Fitoterapia 93 (2014) 39–46

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New clerodane diterpenoid glycosides from the aerial parts of Nannoglottis carpesioides Xian-Hua Meng a, Chuan-Zong Zou b, Xiao-Jie Jin a, Guo-Du Huang a, Yong-Jin Yang a, Xi-Ying Zou a, Xiao-Jun Yao a, Ying Zhu a,⁎ a b

State Key Laboratory of Applied Organic Chemistry, College of Chemistry and Chemical Engineering, Lanzhou University, Lanzhou 730000, PR China Department of Chinese Medicine, Pingliang Medical College, Pingliang 744000, PR China

a r t i c l e

i n f o

Article history: Received 11 September 2013 Accepted in revised form 12 December 2013 Available online 22 December 2013 Keywords: Nannoglottis carpesioides Diterpenoid α-L-arabinose Absolute configurations Markers of chemotaxonomy Chemical compounds studied in the article: (E)-Phytol epoxide (CID: 468705) α-Tocopherol-quinone (CID: 24205) 7,11,15-Trimethyl-3methylidenehexadecane-1,2-diol (CID: 10638889) (23Z)-En-cyclolanlst-3β,25-diol (CID: 10049027) (24R)-Cycloartane-3β,24,25-triol (CID: 5270670) Oleanolic aldehyde (CID: 14423521) Oleanic acid (CID: 10494) Friedelin (CID: 91472)

a b s t r a c t Three new clerodane diterpenoid glycosides with L-arabinose (1–3), together with ten known compounds including phytol-type diterpenes, cycloartane-type, ursane-type, and oleanane-type triterpenes, were isolated from the aerial parts of Nannoglottis carpesioides which a Chinese endemic genus. The structures of the new compounds 1–3 were identified based on chemical and spectroscopic studies, including one- and two-dimensional NMR, HRESIMS, UV, and IR results. Their absolute configurations were determined by the application of theory calculations of optical rotation, which were compared with the experimental data. New aglycone 1a and L-arabinose were obtained by acid hydrolysis of 1 and GC–MS analysis. The cytotoxicities of some isolated compounds against a panel of human cancer cell lines were evaluated by the MTT assay. Clerodane diterpenoides are the characteristic chemical constituents and may be used as chemical markers of the genus Nannoglottis. © 2013 Elsevier B.V. All rights reserved.

1. Introduction The genus Nannoglottis belongs to family Compositae (or Asteraceae), which is a Chinese endemic genus. It contains nine species, mainly growing in the mountain areas of Himalayas and contiguous zone of altitudes 2400–4200 m [1]. So far plant

⁎ Corresponding author. Tel.: +86 13909445175; fax: +86 931 8912582. E-mail address: [email protected] (Y. Zhu). 0367-326X/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.fitote.2013.12.012

taxonomists made studies on the plant taxonomy of Nannoglottis, which was thought to be most closely related to the tribe Astereae [1,2]. The chemical constituents of only two species from the genus Nannoglottis were studied. Two new clerodane diterpenoids and a new dicaffeoyl quinic acid were isolated from the roots of Nannoglottis ravida [3,4]. We previously reported seven new triterpenes and two new neolignans from the roots of Nannoglottis carpesioides (Maxim.) [5]. Our interest on the chemistry of N. carpesioides and for supply useful evidence for chemotaxonomy on Nannoglottis

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8R,9S,10R, 13S)-2-hydroperoxy-13-O-α-L-arabinopyranosyl-3,14 -clerodadiene (3) by comparison of experimental and theoretical calculated optical rotations. Compounds 7–10 were assessed for their cytotoxicity against human promyelocytic leukaemia (HL-60) and human hepatoma (Hep-G2) cell lines using the MTT assay. Compounds 1–3 are first isolated from the Compositae family as clerodane diterpenes with L-arabinoside located at C-13.

prompts us to further investigate the chemical constituents from the aerial parts of this plant. In this paper, we report the isolation and structure elucidation of three new clerodane diterpenoid glycosides (1–3) which possess the same L-arabinose but different aglycones, as well as ten known compounds including three phytol-type diterpenes (4–6), seven triterpenes (7–13): cycloartane-type (7–9), ursane-type (10–11), oleanane-type (12), and A-frideooleanane-type triterpenes (13) (Fig. 1). Their structures were elucidated by means of chemical and extensive spectroscopic methods, including HRESIMS and, 1D- and 2D-NMR. New aglycone 1a and L-arabinose were identified by acid hydrolysis of 1, GC–MS analysis, and spectroscopic methods. The absolute configurations of compounds 1–3 were determined as (2S,5S,8R, 9S,10R,13S)-2-oxo-13-Oα-L-arabinopyranosyl-3,14-clerodadiene (1), (2S,5S,8R,9S,10R, 13S)-13-O-α-L-arabinopyranosyl-3,14-clerodadiene (2), (2S,5S,

2. Experimental 2.1. General Optical rotations were obtained using a Perkin-Elmer-341 polarimeter in CHCl3. IR Spectra were obtained with a Nicolet NEXUS-670 FT-IR spectrometer. UV spectra were collected in

OH O 5' O

OH

3'

13 14 15 20 16 9 10 8 17

2

1 5 18

H

O

H

R1 R2

OH

1' HO

6

R1 R2 1 =O H 2 H 3 OOH H

19

1a

R2 R OH

H

4 R=

OH

O

5 R=

O

R1O

7 R1 = R2 = H, Δ23(Z)

OH O

6 R=

8 R1 = COC13H27 R2 = OH 9 R1 = H R2 = OH

OH OH

H

R1

H

R3

H

H

COOH

H

H R2

H H

HO

10 R1 = R2 = OH R3 = CH3 11 R1 = H R2 = OH R3 = CHO

O H

12

Fig. 1. Compounds 1–13 from Nannoglottis carpesioides.

13

H

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MeOH using a Shimadzu UV-260 spectrometer. 1D and 2D NMR spectra were recorded using a Varian Mercury plus-300 spectrometer at 300 MHz for 1H NMR and 75 MHz for 13C NMR, a Bruker 400 spectrometer at 400 MHz for 1H NMR and 100 MHz for 13C NMR. The chemical shifts are given as δ values in ppm, and J values are reported in Hz with TMS as the internal standard. EIMS were measured on a HP-5988A GC–MS instrument at 70 eV, in m/z. HRESIMS spectra were recorded on a Bruker Daltonics APEX-II mass spectrometer. GC were measured on a Trace DSQ single quadrupole GC–MS using a DB-1701 column (30 m × 0.25 mm × 0.25 μm) with He as carrier gas. Column chromatography was carried out on silica gel (200–300 mesh and Type 60) and TLC was performed on silica gel (GF254, 10–40 μm), with both materials were supplied by Qingdao Marine Chemical Co. Spots were detected on TLC under UV light or by heating after spraying with 5% H2SO4 in C2H5OH (v/v). 2.2. Plant material The aerial parts of N. carpesioides were collected on The North Mountains of Huzhu County in Qinghai province, People’s Republic of China, in August 2006. The plants were taxonomically identified by Prof. Guo-liang Zhang, Department of Life Science, Lanzhou University. A voucher specimen (No. Nc20060801) has been deposited in the State Key Laboratory of Applied Organic Chemistry, Lanzhou University. 2.3. Extraction and isolation Air-dried aerial parts of N. carpesioides (7.0 kg) were powdered and extracted with the petroleum ether (PE)/ Et2O/MeOH for five times [5 × (17.0:17.0:17.0) L] at 25 °C. The extracts were concentrated to dryness and yielded dried residues (340 g) under reduced pressure, which were suspended in water and partitioned sequentially with petrol ether and CHCl3, respectively. The PE-soluble extract was concentrated to give residue (209 g), which was isolated difficultly someday because of containing a lot of wax in it. The PE-soluble extract was subjected to silica gel column chromatography (CC) using a step gradient-elution technique with mixtures of PE/acetone (from 30:1 to 0:1), and finally washed with MeOH, to afford 10 fractions (Fr.1–Fr10). Fraction Fr2 (23.0 g) was subjected to silica gel CC eluted with PE/acetone (10:1), followed by PE/EtOAc (10:1) to give 4 (20 mg); by CHCl3/EtOAc (100:1), CHCl3/acetone (50:1) to give 5 (10 mg), 6 (2 mg), and 10 (3 mg). Fr4 (8.0 g) was subjected to silica gel CC eluted with CHCl3, followed by CHCl3/acetone (50:1) and PE/acetone (20:1) to give 7 (3 mg), 8 (3 mg), 9 (3 mg), and 11 (3 mg). Fr5 (10.0 g) was subjected to CC (silica gel) eluted with CHCl3/EtOAc (40:1), followed by CHCl3/acetone (50:1), PE/acetone (5:1), to yield 12 (2 mg). In order to further obtain glycosides, another portion of the air dried aerial parts of N. carpesioides (4.2 kg) were powdered and extracted with the MeOH for three times (3 × 38.0 L) at 25 °C. The residue (607 g) was obtained after removing the solvent under reduced pressure. The residue was suspended in water (2.5 L) and followed by successive partition with PE (4 × 2.5 L), CHCl3 (4 × 2.0 L), EtOAc (4 × 2.0 L), and n-BuOH (4 × 2.0 L), respectively. The EtOAc– soluble extract was concentrated to give residue (60 g). The residue was subjected to a silica gel CC (silica gel, 700 g)

41

gradient elution with PE/acetone (from 1:0 to 0:1)) and finally washed with MeOH to afford ten fractions, A1–A10. Fraction A1 (1.5 g) was subjected to a silica gel CC eluted with CHCl3, followed by PE/acetone (20:1), to yield 13 (30 mg). A3 (1.2 g) was subjected to silica gel CC eluted with CHCl3/MeOH (30:1), followed by PE/acetone (2:1), to give 2 (10 mg). A7 (2.5 g) was subjected to a silica gel CC eluted with CHCl3/MeOH (20:1), then CHCl3/acetone (3:1), then PE/acetone (2:1), followed by CHCl3/MeOH (15:1), to yield 1 (10 mg) and 3 (15 mg).

2.4. Characteristic data of compounds Compound 1: Colourless oil. [α]20 D +21 (c 0.10, MeOH); UV (MeOH) λmax nm (logε): 202 (0.123), 248 (0.057); IR (film) λmax cm−1: 3397, 2957, 2923, 1711, 1645, 1575, 1410, 1079, 1002, 945, 651; HRESIMS m/z: 437.2901 [M + H]+ (Calcd for 1 13 C NMR (DEPT) see Table 1. C25H41O+ 6 , 437.2898); H- and Compound 2: Colourless oil. [α]20 D +22 (c 0.10, MeOH); IR (film) λmax cm−1: 3403, 2948, 1645, 1445, 1413, 1370, 1079, 1002, 944, 755; HRESIMS m/z: 440.3367 [M + NH4]+ 1 13 (Calcd for C25H46NO+ C NMR (DEPT) 5 , 440.3371); H- and see Table 1. Compound 3: Colourless oil. [α]20 D –7 (c 0.10, MeOH); IR (film) λmax cm−1: 3398, 2924, 1645, 1446, 1413, 1372, 1078, 1002; HRESIMS m/z: 472.3268 [M + NH4]+ (Calcd 1 13 C NMR (DEPT) see for C25H46NO+ 7 , 472.3269); H- and Table 1. 2.5. Acid Hydrolysis and GC–MS analysis Compound 1 (ca. 10 mg) in 5 ml of 2 N CF3COOH was stirred for 4.5 h at 95 °C (reflux). On cooling, the reaction mixture was extracted with CHCl3 three times to afford an aqueous phase and organic layer. The aqueous phase was concentrated under reduced pressure to afford a sugar fraction. Subsequently, the sugar fraction of 1 was dissolved in pyridine (0.5 ml) and stirred with D-cysteine methyl ester hydrochloride (0.08 M) in pyridine (0.6 ml) before 1-trimethylsily-limidazole (0.4 ml) was added, and the mixture was kept at 60 °C for 2 h using the same procedures as in a previous report [6]. The mixture was partitioned between n-hexane and H2O (0.5 ml each). The n-hexane fraction was directly subjected to GC–MS analysis using a DB-1701 column (30 m × 0.25 mm × 0.25 μm) with He as carrier gas. The temperatures of the transfer line and source were 280 and 250 °C, respectively. A temperature gradient system was used for the oven, starting at 80 °C for 1 min and increasing up to 280 °C, at a rate of 20 °C/min. The L-arabinose derivatives was detected by co-injection of the hydrolysate with standard silylated sugars which gave retention times at 12.88 for the standard D-arabinose and 12.82 min, respectively, in GC–MS. The organic layer of 1 was dried completely under reduced pressure and chromatographed on a silica gel column, eluted with a mixture of CHCl3 and MeOH (10:1), to give new aglycone 1a, which was identified as 10βH-13-hydroxy-2-oxo-17α,19β,20α-trimethyl-cleroda-3,14 -diene by 1H NMR (Table 1) and EIMS data. Compound 1a: [α]20 D +2.0 (c 0.5, MeOH); EIMS (probe, 70 eV) m/z (rel. int.): 286 [M–H2O]+ (7), 205 (40), 178 (20), 149 (100), 135 (31),

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Table 1 NMR Data of compounds 1–3 (CDCl3, δ in ppm, J in Hz)a. No.

1b 1

H

1α 1β 2α 2β 3 4 5 6α 6β 7α 7β 8β 9 10β 11α 11β 12α 12β 13 14 15a 15b 16 17α 18 19β 20α Ara-1′α Ara-2′β Ara-3′α Ara-4′α Ara-5′α Ara-5′β OOH a

2.70 dd (18.4, 6.4) 2.55 brd (18.4) – – 5.83 s – – 1.13 brd (11.6) 2.08 dd (14.0, 3.2) 1.19 brd (12.8) 1.32 brdd (12.0, 6.8) 1.44 brd (11.6) – 1.86 brd (6.4) 1.23 brd (9.2) 1.19 brd (12.8) 1.43–1.47 m 1.43–1.47 m – 5.77 dd (17.6, 11.2) 5.27 brd (10.0) 5.20 brd (17.6) 1.36 s 0.75 d (6.4) 1.94 s 1.22 s 0.56 s 4.35 d (5.2) 3.68 brd (8.4) 3.66 brd (6.0) 3.91 brs 3.50 brd (12.8) 3.93 brd (11.6)

2c 13

C

35.4 200.1 128.3 169.3 39.3 36.8 28.2 36.6 39.9 46.9 30.0 35.1 80.6 141.9 116.6 22.8 15.8 20.6 32.1 19.3 98.0 71.7 73.2 68.0 65.1

1

H

1.94–1.96 m 1.72 brd (7.5) 2.04–2.07 m 1.94–1.96 m 5.24 brd (9.9) – – 0.98–1.03 m 1.97 brd (12.9) 1.07 dd (12.3, 3.6) 1.22 brd (14.1) 1.36 brdd (11.1, 8.1) – 1.29 brd (5.4) 1.40 d (14.1, 7.2) 1.15 brd (14.1) 1.44–1.47 m 1.44–1.47 m – 5.76 dd (17.4, 11.1) 5.22 brd (10.8) 5.18 brd (17.4) 1.34 s 0.73 d (6.6) 1.65 s 1.01 s 0.78 s 4.33 d (6.0) 3.66 dd (8.7, 7s.5) 3.63 brd (7.5) 3.90 brd (2.4) 3.46 brd (12.9) 3.89 brd (7.5)

3b 13

C

17.6 24.0 123.1 139.8 36.8 37.7 28.7 37.3 39.8 44.3 31.4 34.5 81.4 142.3 116.3 22.0 15.9 19.7 33.0 17.4 97.9 71.4 73.0 68.0 65.0

1

H

2.24 dd (14.4, 8.4) 2.05 dd (14.4, 7.2) 4.69 brs – 5.42 s – – 1.17 brd (12.0) 1.99 brd (12.8) 1.01–1.11 m 1.19–1.27 m 1.37 brs – 1.62 brd (6.4) 1.24–1.27 m 1.17 brd (12.0) 1.32–1.42 m 1.53–1.56 m – 5.83dd (17.6, 11.2) 5.23 brd (11.6) 5.19 brd (18.0) 1.37 s 0.72 d (6.4) 1.73 s 1.13 s 0.68 s 4.35 d (4.4) 3.71 dd (8.0, 4.4) 3.65 d (8.0) 3.95 brs 3.49 brd (12.8) 3.94 brd (10.4) 9.13 s

1ab 13

C

24.2 80.1 120.8 148.7 37.7 37.4 28.5 37.4 39.4 47.9 31.0 35.0 80.8 142.1 115.9 23.6 16.0 20.1 33.3 18.8 98.2 71.6 73.3 68.3

1

H

2.70 d (6.8) 2.65 d (6.4) – – 5.87 s – – 1.12–1.16 m 2.15 dd (11.2, 4.8) 1.12–1.16 m 1.26–1.35 m 1.45 brd (11.2) – 1.88 brd (7.2) 1.20–1.35 m 1.18 brd (5.2) 1.46–1.50 m 1.46–1.50 m – 6.46 dd (16.0) 6.05 brd (16.4) 5.87 d (4.4) 1.25 s 0.70 s 1.97 d (1.2) 1.25 s 0.76 s

65.4

Assignments made by 1H–1H COSY, HSQC, HMBC, and 1D-NOE experiments. H NMR Recorded at 400 MHz, 13C NMR Recorded at 100 MHz. 1 H NMR Recorded at 300 MHz, 13C NMR Recorded at 75 MHz.

b 1 c

121 (33), 109 (34), 107 (30), 74 (43), 55 (40), 43 (65), 41 (47); 1 H NMR see Table 1.

2.6. Cytotoxicity assay Cytotoxic cytotoxicities of 7–10 were determined with the 3-(4,5-dimethyl-2-thiazoyl)-2, 5-diphenyl-2H-tetrazolium bromide (MTT) method [7]. Promyelocytic leukaemia (HL-60) and hepatoma (Hep-G2) cell lines were cultured in RPMI 1640 medium containing 10% heat inactivated foetal bovine serum supplemented with 100 units/ml penicillin, 100 μg/ml streptomycin at 37 °C, and 5% CO2. Cultures for the assay were grown in 96-well microtitre plates. All tested compounds were dissolved in DMSO. After 24 h of incubation, the compounds underwent serial dilution to give final concentrations of 0–200 μg/ml. Mitomycin was used as a positive control (Sigma, Saint Louis, MO, USA). All experiments involving these samples were replicated three times. The activities were recorded as the IC50 values (the concentration of the tested compound (μM) that inhibits cell growth by 50%).

2.7. Calculations In theoretical calculations, the geometries of the molecules were optimized with Gaussian 09 package at B3LYP/6-31G(d) computational level [8]. The minimum nature of the structure was confirmed by frequency calculations at the same computational level. These geometries were then used to evaluate the optical rotation at B3LYP/6-31G(d) computational level. All optical rotation calculations were performed using PCM solvation model and the MeOH was specified as the solvent which was in agreement with the experimental condition. 3. Results and discussion Compound 1 was obtained as colourless oil. Its molecular formula was determined to be C25H40O6 with six double bond equivalents based on a pseudo-molecular-ion peak observed at m/z 437.2901[M + H]+ (calculated 437.2898) in HRESIMS. The IR spectrum of 1 showed absorption bands at 3397 (OH), 1711 (C_O), and 1645 (C_C) cm−1. The 13C NMR and DEPT spectra for 1 (Table 1) indicated 25 carbon signals including five

X.-H. Meng et al. / Fitoterapia 93 (2014) 39–46

methyls, seven methylenes, eight methines, and five quaternary carbons. The 13C NMR spectrum indicated four olefinic carbons (δC 128.3 d, 169.3 s, 141.9 d, 116.6 t) for a vinyl group and a trisubstituted double bond, a oxygen-bearing quaternary carbon (δC 73.2), and a glycosyl moiety. The 1H NMR spectrum (Table 1) of 1 indicated four olefinic protons (δH 5.83 s, 5.77 dd, 5.27 dd, 5.20 dd) corresponding to a trisubstituted double bond and a vinyl proton an olefinic methyl (δH 1.94), a secondary methyl, three tertiary methyls, and a glycosyl moiety. Besides showed signals of a glycosyl moiety, the NMR data of the remaining 20 carbon resonances suggested that 1 was a clerodane diterpene monoglycoside, which confirmed by a detailed analysis of the 1H–1H COSY and HMBC spectra (Fig. 2). The key HMBC correlations of H-1 at δH 2.55 and 2.70 to C-2, C-5, C-9, and C-10; H-3 to C-1, C-5, and C-18; H3-19 to C-4, C-5, C-6, and C-10, revealed the presence of a cyclohexene ring, in which a ketone group was attached to C-2 and Me-19 to C-5, respectively. HMBC correlations of H-14 at δH 5.77 to C-16 and C-13; H2-15 at δH 5.27 and 5.20 to C-13 and C-14 at δC 141.9, placed a six-carbon side chain with a terminal ethylene unit to C-9. The sugar residue was placed at C-13 by HMBC correlation between C-13 (δC 80.6) and the anomeric proton H-1′ (δH 4.35). The relative stereochemistry of the aglycone for 1 was identical on the basis of the characteristic chemical shifts and NOE correlations of those protons. The 1H- and 13C NMR data (Table 1) of the aglycone of 1 were similar to that of (−)-13-epi-2-oxokolavelool [9]. The major difference between 1 and (−)-13-epi-2-oxokolavelool was the stereochemistry of Me-19. Comparison with the chemical shifts of H-19 and C-19 for the α-orientation Me-19 at δH 1.06 and δC 18.2 resonances reported in the literature [9], the characteristic chemical shifts of the angular Me-19 group at δH 1.22 and δC 32.1 in 1 were shifted to relatively downfield positions. The above signals for 1 revealed the cis-stereochemistry at the junction of the rings A and B [9,10], which were in agreement with those of reported 5β,10β-cis-clerodane analogues [11–13]. Furthermore, the presence of NOE interactions supported the cis-configuration assignment. In NOE experiment, irradiation of H-6 at δH 2.08 led to enhancements of H-18 (+3.35%) and H3-19 at δH 1.22 (+18.33%); irradiation of H3-19 led to enhancements of H-8 (+1.36%) and H-10 at δH 1.86 (+1.47%); irradiation of H3-20 at δH 0.56 led to enhancements of H-11 at δH 1.23 (+7.30%) and H3-17 at δH 0.75 (+4.45%); irradiation of H3-16 at δH 1.36 led to enhancements of H3-17 (+0.63%) and the anomeric proton H-1′ at δH 4.35 (+1.59%).

Fig. 2. Key 1H–1H COSY and HMBC correlations for 1.

43

These data indicated that H3-17, H3-20, H3-16, and H-1′ were in an α-orientation, and H-10 and H3-19 were in a β-orientation. The remaining resonances observed in the 1H- and 13C NMR spectra of 1 were attributable to a glycosyl unit, which showed signals at δH 4.35 (d, J = 5.2 Hz, H-1′), 3.68 (brd, J = 8.4 Hz, H-2′), 3.66 (brd, J = 6.0 Hz, H-3′), 3.91 (brs, H-4′), 3.93 (brd, J = 11.6 Hz, H-5′β), and 3.50 (brd, J = 12.8, H-5′α); δC 98.0 (CH, C-1′), 71.7 (CH, C-2′), 73.2 (CH, C-3′), 68.0 (CH, C-4′), and 65.1 (CH2, C-5′). 1H–1H COSY and HMBC spectra revealed correlations of glycosyl unit (Fig. 2). The 13C NMR spectrum of the sugar moiety was similar to the published spectra of the α-L-arabinose moiety [14,15]. In the 1H NMR spectrum, large coupling constants between H-2′ and H-3′, J = 8.4 Hz, indicated that both were in axial positions. H-4′ was positioned equatorially on the basis of the extremely small coupling among H-4′, H-3′, and H-5′ (J b 1 Hz). In NOE experiments, irradiation of H-1′led to enhancements of H-3′at δH 3.66 (+ 3.33%) and H-5′at δH 3.50 (+ 3.54%); irradiation of H-4′at δH 3.91 led to enhancements of H-3′ (+3.52%) and H-5′ at δH 3.50 (+13.45%). Though the side chain is flexible, H3-17, H3-20, H3-16, and H-1′ of the anomeric proton were assigned as an α-orientation by NOE experiments, H-3′, H-4′ and H-5′ at δH 3.50 were in αorientation. In summation, due to the axial protons at C-1′, C-2′, and C-3′ and equatorial protons at C-4′, the glycone unit was determined to be an α-configuration for the L-arabinose moiety. In order to further confirm the absolute configuration of the arabinose moiety, compound 1 was first hydrolysed, then derivatized by a method described previously, and analysed by GC–MS [6]. The L-arabinose derivative was detected by co-injection of the hydrolysate with standard silylated sugars which gave retention times at 12.88 min for the standard D-arabinose and 12.82 min, respectively, in GC–MS. Consequently, the relative stereochemistry of 1 was elucidated as 10βH-13-O-α-L-arabinopyranosyl-2-oxo-17α,19β, 20α-trimethyl-3,14-clerodadiene. Theory calculation of spectroscopic properties of natural products has been proved to be a powerful tool for the determination of their structures and absolute configurations. In this work, the absolute configuration of 1 was determined by comparison of experimental and theoretical calculated optical rotations [8,16]. Due to the presence of nine stereogenic centres, one of the 81 stereoisomers represents the actual configuration of 1. Considering the relative configuration determined by the NOE date, the specific optical rotations of four selected possible isomers for 1, (5S,8R,9S,10R,13S)-1(1a), (5R,8R,9S,10S,13S)-1 (1b), (5S,8R,9S,10R,13R)-1 (1c), and (5R,8R,9S, 10S,13S)-1 (1d), respectively, have been calculated at the B3LYP/6-31++G (d,p) level. Four selected possible configurations were focused on the stereochemistry of C-5, C-10, and C-13. The specific optical rotations were compared with the experimental data of 1 in MeOH at 589.3 nm of the sodium D line, with the results given in Fig. 3, where the experimental specific rotation for 1 was also listed. In the case of 1, calculated [α]D values for 1a is +26.49, which is consistent with the experimental value of [α]D +21.0 of 1. Though the side chain is flexible, the calculated rotation values (1c +186.6) showed larger difference due to the opposite stereochemistry of C-13. The differences of experimental and calculated values, Δ[α]D = [α]D(calc) − [α]D (expt) for the 5S,8R,9S,5R,10R,13S (1a) and 5S,8R,9S,5R,10R, 13R (1c) isomers, were +4.49 and +165.6, respectively. The

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Fig. 3. Experimental and calculated optical rotations of selected possible isomers for 1 and 3.

calculated [α]D for the 5S,8R,9S,5R,10R,13S is much closer to the experimental [α]D for naturally occurring 1. Therefore, the absolute configuration of 1 was deduced to be 5S,8R,9S,5R,10R, 13S and their stereo-structure was as shown in Fig. 1. By comparison to their NMR spectra (Table 1), compounds 1–3 possess the same glycosyl unit but different aglycones. Compound 2 was obtained as colourless oil. Its molecular formula was determined to be C25H42O5 with five double bond equivalents based on a pseudo-molecular-ion peak at m/z 440.3367 [M + NH4]+ (calculated 440.3371) in HRESIMS. The IR spectrum showed absorption bands at 3403 (OH) and 1645 (C_C) cm−1. The 1H and 13C NMR spectra (Table 1) of 2 were similar to that of 1. The main difference was that the carbonyl carbon at δC 200.1 (C-2) was absent. The appearance of a CH2 at C-2 signals at δC 24.0 was in 2, which were confirmed by HMBC correlations from H-10 at δH1.29 to C-1, C-2, C-5, and C-9, and 1H–1H COSY correlations from H-1 to H-2. The 1H and 13C NMR data of the aglycone of 2 were very similar to that of cis-3, 14-clerodadien-13-ol [10,11]. In NOE experiments, irradiation of H3-19 at δH 1.01 led to enhancements of H-6β (+5.24%), H-10β (+1.46%), H-18 (+1.43%); irradiation of H-1 at δH 1.94–1.96 led to enhancements of H-7 at δH 1.22 (+3.58%), H-11 at δH 1.15 (+7.93%), H-18 (+6.94%); irradiation of H-1 at δH 1.72 led to enhancements of H-10 (+1.98%) and H3-19 (+4.97%); irradiation of H3-19 led to enhancements of H-6 at δH 1.97 (+5.24%) and H-10 (+1.46%). These data indicated that H-10 and H3-19 were in a β-orientation. Therefore, the relative stereochemistry of 2 was elucidated

as 10βH-13-O-α-L-arabinopyranosyl-17α,19β, 20α-trimethyl3,14-clerodadiene. Compounds 2 and 1 possess same stereogenic centres. On comparison with 1, the absolute configuration of 2 was deduced to be 5S,8R,9S,5R,10R,13S. Compound 3 was obtained as colourless oil. Its molecular formula was determined to be C25H42O7 with five double bond equivalents based on a pseudo-molecular-ion peak at m/z 472.3268 [M + NH4]+ (calculated 472.3269) in HRESIMS, which is 18 mass units more than 1. The 1H and 13 C NMR data of 3 were similar to that of 1 (Table 1). The main difference was the presence of a peroxide function linked to C-2, with characteristic signals at δH 4.69 (brs, H-2), 9.13 [brs, H-(OOH)], and δC 80.1 (C-2). These characteristic signals were in agreement with that of the analogue of the clerodane diterpenes with a peroxide function at C-2, (−)-2β-hydroperoxy-kolavelool (δH 4.40 for H-2; 10.19 for H-(OOH); and δC 79.2 for C-2) [9], (2S,5R,8R,9S,10R)-2-hydroperoxy-ent-3-cleroden-l5-oic acid methyl ester (δH 4.28 for H-2 and 9.10 for H-(OOH)) [12], reported in the literature. Comparison with that of the reported clerodane diterpene with a OH group at C-2, (−)-2βhydroxykolavelool (δH 4.15 for H-2 and δC 65.6 for C-2) [9], the chemical shifts of the H-2 and C-2 for 3 were obviously shifted to the downfield, which confirmed the presence of the peroxide function at C-2. The location of 2-OOH group was further confirmed by HMBC correlations from H-1 at δH 2.24 to C-2 (δC 80.1) and C-5; H-1 at δH 2.05 to C-2, C-9, and C-10; H-10 at δH 1.62 to C-1, C-2, C-4, C-5, C-9, C-19, and C-20, and by 1 H–1H COSY correlations from H-2 to H-1, H-3, and H-18.

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In NOE experiments, irradiation of H-2 at δH 4.69 led to enhancements of H-1 (+0.55%) and H3-20 (+1.82%), indicating that the OOH group was in a β-orientation. The 1H and 13C NMR data suggested that 3 was a derivative structurally related to (−)-2β-hydroperoxy-kolavelool [9]. However the main differences were the chemical shift in C-19. This discriminates β-configuration of Me-19 of 3 from the α-configuration of (−)-2β-hydroperoxy-kolavelool. Consequently, the relative stereochemistry of 3 was elucidated as 10βH-13-O-α-L-arabinopyranosyl-2β-hydroperoxyl-17α, 19β,20α-trimethyl-3, 14-clerodadiene. The absolute configuration of 3 was determined by comparison of experimental and theoretical calculated optical rotations. Due to the presence of ten stereogenic centres, one of the 100 stereoisomers represents the actual configuration of 3. Considering the relative configuration determined by the NOE date, the specific optical rotations of four selected possible isomers for 3, (2S,5S,8R,9S,10R,13S)-3 (3a), (2S,5R,8R,9S,10S,13S) -3 (3b), (2S,5S,8R,9S,10R,13R)-3 (3c), and (2S,5R,8R,9S,10S,13S) -3 (3d), respectively, have been calculated at the B3LYP/6-31+ +G (d,p) level. The specific optical rotations were compared with the experimental data of 3 in MeOH at 589.3 nm, with the results given in Fig. 3, where the experimental specific rotation for the naturally occurring 3 was also listed. In the case of 3, calculated [α]D values for 3a is +1.53, which is consistent with the experimental value of [α]D +7.0 of 3. Therefore, the absolute configuration of 3 was deduced to be 2S,5S,8R,9S,10R,13S. As previous reported in the literature [9,12], this kind of hydroperoxy is not stable, and 3 is easily decomposition or might transform into its 2-oxo derivative. By comparison of their physical and spectroscopic data with those reported in the literature, the 10 known compounds were identified as (E)-phytol epoxide (4) [17], α-tocopherol-quinone (5) [18], phytene-3(20)-1,2-diol (6) [19], (23Z)-en-cyclolanlst3β,25-diol (7) [20,21], (24R)-cycloartane-24,25-triol-3β-tetradecanoate (8) [22], (24R)-cycloartane-3β,24,25-triol (9) [22], urs-12-ene-2α,3-diol (10) [23], oleanolic aldehyde (11) [24], oleanolic acid (12) [25,26], and friedelin (13) [27]. In this paper clerodane diterpenoids, cycloartane-type, ursane-type, and oleanane-type triterpenes were isolated from the aerial parts of N. carpesioides, whereas, oleanane-type triterpenes and lignans were isolated from the roots in our previous study [5]. The chemical constituents from the aerial parts of N. carpesioides are clearly differences those from its roots. The new compounds 1–3 were not tested for their cytotoxic activities because they were exhausted or decomposed in the experiment process. The cytotoxic activities of compounds 7– 10 were not reported in previous literatures. Herein, they were evaluated for their cytotoxic activities against two human tumour cell lines, promyelocytic leukaemia HL-60 and hepatoma Hep-G2, using MTT method. Mitomycin was used as a positive control. None of compounds showed cytotoxicity against tested cell lines (IC50 N 20 μM). A few clerodane diterpenes with L-arabinoside were reported from families Compositae and Hymenophyllaceae in the literatures, which were glycosylated at C-6, C-13, C-18 or C-19 [28–30]. Clerodane diterpenes with L-arabinoside from Grindelia scorzonerifolia and Gutierrezia grandis of the family Compositae were genially glycosylated at C-18 or C-19. Only from Trichomanes reniforme of the family Hymenophyllaceae, clerodane diterpenes glycosylated at C-13 with L-arabinoside

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have been isolated [30]. To the best of our knowledge, Clerodane diterpenes with L-arabinoside located at C-13 1–3 are first isolated from the family Compositae. The taxonomic position of the genus Nannoglottis has been the matter of controversies for long time and it has been placed in four different tribes of the family Compositae: Inuleae, Senecioneae, Liabeae, and Astereae [1]. Recent studies supported a close affinity between the genus Nannoglottis and the tribe Astereae by gross-morphology, micromorphology, anatomy, palynology, cytology, ecology, ITS sequences, floral microcharacter, pollen, chromosome, and DNA sequences [1,2]. Qin et al. isolated two clerodane diterpenoides from the roots of N. ravida, which were first considered as a support of chemosystematic classification of Nannoglottis in Astereae based on clerodane-type diterpenoids found in some species of the tribe Astereae [2]. The presence of clerodane diterpenoides from N. carpesioides further supports the chemosystematic position of Nannoglottis in Astereae. 4. Conclusions Three new clerodane diterpenoid glycosides with L-arabinose 1–3 were isolated from the aerial parts of N. carpesioides and their structures were elucidated using extensive spectroscopic studies. Their absolute configurations were determined by the application of theoretical calculated optical rotations, which were compared with the experimental data. To our knowledge, they are first isolated from the family Compositae as clerodane diterpenes with L-arabinoside at C-13. Our study showed that clerodane diterpenoids are characteristic chemical constituents from N. carpesioides and can be used as a chemosystematic marker of the genus Nannoglottis. Conflict of interest The authors declare that they have no conflict of interest. Acknowledgements This research was supported by the Natural Science Foundation of China (NSFC, No. 21272103), the National Science Foundation for Fostering Talents in Basic Research of the National Science Foundation of China (No. J1103307), and the Ministry of Science and Technology of the People’s Republic of China. References [1] Gao TG, Chen YL. Microcharacters in the ligules of Nannoglottis (Compositae) and their systematic significance. Acta Phytotax Sin 2005;43:12–21. [2] Liu JQ, Ho TN, Liu SW. Systematic position of Nannoglottis Maxim. s.l. (Asteraceae): karyomorphological data. Acta Phytotaxon Sin 2000;38:236–41. [3] Qin HL, Li ZH. Clerodane-type diterpenoids from Nannoglottis ravida. Phytochemistry 2004;65:2533–7. [4] Jiang J, Zhang BB, Liao ZX. A new dicaffeoyl quinic acid from Nannoglottis ravida. Chin Chem Lett 2010;21:203–5. [5] Xue CB, Chai DW, Jin XJ, Bi YR, Yao XJ, Wu WS, et al. Triterpenes and neolignans from the roots of Nannoglottis carpesioides. Phytochemistry 2011;72:1804–13. [6] Li W, Fu HW, Bai H, Sasaki T, Kato H, Koike K. Triterpenoid saponins from Rubus ellipticus var. obcordatus. J Nat Prod 2009;72:1755–60. [7] Hussain RF, Nouri AME, Oliver RTD. A new approach for measurement of cytotoxicity using colorimetric assay. J Immunol Methods 1993;160:89–96.

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New clerodane diterpenoid glycosides from the aerial parts of Nannoglottis carpesioides.

Three new clerodane diterpenoid glycosides with L-arabinose (1-3), together with ten known compounds including phytol-type diterpenes, cycloartane-typ...
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