Original Papers
925
Authors
Wei Huang, Wen-Jing Zhang, Yi-Qing Cheng, Rong Jiang, Wei Wei, Chao-Jun Chen, Gang Wang, Rui-Hua Jiao, Ren-Xiang Tan, Hui-Ming Ge
Affiliation
Institute of Functional Biomolecules, State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing, China
Key words " Cryptocarya concinna l " Lauraceae l " flavonoid l " cytotoxic activity l " antimicrobial activity l
Abstract
received revised accepted
Nov. 1, 2013 May 19, 2014 May 20, 2014
Bibliography DOI http://dx.doi.org/ 10.1055/s-0034-1368613 Published online July 16, 2014 Planta Med 2014; 80: 925–930 © Georg Thieme Verlag KG Stuttgart · New York · ISSN 0032‑0943 Correspondence Dr. Hui-Ming Ge Institute of Functional Biomolecules State Key Laboratory of Pharmaceutical Biotechnology Nanjing University 22 Hankou Road Nanjing 210093 China Phone: + 86 25 83 59 32 01 Fax: + 86 25 83 59 32 01 [email protected] Correspondence Dr. Ren-Xiang Tan Institute of Functional Biomolecules State Key Laboratory of Pharmaceutical Biotechnology Nanjing University 22 Hankou Road Nanjing 210093 China Phone: + 86 25 83 59 32 01 Fax: + 86 25 83 59 32 01 [email protected]
!
Five new flavonoids, cryptoconones A–E (1–5), along with six known compounds (6–11), were isolated from the stems of Cryptocarya concinna. The structures of these compounds were elucidated on the basis of spectroscopic data interpretation, and the absolute configurations were determined via circular dichroism spectra and X‑ray crystal analysis. The cytotoxic and antimicrobial activities of these compounds were also eval-
Introduction !
The genus Cryptocarya (Lauraceae) consists of about 250 species distributed mainly in the tropical and subtropical regions of the world. Earlier studies on this genus reported flavonoids [1–8], pyrones [6, 9–12], and alkaloids [13–17] as the main secondary metabolite constituents with varied biological activities. In our previous study, several flavonoids with a variety of bioactivities from C. maclurei and C. chingii were found [1, 2]. As a part of our continuing search for bioactive constituents from tropical plants [1, 2, 18–20], a 95 % EtOH extract of the stems of Cryptocarya concinna Hance with no previous literature reports on its chemistry has been investigated with five new (1–5) and six known flavonoids (6–11) obtained. Reported herein are the isolation, structure elucidation, and biological activities of these compounds.
Results and Discussion !
Repeated column chromatography of the EtOH extract (95%) of the stems afforded five new flavonoids, 1–5, along with six known flavonoids, cryptochinone A (6) [4], cryptogione C (7) [1], cryptocaryanone A (8) [8], cryptocaryone (9) [8],
uated. Compounds 9 and 10 exhibited moderate cytotoxic activities against HCT116, HT-29, SW480, and MDA‑MB‑231 cell lines with IC50 values ranging from 6.25 to 9.35 µM. Compounds 8 and 11 exhibited antimicrobial activity against Fusarium moniliforme and Botrytis cinerea, respectively, with the same minimum inhibitory concentration of 5 µg/mL. Supporting information available online at http://www.thieme-connect.de/products
kurzichalcolactone A (10) [21], and kurzichalcolactone B (11) [7]. The known compounds were determined by comparing their physical and spectroscopic properties to literature values. Compound 1 was obtained as a yellowish oil. The " Table 1) for 1 were almost the NMR signals (l same as those of cryptogione E [1], except for the absence of a methoxyl group. This was further supported by HRESIMS, which gave an [M + Na]+ ion peak at m/z 307.0941 (calcd. for C17H16NaO4, 307.0941), being 14 mass units less than that of cryptogione E [1]. The 2R configuration of 1 was evidenced by a negative Cotton effect at 349 nm due to the n → π* transition [1, 22, 23]. Moreover, 1 showed a similar circular dichroism (CD) spectrum compared to that of cryptogione E [1], indicating that the absolute configuration of 1 is 2R, 5S. Thus, the structure of 1 was elucidated as " Fig. 1 and further confirmed by 1Hshown in l 1 H COSY, DEPT, HSQC, HMBC, and NOESY " Fig. 2) experiments. (l Compound 2 appeared as a yellowish oil with a molecular formula of C17H16O4 deduced from the observed [M + Na]+ peak at m/z 307.0934 in the HRESIMS spectrum. The 1H and 13C NMR spectra " Table 1) of 2 were closely similar to those of 1, (l indicating it as an epimer of 1, which was further " Fig. 2). The confirmed by 2D NMR correlations (l 2S configuration was assigned by a positive Cot-
Huang W et al. Cytotoxic and Antimicrobial …
Planta Med 2014; 80: 925–930
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Cytotoxic and Antimicrobial Flavonoids from Cryptocarya concinna
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1a δH (J in Hz)
2 3 4 5 6 7 8 9 10 11 12 1′ 2′,6′ 3′,5′ 4′ a
5.43 dd (14.0, 3.5) 2.90 dd (16.5, 14.0) 2.71 dd (16.5, 3.5) 3.49 m 2.49 m 6.40 m 6.06 d (10.0)
2.37 m
7.41 m 7.41 m 7.41 m
2a δC 80.3 42.5 190.7 25.5 28.1 138.9 122.7 166.0 111.0 35.3 176.6 138.1 126.2 128.8 128.8
δH (J in Hz) 5.42 dd (14.5, 3.0) 2.91 dd (16.5, 14.5) 2.68 dd (16.5, 3.0) 3.50 m 2.49 m 2.58 m 6.43 ddd (10.0, 6.0, 2.5) 6.01 dd (10.0, 2.5)
2.47 m
7.42 m 7.42 m 7.42 m
δC
Table 1 1 and 2.
1
H and 13C NMR data for
80.4 42.9 190.8 25.4 28.8 139.9 122.4 165.7 111.3 37.7 176.2 138.2 126.2 128.8 128.8
Recorded in CDCl3 at 500 MHz for 1H NMR, 125 MHz for 13C NMR, δ in ppm, J in Hz
Fig. 1
ton effect at 325 nm due to the n → π* transition [8, 22, 23]. Therefore the absolute configuration of 2 was confirmed as 2S, 5S when compared to 1. Compound 3 was obtained as a colorless powder. HRESIMS data was used to determine the molecular formula as C17H14O5, the same as that of co-occurring cryptogione C (7) [1]. From its spec" Table 2), compound 3 was found to be similar to troscopic data (l 7. The major difference was that the hydroxyl group coupled at C6 in 3 instead of C-8 in 7, which was supported by the HMBC correlations between H-11 (δH 4.22, 4.17) and C-6 (δC 150.9), C-10 (δC 120.9); H-7 (δH 7.18) and C-9 (δC 157.1), C-5 (δC 122.9); H-8 " Fig. 3). The abso(δH 6.91) and C-6 (δC 150.9), C-9 (δC 157.1) (l lute configuration of 3 was also confirmed as 2S by CD, as in the case of 2. Thus, the structure of 3 was identified as (S)-2-(6-hydroxy-4-oxo-2-phenylchroman-5-yl) acetic acid.
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Planta Med 2014; 80: 925–930
Structures of 1–11.
Compound 4 was isolated as colorless needles and gave a molecular formula of C17H16O5 by HRESIMS, the same as that of co-oc" Table 2) of 4 curring cryptochinone A (6) [4]. The NMR spectra (l were quite similar to those of 6, except that the 7β-configuration of 6 was replaced by 7α-configuration of 4. The relative configuration of 4 was established as rel-2S,5R,6R,7R by X‑ray crystal " Fig. 4). The 2S configuration was determined by a posanalysis (l " Fig. 5) itive Cotton effect at 340 nm due to the n → π* transition (l [4, 22, 23]. Compound 5, a colorless oil, exhibited the same molecular formula as compound 4 (C17H16O5) from the HRESIMS data. Com" Table 2) of 4 and 5 indiparison of the 1H and 13C NMR data (l cated that they are different only in relative stereochemistry. " Fig. 6) and 1H NMR coupling constants The NOESY interactions (l were used to determine the relative configuration of ring A in 5, as in the case of cryptogiones G and H [2]. H-6 was deduced to be
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Position
of 8.0 Hz. The 2R configuration was identified by a negative Cot" Fig. 5), as in the case of 1. The chirality at ton effect at 316 nm (l C-5 dominated the CD spectra at 200–240 nm by comparison of " Fig. 5) of 4, 6 [4], and cryptogione D [1], which the CD spectra (l presented similar cyclohexene units. Thus, the absolute configuration at C-5 of 5 was determined as 5S by the reversed CD spectrum of 5 at 200–240 nm compared to that of 4. Therefore, com" Fig. 1. pound 5 was established as shown in l All the isolates were tested for cytotoxic activity against HCT116, HT-29, SW480, and MDA‑MB‑231 cell lines. Compounds 9 and 10 " Table 3), while others exhibited moderate cytotoxic activity (l were inactive (IC50 > 50 µM). These compounds were also evaluated for antibacterial (Xanthomonas oryzae pv. oryzae, Xanthomonas oryzae pv. oryzicola, Acidovorax avenae subsp. citrulli, and Erwinia amylovora) and antifungal (Phytophthora capsici, Fusarium moniliforme, Alternaria solani, and Botrytis cinerea) activities. Only compounds 8, 10, and 11 showed significant antimi" Table 4). The purity of the tested compounds crobial activity (l was determined to be over 95 % by using the HPLC‑DAD method. Fig. 2 Key HMBC (→), COSY (—), and NOESY (↔) correlations of 1 and 2. (Color figure available online only.)
Materials and Methods !
General experimental procedures axial according to the NOE correlations between H-6 and H-8α. Therefore, H-7 and H-5 were determined to be equatorial and axial, respectively, because of the 3J6,7 value of 3.2 Hz and 3J5,6 value
Table 2
1
H and 13C NMR data for 3–5.
Position
3a δH (J in Hz)
2 3 4 5 6 7 8 9 10 11 12 1′ 2′,6′ 3′,5′ 4′ a
Optical rotations were obtained on a Rudolph Autopol III automatic polarimeter. UV spectra were recorded on a Hitachi U3000 spectrophotometer. CD spectra were obtained on a Jasco J810 spectrometer, and the IR spectra (KBr) were performed on a
5.49 dd (13.2, 2.8) 3.06 dd (16.4, 13.2) 2.78 dd (16.4, 2.8)
7.18 d (8.8) 6.91 d (8.8)
4.22 d (16.8) 4.17 d (16.8)
7.58 m 7.44 m 7.38 m
4b δC 79.9 46.8 194.3 122.9 150.9 123.9 118.3 157.1 120.9 32.4 172.5 140.8 127.3 129.5 129.3
5b
δH (J in Hz)
δC
5.38 dd (14.4, 3.2) 2.69 dd (16.8, 3.2) 2.93 dd (16.8, 14.4) 3.58 m 4.62 t (6.4) 4.37 m 2.53 dd (18.4, 4.8) 2.78 dd (18.4, 2.2)
3.06 dd (18.0, 8.8) 2.47 dd (18.0, 4.4)
7.41 m 7.41 m 7.41 m
80.8 42.8 190.8 31.4 79.4 65.4 32.4 168.3 110.8 36.1 176.2 137.7 126.4 129.1 129.3
δH (J in Hz) 5.42 dd (14.0, 3.2) 2.71 dd (16.4, 3.2) 2.97 dd (16.4, 14.0) 3.60 dt (9.2, 8.0) 4.71 dd (8.0, 3.2) 4.31 br s 2.64 m (β) 2.68 m (α)
3.02 dd (18.0, 9.2) 2.48 dd (18.0, 8.0)
7.40 m 7.41 m 7.41 m
δC 81.0 42.8 190.8 32.1 78.5 66.4 33.5 167.4 110.8 34.9 177.1 137.7 126.1 128.9 129.1
Recorded in acetone-d6 at 400 MHz for 1H NMR, 100 MHz for 13C NMR, δ in ppm, J in Hz; b Recorded in CDCl3 at 400 MHz for 1H NMR, 100 MHz for 13C NMR, δ in ppm, J in Hz
Fig. 3 Key HMBC (→) and COSY (—) correlations of 3–5. (Color figure available online only.)
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Original Papers
Original Papers
with a voucher specimen (IFB-20101202) conserved at the Institute of Functional Biomolecules, Nanjing University.
Extraction and isolation
Fig. 4
X‑ray crystal analysis of 4.
Nexus 870 FT‑IR spectrometer. Using solvent signals as internal standard, NMR data were acquired on a Bruker DRX-500 or DRX-400 NMR spectrometer. HRESIMS spectra were recorded on an Agilent 6210 TOF LC‑MS spectrometer. Silica gel (200–300 mesh) for column chromatography was produced by Qingdao Marine Chemical Factory. Sephadex LH-20 was purchased from Pharmacia Biotech. ODS‑A gel (AA12S50) was purchased from YMC Co., Ltd. Semipreparative HPLC analysis was performed on an Agilent 1260 HPLC system equipped with a Hypersil ODS column (250 mm × 10 mm, 5 µm, Thermo Fisher Scientific).
Plant material C. concinna was collected in December 2010 from the Botanical Garden at Jianfeng Town, Ledong County, Hainan Island (China). Identification was provided by Prof. X. J. Tian (Nanjing University)
Air-dried and powdered stem woods of C. concinna (25 kg) were extracted with EtOH‑H2O (v/v, 95 : 5, 4 × 50 L) at room temperature and concentrated in vacuo to give a crude extract (716 g), which was subjected to silica gel (3.5 kg, 200–300 mesh) column chromatography (12 × 120 cm) (CHCl3/MeOH, 1 : 0 → 0 : 1, each 10 L) to afford fractions A–T. Fraction D (CHCl3/MeOH, 100 : 4) (24 g) was refined over a reversed-phase silica gel column (5 × 50 cm), eluting with a MeOH/H2O gradient (20% → 100 % MeOH, each 2 L), to afford 16 fractions (D1–D16). Fraction D3 (MeOH/H2O, 30 : 70) (721 mg) was further separated over Sephadex LH-20 column chromatography (2.5 × 60 cm), eluting with MeOH (2 L), to give four subfractions (D3a-D3d). Subfraction D3c (700–1000 mL, 62 mg) was purified by semipreparative reversedphase HPLC (MeOH/H2O, 36 : 64, 2 ml/min) to obtain 4 (10 mg, tR = 49.0 min), 5 (5 mg, tR = 38.5 min), and 6 (12 mg, tR = 43.8 min). Fraction D4 (MeOH/H2O, 35 : 65) (2020 mg) was purified over Sephadex LH-20 CC (2.5 × 60 cm) (MeOH, 2 L) to give 8 (627 mg). Fraction D5 (MeOH/H2O, 40 : 60) (728 mg) was separated over Sephadex LH-20 CC (2.5 × 60 cm) (MeOH, 2 L) to give four subfractions (D5a-D5d). Subfraction D5b (400–600 mL, 70 mg) was purified by semipreparative reversed-phase HPLC (MeCN/H2O, 47 : 53, 2 ml/min), to obtain 3 (4 mg, tR = 14.5 min) and 7 (6 mg, tR = 19.7 min). Fraction D7 (MeOH/H2O, 50 : 50) (1143 mg) was chromatographed on silica gel (100 g, 200–300 mesh) CC (3 × 60 cm) eluting with petroleum ether/acetone (7 : 1, 10 L) to obtain 10 (4–5 L, 110 mg) and 11 (6–7.5 L, 170 mg). Fraction E (CHCl3/MeOH, 100 : 6) (28 g) was subjected to separation on a reversed-phase silica gel column (5 × 50 cm) to give 16 fractions (E1–E16). Fraction E6 (MeOH/H2O, 45 : 55) (540 mg) was further separated over Sephadex LH-20 CC (2.5 × 60 cm) (MeOH, 2 L) to afford four subfractions (E6a-E6d). Subfraction E6a (0–400 mL, 16 mg) was purified by semipreparative reversed-phase HPLC (MeOH/H2O, 52 : 48, 2 mL/min) to obtain 1 (2 mg, tR = 48.5 min) and 2 (2 mg, tR = 48.6 min). Fraction E7 (MeOH/H2O, 50 : 50) (870 mg) was passed over Sephadex LH-20 CC (2.5 × 60 cm) (MeOH, 2 L) to give three subfractions (E7a-E7c). Subfraction
Fig. 5 CD spectra of 4, 5, 6, and cryptogione D. (Color figure available online only.)
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Planta Med 2014; 80: 925–930
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E7b (300–800 mL, 329 mg) was chromatographed on a silica gel (20 g, 200–300 mesh) column (2.5 × 60 cm), eluting with CH2Cl2/ MeOH (300 : 1, 4 L) to obtain 9 (500–800 mL, 12 mg).
Cryptoconone D (4): Colorless needles; UV (MeOH) λmax (log ε) 269 (4.06) nm; [α]30 D + 100 (c 0.050, MeOH); CD (MeOH) λext (θ) 208 (+ 6.82), 220 (+ 11.79), 241 (+ 2.98), 261 (+ 6.70), 297 (− 3.08), 340 (+ 0.29) nm; IR (KBr) νmax 3398, 2924, 1780, 1658, 1613, 1422, 1172 cm−1; 1H NMR (CDCl3, 400 MHz) and 13C NMR " Table 2; crystallo(CDCl3, 100 MHz) spectroscopic data, see l graphic data, see Table 1S (in Supporting Information); HRESIMS (positive) m/z 301.1073 [M + H]+ (calcd. for C17H17O5, 301.1072). Cryptoconone E (5): Colorless oil; UV (MeOH) λmax (log ε) 271 (3.95) nm; [α]30 D − 93 (c 0.056, MeOH); CD (MeOH) λext (θ) 204 (− 3.25), 221 (− 7.75), 274 (+ 4.23), 316 (− 1.76) nm; IR (KBr) νmax 3424, 2920, 1773, 1662, 1612, 1423, 1176 cm−1; 1H NMR (CDCl3, 400 MHz) and 13C NMR (CDCl3, 100 MHz) spectroscopic data, see " Table 2; HRESIMS (positive) m/z 323.0894 [M + Na]+ (calcd. for l C17H16NaO5, 323.0890).
Cytotoxic activity assay Isolates Cryptoconone A (1): Yellowish oil; UV (MeOH) λmax (log ε) 320 (3.85) nm; [α]30 D − 87 (c 0.057, MeOH); CD (MeOH) λext (θ) 221 (− 20.52), 245 (− 0.01), 259 (− 0.90), 309 (+ 5.90), 349 (− 11.77) nm; IR (KBr) νmax 3434, 2924, 1723, 1651, 1566, 1426, 1382 cm−1; 1 H NMR (CDCl3, 500 MHz) and 13C NMR (CDCl3, 125 MHz) spec" Table 1; HRESIMS (positive) m/z 307.0941 troscopic data, see l + [M + Na] (calcd. for C17H16NaO4, 307.0941). Cryptoconone B (2): Yellowish oil; UV (MeOH) λmax (log ε) 325 (3.85) nm; [α]30 D + 90 (c 0.061, MeOH); CD (MeOH) λext (θ) 226 (− 14.96), 249 (+ 4.79), 273 (+ 1.45), 325 (+ 5.69) nm; IR (KBr) νmax 3427, 2924, 1722, 1636, 1565, 1427 cm−1; 1H NMR (CDCl3, 500 MHz) and 13C NMR (CDCl3, 125 MHz) spectroscopic data, see " Table 1; HRESIMS (positive) m/z 307.0934 [M + Na]+ (calcd. for l C17H16NaO4, 307.0941). Cryptoconone C (3): Colorless powder; UV (MeOH) λmax (log ε) 260 (4.12), 358 (3.83) nm; [α]30 D + 112 (c 0.078, MeOH); CD (MeOH) λext (θ) 215 (+ 5.74), 260 (− 1.93), 280 (+ 0.08), 327 (− 4.75), 369 (+ 3.97) nm; IR (KBr) νmax 3384, 2923, 1664, 1613 cm−1; 1H NMR (acetone-d6, 400 MHz) and 13C NMR (ace" Table 2; HRESIMS tone-d6, 100 MHz) spectroscopic data, see l (positive) m/z 321.0736 [M + Na]+ (calcd. for C17H14NaO5, 321.0733).
Compound 9 10 Doxorubicina a
Antimicrobial activity assay The antimicrobial activities were evaluated against four bacterial strains (X. oryzae pv. oryzae, X. oryzae pv. oryzicola, A. avenae subsp. citrulli, and E. amylovora) and four fungal strains (P. capsici, F. moniliforme, A. solani, and B. cinerea), which were obtained from College of Plant Protection, Nanjing Agricultural University. The antimicrobial assay was performed as described previously [24, 25]. The broth dilution method using 96-well microtiter
IC50 (µM)
Table 3 Cytotoxicity of 9 and 10.
HCT116
HT-29
SW480
MDA‑MB‑231
9.35 ± 0.28 6.49 ± 0.19 1.48 ± 0.11
7.23 ± 0.44 6.25 ± 0.57 1.87 ± 0.26
6.52 ± 0.30 7.44 ± 0.27 2.34 ± 0.38
9.03 ± 0.34 8.46 ± 0.39 2.59 ± 0.23
Positive control
Target microorganism
Antimicrobial activity (MIC in µg/mL) 8
Xanthomonas oryzae pv. oryzae a Xanthomonas oryzae pv. oryzicola a Acidovorax avenae subsp. citrulli a Erwinia amylovora a Phytophthora capsici b Fusarium moniliforme b Alternaria solani b Botrytis cinerea b a
HCT116 (human colon cancer), HT-29 (human colorectal carcinoma), SW480 (human colon cancer), and MDA‑MB‑231 (human breast cancer) cells (Jiangsu Provincial Center for Disease Prevention and Control; 104 cells/ml) were seeded in 96-well tissue culture plates for 24 h. Then, cells were treated with different concentrations of test compounds (final concentration 40, 20, 10, 5, 2.5, and 1 µM) for 48 h. After the incubation period, MTT-solution (in PBS) was added to each well and cells were further incubated for 4 h at 37 °C. The reaction product, formazan, was extracted with DMSO, and the absorbance was read at 570 nm. The results were expressed as concentrations of compound producing 50 % toxicity (IC50 value). Each assay was performed in triplicate. Doxorubicin (≥ 98 %, Sigma-Aldrich) was used as a positive control.
20 20 20 20 10 5 > 40 40
10
11
10 10 10 10 > 40 40 > 40 20
10 10 10 10 > 40 > 40 20 5
Positive control
Table 4 Antimicrobial activity of 8, 10, and 11.
2.5 2.5 2.5 1.25 5 10 10 20
Kanamycin as positive control; b nystatin as positive control
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Planta Med 2014; 80: 925–930
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Fig. 6 Relative configuration of A ring in 5. (Color figure available online only.)
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plates was applied, and the final concentration of test compounds ranged from 40 to 0.16 µg/mL. Kanamycin (≥ 98 %, Sangon Biotech) and nystatin (≥ 4,400 USP units/mg, Aladdin) were used as positive controls. The minimum inhibitory concentration (MIC) was determined for three times as the lowest concentration at which no growth was observed.
Supporting information 1D and 2D NMR spectra of compounds 1–5 and CD spectra of compounds 1–3 are available as Supporting Information. Crystallographic data for compound 4 (Table 1S) has been deposited at the Cambridge Crystallographic Data Center (deposition No. CCDC-952074). These data can be obtained free of charge at www.ccdc.caam.ac.uk/conts/retrieving.html.
Acknowledgments !
This work was co-financed by the NSFC (81172948, 81121062, and 21132004) and Project 863(2013AA092903).
Conflict of Interest !
All authors here declare no conflicts of interest.
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9 Grkovic T, Blees JS, Colburn NH, Schmid T, Thomas CL, Henrich CJ, McMahon JB, Gustafson KR. Cryptocaryols A–H, α-pyrone-containing 1,3polyols from Cryptocarya sp. implicated in stabilizing the tumor suppressor pdcd4. J Nat Prod 2011; 74: 1015–1020 10 Davis RA, Demirkiran O, Sykes ML, Avery VM, Suraweera L, Fechner GA, Quinn RJ. 7′,8′-dihydroobolactone, a trypanocidal α-pyrone from the rainforest tree Cryptocarya obovata. Bioorg Med Chem Lett 2010; 20: 4057–4059 11 Meragelman TL, Scudiero DA, Davis RE, Staudt LM, McCloud TG, Cardellina II JH, Shoemaker RH. Inhibitors of the NF-κB activation pathway from Cryptocarya rugulosa. J Nat Prod 2009; 72: 336–339 12 Sturgeon CM, Cinel B, Marrero ARD, McHardy LM, Ngo M, Andersen RJ, Roberge M. Abrogation of ionizing radiation-induced G2 checkpoint and inhibition of nuclear export by Cryptocarya pyrones. Cancer Chemother Pharmacol 2008; 61: 407–413 13 Wu TS, Sua CR, Lee KH. Cytotoxic and anti-HIV phenanthroindolizidine alkaloids from Cryptocarya chinensis. Nat Prod Commun 2012; 7: 725– 727 14 Awang K, Hadi AHA, Saidi N, Mukhtar MR, Morita H, Litaudon M. New phenanthrene alkaloids from Cryptocarya crassinervia. Fitoterapia 2008; 79: 308–310 15 Toribio A, Bonfils A, Delannay E, Prost E, Harakat D, Henon E, Richard B, Litaudon M, Nuzillard JM, Renault JH. Novel seco-dibenzopyrrocoline alkaloid from Cryptocarya oubatchensis. Org Lett 2006; 8: 3825–3828 16 Lin FW, Wang JJ, Wu TS. New pavine n-oxide alkaloids from the stem bark of Cryptocarya chinensis Hemsl. Chem Pharm Bull 2002; 50: 157–159 17 Lin FW, Wu PL, Wu TS. Alkaloids from the leaves of Cryptocarya chinensis Hemsl. Chem Pharm Bull 2001; 49: 1292–1294 18 Ge HM, Sun H, Jiang N, Qin YH, Dou H, Yan T, Hou YY, Griesigner C, Tan RX. Relative and absolute configuration of vatiparol (1 mg): a novel anti-inflammatory polyphenol. Chem Eur J 2012; 18: 5213–5221 19 Yan T, Wang T, Wei W, Jiang N, Qin YH, Tan RX, Ge HM. Polyphenolic acetylcholinesterase inhibitors from Hopea chinensis. Planta Med 2012; 78: 1015–1019 20 Qin YH, Zhang J, Cui JT, Guo ZK, Jiang N, Tan RX, Ge HM. Oligostilbenes from Vatica mangachapoi with xanthine oxidase and acetylcholinesterase inhibitory activities. RSC Adv 2011; 1: 135–141 21 Fu X, Sévenet T, Remy F, Païs M, Hadi AHA, Zeng LM. Flavanone and chalcone derivatives from Cryptocarya kurzii. J Nat Prod 1993; 56: 1153– 1163 22 Gaffield W. Circular dichroism, optical rotatory dispersion, and absolute configuration of flavanones, 3-hydroxyflavanones, and their glycosides, determination of aglycone chirality in flavanone glycosides. Tetrahedron 1970; 26: 4093–4108 23 Slade D, Ferreira D, Marais JPJ. Circular dichroism, a powerful tool for the assessment of absolute configuration of flavonoids. Phytochemistry 2005; 66: 2177–2215 24 Taglialatela-Scafati O, Pollastro F, Chianese G, Minassi A, Gibbons S, Arunotayanun W, Mabebie B, Ballero M, Appendino G. Antimicrobial phenolics and unusual glycerides from Helichrysum italicum subsp. microphyllum. J Nat Prod 2013; 76: 346–353 25 Chakthong S, Weaaryee P, Puangphet P, Mahabusarakam W, Plodpai P, Voravuthikunchai SP, Kanjana-Opas A. Alkaloid and coumarins from the green fruits of Aegle marmelos. Phytochemistry 2012; 75: 108–113
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925
Authors
Wei Huang, Wen-Jing Zhang, Yi-Qing Cheng, Rong Jiang, Wei Wei, Chao-Jun Chen, Gang Wang, Rui-Hua Jiao, Ren-Xiang Tan, Hui-Ming Ge
Affiliation
Institute of Functional Biomolecules, State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing, China
Key words " Cryptocarya concinna l " Lauraceae l " flavonoid l " cytotoxic activity l " antimicrobial activity l
Abstract
received revised accepted
Nov. 1, 2013 May 19, 2014 May 20, 2014
Bibliography DOI http://dx.doi.org/ 10.1055/s-0034-1368613 Published online July 16, 2014 Planta Med 2014; 80: 925–930 © Georg Thieme Verlag KG Stuttgart · New York · ISSN 0032‑0943 Correspondence Dr. Hui-Ming Ge Institute of Functional Biomolecules State Key Laboratory of Pharmaceutical Biotechnology Nanjing University 22 Hankou Road Nanjing 210093 China Phone: + 86 25 83 59 32 01 Fax: + 86 25 83 59 32 01 [email protected] Correspondence Dr. Ren-Xiang Tan Institute of Functional Biomolecules State Key Laboratory of Pharmaceutical Biotechnology Nanjing University 22 Hankou Road Nanjing 210093 China Phone: + 86 25 83 59 32 01 Fax: + 86 25 83 59 32 01 [email protected]
!
Five new flavonoids, cryptoconones A–E (1–5), along with six known compounds (6–11), were isolated from the stems of Cryptocarya concinna. The structures of these compounds were elucidated on the basis of spectroscopic data interpretation, and the absolute configurations were determined via circular dichroism spectra and X‑ray crystal analysis. The cytotoxic and antimicrobial activities of these compounds were also eval-
Introduction !
The genus Cryptocarya (Lauraceae) consists of about 250 species distributed mainly in the tropical and subtropical regions of the world. Earlier studies on this genus reported flavonoids [1–8], pyrones [6, 9–12], and alkaloids [13–17] as the main secondary metabolite constituents with varied biological activities. In our previous study, several flavonoids with a variety of bioactivities from C. maclurei and C. chingii were found [1, 2]. As a part of our continuing search for bioactive constituents from tropical plants [1, 2, 18–20], a 95 % EtOH extract of the stems of Cryptocarya concinna Hance with no previous literature reports on its chemistry has been investigated with five new (1–5) and six known flavonoids (6–11) obtained. Reported herein are the isolation, structure elucidation, and biological activities of these compounds.
Results and Discussion !
Repeated column chromatography of the EtOH extract (95%) of the stems afforded five new flavonoids, 1–5, along with six known flavonoids, cryptochinone A (6) [4], cryptogione C (7) [1], cryptocaryanone A (8) [8], cryptocaryone (9) [8],
uated. Compounds 9 and 10 exhibited moderate cytotoxic activities against HCT116, HT-29, SW480, and MDA‑MB‑231 cell lines with IC50 values ranging from 6.25 to 9.35 µM. Compounds 8 and 11 exhibited antimicrobial activity against Fusarium moniliforme and Botrytis cinerea, respectively, with the same minimum inhibitory concentration of 5 µg/mL. Supporting information available online at http://www.thieme-connect.de/products
kurzichalcolactone A (10) [21], and kurzichalcolactone B (11) [7]. The known compounds were determined by comparing their physical and spectroscopic properties to literature values. Compound 1 was obtained as a yellowish oil. The " Table 1) for 1 were almost the NMR signals (l same as those of cryptogione E [1], except for the absence of a methoxyl group. This was further supported by HRESIMS, which gave an [M + Na]+ ion peak at m/z 307.0941 (calcd. for C17H16NaO4, 307.0941), being 14 mass units less than that of cryptogione E [1]. The 2R configuration of 1 was evidenced by a negative Cotton effect at 349 nm due to the n → π* transition [1, 22, 23]. Moreover, 1 showed a similar circular dichroism (CD) spectrum compared to that of cryptogione E [1], indicating that the absolute configuration of 1 is 2R, 5S. Thus, the structure of 1 was elucidated as " Fig. 1 and further confirmed by 1Hshown in l 1 H COSY, DEPT, HSQC, HMBC, and NOESY " Fig. 2) experiments. (l Compound 2 appeared as a yellowish oil with a molecular formula of C17H16O4 deduced from the observed [M + Na]+ peak at m/z 307.0934 in the HRESIMS spectrum. The 1H and 13C NMR spectra " Table 1) of 2 were closely similar to those of 1, (l indicating it as an epimer of 1, which was further " Fig. 2). The confirmed by 2D NMR correlations (l 2S configuration was assigned by a positive Cot-
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Cytotoxic and Antimicrobial Flavonoids from Cryptocarya concinna
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1a δH (J in Hz)
2 3 4 5 6 7 8 9 10 11 12 1′ 2′,6′ 3′,5′ 4′ a
5.43 dd (14.0, 3.5) 2.90 dd (16.5, 14.0) 2.71 dd (16.5, 3.5) 3.49 m 2.49 m 6.40 m 6.06 d (10.0)
2.37 m
7.41 m 7.41 m 7.41 m
2a δC 80.3 42.5 190.7 25.5 28.1 138.9 122.7 166.0 111.0 35.3 176.6 138.1 126.2 128.8 128.8
δH (J in Hz) 5.42 dd (14.5, 3.0) 2.91 dd (16.5, 14.5) 2.68 dd (16.5, 3.0) 3.50 m 2.49 m 2.58 m 6.43 ddd (10.0, 6.0, 2.5) 6.01 dd (10.0, 2.5)
2.47 m
7.42 m 7.42 m 7.42 m
δC
Table 1 1 and 2.
1
H and 13C NMR data for
80.4 42.9 190.8 25.4 28.8 139.9 122.4 165.7 111.3 37.7 176.2 138.2 126.2 128.8 128.8
Recorded in CDCl3 at 500 MHz for 1H NMR, 125 MHz for 13C NMR, δ in ppm, J in Hz
Fig. 1
ton effect at 325 nm due to the n → π* transition [8, 22, 23]. Therefore the absolute configuration of 2 was confirmed as 2S, 5S when compared to 1. Compound 3 was obtained as a colorless powder. HRESIMS data was used to determine the molecular formula as C17H14O5, the same as that of co-occurring cryptogione C (7) [1]. From its spec" Table 2), compound 3 was found to be similar to troscopic data (l 7. The major difference was that the hydroxyl group coupled at C6 in 3 instead of C-8 in 7, which was supported by the HMBC correlations between H-11 (δH 4.22, 4.17) and C-6 (δC 150.9), C-10 (δC 120.9); H-7 (δH 7.18) and C-9 (δC 157.1), C-5 (δC 122.9); H-8 " Fig. 3). The abso(δH 6.91) and C-6 (δC 150.9), C-9 (δC 157.1) (l lute configuration of 3 was also confirmed as 2S by CD, as in the case of 2. Thus, the structure of 3 was identified as (S)-2-(6-hydroxy-4-oxo-2-phenylchroman-5-yl) acetic acid.
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Structures of 1–11.
Compound 4 was isolated as colorless needles and gave a molecular formula of C17H16O5 by HRESIMS, the same as that of co-oc" Table 2) of 4 curring cryptochinone A (6) [4]. The NMR spectra (l were quite similar to those of 6, except that the 7β-configuration of 6 was replaced by 7α-configuration of 4. The relative configuration of 4 was established as rel-2S,5R,6R,7R by X‑ray crystal " Fig. 4). The 2S configuration was determined by a posanalysis (l " Fig. 5) itive Cotton effect at 340 nm due to the n → π* transition (l [4, 22, 23]. Compound 5, a colorless oil, exhibited the same molecular formula as compound 4 (C17H16O5) from the HRESIMS data. Com" Table 2) of 4 and 5 indiparison of the 1H and 13C NMR data (l cated that they are different only in relative stereochemistry. " Fig. 6) and 1H NMR coupling constants The NOESY interactions (l were used to determine the relative configuration of ring A in 5, as in the case of cryptogiones G and H [2]. H-6 was deduced to be
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Position
of 8.0 Hz. The 2R configuration was identified by a negative Cot" Fig. 5), as in the case of 1. The chirality at ton effect at 316 nm (l C-5 dominated the CD spectra at 200–240 nm by comparison of " Fig. 5) of 4, 6 [4], and cryptogione D [1], which the CD spectra (l presented similar cyclohexene units. Thus, the absolute configuration at C-5 of 5 was determined as 5S by the reversed CD spectrum of 5 at 200–240 nm compared to that of 4. Therefore, com" Fig. 1. pound 5 was established as shown in l All the isolates were tested for cytotoxic activity against HCT116, HT-29, SW480, and MDA‑MB‑231 cell lines. Compounds 9 and 10 " Table 3), while others exhibited moderate cytotoxic activity (l were inactive (IC50 > 50 µM). These compounds were also evaluated for antibacterial (Xanthomonas oryzae pv. oryzae, Xanthomonas oryzae pv. oryzicola, Acidovorax avenae subsp. citrulli, and Erwinia amylovora) and antifungal (Phytophthora capsici, Fusarium moniliforme, Alternaria solani, and Botrytis cinerea) activities. Only compounds 8, 10, and 11 showed significant antimi" Table 4). The purity of the tested compounds crobial activity (l was determined to be over 95 % by using the HPLC‑DAD method. Fig. 2 Key HMBC (→), COSY (—), and NOESY (↔) correlations of 1 and 2. (Color figure available online only.)
Materials and Methods !
General experimental procedures axial according to the NOE correlations between H-6 and H-8α. Therefore, H-7 and H-5 were determined to be equatorial and axial, respectively, because of the 3J6,7 value of 3.2 Hz and 3J5,6 value
Table 2
1
H and 13C NMR data for 3–5.
Position
3a δH (J in Hz)
2 3 4 5 6 7 8 9 10 11 12 1′ 2′,6′ 3′,5′ 4′ a
Optical rotations were obtained on a Rudolph Autopol III automatic polarimeter. UV spectra were recorded on a Hitachi U3000 spectrophotometer. CD spectra were obtained on a Jasco J810 spectrometer, and the IR spectra (KBr) were performed on a
5.49 dd (13.2, 2.8) 3.06 dd (16.4, 13.2) 2.78 dd (16.4, 2.8)
7.18 d (8.8) 6.91 d (8.8)
4.22 d (16.8) 4.17 d (16.8)
7.58 m 7.44 m 7.38 m
4b δC 79.9 46.8 194.3 122.9 150.9 123.9 118.3 157.1 120.9 32.4 172.5 140.8 127.3 129.5 129.3
5b
δH (J in Hz)
δC
5.38 dd (14.4, 3.2) 2.69 dd (16.8, 3.2) 2.93 dd (16.8, 14.4) 3.58 m 4.62 t (6.4) 4.37 m 2.53 dd (18.4, 4.8) 2.78 dd (18.4, 2.2)
3.06 dd (18.0, 8.8) 2.47 dd (18.0, 4.4)
7.41 m 7.41 m 7.41 m
80.8 42.8 190.8 31.4 79.4 65.4 32.4 168.3 110.8 36.1 176.2 137.7 126.4 129.1 129.3
δH (J in Hz) 5.42 dd (14.0, 3.2) 2.71 dd (16.4, 3.2) 2.97 dd (16.4, 14.0) 3.60 dt (9.2, 8.0) 4.71 dd (8.0, 3.2) 4.31 br s 2.64 m (β) 2.68 m (α)
3.02 dd (18.0, 9.2) 2.48 dd (18.0, 8.0)
7.40 m 7.41 m 7.41 m
δC 81.0 42.8 190.8 32.1 78.5 66.4 33.5 167.4 110.8 34.9 177.1 137.7 126.1 128.9 129.1
Recorded in acetone-d6 at 400 MHz for 1H NMR, 100 MHz for 13C NMR, δ in ppm, J in Hz; b Recorded in CDCl3 at 400 MHz for 1H NMR, 100 MHz for 13C NMR, δ in ppm, J in Hz
Fig. 3 Key HMBC (→) and COSY (—) correlations of 3–5. (Color figure available online only.)
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Original Papers
Original Papers
with a voucher specimen (IFB-20101202) conserved at the Institute of Functional Biomolecules, Nanjing University.
Extraction and isolation
Fig. 4
X‑ray crystal analysis of 4.
Nexus 870 FT‑IR spectrometer. Using solvent signals as internal standard, NMR data were acquired on a Bruker DRX-500 or DRX-400 NMR spectrometer. HRESIMS spectra were recorded on an Agilent 6210 TOF LC‑MS spectrometer. Silica gel (200–300 mesh) for column chromatography was produced by Qingdao Marine Chemical Factory. Sephadex LH-20 was purchased from Pharmacia Biotech. ODS‑A gel (AA12S50) was purchased from YMC Co., Ltd. Semipreparative HPLC analysis was performed on an Agilent 1260 HPLC system equipped with a Hypersil ODS column (250 mm × 10 mm, 5 µm, Thermo Fisher Scientific).
Plant material C. concinna was collected in December 2010 from the Botanical Garden at Jianfeng Town, Ledong County, Hainan Island (China). Identification was provided by Prof. X. J. Tian (Nanjing University)
Air-dried and powdered stem woods of C. concinna (25 kg) were extracted with EtOH‑H2O (v/v, 95 : 5, 4 × 50 L) at room temperature and concentrated in vacuo to give a crude extract (716 g), which was subjected to silica gel (3.5 kg, 200–300 mesh) column chromatography (12 × 120 cm) (CHCl3/MeOH, 1 : 0 → 0 : 1, each 10 L) to afford fractions A–T. Fraction D (CHCl3/MeOH, 100 : 4) (24 g) was refined over a reversed-phase silica gel column (5 × 50 cm), eluting with a MeOH/H2O gradient (20% → 100 % MeOH, each 2 L), to afford 16 fractions (D1–D16). Fraction D3 (MeOH/H2O, 30 : 70) (721 mg) was further separated over Sephadex LH-20 column chromatography (2.5 × 60 cm), eluting with MeOH (2 L), to give four subfractions (D3a-D3d). Subfraction D3c (700–1000 mL, 62 mg) was purified by semipreparative reversedphase HPLC (MeOH/H2O, 36 : 64, 2 ml/min) to obtain 4 (10 mg, tR = 49.0 min), 5 (5 mg, tR = 38.5 min), and 6 (12 mg, tR = 43.8 min). Fraction D4 (MeOH/H2O, 35 : 65) (2020 mg) was purified over Sephadex LH-20 CC (2.5 × 60 cm) (MeOH, 2 L) to give 8 (627 mg). Fraction D5 (MeOH/H2O, 40 : 60) (728 mg) was separated over Sephadex LH-20 CC (2.5 × 60 cm) (MeOH, 2 L) to give four subfractions (D5a-D5d). Subfraction D5b (400–600 mL, 70 mg) was purified by semipreparative reversed-phase HPLC (MeCN/H2O, 47 : 53, 2 ml/min), to obtain 3 (4 mg, tR = 14.5 min) and 7 (6 mg, tR = 19.7 min). Fraction D7 (MeOH/H2O, 50 : 50) (1143 mg) was chromatographed on silica gel (100 g, 200–300 mesh) CC (3 × 60 cm) eluting with petroleum ether/acetone (7 : 1, 10 L) to obtain 10 (4–5 L, 110 mg) and 11 (6–7.5 L, 170 mg). Fraction E (CHCl3/MeOH, 100 : 6) (28 g) was subjected to separation on a reversed-phase silica gel column (5 × 50 cm) to give 16 fractions (E1–E16). Fraction E6 (MeOH/H2O, 45 : 55) (540 mg) was further separated over Sephadex LH-20 CC (2.5 × 60 cm) (MeOH, 2 L) to afford four subfractions (E6a-E6d). Subfraction E6a (0–400 mL, 16 mg) was purified by semipreparative reversed-phase HPLC (MeOH/H2O, 52 : 48, 2 mL/min) to obtain 1 (2 mg, tR = 48.5 min) and 2 (2 mg, tR = 48.6 min). Fraction E7 (MeOH/H2O, 50 : 50) (870 mg) was passed over Sephadex LH-20 CC (2.5 × 60 cm) (MeOH, 2 L) to give three subfractions (E7a-E7c). Subfraction
Fig. 5 CD spectra of 4, 5, 6, and cryptogione D. (Color figure available online only.)
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E7b (300–800 mL, 329 mg) was chromatographed on a silica gel (20 g, 200–300 mesh) column (2.5 × 60 cm), eluting with CH2Cl2/ MeOH (300 : 1, 4 L) to obtain 9 (500–800 mL, 12 mg).
Cryptoconone D (4): Colorless needles; UV (MeOH) λmax (log ε) 269 (4.06) nm; [α]30 D + 100 (c 0.050, MeOH); CD (MeOH) λext (θ) 208 (+ 6.82), 220 (+ 11.79), 241 (+ 2.98), 261 (+ 6.70), 297 (− 3.08), 340 (+ 0.29) nm; IR (KBr) νmax 3398, 2924, 1780, 1658, 1613, 1422, 1172 cm−1; 1H NMR (CDCl3, 400 MHz) and 13C NMR " Table 2; crystallo(CDCl3, 100 MHz) spectroscopic data, see l graphic data, see Table 1S (in Supporting Information); HRESIMS (positive) m/z 301.1073 [M + H]+ (calcd. for C17H17O5, 301.1072). Cryptoconone E (5): Colorless oil; UV (MeOH) λmax (log ε) 271 (3.95) nm; [α]30 D − 93 (c 0.056, MeOH); CD (MeOH) λext (θ) 204 (− 3.25), 221 (− 7.75), 274 (+ 4.23), 316 (− 1.76) nm; IR (KBr) νmax 3424, 2920, 1773, 1662, 1612, 1423, 1176 cm−1; 1H NMR (CDCl3, 400 MHz) and 13C NMR (CDCl3, 100 MHz) spectroscopic data, see " Table 2; HRESIMS (positive) m/z 323.0894 [M + Na]+ (calcd. for l C17H16NaO5, 323.0890).
Cytotoxic activity assay Isolates Cryptoconone A (1): Yellowish oil; UV (MeOH) λmax (log ε) 320 (3.85) nm; [α]30 D − 87 (c 0.057, MeOH); CD (MeOH) λext (θ) 221 (− 20.52), 245 (− 0.01), 259 (− 0.90), 309 (+ 5.90), 349 (− 11.77) nm; IR (KBr) νmax 3434, 2924, 1723, 1651, 1566, 1426, 1382 cm−1; 1 H NMR (CDCl3, 500 MHz) and 13C NMR (CDCl3, 125 MHz) spec" Table 1; HRESIMS (positive) m/z 307.0941 troscopic data, see l + [M + Na] (calcd. for C17H16NaO4, 307.0941). Cryptoconone B (2): Yellowish oil; UV (MeOH) λmax (log ε) 325 (3.85) nm; [α]30 D + 90 (c 0.061, MeOH); CD (MeOH) λext (θ) 226 (− 14.96), 249 (+ 4.79), 273 (+ 1.45), 325 (+ 5.69) nm; IR (KBr) νmax 3427, 2924, 1722, 1636, 1565, 1427 cm−1; 1H NMR (CDCl3, 500 MHz) and 13C NMR (CDCl3, 125 MHz) spectroscopic data, see " Table 1; HRESIMS (positive) m/z 307.0934 [M + Na]+ (calcd. for l C17H16NaO4, 307.0941). Cryptoconone C (3): Colorless powder; UV (MeOH) λmax (log ε) 260 (4.12), 358 (3.83) nm; [α]30 D + 112 (c 0.078, MeOH); CD (MeOH) λext (θ) 215 (+ 5.74), 260 (− 1.93), 280 (+ 0.08), 327 (− 4.75), 369 (+ 3.97) nm; IR (KBr) νmax 3384, 2923, 1664, 1613 cm−1; 1H NMR (acetone-d6, 400 MHz) and 13C NMR (ace" Table 2; HRESIMS tone-d6, 100 MHz) spectroscopic data, see l (positive) m/z 321.0736 [M + Na]+ (calcd. for C17H14NaO5, 321.0733).
Compound 9 10 Doxorubicina a
Antimicrobial activity assay The antimicrobial activities were evaluated against four bacterial strains (X. oryzae pv. oryzae, X. oryzae pv. oryzicola, A. avenae subsp. citrulli, and E. amylovora) and four fungal strains (P. capsici, F. moniliforme, A. solani, and B. cinerea), which were obtained from College of Plant Protection, Nanjing Agricultural University. The antimicrobial assay was performed as described previously [24, 25]. The broth dilution method using 96-well microtiter
IC50 (µM)
Table 3 Cytotoxicity of 9 and 10.
HCT116
HT-29
SW480
MDA‑MB‑231
9.35 ± 0.28 6.49 ± 0.19 1.48 ± 0.11
7.23 ± 0.44 6.25 ± 0.57 1.87 ± 0.26
6.52 ± 0.30 7.44 ± 0.27 2.34 ± 0.38
9.03 ± 0.34 8.46 ± 0.39 2.59 ± 0.23
Positive control
Target microorganism
Antimicrobial activity (MIC in µg/mL) 8
Xanthomonas oryzae pv. oryzae a Xanthomonas oryzae pv. oryzicola a Acidovorax avenae subsp. citrulli a Erwinia amylovora a Phytophthora capsici b Fusarium moniliforme b Alternaria solani b Botrytis cinerea b a
HCT116 (human colon cancer), HT-29 (human colorectal carcinoma), SW480 (human colon cancer), and MDA‑MB‑231 (human breast cancer) cells (Jiangsu Provincial Center for Disease Prevention and Control; 104 cells/ml) were seeded in 96-well tissue culture plates for 24 h. Then, cells were treated with different concentrations of test compounds (final concentration 40, 20, 10, 5, 2.5, and 1 µM) for 48 h. After the incubation period, MTT-solution (in PBS) was added to each well and cells were further incubated for 4 h at 37 °C. The reaction product, formazan, was extracted with DMSO, and the absorbance was read at 570 nm. The results were expressed as concentrations of compound producing 50 % toxicity (IC50 value). Each assay was performed in triplicate. Doxorubicin (≥ 98 %, Sigma-Aldrich) was used as a positive control.
20 20 20 20 10 5 > 40 40
10
11
10 10 10 10 > 40 40 > 40 20
10 10 10 10 > 40 > 40 20 5
Positive control
Table 4 Antimicrobial activity of 8, 10, and 11.
2.5 2.5 2.5 1.25 5 10 10 20
Kanamycin as positive control; b nystatin as positive control
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Fig. 6 Relative configuration of A ring in 5. (Color figure available online only.)
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plates was applied, and the final concentration of test compounds ranged from 40 to 0.16 µg/mL. Kanamycin (≥ 98 %, Sangon Biotech) and nystatin (≥ 4,400 USP units/mg, Aladdin) were used as positive controls. The minimum inhibitory concentration (MIC) was determined for three times as the lowest concentration at which no growth was observed.
Supporting information 1D and 2D NMR spectra of compounds 1–5 and CD spectra of compounds 1–3 are available as Supporting Information. Crystallographic data for compound 4 (Table 1S) has been deposited at the Cambridge Crystallographic Data Center (deposition No. CCDC-952074). These data can be obtained free of charge at www.ccdc.caam.ac.uk/conts/retrieving.html.
Acknowledgments !
This work was co-financed by the NSFC (81172948, 81121062, and 21132004) and Project 863(2013AA092903).
Conflict of Interest !
All authors here declare no conflicts of interest.
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