Phytochemistry 96 (2013) 378–388

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Scapiformolactones A–I: Germacrane sesquiterpenoids with an unusual D3-15,6-lactone moiety from Salvia scapiformis Yongji Lai a,1, Yongbo Xue a,1, Mengke Zhang a, Jinwen Zhang b, Wei Tang c, Junjun Liu a, Liang Lei a, Juming Yan d, Zengwei Luo a, Jianping Zuo c, Yan Li d, Guangmin Yao a,⇑, Yonghui Zhang a,⇑ a Hubei Key Laboratory of Natural Medicinal Chemistry and Resource Evaluation, School of Pharmacy, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, PR China b Tongji Hospital Affiliated to Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, PR China c State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, PR China d State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650204, PR China

a r t i c l e

i n f o

Article history: Received 9 November 2012 Received in revised form 17 May 2013 Available online 1 November 2013 Keywords: Salvia scapiformis Lamiaceae Germacrane sesquiterpene Conformation Chemical transformation Quantum mechanical calculation Cytotoxicity Immunomodulatory activity

a b s t r a c t Nine germacrane sesquiterpenoids with an unusual D3-15,6-lactone moiety, scapiformolactones A–I (1– 9), and one known seco-germacrane sesquiterpenoid, 3,7,11-trimethyldodeca-l,6,9-triene-3,11-diol (10), were isolated from whole plants of Salvia scapiformis Hance. Their structures were elucidated by spectroscopic methods including HR-ESIMS, IR, UV, NMR, and CD, as well as by quantum mechanical calculations and chemical transformations. Structures of compounds 1–3 were also confirmed by single-crystal X-ray diffraction analysis. Six germacrane 6,15-diol derivatives (11–16) were obtained by chemical transformation. Compounds 1–9 and 11–16 were evaluated for their in vitro immunomodulatory effects on T and B cells, as well as their in vitro cytotoxicity against five human cancer cell lines, HL-60, SMMC-7721, A-549, MCF-7, and SW480. Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction Salvia is one of the largest genera of the family Lamiaceae (formerly Labiatae). It comprises of over 1000 species and is distributed throughout tropical and temperate zones of the world; there are 84 species in China (Li and Ian, 1994). More than 1000 bioactive natural products including sesquiterpenoids, diterpenoids, sesterterpenoids, steroids, triterpenoids, and phenolic compounds have also been isolated from 134 Salvia species (Wu et al., 2012). Germacrane-type sesquiterpenoids are quite rare in Salvia species, however, although they are the most abundant among all known sesquiterpenoids. To date, there have been 24 germacrane sesquiterpenoids reported belonging to four structural subtypes of germacrane sesquiterpenes (Li et al., 2003; Pan et al., 2010; Wang et al., 2008; Xu et al., 2008), germacrane furanosesquiterpenoids (Li et al., 2003; González et al., 1989; Rustaiyan et al., 1992; Xu et al., 2005, 2008), 12,6-germacranolides (Liu et al., 2009), and 12,8-germacranolides (Ali et al., 2007; Xu et al., 2008) from seven ⇑ Corresponding authors. Tel.: +86 27 83692311; fax: +86 27 83691325. E-mail addresses: [email protected] (G. Yao), [email protected] (Y. Zhang). 1 These authors contribute equally to this work. 0031-9422/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.phytochem.2013.10.003

Salvia species, S. palaefolia (González et al., 1989), S. glutinosa (Rustaiyan et al., 1992), S. roborowskii (Li et al., 2003; Liu et al., 2009), S. nubicola (Ali et al., 2007), S. castanea (Xu et al., 2005, 2008), S. chinensis (Wang et al., 2008), and S. trijuga (Pan et al., 2010). Salvia scapiformis Hance is widely distributed in Fujian, Guangdong, Guangxi, Guizhou, Hunan, Jiangxi, Zhejiang, Taiwan, and as well as the Philippines (Li and Ian, 1994). Whole plants of S. scapiformis, known as a folk medicine ‘‘Bai-Bu-Yao’’ in China, are used to treat cough, hemoptysis, traumatic injuries, traumatic hemorrhage, dysentery, and furunculosis (Fang and Liao, 2006). However, there have been no phytochemical investigations reported on any part of this plant. In the course of searching for bioactive natural products from Chinese folk medicines, nine new germacrane sesquiterpenoids with an unusual D3-15,6-lactone moiety, scapiformolactones A–I (1–9) (Fig. 1), and one known seco-germacrane sesquiterpene, 3,7,1l-trimethyldodeca-l,6,9-triene-3,ll-diol (10) (Grande et al., 1992), were isolated from the acetone extract of the whole plants of S. scapiformis. Their structures were determined by means of spectroscopic methods including HR-ESIMS, IR, UV, NMR, CD, and single-crystal X-ray diffraction analysis, as well as by quantum mechanical calculations and chemical transformation. The known seco-germacrane sesquiterpene 10 was

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Y. Lai et al. / Phytochemistry 96 (2013) 378–388

Fig. 1. Chemical structures of compounds 1–16.

isolated from the Salvia genus for the first time. Six new germacrane derivatives with a 6,15-diol moiety 11–16 were obtained by chemical transformation. In this paper, the isolation, structural elucidation, and in vitro immunomodulatory effects on murine T and B cells, as well as in vitro cytotoxicity against five human cancer cell lines, HL-60, SMMC-7721, A-549, MCF-7, and SW480, of compounds 1–9 and 11–16, are reported.

2. Results and discussion Compound 1 was obtained as colorless crystals, m.p. 130.0– 131.0 °C. Its molecular formula was deduced as C22H32O7 from (+)-HRESIMS at m/z 409.2199 [M+H]+ (calcd 409.2226), inferring seven degrees of unsaturation. In the UV spectrum, an absorption maximum at 229 nm suggests the presence of an a,b-unsaturated ester carbonyl. The IR spectrum showed the presence of an unsaturated lactone (1766 cm1), and ketone carbonyl (1715 cm1), and double bonds (1680 cm1) functionalities. The 1H NMR spectrum

(Table 1) showed resonances of five methyl doublets at dH 0.92 (J = 5.9 Hz), 1.10 (J = 7.0 Hz), 1.12 (J = 5.9 Hz), 1.17 (J = 6.9 Hz), and 1.22 (J = 6.4 Hz), one methyl singlet of acetyl group at dH 1.98, one vinyl triplet at dH 6.95 (t, 4.4), and three oxygen-bearing methine protons at dH 5.05 (dd, J = 9.0, 1.6 Hz), 5.29 (m), and 5.41 (d, J = 7.7 Hz). The 13C NMR (Table 3) and DEPT spectrum displayed six methyls, three methylenes, eight methines (one olefinic and three oxygenated), and five quaternary carbons (one olefinic, three ester carbonyls, and one ketone carbonyl). One double bond, three ester carbonyls, and one ketone carbonyl account for five degrees of unsaturation, with the remaining two degrees of unsaturation suggesting existence of two rings in 1. The gross structure of 1 was deduced from extensive analyses of the 2D NMR spectroscopic data, including 1H–1H COSY, HSQC, and HMBC data (Fig. 2). Analysis of the 1H–1H COSY and HSQC spectra suggested the existence of three partial structures: (I) C-2/C-3, (II) C-5/C-6(O)/C-7[C-11(C-12)/C-13]/C-8(O)/C-9/C-10/C-14, and (III) C-50 /C-20 /C-30 /C-40 . HMBC correlations of H-2 (dH 3.34, 3.68), H-9 (dH 1.55, 2.22), H-10 (dH 2.74), and Me-14 (dH 1.17) with C-1 (dC 209.4), and of H-2 (dH 3.34, 3.68), H-5 (dH 2.95, 3.02), and H-6 (dH 5.05) with C-4 (dC 132.5) suggested that fragments I and II were connected by C-1 and C-4, and further established the ten-membered ring of the germacrane skeleton. The connection of partial structures II to III through an ester carbonyl C-10 was deduced by HMBC correlations of H-8 (dH 5.41), H-20 (dH 2.76), and Me-50 (dH 1.10) with C-10 (dC 173.2). Moreover, HMBC correlations of H-3 (dH 6.95), H-5(dH 2.95, 3.20), and H-6 (dH 5.05) with an ester carbonyl C-15 (dC 171.3) moiety suggested that the lactone ring is located between C-6 (dC 77.0) and C-15. Correlations of H-30 (dH 5.29) and acetyl CH3-200 (dH 1.98) with acetyl carbonyl C-100 (dC 170.4) in the HMBC spectrum implied that the O-acetyl group was attached to C-30 . The relative configuration of compound 1 was determined by NOESY analysis (Fig. 2). Assuming H-10 to be b-oriented, the NOESY correlation of H-10 (dH 2.74) and H-8 (dH 5.41) indicated that H-8 was b-oriented; furthermore, correspondingly, the

Table 1 H NMR spectroscopic data of compounds 1–5a (400 MHz).

1

Position

1b

2b

3b

1 2b

3.34 (dd, 16.7, 5.4)

3.28 (dd, 16.7, 5.6)

2a 3

3.68 (dd, 16.7, 11.3) 6.95 (t, 4.4)

3.67 (dd, 16.7, 11.3) 6.87 (m)

3.32 (ddd, 16.6, 5.7, 1.5) 3.67 (dd, 16.6, 11.3) 6.97 (m)

5b 5a 6

2.95 (dd, 15.5, 9.0) 3.20 (d, 15.5) 5.05 (dd, 9.0, 1.6)

2.92 (dd, 15.7, 9.0) 3.20 (d, 15.7) 5.03 (dd, 9.0, 3.1)

2.94 (dd, 15.7, 9.0) 3.20 (d, 15.7) 5.06 (dd, 9.0, 3.2)

7 8 9b

1.84 5.41 1.55 4.2) 2.22 2.74 1.80 0.92 1.12 1.17 2.76 5.29 1.22 1.10 1.98

1.99 5.45 1.61 4.4) 2.16 2.57 1.80 0.86 1.11 1.23 2.69 4.25 1.33 1.18

1.90 (dd, 3.2, 10.2) 5.46 (d, 7.9) 1.56 (ddd, 4.4, 7.9, 16.5) 2.19 (dd, 2.8, 16.5) 2.70 (m) 1.80 (m) 0.90 (d, 6.5) 1.08 (d, 6.5) 1.11 (d, 5.3)

9a 10 11 12 13 14 20 30 40 50 200 a b c d

(m) (d, 7.7) (ddd, 16.6, 7.7, (dd, 16.6, 2.3) (m)d (m) (d, 5.9) (d, 5.9) (d, 6.9) (m)d (m) (d, 6.4) (d, 7.0) (s)

(m) (d, 8.0) (ddd, 16.5, 8.0, (dd, 16.5, 2.6) (m) (m) (d, 6.6) (d, 6.5) (d, 6.6) (m) (m) (d, 6.2) (d, 7.0)

6.94 (m)d 1.64 (dd, 7.1, 1.0) 1.82 (s)

4ab

4bb

5ac

5bc

4.15 (m) 2.69 (m)

4.08 (m) 2.82 (m)d

3.65 (m) 2.49 (m)

3.62 (m)d 2.55 (m)d

(m)d (d, 15.4) (dd, 8.8,

2.71 6.71 2.1) 2.85 3.24 4.63

(m) (d, 8.2) (m)

1.81 (dd, 8.8, 3.5) 5.50 (dd, 11.3, 7.5) 2.02 (m)

2.61 6.85 3.0) 2.75 2.85 4.84 3.7) 1.62 4.89 1.39

(m) (dt, 12.8,

(m)d (d, 15.5) (dd, 9.0,

2.82 6.66 2.0) 2.97 3.69 4.88

(m) (d, 8.0) (m)

1.64 (m) 5.18 (t, 8.6) 1.60 (m)d

(m)d (m)d (m)d (d, 6.1) (d, 6.9) (d, 6.9) (m)d (m)d (d, 6.3) (d, 7.3) (s)

2.27 1.59 2.17 0.90 0.94 1.15 2.77 5.31 1.23 1.12 2.07

1.39 1.41 1.79 0.94 1.07 1.01 2.60 5.03 1.18 1.09 1.96

(m) (m) (m) (d, 6.6) (d, 6.6) (d, 6.3) (m)d (m)d (d, 6.4) (d, 7.3) (s)

1.60 1.37 2.02 0.79 0.91 1.00 2.60 5.03 1.20 1.10 1.98

2.88 6.75 3.1) 2.80 3.18 4.95 3.2) 1.75 5.24 1.49 2.00 1.60 1.71 0.84 1.10 1.28 2.77 5.31 1.23 1.12 1.98

(m)d (dt, 13.3,

(m) (ddd, 11.2, 5.7, (dd, 14.5, 6.9) (d, 14.5) (dd, 8.8, 7.2)

(m) (m) (m) (d, 7.0) (d, 6.9) (d, 6.8) (m) (m) (d, 6.3) (d, 7.0) (s)

J in Hz. 4a and 4b, and 5a and 5b were separately different conformers of compounds 4 and 5, respectively. In pyridine-d5. In CDCl3. Overlapped.

(m)d (ddd, 12.2, 4.8, (m)d (d, 14.5) (dd, 8.9, 7.0)

(m)d (m) (m) (d, 7.0) (d, 6.9) (d, 6.4) (m)d (m)d (d, 6.4) (d, 7.2) (s)

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Y. Lai et al. / Phytochemistry 96 (2013) 378–388

Table 2 H NMR spectroscopic data of compounds 6–9a (400 MHz) at 298 K.

1

Position 6ac 1 2b 2a 3 5b 5a 6 7 8 9b 9a 10 11 12 13 14 20 30 40 50 a b c d

3.99 2.26 2.75 6.46 2.76 2.99 4.87 1.63 4.77 1.28 1.63 1.36 1.85 0.94 1.06 0.98 2.36 3.80 1.16 1.10

6bc (m) (m) (m)d (brd, 12.9) (m)d (d, 15.3) (dd, 8.8, 3.2) (m)d (d, 8.2) (m) (m)d (m)d (m) (d, 6.6) (d, 6.7) (d, 6.7) (m)d (m)d (d, 6.3) (d, 7.2)

7ac

3.86 2.48 2.65 6.39 2.83 3.40 4.64 1.49 5.20 1.67 1.77 1.38 2.03 0.79 0.90 0.99 2.30 3.82 1.18 1.09

d

(m) (m)d (m) (m) (m)d (d, 14.5) (dd, 8.6, 7.4) (m) (dd, 10.6, 8.0) (m)d (m) (m)d (m) (d, 7.0) (d, 6.9) (d, 6.2) (m)d (m)d (d, 6.3) (d, 7.2)

7bc d

8ab d

3.65 2.51 2.63 6.88 2.76 2.85 4.86 1.68 4.93 1.39

(m) (m) (m) (brd, 12.5) (m)d (d, 15.3) (dd, 8.7, 3.4) (m)d (d, 7.3) (m)d

3.65 2.53 2.75 6.72 2.84 3.27 4.64 1.66 5.20 1.65

(m) (m)d (m) (brd, 12.2) (m)d (d, 14.4) (m)d (m)d (dd, 10.8, 7.6) (m)d

1.38 1.81 0.95 1.07 1.02 2.34 3.83 1.17 1.11

(m)d (m)d (d, 6.5) (d, 6.5) (d, 5.9) (m)d (m)d (d, 6.3) (d, 7.3)

1.38 2.07 0.80 0.92 1.01 2.35 3.86 1.19 1.10

(m)d (m)d (d, 7.0) (d, 6.9) (d, 6.2) (m)d (m)d (d, 6.3) (d, 7.2)

4.13 2.70 2.90 6.73 2.82 3.20 4.97 1.84 5.31 1.55 2.01 1.57 1.72 0.83 1.08 1.23

8bb d

(m) (td, 13.3, 8.9) (m)d (m) (m)d (d, 15.5) (dd, 9.0, 3.7) (m)d (d, 7.5) (m)d (m)d (m)d (m)d (d, 6.5) (d, 6.5) (d, 6.8)

4.12 2.87 2.87 6.69 2.95 3.72 4.91 1.84 5.54 2.14 2.29 1.64 2.21 0.90 0.95 1.21

9ac d

(m) (m)d (m)d (m)d (m)d (d, 14.5) (dd, 8.8, 7.2) (m)d (dd, 11.2, 7.5) (m) (dd, 14.8, 11.4) (m)d (m) (d, 7.0) (d, 6.9) (d, 7.4)

6.98 (m) 6.95 (m) 1.65 (dd, 7.1, 1.0) 1.70 (dd, 7.0, 0.9) 1.86 (m)d 1.86 (m)d

3.67 2.50 2.62 6.87 2.76 2.88 4.87 1.75 4.96 1.44 1.44 1.43 1.80 0.96 1.10 0.99

9bc (m) (m) (dd, 13.0, 4.5) (dt, 12.9, 3.2) (m)d (d, 15.2) (dd, 8.8, 3.4) (m)d (d, 7.6) (m) (m) (m)d (m) (d, 6.3) (d, 6.2) (d, 6.3)

6.78 (m)d 1.78 (d, 4.0) 1.75 (d, 7.1)

3.64 2.55 2.75 6.73 2.88 3.35 4.69 1.68 5.20 1.64 1.73 1.43 2.08 0.79 0.93 1.03

(dt, 10.6, 3.1) (m) (m)d (m)d (m)d (d, 14.4) (dd, 8.8, 7.1) (m) (dd, 11.2, 7.4) (m) (m)d (m)d (m) (d, 7.0) (d, 7.0) (d, 6.5)

6.75 (m)d 1.78 (d, 4.0) 1.75 (d, 7.1)

J in Hz. 6a and 6b, 7a and 7b, 8a and 8b, and 9a and 9b were separately different conformers of compounds 6, 7, 8, and 9, respectively. In pyridine-d5. In CDCl3. Overlapped.

Table 3 C NMR spectroscopic data of compounds 1–9a (100 MHz) at 298 K.

13

a b c

Position

1b

2b

3b

4ab

4bb

5ac

5bc

6ac

6bc

7ac

7bc

8ab

8bb

9ac

9bc

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 10 20 30 40 50 100 200

209.4 s 42.6 t 130.8 d 132.5 s 28.3 t 77.0 d 53.7 d 75.3 d 35.2 t 48.0 d 25.9 d 21.7 q 23.9 q 18.0 q 171.3 s 173.2 s 45.7 d 72.2 d 17.4 q 13.4 q 170.4 s 21.5 q

209.5 s 42.5 t 130.5 d 132.6 s 28.4 t 77.2 d 53.3 d 75.1 d 34.8 t 48.4 d 25.9 d 21.7 q 24.0 q 17.7 q 171.4 s 175.0 s 49.4 d 69.4 d 21.6 q 14.0 q

209.3 s 42.5 t 130.5 d 132.5 s 28.2 t 77.1 d 53.3 d 75.0 d 35.1 t 48.0 d 25.8 d 21.6 q 23.8 q 17.7 q 171.3 s 167.2 s 129.3 s 138.0 d 14.6 q 12.5 q

75.6 d 37.5 t 135.3 d 129.7 s 28.7 t 77.1 d 53.9 d 77.1 d 31.9 t 41.3 d 26.1 d 21.8 q 24.0 q 20.9 q 171.5 s 173.0 s 45.7 d 72.2 d 17.4 q 13.4 q 170.5 s 21.6 q

75.2 d 37.1 t 134.1 d 132.5 s 33.5 t 79.8 d 47.4 d 71.0 d 34.7 t 33.6 d 28.7 d 18.2 q 22.2 q 19.6 q 171.5 s 173.5 s 45.5 d 72.5 d 17.5 q 13.2 q 170.5 s 21.6 q

77.6 d 36.3 t 135.4 d 129.2 s 28.4 t 76.3 d 53.1 d 75.8 d 34.1 t 41.1 d 25.7 d 21.5 q 23.8 q 20.9 q 170.6 s 172.3 s 45.0 d 71.7 d 17.0 q 12.8 q 170.4 s 21.5 q

76.3 d 35.0 t 133.5 d 131.8 s 33.1 t 78.9 d 46.4 d 69.8 d 36.9 t 34.3 d 27.8 d 17.7 q 21.9 q 18.3 q 170.9 s 172.6 s 44.7d 71.6 d 16.9 q 12.5 q 170.3 s 21.4 q

75.8 d 35.8 t 134.0 d 129.1 s 28.4 t 76.5 d 52.9 d 76.2 d 30.3 t 40.1 d 25.7 d 21.5 q 23.7 q 19.7 q 170.9 s 175.0 s 47.3 d 69.3 d 20.8 q 14.2 q

75.6 d 35.7 t 132.7 d 132.0 s 33.0 t 79.3 d 46.7 d 70.2 d 33.3 t 32.7 d 27.7 d 17.6 q 21.9 q 18.5 q 171.2 s 175.0 s 47.4 d 69.8 d 21.0 q 14.0 q

77.7 d 36.2 t 135.5 d 129.2 s 28.4 t 76.4 d 52.9 d 75.9 d 33.8 t 41.3 d 25.7 d 21.5 q 23.8 q 20.9 q 170.7 s 175.0 s 47.3 d 69.3 d 20.9 q 14.3 q

76.3 d 34.4 t 133.6 d 131.8 s 33.1 t 78.8 d 46.3 d 69.8 d 36.9 t 34.4 d 27.7 d 17.7 q 21.9 q 18.3 q 171.0 s 175.0 s 47.4 d 69.8 d 21.0 q 14.0 q

75.6 d 37.5 t 135.1 d 129.7 s 28.8 t 77.2 d 53.7 d 76.9 d 32.0 t 41.4 d 26.0 d 21.8 q 24.1 q 20.8 q 171.6 s 167.3 s 129.8 s 137.5 d 14.7 q 12.7 q

75.4 d 37.2 t 134.1 d 132.6 s 33.5 t 79.9 d 47.5 d 70.7 d 34.7 t 33.7 d 28.7 d 17.9 q 22.2 q 19.6 q 171.6 s 167.2 s 129.8 s 138.2 d 14.8 q 12.6 q

77.8 d 36.2 t 135.2 d 129.4 s 28.4 t 76.5 d 52.9 d 75.5 d 34. 1 t 41.2 d 25.8 d 21.6 q 23.7 q 20.9 q 170.7 s 166.9 s 128.9 s 137.4 d 13.3 q 14.6 q

76.4 d 35.0 t 133.5 d 131.9 s 33.0 t 79.1 d 46.6 d 69.5 d 36.9 t 34.4 d 27.8 d 17.4 q 21.9 q 18.3 q 171.0 s 166.9 s 129.0 s 138.0 d 12.2 q 14.6 q

4a and 4b, 5a and 5b, 6a and 6b, 7a and 7b, 8a and 8b, and 9a and 9b were separately different conformers of compounds 4, 5, 6, 7, 8, and 9, respectively. In pyridine-d5. In CDCl3.

O-30 -O-acetyl-20 -methylbutyryl group at C-8 was deduced to be in an a-orientation. The a-orientations of H-9a (dH 2.22), H-5a (dH 3.20), and H-2a (dH 3.68) were determined by the strong NOESY correlations from H-9a to H-5a and H-2a. The geometry of D3(4) double bond was assigned to be E on the basis of NOESY correlation of H-5a and H-2a. However, the NOESY spectrum could not provide sufficient information to establish the orientations of H-6, H-7, H-20 , and H-30 . Hence, other approaches such as application of single-crystal X-ray diffraction were required. After many attempts with different solvents, a suitable single crystal was obtained from MeOH, and subjected to X-ray diffraction analysis on a Bruker APEX-II diffractometer equipped with a graphite-monochromatized Cu Ka radiation (k = 1.54178 Å) at 100 (2) K. The

single crystal X-ray diffraction analysis of 1 (Fig. 3) established not only the relative configuration, but also the absolute configuration by the Flack parameter 0.2 (3) calculated from the given coordinates. Thus, compound 1 was (6S,7S,8S,10R,20 R,30 R)-8-O-(30 -Oacetyl-20 -methylbutyryl)-germacra-3E-en-15,6-lactone-1-one and named scapiformolactone A. Compound 2, a colorless crystal, m.p. 135.8–136.8 °C, gave a quasi-molecular ion peak at m/z 365.1972 [MH] (calcd for C20H29O6, 365.1964) in the ()-HRESIMS, indicating its molecular formula C20H30O6. The NMR spectroscopic data of 2 (Tables 1 and 3) were almost identical to that of 1 except for the absence of signals due to the acetyl group at C-30 in 2, suggesting that it was a 30 deacetyl derivative of 1. The chemical shift of C-30 (dC 69.4) in 2

Y. Lai et al. / Phytochemistry 96 (2013) 378–388

381

Fig. 2. Key 1H–1H COSY, HMBC, and NOESY correlations of 1.

Fig. 4. ORTEP diagram (crystallographic numbering) of 2 with ellipsoids drawn at the 30% probability level.

Fig. 3. ORTEP diagram (crystallographic numbering) of 1 with ellipsoids drawn at the 30% probability level.

moved up-field compared with the chemical shift (dC 72.2) in 1, which further confirmed above deduction. Its structure and relative configuration was elucidated by 2D NMR techniques (1H–1H COSY, HSQC, HMBC, and NOESY), and further confirmed by the single crystal X-ray diffraction analysis (Fig. 4). The CD spectrum of 2, showing a negative Cotton effect at 288 nm and a positive Cotton effect at 238 nm, was also very similar to that of 1. Therefore, the absolute configuration of 2 was same as that of 1. Finally, compound 2 was established as (6S,7S,8S,10R,20 R,30 R)-8-O-(30 -O-hydroxy-20 -methylbutyryl)-germacra-3E-en-15,6-lactone-1-one and named scapiformolactone B. Compound 3 was isolated as a colorless crystal, m.p. 173.2– 174.8 °C. Its molecular formula was determined to be C20H28O5 from the [M+H]+ ion peak at m/z 349.2009 (calcd for C20H29O5, 349.2015) by the (+)-HREIMS, suggesting seven degrees of unsaturation. The signals of the 1H and 13C NMR spectra of 3 (Tables 1 and 3) were similar to those of 2, the major difference being a trisubsti0 0 tuted D2 (3 ) double bond (dH 6.94; dC 129.3, 138.0) in 3, instead of 3 two sp methines (dH 2.69, 4.25; dC 49.4, 69.4) in 2. Its structure was established by 1H–1H COSY, HSQC, HMBC, and NOESY, and

Fig. 5. ORTEP diagram (crystallographic numbering) of 3 with ellipsoids drawn at the 30% probability level.

confirmed by single crystal X-ray diffraction analysis (Fig. 5). The CD spectrum of 3 also showed similar Cotton effects to those of 1 and 2, which suggested that it had the same absolute configuration of 3E, 6S, 7S, 8S, and 10R as those of 1 and 2. Therefore, compound 3 was characterized as (6S,7S,8S,10R)-8-O-tigloyl-germacra-3E-en15,6-lactone-1-one and named scapiformolactone C. Compound 4 had the molecular formula C22H34O7 based on the (+)-HRESIMS at m/z 411.2382 [M+H]+ (calcd for C22H35O7,

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Fig. 6. (A) Superposition of two putative conformers of compound 4 that were obtained from conformational analyses by quantum mechanical calculations at B3LYP/6-31G⁄ level of theory. The carbons in both conformers are colored in green and yellow, respectively. The oxygen atoms are colored in red, and the hydrogen atoms are not shown for clarity. (B) The labeling of carbon atoms in compound 4. (C) The relative free energy profile at B3LYP/6-31G⁄ level of theory for the interconversion of the two conformers of compound 4, including the zero-point and thermal corrections for the system as well as the solvation effects in pyridine obtained by SMD solvation model. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

411.2383), indicating six degrees of unsaturation. Its IR spectrum showed absorptions of hydroxyl (3423 cm1), carbonyl (1737 cm1) and conjugate ester carbonyl (1678 cm1) functionalities. Although compound 4 behaved as a single and pure compound when analyzed by TLC plates with different developing systems and by HPLC using various mobile phases, its 1H NMR spectrum showed two sets of proton signals in a ratio of 1:1 in CDCl3 and a ratio of 1:2 in pyridine-d5. The aforementioned facts implied that 4 was likely a germacrane derivative featuring a mixture of two different conformers in solution (Sorm, 1971; Ugliengo et al., 1990). The NMR spectroscopic data for the major conformer 4a of compound 4 were thus selected to elucidate its structure. The

C NMR spectral data of 4 is very similar to that of 1, the notable difference being, that C-1 in 4 was an oxygenated methine at dC 75.2, instead of a carbonyl group in 1 at dC 209.4. The mass of 4 was 2 amu heavier than that of 1, which further supported this suggestion. The structure and full assignments of all protons and carbons for the two conformers of 4 were accomplished by detailed 2D-NMR spectroscopic analysis (Tables 1 and 3). Its b-orientation was determined by establishment of the cross-peak of H-1 (dH 4.08/4.15) and H-3 (dH 6.66/6.75) in the NOESY spectrum of compound 4. Compound 5 was obtained as a colorless gum, and its molecular formula C22H34O7 was deduced by (+)-HRESIMS at m/z 411.2383 [M+H]+. Its 1H NMR spectrum in CDCl3 showed two sets of proton signals in a ratio of 1:0.88. Great similarities of the NMR spectroscopic data (Tables 1 and 3), IR, UV, and optical rotation of compounds 4 and 5 demonstrated that both compounds had closely related molecular structures, and the only difference was the relative configuration of the hydroxyl group at C-1. The gross structure of 5 was elucidated by comprehensive analysis of 2D NMR (1H–1H COSY, HSQC, HMBC, and NOESY) and by comparison with the NMR spectroscopic data of 4. Hence, compound 5 was a 1b-OH epimer of 4. In order to better understand the conformers, a conformational analysis of compound 4 (Fig. 6) was then carried out by quantum mechanical calculations at B3LYP/6-31G⁄ level of theory. Two stable conformers, as well as the associated transition state (TS), were obtained. The calculated free energy barrier, including the zeropoint and thermal corrections along with the solvation effects in pyridine, was 21 kcal/mol or 20 kcal/mol, suggested the difficulty of one conformer inter-converting with another. As clearly shown in Fig. 6, the biggest difference between the two conformers of 4 is the orientation of C-7 and C-8, whereas other atoms on the ring superimposed very well. Obviously, when one conformer converts into another, it is necessary to move both C-7 and C-8. Thus the dihedrals of C4–C5–C6–C7 and C5–C6–C7–C8 are required to simultaneously rotate. However, due to the presence of the c-lactone ring between C-15 and C-6, the rotation of dihedral C4–C5– C6–C7 (or C15–O–C6–C7) is sterically hindered. Thus, it is difficult to convert from one conformer into another. Accordingly, these two conformers display two sets of NMR signals. When the fivemembered lactone ring is destroyed, however, both conformers are converted into one conformer, and the NMR signals for one of the two conformers will disappear. This hypothesis was further verified by chemical transformation. Compound 4 was reduced by NaBH4 (Scheme 1) to give a new 6,15-diol germacrane derivative 11, which was named scapiformodiol A. As expected, the NMR spectra of 11 showed signals for a single conformer. So, the hindrance for the inter-conversion between two conformers was overcome by cleavage of the

Scheme 1. Chemical transformation of compounds 1–9 and 11–16. Reagents and conditions: (a) NaBH4 (1.0 equiv.), CeCl37H2O (1.0 equiv.), CH3OH, 0 °C; (b) NaBH4 (2.0 equiv.), CeCl37H2O (1.0 equiv.), CH3OH, 0 °C.

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five-membered lactone ring. A similar phenomenon had been observed for isabelin (Yoshioka et al., 1968). In the same manner, compound 5 was transformed with NaBH4 into 12, which also showed signals for a single conformer in the NMR measurements. In order to determine the absolute configuration of 4, 5, 11, and 12, chemical transformations of compound 1 to compounds 4, 5, 11, and 12 were performed. In fact, compound 1 was reduced by

NaBH4 to give not only 4 and 5, but also 11 and 12 (Scheme 1). Therefore, the absolute configurations of compounds 4, 5, 11, and 12 were determined. Hence, compounds 4 and 5 were elucidated as (1S,6S,7S,8S,10R,20 R,30 R)-8-O-(30 -O-acetyl-20 -methylbutyryl)germacra-3E-en-15,6-lactone-1-ol and (1R,6S,7S,8S,10R,20 R, 30 R)-8-O-(30 -O-acetyl-20 -methylbutyryl)-germacra-3E-en-15,6-lactone-1-ol, and named scapiformolactones D and E, respectively.

Table 4 Cytotoxicity against murine splenocyte and effects on murine lymphocyte proliferation induced by Concanavalin A (ConA) (5 mg/mL) or Lipopolysaccharide (LPS) (10 mg/mL) of compounds 1–9 and 11–16. Compounds

Negative control Positive control 1

2

3

4

5

11

12

13

Negative control Positive control 6

7

8

9

14

15

16

a

MTT assay

ConA-induced T cell proliferation

LPS-induced B cell proliferation

Survival rate of splenocyte (%)

[3H] TdR incorporation  103 (cpm)a

[3H] TdR incorporation  103 (cpm)a

Inhibitory/enhanced rate (%)b

0.301 ± 0.056

0.454 ± 0.030

54.329 ± 2.464

37.200 ± 0.218

Inhibitory/enhanced rate (%)b

c

d 7

1  10 1  106 1  105 1  107 1  106 1  105 1  107 1  106 1  105 1  107 1  106 1  105 1  107 1  106 1  105 1  107 1  106 1  105 1  107 1  106 1  105 1  107 1  106 1  105

100 119 85 109 102 80 107 107 103 115 106 97 87 110 112 105 116 103 104 113 109 103 110 107

55.582 ± 3.790 58.083 ± 3.653 57.129 ± 5.972 57.129 ± 0.101 62.688 ± 1.098 60.090 ± 4.468 38.615 ± 9.662* 45.568 ± 12.961* 54.166 ± 8.486 47.436 ± 5.046* 56.587 ± 6.436 55.963 ± 2.435 58.831 ± 3.999 60.316 ± 0.858 59.328 ± 6.711 54.747 ± 8.405 55.558 ± 1.534 23.791 ± 3.512* 58.943 ± 3.068 56.001 ± 5.590 56.734 ± 4.386 56.570 ± 0.684 54.210 ± 1.628 55.651 ± 3.730 0.387 ± 0.060

+2 +7 +6 +5 +15 +11 29 16 ±0 13 +4 +3 +8 +11 +9 +1 +2 56 +8 +3 +4 +4 ±0 +2

64.494 ± 1.709 7

1  10 1  106 1  105 1  107 1  106 1  105 1  107 1  106 1  105 1  107 1  106 1  105 1  107 1  106 1  105 1  107 1  106 1  105 1  107 1  106 1  105

104 113 92 105 105 98 104 106 107 103 105 92 106 105 97 90 94 96 107 113 92

34.475 ± 1.469 31.564 ± 0.096 25.740 ± 1.748* 30.737 ± 1.905* 30.036 ± 4.314* 33.765 ± 3.006 17.363 ± 5.481* 15.866 ± 10.086* 26.065 ± 8.594* 22.194 ± 8.880* 28.218 ± 11.009* 21.080 ± 2.669* 36.549 ± 8.829 36.678 ± 3.435 29.106 ± 8.175* 32.802 ± 1.230 31.402 ± 4.235 10.279 ± 0.906* 27.866 ± 3.302* 20.460 ± 4.446* 30.504 ± 8.137 38.731 ± 1.798 32.889 ± 0.446 31.937 ± 1.246 0.449 ± 0.043

7 15 31 17 19 9 53 57 30 40 24 43 2 1 22 12 16 72 25 45 18 +4 12 14

42.825 ± 0.327 *

40.867 ± 16.343 49.826 ± 19.673* 64.268 ± 9.153 60.609 ± 5.350 61.437 ± 7.013 19.735 ± 7.652* 60.962 ± 7.637 56.391 ± 2.680 56.986 ± 1.581 54.284 ± 0.540* 57.091 ± 1.952 50.854 ± 0.927* 49.167 ± 3.958* 56.438 ± 0.650 60.947 ± 6.942 57.413 ± 8.913 60.202 ± 7.486 63.133 ± 4.841 57.212 ± 4.595 65.107 ± 4.666 61.148 ± 1.827

Results are represented as mean ± SD based on three independent experiments.  Inhibitory effect; + enhanced effect. c Negative control: DMSO. d Positive control: ConA or LPS. p < 0.05, Compared with positive control group. b

*

Concentration (M)

+37 +23 ±0 6 5 69 5 13 12 16 11 21 24 12 5 11 7 2 11 +1 5

24.228 ± 6.024* 25.847 ± 14.622* 30.337 ± 4.769* 36.001 ± 8.914* 35.979 ± 6.517* 20.278 ± 0.954* 33.759 ± 2.755* 32.707 ± 1.470* 29.787 ± 0.333* 38.616 ± 2.853 36.284 ± 1.379 29.687 ± 3.448* 31.056 ± 8.725* 40.537 ± 1.845 35.853 ± 1.990 26.538 ± 7.820* 35.719 ± 2.603 36.327 ± 2.510 47.181 ± 2.729 37.848 ± 1.390 34.005 ± 7.143*

43 40 29 16 16 53 21 24 30 10 15 31 27 5 16 38 17 15 +10 12 21

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Compounds 6 and 7 were both obtained as colorless gums with molecular formula C20H32O6, as deduced from (+)-HRESIMS. Their NMR spectra showed signals in a ratio of 1:0.7 for two conformers closely resembling to those of 4 and 5. Comparison of the NMR spectroscopic data of 6 and 7 (Tables 2 and 3) with that of 2 indicated that 6 and 7 differed structurally from 2 only at C-1 by a hydroxyl instead of a ketone. The relative configuration of 1-OH in 6 was, however, a-oriented and in 7 it was b-oriented, respectively, as deduced from analysis of their NOESY spectra. Similar to 1, compound 2 was reduced by NaBH4 to result in 6 and 7, and further reduction led to two new 6,15-diol germacrane derivatives 13 and 14 (Scheme 1). So, the absolute configurations of 6 and 7 were then established to be the same as that of 2 except for C-1. Thus, compounds 6 and 7 were (1S,6S,7S,8S,10R,20 R,30 R)-8-O-(30 -O-hydroxy-20 -methylbutyryl)-germacra-3E-en-15,6-lactone-1-ol and (1R,6S,7S,8S,10R,20 R,30 R)-8-O-(30 -O-hydroxy-20 -methylbutyryl)-germacra-3E-en-15,6-lactone -1-ol, and named scapiformolactones F and G, respectively. Compounds 8 and 9 were also purified as a colorless gums. The molecular formula C20H30O5 was determined by (+)-HRESIMS. The NMR spectra of 8 and 9 in CDCl3 showed two sets of signals in a ratio of 1:1 and 1:0.88, respectively, while, the NMR spectrum of 8 in pyridine-d5 exhibited two sets of signals in a ratio of 1:2. The core frameworks of compounds 8 and 9 were very similar to those of 6 and 7, respectively. The substituent group of C-8 in 8 and 9 was identical to that in 3, as elucidated from the 1D (Tables 2 and 3) and 2D NMR spectra. Absolute configurations of 8 and 9 were also verified by chemical transformation from 3 (Scheme 1). In this case, 15 and 16 were obtained by reduction of 8 and 9. Similarly, when compound 3 was treated by NaBH4 (Scheme 1), compounds 8, 9, 15, and 16 were achieved, respectively. Therefore, compounds 8 and 9 were elucidated as (1S,6S,7S,8S,10R)-8-O-tigloyl-germacra-3E-en-15,6-lactone-1-ol and (1R,6S,7S,8S,10R)-8-Otigloyl-germacra-3E-en-15,6-lactone-1-ol, and named scapiformolactones H and I, respectively. Generally, the location of the five-membered lactone ring in germacrane sesquiterpenoids is between either C-12 and C-6, or C-12 and C-8. However, the lactone ring formed between C-15 and C-6 is rare. Among them, the double bond of the a,b-unsaturated c-lactone is located at either D4 or D5 (Fraga, 2012; Sorm, 1971). Compounds 1–9 represent the first examples of germacrane sesquiterpenoids with an unusual D3-15,6-lactone moiety. Compounds 1–9 and 11–16 were evaluated for their in vitro immunomodulatory effects (Table 4), compounds 1, 3, 4, 6–9, 11, 12, and 15 significantly inhibited proliferation of LPS-induced murine B cells (p < 0.05), while compounds 7 and 11 significantly inhibited proliferation of ConA-induced murine T cells at 10 lM, (p < 0.05). In addition, these active compounds showed no obvious cytotoxity against murine lymphocytes in the MTT assay. Therefore, the immunosupressive activities of compounds 1, 3, 4, 6–9, 11, 12, and 15 do not involve general cytotoxity. Compounds 2, 5, 13, 14, and 16 showed no significant immunomodulatory effects. The cytotoxicity of compounds 1–9 and 11–16 was tested against five human cancer cell lines, human myeloid leukemias HL-60, human hepatocellular carcinoma SMMC-7721, human lung cancer A549, human breast adenocarcinoma MCF-7, and human colon cancer SW480, as well as the immortalized non-cancerous human bronchial epithelial cell line Beas-2B by the MTT method, in vitro. The results (Table 5) showed that compounds 1–3 had weak cytotoxicity, and compounds 4–9 and 11–16 showed no obvious cytotoxicity (IC50 > 40 lM). 3. Concluding remarks Nine new germacrane sesquiterpene lactones (1–9) and one known seco-germacrane sesquiterpenoid (10) were isolated from

Table 5 Cytotoxicity (IC50 in lM) of compounds 1–9 and 11–16 against five human cancera and one non-cancerous human Beas-2B cell lines. Compounds

HL-60

SMMC-7721

A-549

MCF-7

SW480

BEAS-2B

1 2 3 4 5 6 7 8 9 11 12 13 14 15 16 Cisplatinb Paclitaxelb

33.74 >40 22.89 >40 >40 >40 >40 >40 >40 >40 >40 >40 >40 >40 >40 3.29 40 >40 >40 >40 >40 >40 >40 >40 >40 >40 >40 >40 9.62 40 26.13 >40 >40 >40 >40 >40 >40 >40 >40 >40 >40 >40 >40 9.98 40 >40 34.90 >40 >40 >40 >40 >40 >40 >40 >40 >40 >40 >40 >40 15.92 40 >40 >40 >40 >40 >40 >40 >40 >40 >40 >40 >40 >40 >40 >40 14.43 40 15.44 NT NT NT NT NT NT NT NT NT NT NT NT 11.11 0.88

NT: not tested. a Cell lines: HL-60 acute leukemia; SMMC-7721 liver cancer; A-549 lung cancer; MCF-7 breast cancer; SW480 colon cancer; BEAS-2B normal lung epithelial cells. b Positive control.

S. scapiformis. Compounds 1–9 represent the first examples of germacrane sesquiterpenoids with a D3-15,6-lactone moiety. In addition, six new 6,15-diol germacrane derivatives (11–16) were obtained by chemical transformations. Compounds 1, 3, 4, 6–9, 11, 12, and 15 displayed inhibitory activities against LPS-induced murine B cell proliferation, and compounds 7 and 11 exhibited significant inhibitory activity against ConA-induced murine T cell proliferation. Compounds 1–3 exerted weak cytotoxicity, and compounds 4–9 and 11–16 showed no obvious cytotoxicity (IC50 > 40 lM). 4. Experimental section 4.1. General experimental procedures Melting points (uncorrected) were determined on a Beijing Tech X-5 microscopic melting point apparatus. Optical rotations were measured on a Perkin-Elmer PE-341LC polarimeter. UV spectra were recorded using a Varian Cary 50 spectrophotometer. CD spectra were performed on a JASCO J-810 spectrometer. IR spectra were recorded on a Bruker Vertex 70 FT-IR spectrophotometer. NMR spectra were acquired using Bruker AM-400 spectrometers, with the residual CDCl3 (dH 7.24/dC 77.2) and pyridine-d5 (dH 8.74/dC 150.3) signals as references. HRESIMS data were obtained using an API QSTAR Pulsar spectrometer or Agilent 6520 Accurate-Mass Q-TOF LC/MS. Column chromatography (CC) was performed using silica gel (100–200 mesh and 200–300 mesh, Qingdao Marine Chemical Inc., China), Amberchrom CG161 M (75 lm, Rohm and Haas, America), ODS (50 lm, YMC, Japan), and Sephadex LH-20 (Pharmacia Biotech AB, Uppsala Sweden). Semipreparative HPLC was performed on an Agilent 1100 liquid chromatography with an YMC (250  10 mm, 5 lm) column. Solvents were distilled prior to use, and spectroscopic grade solvents were used. TLC was carried out on precoated silica gel GF254 plates. 4.2. Plant material Whole plants of S. scapiformis were collected at about 1500 m altitude, latitude 30°100 39.4700 North and longitude 109°440 42.9300 East in Changlinggang, Enshi, Hubei Province, People0 s Republic of China, in September 2010, and authenticated by Dr. Jianping Wang of Huazhong University of Technology and Science. A voucher specimen (No. 2010-09-02) is deposited in the herbarium of

Y. Lai et al. / Phytochemistry 96 (2013) 378–388

Hubei Key Laboratory of Natural Medicinal Chemistry and Resource Evaluation, Tongji Medical College, Huazhong University of Technology and Science.

385

1 and 3; (+)-HRESIMS: m/z 409.2199 [M+H]+ (calcd for C22H33O7, 409.2226). 4.6. Scapiformolactone B (2)

4.3. Extraction and isolation Air-dried and powdered whole plants of S. scapiformis (27 kg) were extracted with Me2CO (3  150 L) at room temperature for 7 days. The combined extract was concentrated under reduced pressure. The resulting residue (1300 g) was subjected to a silica gel (100–200 mesh) CC eluting with a gradient of petroleum ether (b.p. 60–90 °C)–Me2CO–MeOH (1:0:0?0:1:0?0:0:1) to give seven fractions (A–G). Fractions C–E were decolorized on Amberchrom GC161M CC (eluted with 70%?100% EtOH–H2O) and subjected to ODS eluting with a gradient of MeOH–H2O (4:6?1:0) to obtain three non-triterpene subfractions C0 –E0 , respectively. Subfraction C0 (12.5 g) was further purified using silica gel (125 g) CC eluting with CHCl3–Me2CO (1:0?10:1) to provide three subfractions (C0 1–C0 3). Subfraction C0 1 was subjected to Sephadex LH-20 eluting with MeOH and then recrystallized in MeOH to afford compounds 1 (2 g, 0.074%) and 3 (500 mg, 0.018%) directly. Compound 10 (15 mg, 0.00055%, tR 30.5 min) was also obtained from subfraction C0 1 by semi-preparative HPLC (MeOH-H2O = 65:35, flow rate: 2 mL/ min). Fraction D0 (8.5 g) was subjected to silica gel CC to afford four subfractions (D0 1–D0 4). Subfraction D0 2 (5.3 g) was purified over Sephadex LH-20 (MeOH) to give subfractions D0 2.1, D0 2.2, D0 2.3, and D0 2.4. Subfraction D0 2.2 (2.1 g) was subjected to repeated chromatography including silica gel CC, ODS, and Sephadex LH-20, and finally semi-preparative HPLC to afford compounds 4 (54 mg, 0.002%), 5 (10 mg, 0.00037%), 8 (28 mg, 0.001%), and 9 (4 mg, 0.00015%). Subfraction D0 3 was subjected to Sephadex LH-20 CC (MeOH) to give subfractions D0 3.1, D0 3.2, and D0 3.3. Compound 2 (800 mg, 0.03%) was obtained from subfraction D0 3 by crystallization from MeOH. Fraction E0 was fractionated by repeated silica gel and Sephadex LH-20 CC, and compounds 6 (100 mg, 0.0037%, tR 25 min) and 7 (8 mg, 0.0003%, tR 29 min) were obtained by semi-preparative HPLC eluted with MeOH–H2O (45:55, flow rate: 2 mL/min).

Colorless crystals (MeOH); m.p. 135.8–136.8 °C; ½a22:5 62.0 (c D 0.10, MeOH); UV (MeOH) kmax (log e) 204 (3.83), 225 (3.87) nm; CD (MeOH) 238 (De +9.83), 288 (De 9.91) nm; IR (film) vmax 3439, 2972, 2937, 1760, 1715, 1679, 1461, 1374, 1329, 1246, 1168, 1070, 983, 743 cm1; for 1H and 13C NMR spectroscopic data, see Tables 1 and 3; ()-HRESIMS: m/z 365.1972 [MH] (calcd for C20H29O6, 365.1964). 4.7. Scapiformolactone C (3) Colorless crystals (MeOH); m.p. 173.2–174.8 °C; ½a22:5 32.2 (c D 0.12, MeOH); UV (MeOH) kmax (log e) 205 (4.21), 218 (4.23); CD (MeOH) 251 (De +2.71), 288 (De 6.59) nm; IR vmax 2930, 1758, 1708, 1676, 1457, 1380, 1337, 1261, 1136, 1069, 981, 733 cm1; for 1H and 13C NMR spectroscopic data, see Tables 1 and 3; (+)HRESIMS: m/z 349.2009 [M+H]+ (calcd for C20H29O5, 349.2015). 4.8. Scapiformolactone D (4) Colorless gum; ½a22:5 +143.8 (c 0.17, MeOH); UV (MeOH) kmax D (log e) 217 (3.95) nm; IR (film) vmax 3423, 2964, 1737, 1679, 1460, 1375, 1241, 1171, 1077, 983, 737 cm1; for 1H and 13C NMR spectroscopic data, see Tables 1 and 3; (+)-HRESIMS: m/z 411.2382 [M+H]+ (calcd for C22H35O7, 411.2383). 4.9. Scapiformolactone E (5) Colorless gum; ½a22:5 +34.7 (c 0.12, MeOH); UV (MeOH) kmax D (log e) 216 (3.94) nm; IR (film) vmax 3448, 2961, 1737, 1678, 1462, 1376, 1316, 1242, 1171, 1078, 1032, 993, 970, 747 cm1; for 1H and 13C NMR spectroscopic data, see Tables 1 and 3; (+)HRESIMS: m/z 411.2383 [M+H]+ (calcd for C22H35O7, 411.2383).

4.4. Reduction of compounds 1–9 Compound 1 (100.8 mg, 0.25 mmol), CeCl37H2O (92.0 mg, 0.25 mmol), and 5 mL MeOH were added to a round bottomed flask, and cooled to 0 °C, then NaBH4 (9.4 mg, 0.25 mmol) was added slowly when stirring. The mixture was stirred at 0 °C for 2 h. After confirmation of the consumption of the starting material by TLC, the reaction was quenched with Me2CO (2 mL) (Fujiwara and Hayashi, 2008). The solvent was evaporated under reduced pressure. The residue was then suspended in H2O and extracted with EtOAc (3 mL  3). The dry combined organic layers were subjected to silica gel CC and semi-preparative HPLC to give the reduction products 4 (39 mg), 5 (11 mg), 11 (33 mg), and 12 (9 mg), respectively. In the same way, reduction of 2 (80 mg) by NaBH4 afforded 6 (30 mg), 7 (7 mg), 13 (24 mg), and 14 (6 mg), whereas, the reduction of 3 (70 mg) by NaBH4 gave 8 (25 mg), 9 (6 mg), 15 (23 mg), and 16 (5.6 mg). Compounds 11–16 were also obtained by reduction of compounds 4–9 with NaBH4, respectively (Scheme 1).

4.10. Scapiformolactone F (6) Colorless gum; ½a22:5 +148.3 (c 0.12, MeOH); UV (MeOH) kmax D (log e) 217 (3.97) nm; IR (film) vmax 3402, 2968, 1751, 1678, 1461, 1377, 1332, 1232, 1182, 1115 1062, 983, 737 cm1; for 1H and 13C NMR spectroscopic data, see Tables 2 and 3; (+)-HRESIMS: m/z 369.2277 [M+H]+ (calcd for C20H33O6, 369.2277). 4.11. Scapiformolactone G (7) Colorless gum; ½a22:5 +26.9 (c 0.13, MeOH); UV (MeOH) kmax D (log e) 216 (3.97) nm; IR (film) vmax 3395, 2969, 1751, 1676, 1461, 1377, 1320, 1265, 1226, 1177, 1108, 1032, 994, 971, 919, 748 cm1; for 1H and 13C NMR spectroscopic data, see Tables 2 and 3; (+)-HRESIMS: m/z 369.2279 [M+H]+ (calcd for C20H33O6, 369.2277). 4.12. Scapiformolactone H (8)

4.5. Scapiformolactone A (1) Colorless crystals (MeOH); m.p. 130.0–131.0 °C; ½a22:5 30.5 (c D 0.23, MeOH); UV (MeOH) kmax (log e) 205 (3.91), 229 (3.89) nm; CD (MeOH) 253 (De +2.60), 288 (De 7.78) nm; IR vmax 2974, 2937, 1766, 1734, 1715, 1680, 1460, 1375, 1336, 1243, 1171, 1071, 984, 742 cm1; for 1H and 13C NMR spectroscopic data, see Tables

Colorless gum; ½a22:5 +184.4 (c 0.15, MeOH); UV (MeOH) kmax D (log e) 214 (4.32) nm; IR (film) vmax 3392, 2961, 1758, 1707, 1650, 1461, 1388, 1337, 1263, 1230, 1143, 1075, 1021, 921, 735 cm1; for 1H and 13C NMR spectroscopic data, see Tables 2 and 3; (+)-HRESIMS: m/z 351.2168 [M+H]+ (calcd for C20H31O5, 351.2171).

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4.13. Scapiformolactone I (9) Colorless gum; ½a22:5 +53.4 (c 0.14, MeOH); UV (MeOH) kmax D (log e) 216 (4.31) nm; IR (film) vmax 3400, 2958, 1759, 1707, 1650, 1463, 1387, 1343, 1263, 1227, 1143, 1075, 1030, 993, 973, 922, 740 cm1; for 1H and 13C NMR spectroscopic data, see Tables 2 and 3; (+)-HRESIMS: m/z 351.2172 [M+H]+ (calcd for C20H31O5, 351.2171). 4.14. (1S,6S,7S,8S,10R,20 R,30 R)-8-O-(30 -O-acetyl-20 -methylbutyryl)1,6,15-trihydroxy-germacra-3E-ene (11) Colorless gum; ½a22:5 +100.7 (c 0.14, MeOH); UV (MeOH) kmax D (log e) 204 (3.72) nm; IR (film) vmax 3347, 2960, 1732, 1460, 1377, 1242, 1197, 1138, 1078, 1034, 988, 899, 856 cm1; 1H NMR (CDCl3, 400 MHz) d 5.47 (1H, dd, J = 11.5, 5.7 Hz, H-3), 5.18 (1H, dd, J = 12.2, 5.5 Hz, H-8), 5.04 (1H, m), 4.38 (1H, m, H-6), 4.04 (1H, d, J = 12.3 Hz, H-15a), 3.99 (1H, d, J = 12.3 Hz, H-15b), 3.66 (1H, dd, J = 9.7, 5.1 Hz, H-1), 3.00–3.09 (1H, overlapped, H5a), 2.61 (1H, m, H-20 ), 2.42 (1H, dd, J = 22.3, 9.7 Hz, H-2a), 2.36 (1H, dd, J = 15.1, 3.2 Hz, H-5b), 2.23 (2H, m, H-2b, 11), 1.97 (3H, s, H-200 ), 1.81 (1H, t, J = 12.2 Hz, H-9a), 1.75 (1H, dd, J = 9.5, 3.7 Hz, H-7), 1.48 (1H, m, H-10), 1.21 (1H, m, H-9b), 1.18 (3H, d, J = 6.4 Hz, H-40 ), 1.08 (3H, d, J = 7.1 Hz, H-50 ), 0.98 (3H, d, J = 6.9 Hz, H-14), 0.91 (3H, d, J = 6.9 Hz, H-13), 0.72 (3H, d, J = 7.0 Hz, H-12); 13C NMR (CDCl3, 100 MHz) d 172.8 (C-10 ), 170.8 (C-100 ), 139.1 (C-4), 126.8 (C-3), 74.7 (C-1), 71.9 (C-30 ), 70.6 (C-8), 69.5 (C-15), 67.7 (C-6), 44.9 (C-20 ), 44.4 (C-7), 37.3 (C-5), 34.0 (C2), 31.7 (C-9), 31.0 (C-10), 26.5 (C-11), 22.1 (C-13), 21.5 (C-200 ), 18.1 (C-12, 14), 16.9 (C-40 ), 12.3 (C-50 ); (+)-HRESIMS: m/z 437.2493 [M+Na]+ (calcd for C22H38O7Na, 437.2515). 4.15. (1R,6S,7S,8S,10R,20 R,30 R)-8-O-(30 -O-acetyl-20 -methylbutyryl)1,6,15-trihydroxy-germacra-3E-ene (12) Colorless gum; ½a22:5 +10.7 (c 0.23, MeOH); UV (MeOH) kmax D (log e) 204 (3.69) nm; IR (film) vmax 3330, 2957, 1734, 1460, 1376, 1242, 1196, 1137, 1079, 1035, 952, 907, 861 cm1; 1H NMR (CDCl3, 400 MHz) d 5.74 (1H, dd, J = 11.3, 5.8 Hz, H-3), 5.18 (1H, dd, J = 12.2, 5.2 Hz, H-8), 5.07 (1H, m, H-30 ), 4.33 (1H, m, H6), 4.12 (1H, d, J = 12.2 Hz, H-15a), 4.02 (1H, d, J = 12.2 Hz, H15b), 3.45 (1H, m, H-1), 2.97 (1H, dd, J = 14.9, 2.8 Hz, H-5a), 2.67 (1H, m, H-2a), 2.63 (1H, m, H-20 ), 2.44 (1H, dd, J = 14.9, 2.0 Hz, H5b), 2.24 (2H, m, H-2b, 11), 1.98 (3H, s, H-200 ), 1.88 (1H, dd, J = 9.6, 2.9 Hz, H-7), 1.66 (1H, t, J = 12.2 Hz, H-9a), 1.42 (1H, m, H10), 1.20 (1H, m, overlapped, H-9b), 1.19 (3H, d, J = 6.4 Hz, H-40 ), 1.10 (3H, d, J = 7.1 Hz, H-50 ), 1.02 (3H, d, J = 6.4 Hz, H-14), 0.92 (3H, d, J = 7.0 Hz, H-13), 0.74 (3H, d, J = 7.0 Hz, H-12); 13C NMR (CDCl3, 100 MHz) d 172.8 (C-10 ), 170.6 (C-100 ), 139.2 (C-4), 126.6 (C-3), 75.9 (C-1), 71.8 (C-30 ), 69.9 (C-8), 69.8 (C-15), 67.6 (C-6), 44.8 (C-20 ), 44.5 (C-7), 37.6 (C-5), 36.0 (C-9), 33.1 (C-10), 32.8 (C2), 26.7 (C-11), 22.0 (C-13), 21.5 (C-200 ), 18.0 (C-12), 17.7(C-14), 16.9 (C-40 ), 12.3 (C-50 ); (+)-HRESIMS: m/z 415.2693 [M+H]+ (calcd for C22H39O7, 415.2696). 4.16. (1S,6S,7S,8S,10R,20 R,30 R)-8-O-(30 -O-hydroxy-20 -methylbutyryl)1,6,15-trihydroxy-germacra-3E-ene (13) Colorless gum; ½a22:5 +81.3 (c 0.14, MeOH); UV (MeOH) kmax D (log e) 204 (3.81) nm; IR (film) vmax 3343, 2964, 1708, 1460, 1382, 1319, 1266, 1194, 1113, 1075, 1034, 988, 895, 855 cm1; 1 H NMR (CDCl3, 400 MHz) d 5.49 (1H, dd, J = 11.6, 5.7 Hz, H-3), 5.24 (1H, dd, J = 12.6, 5.4 Hz, H-8), 4.44 (1H, m, H-6), 4.07 (1H, d, J = 12.4 Hz, H-15a), 4.02 (1H, d, J = 12.4 Hz, H-15b), 3.85 (1H, m, H-30 ), 3.67 (1H, ddd, J = 10.7, 5.1, 1.3 Hz, H-1), 3.06 (1H, dd, J = 15.1, 3.7 Hz, H-5a), 2.45 (1H, m, H-2a), 2.31–2.40 (1H, over-

lapped, H-5b), 2.31 (1H, m, H-20 ), 2.26 (1H, m, H-2b), 2.21 (1H, m, H-11), 1.85 (1H, t, J = 12.6 Hz, H-9a), 1.76 (1H, dd, J = 8.6, 4.0 Hz, H-7), 1.50 (1H, m, H-10), 1.28 (1H, ddd, J = 14.3, 9.3, 5.5 Hz, H-9b), 1.19 (3H, d, J = 6.3 Hz, H-40 ), 1.12 (3H, d, J = 7.2 Hz, H-50 ), 1.02 (3H, d, J = 6.9 Hz, H-14), 0.94 (3H, d, J = 6.9 Hz, H-13), 0.79 (3H, d, J = 7.0 Hz, H-12); 13C NMR (CDCl3, 100 MHz) d 175.1 (C-10 ), 139.0 (C-4), 126.8 (C-3), 75.0 (C-1), 70.8 (C-8), 69.7 (C-30 , 15), 67.8 (C-6), 47.5 (C-20 ), 44.5 (C-7), 37.3 (C-5), 34.2 (C-2), 31.8 (C-9), 31.1 (C-10), 26.5 (C-11), 22.3 (C-13), 21.1 (C-40 ), 18.6 (C12), 18.3(C-14), 14.0 (C-50 ); (+)-HRESIMS: m/z 395.2412 [M+Na]+ (calcd for C20H36O6Na, 395.2410). 4.17. (1R,6S,7S,8S,10R,20 R,30 R)-8-O-(30 -O-hydroxy-20 -methylbutyryl)1,6,15-trihydroxy-germacra-3E-ene (14) Colorless gum; ½a22:5 +4.1 (c 0.10, MeOH); UV (MeOH) kmax D (log e) 204 (3.78) nm; IR (film) vmax 3330, 2963, 1709, 1461, 1383, 1324, 1265, 1191, 1111, 1036, 962, 911 cm1; 1H NMR (CDCl3, 400 MHz) d 5.76 (1H, dd, J = 11.4, 5.8 Hz, H-3), 5.22 (1H, dd, J = 12.4, 5.2 Hz, H-8), 4.35 (1H, m, H-6), 4.14 (1H, d, J = 12.3 Hz, H-15a), 4.04 (1H, d, J = 12.3 Hz, H-15b), 3.87 (1H, m, H-30 ), 3.47 (1H, dt, J = 10.4, 2.9 Hz, H-1), 2.99 (1H, dd, J = 15.8, 2.7 Hz, H-5a), 2.69 (1H, m, H-2a), 2.43 (1H, J = 15.8, 3.4 Hz, H-5b), 2.37 (1H, m, H-20 ), 2.28 (1H, m, H-11), 2.23 (1H, m, H-2b), 1.89 (1H, dd, J = 9.4, 3.3 Hz, H-7), 1.70 (1H, t, J = 12.4 Hz, H-9a), 1.43 (1H, m, H-10), 1.27 (1H, ddd, J = 11.5, 8.4, 3.6 Hz, H-9b), 1.20 (3H, d, J = 6.3 Hz, H-40 ), 1.13 (3H, d, J = 7.2 Hz, H-50 ), 1.04 (3H, d, J = 6.4 Hz, H-14), 0.95 (3H, d, J = 7.0 Hz, H-13), 0.79 (3H, d, J = 7.0 Hz, H-12); 13C NMR (CDCl3, 100 MHz) d 175.1 (C-10 ), 139.1 (C-4), 126.7 (C-3), 75.8 (C-1), 70.2 (C-8), 70.0 (C-15), 69.8 (C-30 ), 67.7 (C-6), 47.5 (C-20 ), 44.6 (C-7), 37.6 (C-5), 36.1 (C-9), 33.2 (C10), 32.8 (C-2), 26.7 (C-11), 22.1 (C-13), 21.0 (C-40 ), 18.3 (C-12), 17.7(C-14), 13.9 (C-50 ); (+)-HRESIMS: m/z 373.2573 [M+H]+ (calcd for C20H36O6Na, 373.2590). 4.18. (1S,6S,7S,8S,10R)-8-O-tigloyl-1,6,15-trihydroxy-germacra-3Eene (15) Colorless crystals (MeOH); m.p. 187.3–188.5 °C; ½aD22:5 +152.5 (c 0.12, MeOH); UV (MeOH) kmax (log e) 205 (4.24) nm; IR (film) vmax 3280, 2962, 1710, 1690, 1652, 1467, 1385, 1344, 1262, 1157, 1113, 1074, 1030, 975, 897, 854, 736 cm1; 1H NMR (MeOD, 400 MHz) d 6.78 (1H, m, H-30 ), 5.56 (1H, dd, J = 11.5, 5.7 Hz, H-3), 5.25 (1H, dd, J = 13.2, 5.3 Hz, H-8), 4.43 (1H, m, H-6), 4.02 (2H, s, H-15), 3.65 (1H, ddd, J = 10.8, 5.1, 1.7 Hz, H-1), 3.18 (1H, dd, J = 14.5, 4.1 Hz, H-5a), 2.51 (1H, dd, J = 24.3, 11.5 Hz, H-2a), 2.35 (1H, dd, J = 14.5, 3.2 Hz, H-5b), 2.32 (1H, m, H-11), 2.23 (1H, m, H-2b), 1.94 (1H, t, J = 13.2 Hz, H-9a), 1.84 (4H, m, H-50 , 7), 1.81 (3H, m, H-40 ), 1.57 (1H, m, H-10), 1.26 (1H, m, H-9b), 1.04 (3H, d, J = 6.9 Hz, H-14), 0.95 (3H, d, J = 7.0 Hz, H-13), 0.75 (3H, d, J = 7.0 Hz, H-12); 13C NMR (MeOD, 100 MHz) d 168.9 (C-10 ), 140.5 (C-4), 138.7 (C-30 ), 130.6 (C-20 ), 127.2 (C-3), 75.5 (C-1), 71.7 (C-8), 69.6 (C-15), 68.4 (C-6), 45.8 (C-7), 38.9 (C-5), 35.0 (C-2), 32.9 (C-9), 32.3 (C-10), 27.9 (C-11), 22.5 (C-13), 18.5 (C-12, 14), 14.5 (C-40 ), 12.3 (C-50 ); (+)-HRESIMS: m/z 355.2475 [M+H]+ (calcd for C20H35O5, 355.2484). 4.19. (1R,6S,7S,8S,10R)-8-O-tigloyl-1,6,15-trihydroxy-germacra-3Eene (16) Colorless crystals (MeOH); m.p. 164.8–165.4 °C; ½a22:5 +44.2 (c D 0.14, MeOH); UV (MeOH) kmax (log e) 204 (4.21) nm; IR (film) vmax 3326, 2957, 1691, 1648, 1469, 1447, 1386, 1341, 1270, 1142, 1074, 1034, 961, 905, 737 cm1; 1H NMR (MeOD, 400 MHz) d 6.85 (1H, m, H-30 ), 5.78 (1H, dd, J = 11.8, 5.8 Hz, H-3), 5.23 (1H, dd, J = 12.3, 5.0 Hz, H-8), 4.38 (1H, m, H-6), 4.08 (1H, d, J = 12.5 Hz, H-15a), 4.03 (1H, d, J = 12.5 Hz, H-15b), 3.39 (1H, m, H-1), 3.13 (1H, dd,

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J = 14.5, 2.3 Hz, H-5a), 2.79 (1H, t, J = 11.8 Hz, H-2a), 2.38 (1H, d, J = 14.5 Hz, H-5b), 2.34 (1H, m, H-11), 2.17 (1H, m, H-2b), 1.98 (1H, dd, J = 9.7, 3.3 Hz, H-7), 1.83 (4H, m, H-50 , 9a), 1.81 (3H, m, H-40 ), 1.46 (1H, m, H-10), 1.21 (1H, m, H-9b), 1.04 (3H, d, J = 6.4 Hz, H-14), 0.95 (3H, d, J = 7.0 Hz, H-13), 0.75 (3H, d, J = 6.9 Hz, H-12); 13C NMR (MeOD, 100 MHz) d 168.8 (C-10 ), 140.3 (C-4), 138.8 (C-30 ), 130.5 (C-20 ), 127.2 (C-3), 76.5 (C-1), 71.1 (C8), 70.0 (C-15), 68.3 (C-6), 45.8 (C-7), 39.0 (C-5), 37.0 (C-9), 34.2 (C-10), 33.8 (C-2), 27.9 (C-11), 22.4 (C-13), 18.4 (C-12), 18.2 (C14) 14.5 (C-40 ), 12.2 (C-50 ); (+)-HRESIMS: m/z 377.2300 [M+H]+ (calcd for C20H34O5Na, 377.2304).

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Dc = 1.193 Mg/m3, F(000) = 376. A total of 4937 reflections were collected in the range 1.92° < h < 25.00°, with 3112 independent reflections [R(int) = 0.0142]; completeness to hmax was 99.8%. The structure was solved by direct methods and refined by full-matrix least-squares on F2, with anisotropic temperature factors for nonhydrogen atoms converging at final R indices [I > 2r(I)], R1 = 0.0343, wR2 = 0.0879; R indices (all data), R1 = 0.0368, wR2 = 0.0905. CCDC 906639 contains the supplementary crystallographic data for the structure of 3. These data can be obtained free of charge via http://www.ccdc.cam.ac.uk/conts/retrieving.html (or from the CCDC, 12 Union Road, Cambridge CB2 1EZ, UK; fax:+44 1223 336033; e-mail: [email protected]).

4.20. X-ray crystallographic analysis of scapiformolactone A (1) A suitable crystal (0.10 mm  0.10 mm  0.05 mm) was used for analysis. The data were measured using a Bruker SMART APEX-II CCD diffractometer, using Cu Ka graphite-monochromated radiation (k = 1.54178 Å). Crystal data: C22H32O7, M = 408.48, monoclinic, space group P21, unit cell dimensions a = 9.8603 (3) Å, b = 9.7544 (4) Å, c = 11.3228 (4) Å, a = 90.00°, b = 90.286 (2)°, c = 90.00°, V = 1088.18 (7) Å3, T = 100(2) K, Z = 2, Dc = 1.247 g/cm3, F(0 0 0) = 440. A total of 4671 reflections were collected in the range 3.91° < h < 60.00°, with 2360 independent reflections [R(int) = 0.0922]; completeness to hmax was 88.9%. The structure was solved by direct methods and refined by full-matrix leastsquares on F2, with anisotropic temperature factors for non-hydrogen atoms converging at final R indices [I > 2r(I)], R1 = 0.0541, wR2 = 0.1372; R indices (all data), R1 = 0.0546, wR2 = 0.1380. The absolute configuration was determined on the basis of the Flack parameter 0.2(3). CCDC 906637 contains the supplementary crystallographic data for the structure of 1. These data can be obtained free of charge via http://www.ccdc.cam.ac.uk/conts/retrieving.html (or from the CCDC, 12 Union Road, Cambridge CB2 1EZ, UK; fax:+44 1223 336033; e-mail: [email protected]). 4.21. X-ray crystallographic analysis of scapiformolactone B (2) A suitable crystal (0.30 mm  0.20 mm  0.10 mm) was used for analysis. The data were measured using a Bruker SMART APEX-II CCD diffractometer, using Mo Ka graphite-monochromated radiation (k = 0.71073 Å). Crystal data: C20H30O6, M = 366.44, monoclinic, space group P21, unit cell dimensions a = 10.709 (2) Å, b = 6.7036 (14) Å, c = 14.532 (3) Å, a = 90.00°, b = 99.015 (2)°, c = 90.00°, V = 1030.3 (4) Å3, T = 298(2) K, Z = 2, Dc = 1.181 Mg/m3, F(0 0 0) = 396. A total of 8390 reflections were collected in the range 2.56° < h < 26.41°, with 4116 independent reflections [R(int) = 0.0200]; completeness to hmax was 99.2%. The structure was solved by direct methods and refined by full-matrix leastsquares on F2, with anisotropic temperature factors for non-hydrogen atoms converging at final R indices [I > 2r(I)], R1 = 0.0337, wR2 = 0.0739; R indices (all data), R1 = 0.440, wR2 = 0.0801. CCDC 906638 contains the supplementary crystallographic data for the structure of 2. These data can be obtained free of charge via http://www.ccdc.cam.ac.uk/conts/retrieving.html (or from the CCDC, 12 Union Road, Cambridge CB2 1EZ, UK; fax:+44 1223 336033; e-mail: [email protected]). 4.22. X-ray crystallographic analysis of scapiformolactone C (3) A suitable crystal (0.45 mm  0.25 mm  0.23 mm) was used for analysis. The data were measured using a Bruker SMART APEX-II CCD diffractometer, using Mo Ka graphite-monochromated radiation (k = 0.71073 Å). Crystal data: C20H28O5, M = 348.42, monoclinic, space group P21, unit cell dimensions a = 9.7972(13) Å, b = 9.3257(12) Å, c = 10.9907 (14) Å, a = 90.00°, b = 105.009 (2)°, c = 90.00°, V = 969.9 (2) Å3, T = 153(2) K, Z = 2,

4.23. Conformational analyses of compound 4 by quantum mechanical calculations The conformational analyses were performed by exhaustively exploring the conformational space. All possible structures of compound 4 were probed by OpenEye OMEGA program (OMEGA, 2012) through considering all possible combinations of single bond rotamers. The root mean square (RMS) distance was used to remove duplicated conformations. All possible conformations were clustered into individual groups in such a way that the RMS distance between any two conformations of the group is less than 0.5 Å (the default in OMEGA program). The conformation with the lowest energy was kept as a unique conformer whereas all other conformations in the same individual group were considered as duplicated conformations and therefore were removed. Two hundred trial conformations were initially generated by OpenEye OMEGA program. All these trial conformations were subjected to the semi-empirical PM3 quantum mechanical optimizations by GAMESS program (version Aug. 11, 2011 R1) (Schmidt et al., 1993), producing a total of 73 trial conformations. All these 73 trial conformations were further optimized at B3LYP/ 6-31G⁄ level of theory by GAMESS program, making a total of 40 stable conformations. By looking into the RMS distances over the atoms on the rings, the 40 stable conformations were classified into two groups, resulting in two conformers concerning the conformation of rings. The transition state associated with the interconversion between the two conformers was obtained by the saddle point search at B3LYP/6-31G⁄ level of theory and was verified by the harmonic normal mode calculations. The solvation effects in pyridine were calculated for the three key conformations by the SMD (Marenich et al., 2009) solvation model implemented in GAMESS program. 4.24. Lymphocyte proliferation test The effects of compounds 1–9 and 11–16 on B and T cell were evaluated according to the method as described by Yao et al. (2005). Statistical analysis: data are presented as mean ± SEM. One-way analysis of variance followed by Student’s t-test was used to determine the difference between two groups where appropriate. A P value < 0.05 was considered significant. 4.25. Cytotoxicity assay The cytotoxicity of compounds 1–9 and 11–16 against HL-60, SMMC-7721, A-549, MCF-7, and SW480 cell lines was assessed using the MTT method as described by Pan et al. (2010). Acknowledgements We are grateful to Dr. J. Wang at Huazhong University of Science and Technology for the authentification of this plant material, and Dr. X. Meng at Central China Normal University and Dr. F. Yin

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at Huaihai Institute of Technology, for their single crystal X-ray diffraction data collection and analysis. This work was financially supported by the Hubei Key Laboratory Foundation of Natural Medicinal Chemistry and Resource Evaluation, Huazhong University of Science and Technology (2010-3), the Fundamental Research Funds for the Central Universities (HUST: 2012QN003), Scientific Research Foundation for the Returned Oversea Chinese Scholars, State Education Ministry of China (2010-1561, 40th), and Program for New Century Excellent Talents in University, State Education Ministry of China (NCET-2008-0224). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.phytochem.2013. 10.003. References Ali, M.S., Ibrahim, S.A., Ahmed, S., Lobkovsky, E., 2007. A new germacranolide and a new ceramide from Salvia nubicola (Lamiaceae). Z. Naturforsch. B 62, 1333–1338. Fang, Z.-X., Liao, C.-L., 2006. Medicinal Flora of Enshi, Hubei, vol. 2. Hubei Science and Technology Press, Wuhan, pp. 292–293. Fraga, B.M., 2012. Natural sesquiterpenoids. Nat. Prod. Rep. 29, 1334–1366. Fujiwara, T., Hayashi, M., 2008. Efficient synthesis of rare sugar d-allal via reversal of diastereoselection in the reduction of protected 1,5-anhydrohex-1-en-3-uloses: protecting group dependence of the stereoselection. J. Org. Chem. 73, 9161–9163. González, A.G., Grillo, T.A., Ravelo, A.G., Luis, J.G., Rodriguez, M.L., Calle, J., Rivera, A., 1989. Study of Salvia palaefolia: absolute configuration of glechomafuran. J. Nat. Prod. 52, 1307–1310. Grande, M., Bellido, I.S., Torres, P., Piera, F., 1992. 9-Hydroxynerolidol esters and bicyclic sesquiterpenoids from Dittrichia viscosa. J. Nat. Prod. 55, 1074–1079. Li, X.-W., Ian, C.H., 1994. Lamiaceae. In: Wu, Z.Y., Raven, P.H., Hong, D.Y. (Eds.), Flora of China, vol. 17. Science Press and Missouri Botanical Garden Press, Beijing, China, and St. Louis, United States, pp. 50–299.

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