Crepidtumines A and B, Two Novel Indolizidine Alkaloids from Dendrobium crepidatum

Two new indolizidine alkaloids crepidatumines A (1) and B (2) together with the stereoisomer of dendrocrepidine B (3) and known analog dendrocrepine (4) were isolated from D. crepidatum. Their structures were determined by HR-ESI-MS, NMR, and Electronic Circular Dichroism (ECD) experiments together with comparison of analogues. Compound (1) possess a (5/6/6/5) tetra-hetero-cyclic ring, whereas compound (2) contains a tricyclic system with an unusual bridged ring, which are the first report in Nature. The biological evaluation revealed that dendrocrepine (4) displayed a potent hypoglycemic effect in vitro.


Identification code 3
Empirical formula C 20 Table 1. Crystal data and structure refinement for compound 3.
indolizidine ring system; the HMBC cross peaks from 3-OH to C-2, C-3, C-4 and C-12 determined the connection of C-3 with C-2, C-4 and C-12, and a hydroxyl group was anchored at C-3; the correlations of 15-Me with C-4, C-14, and of 14-OH with C-4, C-14 and C-15 established the linkage of C-14 with C-4 and C-15, and of C-14 with an hydroxyl group. Considering the chemical shift value of C-14 (δ c = 104.6), degrees of unsaturation and molecular formula, an ether bond must be shaped between C-6 and C-14 to form a unique hemiacetal group. Thus, the planar structure of 1 was characterized. The relative configuration of 1 was determined on the basis of analysis of NOESY correlations and coupling constants (Fig. 3). The correlations from H-5 with H-1, 3-OH, H-7, H-9, and H-2′ (H-6′) implied that H-1, 3-OH, H-5, H-7, and H-9 possessed pesudo-axial bonds, whereas the mono-substituted phenyl and 12-Me possessed pesudo-equatorial bonds; the coupling constant between H-4/H-5 was 3.0 Hz, which revealed that H-4 possessed pesudo-equatorial bond; H-2b was a broad triplet (t, J = 13.2 Hz) and H-2a was a doublet doublet (dd, J = 13.2, 3.6 Hz) confirmed that H-2b possessed pesudo-axial bond and H-2a possessed pesudo-equatorial bond; 15-Me had NOESY correlations with 12-Me, and H-2b had NOESY correlations with 14-OH implied that these groups were close each other in space (Fig. 3). Thus, the relative configuration of 1 was determined. Electronic circular dichroism (ECD) experiments combined with quantum-chemical calculations adopting time-dependent density functional theory (TDDFT) was used to establish the stereochemistry of crepidatumine A (1). The theoretical calculation of ECD was conducted in MeOH using Time-dependent Density functional theory (TD-DFT) at the B3LYP/6-311 + g (d, p) level for all conformers of compounds 1. To get the final spectra, the simulated spectra of the conformers were averaged according to the Boltzmann distribution theory and their relative Gibbs free energy (ΔG). By comparing the experiment spectrum with the calculated ECD spectra, the stereochemistry for 1 was determined to be 1R, 3S, 4R, 5R, 6S, 7R, 9S, 14R (Fig. 4) same as that of its analogue 3, of which stereochemistry was determined by X-ray diffraction experiment.
Compounds 1-4 were tested the anti-inflammatory activity in RAW 264.7 cells without biological effects, whereas the total alkaloids of D. crepidatum (TAD) inhibited the NO production induced by LPS in RAW 264.7 cells in a dose-dependent fashion without cytotoxicity (Fig. 6) 19 . Thus, it is merited to go to mine bioactive natural products from these total alkaloids. In addition, compounds 1-4 were evaluated the cytotoxic activities against three cancer cell lines A459, and HCT116, and gram-positive bacteria Bacillus subtilis 63501, Staphylococcus aureus 29213, and gram-negative bacterium Escherichia coli 25922 without bioactivities. The high glucose model  www.nature.com/scientificreports www.nature.com/scientificreports/ of HepG2 cells was used to evaluate the hypoglycemic effect of compound 4. As a result, compared with the model group, this compound significantly increased the glucose consumption by 29% at the concentrations of 50 μmol/L without cytotoxicity, which implying that this compound might be a potent molecule as a hypoglycemic agent (Fig. 7). conclusion Four indolizidine alkaloids including two new compounds crepidatumines A (1) and B (2) together with the stereoisomer (3) of dendrocrepidine B and known analog dendrocrepine (4) were isolated from D. crepidatum Compound (1) possess a unusual (5/6/6/5) tetra-hetero-cyclic ring, whereas compound (2) contains a unique tricylic system with an unusual bridged ring, which are the first report in Nature. Compounds 1-4 did not display anti-inflammatory, anti-microbial and cytotoxic activities, but compound 4 could increase glucose consumption, implying that 4 might be a potent molecule as a hypoglycemic agent.

Methods
General experimental procedures. Optical rotations were measured on a PerkinElmer 241 polarimeter, and UV data were determined on a ThermoGenesys-10S UV-vis spectrometer 19 . IR data were recorded using a Nicolet IS5FT-IR spectrophotometer. CD spectra were obtained on a JASCO J-810 spectrometer. 1 H and 13 C NMR data were acquired with a Bruker 600 spectrometer using solvent signals (DMSO-d 6 ; δ H 2.50/δc 39.5) as references. the HMQC and HMBC experiments were optimized for 145.0 and 8.0 Hz, respectively. HRESIMS were obtained using a TOF-ESI-MS (Waters Synapt G2, USA). Semipreparative HPLC separation was carried out using a Lumtech instrument packed with a YMC-Pack ODS-A column (5 μm, 250 × 10 mm). Sephadex  Extraction and isolation. The dried stems of D. crepidatum (9.0 kg) were extracted under reflux with 95% ethanol (50 L × 3 h, three times) 19 . The combined extract was suspended with water, and extracted with petroleum ether and CH 2 Cl 2 three times separately. The fraction of CH 2 Cl 2 was concentrated into extractum, and dissolved in 5% hydrochloric acid filtered, then adjusted to pH 10 with ammonia water. Finally, it was extracted by CH 2 Cl 2 three times at room temperature. The CH 2 Cl 2 extract was obtained the total alkaloids 90.    20 . The cells were cultured at 37 °C in DMEM (Invitrogen, California, USA) supplemented with 10% heat-inactivated FBS (Hyclone, Utah, Logan, USA), 2 m ML -glutamine, penicillin G (100 IU/mL) and streptomycin (100 mg/mL) in a humidified atmosphere containing 5% CO 2 and 95% air. The cells were incubated with total alkaloids of D. crepidatum (TAD) at different concentrations and then stimulated with LPS (10 ng/mL) for the indicated time. The stock solutions of TAD were prepared in dimethyl sulfoxide (DMSO), and the final concentration of DMSO was less than 0.5%.

X-ray crystallographic analysis of 3. Upon crystallization from n-Hexane-CH
The level of NO production was monitored by measuring the nitrite level in the culture medium. This was performed by mixing the medium with Griess reagent (1% sulfanilamide in 5% phosphoric acid and 0.1% N-1-naphthylethylenediamine dihydrochloride in water). The absorbance was measured at a wavelength of 540 nm after incubation for 10 min. The nitrite concentration was calculated with reference to a standard curve of sodium nitrite generated from known concentrations. Cell viability was assessed using an MTT assay. www.nature.com/scientificreports www.nature.com/scientificreports/ In Vitro Evaluation of Compound 4. Cell culture: Human hepatoma cells HepG2 were cultured in Dulbecco's modified Eagle's medium (DMEM, HyClone) 19 . DMEM was supplemented with 10% fetal bovine serum (Gibco) and 1% penicillin/streptomycin (HyClone) in a humidified atmosphere of 5% CO 2 and 37 °C.
Assay for cell viability: Before the experiment, the assay for cell viability was determined with the cell counting kit-8 (CCK-8). HepG2 cells were seeded in 96-well plates as 2.5 × 10 3 cells each well. Afterculturing for 24 h,the control group was added with serum-free medium, while the experimental groups were with the medium containingdifferent concentrations (50and 100 μmol/L) of compound 4 or 200 μmol/Lof metformin for another 24 h. Then the cells were treated with CCK-8 for 3 h. Finally, the absorbance was measured at 450 nm. The cell viability was calculated as the absorbance of each treated well divided by the control.
Assay for hypoglycemic activity: For the experiment, the cells were seeded in 96-well plates as 1 × 10 4 cells each well. After culturing for 24 h, the medium containing different concentrations (25 and 50 μmol/L) of compound 4 were added for 24 h. The cells with 200 μmol/L metformin treatment were taken as positive control and the cells with phenol red-free DMEM as control. After the drug treatment, the glucose concentrations of the medium were determined with the glucose oxidase method. The glucose consumption of each well was obtained by subtracting the glucose concentrations of the experimental medium from the control group.
ECD calculations of 1 and 2. Monte Carlo conformational searches were carried out by means of the Spartan's 10 software using Merck Molecular Force Field (MMFF) 19 . The conformers with Boltzmann-population of over 5% were chosen for ECD calculations, and then the conformers were initially optimized at B3LYP/6-31 + g (d, p) level in MeOH using the CPCM polarizable conductor calculation model. The theoretical calculation of ECD was conducted in MeOH using Time-dependent Density functional theory (TD-DFT) at the B3LYP/6-311 + g (d, p) level for all conformers of compounds 1 and 2. Rotatory strengths for a total of 50 excited states were calculated. ECD spectra were generated using the program SpecD is 1.6 (University of Würzburg, Würzburg, Germany) and GraphPad Prism 5 (University of California San Diego, USA) from dipole-length rotational strengths by applying Gaussian band shapes with sigma = 0.3 eV.