New Anti-angiogenic Leading Structure Discovered in the Fruit of Cimicifuga yunnanensis

Cimyunnins A–C (1–3), characterized with an unusual fused cyclopentenone ring G, together with cimyunnin D (4), possessing a highly rearranged γ-lactone ring F, were characterized from the fruit of Cimicifuga yunnanensis. Their structures were elucidated by spectroscopic analysis, X-ray diffraction, and density functional theory calculations. In addition, cimyunnin A exhibited comparable anti-angiogenic activities to those of sunitinib, a clinically-used first-line angiogenesis inhibitor, in the in vitro and ex vivo studies.

The spin system -CH 2 CHCH 3 -due to C-26, C-25, and C-27 in fragment B was also deduced from 1 23). This information coupled with the UV and IR absorptions of l max 266 nm, and n max 1702 and 1655 cm 21 , indicated the existence of a cyclopentenone ring G (C-22 to C-26). Therefore, to fulfill the molecular formula and unsaturation requirement, C-16 and C-23 should be connected by an oxygen atom, which also supported by similar chemical shifts of C-22 (d C 148.9) and C-23 (d C 149.3) due to the electronic effect and conjugative effect from the oxygen atom at C-16 and carbonyl group at C-24, repectively. Finally, fragment B and planar structure of 1 were established.
In the ROESY spectrum ( , and H-26a/ Me-21 were observed, which helped to establish the relative configuration of 1. Our efforts to make fine crystals from 1 failed which precluded the possibility to determine the absolute configuration directly by X-ray crystallography. Therefore, quantum chemical CD calculations were applied 27 . The results showed that spectrum calculated for 1A was nearly identical with the experimental data of 1 (Fig. 3, left) over the whole range of wavelengths under investigation, whereas the spectrum simulated for 1B exhibited very different CD behavior compared with the experimental CD curve of 1 (Fig. 3, right). Therefore, the absolute configuration of 1 was determined as shown.
Cimyunnins B (2) and C (3), obtained as an inseparable mixture. Side-by-side comparison of their NMR data indicated that 2 and 3 were a pair of C-25 epimers and most signals were exchangeable between the two compounds (Table S2) 13 C NMR signals for C-22 and C-23 were successfully assigned based on HMBC correlations between H-24a of compounds 2 and 3 and corresponding carbons.
The 1 H and 13 C NMR spectra (Table S2) of 2 and 3 were very similar to those of 1 with the major differences for signals of ring G. A spin system -CH 2 CHCH 3 -in the ring G was established by the     (Table S3)     On the basis of similar ROESY correlations (Fig. 4), same relative stereochemistries of C-3, C-5, C-8, C-9, C-10, C-12, C-13, C-14, C-16, and C-17 in 4 as in 1 were elucidated. Figure 5B    correlations of H-20/CH 3 -18, CH 3 -21/CH 3 -18, and H-16/H-22b. Finally, the 13 C NMR and OR data of 4 were compared to the calculated results of lactone A (4A) and lactone B (4B) ( Table S9, S10, and S11), which further confirmed the absolute configuration of 4.
To the best of our knowledge, compounds 1-4 stand for two unprecedented classes of CTs. Biosynthetically, 1-3 probably originate from the cycloartane precursors asiaticoside A (5) 18 . The key steps are the formation of intermediates 5A, and 5B through nazarov reaction 28 , and [2 1 2] cycloaddition 29 , respectively. While, compound 4 may derive from 1 through a series of oxidation rearrangements (see Scheme 1 in the Supporting Information).
In vitro and Ex vivo Anti-angiogenic Activities of Compound 1. Although CTs from Cimicifuga spp showed various bioactivities, anti-angiogenic activities were still unknown. In the present study, compound 1 notably inhibited VEGF-induced proliferation of HUVECs at 5.0 mM (due to sample quantity limitation, the activities of compounds 2-4 were not studied), without obvious cytotoxicity against human umbilical vein endothelial cells (HUVECs) (Fig. 6A). Then, the effect of 1 on the motility of HUVECs was studied. As shown in Figure 6B, HUVECs migrated into the clear area when stimulated with 25 ng/ml VEGF. Conversely, compound 1 significantly inhibited the VEGF-induced migration of HUVECs in a time dependent manner at concentration of 2.5 mM. This effect of 1 as strong as that of sunitinib, a clinically-used first-line angiogenesis inhibitor. Subsequently, 1 drastically reduced the new vascular growth density at a dosedependent manner in the chick chorioallantoic membrane (CAM) assay. The maximum reduction of new vascular density was observed at concentration of 10.0 nmol/egg ( Figure 6C), which was comparable to that of sunitinib at the same concentration. The aforementioned results indicated the anti-angiogenic potential of 1 both in vitro and ex vivo.
VEGFR2-signaling pathways is essential for the function of vascular endothelial cells 30 . Finally, to understand the molecular mechanism of 1, we examined the pathways and signaling molecules using western blot. As shown in Figure 7, phosphorylation of VEGFR2 was suppressed by 1 in a dose-dependent manner. Dramatic downregulations of phosph-AKT (Ser473) and phospho-ERK (Thr202/ Tyr204), well-known downstream targets of VEGFR2, were observed at 5.0 and 10.0 mM of 1. However, total VEGFR2, ERK, and AKT remain unchanged. Therefore, compound 1 exerted its anti-angiogenic effect through directly targeting VEGFR2 on the surface of endothelial cells and further antagonizing VEGFR2-mediated downstream signaling cascade. Natural products, due to unrivaled chemical diversity and structural plasticity, are rich sources of novel leading structures for drug discovery 31 . So far, diverse natural compounds, such as anthocyanins, genistein, resveratrol, curcumin, taxol, betulinic acid, and squalamine, showed anti-angiogenic activities both in vitro and in vivo 32 . However, few studies about the anti-angiogenic CTs were reported 33 . Herein, compound 1, with an unprecedented skeleton, showed same level of anti-angiogenic activities as sunitinib both in vitro and ex vivo. Taken together, compound 1 stands for a new type of leading structure of anti-angiogenesis.

Methods
General Experimental Procedures. Optical rotations were obtained with a JASCO P-1020 digital polarimeter, using MeOH as solvent. UV spectra were taken on Shimadzu 2401PC spectrophotometer. CD spectra were obtained by a Chirascan instrument. 1 H, 13 C and 2D NMR experiments were measured on Bruker DRX-500 and Avance III-600 MHz spectrometers (Bruker, Zürich, Switzerland) with the solvent signal as internal reference. Mass spectra were collected from a VG Autospec-3000 spectrometer. BRUKER Tensor-27 instrument were used to record infrared spectra (with KBr pellets). X-ray diffraction was realized on a Bruker SMART APEX Cimyunnins B and C Single Crystal Cultivation. The clear solution of the pair of epimers (Cimyunnins B and C, 1.2 mg) in MeOH (5 mL) was added several drops of water, and then was kept at ambient temperature for slow evaporation to cultivate a single crystal suitable for X-ray crystallographic measurement. ECD Calculation. The theoretical calculations of compound 1 were performed using Gaussian 09 34 . Conformational analysis was initially carried out using Maestro7.5 conformational searching, together with the OPLS_2005 molecular mechanics methods. The optimized conformation geometries and thermodynamic parameters of the predominant conformations were provided (see computational data for 1 in Supplementary Information). The OPLS_2005 conformers were optimized at B3LYP/6-31G(d,p) level. The theoretical calculation of ECD was performed using time dependent Density Functional Theory (TDDFT) at B3LYP/6-31G(d,p) level in methanol with PCM model. The ECD spectra of compound 1 were obtained by weighing the Boltzmann distribution rate of each geometric conformation 35 . The ECD spectra were simulated by overlapping Gaussian functions for each transition according to: The s represented the width of the band at 1/e height, and DE i and R i were the excitation energies and rotational strengths for transition i, respectively. s 5 0.30 eV and R vel had been used in this work.
NMR calculation. For the calculations of 13 C NMR chemical shifts, B3LYP/6-31G(d,p) method was used to optimize the selected conformations. For all optimized structures, vibrational spectra were calculated to ensure that no imaginary frequencies for energy minimum were obtained. NMR calculations were performed at the levels of B3LYP/6-31G(d,p) with the gauge-independent atomic orbital (GIAO) method [36][37][38] . The solvent effect was considered by using pyridine in the calculations to resemble the experimental condition. The polarized continuum model (PCM) of Tomasi et al. was used [39][40][41] . The calculated 13 C NMR chemical shifts were analyzed by subtracting the isotopic shifts for TMS calculated with the same methods [36][37][38] . Different conformers for Lactone A and Lactone B were considered. The 13 C NMR chemical shifts in each compound were considered as the average values of the same atoms in the different conformers (see computational data for 4 in Supplementary  Information). The average values were obtained by the Boltzmann distributions, using the relative Gibbs free energies as weighting factors 35  Cell Proliferaion Assay. HUVECs were seeded in 96-well plates and incubated for 24 h, cells were then starved in M200 medium containing 2% FBS for another 16 h. After starvation, cells were pretreated for 30 min with indicated concentration of compound 1 (1, 10, 20, 30 mmol/L), followed by the stimulation with VEGF (25 ng/ mL) for another 24 h. Cell viability was then determined by MTT assay.
Wound-healing Migration Assay. HUVECs were seeded and grown into full confluence in 6 well plates. Cells were starved with 2% FBS M200 media for 12 h to inactivate cell proliferation and then wounded by pipette tips. Fresh M200 medium with 25 ng/mL VEGF containing vehicle or 2.5 mmol/L sunitinib and compound 1 was added to the scratched monolayers. Images were taken after 0, 6, 12, 24 hours using an inverted microscope (magnification, 103; Nickon). Sunitinib used as a positive control.
CAM Assay in Fertilized Chicken Eggs. The effect of compound 1 on ex vivo angiogenesis was determined by CAM assay. Briefly, fertile leghorn chicken eggs (Poultry Breeding farm, Kunming) were incubated in incubator at 37.8uC with 40% humidity. A small opening was made at the top of live eggs on day 7 under aseptic conditions. Indicated concentrations of compound 1 and sunitinib was mixed with DMSO and tipped on the filter paper, then gently placed on the CAM. The eggs were incubated for 48 h, then fixed with methanol and photographed.