Enmein-type 6,7-seco-ent-Kauranoids from Isodon sculponeatus

Nov 12, 2013 - ABSTRACT: Fourteen enmein-type 6,7-seco-ent-kaurane di- terpenoids, seven new ones (sculponins M−S, 1−7) and seven known compounds ...
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Enmein-type 6,7-seco-ent-Kauranoids from Isodon sculponeatus Hua-Yi Jiang,†,‡ Wei-Guang Wang,† Min Zhou,†,‡ Hai-Yan Wu,†,‡ Rui Zhan,† Xiao-Nian Li,† Xue Du,† Yan Li,† Jian-Xin Pu,*,† and Han-Dong Sun*,† †

State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, People’s Republic of China ‡ University of Chinese Academy of Sciences, Beijing 100049, People’s Republic of China S Supporting Information *

ABSTRACT: Fourteen enmein-type 6,7-seco-ent-kaurane diterpenoids, seven new ones (sculponins M−S, 1−7) and seven known compounds (8−14), were isolated from the aerial parts of Isodon sculponeatus. Compound 1 is the first example of an ent-kauranoid, possessing a 11,12-epoxy group, and compounds 6 and 7 have a rare 3,6-epoxy group. The structures were established primarily by NMR and MS methods, and the absolute configurations of 1, 3, and 6 were determined by single-crystal X-ray diffraction. Compound 14 showed significant cytotoxic activity against five human tumor lines, with IC50 values ranging from 1.0 to 3.5 μM, and it also inhibited NO production in LPS-stimulated RAW264.7 cells, with an IC50 value of 2.2 μM.

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RESULTS AND DISCUSSION Compound 1 had molecular formula C20H26O6, as deduced from HREIMS ([M]+ m/z 362.1734, calcd 362.1729), indicating eight degrees of unsaturation. The IR spectrum showed absorptions at 3475 and 1723 cm−1, consistent with the presence of OH and lactonic carbonyl groups. The 13C NMR and DEPT spectra (Table 1) exhibited 20 carbon signals, including two methyls, five methylenes (one oxygenated and one olefinic), eight methines (five oxygenated), and five quaternary carbons (one olefinic and one carbonyl carbon). The data indicated that compound 1 was a 6,7-seco-entkauranoid-1,8-lactone and that it had structural features similar to the enmein-type ent-kauranoid isodocarpin (14).20 Comparison of the NMR data of compounds 1 and 14 suggested that 1 differed from 14 by the replacement of two methylenes (δC 21.2 and 31.3) by two methines (δC 53.2 and 54.5) and the appearance of an oxygenated methine (δC 76.5, d) in 1 rather than the carbonyl carbon (δC 202.3, s) in 14.20 The structure of compound 1 was supported by the following 2D NMR correlations and its unsaturation degrees: (i) the 1H−1H COSY and HSQC analyses revealed two spin systems, C(1)H− C(2)H2−C(3)H2 and C(9)H−C(11)H−C(12)H−C(13)H− C(14)H2 (bold bonds in Figure 1); (ii) the HMBC correlations from H-9 (δH 3.91), H-13 (δH 3.02), and H2-17 (δH 5.53 and 5.19) to C-15 (δC 76.5, d) implied an OH group at C-15 (Figure 1); (iii) the HMBC correlations from H-11 (δH 4.26) to C-8 (δC 52.8, s) and C-13 (δC 38.9, d), from H-12 (δH 3.33) to C-13 and C-14 (δC 31.5, t), along with the unsaturation degrees of compound 1, disclosed the existence of a three-

he genus Isodon has attracted much attention as a prolific source of ent-kauranoids with diverse structures and biological activities.1 Isodon sculponeatus (Vaniot) Kudo (Lamiaceae), a perennial herb, is widely distributed in southern China and has been used as a folk medicine for treatment of dysentery and beriberi.2,3 Previous phytochemical investigations of this species collected from several different regions of China resulted in the isolation of 36 new diterpenoids.4−15 Sculponin A,15 sculponeatin J,10 and sculponeatin H11 were significantly cytotoxic. Several diterpenoids isolated from I. sculponeatus possess intriguing structures, for instance, 6,7-seco-ent-kauranoids with unprecedented multicyclic skeletons (sculponins A−C),15 dimeric 6,7-seco-ent-kauranoids (sculponins D and E),14 and ent-kauranoids with phenylacetyl substituents (sculponeatins L and M).9 In our ongoing search for bioactive diterpenoids, we investigated the chemical constituents of I. sculponeatus collected from Muli County of Sichuan Province, P. R. China. As a result, a series of enmein-type 6,7-seco-ent-kaurane diterpenoids, including seven new ones, sculponins M−S (1− 7), and seven known ones, enmein (8),16 nodosin (9),17,18 epinodosin (10),16 sculponeatin A (11),4 sculponeatin B (12),4 6β,15α-dihydroxy-6α,20-epoxy-6,7-seco-ent-kaur-16-en-1α,7olide (13),19 and isodocarpin (14),20 were isolated. Compounds 1−4 and 8−14 were evaluated for their cytotoxicity against five human tumor lines (HL-60, SMMC-7721, A-549, MCF-7, and SW-480). Due to the folk use of I. sculponeatus, compounds 9, 11, and 14 were also tested for their inhibitory activity against NO production in LPS-stimulated RAW264.7 cells. © XXXX American Chemical Society and American Society of Pharmacognosy

Received: August 16, 2013

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Table 1. 13C NMR Spectroscopic Data for Sculponins M−S (1−7) (δ in ppm)

a

position

1a,c

2a,c

3a,c

4a,c

5a,c

6a,b

7a,c

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

78.6, CH 23.9, CH2 37.8, CH2 31.0, C 55.9, CH 102.0, CH 174.9, C 52.8, C 39.4, CH 50.2, C 53.2, CH 54.5, CH 38.9, CH 31.5, CH2 76.5, CH 151.8, C 109.0, CH2 33.1, CH3 23.5, CH3 75.3, CH2

77.4, CH 24.1, CH2 37.4, CH2 31.3, C 54.5, CH 102.1, CH 176.1, C 51.2, C 42.4, CH 54.3, C 126.9, CH 136.2, CH 40.3, CH 37.1, CH2 77.1, CH 155.4, C 106.7, CH2 33.1, CH3 23.5, CH3 75.1, CH2

78.6, CH 23.6, CH2 29.7, CH2 42.5, C 54.6, CH 111.8, CH 175.3, C 52.5, C 39.8, CH 50.4, C 67.0, CH 53.1, CH2 76.5, C 41.9, CH2 78.0, CH 162.4, C 108.0, CH2 31.0, CH3 77.2, CH2 72.7, CH2

77.2, CH 32.8, CH2 73.2, CH 38.1, C 55.8, CH 102.2, CH 172.1, C 57.2, C 50.6, CH 49.9, C 65.5, CH 34.2, CH2 41.5, CH 33.2, CH2 212.8, C 78.2, C 20.7, CH3 29.8, CH3 16.3, CH3 74.3, CH2

76.3, CH 24.4, CH2 37.3, CH2 31.8, C 54.6, CH 102.4, CH 176.0, C 56.0, C 45.8, CH 51.1, C 63.3, CH 37.2, CH2 46.5, CH 34.3, CH2 84.2, CH 80.7, C 23.5, CH3 33.2, CH3 23.3, CH3 73.4, CH2

72.0, CH 35.1, CH2 82.0, CH 42.9, C 53.1, CH 109.7, CH 171.6, C 56.7, C 54.5, CH 51.5, C 64.6, CH 34.3, CH2 41.6, CH 31.8, CH2 212.2, C 78.5, C 19.6, CH3 21.3, CH3 28.8, CH3 76.4, CH2

73.6, CH 35.1, CH2 82.6, CH 43.6, C 54.3, CH 109.4, CH 171.6, C 56.6, C 50.2, CH 50.0, C 64.9, CH 34.2, CH2 41.0, CH 33.2, CH2 212.4, C 78.1, C 21.0, CH3 22.8, CH3 29.1, CH3 75.5, CH2

Recorded in C5D5N. bRecorded at 100 MHz. cRecorded at 125 MHz.

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

membered epoxy ring between C-11 and C-12. Thus, 1 was determined to be a 6,7-seco-ent-kauranoid-1,8-lactone possessing a three-membered epoxy ring at a nonclassical position. Furthermore, the OH at C-15 in compound 1 was assigned to be α-oriented due to the upfield shift of C-9 (δC 39.4) caused by the γ-steric compression effect between C-15 OH and H-9α, which was supported by the ROESY correlation between H-15 and H-14α (δH 1.58) (Figure 1). Meanwhile, H1 and H-5 were found to be on the same side and possessed βorientation based on the ROESY correlation of H-1/H-5β. However, ROESY correlations of H2-14 to H-11 and H-12 and of H-13 to H-11 could not be observed. Therefore, it was difficult to determine the relative configuration of the 11,12epoxy group of compound 1 only by analysis of the NMR data. Fortunately, a single crystal of compound 1 was obtained from CHCl3−MeOH (1:10) and an X-ray diffraction experiment was performed. The final refinement on Cu Kα data resulted in a Flack parameter of 0.16(19), and a Hoof parameter of 0.13(11) for 843 Bijvoet pairs allowed unambiguous assignment of the absolute configuration to be 1S, 5R, 6R, 8S, 9S, 10S, 11S, 12R, 13R, and 15R (Figure 2).21,22 Thus, the structure of 1 was established as 6β,15α-dihydroxy-

6α,20:11β,12-diepoxy-6,7-seco-ent-kaur-16-en-1α,7-olide, and it was named sculponin M. Compound 2 possessed the molecular formula C20H26O5 by HREIMS, corresponding to eight degrees of unsaturation. IR absorption bands at 3427, 1737, and 1631 cm−1 indicated the presence of OH, lactonic carbonyl, and double bond groups. On the basis of NMR data (Tables 1 and 2), compound 2 possessed a 6,7-seco-ent-kauranoid-1,8-lactone skeleton similar to that of compound 1, except for the resonances of two olefinic methines (δC 126.9, d; δC 136.2, d) instead of two oxygenated methines (δC 53.2, d; δC 54.5, d) in 1, suggesting a double bond in 2 rather than the three-membered epoxy ring in 1. The corresponding protons of the olefinic methines (δH 6.42 and 6.39) showed 1H−1H COSY correlations with H-9 and H13, respectively, indicating a double bond between C-11 and C12, which was supported by HMBC correlations of H-11 (δH 6.42) with C-9 (δC 42.4), C-10 (δC 54.3), and C-13 (δC 40.3) B

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kauranoid-1,8-lactone similar to sculponeatin B (12).4 The obvious difference between them was the existence of an oxygenated quaternary carbon (C-13, δC 76.5) in 3 instead of the methine (δC 37.6) in sculponeatin B. Thus, an OH was assumed at C-13, and the assumption was verified by the following observations. In its HMBC spectrum (Figure 3), H2-

Figure 2. X-ray crystal structure of 1.

Figure 3. 1H−1H COSY (bold), and selected HMBC (arrow) correlations of 3.

and H-9 (δH 4.24), H-13 (δH 3.08), and H2-14 (δH 2.09 and 1.88) with C-12 (δC 136.2). Comparison of its ROESY spectrum with that of 1 indicated that the relative configurations of the chiral carbons in 2 were identical to those in 1. Thus, compound 2, named sculponin N, was determined to be 6β,15α-dihydroxy-6α,20-epoxy-6,7-seco-entkaur-11(12),16(17)-dien-1α,7-olide. Compound 3, obtained as colorless crystals, displayed a molecular ion peak at m/z 378.1678, in accordance with molecular formula C20H26O7. The 13C NMR and DEPT spectra showed signals for one methyl, seven methylenes (two oxygenated and one olefinic), six methines (four oxygenated), and six quaternary carbons (one oxygenated, one olefinic, and one lactonic carbonyl carbon), suggesting a 6,7-seco-ent-

17 (δH 5.79 and 5.62), H2-14 (δH 3.62 and 2.17), and H2-12 (δH 2.78 and 2.51) correlated to the oxygenated quaternary carbon; C-12 and C-14 shifted downfield to δC 53.1 (Δ 7.9 ppm) and 41.9 (Δ 7.8 ppm), respectively. The ROESY correlations of H-1/H-5β/Me-18 and H-15/H-14α (δH 2.17) disclosed the β-orientation of H-1 and H-15. Moreover, since the enmein-type 6,7-seco-ent-kauranoid with an OH at C-13 is quite unusual, a single crystal diffraction analysis of compound 3 was performed. The values of the Flack [0.01(19)] and Hoof [0.13(10)] parameters for 1264 Bijvoet pairs determined by the X-ray analysis confirmed compound 3 to be 1S, 4R, 5R, 6S, 8S, 9S, 10S, 11S, 13R, and 15R (Figure 4).21,22 Thus, compound 3 was identified as 11β,13β,15α-trihydroxy-6,19:6,20-diepoxy-6,7seco-ent-kaur-16-en-1α,7-olide, and it was named sculponin O.

Table 2. 1H NMR Spectroscopic Data for Sculponins M−S (1−7) (δ in ppm, J in Hz) position

1a,c

1 2a 2b 3a 3b 5 6 9 11 12a 12b 13 14a 14b 15 17a 17b 18 19a 19b 20a 20b

5.12, dd (10.2, 7.1) 1.83, m

4.80, t (8.6) 1.83, m

1.36, m

d (13.5) m br s br s d (9.3) overlap overlap m d (10.8) overlap s s s s s

a

2a,c

2.76, 5.82, 3.91, 4.26, 3.33,

br s br s d (2.7) overlap t (4.7)

1.32, 1.20, 2.48, 5.79, 4.24, 6.42, 6.39,

3.02, 2.79, 1.58, 5.53, 5.53, 5.19, 1.01, 0.97,

m d (11.5) dd (11.5, 4.7) overlap overlap br s s s

3.08, 2.09, 1.88, 5.71, 5.36, 5.05, 1.01, 0.97,

4.24, d (8.6) 4.17, d (8.6)

4.31, d (8.5) 4.25, d (8.5)

3a,c

4a,c

5a,c

5.87, 1.89, 1.79, 1.60,

overlap m m m

6.08, 2.46, 2.37, 4.00,

dd (11.9, 5.8) m overlap m

4.87, dd (10.8, 6.2) 1.87, m

2.98, 6.12, 2.78, 4.62, 2.78, 2.51,

d (5.2) d (5.2) overlap br s overlap dd (14.1, 5.7)

3.62, 2.17, 5.87, 5.79, 5.62, 1.06, 4.01, 3.42, 4.48, 4.24,

d (10.4) d (10.4) overlap br s br s s d (8.7) d (8.7) d (8.8) d (8.8)

2.91, 6.02, 2.99, 5.17, 2.37, 2.07, 2.72, 3.76, 3.15,

br s br s d (3.6) m overlap m m d (10.8) dd (10.8, 4.0)

1.51, s

3.10, 5.75, 3.80, 4.43, 2.71, 2.23, 2.48, 2.47, 2.13, 5.64, 1.77,

1.36, s 1.31, s 4.65, d (8.8) 4.46, d (8.8)

1.34, m br s br s d (9.7) m m dd (14.5, 6.3) overlap overlap d (10.3) s s

6a,b

7a,c

5.41, 2.52, 2.22, 3.88,

t (8.4) m m d (4.4)

5.73, 2.43, 2.20, 3.87,

t (8.5) overlap m d (4.0)

4.09, 5.96, 2.31, 4.52, 2.70, 1.75, 2.58, 2.93, 2.70,

d (3.3) d (3.3) d (10.6) m overlap m m m overlap

2.43, 6.00, 2.01, 4.42, 2.22, 1.97, 2.66, 3.73, 3.09,

overlap br s d (2.2) overlap m dd (15.9, 4.2) m d (11.0) m

1.59, s

1.60, s

1.00, s 0.98, s

1.35, s 1.10, s

1.32, s 1.10, s

4.47, d (8.6) 4.18, d (8.6)

4.81, d (9.2) 4.29, d (9.2)

4.65, d (9.1) 4.38, d (9.1)

Recorded in C5D5N. bRecorded at 400 MHz. cRecorded at 500 MHz. C

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1.35, s) to a methine (δC 82.0), and from a proton at δH 5.96 (d, J = 3.3 Hz) to C-1 (δC 72.0), C-10 (δC 51.5), and C-20 (δC 76.4); (ii) downfield shifts of C-2 and C-4 (Δ 2.3 and 4.8 ppm, respectively); (iii) the requirement for a degree of unsaturation. In the ROESY spectrum, H-11 correlated to H-1, H-5, and H14β (δH 2.70), H-17 had interaction with H-12α, suggesting that the C-11 OH and Me-17 were α-oriented, whereas H-1 and H-5 were β-oriented (Figure 5). C(3)−O and C(6)−O

Figure 4. X-ray crystal structure of 3.

Sculponin P (4) had the molecular formula C20H28O8. The H and 13C NMR spectra (Tables 1 and 2) resembled those of nodosin (9),17,18 but signals of a methyl (δC 20.7, q), an oxygenated quaternary carbon (δC 78.2, s), and an oxygenated methine (δC 73.2, d) were observed instead of the exomethylene and methylene in 9. HMBC correlations from H2-14 (δH 3.76 and 3.15), H-13 (δH 2.72), H2-12 (δH 2.37 and 2.07), and H-17 (δH 1.51) to the oxygenated quaternary carbon C-16 (δC 78.2) implied that an OH was connected to C-16. HMBC correlations from H2-2 (δH 2.46 and 2.37), Me-18 (δH 1.36), and Me-19 (δH 1.51) to the oxygenated methine (δC 73.2), along with the 1H−1H COSY correlation between H-2 and H3, indicated an OH at C-3. The α-, β-, and β-orientations of C-3 OH, C-11 OH, and C-16 OH, respectively, were apparent from the ROESY correlations of H-1/H-3/H-5β/Me-18, of H-9α/H11/H-12α and of Me-17/H-12α. Hence, compound 4 was established as 3α,6β,11β,16β-tetrahydroxy-6α,20-epoxy-6,7seco-ent-kaur-15-one-1α,7-olide, and it was named sculponin P. The molecular formula of compound 5 was determined to be C20H30O7. The 13C NMR signals resembled those of 4, with the apparent differences being the absence of a carbonyl carbon and the appearance of an additional methylene in 5, as well as the downfield shift of a methine (δC 73.2 to δC 84.2). The additional methylene was located at C-3, which was confirmed by 1H−1H COSY correlations of H-1/H2-2/H2-3, as well as HMBC correlations from H2-3 (δH 1.34) to C-1 (δC 76.3), C-4 (δC 31.8), and C-5 (δC 54.6). Similarly, the downfield methine at δC 84.2 was ascribed to C-15, which was linked to an OH on the basis of HMBC correlations from H-15 (δH 5.64) to C-8 (δC 56.0), C-9 (δC 45.8), and C-16 (δC 80.7). ROESY correlations of H-14β (δH 2.13)/H-1β/H-11/H-5β, H-15/H13β and H-9α/Me-17/H-12α established α-orientation of the OH groups at C-11, C-15, and C-17. Thus, 5 was determined to be 6β,11α,15α,16β-tetrahydroxy-6α,20-epoxy-6,7-seco-entkaur-1α,7-olide, and it was named sculponin Q. Compound 6 had the molecular formula C20H26O7, implying eight degrees of unsaturation. Its 1H and 13C NMR spectra (Tables 1 and 2) displayed signals typical for a 6,7-seco-entkauranoid-1,8-lactone, similar to compound 4, with the appearance of a marked downfield methine (at δC 82.0) and an acetal group (at δC 109.7). The NMR and MS data established the linkage of C-3 and C-6 via an oxygen atom, which was supported by (i) HMBC correlations from H2-2 (δH 2.52, m; δH 2.22, m), H-6 (δH 5.96, d, J = 3.3 Hz), Me-18 (δH

Figure 5. 1H−1H COSY (bold), selected HMBC (arrow), and key ROESY correlations of 6.

1

were deduced to be α-orientated due to the stereochemistry of the fused ring system. To support the above-mentioned and to determine the absolute configurations, a single crystal X-ray diffraction analysis of 6 was carried out. The value of the Hooft parameter [0.20(7)] for 1094 Bijvoet pairs verified αorientation of C(3)−O and C(6)−O and assigned the chiral centers of 6 to be 1S, 3R, 5R, 6R, 8S, 9S, 10S, 11S, 13S, and 16S (Figure 6).22 The A-ring in 6 possessed a boat-conformation

Figure 6. X-ray crystal structure of 6.

due to the strain of the fused ring system, which was quite different from the chair-conformation of A-ring in normal enmein-type diterpenoids. Thus, 11α,16β-dihydroxy-3α,6:6,20diepoxy-6,7-seco-ent-kaur-1α,7-olide was assigned to 6, and it was named sculponin R. Enmein-type diterpenoids are usually highly oxygenated, and several of them possess multicyclic skeletons. Only one enmein-type diterpenoid bearing a 3α,6:6,20-diepoxy moiety has been reported previously.23 Compound 7 had the same molecular formula as 6, which was deduced from the HREIMS. The 13C NMR spectra of compounds 6 and 7 were quite similar, except for small differences in the chemical shifts of C-1, C-5, C-9, C-10, C-14, and C-17. Compound 7 differed from 6 only in orientation of the OH group at C-11, as deduced from the 1H NMR and ROESY spectra of 7. ROESY correlations of C-11 OH/H-14β D

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(δH 3.73)/H-1β established the β-orientation of the OH at C11 (δH 6.70), which was supported by the coupling constant of H-9α/H-11 (d, J = 2.2 Hz). ROESY correlations of H-9α/Me17/H-12α (δH 1.97) disclosed the β-orientation of the OH at C-16. Thus, compound 7 was elucidated as 11β,16β-dihydroxy3α,6:6,20-diepoxy-6,7-seco-ent-kaur-1α,7-olide, and it was named sculponin S. Compounds 8−14 were identified by comparison of their physical constant data with data in the literature.4,16−20 Considering the cytotoxic activity of ent-kauranoids previously isolated from plants of the genus Isodon,1 all isolates except compounds 5−7 (due to the sample limitation) were evaluated for their in vitro cytotoxicity against five human cancer cell lines (HL-60, SMMC-7721, A-549, MCF-7, and SW-480) using a previously described method,24 with cisplatin and paclitaxel as positive controls. Compound 14 showed significant cytotoxic activity against all the above tumor cell lines, with IC50 values of 2.8, 3.5, 3.2, 2.6, and 1.0 μM, respectively, and compounds 9 and 11 exhibited cytotoxic activity against the SW-480 cell line, with respective IC50 values of 12.1 and 3.5 μM (Table 3). The activity of compound 14

Table 4. Inhibitory Effects of Compounds 9, 11, and 14 on LPS-Activated NO Production in RAW264.7 Cellsa

a

HL-60

SMMC-7721

A-549

MCF-7

SW480

9 11 14 DDPb paclitaxelb

15.0 12.7 2.8 1.7 2σ (I)]; the final R1 values were 0.0424 (all data); the final wR(F2) values were 0.1221 (all data); the goodness of fit on F2 was 1.059; Flack parameter = 0.01(19); the Hooft parameter is 0.13(10) for 1264 Bijvoet pairs. Crystallographic Data for Sculponin R (6). C20H26O7: M = 378.41, monoclinic, space group P21, Z = 2, a = 8.72300(10) Å, b = 11.3985(2) Å, c = 9.1543(2) Å; α = 90.00°, β = 102.50°, γ = 90.00°, V = 888.63(3) Å3, T = 100(2) K, μ (Cu Kα) = 0.887 mm−1; 6588 reflections measured, 2777 independent reflections (Rint = 0.0435); the final R1 values were 0.0829 [I > 2σ(I)]; the final wR(F2) values were 0.1887 [I > 2σ(I)]; the final R1 values were 0.0830 (all data); the final wR(F2) values were 0.1891 (all data); the goodness of fit on F2 was 1.094; Flack parameter = 0.24(19); the Hooft parameter is 0.20(7) for 1094 Bijvoet pairs. Cytotoxicity Assays. The following human tumor cell lines were used: HL-60, SMMC-7721, A-549, MCF-7, and SW-480, which were obtained from ATCC (Manassas, VA). All the cells were cultured in RPMI-1640 or DMEM medium (Hyclone, Logan, UT), supplemented with 10% fetal bovine serum (Hyclone) at 37 °C in a humidified atmosphere with 5% CO2. Cell viability was assessed by conducting colorimetric measurements of the amount of insoluble formazan formed in living cells based on the reduction of 3-(4,5-dimethylthiazol2-yl)-5 (3-carboxymethoxyphenyl)-2-(4-sulfopheny)-2H-tetrazolium (MTS) (Sigma, St. Louis, MO).24 Briefly, 100 μL of adherent cells were seeded into each well of a 96-well cell culture plate and allowed to adhere for 12 h before drug addition, while suspended cells were seeded just before addition of test compound, both with an initial density of 1 × 105 cells/mL in 100 μL medium. Each tumor cell line was exposed to the test compound at various concentrations in triplicate for 48 h, with cisplatin and paclitaxel (Sigma) as positive controls. After the incubation, MTS (100 μg) was added to each well and the incubation continued at 37 °C for 4 h. The cells were lysed with 100 μL of 20% SDS-50% DMF after removal of 100 μL of medium. The optical density of the lysate was measured at 490 nm in a 96-well microtiter plate reader (Bio-Rad 680). The IC50 value of each compound was calculated by the Reed and Muench’s method.27 Nitric Oxide Production in RAW264.7 Macrophages. Murine monocytic RAW264.7 macrophages were dispensed into 96-well plates (2 × 105 cells/well) containing RPMI 1640 medium (Hyclone) with 10% FBS under a humidified atmosphere of 5% CO2 at 37 °C. After 24 h preincubation, cells were treated with serial dilutions of the compounds, with the maximum concentration of 25 μM, in the presence of 1 μg/mL LPS for 18 h. Each compound was dissolved in DMSO and further diluted in the medium to produce different concentrations. NO production in each well was assessed by adding 100 μL of Griess reagent (reagent A and reagent B, respectively, Sigma) to 100 μL of each supernant from LPS (Sigma) treated or LPS and compound-treated cells in triplicate. After 5 min incubation, the absorbance was measured at 570 nm with 2104 Envision Multilabel Plate Reader (Perkin-Elmer Life Sciences, Inc., Boston, Ma). MG-132 was used as a positive control.28 Determination of Cytotoxic Effects. The cytotoxicity of tested compounds was evaluated by the MTS assay.24 Briefly, RAW264.7 cells, 2 × 105 cells/well, were seeded in 96-well plates. After 24 h incubation, cells were treated with or without tested compounds at given concentrations for 18 h. Then MTS were added to each well and plates kept for 4 h. Test compounds were dissolved in DMSO and absorbance was read at 490 nm. Cytotoxicity was calculated by cell viability of cells without compounds as 100%.

on silica gel (200−300 mesh), eluted with a CHCl3−MeOH gradient, and yielded compounds 6 (2.3 mg) and 7 (2 mg). Sculponin M (1): colorless needle crystals (CHCl3−MeOH 1:10); mp 254−255 °C; [α]22D −269 (c 0.12, CHCl3−MeOH 1:1); UV (MeOH) λmax (log ε) 201 (3.51) nm; IR (KBr) νmax 3475, 1723, 1232 cm−1; 1H and 13C NMR data, see Tables 1 and 2; positive-ion ESIMS m/z 385 [M + Na]+; positive-ion HREIMS [M]+ m/z 362.1734 (calcd for 362.1729). Sculponin N (2): white amorphous powder; [α]22D −226 (c 0.06, MeOH); UV (MeOH) λmax (log ε) 203 (4.05), 254 (3.32) nm; IR (KBr) νmax 3427, 2951, 1737, 1631 cm−1; 1H and 13C NMR data, see Tables 1 and 2; positive-ion ESIMS m/z 369 [M + Na]+; positive-ion HREIMS [M]+ m/z 346.1779 (calcd for 346.1780). Sculponin O (3): colorless needle crystals (CHCl3−MeOH 1:20); mp 276−278 °C; [α]22D −102 (c 0.07, CHCl3−MeOH 1:1); UV (MeOH) λmax (log ε) 202 (3.79), 254 (2.79) nm; IR (KBr) νmax 3409, 1712, 1224 cm−1; 1H and 13C NMR data, see Tables 1 and 2; positiveion ESIMS m/z 401 [M + Na]+; positive-ion HREIMS [M]+ m/z 378.1678 (calcd for 378.1679). Sculponin P (4): white amorphous powder; [α]23D −162 (c 0.05, CHCl3−MeOH 1:1); UV (MeOH) λmax (log ε) 202 (3.24) nm; IR (KBr) νmax 3439, 3432, 1760, 1708, 1632 cm−1; 1H and 13C NMR data, see Tables 1 and 2; positive-ion ESIMS m/z 419 [M + Na]+; positive-ion HREIMS [M]+ m/z 396.1779 (calcd for 396.1784). Sculponin Q (5): white amorphous powder; [α]25D −108 (c 0.10, MeOH); UV (MeOH) λmax (log ε) 202 (3.44) nm; IR (KBr) νmax 3498, 3473, 2941, 1681 cm−1; 1H and 13C NMR data, see Tables 1 and 2; positive-ion ESIMS m/z 405 [M + Na]+; positive-ion HREIMS [M]+ m/z 382.1989 (calcd for 382.1992). Sculponin R (6): colorless needles (MeOH); mp 258−260 °C; [α]24D −59 (c 0.10, MeOH); UV (MeOH) λmax (log ε) 202 (3.69) nm; IR (KBr) νmax 3484, 2938, 1766, 1718 cm−1; 1H and 13C NMR data, see Tables 1 and 2; positive-ion ESIMS m/z 401 [M + Na]+; positive-ion HREIMS [M]+ m/z 378.1679 (calcd for 378.1679). Sculponin S (7): white amorphous powder; [α]24D −80 (c 0.11, MeOH); UV (MeOH) λmax (log ε) 202 (3.30) nm; IR (KBr) νmax 3453, 1758, 1696, 1633 cm−1; 1H and 13C NMR data, see Tables 1 and 2; positive-ion ESIMS m/z 401 [M + Na]+; positive-ion HREIMS [M]+ m/z 378.1682 (calcd for 378.1679). X-ray Crystal Structure Analysis. Colorless crystals of 1 and 3 were obtained from mixed solvent systems of CHCl3−MeOH 1:10 and 1:20, respectively; colorless needles of 6 were obtained in MeOH. Intensity data were collected at 100 K on a Bruker APEX DUO diffractometer equipped with an APEX II CCD using Cu Kα radiation. Cell refinement and data reduction were performed with Bruker SAINT. The structures were solved by direct methods using SHELXS97.26 Refinements were performed with SHELXL-97 using full-matrix least-squares, with anisotropic displacement parameters for all the nonhydrogen atoms. The H-atoms were placed in calculated positions and refined using a riding model. Molecular graphics were computed with PLATON. Crystallographic data (excluding structure factor tables) for the structures reported have been deposited with the Cambridge Crystallographic Data Center as supplementary publications no. CCDC 955513 for 1, CCDC 955514 for 3, and CCDC 955515 for 6. Copies of the data can be obtained free of charge on application to CCDC, 12 Union Road, Cambridge CB 1EZ, U.K. [fax: int. +44(0) (1223) 336 033); e-mail: [email protected]]. Crystallographic Data for Sculponin M (1). C20H26O6: M = 362.41, monoclinic, space group C2, Z = 4, a = 21.7849(9) Å, b = 6.6516(3) Å, c = 12.2025(5) Å; α = 90.00°, β = 97.698(2)°, γ = 90.00°, V = 1752.26(13) Å3, T = 100(2) K, μ (Cu Kα) = 0.831 mm−1; 7111 reflections measured, 2533 independent reflections (Rint = 0.0484); the final R1 values were 0.0423 [I > 2σ(I)]; the final wR(F2) values were 0.1104 [I > 2σ(I)]; the final R1 values were 0.0427 (all data); the final wR(F2) values were 0.1107 (all data); the goodness of fit on F2 was 1.046; Flack parameter = 0.16(19); the Hooft parameter is 0.13(11) for 843 Bijvoet pairs. Crystallographic Data for Sculponin O (3). C20H26O7: M = 378.41, orthorhombic, space group P212121, Z = 4, a = 7.6702(2) Å, b = 10.8110(2) Å, c = 22.0770(4) Å; α = β = γ = 90.00°, V = 1830.68(7)

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ASSOCIATED CONTENT

S Supporting Information *

1

H, 13C NMR, DEPT, HSQC, HMBC, 1H−1H COSY, ROESY, HREIMS, IR, and UV spectra of compounds 1, 3, 6, and 7, and 1 H, 13C NMR, DEPT, and HREIMS spectra of compounds 2, 4, and 5; atomic coordinates and equivalent isotropic F

dx.doi.org/10.1021/np400669t | J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

(16) Han, Q.-B.; Zhang, J.-X.; Shen, Y.-H.; Sun, H.-D. Chin. J. Nat. Med. 2003, 1, 16−20. (17) Fujita, E.; Fujita, T.; Shibuya, M. Tetrahedron Lett. 1966, 7, 3153−3162. (18) Fujita, E.; Fujita, T.; Shibuya, M. Chem. Pharm. Bull. 1968, 16, 1573. (19) Fujita, E.; Fujita, T.; Shibuya, M.; Shingu, T. Tetrahedron 1969, 25, 2517−2530. (20) Yan, F.-L.; Guo, L.-Q.; Bai, S.-P.; Sun, H.-D. J. Chin. Chem. Soc. 2008, 55, 933−936. (21) Flack, H.-D. Acta Crystallogr. 1983, A39, 876−881. (22) Hooft, R. W. W.; Straver, L.-H.; Spek, A.-L. J. Appl. Crystallogr. 2008, 41, 96−103. (23) Liu, X.; Wang, W.-G.; Du, X.; Pu, J.-X.; Sun, H.-D. Fitoterapia 2012, 83, 1451−1455. (24) Monks, A.; Scudiero, D.; Skehan, P.; Shoemaker, R.; Paull, K.; Vistica, D.; Hose, C.; Langley, J.; Cronise, P.; Vaigro-Wolff, A. J. Natl. Cancer Inst. 1991, 83, 757−766. (25) McCartney-Francis, N.-L.; Song, X.; Mizel, D.-E.; Wahl, S.-M. J. Immunol. 2001, 166, 2734−2740. (26) Sheldrick, G.-M.; Schneider, T.-R. Methods Enzymol. 1997, 277, 319−343. (27) Reed, L.-J.; Muench, H. Am. J. Hyg. 1938, 27, 493−497. (28) Fan, J.-T.; Su, J.; Peng, Y.-M.; Li, Y.; Li, J.; Zhou, Y.-B.; Zeng, G.-Z.; Yan, H.; Tan, N.-H. Bioorg. Med. Chem. 2010, 18, 8226−8234.

displacement parameters; bond lengths and angles. This material is available free of charge via the Internet at http:// pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. Tel: (86) 871-65223616. Fax: (86) 871-65216343. *E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This project was supported financially by the National Natural Science Foundation of China (Grants 21322204 and 81172939), the Major State Basic Research Development Program of China (Grant 2009CB522300), the reservationtalent project of Yunnan Province (Grant 2011CI043), the Science and Technology Program of Yunnan Province (Grant 2008IF010), the Major Direction Projection Foundation of CAS Intellectual Innovation Project (Grant KSCX2-EW-J-24), and West Light Foundation of the Chinese Academy of Sciences (J.-X.P.).

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