analysis of HMBC data confirmed the presence of a 5,6-dihydro-4-hydroxy-2-pyrone moiety (a partial structure in mangromicins), based on correlations from H-4 to C-2, C-3 and C-6; from H-5 to C-1, C-3 and C-1′; from H2-15 to C-2; and from H2-16 to C-1, C-2 and C-14. The HMBC correlations from H-4 to C-2′; from H-5 to C-1′; from H2-1′ to C-3, C-4, C-5, and C-2′; from H2-2′ to C-4, C-1′ and C-3′; and from H3-3′ to C-1′ and C-2′ confirmed an n-propyl group linked to the C-4 position. A tetrahydropyran unit was identified, based on HMBC correlations from H2-9 to C-10 and C-11; from H-10 to C-11 and C-14; from H-11 to C-9 and C-13; from H-12 to C-10, C-11, C-13 and C-14. Moreover, the correlations from H-11 and H2-13 to C-12-Me; from H2-13 and H2-15 to C-14-Me; from H3-12-Me to C-11, C-12 and C-13; and from H3-14-Me to C-13, C-14 and C-15 confirmed a secondary methyl and a tertiary methyl linked to the C-12 and C-14 positions, respectively. The HMBC correlations from H2-15 to C-2 and C-14, and from H2-16 to C-1, C-2 and C-14 also showed that the 5,6-dihydro-4-hydroxy-2-pyrone moiety and tetrahydropyran ring were connected by an ethylene bond. Finally, the cyclopentadecane ring was confirmed by the HMBC correlations from H2-6 to C-4, C-5 and C-7; from H-8 to C-9; from H2-9 to C-7, C-10, C-11, and C-8-Me; from H3-8-Me to C-7, C-8 and C-9. Therefore, the planar structure of 1 was elucidated as shown in Figure 2, and it was designated as mangromicin C. Compound 1 was structurally different from all other mangromicin analogs, which have a tetrahydropyran ring instead of a tetrahydrofuran ring.

Figure 2
figure 2

1H-1H COSY (bold) and selected HMBC (arrow) correlations of mangromicin C (1).

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The relative configuration of 1 was estimated by the 1H-1H coupling constant analysis, differential NOE and ROESY experiments compared with mangromicin A.1 The ROESY correlations, the same as mangromicin A, were observed between H-4/H-5, H-4/H-6a, H-5/H-6a, H3-8-Me/H-10, H3-12-Me/H2-13, H2-13/H2-15, H2-13/H3-14-Me and H3-14-Me/H2-16 (Supplementary Figure S7). In addition, the relative configuration of the tetrahydropyran ring was determined by small coupling constant between H-11 and H-10 or H-12 (Table 1), and ROESY correlations between H-11/H3-12-Me and H2-13/H3-12-Me. Moreover, ROESY correlations of H-10/H3-8-Me suggested that H3-8-Me is α-oriented. The coupling constants were observed between H-4 and H-5 (0.4 Hz) and H-5 and H-6b (9.2 Hz). The relative configuration of 1 was proposed as shown in Figure 1.

Compound 1 had more potent scavenging activity against 1,1-diphenyl-2-picrylhydrazyl free radicals (IC50=3.8 μM) than α-tocopherol (IC50=11.4 μM). In addition, 1 had scavenging activity against nitric oxide generated by lipopolysaccharide-stimulated RAW264.7 cells, a murine-macrophage cell line, at final concentration of 100 μM, without cytotoxic activity. Compared to the eight other analogs of mangromicin found, mangromicin C showed moderate anti-oxidant activity against ROS.2 ROS are known to be potentially injurious to living organisms because, in excess, they cause oxidative stress and can damage lipids, proteins and nucleic acids.3, 4, 5 Our results indicate that mangromicin analogs offer potential as ROS scavengers.