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Molecules 2014, 19(3), 3055-3067; doi:10.3390/molecules19033055

Article
Five New Alkaloids from the Stem Bark of Daphniphyllum macropodum
Yunyang Lu 1, Kai Gao 1, Xiaoyang Wang 1, Wei Zhang 1, Ning Ma 1 and Haifeng Tang 1,2,*
1
Department of Pharmacy, Xijing Hospital, Fourth Military Medical University, Xi’an 710032, China; E-Mails: luyunyanggq@163.com (Y.L.); gaokai19881220@163.com (K.G.); steveplum@sina.com (X.W.); zw2214146@163.com (W.Z.); maningtomoto@163.com (N.M.)
2
Institute of Materia Medica, School of Pharmacy, Fourth Military Medical University, Xi’an 710032, China
*
Author to whom correspondence should be addressed; E-Mail: tanghaifeng71@163.com; Tel./Fax: +86-29-8477-4748.
Received: 11 February 2014; in revised form: 25 February 2014 / Accepted: 26 February 2014 /
Published: 10 March 2014

Abstract

: Five new alkaloids, daphnicyclidins M and N (compounds 1 and 2) and calyciphyllines Q–S (compounds 35), along with four known ones, paxiphylline C (6), macropodumine B (7), macropodumine C (8) and daphnicyclidin A (9) were isolated from the stem bark of Daphniphyllum macropodum. Calyciphylline Q (3) is the first calyciphylline A derivative possessing a double bond between C-18 and C-19. Their structures and relative configurations were elucidated on the basis of spectroscopic methods, especially 2D NMR techniques. Compounds 1, 2, 8 and 9 exhibited cytotoxic activity against P-388 cells with IC50 values of 5.7, 6.5, 10.3 and 13.8 µM, respectively. Compounds 1 and 2 also showed cytotoxic activity against SGC-7901 cells with IC50 values of 22.4 and 25.6 µM.
Keywords:
Daphniphyllum macropodum; daphnicyclidins M and N; calyciphyllines Q–S; alkaloids; cytotoxicity

1. Introduction

Plants of genus Daphniphyllum are mainly distributed in southeast of Asia and are well known for producing highly polycyclic and structurally diverse alkaloids, which have drawn a great deal of attention from the biogenetic and synthetic points of view [1,2,3,4]. In recent years, a great number of Daphniphyllum alkaloids have been isolated and identified, and some of them exhibited significant cytotoxic activity against several human cancer cell lines [5,6,7,8,9,10,11,12,13,14,15,16,17,18,19].

In the past years, a series of new bioactive compounds have been studied in our laboratory [20,21,22,23,24]. With the purpose of searching for bioactive and structurally unique Daphniphyllum alkaloids, an investigation of the extracts from the stem bark of Daphniphyllum macropudum was conducted, and this resulted in the isolation of five new alkaloids named daphnicyclidins M and N (compounds 1 and 2) and calyciphyllines Q–S (compounds 35), and four known related alkaloids 69 (Figure 1). More than 20 alkaloids have been isolated from the stem bark of D. macropudum and identified, including various structure types such as yuzurimine-type, daphnicyclidin-type, daphnezomine-type, calyciphylline-type, daphmanidin-type and daphniglaucin-type [25,26,27,28]. Compounds 1 and 2 are daphnicyclidin-type alkaloids, and 35 are calyciphylline-type alkaloids. The analog which shares a similar gross structure with daphnicyclidins M and N has been isolated for only once by Kobayashi [29]. Calyciphylline Q (3) is the first calyciphylline A derivative possessing a double bond between C-18 and C-19. This paper presents the isolation and structural elucidation of the new compounds 15, along with their cytotoxic activities against four tumor cell lines, P-388 (mouse lymphocytic leukemia), A-549 (human lung carcinoma), SGC-7901 (human gastric carcinoma) and HL-60 (human promyelocytic leukemia).

Molecules 19 03055 g001 1024
Figure 1. Structures of compounds 19.

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Figure 1. Structures of compounds 19.
Molecules 19 03055 g001 1024

2. Results and Discussion

Daphnicyclidin M (1) was obtained as light yellow powder. The molecular formula was determined as C23H25NO5 by HREIMS at m/z 418.1632 ([M+Na]+, calcd for C23H25NO5Na, 418.1630), which indicated 12 degrees of unsaturation. 13C-NMR (Table 1) and DEPT spectra revealed 23 carbon signals due to three tetrasubstituted olefins, one disubstituted olefin, two carbonyls, two sp3 quaternary carbons, three sp3 methines, five sp3 methylenes, two sp3 methyls and one methoxy group. Among them, two methylenes (δC = 60.1, δH = 2.37 and 3.07; δC = 53.1, δH = 2.63 and 3.06) and one methine (δC = 67.9, δH = 3.50) were ascribed to those bearing a nitrogen, while two olefin carbons (δC = 168.4 and δC = 146.1, δH = 7.93) and one sp3 quaternary carbon (δC = 77.8) were assigned to those bearing oxygen atoms. Since six out of 12 degrees of unsaturation were accounted for, 1 was inferred to possess six rings.

Table Table 1. 1H-NMR (500 MHz) and 13C-NMR (125 MHz) data for compounds 15 (δ in ppm, J in Hz).

Click here to display table

Table 1. 1H-NMR (500 MHz) and 13C-NMR (125 MHz) data for compounds 15 (δ in ppm, J in Hz).
C1 a2 a3 a4 a5 b
δCδHδCδHδCδHδCδHδCδH
177.8-78.9-209.2-217.6-216.4-
2213.1-212.4-44.42.93 (t, 3.1)43.32.38 (brd, 5.0)41.52.28–2.31 (m)
3a
3b
31.42.26 (d, 3.4)
2.26 (d, 3.4)
33.92.18 (d, 14.8)
2.35 (d, 14.8)
24.41.97 (dt, 14.1, 2.7)
2.26 (dt, 14.2, 3.1)
34.61.86–1.92 (m)
2.76–2.78 (m)
18.62.38–2.40 (m)
2.64–2.66 (m)
467.93.50 (t, 3.3)98.0-64.63.07–3.09 (m)90.44.00 (t, 2.2)87.93.93 (br s)
546.8-53.3-55.9-54.7-52.5-
646.72.56–2.58 (m)42.32.85–2.88 (m)58.01.78–1.85 (m)47.23.01–3.07 c45.22.89 (t, 7.6)
7a
7b
60.12.37 (t, 9.6)
3.06–3.08 (m)
59.42.31 (dd, 8.2, 2.8)
3.13–3.16 (m)
61.92.92 (d, 9.4)
3.28–3.31 (m)
69.63.12 (t, 6.3)
3.51 (t, 13.2)
67.43.04–3.09 (m)
3.26–3.32 (m)
8126.7-128.5-57.3-72.3-69.7-
9108.8-108.2-150.7-139.7-136.9-
10168.4-168.1-151.7-206.1-202.8-
11a
11b
31.02.98–3.01 (m)
3.67–3.73 (m)
31.12.94–2.98 (m)
3.65–3.69 (m)
34.52.06–2.14 (m)
2.36 (d, 18.5)
37.22.32–2.35 (m)
2.32–2.35 (m)
36.02.12–2.15 (m)
2.12–2.15 (m)
12a
12b
28.21.69–1.73 (m)
2.57–2.59 (m)
27.61.67–1.75 (m)
2.54–2.58 (m)
32.11.32–1.37 (m)
1.78–1.85 (m)
19.41.86–1.92 (m)
2.01–2.08 (m)
17.81.71–1.74 (m)
1.91–1.98 (m)
13a
13b
142.0-
-
141.5-
-
47.62.81 (d, 16.5)
3.40 (d, 17.7)
20.22.44–2.50 (m)
2.78–2.82 (m)
32.91.68–1.71 (m)
2.62–2.65 (m)
14a
14b
123.2-
-
123.3-
-
115.9-
-
33.22.64 (t, 8.7)
2.74–2.77 (m)
35.92.32–2.37 (m)
2.50–2.52 (m)
15134.4-134.7-173.5-154.6-156.4-
16a
16b
111.87.70 (d, 5.3)
-
117.77.69 (d, 5.3)
-
26.82.74–2.78 (m)
2.74–2.74 (m)
132.56.89 (dd, 10.8, 6.7)
-
33.32.18–2.23 (m)
2.66–2.70 (m)
17a
17b
146.17.93 (d, 5.3)
-
145.97.92 (d, 5.3)
-
41.72.78–2.80 (m)
2.85–2.87 (m)
122.25.42 (d, 10.8)
5.52 (d, 17.5)
59.13.43–3.45 (m)
3.50–3.53 (m)
1836.92.66–2.70 (m)39.12.49–2.53(m)112.8-33.02.57–2.62(m)31.02.43–2.47 (m)
19a
19b
53.12.61–2.65 (m)
3.02–3.06 (m)
53.82.91 (dd, 15.6, 2.8)
3.17 (dd, 15.2, 2.3)
135.85.77(s)
-
68.23.01–3.07 c
3.63 (dd, 13.3, 7.1)
66.12.99–3.04 (m)
3.55–3.57 (m)
2014.00.81 (3H, d, 6.7)13.30.83 (3H, d, 6.7)19.91.70 (3H, s)19.61.14 (3H, d, 6.7)18.91.03 (3H, d, 6.7)
2128.81.48 (3H, s)25.61.46 (3H, s)27.21.20 (3H, s)23.21.50 (3H, s)22.11.39 (3H, s)
22169.0-169.0-168.1-----
2351.93.83 (3H, s)51.83.82 (3H, s)51.83.70 (3H, s)----
4-OMe--50.03.35 (3H, s)------

a Measured in CD3OD; b Measured in DMSO-d6; c Overlapped.

Four partial structures: a (C-18 to C-19 and C-20), b (C-3 to C-4), c (C-6 to C-7 and C-12, and C-11 to C-12) and d (C-16 to C-17) were deduced from the extensive analysis of the 2D NMR data of 1, including HSQC, 1H-1H COSY and HMBC, as shown in Figure 2. The HMBC correlations from H2-3 to C-1, C-2 and C-13, and H-4 to C-1 indicated C-2, C-3 and C-13 were all connected to C-1; H-19b and H3-20 to C-2 suggested the connectivity between C-18 and C-2. HMBC correlations from H-7b to C-4, H-7a to C-19 and H-19a to C-4 and C-7 gave rise to the connectivity of partial structures a, b and c through a nitrogen atom. The connections between C-4, C-6 and C-8 to C-21 through C-5 were confirmed by the HMBC correlations from H2-3 and H-4 to C-5, H-4 and H-6 to C-8 and H3-21 to C-4, C-5, C-6 and C-8, and this constructed the ring C. HMBC correlations from H2-12 and H2-11 to C-10 and H-11b to C-9 implied that C-11 and C-9 were connected through C-10. The presence of ring F was elucidated by the chemical shifts of C-10 (δC = 168.4) and C-17 (δC = 146.1), and HMBC correlations of H-17 to C-10 and C-15, and H-16 to C-9. The ring E and the methoxy carbonyl group at C-14 were deduced from a comprehensive analysis of the chemical shifts [126.7 (C-8), 108.8 (C-9), 168.4 (C-10), 142.0 (C-13), 123.2 (C-14), 134.4 (C-15)] and HMBC correlation of H-16 to C-14, and H3-23 to C-22. The relative configuration of 1 was elucidated by NOESY correlations as depicted in a computer-generated three-dimensional drawing, as shown in Figure 3.

Molecules 19 03055 g002 1024
Figure 2. Selected 2D NMR correlations of daphnicyclidin M (1).

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Figure 2. Selected 2D NMR correlations of daphnicyclidin M (1).
Molecules 19 03055 g002 1024
Molecules 19 03055 g003 1024
Figure 3. Key NOESY correlations of daphnicyclidin M (1).

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Figure 3. Key NOESY correlations of daphnicyclidin M (1).
Molecules 19 03055 g003 1024

The NOESY correlations of H3-21 to H-4 and H-4 to H-6 indicated that H-4 and H-6 were in the β-orientation. The α-orientation of H-18 was deduced from the NOESY correlations of H-18 to H-7a and H-7b to H-4. Thus, it was clear that the ring B took a boat conformation. In consideration of biosynthetic pathway [20] and the boat conformation of ring B, the OH group at C-1 must be β-oriented. The NOESY correlations of H3-21 to H-11b implied that ring D took a twist-boat conformation, similar to that of daphnicyclidin A [30]. Thus, the structure of daphnicyclidin M was assigned as 1, which is the C-4 dehydroxylated, C-16 and C-17 dehydrogenated derivative of daphnicyclidin K [29].

Daphnicyclidin N (2) showed a molecular formula of C24H27NO6, as determined by HREIMS at m/z 448.1738 ([M+Na]+, calcd. for C24H27NO6Na, 448.1736). The comparison of the 1H-NMR and 13C-NMR (Table 1) data of 2 with those of 1 suggested that the two alkaloids shared the same gross structure. The main difference bteween the two alkaloids was the fact that the molecular weight of 2 was larger than that of 1 by 30 units. Thus, it was proposed that the H-4 was replaced by a methoxy group. This was proved by the chemical shift of C-4 (δC = 98.0) which was shifted downfield ∆δC = +30.1 as compared with that of 1, and the HMBC cross-peak of the H3 signal (δH = 3.35, s) to C-4 (Supporting Information). The relative configuration of 2 was the same as that of 1, thus, OH-1, H-6, CH3-20 and CH3-21 were also β-oriented. Because the chemical shift of C-21 (δC = 25.6) was shifted upfield (∆δC = −2.65) for the γ-steric compression effect from oxygen atom of C-4, the methoxy group at C-4 was also deduced as the β-orientation [31].

Calyciphylline Q (3) was obtained as a light yellow oil, exhibiting a pseudomolecular ion peak at m/z 388 [M+Na]+ in the ESIMS. The molecular formula C23H27NO3 of 3 was established by HRESIMS at m/z 388.1890 ([M+Na]+, calcd. for C23H27NO3Na, 388.1889), corresponding to 11 degrees of unsaturation. The 13C-NMR (Table 1) and DEPT spectra showed 23 carbon signals including two carbonyls, three double bonds, two sp3 quaternary carbons, three sp3 methines, seven sp3 methylenes, two sp3 methyls and one methoxy group. Among them one methylene (δC = 61.9, δH = 2.92 and 3.30), one methine (δC = 64.6, δH = 3.09) and one double bond carbon (δC = 135.8, δH = 5.77) were assigned to those bearing a nitrogen.

The 1H-1H COSY spectrum of 3 revealed the connectivities of three structure fragments a (C-2 to C-4), b (C-6 to C7 and C-12, and C-11 to C-12) and c (C-16 to C-17) as shown in Figure 4. The connection of C-4, C-7 and C-19 to each other through a nitrogen atom was deduced from the HMBC correlations from H-4 to C-19, H-7a to C-4, and H-7b to C-19 and H-19 to C-4. The ring B was elucidated by the HMBC correlations of H-2 to C-18, H-19 to C-2, C-18 and C-20, H3-20 to C-2, C-18 and C-19. A ketone carbonyl at C-1 was revealed from HMBC cross-peaks of H-2 and H-3a to C-1. The HMBC correlations from H3-21 to C-4, C-5 and C-6, from H-3a, H-7a and H-12a to C-5 indicated the connectivities of C-21 to C-4 and C-6 via C-5. The connectivity of fragment b and c through C-10 and the presence of ring F were suggested by the HMBC correlations from H-11a, H2-16 and H-17a to C-10, from H2-11 and H-17b to C-9, and from H2-16 and H-17a to C-15. The linkages of C-13 to C-1, C-5 and C-9 through C-8 were confirmed by the HMBC correlations of H2-13 to C-5, H3-21 to C-5 and C-8, and H-13a to C-1 and C-9. The ring E was elucidated on the basis of the HMBC correlations of H-13a to C-15, H2-16 to C-15 and C-14 and H-17a to C-15. The methoxycarbonyl group at C-14 was deduced from a comprehensive analysis of the chemical shifts [150.7 (C-9), 151.7 (C-10), 115.9 (C-14), 173.5 (C-15)] and HMBC correlation of H-13b to C-22, H2-16 to C-14, and H3-23 to C-22. The relative configuration of 3 was elucidated by NOESY spectrum as shown in Figure 5. The NOESY correlations of H3-21 to H-3b, H-4 and H-6 indicated that H-3b, H-4, H-6 and CH3-21 were all on the same side, and assumed to be in β-orientation just the same as those of daphniyunnine A [32]. The β-orientation of H-2 was implied by the NOESY correlation of H-2 with H-13a. The correlation of H-13b to H3-21 suggested that C-13 was β-oriented. The boat conformation of ring D was deduced from the NOESY correlation of H3-21 to H-12a. Thus, the structure of calyciphylline Q was elucidated as 3.

Molecules 19 03055 g004 1024
Figure 4. Selected 2D NMR correlations of calyciphylline Q (3).

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Figure 4. Selected 2D NMR correlations of calyciphylline Q (3).
Molecules 19 03055 g004 1024
Molecules 19 03055 g005 1024
Figure 5. Key NOESY correlations of calyciphylline Q (3).

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Figure 5. Key NOESY correlations of calyciphylline Q (3).
Molecules 19 03055 g005 1024

Calyciphylline R (4) exhibited a pseudomolecular ion peak at m/z 342 [M+H]+ in the ESIMS, and the molecular formula was established as C21H27NO3 by HRESIMS at m/z 342.2068 ([M+H]+, calcd. for C21H28NO3, 342.2069), corresponding to nine degrees of unsaturation. The 13C-NMR (Table 1) of 4 revealed 21 carbon resonances, which were classified into two carbonyls, two double bonds, two sp3 quaternary carbons, four sp3 methines, seven sp3 methylenes, and two methyl groups. One methine (δC = 90.4, δH = 4.00) and two methylenes (δC = 68.2, δH = 3.04 and 3.63; δC = 69.6, δH = 3.12 and 3.51) were ascribed to those bearing an oxidative nitrogen. Comparison of the NMR (1H-NMR, 13C-NMR, HSQC, 1H-1H COSY and HMBC) spectra of 4 with those of daphlongamine E [33], suggested that the two compounds are closely related. However, significant downfield changes of the chemical shifts of C-4 (δC = 90.4), C-7 (δC = 69.6) and C-19 (δC = 68.2) in relation to those of daphlongamine E (C-4 (δC = 65.5), C-7 (δC = 53.6) and C-19 (δC = 49.8)) indicated that the former one is the N-oxide form of the latter one [34]. Thus, the relative configuration of 4 is the same as daphlongamine E.

Calyciphylline S (5) showed a pseudomolecular ion peak at m/z 360 [M+H]+ in the ESIMS, and the molecular formula was determined as C21H29NO4 by HRESIMS at m/z 360.2172 ([M+H]+, calcd. for C21H30NO4, 360.2175), with 8 degrees of unsaturation. The 13C-NMR (Table 1) and DEPT spectra of 5 revealed 21 carbon signals, ascribed to two carbonyls, one tetrasubstituted olefin, two sp3 quaternary carbons, four sp3 methines, nine sp3 methylenes and two sp3 methyl groups. Among them, one methine (δC = 87.9, δH =3.93) and two methylenes (δC = 66.1, δH = 3.04 and 3.56; δC = 67.4, δH = 3.10 and 3.30) were ascribed to those bearing an oxidative nitrogen. Since three out of eight degrees of unsaturation have been accounted for, 5 was inferred to possess five rings. A comparison of the 13C chemical shifts of C-4 (δC = 87.9), C-7 (δC = 67.4) and C-19 (δC = 66.1) in 5 with those of daphniyunnine B (longeracinphyllin B) indicated the presence of an N-oxide group attached to those C-atoms [32,35]. Thus calyciphylline S was inferred to be the N-oxide form of daphniyunnine B (longeracinphyllin B), which was confirmed by the 2D NMR (HSQC, 1H-1H COSY and HMBC) spectra of 5 (Supporting Information). The structure of calyciphylline S (5) could also be deduced from the comparison of the 1H-NMR and 13C-NMR data (Table 1) of 5 with those of 4. It could be easily inferred that compound 5 was the water addition product of 4 at C-16 and C-17, since one methine (δC = 132.5, δH = 6.89) and one methylene (δC = 122.2, δH = 5.42 and 5.52) disappeared in compound 4 while two methylenes (δC = 33.3, δH = 2.22 and 2.68; δC = 59.1, δH = 3.44 and 3.51) emerged in 5.

Four known alkaloids were identified as paxiphylline C [36], macropodumine B [37], macropodumine C [37] and daphnicyclidin A [30] on the basis of the comparison of their 1H-NMR, 13C-NMR and ESIMS data with that reported.

The cytotoxicity of the new compounds were evaluated against mouse lymphocytic leukemia P-388 cells, human lung carcinoma A-549 cells, human gastric carcinoma SGC-7901 cells and human promyelocytic leukemia HL-60 cells using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay method in vitro [38]. As shown in Table 2, compounds 1, 2, 8 and 9 exhibited cytotoxic activity against P-388 cells with IC50 values of 5.7, 6.5, 10.3 and 13.8 µM, respectively. Compounds 1 and 2 also showed a moderate cytotoxic activity against SGC-7901 cells with IC50 values of 22.4 and 25.6 µM. Compounds 3, 4, 5, 6 and 7 were inactive (IC50 > 50 µM) against to the cell lines above.

Table Table 2. Cytotoxic activity of compounds 19 against four cancer cell lines in vitro.

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Table 2. Cytotoxic activity of compounds 19 against four cancer cell lines in vitro.
Compounds aCytotoxic activity (IC50, µM)
P-388A-549SGC-7901HL-60
15.7>5022.4>50
26.5>5025.6>50
810.3>50>50>50
913.8>50>50>50
Cisplatin b0.30.93.21.1

P-388 = mouse lymphocytic leukemia cell line; A-549 = human lung carcinoma cell line; SGC-7901 = human gastric carcinoma cell line; HL-60 = human promyelocytic leukemia cell line; a Compounds 3, 4, 5, 6 and 7 were inactive (IC50 > 50 µM) against all cell lines; b Cisplatin was used as positive control.

3. Experimental

3.1. General Information

Optical rotations were measured with a Perkin-Elmer 343 polarimeter. 1D and 2D NMR spectra were recorded on a Bruker AVANCE-500 spectrometer with TMS as internal standard. ESIMS and HRESIMS were carried out on a Micromass Quattro mass spectrometer. HPLC was carried out on a Dionex P680 liquid chromatograph equipped with a UV 170 UV/Vis detector using a YMC-Pack R&D ODS A column (20 × 250 mm i.d., 5 μm, YMC Co., Ltd., Kyoto, Japan) and monitored at 225, 250, 275, 300 nm, simultaneously. Column chromatographies were performed on silica gel (200–300 mesh and 300–400 mesh; Qingdao Marine Chemical Inc., Qingdao, P. R. China), reversed phase silica gel (Lichroprep RP-18, 40–63 µm, Merck Inc., New York, NY, USA), and Sephadex LH-20 (40–70 µm, GE-Healthcare, Uppsala, Sweden). Chemical reagents for isolation were of analytical grade and purchased from Tianjin Fuyu Chemical Co. Ltd. (Tianjin, China).

3.2. Plant Material

The stem bark of Daphniphyllum macropodum was collected in Chongqing Province, People’s Republic of China, in November 2012, and identified by associate researcher Maoxiang Lin of the Chongqing Institute of Medicinal Plant Cultivation. A voucher specimen (XJ-T20121215) was deposited in the Herbarium of the Department of Pharmacy, Xijing Hospital, Fourth Military Medical University.

3.3. Extraction and Isolation

The air-dried and powdered stem bark (20.0 kg) of Daphniphyllum macropodum was extracted three times with refluxing 95% EtOH (200 L, 2 h each time). After removal of the solvent under reduced pressure, the extract (2.1 kg) was dispersed in water and adjusted with 1% HCl to pH 2–3, then filtered. The aqueous phase was adjusted to pH 10 with 2 mol∙L−1 NaOH followed by extraction with CHCl3 to get the crude alkaloid (45.0 g). The crude alkaloid was subjected to a silica gel column eluting with a CHCl3/CH3OH (1:0 to 0:1) gradient to obtain four major fractions (A to D). Fraction A (15.5 g) was chromatographed on a silica gel column eluting with a CHCl3/CH3OH (20:1 to 10:1) gradient to give ten further fractions (A1–A10). Fraction A5 was subjected to size exclusion chromatography on a Sephadex LH-20 column equilibrated with CH3OH to remove the pigments and impurities, then was further purified by HPLC to afford compounds 1 (7.7 mg, tR = 20.2 min), 2 (6.2 mg, tR = 21.2 min) and 4 (5.5 mg, tR = 33.6 min) eluting with MeOH/H2O (8:3) at a flow rate of 6 mL/min. Paxiphylline C (5.7 mg, tR = 18.1 min) and compound 3 (4.4 mg, tR = 24.5 min) were obtained from fraction A7 by HPLC eluting with MeOH/H2O (7:3) at a flow rate of 8 mL/min. Fraction A10 was purified by HPLC to give compound 5 (7.2 mg, tR = 22.5 min) eluting with MeOH/H2O (6:4) at a flow rate of 6 mL/min. Fraction B (15.0 g) was subjected to silica gel column chromatography eluting with a CHCl3/CH3OH (10:1 to 0:1) gradient to afford two fractions (B1 and B2). Fraction B1 was purified over a Sephadex LH-20 column equilibrated with CH3OH to yield macropodumine C (15.8 mg). Fraction C (3.4 g) was chromatographed over a Sephadex LH-20 column equilibrated with CH3OH to remove the pigments and impurities, and finally purified by means of HPLC eluting with MeOH/H2O (4:6) to yield daphnicyclidin A (4.8 mg) in 27.5 min and macropodumine B (17.6 mg) in 37.6 min.

Daphnicyclidin M (1): amorphous light yellow powder; Molecules 19 03055 i001 −40.3 (c 0.11, MeOH); UV (MeOH) λmax (log ε) 274 (1.36), 221 (1.26) nm; IR (KBr) νmax 3423, 2915, 1690, 1643, 1616, 1522, 1454, 1375, 1127, 984 cm−1; 1H-NMR and 13C-NMR, see Table 1; positive ESIMS m/z 418 [M+Na]+; positive HRESIMS [M+Na]+ m/z 418.1632 (calcd for C23H25NO5Na, 418.1630).

Daphnicyclidin N (2): amorphous light yellow powder; Molecules 19 03055 i001 −65.5 (c 0.11, MeOH); UV (MeOH) λmax (log ε) 281 (1.14), 204 (5.00) nm; IR (KBr) νmax 3425, 2920, 1686, 1643, 1616, 1525, 1456, 1385, 1126, 991 cm−1; 1H-NMR and 13C-NMR, see Table 1; positive ESIMS m/z 448 [M+Na]+; positive HRESIMS [M+Na]+ m/z 448.1738 (calcd for C24H27NO6Na, 448.1736).

Calyciphylline Q (3): light yellow oil; Molecules 19 03055 i001 −16.8 (c 0.08, MeOH); UV (MeOH) λmax (log ε) 280 (4.96) nm; IR (KBr) νmax 2925, 1709, 1685, 1615, 1440, 1378, 1096 cm−1; 1H-NMR and 13C-NMR, see Table 1; positive ESIMS m/z 388 [M+Na]+; positive HRESIMS [M+Na]+ m/z 388.1890 (calcd for C23H27NO3Na, 388.1889).

Calyciphylline R (4): light yellow oil; Molecules 19 03055 i001 −44.7 (c 0.15, MeOH); UV (MeOH) λmax (log ε) 270.5 (4.23), 204.5 (4.88) nm; IR (KBr) νmax 2920, 1745, 1695, 1575, 1445, 1380 cm−1; 1H-NMR and 13C-NMR, see Table 1; positive ESIMS m/z 342 [M+H]+; positive HRESIMS [M+H]+ m/z 342.2068 (calcd for C21H28NO3, 342.2069).

Calyciphylline S (5): light yellow oil; Molecules 19 03055 i001 −81.2 (c 0.13, DMSO); UV (MeOH) λmax (log ε) 247.5 (4.54) nm; IR (KBr) νmax 3425, 2920, 1701, 1675, 1611, 1438, 1380, 1226, 995, 565 cm−1; 1H-NMR and 13C-NMR, see Table 1; positive ESIMS m/z 360 [M+H]+; positive HRESIMS m/z [M+H]+ 360.2172 (calcd for C21H30NO4, 360.2175).

3.4. Assays for In Vitro Antitumor Activity

The cytotoxicity of compounds 19 against mouse lymphocytic leukemia P-388 cells, human lung carcinoma A-549 cells, human promyelocytic leukemia HL-60 cells and human gastric carcinoma SGC-7901 cells was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay method in vitro. All cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum, 100 U/mL benzyl penicillin, and 100 U/mL streptomycin at 37 C in a humidified atmosphere with 5% CO2. The logarithmic phase cells were seeded on 96-well plates at the concentration of 4 × 103 cell/mL and incubated with various concentrations (100, 80, 60, 40, 20, 10, 1 and 0.25 μM in medium containing less than 0.1% DMSO) of test compounds in triples wells for 48 h, and cisplatin was used as positive control. After that, 20 μL MTT (5 mg/mL) was added to each well, and incubated for another 4 h. The water-insoluble dark blue formazan crystals formed during MTT cleavage in actively metabolizing cells were dissolved in DMSO. The optical density of each well was measured with a Bio-Rad 680 microplate reader at 570 nm. Cytotoxicity was expressed as the concentration of drug inhibiting cell growth by 50% (IC50).

4. Conclusions

Phytochemical investigation of the stem bark of Daphniphyllum macropodum, lead to the isolation of five new Daphniphyllum alkaloids 15, along with four known ones 69. Their structures and relative configurations were elucidated on the basis of spectroscopic methods, especially 2D NMR techniques. All of the compounds were tested for cytotoxic activity against P-388, A-549, HL-60 and SGC-7901 cell lines. P-388 cells were sensitive to compounds 1, 2, 8 and 9, which exhibited selective cytotoxic activity with IC50 values of 5.7, 6.5, 10.3 and 13.8 µM, respectively. Interestingly, compounds 1 and 2 also showed a moderate cytotoxic activity against SGC-7901 cells with IC50 values of 22.4 and 25.6 µM. These preliminary results suggested that the cytotoxicity of these compounds appeared to be structure dependent, indicating that Daphniphyllum alkaloids of the 1 and 2 structural type possessed the potential for further investigation.

Supplementary Materials

Supplementary materials can be accessed at: http://www.mdpi.com/1420-3049/19/3/3055/s1.

Acknowledgments

The authors thank Mingchang Wang, Nuclear Magnetic Resonance Center, Xi’an Modern Chemistry Research Institute, for the NMR measurements, and HuiMin Wang, Mass Measurement Center, Shanghai Institute of Pharmaceutical Industry, for the MS measurements.

Author Contributions

The listed authors contributed to this work as described in the following. Yunyang Lu, Kai Gao and Wei Zhang carried out the extraction and isolation. Yunyang Lu also collected the stem bark of Daphniphyllum macropodum, participated in the structural elucidation. Xiaoyang Wang participated in the structural elucidation. Ning Ma conducted the MTT colorimetric assay and helped interpreting the results. As corresponding author Haifeng Tang organized the study and participated in the structural elucidation. All authors helped preparing the manuscript and approved the final version.

Conflicts of Interest

The authors declare no conflict of interest.

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  • Sample Availability: Samples of the compounds 19 are available from the authors.
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