Pharmaceutical composition and application thereof in preparation of medicine for treating tumors

Abstract

The invention belongs to the technical field of biological medicines, and relates to a pharmaceutical composition and application thereof in preparation of a medicine for treating tumors. The pharmaceutical composition comprises a double-end functional molecule, a catalyst and an alkynyl-modified pharmaceutical molecule, the chemical structure of the double-end functional molecule is shown as a formula I, R is a fluorophore, X is methylene or O, Y is methylene or carbonyl, and n is 1-3; the drug molecule has anti-tumor activity; the catalyst can catalyze an azide group to react with alkynyl, so that a double-end functional molecule is connected with a drug molecule. The pharmaceutical composition provided by the invention can capture drug molecules on the surface of a cell membrane in situ and realize slow release under an enzymatic reaction condition, so that the treatment effect of the drug molecules is improved.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to a pharmaceutical composition and its application in the preparation of drugs for treating tumors. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Cancer is one of the most challenging diseases in medicine. The cell membrane provides a complex landscape rich in potential therapeutic targets that regulate tumorigenesis, progression, and immune evasion. Tissue mapping of the human proteome shows that approximately 59% of predicted drug targets are membrane proteins, including carbonic anhydrase and NINJ1. Furthermore, analysis of the U.S. Food and Drug Administration (FDA) database indicates that approximately 44% of approved small-molecule anticancer drugs act on membrane-associated proteins, highlighting the significant clinical implications of cell surface pharmacology.

[0004] However, many membrane-targeting drugs that exhibit high activity in vitro fail in vivo due to rapid internalization, leading to off-target effects. For example, indomethacin, a drug with potent inhibitory activity against carbonic anhydrase IX (CAIX) in vitro, loses its ability to bind to the cell membrane surface due to rapid internalization in solid tumors, thus completely losing its efficacy. This drug was reclassified from an anticancer candidate for gout treatment not because of insufficient pharmacological efficacy, but because of the lack of a method to maintain membrane binding. Similarly, while chlorpromazine can interfere with membrane signaling in vitro through clathrin-associated lipid rafts, it is rapidly internalized into lysosomes and endoplasmic reticulum in vivo, resulting in the loss of its anticancer activity and limiting its use to the treatment of mental illnesses. This is not an isolated case; many small- and medium-sized molecule drugs designed to act on the cell membrane surface also face the problem of "internalization leading to off-target effects," a predicament that continues to limit the success rate of clinical translation of membrane-targeted tumor therapies. Although the enormous potential of membrane pharmacology in cancer treatment has long been recognized, the field of chemistry has yet to provide an effective way to maintain drug retention in vivo. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention aims to provide a pharmaceutical composition and its application in the preparation of drugs for treating tumors. The pharmaceutical composition provided by the present invention can capture drug molecules in situ on the cell membrane surface and achieve slow release under enzymatic reaction conditions, thereby improving the therapeutic effect of drug molecules.

[0006] To achieve the above objectives, the technical solution of the present invention is as follows: In a first aspect, a pharmaceutical composition comprising a biheaded functional molecule, a catalyst, and an alkynyl-modified pharmaceutical molecule; The chemical structure of the dual-headed functional molecule is shown in Formula I: In this context, R is a fluorescent group, X is methylene or O, Y is methylene or carbonyl, and n is 1 to 3. The drug molecule has anti-tumor activity; The catalyst can catalyze the reaction between azide groups and alkynyl groups, enabling the connection of biheaded functional molecules to drug molecules.

[0007] This invention developed a biheaded functional molecule in previous research. In this molecule, the central saturated long chain enhances the lipophilicity of the probe molecule, enabling targeting of the cell membrane. The introduction of units at both ends strengthens the binding force to the cell membrane surface and induces the long chain to wrap around the phospholipid molecules of the cell membrane, increasing steric hindrance and effectively preventing the internalization of the probe molecule, thus enabling this series of probe molecules to achieve ultra-long residence time on the cell membrane. Based on this biheaded functional molecule, this invention further explores the ability of the azide group of this molecule to react with the alkyne group, constructing a cell membrane-targeted drug delivery and sustained-release platform. Experimental results show that the drug composition can capture drug molecules in situ on the cell membrane surface and achieve sustained-release under enzymatic reaction conditions, significantly improving the therapeutic effect of the drug molecules.

[0008] In some implementations, the alkynyl group is linked to the drug molecule via an ester group. After the biheaded functional molecule is anchored to the cell membrane surface, the alkynyl group reacts with an azide group under the catalysis of a catalyst, allowing the drug molecule to link to the biheaded functional molecule anchored to the cell membrane surface. Then, esterases in the body act on the ester group between the alkynyl group and the drug molecule, thereby achieving sustained release of the drug molecule and improving membrane-targeted drug efficacy.

[0009] On the other hand, the use of a pharmaceutical composition according to the first aspect of the present invention in the preparation of a drug for treating tumors.

[0010] Thirdly, a method for treating tumors, comprising administering a therapeutically effective amount of the pharmaceutical composition described in the first aspect of the present invention.

[0011] The beneficial effects of this invention are as follows: In the pharmaceutical composition provided by the present invention, the dual-headed functional molecule has the ability to remain on the cell membrane for an extended period of time. Based on this, the drug molecule modified with a catalyst and alkyne group can capture the drug in situ on the membrane through a bioorthogonal azido-alkynyl reaction and release it under the action of esterase, thereby improving the membrane-targeted drug efficacy. Attached Figure Description

[0012] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0013] Figure 1 The UV absorption and fluorescence spectra of the Rh-18N3 molecule are shown. Figure 2 The image shows the UV absorption and fluorescence spectra of Alky-IDT drug molecules. Figure 3 This is a confocal fluorescence image of Rh-18N3 cells co-incubated with alkyne-modified indomethacin under copper-free and copper-free catalytic conditions. Figure 4 In vivo experiments using Rh-18N3 molecules to capture drugs. (a) Photographs of tumor tissues excised from mice in different treatment groups 18 days after treatment. (b) Data collection and statistical analysis of relative tumor volume at different time points after treatment in different groups of mice. (c) Data collection and statistical analysis of relative body weight at different time points after treatment in different groups of mice. Group 1: Control group (PBS); Group 2: Control group (1.3 mg / kg indomethacin); Group 3: Control group (1.3 mg / kg Alky-IDT, 5 mg / kg CuAAC); Group 4: Experimental group (5 mg / mL Rh-18N3, 1.3 mg / kg Alky-IDT); Group 5: Experimental group (5 mg / mL Rh-18N3, 1.3 mg / mL Alky-IDT, 5 mg / mL CuAAC). Figure 5 Images of H&E staining of mouse tumor tissue and major organs on day 18. Group 1: Control group (PBS); Group 2: Control group (1.3 mg / kg indomethacin); Group 3: Control group (1.3 mg / kg Alky-IDT, 5 mg / kg CuAAC); Group 4: Experimental group (5 mg / mL Rh-18N3, 1.3 mg / kg Alky-IDT); Group 5: Experimental group (5 mg / mL Rh-18N3, 1.3 mg / mL Alky-IDT, 5 mg / mL CuAAC). Detailed Implementation

[0014] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0015] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of exemplary embodiments according to the invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0016] Given that current anti-tumor drugs are rapidly endocytosed, leading to off-target effects and thus unsatisfactory efficacy, this invention proposes a pharmaceutical composition and its application in the preparation of drugs for treating tumors.

[0017] A typical embodiment of the present invention provides a pharmaceutical composition comprising a biheaded functional molecule, a catalyst, and an alkyne-modified pharmaceutical molecule; The chemical structure of the dual-headed functional molecule is shown in Formula I: In this context, R is a fluorescent group, X is methylene or O, Y is methylene or carbonyl, and n is 1 to 3. The drug molecule has anti-tumor activity; The catalyst can catalyze the reaction between azide groups and alkynyl groups, enabling the connection of biheaded functional molecules to drug molecules.

[0018] In some embodiments, the biheaded functional molecule has the chemical structure shown in Formula II or Formula III;

[0019] X, Y, and n are as described above.

[0020] In some embodiments, the dual-headed functional molecule is selected from the following compounds: .

[0021] In some embodiments, the alkynyl group is linked to the drug molecule via an ester group.

[0022] In some embodiments, the alkynyl-modified drug molecule is: .

[0023] In some embodiments, the catalyst is a monovalent copper salt. The monovalent copper salt may be cuprous chloride, cuprous bromide, cuprous iodide, CuAAC (tris(benzyltriazolylmethyl)amine)cuprous salt, etc.

[0024] The ratio of the biheaded functional molecule, the catalyst, and the alkynyl-modified drug molecule can be any ratio. In some embodiments, the mass ratio of the biheaded functional molecule, the catalyst, and the alkynyl-modified drug molecule is (2~5):(2~5):1.

[0025] In some embodiments, pharmaceutical excipients are also included. Specifically, the pharmaceutical excipients include a pharmaceutical carrier and / or an excipient. The pharmaceutical carrier may be physiological saline, a buffer solution, etc. The excipient may be a binder, filler, preservative, antioxidant, etc.

[0026] In some embodiments, the dosage form is an injection.

[0027] Another embodiment of the present invention provides the use of the above-described pharmaceutical composition in the preparation of a drug for treating tumors.

[0028] In some embodiments, the tumor-treating drug is administered to humans or non-human mammals. Specifically, the non-human mammals include mice, rats, dogs, rabbits, pigs, etc.

[0029] A third embodiment of the present invention provides a method for treating tumors, comprising administering a therapeutically effective amount of the above-described pharmaceutical composition.

[0030] The therapeutically effective amount of the pharmaceutical composition depends on many factors, including, for example, the patient's age and weight, the exact condition requiring treatment and its severity, the nature of the formulation, and the route of administration, and is ultimately determined by the attending physician or veterinarian. In the pharmaceutical composition, each dose unit for oral or parenteral administration preferably contains, as a free base, 0.01 mg to 3000 mg, more preferably 0.5 mg to 1000 mg of the compound of Formula I or a pharmaceutically acceptable salt thereof. Each dose unit for nasal or inhalation administration preferably contains, as a free base, 0.001 mg to 50 mg, more preferably 0.01 mg to 5 mg of the compound of Formula I or a pharmaceutically acceptable salt thereof.

[0031] Specifically, the treatment method for tumors is for mammals, particularly humans.

[0032] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0033] Example 1: The synthetic route for Rh-18N3 is shown below.

[0034]

[0035] Its preparation method specifically includes the following steps: a. Rhodamine B (310 mg, 0.5 mmol) and 1,18-dibromooctadecane (205 mg, 0.5 mmol) were dissolved in 20 mL of redistilled anhydrous ethanol and refluxed at 75 °C for 36 hours. After the reaction was complete, the mixture was cooled to room temperature, and the reaction solution was quenched in ice water and extracted with ethyl acetate (3 × 10 mL). The organic phases were combined, washed, separated, dried over anhydrous sodium sulfate, and the organic solvent was removed by vacuum distillation. The residue was purified by preparative rapid silica gel liquid chromatography (eluent polarity: dichloromethane:methanol = 300:1), and the product with absorption spectra at both 365 nm and 543 nm was collected to give 1-bromooctadecylrhodamine B (Rh-18Br) in 36% yield. The product did not require purification and could be directly used for the next reaction.

[0036] b. Place compound Rh-18Br (100 mg, 0.125 mmol) in a 100 mL open-mouthed double-necked flask, then add 10 mL of acetone to dissolve the reactants and reflux at 80 °C. In a separate beaker, carefully weigh 17 mg of azide TMS (4 eq, 0.5 mmol), add 10 mL of ultrapure water to fully dissolve the azide, and then slowly add it dropwise to the reaction system under reflux at 80 °C while stirring. After the addition is complete, continue stirring the reaction system open for 18 hours, monitoring the reaction progress by thin-layer chromatography until the reactants have completely reacted. After the reaction is complete, cool the system to room temperature, then slowly add 10 mL of 2N dilute hydrochloric acid to quench the reaction, and extract with dichloromethane (3 × 30 mL). Combine the organic phases, wash, separate, dry with anhydrous sodium sulfate, and remove the organic solvent by vacuum distillation. The residue was first crudely purified by recrystallization from a dichloromethane / n-hexane system, and then purified by preparative-grade rapid silica gel liquid chromatography (eluent polarity: dichloromethane:methanol = 200:1). Products exhibiting absorption spectra at both 365 nm and 543 nm were collected to obtain Rh-18N3. Product state: red solid. Yield: 84 mg (89%). Product NMR data: 1H NMR (400 MHz, DMSO-d6) δ 8.23 ​​(dd, J = 7.8, 1.4Hz, 1H), 7.91 (td, J = 7.5, 1.5 Hz, 1H), 7.87 - 7.77 (m, 1H), 7.50 (dd, J =7.5, 1.4 Hz, 1H), 7.10 (dd, J = 9.6, 2.4 Hz, 2H), 7.05 - 6.92 (m, 4H), 3.88 (t, J = 6.2 Hz, 2H), 3.78 - 3.46 (m, 10H), 3.10 (qd, J = 7.3, 4.8 Hz, 4H),1.19 (dd, J = 14.3, 7.1 Hz, 42H). 13 C NMR (101 MHz, DMSO-d6) δ 167.37, 165.53,158.13, 157.61, 155.60, 155.60, 133.54, 133.18, 131.44, 131.33, 131.02,130.97, 130.91, 130.34, 130.09, 115.07, 113.38, 96.32, 65.67, 55.40, 51.07,45.80, 45.69, 29.48, 29.45, 29.42, 29.38, 29.36, 29.23, 29.07, 28.98, 28.69, 28.19, 26.59, 25.71, 12.88. The UV absorption and fluorescence spectra of Rh-18N3 are as follows: Figure 1 As shown.

[0037] Example 2: The synthetic route for Car-20N3 is shown below.

[0038]

[0039] Its preparation method specifically includes the following steps: a. Potassium hydroxide (51.63 mmol, 3 g) solid was dissolved in analytical grade DMF solvent, and the mixture was stirred at room temperature for 10 min. Then, 3-bromocarbazole (8.13 mmol, 2 g) was slowly added to the mixture while stirring. After 30 min, bromoethane (12.3 mmol, 1.38 mL) was slowly added dropwise to the mixture, and stirring continued at room temperature for 18 h. The reaction mixture was filtered to obtain a clear solution. The filtrate was distilled under reduced pressure to obtain N-ethyl-3-bromocarbazole.

[0040] b. Under a nitrogen atmosphere, N-ethyl-3-bromocarbazole (2.73 g, 10 mmol), palladium acetate (2% eq), and tris(o-methyl)phenylphosphine (2.4 g, 7.8 mmol) were dissolved in a mixed solvent of DMF and triethylamine (45 mL, DMF / triethylamine = 2:1), and 4-vinylpyridine (1.05 g, 10 mmol) was added. The mixture was refluxed at 120 °C for 48 h. After the reaction solution was cooled to room temperature, it was quenched in ice water and extracted three times with ethyl acetate (10 mL). Subsequently, the combined organic phases were washed, dried over anhydrous sodium sulfate, and the organic solvent was removed by vacuum distillation. The obtained solid residue was purified by preparative rapid silica gel liquid chromatography (eluent polarity: dichloromethane: n-hexane = 1:5), and the product with absorption spectra at both 254 nm and 365 nm was collected to obtain (E)-3-(4-pyridinylvinyl)-N-ethylcarbazole.

[0041] c. Under a nitrogen atmosphere, (E)-3-(4-pyridinylvinyl)-N-ethylcarbazole (2.98 g, 10 mmol) was dissolved in anhydrous acetonitrile (30 mL), and then 3-bromo-1-propanol (1.39 g, 10 mmol) was added to the reaction system. The mixture was then refluxed at 85 °C for 24 h. After the reactants were slowly cooled to room temperature, the mixture was poured into pre-cooled n-hexane, and the solution was allowed to stand in a refrigerator for 12 h. After crystals precipitated, the solution was filtered to obtain (E)-3-(4-hydroxypropylpyridinylvinyl)-N-ethylcarbazole ammonium bromide in 67% yield.

[0042] Product NMR data: 1 H NMR (400 MHz, DMSO- d 6) δ 8.91 (d, J = 6.5 Hz, 2H),8.61 (s, 1H), 8.29 – 8.17 (m, 4H), 7.90 (d, J = 10.3 Hz, 1H), 7.73 (d,J = 8.6Hz, 1H), 7.66 (d, J = 8.2 Hz, 1H), 7.60 – 7.48 (m, 2H), 7.28 (t, J = 7.5 Hz, 1H),4.79 (s, 1H), 4.57 (t, J = 7.0 Hz, 2H), 4.52 – 4.42 (m, 2H), 3.47 (t, J = 5.8 Hz,2H), 2.13 – 2.02 (m, 2H), 1.33 (t, J = 7.1 Hz, 3H). 13 C NMR (101 MHz, DMSO- d 6) δ153.89, 144.63, 143.02, 141.43, 140.61, 126.93, 126.80, 126.73, 123.44,123.20, 122.61, 121.72, 120.99, 120.45, 120.16, 110.34, 110.20, 57.71, 57.65,37.73, 33.66, 14.25. d. (E)-3-(4-hydroxypropylpyridinylvinyl)-N-ethylcarbazole ammonium bromide (237 mg, 0.5 mmol) and 16-bromohexadecanoic acid (167 mg, 0.5 mmol) were dissolved in dry dichloromethane. Then, 4-dimethylaminopyridine (10 mg) and N,N'-dicyclohexylcarboimide (20 μL) were added to the mixture. After stirring the mixture at room temperature for 14 h, the reaction solution was quenched in ice water. The mixture was extracted three times with dichloromethane (50 mL), and the organic phases were then combined. After washing with saturated brine, drying with anhydrous sodium sulfate, and removing organic solvent by vacuum distillation, the resulting solid residue was purified by preparative rapid silica gel liquid chromatography (eluent polarity: dichloromethane:methanol = 300:1), and the product with absorption spectra at both 254 nm and 365 nm was collected to obtain (E)-16-bromohexadecylpropyl-3-(N-ethylcarbazolevinyl)pyridineammonium bromide, with a yield of 37%.

[0043] e. (E)-16-bromohexadecylpropyl-3-(N-ethylcarbazolevinyl)pyridineammonium bromide (150 mg, 0.2 mmol) was dissolved in a mixture of acetone and water (10 mL, 1:1 v / v), and azido-TMS (17 mg, 4 eq, 0.5 mmol) was slowly added in three portions. The mixture was stirred and refluxed at 80 °C for 18 h. After the reaction solution cooled to room temperature, the product was quenched in ice water and extracted three times with dichloromethane (10 mL). Subsequently, the combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, and then subjected to vacuum distillation to remove the solvent. The resulting solid residue was purified by preparative rapid silica gel liquid chromatography (eluent polarity: dichloromethane:methanol = 200:1). The product exhibiting absorption spectra at both 365 nm and 543 nm was collected to obtain Car-20N3, with a yield of 99%.

[0044] Product NMR data: 1 H NMR (400 MHz, DMSO- d 6) δ 8.97 (d, J = 6.4 Hz, 2H), 8.60 (s, 1H), 8.36 – 8.14 (m, 4H), 7.91 (d, J = 8.6 Hz, 1H), 7.75 (d, J = 8.6 Hz, 1H), 7.68 (d, J = 8.2 Hz, 1H), 7.63 – 7.48 (m, 2H), 7.29 (t, J = 7.4 Hz, 1H), 4.62 (t, J = 6.6 Hz, 2H), 4.50 (q, J = 7.1 Hz, 2H), 4.15 (t, J = 5.7 Hz, 2H), 3.29(t, J = 6.9 Hz, 2H), 2.30 (q, J = 6.2 Hz, 2H), 2.08 (t, J = 7.6 Hz, 2H), 1.49 (p, J =6.9 Hz, 2H), 1.35 (t, J = 7.1 Hz, 5H), 1.26 – 1.01 (m, 23H). 13 C NMR (101 MHz, DMSO- d6) δ 173.01, 154.20, 144.69, 143.30, 141.50, 140.64, 126.92, 126.76,126.69, 123.42, 123.25, 122.64, 121.75, 120.93, 120.29, 120.15, 110.31,110.18, 61.92, 58.01, 51.07, 45.88, 37.73, 33.89, 32.51, 29.74, 29.51, 29.48,29.44, 29.34, 29.27, 29.12, 28.97, 28.69, 26.71, 26.58, 24.85, 14.25. Example 3: The synthetic route for Car-18N3 is shown below.

[0045]

[0046] Its preparation method specifically includes the following steps: Steps a and b are the same as in Example 2.

[0047] c. Under a nitrogen atmosphere, (E)-3-(4-pyridinylvinyl)-N-ethylcarbazole (2.98 g, 10 mmol) was dissolved in anhydrous acetonitrile (30 mL), and then 1,18-dibromooctadecane (4.12 g, 10 mmol) was added to the reaction system. The mixture was then refluxed at 80 °C for 24 h. After the reactants were slowly cooled to room temperature, the mixture was poured into pre-cooled n-hexane, and the solution was allowed to stand in a refrigerator for 12 h. After crystals precipitated, the solution was filtered to obtain (E)-1-bromooctadecyl-3-(N-ethylcarbazolevinyl)pyridineammonium bromide.

[0048] d. (E)-1-bromooctadecyl-3-(N-ethylcarbazolevinyl)pyridine ammonium bromide (142 mg, 0.2 mmol) was dissolved in a mixture of acetone and water (10 mL, 1:1 v / v), and azido-TMS (17 mg, 4 eq, 0.5 mmol) was slowly added in three portions. The mixture was stirred and refluxed at 80 °C for 18 h. After the reaction solution cooled to room temperature, the product was quenched in ice water and extracted three times with dichloromethane (10 mL). Subsequently, the combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, and then subjected to vacuum distillation to remove the solvent. The resulting solid residue was purified by preparative-grade rapid silica gel liquid chromatography (eluent polarity: dichloromethane:methanol = 200:1). The product exhibiting absorption spectra at both 365 nm and 543 nm was collected to obtain Car-18N3 in 99% yield.

[0049] Product NMR data: 1 H NMR (400 MHz, DMSO- d 6) δ 8.87 (d, J = 6.3 Hz, 2H),8.56 (s, 1H), 8.27 – 8.10 (m, 4H), 7.86 (d, J = 8.2 Hz, 1H), 7.68 (d, J = 8.9 Hz, 1H), 7.63 (d, J = 7.6 Hz, 1H), 7.59 – 7.47 (m, 2H), 7.25 (t, J = 7.0 Hz, 1H), 4.58 (t, J = 6.8 Hz, 2H), 4.52 (q, J = 7.3 Hz, 2H), 4.13 (t, J = 5.5 Hz, 2H), 3.18(t, J = 6.7 Hz, 2H), 1.33 (t, J = 2.9 Hz, 7H), 1.26 – 1.01 (m, 31H). 13 C NMR (101MHz, DMSO- d6) δ 155.37, 146.55, 141.47, 141.42, 139.56, 122.91, 121.39,120.27, 119.35, 118.98, 118.51, 117.98, 117.73, 117.02, 115.36, 109.47,108.31, 56.28, 48.96, 35.62, 26.51, 26.24, 25.17, 24.49, 22.08, 21.26, 21.08,20.25, 20.21, 20.03, 18.16, 18.11. Example 4: 3-Hydroxypropyl-4-methylpyridineammonium bromide

[0050] Under a nitrogen atmosphere, 4-methylpyridine (932 mg, 10 mmol) and 3-bromo-1-propanol (1.39 g, 10 mmol) were dissolved in 20 mL of redistilled anhydrous acetonitrile, and stirred overnight at 85 °C. After cooling to room temperature, stirring was continued for 12 hours. The organic solvent and excess 3-bromo-1-propanol were removed from the mixture by vacuum distillation to give 3-hydroxypropyl-4-methylpyridine ammonium bromide. Product state: orange oily liquid. The product requires no purification and can be used directly in subsequent reactions.

[0051] 1-Acetyl-3-hydroxypropylindomethacin-4-methylpyridineammonium bromide

[0052] 3-Hydroxypropyl-4-methylpyridine ammonium bromide (165 mg, 0.5 mmol) and indomethacin (179 mg, 0.5 mmol) were dissolved in 60 mL of redistilled anhydrous dichloromethane. Then, 4-dimethylaminopyridine (10 mg) and N,N'-dicyclohexylcarboimide (20 μL) were added sequentially, and the mixture was stirred at room temperature for 24 hours. After the reaction was complete, the mixture was cooled to room temperature, and the reaction solution was quenched in ice water and extracted with ethyl acetate (3 × 30 mL). The combined organic phases were washed, separated, dried over anhydrous sodium sulfate, and the organic solvent was removed by vacuum distillation. The residue was purified by preparative rapid silica gel liquid chromatography (eluent polarity: dichloromethane:methanol = 200:1). The product exhibiting absorption spectra at both 365 nm and 254 nm was collected to give 1-acetyl-3-hydroxypropylindomethacin-4-methylpyridine ammonium bromide. Product state: white solid. The product requires no purification and can be used directly in subsequent reactions.

[0053] N-Methyl-N-(hydroxyacetyl-1-pentyne)-4-aminobenzaldehyde

[0054] N-Methyl-N-hydroxyethyl-4-aminobenzaldehyde (90 mg, 0.5 mmol) and 4-pentyne-1-acid (49 mg, 0.5 mmol) were dissolved in 60 mL of redistilled anhydrous dichloromethane. Then, 4-dimethylaminopyridine (10 mg) and N,N'-dicyclohexylcarboimide (20 μL) were added sequentially, and the mixture was stirred at room temperature for 24 hours. After the reaction was complete, the mixture was quenched in ice water and extracted with ethyl acetate (3 × 30 mL). The combined organic phases were washed, separated, dried over anhydrous sodium sulfate, and the organic solvent was removed by vacuum distillation. The residue was purified by preparative-grade rapid silica gel liquid chromatography (eluent polarity: dichloromethane:methanol = 200:1). The product with an absorption spectrum at 254 nm was collected to give N-methyl-N-(hydroxyacetyl-1-pentyne)-4-aminobenzaldehyde. Product state: white solid. The product did not require purification and was used directly in subsequent reactions.

[0055] The synthetic route of Alky-IDT is shown below.

[0056]

[0057] Under a nitrogen atmosphere, compounds IND-Py and Alky were dissolved in 5 mL of redistilled anhydrous methanol, and then 3-5 drops of piperidine were added dropwise as a catalyst. The reaction mixture was stirred at 80 °C for 18 hours. After the reaction was complete, the mixture was cooled to room temperature, quenched in ice water, and extracted with ethyl acetate (3 × 30 mL). The combined organic phases were washed, separated, dried over anhydrous sodium sulfate, and the organic solvent was removed by vacuum distillation. The residue was purified by preparative rapid silica gel liquid chromatography (eluent polarity: dichloromethane:methanol = 300:1), and the product exhibiting absorption spectra at both 365 nm and 473 nm was collected to obtain Alky-IDT. Product state: red solid. Yield 59%.

[0058] Product NMR data: 1 H NMR (400 MHz, Chloroform- d ) δ 7.73 - 7.56 (m,5H), 7.45 (d, J = 8.7 Hz, 5H), 7.02 (s, 1H), 6.95 (d, J = 9.0 Hz, 1H), 6.90 (s,1H), 6.85 (d, J= 9.0 Hz, 1H), 6.72 - 6.60 (m, 3H), 3.81 (d, J = 16.3 Hz, 5H), 3.62 (s, 3H), 2.94 (s, 2H), 2.77 (s, 1H), 2.33 (d, J = 14.7 Hz (7H), 1.25 (s, 9H). 13 C NMR (101 MHz, Chloroform- d ) δ 170.76, 156.06, 133.72, 131.25, 131.19,130.80, 130.57, 129.25, 129.18, 111.96, 111.49, 56.06, 55.77, 38.61, 30.50,29.72, 13.55, 13.34. The UV absorption and fluorescence spectra of Alky-IDT are shown below. Figure 2 As shown.

[0059] Application Example 1: Rh-18N3 is used for in situ drug capture in cells.

[0060] like Figure 3 As shown in the figure, cells were first stained with 5 μM Rh-18N3, then washed and simultaneously treated with Cu(I) catalyst (CuAAC) and 1 μM alkynyl-modified indomethacin (Alky-IDT). After co-incubation for 20 min, strong fluorescent colocalization between alkynyl-modified indomethacin (green fluorescence) and Rh-18N3 (red fluorescence) was observed in all three HeLa cell lines. In contrast, almost no colocalization was observed without Cu(I) catalysis. This result indicates that alkynyl-modified indomethacin can be selectively captured by membrane-anchored Rh-18N3, thereby preventing its internalization and significantly enhancing the membrane targeting ability of the drug molecule.

[0061] Application Example 2: Rh-18N3 was used for in situ drug capture in mouse tumors to inhibit tumor growth.

[0062] Specific implementation method: 4T1 cells were cultured in DMEM supplemented with 10% FBS, 100 μg / mL streptomycin, and 100 U / mL penicillin, and maintained at 37°C in a humidified incubator containing 5% CO2. After the cells reached confluence in the culture dishes, 4T1 cells were implanted into BALB / c mice to establish a subcutaneous breast cancer model. Starting on day 6 (when the tumor diameter reached 3 mm), the mice were divided into 5 groups, and the drugs were injected every two days while monitoring the tumor volume. 1. Control (PBS). 2. Control group (1.25 mg / kg indomethacin). 3. Control group (1.25 mg / kg alkynyl-modified indomethacin and 5 mg / kg CuAAC). 4. Experimental group (5 mg / mL Rh-18N3 and 1.25 mg / kg alkynyl-modified indomethacin). 5. Experimental group (5 mg / mL Rh-18N3, 1.25 mg / mL Alky-IDT, and 5 mg / mL CuAAC).

[0063] like Figure 4 As shown in ac, the PBS group ( Figure 4 a-1), indomethacin-only group ( Figure 4 a-2) and the Cu(I)-deficient catalytic group ( Figure 4 a-3 and Figure 4 Tumors of type a-4 grew rapidly. In contrast, the CuAAC catalytic group ( Figure 4 a-5) exhibited significant tumor suppression, with a substantial reduction in volume between day 8 and day 14. This result indicates that Rh-18N3, with its strong membrane retention, can capture alkyne-modified indomethacin in situ and promote sustained-release of the drug through esterase-mediated hydrolysis present in the cytoplasmic membrane, thereby inhibiting tumor growth.

[0064] On day 18, mice were euthanized, and tumor tissue and internal organs (heart, liver, spleen, lungs, and kidneys) were removed, embedded in paraffin, and sectioned. After hematoxylin and eosin (HE) staining, the sections were observed under a regular optical microscope to demonstrate biocompatibility.

[0065] like Figure 5 As shown, HE staining results indicate that there was no damage to the internal organs (heart, liver, spleen, lungs, and kidneys) of the mice. This suggests that simultaneous injection of Rh-18N3, Alky-IDT, and CuAAC does not affect the physiological state of normal tissues, thus demonstrating that this method has high biosafety.

[0066] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A pharmaceutical composition, characterized in that, This includes biheaded functional molecules, catalysts, and alkynyl-modified drug molecules; The chemical structure of the dual-headed functional molecule is shown in Formula I: In this context, R is a fluorescent group, X is methylene or O, Y is methylene or carbonyl, and n is 1 to 3. The drug molecule has anti-tumor activity; The catalyst can catalyze the reaction between azide groups and alkynyl groups, enabling the connection of biheaded functional molecules to drug molecules.

2. The pharmaceutical composition according to claim 1, characterized in that, The dual-headed functional molecule has the chemical structure shown in Formula II or Formula III; 。 3. The pharmaceutical composition according to claim 1, characterized in that, The dual-headed functional molecule is selected from the following compounds: 。 4. The pharmaceutical composition according to claim 1, characterized in that, The alkynyl group is linked to the drug molecule through the ester group.

5. The pharmaceutical composition according to claim 1, characterized in that, Alkyne-modified drug molecules are: 。 6. The pharmaceutical composition of claim 1, characterized in that, The catalyst is a monovalent copper salt.

7. The pharmaceutical composition of claim 1, characterized in that, The mass ratio of the dual-headed functional molecule, the catalyst, and the alkyne-modified drug molecule is (2~5):(2~5):

1.

8. The pharmaceutical composition of claim 1, characterized in that, It also includes pharmaceutical excipients; Alternatively, its dosage form is an injection.

9. The use of a pharmaceutical composition according to any one of claims 1 to 8 in the preparation of a medicament for treating tumors.

10. The application as described in claim 9, characterized in that, The tumor-treating drugs are administered to humans or non-human mammals.

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