Five New Alkaloids from the Stem Bark of Daphniphyllum macropodum

Five new alkaloids, daphnicyclidins M and N (compounds 1 and 2) and calyciphyllines Q–S (compounds 3–5), 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.

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 3-5), and four known related alkaloids 6-9 ( 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 3-5 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 1-5, 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).

Results and Discussion
Daphnicyclidin M (1) was obtained as light yellow powder. The molecular formula was determined as C 23 (Table 1) and DEPT spectra revealed 23 carbon signals due to three tetrasubstituted olefins, one disubstituted olefin, two carbonyls, two sp 3 quaternary carbons, three sp 3 methines, five sp 3 methylenes, two sp 3 methyls and one methoxy group. Among them, two methylenes (δ C = 60.1, δ H = 2.37 and 3.07; δ C = 53.1, δ H = 2.63 and 3.06) and one methine (δ C = 67.9, δ H = 3.50) were ascribed to those bearing a nitrogen, while two olefin carbons (δ C = 168.4 and δ C = 146.1, δ H = 7.93) and one sp 3 quaternary carbon (δ C = 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.  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, 1 H-1 H COSY and HMBC, as shown in Figure 2. The HMBC correlations from H 2 -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 H 3 -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 H 2 -3 and H-4 to C-5, H-4 and H-6 to C-8 and H 3 -21 to C-4, C-5, C-6 and C-8, and this constructed the ring C. HMBC correlations from H 2 -12 and H 2 -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 (δ C = 168.4) and C-17 (δ C = 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 H 3 -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.   [20] and the boat conformation of ring B, the OH group at C-1 must be β-oriented.
The NOESY correlations of H 3 -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 C 24 (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 (δ C = 98.0) which was shifted downfield ∆δ C = +30.1 as compared with that of 1, and the HMBC cross-peak of the H 3 signal (δ H = 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, CH 3 -20 and CH 3 -21 were also β-oriented. Because the chemical shift of C-21 (δ C = 25.6) was shifted upfield (∆δ C = −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].
The 1 H-1 H 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, H 3 -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 H 3 -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, H 2 -16 and H-17a to C-10, from H 2 -11 and H-17b to C-9, and from H 2 -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 H 2 -13 to C-5, H 3 -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, H 2 -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, H 2 -16 to C-14, and H 3 -23 to C-22. The relative configuration of 3 was elucidated by NOESY spectrum as shown in Figure 5. The NOESY correlations of H 3 -21 to H-3b, H-4 and H-6 indicated that H-3b, H-4, H-6 and CH 3 -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 H 3 -21 suggested that C-13 was β-oriented. The boat conformation of ring D was deduced from the NOESY correlation of H 3 -21 to H-12a. Thus, the structure of calyciphylline Q was elucidated as 3.   (Table 1) of 4 revealed 21 carbon resonances, which were classified into two carbonyls, two double bonds, two sp 3 quaternary carbons, four sp 3 methines, seven sp 3 methylenes, and two methyl groups. One methine (δ C = 90.4, δ H = 4.00) and two methylenes (δ C = 68.2, δ H = 3.04 and 3.63; δ C = 69.6, δ H = 3.12 and 3.51) were ascribed to those bearing an oxidative nitrogen. Comparison of the NMR ( 1 H-NMR, 13 C-NMR, HSQC, 1 H-1 H 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 (δ C = 90.4), C-7 (δ C = 69.6) and C-19 (δ C = 68.2) in relation to those of daphlongamine E (C-4 (δ C = 65.5), C-7 (δ C = 53.6) and C-19 (δ C = 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 C 21 (Table 1) and DEPT spectra of 5 revealed 21 carbon signals, ascribed to two carbonyls, one tetrasubstituted olefin, two sp 3 quaternary carbons, four sp 3 methines, nine sp 3 methylenes and two sp 3 methyl groups. Among them, one methine (δ C = 87.9, δ H =3.93) and two methylenes (δ C = 66.1, δ H = 3.04 and 3.56; δ C = 67.4, δ H = 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 13 C chemical shifts of C-4 (δ C = 87.9), C-7 (δ C = 67.4) and C-19 (δ C = 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, 1 H-1 H COSY and HMBC) spectra of 5 (Supporting Information). The structure of calyciphylline S (5) could also be deduced from the comparison of the 1 H-NMR and 13 C-NMR data ( Table 1)  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 1 H-NMR, 13 C-NMR and ESIMS data with that reported.

Plant Material
The

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 CHCl 3 to get the crude alkaloid (45.0 g). The crude alkaloid was subjected to a silica gel column eluting with a CHCl 3 /CH 3 OH (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 CHCl 3 /CH 3 OH (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 CH 3 OH to remove the pigments and impurities, then was further purified by HPLC to afford compounds 1 (7.7 mg, t R = 20.2 min), 2 (6.2 mg, t R = 21.2 min) and 4 (5.5 mg, t R = 33.6 min) eluting with MeOH/H 2 O (8:3) at a flow rate of 6 mL/min. Paxiphylline C (5.7 mg, t R = 18.1 min) and compound 3 (4.4 mg, t R = 24.5 min) were obtained from fraction A7 by HPLC eluting with MeOH/H 2 O (7:3) at a flow rate of 8 mL/min. Fraction A10 was purified by HPLC to give compound 5 (7.2 mg, t R = 22.5 min) eluting with MeOH/H 2 O (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 CHCl 3 /CH 3 OH (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 CH 3 OH to yield macropodumine C (15.8 mg). Fraction C (3.4 g) was chromatographed over a Sephadex LH-20 column equilibrated with CH 3 OH to remove the pigments and impurities, and finally purified by means

Assays for In Vitro Antitumor Activity
The cytotoxicity of compounds 1-9 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% CO 2 . The logarithmic phase cells were seeded on 96-well plates at the concentration of 4 × 10 3 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% (IC 50 ).

Conclusions
Phytochemical investigation of the stem bark of Daphniphyllum macropodum, lead to the isolation of five new Daphniphyllum alkaloids 1-5, along with four known ones 6-9. 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 IC 50 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 IC 50 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.