Anti-inflammatory Grayanane Diterpenoids from the Leaves of

Article Cite This: J. Nat. Prod. XXXX, XXX, XXX−XXX

pubs.acs.org/jnp

Anti-inflammatory Grayanane Diterpenoids from the Leaves of Rhododendron molle Junfei Zhou,† Tingting Liu,† Hanqi Zhang,† Guijuan Zheng,† Yue Qiu,‡ Mengyi Deng,‡ Chun Zhang,‡ and Guangmin Yao*,† †

Hubei Key Laboratory of Natural Medicinal Chemistry and Resource Evaluation, School of Pharmacy, and ‡Department of Nephrology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, People’s Republic of China S Supporting Information *

ABSTRACT: Thirteen new grayanane diterpenoids (1−13), a new dimeric grayanane diterpenoid, bimollfoliagein A (14), and 15 known analogues (15− 29) were isolated from the leaves of Rhododendron molle. The structures of the new compounds (1−14) were determined by extensive spectroscopic data interpretation. The absolute configurations of 1−3, 7, 8, 16, 18, and 24 were defined by single-crystal X-ray diffraction analysis. Mollfoliagein A (1) represents the first example of a 2,3:11,16-diepoxy grayanane diterpenoid, featuring a cis/trans/cis/cis/trans-fused 3/5/7/6/5/5 hexacyclic ring system with a 7,13-dioxahexacyclo[10.3.3.01,11.04,9.06,8.014,17]octadecane scaffold. Diterpenoids 1−29 were evaluated for their anti-inflammatory activities in vitro, and 15, 16, 18, 19, 23−26, 28, and 29 exhibited significant inhibitory activities against nitric oxide production in lipopolysaccharide-induced RAW264.7 mouse macrophages with IC50 values ranging from 2.8 to 35.4 μM. A preliminary structure−activity relationship for the anti-inflammatory activity of diterpenoids 1−29 is discussed.

I

harvested for a long time. In contrast, the leaves of R. molle are renewable and may be collected regularly.9 Therefore, the leaves of R. molle have a definite advantage over the roots as a source. Rhododendron molle, a deciduous shrub distributed mainly in most areas of mainland China, has been used as a traditional Chinese medicine as an anesthetic and anodyne.10 In previous studies, a large number of diterpenoids have been isolated from the flowers,11−18 roots,19−23 and fruits24−27 of R. molle, and some of them exhibited significant anticancer,11 antiviral,19 antinociceptive,20 immunomodulatory,21 and sodium channel antagonistic activities.24 However, phytochemical investigations of the leaves of R. molle are sparse, and only a novel rhodomollane diterpenoid, rhodomollanol A,28a three 2,3:5,6di-seco-grayanane diterpenoids,28b and three grayanane diterpenoids were recently reported by our group.28 In our continuing search for novel anti-inflammatory agents, the leaves of R. molle collected at Qichun, Hubei Province, China, were studied comprehensively, leading to the isolation of 29 diterpenoids, including 13 new grayanane diterpenoids (1−13) and a new dimeric grayanane diterpenoid, bimollfoliagein A (14), together with 15 known grayanane diterpenoids (15−29). Mollfoliagein A (1) represents the first example of a 2,3:11,16-diepoxygrayanane diterpenoid, featuring a cis/trans/cis/cis/transfused 3/5/7/6/5/5 hexacyclic ring system with a 7,13-

nflammation is an emergency reaction to prevent infection, promote tissue repair, and eliminate necrotic cells.1 However, this response must be prompt, steerable, and specific in order to avoid overactivation to inflammatory disorders, which may seriously damage tissues.2 Data have shown that the development of various diseases including obesity, diabetes, thrombosis, and cancer have a close relationship with inflammation.3,4 Although corticosteroids and nonsteroidal drugs possess the most effective therapeutics for a broad spectrum of inflammatory symptoms, these drugs could cause various malignant side effects including a steroid-resistant reaction.5,6 Thus, it is necessary to search for safer and more effective antiinflammatory drugs to improve current therapies. Natural products provide a unique source of inspiration for anti-inflammatory drug exploitation. Compounds originated from natural products or related derivatives occupy 62% of all molecules approved by the FDA from 1981 to 2014, and about 2% of these natural compounds are developed into antiinflammatory drugs.7 Traditional Chinese medicines have a long history of being used as crude plant extracts to treat diseases or disorders. The root extract of Rhododendron molle G. Don (Ericaceae) has been used to treat chronic glomerulonephritis in the clinic,8a and the mechanism of action was reported to downregulate the activation of NF-κB, which plays a pivotal role in the pathogenesis of chronic glomerulonephritis.8b,c However, the antiglomerulonephritis-active components are still unknown. In addition, the roots of R. molle are nonrenewable and cannot be © XXXX American Chemical Society and American Society of Pharmacognosy

Received: September 19, 2017

A

DOI: 10.1021/acs.jnatprod.7b00799 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

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Chart 1

Table 1. 1H (400 MHz) NMR [δ, mult (J in Hz)] Spectroscopic Data for Compounds 1−7 1a

no.

2b

3b

4b

1α 2α 2β 3α 6α 7α

2.81, br s 4.24, d (3.0)

2.33, d (0.6) 3.79, dd (3.0, 0.6)

3.22, d (0.7) 3.65, dd, (2.9, 0.7)

2.41, d (0.6) 3.81, dd (3.0, 0.6)

3.26, d (3.0) 5.31, dd (11.2, 2.2) 2.02, dd (13.6, 2.2)

3.19, d (3.0) 3.87, dd (11.1, 4.5) 1.94, dd (13.6, 4.5)

3.21, d (2.9) 4.06, dd (10.6, 4.3) 1.94, dd (13.0, 4.3)

7β 9β 10α 11α 11β 12α 12β 13α 14α 15α 15β 17 18 19 20 2-OCH3 6-OAc

5b

6b

7b

2.35, d (6.0)

2.63, d (9.0)

2.46, dd (9.8, 4.0)

3.23, d (3.0) 5.01, dd (10.8, 4.9) 1.89, dd (13.4, 4.9)

4.37, dd (6.0, 2.4) 3.43, d (2.4) 5.12, dd (10.6, 4.1) 2.00, dd (13.9, 4.1)

4.15, dd (9.0, 4.0) 3.47, d (4.0) 5.04, dd (11.4, 1.7) 1.38, d (14.2, 1.7)

2.59, dd (13.6, 11.2) 1.87, dd (13.6, 11.1) 1.94, dd (13.0, 10.6) 2.47, m 1.74, d (6.3) 2.52, d (6.6)

1.83, dd (13.4, 10.8) 1.67, d (6.7)

1.91, dd (13.9, 10.6) 1.85, d (6.2)

2.12, dd (14.2, 11.4) 2.66, dd (6.6, 11.9)

3.62, dd (4.0, 3.1) 3.51, d (3.1) 4.93 dd (4.2, 1.8) 2.00, dd (15.7, 4.2) 1.75, dd (15.7, 1.8)

4.91, t (3.3)

1.68, m 1.55, m 2.00, m 1.39, m 2.45, m 4.21, s 4.85, q (1.4)

1.98, m 1.51, m 1.87, m 1.53, m 2.24, d (7.7) 4.55, s 5.10, q (1.2)

1.70, d (1.4) 1.23, s 0.93, s 1.45, s

1.69, m 1.61, m 2.24, m 1.41, m 2.61, m 4.22, s 2.28, overlap 2.28, overlap 4.83, 4.77, s 1.07, s 1.06, s 1.43, s

2.02, s

2.06, s

2.07, s

2.95, d (10.9) 2.27, m 2.50, m 4.60, s 1.78, d (11.0) 2.48, d (11.0) 1.56, s 1.44, s 0.95, s 1.97, s

1.72, m 1.61, m 2.26, m 1.41, m 2.61, m 4.04, s 2.29, overlap 2.29, overlap 4.83, 4.77, s 1.25, s 1.15, s 1.44, s

1.60, overlap 1.60, overlap 1.94, m 1.71, m 2.00, m 4.04, s 1.92, overlap 1.92, overlap 1.35, s 1.27, s 1.18, s 5.37, 5.14, s

2.06, s

1.73, d (1.2) 1.07, s 1.10, s 4.96, 5.06, s

2.73, dq (9.8, 7.5) 1.60, overlap 1.60, overlap 1.52, m 1.32, m 2.26, m 3.46, s 5.50, q (1.5) 1.77, d (1.5) 1.05, s 0.95, s 1.12, d (7.5) 3.37, s 2.10, s

a

Recorded in pyridine-d5. bRecorded in methanol-d4.

dioxahexacyclo[10.3.3.01,11.04,9.06,8.014,17]octadecane scaffold. In this paper, the isolation, structure elucidation, antiinflammatory activities, and structure−activity relationship of 29 diterpenoids (1−29) are reported.

for C22H32O7Na, 431.2046) in the HRESIMS, corresponding to seven indices of hydrogen deficiency. The 1H NMR data (Table 1) of 1 showed resonances attributed to five oxygenated methines (δH 5.31 dd, J = 11.2, 2.2 Hz, H-6α; 4.91, t, J = 3.3 Hz, H-11α; 4.60, s, H-14α; 4.24, d, J = 3.0 Hz, H-2α; 3.26 d, J = 3.0 Hz, H-3α), an acetoxy group (δH 2.06, s, 6-OAc), and four methyls (δH 1.97, s, CH3-20; 1.56, s, CH3-17; 1.44, s, CH3-18; 0.95, s, CH3-19). The 13C NMR and DEPT data (Table 2) indicated the presence of five methyls (δC 33.1, C-20; 24.3, C-

■

RESULTS AND DISCUSSION Mollfoliagein A (1) was obtained as colorless prisms, mp 174− 175 °C. Its molecular formula was determined to be C22H32O7 by the 13C NMR data and [M + Na]+ ion at m/z 431.2025 (calcd B



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Table 2. 13C (100 MHz) NMR Spectroscopic Data for Compounds 1−13 no. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 6-OAc

1a

2b

3b

4b

5b

6b

7b

8b

9b

10b

11b

12b

13b

54.7 59.7 63.6 47.8 79.3 76.3 36.0 51.5 59.9 75.1 78.8 42.0 55.1 80.9 53.2 86.4 24.3 20.4 21.0 33.1 171.5 21.8

54.8 60.7 65.2 48.6 80.5 73.8 42.9 51.0 56.0 78.6 22.6 33.9 56.0 77.0 49.5 155.3 104.6 20.3 21.4 30.5

50.7 60.6 66.2 48.4 80.7 73.3 43.3 51.3 52.4 150.7 25.7 30.1 55.1 82.1 59.8 81.7 23.7 20.6 21.2 116.4

55.0 60.5 65.2 48.5 80.6 77.4 36.2 55.5 50.0 79.2 23.1 24.8 56.8 79.8 131.4 139.2 15.4 20.0 21.3 30.6 171.6 21.5

60.2 81.3 87.3 50.3 84.3 77.6 38.6 51.1 55.9 78.9 22.3 33.8 55.0 77.4 49.6 155.5 104.9 19.8 24.5 29.0 172.2 21.6

51.6 82.0 88.4 48.7 82.1 75.9 33.2 53.0 51.8 148.2 27.1 23.3 51.0 78.5 134.4 144.3 15.3 19.8 26.7 115.6 173.1 21.4

57.0 87.5 86.9 47.0 93.3 72.2 29.3 56.6 88.0 39.1 26.0 21.1 51.9 85.9 128.4 141.1 16.0 19.7 23.8 16.2 172.1 21.6

51.8 92.6 84.7 48.7 85.6 70.9 39.3 57.7 124.0 139.2 27.0 26.7 54.1 89.0 59.3 83.7 23.4 17.2 25.3 19.0 172.4 21.5

43.8 39.1 81.9 55.4 84.1 71.9 44.4 44.9 57.7 151.8 24.1 27.1 47.9 35.1 64.1 81.3 25.8 64.4 20.2 114.4

53.0 94.9 85.4 49.6 84.2 70.0 41.6 50.6 53.0 151.0 25.0 24.6 54.2 81.5 61.2 82.9 24.6 18.7 24.7 113.3

52.4 93.7 85.5 49.4 83.1 75.4 37.0 50.6 56.1 148.9 24.7 24.4 53.6 80.1 61.6 82.6 25.1 19.3 25.0 114.8 173.2 21.4

60.4 92.1 83.7 51.0 85.3 73.7 44.0 52.7 56.5 78.6 22.5 27.2 55.8 80.4 60.4 81.6 23.4 19.9 23.6 29.2

57.6

57.7

58.3

58.6

57.8

60.5 91.4 83.8 50.8 84.7 77.6 39.2 51.5 57.0 78.1 22.6 27.6 55.6 82.1 60.5 80.0 23.9 20.1 23.2 29.4 171.6 21.5 172.9 21.7 57.8

14-OAc 2-OCH3 a

Recorded in pyridine-d5. bRecorded in methanol-d4.

mollfoliagein A (1) was unambiguously determined by singlecrystal X-ray diffraction with Cu Kα radiation (Figure 2). The resulting Flack parameter 0.09(6)30 assigned the absolute configuration of 1 as (1S,2S,3R,5R,6R,8S,9S,10S,11S,13R,14R,16S). Mollfoliagein A (1) represents the first example of a 2,3:11,16-diepoxy grayanane diterpenoid, featuring a cis/trans/cis/cis/trans-fused 3/5/7/6/5/5 hexacyclic ring system with a 7,13-dioxahexacyclo[10.3.3.01,11.04,9.06,8.014,17]octadecane scaffold. Mollfoliagein B (2) was isolated as colorless prisms, mp 209− 211 °C. The 13C NMR data and the sodium-adduct ion detected at m/z 373.1985 in the HRESIMS established the molecular formula of 2 as C20H30O5 (calcd for C20H30O5Na, 373.1991). The NMR data (Tables 1 and 2) of 2 resembled those of rhodojaponin III (15),12 except for the presence of an exocyclic double bond (δH 4.83, 4.77, each s, H2-17; δC 155.3, C-16; 104.6, C-17) in 2 rather than an oxygenated tertiary carbon (δC 81.4, C-16) and a methyl group (δH 1.30, s, CH3-17; δC 23.4, C17) in 15, indicating that compound 2 is a dehydration derivative of 15. The Δ16(17) double bond in 2 was confirmed by the HMBC cross-peaks from H2-17 (δH 4.83, 4.77, each s) to C13 (δC 56.0), C-15 (δC 49.5), and C-16 (δC 155.3). 2D NMR data (Figure S1, Supporting Information) further confirmed the structure of 2. Finally, a single-crystal X-ray diffraction analysis with a Cu Kα radiation experiment proved the structure of 2 (Figure 3) and defined the absolute configuration of 2 as (1S,2S,3R,5R,6R,8R,9R,10R,13S,14R) by the resulting Flack parameter of −0.01(16).30 Therefore, the structure of mollfoliagein B (2) was established as 2β,3β-epoxy5β,6β,10α,14β-tetrahydroxygrayan-16(17)-ene. Mollfoliagein C (3), colorless prisms, mp 196−197 °C, gave a molecular formula of C20H30O5 based on the HRESIMS ion at m/z 373.1981 [M + Na]+ (calcd for C20H30O5Na, 373.1991)

17; 21.0, C-19; 20.4, C-18; 21.8, 6-OAc), three methylenes (δC 53.2, C-15; 42.0, C-12; 36.0, C-7), eight methines including five oxymethines (δC 80.9, C-14; 78.8, C-12; 76.3, C-6; 63.6, C-3; 59.7, C-2), three oxygenated tertiary carbons (δC 86.4, C-16; 79.3, C-5; 75.1, C-10), an ester carbonyl (δC 171.5, 6-OAc), and two quaternary carbons (δC 51.5, C-8; 47.8, C-4). An ester carbonyl group accounts for one index of hydrogen deficiency, and the remaining six indices of hydrogen deficiency suggested the presence of a hexacyclic system in 1. The NMR data of 1 were similar to those of rhodojaponin II (16),29 the major grayanane diterpenoid in this study. The major difference included the presence of an oxygenated methine (δH 4.91, t, J = 3.3 Hz, H-11; δC 78.8, C-11) in 1, instead of a methylene (δH 1.99, m, H-11α; 1.63 m, H-11β; δC 22.8, C-11) in 16. In addition, the oxygenated tertiary carbon C-16 (δC 86.4) in 1 was deshielded by 6.4 ppm compared to that (δC 80.0) of 16. Thus, an oxygen bridge should exist between C-11 and C-16. This deduction was supported by the molecular formula and indices of hydrogen deficiency of 1. The 2D NMR data including 1 H−1H COSY, HSQC, HMBC, and NOESY (Figure 1) further confirmed the structure of 1. Finally, the structure of

Figure 1. 1H−1H COSY, key HMBC, and NOESY correlations of 1. C



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Figure 2. ORTEP drawing of compound 1.


and the 13C NMR data. Comparison of the 1H and 13C NMR data (Tables 1 and 2) of 3 with those of rhodojaponin III (15)12 suggested that the major difference was the presence of an exocyclic double bond (δH 5.37, 5.14, each s, H2-20; δC 150.7, C10; 116.4, C-20) in 3, replacing an oxygenated tertiary carbon (δC 81.4, C-16) and a methyl group (δH 1.30, s, H3-17; δC 23.4, C-17) in 15. Therefore, 3 was also a dehydration derivative of 15. In the HMBC spectrum, correlations from H2-20 (δH 5.37, 5.14, each s) to C-1 (δC 50.7), C-9 (δC 52.4), and C-10 (δC 150.7) suggested the presence of a Δ10(20) double bond in 3. The structure including the relative configuration of 3 was confirmed by 2D NMR data analysis (Figure S2, Supporting Information). The (1R,2S,3R,5R,6R,8S,9S,13R,14R,16R) absolute configuration of 3 was unambiguously established by the X-ray crystallographic diffraction data (Figure 4) with a Flack parameter of 0.1(1)30 and a Hooft parameter of 0.09(4).31


Thus, the structure of mollfoliagein C (3) was identified as 2β,3β-epoxy-5β,6β,10α,14β-tetrahydroxygrayan-10(20)-ene. D



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peaks from 2-OCH3 (δH 3.37) to C-2 (δC 87.5) and from H3-17 (δH 1.77) to C-13 (δC 51.9), C-15 (δC 128.4), and C-16 (δC 141.1) suggested that the methoxy group is located at C-2 and a double bond was located at C-15 and C-16. H-1 was assigned to be α-orientated on the basis of the chemical shift of C-1 (δC 57.0) in 7.16 The NOESY correlations of H-1α to H-3 and H-6 suggested that H-3 and H-6 are both α-oriented, and the NOESY correlations of H-7α to H-10 and H-14 assigned the αorientations of H-10 and H-14. In addition, the β-orientation of H-2 was assigned by the NOESY correlation between H-2 and H3-20. The (1R,2R,3R,5R,6R,8R,9R,10R,13S,14R) absolute configuration of 7 was determined by single-crystal X-ray diffraction analysis (Figure 5) with a Flack parameter of

6-O-Acetylrhodomollein XXXI (4) was isolated as a white, amorphous solid. The molecular formula was assigned as C22H32O6 based on the 13C NMR data and its HRESIMS sodium adduct ion at m/z 415.2084 (calcd for C22H32O6Na, 415.2097). The NMR data of 4 (Tables 1 and 2) were similar to those of rhodomollein XXXI,28a but for the presence of an additional acetyl group (δH 2.02, s, 6-OAc; δC 171.6, 21.5, 6OAc) in 4, indicating that compound 4 was an O-acetyl derivative of rhodomollein XXXI. The HMBC cross-peak of H-6 (δH 5.01, dd, J = 10.8, 4.9 Hz) to the carbonyl (δC 171.6) of the acetoxy group indicated that the acetoxy group was located at C6. The chemical shift of C-1 (δC 55.0) suggested the αorientation of H-1 compared to typical grayanane diterpeniods.20 The NOESY correlation between H-1α and H-6 indicated its α-orientation. 2D NMR data analysis (Figure S3, Supporting Information) further proved the structure of 4 as 6βacetoxy-2β,3β-epoxy-5β,10α,14β-trihydroxygrayan-15(16)ene. Mollfoliagein D (5) was assigned a molecular formula of C22H34O7 by the 13C NMR and the HRESIMS ion at m/z 433.2191 [M + Na]+ (calcd for C22H34O7Na, 433.2202). Comparison of the NMR spectroscopic data of 5 (Tables 1 and 2) with those of rhodomollein XI (25)13 suggested the presence of an exocyclic double bond (δH 4.84, 4.77, each s, H2-17; δC 155.5, C-16; 104.9, C-17) in 5, instead of a methyl group (δH 1.30, s, CH3-17; δC 23.3, C-17) and an oxygenated tertiary carbon (δC 81.4, C-16) in 25. The Δ16(17) double bond was located via the HMBC cross-peaks from H2-17 (δH 4.84, 4.77, each s) to C-13 (δC 55.0), C-15 (δC 49.6), and C-16 (δC 155.5). Therefore, the structure of mollfoliagein D (5) was identified as 6β-acetoxy-2α,3β,5β,10α,14β-pentahydroxygrayan-16(17)-ene by comprehensive 2D NMR data analyses (Figure S4, Supporting Information). 6-O-Acetylrhodomollein XIX (6) possessed an elemental composition of C22H32O6 as established by the [M + Na]+ ion at m/z 415.2092 (calcd for C22H32O6Na, 415.2097) in the HRESIMS and 13C NMR data. The NMR data (Tables 1 and 2) of 6 showed close similarities to rhodomollein XIX, which was obtained from the fruits of R. molle by Qin and co-workers.24 The most noticeable difference was the presence of an acetoxy group (δH 2.07, s, 6-OAc; δC 173.1, 21.4, 6-OAc) in 6 compared to rhodomollein XIX. The HMBC cross-peak from H-6 (δH 5.04, dd, J = 11.4, 1.7 Hz) to 6-OAc (δC, 173.1) suggested that 6 is a 6-O-acetyl derivative of rhodomollein XIX. The αorientation of H-1 was assigned by the chemical shift of C-1 (δC 51.6).20 The NOESY correlation of H-1α to H-6 assigned the α-orientation of H-6. The 2D NMR data analysis (Figure S5, Supporting Information) further confirmed that compound 6 wa s 6 β - a c e t o x y - 2 α , 3 β , 5 β , 1 4β - t e t r a h y d r o x y g r a y a n 10(20),16(17)-diene and named 6-O-acetylrhodomollein XIX. The molecular formula of mollfoliagein E (7) was established as C22H32O6 based on the 13C NMR data and the [M + Na]+ ion at m/z 429.2249 (calcd for C23H34O6Na, 429.2253) in the HRESIMS. The NMR data (Tables 1 and 2) closely resembled those of rhodomollein G, which was isolated from the flowers of R. molle by Li and co-workers.16 Comparing the NMR data (Tables 1 and 2) of 7 with those of rhodomollein G suggested that 7 differs from rhodomollein G by the presence of a methoxy group (δH 3.37, s, 2-OCH3; δC 57.6) and an endocyclic double bond (δH 5.50, q, J = 1.5 Hz, H-15; δC 128.4, C-15; 141.1, C-16) in 7, instead of a methylene (δH 2.88, d, J = 15.6 Hz, H-15α; 2.14, d, J = 15.6 Hz, H-15β; δC 51.3, C-15) and an oxygenated tertiary carbon (δC 81.6, C-16) in rhodomollein G. The HMBC cross-


−0.08(7)30 and a Hooft parameter of −0.06(6).31 Thus, the structure of mollfoliagein E (7) was assigned as 6β-acetoxy5β,9β-epoxy-3β,14β-dihydroxy-2α-methoxygrayan-15(16)-ene. Mollfoliagein F (8) was obtained as colorless prisms, mp 167−168 °C. Its molecular formula was determined to be C23H36O7 by the 13C NMR data and the sodium adduct at m/z [M + Na]+ 447.2347 (calcd for C23H36O7Na, 447.2359) in the HRESIMS. The NMR data (Tables 2 and 3) for 8 were similar to those of rhodomollein XXVI isolated from the roots of R. molle by Yu and co-workers.20 However, 8 possessed a methoxy (δH 3.45, s, 2-OCH3; δC 57.7, 2-OCH3) and an O-acetyl (δH 2.09, s, 6-OAc; δC 172.4, 21.5, 6-OAc) group. The HMBC crosspeaks from 2-OCH3 (δH 3.45, s) to C-2 (δC 92.6) and from the methyl of the acetoxy group (δH 2.09, s, 6-OAc) to C-6 (δC 70.9) indicated that the methoxy and acetoxy groups were located at C-2 and C-6 in 8, respectively. Consequently, single-crystal Xr ay d i ff r a c t i o n an al y s i s (F i g u r e 6 ) s e c u r e d t h e (1R,2R,3R,5R,6R,8S,13R,14R,16R) absolute configuration of 8. Therefore, the structure of mollfoliagein F (8) was defined as 6β-acetoxy-3β,5β,14β,16α-tetrahydroxy-2α-methoxygrayan9(10)-ene. 18-Hydroxygrayanotoxin XVIII (9) exhibited a molecular formula of C20H32O5 according to its 13C NMR data and the [M + Na]+ ion at m/z 375.2146 (calcd for C20H32O5Na, 375.2147) in the HRESIMS. Analysis of the NMR data (Tables 2 and 3) revealed that 9 is a grayanane diterpenoid with a structure close to grayanotoxin XVIII (23).32 An oxygenated methylene group (δH 4.09, 3.49, each d, J = 11.2 Hz, H2-18; δC 64.4, C-18) in 9 replaced the methyl group (δH 1.17, s, CH3-18; δC 18.0, C-18) in E



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Table 3. 1H (400 MHz) NMR [δ, mult (J in Hz)] Spectroscopic Data for Compounds 8−13a no. 1α 2α 2β 3α 6α 7α 7β 9β 11α 11β 12α 12β 13α 14α 15α 15β 17 18 19 20 6-OAc 14-OAc 2-OCH3

8 3.31, d (8.9) 3.94, dd (8.9, 2.2) 3.53, d (2.2) 5.24, d (5.5) 2.37, d (14.6) 2.21, dd (14.6, 5.5) 1.71, m 1.38, m 2.58, m 1.99, m 2.03, m 3.60, s 2.33, d (14.2) 1.72, d (14.2) 1.33, s 0.94, s 1.02, s 1.81, s 2.09, s

9 2.91, t (9.7) 2.26, ddd (14.5, 9.7, 6.8) 1.83, ddd (14.5, 9.7, 3.6) 3.80, dd (6.8, 3.6) 3.73, d (11.4, 2.1) 1.34, d (13.9, 2.1) 2.12, d (13.9, 11.4) 2.40, d (8.9, 7.8) 1.67, m 1.56, overlap 1.56, overlap 1.42, m 1.95, m 1.84, overlap 1.80, d (13.9) 1.71, d (13.9) 1.35, s 4.09, 3.49, d (11.2) 1.10, s 4.93, 5.05, s

3.45, s

10

11

12

13

2.79, d (7.5)

2.74, d (7.9)

2.37, d (4.8)

2.57, d (4.0)

3.74, dd (7.5, 1.5) 3.54, d (1.5) 4.06, dd (8.9, 2.1) 1.84, dd (14.3, 2.1) 1.97, dd (14.3, 8.9) 2.77, m 1.85, m 1.68, overlap 1.67, overlap 1.52, m 1.99, m 4.01, s 2.00, s 2.00, s 1.35, s 1.09, s 1.07, s 5.09, 5.04, s

3.80, dd (7.9, 2.0) 3.56, d (2.0) 5.04, dd (10.2, 1.6) 1.58, dd (14.5, 1.6) 2.18, dd (14.5, 10.2) 2.70, dd (9.0, 6.7) 1.70, m 1.43, m 1.91, m 1.75, m 2.04, m 4.27, s 1.93, s 1.93, s 1.34, s 1.07, s 1.03, s 5.09, 5.07, s 2.09, s

3.91, d (4.8) 3.57, s 3.96, dd (10.8, 3.9) 2.14, dd (13.8, 3.9) 1.86, dd (13.8, 10.8) 2.02, m 1.70, m 1.52, overlap 2.08, m 1.53, overlap 1.78, m 4.29, s 1.97, d (14.8) 1.82, d (14.8) 1.29, s 1.14, s 1.15, s 1.37, s

3.40, s

3.38, s

3.41, s

3.92, d (4.0) 3.57, s 4.83, dd (11.4, 4.6) 1.94, dd (13.7, 4.6) 1.85, dd (13.7, 11.4) 1.83, m 1.76, m 1.57, overlap 2.21, m 1.58, overlap 2.12, m 5.57, s 2.03 (15.6) 1.83 (15.6) 1.33, s 1.10, s 0.93, s 1.39, s 2.03, s 2.18, s 3.39

a

Recorded in methanol-d4.

respectively, except for the presence of an O-methyl group in compounds 10−13. In addition, the chemical shift of C-2 in 10− 13 was deshielded by ca. 10 ppm compared to 19, 20, 24, and 26. Therefore, compounds 10−13 are 2-OCH3 derivatives of 19, 20, 24, and 26. These conclusions were confirmed by the HMBC cross-peaks from OCH3 to C-2 in 10−13. The relative configurations of 10−13 were the same as 19, 20, 24, and 26 based on their coupling constants and NOESY data (Figures S9−S12, Supporting Information). Thus, the structures of 10− 13 were defined as 2-O-methylrhodomolin I (10), 2-Omethylrhodomollein XII (11), 2-O-methylrhodojaponin VI (12), and 2-O-methylrhodojaponin VII (13), respectively. Bimollfoliagein A (14) was obtained as an amorphous powder. The molecular formula of 14 was determined to be C45H72O15 by the HRESIMS ion at m/z 875.4753 [M + Na]+ (calcd for C45H72O15Na, 875.4769) and 13C NMR data. The NMR data of 14 (Table 4) were consistent with the presence of two sets of grayanane-type diterpenoid signals and closely resembled those of birhodomollein B, which was recently obtained from the flowers of R. molle by Ye and co-workers.18 Comparison of the NMR data (Table 4) of 14 with those of birhodomollein B suggested their structural similarity except for the presence of a methoxy group (δH 3.35, s; δC 57.6) in 14. The chemical shift of C-2 (δC 91.4) in 14 was deshielded by 11.1 ppm compared to the oxygenated methine C-2 (δC 80.3) in birhodomollein B,18 indicating that the O-methyl group was located at C-2. The HMBC cross-peak of OCH3 (δC 3.35) to C2 (δC 91.4) supported the above deduction. The α-orientations of H-1 and H-1′ were confirmed by the chemical shifts of C-1 (δC 61.2) and C-1′ (δC 64.1), respectively.18,33 The NOESY correlation between H-1α and H-14 established the αorientation of H-14 in 14. The small coupling constant J = 2.4 Hz between H-1′α and H-2′ indicated that H-2′ was βorientated. The relative configuration of 14 was identical to that of birhodomollein B18 on the basis of their similar coupling


23. Thus, 9 is an 18-hydroxy derivative of grayanotoxin XVIII, which was verified by the HMBC cross-peaks from H2-18 (δH 4.09, 3.49) to C-3 (δC 81.9), C-4 (δC 55.4), C-5 (δC 84.1), and C-19 (δC 20.2). The α-orientation of H-1 was assigned by the chemical shift of C-1 (δC 43.8).33 The NOESY correlation between H-1α and CH3-19 suggested that they were cofacial; correspondingly, 18-CH2OH was assigned a β-orientation. The 2D NMR data (Figure S8, Supporting Information) confirmed the structure of 18-hydroxygrayanotoxin XVIII (9) as 3β,5β,6β,16α,18-pentahydroxygrayan-10(20)-ene, the first example of an 18-hydroxy grayanane-type diterpenoid in the Ericaceae family. Compounds 10−13 were obtained as white, amorphous solids. Their molecular formulas were assigned as C21H34O6, C23H36O7, C21H36O7, and C25H40O9, respectively, based on the 13 C NMR and HRESIMS data. Their NMR data (Tables 2 and 3) are structurally similar to those of 19, 20, 24, and 26, F



Article

Table 4. 1H (400 MHz) and 13C (100 MHz) NMR Spectroscopic Data for Compound 14a δH

no. 1α 2 3 4 5 6 7α 7β 8 9 10 11α 11β 12α 12β 13 14 15α 15β 16 17 18 19 20 6-OAc

2.54, d (3.5) 3.92, d (3.5) 3.63, s

5.22, dd (11.0, 4.1) 2.01, dd (13.6, 4.1) 1.83, dd (13.6, 11.0) 1.85, m 1.72, m 1.57, m 2.04, m 1.63, m 2.38, m 4.41, s 2.07, d (15.7) 1.69, d (15.7) 1.28, s 1.13, s 1.13, s 1.39, s

2.09, s 23.35, s OCH3

δC 1′ 2′ 3′ 4′ 5′ 6′

38.9

7′α 7′β

52.6 56.1 78.9 22.5 27.4 53.7 89.1 62.2 81.2 22.3 19.9 23.9 29.2 172.9

δH

no.

61.2 91.4 83.0 50.9 85.0 78.6

8′ 9′ 10′ 11′α 11′β 12′α 12′β 13′ 14′ 15′α 15′β 16′ 17′ 18′ 19′ 20′ 6′OAc

22.0 57.6

3.01, d (2.4) 4.18, d (2.4) 3.84, s

5.31, dd (11.7, 4.5) 2.12, dd (13.7, 4.5) 1.84, dd (13.7,11.7) 1.85, m 1.61, m 1.57, m 2.43, m 1.41, m 1.83, m 4.51, s 1.99, d (14.9) 1.74, d (14.9)

The known grayanane diterpenoids 15−29 were identified to be rhodojaponin III (15),12 rhodojaponin II (16),29 rhodojaponin V (17),29 rhodojaponin I (18),29 rhodomolin I (19),34 rhodomollein XII (20),13 grayanotoxin II (21),35 grayanotoxin XVI (22),35 grayanotoxin XVIII (23),32 rhodojaponin VI (24),13 rhodomollein XI (25),13 rhodojaponin VII (26),29 rhodomollein XIII (27),13 2-O-methylrhodomollein XI (28),14 and 6-O-acetylrhodomollein XXI (29)14 by HRESIMS and NMR data analyses, as well as comparison with literature data. More importantly, the absolute configurations of rhodojaponin II (16), rhodojaponin I (18), and rhodojaponin VI (24) were confirmed by single-crystal X-ray diffraction data (Figures 7−9) for the first time. This is the first report of the isolation of the grayanotoxin XVI (22) and grayanotoxin XVIII (23) from R. molle.

δC 64.1 89.2 86.0 50.7 85.0 78.9 38.7

51.5 55.8 77.8 23.0 26.0 56.7 79.2 60.4

1.22, s 1.15, s 0.97, s 1.41, s

80.8 22.7 19.9 22.0 29.1 172.3

2.07, s

21.6


a

Recorded in methanol-d4.

Acetylated grayanane diterpenoids are common in nature;33 however, methoxygrayanane diterpenoids are relatively rare. Since the first methoxygrayanane diterpenoid, rhodomolin A, was isolated from the flowers of R. molle in 2005 by Hu and coworkers,15 only five methoxy-containing Ericaceae diterpenoids including three grayanane-type analogues have been re-

constants and NOESY data (Figure S13, Supporting Information). Accordingly, the structure of bimollfoliagein A (14) was defined as 6β-acetoxy-3β,5β,10α,16α-tetrahydroxy-2α-methoxygrayanane-14β-yl 6′β-acetoxy-3′β,5′β,10′α,14′β,16′αpentahydroxygrayanane-2′α-yl ether by 2D NMR analyses.

Figure 7. ORTEP drawing of compound 16. G



Article


significant anti-inflammatory activity (IC50 > 40 μM). In addition, rhodojaponin III (15) exhibited remarkable antiinflammatory activity, with an IC50 value of 7.0 ± 1.0 μM, and was nearly 5-fold more potent than mollfoliagein C (3) (IC50 = 35.4 ± 3.9 μM). Thus, the 10-OH group may be necessary for anti-inflammatory activity, which was supported by the weak anti-inflammatory activity of compounds 5, 8−11, and 19−22, without 10-OH (IC50 > 40 μM). Further structure−activity relationship analysis of 15−18 revealed that the 6-OAc group was essential for the anti-inflammatory activity, and 14-OAc may be a deactiviting group, which was supported by the significant anti-inflammatory activity of 28, with 6-OAc (IC50 = 4.6 ± 0.7 μM), over 12, devoid of 6-OAc, and 13, possessing a 14-OAc (IC50 > 40 μM). Finally, the general cytotoxicities of 1−29 were also evaluated by an MTT method, and none showed cytotoxicity against RAW 264.7 cell lines, either with or without LPS (1 μg/mL), at a concentration of 200 μM. Therefore, the anti-inflammatory activities of compounds 3, 15, 16, 18, 19, 23− 26, 28, and 29 do not result from cytotoxicity. Hitherto, only two grayanane diterpenoids from the Euphorbiaceae plant (Croton tonkinensis), crotonkinensins A and B, showed anti-inflammatory activity, with IC50 values of 7.1 ± 0.2 and 5.5 ± 0.2 μΜ, respectively,40 which are weaker than 16 (IC50 = 2.8 ± 0.5 μΜ), 23 (IC50 = 3.5 ± 0.3 μΜ), and 28 (IC50 = 4.6 ± 0.7 μΜ) in this study. The current study provides useful clues to develop novel anti-inflammatory drugs based on grayanane diterpenoids.

ported.14,15,36,37 The isolation of seven methoxygrayanane diterpenoids (7, 8, and 10−14) in the current study enriched the chemical diversity of grayanane diterpenoids. Diterpenoids 1−29 were tested for their in vitro antiinflammatory activities by measuring the nitric oxide (NO) production in lipopolysacccharide (LPS)-stimulated RAW264.7 mouse macrophages.38,39 Preliminary screening results at 40 μΜ showed that compounds 3, 15, 16, 18, 19, 23−26, 28, and 29 exhibited significant anti-inflammatory activity with inhibition rates of more than 50%. Further screening studies at five different concentrations revealed that these compounds exhibited significant anti-inflammatory activity, with IC50 values ranging from 2.8 to 35.4 μM (Table 5). Rhodojaponin II (16), Table 5. Inhibitory Effects of Diterpenoids 1−29 and Dexamethasone on the LPS-Induced NO Production in RAW 264.7 Mouse Macrophages and the Cell Survival Ratesa survival rates (%) of RAW 264.7 at 200 μM compound

IC50 (μM)b

LPS (0 μg/mL)

LPS (1 μg/mL)

3 15 16 18 19 23 24 25 26 28 29 dexamethasonec

35.4 ± 3.9 7.0 ± 1.0 2.8 ± 0.5 10.1 ± 1.4 8.1 ± 0.8 3.5 ± 0.3 8.0 ± 1.2 9.3 ± 1.3 10.3 ± 0.8 4.6 ± 0.7 5.8 ± 0.6 0.8 ± 0.1

90.3 96.5 101.2 98.9 98.1 96.4 97.0 98.8 97.4 98.3 99.3 97.7

92.0 98.4 93.6 99.7 97.5 93.8 90.4 99.8 93.9 97.3 95.2 97.1

■

EXPERIMENTAL SECTION

General Experimental Procedures. Melting points of single crystals were recorded on a Beijing Tech X-5 microscopic melting point apparatus. Optical rotations were measured in MeOH using a PerkinElmer 341 polarimeter for compounds 1−6, 8, and 10−29 or an Autopol IV automatic polarimeter for compounds 7 and 9. The NMR spectra were determined on a Bruker AM-400, and the 1H and 13 C NMR chemical shifts were referenced to the solvent residual peaks for methanol-d4 at δH 3.31 and δC 49.15, respectively, and pyridine-d5 at δH 8.74, 7.58, 7.22 and δC 150.35, 135.91, 123.87, respectively. HRESIMS data were acquired on a Thermo Fisher LC-LTQ-Orbitrap XL for compounds 1−6, 8, and 10−29 or a Bruker micrOTOF II spectrometer for compounds 7 and 9 in positive ion mode. The crystallographic data were obtained on a Bruker SMART APEX-II CCD diffractometer equipped with graphite-monochromatized Cu Kα radiation (λ = 1.541 78 Å) for compounds 1−3, 8, 16, 18, and 24 or an Agilent Super Nova, Dual, Cu at zero, AtlasS2 diffractometer for

For compounds 1, 2, 4−14, 17, 20−22, and 27, IC50 > 40 μM. Values are expressed as the means ± SD, n = 3. cDexamethasone was used as a positive control.

a

b

possessing a 2β,3β-epoxy moiety and a 16-OH group, showed significant anti-inflammatory activity, with an IC50 value of 2.8 ± 0.5 μΜ. In contrast, new compounds 1, 2, and 4, bearing a 2β,3β-epoxy moiety but lack of 16-OH, did not exhibited significant activity (IC50 > 40 μM), suggesting that the 16-OH group is essential for the anti-inflammatory activity. This may explain why compounds 4−7 without 16-OH did not show H



Article

Mollfoliagein A (1): colorless prisms; mp 174−175 °C; [α]25 D −15 (c 0.1, MeOH); 1H and 13C NMR data, Tables 1 and 2; HRESIMS m/z 431.2025 [M + Na]+ (calcd for C22H32O7Na, 431.2046). Mollfoliagein B (2): colorless prisms; mp 209−211 °C; [α]25 D −20, (c 0.1, MeOH); 1H and 13C NMR data, Tables 1 and 2; HRESIMS m/z 373.1985 [M + Na]+ (calcd for C20H30O5Na, 373.1991). Mollfoliagein C (3): colorless prisms; mp 196−197 °C; [α]25 D −18, (c 0.1, MeOH); 1H and 13C NMR data, Tables 1 and 2; HRESIMS m/z 373.1981 [M + Na]+ (calcd for C20H30O5Na, 373.1991). 6-O-Acetylrhodomollein XXXI (4): white powder; [α]25 D −9 (c 0.1, MeOH); 1H and 13C NMR data, Tables 1 and 2; HRESIMS m/z 415.2084 [M + Na]+ (calcd for C22H32O6Na, 415.2097). Mollfoliagein D (5): amorphous powder; [α]25 D −8 (c 0.1, MeOH); 1 H and 13C NMR data, Tables 1 and 2; HRESIMS m/z 433.2191 [M + Na]+ (calcd for C22H34O7Na, 433.2202). 6-O-Acetylrhodomollein XIX (6): white powder; [α]25 D −18 (c 0.1, MeOH); 1H and 13C NMR data, Tables 1 and 2; HRESIMS m/z 415.2092 [M + Na]+ (calcd for C22H32O6Na, 415.2097). Mollfoliagein E (7): colorless prisms; mp 155−156 °C; [α]25 D −35, (c 0.1, MeOH); 1H and 13C NMR data, Tables 1 and 2; HRESIMS m/z 429.2249 [M + Na]+ (calcd for C23H34O6Na, 429.2253). Mollfoliagein F (8): colorless prisms; mp 167−168 °C; [α]25 D +47 (c 0.1, MeOH); 1H and 13C NMR data, Tables 2 and 3; HRESIMS m/z 447.2347 [M + Na]+ (calcd for C23H36O7Na, 447.2359). 18-Hydroxygrayanotoxin XVIII (9): amorphous powder; [α]25 D −27 (c 0.1, MeOH); 1H and 13C NMR data, Tables 2 and 3; HRESIMS m/z 375.2146 [M + Na]+ (calcd for C20H32O6Na, 375.2147). 2-O-Methylrhodomolin I (10): amorphous powder; [α]25 D −7 (c 0.1, MeOH); 1H and 13C NMR data, Tables 2 and 3; HRESIMS m/z 405.2243 [M + Na]+ (calcd for C21H34O6Na, 405.2253). 2-O-Methylrhodomollein XII (11): amorphous powder; [α]25 D −14 (c 0.1, MeOH); 1H and 13C NMR data, Tables 2 and 3; HRESIMS m/z 447.2346 [M + Na]+ (calcd for C23H36O7Na, 447.2359). 2-O-Methylrhodojaponin VI (12): amorphous powder; [α]25 D −9 (c 0.1, MeOH); 1H and 13C NMR data, Tables 2 and 3; HRESIMS m/z 423.2332 [M + Na]+ (calcd for C21H36O7Na, 423.2359). 2-O-Methylrhodojaponin VII (13): amorphous powder; [α]25 D −25 (c 0.1, MeOH); 1H and 13C NMR data, Tables 2 and 3; HRESIMS m/z 507.2569 [M + Na]+ (calcd for C25H40O9Na, 507.2570). Bimollfoliagein A (14): amorphous powder; [α]25 D −16 (c 0.1, MeOH); 1H and 13C NMR data, Table 4; HRESIMS m/z 875.4753 [M + Na]+ (calcd for C45H72O15Na, 875.4769). Single-Crystal X-ray Diffraction Analysis. The intensity data collection, structure solution, reduction, and hydrogen atom treatment for compounds 1−3, 7, 8, 16, 18, and 24 were performed as described in a previous paper.33 The crystallographic data of 1−3, 7, 8, 16, 18, and 24 have been deposited in the Cambridge Crystallographic Data Centre with deposit numbers CCDC 1573695−1573702, and their ORTEP drawing crystal structures are shown in Figures 2−9, respectively. Crystallographic Data for 1. C22H32O7·H2O, M = 426.49, T = 296(2) K, monoclinic, P21, a = 12.5466(6) Å, b = 6.2439(3) Å, c = 13.9222(6) Å, α = 90°, β = 101.124(2)°, γ = 90°, V = 1070.17(9) Å3, Z = 2, Dcalcd = 1.324 mg/cm3, absorption coefficient 0.828 mm−1, F(000) = 460, 0.12 × 0.10 × 0.10 mm3, theta range for data collection 3.235° to 65.976°, −11 ≤ h ≤ 14, − 7 ≤ k ≤ 7, −16 ≤ l ≤ 16, reflections collected 12 264, independent reflections 3590 [R(int) = 0.0299], completeness to θ = 65.976°, largest diff peak and hole 0.186 and −0.170 e·Å−3, F2 = 1.086, data/restraints/parameters 3590/4/285, final R indices [I > 2σ(I)] R1 = 0.0316, wR2 = 0.0923, R indices (all data) R1 = 0.0322, wR2 = 0.0931, Flack parameter 0.09(6). Crystallographic Data for 2. C20H30O5, M = 350.44, T = 293(2) K, monoclinic, P21, a = 7.9231(3) Å, b = 10.9643(4) Å, c = 21.6231(7) Å, α = 90°, β = 95.0830(10)°, γ = 90°, V = 1871.04(12) Å3, Z = 4, Dcalcd = 1.244 mg/cm3, absorption coefficient 0.714 mm−1, F(000) = 760, 0.05 × 0.04 × 0.03 mm3, theta range for data collection 4.525° to 68.275°, −9 ≤ h ≤ 9, −13 ≤ k ≤ 13, − 26 ≤ l ≤ 26, reflections collected 20 899, independent reflections 6789 [R(int) = 0.0365], completeness to θ = 67.679°, largest diff peak and hole 0.150 and −0.161 e·Å−3, F2 = 1.015, data/restraints/parameters 6789/1/466, final R indices [I > 2σ(I)] R1 =

compound 7. Samples were purified by semipreparative HPLC using an Agilent 1200 or Dionex P680 quaternary system with a UV detector and a semipreparative column (5 μm, 10 × 250 mm, Welch Ultimate XBC18 or XB-phenyl) at a flow rate of 1.5 mL/min. Plant Material. The leaves of R. molle were collected in Qichun, Hubei Province, China, in May 2014, and identified by Prof. Jianping Wang at Huazhong University of Science and Technology (HUST). A voucher specimen (No. 20140520) has been deposited at HUST. Extraction and Isolation. The air-dried leaves (25.0 kg) of R. molle were powered and extracted five times using 95% EtOH at room temperature (7 days each). A dark brown crude extract (3.7 kg) was obtained by concentration of the combined percolates under reduced pressure. The extract (3.7 kg) was suspended in 30 L of H2O and extracted excessively with petroleum ether and CHCl3. The combined CHCl3 extract (1190.5 g) was loaded on an MCI gel column utilizing a MeOH−H2O gradient (9:1 to 10:0, v/v) to get two fractions, A and B. Fraction A (eluted with MeOH−H2O 90%) was separated using a silica gel column (100−200 mesh) (eluted with CH2Cl2−acetone, 20:1−1:1, v/v) to yield five fractions (AA−AE). Subfraction AC was separated on an RP C18 column (MeOH−H2O, 10:90 to 100:0) to give four fractions (ACa−ACd). Subfractions ACa (eluted with MeOH−H2O, 10:90) and ACb (eluted with MeOH−H2O, 20:80) were chromatographed on a Sephadex LH-20 column (MeOH) to give five subfractions, ACa1− ACa3, ACb1, and ACb2, respectively. Similarly, subfraction ADa was separated on an RP C18 column eluting with MeOH−H2O from 10% to 100% to yield four fractions, ADa1−ADa4. Subfraction ACa1 was subjected to a silica gel column (200−300 mesh) to obtain four subfractions, ACa1a−ACa1d. Subfraction ACa1a was purified by semipreparative RP C18 HPLC eluting with a gradient of CH3CN in H2O 35% to yield compounds 1 (4.3 mg, tR = 32.4 min) and 7 (3.7 mg, tR = 36.8 min). Compounds 26 (12.7 mg) and 28 (14.6 mg) were obtained by an RP C18 column with 15% MeOH−H2O from subfraction ACa1b. Compounds 2 (9.6 mg, tR = 58.2 min) and 4 (3.3 mg, tR = 68.5 min) were isolated from subfraction ACa1c, using semipreparative HPLC with an RP phenyl column (eluted with a gradient of MeOH in H2O 60%), and compound 24 (35.7 mg) was crystallized in MeOH from subfraction ACa1d. Subfraction ACa1d was subjected to chromatography on an RP C18 column with 20% MeOH− H2O to afford 27 (7.5 mg). Subfraction ACb1 was subjected to a silica gel column (200−300 mesh) to obtain three subfractions, ACb1a− ACb1c, and 11 (9.3 mg), and subfraction ACb1a was resolved on an RP C18 ODS column eluting with a gradient of MeOH−H2O (2:8, v/v) to afford 14 (4.7 mg) and 18 (16.6 mg). Compound 16 (826.5 mg) was crystallized in MeOH−H2O (9:1, v/v) from subfraction ACb1b. The residue of subfraction ACb1b was loaded onto an RP C18 column eluting with MeOH−H2O (15:85, v/v) to afford 17 (8.4 mg). Fraction ACb1c was purified by semipreparative HPLC eluting with MeOH− H2O (65:35, v/v) to produce 11 (4.3 mg, tR = 34.7 min) and 12 (3.7 mg, tR = 28.3 min). Fraction ADa1 was subjected to silica gel column (200−300 mesh) chromatography (petroleum ether−acetone, 3:1− 1:1, v/v) to obtain four major fractions, ACa1a−ACa1c and 19 (9.7 mg), and fraction ADa1a was subjected to semipreparative HPLC (MeOH−H2O, 5:5, v/v) to obtain 6 (2.3 mg, tR = 49.4 min) and 8 (6.7 mg, tR = 53.8 min). A silica gel column (300−400 mesh) was applied to purify subfraction ADa1b into 3 (5.5 mg) and 20 (6.5 mg). Subfraction ADa1c was purified using semipreparative HPLC eluted with MeOH− H2O (11:9, v/v) to yield 5 (6.2 mg, tR = 39.5 min) and 21 (3.7 mg, tR = 43.3 min). In the same manner, fraction ADa2 was separated on a silica gel column to yield three major subfractions, ADa2a−ADa2c, and compound 29 (12.5 mg), which was crystallized from MeOH−H2O (9:1, v/v). Subfraction ADa2a was purified by semipreparative HPLC with MeCN−H2O (25:75, v/v) to yield compounds 13 (5.2 mg, tR = 34.6 min) and 22 (5.6 mg, tR = 29.9 min). Compounds 10 (4.3 mg, tR = 41.8 min) and 23 (3.7 mg, tR = 45.1 min) were isolated from subfraction ADa2b by semipreparative HPLC eluting with MeOH−H2O (6:4, v/ v). Subfraction ADa2c was purified by semipreparative HPLC eluating with MeCN−H2O (2:8, v/v) to yield 9 (9.5 mg, tR = 28.7 min) and 25 (6.6 mg, tR = 24.2 min). I



Article

Anti-inflammatoryActivity Evaluation. The anti-inflammatory activity of compounds 1−29 in vitro was evaluated by measuring the amount of NO production in LPS-induced RAW 264.7 mouse macrophages, as described previously.38,39 The general cytotoxity of the active compounds at 200 μM against RAW 264.7 mouse macrophages with or without 1 μg/mL LPS was measured according to the MTT method.38,39

0.0336, wR2 = 0.0866, R indices (all data) R1 = 0.0425, wR2 = 0.0919, Flack parameter −0.01(16). Crystallographic Data for 3. 2(C20H30O5)·H2O, M = 718.9, T = 173(2) K, monoclinic, P21, a = 6.2989(2) Å, b = 27.0429(6) Å, c = 11.3517(3) Å, α = 90°, β = 104.0960(10)°, γ = 90°, V = 1875.43(9) Å3, Z = 2, Dcalcd = 1.273 mg/cm3, absorption coefficient 0.744 mm−1, F(000) = 780, 0.18 × 0.14 × 0.12 mm3, theta range for data collection 4.02° to 66.96°, −6 ≤ h ≤ 7, −32 ≤ k ≤ 32, −13 ≤ l ≤ 13, reflections collected 19 443, independent reflections 6531 [R(int) = 0.0211], completeness to θ = 66.69°, largest diff peak and hole 0.148 and −0.144 e·Å−3, F2 = 1.047, data/restraints/parameters 6531/1/498, final R indices [I > 2σ(I)] R1 = 0.0325, wR2 = 0.0825, R indices (all data) R1 = 0.0341, wR2 = 0.0836, Flack parameter 0.1(1). Crystallographic Data for 7. C23H34O6, M = 406.50, T = 100(10) K, monoclinic, P21, a = 9.81810(10) Å, b = 16.3996(2) Å, c = 13.9736(2) Å, α = 90°, β = 107.0090(10)°, γ = 90°, V = 2151.52(5) Å3, Z = 4, Dcalcd = 1.255 mg/cm3, absorption coefficient 0.728 mm−1, F(000) = 880, 0.18 × 0.15 × 0.11 mm3, theta range for data collection 8.536° to 147.134°, −11 ≤ h ≤ 12, −20 ≤ k ≤ 19, − 17 ≤ l ≤ 17, reflections collected 21 222, independent reflections 8055 [R(int) = 0.0325], completeness to θ = 147.134, largest diff peak and hole 0.23 and −0.29 e·Å−3, F2 = 1.039, data/restraints/parameters 8055/1/542, final R indices [I > 2σ(I)] R1 = 0.0371, wR2 = 0.0986, R indices (all data) R1 = 0.0379, wR2 = 0.0998, Flack parameter −0.08(7), Hooft parameter −0.06(6). Crystallographic Data for 8. C23H36O7·H2O, M = 442.53, T = 173(2) K, orthorhombic, P21 21 21, a = 8.6611(9) Å, b = 13.5236(5) Å, c = 18.6939(12) Å, α = 90°, β = 90°, γ = 90°, V = 2189.6(3) Å3, Z = 4, Dcalcd = 1.342 mg/cm3, absorption coefficient, 0.827 mm−1, F(000) = 960, crystal size 0.16 × 0.13 × 0.12 mm3, theta range for data collection 2.36° to 66.58°, −10 ≤ h ≤ 9, −14 ≤ k ≤ 16, −22 ≤ l ≤ 20, reflections collected 15 760, independent reflections 3846 [R(int) = 0.1036], completeness to θ = 66.58, largest diff peak and hole 0.237 and −0.263 e·Å−3, F2 = 1.058, data/restraints/parameters 3846/0/288, final R indices [I > 2σ(I)], R1 = 0.0424, wR2 = 0.1113, R indices (all data) R1 = 0.0441, wR2 = 0.1127, Flack parameter −0.2(2). Crystallographic Data for 16. C22H34O7·CH3OH, M = 442.53, T = 296(2) K, orthorhombic, P2(1)2(1)2(1), a = 9.5407(4) Å, b = 11.7224(5) Å, c = 19.9776(9) Å, α = 90°, β = 90°, γ = 90°, V = 2234.29(17) Å3, Z = 4, Dcalcd = 1.316 mg/cm3, absorption coefficient 0.810 mm−1, F(000) = 960, crystal size 0.20 × 0.20 × 0.20 mm3, theta range for data collection 4.37° to 67.43°, −11 ≤ h ≤ 11, −12 ≤ k ≤ 13, −23 ≤ l ≤ 21, reflections collected 12 227, independent reflections 3903 [R(int) = 0.0244], completeness to θ = 67.43°, largest diff peak and hole 0.351 and −0.291 e·Å−3, F2 = 1.101, data/restraints/parameters 3903/0/291, final R indices [I > 2σ(I)] R1 = 0.0418, wR2 = 0.1128, R indices (all data) R1 = 0.0436, wR2 = 0.1165, Flack parameter 0.0(2). Crystallographic Data for 18. C24H36O8, M = 452.53, T = 298(2) K, monoclinic, P21, a = 8.7254(2) Å, b = 13.1452(3) Å, c = 20.1229(5) Å, α = 90°, β = 98.5930(10)°, γ = 90°, V = 2282.13(9) Å3, Z = 4, Dcalcd = 1.317 mg/cm3, absorption coefficient 0.809 mm−1, F(000) = 976, crystal size 0.30 × 0.20 × 0.10 mm3, theta range for data collection 2.22° to 66.63°, −10 ≤ h ≤ 10, −15 ≤ k ≤ 15, −23 ≤ l ≤ 23, reflections collected 28 647, independent reflections 8005 [R(int) = 0.0197], completeness to θ = 66.63°, largest diff peak and hole 0.379 and −0.387 e·Å−3, F2 = 1.055, data/restraints/parameters 8005/1/578, final R indices [I > 2σ(I)] R1 = 0.034, wR2 = 0.0935, R indices (all data) R1 = 0.0345, wR2 = 0.0939, Flack parameter 0.08(9). Crystallographic Data for 24. C20H34O7·C2H5O, M = 431.53, T = 173(2) K, orthorhombic, P21 21 21, a = 10.3826(3) Å, b = 10.5016(3) Å, c = 19.1653(6) Å, α = 90°, β = 90°, γ = 90°, V = 2089.67(11) Å3, Z = 4, Dcalcd = 1.372 mg/cm3, absorption coefficient 0.849 mm−1, F(000) = 940, crystal size 0.20 × 0.20 × 0.20 mm3, theta range for data collection 4.614° to 68.261°, −12 ≤ h ≤ 12, − 12 ≤ k ≤ 12, −23 ≤ l ≤ 23, reflections collected 18 724, independent reflections 3822 [R(int) = 0.0238], completeness to θ = 67.679°, largest diff peak and hole 0.481 and −0.635 e·Å−3, F2 = 1.008, data/restraints/parameters 3822/0/301, final R indices [I > 2σ(I)] R1 = 0.0369, wR2 = 0.1048, R indices (all data) R1 = 0.0377, wR2 = 0.1059, Flack parameter 0.00(3).

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.7b00799. (+)-HRESIMS and 1D and 2D NMR spectra of the new compounds 1−14; 2D NMR analysis of the new compounds 2−14 (PDF) CIF files for compounds 1−3, 7, 8, 16, 18, and 24 (CIF) (CIF) (CIF) (CIF) (CIF) (CIF) (CIF) (CIF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Guangmin Yao: 0000-0002-8893-8743 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the Fundamental Research Funds for the Central Universities (HUST: 2016YXMS148 and 2016YXZD049) and the National Natural Science Foundation of China (81001368 and 31170323). We thank the Analytical and Testing Center at Huazhong University of Science and Technology for the spectroscopic data collection and the Instrumental Analysis Center of Shanghai Jiao Tong University for X-ray crystallographic analysis.

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REFERENCES

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Anti-inflammatory Grayanane Diterpenoids from the Leaves of

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