1870
J. Nat. Prod. 2004, 67, 1870-1875
Prenylated Benzophenones and Xanthones from Hypericum scabrum Naonobu Tanaka,† Yoshihisa Takaishi,*,† Yasuhiro Shikishima,† Yuka Nakanishi,‡ Kenneth Bastow,‡ Kuo-Hsiung Lee,‡ Gisho Honda,§ Michiho Ito,§ Yoshio Takeda,⊥ Olimjon K. Kodzhimatov,| and Ozodbek Ashurmetov| Faculty of Pharmaceutical Sciences, University of Tokushima, Shomachi 1-78, Tokushima, 770-8505, Japan, Natural Products Laboratory, School of Pharmacy, University of North Carolina, Chapel Hill, North Carolina 27599, Graduate School of Pharmaceutical Sciences, Kyoto University, Yoshida, Sakyo-ku, Kyoto, 606-8501, Japan, Faculty of Arts and Sciences, University of Tokushima, Minamijosanjima, Tokushima 770-8502, Japan, and Institute of Botany and Botanical Garden, F. Khodzhaev, st. 32, 700143, Tashkent, Uzbekistan Received January 30, 2004
Two new polyprenylated benzophenones, a new polyprenylated phloroglucinol, and six new xanthone derivatives were isolated from the aerial parts of the Uzbekistan medicinal plant Hypericum scabrum. Their structures were elucidated on the basis of spectroscopic evidence. The isolated compounds showed moderate cytotoxicity for human tumor cells. The recent widespread interest in the antidepressant activity of Hypericum perforatum (St. John’s wort) has encouraged the investigation of secondary metabolites from other Hypericum species.1 The genus Hypericum occurs widely in temperate regions of the world and has been used as traditional medicinal plants in various parts of the world. It produces various types of secondary metabolites, including flavonoids, biflavonoids, xanthones, naphthodianthorones, and prenylated phloroglucinols.2 These compounds show diverse biological activity.3-8,11-13 H. scabrum is one of the most popular medicinal herbs in Uzbekistan and is used in the treatment of bladder, intestinal, and heart diseases, rheumatism, and cystitis.12,13 The volatile oil constituents of H. scabrum have been studied,14,15 and we previously reported nine novel polyprenylated benzoylphloroglucinol derivatives named hyperibones A-I.16 As part of our continuing study of the chemical constituents of medicinal plants in Uzbekistan, we have examined the aerial parts of H. scabrum and isolated nine new compounds [hyperibones J-L (1-3), hyperxanthones A-F (49)] and 16 known compounds (10-25). In this paper, we describe the isolation, structure elucidation, and cytotoxicity of the isolated compounds for human tumor cells. Results and Discussion The methanol extract of air-dried aerial parts of H. scabrum was partitioned between H2O and EtOAc. The EtOAc layer was separated by column chromatography (CC) to afford nine new (1-9) and 16 known (10-25) compounds. Hyperibone J (1)17 was assigned a molecular formula of C31H46O5 on the basis of the positive-ion HRFABMS, and its IR spectrum showed a hydroxyl band (3323 cm-1) and three carbonyl bands (1777, 1742, and 1678 cm-1). The 1H NMR spectrum of 1 indicated the presence of a hydroxyl proton [δH 7.70 (1H, brs)], three protons attached to double bonds [δH 5.02 (1H, t, J ) 7.3 Hz), 4.96 (1H, t, J ) 5.3 Hz), and 4.90 (1H, t, J ) 6.8 Hz)], one isopropyl group [δH 3.18 (1H, sept, J ) 6.6 Hz), 1.08, 0.91 (each 3H, d, J ) 6.6 Hz)], * To whom correspondence should be addressed. Tel: 81-88-6337275. Fax: 81-88-6339501. E-mail:
[email protected]. † Faculty of Pharmaceutical Sciences, University of Tokushima. ‡ University of North Carolina. § Kyoto University. ⊥ Faculty of Arts and Sciences, University of Tokushima. | Uzbekistan Institute of Botany.
10.1021/np040024+ CCC: $27.50
five methylenes, and eight methyl singlets. The 13C NMR spectrum of 1 showed three carbonyl carbons [δC 217.9, 209.9, and 207.0], three double-bond carbons [δC 136.1, 133.6, 131.9, 124.2, 122.1, and 115.9], two oxygenated quaternary carbons [δC 107.9 and 97.3], and three other quaternary carbons, two methine carbons, five methylene carbons, and 10 methyl carbons. On the basis of these data, 1 was assumed to be a prenylated phloroglucinol derivative. The 13C NMR spectrum of 1 was very similar to that of 9-hydroxyhyperforin-9,3-hemiacetal18 except for the chemical shifts of C-5 and C-19-C-23. The structure of 1 was presumed to be a C-5-substituted compound of 9-hydroxyhyperforin-9,3-hemiacetal. In the HMBC spectrum of 1, the signal of H3-19 (δH 1.12) was correlated with the carbon signals of C-4 (δC 209.9), C-5 (δC 51.5), C-6 (δC 34.3), and C-9 (δC 107.9). This indicated that a methyl group is located at C-5 of 1 in place of the isopentenyl group in 9-hydroxyhyperforin-9,3-hemiacetal. The relative configuration of C-7 and C-8 was determined on the basis of the following NOESY results: OH-9 [δH 7.70 (1H, s)] with H3-25 [δH 0.98 (3H, s)], H3-25 with H-6ax [δH 1.28 (1H, m)]; H-6eq [δH 1.86 (1H, dd, J ) 14.3, 3.4)] with H-7 [δH 1.04 (1H, m)]. Thus, the structure of 1 (hyperibone J) was assigned as shown in Figure 1. Hyperibone K (2) has a molecular formula of C33H40O4 on the basis of negative-ion HRFABMS and showed a carbonyl absorption at 1701 cm-1 in its IR spectrum, and its UV spectrum showed the presence of an aromatic moiety (246 nm). The 1H NMR spectrum revealed the presence of a benzene ring [δH 7.44 (1H, t, J ) 7.3 Hz), 7.29 (2H, dd, J ) 7.5, 7.3 Hz), and 7.22 (2H, d, J ) 7.5 Hz)], three vinyl protons [δH 5.20 (1H, t, J ) 7.3 Hz), 5.01 (1H, d, J ) 8.1), and 4.93 (1H, t, J ) 6.3 Hz)], two methines [δH 4.20 (1H, brd, J ) 8.1 Hz) and 1.72 (1H, m)], three methylenes [δH 2.53 (2H, d, J ) 7.3 Hz), 2.47 (2H, m), 2.48 and 2.42 (each 1H, m)], and eight methyls [δH 1.77, 1.75, 1.70, 1.68, 1.66, 1.63, 1.24, and 1.16 (each 3H, s)]. The 13C NMR spectrum indicated that the presence of three carbonyl carbons [δC 203.6, 201.6, and 201.1], a benzoyl group [δC 193.4, 135.1, 132.6, 129.3 × 2, and 128.1 × 2], three double bonds [δC 135.0, 134.8, 134.5, 120.7, 119.5, and 118.8], four quaternary carbons [δC 79.5, 77.2, 69.2, and 53.6], two methines, three methylenes, and eight methyls. On the basis of these data, 2 was assumed to be a prenylated benzophenone derivative that has three 3-methyl-2-butenyl units. In the
© 2004 American Chemical Society and American Society of Pharmacognosy Published on Web 11/09/2004
Prenylated Benzophenones from Hypericum
Figure 1.
Figure 2. NOE correlations of compound 2. 1H-1H
COSY spectrum, the signal of H-27 (δH 4.20) correlated with the signals of H-28 (δH 5.01) and H-7 (δH 1.72). Moreover, the long-range correlations of H-27 with C-3 (δC 79.5), C-4 (δC 201.6), C-6 (δC 41.2), and C-7 (δC 47.9) and H-7 with C-1 (δC 77.2), C-3, and C-5 (δC 69.2) were observed in the HMBC spectrum. These facts indicated that the methine carbon (C-27) was connected to three carbons (C-28, C-7, and C-3). The long-range correlations of H2-10 (δH 2.47) with C-1 and H2-22 (δH 2.53) with C-9 (δC 203.6), C-4, C-5, and C-6 (δC 41.2) showed that the two remaining 3-methyl-2-butenyl units were located at C-1 and C-5. Therefore, the benzoyl group must be located at C-3. The relative configuration of C-27 and C-8 was determined on the basis of the NOESY spectrum shown in Figure 2. Thus, the structure of 2 (hyperibone K) was assigned as shown. Compound 3 showed a hydroxyl absorption (3349 cm-1) and two carbonyl absorptions (1732 and 1673 cm-1) in its IR spectrum and displayed a molecular ion peak at m/z 448.2589 ([M]+, calcd 448.2614) in the HREIMS. These data indicated the molecular formula as C29H36O4. However, there were duplicate signals in its 1H and 13C NMR spectra (in CDCl3) in the ratio of approximately 2:1, and
Journal of Natural Products, 2004, Vol. 67, No. 11 1871
two proton signals of the enolic OH group (δH 17.70 and 15.48) were observed. 3 was thus considered to be a mixture of the enol tautomers of 3a and 3b (ca 2:1). In comparison with compounds isolated from Guttiferae, the 13C NMR data of 3 were very similar to that of 7-epiclusianone (10), which was observed as a mixture of two enolic tautomers (5:4, in CDCl3) in the NMR spectrum,19 except for the C-10 methyl signal of 3. In the HMBC spectrum of 3, H3-10 (δH 1.42) was correlated with the carbon signals of C-2 (δC 197.8), C-1 (δC 62.0), C-8 (δC 48.5), and C-9 (δC 209.2) in the major tautomer 3a, and H3-10 (δH 1.29) was correlated with the carbon signals of C-2 (δC 194.7), C-1 (δC 65.3), C-8 (δC 47.6), and C-9 (δC 209.2) in the minor tautomer 3b. These facts indicated that the methyl group (C-10) was located at C-1. The assignment of other carbons was done by comparison of the 13C NMR data with those of 10. Thus, the structures of 3a (hyperibone L-a) and 3b (hyperibone L-b) were determined as shown. Hyperxanthone A (4) showed hydroxy and conjugated carbonyl bands at 3423 and 1650 cm-1 in its IR spectrum. The HREIMS of 4 gave a molecular ion at m/z 344.0913 ([M]+, calcd 344.0896), suggesting the molecular formula of C18H16O7. The 1H NMR spectrum of 4 showed the presence of a hydrogen-bonded hydroxyl proton [δH 13.19 (1H, s)], one singlet aromatic proton [δH 6.75 (1H, s)], two meta-coupled aromatic protons [δH 6.25 (1H, d, J ) 1.6 Hz) and 6.19 (1H, d, J ) 1.6 Hz)], an oxygenated methine proton [δH 4.80 (1H, t, J ) 9.2 Hz)], one methylene group attached to the aromatic ring [δH 3.72 (2H, d, J ) 9.2 Hz)], and two methyl protons [δH 1.30 and 1.26 (each 3H, s)]. Its 13C NMR spectrum (Table 3) revealed the presence of 18 carbons including one conjugated carbonyl carbon (δC 182.1), 12 aromatic carbons, one oxygenated quaternary carbon, one oxygenated methine carbon, one methylene carbon, and two methyl carbons. These data suggested that 4 is a xanthone derivative having a 2,3-dihydroxy-3methylbutyl side chain. In the HMBC spectrum, the following key long-range correlations were observed: OH-1 with C-1, C-2, and C-9a; H-2 with C-1, C-3, C-4, and C-9a; H-4 with C-2, C-3, C-4a, and C-9a; H-5 with C-8a, C-4b, C-6, and C-7. These correlations indicated that four hydroxyl functions were located at C-1, C-3, C-6, and C-7. The long-range correlations of H2-1′ with C-7 and C-8 revealed that the 2,3-dihydroxy-3-methylbutyl side chain is located at C-8. Furthermore, the downfield shifted carbon signal of C-2′ (δC 91.5) pointed out the existence of a fivemembered ring located at C-7 and C-8. Hyperxanthone B (5) had a molecular formula of C18H16O8 on the basis of HREIMS (m/z 360.0852 [M]+). The 1H and 13C NMR data of 5 were similar to those of 4, except for H-1′ [5: δH 5.89 (1H, d, J ) 5.5 Hz), 4: δH 3.72 (2H, d, J ) 9.2 Hz)], H-2′ [5: δH 4.47 (1H, d, J ) 5.5 Hz), 4: δH 4.80 (1H, d, J ) 9.2 Hz)], C-1′ [5: δC 73.9, 4: δC 32.9], and C-2′ [5: δC 97.1, 4: δC 91.5]. In the HMBC spectrum, H3-4′ and H3-5′ were correlated with C-2′ (δC 97.1) and C-3′ (δC 70.7), and H-2′ was correlated with C-1′. These findings indicated the presence of a hydroxymethine at C-1′ in 5 instead of methylene in 4. Thus, the structure of 5 was determined as shown. Hyperxanthone C (6), C18H16O7, exhibited a hydrogenbonded hydroxyl proton [δH 13.45 (1H, s)], aromatic protons [δH 6.81 (1H, s), 6.31 and 6.19 (each 1H, brs)], and a 2-hydroxy-3-methyl-3-butenyl side chain [δH 5.14, 4.88 (each 1H, s), 4.53 (1H, dd, J ) 10.2, 1.8 Hz), 4.20 (1H, dd, J ) 13.2, 1.8 Hz), 3.10 (1H, dd, J ) 13.2, 10.2 Hz), and 1.96 (3H, s)] in its 1H NMR spectrum. Its 13C NMR data were very similar to that of 4, except for the chemical shifts
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Journal of Natural Products, 2004, Vol. 67, No. 11
Table 1.
13C
NMR Data for 1 and 9-Hydroxyhyperforin-9,3-hemiacetal 9-hydroxyhyperforin9,3-hemiacetalb
1a
a
Tanaka et al.
9-hydroxyhyperforin9,3-hemiacetalb
1a
position
δC
position
δC
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
71.1 207.0 97.3 209.9 51.5 34.3 41.8 47.3 107.9 217.9 39.4 17.7 19.2 24.0 115.9 136.1 18.1 25.8 16.8
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
71.9 207.6 97.7 209.1 55.2 33.5 41.7 47.1 108.8 218.3 39.5 17.5 19.1 24.4 116.9 135.7 17.9 25.9 31.7
position
20 21 22 23 24 25 26 27 28 29 30 31
δC
position
δC
28.6 122.1 133.6 18.0 25.9 15.5 37.0 23.8 124.2 131.9 17.9 25.9
20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35
119.9 133.5 17.7 26.1 28.9 122.6 133.4 17.9 25.7 15.8 37.4 24.3 124.8 131.7 17.8 25.8
Measured in CDCl3. b Measured in C6D6.
Table 2. NMR Data for 2a position 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33
1H
(δH)
HMBC
(13C
2.48 [1H, m] 2.42 [1H, m] 1.72 [1H, m]
4, 5, 8, 9
2.47 [2H, m] 4.93 [1H, t (6.3)]
1, 9, 11, 12 13, 14
1.63 [3H, s] 1.66 [3H, s]
11, 12, 14 11, 12, 13
No.)
1, 3, 5
7.22 [1H, d (7.5)] 7.29 [1H, dd (7.5, 7.3)] 7.44 [1H, t (7.3)] 7.29 [1H, dd (7.5, 7.3)] 7.22 [1H, d (7.5)] 2.53 [2H, d (7.3)] 5.20 [1H, t (7.3)]
15, 16, 18, 19, 21 16, 17, 19, 20 17, 18, 20, 21 16, 18, 19, 21 16, 17, 19, 20 4, 5, 6, 9, 23, 24 25, 26
1.68 [3H, s] 1.70 [3H, s] 4.20 [1H, brd (8.1)] 5.01 [1H, d (8.1)]
23, 24, 26 23, 24, 25 3, 4, 6, 7, 28, 29 3, 27, 30, 31
1.75 [3H, s] 1.77 [3H, s] 1.24 [3H, s] 1.16 [3H, s]
28, 29, 31 28, 29, 30 1, 7, 8, 33 1, 7, 8, 32
13C
(δC)
77.2 201.1 79.5 201.6 69.2 41.2 47.9 53.6 203.6 23.5 119.5 134.5 18.2 26.1 193.4 135.1 129.3 128.1 132.6 128.1 129.3 27.6 118.8 135.0 18.2 26.1 51.4 120.7 134.8 18.5 26.1 22.9 23.6
a Measured in CDCl . Coupling constants given (J in Hz) in 3 parentheses.
of C-6, C-7, and the side chain (Table 4). In the HMBC spectrum of 6, H-5 was correlated to C-4b, C-6, C-7, and C-8a, and H2-1′ was correlated with C-7 and C-8. These facts indicated that 6 is a 1,3,6,7-tetrahydroxyxanthone having a 2-hydroxyl-3-methyl-3-butenyl side chain at C-8. The positive FABMS of hyperxanthone D (7) gave the quasi-molecular ion at m/z 329.0987 ([M + H]+ calcd 329.1025), suggesting the molecular formula of C18H16O6. The 13C NMR data of 7 were similar to those of 6 except for the chemical shifts of C-4b-C-8a (Table 4). The 1H NMR
Table 3. 13C NMR Data (δC) for Enolic Tautomers of 3 (3a/3b) and 10 (10a/10b)a position
3a
position
10a
position
3b
position
10b
1 2 3 4 5 6 7 8 9 10
62.0 197.8 114.8 194.4 62.7 40.4 46.5 48.5 209.2 12.9
65.3 194.7 114.8 198.3 58.3 38.7 46.1 47.6 209.2 13.4
197.0 137.0 129.0 128.0 132.8 128.0 129.0 31.6 119.9 134.9 18.3 26.2 29.2 123.8 133.0 17.9 25.9 27.2 23.1
66.1 197.8 116.5 193.2 63.4 40.6 46.9 48.9 207.9 27.2 119.4 135.0 18.1 26.1 196.8 137.3 129.5 127.9 132.5 127.9 129.5 31.9 120.6 134.8 18.3 26.3 29.2 124.2 132.7 17.8 25.8 27.0 23.0
1 2 3 4 5 6 7 8 9 10
11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33
11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29
196.7 136.8 128.9 128.1 132.8 128.1 128.9 32.1 119.0 135.1 18.3 26.2 29.3 124.1 133.1 18.0 26.0 27.0 22.5
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33
69.0 193.5 116.5 198.3 58.9 39.4 46.7 48.3 207.9 26.6 119.8 135.0 18.3 26.0 197.9 137.3 126.6 127.7 132.9 127.7 126.6 31.2 120.9 134.8 18.4 26.2 29.4 124.8 132.7 17.8 25.9 26.9 22.5
a
Measured in CDCl3.
spectrum of 7 showed ortho-coupling protons at δH 7.32 and 7.26 (each 1H, d, J ) 8.9 Hz). On consideration of the molecular formula of 6 and 7, and the 1H NMR differences, the structure of 7 was deduced to be a dehydroxy compound of 6. The HMBC spectrum revealed the following correlations: H-5 (δH 7.26) with C-8a (δC 119.4) and C-7 (δC 153.5); H-6 (δH 7.32) with C-4b (δC 151.9) and C-8 (δC 126.8); H21′ (δH 4.10, 3.11) with C-7, C-8, and C-8a. On the basis of these data, the structure of 7 was determined as shown. Hyperxanthone E (8), C18H16O6, showed hydroxyl (3421 cm-1), conjugated carbonyl (1650 cm-1), and aromatic (1613
Prenylated Benzophenones from Hypericum
Journal of Natural Products, 2004, Vol. 67, No. 11 1873
Figure 3.
Table 4. 11)
13C
NMR Data (δC) for Xanthone Derivatives (4-9,
position
4a
5a
6a
7a
8b
9a
11c
1 2 3 4 4a 4b 5 6 7 8 8a 9 9a 1′ 2′ 3′ 4′ 5′ OMe
164.2 98.1 165.0 93.9 158.4 152.9 102.8 148.3 145.8 126.9 110.7 182.1 103.2 32.9 91.5 71.2 25.4 25.1
163.9 98.5 165.6 94.1 158.4 153.6 104.3 149.9 146.3 127.9 110.6 181.1 103.2 73.9 97.1 70.7 25.6 25.0
164.3 98.1 165.0 93.3 157.6 153.8 101.7 154.0 142.7 127.2 111.5 182.7 103.3 33.9 77.9 148.7 109.7 18.1
164.2 98.7 167.7 93.8 157.9 151.9 117.2 124.7 153.5 126.8 119.4 183.3 103.1 33.9 77.5 148.9 109.6 18.1
164.7 98.5 165.7 93.9 158.5 154.3 101.1 153.5 142.0 131.0 112.1 183.5 104.0 44.3 23.0 72.1 29.1 29.1
164.1 98.1 165.1 94.1 158.3 153.3 103.1 148.5 143.8 125.0 110.6 181.0 103.1 38.7 109.2
164.4 98.3 165.1 93.5 157.8 153.3 103.1 153.7 138.4 120.5 108.0 182.7 103.5 121.1 133.2 76.4 26.7 26.7
a
55.4 b
Measured in acetone-d6. Measured in methanol-d4. c Measured in DMSO-d6.
cm-1) absorption bands in its IR spectrum. The 1H and 13C NMR data of 8 were similar to those of toxyloxanthone B (11)20-22 except for the side chain (Table 4). The side chain of 8 is a pyran ring fused between C-7 and C-8 [δH 3.45 (2H, t, J ) 8.0 Hz) 1.81 (2H, t, J ) 8.0 Hz), 1.35 (6H, s); δC 72.1 44.3, 29.1 × 2, 23.0]. On the basis of these data the structure of 8 was determined as shown. The 1H and 13C NMR spectroscopic data of hyperxanthone F (9), C16H12O7, were very similar to those of 4 except for the side chain. The 1H NMR data of the side chain showed the presence of one oxygenated methine signal [δH 5.86 (1H, dd, J ) 6.6, 2.2 Hz)], one methylene signal attached to the aromatic ring [δH 3.77 (1H, dd, J ) 18.3, 6.6 Hz) and 3.58 (1H, dd, J ) 18.3, 2.2 Hz)], one methoxy signal [δH 3.51 (3H, s)], and one acetal carbon signal (δC 109.2). In the HMBC spectrum, H2-1′ (δH 3.77 and 3.58) was correlated with C-8, H-2′ (δH 5.86) was correlated with C-7 (δC 143.8), and H3-OMe (δH 3.51) was correlated with C-2′ (δC 109.2). Thus, the structure of 9 was determined as shown. The following known compounds were identified by comparison with literature data: 7-epiclusianone (10),19 toxyloxanthone B (11),20-22 1,3,6,7-tetrahydroxy-8-(3-meth-
Table 5. Cytotoxicity Data for Compounds against Human Tumor Cells cell lines (IC50, mcg/mL)a A549b
compound 1 2 3 4 6 7 8 10 11 12 13 15 16 17 18 19 20 21 22 23 24 25
>20 (49)c 13.7 9.2 >20 (26) 9.3 >20 (43) 18.5 13.7 >20 (28) 18.5 14.5 8.5 10.0 12.9 >20 (21) NAd >20 (12) NA >20 (11) 14.8 >20 (13) >20 (27)
MCF-7b 17.8 10.0 15.0 >20 (27) 11.2 >20 (34) 19.3 10.0 >20 (17) 18.4 19.5 15.2 9.3 14.3 19.5 NA >20 (18) NA >20 (17) 4.7 11.2 >20 (26)
a IC 50 ) concentration that causes a 50% reduction in absorbance at 562 nm relative to untreated cells using the SRB assay. b A549, lung; MCF-7, breast. c If inhibition is
