Fitoterapia 99 (2014) 153–158
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Bioactive metabolites from the endophytic fungus Alternaria alternata Ying Wang, Ming-Hua Yang ⁎, Xiao-Bing Wang, Tian-Xiao Li, Ling-Yi Kong ⁎⁎ State Key Laboratory of Natural Medicines, Department of Natural Medicinal Chemistry, China Pharmaceutical University, 24 Tong Jia Xiang, Nanjing 210009, People's Republic of China
a r t i c l e
i n f o
Article history: Received 16 August 2014 Accepted 18 September 2014 Available online 28 September 2014 Keywords: Alternaria alternata Altenuene enantiomers Isocoumarin Antimicrobial
a b s t r a c t Two altenuene derivatives (1–2) and one isocoumarin (3), together with six known compounds (4–9) were isolated from solid cultures of an endophytic fungus Alternaria alternata, obtained from the fresh branches of Camellia sinensis. Chiral analysis revealed the racemic nature of 1 and 2, which were subsequently resolved into two pairs of enantiomers [(+)-1 and (−)-1, (+)-2 and (−)-2]. Structures of all the isolates were identified through spectroscopic data. Absolute configurations of the two pairs of enantiomers were determined by electronic circular dichroism (ECD) calculation and the chiral center of C-10 in 3 was deduced via [Rh2(OCOCF3)4]-induced CD experiment. All the isolates were evaluated for their antimicrobial abilities against the pathogenic bacteria and fungi as well as cytotoxic activities against two human tumor cell lines. Compound 5 was the most active against Bacillus subtilis with MIC80 of 8.6 μg/ml, and compounds 1–3, 6–7 and 9 exhibited moderate to weak inhibition towards the test pathogenic microorganism. Compound 4 showed mild cytotoxic activity against human osteosarcoma cells U2OS with IC50 of 28.3 μM. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Alternaria species have been identified as a prolific fungal source of new pharmacologically active metabolites such as steroids, terpenoids, pyrones, quinones, phenolics and nitrogen-containing compounds, which were suitable for specific medicinal or agricultural applications [1]. Dibenzopyranones with polyketide origin, such as alternariols and altenuenes were typical isolates from the genus Alternaria. These dibenzopyranone derivatives were examined to have various biological activities and functions, mainly including cytotoxicity [2,3] and antimicrobial [4,5] properties. During the investigation of bioactive natural products from plant endophytes, our attention was drawn to a strain of Alternaria sp.,
⁎ Corresponding author. Tel.: +86 25 8618 5039. ⁎⁎ Corresponding author. Tel./fax: +86 25 8327 1405. E-mail addresses: [email protected] (M.-H. Yang), [email protected] (L.-Y. Kong).
http://dx.doi.org/10.1016/j.fitote.2014.09.015 0367-326X/© 2014 Elsevier B.V. All rights reserved.
whose crude extract displayed potent inhibitory abilities against Staphylococcus aureus and Bacillus subtilis with inhibition zones of 19.5 and 25.3 mm respectively in the preliminary agar diffusion screening (37.7 mm of the positive control penicillin). Therefore, a systematic chemical study was performed and resulted in the isolation of altenuene-2-acetoxy ester (1), altenuene-3-acetoxy ester (2) and a new isocoumarin (+)-(10R)-7-hydroxy-3-(2-hydroxy-propyl)-5,6-dimethylisochromen-1-one (3), along with six known dibenzopyranone derivatives, alternariol 9-methyl ether (4) [5], alternariol (5) [5], phialophoriol (6) [6], altenuene (7) [7], 5′-epialtenuene (8) [8] and alterlactone (9) [2]. Chiral analysis of 1 and 2 revealed the presence of racemic mixture, and subsequent racemic resolution resolved into two pairs of enantiomers (+)-1 [(+)(2S, 3S, 4aS)-altenuene-2-acetoxy ester], (−)-1 [(−)-(2R, 3R, 4aR)-altenuene-2-acetoxy ester], (+)-2 [(+)-(2S, 3S, 4aS)altenuene-3-acetoxy ester] and (−)-2 [(−)-(2R, 3R, 4aR)altenuene-3-acetoxy ester]. Details of the isolation, structure elucidation and bioactive evaluation of these compounds are discussed below.
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2. Experimental 2.1. General Optical rotations were determined on a JASCO P-1020 polarimeter at room temperature. CD spectra were performed on a JASCO J-810 spectrometer. UV spectra were recorded on a Shimadzu UV-2501 spectrophotometer. IR spectra were measured by a Bruker Tensor 27 spectrometer. 1D and 2D NMR were carried out on a Bruker Avance III NMR (1H: 500 MHz, 13C: 125 MHz) instrument at 300 K, with TMS as internal standard. ESIMS was obtained on an Agilent 1100 Series LC/MSD ion-trap mass spectrometer and HRESIMS was recorded on an Agilent 6520B UPLC-Q-TOF mass spectrometer, respectively. Chiral HPLC was done with a Daicel Chiralpak AD-H column (250 × 4.6 mm, 5 μm). Preparative HPLC was performed by a Shimadzu LC-10A equipped with a Shim-pack RP-C18 column (20 × 200 mm) and a Shimadzu SPD-20A detector. Column chromatography (CC) was performed with silica gel and Sephadex LH20. All the solvents used were of analytical grade.
afford compounds 7 (1.0 mg) and 8 (1.1 mg) with MeOH–H2O (65:35, 10 ml/min) as mobile phase. Compound 3 (2.5 mg) was obtained from fraction D4 (0.2 g) by preparative HPLC using methanol in water (60:40, 10 ml/min), and compound 9 (2.0 mg) was purified from fraction F (0.2 g) eluting with MeOH–H2O (55:45, 10 ml/min). Compounds (+)-1 (3.7 mg) and (−)-1 (0.9 mg) were separated from 1 by a chiral column using n-hexane–isopropanol (90:10, 0.80 ml/min), and compounds (+)-2 (1.5 mg) and (−)-2 (1.2 mg) were isolated from 2 (n-hexane/isopropanol, 80:20, 0.75 ml/min) by the similar procedure of 1. Compound 1: white solid; UV (MeOH) λmax (logε): 193 (3.86), 200 (3.91), 241 (4.27), 278 (3.78), 319 (3.53) nm; IR (KBr) νmax: 3444, 2922, 2349, 1627, 1384, 1263, 1163, 1117 cm−1; ESIMS positive m/z: 335 [M + H]+; HRESIMS m/z: 335.1124 [M + H]+ (calcd for C17H19O7, 335.1125); and 1H and 13 C NMR data (CDCl3), see Table 1. (+)-1 (−)-1
2.2. Fungal material The title strain was isolated from the branches of Camellia sinensis, collected from the suburb of Nanjing, Jiangsu Province, People's Republic of China, in October 2012. The culture was grown on potato dextrose agar (PDA) and distinguished morphologically as Alternaria sp., which was further reinforced by 18S rDNA sequence with a 100% identity to Alternaria alternata, conducted by the fungal identification service (Shanghai Majorbio Bio-pharm Technology). The fungal strain was cultivated on PDA medium at 28 °C until sufficient growth was observed (about 5 days). Then plugs of agar supporting mycelia growth were cut into small pieces and transferred aseptically into a 1000 ml Erlenmeyer flask containing 300 ml potato dextrose broth, and incubated on a rotary shaker at 180 rpm for 7 days to prepare the seed culture. Fermentation was carried out in ten 500 ml Erlenmeyer flasks, each containing 80 g of rice and soaked with 120 ml distilled water overnight before autoclaving. Each flask was inoculated with 10 ml seed liquid, and cultivated under stationary conditions at 28 °C for 30 days. 2.3. Extraction and isolation The fermented substrate was saturated with methanol and filtrated. After removal of the solvent under reduced pressure, the crude extract (8.6 g) was subjected to a silica gel column, eluted with a gradient of petroleum ether and acetone mixture from 20:1 to 1:1 (v/v) to generate six fractions (A–F). Fraction C (1.6 g) gave the main compounds 4 (19.0 mg) and 5 (9.5 mg) after Sephadex LH-20 CC (CH2Cl2–MeOH, 1:1). Fraction E (1.3 g) was chromatographed over silica gel CC (CH2Cl2– Acetone, 25:1, 15:1, 5:1, v/v) and further purified by preparative HPLC using MeOH–H2O (60:40, 10 ml/min) to yield 1 (5.7 mg) and 2 (3.5 mg). Fraction D (2.0 g) was applied onto a silica gel CC, and eluted with CH2Cl2–MeOH (20:1, 10:1, 5:1, v/v) to acquire five major subfractions (D1–D5). Subfraction D2 (0.7 g) gave compound 6 (2.7 mg) after CC on Sephadex LH-20 (MeOH). Subfraction D3 (0.9 g) displayed in the same way as subfraction D2, and further purified by preparative HPLC to
−4 , MeOH) [α]25 D + 13.3 (c 0.17, MeOH); CD (2.0 × 10 λmax nm (Δε) 241 (−7.43), 280 (+4.84). −4 , MeOH) [α]25 D − 13.3 (c 0.11, MeOH); CD (1.0 × 10 λmax nm (Δε) 241 (+3.67), 280 (−2.49).
Compound 2: white solid; UV (MeOH) λmax (logε): 194 (3.81), 200 (3.86), 240 (4.19), 277 (3.72), 317 (3.46) nm; IR (KBr) νmax: 3450, 2923, 2852, 1642, 1384, 1163 cm−1; ESIMS positive m/z: 335 [M + H]+; HRESIMS m/z: 335.1127 [M + H]+ (calcd for C17H19O7, 335.1125); and 1H and 13C NMR data (CDCl3) see Table 1. (+)-2 (−)-2
−4 , MeOH) [α]25 D + 11.6 (c 0.11, MeOH); CD (2.0 × 10 λmax nm (Δε) 241 (−2.34), 280 (+1.45). −4 , MeOH) [α]25 D − 11.6 (c 0.12, MeOH); CD (2.0 × 10 λmax nm (Δε) 241 (+2.35), 280 (−1.46).
Compound 3: white solid; [α]25 D + 35.3 (c 0.09, MeOH); UV (MeOH) λmax (log ε): 213 (3.88), 244 (3.65), 252 (3.61), 292 (3.51) nm; CD (2 × 10−4, MeOH) λmax nm (Δε) 365 (+3.67), 426 (−2.92); IR (KBr) νmax: 3448, 2927, 1645, 1550, 1463, 1384, 1270, 1115 cm−1; ESIMS positive m/z: 249 [M + H]+; HRESIMS m/z: 249.1120 [M + H]+ (C14H17O4, calcd for 249.1121); and 1H and 13C NMR data (Acetone-d6) see Table 1. 2.4. Absolute configuration of the secondary alcohol in 3 A solid [Rh2(OCOCF3)4] (2.0 mg) was added into compound 3 (0.7 mg) dissolving in CH2Cl2. After mixing, the first CD spectrum was recorded at once, and the time evolution was monitored until stationary (15 min). The inherent CD spectrum was subtracted. The metal complexes gave an induced CD spectrum, in which the sign of the E band around 350 nm was correlated to the absolute configuration of 3 [9,10]. 2.5. Biological assays The antibacterial activities against Gram-positive S. aureus ATCC 25923 (S. aureus), B. subtilis ATCC 6633 (B. subtilis) and Gram-negative Escherichia coli ATCC 25922 (E. coli) were initially performed by disk diffusion assay [11]. Each paper disk (6 mm diameter) permeating with 10 μl of test sample
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Table 1 1 H NMR (500 MHz) and 13C NMR (125 MHz) data for 1–3. No.
1a δH (multi, J in Hz)
δC
δH (multi, J in Hz)
δC
1 2 3 4
6.04 (d, 2.7) 5.23 (dd, 2.7, 5.7) 4.10 (m) He:2.54 (dd, 4.1, 14.6) Ha:2.09 (dd, 8.6, 14.6)
124.7 74.0 67.6 39.9
6.19 (d, 3.3) 4.29 (dd, 3.0, 5.2) 5.05 (m) He:2.60 (dd, 4.1,14.6) Ha:2.13 (dd, 8.7,14.7)
127.7 69.3 72.6 37.1
4a 6 7 7a 8 9 10 10a 10b 4a-CH3 9-OCH3 7-OH 2/3-OAc
6.45 (d, 2.2) 6.52 (d, 2.2)
1.56 (s) 3.85 (s) 11.26 2.13 (s)
a b
2a
80.7 168.8 164.4 103.3 100.8 166.4 101.5 138.5 136.4 28.1 56.0 171.2 21.3
6.46 (d, 2.3) 6.54 (d, 2.3)
1.54 (s) 3.86 (s) 11.31 (s) 2.11 (s)
No.
80.3 168.5 164.6 100.8 101.4 166.4 103.3 138.4 135.0 27.9 55.9
3b δH (multi, J in Hz)
1 3 4 4a 5 6 7 8 8a 9 10 11 7-OH 10-OH 5-CH3 6-CH3
5.95 (s)
6.78 (s) 2.62 (dd, 7.5, 14.3) 2.65 (dd, 5.4, 14.3) 4.20 (m) 1.23 (d, 6.2)
2.78 (s) 2.18 (s)
δC 180.4 165.3 113.6 116.8 141.2 123.8 160.6 101.5 159.1 44.8 66.3 24.4
17.8 12.1
171.0 21.3
Recorded in CDCl3. Recorded in Acetone-d6.
(100 μg/ml methanol) was dried and placed on Mueller Hinton agar plate containing bacterial inoculum. Following incubation (37 °C, 24 h), diameter (in mm) of the growth inhibition zone was recorded to evaluate antibacterial potential. For further determination of the minimal inhibition concentration (MIC), broth microdilution [12] in sterile 96-well plates was performed. The test bacteria was inoculated on nutrient agar for 18–24 h, then a colony approximately 1 mm in diameter was collected with a sterile loop and dissolved into Mueller Hinton broth. The bacterial cell suspension was adjusted to 1.0 × 104– 1.0 × 106 cells/ml before use. Each test compound was firstly dissolved in DMSO, then diluted by broth to different concentrations (0–50.0 μg/ml, DMSO b 1%). Test well was introduced into 100 μl bacterial suspension and 100 μl individual compounds. Blank well was incubated with only medium under the same condition. Optical density (OD) measurement was recorded at 578 nm after incubation for 24 h at 37 °C. MIC was determined by identifying the drug concentration at which 80% of bacterial growth was inhibited. All experiments were performed in three replicates and with penicillin (b 2.0 μg/ml) as the positive control. Antifungal activities against Candida albicans ATCC 24433 (C. albicans) and Trichophyton rubrum ATCC 28189 (T. rubrum) were conducted in triplicate by the microdilution method [13,14]. The preparation of Candida inocula was done by picking 24-hour-old colonies (about 1 mm in diameter) from the PDA plate and aseptically transfered into 3 ml sterile 0.85% saline solution. The solution was adjusted to 5.0 × 102–2.5 × 103 cells/ml with RPMI-1640 medium by a hemocytometer to prepare for the cell suspension. Similarly, the culture of T. rubrum was grown on PDA plate for 5 days and diluted to a final spore density of 0.4 × 104–5.0 × 104 cells/ml after discarding mycelium. Each test compound was diluted to different concentrations (0–50.0 μg/ml, DMSO b 1%) using the method described above for bacteria. Each test well was introduced into 100 μl cell suspension and 100 μl respective compounds. Blank well with only medium was used to ensure medium sterility.
Fluconazole was the positive control for yeast (b1.0 μg/ml) assay and filamentous fungi (b4.0 μg/ml) test. The 96-well plates for C. albicans and T. rubrum were incubated at 35 °C for 24 h and 72 h, respectively. OD measurement was made at 595 nm and MIC was determined as described above for bacteria. Moreover, all the isolated compounds were further tested in vitro for their cytotoxicities against two human tumor cell lines U2OS and HepG2 by using the MTT method [15] with cisplatinum as positive control. Absorbance was recorded at 570 nm by a microplate reader to detect cell viability. All experiments were performed in three replicates. 3. Results and discussion Compound 1, white solid, had a molecular formula of C17H18O7 as HRESIMS exhibited a quasi-molecular ion at m/z 335.1124 [M + H]+ (calcd for C17H19O7, 335.1125), giving nine degrees of unsaturation. The 1H and 13C NMR together with HSQC spectra revealed one methyl [δH/δC, 1.56 (3H, s)/28.1], one methylene [δH/δC, 2.54 (1H, dd, 4.1, 14.6), 2.09 (1H, dd, 8.6, 14.6)/39.9], two oxygenated methines [δH/δC, 4.10 (1H, m)/ 67.6; 5.23 (1H, dd, 2.7, 5.7)/74.0], one protonated olefinic group [δH/δC, 6.04 (1H, d, 2.7)/124.7], one ester carbonyl (δC 168.8), one sp3 quaternary carbon (δC 80.7), one sp2 quaternary carbon (δC 136.4) and a tetrasubstituted benzene ring with a pair of meta-coupled aromatic protons [δH/δC 6.45 (1H, d, 2.2), 6.52 (1H, d, 2.2)/164.4, 103.3, 101.5, 138.5, 166.4, 100.8]. These data implied the altenuene core of 1 [7], which was mainly confirmed by HMBC correlations from H-8 (δH 6.45) to C-9 (δC 166.4), C-7 (δC 164.4) and C-10 (δC 101.5), from H-1 (δH 6.04) to C-2 (δC 74.0), C-10a (δC 138.5) and C-10b (δC 136.4), from Ha-4 (δH 2.09) to C-2, C-3 (δC 67.6) and C-4a (δC 80.7) and from methyl protons (δH 1.56) to C-4a, C-10b and C-4 (δC 39.9). The remaining 8 protons and 3 carbons attributed to a phenolic hydroxyl group [δH 11.26 (1H, s)], a methoxyl group [δH/δC, 3.85 (3H, s)/56.0], an O-acetyl unit [δH/δC, 2.13
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(3H, s)/21.3, 171.2] and an undetected hydroxyl group. The relatively downfield chemical shift of C-3 (δC 67.6) and the HMBC correlations from H-3 to C-2 and C-4 placed a hydroxyl at C-3. HMBC signals from hydroxyl proton at δH 11.26 to C-7 and C-8, from methoxyl protons at δH 3.85 to C-9 and C-10 confirmed the attachment of the phenolic hydroxyl and the methoxyl group at C-7 and C-9, respectively. In addition, the acetoxyl group was located at C-2 based on the HMBC correlation between H-2 and its ester carbonyl carbon. Thus, the planar structure of 1 was determined as in Fig. 1. The relative stereochemistry of 1 was mainly established by J values and ROESY correlations of relevant protons. An apparent axial– axial relationship existed between Ha-4 and H-3 as judged from the large coupling constant (J = 8.6 Hz) between them. ROESY correlations between Ha-4, H-2 (δH 5.23) and the methyl protons at δH 1.56 (H3–4a) lead to the assignment of these protons on the same face of the molecule and a pseudoequatorial orientation of the C-2 O-acetyl group. Remarkably, the optical rotation of compound 1 was approximate to zero, suggesting the mixture of enantiomers. A follow-up chiral separation yielded a pair of enantiomers (+)-1 and (−)-1 (Fig. 3), which exhibited similar signal intensity but opposite Cotton effects in their CD spectra (Fig. 4), further confirming their enantiomeric relationship. To determine absolute configuration, their experimental CD spectra were compared with those predicted from quantum mechanical time-dependent density functional theory (TDDFT) calculations. As a result, the calculated ECD spectrum for the (2S, 3S, 4aS) stereoisomer matched well with the experimental CD data of (+)-1 (Fig. 4.b), which designated the configuration of (+)-1 as (2S, 3S, 4aS)-altenuene-2-acetoxy ester. As a consequence, (−)-1, with optical value of the same magnitude to (+)-1, was assigned as (2R, 3R, 4aR)-altenuene-2-acetoxy ester. Compound 2, an isomer of 1, also had a molecular formula of C17H18O7 on the basis of a quasi-molecular ion at m/z 335.1127 [M + H]+ (calcd for C17H19O7, 335.1125). The UV, IR and NMR spectra of 2 resembled those of 1, demonstrating the
Fig. 2. Selected HMBC and ROESY correlations of 1 and 3.
same altenuene core of 2. Compared with 1, the obvious differences were the chemical shifts of H-2 and H-3, which were upfield-shifted from δH 5.23 to δH 4.29 and downfield-shifted from δH 4.10 to δH 5.05, respectively. This, taken together with HMBC correlation between the ester carbonyl carbon (δC 171.0) of the O-acetyl group and H-3 (δH 5.05) readily located the O-acetyl group at C-3 rather than at C-2 in 1. The relative configuration of 2 was determined as the same with 1, for that the analogous coupling constant (J = 8.7 Hz) between Ha-4 and H-3 as well as identical ROESY correlations of key protons were observed. Compound 2 was also optically inactive and resolved into a pair of enantiomers (+)-2 and (−)-2 by a chiral column (Fig. 3). The CD spectrum of (+)-2 and (−)-2 was identical with (+)-1 and (−)-1 respectively (Fig. 4), revealing the same absolute configurations of the two pairs of enantiomers. Based on the observation above, structures of (+)-2 and (−)-2 were determined to be (+)-(2S, 3S, 4aS)-altenuene3-acetoxy ester and (−)-(2R, 3R, 4aR)-altenuene-3-acetoxy ester, respectively. Compound 3 was obtained as a white powder. HRESIMS ion at m/z 249.1120 [M + H]+ revealed its molecular formula to be C14H16O4 (calculated for C14H17O4, 249.1121), indicating seven double-bond equivalents. The IR spectrum of 3 showed a phenolic hydroxyl (3448 cm−1) and a characteristic absorption band of benzene ring (1645, 1550, 1463 cm−1). The 1H, 13C
Fig. 1. Structures of compounds 1–9.
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Fig. 3. Chiral HPLC analysis of 1 (upper) and 2 (down).
NMR and HSQC spectrum displayed 14 carbons and 14 protons (Table 1). Among them, 11 carbons and their corresponding protons revealed two isolated methyls [δH/δC, 2.18 (3H, s)/12.1; 2.78 (3H, s)/17.8], one protonated olefinic group [δH/δC, 5.95 (1H, s)/113.6], one sp2 quaternary carbon (δC 165.3), one easter carbonyl (δC 180.4) and a pentasubstituted benzene ring (δC 101.5, 116.8, 123.8, 141.2, 159.1, 160.6) with an aromatic singlet proton [δH 6.78 (1H, s)]. Such data indicated that compound 3 was an isocoumarin [16]. Analysis of HMBC correlations (Fig. 2) confirmed this deduction and further positioned two methyls and a hydroxyl to C-5, C-6 and C-7, respectively. The remaining 3 carbons and 6 protons attributed to a methylene [δH/δC, 2.62 (1H, dd, 7.5, 14.3), 2.65(1H, dd, 5.4, 14.3)/44.8], an oxymethine [δH/δC, 4.20 (1H, m)/66.3] and a methyl [δH/δC, 1.23 (3H, d, 6.2)/24.4], which indicated a 2hydroxyl-propanyl fragment. Moreover, HMBC correlations from the methylene to C-3 and C-4 attached the 2-hydroxylpropanyl chain to C-3. The absolute configuration at C-10, bearing a secondary-alcohol group, was determined as R by the in-situ [Rh2 (OCOCF3)4] method according to the notable negative Cotton effect around 350 nm in the induced CD spectrum (Fig. S10, Supporting information). Consequently, the structure of 3 was determined as depicted. Initial agar diffusion assay revealed that the isolates had positive bacteriostatic effect against Gram-positive bacteria (S. aureus and B. subtilis) with inhibition zones of 10–25 mm but
Fig. 4. a. CD spectra of (+)-1, (−)-1, (+)-2 and (−)-2 (in methanol). b. Calculated and experimental CD spectra of (+)-1.
insensitive to Gram-negative bacteria (E. coli). Further microdilution assessment was carried out with the results listed in Table 2. In agreement with literature reports [4,5], altenuene analogs 1–3, 6 and 7 as well as alternariol derivatives 5 and 9
Table 2 Minimum inhibitory concentration (MIC80, μg/ml) of 1–9a. Samples
Sab
(+)-1 (−)-1 (+)-2 (−)-2 3 4 5 6 7 8 9 Fluconazole Penicillin
17.1 ± 15.4 ± 46.8 ± 45.0 ± N50.0 N50.0 N50.0 N50.0 38.9 ± N50.0 N50.0
a
1.2 1.1 2.0 1.7
1.5
1.2 ± 0.1
Bsb
Cab
Trb
N50.0 N50.0 N50.0 N50.0 19.7 ± 1.0 N50.0 8.6 ± 0.7 16.7 ± 1.2 N50.0 N50.0 N50.0
19.5 ± 1.5 48.8 ± 1.2 24.0 ± 1.0 N50.0 N50.0 N50.0 N50.0 N50.0 13.7 ± 0.7 N50.0 17.1 ± 1.8 0.6 ± 0.1
N50.0 N50.0 N50.0 N50.0 32.0 ± 2.1 N50.0 N50.0 N50.0 N50.0 N50.0 35.5 ± 2.2 1.0 ± 0.2
0.9 ± 0.1
A compound was regarded as inactive when MIC80 N 50.0 μg/ml. b Sa, Bs, Ca, Tr represented Staphylococcus aureus ATCC 25923, Bacillus subtilis ATCC 6633, Candida albicans ATCC 24433 and Trichophyton rubrum ATCC 28189.
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showed moderate to weak antimicrobial activities. However, it is difficult to clarify a common structural characteristic which might explain their abilities as antibacterial or antifungal agents. But interestingly, enantiomeric altenuene-2-acetoxy ester [(+)-1 and (−)-1] displayed similar moderate inhibitory effects against S. aureus with MIC values of 17.1 and 15.4 μg/ml, whereas the analogs altenuene-3-acetoxy ester [(+)-2 and (−)-2], altenuene (7) and 5′-epialtenuene (8) showed no significant effect. These results indicated that the enantiomeric difference might have a negligible effect against S. aureus but acetylation of 2-OH could improve the ability. Moreover, (+)-1 and (+)-2 exhibited efficient growth inhibition of C. albicans while (−)-1 and (−)-2 were less active, suggesting the different antifungal abilities between enantiomers. Additionally, alternariol (5) was the most active against B. subtilis with MIC value of 8.6 μg/ml while alternariol 9-methyl ether (4) was not sensitive at the concentration of 50.0 μg/ml, indicating methylation of 9-OH may be detrimental for the antibacterial activity. Alternariol derivatives have been detected to have pronounced cytotoxicity against mouse lymphoma cell [2]. But in our cytotoxic test, all the isolates were inactive at 50.0 μM against two human tumor cell lines U2OS and HepG2 except that alternariol 9-methyl ether (4) showed temperate inhibition of human osteosarcoma cell U2OS with IC50 28.3 μM. Acknowledgments This research work was funded by the National Natural Science Foundation of China (81073009), the Program for Changjiang Scholars and Innovative Research Team in University (IRT1193), the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD), the youth fund project of basic research program of Jiangsu Province (Natural Science Foundation, BK20130651), and the Fundamental Research Funds for the Central Universities (JKQZ2013016). Appendix A. Supplementary data HRESIMS, 1D NMR spectrum of compounds 1–3 and CD spectra of compound 3 are available in Supporting information. Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.fitote.2014.09.015.
References [1] Lou JF, Fu LY, Peng YL, Zhou LG. Metabolites from Alternaria fungi and their bioactivities. Molecules 2013;18:5891–935. [2] Aly AH, Edrada-Ebel R, Indriani ID, Wray V, Muller WE, Totzke F, et al. Cytotoxic metabolites from the fungal endophyte Alternaria sp. and their subsequent detection in its host plant Polygonum senegalense. J Nat Prod 2008;71:972–80. [3] Schreck I, Deigendesch U, Burkhardt B, Marko D, Weiss C. The Alternaria mycotoxins alternariol and alternariol methyl ether induce cytochrome P450 1A1 and apoptosis in murine hepatoma cells dependent on the aryl hydrocarbon receptor. Arch Toxicol 2012;86:625–32. [4] Jiao P, Gloer JB, Campbell J, Shearer CA. Altenuene derivatives from an unidentified freshwater fungus in the family Tubeufiaceae. J Nat Prod 2006;69:612–5. [5] Gu W. Bioactive metabolites from Alternaria brassicicola ML-P08, an endophytic fungus residing in Malus halliana. World J Microbiol Biotechnol 2009;25:1677–83. [6] Ye F, Chen GD, He JW, Li XX, Sun X, Guo LD, et al. Xinshengin, the first altenusin with tetracyclic skeleton core from Phialophora spp. Tetrahedron Lett 2013;54:4551–4. [7] He JW, Chen GD, Gao H, Yang F, Li XX, Peng T, et al. Heptaketides with antiviral activity from three endolichenic fungal strains Nigrospora sp., Alternaria sp. and Phialophora sp. Fitoterapia 2012;83:1087–91. [8] Bradburn N, Coker RD, Blunden G, Turner CH, Crabb TA. 5′-Epialtenuene and neoaltenuene, dibenzo-α-pyrones from Alternaria Alternata cultured on rice. Phytochemistry 1994;35:665–9. [9] Frelek J, Szczepek WJ. [Rh2(OCOCF3)4] as an auxiliary chromophore in chiroptical studies on steroidal alcohols. Tetrahedron Asymmetry 1999; 10:1507–20. [10] Frelek J. Configurational assignment of vic-amino alcohols from their circular dichroism spectra with dirhodium tetraacetate as an auxiliary chromophore. Tetrahedron Asymmetry 1999;10:2809–16. [11] Fiedler HP, Bruntner C, Riedlinger J, Bull AT, Knutsen G, Goodfellow M, et al. Proximicin A, B and C, novel aminofuran antibiotic and anticancer compounds isolated from marine strains of the actinomycete Verrucosispora. J Antibiot 2008;61:158–63. [12] Qin XJ, Lunga PK, Zhao YL, Li JL, Yang XW, Liu YP, et al. Antibacterial prenylbenzoic acid derivatives from Anodendron formicinum. Fitoterapia 2014;92:238–43. [13] Li RX, Chen SX, Niu SB, Guo LD, Yin J, Che YS. Exserolides A–F, new isocoumarin derivatives from the plant endophytic fungus Exserohilum sp. Fitoterapia 2014;96:88–94. [14] Koroishi AM, Sehn E, Baesso ML, Ueda-Nakamura T, Nakamura CV, Cortez DAG. Antifungal activity and nail permeation of nail lacquer containing Piper regnellii (Miq.) C. CD. var. pallescens (C. DC.) Yunck (Piperaceae) leave extracts and derivatives. Molecules 2010;15:3920–31. [15] Alley MC, Scudiero DA, Monks A, Hursey ML, Czerwinski MJ, Fine DL, et al. Feasibility of drug screening with panels of human tumor cell lines using a microculture tetrazolium assay. Cancer Res 1988;48:589–601. [16] Wang QX, Bao L, Yang XL, Guo H, Yang RN, Ren B, et al. Polyketides with antimicrobial activity from the solid culture of an endolichenic fungus Ulocladium sp. Fitoterapia 2012;83:209–14.
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Fitoterapia journal homepage: www.elsevier.com/locate/fitote
Bioactive metabolites from the endophytic fungus Alternaria alternata Ying Wang, Ming-Hua Yang ⁎, Xiao-Bing Wang, Tian-Xiao Li, Ling-Yi Kong ⁎⁎ State Key Laboratory of Natural Medicines, Department of Natural Medicinal Chemistry, China Pharmaceutical University, 24 Tong Jia Xiang, Nanjing 210009, People's Republic of China
a r t i c l e
i n f o
Article history: Received 16 August 2014 Accepted 18 September 2014 Available online 28 September 2014 Keywords: Alternaria alternata Altenuene enantiomers Isocoumarin Antimicrobial
a b s t r a c t Two altenuene derivatives (1–2) and one isocoumarin (3), together with six known compounds (4–9) were isolated from solid cultures of an endophytic fungus Alternaria alternata, obtained from the fresh branches of Camellia sinensis. Chiral analysis revealed the racemic nature of 1 and 2, which were subsequently resolved into two pairs of enantiomers [(+)-1 and (−)-1, (+)-2 and (−)-2]. Structures of all the isolates were identified through spectroscopic data. Absolute configurations of the two pairs of enantiomers were determined by electronic circular dichroism (ECD) calculation and the chiral center of C-10 in 3 was deduced via [Rh2(OCOCF3)4]-induced CD experiment. All the isolates were evaluated for their antimicrobial abilities against the pathogenic bacteria and fungi as well as cytotoxic activities against two human tumor cell lines. Compound 5 was the most active against Bacillus subtilis with MIC80 of 8.6 μg/ml, and compounds 1–3, 6–7 and 9 exhibited moderate to weak inhibition towards the test pathogenic microorganism. Compound 4 showed mild cytotoxic activity against human osteosarcoma cells U2OS with IC50 of 28.3 μM. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Alternaria species have been identified as a prolific fungal source of new pharmacologically active metabolites such as steroids, terpenoids, pyrones, quinones, phenolics and nitrogen-containing compounds, which were suitable for specific medicinal or agricultural applications [1]. Dibenzopyranones with polyketide origin, such as alternariols and altenuenes were typical isolates from the genus Alternaria. These dibenzopyranone derivatives were examined to have various biological activities and functions, mainly including cytotoxicity [2,3] and antimicrobial [4,5] properties. During the investigation of bioactive natural products from plant endophytes, our attention was drawn to a strain of Alternaria sp.,
⁎ Corresponding author. Tel.: +86 25 8618 5039. ⁎⁎ Corresponding author. Tel./fax: +86 25 8327 1405. E-mail addresses: [email protected] (M.-H. Yang), [email protected] (L.-Y. Kong).
http://dx.doi.org/10.1016/j.fitote.2014.09.015 0367-326X/© 2014 Elsevier B.V. All rights reserved.
whose crude extract displayed potent inhibitory abilities against Staphylococcus aureus and Bacillus subtilis with inhibition zones of 19.5 and 25.3 mm respectively in the preliminary agar diffusion screening (37.7 mm of the positive control penicillin). Therefore, a systematic chemical study was performed and resulted in the isolation of altenuene-2-acetoxy ester (1), altenuene-3-acetoxy ester (2) and a new isocoumarin (+)-(10R)-7-hydroxy-3-(2-hydroxy-propyl)-5,6-dimethylisochromen-1-one (3), along with six known dibenzopyranone derivatives, alternariol 9-methyl ether (4) [5], alternariol (5) [5], phialophoriol (6) [6], altenuene (7) [7], 5′-epialtenuene (8) [8] and alterlactone (9) [2]. Chiral analysis of 1 and 2 revealed the presence of racemic mixture, and subsequent racemic resolution resolved into two pairs of enantiomers (+)-1 [(+)(2S, 3S, 4aS)-altenuene-2-acetoxy ester], (−)-1 [(−)-(2R, 3R, 4aR)-altenuene-2-acetoxy ester], (+)-2 [(+)-(2S, 3S, 4aS)altenuene-3-acetoxy ester] and (−)-2 [(−)-(2R, 3R, 4aR)altenuene-3-acetoxy ester]. Details of the isolation, structure elucidation and bioactive evaluation of these compounds are discussed below.
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2. Experimental 2.1. General Optical rotations were determined on a JASCO P-1020 polarimeter at room temperature. CD spectra were performed on a JASCO J-810 spectrometer. UV spectra were recorded on a Shimadzu UV-2501 spectrophotometer. IR spectra were measured by a Bruker Tensor 27 spectrometer. 1D and 2D NMR were carried out on a Bruker Avance III NMR (1H: 500 MHz, 13C: 125 MHz) instrument at 300 K, with TMS as internal standard. ESIMS was obtained on an Agilent 1100 Series LC/MSD ion-trap mass spectrometer and HRESIMS was recorded on an Agilent 6520B UPLC-Q-TOF mass spectrometer, respectively. Chiral HPLC was done with a Daicel Chiralpak AD-H column (250 × 4.6 mm, 5 μm). Preparative HPLC was performed by a Shimadzu LC-10A equipped with a Shim-pack RP-C18 column (20 × 200 mm) and a Shimadzu SPD-20A detector. Column chromatography (CC) was performed with silica gel and Sephadex LH20. All the solvents used were of analytical grade.
afford compounds 7 (1.0 mg) and 8 (1.1 mg) with MeOH–H2O (65:35, 10 ml/min) as mobile phase. Compound 3 (2.5 mg) was obtained from fraction D4 (0.2 g) by preparative HPLC using methanol in water (60:40, 10 ml/min), and compound 9 (2.0 mg) was purified from fraction F (0.2 g) eluting with MeOH–H2O (55:45, 10 ml/min). Compounds (+)-1 (3.7 mg) and (−)-1 (0.9 mg) were separated from 1 by a chiral column using n-hexane–isopropanol (90:10, 0.80 ml/min), and compounds (+)-2 (1.5 mg) and (−)-2 (1.2 mg) were isolated from 2 (n-hexane/isopropanol, 80:20, 0.75 ml/min) by the similar procedure of 1. Compound 1: white solid; UV (MeOH) λmax (logε): 193 (3.86), 200 (3.91), 241 (4.27), 278 (3.78), 319 (3.53) nm; IR (KBr) νmax: 3444, 2922, 2349, 1627, 1384, 1263, 1163, 1117 cm−1; ESIMS positive m/z: 335 [M + H]+; HRESIMS m/z: 335.1124 [M + H]+ (calcd for C17H19O7, 335.1125); and 1H and 13 C NMR data (CDCl3), see Table 1. (+)-1 (−)-1
2.2. Fungal material The title strain was isolated from the branches of Camellia sinensis, collected from the suburb of Nanjing, Jiangsu Province, People's Republic of China, in October 2012. The culture was grown on potato dextrose agar (PDA) and distinguished morphologically as Alternaria sp., which was further reinforced by 18S rDNA sequence with a 100% identity to Alternaria alternata, conducted by the fungal identification service (Shanghai Majorbio Bio-pharm Technology). The fungal strain was cultivated on PDA medium at 28 °C until sufficient growth was observed (about 5 days). Then plugs of agar supporting mycelia growth were cut into small pieces and transferred aseptically into a 1000 ml Erlenmeyer flask containing 300 ml potato dextrose broth, and incubated on a rotary shaker at 180 rpm for 7 days to prepare the seed culture. Fermentation was carried out in ten 500 ml Erlenmeyer flasks, each containing 80 g of rice and soaked with 120 ml distilled water overnight before autoclaving. Each flask was inoculated with 10 ml seed liquid, and cultivated under stationary conditions at 28 °C for 30 days. 2.3. Extraction and isolation The fermented substrate was saturated with methanol and filtrated. After removal of the solvent under reduced pressure, the crude extract (8.6 g) was subjected to a silica gel column, eluted with a gradient of petroleum ether and acetone mixture from 20:1 to 1:1 (v/v) to generate six fractions (A–F). Fraction C (1.6 g) gave the main compounds 4 (19.0 mg) and 5 (9.5 mg) after Sephadex LH-20 CC (CH2Cl2–MeOH, 1:1). Fraction E (1.3 g) was chromatographed over silica gel CC (CH2Cl2– Acetone, 25:1, 15:1, 5:1, v/v) and further purified by preparative HPLC using MeOH–H2O (60:40, 10 ml/min) to yield 1 (5.7 mg) and 2 (3.5 mg). Fraction D (2.0 g) was applied onto a silica gel CC, and eluted with CH2Cl2–MeOH (20:1, 10:1, 5:1, v/v) to acquire five major subfractions (D1–D5). Subfraction D2 (0.7 g) gave compound 6 (2.7 mg) after CC on Sephadex LH-20 (MeOH). Subfraction D3 (0.9 g) displayed in the same way as subfraction D2, and further purified by preparative HPLC to
−4 , MeOH) [α]25 D + 13.3 (c 0.17, MeOH); CD (2.0 × 10 λmax nm (Δε) 241 (−7.43), 280 (+4.84). −4 , MeOH) [α]25 D − 13.3 (c 0.11, MeOH); CD (1.0 × 10 λmax nm (Δε) 241 (+3.67), 280 (−2.49).
Compound 2: white solid; UV (MeOH) λmax (logε): 194 (3.81), 200 (3.86), 240 (4.19), 277 (3.72), 317 (3.46) nm; IR (KBr) νmax: 3450, 2923, 2852, 1642, 1384, 1163 cm−1; ESIMS positive m/z: 335 [M + H]+; HRESIMS m/z: 335.1127 [M + H]+ (calcd for C17H19O7, 335.1125); and 1H and 13C NMR data (CDCl3) see Table 1. (+)-2 (−)-2
−4 , MeOH) [α]25 D + 11.6 (c 0.11, MeOH); CD (2.0 × 10 λmax nm (Δε) 241 (−2.34), 280 (+1.45). −4 , MeOH) [α]25 D − 11.6 (c 0.12, MeOH); CD (2.0 × 10 λmax nm (Δε) 241 (+2.35), 280 (−1.46).
Compound 3: white solid; [α]25 D + 35.3 (c 0.09, MeOH); UV (MeOH) λmax (log ε): 213 (3.88), 244 (3.65), 252 (3.61), 292 (3.51) nm; CD (2 × 10−4, MeOH) λmax nm (Δε) 365 (+3.67), 426 (−2.92); IR (KBr) νmax: 3448, 2927, 1645, 1550, 1463, 1384, 1270, 1115 cm−1; ESIMS positive m/z: 249 [M + H]+; HRESIMS m/z: 249.1120 [M + H]+ (C14H17O4, calcd for 249.1121); and 1H and 13C NMR data (Acetone-d6) see Table 1. 2.4. Absolute configuration of the secondary alcohol in 3 A solid [Rh2(OCOCF3)4] (2.0 mg) was added into compound 3 (0.7 mg) dissolving in CH2Cl2. After mixing, the first CD spectrum was recorded at once, and the time evolution was monitored until stationary (15 min). The inherent CD spectrum was subtracted. The metal complexes gave an induced CD spectrum, in which the sign of the E band around 350 nm was correlated to the absolute configuration of 3 [9,10]. 2.5. Biological assays The antibacterial activities against Gram-positive S. aureus ATCC 25923 (S. aureus), B. subtilis ATCC 6633 (B. subtilis) and Gram-negative Escherichia coli ATCC 25922 (E. coli) were initially performed by disk diffusion assay [11]. Each paper disk (6 mm diameter) permeating with 10 μl of test sample
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Table 1 1 H NMR (500 MHz) and 13C NMR (125 MHz) data for 1–3. No.
1a δH (multi, J in Hz)
δC
δH (multi, J in Hz)
δC
1 2 3 4
6.04 (d, 2.7) 5.23 (dd, 2.7, 5.7) 4.10 (m) He:2.54 (dd, 4.1, 14.6) Ha:2.09 (dd, 8.6, 14.6)
124.7 74.0 67.6 39.9
6.19 (d, 3.3) 4.29 (dd, 3.0, 5.2) 5.05 (m) He:2.60 (dd, 4.1,14.6) Ha:2.13 (dd, 8.7,14.7)
127.7 69.3 72.6 37.1
4a 6 7 7a 8 9 10 10a 10b 4a-CH3 9-OCH3 7-OH 2/3-OAc
6.45 (d, 2.2) 6.52 (d, 2.2)
1.56 (s) 3.85 (s) 11.26 2.13 (s)
a b
2a
80.7 168.8 164.4 103.3 100.8 166.4 101.5 138.5 136.4 28.1 56.0 171.2 21.3
6.46 (d, 2.3) 6.54 (d, 2.3)
1.54 (s) 3.86 (s) 11.31 (s) 2.11 (s)
No.
80.3 168.5 164.6 100.8 101.4 166.4 103.3 138.4 135.0 27.9 55.9
3b δH (multi, J in Hz)
1 3 4 4a 5 6 7 8 8a 9 10 11 7-OH 10-OH 5-CH3 6-CH3
5.95 (s)
6.78 (s) 2.62 (dd, 7.5, 14.3) 2.65 (dd, 5.4, 14.3) 4.20 (m) 1.23 (d, 6.2)
2.78 (s) 2.18 (s)
δC 180.4 165.3 113.6 116.8 141.2 123.8 160.6 101.5 159.1 44.8 66.3 24.4
17.8 12.1
171.0 21.3
Recorded in CDCl3. Recorded in Acetone-d6.
(100 μg/ml methanol) was dried and placed on Mueller Hinton agar plate containing bacterial inoculum. Following incubation (37 °C, 24 h), diameter (in mm) of the growth inhibition zone was recorded to evaluate antibacterial potential. For further determination of the minimal inhibition concentration (MIC), broth microdilution [12] in sterile 96-well plates was performed. The test bacteria was inoculated on nutrient agar for 18–24 h, then a colony approximately 1 mm in diameter was collected with a sterile loop and dissolved into Mueller Hinton broth. The bacterial cell suspension was adjusted to 1.0 × 104– 1.0 × 106 cells/ml before use. Each test compound was firstly dissolved in DMSO, then diluted by broth to different concentrations (0–50.0 μg/ml, DMSO b 1%). Test well was introduced into 100 μl bacterial suspension and 100 μl individual compounds. Blank well was incubated with only medium under the same condition. Optical density (OD) measurement was recorded at 578 nm after incubation for 24 h at 37 °C. MIC was determined by identifying the drug concentration at which 80% of bacterial growth was inhibited. All experiments were performed in three replicates and with penicillin (b 2.0 μg/ml) as the positive control. Antifungal activities against Candida albicans ATCC 24433 (C. albicans) and Trichophyton rubrum ATCC 28189 (T. rubrum) were conducted in triplicate by the microdilution method [13,14]. The preparation of Candida inocula was done by picking 24-hour-old colonies (about 1 mm in diameter) from the PDA plate and aseptically transfered into 3 ml sterile 0.85% saline solution. The solution was adjusted to 5.0 × 102–2.5 × 103 cells/ml with RPMI-1640 medium by a hemocytometer to prepare for the cell suspension. Similarly, the culture of T. rubrum was grown on PDA plate for 5 days and diluted to a final spore density of 0.4 × 104–5.0 × 104 cells/ml after discarding mycelium. Each test compound was diluted to different concentrations (0–50.0 μg/ml, DMSO b 1%) using the method described above for bacteria. Each test well was introduced into 100 μl cell suspension and 100 μl respective compounds. Blank well with only medium was used to ensure medium sterility.
Fluconazole was the positive control for yeast (b1.0 μg/ml) assay and filamentous fungi (b4.0 μg/ml) test. The 96-well plates for C. albicans and T. rubrum were incubated at 35 °C for 24 h and 72 h, respectively. OD measurement was made at 595 nm and MIC was determined as described above for bacteria. Moreover, all the isolated compounds were further tested in vitro for their cytotoxicities against two human tumor cell lines U2OS and HepG2 by using the MTT method [15] with cisplatinum as positive control. Absorbance was recorded at 570 nm by a microplate reader to detect cell viability. All experiments were performed in three replicates. 3. Results and discussion Compound 1, white solid, had a molecular formula of C17H18O7 as HRESIMS exhibited a quasi-molecular ion at m/z 335.1124 [M + H]+ (calcd for C17H19O7, 335.1125), giving nine degrees of unsaturation. The 1H and 13C NMR together with HSQC spectra revealed one methyl [δH/δC, 1.56 (3H, s)/28.1], one methylene [δH/δC, 2.54 (1H, dd, 4.1, 14.6), 2.09 (1H, dd, 8.6, 14.6)/39.9], two oxygenated methines [δH/δC, 4.10 (1H, m)/ 67.6; 5.23 (1H, dd, 2.7, 5.7)/74.0], one protonated olefinic group [δH/δC, 6.04 (1H, d, 2.7)/124.7], one ester carbonyl (δC 168.8), one sp3 quaternary carbon (δC 80.7), one sp2 quaternary carbon (δC 136.4) and a tetrasubstituted benzene ring with a pair of meta-coupled aromatic protons [δH/δC 6.45 (1H, d, 2.2), 6.52 (1H, d, 2.2)/164.4, 103.3, 101.5, 138.5, 166.4, 100.8]. These data implied the altenuene core of 1 [7], which was mainly confirmed by HMBC correlations from H-8 (δH 6.45) to C-9 (δC 166.4), C-7 (δC 164.4) and C-10 (δC 101.5), from H-1 (δH 6.04) to C-2 (δC 74.0), C-10a (δC 138.5) and C-10b (δC 136.4), from Ha-4 (δH 2.09) to C-2, C-3 (δC 67.6) and C-4a (δC 80.7) and from methyl protons (δH 1.56) to C-4a, C-10b and C-4 (δC 39.9). The remaining 8 protons and 3 carbons attributed to a phenolic hydroxyl group [δH 11.26 (1H, s)], a methoxyl group [δH/δC, 3.85 (3H, s)/56.0], an O-acetyl unit [δH/δC, 2.13
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(3H, s)/21.3, 171.2] and an undetected hydroxyl group. The relatively downfield chemical shift of C-3 (δC 67.6) and the HMBC correlations from H-3 to C-2 and C-4 placed a hydroxyl at C-3. HMBC signals from hydroxyl proton at δH 11.26 to C-7 and C-8, from methoxyl protons at δH 3.85 to C-9 and C-10 confirmed the attachment of the phenolic hydroxyl and the methoxyl group at C-7 and C-9, respectively. In addition, the acetoxyl group was located at C-2 based on the HMBC correlation between H-2 and its ester carbonyl carbon. Thus, the planar structure of 1 was determined as in Fig. 1. The relative stereochemistry of 1 was mainly established by J values and ROESY correlations of relevant protons. An apparent axial– axial relationship existed between Ha-4 and H-3 as judged from the large coupling constant (J = 8.6 Hz) between them. ROESY correlations between Ha-4, H-2 (δH 5.23) and the methyl protons at δH 1.56 (H3–4a) lead to the assignment of these protons on the same face of the molecule and a pseudoequatorial orientation of the C-2 O-acetyl group. Remarkably, the optical rotation of compound 1 was approximate to zero, suggesting the mixture of enantiomers. A follow-up chiral separation yielded a pair of enantiomers (+)-1 and (−)-1 (Fig. 3), which exhibited similar signal intensity but opposite Cotton effects in their CD spectra (Fig. 4), further confirming their enantiomeric relationship. To determine absolute configuration, their experimental CD spectra were compared with those predicted from quantum mechanical time-dependent density functional theory (TDDFT) calculations. As a result, the calculated ECD spectrum for the (2S, 3S, 4aS) stereoisomer matched well with the experimental CD data of (+)-1 (Fig. 4.b), which designated the configuration of (+)-1 as (2S, 3S, 4aS)-altenuene-2-acetoxy ester. As a consequence, (−)-1, with optical value of the same magnitude to (+)-1, was assigned as (2R, 3R, 4aR)-altenuene-2-acetoxy ester. Compound 2, an isomer of 1, also had a molecular formula of C17H18O7 on the basis of a quasi-molecular ion at m/z 335.1127 [M + H]+ (calcd for C17H19O7, 335.1125). The UV, IR and NMR spectra of 2 resembled those of 1, demonstrating the
Fig. 2. Selected HMBC and ROESY correlations of 1 and 3.
same altenuene core of 2. Compared with 1, the obvious differences were the chemical shifts of H-2 and H-3, which were upfield-shifted from δH 5.23 to δH 4.29 and downfield-shifted from δH 4.10 to δH 5.05, respectively. This, taken together with HMBC correlation between the ester carbonyl carbon (δC 171.0) of the O-acetyl group and H-3 (δH 5.05) readily located the O-acetyl group at C-3 rather than at C-2 in 1. The relative configuration of 2 was determined as the same with 1, for that the analogous coupling constant (J = 8.7 Hz) between Ha-4 and H-3 as well as identical ROESY correlations of key protons were observed. Compound 2 was also optically inactive and resolved into a pair of enantiomers (+)-2 and (−)-2 by a chiral column (Fig. 3). The CD spectrum of (+)-2 and (−)-2 was identical with (+)-1 and (−)-1 respectively (Fig. 4), revealing the same absolute configurations of the two pairs of enantiomers. Based on the observation above, structures of (+)-2 and (−)-2 were determined to be (+)-(2S, 3S, 4aS)-altenuene3-acetoxy ester and (−)-(2R, 3R, 4aR)-altenuene-3-acetoxy ester, respectively. Compound 3 was obtained as a white powder. HRESIMS ion at m/z 249.1120 [M + H]+ revealed its molecular formula to be C14H16O4 (calculated for C14H17O4, 249.1121), indicating seven double-bond equivalents. The IR spectrum of 3 showed a phenolic hydroxyl (3448 cm−1) and a characteristic absorption band of benzene ring (1645, 1550, 1463 cm−1). The 1H, 13C
Fig. 1. Structures of compounds 1–9.
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157
Fig. 3. Chiral HPLC analysis of 1 (upper) and 2 (down).
NMR and HSQC spectrum displayed 14 carbons and 14 protons (Table 1). Among them, 11 carbons and their corresponding protons revealed two isolated methyls [δH/δC, 2.18 (3H, s)/12.1; 2.78 (3H, s)/17.8], one protonated olefinic group [δH/δC, 5.95 (1H, s)/113.6], one sp2 quaternary carbon (δC 165.3), one easter carbonyl (δC 180.4) and a pentasubstituted benzene ring (δC 101.5, 116.8, 123.8, 141.2, 159.1, 160.6) with an aromatic singlet proton [δH 6.78 (1H, s)]. Such data indicated that compound 3 was an isocoumarin [16]. Analysis of HMBC correlations (Fig. 2) confirmed this deduction and further positioned two methyls and a hydroxyl to C-5, C-6 and C-7, respectively. The remaining 3 carbons and 6 protons attributed to a methylene [δH/δC, 2.62 (1H, dd, 7.5, 14.3), 2.65(1H, dd, 5.4, 14.3)/44.8], an oxymethine [δH/δC, 4.20 (1H, m)/66.3] and a methyl [δH/δC, 1.23 (3H, d, 6.2)/24.4], which indicated a 2hydroxyl-propanyl fragment. Moreover, HMBC correlations from the methylene to C-3 and C-4 attached the 2-hydroxylpropanyl chain to C-3. The absolute configuration at C-10, bearing a secondary-alcohol group, was determined as R by the in-situ [Rh2 (OCOCF3)4] method according to the notable negative Cotton effect around 350 nm in the induced CD spectrum (Fig. S10, Supporting information). Consequently, the structure of 3 was determined as depicted. Initial agar diffusion assay revealed that the isolates had positive bacteriostatic effect against Gram-positive bacteria (S. aureus and B. subtilis) with inhibition zones of 10–25 mm but
Fig. 4. a. CD spectra of (+)-1, (−)-1, (+)-2 and (−)-2 (in methanol). b. Calculated and experimental CD spectra of (+)-1.
insensitive to Gram-negative bacteria (E. coli). Further microdilution assessment was carried out with the results listed in Table 2. In agreement with literature reports [4,5], altenuene analogs 1–3, 6 and 7 as well as alternariol derivatives 5 and 9
Table 2 Minimum inhibitory concentration (MIC80, μg/ml) of 1–9a. Samples
Sab
(+)-1 (−)-1 (+)-2 (−)-2 3 4 5 6 7 8 9 Fluconazole Penicillin
17.1 ± 15.4 ± 46.8 ± 45.0 ± N50.0 N50.0 N50.0 N50.0 38.9 ± N50.0 N50.0
a
1.2 1.1 2.0 1.7
1.5
1.2 ± 0.1
Bsb
Cab
Trb
N50.0 N50.0 N50.0 N50.0 19.7 ± 1.0 N50.0 8.6 ± 0.7 16.7 ± 1.2 N50.0 N50.0 N50.0
19.5 ± 1.5 48.8 ± 1.2 24.0 ± 1.0 N50.0 N50.0 N50.0 N50.0 N50.0 13.7 ± 0.7 N50.0 17.1 ± 1.8 0.6 ± 0.1
N50.0 N50.0 N50.0 N50.0 32.0 ± 2.1 N50.0 N50.0 N50.0 N50.0 N50.0 35.5 ± 2.2 1.0 ± 0.2
0.9 ± 0.1
A compound was regarded as inactive when MIC80 N 50.0 μg/ml. b Sa, Bs, Ca, Tr represented Staphylococcus aureus ATCC 25923, Bacillus subtilis ATCC 6633, Candida albicans ATCC 24433 and Trichophyton rubrum ATCC 28189.
158
Y. Wang et al. / Fitoterapia 99 (2014) 153–158
showed moderate to weak antimicrobial activities. However, it is difficult to clarify a common structural characteristic which might explain their abilities as antibacterial or antifungal agents. But interestingly, enantiomeric altenuene-2-acetoxy ester [(+)-1 and (−)-1] displayed similar moderate inhibitory effects against S. aureus with MIC values of 17.1 and 15.4 μg/ml, whereas the analogs altenuene-3-acetoxy ester [(+)-2 and (−)-2], altenuene (7) and 5′-epialtenuene (8) showed no significant effect. These results indicated that the enantiomeric difference might have a negligible effect against S. aureus but acetylation of 2-OH could improve the ability. Moreover, (+)-1 and (+)-2 exhibited efficient growth inhibition of C. albicans while (−)-1 and (−)-2 were less active, suggesting the different antifungal abilities between enantiomers. Additionally, alternariol (5) was the most active against B. subtilis with MIC value of 8.6 μg/ml while alternariol 9-methyl ether (4) was not sensitive at the concentration of 50.0 μg/ml, indicating methylation of 9-OH may be detrimental for the antibacterial activity. Alternariol derivatives have been detected to have pronounced cytotoxicity against mouse lymphoma cell [2]. But in our cytotoxic test, all the isolates were inactive at 50.0 μM against two human tumor cell lines U2OS and HepG2 except that alternariol 9-methyl ether (4) showed temperate inhibition of human osteosarcoma cell U2OS with IC50 28.3 μM. Acknowledgments This research work was funded by the National Natural Science Foundation of China (81073009), the Program for Changjiang Scholars and Innovative Research Team in University (IRT1193), the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD), the youth fund project of basic research program of Jiangsu Province (Natural Science Foundation, BK20130651), and the Fundamental Research Funds for the Central Universities (JKQZ2013016). Appendix A. Supplementary data HRESIMS, 1D NMR spectrum of compounds 1–3 and CD spectra of compound 3 are available in Supporting information. Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.fitote.2014.09.015.
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