Diterpenoids from the Flowers of Rhododendron molle

May 2, 2014 - Medica, Chinese Academy of Sciences and the Chinese University of Hong Kong. §. School of ... of plants belonging to the Ericaceae fami...
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Diterpenoids from the Flowers of Rhododendron molle Shuai-Zhen Zhou,†,‡ Sheng Yao,†,‡ Chunping Tang,†,‡ Changqiang Ke,†,‡ Lu Li,†,‡ Ge Lin,‡,§ and Yang Ye*,†,‡,⊥ †

State Key Laboratory of Drug Research & Natural Products Chemistry Department, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zu-Chong-Zhi Road, Zhangjiang Hi-Tech Park, Shanghai 201203, People’s Republic of China ‡ Joint Research Laboratory for Promoting Globalization of Traditional Chinese Medicines between Shanghai Institute of Materia Medica, Chinese Academy of Sciences and the Chinese University of Hong Kong § School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong SAR, People’s Republic of China ⊥ School of Life Science and Technology, ShanghaiTech University, Shanghai, People’s Republic of China S Supporting Information *

ABSTRACT: A new seco-kalmane-type diterpenoid, seco-rhodomollone (1), five new grayanane-type diterpenoids, rhodomollein XXI (2), 6-O-acetylrhodomollein XXI (3), 6,14-di-O-acetylrhodomollein XXI (4), rhodomollein XXII (5), and 2-Omethylrhodomollein XI (6), and two new kalmane-type diterpenoids, rhodomolleins XXIII (7) and XXIV (8), together with seven known compounds, were isolated from the flowers of Rhododendron molle collected in Guangxi Province, China. The absolute configurations of 1 and 3 were defined by single-crystal X-ray diffraction experiments. Compound 1 possesses an unprecedented 1,5-seco-kalmane skeleton presumably derived by cleavage of the C-1−C-5 bond of the kalmane skeleton. Compounds 2−4 represent the first examples from a natural source of grayanane-type diterpenoids with a chlorine substituent.

D

rhodomollein XXII (5), and 2-O-methylrhodomollein XI (6), and two new kalmane-type diterpenoids, named rhodomolleins XXIII (7) and XXIV (8). Seven known diterpenoids were also obtained and identified as rhodomolleins XI and XIV3 and rhodojaponins I, II,13 III,14 VI,15 and VII.16 Herein, we report the isolation and structural elucidation of the eight new compounds. The absolute configurations of compounds 1 and 3 were confirmed by single-crystal X-ray diffraction experiments. A plausible biosynthesis pathway for seco-rhodomollone (1) is also proposed.

iterpenoids are the characteristic secondary metabolites of plants belonging to the Ericaceae family. Nine types of diterpenoid skeleton have been reported from the ericaceous species: the grayananes,1 kalmanes,2 leucothanes,3 4,5-seco-entkauranes,4 1,5-seco-grayananes,5 3,4-seco-grayananes,6 9,10-secograyananes,7 1,10:2,3-disecograyananes,8 and micranthanes.9 All of these diterpenoid types are assumed to originate from a common ent-kaurane skeleton.9,10 To date, over 100 diterpenoids have been reported from the Ericaceae family, with about 40 of them from Rhododendron molle G. Don.11 R. molle is a well-known poisonous plant widely distributed in China, with flowers that possess narcotic and insecticidal activity, as noted in many traditional medicine books.12 Recently, two novel skeletons were reported for compounds from this plant,6,8 which prompted us to probe for other unique and interesting structures. With this aim of searching for diversified diterpenoid structures, we have investigated the flowers of R. molle collected in Guangxi Province, China. This resulted in the isolation of the first compound with a 1,5-seco-kalmane-type skeleton, designated as seco-rhodomollone (1), five new grayanane-type diterpenoids, named rhodomollein XXI (2), 6-O-acetylrhodomollein XXI (3), 6,14-di-O-acetylrhodomollein XXI (4), © 2014 American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION The 95% EtOH extract of the flowers of R. molle was concentrated, suspended in H2O, and partitioned with petroleum ether, CH2Cl2, EtOAc, and then n-BuOH. The CH2Cl2, EtOAc, and n-BuOH extracts were subjected to repeated column chromatography over silica gel, Sephadex LH20, and MCI gel, which afforded eight new diterpenoids (1−8) and seven known compounds. Received: January 24, 2014 Published: May 2, 2014 1185

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revealed the existence of 20 carbon resonances including four methyl, five methylene, six methine (one olefinic at δC 122.2 and two oxygenated at δC 71.7 and 80.9), and five quaternary carbons (one carbonyl at δC 218.1, one olefinic at δC 138.9, and two oxygenated at δC 84.6 and 82.0). The olefinic proton and two olefinic carbons suggested a trisubstituted double bond. Two of the five indices of hydrogen deficiency were ascribed to the carbonyl group and the double bond, with the remaining three indicating the presence of a tricyclic system. The 1H−1H COSY (Figure 1a, bold lines) and HSQC data revealed the partial structures −HC(1)−C(2)H 2−C(3)H(OH)− and −C(6)H(OH)−C(7)H 2 − and a five-membered ring, −C(9)H−C(11)H2−C(12)H2−C(13)H−C(14)H−C(9)H−. The HMBC data (arrows in Figure 1a) showed correlations from H-3 and H-7 to C-5, from H-6 and H-9 to C-8, from H-14 to C-10, and from H-9 to C-1 that established an 11-membered ring. Correlations from H-13 and H-14 to C-8 and C-15 suggested a second five-membered ring. The two fivemembered rings are fused through the C-13 and C-14 bond, which was deduced from the correlations from H-14 to C-12, C-15, and C-16 and from H-13 to C-8. The connection of the 11-membered ring to the two fused five-membered rings was established by the HMBC correlations from H-6 to C-8, from H-9 to C-1, and from H-14 to C-10. The four methyl groups were placed at C-4, C-10, and C-16, inferred from the H3-20 to C-1, H3-18(19) to C-5, and H3-17 to C-13 correlations. The hydroxy groups were located at C-3, C-6, C-8, and C-16, deduced from the low-field chemical shifts of the respective carbons. Thus, the planar structure of 1 was constructed. The relative configuration of 1 was inferred from a ROESY experiment (dashed arrows in Figure 1b). The cross-peaks of H-9/H3-17 and H-12b/H3-17 indicated that H-9, H-12b, and H3-17 were cofacial. The correlations of H-12a/H-13 and H12a/H-14 suggested that these protons were on the other face.

Compound 1 was obtained as optically active, colorless needles from MeOH. The HRESIMS of 1 showed a quasimolecular ion at m/z 351.2169 [M − H]−, which, combined with its 13C NMR spectrum, suggested a molecular formula of C20H32O5 with five indices of hydrogen deficiency. The IR spectrum indicated the presence of hydroxy (3423 cm−1) and carbonyl (1697 cm−1) groups. The 1H NMR data (Table 1) displayed four methyl singlets (δH 1.28, 1.29, 1.37, 1.69), two oxygenated methine groups (δH 3.68, 4.54), and one olefinic proton (δH 5.00). The 13C NMR and DEPT data (Table 2)

Table 1. 1H NMR Spectroscopic Data (500 MHz, Methanol-d4) for Compounds 1−5 1a position 1 2a 2b 3 6 7a 7b 9 11a 11b 12a 12b 13 14 15a 15b 17 18 19 20 6-Ac 14-Ac a

δH (J in Hz) 5.00, 2.57, 2.19, 3.68, 4.54, 2.11, 1.85, 2.01, 1.54, 1.42, 1.83, 0.96, 2.57, 2.63, 1.70, 1.70, 1.28, 1.37, 1.29, 1.69,

d (9.2) m d (14.9) br s d (8.1) d (15.6) dd (8.1, 15.6) m m m m m m m m m s s s s

2

3

δH (J in Hz)

δH (J in Hz)

4

5

δH (J in Hz)

δH (J in Hz)

2.82, d (4.9)

2.90, d (4.7)

3.10, d (4.4)

4.48, 3.70, 3.99, 2.15, 1.86, 1.79, 1.69, 1.52, 2.12, 1.54, 2.01, 4.29, 1.98, 1.82, 1.27, 1.14, 1.29, 1.36,

4.51, 3.72, 5.18, 2.15, 1.84, 1.81, 1.69, 1.51, 2.13, 1.55, 2.05, 4.31, 1.98, 1.75, 1.28, 1.17, 1.13, 1.40, 2.06,

4.51, 3.71, 4.88, 1.94, 1.85, 1.84, 1.75, 1.58, 2.24, 1.60, 2.12, 5.61, 2.05, 1.82, 1.33, 1.12, 1.11, 1.40, 2.03, 2.20,

d (4.9) s dd (4.2, 10.8) dd (4.2, 13.8) dd (10.8, 13.8) d (7.0) m m m m br s s d (15.1) d (15.1) s s s s

d (4.7) s dd (4.3, 11.0) dd (4.3, 13.6) dd (11.0, 13.6) d (6.1) m m m m br s s d (14.8) d (14.8) s s s s s

d (4.4) s dd (4.6, 11.6) dd (4.6, 13.7) m m m m m m br s s d (15.1) d (15.1) s s s s s s

2.33, 4.63, 2.74, 1.95, 2.61, 1.81, 1.47, 2.18, 1.47, 1.99, 4.33, 2.21, 1.80, 1.29, 1.24, 1.29, 1.47,

d (1.2) dd (3.3, 4.8) dd (3.3, 15.8) dd (4.8, 15.8) d (7.1) m m m m br s s d (15.0) d (15.0) s s s s

In CDCl3. 1186

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Table 2. 13C NMR Spectroscopic Data (500 MHz, Methanol-d4) for Compounds 1−5 position 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 6-Ac

1a 122.2, 32.9, 80.9, 49.2, 218.1, 71.7, 37.9, 84.6, 55.5, 138.9, 33.1, 30.3, 57.2, 55.8, 49.6, 82.0, 24.1, 21.2, 26.5, 12.0,

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

2 63.9, 66.6, 89.5, 51.6, 86.1, 73.5, 43.7, 52.5, 55.4, 78.9, 22.4, 27.0, 56.3, 80.1, 60.2, 81.4, 23.3, 24.4, 19.8, 28.1,

3

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

64.3, 66.0, 89.4, 51.5, 85.6, 77.5, 39.4, 52.4, 55.7, 78.7, 22.4, 26.9, 56.4, 79.5, 59.9, 81.2, 23.2, 24.3, 19.9, 28.2, 21.4, 171.6,

14-Ac a

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

63.8, 65.9, 89.4, 51.5, 85.5, 77.4, 38.9, 51.4, 56.8, 78.5, 22.6, 27.5, 55.6, 81.8, 60.3, 79.9, 23.7, 24.2, 20.0, 28.3, 21.3, 171.4, 21.6, 172.9,

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

143.2, 212.6, 52.2, 41.1, 181.7, 65.3, 42.2, 52.8, 51.2, 76.9, 21.1, 26.9, 56.2, 80.1, 60.8, 81.2, 23.3, 27.1, 26.5, 31.6,

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

In CDCl3.

Δ1(10)-1,5-seco-3,6,8,16-tetrahydroxykalm-5-one and named seco-rhodomollone (1).

Figure 2. Perspective ORTEP drawing of 1. Figure 1. (a) Important 1H−1H COSY () and HMBC (H → C) correlations of 1. (b) Key ROESY correlations and the possible conformation of 1 (generated by computer).

Compound 2 was obtained as an amorphous powder. The quasi-molecular ion peak at m/z 403 [M − H]− in the ESIMS and an isotopic ion at m/z 405 [M + 2 − H]− with ca. 30% intensity indicated a chloro-substituted compound. The quasimolecular ion at m/z 427.1855 [M + Na]+ in the HRESIMS, combined with the 13C NMR data, suggested a molecular formula of C20H33O6Cl, corresponding to four indices of hydrogen deficiency. The IR absorption bands at 3334 cm−1 indicated the presence of hydroxy groups. The 1H NMR data of 2 (Table 1) showed resonances for four methyl singlets (δH 1.14, 1.27, 1.29, 1.36) and four methine groups bearing an electrophilic atom (δH 3.70, 3.99, 4.29, 4.48). Twenty carbon resonances were observed in the 13 C NMR and DEPT data (Table 2) and ascribed to four methyl, four methylene, seven methine (three oxygenated at δC 73.5, 80.1, 89.5), and five quaternary carbons (three oxygenated

The cross-peaks between H3-20/H-2 revealed an E-configuration of the C-1/C-10 double bond. The relative configuration of C-3, C-6, and C-8 could not be determined by the ROESY experiment due to the flexible macrocyclic system, but was tentatively assigned as for the known kalmanol,2 considering the presumed biogenetic relationship between these two compounds. The absolute configuration of 1 was unambiguously determined by an X-ray crystallographic diffraction experiment with Cu Kα radiation on a single crystal of 1 obtained from MeOH (Figure 2). Accordingly, the structure of 1 was defined as (3S,6S,8S,9S,13R,14S,16R,E)1187

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at δC 78.9, 81.4, 86.1). The 1H−1H COSY correlations (Figure 3) revealed the presence of the partial structures −C(1)H−

Table 4. 13C NMR Spectroscopic Data (500 MHz, Methanold4) for Compounds 6−9 position 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 6-Ac

Figure 3. Important 1H−1H COSY () and HMBC (H→C) correlations of 2.

C(2)H−C(3)H(OH)−, −C(6)H(OH)−C(7)H 2 −, and −C(9)H−C(11)H2−C(12)H2−C(13)H−. The NMR data for 2, featuring a grayanane-type diterpenoid with six oxygenation sites, resembled those of rhodomollein XI (9).3 There was, however, two major difference in these two compounds; that is, there is an additional acetyl group in 9, as well as the difference for C-2, with a shift from δC 80.7 in 9 (1H and 13C NMR data in methanol-d4 are given in Tables 3 and 4 for ease of Table 3. 1H NMR Spectroscopic Data (500 MHz, Methanold4) for Compounds 6−9 position 1 2a 2b 3 6

6

7

8

9

δH (J in Hz)

δH (J in Hz)

δH (J in Hz)

δH (J in Hz)

3.14, dd (4.7, 11.8) 2.14, m 1.90, dd (4.7, 14.9) 3.64, d (5.1) 4.17, d (9.2)

3.10, dd (3.8, 11.4) 2.24, m 1.96, dd (3.8, 14.5) 3.57, d (4.3) 4.08, d (10.2)

2.44, dd (14.6, 9.2) 1.45, d (14.6)

2.35, m

2.43, d (4.3)

3.91, d (4.3)

9 11a 11b 12a 12b 13

3.59, s 5.13, dd (4.4, 11.1) 2.12, dd (4.4, 13.6) 1.83, dd (11.1, 13.6) 1.79, d (6.6) 1.70, m 1.51, m 2.10, m 1.54, m 2.04, br s

14 15a 15b 17 18 19 20

4.31, 1.96, 1.74, 1.28, 1.00, 1.11, 1.39,

6-Ac 2-OMe

2.06, s 3.39, s

7a 7b

s d (14.9) d (14.9) s s s s

2.20, m 2.10, m 1.60, m 1.56, m 2.68, dd (8.2, 14.4) 3.21, d (8.2) 1.67, d (13.8) 1.58, d (13.8) 1.23, s 1.04, s 1.22, s 1.79, s

1.66, d (15.0) 2.44, m 2.01, m 1.32, m 1.84, m 1.26, m 2.57, dd (9.2, 17.4) 2.38, d (9.2) 1.83, d (13.9) 1.62, d (13.9) 1.22, s 1.08, s 1.23, s 5.08, 5.22, each s

2-OMe

6 60.8, 91.4, 83.4, 50.8, 84.6, 77.7, 39.5, 52.5, 55.9, 78.2, 22.4, 27.0, 56.4, 79.6, 59.9, 81.2, 23.1, 23.1, 19.8, 29.0, 21.5, 171.7, 57.7,

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

48.3, 35.2, 85.0, 51.6, 89.0, 71.3, 43.0, 85.3, 141.6, 129.6, 31.7, 29.7, 58.8, 59.6, 55.3, 82.1, 23.7, 23.8, 19.7, 19.2,

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

47.2, 37.5, 84.2, 52.8, 85.5, 72.0, 42.6, 83.2, 50.3, 152.6, 36.1, 30.6, 58.8, 57.8, 53.1, 81.6, 23.3, 24.0, 20.5, 112.9,

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

60.2, 80.7, 87.0, 50.1, 84.1, 77.9, 39.8, 52.5, 55.8, 78.7, 22.2, 27.1, 56.4, 79.7, 59.9, 81.2, 23.1, 24.3, 19.8, 28.8, 21.5, 171.8,

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

2.32, d (6.0)

15 (Figure 3). In a ROESY experiment, the cross-peaks of H-1/ H-14, H-1/H3-18, H-3/H3-18, and H-1/H-6 indicated that H1, H-3, H-14, H-6, and H3-18 were cofacial. The correlations of H3-20/H-2 and H3-20/H-9 indicated that H3-20, H-9, and H-2 occupied opposite faces, which suggested that Cl-2 has an αorientation. Analysis of HSQC, HMBC, and ROESY experiments enabled full assignment of all proton and carbon atoms. Thus, the structure of compound 2 was established as 2αchloro-3β,5β,6β,10α,14β,16α-hexahydroxygrayanane and named rhodomollein XXI. Compound 3 was obtained as optically active, colorless crystals from MeOH. The quasi-molecular ion at m/z 445 [M − H]− and an isotopic ion at m/z 447 [M + 2 − H]− with ca. 30% intensity in the ESIMS again suggested a chloro substitutent in the molecule. The molecular formula was determined to be C22H35O7Cl (C2H2O mass units more than that of 2) on the basis of its HRESIMS and 13C NMR data. The IR spectrum indicated the presence of hydroxy and carbonyl groups (3415 and 1743 cm−1). The 1H and 13C NMR data for 3 (Tables 1 and 2) showed high similarity to those for compound 2, except for an additional acetyl group, suggesting that 3 was an acetylated derivative of 2. The chemical shifts of H-6 and C-6 for 3 (δH 5.18, δC 77.5), relatively low-field compared to those of 2 (δH 3.99, δC 73.5), further confirmed the deduction. The full structure including the absolute configuration (1S,2R,3R,5R,6R,8S,9R,10R,13R,14R,16R) was confirmed by X-ray diffraction analysis (Figure 4). Therefore, the structure of 3 was defined as 6β-acetoxy-2α-chloro3β,5β,10α,14β,16α-pentahydroxygrayanane and named 6-Oacetylrhodomollein XXI. Compound 4 was also characterized as a chloro-substituted compound by the quasi-molecular ion and its isotopic ion with a ca. 3:1 intensity ratio in the ESIMS. The HRESIMS and 13C NMR data indicated a molecular formula of C24H37O8Cl, implicating six indices of hydrogen deficiency. The 1H and 13C

4.36, dd (2.8, 6.0) 3.42, d (2.8) 5.15, dd (4.3, 10.9) 2.15, dd (4.3, 13.7) 1.84, dd (10.9, 13.7) 1.78, d (7.1) 1.70, m 1.50, m 2.10, m 1.53, m 2.04, br s 4.34 s 1.96, d (15.0) 1.75, d (15.0) 1.28, s 1.05, s 1.06, s 1.40, s 2.05, s

comparison) to δC 66.6 in 2. This suggested that the chlorine atom might be located at C-2. Thus, 2 was deduced as being the C-2 chloro-substituted and 6-deacetylated derivative of 9. The proposed structure was further confirmed by HMBC correlations from H-2 to C-4 and C-10, from H-3 to C-5, from H-1 to C-6, from H-6 to C-8, from H-7 to C-9 and C-15, from H2-11 to C-10, from H-14 to C-12, C-15, and C-16, from H3-20 to C-9, from H3-18(19) to C-5, and from H3-17 to C-13 and C1188

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3.59, 3.91, 4.31, 5.13), along with a methoxy group (δH 3.39). Apart from the carbons assigned to the acetyl (δC 21.5, 171.7) and the methoxy group (δC 57.7), the remaining 20 carbon resonances (Table 4) comprised four methyl, four methylene, seven methine (four oxygenated at δC 77.7, 79.6, 83.4, 91.4), and five quaternary carbons (three oxygenated at δC 78.2, 81.2, 84.6). These data suggested that 6 was a grayanane-type diterpenoid with seven sites of oxygenation. Further analysis of the NMR data (Tables 3 and 4) revealed a close resemblance to the data for 9, except that an additional O-methyl resonance at δH 3.39 was observed along with chemical shift changes at C-2 and C-3. Thus, the C-2 resonance of 6 was deshielded to δC 91.4 (10.7 ppm relative to that of 9), suggesting that the methoxy group was located at C-2. The proposed structure was confirmed by HMBC correlations from the methoxy protons to C-2. The relative configuration of 6 was proved the same as that of 9 by the ROESY experiment. The cross-peaks of H-1/ H-14, H-1/H3-18, H-3/H3-18, and H-1/H-6 indicated that H1, H-3, H-14, H-6, and H3-18 were cofacial. The correlations of H3-20/H-2 and H3-20/H-9 indicated that H3-20, H-9, and H-2 occupied opposite sides. Therefore, the structure of 6 was deduced as being 6β-acetoxy-3β,5β,10α,14β,16α-pentahydroxy2α-methoxygrayanane and named 6-O-methylrhodomollein XI. Compound 7 was obtained as an amorphous powder. The quasi-molecular ion at m/z 375.2149 [M + Na]+ in the HRESIMS, together with the 13C NMR data, suggested a molecular formula of C20H32O5, corresponding to five indices of hydrogen deficiency. The IR absorption band at 3426 cm−1 indicated the presence of hydroxy groups. The 1H NMR data (Table 3) showed resonances of four methyl singlets (δH 1.04, 1.22, 1.23, 1.79) and two oxygenated methine groups (δH 3.64, 4.17). A total of 20 carbon resonances were observed in the 13C NMR and DEPT data (Table 4), including four methyl, five methylene, five methine (two oxygenated at δC 71.3, 85.0), and six quaternary carbons (two olefinic at δC 129.6, 141.6 and three oxygenated at δC 82.1, 85.3, 89.0). The 1H−1H COSY data (Figure 5) revealed the presence of three spin systems,

Figure 4. Perspective ORTEP drawing of 3.

NMR data for 4 (Tables 1 and 2) resembled those for 3, except for the additional resonances of an O-acetyl group, suggesting that 4 was an acetyl derivative of 3. The downfield shift of H-14 (δH 5.61 in 4 vs δH 4.31 in 3) and C-14 (δC 81.8 in 4 vs δC 79.5 in 3) indicated that the additional O-acetylation occurred at C14. The proposed structure was confirmed by an HMBC correlation between H-14 and the carbonyl carbon at δC 172.9. Thus, 4 was established as 6β,14β-diacetoxy-2α-chloro3β,5β,10α,16α-tetrahydroxygrayanane and named 6,14-di-Oacetylrhodomollein XXI. The molecular formula of compound 5, an amorphous powder, was established as C20H30O5 according to the 13C NMR data and the quasi-molecular ion at m/z 373.1969 [M + Na]+ in the HRESIMS. The IR spectrum indicated the presence of hydroxy (3436 cm−1) and α,β-unsaturated carbonyl groups (1664, 1645 cm−1). The 1H NMR data (Table 1) displayed four methyl singlets (δH 1.24, 1.29, 1.29, 1.47) and two oxygenated methine groups (δH 4.33, 4.63). The 13C NMR and DEPT data (Table 2) indicated 20 carbon resonances ascribed to four methyl, five methylene, four methine (two oxygenated at δC 65.3, 80.1), and seven quaternary carbons (one carbonyl at δC 212.6, two olefinic at δC 143.2, 181.7, and two oxygenated at δC 76.9, 81.2). The 1H−1H COSY data revealed two spin systems, namely, −C(6)H(OH)−C(7)H2− and −C(9)H− C(11)H2−C(12)H2−C(13)H−. The 13C NMR data of 5 closely parallel those of the known compound rhodomollein XX,17 except that a methylene carbon (δC 52.2) rather than an oxygenated methine carbon (C-3 of rhodomollein XX) was observed in 5. Thus, 5 was assumed to be the 3-deoxygenated derivative of rhodomollein XX, which was verified by HMBC correlations from H2-3 to C-1, C-2, and C-5. The relative configuration of 5 was elucidated by the ROESY experiment (Supporting Information). Consequently, the structure of compound 5 was defined as Δ1(5)-6β,10α,14β,16α-tetrahydroxygrayan-2-one and named rhodomollein XXII. Compound 6 was obtained as an amorphous powder. The HRESIMS, combined with the 13C NMR data, suggested a molecular formula of C23H38O8 on the basis of the quasimolecular ion at m/z 441.2470 [M − H]−. The IR spectrum indicated the presence of hydroxy (3394 cm−1) and carbonyl groups (1716 cm−1). The 1H NMR data (Table 3) displayed four methyl singlets (δH 1.00, 1.11, 1.28, 1.39), one acetyl methyl (δH 2.06), and four oxygenated methine groups (δH

Figure 5. Important 1H−1H COSY () and HMBC (H→C) correlations of 7.

namely, −C(1)H−C(2)H2−C(3)H(OH)−, −C(6)H(OH)− C(7)H2−, and −C(11)H2−C(12)H2−C(13)H−C(14)H−. Analysis of HSQC, HMBC, and ROESY data enabled assignment of all proton and carbon atoms. The 13C NMR data for 7 resembled those of kalmanol (see Scheme 1), a known compound reported from the same plant,18 suggesting a kalmane-type diterpenoid. However, the resonances for C-9 and C-10 in kalmanol (δC 52.8, 75.7) were shifted to δC 141.6 and 129.6, respectively, for compound 7. This suggested a C-9/ C-10 intra-annular double bond, which was confirmed by HMBC correlations from H2-2 to C-4 and C-10, from H-1 to C-6 and C-9, from H2-12 to C-9, C-14, and C-16, and from H320 to C-9 (Figure 5). The ROESY correlations of H-1/H-6, H1189

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were recorded with a Nicolet Magna FTIR-750 spectrometer. Analytical HPLC and ESIMS spectra were performed on a Waters 2695 instrument with a 2998 PDA coupled with a Waters Acquity ELSD and a Waters 3100 SQDMS detector. All HPLC analyses were carried out on a Waters Sunfire RP C18, 3.5 μm, 4.6 mm × 100 mm column, eluted with a gradient of CH3CN−H2O (5 to 95%) with 0.1% HOAc. HRESIMS were recorded on a Waters Xevo QTof mass detector. 1H, 13C, and 2D NMR spectra were recorded using a Bruker Avance III 500 instrument with solvent resonances [methanol-d4 (δH 3.31; δC 49.00) and CDCl3 (δH 7.26; δC 77.16)] as internal standards. Chemical shifts are reported in ppm (δ), and coupling constants (J) in hertz. Preparative HPLC was performed on a Varian PrepStar system with an Alltech 3300 ELSD. Chromatographic separations were carried out on a Waters Sunfire RP C18, 5 μm, 30 mm × 150 mm column and a Waters Sunfire RP C18, 5 μm, 19 mm × 150 mm column, using a gradient solvent system composed of H2O and CH3CN, with a flow rate of 25.0 and 10.0 mL/min, respectively. Silica gel for flash chromatography was produced by Qingdao Marine Chemical Industrials. MCI gel CHP20P (75−150 μm, Mitsubishi Chemical Industries, Japan) and Sephadex LH-20 (Pharmacia Biotech AB, Uppsala, Sweden) were used for column chromatography. TLC was carried out on precoated silica gel 60 F254 aluminum sheets (Merck, Germany), and the TLC spots were viewed at 254 nm and visualized with 5% sulfuric acid in alcohol containing 10 mg/mL vanillin. X-ray crystallographic analysis was carried out on a Bruker Smart Apex CCD diffractometer with graphite-monochromated Mo Kα radiation (λ = 0.71073 Å) or Cu Kα radiation (λ = 1.54178 Å). Plant Material. The flowers of R. molle were collected in Jinxiu County, Guangxi Province, China, in 2010 and identified by Prof. JinGui Shen from the Shanghai Institute of Materia Medica. A voucher specimen (No. 20100811) has been deposited at the Herbarium of the Shanghai Institute of Materia Medica, Chinese Academy of Sciences. Extraction and Isolation. The air-dried flowers of R. molle (5 kg) were ground into a powder and extracted with 95% EtOH (3 × 25 L) at room temperature (72 h each). Concentration of the combined percolates under reduced pressure yielded a dark brown, crude extract (400 g). The extract was suspended in H2O and partitioned with petroleum ether (PE), CH2Cl2, EtOAc, and n-BuOH, successively, yielding fractions PE (90 g), CH2Cl2 (90 g), EtOAc (30 g), and nBuOH (45 g). The CH2Cl2 extract was applied to an MCI gel column (eluted with MeOH in H2O, 50, 60, 70, 80, and 95%, successively) to afford nine fractions (CA−CI) on the basis of TLC analysis. Fraction CF was subjected to chromatography on a Sephadex LH-20 column (eluted with MeOH) to give subfractions CF1−CF4. CF2 was chromatographed on a silica gel column (200−300 mesh) eluted with CHCl3− MeOH (120:1) to give subfractions CF2a−CF2e. Subfraction CF 2d was separated on a silica gel column (eluted with PE−acetone, 2:1) to afford 1 (20 mg). Fraction CE was separated on a Sephadex LH-20 column (eluted with MeOH) to give subfractions CE1−CE4. Subfraction CE1 was subjected to chromatography on a silica gel column (300−400 mesh, eluted with CHCl3−MeOH from 150:1 to 100:1), giving 4 (105 mg) and rhodojaponin I (47 mg). Fraction CD was applied to a Sephadex LH-20 column (eluted with MeOH) to yield five subfractions (CD1−CD5). Subfraction CD3 was separated on a silica gel column (200−300 mesh, eluted with CHCl3−MeOH from 100:1 to 20:1) to yield rhodojaponin VII (102 mg) and subfractions CD3a to CD3d. Subfraction CD3a was chromatographed on a silica gel column (200−300 mesh, eluted with PE−acetone, 1:1) to afford 3 (526 mg) and rhodojaponin II (156 mg). Subfraction CD3b was separated on a silica gel column (300−400 mesh, eluted with PE−acetone, 1:1) to yield 5 (13 mg), rhodojaponin III (3 mg), and subfractions CD3b1−CD3b3. Subfraction CD3b3 was further purified by chromatography on a Sephadex LH-20 column (eluted with MeOH) to afford 6 (44 mg). The EtOAc extract was subjected to chromatography on an MCI gel column (eluted with MeOH in H2O, 50, 60, 70, 80, and 95%, successively) to afford eight fractions (EA−EH) on the basis of TLC analysis. Fraction EB was applied to a Sephadex LH-20 column (eluted with MeOH) to give subfractions EB1−EB3. Subfraction EB1 was

Scheme 1. Proposed Biosynthesis Pathway for secoRhodomollone (1)

1/H3-18, H-3/H3-18, and H-13/H-14 also indicated that 7 has the same relative configuration as kalmanol. Therefore, the structure of 7 was defined as Δ9(10)-3β,5β,6β,8α,16α-pentahydroxykalmene and named rhodomollein XXIII. Compound 8 was obtained as an amorphous powder. The quasi-molecular ion at m/z 375.2144 [M + Na]+ in the HRESIMS and its 13C NMR data suggested a molecular formula of C20H32O5, the same as that of 7. The IR spectrum indicated the presence of hydroxy groups (3418 cm−1). The 13 C NMR data for 8 (Table 4) showed similarities to those of 7, except that resonances of an exocyclic double bond (δH 5.08, 5.22; δC 112.9, 152.6) rather than an intra-annular double bond were observed for 8. The position was confirmed at C-10/C-20 by HMBC correlations from H-14 to C-10 and from H-20 to C-1 and C-9. In the ROESY experiment, cross-peaks of H-1/H6, H-1/H3-18, H-3/H3-18, and H-1/H-14 confirmed the relative configuration to be the same as 7. Accordingly, the structure of 8 was established as Δ10(20)-3β,5β,6β,8α,16αpentahydroxykalmene and named rhodomollein XXIV. seco-Rhodomollone (1) contains an unprecedented 1,5-secokalmane skeleton, which likely originates biosynthetically from the kalmane skeleton. A plausible biosynthetic pathway for compound 1 is shown in Scheme 1. The 9,10 double bond of rhodomollein XXVI (7) is presumably hydrated to produce kalmanol. Deprotonation of the 5-OH group of kalmanol, loss of the 10-OH group, along with concomitant cleavage of the 1,5-carbon bond, and formation of the 1,10-double bond would afford seco-rhodomollone (1). In summary, a total of 15 diterpenoids, including eight new compounds, were isolated from the flowers of R. molle. These compounds possess two types of skeletons, namely, the grayanane- and the kalmane-type skeletons. Rhodomollein XXI (2), 6-O-acetylrhodomollein XXI (3), and 6,14-di-Oacetylrhodomollein XXI (4) are the first examples of chlorosubstituted grayanane-type diterpenoids of natural origin. secoRhodomollone (1) was identified as a new seco-kalmane-type diterpenoid, while rhodomolleins XXIII (7) and XXIV (8) are two new examples of the rare kalmane-type skeleton, of which only three other examples have been reported.2,3,18

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were obtained on a Rudulph Autopol VI Automatic polarimeter. IR spectra 1190

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separated on a silica gel column (200−300 mesh, eluted with CHCl3− MeOH, 20:1) to yield rhodomollein XI (9, 865 mg) and rhodojaponin VI (372 mg) and subfractions EB1a−EB1c. Subfraction EB1c was separated on a Sephadex LH-20 column (eluted with MeOH) and then purified by preparative HPLC (eluted with a gradient of CH3CN in H2O from 5% to 45%, Waters Sunfire RP C18, 5 μm, 30 mm × 150 mm column, flow rate 25.0 mL/min, 100 min) to yield 2 (44 mg). The n-BuOH extract was applied to an MCI gel column (eluted with EtOH in H2O, 30, 40, 50, and 95%, successively) to afford four fractions (BA−BD) on the basis of TLC analysis. Fraction BB was subjected to chromatography on a silica gel column (300−400 mesh, eluted with CH2Cl2−MeOH, 20:1, 15:1, and 10:1) to afford five subfractions (BB1−BB5) and rhodomollein XIV (17 mg). Subfraction BB1 was further purified by preparative HPLC (eluted with a gradient of CH3CN in H2O from 5% to 15%, Waters Sunfire RP C18, 5 μm, 19 mm × 150 mm column, flow rate 10.0 mL/min, 90 min) to yield 7 (16 mg) and 8 (3 mg). seco-Rhodomollone (1): colorless needles from MeOH; mp 152− 155 °C; [α]25D +108 (c 0.1, CH2Cl2); IR (KBr) νmax 3423, 2949, 1697, 1683, 1446, 1057 cm−1; 1H and 13C NMR data, see Tables 1 and 2; ESIMS m/z 351 [M − H]−; HRESIMS m/z 351.2169 [M − H]− (calcd for C20H31O5, 351.2171). Rhodomollein XXI (2): amorphous powder; [α]20D −28 (c 0.1, MeOH); IR (KBr) νmax 3334, 2946, 1041 cm−1; 1H and 13C NMR data, see Tables 1 and 2; ESIMS m/z 405 [M + 2 − H]− (33), m/z 403 [M − H]− (100); HRESIMS m/z 427.1855 [M + Na]+ (calcd for C20H33O6ClNa, 427.1863). 6-O-Acetylrhodomollein XXI (3): colorless crystals from MeOH; mp 129−131 °C; [α]20D −21 (c 0.1, MeOH); IR (KBr) νmax 3415, 2960, 1743, 1238, 1024 cm−1; 1H and 13C NMR data, see Tables 1 and 2; ESIMS m/z 447 [M + 2 − H]− (39), m/z 445 [M − H]− (100); HRESIMS m/z 469.1958 [M + Na]+ (calcd for C22H35O7ClNa, 469.1969). 6,14-Di-O-acetylrhodomollein XXI (4): amorphous powder; [α]20D −22 (c 0.1, acetone); IR (KBr) νmax 3450, 2958, 1728, 1699, 1245, 1047 cm−1; 1H and 13C NMR data, see Tables 1 and 2; ESIMS m/z 489 [M + 2 − H]− (36), m/z 487 [M − H]− (100); HRESIMS m/z 487.2112 [M − H]− (calcd for C24H36O8Cl, 487.2099). Rhodomollein XXII (5): amorphous powder; [α]20D −22 (c 0.1, MeOH); IR (KBr) νmax 3436, 2927, 1664, 1645, 1106, 1039 cm−1; 1H and 13C NMR data, see Tables 1 and 2; ESIMS m/z 349 [M − H]−; HRESIMS m/z 373.1969 [M + Na]+ (calcd for C20H30O5Na, 373.1991). 2-O-Methylrhodomollein XI (6): amorphous powder; [α]20D −12 (c 0.1, MeOH); IR (KBr) νmax 3394, 2935, 1716, 1372, 1257, 1099, 1049 cm−1; 1H and 13C NMR data, see Tables 3 and 4; ESIMS m/z 441 [M − H]−; HRESIMS m/z 441.2470 [M − H]− (calcd for C23H37O8, 441.2488). Rhodomollein XXIII (7): amorphous powder; [α]20D +92 (c 0.1, MeOH); IR (KBr) νmax 3426, 2923, 1637, 1444, 1420, 1016, 922, 856 cm−1; 1H and 13C NMR data, see Tables 3 and 4; ESIMS m/z 351 [M − H]−; HRESIMS m/z 375.2149 [M + Na]+ (calcd for C20H32O5Na, 375.2147). Rhodomollein XXIV (8): amorphous powder; [α]20D +56 (c 0.1, MeOH); IR (KBr) νmax 3418, 2928, 1630, 1449, 1411, 1378, 1056, 891, 850 cm−1; 1H and 13C NMR data, see Tables 3 and 4; ESIMS m/ z 351 [M − H]−; HRESIMS m/z 375.2144 [M + Na]+ (calcd for C20H32O5Na, 375.2147). Crystallographic Data for Compound 1. Compound 1 was obtained by recrystallization from MeOH: colorless, formula (C20H32O5)2, 7(H2O), fw 415.51, monoclinic, crystal size 0.30 × 0.03 × 0.02 mm, space group C2, a = 30.6177(19) Å, b = 6.6765(4) Å, c = 10.7301(7) Å, V = 2178.7(2) Å3, Z = 4, Dcalcd = 1.267 mg/m3, F(000) = 908, reflections collected 9496, reflections unique 3333 (Rint = 0.0685), final R indices for I > 2σ(I), R1 = 0.0521, wR2 = 0.1255, R indices for all data R1 = 0.0574, wR2 = 0.1302, completeness to θ = 69.24, maximum transmission 0.9840, minimum transmission 0.7933, absolute structure parameter −0.1(2). Unit cell dimensions were measured at 140(2) K using Cu Kα radiation (λ = 1.54178 Å). The structure was solved by direct methods using the program SHELXS-

97. The refinement method was full-matrix least-squares on F2, with goodness-of-fit on F2 = 1.086. The X-ray diffraction material has been deposited at the Cambridge Crystallographic Data Centre (CCDC 940591).19 Crystallographic Data for Compound 3. Compound 3 was obtained by recrystallization from MeOH: colorless, formula C22H35O7Cl, fw 446.95, monoclinic, crystal size 0.25 × 0.22 × 0.17 mm, space group P2(1), a = 10.8838(2) Å, b = 56.5355(10) Å, c = 11.1692(2) Å, V = 6105.48(19) Å3, Z = 10, Dcalcd = 1.234 mg/m3, F(000) = 2400, reflections collected 34 956, reflections unique 20 391 (Rint = 0.0344), final R indices for I > 2σ(I), R1 = 0.0615, wR2 = 0.1584, R indices for all data R1 = 0.0650, wR2 = 0.1609, completeness to θ = 26.00, maximum transmission 0.9674, minimum transmission 0.9526, absolute structure parameter 0.03(5). The structure was solved by direct methods using the program SHELXS-97. The refinement method was full-matrix least-squares on F2, with goodness-of-fit on F2 = 1.041. The X-ray diffraction material has been deposited at the Cambridge Crystallographic Data Centre (CCDC 940592).19

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ASSOCIATED CONTENT

S Supporting Information *

The HMBC correlations of compounds 1−8, flowcharts of extraction and isolation, 1H and 13C NMR, 1H−1H COSY, HSQC, HMBC, and ROESY spectra of compounds 1, 2, 5, and 7, 1H and 13C NMR, HMBC, and ROESY spectra of compounds 6 and 8, 1H and 13C NMR and HMBC spectra of compounds 3 and 4, and CIF files for compounds 1 and 3. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel: 86-21-50806726. Fax: 86-50806726. E-mail: yye@mail. shcnc.ac.cn. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We are thankful for the financial support of the National Science & Technology Major Project “Key New Drug Creation and Manufacturing Program” (No. 2012ZX09301001-001). We thank the National Natural Science Funds (No. 81302657), the Ministry of Science and Technology (2010DFA30980), Chinese Academy of Sciences (KSZD-EW-Z-004-01), and the Shanghai Commission of Science and Technology (11DZ1970700, 12JC1410300).

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REFERENCES

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