Bafilomycins and Odoriferous Sesquiterpenoids from Streptomyces

Article pubs.acs.org/jnp

Bafilomycins and Odoriferous Sesquiterpenoids from Streptomyces albolongus Isolated from Elephas maximus Feces Nan Ding,†,‡ Yi Jiang,§ Li Han,† Xiu Chen,† Jian Ma,† Xiaodan Qu,† Yu Mu,† Jiang Liu,† Liya Li,† Chenglin Jiang,§ and Xueshi Huang*,† †

Institute of Microbial Pharmaceuticals, College of Life and Health Sciences, Northeastern University, Shenyang 110819, People’s Republic of China ‡ Laboratory of Metabolic Disease Research and Drug Development, China Medical University, Shenyang 110001, People’s Republic of China § Yunnan Institute of Microbiology, Yunnan University, Kunming 650091, People’s Republic of China S Supporting Information *

ABSTRACT: From a fermentation broth of Streptomyces albolongus obtained from Elephas maximus feces, nine bafilomycins (1−9) and seven odoriferous sesquiterpenoids (10−16) were isolated. The structures of the new compounds, including three bafilomycins, 19-methoxybafilomycin C1 amide (1), 21-deoxybafilomycin A1 (2), and 21-deoxybafilomycin A2 (3), and two sesquiterpenoid degradation products, (1β,4β,4aβ,8aα)-4,8adimethyloctahydronaphthalene-1,4a(2H)-diol (10) and (1β,4β,4aβ,7α,8aα)-4,8a-dimethyloctahydronaphthalene-1,4a,7(2H)-triol (11), were elucidated by comprehensive spectroscopic data analysis. The cytotoxicity activity against four human cancer cell lines and antimicrobial activities against a panel of bacteria and fungi of all compounds isolated were evaluated. Compounds 1, 7, and 8 were cytotoxic, with IC50 values ranging from 0.54 to 5.02 μM. Compounds 2, 7, 8, and 10 showed strong antifungal activity against Candida parapsilosis, with MIC values of 3.13, 1.56, 1.56, and 3.13 μg/mL respectively.

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known compounds, bafilomycin A1 (4),11 bafilomycin A2 (5),12 bafilomycin G (6),13 bafilomycin C1 (7),14 bafilomycin C1 amide (8),14 bafilomycin D (9),15 (3β,4β,4aβ,8aα)-4,8adimethyloctahydronaphthalene-3,4a(2H)-diol (12),16 caryolan1-ol (13),17 caryolan-1,9β-diol (14),18 1(10)E,5E-germacradiene-3,11-diol (15),19 and epicubenol (16).20 The structures of these compounds were determined based on detailed spectroscopic analysis. The spectroscopic data of 12 are reported for the first time. The proposed biosynthetic pathway for odoriferous sesquiterpenoids 10−16 is discussed. Finally, the results of biological assays to evaluate the cytotoxic, antibacterial, and antifungal activities of these isolated compounds are presented.

ll animals have a large numbers of microbes present in their gastrointestinal tracts and feces. The intestinal microbial community, comprising 1013 to 1014 microorganisms, is an important resource for identifying new microbial species and new bioactive natural products.1−3 Over the past few years, we have studied the diversity and bioactivities of cultivable actinobacteria isolated from the fecal samples of almost 50 species of animals that were collected from Yunnan Wild Animal Park in China.4−7 Several new microbial species and new natural products have been isolated from animal feces.8−10 In an ongoing search for new bioactive metabolites from microbial sources associated with animal feces, the chemical constituents of Streptomyces albolongus (YIM 101047) were systematically investigated. S. albolongus was isolated from healthy adult Elephas maximus fecal samples. An extract from a 100 mL culture broth of S. albolongus showed promising cytotoxic and antimicrobial activities and diverse constituents, which were detected using HTLC and HPLC-MS. In the present study, we report the isolation, structural elucidation, and cytotoxic and antimicrobial activities of chemical constituents obtained from a fermentation broth of S. albolongus. We identified three new bafilomycins, 19methoxybafilomycin C1 amide (1), 21-deoxybafilomycin A1 (2), and 21-deoxybafilomycin A2 (3); two new sesquiterpenoid degradations, (1β,4β,4aβ,8aα)-4,8a-dimethyloctahydronaphthalene-1,4a(2H)-diol (10) and (1β,4β,4aβ,7α,8aα)-4,8adimethyloctahydronaphthalene-1,4a,7(2H)-triol (11); and 11 © XXXX American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION

The fermentation broth (70 L) of S. albolongus was centrifuged, and the culture supernatant was collected. The supernatant was treated with the polymeric resin Amberlite XAD-16, and bound compounds were eluted with an ethanol−water gradient. Compounds in the fraction were isolated by sequential chromatography over Sephadex LH-20, silica gel, and ODS to isolate pure bafilomycins 1−9 and odoriferous sesquiterpenoids 10−16. Received: September 14, 2015

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DOI: 10.1021/acs.jnatprod.5b00827 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

Bafilomycins are a family of compounds characterized by the presence of 16-membered macrolide rings that are produced by Streptomyces species. Bafilomycins possess a broad spectrum of biological activities, including antibacterial, antifungal, nematocidal, and cytotoxic activities, and can inhibit vacuolar H+ATPases.21 Known bafilomycins 4−9 were identified by comparing their spectroscopic data with values found in the literature. Compound 1 was obtained as a colorless solid and was assigned a molecular formula of C40H63NO11 based on HRESIMS and 13C NMR data. Its IR spectrum revealed the presence of hydroxy groups (3430 cm−1), carbonyl groups (1713 cm−1), and double bonds (1650, 1619 cm−1). The 1H NMR data revealed seven olefinic protons (δH 6.97, 6.55, 6.53, 6.50, 5.82, 5.72, 5.17), six oxygenated methine protons (δH 5.13, 4.89, 3.95, 3.40, 3.16, 3.13), two singlet methyls (δH 1.88, 1.75), and seven doublet methyls (δH 0.98, 0.94, 0.88, 0.87, 0.84, 0.83, 0.75). The 13C NMR spectrum of 1 showed all 40 signals indicated by the molecular formula, including two ester carbonyl carbons, one amide carbonyl carbon, 10 olefinic carbons, one ketal, six oxygenated methine carbons, three methoxyls, six methines, two methylenes, and nine methyls, as determined in an HSQC experiment. The 1H and 13C NMR data were almost identical to the known bafilomycin C1 amide (8),14 which was also isolated from the same strain. The major difference was the addition of a methoxy group (δC 46.3, δH 2.98) in 1. HMBC correlation between the methoxy group (δH 2.98) and C-19 (δC 102.8) revealed that the methoxy group was located at C-19. This result was also confirmed by the carbon chemical shifts of C-18 (ΔδC −4.4 ppm), C-19 (ΔδC 4.0 ppm), and C-20 (ΔδC −3.3 ppm) compared to the 13C NMR data of 8. Thus, the planar structure of 1 was elucidated as a 19methoxybafilomycin C1 amide. The absolute configuration of typical bafilomycin A1 (4) was determined using X-ray11 and total synthesis.22−24 The similar 13C NMR data between 1 and bafilomycin A1 indicated that 1 possessed the same relative configuration as bafilomycin A1. NOE correlations helped reconfirm the relative configuration of 1 (Figure 1). Compound 2 was isolated as a colorless solid, and its molecular formula was deduced as C35H58O8 based on HRESIMS and 13C NMR data. 1H and 13C NMR data indicated that 2 bore a close resemblance to the known compound bafilomycin A1 (4). The only difference was that one oxygenated methine was absent and one more methylene was present (δC 28.4, δH 1.46, m, 1.40, m) in 2. 1H−1H COSY correlations from H-21a (δH 1.46, m) to H-20 (δH 1.70, m, 1.23, m) and H-22 (δH 1.33, m) and from H-21b (δH 1.40, m)

Figure 1. Key NOE correlations of fragments A and B of 1.

to H-20 (δH 1.70, m, 1.23, m) revealed that 2 was 21deoxybafilomycin A1. 13C NMR data for 2 together with key NOE correlations revealed that 2 possessed the same relative configuration as bafilomycin A1. Compound 3 was isolated as a colorless solid, and its molecular formula was deduced as C36H60O8 by its HRESIMS at m/z 643.4179 [M + Na]+ and its 13C NMR spectrum. The 1 H and 13C NMR data of 3 were very similar to those of 2, except that an additional methoxy group (δC 46.1, δH 2.96) was found in 3. The different chemical shifts of C-18, C-19, and C20 suggested that a methoxyl was located at C-19, and this was confirmed by HMBC correlations between methoxy group protons (δH 2.96) and C-19 (δC 101.3). We speculate that 3 has the same relative configuration as 2 according to their closely similar 13C NMR data and their assumed same bafilomycin biogenetic pathway.25 Compared to the compounds 1 and 4−9, the absence of C21 hydroxy group in compounds 2 and 3 indicated that the KR and DH domains of the PKS module responsible for the biosynthesis of this extender unit were conditionally active in the organism.25 The 13C NMR spectrum of 10 exhibited 12 carbon signals, which were assigned to two methyl groups, six methylenes, two methines, one aliphatic quaternary carbon, and one oxygenated sp3 nonprotonated carbon (Table 2). The 1H and 13C NMR data of 10 suggested that it was very similar to a related degraded sesquiterpene, geosmin, which has a characteristic odor of moist soil, possesses a degraded sesquiterpene skeleton, and has been isolated from miscellaneous microorganisms.26 Compared to the NMR data from geosmin,27,28 one methylene at C-1 in geosmin was absent and an additional oxygenated methine signal (δC 76.0, δH 3.27) was present in 10, which indicated hydroxylation at C-1 of 10. HMBC correlations between H-1 (δH 3.27) and C-2 (δC 28.8), C-8a (δC 39.6), and C-9 (δC 20.3) and between H-9 (δH 0.89) and C-1 (δC 76.0), C-8 (δC 30.6), and C-8a (δC 39.6) reconfirmed that the hydroxy was located at C-1. COSY correlations further supported the result. The relative configuration of 10 was determined in a NOESY experiment. NOE interactions from B



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Table 1. 1H NMR (600 MHz) and 13C NMR (150 MHz) Data for Compounds 1−3 in DMSO-d6 1 position

δC, type

1 2 3 4 5 6 7 8 9

164.6, 141.3, 132.1, 130.9, 144.4, 37.6, 78.8, 39.7, 41.6,

C C CH C CH CH CH CH CH2

10 11 12 13 14 15 16 17 18 19 20

142.7, 124.4, 132.0, 126.0, 83.6, 76.1, 39.9, 69.3, 38.2, 102.8, 35.5,

C CH CH CH CH CH CH CH CH C CH2

21 22 23 24 25 26 27 28 29 30 31 32 33 2-OMe 14-OMe 19-OMe 1′ 2′ 3′ 4′ 7-OH 17-OH 19-OH 4′-NH2

74.2, CH 37.4, 76.5, 28.1, 20.8, 13.9, 18.4, 23.1, 19.4, 11.6, 7.7, 12.4, 14.4, 59.9, 55.6, 46.3, 165.1, 129.1, 138.4, 164.9,

CH CH CH CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 C CH CH C

2 δH (J in Hz)

6.53, s 5.82, 2.40, 3.16, 1.71, 2.07, 1.86,

d (8.7) m m m dd (14.1, 11.5) brd (14.1)

5.72, 6.50, 5.17, 3.95, 5.13, 1.77, 3.40, 1.98,

d (10.7) dd (15.0, 10.7) dd (15.0, 7.9) t (7.0) brd (6.0) m dd (9.4, 7.7) brq (7.0)

2.24, dd (13.0, 4.9) 1.44, brt (13.0) 4.89, m 1.53, 3.13, 1.91, 0.98, 1.88, 0.94, 0.88, 1.75, 0.83, 0.87, 0.75, 0.84, 3.56, 3.14, 2.98,

m m m d (6.8) s d (6.8) d (7.0) s d (7.0) d (7.0) d (6.6) d (7.0) s s s

δC, type 164.8, 141.3, 132.4, 131.0, 144.4, 37.5, 78.8, 39.5, 41.6,


143.0, 124.3, 132.1, 126.0, 83.4, 75.7, 38.8, 70.3, 42.8, 97.6, 33.7,


28.4, CH2 31.9, 77.8, 28.1, 21.0, 14.0, 18.3, 22.9, 19.6, 11.0, 7.2, 17.6, 14.9, 59.7, 55.5,

3 δH (J in Hz)

CH CH CH CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3

6.52, s 5.81, 2.40, 3.16, 1.71, 2.03, 1.88,

d (8.6) m m m dd (14.3, 11.3) m

5.70, 6.51, 5.12, 3.95, 5.11, 1.85, 4.00, 1.57,

d (11.1) dd (15.0, 11.1) dd (15.0, 8.5) dd (7.7, 6.7) m m dd (10.4, 6.4) brq (6.6)

1.70, 1.23, 1.46, 1.40, 1.33, 3.33, 1.79, 0.84, 1.88, 0.95, 0.87, 1.77, 0.75, 0.86, 0.74, 0.70, 3.53, 3.14,

m m m m m m m d (7.0) s d (7.0) d (7.0) s d (7.0) d (7.0) d (6.6) d (6.8) s s

δC, type 164.7, 141.3, 132.1, 130.9, 144.4, 37.6, 78.8, 39.5, 41.6,


142.7, 124.4, 132.1, 126.0, 83.7, 76.2, 39.9, 69.5, 38.3, 101.3, 29.4,


28.2, CH2 31.4, 78.9, 28.4, 20.7, 13.9, 18.4, 23.1, 19.3, 11.6, 7.9, 17.7, 14.7, 59.9, 55.6, 46.1,

CH CH CH CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3

δH (J in Hz)

6.53, s 5.82, brd (9.0) 2.40, m 3.17, m 1.71, m 2.07 dd (14.3, 11.1) 1.85, m 5.72, 6.50, 5.17, 3.96, 5.14, 1.77, 3.39, 1.91,

d (11.1) dd (14.9, 10.9) dd (14.9, 7.9) dd (7.2, 6.8) dd (6.0, 2.4) m dd (10.0, 7.5) brq (7.0)

1.76, 1.47, 1.42, 1.33, 1.31, 2.95, 1.84, 0.94, 1.88, 0.95, 0.88, 1.75, 0.82, 0.85, 0.74, 0.81, 3.56, 3.14, 2.96,

m m m m m m m d (7.0) s d (7.0) d (7.0) s d (6.8) d (6.8) d (6.0) d (6.8) s s s

6.55, d (15.6) 6.97, d (15.6) 4.88, d (5.3) 4.29, d (7.2)

4.90, d (5.5) 4.49, d (5.3) 5.32, brs

4.88, d (5.5) 4.14, d (7.0)

7.89, brs 7.50, brs

structural relationship to 10, except for the presence of an additional oxygenated methine signal at δC 65.5, δH 3.96 (1H, m) and the absence of a methylene. COSY correlations from δH 3.96 (H-7) to δH 2.25 and 0.99 (H-8) and from H-7 to δH 1.93 and 1.41 (H-6) suggested that an additional hydroxy was located at C-7. HMBC correlations between H-7 (δH 3.96) and C-6 (δC 28.9), C-8 (δC 36.9), and C-8a (δC 39.4) also confirmed the position of the hydroxy group. A NOESY experiment helped to establish the relative configuration of 11. NOE interactions from H-8 (δH 2.25) to 1-OH (δH 5.40) and

H-8 (δH 2.21) to 1-OH (δH 5.38) and 4a-OH (δH 4.75), from 4a-OH (δH 4.75) to H-10 (δH 0.70), and from H-9 (δH 0.89) to H-1 (δH 3.27) and H-4 (δH 1.61) indicated that 1-OH, 4-CH3, and 4a-OH were on the same side, while H-1, H-4, and 8a-CH3 were located on the opposite side (Figure 2). Therefore, 10 was determined to be (1β,4β,4aβ,8aα)-4,8a-dimethyloctahydronaphthalene-1,4a(2H)-diol. Compound 11 has the molecular formula C12H22O3, which was established by its HRESIMS and 13C NMR data. Analysis of its 1H and 13C NMR data indicated that 11 possesses a close C



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Table 2. 1H NMR (600 MHz) and 13C NMR (150 MHz) Data for Compounds 10−12 in DMSO-d6 10 position

δC, type

11 δH (J in Hz)

δC, type

12 δH (J in Hz)

δC, type

1

76.0, CH

3.27, m

75.7, CH

3.25, m

35.4, CH2

2

28.8, CH2

4 4a 5

34.3, CH 75.1, C 30.1, CH2

1.92, 1.45, 1.58, 1.20, 1.68,

m m m m m

29.3, CH2

25.5, CH2

m m m m m

27.9, CH2

3

1.89, 1.49, 1.60, 1.19, 1.61,

6

21.2, CH2

7

20.6, CH2

m m m m m

30.6, CH2

m brt (13.5) dt (13.2, 4.9) m m m td (13.0, 4.7) brd (13.0)

1.39, 1.29, 1.93, 1.41, 3.96,

8

1.40, 1.13, 1.67, 1.36, 1.51, 1.44, 2.21, 0.78,

8a 9 10 1-OH 3-OH 4a-OH 7-OH

39.6, C 20.3, CH3 15.3, CH3

0.89, s 0.70, d (6.0) 5.38, d (4.5)

25.6, CH2 33.9, CH 75.4, C 25.8, CH2 28.9, CH2 65.5, CH 36.9, CH2

2.25, dd (14.1, 4.0) 0.99, brd (14.1)

39.4, C 23.1, CH3 15.4, CH3

1.12, s 0.73, d (6.4) 5.40, d (4.5)

4.75, brs

4.79, brs 4.20, d (2.5)

72.5, CH 36.3, CH 75.5, C 29.6, CH2 20.7, CH2 20.8, CH2 30.4, CH2 37.7, C 20.5, CH3 12.3, CH3

δH (J in Hz) 1.71, 0.85, 1.80, 1.58, 3.70,

td (12.8, 3.8) brd (12.8) m m m

1.58, m 1.41, 1.20, 1.61, 1.34, 1.49, 1.34, 1.78, 0.76,

brd (13.0) td (13.0, 4.3) m m m m m m

0.95, s 0.90, d (7.0) 5.27, d (4.5) 4.71, brs

Figure 2. Key NOE correlations of 10 and 11.

Figure 3. Proposed biosynthetic pathway of 10−16.

4a-OH (δH 4.79), from 4a-OH (δH 4.79) to H-10 (δH 0.73), and from H-9 (δH 1.12) to H-1 (δH 3.25), H-4 (δH 1.68), and

7-OH (δH 4.20) indicated that 1-OH, 4-CH3, and 4a-OH had a cis configuration; the 7-OH and 8a-CH3 were cis (Figure 2). D



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8 vs 1; 3 vs 2). Among the tested bafilomycins, only 7, which contained a hydroxy at C-19 and a fumaric acid moiety at C-21, showed antibacterial activity against the Gram-positive bacteria B. subtilis and S. aureus, with MIC values of 12.5 μg/mL. Bafilomycins 2, 7, and 8 demonstrated antifungal activity against three fungi, while the other bafilomycins were inactive against all of the above test organisms at 100 μg/mL. Sesquiterpenoids 13−16 did not show any cytotoxic, antibacterial, or antifungal activities. Degraded sesquiterpenoids 10−12 were marginally cytotoxic with IC50 values that ranged from 34.55 to 100 μM. Compound 10 showed strong antifungal activity against C. albicans and C. parapsilosis, with MIC values of 12.5 and 3.13 μg/mL, respectively. Compound 11 showed weak antifungal activity against C. albicans and C. parapsilosis, while both 10 and 11 were inactive against C. neoformans at 100 μg/mL.

Thus, the relative configuration of 11 was assigned as 1β,4β,4aβ,7α,8aα, and the compound was named (1β,4β,4aβ,7α,8aα)-4,8a-dimethyloctahydronaphthalene-1,4a,7(2H)triol. The structure of 12 was elucidated as (3β,4β,4aβ,8aα)-4,8adimethyloctahydronaphthalene-3,4a(2H)-diol based on 1D and 2D NMR spectra. As a synthesized intermediate, 12 was reported by Gosselin et al.16 Given the lack of NMR data for 12, we assigned its 1H and 13C NMR data, which are listed in Table 2. Structures of sesquiterpenoids 13−16 were elucidated by comparing their NMR data with published data.17−20 Although many terpenoid synthase genes are generally considered to be silent in bacteria, the biosynthesis of odoriferous sesquiterpenoids has been demonstrated in heterologous bacteria.29−32 On the basis of the structural analyses of 10−16 and on biosynthesis information found in the literature,25,29−31 we suggest that sesquiterpenoids 10−16 in S. albolongus were synthesized from the same precursor, farnesyl pyrophosphate, in the organism. The presumptive biosynthetic pathway for 10−16 is shown in Figure 3. Compounds 1−16 were evaluated for their cytotoxic activity against four human cancer cell lines via the MTT assay (Table 3). Antimicrobial assays of 1−16 against three bacteria and

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Table 3. Cytotoxic (IC50 in μM) Activities of Compounds 1− 12 compound

BGC-823

Caco-2

H460

SMMC-7721

1 2 3 4 5 6 7 8 9 10 11 12 adriamycin

3.58 11.82 46.94 6.47 22.40 69.20 1.83 0.99 9.46 34.55 47.19 49.30 1.48

2.81 46.25 >100 5.49 10.15 >100 1.89 2.30 14.61 85.50 42.50 >100 0.97

5.02 44.10 >100 12.41 26.70 >100 3.43 2.34 49.80 >100 >100 >100 0.98

1.36 10.14 13.60 2.03 9.42 8.67 0.54 1.05 9.79 41.7 > 100 > 100 2.24

EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were determined using an Anton Paar MCP200 automatic polarimeter. Ultraviolet spectra were measured with a Beckman Coulter DU 730 nucleic acid/protein analyzer. IR spectra were recorded with a Bruker Tensor 27 FT-IR spectrometer (film). 1D and 2D NMR spectra were collected on a Bruker AV-600 spectrometer, δ in ppm relative to TMS, J in Hz. ESIMS were recorded on an Agilent 1290-6420 Triple Quadrupole LC-MS spectrometer. HRESIMS were measured with an Agilent G6230 TOF mass spectrometer. Silica gel (100−200 mesh, 200−300 mesh, Qingdao Marine Chemical Ltd., Qingdao, China), Sephadex LH-20 (GE Healthcare Biosciences AB, Uppsala, Sweden), YMC*GEL ODS-A (S-50 μm, 12 nm) (YMC Co., Ltd., Kyoto, Japan), and Amberlite XAD-16 polymeric resin (Rohm and Hass Shanghai Chemical Industry Co., Ltd., Shanghai, China) were used for column chromatography. MTT and antimicrobial assays were analyzed using a microplate reader (BioTek Synergy H1, BioTek Instruments, Inc., Vermont, USA). Microbial Material. The producing organism was isolated from fresh fecal samples excreted by healthy adult Elephas maximus living in Xishuangbanna National Nature Reserve, Xishuangbanna, Yunnan Province, P. R. China, in October 2009. The strain was identified as Streptomyces albolongus by Y.J. based on morphological characteristics and 16S rRNA gene sequences. The BLAST result showed that the sequence was most similar (99.89%) to the sequence of S. albolongus (strain: NBRC 13465T, GenBank accession no. AB184425). The strain (No. YIM 101047) was deposited at the Yunnan Institute of Microbiology, Yunnan University, China. Fermentation, Extraction, and Isolation. A slant culture of the strain was used to inoculate 500 mL Erlenmeyer flasks containing 100 mL of seed medium composed of 4 g L−1 yeast extract, 4 g L−1 glucose, 5 g L−1 malt extract, 1.0 mL of multiple vitamin solution, and 1.0 mL of trace element solution at a pH of 7.2 with no adjustment. The flasks were cultured for 2 days at 28 °C on a rotary shaker at 180 rpm. This seed culture was used to inoculate fermentation medium with a 10% volume. The fermentation was carried out in a 500 mL Erlenmeyer flask containing 100 mL of fermentation medium composed of 10 g L−1 soybean meal, 2 g L−1 peptone, 20 g L−1

three fungi were carried out using a micro broth dilution method (Table 4). All of these bafilomycins showed different levels of cytotoxic activities that were influenced by the presence of oxygenic functional substituents on C-19 and C-21. A fumaric ester/amide moiety on C-21 promoted cytotoxic activity (1, 7, and 8 vs 4), which is in agreement with a previous report.14 No oxygenic functional substituent on C-21 decreased cytotoxic activity (2 vs 4; 3 vs 5). Replacing the 19-methoxyl on the skeleton with a hydroxy increased cytotoxic activity (4 vs 5;

Table 4. Antimicrobial Activities of 2, 7, 8, 10, and 11 (MIC μg/mL) MIC B. subtilis S. aureas E. coli C. albicans C. parapsilosis C. neoformans a

2

7

8

10

11

control

100 100 >100

0.25a 0.13a 0.13a 1.0b 2.0b 2.0b

12.5 12.5 100 3.13 25

1.56 1.56 1.56

100 1.56 25

12.5 3.13 >100

Ciprofloxacin. bAmphotericin B. E



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glucose, 5 g L−1 soluble starch, 2 g L−1 yeast extract, 4 g L−1 NaCl, 0.5 g L−1 K2HPO4, 0.5 g L−1 MgSO4·7H2O, and 2 g L−1 CaCO3 at a pH of 7.8 with no adjustment, and it was incubated for 7 days at 28 °C on a rotary shaker at 180 rpm. The completed fermentation broth (70 L) was separated into filtrate and mycelium by centrifugation. Compounds in the culture filtrate were absorbed onto Amberlite XAD-16 polymeric resin. After washing with water and 20% EtOH, the absorbed organic material was eluted with 95% EtOH to yield 28 g of dried extract after removal of the solvent in vacuo. The dried extract was subjected to silica gel column chromatography elution with a gradient of CH2Cl2−MeOH to yield 13 fractions. Fraction 2 was subjected to Sephadex LH-20 chromatography (MeOH) to produce five subfractions. Fraction 2.2 was separated by silica gel column chromatography with petroleum ether−EtOAc (10:1) to yield 13 (3.0 mg). Fraction 3 was subjected to Sephadex LH-20 chromatography (MeOH) to produce six subfractions. Fraction 3.1 was repeatedly separated using silica gel column chromatography eluting with petroleum ether−EtOAc (8:1) to yield 2 (3.0 mg) and 3 (4.5 mg) and eluting with petroleum ether−EtOAc (3:1) to yield 4 (14.5 mg), 5 (11.0 mg), 6 (14.0 mg), and 9 (18.5 mg). Subfraction 3.2 was separated by ODS column chromatography, eluting with methanol−water (80:20), to yield 15 (9.8 mg) and 16 (21.0 mg). Subfraction 3.3 was subjected to ODS column chromatography, eluting with methanol−water (70:30), to yield 10 (3.5 mg) and 12 (6.5 mg). Fraction 4 was subjected to Sephadex LH20 column chromatography (MeOH) to produce four subfractions. Fraction 4.1 was separated by silica gel column chromatography, eluting with petroleum ether−acetone (3:1), to yield 1 (16.8 mg), 7 (13.5 mg), and 8 (14.0 mg). Fraction 4.4 was subjected to ODS column chromatography, eluting with methanol−water (60:40), to yield 11 (2.5 mg). Fraction 5 was subjected to a Sephadex LH-20 column (MeOH) to produce three subfractions. Fraction 5.2 was purified by silica gel column chromatography (petroleum ether− EtOAc, 2:1) to yield 14 (3.0 mg). 19-Methoxybafilomycin C1 amide (1): colorless, amorphous solid; [α]20 D +28.0 (c 1.0, MeOH); UV (MeOH) λmax (log ε) 241 (2.66), 277 (2.19) nm; IR (film) νmax 3430, 3377, 2964, 2931, 2874, 2359, 2341, 1713, 1650, 1619, 1449, 1381, 1196, 1102 cm−1; 1H NMR and 13C NMR data, see Table 1; ESIMS m/z 756 [M + Na]+, 772 [M + K]+; HRESIMS m/z 756.4299 [M + Na]+ (calcd for C40H63NO11Na, 756.4299). 21-Deoxybafilomycin A1 (2): colorless, amorphous solid; [α]20 D +16.0 (c 1.0, MeOH); UV (MeOH) λmax (log ε) 245 (3.16), 285 (2.70) nm; IR (film) νmax 3465, 2958, 2929, 1687, 1647, 1621, 1457, 1361, 1244, 1101, 1099 cm−1; 1H NMR and 13C NMR data, see Table 1; ESIMS m/z 629 [M + Na]+, 645 [M + K]+; HRESIMS m/z 629.4026 [M + Na]+, (calcd for C35H58O8Na, 629.4029). 21-Deoxybafilomycin A2 (3): colorless, amorphous solid; [α]20 D +61.5 (c 0.26, MeOH); UV (MeOH) λmax (log ε) 244 (2.81), 284 (2.60) nm; IR (film) νmax 3419, 2957, 2926, 1687, 1647, 1623, 1457, 1361, 1258, 1097, 1031, 1010 cm−1; 1H NMR and 13C NMR data, see Table 1; ESIMS m/z 643 [M + Na], 659 [M + K]+; HRESIMS m/z 643.4179 [M + Na]+ (calcd for C36H60O8Na, 643.4186). (1β,4β,4aβ,8aα)-4,8a-Dimethyloctahydronaphthalene-1,4a(2H)diol (10): colorless oil; [α]20 D +60.0 (c 0.2, MeOH); IR (film) νmax 3331, 2931, 2862, 1460, 1445, 1259, 1095 cm−1; 1H NMR and 13C NMR data, see Table 2; ESIMS m/z 221 [M + Na]+, 419 [2M + Na]+; HRESIMS m/z 221.1513 [M + Na]+ (calcd for C12H22O2Na, 221.1518). (1β,4β,4aβ,7α,8aα)-4,8a-Dimethyloctahydronaphthalene1,4a,7(2H)-triol (11): colorless oil; [α]20 D +28.5 (c 0.28, MeOH); IR (film) νmax 3321, 2937, 2923, 1453, 1430, 1260, 1090, 1054, 1029 cm−1; 1H NMR and 13C NMR data, see Table 2; ESIMS m/z 237 [M + Na]+, 253 [M + K]+; HRESIMS m/z 237.1461 [M + Na]+ (calcd for C12H22O3Na, 237.1467). Cytotoxicity Assay. The cytotoxicities of 1−16 were evaluated in a human gastric carcinoma cell line (BGC-823), a human colon adenocarcinoma cell line (Caco-2), a human large-cell lung carcinoma cell line (H460), and a human hepatocellular carcinoma cell line (SMMC-7721) in an MTT assay as previously reported.33 IC50 was

defined as a 50% reduction of absorbance from the control assay. Adriamycin was used as a positive control. Antimicrobial Assay. A micro broth dilution34 assay was used to evaluate the minimum inhibitory concentrations (MICs) of 1−16 against three bacteria (Bacillus subtilis ATCC 6633, Staphylococcus aureus ATCC 25923, and Escherichia coli ATCC 25922) and three fungi (Candida albicans ATCC MYA-2876, Candida parapsilosis ATCC 22019, and Cryptococcus neoformans ATCC 208821). Antibacterial and antifungal tests were performed in Luria−Bertani (LB) medium (5 g L−1 yeast extract, 10 g L−1 peptone, 5 g L−1 NaCl, pH 7.0) and RPMI-1640 broth (10.4 g L−1 RPMI-1640, 2 g L−1 NaHCO3, 34.53 g L−1 MOPs, pH 7.0). Test compounds were dissolved in DMSO and 2-fold serially diluted to eight different concentrations (11.0−0.086 mg/mL), and each solution was 10-fold diluted using LB/RPMI-1640 culture medium (1.1−0.0086 mg/mL). Test sample solutions (10 μL) and 100 μL of prepared bacterial suspensions containing 1 × 106 cfu/mL of bacteria (2 × 103 cfu/mL for C. albicans and C. parapsilosis and 5 × 104 cfu/mL for C. neoformans) were added to each well of 96-well microtiter plates, and the plates were incubated for 24 h at 28 °C for bacteria, 48 h at 28 °C for C. albicans and C. parapsilosis, and 72 h at 28 °C for C. neoformans. MTT (10 μL of 5 mg/mL solution) was added into each well and incubated for another 4 h, after which the liquid in the wells was removed. DMSO (100 μL) was added to each well. The absorbance was recorded on a microplate reader at a wavelength of 570 nm. The MIC was defined as the lowest concentration of the antimicrobial agent that completely inhibited visual growth of an organism. Ciprofloxacin and amphotericin B were used as positive controls against bacteria and fungi, respectively.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.5b00827. 1D and 2D NMR, HRESIMS spectra of compounds 1− 3, 10, and 11 (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Tel: 0086-24-83656122. Fax: 0086-24-83656122. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was funded by National Natural Science Foundation of China (Nos. 81072553, 31270001, and 31460005), Basic Scientific Research Fund of Northeastern University, China (Nos. N120820002, N142002001, and N142004004), and Yunnan Provincial Society Development Project (2014BC006).

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