During our studies on the search for bioactive natural products from various sources,1 we have investigated bioactive metabolites of actinomycete strains and isolated a new tyramine derivative2 or teleocidin derivative3 in our screening studies targeting tumor necrosis-factor-related apoptosis-inducing ligand signaling.4 Conversely, we recently investigated the chemical constituents of Kitasatospora sp. IFM10917, based on TLC examination using anisaldehyde as a spray reagent, and isolated new carbamate- and pyridine-containing compounds, named fuzanins A–D.5 We further investigated the culture of Kitasatospora sp. IFM10917 and performed chemical studies on its extract, resulting in the isolation of further carbamate-containing metabolites, named fuzanins E–I (1–5) (Figure 1). Herein we describe the isolation and structural elucidation of these new compounds.

Figure 1
figure 1

Structures of 1–6.

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The cultured supernatant of Kitasatospora sp. IFM10917 was partitioned between ethyl acetate (EtOAc) and water. The EtOAc-soluble fraction was subjected to silica gel column chromatography, followed by purification with Sephadex LH-20 column (GE Healthcare Bio-Sciences, Uppsala, Sweden) and reversed-phase HPLC on ODS to give five new compounds, fuzanins E–I (1–5).

Fuzanin E (1) was revealed to have the molecular formula of C14H21NO4 on the basis of high-resolution fast atom bombardment mass spectroscopy (HRFABMS) at m/z 268.1560 ((M+H)+, Δ +1.1 mmu). Absorption at 3360 and 1740 cm−1 in the IR spectrum suggested the presence of hydroxyl and carbonyl groups, respectively. The 13C NMR spectrum (Table 1) showed 14 signals, which were assigned to two methyls (δC 23.7 and 18.0), three aliphatic methylenes (δC 28.2, 37.9 and 44.6), four heteroatom-bearing methines (δC 60.5, 62.9, 67.4 and 80.6), three sp2 methines (δC 127.7, 126.8 and 137.8), one sp2 quaternary carbon (δC 133.2) and one carbonyl carbon (δC 156.4); the 13C chemical shift of this carbonyl group indicated that it was located between oxygen and nitrogen atoms, suggesting that 1 had a carbamate moiety. The 1H NMR spectrum of 1 (Table 1) revealed signals due to two methyls (δH 1.21 (3H, d, J=6.4 Hz) and 1.69 (3H, s)), one heteroatom-bearing methylene (δH 2.69 and 4.20), four heteroatom-bearing methines (δH 3.82, 3.88, 4.35 and 4.48) and three olefinic protons (δH 5.58, 5.64 and 5.94). These 1H and 13C NMR spectral data were similar to those of fuzanin B (6) (Figure 1), except for the number of double bonds. Fuzanin B (6) possessed a two disubstituted diene, whereas fuzanin E (1) had one disubstituted olefin and two more sp3 methylene groups. The 1H-1H COSY spectrum of 1 revealed 1H-connectivities from H2-2 to H-3 and from H-6 to H-13, and the disubstituted olefin was shown to be at C-8 and C-9 and a secondary hydroxyl group at C-12 position from the COSY correlation data. The large coupling constants (J8,9=14.9 Hz) suggested the E-configurations of Δ8,9-double bonds. The HMBC spectrum of 1 also suggested that fuzanin E (1) had the same bicyclic ring system as fuzanin B (6) containing a carbamate group (H2-2/C-3, H2-2/C-4, H2-2/C-6, H2-2/C-15, H-4/C-6, H-7/C-5, H-7/C-8, H2-10/C-8, H2-10/C-9, H2-10/C-11, H2-10/C-12, H3-13/C-11, H3-13/C-12, H3-14/C-4, H3-14/C-5 and H3-14/C-6). The relative configuration of 1 was suggested by 1H-1H coupling constants and NOE experiments. A large vicinal coupling constant (J=9.4 Hz) was observed between H-2β and H-3, indicating that H-2β and H-3 were trans-diaxial. NOE correlations were observed for H-2α/H-3, H-2β/H-6 and H-6/H-8 revealed that H-2β, H-6 and H-8 were on the same side of the plane, whereas H-2α and H-3 were on the other side. The absolute configurations at C-3 and C-12 of 1 were elucidated by applying the modified Mosher’s method.6 Differences in the chemical shifts (Δδ values: δS–δR) of the 1H NMR spectra of (S)- and (R)-MTPA (α-methoxy-α-(trifluoromethyl)phenylacetic acid) diesters are shown in Table 2. Diagnostic positive Δδ values were observed for H-4, H3-13 and H3-14, and negative Δδ values for H-2α, H-2β and H2-11. These findings led to assignment of the 3R,12R-configuration. On the basis of these results, the structure of fuzanin E was concluded as 1, including the absolute configurations assigned as 3R, 6S, 7S, 12R.

Table 1 1H and 13C NMR spectral data for fuzanins E (1), F (2), G (3), H (4) and I (5) in CDCl3
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Table 2 Comparison of the 1H NMR chemical shifts of H-6, H-7 and H-8, and assignment of relative configurations between H-6 and H-7
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The high-resolution electron spray ionization mass spectra (HRESIMS) of fuzanin F (2) exhibited a quasi-molecular ion peak at m/z 290.1358 ((M+Na)+, Δ –0.5 mmu), indicating the molecular formula C14H21NO4. The 1H and 13C NMR spectra of 2 (Table 1) were almost identical to those of 1, except for the signals of positions C-6, C-7 and C-8. The 1H NMR chemical shifts due to H-6 (δH 4.31), H-7 (δH 4.98) and H-8 (δH 5.33) for 2 were significantly different from those for 1 (δH 3.88 (H-6), δH 4.48 (H-7) and δH 5.64 (H-8)) (Table 2), whereas the three carbon signals at δC 58.0 (C-6), δC 78.6 (C-7) and δC 122.9 (C-8) in 2 resonated in higher fields than those of 1 (δC 60.5 (C-6), δC 80.6 (C-7) and δC 126.8 (C-8)). From these findings, 2 was suggested as a stereoisomer of 1 at position C-7, which was also supported by the significant NOE correlations between H-6 and H-7 for 2, instead of the correlation between H-6 and H-8 observed for 1. The absolute configuration of 2 was determined as 3R,12R configurations using the modified Mosher’s method (Table 3). From these results, the structure of fuzanin F was revealed as 2 with 3R,6S,7S,12R configurations.

Table 3 Δδ values (δS−δR) of (S)- and (R)-MTPA esters of 1–4
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Fuzanin G (3) was shown to have a molecular formula of C14H19NO4 from HRFABMS data at m/z 288.1199 ((M+Na)+, Δ −2.2 mmu). The 1H and 13C NMR spectra of 3 (Table 1) were similar to those of 1 except for the presence of a ketone (δC 207.3) for 3 in place of an oxymethine at C-12 for 1. The C-13 methyl signal of 3 was observed as a singlet (δH 2.15, (3H, s)). Thus, the structure of fuzanin G (3) was inferred as a 12-dehydro derivative of fuzanin E (1), which was further supported by the 1H-1H COSY and HMBC spectra (data not shown). The relative configuration between H-6 and H-7 of 3 was deduced as anti from the comparison of the 1H chemical shifts of H-6 (δH 3.87), H-7 (δH 4.46) and H-8 (δH 5.63) as discussed above in the case of fuzanin E (1) and F (2), as shown in Table 2. This configuration was also consistent with the observation of significant NOE from H-6 to H-8 for 3. The absolute configuration was determined as 3R based on the modified Mosher’s method (Table 3). Thus, the structure 3 was assigned to fuzanin G.

The molecular formula of fuzanin H (4), C14H19NO4, determined by the HRESIMS data at m/z 288.1201 ((M+Na)+, Δ –0.5 mmu) was the same as that of fuzanin G (3). The 1H and 13C NMR spectral data of 4 (Table 1) were almost identical to those of 3, except for the chemical shifts of H-6, H-7 and H-8 positions, which were diagnostic for the configuration for H-6/H-7 (vide supra). On the basis of the comparison data in Table 2, the relative configuration of H-6/H-7 was suggested as syn. Thus, fuzanin H (4) was deduced as a C-7 epimer of fuzanin G (3), and the absolute configuration was determined as 3R from the modified Mosher’s method (Table 3).

Fuzanin I (5) had a molecular formula of C14H23NO4, revealed from the HRESIMS data at m/z 292.1513 ((M+Na)+, Δ −1.2 mmu), having two more hydrogen atoms than fuzanin E (1) or F (2). The 1H and 13C NMR data of fuzanin I (5) (Table 1) showed no signals due to an olefinic methyl group corresponding to C-14 of 1–4, whereas two doublet methyl signals were observed and were assigned to C-13 and C-14 positions. The 1H-1H COSY spectrum showed that the methylene protons on C-2 (H2-2, δH 3.71 and 3.22) were connected to another methylene group (H2-3, δH 1.81 and 1.72), which in turn was coupled to an sp3 oxymethine proton at C-4 (H-4, δH 3.97). The HMBC spectrum revealed correlations from the methyl protons on C-14 (H3-14, δH 0.98) to the oxymethine carbon (C-4, δC 68.2) and two other sp3 methines (C-5, δC 42.1; C-6, δC 60.4). From these observations, it was suggested that the C-4/C-5 position of 5 was saturated and a secondary hydroxyl group was located at the C-4 position instead of C-3 position. The 1H-1H COSY and HMBC spectra indicated that the structure of the rest of the molecule of 5 (C-6 to C-13 part) was identical to that of fuzanin E (1). The coupling constant between H-5 and H-6 was large (J=10.5 Hz), suggesting that these two hydrogens were both axial. The oxymethine proton (H-4) was observed as a broad singlet, thus implying that this hydrogen had small coupling constants with neighboring hydrogens and was therefore equatorial. These observations revealed 4β-OH, 5β-CH3 and 6β-H configurations for 5. The H-6/H-7 configuration was suggested as anti from the diagnostic 1H NMR chemical shifts of H-7 and H-8, as shown in Table 2, although the chemical shift of H-6 of 5 was slightly different from those of 1 or 3 due to the C-5 position being sp3 or sp2. This configuration was also supported by the observation of significant NOE from H-6 to H-8 for 5. From these results, the structure 5 was assigned to fuzanin I. The absolute configuration of 5 remained undefined because the modified Mosher’s method could not be applied on 5 due to small quantity.

We examined the bioactivity of fuzanins E–H (1–4) using cell-based assay systems constructed in our laboratories targeting Wnt signaling pathways, but these compounds did not exhibit inhibition of Wnt signal transcription activity even at 100 μM using a luciferase reporter gene assay in SuperTOP-Flash transfected cells.7 Fuzanins E–I (1–5) also did not show antimicrobial activity at 100 μg per disc against Bacillus subtilis.

Materials and methods

General

Optical rotations were measured with a JASCOP-1020 polarimeter (JASCO, Tokyo, Japan). IR spectra were measured on ATR (attenuated total reflection) on a JASCO FT-IR 230 spectrophotometer. UV spectra were measured on a Shimadzu UV mini-1240 spectrometer (Shimadzu, Kyoto, Japan). NMR spectra were recorded on JEOL JNM-A400 and JEOL JNM-ECP600 spectrometers (JEOL, Tokyo, Japan) with a deuterated solvent, the chemical shift of which was used as an internal standard. FABMS were obtained on a JEOL AX-500 spectrophotometer, and HRFABMS were obtained on a JEOL HX-110 mass spectrometer. HRESIMS were obtained on an Exactive (Thermo Scientific, Kanagawa, Japan).

Microbial strain

Kitasatospora sp. IFM 10917 was separated from a soil sample collected in Toyama City, Japan in March 2007, as described previously.5 The identification was carried out by Professor Yuzuru Mikami of the Medical Mycology Research Center, Chiba University, where a voucher specimen is deposited with code IFM 10917.

Extraction and isolation

The culture broth (6 l total), obtained as described previously,5 was harvested and centrifuged to separate the mycelia and supernatant. The supernatant was concentrated under reduced pressure to 500 ml and partitioned between EtOAc (500 ml × 3) to give the EtOAc-soluble fraction (937 mg). This material was subjected to silica gel column chromatography (25 × 450 mm) eluted with a gradient of mixtures (hexane/EtOAc=1:1, 4:6, 3:7, 1:4, and MeOH) to give 14 fractions 1A to 1N, which were subjected to TLC examination, and three fractions (1I, 1J and 1K) were selected as anisaldehyde-positive fractions, which were suggested to contain previously isolated fuzanins A–D5 and other related compounds. Fraction 1I (26.8 mg) eluted with hexane/EtOAc (2:8) was purified by Sephadex LH-20 column chromatography (10 × 450 mm) eluted with MeOH, followed by preparative HPLC (YMC-Pack ODS-AM, 10 × 250 mm; eluent, 50% MeOH; flow rate, 1.0 ml min−1; UV detection at 254 nm; YMC Co Ltd, Kyoto, Japan) to afford fuzanin G (3, 2.2 mg, Rt (retention time) 24.0 min) and fuzanin H (4, 2.4 mg, Rt 20.5 min). Fraction 1J (27.1 mg) eluted with hexane/EtOAc (2:8) was fractionated by Sephadex LH-20 column chromatography (10 × 450 mm) eluted with MeOH, followed by preparative HPLC (YMC-Pack ODS-AM, 10 × 250 mm; eluent, 50% MeOH; flow rate, 1.0 ml min−1; UV detection at 254 nm) to give fuzanin E (1, 3.5 mg, Rt 27.0 min), fuzanin F (2, 2.2 mg, Rt 22.0 min) and fuzanin H (4, 0.9 mg, Rt 20.0 min). Fraction 1K (25.6 mg) eluted with hexane/EtOAc (0:1) was separated by Sephadex LH-20 column chromatography (13 × 400 mm) eluted with MeOH, followed by preparative HPLC (Develosil ODS UG-5, 10 × 250 mm; eluent, 50% MeOH; flow rate, 1.0 ml min−1; UV detection at 254 nm) to afford fuzanin F (2, 2.4 mg, Rt 23.0 min) and fuzanin I (5, 1.2 mg, Rt 31.5 min).

Fuzanin E (1)

Colorless oil, [α]D24 –2.9 (c 1.0, MeOH); IR νmax (ATR) 3360 and 1736 cm−1; 1H and 13C NMR data in Table 1; FABMS m/z 268 (M+H)+ and 306 (M+K)+; HRFABMS m/z 268.1560 (M+H)+, calcd for C14H22NO4, 268.1549.

Fuzanin F (2)

Colorless oil, [α]D16 +10.6 (c 1.0, MeOH); IR νmax (ATR) 3368 and 1734 cm−1; 1H and 13C NMR data in Table 1; HRESIMS m/z 290.1358 (M+Na)+, calcd for C14H21NO4Na, 290.1363.

Fuzanin G (3)

Colorless oil, [α]D24 –2.5 (c 1.0, MeOH); IR νmax (ATR) ca. 3300, 1739 and 1713 cm−1; 1H and 13C NMR data in Table 1; FABMS m/z 265 [M+] and 304 (M+K)+; HRFABMS m/z 288.1199 (M+Na)+, calcd for C14H19NO4Na, 288.1212.

Fuzanin H (4)

Colorless oil, [α]D16 +20.2 (c 1.0, MeOH); IR νmax (ATR) ca. 3300, 1741 and 1715 cm−1; 1H and 13C NMR data in Table 1; HRESIMS m/z 288.1201 (M+Na)+, calcd for C14H19NO4Na, 288.1212.

Fuzanin I (5)

Colorless oil, [α]D24 –4.8 (c 1.0, MeOH); IR νmax (ATR) 3383 and 1732 cm−1; 1H and 13C NMR data in Table 1; HRESIMS m/z 292.1513 (M+Na)+, calcd for C14H23NO4Na, 292.1525.

Preparation of Mosher’s esters derivatives

Fuzanin E (1) (1.1 mg) was treated with (R)-MTPA-Cl (8 μl, 10.4 mg) in pyridine (8 μl) at room temperature for 24 h, and the mixture was evaporated under reduced pressure. The residue was purified by silica gel chromatography (7 × 65 mm, hexane/EtOAc=1:1) to give the (S)-MTPA diester of 1 (2.8 mg): ESIMS, m/z 722 (M+Na)+. Fuzanin E (1) was also treated with (S)-MTPA-Cl by the same procedures as above to afford (R)-MTPA diester of 1: ESIMS, m/z 722 (M+Na)+. Corresponding (S)- and (R)-MTPA esters of 2–5 were prepared by the same procedures as above. (S)-MTPA ester of 2: ESIMS, m/z 722 (M+H)+. (R)-MTPA ester of 2: ESIMS, m/z 722 (M+Na)+. (S)-MTPA ester of 3: ESIMS, m/z 504 (M+Na)+. (R)-MTPA ester of 3: ESIMS, m/z 504 (M+Na)+. (S)-MTPA ester of 4: ESIMS, m/z 504 (M+Na)+. (R)-MTPA ester of 4: ESIMS, m/z 504 (M+Na)+.