Antiproliferative Sesquiterpenoids from Ligularia rumicifolia with

Article Cite This: J. Nat. Prod. 2018, 81, 1992−2003

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Antiproliferative Sesquiterpenoids from Ligularia rumicifolia with Diverse Skeletons Ye Ye,†,‡,∇ Drolma Dawa,§,∇ Guang-Hui Liu,†,‡ Min Zhao,† Dorje Tseden,§ Yu-Cheng Gu,∥ Li-Sheng Ding,† Zhi-Xing Cao,*,⊥ and Yan Zhou*,† †

Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu 610041, People’s Republic of China University of Chinese Academy of Sciences, Beijing 100049, People’s Republic of China § Tibet Autonomous Region Institute for Food and Drug Control, Lhasa 850000, People’s Republic of China ∥ Syngenta Jealott’s Hill International Research Centre, Berkshire RG42 6EY, U.K. ⊥ Pharmacy College, Chengdu University of Traditional Chinese Medicine, Chengdu 611137, People’s Republic of China

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‡

S Supporting Information *

ABSTRACT: Twenty-two new sesquiterpenoids with four skeletal types and 15 known analogues were isolated from the whole plants of Ligularia rumicifolia. The structures of the isolates were elucidated based on comprehensive spectroscopic data analysis. Compound 1 is a C14 nor-sesquiterpenoid featuring a 6/6/6 tricyclic skeleton with a 9,13-ether bridge. The absolute configuration of 2 was established through single-crystal X-ray diffraction data. Compounds 13−16 exhibited in vitro antiproliferative activity against the four human tumor cell lines A-549, HGC-27, HeLa, and MV4-11. Specifically, compounds 13 and 16 showed antiproliferative activity against the MV4-11 cell line with IC50 values of 0.5 ± 0.2 and 1.1 ± 0.5 μM, respectively.

S

sore throat and as an anti-inflammatory medicine among the Lhoba people in Milin County, Tibet.10 Previous phytochemical investigations of this genus from Xinjiang, Sichuan, Yunnan, and Hubei Provinces of China, led to the discovery of a multitude of sesquiterpenoids with multiple biological activities. In searching for more bioactive and structurally diverse sesquiterpenoids, as well as in consideration of the peculiar habitat of L. rumicifolia, collected from the Lhasa County at an altitude of 5000 m in Tibet, a systematic study of sesquiterpenoids metabolites from L. rumicifolia and their biological activities was carried out. As a result, 22 new and 15 known sesquiterpenoids involving four different skeletons were obtained. Compound 1 is a rare C14 noreudesmane sesquiterpenoid featuring a 6/6/6 tricyclic skeleton with an oxygen bridge. Compounds 13−16 and 29 showed significant in vitro antiproliferative activity against the human tumor cell lines HGC-27, A-549, HeLa, and MV4-11. Herein,

esquiterpenoids, possessing a variety of different carbon cores, including eremophilanes, bisabolanes, oplopanes, benzofurans, and germacrenes, were investigated as the main components in the genus Ligularia (Asteraceae).1−3 More than 500 eremophilane sesquiterpenoids have been isolated from this genus.1 These sesquiterpenoids also displayed a variety of bioactivities including cytotoxic, anti-inflammatory, antibacterial, insecticidal, and antiproliferative activities.4,5 Both structural diversity and unique biological activities have attracted scientists’ attention, and extensive phytochemical and biological investigations aiming at sesquiterpenoids from the genus Ligularia have been carried out by several Asian research groups over recent decades.4,6−8 Of course, many other scientists have also made great contributions to the research of sesquiterpenoids in this genus.9 Therefore, further studies toward the discovery of new and medicinally significant sesquiterpenoids are warranted. Ligularia rumicifolia (Drumm.) S. W. Liu, a perennial herbaceous plant distributed in Tibet, has been used to treat © 2018 American Chemical Society and American Society of Pharmacognosy

Received: March 13, 2018 Published: September 13, 2018 1992

DOI: 10.1021/acs.jnatprod.8b00197 J. Nat. Prod. 2018, 81, 1992−2003

Journal of Natural Products

Article

Chart 1

Table 1. 1H and 13C NMR Spectroscopic Data of Compound 1 (δ in ppm, J in Hz)a,b δH

position 1 2 3 4 5 6

7.06, d (8.1) 6.72, d (8.1)

2.99, dd (17.5, 6.3) 2.74, d (17.5) 3.06, m

7 a

Recorded in CDCl3.

δC

position

δH

δC

128.6, CH 113.2, CH 153.6, C 121.1, C 138.0, C 34.2, CH2

8

2.30, dt (12.4, 2.8) 1.90, d (12.4) 4.83, m

32.2, CH2

9 10 11 12

35.0, CH

13 14

4.85, s 4.75, s 3.90, d (4.7) 2.15, s

70.1, CH 126.5, C 149.0, C 107.9, CH2 63.7, CH2 10.8, CH3

b1

H NMR data recorded at 400 Hz and 13C NMR data recorded at 400 Hz.

the isolation, structural elucidation, and antiproliferative activity of compounds 1−37 are described.

■

RESULTS AND DISCUSSION The MeOH extract of the air-dried whole plant of L. rumicifolia was applied to purification using RP-18 silica gel CC, silica gel CC, semipreparative HPLC, and Sephadex LH-20 to afford a new nor-eudesmane-type, 10 new oplopane-type, four new eremophilane-type, and seven new bisabolane-type sesquiterpenoids, as well as 15 known analogues (Chart 1). These known

Figure 1. 1H−1H COSY (bold) and selected HMBC (arrow) correlations of 1.

1993



Article

requirement of one index of hydrogen deficiency (IOHD) and the presence of two additional oxygenated carbons (δC 70.1 and 63.7) in 1 compared with those of ligudentatol22 suggested an ether linkage from C-9 to C-13. This was corroborated via the key HMBC correlation of H-9 with C-13. Thus, the structure of rumicifoline A (1) was defined as the rare C14 nor-eudesmane sesquiterpenoid featuring a 6/6/6 tricyclic skeleton with an oxygen bridge. Rumicifoline B (2) was crystallized from MeOH as colorless needles. Its molecular formula C37H54O12 was assigned from the (+)-HRESIMS ion at m/z 713.35123 [M + Na]+ (calcd for C37H54O12Na, 713.35130), indicative of 11 IOHDs. The presence of hydroxy (3565 cm−1), ester carbonyl (1728 and 1715 cm−1), and olefinic (1646 cm−1) functional groups was observed in the IR data. The DEPT and 13C NMR data (Table 3) indicated five ester carbonyl resonances at δC 177.7, 172.2, 171.3, 168.3, 166.7 and six vinylic carbons at δC 164.6, 140.5, 140.2, 128.8, 115.0, and 114.9. Based on the 1D and 2D NMR data, the five ester groups were identified as an acetoxy (AcO), a 2-methylbutanoyloxy (MebuO), an angeloyloxy (AngO), a 4methylsenecioyloxy (MeSenO), and a 3-hydroxybutanoyloxy group. Apart from the five ester groups, the remaining 15 carbons (Table 3) comprise an sp2 quaternary carbon, an oxygenated sp3 tertiary carbon, nine methines (five oxygenated), two methylenes (one olefinic and one oxygenated), and two methyl carbons that should be part of the parent nucleus with four IOHDs. The 2D spectra of 2 showed the presence of a five- and a sixmembered carbocycle via the 1H−1H COSY correlations (Figure 2). The HMBC correlation networks of H-1/C-10; H9/C-1, C-7, and C-10; and H-14/C-1, C-9, and C-10, indicated a 5/6 bicyclic carbon skeleton with a terminal double bond. The 1 H−1H COSY correlations of Me-15/H-4/H-5 and the HMBCs of Me-15/C-5 demonstrated the presence of a substituted ethyl group at C-5. Based on the HMBC correlations between Me-13/ C-7, C-11, and C-12; H-7/C-11; and H-12/C-7 and C-11, one IOHD is contributed by the 11,12-epoxide moiety. These results indicated that 2 is an oplopane derivative. The five ester groups, MebuO, AcO, 3-hydroxybutanoyloxy, AngO, and MeSenO, were connected to C-2, C-3, C-4, C-8, and C-9, respectively, based on the HMBCs of H-2 (δH 5.66)/C-1′ (δC 177.7), H-3 (δH 5.64)/C-1″ (δC 171.3), H-4 (δH 5.29)/C-1‴ (δC 172.2), H8 (δH 5.11)/C-1⁗ (δC 168.3), and H-9 (δH 5.73)/C-1′′′′′ (δC 166.7) (Figure 2). The relative configuration of 2 was established via ROESY correlation networks and the 1H−1H coupling constants. On the basis of the large J1,6 (12.9 Hz) coupling constants, the orientations of H-1 and H-6 should be opposite, thus indicative of a trans-bicyclic moiety in accordance with the known configuration.8a,11 Furthermore, J1,2 (2.8 Hz), J3,5 (4.1 Hz), J5,6 (10.0 Hz), J7,8 (9.5 Hz), and J8,9 (3.2 Hz), combined with the ROESY cross peaks of H-1/H-7, indicated that H-1, H-2, H-3, H-5, and H-7 were cofacial in 2. Therefore, the relative configuration of 2 was arbitrarily assigned with β-orientations for H-6, H-8, and H-9 and α-orientations for H-1, H-2, H-3, H-5, and H-7. However, the orientations of H-4 and H-3‴ were difficult to determine by interpretation of the ROESY correlations. However, a single crystal of 2 obtained from MeOH was subjected to X-ray diffraction experiments using Cu Kα radiation. The crystallography data of 2 permitted definition of the configurations of C-4 and C-3‴ and further permitted unambiguous assignment of the absolute configuration as (1R,2S,3R,4R,5R,6R,7S,8R,9S,11S,2′S,3‴R) with a Flack param-

Figure 2. 1H−1H COSY (bold) and selected HMBC (arrow) correlations of 2.

Figure 3. ORTEP drawing of compound 2.

compounds were identified as (1R,2S,4Z,6R,7S,8R,9S,11S,2″S,2‴E,2⁗S)-11,12-epoxy-2,8di(2-methylbutanoyloxy)-9-(3‴-methyl-2‴-pentenoyloxy)10(14)-oplopen-3-one (12),11 petasin (17),12 3α-tigloyloxyeremophila-9,11-dien-8-one (18), 13 19, 14 petasol (20),15 (1R,2R,3S,5S,6R)-5-acetoxy-2,8-diangeloyloxy-1,3-dihydroxybisabola-7(14),10-den-4-one (21),16 fararone A (25),17 (1R,3R,4R,5S,6S)-1-acetoxy-8-angeloyloxy-3,4-epoxy-5-hydroxybisabola-7(14),10-dien-2-one (30),18 songaricalarin F (31),19 (4R,6E)-2-acetoxy-8-angeloyloxy-4-hydroxybisabola-2,6,10trien-1-one (32),20 tussfararin F (33),21 altaicalarin B (34),7 ligudentatol (35),22 7α-H-3α-angeloyloxy-9(10)-en-11,12epoxy-8-oxoeremophilane (36),23 and 7β-H-3α-angeloyloxy9(10)-en-11,12-epoxy-8-oxoeremophilane (37)23 by comparing their observed and reported spectroscopic data. Rumicifoline A (1), obtained as colorless oil, was assigned a molecular formula as C14H16O2 based on its 13C NMR data and the (−)-HRESIMS ion at m/z 215.10725 [M − H]− (calcd for C14H15O2, 215.10720). The presence of hydroxy (3575 cm−1), olefinic (1645 cm−1), and aromatic (1600 cm−1) functionalities was observed in the IR spectrum. The 1H NMR data (Table 1) contained signals characteristic of two aromatic ring protons (δH 7.06 and 6.72, each 1H, d, J = 8.1 Hz) and two olefinic protons (δH 4.85, 4.75). The DEPT and 13C NMR spectra displayed 14 carbon resonances attributed to four quaternary carbons, an oxygenated sp2 tertiary carbon, four methines (one oxygenated), four methylenes (one oxygenated and one olefinic), and a methyl carbon. The similar NMR data of 1 and ligudentatol22 suggested that 1 was also a C14 nor-eudesmane sesquiterpenoid. This conclusion was verified via the 1H−1H COSY cross peaks of H-6/H-7/H-8/H-9 and H-1/H-2 and combined with the HMBC correlations of H-1 with C-3/C-5/C-9; H-2 with C-4/ C-10; H-6 with C-4/C-5/C-10; H-8 with C-6/C-10; H-12 with C-7/C-11/C-13; and Me-14 with C-3/C-4/C-5 (Figure 1). The 1994



Article

Table 2. 1H NMR Spectroscopic Data of Compounds 2−6 (δ in ppm, J in Hz) position 1 2 3 4a 4b 5 6 7 8 9 12a 12b 13 14a 14b 15 OMe 22′ 3′a 3′b 4′ 5′ 32″ 42‴a 2‴b 3‴ 4‴ 82⁗ 3⁗a 3⁗b 4⁗a 4⁗b 5⁗ 6⁗ 92′′′′′ 4′′′′′ 5′′′′′ 6′′′′′

2a

3b

4b

5b

2.74, dd (12.9, 2.8) 5.66, dd (4.1, 2.8) 5.64, dd (5.7, 4.1) 5.29, dq (6.8, 4.1)

2.48, dd (11.4, 2.9) 5.63, dd (3.7,2.9) 5.51, dd (3.7, 3.7) 5.24, m

2.54, dd (10.8, 3.6) 5.28, d (3.6)

2.51, dd (11.4, 3.7) 5.28, d (3.7)

2.64, overlap 5.52, d (3.9)

3.59, m

3.58, m

2.83, ddd (10.0, 5.7, 4.1) 2.09, ddd(12.9, 10.0, 9.5) 1.91, dd (9.5, 7.8) 5.11, dd (9.5, 3.2) 5.73, d (3.2) 2.85, d (4.2) 2.68, d (4.2) 1.24, s 5.25, s 4.86, s 1.51, d (6.8)

2.71, m 1.96, m 1.75, m 5.06, dd (9.8, 3.1) 5.60, d (3.1) 2.79, d (3.6) 2.71, d (3.6) 1.21, s 5.22, s 4.80, s 1.43, d (6.8)

2.17, m 2.56, m 1.86, dd (11.6, 9.7) 5.25, dd (11.6, 3.0) 5.76, d (3.0) 2.52, overlap

2.15, m 2.57, m 1.83, t (10.9) 5.17, dd (9.5, 3.1) 5.73, d (3.1) 2.55, m

MebuO 2.50, m 1.77, m 1.52, m 1.00, t (7.4) 1.22, d (7.1) AcO 2.04, s

MebuO 2.38, m 1.73, m 1.45, m 0.94, t (7.4) 1.16, d (7.0) AcO 2.01, s

1.28, s 5.30, s 4.90, s 1.36, d (6.4) 3.24, s MebuO 2.37, m 1.68, m 1.44, m 0.86, t (7.4) 1.12, d (7.0)

1.26, s 5.29, s 4.89, s 1.37, d (6.4) 3.24, s MebuO 2.38, m 1.69, m 1.45, m 0.87, t (7.4) 1.12, d (7.0)

2.05, m 1.63, m 2.26, overlap 2.21, m 1.91, overlap 5.28, dd (10.2, 2.7) 5.82, d (2.7) 2.65, m 2.61, m 1.32, s 5.34, s 4.93, s 0.94, t (7.3)

2.37, t (6.1)

2.45, m 2.26, m 4.10, q (6.3) 1.20, d (6.3) MevalO 2.14, overlap 1.86, m

AngO

AngO

1.97, dq (7.2, 1.0)

MebuO 2.38, overlap 1.74, m 1.45, overlap 0.89, t (7.4)

1.86, q (1.0)

1.15, d (7.0)

1.92, q (1.5)

MeSenO 5.59, brs 2.12, q (7.4) 1.02, t (7.4) 2.08, brs

MeSenO 5.60, brs 2.15, q (7.4) 1.04, t (7.4) 2.09, brs

MeSenO 5.65, brs 2.17, q (7.4) 1.07, t (7.4) 2.13, brs

4.12, q (6.3) 1.20, d (6.3) AngO 6.13, qq (7.3, 1.4) 1.98, dq (7.3, 1.4) 1.84, q (1.4) MeSenO 5.68, brs 2.19, q (7.4) 1.06, t (7.4) 2.10, brs

6.07, qq (7.2, 1.0)

1.34, m 1.16, m 0.83, t (7.4) 0.89, d (6.7) MeSenO 5.58, brs 2.14, q (7.4) 1.04, t (7.4) 2.08, brs

6b

MebuO 2.41, m 1.70, m 1.48, m 0.91, t (7.2) 1.17, d (7.0)

6.13, qq (7.2, 1.5) 2.03, dq (7.2, 1.5)

a

Recorded in methanol-d4 at 400 Hz. bRecorded in CDCl3 at 400 Hz.

MeSenO groups. The remaining 15 carbon signals of the tricyclic core were similar to those of 2, which suggested that 4 was an oplopane derivative. The distinction was the replacement of an oxygenated methine (C-3, δ C 73.8) in 2 by a nonconjugated carbonyl (C-3, δC 206.9) in 4. This was verified via the HMBC correlations of H-4/C-3 and H-2/C-3. The positions of the MebuO, MeO, AngO, and MeSenO groups were determined via the HMBC correlations of H-2, MeO-4, H8, and H-9 with the corresponding carbons at δC 175.0, 75.5, 166.9, and 165.2, respectively. The ROESY correlations of H-1 with H-5, H-1 with H-7, H-2 with H-6, and H-6 with H-8, in combination with J1,2 (3.6 Hz), J1,6 (10.8 Hz), and J8,9 (3.0 Hz), proved that 4 has the same relative configuration as 2. Hence, these results allowed the definition of the structure of rumicifoline D (4), as shown in Chart 1.

eter of 0.05(3) (Figure 3). Thus, the structure of 2, rumicifoline B, was defined as shown in Chart 1. The (+)-HRESIMS data permitted the molecular formula of rumicifoline C (3) to be assigned as C38H58O12. The 1D NMR data of 3 were comparable to those of 2, indicating that they are quite similar, except for the replacement of the AngO group in 2 by a 3-methylpentanoyloxy (MevalO) group in 3. This was verified based on the HMBC correlation of H-8 with C-1⁗ (δC 172.7). Similar ROESY cross peaks and 1H−1H coupling constants for 3 and 2 showed that the relative configurations of these compounds were identical. Hence, the structure of rumicifoline C (3) was deduced as shown in Chart 1. The (+)-HRESIMS and 13C NMR data of 4 were indicative of a molecular formula of C32H46O9. The NMR data (Tables 2 and 3) demonstrated the presence of MeO, MebuO, AngO, and 1995



Article

Table 3. 13C NMR Spectroscopic Data of Compounds 2−6 (δ in ppm) position

2a

3b

4b

5b

6b

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 OMe 21′ 2′ 3′ 4′ 5′ 31″ 2″ 41‴ 2‴ 3‴ 4‴ 81⁗ 2⁗ 3⁗ 4⁗ 5⁗ 6⁗ 91′′′′′ 2′′′′′ 3′′′′′ 4′′′′′ 5′′′′′ 6′′′′′

45.8, CH 72.8, CH 73.8, CH 71.2, CH 46.3, CH 44.9, CH 50.2, CH 73.9, CH 74.0, CH 140.5, C 56.4, C 53.9, CH2 17.9, CH3 115.0, CH2 17.9, CH3

44.5, CH 71.0, CH 72.3, CH 69.9, CH 45.2, CH 43.6, CH 48.9, CH 72.2, CH 72.7, CH 137.9, C 55.1, C 53.0, CH2 16.2, CH3 114.9, CH2 16.2, CH3 MebuO 176.0, C 41.6, CH 26.5, CH2 11.9, CH3 17.4, CH3 AcO 170.1, C 21.2, CH3

44.9, CH 71.8, CH 206.9, C 75.5, CH 58.0, CH 40.1, C 49.2, C 72.3, CH 72.9, CH 137.5, C 55.5, C 52.5, C 16.2, CH3 115.8, CH2 16.5, CH3 56.3, CH3 MebuO 175.0, C 40.9, CH 26.4, CH2 11.4, CH3 16.1, CH3

45.6, CH 71.1, CH 210.4, C 21.9, CH2 54.1, CH 40.9, CH 49.5, CH 71.9, CH 73.1, CH 137.5, C 55.4, C 52.4, CH2 16.5,CH3 115.8, C 10.2, CH3

MebuO 177.7, C 42.9, CH 27.6,CH2 12.4,CH3 19.1, CH3 AcO 171.3, C 21.4, CH3

44.9, CH 71.8, CH 206.9, C 75.5, CH 58.1, CH 40.0, CH 49.2, CH 72.0, CH 73.1, CH 137.4, C 55.5, C 52.6, CH2 16.5, CH3 115.8, CH2 16.5, CH3 56.3, CH3 MebuO 175.0, C 41.0, CH 26.4, CH2 11.4, CH3 16.1, CH3

172.2, C 44.9, CH2 65.4, CH 23.3, CH3 AngO 168.3, C 128.8, C 140.2, CH 16.9, CH3 20.6, CH3

171.9, C 43.0, CH2 64.6, CH 22.9, CH3 MevalO 172.7, C 41.5, CH2 31.5, CH 29.1, CH2 11.1, CH3 19.1, CH3 MeSenO 165.2, C 113.9, CH 163.0, C 33.7,CH2 11.7,CH3 18.9, CH3

AngO 166.9, C 127.4, C 139.2, CH 15.7,CH3 20.3, CH3

MebuO 176.0, C 41.2, CH 26.3, CH2 11.6, CH3 16.1, CH3

AngO 166.9, C 127.4, C 139.3, CH 15.7,CH3 20.3, CH3

MeSenO 165.2, C 113.9, CH 163.0, C 33.7, CH2 11.8,CH3 18.9, CH3

MeSenO 165.2, C 113.9, CH 163.0, C 33.8, CH2 11.8, CH3 18.9, CH3



HMBC correlation of H-8 with C-1⁗ (δC 176.0) was clearly observed. Thus, the structure of rumicifoline E (5) was defined as shown in Chart 1. Rumicifoline F (6) was assigned a molecular formula of C31H44O8, as established via the 13C NMR and (+)-HRESIMS data, with 30 mass units less than 4. Additionally, the absence of a methoxy group (δC 56.3) and an oxygenated methine (δC 75.5) and the presence of a methylene (δC 21.9) in 6 indicated the presence of a C-4 methylene group. This was verified by the 1 H−1H COSY correlations of H-5/H2-4/Me-15. Therefore, the structure of rumicifoline F (6) was established as shown in Chart 1. The 13C NMR and (+)-HRESIMS data of 7 and 8 implied that these compounds shared the same molecular formula (C31H42O8), with 2 mass units less than 6. Comparing the NMR data of 7 with those of 6 revealed many similarities, except for the presence of two additional vinylic carbons in the 13C spectra of 7. The observation of a doublet of doublets splitting pattern of the methyl signal at δH 2.02 (dd, J = 7.7, 2.4 Hz, H-15) in the 1H NMR data of 7 instead of the triplet at δH 0.94 (t, J = 7.3 Hz, H15) in 6 revealed the presence of a Δ4(5) double bond in 7 (Figure 4). This was confirmed by the 1H−1H COSY interaction of H-4 (δH 6.78, qd, J = 7.7, 2.8 Hz)/H-15 and the HMBC correlations of H-4/C-3, C-5, and C-6, H-15/C-4, and C-5. The E configuration of the double bond in 7 was defined by the ROESY correlations of Me-15/H-7 and Me-15/H-6. Similarly, compound 8 was assigned as the Z geometrical isomer of 7. Thus, the structures of (4E)-rumicifoline G (7) and (4Z)rumicifoline G (8) were defined as shown in Chart 1. Similar 13C NMR and (+)-HRESIMS data indicated that compounds 9 and 10 possessed the same molecular formula of C32H46O8. The NMR data of 9 and 10 showed similar resonances to those of 7 and 8 except for the replacement of the AngO group in 7 and 8 by a MevalO group in 9 and 10. Thus, the structures of (4E)-rumicifoline H (9) and (4Z)rumicifoline H (10) were defined as shown in Chart 1. Rumicifoline I (11) gave a molecular formula of C31H44O8 based on the (+)-HRESIMS and 13C NMR data. The NMR data (Tables 4 and 5) of 11 resembled those of 12, except for the E configuration of Δ4(5) in 11. Thus, the structure of rumicifoline I (11) was assigned as shown in Chart 1. Rumicifoline J (13) gave a molecular formula of C20H28O4, as deduced from the (+)-HRESIMS ion at m/z 355.18810 [M + Na]+ (calcd for C20H28O4Na, 355.18853), indicative of seven IOHDs. The presence of ester carbonyl (1710 cm−1) and hydroxy (3475 cm−1) functionalities was observed in the IR spectrum. The NMR data (Table 6) displayed resonances for an AngO group, and the remaining 15 carbon resonances were attributed to an eremophilane sesquiterpenoid with a terminal double bond (δH 4.99, s, 1H and 4.80, s, 1H; δC 114.5, CH2), an α,β-unsaturated carbonyl moiety (δH 6.21, s, 1H; δC 198.5, C, 120.7, CH, and 167.7, C), and two methyl groups (δH 1.21, s, 3H, and 0.96, d, J = 6.7 Hz, 3H). The eremophiane core of 13 was further verified via 2D NMR analysis (Figure 5). Additionally, the AngO group was connected to C-3, and a hydroxy group was linked to C-1 by the HMBC cross-peaks. The ROESY correlations (Figure 5) between H-1 and H-3, H1 and Me-14, H-7 and Me-14, and H-3 and Me-15 indicated that the AngO and hydroxy groups were α orientated, and H-7, Me14, and Me-15 were β orientated. Accordingly, the structure of rumicifoline J (13) was defined as 1α-hydroxypetasin.12 Compound 14 possessed an identical molecular formula (C20H28O4) as 13, as concluded from the similar (+)-HRESIMS

MebuO 174.9, C 41.0, CH 26.7, CH2 11.5, CH3 16.5, CH3

a Recorded in methanol-d4 at 100 MHz. bRecorded in CDCl3 at 100 MHz.

Figure 4. 1H−1H COSY (bold) and HMBC (arrow) correlations of 7.

Rumicifoline E (5) (C32H48O9) was also an oplopane analogue of 4 by comparison of their NMR data, but the AngO group in 4 was replaced by a MebuO group in 5. The 1996



Article

Table 4. 1H NMR Spectroscopic Data of Compounds 7−11 (δ in ppm, J in Hz) position

7a

8a

9a

10a

11b

1 2 4 6 7 8 9 12a 12b 13 14a 14b 15 22′ 3′a 3′b 4′ 5′ 82″a 2″b 3″ 4″

3.10, m 5.61, d (5.0) 6.78, qd (7.7, 2.8) 2.47, m 2.66, dd (12.0,10.2) 5.42, dd (10.2, 2.2) 5.63, d (2.2) 2.95, d (3.6) 2.77, d (3.6) 1.65, s 5.20, brs 5.01,brs 2.02, dd (7.7, 2.4) MebuO 2.38, m 1.59, m 1.45, m 0.85, t (7.5) 1.07, d (7.0) AngO

2.91, dd (13.3, 3.6) 5.52, d (4.4) 6.49, qd (7.3, 2.4) 2.59, m 2.29, dd (11.0, 9.3) 5.26, dd (9.3, 2.0) 5.67, d (2.0) 2.83, d (4.2) 2.66, d (4.2) 1.49, s 5.25, brs 4.94, brs 2.16, dd (7.3, 2.6) MebuO 2.40, m 1.64, m 1.46, m 0.87, t (7.5) 1.10, d (7.0) AngO

6.14, qq (7.2, 1.4) 1.95, dq (7.2, 1.4)

5″ 6″ 92‴ 4‴ 5‴ 6‴

1.83, q (1.4)

1.83, q (1.4)

MesenO 5.69, brs 2.21, q (7.5) 1.07, t (7.5) 2.17, brs

MesenO 5.69, brs 2.20, q (7.5) 1.06, t (7.5) 2.13, brs

2.90, m 5.51, d (4.4) 6.48, qd (7.3, 2.4) 2.57, m 2.25, dd (11.8, 10.3) 5.19, dd (10.3, 1.9) 5.61, d (1.9) 2.82, d (4.2) 2.67, d (4.2) 1.51, s 5.23, brs 4.94, brs 2.17, dd (7.3, 2.6) MebuO 2.40, m 1.62, m 1.42, m 0.86, t (7.5) 1.10, d (6.9) MevalO 2.32, dd (14.7, 6.2) 2.10, dd (14.7, 8.0) 1.84, m 1.36, m 1.20, m 0.88, t (7.5) 0.91, d (6.7) MesenO 5.69, brs 2.21, q (7.5) 1.07, t (7.5) 2.15, brs

3.22, m 5.67, d (4.9) 6.73, qd (7.7, 2.8) 2.54, m 2.77, dd (11.7, 10.1) 5.39, dd (10.1, 1.9) 5.65, d (1.9) 2.98, d (3.7) 2.77, d (3.7) 1.65, s 5.21, brs 5.03, brs 2.04, dd (7.7, 2.5) MebuO 2.40, m 1.60, m

6.16, qq (7.2, 1.4) 1.94, dq (7.2, 1.4)

3.10, m 5.61, d (4.9) 6.78, qd (7.7, 2.7) 2.45, m 2.62, dd (11.7, 10.1) 5.36, dd (10.1, 2.1) 5.57, d (2.1) 2.92, d (3.5) 2.77, d (3.5) 1.61, s 5.19, brs 5.00, brs 2.01, dd (7.7, 2.4) MebuO 2.38, m 1.57, m 1.42, m 0.84, t (7.5) 1.07, d (6.9) MevalO 2.30, m 2.14, m 1,81, m 1.36, m 1.23, m 0.87, t (7.5) 0.92, d (6.7) MesenO 5.68, brs 2.22, q (7.5) 1.07, t (7.5) 2.18, brs

0.85, t (7.4) 1.07, d (7.0) MebuO 2.40, m 1.47, m 0.89, t (7.4) 1.13, d (7.0) MesenO 5.72, brs 2.24, q (7.5) 1.09, t (7.5) 2.20, brs

a

Recorded in methanol-d4 at 400 MHz. bRecorded in acetone-d6 at 400 MHz.

22, which accounted for three IOHDs. Therefore, compound 22 should be monocyclic and thus a bisabolane sesquiterpenoid derivative, which was supported by the 1H−1H COSY cross peaks of H-2/H-1/H-6/H-5 and H-8/H-9/H-10 and the HMBC correlations of H-2/C-4; H-5/C-4 and C-7; Me-12/ C-10, C-11, and C-13; H-14/C-6, C-7, and C-8; and Me-15/C2, C-3, and C-4 (Figure 6). The TigO, AcO, and AngO groups were connected to C-2, C-5, and C-8, respectively, based on the HMBC analysis. The relative configuration of 22 was determined via the ROESY correlations, combined with 1H−1H coupling constants. If H-6 has an α-orientation, H-1 and H-5 should have βorientations according to the large J1,6 (10.3 Hz) and J5,6 (12.3 Hz) values. Furthermore, J1,2 (2.7 Hz) indicated that H-1 and H2 were cofacial. Thus, the structure of rumicifoline M (22) was defined as shown in Chart 1. Compound 23 has the molecular formula of C23H34O7, as established via the 13C NMR and (+)-HRESIMS data. Its NMR data were highly similar to those of fararone A (25)19 except for the replacement of the 2-chloro and 3-hydroxy groups in 25 by a hydroxy and a methoxy (δC 52.4) group in 23, respectively. Therefore, the structure of rumicifoline N (23) was assigned as shown. The structure of rumicifoline O (24) was highly similar to that of 23, but the signals of an AngO group at C-3 in 24 were observed instead of those of an AcO group in 23. By combining

data. A comparison of the 1D and 2D NMR data of 14 and 13 revealed that these compounds possessed the same 2D structure. The only difference was that the HO-1 of 14 was β orientated. Thus, compound 14 was structurally characterized as 1-epirumicifoline J. The molecular formulas of 15 and 16 were assigned as C20H30O3 and C21H32O3, respectively, by the (+)-HRESIMS data. The NMR data (Table 6) of 15 and 16 resembled those of petasin (17),12 except for the different substituents at C-3. A 3methylbutanoyloxy (ValO) group (δH 2.21, 2H, d, J = 7.1 Hz; 2.10, 1H, m; 0.97, 6H, d, J = 6.6 Hz; δC 174.3; 44.5; 27.0; 22.8; 22.7) was linked to C-3 in 15, and a MevalO group was connected to C-3 in 16. This was verified via HMBC data analysis. Thus, the structures of rumicifolines K (15) and L (16) were defined as shown in Chart 1. Rumicifoline M (22) displayed a molecular formula of C27H38O9 based on the (+)-HRESIMS data, indicative of nine IOHDs. Its NMR data (Tables 7 and 8) showed resonances for an AngO, a tigloyloxy (TigO), and an AcO group, accounting for five IHODs. Apart from these ester groups, the remaining 15 carbon resonances comprise two sp2 quaternary carbons, a carbonyl carbon, an oxygenated sp3 tertiary carbon, six methines (one olefinic and four oxygenated), two methylenes (one olefinic), and three methyl carbons. The 1D NMR data displayed signals characteristic of a methyl, a carbonyl, an isopentenyl, and a terminal double bond in the carbon core of 1997



Article

Table 5. 13C NMR Spectroscopic Data of Compounds 7−11 (δ in ppm) position

7a

8a

9a

10a

11b

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 21′ 2′ 3′ 4′ 5′ 81″ 2″ 3″ 4″ 5″ 6″ 91‴ 2‴ 3‴ 4‴ 5‴ 6‴

46.2, CH 71.5, CH 200.0, C 136.8, CH 140.2, C 37.8, CH 48.6, CH 73.5, CH 71.3, CH 139.1, C 56.7, C 50.0, CH2 22.6, CH3 112.2, CH2 14.9, CH3 MebuO 176.9, C 42.2, CH 27.9, CH2 11.8, CH3 17.3, CH3 AngO 168.1, C 128.5, C 140.5, CH 16.1, CH3 20.6, CH3

46.0, CH 73.4, CH 200.9, C 140.9, CH 138.7, C 42.2, CH 48.6, CH 74.4, CH 73.4, CH 139.5, C 57.0, C 52.4, CH2 22.0, CH3 114.2, CH2 15.2, CH3 MebuO 176.9, C 42.2, CH 28.0, CH2 11.8, CH3 17.1, CH3 AngO 168.0, C 128.6, C 140.2, CH 16.1, CH3 20.6, CH3 MesenO 166.9, C 114.7, CH 165.1, C 34.7, CH2 12.4, CH3 19.1, CH3

45.9, CH 73.4, CH 200.9, C 140.9, CH 138.7, C 42.2, CH 49.0, CH 74.4, CH 73.3, CH 139.4, C 57.1, C 52.4, CH2 21.9, CH3 114.1, CH2 15.2, CH3 MebuO 177.0, C 42.2, CH 28.0, CH2 11.7, CH3 17.1, CH3 MevalO 173.8, C 42.4, CH2 33.1, CH 30.2, CH2 11.6, CH3 19.7, CH3 MesenO 167.0, C 114.7, CH 165.1, C 34.7, CH2 12.3, CH3 19.1, CH3

44.9, CH 69.9, CH 197.9, C 134.4, CH 139.3, C 36.3, CH 47.4, CH 71.9, CH 69.7, CH 138.0, C 55.2, C 48.6, CH2 21.7, CH3 110.6, CH2 13.7, CH3 MebuO 174.9, C 41.0, CH 26.6, CH2 10.9, CH3 16.4, CH3 MebuO 174.7, C 40.8, CH 26.4, CH3 10.9, CH3 16.1, CH3

MesenO 167.3, C 114.4, CH 165.6, C 34.8, CH2 12.3, CH3 19.2, CH3

46.1, CH 71.5, CH 200.0, C 136.7, CH 140.3, C 37.6, CH 48.6, CH 73.3, CH 71.1, CH 138.9, C 56.6, C 50.0,CH2 22.5, CH3 112.1, CH2 14.8, CH3 MebuO 176.9, C 42.2, CH 28.0, CH2 11.8, CH3 17.3, CH3 MevalO 173.9, C 42.4, CH2 33.4, CH 30.1, CH2 11.6, CH3 19.6, CH3 MesenO 167.2, C 114.3, CH 165.7, C 34.7, CH2 12.3, CH3 19.2, CH3

methyl carbons (δC 29.5 and 29.1). This was verified by the H−1H COSY cross peaks of H-8/H-9/H-10 and the HMBC correlations of Me-12/Me-13 with C-10/C-11. The large J9,10 (15.5 Hz) value indicated the E configuration of the Δ9(10) double bond. The J1,6 (8.0 Hz) and J5,6 (12.0 Hz) coupling constants, combined with the ROESY interactions between H-2 and H-6, showed that H-1, H-2, H-6, and Me-15 were cofacial, while H-5 was on the opposite side. Accordingly, the structure of rumicifoline Q (27) was elucidated as shown in Chart 1. Rumicifoline R (28) had a molecular formula of C22H30O6 based on the 13C NMR and (+)-HRESIMS data. Based on its 1D NMR data (Tables 7 and 8), the signals characteristic of the AcO and AngO groups and the remaining 15 carbons indicated a typical bisabolane sesquiterpenoid with an α,β-unsaturated carbonyl moiety. The 1H−1H COSY cross peaks of H-4/H-5/ H-6, along with the HMBCs of H-5 with C-1/C-3, and Me-15 (δH 1.93) with C-2/C-3/C-4, defined the presence of a Δ2(3) double bond (Figure 6). The 1H−1H COSY correlations of OH (δH 2.55)/H-4 (δH 4.55) are indicative of the position of the hydroxy at C-4. The AngO group was connected to C-8, based on the correlations between H-8 (δH 3.20) and the carbonyl carbon at δC 167.6 in the HMBC data. Therefore, the AcO group was assigned to C-2. The ROESY interactions of H-4 with H-6 showed that H-4 and H-6 were cofacial. Thus, the structure of rumicifoline R (28) was defined as shown in Chart 1. Compound 29 (C27H36O8) possessed an analogous structure to 28 by comparison of their NMR data, except for the presence of an additional AngO group in 29. This conclusion was corroborated via the key HMBC correlation of H-5/C-1″ (δC 168.1). The ROESY interaction of H-4/H-6 indicated that H-4 and H-6 were cofacial. The orientation of H-5 should be opposite to that of H-6 due to the large J5,6 (11.9 Hz) coupling constant. Thus, the structure of 5α-angeloyloxyrumicifoline R (29) was defined as shown in Chart 1. The new compounds were evaluated for their in vitro antiproliferative activities against four human tumor cell lines HGC-27, A-549, HeLa, and MV4−11, using the 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) asssy. 24 Compounds 13−16 and 29 exhibited antiproliferative activity against the four human tumor cell lines (Table 9). Specifically, the two eremophilane-type sesquiterpenoids 13 and 16 showed antiproliferative activity against the MV4−11 cell line with IC50 values of 0.5 ± 0.2 and 1.1 ± 0.5 μM, respectively. Compound 2 showed moderate antiproliferative activities against the human tumor cell lines, and the remaining compounds were inactive in these assay systems. The four new eremophilane-type sesquiterpenoids exhibited antiproliferative activity against the four human tumor cell lines, and only some of the oplopane-type and bisabolanetype sesquiterpenoids showed antiproliferative activity. Furthermore, the known eremophilane-type compounds petasin (17), and 18, and petasol (20) exhibited significant cytotoxic activity against human neuroblastoma cells.25 The eremophilane-type sesquiterpenoids showed more potent inhibitory effects on antiproliferative activity. 1

MesenO 165.5, C 113.5, CH 163.5, C 33.4, CH2 11.4, CH3 18.1, CH3

a Recorded in methanol-d4 at 100 MHz. bRecorded in acetone-d6 at 100 MHz.

the 2D NMR data, compound 24 possessed a relative configuration identical to that of 23. Compound 26 possessed a molecular formula of C22H30O6, as deduced from the (+)-HRESIMS data. The NMR data of 26 and 23 implied that the structures of these compounds were quite similar, expect for the absence of a hydroxy and a methoxy group in 26. The shielding of C-2 (from δC 73.7 to 62.9) and C-3 (from δC 85.6 to 58.5) revealed the presence of a 2,3-epoxide group in 26. The ROESY interactions of H-6 with H-2 and Me-15 with H-5, coupled with the small J5,6 (2.6 Hz) coupling constant, indicated that H-2, H-5, H-6, and Me-15 were cofacial. Therefore, the structure of rumicifoline P (26) was defined as shown in Chart 1. Compound 27 had a molecular formula of C27H36O 9 according to the (+)-HRESIMS data. By comparing the NMR data of 27 with those of 26, their structures were closely analogous, except for an additional AngO group at C-1 in 27 based on the HMBC data analysis. Furthermore, the isoprenyl group in 27 should be oxygenated based on the presence of the following carbon resonances: an oxygenated carbon (δC 70.3), two olefinic methine carbons (δC 124.1 and 143.3), and two

■

EXPERIMENTAL SECTION

General Experimental Procedures. The general experiments were included in the Supporting Information. Plant Material. The whole plants of L. rumicifolia were collected in August 2015 from Lhasa, Tibet Autonomous Region, People’s Republic of China, and identified by Dr. Drolma Dawa (Tibet Autonomous Region Institute for Food and Drug control, Lhasa, Tibet). A voucher 1998



Article

Table 6. 1H and 13C NMR Spectroscopic Data of Compounds 13−16 (δ in ppm, J in Hz) 13a,c

14a,c

position

δH

1a 1b 2a 2b 3 4 5 6a 6b 7 8 9 10 11 12a 12b 13 14 15 1′ 2′a 2′b 3′ 4′a 4′b 5′ 6′

4.42, ddd (12.4, 4.6, 1.9)

66.2, CH

4.48, t (3.0)

73.2, CH

2.57, m 1.55, m 4.96, td (11.2, 4.2) 1.64, m

40.7, CH2

2.41, m 1.70, m 5.33, td (11.1, 4.1) 1.66, m

38.6, CH2

2.02, m 3.13, dd (14.3, 4.6) 6.21, s

4.99, s 4.80, s 1.72, s 1.21, s 0.96, d (6.7)

δC

15b,c

70.9, CH 47.7, CH 40.3, C 42.3, CH2 50.4, CH 198.5, C 120.7, CH 167.7, C 143.1, C 114.5, CH2 20.1, CH3 17.9, CH3 10.4, CH3 167.5, C 127.6, C

δH

2.00, m 3.21, dd (14.2, 4.6) 5.87, s

4.97, s 4.81, s 1.70, s 1.42, s 0.99, d (6.7)

δC

δH

70.0, CH 47.3, CH 39.4, C 43.1, CH2 50.8, CH 199.5, C 126.8, CH 164.7, C 143.0, C 114.7, CH2 20.0, CH3 19.4, CH3 10.5, CH3 167.5, C 127.8, C

2.60, m 2.40, m 2.16, m 1.49, m 4.92, m 1.61, m 2.07, m 1.96, m 3.23, dd (14.4, 4.7) 5.75, s

4.93, s 4.81, s 1.71, s 1.27, s 0.97, d (6.6)

16b,c δC 31.5, CH2 32.7, CH2 74.6, CH 48.5, CH 41.4, C 42.9, CH2 51.4, CH 201.1, C 125.1, CH 170.1, C 144.7, C 114.9, CH2

2.21, d (7.1)

20.2, CH3 17.3, CH3 10.8, CH3 174.3, C 44.5, CH2

6.08, qq (7.2, 1.2) 1.96, dq (7.2, 1.2)

138.5, CH 15.8, CH3

6.06, qq (7.2, 1.3) 1.97, dq (7.2, 1.3)

138.1, CH 15.7, CH3

2.10, m 0.97, d (6.6)

27.0, CH 22.8, CH3

1.86, q (1.2)

20.5, CH3

1.87, q (1.3)

20.6, CH3

0.97, d (6.6)

22.7, CH3

δH 2.59, m 2.40, m 2.19, m 1.49, m 4.92, m 1.63, m 2.06, m 1.96, m 3.23, dd (14.3, 4.7) 5.76, s

4.94, s 4.82, s 1.71, s 1.28, s 0.97, d (6.7) 2.34, dd (14.6, 6.2) 2.13, dd (14.6, 8.1) 1.88, m 1.36, m 1.25, m 0.92, t (7.5) 0.95, d (6.7)

δC 31.5, CH2 32.7, CH2 74.6, CH 48.5, CH 41.4, C 42.9, CH2 51.4, CH 201.1, C 125.1, CH 170.1, C 144.7, C 114.9, CH2 20.2, CH3 17.3, CH3 10.8, CH3 174.6, C 42.6, CH2 33.3, CH 30.4, CH2 11.6, CH3 19.6, CH3

a

Recorded in CDCl3. bRecorded in methanol-d4. c1H NMR data recorded at 400 Hz and 13C NMR data recorded at 100 Hz. NMR data (400 Hz, CDCl3), Table 2; and 13C NMR data (100 Hz, CDCl3), Table 3. Rumicifoline D (4). colorless oil; [α]20D − 109 (c 0.2 in MeOH); IR (KBr) υmax 2967, 1709, 1645, 1449, 1381, 1229, 1138, 933 cm−1; UV (MeOH) λmax (log ε) 219 (4.5) nm; positive (+)-HRESIMS [M + Na]+ m/z 597.30362 (calcd for C32H46O9Na, 597.30395); 1H NMR data (400 Hz, CDCl3), Table 2; and 13C NMR data (100 Hz, CDCl3), Table 3. Rumicifoline E (5). colorless oil; [α]20D − 135 (c 0.2 in MeOH); IR (KBr) υmax 2967, 1711, 1648, 1450, 1381, 1229, 1139, 932 cm−1; UV (MeOH) λmax (log ε) 219 (4.3) nm; positive (+)-HRESIMS [M + Na]+ m/z 599.31943 (calcd for C32H48O9Na, 599.31960); 1H NMR data (400 Hz, CDCl3), Table 2; and 13C NMR data (100 Hz, CDCl3), Table 3. Rumicifoline F (6). colorless oil; [α]20D − 102 (c 0.2 in MeOH); IR (KBr) υmax 2967, 1707, 1645, 1449, 1380, 1229, 1136, 933 cm−1; UV (MeOH) λmax (log ε) 221 (4.5) nm; positive (+)-HRESIMS [M + Na]+ m/z 567.29406 (calcd for C31H44O8Na, 567.29339); 1H NMR data (400 Hz, CDCl3), Table 2; and 13C NMR data (100 Hz, CDCl3), Table 3. (4E)-Rumicifoline G (7). colorless oil; [α]20D − 67 (c 0.4 in MeOH); IR (KBr) υmax 2959, 1707, 1645, 1445, 1380, 1226, 1139, 931 cm−1; UV (MeOH) λmax (log ε) 220 (4.4) nm; positive (+)-HRESIMS [M + Na]+ m/z 565.27772 (calcd for C31H42O8Na, 565.27774); 1H NMR data (400 Hz, methanol-d4), Table 4; 13C NMR data (100 Hz, methanol-d4), Table 5. (4Z)-Rumicifoline G (8). colorless oil; [α]20D − 117 (c 0.3 in MeOH); IR (KBr) υmax 2970, 1709, 1646, 1446, 1381, 1225, 1138, 935 cm−1; UV (MeOH) λmax (log ε) 219 (4.4) nm; positive (+)-HRESIMS [M + Na]+ m/z 565.27778 (calcd for C31H42O8Na, 565.27774); 1H NMR data (400 Hz, methanol-d4), Table 4; 13C NMR data (100 Hz, methanol-d4), Table 5.

Figure 5. 1H−1H COSY (bold), selected HMBC (arrow), and key NOESY (double arrow) correlations of 13.

specimen (No. CIB20150823T1) was deposited at Chengdu Institute of Biology, Chinese Academy of Sciences. Extraction and Isolation. The whole plants of L. rumicifolia (10.0 kg) were air-dried and powdered, and extracted with MeOH (3 × 40 L, 3 days each) at rt. The filtered solution was concentrated in vacuo to afford a crude extract (2.1 kg). The detailed process of separation and purification is described in the Supporting Information. Rumicifoline A (1). colorless oil; [α]20D + 211 (c 0.7 in MeOH); IR (KBr) υmax 3575, 3100, 1645, 1600, 895 cm−1; UV (MeOH) λmax (log ε) 206 (4.5) nm; (−)-HRESIMS [M − H]− m/z 215.10725 (calcd for C14H15O2, 215.10720); 1H NMR data (400 Hz, CDCl3), Table1; 13C NMR data (100 Hz, CDCl3), Table 1. Rumicifoline B (2). colorless crystals; mp 121−122 °C; [α]20D + 45 (c 0.1 in MeOH); IR (KBr) υmax 3565, 2971, 1728, 1715, 1646, 1457, 1384, 1234, 1143, 1087, 931 cm−1; UV (MeOH) λmax (log ε) 219 (4.5) nm; positive (+)-HRESIMS [M + Na]+ m/z 713.35123 (calcd for C37H54O12Na, 713.35130); 1H NMR data (400 Hz, methanol-d4), Table 2; 13C NMR data (100 Hz, methanol-d4), Table 3. Rumicifoline C (3). colorless oil; [α]20D + 70 (c 0.2 in MeOH); IR (KBr) υmax 3561, 2968, 1710, 1650, 1451, 1385, 1231, 1140, 933 cm−1;UV (MeOH) λmax (log ε) 219 (4.5) nm; positive (+)-HRESIMS [M + Na]+ m/z 729.38414 (calcd for C38H58O12Na, 729.38260); 1H 1999



Article

Table 7. 1H NMR Spectroscopic Data of Compounds 22−24 and 26−29 (δ in ppm, J in Hz) 22a

position

23a

24a

1a 1b

4.58, dd (10.3, 2.7)

2.29, m 1.77, m

2.43, m 1.79, m

2

5.50, d (2.7)

3.78, ddd (13.7, 4.6, 2.0)

3.80, dd (11.8, 4.6)

4 5a

5.97, d (12.3)

5.29, d (12.8)

5.46, d (12.2)

2.68, dd (12.3, 10.3) 4.81, dd (8.3, 3.0) 2.54, m

2.38, m

2.41, m

5.13, t (7.0) 1.67, s 1.61, s 5.30, s 5.22, s 1.26, s 3.39, s

5.04, t (7.0) 1.66, s 1.60, s 5.28, s 5.08, s 1.50, s 2.24, d (2.0) 3.45, s 5-AcO 2.02, s

5b 6 8 9 10 12 13 14a 14b 15 OH OMe

2-TigO 2′ 3′ 4′ 5′ 2″ 3″ 4″ 5″ 83‴ 4‴ 5‴

5.17, t (6.6) 2.35, m

6.77, qq (6.9, 0.8) 1.78, dq (6.9, 0.8) 1.76, q (0.8) 5-AcO 2.10, s

AngO 6.13, qq (7.3, 1.1) 1.94, dq (7.3,1.1) 1.86, q (1.1)

26a 2.49, dd (15.3, 10.0) 2.17, ddd (15.3, 6.4, 4.4) 3.37, d (4.1)

5.59, d (8.0)

4.77, d (2.6)

5.76, d (12.0)

3.46, s 5-AngO

5-AcO 2.08, s

AngO 6.05, qq (7.2, 1.4) 1.94, dq (7.2, 1.4) 1.84, q (1.4)

28a

1-AngO

4.55, m 2.47, m

4.60, dd (8.4, 1.0) 5.58, dd (11.9, 8.4)

2.22, m 3.20, dd (4.5, 11.4) 5.13, dd (4.7, 8.2) 2.43, m

AngO 6.05, qd (7.2, 1.2) 1.94, dq (7.2, 1.2) 1.87, q (1.2)

3.59, d (11.9) 5.35, dd (8.6, 5.1) 2.34, m

5.10, t (7.2) 1.66, s 1.60, s 5.28, s 5.05, s 1.93, s 2.55, d (6.9)

5.09, d (7.1) 1.66, s 1.62, s 5.38, s 5.12, s 1.97, s

2-AcO 2.23, s

2-AcO 2.22, s

6.18, qd (7.2, 1.2) 1.98, dq (7.2, 1.2) 1.87, q (1.2) 5-AcO 2.10, s

AngO 6.05, qd (7.2, 1.4) 1.95, dq (7.2, 1.4) 1.86, q (1.4)

29b

3.41, s

2.77, ddd (9.4, 6.4, 2.6) 2.97, dd (12.0, 8.0) 5.18, dd (7.7, 5.1) 5.13, t (6.8) 5.70, d (8.0) 2.37, m 2.33, m 5.55, dd (15.5, 8.0) 5.04, t (7.0) 4.99, t (7.1) 5.96, d (15.5) 1.64, s 1.66,s 1.32, s 1.58, s 1.59, s 1.28, s 5.26, s 5.28, s 5.36, s 5.07, s 5.19, s 5.19, s 1.53, s 1.41, s 1.46, s

6.05, qq (7.2, 1.4) 1.94, dq (7.2, 1.4) 1.86, q (1.4)

AngO 6.06, qd (7.2, 1.3) 1.96, dq (7.2, 1.3) 1.86, q (1.3)

27a

5-AngO

AngO 6.03, qq (7.2, 1.2) 1.94, dq (7.2, 1.2) 1.83, q (1.2)

6.10, qq (7.2, 1.4) 1.94, dq (7.2, 1.4) 1.88, q (1.4) AngO 6.04, qq (7.2, 1.4) 1.91, dq (7.2, 1.4) 1.79, q (1.4)

a

Recorded in CDCl3 at 400 MHz. bRecorded in methanol-d4 at 400 MHz. cm−1; UV (MeOH) λmax (log ε) 207 (4.1), 225 (4.3) nm; positive (+)-HRESIMS [M + Na]+ m/z 355.18824 (calcd for C20H28O4Na, 355.18853); 1H NMR data (400 Hz, CDCl3), Table 6; 13C NMR data (100 Hz, CDCl3), Tables 6. Rumicifoline K (15). colorless oil; [α]20D + 40 (c 0.3 in MeOH); IR (KBr) υmax 2960, 2872, 1733, 1677, 1445, 1374, 1294, 1187, 1004, 893 cm−1; UV (MeOH) λmax (log ε) 210 (3.79), 232 (4.0) nm; positive (+)-HRESIMS [M + Na]+ m/z 341.20786 (calcd for C20H30O3Na, 341.20926); 1H NMR data (400 Hz, methanol-d4), Table 6; 13C NMR data (100 Hz, methanol-d4), Table 6. Rumicifoline L (16). colorless oil; [α]20D + 30 (c 0.3 in MeOH); IR (KBr) υmax 2957, 2874, 1726, 1666, 1458, 1380, 1253, 1184, 985 cm−1; UV (MeOH) λmax (log ε) 210 (3.80), 232 (4.0) nm; positive (+)-HRESIMS [M + Na]+ m/z 355.22412 (calcd for C21H32O3Na, 355.22491); 1H NMR data (400 Hz, methanol-d4), Table 6; 13C NMR data (100 Hz, methanol-d4), Table 6. Rumicifoline M (22). colorless oil; [α]20D + 14 (c 0.1 in MeOH); IR (KBr) υmax 3465, 2955, 2930, 1745, 1720, 1650, 1450, 1230, 1035 cm−1; UV (MeOH) λmax (log ε) 216 (4.4) nm; positive (+)-HRESIMS [M + Na]+ m/z 529.24204 (calcd for C27H38O9Na, 529.24135); 1H NMR data (400 Hz, CDCl3), Table 7; and 13C NMR data (100 Hz, CDCl3), Tables 8. Rumicifoline N (23). colorless oil; [α]20D − 18 (c 0.2 in MeOH); IR (KBr) υmax 3448, 2927, 1715, 1645, 1450, 1385, 1230, 1038 cm−1; UV (MeOH) λmax (log ε) 205 (4.2) nm; positive (+)-HRESIMS [M + Na]+ m/z 445.22087 (calcd for C23H34O7Na, 455.22022); 1H NMR data

(4E)-Rumicifoline H (9). colorless oil; [α]20D − 105 (c 0.2 in MeOH); IR (KBr) υmax 2967, 1709, 1645, 1445, 1381, 1226, 1138, 933 cm−1; UV (MeOH) λmax (log ε) 219 (4.3) nm; positive (+)-HRESIMS [M + Na]+ m/z 581.30912 (calcd for C32H46O8Na, 581.30904); 1H NMR data (400 Hz, methanol-d4), Table 4; 13C NMR data (100 Hz, methanol-d4), Table 5. (4Z)-Rumicifoline H (10). colorless oil; [α]20D − 165 (c 0.2 in MeOH); IR (KBr) υmax 2968, 1708, 1645, 1447, 1378, 1231, 1135, 930 cm−1; UV (MeOH) λmax (log ε) 219 (4.3) nm; positive (+)-HRESIMS [M + Na]+ m/z 581.30930 (calcd for C32H46O8Na, 581.30904); 1H NMR data (400 Hz, methanol-d4), Table 4; 13C NMR data (100 Hz, methanol-d4), Table 5. Rumicifoline I (11). colorless oil; [α]20D − 176 (c 0.3 in MeOH); IR (KBr) υmax 2970, 1704, 1645, 1449, 1381, 1229, 1131, 927 cm−1; UV (MeOH) λmax (log ε) 222 (4.4) nm; positive (+)-HRESIMS [M + Na]+ m/z 567.29269 (calcd for C31H44O8Na, 567.29339); 1H NMR data (400 Hz, acetone-d6), Table 4; 13C NMR data (100 Hz, acetone-d6), Table 5. Rumicifoline J (13). colorless oil; [α]20D + 24 (c 0.2 in MeOH); IR (KBr) υmax 3475, 2940, 1710, 1655, 1440, 1250, 950, 900 cm−1; UV (MeOH) λmax (log ε) 207 (4.1), 228 (4.3) nm; positive (+)-HRESIMS [M + Na]+ m/z 355.18810 (calcd for C20H28O4Na, 355.18853); 1H NMR data (400 Hz, methanol-d4), Table 6; 13C NMR data (100 Hz, methanol-d4), Table 6. 1-epi-Rumicifoline J (14). colorless oil; [α]20D + 15 (c 0.2 in MeOH); IR (KBr) υmax 3475, 2945, 1700, 1650, 1445, 1250, 950, 900 2000



Article

Table 8. 13C NMR Spectroscopic Data of Compounds 22−24 and 26−29 (δ in ppm) position

22a

23a

24a

26a

27a

28a

29b

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 OMe

71.8, CH 77.5, CH 76.1, C 203.3, C 73.9, CH 47.6, CH 147.0, C 77.9, CH 31.3, CH2 119.3, CH 134.4, C 25.7, CH3 17.9, CH3 111.0, CH2 19.5, CH3

34.6, CH2 73.7, CH 85.6, C 203.1, C 76.7, CH 39.1, CH 147.0, C 75.2, CH 32.3, CH2 118.9, CH 134.6, C 25.8, CH3 18.0, CH3 113.0, CH2 14.2, CH3 52.4, CH3 5-AcO 170.0, C 20.2, CH3

34.2, CH2 73.8, CH 85.7, C 203.1, C 75.9, CH 40.7, CH 147.2, C 74.3, CH 32.6, CH2 119.1, CH 134.5, C 25.7, CH3 17.9, CH3 112.2, CH2 14.2, CH3 52.4, CH3 5-AngO 166.9, C 127.8, C 139.0, CH 15.8, CH3 20.5, CH3

25.9, CH2 62.9, CH 58.5, C 201.5, C 77.6, CH 42.2, CH 145.3, C 75.9, CH 32.7, CH2 118.7, CH 134.8, C 25.7, CH3 18.0, CH3 115.9, CH2 15.2, CH3

69.4, CH 65.5, CH 61.4, C 199.6, C 71.6, CH 51.0, CH 143.2, C 73.6, CH 124.1, CH 143.3, CH 70.3, C 29.5, CH3 29.1, CH3 116.6, CH2 14.3, CH3

190.1, C 141.8, C 148.3, C 68.8, CH 37.5, CH2 48.0, CH 146.4, C 75.8, CH 32.4, CH2 119.4, CH 134.4, C 25.7,CH3 18.0, CH3 114.4, CH2 13.6, CH3

189.7, C 142.6, C 149.6, C 73.1, CH 75.3, CH 54.6, CH 143.2, C 76.9, CH 33.1, CH2 120.7, CH 135.3, C 26.0, CH3 18.1, CH3 117.5, CH2 13.4, CH3

5-AcO 170.3, C 20.4, CH3

1-AngO 165.9, C 126.5, C 141.3, CH 16.0, CH3 20.4, CH3 AcO 169.8, C 20.5, CH3

2-AcO 168.5, C 20.2, CH3

2-AcO 170.0, C 20.1, CH3

AngO 166.5, C 127.6, C 138.7, CH 15.7, CH3 20.5, CH3

AngO 167.6, C 127.9, C 138.1, CH 15.7, CH3 20.5, CH3

AngO 168.1, C 129.0, C 139.4, CH 15.9, CH3 20.7, CH3 AngO 168.5, C 129.4, C 138.5, CH 16.1, CH3 20.7, CH3

2-TigO 166.6, C 128.0, C 138.5, CH 14.5, CH3 12.1, CH3 AcO 170.5, C 20.5, CH3

1′ 2′ 3′ 4′ 5′ 51″ 2″ 3″ 4″ 5″ 81‴ 2‴ 3‴ 4‴ 5‴

AngO 168.2, C 127.2, C 140.2, CH 15.8, CH3 20.4, CH3

AngO 166.8, C 127.9, C 138.6, CH 17.5, CH3 20.6, CH3

AngO 166.8, C 127.1, C 138.3, CH 15.7, CH3 20.4, CH3

AngO 167.0, C 127.7,C 138.5, CH 15.7, CH3 20.6, CH3

a

Recorded in CDCl3 at 100 MHz. bRecorded in methanol-d4 at 100 MHz.

Figure 6. 1H−1H COSY (bold) and selected HMBC (arrow) correlations of 22, 23, and 28.

Table 9. Antiproliferative Activity of Compounds 2, 13−16, and 29 against Four Human Tumor Cell Linesa compound

A-549

HGC-27

HeLa

MV4−11

2 13 14 15 16 29 taxol

12.5 ± 5.4 6.3 ± 1.8 13.5 ± 7.2 9.7 ± 3.8 6.6 ± 1.5 9.8 ± 4.7 0.051 ± 0.016

6.1 ± 2.6 2.1 ± 0.9 7.2 ± 2.7 5.3 ± 1.89 3.6 ± 0.9 2.2 ± 1.0 0.028 ± 0.007

8.4 ± 3.2 2.4 ± 0.9 5.7 ± 3.3 6.6 ± 1.6 3.3 ± 1.2 6.3 ± 2.9 0.046 ± 0.022

9.0 ± 3.6 0.5 ± 0.2 2.5 ± 0.8 7.5 ± 2.5 1.1 ± 0.5 2.9 ± 0.6 0.017 ± 0.006

Results are expressed as IC50 values in μM. Cell lines: A-549, lung cancer; HGC-27, stomach cancer; HeLa, cervical cancer; MV4−11, leukemia.

a

(400 Hz, CDCl3), Table 7; and Tables 8.

Rumicifoline O (24). colorless oil; [α]20D − 6 (c 0.2 in MeOH); IR (KBr) υmax 3451, 2927, 1716, 1646, 1456, 1384, 1230, 1154, 1040

13

C NMR data (100 Hz, CDCl3), 2001



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cm−1; UV (MeOH) λmax (log ε) 205 (4.2) nm; positive (+)-HRESIMS [M + Na]+ m/z 485.25130 (calcd for C26H38O7Na, 485.25152); 1H NMR data (400 Hz, CDCl3), Table 7; and 13C NMR data (100 Hz, CDCl3), Tables 8. Rumicifoline P (26). colorless oil; [α]20D + 9 (c 0.2 in MeOH); IR (KBr) υmax 2930, 1720, 1645, 1443, 1376, 1135, 1030 cm−1; UV (MeOH) λmax (log ε) 205 (4.2) nm; positive (+)-HRESIMS [M + Na]+ m/z 413.19384 (calcd for C22H30O6Na, 413.19401); 1H NMR data (400 Hz, CDCl3), Table 7; and 13C NMR data (100 Hz, CDCl3), Tables 8. Rumicifoline Q (27). colorless oil; [α]20D + 40 (c 0.2 in MeOH); IR (KBr) υmax 3450, 2926, 1745, 1720, 1645, 1445, 1377, 1229, 1143, 1033 cm−1; UV (MeOH) λmax (log ε) 205 (4.3) nm; positive (+)-HRESIMS [M + Na]+ m/z 527.22697 (calcd for C27H36O9Na, 527.22570); 1H NMR data (400 Hz, CDCl3), Table 7; and 13C NMR data (100 Hz, CDCl3), Tables 8. Rumicifoline R (28). colorless oil; [α]20D − 19 (c 0.2 in MeOH); IR (KBr) υmax 3449, 2925, 1712, 1645, 1452, 1381, 1229, 1152, 1037 cm−1; UV (MeOH) λmax (log ε) 223 (4.2) nm; positive (+)-HRESIMS [M + Na]+ m/z 413.19365 (calcd for C22H30O6Na, 413.19401); 1H NMR data (400 Hz, CDCl3), Table 7; and 13C NMR data (100 Hz, CDCl3), Tables 8. 5α-Angelouloxyrumicifoline R (29). colorless oil; [α]20D − 41 (c 0.2 in MeOH); IR (KBr) υmax 3452, 2927, 1716, 1646, 1456, 1384, 1231, 1154, 1040 cm−1; UV (MeOH) λmax (log ε) 218 (4.4) nm; positive (+)-HRESIMS [M + Na]+ m/z 511.23145 (calcd for C27H36O8Na, 511.23079); 1H NMR data (400 Hz, methanol-d4), Table 7; 13C NMR data (100 Hz, methanol-d4), Table 8. X-ray Crystallographic Analysis. Crystals of 2 were obtained from MeOH. The details of the protocols are described in the Supporting Information (Crystallographic Analysis). Crystallographic data of 2 [Flack parameter = 0.05(3)] has been deposited at the Cambridge Crystallographic Data Centre (Deposition number: CCDC 1826419). Antiproliferation Assays. The following human tumor cell lines were used: HGC-27 (stomach cancer), A-549 (lung cancer), HeLa (cervical cancer), and MV4-11 (leukemia). Standard MTT assays were carried out with taxol as positive control. The detailed process is described in the Supporting Information. Statistical Analysis. All antiproliferation assays were repeated more than three times. The IC50 value was calculated via Reed and Muench’s method, and the data are expressed as mean ± SD.

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ACKNOWLEDGMENTS This project was supported financially by the National Natural Science Foundation of China (Grants 21402157, 21572221, and 21571119).

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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.8b00197. General experimental procedures, extraction and isolation, antiproliferation assays, original HRESIMS and NMR spectra of compounds 1−11, 13−16, 22−24, and 26−29, and X-ray crystallographic data of 2 (PDF) Crystallographic file for 2 (CIF)

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Article

AUTHOR INFORMATION

Corresponding Authors

*Z.-X. Cao E-mail: [email protected]. Tel: (86) 02861800231. *Y. Zhou E-mail: [email protected]. Tel: (86) 028-82890810. ORCID

Yan Zhou: 0000-0002-3015-724X Author Contributions ∇

Y. Ye and D. Dawa contributed equally.

Notes

The authors declare no competing financial interest. 2002



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2003


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