Natural Product Research, 2014 Vol. 28, No. 8, 563–567, http://dx.doi.org/10.1080/14786419.2013.867856
Two new natural products from Croton kongensis Gagnep. Lin Sunab, Zhaoqing Mengab, Zhaoliang Liab, Biao Yangab, Zhenzhong Wangab, Gang Dingab and Wei Xiaoab* a Jiangsu Kanion Pharmaceutical Co. Ltd, 58 South Haichang Road, Lianyungang 222001, People’s Republic of China; bState Key Laboratory of New-tech for Chinese Medicine Pharmaceutical Process, Lianyungang 222001, People’s Republic of China
(Received 6 October 2013; final version received 18 November 2013) A new diterpenoid, 14b-hydroxy-3-oxo-ent-kaur-16-ene (1), and a new nor-lignan, 8S-(2 )-8-(4-hydroxy-3-methoxybenzoyl)-dihydrofuran-8(80 H)-one (4), along with five known compounds were isolated from the twigs and leaves of Croton kongensis Gagnep. Their isolations were clarified and structures were elucidated by the extensive spectroscopic analyses, especially 2D NMR experiments. Keywords: Croton kongensis Gagnep.; Euphorbiaceae; diterpenoid; nor-lignan; structural elucidation
1. Introduction The genus Croton (Euphorbiaceae) is widely distributed in the tropical and subtropical areas (Chen et al. 1996). Previous studies about this genus reported a series of diterpenoids (Thongtan et al. 2003), triterpenoids (Barbosa et al. 2003), alkaloids (Aboagye et al. 2000) and flavonoids (Charris et al. 2000). Croton kongensis Gagnep. (Euphorbiaceae) is a small shrub and used in folk medicine for dysmenorrhoea in Thailand (Thongtan et al. 2003). The research was carried out on the twigs and leaves of C. kongensis Gagnep., which were collected from Hainan province, People’s Republic of China, which led to the isolation of three diterpenoids (1 – 3) and four lignan derives (4– 7). Two new compounds, 14b-hydroxy-3-oxo-ent-kaur-16-ene (1), and 8S-(2 )-8-(4hydroxy-3-methoxybenzoyl)-dihydrofuran-8(80 H)-one (4), together with the known compounds, were determined by extensive spectroscopic analyses, especially 2D NMR experiments.
2. Results and discussion Compound 1 was isolated as a white amorphous powder. High-resolution EI-MS spectrometry led to its molecular formula C20H30O2 by molecular ion peak at m/z 302.2245, incorporating six double bond equivalents (DBEs). In the 1H NMR spectrum, three methyls (dH 1.09, s, H3-18; 1.04, s, H3-19; 1.03, s, H3-20), two exocyclic double bond protons (dH 4.96, m, H2-17) and an oxygenated proton (dH 4.13, d, J ¼ 5.0 Hz, H-14) were observed. In total, 20 carbon resonances were observed in the 13C NMR and DEPT spectra (CDCl3) comprising three methyl groups (dC 27.7, 21.0, 18.5), an oxo carbonyl group (dC 218.2) an exocyclic double bond (dC 107.28, 152.3), seven methylenes, four methines (one oxygenated) and three quaternary carbons. The aforementioned descriptions accounted for two DBEs of compound 1, and remaining four required that compound 1 possessed a tetracyclic core.
*Corresponding author. Email: [email protected]
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On the basis of the detailed analysis of 2D NMR spectra and a comparison with those of classic ent-kaurane diterpenoids, compound 1 was found to be similar to 15b-hydroxy-3-oxoent-kaur-16-ene (Fraga et al. 2004), with only difference in the location of the hydroxyl group. In the HMBC spectrum (Figure S17A), the correlations of H-14 (dH 4.13, d, J ¼ 5.0 Hz)/C-15, C16 and C-12 assigned the hydroxyl group to C-14. Thus, the planar structure of 1 was determined as shown. The ROESY spectrum (Figure S17B) revealed that H-14 had strong cross-peak with H-13 and H3-20 indicating that H-14, H-13 and Me-20 were cofacial and arbitrarily assigned to be a-oriented. Subsequently, the ROESY correlations of H-1b/H-9, H-1b/H-5, H-5/H-9, H-7b/ H-9 and H-9/H-15b indicated that these protons were b-oriented. Therefore, compound 1 was characterised as shown and named as 14b-hydroxy-3-oxo-ent-kaur-16-ene. Compound 4 was obtained as a yellowish oil. Its molecular formula C12H12O5 was determined by HR-ESI-MS at m/z 235.0599 (calcd 235.0606), incorporating seven DBEs. The IR spectrum revealed absorptions of carbonyl moieties at 1772.3 cm21 and a hydroxyl group at 3426 cm21. In the 1H NMR spectrum, three low-field proton signals (dH 7.58, d, J ¼ 2.0 Hz, H2; 7.47, dd, J ¼ 8.4, 2.0 Hz, H-6; 7.02, d, J ¼ 8.4 Hz, H-5) indicated a typical ABX system aromatic ring. The 13C NMR and DEPT spectra (CDCl3) revealed 12 carbon signals, including a conjugated oxo carbonyl (dC 194.8), an ester carbonyl (dC 175.6), a trisubstituted aromatic ring (dC 151.6, 147.3, 128.1, 123.8, 114.3, and 110.4), an oxygenated methylene (dC 69.5), a methoxy group (dC 56.3), a methylene (dC 31.3) and a methine (dC 41.8) carbon. This accounted for six DBEs and the remaining one assigned to be an extra ring. Detailed analysis 2D spectra revealed the connections of these functionalities. The oxo carbonyl was deduced to conjugate with the aromatic by using the HMBC correlations of H-2/C-7 and H-6/C-7 (Figure S18). The fivemembered lactone ring was rationalised by the HMBC correlations of H2-9/C-90 , H-80 /C-90 , H-8/ C-9 and H-8/C-80 . The methoxy group and the hydroxyl group were assigned as C-3 and C-4, respectively, by the HMBC correlations of CH3O/C-3, H-5/C-3, H-6/C-4 and H-2/C-4. A further comparison of the NMR data with that of 4S-(2 )-4-benzoyl-4,5-dihydrofuran-2(3H)-one (Ender et al. 1996) indicated that compound 4 was a nor-lignan elucidated as shown. The optical rotation value of compound 4 was determined to be 2 4.00, indicating that the absolute configuration at C-8 was S (Ender et al. 1996). Thus, the structure of 4 was determined, and named as 8S-(2 )-8-(4-hydroxy-3-methoxybenzoyl)-dihydrofuran-8(80 H)-one. Five known compounds, ent-7a,14b-dihydroxykaur-16-en-15-one (Perry et al. 1999), ent18-acetoxy-7a,14b-dihydroxykaur-16-en-15-one (Giang et al. 2003), (1R*,5R*,6S*)-6-(4hydroxy-3-methoxypheyl)-3,7-dioxabicyclo[3.3.0]octan-2-one (Yamauchi et al. 2004), pinoresinol (Brenes et al. 2000) and matairesinol (Yang et al. 2013) were also obtained. Their structures were determined by comparing with the literature data (Figure 1). 3. Experimental 3.1. General experimental procedures NMR spectra were recorded on Varian Mercury-400 (Varian Medical Systems, Inc., United States), Bruker Avance III 500 spectrometers (Bruker Scientific Technology Co. Ltd, United States), with trimethylsilane as the internal standard. ESI-MS was carried out on a Bruker Daltonics esquire 3000plus instrument (Bruker Scientific Technology Co. Ltd, United States). HR-EI-MS spectra were recorded on a Thermo-DFS EI-HR (Thermo Fisher Scientific Co. Ltd, United States). HR-ESI-MS spectra were measured on an LCT Premier XE (Waters) mass spectrometer (Waters Technology Ltd, United States). UV spectra were recorded on a Shimadzu UV-2550 UV – visible spectrophotometer (Shimadzu Co. Ltd, Japan). Optical rotations were measured on a Perkin-Elmer 341 polarimeter (PerkinElmer Inc, United States) at room temperature. IR spectra were measured on a Perkin-Elmer 577 IR spectrometer (PerkinElmer Inc, United States); KBr discs. Semi-preparative HPLC was performed on Waters 1525 pump,
Natural Product Research 12 13 16 17 14 2 OH 8 10 3 5 H 15 4 7 O H 6 18 19 11 20 1 9
11 20 1 9
15 O OH
H 6 R1 18 19 R1 2 CH3 3 CH2OH
7 H 9
O 9' O
O OCH3 H O
Figure 1. Structures of compounds 1 – 7.
Waters 2489 detector and a YMC-Pack ODS-A column (250 £ 10 mm, S-5 mm, 12 nm). Column chromatography (CC) was performed on silica gel (300 – 400 mesh), Sephadex LH-20 (Amersham Biosciences, Piscataway, United States) and C18 reverse-phased silica gel (150 – 200 mesh, Merck, Hunterdon, United States), and pre-coated silica gel GF254 plates (Qingdao Marine Chemical Plant, Qingdao, People’s Republic of China) were used for thin-layer chromatography. All solvents were of analytical grade (Shanghai Chemical Reagents Company, Ltd, Shanghai, People’s Republic of China), and the solvents used in HPLC were of HPLC grade (J&K Scientific Ltd, Shanghai, People’s Republic of China). 3.2. Plant materials The twigs and leaves of C. kongensis Gagnep. were collected from Hainan province, People’s Republic of China. The specimen was identified by Zhaoqing Meng, Jiangsu Kanion Pharmaceutical Co. Ltd, Lianyungang, People’s Republic of China, where a voucher specimen (No. CKG 20120820G) has been deposited. 3.3. Extraction and isolation The air-dried and powdered twigs and leaves of C. kongensis Gagnep. (3.0-kg) were percolated three times with 95% ethanol (4 L each time) at room temperature. The solvent was removed in vacuo to yield 300 g crude extract. The crude was suspended with hot water (0.5 L) and then extracted with EtOAc four times (0.6 L each) to yield the crude extract (250 g). The EtOAc extract was chromatographed on a silica gel CC eluted with petroleum ether/acetone (20:1 to 1:1) to yield three fractions A – C. Fraction A (22 g) was separated by the silica gel CC (CHCl3/ MeOH, 100:1 to 5:1) to yield three fractions A1 – A3. Fraction A2 (800 mg) was further separated by passage over Sepadex LH-20 column (MeOH) to yield three fractions A2a – A2c. A2a (25 mg) was then separated using semi-preparative HPLC (eluent: CH3CN/H2O, 60%) to obtain compound 6 (5 mg). Compound 7 (8 mg) was purified at fraction A2b (30 mg) by semipreparative HPLC (eluent: CH3CN/H2O, 60%). Fraction A2c was separated by using C18 reverse-phased silica (MeOH/H2O, 50 –80%) to give three fractions A2c1 – A2c3. Compounds 4 (3 mg) and 5 (4 mg) were purified from fraction A2c2 by semi-preparative HPLC (eluent:
L. Sun et al.
CH3CN/H2O, 65%). Using the same procedures, fraction B (12 g) was re-chromatographed on silica gel (CHCl3/MeOH, 100:1 to 5:1) to yield five subfractions B1 – B5. Fraction B3 was then subjected to C18 reverse-phased silica and Sepadex LH-20 to yield subfractions B3a –B3d. Subfraction B3c was a combination of compounds 1 –3 (4, 8 and 25 mg, respectively), which were finally purified by semi-preparative HPLC.
3.3.1. 14b-Hydroxy-3-oxo-ent-kaur-16-ene (1) White amorphous powder; ½a20 D 2 75:4 (c ¼ 0.14, MeOH); UV (MeOH) lmax 230 nm; IR (KBr) nmax 3440, 2925, 2854, 1687, 1462, 1385, 1066, 872 cm21; 1H NMR (CDCl3): dH 1.46 (m, H1a), 1.97 (m, H-1b), 2.46 (m, H2-2), 1.51 (m, H2-5), 1.51 (m, H2-6), 2.23 (m, H-7a), 1.38 (m, H9), 1.46 (m, H-11a), 1.66 (m, H-11b), 1.67 (m, H-12a), 1.80 (m, H-12b), 2.62 (m, H-13), 4.13 (d, J ¼ 5.0 Hz, H-14), 2.34 (d, J ¼ 17.3 Hz, H-15a), 2.04 (d, J ¼ 17.3 Hz, H-15b), 4.96 (m, H2-17), 1.09 (s, H3-18), 1.04 (s, H3-19) and 1.03 (s, H3-20); 13C NMR (CDCl3): dC 39.4 (C-1), 34.2 (C2), 218.2 (C-3), 47.2 (C-4), 54.4 (C-5), 21.1 (C-6), 31.8 (C-7), 49.2 (C-8), 57.5 (C-9), 38.6 (C10), 18.3 (C-11), 32.9 (C-12), 51.8 (C-13), 76.2 (C-14), 44.4 (C-15), 152.3 (C-16), 107.3 (C-17), 27.7 (C-18), 21.0 (C-19) and 18.5 (C-20); HR-EI-MS m/z 302.2245 for C20H30O2.
3.3.2. 8S-(2 )-8-(4-hydroxy-3-methoxybenzoyl)-dihydrofuran-8(8 0 H)-one (4) Yellowish oil; ½a20 D 2 4:0 (c ¼ 0.05, MeOH); UV (MeOH) lmax: 209, 228 and 281 nm; IR (KBr) nmax 3426, 2922, 1772, 1668, 1591, 1516, 1429, 1275, 1164 and 1028 cm21; 1H NMR (CDCl3) dH 7.58 (d, J ¼ 2.0 Hz, H-2), 7.48 (d, J ¼ 8.4 Hz, H-5), 7.02 (d, J ¼ 8.4, 2.0 Hz, H-6), 4.37 (m, H-8), 4.63 (dd, J ¼ 9.0, 8.6 Hz, H-9a), 4.48 (dd, J ¼ 9.0, 6.9 Hz, H-9b), 3.06 (dd, J ¼ 17.8, 7.8 Hz, H-80 a), 2.79 (dd, J ¼ 17.8, 9.4 Hz, H-80 b) and 4.00 (s, CH3O); 13C NMR (CDCl3) dC 128.1 (C-1), 110.4 (C-2), 147.3 (C-3), 151.6 (C-4), 114.3 (C-5), 123.8 (C-6), 194.8 (C-7), 41.8 (C-8), 69.5 (C-9), 31.3 (C-80 ), 175.6 (C-90 ), 56.3 (C-C H3O); HR-ESI-MS m/z 235.0599 ([M –H] – , calculated for C12H11O5, 235.0606).
4. Conclusion Two new compounds, 14b-hydroxy-3-oxo-ent-kaur-16-ene (1) and 8S-( –)-8-(4-hydroxy-3methoxybenzoyl)-dihydrofuran-8(80 H)-one (4), and five known compounds, ent-7a,14bdihydroxykaur-16-en-15-one (2), ent-18-acetoxy-7a,14b-dihydroxykaur-16-en-15-one (3), (1R*,5R*,6S*)-6-(4-hydroxy-3-methoxypheyl)-3,7-dioxabicyclo[3.3.0]octan-2-one (5), pinoresinol (6) and matairesinol (7), were isolated from the twigs and leaves of C. kongensis Gagnep. Their structures were elucidated by extensive spectroscopic analyses and comparison with the literature data.
Supplementary material Supplementary figures relating to this article are available online.
Acknowledgements Financial support from the National Science and Technology Major Project ‘Key New Drug Creation and Manufacturing Program’ (Grant no. 2013ZX09402203) of the People’s Republic of China is gratefully acknowledged.
Natural Product Research
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