Cytotoxic ent-Kaurane Diterpenoids from Salvia ... - ACS Publications

Article pubs.acs.org/jnp

Cytotoxic ent-Kaurane Diterpenoids from Salvia cavaleriei Heng Zheng,†,‡ Qiong Chen,†,‡ Mengke Zhang,† Yongji Lai,† Liang Lei,† Penghua Shu,† Jinwen Zhang,‡ Yongbo Xue,† Zengwei Luo,† Yan Li,§ Guangmin Yao,*,† and Yonghui Zhang*,† †

Hubei Key Laboratory of Natural Medicinal Chemistry and Resource Evaluation, School of Pharmacy, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, People’s Republic of China ‡ Tongji Hospital Affiliated to Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, People’s Republic of China § State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650204, People’s Republic of China S Supporting Information *

ABSTRACT: Fifteen new ent-kaurane diterpenoids, compounds 1−15, and two known analogues, 4-epi-henryine A (16) and leukamenin E (17), were isolated from the whole plants of Salvia cavaleriei. The structures of the new compounds were established by spectroscopic methods, and their absolute configurations were determined by electronic circular dichroism and singlecrystal X-ray diffraction analyses with Cu Kα radiation. Compounds 1−15 were evaluated for their cytotoxicity against five human cancer cell lines, HL-60, SMMC-7721, A-549, MCF-7, and SW480, as well as the noncancerous Beas-2B cell line. Compounds 1−10, 12, 14, and 15 showed broad-spectrum cytotoxicity, with compounds 1, 3, 6−10, 12, and 15 exhibiting more potent cytotoxicity than the positive control, cis-platin, with IC50 values ranging from 0.65 to 6.4 μM. active components. In the process, 15 new ent-kaurane diterpenoids, compounds 1−15, and two known ent-kaurane diterpenoids, 4-epi-henryine A (16)14 and leukamenin E (17),15 were isolated. Here, we describe the isolation, structure elucidation, and cytotoxicity of compounds 1−15.

Salvia L., the largest genus of the family Lamiaceae, comprises over 1000 species, which are widely distributed in tropical and temperate regions of the world,1 and some species have been cultivated for use as herbal medicines and ornamentals.2,3 Studies on the chemical constituents of Salvia revealed the presence of polyphenolics2 and terpenoids.3,4 The identified diterpenoids mainly belong to seven skeletal types, abietane, clerodane, pimarane, labdane, ent-kaurane, icetexane, and apianane.3,4 Collectively, these compounds possess antibacterial, antileishmanial, antimicrobial, antioxidant, antispasmolytic, antituberculosis, and antitumor activities.3−6 Interestingly, although ent-kaurane diterpenoids are common in plants of the genus Isodon (Lamiaceae), 7 only one ent-kaurane diterpenoid has been previously reported from the Salvia genus.8 Salvia cavaleriei Lévl is endemic to China and is distributed in the Guizhou, Sichuan, Guangdong, Guangxi, Hubei, Hunan, Jiangxi, Shaanxi, and Yunnan provinces.9 The whole plant of S. cavaleriei is used as a Chinese herbal medicine to treat hematemesis, metrorrhagia, dysentery with bloody stools, and traumatic hemorrhage.10 Previous phytochemical investigations on S. cavaleriei var. cavaleriei 11 and S. cavaleriei var. simplicifolia12 have resulted in the isolation of depsides, phenolic glycosides, triterpenoids, and steroids. In our search for anticancer agents from Chinese herbal medicines,13 we found that the 95% EtOH extract of the whole plants of S. cavaleriei showed significant cytotoxicity against the HL-60 cell line, which encouraged us to pursue the characterization of the © 2013 American Chemical Society and American Society of Pharmacognosy

■

RESULTS AND DISCUSSION 11α-Hydroxyleukamenin E (1) was obtained as colorless needles, mp 195−197 °C. The molecular formula of 1 was assigned as C22H32O6 by the HRESIMS of the pseudomolecular ion [M + Na]+ at m/z 415.2067 (calcd for C22H32O6Na, 415.2097), indicating seven indices of hydrogen deficiency. The UV absorption maximum at 234 nm was consistent with the presence of an α,β-unsaturated carbonyl group. The IR spectrum suggested the presence of hydroxy (3394 cm−1), an ester carbonyl (1725 cm−1), a double bond (1647 cm−1), and conjugated carbonyl (1705 cm−1) functionalities. The 1H NMR spectrum (Table 1) showed resonances for three methyls (δH 0.92, H3-18; 0.99, H3-19; 1.34 H3-20), an acetyl (δH 2.07), four oxymethines (δH 3.98, ddd, H-11β; 4.07, dd, H-7β; 4.62, t, H3α; 5.10, s, H-14α), and two olefinic protons arising from an exocyclic double bond (δH 6.04, s, H-17a; 5.42, s, H-17b). The 13 C NMR and DEPT spectra (Table 4) of 1 displayed a total of 22 carbon resonances assignable to a ketocarbonyl (δC 209.0, C-15), an acetyl (δC 172.7, 21.3), an exocyclic double bond (δC Received: July 24, 2013 Published: November 20, 2013 2253

dx.doi.org/10.1021/np400600c | J. Nat. Prod. 2013, 76, 2253−2262

Journal of Natural Products

Article

dichroism (ECD) spectrum.17 The NOESY correlations of H11 to H-9β and H-12β and the large coupling constants J = 13.0, 8.0, and 6.0 Hz of H-11 (δH 3.98, ddd) with H-9 and H12 established the β-orientation of H-11. Analyses of the 2D NMR data, including HSQC, 1H−1H COSY, HMBC, and NOESY (Figure 1), defined compound 1 as 7α,11α,14βtrihydroxy-3β-acetoxy-ent-kaur-16-en-15-one. The structure was confirmed by single-crystal X-ray diffraction analysis, and its absolute configuration was assigned as 3S,5S,7R,8R,9S,10R,11R,13S,14R based on a Flack parameter of 0.19(14)18 (Figure 2). 11β-Hydroxyleukamenin E (2) exhibited a pseudomolecular ion at m/z 415.2065 [M + Na]+ in the HRESIMS, which is in agreement with the molecular formula C22H32O6 (calcd for C22H32O6Na, 415.2097). Compound 2 has the same molecular formula as 1, and the NMR resonances for 2 (Tables 1 and 4) resemble those of 1. The obvious difference is that the multiplicity and coupling constants of H-11 are a doublet (J = 4.5 Hz) in 2 instead of a doublet of doublets of doublets (J = 13.0, 8.0, 6.0 Hz) in 1. Correspondingly, C-11 (δC 65.5) in 2 was shifted upfield compared to the corresponding signal in 1 (δC 69.5). These differences suggest that 2 is the 11-epimer of 1. The α-orientation of H-11 in 2 was determined by the NOESY correlations between H-11 and both H-1α and H320α. Detailed 2D NMR analyses confirmed the identification of 2 as 7α,11β,14β-trihydroxy-3β-acetoxy-ent-kaur-16-en-15-one. The absolute configuration of 2 was determined as 3S,5S,7R,8R,9S,10R,11S,13S,14R by single-crystal X-ray diffraction with Cu Kα radiation (Figure 3). The molecular formula of 11-oxoleukamenin E (3) was determined to be C22H30O6 by the HRESIMS ion at m/z 413.1923 [M + Na]+ (calcd for C22H30O6Na, 413.1940). Comparison of the NMR data of 3 (Tables 1 and 4) with those of 2 indicated that a C-11 oxo (δC 206.8) group in 3 replaced the oxymethine function in 2 (δC 65.5). This suggestion was

150.2, C-16; 117.1, C-17), three methyls (δC 29.2, C-18; 22.5, C-19; 19.3, C-20), four methylenes (δC 41.1, C-12; 36.2, C-1; 29.3, C-6; 24.0, C-2), four oxymethines (δC 79.2, C-3; 76.3, C14; 76.0, C-7; 69.5, C-11), three methines (δC 61.2, C-9; 49.3, C-5; 47.4, C-13), and three quaternary carbons (δC 62.8, C-8; 42.6, C-10; 38.1, C-4). The 13C NMR spectrum of 1 is similar to that of the co-occurring known compound 17 (leukamenin E),15 except for a C-11 oxymethine group in 1 replacing a methylene in 17. Thus, 1 is the 11-OH derivative of 17. 1H−1H COSY correlations from H-11 to both H-9 and H-12, together with HMBC correlations of H-11 to C-8 and C-10, supported the above assignment. The NOESY correlations (Figure 1) of H-14/H3-20 and H-5/H-9 allowed assignment of 1 as an entkaurane diterpenoid,16 which was further supported by the negative Cotton effect at λmax 342 nm in the electronic circular

Table 1. 1H NMR [δ, mult (J in Hz)] Data for Compounds 1−5 (400 MHz) 1a

position 1α 1β 2α 2β 3α 5β 6α 6β 7β 9β 11α 11β 12α 12β 13α 14α 17a 17b 18 19 20 OAc a

2.52 1.37 1.97 1.54 4.62 1.47 1.70 1.88 4.07 1.69

ddd (14.0, 3.5, 3.3) ddd (14.0, 13.3, 3.8) overlap dddd (15.3, 3.4, 3.4, 3.3) t (2.6) dd (12.2, 1.5) ddd (12.8, 12.2, 12.0) ddd (12.8, 3.9, 1.5) dd (12.0, 4.0) d (8.0)

3.98 2.24 1.98 3.05 5.10 6.04 5.42 0.92 0.99 1.34 2.07

ddd (13.0, 8.0, 6.0) ddd (13.0, 12.8, 2.5) overlap br s s s s s s s s

2a

3b

1.64 1.40 2.00 1.64 4.66 1.48 1.70 1.90 4.21 1.48 3.96

overlap ddd (14.0, 13.5, 3.0) m overlap t (2.8) dd (12.5, 1.4) ddd (12.7, 12.5, 12.0) ddd (12.7, 4.2, 1.4) dd (12.0, 4.2) br s d (4.5)

1.32 1.41 1.74 1.59 4.61 1.43 1.82 1.93 4.37 1.81

ddd (13.8, 3.3, 3.3) ddd (13.8, 12.1, 3.0) m dddd (15.3, 3.3, 3.0, 3.0) t (2.6) dd (12.9, 1.4) ddd (12.9, 12.4, 11.9) dd (12.4, 2.6) dd (11.9, 4.3) br s

2.22 2.05 2.96 4.90 5.95 5.34 0.94 0.98 1.08 2.06

ddd (14.7, 4.5, 3.1) ddd (14.7, 3.8, 2.2) br s s s s s s s s

2.74 2.57 3.19 5.29 6.20 5.53 0.87 0.91 1.11 2.01

dd (16.5, 3.2) br d (16.5) br s s s s s s s s

4a 2.57 1.81 2.25 2.70

ddd ddd ddd ddd

1.96 1.76 1.89 4.09 1.84 3.98 2.16 1.99 3.06 5.01 6.05 5.43 1.15 1.10 1.21

(14.5, (14.5, (15.5, (15.5,

5a 8.5, 6.6) 10.5, 4.8) 8.5, 4.8) 10.5, 6.6)

dd (13.0, 2.0) ddd (13.0, 12.8, 11.9) ddd (12.8, 3.4, 2.0) dd (11.9, 3.4) d (8.0)

1.58 1.24 1.90 1.53 3.36 1.49 1.84 1.92 4.22 1.75

overlap m overlap overlap t (3.5) dd (12.4, 1.9) overlap overlap dd (11.9, 4.6) br s

ddd (12.8, 8.0, 5.6) ddd (12.8, 12.8, 1.8) ddd (12.8, 5.2, 5.6) br s s s s s s s

2.94 2.53 3.22 5.39 6.19 5.61 0.99 0.91 1.17

dd (16.5, 3.5) ddd (16.5, 3.0, 1.8) br s br s s s s s s

Recorded in methanol-d4. bRecorded in CDCl3. 2254



Article

Table 2. 1H NMR [δ, mult (J in Hz)] Data for Compounds 6−10 (400 MHz) 6a

position 1α 1β 2α 2β 3α 3β 5β 6α 6β 7β 9β 11α 11β 12α 12β 13α 14α 16α 17a 17b 18a 18b 19 20 OAc OMe a

1.84 1.71 2.48 2.48

dd dd dd dd

1.75 1.92 1.98 4.22 1.84

2.95 2.55 3.24 5.37

(14.1, 5.8) (14.1, 9.0) (9.0, 5.8) (9.0, 5.8)

7a

8a

9a

1.51 1.33 1.98 1.62 4.66

m ddd (13.5, 13.5, 3.6) m dddd (15.2, 3.3, 3.2, 3.2) t (2.6)

1.77 1.53 2.01 1.67 4.69

ddd (13.0, 3.8, 2.6) ddd (13.5, 13.0, 3.7) m dddd (15.0, 3.5, 3.3, 3.2) t (2.7)

dd (12.4, 7.0) ddd (13.0, 12.4, 9.5) ddd (13.0, 7.0, 6.8) dd (9.5, 6.8) br s

1.51 1.71 1.91 4.32 1.84 5.47

dd (12.4, 1.2) ddd (12.4, 12.4, 12.1) ddd (12.4, 4.6, 1.2) dd (12.1, 4.6) dd (3.6, 1.1) dd (9.6, 3.6)

1.39 1.75 1.86 4.39 1.41

dd (9.8, 1.8) ddd (12.7, 12.2, 9.8) ddd (12.7, 5.3, 1.8) dd (12.2, 5.3) d (4.2)

dd (16.6, 3.6) ddd (16.6, 3.5, 2.0) ddd (3.6, 3.5, 1.1) d (1.1)

6.05 ddd (9.6, 6.9, 1.4)

1.54 1.07 1.62 1.49 1.43 1.36 1.34 1.85 1.92 4.16 1.71

m ddd (13.4, 12.7, 2.8) m m m m dd (11.6, 2.4) ddd (12.4, 11.6, 11.6) ddd (12.4, 4.8, 2.4) dd (11.6, 4.8) br s

2.93 2.53 3.21 5.38

dd (16.5, 3.6) ddd (16.5, 3.2, 1.8) dd (3.6, 3.2) s

6.18 5.60 3.97 3.65 0.91 1.21 2.09

s s d (11.1) d (11.1) s s s

10b 1.40 1.40 1.75 1.58 4.60

m m m dddd (15.2, 3.2, 3.1, 3.0) dd (2.7, 2.3)

1.43 1.78 1.89 4.23 1.71

dd (11.6, 1.5) ddd (12.6, 11.9, 11.6) ddd (12.6, 4.3, 1.5) dd (11.9, 4.3) br s

2.49 2.66 2.79 5.34 3.23 3.59 3.36 0.85

dd (17.6, 3.9) ddd (17.6, 2.4, 2.0) dd (3.9, 2.4) s dd (9.4, 4.7) dd (9.9, 4.7) dd (9.9, 9.4) s

0.89 1.08 2.00 3.25

s s s s

3.10 dd (4.2, 4.1)

3.28 br d (6.9) 5.03 d (1.4)

3.14 dd (3.8, 3.8) 3.39 dd (3.8, 1.3) 5.04 d (1.3)

6.18 s 5.61 s 1.15 s

5.77 s 5.18 s 0.95 s

6.11 s 5.56 s 0.94 s

1.11 s 1.20 s

0.98 s 1.07 s 2.07 s

1.01 s 1.40 s 2.08 s

Recorded in methanol-d4. bRecorded in CDCl3.

Table 3. 1H NMR [δ, mult (J in Hz)] Data for Compounds 11−15 (400 MHz) in Methanol-d4 position 1α 1β 2α 2β 3α 3β 5β 6α 6β 7β 9β 12α 12β 13α 14α 16α 16β 17a 17b 18a 18b 19 20 OAc OMe OEt

11

12

13

1.35 1.44 1.91 1.59 4.64

ddd (13.6, 3.4, 3.2) overlap overlap dddd (15.2, 3.4, 3.4, 3.2) t (2.6)

1.42 1.42 1.91 1.59 4.64

m m overlap dddd (15.4, 3.3, 3.2, 3.2) t (2.7)

1.85 1.72 2.47 2.47

overlap overlap dd (9.0, 5.8) dd (9.0, 5.8)

1.43 1.82 1.92 4.11 1.70 2.88 2.53 2.69 5.37

overlap ddd (12.6, 12.4, 12.0) overlap dd (12.0, 4.6) d (1.4) dd (16.6, 3.6) ddd (16.6, 3.3, 1.4) dd (3.6, 3.3) s

1.46 1.82 1.93 4.07 1.65 2.69 2.69 2.78 5.44 3.22

dd (12.6, 1.6) ddd (12.6, 12.5, 12.0) overlap dd (12.0, 4.4) s d (3.5) d (3.5) m br s m

1.71 1.93 1.93 4.11 1.81 2.89 2.55 2.70 5.35

overlap overlap overlap dd (10.3, 6.1) d (1.4) dd (16.6, 3.6) ddd (16.6, 3.3, 1.4) dd (3.6, 3.3) s

2.32 3.74 3.74 0.93

t (7.0) d (7.0) d (7.0) s

2.34 3.69 3.69 1.14

t (7.1) d (7.1) d (7.1) s

3.71 dd (10.0, 4.6) 3.42 overlap 0.93 s

0.99 s 1.19 s 2.06 s

0.99 s 1.17 s 2.07 s

3.50 m 1.18 t (7.0)

3.48 m 1.15 t (7.0)

1.10 s 1.19 s 3.34 s

2255

14 1.56 1.08 1.63 1.51 1.36 1.43 1.30 1.80 1.89 4.05 1.68 2.87 2.52 2.67 5.36

overlap m overlap overlap overlap overlap dd (12.0, 1.8) ddd (12.8, 12.0, 11.8) ddd (12.8, 4.7, 1.8) dd (11.8, 4.7) br s dd (16.5, 3.8) ddd (16.5, 3.2, 1.9) dd (3.8, 3.2) s

2.31 3.68 3.68 3.95 3.64 0.91 1.20 2.08 3.33

t (7.1) d (7.1) d (7.1) d (11.2) d (11.2) s s s s

15 1.64 1.06 1.49 1.61 1.42 1.35 1.32 1.80 1.88 4.01 1.63 2.66 2.62 2.76 5.44 3.22

overlap ddd (14.2, 13.3, 4.2) overlap overlap overlap overlap overlap ddd (12.5, 12.3, 11.8) ddd (12.5, 4.5, 2.3) dd (11.8, 4.5) br s m m m br s m

3.63 3.37 3.95 3.64 0.91 1.18 2.08 3.29

d d d d s s s s

(10.1) (10.1) (11.1) (11.1)



Article

Table 4. 13C NMR Data for Compounds 1−15 (100 MHz) position

1a

2a

3b

4a

5a

6a

7a

8a

9a

10b

11a

12a

13a

14a

15a

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

36.2 24.0 79.2 38.1 49.3 29.3 76.0 62.8 61.2 42.6 69.5 41.1 47.4 76.3 209.0 150.2 117.1 29.2 22.5 19.3 172.7 21.3

34.2 23.9 79.2 37.9 48.8 29.4 75.7 60.5 66.9 39.6 65.5 40.7 46.7 76.8 208.2 150.4 116.0 28.7 22.3 18.4 172.5 21.2

32.9 22.6 77.0 36.9 47.1 27.5 73.3 60.3 67.3 39.8 206.8 49.8 44.5 74.0 204.7 146.0 122.4 28.2 21.9 19.1 170.8 21.4

41.9 34.9 221.2 48.3 53.1 31.2 75.0 62.5 59.8 41.0 68.6 40.9 47.3 76.1 208.5 150.1 117.3 28.8 21.0 20.3

33.7 26.2 75.9 38.8 47.1 29.4 74.2 61.2 69.7 41.0 209.1 51.0 46.2 75.4 205.6 148.6 121.5 29.3 22.8 19.5

38.6 34.9 218.4 48.3 52.6 30.9 73.5 60.8 68.0 39.9 209.0 50.8 46.1 75.4 205.4 148.4 121.7 27.5 22.1 19.1

34.1 23.7 79.1 37.8 47.7 29.5 75.6 60.4 59.9 39.8 126.4 133.7 48.8 74.3 209.8 150.5 113.0 28.5 22.4 17.8 172.6 21.2

34.0 23.5 79.0 37.5 48.1 29.9 75.5 58.8 53.3 41.8 50.9 55.6 49.2 73.7 208.9 145.7 119.1 28.7 22.8 16.8 172.6 21.2

40.2 18.7 36.3 37.8 47.9 29.7 73.8 61.2 69.9 41.0 209.1 51.0 46.2 75.4 205.7 148.4 121.8 73.3 18.2 20.0 173.1 20.9

32.7 22.6 77.0 36.9 47.2 27.9 73.7 60.7 66.8 39.8 207.5 44.6 39.7 74.6 216.9 50.0 68.3 28.2 21.8 19.0 170.8 21.4 59.0

34.4 23.7 78.7 38.0 48.5 29.5 73.9 62.6 69.1 40.7 209.7 50.8 41.2 77.7 217.3 54.2 72.6 28.7 22.3 19.2 172.5 21.2

34.2 23.6 78.7 38.0 48.7 29.4 74.6 61.5 69.1 40.8 209.8 45.7 41.3 75.9 217.6 51.4 67.2 28.6 22.3 19.3 172.5 21.2

38.6 34.9 218.5 48.3 52.6 31.1 73.3 62.3 67.5 39.8 209.8 50.6 41.1 77.7 217.2 54.1 74.7 27.6 22.1 19.0

40.3 18.7 36.3 37.8 47.9 29.9 73.6 62.7 69.4 40.9 209.8 50.7 41.1 77.8 217.3 54.0 74.8 73.3 18.2 19.9 173.1 20.9 58.9

40.1 18.7 36.3 37.8 48.2 29.8 74.4 61.6 69.4 41.0 209.8 45.7 41.3 75.9 217.4 51.4 69.3 73.3 18.1 20.0 172.9 20.9 59.2

67.5 15.5

67.8 15.5

OMe OEt a

59.0

Recorded in methanol-d4. bRecorded in CDCl3

Figure 1. 1H−1H COSY, key HMBC, and NOESY correlations of compound 1.

Figure 3. X-ray ORTEP drawing of compound 2. Figure 2. X-ray ORTEP drawing of compound 1.

tal X-ray diffraction analysis by Cu Kα radiation (Figure 4) e s t a b li s h e d t h e a b s o lu t e c o n fi g u r a t i o n o f 3 a s 3S,5S,7R,8R,9S,10R,13S,14R. 3-Oxo-11α-hydroxyleukamenin E (4) was obtained as colorless needles. Its molecular formula, C20H28O5, was inferred

verified by the HMBC correlations from H-9 (δH 1.81, br s), H2-12 (δH 2.74, dd; 2.57, br d), and H-13 (δH 3.19, br s) to the C-11 carbonyl. Consequently, 3 was characterized as 7α,14βdihydroxy-3β-acetoxy-ent-kaur-16-ene-11,15-dione. Single-crys2256



Article

the HRESIMS determined the molecular formula of 6 as C20H26O5 (calcd for C20H26O5Na, 369.1678). The 13C NMR spectrum (Table 4) of 6 differed from that of 5 in the presence of a carbonyl (δC 218.4) at C-3 in 6, instead of an oxymethine in 5 (δC 75.9). The assignment of the C-3 carbonyl group was further supported by the HMBC correlations from H2-1 (δH 1.84 and 1.71), H2-2 (δH 2.48), H3-18 (δH 1.15), and H3-19 (δH 1.11) to the carbonyl (δC 218.4). Compound 6 was identified as 7α,14β-dihydroxy-ent-kaur-16-ene-3,11,15-trione by detailed 2D NMR analyses. The similarity of the ECD spectra of 6 and 5 indicates that 6 possesses the same absolute configuration as that of 5, except at C-3. 11,12-Didehydroleukamenin E (7) had the molecular formula C22H30O5 by HRESIMS (m/z 397.1976 [M + Na]+, calcd for C22H30O5Na, 397.1991), implying eight indices of hydrogen deficiency. The NMR data of 7 (Tables 2 and 4) resembled that of 1, except for the presence of a double bond (δH 6.05, ddd, H-12; 5.47, dd, H-11; δC 133.7, C-12; 126.4, C11) in 7, instead of an oxymethine (δH 3.98, ddd, H-11; δC 69.5, C-11) and a methylene (δH 2.24, H-12α; 1.98, H-12β; δC 41.1, C-12) in 1. Correlations from the olefinic protons to H-9 and H-13 in the 1H−1H COSY spectrum indicated that the double bond was located at C-11. This was confirmed by the HMBC correlations from H-9, H-13, and H-14 to C-11 and C12. Two-dimensional NMR analyses allowed the unambiguous identification of 7 as 7α,14β-dihydroxy-3β-acetoxy-ent-kaur11(12),16(17)-dien-15-one. In consideration of its biosynthetic origin, the absolute configuration of compound 7, except at C11, is the same as that of compound 1. The molecular formula of 11α,12α-epoxyleukamenin E (8) was assigned as C22H30O6 based on HRESIMS (m/z 413.1926 [M + Na]+, calcd for C22H30O6Na, 413.1940), indicating eight indices of hydrogen deficiency. The NMR spectra (Tables 2 and 4) of 8 were similar to those of 1. The significant differences were H-11 (δH 3.10, dd, J = 4.2, 4.1 Hz) and C-11 (δC 50.9) in 8 were shifted upfield compared to 1 (δH 3.98, ddd, J = 13.0, 8.0, 6.0 Hz, H-11; δC 69.5, C-11), and an oxymethine (δH 3.14, H-12; δC 55.6, C-12) in 8 replaces the C12 methylene (δC 41.1) in 1. Compound 8 possesses one more index of hydrogen deficiency than 1, which, in view of the chemical shift differences, is consistent with the presence of an 11,12-epoxide. This was confirmed by the HMBC cross-peaks for 8 from H-11 (δH 3.10) to C-8 (δC 58.8) and C-9 (δC 53.3) and from H-12 (δH 3.14) to C-14 (δC 73.7). The α-orientation of the 11,12-epoxy moiety was determined on the basis of the NOESY correlations between H-11 and H-9β and H2-1 and between H-12 and H2-17. Thus, the structure of 8 was established as 7α,14β-dihydroxy-3β-acetoxy-11α,12α-epoxy-entkaur-16-en-15-one. Its structure was confirmed and its absolute configuration defined as 3S,5S,7R,8R,9S,10R,11S,12R,13S,14R by single-crystal X-ray diffraction analysis (Figure 6). 18-Deacetyl-4-epi-henryine A (9) was obtained as a white, amorphous powder. Its molecular formula was determined as C22H30O6 by HRESIMS at m/z 413.1923 [M + Na]+ (calcd for C22H30O6Na, 413.1940). Compound 9 has the same molecular formula as that of 3, but shows several differences in its NMR data (Tables 2 and 4). First, 9 shows signals for two methyls (δH 1.21, s, H3-20; 0.91, s, H3-19; δC 20.0, C-20; 18.2, C-19), one fewer than in 3. Second, signals for an oxymethylene (δH 3.97 and 3.65, d, H2-18; δC 73.3, C-18) and a methylene (δH 1.43 and 1.36, H2-3; δC 36.6, C-3) are observed in 9, replacing a methyl (δH 0.87, s, H3-18; δC 28.2, C-18) and an oxymethine (δH 4.61, t, H-3; δC 77.0, C-3) in 3. HMBC correlations of H2-

Figure 4. X-ray ORTEP drawing of compound 3.

from the pseudomolecular ion peak at m/z 371.1822 [M + Na]+ in the HRESIMS (calcd for C20H28O5Na, 371.1834). Comparison of the NMR spectra of 4 (Tables 1 and 4) with those of 1 indicated that the acetyl signals in 1 were absent in 4, and the C-3 oxymethine function in 1 was replaced by a carbonyl (δC 221.2) in 4. HMBC correlations from H2-1 (δH 2.57 and 1.81), H2-2 (δH 2.25 and 2.70), H3-18 (δH 1.15), and H3-19 (δH 1.10) to the carbonyl established the carbonyl placement at C-3. Comprehensive 2D NMR analyses supported the assignment of the structure of 4 as 7α,11α,14β-trihydroxyent-kaur-16-ene-3,15-dione. The absolute configuration of 4 was illustrated as 5S,7R,8R,9S,10R,11R,13S,14R by singlecrystal X-ray diffraction analysis with Cu Kα radiation (Figure 5).


3-Deacetyl-11-oxoleukamenin E (5) possessed the molecular formula C20H28O5, confirmed by the HRESIMS ion at m/z 371.1821 [M + Na]+ (calcd for C20H28O5Na, 371.1834). Analysis of its NMR spectra (Tables 1 and 4) and comparison with those of 3 showed that 5 lacked an acetyl group compared with 3. The upfield shift of H-3 in 5 (δH 3.36, t) compared with that of 3 (δH 4.61, t) suggested that the 3-acetoxy group in 3 was replaced with a hydroxy group in 5. The cross-peaks from H-3 to H2-2, H3-18, and H3-19 in the NOESY spectrum of 5, as well as the smaller coupling constant J = 2.6 Hz between H-3 and H2-2, indicated that H-3 is α-oriented. Subsequently, the structure of 5 was determined as 3β,7α,14β-trihydroxy-ent-kaur16-ene-11,15-dione. The absolute configuration of 5 is the same as that of 3, based on similar ECD spectra. 3,11-Dioxoleukamenin E (6) was isolated as a white, amorphous powder. The [M + Na]+ ion at m/z 369.1665 in 2257



Article

Compound 11 was isolated as colorless crystals. The molecular formula C24H36O7 was determined from the HRESIMS ion at m/z 459.2324 [M + Na]+ (calcd for C24H36O7Na, 459.2359). The NMR data of 11 (Tables 3 and 4) were similar to those of 10, differing by an ethoxy group (δH 1.18, t, 3H; 3.50, m, 2H; δC 67.5, 15.5) in 11 replacing the methoxy group in 10. HMBC correlations of H2-17 (δH 3.74, d) to the oxymethylene (δC 67.5) of the ethoxy group, and the oxymethylene protons (δH 3.50, m) of the ethoxy group to C17 (δC 72.6), confirmed the ethoxy group was located at C-17. The NOESY correlations between H-17 and H-13α (δH 2.69, dd), between H-16 (δH 2.32, t) and H-12β (δH 2.53, ddd), and between H-12α (δH 2.88, dd) and H3-20 (δH 1.19, s) established the α-orientation of CH2-17. Detailed 2D NMR analyses established the structure of 11 as 7α,14β-dihydroxy17α-ethoxymethyl-3β-acetoxy-ent-kaur-11,15-dione. The absolute configuration was determined as 3S,5S,7R,8R,9S,10R,13S,14R,16R from single-crystal X-ray diffraction analysis by Cu Kα radiation (Figure 8).


18 with C-3 (δC 36.3), C-4 (δC 37.8), C-5 (δC 47.9), and C-19 (δC 18.2) indicated that the oxymethylene is located at C-18. In fact, further correlations of H2-18 to the carbonyl (δC 173.1) place an acetoxy group at C-18. The C-18 acetoxy methylene group is β-oriented according to NOESY correlations between H2-18 and both H-5β and H-6β. As confirmed by 2D NMR data analyses, 9 was identified as 7α,14β-dihydroxy-18βacetoxy-ent-kaur-16-ene-11,15-dione. The absolute configuration of 9 was assigned as 5S,7R,8R,9S,10R,13S,14R, based on the similarity of the ECD spectra of 9 and 3. The HRESIMS ion at m/z 445.2185 [M + Na]+ indicates a C23H34O7 (calcd for C23H34O7Na, 445.2202) molecular formula for compound 10. The 1H and 13C NMR data (Tables 2 and 4) of 10 resemble those of 3, except for an additional methoxy group (δH 3.25, s, 3H; δC 59.0), an oxymethylene (δH 3.59, dd, H-17a; 3.36, dd, H-17b; δC 68.3, C-17), and a methine (δH 3.23, dd, H-16; δC 50.0, C-16) in 10, replacing a C-16−C17 exocyclic double bond in 3. The cross-peaks between H-13/ H-16/H-17 in the 1H−1H COSY spectrum and the HMBC correlations from H2-17 and H-16 to C-13 (δC 39.7) and C-15 (δC 216.9) supported the assignment of the oxymethylene at C17 and the methine at C-16 in the five-membered ring. The methoxy group explains the oxygenation at C-17, and its position is assigned based on the HMBC correlations from H217 to the OCH3 carbon and from the methoxy protons to C-17. NOESY correlations between H-17b and H-12β, between H12α and H3-20, and between H-16 and H-13α suggested that the C-17 methoxymethyl group is β-oriented. Consequently, the structure of 10 was elucidated as 7α,14β-dihydroxy-17βmethoxymethyl-3β-acetoxy-ent-kaur-11,15-dione. Analysis of single-crystal X-ray diffraction (Figure 7) confirmed the structure and established the absolute configuration of 10 as 3S,5S,7R,8R,9S,10R,13S,14R,16S.


The molecular formulas of compounds 12−15 were determined as C 24 H 36 O 7 , C 21 H 30 O 6 , C 23 H 34 O 7 , and C23H34O7, respectively, according to their HRESIMS data. Comparison of their NMR data with those of compounds 3, 6, and 9, respectively, suggested that compound 12 was an EtOH addition product of compound 3, compound 13 was a MeOH addition product of compound 6, and compounds 14 and 15 were MeOH addition products of compound 9. Detailed 2D NMR data and ECD analyses assigned compounds 12−15 as 7α,14β-dihydroxy-17β-ethyoxymethyl-3β-acetoxy-ent-kaur11,15-dione, 7α,14β-dihydroxy-17α-methoxymethyl-ent-kaur3,11,15-trione, 7α,14β-dihydroxy-17α-methoxymethyl-18β-acetoxy-ent-kaur-11,15-dione, and 7α,14β-dihydroxy-17β-methoxymethyl-18β-acetoxy-ent-kaur-11,15-dione, respectively. Since EtOH and MeOH were used during the extraction and isolation, compounds 10−15 may be artifacts of compounds 3, 6, and 9, respectively. In order to verify this, compounds 3, 6, and 9 were separately dissolved in MeOH and EtOH and stirred overnight. Co-TLC detected the presence of compounds 10−15 in the solution of compounds 3, 6, and 9, respectively. To date, seven diterpene skeletal types, abietane, clerodane, pimarane, labdane, ent-kaurane, icetexane, and apianane, have been isolated from Salvia species. Among them, abietane and clerodane diterpenoids are the most common. Interestingly, abietane diterpenoids are mainly reported from Asian and European Salvia species, while clerodane diterpenoids were found commonly in American Salvia species.3,4 However, the ent-kaurane diterpenoid skeleton is rare in Salvia species. Only one ent-kaurane diterpene, verbenacine, was reported from a Salvia species (S. verbenaca).8 The isolation of the ent-kaurane

Figure 7. X-ray ORTEP drawing of compound 10. 2258



Article

Table 5. Cytotoxicity of Compounds 1−15 (IC50 in μM) against Five Human Cancer Cell Lines and the Noncancerous Human Beas-2B Cell Line compound

HL-60

SMMC-7721

A-549

MCF-7

SW480

BEAS-2B

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 DDP (cis-platin) paclitaxel

2.5 9.7 2.8 >10 >10 3.3 2.1 0.65 0.71 3.7 >10 3.3 >10 >10 2.8 1.2 10 >10 4.9 2.8 1.3 0.96 4.0 >10 3.5 >10 >10 3.5 6.4 10 2.7 >10 >10 6.4 3.1 1.1 0.94 2.9 >10 3.5 >10 >10 3.0 9.2 10 2.5 >10 >10 3.1 1.7 3.0 1.8 2.5 >10 3.1 >10 >10 2.0 15.9 10 3.0 >10 6.7 2.6 13.4 10 2.0 >10 >10 3.1 0.88 0.73 0.87 2.5 >10 2.8 >10 >10 3.0 11.1 0.58

diterpenoids, compounds 1−17, suggests that S. cavaleriei expresses a nontypical chemical profile and may taxonomically be related to S. verbenaca. Compounds 1−15 were evaluated for their cytotoxicity against the human cancer cell lines HL-60, SMMC-7721, A549, MCF-7, and SW480 and an immortalized noncancerous cell line, Beas-2B, by the reported MTS method.13a,19 cis-Platin and paclitaxel were used as positive controls. As shown in Table 5, compounds 1−10, 12, 14, and 15 showed cytotoxicity, while compounds 11 and 13 were inactive (IC50 > 10 μM). Compounds 1, 3, 6−10, 12, and 15 exhibited more potent cytotoxicity than cis-platin. Among them, compound 9 showed the strongest activity, with IC50 values against HL-60, SMMC7721, A549, MCF-7, and SW480 cells lines ranging from 0.71 to 1.8 and 1.7 μM. However, compounds 1, 3, 6−10, 12, and 15 also showed strong cytotoxicity against the noncancerous Beas-2B cell line and did not exhibit selective cytotoxicity against the cancer cell lines tested. Compound 16, the 18deacetyl derivative of 9, was reported to be noncytotoxic to the A549 and MCF-7 cell lines,20 which suggested that the 18-Oacetyl group in 9 may be required for activity. Compounds 2, 4, 5, and 14 exhibited higher IC50 values against the noncancerous Beas-2B cell line (IC50 > 10 μM) than against the SW480 cell line, with IC50 values of 5.5, 8.1, 7.6, and 6.7 μM, respectively, indicating at least a small selectivity in inhibiting SW480 cells (Table 5). In addition, compound 2 showed selective cytotoxicity against the SMMC-7721 cell line compared with the Beas-2B cell line, with an IC50 value of 3.8 and >10 μM, respectively. Comparison of the activities of 1 and 2 suggests that α-orientation of the 11-OH increases cytotoxicity compared with the β-orientation. Although 4 possesses an 11α-OH, its lower cytotoxicity compared with that of 1 may be due to the lack of a 3-acetoxy group. In fact, compound 3 possesses more potent cytotoxicity than 5, a further indication that the 3-acetoxy group is important for the cytotoxicity of these compounds. Compound 17 was reported to be noncytotoxic to the A-549 and MCF-7 cell lines;21 however, compounds 1, 3, 7, and 8 had significant cytotoxicity against these two cell lines. Thus, a Δ11(12) double bond or a C-11−C12 epoxy group may be essential for activity. Interestingly, compounds 10, 12, and 15, which possess a 16S configuration,

showed more potent cytotocixity than compounds 11, 13, and 14, having a 16R configuration, thus suggesting that a 16S configuration in ent-kaurane diterpenoids may enhance the cytotocixity. As a result, a cyclopentanone conjugated with an exomethylene group, a 3-acetoxy group, an 11α-OH, a Δ11(12) double bond, an 11α,12α-epoxide, and 16S configuration in entkaurane diterpenoids appear to enhance cytotoxicity.

■

EXPERIMENTAL SECTION

General Experimental Procedures. Melting points were measured on an X-5 micromelting point apparatus without correction (Beijing Tech Instrument Co. Ltd.). Optical rotations were determined in MeOH on a Perkin-Elmer 341 polarimeter. UV spectra were recorded on a Varian Cary 50 spectrometer. ECD spectra were obtained on a JASCO J-810 spectrometer. IR spectra were determined on a Bruker Vertex 70 instrument. NMR spectra were recorded on a Bruker AM-400 spectrometer, and the 1H and 13C NMR chemical shifts were referenced to the solvent peaks for CDCl3 at δH 7.24 and δC 77.23 or for methanol-d4 at δH 3.31 and δC 49.15. HRESIMS were conducted in the positive-ion mode on a Thermo Fisher LC-LTQOrbitrap XL spectrometer. The crystallographic data were obtained on a Bruker SMART APEX-II CCD diffractometer equipped with graphite-monochromatized Cu Kα radiation (λ = 1.54178 Å). Column chromatography (CC) was carried out on silica gel (100−200 mesh, 200−300 mesh, and 400 mesh, Qingdao Ocean Chemical Industry Co. Ltd., People’s Republic of China), RP C18 silica gel (ODS-A-HG, YMC Co. Ltd., Japan), and Sephadex LH-20 (GE Healthcare BioSciences AB, Sweden). HPLC was conducted on an Agilent 1200 instrument with detection at 210 or 230 nm using an RP C18 (5 μm, 250 × 10 mm, YMC-pack ODS-A) column and MeOH−H2O or MeCN−H2O as the mobile phase at a flow rate of 1.5 mL/min. MPLC was carried out with an EZ Purifier III chromatography system. TLC was performed with silica gel 60 F254 (Yantai Chemical Industry Research Institute, Yantai, China) and RP-C18 F254 plates (Merck). Plant Material. The whole plants of S. cavaleriei were collected at Enshi, Hubei Province, People’s Republic of China, in September 2010. The plant material was authenticated by Dr. Jianping Wang at the School of Pharmacy, Tongji Medical College, Huazhong University of Science and Technology. A voucher specimen (No. 20100901) has been deposited at Hubei Key Laboratory of Natural Medicinal Chemistry and Resource Evaluation, School of Pharmacy, Tongji Medical College, Huazhong University of Science and Technology. Extraction and Isolation. The dried whole plants of S. cavaleriei (20 kg) were extracted four times in 95% aqueous EtOH at room 2259



Article

ECD (MeOH) λmax (Δε) 352 (−0.1), 311 (+1.5)228 (+2.2) nm; IR (KBr) νmax 3381, 2955, 2877, 1733, 1698, 1645, 1460, 1388, 1142, 1090, 1071, 992, 775 cm−1; 1H NMR data see Table 1; 13C NMR data see Table 4; positive HRESIMS m/z 371.1821 [M + Na]+ (calcd for C20H28O5Na, 371.1834). 3,11-Dioxoleukamenin E (6): white, amorphous powder; [α]20D +4 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 232 (3.93) nm; ECD (MeOH) λmax (Δε) 352 (−0.1), 313 (+1.2), 224 (+2.4) nm; IR (KBr) νmax 3360, 2985, 2963, 1727, 1704, 1672, 1646, 1457, 1386, 1234, 1198, 1134, 1079, 1042, 968, 777, 654 cm−1; 1H NMR data see Table 2; 13C NMR data see Table 4; positive HRESIMS m/z 369.1665 [M + Na]+ (calcd for C20H26O5Na, 369.1678). 11,12-Didehydroleukamenin E (7): colorless needles (MeOH); mp 212−214 °C; [α]20D −182 (c 0.2, MeOH); UV (MeOH) λmax (log ε) 228 (3.75) nm; ECD (MeOH) λmax (Δε) 244 (−1.1), 220 (+2.7), 205 (−13.2) nm; IR (KBr) νmax 3435, 2959, 2869, 1728, 1652, 1460, 1381, 1250, 1180, 1161, 1094, 1027, 984, 804, 671 cm−1; 1H NMR data see Table 2; 13C NMR data see Table 4; positive HRESIMS m/z 397.1976 [M + Na]+ (calcd for C22H30O5Na, 397.1991). 11α,12α-Epoxyleukamenin E (8): colorless needles (MeOH); mp 197−198 °C; [α]20D −87 (c 0.4, MeOH); UV (MeOH) λmax (log ε) 227 (3.84) nm; ECD (MeOH) λmax (Δε) 335 (−0.1), 226 (−2.2), 200 (+0.8) nm; IR (KBr) νmax 3262, 2944, 2871, 1722, 1650, 1376, 1251, 1210, 1186, 1114, 1077, 1017, 978, 869, 660, 563 cm−1; 1H NMR data see Table 2; 13C NMR data see Table 4; positive HRESIMS m/z 413.1926 [M + Na]+ (calcd for C22H30O6Na, 413.1940). 18-Deacetyl-4-epi-henryine A (9): white, amorphous powder; [α]20D +8 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 228 (3.71) nm; ECD (MeOH) λmax (Δε) 351 (−0.1), 312 (+1.4), 226 (+2.4) nm; IR (KBr) νmax 3450, 2952, 2853, 1729, 1706, 1648, 1466, 1384, 1254, 1138, 1094, 1041, 676 cm−1; 1H NMR data see Table 2; 13C NMR data see Table 4; positive HRESIMS m/z 413.1923 [M + Na]+ (calcd for C22H30O6Na, 413.1940). 7α,14β-Dihydroxy-17β-methoxymethyl-3β-acetoxy-ent-kaur11,15-dione (10): colorless needles (MeOH); mp 165−167 °C; [α]20D +12 (c 1.1, MeOH); ECD (MeOH) λmax (Δε) 306 (+0.7) nm; IR (KBr) νmax 3381, 2951, 1734, 1704, 1476, 1392, 1313, 1244, 1153, 1087, 1058, 969, 776 cm−1; 1H NMR data see Table 2; 13C NMR data see Table 4; positive HRESIMS m/z 445.2185 [M + Na]+ (calcd for C23H34O7Na, 445.2202). 7α,14β-Dihydroxy-17α-ethoxymethyl-3β-acetoxy-ent-kaur11,15-dione (11): colorless needles (MeOH); mp 198−200 °C; [α]20D +71 (c 0.1, MeOH); ECD (MeOH) λmax (Δε) 308 (+2.0), 218 (−0.3) nm; IR (KBr) νmax 3283, 2968, 2866, 1736, 1703, 1461, 1372, 1243, 1103, 1061, 979, 879, 505 cm−1; 1H NMR data see Table 3; 13C NMR data see Table 4; positive HRESIMS m/z 459.2324 [M + Na]+ (calcd for C24H36O7Na, 459.2359). 7α,14β-Dihydroxy-17β-ethyoxymethyl-3β-acetoxy-ent-kaur11,15-dione (12): white, amorphous powder; [α]20D −2 (c 1.0, MeOH); ECD (MeOH) λmax (Δε) 306 (+0.6) nm; IR (KBr) νmax 3377, 2958, 2877, 1733, 1706, 1451, 1376, 1248, 1095, 1025, 982, 886, 844, 776, 608, 510 cm−1; 1H NMR data see Table 3; 13C NMR data see Table 4; positive HRESIMS m/z 459.2324 [M + Na]+ (calcd for C24H36O7Na, 459.2359). 7α,14β-Dihydroxy-17α-methoxymethyl-ent-kaur-3,11,15-trione (13): colorless needles (MeOH); mp 180−182 °C; [α]20D −5 (c 0.1, MeOH); ECD (MeOH) λmax (Δε) 310 (+2.1), 206 (+0.8) nm; IR (KBr) νmax 3227, 2930, 2853, 1732, 1702, 1456, 1381, 1204, 1160, 1111, 1067, 950, 754, 555 cm−1; 1H NMR data see Table 3; 13C NMR data see Table 4; positive HRESIMS m/z 401.1913 [M + Na]+ (calcd for C21H30O6Na, 401.1940). 7α,14β-Dihydroxy-17α-methoxymethyl-18β-acetoxy-ent-kaur11,15-dione (14): colorless needles (MeOH); mp 199−200 °C; [α]20D +32 (c 0.1, MeOH); ECD (MeOH) λmax (Δε) 308 (+2.7), 220 (−0.3), 204 (+0.6) nm; IR (KBr) νmax 3464, 3396, 2952, 2934, 2898, 1740, 1707, 1468, 1440, 1382, 1229, 1116, 1075, 1041, 965, 905, 755, 585 cm−1; 1H NMR data see Table 3; 13C NMR data see Table 4; positive HRESIMS m/z 445.2169 [M + Na]+ (calcd for C23H34O7Na, 445.2202).

temperature. The filtrate was combined and concentrated under reduced pressure to afford a residue (1.5 kg). The residue was suspended in H2O and extracted with petroleum ether, CHCl3, EtOAc, and n-BuOH, successively. The active CHCl3 extract (252.5 g) was subjected to CC over silica gel (100−200 mesh) eluting with a petroleum ether−acetone step gradient system (8:1, 5:1, 3:1, 2:1, 1:1, 0:1) to give fractions A−F. Fraction B was separated on an RP C18 column (from 30:70 to 80:20 MeOH−H2O) by MPLC to afford subfractions B1−B4. Fraction B3 was subjected to CC on silica gel using a gradient system of petroleum ether−acetone (5:1) providing three subfractions, B3A−B3C. Compounds 7 (6.0 mg, tR 46.2 min) and 17 (12.0 mg, tR 50.6 min) were purified from subfraction B3B by HPLC (60:40 MeOH−H2O). Separation of fraction C by MPLC RP C18 eluted with a MeOH−H2O (20:80−100:0) gradient system yielded five main subfractions, C1−C5. Subfraction C2 was chromatographed on Sephadex LH-20 (MeOH) and then purified by silica gel CC, eluted with CHCl3−acetone (300:1), to yield compounds 3 (25.0 mg) and 10 (20.0 mg). Subfraction C3 was subjected to repeated chromatography over silica gel (petroleum ether−acetone, from 5:1 to 0:1) to give subfractions C3A−C3C. Subfraction C3A afforded compounds 8 (13.0 mg, tR 32.7 min) and 11 (4.0 mg, tR 45.2 min) by HPLC (40:60 MeCN−H2O) and 12 (29.0 mg) by silica gel column chromatography (CH2Cl2−acetone, 5:1). Compound 14 (3.0 mg) was separated from the subfraction C3C by recrystallization from petroleum ether−acetone (3:1). HPLC (60:40 MeOH−H2O) was applied to furnish compounds 9 (6.0 mg, tR 46.4 min) and 15 (5.0 mg, tR 61.1 min) from the remainder of subfraction C3C. Fraction D was divided into four subfractions (D1−D4) by Sephadex LH-20 (MeOH). Compounds 6 (7.0 mg) and 13 (3.0 mg) were crystallized from subfraction D2. The remainder of fraction D2 was separated further by silica gel (petroleum ether−acetone, 3:1), followed by HPLC (25:75 MeCN−H2O), to obtain compound 5 (9.0 mg, tR 38.9 min). Separation of subfraction D3 by silica gel column chromatography (petroleum ether−acetone, from 6:1 to 4:1) afforded compounds 1 (49.0 mg) and 2 (10.0 mg). Compound 16 (6.0 mg, tR 105.5 min) was obtained by HPLC (40:60 MeOH/H2O) from the remainder of subfraction D3. Fraction E gave compound 4 (12.0 mg) by Sephadex LH-20 (MeOH) and silica gel column chromatography (petroleum ether−acetone, 3:1). 11α-Hydroxyleukamenin E (1): colorless needles (MeOH); mp 195−197 °C; [α]20D −36 (c 1.6, MeOH); UV (MeOH) λmax (log ε) 234 (3.92) nm; ECD (MeOH) λmax (Δε) 344 (−0.3), 242 (+1.1), 209 (−4.8) nm; IR (KBr) νmax 3394, 2937, 2873, 1725, 1705, 1647, 1450, 1375, 1248, 1182, 1069, 979, 843, 713 cm−1; 1H NMR data see Table 1; 13C NMR data see Table 4; positive HRESIMS m/z 415.2067 [M + Na]+ (calcd for C22H32O6Na, 415.2097). 11β-Hydroxyleukamenin E (2): colorless needles (MeOH); mp 201−202 °C; [α]20D −48 (c 0.3, MeOH); UV (MeOH) λmax (log ε) 236 (3.90) nm; ECD (MeOH) λmax (Δε) 327 (−0.3), 246 (−1.0), 203 (−2.1) nm; IR (KBr) νmax 3380, 2939, 2863, 1732, 1694, 1654, 1471, 1380, 1273, 1078, 1029, 993, 965, 594 cm−1; 1H NMR data see Table 1; 13C NMR data see Table 4; positive HRESIMS m/z 415.2065 [M + Na]+ (calcd for C22H32O6Na, 415.2097). 11-Oxoleukamenin E (3): colorless needles (MeOH); mp 170−172 °C; [α]20D +38 (c 0.2, MeOH); UV (MeOH) λmax (log ε) 230 (3.77) nm; ECD (MeOH) λmax (Δε) 351 (−0.1), 312 (+1.1), 226 (+2.2) nm; IR (KBr) νmax 3370, 2957, 1727, 1706, 1646, 1438, 1380, 1250, 1138, 1093, 1022, 982, 658 cm−1; 1H NMR data see Table 1; 13C NMR data see Table 4; positive HRESIMS m/z 413.1923 [M + Na]+ (calcd for C22H30O6Na, 413.1940). 3-Oxo-11α-hydroxyleukamenin E (4): colorless needles (MeOH); mp 256−258 °C; [α]20D −184 (c 0.4, MeOH); UV (MeOH) λmax (log ε) 234 (3.88) nm; ECD (MeOH) λmax (Δε) 340 (−0.4), 290 (−1.8), 242 (+0.9), 200 (−4.4), nm; IR (KBr) νmax 3259, 2966, 2938, 2895, 2865, 1731, 1704, 1650, 1486, 1460, 1366, 1242, 1130, 1062, 1022, 948 cm−1; 1H NMR data see Table 1; 13C NMR data see Table 4; positive HRESIMS m/z 371.1822 [M + Na]+ (calcd for C20H28O5Na, 371.1834). 3-Deacetyl-11-oxoleukamenin E (5): white, amorphous powder; [α]20D +22 (c 0.3, MeOH); UV (MeOH) λmax (log ε) 227 (3.50) nm; 2260



Article

reflections [R(int) = 0.0205]; final R indices [I > 2σ (I)] R1 = 0.0331, wR2 = 0.1081; R indices (all data) R1 = 0.0348, wR2 = 0.1177; Flack parameter 0.1(3). Crystallographic data of 11: C24H36O7, formula weight 436.53, crystal size 0.26 × 0.20 × 0.20 mm3, crystal system orthorhombic space group P21, a = 12.0609(2) Å, b = 6.82630(10) Å, c = 29.7481(5) Å, α = γ = 90°, β = 98.0820(10)°, V = 2424.87(7) Å3, Z = 4, Dc = 1.196 Mg/m3, μ(Cu Kα) = 0.733 mm−1, F(000) = 944, absorption coefficient 0.711 mm−1; total 18 261 reflections, 6586 independent reflections [R(int) = 0.0230]; final R indices [I > 2σ (I)] R1 = 0.0370, wR2 = 0.1032; R indices (all data) R1 = 0.0373, wR2 = 0.1037; Flack parameter −0.03(15). Crystallographic data for the structures of compounds 1−4, 8, 10, and 11 reported in this paper have been deposited with the Cambridge Crystallographic Data Centre (deposit numbers CCDC 915525− 915531, respectively). Copies of these data can be obtained, free of charge, on application to the Director, CCDC, 12 Union Road, Cambridge CB2 1EZ, UK (fax: +44-(0)1223-336033 or e-mail: [email protected]). Cytotoxicity Assay. The cytotoxicity assay was performed according to the MTS method in 96-well microplates, as reported previously.13a,19 The assay is based on reduction of the tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfopheny)-2H-tetrazolium (MTS) in metabolically active cells to produce the corresponding formazan product, accomplished by NADPH- or NADH-dependent dehydrogenase enzymes. After incubation with test substances and controls, this is a rapid colorimetric assay to measure cellular proliferation and cytotoxicity. Five human cancer cell lines, HL-60 human myeloid leukemia, SMMC-7721 human hepatocellular carcinoma, A-549 lung cancer, MCF-7 breast cancer, and SW480 human colon cancer, together with one noncancerous human pulmonary epithelial cell line, BEAS-2B, were assayed. Cells were cultured in RPMI-1640 or in DMEM medium (Hyclone, Logan, UT, USA) and supplemented with 10% fetal bovine serum (Hyclone, Logan, UT, USA) in 5% CO2 at 37 °C. A volume of 100 μL of adherent cells was seeded into each well of the 96-well culture plates with initial density of 1 × 105 cells/mL, and cells were allowed to adhere for 12 h before addition of test compounds. Each cancer cell line was exposed to test compounds at concentrations of 0.064, 0.32, 1.6, 8, and 40 μM in triplicate for 48 h at 37 °C in 200 μL of media. Wells with DDP (cis-platin, Sigma, St. Louis, MO, USA) and paclitaxel (Sigma, St. Louis, MO, USA) were used as positive controls. Then, 100 μL of media and 20 μL of MTS (Sigma, St. Louis, MO, USA) were added to each well and cultured for 4 h. After compound treatment, cell viability was detected by a Bio-Rad 680 at λ = 595 nm, and a cell growth curve was graphed. IC50 values were calculated by Reed and Muench’s method.22

7α,14β-Dihydroxy-17β-methoxymethyl-18β-acetoxy-ent-kaur11,15-dione (15): white, amorphous powder; [α]20D −13 (c 0.1, MeOH); ECD (MeOH) λmax (Δε) 308 (+0.6) nm; IR (KBr) νmax 3406, 2938, 2910, 2852, 1744, 1721, 1467, 1389, 1367, 1229, 1145, 1102, 1038, 963, 782, 526 cm−1; 1H NMR data see Table 3; 13C NMR data see Table 4; positive HRESIMS m/z 445.2180 [M + Na]+ (calcd for C23H34O7Na, 445.2202). Single-Crystal X-ray Diffraction Analysis and Crystallographic Data of Compounds 1−4, 8, 10, and 11. Diffraction intensity data for compounds 1−4, 8, 10, and 11 were acquired on a Bruker APEX-II diffractometer employing graphite-monochromatized Cu Kα radiation (λ = 1.54178 Å) at 298(2) K. Data were collected by Bruker APEX2 software. Data reduction was conducted with Bruker SAINT. Structure solution and refinement were performed with the SHELXTL program package. All non-hydrogen atoms were refined anisotropically. The hydrogen atom positions were geometrically idealized and allowed to ride on their parent atoms. The crystal structures of compounds 1−4, 8, 10, and 11 were drawn by ORTEP 3 for windows (version 2.02) and are shown in Figures 2−8, respectively. Crystallographic data of 1: C22H32O6·2H2O, formula weight 428.51, crystal size 0.15 × 0.12 × 0.11 mm3, crystal system monoclinic space group P2(1), a = 8.5553(2) Å, b = 7.0580(2) Å, c = 18.5846(5) Å, α = γ = 90°, β = 94.6680(10)°, V = 1118.48(5) Å3, Z = 2, Dc = 1.272 Mg/m3, μ(Cu Kα) = 0.733 mm−1, F(000) = 464, absorption coefficient 0.793 mm−1; total 12 021 reflections, 3535 independent reflections [R(int) = 0.0210]; final R indices [I > 2σ(I)] R1 = 0.0310, wR2 = 0.0872; R indices (all data) R1 = 0.0312, wR2 = 0.0875; Flack parameter 0.19(14). Crystallographic data of 2: C22H32O6·CH3OH·H2O, formula weight 442.53, crystal size 0.12 × 0.12 × 0.11 mm3, crystal system orthorhombic, space group P2(1)2(1)2(1), a = 8.4307(2) Å, b = 11.7779(3) Å, c = 23.1333(5) Å, α = β = γ = 90°, V = 2297.04(9) Å3, Z = 4, Dc = 1.280 Mg/m3, μ(Cu Kα) = 0.733 mm−1, F(000) = 960, absorption coefficient 0.788 mm−1; total 11 826 reflections, 3364 independent reflections [R(int) = 0.0260]; final R indices [I > 2σ(I)] R1 = 0.0291, wR2 = 0.0793; R indices (all data) R1 = 0.0294, wR2 = 0.0795; Flack parameter −0.04(16). Crystallographic data of 3: C22H30O6·H2O, formula weight 408.48, crystal size 0.32 × 0.20 × 0.20 mm3, crystal system orthorhombic space group P2(1)2(1)2(1), a = 7.57840(10) Å, b = 10.6565(2) Å, c = 26.4145(5) Å, α = β = γ = 90°, V = 2133.21(6) Å3, Z = 4, Dc = 1.272 Mg/m3, μ(Cu Kα) = 0.733 mm−1, F(000) = 880, absorption coefficient 0.774 mm−1; total 13 794 reflections, 3390 independent reflections [R(int) = 0.0296]; final R indices [I > 2σ(I)] R1 = 0.0335, wR2 = 0.0923; R indices (all data) R1 = 0.0340, wR2 = 0.0928; Flack parameter −0.1(2). Crystallographic data of 4: C20H28O5, formula weight 348.42, crystal size 0.15 × 0.12 × 0.10 mm3, crystal system orthorhombic space group P2(1)2(1)2(1), a = 6.506 Å, b = 13.700 Å, c = 20.624 Å, α = β = γ = 90°, V = 1838.3 Å3, Z = 4, Dc = 1.259 Mg/m3, μ(Cu Kα) = 0.733 mm−1, F(000) = 752, absorption coefficient 0.089 mm−1; total 8460 reflections, 1821 independent reflections [R(int) = 0.1119]; final R indices [I > 2σ(I)] R1 = 0.0388, wR2 = 0.1037; R indices (all data) R1 = 0.0413, wR2 = 0.1043; Flack parameter 0(10). Crystallographic data of 8: C22H30O6·H2O, formula weight 408.48, crystal size 0.23 × 0.10 × 0.10 mm3, crystal system orthorhombic space group P2(1)2(1)2(1), a = 7.5766(2) Å, b = 10.6290(2) Å, c = 26.1402(5) Å, α = β = γ = 90°, V = 2105.11(8) Å3, Z = 4, Dc = 1.289 Mg/m3, μ(Cu Kα) = 0.733 mm−1, F(000) = 880, absorption coefficient 0.784 mm−1; total 11 433 reflections, 3047 independent reflections [R(int) = 0.0215]; final R indices [I > 2σ (I)] R1 = 0.0336, wR2 = 0.0944; R indices (all data) R1 = 0.0339, wR2 = 0.0949; Flack parameter 0.03(19). Crystallographic data of 10: C23H34O7, formula weight 422.50, crystal size 0.20 × 0.10 × 0.10 mm3, crystal system orthorhombic space group P2(1)2(1)2(1), a = 6.71180(10) Å, b = 13.4388(2) Å, c = 23.6639(4) Å, α = β = γ = 90°, V = 2134.45(6) Å3, Z = 4, Dc = 1.315 Mg/m3, μ(Cu Kα) = 0.733 mm−1, F(000) = 912, absorption coefficient 0.790 mm−1; total 7137 reflections, 1998 independent

■

ASSOCIATED CONTENT

S Supporting Information *

(+)-HRESIMS, UV, IR, ECD, and 1D and 2D NMR spectra for compounds 1−15 and X-ray crystallographic data (CIF file) for compounds 1−4, 8, 10, and 11. This material is available free of charge via the Internet at http://pubs.acs.org.

■

AUTHOR INFORMATION

Corresponding Authors

*(G.Y.) Tel: 86-27-83692311. Fax: 86-27-83692762. E-mail: [email protected]. *(Y.Z.) E-mail: [email protected]. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS We thank Professor F. D. Horgen at Hawaii Pacific University for editing the manuscript. We are grateful to Dr. J.-P. Wang at Huazhong University of Science and Technology for the 2261



Article

(21) Zhao, Y.; Pu, J. X.; Huang, S. X.; Ding, L. S.; Wu, Y. L.; Li, X.; Yang, L. B.; Xiao, W. L.; Chen, G. Q.; Sun, H. D. J. Nat. Prod. 2009, 72, 988−993. (22) Reed, L. J.; Muench, H. Am. J. Hyg. 1938, 27, 493−497.

authentication of the plant material, and Dr. X.-G. Meng at Central China Normal University for single-crystal X-ray diffraction analysis. This project was financially supported by the Hubei Key Laboratory Foundation of Natural Medicinal Chemistry and Resource Evaluation, Huazhong University of Science and Technology (2010-3, to G.Y.), the Fundamental Research Funds for the Central Universities (HUST: 2012QN003, to G.Y.), Scientific Research Foundation for the Returned Oversea Chinese Scholars, State Education Ministry of China (2010-1561, 40th, to G.Y.), Program for Youth Chutian Scholar of Hubei Province of China (to G.Y.), and Program for New Century Excellent Talents in University, State Education Ministry of China (NCET-2008-0224, to Y.Z).

■

REFERENCES

(1) Li, M. H.; Chen, J. M.; Peng, Y.; Xiao, P. G. Mode. Tradit. Chin. Med. Mater. Med. 2008, 10, 46−52. (2) Lu, Y.; Foo, Y. L. Phytochemistry 2002, 59, 117−140. (3) Wu, Y. B.; Ni, Z. Y.; Shi, Q. W.; Dong, M.; Kiyota, H.; Gu, Y. C.; Cong, B. Chem. Rev. 2012, 112, 5967−6026. (4) Pan, Z. H.; Xu, G.; Zhao, Q. S. Guangxi Zhiwu 2010, 30, 781− 790. (5) Kabouche, A.; Kabouche, Z. Stud. Nat. Prod. Chem. 2008, 35, 753−833. (6) Ö ztekin, N.; Başkan, S.; Evrim Kepekçi, S.; Erim, F. B.; Topçu, G. J. Pharmaceut. Biomed. Anal. 2010, 51, 439−442. (7) Zhao, W.; Pu, J. X.; Du, X.; Su, J.; Li, X. N.; Yang, J. H.; Xue, Y. B.; Li, Y.; Xiao, W. L; Sun, H. D. J. Nat. Prod. 2011, 74, 1213−1220. (8) Ahmed, B.; AI-Howiriny, T. A.; Al-Rehaily, A. J.; Mossa, J. S. Z. Naturforsch. C 2004, 59, 9−14. (9) Li, X. W.; Ian, C. H. Lamiaceae. In Flora of China; Wu, Z. Y., Raven, P. H., Hong, D. Y., Eds.; Science Press: Beijing, China, and Missouri Botanical Garden Press: St. Louis, MO, USA, 1994; Vol. 17, pp 50−299. (10) Fang, Z. X., Liao, C. L. Medicinal Flora of Enshi, Hubei Province; Hubei Science and Technology Press: Wuhan, 2006. pp 288−289. (11) Wang, H. Y.; Hu, D. Y.; Xue, W.; Yang, S.; Song, B. A. Nat. Prod. Res. Dev. 2011, 23, 63−65. (12) (a) Zhao, L. M.; Liang, X. T.; Li, L. N. J. Chin. Pharm. Sci. 1997, 6, 111−112. (b) Zhang, H. J.; Li, L. N. Planta Med. 1994, 60, 70−72. (c) Li, L. N. Chin. J. Org. Chem. 1993, 13, 303−304. (13) (a) Luo, Z. W.; Wang, F. Q.; Zhang, J. W.; Li, X. Y.; Zhang, M. K.; Hao, X. C.; Xue, Y. B.; Li, Y.; Horgen, F. D.; Yao, G. M.; Zhang, Y. H. J. Nat. Prod. 2012, 75, 2113−2120. (b) Shu, P. H.; Wei, X. L.; Xue, Y. B.; Li, W. J.; Zhang, J. W.; Xiang, M.; Zhang, M. K.; Luo, Z. W.; Li, Y.; Yao, G. M.; Zhang, Y. H. J. Nat. Prod. 2013, 76, 1303−1312. (c) Guo, J. R.; Zhang, J. W.; Shu, P. H.; Kong, L. M.; Hao, X. C.; Xue, Y. B.; Luo, Z. W.; Li, G.; Li, Y.; Yao, G. M.; Zhang, Y. H. Molecules 2012, 17, 6424−6433. (14) Zhou, B. N.; Chen, J.-B.; Wang, C. Y.; Blasko, G.; Cordell, G. A. Phytochemisty 1989, 28, 3536−3538. (15) Takeda, Y.; Fujita, T.; Ueno, A. Chem. Lett. 1981, 10, 1229− 1232. (16) Sun, H. D.; Lin, Z. W.; Shen, X. Y.; Takeda, Y.; Fujita, T. Phytochemistry 1991, 30, 603−606. (17) (a) Hong, S. S.; Lee, S. A.; Han, X. H.; Jin, H. Z.; Lee, J. H.; Lee, D.; Lee, J. J.; Hong, J. T.; Kim, Y.; Ro, J. S.; Hwang, B. Y. J. Nat. Prod. 2007, 70, 632−636. (b) Li, L. M.; Weng, Z. Y.; Huang, S. X.; Pu, J. X.; Li, S. H.; Huang, H.; Yang, B. B.; Han, Y.; Xiao, W. L.; Li, M. L.; Han, Q. B.; Sun, H. D. J. Nat. Prod. 2007, 70, 1295−1301. (18) (a) Flack, H. D. Acta Crystallogr. 1983, A39, 876−881. (b) Flack, H. D.; Bernardinelli, G. Chirality 2008, 20, 681−690. (19) Cory, A. H.; Owen, T. C.; Barltrop, J. A.; Cory, J. G. Cancer Commun. 1991, 3, 207−212. (20) Zhao, Y.; Pu, J. X.; Huang, S. X.; Wu, Y. L.; Yang, L. B.; Xiao, W. L.; Han, Q. B.; Chen, G. Q.; Sun, H. D. J. Nat. Prod. 2009, 72, 125− 129. 2262


Cytotoxic ent-Kaurane Diterpenoids from Salvia ... - ACS Publications

Recommend Documents