Grayanane Diterpenoids from the Leaves of Rhododendron

Jun 27, 2019 - Twenty-four grayanane diterpenoids (1–24) including 12 new ones (1–12) were isolated from Rhododendron auriculatum. The structures ...
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Cite This: J. Nat. Prod. XXXX, XXX, XXX−XXX

Grayanane Diterpenoids from the Leaves of Rhododendron auriculatum and Their Analgesic Activities Na Sun,† Guijuan Zheng,† Meijun He,‡ Yuanyuan Feng,† Junjun Liu,† Meicheng Wang,† Hanqi Zhang,† Junfei Zhou,† and Guangmin Yao*,† †

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Hubei Key Laboratory of Natural Medicinal Chemistry and Resource Evaluation, School of Pharmacy, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, People’s Republic of China ‡ Institute of Chinese Herbal Medicines, Hubei Academy of Agricultural Sciences, Enshi 445500, People’s Republic of China S Supporting Information *

ABSTRACT: Twenty-four grayanane diterpenoids (1−24) including 12 new ones (1−12) were isolated from Rhododendron auriculatum. The structures of the new grayanane diterpenoids (1−12) were defined via extensive spectroscopic data analysis. The absolute configurations of compounds 2−4, 10−12, 14, and 16 were established by single-crystal X-ray diffraction analysis, and electronic circular dichroism data were used to define the absolute configurations of auriculatols D (8) and E (9). Auriculatol A (1) is the first example of a 5,20-epoxygrayanane diterpenoid bearing a 7-oxabicyclo[4.2.1]nonane motif and a trans/cis/cis/cis-fused 5/5/7/6/5 pentacyclic ring system. Auriculatol B (2) is the first example of a 3α,5α-dihydroxy-1-βHgrayanane diterpenoid. 19-Hydroxy-3-epi-auriculatol B (6) and auriculatol C (7) represent the first examples of 19hydroxygrayanane and grayan-5(6)-ene diterpenoids, respectively. Diterpenoids 1−24 showed analgesic activities in the writhing test induced by HOAc, and 2, 6, 10, 13, 19, and 24 at a dose of 5.0 mg/kg exhibited significant analgesic effects (inhibition rates >50%). Grayanane diterpenoids grayanotoxins I (19) and IV (24) at doses of 0.2 and 0.04 mg/kg showed more potent analgesic activities than morphine.

T

Rhododendron auriculatum Hemsl., an Ericaceous evergreen small tree or shrub endemic to China, is mainly distributed in western Hubei, northern Guizhou, southern Shanxi, and eastern Sichuan of China. As a folk medicine, the bark and roots are used to treat cough.9 To date, there is no report of any diterpenoids from R. auriculatum, although seven known compounds including two triterpenoids and five phenolic compounds have been reported from the branches of R. auriculatum.9 In a search for new diterpenoids from Ericaceae plants,5,6,8,10−14 the leaves of R. auriculatum were collected and studied, and diterpenoids 1−24 were isolated. Among them, compounds 1−12 are new grayanane diterpenoids. Auriculatol A (1) represents the first 5,20-epoxygrayanane diterpenoid bearing a 7-oxabicyclo[4.2.1]nonane motif and a trans/cis/cis/cis-fused 5/5/7/6/5 pentacyclic ring system, and auriculatol B (2) is the first example of a

he Ericaceae family is one of the bigger families in the world and comprises 127 genera and 5583 species, which mainly grow in temperate and subarctic regions, as well as high elevations in tropical regions. Among them, Rhododendron L. is the largest genus of Ericaceae, and it is widely distributed in North America, Europe, and Asia.1 Many Rhododendron plants are used not only as ornamentals but also as folk medicines for the treatment of cold, cough, pain, and inflammation.2 Previous phytochemical studies revealed that triterpenoids, diterpenoids,3 megastigmane sesquiterpenoids, flavonoids, iridoid glycosides, and phenolic compounds are the main chemical constituents in Rhododendron species.2,4 In recent years, grayanane and related diterpenoids from Ericaceae received attention due to their novel structure and fascinating bioactivities such as anti-HIV,3 anti-inflammatory,5 antimicrobial,2 immunomodulatory,6 antinociceptive,7 and PTP1B inhibitory activities.8 © XXXX American Chemical Society and American Society of Pharmacognosy

Received: February 1, 2019

A

DOI: 10.1021/acs.jnatprod.9b00095 J. Nat. Prod. XXXX, XXX, XXX−XXX

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grayanotoxins I (19),20 II (23),21 IV (24),20 IX (18),22 and XVIII (21),23 6-deoxygrayanotxin XVII (20),6 and pieristoxin S (22),24 by NMR spectroscopic data analysis and comparison with literature data. Single-crystal X-ray diffraction analysis defined the structures of rhododecorumins X (14) (Figure 1) and VIII (16) (Figure 2) for the first time, and the calculated Flack parameters of −0.01(15) and −0.06(12)25 assigned their absolute configurations as (1S,2S,3R,5R,6R,8R,9R,13S,16R) and (1R,2R,3R,5R,6R,8R,9R,13S,16R), respectively. Auriculatol A (1) was obtained as a colorless oil. The 13C NMR data and the sodium adduct ion at m/z 405.2260 [M + Na]+ in the HRESIMS established the molecular formula of 1 as C21H34O6 (calcd for C21H34O6Na, 405.2253), indicating five indices of hydrogen deficiency. The 1H NMR data (Table 1) showed resonances for one acetal (δH 4.98, s, H-20), two oxymethines (δH 3.48, d, H-3; 3.70, dd, H-6), three methines (δH 2.61, dd, H-1; 2.08, s, H-10; 1.69, m, H-9), three methyls (δH 1.03, s, H3-19; 1.30, s, H3-18; 1.22, s, H3-17), and a methoxy group (δH 3.41, s, −OCH3). Twenty-one carbon resonances in the 13C NMR data (Table 2) were assigned to three oxygenated tertiary carbons (δC 78.8, C-13; 81.0, C-16; 103.9, C-5), two quaternary carbons (δC 39.9, C-8; 52.8, C-4), one acetal (δC 117.8, C-20), two oxymethines (δC 85.9, C-3; 74.0, C-6), three methines (δC 61.5, C-10; 49.2, C-9; 43.0, C-1), six methylenes (δC 61.5, C-15; 32.6, C-12; 41.3, C-2; 41.3, C-14; 48.1, C-7; 29.2, C-11), a methoxy group (δC 56.4, −OCH3), and three methyls (δC 24.8, C-19; 21.5, C-17; 20.5, C-18) by DEPT and HSQC spectra. Based upon the absence of olefinic and carbonyl functionalities, the five indices of hydrogen deficiency deduced from the molecular formula suggested that auriculatol A (1) contains five rings. The NMR data of auriculatol A (1) showed a similarity to those of pieristoxin S (22),24 and the major differences were the presence of a methoxy group (δH 3.41, s; δC 56.4, −OCH3), an acetal (δH 4.98, s, H-20; δC 117.8, C-20), and a methine (δH 2.08, s, H-10; δC 61.5, C-10) in auriculatol A (1), which replaced the exocyclic double bond (δH 5.08, 5.00, s, H220; δC 153.2, C-10, 113.9, C-20) in pieristoxin S (22). The HMBC correlations from the acetal H-20 (δH 4.98, s) to C-10 (δC 61.5), C-9 (δC 49.2), and C-1 (δC 43.0) defined the location of the acetal group at C-20. The chemical shifts of H-20 (δH 4.98), C-20 (δC 117.8), and C-5 (δC 103.9) indicated that there is an oxygen bridge between C-20 and C-5 in auriculatol A (1), forming the fifth ring as required by the molecular formula. The HMBC correlation of H-20 (δH 4.98) to C-5 (δC 103.9) proved this conclusion. HMBC correlations from the methoxy group (δH 3.41, s) to C-20 (δC 117.8) and from H-20 (δH 4.98, s) to the methoxy group (δC 56.4) revealed the location of the methoxy group at C-20. The 1H−1H COSY, HSQC, and HMBC data analysis (Figure 3) assigned the 2D structure of auriculatol A (1). The chemical shifts of H-1 (δH 2.61, dd) and C-1 (δC 43.0) revealed the α-orientation of H-1.6 NOESY correlations (Figure 3) from H-6 to H-1α/H3-19 and from H-3 to H3-19 established the α-orientations of H-3, H-6, and H3-19. The β-orientation of the 5,20-epoxy group and the α-orientation of the oxymethyl group were supported by the NOESY correlations between H20 and H-9 and between H-1 and H-10. The relative configurations of the remaining stereocenters of auriculatol A (1) are the same as in pieristoxin S (22). The cis-junction of rings C and D and the β-orientation of H3-17 were confirmed by the NOESY correlations between H-12β and H3-17, and the αorientation of HO-13 was assigned by the NOESY correlations between H-9β and H-15β and between H-12β and H3-17. Thus,

3α,5α-dihydroxy-1-βH-grayanane diterpenoid. 19-Hydroxy-3epi-auriculatol B (6) and auriculatol C (7) represent the first examples of 19-hydroxygrayanane and grayan-5(6)-ene diterpenoids, respectively.

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RESULTS AND DISCUSSION The concentrated 95% EtOH extract of the leaves of R. auriculatum was extracted with petroleum ether to remove the essential oils and then extracted with CHCl3. The CHCl3 fraction was fractionated and further separated by silica gel, Sephadex LH-20, reversed-phase C18, and finally HPLC to give 12 new grayanane diterpenoids (1−12) and 12 known grayanane analogues (13−24), which were identified as craiobiotoxin I (13),15 rhododecorumins X (14) and VIII (16),16 pierisformosin A (15),17,18 grayathol A (17),19 B

DOI: 10.1021/acs.jnatprod.9b00095 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Figure 1. ORTEP drawing of rhododecorumin X (14).

Figure 2. ORTEP drawing of rhododecorumin VIII (16).

micranthanoside A. Thus, the 2D structure of auriculatol B (2) is the same as the aglucone of micranthanoside A. 1H−1H COSY, HSQC, and HMBC data (Figure S1, Supporting Information) assigned the structure of auriculatol B (2). The chemical shift of C-1 (δC 52.1) revealed the β-orientation of H-1 in auriculatol B (2).6 The β-orientations of H-3, H3-18, and H-9 were supported by NOESY correlations (Figure S1, Supporting Information) from H-9/H3-18 to H-1β and from H-3 to H3-18. Finally, the structure of auriculatol B (2) (Figure 4) was defined as 3α,5α,16α-trihydroxy-1-βH-grayan-10(20)-ene by single-crystal X-ray diffraction, and the absolute configuration was assigned as (1R,3R,5S,8S,9R,13R,16R) by the calculated Flack parameter of 0.05(4).25 Auriculatol B (2) represents the first 3α,5αdihydroxy-1-βH-grayanane diterpenoid. 3-epi-Grayanotoxin XVIII (3) (colorless prisms, mp 192−193 °C (MeOH)) possesses the same molecular formula, C20H32O4 (calcd for C20H32O4Na, 359.2198; calcd for C40H64O8Na, 695.4499), as grayanotoxin XVIII (21),23 which was deduced from the HRESIMS ions at m/z359.2210 [M + Na]+ and 695.4523 [2 M + Na]+ as well as the 13C NMR data. The NMR data of 3-epi-grayanotoxin XVIII (3) were similar to those of grayanotoxin XVIII (21) (Tables 1 and 2),23 except that C-3 (δC 79.5) in 3 was shielded compared to 21 (δC 81.6), while H-3 (δH

the structure of auriculatol A (1) was defined as 3β,6β,13α,16αtetrahydroxy-5β,20β-epoxy-20α-methoxygrayanane. The grayananes are the major type of Ericaceae diterpenoids,3 while epoxygenated grayanane diterpenoids are relatively rare. To date, approximately 55 epoxygrayanane diterpenoids have been reported, including 28 2,3-epoxygrayananes,3,5,24 a 2,3:11,16-diepoxygrayanane,5 11 5,9-epoxygrayananes,3,5,26,27 five 6,10-epoxygrayananes,6,16,28 a 9,10-epoxygrayanane,27 a 1,7-epoxygrayanane,28 a 2,10-epoxygrayanane,29 a 15,16-epoxygrayanane,30 an 11,16-epoxygrayanane,31 and five 10,14epoxygrayananes.28 However, there is no report of 5,20epoxygrayanane diterpenoids. Thus, auriculatol A (1) represents the first 5,20-epoxygrayanane diterpenoid. Auriculatol B (2) was obtained as colorless prisms, mp 186− 187 °C (MeOH). Its molecular formula was assigned as C20H32O3 by the HRESIMS ion at m/z 343.2240 [M + Na]+ (calcd for C20H32O3Na, 343.2249) and the 13C NMR data. The NMR data of 2 (Tables 1 and 2) resembled those of the grayanane glucoside micranthanoside A,13 which was recently isolated from Rhododendron micranthum by our group, and their major differences were the absence of a β-D-glucopyranosyl unit and the shielding of C-3 (δC 82.0) and H-3 (δH 3.66, dd) in 2, compared to those [C-3 (δC 89.4) and H-3 (δH 4.15, dd)] in C

DOI: 10.1021/acs.jnatprod.9b00095 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Table 1. 1H NMR (400 MHz) [δ, mult. (J in Hz)] Spectroscopic Data for Compounds 1−6 1a

no. 1α 1β 2α 2β 3α 3β 6α

2a

3a

2.61, dd (12.0, 3.5) 2.71, ddd (14.8, 12.0, 6.7) 1.55, dd (14.8, 3.5)

2.77, t (10.0) 2.28, ddd (14.4, 10.0, 7.4) 1.69, ddd (14.4, 10.0, 2.7)

4b

3.07, dd (11.1, 8.0)

3.07, t (9.6)

1.70, ddd (13.9, 11.1, 7.2) 2.09, ddd (13.9, 9.4, 8.0)

2.68, ddd (14.5, 9.6, 7.1)

3.48, d (6.7)

2.15, ddd (14.5, 9.6, 1.5) 4.03, dd (7.1, 1.5)

3.70, dd (11.0, 3.6)

6β 7α

1.52, dd (14.0, 3.6)

7β

1.97, dd (14.0, 11.0)

9β 10 11α 11β 12α 12β 13α 14α 14β 15α 15β 17

1.69, m 2.08, s 1.60, m 1.69, m 1.67, m 1.85, m

3.66, dd (7.4, 2.7) 1.90, ddd (15.8, 6.2, 2.1) 1.32, ddd (15.8, 13.1, 2.4) 1.75, ddd (14.8, 13.1, 2.1) 1.53, ddd (14.8, 6.2, 2.4) 2.07, d (4.8)

4.07, dd (9.4, 7.2) 3.85, dd (10.5, 2.3)

1.77, m 1.85, m

5a

6a

2.98, t (10.1) 2.20, ddd (13.7, 10.1, 9.8) 1.53, ddd (13.7, 10.1, 4.8) 4.12, dd (9.8, 4.8)

2.93, t (10.1) 2.28, ddd (13.6, 10.1, 9.5)

1.30, ddd (15.8, 13.2, 2.4) 1.94, ddd (15.8, 6.4, 2.1) 1.70, ddd (14.2, 6.4, 2.4) 1.54, ddd (14.2, 13.2, 2.1) 2.08, d (5.9)

1.44, ddd (15.3, 13.2, 2.3)

1.54, ddd (13.6, 10.1, 3.9) 4.45, dd (9.5, 3.9)

2.11, ddd (15.3, 6.8, 1.8)

1.37, dd (14.2, 2.3)

1.65, m

1.74, (14.2, 6.8, 2.3)

2.01, dd (14.2, 10.5)

2.06, dd (13.0, 9.6)

2.43, t (7.2)

3.12, br s

1.77, d (11.6) 1.90, d (11.6) 1.68, d (14.5) 1.80, d (14.5) 1.22, s

1.58, m 1.78, m 1.73, m 1.81, m 1.86, m 1.57, d (10.0) 1.69, dd (10.0, 2.6) 1.88, d (14.5) 1.58, d (14.5) 1.36, s

1.58, m 1.44, m 1.70, m 1.55, m 1.92, m 1.77, d (11.8) 1.87, d (11.8, 3.5) 1.69, d (14.5) 1.81, d (14.5) 1.34, s

1.73, m 1.81, m 1.78, m 1.58, m 1.86, m 1.55, d (10.0) 1.77, dd (10.0, 2.6) 1.59, d (14.4) 1.88, d (14.4) 1.34, s

1.73, m 1.81, m 1.53, m 1.63 m 1.85, m 1.56, d (11.1) 1.68, dd (11.1, 4.6) 1.59, d (13.4) 1.87, d (13.4) 1.34, s

18 19

1.30, s 1.03, s

0.87, s 0.96, s

1.12, s 0.97, s

1.71, overlap 1.78, overlap 1.76, overlap 1.87, overlap 2.53, m 1.78, d (11.0) 2.27, dd (11.0, 4.6) 1.94, d (14.0) 2.15, d (14.0) 4.17, d (10.9); 4.05, d (10.9) 1.31, s 0.87, s

0.86, s 0.97, s

20 21

4.98, s 3.41, s

5.23, s; 5.11, s

5.04, s; 4.87, s

5.14, s; 5.11, s

5.21, s; 5.09, s

0.85, s 3.63, d (11.3); 3.56, d (11.3) 5.20, s; 5.08, s

1.54, (14.2, 13.2, 1.8) 2.09, d (6.4)

a

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

Table 2. 13C NMR (100 MHz) Spectroscopic Data for Compounds 1−12 no.

1a

2a

3a

4b

5a

6a

7a

8a

9a

10a

11a

12a

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

43.0 41.3 85.9 52.8 103.9 74.0 48.1 39.9 49.2 61.5 29.2 32.6 78.8 41.3 61.5 81.0 21.5 20.5 24.8 117.8 56.4

52.1 38.0 82.0 51.2 83.9 32.7 40.7 48.1 55.4 151.0 26.7 25.4 49.9 36.8 58.9 81.6 24.2 24.6 17.4 111.9

42.9 37.4 79.5 51.3 82.7 72.4 46.5 45.0 56.0 153.6 26.7 24.4 48.1 35.9 63.4 81.4 25.5 22.0 18.2 113.2

47.4 39.2 80.6 50.5 84.0 31.7 38.2 46.6 50.0 154.3 25.0 24.5 45.6 37.6 56.1 83.8 66.6 17.8 24.3 111.7

51.3 36.2 80.2 51.6 83.7 33.5 36.9 48.1 55.4 151.8 25.4 26.8 49.8 40.8 58.9 81.6 24.3 19.1 20.5 111.5

52.2 36.6 74.7 54.6 85.4 34.2 40.6 48.1 55.4 150.7 25.4 26.8 49.8 36.9 58.8 81.6 24.3 14.7 66.9 111.9

52.9 34.4 81.2 46.9 155.5 122.0 43.5 48.2 61.0 76.3 22.0 27.4 49.8 37.1 59.9 80.5 24.1 24.8 27.3 21.7

138.0 43.4 79.9 51.7 140.1 119.9 143.5 51.6 47.4 150.6 25.7 27.0 51.1 41.6 57.6 81.7 24.0 25.5 21.1 114.5

133.0 37.9 79.1 47.9 64.5 214.9 52.6 49.4 149.9 131.3 122.3 31.8 48.4 41.1 59.8 82.2 27.7 15.7 26.1 23.9

45.7 37.2 81.7 49.9 85.9 69.5 47.1 46.4 123.8 138.5 27.8 35.7 80.0 53.8 55.4 80.1 21.2 17.7 24.7 20.6

52.8 82.2 87.8 47.6 85.1 69.1 46.9 46.3 122.7 138.7 27.5 35.9 80.0 53.1 56.9 80.2 21.1 17.8 25.1 18.6

59.3 36.9 84.9 51.6 87.5 71.8 48.7 42.5 55.2 154.0 28.6 34.2 78.4 41.7 55.3 81.8 21.2 20.6 23.0 111.6

a

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

D

DOI: 10.1021/acs.jnatprod.9b00095 J. Nat. Prod. XXXX, XXX, XXX−XXX

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and the NOESY analysis (Figure S3, Supporting Information) established its relative configuration. Finally, the structure of 6deoxycraiobiotoxin I (4) was defined as 3β,5β,16α,17tetrahydroxygrayan-10(20)-ene by single-crystal X-ray diffraction (Figure 6), and the calculated Flack parameter of 0.01(5)25 permitted assignment of the (1S,3S,5R,8S,9R,13R,16R) absolute configuration of 6-deoxycraiobiotoxin I (4). 6-Deoxycraiobiotoxin I (4) represents the second example of a 17hydroxygrayanane diterpenoid.15 A molecular formula of C20H32O3 (calcd for C20H32O3Na, 343.2249) was suggested for 3-epi-auriculatol B (5) on the basis of the HRESIMS ion at m/z 343.2251 [M + Na]+ and 13C NMR data, which is the same as that of auriculatol B (2). The NMR data of 3-epi-auriculatol B (5) resembled those of auriculatol B (2) (Tables 1 and 2), except for the shielding of C-3 (δC 80.2) and the deshielding of H-3 (δH 4.12) in 5 compared to those (δH 3.66, δC 82.0) in 2. Thus, 3-epi-auriculatol B (5) is the 3-epimer of auriculatol B (2). The HSQC, 1H−1H COSY, and HMBC data (Figure S4, Supporting Information) supported this conclusion. The β-orientation of H-1 in 3-epi-auriculatol B (5) was supported by the chemical shift of C-1 (δC 51.3), which is similar to 5α-OH-1-βH-grayanane diterpenoids,13 and the βorientation of H-9 was assigned by the NOESY correlations between H-9 and H-1β. The orientation of H-3α was indicated by the strong NOESY correlation between H-3 and H3-19α as well as the weak NOESY correlation between H-3 and H3-18β. Comparison of their NMR data revealed the same configurations of 5 and 2, except for C-3. Therefore, the structure of 5 was defined as 3β,5α,16α-trihydroxy-1-βH-grayan-10(20)-ene. The 13C NMR data and the HRESIMS ion at m/z 359.2211 [M + Na]+ indicated a molecular formula of C20H32O4 (calcd for C20H32O4Na, 359.2198) for 19-hydroxy-3-epi-auriculatol B (6), having one more oxygen atom than 3-epi-auriculatol B (5). The NMR data of 19-hydroxy-3-epi-auriculatol B (6) were similar to those of 3-epi-auriculatol B (5) (Tables 1 and 2), except for an oxygenated methylene (δC 66.9, C-19; δH 3.63, d; 3.56, d, H219) in 6, which replaced the methyl group (δH 0.97, s, H3-19; δC 20.5, C-19) in 5. Thus, 19-hydroxy-3-epi-auriculatol B (6) is the 19-hydoxy derivative of 3-epi-auriculatol B (5). HMBC correlations from H2-19 (δH 3.63, d; 3.56, d) to C-18 (δC 14.7), C-4 (δC 54.6), and C-3 (δC 74.7) and from H-3 (δH 4.45)/H3-18 (δH 0.85) to C-19 (δC 66.9) supported this conclusion. The β-orientation of H-1 in 19-hydroxy-3-epiauriculatol B (6) was supported by the chemical shift of C-1 (δC 52.2), which is similar to 5α-OH-1-βH-grayanane diterpenoids.13 NOESY correlations (Figure S5, Supporting Information) from H-9/H3-18 to H-1β assigned the β-orientations of

Figure 3. 1H−1H COSY, key HMBC, and NOESY correlations of auriculatol A (1).

4.07, dd) in 3 was deshielded compared to 21 (δH 3.79, dd). Thus, compound 3 is the 3-epimer of grayanotoxin XVIII (21). The chemical shifts of C-1 (δC 42.9) and H-1 (δH 3.07, dd) indicated the α-orientation of H-1 in 3-epi-grayanotoxin XVIII (3).6 NOESY correlations (Figure S2, Supporting Information) from H-6/H3-19 to H-1α established the α-orientations of H319 and H-6. The weak NOESY correlation between H-3 and H319α as well as the strong NOESY correlation between H-3 and H3-18β suggested the β-orientation of H-3. Finally, the structure of 3-epi-grayanotoxin XVIII (3) was defined as 3α,5β,6β,16αtetrahydroxygrayan-10(20)-ene by single-crystal X-ray diffraction (Figure 5), and the resulting Flack parameter of 0.05(6)25 permitted assignment of its absolute configuration as (1S,3R,5R,6R,8R,9R,13R,16R). To date, only two 3α-oxygrayanane diterpenoids, pierisformosid E from Pieris formosa27 and 3-epi-grayanoside B from Rhododendron micranthum,13 have been reported. Auriculatol B (2) and 3-epi-grayanotoxin XVIII (3) are the third and fourth 3α-oxygrayanane diterpenoids. 13 C NMR data and the HRESIMS ions at m/z 695.4502 [2 M + Na]+ and 359.2210 [M + Na]+ assigned the molecular formula of 6-deoxycraiobiotoxin I (4) as C20H32O 4 (calcd for C20H32O4Na, 359.2198, C40H64O8Na, 695.4499), possessing one less oxygen atom than craiobiotoxin I (13).15 The NMR data of 6-deoxycraiobiotoxin I (4) and craiobiotoxin I (13) were highly similar (Tables 1 and 2), except for the shielding of C-6 (δC 31.7) in 4 compared to craiobiotoxin I (13) (δC 72.5). Therefore, compound 4 is the 6-deoxy derivative of craiobiotoxin I (13). This deduction was supported by the 1 H−1H COSY correlations of H2-6 (δH 1.85, 1.77) to H2-7 (δH 2.06, 1.65) as well as HMBC correlations of H2-6 to C-8 (δC 46.6), C-5 (δC 84.0), and C-1 (δC 47.4) (Figure S3, Supporting Information). Similar to 3, the chemical shift of C-1 (δC 47.4) in 6-deoxycraiobiotoxin I (4) supported the α-orientation of H-1,6

Figure 4. ORTEP drawing of auriculatol B (2). E

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Figure 5. ORTEP drawing of 3-epi-grayanotoxin XVIII (3).

Figure 6. ORTEP drawing of 6-deoxycraiobiotoxin I (4).

H3-18 and H-9. Correspondingly, H2-19 is α-oriented. The weak NOESY correlation of H3-18β to H-3 as well as the strong NOESY correlation from H-3 to H2-19α suggested that H-3 was α-oriented. Thus, the structure of 19-hydroxy-3-epi-auriculatol B (6) was defined as 3β,5α,16α,19α-tetrahydroxy-1-βH-grayan10(20)-ene. 19-Hydroxy-3-epi-auriculatol B (6) represents the first example of a 19-hydroxygrayanane diterpenoid. Auriculatol C (7) was suggested to have a molecular formula of C20H32O3 (calcd for C20H32O3Na, 343.2249) by the 13C NMR data and HRESIMS ion at m/z 343.2209 [M + Na]+. The NMR data of auriculatol C (7) had similarities to those of grayanotoxin I (19) (Tables 2 and 3),20 except for the presence of a trisubstituted double bond (δC 155.5, C-5; 122.0, C-6; δH 5.61, ddd, J = 8.6, 4.4, 2.5 Hz, H-6) in 7, which replaced the oxygenated tertiary carbon (δC 85.1, C-5) and oxymethine (δC 74.3, C-6; δH 3.56, d, H-6) in 19. In addition, a methylene group (δC 37.1, C-14; δH 1.69, dd; 1.51, d, J = 11.2 Hz, H2-14) in 7 replaced the oxymethine (δC 83.8, C-14; δH 5.46, s, H-14) and acetoxy group (δC 21.5, 172.6, AcO-14; δH 2.10) in grayanotoxin I (19). 1H−1H COSY, HSQC, and HMBC data defined the structure of auriculatol C (7) (Figure S6, Supporting Information). The β-orientation of H-1 in auriculatol C (7) was supported by the large chemical shift of C-1 (δC 52.9).6 The β-orientations of H3-18 and H-9 were assigned by the NOESY correlations from H-9 (δH 1.53)/H3-18 (δH 0.94) to H-1β (δH

2.93, dd). The α-orientation of H-3 was defined by the strong NOESY correlation between H3-19α and H-3 (Figure S6, Supporting Information). Auriculatol C (7) represents the first grayan-5(6)-ene diterpenoid. On the basis of the HRESIMS ion at m/z 323.1965 [M + Na]+ and 13C NMR data, auriculatol D (8) was suggested to have a molecular formula of C20H28O2 (calcd for C20H28O2Na, 323.1987). NMR data analysis indicated that auriculatol D (8) (Tables 2 and 3) resembled the known compound pierisformoside C,32 and the major differences were the absence of a β-Dglucopyranosyl unit and the shielding of C-3 (δC 79.9) in auriculatol D (8) compared to that (δC 87.4) in pierisformoside C. Thus, auriculatol H (8) should be the aglucone of pierisformoside C. HSQC, 1H−1H COSY, NOESY, and HMBC data analysis defined the structure of auriculatol D (8) as 3β,16α-dihydroxygrayan-1(5),6(7),10(20)-triene. The electronic circular dichroism (ECD) spectra of (3S,8S,9R,13R,16R)8 and its enantiomer were calculated as described previously.14 As shown in Figure 7, the measured ECD spectrum of auriculatol D (8) is opposite of the calculated ECD spectrum of (3R,8R,9S,13S,16S)-8 and fits well with that of (3S,8S,9R,13R,16R)-8. Therefore, the absolute configuration of 8 was defined as (3S,8S,9R,13R,16R). Auriculatol D (8) is the second grayan-1(5),6(7),10(20)-triene diterpenoid. F

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Table 3. 1H NMR [δ, mult. (J in Hz)] Spectroscopic Data for Compounds 7−12 in Methanol-d4 (400 MHz) no. 1α 1β 2α 2β 3α 5α 6α 7α 7β 9β 11α 11β 12α 12β 13α 14α 14β 15α 15β 17 18 19 20

7

8

9

10 3.34, t (9.5)

2.93, dd (10.1, 4.6) 1.74, ddd (14.0, 10.1, 3.06, dd (16.6, 6.6) 6.3) 2.26, ddd (14.0, 6.6, 4.6) 2.22, dd (16.6, 3.8) 3.83, t (6.6) 3.82, dd (6.3, 3.8) 5.61, ddd (8.6, 4.4, 2.5) 2.40, dd (14.1, 4.4) 2.00, dd (14.1, 8.6) 1.53, m 1.51, m 1.59, m 1.58, m

5.55, d (12.2) 5.49, d (12.2)

1.52, m 1.82, m 1.51, d (11.2) 1.69, dd (11.2, 4.5) 1.57, d (15.4) 1.73, d (15.4) 1.32, s 0.94, s 1.05, s 1.09, s

1.59, m 1.93, m 1.47, d (10.8) 1.96, br d (10.8) 1.90, d (15.4) 1.95, d (15.4) 1.38, s 0.97, s 1.00, s 5.33, s; 5.22, s

2.64, br s 1.81, m 1.88, m 1.83, m

11

12

3.14, d (8.9) 2.78, t (9.3) 2.24, ddd (14.4, 9.3, 4.5) 1.85, ddd (14.4, 9.3, 4.5) 3.70, d (4.5)

2.66, dd (16.0, 7.9)

2.28, ddd (14.0, 9.5, 6.6)

2.38, dd (16.0, 10.3) 3.69, dd (10.3, 7.9) 2.65, br s

2.01, ddd (14.0, 9.5, 3.4)

4.26, dd (8.9, 3.6)

3.57, dd (6.6, 3.4)

3.38, d (3.6)

3.96, dd (4.3, 2.7) 2.25, dd (15.2, 2.7) 1.77, dd (15.2, 4.3)

3.93, dd (5.0, 1.6) 2.31, dd (15.4, 1.6) 1.83, dd (15.4, 5.0)

2.74, ddd (15.4, 13.0, 7.2) 2.02, ddd (15.4, 9.5, 3.2) 1.70, dddd (13.0, 9.5, 3.2, 3.0) 1.53, ddd (13.0, 13.0, 7.2)

2.73, ddd (15.4, 12.9, 6.5) 2.07, ddd (15.4, 13.0, 6.0) 1.72, dddd (12.9, 13.0, 6.0, 3.0) 1.53, ddd (12.9, 12.9, 6.5)

1.33, d (10.5) 2.08, dd (10.5, 3.0) 2.06, d (15.4) 2.11, d (15.4) 1.20, s 0.99, s 0.88, s 1.73, s

1.38, d (10.5) 2.11, dd (10.5, 3.0) 2.16, d (15.4) 1.96, d (15.4) 1.20, s 0.90, s 0.99, s 1.85, s

1.90, d (11.0) 3.36, d (11.0) 5.25, t (3.6) 2.21, m 2.21, m 2.09, m 2.07, d (10.1) 1.61, dd (10.1, 2.6) 1.63, d (13.1) 1.98, dd (13.1, 2.6) 1.34, s 0.76, s 1.10, s 1.91, s

3.54, d (10.1) 1.27, d (14.0) 2.63, dd (14.0, 10.1) 1.97, d (6.7) 1.70, m 1.85, m 1.82, m 1.68, m 1.77, d (11.0) 1.83, d (11.0) 1.74, d (15.4) 1.70, d (15.4) 1.19, s 1.20, s 0.95, s 5.21, s; 5.19, s

Figure 7. Experimental and calculated ECD spectra of compounds 8 and 9 and their enantiomers in MeOH.

Auriculatol E (9) possesses a molecular formula of C20H28O3 (calcd for C20H28O3Na, 339.1936) as indicated by the 13C NMR data and the HRESIMS [M + Na]+ ion at m/z 339.1958. The NMR data of auriculatol E (9) were similar to those of rhodomicranoside F (Tables 3 and 2), which was isolated from Rhododendron micranthum,14 except for the absence of a β-Dglucopyranosyl unit and the shielding of C-3 (δC 79.1) in 9 compared to that (δC 87.8) in rhodomicranoside F. Thus, auriculatol I (9) should be the aglucone of rhodomicranoside F. 2D NMR data (Figure S8, Supporting Information) established the structure of auriculatol E (9) as 3β,16α-dihydroxy-5-αHgrayan-1(10)-9(11)-dien-6-one. The calculated ECD spectrum of (3S,5R,8S,13R,16R)-9 matched the experimental ECD spectrum of auriculatol I (9), assigning the absolute configuration of auriculatol E (9) as (3S,5R,8S,13R,16R) (Figure 7). Auriculatol E (9) is the third example of a 5-αHgrayan-1(10),9(11)-dien-6-one diterpenoid.

Auriculatol F (10) was obtained as colorless prisms (MeOH), mp 152−153 °C. Its molecular formula was established as C20H32O5 (calcd for C20H32O5Na, 375.2147) by the 13C NMR data and HRESIMS ion at m/z 375.2158 [M + Na]+. The NMR data of auriculatol F (10) (Tables 2 and 3) resembled those of pieristoxin S (22),24 and the major difference was the presence of a tetrasubstituted endocyclic double bond (δC 123.8, C-9; 138.5, C-10) in auriculatol F (10) replacing the exocyclic double bond (δC 153.2, C-10; 113.9, C-20; δH 5.08, 5.00, s, H2-20) in pieristoxin S (22). The HMBC correlations (Figure S9, Supporting Information) of H3-20 (δH 1.73, s) to C-10 (δC 138.5), C-9 (δC 123.8), and C-1 (δC 45.7) supported the location of the Δ9(10) tetrasubstituted double bond. The structure of auriculatol F (10) (Figure 8) was defined as 3β,5β,6β,13α,16α-pentahydroxygrayan-9(10)-ene by singlecrystal X-ray diffraction, and the Flack parameter of 0.04(4)25 G

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Figure 8. ORTEP drawing of auriculatol F (10).

Figure 9. ORTEP drawing of 2α-hydroxyauriculatol F (11).

Figure 10. ORTEP drawing of 1-epi-pieristoxin S (12).

indicating one more oxygen atom than auriculatol F (10). The NMR data of 2α-hydroxyauriculatol F (11) were similar to those of auriculatol F (10) (Tables 2 and 3), except for an oxygenated methine (δH 4.26, dd, H-2; δC 82.2, C-2) in 11 that replaced the methylene (δH 2.28, ddd, 2.01, ddd, H2-2; δC 37.2, C-2) in 10.

permitted assignment of the (1S,3S,5R,6R,8R,13S,16R) absolute configuration. The 13C NMR data and the HRESIMS ion at m/z 391.2079 [M + Na]+ suggested a molecular formula of C20H32O6 (calcd for C20H32O6Na, 391.2097) for 2α-hydroxyauriculatol F (11), H

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Figure 11. Antinociceptive activities of compounds 1−24 in the acetic acid-induced writhing test. Data represent the mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001, vs vehicle (veh). Morphine (morph) was used as a positive control.

Grayanotoxin IV (24) with AcO-14 exhibited a more significant analgesic effect than 20−23 without AcO-14, suggesting that AcO-14 is the activating group in the grayan-10(20)-ene diterpenoids. The more significant analgesic effect of grayanotoxin IX (18) with AcO-14 than grayathol A (17) without AcO-14 indicated the importance of the AcO-14 group in the grayan-10(20),15(16)-diene. The best analgesic effects of grayanotoxin I (19) with AcO-14 further supported that AcO-14 was an activating group. 6-Deoxycraiobiotoxin I (4) and craiobiotoxin I (13), bearing HO-17, exhibited more significant analgesic effects than 6-deoxygrayanotxin XVII (20) and grayanotoxin XVIII (21) without HO-17, implying that HO17 is an activating group in the grayanane diterpenoids. The conclusion that HO-19 is also an activating group could be drawn from the more significant analgesic effect of auriculatol F (6) with HO-19 than auriculatol E (5) without HO-19. The orientation of the 3-hydroxy group in the 5α-OH-1-βH-grayan10(20)-ene-type diterpenoids may play an important role in the analgesic activity because auriculatol B (2) with 3α-OH showed stronger activity than auriculatol E (5) with 3β-OH. Compared to 22, the 5,20-epoxy and the methoxy group in 1 slightly increased the analgesic effect. This preliminary structure− activity relationship may give some hints for the design of novel analgesic agents based on grayanane skeleton diterpenoids.

Thus, 2α-hydroxyauriculatol F (11) is a 2-hydroxy derivative of auriculatol F (10). HMBC correlations from H-2 (δH 4.26, dd) to C-3 (δC 87.8) and C-1 (δC 52.8) as well as 1H−1H COSY correlations from H-2 (δH 4.26, dd) to H-1 (δH 3.14, d) and H-3 (δH 3.38, d) in 2α-hydroxyauriculatol F (11) (Figure S10, Supporting Information) supported this conclusion. The structure of 2α-hydroxyauriculatol F (11) was defined as 2α,3β,5β,6β,13α,16α-hexahydroxygrayan-9(10)-ene by singlecrystal X-ray diffraction (Figure 9), and the resulting Flack parameter of 0.08(12) permitted assignment of the (1R,2R,3R,5R,6R,8R,13S,16R) absolute configuration.25 1-epi-Pieristoxin S (12) had the same molecular formula of C20H32O5 (calcd for C20H32O5Na, 375.2147) as pieristoxin S (22),24 which was defined by the 13C NMR data and HRESIMS ion at m/z 375.2156 [M + Na]+. The NMR data of 1-epipieristoxin S (12) were also similar to those of pieristoxin S (22) (Tables 3 and 2), and the major difference was that C-1 (δC 59.3) in 12 was deshielded compared to 22 (δC 45.4). Thus, 1epi-pieristoxin S (12) is the 1-epimer of pieristoxin S (22). The chemical shift of C-1 in 12 (δC 59.3) indicated the β-orientation of H-1,6 and NOESY data analysis (Figure S11, Supporting Information) supported this conclusion. Finally, the structure of 1-epi-pieristoxin S (12) was defined as 3β,5β,6β,13α,16αpentahydroxy-1-βH-grayan-10(20)-ene by single-crystal X-ray diffraction (Figure 10), and a Flack parameter of 0.09(6)25 permitted assignment of the (1R,3S,5R,6R,8R,9R,13S,16R) absolute configuration. Grayanane diterpenoids 1−24 were evaluated for their analgesic activities using a writhing test induced by HOAc.7,13,14,24,27 The results revealed that compounds 1−24 showed analgesic activities (Figure 11). Compounds 1, 4, 7, 11, 12, and 23 expressed moderate analgesic effects at 5.0 mg/kg, and 2, 6, 10, and 13 at a dose of 5.0 mg/kg exhibited significant analgesic activities (p < 0.001) with inhibition rates of 64.1%, 58.8%, 52.3%, and 53.7%, respectively. Owing to the limited amount of material, auriculatol D (8) and grayanotoxin IX (18) were evaluated for their analgesic activities at a lower dose of 1.0 mg/kg, and both also showed moderate analgesic activities. More importantly, grayanotoxins I (19) and IV (24) showed more potent analgesic activities at a low dose of 0.2 mg/kg than morphine, the positive control, and showed significant analgesic effects at an even lower dose of 0.04 mg/kg, which was consistent with the results recently reported by our group.14 Analysis of the structures and analgesic activity relationships could allow some preliminary conclusions to be drawn.

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EXPERIMENTAL SECTION

General Experimental Procedures. The NMR, single-crystal Xray diffraction, melting points, optical rotations, HRESIMS, UV, IR, and ECD data were recorded using the same instruments listed in a previously published paper.13 Compounds were purified using a Dionex P680 HPLC over a Welch Ultimate XB-C18 HPLC column (5 μm, 10 × 250 mm) at a rate of 1.5 mL/min. Plant Material. The leaves of R. auriculatum Hemsl. were collected in June 2013 at Enshi, Hubei Province, People’s Republic of China, and identified by Prof. Yansong Peng at Lushan Botanical Garden, Jiangxi Province and Chinese Academy of Sciences. A voucher specimen (No. 20130630) was deposited at School of Pharmacy, Tongji Medical College, Huazhong University of Science and Technology. Extraction and Isolation. The leaves of R. auriculatum (50.4 kg) were air-dried, powdered, and extracted with 95% EtOH three times. The concentrated 95% EtOH extract (8500 g) was suspended in H2O and extracted with petroleum ether to remove the essential oils and then extracted with CHCl3. The CHCl3 extract (2100 g) was separated on a MCI column eluting with 70% MeOH to yield a 70% MeOH fraction (460 g), from which eight fractions, A−H, were obtained by a silica gel CC (CHCl3−MeOH, 15:1 to 3:1). Three subfractions B1−B3 were obtained from fraction B by a Sephadex LH-20 column (100% MeOH). I

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Auriculatol D (8): pale yellow oil; [α]20D +78 (c 0.1, MeOH); ECD (MeOH) 221 (Δε, −7.39); 295 (Δε, +6.44) nm; UV (MeOH) λmax (log ε) 210 (3.4), 297 (3.1) nm; IR (KBr) νmax 3351, 2928, 2864, 1755, 1590,1455, 1383, 1129, 1065, 888 cm−1; 1H and 13C NMR data, Tables 3 and 2; HRESIMS m/z 323.1965 [M + Na]+ (calcd for C20H28O2Na, 323.1987). Auriculatol E (9): pale yellow oil; [α]20D +21 (c 0.2, MeOH); ECD (MeOH) 242 (Δε, +5.44); 335(Δε, +0.39) nm; UV (MeOH) λmax (log ε) 202 (3.9), 232 (3.8) nm; IR (KBr) νmax 3380, 2933, 2879, 1708, 1655,1382, 1077, 1040 cm−1; 1H and 13C NMR data, Tables 3 and 2; HRESIMS m/z 339.1958. Auriculatol F (10): colorless prisms, mp 152−153 °C (MeOH); [α]20D +59 (c 0.2, MeOH); 1H and 13C NMR data, Tables 3 and 2; HRESIMS m/z 375.2158 (calcd for C20H32O5Na, 375.2147). 2α-Hydroxyauriculatol F (11): colorless prisms, mp 234−235 °C (MeOH); [α]20D +76 (c 1.0, MeOH); 1H and 13C NMR data, Tables 3 and 2; HRESIMS m/z 391.2079 (calcd for C20H32O6Na, 391.2097). 1-epi-Pieristoxin S (12): colorless prisms, mp 199−200 °C (MeOH); [α]20D +21 (c 0.2, MeOH); 1H and 13C NMR data, Tables 3 and 2; HRESIMS m/z 375.2156 (calcd for C20H32O5Na, 375.2147). Single-Crystal X-ray Diffraction Analysis. The crystallographic data for compounds 2−4, 10−12, 14, and 16 (CCDC 1894991− 1894998) were collected by a Bruker SMART APEX-II CCD diffractometer, and the structures were refined and solved by the procedures as previously described.33 Crystallographic data of auriculatol B (2): C20H32O3, M = 320.45, T = 296(2) K, V = 3503.9(12) Å3, Z = 8, calculated density 1.215 mg/ m3, orthorhombic, P212121, α = β = γ = 90°, a = 12.043(2) Å, b = 12.546(3) Å, c = 23.192(5) Å, F(000) = 1408, F2 (goodness-of-fit) = 1.050, 3.812° < θ < 64.966°, −12 ≤ h ≤ 13, −27 ≤ l ≤ 26, −14 ≤ k ≤ 14, data/restraints/parameters 5896/0/428, 23 903 reflections collected/ 5896 reflections unique [R(int) = 0.0284], R indices (all data) R1 = 0.0323 (wR2 = 0.0970), final R indices R1 = 0.0318 (wR2 = 0.0959) [I > 2σ(I)], Flack parameter 0.05(4). Crystallographic data of 3-epi-grayanotoxin XVIII (3): C20H32O4, M = 336.45, T = 100(10) K, V = 1773.41(3) Å3, Z = 4, calculated density 1.260 mg/m3, orthorhombic, P212121, α = β = γ = 90°, a = 7.18422(8) Å, b = 9.88369(10) Å, c = 24.9753(3) Å, F(000) = 736, F2 (goodness-of-fit) = 1.077, 3.539° < θ < 73.508°, −5 ≤ h ≤ 8, −11 ≤ k ≤ 9, −25 ≤ l ≤ 30, data/restraints/parameters 3473/0/235, 7665 reflections collected/3439 reflections unique [R(int) = 0.0311], R indices (all data) R1 = 0.0316 (wR2 = 0.0789), final R indices R1 = 0.0786 (wR2 = 0.0789) [I > 2σ(I)], Flack parameter 0.05(6). Crystallographic data of 6-deoxycraiobiotoxin I (4): C20H32O4, M = 336.46, T = 100(10) K, V = 1743.285(19) Å3, Z = 4, calculated density 1.282 mg/m3, orthorhombic, P212121, α = β = γ = 90°, a = 6.19529(4) Å, b = 12.69397(8) Å, c = 22.16712(14) Å, F(000) = 736, F2 (goodness-of-fit) = 1.017, 3.9780° < θ < 73.5650°, −4 ≤ h ≤ 7, −15 ≤ k ≤ 15, −26 ≤ l ≤ 27, data/restraints/parameters 3428/0/231, 7630 reflections collected/3343 reflections independent [R(int) = 0.0264], R indices (all data) R1 = 0.0273 (wR2 = 0.0695), final R indices R1 = 0.0264 (wR2 = 0.0690) [I > 2σ(I)], Flack parameter 0.01(5). Crystallographic data of auriculatol F (10): C20H32O5, M = 352.45, T = 100(10) K, V = 1732.12(3) Å3, Z = 4, calculated density 1.352 mg/ m3, orthorhombic, P212121, α = β = γ = 90°, a = 7.46660(10) Å, b = 9.33390(10) Å, c = 24.8537(2) Å, F(000) = 768, F2 (goodness-of-fit) = 1.048, 3.5590° < θ < 73.5390°, −9 ≤ h ≤ 8, −11 ≤ k ≤ 11, − 28 ≤ l ≤ 30, data/restraints/parameters 3434/0/262, 16 932 reflections collected/3332 reflections unique [R(int) = 0.0265], R indices (all data) R1 = 0.0275 (wR2 = 0.0695), final R indices R1 = 0.0265 (wR2 = 0.0690) [I > 2σ(I)], Flack parameter 0.04(4). Crystallographic data of 2α-hydroxyauriculatol F (11): C20H32O6, M = 368.46, T = 173(2) K, V = 1835.37(9) Å3, Z = 4, calculated density 1.333 mg/m3, orthorhombic, P212121, α = β = γ = 90°, a = 7.0243(2) Å, b = 10.7505(3) Å, c = 24.3048(7) Å, F(000) = 814, F2 (goodness-of-fit) = 1.064, 3.637° < θ < 66.660°, −8 ≤ h ≤ 6, −12 ≤ k ≤ 11, −22 ≤ l ≤ 28, data/restraints/parameters 3084/0/245, 7404 reflections collected/ 3084 reflections independent [R(int) = 0.0377], R indices (all data) R1 = 0.0425 (wR2 = 0.0879), final R indices R1 = 0.0371 (wR2 = 0.0847) [I > 2σ(I)], Flack parameter 0.08(12).

Five subfractions, B1a−B1e, were obtained from fraction B1 by an RP C18 CC (MeOH−H2O, from 10:90 to 100:0). Subfraction B1b was purified by RP HPLC (50% CH3CN−H2O) to give auriculatol B (2, tR = 30.7 min, 5.1 mg). 3-epi-Auriculatol B (5, tR = 29.5 min, 18.2 mg) was obtained from fraction B1c by RP HPLC (50% CH3CN), and 3-epigrayanotoxin XVIII (3, tR = 29.5 min, 14.3 mg) was yielded from fraction B1d by an RP HPLC (45% MeOH). Four subfractions, C1− C4, were obtained from fraction C by an RP C18 CC (MeOH−H2O, from 10:90 to 60:40). Three subfractions, C1a−C1c, were obtained from fraction C1 by a Sephadex LH-20 column (100% MeOH). Auriculatol E (9, tR = 29.8 min, 4.4 mg) was isolated from fraction C1a by an RP HPLC (45% CH3CN). Similarly, grayanotoxins IX (18, tR = 39.5 min, 5.0 mg) and 6-deoxygrayanotxin XVII (20, tR = 33.6 min, 8.2 mg) were isolated from fractions C1b and C1c, respectively, by an RP HPLC (48% and 45% CH3CN, respectively). Two subfractions, C2a and C2c, were obtained from fraction C2 by a silica gel CC. Fraction C2a was purified by a Sephadex LH-20 column (MeOH) to give pieristoxin S (22, 30.5 mg). Grayanotoxin I (19, tR = 26.2 min, 4.2 mg) was obtained by an RP HPLC (20% CH3CN) from fraction C2c. Fraction C2 was purified by a Sephadex LH-20 column (100% MeOH) and then an RP HPLC (65% CH3CN−H2O) to afford grayathol A (17, tR = 28.0 min, 4.0 mg). Three subfractions, D1−D3, were obtained from fraction D by a silica gel column (CHCl3−MeOH, 30:1−5:1) and then a Sephadex LH-20 column (100% MeOH). Fraction D1 was purified by an RP C18 CC (MeOH−H2O, from 10:90 to 60:40) to give grayanotoxin XVIII (21, 100.2 mg). Fraction D2 was purified by an RP HPLC (28% CH3CN) to yield grayanotoxins II (23, tR = 37.0 min, 10.6 mg) and IV (24, tR = 26.8 min, 4.8 mg). Subfractions E1 and E2 were obtained from fraction E by a Sephadex LH-20 column (100% MeOH), and auriculatol F (10, tR = 34.2 min, 11.2 mg) was isolated from fraction E1 by an RP HPLC (52% MeOH). Subfractions E2a− E2c were obtained from fraction E2 by a silica gel CC, pierisformosin A (15, 34.4 mg) was isolated from fraction E2b by Sephadex LH-20, auriculatol D (8, tR = 46.4 min, 5.1 mg) was obtained from fraction E2a by an RP HPLC (70% MeOH), and 1-epi-pieristoxin S (12, tR = 35.4 min, 4.6 mg) and craiobiotoxin I (13, tR = 50.2 min, 6.8 mg) were obtained by an RP HPLC (50% MeOH) from fraction E2c. In a similar way, fraction F was fractionated and further purified by an RP HPLC (38% CH3CN) to afford 19-hydroxy-3-epi-auriculatol B (6, tR = 30.2 min, 5.0 mg) and auriculatol C (7, tR = 34.4 min, 4.6 mg). Auriculatol A (1, 14.4 mg), 6-deoxycraiobiotoxin I (4, 20.1 mg), and rhododecorumin X (14, 15.6 mg) were obtained from fraction G. Fraction H was repeatedly fractionated and afforded rhododecorumin VIII (16, 10.0 mg, 30% MeOH/H2O, tR = 20.2 min) and 2α-hydroxyauriculatol F (11, 17.6 mg, 45% MeOH/H2O, tR = 25.0 min) by RP HPLC. Auriculatol A (1): colorless oil; [α]20D +3 (c 0.5, MeOH); 1H and 13C NMR data, Tables 1 and 2; HRESIMS m/z 405.2260 [M + Na]+ (calcd for C21H34O6Na, 405.2253). Auriculatol B (2): colorless prisms, mp 186−187 °C (MeOH); [α]20D −43 (c 0.2, MeOH); 1H and 13C NMR data, Tables 1 and 2; HRESIMS m/z 343.2240 [M + Na]+ (calcd for C20H32O3Na, 343.2249). 3-epi-Grayanotoxin XVIII (3): colorless prisms, mp 192−193 °C (MeOH); [α]20D −27 (c 1.0, MeOH); 1H and 13C NMR data, Tables 1 and 2; HRESIMS m/z 695.4523 [2 M + Na]+ (calcd for C40H64O8Na, 695.4499), 359.2210 [M + Na]+ (calcd for C20H32O4Na, 359.2198). 6-Deoxycraiobiotoxin I (4): colorless prisms, mp 211−213 °C (MeOH); [α]20D −29 (c 0.1, MeOH); 1H and 13C NMR data, Tables 1 and 2; HRESIMS m/z 695.4502 [2 M + Na]+ (calcd for C40H64O8Na, 695.4499), 359.2210 [M + Na]+ (calcd for C20H32O4Na, 359.2198). 3-epi-Auriculatol B (5): white, amorphous powder; [α]20D −24 (c 0.5, MeOH); 1H and 13C NMR data, Tables 1 and 2; HRESIMS m/z 343.2251. 19-Hydroxy-3-epi-auriculatol B (6): white, amorphous powder; [α]20D −2 (c 0.3, MeOH); HRESIMS m/z 359.2211 [M + Na]+ (calcd for C20H32O4Na, 359.2198); 1H and 13C NMR data, Tables 1 and 2. Auriculatol C (7): pale yellow oil; [α]20D −32 (c 0.5, MeOH); 1H and 13 C NMR data, Tables 3 and 2; HRESIMS m/z 343.2209 [M + Na]+ (calcd for C20H32O3Na, 343.2249). J

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Crystallographic data of 1-epi-pieristoxin S (12): C320H582O115, M = 6269.82, T = 173(2) K, V = 8456.3(9) Å3, Z = 1, calculated density 1.231 mg/m3, monoclinic, C2, α = γ = 90°, β = 126.751(2)°, a = 34.793(2) Å, b = 13.6374(7) Å, c = 22.2430(13) Å, F(000) = 3422, F2 (goodness-of-fit) = 1.196, 2.479° < θ < 66.709°, −41 ≤ h ≤ 41, −16 ≤ k ≤ 16, −26 ≤ l ≤ 26, data/restraints/parameters 14 748/1/1016, 46 020 reflections collected/14 748 reflections unique [R(int) = 0.0590], R indices (all data) R1 = 0.0867 (wR2 = 0.2619), final R indices R1 = 0.0828 (wR2 = 0.2517) [I > 2σ(I)], Flack parameter = 0.09(6) for 5818 quotients. Crystallographic data of rhododecorumin X (14). C20H32O6, M = 368.46, T = 293(2) K, V = 941.39(3) Å3, calculated density 1.300 mg/ m3, Z = 2, monoclinic, P21, α = γ = 90°, β = 100.3517(16)°, a = 7.21196(13) Å, b = 11.84870(16) Å, c = 11.19881(17) Å, F(000) = 400, F2 (goodness-of-fit) = 1.044, 8.02° < θ < 133.2°, − 8 ≤ h ≤ 8, −14 ≤ k ≤ 14, −13 ≤ l ≤ 13, data/restraints/parameters 3345/1/250, 18 702 reflections collected/3345 reflections unique [R(int) = 0.0295], R indices (all data) R1 = 0.0404 (wR2 = 0.1071), final R indices R1 = 0.0401 (wR2 = 0.1067) [I > 2σ(I)], Flack parameter −0.01(15). Crystallographic data of rhododecorumin VIII (16): C41H77O21, M = 906.02, T = 173(2) K, V = 2367.56(11) Å3, Z = 2, calculated density 1.271 mg/m3, monoclinic, P21, α = γ = 90°, β = 110.5150(10)°, a = 18.1477(5) Å, b = 7.1529(2) Å, c = 19.4739(5) Å, F(000) = 982, F2 = 1.036, 2.422° < θ < 66.674°, − 21 ≤ h ≤ 21, − 8 ≤ k ≤ 8, − 23 ≤ l ≤ 22, data/restraints/parameters 7937/155/633, 15 517 reflections collected/7937 reflections unique [R(int) = 0.0481], R indices (all data) R1 = 0.0849 (wR2 = 0.1889), final R indices R1 = 0.0649 (wR2 = 0.1713) [I > 2σ(I)], Flack parameter −0.06(12). Analgesic Activity Evaluation. The analgesic activities of diterpenoids 1−24 were evaluated in a writhing test induced by HOAc as previously described.13,14

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and Dr. Y. Cai at the Analysis and Measurement Centre of Huazhong University of Science and Technology for crystallographic data collection, and Dr. T. Zeng at the Analysis and Measurement Centre of Huazhong University of Science and Technology for ECD, IR, and UV data collection.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.9b00095. 2D NMR analysis of new compounds 2−12; NMR and HRESIMS spectra for new compounds 1−12; UV and IR spectra for compounds 8 and 9 (PDF) Crystallographic data for compound 2 (CIF) Crystallographic data for compound 3 (CIF) Crystallographic data for compound 4 (CIF) Crystallographic data for compound 10 (CIF) Crystallographic data for compound 11 (CIF) Crystallographic data for compound 12 (CIF) Crystallographic data for compound 14 (CIF) Crystallographic data for compound 16 (CIF)

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REFERENCES

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Junjun Liu: 0000-0001-9953-8633 Guangmin Yao: 0000-0002-8893-8743 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by the Fundamental Research Funds for the Central Universities (HUST: 2016YXMS148) and National Natural Science Foundation of China (U1703109 and 81001368). We thank Dr. L. Li at the Instrumental Analysis Center of Shanghai Jiao Tong University K

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(33) Shu, P.; Wei, X.; Xue, Y.; Li, W.; Zhang, J.; Xiang, M.; Zhang, M.; Luo, Z.; Li, Y.; Yao, G.; Zhang, Y. J. Nat. Prod. 2013, 76, 1303−1312.

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DOI: 10.1021/acs.jnatprod.9b00095 J. Nat. Prod. XXXX, XXX, XXX−XXX