Leonurusoleanolides E–J, Minor Spirocyclic Triterpenoids from

Dec 13, 2013 - ABSTRACT: Six new (leonurusoleanolides E−J, 1−6) and five known (7−11) nortriterpenoids were isolated and characterized from the ...
1 downloads 0 Views 440KB Size
Download PDF
Note pubs.acs.org/jnp

Leonurusoleanolides E−J, Minor Spirocyclic Triterpenoids from Leonurus japonicus Fruits Miao Ye,†,‡,§ Juan Xiong,† Jing-Jing Zhu,§ Jun-Lin Hong,⊥ Yun Zhao,§ Hui Fan,§ Guo-Xun Yang,† Gang Xia,§ and Jin-Feng Hu*,†,§ †

Department of Natural Products Chemistry, School of Pharmacy, Fudan University, Shanghai 201203, People’s Republic of China School of Pharmacy, Fujian University of Traditional Chinese Medicine, Fuzhou 350122, People’s Republic of China § Department of Chemistry and Institutes for Advanced Interdisciplinary Research, East China Normal University (ECNU), Shanghai 200062, People’s Republic of China ⊥ College of Chemistry and Materials Science, Sichuan Normal University, Chengdu 610068, People’s Republic of China ‡

S Supporting Information *

ABSTRACT: Six new (leonurusoleanolides E−J, 1−6) and five known (7−11) nortriterpenoids were isolated and characterized from the dried fruits of Leonurus japonicus. They all contain a distinctive 19(18→17)-abeo-28-noroleanane-type spirocylclic skeleton with a trans or a cis acyl substituent at C-3 or C-23. Similar to the previously known leonurusoleanolides A/B (7/8) and C/D (9/10), compounds 1/2 and 3/4 were also found to exist as equilibrium mixtures of trans and cis isomers. The isolated pure compounds and mixtures were evaluated for their cytotoxicity against a small panel of human cancer cell lines (BGC-823 and KE-97 gastric carcinoma, Huh-7 hepatocarcinoma, Jurkat T cell lymphoblasts, and MCF7 breast adenocarcinoma) using the CellTiter-Glo luminescent cell viability assay method. Among them, (2α,3β,17R*,18β)-3-O(trans-caffeoyl)-19(18→17)-abeo-28-norolean-12-ene-2,18,23-triol (leonurusoleanolide J, 6) showed the most potent cytotoxic activity, with IC50 values less than 10 μM.

A

ir-dried ripe fruits of the herbaceous plant Leonurus japonicus Houtt. (syn. L. heterophyllus Sweet,1,2 family Lamiaceae), known as “Chong-Wei-Zi” in Chinese, motherwort fruit, and Fructus Leonuri, have been found previously to yield several unusual 28-noroleanane-type spirocyclic triterpenoids3,4 with interesting nerve growth factor (NGF)-potentiating activity.3 Additionally, essential oil5 and cyclic nonapeptides6 were also reported from the motherwort fruit. As part of our ongoing research effort to discover new bioactive triterpenoids from higher plants,7−9 the fruits of L. japonicus were phytochemically reinvestigated. In this study, six new representatives of 28-noroleanane-derived spirocyclic triterpenoids (1−6) were identified. In addition, the related known nortriterpenoids leonurusoleanolides A−D (7−10)3 and phlomistetraol B (11)4,10 were also obtained. Reported herein are the isolation and structure elucidation of the new compounds and the evaluation of their cytotoxicity against five human cancer cell lines. Compound 1 was obtained as a white, amorphous powder. Its molecular formula was established as C39H56O7 based on a pseudomolecular ion peak [M + Na]+ at m/z 659.3917 (calcd for C39H56O7Na, 659.3918) in its HRESIMS. The 1H NMR spectrum (Table 1) of 1 displayed signals of six tertiary methyls [δ 1.11 (Me-25), 1.04 (Me-27), 1.02 (Me-30), 1.00 (Me-29), 0.97 (Me-26), and 0.82 (Me-24)], three oxymethines [δ 3.75 (1H, ddd, J = 10.6, 9.6, 5.5 Hz, H-2β), 3.38 (1H, d, J = 9.6 Hz, © XXXX American Chemical Society and American Society of Pharmacognosy

H-3α), and 3.86 (1H, br s, H-18α)], a hydroxymethylene group [δ 4.12 and 4.05 (ABq, J = 11.3 Hz, H2-23)], and an olefinic proton at δ 5.77 (br s, H-12). Additionally, a typical transferuloyl moiety was observed, which included a trans-double bond [δ 7.62 and 6.37 (each 1H, d, J = 15.9 Hz, H-7′ and H8′)], a benzene ring with an ABX system [δ 7.18 (1H, d, J = 1.6 Received: October 8, 2013

A

dx.doi.org/10.1021/np400838a | J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Note

Table 2. 13C NMR Data of Compounds 1−6 (125 MHz)a

Table 1. 1H NMR Data of Compounds 1−4 (500 MHz, CD3OD, J in Hz) position 1 2 3 5 6 7

1 2.06, dd, 12.7, 5.5 0.94, dd, 12.7, 10.6 3.75, ddd, 10.6, 9.6, 5.5 3.38, d, 9.6

2

24 25 26 27 29 30 8′ 7′

7.62, d, 15.9

6.84, d, 12.9

2′

7.18, d, 1.6

7.76, d, 1.5

5′

6.81, d, 8.2

6.76, d, 8.2

6′

7.05, dd, 8.2, 1.6

7.09, dd, 8.2, 1.5

OMe

3.88, s

3.86, 3H, s

16 18 19 21 22 23

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 29 30 1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′ 9′ 3′-OMe

2.06, dd, 12.6, 5.5 2.06, dd, 12.6, 4.5 0.93, dd, 12.6, 11.3 0.93, m 3.75, ddd, 11.3, 3.74, ddd, 10.4, 9.6, 4.5 10.0, 5.5 3.38, d, 9.6 3: 3.38, d, 9.6; 4: 3.27, d, 9.6 1.30, m 1.29, m 1.46, m 3: 1.38, m; 4: 1.30, m 1.53, m 1.53, m 1.43, m 1.44, m 1.58, m 1.55, m 2.00, m 2.00, m 5.73, br s 3: 5.77, br s; 4: 5.74, br s 1.73, m 1.73, m

1.32, m 1.46, m 1.53, m 1.43, m 1.57, m 2.00, m 5.77, br s 1.74, ddd, 11.4, 10.7, 5.4 1.01, m 1.54, m 3.86, br s 1.95, d, 12.9 1.11, d, overlapped 1.46, m 1.36, m 1.57, m 1.31, m 4.12, d, 11.3 4.05, d, 11.3 0.82, s 1.11, s 0.97, s 1.04, s 1.00, s 1.02, s 6.37, d, 15.9

9 11 12 15

position

3/4

1.01, m 1.53, m 3.87, br s 1.94, d, 12.8 1.11, d, overlapped 1.45, m 1.36, m 1.56, m 1.31, m 4.12, d, 11.4 4.05, d, 11.4 0.79, s 1.07, s 0.93, s 0.93, s 0.99, s 1.02, s 5.79, d, 12.9

3′

1.01, m 1.58, m 3.85, br s 1.94, d, 13.0 1.09, d, overlapped 1.46, m 1.36, m 1.54, m 1.36, m 4.12, d, 11.4 4.05, d, 11.0 3: 0.82, s; 4: 0.79, s 3: 1.11, s; 4: 1.06, s 3: 0.97, s; 4: 0.93, s 3: 1.03, s; 4: 0.92, s 3: 0.99, s; 4: 1.00, s 1.02, s 3: 6.32, d, 15.9; 4: 5.78, d, 12.7 3: 7.62, d, 15.9; 4: 6.86, d, 12.7 3: 7.43, d, 8.5; 4: 7.59, d, 8.7 3: 6.81, d, 8.6; 4: 6.75, d, 8.8 3: 6.81, d, 8.6; 4: 6.75, d, 8.8 3: 7.43, d, 8.5; 4: 7.59, d, 8.7

1b

2b d

49.0 69.7 77.9 44.3 49.7d 19.9 35.1 41.3 49.6d 39.3 24.5 119.3 143.8 45.6 28.3 37.5 51.4 75.9 53.6 40.1 42.9 29.8 67.3 14.3 18.4 18.5 23.9 30.7 30.7 127.8 112.1 149.9 151.2 116.9 124.6 147.2 115.8 169.1 57.1

3b e

49.0 69.5 78.0 44.0 49.7e 19.4 34.8 40.8 49.6e 39.0 24.2 119.2 143.6 45.2 28.1 37.0 51.2 76.4 53.1 39.8 43.1 29.6 66.7 14.0 18.2 18.0 23.4 30.4 30.5 126.4 114.8 147.0 149.6 116.0 125.6 144.2 116.6 169.0 56.6

4b f

48.5 69.4 77.9 43.9 48.9f 19.2 34.8 40.7 48.9f 38.9 24.0 119.0 143.9 45.0 28.0 36.9 51.1 76.2 53.0 39.7 43.0 29.5 66.6 13.8 18.0 18.1 23.5 30.3 30.4 127.1 131.1 116.9 161.4 116.9 131.1 146.6 115.2 168.9

g

48.5 69.4 77.7 43.6 48.3g 19.1 34.6 40.6 48.9g 38.8 24.0 119.0 143.5 44.9 27.9 36.9 51.1 76.2 53.0 39.7 43.0 29.5 66.4 13.9 17.9 18.0 23.4 30.3 30.4 127.1 133.4 116.0 161.4 116.0 133.4 144.3 116.7 168.9

5c

6c

48.0 68.3 77.2 43.0 48.1 18.5 34.0 39.7 48.4 38.0 23.3 118.2 143.1 44.0 27.4 35.9 50.3 74.7 52.3 39.0 42.3 29.0 66.2 13.9 17.7 17.8 22.8 29.9 30.1 126.7 115.7 147.5 150.3 116.5 121.8 145.5 115.0 167.2

48.4 66.3 79.5 43.5 47.0 17.9 33.7 39.5 47.5 37.7 23.1 118.0 142.9 43.9 27.3 35.8 50.0 74.5 52.1 38.8 42.1 28.8 64.4 14.5 17.5 17.5 22.7 29.7 29.9 126.7 115.5 147.3 150.0 116.3 122.0 145.4 115.2 168.2

a

Assignments were made by a combination of 1D- and 2D- (COSY, HSQC, HMBC) NMR experiments. b Measured in CD 3OD. c Measured in C5D5N. d−gOverlapped with the same superscript in the same column.

Hz, H-2′), 6.81 (1H, d, J = 8.2 Hz, H-5′), 7.05 (1H, dd, J = 8.2, 1.6 Hz, H-6′)], and a methoxy group [δ 3.88 (3H, s, OMe-3′)]. Apart from 10 typical carbon signals (δ 57.1, 112.1, 115.8, 116.9, 124.6, 127.8, 147.2, 149.9, 151.2, 169.1) (Table 2) assigned for the trans-feruloyl unit, the 13C and DEPT NMR spectroscopic data of 1 exhibited only 29 resonances attributable to the core structure [six sp3 methyl, 10 sp3 methylene (one oxygenated at δ 67.3), five sp3 (three oxygenated at δ 69.7, 75.9, 77.9) and one sp2 (δ 119.3) methine, six sp3 and one sp2 (δ 143.8) quaternary carbon (Table 2)]. The above data showed general features resembling those of leonurusoleanolide C (9), a known pentacyclic 28noroleanane-type spirocyclic triterpenoid derivative previously isolated from L. heterophyllus3 (syn. L. japonicus1,2). Compound 1 has a 19(18→17)-abeo-28-norolean-12-ene framework with oxygenated substituents at C-2, C-3, C-18, and C-23, which was

confirmed by 2D-NMR experiments (COSY, HSQC, and HMBC). Indeed, the only difference between compounds 1 and 9 is that the trans-feruloyl moiety is located at C-23 in 1, and hence key HMBC correlations from H2-23 (δ 4.12/4.05) to C-3 (δ 77.9), C-5 (δ 49.7), and C-9′ (δ 169.1) were observed (Figure 1). Similar to compound 9,3 the relative configuration of 1 was determined by analysis of the coupling constants (Table 1) and NOE correlations (Figure 2) together with biogenetic considerations. In particular, the large value of JH‑2,3 (9.6 Hz) indicated that both H-2 and H-3 are at axial positions. Clear NOE correlations were observed between H-2 (δ 3.75) and Me-24 (δ 0.82), between Me-24 and Me-25 (δ 1.11), between H-18 (δ 3.86) and Me-27 (δ 1.04), and between H-18 and H19 (δ 1.95). These data revealed that H-2, Me-24, and Me-25 are β-oriented, whereas H-18, Me-27, and the C17−C19 bond B

dx.doi.org/10.1021/np400838a | J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Note

and 2) were almost superimposable on those of compound 1. In the low-field region of the 1H NMR spectrum, two sets of well-separated signals for the p-coumaroyl moiety were present: δ 7.62 (1H, d, J = 15.9 Hz, H-7′), 6.32 (1H, d, J = 15.9 Hz, H8′), 7.43 (2H, d, J = 8.5 Hz, H-2′, 6′), and 6.81 (2H, d, J = 8.6 Hz, H-3′, 5′) for the E-isomer (3) and δ 6.86 and 5.78 (each 1H, d, J = 12.7 Hz, H-7′ and H-8′), 7.59 (2H, d, J = 8.7 Hz, H2′, H-6′), and 6.75 (2H, d, J = 8.8 Hz, H-3′, H-5′) for the Zisomer (4). Compounds 3 and 4 are closely related to leonurusoleanolides A/B (7/8), another pair of inseparable analogues with a trans- or a cis-p-coumaroyl moiety at C-3.3 The ester at C-23 in compounds 3 and 4 was confirmed by the HMBC NMR spectrum. Thus, the structures of 3 and 4 were established as (2α,3β,17R*,18β)-23-O-(trans-p-coumaroyl)19(18→17)-abeo-28-norolean-12-ene-2,3,18-triol (leonurusoleanolide G, 3) and (2α,3β,17R*,18β)-23-O-(cis-p-coumaroyl)-19(18→17)-abeo-28-norolean-12-ene-2,3,18-triol (leonurusoleanolide H, 4), respectively. The molecular formula of leonurusoleanolide I (5) was assigned as C38H54O7 on the basis of a pseudomolecular ion peak at m/z 645.3808 [M + Na]+ (calcd for C38H54O7Na, 645.3762) in its HRESIMS. Comparison of the 1H and 13C NMR data of 5 (Tables 2 and 3) with those of compounds 1−4 revealed that 5 is also a 19(18→17)-abeo-28-norolean-12-ene derivative bearing a different acyl unit at C-23. The proton signals of a 1,3,4-substituted aromatic ring [δ 7.57 (1H, br s, H2′), 7.23 (1H, d, J = 8.0 Hz, H-5′), 7.14 (1H, br d, J = 8.0 Hz,, H-6′)] and a trans-double bond [δ 7.98 and 6.67 (each, 1H, d, J = 15.8 Hz, H-7′ and H-8′, respectively)] were indicative of a trans-caffeoyl moiety. Thus, compound 5 was identified as (2α,3β,17R*,18β)-23-O-(trans-caffeoyl)-19(18→17)-abeo-28norolean-12-ene-2,3,18-triol. In a similar manner, leonurusoleanolide J (6) was elucidated as (2α,3β,17R*,18β)-3-O-(trans-caffeoyl)-19(18→17)-abeo-28norolean-12-ene-2,18,23-triol, by comparison of its HRESIMS and 1H and 13C NMR data with those of compounds 5 and 9.3 In particular, the downfield shift of H-3 (5: δ 3.99; 6: δ 5.81) (Table 3) suggested that the 3-hydroxy group is esterified. The trans-caffeoyl moiety at C-3 in 6 was confirmed by the corresponding HMBC NMR experiments. The identified nortriterpenoids (1−11) possess a unique 28noroleanane-type spirocyclic skeleton. This class of compounds has been reported mainly from plants in the family Lamiaceae, including Phlomis umbrosa,10 Phlomis viscosa,11 Notochaete hamosa,12 Gomphostemma parvif lorum,13 and Rostrinucula dependens.14 Interestingly, their ester derivatives have been found so far only from the fruits of L. japonicus.3,4 All the purified compounds and mixtures were tested for their cytotoxic potency against a small panel of human cancer cell lines (BGC-823 and KE-97 gastric carcinoma, Huh-7 hepatocarcinoma, Jurkat T cell lymphoblasts, and MCF-7 breast adenocarcinoma). As shown in Table 4, leonurusoleanolides I (5) and J (6), each with a trans-caffeoyl group at either C-23 or C-3, exhibited the most potent cytotoxic effects against all five tumor cell lines, with IC50 values below 10 μM. The remaining samples (1−4 and 7−10) were found to be inactive (IC50’s > 10 μM). Thus, the trans-caffeoyl moiety may be essential for the potent cytotoxicities of the nortriterpenoids, which is consistent with a previous report.15

Figure 1. Observed key HMBC correlations of 1.

Figure 2. Observed key NOE correlations of 1.

are α-oriented, as depicted in Figure 2. Accordingly, the R* configuration of C-17 in 1 was determined to be the same as that of compound 9.3 Consequently, compound 1 was elucidated as (2α,3β,17R*,18β)-23-O-(trans-feruloyl)-19(18→ 17)-abeo-28-norolean-12-ene-2,3,18-triol (leonurusoleanolide E). Leonurusoleanolide F (2) could be separated from 1 by reversed-phase HPLC (see Experimental Section); however, 2 was found to be unstable and quickly interconverted to 1 even at room temperature. Compound 2 was eventually obtained as an equilibrium mixture (1:2: ca. 4:1 ratio). Both 2 and 1 have the same molecular formula (C39H56O7) based on their HRESIMS data. The 1H NMR data of the mixture showed general features very similar to those of 1, but also displayed an additional set of signals in the range δH 5.79 to 7.76 ppm, characteristic of a cis-feruloyl moiety [e.g., a cis-double bond at δ 6.84 (1H, d, J = 12.9 Hz, H-7′) and 5.79 (1H, d, J = 12.9 Hz, H-8′), a benzene ring with an ABX system at δ 7.76 (1H, d, J = 1.5 Hz, H-2′), 6.76 (1H, d, J = 8.2 Hz, H-5′), and 7.09 (1H, dd, J = 8.2, 1.5 Hz,, H-6′), and a methoxy group at δ 3.86 (3H, s, OMe-3′)] (Table 1). Thus, compound 2 was deduced to be (2α,3β,17R*,18β)-23-O-(cis-feruloyl)-19(18→17)-abeo-28-norolean-12-ene-2,3,18-triol, as was confirmed by detailed 2DNMR experiments. Like the above equilibrium mixture, compounds 3 and 4 could be separated by HPLC (see Experimental Section), but they interconverted within 2 h at room temperature. Compounds 3 and 4 were finally obtained as a pair of inseparable isomers with a trans- or a cis-p-coumaroyl (rather than feruloyl) moiety at C-23 (3:4: ca. 4:1 ratio). The molecular formula (C38H54O6) of 3/4 was determined by HRESIMS. The 1H and 13C NMR spectroscopic data (Tables 1

■

EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured on a Perkin-Elmer 341 polarimeter (Perkin-Elmer, Waltham,

C

dx.doi.org/10.1021/np400838a | J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Note

Table 3. 1H NMR Data of Compounds 5 and 6 (500 MHz, C5D5N, J in Hz) position 1 2 3 5 6 7 9 11 12 15 16 18 19 21 22 23 24 25 26 27 29 30 8′ 7′ 2′ 5′ 6′

5 2.39, 1.34, 4.27, 3.99, 1.62, 1.65, 1.44, 1.67, 1.45, 1.70, 2.06, 6.28, 1.74, 0.96, 1.58, 4.15, 2.32, 1.23, 1.81, 1.48, 1.97, 1.39, 4.69, 4.46, 1.08, 1.12, 1.04, 1.08, 1.09, 1.17, 6.67, 7.98, 7.57, 7.23, 7.14,

Gold 168 and a Sedex 80 (Sedere, Alfortville, France) evaporative light-scattering detector (ELSD), and a YMC-Pack ODS-A column (YMC Co., Ltd., Kyoto, Japan) (250 × 10 mm, 5 μm). Column chromatography (CC) was carried out on silica gel (200−300 mesh, Kang-Bi-Nuo Silysia Chemical Ltd., Yantai, People’s Republic of China), MCI gel CHP20P (75−150 μm, Mitsubishi Chemical Industries, Tokyo, Japan), and TLC on silica gel (GF254, 0.25 mm, Kang-Bi-Nuo Silysia Chemical Ltd., Yantai, People’s Republic of China). Spots were visualized using UV light (254 nm) and 15% H2SO4−EtOH. Plant Material. The fruits of L. japonicus were purchased from Shanghai Jiu-Zhou-Tong Medicine Co., Ltd., and were originally collected in August 2009 from Bozhou City, Anhui Province, People’s Republic of China. The plant material was identified by Prof. BaoKang Huang (Department of Pharmacognosy, the Second Military Medical University, Shanghai, People’s Republic of China). A voucher specimen (No. 100126) was deposited at the Herbarium of the Department of Natural Products Chemistry, School of Pharmacy, Fudan University. Extraction and Isolation. Powdered and dried fruits of L. japonicus (20.0 kg) were extracted with methanol (15 L × 4) under reflux for 4 h. The solvent was removed at reduced pressure to give a dark brown residue (ca. 832.6 g), which was then suspended in H2O (2 L) and extracted with EtOAc three times (3 × 2 L). The entire EtOAc-soluble extract (ca. 340.2 g) was subjected to flash CC over silica gel eluting with gradients of petroleum ether (PE)−EtOAc− MeOH (2:1:0 → 0:0:1, v/v/v) to yield 10 fractions (Fr. 1−Fr. 10). Fr. 3 (PE−EtOAc, 1:1, 32.7 g) was fractionated on a silica gel column eluted with CH2Cl2−MeOH (35:1) to give six subfractions (Fr. 3A− Fr. 3F). Fr. 3D (355.3 mg) was purified by CC on silica gel (CH2Cl2− MeOH, 30:1) and then semipreparative HPLC (MeOH−H2O, 95:5, v/v) to afford compounds 7 (tR = 10.3 min) and 8 (tR = 8.0 min). Similar to a previous finding,3 these two compounds readily interconverted and reached equilibrium within 2 h (the amount obtained of the mixture 7/8 was 15.6 mg). A mixture of compounds 9 and 10 (21.8 mg) was obtained from Fr. 3E (78.5 mg) by crystallization (CH2Cl2−MeOH, 30:1). Fr. 4 (neat EtOAc, 45.7 g) was subjected to silica gel CC (CH2Cl2−MeOH, 35:1) to generate Fr. 4A to Fr. 4E. Fr. 4B (5.3 g) was applied to CC over MCI gel using a gradient of MeOH−H2O (from 70% to 100%, v/v), and the fraction eluted with 80% MeOH−H2O was further purified by semipreparative HPLC to yield compounds 1 (8.4 mg, tR = 9.6 min) and 2 (tR = 7.9 min). The method was set as a linear gradient of MeOH in H2O [with 0.1% TFA (trifluoroacetic acid) as buffer] from 97% to 98% for 15 min. Differing from compounds 7/8, 1 was rather stable (less than 5% of 1 was found to be interconverted to 2 in about one week), but 2 rapidly interconverted to 1 and reached equilibrium within 2 h (the amount obtained of the mixture 2/1 was 6.4 mg). Compounds 3 (tR = 21.5 min) and 4 (tR = 13.5 min) were obtained by semipreparative HPLC from Fr. 4C (2.3 g). The method used was 92% MeOH in H2O (0.1% TFA as buffer) for 15 min; then a linear gradient of MeOH was applied from 92% to 96% for 1 min, followed by 96% MeOH for 22 min. Similar to 7/8, compounds 3/4 eventually existed as a mixture (19.6 mg). Compound 6 (14.8 mg, tR = 17.0 min) was obtained from Fr. 4D (553.8 mg) by semipreparative HPLC (MeCN−H2O, 86:14, v/ v). Fr. 5 (EtOAc−MeOH, 20:1, 18.2 g) was separated by repeated CC

6

dd, 12.4, 4.1 dd, 11.9, 11.0 ddd, 11.0, 10.6, 4.8 d, 9.3 m m m m m m m br s m m m br s d, 12.7 d, 12.6 m m m m d, 11.1 d, 11.1 s s s s s s d, 15.8 d, 15.8 br s 1H, d, 8.0 1H, br d, 8.0

2.42, 1.40, 4.47, 5.81, 1.97, 1.76, 1.44, 1.76, 1.45, 1.71, 2.05, 6.29, 1.79, 0.99, 1.63, 4.20, 2.35, 1.27, 1.81, 1.49, 2.00, 1.43, 3.63, 3.48, 0.95, 1.15, 1.07, 1.08, 1.10, 1.18, 6.67, 7.97, 7.57, 7.23, 7.11,

dd, 12.4, 4.0 dd, 12.5, 10.2 ddd, 10.2, 10.0, 4.2 d, 9.9 m m m m m m m br s m m m br s d, 12.7 d, 12.7 m m m m d, 11.4 d, 11.7 s s s s s s d, 15.8 d, 15.8 br s d, 8.0 br d, 8.0

MA, USA). UV absorptions were obtained on a Libra S35 PC UV−vis recording spectrophotometer (Biochrom Ltd., Cambridge, UK), and IR spectra were measured on an Avatar 360 FT-IR spectrophotometer (Thermo Scientific, Waltham, MA, USA), using KBr pellets. NMR spectra were recorded on a Bruker Avance DRX-500 spectrometer (Bruker Daltonics, Boston, MA, USA). Chemical shifts are expressed in δ (ppm) and are referenced to the residual solvent signals. HRESIMS were recorded on a Bruker Daltonics micrOTOF-Q mass spectrometer (Bruker Daltonics, Boston, MA, USA). Semipreparative HPLC was performed on a Beckman System (Beckman Coulter, Krefeld, Germany) consisting of a Beckman Coulter System Gold 508 autosampler, Gold 126 gradient HPLC pumps with a Beckman System

Table 4. Cytotoxicities of Compounds 5, 6, and 11 IC50 (mean ± SEM, μM)a compound

BGC-823

KE-97

Huh-7

Jurkat

5b 6b 11b staurosporineb

>10 7.5 ± 1.3 >10 0.38 ± 0.02

>10 3.0 ± 0.3 >10 0.13 ± 0.01

9.7 ± 0.7 1.9 ± 0.5 >10 0.23 ± 0.07

9.7 ± 5.7 3.9 ± 0.9 >10 0.14 ± 0.02

MCF-7 4.3 4.6 8.5 0.52

± ± ± ±

0.1 0.08 0.3 0.1

a

IC50 values refer to the 50% inhibition concentration and were calculated from regression using 10 different concentrations with triplicate experiments. bThe purity of the tested compounds and the positive control ranged from 95.1% to 99.5% as determined by analytical HPLC with ELSD detection. Staurosporine was used as a positive control. D

dx.doi.org/10.1021/np400838a | J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

■

(silica gel, PE−acetone, 1:1; MCI gel, MeOH−H2O, 60% to 100%, v/ v) to yield compounds 5 (54.8 mg) and 11 (34.8 mg). Leonurusoleanolide E (1): amorphous, white powder; [α]22D +21.6 (c 0.25, MeOH); IR (KBr) νmax 3350, 2970, 2937, 1673, 1599, 1523, 1266, 1145, 1097, 1035, 969 cm−1; UV (MeOH) λmax (log ε) 328 (3.51), 302 (3.34), 237 (3.30) nm; 1H (500 MHz) and 13C (125 MHz) NMR data (CD3OD), see Tables 1 and 2; (+)-ESIMS m/z 659 [M + Na]+; (+)-HRESIMS m/z 659.3917 [M + Na]+ (calcd for C39H56O7Na, 659.3918). Leonurusoleanolides F (2) and E (1): amorphous, white powder; [α]22D +46.2 (c 0.25, MeOH); IR (KBr) νmax 3449, 2946, 1665, 1604, 1515, 1014, 966, 742 cm−1; UV λmax (MeOH) (log ε) 330 (3.63), 303 (3.84), 235 (3.39) nm; 1H (500 MHz) and 13C (125 MHz) NMR data (CD3OD), see Tables 1 and 2; (+)-ESIMS m/z 659 [M + Na]+; (+)-HRESIMS m/z 659.3915 [M + Na]+ (calcd for C39H56O7Na, 659.3918). Leonurusoleanolides G (3) and H (4): amorphous, white powder; [α]22D +28.7 (c 0.31, MeOH); IR (KBr) νmax 3435, 2915, 2360, 2353, 1660, 1583, 1380, 1041, 679, 641 cm−1; UV λmax (MeOH) (log ε) 332 (3.76), 304 (3.38), 237 (3.26) nm; 1H (500 MHz) and 13C (125 MHz) NMR data (CD3OD), see Tables 1 and 2; (+)-ESIMS m/z 629 [M + Na]+; (+)-HRESIMS m/z 629.3807 [M + Na]+ (calcd for C38H54O6Na, 629.3813). Leonurusoleanolide I (5): amorphous, white powder; [α]22D +19.1 (c 0.25, MeOH); IR (KBr) νmax 3444, 2968, 2893, 2363, 1633, 1457, 668 cm−1; UV λmax (MeOH) (log ε) 332 (3.87), 304 (3.90), 244 (3.76) nm; 1H (500 MHz) and 13C (125 MHz) NMR data (C5D5N), see Tables 2 and 3; (+)-ESIMS m/z 645 [M + Na]+; (+)-HRESIMS m/z 645.3806 [M + Na]+ (calcd for C38H54O7Na, 645.3762). Leonurusoleanolide J (6): amorphous, white powder; [α]22D +36.2 (c 0.22, MeOH); IR (KBr) νmax 3441, 2968, 2893, 2363, 1635, 1457, 1104, 668, 555 cm−1; UV λmax (MeOH) (log ε) 330 (4.01), 302 (3.90), 247 (3.79) nm; 1H (500 MHz) and 13C (125 MHz) NMR data (C5D5N), see Tables 2 and 3; (+)-ESIMS m/z 645 [M + Na]+; (+)-HRESIMS m/z 645.3760 [M + Na]+ (calcd for C38H54O7Na, 645.3762). Cytotoxicity Assays. Five human cancer cell lines (BGC-823 and KE-97 gastric carcinoma, Huh-7 hepatocarcinoma, Jurkat T cell lymphoma, A549 lung adenocarcinoma) were purchased from the cell bank of the Shanghai Institute of Cell Biology. The BGC-823, KE-97, Jurkat, and A549 cell lines were cultured in Roswell Park Memorial Institute-1640 (RPMI-1640) medium, while the Huh-7 cell line was cultured in Dulbecco’s modified Eagle medium. All media were supplemented with 10% fetal bovine serum with 100 units/mL penicillin/streptomycin (Invitrogen, Carlsbad, CA, USA). The cells were maintained at 37 °C in a humidified environment with 5% CO2. The cell viability was determined by using the CellTiter Glo luminescent cell viability assay.7,8,16 Similar to an earlier study,7 the operation process was kept away from bright light and the cells were incubated in a dark incubator in order to exclude phototoxicity. Cells were also incubated in fresh cell culture medium and washed carefully to avoid false positive results.7 Briefly, the cancer cells were seeded into 384-well plates at an initial density of 1000 cells/well in 40 μL of medium. Then, the cells were treated with serially diluted compounds or the positive control staurosporine (Sigma-Aldrich, St. Louis, MO, USA, catalog no. S6942-200UL). After incubation for 72 h, 10% of CellTiter Glo reagent (Promega, Madison, WI, USA) was added, and luminescent signals were read on a VeriScan reader (Thermo Fisher Scientific, Waltham, MA, USA). The IC50 value was calculated from the curves generated by plotting the percentage of the viable cells versus test concentrations on a logarithmic scale using SigmaPlot 10.0 software.

■

Note

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected] (J.-F. Hu). Tel/Fax: +86 21 51980172. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS The authors thank Prof. B.-K. Huang (Department of Pharmacognosy, the Second Military Medical University of China) for the plant identification. This work was supported by NSFC grants (nos. 81273401, 81202420), an STCSM grant (no. 11DZ1921203), grants from the Ph.D. Programs Foundation of Ministry of Education (MOE) of China (nos. 20120071110049, 20120071120049), a MOST grant (no. 2011ZX09307-002-01), and the National Basic Research Program of China (973 Program, grant no. 2013CB530700).

■

REFERENCES

(1) Funk, V.; Hollowell, T.; Berry, P.; Kelloff, C.; Alexander, S. N. Contrib. US Natl. Herb 2007, 55, 322. (2) Romero-Gonzάlez, R. R.; Á vila-Núñez, J. L.; Aubert, L.; AlonsoAmelot, M. E. Phytochemistry 2006, 67, 965−970. (3) Liu, Y.; Kubo, M.; Fukuyama, Y. J. Nat. Prod. 2012, 75, 1353− 1358. (4) Zheng, Y.-Q.; Yan, H.; Han, J.; Tan, N.-H. Chin. J. Chin. Mat. Med 2012, 37, 2088−2091. (5) Xiong, L.; Peng, C.; Zhou, Q.-M.; Wan, F.; Xie, X.-F.; Guo, L.; Li, X.-H.; He, C.-J.; Dai, O. Molecules 2013, 18, 963−973. (6) (a) Morita, H.; Iizuka, T.; Gonda, A.; Itokawa, H.; Takeya, K. J. Nat. Prod. 2006, 69, 839−841. (b) Morita, H.; Gonda, A.; Takeya, K.; Itokawa, H. Bioorg. Med. Chem. Lett. 1996, 6, 767−770. (7) Hong, Z.-L.; Xiong, J.; Wu, S.-B.; Zhu, J.-J.; Hong, J.-L.; Zhao, Y.; Xia, G.; Hu, J.-F. Phytochemistry 2013, 86, 159−167. (8) Wu, S.-B.; Bao, Q.-Y.; Wang, W.-X.; Zhao, Y.; Xia, G.; Zhao, Z.; Zeng, H.-Q.; Hu, J.-F. Planta Med. 2011, 77, 922−928. (9) Wu, S.-B.; Su, J.-J.; Sun, L.-H.; Wang, W.-X.; Zhao, Y.; Li, H.; Zhang, S.-P.; Dai, G.-H.; Wang, C.-G.; Hu, J.-F. J. Nat. Prod. 2010, 73, 1898−1906. (10) Liu, P.; Yao, Z.; Zhang, W.; Takaishi, Y.; Duan, H.-Q. Chem. Pharm. Bull. 2008, 56, 951−955. (11) Ç alış, I.̇ ; Kırmızıbekmez, H.; Taşdemir, D.; Rüedi, P. Helv. Chim. Acta 2004, 87, 611−619. (12) Luo, Y. G.; Feng, C.; Tian, Y. J.; Li, B. G.; Zhang, G. L. Tetrahedron 2003, 59, 8227−8232. (13) Wang, Q.; Luo, S.-D.; Ju, P.; Wang, Y.-F. Helv. Chim. Acta 2007, 90, 1360−1365. (14) Peng, S. L.; Li, R.; Bao, T.; Liao, X.; Ding, L. S. Fitoterapia 2005, 76, 625−628. (15) Kurata, R.; Adachi, M.; Yamakawa, O.; Yoshimoto, M. J. Agric. Food Chem. 2007, 55, 185−190. (16) Ye, M.; Su, J.-J.; Liu, S.-T.; Cao, L.; Xiong, J.; Zhao, Y.; Fan, H.; Yang, G.-X.; Xia, G.; Hu, J.-F. Nat. Prod. Res. 2013, 27, 1136−1140.

ASSOCIATED CONTENT

S Supporting Information *

The 1D- and 2D-NMR spectra and HRESIMS of compounds 1−6 are available free of charge via the Internet at http://pubs. acs.org. E

dx.doi.org/10.1021/np400838a | J. Nat. Prod. XXXX, XXX, XXX−XXX