Forsygenin glucosamine and preparation method and application thereof
By designing and synthesizing Forsythiamin glucosamine, the existing osteoarthritis drugs have large toxic side effects, large individual differences in efficacy, obvious gastrointestinal side effects and insufficient stability have been solved, and the grip and cartilage tissue regeneration have been significantly improved in the osteoarthritis mouse model, with better pro-chondrogenic activity and anti-inflammatory activity.
Patent Information
- Application Number
- CN202510061854.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-15
- Publication Date
- 2025-05-13
- Estimated Expiration
- Not applicable · inactive patent
AI Technical Summary
The existing drugs used for osteoarthritis treatment have problems such as large toxic side effects, large individual efficacy differences, obvious gastrointestinal side effects and are not suitable for long-term use. There are also shortcomings in the stability and water solubility of existing small molecule drugs.
By designing and synthesizing forsythia lipolytic glucosamine, lipophilic forsythia lipolytic and hydrophilic acetyl glucosamine are spliced by using glycosylation reactions to form compounds with good pharmacokinetic characteristics and physical and chemical properties, which are used for the treatment of osteoarthritis.
In the mouse model of osteoarthritis, forsythia glucosamine significantly improved the grip of mice, enhanced cartilage tissue regeneration, and had better prochondrogenic activity and anti-inflammatory activity than existing forsythia glucosamine and glucosamine.
Smart Images
Figure CN119978041A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of medicine, and in particular relates to forsythiaside aminoglucosidase and a preparation method thereof, as well as application in the treatment of osteoarthritis. Background Art
[0002] Osteoarthritis (OA) is a degenerative disease characterized by the destruction or damage of articular cartilage, manifested as structural lesions in articular cartilage, subchondral bone, ligaments, joint capsule, synovium and periarticular muscles, which can lead to joint pain and joint dysfunction in the knee, hip, ankle, elbow, spine and other joints. Osteoarthritis is more common in the adult population, especially in the elderly. Statistics show that there are more than 500 million osteoarthritis patients worldwide, and more than 130 million in my country, with an overall incidence rate of more than 10%.
[0003] The chronic and progressive nature of the disease makes the safety requirements for related drugs very high. However, among the existing small molecule drugs, glucosamine cannot fight inflammation, and the individual differences in efficacy are large; non-steroidal anti-inflammatory drugs and diacerein, which have been newly launched in recent years, 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 Phase II stage, and the progress is not smooth. Whether they can be marketed is still uncertain. Therefore, there is an urgent need for chemical small molecule drugs with low toxicity and side effects and suitable for long-term use in clinical practice.
[0004]
[0005] Phillygenin is a lignin monomer compound derived from the traditional Chinese medicinal material Forsythia suspensa. It mainly exists in the form of phillyrin and can be easily prepared by hydrolysis of phillyrin. Studies have shown that this compound has anti-inflammatory and antioxidant activities, and can reduce the occurrence of chondrocyte inflammation and effectively improve the symptoms of osteoarthritis in mice by inhibiting the NRF2 / NF-κB pathway. However, its low water solubility and poor stability restrict its direct application in the creation of related new drugs.
[0006] Patent CN117720597A uses the twin drug combination principle to splice lipophilic forsythiaside and hydrophilic acetylglucosamine through glycosylation reaction to design and synthesize forsythiaside acetylglucosamine. The compound has reasonable pharmacokinetic characteristics and physicochemical properties, and has both targeted cartilage repair and anti-inflammatory functions, showing good potential in the development of osteoarthritis treatment drugs.
[0007] In order to further improve the therapeutic effect of forsythiaside derivatives on osteoarthritis, the present invention designs a new synthetic route to further investigate the application prospects of the corresponding glycoside compounds in the treatment of osteoarthritis. Summary of the invention
[0008] The invention relates to the design and synthesis of forsythiaside aminoglucosidase, and studies the therapeutic effect thereof on an osteoarthritis mouse model.
[0009] In order to achieve the above-mentioned object, the first aspect of the present invention discloses a forsythiaside aminoglycoside compound, the general structural formula of which is shown as follows:
[0010] In the general structural formula, R 1 is amino or phthalamido, R 2 , R 3 , R 4 The group represents a hydroxyl group or an alkanoyloxy group, The alkanoyloxy group is a straight-chain alkanoyloxy group or a deuterated alkanoyloxy group.
[0011] Preferably, the alkanoyloxy group is a C1-C6 alkanoyloxy group or a deuterated alkanoyloxy group.
[0012] The specific compound structure is shown below: .
[0013] The second aspect of the present invention discloses a method for preparing the forsythiaside aminoglycoside compound, and the specific steps are as follows:
[0014] Forsythiaside I and anomeric bromoglucosamine II (hydroxyacetyl protection / aminophthaloyl protection) undergo nucleophilic substitution reaction under the action of phase transfer catalyst tetrabutylammonium chloride and strong base sodium hydroxide to generate forsythiaside aminoglucoside S1 (hydroxyacetyl protection / aminophthaloyl protection); Compound S1 was stirred in 7M ammonia methanol solution and deacetylated to obtain forsythiaside aminoglucosidone S2 (aminophthaloyl protected), which was further heated at 70 o C is treated with hydrazine hydrate to remove the phthaloyl group to obtain forsythiaside aminoglucoside S3.
[0015] The third aspect of the present invention discloses the use of the forsythiaside aminoglycoside compound in the preparation of a drug for treating osteoarthritis.
[0016] Compared with the prior art, the present invention has the following advantages: At the same dosage, the forsythiaside amino glucosinolate synthesized by the present invention is much better than forsythiaside and glucosamine in improving the grip of mice and enhancing the regeneration of cartilage tissue in osteoarthritis mice, and has unexpected technical effects; and compared with the compound S2 disclosed in patent CN117720597A, it also shows better chondrogenesis and anti-inflammatory activities in the osteoarthritis mouse model. BRIEF DESCRIPTION OF THE DRAWINGS
[0017] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the existing methods and experiments, the following is a brief introduction to the drawings required for use in the embodiments. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without paying creative work.
[0018] Figure 1 This is a diagram of the preparation process of compounds S1-S3 in Examples 1-2.
[0019] Figure 2 The grip strength of osteoarthritis model mice was measured. * P < 0.05 (statistically significant), ns P > 0.05 (no statistical difference).
[0020] Figure 3 H&E staining and safranin fast green staining images of knee joint sections of osteoarthritis model mice.
[0021] Figure 4 The Mankin's score statistical results of the experiment on cartilage regeneration in osteoarthritis model mice, * P<0.05 (statistically significant), ns P>0.05 (no statistical difference).
[0022] Figure 5 Figure 2 shows the infiltration status of inflammatory cells. * P < 0.05 (statistically significant), ns P > 0.05 (no statistical difference).
[0023] Figure 6 The graph shows the determination of inflammatory factors in joint tissues, * P<0.05 (statistically significant), ns P>0.05 (no statistical difference). DETAILED DESCRIPTION
[0024] The technical scheme of the present invention is described in detail below in conjunction with the accompanying drawings and embodiments, but the present invention is not limited to the scope of the embodiments. The experimental methods and techniques in the following embodiments without specifying specific conditions are usually carried out according to conventional conditions in the field or according to the conditions recommended by the manufacturer.
[0025] Example 1 Synthesis of Compound S1: Weigh forsythiaside I (136.2 mg, 0.37 mmol, 1.0 eq.) and add it to 2 mL of sodium hydroxide aqueous solution (1M NaOH), and stir vigorously at room temperature for 30 min. Weigh anomeric bromoglucosamine II (230.0 mg, 0.46 mmol, 1.2 eq.) and tetrabutylammonium bromide (148.3 mg, 0.46 mmol, 1.2 eq.), respectively, and add them to the alkaline aqueous solution of forsythiaside, then add 2 mL of chloroform solution, and stir at 35°C for 18 h. After the reaction is completed, dichloromethane and aqueous solution are added to dilute, and the aqueous phase is extracted with dichloromethane three times. The organic phases are combined, dried, filtered, and dried, and the crude product is separated by silica gel column chromatography (eluent is PE: EA = 1: 1, R f = 0.35) to give a white solid S1 (195.7 mg, yield 67%). 1 H NMR (400 MHz, CDCl 3 ) 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 = 2.9Hz, 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. Example 2 Synthesis of Compounds S2 and S3: Add S1 (80.0 mg, 0.1 mmol) and a stirrer to a dry round-bottom flask, then add 5 mL of ammonia methanol solution (7 M) and stir at room temperature for 12 h. After the reaction is completed, spin dry the solvent to obtain the crude product S2. Transfer the crude product to a Shrek tube, replace nitrogen three times, add 1.5 mL of ethanol solution and 100 μL of hydrazine hydrate solution (85% in H 2 O), placed at 70 o C and stirred for 20 h. After the reaction, the crude product was dried by spin drying. The crude product was dispersed in dichloromethane and ultrasonically beaten until the solution was a white emulsion. Filtered, the filter cake was washed with dichloromethane several times, and the filtrate was collected and dried by spin drying. The obtained crude product was dispersed in ethyl acetate, ultrasonically beaten until the solution was a white emulsion, filtered, and the filter cake was washed with a small amount of ethyl acetate. The obtained filter cake was dried to obtain forsythiaside aminoglycoside S3 (36.8 mg, yield 69%). 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] The specific experimental operations and experimental results are as follows: Animal experiment operation steps: Thirty-six 8-week-old clean-grade male BALB / c mice (purchased from Shanghai Jiesijie Experimental Animal Co., Ltd.) were selected and fed with conventional maintenance mouse diet (Jiangsu Collaborative Pharmaceutical Bioengineering Co., Ltd.). After one week of adaptation in the animal experimental facility, the mice were divided into wild type (WT, injected with normal saline), osteoarthritis (OA, injected with papain but not treated), phillyrin (Phillyrin, treated with phillyrin after papain injection), glucosamine (Glucosamine, treated with glucosamine after papain injection), S3 (treated with S3 after papain injection) and III group: The specific structure of S2 indicated in the acetylglucosamine of phillyrin in patent CN117720597A is as follows (treated with III after papain injection), and osteoarthritis modeling was started.
[0027]
[0028] Osteoarthritis modeling and drug administration: The total duration of this experiment was 35 days. On the 1st, 3rd, and 5th days of the experiment, a mixed solution of 0.8% (w / v) papain and 0.003 mol / L L-cysteine was injected into the right knee joint cavity, and the injection dose was 1.25mL / kg or an equal amount of normal saline (WT). Before injection, the experimental mice were anesthetized with 45 mg / kg sodium pentobarbital intraperitoneally, and then the right knee was depilated and disinfected with 75% ethanol. The right lower limb of the mouse was held tightly with the left hand to flex it 60 degrees. The needle of the syringe was held in the right hand and the outer edge of the patellar tendon below the patella was used as the needle point. The needle was inserted slightly inward and downward. After passing through the joint capsule, a sense of emptiness was felt. At this time, the corresponding concentration of papain mixed solution or an equal amount of normal saline was injected to complete the injection. 24-48 hours after injection, the modeling side knee joint was compared with the healthy side for observation. Swelling, flexion and extension dysfunction, skipping steps, dragging steps, and reduced activity on the modeling side indicated the presence of synovial inflammation and cartilage damage. After successful modeling (starting from the 5th day), oral administration was performed at noon every day. The daily dose of forsythiaside, glucosamine, S3 and III was 100 mg / kg. After 30 days of continuous oral administration, the osteoarthritis disease status was analyzed by mouse grip strength test, knee joint section and knee joint tissue protein ELISA experiment.
[0029] Example 3 Experimental results: 1. Effect of forsythiaside aminoglycoside S3 on the grip strength of osteoarthritis mice The ZL-010 rat grip tester (Anhui Yaokun Biotechnology Co., Ltd.) was used to test the grip of the right hind limbs of the experimental mice. The mice to be tested were caught and their right hind limbs grasped the elastic metal strip on the grip test board and gradually pulled outward until the mice could no longer maintain their grip and the metal strip fell off their claws. The average value was taken as the experimental result of each mouse for three consecutive measurements, and the groups were statistically analyzed and plotted. The results showed that compared with the OA group, glucosamine, S3 and III all had a certain improvement effect on the grip of mice, while forsythiaside had no obvious improvement; forsythiaside aminoglycoside S3 was superior to forsythiaside acetyl glucosamine III ( Figure 2 ).
[0030] 2. Effect of forsythiaside aminoglycoside S3 on cartilage regeneration in osteoarthritis mice The experimental mice were killed by cervical dislocation, and the joints were cut off with a scalpel at 0.3 cm outside the modeling knee joint, and the joint samples were removed in one piece. The removed joint samples were fixed with 10% formaldehyde for 48 h, and then decalcified with formalin-nitric acid decalcification solution for 24 h. The decalcified specimens were dehydrated, transparent, wax-impregnated and embedded to make paraffin specimens. The paraffin specimens were cut into 3 μm thick paraffin slices using an HS-2205 rotary slicer (Jinhua Huasu). After dewaxing and rinsing with distilled water for a while, they were stained with hematoxylin and eosin (H&E, staining the whole tissue of the knee joint) and safranin fast green staining (staining cartilage). The slices were observed and photographed using a Nikon Ti-S inverted microscope, and the Mankin's score was calculated to determine whether S3 had a protective effect on cartilage tissue and knee joint inflammation. The results showed that S3, III and glucosamine had similar alleviating effects on cartilage damage caused by osteoarthritis and could enhance cartilage tissue regeneration in osteoarthritis mice, while forsythiaside had no significant alleviating effect; the alleviating effect of forsythiaside aminoglycoside S3 was better than that of forsythiaside acetyl glucosamine III ( Figure 3 and 4 ).
[0031] 3. Effect of forsythiaside aminoglycoside S3 on inflammation in osteoarthritis mice.
[0032] based on Figure 3 In the H&E stained images, the inflammatory cells at the joint joints were counted using the pathological image analysis software QuPath to analyze the progression of knee joint inflammation and the infiltration of inflammatory cells. Part of the mouse knee joint tissue was placed in RIPA lysis buffer, homogenized with a Labgic L-HOB homogenizer, and then lysed with a Hannuo HN98-IIB tissue cell ultrasonic crusher. The total protein extract was obtained after centrifugation. After the extracted protein concentration was quantified using the Biyuntian BCA protein quantification kit, the inflammatory-related cytokines in the sample were quantified using the Abcam mouse TNF-α, IL-1β, IL-6, and MCP-1 protein ELISA detection kit to determine the level of inflammation in the knee joint tissue. The results showed that forsythin, S3 and III all had good anti-inflammatory effects and could effectively alleviate the inflammatory condition of the knee joints of osteoarthritis mice, while the anti-inflammatory effect of glucosamine was not obvious; S3 and III had comparable anti-inflammatory effects, and both were able to reduce the infiltration of inflammatory cells and the expression of inflammatory-related cytokines while enhancing the regeneration of cartilage tissue in osteoarthritis mice, thereby alleviating osteoarthritis ( Figure 5 and Figure 6 ).
[0033] 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 other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principle of the present invention should be equivalent replacement methods. In addition, the hydrochloride, sulfate, phosphate, citrate, maleate, etc. of forsythiaside aminoglucosidase also belong to the protection scope of the present invention and are included in the protection scope of the present invention.
Claims
1. A forsythiaside aminoglycoside compound, characterized in that: The general structural formula of the compound is shown below: ； In the general structural formula, R1 is amino or phthaloylamino, and R2, R3, and R4 groups represent hydroxyl or alkanoyloxy groups. The alkanoyloxy group is a straight-chain alkanoyloxy group or a deuterated alkanoyloxy group.
2. The forsythiaside aminoglycoside compound according to claim 1, characterized in that: The alkanoyloxy group is a C1-C6 alkanoyloxy group or a deuterated alkanoyloxy group.
3. The forsythiaside aminoglycoside compound according to claim 1, characterized in that: The specific compound structure is shown below: 。 4. The method for preparing the forsythiaside aminoglycoside compound according to any one of claims 1 to 3, characterized in that: The specific steps are as follows: ； Forsythiaside I and anomeric bromoglucosamine II (hydroxyacetyl protection / aminophthaloyl protection) undergo nucleophilic substitution reaction under the action of phase transfer catalyst tetrabutylammonium chloride and strong base sodium hydroxide to generate forsythiaside aminoglucoside S1 (hydroxyacetyl protection / aminophthaloyl protection); Compound S1 was stirred in 7M ammonia methanol solution and deacetylated to obtain forsythiaside aminoglucosidone S2 (aminophthaloyl protected), which was further heated at 70 o C is treated with hydrazine hydrate to remove the phthaloyl group to obtain forsythiaside aminoglucoside S3.
5. Use of the forsythiaside aminoglycoside compound according to any one of claims 1 to 3 in the preparation of a drug for treating osteoarthritis.