Bioactive Polyprenylated Acylphloroglucinol

Article pubs.acs.org/jnp

Bioactive Polyprenylated Acylphloroglucinol Derivatives from Hypericum cohaerens Xia Liu,†,‡ Xing-Wei Yang,†,‡ Chao-Qun Chen,† Chun-Yan Wu,† Jing-Jing Zhang,†,‡ Jun-Zeng Ma,† Huan Wang,† Li-Xin Yang,† and Gang Xu*,† †

State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, People’s Republic of China ‡ University of Chinese Academy of Sciences, Beijing 100039, People’s Republic of China S Supporting Information *

ABSTRACT: Nine new polyprenylated acylphloroglucinol derivatives, hypercohins B−J (1−9), and nine known analogues were isolated from the aerial parts of Hypericum cohaerens. The structures of 1−9 were elucidated based on spectroscopic analysis, and the absolute configuration of 1 was confirmed by X-ray crystallographic analysis. The inhibitory activities of these isolates on acetylcholinesterase and five human tumor cell lines were tested, and hypercohins B−D (1−3) exhibited moderate inhibitory activity (IC50 5.8−17.9 μM) against the tested tumor cell lines.

P

(pentamethyltetrahydrofuran-3-yl)methanol] at C-3. In addition, acylphloroglucinol derivatives are usually isolated as oils, which makes elucidation of their configurations, particularly absolute configurations, difficult. However, we obtained a single crystal of 1 in MeOH−acetone−H2O, which confirmed not only the structure including the unusual substituent at C-3 but also the absolute configuration. Considering several cytotoxic polycyclic acylphloroglucinols have been isolated in our laboratory previously,10,11 together with the fact that this type of metabolite was closely associated with neurodegenerative diseases such as Alzheimer’s disease,1 we examined the inhibitory activities of these compounds against acetylcholinesterase (AChE) and five human tumor cell lines accordingly.

olyprenylated polycyclic acylphloroglucinols have a highly oxygenated and densely substituted bicyclo[3.3.1]nonane2,4,9-trione or similar skeleton substituted with prenyl or geranyl side chains. These compounds are reported mainly from plants of the family Guttiferae.1 They are also reported to possess a wide variety of bioactivities such as tumor inhibitory, antimicrobial, HIV preventive, and antioxidant activities. In the CNS, they act as modulators of neurotransmitters associated with the symptoms of neuronal damage and depression.2−7 Hyperforin, an acylphloroglucinol metabolite from H. perforatum (St. John’s Wort), possesses antidepressant, anticancer, and antibiotic activities.8 Since the isolation of this compound in 1975,9 the structures and biological activities of acylphloroglucinols, especially polycyclic acylphloroglucinols with prenyl side chains, have attracted wide attention in the natural product and synthetic chemistry fields.10−14 Until 2005, 119 analogues of hyperforin with polycyclic acylphloroglucinol cores have been isolated from different plants within the Guttiferae, 57 of which were from Hypericum.1 Distributed in temperate regions throughout the world, Hypericum has been used as a traditional medicine in many countries.15 H. cohaerens N. Robson is endemic to Guizhou and Yunnan Provinces, People’s Republic of China.16 A previous phytochemical investigation of H. cohaerens identified hypercohin A, a polyprenylated acylphloroglucinol derivative with a bicyclo[5.3.1]hendecane core.17 In the present study, nine new acylphloroglucinols, hypercohins B−J (1−9), were isolated from H. cohaerens along with nine known analogues. Previously, no natural acylphloroglucinols have been reported with a 10carbon moiety at C-3. Therefore, it is noteworthy that compounds 1−3 share the 10-carbon fragment [2,2,4,4,5© XXXX American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION The MeOH extract of the air-dried and powdered aerial parts of H. cohaerens was subjected to silica gel column chromatography eluted with a petroleum ether−acetone gradient to afford five fractions, A−E. Fraction B was subjected to a series of chromatographic methods, which led to the isolation of nine new polyprenylated acylphloroglucinol derivatives, hypercohins B−J (1−9), and nine known substances, furohyperforin,18 furoadhyperforin,19 uraloidin A,20 uraloidin B,21 uraloidin C,21 27-epifurohyperforin isomer 1,22 furohyperforin isomer 1,22 furoadhyperforin isomer A,22 and pyrano[7,28-b]hyperforin.23 The absolute configuration of 1 was confirmed by X-ray diffraction studies (CCDC 930811). Received: April 11, 2013

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dx.doi.org/10.1021/np400287r | J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

The relative configuration of 1 was elucidated by analysis of the ROESY spectrum. Diagnostic cross-peaks between H-32/ H-27, H-27/H-6β, and H-6α/H-23 showed that 1 has the same relative configuration at C-7, C-8, and C-23 as furohyperforin (Figure 1). The ROESY correlations from H-18 to H-11, Me20, and Me-42 indicated that H-18, Me-20, and Me-42 were βoriented. H-39 was determined to be α-oriented by its correlations with Me-21 and Me-43. To determine the absolute configuration and confirm the structure, particularly the unusual substituent at C-3, compound 1 was subjected to an X-ray diffraction study using Cu Kα radiation (Figure 2), which unambiguously confirmed the structure and clarified the absolute configuration as 1R, 5S, 7S, 8R, 18S, 23S, and 39R. Thus, the structure of 1 was characterized and the compound named hypercohin B. Hypercohin C (2) was isolated as a colorless oil and yielded a pseudomolecular ion peak at m/z 711.4239 ([M + Na]+, calcd 711.4236) in the HREIMS analysis, indicative of the molecular formula C43H60O7. Comparison of the 1D and 2D NMR data indicated that the structures of 1 and 2 were similar (Tables 1 and 2). However, the signals for the isopropyl group in 1 were replaced by signals for a phenyl group in 2, which was confirmed by HMBC correlations from H-12 and H-16 to C-10 (δC 195.4) and the proton spin system of H-12/H-13/H-14/H15/H-16 from the 1H−1H COSY spectrum. The ROESY correlations of Me-32/H-27, Me-32/H-6β, and H-6α/H-23 showed that 2 had the same relative configurations as 1 at C-7, C-8, and C-23. In addition, the ROESY cross-peaks of H-18/H12, H-18/Me-20, H-18/Me-42, H-39/Me-21, and H-39/Me-43 in 2 were all present, which suggested the configurations of C18 and C-39 in 2 were also the same as in 1. Hypercohin D (3) possesses the same molecular formula as 1 according to HREIMS analysis. The 1H and 13C NMR spectra of 1 and 3 were similar except for the resonances in the vicinity of C-18 and C-39 (Tables 1 and 2). Compared with those of 1, the chemical shifts of C-17 (δC 27.3), C-20 (25.6), and C-42 (32.9) were upfield, while C-21 (δC 23.8) and C-43 (24.0) were downfield, which implied that 3 was stereoisomeric with 1 in C18 and C-39. The relative configurations of C-1, C-5, C-7, C-8, and C-23 in 3 are the same as in 1, as determined by the NOE correlations of Me-32/H-27, Me-32/H-6β, and H-6α/H-23 observed in the ROESY experiment. The ROESY correlations of H-7/H-18, H-18/Me-21, and H-39/Me-20 indicated that the orientations of H-18 and H-39 are α and β, respectively. Therefore, the structure of 3 was determined and named hypercohin D. The molecular formulas of hypercohins E (4) and F (5) were determined as C35H52O5 and C36H54O5, respectively, according to HRESIMS analysis. The 1D and 2D NMR data of 4 were similar to those of furohyperforin (Tables 1 and 3),18 indicating these two compounds share the same planar structure. Comparison of their NMR data revealed the replacement of the isopropyl group at C-10 in 4 with an isobutyl group (δC 49.7, C-11; 16.9, Me-12; 28.5, C-13; 11.9, Me-14) in 5. The configurations of the core structures of 4 and 5 were also found to be the same as furohyperforin by analysis of their ROESY spectra, which were confirmed by the correlations of Me-32/H27, Me-32/6β, and H-6α/H-22α. The orientations of H-23 in 4 and 5 were determined to be β by the ROESY correlations from both Me-25 and Me-26 to H-6α (Figure 3). Therefore, the structures of 4 and 5 were established as shown. Hypercohins G (6) and H (7) were assigned the molecular formulas C35H52O4 and C38H50O4 by HREIMS and HRESIMS

Hypercohin B (1) was obtained as colorless crystals. Its molecular formula, C40H62O7, was established by positive HREIMS (m/z 654.4503, [M]+, calcd 654.4496), indicating 10 indices of hydrogen deficiency. The IR spectrum showed absorption bands for hydroxy (3446 cm−1) and carbonyl (1728 and 1667 cm−1) groups. Analysis of 13C and DEPT NMR data (Table 1) indicated the characteristic signals for an acylphloroglucinol with two nonconjugated carbonyl groups (δC 205.9, C-9; 211.2, C-10), an enolized 1,3-diketo group (δC 194.7, C-2; 115.8, C-3; 177.0, C-4), three quaternary carbons at δC 84.7 (C-1), 61.4 (C-5), and 49.9 (C-8), a methine (δC 44.3, C-7), and a methylene (δC 39.8, C-6) carbon.18−23 We also observed signals for two olefinic protons of isoprenyl groups (δH 5.04, 2H, m), 11 methyls (δH 0.991−1.69, s), and an isopropyl group (δH 0.98, 3H, d, J = 6.6 Hz; 1.04, 3H, d, J = 6.6 Hz; 1.98, 1H, sept, J = 6.6 Hz) (Table 2). On the basis of these observations, 1 was shown to be an acylphloroglucinol-type derivative. Comparison of the NMR data of 1 with those of furohyperforin revealed that they are structurally similar except that the signal for the isoprenyl group at C-3 in furohyperforin was replaced in 1 by a 10-carbon unit composed of four methyl (C-20, C-21, C-42, and C-43), two methylene (C-17 and C40), two methine (C-18 and C-39), and two quaternary carbons (C-19 and C-41).18 The structure of this 10-carbon unit, as shown in Figure 1, was deduced by the 1H−1H COSY correlations of H-17/H-18 and H-39/H-40, with the HMBC correlations from both Me-20 and Me-21 to C-18, C-19, and C39, and Me-42 and Me-43 to C-39 and C-41. A five-membered ether ring was formed by the connection of C-18 and C-41 through an oxygen atom, as elucidated by the HMBC correlations of H-18 (δH 3.53, 1H, m) with C-41 (δC 81.8) coupled with the indices of hydrogen deficiency. In addition, the HMBC correlations from H2-17 (δH 2.57, 1H, dd, J = 13.8, 10.2 Hz; δH 2.48, 1H, dd, J = 13.8, 2.6 Hz) to C-2 (δC 194.7), C-3 (δC 115.8), and C-4 (δC 177.0) deduced the linkage of this unit to the core structure. B



Article

Table 1. 13C NMR Data for Compounds 1−7 (δ in ppm, 150 MHz) position 1 2 3 4 5 6 7 8 9 10 11 12(16) 13(15) 14 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 a

1a 84.7 194.7 115.8 177.0 61.4 39.8 44.3 49.9 205.9 211.2 43.1 22.1 20.9

C C C C C CH2 CH C C C CH CH3 CH3

24.8 CH2 85.2 CH 43.7 C 22.5 CH3 26.9 CH3 30.2 CH2 91.8 CH 72.2 C 27.2 CH3 24.4 CH3 28.3 CH2 123.6 CH 134.4 C 26.05 CH3 18.1 CH3 14.5 CH3 37.7 CH2 26.11 CH2 125.9 CH 132.0 C 26.0 CH3 17.9 CH3 61.1 CH 60.9 CH2 81.8 C 21.6 CH3 31.6 CH3

2a 81.0 C 195.3 C 115.1 C 176.6 C 61.9 C 40.3 CH2 43.9 CH 51.1 C 206.5 C 195.4 C 138.4 C 129.2 CH 129.0 CH 133.1 CH 25.2 CH2 85.2 CH 44.0 C 22.9 CH3 26.9 CH3 30.3 CH2 91.8 CH 72.0 C 27.4 CH3 24.5 CH3 28.5 CH2 123.6 CH 134.5 C 26.1 CH3 18.2 CH3 15.0 CH3 37.9 CH2 26.2 CH2 125.8 CH 132.1 C 26.0 CH3 17.9 CH3 60.89 CH 60.88 CH2 82.1 C 22.5 CH3 31.8 CH3

3a

4b

85.0 195.1 115.2 177.4 61.3 39.3 42.8 49.6 206.0 211.8 42.9 22.1 20.9


83.7 193.4 117.2 175.1 60.2 40.8 44.5 47.9 205.4 210.1 42.1 21.8 20.7


27.3 84.6 44.7 25.6 23.8 30.6 91.8 71.2 26.7 25.4 28.4 124.0 134.0 26.1 18.1 15.1 37.7 25.8 126.0 131.9 25.9 18.0 59.8 60.8 82.4 32.9 24.0

CH2 CH C CH3 CH3 CH2 CH C CH3 CH3 CH2 CH C CH3 CH3 CH3 CH2 CH2 CH C CH3 CH3 CH CH2 C CH3 CH3

23.1 122.3 132.4 25.8 17.8 29.0 93.0 70.7 26.8 26.7 27.8 123.4 133.2 25.9 18.0 14.2 37.1 25.7 125.9 131.3 25.8 17.7

CH2 CH C CH3 CH3 CH2 CH C CH3 CH3 CH2 CH C CH3 CH3 CH3 CH2 CH2 CH C CH3 CH3

5a 84.3 C 194.8 C 118.1 C 176.2 C 60.8 C 41.6 CH2 45.5 CH 49.6 C 205.7 C 211.1 C 49.7 CH 16.9 CH3 28.5 CH2 11.9 CH3 23.4 CH2 122.5 CH 133.3 C 25.9 CH3 18.04 CH3 29.3 CH2 93.7 CH 71.3 C 26.5 CH3 26.4 CH3 28.2 CH2 123.5 CH 134.1 C 26.0 CH3 17.97 CH3 14.2 CH3 37.8 CH2 26.2 CH2 125.9 CH 132.0 C 26.0 CH3 17.8 CH3

6c 83.2 192.7 116.3 173.1 59.4 37.8 43.2 48.1 205.1 209.8 41.8 21.3 20.3


22.2 CH2 121.1 CH 132.3 C 25.6 CH3 17.74 CH3 33.5 CH2 89.8 CH 33.2 CH 18.9 CH3 17.68 CH3 27.1 CH2 122.3 CH 133.4 C 25.9 CH3 17.9 CH3 13.4 CH3 36.3 CH2 25.2 CH2 124.8 CH 131.0 C 25.7 CH3 17.72 CH3

7b 80.2 C 193.7 C 115.8 C 173.9 C 60.9 C 38.7 CH2 43.8 CH 50.1 C 205.8 C 194.9 C 137.9 C 128.9 CH 128.6 CH 132.8 CH 22.9 CH2 121.7 CH 132.6 C 25.9 CH3 17.8 CH3 33.6 CH2 90.8 CH 33.7 CH 18.6 CH3 17.8 CH3 27.9 CH2 123.3 CH 133.8 C 25.8 CH3 18.0 CH3 14.5 CH3 37.3 CH2 25.85 CH2 125.6 CH 131.4 C 25.90 CH3 17.9 CH3

Recorded in methanol-d4. bRecorded in acetone-d6. cRecorded in CDCl3.

(m/z 536.3855 [M]+ and 593.3606 [M + Na]+), respectively. Comparison of the NMR data (Tables 1 and 3) of 6 and 7 with those of furohyperforin indicated that they are comparable.18 The only significant difference observed between furohyperforin and 6 was that the oxygenated quaternary carbon (δC 71.2, C-24) in furohyperforin was replaced by a methine (δC 33.2, C24) in 6, indicating that 6 is the deoxy derivative of furohyperforin. This was confirmed by the 1H−1H COSY correlations between Me-25/H-24, H-24/Me-26, H-24/H-23, and H-23/H2-22. The main difference between 6 and 7 is that the isopropyl group at C-10 in 6 is replaced by a phenyl group in 7. This was confirmed by the HMBC correlations from the two aromatic protons at δH 7.46 (2H, d, J = 8.3, H-12 and H16) to C-10 (δC 194.9) and the proton spin system of H-12/H13/H-14/H-15/H-16 observed in the 1H−1H COSY spectrum. The relative configurations of 6 and 7 were suggested to be the same as those of furohyperforin based on the marked similarity

of their 13C NMR data, which was confirmed by the diagnostic ROESY correlations of Me-32/H-27, Me-32/6β, and H-6α/H23. The HRESIMS of compound 8 showed a protonated molecular ion at m/z 549.3956 [M + H]+, which was used to establish its molecular formula as C36H52O4. Evidence from the UV, IR, and NMR data indicated that 8 is an analogue of pyrano[7,28-b]hyperforin.23 Comparison of the NMR spectroscopic data of 8 with those of pyrano[7,28-b]hyperforin revealed that the only difference was at C-10. Instead of two methyl doublets (δH 1.12, d, J = 6.6 Hz; 1.05, d, J = 6.6 Hz), 8 contained a triplet (δH 0.78) of a methyl group (Me-14) next to a methylene (δH 1.71 and 1.29, H2-13) and a methyl doublet at δH 1.09 (Me-12), characteristic of an isobutyl moiety (Table 4). In the HMBC spectrum, correlations from Me-12 to C-10, C11, and C-13 and from Me-14 to C-11 and C-13 were observed. In the ROESY spectrum, the correlations of Me-32/H-27, MeC



Article

Table 2. 1H NMR Data for Compounds 1−3 (600 MHz, δ in ppm, J in Hz) 1a

position 6α 6β 7 11 12(16) 13(15) 14 17 18 20 21 22 23 25 26 27 28 30 31 32 33 34 35 37 38 39 40 42 43 a

2.11, 1.64, 1.71, 1.98, 0.98, 1.04,

dd (13.2, 4.2) mb m sept (6.6) d (6.6) d (6.6)

2.57, dd (13.8, 10.2) 2.48, dd (13.8, 2.6) 3.53, mb 0.991, s 1.06, s 2.74, dd (12.9, 9.6) 1.86, dd (12.9, 6.6) 4.72, dd (9.6, 6.6) 1.38, s 1.16, s 2.16, m 1.82, m 5.05, mb 1.69, s 1.59, s 1.02, s 1.92, m 1.42, m 2.07, m 1.95, mb 5.03, mb 1.65, s 1.61, s 1.75, dd (9.0, 7.2) 3.64, dd (10.8, 7.2) 3.51, mb 0.994, s 1.27, s

2a b

2.19, m 1.72, mb 1.97, m 7.44, d (8.6) 7.27, t (8.6, 7.9) 7.41, t (7.9) 2.58, dd (13.8, 10.2) 2.47, dd (13.8, 2.4) 3.56, dd (10.2, 2.4) 0.91, s 1.05, s 2.67, dd (13.2, 9.6) 1.95, dd (13.2, 6.6) 4.79, dd (9.6, 6.6) 1.44, s 1.18, s 2.20, m 1.91, m 5.10, t (6.9) 1.71, s 1.63, s 1.15, s 2.01, overlap 1.58, m 2.18, mb 2.01, overlap 5.05, t (6.4) 1.66, s 1.62, s 1.77, dd (8.1, 6.9) 3.62, dd (10.8, 6.9) 3.51, dd (10.8, 8.1) 1.12, s 1.31, s

3a 2.14, 1.61, 1.97, 2.06, 0.97, 1.05,

dd (13.8, 4.2) mb mb sept (6.3) d (6.3) d (6.3)

2.64, dd (13.6, 11.4) 2.32, dd (13.6, 3.0) 3.95, dd (11.4, 3.0) 1.09, s 1.034, s 2.62, m 1.85, mb 4.64, dd (10.8, 5.4) 1.34, s 1.18, s 2.12, m 1.84, mb 5.11, t (6.9) 1.70, s 1.59, s 1.031, s 1.83, mb 1.55, m 2.07, mb 1.95, mb 5.01, t (7.2) 1.65, s 1.60, s 1.92, dd (8.4, 6.3) 3.68, dd (10.8, 6.3) 3.60, dd (10.8, 8.4) 1.28, s 1.14, s

Figure 2. Single-crystal structure of 1.

their 1H and 13C NMR data. When H-7 is α-oriented, the chemical shift of C-7 is δC 41−44, and the difference in chemical shifts of H-6β and H-6α is always 0.3−1.2 ppm, regardless of NMR solvent.1,18−23 Otherwise, the difference in chemical shifts of H-6β and H-6α is 0.0−0.2 ppm and the chemical shift of C-7 is always 45−49 ppm. Therefore, the chemical shifts of C-7 (δC 43.5) and the difference in chemical shifts of H-6β and H-6α (0.43 ppm) suggested that H-7 was αoriented, which was confirmed with ROESY analysis. Thus, the structure of 8 was elucidated and named hypercohin I. Hypercohin J (9) gave a molecular formula of C38H50O6 from HREIMS analysis, 16 mass units more than uraloidin C,21 indicating the presence of an additional hydroxy group. The 1D NMR data of 9 were similar to those of uraloidin C. However, the signal for the methylene at C-17 (δC 28.6) in uraloidin C was replaced with an oxygenated methine (δC 71.2) in 9 (Table 4). This observation suggested that 9 is the 17-OH derivative of uraloidin C, which was supported by the HMBC correlations observed from H-17 (δH 5.24, 1H, d, J = 3.0 Hz) to C-2 (δC 190.4), C-3 (122.7), C-4 (180.7), C-18 (102.5), and C-19 (71.5). The ROESY correlations of Me-32/H-27, H-27/H-6β, H-6β/H-22, and H-16/H-22 showed that 9 had the same relative configurations at C-1, C-5, C-7, and C-8 as uraloidin C. Both H-17 and H-18 were elucidated to be β-oriented by the ROESY correlations of H-12/H-17 and H-17/H-18 (Figure 5). The coupling constant of H-17 and H-18 (3.0 Hz) also suggested a cis relationship of these two hydrogens. Accordingly, 9 was established as the 17-OH derivative of uraloidin C and named hypercohin J. The AChE inhibitory activity of all new and known compounds isolated was assayed using the method developed by Ellman et al.24 Only 7 and 9 showed moderate inhibitory activities (inhibition percentage was 31.7% and 23.0%, respectively) at a concentration of 100 μM. In addition, the 18 isolates were also tested for their cytotoxic effects against five human cancer cell lines, HL-60, A-549, SMMC-7721, MCF-7, and SW480, using the MTT method.25 Interestingly, compounds 1−3, which share the unusual 10-carbon fragment [2,2,4,4,5-(pentamethyltetrahydrofuran-3-yl)methanol] at C-3, showed moderate toxicities, while other isolates exhibited no inhibitory activities (Table 5).

Recorded in methanol-d4. bSignal partially obscured.

Figure 1. Key HMBC (→), 1H−1H COSY (−), and ROESY (↔) correlations of 1.

32/6β, H-6β/H-22, and H-22/H-11 indicated that the relative configuration of 8 was the same as in pyrano[7,28-b]hyperforin (Figure 4). Because the signals of H-7 overlapped with several other signals such as Me-25, Me-26, Me-30, and Me-38 in the δH 1.60−1.70 region, the ROESY correlations of these protons were also crowded in the ROESY spectrum. Therefore, we sought other evidence for further elucidation of the orientation of H-7. The orientation of H-7 in polyprenylated polycyclic acylphloroglucinols can be readily distinguished by examining D



Article

Table 3. 1H NMR Data for Compounds 4−7 (600 MHz, δ in ppm, J in Hz) 4a

5b 2.57, brd (9.6)

6β

2.74, dd (13.8, 4.2) 1.51, m

1.42, md

1.46, m

7 11 12(16) 13(15)

1.58, 1.96, 0.94, 1.02,

1.53, 1.71, 1.03, 1.68,

1.61, 1.97, 0.98, 1.06,

position 6α

14 17

18 20 21 22

m md d (6.6) d (6.6)

3.05, dd (13.8, 7.2) 2.99, md 5.14, 1.60, 1.68, 2.97,

t (7.2) s s md

1.94, m 23 24 25 26 27 28 30 31 32 33 34 35 37 38

d

4.55, dd (10.2, 4.2) 1.37, 1.22, 2.13, 1.78, 4.99, 1.60, 1.55, 1.01, 2.00, 1.39, 2.09, 1.99, 5.04, 1.63, 1.60,

s s m m t (7.2) s s s m md m md t (6.3) s s

m overlap d (6.6) md

1.18, md 0.76, t (7.5) 3.08, dd (13.8, 7.5) 3.03, dd (13.8, 7.5) 5.12, t (7.5) 1.63, s 1.71, s 2.98, dd (13.5, 10.2) 1.85, dd (13.5, 4.8) 4.47, dd (10.2, 4.8) 1.37, 1.20, 2.14, 1.76, 5.02, 1.67, 1.57, 1.01, 1.97, 1.30, 2.09, 1.96, 5.03, 1.66, 1.61,

s s m m md s s s md m m md md s s

6c 2.00, m

md m d (6.4) d (6.4)

7a position

7.46, d (8.3) 7.27, t (8.3, 7.5)

4.36, m 1.86, 1.08, 0.94, 2.15, 1.74, 4.92, 1.67, 1.54, 1.02, 2.06, 1.28, 2.12, 1.90, 5.05, 1.62, 1.58,

1.94, 1.08, 0.98, 2.24, 1.92, 5.10, 1.64, 1.62, 1.16, 2.15, 1.50, 2.22, 2.04, 5.06, 1.64, 1.62,

m d (6.8) d (6.8) m m t (6.8) s s s m m m m md s s

8a

2.28, dd (13.2, 4.2) 1.68, dd (13.2, 12.6) 1.89, m

7.45, t (7.5) 3.05, dd (13.8, 7.2) 2.99, dd (13.8, 7.2) 5.12, md 1.60, s 1.67, s 2.32 dd (13.2, 10.5) 2.09 dd (13.2, 5.4) 4.67, m

3.06, dd (13.8, 7.4) 2.98, dd (13.8, 7.4) 5.08, md 1.62, s 1.66, s 2.36, dd (13.0, 11.0) 1.82, m

Table 4. 1H and 13C NMR Data (δ in ppm) for Compounds 8 and 9

m d (6.6) d (6.6) md m md s s s m m md md t (7.2) s s

a Recorded in acetone-d6. bRecorded in methanol-d4. cRecorded in CDCl3. dSignal partially obscured.

δC

1 2 3 4 5 6α

84.2 188.5 114.6 170.8 56.5 38.7

C C C C C CH2

6β 7 8 9 10 11 12(16) 13(15)

43.5 49.0 206.2 209.0 49.0 16.5 27.3

CH C C C CH CH3 CH2

14 17 18 19 20 21 22

11.6 115.4 123.7 81.8 28.6 28.4 28.9

CH CH CH C CH3 CH3 CH2

23 24 25 26 27

119.2 133.8 25.9 18.1 27.1

CH C CH3 CH3 CH2

28 29 30 31 32 33

122.6 133.3 25.8 17.9 13.4 36.5

CH C CH3 CH3 CH3 CH2

34 35 36 37 38

δH (J in Hz)

1.84, overlap 1.41, mc 1.59, mc

1.84, 1.09, 1.71, 1.29, 0.78, 6.44, 5.32,

CH C CH3 CH3

1.64, 1.64, 2.07, 1.72, 4.94,

s s m mc t (7.2)

1.64, 1.54, 0.99, 1.86, 1.36, 2.08, 1.88, 5.03,

s s s mc mc m mc t

1.61, s 1.56, s

a Recorded in CDCl3. obscured.

b

δC 81.0 190.4 122.7 180.7 57.0 40.7

C C C C C CH2

42.8 51.0 207.8 195.2 138.1 129.4 128.9

CH C C C C CH CH

133.3 71.2 102.5 71.5 26.0 24.8 30.0

CH CH CH C CH3 CH3 CH2

121.0 135.4 26.2 18.3 28.8

CH C CH3 CH3 CH2

123.6 134.5 26.1 18.2 15.0 38.0

CH C CH3 CH3 CH3 CH2

26.0 CH2 125.9 132.0 26.0 17.9

CH C CH3 CH3

δH (J in Hz)

2.09, dd (13.5, 4.5) 1.59, mc 2.22, m

7.49, d (8.2) 7.25, t (8.2, 7.2) 7.42, t (7.2) 5.24, d (3.0) 4.50, d (3.0) 1.31, s 1.27, s 2.56, dd (15.0, 6.9) 2.51, dd (15.0, 6.9) 5.18, t (6.9) 1.65, 1.65, 2.15, 1.93, 5.13,

s s m m t (6.9)

1.72, 1.61, 1.11, 1.97, 1.77, 2.17, 2.05, 5.04,

s s s m m mc m t (7.2)

1.65, s 1.61, s

Recorded in methanol-d4. cSignal partially

detected on a Shamashim UV 2401 spectrometer. IR spectra were determined on a Bruker Tensor-27 infrared spectrophotometer with KBr disks. 1D and 2D NMR spectra were recorded on Bruker AM400, Bruker DRX-500, and DRX-600 spectrometers using TMS as an internal standard. Unless otherwise specified, chemical shifts (δ) were expressed in ppm with reference to the solvent signals. HRESIMS analysis and HREIMS were carried out on an API QSTAR time-offlight spectrometer and on a Waters Auto Spec Premier P776 mass spectrometer. Semipreparative HPLC was performed on an Agilent 1100 HPLC with a Razorback SB-C18 (9.4 mm × 25 cm) column. Silica gel (100−200 and 200−300 mesh, Qingdao Mak all Group Co., Ltd.), Amphichroic RP-18 gel (40−63 μm, Merck, Darmstadt,

Figure 3. Key HMBC (→), 1H−1H COSY (−), and ROESY (↔) of 4.

■

overlap d (6.6) mc m t (7.5) d (9.9) d (9.9)

1.37, s 1.43, s 2.45, dd (14.4, 6.9) 2.39, dd (14.4, 6.9) 4.97, t (6.9)

24.9 CH2 124.7 131.1 25.7 17.6

9b

EXPERIMENTAL SECTION

General Experimental Procedures. Melting points were obtained on an X-4 micro melting point apparatus. Optical rotations were measured on a JASCO P-1020 polarimeter. UV spectra were E



Article

(3 mg), 5 (4 mg), 7 (3 mg), furoadhyperforin (60 mg), and furohyperforin (300 mg). Uraloidin A (16 mg), uraloidin B (21 mg), and uraloidin C (14 mg) were obtained from Fr. B2a3 separated by semipreparative HPLC with 92.5% CH3CN−H2O. After purification by semipreparative HPLC (RP-18, 90% CH3CN−H2O), Fr. B2a4 afforded 27-epifurohyperforin isomer 1 (14 mg), furohyperforin isomer 1 (18 mg), and furohyperforin isomer A (15 mg). Fr. B3 was separated over an MCI gel column (MeOH−H2O from 85:15 to 100:0) to obtain five fractions (Fr. B3a−B3e). Fr. B3b was then chromatographed on a silica gel column, eluted with petroleum ether− acetone (from 9:1 to 7:3), to yield seven fractions (Fr. B3b1−B3b7). Compounds 1 (5 mg), 2 (4 mg), 3 (13 mg), and 9 (5 mg) were isolated from Fr. B3b2 (800 mg) by chromatography on C18 silica gel columns, silica gel columns, and repeated semipreparative HPLC. Hypercohin B (1): colorless crystals; mp 156−157 °C; [α]24D +30.8 (c 0.15, CH3OH); UV (MeOH) λmax (log ε) 272 (3.47), 202 (3.51) nm; IR (KBr) νmax 3446, 2974, 2926, 2874, 1728, 1667, 1621, 1468, 1368 cm−1; 1H and 13C NMR data, see Tables 1 and 2; positive ESIMS m/z 677 [M + Na]+; HREIMS m/z 654.4503 [M]+ (calcd for C40H62O7, 654.4496). Hypercohin C (2): colorless oil; [α]15D −43.6 (c 0.10, CH3OH); UV (MeOH) λmax (log ε) 274 (3.33), 249 (3.39), 201 (3.68) nm; IR (KBr) νmax 3433, 2971, 2926, 1728, 1695, 1627, 1447, 1368 cm−1; 1H and 13C NMR data, see Tables 1 and 2; positive ESIMS m/z 711 [M + Na]+; HREIMS m/z 711.4239 [M + Na]+ (calcd for C43H60O7 Na, 711.4236). Hypercohin D (3): colorless oil; [α]14D +81.5 (c 0.10, CH3OH); UV (MeOH) λmax (log ε) 273 (3.39), 232 (3.10), 201 (3.50) nm; IR (KBr) νmax 3431, 2972, 2927, 2874, 1730, 1623, 1451, 1366 cm−1; 1H and 13C NMR data, see Tables 1 and 2; positive ESIMS m/z 677 [M + Na]+; HREIMS m/z 677.4376 [M + Na]+ (calcd for C40H62O7 Na, 677.4393). Hypercohin E (4): colorless oil; [α]21D +17.9 (c 0.10, CH3OH); UV (MeOH) λmax (log ε) 273 (3.29), 205 (3.37) nm; IR (KBr) νmax 3434, 2968, 2926, 2873, 1729, 1624, 1450, 1377 cm−1; 1H and 13C NMR data, see Tables 1 and 3; positive ESIMS m/z 575 [M + Na]+; HRESIMS m/z 553.3899 [M + H]+ (calcd for C35H53O5, 553.3893). Hypercohin F (5): colorless oil; [α]21D +18.6 (c 0.15 CH3OH); UV (MeOH) λmax (log ε) 273 (3.29), 205 (3.35) nm; IR (KBr) νmax 3442, 2967, 2926, 2876, 1729, 1625, 1451, 1377 cm−1; 1H and 13C NMR data, see Tables 1 and 3; positive ESIMS m/z 589 [M + Na]+; HRESIMS m/z 567.4046 [M + H]+ (calcd for C36H55O5, 567.4049). Hypercohin G (6): colorless oil; [α]21D +42.4 (c 0.20, CH3OH); UV (MeOH) λmax (log ε) 271 (3.10), 204 (3.14) nm; IR (KBr) νmax 2967, 2930, 2874, 1729, 1622, 1450, 1378 cm−1; 1H and 13C NMR data, see Tables 1 and 3; positive ESIMS m/z 537 [M + H]+; HREIMS m/z 536.3855 [M]+ (calcd for C35H52O4, 536.3866). Hypercohin H (7): colorless oil; [α]24D −19.0 (c 0.11, CH3OH); UV (MeOH) λmax (log ε) 274 (3.35), 248 (3.42), 206 (3.56) nm; IR (KBr) νmax 2962, 2925, 2856, 1727, 1695, 1626, 1448, 1373 cm−1; 1H and 13C NMR data, see Tables 1 and 3; positive ESIMS m/z 593 [M + Na]+; HRESIMS m/z 593.3613 [M + Na]+ (calcd for C38H50O4 Na, 593.3606). Hypercohin I (8): colorless oil; [α]24D +60.0 (c 0.22, CH3OH); UV (MeOH) λmax (log ε) 314 (2.91), 254 (3.11), 204 (3.55) nm; IR (KBr) νmax 2969, 2926, 2876, 1764, 1727, 1638, 1586, 1451, 1413, 1365 cm−1; 1H and 13C NMR data, see Table 4; positive ESIMS m/z 549 [M + H]+; HRESIMS m/z 549.3956 [M + H]+ (calcd for C36H53O4, 549.3943). Hypercohin J (9): colorless oil; [α]24D −4.8 (c 0.09, CH3OH); UV (MeOH) λmax (log ε) 274 (3.53), 249 (3.62), 202 (3.89) nm; IR (KBr) νmax 3440, 2970, 2925, 1726, 1698, 1625, 1447, 1411; 1384 cm−1; 1H and 13C NMR data, see Table 4; positive ESIMS m/z 625 [M + Na]+; HREIMS m/z 602.3600 [M]+ (calcd for C38H50O6, 602.3607). X-ray Crystallographic Analysis. Colorless crystals of 1 were obtained from MeOH−Me2CO−H2O. Crystal data were obtained on a Bruker APEX DUO with a graphite monochromator with Cu Kα radiation (λ = Å). The structures were solved by direct methods using SHELXS-97.26 Crystal data of 1: C40H62O7 (M = 654.90);



Table 5. Cytotoxcity of the Isolates on Five Cancer Cell Lines with IC50 Values (μM) compounda

HL-60

SMMC-7721

A-549

MCF-7

SW480

1 2 3 DDPb paclitaxelb

5.8 12.6 8.8 1.8 20 8.2 9.5 15.6 20 9.3 18.7 20 μM) for all cell lines. bDDP (cis-platin) and paclitaxel were used as positive controls.

Germany), and MCI gel (75−150 μm, Mitsubishi Chemical Corporation, Tokyo, Japan) were used for column chromatography. Plant Material. The aerial parts of H. cohaerens N. Robson were collected in Daguan Prefecture, Yunnan Province, People’s Republic of China, in October 2009. The plant was identified by Dr. En-De Liu, Kunming Institute of Botany, Kunming, People’s Republic of China. A voucher specimen was deposited at the Kunming Institute of Botany with identification number 200910H01. Extraction and Isolation. The aerial parts of H. cohaerens (10.0 kg) were powdered and percolated with MeOH at room temperature and filtered. The filtrate was concentrated in vacuo. The crude extract was subjected to silica gel column chromatography eluted with a petroleum ether−acetone gradient (1:0, 8:1, 4:1, 2:1, and 0:1) to afford five fractions, A−E. Fraction B (86.4 g) was separated over an MCI gel column (MeOH−H2O from 8:2 to 10:0) to obtain five fractions (Fr. B1−B4). Fr. B1 (18.5 g) was chromatographed on a silica gel column, eluted with petroleum ether−acetone (from 50:1 to 10:1), to yield four fractions (Fr. B1a−B1d). Fr. B1a (9.3 g) was repeatedly subjected to silica gel columns eluted with petroleum ether−EtOAc (from 50:1 to 6:1) and was further purified by semipreparative HPLC (MeOH−H2O, 95:5) to afford 6 (4 mg), 8 (20 mg), and pyrano[7,28-b]hyperforin (30 mg). Fr. B2 (22.0 g) was isolated over an MCI gel column (MeOH−H2O from 85:15 to 100:0) to obtain four fractions (Fr. B2a−B2d). Fr. B2a (5.0 g) was separated on a silica gel column, eluted with petroleum ether−acetone (from 50:1 to 8:2), to yield six fractions (B2a1−B2a6). Fr. B2a2 was purified by repeated silica gel columns and semipreparative HPLC to afford 4 F



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orthorhombic crystal (0.05 × 0.06 × 0.60 mm); space group P212121; unit cell dimensions a = 12.2253(2) Å, b = 16.6218(2) Å, c = 18.6036(3) Å, V = 3780.37(10) Å3; Z = 4; ρcalcd = 1.151 mg/m3; μ = 0.610 mm−1, F(000) = 1432; 6305 unique reflections (Rint = 0.0312), which were used in all calculations; the final refinement gave R1 = 0.0384 (>2σ(I)) and wR2 = 0.1016 (all data); Flack parameter = −0.1(3). Crystallographic data for 1 have been deposited in the Cambridge Crystallographic Data Centre (deposition number: CCDC 930811). Copies of the data can be obtained free of charge via www. ccdc.cam.ac.uk. Acetylcholinesterase Inhibitory Activity. Acetylcholinesterase inhibitory activity of the compounds was assayed by the spectrophotometric method developed by Ellman et al.24 Acetylthiocholine iodide (Sigma) was used as substrate in the assay. Compounds were dissolved in DMSO. The reaction mixture contained 1100 μL of phosphate buffer (pH 8.0), 10 μL of test compound solution (100 μM), and 40 μL of acetyl cholinesterase solution (0.04 U/100 μL), and the mixture was incubated for 20 min (30 °C). The reaction was initiated by the addition of 20 μL of DTNB (6.25 mM) and 20 μL of acetylthiocholine. The hydrolysis of acetylthiocholine was monitored at 405 nm after 30 min. Tacrine was used as positive control. All the reactions were performed in triplicate. The percentage inhibition was calculated as follows: % inhibition = [(E − S)/E] × 100 (E is the activity of the enzyme without test compound and S the activity of enzyme with test compound). Cytotoxcity Assays. The human tumor cell lines used were HL60, SMMC-7721, A-549, MCF-7, and SW-480, which were obtained from ATCC (Manassas, VA, USA). All cells were cultured in RPMI1640 or DMEM medium (Hyclone, Logan, UT, USA), supplemented with 10% fetal bovine serum (FBS, Hyclone) at 37 °C in a humidified atmosphere with 5% CO2. Cell viability was assessed by conducting colorimetric measurements of the amount of insoluble formazan formed in living cells based on the reduction of 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Sigma, St. Louis, MO, USA).17 Briefly, 100 μL of adherent cells was seeded into each well of a 96-well cell culture plate and allowed to adhere for 12 h before test compound addition, while suspended cells were seeded just before test compound addition, both with an initial density of 1 × 105 cells/mL in 100 μL of medium. Each tumor cell line was exposed to the test compound at various concentrations in triplicate for 48 h, with cisplatin and paclitaxel (Sigma) as positive control. After the incubation, MTT (100 μg) was added to each well, and the incubation continued for 4 h at 37 °C. The cells were lysed with 100 μL of 20% SDS−50% DMF after removal of 100 μL of medium. The optical density of the lysate was measured at 595 nm in a 96-well microtiter plate reader (Bio-Rad 680). The IC50 value of each compound was calculated by Reed and Muench’s method.25

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ASSOCIATED CONTENT

* Supporting Information S

This material (1H, 13C NMR, DEPT, HSQC, HMBC, COSY, ROESY, MS, IR, and UV spectroscopic data for compounds 1− 9 and X-ray data of compound 1) is available free of charge via the Internet at http://pubs.acs.org.

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Article

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel/Fax: (86) 87165217971. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by the NSFC (20972167), the Young Academic Leader Raising Foundation of Yunnan Province (No. 2009CI073), and CAS (G.X.). G


Bioactive Polyprenylated Acylphloroglucinol

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