Glaulactams A–C, daphniphyllum alkaloids from Daphniphyllum glaucescens

Glaulactams A–C (1–3), which possess a novel skeleton, as well as the known compound daphmanidin B (4), were isolated from the leaves of Daphniphyllum glaucescens and separated using ion-exchange chromatography aided by NMR fingerprinting. Their structures, including their absolute configurations, were elucidated by spectroscopic analyses and time-dependent density-functional-theory-calculated electronic circular dichroism spectra; the data were subsequently analyzed to gain insight into the respective biogenetic relationships between the isolates, which exhibited anti-H1N1 and immunosuppressive activities.

The first daphniphylline-type alkaloid, daphniphylline, which possesses a polycyclic, C 30 -aliphatic structure, was discovered in 1966 1 . A number of novel structures from the Daphniphyllaceae family of plants were recently reported, including himalensine A, with its 13,14,22-trinorcalyciphylline A backbone 2 , himalensine B with its 22-nor-1,13-secodaphnicyclidin framework 2 , and macropodumines A-C with fused pentacyclic ring systems 3 . The complex and fascinating structures of these alkaloids render them synthetically challenging 4 . Many types of alkaloid are reported to be produced by Daphniphyllum glaucescens, including daphniglaucin C, which has a novel structure that contains hexahydroazulene and octahydroindole ring systems 5 and daphniglaucins A and B, which have unique 1-azoniatetracyclo[5.2.2.0 1,6 0. 4,9 ]undecane motifs 6 . In addition, daphniglaucins D-H possess fused hexacyclic skeletons, and daphniglaucins J and K are yuzurimine-type alkaloids that have previously been reported from this species 7 .
Natural product chemists are very interested in the discovery of novel structures with unique properties 8 . To date, a variety of methods have been used to facilitate the discovery of novel natural products, with examples including NMR-fingerprinting 8 and LC-MS-guided methods 9 . Daphnezomine-F-type 10,11 and daphmanidin-C-type 12 alkaloids are rare groups of daphniphyllum alkaloids; they each possess a lactam functional group that is biogenetically derived through the oxidative cleavage of a yuzurimine C-C bond. The presence of lactams in these daphniphyllum compounds inspired us to develop a new method that combines ion-exchange chromatography (IXC) and NMR fingerprinting to screen for new alkaloids. Hence, in this article, we report the isolation, structural characterization, and biological evaluation of glaulactams A-C (1-3) from the leaves of Daphniphyllum glaucescens.

Results and Discussion
Air-dried D. glaucescens leaves (1.53 kg) were extracted with MeOH and concentrated to obtain a crude extract, which was dispersed in 80% aqueous MeOH to give a methanolic suspension. This suspension was then partitioned with hexane (4 × 2 L) to remove the lipid constituents. The methanolic extract was filtered and passed through a cation-exchange resin. The eluent containing the acidic and neutral compounds was concentrated under reduced pressure and subjected to Diaion HP20 column chromatography, followed by vacuum liquid chromatography (VLC) over silica gel to yield 26 fractions (4A-4Z); among these fractions, the 1 H NMR spectra of fractions 4V, 4W, and 4Z were found to exhibit signals characteristic of daphniphyllum alkaloids (see the Extraction and Isolation section). As a result, these three fractions were selected for repeated column chromatography over silica gel and RP-18 stationary phases to yield three compounds that had structures that were consistent with 1 (4.2 mg), 2 (29.2 mg), and 3 (3.0 mg) (Fig. 1 Fig. 2. Subsequent HMBC spectral analysis revealed where these three structural fragments are linked. The C-1 carbonyl (δ C 180.5) and C-19 (δ C 51.4, δ H 3.16, m, 2 H) of fragment I were speculated to be due to a γ-lactam ring, which was verified by the observed HMBC-derived H 2 -3/C-1 and H 2 -19/C-1 correlations. HMBC correlations between H 2 -21 and C-4, C-5, C-6, and C-8, and between H 2 -7 and C-1 and C-19 linked fragment I to II, resulting in an amide-bridged bicyclic system (C-1 to C-7 and the N atom, and C-18 to C-19). The link between the methyl carboxylate and C-14 in fragment III was made on the basis of the HMBC correlations between both H-14 and H 3 -23, and C-22, while C-13, C-15, and C-17 are attached to C-8, C-9, and C-10 of the conjugated diene on the basis of the HMBC correlations between H-14 and C-8 and C-9, and between H-11 and C-9 and C-17. The acetoxy groups at C-4 and C-21 were assigned on the basis of HMBC correlations between H-4 and H 2 -21 and the respective acetyl carbonyls at δ C 168.9 and 170.5. Hence, the planar structure of 1 was assigned to be a fused pentacyclic ring with a γ-lactam functionality (Fig. 2).
The absolute configurations of compounds 1-3 were determined by comparing the experimental electronic circular dichroism (ECD) spectra to those calculated theoretically. Compounds 1-3 were subjected to standard conformational analysis as implemented in the Confab program 13 . The generated lowest-energy structures following further B3LYP/6-31G(d) optimizations were then used to calculate ECD spectra by time-dependent density functional theory (TDDFT) at the TD-CAM-B3LYP/def2TZVP level. All calculations were performed no. using the Gaussian 09 Rev. D program package using the "ultrafine grid" option, Integral (Grid = UltraFine), with solvent effects accounted for using the IEFPCM method 14 . The ECD spectrum of each compound 1-3 was finally generated as the Boltzmann-weighted sum of the spectra generated for the various conformers in each case, which resulted in the establishment of the absolute configurations of glaulactams A-C were, as illustrated by structures 1-3, since the calculated spectra are in good agreement with those acquired experimentally ( Fig. 4; Supplementary Fig. S5). It is possible to describe the biosynthetic origins of 1-4 on the basis of the lactam formation mechanism of hemiaminals, the structure of yuzurimine E 15 and a proposed compound i. In the proposed mechanism (Fig. 5), oxidative cleavage of the C-1-C-2 bond in yuzurimine E via pathway a 16  In preliminary biological screening, the extract of D. glaucescens exhibited antiviral activity against the influenza virus and was immunosuppressive in lipopolysaccharide (LPS)-stimulated murine dendritic cells (DCs). Compounds 1-4 were tested for their anti-influenza virus (i.e., anti-H1N1) activities in Madin-Darby canine kidney (MDCK) cells using the plaque assay with betulinic acid as the positive control 17 . Cytotoxicity testing revealed that the isolates were not toxic to uninfected host MDCK cells at a concentration of 100 μM (Fig. 6a). However, at a concentration of 50 μM, compounds 1 and 4 were found to substantially inhibit plaque formation of MDCK cells by H1N1 virus infection, to values of 24.4% and 28.0%, respectively. Although less effective under the same treatment conditions, compounds 2 and 3 were found to moderately inhibit plaque formation (69.1% and 63.5%, respectively) (Fig. 6b). In addition, compounds 1-3 were also evaluated for their immunosuppressive activities. Mouse bone-marrow DCs were treated with compounds 1-3, and the immunosuppressive agent quercetin was used as the positive control 18 . In preliminary studies, compounds 1-3 (50 μg/mL) and quercetin (50 μM) were found to have no significant cytotoxic effects on murine DCs in the presence of LPS (100 ng/ mL) (Fig. 7). However, subsequent experiments revealed that compounds 1-3 significantly suppress the levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), IL-12p70, and nitric oxide (NO) in LPS-stimulated murine DCs (Fig. 7). These results confirm that the immunosuppressive properties of compounds 1-3 are not due to their cytotoxicities in DCs, and that the observed effects are similar to that induced by quercetin.
In summary, IXC and NMR fingerprinting were used to identify and isolate three novel daphniphyllum alkaloids, whose anti-influenza and immunosuppressive activities were then explored. The method described herein can be implemented as a convenient alternative to existing methods commonly employed to extract unique compounds from a complex array of natural products.

Ethical Statement. The Institutional Animal Care and Use Committee (IACUC) of National Chung Hsing
University approved the experimental procedures (approved protocol no. NCHU-IACUC-104-027). The methods were performed in accordance with the approved guidelines.
General experimental procedures. Optical rotations were measured on a JASCO P2000 digital polarimeter and IR spectra were acquired on a Shimadzu IR Prestige-21 FT-IR spectrometer. NMR spectra, in pyridine-d 5 , were recorded on a 500 MHz Avance III spectrometer (Bruker, Rheinstetten, Germany). The 1 H and 13 C NMR chemical shifts were referenced to the solvent residual peaks at δ H 7.58 and δ C 135.5 for pyridine-d 5     Extraction and Isolation. Air-dried leaves of D. glaucescens (10.38 kg) were exhaustively minced and extracted with MeOH (4 × 20 L). The solvent was concentrated, and 20% water was added to yield an aqueous suspension, to which hexane (4 × 2 L) was added to removed lipids and chlorophylls. The methanolic solution   Plaque-reduction assay. Monolayer MDCK cells were seeded in six-well plates (5 × 10 5 cells/well) for 24 h.
The influenza virus A/WSN/33(H1N1) (100 plaque-forming units (PFU) per well) was mixed with each of the indicated compounds for 30 min at room temperature. The mixtures were subsequently adsorbed to the preseeded cells for 1 h at 37 °C. After removal of the medium, the cells were washed with PBS (three times) and then overlaid with 0.3% agarose containing the indicated compounds for an additional 48 h at 37 °C, after which the cells were fixed with 10% formaldehyde for 1 h. Viral plaques were counted by staining with 0.5% crystal violet.

Mice and the preparation of bone marrow-derived murine dendritic cell (DC).
According to the published method 19 , the murine bone-marrow-derived DCs were prepared from female C57BL/6 mice housed under controlled-temperature (22 ± 2 °C) and humidity (45-65%) conditions, with a 12-h light/dark cycle and free access to food and water. The animals were treated according to the requirements of the Institutional Animal Care and Use Committee of National Chung-Hsing University.
Computational details. Since different conformers of a specific stereochemical configuration can give different ECD spectra, it is critical to identify all relevant conformers in order to accurately predict the ECD spectrum. Therefore, standard conformational analyses were performed using the Confab program 13 . All optimized conformations were in a 10 kcal/mol energy window, with a root mean square (RMS) step-size of 0.2 Å. These conformers were then re-optimized at the B3LYP/6-31G(d) level of theory and verified to be true minima on the potential energy surface by frequency analyses. Then resulting geometries were subsequently used for three single-point calculations. CAM-B3LYP/TZVP 21 calculations in the vacuum state were carried out in order to obtain converged wavefunctions for the ground states, which were used in the next two calculations. Energies were calculated at the M062x/Def2TZVP level with ethanol as the solvent, as this method provides more precise energies for conformational ordering 22 . The series of conformers was restricted by removing duplicates (RMSD < 0.2) and conformations outside of a 4 kcal/mol energy window, based on the Gibbs free energy at the M062x/Def2TZVP//B3LYP/6-31G(d) level in ethanol. The resulting structures were used in TDDFT ECD calculations, in ethanol as the solvent, at the TD-CAM-B3LYP/def2TZVP level by considering the 100 lowest-energy states. All calculation were performed using the Gaussian 09 program package with the "ultrafine grid" option, Integral (Grid = UltraFine), and all solvent effects were accounted for using the IEFPCM method 14,23 . Finally, the overall ECD spectra were combined following Boltzmann weighting on the basis of the Gibbs free energies of the corresponding conformer calculated at the M062x/Def2TZVP//B3LYP/6-31G (d) level. All ECD spectra were processed with SpecDis and simulated by Gaussian functions with bandwidths (σ) of 0.16 eV and by considering velocity representations 24 .