New Dimeric and seco-Abietane Diterpenoids from Salvia wardii

Abstract

Two dimeric abietane diterpenoids, salviwardins A and B (1 and 2), and a seco-abietane diterpenoid salviwardin C (3), along with five known analogues (4-8), were isolated from the roots of Salvia wardii. The structures of these isolates were elucidated by extensive spectroscopic methods. The inhibitory activities of these isolates against five human cancer cell lines in vitro were also tested.

Nat. Prod. Bioprospect. (2015) 5:7782 DOI 10.1007/s13659-015-0054-6

ORIGINAL ARTICLE

New Dimeric and seco-Abietane Diterpenoids from Salvia wardii

Qiu-Li Xiao Fan Xia Xing-Wei Yang

Yang Liao Li-Xin Yang Yu-Kun Wei

Xian Li Gang Xu

Received: 26 January 2015 / Accepted: 14 February 2015 / Published online: 8 April 2015 The Author(s) 2015. This article is published with open access at Springerlink.com

Abstract Two dimeric abietane diterpenoids, salviwardins A and B (1 and 2), and a seco-abietane diterpenoid salviwardin C (3), along with ve known analogues (48), were isolated from the roots of Salvia wardii. The structures of these isolates were elucidated by extensive spectroscopic methods. The inhibitory activities of these isolates against ve human cancer cell lines in vitro were also tested.

Keywords Salvia wardii Dimeric abietane diterpenoids seco-abietane

1 Introduction

The genus Salvia is a rich source of diterpenoids with structural diversity [14]. Hundreds of diterpenoids with interesting bioactivities, such as tanshinone IIA (treat cardiovascular diseases), salvicine (a signicant antitumor agent), neotanshinlactone (inhibition of breast cancer), and salvinorin A (the rst non-nitrogenated opium receptor

agonist), have been characterized from the plants within this genus [58]. Many species of this genus, such as S. miltiorrhiza, S. yunnanensis, and S. przewalskii, are used to treat cardiovascular diseases [911], and S. prionitis is used in Chinese folk medicine for the treatment of tonsillitis, pharyngitis, and bacillary dysentery [12].

Salvia wardii, a herb with violet owers distributed in east of Tibet, has not been chemically studied before [13]. Aiming at searching for structurally interesting and bioactive diterpenoids from the Salvia plants, we chemically investigated S. wardii and isolated three new abietane diterpenoids, salviwardins AC (13), and ve known analogues (48). The inhibitory activities of these isolates against ve human cancer cell lines in vitro were also tested.

2 Results and Discussion

The acetone extract of the air-dried and powdered the roots of S. wardii (33 kg) was subjected to a silica gel column to afford fractions AG. Fraction B was subjected to a series of chromatographic methods, and led to the isolation of three new abietane derivatives, salviwardins AC (13), together with ve knows analogues, including prionitin (4) [14], sahandol (5) [15], salvilenone (6) [16], microstegiol (7) [17], and ferruginol (8) [18].

Electronic supplementary material The online version of this

article (doi:http://dx.doi.org/10.1007/s13659-015-0054-6

Web End =10.1007/s13659-015-0054-6 ) contains supplementary

material, which is available to authorized users.

Q.-L. Xiao F. Xia X. Li (&)

School of Pharmaceutical Science and Yunnan Key Laboratory of Pharmacology of Natural Products, Kunming Medical University, Kunming 650500, Yunnan, Peoples Republic of Chinae-mail: [email protected]

X.-W. Yang Y. Liao L.-X. Yang G. Xu (&)

State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, Peoples Republic of China e-mail: [email protected]

Y.-K. WeiShanghai Chenshan Plant Science Research Center, Chinese Academy of Sciences, Beijing, Peoples Republic of China

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Salviwardin A (1) was obtained as orange powder. Its molecular formula C40H54O4 was established by its

13C NMR and HREIMS (m/z 598.4029, [M]?) data, indicating 14 degrees of unsaturation. The IR absorption at 3440 and 1624 cm-1 implied the existence of hydroxyl and carbonyl groups. The 13C and DEPT NMR (Table 1) spectroscopic data of 1 revealed 40 carbon signals, comprising fteen quaternary carbons (one carbonyl, nine olenic, and one oxygenated group), seven methines (one oxygenated and three olenic ones), eight methylenes and ten methyls. The 1H NMR (Table 2) spectrum of 1 showed the presence of two isopropyl groups and six singlet methyls. The 13C and

DEPT NMR spectroscopic data showed four noticeable quaternary signals for abietane diterpenoid at dC 40.1 (s,

C-4), dC 46.7 (s, C-10), dC 33.8 (s, C-40), dC 39.3 (s, C-100) [19, 20]. These evidences indicated that compound 1 should be a dimer of two abietane diterpenoids units.

Analysis of the 1D and 2D NMR spectra distinguished two sets of diterpenoid signals, C-1C-20 and C-10C-200, respectively. According to the characteristic signals for normal abietane diterpenoids at dC 40.1 (s, C-4), dC 46.7 (s, C-10), dC 24.3 (q, C-18), dC 30.8 (q, C-19), dC 24.2 (q,

C-20), and an isopropyl group at dC 26.9 (d, C-15), dC21.5 (q, Me-16), and dC 21.0 (q, Me-17), the structure of unit 1 can be ascribed to be an abietane diterpenoid [21 23]. The HMBC correlations (Fig. 1) from Me-20 (dH 1.40) to C-1 (dC 31.7), C-5 (dC 80.2), C-9 (dC 124.7), and C-10; from Me-18 (dH 1.32) and Me-19 (dH 1.02) to

C-3 (dC 38.9), C-4, and C-5; from H-15 (dH 26.9) to C-12 (dC 181.8), C-13 (dC 141.3), and C-14 (dC 135.0); from H-14 (dH 6.55) to C-7 (dC 141.2), C-9, and C-12; and from H-6 (dH 5.42) to C-8 (dC 140.8), together with proton spin systems H-1/H-2/H-3 and H-6/H-7 obtained from the 1H1H COSY spectrum (Fig. 1), established the structure of the unit 1.

The other unit was also deduced to be an abietane diterpenoid based on the characteristic quaternary signals at dC 33.8 (s, C-40) and dC 39.3 (s, C-100), the typical methyls at dC 33.9, dC 22.2, dC 20.4 for Me-180, Me-190, and Me-200, respectively, and the isopropyal group at dC27.6 (C-150), dC 22.3 (Me-160), and dC 21.5 (Me-170). The planar structure of this unit was elucidated to be almost identical with that of the known analogue, dethdroabietane-11,12-diol [24], based on the comparative analysis of their NMR spectral data and the HMBC correlations from Me-200 (dH 1.43) to C-10 (dC 36.9), C-50 (dC 53.0), C-90 (dC 135.0), and C-100; from Me-180 (dH 0.95) and Me-190 (dH 0.95) to C-30, C-40, and C-50; from H-150 (dH 27.6) to C-120 (dC 133.8), C-130 (dC 132.8), and C-140 (dC 119.5);

from H-140 (dH 6.36) to C-70 (dC 32.4), C-90, and C-120; and from H-50 (dH 1.26) to C-40 and C-60 (dC 19.3), together with two proton spin systems, H-10/H-20/H-30 and H-60/H-70, observed from the 1H1H COSY spectrum (Fig. 1).

The two units account for 13 degrees of unsaturation. Since the totally degrees of unsaturation were 14, the remained one degree of unsaturation should be ascribed to the linkage between the two units through C-11/C-5/C-6 to C-110/C-120 to create an additional ring. The HMBC of

H-6/C-120 conrmed the linkage of C-6/C-120 through an

Table 1 13C NMR data for 1 and 2 in CDCl3 (100 MHz, d in ppm,

J in Hz)

Position 1 2

1 31.7, CH2 31.7, CH2 2 18.5, CH2 18.5, CH2 3 38.9, CH2 38.8, CH2 4 40.1, C 40.1, C5 80.2, C 82.7, C6 70.0, CH 69.8, CH 7 141.2, CH 140.9, CH 8 140.4, C 133.9, C9 124.7, C 124.5, C 10 46.7, C 46.7, C 11 144.1, C 144.2, C 12 181.8, C 181.8, C 13 141.3, C 141.5, C 14 135.0, CH 134.8, CH 15 26.9, CH 26.9, CH 16 21.5, CH3 21.7, CH3 17 21.0, CH3 21.5, CH3 18 24.3, CH3 24.2, CH3 19 30.8, CH3 30.9, CH3 20 24.2, CH3 24.3, CH3 10 36.9, CH2 36.8, CH2 20 19.4, CH2 19.3, CH2 30 41.5, CH2 40.8, CH2 40 33.8, C 33.3, C50 53.0, CH 51.6, CH 60 19.3, CH2 126.9, CH 70 32.4, CH2 128.0, CH 80 127.2, C 125.8, C90 135.0, C 132.7, C 100 39.3, C 41.3, C 110 141.6, C 143.1, C 120 133.8, C 139.5, C 130 132.8, C 132.9, C 140 119.5, CH 118.0, CH 150 27.6, CH 27.3, CH 160 22.3, CH3 22.5, CH3 170 21.5, CH3 22.2, CH3 180 33.9, CH3 33.0, CH3 190 22.2, CH3 20.9, CH3 200 20.4, CH3 18.3, CH3

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New Dimeric and seco-Abietane Diterpenoids 79

Table 2 1H NMR data for compounds 1 and 2 in CDCl3 (400 MHz, d in ppm, J in Hz)

No. 1 2

1 2.42, td (5.1, 16.1) 2.40, td (5.5, 16.9,)2.79, m 2.77, br.d (16.9)2 1.72, m 1.72, m3 1.86, m 1.87, td (4.9, 15.9)1.29, overlap 1.29, m6 5.42, d (3.4) 5.37, d (3.5)7 6.07, d (3.4) 5.98, d (3.5) 14 6.55, s 6.51, s15 2.95, sept (8.7) 2.95, sept (8.3) 16 1.03, d (8.7) 1.06, d (8.3) 17 1.07, d (8.7) 1.08, d (8.3) 18 1.32, s 1.32, s19 1.02, s 1.05, s20 1.40, s 1.39, s10 1.20, m 1.72, m3.11, br.d (16.3) 3.03, br.d (16.4) 20 1.54, m 1.77, m1.79, m 1.61, m 30 1.20, m 1.25, m1.45, m 1.47, br.d (17.3) 50 1.26, m 2.14, t (3.7)

60 1.79, m 5.82, dd (3.6, 11.9)1.54, m70 2.75, m 6.37, dd (3.8, 11.9)

140 6.36, s 6.41, s150 2.95, sept (8.5) 2.95, sept (8.6)

160 0.84, d (8.5) 0.86, d (8.6) 170 1.02, d (8.5) 1.04, d (8.6)

180 0.95, s 1.07, s 190 0.95, s 0.96, s 200 1.43, s 1.18, s

oxygen atom. In the 1H NMR spectrum of 1, an obvious OH signal can be found at dH 7.50, and this OH group can be deduced to be attached at C-11 as evidenced by its

HMBC correlations with C-9 (dC 124.7), C-11 (dC 144.1), and C-12 (dC 181.8). Then, the remained oxygenated quaternary at (C-5) and the oxygenated aromatic quaternary carbon (C-110) were deduced to linked through ether bridge.

In the NOESY spectrum (Fig. 1), diagnostic cross-peaks of H-6/Me-20, Me-19/H-6, and Me-19/Me-20 indicated the b-orientation of H-6, Me-19, and Me-20. The NOE correlations of Me-200/Me-190 and Me-180/H-50 indicated that

H-50 was a-oriented. In addition, the a-substitution of O-5 was suggested by analysis of the molecular model of 1, otherwise the NOE correlation of H-6/Me-19 should be unobservable.. Thus, the structure of 1 was elucidated and named salviwardin A.

The molecular formula of salviwardin B (2) was determined to be C40H52O4 from its 13C NMR and HRESIMS spectral data, indicating one more unsaturation than 1.

Comparison of their 1D and 2D NMR data indicated that the structures of 1 and 2 were similar to each other (Tables 1, 2). The difference lied in that the two methylenes (C-60 dC 19.3 and C-70 dC 32.4) in 1 were replaced by a double bond (C-60 dC 126.9 and C-70 dC 128.0) in 2, which indicated that 2 was a 60,70-dehydrogen derivative of 1. This was conrmed by HMBC correlations from H-60 (dH 5.82) to C-40 (dC 33.3), C-50 (dC 51.6) and

C-80 (dC 125.8), and from H-70 (dH 6.37) to C-50and C-140 (dC 118.0). By detailed analysis of its ROESY (Fig. 2) spectrum, the relative conguration of 2 was also elucidated to be the same as that of 1. Ultimately, the structure of 2 was determined and named salviwardin B.

Fig. 1 Key HMBC ( ), 1H-1H COSY ( ), and ROESY ( ) correlations of 1

Fig. 2 Key HMBC ( ) and 1H-1H COSY ( ) correlations of 2

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Table 3 1H and 13C NMR data for compound 3 in CDCl3

Position dCa dH (J in Hz)b Position dCa dH (J in Hz)b

1 26.0, CH2 3.10, td (3.9,13.8) 11 141.0, C3.41, dt (5.2,13.8) 12 143.7, C2 33.6, CH2 2.26, m 13 135.6, C3 86.5, CH 4.50, dd (7.2, 9.0) 14 119.6, CH 7.26, s4 145.5, C 15 28.0, CH 3.34, sept (6.9) 5 131.6, C 16 23.0, CH3 1.29, d (6.9)

6 127.1, CH 7.02, d (8.3) 17 22.4, CH3 1.33, d (6.9) 7 125.9, CH 7.42, d (8.3) 18 111.0, CH2 4.90, s8 128.2, C 5.10, s9 126.7, C 19 18.8, CH3 1.85, s10 132.3, C 20 20.3, CH3 2.42, s

a Recorded at 150 MHz

b Recorded at 600 MHz

Fig. 3 Key HMBC ( ) and 1H-1H COSY ( ) correlations of 3

Salviwardin C (3) was assigned the molecular formula C20H24O2 by analysis of its

13C NMR and HREIMS (m/z 296.1775, [M]?). Comparing of NMR spectroscopic data of 3 (Table 3) with that of naphth-[1,8-bc]-oxocin-11-ol,2,3,4,5tetrhydro-2,2,6-trimethyl-10-(1-methlethyl) indicated that the two compounds are similar to each other. The difference lied in that the C-3, C-4, and Me-18 in the known compound were replaced by an oxygenated methine at dC 86.5 (C-3) and a part of double-bond (C-4, dC 145.5 and C-18, dC 111.0) in 3 [25]. The HMBC correlations from H-3 (dH 4.50) to C-1 (dC 26.0), C-2 (dC 33.6), C-4 (dC 145.5), C-11 (dC 141.0), C-18 (dC 111.0) and C-19 (dC18.8), and from H-18 (dH 5.10 and 4.90) to C-3 (dC 86.5), C-4 (dC 145.5), C-19, together with the proton spin systems

H-1/H-2/H-3, determined the structure of A ring of 3 as shown in Fig. 3. Other parts of 3 were identical to those of the known compound by detailed analysis of the 1H1H COSY and HMBC correlations (Fig. 3). Therefore, the structure of 3 was determined and named salviwardin C.

All isolates were tested for their in vitro inhibitory activities against HL-60, SMMC-7721, A549, MCF-7, and SW480 human tumor cell lines using the MTT method described previously [26]. The results indicated that all the compounds were inactive with IC50 [ 30 lM.

3 Experiment Section

3.1 General Experimental Procedures

Optical rotations were obtained with a Jasco P-1020 polarimeter. UV spectra were measured on Shimadzu UV-2401A spectraphotometer. IR spectra were detected on a Bruker Tensor-27 infrared spectrophotometer with KBr pellets. 1D and 2D NMR spectra were recorded on Bruker

AV-400, and Avance III-600 MHz spectrometers with TMS as the internal standard. Chemical shifts (d) were expressed in ppm with reference to the solvent signals. HRESIMS analysis and HREIMS were determined on API QSTAR time-of-ight spectrometer and on Waters Auto spec Premier P776 mass spectrometer. Semi-preparative HPLC was performed on an Agilent 1100 liquid chromatography with a Zorbax SB-C18 (9.4 mm 9 25 cm) column. Column chromatography was performed on Sephadex LH-20 (GE Healthcare), Silica gel (100200 and 200300 mesh, Qingdao Marine Chemical Co., Ltd., Qingdao, China), and Amphichroic RP-18 gel (4063 lm,

Merck, Darmstadt, Germany) and MCI gel (75150 lm, Mitsubishi Chemical Corporation, Tokyo, Japan). Fractions were monitored by TLC and spots were visualized by heating silica gel plates sprayed with 10 % H2SO4 in

EtOH.

3.2 Plant Material

The roots parts of S. wardii were collected in Zuogong prefecture Tibet, China, in July 2011. The plant was

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New Dimeric and seco-Abietane Diterpenoids 81

identied by Dr. Yu-Kun Wei, Shanghai Chenshan Plant Science Research Center, Chinese Academy of Sciences. A voucher specimen was deposited in Kunming Institute of Botany,Chinese Academy of Sciences with identication number 20110712.

3.3 Extraction and Isolation

The roots parts of the air-dried S. wardii (33 kg) were powdered and percolated with acetone at room temperature and ltered. The ltrate was evaporated in vacuo to be concentrated. The crude extract (1.6 kg) was subjected to silica gel column chromatography eluted with CHCl3,

EtoAc and MeOH, respectively. The fraction CHCl3 with petroleum-CHCl3-EtoAc gradient (50:1:1, 20:1:1, 10:1:1, and 5:1:1) to produce seven fractions, AG. Fraction B (223 g) was separated over a MCI gel column (MeOH-H2O

from 70:30 to 100:0) to obtain eight fractions (Fr. B1B8). Fr. B1 (12 g) was isolated over an RP-18 gel column (MeOHH2O from 85:15 to 100:0) to obtain seven fractions (Fr. B2aB2g). Fr. B2a (2 g) was separated on a silica gel column, eluted with petroleum ether-acetone (from 100:1 to 50:1), to yield six fractions (B2a1B2a6). Fr. B2a2 was puried by repeated silica gel columns and semipreparative HPLC (RP-18, 98 % MeCN-H2O) and

TLC to afford 1 (80 mg), 2 (13 mg). Fr. B3 (27 g) was separated over a MCI-gel column (MeOH-H2O from 85:15 to 100:0) to obtain six fractions (Fr. B3aB3f). Fr. B3e (5 g) was then chromatographed on a silica gel column, eluted with petroleum ether-acetone (from 50:1 to 5:1), to yield eight fractions (Fr. B3e1B3e8). Subfraction B3e1 (110 mg) was chromatographed by semipreparative HPLC (89 % MeOH-H2O) to afford two fractions (Fr. B3e1a

Fr. B3e1b). Fr. B3e1a (15 mg) was chromatographed by semipreparative HPLC (92 % MeCN-H2O) to afford 3 (10 mg). Subfraction B3e1 (1.7 g) was chromatographed by semipreparative HPLC (83 % MeOH-H2O) to afford two fractions (Fr. B3e2aFr. B3e1f). Fr. B3e1a (15 mg)

was chromatographed by semipreparative HPLC (92 % MeCN-H2O) and chromatographed on a silica gel column, eluted with petroleum ether-acetone (100:1) to afford prionitin (4, 100 mg). Fr. B3f (3.3 g) was then chromatographed on a silica gel column, eluted with petroleum ether-acetone (from 50:1 to 5:1), to yield eight fractions (Fr. B3f1B3f7). Fr. B3f1 (726 mg) was then chromatographed on a silica gel column, eluted with petroleum ether-acetone (50:1), to yield eight fractions (Fr. B3f1a B3f1f). Fr. Bf1a, Fr. Bf1c, and Fr. Bf1f were puried by semipreparative TLC and chromatographed by semi-preparative HPLC (92 % MeCN-H2O) to yield sahandol (5, 49 mg), salvilenone (6, 10 mg), microstegiol (7, 6 mg), and ferruginol (8, 120 mg).

3.4 Salviwardin A (1)

Orange powder; a 16D ? 325 (c 0.17, CDCl3); UV (CDCl3)

kmax (log e) 374.5 (3.68) nm; IR (KBr) mmax 3441, 2939, 1624, 1476, 1417, 1364, 1296, 1134, 1121, 1011 cm-1; 1H

and 13C NMR data, see Tables 1 and 2; positive ESIMS m/z 599 [M ? H]?; positive HREIMS m/z 598.4029 (calcd for C40H54O4 [M]?, 598.4022).

3.5 Salviwardin B (2)

Orange powder; a 20D ? 184 (c 0.19, MeOH); UV (MeOH)

kmax (log e) 476 (3.25) nm; IR (KBr) mmax 3442, 2927, 2870, 1626, 1579, 1467, 1394, 1291, 1172, 1088, 1008 cm-1; 1H and 13C NMR data, see Tables 1 and 2; positive ESIMS m/z 619 [M ? Na]?; positive HRESIMS m/z 597.3941 (calcd for C40H53O4 [M ? H]?, 597.3944).

3.6 Salviwardin C (3)

Colorless oil; a 26D -2 (c 0.26, CDCl3); UV (CDCl3) kmax

(log e) 336.5 (3.35) nm; IR (KBr) mmax 3430, 2927, 2871, 1722, 1633, 1423, 1330, 1271, 1172, 1023 cm-1; 1H and

13C NMR data see Table 3; positive EIMS m/z 296 ([M]?,77), 268 (26), 267 (94), 265 (100), 255 (18), 241 (35), 213(54); positive HREIMS m/z 296.1775 (calcd for C20H24O2

[M]?, 296.1776).

3.7 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, USA). All cells were cultured in RPMI-1640 or DMEM medium (Hyclone, Logan, UT, USA), supplemented with 10 % fetal bovine serum (FBS, Hyclone) at 37 C in a humidied 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-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Sigma, St. Louis, MO, USA). Briey, 100 lL of adherent cells was seeded into each well of a 96-well cell culture plate and allowed to adhere for 12 h before test compound addition, while suspended cells were seeded just before test compound addition, both with an initial density of 1 9 105 cells/mL in 100 lL of medium. Each tumor cell line was exposed to the test compound at various concentrations in triplicate for 48 h, with cis-platin and paclitaxel (Sigma) as positive control. After the incubation, MTT (100 lg) was added to each well, and the incubation continued for 4 h at 37 C. The cells were lysed with 100 lL of 20 % SDS -50 % DMF after removal of

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100 lL of medium. The optical density of the lysate was measured at 595 nm in a 96-well microtiter plate reader (Bio-Rad 680). The IC50 value of each compound was calculated by Reed and Muenchs method [25].

Acknowledgments The work was nancially supported by the foundations from NSFC (81373291), the National Science and Technology Support Program of China (2013BAI11B02), the Young Academic Leader Raising Foundation of Yunnan Province (No. 2009CI073), and the foundation from CAS to Dr. G. Xu.

Conict of Interest All authors declare no conict of interest.

Open Access This article is distributed under the terms of the Creative Commons Attribution License which permits any use, distribution, and reproduction in any medium, provided the original author(s) and the source are credited.

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