Planta Med 2015; 81(18): 1712-1718
DOI: 10.1055/s-0035-1557743
Natural Product Chemistry
Original Papers
Georg Thieme Verlag KG Stuttgart · New York

Four New Amaryllidaceae Alkaloids from Lycoris radiata and Their Cytotoxicity

Song Ang*
1   Institute of Traditional Chinese Medicine and Natural Products, Jinan University, Guangzhou, PR China
2   JNU-HKUST Joint Laboratory for Neuroscience and Innovative Drug Research, Jinan University, Guangzhou, PR China
,
Xia-Mei Liu*
1   Institute of Traditional Chinese Medicine and Natural Products, Jinan University, Guangzhou, PR China
2   JNU-HKUST Joint Laboratory for Neuroscience and Innovative Drug Research, Jinan University, Guangzhou, PR China
,
Xiao-Jun Huang
1   Institute of Traditional Chinese Medicine and Natural Products, Jinan University, Guangzhou, PR China
2   JNU-HKUST Joint Laboratory for Neuroscience and Innovative Drug Research, Jinan University, Guangzhou, PR China
,
Dong-Mei Zhang
1   Institute of Traditional Chinese Medicine and Natural Products, Jinan University, Guangzhou, PR China
,
Wei Zhang
1   Institute of Traditional Chinese Medicine and Natural Products, Jinan University, Guangzhou, PR China
,
Lei Wang
1   Institute of Traditional Chinese Medicine and Natural Products, Jinan University, Guangzhou, PR China
2   JNU-HKUST Joint Laboratory for Neuroscience and Innovative Drug Research, Jinan University, Guangzhou, PR China
,
Wen-Cai Ye
1   Institute of Traditional Chinese Medicine and Natural Products, Jinan University, Guangzhou, PR China
2   JNU-HKUST Joint Laboratory for Neuroscience and Innovative Drug Research, Jinan University, Guangzhou, PR China
› Author Affiliations
Further Information

Correspondence

Prof. Dr. Wen-Cai Ye
Institute of Traditional Chinese Medicine and Natural Products, College of Pharmacy, Jinan University
601 West Huangpu Avenue
Guangzhou 510632
P. R. China
Phone: +86 20 85 22 09 36   
Fax: +86 20 85 22 15 59   

 


Dr. Lei Wang
Institute of Traditional Chinese Medicine and Natural Products, College of Pharmacy, Jinan University
601 West Huangpu Avenue
Guangzhou 510632
P. R. China
Phone: +86 20 85 22 35 53   
Fax: +86 20 85 22 15 59   

Publication History

received 27 December 2014
revised 05 June 2015

accepted 10 June 2015

Publication Date:
07 August 2015 (online)

 

Abstract

Four new Amaryllidaceae alkaloids, named lycoranines C–F (14), together with seven known ones (511) were isolated from the bulbs of Lycoris radiata. Their structures with absolute configurations were elucidated by nuclear magnetic resonance, high-resolution electrospray ionization mass spectrometry, circular dichroism spectra, modified Mosherʼs method, and molecular modeling calculation. Compounds 6, 7, 10, and 11 exhibited a potent inhibitory effect on A549 and LoVo cells with IC50 values ranging from 3.97 ± 0.36 to 17.37 ± 1.57 µM.


#

Introduction

The plant Lycoris radiata (LʼHér.) Herb. (Amaryllidaceae) is mainly distributed in eastern Asia, particularly in China and Japan. The bulbs of this plant have been used as a traditional Chinese medicine in the treatment of laryngeal trouble, suppurative wounds, and furuncles [1]. Previous phytochemical studies on L. radiata revealed that Amaryllidaceae alkaloids are the characteristic constituents of this plant. To date, more than 50 Amaryllidaceae alkaloids have been isolated from L. radiata [2], which showed cytotoxic, acetylcholinesterase inhibitory, antiviral, and antimalarial activities [3], [4], [5]. Our previous search for bioactive constituents from the bulbs of L. radiata resulted in the isolation and identification of two new alkaloids [6] and several known ones [7]. Further investigation on the ethanol extract of the plant has led to the isolation of four new alkaloids, named lycoranines C–F (14), as well as seven known ones (511) ([Fig. 1]). The structures of the new compounds were determined with the aid of NMR and high-resolution electrospray ionization mass spectrometry (HRESIMS) data. To determine their absolute configurations, the modified Mosherʼs method, circular dichroism (CD) spectroscopy, and molecular modeling calculation were applied. In addition, the cytotoxic effects of these alkaloids were evaluated in vitro. Compounds 6, 7, 10, and 11 exhibited a potent inhibitory effect against A549 and LoVo cells with IC50 values ranging from 3.97 ± 0.36 to 17.37 ± 1.57 µM. This paper describes the isolation, structure elucidation, and cytotoxicity of these Amaryllidaceae alkaloids.

Zoom Image
Fig. 1 Chemical structures of 111.

#

Results and Discussion

The molecular formula of lycoranine C (1) was determined to be C16H21NO3 on the basis of a quasi-molecular ion peak at m/z 276.1598 [M + H]+ (calcd. for C16H22NO3: 276.1594) in its HRESIMS. The IR spectrum showed characteristic absorptions attributable to an aromatic ring (1507, 1462 cm−1) and a hydroxyl group (3319 cm−1). The 1H NMR spectra ([Table 1]) indicated the existence of two aromatic protons coupled in an AX system [δ H 7.06 (1H, d, J = 8.0 Hz, H-12), 6.68 (1H, d, J = 8.0 Hz, H-11)], two oxygenated protons [δ H 4.45 (1H, br s, H-16), 4.30 (1H, dddd, J = 8.8, 8.8, 4.3, 4.3 Hz, H-2)], and a methyl group [δ H 2.39 (3H, s, N-CH3)]. The 13C NMR and distortionless enhancement by the polarization transfer (DEPT) spectrum ([Table 1]) displayed sixteen carbon signals, including a methyl group, six methylene, four methine, and five quaternary carbons. Assignment of the 1H and 13C NMR data ([Table 1]) of 1 was completed with the aid of 1H-1H correlation spectroscopy (COSY), heteronuclear single quantum correlation (HSQC), heteronuclear multiple-bond correlation (HMBC), and rotating-frame nuclear Overhauser effect spectroscopy (ROESY) data ([Fig. 2]). Comparison of the NMR data of 1 with those of the known compound O-demethyllycoramine revealed that the signals of 1 were almost identical to those of O-demethyllycoramine, except for some differences observed at the C-1, C-3, and C-4 positions [7]. These findings suggested that the hydroxyl at the C-2 position of 1 could be α-oriented, which was confirmed by the J values between H-2 and H-1α/H-3α (J > 8.0 Hz), as well as by the ROESY correlation between H-2 (δ H 4.30) and H-4β (δ H 1.53) ([Fig. 3]). Based on the X-ray crystallographic data of lycoramine hydrobromide (CCDC 643 823) and molecular modeling calculations (see Supporting Information), the conformation of 1 was determined ([Fig. 3]). The assignment of α and β proton signals was completed by detail analysis of the coupling constants and the ROESY spectrum. The peak shape and coupling constants of H-4β (br t, J = 12.7 Hz), H-6β (br d, J = 14.1 Hz), H-7α (br d, J = 14.5 Hz), and H-7β (br t, J = 12.6 Hz) as well as the ROESY correlations between H-7β and H-9β/H-4α, between H-2 and H-4β, between H-1α and H-3α, and between H-16 and H-6α indicated the signal assignments as shown in [Table 1]. In addition, the 1H NMR assignments of 1 were further confirmed by comparison with the literature data of similar compounds [8], [9] and by molecular modeling calculations using the Gaussian09 program (see Supporting Information).

Zoom Image
Fig. 2 Selected 1H-1H COSY and HMBC correlations for 14. (Color figure available online only.)
Zoom Image
Fig. 3 Key ROESY correlations for 1 and 3. (Color figure available online only.)

Table 11H- and 13C-NMR data of 1–4 (δ in ppm, J in Hz). Overlapped signals were reported without designating multiplicity; *recorded in C5D5N; **recorded in CDCl3; ***recorded in DMSO-d 6.

1*

2**

3***

4*

δ H

δ C

δ H

δ C

δ H

δ C

δ H

δ C

1

α 2.84 m
β 1.95

35.9 (t)

1

α 3.07
β 3.28 dd (16.9, 4.2)

38.1 (t)

2

α 3.64
β 3.80

67.8 (t)

α 3.13 m
β 2.83 dd (18.1, 8.3)

44.9 (t)

2

4.30 dddd (8.8, 8.8, 4.3, 4.3)

66.0 (d)

2

3.79 m

75.4 (d)

3

α 2.80 m
β 2.70 m

25.2 (t)

2.43 m

30.9 (t)

3

α 1.58 m
β 1.95

30.4 (t)

3

α 2.02 td (13.7, 7.3)
β 2.14 m

27.3 (t)

3a

137.8 (s)

141.3 (s)

4

α 2.26 m
β 1.53 br t (12.7)

27.9 (t)

4

α 3.05
β 2.91 m

22.5 (t)

4

5.77 s

122.4 (d)

5.44 d (2.2)

114.8 (d)

5

47.0 (s)

4a

146.7 (s)

5

4.12

65.5 (d)

2.58 m

32.4 (t)

6

α 1.95
β 1.76 br d (14.1)

30.1 (t)

6

8.78 s

148.7 (d)

5a

4.53 s

81.9 (d)

4.93 d (2.8)

76.1 (d)

7

α 2.98 br d (14.5)
β 3.19 br t (12.6)

54.3 (t)

6a

133.5 (s)

7

164.4 (s)

166.0 (s)

9

α 3.71 d (15.0)
β 4.06 d (15.0)

60.9 (t)

7

7.11 s

103.9 (d)

7a

112.0 (s)

117.3 (s)

10

128.3 (s)

8

147.6 (s)

8

7.31 s

112.2 (d)

7.83 s

113.0 (d)

11

6.68 d (8.0)

121.7 (d)

9

151.3 (s)

9

148.7 (s)

149.7 (s)

12

7.06 d (8.0)

116.1 (d)

10

7.12 br s

99.0 (d)

10

155.6 (s)

154.5 (s)

13

142.4 (s)

10a

124.4 (s)

11

7.23 s

115.0 (d)

7.12 s

111.2 (d)

14

146.3 (s)

10b

123.8 (s)

11a

135.8 (s)

139.1 (s)

15

137.8 (s)

OCH3

3.43 s

56.2 (q)

11b

3.59

32.3 (d)

2.74 dd (9.7, 2.3)

43.8 (d)

16

4.45 br s

91.6 (d)

O-CH2-O

6.05 s

101.7 (t)

11c

4.14

76.1 (d)

3.46 d (8.0)

59.8 (d)

N-CH3

2.39 s

41.6 (q)

9-OCH3

3.77 s

55.1 (q)

3.79 s

56.2 (q)

10-OCH3

3.80 s

56.3 (q)

N-CH3

2.98 s

54.4 (q)

To determine the absolute configuration of C-2, the modified Mosherʼs method was applied [10]. The (R)- and (S)-Mosherʼs acid (MTPA) esters (1a and 1b) of 1 were synthesized. The differences of the chemical shift values (Δδ = δ Sδ R, ppm) are shown in [Fig. 4]. The Δδ values for protons at C-1 were negative, while positive values were observed for protons at C-3 and C-4, revealing that C-2 has an R configuration. Thus, the structure of 1 was determined to be 2-epi-O-demethyllycoramine.

Zoom Image
Fig. 4Δδ values (δS–δR ) for the MTPA ester of 1.

The molecular formula of lycoranine D (2) was determined to be C15H15NO3 by its HRESIMS data (m/z 258.1119 [M + H]+, calcd. for C15H16NO3: 258.1125). The IR spectrum revealed the presence of a methylenedioxy group (1246, 939 cm−1) and an aromatic ring (1590, 1461 cm−1). The UV spectrum of 2 showed absorption maxima at 204 and 237 nm. The 1H NMR spectrum showed signals for three aromatic protons at δ H 7.11 (1H, s, H-7), 7.12 (1H, s, H-10), and 8.78 (1H, s, H-6), a methoxyl at δ H 3.43 (3H, s, O-CH3), and a methylenedioxy group at δ H 6.05 (2H, s, O-CH2-O) ([Table 1]). The 13C NMR and DEPT spectra of 2 displayed 15 signals, including a methoxy group (δ C 56.2) and a methylenedioxy unit (δ C 101.7) ([Table 1]), which suggested that 2 possessed a skeleton with 13 C-atoms. Comparison of the 13C NMR data of 2 with those of the known compound 1,2,3,4-tetrahydro[1, 3]dioxolo[4,5-j]phenanthridine revealed that their signals were similar, except for the appearance of the signals for a methoxyl (δ C 56.2) and an oxygenated methine as well as the disappearance of the signal for a methylene in 2 [11], [12]. Thus, 2 was proposed to be a methoxyl substituted derivative of the known compound. The HMBC correlation between methoxyl (δ H 3.43) and C-2 (δ C 75.4) as well as the ROESY cross-peaks between methoxyl (δ H 3.43) and H-1 (δ H 3.07, 3.28)/H-3 (δ H 2.02, 2.14) indicated that the methoxy group was connected to the C-2 position ([Fig. 2]). With the aid of 1H-1H COSY, HSQC, HMBC, and ROESY experiments ([Fig. 2]), the planar structure of 2 was determined. The peak shape and coupling constants of H-1β (dd, J = 16.9, 4.2 Hz) and H-3α (td, J = 13.7, 7.3 Hz) as well as the ROESY correlations between H-1α and H-3α, and between H-2β and H-4β allowed the assignment of these signals, which was further confirmed by molecular modeling calculations (see Supporting Information). The absolute configuration of 2 was determined to be 2R by the calculation of the optical rotation values at three different levels (theory-B3LYP, B3P86, and HF) using the 6–311++G(2 d, p) and aug-cc-pVDZ basis sets in Gaussian 09 software (see Supporting Information) [13]. The calculated specific rotation values and the experimental data for 2 are summarized in [Table 2]. These results indicated that the absolute configuration at C-2 in 2 was R. Therefore, the structure of 2 was elucidated as (2R)-2-methoxy-1,2,3,4-tetrahydro[1, 3]dioxolo[4,5-j]phenanthridine.

Table 2 Calculated and experimental specific rotation values of 2.

Calcd. (in deg. [dm g/cm−3]−1)

Exptl.

Methods

R

S

HF/aug-cc-pVDZ

+ 182.39

− 182.39

+ 126.1

B3LYP/aug-cc-pVDZ

+ 267.95

− 267.95

B3LYP/6–311++G(2 d,p)

+ 267.72

− 267.72

B3P86/6–311++G(2 d,p)

+ 277.24

− 277.24

Lycoranine E (3) was shown to have the molecular formula C17H19NO6 by its HRESIMS data (m/z 334.1293 [M + H]+, calcd. for C17H20NO6: 334.1285). The UV spectrum showed maxima absorptions at 205, 229, 272, and 302 nm. The IR spectra revealed the characteristic absorptions for hydroxyl (3364 cm−1), carbonyl (1706 cm−1), and aromatic rings (1515, 1463 cm−1). The 1H NMR spectrum displayed the signals for two aromatic protons [δ H 7.23 (1H, s, H-11), 7.31 (1H, s, H-8)], an olefinic proton [δ H 5.77 (1H, s, H-4)], a methoxy group [δ H 3.77 (3H, s, OCH3)], and an N-methyl group [δ H 2.98 (3H, s, N-CH3)] ([Table 1]). The 13C NMR and DEPT spectra displayed 17 carbon signals including a methoxyl (δ C 55.1) and an N-methyl (δ C 54.4) ([Table 1]), which suggested that compound 3 possessed a skeleton with 15 C-atoms. The olefinic carbon signals at δ C 137.8 and 122.4 indicated 3 could be a homolycorine-type alkaloid [14], [15]. With the aid of 1H-1H COSY, HSQC, HMBC, and ROESY experiments, all the 1H and 13C NMR signals were assigned as shown in [Table 1]. Comparison of the NMR data of 3 with the known compound 9-O-demethylhomolycorine N-oxide suggested that their NMR signals were similar but not identical [16]. The main difference was a hydroxyl group instead of a hydrogen atom located at the C-5 position in 3, which was further confirmed by the HMBC correlations between H-5 (δ H 4.12) and C-3a (δ C 137.8)/C-5a (δ C 81.9)/C-11b (δ C 32.3), as well as the 1H-1H COSY cross-peak between H-5 (δ H 4.12) and H-4 (δ H 5.77)/H-5a (δ H 4.53) ([Fig. 2]). In addition, the HMBC correlation between the methoxyl (δ H 3.77) and C-9 (δ C 148.7), as well as the ROESY cross-peak between methoxyl (δ H 3.77) and H-8 (δ H 7.31) revealed that the methoxyl was located at the C-9 position. The relative configuration of the hydroxyl group at C-5 was deduced to be β on the basis of the ROESY correlations between H-5 (δ H 4.12) and H-11b (δ H 3.59)/H-5a (δ H 4.53) ([Fig. 3]). To determine the absolute configuration of 3, a CD measurement was applied. The CD spectrum of 3 showed negative Cotton effects at 273 and 228 nm, as well as positive ones at 248 and 204 nm, which was in accordance with the homolycorine-type alkaloids with 5R, 5aS, 11bS, and 11cS configurations ([Fig. 5]) [17]. Thus, the structure of 3 was determined as 5β-hydroxy-10-O-demethylhomolycorine N-oxide.

Zoom Image
Fig. 5 Circular dichroism spectra of 3 and 4 (in MeOH). (Color figure available online only.)

The molecular formula of lycoranine F (4) was established as C17H19NO4 by a quasi-molecular ion peak at m/z 302.1381 [M + H]+ (calcd. for C17H20NO4: 302.1387) in its HRESIMS. The UV and IR spectra of 4 showed similar absorptions to those of 3. The 1H NMR spectrum displayed two aromatic protons [δ H 7.83 (1H, s, H-8), 7.12 (1H, s, H-11)], an olefinic proton [δ H 5.44 (1H, d, J = 2.2 Hz, H-4)], and two methoxy groups [δ H 3.80 (3H, s, OCH3), 3.79 (3H, s, OCH3)] ([Table 1]). Comparison of the 13C NMR data of 4 with those of the known compound homolycorine (5) revealed that their signals were similar, except for the resonance changes for C-2 and C-11c and the absence of N-methyl signal. Thus, 4 was proposed as an N-demethyl derivative of homolycorine (5), which was confirmed by the analysis of 1H-1H COSY, HSQC, HMBC, and ROESY data ([Fig. 2]). The absolute configuration of 4 was determined as 5aR, 11bS, and 11cS by comparison of its CD spectrum with that of homolycorine (5) ([Fig. 5]). Based on the above evidences, the structure of 4 was established as N-demethylhomolycorine.

Other Amaryllidaceae alkaloids, homolycorine (5) [14], [15], haemanthidine (6) [18], haemanthamine (7) [19], α-dihydrolycorine (8) [20], galanthine (9) [7], lycorine (10) [20], [21] and pseudolycorine (11) [22], were also isolated from L. radiata. Their structures ([Fig. 1]) were established by comparing their spectroscopic data with those reported in literature.

Amaryllidaceae alkaloids have attracted much attention due to their multiple bioactivities, such as antitumor, antiviral, and acetylcholinesterase-inhibitory effects [3], [4], [5]. To further investigate the bioactivity of the isolated alkaloids, the cytotoxicities of compounds 1–11 against A549 and LoVo cells were evaluated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay using doxorubicin as a positive control. As shown in [Table 3], compounds 6, 7, 10, and 11 showed potent cytotoxic activities against A549 and LoVo cells with IC50 values in the ranges of 3.97–11.36 µM and 8.98–17.37 µM, respectively. Itʼs noteworthy to mention that for lycorine-type alkaloids, lycorine (10) and pseudolycorine (11) exhibited potent cytotoxic effects, while α-dihydrolycorine (8) and galanthine (9) were nearly inactive on A549 and LoVo cell lines. Since the opened ring in pseudolycorine (11) resulted in complete retention of its antiproliferative activity, it was likely that the hydrogenation of the olefinic bond in α-dihydrolycorine (8) and the methylation of the C-2 hydroxy group in galanthine (9) were responsible for the absence of activity of 8 and 9. These results indicated that the substructure of the C ring plays an important role in the cytotoxic activity of lycorine-type alkaloids.

Table 3 The IC50 values of cytotoxic activities against A549 and LoVo cells; (72 h, X ± SD).

Compounds

Cytotoxicity (IC50, µM)

A549

LoVo

1

> 20

> 20

2

> 20

> 20

3

> 20

> 20

4

> 20

> 20

5

> 20

> 20

6

11.36 ± 0.63

17.37 ± 1.57

7

4.48 ± 0.46

9.02 ± 0.45

8

> 20

> 20

9

> 20

> 20

10

9.55 ± 0.40

17.36 ± 1.26

11

3.97 ± 0.36

8.98 ± 0.72

Doxorubicin

0.21 ± 0.10

0.85 ± 0.98


#

Materials and Methods

General experimental procedures

UV spectra were measured on a Jasco V-550 UV/VIS spectrophotometer. IR spectra were obtained on a Jasco FI/IR-480 plus infrared spectrometer with KBr discs. Optical rotations were detected on a Jasco P-1020 polarimeter. CD spectrum was taken on a Jasco J-810 circular dichroism spectrometer at room temperature using a 0.2-cm standard cell. HRESIMS data were determined on Agilent 6210 ESI/TOF mass spectrometer (Agilent Technologies). ESIMS spectra were recorded on a Finnigan LCQ Advantage Max ion trap mass spectrometer (Thermo Electron). NMR spectra were measured on a Bruker AV-400 spectrometer. Chemical shift values were expressed in δ (ppm) relative to tetramethylsilane (TMS) as the internal standard. Column chromatographies (CC) were carried out with silica gel (200–300 mesh, Qingdao Marine Chemical Factory), ODS (YMC-gel ODS-A, S-150 µm, YMC Co. Ltd.), and Sephadex LH-20 (Pharmacia Biotec AB). TLC was performed using precoated silica gel GF254 plates (Yantai Chemical Industry Research Institute). High-performance liquid chromatography (HPLC) was carried out on an Agilent chromatograph 1260 equipped with a G1310B pump and a G1365D UV detector coupled with analytical and preparative Capcell Pak MG II C18 columns (250 × 4.6 mm and 250 × 20 mm, respectively). All solvents used in column chromatography and HPLC were of analytical grade (Tianjin Damao Chemical Plant) and chromatographic grade (Fisher), respectively.


#

Plant material

The bulbs of L. radiata were collected in September of 2008 in Lanxi City, Zhejiang province of China, and identified by Prof. Guang-Xiong Zhou, College of Pharmacy, Jinan University, Guangzhou, PR China. A voucher specimen (No. 2 008 092 301) was deposited in the herbarium of the Institute of Traditional Chinese Medicine and Natural Products, Jinan University, Guangzhou, PR China.


#

Extraction and isolation

The air-dried and powdered bulbs of L. radiata (40 kg) were extracted with 95 % EtOH (80 L × 4, two days each) at room temperature. The ethanol extract was evaporated under reduced pressure (45 °C) to afford a brownish residue (4.1 kg), which was dissolved in water (10 L), basified to pH 9–10 with 10 % aqueous ammonia and extracted with chloroform (4 × 10 L, room temperature). The chloroform solution was then extracted with 5 % hydrochloric acid (4 × 10 L, room temperature). The acidic water solution was basified to pH 8–9 with Na2CO3 and extracted with EtOAc four times (each 10 L, room temperature), and then was concentrated under reduced pressure (40 °C) to yield the total alkaloids (190 g). An alkaloid fraction (180 g) was subjected to a silica gel column (200–300 mesh, 10 × 150 cm, 4.0 kg) using a gradient CHCl3-MeOH system (100 : 0, 100 : 1, 100 : 2, 100 : 3, 100 : 5, 100 : 7, 100 : 10, 100 : 20, 100 : 30, 100 : 50, 100 : 100, each 15 L) to afford 11 fractions (Fr. 1 – Fr. 11). Fr. 2 (2.5 g) was chromatographed on silica gel (200–300 mesh, 5 × 40 cm, 60 g) eluting with petroleum ether-acetone (100 : 0, 100 : 1, 100 : 1.5, 100 : 2, 100 : 3, 100 : 4, 100 : 5, 100 : 7, 100 : 10, 100 : 20, 100 : 30, 100 : 50, 100 : 100, each 1 L) to obtain 13 subfractions (Fr. 2.1 – Fr. 2.13). Fr. 2.3 (80 mg) was further purified by preparative HPLC using MeOH-H2O (60 : 40, 0.01 % diethylamine, 8 mL/min) as the eluent to afford 1 (8 mg, t R = 48 min) and 9 (6 mg, t R = 60 min). Fr. 3 (3.6 g) was further separated by a silica gel column (200–300 mesh, 5 × 60 cm, 100 g) using CHCl3-MeOH (100 : 5, 100 : 10, 100 : 20, 100 : 100, each 1.5 L) as the eluent to give four subfractions (Fr. 3.1 – Fr. 3.4). Fr. 3.2 (200 mg) was purified by a Sephadex LH-20 column (1.2 × 100 cm, 30 g), eluted by CHCl3-MeOH (1 : 1, 1 L), and then further purified by preparative HPLC using MeOH-H2O (45 : 55, 0.01 % diethylamine, 6 mL/min) as the mobile phase to yield 3 (18 mg, t R = 30 min). Fr. 6 (15 g) was subjected to silica gel CC (200–300 mesh, 8 × 60 cm, 400 g; CHCl3-MeOH, 100 : 1, 100 : 2, 100 : 5, 100 : 10, 100 : 20, 100 : 100, each 3 L) to afford six subfractions (Fr. 6.1 – Fr. 6.6). Subfraction Fr. 6.3 (3.3 g) was further purified by RP-18 CC (3 × 40 cm, 100 g; MeOH-H2O, 40 : 60, 2.5 L) to yield 5 (10 mg) and 8 (21 mg). Fr. 6.5 (200 mg) was purified by Sephadex LH-20 CC (1.2 × 100 cm, 30 g; CHCl3-MeOH, 1 : 1, 1 L) to afford 10 (13 mg) and a mixture, which was further separated by preparative C18 HPLC with MeOH-H2O (40 : 60, 0.01 % diethylamine, 6 mL/min) as the eluent to give 11 (5 mg, t R = 35 min). Fr. 8 (150 mg) was chromatographed on silica gel (300–400 mesh, 2.0 × 25 cm, 10 g) eluting with CHCl3-MeOH (10 : 1, 0.8 L) to obtain 2 (3 mg) and 6 (12 mg). Fr. 10 (2.6 g) was subjected to ODS column (3 × 40 cm, 100 g) eluting with MeOH-H2O (35 : 65, 2 L) to obtain 4 (14 mg) and 7 (22 mg).


#

Cytotoxicity assay

The human lung carcinoma cell line A549 and human colon carcinoma cell line LoVo were purchased from American Type Culture Collection (ATCC). All the cells were cultured in RPMI-1640 medium (Sigma) containing 10 % new bovine serum or fetal bovine serum and 1 % penicillin-streptomycin in 5 % CO2 at 37 °C. The cytotoxicities of all compounds (111, purities > 90 %) against A549 and LoVo cells were measured according to the MTT assay. The absorbance was measured at 570 nm using a microplate spectrophotometer (Thermo scientific multiskan MK3). The concentration giving 50 % inhibition (IC50) was determined from the dose-response curves using Prism software and expressed as the mean ± S. D. Doxorubicin (Sigma, purity ≥ 98 %) was used as a positive control.


#

Preparation of Mosherʼs acid esters of 1

Compound 1 (2 × 2.5 mg) in anhydrous pyridine (0.5 mL) was reacted with (S)-α-methoxy-α-(trifluoromethyl) phenylacetic chloride (10 µL) and (R)-α-methoxy-α-(trifluoromethyl) phenylacetic chloride (10 µL), respectively. Each mixture was stirred at room temperature for 6 h, then evaporated to dryness and purified by column chromatography (Sephadex LH-20, CHCl3/MeOH, 1 : 1) to yield the (R)-MTPA ester (1a, 2.2 mg) and the (S)-MTPA ester (1b, 2.3 mg).

Lycoranine C (1): amorphous powder; [α]D 25 − 40.9 (c = 0.33, MeOH); IR (KBr) v max · cm−1: 3319, 2935, 1507, 1462, 1308, 1067; UV λ max nm (log ε): 208 (3.67), 231 (2.94), 287 (2.58); HRESIMS m/z 276.1598 [M + H]+ (calcd. for C16H22NO3: 276.1594); 1H (400 MHz, pyridine-d 5) and 13C NMR (100 MHz, pyridine-d 5) data, see [Table 1].

Lycoranine D (2): amorphous powder; [α]D 25 + 126.1 (c = 0.35, MeOH); IR (KBr) v max · cm−1: 2930, 1590, 1461, 1246, 1095, 1037, 939, 834; UV λ max nm (log ε): 204 (2.31), 237 (3.59); HRESIMS m/z 258.1119 [M + H]+ (calcd. for C15H16NO3: 258.1125); 1H (400 MHz, CDCl3) and 13C NMR (100 MHz, CDCl3) data, see [Table 1].

Lycoranine E (3): amorphous powder; [α]D 25 − 8.2 (c = 0.36, C5H5 N); IR (KBr) v max · cm−1: 3364, 1706, 1515, 1463, 1297, 1071; UV λ max nm (log ε): 205 (3.65), 229 (3.68), 272 (3.22), 302 (3.05); HRESIMS m/z 334.1293 [M + H]+ (calcd. for C17H20NO6: 334.1285); CD (MeOH) λ max (Δε): 272.9 (− 14.3), 247.5 (+ 20.1), 227.8 (− 35.9), 204.3 (+ 36.1); 1H (400 MHz, DMSO-d 6) and 13C NMR (100 MHz, DMSO-d 6) data, see [Table 1].

Lycoranine F (4): amorphous powder; [α]D 25 + 33.5 (c = 0.34, MeOH); IR (KBr) v max · cm−1: 3334, 2933, 1698, 1603, 1511, 1459, 1361, 1304, 1263, 1080, 777; UV λ max nm (log ε): 205 (3.66), 227 (3.78), 268 (3.41), 300 (3.19); HRESIMS m/z 302.1381 [M + H]+ (calcd. for C17H20NO4: 302.1387); CD (MeOH) λ max (Δε): 269.3 (− 9.7), 247.8 (+ 12.6), 228.5 (− 22.8), 204.3 (+ 31.2); 1H (400 MHz, pyridine-d 5) and 13C NMR (100 MHz, pyridine-d 5) data, see [Table 1].


#

Supporting information

HR-ESI-MS, IR, UV, and NMR spectra of lycoranines C–F (14) as well as NMR spectra of the MTPA esters of 1 are available as Supporting Information.


#
#

Acknowledgments

This work was supported financially by the Program for National Natural Science Foundation of China (No. 81 273 391), the Ministry of Science and Technology of the Peopleʼs Republic of China (Nos. 2013DFM30 080, 2013BAI11B05, 2012ZX09 103 201–056), and the Program of Pearl River Young Talents of Science and Technology in Guangzhou, China (2013J2 200 058).


#
#

Conflict of Interest

The authors declare no conflict of interest.

* These authors contributed equally to this work.


Supporting Information

  • References

  • 1 Zhongguo Kexueyuan. Flora Republicae Popularis Sinicae (Zhongguo Zhiwu Zhi), Part 1. Beijing: Science Press; 1985: 16-18
  • 2 Feng T, Wang YY, Su J, Li Y, Cai XH, Luo XD. Amaryllidaceae alkaloids from Lycoris radiata . Helv Chim Acta 2011; 94: 178-183
  • 3 Weniger B, Italiano L, Beck JP, Bastida J, Bergonon S, Codina C, Lobstein A, Anton R. Cytotoxic activity of amaryllidaceae alkaloids. Planta Med 1995; 61: 77-79
  • 4 Szlavik L, Gyuris A, Minarovits J, Forgo P, Molnar J, Hohmann J. Alkaloids from Leucojum vernum and antiretroviral activity of Amaryllidaceae alkaloids. Planta Med 2004; 70: 871-873
  • 5 He J, Qi WB, Wang L, Tian J, Jiao PR, Liu GQ, Ye WC, Liao M. Amaryllidaceae alkaloids inhibit nuclear-to-cytoplasmic export of ribonucleoprotein (RNP) complex of highly pathogenic avian influenza virus H5N1. Influenza Other Respir Viruses 2012; 7: 922-931
  • 6 Wang L, Zhang XQ, Yin ZQ, Wang Y, Ye WC. Two new Amaryllidaceae alkaloids from the bulbs of Lycoris radiata . Chem Pharm Bull 2009; 57: 610-611
  • 7 Wang L, Yin ZQ, Cai Y, Zhang XQ, Yao XS, Ye WC. Amaryllidaceae alkaloids from the bulbs of Lycoris radiata . Biochem Syst Ecol 2010; 38: 444-446
  • 8 Forgo P, Hohmann J. Leucovernine and acetylleucovernine, alkaloids from Leucojum vernum . J Nat Prod 2005; 68: 1588-1591
  • 9 Kobayashi S, Satoh K, Numata A, Shingu T, Kihara M. Alkaloid N-oxides from Lycoris sanguinea . Phytochemistry 1991; 30: 675-677
  • 10 Wang L, Yin ZQ, Wang Y, Zhang XQ, Li YL, Ye WC. Perisesaccharides A–E, New oligosaccharides from the root barks of Periploca sepium . Planta Med 2010; 76: 909-915
  • 11 Kumemura T, Choshi T, Yukawa J, Hirose A, Nobuhiro J, Hibino S. A highly efficient synthesis of trispheridine through a construction of tetrahydrophenanthridine based on a microwave-assisted thermal electrocyclic reaction of an aza 6π-electron system. Heterocycles 2005; 66: 87-90
  • 12 Pandey G, Balakrishnan M. Suzuki cross-coupling/reductive debenzyloxycarbonylation sequence for the syntheses of [c] annulated isoquinolines: application for the syntheses of pancratistatin-like isoquinolines. J Org Chem 2008; 73: 8128-8131
  • 13 Wang WJ, Wang L, Huang XJ, Jiang RW, Yang XL, Zhang DM, Chen WM, Tang BQ, Wang Y, Zhang XQ, Ye WC. Two pairs of new benzofuran enantiomers with unusual skeletons from Eupatorium chinense . Tetrahedron Lett 2013; 54: 3321-3324
  • 14 Bastida J, Llabrés JM, Viladomat F, Codina C, Rubiralta M, Feliz M. Narcissus alkaloids, III. 9-O-demethylhomolycorine from Narcissus confusus . J Nat Prod 1987; 50: 199-202
  • 15 Jeffs PW, Abou-Donia A, Campau D, Staiger D. Structures of 9-O-demethylhomolycorine and 5α-hydroxyhomolycorine. Alkaloids of Crinum defixum, C. scabrum, and C. latifolium. Assignment of aromatic substitution patterns from 1H-coupled 13C spectra. J Org Chem 1985; 50: 1732-1737
  • 16 Suau R, Rico R, García AI, Gomez AI. New Amaryllidaceae alkaloids from Narcissus Papyraceus Ker-Gawler. Heterocycles 1990; 31: 517-522
  • 17 Wagner J, Pharm HL, Döpke W. Alkaloids from Hippeastrum equestre Herb. − 5. Circular dichroism studies. Tetrahedron 1996; 52: 6591-6600
  • 18 Hohmann J, Forgo P, Molnar J, Wolfard K, Molnar A, Thalhammer T, Mathe I, Sharples D. Antiproliferative amaryllidaceae alkaloids isolated from the bulbs of Sprekelia formosissima and Hymenocallis × festalis . Planta Med 2002; 68: 454-457
  • 19 Kobayashi S, Ishikawa H, Kihara M, Shingu T, Hashimoto T. Isolation of carinatine and pretazettine from the bulbs of Zephyranthes carinate Herb (Amaryllidaceae). Chem Pharm Bull 1977; 25: 2244-2248
  • 20 Evidente A, Cicala MR, Giudicianni I, Randazzo G, Riccio R. 1H and 13C NMR analysis of lycorine and α-dehydrolycorine. Phytochemistry 1983; 22: 581-584
  • 21 Youssef DTA, Frahm AW. Alkaloids of the flowers of Pancratium maritimum . Planta Med 1998; 64: 669-670
  • 22 Llabres JM, Viladomat F, Bastida J, Codina C, Serrano M, Feliz M. Two alkaloids from Narcissus requienii . Phytochemistry 1986; 25: 1453-1459

Correspondence

Prof. Dr. Wen-Cai Ye
Institute of Traditional Chinese Medicine and Natural Products, College of Pharmacy, Jinan University
601 West Huangpu Avenue
Guangzhou 510632
P. R. China
Phone: +86 20 85 22 09 36   
Fax: +86 20 85 22 15 59   

 


Dr. Lei Wang
Institute of Traditional Chinese Medicine and Natural Products, College of Pharmacy, Jinan University
601 West Huangpu Avenue
Guangzhou 510632
P. R. China
Phone: +86 20 85 22 35 53   
Fax: +86 20 85 22 15 59   

  • References

  • 1 Zhongguo Kexueyuan. Flora Republicae Popularis Sinicae (Zhongguo Zhiwu Zhi), Part 1. Beijing: Science Press; 1985: 16-18
  • 2 Feng T, Wang YY, Su J, Li Y, Cai XH, Luo XD. Amaryllidaceae alkaloids from Lycoris radiata . Helv Chim Acta 2011; 94: 178-183
  • 3 Weniger B, Italiano L, Beck JP, Bastida J, Bergonon S, Codina C, Lobstein A, Anton R. Cytotoxic activity of amaryllidaceae alkaloids. Planta Med 1995; 61: 77-79
  • 4 Szlavik L, Gyuris A, Minarovits J, Forgo P, Molnar J, Hohmann J. Alkaloids from Leucojum vernum and antiretroviral activity of Amaryllidaceae alkaloids. Planta Med 2004; 70: 871-873
  • 5 He J, Qi WB, Wang L, Tian J, Jiao PR, Liu GQ, Ye WC, Liao M. Amaryllidaceae alkaloids inhibit nuclear-to-cytoplasmic export of ribonucleoprotein (RNP) complex of highly pathogenic avian influenza virus H5N1. Influenza Other Respir Viruses 2012; 7: 922-931
  • 6 Wang L, Zhang XQ, Yin ZQ, Wang Y, Ye WC. Two new Amaryllidaceae alkaloids from the bulbs of Lycoris radiata . Chem Pharm Bull 2009; 57: 610-611
  • 7 Wang L, Yin ZQ, Cai Y, Zhang XQ, Yao XS, Ye WC. Amaryllidaceae alkaloids from the bulbs of Lycoris radiata . Biochem Syst Ecol 2010; 38: 444-446
  • 8 Forgo P, Hohmann J. Leucovernine and acetylleucovernine, alkaloids from Leucojum vernum . J Nat Prod 2005; 68: 1588-1591
  • 9 Kobayashi S, Satoh K, Numata A, Shingu T, Kihara M. Alkaloid N-oxides from Lycoris sanguinea . Phytochemistry 1991; 30: 675-677
  • 10 Wang L, Yin ZQ, Wang Y, Zhang XQ, Li YL, Ye WC. Perisesaccharides A–E, New oligosaccharides from the root barks of Periploca sepium . Planta Med 2010; 76: 909-915
  • 11 Kumemura T, Choshi T, Yukawa J, Hirose A, Nobuhiro J, Hibino S. A highly efficient synthesis of trispheridine through a construction of tetrahydrophenanthridine based on a microwave-assisted thermal electrocyclic reaction of an aza 6π-electron system. Heterocycles 2005; 66: 87-90
  • 12 Pandey G, Balakrishnan M. Suzuki cross-coupling/reductive debenzyloxycarbonylation sequence for the syntheses of [c] annulated isoquinolines: application for the syntheses of pancratistatin-like isoquinolines. J Org Chem 2008; 73: 8128-8131
  • 13 Wang WJ, Wang L, Huang XJ, Jiang RW, Yang XL, Zhang DM, Chen WM, Tang BQ, Wang Y, Zhang XQ, Ye WC. Two pairs of new benzofuran enantiomers with unusual skeletons from Eupatorium chinense . Tetrahedron Lett 2013; 54: 3321-3324
  • 14 Bastida J, Llabrés JM, Viladomat F, Codina C, Rubiralta M, Feliz M. Narcissus alkaloids, III. 9-O-demethylhomolycorine from Narcissus confusus . J Nat Prod 1987; 50: 199-202
  • 15 Jeffs PW, Abou-Donia A, Campau D, Staiger D. Structures of 9-O-demethylhomolycorine and 5α-hydroxyhomolycorine. Alkaloids of Crinum defixum, C. scabrum, and C. latifolium. Assignment of aromatic substitution patterns from 1H-coupled 13C spectra. J Org Chem 1985; 50: 1732-1737
  • 16 Suau R, Rico R, García AI, Gomez AI. New Amaryllidaceae alkaloids from Narcissus Papyraceus Ker-Gawler. Heterocycles 1990; 31: 517-522
  • 17 Wagner J, Pharm HL, Döpke W. Alkaloids from Hippeastrum equestre Herb. − 5. Circular dichroism studies. Tetrahedron 1996; 52: 6591-6600
  • 18 Hohmann J, Forgo P, Molnar J, Wolfard K, Molnar A, Thalhammer T, Mathe I, Sharples D. Antiproliferative amaryllidaceae alkaloids isolated from the bulbs of Sprekelia formosissima and Hymenocallis × festalis . Planta Med 2002; 68: 454-457
  • 19 Kobayashi S, Ishikawa H, Kihara M, Shingu T, Hashimoto T. Isolation of carinatine and pretazettine from the bulbs of Zephyranthes carinate Herb (Amaryllidaceae). Chem Pharm Bull 1977; 25: 2244-2248
  • 20 Evidente A, Cicala MR, Giudicianni I, Randazzo G, Riccio R. 1H and 13C NMR analysis of lycorine and α-dehydrolycorine. Phytochemistry 1983; 22: 581-584
  • 21 Youssef DTA, Frahm AW. Alkaloids of the flowers of Pancratium maritimum . Planta Med 1998; 64: 669-670
  • 22 Llabres JM, Viladomat F, Bastida J, Codina C, Serrano M, Feliz M. Two alkaloids from Narcissus requienii . Phytochemistry 1986; 25: 1453-1459

Zoom Image
Fig. 1 Chemical structures of 111.
Zoom Image
Fig. 2 Selected 1H-1H COSY and HMBC correlations for 14. (Color figure available online only.)
Zoom Image
Fig. 3 Key ROESY correlations for 1 and 3. (Color figure available online only.)
Zoom Image
Fig. 4Δδ values (δS–δR ) for the MTPA ester of 1.
Zoom Image
Fig. 5 Circular dichroism spectra of 3 and 4 (in MeOH). (Color figure available online only.)