Calycindaphines A–J, Daphniphyllum alkaloids from the roots of Daphniphyllum calycinum

Ten new Daphniphyllum alkaloids, calycindaphines A–J (1–10), together with seventeen known alkaloids were isolated from the roots of Daphniphyllum calycinum. Their structures were established by extensive spectroscopic methods and compared with data from literature. Compound 1 is a novel alkaloid with a new rearrangement C22 skeleton with the 5/8/7/5/5 ring system. Compound 2 represents the second example of calyciphylline G-type alkaloids. Compound 10 is the first example of secodaphniphylline-type alkaloid absent of the oxygen-bridge between C-25/C-29. The possible biogenetic pathways of 1 and 2 were also proposed. All the isolated compounds were evaluated for their bioactivities in three cell models. Compounds 22, 23, and 26 showed significant NF-κB transcriptional inhibitory activity at a concentration of 50 μM. Compounds 16 and 18 exhibited significant TGF-β inhibitory activity in HepG2 cells. Compounds 24 and 26 induced autophagic puncta and mediated the autophagic marker LC3-II conversion in HEK293 cells.

In the early report, the possible biogenetic pathway of daphhimalenine A was proposed that its precursor daphhimalenine B underwent multi-step oxidation to lose the C-21 and rearranged to form 1-azabicyclo[5.2.1]decane ring system. 14 Calycindaphine A (1) has the same ring system with daphhimalenine A but reserving the key C-21 methyl. According to the structure of 1, another possible biogenetic pathway to form 1azabicyclo[5.2.1]decane ring system was proposed, and shown in Scheme 1. The biogenetic origin of 1 and daphhimalenine A seems to be modied from a yuzurimine-type alkaloid, yunnandaphnine A (13). 7 Yunnandaphnine A might undergo the oxidation of C-1 and the breakdown of C-1/C-8 bond to form 1azabicyclo [5.2.1]decane ring. Then, the intermediate I should undergo the dehydrogenation and sigmatropic rearrangement procedures to yield 1 (C 22 skeleton). The C-21 methyl of 1 should further be oxidated to give II. Then the oxidative  Table 2. Comparing with the known Daphniphyllum alkaloids, the spectroscopic data of 2 is similar to those of calyciphylline G 15 except for the absence of D 2,18 and D 19,N and the presence of additional carbonyl and hydroxyl groups at C-2 and C-4 in 2, respectively. These function groups were assigned by the HMBC correlations from H-3/H-18/H-19/H-20 to C-2 and H-3/H-6/H-21 to C-4 as well as the chemical shi of C-4 (Fig. 2). The relative stereochemistry of 2 was elucidated by the NOESY experiment.  The correlations of H-4/H-21/H-13b and H-21/H-6 suggested that they are on the same side and b-oriented (Fig. 3), which is the same as those of calyciphylline G. 15 Accordingly, the hydroxyl group at C-4 was placed at a-orientation. Thus, the structure of 2 was determined as shown in Fig. 1 and named calycindaphine B.
The calyciphylline G was isolated as a quaternary amine alkaloid containing a 5-azatricyclo [6.2.1.01,5]undecane ring in 2007. 15 However, the possible biogenetic pathway of this unprecedented fused-hexacyclic skeleton has not been described. Comparison of the structural features of calyciphylline G and 2 suggested that the calyciphylline G might be regarded as the key intermediate for 2. A plausible biogenetic pathway for this fused-hexacyclic skeleton is proposed as shown in Scheme 1. Calycindaphine B (2) and calyciphylline G might also be generated from yunnandaphnine A, 7 which might be dehydrated in ring E to form intermediated III. Then, the intermediated III should be reduced and undergo the Wagner-Meerwein rearrangement to yield calyciphylline G. Following, hydrogenation of D 2,18 /D 19,N and oxidation of C-2 and C-4 result in the formation of 2.
Compound 3 has a molecular formula of C 24 H 33 O 4 N with nine degrees of unsaturation. Analysis of spectroscopic data of 3 suggested that 3 have the same skeleton as that of calyciphylline E (11). 16 The major difference is the presence of an additional methoxy group in 3. Based on the HMBC correlation from Hmethoxy (d H 3.25, s) to C-1 (d C 98.3), the methoxy group was placed at C-1 (Fig. 2). The NOESY correlations (Fig. 3) between H-methoxy to H-2/H-18 suggest that they are on the same side and assigned as an a-orientation. Consequently, the structure of 3 was identied as shown in Fig. 1, and named calycindaphine C.
The molecular formula of compound 4 was deduced as C 22 H 29 O 4 N on the basis of its HRESIMS data. The NMR spectroscopic data of 4 were closely related to that of oldhamiphylline A 17 except that the hydroxylated methine at d C 75.8, the methylene at d C 37.1, and the N-substituted methylene at d C 61.34 in oldhamiphylline A are replaced by a methylene at d C 25.4 (C-11), a hydroxylated quaternary carbon at d C 77.4 (C-18), and a lactam carbonyl carbon at d C 174. 4 (C-19) in 4, respectively. These changes were proved by the HMBC correlations from H-10/H-12 to C-11, H-1/H-2/H-3/H-20 to C-18, and H-1/H-7/ H-20 to C-19 (Fig. 2). The signicant NOESY cross-peak of H-21/ H-1 demonstrated the H-21 and H-1 were cofacial and placed C-21 to a-orientation (Fig. 3). Furthermore, the NOESY correlation from H-20 to H-3 indicated there are on the same side and placed C-20 to b-orientation. Accordingly, the hydroxyl group attaching to C-18 was assigned as b-orientation. Therefore, the structure of 4 was identied as shown in Fig. 1, and named calycindaphine D.
Calycindaphines E-G (5-7) possess the molecular formula C 23 H 33 O 4 N, C 23 H 31 O 5 N, and C 23 H 31 O 4 N, respectively. Their NMR data analysis suggested that compounds 5-7 belong to daphnezomine F-type skeleton. 18 The NMR data of 5 are similar to those of daphlongeranine C 18 except that the hydroxy methylene at C-21 in daphlongeranine C was replaced by a singlet methyl in 5. This change was supported by the HMBC correlations from H-21 to C-4/C-5/C-6/C-8 and H-4/H-6 to C-5/C-21. Compound 6 is structurally like 5 except that the C-9/C-10  double bond, the methylene at C-7, and the O-methine at C-2 in 5 are absented in 6, and an acylamide, two oxygenated quaternary carbons, and a methylene were presented in 6, respectively. The oxygenated C-9/C-10 in 6 were devised as an epoxy threemembered ring by the chemical shis of C-9 and C-10 combined with the exclusive molecular formula from the exact result of HRESIMS. In addition, the HMBC correlations from H-6/H-12/H-19 to the extra acylamide (C-7), H-15/H-16/H-17 to the pair of oxygenated quaternary carbons (C-9/C-10), and H-20/H-18/H-4/H-3 to C-2 supported the above conjectures. Comparison of the chemical shis of C-9 and C-10 with those of alkaloids containing epoxy group at C-9 and C-10 suggested that the epoxy group in 6 is a-oriented. [19][20][21] Careful analysis of NMR data of 7 indicated that 7 is a daphnezomine Ftype alkaloid with two double bonds and a hydroxylated quaternary carbon. HMBC correlations from H-7 to C-1/C-5/C-6/ C-12/C-19, H-4/H-12/H-21 to C-6, H-15/H-16/H-11 to C-10/C-17, and H-14/H-15 to C-9 implied that two double bonds were placed at C-7/C-6 and C-10/C-17, and the hydroxylated quaternary carbon was xed at C-9. Thus, the structures of 5-7 were determined as shown (Fig. 1).
The 1D NMR data of 8 suggested that compound 8 was closely related to 23. 11 The major differences between 8 and 23 were that the chemical shi of H-22 was down-shielded from d H 3.33 (in 23) to d H 3.94 (in 8), which might be caused by the different conguration of C-22. Furthermore, analysis of the 1 H-1 H COSY and HMBC spectra implied that 8 and 23 have same planar structure. The NOESY cross-peak (Fig. 3) between H-22/H-24 in 8 illustrating that these protons are in cofacial and assigned to be b-orientation, which is opposite to that of 23. Furthermore, the optical value of 8 and 23 was measured as [a] 22.5 D +26.8 (c ¼ 0.5,MeOH) and [a] 22.5 D À49.4 (c ¼ 0.5,MeOH) respectively, which also provided evidence for the different conguration of C-22 in 8 and 23. Accordingly, compound 8 was elucidated as shown in Fig. 1 and named calycindaphine H. Calycindaphine I (9) has a molecular formula C 30 H 47 O 4 N. Comparison of its 1D NMR spectra with those of 8 showed that the hydroxylated methine at d C 75. 5 (C-22) and methyl at d C 21.3 (C-21) in 8 are replaced by a carbonyl at d C 213.0 (C-22) and a hydroxylated methylene carbon at d C 66.2 (C-21) in 9, respectively. These changes were further conrmed by the HMBC correlations from H-21 to C-4/C-5/C-6/C-8 and H-13/H-14/H-24 to C-22 (Fig. 2). Thus, the structure of 9 was determined as shown in Fig. 1.
Compound 10 showed a protonated [M + H] + 29,22,23 which supported that the specic linkage of C-25-O-C-29 in secodaphniphylline-type alkaloids is broken to form a hydroxy at C-25 and C-29 in 10, respectively. The above spectroscopic evidence deduced the structure of 10 as depicted in Fig. 1, and named calycindaphine J.

Conclusions
In conclusion, ten new Daphniphyllum alkaloids, calycindaphines A-J (1-10), together with 17 known alkaloids were isolated from the roots of D. calycinum. Compound 1 is a novel alkaloid with a new C 22 skeleton with a rare 5/8/7/5/5 ring system containing a unique 1-azabicyclo [5.2.1]decane. Compound 2 is the second example for the unique skeleton with 5/6/5/8/5/5 ring system. Compound 10 is the rst example of secodaphniphylline-type alkaloid absent of the oxygen-bridge between C-25/C-29.
Compounds 16,18,[22][23][24], and 26 exhibited their potential bioactivities on NF-kB or TGF-b inhibition and/or cell autophagic induction. Our ndings not only revealed the chemicals from the roots of D. calycinum for the rst time, but also give a new insight into the complex polycyclic skeletons and structural diversity of Daphniphyllum alkaloids.

General experimental procedures
Optical rotations were measured on a Rudolph Research Analytical Autopol I automatic polarimeter. Ultraviolet (UV) and CD spectra were recorded on a Jasco J-1500 Circular Dichroism Spectrometer. IR spectra were carried on an Agilent Cary 660 series FT-IR spectrometer (KBr). HRESIMS spectra were obtained on an Agilent 6230 HRESIMS spectrometer. 1D and 2D NMR spectra were performed on a Bruker Ascend 600 NMR spectrometer. The chemical shis were expressed in d (ppm) with TMS as an internal reference. Column chromatography was performed on Silica gel (40-60 mesh, Grace, USA) column. Thin layer chromatography was carried on precoated silica gel 60 F 254 plates (200 mm thick, Merck KGaA, Germany). MPLC was performed using a Buchi Sepacore ash system with a RP-18 column (SilicBond C 18 , 36 Â 460 mm ID, 40-63 mm particle size). Semi-preparative HPLC was conducted on an Agilent 1100/1200 liquid chromatography instrument with a Waters Xbridge Prep C 18 column (10 Â 250 mm, 5 mm) or Xbridge Prep C 8 column (10 Â 250 mm, 5 mm). UHPLC analyses were conducted on an Agilent 1290 system using a ZORBAX RRHD Eclipse Plus C 18 column (1.8 mm, 2.1 Â 50 mm, Agilent).

Plant material
The

Extraction and isolation
The air-dried, powdered roots (30 kg) of D. calycinum were extracted three times with 80% EtOH. The combined ltrates were concentrated under vacuum to afford a dark extract, which was adjusted to pH 2 with HCl. The acidic mixture was centrifuged to remove dark brown precipitates. The aqueous layer was basied to pH 10 with NaHCO 3 and then exhaustively extracted with EtOAc to afford the crude alkaloids (170.0 g). The crude alkaloids were subjected to a Silica gel column (CHCl 3 /MeOH, 1 : 0-0 : 1) to obtain 10 fractions (Fr.A-Fr.F).

ECD calculations of compound 1
Conformational analyses were carried out via random searching in the Sybyl-X 2.0 using the MMFF94S force eld with an energy cutoff of 2.5 kcal mol À1 . The results showed the nine lowest energy conformers for both compounds. Subsequently, the conformers were re-optimized using DFT at the PBE0-D3(BJ)/ def2-SVP level in MeOH using the polarizable conductor calculation model (SMD) by the ORCA4.2.1 program. The energies, oscillator strengths, and rotational strengths (velocity) of the rst 60 electronic excitations were calculated using the TDDFT methodology at the PBE0/def2-TZVP level in MeOH. The ECD spectra were simulated by the overlapping Gaussian function (half the bandwidth at 1/e peak height, sigma ¼ 0.30 for all). To get the nal spectra, the simulated spectra of the conformers were averaged according to the Boltzmann distribution theory and their relative Gibbs free energy (DG).

Cell lines and cell cultures
The HepG2-NF-kB-Luc cell line is stably transfected with the NF-kB-luciferase gene, which was generously provided by Dr C. H. Leung (University of Macau). Cells were cultivated with DMEM medium supplemented with 10% fetal bovine serum (FBS), 100 U mL À1 penicillin, and 100 mg mL À1 streptomycin at 37 C with 5% CO 2 and 95% air incubator. SMAD 2/3 responsive luciferase reporter HepG2 stable cell line was purchased from Signosis. Cells were cultivated with DMEM medium supplemented with 5% FBS, 100 U mL À1 penicillin, 100 mg mL À1 streptomycin, and 100 mg mL À1 hygromycin B at 37 C with 5% CO 2 and 95% air incubator.
HEK293 cell line stable transfected with GFP-LC3 was kindly provided by Dr X. M. Zhu (Macau University of Science and Technology). The cells were cultured in an a-MEM medium supplemented with 10% FBS under a humidied atmosphere containing 5% CO 2 at 37 C.

NF-kB luciferase assay
The NF-kB activity was determined by NF-kB luciferase assay as described in our previous publication with a slight modication. 32 Briey, HepG2-NF-kB-Luc cells were seeded on a 96-well microplate with 1 Â 10 4 cells per well and cultured at 37 C with 5% CO 2 incubator for 18 h. Then, cells were pretreated with compounds (6, 12.5, and 50 mM) for 12 h and induced with TNFa (10 ng mL À1 ) for 4 h. Ammonium pyrrolidinedithiocarbamate (PDTC) was used as the positive control. The rey luciferase signal was measured with the Bright-Glo Luciferase Reporter Assay System (Promega, Madison, WI) according to the manufacturer's instruction using a multimode reader (SpectraMax iD5, Danaher).

TGF-b induced SMAD luciferase assay
The effects of compounds on the TGF-b/SMAD were determined by TGF-b/SMAD luciferase assay. Briey, HepG2/SMAD-Luc cells were seeded on a 96-well microplate with 1 Â 10 4 cells per well and cultured at 37 C with 5% CO 2 incubator for 18 h. Aer adhesion, the cells were pretreated with compounds at different concentration for 6 h and induced with TGF-b (10 ng mL À1 ) for 18 h. Meanwhile, SB-431542, a specic inhibitor of TGFb Receptor Kinase, was applied as the positive control. The rey luciferase signal was measured with the Bright-Glo Luciferase Reporter Assay System (Promega, Madison, WI) according to the manufacturer's instruction using a multimode reader (SpectraMax iD5, Danaher).

LC3 puncta counting
The HEK293-GFP-LC3 cells were applied for visualizing autophagosome formation aer treatment with various compounds for 24 hours. During autophagosome formation, GFP-LC3 is processed and recruited to the autophagosome membrane, where it can be imaged as cytophasmic puncta by IncuCyte ZOOM live cell imaging (Olympus, coupled with Hamama Tsu ORCA-Flash 40 LT Plus Scientic CMOS Digital Camera). The percentage of GFP-LC3 positive cells can be determined and is indicative of autophagosome formation.
The cells were seeded onto 6-well plates with a-MEM medium and cultured for 24 hours. Aer treating with various concentrations of compounds 24 and 26, cells were collected and lysed in lysis buffer on ice for 30 minutes. Protein samples were electrophoresed using 15% SDS-PAGE gel and transferred onto a nitrocellulose membrane (NC membrane). The membranes were blocked by a 5% BSA and incubated with the primary antibody overnight at 4 C, followed by the secondary antibody for 1 hour at room temperature. Protein bands were detected by the LI-COR Odyssey imaging system (Lincoln, NE).

Conflicts of interest
There are no conicts to declare.