Original Papers
Bisbenzylisoquinoline Alkaloids from the Roots of Cyclea tonkinensis
Authors
Jian-zhong Wang, Jing Liao, Wen-long Xu, Xiao-bing Chen
Affiliation
West China College of Pharmacy, Sichuan University, Chengdu, P. R. China
Key words " Cyclea tonkinensis l " Menispermaceae l " bisbenzylisoquinoline l alkaloid " cissampentine A l " cissampentine B l " cissampentine C l " cissampentine D l " cytotoxic activity l
Abstract !
Cissampentine A (1), an enantiomer of cissampentin, three new cycleatjehenine-type bisbenzylisoquinoline alkaloids, cissampentine B–D (2–4), and five known alkaloids were isolated from the roots of Cyclea tonkinensis. Their structures were established by interpretation of NMR, high-resolution ESI‑MS data, and CD spectra. In vitro stud-
Introduction !
received revised accepted
July 9, 2014 February 25, 2015 February 26, 2015
Bibliography DOI http://dx.doi.org/ 10.1055/s-0035-1545882 Published online April 9, 2015 Planta Med 2015; 81: 600–605 © Georg Thieme Verlag KG Stuttgart · New York · ISSN 0032‑0943 Correspondence Associate Prof. Dr. Jian-zhong Wang West China College of Pharmacy Sichuan University Department Medicinal Natural Products No. 17, Duan 3, Renmin Nan Road Chengdu 610041 Peopleʼs Republic of China Phone: + 86 28 85 50 37 70 [email protected]
Natural bisbenzylisoquinoline (BBI) alkaloids possess a number of interesting biological activities, including hypotensive, muscle relaxant, cytotoxic, antiplasmodial, and reverse multidrug-resistant activity [1, 2]. BBI alkaloids represent the largest subgroup of isoquinoline alkaloids, with over 470 members [3, 4], and are derived biogenetically via phenol-oxidative tail-to-tail or head-totail coupling of two units of benzylisoquinoline alkaloids. The two moieties are usually bound by one diaryl ether bridge or more. However, in rare cases, a methyleneoxy bridge or carbon-carbon bridges are present. Cyclea tonkinensis Gagnep (Menispermaceae) is a creeper growing in the southwest of China [5], whose roots are used in Chinese traditional medicine for the treatment of stomachaches and tooth pain. This paper investigated the chemical ingredients of the roots of C. tonkinensis and conducted a preliminary biological study. The chemical investigation led to the discovery of cissampentine A (1), an enantiomer of cissampentin, three new cycleatjehenine-type bisbenzylisoquinoline alkaloids, cissampentine " Fig. 1), and five known alkaloids, inB–D (2–4) (l cluding 7-O-methylhayatidine (5), (−)-curine (6), wattisine A (7), α-cyclanoline (8), and steponine (9) [6–8]. Cissampentin, incorporating an unusual methyleneoxy bridge, was reported by D. L. Galinis as a
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Planta Med 2015; 81: 600–605
ies indicated that compounds 1 and 4 exhibited cytotoxicity against the HCT-8 tumor cell line (IC50 values of 8.97 and 9.73 µM, respectively), and compound 4 was also active against the Bel7402 tumor cell line (IC50 value of 5.36 µM). Supporting information available online at http://www.thieme-connect.de/products
racemic mixture from Cissampelos fasciculate that exhibited significant activity as a repellent for leafcutter ants [9]. Unfortunately, the configurations of this mixture [(1-R, 1′-R), (1-S, 1′-S), or (1S, 1′-R), (1-R, 1′-S)] has not been determined. To date, only four cycleatjehenine-type alkaloids, including cissampentin, have been reported, and the stereochemistry of C-1 and C-1′ remain undetermined [3, 9]. The present study focused on the elucidation of the structures of cissampentines A– D (1–4) by 1D and 2D NMR techniques and highresolution ESI‑MS spectra. The absolute configurations of compounds 1–4 were confirmed by CD spectra. We also tested 1–4 for their cytotoxicity against the HCT-8, Bel-7402, and A2780 tumor cell lines.
Results and Discussion !
Compound 1 was isolated as a white powder. HRESIMS indicated [M + H]+ at m/z 609.2969 (calcd. 609.2965), corresponding to the molecular formula C37H41 N2O6. The IR spectrum suggested the presence of a hydroxyl group (3508 cm−1), aromatic rings (1616 and 1510 cm−1), and ether bonds (1274 and 1215 cm−1). The 13C NMR spectra of 1 in conjunction with the hydrogen data in " Table 1 revealed the presence of 14 quaternary, l 12 methine, 7 methylene, and 4 methyl signals for a total of 37 carbon resonances. Thirteen nonaro-
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matic carbon resonances were assigned to two N-methyl groups (δC 42.9; δH 2.27, s, NMe-2; δC 43.8; δH 2.48, s, NMe-2′), two aromatic methoxyl groups (δC 55.9; δH 3.87, s, OMe-6; δC 55.8; δH 3.79, s, OMe-6′), two aliphatic methines, and seven aliphatic methylene units [one oxymethylene (δH 4.58, 1H, d, J = 12.4 Hz; δH 5.06, 1H, d, J = 12.4 Hz; δC 76.8)]. These data suggested the presence of two tetrahydrobenzylisoquinoline units in 1. The ESIMS fragmentation peaks at m/z 312, 311, 298, 297, and 192, due to the fragment after the facile cleavage of the two benzylic bonds, were characteristics of head-to-tail BBI alkaloids (Fig. 38S, Supporting Information) [10]. The two tetrahydrobenzylisoquinoline moieties accounted for 18 degrees, and the one remaining degree of unsaturation was the result of an internal macrocycle caused by the formation of two bridges. The 1H NMR spectrum revealed the signals for one p-disubstituted benzene ring (δH 7.31, 2H, br.d, 8.0 Hz; δH 7.08, 2H, br. d, 8.0 Hz) and a 1,2,4-trisubstituted benzene ring (δH 6.78, 1H, d, 1.2 Hz; δH 6.86, 1H, d, 8.4 Hz; δH 6.90, 1H, dd, 8.4, 1.2 Hz). Another three isolated aromatic protons (δH 6.21, 6.63, 6.80, each 1H, s) could be assigned to two remaining tetrahydroisoquinoline moieties. The methine carbon signals at δC 61.3 and 63.7, characterized as C-1 and C-1′, implied that C-8 was oxygenated and that C-8′ bore a hydrogen; the δ values of C-1 were shifted to a higher field due to the C-8 oxygen γ-gauche effect [11]. With respect to the ether linkage involving a 7′-11 or a 7′-12, the key NOE correlations " Fig. 2) between H-10 and H-8′ implied that compound 1 pos(l sessed a 7′-11 ether linkage. This structure could be produced by a gradual rotation of ring C placing C-12 at the external part of the molecule, which creates a less hindered conformation and allows H-10 access to H-8′ [12]. The presence of a methyleneoxy bridge was indicated by the diagnostic AB system at δH 4.58 and 5.06 (J = 12.4 Hz). This linkage was assigned to C-7 and C-15′ due to the HMBC correlations between the signals at δH 4.58 and 5.06 and the δC 132.5 resonance. The methoxyl signal at δH 3.87 (δC 55.9) was assigned to OCH3-6, and another methoxyl hydrogen
δH 3.79 (δC 55.8) should be attached to C-6′ due to long-range correlations from 6-OCH3 to C-6 and from 6′-OCH3 to C-6′. For key " Fig. 2. HMBC correlations, see l Comparing the 13C‑NMR with those of the reported cissampentin compounds revealed that the compounds shared the same carbon signals [9]. Compound 1 exhibited negative specific rotations ([α]20 D − 85, CDCl3), whereas cissampentin was observed to be a racemic mixture, suggesting that compound 1 was an enantiomer of cissampentin. We collected the CD spectral data of headto-tail bisbenzylisoquinoline alkaloids with two ether bridges [3, 6, 7]. The compounds with the structures (1-R, 1′-R) and (1-S, 1′R) all exhibited a strong negative Cotton effect at 210–220 nm and had a weak negative Cotton effect at 285–295 nm. The compound with the 1-R, 1′-R exhibited a weak positive Cotton effect at 272–280 nm, whereas a weak shoulder negative Cotton effect curve was present in the same area for the compound with the 1S, 1′-R configuration. The Cotton effect curve of 1 closely resembled that of (−)-curine (1-R, 1′-R) [7], with a negative Cotton effect at 218 nm and a positive Cotton effect at 279 nm. Thus, we concluded that compound 1 had a 1-R, 1′-R configuration and named it cissampentine A. Therefore, we deduced that cissampentin was a racemic mixture with configurations of (1-R, 1′-R) and (1-S, 1′-S). Compound 2 was isolated as a white powder, [α]20 D − 90 (c 0.34, CHCl3). Its HR-ESIMS indicated [M + H]+ at m/z 623.3117 (calcd. for C38H43 N2O6, 623.3121). The IR spectrum indicated the presence of a hydroxyl group (3415 cm−1), aromatic rings (1618 and 1513 cm−1), and ethers (1219 and 1271 cm−1). ESIMS peaks at m/ z 342, 312, 311, 298, 297, 192, and 176 suggested a BBI dimer in" Tables 1 volving head-to-tail coupling. The NMR profiles of 2 (l and 2) were similar to those of 1, featuring a para-disubstituted benzene system (δH 7.18, 2H, br.d, 8.0 Hz; δH 6.95, 2H, br.d, 8.0 Hz) and a 1,2,4-trisubstituted benzene substructure (δH 6.77, 1H, d, 2.0 Hz; δH 6.78, 1H, d, 8.0 Hz; δH 6.85, 1H, dd, 8.0, 2.0 Hz). However, a notable difference included the absence of a threeproton singlet at δH 3.77 attributable to the OCH3 group at C-12
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Fig. 1 Structures of cissampentine A (1), cissampentine B (2), cissampentine C (3), and cissampentine D (4).
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1 3 4 4a 5 6 7 8 8a α 9 10 11 12 13 14 2-NMe 6-OMe 12-OMe 1′ 3′ 4′ 4a’ 5′ 6′ 7′ 8′ 8a’ α′ 9′ 10′ 11′ 12′ 13′ 14′ 15′ 2′-NMe 6′-OMe
61.3 d 44.9 t 24.1 t 129.6 s 102.8 d 150.5 s 132.5 s 146.6 s 119.4 s 40.7 t 135.1 s 117.9 d 144.2 s 144.8 s 114.9 d 124.4 d 42.9 q 55.9 q
60.9 d 44.8 t 24.3 t 129.6 s 102.6 d 150.5 s 132.4 s 146.2 s 118.8 s 40.0 t 135.1 s 111.6 d 145.8 s 148.0 s 118.4 d 123.7 d 42.6 q 55.9 q 55.5 q 63.7 d 52.7 t 29.5 t 130.9 s 112.0 d 148.3 s 143.3 s 116.1 d 129.7 s 37.5 t 139.3 s 130.3 d 128.5 d 134.4 s 128.5 d 130.3 d 76.6 t 43.7 q 55.5 q
75.4 d 56.8 t 24.2 t 125.4 s 102.4 d 151.4 s 132.5 s 146.1 s 115.3 s 41.3 t 130.8 s 119.9 d 144.0 s 146.8 s 115.9 d 124.9 d 58.1 q 55.5 q
60.7 d 46.9 t 26.6 t 130.1 s 103.1 d 150.4 s 131.0 s 146.4 s 117.9 s 37.9 t 133.5 s 122.6 d 145.3 s 146.5 s 114.8 d 127.2 d 42.9 q 55.7 q
63.3 d 52.3 t 28.7 t 130.3 s 112.1 d 147.5 s 144.3 s 114.5 d 129.2 s 34.9 t 139.0 s 130.3 d 128.4 d 134.2 s 128.4 d 130.3 d 76.3 t 43.3 q 55.5 q
64.5 d 47.4 t 26.6 t 130.7 s 112.3 d 148.7 s 144.3 s 120.6 d 131.4 s 40.6 t 140.6 s 129.2 d 129.9 d 135.3 s 129.9 d 129.2 d 74.6 t 42.5 q 56.0 q
63.7 d 52.6 t 29.6 t 131.6 s 112.4 d 148.6 s 143.3 s 117.2 d 129.8 s 36.4 t 139.6 s 130.3 d 128.7 d 134.8 s 128.7 d 130.3 d 76.8 t 43.8 q 55.8 q
Table 1 13C (100 MHz) NMR data of compounds 1, 2, 3, and 4 (δ ppm, J in Hz).
CDCl3; b CDCl3+CD3OD
Fig. 2 Key HMBC and NOE correlations of cissampentine A (1).
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H (400 MHz) NMR data of compounds 1, 2, 3, and 4 (δ ppm, J in Hz).
No.
1a
2a
3b
4a
1 3 4 5 α
3.57 d (8.0) 3.26 m, 2.88 m 2.84 m, 2.43 m 6.21 s 2.70 dd (14.4, 8.0), 2.20 d (14.4) 6.78 d (1.2) 6.86 d (8.4) 6.90 dd (8.4, 1.2) 2.27 s 3.87 s
3.59 d (8.4) 3.18 m, 2.72 m 2.83 m, 2.42 m 6.15 s 2.65 dd (14.4, 8.4), 2.18 d (14.4) 6.77 d (2.0) 6.78 d (8.0) 6.85 dd (8.0, 2.0) 2.16 s 3.87 s 3.77 s 3.56 dd (3.6, 3.2) 3.01 m, 2.44 m 2.96 m, 2.50 m 6.53 s 6.61 s 3.30 dd (16.4, 3.6), 2.88 dd (16.4, 3.2) 7.18 br.d (8.0) 6.95 br.d (8.0) 6.95 br.d (8.0) 7.18 br.d (8.0) 4.98 d (12.0), 4.43 d (12.0) 2.40 s 3.70 s
4.38 d (8.0) 3.75 m, 3.40 m 3.45 m, 2.78 m 6.30 s 2.62 dd (17.6, 8.0), 2.88 d (17.6) 6.94 d (1.2) 6.85 d (8.0) 6.72 dd (8.0, 1.2) 2.96 s 3.85 s
3.76 dd (5.6, 2.8) 2.92 m, 2.58 m 2.66 m, 2.21 m 6.17 s 2.89 dd (14.8, 2.8), 2.75 dd (14.8, 5.6) 6.57 d (2.0) 6.74 d (8.0) 6.88 dd (8.0, 2.0) 2.37 s 3.88 s
3.60 dd (4.0, 2.8) 3.10 m, 2.52 m 3.08 m, 2.60 m 6.64 s 6.58 s 3.33 dd (16.8, 4.0), 2.92 dd (16.8, 2.8) 7.31 br.d (8.0) 7.09 br.d (8.0) 7.09 br.d (8.0) 7.31 br.d (8.0) 4.98 d (12.0), 4.55 d (12.0) 2.40 s 3.82 s
3.64 dd (7.8, 2.0) 3.13 m, 2.76 m 2.90 m, 2.86 m 6.67 s 6.38 s 3.26 dd (13.6, 2.0), 2.93 (13.6, 7.8) 7.29 br.d (8.0) 7.12 br.d (8.0) 7.12 br.d (8.0) 7.29 br.d (8.0) 5.25 d (12.4), 4.98 d (12.4) 2.51 s 3.90 s
10 13 14 2-NMe 6-OMe 12-OMe 1′ 3′ 4′ 5′ 8′ α′ 10′ 11′ 13′ 14′ 15′ 2′-NMe 6′-OMe a
3.66 dd (4.0, 2.8) 3.10 m, 2.56 m 3.08 m, 2.60 m 6.63 s 6.80 s 3.35 dd (16.4, 4.0), 3.03 dd (16.4, 2.8) 7.31 br.d (8.0) 7.08 br.d (8.0) 7.08 br.d (8.0) 7.31 br.d (8.0) 5.06 d (12.4), 4.58 d (12.4) 2.48 s 3.79 s
CDCl3; b CDCl3+CD3OD
in 1, implying that the OH in 1 was replaced by an OCH3 at C-12 in 2. These assignments were confirmed by NOEDS. Irradiation of 12-OCH3 (δH 3.77) produced an enhancement of the H-13 signals at δH 6.78. In turn, irradiation at δH 6.78 resulted in an increase of δH 3.77. Further COSY, HMQC, and HMBC correlation experiments led to the complete assignment of all protons and carbon signals of 2 (Fig. 39S, Supporting Information). The absolute configuration of 2 was determined to be 1-R, 1′-R, based on the similarity of CD spectrum with that of 1. Compound 2 was named cissampentine B. Compound 3 was isolated as a white powder, [α]20 D − 75 (c 0.14, MeOH). HR-ESIMS indicated [M + H]+ at m/z 625.2919 (calcd. for C37H41 N2O7, 625.2913). The 1H NMR spectrum was very similar to that of 1 with respect to the aromatic protons and the aromatic substituents. However, a remarkable difference was observed with signals for the 2-N-methyl group and the adjoining H-1, which were both shifted downfield. This result indicates that 3 was the 2-N-oxide of 1, which was further evidenced by the 16 additional mass units in the mass spectrum of 3. The 2-N-methyl singlet at δH 2.96 and the H-1 doublet signal at δH 4.38 were characteristic of a trans-relationship between the N-oxide oxygen and H-1 [13]. This trans-relationship was confirmed by a NOESY correlation between the δH 2.96 N-methyl singlet and the H-1 signal at δH 4.38 (Fig. 40S, Supporting Information). The CD spectrum of 3 was very similar to that of 1, implying that the absolute configuration of 3 was 1-R, 1′-R. The 1H NMR and 13C NMR assign" Tables 1 and 2) were completed by interpretation of the ments (l 2D NMR spectra, and compound 3 was named cissampentine C. Compound 4 was obtained as a white powder with [α]20 D -− 129, (c 0.33, CHCl3). HR-ESIMS indicated the [M + H]+ at m/z 609.2969 corresponding to C37H41 N2O6, and indicated 19 degrees of unsat-
uration. The IR spectrum revealed the presence of similar functional groups; a hydroxyl group (3417 cm−1), aromatic rings (1615 and 1507 cm−1), and ethers (1215 and 1271 cm−1). The head-to-tail coupling pattern was deduced from the ESIMS peaks at m/z 314, 312, 311, 298, 229, 208, and 192. The 1H NMR spectrum and the 13C NMR of 4 exhibited one p-disubstituted benzene ring (δH 7.29, 2H, br. d, 8.0 Hz; δH 7.12, 2H, br. d, 8.0 Hz), one 1,2,4-trisubstituted benzene ring (δH 6.57, 1H, d, 2.0 Hz; δH 6.74, 1H, d, 8.0 Hz; δH 6.88, 1H, dd, 8.0, 2.0 Hz), three aromatic singlet protons at δH 6.17, 6.38, and 6.67, two N-methyl groups (δC 42.9; δH 2.37, s, NMe-2; δC 42.5; δH 2.51, s, NMe-2′), and two aromatic methoxyl groups (δC 55.7; δH 3.88, s, OMe-6; δC 56.0; δH 3.90, s, OMe-6′). The methine carbon signals at δC 60.7 and 64.5, characterized as C-1 and C-1′, implied that C-8 was oxygenated and that C-8′ bore a hydrogen. The NOESY correlations between H-10 and H-8′ revealed the ether linkage at C-7′/ C-11 (Fig. 41S, Supporting Information). A methyleneoxy bridge of compound 4 was indicated by the diagnostic AB system at δH 5.25 and 4.98 (J = 12.4 Hz). This linkage was assigned to C-7 and C-15′ due to the HMBC correlations between the signals at δH 4.98 and 5.25 and δC 131.0. The unambiguous assignments of the 1H and 13C NMR data of 4 were achieved by 2D NMR techniques (1H-1H COSY, HMQC, HMBC, and NOESY). Compound 4 exhibited the same molecular formula as compound 1, and the C-1 proton coupling constant (δH 3.76, 1H, dd, 5.6, 2.8 Hz) revealed a significant difference compared with that of 1 (δH 3.57, 1H, d, 8.0 Hz). This evidence implies that compound 4 was the C-1 isomer of compound 1. The CD spectrum of 4 was similar to that of wattsine A (1-S, 1′-R), exhibiting a strong negative Cotton effect at 214 nm and a weak shoulder negative curve
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Table 2
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at 272–280 nm [3, 7]. Thus, the absolute configuration of 4 was 1S, 1′-R, and the compound was named cissampentine D. Compounds 1–4 were evaluated for their cytotoxic activities against Bel-7402, HCT-8, and A2780 cancer cell lines in vitro by the MTT assay using paclitaxel as a positive control, as shown in " Table 3. Cissampentine A (1) exhibited cytotoxic activities with l an IC50 value of 8.97 µM against HCT-8. Cissampentine D (4) exhibited cytotoxic activities with an IC50 value of 9.73 µM against HCT-8, and 5.36 µM against Bel-7402.
Table 3 Cytotoxicity of cissampentine A (1), cissampentine B (2), cissampentine C (3), and cissampentine D (4) against cultured HCT-8, Bel-7402, and A2780 cancer cell lines. Compounds
1 2 3 4 Paclitaxel
Growth inhibition constant (IC50) (µM) HCT-8
Bel-7402
A2780
8.97 > 10 > 10 9.73 0.72
> 10 > 10 > 10 5.36 1.43
> 10 > 10 > 10 > 10 0.35
Materials and Methods !
General Optical rotations were measured in CHCl3 or MeOH using a Perkin-Elmer 341 polarimeter. Melting points were taken on a micro-melting point apparatus (Kexing-X4) without correction. UV spectra were measured on a PuXi Tu 1800 PC spectropolarimeter. IR spectra were obtained on a Nicolet FT‑IR 200 SXV spectrophotometer. CD spectra were obtained with a JASCO J-810 spectropolarimeter. NMR spectra were recorded on a Varian Unity INOVA 400/45 NMR spectrometer with standard pulse sequences operating at 400 MHz in 1H NMR and 100 MHz in 13C NMR in CDCl3 or CD3OD. Mass spectra were measured on Waters Q‑TOF-Premier spectrometers equipped with an APCI/ESI multimode ion source detector. Authentic samples were isolated by our research group. Silica gel H (Qindao Marine Chemical Factory) was used for column chromatography. Zones on TLC plates (silica gel G) were detected with modified Dragendorffʼs reagent (Shanghai Puzhen Biotech).
Plant material The roots of C. tonkinensis were collected in Guangxi Province, China (voucher No. 200 802/T) in February 2008 and were identified by Dr. Hong Zhang, Sichuan Institute for Food and Drug Control. Voucher specimens were deposited in the West China College of Pharmacy, Sichuan University.
nol (95 : 5, 800 mL; 90 : 10, 1000 mL; and 7 : 3, 200 mL) as the eluent afforded wattisine A (7) (33 mg) and (−) curine (6) (442 mg). Further separation of subfraction D (3.1 g) by silica gel column chromatography (45–75 µm, 3 × 25 cm) using chloroform-methanol (9 : 1, 1000 mL; 8 : 2, 300 mL; and 6 : 4, 200 mL) as the eluent provided cissampentine C (3) (15 mg). Column chromatography of subfraction F (650 mg) over silica gel (45–75 µm, 2 × 10 cm) using methanol-water (98 : 2, 30 mL; 9 : 1, 100 mL; and 8 : 2, 100 mL) as the eluent gave α-cyclanoline (8) (16 mg) and steponine (9) (9 mg). Compounds 5–9 were identified by 1H and 13C NMR spectral data [6–8] as well as by direct comparison with authentic samples of 7-O-methylhayatidine, (−)-curine, wattisine A, α-cyclanoline, and steponine, respectively.
Cissampentine A (1) White amorphous powder, m. p. 168–171 °C. [α]20 D −85 (c 0.28, CHCl3). UV (MeOH) λmax (log ε) nm: 210 (4.33), and 280 (3.80), CD (MeOH) λmax (Δ ε) nm: 218 (− 27.3), 250 (− 4.89), 279 (+ 1.25), and 297 (− 2.37). IR (KBr) νmax · cm−1: 3508, 2930, 1616, 1510, 1444, 1274, 1215, 1120, 1021, 975, and 802. 1H‑NMR " Table 2. 13C‑NMR (100 MHz, CDCl ), see (400 MHz, CDCl3), see l 3 " l Table 1. ESI‑MS m/z: 609 [M + H]+, 472, 386, 314, 312, 311, 298, 297, 192, and 175; HR-ESIMS m/z: 609.2969 [M + H]+ (calcd. for C37H41 N2O6, 609.2965).
Extraction and isolation The air-dried and powdered roots (8.0 kg) of C. tonkinensis were extracted with 95 % EtOH (80 L × 3) three times at room temperature. After removal of the solvent of extraction, the crude residue (408 g) was suspended in 2.5 L of H2O and acidified with 2 N hydrochloric acid to pH 3. The acidic mixture was defatted with ethyl acetate (1500 mL × 2) and then basified with 10 % aqueous NH4OH to pH 10. Extraction of the subsequent mixture with CHCl3 (1500 mL × 3) afforded crude alkaloids (42.0 g), which were subjected to silica gel column chromatography (45–75 µm, 10 × 90 cm) using a step gradient of CHCl3-MeOH-diethylamine 99 : 1 : 0.5 (2000 mL), 98 : 2 : 0.5 (2000 mL), 95 : 5 : 0.5 (8000 mL), 90 : 10 : 0.5 (4000 mL), and 30 : 10 : 0.5 (2000 mL). Fractions of 200 mL were collected and monitored by TLC, resulting in five major fractions (F1–F5). Fraction F2 (17.0 g) was further chromatographed on a silica gel column (45–75 µm, 8 × 35 cm) employing CHCl3-MeOH 95 : 5 (5500 mL), 90 : 10 (2000 mL), and 70 : 30 (2000 mL) as the eluent to afford six subfractions (A–F). Purification of subfraction A (3.0 g) by a silica gel column (45–75 µm, 4 × 20 cm), eluting with CHCl3-MeOH (95 : 5, 1000 mL) yielded cissampentine A (1) (28 mg), cissampentine B (2) (13 mg), cissampentine D (4) (160 mg), and 7-O-methylhayatidine (5) (106 mg). Column chromatography of subfraction B (9.0 g) over a silica gel column (45–75 µm, 8 × 30 cm) using chloroform-metha-
Wang J-z et al. Bisbenzylisoquinoline Alkaloids from …
Cissampentine B (2) White amorphous powder, m. p. 185–188 °C. [α]20 D −90 (c 0.34, CHCl3). UV (MeOH) λmax (log ε) nm: 210 (4.41), and 279 (3.87). CD (MeOH) λmax (Δ ε) nm: 216 (− 37.8), 250 (− 6.52), 279 (+ 2.74), and 295 (− 3.75). IR (KBr) νmax · cm−1: 3415, 2924, 1618, 1513, 1459, 1371, 1271, 1219, 1123, 1019, and 972. 1H‑NMR " Table 2. 13C‑NMR (100 MHz, CDCl ), see (400 MHz, CDCl3), see l 3 " Table 1. NOEDS: H-13 to 12-OMe (10%), 12-OMe to H-13 (6 %). l ESIMS m/z: 623 [M + H]+, 342, 312, 311, 298, 297, 192, and 176. HR-ESIMS m/z: 623.3117 [M + H]+ (calcd. for C38H43 N2O6, 623.3121).
Cissampentine C (3) White amorphous powder, m. p. 234–236 °C. [α]20 D -75 (c 0.14, MeOH). UV (MeOH) λmax (log ε) nm: 210 (4.31), and 280 (3.79). CD (MeOH) λmax (Δ ε) nm: 216 (− 20.2), 254 (− 3.27), 279 (+ 1.47), and 298 (− 1.79). IR (KBr) νmax · cm−1: 3425, 2933, 1607, 1506, 1450, 1268, 1221, 1116, and 1018. 1H‑NMR (400 MHz, " Table 2.13C‑NMR (100 MHz, CDCl CDCl3 + CD3OD), see l 3 + " Table 1. ESIMS m/z: 625 [M + H]+, 417, 344, 327, CD3OD), see l 314, 312, 297, 296, 208, and 192. HR-ESIMS m/z: 625.2919 [M + H]+ (calcd. for C37H41 N2O7, 625.2913).
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Cissampentine D (4)
Conflict of Interest
White amorphous powder, m. p. 178–181 °C. [α]20 D −129 (c 0.33, CHCl3). UV (MeOH) λmax (log ε) nm: 213 (4.28), and 280 (3.67). CD (MeOH) λmax (Δ ε) nm: 214 (− 44.5), 258 (− 0.67), 284 (− 1.85), and 297 (− 0.85). IR (KBr) νmax · cm−1: 3417, 2934, 1615, 1507, 1443, 1367, 1271, 1215, 1120, 1019, and 828. 1H‑NMR " Table 2.13C‑NMR (100 MHz, CDCl ), see (400 MHz, CDCl3), see l 3 " l Table 1. ESIMS m/z: 609 [M + H]+, 462, 314, 312, 311, 298, 229, 208, and 192. HR-ESIMS m/z: 609.2969 [M + H]+ (calcd. for C37H41N2O6, 609.2965).
!
The cytotoxic activities of the compounds 1–4 (purity > 90 % by TLC) and paclitaxel (purity > 99 %, Hainan Shunyuan Chemotech) were evaluated in vitro by measuring cell viability using the MTT assay. HCT-8, Bel-7402, and A2780 cells (Shanghai Bioleaf Biotech) were maintained in RPMI-1640 medium supplied with 5 % FBS. Cells in the logarithmic phase were cultured at a density of 50 000 cells/ml per well in a 24-well plate. The cells were exposed to various concentrations of the test compounds for 72 h, and each compound was tested in triplicate at every concentration. The methylene blue dye assay was used to evaluate the effects of the tested compounds on cell growth. The IC50 value resulting from 50 % inhibition of cell growth was calculated graphically as a comparison with the control.
Supporting information The isolation and purification of the compounds, HR‑MS, CD, 1H and 13C NMR as well as 2D‑NMR spectra for compounds 1–4 are available as Supporting Information.
Acknowledgements !
The authors declare no conflicts of interest.
References 1 Angerhofer CK, Guinaudeau H, Wongpanich V, Pezzuto JM, Cordell GA. Antiplasmodial and cytotoxic activity of natural bisbenzylisoquinoline alkaloids. J Nat Prod 1999; 62: 59–66 2 Hall AM, Chang CJ. Multidrug-resistance modulators from Stephania japonica. J Nat Prod 1997; 60: 1193–1195 3 Schiff PL jr. Bisbenzylisoquinoline alkaloids. J Nat Prod 1997; 60: 934– 953 4 Ndaya TJ, Wright AD, Konig GM. Hplc isolation of the anti-plasmodially active bisbenzylisoquinoline alkaloids present in roots of Cissampelos mucronata. Phytochem Anal 2003; 14: 13–22 5 Luo XR, Zhao SY. Medicinal plant of Menispermaceae in China. Guihaia 1986; 6: 49–61 6 Wang JZ, Liao J, Lei Y. Study on the chemical constituents of Cycle wattii and structure of curine-type alkaloids. West China J Pharm Sci 2014; 29: 11–13 7 Wang JZ, Liu XY, Wang FP. Two new curine-type bisbenzylisoquinoline alkaloids from the roots of Cyclea wattii with cytotoxic activities. Chem Pharm Bull 2010; 58: 986–988 8 Tanahashi T, Su Y, Nagakura N, Nayeshiro H. Quaternary isoquinoline alkaloids from Stephania cepharantha. Chem Pharm Bull 2000; 48: 370–373 9 Galinis DL, Wiemer DF, Cazin J jr. Cissampentin: a new bisbenzylisoquinoline alkaloid from Cissampelos fasiculata. Tetrahedron 1993; 49: 1337–1342 10 Tomita M, Kikuchi T, Fujitani K, Kato A, Furukawa H, Aoyagi Y, Kitano M, Ibuka T. Mass spectrometry of bisbenzylisoquinoline alkaloids. Tetrahedron Lett 1966; 8: 857–864 11 Koike L, Marsaioli AJ, Reis F. Proton and carbon-13 nuclear magnetic resonance spectroscopy and conformational aspects of the curine class of bisbenzylisoquinoline alkaloids. J Org Chem 1981; 46: 2385–2389 12 Koike L, Marsaioli AJ, Ruveda EA, Reis F. Stereochemical aspects and 13C NMR spectroscopy of the berbamine class of bisbenzylisoquinoline alkaloids. Tetrahedron Lett 1979; 20: 3765–3768 13 Guinaudeau H, Freyer AJ, Shamma M. Spectral characteristics of the bisbenzylisoquinoline alkaloids. Nat Prod Rep 1986; 476–488
The authors sincerely thank Dr. Hong Zhang, Sichuan Institute for Food and Drug Control, for supplying plant material and Ms. KaiLei Lin, Shanghai Institute of Materia Medica, for recording the CD spectra.
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Bisbenzylisoquinoline Alkaloids from the Roots of Cyclea tonkinensis
Authors
Jian-zhong Wang, Jing Liao, Wen-long Xu, Xiao-bing Chen
Affiliation
West China College of Pharmacy, Sichuan University, Chengdu, P. R. China
Key words " Cyclea tonkinensis l " Menispermaceae l " bisbenzylisoquinoline l alkaloid " cissampentine A l " cissampentine B l " cissampentine C l " cissampentine D l " cytotoxic activity l
Abstract !
Cissampentine A (1), an enantiomer of cissampentin, three new cycleatjehenine-type bisbenzylisoquinoline alkaloids, cissampentine B–D (2–4), and five known alkaloids were isolated from the roots of Cyclea tonkinensis. Their structures were established by interpretation of NMR, high-resolution ESI‑MS data, and CD spectra. In vitro stud-
Introduction !
received revised accepted
July 9, 2014 February 25, 2015 February 26, 2015
Bibliography DOI http://dx.doi.org/ 10.1055/s-0035-1545882 Published online April 9, 2015 Planta Med 2015; 81: 600–605 © Georg Thieme Verlag KG Stuttgart · New York · ISSN 0032‑0943 Correspondence Associate Prof. Dr. Jian-zhong Wang West China College of Pharmacy Sichuan University Department Medicinal Natural Products No. 17, Duan 3, Renmin Nan Road Chengdu 610041 Peopleʼs Republic of China Phone: + 86 28 85 50 37 70 [email protected]
Natural bisbenzylisoquinoline (BBI) alkaloids possess a number of interesting biological activities, including hypotensive, muscle relaxant, cytotoxic, antiplasmodial, and reverse multidrug-resistant activity [1, 2]. BBI alkaloids represent the largest subgroup of isoquinoline alkaloids, with over 470 members [3, 4], and are derived biogenetically via phenol-oxidative tail-to-tail or head-totail coupling of two units of benzylisoquinoline alkaloids. The two moieties are usually bound by one diaryl ether bridge or more. However, in rare cases, a methyleneoxy bridge or carbon-carbon bridges are present. Cyclea tonkinensis Gagnep (Menispermaceae) is a creeper growing in the southwest of China [5], whose roots are used in Chinese traditional medicine for the treatment of stomachaches and tooth pain. This paper investigated the chemical ingredients of the roots of C. tonkinensis and conducted a preliminary biological study. The chemical investigation led to the discovery of cissampentine A (1), an enantiomer of cissampentin, three new cycleatjehenine-type bisbenzylisoquinoline alkaloids, cissampentine " Fig. 1), and five known alkaloids, inB–D (2–4) (l cluding 7-O-methylhayatidine (5), (−)-curine (6), wattisine A (7), α-cyclanoline (8), and steponine (9) [6–8]. Cissampentin, incorporating an unusual methyleneoxy bridge, was reported by D. L. Galinis as a
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ies indicated that compounds 1 and 4 exhibited cytotoxicity against the HCT-8 tumor cell line (IC50 values of 8.97 and 9.73 µM, respectively), and compound 4 was also active against the Bel7402 tumor cell line (IC50 value of 5.36 µM). Supporting information available online at http://www.thieme-connect.de/products
racemic mixture from Cissampelos fasciculate that exhibited significant activity as a repellent for leafcutter ants [9]. Unfortunately, the configurations of this mixture [(1-R, 1′-R), (1-S, 1′-S), or (1S, 1′-R), (1-R, 1′-S)] has not been determined. To date, only four cycleatjehenine-type alkaloids, including cissampentin, have been reported, and the stereochemistry of C-1 and C-1′ remain undetermined [3, 9]. The present study focused on the elucidation of the structures of cissampentines A– D (1–4) by 1D and 2D NMR techniques and highresolution ESI‑MS spectra. The absolute configurations of compounds 1–4 were confirmed by CD spectra. We also tested 1–4 for their cytotoxicity against the HCT-8, Bel-7402, and A2780 tumor cell lines.
Results and Discussion !
Compound 1 was isolated as a white powder. HRESIMS indicated [M + H]+ at m/z 609.2969 (calcd. 609.2965), corresponding to the molecular formula C37H41 N2O6. The IR spectrum suggested the presence of a hydroxyl group (3508 cm−1), aromatic rings (1616 and 1510 cm−1), and ether bonds (1274 and 1215 cm−1). The 13C NMR spectra of 1 in conjunction with the hydrogen data in " Table 1 revealed the presence of 14 quaternary, l 12 methine, 7 methylene, and 4 methyl signals for a total of 37 carbon resonances. Thirteen nonaro-
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matic carbon resonances were assigned to two N-methyl groups (δC 42.9; δH 2.27, s, NMe-2; δC 43.8; δH 2.48, s, NMe-2′), two aromatic methoxyl groups (δC 55.9; δH 3.87, s, OMe-6; δC 55.8; δH 3.79, s, OMe-6′), two aliphatic methines, and seven aliphatic methylene units [one oxymethylene (δH 4.58, 1H, d, J = 12.4 Hz; δH 5.06, 1H, d, J = 12.4 Hz; δC 76.8)]. These data suggested the presence of two tetrahydrobenzylisoquinoline units in 1. The ESIMS fragmentation peaks at m/z 312, 311, 298, 297, and 192, due to the fragment after the facile cleavage of the two benzylic bonds, were characteristics of head-to-tail BBI alkaloids (Fig. 38S, Supporting Information) [10]. The two tetrahydrobenzylisoquinoline moieties accounted for 18 degrees, and the one remaining degree of unsaturation was the result of an internal macrocycle caused by the formation of two bridges. The 1H NMR spectrum revealed the signals for one p-disubstituted benzene ring (δH 7.31, 2H, br.d, 8.0 Hz; δH 7.08, 2H, br. d, 8.0 Hz) and a 1,2,4-trisubstituted benzene ring (δH 6.78, 1H, d, 1.2 Hz; δH 6.86, 1H, d, 8.4 Hz; δH 6.90, 1H, dd, 8.4, 1.2 Hz). Another three isolated aromatic protons (δH 6.21, 6.63, 6.80, each 1H, s) could be assigned to two remaining tetrahydroisoquinoline moieties. The methine carbon signals at δC 61.3 and 63.7, characterized as C-1 and C-1′, implied that C-8 was oxygenated and that C-8′ bore a hydrogen; the δ values of C-1 were shifted to a higher field due to the C-8 oxygen γ-gauche effect [11]. With respect to the ether linkage involving a 7′-11 or a 7′-12, the key NOE correlations " Fig. 2) between H-10 and H-8′ implied that compound 1 pos(l sessed a 7′-11 ether linkage. This structure could be produced by a gradual rotation of ring C placing C-12 at the external part of the molecule, which creates a less hindered conformation and allows H-10 access to H-8′ [12]. The presence of a methyleneoxy bridge was indicated by the diagnostic AB system at δH 4.58 and 5.06 (J = 12.4 Hz). This linkage was assigned to C-7 and C-15′ due to the HMBC correlations between the signals at δH 4.58 and 5.06 and the δC 132.5 resonance. The methoxyl signal at δH 3.87 (δC 55.9) was assigned to OCH3-6, and another methoxyl hydrogen
δH 3.79 (δC 55.8) should be attached to C-6′ due to long-range correlations from 6-OCH3 to C-6 and from 6′-OCH3 to C-6′. For key " Fig. 2. HMBC correlations, see l Comparing the 13C‑NMR with those of the reported cissampentin compounds revealed that the compounds shared the same carbon signals [9]. Compound 1 exhibited negative specific rotations ([α]20 D − 85, CDCl3), whereas cissampentin was observed to be a racemic mixture, suggesting that compound 1 was an enantiomer of cissampentin. We collected the CD spectral data of headto-tail bisbenzylisoquinoline alkaloids with two ether bridges [3, 6, 7]. The compounds with the structures (1-R, 1′-R) and (1-S, 1′R) all exhibited a strong negative Cotton effect at 210–220 nm and had a weak negative Cotton effect at 285–295 nm. The compound with the 1-R, 1′-R exhibited a weak positive Cotton effect at 272–280 nm, whereas a weak shoulder negative Cotton effect curve was present in the same area for the compound with the 1S, 1′-R configuration. The Cotton effect curve of 1 closely resembled that of (−)-curine (1-R, 1′-R) [7], with a negative Cotton effect at 218 nm and a positive Cotton effect at 279 nm. Thus, we concluded that compound 1 had a 1-R, 1′-R configuration and named it cissampentine A. Therefore, we deduced that cissampentin was a racemic mixture with configurations of (1-R, 1′-R) and (1-S, 1′-S). Compound 2 was isolated as a white powder, [α]20 D − 90 (c 0.34, CHCl3). Its HR-ESIMS indicated [M + H]+ at m/z 623.3117 (calcd. for C38H43 N2O6, 623.3121). The IR spectrum indicated the presence of a hydroxyl group (3415 cm−1), aromatic rings (1618 and 1513 cm−1), and ethers (1219 and 1271 cm−1). ESIMS peaks at m/ z 342, 312, 311, 298, 297, 192, and 176 suggested a BBI dimer in" Tables 1 volving head-to-tail coupling. The NMR profiles of 2 (l and 2) were similar to those of 1, featuring a para-disubstituted benzene system (δH 7.18, 2H, br.d, 8.0 Hz; δH 6.95, 2H, br.d, 8.0 Hz) and a 1,2,4-trisubstituted benzene substructure (δH 6.77, 1H, d, 2.0 Hz; δH 6.78, 1H, d, 8.0 Hz; δH 6.85, 1H, dd, 8.0, 2.0 Hz). However, a notable difference included the absence of a threeproton singlet at δH 3.77 attributable to the OCH3 group at C-12
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Fig. 1 Structures of cissampentine A (1), cissampentine B (2), cissampentine C (3), and cissampentine D (4).
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2a
3b
4a
1 3 4 4a 5 6 7 8 8a α 9 10 11 12 13 14 2-NMe 6-OMe 12-OMe 1′ 3′ 4′ 4a’ 5′ 6′ 7′ 8′ 8a’ α′ 9′ 10′ 11′ 12′ 13′ 14′ 15′ 2′-NMe 6′-OMe
61.3 d 44.9 t 24.1 t 129.6 s 102.8 d 150.5 s 132.5 s 146.6 s 119.4 s 40.7 t 135.1 s 117.9 d 144.2 s 144.8 s 114.9 d 124.4 d 42.9 q 55.9 q
60.9 d 44.8 t 24.3 t 129.6 s 102.6 d 150.5 s 132.4 s 146.2 s 118.8 s 40.0 t 135.1 s 111.6 d 145.8 s 148.0 s 118.4 d 123.7 d 42.6 q 55.9 q 55.5 q 63.7 d 52.7 t 29.5 t 130.9 s 112.0 d 148.3 s 143.3 s 116.1 d 129.7 s 37.5 t 139.3 s 130.3 d 128.5 d 134.4 s 128.5 d 130.3 d 76.6 t 43.7 q 55.5 q
75.4 d 56.8 t 24.2 t 125.4 s 102.4 d 151.4 s 132.5 s 146.1 s 115.3 s 41.3 t 130.8 s 119.9 d 144.0 s 146.8 s 115.9 d 124.9 d 58.1 q 55.5 q
60.7 d 46.9 t 26.6 t 130.1 s 103.1 d 150.4 s 131.0 s 146.4 s 117.9 s 37.9 t 133.5 s 122.6 d 145.3 s 146.5 s 114.8 d 127.2 d 42.9 q 55.7 q
63.3 d 52.3 t 28.7 t 130.3 s 112.1 d 147.5 s 144.3 s 114.5 d 129.2 s 34.9 t 139.0 s 130.3 d 128.4 d 134.2 s 128.4 d 130.3 d 76.3 t 43.3 q 55.5 q
64.5 d 47.4 t 26.6 t 130.7 s 112.3 d 148.7 s 144.3 s 120.6 d 131.4 s 40.6 t 140.6 s 129.2 d 129.9 d 135.3 s 129.9 d 129.2 d 74.6 t 42.5 q 56.0 q
63.7 d 52.6 t 29.6 t 131.6 s 112.4 d 148.6 s 143.3 s 117.2 d 129.8 s 36.4 t 139.6 s 130.3 d 128.7 d 134.8 s 128.7 d 130.3 d 76.8 t 43.8 q 55.8 q
Table 1 13C (100 MHz) NMR data of compounds 1, 2, 3, and 4 (δ ppm, J in Hz).
CDCl3; b CDCl3+CD3OD
Fig. 2 Key HMBC and NOE correlations of cissampentine A (1).
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H (400 MHz) NMR data of compounds 1, 2, 3, and 4 (δ ppm, J in Hz).
No.
1a
2a
3b
4a
1 3 4 5 α
3.57 d (8.0) 3.26 m, 2.88 m 2.84 m, 2.43 m 6.21 s 2.70 dd (14.4, 8.0), 2.20 d (14.4) 6.78 d (1.2) 6.86 d (8.4) 6.90 dd (8.4, 1.2) 2.27 s 3.87 s
3.59 d (8.4) 3.18 m, 2.72 m 2.83 m, 2.42 m 6.15 s 2.65 dd (14.4, 8.4), 2.18 d (14.4) 6.77 d (2.0) 6.78 d (8.0) 6.85 dd (8.0, 2.0) 2.16 s 3.87 s 3.77 s 3.56 dd (3.6, 3.2) 3.01 m, 2.44 m 2.96 m, 2.50 m 6.53 s 6.61 s 3.30 dd (16.4, 3.6), 2.88 dd (16.4, 3.2) 7.18 br.d (8.0) 6.95 br.d (8.0) 6.95 br.d (8.0) 7.18 br.d (8.0) 4.98 d (12.0), 4.43 d (12.0) 2.40 s 3.70 s
4.38 d (8.0) 3.75 m, 3.40 m 3.45 m, 2.78 m 6.30 s 2.62 dd (17.6, 8.0), 2.88 d (17.6) 6.94 d (1.2) 6.85 d (8.0) 6.72 dd (8.0, 1.2) 2.96 s 3.85 s
3.76 dd (5.6, 2.8) 2.92 m, 2.58 m 2.66 m, 2.21 m 6.17 s 2.89 dd (14.8, 2.8), 2.75 dd (14.8, 5.6) 6.57 d (2.0) 6.74 d (8.0) 6.88 dd (8.0, 2.0) 2.37 s 3.88 s
3.60 dd (4.0, 2.8) 3.10 m, 2.52 m 3.08 m, 2.60 m 6.64 s 6.58 s 3.33 dd (16.8, 4.0), 2.92 dd (16.8, 2.8) 7.31 br.d (8.0) 7.09 br.d (8.0) 7.09 br.d (8.0) 7.31 br.d (8.0) 4.98 d (12.0), 4.55 d (12.0) 2.40 s 3.82 s
3.64 dd (7.8, 2.0) 3.13 m, 2.76 m 2.90 m, 2.86 m 6.67 s 6.38 s 3.26 dd (13.6, 2.0), 2.93 (13.6, 7.8) 7.29 br.d (8.0) 7.12 br.d (8.0) 7.12 br.d (8.0) 7.29 br.d (8.0) 5.25 d (12.4), 4.98 d (12.4) 2.51 s 3.90 s
10 13 14 2-NMe 6-OMe 12-OMe 1′ 3′ 4′ 5′ 8′ α′ 10′ 11′ 13′ 14′ 15′ 2′-NMe 6′-OMe a
3.66 dd (4.0, 2.8) 3.10 m, 2.56 m 3.08 m, 2.60 m 6.63 s 6.80 s 3.35 dd (16.4, 4.0), 3.03 dd (16.4, 2.8) 7.31 br.d (8.0) 7.08 br.d (8.0) 7.08 br.d (8.0) 7.31 br.d (8.0) 5.06 d (12.4), 4.58 d (12.4) 2.48 s 3.79 s
CDCl3; b CDCl3+CD3OD
in 1, implying that the OH in 1 was replaced by an OCH3 at C-12 in 2. These assignments were confirmed by NOEDS. Irradiation of 12-OCH3 (δH 3.77) produced an enhancement of the H-13 signals at δH 6.78. In turn, irradiation at δH 6.78 resulted in an increase of δH 3.77. Further COSY, HMQC, and HMBC correlation experiments led to the complete assignment of all protons and carbon signals of 2 (Fig. 39S, Supporting Information). The absolute configuration of 2 was determined to be 1-R, 1′-R, based on the similarity of CD spectrum with that of 1. Compound 2 was named cissampentine B. Compound 3 was isolated as a white powder, [α]20 D − 75 (c 0.14, MeOH). HR-ESIMS indicated [M + H]+ at m/z 625.2919 (calcd. for C37H41 N2O7, 625.2913). The 1H NMR spectrum was very similar to that of 1 with respect to the aromatic protons and the aromatic substituents. However, a remarkable difference was observed with signals for the 2-N-methyl group and the adjoining H-1, which were both shifted downfield. This result indicates that 3 was the 2-N-oxide of 1, which was further evidenced by the 16 additional mass units in the mass spectrum of 3. The 2-N-methyl singlet at δH 2.96 and the H-1 doublet signal at δH 4.38 were characteristic of a trans-relationship between the N-oxide oxygen and H-1 [13]. This trans-relationship was confirmed by a NOESY correlation between the δH 2.96 N-methyl singlet and the H-1 signal at δH 4.38 (Fig. 40S, Supporting Information). The CD spectrum of 3 was very similar to that of 1, implying that the absolute configuration of 3 was 1-R, 1′-R. The 1H NMR and 13C NMR assign" Tables 1 and 2) were completed by interpretation of the ments (l 2D NMR spectra, and compound 3 was named cissampentine C. Compound 4 was obtained as a white powder with [α]20 D -− 129, (c 0.33, CHCl3). HR-ESIMS indicated the [M + H]+ at m/z 609.2969 corresponding to C37H41 N2O6, and indicated 19 degrees of unsat-
uration. The IR spectrum revealed the presence of similar functional groups; a hydroxyl group (3417 cm−1), aromatic rings (1615 and 1507 cm−1), and ethers (1215 and 1271 cm−1). The head-to-tail coupling pattern was deduced from the ESIMS peaks at m/z 314, 312, 311, 298, 229, 208, and 192. The 1H NMR spectrum and the 13C NMR of 4 exhibited one p-disubstituted benzene ring (δH 7.29, 2H, br. d, 8.0 Hz; δH 7.12, 2H, br. d, 8.0 Hz), one 1,2,4-trisubstituted benzene ring (δH 6.57, 1H, d, 2.0 Hz; δH 6.74, 1H, d, 8.0 Hz; δH 6.88, 1H, dd, 8.0, 2.0 Hz), three aromatic singlet protons at δH 6.17, 6.38, and 6.67, two N-methyl groups (δC 42.9; δH 2.37, s, NMe-2; δC 42.5; δH 2.51, s, NMe-2′), and two aromatic methoxyl groups (δC 55.7; δH 3.88, s, OMe-6; δC 56.0; δH 3.90, s, OMe-6′). The methine carbon signals at δC 60.7 and 64.5, characterized as C-1 and C-1′, implied that C-8 was oxygenated and that C-8′ bore a hydrogen. The NOESY correlations between H-10 and H-8′ revealed the ether linkage at C-7′/ C-11 (Fig. 41S, Supporting Information). A methyleneoxy bridge of compound 4 was indicated by the diagnostic AB system at δH 5.25 and 4.98 (J = 12.4 Hz). This linkage was assigned to C-7 and C-15′ due to the HMBC correlations between the signals at δH 4.98 and 5.25 and δC 131.0. The unambiguous assignments of the 1H and 13C NMR data of 4 were achieved by 2D NMR techniques (1H-1H COSY, HMQC, HMBC, and NOESY). Compound 4 exhibited the same molecular formula as compound 1, and the C-1 proton coupling constant (δH 3.76, 1H, dd, 5.6, 2.8 Hz) revealed a significant difference compared with that of 1 (δH 3.57, 1H, d, 8.0 Hz). This evidence implies that compound 4 was the C-1 isomer of compound 1. The CD spectrum of 4 was similar to that of wattsine A (1-S, 1′-R), exhibiting a strong negative Cotton effect at 214 nm and a weak shoulder negative curve
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at 272–280 nm [3, 7]. Thus, the absolute configuration of 4 was 1S, 1′-R, and the compound was named cissampentine D. Compounds 1–4 were evaluated for their cytotoxic activities against Bel-7402, HCT-8, and A2780 cancer cell lines in vitro by the MTT assay using paclitaxel as a positive control, as shown in " Table 3. Cissampentine A (1) exhibited cytotoxic activities with l an IC50 value of 8.97 µM against HCT-8. Cissampentine D (4) exhibited cytotoxic activities with an IC50 value of 9.73 µM against HCT-8, and 5.36 µM against Bel-7402.
Table 3 Cytotoxicity of cissampentine A (1), cissampentine B (2), cissampentine C (3), and cissampentine D (4) against cultured HCT-8, Bel-7402, and A2780 cancer cell lines. Compounds
1 2 3 4 Paclitaxel
Growth inhibition constant (IC50) (µM) HCT-8
Bel-7402
A2780
8.97 > 10 > 10 9.73 0.72
> 10 > 10 > 10 5.36 1.43
> 10 > 10 > 10 > 10 0.35
Materials and Methods !
General Optical rotations were measured in CHCl3 or MeOH using a Perkin-Elmer 341 polarimeter. Melting points were taken on a micro-melting point apparatus (Kexing-X4) without correction. UV spectra were measured on a PuXi Tu 1800 PC spectropolarimeter. IR spectra were obtained on a Nicolet FT‑IR 200 SXV spectrophotometer. CD spectra were obtained with a JASCO J-810 spectropolarimeter. NMR spectra were recorded on a Varian Unity INOVA 400/45 NMR spectrometer with standard pulse sequences operating at 400 MHz in 1H NMR and 100 MHz in 13C NMR in CDCl3 or CD3OD. Mass spectra were measured on Waters Q‑TOF-Premier spectrometers equipped with an APCI/ESI multimode ion source detector. Authentic samples were isolated by our research group. Silica gel H (Qindao Marine Chemical Factory) was used for column chromatography. Zones on TLC plates (silica gel G) were detected with modified Dragendorffʼs reagent (Shanghai Puzhen Biotech).
Plant material The roots of C. tonkinensis were collected in Guangxi Province, China (voucher No. 200 802/T) in February 2008 and were identified by Dr. Hong Zhang, Sichuan Institute for Food and Drug Control. Voucher specimens were deposited in the West China College of Pharmacy, Sichuan University.
nol (95 : 5, 800 mL; 90 : 10, 1000 mL; and 7 : 3, 200 mL) as the eluent afforded wattisine A (7) (33 mg) and (−) curine (6) (442 mg). Further separation of subfraction D (3.1 g) by silica gel column chromatography (45–75 µm, 3 × 25 cm) using chloroform-methanol (9 : 1, 1000 mL; 8 : 2, 300 mL; and 6 : 4, 200 mL) as the eluent provided cissampentine C (3) (15 mg). Column chromatography of subfraction F (650 mg) over silica gel (45–75 µm, 2 × 10 cm) using methanol-water (98 : 2, 30 mL; 9 : 1, 100 mL; and 8 : 2, 100 mL) as the eluent gave α-cyclanoline (8) (16 mg) and steponine (9) (9 mg). Compounds 5–9 were identified by 1H and 13C NMR spectral data [6–8] as well as by direct comparison with authentic samples of 7-O-methylhayatidine, (−)-curine, wattisine A, α-cyclanoline, and steponine, respectively.
Cissampentine A (1) White amorphous powder, m. p. 168–171 °C. [α]20 D −85 (c 0.28, CHCl3). UV (MeOH) λmax (log ε) nm: 210 (4.33), and 280 (3.80), CD (MeOH) λmax (Δ ε) nm: 218 (− 27.3), 250 (− 4.89), 279 (+ 1.25), and 297 (− 2.37). IR (KBr) νmax · cm−1: 3508, 2930, 1616, 1510, 1444, 1274, 1215, 1120, 1021, 975, and 802. 1H‑NMR " Table 2. 13C‑NMR (100 MHz, CDCl ), see (400 MHz, CDCl3), see l 3 " l Table 1. ESI‑MS m/z: 609 [M + H]+, 472, 386, 314, 312, 311, 298, 297, 192, and 175; HR-ESIMS m/z: 609.2969 [M + H]+ (calcd. for C37H41 N2O6, 609.2965).
Extraction and isolation The air-dried and powdered roots (8.0 kg) of C. tonkinensis were extracted with 95 % EtOH (80 L × 3) three times at room temperature. After removal of the solvent of extraction, the crude residue (408 g) was suspended in 2.5 L of H2O and acidified with 2 N hydrochloric acid to pH 3. The acidic mixture was defatted with ethyl acetate (1500 mL × 2) and then basified with 10 % aqueous NH4OH to pH 10. Extraction of the subsequent mixture with CHCl3 (1500 mL × 3) afforded crude alkaloids (42.0 g), which were subjected to silica gel column chromatography (45–75 µm, 10 × 90 cm) using a step gradient of CHCl3-MeOH-diethylamine 99 : 1 : 0.5 (2000 mL), 98 : 2 : 0.5 (2000 mL), 95 : 5 : 0.5 (8000 mL), 90 : 10 : 0.5 (4000 mL), and 30 : 10 : 0.5 (2000 mL). Fractions of 200 mL were collected and monitored by TLC, resulting in five major fractions (F1–F5). Fraction F2 (17.0 g) was further chromatographed on a silica gel column (45–75 µm, 8 × 35 cm) employing CHCl3-MeOH 95 : 5 (5500 mL), 90 : 10 (2000 mL), and 70 : 30 (2000 mL) as the eluent to afford six subfractions (A–F). Purification of subfraction A (3.0 g) by a silica gel column (45–75 µm, 4 × 20 cm), eluting with CHCl3-MeOH (95 : 5, 1000 mL) yielded cissampentine A (1) (28 mg), cissampentine B (2) (13 mg), cissampentine D (4) (160 mg), and 7-O-methylhayatidine (5) (106 mg). Column chromatography of subfraction B (9.0 g) over a silica gel column (45–75 µm, 8 × 30 cm) using chloroform-metha-
Wang J-z et al. Bisbenzylisoquinoline Alkaloids from …
Cissampentine B (2) White amorphous powder, m. p. 185–188 °C. [α]20 D −90 (c 0.34, CHCl3). UV (MeOH) λmax (log ε) nm: 210 (4.41), and 279 (3.87). CD (MeOH) λmax (Δ ε) nm: 216 (− 37.8), 250 (− 6.52), 279 (+ 2.74), and 295 (− 3.75). IR (KBr) νmax · cm−1: 3415, 2924, 1618, 1513, 1459, 1371, 1271, 1219, 1123, 1019, and 972. 1H‑NMR " Table 2. 13C‑NMR (100 MHz, CDCl ), see (400 MHz, CDCl3), see l 3 " Table 1. NOEDS: H-13 to 12-OMe (10%), 12-OMe to H-13 (6 %). l ESIMS m/z: 623 [M + H]+, 342, 312, 311, 298, 297, 192, and 176. HR-ESIMS m/z: 623.3117 [M + H]+ (calcd. for C38H43 N2O6, 623.3121).
Cissampentine C (3) White amorphous powder, m. p. 234–236 °C. [α]20 D -75 (c 0.14, MeOH). UV (MeOH) λmax (log ε) nm: 210 (4.31), and 280 (3.79). CD (MeOH) λmax (Δ ε) nm: 216 (− 20.2), 254 (− 3.27), 279 (+ 1.47), and 298 (− 1.79). IR (KBr) νmax · cm−1: 3425, 2933, 1607, 1506, 1450, 1268, 1221, 1116, and 1018. 1H‑NMR (400 MHz, " Table 2.13C‑NMR (100 MHz, CDCl CDCl3 + CD3OD), see l 3 + " Table 1. ESIMS m/z: 625 [M + H]+, 417, 344, 327, CD3OD), see l 314, 312, 297, 296, 208, and 192. HR-ESIMS m/z: 625.2919 [M + H]+ (calcd. for C37H41 N2O7, 625.2913).
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Cissampentine D (4)
Conflict of Interest
White amorphous powder, m. p. 178–181 °C. [α]20 D −129 (c 0.33, CHCl3). UV (MeOH) λmax (log ε) nm: 213 (4.28), and 280 (3.67). CD (MeOH) λmax (Δ ε) nm: 214 (− 44.5), 258 (− 0.67), 284 (− 1.85), and 297 (− 0.85). IR (KBr) νmax · cm−1: 3417, 2934, 1615, 1507, 1443, 1367, 1271, 1215, 1120, 1019, and 828. 1H‑NMR " Table 2.13C‑NMR (100 MHz, CDCl ), see (400 MHz, CDCl3), see l 3 " l Table 1. ESIMS m/z: 609 [M + H]+, 462, 314, 312, 311, 298, 229, 208, and 192. HR-ESIMS m/z: 609.2969 [M + H]+ (calcd. for C37H41N2O6, 609.2965).
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The cytotoxic activities of the compounds 1–4 (purity > 90 % by TLC) and paclitaxel (purity > 99 %, Hainan Shunyuan Chemotech) were evaluated in vitro by measuring cell viability using the MTT assay. HCT-8, Bel-7402, and A2780 cells (Shanghai Bioleaf Biotech) were maintained in RPMI-1640 medium supplied with 5 % FBS. Cells in the logarithmic phase were cultured at a density of 50 000 cells/ml per well in a 24-well plate. The cells were exposed to various concentrations of the test compounds for 72 h, and each compound was tested in triplicate at every concentration. The methylene blue dye assay was used to evaluate the effects of the tested compounds on cell growth. The IC50 value resulting from 50 % inhibition of cell growth was calculated graphically as a comparison with the control.
Supporting information The isolation and purification of the compounds, HR‑MS, CD, 1H and 13C NMR as well as 2D‑NMR spectra for compounds 1–4 are available as Supporting Information.
Acknowledgements !
The authors declare no conflicts of interest.
References 1 Angerhofer CK, Guinaudeau H, Wongpanich V, Pezzuto JM, Cordell GA. Antiplasmodial and cytotoxic activity of natural bisbenzylisoquinoline alkaloids. J Nat Prod 1999; 62: 59–66 2 Hall AM, Chang CJ. Multidrug-resistance modulators from Stephania japonica. J Nat Prod 1997; 60: 1193–1195 3 Schiff PL jr. Bisbenzylisoquinoline alkaloids. J Nat Prod 1997; 60: 934– 953 4 Ndaya TJ, Wright AD, Konig GM. Hplc isolation of the anti-plasmodially active bisbenzylisoquinoline alkaloids present in roots of Cissampelos mucronata. Phytochem Anal 2003; 14: 13–22 5 Luo XR, Zhao SY. Medicinal plant of Menispermaceae in China. Guihaia 1986; 6: 49–61 6 Wang JZ, Liao J, Lei Y. Study on the chemical constituents of Cycle wattii and structure of curine-type alkaloids. West China J Pharm Sci 2014; 29: 11–13 7 Wang JZ, Liu XY, Wang FP. Two new curine-type bisbenzylisoquinoline alkaloids from the roots of Cyclea wattii with cytotoxic activities. Chem Pharm Bull 2010; 58: 986–988 8 Tanahashi T, Su Y, Nagakura N, Nayeshiro H. Quaternary isoquinoline alkaloids from Stephania cepharantha. Chem Pharm Bull 2000; 48: 370–373 9 Galinis DL, Wiemer DF, Cazin J jr. Cissampentin: a new bisbenzylisoquinoline alkaloid from Cissampelos fasiculata. Tetrahedron 1993; 49: 1337–1342 10 Tomita M, Kikuchi T, Fujitani K, Kato A, Furukawa H, Aoyagi Y, Kitano M, Ibuka T. Mass spectrometry of bisbenzylisoquinoline alkaloids. Tetrahedron Lett 1966; 8: 857–864 11 Koike L, Marsaioli AJ, Reis F. Proton and carbon-13 nuclear magnetic resonance spectroscopy and conformational aspects of the curine class of bisbenzylisoquinoline alkaloids. J Org Chem 1981; 46: 2385–2389 12 Koike L, Marsaioli AJ, Ruveda EA, Reis F. Stereochemical aspects and 13C NMR spectroscopy of the berbamine class of bisbenzylisoquinoline alkaloids. Tetrahedron Lett 1979; 20: 3765–3768 13 Guinaudeau H, Freyer AJ, Shamma M. Spectral characteristics of the bisbenzylisoquinoline alkaloids. Nat Prod Rep 1986; 476–488
The authors sincerely thank Dr. Hong Zhang, Sichuan Institute for Food and Drug Control, for supplying plant material and Ms. KaiLei Lin, Shanghai Institute of Materia Medica, for recording the CD spectra.
Wang J-z et al. Bisbenzylisoquinoline Alkaloids from …
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