Drug targeting delivery system with biological orthogonal property and application thereof

Abstract

The invention discloses a drug targeting delivery system with biological orthogonality and application thereof, and relates to the technical field of biological medicine. The drug targeted delivery system comprises mutant galactosidase and a prodrug, the amino acid sequence of the mutant galactosidase is as shown in SEQ ID NO. 6; the structural formula of the prodrug is shown in the specification; wherein R1 is phenyl, benzyl or phenethyl; and R2 is a drug loading group. According to the present invention, the combination of the novel mutant galactosidase and the specific structure prodrug is constructed, such that the efficient and accurate drug delivery is achieved, the problems of narrow substrate application range, insufficient safety, limited drug delivery mode and the like of the existing enzyme-prodrug system are solved, and the flexible, safe and efficient new strategy is provided for the drug targeting delivery.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a drug targeted delivery system with bioorthogonal properties and its applications. Background Technology

[0002] Enzymatic prodrug therapy is one of the emerging and popular methods for treating tumors in recent years. Its design principle is as follows: Figure 1 As shown, under the catalysis of hydrolytic enzymes highly expressed in tumor tissue, inactive or weakly active prodrugs are hydrolyzed, releasing active drugs with high selectivity, thereby exerting anti-tumor effects and improving drug targeting while reducing drug toxicity to some extent. However, most prodrug-responsive enzymes are currently endogenous, which can cause non-specific release of drugs in the body, leading to toxic side effects on normal organs.

[0003] To improve the targeting of cytotoxic drugs to tumor tissues, antibody-drug conjugates (ADCs) have emerged. ADCs consist of a monoclonal antibody (mAb), a cytotoxic drug (payload), and a linker, and their structure is as follows: Figure 2 As shown in the illustration (using DS-8201). Since the linker requires hydrolysis to release cytotoxic drugs into tumor tissue and kill tumor cells, ADC (Antibody-Drug Conversion) is broadly considered a more targeted prodrug therapy strategy. Although the introduction of antibodies greatly improves drug targeting, the linker is still designed to target endogenous enzymes. Therefore, optimizing the linker structure is crucial in ADC drug development. It's essential to ensure efficient drug hydrolysis at the tumor site while minimizing non-specific drug release into normal tissues. After extensive screening, several ADC drugs are now used clinically, benefiting patients. Therefore, optimizing the structure of the response group to achieve controlled drug release and targeted delivery can effectively solve the off-target toxicity problem caused by highly active drugs.

[0004] Considering the toxic side effects caused by non-specific drug release in normal organs due to prodrug design targeting endogenous hydrolases, and influenced by the chemical design principles of ADC drug linkers, Professor Wang Peng's team designed a drug delivery system with bioorthogonal properties in 2019. The design principle is as follows: Figure 3As shown, 6-methylgalactose (6-Me-Gal) was prepared by methylation modification at the six-position of galactose (Gal). Studies have shown that wild-type galactosidase cannot hydrolyze this substrate. Therefore, although there are many endogenous galactosidases in vivo, using 6-Me-Gal for nitric oxide (NO) delivery can effectively avoid the increase in heart rate caused by non-specific release of NO in normal tissues. This design strategy ensures the safety of the prodrug in vivo. In addition, the sterically hindered histidine at position 363 of the wild-type galactosidase (Wild-type A4-β-Gal) catalytic pocket was mutated to a sterically hindered alanine. The resulting mutant galactosidase (Mutant A4-β-Gal) can efficiently recognize and hydrolyze the 6-methylgalactosidase substrate, achieving targeted delivery of NO and promoting angiogenesis in mice with lower limb ischemia, thus verifying the in vivo therapeutic effect of this delivery strategy.

[0005] Professor Wang Peng's team developed a bioorthogonal hydrolysis system consisting of a mutant galactosidase (Mutant A4-β-Gal) and a chemically modified galactoside substrate (6-Me-Gal), achieving targeted drug delivery. However, Mutant A4-β-Gal can only recognize 6-Me-Gal and exhibits poor hydrolytic activity towards galactose modified with other groups. This directly limits the use of 6-Me-Gal in prodrug design, significantly restricting the flexible modification of glycoside prodrug structures and consequently limiting the regulation of prodrug pharmacokinetic parameters. In addition, Mutant A4-β-Gal still has a strong hydrolytic ability towards Gal, which may cause non-specific hydrolysis of galactose residues on biomolecules in vivo, raising concerns about its in vivo safety. Furthermore, this type of glycoside prodrug is highly water-soluble and often requires injection to be effective, causing significant discomfort to patients and reducing patient compliance.

[0006] Therefore, there is a need to develop a new generation of glycosidase-substrate hydrolysis reaction systems that allow for flexible modification of the structure of glycoside prodrugs, have higher safety profiles, and are orally administered for precise drug delivery. Summary of the Invention

[0007] The purpose of this invention is to provide a drug-targeted delivery system with bioorthogonal properties and its applications, thereby addressing the problems existing in the prior art. This drug-targeted delivery system enables efficient and precise drug delivery, providing a flexible, safe, and efficient new strategy for drug-targeted delivery.

[0008] To achieve the above objectives, the present invention provides the following solution:

[0009] This invention provides a drug-targeted delivery system with bioorthogonal properties, comprising a mutant galactosidase and a prodrug;

[0010] The amino acid sequence of the mutant galactosidase is shown in SEQ ID NO. 6;

[0011] The structural formula of the prodrug is: Wherein, R1 is phenyl, benzyl, or phenethyl; R2 is the drug-loaded group.

[0012] Preferably, R1 is phenylethyl.

[0013] Furthermore, the drug-loaded group corresponds to a fluorescent reporter molecule.

[0014] Furthermore, the drug-loaded group mentioned above corresponds to a cancer treatment drug.

[0015] Furthermore, the cancer treatment drug is SN38.

[0016] The present invention also provides the application of a drug-targeted delivery system with bioorthogonal properties in the preparation of a drug for treating diseases, wherein the drug-targeted delivery system comprises a mutant galactosidase and a prodrug;

[0017] The amino acid sequence of the mutant galactosidase is shown in SEQ ID NO. 6;

[0018] The structural formula of the prodrug is: Wherein, R1 is phenyl, benzyl, or phenethyl; R2 is the drug-loaded group.

[0019] Furthermore, R1 is phenylethyl.

[0020] Furthermore, when the disease treatment drug is a breast cancer treatment drug, the loading drug corresponding to the loading drug group is SN38.

[0021] The present invention also provides a breast cancer treatment drug, including a drug targeted delivery system;

[0022] The drug-targeted delivery system includes a mutant galactosidase and a prodrug;

[0023] The amino acid sequence of the mutant galactosidase is shown in SEQ ID NO. 6;

[0024] The structural formula of the prodrug is: Wherein, R1 is phenylethyl; R2 is the drug-loaded group;

[0025] The drug loading group corresponds to the drug loading drug SN38.

[0026] Furthermore, the breast cancer treatment drug also includes pharmaceutically acceptable excipients.

[0027] The present invention discloses the following technical effects:

[0028] This invention provides a drug-targeted delivery system with bioorthogonal properties. By constructing a novel combination of a mutant galactosidase and a prodrug with a specific structure, efficient and precise drug delivery is achieved. This drug-targeted delivery system has the following outstanding advantages:

[0029] (1) The mutant enzyme selected in this invention has significantly reduced hydrolytic activity on natural galactosidase substrates, effectively avoiding non-specific drug release caused by endogenous galactosidase in vivo, thereby significantly improving the stability and safety of the prodrug in vivo.

[0030] (2) This mutant enzyme can efficiently recognize and hydrolyze benzene ring-modified galactoside prodrugs, enabling the controlled release of active drugs at the target site, thus improving the precision and efficacy of treatment.

[0031] (3) The groups introduced into the prodrug structure effectively improved its lipid-water partition coefficient, significantly increased oral bioavailability, made oral administration possible, and enhanced patient compliance. In a breast cancer treatment model, the system successfully achieved targeted release of the loaded drug (SN38), significantly inhibited tumor growth, and showed good clinical application prospects.

[0032] This invention addresses the problems of narrow substrate applicability, insufficient safety, and limited administration methods in existing enzyme-prodrug systems, providing a flexible, safe, and efficient new strategy for targeted drug delivery. Attached Figure Description

[0033] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0034] Figure 1 Design schematic diagram for prodrug;

[0035] Figure 2 Diagrams showing the structure of ADC drugs and their protease hydrolysis.

[0036] Figure 3 Schematic diagram of the design of mutant galactosidase and 6-Me-Gal substrate;

[0037] Figure 4 The structural formula of the galactoside substrate;

[0038] Figure 5 The graph shows the results of detecting the hydrolytic effect of wild-type galactosidase on different galactoside substrates.

[0039] Figure 6 The crystal structure diagram of galactosidase 1KWK;

[0040] Figure 7 The graph shows the results of detecting the hydrolytic effects of mutant enzymes 1-6 and wild-type galactosidase (7) on different galactoside substrates.

[0041] Figure 8 The results of serum stability assays for different galactoside substrates are shown in the figure.

[0042] Figure 9 The images show the results of targeting and releasing different galactoside substrates in vivo. A is a fluorescence image of mice after the near-infrared fluorescent molecule Gal-DAO is injected into them; B is a fluorescence image of mice after the near-infrared fluorescent molecule 3-C-Gal-DAO is injected into them; and C is a fluorescence image of mice with enzyme 5 fixed in the hind limbs after the near-infrared fluorescent molecule 3-C-Gal-DAO is injected into them.

[0043] Figure 10 The graph shows the oral pharmacokinetic test results for Gal-DAO and 3-C-Gal-DAO.

[0044] Figure 11 A statistical graph showing the tumor volume of mice in different groups;

[0045] Figure 12 Fluorescence imaging of tumor tissues from different groups of mice. Detailed Implementation

[0046] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0047] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0048] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0049] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This specification and embodiments are merely exemplary.

[0050] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0051] The method for synthesizing the compound involved in this invention is as follows:

[0052] Synthesis Route 1:

[0053] Synthesis of compound 2:

[0054] Compound 1 (10 g, 0.055 mol) was weighed into a round-bottom flask, anhydrous acetone was added, and a magnetic stir bar was placed. 2,2-Dimethoxypropane (27.28 mL, 0.222 mol) was added dropwise with stirring; the reaction system was initially turbid. The pH of the reaction system was adjusted to 1–3 using p-toluenesulfonic acid, and the mixture was stirred at room temperature. As the reaction proceeded, the system gradually became clear. The reaction was monitored by thin-layer chromatography (TLC) with petroleum ether:ethyl acetate = 1:2 as the developing solvent and CMC as the colorimetric reagent. The Rf value of the target compound was 0.4. After approximately 12 hours, the conversion of the starting material was detected as complete. The reaction was quenched with triethylamine, concentrated under reduced pressure, and purified by rapid column chromatography (petroleum ether:ethyl acetate = 2:1). The yield was 96.8%.

[0055] Synthesis of compound 3:

[0056] Compound 2 (14 g, 0.054 mol) was weighed into a round-bottom flask, a magnetic stir bar was placed inside, and the substrate was dissolved in dichloromethane. While stirring, 27.38 g, 0.064 mol, of Dys-Martin oxidant was slowly added to the reaction system at room temperature under inert gas protection. The reaction was monitored by thin-layer chromatography (TLC) with dichloromethane:ethyl acetate as the developing solvent (3:1) and CMC as the colorimetric reagent. The Rf value of the target compound was 0.6. After approximately 10 hours, the conversion of the starting material was confirmed to be complete. Saturated sodium bicarbonate solution was slowly added to the system to quench the reaction, at which point a white solid precipitated. The system was filtered, and the filtrate was collected. The lower aqueous phase was separated, washed 2-3 times with dichloromethane, and the organic phases were combined. The organic phases were washed 2-3 times each with saturated sodium bicarbonate solution and saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. Rapid column chromatography was used for separation and purification (dichloromethane:ethyl acetate = 15:1). The yield was 63.2%.

[0057] Synthesis Route 2:

[0058]

[0059] Synthesis of compound 4:

[0060] Compound 3 (10 g, 0.039 mol) was weighed into a round-bottom flask, cooled to 0 °C in an ice bath, and the substrate was dissolved in tetrahydrofuran. A magnetic stir bar was placed in the flask, and the mixture was kept under inert gas protection. Using a syringe with a long needle, 9.3 mL of phenylmagnesium bromide (0.058 mol) was slowly added dropwise to the stirred system. The mixture was stirred at 0 °C under inert gas protection. Thin-layer chromatography (TLC) was used to monitor the reaction. The developing solvent was petroleum ether:ethyl acetate = 3:1. CMC was used as the colorimetric reagent. The Rf value of the target compound was 0.4, and the conversion of the starting material was detected after approximately 2 hours. The reaction mixture was extracted with ethyl acetate into a separatory funnel. The mixture was washed 2-3 times with water and saturated sodium chloride, and the aqueous phase was re-extracted with ethyl acetate. The organic phases were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. Rapid column chromatography was used for separation and purification, with petroleum ether:ethyl acetate = 6:1 as the eluent. The yield was 71.5%.

[0061] Compound 4: 1H NMR (400 MHz, CDCl3) δ 7.43 (d, J = 7.4 Hz, 2H), 7.37 (t,J = 7.5 Hz, 2H), 7.29 (t, J = 7.2 Hz, 1H), 5.57 (d, J = 5.0 Hz, 1H), 4.95 (t,J = 5.1 Hz, 1H), 4.57 (dd, J = 8.0, 2.2 Hz, 1H), 4.37 (d, J = 8.0 Hz, 1H), 4.31 (dd, J = 5.0, 2.3 Hz, 1H), 3.93 (d, J = 6.4 Hz, 1H), 3.38 (d, J = 5.8Hz, 1H), 1.55 (s, 3H), 1.49 (s, 3H), 1.36 (s, 3H), 1.30 (s, 3H).

[0062] Synthesis of compound 5:

[0063] Compound 4 (9.4 g, 0.028 mol) was weighed into a round-bottom flask and dissolved in anhydrous tetrahydrofuran. A magnetic stir bar was added, and the mixture was cooled to 0 °C in an ice bath. While stirring, sodium hydride (1 g, 0.042 mol) was weighed and slowly added to the system. After stirring for 15 minutes, carbon disulfide (2.5 mL, 0.042 mol) was slowly added dropwise. After stirring for 15 minutes, iodomethane (5.2 mL, 0.084 mol) was added, and the mixture was stirred at 0 °C. The reaction was monitored by thin-layer chromatography using petroleum ether:ethyl acetate as the developing solvent (5:1) and CMC as the colorimetric reagent. The Rf value of the target compound was 0.6, and the conversion of the starting material was detected after approximately 6 hours. The reaction mixture was extracted with ethyl acetate into a separatory funnel. The mixture was washed with water and saturated sodium chloride, and the aqueous phase was re-extracted with ethyl acetate. The combined organic phases were dried over anhydrous sodium sulfate and concentrated under reduced pressure. The product was directly used in the next step after separation and purification by rapid column chromatography (petroleum ether:ethyl acetate = 14:1). The obtained compound was dissolved in toluene, and tri-n-butyltin hydride (15 mL, 0.056 mol) and azobisisobutyronitrile (6.2 mL, 0.042 mol) were slowly added with stirring under reflux protection. The reaction was monitored by thin-layer chromatography (TLC) with petroleum ether:ethyl acetate = 7:1 as the developing solvent (two runs were required because the Rf values ​​of the substrate and target compound were similar). CMC was used for color development; the Rf value of the target compound was 0.6, and the conversion of the starting material was detected after approximately 10 hours. The mixture was concentrated under reduced pressure and then separated and purified by rapid column chromatography with petroleum ether:ethyl acetate = 20:1 as the eluent. The combined yield of the two steps was 81.1%.

[0064] Compound 5: 1H NMR (600 MHz, CDCl3) δ 7.35 - 7.27 (m, 4H), 7.21 (ddd, J =8.5, 6.1, 2.3 Hz, 1H), 5.56 (d, J = 5.0 Hz, 1H), 4.55 (dd, J = 8.0, 2.2 Hz,1H), 4.29 (dd, J = 5.0, 2.3 Hz, 1H), 4.06 (dd, J = 8.0, 1.6 Hz, 1H), 4.00(td, J = 7.1, 1.4 Hz, 1H), 3.05 - 2.90 (m, 2H), 1.52 (s, 3H), 1.50 (s, 3H),1.35 (s, 3H), 1.32 (s, 3H).

[0065] Synthesis of compound 6:

[0066] Compound 5 (7.3 g, 0.023 mol) was weighed into a round-bottom flask, and an acetic acid / water solution (v / v = 80%) was added. A magnetic stir bar was placed, and the mixture was stirred under reflux. Thin-layer chromatography (TLC) was used to monitor the reaction, with dichloromethane:methanol = 5:1 as the developing solvent. CMC was used for color development, and the Rf value of the target compound was 0.4. The conversion of the starting material was detected after approximately 7 hours. An equal volume of purified water was added, and the mixture was concentrated under reduced pressure. The product was then removed by pyridine 2-3 times under high vacuum and used directly in the next step. The obtained compound was dissolved in pyridine, and an acetic anhydride (21.3 mL, 0.227 mol) was added under a magnetic stir bar. The mixture was stirred overnight at room temperature under nitrogen protection. TLC was used to monitor the reaction, with petroleum ether:ethyl acetate = 1:1 as the developing solvent. CMC was used for color development, and the Rf value of the target compound was 0.68. The conversion of the starting material was detected after approximately 12 hours. The reaction mixture was extracted using ethyl acetate in a separatory funnel. The solution was washed with water, saturated sodium bicarbonate, and saturated sodium chloride, respectively. The aqueous phase was then extracted with ethyl acetate, and the organic phases were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. Rapid column chromatography was used for separation and purification, with petroleum ether:ethyl acetate as the eluent in a 5:1 ratio. The overall yield was 74.2%.

[0067] Synthesis of compound 7:

[0068] Compound 6 (7 g, 0.017 mol) was weighed into a round-bottom flask, dissolved in dichloromethane, and cooled to 0°C in an ice bath using a magnetic stir bar. A hydrobromic acid-acetic acid solution (v / v = 33%) was added dropwise with stirring. The reaction was monitored by thin-layer chromatography using petroleum ether:ethyl acetate as the developing solvent (3:1) and CMC reagent. The Rf value of the target compound was 0.4. The conversion of the starting material was detected after approximately 2 hours. The system was concentrated under reduced pressure and reconstituted with ethyl acetate. This step was repeated until no obvious sour taste was detected, and a sample was collected for the next step.

[0069] Synthesis of compound 8:

[0070] Compound 7 (6.9 g, 0.016 mol) was dissolved in anhydrous acetonitrile and placed in a magnetic stirrer. While stirring, p-nitrophenol (4.4 g, 0.032 mol) was added first, followed by potassium carbonate (6.6 g, 0.048 mol), and the mixture was stirred at room temperature. Thin-layer chromatography (TLC) was used to monitor the reaction, with petroleum ether:ethyl acetate as the developing solvent and CMC as the colorimetric reagent. The Rf value of the target compound was 0.42. The conversion of the starting material was detected after approximately 8 hours. The reaction mixture was extracted with ethyl acetate in a separatory funnel, washed with water and saturated sodium chloride, and the aqueous phase was re-extracted with ethyl acetate. The organic phases were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. Rapid column chromatography was used for separation and purification, with petroleum ether:ethyl acetate as the eluent (4:1). The overall yield was 84.0%.

[0071] Synthesis of compound 9:

[0072] Compound 8 was weighed and placed in a round-bottom flask. It was dissolved in a methanol:dichloromethane solution (1:1 volume ratio) and stirred at room temperature. The reaction was monitored by thin-layer chromatography (TLC) with dichloromethane:methanol (5:1) as the developing solvent and CMC as the colorimetric reagent. The Rf value of the target compound was 0.39. After approximately 3 hours, the conversion of the starting material was detected as complete. The system was neutralized using a cation exchange resin, filtered, and concentrated under reduced pressure. Rapid column chromatography was used for separation and purification with dichloromethane:methanol (8:1) as the eluent. The overall yield was 98.6%.

[0073] Compound 9: ¹H NMR (600 MHz, MeOD) δ 8.18 - 7.86 (m, 2H), 7.36 - 7.19 (m, 5H), 6.93 - 6.66 (m, 2H), 4.78 (d, J = 7.7 Hz, 1H), 3.88 (dd, J = 9.6, 4.4 Hz, 1H), 3.86 - 3.81 (m, 2H), 3.59 (dd, J = 9.7, 3.5 Hz, 1H), 3.03 (dd, J = 13.7, 9.6 Hz, 1H), 2.90 (dd, J = 13.8, 4.4 Hz, 1H).

[0074] Synthesis Route 3:

[0075]

[0076] Synthesis of compound 10:

[0077] Potassium tert-butoxide (11.48 g, 0.102 mol) was dissolved in tetrahydrofuran and placed in a magnetic bath to cool to 0°C. Benzyltriphenylphosphine chloride (36.16 g, 0.093 mol) was added with stirring, and the temperature was gradually raised to room temperature while stirring for 20 minutes. Compound 3 (8 g, 0.031 mol) in tetrahydrofuran solution was slowly added while stirring at room temperature. Thin-layer chromatography was used for monitoring; the developing solvent was petroleum ether:ethyl acetate = 3:1, and CMC was used for color development, with an Rf value of 0.56. The conversion of the starting material was detected after approximately 2 hours. The reaction mixture was extracted with ethyl acetate into a separatory funnel, washed with water and saturated sodium chloride, and the aqueous phase was re-extracted with ethyl acetate. The organic phases were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. Rapid column chromatography was used for separation and purification; the eluent was petroleum ether:ethyl acetate = 17:1. The yield was 62.1%.

[0078] Compound 10: 1H NMR (400 MHz, CDCl3) δ 7.36 - 7.28 (m, 4H), 7.23 (dd, J =6.2, 2.9 Hz, 1H), 6.75 (d, J = 11.7 Hz, 1H), 5.94 (dd, J = 11.6, 9.3 Hz, 1H), 5.60 (d, J = 5.2 Hz, 1H), 4.70 (d, J = 9.2 Hz, 1H), 4.60 (dd, J = 7.9, 2.2Hz, 1H), 4.31 (dd, J = 5.2, 2.3 Hz, 1H), 4.21 (dd, J = 7.9, 1.8 Hz, 1H), 1.52(s, 3H), 1.36 (s, 3H), 1.30 (s, 3H), 1.27 (s, 3H).

[0079] Synthesis of compound 11:

[0080] Compound 10 (6.37 g, 0.019 mol) was dissolved in anhydrous ethanol, and a catalytic amount of palladium (10% supported on carbon) was added. The mixture was stirred under hydrogen atmosphere at 4.5 atm. Thin-layer chromatography was used for monitoring; the developing solvent was petroleum ether:ethyl acetate = 3:1, and potassium permanganate was used as the colorimetric reagent, with an Rf value of 0.56. The conversion of the starting material was detected after approximately 16 hours. The mixture was filtered, and the filtrate was concentrated under reduced pressure. The yield was 97.7%.

[0081] Compound 11: ¹H NMR (600 MHz, CDCl₃) δ 7.31 - 7.29 (m, 3H), 7.23 - 7.19 (m, 1H), 5.56 (d, J = 5.0 Hz, 1H), 4.55 (dd, J = 8.0, 2.2 Hz, 1H), 4.29 (dd, J = 5.0, 2.3 Hz, 1H), 4.06 (dd, J = 8.0, 1.6 Hz, 1H), 4.00 (td, J = 7.1, 1.4 Hz, 1H), 3.03 - 2.90 (m, 2H), 1.52 (s, 3H), 1.50 (s, 3H), 1.35 (s, 3H), 1.32 (s, 2H). 3H).

[0082] Synthesis of compound 12:

[0083] Compound 11 (6.2 g, 0.018 mol) was weighed and placed in a round-bottom flask. An acetic acid / water solution (v / v = 80%) was added, and the mixture was refluxed under a magnetic stir bar. The reaction was monitored by thin-layer chromatography (TLC) with dichloromethane:methanol (5:1) as the developing solvent and CMC reagent as the colorimetric reagent. The Rf value of the target compound was 0.4. The reaction was considered complete after approximately 7 hours. An equal volume of purified water was added, and the mixture was concentrated under reduced pressure. The product was then removed by pyridine 2-3 times under high vacuum and used directly in the next step. The obtained compound was dissolved in pyridine, and acetic anhydride (17 mL, 0.18 mol) was added under a magnetic stir bar. The mixture was stirred overnight at room temperature under nitrogen protection. The reaction was monitored by TLC with petroleum ether:ethyl acetate (1:1) as the developing solvent and CMC reagent as the colorimetric reagent. The Rf value of the target compound was 0.68. The reaction was considered complete after approximately 12 hours. The reaction mixture was extracted using ethyl acetate in a separatory funnel. The solution was washed with water, saturated sodium bicarbonate, and saturated sodium chloride, respectively. The aqueous phase was then extracted with ethyl acetate, and the organic phases were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. Rapid column chromatography was used for separation and purification, with petroleum ether:ethyl acetate as the eluent in a 5:1 ratio. The overall yield was 73.6%.

[0084] Synthesis of compound 13:

[0085] Compound 12 (5.6 g, 0.013 mol) was weighed into a round-bottom flask, dissolved in dichloromethane, and cooled to 0°C in an ice bath using a magnetic stir bar. A 33% hydrobromic acid-acetic acid solution was added dropwise with stirring. The reaction was monitored by thin-layer chromatography using petroleum ether:ethyl acetate as the developing solvent (3:1) and CMC reagent. The Rf value of the target compound was 0.4. The conversion of the starting material was detected after approximately 2 hours. The system was concentrated under reduced pressure and reconstituted with ethyl acetate. This step was repeated until no obvious sour taste was detected, and a sample was collected for the next step.

[0086] Synthesis of compound 14:

[0087] Compound 13 (5.4 g, 0.012 mol) was dissolved in anhydrous acetonitrile and placed in a magnetic stirrer. While stirring, p-nitrophenol (3.34 g, 0.024 mol) was added first, followed by potassium carbonate (5 g, 0.036 mol), and the mixture was stirred at room temperature. Thin-layer chromatography (TLC) was used to monitor the reaction, with petroleum ether:ethyl acetate as the developing solvent and CMC as the colorimetric reagent. The Rf value of the target compound was 0.42. The conversion of the starting material was detected after approximately 8 hours. The reaction mixture was extracted with ethyl acetate in a separatory funnel, washed with water and saturated sodium chloride, and the aqueous phase was re-extracted with ethyl acetate. The organic phases were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. Rapid column chromatography was used for separation and purification, with petroleum ether:ethyl acetate as the eluent (4:1). The overall yield was 85.3%.

[0088] Compound 14: 1H NMR (600 MHz, CDCl3) δ 8.23 ​​(d, J = 8.9 Hz, 2H), 7.31 (t,J = 7.4 Hz, 2H), 7.23 (t, J = 7.7 Hz, 1H), 7.14 (d, J = 7.5 Hz, 2H), 7.07 (d,J = 8.8 Hz, 2H), 5.57 - 5.46 (m, 1H), 5.39 (s, 1H), 5.13 (d, J = 7.9 Hz, 1H), 5.09 (dd, J = 10.4, 2.9 Hz, 1H), 3.78 (dd, J = 9.1, 3.7 Hz, 1H), 2.80 (ddd, J= 14.0, 8.6, 5.6 Hz, 1H), 2.71 - 2.62 (m, 1H), 2.19 (s, 3H), 2.05 (s, 4H), 2.01 (s, 3H), 1.25 (s, 2H).

[0089] Synthesis of compound 15:

[0090] Compound 14 was weighed and placed in a round-bottom flask. It was dissolved in a methanol:dichloromethane solution (1:1 volume ratio). The pH of the system was adjusted to 9-10 using sodium methoxide, and the mixture was stirred at room temperature. Thin-layer chromatography (TLC) was used to monitor the reaction, with dichloromethane:methanol (5:1) as the developing solvent. CMC was used for color development, and the Rf value of the target compound was 0.39. After approximately 3 hours, the conversion of the starting material was detected as complete. The system was then adjusted to neutral using a cation exchange resin, filtered, and concentrated under reduced pressure. Rapid column chromatography was used for separation and purification, with dichloromethane:methanol (8:1) as the eluent. The overall yield was 97.9%.

[0091] Compound 15: 1H NMR (600 MHz, MeOD) δ 8.24 (d, J = 9.2 Hz, 2H), 7.29 -7.15 (m, 7H), 5.00 (d, J = 7.7 Hz, 1H), 3.83 (dd, J = 9.6, 7.8 Hz, 1H), 1.97 - 1.83 (m, 1H).

[0092] Synthesis Route 4:

[0093]

[0094] Synthesis of compound 16:

[0095] Potassium tert-butoxide (14.4 g, 0.129 mol) was dissolved in tetrahydrofuran, placed in a magnetic bath, and cooled to 0°C in an ice bath. (Methoxymethyl)triphenylphosphine chloride (39.8 g, 0.116 mol) was added with stirring, and the temperature was gradually raised to room temperature while stirring for 20 minutes. Compound 3 (10 g, 0.039 mol) in tetrahydrofuran solution was slowly added, and the mixture was stirred at room temperature. Thin-layer chromatography was used for monitoring; the developing solvent was petroleum ether:ethyl acetate = 5:1, and CMC was used for color development. The Rf value of the target compound was 0.4. The conversion of the starting material was detected after approximately 2 hours. The reaction mixture was extracted with ethyl acetate in a separatory funnel, washed with water and saturated sodium chloride, and the aqueous phase was re-extracted with ethyl acetate. The organic phases were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. Rapid column chromatography was used for separation and purification; the eluent was petroleum ether:ethyl acetate = 8:1. The obtained product was used directly in the next step. The obtained product was dissolved in acetone, a magnetic stir bar was added, and 1N hydrochloric acid was added dropwise while stirring to adjust the pH to 3-4. Thin-layer chromatography was used for sampling and monitoring. The original target compound product spot gradually disappeared, and a new spot with slightly higher polarity appeared below it. The developing solvent was petroleum ether:ethyl acetate = 5:1. CMC colorimetric reagent was used for color development, and the Rf value of the new product was 0.28. The overall yield was 70.2%.

[0096] Synthesis of compound 17:

[0097] Potassium tert-butoxide (10 g, 0.089 mol) was dissolved in tetrahydrofuran and placed in a magnetic bath to cool to 0°C. Benzyltriphenylphosphine chloride (31.5 g, 0.081 mol) was added with stirring, and the temperature was gradually raised to room temperature while stirring for 20 minutes. Compound 16 (7.4 g, 0.027 mol) in tetrahydrofuran solution was slowly added while stirring at room temperature. Thin-layer chromatography was used for monitoring; the developing solvent was petroleum ether:ethyl acetate = 3:1, and CMC was used for color development, with an Rf value of 0.55. The conversion of the starting material was detected after approximately 2 hours. The reaction mixture was extracted with ethyl acetate into a separatory funnel, washed with water and saturated sodium chloride, and the aqueous phase was re-extracted with ethyl acetate. The organic phases were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. Rapid column chromatography was used for separation and purification; the eluent was petroleum ether:ethyl acetate = 17:1. The yield was 60.4%.

[0098] Compound 17: 1H NMR (600 MHz, CDCl3) δ 7.36 (d, J = 7.7 Hz, 2H), 7.29 (t,J = 7.5 Hz, 2H), 7.20 (t, J = 7.4 Hz, 1H), 6.54 (d, J = 15.9 Hz, 1H), 6.33 -6.21 (m, 1H), 5.56 (d, J = 5.0 Hz, 1H), 4.60 (dd, J = 7.9, 1.8 Hz, 1H), 4.31(dd, J = 4.7, 1.9 Hz, 1H), 4.20 (d, J = 7.9 Hz, 1H), 3.86 (t, J = 6.9 Hz,1H), 2.62 - 2.46 (m, 2H), 1.53 (s, 3H), 1.50 (s, 3H), 1.37 (s, 3H), 1.34 (s, 3H).

[0099] Synthesis of compound 18:

[0100] Compound 17 was dissolved in anhydrous ethanol, and a catalytic amount of palladium (10% supported on carbon) was added. The mixture was stirred under hydrogen conditions at 4.5 atm. Thin-layer chromatography was used for monitoring; the developing solvent was petroleum ether:ethyl acetate = 3:1, and potassium permanganate was used as the colorimetric reagent, with an Rf value of 0.56. The conversion of the starting material was detected after approximately 12 hours. The mixture was filtered, and the filtrate was concentrated under reduced pressure. The yield was 98.1%.

[0101] Compound 18: 1H NMR (600 MHz, CDCl3) δ 7.29 - 7.24 (m, 2H), 7.17 (dd, J =16.0, 7.5 Hz, 3H), 5.53 (d, J = 5.1 Hz, 1H), 4.57 (dd, J = 7.9, 2.0 Hz, 1H), 4.28 (dd, J = 5.1, 2.1 Hz, 1H), 4.11 (dd, J = 7.9, 1.2 Hz, 1H), 3.73 (dd, J =8.3, 4.2 Hz, 1H), 2.64 (t, J = 7.8 Hz, 2H), 1.83 (ddd, J = 10.2, 7.8, 4.5 Hz,1H), 1.74 (ddd, J = 13.5, 9.0, 4.4 Hz, 1H), 1.71 - 1.64 (m, 1H), 1.62 - 1.56 (m, 1H), 1.51 (s, 3H), 1.46 (s, 3H), 1.33 (d, J = 10.3 Hz, 6H).

[0102] Synthesis of compound 19:

[0103] Compound 18 (5.57 g, 0.016 mol) was weighed and placed in a round-bottom flask. An acetic acid / water solution (v / v = 80%) was added, and the mixture was refluxed under a magnetic stir bar. The reaction was monitored by thin-layer chromatography (TLC) with dichloromethane:methanol (5:1) as the developing solvent and CMC reagent as the colorimetric reagent. The Rf value of the target compound was 0.4. The reaction was considered complete after approximately 7 hours. An equal volume of purified water was added, and the mixture was concentrated under reduced pressure. The product was then removed by pyridine 2-3 times under high vacuum and used directly in the next step. The obtained compound was dissolved in pyridine, placed under a magnetic stir bar, and acetic anhydride (15 mL, 0.16 mol) was added. The mixture was stirred overnight at room temperature under nitrogen protection. The reaction was monitored by TLC with petroleum ether:ethyl acetate (1:1) as the developing solvent and CMC reagent as the colorimetric reagent. The Rf value of the target compound was 0.68. The reaction was considered complete after approximately 12 hours. The reaction mixture was extracted using ethyl acetate in a separatory funnel. The solution was washed with water, saturated sodium bicarbonate, and saturated sodium chloride, respectively. The aqueous phase was then extracted with ethyl acetate, and the organic phases were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. Rapid column chromatography was used for separation and purification, with petroleum ether:ethyl acetate as the eluent in a 5:1 ratio. The overall yield was 72.3%.

[0104] Synthesis of compound 20:

[0105] Compound 19 (5 g, 0.012 mol) was weighed into a round-bottom flask, dissolved in dichloromethane, and cooled to 0°C in an ice bath using a magnetic stir bar. A 33% hydrobromic acid-acetic acid solution was added dropwise with stirring. The reaction was monitored by thin-layer chromatography using petroleum ether:ethyl acetate (3:1) as the developing solvent and CMC reagent as the colorimetric reagent. The Rf value of the target compound was 0.4. The conversion of the starting material was detected after approximately 2 hours. The system was concentrated under reduced pressure and reconstituted with ethyl acetate. This step was repeated until no obvious sour taste was detected, and a sample was collected for the next step.

[0106] Synthesis of Compound 21

[0107] Compound 20 (5 g, 0.011 mol) was dissolved in anhydrous acetonitrile and placed in a magnetic stirrer. While stirring, p-nitrophenol (3 g, 0.022 mol) was added first, followed by potassium carbonate (4.6 g, 0.033 mol), and the mixture was stirred at room temperature. Thin-layer chromatography (TLC) was used to monitor the reaction, with petroleum ether:ethyl acetate as the developing solvent and CMC as the colorimetric reagent. The Rf value of the target compound was 0.42. The conversion of the starting material was detected after approximately 8 hours. The reaction mixture was extracted with ethyl acetate in a separatory funnel, washed with water and saturated sodium chloride, and the aqueous phase was re-extracted with ethyl acetate. The organic phases were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. Rapid column chromatography was used for separation and purification, with petroleum ether:ethyl acetate as the eluent (4:1). The overall yield was 84.0%.

[0108] Synthesis of compound 22:

[0109] Compound 21 was weighed and placed in a round-bottom flask. It was dissolved in a methanol:dichloromethane solution (1:1 volume ratio). The pH of the system was adjusted to 9-10 using sodium methoxide, and the mixture was stirred at room temperature. Thin-layer chromatography (TLC) was used to monitor the reaction, with dichloromethane:methanol (5:1) as the developing solvent. CMC was used for color development, and the Rf value of the target compound was 0.39. After approximately 3 hours, the conversion of the starting material was detected as complete. The system was neutralized using a cation exchange resin, filtered, and concentrated under reduced pressure. Rapid column chromatography was used for separation and purification, with dichloromethane:methanol (8:1) as the eluent. The overall yield was 96.6%.

[0110] Compound 22: ¹H NMR (600 MHz, MeOD) δ 8.23–8.15 (m, 2H), 7.24 (t, J = 7.6 Hz, 2H), 7.22–7.12 (m, 5H), 5.02 (d, J = 7.7 Hz, 1H), 3.81 (dd, J = 9.7, 7.7 Hz, 1H), 3.76–3.70 (m, 2H), 3.60 (dd, J = 9.7, 3.4 Hz, 1H), 2.72–2.58 (m, 2H), 1.87–1.72 (m, 3H), 1.61 (ddd, J = 13.5, 9.9, 5.7 Hz, 1H).

[0111] Synthesis Route 5:

[0112] Synthesis of compound 23 (3-C-Gal-MU):

[0113] Compound 20 was dissolved in dichloromethane. Sodium hydroxide (53.35 mg, 1.33 mmol) was weighed and dissolved in 1 mL of pure water. 4-Methylumbelliferone (180.75 mg, 1.03 mmol) and tetrabutylammonium bromide (189.48 mg, 0.513 mmol) were added to the sodium hydroxide solution, and then added dropwise to the dichloromethane solution of compound 20 (234 mg, 0.513 mmol). An equal volume of pure water was added, and the mixture was stirred at room temperature. The reaction was monitored by thin-layer chromatography with petroleum ether:ethyl acetate as the developing solvent. The Rf value of the product was 0.32. After the conversion of the starting materials, the reaction solution was extracted using dichloromethane in a separatory funnel. The mixture was washed with water and saturated sodium chloride, and the aqueous phase was re-extracted using dichloromethane. The organic phases were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The product was dissolved in a dichloromethane / methanol (v / v = 1 / 1) solution. The pH of the system was adjusted to 9-10 using sodium methoxide, and the mixture was stirred at room temperature. Thin-layer chromatography (TLC) was used to monitor the reaction, with dichloromethane:methanol = 5:1 as the developing solvent. CMC was used for color development. After approximately 1 hour, the conversion of the starting material was detected. The system was neutralized using a cation exchange resin, filtered, and concentrated under reduced pressure. Rapid column chromatography was then used for separation and purification, with dichloromethane:methanol = 12:1 as the eluent. The overall yield was 72.3%.

[0114] Compound 23: ¹H NMR (400 MHz, MeOD) δ 7.68 (d, J = 8.7 Hz, 1H), 7.32–7.01 (m, 7H), 6.22 (s, 1H), 5.03 (d, J = 7.6 Hz, 1H), 4.63 (s, 1H), 3.84 (t, J = 8.5 Hz, 1H), 3.76 (s, 2H), 3.64 (d, J = 8.0 Hz, 1H), 3.38 (s, 1H), 2.68 (s, 2H), 2.46 (s, 2H), 1.97–1.70 (m, 3H).

[0115] Synthesis Route 6:

[0116] Synthesis of compound 24 (3-C-Gal-DAO):

[0117] 1 g of molecular sieve was weighed and added to a round-bottom flask. Compound 20 (234 mg, 0.513 mmol) was dissolved in 10 mL of anhydrous acetonitrile and injected into the flask. A magnetic stir bar was placed in the flask, and DAO (122.6 mg, 0.513 mmol) and silver oxide (142.2 mg, 0.616 mmol) were added with stirring. Then, 0.3 mL of 2,6-lutidine was injected into the system. The mixture was stirred at room temperature, and the reaction was monitored by thin-layer chromatography using petroleum ether:ethyl acetate (1:1) as the developing solvent. CMC was used for color development, and the Rf value of the target compound was 0.28. After the starting material conversion was complete, the mixture was filtered through silica gel and concentrated under reduced pressure. The product was dissolved in a dichloromethane / methanol (v / v = 1 / 1) solution, and the pH of the system was adjusted to 9-10 using sodium methoxide. The mixture was stirred at room temperature. The reaction was monitored by thin-layer chromatography (TLC) with dichloromethane:methanol = 5:1 as the developing solvent. CMC was used for color development, and the conversion of the starting material was detected after approximately 1 hour. The system was neutralized using cation exchange resin, filtered, and concentrated under reduced pressure. Rapid column chromatography was then used for separation and purification with dichloromethane:methanol = 10:1 as the eluent. The overall yield was 68.6%.

[0118] Compound 24: 1H NMR (600 MHz, MeOD) δ 7.49 (d, J = 8.7 Hz, 1H), 7.34 (d,J = 9.8 Hz, 1H), 7.17 (d, J = 2.6 Hz, 1H), 7.12 (t, J = 7.5 Hz, 2H), 7.03(dd, J = 13.8, 7.1 Hz, 2H), 6.99 (dd, J = 8.7, 2.6 Hz, 1H), 6.57 (d, J = 2.0Hz, 1H), 6.53 (dd, J = 9.8, 2.0 Hz, 1H), 4.88 (d, J = 7.7 Hz, 2H), 4.48 (s,1H), 3.71 (dd, J = 9.7, 7.7 Hz, 1H), 3.64 (t, J = 3.0 Hz, 1H), 3.51 (dd, J =9.7, 3.4 Hz, 1H), 3.25 (s, 1H), 2.63 - 2.43 (m, 2H), 1.83 - 1.69 (m, 2H),1.69 - 1.59 (m, 1H), 1.56 - 1.45 (m, 1H), 1.38 (s, 2H), 1.26 (s, 2H), 0.83 -0.71 (m, 1H).

[0119] Synthesis Route 7:

[0120]

[0121] The synthesis of compound 26 is referenced to compound 20.

[0122] Synthesis of compound 27:

[0123] Compound 26 (0.024 mol) was dissolved in anhydrous acetonitrile and placed in a magnetic stirrer. 4-hydroxybenzaldehyde (0.049 mol) was added first, followed by potassium carbonate (0.073 mol), while stirring at room temperature. The reaction was monitored by thin-layer chromatography using CMC reagent. The conversion of the starting material was detected after approximately 8 hours. The reaction mixture was extracted with ethyl acetate in a separatory funnel, washed with water and saturated sodium chloride, and the aqueous phase was re-extracted with ethyl acetate. The organic phases were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. Rapid column chromatography was used for separation and purification.

[0124] Compound 27b: NMR (600 MHz, ) δ 9.91 (s, 1H), 7.84 (dd, J = 6.4, 4.7 Hz, 2H), 7.27 (t, J = 7.5 Hz, 2H), 7.19 (t, J = 7.4 Hz, 1H), 7.12 (d, J = 7.1 Hz,2H), 7.08 (d, J = 8.7 Hz, 2H), 5.49 (dd, J = 10.4, 7.9 Hz, 1H), 5.36 (d, J =3.1 Hz, 1H), 5.15 (d, J = 7.9 Hz, 1H), 5.11 (dd, J = 10.5, 3.5 Hz, 1H), 3.80(dd, J = 7.9, 5.4 Hz, 1H), 2.63 (dtd, J = 21.5, 14.0, 7.8 Hz, 2H), 2.13 (s,3H), 2.05 (s, 3H), 2.01 (s, 3H), 1.83 - 1.48 (m, 4H).

[0125] Synthesis of compound 28:

[0126] Compound 27 (0.02 mol) was dissolved in dichloromethane / methanol (v / v) = 1:1. A magnetic stir bar was placed in the solution, and the system was cooled to 0°C. Sodium borohydride (0.01 mol) was slowly added in portions to the stirred system. The reaction was monitored by thin-layer chromatography. After the reactants were converted, the reaction mixture was extracted using ethyl acetate in a separatory funnel. The mixture was washed with water and saturated sodium chloride, and the aqueous phase was re-extracted using ethyl acetate. The organic phases were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. Rapid column chromatography was used for separation and purification.

[0127] Compound 28b: NMR (600 MHz, ) δ 7.27 - 7.16 (m, 4H), 7.11 (t, J = 7.3 Hz, 1H), 7.05 (d, J = 7.5 Hz, 2H), 6.90 (d, J = 8.4 Hz, 2H), 5.38 (dd, J = 10.4,8.0 Hz, 1H), 5.26 (d, J = 3.3 Hz, 1H), 5.00 (dd, J = 10.4, 3.4 Hz, 1H), 4.92(d, J = 7.9 Hz, 1H), 4.56 (s, 2H), 3.66 (dd, J = 8.2, 5.2 Hz, 1H), 2.54 (dtd,J = 21.6, 14.1, 7.8 Hz, 2H), 2.05 (s, 3H), 1.98 (s, 3H), 1.93 (s, 3H), 1.74 -1.56 (m, 4H).

[0128] Synthesis of compound 29:

[0129] Compound 28 (0.018 mol) was dissolved in dichloromethane, and the system was cooled to 0°C in an ice bath. A magnetic syringe was placed in the system, and phosphorus tribromide (0.009 mol) was slowly added dropwise using a microsyringe. The reaction was monitored by thin-layer chromatography. After the reactants were converted, the reaction was quenched with saturated sodium bicarbonate solution, and the mixture was transferred to a separatory funnel for extraction. The mixture was washed with water and saturated sodium chloride, and the aqueous phase was extracted with ethyl acetate. The organic phases were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. Rapid column chromatography was used for separation and purification.

[0130] Synthesis of compound 30:

[0131] Compound 29 (0.016 mol), SN38 (0.02 mol), and potassium carbonate (0.032 mol) were weighed and added to a round-bottom flask. A magnetic stir bar was placed in the flask, and an argon-protected three-way valve was connected. The system was evacuated using an oil pump. Under argon protection and an ice bath, the substrate was dissolved using ultra-dry N,N-dimethylformamide. After stirring for 2 minutes, the ice bath was removed, and the mixture was stirred at room temperature. The reaction was monitored by thin-layer chromatography. After the reactants were converted, the system was extracted using dichloromethane in a separatory funnel. The mixture was washed with water and saturated sodium chloride, and the aqueous phase was extracted back with dichloromethane. The organic phases were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. Rapid column chromatography was used for separation and purification.

[0132] Synthesis of compound 31:

[0133] Compound 30 was weighed and placed in a round-bottom flask. It was dissolved in a 1:1 methanol / dichloromethane solution. The pH of the system was adjusted to 9-10 using sodium methoxide, and the mixture was stirred at room temperature. The reaction was monitored by thin-layer chromatography using CMC reagent. After approximately 1 hour, the conversion of the starting material was detected. The system was neutralized using a cation exchange resin, filtered, and concentrated under reduced pressure. Rapid column chromatography was then used for separation and purification.

[0134] Compound 31b (3-C-Gal-SN38): NMR (600 MHz, ) δ 7.60 (d, J = 2.4 Hz, 3H), 7.55 (dd, J = 9.2, 2.5 Hz, 3H), 7.48 (d, J = 8.6 Hz, 6H), 7.27 (s, 3H), 7.07(d, J = 8.6 Hz, 6H), 6.49 (s, 3H), 5.42 (s, 5H), 5.29 (d, J = 4.3 Hz, 12H), 5.15 (d, J = 5.2 Hz, 3H), 4.84 (dd, J = 6.6, 4.6 Hz, 6H), 4.63 (t, J = 5.3Hz, 3H), 4.49 (d, J = 4.7 Hz, 3H), 4.34 (t, J = 5.1 Hz, 1H), 3.70 (t, J = 4.0Hz, 3H), 3.61 - 3.51 (m, 9H), 3.51 - 3.37 (m, 8H), 3.18 (dd, J = 14.8, 6.8Hz, 6H), 1.86 (ddt, J = 21.5, 14.1, 7.2 Hz, 6H), 1.28 (t, J = 7.6 Hz, 9H), 1.06 (t, J = 7.0 Hz, 3H), 0.88 (t, J = 7.3 Hz, 9H).

[0135] Synthesis Route 8:

[0136] The synthesis of compound 32 (Gal-DAO) is referenced to compound 24.

[0137] Compound 32: 1H NMR (600 MHz, MeOD) δ 7.60 (d, J = 8.7 Hz, 1H), 7.43 (d,J = 9.8 Hz, 1H), 7.37 (d, J = 2.6 Hz, 1H), 7.14 (dd, J = 8.7, 2.6 Hz, 1H), 6.74 (d, J = 2.1 Hz, 1H), 6.62 (dd, J = 9.8, 2.1 Hz, 1H), 5.00 (d, J = 7.7Hz, 1H), 3.93 (d, J = 3.4 Hz, 1H), 3.87 - 3.78 (m, 3H), 3.64 (dd, J = 9.7,3.4 Hz, 1H), 3.32 - 3.30 (m, 2H), 1.55 (d, J = 2.7 Hz, 5H).

[0138] Example 1

[0139] 1. Design of chemically modified galactoside substrates

[0140] Based on the structure of the natural galactosidase substrate (Gal-PNP), the 6-position of galactose was chemically modified by introducing different substituent groups to prepare three non-natural galactosidase substrates (1-C-Gal-PNP, 2-C-Gal-PNP, and 3-C-Gal-PNP) that are not recognized by endogenous galactosidases. Figure 4 As shown.

[0141] The introduction of these three groups not only modulates the steric hindrance at the 6-position of galactose but also regulates the electronic effects of the sugar ring. Wild-type galactosidase was used to hydrolyze Gal-PNP, 1-C-Gal-PNP, 2-C-Gal-PNP, and 3-C-Gal-PNP, respectively, as follows:

[0142] Wild-type galactosidase was co-incubated with three compounds. The total reaction volume was 400 μL, containing 20 μL of enzyme (stock solution concentration of 0.9 mg / mL), 40 μL of compound substrate solution (stock solution concentration of 500 μM), and 340 μL of PBS buffer (pH 7.4). After incubation at 37°C for 30 minutes, 400 μL of stop solution (0.75 g glycine dissolved in 20 mL of pure water, 18.4 mL of 0.5 mol / L NaOH solution added, pH adjusted to 10.4, and filtered through a membrane) was added to terminate the enzyme reaction. Each experimental condition was performed in triplicate, and the absorbance was measured at 405 nm using a microplate reader.

[0143] The results of the hydrolysis reaction are shown in Figure 5 The results showed that the three designed substrates were completely unaffected by wild-type galactosidase, thus avoiding the problem of non-specific release of prodrugs designed using this chemically modified substrate.

[0144] 2. Design and expression of mutant galactosidase

[0145] Using molecular biology techniques, a highly thermostable galactosidase (A4-β-Gal, 1KWK) derived from previously screened A4 thermophilic bacteria was analyzed. Figure 6 To engineer the catalytic pocket, the six loops (yellow parts) near the catalytic pocket were truncated and mutated to make the catalytic pocket more open. The mutation sequences are shown in Table 1. At the same time, plasmids were constructed and transformed into E. coli to express and purify the six designed mutant galactosidases.

[0146] Table 1 Mutation design of galactosidase 1KWK

[0147]

[0148] The amino acid sequence of galactosidase 1KWK is shown in SEQ ID NO.1. The amino acid sequences of mutant enzymes 1-6 are shown in SEQ ID NO.2-7, respectively.

[0149] 3. Hydrolysis experiment of mutant galactosidase with substrate

[0150] Hydrolysis experiments were conducted using Gal-PNP, 1-C-Gal-PNP, 2-C-Gal-PNP, and 3-C-Gal-PNP as substrates, respectively, with mutant enzymes 1-6 or wild-type galactosidase (7):

[0151] The total reaction volume was 400 μL, containing 20 μL of enzyme (stock solution concentration of 0.9 mg / mL), 40 μL of compound substrate solution (stock solution concentration of 500 μM), and 340 μL of PBS buffer (pH 7.4). After incubation at 37°C for 30 minutes, 400 μL of stop solution (composed of 0.75 g glycine dissolved in 20 mL of pure water, 18.4 mL of 0.5 mol / L NaOH solution, pH adjusted to 10.4, and filtered through a membrane) was added to terminate the enzyme reaction. Each experimental condition was performed in triplicate, and absorbance was measured at 405 nm using a microplate reader.

[0152] The hydrolysis experiment results are shown in Figure 7The results showed that neither mutant enzymes 1-6 nor the wild-type galactosidase (enzyme 7) could hydrolyze 1-C-Gal-PNP, while 2-C-Gal-PNP and 3-C-Gal-PNP could be significantly hydrolyzed by the mutant enzymes, verifying the necessity of replacing OH with CH2. The hydrolytic ability of 3-C-Gal-PNP was significantly stronger than that of 2-C-Gal-PNP. Among them, enzyme 5 showed the most significant effect in hydrolyzing 3-C-Gal-PNP, and enzyme 5 was relatively weak in hydrolyzing Gal-PNP.

[0153] Through this experiment, the present invention screened out an orthogonal reaction pair between enzyme No. 5 and 3-C-Gal-PNP.

[0154] Example 2

[0155] The structural formula of Gal-Mu is: The structural formula of 3-C-Gal-Mu is: .

[0156] The serum stability of 3-C-Gal-MU and Gal-MU was compared. The serum stability experiment is as follows:

[0157] A 400 μL reaction system was used, containing 80 μL C57BL / 6 mouse serum, 296 μL PBS buffer, 20 μL DMSO, and 4 μL of substrate 3-C-Gal-MU or Gal-MU (stock solution concentration 1 mM). The reaction system was incubated at 37 °C for 0 h, 2 h, 6 h, 12 h, and 24 h, respectively. Detection was performed using a microplate reader. The reader was preheated for 3 minutes before detection, and the Blue mode was selected, with an excitation wavelength of 360 nm and an absorption wavelength of 460 nm using a filter.

[0158] Incubation of the substrate with serum revealed that wild-type galactosidase in serum could hydrolyze Gal-Mu in a time-dependent manner, with fluorescence increasing over time; however, 3-C-Gal-Mu showed good stability in serum, with no change in fluorescence over time (e.g., ...). Figure 8 (As shown). This demonstrates that the phenethyl-modified galactoside substrate remains stable in serum, which significantly reduces non-specific release of the prodrug and ensures targeted delivery of the prodrug.

[0159] Example 3

[0160] The structural formula of Gal-DAO is: The structural formula of 3-C-Gal-DAO is:

[0161] .

[0162] The specificity and catalytic efficiency of enzyme 5 for the 3-C-Gal-DAO probe were evaluated using a site-specific release assay in the hind limb. In mouse hind limb surgery, after exposing the corresponding area, 50 μL of hydrogel containing enzyme 5 was injected subcutaneously and cured by irradiation with 405 nm UV light for 30 seconds, followed by wound closure. Finally, 50 μL of 5 mM 3-C-Gal-DAO solution (DMSO:PBS = 1:1 solvent) was injected via the tail vein. Images were acquired using an IVIS in vivo imaging system.

[0163] The preparation method of hydrogel containing enzyme No. 5 is as follows: First, 25 mg of lithium phenyl-2,4,6-trimethylbenzoyl phosphate (LAP) was dissolved in 10 mL of PBS to prepare an initiator solution; 171.48 μL of the initiator solution was taken, and 10 mg of HAMA and enzyme No. 5 were added to finally prepare a hydrogel system with an enzyme working concentration of 0.5 mg / mL.

[0164] Near-infrared fluorescent molecule Gal-DAO was injected into mice, and imaging revealed that fluorescence was dispersed throughout the mice's bodies. Figure 9 (A), but when 3-C-Gal-DAO was injected into mice, no significant fluorescence signal distribution was observed ( Figure 9 (B), which demonstrates the stability of 3-C-Gal-DAO in small animals; in addition, when enzyme 5 was immobilized on the hind limb of a mouse using a hydrogel, and then 3-C-Gal-DAO was injected into the mouse, a clear fluorescent signal was observed at the immobilized enzyme site. Figure 9 (C), which fully demonstrates in vivo that enzyme 5 can specifically hydrolyze 3-C-Gal-DAO to achieve targeted delivery of prodrugs.

[0165] Near-infrared fluorescent molecules Gal-DAO and 3-C-Gal-DAO were administered via gavage. Blood samples were collected from the canthus of the eye at different time points, and serum was obtained by centrifugation, enzymatic hydrolysis, and the fluorescence intensity of DAO in the serum was measured. The results are as follows: Figure 10 As shown in the figure. Analysis of the experimental results revealed that the oral bioavailability of 3-C-Gal-DAO is higher than that of Gal-DAO, demonstrating that the benzene ring structure introduced in this invention has a strong ability to regulate the lipid-water partition coefficient and thus a strong regulatory effect on oral absorption.

[0166] Example 4

[0167] Gal-SN38 is compound 31a; 3-C-Gal-SN38 is compound 31b.

[0168] Treatment efficacy verification experiment:

[0169] 1. Experimental Methods

[0170] A mouse tumor model was constructed using 4T1 breast cancer cells. Tumor-bearing mice were randomly divided into two groups: a control group (Ctrl) and a treatment group (3-C-Gal-SN38). All mice underwent partial tumor resection surgery, and a mutant galactosidase was pre-embedded in the tumor tissue. This was achieved by mixing the mutant galactosidase with a thermosensitive hydrogel, applying it evenly to the tumor resection wound, and then suturing the skin. The thermosensitive hydrogel could solidify at body temperature (37 °C).

[0171] Dosage regimen:

[0172] Control group: Normal saline was administered by gavage on days 1, 4, 7 and 10.

[0173] In the treatment group, 3-C-Gal-SN38 was administered by gavage at the same time point at a dose of 20 mg / kg.

[0174] Tumor monitoring and imaging:

[0175] Tumor volume was measured and recorded on days 1, 4, 7, 10, 13, 16, and 19. On day 19, all mice were intraperitoneally injected with fluorescein for in vivo fluorescence imaging to visually compare the size and activity of tumors in the two groups.

[0176] 2. Experimental Results

[0177] like Figure 11 As shown, the tumor volume in the treatment group (3-C-Gal-SN38) was significantly smaller than that in the control group throughout the experiment. From day 7 onwards, the difference between the two groups gradually became more obvious and continued to widen over time, indicating that 3-C-Gal-SN38 effectively inhibited tumor growth.

[0178] Fluorescence imaging results on day 19 ( Figure 12 Further confirmation showed that the tumor fluorescence signal intensity in the treatment group was significantly lower than that in the control group, indicating that the tumor volume was smaller and the activity was lower, which was consistent with the tumor volume measurement results.

[0179] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A drug-targeted delivery system having bioorthogonal properties, characterized in that, The drug targeting delivery system comprises a mutant galactosidase and a prodrug; The amino acid sequence of the mutant galactosidase is shown as SEQ ID NO. 6; The structural formula of the prodrug is: ; wherein R1 is phenyl, benzyl or phenethyl; and R2 is a payload drug group.

2. The drug targeting delivery system according to claim 1, wherein, The R1 is phenethyl.

3. The drug targeting delivery system according to claim 2, wherein, The load drug corresponding to the load drug group is a fluorescent reporter molecule.

4. The drug targeting delivery system according to claim 2, wherein, The load drug corresponding to the load drug group is a cancer treatment drug.

5. The drug targeting delivery system according to claim 4, wherein, The cancer treatment drug is SN38.

6. Use of a drug targeting delivery system having bioorthogonal properties for the manufacture of a medicament for the treatment of a disease, characterized in that, The drug targeting delivery system comprises a mutant galactosidase and a prodrug; The amino acid sequence of the mutant galactosidase is shown as SEQ ID NO. 6; The structural formula of the prodrug is: ; wherein R1 is phenyl, benzyl or phenethyl; and R2 is a payload drug group.

7. Use according to claim 6, characterized in that, The R1 is phenethyl.

8. Use according to claim 7, characterized in that, When the disease treatment drug is a breast cancer treatment drug, the load drug corresponding to the load drug group is SN38.

9. A therapeutic agent for breast cancer, characterized by comprising the compound or salt according to claim 1. The drug targeting delivery system comprises a mutant galactosidase and a prodrug; The amino acid sequence of the mutant galactosidase is shown as SEQ ID NO. 6; The load drug corresponding to the load drug group is SN38. The structural formula of the prodrug is: ; wherein R1 is phenethyl; R2 is a payload group; The breast cancer treatment drug further comprises a pharmaceutically acceptable excipient.

10. The breast cancer therapeutic drug according to claim 9, characterized by, ​

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