Forsygenin glucosamine and preparation method and application thereof

Abstract

The invention belongs to the technical field of medicines, and particularly relates to forsythiaside glucosamine, a preparation method thereof and application of the forsythiaside glucosamine in treatment of osteoarthritis. The invention relates to design and synthesis of forsythiaside glucosamine, and researches on the treatment effect of forsythiaside glucosamine on an osteoarthritis mouse model. In the structural general formula of the compound, R1 is an amino group, a phthaloyl amino group or an allyloxy amido group, R2, R3 and R4 groups represent a hydroxyl group or an alkanoyloxy group, and the configurations of different head positions are alpha and beta. Wherein the alkanoyloxy group is a C1-C6 alkanoyloxy group or a deuterated alkanoyloxy group. The synthesized forsythiaside glucosamine is used as a twin drug, and shows better cartilage generation promoting activity and anti-inflammatory activity compared with combined use of forsythiaside and glucosamine on an osteoarthritis mouse model. In addition, under the same administration dosage, the activity of promoting cartilage generation and the like is obviously superior to that of forsythiaside acetylglucosamine in the prior art.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of medicine, and particularly relates to phillygenin glucosamine and a preparation method thereof and application thereof in the treatment of osteoarthritis. BACKGROUND

[0002] Osteoarthritis (OA) is a degenerative disease characterized by destruction or damage of articular cartilage, and is manifested as structural lesions of articular cartilage, subchondral bone, ligament, joint capsule, synovial membrane and joint surrounding muscle, which can cause joint pain and joint dysfunction of knee, hip, ankle, elbow, spine and other joints. Osteoarthritis is relatively common in the adult population, especially in the elderly.

[0003] The chronic and progressive nature of the disease makes the safety of related drugs very high. However, among existing small molecule drugs, glucosamine cannot resist inflammation, and the therapeutic effect varies greatly among individuals; non-steroidal anti-inflammatory drugs and recently marketed diacereine have gastrointestinal side effects and are not suitable for long-term use; and some drugs in the clinical research stage, such as SM04690 and GLPG1972, are still in the clinical research stage and are not progressing smoothly. Whether they can be marketed is still uncertain. Therefore, there is an urgent need in the clinic for chemical small molecule drugs with small toxic and side effects and suitable for long-term use. ; Phillygenin is a lignin monomer compound derived from traditional Chinese medicinal material Forsythia, mainly existing in the form of Phillyrin, and can be easily prepared by hydrolysis of Phillyrin. Studies have shown that the compound has anti-inflammatory, antioxidant and other activities, can reduce the occurrence of cartilage cell inflammation by inhibiting the NRF2 / NF-κB pathway, and effectively improve the symptoms of osteoarthritis in mice. However, its water solubility is relatively low and its stability is not good, which restricts its direct application in related new drug development.

[0004] Patent CN117720597A designs and synthesizes phillygenin glucosamine by splicing lipophilic phillygenin and hydrophilic acetyl glucosamine through glycosylation. The compound has reasonable pharmacokinetic characteristics and physicochemical properties, and has dual functions of targeting cartilage repair and anti-inflammatory, showing good potential in the development of osteoarthritis treatment drugs.

[0005] In order to further improve the therapeutic effect of phillygenin derivatives on osteoarthritis and clarify the structure-activity relationship, the present application further designs and synthesizes phillygenin acetyl glucosamine ( α configuration), phillygenin glucosamine ( α configuration and β configuration), and phillygenin butyryl glucosamine ( β(Configuration), to further investigate the application prospects of this type of glycoside compound in the treatment of osteoarthritis. Summary of the Invention

[0006] This invention relates to the design and synthesis of forsythoside glucosinolate compounds, and to the study of their therapeutic effects in a mouse model of osteoarthritis. The forsythoside glucosinolate synthesized in this invention, as a twin drug, exhibited superior chondrogenic and anti-inflammatory activities in a mouse model of osteoarthritis compared to the combined use of forsythoside and glucosamine. Furthermore, at the same dosage, it also significantly outperformed the existing forsythoside acetylglucosinolate in terms of chondrogenic and other activities.

[0007] To achieve the above objectives, the first aspect of the present invention discloses a forsythoside glucosinolate compound, the general structural formula of which is shown below: , In the general structural formula, R1 is an amino group, phthalamide group, or allyloxyamide group, and R2, R3, and R4 groups represent hydroxyl or alkanoyloxy groups. The configurations at the anomeric positions are α and β.

[0008] The alkyl oxy group is a straight-chain alkyl oxy group or a deuterated alkyl oxy group.

[0009] Preferably, the alkanoyloxy group is a C1-C6 alkanoyloxy group or a deuterated alkanoyloxy group.

[0010] The specific compound structural formula is shown below: ;

[0011] The second aspect of this invention discloses a method for preparing the above-mentioned forsythoside glucosinolate compound, the specific steps of which are as follows: 1) Synthesis of S1-S3: ; i. Forsythoside I and anomeric bromoglucosamine II (hydroxyacetyl-protected / aminophthaloyl-protected) undergo a nucleophilic substitution reaction under the action of the phase-transfer catalyst tetrabutylammonium bromide and the strong base sodium hydroxide to generate forsythoside S1 (hydroxyacetyl-protected / aminophthaloyl-protected). α Configuration); ii. Compound S1, under stirring in a 7M ammonia-methanol solution, undergoes deacetylation to yield forsythoside S2 (aminophthaloyl protected). α Configuration), further in 70 o C is dephthaloylated with hydrazine hydrate to obtain forsythoside S3 ( α Configuration); 2) Synthesis of S4-S6: ; i. Acetyl-protected glucosamine III undergoes acylation with allyloxycarbonyl chloride in the presence of sodium bicarbonate to generate intermediate IV, which is then anolybrodized in the presence of hydrobromic acid to generate intermediate V. ii. Forsythoside and intermediate V undergo anophilic substitution at the anomeric position in the presence of the phase-transfer catalyst tetrabutylammonium bromide and the strong base sodium hydroxide, generating compound S4 (hydroxyacetyl protected / aminoallyloxycarbonyl protected). β Configuration); iii. Using N,N-dimethylpyrimidinetrione as a promoter, S4 underwent palladium-catalyzed deallyoxycarbonylation to yield compound S5 (hydroxyacetyl protected). β Configuration), then stirred in 7M ammonia-methanol solution, deacetylated to obtain forsythoside S6 ( β Configuration).

[0012] 3) S7 synthesis: ; i. Using acetyl chloride as the acylation reagent and triethylamine as the base, S3 undergoes an acetylation reaction to produce S7.

[0013] 4) Synthesis of S8-S9: ; i. Acetyl-protected glucosamine III undergoes acylation with butyryl chloride under the action of triethylamine to generate intermediate VI, which is then anolybrodized under the action of hydrobromic acid to generate intermediate VII; ii. Forsythoside and intermediate V undergo anophilic substitution at the anomeric position in the presence of the phase-transfer catalyst tetrabutylammonium bromide and the strong base sodium hydroxide, generating compound S8 (hydroxyacetyl protected / aminobutyryl protected). β Configuration); iii. S8 was stirred in a 7M ammonia-methanol solution to deacetylate and obtain forsythoside S9. β Configuration).

[0014] The third aspect of this invention discloses the use of the above-mentioned forsythoside glucosinolate compound in the preparation of a drug for treating osteoarthritis.

[0015] Compared with the prior art, the present invention has the following advantages: Based on patent CN117720597A, this invention has thoroughly studied the structure-activity relationship and found that forsythoside glucosinolate (a twin drug formed by glucosamine and forsythoside through the formation of an ester bond), especially the β configuration, shows significantly superior cartilage regeneration and anti-inflammatory activities in a mouse model of osteoarthritis compared to compound S2 (a twin drug formed by forsythoside and acetylglucosamine through the formation of an ester bond) disclosed in patent CN117720597A. Compared with the combined use of forsythoside and glucosamine, it is significantly better in improving grip strength and enhancing cartilage tissue regeneration in mice with osteoarthritis, showing unexpected technical effects and demonstrating the effectiveness of the twin drug design. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or existing methods and experiments, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation of the scope. For those skilled in the art, other related drawings can be obtained from these drawings without creative effort.

[0017] Figure 1 The diagram shows the preparation process of compounds S1-S9 in Examples 1-5.

[0018] Figure 2 For grip strength determination in osteoarthritis model mice, * P < 0.05 (statistically significant), ns P > 0.05 (no statistically significant difference).

[0019] Figure 3 Images of H&E staining and Safranin-Fix-Green staining of knee joint sections from a mouse model of osteoarthritis.

[0020] Figure 4 The statistical results of the Mankin's score for the effect of osteoarthritis model mice on cartilage regeneration are shown. * P < 0.05 (statistically significant), ns P > 0.05 (no statistically significant difference).

[0021] Figure 5 The image shows the inflammatory cell infiltration status. * P < 0.05 (statistically significant), ns P > 0.05 (no statistically significant difference).

[0022] Figure 6 The graph shows the inflammatory factors measured in joint tissue. * P < 0.05 (statistically significant), ns P > 0.05 (no statistically significant difference). Detailed Implementation

[0023] The technical solution of the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but this does not limit the present invention to the scope of the described embodiments. Experimental methods and techniques in the following embodiments that do not specify specific conditions are generally performed according to conventional conditions in the relevant art or according to the conditions recommended by the manufacturer.

[0024] Example 1: Synthesis of compound S1: Forsythoside I (136.2 mg, 0.37 mmol, 1.0 eq.) was weighed and added to 2 mL of sodium hydroxide aqueous solution (1M NaOH), and stirred vigorously at room temperature for 30 min. Angiotensin II (230.0 mg, 0.46 mmol, 1.2 eq.) and tetrabutylammonium bromide (148.3 mg, 0.46 mmol, 1.2 eq.) were weighed and added separately to the forsythoside I aqueous solution, followed by 2 mL of chloroform solution. The mixture was stirred at 35 °C for 18 h. After the reaction was complete, the mixture was diluted with dichloromethane and aqueous solution. The aqueous phase was extracted three times with dichloromethane. The organic phases were combined, dried, filtered, and evaporated to dryness. The crude product was separated by silica gel column chromatography (elution buffer: PE:EA = 1:1) to give a white solid S1 (195.7 mg, yield 67%, α configuration). 1 H NMR (400MHz, CDCl3) δ 7.90 (dd, J = 5.5, 3.1 Hz, 2H), 7.76 (dd, J = 5.5, 3.1 Hz, 2H), 6.97 (d, J = 8.1 Hz, 1H), 6.94 – 6.88 (m, 2H), 6.85 (d, J = 11.0 Hz, 2H), 6.79(dd, J = 8.2, 1.8 Hz, 1H), 6.75 (d, J = 1.8 Hz, 1H), 5.39 (d, J = 3.7 Hz, 1H), 5.19– 5.09 (m, 1H), 4.84 (d, J = 5.1 Hz, 1H), 4.71 – 4.61 (m, 2H), 4.40 (q, J = 5.2,4.5 Hz, 2H), 4.20 – 4.16 (m, 1H), 4.09 (d, J = 9.2 Hz, 1H), 3.91 (d, J= 2.9 Hz,1H), 3.89 (s, 3H), 3.87 (s, 3H), 3.85 – 3.74 (m, 2H), 3.32 – 3.24 (m, 2H),3.22 (s, 3H), 2.80 (q, J = 7.1 Hz, 1H), 2.11 (s, 3H), 2.09 (s, 3H), 1.94 (s,3H); HRMS (ESI) Calcd. for C 41 H 44 NO 15 [(M+H) + 790.2705, measured value 790.2706.

[0025] Example 2 Synthesis of compounds S2 and S3: Add S1 (80.0 mg, 0.1 mmol) and a stir bar to a dry round-bottom flask, followed by 5 mL of ammonia-methanol solution (7 M). Stir for 12 h at room temperature. After the reaction is complete, evaporate the solvent to obtain crude S2. Transfer the crude product to a Shrek tube, purge with nitrogen three times, add 1.5 mL of ethanol solution and 100 μL of hydrazine hydrate solution (85% in H2O) to the tube, and place at 70°C. o The reaction was stirred at C for 20 h. After the reaction was complete, the solution was evaporated to dryness to obtain the crude product. The crude product was dispersed in dichloromethane and sonicated until the solution was a white emulsion. The solution was filtered, and the filter cake was washed several times with dichloromethane. The filtrate was collected and evaporated to dryness. The obtained crude product was dispersed in ethyl acetate and sonicated until the solution was a white emulsion. The solution was filtered, and the filter cake was washed with a small amount of ethyl acetate. The obtained filter cake was dried to obtain forsythoside S3 (36.8 mg, yield 69%, α configuration). 1 H NMR (400 MHz, DMSO-) d 6) δ 7.16 (d, J = 8.2 Hz, 1H), 6.99 (d, J = 1.9 Hz, 1H), 6.93(dd, J = 5.1, 3.3 Hz, 2H), 6.88 (dt, J = 8.4, 2.4 Hz, 2H), 5.21 (d, J = 3.4 Hz,1H), 5.00 (s, 1H), 4.97 (d, J = 5.7 Hz, 1H), 4.80 (d, J = 5.9 Hz, 1H), 4.49 (t,J =5.9 Hz, 1H), 4.38 (d, J = 6.8 Hz, 1H), 4.11 (d, J = 9.3 Hz, 1H), 3.78 (s, 3H), 3.75 (s, 3H), 3.74 (s, 3H), 3.64 – 3.55 (m, 2H), 3.52 – 3.47 (m, 1H), 3.45 –3.38 (m, 2H), 3.20 – 3.14 (m, 1H), 3.09 (t, J = 8.6 Hz, 1H), 2.88 – 2.82 (m,1H), 2.55 (dd, J = 10.0, 3.5 Hz, 1H); HRMS (ESI) Calcd. for C 27 H 36 NO 10 [(M+H) + ]534.2334, measured value 534.2336.

[0026] Example 3 Synthesis of compounds S4-S6: Acetyl-protected glucosamine III (11.51 g, 30.0 mmol) was dissolved in 115 mL of H₂O, and NaHCO₃ (9.66 g, 115 mmol) was added. Then, 115 mL of chloroform solution containing Alloc-Cl (4.77 mL, 45.0 mmol) was slowly added, and the reaction was stirred for 4 h. After the reaction was complete, the aqueous phase was extracted twice with chloroform, and the organic phase was washed with saturated brine and dried over anhydrous sodium sulfate. The crude product was separated by silica gel column chromatography (elution buffer: PE:EA = 1:1, R...). f= 0.45), yielding a white solid compound IV (9.42 g, yield 90.6%) for later use. Compound IV (9.42 g, 27.2 mmol) was dissolved in 100 mL DCM, and 51.8 mL HBr (33% in AcOH) was added. The mixture was stirred at room temperature for 3 h. After the reaction was complete, ice-cold water and chloroform were added to the reaction solution for extraction and separation. The organic phase was retained, and the pH was adjusted to 7 by adding ice-cold dilute sodium bicarbonate solution. The organic phase was washed with water, dried over anhydrous sodium sulfate, filtered, and evaporated to dryness at low temperature to obtain compound V (1.34 g, yield 13.5%) for later use. Forsythoside I (0.89 g, 2.39 mmol) was dissolved in 21 mL sodium hydroxide aqueous solution (210 mg, 5.25 mmol), and stirred for 15 min. Then, V (1.34 g, 3.66 mmol), TBAB (504 mg, 1.57 mmol) and 21 mL chloroform were added, and the mixture was stirred overnight at 40 °C. After the reaction was complete, the mixture was extracted and separated, dried over anhydrous sodium sulfate, and then passed through a column chromatography with a PE:EA ratio of 1:1 to obtain compound S4 (893 mg, yield 54.4%) for later use. Compound S4 (893 mg, 1.36 mmol) was placed in a three-necked flask, and tetrakis(triphenylphosphine)palladium (235 mg, 0.204 mmol) and 1,3-dimethylbarbituric acid (849 mg, 5.44 mmol) were added. After N2 replacement, 80 mL of DCM solvent was added, and the mixture was reacted at 40 °C for 4 h. The pH was adjusted to 7 with sodium bicarbonate aqueous solution, extracted and separated, dried and filtered, and then passed through a column chromatography with a DCM:MeOH ratio of 20:1 to obtain compound S5 (197 mg, yield 22.0%) for later use. Compound S5 (197 mg, 0.299 mmol) was stirred overnight in a methanol solution of 7 M NH3, evaporated to dryness, and purified by repeated slurrying with EA to obtain a white solid β-forsythoside glucosinolate S6 (102 mg, yield 64.0%). 1 HNMR (400 MHz, DMSO- d 6) δ 7.11 (d, J = 8.3 Hz, 1H), 7.00 – 6.84 (m, 6H), 5.13(s, 1H), 5.01 (d, J = 4.2 Hz, 1H), 4.80 (d, J = 5.9 Hz, 1H), 4.66 (d, J = 8.0 Hz, 1H), 4.58 (t, J = 5.8 Hz, 1H), 4.38 (d, J = 6.8 Hz, 1H), 4.10 (d, J= 9.3 Hz, 1H), 3.79 – 3.73 (m, 12H), 3.70 – 3.65 (m, 1H), 3.48 (dt, J = 11.7, 5.9 Hz, 1H), 3.09 (t, J = 8.6 Hz, 1H), 2.84 (dt, J = 9.2, 6.4 Hz, 1H), 2.68 (t, J = 8.4 Hz, 1H); MS (ESI-TOF)m / z:[M+H] + Calcd for C 21 H 25 NO5534.2, found 534.2.

[0027] Example 4 Synthesis of compound S7: Compound S3 (36.8 mg, 0.069 mmol) was dissolved in dichloromethane, and triethylamine (10.55 μL, 0.076 mmol) was added. Acetyl chloride (5.42 μL, 0.076 mmol) was added dropwise at 0 °C, and the mixture was then transferred to room temperature for 3 h. After the reaction was complete, the mixture was extracted with dichloromethane and water, and the organic phase was collected, dried over anhydrous sodium sulfate, filtered, and evaporated to dryness to give S7 (31.06 mg, 71% yield). 1 H NMR (400 MHz, DMSO- d 6) δ 7.86 (d, J = 7.2 Hz, 1H), 7.01 (d, J = 8.3Hz, 2H), 6.96 – 6.85 (m, 5H), 5.27 (d, J = 3.2 Hz, 1H), 5.13 (d, J = 5.4 Hz, 1H), 4.87 (d, J = 5.2 Hz, 1H), 4.80 (d, J = 5.9 Hz, 1H), 4.48 (d, J = 6.0 Hz, 1H), 4.39(d, J = 6.8 Hz, 1H), 4.11 (d, J = 9.2 Hz, 1H), 3.75 (m, 11H), 3.55 (m, 2H), 3.27(m, 1H), 3.09 (t, J= 8.6 Hz, 1H), 2.91 – 2.79 (m, 1H); MS (ESI-TOF)m / z:[M+Na] + Calcd for C 29 H 37 NO 11 598.2, found 598.2.

[0028] Example 5 Synthesis of compounds S8-S9: Acetyl-protected glucosamine III (1 g, 2.61 mmol) was dissolved in DCM, and triethylamine (750 μL, 5.48 mmol) was added. Butyryl chloride (300 μL, 2.87 mmol) was slowly added dropwise at 0 °C, and the mixture was stirred at room temperature for 3 h. After the reaction was complete, the mixture was extracted with DCM and water, and the organic phase was collected, dried over anhydrous sodium sulfate, filtered, and evaporated to dryness to obtain a white solid VI (982 mg, 90% yield) for later use. Compound VI (982 mg, 2.35 mmol) obtained from the reaction was dissolved in DCM, and 6 mL of HBr (33% inAcOH) was added. The mixture was stirred at room temperature for 3 h. After the reaction was complete, the reaction mixture was extracted with ice-cold water and chloroform, and the organic phase was retained. The pH was adjusted to 7 by adding ice-cold dilute sodium bicarbonate solution, the organic phase was washed with water, dried over anhydrous sodium sulfate, filtered, and evaporated to dryness at low temperature to obtain compound VII (878.5 mg, 87.4% yield) for later use. 21 mL of H₂O and NaOH (210 mg, 5.25 mmol) were added to compound I (0.89 g, 2.39 mmol), and the mixture was stirred for 15 min. Then, compound VII (878.5 mg, 2.01 mmol), TBAB (504 mg, 1.57 mmol), and 21 mL of chloroform were added, and the mixture was stirred overnight at 40 °C. After the reaction was complete, the mixture was extracted and separated. After drying with anhydrous sodium sulfate, the solution was purified by column chromatography (PE:EA = 1:1) to obtain compound S8 (763.7 mg, yield 52.2%) for later use. Compound 6 (763.7 mg, 1.05 mmol) was stirred overnight in a methanol solution of 7 M NH₃, evaporated to dryness, and purified by repeated slurry washing with EA to obtain a white solid S9 (426 mg, yield 67.2%). 1 H NMR (400 MHz, DMSO- d 6) δ 7.71 (d, J = 8.8 Hz, 1H), 7.06(d, J = 8.3 Hz, 1H), 6.98 – 6.83 (m, 5H), 5.08 (d, J = 5.1 Hz, 1H), 5.01 (d, J=5.4 Hz, 1H), 4.97 (d, J = 8.4 Hz, 1H), 4.80 (d, J = 5.9 Hz, 1H), 4.60 (d, J = 5.7Hz, 1H), 4.37 (d, J = 6.8 Hz, 1H), 4.09 (d, J = 9.3 Hz, 1H), 3.79 – 3.69 (m,11H), 3.63 (m, 1H), 3.54 – 3.37 (m, 1H), 3.21 (m, 2H), 3.08 (t, J = 8.6 Hz, 1H), 2.83 (q, J = 7.9, 7.3 Hz, 1H), 2.03 (m, 2H), 1.76 (s, 1H), 1.57 – 1.47 (m,1H), 0.83 (t, J = 7.4 Hz, 3H); MS (ESI-TOF)m / z:[M+H] + Calcd for C 31 H 41 NO 11 604.3, found 604.3.

[0029] Example 6: The specific experimental procedures and results are as follows: Animal experiment procedure: Forty 8-week-old clean-grade male BALB / c mice (purchased from Shanghai Jiesijie Laboratory Animal Co., Ltd.) were selected and fed with routine maintenance rodent diet (Jiangsu Xietong Pharmaceutical Biotechnology Co., Ltd.). After acclimatizing in the animal experimental facility for one week, the mice were divided into wild-type (WT, injected with physiological saline), osteoarthritis (OA, injected with papain but not treated), forsythoside + glucosamine (Phi + Glu, injected with papain and treated with forsythoside and glucosamine), S3 (injected with papain and treated with S3), S6 (injected with papain and treated with S6), S7 (injected with papain and treated with S7), S0 (injected with papain and treated with S0), and S9 (injected with papain and treated with S9). The specific structure of S2 in the forsythoside acetylglucosamine in patent CN117720597A is as follows (injected with papain and treated with S0). Osteoarthritis modeling was then initiated.

[0030]

[0031] S0; Modeling and Administration of Osteoarthritis: The total duration of this experiment was 35 days. On days 1, 3, and 5 of the experiment, mice were injected intraperitoneally with a mixture of 0.8% (w / v) papain and 0.003 mol / L L-cysteine ​​at a dose of 1.25 mL / kg or an equivalent volume of physiological saline (WT). Before injection, the mice were anesthetized with an intraperitoneal injection of 45 mg / kg sodium pentobarbital. The hair on the right knee was removed, and the area was disinfected with 75% ethanol. The left hand held the mouse's right hind limb firmly, flexing it to 60 degrees. The right hand held the syringe needle, using the outer edge of the patellar tendon below the patella as the injection point, and inserted it slightly inward and downward. After passing through the joint capsule, a feeling of loss of contact was felt. At this point, the appropriate concentration of papain mixture or an equivalent volume of physiological saline was injected to complete the injection. 24-48 hours after injection, the knee joint of the modeled side was compared with the healthy side. Swelling, flexion and extension dysfunction, hopping, dragging, and reduced activity on the modeled side indicated the presence of synovial inflammation and cartilage damage. After successful modeling (starting from day 5), mice were administered the drug by gavage at noon every day. The daily dose of Phi + Glu (equal molecular weight mixture), S3, S6, S7, S9 and S0 was 100 mg / kg. After 30 days of continuous gavage treatment, the osteoarthritis disease status was analyzed by mouse grip strength test, knee joint slices and knee joint tissue protein ELISA experiment.

[0032] Experimental results: 1. Effects of Forsythoside S3, S6, S7, S0, and S9 on grip strength in mice with osteoarthritis The grip strength of the right hind limb of experimental mice was tested using the ZL-010 rat and mouse grip strength tester (Anhui Yaokun Biotechnology Co., Ltd.). The mice were grasped and their right hind limbs were made to grip the elastic metal strip on the grip strength test plate. The strip was then gradually pulled outwards until the mouse could no longer maintain its grip and the metal strip detached from its paw. Three consecutive measurements were taken, and the average value was used as the experimental result for each mouse. The results were then statistically analyzed and plotted in groups. The results showed that ( Figure 2 All drug administration groups showed some improvement in grip strength in mice. The grip strength improvement effect of forsythoside glucosinolate (S3 and S6) was more significant, which was better than that of forsythoside acetylglucosinolate (S7 and S0) and forsythoside butyryl glucosinolate (S9). It was also better than the combined administration of forsythoside and glucosamine (Phi+Glu), which proved the rationality of the twin drug design. Among them, the β configuration of S6 was better.

[0033] 2. Effects of Forsythoside S3, S6, S7, S0, and S9 on cartilage regeneration in osteoarthritis mice. The experimental mice were euthanized by cervical dislocation, and the knee joint was surgically severed 0.3 cm lateral to the model joint, and the entire joint sample was removed. The removed joint sample was fixed in 10% formaldehyde for 48 h, and then decalcified in formalin-nitric acid decalcification solution for 24 h. The decalcified specimen was dehydrated, cleared, paraffin-embedded, and embedded to prepare paraffin specimens. The paraffin specimens were cut into 3 μm thick paraffin sections using an HS-2205 rotary microtome (Jinhua Huasu). After dewaxing and rinsing briefly with distilled water, the sections were stained with hematoxylin and eosin (H&E, for staining the entire knee joint tissue) and safranin and fast green (for staining cartilage), respectively. The sections were observed and photographed using a Nikon Ti-S inverted microscope, and Mankin's score was calculated to determine whether the compound had a protective effect against cartilage tissue and knee joint inflammation. The results showed that all treatment groups (including the combined administration of forsythoside and glucosamine) had a certain ameliorative effect on cartilage damage caused by osteoarthritis in mice and could enhance cartilage tissue regeneration in mice with osteoarthritis. Among them, the β-configuration forsythoside glucosamine S6 had the most significant effect. Figure 3 and Figure 4 ).

[0034] 3. Effects of forsythoside glucosinolates S3, S6, S7, S0, and S9 on inflammation in mice with osteoarthritis.

[0035] based on Figure 3 Images stained with H&E were used to count inflammatory cells at the joint joint using the pathological image analysis software QuPath, analyzing the progression of knee joint inflammation and the status of inflammatory cell infiltration. Partial knee joint tissue from mice was placed in RIPA lysis buffer, homogenized using a Labgic L-HOB homogenizer, and then lysed using a Hanno HN98-IIB tissue cell ultrasonic disruptor. After centrifugation, total protein extract was obtained. The concentration of the extracted protein was quantified using the Beyotime BCA protein quantification kit, and the inflammation-related cytokines in the samples were quantified using the Abcam mouse TNF-α, IL-1β, IL-6, and MCP-1 protein ELISA kit to determine the level of inflammation in the knee joint tissue. Results showed that all treatment groups (including co-administration of forsythoside S6 and glucosamine) had good anti-inflammatory effects in osteoarthritis model mice, enhancing cartilage tissue regeneration while reducing inflammatory cell infiltration and the expression of inflammation-related cytokines, thus alleviating osteoarthritis. Among them, the anti-inflammatory effect of β-form forsythoside S6 was the most significant. Figure 5 and Figure 6 ).

[0036] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be equivalent substitutions. In addition, hydrochloride, sulfate, phosphate, citrate, maleate, etc. of forsythoside glucosinolate are also within the protection scope of the present invention and are included within the protection scope of the present invention.

Claims

1. A forsythoside glucosinolate compound, characterized in that, The general structural formula of the compound is shown below: ， In the general structural formula, R1 is an amino group, phthalamide group or allyloxyamide group, R2, R3 and R4 groups represent hydroxyl or alkanoyloxy groups, and the configuration of the anomeric position is α and β. The alkyl oxy group is a straight-chain alkyl oxy group or a deuterated alkyl oxy group.

2. The forsythoside glucosinolate compound according to claim 1, characterized in that, The alkyl oxy group is a C1-C6 alkyl oxy group or a deuterated alkyl oxy group.

3. The forsythoside glucosinolate compound according to claim 1, characterized in that, The specific compound structural formula is shown below: ； 。 4. The method for preparing the forsythoside glucosinolate compound according to any one of claims 1-3, characterized in that, The specific steps are as follows: 1) Synthesis of S1-S3: ； i. Forsythoside I and anomeric bromoglucosamine II (hydroxyacetyl protected / aminophthaloyl protected) undergo a nucleophilic substitution reaction under the action of phase transfer catalyst tetrabutylammonium bromide and strong base sodium hydroxide to generate forsythoside glucosamine S1 (hydroxyacetyl protected / aminophthaloyl protected, α configuration). ii. Compound S1, under stirring in a 7M ammonia-methanol solution, is deacetylated to give forsythoside S2 (aminophthaloyl protected, α-configuration), further subjected to 70... o C is dephthaloylated with hydrazine hydrate to give forsythoside S3 (α configuration). 2) Synthesis of S4-S6: ； i. Acetyl-protected glucosamine III undergoes acylation with allyloxycarbonyl chloride in the presence of sodium bicarbonate to generate intermediate IV, which is then anolylated by hydrobromic acid to generate intermediate V; ii. Forsythoside and intermediate V undergo anophilic substitution at the anomeric position in the presence of the phase-transfer catalyst tetrabutylammonium bromide and the strong base sodium hydroxide to generate compound S4 (hydroxyacetyl protected / aminoallyloxycarbonyl protected, β configuration). iii. Using N,N-dimethylpyrimidinetrione as a promoter, S4 was deacetylated by palladium catalysis to obtain compound S5 (hydroxyacetyl protected, β configuration), which was then deacetylated in 7M ammonia-methanol solution to obtain forsythoside glucosinolate S6 (β configuration).

5. The use of the forsythoside glucosinolate compound as described in any one of claims 1-3 in the preparation of a medicament for treating osteoarthritis.

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