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. 2026 Feb 16;19(2):327. doi: 10.3390/ph19020327

Design, Synthesis and Biological Evaluation of Novel Furanocoumarin Derivatives: Validation of Anti-Osteoporotic Efficacy In Vitro and In Vivo

Xiaoming Chen 1, Shuirong Chen 2,3, Qinhan Gao 3, Gang Li 3, Yan Geng 3, Xudong Qian 3, Hongliang Yao 3,*, Qibiao Wu 1,4,*, Jingjing Zhang 1,5,*
Editor: Emmanuel Moreau
PMCID: PMC12944601  PMID: 41754866

Abstract

Background/Objectives: Osteoporosis is a metabolic bone disease characterized by reduced bone mass and impaired bone microarchitecture. It has become a major clinical challenge due to the limitations of current therapeutic approaches. Isoimperatorin (ISO), a naturally occurring and biologically active furanocoumarin extracted from various traditional herbals, exhibits therapeutic potential in combating osteoporosis. However, toxicity limits its application. Methods: In vivo, compound B15 was evaluated in an Ovariectomy (OVX) mice model, where treatment was associated with changes in bone microarchitecture parameters, modulation of serum bone metabolism markers, and alterations in the histopathological features of bone tissue. Results: In this study, a new series of furanocoumarin derivatives was designed and synthesized for the treatment of osteoporosis. Compared with ISO, compound B15 has less toxicity and better ability to inhibit osteoclast formation in vitro. Compound B15 could decelerate the progression of osteoporosis in ovariectomized mice. It is worth mentioning that the expression of estradiol in the serum of mice with excised ovaries was significantly increased by compound B15. Conclusions: These results imply that the novel furanocoumarin derivative B15 is a promising therapeutic option for osteoporosis.

Keywords: osteoporosis, osteoclastogenesis, furanocoumarin derivatives

1. Introduction

Osteoporosis (OP) is the most common metabolic disease of the skeletal system and is characterized by decreased bone mineral density and deterioration of bone microarchitecture, leading to an increased risk of fragility fractures [1,2]. With the acceleration of global population aging, the incidence of osteoporosis continues to rise, and osteoporotic fractures are associated with substantial morbidity, mortality, and a heavy socioeconomic burden [3,4]. Osteoporosis represents an intricate and multifaceted disease, and its therapeutic approach encompasses a broad spectrum of small-molecule medications. At present, pharmacological treatment of osteoporosis mainly relies on anti-resorptive agents and bone-forming agents [5,6,7,8,9]. Currently, a diverse range of pharmacological agents are employed in the treatment of osteoporosis, including vitamin D, calcium, estrogen, parathyroid hormone, bisphosphonates, selective estrogen receptor modulators (SERMs), and calcitonin. Although these therapies are effective in reducing fracture risk, their long-term application is limited by safety concerns and poor patient adherence. Bisphosphonate drugs have gastrointestinal irritation and serious side effects. Selective estrogen receptor modulators increase the risk of venous thrombosis and are only applicable to women. Hormone replacement therapy increases the risks of cancer and cardiovascular diseases. Therefore, the development of alternative therapeutic strategies with improved safety and efficacy remains an important objective.

Plant-derived bioactive compounds have long been recognized as an important resource for drug discovery owing to their favorable efficacy and safety profiles [10]. Among them, isoimperatorin (ISO) is a linear furanocoumarin predominantly found in medicinal plants, such as Salvia miltiorrhiza and Angelica dahurica, and has attracted increasing attention due to its diverse pharmacological activities, including anti-inflammatory, antioxidant, and immunomodulatory effects [11,12,13]. Previous studies have demonstrated that ISO inhibits osteoclast differentiation and function, alleviates excessive bone resorption, and prevents estrogen deficiency-induced bone loss [14].

In this study, 36 compounds were designed and synthesized with ISO as the lead compound. Among them, compound B15 was superior to ISO in terms of safety and inhibition of osteoclast formation. It is worth mentioning that the compound was able to increase the level of Estradiol (E2) in the serum of Ovariectomy (OVX) mice, which may provide a new direction for the treatment of postmenopausal osteoporosis in clinical practice.

2. Results

2.1. Structure-Based Molecular Design

Previous research has revealed that isoimperatorin was found to diminish the secretion of proinflammatory factors and suppress receptor activator for nuclear factor-κB ligand-triggered osteoclast differentiation in periodontal membrane cells. In this work, we took the furanocoumarin of isoimperatorin as the scaffold and changed the alkyl group of isoimperatorin at different positions with various flexible chains or rigid chains, leading to the design of two series of isoimperatorin derivatives (Figure 1). Compared with ISO, compound B15 has been shown to have less toxicity and better bioactivity. In addition, compound B15 has the potential to decelerate the progression of osteoporosis in ovariectomized mice.

Figure 1.

Figure 1

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Fragment-based molecular design of novel furanocoumarin derivatives.

2.2. Chemistry

All target compounds A1–A19 and B1–B17 were successfully prepared through the synthetic protocols presented in Scheme 1. Intermediate 3 was obtained by demethylation of starting material 2. The spectra including 1H NMR, 13C NMR and HRMS are summarized in in Figure S1 of the Supplementary Materials. The purity of synthesized compounds was higher than 95%, as analyzed by High-Performance Liquid Chromatography (HPLC, Thermo-UltiMate 3000, UV detection at 254 nm) on a C18 column (4.6 mm × 250 mm, 5 μm).

Scheme 1.

Scheme 1

Scheme 1

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Synthetic routes of A1–A19 and B1–B17. Reagents and conditions: (a) RBr, K2CO3, DMF, 80 °C, 8 h; (b) BBr3, DCM, N2, 0 °C, 1 h, 0 °C to rt, 3 h.

2.3. Initial Screening of Cellular Activity of the Synthesized Compounds

To evaluate compound toxicity, cell viability was measured, and BDMDs were isolated from the bone marrow of healthy C57BL/6 mice. As shown in Figure 2A, except for 10 compounds (including A2, A8, and others), which were slightly toxic, the rest of the compounds had no obvious toxicity. It was found that methyl carbonate or a larger quinoline ring in series A, as well as cyclopentylmethoxyl, tetrahydropyranylmethoxyl, pyridinylmethoxyl, pyrrolidinonemethoxyl, methoxy-benzyloxyl and long-chain acid ether groups in series B, resulted in relatively high toxicity. At the same time, it was observed that compound B5 with an allyl group in R2, which shares a similar molecular structure with ISO, exhibited influence on cell viability. On the contrary, compound A5 demonstrated good safety.

Figure 2.

Figure 2

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Initial screening of cellular activity of the synthesized compounds. (A) Potential toxicity of different compounds (10 μM) in BMDMs. (B) qRT-PCR analysis of c-fos mRNA expression after being treated with Alendronate (Alen-Na, 1 μM), ISO (1 μM) or different synthesized compounds (10 μM) in RANKL-induced BMDMs. *** p < 0.001, ** p < 0.01, * p < 0.05 vs. the Control group; ### p < 0.001 vs. the RANKL group. Data are presented as mean ± SEM (n = 3).

Subsequently, non-toxic compounds were detected for the expression of c-fos mRNA (Figure 2B). Among the remaining compounds in series A, compounds with flexible linear amine had good activity in RANKL-induced BMDMs. And pyridinylmethoxyl, tetrahydropyranylmethoxyl groups also contributed to some extent. Regarding series B, even though the linear acetate group in compound B12 increased activity slightly, compound B15 with boc-piperidine showed the best ability to inhibit the expression of c-fos. The specific values are shown in Table S2.

Studies have shown that the expression of TNF-α is increased in osteoporosis [15,16,17,18]. In addition, some work reported that ISO can effectively reduce the expression of TNF-α [19,20,21]. In order to select the compound for further research, the expression of TNF-α was detected by qRT-PCR. As shown in Figure S2, the expression of TNF-α could be effectively reduced by compound B15, but the effect of A16 is minimal. Combined with the results of CCK8 and qRT-PCR, compound B15 was selected for further evaluation.

2.4. Cellular Activity of Compound B15

To explore the safety of compounds, cell viability of precursor compound ISO and compound B15 was compared (Figure 3A,B). No significant toxicity was observed for compound B15, but ISO showed significant toxicity at a dose of 5 μM in BDMDs.

Figure 3.

Figure 3

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Cellular activity of compound B15. (A) Potential toxicity of compound B15 at concentrations of 0, 0.01, 0.1, 0.5, 1, 5, 10 and 15 μM in BMDMs. (B) Potential toxicity of compound ISO at concentrations of 0, 0.01, 0.1, 0.5, 1, 5, 10 and 15 μM in BMDMs. (C) TRAP staining of BMDMs treated with compound B15 at concentrations of 0, 10, 20, 30, 35, 40, 45 and 50 nM. (D) The area of osteoclast treated with compound B15 in RANKL-induced BMDMs. (E) Inhibition of osteoclast formation by B15 in RANKL-stimulated BMDMs. (F) TRAP staining of BMDMs treated with ISO (0, 10, 20, 30, 35, 40, 45 and 50 nM). (G) The area of osteoclast treated with ISO in RANKL-induced BMDMs. (H) Inhibition of osteoclast formation by ISO in RANKL-stimulated BMDMs. Data are presented as mean ± SEM (n = 3).

Osteoclast hyperfunction is one of the main pathological features of osteoporosis, and its increased activity leads to increased bone resorption and decreased bone mass, which provokes osteoporosis [14]. Tartaric acid phosphatase (TRAP) is a marker enzyme of osteoclasts. It is specifically distributed in and unique to osteoclasts and is usually used as an important marker for their identification [22]. To explore the ability to inhibit osteoclast formation, TRAP staining was performed. As shown in Figure 3C–H, the area of osteoclasts was significantly reduced by compound B15, and the IC50 of compound B15 for inhibiting osteoclast formation was 37.86 nM, while the IC50 of ISO for inhibiting osteoclast formation was greater than 50 nM. The results show that compound B15 has a better safety profile and stronger ability to inhibit osteoclast formation than ISO. Based on the IC50 value determined from TRAP staining analysis, compound B15 was applied at a concentration of 40 nM in subsequent experiments.

2.5. The Ability of Compound B15 to Inhibit Osteoclast Formation In Vitro

Matrix Metalloproteinase-9 (MMP9) is a group of zinc-containing intraionic peptidases activated by calcium ions [12]. MMP9 is specifically expressed in osteoclasts and promotes osteoporosis formation, which may play an important role in osteoclast bone resorption [23]. Elevated expression of dendritic cell-specific transmembrane protein (Dc-stamp) and Nuclear Factor Activated T Cells 1 (NFATc1) is one of the causes of osteoporosis [24]. To further explore the ability of compound B15 to inhibit osteoclast formation, the mRNA expression levels of osteoclast markers were detected.

As shown in Figure 4A–C, the increases in MMP9, Dc-stamp and NFATc1 expressions in BMDMs caused by RANKL were significantly reduced by B15. F-actin fluorescence staining was also detected (Figure 4D–F). Compared with the Control group, the actin ring in the RANKL group exhibited a larger ring structure and a higher number of nuclei within the rings. The area of the actin ring in the B15 group was significantly reduced, and the number of nuclei in the rings was also reduced. The results show that the formation of osteoclasts was significantly inhibited by compound B15.

Figure 4.

Figure 4

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The ability of compound B15 to inhibit osteoclast formation in vitro. qRT-PCR analysis of MMP9 (A), Dc-stamp (B) and NFATc1 (C) mRNA expression after being treated with Alen-Na (1 μM), ISO (1 μM) or compound B15 (40 nM) in RANKL-stimulated BMDMs. (D–F) F-actin ring staining results and statistics. *** p < 0.001, * p < 0.05 vs. the Control group; ### p < 0.001, # p < 0.05 vs. the RANKL group. Data are presented as mean ± SEM (n = 3).

2.6. Effects of Compound B15 in the Ovariectomy (OVX) Mice Model

In vivo, compound B15 was evaluated in an Ovariectomy (OVX) mice model, where treatment was associated with changes in bone microarchitecture parameters, modulation of serum bone metabolism markers, and alterations in the histopathological features of bone tissue.

The model was established by removing the ovaries of mice, and the experiment process is shown in the Figure 5A. There was no significant change in the body weight of the mice in each group (Figure 5B). After the mice were sacrificed, micro-CT of the femurs was performed, and the results are shown in Figure 5C. According to the statistical results of micro-CT (Figure 5D–F), BV/TV and Tb.Th were decreased, and BS/BV was increased after the ovaries were removed from the mice. These changes were reversed by compound B15. This indicates that the progression of osteoporosis could be delayed by compound B15 in vivo.

Figure 5.

Figure 5

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Effects of compound B15 in the OVX mice model. (A) The flow chart for modeling and treatment of OVX mice. After 7 days of adaptive feeding, the OVX mice model was established. Two weeks later, gavage treatment was carried out for 4 weeks (once a day). Finally, the tissue of mice was collected and tested. (B) The weight of mice. (C–F) Images and evaluation of micro-CT of mice femur. * p < 0.05 vs. the Sham group; ### p < 0.001, ## p < 0.01, #p < 0.05 vs. the OVX group. Data are presented as mean ± SEM (n = 7).

2.7. Biochemical Test Results of Mice Serum from OVX Mice

The biochemistry of mice was tested to explore the potential toxicity of compound B15. As shown in Figure 6A–F, the expressions of alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), cardiac troponin (cTN1) and C-reactive protein (CRP) were not affected by compound B15. However, the expression of Estradiol (E2) was increased after being treated with compound B15 (Figure 6G). This suggests that compound B15 may be effective in treating osteoporosis by increasing the expression of estrogen.

Figure 6.

Figure 6

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Biochemical test results of mice serum from OVX mice. (A) ALT: alanine aminotransferase, (B) AST: aspartate aminotransferase, (C) BUN: blood urea nitrogen, (D) CRP: C-reactive protein, (E) CK-MB: Creatine Kinase MB Isoenzyme, (F) cTnI: cardiac troponin, (G) E2: Estradiol. # p < 0.05 compared to OVX group. Each value is represented as mean ± SEM (n = 7).

3. Discussion

3.1. Initial Screening of Cellular Activity of the Synthesized Compounds

Osteoporosis is a chronic bone disease characterized by reduced bone tissue quality and density, leading to bone fragility and an increased risk of fractures, especially in the elderly, further increasing mortality [25,26]. Therefore, treatment of osteoporosis is urgently needed. In the pathogenesis of osteoporosis, osteoclast formation is considered as one of the main triggers [22].

Isoimperatorin (ISO) is a linear furanocoumarin predominantly found in medicinal plants, such as Salvia miltiorrhiza and Angelica dahurica, and has attracted increasing attention due to its diverse pharmacological activities, including anti-inflammatory, antioxidant, and immunomodulatory effects. Previous studies have demonstrated that ISO inhibits osteoclast differentiation and function, alleviates excessive bone resorption, and prevents estrogen deficiency–induced bone loss [14].

According to ISO, 36 compounds were designed and synthesized. The premise of treating osteoporosis is that the compound has a good safety profile. Cell viability assays were performed to exclude compounds with cytotoxic effects (Figure 2A), after which the remaining derivatives were evaluated in BMDMs. A total of 23 compounds without significant toxicity were tested for osteoporosis-related genes.

Cellular proto-oncogene FOS (c-fos) is an immediate early gene that is usually rapidly expressed after cell stimulation. It plays an important role in cell proliferation, differentiation, and apoptosis [27]. In patients, the expression level of the c-fos gene was significantly increased. This suggests that c-fos is closely related to osteoporosis and plays an important role in its pathogenesis. Quantitative real-time PCR analysis demonstrated that treatment with selected compounds reduced the mRNA expression level of c-fos (Figure 2B). These results suggest that compound B15 exhibits good safety and bioactivity to reduce c-fos.

3.2. Cellular Activity of Compound B15

Compounds B15 and ISO were simultaneously subjected to cytotoxicity experiments. The results show that compound B15 is safer, which may reduce the probability of harm to the human body and reduce side effects when treating osteoporosis, relative to ISO (Figure 3A,B). Osteoclasts are multinucleated cells in bone tissue that perform the function of bone resorption [28]. In the pathogenesis of osteoporosis, increased osteoclast activity and number, which lead to increased bone resorption, are key factors [29,30]. The occurrence of osteoporosis is associated with abnormal osteoclast activity. Overactive osteoclasts accelerate the breakdown of bone tissue, reducing the deposition of minerals like calcium, leading to reduced bone density [29,30].

TRAP is the signature enzyme of osteoclasts. In this study, TRAP staining was used to explore the ability of compounds to inhibit osteoclast formation. As shown in Figure 3C–E, the area of osteoclasts was significantly reduced by compound B15. The results show that the IC50 of compound B15 for inhibiting osteoclasts was 37.86 nM. However, the IC50 of ISO for inhibiting osteoclasts was greater than 50 nM (Figure 3F–H). This suggests that the modified compound has a stronger ability to inhibit osteoclasts.

3.3. The Ability of Compound B15 to Inhibit Osteoclast Formation In Vitro

Osteoclasts are responsible for bone resorption. When osteoclasts attach to the bone surface, the actin ring formed by cytoskeletal reorganization is closely connected to the bone surface. Osteoclasts then transport osteoclast-related functional proteins, such as MMP9, and internalize bone degradation products through vesicles [31,32]. Dc-stamp is a key protein that regulates cell fusion during osteoclast production [33]. Disruption of the Dc-stamp domain impairs its function, leading to an increase in bone mass. The role of nFATc1 in osteoporosis is mainly reflected in its role as a major regulator of osteoclast differentiation and gene expression [24]. Its expression and activity were regulated by RANK-RANKL, NF-κB, MAPK, Ca2+, etc. [34]. Inhibition of NFATc1 expression reduces osteoclast formation and aids in the treatment of osteoporosis. In this work, the mRNA expressions of MMP9, Dc-stamp and nFATc1 were detected. The results show that the expression of MMP9, Dc-stamp and nFATc1 could be decreased by compound B15 in RANKL-induced BMDMs (Figure 4A–C).

F-actin forms actin rings in osteoclasts and plays a decisive role in their bone resorption activity [35]. Disruption of the occlusive band of osteoclasts has been reported to lead to inhibition of osteoclast bone resorption function. The area of the actin ring was significantly reduced and the number of nuclei within the ring was also reduced by compound B15 (Figure 4D–F). It is worth mentioning that compared to Alen-Na, compound B15 showed a better ability to inhibit osteoclast formation, even though the dose of compound B15 was lower in the experiment. This further suggests that compound B15 could inhibit osteoclast formation.

3.4. Effects of Compound B15 in the OVX Mice Model

OVX model mainly reflects the characteristics of bone loss and bone metabolism in the early stages of postmenopausal osteoporosis in women. It has now become a classic model for studying postmenopausal osteoporosis (PMOP) and is widely used for preclinical research. After the osteoporosis model was established, the effects of different compounds on mice were roughly observed by measuring their weight. Compared to Alen-Na, compound B15 showed better safety, according to the weight results of mice (Figure 5B). Micro-CT is one of the most intuitive and accurate methods for detecting osteoporosis. BV/TV and BS/TV reflect bone mass. Tb.Th is one of the main indicators for evaluating the spatial morphological structure of trabecular bones. In osteoporosis, bone catabolism is greater than bone anabolism, BV/TV and BS/TV are increased, and Tb.Th is decreased [36]. This situation was partially reversed by compound B15 (Figure 5C–F). In terms of restoring bone structure, the effect of compound B15 was comparable to Alen-Na in some indicators, such as BS/TV. These results indicate that osteoporosis caused by removal of the ovaries could be treated by compound B15.

3.5. Biochemical Test Results of Mice Serum from OVX Mice

Potential toxicity is not allowed in drug development [37]. Thus, hematological tests were completed. ALT is mainly found in liver cells and is one of the important indicators for evaluating liver function and detecting liver damage. AST is mainly used to assess the health of tissues such as the liver and heart muscle. BUN is an indicator that reflects kidney function. CRP plays an important role in the inflammatory response and is one of the commonly used inflammatory markers in clinical practice. CK-MB is one of the most sensitive indicators for early diagnosis of acute myocardial infarction in clinical practice. cTNI is a biomarker of myocardial damage. Relevant indicators in serum were detected (Figure 6A–F). The results show that compound B15 had little effect on various indicators in mice, indicating that compound B15 has good safety in vivo.

E2 is a hormone secreted by the ovaries and plays a vital role in the female reproductive system. The expression of E2 was increased by compound B15 (Figure 6G), suggesting that compound B15 may be helpful in treating postmenopausal osteoporosis.

4. Materials and Methods

4.1. Reagents and Equipments

Reagents and solvents were commercially purchased. The progress of the reaction was monitored by thin-layer chromatography analysis using silica gel G plates through a UV chamber at 254 nm for visualization of TLC spots. The mixtures were purified by flash column chromatography using silica gel. Both 1H NMR and 13C NMR were recorded on Bruker 400, 500, or 600 spectrometers (German Bruker Company). Abbreviations for signal coupling are as follows: s, singlet; d, doublet; t, triplet; m, multiplet, and br, broad. High-resolution mass spectra were measured using AB Sciex Triple TOF™ 5600 plus (AB SCIEX USA).

Synthesis of Intermediate 3: First, 4.85 g (22.43 mmol, 1.0 equiv) of 4-methoxy-7H-furo[3,2-g]chromen-7-one (compound 2) was dissolved in 50 mL anhydrous dichloromethane. Then, BBr3 (67.30 mmol 3.0 equiv) was added slowly under nitrogen protection at 0 °C for 1 h, and the reaction was stirred at room temperature for 3 h. After the thin-layer chromatography monitoring reaction was completed, the reaction solution was slowly poured into pre-cooled water in a fume hood to precipitate the solid to obtain compound 3 (4.5 g, 22.26 mmol) as a yellow solid.

4-hydroxy-7H-furo[3,2-g]chromen-7-one (3): Yellow solid; Yield: 99%; 1H NMR (400 MHz, CD3OD) δ 8.32 (d, J = 9.6 Hz, 1H), 7.70 (d, J = 2.4 Hz, 1H), 7.05 (dd, J = 2.4, 1.2 Hz, 1H), 7.02 (s, 1H), 6.22 (d, J = 9.6 Hz, 1H). 13C NMR (101 MHz, CD3OD) δ 163.8, 159.3, 154.4, 149.4, 146.0, 141.6, 114.1, 111.6, 105.5, 105.0, 92.3. ESI-HRMS m/z: calculated for C11H7O4+ [M + H]+, 203.0266; found, 203.0335.

General Procedure for Synthesis of A1–A19 and B1–B17: To a solution of 9-hydroxy-7H-furo[3,2-g]chromen-7-one (compound 1, 50 mg, 0.25 mmol, 1.0 equiv) in DMF (2 mL), potassium carbonate (136.7 mg, 0.99 mmol, 4.0 equiv) and the corresponding bromide (0.27 mmol, 1.1 equiv) were added. The resulting mixture was stirred for 8 h at 80 °C. Water (5 mL) was added and diluted by addition of ethyl acetate (15 mL). The organic layer was separated, and the aqueous layer was extracted with ethyl acetate (3 × 15 mL). The combined organic fractions were washed with brine, dried with anhydrous Na2SO4, and concentrated under reduced pressure. The residue was purified by flash chromatography (PE/EA = 9:1) on silica gel to give compounds A1–A19. Compounds B1–B17 were synthesized following an analogous procedure (PE/EA = 9:1), employing intermediate 3 as the starting material in place of compound 1.

9-(cyclopentylmethoxy)-7H-furo[3,2-g]chromen-7-one (A1): White solid; yield: 43%; 1H NMR (400 MHz, CDCl3) δ 7.76 (d, J = 9.6 Hz, 1H), 7.69 (d, J = 2.4 Hz, 1H), 7.35 (s, 1H), 6.81 (d, J = 2.4 Hz, 1H), 6.37 (d, J = 9.6 Hz, 1H), 4.35 (d, J = 7.2 Hz, 2H), 2.51–2.39 (m, 1H), 1.93–1.84 (m, 2H), 1.68–1.63 (m, 2H), 1.61–1.56 (m, 2H), 1.51–1.42 (m, 2H). 13C NMR (101 MHz, CDCl3) δ 160.8, 148.5, 146.8, 144.5, 143.7, 132.4, 126.1, 116.7, 114.9, 113.1, 106.8, 78.4, 40.0, 29.4 (2 × C), 25.6 (2 × C). ESI-HRMS m/z: calculated for C17H17O4+ [M + H]+, 285.1121; found, 285.1120. HPLC: tR 6.053 min, purity 99.4%.

9-(cyclopropylmethoxy)-7H-furo[3,2-g]chromen-7-one (A2): White solid; yield: 66%; 1H NMR (400 MHz, CD3OD) δ 8.00 (d, J = 9.6 Hz, 1H), 7.86 (d, J = 2.4 Hz, 1H), 7.52 (s, 1H), 6.93 (d, J = 2.4 Hz, 1H), 6.36 (d, J = 9.6 Hz, 1H), 4.26 (d, J = 7.2 Hz, 2H), 1.35–1.28 (m, 1H), 0.60–0.53 (m, 2H), 0.37–0.30 (m, 2H). 13C NMR (101 MHz, CD3OD) δ 162.8, 149.7, 148.4, 146.7, 144.7, 132.8, 127.8, 118.0, 114.9 (2 × C), 107.9, 79.6, 11.9, 3.5 (2 × C). ESI-HRMS m/z: calculated for C15H13O4+ [M + H]+, 257.0808; found, 257.0808. HPLC: tR 4.710 min, purity 100.0%.

methyl 2-((7-oxo-7H-furo[3,2-g]chromen-9-yl)oxy)acetate (A3): White solid; yield: 56%; 1H NMR (400 MHz, DMSO-d6) δ 8.11 (d, J = 9.6 Hz, 1H), 8.07 (d, J = 2.4 Hz, 1H), 7.63 (s, 1H), 7.05 (d, J = 2.4 Hz, 1H), 6.41 (d, J = 9.6 Hz, 1H), 5.16 (s, 2H), 3.66 (s, 3H). 13C NMR (101 MHz, DMSO-d6) δ 169.6, 160.0, 148.4, 146.4, 145.7, 142.1, 130.6, 126.3, 116.8, 114.7, 114.3, 107.5, 68.9, 52.4. ESI-HRMS m/z: calculated for C14H11O6+ [M + H]+, 275.0550; found, 275.0551. HPLC: tR 3.190 min, purity 100.0%.

9-((4-methoxybenzyl)oxy)-7H-furo[3,2-g]chromen-7-one (A4): White solid; yield: 38%; 1H NMR (400 MHz, DMSO-d6) δ 8.15–8.11 (m, 2H), 7.67 (s, 1H), 7.44–7.40 (m, 2H), 7.09 (d, J = 2.4 Hz, 1H), 6.94–6.89 (m, 2H), 6.43 (d, J = 9.6 Hz, 1H), 5.39 (s, 2H), 3.73 (s, 3H). 13C NMR (101 MHz, DMSO-d6) δ 159.8, 159.3, 147.9, 147.5, 145.3, 143.0, 130.5, 130.1 (2 × C), 128.6, 125.7, 116.4, 114.3, 114.2, 113.8 (2 × C), 107.1, 74.4, 55.1. ESI-HRMS m/z: calculated for C19H15O5+ [M + H]+, 323.0914; found, 323.0911. HPLC: tR 5.310 min, purity 99.6%.

9-(allyloxy)-7H-furo[3,2-g]chromen-7-one (A5): White solid; yield: 69%; 1H NMR (400 MHz, DMSO-d6) δ 8.14 (d, J = 14.0 Hz, 1H), 8.13 (d, J = 2.4 Hz, 1H), 7.69 (s, 1H), 7.10 (d, J = 2.0 Hz, 1H), 6.44 (d, J = 10.0 Hz, 1H), 6.16–6.03 (m, 1H), 5.45–5.37 (m, 1H), 5.27–5.21 (m, 1H), 4.95 (t, J = 1.6 Hz, 1H), 4.93 (t, J = 1.6 Hz, 1H). 13C NMR (101 MHz, DMSO-d6) δ 159.8, 147.9, 147.4, 145.3, 142.9, 133.7, 130.4, 125.8, 118.6, 116.4, 114.3, 114.2, 107.1, 73.7. ESI-HRMS m/z: calculated for C14H11O4+ [M + H]+, 243.0652; found, 243.0641. HPLC: tR 4.990 min, purity 97.3%.

9-((6-methylpyridin-2-yl)methoxy)-7H-furo[3,2-g]chromen-7-one (A6): White solid; yield: 80%; 1H NMR (400 MHz, DMSO-d6) δ 8.16 (d, J = 9.6 Hz, 1H), 8.13 (d, J = 2.4 Hz, 1H), 7.76 (t, J = 7.6 Hz, 1H), 7.71 (s, 1H), 7.51 (d, J = 7.6 Hz, 1H), 7.22 (d, J = 7.6 Hz, 1H), 7.11 (d, J = 2.4 Hz, 1H), 6.45 (d, J = 9.6 Hz, 1H), 5.50 (s, 2H), 2.44 (s, 3H). 13C NMR (101 MHz, DMSO-d6) δ 159.7, 157.4, 155.7, 148.0, 147.2, 145.3, 142.8, 137.4, 130.7, 125.9, 122.6, 118.8, 116.5, 114.5, 114.3, 107.1, 75.4, 23.8. ESI-HRMS m/z: calculated for C18H14O4N+ [M + H]+, 308.0917; found, 308.0926. HPLC: tR 2.737 min, purity 97.5%.

9-((tetrahydro-2H-pyran-4-yl)methoxy)-7H-furo[3,2-g]chromen-7-one (A7): White solid; yield: 57%; 1H NMR (400 MHz, DMSO-d6) δ 8.14 (d, J = 12.0 Hz, 1H), 8.13 (s, 1H), 7.68 (s, 1H), 7.10 (d, J = 2.4 Hz, 1H), 6.43 (d, J = 9.6 Hz, 1H), 4.25 (d, J = 6.4 Hz, 2H), 3.93–3.84 (m, 2H), 3.34 (dd, J = 11.6, 2.4 Hz, 1H), 3.33 (d, J = 2.4 Hz, 1H), 2.09–1.97 (m, 1H), 1.81–1.72 (m, 2H), 1.45–1.31 (m, 2H). 13C NMR (101 MHz, DMSO-d6) δ 159.8, 147.9, 147.3, 145.3, 142.8, 131.2, 125.8, 116.5, 114.2, 114.1, 107.1, 78.0, 66.6 (2 × C), 35.4, 29.0 (2 × C). ESI-HRMS m/z: calculated for C17H17O5+ [M + H]+, 301.1071; found, 301.1072. HPLC: tR 4.337 min, purity 99.9%.

methyl 3-(((7-oxo-7H-furo[3,2-g]chromen-9-yl)oxy)methyl)benzoate (A8): White solid; yield: 24%; 1H NMR (400 MHz, DMSO-d6) δ 8.17–8.12 (m, 3H), 7.94–7.89 (m, 1H), 7.80–7.76 (m, 1H), 7.71 (s, 1H), 7.55 (t, J = 7.6 Hz, 1H), 7.11 (d, J = 2.4 Hz, 1H), 6.45 (d, J = 9.6 Hz, 1H), 5.54 (s, 2H), 3.86 (s, 3H). 13C NMR (101 MHz, DMSO-d6) δ 166.0, 159.7, 148.0, 147.4, 145.3, 143.0, 137.6, 132.8, 130.4, 129.8, 129.0 (2 × C), 128.6, 125.8, 116.5, 114.7, 114.3, 107.2, 74.1, 52.3. ESI-HRMS m/z: calculated for C20H15O6+ [M + H]+, 351.0863; found, 351.0861. HPLC: tR 6.460 min, purity 96.0%.

9-(pyridin-3-ylmethoxy)-7H-furo[3,2-g]chromen-7-one (A9): White solid; yield: 31%; 1H NMR (400 MHz, DMSO-d6) δ 8.69 (d, J = 2.0 Hz, 1H), 8.54 (dd, J = 4.8, 1.6 Hz, 1H), 8.15–8.11 (m, 2H), 7.93 (dt, J = 7.6, 2.0 Hz, 1H), 7.70 (s, 1H), 7.41 (ddd, J = 8.0, 4.8, 2.0 Hz, 1H), 7.09 (d, J = 2.4 Hz, 1H), 6.44 (d, J = 9.6 Hz, 1H), 5.50 (s, 2H). 13C NMR (101 MHz, DMSO-d6) δ 159.8, 149.7, 149.5, 148.1, 147.5, 145.4, 143.1, 136.2, 132.4, 130.4, 125.8, 123.7, 116.5, 114.8, 114.3, 107.3, 72.5. ESI-HRMS m/z: calculated for C17H12O4N+ [M + H]+, 294.0761; found, 294.0742. HPLC: tR 2.787 min, purity 99.5%.

3-(((7-oxo-7H-furo[3,2-g]chromen-9-yl)oxy)methyl)benzaldehyde (A10): White solid; yield: 16%; 1H NMR (400 MHz, DMSO-d6) δ 10.02 (s, 1H), 8.18–8.10 (m, 2H), 8.06 (t, J = 2.0 Hz, 1H), 7.87 (ddt, J = 13.6, 7.6, 1.6 Hz, 2H), 7.70 (s, 1H), 7.63 (t, J = 7.6 Hz, 1H), 7.10 (d, J = 2.0 Hz, 1H), 6.44 (d, J = 9.6 Hz, 1H), 5.56 (s, 2H). 13C NMR (101 MHz, DMSO-d6) δ 193.1, 159.8, 148.1, 147.5, 145.4, 143.1, 138.1, 136.4, 134.1, 130.5, 129.6, 129.5, 128.8, 125.9, 116.6, 114.8, 114.4, 107.3, 74.1. ESI-HRMS m/z: calculated for C19H13O5+ [M + H]+, 321.0758; found, 321.0755. HPLC: tR 4.223 min, purity 96.2%.

9-((3-methylbenzyl)oxy)-7H-furo[3,2-g]chromen-7-one (A11): White solid; yield: 70%; 1H NMR (400 MHz, DMSO-d6) δ 8.15–8.11 (m, 2H), 7.67 (s, 1H), 7.34 (s, 1H), 7.30–7.22 (m, 2H), 7.13 (d, J = 8.0Hz, 1H), 7.09 (d, J = 2.0 Hz, 1H), 6.43 (d, J = 9.6 Hz, 1H), 5.41 (s, 2H), 2.29 (s, 3H). 13C NMR (101 MHz, DMSO-d6) δ 159.9, 148.0, 147.6, 145.4, 143.1, 137.6, 136.7, 130.7, 129.0, 128.9, 128.4, 125.9, 125.3, 116.5, 114.5, 114.3, 107.2, 74.8, 21.1. ESI-HRMS m/z: calculated for C19H15O4+ [M + H]+, 307.0965; found, 307.0966. HPLC: tR 7.157 min, purity 99.0%.

2-((7-oxo-7H-furo[3,2-g]chromen-9-yl)oxy)ethyl acetate (A12): White solid; yield: 59%; 1H NMR (400 MHz, DMSO-d6) δ 8.15 (d, J = 9.6 Hz, 1H), 8.13 (d, J = 2.2 Hz, 1H), 7.70 (s, 1H), 7.10 (d, J = 2.0 Hz, 1H), 6.44 (d, J = 9.6 Hz, 1H), 4.62–4.57 (m, 2H), 4.37–4.32 (m, 2H), 1.98 (s, 3H). 13C NMR (101 MHz, DMSO-d6) δ 170.4, 159.8, 148.1, 147.5, 145.4, 143.0, 130.6, 125.9, 116.6, 114.6, 114.3, 107.3, 71.3, 63.2, 20.7. ESI-HRMS m/z: calculated for C15H13O6+ [M + H]+, 289.0707; found, 289.0704. HPLC: tR 4.023 min, purity 100.0%.

ethyl 2-((7-oxo-7H-furo[3,2-g]chromen-9-yl)oxy)acetate (A13): Yellowish solid; yield: 52%; 1H NMR (400 MHz, DMSO-d6) δ 8.14 (d, J = 9.6 Hz, 1H), 8.09 (d, J = 2.2 Hz, 1H), 7.65 (s, 1H), 7.08 (d, J = 2.0 Hz, 1H), 6.44 (d, J = 9.6 Hz, 1H), 5.17 (s, 2H), 4.15 (q, J = 7.2 Hz, 2H), 1.16 (t, J = 7.2 Hz, 3H). 13C NMR (101 MHz, DMSO-d6) δ 168.7, 159.7, 148.0, 146.0, 145.4, 141.7, 130.3, 126.0, 116.5, 114.4, 113.8, 107.1, 68.6, 60.8, 14.1. ESI-HRMS m/z: calculated for C15H13O6+ [M + H]+, 289.0707; found, 289.0704. HPLC: tR 5.690 min, purity 96.1%.

9-(quinolin-8-ylmethoxy)-7H-furo[3,2-g]chromen-7-one (A14): Red solid; yield: 61%; 1H NMR (400 MHz, DMSO-d6) δ 8.89 (dd, J = 4.4, 2.0 Hz, 1H), 8.41 (dd, J = 8.4, 2.0 Hz, 1H), 8.15 (d, J = 9.6 Hz, 1H), 8.11 (d, J = 2.4 Hz, 1H), 8.08 (d, J = 7.2 Hz, 1H), 7.98 (d, J = 8.4 Hz, 1H), 7.70–7.64 (m, 2H), 7.57 (dd, J = 8.0, 4.0 Hz, 1H), 7.10 (d, J = 2.4 Hz, 1H), 6.44 (d, J = 9.6 Hz, 1H), 6.18 (s, 2H). 13C NMR (101 MHz, DMSO-d6) δ 159.9, 150.2, 148.0, 147.3, 145.4, 145.2, 142.9, 136.5, 134.8, 131.3, 128.3, 128.0, 127.8, 126.4, 126.0, 121.8, 116.6, 114.3, 114.2, 107.2, 71.1. ESI-HRMS m/z: calculated for C21H14NO4+ [M + H]+, 344.0917; found, 344.0918. HPLC: tR 5.243 min, purity 98.3%.

tert-butyl 4-(((7-oxo-7H-furo[3,2-g]chromen-9-yl)oxy)methyl)piperidine-1-carboxylate (A15): White solid; yield: 64%; 1H NMR (400 MHz, DMSO-d6) δ 8.14–8.09 (m, 2H), 7.66 (s, 1H), 7.08 (d, J = 2.4 Hz, 1H), 6.42 (d, J = 9.6 Hz, 1H), 4.23 (d, J = 6.4 Hz, 2H), 4.03–3.91 (m, 2H), 2.74 (s, 2H), 1.99–1.91 (m, 1H), 1.83 (dd, J = 12.8, 3.6 Hz, 2H), 1.38 (s, 9H), 1.26–1.14 (m, 2H). 13C NMR (101 MHz, DMSO-d6) δ 159.9, 154.0, 148.0, 147.4, 145.4, 142.9, 131.2, 125.9, 116.6, 114.3, 114.2, 107.2, 78.7, 77.8, 43.3 (2 × C), 42.7 (2 × C), 36.3, 28.2 (3 × C). ESI-HRMS m/z: calculated for C22H26NO6+ [M + H]+, 400.1754; found, 400.1739. HPLC: tR 4.490 min, purity 96.6%.

9-(2-(diethylamino)ethoxy)-7H-furo[3,2-g]chromen-7-one (A16): Yellowish solid; yield: 13%; 1H NMR (400 MHz, DMSO-d6) δ 8.14–8.09 (m, 2H), 7.66 (s, 1H), 7.08 (d, J = 2.0 Hz, 1H), 6.42 (d, J = 9.6 Hz, 1H), 4.48 (t, J = 6.0 Hz, 2H), 2.90 (t, J = 6.0 Hz, 2H), 2.58 (q, J = 7.2 Hz, 4H), 0.92 (t, J = 7.2 Hz, 6H). 13C NMR (101 MHz, DMSO-d6) δ 160.3, 148.3, 147.6, 145.8, 143.2, 131.4, 126.2, 116.8, 114.6, 114.4, 107.6, 71.6, 52.5, 47.4 (2 × C), 11.9 (2 × C). ESI-HRMS m/z: calculated for C17H20NO4+ [M + H]+, 302.1387; found, 302.1395. HPLC: tR 2.307 min, purity 96.5%.

ethyl 6-((7-oxo-7H-furo[3,2-g]chromen-9-yl)oxy)hexanoate (A17): Yellow oil; yield: 69%; 1H NMR (400 MHz, DMSO-d6) δ 8.12 (d, J = 8.0 Hz, 1H), 8.09 (s, 1H), 7.66 (s, 1H), 7.08 (s, 1H), 6.41 (d, J = 9.6 Hz, 1H), 4.39–4.34 (m, 2H), 4.04–3.99 (m, 2H), 2.29 (t, J = 7.2 Hz, 2H), 1.74 (q, J = 7.2 Hz, 2H), 1.58 (p, J = 7.2 Hz, 2H), 1.48 (p, J = 8.4 Hz, 2H), 1.14 (td, J = 7.2, 1.2 Hz, 3H). 13C NMR (101 MHz, DMSO-d6) δ 173.0, 159.9, 147.9, 147.5, 145.4, 143.0, 131.1, 125.9, 116.6, 114.3, 114.2, 107.2, 73.4, 59.8, 33.6, 29.3, 24.9, 24.3, 14.2. ESI-HRMS m/z: calculated for C19H21O6+ [M + H]+, 345.1333; found, 345.1326. HPLC: tR 7.583 min, purity 98.8%.

5-(((7-oxo-7H-furo[3,2-g]chromen-9-yl)oxy)methyl)pyrrolidin-2-one (A18): Yellow solid; yield: 19%; 1H NMR (400 MHz, DMSO-d6) δ 8.17–8.11 (m, 2H), 7.75 (s, 1H), 7.69 (s, 1H), 7.10 (d, J = 2.0 Hz, 1H), 6.43 (d, J = 9.6 Hz, 1H), 4.32 (h, J = 4.8 Hz, 2H), 3.92 (dt, J = 8.4, 4.4 Hz, 1H), 2.40–2.32 (m, 1H), 2.27–2.19 (m, 1H), 2.18–2.12 (m, 1H), 2.10–2.04 (m, 1H). 13C NMR (101 MHz, DMSO-d6) δ 177.1, 159.8, 148.0, 147.3, 145.4, 142.8, 131.1, 125.9, 116.6, 114.4, 114.3, 107.3, 75.9, 53.2, 29.6, 23.0. ESI-HRMS m/z: calculated for C16H14NO5+ [M + H]+, 300.0867; found, 300.0879. HPLC: tR 3.647 min, purity 97.4%.

ethyl 5-((7-oxo-7H-furo[3,2-g]chromen-9-yl)oxy)pentanoate (A19): Clear oil; yield: 25%; 1H NMR (400 MHz, DMSO-d6) δ 8.15 (d, J = 9.6 Hz, 1H), 8.12 (d, J = 2.4 Hz, 1H), 7.69 (s, 1H), 7.10 (d, J = 2.2 Hz, 1H), 6.44 (d, J = 9.6 Hz, 1H), 4.42–4.36 (m, 2H), 4.04 (q, J = 7.2 Hz, 2H), 2.42–2.35 (m, 2H), 1.80–1.72 (m, 4H), 1.16 (t, J = 7.2 Hz, 3H). 13C NMR (101 MHz, DMSO-d6) δ 172.8, 159.8, 147.9, 147.4, 145.3, 142.9, 130.9, 125.8, 116.5, 114.2, 114.1, 107.1, 73.0, 59.7, 33.1, 28.9, 21.0, 14.1. ESI-HRMS m/z: calculated for C18H19O6 + [M + H]+, 331.1176; found, 331.1175. HPLC: tR 5.153 min, purity 99.3%.

4-(cyclopentylmethoxy)-7H-furo[3,2-g]chromen-7-one (B1): White solid; yield: 23%; 1H NMR (400 MHz, DMSO-d6) δ 8.18 (d, J = 9.6 Hz, 1H), 8.03 (d, J = 2.0 Hz, 1H), 7.34 (s, 1H), 7.32 (dd, J = 2.4, 1.0 Hz, 1H), 6.34 (d, J = 9.6 Hz, 1H), 4.38 (d, J = 6.8 Hz, 2H), 2.43–2.31 (m, 1H), 1.86–1.76 (m, 2H), 1.66–1.52 (m, 4H), 1.44–1.34 (m, 2H). 13C NMR (101 MHz, DMSO-d6) δ 160.2, 157.7, 152.1, 148.9, 146.0, 139.4, 113.1, 112.4, 106.0, 105.7, 93.2, 76.5, 40.2, 28.8 (2 × C), 25.1 (2 × C). ESI-HRMS m/z: calculated for C17H17O4+ [M + H]+, 285.1121; found, 285.1126. HPLC: tR 7.757 min, purity 99.0%.

4-(cyclopropylmethoxy)-7H-furo[3,2-g]chromen-7-one (B2): White solid; yield: 28%; 1H NMR (400 MHz, DMSO-d6) δ 8.21 (dd, J = 9.6, 0.8 Hz, 1H), 8.02 (d, J = 2.4 Hz, 1H), 7.34 (t, J = 0.8 Hz, 1H), 7.27 (dd, J = 2.4, 1.0 Hz, 1H), 6.33 (d, J = 9.6 Hz, 1H), 4.29 (d, J = 7.2 Hz, 2H), 1.31–1.23 (m, 1H), 0.60–0.54 (m, 2H), 0.41–0.33 (m, 2H). 13C NMR (101 MHz, DMSO-d6) δ 160.3, 157.6, 152.2, 148.8, 146.1, 139.7, 113.8, 112.6, 106.7, 105.6, 93.6, 77.4, 10.8, 3.1 (2 × C). ESI-HRMS m/z: calculated for C15H13O4+ [M + H]+, 257.0808; found, 257.0806. HPLC: tR 6.187 min, purity 99.7%.

methyl 2-((7-oxo-7H-furo[3,2-g]chromen-4-yl)oxy)acetate (B3): White solid; yield: 8%; 1H NMR (400 MHz, DMSO-d6) δ 8.31 (dd, J = 9.6, 0.8 Hz, 1H), 8.05 (d, J = 2.4 Hz, 1H), 7.44 (t, J = 0.8 Hz, 1H), 7.23 (dd, J = 2.4, 1.2 Hz, 1H), 6.39 (d, J = 9.6 Hz, 1H), 5.22 (s, 2H), 3.70 (s, 3H).13C NMR (101 MHz, DMSO-d6) δ 169.2, 160.2, 157.5, 152.0, 147.8, 146.7, 139.8, 113.7, 113.0, 107.0, 104.8, 94.6, 68.9, 52.2. ESI-HRMS m/z: calculated for C14H11O6+ [M + H]+, 321.0758; found, 321.0756. HPLC: tR 3.650 min, purity 96.3%.

4-((4-methoxybenzyl)oxy)-7H-furo[3,2-g]chromen-7-one (B4): Red solid; yield: 68%; 1H NMR (400 MHz, DMSO-d6) δ 8.09 (d, J = 9.6 Hz, 1H), 8.06 (d, J = 2.4 Hz, 1H), 7.45–7.42 (m, 3H), 7.38 (s, 1H), 6.95 (d, J = 8.4 Hz, 2H), 6.29 (d, J = 9.6Hz, 1H), 5.48 (s, 2H), 3.75 (s, 3H). 13C NMR (101 MHz, DMSO-d6) δ 160.2, 159.5, 157.7, 152.2, 148.5, 146.2, 139.7, 130.2 (2 × C), 128.5, 114.0, 113.9 (2 × C), 112.6, 106.7, 105.8, 93.8, 74.1, 55.2. ESI-HRMS m/z: calculated for C19H15O5+ [M + H]+, 323.0914; found, 323.0906. HPLC: tR 5.230 min, purity 97.8%.

4-(allyloxy)-7H-furo[3,2-g]chromen-7-one (B5): White solid; yield: 60%; 1H NMR (400 MHz, DMSO-d6) δ 8.19 (d, J = 9.6 Hz, 1H), 8.02 (d, J = 2.4 Hz, 1H), 7.39–7.27 (m, 2H), 6.32 (d, J = 10.0 Hz, 1H), 6.19–6.08 (m, 1H), 5.47 (dd, J = 17.2, 1.6Hz, 1H), 5.30 (dd, J = 10.8, 1.6 Hz, 1H), 5.05–5.00 (m, 2H).13C NMR (101 MHz, DMSO-d6) δ 160.2, 157.6, 152.2, 148.3, 146.2, 139.6, 133.5, 118.3, 113.5, 112.6, 106.5, 105.6, 93.7, 73.0. ESI-HRMS m/z: calculated for C14H11O4+ [M + H]+, 243.0652; found, 243.0641. HPLC: tR 6.327 min, purity 98.9%.

methyl 4′-(((7-oxo-7H-furo[3,2-g]chromen-4-yl)oxy)methyl)-[1,1′-biphenyl]-2-carboxylate (B6): White solid; yield: 75%; 1H NMR (400 MHz, DMSO-d6) δ 8.19 (d, J = 10.0 Hz, 1H), 8.08 (d, J = 2.4 Hz, 1H), 7.73 (d, J = 7.6 Hz, 1H), 7.62 (t, J = 7.6 Hz, 1H), 7.58 (s, 1H), 7.56 (s, 1H), 7.51–7.47 (m, 1H), 7.46–7.42 (m, 2H), 7.40 (s, 1H), 7.33 (s, 1H), 7.31 (s, 1H), 6.33 (d, J = 10.0 Hz, 1H), 5.62 (s, 2H), 3.55 (s, 3H). 13C NMR (101 MHz, DMSO-d6) δ 168.6, 160.2, 157.7, 152.2, 148.4, 146.3, 140.9, 140.6, 139.7, 135.6, 131.7, 130.9, 130.6, 129.5, 128.4 (2 × C), 128.1 (2 × C), 127.7, 113.7, 112.7, 106.7, 105.8, 93.9, 73.9, 52.0. ESI-HRMS m/z: calculated for C26H19O6+ [M + H]+, 427.1176; found, 427.1172. HPLC: tR 3.370 min, purity 97.3%.

4-((tetrahydro-2H-pyran-4-yl)methoxy)-7H-furo[3,2-g]chromen-7-one (B7): White solid; yield: 34%; 1H NMR (400 MHz, DMSO-d6) δ 8.20 (d, J = 10.4 Hz, 1H), 8.03 (d, J = 2.4 Hz, 1H), 7.34 (s, 1H), 7.33 (dd, J = 2.4, 1.0 Hz, 1H), 6.33 (d, J = 9.6 Hz, 1H), 4.36 (d, J = 6.4Hz, 2H), 3.90 (dd, J = 11.2, 4.8 Hz, 2H), 3.38–3.31 (m, 2H), 2.15–2.03 (m, 1H), 1.74 (dd, J = 12.8, 2.0 Hz, 2H), 1.49–1.34 (m, 2H). 13C NMR (101 MHz, DMSO-d6) δ 160.3, 157.8, 152.2, 148.9, 146.1, 139.5, 113.0, 112.6, 106.0, 105.8, 93.4, 76.8, 66.8 (2 × C), 35.4, 29.1 (2 × C). ESI-HRMS m/z: calculated for C17H17O5+ [M + H]+, 301.1071; found, 301.1073. HPLC: tR 6.020 min, purity 100.0%.

methyl 3-(((7-oxo-7H-furo[3,2-g]chromen-4-yl)oxy)methyl)benzoate (B8): White solid; yield: 73%; 1H NMR (400 MHz, DMSO-d6) δ 8.16 (d, J = 9.6 Hz, 1H), 8.13 (s, 1H), 8.07 (d, J = 2.4 Hz, 1H), 7.95 (d, J = 9.6 Hz, 1H), 7.84 (d, J = 6.4 Hz, 1H), 7.59 (d, J = 7.6 Hz, 1H), 7.44 (d, J = 2.0 Hz, 1H), 7.41 (s, 1H), 6.32 (s, 1H), 5.66 (s, 2H), 3.86 (s, 3H). 13C NMR (101 MHz, DMSO-d6) δ 166.1, 160.2, 157.7, 152.2, 148.3, 146.3, 139.5, 137.5, 132.9, 130.0, 129.3, 129.1, 128.7, 113.6, 112.8, 106.5, 105.8, 94.0, 73.4, 52.4. ESI-HRMS m/z: calculated for C20H15O6+ [M + H]+, 351.0863; found, 351.0862. HPLC: tR 6.607 min, purity 99.3%.

4-(pyridin-3-ylmethoxy)-7H-furo[3,2-g]chromen-7-one (B9): White solid; yield: 43%; 1H NMR (400 MHz, DMSO-d6) δ 8.74 (d, J = 2.4 Hz, 1H), 8.58 (dd, J = 4.8, 2.0 Hz, 1H), 8.15 (d, J = 9.6 Hz, 1H), 8.07 (d, J = 2.4 Hz, 1H), 7.97 (dt, J = 8.0, 2.0 Hz, 1H), 7.47–7.42 (m, 2H), 7.39 (s, 1H), 6.30 (d, J = 10.0 Hz, 1H), 5.61 (s, 2H).13C NMR (101 MHz, DMSO-d6) δ 160.2, 157.6, 152.1, 149.7, 149.5, 148.1, 146.4, 139.6, 136.2, 132.2, 123.8, 113.7, 112.8, 106.6, 105.7, 94.0, 71.8. ESI-HRMS m/z: calculated for C17H12NO4+ [M + H]+, 294.0761; found, 294.0764. HPLC: tR 2.990 min, purity 98.8%.

3-(((7-oxo-7H-furo[3,2-g]chromen-4-yl)oxy)methyl)benzaldehyde (B10): Yellow solid; yield: 47%; 1H NMR (400 MHz, DMSO-d6) δ 10.04 (s, 1H), 8.18 (d, J = 9.6 Hz, 1H), 8.10–8.04 (m, 2H), 7.94–7.85 (m, 2H), 7.66 (t, J = 7.6 Hz, 1H), 7.44 (d, J = 2.0 Hz, 1H), 7.39 (s, 1H), 6.31 (d, J = 9.6 Hz, 1H), 5.67 (s, 2H). 13C NMR (101 MHz, DMSO-d6) δ 193.2, 160.2, 157.7, 152.2, 148.3, 146.3, 139.6, 137.9, 136.5, 134.0, 129.7, 129.6, 128.7, 113.6, 112.8, 106.5, 105.8, 94.0, 73.3. ESI-HRMS m/z: calculated for C19H13O5+ [M + H]+, 321.0758; found, 321.0756. HPLC: tR 4.807 min, purity 98.2%.

4-((3-methylbenzyl)oxy)-7H-furo[3,2-g]chromen-7-one (B11): White solid; yield: 48%; 1H NMR (400 MHz, DMSO-d6) δ 8.12 (d, J = 10.0 Hz, 1H), 8.04 (d, J = 2.4 Hz, 1H), 7.40 (s, 1H), 7.36 (s, 1H), 7.33 (s, 1H), 7.30–7.26 (m, 2H), 7.17 (d, J = 8.0 Hz, 1H), 6.30 (d, J = 9.6 Hz, 1H), 5.50 (s, 2H), 2.31 (s, 3H). 13C NMR (101 MHz, DMSO-d6) δ 160.2, 157.7, 152.2, 148.5, 146.2, 139.6, 137.9, 136.5, 129.1, 128.7, 128.6, 125.3, 113.6, 112.7, 106.6, 105.8, 93.8, 74.2, 21.1. ESI-HRMS m/z: calculated for C19H15O4+ [M + H]+, 307.0965; found, 307.0965. HPLC: tR 3.530 min, purity 97.5%.

2-((7-oxo-7H-furo[3,2-g]chromen-4-yl)oxy)ethyl acetate (B12): White solid; yield: 36%; 1H NMR (400 MHz, DMSO-d6) δ 8.17 (d, J = 10.4 Hz, 1H), 8.05 (d, J = 2.4 Hz, 1H), 7.39 (s, 1H), 7.30 (dd, J = 2.4, 1.2 Hz, 1H), 6.36 (d, J = 9.6 Hz, 1H), 4.69–4.62 (m, 2H), 4.43–4.37 (m, 2H), 2.03 (s, 3H). 13C NMR (101 MHz, DMSO-d6) δ 170.5, 160.2, 157.6, 152.1, 148.3, 146.4, 139.4, 113.7, 112.8, 106.6, 105.3, 94.0, 71.1, 62.9, 20.8. ESI-HRMS m/z: calculated for C15H13O6+ [M + H]+, 289.0707; found, 289.0706. HPLC: tR 3.850 min, purity 99.1%.

ethyl 2-((7-oxo-7H-furo[3,2-g]chromen-4-yl)oxy)acetate (B13): White solid; yield: 34%; 1H NMR (400 MHz, DMSO-d6) δ 8.31 (dd, J = 10.0, 0.8 Hz, 1H), 8.05 (d, J = 2.4 Hz, 1H), 7.43 (t, J = 0.8 Hz, 1H), 7.24 (dd, J = 2.4, 0.8 Hz, 1H), 6.39 (d, J = 10.0 Hz, 1H), 5.20 (s, 2H), 4.16 (q, J = 7.2 Hz, 2H), 1.19 (t, J = 7.2 Hz, 3H). 13C NMR (101 MHz, DMSO-d6) δ 168.7, 160.2, 157.5, 152.0, 147.8, 146.7, 139.8, 113.6, 112.9, 106.9, 104.8, 94.5, 68.9, 61.1, 14.1. ESI-HRMS m/z: calculated for C15H13O6+ [M + H]+, 289.0707; found, 289.0706. HPLC: tR 4.167 min, purity 95.8%.

4-(quinolin-8-ylmethoxy)-7H-furo[3,2-g]chromen-7-one (B14): White solid; yield: 58%; 1H NMR (600 MHz, CDCl3) δ 8.95 (dd, J = 4.2, 1.8 Hz, 1H), 8.23 (dd, J = 8.4, 1.8 Hz, 1H), 8.21 (d, J = 9.6 Hz, 1H), 7.89 (d, J = 7.2 Hz, 1H), 7.87 (d, J = 8.4 Hz, 1H), 7.59 (t, J = 7.8 Hz, 1H), 7.55 (d, J = 2.4 Hz, 1H), 7.49 (dd, J = 8.4, 4.2 Hz, 1H), 7.19 (s, 1H), 7.06 (d, J = 2.4 Hz, 1H), 6.23 (d, J = 9.6 Hz, 1H), 6.19 (s, 2H). 13C NMR (151 MHz, CDCl3) δ 161.3, 158.2, 152.6, 150.0, 149.1, 145.7, 144.9, 139.6, 136.4, 134.5, 128.4, 128.2, 128.1, 126.3, 121.6, 114.2, 112.6, 107.4, 105.3, 94.4, 71.4. ESI-HRMS m/z: calculated for C21H14NO4+ [M + H]+, 344.0917; found, 344.0918. HPLC: tR 4.583 min, purity 95.9%.

tert-Butyl 4-(((7-oxo-7H-furo[3,2-g]chromen-4-yl)oxy)methyl)piperidine-1-carboxylate (B15): White solid; yield: 60%; 1H NMR (400 MHz, DMSO-d6) δ 8.17 (d, J = 10.0 Hz, 1H), 8.02 (d, J = 2.4 Hz, 1H), 7.32 (s, 1H), 7.30 (dd, J = 2.4, 1.2 Hz, 1H), 6.31 (d, J = 10.0 Hz, 1H), 4.34 (d, J = 6.4 Hz, 2H), 4.00 (d, J = 12.8 Hz, 2H), 2.75 (s, 2H), 2.06–1.95 (m, 1H), 1.85–1.77 (m, 2H), 1.39 (s, 9H), 1.28–1.20 (m, 2H). 13C NMR (101 MHz, DMSO-d6) δ 160.2, 157.7, 154.0, 152.2, 148.8, 146.1, 139.5, 113.0, 112.5, 106.0, 105.8, 93.4, 78.6, 76.6, 43.7 (2 × C), 42.8 (2 × C), 36.3, 28.2 (3 × C). ESI-HRMS m/z: calculated for C22H26NO6+ [M + H]+, 400.1755; found, 400.1773. HPLC: tR 6.077 min, purity 99.7%.

5-(((7-oxo-7H-furo[3,2-g]chromen-4-yl)oxy)methyl)pyrrolidin-2-one (B16): White solid; yield: 91%; 1H NMR (600 MHz, DMSO-d6) δ 8.34 (d, J = 10.2 Hz, 1H), 8.10 (s, 1H), 8.04 (d, J = 2.4 Hz, 1H), 7.35 (d, J = 3.6 Hz, 2H), 6.32 (d, J = 10.2 Hz, 1H), 4.48 (dd, J = 9.0, 3.6 Hz, 1H), 4.35 (dd, J = 9.6, 6.6 Hz, 1H), 4.00–3.96 (m, 1H), 2.31–2.26 (m, 1H), 2.25–2.20 (m, 1H), 2.17–2.14 (m, 1H), 1.98–1.92 (m, 1H). 13C NMR (151 MHz, DMSO-d6) δ 177.2, 160.2, 157.7, 152.2, 148.6, 146.1, 140.0, 113.0, 112.3, 106.1, 105.7, 93.6, 75.5, 53.0, 29.6, 22.5. ESI-HRMS m/z: calculated for C16H14NO5+ [M + H]+, 300.0867; found, 300.0879. HPLC: tR 3.473 min, purity 97.7%.

ethyl 6-((7-oxo-7H-furo[3,2-g]chromen-4-yl)oxy)hexanoate (B17): White solid; yield: 73%; 1H NMR (400 MHz, DMSO-d6) δ 8.16 (dd, J = 9.6, 0.6 Hz, 1H), 8.01 (d, J = 2.4 Hz, 1H), 7.31 (t, J = 0.8 Hz, 1H), 7.29 (dd, J = 2.4, 0.8 Hz, 1H), 6.30 (d, J = 10.0 Hz, 1H), 4.46 (t, J = 6.4 Hz, 2H), 4.03 (q, J = 7.2 Hz, 2H), 2.31 (t, J = 7.2 Hz, 2H), 1.79 (dt, J = 14.0, 6.4 Hz, 2H), 1.63–1.56 (m, 2H), 1.52–1.44 (m, 2H), 1.15 (t, J = 7.2 Hz, 3H). 13C NMR (101 MHz, DMSO-d6) δ 173.0, 160.2, 157.7, 152.2, 148.8, 146.0, 139.6, 113.0, 112.4, 106.1, 105.7, 93.3, 72.5, 59.8, 33.5, 29.1, 25.0, 24.3, 14.2. ESI-HRMS m/z: calculated for C19H21O6+ [M + H]+, 345.1333; found, 345.1326. HPLC: tR 4.493 min, purity 98.6%.

4.2. Cell Culture and Viability Assay

Bone marrow-derived macrophages (BMDMs) of mice were extracted from their femurs. The mice were sacrificed, followed by α-MEM flushing of their femurs. Finally, the cells were cultured in α-MEM supplemented with 10% fetal bovine serum under 95% humidified air and 5% CO2 at 37 °C.

Cell viability was evaluated using a CCK-8 assay. BMDMs (106 cells/well) were seeded into 96-well plates. After 24 h, the medium was replaced with fresh medium containing different compounds for 24 h. Then, 10 μL of CCK-8 reagent was added to each well for 1 h. Absorbance at 450 nm was measured.

4.3. TRAP Staining

BMDMs were seeded into 24-well plates (106 cells/well) for 24 h, and then cultured in medium supplemented with 50 ng/mL M-CSF, 100 ng/mL RANKL and compound B15 or isoimperatorin (0, 10, 20, 30, 35, 40, 45, 50 nM) to induce osteoclast differentiation for 7 days. Then, TRAP staining was performed, and TRAP-positive multinucleated cells (≥3 nuclei) were identified under a light microscope and quantified using ImageJ 1.46r software.

4.4. F-Actin Ring Formation Assay

BMDMs were induced by M-CSF and RANKL, as well as compound B15 (40 nM) or ISO (1 μM) or Alendronate (Alen-Na, 1 μM) treatment for 7 days. Cells were fixed with 4% paraformaldehyde (PFA) and permeabilized using 0.1% (v/v) Triton X-100. Subsequently, F-actin rings were stained for 30 min with tetramethylrhodamine isothiocyanate (TRITC)-conjugated phalloidin according to the manufacturer’s instructions, and cell nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI) for 5 min. F-actin ring formation was visualized and imaged using a fluorescence microscope, and quantitative analysis was performed with ImageJ software.

4.5. Quantitative Real-Time PCR Analysis (qRT-PCR)

BMDMs (106 cells/well) were seeded into 12-well plates and treated with different compounds, followed by exposure to M-CSF and RANKL for 5 days. Then, total cellular RNA was extracted and determined. The isolated RNA was then reverse-transcribed into complementary DNA (cDNA). qRT-PCR was subsequently performed using SYBR Premix Ex Taq II (Takara) on a PCR thermal cycler (Bio-Rad, CA, USA). The used primer sequences are listed in Table S1.

4.6. Animal Experiments

A total of 42 female C57BL/6 mice (18–22 g) were obtained commercially from Zhuhai Bestest Biotechnology Co., Ltd. (Certificate No. 2025-00512025527287, Guangdong, China) and housed under controlled conditions (24 ± 1 °C, 60–70% humidity). Water and food were provided ad libitum throughout the experiment. All procedures were approved by the Institutional Ethical Committee for Animal Research of the Institute of Zoology, Guangdong Academy of Science (No. GIZ20251011, approved on 11 October 2025).

Mice were randomly assigned to six groups: Sham group, OVX, Alen-Na (10 mg/kg), B15-L (5 mg/kg), B15-M (10 mg/kg) and B15-H (20 mg/kg), with 7 mice in each group. The OVX mice model was established via bilateral ovariectomy. The specific operation was as follows: The mice were anesthetized, and their fur was shaved off. An incision was made along the midline of the abdomen, followed by removal of the ovaries and surrounding adipose tissue. In the Sham group, only the periovarian fat was removed. All mice were injected with antibiotics postoperatively. Two weeks after surgery, the mice were treated with Alen-Na or B15 for 4 weeks. Then, the mice were euthanized, and their femurs were harvested and fixed in 4% paraformaldehyde for further analysis.

4.7. Micro-CT

The femora were mounted in scanning tubes for imaging and subjected to high-resolution micro-computed tomography (micro-CT) scanning of the distal femur. Three-dimensional reconstructed images were generated using NRecon software, and morphological parameters were quantified from the scan data, including Bone Volume (BV), Tissue Volume (TV), Bone Scintigraphy (BS) and trabecular thickness (Tb.Th).

4.8. Biochemical Testing

The serum of the mice was collected and tested. The detection indicators included aspartate aminotransferase (AST), alanine aminotransferase (ALT), blood urea nitrogen (BUN), cardiac troponin (cTN1), C-reaction protein (CRP), Creatine Kinase MB Isoenzyme (CK-MB) and Estradiol (E2).

4.9. Statistical Analyses

All experiments were performed in triplicate to ensure reproducibility. Data are presented as the mean ± standard deviation (SD). Data normality was assessed using the Shapiro–Wilk test. When assumptions of normality and homogeneity of variance were satisfied, differences between groups were analyzed using Student’s t-test for two-group comparisons and one-way analysis of variance (ANOVA), followed by Tukey’s post hoc test. Data was processed with GraphPad Prism version 8.0.3. Statistical significance was defined as *** p or ### p < 0.001, ** p or ## p < 0.01, * p or # p < 0.05.

5. Conclusions

Compared with ISO, compound B15 exhibited favorable safety profiles in cellular and animal levels. The IC50 value of compound B15 that inhibits the formation of osteoclasts was 37.86 nM, while that of ISO was over 50 nM. Compound B15 reduced the expressions of c-fos, MMP9, Dc-stamp and NFATc1. It is worth mentioning that through the F-actin ring, compound B15 was more effective than Alen-Na in inhibiting the formation of osteoclasts. The bone tissue morphology of mice was restored to a certain extent, similar to the effect of Alen-Na. In addition, compound B15 could decelerate the progression of osteoporosis in ovariectomized mice by increasing their E2 levels [38,39]. These findings suggest that compound B15 is a promising therapeutic candidate for osteoporosis and warrants further investigation.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/ph19020327/s1, Figure S1: 1H NMR, 13C NMR Spectra, HRMS, HPLC Spectra; Figure S2: The ability of compounds to inhibit TNF expression; Table S1: Primer sequences used for quantitative PCR: Table S2: Cytotoxicity and c-fos inhibition of synthesized compounds.

pharmaceuticals-19-00327-s001.zip (6.9MB, zip)

Author Contributions

X.C.: Writing—review and editing, conceptualization, data curation. S.C.: In vivo and in vitro experiment. Q.G.: Synthesis. G.L.: Synthesis, funding acquisition. Y.G.: In vivo and in vitro experiment. X.Q.: Synthesis. H.Y.: Writing—review and editing, conceptualization, data curation, funding acquisition. Q.W.: Writing—review and editing, conceptualization, data curation. J.Z.: Writing—review and editing, data curation, funding acquisition. All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement

All the mice were obtained commercially from Zhuhai Bestest Biotechnology Co., Ltd. (Certificate No. 2025-00512025527287, Guangdong, China). All procedures were approved by the Institutional Ethical Committee for Animal Research of the Institute of Zoology, Guangdong Academy of Science (No. GIZ20251011) approved on 11 October 2025.

Informed Consent Statement

Not applicable.

Data Availability Statement

The original contributions presented in this study are included in the article/Supplementary Materials. Further inquiries can be directed to the corresponding authors.

Conflicts of Interest

The authors declare no conflicts of interest.

Funding Statement

This study was funded by the Science and Technology Development Fund, Macau SAR (0048/2023/AFJ and 0164/2023/RIA3); GDAS’ Project of Science and Technology Development (2022GDASZH-2022010110).

Footnotes

Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

pharmaceuticals-19-00327-s001.zip (6.9MB, zip)

Data Availability Statement

The original contributions presented in this study are included in the article/Supplementary Materials. Further inquiries can be directed to the corresponding authors.

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