Discovery of a Potent Antiosteoporotic Drug Molecular Scaffold Derived from Angelica sinensis and Its Bioinspired Total Synthesis

Abstract

Angelica sinensis, commonly known as Dong Quai in Europe and America and as Dang-gui in China, is a medicinal plant widely utilized for the prevention and treatment of osteoporosis. In this study, we report the discovery of a new category of phthalide from Angelica sinensis, namely falcarinphthalides A and B (1 and 2), which contains two fragments, (3R,8S)-falcarindiol (3) and (Z)-ligustilide (4). Falcarinphthalides A and B (1 and 2) represent two unprecedented carbon skeletons of phthalide in natural products, and their antiosteoporotic activities were evaluated. The structures of 1 and 2, including their absolute configurations, were established using extensive analysis of NMR spectra, chemical derivatization, and ECD/VCD calculations. Based on LC-HR-ESI-MS analysis and DFT calculations, a production mechanism for 1 and 2 involving enzyme-catalyzed Diels-Alder/retro-Diels-Alder reactions was proposed. Falcarinphthalide A (1), the most promising lead compound, exhibits potent in vitro antiosteoporotic activity by inhibiting NF-κB and c-Fos signaling-mediated osteoclastogenesis. Moreover, the bioinspired gram-scale total synthesis of 1, guided by intensive DFT study, has paved the way for further biological investigation. The discovery and gram-scale total synthesis of falcarinphthalide A (1) provide a compelling lead compound and a novel molecular scaffold for treating osteoporosis and other metabolic bone diseases.

Figure 1

Chemical structures of 1−4.

Figure 2

Key ¹H−¹H COSY and HMBC correlations of 1.

Figure 3

(A) Experimental ECD spectrum of 1, and calculated ECD spectra of (3′R,8′S)-1′, (3′R,8′R)-1′, (3′S,8′R)-1′, and (3′S,8′S)-1′ (UV correction = −18 nm, bandwidth σ = 0.3 eV). (B) Experimental VCD spectra of 1 and 1a and calculated VCD spectra of (3′R,8′S)-1′a and (3′R,8′R)-1′a in the CDCl₃ solution.

Figure 4

(A) The plausible modes of formation of compound 1. (B) DFT-calculated energies profiles for Diels−Alder/retro-Diels−Alder reactions of 1 via TS-1 and TS-2. Free energies are given in kcal mol⁻¹.

Figure 5

Compounds 1, 3, and 4, but not 2, inhibit osteoclast formation and bone resorption via suppressing osteoclastogenesis. (A) Typical images of tartrate-resistant acid phosphatase (TRAP) staining cells treated with different dosages of 1−4 and positive control (pamidronate; 20 μM) for receptor activator of nuclear factor-κB ligand (RANKL) and macrophage colony stimulating factor (M-CSF) induced 4 days. TRAP staining-positive (TRAP⁺) multinucleated cells (MNCs, nuclei ≥3) were counted as osteoclasts. Scale bar = 100 μm. Red arrow: osteoclast. (B) Number of TRAP⁺ MNCs in each field. (C) The total area of MNCs per well relative to control. (D) Typical images of F-actin ring treated with 1−4 (10 μM) and positive control (pamidronate; 20 μM). Scale bar = 100 μm. White arrow: F-actin ring. (E) Quantitative analyses of size of F-actin rings in each field. (F) Typical images of resorption pits per well (96 well plate) treated with 1−4 (10 μM) and positive control (pamidronate; 20 μM). Red arrow: resorbed pits. Scale bar = 1000 μm. (G) Quantitative analyses of the percentage of the area of pits resorbed by osteoclasts in each well relative to control. The data were expressed as mean ± SD * P < 0.05, ** P < 0.01, ***P < 0.001 vs Control; n ≥ 3. NC: Control without RANKL-induced; C: Control with RNAKL and M-CSF induced; TRAP: tartrate-resistant acid phosphatase; Pam: pamidronate; C1−C4: Compounds 1−4.

Figure 6

Compounds 1, 3, and 4 block osteoclastogenesis via suppressing RANKL-induced activation of of NF-κB/c-Fos signaling. (A) Analysis of protein expression and (B−E) relative mRNA levels of osteoclastogenesis-related markers in RAW264.7 cells cultured in the presence or absence of compounds 1−4 with RANKL and M-CSF. (F) Confocal microscopy images showing the nuclear translocation of NF-κB p65 in RANKL-induced RAW264.7 cells after 1 h incubation, with or without pretreatment using compounds 1−4 (10 μM) for 4 h. NF-κB p65 is represented in green, while cell nuclei (DAPI) are shown in blue. Scale bar = 20 μm. (G) Quantification of mean per-pixel fluorescence intensity (MFI) of NF-κB p65 in the nucleus. (H) A proposed scheme illustrating the inhibitory effects of compounds 1, 3, and 4 on osteoclastogenesis. Data are expressed as mean ± SD *P < 0.05, **P < 0.01, ***P < 0.001 vs control. n ≥ 3.

Figure 7

(A) Comparison of esteryl and silanyl activations. (B) DFT-calculated transition states for the proposed Diels−Alder reaction (only the transition states with β-face selectivity are shown for clarity). (C) Gram-scale total synthesis of falcarinphthalide A (1).