Original Papers
695
Authors
Van Trinh Thi Thanh 1, Van Cuong Pham 1, Huong Doan Thi Mai 1, Marc Litaudon 2, Françoise Guéritte 2, Van Hung Nguyen 1, Van Minh Chau 1
Affiliations
1 2
Key words " Euphorbiaceae l " Cleistanthus indochinensis l " lignans l " glycosides l " cytotoxicity l
received revised accepted
February 7, 2014 February 11, 2014 April 22, 2014
Bibliography DOI http://dx.doi.org/ 10.1055/s-0034-1368505 Published online June 4, 2014 Planta Med 2014; 80: 695–702 © Georg Thieme Verlag KG Stuttgart · New York · ISSN 0032‑0943 Correspondence Dr. Van Cuong Pham Institute of Marine Biochemistry Vietnam Academy of Science and Technology 18, Hoang Quoc Viet Road Caugiay, Hanoi Vietnam Phone: + 84 04 37 56 49 95 Fax: + 84 04 37 91 70 54 [email protected] Correspondence Prof. Dr. Van Minh Chau Institute of Marine Biochemistry Vietnam Academy of Science and Technology 18, Hoang Quoc Viet Road Caugiay, Hanoi Vietnam Phone: + 84 04 37 56 49 95 Fax: + 84 04 37 91 70 54 [email protected]
Institute of Marine Biochemistry of the Vietnam Academy of Science and Technology (VAST), Cau Giay, Hanoi, Vietnam Institut de Chimie des Substances Naturelles, Gif-sur Yvette, France
Abstract !
Eight new aryltetralin lignans, cleisindosides A–F (1–6), picroburseranin (7), and 7-hydroxypicropolygamain (8), were isolated from the fruits of Cleistanthus indochinensis (Euphorbiaceae). The structures of the isolates were established on the basis of their one- and two-dimensional NMR spectral data, as well as their mass spectrometric data. Compound 7 was found to have potent cytotoxicity against oral epidermoid carcinoma cells with an IC50 value of 0.062 µM, whereas glycosylation to 3 (IC50 7.5 µM) and stereochemical changes to 8 (IC50 10.8 µM) led to marked decreases in biological activity. Thus, it was deter-
Introduction !
Cleistanthus is a plant genus of the Euphorbiaceae family and comprises approximately 140 species that are distributed across a variety of locations from Africa to the Pacific Islands [1]. Many plants of this genus are known for their toxic properties [2–6]. Cleistanthus has been shown to contain bioactive lignan compounds such as cleistanthins A and B (from Cleistanthus collinus); these were found to be cytotoxic against several tumor cell lines [7, 8]. Also, cleistanthins A and B inhibited the actions of the alpha adrenergic receptor and the nicotinic cholinergic receptor [9]. In Vietnam, 14 species of Cleistanthus have been identified and, importantly, some of them were used in traditional medicine [10]. Previously, we reported the bioassay-guided purification of two new cytotoxic lignans, cleistantoxin and neocleistantoxin, from the dichloromethane extract of the fruits of Cleistanthus indochinensis [11]. Cleistantoxin was found to be significantly active against several cancer cell lines and its amide derivatives were synthesized and evaluated for their cytotoxicity. In the continuation of research on more polar
mined that the C-7 and C-8′ positions are critical for the biological activity of the lignans from this plant.
Abbreviations !
ATCC: MTT: FBS:
American Type Cell Culture Collection 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide fetal bovine serum
Supporting information available online at http://www.thieme-connect.de/products
fractions, the present paper describes the bioassay-guided purification of eight new lignans from the methanol extract of the fruits of C. indochinensis, which inhibits the growth of oral epidermoid carcinoma (KB) cells (73% at a concentration of 10 µg/mL).
Results and Discussion !
The dried and milled fruits of C. indochinensis (100 g) were extracted successively with CH2Cl2 and MeOH at room temperature. The MeOH soluble secondary metabolites were separated using open column chromatography (CC) on silica gel. The resulting fractions were tested against KB cells and the active fractions were further purified " Fig. 1). by CC to afford compounds 1–8 (l Compound 1 was obtained as a white microcrystalline solid and was optically active, [α]30 D − 50.0 (c 0.3, MeOH). Its 1H NMR spectrum presented signals of two singlet aromatic protons at δH = 6.50 (H-3) and 7.04 (H-6), and an ABX ring system, which was characterized by three aromatic protons at δH = 6.54 (H-2′), 6.75 (H-5′), and
Trinh-Thi-Thanh V et al. Cytotoxic Aryltetralin Lignans …
Planta Med 2014; 80: 695–702
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Cytotoxic Aryltetralin Lignans from Fruits of Cleistanthus indochinensis
Original Papers
Fig. 1
6.37 (H-6′). Also, signals of a sugar moiety, two methylenedioxy groups, a methylene, and three methines were noted in the aliphatic region. The 13C NMR and DEPT spectra showed the resonances corresponding to the functional groups observed in the 1 H NMR spectrum, with additional signals of a carboxylate group and seven aromatic quaternary carbons. The chemical shifts of C4, C-5, C-7, C-9, C-3′, and C-4′ suggested their linkage to an oxygen atom. Analysis of the 1H-1H COSY spectrum revealed the spin-spin coupling systems as follows: connection of sugar protons; correlations of the ABX system; and cross-peaks of a proton at δH = 2.85 (H-8) to the protons at δH = 5.02 (H-7), 4.32 and 4.36 (CH2-9), and 3.39 (H-8′). The resonance for H-8′ correlated to the proton at δH = 4.52 (H-7′). The planar structure of 1 was then established by analysis of its HMBC spectrum. The ABX aromatic ring (A-ring) was bonded to C-7′, as indicated by correlations of the carbon resonance at δC = 42.6 (C-7′) with protons H-2′ and H-6′. Correlations of the carbonyl carbon resonance at δC = 174.8 (C-9′) with protons CH2-9 and H-7′ indicated the presence of a lactone ring. The location of the methylenedioxy at C-5 and C-4 was revealed by correlations of protons at δH = 6.01 and 6.02 (CH2-10) with C-5 at δC = 147.8 and C-4 at δC = 146.1. Similarly, the linkage of the methylenedioxy at δH = 5.94 and 5.95 (CH210′) to carbons C-3′ and C-4′ was determined from their HMBC cross-peaks. Finally, the sugar moiety was linked to C-7, as determined by a correlation of C-7 with the anomeric proton at δH = 4.25 (H-1′′). The relative configuration of 1 was then established by the analysis of coupling constants and a NOESY experiment. Proton H-8′ afforded a strong (J = 14.5 Hz) and a small (J = 5.5 Hz) coupling constant while H-7′ appeared as a doublet (J = 5.5 Hz), depicting a trans-pseudodiaxial relationship between H-8 and H-8′ and a pseudoequatorial orientation for H-7′. In addition, H-7 had a gauche (J = 3.5 Hz) coupling constant, and thus a pseudoequatorial disposition. The β-glucopyranose was assigned to the sugar from coupling constant analysis of all sugar protons " Table 1). This was confirmed by acidic hydrolysis of 1 (l " Fig. 2). The hydrolyzed sugar had a positive optical rotation (l value [α]28 D + 51.0 (c 0.15, H2O) and an identical Rf value with the standard D-(+)-glucose, revealing the D-configuration for the glucose moiety of 1. This compound was named cleisindoside A. The 1D NMR spectra of compound 2 displayed signals similar to those of 1, with additional signals of a second carbohydrate moiety at δC = 111.1 (d, C-1′′′), 78.3 (d, C-2′′′), 80.5 (s, C-3′′′), 75.0 (d, C4′′′), and 65.6 (t, C-5′′′) that strongly suggested the presence of an apiofuranose moiety. Analyses of 2D NMR experiments and pro-
Trinh-Thi-Thanh V et al. Cytotoxic Aryltetralin Lignans …
Structures of compounds 1–8.
" Table 1) depicted structural similarton coupling constants (l ities with 1, namely the lignan aglycone and the β-glucopyranose moieties. The bonding of O-β-glucopyranose to C-7 was established from an HMBC cross-peak of the anomeric carbon at δC = 101.8 (C-1′′) to the proton at δH = 5.10 (H-7), and the linkage of the O-apiofuranose to C-6′′ of the glucopyranose by the correlation of C-6′′ at δC = 69.3 to H-1′′′ at δH 5.07. The downfield chemical shift of the anomeric carbon at δC = 111.1 (C-1′′′) suggested a β-form for the apiofuranose [12, 13]. In addition, the glucose and apiose from the hydrolysis of 2 gave positive specific rotations, indicating their D-configuration. This aryltetralin lignan glycoside was named cleisindoside B. Some similarities existed between the 1H and 13C NMR spectra of compound 3 with those of 1 and 2. In particular, differences existed in the saccharide side chain. Analyses of 2D NMR spectra of 3 revealed an identical lignan aglycone and a β-glucopyranose moiety, while the remaining signals were distributed to a β-xylopyranose linking to C-6′′ of the β-glucopyranose, as indicated by an HMBC cross-peak of a carbon at δC = 69.4 (C-6′′) with a proton at δH = 4.19 (H-1′′′). The hydrolyzed glucose and xylose had a positive specific optical rotation, thus assigning the D-configuration for both the glucopyranose and xylopyranose moieties of 3 (cleisindoside C). Comparison of the 1H NMR spectrum of 4 with that of cleistantoxin [11] revealed a structural similarity with additional signals of a β-glucopyranose unit [δH = 4.34 (d, J = 7.5 Hz, H-1′′), 3.28 (dd, J = 7.5 and 8.5 Hz, H-2′′), 3.39 (dd, J = 8.5 and 8.5 Hz, H-3′′), 3.41 (dd, J = 8.5 and 8.5 Hz, H-4′′), 3.18 (m, H-5′′), and 3.67 (m, CH26′′)]. Analysis of its HMBC spectrum determined the linkage of β-glucopyranose to C-7 by a cross-peak of C-1′′ at δC = 99.4 with H-7 at δH = 5.28. A trans-pseudodiaxial relationship between H7 and H-8 was established from their anti-coupling constant (J = 8.5 Hz). H-8′ appeared as a doublet of doublets with strong (J = 15.0 Hz) and small (J = 4.0 Hz) coupling constants, whereas H-7′ had a gauche coupling constant (J = 4.0 Hz). This indicated that H-7′ contained a pseudoequatorial orientation and H-8′ was pseudoaxial. Thus, in comparison with compounds 1–3, an epimerization at C-7 was observed for 4. The D-configuration was assigned for the glucopyranose in 4, since its hydrolyzed glucose had a positive optical rotation value. This compound was reported here for the first time and named cleisindoside D. The 1D NMR signals of compound 5 were similar to those of 4. The significant differences were the presence of an additional acetyl group and the CH2-6′′ signals of the sugar moiety being
Planta Med 2014; 80: 695–702
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696
Original Papers
3 6 7 8 9 10 2′ 5′ 6′ 7′ 8′ 10′ 1′′ 2′′ 3′′ 4′′ 5′′ 6′′
1a
2b
3a
8c
6.50 s 7.04 s 5.02 d (3.5) 2.85 dddd (3.5, 8.5, 9.0, 14.5) 4.32 dd (8.5, 8.5) 4.36 dd (8.5, 9.0) 6.01 s 6.02 s 6.54 d (1.5) 6.75 d (8.0) 6.37 dd (1.5, 8.0) 4.52 d (5.5) 3.39 dd (5.5, 14.5) 5.94 s 5.95 s 4.25 d (8.0) 3.01 ddd (4.0, 8.0, 8.0) 3.11 m 3.03 ddd (4.0, 8.0, 9.0) 3.12 m 3.46 dd (6.0, 11.0) 3.76 dd (6.0, 11.0)
6.48 s 7.09 s 5.10 d (3.5) 3.02 dddd (3.5, 8.2, 10.5, 14.3) 4.38 dd (8.2, 8.2) 4.52 dd (8.2, 10.5) 5.96 d (1.0) 5.97 d (1.0) 6.57 d (1.5) 6.67 d (8.0) 6.48 dd (1.5, 8.0) 4.56 d (5.7) 3.53 dd (5.7, 14.3) 5.88 d (1.0) 5.89 d (1.0) 4.48 d (8.0) 3.24 dd (8.0, 9.0) 3.35 dd (9.0, 9.0) 3.29 dd (9.0, 9.0) 3.47 ddd (2.0, 7.0, 9.0) 3.63 dd (7.0, 11.5) 4.10 dd (2.0, 11.5) 5.07 d (2.7) 3.98 d (2.7)
6.49 s 7.08 s 4.98 d (3.0) 2.83 m
6.11 s 6.99 s 4.26 d (10.0) 2.47 m
4.31 dd (8.0, 8.0) 4.38 dd (8.0, 8.0) 6.01 br. s 6.02 br. s 6.54 d (1.5) 6.75 d (8.0) 6.38 dd (1.5, 8.0) 4.51 d (5.5) 3.35 dd (5.5, 14.5) 5.94 br. s 5.95 br. s 4.33 d (8.0) 3.00 m 3.13 dd (9.0, 9.0) 3.02 m 3.32 m 3.51 dd (8.0, 11.0) 4.05 br. d (11.0) 4.19 d (7.5) 3.01 m 3.11 dd (9.0, 9.0) 3.31 m
4.28 dd (6.2, 9.5) 4.49 dd (1.0, 9.5) 5.75 d (1.5) 5.76 d (1.5) 6.57 d (1.5) 6.68 d (8.0) 6.62 dd (1.5, 8.0) 3.83 d (6.2) 3.00 dd (6.2, 9.0) 5.82 br. s 5.83 br. s
1′′′ 2′′′ 3′′′ 4′′′
3.83 d (10.0) 4.05 d (10.0) 3.62 m
5′′′′ OH-2′′ OH-3′′ OH-4′′ OH-6′′ a
Table 1 1H NMR data for compounds 1–3 and 8 (500 MHz).
3.05 dd (11.5, 11.5) 3.73 dd (5.5, 11.5)
4.97 d (4.0) 4.89 m 4.89 m 4.63 t (6.0)
DMSO-d6; b CD3OD; c CDCl3+CD3OD
shifted downfield. Careful analysis of the 2D NMR spectra allowed for the determination of the structure of 5, which was an acetylated derivative of 4. The location of the acetyl group at C-6′′ was defined by HMBC correlations between the carbonyl of the acetyl group at δC = 171.1 with protons at δH = 4.16 and 4.27 (CH2-6′′). Analysis of the proton coupling constants of 5 indicated an identical stereochemistry with 4. On the basis of these data, " Fig. 1 and was the structure of 5 was identified as indicated in l named cleisindoside E. The 1H and 13C NMR spectra of 6 also indicated the presence of a β-glucopyranose unit characterized from carbons at δC = 103.0 (d, C-1′′), 73.2 (d, C-2′′), 75.9 (d, C-3′′), 69.8 (d, C-4′′), 76.9 (d, C-5′′), and 60.6 (t, C-6′′). Comparison of the 1D NMR signals of the aglycon moiety of 6 with those of 1–3 depicted the presence of a double bond and a methylene as opposed to three sp3 methines and the absence of a methylenedioxy group. Furthermore, examination of 1H-1H COSY data allowed the assignment of a spin-spin coupling system from CH2-7 (δH = 2.69 and 2.71) to CH2-9 (δH = 3.97 and 4.64) via H-8 (δH = 3.31). These data suggested the presence of the double bond at C-7′ and C-8′ in the structure of 6. " Fig. 3), Analysis of 2D NMR data established the structure of 6 (l in which the β-glucopyranose moiety was linked to C-3′ of the Aring, as indicated by an HMBC cross-peak of C-3′ at δC = 144.4 with the anomeric proton H-1′′ at δH = 4.67. The D-configuration for the glucopyranose moiety of 6 was assigned from the positive
Trinh-Thi-Thanh V et al. Cytotoxic Aryltetralin Lignans …
Fig. 2 Key HMBC correlations for 1.
Planta Med 2014; 80: 695–702
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Pos.
697
698
Original Papers
specific optical rotation of its hydrolyzed glucose. This new lignan glucoside was named cleisindoside F. Analysis of the 1H and 13C NMR spectra of 7 revealed the same planar structure with burseranin, which was previously isolated from Bursera graveolens [14]. However, H-8′ of 7 presented small (J = 4.5 Hz) and strong (J = 14.0 Hz) coupling constants, whereas these values were J = 3.0 and 9.5 Hz for burseranin (C/D cis-fused junction). This observation clearly suggested a C/D trans-fusion " Table 2) for 7 [15]. Analysis of the proton coupling constants (l and NOESY data indicated that 7 contained the same stereochemistry at C-8, C-8′, and C-7′ as compounds 1–5, and was named picrobursenin, as it appeared to be an 8′-epimer of burseranin.
Compound 8 was obtained as a white microcrystalline material and was optically active [α]30 D + 14.0 (c 0.5, CHCl3). Its HRESI mass spectrum showed the pseudomolecular [M + Na]+ ion at m/z 391.0786, indicating the molecular formula of C20H16O7 (calcd. 391.0794 for C20H16NaO7). The planar structure of 8 was elucidated by analysis of 2D NMR experiments, in which the loss of a methoxy group at C-6 was observed for 8, as compared to cleistantoxin. Coupling constant analysis of the aliphatic protons demonstrated that H-7 had an anti-coupling (J = 10.0 Hz) with H-8, suggesting that their trans-pseudodiaxial relationship was identical to cleistantoxin. In contrast, H-7′ had a gauche coupling (J = 6.2 Hz) and H-8′ was a doublet of doublets with two coupling constants (J = 9.0 and 6.2 Hz). Thus, the coupling constant between H-8′ and H-8 was J = 9.0 Hz (this value was J = 15.0 Hz for cleistantoxin), assigning a cis-fused junction for the C/D rings
4c
5d
6e
7f
3 6 7
6.26 s
6.34 s
6.24 s
5.28 d (8.5)
5.27 d (10.5)
8 9
3.04 m 4.07 dd (8.2, 8.2) 4.64 dd (8.2, 8.2) 5.89 s 5.93 s 6.65 m 6.69 m 6.65 m 4.45 d (4.0) 2.70 dd (4.0, 15.0) 5.83 s 5.84 s 4.34 d (7.5) 3.28 dd (7.5, 8.5) 3.39 dd (8.5, 8.5) 3.41 dd (8.5, 8.5) 3.18 m 3.67 m
3.20 m 4.06 dd (1.5, 8.0) 4.65 dd (8.0, 8.0) 5.99 d (1.0) 5.98 (1.0) 6.65 d (1.5) 6.73 d (8.0) 6.76 dd (1.5, 8.0) 4.54 d (4.5) 2.68 dd (4.5, 14.5) 5.92 s
6.39 s 6.94 s 2.69 dd (6.5, 15.0) 2.71 dd (15.0, 15.0) 3.31 m 3.97 dd (8.5, 8.5) 4.64 dd (8.5, 8.5) 5.99 d (1.0) 6.00 d (1.0) 7.11 d (1.5) 6.86 d (8.0) 6.74 dd (1.5, 8.0)
Pos.
10 2′ 5′ 6′ 7′ 8′ 10′ 1′′ 2′′ 3′′ 4′′ 5′′ 6′′ 2′′′ OMe c
Fig. 4 Key NOE correlations for 8.
3.99 s
4.38 d (7.5) 3.39 m 3.53 dd (8.5, 8.5) 3.35 dd (8.5, 8.5) 3.38 m 4.16 dd (7.0, 11.0) 4.27 dd (2.0, 11.0) 1.65 s 4.07 s
4.67 d (8.0) 3.33 dd (8.0, 8.0) 3.28 m 3.24 m 3.21 m 3.48 m 3.64 m
CDCl3+CD3OD; d CDCl3; e DMSO-d6, 343 K; f acetone-d6
Trinh-Thi-Thanh V et al. Cytotoxic Aryltetralin Lignans …
2.49 dd (11.0, 16.2) 3.19 dd (5.5, 16.2) 2.63 m 4.00 dd (8.5, 10.0) 4.46 dd (7.5, 8.5) 5.94 d (1.0) 5.95 d (1.0) 6.65 d (1.5) 6.68 d (8.5) 6.53 dd (1.5, 8.5) 4.53 d (4.5) 2.82 dd (4.5, 14.0) 5.92 d (1.0) 5.93 d (1.0)
Planta Med 2014; 80: 695–702
4.03 s
Table 2 1H NMR data for compounds 4–7 (500 MHz).
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Fig. 3 Selected HMBC cross-peaks for 6.
Original Papers
699
[15], which was clearly confirmed by a strong interaction be" Fig. 4). In compartween H-8 and H-8′ in the NOESY spectrum (l ison with other compounds from this plant, an epimerization at C-8′ was observed for 8. This compound was named 7-hydroxypicropolygamain as it had a structural similarity with picropolygamain [16, 17]. The absolute configuration at C-7′ was established by examination of the circular dichroism (CD) spectra of compounds 2, 4, 7, and 8, which afforded positive Cotton effects at 293–297 nm (Fig. 68S, Supporting Information). Since the sign of the first couplet is determined by the configuration of the aryl substituent at C-7′, namely negative for 7′S and positive for 7′R, C-7′ were assigned as having the R-configuration for compounds 2, 4, 7, and 8 [18–20]. The isolates were tested for in vitro biological activity against KB cells. The most active compound was 7 with an IC50 of 0.062 µM, followed by 3 (IC50 7.5 µM) and 8 (IC50 10.8 µM). The remaining compounds exhibited weaker cytotoxicity (IC50 > 20.0 µM). Taxotere was used as a positive control and had an IC50 of 0.12 nM. A comparison of cytotoxicity values between cleistantoxin (IC50 0.022 µM) and 7 revealed that the presence of an OH group at C7 seems to slightly increase biological activity. On the contrary, the presence of sugar moieties at C-7 significantly decreased their cytotoxicity, especially when comparing cytotoxicity values of cleistantoxin and 7 with those of 4 and 5. Finally, it is apparent that a stereochemical change at C-8′ of the lactone ring led to a major decrease in biological activity, indicating the importance of this moiety (via a comparison of cleistantoxin and 7 cytotoxicities with those of 6 and 8).
Concentrations of cleistantoxin and cleisindoside B (2) in the fruit of C. indochinensis were analyzed by means of HPLC/DAD. The compounds were identified through comparison of their retention times and corresponding UV/DAD absorption spectrum with " Fig. 5). The results indicated that those of the pure compounds (l the cleistantoxin content was 178 mg/g in the CH2Cl2 crude extract, while the content of cleisindoside B (2) was 8.56 mg/g in the MeOH crude extract.
Materials and Methods !
General Melting points were recorded on a Buchi B-545 instrument and are uncorrected. Optical rotations were recorded on a Polax-2L polarimeter in CHCl3. UV spectra were recorded on a UV-1601 spectrometer. IR spectra were measured on a Nicolet Impact410 FT‑IR spectrometer. CD spectra were measured on a JASCO J-810 spectrophotometer. Quantitative analysis of cleistantoxin from the CH2Cl2 extract and from 2 in the MeOH extract was carried out by an Agilent 1200 Series HPLC‑DAD instrument. 1H and 13 C NMR spectra were recorded on a Bruker AM500 FT‑NMR spectrometer as indicated with either the CDCl3 (δH 7.24 ppm, δC 77.0 ppm), CD3OD (δH 3.30 ppm, δC 49.0 ppm), DMSO-d6 (δH 2.49 ppm, δC 39.5 ppm), or acetone-d6 (δH 2.04 ppm, δC 29.8 and 206.0 ppm) signal as the internal standard. J values are expressed in Hz. The HMBC measurements were optimized to 7.0 Hz longrange couplings, and NOESY experiments were run with a 150ms mixing time. High-resolution ESIMS were measured on a VARIAN 910 spectrometer.
Trinh-Thi-Thanh V et al. Cytotoxic Aryltetralin Lignans …
Planta Med 2014; 80: 695–702
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Fig. 5 HPLC chromatograms: A CH2Cl2 crude extract, B MeOH crude extract. (Color figure available online only.)
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Original Papers
a
13
C NMR data for compounds 1–8 (125 MHz).
Position
1a
2b
3a
4c
5d
6e
7f
8c
1 2 3 4 5 6 7 8 9 10 1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′ 9′ 10′ 1′′ 2′′ 3′′ 4′′ 5′′ 6′′ 1′′′ 2′′′ 3′′′ 4′′′ 5′′′ OMe
128.2 133.0 109.9 146.1* 147.8* 110.2 70.4 37.2 67.8 101.3 134.3 110.8 145.9 146.6 107.4 123.5 42.6 40.1 174.8 100.8 99.8 73.6 76.6 70.4 77.0 61.3
134.6 129.8 111.2 150.0* 148.3* 111.2 73.2 39.1 69.9 102.8 135.4 112.0 148.6 147.9 108.4 125.0 44.7 42.1 177.7 102.3 101.8 75.1 78.0 72.0 76.9 69.3 111.1 78.3 80.5 75.0 65.6
126.8 132.8 109.7 147.7* 146.2* 110.3 71.0 37.1 67.9 101.2 134.2 110.7 146.6 145.9 107.4 123.5 42.5 40.2 174.7 100.8 100.3 73.4 76.5 70.5 75.2 69.4 104.4 73.4 76.6 69.5 65.6
122.0 135.3 104.8 149.9 136.8 142.1 75.2 38.4 72.0 101.5 132.8 107.6 147.3 146.6 110.7 123.8 44.0 45.5 174.2 100.9 99.4 73.5 76.3 69.8 76.0 61.5
121.7 136.1 105.5 150.4 137.1 142.0 77.0 37.6 72.0 101.8 133.1 110.8 147.6 147.0 108.0 124.1 44.4 45.6 174.0 101.3 98.1 73.4 76.7 70.2 74.2 63.3 171.1 20.1
131.1 129.3 108.0 147.9* 145.9* 108.4 32.0 35.0 70.2 125.3 125.3 119.9 144.4 147.2 115.1 124.6 145.4 120.0 167.5
122.5 133.2 105.0 149.1 135.9 141.8 27.7 33.1 72.8 101.8 136.1 111.9 148.0 147.2 108.0 124.9 44.2 47.3 175.3 101.9
133.4 130.7 108.3 146.4 146.4 104.4 67.7 42.5 69.6 100.7 137.0 108.8 146.5 147.9 108.2 122.1 43.1 45.4 178.6 100.9
59.9
60.2
b
c
d
e
103.0 73.2 75.9 69.8 76.9 60.6
59.7
f
DMSO-d6; CD3OD; CDCl3+CD3OD; CDCl3; DMSO-d6, 343 K; acetone-d6; * chemical shifts should be exchanged with each other in the same column
Plant material The fruits of C. indochinensis were collected at Quytrau – Nghean (Vietnam) in May 2003, and a specimen (VN 1086) was deposited at the Institute of Ecology and Natural Resources, Vietnam Academy of Science and Technology (VAST). The plant was identified by Dr. Nguyen Quoc Binh of the Institute of Ecology and Natural Resources, VAST.
Extraction and isolation Dried and ground fruits of C. indochinensis (100 g) were extracted successively with CH2Cl2 (4 × 300 mL, 1 h each) and MeOH (4 × 300 mL, 1 h each) at room temperature in a sonicator. The solvents were removed under diminished pressure to give residues of 4.0 g (CH2Cl2) and 5.5 g (MeOH). The MeOH soluble secondary metabolites (5.5 g) were separated using reversed-phase (RP-18) CC (5 × 60 cm), eluted with a mixture of MeOH/H2O (2.5 L, 10 to 100 % MeOH in H2O) to provide 11 fractions. A cytotoxicity assay against KB cells revealed two active fractions (4 and 8). Fraction 4 (250 mL, 335 mg) was washed with MeOH to obtain 3 (9 mg). The filtrate (35 mL) was concentrated to dryness and purified by CC on silica gel (40–63 µm, 2 × 50 cm, 5% MeOH in CH2Cl2), providing 4 subfractions. Subfraction 1 (150 mL, 65 mg) was subjected to a Sephadex LH-20 column (1 × 50 cm, MeOH) to yield 4 (18 mg). Subfraction 2 (110 mL, 124 mg) was washed with MeOH affording 1 (10 mg) and the filtrate (20 mL) was purified by CC on Sephadex LH-20 (1 × 50 cm, MeOH) to provide 5 (13 mg).
Trinh-Thi-Thanh V et al. Cytotoxic Aryltetralin Lignans …
Subfraction 3 (150 mL, 75 mg) was recrystallized in MeOH (1 mL) to provide 6 (9 mg) and subfraction 4 (240 mL, 83 mg) was chromatographed on Sephadex LH-20 column (1 × 50 cm, 80 mL, MeOH) to obtain 2 (11 mg). Fraction 8 (200 mL, 106 mg) was subjected to CC on silica gel (1 × 50 cm, 1–20% MeOH in CH2Cl2) affording 7 (15 mg) and 8 (5 mg). Cleisindoside A (1): White microcrystalline (acetone/MeOH), mp −1 261–262 °C; [α]30 D − 50.0 (c 0.3, MeOH); IR (KBr), νmax (cm ): 3444, 2910, 1773, 1626, 1491, 1244, 1382, 1080, 1034; UV (MeOH) λmax nm (log ε): 215.4 (4.32), 287.5 (3.98); HRESI MS (positive ion mode) m/z 553.1323 [M + Na]+ (calcd. 553.1322 for " Tables 1 and 3. C26H26NaO12); NMR data see l Cleisindoside B (2): White microcrystalline (acetone/MeOH), mp −1 248–249 °C; [α]30 D − 52.0 (c 1.0, MeOH); IR (KBr), νmax (cm ): 3421, 2929, 1764, 1624, 1490, 1244, 1039; UV (MeOH) λmax nm (log ε): 215.0 (4.36), 238.0 (3.96), 288.8 (3.96); HRESI MS (positive ion mode) m/z 685.1746 [M + Na]+ (calcd. 685.1745 for " Tables 1 and 3. C31H34NaO16); NMR data see l Cleisindoside C (3): White microcrystalline (acetone/MeOH), mp −1 278–279 °C; [α]30 D − 65.0 (c 0.9, MeOH); IR (KBr), νmax (cm ): 3418, 2916, 1752, 1624, 1487, 1234, 1036; UV (MeOH) λmax nm (log ε): 216.0 (3.86), 235.0 (3.15), 286.8 (3.12); HRESI MS (positive ion mode) m/z 685.1752 [M + Na]+ (calcd. 685.1745 for " Tables 1 and 3. C31H34NaO16); NMR data see l Cleisindoside D (4): White microcrystalline (acetone/MeOH), mp −1 261–262 °C; [α]30 D − 188.0 (c 0.5, MeOH); IR (KBr), νmax (cm ):
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Table 3
Original Papers
Acid hydrolysis of 1–6 Each solution of 1–6 (each 5 mg) was heated at 95 °C for 5 h in 1 N HCl (dioxane – H2O, 1 : 1, 2.5 mL). The filtrates from the hydrolysate were neutralized with DOWEX HCR‑S ion-exchange resin and filtered. A portion of the filtrate was concentrated under reduced pressure and examined for carbohydrates by silica gel TLC [Kieselgel 60 (Merck Art 5554), i-PrOH/Me2CO/H2O (5 : 3 : 1)] using authentic samples. The Rf values of each sugar were as follows: glucose, 0.41; xylose, 0.48; and apiose, 0.53. The remaining filtrate was concentrated under reduced pressure and purified by preparative TLC. The sugars were identified as D-glucose {[α]28 D + 51 (c 0.15, H2O)}, D-apiose {[α]28 D + 12 (c 0.08, H2O)}, and D-xylose {[α]28 D + 25 (c 0.09, H2O)}.
Cytotoxic activity assay The human KB tumor (oral epidermoid carcinoma) cell line was obtained from ATCC. KB cells were maintained in Dulbeccoʼs D‑MEM medium, supplemented with 10% fetal calf serum, L-glutamine (2 mM), penicillin G (100 UI/mL), streptomycin (100 µg/ mL), and gentamicin (10 µg/mL). Stock solutions of compounds were prepared in DMSO/H2O (1/9), and cytotoxicity assays were carried out in 96-well microtiter plates against human nasopharynx carcinoma KB cells (3 × 103 cells/mL) using a modification of the published method [21]. After 72 h incubation at 37 °C in air/ CO2 (95 : 5) with or without the test compounds, cell growth was estimated by colorimetric measurement of stained living cells by neutral red. Optical density was determined at 540 nm with a Titertek Multiscan photometer. The IC50 value was defined as the concentration of sample necessary to inhibit the cell growth to 50 % of the control. Taxotere (in-house product, purity > 99 %) was used as a reference compound. The purity of the tested compounds was > 95 % as observed from their 1H NMR spectra (Supporting Information).
HPLC‑DAD quantitative analysis of cleistantoxin and cleisindoside B (2) High-performance liquid chromatography was performed with an Agilent 1200 Series. UV‑VIS detector DAD and Software Agilent ChemStation were used. Reverse-phase chromatography analyses were carried out using a Zobax eclipse XDB C-18 column (4.6 mm × 250 mm) packed with 5 µm diameter particles. The volume injection was 5 µL and the gradient elution was conducted according to the Evaristo and Leitao method, with minor modifications [22]. The mobile phase consisted of water (solvent A) and methanol (solvent B). Stock solutions of cleistantoxin and 2 were prepared in methanol in the range of 6.25–1000 µg/mL. Samples and standard solutions, as well as the mobile phase, were degassed and filtered through a 0.45-µm membrane filter (Millipore). Identification of the compounds was done by comparison of their retention times and UV absorption spectrum with those of the pure compounds cleistantoxin and 2.
Supporting information HRESI MS and 1D and 2D NMR spectra of compounds 1–8 are available as Supporting Information.
Acknowledgements !
The authors thank Mr. Dao Dinh Cuong and Dr. Nguyen Quoc Binh (VAST – Vietnam) for plant collection and botanical determination. The Centre National de la Recherche Scientifique (CNRS, France) is gratefully acknowledged for the French Vietnamese Laboratory of Natural Products Chemistry (NATPROCHEMLAB) and The Vietnam National Foundation for Science and Technology Development (NAFOSTED) is acknowledged for financial support (Grant No: 104.01.75.09).
Conflict of Interest !
The authors declare no conflict of interest.
References 1 Pinho PMM, Kijjoa A. Chemical constituents of the plants of the genus Cleistanthus and their biological activity. Phytochem Rev 2007; 6: 175– 182 2 Ramesh C, Ravindranath N, Ram TS, Das B. Arylnaphthalide lignans from Cleistanthus collinus. Chem Pharm Bull 2003; 51: 1299–1300 3 Eswarappa S, Chakraborty AR, Palatty BU, Vasnaik M. Cleistanthus collinus poisoning: case reports and review of the literature. J Toxicol Clin Toxicol 2003; 41: 369–372 4 Govindachari TR, Sathe SS, Viswanathan N, Pai BR, Srinivasan M. Chemical constituents of Cleistanthus collinus (Rox.). Tetrahedron 1969; 25: 2815–2821 5 Anjaneyulu ASR, Ramaiah PR, Row LR, Venkateswarlu R, Pelter A, Ward RS. New lignans from the heartwood of Cleistanthus collinus. Tetrahedron 1981; 37: 3641–3652 6 Sastry KV, Rao EV, Buchanan JG, Sturgeon RJ. Cleistanthoside B, a diphyllin glycoside from Cleistanthus patulus heartwood. Phytochemistry 1987; 26: 1153–1154 7 Pradheepkumar CP, Shanmugam G. Anticancer potential of cleistanthin A isolated from the tropical plant Cleistanthus collinus. Oncol Res 1999; 11: 225–232 8 Prabhakaran C, Kumar P, Panneerselvam N, Rajesh S, Shanmugam G. Cytotoxic and genotoxic effects of cleistanthin B in normal and tumour cells. Mutagenesis 1996; 11: 553–557 9 Parasuraman S, Raveendran R. Effect of cleistanthin A and B on adrenergic and cholinergic receptors. Pharmacogn Mag 2011; 7: 243–247
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3419, 2932, 1772, 1619, 1475, 1235, 1073, 1035; UV (MeOH) λmax nm (log ε): 222.7 (4.37), 286.7 (3.82); HRESI MS (positive ion mode) m/z 583.1409 [M + Na]+ (calcd. 583.1428 for " Tables 2 and 3. C27H28NaO13); NMR data see l Cleisindoside E (5): White microcrystalline (acetone/MeOH), mp −1 265–266 °C; [α]30 D − 196.0 (c 0.5, MeOH); IR (KBr), νmax (cm ): 3427, 2925, 1775, 1740, 1615, 1479, 1373, 1236, 1077, 1035; UV (MeOH) λmax nm (log ε): 212.2 (3.59), 239.8 (4.19), 287.5 (3.82); HRESI MS (positive ion mode) m/z 625.1523 [M + Na]+ (calcd. " Tables 2 and 3. 625.1533 for C29H30NaO14); NMR data see l Cleisindoside F (6): White microcrystalline (acetone/MeOH), mp −1 261–262 °C; [α]30 D − 50.0 (c 0.3, MeOH); IR (KBr), νmax (cm ): 3463, 2899, 1725, 1636, 1598, 1508, 1359, 1207, 1074, 1036; UV (MeOH) λmax nm (log ε): 250.0 (3.71), 348.1 (3.33); HRESI MS (positive ion mode) m/z 523.1225 [M + Na]+ (calcd. 523.1216 for " Tables 2 and 3. C25H24NaO11); NMR data see l Picrobursenin (7): White microcrystalline (acetone), mp 186– −1 188 °C; [α]30 D − 100.5 (c 0.55, CHCl3); IR (KBr), νmax (cm ): 3461, 2894, 2845, 1773, 1616, 1474, 1387, 1248, 1038; UV (MeOH) λmax nm (log ε): 227.8 (2.19), 286.3 (1.77); HRESI MS (positive ion mode) m/z 405.0948 [M + Na]+ (calcd. 405.0950 for " Tables 2 and 3. C21H18NaO7); NMR data see l 7-Hydroxypicropolygamain (8): White microcrystalline (acetone), mp 259–260 °C; [α]30 D + 14.0 (c 0.5, CHCl3); IR (KBr), νmax (cm−1): 3494, 3444, 1750, 1624, 1479, 1444, 1253, 1179, 1036; UV (CHCl3) λmax nm (log ε): 215.4 (3.38), 240.4 (4.11), 290.5 (4.05); HRESI MS (positive ion mode) m/z 391.0786 [M + Na]+ " Tables 1 and 3. (calcd. 391.0794 for C20H16NaO7); NMR data see l
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10 Pham HH. An illustrated flora of Vietnam, 2nd ed. Volume II. Vietnam: NXB Tre, TP Ho CHi Minh; 2003: 231–235 11 Thanh VT, Pham VC, Doan TMH, Litaudon M, Gueritte F, Retailleau P, Nguyen VH, Chau VM. Cytotoxic lignans from fruits of Cleistanthus indochinensis: synthesis of cleistantoxin derivatives. J Nat Prod 2012; 75: 1578–1583 12 Ishii T, Yanagisawa M. Synthesis, separation and NMR spectral analysis of methyl apiofuranosides. Carbohydr Res 1998; 313: 189–192 13 Kitagawa I, Hori K, Sakagami M, Hashiuchi F, Yoshikawa M, Ren J. Saponin and sapogenol. XLIX. On the constituents of the roots of Glycyrrhiza inflata Batalin from Xinjiang, China. Characterization of two sweet oleanane-type triterpene oligoglycosides, apioglycyrrhizin and araboglycyrrhizin. Chem Pharm Bull 1993; 41: 1350–1357 14 Nakanishi T, Inatomi Y, Murata H, Shigeta K, Iida N, Inada A, Murata J, Farrera MA, Iinuma M, Tanaka T, Tajima S, Oku N. A new and known cytotoxic aryltetralin-type lignans from stems of Bursera graveolens. Chem Pharm Bull 2005; 53: 229–231 15 Silva R, Heleno VCG, Albuquerque S, Bastos JK, Silva MLA, Donate PM, Silva GV. Spectral assignments and reference data. Magn Reson Chem 2004; 42: 985–989
Trinh-Thi-Thanh V et al. Cytotoxic Aryltetralin Lignans …
16 Peraza-Sanchez SR, Pena-Rodriquez LM. Isolation of picropolygamain from the resin of Bursera simaruba. J Nat Prod 1992; 55: 1768–1771 17 Sheriha GM, Abouamer K, Elshtaiwi BZ, Ashour AS, Abed FA, Alhallaq HH. Quinoline alkaloids and cytotoxic lignans from Haplophyllum tuberculatum. Phytochemistry 1987; 26: 3339–3341 18 Klyne W, Stevenson R, Swan RJ. Optical rotatory dispersion. Part XXVIII. The absolute configuration of otobain and derivatives. J Chem Soc C 1966; 893–896 19 Zhao C, Nagatsu A, Hatano K, Shirai N, Kato S, Ogihara Y. New lignan glycosides from Chinese medicinal plant, Sinopodophillum emodi. Chem Pharm Bull 2003; 51: 255–261 20 Gu JQ, Park EJ, Totura S, Riswan S, Fong HH, Pezzuto JM, Kinghorn AD. Constituents of the twigs of Hernandia ovigera that inhibit the transformation of JB6 murine epidermal cells. J Nat Prod 2002; 65: 1065– 1068 21 Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Meth 1983; 65: 55–63 22 Evaristo IM, Leitao MC. Identification and quantification by DAD-HPLC of the phenolic fraction contained in the leaves of Quercus suber L. Silva Lusitana 2001; 9: 135–141
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695
Authors
Van Trinh Thi Thanh 1, Van Cuong Pham 1, Huong Doan Thi Mai 1, Marc Litaudon 2, Françoise Guéritte 2, Van Hung Nguyen 1, Van Minh Chau 1
Affiliations
1 2
Key words " Euphorbiaceae l " Cleistanthus indochinensis l " lignans l " glycosides l " cytotoxicity l
received revised accepted
February 7, 2014 February 11, 2014 April 22, 2014
Bibliography DOI http://dx.doi.org/ 10.1055/s-0034-1368505 Published online June 4, 2014 Planta Med 2014; 80: 695–702 © Georg Thieme Verlag KG Stuttgart · New York · ISSN 0032‑0943 Correspondence Dr. Van Cuong Pham Institute of Marine Biochemistry Vietnam Academy of Science and Technology 18, Hoang Quoc Viet Road Caugiay, Hanoi Vietnam Phone: + 84 04 37 56 49 95 Fax: + 84 04 37 91 70 54 [email protected] Correspondence Prof. Dr. Van Minh Chau Institute of Marine Biochemistry Vietnam Academy of Science and Technology 18, Hoang Quoc Viet Road Caugiay, Hanoi Vietnam Phone: + 84 04 37 56 49 95 Fax: + 84 04 37 91 70 54 [email protected]
Institute of Marine Biochemistry of the Vietnam Academy of Science and Technology (VAST), Cau Giay, Hanoi, Vietnam Institut de Chimie des Substances Naturelles, Gif-sur Yvette, France
Abstract !
Eight new aryltetralin lignans, cleisindosides A–F (1–6), picroburseranin (7), and 7-hydroxypicropolygamain (8), were isolated from the fruits of Cleistanthus indochinensis (Euphorbiaceae). The structures of the isolates were established on the basis of their one- and two-dimensional NMR spectral data, as well as their mass spectrometric data. Compound 7 was found to have potent cytotoxicity against oral epidermoid carcinoma cells with an IC50 value of 0.062 µM, whereas glycosylation to 3 (IC50 7.5 µM) and stereochemical changes to 8 (IC50 10.8 µM) led to marked decreases in biological activity. Thus, it was deter-
Introduction !
Cleistanthus is a plant genus of the Euphorbiaceae family and comprises approximately 140 species that are distributed across a variety of locations from Africa to the Pacific Islands [1]. Many plants of this genus are known for their toxic properties [2–6]. Cleistanthus has been shown to contain bioactive lignan compounds such as cleistanthins A and B (from Cleistanthus collinus); these were found to be cytotoxic against several tumor cell lines [7, 8]. Also, cleistanthins A and B inhibited the actions of the alpha adrenergic receptor and the nicotinic cholinergic receptor [9]. In Vietnam, 14 species of Cleistanthus have been identified and, importantly, some of them were used in traditional medicine [10]. Previously, we reported the bioassay-guided purification of two new cytotoxic lignans, cleistantoxin and neocleistantoxin, from the dichloromethane extract of the fruits of Cleistanthus indochinensis [11]. Cleistantoxin was found to be significantly active against several cancer cell lines and its amide derivatives were synthesized and evaluated for their cytotoxicity. In the continuation of research on more polar
mined that the C-7 and C-8′ positions are critical for the biological activity of the lignans from this plant.
Abbreviations !
ATCC: MTT: FBS:
American Type Cell Culture Collection 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide fetal bovine serum
Supporting information available online at http://www.thieme-connect.de/products
fractions, the present paper describes the bioassay-guided purification of eight new lignans from the methanol extract of the fruits of C. indochinensis, which inhibits the growth of oral epidermoid carcinoma (KB) cells (73% at a concentration of 10 µg/mL).
Results and Discussion !
The dried and milled fruits of C. indochinensis (100 g) were extracted successively with CH2Cl2 and MeOH at room temperature. The MeOH soluble secondary metabolites were separated using open column chromatography (CC) on silica gel. The resulting fractions were tested against KB cells and the active fractions were further purified " Fig. 1). by CC to afford compounds 1–8 (l Compound 1 was obtained as a white microcrystalline solid and was optically active, [α]30 D − 50.0 (c 0.3, MeOH). Its 1H NMR spectrum presented signals of two singlet aromatic protons at δH = 6.50 (H-3) and 7.04 (H-6), and an ABX ring system, which was characterized by three aromatic protons at δH = 6.54 (H-2′), 6.75 (H-5′), and
Trinh-Thi-Thanh V et al. Cytotoxic Aryltetralin Lignans …
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Cytotoxic Aryltetralin Lignans from Fruits of Cleistanthus indochinensis
Original Papers
Fig. 1
6.37 (H-6′). Also, signals of a sugar moiety, two methylenedioxy groups, a methylene, and three methines were noted in the aliphatic region. The 13C NMR and DEPT spectra showed the resonances corresponding to the functional groups observed in the 1 H NMR spectrum, with additional signals of a carboxylate group and seven aromatic quaternary carbons. The chemical shifts of C4, C-5, C-7, C-9, C-3′, and C-4′ suggested their linkage to an oxygen atom. Analysis of the 1H-1H COSY spectrum revealed the spin-spin coupling systems as follows: connection of sugar protons; correlations of the ABX system; and cross-peaks of a proton at δH = 2.85 (H-8) to the protons at δH = 5.02 (H-7), 4.32 and 4.36 (CH2-9), and 3.39 (H-8′). The resonance for H-8′ correlated to the proton at δH = 4.52 (H-7′). The planar structure of 1 was then established by analysis of its HMBC spectrum. The ABX aromatic ring (A-ring) was bonded to C-7′, as indicated by correlations of the carbon resonance at δC = 42.6 (C-7′) with protons H-2′ and H-6′. Correlations of the carbonyl carbon resonance at δC = 174.8 (C-9′) with protons CH2-9 and H-7′ indicated the presence of a lactone ring. The location of the methylenedioxy at C-5 and C-4 was revealed by correlations of protons at δH = 6.01 and 6.02 (CH2-10) with C-5 at δC = 147.8 and C-4 at δC = 146.1. Similarly, the linkage of the methylenedioxy at δH = 5.94 and 5.95 (CH210′) to carbons C-3′ and C-4′ was determined from their HMBC cross-peaks. Finally, the sugar moiety was linked to C-7, as determined by a correlation of C-7 with the anomeric proton at δH = 4.25 (H-1′′). The relative configuration of 1 was then established by the analysis of coupling constants and a NOESY experiment. Proton H-8′ afforded a strong (J = 14.5 Hz) and a small (J = 5.5 Hz) coupling constant while H-7′ appeared as a doublet (J = 5.5 Hz), depicting a trans-pseudodiaxial relationship between H-8 and H-8′ and a pseudoequatorial orientation for H-7′. In addition, H-7 had a gauche (J = 3.5 Hz) coupling constant, and thus a pseudoequatorial disposition. The β-glucopyranose was assigned to the sugar from coupling constant analysis of all sugar protons " Table 1). This was confirmed by acidic hydrolysis of 1 (l " Fig. 2). The hydrolyzed sugar had a positive optical rotation (l value [α]28 D + 51.0 (c 0.15, H2O) and an identical Rf value with the standard D-(+)-glucose, revealing the D-configuration for the glucose moiety of 1. This compound was named cleisindoside A. The 1D NMR spectra of compound 2 displayed signals similar to those of 1, with additional signals of a second carbohydrate moiety at δC = 111.1 (d, C-1′′′), 78.3 (d, C-2′′′), 80.5 (s, C-3′′′), 75.0 (d, C4′′′), and 65.6 (t, C-5′′′) that strongly suggested the presence of an apiofuranose moiety. Analyses of 2D NMR experiments and pro-
Trinh-Thi-Thanh V et al. Cytotoxic Aryltetralin Lignans …
Structures of compounds 1–8.
" Table 1) depicted structural similarton coupling constants (l ities with 1, namely the lignan aglycone and the β-glucopyranose moieties. The bonding of O-β-glucopyranose to C-7 was established from an HMBC cross-peak of the anomeric carbon at δC = 101.8 (C-1′′) to the proton at δH = 5.10 (H-7), and the linkage of the O-apiofuranose to C-6′′ of the glucopyranose by the correlation of C-6′′ at δC = 69.3 to H-1′′′ at δH 5.07. The downfield chemical shift of the anomeric carbon at δC = 111.1 (C-1′′′) suggested a β-form for the apiofuranose [12, 13]. In addition, the glucose and apiose from the hydrolysis of 2 gave positive specific rotations, indicating their D-configuration. This aryltetralin lignan glycoside was named cleisindoside B. Some similarities existed between the 1H and 13C NMR spectra of compound 3 with those of 1 and 2. In particular, differences existed in the saccharide side chain. Analyses of 2D NMR spectra of 3 revealed an identical lignan aglycone and a β-glucopyranose moiety, while the remaining signals were distributed to a β-xylopyranose linking to C-6′′ of the β-glucopyranose, as indicated by an HMBC cross-peak of a carbon at δC = 69.4 (C-6′′) with a proton at δH = 4.19 (H-1′′′). The hydrolyzed glucose and xylose had a positive specific optical rotation, thus assigning the D-configuration for both the glucopyranose and xylopyranose moieties of 3 (cleisindoside C). Comparison of the 1H NMR spectrum of 4 with that of cleistantoxin [11] revealed a structural similarity with additional signals of a β-glucopyranose unit [δH = 4.34 (d, J = 7.5 Hz, H-1′′), 3.28 (dd, J = 7.5 and 8.5 Hz, H-2′′), 3.39 (dd, J = 8.5 and 8.5 Hz, H-3′′), 3.41 (dd, J = 8.5 and 8.5 Hz, H-4′′), 3.18 (m, H-5′′), and 3.67 (m, CH26′′)]. Analysis of its HMBC spectrum determined the linkage of β-glucopyranose to C-7 by a cross-peak of C-1′′ at δC = 99.4 with H-7 at δH = 5.28. A trans-pseudodiaxial relationship between H7 and H-8 was established from their anti-coupling constant (J = 8.5 Hz). H-8′ appeared as a doublet of doublets with strong (J = 15.0 Hz) and small (J = 4.0 Hz) coupling constants, whereas H-7′ had a gauche coupling constant (J = 4.0 Hz). This indicated that H-7′ contained a pseudoequatorial orientation and H-8′ was pseudoaxial. Thus, in comparison with compounds 1–3, an epimerization at C-7 was observed for 4. The D-configuration was assigned for the glucopyranose in 4, since its hydrolyzed glucose had a positive optical rotation value. This compound was reported here for the first time and named cleisindoside D. The 1D NMR signals of compound 5 were similar to those of 4. The significant differences were the presence of an additional acetyl group and the CH2-6′′ signals of the sugar moiety being
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Original Papers
3 6 7 8 9 10 2′ 5′ 6′ 7′ 8′ 10′ 1′′ 2′′ 3′′ 4′′ 5′′ 6′′
1a
2b
3a
8c
6.50 s 7.04 s 5.02 d (3.5) 2.85 dddd (3.5, 8.5, 9.0, 14.5) 4.32 dd (8.5, 8.5) 4.36 dd (8.5, 9.0) 6.01 s 6.02 s 6.54 d (1.5) 6.75 d (8.0) 6.37 dd (1.5, 8.0) 4.52 d (5.5) 3.39 dd (5.5, 14.5) 5.94 s 5.95 s 4.25 d (8.0) 3.01 ddd (4.0, 8.0, 8.0) 3.11 m 3.03 ddd (4.0, 8.0, 9.0) 3.12 m 3.46 dd (6.0, 11.0) 3.76 dd (6.0, 11.0)
6.48 s 7.09 s 5.10 d (3.5) 3.02 dddd (3.5, 8.2, 10.5, 14.3) 4.38 dd (8.2, 8.2) 4.52 dd (8.2, 10.5) 5.96 d (1.0) 5.97 d (1.0) 6.57 d (1.5) 6.67 d (8.0) 6.48 dd (1.5, 8.0) 4.56 d (5.7) 3.53 dd (5.7, 14.3) 5.88 d (1.0) 5.89 d (1.0) 4.48 d (8.0) 3.24 dd (8.0, 9.0) 3.35 dd (9.0, 9.0) 3.29 dd (9.0, 9.0) 3.47 ddd (2.0, 7.0, 9.0) 3.63 dd (7.0, 11.5) 4.10 dd (2.0, 11.5) 5.07 d (2.7) 3.98 d (2.7)
6.49 s 7.08 s 4.98 d (3.0) 2.83 m
6.11 s 6.99 s 4.26 d (10.0) 2.47 m
4.31 dd (8.0, 8.0) 4.38 dd (8.0, 8.0) 6.01 br. s 6.02 br. s 6.54 d (1.5) 6.75 d (8.0) 6.38 dd (1.5, 8.0) 4.51 d (5.5) 3.35 dd (5.5, 14.5) 5.94 br. s 5.95 br. s 4.33 d (8.0) 3.00 m 3.13 dd (9.0, 9.0) 3.02 m 3.32 m 3.51 dd (8.0, 11.0) 4.05 br. d (11.0) 4.19 d (7.5) 3.01 m 3.11 dd (9.0, 9.0) 3.31 m
4.28 dd (6.2, 9.5) 4.49 dd (1.0, 9.5) 5.75 d (1.5) 5.76 d (1.5) 6.57 d (1.5) 6.68 d (8.0) 6.62 dd (1.5, 8.0) 3.83 d (6.2) 3.00 dd (6.2, 9.0) 5.82 br. s 5.83 br. s
1′′′ 2′′′ 3′′′ 4′′′
3.83 d (10.0) 4.05 d (10.0) 3.62 m
5′′′′ OH-2′′ OH-3′′ OH-4′′ OH-6′′ a
Table 1 1H NMR data for compounds 1–3 and 8 (500 MHz).
3.05 dd (11.5, 11.5) 3.73 dd (5.5, 11.5)
4.97 d (4.0) 4.89 m 4.89 m 4.63 t (6.0)
DMSO-d6; b CD3OD; c CDCl3+CD3OD
shifted downfield. Careful analysis of the 2D NMR spectra allowed for the determination of the structure of 5, which was an acetylated derivative of 4. The location of the acetyl group at C-6′′ was defined by HMBC correlations between the carbonyl of the acetyl group at δC = 171.1 with protons at δH = 4.16 and 4.27 (CH2-6′′). Analysis of the proton coupling constants of 5 indicated an identical stereochemistry with 4. On the basis of these data, " Fig. 1 and was the structure of 5 was identified as indicated in l named cleisindoside E. The 1H and 13C NMR spectra of 6 also indicated the presence of a β-glucopyranose unit characterized from carbons at δC = 103.0 (d, C-1′′), 73.2 (d, C-2′′), 75.9 (d, C-3′′), 69.8 (d, C-4′′), 76.9 (d, C-5′′), and 60.6 (t, C-6′′). Comparison of the 1D NMR signals of the aglycon moiety of 6 with those of 1–3 depicted the presence of a double bond and a methylene as opposed to three sp3 methines and the absence of a methylenedioxy group. Furthermore, examination of 1H-1H COSY data allowed the assignment of a spin-spin coupling system from CH2-7 (δH = 2.69 and 2.71) to CH2-9 (δH = 3.97 and 4.64) via H-8 (δH = 3.31). These data suggested the presence of the double bond at C-7′ and C-8′ in the structure of 6. " Fig. 3), Analysis of 2D NMR data established the structure of 6 (l in which the β-glucopyranose moiety was linked to C-3′ of the Aring, as indicated by an HMBC cross-peak of C-3′ at δC = 144.4 with the anomeric proton H-1′′ at δH = 4.67. The D-configuration for the glucopyranose moiety of 6 was assigned from the positive
Trinh-Thi-Thanh V et al. Cytotoxic Aryltetralin Lignans …
Fig. 2 Key HMBC correlations for 1.
Planta Med 2014; 80: 695–702
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Pos.
697
698
Original Papers
specific optical rotation of its hydrolyzed glucose. This new lignan glucoside was named cleisindoside F. Analysis of the 1H and 13C NMR spectra of 7 revealed the same planar structure with burseranin, which was previously isolated from Bursera graveolens [14]. However, H-8′ of 7 presented small (J = 4.5 Hz) and strong (J = 14.0 Hz) coupling constants, whereas these values were J = 3.0 and 9.5 Hz for burseranin (C/D cis-fused junction). This observation clearly suggested a C/D trans-fusion " Table 2) for 7 [15]. Analysis of the proton coupling constants (l and NOESY data indicated that 7 contained the same stereochemistry at C-8, C-8′, and C-7′ as compounds 1–5, and was named picrobursenin, as it appeared to be an 8′-epimer of burseranin.
Compound 8 was obtained as a white microcrystalline material and was optically active [α]30 D + 14.0 (c 0.5, CHCl3). Its HRESI mass spectrum showed the pseudomolecular [M + Na]+ ion at m/z 391.0786, indicating the molecular formula of C20H16O7 (calcd. 391.0794 for C20H16NaO7). The planar structure of 8 was elucidated by analysis of 2D NMR experiments, in which the loss of a methoxy group at C-6 was observed for 8, as compared to cleistantoxin. Coupling constant analysis of the aliphatic protons demonstrated that H-7 had an anti-coupling (J = 10.0 Hz) with H-8, suggesting that their trans-pseudodiaxial relationship was identical to cleistantoxin. In contrast, H-7′ had a gauche coupling (J = 6.2 Hz) and H-8′ was a doublet of doublets with two coupling constants (J = 9.0 and 6.2 Hz). Thus, the coupling constant between H-8′ and H-8 was J = 9.0 Hz (this value was J = 15.0 Hz for cleistantoxin), assigning a cis-fused junction for the C/D rings
4c
5d
6e
7f
3 6 7
6.26 s
6.34 s
6.24 s
5.28 d (8.5)
5.27 d (10.5)
8 9
3.04 m 4.07 dd (8.2, 8.2) 4.64 dd (8.2, 8.2) 5.89 s 5.93 s 6.65 m 6.69 m 6.65 m 4.45 d (4.0) 2.70 dd (4.0, 15.0) 5.83 s 5.84 s 4.34 d (7.5) 3.28 dd (7.5, 8.5) 3.39 dd (8.5, 8.5) 3.41 dd (8.5, 8.5) 3.18 m 3.67 m
3.20 m 4.06 dd (1.5, 8.0) 4.65 dd (8.0, 8.0) 5.99 d (1.0) 5.98 (1.0) 6.65 d (1.5) 6.73 d (8.0) 6.76 dd (1.5, 8.0) 4.54 d (4.5) 2.68 dd (4.5, 14.5) 5.92 s
6.39 s 6.94 s 2.69 dd (6.5, 15.0) 2.71 dd (15.0, 15.0) 3.31 m 3.97 dd (8.5, 8.5) 4.64 dd (8.5, 8.5) 5.99 d (1.0) 6.00 d (1.0) 7.11 d (1.5) 6.86 d (8.0) 6.74 dd (1.5, 8.0)
Pos.
10 2′ 5′ 6′ 7′ 8′ 10′ 1′′ 2′′ 3′′ 4′′ 5′′ 6′′ 2′′′ OMe c
Fig. 4 Key NOE correlations for 8.
3.99 s
4.38 d (7.5) 3.39 m 3.53 dd (8.5, 8.5) 3.35 dd (8.5, 8.5) 3.38 m 4.16 dd (7.0, 11.0) 4.27 dd (2.0, 11.0) 1.65 s 4.07 s
4.67 d (8.0) 3.33 dd (8.0, 8.0) 3.28 m 3.24 m 3.21 m 3.48 m 3.64 m
CDCl3+CD3OD; d CDCl3; e DMSO-d6, 343 K; f acetone-d6
Trinh-Thi-Thanh V et al. Cytotoxic Aryltetralin Lignans …
2.49 dd (11.0, 16.2) 3.19 dd (5.5, 16.2) 2.63 m 4.00 dd (8.5, 10.0) 4.46 dd (7.5, 8.5) 5.94 d (1.0) 5.95 d (1.0) 6.65 d (1.5) 6.68 d (8.5) 6.53 dd (1.5, 8.5) 4.53 d (4.5) 2.82 dd (4.5, 14.0) 5.92 d (1.0) 5.93 d (1.0)
Planta Med 2014; 80: 695–702
4.03 s
Table 2 1H NMR data for compounds 4–7 (500 MHz).
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Fig. 3 Selected HMBC cross-peaks for 6.
Original Papers
699
[15], which was clearly confirmed by a strong interaction be" Fig. 4). In compartween H-8 and H-8′ in the NOESY spectrum (l ison with other compounds from this plant, an epimerization at C-8′ was observed for 8. This compound was named 7-hydroxypicropolygamain as it had a structural similarity with picropolygamain [16, 17]. The absolute configuration at C-7′ was established by examination of the circular dichroism (CD) spectra of compounds 2, 4, 7, and 8, which afforded positive Cotton effects at 293–297 nm (Fig. 68S, Supporting Information). Since the sign of the first couplet is determined by the configuration of the aryl substituent at C-7′, namely negative for 7′S and positive for 7′R, C-7′ were assigned as having the R-configuration for compounds 2, 4, 7, and 8 [18–20]. The isolates were tested for in vitro biological activity against KB cells. The most active compound was 7 with an IC50 of 0.062 µM, followed by 3 (IC50 7.5 µM) and 8 (IC50 10.8 µM). The remaining compounds exhibited weaker cytotoxicity (IC50 > 20.0 µM). Taxotere was used as a positive control and had an IC50 of 0.12 nM. A comparison of cytotoxicity values between cleistantoxin (IC50 0.022 µM) and 7 revealed that the presence of an OH group at C7 seems to slightly increase biological activity. On the contrary, the presence of sugar moieties at C-7 significantly decreased their cytotoxicity, especially when comparing cytotoxicity values of cleistantoxin and 7 with those of 4 and 5. Finally, it is apparent that a stereochemical change at C-8′ of the lactone ring led to a major decrease in biological activity, indicating the importance of this moiety (via a comparison of cleistantoxin and 7 cytotoxicities with those of 6 and 8).
Concentrations of cleistantoxin and cleisindoside B (2) in the fruit of C. indochinensis were analyzed by means of HPLC/DAD. The compounds were identified through comparison of their retention times and corresponding UV/DAD absorption spectrum with " Fig. 5). The results indicated that those of the pure compounds (l the cleistantoxin content was 178 mg/g in the CH2Cl2 crude extract, while the content of cleisindoside B (2) was 8.56 mg/g in the MeOH crude extract.
Materials and Methods !
General Melting points were recorded on a Buchi B-545 instrument and are uncorrected. Optical rotations were recorded on a Polax-2L polarimeter in CHCl3. UV spectra were recorded on a UV-1601 spectrometer. IR spectra were measured on a Nicolet Impact410 FT‑IR spectrometer. CD spectra were measured on a JASCO J-810 spectrophotometer. Quantitative analysis of cleistantoxin from the CH2Cl2 extract and from 2 in the MeOH extract was carried out by an Agilent 1200 Series HPLC‑DAD instrument. 1H and 13 C NMR spectra were recorded on a Bruker AM500 FT‑NMR spectrometer as indicated with either the CDCl3 (δH 7.24 ppm, δC 77.0 ppm), CD3OD (δH 3.30 ppm, δC 49.0 ppm), DMSO-d6 (δH 2.49 ppm, δC 39.5 ppm), or acetone-d6 (δH 2.04 ppm, δC 29.8 and 206.0 ppm) signal as the internal standard. J values are expressed in Hz. The HMBC measurements were optimized to 7.0 Hz longrange couplings, and NOESY experiments were run with a 150ms mixing time. High-resolution ESIMS were measured on a VARIAN 910 spectrometer.
Trinh-Thi-Thanh V et al. Cytotoxic Aryltetralin Lignans …
Planta Med 2014; 80: 695–702
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Fig. 5 HPLC chromatograms: A CH2Cl2 crude extract, B MeOH crude extract. (Color figure available online only.)
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a
13
C NMR data for compounds 1–8 (125 MHz).
Position
1a
2b
3a
4c
5d
6e
7f
8c
1 2 3 4 5 6 7 8 9 10 1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′ 9′ 10′ 1′′ 2′′ 3′′ 4′′ 5′′ 6′′ 1′′′ 2′′′ 3′′′ 4′′′ 5′′′ OMe
128.2 133.0 109.9 146.1* 147.8* 110.2 70.4 37.2 67.8 101.3 134.3 110.8 145.9 146.6 107.4 123.5 42.6 40.1 174.8 100.8 99.8 73.6 76.6 70.4 77.0 61.3
134.6 129.8 111.2 150.0* 148.3* 111.2 73.2 39.1 69.9 102.8 135.4 112.0 148.6 147.9 108.4 125.0 44.7 42.1 177.7 102.3 101.8 75.1 78.0 72.0 76.9 69.3 111.1 78.3 80.5 75.0 65.6
126.8 132.8 109.7 147.7* 146.2* 110.3 71.0 37.1 67.9 101.2 134.2 110.7 146.6 145.9 107.4 123.5 42.5 40.2 174.7 100.8 100.3 73.4 76.5 70.5 75.2 69.4 104.4 73.4 76.6 69.5 65.6
122.0 135.3 104.8 149.9 136.8 142.1 75.2 38.4 72.0 101.5 132.8 107.6 147.3 146.6 110.7 123.8 44.0 45.5 174.2 100.9 99.4 73.5 76.3 69.8 76.0 61.5
121.7 136.1 105.5 150.4 137.1 142.0 77.0 37.6 72.0 101.8 133.1 110.8 147.6 147.0 108.0 124.1 44.4 45.6 174.0 101.3 98.1 73.4 76.7 70.2 74.2 63.3 171.1 20.1
131.1 129.3 108.0 147.9* 145.9* 108.4 32.0 35.0 70.2 125.3 125.3 119.9 144.4 147.2 115.1 124.6 145.4 120.0 167.5
122.5 133.2 105.0 149.1 135.9 141.8 27.7 33.1 72.8 101.8 136.1 111.9 148.0 147.2 108.0 124.9 44.2 47.3 175.3 101.9
133.4 130.7 108.3 146.4 146.4 104.4 67.7 42.5 69.6 100.7 137.0 108.8 146.5 147.9 108.2 122.1 43.1 45.4 178.6 100.9
59.9
60.2
b
c
d
e
103.0 73.2 75.9 69.8 76.9 60.6
59.7
f
DMSO-d6; CD3OD; CDCl3+CD3OD; CDCl3; DMSO-d6, 343 K; acetone-d6; * chemical shifts should be exchanged with each other in the same column
Plant material The fruits of C. indochinensis were collected at Quytrau – Nghean (Vietnam) in May 2003, and a specimen (VN 1086) was deposited at the Institute of Ecology and Natural Resources, Vietnam Academy of Science and Technology (VAST). The plant was identified by Dr. Nguyen Quoc Binh of the Institute of Ecology and Natural Resources, VAST.
Extraction and isolation Dried and ground fruits of C. indochinensis (100 g) were extracted successively with CH2Cl2 (4 × 300 mL, 1 h each) and MeOH (4 × 300 mL, 1 h each) at room temperature in a sonicator. The solvents were removed under diminished pressure to give residues of 4.0 g (CH2Cl2) and 5.5 g (MeOH). The MeOH soluble secondary metabolites (5.5 g) were separated using reversed-phase (RP-18) CC (5 × 60 cm), eluted with a mixture of MeOH/H2O (2.5 L, 10 to 100 % MeOH in H2O) to provide 11 fractions. A cytotoxicity assay against KB cells revealed two active fractions (4 and 8). Fraction 4 (250 mL, 335 mg) was washed with MeOH to obtain 3 (9 mg). The filtrate (35 mL) was concentrated to dryness and purified by CC on silica gel (40–63 µm, 2 × 50 cm, 5% MeOH in CH2Cl2), providing 4 subfractions. Subfraction 1 (150 mL, 65 mg) was subjected to a Sephadex LH-20 column (1 × 50 cm, MeOH) to yield 4 (18 mg). Subfraction 2 (110 mL, 124 mg) was washed with MeOH affording 1 (10 mg) and the filtrate (20 mL) was purified by CC on Sephadex LH-20 (1 × 50 cm, MeOH) to provide 5 (13 mg).
Trinh-Thi-Thanh V et al. Cytotoxic Aryltetralin Lignans …
Subfraction 3 (150 mL, 75 mg) was recrystallized in MeOH (1 mL) to provide 6 (9 mg) and subfraction 4 (240 mL, 83 mg) was chromatographed on Sephadex LH-20 column (1 × 50 cm, 80 mL, MeOH) to obtain 2 (11 mg). Fraction 8 (200 mL, 106 mg) was subjected to CC on silica gel (1 × 50 cm, 1–20% MeOH in CH2Cl2) affording 7 (15 mg) and 8 (5 mg). Cleisindoside A (1): White microcrystalline (acetone/MeOH), mp −1 261–262 °C; [α]30 D − 50.0 (c 0.3, MeOH); IR (KBr), νmax (cm ): 3444, 2910, 1773, 1626, 1491, 1244, 1382, 1080, 1034; UV (MeOH) λmax nm (log ε): 215.4 (4.32), 287.5 (3.98); HRESI MS (positive ion mode) m/z 553.1323 [M + Na]+ (calcd. 553.1322 for " Tables 1 and 3. C26H26NaO12); NMR data see l Cleisindoside B (2): White microcrystalline (acetone/MeOH), mp −1 248–249 °C; [α]30 D − 52.0 (c 1.0, MeOH); IR (KBr), νmax (cm ): 3421, 2929, 1764, 1624, 1490, 1244, 1039; UV (MeOH) λmax nm (log ε): 215.0 (4.36), 238.0 (3.96), 288.8 (3.96); HRESI MS (positive ion mode) m/z 685.1746 [M + Na]+ (calcd. 685.1745 for " Tables 1 and 3. C31H34NaO16); NMR data see l Cleisindoside C (3): White microcrystalline (acetone/MeOH), mp −1 278–279 °C; [α]30 D − 65.0 (c 0.9, MeOH); IR (KBr), νmax (cm ): 3418, 2916, 1752, 1624, 1487, 1234, 1036; UV (MeOH) λmax nm (log ε): 216.0 (3.86), 235.0 (3.15), 286.8 (3.12); HRESI MS (positive ion mode) m/z 685.1752 [M + Na]+ (calcd. 685.1745 for " Tables 1 and 3. C31H34NaO16); NMR data see l Cleisindoside D (4): White microcrystalline (acetone/MeOH), mp −1 261–262 °C; [α]30 D − 188.0 (c 0.5, MeOH); IR (KBr), νmax (cm ):
Planta Med 2014; 80: 695–702
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Table 3
Original Papers
Acid hydrolysis of 1–6 Each solution of 1–6 (each 5 mg) was heated at 95 °C for 5 h in 1 N HCl (dioxane – H2O, 1 : 1, 2.5 mL). The filtrates from the hydrolysate were neutralized with DOWEX HCR‑S ion-exchange resin and filtered. A portion of the filtrate was concentrated under reduced pressure and examined for carbohydrates by silica gel TLC [Kieselgel 60 (Merck Art 5554), i-PrOH/Me2CO/H2O (5 : 3 : 1)] using authentic samples. The Rf values of each sugar were as follows: glucose, 0.41; xylose, 0.48; and apiose, 0.53. The remaining filtrate was concentrated under reduced pressure and purified by preparative TLC. The sugars were identified as D-glucose {[α]28 D + 51 (c 0.15, H2O)}, D-apiose {[α]28 D + 12 (c 0.08, H2O)}, and D-xylose {[α]28 D + 25 (c 0.09, H2O)}.
Cytotoxic activity assay The human KB tumor (oral epidermoid carcinoma) cell line was obtained from ATCC. KB cells were maintained in Dulbeccoʼs D‑MEM medium, supplemented with 10% fetal calf serum, L-glutamine (2 mM), penicillin G (100 UI/mL), streptomycin (100 µg/ mL), and gentamicin (10 µg/mL). Stock solutions of compounds were prepared in DMSO/H2O (1/9), and cytotoxicity assays were carried out in 96-well microtiter plates against human nasopharynx carcinoma KB cells (3 × 103 cells/mL) using a modification of the published method [21]. After 72 h incubation at 37 °C in air/ CO2 (95 : 5) with or without the test compounds, cell growth was estimated by colorimetric measurement of stained living cells by neutral red. Optical density was determined at 540 nm with a Titertek Multiscan photometer. The IC50 value was defined as the concentration of sample necessary to inhibit the cell growth to 50 % of the control. Taxotere (in-house product, purity > 99 %) was used as a reference compound. The purity of the tested compounds was > 95 % as observed from their 1H NMR spectra (Supporting Information).
HPLC‑DAD quantitative analysis of cleistantoxin and cleisindoside B (2) High-performance liquid chromatography was performed with an Agilent 1200 Series. UV‑VIS detector DAD and Software Agilent ChemStation were used. Reverse-phase chromatography analyses were carried out using a Zobax eclipse XDB C-18 column (4.6 mm × 250 mm) packed with 5 µm diameter particles. The volume injection was 5 µL and the gradient elution was conducted according to the Evaristo and Leitao method, with minor modifications [22]. The mobile phase consisted of water (solvent A) and methanol (solvent B). Stock solutions of cleistantoxin and 2 were prepared in methanol in the range of 6.25–1000 µg/mL. Samples and standard solutions, as well as the mobile phase, were degassed and filtered through a 0.45-µm membrane filter (Millipore). Identification of the compounds was done by comparison of their retention times and UV absorption spectrum with those of the pure compounds cleistantoxin and 2.
Supporting information HRESI MS and 1D and 2D NMR spectra of compounds 1–8 are available as Supporting Information.
Acknowledgements !
The authors thank Mr. Dao Dinh Cuong and Dr. Nguyen Quoc Binh (VAST – Vietnam) for plant collection and botanical determination. The Centre National de la Recherche Scientifique (CNRS, France) is gratefully acknowledged for the French Vietnamese Laboratory of Natural Products Chemistry (NATPROCHEMLAB) and The Vietnam National Foundation for Science and Technology Development (NAFOSTED) is acknowledged for financial support (Grant No: 104.01.75.09).
Conflict of Interest !
The authors declare no conflict of interest.
References 1 Pinho PMM, Kijjoa A. Chemical constituents of the plants of the genus Cleistanthus and their biological activity. Phytochem Rev 2007; 6: 175– 182 2 Ramesh C, Ravindranath N, Ram TS, Das B. Arylnaphthalide lignans from Cleistanthus collinus. Chem Pharm Bull 2003; 51: 1299–1300 3 Eswarappa S, Chakraborty AR, Palatty BU, Vasnaik M. Cleistanthus collinus poisoning: case reports and review of the literature. J Toxicol Clin Toxicol 2003; 41: 369–372 4 Govindachari TR, Sathe SS, Viswanathan N, Pai BR, Srinivasan M. Chemical constituents of Cleistanthus collinus (Rox.). Tetrahedron 1969; 25: 2815–2821 5 Anjaneyulu ASR, Ramaiah PR, Row LR, Venkateswarlu R, Pelter A, Ward RS. New lignans from the heartwood of Cleistanthus collinus. Tetrahedron 1981; 37: 3641–3652 6 Sastry KV, Rao EV, Buchanan JG, Sturgeon RJ. Cleistanthoside B, a diphyllin glycoside from Cleistanthus patulus heartwood. Phytochemistry 1987; 26: 1153–1154 7 Pradheepkumar CP, Shanmugam G. Anticancer potential of cleistanthin A isolated from the tropical plant Cleistanthus collinus. Oncol Res 1999; 11: 225–232 8 Prabhakaran C, Kumar P, Panneerselvam N, Rajesh S, Shanmugam G. Cytotoxic and genotoxic effects of cleistanthin B in normal and tumour cells. Mutagenesis 1996; 11: 553–557 9 Parasuraman S, Raveendran R. Effect of cleistanthin A and B on adrenergic and cholinergic receptors. Pharmacogn Mag 2011; 7: 243–247
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Planta Med 2014; 80: 695–702
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3419, 2932, 1772, 1619, 1475, 1235, 1073, 1035; UV (MeOH) λmax nm (log ε): 222.7 (4.37), 286.7 (3.82); HRESI MS (positive ion mode) m/z 583.1409 [M + Na]+ (calcd. 583.1428 for " Tables 2 and 3. C27H28NaO13); NMR data see l Cleisindoside E (5): White microcrystalline (acetone/MeOH), mp −1 265–266 °C; [α]30 D − 196.0 (c 0.5, MeOH); IR (KBr), νmax (cm ): 3427, 2925, 1775, 1740, 1615, 1479, 1373, 1236, 1077, 1035; UV (MeOH) λmax nm (log ε): 212.2 (3.59), 239.8 (4.19), 287.5 (3.82); HRESI MS (positive ion mode) m/z 625.1523 [M + Na]+ (calcd. " Tables 2 and 3. 625.1533 for C29H30NaO14); NMR data see l Cleisindoside F (6): White microcrystalline (acetone/MeOH), mp −1 261–262 °C; [α]30 D − 50.0 (c 0.3, MeOH); IR (KBr), νmax (cm ): 3463, 2899, 1725, 1636, 1598, 1508, 1359, 1207, 1074, 1036; UV (MeOH) λmax nm (log ε): 250.0 (3.71), 348.1 (3.33); HRESI MS (positive ion mode) m/z 523.1225 [M + Na]+ (calcd. 523.1216 for " Tables 2 and 3. C25H24NaO11); NMR data see l Picrobursenin (7): White microcrystalline (acetone), mp 186– −1 188 °C; [α]30 D − 100.5 (c 0.55, CHCl3); IR (KBr), νmax (cm ): 3461, 2894, 2845, 1773, 1616, 1474, 1387, 1248, 1038; UV (MeOH) λmax nm (log ε): 227.8 (2.19), 286.3 (1.77); HRESI MS (positive ion mode) m/z 405.0948 [M + Na]+ (calcd. 405.0950 for " Tables 2 and 3. C21H18NaO7); NMR data see l 7-Hydroxypicropolygamain (8): White microcrystalline (acetone), mp 259–260 °C; [α]30 D + 14.0 (c 0.5, CHCl3); IR (KBr), νmax (cm−1): 3494, 3444, 1750, 1624, 1479, 1444, 1253, 1179, 1036; UV (CHCl3) λmax nm (log ε): 215.4 (3.38), 240.4 (4.11), 290.5 (4.05); HRESI MS (positive ion mode) m/z 391.0786 [M + Na]+ " Tables 1 and 3. (calcd. 391.0794 for C20H16NaO7); NMR data see l
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10 Pham HH. An illustrated flora of Vietnam, 2nd ed. Volume II. Vietnam: NXB Tre, TP Ho CHi Minh; 2003: 231–235 11 Thanh VT, Pham VC, Doan TMH, Litaudon M, Gueritte F, Retailleau P, Nguyen VH, Chau VM. Cytotoxic lignans from fruits of Cleistanthus indochinensis: synthesis of cleistantoxin derivatives. J Nat Prod 2012; 75: 1578–1583 12 Ishii T, Yanagisawa M. Synthesis, separation and NMR spectral analysis of methyl apiofuranosides. Carbohydr Res 1998; 313: 189–192 13 Kitagawa I, Hori K, Sakagami M, Hashiuchi F, Yoshikawa M, Ren J. Saponin and sapogenol. XLIX. On the constituents of the roots of Glycyrrhiza inflata Batalin from Xinjiang, China. Characterization of two sweet oleanane-type triterpene oligoglycosides, apioglycyrrhizin and araboglycyrrhizin. Chem Pharm Bull 1993; 41: 1350–1357 14 Nakanishi T, Inatomi Y, Murata H, Shigeta K, Iida N, Inada A, Murata J, Farrera MA, Iinuma M, Tanaka T, Tajima S, Oku N. A new and known cytotoxic aryltetralin-type lignans from stems of Bursera graveolens. Chem Pharm Bull 2005; 53: 229–231 15 Silva R, Heleno VCG, Albuquerque S, Bastos JK, Silva MLA, Donate PM, Silva GV. Spectral assignments and reference data. Magn Reson Chem 2004; 42: 985–989
Trinh-Thi-Thanh V et al. Cytotoxic Aryltetralin Lignans …
16 Peraza-Sanchez SR, Pena-Rodriquez LM. Isolation of picropolygamain from the resin of Bursera simaruba. J Nat Prod 1992; 55: 1768–1771 17 Sheriha GM, Abouamer K, Elshtaiwi BZ, Ashour AS, Abed FA, Alhallaq HH. Quinoline alkaloids and cytotoxic lignans from Haplophyllum tuberculatum. Phytochemistry 1987; 26: 3339–3341 18 Klyne W, Stevenson R, Swan RJ. Optical rotatory dispersion. Part XXVIII. The absolute configuration of otobain and derivatives. J Chem Soc C 1966; 893–896 19 Zhao C, Nagatsu A, Hatano K, Shirai N, Kato S, Ogihara Y. New lignan glycosides from Chinese medicinal plant, Sinopodophillum emodi. Chem Pharm Bull 2003; 51: 255–261 20 Gu JQ, Park EJ, Totura S, Riswan S, Fong HH, Pezzuto JM, Kinghorn AD. Constituents of the twigs of Hernandia ovigera that inhibit the transformation of JB6 murine epidermal cells. J Nat Prod 2002; 65: 1065– 1068 21 Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Meth 1983; 65: 55–63 22 Evaristo IM, Leitao MC. Identification and quantification by DAD-HPLC of the phenolic fraction contained in the leaves of Quercus suber L. Silva Lusitana 2001; 9: 135–141
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