Dibenzoylated withanolide a and synthetic method and application thereof

Abstract

The application discloses a kind of diphthaloyl solasodine A and its synthesis method and application, belong to medical technical field.The experimental results of applicant show that, the weight loss activity of the diphthaloyl solasodine A described in the application is reduced relative to solasodine A, but still maintains the remarkable weight loss effect, can be used as the candidate drug for treating obesity weight loss.

Description

Technical Field

[0001] This invention relates to bisbenzoylated solanine A, its synthesis method, and its application, belonging to the field of pharmaceutical technology. Background Technology

[0002] Obesity and its cardiometabolic sequelae have become a hallmark public health challenge of the 21st century. In 2022, more than one billion people worldwide were obese, and its prevalence continues to rise across all age groups. Obesity significantly increases the risk of type 2 diabetes, non-alcoholic fatty liver disease, and cardiovascular disease, all driven by chronic energy surplus, insulin resistance, ectopic lipid accumulation, and low-grade inflammation, forming an interconnected cardiometabolic continuum. The global burden of diabetes now exceeds 500 million adults and is projected to increase further in the coming decades, with type 2 diabetes accounting for the majority. Therefore, there remains a significant need for treatments that can safely achieve sustainable weight loss and further improve glycemic control.

[0003] Withaferin A (WA) is the most active steroidal endose compound from Ashwagandha, possessing a wide range of pharmacological activities, including anti-inflammatory, anticancer, neuroprotective, antioxidant, anti-obesity, and hepatoprotective effects. Existing studies have shown that withaferin A can reduce body weight in diet-induced obese (DIO) mice with hyperleptinemia, but its effect is minimal in ob / ob or db / db models.

[0004] Patent application JP2016537431A discloses a compound defined by Formula I or a pharmaceutically acceptable salt or prodrug thereof.

[0005] ;

[0006] In the formula, R1 to R7 are independently hydrogen, carboxylic acid, formyl, primary amide; sulfinyl, halogen, nitrile, or CF3; or alkyl, cycloalkyl, heterocycloalkyl, alkylaryl, alkenyl, alkynyl, aryl, or heteroaryl; when R8 is present, it is individually alkyl, cyclopropyl, cyclobutyl ether, amine, halogen, hydroxyl, ether, nitrile, CF3, ester, amide, carbamate, thioether, carboxylic acid, and aryl in each occurrence; the dashed line indicates a single or double bond. This invention indicates that the compound can induce weight loss in patients with mild obesity, obesity, or morbid obesity, and reduce body fat in patients with mild obesity, obesity, or morbid obesity, thereby reducing food intake in patients with mild obesity, obesity, or morbid obesity, improving glucose homeostasis in patients with mild obesity, obesity, or morbid obesity, or a combination thereof. In some embodiments, the compound is Witherin A, or a Michael addition product of a compound of formula I. The experimental results of this invention show that Witherin A does not cause weight loss or inhibit food intake in lean mice; its appetite-reducing effect is limited to obese animals, and it can lower blood glucose levels in obese mice. When Witherin A is administered to the db / db mouse model, blood glucose levels tend to decrease at the end of 3 weeks of treatment, but this does not reach a statistically significant level. No specific experiments have been conducted in the literature on the Michael addition product of compound I in reducing obesity and food intake. To the knowledge of the inventors of this application, there are few reports on the in vivo metabolic endpoints of semi-synthetic witherin A derivatives in diet-induced obesity models. This uncertainty complicates target-based optimization studies and makes phenotype-guided in vivo assessments particularly important for preferential selection of witherin A derivatives and defining the structural features required to retain metabolic efficacy. Summary of the Invention

[0007] The technical problem to be solved by the present invention is to provide a novel bisbenzoylated solanacolin A with good weight loss effect in diet-induced obese mice, as well as its synthesis method and application.

[0008] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0009] The bisbenzoylated solanine A of the present invention is a salt having the following formula 6 or a pharmaceutically acceptable salt thereof:

[0010] .

[0011] The method for synthesizing bisbenzoylated solanacolin A according to the present invention includes: dissolving solanacolin A in an organic solvent, adding benzoic anhydride in the presence of triethylamine to react and obtain the crude target compound.

[0012] Furthermore, the organic solvent may be 1,2-dichloroethane, dichloromethane, chloroform, or acetone, etc.

[0013] Furthermore, the reaction was carried out under non-heating conditions, such as at room temperature. The completeness of the reaction was monitored using thin-layer chromatography.

[0014] Furthermore, the amount of triethylamine used is 3 to 10 times the molar amount of solanine A, and the amount of benzoic anhydride used is 1.2 to 5 times the molar amount of solanine A.

[0015] Furthermore, the synthesis method of the present invention further includes a step of purifying the obtained crude target compound. Existing conventional purification methods can be used to purify the crude target compound to improve its purity. In this application, silica gel column chromatography is used to purify the obtained crude product. The eluent used for elution during column chromatography is preferably a mixed solvent composed of ethyl acetate (EtOAc) and petroleum ether (PE). Further, the volume ratio of ethyl acetate to petroleum ether is preferably 1:50 to 1:20, more preferably 1:15 to 1:8.

[0016] The applicant discovered through experiments that the bisbenzoylated solanacone A described in this invention has a relatively good weight loss effect. Therefore, this invention also includes the use of bisbenzoylated solanacone A or a pharmaceutically acceptable salt thereof in the preparation of a medicament for the prevention or treatment of obesity.

[0017] Furthermore, the present invention also provides a pharmaceutical composition containing a therapeutically effective dose of the above-mentioned bisbenzoylated solanine A or a pharmaceutically acceptable salt thereof.

[0018] Compared with the prior art, the present invention provides a novel bisbenzoylated solanacone A and its synthesis method. The applicant's weight loss experiment on diet-induced obese mice showed that although the weight loss activity of the bisbenzoylated solanacone A of the present invention is reduced compared with solanacone A, it still maintains a significant weight loss effect. Attached Figure Description

[0019] Figure 1 The effects of withaferin A and its derivatives on body weight in DIO mice. (A) Time course of body weight change in diet-induced obese (DIO) mice during administration. Mice were treated with solvent control (DMSO), withaferin A (compound 1), and withaferin A derivatives (compounds 2–15), respectively. Body weight is expressed as change relative to baseline (day 0). Data are presented as mean ± SEM (solvent control group n = 3; each treatment group n = 5). (B) Endpoint body weight change at day 21 for each treatment group. Data are presented as mean ± SD (solvent control group n = 3; each treatment group n = 5). Statistical significance of each treatment group relative to the solvent control group was determined using one-way ANOVA and Dunnett's multiple comparison test. p < 0.05, p < 0.01, p < 0.001; ns, not significant).

[0020] Figure 2 The effects of withaferin A and its derivatives on food intake in DIO mice. (A) Cumulative food intake in diet-induced obese (DIO) mice during the administration period. Mice were treated with solvent control (DMSO), withaferin A (1), and methanolide analogs (compounds 2–15), respectively. Data are presented as mean ± SEM (solvent control group n = 2; each treatment group n = 3). (B) Total food intake on day 21. Data are presented as mean ± SD (solvent control group n = 2; each treatment group n = 3). One-way ANOVA and Dunnett's multiple comparison test were used to determine the statistical significance of each treatment group relative to the solvent control group. p < 0.05, p < 0.01, p < 0.001; ns, not significant).

[0021] Figure 3 To investigate the effects of withaferin A and its derivatives on glycemic control in DIO mice. (A) Glycemic levels in diet-induced obese (DIO) mice during administration. Mice were treated with solvent control (DMSO), withaferin A (1), and its derivatives (compounds 2–15). Data are presented as mean ± SEM (solvent control group n = 3; each treatment group n = 5). (B) Glycemic levels on day 21. Data are presented as mean ± SD (solvent control group n = 3; each treatment group n = 5). One-way ANOVA and Dunnett's multiple comparison test were used to determine the statistical significance of each treatment group relative to the solvent control group. p < 0.05, p < 0.01, p < 0.001; ns, not significant). Detailed Implementation

[0022] To better explain the technical solution of the present invention, the present invention will be further described in detail below with reference to the embodiments, but the implementation of the present invention is not limited thereto.

[0023] The structures of compounds 1-15 appearing in the following examples are shown below, wherein compound 1 represents the compound with the structure shown in Formula 1, i.e., withaferin A; compound 2 represents the compound with the structure shown in Formula 2; compound 3 represents the compound with the structure shown in Formula 3; compound 4 represents the compound with the structure shown in Formula 4; compound 5 represents the compound with the structure shown in Formula 5; compound 6 represents the compound with the structure shown in Formula 6; compound 7 represents the compound with the structure shown in Formula 7; compound 8 represents the compound with the structure shown in Formula 8; compound 9 represents the compound with the structure shown in Formula 9; compound 10 represents the compound with the structure shown in Formula 10; compound 11 represents the compound with the structure shown in Formula 11; compound 12 represents the compound with the structure shown in Formula 12; compound 13 represents the compound with the structure shown in Formula 13; compound 14 represents the compound with the structure shown in Formula 14; and compound 15 represents the compound with the structure shown in Formula 15.

[0024]

[0025] Example 1

[0026] 1. Experimental Section

[0027] 1.1 Chemical synthesis.

[0028] Reagents and solvents were purchased from commercial suppliers and used directly (usually nominally >95% pure) unless otherwise specified. Reactions were carried out with magnetic stirring. Reactions sensitive to moisture or air were carried out under a nitrogen atmosphere using dried glassware and anhydrous solvents. Temperatures referred to are external bath temperatures. Reactions at 0°C were carried out in a Dewar bath containing an ice-water mixture. The reaction progress was monitored by thin-layer chromatography on silica gel plates (Qingdao, 60F-254, 0.25 mm, glass plate) and developed under UV light (254 nm). Rapid column chromatography was performed on silica gel (Qingdao, 60; particle size 0.040–0.063 mm). Unless otherwise specified, reported yields correspond to the products isolated after column chromatography purification. NMR spectra were recorded on a Bruker Avance 600 spectrometer (1H, 600 MHz; 13C, 150 MHz). Chemical shifts are reported in ppm and referenced to the residual solvent signal of CDCl3 (δH = 7.26 ppm, δC = 77.16 ppm) unless otherwise stated. High-resolution mass spectrometry data were recorded on a Thermo Ultimate 3000 Q-Exactive Focus mass spectrometer.

[0029] All proportions mentioned below for the preparation of each compound using silica gel rapid column chromatography purification are volume ratios.

[0030] Preparation of compound 6. Triethylamine (0.60 mL, 1.06 mmol, 10 equivalents) was added to a stirred solution of withaferin A (50.0 mg, 0.106 mmol, 1.0 equivalents) in anhydrous CH2Cl2 (2.5 mL), followed by benzoic anhydride (28.7 mg, 0.127 mmol, 1.2 equivalents) at room temperature. The reaction mixture was stirred at room temperature for 4 hours. The solvent was removed directly under reduced pressure, and the crude product was purified by silica gel column chromatography (eluent: EtOAc / PE, 1:15 to 1:8) to give compound 6 (45.3 mg, yield: 63%) as a pale yellow solid.

[0031] Compound 6: 1 H NMR (600 MHz, Chloroform-d) δ 8.01 (d, J = 7.8 Hz, 2H), 7.94 (d, J = 7.8 Hz, 2H), 7.57 – 7.51 (m, 2H), 7.41 (q, J = 7.4 Hz, 4H), 7.14(dd, J = 9.9, 6.1 Hz, 1H), 6.29 (d, J = 9.8 Hz, 1H), 5.14 (s, 2H), 4.93 (d, J= 6.1 Hz, 1H), 4.42 (d, J = 13.3 Hz, 1H), 3.29 (s, 1H), 2.53 (dd, J = 18.0,13.2 Hz, 1H), 2.21 – 2.16 (m, 1H), 2.13 (s, 3H), 2.07 – 1.91 (m, 3H), 1.74 –1.59 (m, 3H), 1.51 (s, 3H), 1.40 – 1.28 (m, 4H), 1.14 (dd, J = 12.2, 5.5 Hz,1H), 1.07 (dd, J = 12.1, 9.3 Hz, 1H), 1.00 (d, J = 6.6 Hz, 3H), 0.93 (dq, J =16.0, 6.8, 5.6 Hz, 2H), 0.86 (t, J = 6.9 Hz, 1H), 0.70 (s, 3H). 13C NMR (150MHz, CDCl3) δ 201.29, 166.52, 165.91, 165.36, 157.19, 139.91, 134.19, 133.49,133.10, 130.10, 129.84, 129.81, 129.40, 128.60, 128.42, 122.14, 78.31, 72.69,61.26, 60.48, 58.55, 56.19, 52.04, 48.27, 44.30, 42.70, 39.30, 38.89, 31.25,30.29, 29.79, 29.72, 29.41, 27.43, 24.35, 21.50, 20.83, 16.01, 13.46, 11.69.HRMS (ESI): m / z calcd for C 42 H 47 O8 + [M + H] + : 679.3265; found: 679.3247.

[0032] Preparation of compounds 2, 5, 14, and 15. Using withaferin A as the starting material, preparations were carried out according to existing literature (Wang, Y.; Lu, M.; Li, Y.; Huang, X.; Liang, Z.; Zhao, Y.; Huang, H.; Yi, T.; Su, D.; Yan, ZJ Nat. Prod. 2025, 88, 2151-2157.). The preparation of compound 2 is described below as an example:

[0033] Under nitrogen protection, withaferin A (180.0 mg, 0.38 mmol, 1.0 equivalent) was dissolved in dichloromethane (CH2Cl2, 3 mL), and manganese dioxide (MnO2, 665.0 mg, 7.65 mmol, 20.0 equivalent) was added. The reaction mixture was stirred at room temperature for 3 h. After the reaction was complete, the mixture was filtered through a layer of diatomaceous earth (Celite), and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: ethyl acetate (EtOAc) / petroleum ether (PE), 1 / 4 to 4 / 1) to give compound 2 (138.7 mg, 78% yield) as a pale yellow solid.

[0034] Compound 2: 1H NMR (600 MHz, Chloroform-d) δ 6.91 – 6.82 (m, 2H), 4.50– 4.25 (m, 3H), 3.42 (d, J = 2.6 Hz, 1H), 2.89 (d, J = 6.7 Hz, 1H), 2.50 (dd,J = 17.8, 13.3 Hz, 1H), 2.16 (dd, J = 15.0, 2.9 Hz, 1H), 2.04 (s, 3H), 2.02 –1.93 (m, 3H), 1.66 (q, J = 8.4, 3H), 1.64 – 1.55 (m, 2H), 1.48 – 1.41 (m,2H), 1.37 (s, 3H), 1.36 – 1.23 (m, 3H), 1.20 – 1.09 (m, 2H), 1.01 (dd, J =6.7, 1.6 Hz, 3H), 0.72 (d, J = 1.6 Hz, 3H). 13 C NMR (150 MHz, Chloroform-d) δ202.27, 194.00, 167.12, 152.95, 141.73, 139.28, 125.82, 78.81, 64.09, 63.65,57.58, 55.71, 52.20, 50.00, 43.78, 42.80, 39.55, 38.88, 30.65, 29.94, 29.76,27.31, 24.37, 23.58, 20.16, 19.32, 13.46, 11.92. HRMS (ESI): m / z calcd forC28H37O6 + [M + H] + : 469.2585; found: 469.2580.

[0035] Preparation of compounds 3 and 11. H2O2 (wt = 30%, 0.3 mL) was added to a solution of withaferin A (50.0 mg, 0.106 mmol, 1.0 equivalent) in CH2Cl2 (2.5 mL) / MeOH (5 mL), followed by NaOH (4N, 0.1 mL). The reaction mixture was stirred at room temperature for 2 min. H2O (5 mL) was added and the mixture was extracted with CH2Cl2 (3 × 5 mL). The combined organic phases were dried over anhydrous Na2SO4, filtered, and concentrated. The residues were purified by rapid silica gel chromatography (eluent: EtOAc / PE, 1 / 6 to 1 / 1) to give compounds 3 (22 mg, yield: 43%) and 11 (12 mg, yield: 23%), both pale yellow solids.

[0036] Compound 3: 1 H NMR (600 MHz, Chloroform-d) δ 4.44 – 4.31 (m, 3H), 3.91(d, J = 3.7 Hz, 1H), 3.72 (t, J = 4.2 Hz, 1H), 3.57 (d, J = 4.5 Hz, 1H), 3.10(d, J = 2.1 Hz, 1H), 3.04 – 2.92 (m, 2H), 2.49 (dd, J = 17.9, 13.3 Hz, 1H), 2.23 – 2.15 (m, 1H), 2.03 (s, 3H), 2.02 – 1.95 (m, 2H), 1.94 – 1.88 (m, 1H),1.70 – 1.61 (m, 2H), 1.42 – 1.31 (m, 5H), 1.22 (s, 3H), 1.19 – 1.09 (m, 3H), 1.09 – 1.04 (m, 2H), 0.98 (d, J = 6.7 Hz, 3H), 0.65 (s, 3H). 1313C NMR (150 MHz, Chloroform-d) δ 205.41, 166.16, 152.14, 124.76, 77.84, 73.99, 62.93, 58.80, 56.52, 55.14, 54.53, 53.92, 50.96, 47.74, 41.78, 39.83, 37.93, 37.88, 30.13, 28.94, 28.65, 26.45, 23.37, 19.25, 19.17, 13.76, 12.49, 10.59. HRMS (ESI): m / z calcd for C 28 H 39 O7 + [M + H] + : 487.2690; found: 487.2687.

[0037] Compound 11: 1 1H NMR (600 MHz, Chloroform-d) δ 4.55 (dt, J = 11.4, 3.7 Hz, 1H), 4.13 (d, J = 12.5 Hz, 1H), 3.91 (d, J = 3.8 Hz, 1H), 3.79 (d, J = 12.6 Hz, 1H), 3.72 (t, J = 4.1 Hz, 1H), 3.57 (d, J = 4.5 Hz, 1H), 3.11 (d, J = 1.4 Hz, 1H), 2.21 – 2.16 (m, 1H), 2.05 – 1.96 (m, 2H), 1.93 – 1.86 (m, 2H), 1.73 (ddd, J = 12.9, 6.7, 3.8 Hz, 1H), 1.68 – 1.62 (m, 1H), 1.56 (s, 3H), 1.41 – 1.31 (m, 6H), 1.21 (s, 3H), 1.18 – 1.10 (m, 3H), 1.03 (dd, J = 12.2, 9.1 Hz, 3H), 0.90 (d, J = 6.6 Hz, 3H), 0.63 (s, 3H). 13C NMR (150 MHz, Chloroform-d) δ 205.41, 169.25, 75.86, 74.02, 62.94, 62.37, 59.69, 59.31,58.83, 55.12, 54.51, 53.92, 51.00, 47.73, 41.79, 39.82, 37.91, 37.61, 30.11,28.65, 28.28, 26.37, 23.33, 19.25, 16.55, 13.77, 12.14, 10.59.HRMS (ESI): m / zcalcd for C 28 H 39 O8 + [M + H] + : 503.2639; found: 503.2634.

[0038] Preparation of Compound 4. 4-Methylmorpholine N-oxide (NMO, 18.0 mg, 0.106 mmol, 1.0 equivalent) and citric acid (40.7 mg, 0.212 mmol, 2.0 equivalent) were added to a stirred solution of withaferin A (50.0 mg, 0.106 mmol, 1.0 equivalent) in tBuOH (0.8 mL) and H₂O (0.4 mL). After stirring at room temperature for 5 min, K₂O₄·2H₂O (0.8 mg, 0.002 mmol, 0.02 equivalent) was added, and the reaction mixture was stirred at room temperature for 2 h. After the reaction was complete, the reaction was quenched with saturated Na₂S₂O₃ aqueous solution (0.5 mL), and stirring was continued for 30 min. The mixture was diluted with EtOAc (3 mL) and partitioned. The aqueous layer was extracted with EtOAc (3 × 3 mL). The combined organic phases were dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. The crude product was purified by silica gel rapid column chromatography (eluent: EtOAc) to give compound 4 (35.8 mg, yield: 67%) as a white solid.

[0039] Compound 4: 1H NMR (600 MHz, Chloroform-d) δ 4.95 (t, J = 2.9 Hz, 1H),4.42 (dt, J = 13.3, 3.5 Hz, 1H), 4.35 (s, 2H), 4.31 (t, J = 3.5 Hz, 1H), 3.76(d, J = 3.4 Hz, 1H), 3.58 (d, J = 3.9 Hz, 1H), 3.28 (d, J = 2.6 Hz, 1H), 2.85(s, 1H), 2.56 (dd, J = 17.8, 13.3 Hz, 1H), 2.10 (d, J = 14.8 Hz, 1H), 2.03(s, 3H), 2.01 – 1.91 (m, 3H), 1.62 (d, J = 2.5 Hz, 2H), 1.47 (s, 3H), 1.45 –1.41 (m, 1H), 1.37 – 1.30 (m, 2H), 1.28 – 1.22 (m, 3H), 1.17 – 1.09 (m, 2H),1.03 (d, J = 6.6 Hz, 3H), 0.99 – 0.94 (m, 1H), 0.69 (s, 3H). 13 C NMR (150 MHz,Chloroform-d) δ 211.55, 166.34, 152.45, 124.64, 77.90, 75.59, 71.53, 70.56,65.07, 63.36, 56.50, 54.96, 51.24, 48.65, 42.15, 41.81, 38.66, 37.95, 30.05,28.92, 27.56, 26.32, 23.39, 22.18, 19.20, 17.07, 12.42, 10.84. HRMS (ESI): m / z calcd for C 28 H 41 O8 + [M + H] + : 505.2796; found: 505.2791.

[0040] Preparation of compound 7. Triethylamine (0.30 mL, 2.12 mmol, 20 equivalents) was added to a stirred solution of withaferin A (50.0 mg, 0.106 mmol, 1.0 equivalents) in anhydrous CH2Cl2 (2.5 mL), followed by triethylchlorosilane (19.1 mg, 0.127 mmol, 1.2 equivalents) at room temperature. The reaction mixture was stirred at room temperature for 3 hours. The solvent was removed directly under reduced pressure, and the crude product was purified by rapid silica gel column chromatography (eluent: EtOAc / PE, 1:15 to 1:10) to give compound 7 (52.5 mg, yield: 71%) as a pale yellow solid.

[0041] Compound 7: 1 H NMR (600 MHz, Chloroform-d) δ 6.89 (dd, J = 10.4, 6.0Hz, 1H), 6.13 (d, J = 9.3 Hz, 1H), 4.49 (d, J = 11.7 Hz, 1H), 4.36 (d, J =12.0 Hz, 2H), 3.56 (d, J = 6.0 Hz, 1H), 3.06 (s, 1H), 2.44 (dd, J = 17.7,13.4 Hz, 1H), 2.17 – 2.09 (m, 1H), 2.05 (s, 3H), 1.99 – 1.89 (m, 4H), 1.70 –1.57 (m, 4H), 1.50 – 1.41 (m, 2H), 1.38 (s, 3H), 1.35 (s, 2H), 1.26 (s, 3H), 0.61 (q, J = 8.1 Hz, 6H), 0.58 – 0.54 (m, 6H). 13C NMR (150 MHz, CDCl3) δ202.30, 165.89, 154.68, 143.74, 132.18, 126.01, 78.23, 71.22, 63.51, 59.85,56.73, 56.26, 52.07, 48.20, 44.43, 42.65, 39.37, 38.88, 32.02, 31.59, 31.54,30.22, 30.16, 29.90, 29.79, 29.45, 29.41, 27.40, 24.40, 22.78, 21.22, 20.56,16.33, 14.21, 13.43, 11.61, 6.86, 6.73, 4.96, 4.43. HRMS (ESI): m / z calcd forC 40 H 66 NaO6Si2 + [M + Na] + : 721.4290; found: 721.5064.

[0042] Preparation of compound 8. Triethylamine (0.15 mL, 1.06 mmol, 10 equivalents) was added to a stirred solution of withaferin A (50.0 mg, 0.106 mmol, 1.0 equivalents) in anhydrous CH2Cl2 (2.5 mL) at room temperature. Then, trichlorotrihexylsilane (43.9 mg, 0.138 mmol, 1.3 equivalents) was added dropwise, and the reaction mixture was stirred at room temperature until complete (TLC monitoring). The solvent was removed directly under reduced pressure, and the crude product was purified by rapid silica gel column chromatography (eluent: EtOAc / PE, 1:30 to 1:10) to give compound 8 (43.7 mg, yield: 54%) as a pale yellow oil.

[0043] Compound 8: 1H NMR (600 MHz, Chloroform-d) δ 6.92 (dd, J = 10.0, 5.9Hz, 1H), 6.19 (d, J = 10.0 Hz, 1H), 4.48 (d, J = 11.7 Hz, 1H), 4.36 (t, J =13.2 Hz, 2H), 3.74 (d, J = 5.9 Hz, 1H), 3.22 (s, 1H), 2.45 (dd, J = 17.7,13.2 Hz, 1H), 2.14 (dt, J = 15.2, 2.9 Hz, 1H), 2.05 (s, 3H), 1.96 (ddt, J =21.0, 13.1, 3.7 Hz, 3H), 1.81 (dt, J = 14.4, 3.7 Hz, 1H), 1.70 – 1.59 (m,2H), 1.53 – 1.43 (m, 2H), 1.40 (s, 3H), 1.27 (m, 30H), 1.17 – 1.03 (m, 3H),0.97 (d, J = 6.7 Hz, 3H), 0.86 (t, J = 6.8 Hz, 9H), 0.69 (s, 3H), 0.60 – 0.59(m, 6H). 13 C NMR (150 MHz, Chloroform-d) δ 202.40, 165.89, 154.55, 142.07,132.44, 126.06, 78.21, 70.03, 64.01, 62.55, 56.78, 56.19, 52.13, 47.82,44.26, 42.68, 39.45, 38.91, 33.45, 31.68, 31.29, 30.18, 29.89, 27.41, 24.39,23.25, 22.74, 22.19, 20.64, 17.47, 14.27, 13.64, 13.46, 11.72. HRMS (ESI): m / z calcd for C 46 H 77 O6Si + [M + H] + : 753.5484; found: 753.5481.

[0044] Preparation of compound 9. Under argon protection, thionyl chloride (12 μL, 0.12 mmol) was slowly added to a solution of withaferin A (40 mg, 0.08 mmol) in anhydrous dichloromethane (7 mL) at 0 °C. The reaction mixture was stirred at room temperature for 3 hours. Subsequently, the reaction was quenched by adding ice water (15 mL) and extracted with dichloromethane (3 × 15 mL). The organic phases were combined, washed with saturated brine, and dried over anhydrous sodium sulfate. The solvent was removed by vacuum distillation to give an oily product. The crude product was purified by preparative thin-layer chromatography (electrolyte: PE / EtOAc = 4 / 1) to give compound 9 (6.6 mg, yield 17%).

[0045] Compound 9: 1 H NMR (600 MHz, Chloroform-d) δ 6.48 (dd, J = 10.4, 2.5 Hz,1H), 6.01 (dd, J = 10.4, 2.1 Hz, 1H), 4.49 – 4.37 (m, 3H), 3.74 (s, 1H), 3.14(d, J = 4.7 Hz, 1H), 2.51 (dd, J = 18.0, 13.1 Hz, 1H), 2.36 – 2.28 (m, 1H), 2.10 (s, 3H), 2.03 – 1.97 (m, 2H), 1.95 – 1.88 (m, 1H), 1.70 – 1.63 (m, 3H),1.44 – 1.33 (m, 4H), 1.26 (s, 3H), 1.25 (dd, J = 4.4, 2.7 Hz, 3H), 1.11 –1.06 (m, 2H), 0.97 (d, J = 6.7 Hz, 3H), 0.68 (s, 3H). 13 C NMR (150 MHz, Chloroform-d) δ 200.25, 164.78, 156.22, 142.94, 127.86, 124.22, 78.26, 66.27, 58.60, 57.34, 55.44, 51.88, 45.98, HRMS (ESI): m / z calcd for C 28 H 38ClO5 + [M + H] + : 489.2402; found: 489.2397.

[0046] Preparation of compound 10. Triethylamine (0.30 mL, 2.12 mmol, 20 equivalents) was added to a stirred solution of withaferin A (50.0 mg, 0.106 mmol, 1.0 equivalents) in anhydrous CH2Cl2 (2.5 mL), followed by acetic anhydride (54.0 mg, 0.530 mmol, 5.0 equivalents) at room temperature. The reaction mixture was stirred at room temperature for 2 hours. The solvent was removed directly under reduced pressure, and the crude product was purified by rapid silica gel column chromatography (eluent: EtOAc / PE, 1:15 to 1:6) to give compound 10 (48.0 mg, yield: 81%) as a white solid.

[0047] Compound 10: 1 H NMR (600 MHz, Chloroform-d) δ 7.02 (ddd, J = 9.9, 6.1,1.0 Hz, 1H), 6.24 (dd, J = 9.8, 1.0 Hz, 1H), 4.93 – 4.79 (m, 2H), 4.65 (d, J= 6.1 Hz, 1H), 4.39 (dt, J = 13.2, 3.5 Hz, 1H), 3.21 (s, 3H), 2.50 (dd, J =17.9, 13.2 Hz, 1H), 2.16 (dt, J = 14.9, 2.7 Hz, 1H), 2.06 (s, 3H), 2.04 (s,6H), 2.02 – 1.98 (m, 2H), 1.96 – 1.92 (m, 1H), 1.70 – 1.61 (m, 3H), 1.50 –1.43 (m, 2H), 1.38 (s, 3H), 1.15 (dd, J = 12.3, 5.5 Hz, 1H), 1.08 – 1.02 (m,2H), 0.98 (d, J = 6.6 Hz, 3H), 0.94 – 0.89 (m, 1H), 0.88 – 0.84 (m, 1H), 0.69(s, 3H). 13C NMR (150 MHz, Chloroform-d) δ 201.30, 171.03, 170.23, 165.40,157.10, 139.92, 134.01, 121.99, 78.30, 72.28, 61.11, 60.39, 58.13, 56.20,52.02, 48.24, 44.25, 42.69, 39.29, 38.88, 31.24, 30.22, 29.69, 27.43, 24.36,21.39, 21.04, 20.93, 20.72, 15.81, 13.45, 11.69. HRMS (ESI): m / z calcd forC 32 H 43 O8 + [M + H] + : 555.2952; found: 555.2950.

[0048] Preparation of compound 12. Jones' reagent (CrO3 in H2SO4, 2.0 M, 0.32 mL, 0.636 mmol, 6.0 equivalent) was added dropwise to a stirred solution of withaferin A (50.0 mg, 0.106 mmol, 1.0 equivalent) in acetone (3.0 mL) at 0°C. The reaction mixture was allowed to warm naturally to room temperature and stirred for 72 hours. The reaction was quenched by adding i-PrOH (isopropanol) until the orange color disappeared. The mixture was filtered through a short diatomaceous earth mat. The filtrate was concentrated under reduced pressure, and the residue was purified by rapid silica gel column chromatography (eluent: MeOH / CH2Cl2, 1:50 to 1:25) to give compound 12 (16.9 mg, yield: 33%) as a pale yellow solid.

[0049] Compound 12: 1H NMR (600 MHz, Chloroform-d) δ 6.92 – 6.80 (m, 2H), 4.48(dt, J = 13.5, 3.4 Hz, 1H), 3.43 (d, J = 2.5 Hz, 1H), 2.72 (t, J = 16.4 Hz, 1H), 2.32 (dd, J = 14.9, 6.9 Hz, 1H), 2.25 – 2.14 (m, 2H), 2.08 – 1.95 (m,4H), 1.71 – 1.56 (m, 6H), 1.48 – 1.41 (m, 3H), 1.38 (s, 3H), 1.03 (d, J = 6.7Hz, 3H), 0.87 (t, J = 7.1 Hz, 3H), 0.73 (s, 3H). 13 C NMR (150 MHz, Chloroform-d) δ 202.28, 193.93, 141.71, 139.33, 130.15, 130.02, 78.96, 70.67, 64.08,63.62, 55.67, 52.05, 50.00, 43.76, 42.90, 39.55, 38.59, 30.63, 27.36, 24.36,23.60, 22.83, 19.36, 14.27, 13.40, 11.96. HRMS (ESI): m / z calcd for C 28 H 35 O7 + [M + H] + : 483.2377; found: 483.2370.

[0050] Preparation of compound 13. At room temperature, K₂CO₃ (19.9 mg, 0.144 mmol, 3.0 equivalent) was added to a stirred solution of compound 12 (23.0 mg, 0.048 mmol, 1.0 equivalent) in acetone (1.5 mL). Dimethyl sulfate was then added dropwise, and the reaction mixture was stirred at room temperature until complete (TLC monitoring). The reaction mixture was filtered to remove inorganic salts and concentrated under reduced pressure. The residue was diluted with EtOAc, and the aqueous layer was extracted with EtOAc (3 × 5 mL). The combined organic phases were dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. The crude product was purified by rapid silica gel column chromatography (eluent: EtOAc / petroleum ether, 1:3 to 2:1) to give compound 13 (11.2 mg, yield: 47%) as a pale yellow solid.

[0051] Compound 13: 1 H NMR (600 MHz, Chloroform-d) δ 6.89 – 6.82 (m, 2H), 4.45 (dt, J = 13.1, 3.4 Hz, 1H), 3.85 (s, 3H), 3.42 (d, J = 2.5 Hz, 1H), 2.52 (dd,J = 18.0, 13.0 Hz, 1H), 2.21 (t, J = 7.7 Hz, 1H), 2.16 (d, J = 15.1 Hz, 1H), 2.08 (s, 3H), 2.07 – 1.94 (m, 6H), 1.68 – 1.57 (m, 3H), 1.46 – 1.41 (m, 2H),1.37 (s, 3H), 1.01 (d, J = 6.6 Hz, 3H), 0.87 (t, J = 6.9 Hz, 3H), 0.71 (s, 3H). 13 C NMR (150 MHz, CDCl3) δ 202.26, 193.98, 165.59, 162.45, 157.84, 141.72,139.28, 123.46, 78.94, 64.07, 63.63, 55.70, 52.67, 52.18, 49.99, 43.77,42.82, 39.54, 38.79, 30.64, 27.33, 24.37, 23.57, 22.81, 21.64, 19.32, 14.25,13.41, 11.91.HRMS (ESI): m / z calcd for C 29 H 37 O7 + [M + H] + : 497.2534; found:497.2527.

[0052] 1.2 Bioactivity experiment.

[0053] All animal experimental procedures were performed in accordance with the Guidelines for the Care and Use of Laboratory Animals and were approved by the relevant institutional ethics committees. Male C57BL / 6J mice were housed under standard conditions (12-hour light / dark cycle, 22–25°C) with free access to water and feed unless otherwise specified. A diet-induced obesity model was established by feeding mice a 60 kcal high-fat diet (DIO; Research Diets D12492) for 16–18 weeks. Mice reaching a weight of 40–50 g were included in the study and randomly assigned to groups based on baseline weight (and, where applicable, fasting blood glucose). Withaferin A and its derivatives (compounds 2–15) were dissolved in DMSO and administered intraperitoneally once daily at a dose of 1.2 mg / kg for 21 consecutive days; the solvent control group received the corresponding volume of DMSO. Body weight was monitored throughout the administration period. For food intake measurements, mice were housed individually and provided with pre-weighed food; leftover food and spillage were recorded to calculate cumulative intake. For glycemic control assessment, mice were fasted for 14-16 hours before blood glucose measurement. Mice were gently restrained, their tails were cut open with sterile scissors, the first drop of blood was wiped away, and blood glucose levels were measured from the second drop using a handheld glucometer. Data are presented as mean ± SEM (time-major graph) and mean ± SD (endpoint on day 21); the size of each group is indicated in the corresponding legend. Figure 1 and Figure 3 Solvent control group n = 3; each drug administration group n = 5; Figure 3 (Solvent control group n = 2; each drug administration group n = 3). Statistical analysis was performed using GraphPad Prism; endpoint comparisons with the solvent control group were performed using one-way ANOVA followed by Dunnett's multiple comparison test, and p < 0.05 was considered statistically significant.

[0054] 2. Results and Discussion

[0055] 2.1 Effects of withaferin A and its derivatives on body weight in DIO mice

[0056] To assess how withaferin A derivatives affect in vivo weight loss, withaferin A (1) and its derivative series (compounds 2–15) were evaluated in a DIO mouse model under the same dosing regimen (see Experimental Section). Consistent with previous reports, in the context of DIO hyperleptinemia, compound 1, as a leptin-sensitizing linaloolone, produced rapid and sustained weight loss throughout the dosing period compared to the solvent control group. Figure 1 A). Endpoint analysis on day 21 ( Figure 1B) This trend was confirmed, confirming that compound 1 is the best performing member of the series under the current experimental conditions.

[0057] Among the diverse derivatives, the weight change trajectory generally showed a weakening effect or greater volatility compared to the parent skeletal structure. Although some compounds exhibited a partial decreasing trend over time ( Figure 1 (A), but statistics from day 21 showed that only a limited number of analogues retained a significant weight-loss benefit relative to the solvent control group—namely compounds 2, 6, 10, and 12 ( Figure 1 B), and the data for the remaining derivatives are closer to the solvent control group. In particular, most modifications made in the electrophilic core region (focused on modifications of the A / B rings) reduced the in vivo weight-loss activity (e.g., compounds 3–5, 11, and 14), with compound 2 being the only core-modified analog that retained measurable activity. In contrast, some transformations at the C-27 / side chain site were partially tolerated: simple esterification (compounds 6 and 10) and oxidation to the corresponding carboxylic acid (compound 12) still maintained significant (albeit reduced) weight-loss effects, while other side-chain / lactone modifications (compounds 7–9, 13, and deoxygenated reference 15) did not yield statistically significant benefits over the solvent control group. Notably, no derivative was more effective than compound 1, suggesting that the natural functional group combination of withaferin A (1) still exhibits an unusually high degree of optimization in the DIO weight-loss phenotype of this study.

[0058] 2.2 Effects of withaferin A and its derivatives on food intake in DIO mice

[0059] Given that the weight loss induced by withaferin A (1) in hyperleptinemic DIO mice was associated with reduced calorie intake, we then quantified food consumption throughout the administration period. Figure 2 Consistent with its significant weight-weight effect, 1 produced the most significant and sustained cumulative food intake inhibition over 21 days compared to the solvent control (DMSO). Figure 3 A) showed differences early in the study and remained lower than all other treatment groups throughout the study.

[0060] In contrast, most derivatives showed only modest and heterogeneous effects on food intake. While some analogs showed a trend toward reducing cumulative intake compared to solvent controls (…),… Figure 2 (A), but none could surpass the inhibitory magnitude or consistency observed with compound 1. Endpoint analysis on day 21 reinforced this pattern: compared to the solvent control, compound 1 significantly reduced total food intake (A). Figure 2(B) In this study design, none of the other analogues achieved statistically significant inhibitory effects.

[0061] In summary, these feeding phenotypes are consistent with the structure-dependent pharmacodynamic trends observed in the weight outcomes, further supporting withaferin A (1) as a rigorous benchmark in this model. Most withaferin A derivatives generally exhibit attenuated or fluctuating feeding inhibition, suggesting that maintaining the natural functional group arrangement of compound 1 is crucial for preserving the appetite-related portion of its anti-obesity activity.

[0062] 2.3 Effects of withaferin A and its derivatives on glycemic control in DIO mice

[0063] In the same batch of DIO mice, glycemic control was monitored longitudinally (days 0, 7, 14, and 21). Figure 3 A), and a summary was made at the end of day 21 (A). Figure 3 B). Under our administration conditions, withaferin A (1) showed the clearest and most sustained hypoglycemic effect, with an early decrease in blood glucose on day 7 and sustained until day 21, reaching the lowest endpoint blood glucose value among the withaferin A derivatives tested. In contrast, most derivatives showed only slight or fluctuating deviations from the solvent control group throughout the time period and failed to achieve the sustained effect of compound 1 (B). Figure 3 ).

[0064] From a structure-activity relationship perspective, these curves indicate that modifications to withaferin A generally weaken the durability of its glycemic benefit. Core-modified A / B ring variants (e.g., epoxide derivatives, i.e., compounds 3 / 11 and ortho-diols, i.e., compounds 4) and side-chain / δ-lactone modifications (benzoyl / acetyl homologues, i.e., compounds 6 / 10, and lactone / side-chain modified acids / esters, i.e., compounds 12 / 13) showed data points largely closer to the solvent control group by day 21. Notably, the effects of referencing asholinone 4-epi-5,6-deoxywithaferin A (compound 14) and 27-deoxywithaferin A (compound 15) also did not surpass those of compound 1, further confirming that the parent scaffold remains a stringent benchmark for glycemic control in DIO mice.

[0065] In summary, in the DIO mouse model, withaferin A consistently produced the most significant and durable reductions in body weight, food intake, and glycemic index, while most withaferin A derivatives exhibited diminished and / or more volatile efficacy, none surpassing the parent natural product withaferin A. Only a few derivatives (compounds 2, 6, 10, and 12) retained some endpoint benefits, highlighting a steep activity cliff and the stringent structural requirements for maintaining the in vivo metabolic phenotype. In conclusion, these negative but informative results define the practical structure-activity relationship boundaries of withaferin A derivatizations and lay the foundation for the development of next-generation ashinaferone small molecules for obesity / T2DM intervention.

Claims

1. A bisbenzoylsodium benzoate A or a pharmaceutically acceptable salt thereof having the structure shown in Formula 6 below: 。 2. The method for synthesizing bisbenzoylated solanine A according to claim 1, characterized in that, Solanine A was dissolved in an organic solvent, and benzoic anhydride was added in the presence of triethylamine to react and obtain the crude product of the target compound.

3. The synthesis method according to claim 2, characterized in that, The organic solvent is 1,2-dichloroethane, dichloromethane, chloroform, or acetone.

4. The synthesis method according to claim 2, characterized in that, The reaction was carried out without heating.

5. The synthesis method according to any one of claims 2 to 4, characterized in that, It also includes a step of purifying the crude target compound obtained.

6. The use of the bisbenzoylated solanine A or a pharmaceutically acceptable salt thereof as described in claim 1 in the preparation of a medicament for the prevention or treatment of obesity.

7. A pharmaceutical composition comprising a therapeutically effective dose of the bisbenzoylated solanine A of claim 1 or a pharmaceutically acceptable salt thereof.

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