Cytotoxic Oxepinochromenone and Flavonoids from the Flower Buds

Sep 24, 2013 - ... ECD spectrum (Figure S7) permitted the assignment of 5R and 6S ... Long-range correlations of the hydroxy proton (δH 11.30) with C...
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Cytotoxic Oxepinochromenone and Flavonoids from the Flower Buds of Rosa rugosa Qiu-Fen Hu,†,‡ Bin Zhou,†,§ Jian-Ming Huang,‡,⊥ Zhi-Yong Jiang,† Xiang-Zhong Huang,† Li-Ying Yang,† Xue-Mei Gao,*,† Guang-Yu Yang,*,†,§ and Chun-Tao Che‡ †

Key Laboratory of Chemistry in Ethnic Medicinal Resources, State Ethnic Affairs Commission and Ministry of Education, Yunnan University of Nationalities, Kunming 650031, Yunnan, People’s Republic of China ‡ Department of Medicinal Chemistry and Pharmacognosy and WHO Collaborating Center for Traditional Medicine, College of Pharmacy, University of Illinois at Chicago, Chicago, Illinois 60612, United States § Key Laboratory of Tobacco Chemistry of Yunnan Province, Yunnan Academy of Tobacco Science, Kunming 650106, Yunnan, People’s Republic of China ⊥ Department of Pharmacognosy, School of Pharmacy, Fudan University, Shanghai 201203, People’s Republic of China S Supporting Information *

ABSTRACT: A new oxepinochromenone, rugosachromenone A (1), seven new flavonoids, rugosaflavonoids A−G (2−8), and 11 known compounds (9−19) were isolated from the flower buds of Rosa rugosa. Compound 1 is found from Nature for the first time. Compound 2 displayed cytotoxicity against NB4, SHSY5Y, and MCF7 cells with IC50 values of 2.2, 2.5, and 2.3 μM, respectively, and 3 was toxic to A549 and MCF7 cells with IC50 values of 1.2 and 2.8 μM, respectively.

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Rosa rugosa Thunb. (Rosaceae) is a common ornamental flower distributed in the temperate regions of eastern Asia and widely cultivated in Yunnan Province.1,2 The petals and buds of R. rugosa are often used as food, incense, and Chinese medicinal materials for the treatment of stomachache, diarrhea, and gynecological problems.3 Previous studies have shown the presence of tannins,4 terpenoids,5−7 and flavonoids8,9 in this genus. Anti-inflammatory and cytotoxic activities have been observed with selected chemical ingredients isolated from R. rugosa.7,10 Previous studies in our laboratories on this plant have led to the isolation of three new aurones that possess antiHIV-1 and cytotoxic properties.9 Continuing the search for novel and bioactive metabolites from medicinal plants, we now report the isolation and characterization of a new oxepinochromenone (1), seven new flavonoids (2−8), and 11 known structures (9−19) from the flower buds of R. rugosa. Compound 1 is the first example of an oxepinochromenone found in Nature. Described in this paper are the structure elucidation of 1−8 and biological evaluation of the compounds isolated. © XXXX American Chemical Society and American Society of Pharmacognosy

RESULTS AND DISCUSSION The flower buds of R. rugosa were extracted with 95% MeOH, followed by repeated column chromatography on silica gel, Sephadex LH-20, and RP-18 silica gel. Final purification by semipreparative RP-HPLC afforded a new oxepinochromenone and 18 flavonoids. The new structures 1−8 are shown in Figure 1, and their 1H and 13C NMR data are given in Tables 1−4. The known compounds (Supporting Information, Figure S1) were identified as catechin (9),11 kaempferol (10),11 quercetin (11),11 candenatenin E (12),12 (2R,3R)-3,5-dihydroxy-7methoxyflavanone (13),13 (3S,4R)-6,3′-dimethoxyisoflavan-4ol (14),14 7,8,3′-trihydroxy-4′-methoxyisoflavone (15),15 7,8,3′trihydroxy-6,4′-dimethoxyisoflavone (16),16 glyinflanin B (17),17 hamilcone (18),18 and (2R)-2′,4′-dihydroxy-7-methoxy-8-(2-hydroxyethyl)flavan (19).19 Compound 1 was obtained as a pale yellow gum. The HRESIMS pseudomolecular ion [M − H]− at m/z 345.0615 is Received: May 21, 2013

A

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Figure 1. Structures of new flavonoids from R. rugosa.

Table 1. 1H and 13C NMR Data of Compounds 1−3 (δ in ppm, in pyridine-d5, 500 and 125 MHz) compound 1 δC (mult)

no. 2 3 4 5 6 7 8 9 10 11 12 1′ 2′ 3′ 4′ 5′ 6′ 6-OMe 7-OMe 11-OMe 4′-OMe 3′-OH 4′-OH

166.2 111.9 185.4 78.1 70.1 121.8 139.4 169.6 105.5 171.8

s d s s d d d s s s

122.6 131.2 116.5 158.5 116.5 131.2

s d d s d d

52.5 q

compound 2

δH (mult, J, Hz) 6.60 s

4.64 d (7.2) 5.64 dd (6.2, 7.2) 6.59 d (6.2)

7.80 d (8.8) 6.87 d (8.8) 6.87 d (8.8) 7.80 d (8.8)

δC (mult) 163.2 105.2 181.5 136.8 113.1 165.0 103.8 158.8 107.9 168.3

s d s s d s d s s s

122.9 131.0 115.6 161.1 115.6 131.0

s d d s d d

3.69 s

52.4 q 55.6 q

2 3 4 5 6 7 8 9 10 1′ 2′, 6′ 3′, 5′

δC (mult) 158.4 135.2 179.4 162.9 100.1 166.4 94.9 159.3 105.5 122.7 131.2 116.0

s s s s d s d s s s d d

δH (mult, J, Hz)

6.13 s 6.30 s

7.99 d (8.7) 6.81 overlap

δH (mult, J, Hz) 6.68 s

6.89 d (1.8) 6.74 d (1.8)

7.76 d (8.8) 7.00 d (8.8) 7.00 d (8.8) 7.76 d (8.8)

δC (mult) 164.2 105.5 179.0 122.1 146.3 159.1 113.6 151.8 120.2 199.9 29.0 122.9 114.0 145.4 148.9 116.3 119.5 55.9 61.0

s d s d s s s s s s q s d s s d d q q

δH (mult, J, Hz) 6.64 s 7.39 s

2.50 s 7.23 d (1.8)

7.04 7.32 3.79 3.81

d (8.1) dd (1.8, 8.1) s s

3.95 s 3.80 s 10.81 brs 11.09 brs

11.50 s

Table 2. 1H and 13C NMR Data of Compound 4 (δ in ppm, in methanol-d4, 400 and 100 MHz) position

compound 3

position

δC (mult)

4′ 1″ 2″ 3″ 4″ 5″ 6″ 1‴ 2‴, 6‴ 3‴, 5‴ 4‴ 7‴

161.5 104.0 75.7 78.0 71.7 75.8 64.3 122.0 132.2 116.8 161.2 168.8

s d d d d d t s d d s s

consistent with the molecular formula C17H14O8 (11 indices of hydrogen deficiency). Analysis of the 1H, 13C, and HSQC NMR data (Table 1) revealed the presence of a phenolic hydroxy proton (δH 11.50), a methoxycarbonyl group (δC 171.8, 52.5; δH 3.69), an oxymethine (δC 70.1, δH 4.64), an O-bearing quaternary carbon (δC 78.1), 12 olefinic/aromatic carbons (seven of which are protonated), and a carbonyl group (δC 185.4). The 1H and 13C NMR data of C-2 to C-10 of 1 are comparable to those of the dihydrooxepine moiety of conioxepinol C.20 Key HMBC correlations (Figure 2) between H-8 (δH 5.64) and C-9 (δC 169.6), as well as between H-6 (δH 4.64) and C-10 (δC 105.5), led to the proposal of the dihydrooxepine moiety being connected to C-9 and C-10. Assignment of a methoxycarbonyl group at C-5 was based on a long-range HMBC correlation observed between H-6 (δH 4.64) and the ester carbonyl carbon (δC 171.8). Finally, HMBC correlations of the phenolic hydroxy proton (δH 11.50) with C-

δH (mult, J, Hz) 5.24 d (7.0) 3.48 overlap 3.46 overlap 3. 34 m 3.48 overlap 4.18, 4.30 m 7.92 d (8.9) 6.81 overlap

B

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Table 3. 1H NMR and 13C NMR Data of Compounds 5 and 6 (δ in ppm, in pyridine-d5, 500 and 125 MHz) compound 5 no. 2α

δC (mult)

δH (mult, J, Hz)

66.6 t

2β 3 4 5 6 7 8 9 10 1′ 2′ 3′ 4′ 5′ 6′ 7-OMe 4′-OMe 4′-OH 6-OH

39.0 78.1 113.1 142.8 147.8 104.9 149.4 134.8 130.3 127.1 114.0 160.0 114.0 127.1 55.7

3.65 dd (10.2, 11.0) 4.21 dd (5.2, 10.2) 3.50 m 5.31 d (6.6) 7.69 s

d d d s s d s s s s d s d s q

7.36 s

7.41 d (8.6) 6.94 d (8.6) 6.94 d (8.6) 7.41 d (8.6) 3.73 s

compound 6 δC (mult)

δH (mult, J, Hz)

67.0 t

38.8 78.5 115.4 139.0 149.3 103.2 149.0 134.5 128.6 126.8 113.8 163.2 113.8 126.8 55.8 55.9

3.57 dd (10.2, 11.2) 4.34 dd (5.2, 10.8) 3.46 m 5.33 d (6.9) 7.73 s

d d d s s d s s s s d s d s q q

7.28 s

Figure 2. Selected HMBC (↷) correlations of 1, 3, 5, and 7.

generated as an auxiliary chromophore. The negative Cotton effects observed at around 302 and 408 nm in the induced ECD spectrum (Figure S7) permitted the assignment of 5R and 6S configurations according to the empirical rule proposed by Snatzke and exemplified by Wang.20 All available data suggested rugosachromenone A (1) to be a new oxepinochromenone, as shown in Figure 1. Compound 2 was obtained as an orange gum. A molecular formula of C18H14O6 was inferred from HRESIMS (m/z 349.0682 [M + Na]+, calcd 349.0688). The 1H and 13C NMR data of 2 (Table 1) revealed 18 carbon and 14 proton signals. The structure was likely a flavone11 bearing a methoxycarbonyl group (δC 168.3, 52.4; δH 3.95), a methoxy group (δC 55.6, δH 3.80), a phenolic hydroxy group (δH 11.30), and seven aromatic protons. The HMBC correlation between the methoxy protons (δH 3.80) and C-4′ (δC 161.1) suggested a methoxy group at C4′. Long-range correlations of the hydroxy proton (δH 11.30) with C-6 (δC 113.1), C-7 (δC 165.0), and C-8 (δC 103.8) led to the assignment of the hydroxy group at C-7. The methoxycarbonyl group at C-5 was supported by HMBC correlations of H-6 (δH 6.89) with the ester carbonyl carbon (δC 168.3), and no correlation was observed between H-8 (δH 6.74) and the carbonyl carbon. The typical proton signal [δH 6.89 d (1.8 Hz), 6.74 d (1.8 Hz), 7.76 (2H) d (8.8 Hz), 7.00 (2H) d (8.8 Hz)] also supported a 5,7-disubstituted ring A and a 4′monosubstituted ring B. The structure of rugosaflavonoid A (2) was established as shown in Figure 1. Compounds 1 and 2 may be methyl ester artifacts due to the extraction using methanol. Therefore, we extracted the samples with acetonitrile and determined the presence of 1 and 2 by HPLC. The chromatogram (Figure S25) showed that compounds 1 and 2 are indeed natural products. Compound 3 was obtained as an orange-yellow gum. A molecular formula of C19H16O7 was assigned from HRESIMS (m/z 379.0797 [M + Na]+, calcd 379.0794). The 1H and 13C NMR data of 3 (Table 1) displayed 19 carbon and 16 proton signals, corresponding to a flavonoid nucleus11 (δC 164.2, 105.5, 179.0, 122.1, 146.3, 159.1, 113.6, 151.8, 120.2, 122.9, 114.0, 145.4, 148.9, 116.3, 119.5), one acetyl group (δC 199.9, 29.0; δH 2.50), two methoxy groups (δC 55.9, 61.0, δH 3.79, 3.81), and two phenolic hydroxy protons (δH 10.81, 11.09). The typical proton signals at δH 7.23 d (1.8 Hz) 1H, δH 7.04 d (8.1 Hz) 1H, and δH 7.32 dd (1.8, 8.1 Hz) 1H revealed 3,4disubstitution on ring B, and the HMBC correlations (Figure 2) of the hydroxy proton at δH 10.81 with C-2′ (δC 114.0), C-3′ (δC 145.4), and C-4′ (δC 148.9) and the other hydroxy signal at

7.43 d (8.6) 6.99 d (8.6) 6.99 7.43 3.80 3.82

d (8.6) d (8.6) s s

11.00 brs 11.35 brs

Table 4. 1H NMR and 13C NMR Data of Compounds 7 and 8 (δ in ppm, in CDCl3, 500 and 125 MHz) compound 7 no.

δC (mult)

1 2 3

33.0 t 125.7 d 130.9 d

1′ 2′ 3′ 4′ 5′ 6′ 1″ 2″ 3″ 4″ 5″ 6″ 5′-OMe 2″-OMe 3″-OMe −OCH2O− 4″-OH

127.8 113.0 146.3 147.5 119.2 115.2 126.3 151.4 140.0 149.0 110.9 124.3

s d s s d d s s s s d d

56.1 q 60.8 q 101.4 t

δH (mult, J, Hz) 3.46 dd (6.9, 1.5) 6.23 m 6.72 dd (15.9, 1.6) 6.92 d (2.4)

6.52 d (8.6) 6.75 dd (2.4, 8.6)

6.63 d (8.5) 6.81 d (8.5) 3.77 s 3.85 s 5.81, 5.87 s 9.82 brs

compound 8 δC (mult) 33.2 t 125.7 d 131.0 d 133.0 106.1 147.3 136.0 149.3 103.8 126.8 151.0 140.2 148.5 110.5 124.0 55.8 56.0 60.8 101.2

s d s s s d s s s s d d q q q t

δH (mult, J, Hz) 3.44 dd (6.9, 1.5) 6.22 m 6.73 dd (15.9, 1.6) 6.79 s

6.66 s

6.61 d (8.4) 6.83 d (8.4) 3.80 s 3.78 s 3.84 s 5.82, 5.89 s 9.88 brs

4′ (δC 158.5) and C-3′, 5′ (δC 116.5) allowed the assignment of a phenolic hydroxy group at C-4′. The relative configuration of 1 was determined by a NOESY experiment, in which a correlation between H-6 and 11-OMe suggested a cis relationship for the 5,6-diol moiety. The absolute configuration was then determined by the in situ dimolybdenum electronic circular dichroic (ECD) method.20,21 Thus, upon addition of Mo2(OAc)4 to a solution of 1 in DMSO, a metal complex was C

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δH 11.09 with C-3′ (δC 145.4), C-4′ (δC 148.9), and C-5′ (δC 116.3) suggested the locations of the two phenolic hydroxy groups at C-3′ and C-4′. The HMBC correlation of the methyl proton signal at δH 2.50 with C-8 (δC 113.6) suggested the presence of an acetyl group at C-8. The assignment of two methoxy groups at C-6 and C-7 was supported by the HMBC correlation of the methoxy proton signal at δH 3.79 with C-6 (δC 146.3) and the signal at δH 3.81 with C-7 (δC 159.1). The HMBC correlations between H-5 (δH 7.39 s) and C-6 (δC 146.3), C-7 (δC 159.1), C-4 (δC 179.0), and C-10 (δC 120.2) and a ROESY correlation between H-5 (δH 7.39 s) and MeO-6 (δH 3.79) also supported the 6,7,8-trisubstitution of ring A. Thus, the structure of 3 was established and given the trivial name rugosaflavonoid B. Compound 4 was obtained as a pale yellow gum. Its HRESIMS spectrum in the negative mode revealed an [M − H]− signal at m/z 567.1130, consistent with the molecular formula C28H24O13 (17 indices of hydrogen deficiency). Its IR spectrum exhibited the presence of hydroxy (3465 cm−1), carbonyl (1687, 1658 cm−1), and aromatic (1615, 1541, 1502 cm−1) functionalities. Its 13C and DEPT NMR spectra displayed signals for 28 carbons corresponding to a 5,7,4′substituted flavonol22 [δC 158.4, 135.2, 179.4, 162.9, 100.1, 166.4, 94.9, 159.3, 105.5, 122.7, 131.2 (2C), 116.0 (2C), 161.5], a glucosyl moiety11 (δC 104.0, 75.7, 78.0, 71.7, 75.8, 64.3), and a 4-substituted benzoyloxy group11 [δC 122.0, 132.2 (2C), 116.8 (2C), 161.2, 168.8]. The 1H NMR signals (Table 2) also supported a 5,7,4′-substituted flavonol skeleton bearing a glucosyl and a 4-substituted benzoyloxy group. The longrange correlation between H-1″ (δH 5.24) and C-3 (δC 135.2) indicated the glucosyl group was located at C-3. The glucosyl group should be in a β-configuration due to the coupling constant of H-1″ (7.0 Hz)23 and by comparing the 1H and 13C NMR data with those of glucose.22 Furthermore, this was also confirmed by ROESY correlations of H-1″ with H-3″ and H-5″ and of H-2″ with H-4″. The attachment of the 4-substituted benzoyloxy group at C-6″ of glucose was supported by the HMBC correlations of H-6″ (δH 4.18, 4.30) with C-7‴ (δC 168.8). On the basis of the above evidence, the structure of rugosaflavonoid C was established as shown in Figure 1. Compounds 5 and 6 were obtained as orange gums, and they show molecular ions at m/z 301.1078, and 301.1062, respectively, in the negative HRESIMS, corresponding to the same molecular formula, C17H18O5. The 1H and 13C NMR data (Table 3) of compound 5 exhibited signals for 17 carbons and 18 protons, including two aromatic rings with six aromatic protons (δH 7.69, 7.36, 7.41 2H, and 6.94 2H), an oxygenated methine (δC 78.1), a methine (δC 39.0), an oxymethylene (δC 66.6), and two methoxy groups (δC 55.7, 56.0). Strong absorption bands accounting for hydroxy (3396 cm−1) and aromatic groups (1618, 1536, 1475 cm−1) were observed in the IR spectrum. The UV spectrum of 5 displayed an absorption at 298 nm, confirming the existence of aromatic groups. The 1H NMR spectrum showed a pair of doublets at δH 3.65 (10.2, 11.0 Hz) and δH 4.21 (5.2, 10.2 Hz), a multiplet at δH 3.50, and a doublet at δH 5.31 (6.6 Hz). These signals were assignable to H-2, H-3, and H-4 of a 4-hydroxyisoflavan skeleton, respectively.24 The typical proton signals at δH 7.69 s, 1H and δH 7.36 s, 1H revealed a 6,7-disubstituted ring A.11 Proton signals at δH 7.41 d (8.6 Hz), 2H and δH 6.94 d (8.6 Hz), 2H revealed that ring B was 4′-substituted.11 The HMBC correlations (Figure 2) of the phenolic hydroxy proton (δH 11.00) with C-3′ (δC 114.0) and C-4′ (δC 160.0) indicated that

the phenolic hydroxy group was located at C-4′. The assignment of methoxy groups to C-6 and C-7 was supported by the HMBC correlations of the methoxy proton signals (δH 3.88, 3.73) with C-6 (δC 142.8) and C-7 (δC 147.8), respectively. This was further confirmed by the NOESY correlations (Figure 3) of OMe-6 (δH 3.88) with H-5 (δH

Figure 3. Key NOESY correlations (↔) of 5.

7.69) and of OMe-7 (δH 3.73) with H-8 (δH 7.36). The coupling constant between H-2β and H-3 inferred an α and pseudoaxial orientation of H-3. Moreover, the large coupling constants between H-3 and H-4 revealed their trans relationship, indicating the β-orientation of H-4. The relative configuration was confirmed by NOESY results (Figure 3). Finally, the 1H and 13C NMR chemical shifts of C-2, C-3, and C-4 displayed agreement with those of conferol A24 and bolusanthol A.25 Thus, compound 5 was elucidated as shown in Figure 1 and given the trivial name rugosaflavonoid D. The 1H and 13C NMR spectra of 6 were similar to those of 5, the most obvious differences being a downshift of C-4′ from δC 160.0 to δC 163.2 and an upshift of C-6 from δC 142.8 to δC 139.0. The HMBC correlations of the phenolic hydroxy proton (δH 11.35) with C-5 (δC 115.4), C-6 (δC 139.0), and C-7 (δC 149.3) indicated that the phenolic hydroxy group was located at C-6. Moreover, the assignment of methoxy groups to C-7 and C-4′ was supported by the HMBC correlations of the methoxy proton signals (δH 3.80, 3.82) with C-7 (δC 149.3) and C-4′ (δC 163.2), respectively. The relative configuration at C-3 and C-4 was confirmed by NOESY results. Thus, the structure of compound 6 (rugosaflavonoid E) was established as shown in Figure 1. Rugosaflavonoid F (7) was obtained as a pale yellow gum. The HREIMS exhibited [M + Na]+ at m/z 337.1044, consistent with the molecular formula C18H18O5 (10 indices of hydrogen deficiency). Its 13C and DEPT NMR spectra (Table 4) displayed signals for 18 carbons, corresponding to two aromatic rings, a methylene (δC 33.0), a pair of olefinic carbons (δC 125.7, 130.9), a methylenedioxy (δC 101.4), and two methoxy groups (δC 56.1, 60.8). The IR spectrum showed the presence of hydroxy (3398 cm−1) and aromatic (1592, 1496, 1453 cm−1) functionalities. The UV spectrum displayed absorptions at λmax 210 and 261 nm, suggesting the presence of a conjugated aromatic chromophore. The 1H NMR spectroscopic data (Table 4) displayed signals for ABC-type protons at δH 6.92 d (2.4 Hz), H-2′, δH 6.52 d (8.6 Hz), H-5′, and δH 6.75 dd (2.4, 8.6 Hz), H-6′, as well as ortho-coupled AB-type protons at δH 6.81 d (8.5 Hz), H-6 and δH 6.63 d (8.5 Hz), H-5, suggesting the presence of a 1′,3′,4′-trisubstituted and a 1″,2″,3″,4″tetrasubstituted aromatic ring, respectively. The signals of the ABX2-type protons appearing at δH 6.72 1H dd (15.9, 1.6 Hz), H-3, 6.23 1H (m), H-2, and 3.46 2H dd (6.9, 1.5 Hz), H-1 were consistent with a 1,3-diarylpropene structure.11 In addition, a methylenedioxy (δH 5.81, 5.87) and two methoxy groups (δH 3.77, 3.85), along with a low-field hydroxy proton (δH 9.82), were present. These data indicated that the structure of 7 was closely related to that of candenatenin A,12 with a D

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(A−F). Further separation of fraction B (32.8 g) by silica gel column chromatography, eluted with petroleum ether−acetone (9:1−1:2), yielded subfractions B1−B6. Subfraction B3 (6.47 g) was loaded onto another silica gel column using petroleum ether−EtOAc elution and, finally, semipreparative HPLC (50−55% MeOH−H2O, flow rate 12 mL/min) to afford 2 (13.6 mg), 7 (17.5 mg), 8 (18.4 mg), 13 (22.3 mg), and 14 (14.0 mg). Subfraction B4 (7.26 g) was separated on a silica gel column eluted by petroleum ether−EtOAc, followed by semipreparative HPLC (42−50% MeOH−H2O, flow rate 12 mL/ min), to give 1 (9.22 mg), 3 (8.60 mg), 5 (9.82 mg), 6 (12.8 mg), 12 (14.3 mg), and 17 (17.5 mg). Subsequent separation of fraction D (28.2 g) by silica gel column chromatography, eluted with petroleum ether−acetone (8:2−1:2), yielded subfractions D1−D5. Subfraction D3 (11.8 g) was purified by silica gel column chromatography using petroleum ether−EtOAc mixtures and semipreparative HPLC (35− 42% MeOH−H2O, flow rate 12 mL/min) to afford 15 (6.38 mg), 16 (11.6 mg), 18 (16.3 mg), and 19 (18.2 mg). Subfraction D4 (8.26 g) was subjected to silica gel column chromatography eluted by petroleum ether−acetone followed by semipreparative HPLC (30− 35% MeOH−H2O, flow rate 12 mL/min) to give 4 (13.6 mg), 9 (49.7 mg), 10 (26.5 mg), and 11 (33.6 mg). −58.6 (c 0.20, Rugosachromenone A (1): yellow gum; [α]22.5 D MeOH); ECD (in DMSO containing Mo2(OAc)4 with the inherent ECD spectrum subtracted, c 0.25) Δε 302 −3.82, Δε 342 +4.22; Δε 408 −5.22; UV (MeOH) λmax (log ε) 338 (2.87), 272 (3.92), 210 (4.27) nm; IR (KBr) νmax 3448, 2963, 1742, 1658, 1602, 1548, 1486, 1439, 1285, 1164, 1098, 857 cm−1; 13C NMR and 1H NMR data (in pyridine-d5) see Table 1; negative ESIMS m/z 345 [M − H]−; negative HRESIMS m/z 345.0615 [M − H]− (calcd for C17H13O8, 345.0610). Rugosaflavonoid A (2): orange gum; UV (MeOH) λmax (log ε) 365 (4.11), 257 (4.28), 210 (4.65) nm; IR (KBr) νmax 3416, 1702, 1657, 1610, 1565, 1456, 1432, 1287, 1182, 1028, 893 cm−1; 13C NMR and 1 H NMR data (in pyridine-d5) see Table 1; positive ESIMS m/z 349 [M + Ma]+; HRESIMS m/z 349.0682 [M + Na]+ (calcd for C18H14NaO6, 349.0688). Rugosaflavonoid B (3): orange-yellow gum; UV (MeOH) λmax (log ε) 368 (4.08), 283 (3.86), 257 (4.32), 210 (4.73) nm; IR (KBr) νmax 3468, 1695, 1657, 1614, 1537, 1483, 1459, 1124, 1090, 936, 865 cm−1; 13 C NMR and 1H NMR data (in pyridine-d5) see Table 1; positive ESIMS m/z 379 [M + Ma]+; HRESIMS m/z 379.0797 [M + Na]+ (calcd for C19H16NaO7, 379.0794). −62.3 (c 0.15, Rugosaflavonoid C (4): pale yellow gum; [α]24.8 D MeOH); UV (MeOH) λmax (log ε) 355 (4.10), 314 (4.36), 265 (4.28), 210 (4.76) nm; IR (KBr) νmax 3465, 1687, 1658, 1615, 1541, 1502, 1462, 1353, 1218, 1154, 875, 762 cm−1; 1H and 13C NMR data (in CD3OD), see Table 2; negative ESIMS m/z 567 [M − H]−; negative HRESIMS m/z 567.1130 [M − H]− (calcd for C28H23O13, 567.1139). −73.2 (c 0.20, Rugosaflavonoid D (5): pale orange gum; [α]22.5 D MeOH); UV (MeOH) λmax (log ε) 345 (2.92), 298 (4.22), 210 (4.76) nm; IR (KBr) νmax 3396, 2943, 2884, 1618, 1536, 1475, 1422, 1360, 1169, 1037, 865 cm−1; 1H NMR and 13C NMR data (in pyridine-d5), Table 3; ESIMS (negative ion mode) m/z 301 [M − H]−; HRESIMS (negative ion mode) m/z 301.1078 [M − H]− (calcd for C17H17O5, 301.1076). Rugosaflavonoid E (6): orange gum; [α]22.5 D −68.5 (c 0.20, MeOH); UV (MeOH), λmax (log ε) 345 (2.86), 230 (4.23), 210 (4.68) nm; IR (KBr) νmax 3394, 2947, 2880, 1615, 1534, 1471, 1426, 1363, 1167, 1035, 862 cm−1; 1H NMR and 13C NMR data (in pyridine-d5), Table 3; ESIMS (negative ion mode) m/z 287 [M − H]−; HRESIMS (negative ion mode) m/z 301.1062 [M − H]− (calcd for C17H17O5, 301.1076). Rugosaflavonoid F (7): pale yellow gum; UV (MeOH) nm (log ε) 261 (3.78); 210 (4.32), IR (KBr) cm−1 3398, 2932, 2847, 1592, 1496, 1453, 1162, 1076, 908, 864; 1H and 13C NMR (in CDCl3) see Table 4; ESIMS m/z 337 [M + Ma]+; HRESIMS m/z 337.1044 [M + Na]+ (calcd for C18H18NaO5, 337.1052). Rugosaflavonoid G (8): pale yellow gum; UV (MeOH) nm (log ε) 264 (3.83); 210 (4.36); IR (KBr) cm−1 3402, 2936, 2851, 1595, 1498,

methoxy and hydroxy group in candenatenin A being substituted by a methylenedioxy group in 7 on ring B. The HMBC correlations of the two methoxy proton signals (δH 3.77, 3.85) with C-2″ (δC 151.4) and C-3″ (δC 140.0) led to the assignment of the methoxy groups at C-2″ and C-3″. The assignment of the methylenedioxy group at C-3′ and C-4′ was supported by the HMBC correlation of the methylenedioxy protons (δH 5.81, 5.87) with C-3′ (δC 146.3) and C-4′ (δC 147.5). Finally, the HMBC correlations of the hydroxy proton at δH 9.82 with C-3″ (δC 140.0), C-4″ (δC 149.0), and C-5″ (δC 110.9) suggested the position of the hydroxy group at C-4″. Thus, the structure of rugosaflavonoid F (7) was determined as shown in Figure 1. Compound 8 was obtained as pale yellow gum, exhibiting a quasimolecular ion at m/z 367.1165 [M + Na]+ in the HRESIMS (calcd m/z 367.1158), consistent with the molecular formula C19H20O6. A comparison of the 1H and 13C NMR data between 8 and 7 (Table 4) revealed the presence of an additional methoxy group (δC 55.8, δH 3.80) on ring B. The location of the methoxy group at C-5′ was supported by the HMBC correlation of the methoxy proton (δC 3.80) with C-5′ (δC 147.3). The structure of 8 (rugosaflavonoid G) was determined as shown in Figure 1. Many flavonoid derivatives are known to be cytotoxic,9,26,27 and antitumor activities have been reported for R. rugosa.7,9,10 The new compounds 1−8, together with 19, were tested for their cytotoxicity against five human tumor cell lines (NB4, A549, SHSY5Y, PC3, and MCF7) using the MTT method, with paclitaxel as the positive control.28 Compound 2 exhibited weak cytotoxicity against NB4, SHSY5Y, and MCF7 cells with IC50 values of 2.2, 2.5, and 2.3 μM, respectively (Table 4). Compound 3 also showed cytotoxicity against A549 and MCF7 cells with IC50 values of 1.2 and 2.8 μM, respectively.

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured with a Horiba SEPA-300 polarimeter. UV spectra were obtained using a Shimadzu UV-2401A spectrophotometer. ECD spectra were measured on a JASCO J-810 spectropolarimeter. A Tenor 27 spectrophotometer was used for scanning IR spectra. 1D and 2D NMR spectroscopic data were recorded on a DRX-500 or DRX-400 NMR spectrometer with TMS as internal standard. Chemical shifts (δ) are expressed in ppm with reference to the TMS signal. HRESIMS was performed on a VG Autospec-3000 spectrometer. Semipreparative HPLC was performed on a Shimadzu LC-8A preparative liquid chromatograph with Zorbax PrepHT GF (21.2 mm × 25 cm) or Venusil MP C18 (20 mm × 25 cm) columns. Column chromatography was performed using silica gel (200−300 mesh, Qing-dao Marine Chemical, Inc., Qingdao, China), Lichroprep RP-18 gel (40−63 μm, Merck, Darmstadt, Germany), Sephadex LH-20 (Sigma-Aldrich, Inc., USA), or MCI gel (75−150 μm, Mitsubishi Chemical Corporation, Tokyo, Japan). Column fractions were monitored by TLC visualized by spraying with 5% H2SO4 in EtOH and heating. Plant Material. The air-dried flower buds of R. rugosa were purchased from Kunming Juhuacun Chinese Herb Medicine Market in September 2011. The plants were grown in Dounan village, Chenggong County of Kunming, and harvested in June 2011. The species was identified by Prof. Y. J. Chen. A voucher specimen (YNNI 11-9-47) was deposited in the herbarium of the Yunnan University of Nationalities. Extraction and Isolation. The samples (8.0 kg) were crushed onto a 30 mesh, and the powders were extracted with 95% aqueous MeOH (4 × 10 L) at room temperature and filtered. The filtrate was evaporated under reduced pressure, and the crude extract (522 g) was applied to a silica gel (150−200 mesh) column eluted with CHCl3− MeOH gradients (20:1, 9:1, 8:2, 7:3, 6:4, 5:5) to afford six fractions E

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1450, 1168, 1072, 904, 862; 1H and 13C NMR (in CDCl3) see Table 4; ESIMS m/z 367 [M + Ma]+; HRESIMS m/z 367.1165 [M + Na]+ (calcd for C19H20NaO6, 367.1158). Bioassay. The IC50 values of compounds were measured using the MTT assay. The MTT assay is a colorimetric assay for measuring the activity of cellular enzymes that reduce the tetrazolium dye MTT (3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, a yellow tetrazole) to its insoluble formazan, giving a purple color. First, 2500 cells suspended in 100 μL of MEM medium were seeded, respectively, in a 96-well plate. After 24 h incubation, fresh medium containing various concentrations of each compound was added into the 96-well plate to replace the old medium. The concentrations ranged from 100 to 1.5625 μM, which was achieved by doing 2-fold dilutions six times. The OD595 values of the control groups at 0 and 72 h together with the compound-treated groups at 72 h from the MTT assay were measured using a plate reader. IC50 is the concentration of a compound inhibiting 50% of the cell growth.

(2) Lu, J. L. China Flowers Hortic. 2012, 11, 26−28. (3) Putian, L.; Jiang, Y., Eds. Flora of China; Chinese Science Press: Beijing, 1977. (4) Ochir, S.; Park, B.; Nishizawa, M.; Kanazawa, T.; Funaki, M.; Yamagishi, T. J. Nat. Med. 2010, 64, 383−387. (5) Hashidoko, Y.; Tahara, S.; Mizutani, J. Phytochemistry 1993, 32, 387−390. (6) Hashidoko, Y. Phytochemistry 1996, 43, 535−549. (7) An, H. J.; Kim, I. T.; Park, H. J.; Kim, H. M.; Choid, J. H.; Lee, K. T. Int. Immunopharm. 2011, 11, 504−510. (8) Xiao, Z. P.; Wu, H. K.; Wu, T.; Shi, H.; Hang, B.; Aisa, H. A. Chem. Nat. Compd. 2006, 42, 736−737. (9) Gao, X. M.; Yang, L. Y.; Shu, L. D.; Shen, Y. Q.; Zhang, Y. J.; Hu, Q. F. Heterocycles 2012, 85, 1925−1931. (10) Ma, M. H.; Cui, B.; Yu, H. F.; Yuan, C. Z. J. Shandong Inst. Light. Ind. 2008, 22, 38−42. (11) Yu, D. Q.; Yang, J. S. Handbook of Analytical Chemistry, 2nd ed. (Vol. 7); Nuclear Magnetic Resonance Spectroscopy; Chinese Chemical Industry Press: Beijing, 1999; pp 299−312. (12) Sarot, C.; Chatchanok, K.; Chanita, P.; Akkharawit, K. J. Nat. Prod. 2009, 72, 1395−1398. (13) Rossi, M. H.; Yoshida, M.; Maia, J. G. S. Phytochemistry 1997, 45, 1263−1269. (14) Tezuka, Y.; Morikawa, K.; Li, F.; Auw, L.; Awale, S.; Nobukawa, T.; Kadota, S. J. Nat. Prod. 2011, 74, 102−105. (15) Bezuidenhout, S. C.; Bezuidenhoudt, B. C. B.; Ferreira, D. Phytochemistry 1988, 27, 2329−2334. (16) Socorro, M. P.; Pinto, A. C.; Kaiser, C. R. Z. Naturforsch., B: Chem. Sci. 2003, 58b, 1206−1209. (17) Zheng, L.; Fukai, T.; Kaneki, T.; Nomura, T.; Zhang, R. Y.; Lou, Z. C. Heterocycles 1992, 34, 85−97. (18) Huang, L.; Wall, M. E.; Wani, M. C.; Navarro, H.; Santisuk, T.; Reutrakul, V.; Seo, E. K.; Farnsworth, N. R.; Kinghorn, A. D. J. Nat. Prod. 1998, 61, 446−450. (19) Zhang, Y. F.; Shao, Q.; Fan, X. H.; Cheng, Y. Y. CN Patent, 102161649, 2011. (20) Wang, Y. C.; Zheng, Z. H.; Liu, S. C.; Zhang, H.; Li, E. W.; Guo, L, D.; Che, Y. S. J. Nat. Prod. 2010, 73, 920−924. (21) Górecki, M.; Jabłońska, E.; Kruszewska, A.; Suszczyńska, A.; Urbańczyk-Lipkowska, Z.; Gerards, M.; Morzycki, J. W.; Szczepek, W. J.; Frelek, J. J. Org. Chem. 2007, 72, 2906−2916. (22) Maheswara, M.; Rao, Y. K.; Rao, V. M.; Rao, C. V. Asian J. Chem. 2006, 18, 2761−2765. (23) Schwikkard, S.; Zhou, B. N.; Glass, T. E.; Sharp, J. L.; Mattern, M. R.; Johnson, R. K.; Kingston, D. G. J. Nat. Prod. 2000, 63, 457− 460. (24) Khan, R.; Malik, A.; Adhikari, A.; Qadir, M. I.; Choudhary, M. I. Chem. Pharm. Bull. 2009, 57, 415−417. (25) Bojase, G.; Wanjala, C. C. W.; Majinda, R. R. T. Phytochemistry 2001, 56, 837−841. (26) Elisa, T.; Maurizio, L. G.; Santo, G.; Danila, D. M.; Marco, G. Food Chem. 2007, 104, 466−479. (27) Seibert, H.; Maser, E.; Schweda, K.; Seibert, S.; Gülden, M. Food Chem. Toxicol. 2011, 49, 2398−2407. (28) Mosmann, T. J. Immunol. Methods 1983, 65, 55−63.

Table 5. Cytotoxicity Data for the Compounds from R. rugosa compound

NB4

A549

SHSY5Y

PC3

MCF7

1 2 3 4 5 6 7 8 19 paclitaxel

>10 2.2 3.6 4.7 >10 >10 >10 >10 >10 0.03

7.2 6.7 1.2 >10 >10 >10 >10 >10 >10 0.02

>10 2.5 4.3 8.2 >10 >10 >10 >10 >10 0.2

6. 8 4.8 8.4 >10 >10 >10 >10 >10 >10 0.2

>10 2.3 2.8 7.3 >10 >10 >10 >10 >10 0.1

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ASSOCIATED CONTENT

S Supporting Information *

Structure of known compounds; 1H, 13C NMR, HSQC, HMBC, and HRESIMS spectra of 1 and 4; 1H and 13C NMR spectra of 2−3 and 5−8; and CD of 1; the chromatogram of compounds 1 and 2 are available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Authors

*Tel: 86-871-5913043. E-mail: [email protected] (X. M. Gao). *E-mail: [email protected] (G.-Y. Yang). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Q.F.H. and J.M.H. acknowledge the receipt of visiting scholarships to work at the University of Illinois at Chicago. This project was supported partially by the Excellent Scientific and Technological Team of Yunnan High School (2010CI08), the Yunnan University of Nationalities Green Chemistry and Functional Materials Research for Provincial Innovation Team (2011HC008), and Open Research Fund Program of the Key Laboratory of Ethnic Medicine Resource Chemistry (Yunnan University of Nationalities) (2010XY08).

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REFERENCES

(1) Gault, S. M.; Synge, P. M., Eds. The Dictionary of Roses in Colour; Ebury Press: London, 1971. F

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