Note pubs.acs.org/jnp
Nabscessins A and B, Aminocyclitol Derivatives from Nocardia abscessus IFM 10029T Shoko Hara,†,§ Naoki Ishikawa,†,§ Yasumasa Hara,† Tatsuo Nehira,‡ Kanae Sakai,⊥ Tohru Gonoi,⊥ and Masami Ishibashi*,† †
Graduate School of Pharmaceutical Sciences, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8675, Japan Graduate School of Integrated Arts and Sciences, Hiroshima University, 1-7-1 Kagamiyama, Higashi-hiroshima, 739-8521, Japan ⊥ Medical Mycology Research Center, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8673, Japan ‡
S Supporting Information *
ABSTRACT: Two new aminocyclitol amide derivatives, nabscessins A (1) and B (2), were isolated from the culture broth of a pathogenic actinomycete species, Nocardia abscessus IFM 10029T. The structures of nabscessins A and B were elucidated by spectral studies, and the compounds showed antifungal activity against Cryptococcus neoformans, with IC50 values of 32 and 16 μg/mL, respectively.
ctinomycetes bacteria of the genus Nocardia are mostly opportunistic pathogens, and there has been an increasing number of studies on metabolites produced by members of this genus.1,2 Nocardia belongs to the same actinomycetes family as the genus Streptomyces; however, although the genus Streptomyces is known as a rich source of bioactive secondary metabolites, there have been far fewer studies on the bioactive secondary metabolites of Nocardia. A recent genome-based analysis showed that the number of secondary metabolite gene clusters in Nocardia is comparable to that in Streptomyces, suggesting that Nocaridia is also likely a valuable source of bioactive secondary metabolites.2 In our continuing search for bioactive compounds from Actinomycetes,3,4 we recently initiated a program investigating Nocardia species as unique microbial sources of natural products. Here we describe the isolation and structure elucidation of two new aminocyclitol amide derivatives, nabscessins A (1) and B (2), from the culture broth of a pathogenic actinomycete species, Nocardia abscessus IFM 10029T, which was isolated from an intraarticular abscess of the knee of a human patient.
A
(NB),6 Waksman,7 and yeast-malt-glucose (YMG) media8] on a small scale, and each broth analyzed by LCMS. Unique peaks as determined by the photodiode array data were observed from the mCD culture broth. A large-scale culture of N. abscessus IFM 10029T in mCD medium was then carried out. The supernatant and the methanol extract of the mycelia were combined, and the combined materials were partitioned between ethyl acetate and water. The ethyl acetate-soluble fraction was then subjected to fractionation using silica gel column chromatography, followed by reversed-phase HPLC separation to give two new compounds (1 and 2), named nabscessins A and B, respectively. Nabscessin A (1) was shown to have the molecular formula C22H24N2O9 by HRESIMS. The 1H NMR data of 1 in acetoned6 showed seven aromatic hydrogens (δH 7.41−6.75), five sp3 methines (δH 5.31−3.74), one methylene (δH 2.44−1.75), one methyl (δH 2.54), and two broad signals [δH 7.67 (1H) and 5.87 (2H)]. The 13C NMR spectrum of 1 exhibited 22 peaks (Table 1), which were assignable to seven aromatic methines, eight non-hydrogen-bearing carbons, five sp3 methines, one methylene, and one methyl, based on analysis of the HMQC data. Cross-peaks observed in the 1H−1H COSY data of 1 suggested the presence of three partial structures: A, B, and C (Figure 1). The COSY data indicated that partial structure C constructed a cyclohexane ring and the presence of an amide proton (δH 7.67, 1H, d, J = 8.3 Hz) at the C-1 position. The 1H NMR chemical shifts of H-1−H-5 suggested that C-2−C-5 were consecutive sp3 oxymethine carbons (Figure 1), and all substituents in partial structure C were elucidated to have
Nocardia abscessus IFM 10029T strain was grown in four liquid media [modified Czapek-Dox (mCD),5 nutrient broth
Received: October 12, 2016 Published: January 23, 2017
© 2017 American Chemical Society and American Society of Pharmacognosy
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DOI: 10.1021/acs.jnatprod.6b00935 J. Nat. Prod. 2017, 80, 565−568
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Table 1. 1H and 13C NMR Spectral Data of 1 and 2 in Acetone-d6 1 position 1 2 3 4 5 6 1-NH 1′ 2′ 3′ 4′ 5′ 6′ 7′ 1″ 2″ 3″ 4″ 5″ 6″ 7″ 8″ 1‴ 1‴-NH2
δC (ppm) 50.4 75.8 74.8 77.6 70.3 34.1
137.4 115.3 158.3 118.9 130.1 119.0 167.7 115.0 162.2 115.8 134.4 123.4 141.8 171.0 23.4 156.7
δH (ppm)/J (Hz) 4.14 3.74 3.80 5.31 5.04 1.75 2.44 7.67
m t (9.6) t (9.6) t (9.6) ddd (12.0, 9.6, 4.7) q (12.0) dt (12.0, 4.7) d (8.3)
2 δC (ppm)
HMBC
47.9 78.3 74.5 75.9 72.7 34.0
1, 3 3, 5, 7″ 1‴ 1, 4, 5 1, 5 7′
7.41 dd (2.4, 1.4)
3′, 4′, 7′
6.96 ddd (8.0, 2.4, 1.0) 7.24 t (8.0) 7.35 ddd (8.0, 1.4, 1.0)
2′, 6′ 1′, 3′ 2′, 4′, 7′
6.77 d (7.8) 7.29 t (7.8) 6.75 d (7.8)
2″, 5″ 2″, 6″ 3″, 8″
2.54 s
1″, 5″, 6″
137.2 115.0 158.2 118.9 130.1 118.8 166.9 115.2 161.7 115.7 134.3 123.2 141.7 171.3 23.1 157.3
5.87 br
δH (ppm)/J (Hz) 4.55 5.33 3.83 3.58 4.75 1.88 2.33 7.62
HMBC
m t (9.6) t (9.6) t (9.6) ddd (12.4, 9.6, 4.6) q (12.4) dt, (12.4, 4.6) d (8.9)
2 1, 2, 3, 4, 1, 1, 1,
3, 7″ 4 5 1‴ 2, 4, 5 2, 4, 5 7′
7.23 m
1′, 3′, 6′, 7′
6.91 ddd (6.9, 2.8, 0.9) 7.19 m 7.18 m
6′ 3′ 1′, 4′, 7′
6.70 d (8.2) 7.22 m 6.68 d (8.2)
2″, 5″ 2″, 6″ 4″, 6″, 8″
2.45 s
1″, 5″, 6″
5.89 br
equatorial orientations based on the 1H−1H coupling constants between the ring hydrogens (H-1−H-5): J1,2 = J2,3 = J3,4 = J4,5 = 9.6 Hz (all axial−axial relationships). Partial structure A was consistent with 3-hydroxybenzoic acid (3-HBA) based on the 1 H−1H COSY and HMBC correlations (Figures 1 and 2). H-2′ and H-6′ in partial structure A and the amide proton (1-NH, δH 7.67) in partial structure C showed long-range correlations to a carbonyl carbon (C-7′, δC 167.7), suggesting that partial structure A was connected at the C-1 position through an amide bond. Partial structure B was consistent with 6methylsalicylic acid (6-MSA), given the HMBC correlations (Figure 2) and based on comparison of the 13C NMR chemical shifts in the literature [6-MSA part of ampelomin G:9 δC 117.1, 160.9, 115.6, 133.9, 123.5, 141.4, 171.7, and 22.7, corresponding to C-1″−C-8″ of 1 (Table 1), respectively]. The HMBC correlation observed from H-4 (δH 5.31) in partial structure C to the C-7″ carbonyl (δC 171.0) in the 6-MSA moiety revealed the location of the 6-MSA ester group to be on C-4. The broad 1 H NMR signal [δH 5.87 (2H), 1‴-NH2] and the 13C NMR signal at δC 156.7 (C-1‴) were suggestive of the presence of a carbamate group (−OCONH2), and this was supported by consideration of the molecular formula of 1. The HMBC correlation observed from H-5 (δH 5.04) to the carbamate carbonyl carbon (δC 156.7, C-1‴) suggested that the carbamate (OCONH2) group was attached on C-5. The remaining two oxymethines (C-2 and C-3) were deduced to each bear a hydroxy group, consistent with the 1H NMR chemical shifts of H-2 and H-3 (δH 3.74 and 3.80, respectively). From these results, the structure of nabscessin A was concluded to be 1 and to have an aminocyclitol nucleus with amide, ester, and carbamate substituents. Nabscessin B (2) had the same molecular formula as 1 (C22H24N2O9), as indicated by the HRESIMS data. Analysis of
the 1D and 2D NMR data (Table 1) revealed that 2 comprised the same partial structures A−C, including the configurations of the substituents in C, but the locations of the substituents of the cyclohexane ring were different from that of 1. The 1H−1H COSY cross-peak between H-1 (δH 4.55) and 1-NH (δH 7.62), along with the HMBC correlations for 1-NH (δH 7.62)/C-7′ (δC 166.9) and 1-NH (δH 7.62)/C-1 (δC 47.9), indicated that the 3-HBA amide group was attached on C-1, whereas the HMBC correlation from H-5 (δH 4.75) to the carbamate carbonyl carbon (δC 157.3, C-1‴) implied the presence of the carbamate (OCONH2) on C-5. The 1H−1H COSY correlations revealed that the 1H NMR chemical shifts of H-2, H-3, and H-4 were δH 5.33, 3.83, and 3.58, respectively. These assignments suggested the location of hydroxy groups on C-3 and C-4 and attachment of the 6-MSA ester group on C-2, consistent with the HMBC correlation from H-2 (δH 5.33) to the C-7″ carbonyl carbon (δC 171.3) of 6-MSA. Thus, the structure of nabscessin B was revealed to be 2. The absolute configuration of 1 and 2 remained undefined. Nabscessins A (1) and B (2) contain an aminocyclitol nucleus, 2-deoxy-scyllo-inosamine,10,11 and related aminocyclitols are key aglycons of clinically important aminoglycoside antibiotics.12 The antimicrobial activities of nabscessins A (1) and B (2) were examined and revealed that 1 and 2 showed antifungal activity against Cryptococcus neoformans, with IC50 values of 32 and 16 μg/mL, respectively.
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EXPERIMENTAL SECTION
General Experimental Procedures. NMR spectra were recorded on JEOL ECZ600 and ECA600 NMR spectrometers with a deuterated solvent whose chemical shifts were used as an internal standard. HRESIMS were obtained on a JEOL JMS-T100LP mass spectrometer. Column chromatography was performed using silica gel 60N (Fuji 566
DOI: 10.1021/acs.jnatprod.6b00935 J. Nat. Prod. 2017, 80, 565−568
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Center. The strain was reidentified as Nocardia abscessus by sequencing 16S rRNA gene. Small-Scale Culture. The strain was cultivated in 5 mL of BHI+ liquid medium consisting of Bacto brain heart infusion (3.7 g/100 mL), glucose (1 g/100 mL), and glycerol (1 mL/100 mL) in a 10 mL Erlenmeyer flask for 5 days at 28 °C with shaking (160 rpm). A portion (500 μL) of this culture was inoculated into each of four liquid media (25 mL) in 50 mL Erlenmeyer flasks for 7 days at 28 °C with shaking (160 rpm). The four culture media were (1) modified CzapekDox medium consisting of sucrose (3 g/100 mL), NaNO3 (0.3 g/100 mL), KH2PO4 (0.1 g/100 mL), KCl (0.05 g/100 mL), MgSO4·7H2O (0.05 g/100 mL), and FeSO4·7H2O (0.001 g/100 mL); (2) nutrient broth medium consisting of Difco nutrient broth (1.6 g/100 mL); (3) Waksman medium consisting of glucose (2.0 g/100 mL), meat extract (0.5 g/100 mL), peptone (0.5 g/100 mL), dried yeast (0.3 g/100 mL), NaCl (0.5 g/100 mL), and CaCO3 (0.3 g/100 mL); and (4) yeastmalt-glucose medium consisting of yeast extract (0.4 g/100 mL), malt extract (1.0 g/100 mL), and glucose (0.4 g/100 mL). The pH of all media was adjusted to 7.3 with NaOH. The cells from each of the four cultures were extracted with methanol and subjected to LCMS analysis [Shimadzu LCMS-2020; column: COSMOSIL 5C18-AR-II (⦶ 2.0 × 150 mm) (Nacalai Tesque Co. Ltd., Kyoto, Japan); solvent: 10−100% MeOH in 0.1% HCOOH, 0−30 min, linear gradient, and 100% MeOH isocratic 30−60 min; flow rate: 0.2 mL/min; UV detection: photodiode array (190−600 nm); MS detection: ESI (positive and negative) (m/z 100−2000)]. Fermentation. The strain N. abscessus IFM 10029T was cultured for 2 days at 30 °C and 1 day at 37 °C on BHI+ agar medium. To prepare the seed culture, pieces (1 cm × 1 cm) of the well-grown agar culture were inoculated into a 200 mL Erlenmeyer flask containing 100 mL of BHI+ liquid medium together with 16 glass beads for 5 days at 28 °C with shaking (160 rpm); then 15 mL of this culture was added to each of five Erlenmeyer flasks (1 L) containing 500 mL of mCD medium, which were incubated for 7 days at 28 °C with shaking (160 rpm). The culture broth (2.5 L) was centrifuged at 3000 rpm for 20 min to give the supernatant and mycelial cake; the mycelial cake was extracted with MeOH (900 mL × 3). The MeOH extract was combined with the supernatant obtained above, and the combined materials were partitioned with ethyl acetate (2.5 L × 3) and water. A part of the ethyl acetate-soluble fraction (104 mg) was subjected to silica gel 60N column chromatography (⦶ 2.5 × 30 cm), eluted with 0−100% MeOH in CHCl3, to afford fractions 1A to 1L. Fraction 1I (16.7 mg), eluted with 20% MeOH in CHCl3, was further separated by silica gel 60N column chromatography (⦶ 1.5 × 10 cm) by eluting with 0−100% MeOH in CHCl3 to afford a fraction (7.4 mg) that eluted in 10−20% MeOH in CHCl3. This fraction was purified by reversed-phase HPLC [COSMOSIL 5C18-AR-II (⦶ 10.0 × 250 mm); eluant: 40% MeOH in H2O; flow rate: 5 mL/min; UV detection: photodiode array (190−400 nm)] to give nabscessins A (1, 2.7 mg, tR 5.8 min) and B (2, 0.8 mg, tR 10.4 min). Fraction 1J (15.8 mg), eluted with 50−100% MeOH in CHCl3 from the first silica gel column (vide supra), was also separated by silica gel 60N column chromatography (⦶ 1.5 × 10 cm) by elution with 0−100% MeOH in CHCl3 to give nabscessin B (2, 5.8 mg) in the fraction eluted with 14% MeOH in CHCl3. Nabscessin A (1): colorless solid; [α]21D −10.8 (c 1.0, MeOH); UV (MeOH) λmax (log ε) 241 (4.2) and 292 (3.8) nm; IR νmax (ATR) 3347, 1715, 1584, and 1058 cm−1; CD (MeOH) λnm (Δε) 207 (+0.8), 219 (−2.7), 227 (−1.9), 240 (−3.6), 280 (−1.1), and 314 (−0.4); 1H and 13C NMR data in Table 1; HRESIMS m/z 483.1415 [M + Na]+ (calcd for C22H24N2O9Na, 483.1380). Nabscessin B (2): colorless solid; [α]21D +15.7 (c 1.0, MeOH); UV (MeOH) λmax (log ε) 242 (4.1) and 292 (3.7) nm; IR νmax (ATR) 3402, 1712, and 1335 cm−1; CD (MeOH) λnm (Δε) 205 (−14.7), 219 (+10.6), 234 (−0.2), 248 (+6.9), and 318 (−1.1); 1H and 13C NMR data in Table 1; HRESIMS m/z 483.1404 [M + Na]+ (calcd for C22H24N2O9Na, 483.1380). Antimicrobial Testing. Antimicrobial assays were carried out as described previously,13 and MIC and IC50 values of compounds 1 and 2 were as follows: Escherichia coli (MIC >32 and >32 μg/mL,
Figure 1. Partial structures A−C and 1H−1H COSY correlations for 1.
Figure 2. HMBC correlations for 1. Silysia Chemical Co., Kasugai, Japan) and Chromatorex ODS (Fuji Silysia Chemical Co., Kasugai, Japan). Preparative HPLC was performed using COSMOSIL 5C18-AR-II (⦶ 10.0 × 250 mm) (Nacalai Tesque Co. Ltd., Kyoto, Japan). Optical rotation was determined with a JASCO P-2200 polarimeter. Circular dichroism was measured with a JASCO J-1100 CD spectrometer. Infrared spectroscopy was conducted using a JASCO FT/IR-4700 spectrophotometer. UV−vis spectra were measured with a Shimadzu UVmini 1240 UV− vis spectrophotometer. Microbial Strain. Nocardia abscessus IFM 10029T strain was originally isolated from an intra-articular abscess of the knee after total joint replacement surgery in a 56-year-old male patient and was stored in a freeze-dried state at Chiba University Medical Mycology Research 567
DOI: 10.1021/acs.jnatprod.6b00935 J. Nat. Prod. 2017, 80, 565−568
Journal of Natural Products
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respectively), Staphylococcus aureus (MIC >32 and >32 μg/mL, respectively), Bacillus subtilis (MIC >32 and >32 μg/mL, respectively), Micrococcus luteus (MIC >32 and >32 μg/mL, respectively), Aspergillus niger (IC50 >32 and >32 μg/mL, respectively), Candida albicans (IC50 >32 and >32 μg/mL, respectively), Trichophyton mentagrophytes (IC50 >32 and >32 μg/mL, respectively), and Cryptococcus neoformans (IC50 32 and 16 μg/mL, respectively). Amphotericin B, micafungin, hygromycin B, and kanamycin were used as positive controls for antimicrobial activities against E. coli (MIC >31.2, >62.5, 2, and 31.2, 0.5, 0.5, and 31.2, 0.5, 2, and 31.2, 7.8, 1, 1 μg/mL, respectively), A. niger (IC50 1.8,
