Two new alkaloids from Cephalotaxus hainanensis Li

Phytochemistry Letters 34 (2019) 1–4

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Two new alkaloids from Cephalotaxus hainanensis Li a,1

a,1

a

a

a

a

Miao Yu , Hao Wang , Pei Wang , Shengzhuo Huang , Caihong Cai , Fandong Kong , ⁎ ⁎ Youle Qub, Limin Liuc, Wenli Meia, , Haofu Daia, a b c

T

Hainan Key laboratory for research and development of natural products from Li folk medicine, 571101, Haikou, PR China College of Food and Medicine, Zhejiang Ocean University, Zhoushan, PR China School of Chemistry and Chemical Engineering, Jinggangshan University, Ji’an, PR China

ARTICLE INFO

ABSTRACT

Keywords: Cephalotaxus hainanensis Li Alkaloids Cytotoxic activity

Two new alkaloids (1–2), and four known ones (3–6) were isolated from branches of Cephalotaxus hainanensis Li. The structures of new alkaloids were elucidated by extensive analysis of HRESIMS, 1D and 2D NMR data. The absolute configuration of compound 2 was established on the basis of experimental and computed ECD data. All compounds were evaluated for their cytotoxic activity against K562, BEL-7402, SGC-7901, A549, and Hela human cancer cell lines. Compound 6 exhibited significant activity with IC50 values ranging from 4.86 to 26.56 μM, respectively.

1. Introduction

2. Results and discussion

Cephalotaxus, considered as the sole genus of the family Cephalotaxaceae, has been reported as a source of ester alkaloids with antitumor activity, such as harringtonine, homoharringtonine, and isoharringtonine etc(Tang et al., 1992). For example, homoharringtonine was used for the treatment of chronic or accelerated phase chronic myeloid leukemia (Abdelkafi et al., 2012). In the last decades, new structurally and biologically interesting secondary metabolites, particularly alkaloids from the genus Cephalotaxus have attracted researchers’ attention (Ni et al., 2016). Cephalotaxus hainanensis Li occurs in China, in the areas of Hainan, Guangxi, and Yunnan (Fu, 1999), is the main raw material for the production of the anticancer drug homoharringtonine. The stems and leaves of C. hainanensis contain a variety of chemical components, including alkaloids, lactones, phenylpropanoids and lignans (Xue et al., 1981; Lin et al., 1985; Liu et al., 2008a,b; Mei et al., 2006). The harringtonine and homoharringtonine, clinically used for the treatment of chronic myeloid leukemia, were also identified in C. hainanensis Li (Abdelkafi et al., 2012). In our effects to attain new alkaloids from the genus Cephalotaxus, two new alkaloids and four known ones (Fig. 1) were isolated from the crude extract of the branches of C. hainanensis guided by HPLC and LC–MS analysis. This paper describes the isolation, structural elucidation and cytotoxic activity screening of alkaloids.

Compound 1 was a white power. Its molecular formula was deduced as C20H23NO4 with 10 degrees of unsaturation based on the HRESIMS ion peaks at m/z 342.1700 [M + H]+ (C20H24NO4, calcd. for 342.1705). The 1H NMR data (Table 1) of 1 revealed the presence of 1,2,4-trisubstituted benzene ring (δH 7.00, H-14; 6.99, H-18; 6.86, H17), 1,2,3,4-tetrasubstituted benzene ring (δH 6.77, H-6; 6.65, H-5), and two singlet methoxys at δH 3.88 (s, 6 H) and 3.82 (s, 3 H). The 13C NMR data (Table 1) revealed the presence of 20 carbons attributed to seven sp2 quaternary carbons, five sp2 methines, two sp3 methines, three sp3 methylenes, and three methoxy groups as edited by the DEPT and HSQC spectra. The above spectroscopic data accounted for eight degrees of unsaturation, and the remaining two degrees of unsaturation required 1 possessing two other rings. The 1H-1H COSY correlations between H2-2 (δH 3.14 and 3.37)/H2-3 (δH 2.78 and 2.91) and H-10 (δH 4.15)/H2-11 (δH 1.97 and 2.33)/H-12 (δH 5.28) suggested two spin-coupled systems of C-2 (δC 44.0)/C-3 (δC 27.8) and C-10 (δC 52.1)/C-11 (δC 37.4)/C-12 (δC 78.0) (Fig. 2). The linkage of these two sequences through C-2–N–C10 was determined by HMBC correlation of H-2 to C-10 and by their chemical shifts at the downfield region. The HMBC correlations from H14 and H-18 to C-12 and C-16 (δC 149.0), and from H-17 to C-13 (δC 134.0) and C-15 (δC 149.2) suggested the connectivity between C-12 and C-13. Two methoxy groups located at C-15 and C-16 were revealed by HMBC correlations from two methoxy groups at 3.88 ppm to the corresponding carbons C-15 and C-16 respectively, and further

Corresponding authors. E-mail addresses: [email protected] (W. Mei), [email protected] (H. Dai). 1 These authors contributed equally to this work. ⁎

https://doi.org/10.1016/j.phytol.2019.08.008 Received 26 April 2019; Received in revised form 3 August 2019; Accepted 28 August 2019 1874-3900/ © 2019 Published by Elsevier Ltd on behalf of Phytochemical Society of Europe.

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Fig. 1. Structures of alkaloids 1–6 isolated from C. hainanensis. Table 1 The 1H (500 MHz) and ppm). Pos.

13

1a

2b

δC

δH, (J in Hz)

1 2

44.0, CH2

3

27.8, CH2

4

126.2, C

3.14, 5.3); 3.37, 2.78, 2.91, 7.0)

5 6 7 8 9 10 11

120.0, CH 111.3, CH 146.0, C 142.6, C 122.7, C 52.1, CH 37.4, CH2

12 13 14 15 16 17 18 3-OCH3 7-OCH3 15-OCH3 16-OCH3 17-OCH3

78.0, CH 134.0, C 109.6, CH 149.2, C 149.0, C 111.2, CH 118.8, CH

a b

56.4, CH3 56.1, CH3 56.1, CH3

group present at C-7 (δC 146.0) was revealed by HMBC correlations from methoxy protons at 3.82 ppm and H-5 to C-7, and by NOE correlation between H-6 and 7−OCH3. Based on its molecular formula, one oxygen atom and one ring were left. The remaining oxygenated aromatic carbon C-8 (δC 142.6) was expected to form an ether linkage with C-12 resulting in a 3,4-dihydro-2H-pyran ring. Thus, the planar structure of 1 was elucidated as shown by extensive analysis of 2D NMR. The relative configuration was established by NOE of H-10 and H-12 in the ROESY spectrum, together with large axial-axial coupling constants (3J10,11a = 11.7 Hz; 3J11a,12 = 12.0 Hz), which assigned the same orientations of H-10 and H-12. Compound 2 was obtained as a yellow oil. Its molecular formula was deduced as C19H25NO4 with 8 degrees of unsaturation based on the HRESIMS ion peak at m/z 354.1653 [M + Na]+. The 13C NMR data (Table 1) revealed the presence of 19 carbons attributed to five nonprotonated sp2 carbons, one quaternary sp3 carbon, three sp2 methines, two sp3 methines six sp3 methylenes, and two methoxy groups as deduced from the DEPT and HSQC spectra. The 1D NMR data were similar to those of 2-hydroxyisotaxodine (Panichanun et al., 1984). Detailed analysis of its 1H-1H COSY correlations and HMBC correlations (Fig. 2) revealed that 2 had the same planar structure to 2-hydroxyisotaxodine. However, the signal of 3-OMe shifted upfield to 2.74 ppm, similar to that of schelhammericine (Tsuda et al., 1996), which indicated the βorientated configuration of the 3-OMe. The relative configuration of 2−OH was deduced to be β-orientated by NOE of H-2 and H-3. The absolute configuration of 2 was determined by comparison of the experimental and simulated electronic circular dichroism (ECD) spectra generated by time-dependent density functional theory (TDDFT) (Zhu et al., 2018). Boltzmann distributions were estimated from the B3LYP and the CAM-B3LYP energies. The experimental (exptl.) ECD curve matched well with the calculated (cald.) one (Fig. 3). Thus, the structure of 2 was established as shown in Fig. 1 and named as 3-epi-2-hydroxyisotaxodine. The four known alkaloids were identified as taxodine (3) (Qiu et al., 1997), isocephalotaxine (4) (Bocar et al., 2003), cephalezomine H (5) (Morita et al., 2002), drupacine (6) (Weisleder et al., 1980) after analysis of their 1D and 2D NMR and ESIMS data (Fig. 1). Homoerythrina-type (2–3) and cephalotaxine-type (4–6) alkaloids identified from C. hainanensis present two main types of alkaloids existing in the genus Cephalotaxus. Both types were proposed to be derived from the common precursor, a derivative of 1-phenylethyl-

C (125 MHz) NMR data of compounds 1 and 2 (δ in

ddd (12.5, 11.2, dd (12.5, 7.0) dd (17.9, 5.3); ddd (17.9, 11.2,

δC

δH, (J in Hz)

121.0, CH 69.7, CH

5.64, s 4.04, s

81.4, CH

3.49, m

30.7, CH2

2.78, overlapped 1.98, dd (14.0, 3.5) 6.65, d (8.2)

6.65, d (8.2) 6.77, d (8.2)

67.0, C 146.3, C 28.9, CH2 46.7, CH2

4.15, dd (11.7, 4.7) 1.97, ddd (12.8, 12.0, 11.7); 2.33, ddd (12.8, 4.7, 2.4) 5.28, dd (12.0, 2.4)

51.1, CH2 24.7, CH2

3.52, m; 3.16, m 1.87, m; 1.62, m

37.4, CH2 134.3, C 134.3, C 119.7, CH 144.2, C 147.1, C 115.2, CH 55.7, CH3

3.18, m; 2.77, m

56.4, CH3

3.77, s

7.00, s 6.86, d (8.1) 6.98, d (8.1) 3.82, s 3.88, s 3.88, s

2.42, m; 2.49, m 2.72, m

6.68, s 6.63, s 2.74, s

Recorded in CDCl3. Recorded in CD3OD.

confirmed by NOE correlations between H-14 and 15−OCH3, H-17 and 16−OCH3. In addition, the connectivity between C-3 and C-4 (δC 125.9) was deduced by HMBC correlations from H2-3 and H-6 to C-4, and from H-5 to C-3. A piperidine ring was identified by the HMBC cross-peaks of H-3 and H-10 to C-9 (δC 122.8). The remaining methoxy 2

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Fig. 2. Key 2D NMR correlations of compounds 1 and 2.

IR instrument (Thermo, America) using KBr pellets. ECD spectra were measured on a JASCO J-715 spectrophotometer (JASCO, Japan). Column chromatography was carried out with silica gel (60–80, 200–300 mesh, Qingdao Haiyang Chemical Co. Ltd., China), ODS gel (20–45 μm, Fuji Silysia Chemical Co. Ltd., Japan), and Sephadex LH-20 (Merck Co.Ltd., Germany). TLC was carried out on silica gel G precoated plates (Qingdao Haiyang Chemical Co. Ltd., China), and peaks were detected under UV light at 254 nm and then by spraying with 5% H2SO4 in EtOH followed by heating and potassium iodide chromogenic developer. HPLC analysis was performed with an Agilent Technologies 1260 Infinity equipped with an Agilent DAD G1315D detector. UPLCMS were API QSTAR Pulsar HR-ESI-MS (Bruke, Germany) and Dionex UltiMate 3000 (Agilent, America). 3.2. Plant materials The plant material was collected in Haikou, Hainan, in China in September 2017, and identified as Cephalotaxus hainanensis Li by Dr. Jun Wang. A voucher specimen (No. HUANG00020) was deposited at the Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Sciences.

Fig. 3. Experimental and calculated curves for 2.

1,2,3,4-tetrahydroisoquinoline (Parry et al., 1980; Abdelkafi and Nay, 2012). The first identification of cephahainanine (1) from C. hainanensis supports the previously proposed biosynthesis pathway. Compounds (1–6) were tested for cytotoxicity against human cancer cell lines (K562, SGC-7901, BEL-7402, A549 and Hela). Compound 6 exhibited significant activity against all five cell lines with IC50 values of 4.86, 9.70, 26.56, 22.99, and 11.00 μM respectively (Table 2).

3.3. Extraction and isolation The air-dried and pulverized branches of C. hainanensis (100 kg) was extracted with 80% ethanol (3 × 350 L, one week each) at room temperature, respectively, and the solvent was evaporated in vacuo. The extract (330 g) was dissolved in 1% HCl solution (v/v) to pH 2–3, basified with 10% ammonia solution (v/v) to pH 7–8, and partitioned with EtOAc. The obtained EtOAc crude extract (28.2 g) was fractionated into 18 fractions (Fr.1-Fr.18) on silica gel and eluted with a gradient elution of EtOAc-petroleum ether (10–100%), finally, eluted with acetone. Fr.8 (1.3 g) was fractionated by RP-18 column chromatography with a gradient of H2O-MeOH to give 20 fractions (Fr.8.1Fr.8.20). Fr.8.7 was a single compound 1 (8.9 mg). Fr.8.5 (178.1 mg) was purified on a vacuum silica gel column with petroleum-acetone (8:1 to 1:1, v/v) to afford 5 (7.0 mg) and 6 (14.5 mg). Fr.8.17 (67.7 mg) was purified by a Sephadex LH-20 column and eluted with MeOH to

3. Experimental 3.1. General experimental procedures The NMR spectra were measured with a Bruker AV III spectrometer (Bruker, Germany), operated at 500 MHz for 1H NMR and 125 MHz for 13 C NMR. Chemical shifts were referenced to the solvent residual peaks. The UV spectra were recorded on a Shimadzu UV-2550 spectrometer (Shimadzu, Japan). IR absorptions were obtained on a Nicolet 380 FTTable 2 The results of cytotoxicity assay of alkaloids 1to 6. Compound

1–5 6 doxorubicin hydrochloridea a

IC50 (μM) K562

BEL-7402

SGC-7901

A549

Hela

> 50 4.86 ± 0.62 7.93 ± 0.45

> 50 9.70 ± 0.75 6.47 ± 0.33

> 50 26.56 ± 1.38 3.62 ± 0.12

> 50 22.99 ± 1.45 14.77 ± 0.85

> 50 11.00 ± 1.11 6.29 ± 0.40

Positive control. 3

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yield compound 4 (3.2 mg). Fr.8.13 (33.6 mg) was separated by silica gel and eluted with CHCl3-MeOH (30:1, v/v) to yield compound 3 (2.0 mg) and 2 (4.3 mg).

interest Scientific Institution Basal Research Fund for Innovative Research Team Program of CATAS (17 CXTD-15), and China Agriculture Research System (CARS-21).

3.3.1. Cephahainanine (1) White power; [α]D25 +61.9 (c 1.0, CHCl3) ; UV (CHCl3) λmax (log ε): 279 (2.75) nm; IR (KBr) : 3449, 3383, 2928, 1632, 1508,1448, 1264, 1101, 1029 cm−1; 1H and 13C NMR data see Table 1 (CDCl3), see Table 1; HRESIMS m/z 342.1695 [M + H]+ (calcd for C20H24NO4: 342.1700).

Appendix A. Supplementary data

3.3.2. 3-Epi-2-hydroxyisotaxodine (2) Yellow oil; [α]D25 +241.5 (c 2.0, CH3OH) ; UV (CH3OH) λmax (log ε): 283 (3.25) nm; IR (KBr) : 3507, 3423, 3334, 2923, 2851, 1610, 1510, 1421, 1150, 1110 cm−1; ECD (c 0.04 mg mL−1, MeOH) λmax (mdeg): 205 (+28.94), 224 (−1.51), 241 (+1.67); 1H and 13C NMR data see Table 1 (CD3OD), see Table 2; HRESIMS m/z 354.1653 [M + Na]+ (calcd for C19H25NO4Na: 354.1676).

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Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.phytol.2019.08.008. References

3.4. Cytotoxicity assay Cytotoxicity against K562, BEL-7402, SGC-7901, A549 and Hela tumor cell lines were tested by MTT method as described previously (Mosmann, 1983; Mi et al., 2019). K562 human myeloid leukemia cell line, BEL-7402 Human hepatocellular carcinoma cell line, SGC-7901 human gastric cell line, A549 human lung adenocarcinoma cell line, and Hela human cervical cancer cell line were obtained from the Cell Bank of Type Culture Collection of the Chinese Academy of Sciences, Shanghai Institute of Cell Biology. Doxorubicin hydrochloride was performed as a positive control with IC50 values between 3.6214.77 μM. 4. Conclusion In the study, two new alkaloids, along with four known ones were isolated from C. hainanensis. The MTT assay was used to evaluate the cytotoxicity of compounds 1–6 against K562, SGC-7901, BEL-7402, A549 and Hela human cancer cell lines. Drupacine (6) exhibited significant cytotoxic activity against five human cancer cell lines with IC50 values ranging from 4.86 to 26.56 μM. Acknowledgments This work was supported by the Hainan Natural Science Foundation Innovation Research Team Project (2017CXTD020), Central Public-

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