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Four new sesquiterpene polyol esters from Celastrus angulatus
Phytochemistry Letters 7 (2014) 101–106
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Four new sesquiterpene polyol esters from Celastrus angulatus Hai-Yan Zhang a, Tian-Zeng Zhao a,*, Jian-Jun Dong a, Rong-Feng Chen a, Zhi-Hong Li b, Hai-Lin Qin b,** a
Key Laboratory of Natural Products, Henan Academy of Science, Zhengzhou 450002, China State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Key Laboratory of Polymorphic Drugs of Beijing, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 17 June 2013 Received in revised form 10 August 2013 Accepted 3 October 2013 Available online 22 October 2013
Celastrus species, such as Celastrus angulatus, has been demonstrated to be very rich in natural sesquiterpene polyol esters sharing the b-dihydroagarofuran skeleton, some of which showed various biological activities. In this paper, four new sesquiterpene polyol esters, named as angulatins K-N (1–4), along with three known ones, 1b-acetoxy-9b-benzoxy-4a, 6a-dihydroxy-8a, 15-diisobutanoyloxy-2b(a-methyl)-butanoyloxy-b-dihydroagarofuran, angulatin A, and celangulin III, were isolated from the root bark of C. angulatus. The structures of the new compounds were elucidated by extensive spectroscopic methods, mainly including HR-MS and 1D and 2D NMR techniques. ß 2013 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.
Keywords: Celastrus angulatus Sesquiterpene polyol esters Angulatins K-N
1. Introduction Some species belonging to the plant family Celastraceae have been used throughout South America and China to protect cultivated vegetables and crops from insect damage for centuries, and also to treat some human diseases in traditional Chinese medicine (TCM), such as stomach complaint, fever, rheumatoid arthritis, and cancer (Spivey et al., 2002; Bru¨ning and Wagner, 1978; Sua et al., 2009). Phytochemically, these plants have been documented to produce a large spectrum of structurally and biogenetically diverse secondary metabolites, such as sesquiterpenes, alkaloids, triterpenes, diterpines, and flavonoids (Spivey et al., 2002; Bru¨ning and Wagner, 1978; Sua et al., 2009). In particularly, sesquiterpene polyol esters with b-dihydroagarofuran skeleton are very rich in Celastrus species of the family. Many of these sesquiterpene polyol esters exhibit insect antifeedant activity. A couple of these compounds were also found to show moderate or significant cytotoxicity (Spivey et al., 2002; Sua et al., 2009). In our previous studies, some new sesquiterpene polyol esters with b-dihydroagarofuran skeleton were isolated from the root barks and leaves of Celastrus angulatus Maxim., a wild species widely distributed in north-western and central China (Wang et al., 1991; Yin et al., 1999; Yin et al., 1999; Qin et al., 1999; Wu et al.,
* Corresponding author. Tel.: +86 371 65353128; fax: +86 371 65353099. ** Corresponding author. Tel.: +86 010 83172503; fax: +86 010 63017757. E-mail addresses: [email protected] (H.-Y. Zhang), [email protected] (T.-Z. Zhao), [email protected] (H.-L. Qin).
2004; Zhang et al., 2011). As part of our ongoing search for new bioactive natural compounds from Celastraceae plants, four new sesquiterpene polyol esters with b-dihydroagarofuran skeleton, named as angulatins K-N (1–4), respectively, as well as three known ones, 1b-acetoxy-9b-benzoxy-4a, 6a-dihydroxy-8a, 15diisobutanoyloxy-2b-(a-methyl)-butanoyloxy-b-dihydroagarofuran (5) (Wei et al., 2009), angulatin A (6) (Wang et al., 1991), and celangulin III (7) (Wu et al., 1992), were isolated from the root barks of title plant collected in suburban Xian city, Shanxi Province, P.R. China (Fig. 1). This paper describes the isolation and structural determination of these new compounds. 2. Results and discussion Angulatin K (1) was isolated as a white amorphous powder. It was assigned to possess a molecular formula C37H42O14 on the basis of positive HRESI-MS, which indicated 17 degrees of unsaturation. The IR spectrum indicated the presence of ester carbonyl (1750 cm1) and hydroxyl (3434 cm1). The 1H and 13C NMR spectroscopic data of 1 were very similar with those of some b-dihydroagrofuran sesquiterpene polyol esters isolated from Celastrus species, such as angulatin A (6) (Wang et al., 1991), with diagnostic signals of b-dihydroagrofuran skeleton being detected to resonate at dH 1.51 (3H, s, Me-14), 1.59 (3H, s, Me-12), 1.74 (3H, s, Me-13), 4.78 and 4.87 (2H, ABq, J = 13.2 Hz, H2-15) in the 1H NMR spectrum, which correlated to the corresponding three tert-methyl carbon signals at dC 24.5 (C-14), 29.7 (C-12), and 25.7 (C-13), and to the methyleneoxy group at dC 61.4 (C-15), respectively, in the HSQC spectrum, and at dC 84.4 (C-11) and 92.0 (C-5) in the 13C NMR
1874-3900/$ – see front matter ß 2013 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.phytol.2013.10.002
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Fig. 1. Structures of compounds 1–7.
spectrum. In addition, the 1H NMR spectrum (Table 1) also indicated the presence of five oxygen-bearing methine groups at dH 5.49 (1H, d, J = 2.6 Hz), 5.39 (1H, br d, J = 2.6 Hz), 6.71 (1H, s), 5.88 (1H, dd, J = 2.9, 9.6 Hz), and 6.08 (1H, d, J = 9.6 Hz), and one hydroxyl group at dH 2.96 (1H, s) which was confirmed by interchange of the labile proton with deuterium. Four acetoxy and two benzoxy groups were also exhibited by the four methyl groups at dH 1.55, 1.88, 2.08, and 2.46 as four singlets and the ten aromatic protons at dH 7.44–8.19 (10H, m). The 13C NMR and DEPT spectra indicated that 1 contained 37 carbon signals, including twelve quaternary carbon atoms, seven methyls, two methylenes, and sixteen methimes (Table 2). After the one-bond 1H–13C connectivities were established by HSQC experiment, the linkages of the ester functionalities to b-dihydroagarofuran skeleton were determined by HMBC experiment. The long-range 1H–13C correlations from H-6 and H-9 to the carbonyl signals of the two benzoate groups at dC 165.7, from OH-4 to C-4 at dC 69.7, as well as from H-1, H-2, H-8, and H2-15 to the carbonyl signals of four acetate groups at dC 169.6, 169.5, 169.8, and 170.4, respectively, indicated the 1, 2, 8, 15-tetraacetoxy-6, 9-dibenzoxy-4-hydroxy-b-dihydroagrofuran connectivities (Fig. 2). The relative configuration of 1 was elucidated by the 2D NOESY experiment, which showed the NOE correlations between H-1 and H-9, H-1 and H-3ax, H-6 and H8, H-6 and H-14, H-7 and H-12, H-9 and H-13, H-15 and H-14 (Fig. 3). These results confirmed not only the above assignment of each hydrogen atom, but also the a-orientations for H-1, H-3ax, H9 and OH-4, and the b-configurations for H-6 and H-8. The characteristic fragments in its EI-MS at m/z 43 [CH3CBBO]+, 105 [(C6H5)CBBO]+, 202 [(CH3)2CHCH5 5CHCH2OBu]+, and 710 [M]+ further confirmed the above results (Wang et al., 1991; Wakakayashi et al., 1988). Thus, the structure of 1 was elucidated to be 1b, 2b, 8a, 15-tetraacetoxy-6a, 9b-dibenzoxy-4a-hydroxy-bdihydroagrofuran, and it was given the trivial name angulatin K. Angulatin L (2) was isolated as a white amorphous powder. The molecular formula was determined to be C33H38O15 by the pseudomolecular ion peak in the positive HRESI-MS. The IR spectrum indicated the presence of ester carbonyl groups (1745 cm1), with no hydroxyl being observed. The 13C NMR
spectrum displayed 33 carbon signals, which were assigned by DEPT experiments as seven methyls, two methylenes, thirteen methines, and eleven quaternary carbons (Table 2). The features of b-dihydroagarofuran sesquiterpene were indicated by the three tert-methyl carbon signals at dC 16.8 (C-14), 30.3 (C-12), and 25.7 (C-13), one methyleneoxy carbon signal at dC 65.6 (C-15), and two quaternary oxygen-bearing carbon signals at dC 81.1 (C-11) and 89.6 (C-5) in the 13C NMR spectrum, and the substitution of four acetates and two furoates were also determined from the remaining signals (Wu et al., 1992). This conclusion was confirmed by the 1H NMR spectrum which showed signals due to six methyls linked at quaternary carbons at dH 1.46, 1.59, 1.65, 2.13 (each 3H, s), and 2.08 (6H, s) and one methyl at dH 1.16 (d, J = 7.6 Hz) linked to methine, one oxygen-bearing methylene at dH 4.62, 5.06 (2H, ABq, J = 12.8 Hz), and five oxygen-bearing methines at dH 5.64 (1H, d, J = 3.8 Hz), 5.59 (1H, m), 6.42 (1H, s), 5.41 (1H, d, J = 2.7 Hz), and 5.60 (1H, s) (Table 1). The NMR data of 2 was very similar to those of Celahin D, a b-dihydroagrofuran sesquiterpene polyol ester isolated from C. hindsii (Wakakayashi et al., 1988). Their main difference was the absence of the signals of two sets of benzoate groups in the former compared to the latter, yet, two furoate groups indicated by the signals at dH 8.21 (1H, s), 6.86 (1H, d, J = 1.4 Hz), 7.46 (1H, d, J = 1.4 Hz), and 8.01 (1H, br s), 6.73 (1H, d, J = 1.5 Hz), 7.43 (1H, br d, J = 1.5 Hz) were evident in the former, which were correlated to their corresponding carbon signals by means of HSQC experiment. The long-range 1H–13C correlations in HMBC spectrum revealed that the four acetoxyls were linked at C1, C-2, C-6, and C-15, and the two furoate groups were linked at C-8 and C-9, respectively (Fig. 2). The key NOESY correlations of H-1 with H-3ax, H-6 with H-14, and H-7 with H-12 (Fig. 3) confirmed not only the assignment of relevant hydrogen atom, but also the aorientation of H-1, H-2 and H-8 and the b-orientation of H-6 and H9. Therefore, the structure of 2 was inferred to be 1b, 2b, 6a, 15tetraacetoxy-8b, 9a-difuroyloxy-b-dihydroagrofuran, and it was given the trivial name angulatin L. Angulatin M (3) was isolated as a white amorphous powder. It had the molecular formula C34H46O13 according to its positive HRESI-MS. The IR spectrum showed the presence of ester carbonyl
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Table 1 1 H NMR spectroscopic data for compounds 1–4 (400 MHz, CDCl3). Position
1
1 2 3eq 3ax 4 6 7 8 9 12 13 14 15
5.49 5.39 2.01 2.15
d (2.6) br d (2.6) br d (13.1) br d (13.1)
6.71 2.66 5.88 6.08 1.59 1.74 1.51 4.78 4.87 1.55 2.08
s d (2.9) dd (2.9, 9.6) d (9.6) s s s d (13.2) d (13.2) s s
1.88 2.46 7.92 7.44 7.60
s s ma m m
OAc-1 OAc-2 OAc-6 OAc-8 OAc-15 OBz-6
20 ,60 30 ,50 40
2 5.64 5.59 1.77 2.50 2.39 6.42 2.46 5.41 5.60 1.46 1.59 1.16 4.62 5.06 1.65 2.08 2.13
3 d (3.8) m m m m s d (2.7) d (2.7) s s s d (7.6) d (12.8) d (12.8) s s s
2.08 s
4
5.49 5.42 2.02 2.10
d (3.2) m dd (2.5, 14.9) m
5.54 5.54 1.98 2.22
s m dd (2.0, 14.8) dd (2.8, 14.8)
5.46 5.36 1.98 2.09
d (3.4) m dd (2.8, 14.9) m
5.22 2.57 5.64 6.07 1.62 1.72 1.79 4.76
br d (5.1) br d (3.2) dd (3.2, 9.8) d (9.8) s s br s s
6.68 2.48 5.50 5.70 1.62 1.71 1.48 4.67 5.08 1.66 2.11 2.13
br s br d (2.6) d (2.6) s s s s d (13.0) d (13.0) s sb sb
5.22 2.55 5.59 6.04 1.59 1.70 1.75 4.63 4.85 1.52 2.07
s d (3.2) dd (3.2, 9.8) d (9.8) s s s d (13.2) d (13.2) s s
1.53 s
2.35 s
OBz-8
OBz-9
OFu-8
OFu-9
OiBu-2
20 ,60 30 ,50 40 20 40 50 20 40 50 10 20 10 20
OiBu-15
10 20
a,b
2.04 s
8.17 m 7.48 m 7.60 m
OiBu-8
OH-4 OH-6
Angulatin A
8.19 ma 7.44 m 7.60 m
7.84 m 7.42 m 7.55 m 8.21 6.86 7.46 8.01 6.73 7.43
s d (1.4) d (1.4) br s d (1.5) br d (1.5)
8.03 br s 6.74 br d (1.1) 7.44 br d (1.1) 2.36 0.89 0.94 2.57 1.18 1.19
2.96 s
7.83 m 7.39 m 7.52 m
sept (7.0) d (7.0) d (7.0) sept (7.0) d (7.0) d (7.0)
3.11 br s 5.25 d (5.1)
2.72 s
2.35 sept (7.0) 0.89 d (7.0) 0.93 d (7.0) 2.81 sept (7.0) 1.321 d (7.0) 1.324 d (7.0) 3.12 br s 5.22 br s
Interchangeable signals within the same letters.
(1736 cm1) and hydroxyl (3433 cm1). From 1D (1H, 13C, and DEPT) and 2D (1H–1H COSY, HSQC and HMBC) NMR spectra of 3, as well as their comparisons with those of angulatin A (Wang et al., 1991), it was indicated that the similarities between the two compounds were very apparent, with compound 3 showing almost the same categories of signals as those of the latter (see Tables 1 and 2). The main differences were determined by the chemical shifts to be the different linkages of ester groups in 3 from angulatin A, which were confirmed by the long-range 1 H–13C correlations in HMBC spectrum between H-2 at dH 5.42 (m) and H-8 at dH 5.64 (dd, J = 3.2, 9.8 Hz) with the carbonyl signals of isobutanoyloxy groups at dC 175.7 and 175.9, respectively. Other key long-range 1H–13C correlations in HMBC spectrum see Fig. 2. The NOE correlations of H-1 (dH 5.49 (d, J = 3.2 Hz)) with H-3ax (dH 2.10 (m)), H-1 with H-9 (dH 6.07 (d, J = 9.8 Hz)), H-6 (dH 5.22 (br d, J = 5.1 Hz)) with H-8, H-6 with H14 (dH 1.79 (br s)), H-7 (dH 2.57 (br d, J = 3.2 Hz)) with H-12 (dH 1.62 (s)), and H-9 with H-13 (dH 1.72 (s)) (Fig. 3) confirmed not only the assignment of relevant hydrogen atom, but also the aorientations of H-1, H-2, and H-9 and the b-orientations of H-6 and H-8. As such, the structure of 3 was determined to be 1b,
15-diacetoxy-9b-benzoxy-2b,8a-diisobutanoyloxy-b-dihydroagrofuran, and it was given the trivial name angulatin M. Angulatin N (4) was isolated as a white amorphous powder. It had the molecular formula C35H40O15 according to its positive HRESI-MS. The IR spectrum showed the presence of ester carbonyl (1746 cm1) and hydroxyl (3433 cm1) groups. The 1H NMR spectrum (Table 1) showed seven methyls at dH 1.62, 1.71, 1.48, 1.66, 2.11, 2.13, and 2.04 as singlets, of which, four acetoxyls were evident from the 1H/13C (DEPT) NMR data at dH/dC 1.66 (s)/169.4 (s) and 20.5 (q), 2.11 (s)/169.8 (s) and 21.1 (q), 2.13 (s)/169.8 (s) and 21.2 (q), and 2.04 (s)/170.6 (s) and 21.5 (q)) assigned by the combination of HSQC and HMBC experiments. Besides, one furoyloxy unit confirmed by both dH 8.03 (1H, br s), 6.74 (1H, br d, J = 1.1 Hz), and 7.44 (1H, br d, J = 1.1 Hz) and dC 160.6 (s), 148.9 (d), 118.0 (s), 109.8 (d), and 144.0 (d) and one benzoxy unit characterized by dH 8.17 (2H, m), 7.48 (2H, m), and 7.60 (1H, m) and dC 165.4 (s), 129.6 (s), 130.0 (d), 128.4 (d), and 133.5 (d) were also evident on the basis of the one-bond 1H–13C connectivities from the HSQC experiment. These consequences were compatible with the long-range 1H–13C correlations from HMBC experiments, including those from H-8 at dH 5.50 (1H, d, J = 2.6 Hz) to dC 165.45
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Table 2 13 C and DEPT NMR spectroscopic data for compounds 1–4 (100 MHz, CDCl3). Position
1
2
3
4
Angulatin A
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 OAc-1
75.1 (d) 67.7 (d) 42.4 (t) 69.7 (s) 92.0 (s) 76.0 (d) 52.2 (d) 73.6 (d) 75.6 (d) 51.6 (s) 84.4 (s) 29.7 (q) 25.7 (q) 24.5 (q) 61.4 (t) 169.6 (s) 20.5 (q) 169.5 (s) 21.2 (q)
71.5 (d) 69.2 (d) 31.1 (t) 32.8 (d) 89.6 (s) 75.0 (d) 53.6 (d) 77.0 (d) 72.5 (d) 52.6 (s) 81.4 (s) 30.3 (q) 25.7 (q) 16.8 (q) 65.6 (t) 169.5 (s) 20.5 (q) 170.6 (s) 21.2 (q)c 169.7 (s) 21.3 (q)
74.9 (d) 67.0 (d) 41.3 (t) 72.1 (s) 91.5 (s) 77.2 (d) 53.6 (d) 73.4 (d) 75.5 (d) 50.6 (s) 84.6 (s) 30.1 (q) 26.3 (q) 24.2 (q) 61.8 (t) 169.4 (s) 20.4 (q)
70.5 (d) 67.8 (d) 41.9 (t) 69.9 (s) 91.4 (s) 75.2 (d) 53.7 (d) 77.2 (d) 72.2 (d) 54.2 (s) 83.2 (s) 29.5 (q) 25.6 (q) 24.2 (q) 65.8 (q) 169.4 (s) 20.5 (q) 169.8 (s) 21.1 (q)d 169.8 (s) 21.2 (q)d
75.04 67.33 41.13 72.06 91.49 76.91 53.59 73.76 75.23 50.64 84.44 30.00 26.67 24.19 61.68 169.30 20.37 169.40 20.98
170.0 (s) 21.3 (q)c
170.4 (s) 21.5 (q)
170.6 (s) 21.5 (q)d
OAc-2 OAc-6 OAc-8 OAc-15 OBz-6
OBz-8
OBz-9
OFu-8
OFu-9
OiBu-2
C5 5O 10 20 ,60 30 ,50 40 C5 5O 10 20 ,60 30 ,50 40 C5 5O 10 20 ,60 30 ,50 40 C5 5O 20 30 40 50 C5 5O 20 30 40 50 C5 5O 10 20
OiBu-8
C5 5O 10 20
OiBu-15
C5 5O 10 20
a,b,c,d
169.8 (s) 20.8 (q) 170.4 (s) 21.4 (q) 165.7 (s) 129.2 (s) 129.6 (d)a 128.7 (d)b 133.5 (d)
165.4 129.6 130.0 128.4 133.5 165.7 129.2 130.1 128.7 133.5
(s) (s) (d)a (d)b (d)
165.8 129.5 129.4 128.7 133.5 161.7 148.6 119.0 110.0 143.9 160.6 148.8 118.2 109.8 143.9
(s) (s) (d) (d) (d)
(s) (s) (d) (d) (d)
(s) (d) (s) (d) (d) (s) (d) (s) (d) (d)
165.59 129.40 129.40 128.53 133.30
160.6 148.9 118.0 109.8 144.0 175.7 (s) 34.1 (d) 18.5 (q) 18.6 (q) 175.9 (s) 34.3 (d) 18.9 (q) 18.9 (q)
(s) (d) (s) (d) (d)
175.65 34.03 18.40 18.56 176.40 34.28 18.96 19.08
Interchangeable signals within the same letters.
(C5 5O), and from H-9 at dH 5.70 (1H, s) to dC 160.6 (C5 5O), which are indicators that the benzoate group was located at C-8 and the furoate group at C-9 (Fig. 2). Other key long-range 1H–13C correlations in HMBC spectrum of 4 see Fig. 2. The characteristic fragments in its EI-MS at m/z 43 [CH3CBBO+], 95 [(C4H3O)CBBO]+, 105 [(C6H5)CBBO]+, and 192 [(CH3)2CHCH5 5CHCH2OFu]+ further confirmed the above results. The remaining 1H and 13C NMR
spectroscopic data of 4 were very similar to those of its counterparts in the known celangulatin E (Huang et al., 2000), indicating that compound 4 possesses the same skeleton of bdihydroagarofuran as the latter. The NOE correlations of H-1 at dH 5.54 (s) with H-3ax at dH 2.22 (dd, J = 2.8, 14.8 Hz), H-6 with H-14 at dH 1.48 (s), and H-7 with H-12 at dH 1.62 (s) suggested the aorientation of H-1, H-2, and H-8 and the b-orientation of H-6 and
H.-Y. Zhang et al. / Phytochemistry Letters 7 (2014) 101–106
105
O
O
O
O
O
O O
O
O
O O
O O
OH
O
O O
O
1
2 O
O
O
O
O O
O
O
O
O O
O O
O
O
O
O
O
O
O
O O
O
O
O
O
O
O O
O
O
O
O
O
OH
O
OH
OH
O
3
4 Fig. 2. Key HMBC correlations in 1–4.
H-9 (Fig. 3). On the basis of the above results, the structure of 4 was elucidated to be 1b,2b,6a,15-tetraacetoxy-8b-benzoxy-9a-furoyloxy-4a-hydroxy-b-dihydroagrofuran, and it was given the trivial name angulatin N.
Henan Agriculture University. A voucher specimen (CA08) is deposited at the Key Laboratory of Natural Products, Henan Academy of Science, China. 3.3. Extraction and isolation
3. Experimental 3.1. General experimental procedures Optical rotations were obtained on a Perkin Elemer 341 polarimeter. IR (KBr) spectra were recorded on a Testscan Shimadzu FTIR 8000 series HYPER infrared spectrometer. 1D and 2D NMR spectra were taken on a DRX-400 spectrometer with tetramethylsilane as internal standard and CDCl3 as solvent. EI-MS were measured on an Autospec-UltimaETOF spectrometer, in m/z (rel.%). HRESI-MS were performed on a Q-Tof MicroTM instrument. TLC were carried out on glass plate precoated with silica gel G (Qingdao Marine Chemical Factory), and petroleum ether (PE)/acetone 3:1 was used as eluent with visualization being done under UV light at l 365 nm, followed by spraying with 10% aq. H2SO4 and heating at 110 8C. Column chromatographies were undertaken over silica gel (200–300 mesh; Qingdao Marine Chemical Factory). 3.2. Plant material The root bark of C. angulatus were collected in suburban Xian City, Shanxi Province, P. R. China, in June 2008, and taxonomically authenticated by Prof. Chang-Shan Zhu, the Department of Botany,
Dried and powdered root bark (4.5 kg) of C. angulatus were extracted for three times with petroleum ether (2 h, 1 h, 1 h) under reflux condition. After evaporation of the filtrate under vacuum condition, a yellow semisolid residue (105 g) was obtained. The residue was dissolved in 80% methanol and extracted with petroleum ether for several times until the upper solvent being very transparent. The 80% methanol soluble portions were evaporated under vacuum condition to afford another yellow residue (71 g), which was chromatographed on a silica gel column eluting with a gradient system of petroleum ether/EtOAc (10:1, 9:1, 7:1, 6:1, 5:1, 4:1, 7:3, 6:4, 4:6) to give 300 fractions (each 500 mL). Fraction 85 was subjected to reversed RP-18 column eluted with MeOHH2O = 65:35 to afford compounds 1 (9.5 mg) and 2 (5 mg), both as white amorphous powder. Fractions 110–113 was collected according to TLC detection and subjected to reversed RP-18 column (MeOHH2O, 65:35) to yield compound 3 (6 mg). Fraction 240 was separated on RP-18 gel column (MeOHH2O, 65:35) to give compound 4 (15 mg). Compound 5 (7 mg) was obtained from fraction 66 by purification with preparative HPLC (RP-18, MeOHH2O, 65:35). Compounds 6 (60 mg) and 7 (8 mg) were crystallized in methanol from fraction 121 and fraction 212, respectively.
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Fig. 3. Key NOE correlations in 1–4.
3.3.1. Angulatin K (1) White amorphous powder; +62.5 (MeOH; c 0.016); IR, nmax cm1: 3434, 2930, 1750, 1601, 1451, 1369, 1273, 1228, 1153, 1107, 1070, 1045, 860, 714 cm; 1H and 13C NMR, see Tables 1 and 2; EIMS m/z: 710 (0.3%), 650 (1.8%), 528 (8.4%), 395 (8.0%), 336 (31.9%), 268 (10.5%), 244 (18.0%), 202 (26.0%), 164 (10.2%), 137 (9.9%), 105 (100%), 77 (34.7%), 43 (31.0%); HRESIMS (positive-ion mode) m/z: 733.2476 [M+Na]+, (calcd for C37H42NaO14, 733.2470). 3.3.2. Angulatin L (2) White amorphous powder; +10.0 (MeOH; c 0.01); IR, nmax cm1: 2923, 2852, 1745, 1508, 1369, 1311, 1231, 1160, 1081, 874, 760, 604; 1H and 13C NMR, see Tables 1 and 2; EIMS m/z: 632 (7.7%), 590 (2.2%), 243 (15.8%), 237 (10.5%), 218 (8.6%), 201 (9.3%), 189 (5.0%), 159 (8.0%), 135 (11.4%), 95 (100%), 83 (21.0%), 77 (3.1%), 43 (25.4%); HRESIMS (positive-ion mode) m/z: 697.2110 [M+Na]+, (calcd for C33H38NaO15, 697.2106). 3.3.3. Angulatin M (3) White amorphous powder; 18.0 (MeOH; c 0.10); IR, nmax cm1: 3433, 2917, 2866, 1736, 1637, 1384, 1274, 1228, 1142, 1108, 1026, 879, 714; 1H and 13C NMR, see Tables 1 and 2; EIMS m/z: 261 (2.2%), 244 (5.9%), 229 (7.1%), 202 (15.5%), 164 (11.1%), 105 (100%), 77 (17.0%), 43 (19.2%); HRESIMS (positive-ion mode) m/z: 685.2833 [M+Na]+, (calcd for C34H46NaO13, 685.3408). 3.3.4. Angulatin N (4) White amorphous powder; 5.9 (MeOH; c 0.07); IR, nmax cm1:3433, 2924, 2852, 1746, 1369, 1309, 1230, 1159, 874, 760, 716, 604; 1H and 13C NMR, see Tables 1 and 2; EIMS m/z: 262 (4.3%), 244 (6.8%), 192 (50.8%), 164 (9.6%), 137 (5.0%), 105 (100%), 95 (81.4%), 77 (8.4%), 43 (24.8%). HRESIMS (positive-ion mode) m/z: 723.2256 [M+Na]+, (calcd for C35H40NaO15, 723.2261).
Acknowledgements We are grateful to Mr. W.G. Zhu, Y.X. Xie and Ms. S.M. Wang for the NMR and MS measurements. This work was supported by grants from National Science and Technology Project of China (2011ZX09307-002-01, 2012ZX09301002-002) References ¨ bersicht u¨ber die celastraceen-inhaltsstoffe: chemie, Bru¨ning, R., Wagner, H., 1978. U chemotaxonomie, biosynthese, pharmakologie. Phytochemistry 17, 1821–1858. Huang, H.C., Shen, C.C., Chen, C.F., Wu, Y.C., Ku, Y.H., 2000. A novel agarofuran sesquiterpene, Celahin D from Celastrus hindsii Benth. Chem. Pharm. Bull. 48, 1079–1080. Qin, H.L., Zhao, T.Z., Shang, Y.J., 1999. A new sesquiterpene from Celastrus angulatus. Chin. Chem. Lett. 10, 825–826. Spivey, A.C., Weston, M., Woodhead, S., 2002. Celastraceae sesquiterpenoids: biological activity and synthesis. Chem. Soc. Rev. 31, 43–59. Sua, X.H., Zhang, M.L., Zhan, W.H., Huo, C.H., Shi, Q.W., Gu, Y.C., 2009. Chemical and pharmacological studies of the plants from genus Celastrus. Chem. Biodivers. 6, 146–161. Wakakayashi, N., Wu, W.J., Waters, R.M., Redfern, R.E., Mills Jr., G.D., Demilo, A.B., 1988. Celangulin: a nonalkaloidal insect antifeedant from Chinese bittersweet, Celastrus angulatus. J. Nat. Prod. 51, 537–542. Wang, M.T., Qin, H.L., Kong, M., Li, Y.Z., 1991. Insecticidal sesquiterpene polyol ester from Celastrus angulatus. Phytochemistry 30, 3931–3933. Wei, S., Wang, M., Zhang, J., Qian, Y., Ji, Z., Wu, W., 2009. Three novel sesquiterpene esters from Celastrus angulatus. Nat. Prod. Commun. 4, 461–466. Wu, W.J., Tu, Y.Q., Liu, H.X., Zhu, J.B., 1992. Celangulins II, III, and IV: new insecticidal sesquiterpenoids from Celastrus angulatus. J. Nat. Prod. 55, 1294–1298. Wu, M.J., Zhao, T.Z., Shang, Y.J., Fu, J.G., Zhang, H.Y., 2004. A new sesquiterpene polyol ester from Celastrus angulatus. Chin. Chem. Lett. 15, 41–42. Yin, W.P., Zhao, T.Z., Gao, L.J., Zou, D.P., Kang, J.X., 1999. A novel sesquiterpene polyol ester from Celastrus angulatus and its antitumor activities. Chin. Chem. Lett. 10, 487–490. Yin, W.P., Zhao, T.Z., Gao, L.J., Qin, H.L., Zou, D.P., Kang, J.X., 1999. Structure determination of two new derivatives of sesquiterpenes by NMR techniques. Chin. J. Magn. Reson. 16, 225–229. Zhang, H.Y., Zhao, T.Z., Wei, Y., Liu, H.M., 2011. Two new sesquiterpene polyol esters from the root barks of Celastrus angulatus. J. Asian Nat. Prod. Res. 13, 304–311.
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Phytochemistry Letters journal homepage: www.elsevier.com/locate/phytol
Four new sesquiterpene polyol esters from Celastrus angulatus Hai-Yan Zhang a, Tian-Zeng Zhao a,*, Jian-Jun Dong a, Rong-Feng Chen a, Zhi-Hong Li b, Hai-Lin Qin b,** a
Key Laboratory of Natural Products, Henan Academy of Science, Zhengzhou 450002, China State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Key Laboratory of Polymorphic Drugs of Beijing, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 17 June 2013 Received in revised form 10 August 2013 Accepted 3 October 2013 Available online 22 October 2013
Celastrus species, such as Celastrus angulatus, has been demonstrated to be very rich in natural sesquiterpene polyol esters sharing the b-dihydroagarofuran skeleton, some of which showed various biological activities. In this paper, four new sesquiterpene polyol esters, named as angulatins K-N (1–4), along with three known ones, 1b-acetoxy-9b-benzoxy-4a, 6a-dihydroxy-8a, 15-diisobutanoyloxy-2b(a-methyl)-butanoyloxy-b-dihydroagarofuran, angulatin A, and celangulin III, were isolated from the root bark of C. angulatus. The structures of the new compounds were elucidated by extensive spectroscopic methods, mainly including HR-MS and 1D and 2D NMR techniques. ß 2013 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.
Keywords: Celastrus angulatus Sesquiterpene polyol esters Angulatins K-N
1. Introduction Some species belonging to the plant family Celastraceae have been used throughout South America and China to protect cultivated vegetables and crops from insect damage for centuries, and also to treat some human diseases in traditional Chinese medicine (TCM), such as stomach complaint, fever, rheumatoid arthritis, and cancer (Spivey et al., 2002; Bru¨ning and Wagner, 1978; Sua et al., 2009). Phytochemically, these plants have been documented to produce a large spectrum of structurally and biogenetically diverse secondary metabolites, such as sesquiterpenes, alkaloids, triterpenes, diterpines, and flavonoids (Spivey et al., 2002; Bru¨ning and Wagner, 1978; Sua et al., 2009). In particularly, sesquiterpene polyol esters with b-dihydroagarofuran skeleton are very rich in Celastrus species of the family. Many of these sesquiterpene polyol esters exhibit insect antifeedant activity. A couple of these compounds were also found to show moderate or significant cytotoxicity (Spivey et al., 2002; Sua et al., 2009). In our previous studies, some new sesquiterpene polyol esters with b-dihydroagarofuran skeleton were isolated from the root barks and leaves of Celastrus angulatus Maxim., a wild species widely distributed in north-western and central China (Wang et al., 1991; Yin et al., 1999; Yin et al., 1999; Qin et al., 1999; Wu et al.,
* Corresponding author. Tel.: +86 371 65353128; fax: +86 371 65353099. ** Corresponding author. Tel.: +86 010 83172503; fax: +86 010 63017757. E-mail addresses: [email protected] (H.-Y. Zhang), [email protected] (T.-Z. Zhao), [email protected] (H.-L. Qin).
2004; Zhang et al., 2011). As part of our ongoing search for new bioactive natural compounds from Celastraceae plants, four new sesquiterpene polyol esters with b-dihydroagarofuran skeleton, named as angulatins K-N (1–4), respectively, as well as three known ones, 1b-acetoxy-9b-benzoxy-4a, 6a-dihydroxy-8a, 15diisobutanoyloxy-2b-(a-methyl)-butanoyloxy-b-dihydroagarofuran (5) (Wei et al., 2009), angulatin A (6) (Wang et al., 1991), and celangulin III (7) (Wu et al., 1992), were isolated from the root barks of title plant collected in suburban Xian city, Shanxi Province, P.R. China (Fig. 1). This paper describes the isolation and structural determination of these new compounds. 2. Results and discussion Angulatin K (1) was isolated as a white amorphous powder. It was assigned to possess a molecular formula C37H42O14 on the basis of positive HRESI-MS, which indicated 17 degrees of unsaturation. The IR spectrum indicated the presence of ester carbonyl (1750 cm1) and hydroxyl (3434 cm1). The 1H and 13C NMR spectroscopic data of 1 were very similar with those of some b-dihydroagrofuran sesquiterpene polyol esters isolated from Celastrus species, such as angulatin A (6) (Wang et al., 1991), with diagnostic signals of b-dihydroagrofuran skeleton being detected to resonate at dH 1.51 (3H, s, Me-14), 1.59 (3H, s, Me-12), 1.74 (3H, s, Me-13), 4.78 and 4.87 (2H, ABq, J = 13.2 Hz, H2-15) in the 1H NMR spectrum, which correlated to the corresponding three tert-methyl carbon signals at dC 24.5 (C-14), 29.7 (C-12), and 25.7 (C-13), and to the methyleneoxy group at dC 61.4 (C-15), respectively, in the HSQC spectrum, and at dC 84.4 (C-11) and 92.0 (C-5) in the 13C NMR
1874-3900/$ – see front matter ß 2013 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.phytol.2013.10.002
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Fig. 1. Structures of compounds 1–7.
spectrum. In addition, the 1H NMR spectrum (Table 1) also indicated the presence of five oxygen-bearing methine groups at dH 5.49 (1H, d, J = 2.6 Hz), 5.39 (1H, br d, J = 2.6 Hz), 6.71 (1H, s), 5.88 (1H, dd, J = 2.9, 9.6 Hz), and 6.08 (1H, d, J = 9.6 Hz), and one hydroxyl group at dH 2.96 (1H, s) which was confirmed by interchange of the labile proton with deuterium. Four acetoxy and two benzoxy groups were also exhibited by the four methyl groups at dH 1.55, 1.88, 2.08, and 2.46 as four singlets and the ten aromatic protons at dH 7.44–8.19 (10H, m). The 13C NMR and DEPT spectra indicated that 1 contained 37 carbon signals, including twelve quaternary carbon atoms, seven methyls, two methylenes, and sixteen methimes (Table 2). After the one-bond 1H–13C connectivities were established by HSQC experiment, the linkages of the ester functionalities to b-dihydroagarofuran skeleton were determined by HMBC experiment. The long-range 1H–13C correlations from H-6 and H-9 to the carbonyl signals of the two benzoate groups at dC 165.7, from OH-4 to C-4 at dC 69.7, as well as from H-1, H-2, H-8, and H2-15 to the carbonyl signals of four acetate groups at dC 169.6, 169.5, 169.8, and 170.4, respectively, indicated the 1, 2, 8, 15-tetraacetoxy-6, 9-dibenzoxy-4-hydroxy-b-dihydroagrofuran connectivities (Fig. 2). The relative configuration of 1 was elucidated by the 2D NOESY experiment, which showed the NOE correlations between H-1 and H-9, H-1 and H-3ax, H-6 and H8, H-6 and H-14, H-7 and H-12, H-9 and H-13, H-15 and H-14 (Fig. 3). These results confirmed not only the above assignment of each hydrogen atom, but also the a-orientations for H-1, H-3ax, H9 and OH-4, and the b-configurations for H-6 and H-8. The characteristic fragments in its EI-MS at m/z 43 [CH3CBBO]+, 105 [(C6H5)CBBO]+, 202 [(CH3)2CHCH5 5CHCH2OBu]+, and 710 [M]+ further confirmed the above results (Wang et al., 1991; Wakakayashi et al., 1988). Thus, the structure of 1 was elucidated to be 1b, 2b, 8a, 15-tetraacetoxy-6a, 9b-dibenzoxy-4a-hydroxy-bdihydroagrofuran, and it was given the trivial name angulatin K. Angulatin L (2) was isolated as a white amorphous powder. The molecular formula was determined to be C33H38O15 by the pseudomolecular ion peak in the positive HRESI-MS. The IR spectrum indicated the presence of ester carbonyl groups (1745 cm1), with no hydroxyl being observed. The 13C NMR
spectrum displayed 33 carbon signals, which were assigned by DEPT experiments as seven methyls, two methylenes, thirteen methines, and eleven quaternary carbons (Table 2). The features of b-dihydroagarofuran sesquiterpene were indicated by the three tert-methyl carbon signals at dC 16.8 (C-14), 30.3 (C-12), and 25.7 (C-13), one methyleneoxy carbon signal at dC 65.6 (C-15), and two quaternary oxygen-bearing carbon signals at dC 81.1 (C-11) and 89.6 (C-5) in the 13C NMR spectrum, and the substitution of four acetates and two furoates were also determined from the remaining signals (Wu et al., 1992). This conclusion was confirmed by the 1H NMR spectrum which showed signals due to six methyls linked at quaternary carbons at dH 1.46, 1.59, 1.65, 2.13 (each 3H, s), and 2.08 (6H, s) and one methyl at dH 1.16 (d, J = 7.6 Hz) linked to methine, one oxygen-bearing methylene at dH 4.62, 5.06 (2H, ABq, J = 12.8 Hz), and five oxygen-bearing methines at dH 5.64 (1H, d, J = 3.8 Hz), 5.59 (1H, m), 6.42 (1H, s), 5.41 (1H, d, J = 2.7 Hz), and 5.60 (1H, s) (Table 1). The NMR data of 2 was very similar to those of Celahin D, a b-dihydroagrofuran sesquiterpene polyol ester isolated from C. hindsii (Wakakayashi et al., 1988). Their main difference was the absence of the signals of two sets of benzoate groups in the former compared to the latter, yet, two furoate groups indicated by the signals at dH 8.21 (1H, s), 6.86 (1H, d, J = 1.4 Hz), 7.46 (1H, d, J = 1.4 Hz), and 8.01 (1H, br s), 6.73 (1H, d, J = 1.5 Hz), 7.43 (1H, br d, J = 1.5 Hz) were evident in the former, which were correlated to their corresponding carbon signals by means of HSQC experiment. The long-range 1H–13C correlations in HMBC spectrum revealed that the four acetoxyls were linked at C1, C-2, C-6, and C-15, and the two furoate groups were linked at C-8 and C-9, respectively (Fig. 2). The key NOESY correlations of H-1 with H-3ax, H-6 with H-14, and H-7 with H-12 (Fig. 3) confirmed not only the assignment of relevant hydrogen atom, but also the aorientation of H-1, H-2 and H-8 and the b-orientation of H-6 and H9. Therefore, the structure of 2 was inferred to be 1b, 2b, 6a, 15tetraacetoxy-8b, 9a-difuroyloxy-b-dihydroagrofuran, and it was given the trivial name angulatin L. Angulatin M (3) was isolated as a white amorphous powder. It had the molecular formula C34H46O13 according to its positive HRESI-MS. The IR spectrum showed the presence of ester carbonyl
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Table 1 1 H NMR spectroscopic data for compounds 1–4 (400 MHz, CDCl3). Position
1
1 2 3eq 3ax 4 6 7 8 9 12 13 14 15
5.49 5.39 2.01 2.15
d (2.6) br d (2.6) br d (13.1) br d (13.1)
6.71 2.66 5.88 6.08 1.59 1.74 1.51 4.78 4.87 1.55 2.08
s d (2.9) dd (2.9, 9.6) d (9.6) s s s d (13.2) d (13.2) s s
1.88 2.46 7.92 7.44 7.60
s s ma m m
OAc-1 OAc-2 OAc-6 OAc-8 OAc-15 OBz-6
20 ,60 30 ,50 40
2 5.64 5.59 1.77 2.50 2.39 6.42 2.46 5.41 5.60 1.46 1.59 1.16 4.62 5.06 1.65 2.08 2.13
3 d (3.8) m m m m s d (2.7) d (2.7) s s s d (7.6) d (12.8) d (12.8) s s s
2.08 s
4
5.49 5.42 2.02 2.10
d (3.2) m dd (2.5, 14.9) m
5.54 5.54 1.98 2.22
s m dd (2.0, 14.8) dd (2.8, 14.8)
5.46 5.36 1.98 2.09
d (3.4) m dd (2.8, 14.9) m
5.22 2.57 5.64 6.07 1.62 1.72 1.79 4.76
br d (5.1) br d (3.2) dd (3.2, 9.8) d (9.8) s s br s s
6.68 2.48 5.50 5.70 1.62 1.71 1.48 4.67 5.08 1.66 2.11 2.13
br s br d (2.6) d (2.6) s s s s d (13.0) d (13.0) s sb sb
5.22 2.55 5.59 6.04 1.59 1.70 1.75 4.63 4.85 1.52 2.07
s d (3.2) dd (3.2, 9.8) d (9.8) s s s d (13.2) d (13.2) s s
1.53 s
2.35 s
OBz-8
OBz-9
OFu-8
OFu-9
OiBu-2
20 ,60 30 ,50 40 20 40 50 20 40 50 10 20 10 20
OiBu-15
10 20
a,b
2.04 s
8.17 m 7.48 m 7.60 m
OiBu-8
OH-4 OH-6
Angulatin A
8.19 ma 7.44 m 7.60 m
7.84 m 7.42 m 7.55 m 8.21 6.86 7.46 8.01 6.73 7.43
s d (1.4) d (1.4) br s d (1.5) br d (1.5)
8.03 br s 6.74 br d (1.1) 7.44 br d (1.1) 2.36 0.89 0.94 2.57 1.18 1.19
2.96 s
7.83 m 7.39 m 7.52 m
sept (7.0) d (7.0) d (7.0) sept (7.0) d (7.0) d (7.0)
3.11 br s 5.25 d (5.1)
2.72 s
2.35 sept (7.0) 0.89 d (7.0) 0.93 d (7.0) 2.81 sept (7.0) 1.321 d (7.0) 1.324 d (7.0) 3.12 br s 5.22 br s
Interchangeable signals within the same letters.
(1736 cm1) and hydroxyl (3433 cm1). From 1D (1H, 13C, and DEPT) and 2D (1H–1H COSY, HSQC and HMBC) NMR spectra of 3, as well as their comparisons with those of angulatin A (Wang et al., 1991), it was indicated that the similarities between the two compounds were very apparent, with compound 3 showing almost the same categories of signals as those of the latter (see Tables 1 and 2). The main differences were determined by the chemical shifts to be the different linkages of ester groups in 3 from angulatin A, which were confirmed by the long-range 1 H–13C correlations in HMBC spectrum between H-2 at dH 5.42 (m) and H-8 at dH 5.64 (dd, J = 3.2, 9.8 Hz) with the carbonyl signals of isobutanoyloxy groups at dC 175.7 and 175.9, respectively. Other key long-range 1H–13C correlations in HMBC spectrum see Fig. 2. The NOE correlations of H-1 (dH 5.49 (d, J = 3.2 Hz)) with H-3ax (dH 2.10 (m)), H-1 with H-9 (dH 6.07 (d, J = 9.8 Hz)), H-6 (dH 5.22 (br d, J = 5.1 Hz)) with H-8, H-6 with H14 (dH 1.79 (br s)), H-7 (dH 2.57 (br d, J = 3.2 Hz)) with H-12 (dH 1.62 (s)), and H-9 with H-13 (dH 1.72 (s)) (Fig. 3) confirmed not only the assignment of relevant hydrogen atom, but also the aorientations of H-1, H-2, and H-9 and the b-orientations of H-6 and H-8. As such, the structure of 3 was determined to be 1b,
15-diacetoxy-9b-benzoxy-2b,8a-diisobutanoyloxy-b-dihydroagrofuran, and it was given the trivial name angulatin M. Angulatin N (4) was isolated as a white amorphous powder. It had the molecular formula C35H40O15 according to its positive HRESI-MS. The IR spectrum showed the presence of ester carbonyl (1746 cm1) and hydroxyl (3433 cm1) groups. The 1H NMR spectrum (Table 1) showed seven methyls at dH 1.62, 1.71, 1.48, 1.66, 2.11, 2.13, and 2.04 as singlets, of which, four acetoxyls were evident from the 1H/13C (DEPT) NMR data at dH/dC 1.66 (s)/169.4 (s) and 20.5 (q), 2.11 (s)/169.8 (s) and 21.1 (q), 2.13 (s)/169.8 (s) and 21.2 (q), and 2.04 (s)/170.6 (s) and 21.5 (q)) assigned by the combination of HSQC and HMBC experiments. Besides, one furoyloxy unit confirmed by both dH 8.03 (1H, br s), 6.74 (1H, br d, J = 1.1 Hz), and 7.44 (1H, br d, J = 1.1 Hz) and dC 160.6 (s), 148.9 (d), 118.0 (s), 109.8 (d), and 144.0 (d) and one benzoxy unit characterized by dH 8.17 (2H, m), 7.48 (2H, m), and 7.60 (1H, m) and dC 165.4 (s), 129.6 (s), 130.0 (d), 128.4 (d), and 133.5 (d) were also evident on the basis of the one-bond 1H–13C connectivities from the HSQC experiment. These consequences were compatible with the long-range 1H–13C correlations from HMBC experiments, including those from H-8 at dH 5.50 (1H, d, J = 2.6 Hz) to dC 165.45
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Table 2 13 C and DEPT NMR spectroscopic data for compounds 1–4 (100 MHz, CDCl3). Position
1
2
3
4
Angulatin A
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 OAc-1
75.1 (d) 67.7 (d) 42.4 (t) 69.7 (s) 92.0 (s) 76.0 (d) 52.2 (d) 73.6 (d) 75.6 (d) 51.6 (s) 84.4 (s) 29.7 (q) 25.7 (q) 24.5 (q) 61.4 (t) 169.6 (s) 20.5 (q) 169.5 (s) 21.2 (q)
71.5 (d) 69.2 (d) 31.1 (t) 32.8 (d) 89.6 (s) 75.0 (d) 53.6 (d) 77.0 (d) 72.5 (d) 52.6 (s) 81.4 (s) 30.3 (q) 25.7 (q) 16.8 (q) 65.6 (t) 169.5 (s) 20.5 (q) 170.6 (s) 21.2 (q)c 169.7 (s) 21.3 (q)
74.9 (d) 67.0 (d) 41.3 (t) 72.1 (s) 91.5 (s) 77.2 (d) 53.6 (d) 73.4 (d) 75.5 (d) 50.6 (s) 84.6 (s) 30.1 (q) 26.3 (q) 24.2 (q) 61.8 (t) 169.4 (s) 20.4 (q)
70.5 (d) 67.8 (d) 41.9 (t) 69.9 (s) 91.4 (s) 75.2 (d) 53.7 (d) 77.2 (d) 72.2 (d) 54.2 (s) 83.2 (s) 29.5 (q) 25.6 (q) 24.2 (q) 65.8 (q) 169.4 (s) 20.5 (q) 169.8 (s) 21.1 (q)d 169.8 (s) 21.2 (q)d
75.04 67.33 41.13 72.06 91.49 76.91 53.59 73.76 75.23 50.64 84.44 30.00 26.67 24.19 61.68 169.30 20.37 169.40 20.98
170.0 (s) 21.3 (q)c
170.4 (s) 21.5 (q)
170.6 (s) 21.5 (q)d
OAc-2 OAc-6 OAc-8 OAc-15 OBz-6
OBz-8
OBz-9
OFu-8
OFu-9
OiBu-2
C5 5O 10 20 ,60 30 ,50 40 C5 5O 10 20 ,60 30 ,50 40 C5 5O 10 20 ,60 30 ,50 40 C5 5O 20 30 40 50 C5 5O 20 30 40 50 C5 5O 10 20
OiBu-8
C5 5O 10 20
OiBu-15
C5 5O 10 20
a,b,c,d
169.8 (s) 20.8 (q) 170.4 (s) 21.4 (q) 165.7 (s) 129.2 (s) 129.6 (d)a 128.7 (d)b 133.5 (d)
165.4 129.6 130.0 128.4 133.5 165.7 129.2 130.1 128.7 133.5
(s) (s) (d)a (d)b (d)
165.8 129.5 129.4 128.7 133.5 161.7 148.6 119.0 110.0 143.9 160.6 148.8 118.2 109.8 143.9
(s) (s) (d) (d) (d)
(s) (s) (d) (d) (d)
(s) (d) (s) (d) (d) (s) (d) (s) (d) (d)
165.59 129.40 129.40 128.53 133.30
160.6 148.9 118.0 109.8 144.0 175.7 (s) 34.1 (d) 18.5 (q) 18.6 (q) 175.9 (s) 34.3 (d) 18.9 (q) 18.9 (q)
(s) (d) (s) (d) (d)
175.65 34.03 18.40 18.56 176.40 34.28 18.96 19.08
Interchangeable signals within the same letters.
(C5 5O), and from H-9 at dH 5.70 (1H, s) to dC 160.6 (C5 5O), which are indicators that the benzoate group was located at C-8 and the furoate group at C-9 (Fig. 2). Other key long-range 1H–13C correlations in HMBC spectrum of 4 see Fig. 2. The characteristic fragments in its EI-MS at m/z 43 [CH3CBBO+], 95 [(C4H3O)CBBO]+, 105 [(C6H5)CBBO]+, and 192 [(CH3)2CHCH5 5CHCH2OFu]+ further confirmed the above results. The remaining 1H and 13C NMR
spectroscopic data of 4 were very similar to those of its counterparts in the known celangulatin E (Huang et al., 2000), indicating that compound 4 possesses the same skeleton of bdihydroagarofuran as the latter. The NOE correlations of H-1 at dH 5.54 (s) with H-3ax at dH 2.22 (dd, J = 2.8, 14.8 Hz), H-6 with H-14 at dH 1.48 (s), and H-7 with H-12 at dH 1.62 (s) suggested the aorientation of H-1, H-2, and H-8 and the b-orientation of H-6 and
H.-Y. Zhang et al. / Phytochemistry Letters 7 (2014) 101–106
105
O
O
O
O
O
O O
O
O
O O
O O
OH
O
O O
O
1
2 O
O
O
O
O O
O
O
O
O O
O O
O
O
O
O
O
O
O
O O
O
O
O
O
O
O O
O
O
O
O
O
OH
O
OH
OH
O
3
4 Fig. 2. Key HMBC correlations in 1–4.
H-9 (Fig. 3). On the basis of the above results, the structure of 4 was elucidated to be 1b,2b,6a,15-tetraacetoxy-8b-benzoxy-9a-furoyloxy-4a-hydroxy-b-dihydroagrofuran, and it was given the trivial name angulatin N.
Henan Agriculture University. A voucher specimen (CA08) is deposited at the Key Laboratory of Natural Products, Henan Academy of Science, China. 3.3. Extraction and isolation
3. Experimental 3.1. General experimental procedures Optical rotations were obtained on a Perkin Elemer 341 polarimeter. IR (KBr) spectra were recorded on a Testscan Shimadzu FTIR 8000 series HYPER infrared spectrometer. 1D and 2D NMR spectra were taken on a DRX-400 spectrometer with tetramethylsilane as internal standard and CDCl3 as solvent. EI-MS were measured on an Autospec-UltimaETOF spectrometer, in m/z (rel.%). HRESI-MS were performed on a Q-Tof MicroTM instrument. TLC were carried out on glass plate precoated with silica gel G (Qingdao Marine Chemical Factory), and petroleum ether (PE)/acetone 3:1 was used as eluent with visualization being done under UV light at l 365 nm, followed by spraying with 10% aq. H2SO4 and heating at 110 8C. Column chromatographies were undertaken over silica gel (200–300 mesh; Qingdao Marine Chemical Factory). 3.2. Plant material The root bark of C. angulatus were collected in suburban Xian City, Shanxi Province, P. R. China, in June 2008, and taxonomically authenticated by Prof. Chang-Shan Zhu, the Department of Botany,
Dried and powdered root bark (4.5 kg) of C. angulatus were extracted for three times with petroleum ether (2 h, 1 h, 1 h) under reflux condition. After evaporation of the filtrate under vacuum condition, a yellow semisolid residue (105 g) was obtained. The residue was dissolved in 80% methanol and extracted with petroleum ether for several times until the upper solvent being very transparent. The 80% methanol soluble portions were evaporated under vacuum condition to afford another yellow residue (71 g), which was chromatographed on a silica gel column eluting with a gradient system of petroleum ether/EtOAc (10:1, 9:1, 7:1, 6:1, 5:1, 4:1, 7:3, 6:4, 4:6) to give 300 fractions (each 500 mL). Fraction 85 was subjected to reversed RP-18 column eluted with MeOHH2O = 65:35 to afford compounds 1 (9.5 mg) and 2 (5 mg), both as white amorphous powder. Fractions 110–113 was collected according to TLC detection and subjected to reversed RP-18 column (MeOHH2O, 65:35) to yield compound 3 (6 mg). Fraction 240 was separated on RP-18 gel column (MeOHH2O, 65:35) to give compound 4 (15 mg). Compound 5 (7 mg) was obtained from fraction 66 by purification with preparative HPLC (RP-18, MeOHH2O, 65:35). Compounds 6 (60 mg) and 7 (8 mg) were crystallized in methanol from fraction 121 and fraction 212, respectively.
106
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Fig. 3. Key NOE correlations in 1–4.
3.3.1. Angulatin K (1) White amorphous powder; +62.5 (MeOH; c 0.016); IR, nmax cm1: 3434, 2930, 1750, 1601, 1451, 1369, 1273, 1228, 1153, 1107, 1070, 1045, 860, 714 cm; 1H and 13C NMR, see Tables 1 and 2; EIMS m/z: 710 (0.3%), 650 (1.8%), 528 (8.4%), 395 (8.0%), 336 (31.9%), 268 (10.5%), 244 (18.0%), 202 (26.0%), 164 (10.2%), 137 (9.9%), 105 (100%), 77 (34.7%), 43 (31.0%); HRESIMS (positive-ion mode) m/z: 733.2476 [M+Na]+, (calcd for C37H42NaO14, 733.2470). 3.3.2. Angulatin L (2) White amorphous powder; +10.0 (MeOH; c 0.01); IR, nmax cm1: 2923, 2852, 1745, 1508, 1369, 1311, 1231, 1160, 1081, 874, 760, 604; 1H and 13C NMR, see Tables 1 and 2; EIMS m/z: 632 (7.7%), 590 (2.2%), 243 (15.8%), 237 (10.5%), 218 (8.6%), 201 (9.3%), 189 (5.0%), 159 (8.0%), 135 (11.4%), 95 (100%), 83 (21.0%), 77 (3.1%), 43 (25.4%); HRESIMS (positive-ion mode) m/z: 697.2110 [M+Na]+, (calcd for C33H38NaO15, 697.2106). 3.3.3. Angulatin M (3) White amorphous powder; 18.0 (MeOH; c 0.10); IR, nmax cm1: 3433, 2917, 2866, 1736, 1637, 1384, 1274, 1228, 1142, 1108, 1026, 879, 714; 1H and 13C NMR, see Tables 1 and 2; EIMS m/z: 261 (2.2%), 244 (5.9%), 229 (7.1%), 202 (15.5%), 164 (11.1%), 105 (100%), 77 (17.0%), 43 (19.2%); HRESIMS (positive-ion mode) m/z: 685.2833 [M+Na]+, (calcd for C34H46NaO13, 685.3408). 3.3.4. Angulatin N (4) White amorphous powder; 5.9 (MeOH; c 0.07); IR, nmax cm1:3433, 2924, 2852, 1746, 1369, 1309, 1230, 1159, 874, 760, 716, 604; 1H and 13C NMR, see Tables 1 and 2; EIMS m/z: 262 (4.3%), 244 (6.8%), 192 (50.8%), 164 (9.6%), 137 (5.0%), 105 (100%), 95 (81.4%), 77 (8.4%), 43 (24.8%). HRESIMS (positive-ion mode) m/z: 723.2256 [M+Na]+, (calcd for C35H40NaO15, 723.2261).
Acknowledgements We are grateful to Mr. W.G. Zhu, Y.X. Xie and Ms. S.M. Wang for the NMR and MS measurements. This work was supported by grants from National Science and Technology Project of China (2011ZX09307-002-01, 2012ZX09301002-002) References ¨ bersicht u¨ber die celastraceen-inhaltsstoffe: chemie, Bru¨ning, R., Wagner, H., 1978. U chemotaxonomie, biosynthese, pharmakologie. Phytochemistry 17, 1821–1858. Huang, H.C., Shen, C.C., Chen, C.F., Wu, Y.C., Ku, Y.H., 2000. A novel agarofuran sesquiterpene, Celahin D from Celastrus hindsii Benth. Chem. Pharm. Bull. 48, 1079–1080. Qin, H.L., Zhao, T.Z., Shang, Y.J., 1999. A new sesquiterpene from Celastrus angulatus. Chin. Chem. Lett. 10, 825–826. Spivey, A.C., Weston, M., Woodhead, S., 2002. Celastraceae sesquiterpenoids: biological activity and synthesis. Chem. Soc. Rev. 31, 43–59. Sua, X.H., Zhang, M.L., Zhan, W.H., Huo, C.H., Shi, Q.W., Gu, Y.C., 2009. Chemical and pharmacological studies of the plants from genus Celastrus. Chem. Biodivers. 6, 146–161. Wakakayashi, N., Wu, W.J., Waters, R.M., Redfern, R.E., Mills Jr., G.D., Demilo, A.B., 1988. Celangulin: a nonalkaloidal insect antifeedant from Chinese bittersweet, Celastrus angulatus. J. Nat. Prod. 51, 537–542. Wang, M.T., Qin, H.L., Kong, M., Li, Y.Z., 1991. Insecticidal sesquiterpene polyol ester from Celastrus angulatus. Phytochemistry 30, 3931–3933. Wei, S., Wang, M., Zhang, J., Qian, Y., Ji, Z., Wu, W., 2009. Three novel sesquiterpene esters from Celastrus angulatus. Nat. Prod. Commun. 4, 461–466. Wu, W.J., Tu, Y.Q., Liu, H.X., Zhu, J.B., 1992. Celangulins II, III, and IV: new insecticidal sesquiterpenoids from Celastrus angulatus. J. Nat. Prod. 55, 1294–1298. Wu, M.J., Zhao, T.Z., Shang, Y.J., Fu, J.G., Zhang, H.Y., 2004. A new sesquiterpene polyol ester from Celastrus angulatus. Chin. Chem. Lett. 15, 41–42. Yin, W.P., Zhao, T.Z., Gao, L.J., Zou, D.P., Kang, J.X., 1999. A novel sesquiterpene polyol ester from Celastrus angulatus and its antitumor activities. Chin. Chem. Lett. 10, 487–490. Yin, W.P., Zhao, T.Z., Gao, L.J., Qin, H.L., Zou, D.P., Kang, J.X., 1999. Structure determination of two new derivatives of sesquiterpenes by NMR techniques. Chin. J. Magn. Reson. 16, 225–229. Zhang, H.Y., Zhao, T.Z., Wei, Y., Liu, H.M., 2011. Two new sesquiterpene polyol esters from the root barks of Celastrus angulatus. J. Asian Nat. Prod. Res. 13, 304–311.