Glycopeptide compound as well as preparation method and application thereof

Abstract

The invention discloses a glycopeptide compound as well as a preparation method and application thereof, and belongs to the technical field of medicinal chemistry. The glycopeptide compound has a structure as shown in a formula (I) and comprises three components, namely a B cell epitope, a regulatory T cell epitope and rapamycin, and the three components are coupled through a KK protein joint. The invention further provides application of the glycopeptide compound in preparation of a medicine for treating anti-MAG neuropathy autoimmune diseases and a medicine composition. # imgabs0 #

Description

Technical Field

[0001] The present invention relates to the technical field of medicinal chemistry, and in particular to a glycopeptide compound, a preparation method and an application thereof. Background Art

[0002] Autoimmune diseases (AIDs) are a series of illnesses caused by an abnormal immune response to self-antigens, resulting in an inability to tolerate one's own cells and tissues, leading to tissue damage or dysfunction. Abnormal post-translational modifications of certain proteins can alter peptide epitopes, creating new self-epitopes that are recognized and attacked by the immune system, thus inducing autoimmune diseases.

[0003] Glycosylation is the most common type of post-translational modification, and many glycopeptide epitopes are also associated with some autoimmune diseases, such as IgA nephritis, systemic lupus erythematosus, and rheumatoid arthritis. In addition to these glycopeptide self-epitopes, some oligosaccharide epitopes can also become targets of attack in autoimmune diseases, which are often seen in autoimmune neuropathy. For example, antibodies against the trisaccharide epitope of HNK-1 (human natural killer 1) contained in myelin-associated glycoprotein (MAG) can induce anti-MAG neuropathy.

[0004] Currently, the main treatments for anti-MAG neuropathy include plasma exchange therapy and intravenous immunoglobulin (IVIg) injection, but the treatment effect is usually poor and the recurrence rate is high. Therefore, there is an urgent need to develop drugs with more reliable efficacy and fewer side effects. Summary of the Invention

[0005] In view of this, the present invention provides a glycopeptide compound and a preparation method thereof, wherein the glycopeptide compound can be used for the treatment of MAG neuropathy.

[0006] In a first aspect, the present invention provides a glycopeptide compound, the structural formula of which is shown in formula (I):

[0007]

[0008] Wherein, the structural formula of R1 is shown in formula (II), and the structural formula of R2 is shown in formula (III);

[0009]

[0010] The glycopeptide compound of the present invention comprises three components: a B cell epitope, a regulatory T cell epitope and rapamycin, and the three components are coupled via a KK protein linker.

[0011] The B cell epitope is the HNK-1 (HSO3-3GlcAβ1-3Galβ1-4GlcNAc) trisaccharide antigen epitope, which can specifically bind to the B cell antigen receptor (BCR) that secretes anti-MAG autoantibodies, thereby allowing the glycopeptide compound to be specifically taken up and internalized by autoreactive B cells in the body through the BCR-mediated antigen endocytosis pathway.

[0012] The regulatory T cell epitope is a peptide hCDR1 based on the complementarity determining region (CDR) 1 sequence of a human monoclonal anti-DNA autoantibody with the 16 / 6 idiotype (16 / 6Id). hCDR1 can upregulate CD4 + CD25 + Immunomodulatory cell populations, thereby regulating autoimmunity.

[0013] The KK protein linker is a dipeptide sequence formed by two lysines, which can be specifically recognized and cleaved by cathepsin B in endosomes and lysosomes. In this way, the regulatory T cell epitope can be presented to regulatory T cells through MHC II molecules, thereby enhancing the effect of the regulatory T cell immune response.

[0014] Rapamycin is a common immunosuppressant that is often used for immunosuppression after organ transplantation. Its role in this glycopeptide compound is to prevent the immune activation response caused in the above process.

[0015] In the above-mentioned glycopeptide compound, the HNK-1 trisaccharide antigen epitope at one end is hydrophilic, while the rapamycin at the other end is lipophilic. Therefore, the glycopeptide can self-assemble under certain conditions, arranging the hydrophilic end on the outside and the lipophilic end on the inside. This is more conducive to the multivalent binding of the HNK-1 trisaccharide antigen epitope and BCR, increasing its affinity with BCR, and thus enhancing the efficiency of B cell uptake.

[0016] In a second aspect, the present invention provides a method for preparing the above-mentioned glycopeptide compound, comprising the following steps:

[0017] Compound 5 reacts with compound 6 to generate compound 7;

[0018] Compound 32 reacts with compound 9 to form compound 33 through a thiol-double bond addition reaction;

[0019] Compound 33 reacts with compound 7 to undergo an azide-alkynyl cycloaddition reaction to obtain;

[0020]

[0021]

[0022] Preferably, the compound 5 is obtained by sequentially subjecting the compound 3 to sulfation reaction and reduction reaction;

[0023]

[0024] Furthermore, the compound 3 is obtained by sequentially coupling compound 1 with galactose and glucuronic acid;

[0025]

[0026] Preferably, the preparation method of compound 6 is: reacting aminotris(carboxyethoxymethyl)methane with dibenzocyclooctene-succinimide ester to produce compound 25, and then esterifying compound 25 with N-hydroxysuccinimide to obtain the compound;

[0027]

[0028] Preferably, the preparation method of compound 9 is: reacting compound 29 with 4-pentynoic acid succinimidyl ester to generate compound 31, and then reacting the compound 31 with compound 8 through an azide-alkynyl cycloaddition reaction to obtain compound 9;

[0029]

[0030] Furthermore, the compound 8 is obtained by reacting rapamycin with succinic anhydride, N-hydroxysuccinimide and azide-triethylene glycol-amino in sequence.

[0031] In a third aspect, the present invention provides a pharmaceutical composition comprising a compound represented by formula (I) and a pharmaceutically acceptable carrier, diluent or excipient.

[0032] Preferably, the pharmaceutical composition is suitable for the treatment of anti-MAG neuropathy autoimmune diseases.

[0033] The present invention has achieved the following beneficial effects:

[0034] The present invention prepares a glycopeptide compound as shown in formula (I) by coupling a B cell epitope, a regulatory T cell epitope, and rapamycin with a KK protein linker. This glycopeptide compound can specifically bind to the BCR on the surface of B cells, leading to internalization by the B cells. It then presents the regulatory epitope to regulatory T cells, stimulating them to secrete regulatory cytokines that act on effector T cells and effector B cells, thereby achieving an immunosuppressive effect. The preparation of this compound has important implications for the development of treatments for other autoimmune diseases, particularly those associated with glycoepitopes. DETAILED DESCRIPTION

[0035] It should be noted that the following detailed descriptions are exemplary and intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention belongs.

[0036] The technical solution of the present invention is further described below with reference to specific examples. The present invention has no particular limitation on the source of the reagents, and commercially available products known to those skilled in the art can be used.

[0037] Example 1 Synthesis of Compound 1

[0038] The reaction equation is as follows:

[0039]

[0040] To a 1L round-bottom flask, glucosamine hydrochloride (20.0 g, 92.75 mmol) and sodium bicarbonate (23.37 g, 278.25 mmol) were added and dissolved in water (300 ml). 2,2,2-Trichloroethyl chloroformate (19.15 ml, 139.13 mmol) was added at 0°C and stirred overnight. After completion of the reaction, the mixture was filtered using a Buchner funnel and dried to afford Compound 12 (31.1 g, 95% yield) as a white solid.

[0041] Compound 12 (20.0 g, 56.66 mmol) was added to a 500 mL round-bottom flask and dissolved in pyridine (160 mL) in an ice bath. Acetic anhydride (38 mL) was then slowly added and stirred at room temperature overnight. After the reaction was complete, thin-layer chromatography (petroleum ether:ethyl acetate = 1:1) was performed and the mixture was concentrated by rotary evaporation. The concentrated reaction solution was dissolved in dichloromethane and extracted with dilute hydrochloric acid and saturated sodium bicarbonate aqueous solution, respectively. The organic phase was collected and dried over anhydrous sodium sulfate. Finally, the mixture was filtered through a Buchner funnel and evaporated to dryness to obtain compound 13 (28.9 g, 98%) as a white solid.

[0042] To a 500 mL round-bottom flask, compound 13 (8.13 g, 15.60 mmol), hydrazine acetate (1.44 g, 15.60 mmol), and N,N-dimethylformamide (DMF 150 ml) were added and reacted at 50°C for 45 min. After completion of the reaction, the mixture was concentrated by rotary evaporation after thin-layer chromatography (petroleum ether:ethyl acetate = 1:1). The concentrated reaction solution was dissolved in dichloromethane and extracted three times with water. The organic phase was dried over anhydrous sodium sulfate and concentrated by rotary evaporation to obtain compound 14 (6.1 g, 82% yield) as a white solid.

[0043] To a 250 mL round-bottom flask, compound 14 (6.13 g, 12.80 mmol), trichloroacetonitrile (3.91 mL, 38.39 mmol), and 1,8-diazacyclo[5,4,0]undecene-7 (0.58 mL, 3.84 mmol) were added, along with ultra-dry dichloromethane (100 mL). The mixture was stirred at room temperature for 1 hour. After completion of the reaction, the mixture was concentrated by rotary evaporation and purified by flash column chromatography (petroleum ether:ethyl acetate = 3:1) to obtain compound 15 (6.6 g, 83.4% yield) as a white solid.

[0044] Compound 15 (487 mg, 0.78 mmol), 3-chloro-1-propanol (131 μL, 1.56 mmol), ultra-dry dichloromethane (10 mL), and 4A molecular sieves (1 g) were added to a 50 mL three-necked flask. The mixture was stirred at room temperature for 30 min, then on ice for 10 min. Trimethylsilyl trifluoromethanesulfonate (28 μL, 1.56 mmol) was added and allowed to react for 1 h. Thin-layer chromatography (petroleum ether:ethyl acetate = 2:1) confirmed the reaction was complete and quenched with a few drops of triethylamine. The mixture was filtered through celite, concentrated by rotary evaporation, and purified by flash column chromatography (petroleum ether:ethyl acetate = 3:1) to obtain compound 16 (0.40 g, 93% yield) as a white solid.

[0045] Compound 16 (1.22 g, 2.19 mmol), acetic acid (20 mL), and Zn powder (4.7 g, 72.41 mmol) were added to a 100 mL round-bottom flask and allowed to react at room temperature for 4 h. After completion of the reaction, as determined by thin-layer chromatography (dichloromethane:methanol = 14:1), the product was filtered through celite, rinsed with methanol, concentrated by rotary evaporation, and purified by flash column chromatography (dichloromethane:methanol = 50:1) to obtain compound 17 (792.92 g, 95% yield) as a white solid.

[0046] Compound 17 (4.2 g, 11.02 mmol), pyridine (30 mL), and anhydride (7.3 mL) were added to a 100 mL round-bottom flask and stirred overnight at room temperature. After completion of the reaction, as determined by thin-layer chromatography (petroleum ether:ethyl acetate = 1:4), the mixture was concentrated by rotary evaporation and purified by flash column chromatography (petroleum ether:ethyl acetate = 1:2). Compound 18 (4.7 g, 98% yield) was obtained as a white solid.

[0047] To a 100 mL round-bottom flask, compound 18 (4.66 g, 11.01 mmol), sodium azide (2.86 g, 44.05 mmol), tetrabutylammonium iodide (0.80 g, 2.20 mmol), and N,N-dimethylformamide (30 mL) were added. The mixture was heated to 110°C and stirred for 12 h. After completion of the reaction, thin-layer chromatography (petroleum ether:ethyl acetate = 1:4) was performed. The mixture was filtered through celite, concentrated by rotary evaporation, and purified by flash column chromatography (petroleum ether:ethyl acetate = 1:2). Compound 17 (4.62 g, 98% yield) was obtained as a white solid.

[0048] Compound 17 (2.28 g, 5.32 mmol) and anhydrous methanol (85 mL) were added to a 100 mL round-bottom flask. Sodium methoxide was gradually added until the pH was in the range of 9.0-10.0, and the mixture was stirred at room temperature overnight. After completion of the reaction by thin-layer chromatography (petroleum ether:ethyl acetate = 1:4), the pH was adjusted to neutral with dilute hydrochloric acid, and the mixture was concentrated by rotary evaporation. After separation and purification via P-2 gel chromatography column and DEAE anion exchange column, a white solid compound 1 (1.38 g, 85% yield) was obtained. The NMR analysis results of compound 1 are as follows: 1 H NMR(600MHz,D2O)δ4.47(d,J=8.4Hz,1H),3.94(dt,J=10.8,5.6Hz,1H),3.89(dd,J=12.4,1.8Hz,1H),3.7 3-3.68(m,1H),3.68-3.60(m,2H),3.53-3.48(m,1H),3.43-3.31(m,4H),2.01(s,3H),1.83-1.78(m,2H).

[0049] Example 2 Synthesis of Compound 2

[0050] The disaccharide compound 2 was synthesized by assembling modules using a multi-enzyme cascade enzymatic method. The reaction equation is as follows:

[0051]

[0052] Compound 1 (1 g, 3.29 mmol), galactose (Gal) (0.71 g, 3.95 mmol), ATP (2.08 g, 3.95 mmol), UTP (1.91 g, 3.95 mmol), Tris (100 mM), and MgCl2 (100 mM) were dissolved in 50 mL of double-distilled water. The pH was adjusted to 7.5 with 1 M hydrochloric acid. Galactokinase (EcGalK) (25 mL, approximately 2 mg / mL), uridine diphosphate glycoside pyrophosphatase (BLUSP) (30 mL, approximately 2 mg / mL), and β1,4-galactose glycosyltransferase (NmLgtB) (50 mL, approximately 2 mg / mL) were then added. The mixture was incubated at 37°C in a shaker for 12 h. The reaction was detected by thin-layer chromatography (ethyl acetate:methanol:water:acetic acid = 8:3:1:0.5, v / v). After completion, an equal volume of glacial ethanol was added to terminate the reaction. The reaction mixture was mixed with icy ethanol and placed in a -20°C refrigerator for at least 30 minutes. The mixture was then centrifuged at 12,000 rpm and 4°C for 30 minutes. The supernatant was collected and evaporated to dryness. The evaporated product was separated and purified by P-2 gel chromatography and a DEAE anion exchange column to obtain compound 2 (1.5 g, 95% yield) as a white solid. The parameters were as follows: 1 H NMR(600MHz,D2O)δ4.54(d,J=7.5Hz,1H),4.48(d,J=7.8Hz,1H),3.98(td,J=11.7,3. 9Hz,2H),3.93(d,J=3.4Hz,1H),3.83(dd,J=12.3,5.1Hz,1H),3.79–3.69(m,6H),3.69 –3.67(m,1H),3.67–3.66(m,1H),3.60(ddt,J=7.3,5.0,2.3Hz,1H),3.54(dd,J=9.9, 7.8Hz, 1H), 3.38 (td, J = 6.5, 3.1Hz, 2H), 2.05 (d, J = 0.9Hz, 3H), 1.85 (p, J = 6.3Hz, 2H).

[0053] Example 3 Synthesis of Compound 3

[0054] The trisaccharide compound 3 was synthesized by assembling modules using a multi-enzyme cascade enzymatic method. The reaction equation is as follows:

[0055]

[0056] Compound 2 (100 mg, 0.21 mmol), glucuronic acid (GlcA) (49.95 mg, 0.2 mmol), ATP (137 mg, 0.26 mmol), UTP (125.88 mg, 0.26 mmol), Tris (100 mM), and MgCl2 (20 mM) were dissolved in 5 mL of double-distilled water. The pH was adjusted to 7.5 with 1 M hydrochloric acid. Appropriate amounts of glucuronide kinase (AtGlcAK) (1 mL, approximately 1 mg / mL), uridine diphosphate glycoside pyrophosphatase (BLUSP) (1 mL, approximately 2 mg / mL), and β1,3-glucuronosyltransferase (GlcAT-P) (5 mL, approximately 0.5 mg / mL) were added and the mixture was allowed to react in a shaker at 37°C for 36 h. The reaction was determined by thin-layer chromatography (ethyl acetate:methanol:water:acetic acid = 4:2:1:0.5, v / v). After completion of the reaction, an equal volume of glacial ethanol was added to terminate the reaction. The reaction mixture was mixed with icy ethanol and placed in a -20°C refrigerator for at least 30 minutes. The mixture was then centrifuged at 12,000 rpm and 4°C for 30 minutes. The supernatant was collected and evaporated to dryness. The evaporated product was separated and purified by P-2 gel chromatography and a DEAE anion exchange column to obtain white compound 3 (132 mg, 96% yield). The parameters were as follows: 1 HNMR(600MHz,D2O)δ4.65(d,J=7.8Hz,1H),4.50(dd,J=7.9,1.2Hz,2H),4.16(d,J=3.2Hz,1H),3.99-3.93(m,2H),3.83-3.77(m,2 H),3.76-3.61(m,10H),3.57(ddd,J=9.5,5.2,2.2Hz,1H),3.52-3.45(m,2H),3.41-3.31(m,3H),2.02(s,3H),1.84-1.79(m,2H).

[0057] Example 4 Synthesis of Compound 4

[0058] The reaction equation is as follows:

[0059]

[0060] Compound 3 (1 g, 1.56 mmol) was completely dissolved in 20 mL of ultra-dry N,N-dimethylformamide, and acetic anhydride (6.1 mL, 65.38 mmol) was added. The mixture was allowed to react at 80°C for 3 h under argon. The reaction progress was monitored by thin-layer chromatography (ethyl acetate:methanol:water:acetic acid = 4:2:1:0.5). After the complete disappearance of the starting material, the reaction solution was cooled to room temperature. Ultra-dry pyridine (20 mL) was then added to the reaction solution, and the mixture was stirred at room temperature overnight. The mixture was then analyzed by thin-layer chromatography (ethyl acetate:methanol:water:acetic acid = 4:2:1:0.5). After the reaction was complete, the mixture was concentrated by rotary evaporation and purified by flash column chromatography (petroleum ether:ethyl acetate = 1:4) to obtain compound 20 (1.36 g, 95% yield).

[0061] Compound 20 (100 mg, 0.11 mmol) was completely dissolved in 5 mL of anhydrous methanol, and anhydrous sodium acetate (10.71 mg, 0.13 mmol) was added and allowed to react at room temperature for 3 h. Thin layer chromatography (ethyl acetate) showed that the reaction was complete. + The reaction solution was neutralized to a neutral pH by using a cation exchange resin, filtered, concentrated by rotary evaporation, and purified by flash column separation (petroleum ether:ethyl acetate=1:4) to obtain compound 21 (92.3 mg, yield 89%).

[0062] Compound 21 (100 mg, 0.10 mmol) was completely dissolved in 5 mL of ultra-dry N,N-dimethylformamide, and sulfur trioxide trimethylamine complex (732 mg, 5.24 mmol) was added. The mixture was reacted at 55°C under argon for 4 h. Thin-layer chromatography (dichloromethane:methanol = 10:1) confirmed the reaction was complete. The reaction solution was cooled to room temperature and quenched by adding excess anhydrous sodium bicarbonate and stirring for 2 h. The mixture was filtered through celite, concentrated by rotary evaporation, and purified by flash column chromatography (dichloromethane:methanol = 15:1) to obtain compound 22 (92.15 mg, 85% yield).

[0063] Compound 22 (100 mg, 0.1 mmol) was completely dissolved in 2 mL of anhydrous methanol, and sodium methoxide was gradually added until the pH value was within the range of 9.0-10.0. After the reaction was allowed to react at room temperature for 4 h, water equal in volume to anhydrous methanol was added and the reaction was continued for 1 h. After the reaction was complete, H + The reaction solution was neutralized to a neutral pH using a cation exchange resin, filtered, concentrated by rotary evaporation, and purified by DEAE anion exchange column and P-2 gel chromatography to obtain compound 4 (70.1 mg, yield 89%) as a white solid. The parameters are as follows: 1H NMR(600MHz,D2O)δ4.50(dt,J=8.2,1.7Hz,2H),4.30(td,J=9.1,1.4Hz,1H),4.16(d,J=3.2Hz,1H),3.95(td,J=11.8,10 .8,7.8Hz,3H),3.83-3.62(m,14H),3.60-3.54(m,2H),3.39-3.31(m,3H),2.02(d,J=1.3Hz,3H),1.82(p,J=6.4Hz,2H).

[0064] Example 5 Synthesis of Compound 5

[0065] The reaction equation is as follows:

[0066]

[0067] Compound 4 (100 mg, 0.16 mmol) was completely dissolved in pyridine (5 mL) and water (1 mL). Triethylamine (1.08 mL, 7.79 mmol) and 1,3-propanedithiol (0.78 mL, 7.79 mmol) were added and reacted at 40°C for 1 hour. The reaction was complete as determined by thin-layer chromatography (ethyl acetate:methanol:water:acetic acid = 2:1:1:0.5). The mixture was concentrated by rotary evaporation and purified by RP-18 silica gel column and P-2 gel chromatography to obtain compound 5 (86.3 mg, 90% yield). The parameters are as follows: 1 H NMR(600MHz,D2O)δ4.73(d,J=7.9Hz,1H),4.49(dd,J=8.0,5.2Hz,2H),4.30(t,J=9.1Hz,1H),4.15(d,J=3.3Hz,1H),4.02-3.9 6(m,2H),3.84-3.78(m,2H),3.77-3.64(m,12H),3.59-3.54(m,2H),3.05(t,J=7.0Hz,2H),2.02(s,3H),1.92(p,J=6.9Hz,2H).

[0068] Example 6 Synthesis of Compound 6

[0069] The reaction equation is as follows:

[0070]

[0071] Aminotris(carboxyethoxymethyl)methane (57 mg, 0.17 mmol) was dissolved in 0.5 mL of ultra-dry N,N-dimethylformamide. N,N-diisopropylethylamine (0.29 mL, 1.69 mmol) was then added. After stirring at room temperature for 5 min, dibenzocyclooctene succinimide ester (85 mg, 0.21 mmol) was added and stirred at room temperature for 40 h. Thin-layer chromatography (ethyl acetate:methanol:water:acetic acid = 8:3:1:0.5) confirmed the reaction was complete. The mixture was concentrated by rotary evaporation and purified by flash column chromatography (dichloromethane:methanol = 2:3) to afford compound 25 (68.67 mg, 65% yield). The parameters were as follows: 1 H NMR (600MHz, CD3OD) δ7.53 (d, J=7.6Hz, 1H), 7.48 (dd, J=7.7, 1.7Hz, 1H), 7.40-7.29 (m, 3H), 7.23 (m,J=16.5,14.8,9.4,7.8Hz,2H),7.13(dd,J=7.3,1.7Hz,1H),5.01(d,J=13.7Hz,1H),3.62-3.53 (m,3H),3.51-3.44(m,9H),2.53(dt,J=15.7,7.6Hz,1H),2.36(td,J=6.2,1.7Hz,6H),2.29-2.21( m,1H),2.02(ddd,J=14.8,8.2,6.0Hz,1H),1.82(ddd,J=17.0,8.3,6.0Hz,1H),1.27-1.14(m,1H).

[0072] Compound 25 (9 mg, 0.014 mmol) was dissolved in ultra-dry N,N-dimethylformamide (0.5 mL), and N-hydroxysuccinimide (8.28 mg, 0.072 mmol) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (13.80 mg, 0.072 mmol) were added. The mixture was protected by nitrogen and stirred at room temperature for 15 h. The reaction was complete as determined by thin-layer chromatography (ethyl acetate: methanol: water = 16:3:1). A large amount of dichloromethane was added to dilute the reaction solution, and the organic phase was extracted three times with a saturated sodium chloride aqueous solution, and the organic phase was collected. The mixture was concentrated by rotary evaporation and purified by flash column separation (dichloromethane: methanol = 25:1) to obtain compound 6 (11.2 mg, yield 85%). The parameters are as follows: 1H NMR (600MHz, CDCl3) δ7.65 (d, J = 7.4Hz, 1H), 7.47-7.44 (m, 1H), 7.41-7.27 (m, 5H) ,7.24(d,J=7.4Hz,1H),6.05(s,1H),5.30(s,1H),5.12(d,J=14.0Hz,1H),3.72(t ,J=6.0Hz,6H),3.70-3.62(m,7H),2.88-2.65(m,19H),2.39(ddd,J=15.7,9.7,6. 1Hz, 1H), 2.07 (ddd, J=15.3, 9.5, 5.3Hz, 1H), 1.96 (ddd, J=15.7, 9.6, 5.4Hz, 2H).

[0073] Example 7 Synthesis of Compound 7

[0074] The reaction equation is as follows:

[0075]

[0076] Wherein, the structural formula of R1 is shown in formula (II):

[0077]

[0078] Compound 6 (11 mg, 0.012 mmol) and compound 5 (50.13 mg, 0.072 mmol) were dissolved in ultra-dry dimethyl sulfoxide (0.2 mL). The pH of the reaction solution was adjusted to 7.5 with N,N-diisopropylethylamine and stirred at 35°C for 72 h. The reaction was confirmed to be complete by thin-layer chromatography (ethyl acetate:methanol:water = 2:2:1). Compound 7 (25.5 mg, 80% yield) was isolated and purified by RP-18 silica gel column and P-2 gel chromatography.

[0079] Example 8 Synthesis of Compound 8

[0080] The reaction equation is as follows:

[0081]

[0082] Rapamycin (500 mg, 0.55 mmol), anhydrous toluene (10 mL), succinic anhydride (250 mg, 2.50 mmol), and lipase (1.125 g) were added to a 50 mL round-bottom flask and reacted at 45°C under argon for 8 h. After completion of the reaction, as determined by thin-layer chromatography (dichloromethane:methanol = 13:1), the filtrate was filtered, collected, and evaporated to dryness using a rotary evaporator. Purification was then performed using a flash column chromatography (dichloromethane:methanol = 30:1) to afford Compound 27 as a white solid (501.7 mg, 90% yield).

[0083] Compound 27 (300 mg, 0.30 mmol) was added to a 50 mL eggplant-shaped flask and completely dissolved in ultra-dry dichloromethane (10 mL). N-hydroxysuccinimide (102.35 mg, 0.89 mmol) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (170.58 mg, 0.89 mmol) were then added. The mixture was reacted under nitrogen atmosphere at room temperature for 6 h. After completion of the reaction, thin-layer chromatography (dichloromethane:methanol = 13:1) was performed, followed by dilution with a large amount of dichloromethane and extraction three times with saturated sodium chloride. The organic phase was collected and concentrated by rotary evaporation and purified by flash column chromatography (dichloromethane:methanol = 80:1) to afford compound 28 (263 mg, 80% yield).

[0084] Compound 28 (50 mg, 0.045 mmol) and azide-triethylene glycol-amino group (19.6 mg, 0.09 mmol) were dissolved in ultra-dry N,N-dimethylformamide (2 mL). The pH of the reaction solution was adjusted to 7.5 with N,N-diisopropylethylamine and stirred at room temperature for 48 h. After completion of the reaction by thin-layer chromatography (dichloromethane:methanol = 9:1), the mixture was concentrated by rotary evaporation and purified by flash column chromatography (dichloromethane:methanol = 50:1) to afford compound 8 (46.4 mg, 85% yield).

[0085] Example 2 Synthesis of Compound 9

[0086] The reaction equation is as follows:

[0087]

[0088] Compound 29 (20 mg, 0.02 mmol) and 4-pentynoic acid succinimidyl ester (40.32 mg, 0.21 mmol) were dissolved in a mixture of dimethyl sulfoxide (DMSO) and phosphate buffer (pH 7) at a ratio of 1:4 and stirred at room temperature for 16 h. The emulsion was then added with 5 mL of 0.01 M phosphate buffer to redissolve the suspended protein. The mixture was centrifuged at 12,000 g for 5 min at 4°C to remove insoluble impurities. The supernatant was collected and evaporated to dryness. Compound 31 was isolated and purified by high-performance liquid chromatography.

[0089] Compound 31 (5 mg, 0.004 mmol) and compound 8 (50.25 mg, 0.04 mmol) were dissolved in 2 mL of dimethyl sulfoxide. Copper sulfate (0.25 mM), tris(3-hydroxypropyltriazolemethyl)amine (1.25 mM), and sodium ascorbate (1.0 mM) were added, and the mixture was stirred at room temperature for 2 h. Compound 9 was isolated and purified by HPLC.

[0090] Example 10 Synthesis of Glycopeptide Compound of Formula (I)

[0091] The reaction equation is as follows:

[0092]

[0093] Wherein, the structural formula of R2 is shown in formula (III):

[0094]

[0095] Compound 9 (5 mg, 1 μmol) and compound 32 (3.18 mg, 1 μmol) were dissolved in dimethyl sulfoxide (1 mL), gently stirred at room temperature for 24 h under argon protection, and separated and purified by high performance liquid chromatography to obtain compound 33.

[0096] Compound 33 (5 mg, 0.63 μmol) and compound 7 (1.68 mg, 0.63 μmol) were dissolved in 200 μL dimethyl sulfoxide, and copper sulfate (0.25 mM), tris(3-hydroxypropyltriazolemethyl)amine (1.25 mM) and sodium ascorbate (1.0 mM) were added. The mixture was stirred at room temperature for 2 h and separated and purified by high performance liquid chromatography to obtain a glycopeptide compound of formula (I).

[0097]

[0098] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. A glycopeptide compound, characterized in that Its structural formula is shown in formula (I): Wherein, the structural formula of R1 is shown in formula (II), and the structural formula of R2 is shown in formula (III); 2. The method for preparing the glycopeptide compound according to claim 1, wherein The method comprises the following steps: compound 5 reacts with compound 6 to generate compound 7; Compound 32 reacts with compound 9 to form compound 33 through a thiol-double bond addition reaction; Compound 33 reacts with compound 7 to undergo an azide-alkynyl cycloaddition reaction to obtain; 3. The preparation method according to claim 2, wherein The compound 5 is obtained by sequentially subjecting the compound 3 to sulfation reaction and reduction reaction; 4. The preparation method according to claim 3, wherein The compound 3 is obtained by sequentially coupling compound 1 with galactose and glucuronic acid; 5. The preparation method according to claim 2, wherein The preparation method of compound 6 is as follows: aminotris(carboxyethoxymethyl)methane is reacted with dibenzocyclooctene-succinimide ester to generate compound 25, and then compound 25 is esterified with N-hydroxysuccinimide to obtain the compound; 6. The preparation method according to claim 2, wherein The preparation method of the compound 9 is as follows: compound 29 is reacted with 4-pentynoic acid succinimidyl ester to generate compound 31, which is then reacted with compound 8 to undergo an azide-alkynyl cycloaddition reaction to obtain compound 31; 7. The preparation method according to claim 6, wherein The compound 8 is prepared by sequentially reacting rapamycin with succinic anhydride, N-hydroxysuccinimide and azide-triethylene glycol-amino groups.

8. Use of the glycopeptide compound according to claim 1 in the preparation of a medicament for treating anti-MAG neuropathy autoimmune diseases.

9. A pharmaceutical composition, characterized in that The composition comprises the glycopeptide compound according to claim 1 and a pharmaceutically acceptable carrier, diluent or excipient.

10. The pharmaceutical composition according to claim 9, wherein The pharmaceutical composition is suitable for the treatment of anti-MAG neuropathy autoimmune diseases.

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