Isflavone compound, preparation method and application thereof

Abstract

This invention discloses an isoflavone compound, its preparation method, and its applications. The compounds are derived by directional esterification modification of all phenolic hydroxyl groups with allyloxycarbonyl chloride, o-chlorobenzoyl chloride, or cinnamoyl chloride, using 4',7-dihydroxyisoflavone or 4',5,7-trihydroxyisoflavone as the parent nucleus. The invention also provides a mild and efficient preparation method for these compounds and confirms their significant anti-inflammatory pharmacological activity. Pharmacological studies show that compounds B and E, in particular, exhibit excellent in vitro and in vivo anti-inflammatory activity, with compound E showing superior activity compared to the positive control drug dexamethasone. These compounds can be used to prepare drugs for treating inflammatory diseases such as pneumonia and colitis.

Description

Technical Field

[0001] This invention relates to the field of medicinal chemistry, and more particularly to an isoflavone compound, its preparation method, and its application. Background Technology

[0002] Isoflavones are a class of natural polyphenols widely found in legumes, possessing the basic skeletal structure of benzo-γ-pyranone. Among them, 4',7-dihydroxyisoflavone (daidzein) and 4',5,7-trihydroxyisoflavone (genistein) are two of the most representative natural isoflavone monomers, known to possess various biological activities such as antioxidant, anti-inflammatory, antitumor, and bone metabolism regulation.

[0003] Although natural isoflavones have shown some anti-inflammatory potential in in vitro experiments, their clinical translation faces three major bottlenecks: First, their bioavailability is extremely low. Natural isoflavone molecules contain multiple free phenolic hydroxyl groups, which are highly polar and poorly lipid-soluble, making it difficult for them to effectively penetrate cell membranes and biological barriers (such as the blood-brain barrier and intestinal mucosal barrier).

[0004] Second, their anti-inflammatory activity is limited. Multiple studies have shown that natural isoflavones have a weak inhibitory effect on inflammatory factors, with a low half-maximal inhibitory concentration (IC50). 50 The concentration of natural isoflavones is typically above 50-100 μmol / L, far exceeding that of commonly used anti-inflammatory drugs (such as dexamethasone). This means that to achieve the same efficacy, natural isoflavones require extremely high dosages, which not only increases the cost of medication but may also lead to potential toxic side effects due to high doses.

[0005] Third, structural modification strategies have limitations. To improve the medicinal properties of natural isoflavones, various structural modification methods have been explored in existing technologies, mainly including: Glycosylation modification: Although it can improve water solubility, it often further reduces cell membrane permeability, and glycosidic bonds are easily broken down by enzymes in vivo; Single-position alkylation / acylation: only partially modifies the hydroxyl group, resulting in limited improvement and potentially disrupting the spatial conformation of the pharmacophore; Full methylation modification: Although it can significantly improve lipophilicity, the methyl group is small in size and it is difficult to generate additional target binding, so the activity improvement is not obvious; Modification with sterically hindered groups: Some studies have attempted to introduce large-volume groups, but these often hinder the binding of compounds to target proteins due to steric hindrance, resulting in loss of activity.

[0006] In addition, existing synthetic processes for isoflavone derivatives mostly employ traditional acylation methods, which require high-temperature reactions under strong base (such as pyridine, triethylamine) or strong acid catalysis. These harsh conditions can easily lead to the destruction of the isoflavone core skeleton or side reactions, resulting in low product yields and difficulty in ensuring purity, which is not conducive to industrial production.

[0007] In summary, developing a class of isoflavone derivatives that can significantly improve physicochemical properties and bioavailability, greatly enhance anti-inflammatory activity, and have a mild and efficient synthesis process remains a pressing technical challenge in this field. Summary of the Invention

[0008] This invention addresses the key technical bottlenecks in natural isoflavone compounds, such as their inherent defects (excessive water solubility, poor lipid solubility, low cell membrane permeability, and rapid metabolism in vivo due to polyphenolic hydroxyl groups), which lead to weak anti-inflammatory activity and difficulty in drug formulation. It proposes an isoflavone compound, its preparation method, and its application.

[0009] To solve the above-mentioned technical problems, the technical solution of the present invention is as follows: an isoflavone compound, wherein the compound is selected from one or more of compounds A-F, and the structural formulas are shown as AF respectively: .

[0010] Preferably, the compound is selected from compound B or compound E, and the preferred compound has stronger anti-inflammatory activity, even superior to the positive control drug.

[0011] The present invention also provides a method for preparing the isoflavone compounds, comprising the following steps: Step (1) Select 4',7-dihydroxyisoflavone or 4',5,7-trihydroxyisoflavone as the parent substrate; Step (2) Select the corresponding acyl chloride reagent according to the target product: allyloxycarbonyl chloride, o-chlorobenzoyl chloride or cinnamoyl chloride; Step (3) Dissolve the parent substrate and phase transfer catalyst in an organic solvent (such as DCM) at 10-40℃, add an inorganic base aqueous solution, and add acyl chloride reagent dropwise while stirring for 6-16 hours; Step (4) After the reaction is complete, the target compound is obtained by extraction, washing, drying and column chromatography purification.

[0012] Preferably, the phase transfer catalyst is Aliquat 336; the molar ratio of the parent substrate to the acyl chloride reagent is 1:2-5.

[0013] The present invention also provides a pharmaceutical composition comprising the isoflavone compound or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or excipient.

[0014] The present invention also provides the use of the isoflavone compounds or the pharmaceutical compositions in the preparation of medicaments for treating inflammatory diseases.

[0015] Preferably, the inflammatory disease is selected from pneumonia, colitis, sepsis, rheumatoid arthritis, or asthma.

[0016] As a further description of the above scheme: the drug exerts its anti-inflammatory effect by inhibiting the TLR4 / MyD88 / NF-κB signaling pathway; and exerts its tissue repair effect by regulating Bcl-2 / Bax-mediated apoptosis and Beclin1 / LC3-mediated autophagy.

[0017] This invention represents the first systematic approach to simultaneously, efficiently, and selectively esterify all phenolic hydroxyl groups on the core of 4',7-dihydroxy or 4',5,7-trihydroxy isoflavones, rather than modifying only certain sites or employing traditional methods such as glycosylation or methylation. This strategy, while preserving the basic skeleton of the core, fundamentally alters its polarity distribution, significantly improving lipophilicity and cellular uptake efficiency, providing a novel pathway to overcome the low bioavailability of natural isoflavones.

[0018] Although the acyl chloride reagents used (such as allyloxycarbonyl chloride, o-chlorobenzoyl chloride, and cinnamoyl chloride) have been reported in organic synthesis, only combinations of a specific core (trihydroxyisoflavones) and a specific substituent (o-chlorobenzoyl) can produce breakthrough anti-inflammatory activity. Experimental results show that other combinations (such as dihydroxy core + o-chlorobenzoyl, or trihydroxy core + cinnamoyl) do not enhance activity as much as compound E.

[0019] Of the compounds obtained, compound E exhibited an IC50 inhibition of LPS-induced NO release in an in vitro RAW264.7 cell model. 50 The concentration reached 4.465 μmol / L, significantly better than dexamethasone (IC50), a first-line clinical drug. 50 = 7.971 μmol / L), while the original parent nucleus has extremely weak activity (IC50). 50 (>50 μmol / L), this order-of-magnitude increase in activity achieved unexpected technical results.

[0020] Compound E not only exhibits excellent in vitro performance but also demonstrates significant therapeutic effects in animal models of acute pneumonia and ulcerative colitis. Mechanistic studies reveal that it achieves multi-target anti-inflammatory effects through dual regulation—inhibiting the TLR4 / MyD88 / NF-κB inflammatory signaling pathway while simultaneously modulating the balance between Bcl-2 / Bax-mediated apoptosis and Beclin1 / LC3-mediated autophagy. This mechanism differs from that of simple glucocorticoid drugs, providing a new approach for developing safe and highly effective novel anti-inflammatory drugs.

[0021] Compared with the prior art, the present invention has the following beneficial effects: This invention effectively reduces molecular polarity and significantly improves the lipophilicity and transmembrane ability of the compound by modifying all phenolic hydroxyl groups in the 4',7-dihydroxy and 4',5,7-trihydroxy isoflavone cores with hydrophobic groups, thus laying a solid physicochemical foundation for its targeting of inflammatory sites and exertion of pharmacological effects in vivo.

[0022] The modified compounds, especially compounds B and E, exhibited in vitro anti-inflammatory activity (inhibition of IC50 by NO). 50 (Measured) The activity was several times higher than that of the unmodified natural nucleus. Among them, compound E even surpassed the activity of the positive control drug dexamethasone, showing great potential to become a new generation of highly effective anti-inflammatory drugs.

[0023] In vivo experiments demonstrated that compound E can effectively alleviate inflammatory damage in the lungs and intestines by inhibiting the key TLR4 / MyD88 / NF-κB inflammatory signaling pathway and regulating Bcl-2 / Bax-mediated apoptosis and Beclin1 / LC3-mediated autophagy. This provides strong experimental evidence for its application in the treatment of various inflammatory diseases such as pneumonia and colitis.

[0024] The preparation method provided by this invention has mild reaction conditions, does not require high temperature, high pressure or precious metal catalysts, has simple post-processing, and produces high product yield and good purity. It is very suitable for laboratory research and future industrial production, and has good application prospects and economic value. Attached Figure Description

[0025] Figure 1 This is the mass spectrum of compound A; Figure 2 The hydrogen NMR spectrum of compound A; Figure 3 Here is the carbon NMR spectrum of compound A; Figure 4 This is the mass spectrum of compound B; Figure 5 The hydrogen NMR spectrum of compound B; Figure 6 This is the carbon NMR spectrum of compound B; Figure 7 This is the mass spectrum of compound C; Figure 8 The hydrogen NMR spectrum of compound C; Figure 9 This is the carbon NMR spectrum of compound C; Figure 10 This is the mass spectrum of compound D; Figure 11 The hydrogen NMR spectrum of compound D; Figure 12The carbon NMR spectrum of compound D; Figure 13 This is the mass spectrum of compound E; Figure 14 The hydrogen NMR spectrum of compound E; Figure 15 The carbon NMR spectrum of compound E; Figure 16 Mass spectrum of compound F; Figure 17 The hydrogen NMR spectrum of compound F; Figure 18 The carbon NMR spectrum of compound F; Figure 19 Pathological sections of mouse lung tissue from each group in Example 9 (HE staining, ×200); Figure 20 The levels of TNF-α (A), IL-1β (B), and IL-6 (C) in the bronchoalveolar lavage fluid of mice in each group in Example 9; Figure 21 Electrophoresis images of various proteins in the lung tissue of mice in each group in Example 9 (A), and comparison of the protein levels of TLR4 (B), MyD88 (C), and NF-κBp65 (D); (x ± s, n=3), compared with the model group, *P<0.05, **P<0.01; Figure 22 Pathological sections of colon tissue from each group of mice in Example 10 (HE staining, ×200). Figure 23 The levels of key inflammatory factors such as TNF-α (A), IL-6 (B), IL-1β (C), and IL-12 (D) in the serum of mice in each group of Example 10 were recorded. Figure 24 Electrophoresis images of various proteins in the colon tissue of mice in Example 10 (A), and the protein expression levels of Bcl-2 (B), Bax (C), Beclin1 (D), and LC3 (E); (x ± s, n=3). Compared with the model group, *P<0.05, **P<0.01. Detailed Implementation

[0026] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. However, the present invention is not limited to the following technical solutions. Unless otherwise specified, all reagents used in the present invention are purchased from reagent companies.

[0027] Example 1 Preparation of Compound A

[0028] 4',7-Dihydroxyisoflavone (1270 mg, 5 mmol) was dissolved in 50 mL of DCM at room temperature (15-25℃), and NaOH (3M, 10 mL) and 1 drop of Aliquat 336 were added and stirred until homogeneous. Allyl chloroformate (1920 mg, 16 mmol, 3.2 eq) was then added dropwise, and the reaction was allowed to proceed overnight. The reaction mixture was washed twice with 100 mL of water, and the organic phase was collected and dried over anhydrous sodium sulfate. The solvent was removed by rotary evaporation under vacuum, and the mixture was separated by rapid silica gel column chromatography. Recrystallization from PE yielded 1.332 g of a white solid, with a recovery rate of 63.1%.

[0029] Structural analysis revealed that the white solid was compound A (4',7-di-O-(allyloxycarbonyl) isoflavone), as shown below. Figure 1-3 As shown. ESI-HRMS: m / z [M + Na] + calcd for C 23 H 18 O8Na:445.0899; found: 445.0903. 1 HNMR (500 MHz, CDCl3) δ 8.33 (d, J = 8.7 Hz, 1H), 8.01 (s, 1H), 7.67- 7.52 (m,2H), 7.41 (d, J = 2.2 Hz, 1H), 7.30- 7.26 (m, 3H), 6.01 (ddtd, J = 17.2,10.4, 5.9, 2.4 Hz, 2H), 5.53- 5.27 (m, 4H), 4.77 (ddt, J = 15.4, 5.9, 1.3 Hz, 4H). 13 C NMR (125MHz, CDCl3) δ 175.35, 156.63, 154.75, 153.37, 153.28, 152.46,151.14, 131.07, 131.07, 130.69, 130.12, 129.38, 128.06, 124.75, 122.40,121.23, 121.23, 120.13, 119.67, 119.01, 110.38, 69.71, 69.29.

[0030] Example 2 Preparation of Compound B

[0031] 4',7-Dihydroxyisoflavone (1270 mg, 5 mmol) was dissolved in 50 mL of DCM at room temperature. NaOH (3 M, 10 mL) and 1 drop of Aliquat 336 were added and stirred until homogeneous. Then, o-chlorobenzoyl chloride (2450 mg, 14 mmol, 2.8 eq) was added dropwise, and the reaction was allowed to proceed overnight. The reaction solution was washed twice with 100 mL of water, and the organic phase was collected and dried over anhydrous sodium sulfate. The solvent was removed by rotary evaporation under vacuum. The solution was separated by rapid silica gel column chromatography, recrystallized from PE, and a white solid of 1.374 g was collected (recovery: 51.8%).

[0032] Structural analysis revealed that the white solid was compound B (4',7-di-O-(o-chlorobenzoyl) isoflavone), such as Figure 4-6 As shown. EI-HRMS: m / z [M + Na] + calcd for C 29 H 16 Cl2O6Na:553.0222; found: 553.0225. 1 HNMR (500 MHz, CDCl3) δ 8.40 (d, J = 8.7 Hz, 1H), 8.13 - 8.05 (m, 3H), 7.71-7.63 (m, 2H), 7.57- 7.49 (m, 5H), 7.45- 7.40 (m, 2H), 7.35 (dd, J = 8.6, 1.8Hz, 3H). 13 C NMR (125 MHz, CDCl3) δ 175.48, 164.01, 163.10, 156.73, 154.52,153.33, 150.76, 134.79, 134.48, 133.80, 133.29, 132.15, 131.97, 131.61,131.40, 130.20, 130.20, 129.49, 129.22, 128.37, 128.05, 126.93, 126.80,124.85, 122.54, 121.81, 121.81, 119.69, 111.17.

[0033] Example 3 Preparation of Compound C

[0034] 4',7-Dihydroxyisoflavone (1270 mg, 5 mmol) was dissolved in 50 mL of DCM at room temperature. NaOH (3 M, 10 mL) and 1 drop of Aliquat 336 were added and stirred until homogeneous. Cinnamyl chloride (2324 mg, 14 mmol, 2.8 eq) was then added dropwise and the reaction mixture was allowed to react overnight. The reaction solution was washed twice with 100 mL of water, and the organic phase was collected and dried over anhydrous sodium sulfate. The solvent was removed by rotary evaporation under vacuum. The mixture was separated by rapid silica gel column chromatography, recrystallized from PE, and a white solid of 1.432 g was collected (recovery: 55.7%).

[0035] Structural analysis revealed that the white solid was compound C (4',7-di-O-(cinnamoyl) isoflavone), such as Figure 7-9 As shown. EI-HRMS: m / z [M + Na] + calcd for C 33 H 22 O6Na:537.1314; found: 537.1314. 1 H NMR (500 MHz, CDCl3) δ 8.37 (d, J = 8.7 Hz, 1H), 8.05 (s, 1H), 7.92 (dd, J =17.5, 15.9 Hz, 2H), 7.62 (dtd, J = 8.4, 6.1, 2.5 Hz, 6H), 7.50- 7.39 (m, 7H), 7.30- 7.26 (m, 3H), 6.66 (dd, J = 16.0, 1.7 Hz, 2H). 13 C NMR (125 MHz, CDCl3)δ 175.54, 165.39, 164.54, 156.73, 154.78, 153.24, 150.88, 147.86, 146.83,134.17, 133.89, 131.14, 130.79, 130.10, 130.10, 129.20, 129.12, 129.12,129.04, 129.04, 128.49, 128.49, 128.37, 128.37, 127.89, 124.85, 122.28,121.83, 121.83, 119.73, 117.19, 116.41, 111.04.

[0036] Example 4 Preparation of Compound D

[0037] 4',5,7-Trihydroxyisoflavone (1350 mg, 5 mmol) was dissolved in 50 mL of DCM at room temperature. NaOH (3 M, 10 mL) and 1 drop of Aliquat 336 were added and stirred until homogeneous. Allyl chloroformate (2880 mg, 24 mmol, 4.8 eq) was then added dropwise, and the reaction was allowed to proceed overnight. The reaction mixture was washed twice with 100 mL of water, and the organic phase was collected and dried over anhydrous sodium sulfate. The solvent was removed by rotary evaporation under vacuum. The mixture was separated by rapid silica gel column chromatography, recrystallized from PE, and a white solid of 1.339 g was collected (recovery: 51.3%).

[0038] Structural analysis revealed that the white solid was compound D (5,7,4'-tris-O-(allyloxycarbonyl) isoflavone), such as Figure 10-12 As shown. ESI-HRMS: m / z [M + Na] + calcd for C 27 H 22 O 11 Na:545.1060; found:545.1064. 1 HNMR (500MHz, CDCl3) δ 7.94 (s, 1H), 7.60- 7.47 (m, 2H), 7.40 (d, J = 2.3 Hz, 1H), 7.27 (dd, J = 8.8, 6.7 Hz, 2H), 7.07 (d, J = 2.4 Hz, 1H), 6.12- 5.91 (m, 3H), 5.57-5.21 (m, 6H), 4.88-4.67 (m, 6H). 13 C NMR (125MHz, CDCl3) δ 174.24,157.63, 154.00, 153.35, 152.51, 152.18, 151.87, 151.19, 150.75, 131.20,131.07, 130.54, 130.33, 130.33, 128.92, 125.62, 121.16, 121.16, 120.30,119.67, 119.46, 115.90, 113.06, 108.75, 69.86, 69.83, 69.28.

[0039] Example 5 Preparation of Compound E

[0040] 4',5,7-Trihydroxyisoflavone (1350 mg, 5 mmol) was dissolved in 50 mL of DCM at room temperature. NaOH (3 M, 10 mL) and 1 drop of Aliquat 336 were added and stirred until homogeneous. Then, o-chlorobenzoyl chloride (3675 mg, 21 mmol, 4.2 eq) was added dropwise, and the reaction was allowed to proceed overnight. The reaction solution was washed twice with 100 mL of water, and the organic phase was collected and dried over anhydrous sodium sulfate. The solvent was evaporated to dryness under vacuum, and the mixture was separated by rapid silica gel column chromatography. Recrystallization from PE yielded 1.478 g of a white solid, with a recovery rate of 43.2%.

[0041] Structural analysis revealed that the white solid was compound E (5,7,4'-tri-O-(o-chlorobenzoyl) isoflavone), such as Figure 13-15 As shown. EI-HRMS: m / z [M + Na] + calcd for C 36 H 19 Cl3O8Na: 707.0043; found: 707.0042. 1 H NMR (500 MHz, CDCl3) δ 8.37 (dd, J = 7.8, 1.6 Hz, 1H), 8.11- 8.06(m, 1H), 8.04 (dd, J = 7.8, 1.6 Hz, 1H), 7.98 (s, 1H), 7.59- 7.54 (m, 4H), 7.52- 7.48 (m, 4H), 7.48 - 7.36 (m, 4H), 7.32 - 7.28 (m, 2H), 7.18 (d, J =2.3 Hz, 1H). 13C NMR (125 MHz, CDCl3) δ 174.15, 163.98, 163.35, 162.54,157.82, 153.84, 152.17, 150.79, 150.56, 134.94, 134.57, 134.46, 133.96,133.27, 133.18, 133.01, 132.23, 131.94, 131.67, 131.37, 131.02, 130.44,130.44, 129.22, 129.13, 128.80, 128.04, 126.97, 126.91, 126.78, 125.80, 121.76, 121.76, 116.05, 114.37, 109.61.

[0042] Example 6 Preparation of compound F

[0043] 4',5,7-Trihydroxyisoflavone (1350 mg, 5 mmol) was dissolved in 50 mL of DCM at room temperature. NaOH (3 M, 10 mL) and 1 drop of Aliquat 336 were added and stirred until homogeneous. Cinnamyl chloride (3486 mg, 21 mmol, 4.2 eq) was then added dropwise and the reaction mixture was allowed to react overnight. The reaction solution was washed twice with 100 mL of water, and the organic phase was collected and dried over anhydrous sodium sulfate. The solvent was evaporated to dryness under vacuum, and the mixture was separated by rapid silica gel column chromatography. Recrystallization from PE yielded 1.486 g of a white solid, with a recovery rate of 45.0%.

[0044] Structural analysis revealed that the white solid was compound F (5,7,4'-tri-O-(cinnamoyl) isoflavone), such as Figure 16-18 As shown. EI-HRMS: m / z [M + Na] + calcd for C 42 H 28 O8Na: 683.1682; found: 683.1682. 1 H NMR (500 MHz, CDCl3) δ 7.97- 7.82 (m, 4H), 7.60 (ddd, J = 14.6, 6.4, 3.0 Hz, 6H),7.54 (d, J = 8.3 Hz, 2H), 7.48- 7.38 (m, 10H), 7.21 (d, J = 8.4 Hz, 2H), 7.05(d,J = 2.3 Hz, 1H), 6.76 (d, J = 16.0 Hz, 1H), 6.63 (dd, J = 16.0, 3.6 Hz, 2H). 13 C NMR (125 MHz, CDCl3) δ 174.31, 165.35, 165.13, 164.05, 157.83,154.12, 152.08, 150.88, 150.78, 148.16, 147.19, 146.76, 134.29, 134.16,133.82, 131.22, 130.76, 130.63, 130.36, 129.13, 129.13, 129.06, 129.02,129.02, 128.89, 128.74, 128.62, 128.54, 128.54, 128.51, 128.41, 128.35, 128.35, 125.81, 121.78, 121.78, 117.19, 117.00, 116.19, 115.88, 114.27, 109.21.

[0045] Example 7: Study on the physicochemical properties and cellular uptake of the compound 1. Determination of the n-octanol-water partition coefficient (Log P) Log P is a key indicator for evaluating the lipid-water partition properties of compounds and is closely related to membrane permeability and bioavailability. This study used the shake-flask method to determine the Log P values ​​of two natural isoflavone cores and six esterified derivatives, exploring the effects of the number of hydroxyl groups in the core and the type of esterified substituents on their lipid solubility. The results are shown in Table 1.

[0046] The number of hydroxyl groups in the parent nucleus is a core factor affecting lipophilicity. 4',7-Dihydroxyisoflavone (Log P=3.417) has significantly higher lipophilicity than 4',5,7-Trihydroxyisoflavone (Log P=2.593), with a difference of 0.824, indicating that the number of hydroxyl groups is negatively correlated with lipophilicity. The more hydroxyl groups, the stronger the hydrophilicity and the lower the lipophilicity, which is consistent with the structure-activity relationship of isoflavones.

[0047] Esterification significantly improves the lipophilicity of isoflavones, with the increase being greater after modification of the trihydroxy nucleus. The Log P values ​​of dihydroxy nucleus derivatives A, B, and C increased by 1.116, 2.206, and 2.043 compared to the nucleus, respectively; while those of trihydroxy nucleus derivatives D, E, and F increased by 1.834, 3.837, and 2.890, respectively. Due to the lower initial lipophilicity of the trihydroxy nucleus, the modification effect is more significant.

[0048] Substituent type affects the enhancement of lipophilicity: allyloxycarbonyl (aliphatic) modification has the lowest Log P values ​​for A and D, indicating the weakest enhancement effect; cinnamoyl and o-chlorobenzoyl (aromatic) modifications are more effective, as the hydrophobic skeleton of the aromatic ring can enhance lipophilicity. Furthermore, there is a synergistic effect between the substituent and the parent nucleus hydroxyl group. Among the trihydroxy parent nucleus, cinnamoyl (compound E, Log P = 6.430) shows the best effect, while among the dihydroxy parent nucleus, o-chlorobenzoyl (compound B, Log P = 5.623) is more advantageous.

[0049] In summary, the lipid solubility of isoflavones is determined by both the number of hydroxyl groups in the parent nucleus and the type of esterification substituents: the more hydroxyl groups, the lower the lipid solubility; aromatic acyl modifications have a better effect on improving lipid solubility than aliphatic modifications, and this effect is positively correlated with the number of hydroxyl groups. This study provides experimental evidence for the targeted structural modification and bioactivity research of isoflavones.

[0050] Table 1. Results of Log P measurement (x ± s)

[0051] 2. Artificial Membrane Permeability Experiment Parallel artificial membrane permeability assay (PAMPA) is a core in vitro model simulating the intestinal or biomembrane barrier. It accurately reflects the transmembrane transport capacity and oral absorption potential of molecules by detecting the apparent permeability coefficient (Pe) of compounds. This study used the PAMPA model to systematically evaluate the transmembrane properties of two natural isoflavone cores and six esterified derivatives. The results are shown in Table 2.

[0052] The basal permeability of the natural isoflavone nucleus is extremely low and strictly dependent on the configuration of the nucleus's hydroxyl groups. The Pe value of 4',7-dihydroxyisoflavone (natural nucleus 1) is 0.827 × 10⁻⁶. -6 cm / s, significantly higher than that of 4',5,7-trihydroxyisoflavones containing three hydroxyl groups (natural parent nucleus 2, 0.533×10 cm / s). -6 This result is in high agreement with the Log P law, confirming that the increased number of hydroxyl groups leading to strong hydrophilicity is a key factor hindering molecules from penetrating hydrophobic lipid membranes.

[0053] All esterification modifications significantly increased the Pe value of the compounds, verifying the decisive role of improved lipophilicity in membrane transport. Compared to the corresponding parent nucleus, the permeability of the derivatives increased by 4-11 times. Among them, aromatic acyl modifications were generally more effective than aliphatic modifications: in the dihydroxy parent nucleus system, compound B modified with o-chlorobenzoyl (5.707 × 10⁻⁶) showed the best permeability increase. -6 (cm / s) and cinnamoyl-modified compound C (5.483×10) -6The permeability (cm / s) was similar, both at a high level; however, in the trihydroxy core system, the cinnamon yl modified compound E performed best, with a Pe value as high as 6.147 × 10⁻⁶. -6 cm / s, the highest among all tested compounds.

[0054] In summary, esterification modification effectively overcomes the membrane permeation bottleneck of natural isoflavones by improving lipophilicity. These results clarify the structure-activity relationship of "structural modification-enhanced lipophilicity-improved permeability," providing direct experimental support for designing high-bioavailability isoflavone neuroprotective drug candidates.

[0055] Table 2. Results of apparent permeability coefficient determination of the compounds (x ± s)

[0056] 3. Cellular uptake assay: Determination of intracellular drug concentration in RAW264.7 cells. Cellular uptake capacity is a key indicator for evaluating drug bioavailability and efficacy, directly determining the effective concentration of the drug in target cells. In this experiment, RAW264.7 cells were used as a model. Cells were co-incubated with the same concentration (10 μM) of natural isoflavone cores and esterified derivatives for a certain period. Cells were then lysed, and intracellular drug concentrations were accurately detected using HPLC / MS to investigate the effect of structural modification on the cellular uptake capacity of isoflavone compounds. The experimental results are shown in Table 3.

[0057] Experimental results showed that the cellular uptake capacity of natural isoflavone nuclei was extremely low and negatively correlated with the number of hydroxyl groups in the nucleus. The intracellular drug concentration of 4',7-dihydroxyisoflavone (natural nucleus 1) was 12.720 nmol / L, while the intracellular concentration of 4',5,7-trihydroxyisoflavone (natural nucleus 2) was only 8.567 nmol / L, the latter being 32.6% lower than the former. This suggests that the more hydroxyl groups in the nucleus, the stronger the hydrophilicity, and the more difficult it is for the nucleus to penetrate the cell membrane and enter the cell.

[0058] The intracellular drug concentrations of all esterified derivatives were significantly higher than those of their corresponding natural parent nuclei, confirming that esterification modification can effectively enhance the cellular uptake of isoflavones. Among them, aromatic acyl-modified derivatives showed significantly better uptake effects than aliphatic allyloxycarbonyl-modified derivatives; and the cellular uptake enhancement of derivatives modified with trihydroxy parent nuclei was generally greater than that of derivatives modified with dihydroxy parent nuclei.

[0059] Compound E (trihydroxy+cinnamyl) exhibited the highest intracellular drug concentration at 188.243 nmol / L, which was 22.09 times and 14.79 times higher than that of the natural parent nucleus 2 and parent nucleus 1, respectively. Compound F (165.417 nmol / L) and compound B (158.093 nmol / L) followed. In summary, cinnamyl modification showed the best effect in enhancing the cellular uptake of isoflavones, providing experimental evidence for the subsequent design of highly bioavailable isoflavone derivatives.

[0060] Table 3 Intracellular drug concentrations of compounds (nmol / L, x ± s)

[0061] Example 8: In vitro anti-inflammatory activity of compound AF and comparative study with its natural parent nucleus. This experiment used RAW264.7 macrophages in the logarithmic growth phase, at a concentration of 5 × 10⁻⁶ cells / cells. 4 One sample per well was seeded into a 96-well plate, divided into a blank control group, an LPS model group, experimental groups with different concentrations of compound AF, a natural parent nucleus control group, and a dexamethasone positive control group. Except for the blank control group, the other groups were treated with LPS (1 μg / mL) to induce inflammation. After 24 hours of culture, the supernatant was collected, and NO production was detected using Griess' reagent. The half-maximal inhibitory concentration (IC50) of each compound against NO was calculated. 50 The results are shown in Table 4.

[0062] Table 4. Half-maximal inhibitory concentrations (IC50) of each compound and its natural parent nucleus against NO. 50 Comparison (x ± s)

[0063] IC 50 IC50 is a key indicator for evaluating the inhibitory activity of compounds. 50 The lower the value, the stronger the inhibitory activity of the compound against NO. This study found that the IC50 values ​​for NO in each group of cells... 50 The values ​​(μmol / L) are sorted from lowest to highest as follows: Compound E (4.465) < Dexamethasone (7.971) < Compound B (8.680) < Compound D (14.383) < Compound F (17.090) < Compound C (19.446) < Compound A (23.094) < 4',7-dihydroxyisoflavone (54.954) < 4',5,7-trihydroxyisoflavone (69.728). It is evident that modification of the parent nucleus enhances its anti-inflammatory activity, with Compound E exhibiting the best anti-inflammatory activity and its IC50 value being [missing value]. 50The value was 4.465 μmol / L, the lowest among all tested compounds, and significantly lower than the positive control drug dexamethasone (7.971 μmol / L), indicating that it has a strong inhibitory effect on NO and is the candidate compound with the strongest anti-inflammatory activity in this invention. The anti-inflammatory activity of compound B is close to that of the positive control drug dexamethasone (8.680 μmol / L), indicating that its anti-inflammatory activity is comparable to that of the positive control drug and it has good development potential. In summary, compounds E and B have significant inhibitory activity against inflammatory factors and NO, especially compound E, whose activity is superior to that of the positive control drug dexamethasone, and can be considered as key research targets for subsequent anti-inflammatory drug development.

[0064] Example 9: In vivo anti-inflammatory effect of compound E on mice with acute pneumonia. In this study, BALB / c mice were randomly divided into a control group (blank group), a model group, a positive control group (azithromycin 20 mg / kg / d), and low (20 mg / kg), medium (40 mg / kg), and high (60 mg / kg) groups of compound E, with 8 mice in each group. Before the experiment, the mice were anesthetized with isoflurane. Subsequently, lipopolysaccharide (LPS) was dripped onto the posterior pharyngeal wall of the model and drug-treated groups to establish an acute pneumonia model, while the control group was dripped with physiological saline. After 5 consecutive days of gavage administration, the anti-inflammatory effect of compound E was measured by evaluating lung tissue pathological scores, the levels of inflammatory factors in bronchoalveolar lavage fluid, and lung tissue protein expression.

[0065] The experimental results showed that, compared with the model group, alveolar structural disorder and inflammatory cell infiltration were significantly increased in the model group. However, in the high-dose compound E group, the pathological morphology was similar to that of the control group, and the pathological score was significantly reduced. Figure 19 Meanwhile, the levels of TNF-α, IL-6, and IL-1β in the bronchoalveolar lavage fluid of mice in compound E group were significantly lower than those in the model group. Figure 20 Furthermore, in the lung tissue of mice in the high-dose group of compound E, the expression levels of TLR4, MyD88, and NF-κB p65 proteins were significantly reduced, and this effect showed a clear dose-dependent effect. Figure 21 Based on the above results, we can conclude that compound E can effectively reduce lung inflammation in mice with acute pneumonia by inhibiting the TLR4 / MyD88 / NF-κB signaling pathway, demonstrating a good anti-inflammatory effect.

[0066] Example 10: In vivo anti-inflammatory effect of compound E on mice with ulcerative colitis

[0067] In this embodiment, male BALB / c mice were selected as experimental subjects and randomly divided into a normal group (blank group), a model group, high, medium, and low dose groups of compound E, and a sulfasalazine group, with 8 mice in each group. The model group and each treatment group were administered 2.5% diethylnitrosamine (DSS) solution by gavage to establish an ulcerative colitis model, while the normal group was administered an equal volume of distilled water by gavage. After modeling, each group was administered the drugs continuously for 7 days. The normal group and the model group were administered physiological saline by gavage, while the treatment groups were administered different doses of compound E solution (20, 40, and 60 mg / kg) and sulfasalazine (100 mg / kg) by gavage, respectively.

[0068] This study comprehensively evaluated the anti-inflammatory effect of compound E by observing pathological changes in colonic tissue, analyzing serum inflammatory factor levels, and detecting colonic tissue protein expression. The experimental results showed that all dose groups of compound E could alleviate intestinal inflammation and promote tissue repair to varying degrees, exhibiting a dose-dependent effect. Figure 22 Furthermore, compound E significantly reduced the levels of key inflammatory factors such as TNF-α, IL-6, IL-1β, and IL-12 in serum (Figure 23). At the colonic tissue level, compound E effectively alleviated colonic mucosal damage in mice with ulcerative colitis by upregulating the expression of the anti-apoptotic protein Bcl-2, downregulating the expression of the pro-apoptotic protein Bax, and increasing the expression of autophagy-related proteins Beclin1 and LC3. Figure 24 These findings reveal that compound E can demonstrate significant anti-inflammatory potential in alleviating symptoms of ulcerative colitis in mice by regulating apoptosis and autophagy signaling pathways.

[0069] It should be noted that the embodiments described above are only for explaining the present invention and do not constitute any limitation on the present invention. The present invention has been described with reference to typical embodiments, but it should be understood that the words used therein are descriptive and explanatory terms, not limiting terms. Modifications can be made to the present invention within the scope of the claims, and revisions can be made to the present invention without departing from the scope and spirit of the present invention. Although the present invention described herein relates to specific methods, materials, and embodiments, it does not mean that the present invention is limited to the specific examples disclosed herein; on the contrary, the present invention extends to all other methods and applications having the same function.

Claims

1. An isoflavone compound, characterized in that, The compound is selected from one or more of compounds A to F, and the structural formulas are shown as AF respectively: 。 2. The isoflavone compound according to claim 1, characterized in that, The compound is selected from compound B or compound E.

3. The method for preparing the isoflavone compound according to claim 1, characterized in that, Includes the following steps: Step (1) Select 4',7-dihydroxyisoflavone or 4',5,7-trihydroxyisoflavone as the parent substrate; Step (2) Select the corresponding acyl chloride reagent according to the target product: allyloxycarbonyl chloride, o-chlorobenzoyl chloride or cinnamoyl chloride; Step (3) Dissolve the parent substrate and phase transfer catalyst in an organic solvent at 10-40℃, add an inorganic base aqueous solution, and add acyl chloride reagent dropwise while stirring for 6-16 hours; Step (4) After the reaction is complete, the target compound is obtained by extraction, washing, drying and column chromatography purification.

4. The preparation method according to claim 3, characterized in that, The phase transfer catalyst is Aliquat 336; the molar ratio of the parent substrate to the acyl chloride reagent is 1:2-5.

5. A pharmaceutical composition, characterized in that, It comprises the isoflavone compound of claim 1 or 2 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or excipient.

6. The use of the isoflavone compound of claim 1 or 2 or the pharmaceutical composition of claim 5 in the preparation of a medicament for treating inflammatory diseases.

7. The application according to claim 6, characterized in that, The inflammatory disease is selected from pneumonia, colitis, sepsis, rheumatoid arthritis, or asthma.

8. The application according to claim 6, characterized in that, The drug exerts its anti-inflammatory effect by inhibiting the TLR4 / MyD88 / NF-κB signaling pathway; and exerts its tissue repair effect by regulating Bcl-2 / Bax-mediated apoptosis and Beclin1 / LC3-mediated autophagy.

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