Three-branch load system as well as preparation method and application thereof

Abstract

The invention discloses a three-branch load system as well as a preparation method and application thereof, and relates to the technical field of medicinal chemistry. A structure containing terminal alkynyl is introduced into an original trimethyl lock structure to serve as a third connecting site, and the site can be connected with a targeting group with the tail end connected with an azide group through click reaction; therefore, the parallel connection of the three modules of the triggering group, the targeting group and the drug / probe is realized; if the modules, especially the targeting group and the triggering group, need to be replaced, the required operation steps are fewer than the traditional mode, the operation steps are simplified, and the working efficiency is improved.

Description

Technical Field

[0001] The present invention relates to the technical field of pharmaceutical chemistry, and particularly relates to a three-branch loading system, a preparation method thereof, and an application thereof. Background Art

[0002] Figure 1 As shown in Figure A, the trimethyl lock structure is composed of derivatives of o-hydroxycinnamic acid substituted by three methyl groups. Due to the mutual repulsion between the three methyl groups, the phenolic hydroxyl group and the carboxyl group are promoted to undergo an intramolecular nucleophilic addition-elimination reaction to form a lactone, and this reaction can proceed spontaneously rapidly.

[0003] Figure 1 As shown in Figure B, under certain specific conditions, such as the presence of a certain overexpressed enzyme in the body, a specific pH value, overexpressed reactive oxygen species, light of a certain wavelength, etc., the ether bond at the trigger group R1 will break, and while R1 is removed, the phenolic hydroxyl group is exposed, which will rapidly trigger intramolecular cyclization and simultaneously release the group R2 (which can be a drug molecule or a chemical probe, etc.) it carries. This plays a role in releasing drugs or probes under specific conditions and can achieve the purpose of precise drug or probe delivery.

[0004] The traditional trimethyl lock structure has only two sites (or branches) that can be used to connect other groups ( Figure 2 as shown in Figure A). Therefore, the targeting group can only be connected to the trigger group in series first and then connected to the phenolic hydroxyl group of the trimethyl lock.

[0005] In this regard, the inventor believes that this connection method of series connection of the targeting group and the trigger group has poor flexibility. If different targeting groups or trigger groups want to be changed, the required operation steps are also relatively complicated. Therefore, how to solve the above technical problems is an urgent technical problem for those skilled in the art.

[0006] The information disclosed in this background art section is only intended to increase the understanding of the overall background of the present invention and should not be regarded as an admission or any form of implication that this information constitutes the prior art already known to those of ordinary skill in the art. Summary of the Invention

[0007] In view of the above technical problems, an embodiment of the present invention provides a three-branch loading system, a preparation method thereof, and an application thereof to solve the problems raised in the above background art.

[0008] A three-branch loading system, the structural formula of which is as shown in Figure 3 formula (I) therein.

[0009] Among them, the three-branch loading system shown in formula (I) is a polysubstituted phenol structure. The 1-position of the benzene ring is a phenolic hydroxyl group, the 2-position is a 4'-hydroxy-2'-methylbutan-2'-yl group, the 3-position and 5-position of the benzene ring are methyl groups, and the 4-position of the benzene ring is a 3''-alkynyl-1''-butyl ether.

[0010] A method for synthesizing the three-branch loading system as described above, and the synthetic reaction route is shown in Figure 4 .

[0011] Preferably, the method for synthesizing the three-branch loading system includes the following steps: In the first step, 2,6-dimethyl-1,4-benzenediol reacts with methyl 3,3-dimethylacrylate under the catalysis of methanesulfonic acid to generate compound III; In the second step, compound III reacts with 3-alkyn-1-ol under the action of diethyl azodicarboxylate and triphenylphosphine to generate compound II; In the third step, the lactone ring on compound II is reductively ring-opened under the action of a reducing agent to generate compound I; the reducing agent is lithium aluminum hydride.

[0012] An application of the three-branch loading system as described above in the preparation of targeted drugs or targeted probes.

[0013] Preferably, the targeted drugs include drugs for treating tumors, inflammation or neurodegenerative diseases; the targeted probes include probes for diagnosing tumors, inflammation or neurodegenerative diseases.

[0014] Preferably, a trigger group, a drug or a probe, and a targeting group are connected to compound I.

[0015] Preferably, compound I has 3 group connection sites, including: the phenolic hydroxyl group site at the 1-position, the alcoholic hydroxyl group site at the 4'-position, and the alkynyl site at the end of the ether bond at the 4''-position; Among them, the phenolic hydroxyl group site at the 1-position is used to connect the trigger group, the hydroxyl group on the alcoholic hydroxyl group site at the 4'-position is oxidized to a carboxyl group and then used to connect the drug or the probe, and the alkynyl site at the end of the ether bond at the 4''-position is used to connect the targeting group.

[0016] Preferably, after the targeted drug or the targeted probe reaches the target position of the organism or cell, its action mode includes: The targeting group on compound I binds to the target protein. Under the microenvironment near the target, the phenolic hydroxyl group at the 1-position is released through the trigger group, and then the intramolecular spontaneous cyclization of compound I is triggered, releasing the drug or the probe for targeted treatment or targeted diagnosis of the disease.

[0017] A method for preparing a targeted drug or a targeted probe using the three-branch loading system according to claim 1: In the first step, a silyl ether protecting group of an alcohol hydroxyl group is introduced at the 4'-position of Compound I; In the second step, a triggering group is connected to the phenolic hydroxyl group at the 1-position of Compound I; In the third step, the silyl ether protecting group at the 4'-position of Compound I is removed; In the fourth step, the alcohol hydroxyl group at the 4'-position of Compound I is oxidized to a carboxyl group, and a drug or a probe is connected to the carboxyl group at the 4'-position; In the fifth step, a targeting group is connected to the alkyne group at the end of the ether bond at the 4''-position of Compound I.

[0018] Preferably, the triggering group includes a group that can be cleaved in the in vivo microenvironment; such as a disulfide bond, a hydrazone bond, etc. that are sensitive to the tumor microenvironment.

[0019] A three-branched loading system provided by an embodiment of the present invention, its preparation method and application have the following beneficial effects: The present invention proposes a new three-branched trimethyl lock loading system, which can realize the parallel connection of three modules of a triggering group, a targeting group and a drug / probe, and the connection method of the modules is flexible and the replacement is simple. Description of the Drawings

[0020] Figure 1 It is a schematic diagram of the spontaneous cyclization and release of the trimethyl lock; Figure 2 A is a schematic diagram of the traditional trimethyl lock connection form; Figure 2 B is a schematic diagram of the improved connection form of the present invention; Figure 3 It is the structural formula of the three-branched loading system in the present invention; Figure 4 It is the synthesis route diagram of the three-branched loading system in the present invention; Figure 5 It is a schematic diagram of the three-branched loading system loading specific triggering groups, drugs and targeting groups; Figure 6 It is the nuclear magnetic resonance hydrogen spectrum diagram of the three-branched loading system; Figure 7 It is the nuclear magnetic resonance carbon spectrum diagram of the three-branched loading system; Figure 8 It is the high-resolution mass spectrum diagram of the three-branched loading system. Detailed Embodiments

[0021] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative efforts belong to the scope of protection of the present invention.

[0022] In view of the above technical problems, an embodiment of the present invention provides a three-branch load system, a preparation method thereof and an application, so as to solve the problems raised in the above background art.

[0023] Example 1: Preparation experiment of the three-branch load system The synthesis route is as Figure 4 shown: The specific preparation method includes: First step, using commercially available 2,6-dimethyl-1,4-benzenediol as the raw material. Methanesulfonic acid (50.0 mL, 770.6 mmol) was added to a mixture of 2,6-dimethyl-1,4-benzenediol (10.0 g, 72.4 mmol) and methyl 3,3-dimethylacrylate (11.5 mL, 94.1 mmol), and then the mixture was stirred at 70 °C for 90 min. After the reaction was completed, the mixture was allowed to return to room temperature, and cold water and ethyl acetate were added for extraction. The organic layer was collected and washed with saturated sodium bicarbonate. The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated, and concentrated under reduced pressure to obtain a crude product. Recrystallization was carried out with a mixed solution of ethyl acetate:n-hexane = 1:10 to obtain 11.2 g of pure product III with a yield of 70.3%. 1H NMR (400 MHz, CDCl3) δ 6.73 (s, 1H), 4.95 (s, 1H), 2.59 (s,2H), 2.40 (s, 3H), 2.26 (s, 3H), 1.48 (s, 6H). HRMS (ESI) m / z calcd forC13H17O3 [M+H]+: 221.1172, found: 221.1170. Second step, compound III (5.0 g, 22.7 mmol), triphenylphosphine (8.9 g, 34.1 mmol) and 3-butyn-1-ol (3.4 mL, 45.4 mmol) were dissolved in 30.0 mL of THF, and diethyl azodicarboxylate (5.3 mL, 34.1 mmol) was added dropwise at 0 °C. After stirring for 30 min, the mixture was transferred to 70 °C for heating under reflux for 24 h. After monitoring the reaction by TLC and completion, THF was removed by concentration under reduced pressure. The residue was separated by column chromatography (pe:ea = 6:1) to obtain 2.9 g of product II with a yield of 47.4%. 1 1H NMR(400 MHz, CDCl3) δ 6.77 (s, 1H), 3.85 (t, J J = 6.8 Hz, 2H), 2.72 (td, J J = 6.8,2.7 Hz, 2H), 2.60 (s, 2H), 2.45 (s, 3H), 2.30 (s, 3H), 2.09 (t, J= 2.6 Hz, 1H), 1.47 (s, 6H). Step 3: Under nitrogen protection, lithium aluminum hydride (278.7 mg, 7.3 mmol) was added to a two-necked flask containing 5.0 mL of THF at 0 °C. A 15.0 mL THF solution of compound II (2.0 g, 7.3 mmol) was added dropwise to the two-necked flask, and then the mixture was heated and stirred at 46 °C for 4 h, and then stirred at room temperature for 12 h. It was concentrated under reduced pressure, and a mixed solution of ethyl acetate and water was added. The organic layer was separated and collected by liquid separation. After the organic layer was washed with saturated brine, it was dried over anhydrous sodium sulfate, concentrated under reduced pressure, and separated by column chromatography (pe:ea = 1:1) to obtain 1.8 g of product I with a yield of 90.5%. 1 H NMR (400 MHz, CDCl3) δ 6.38 (s, 1H), 3.80 (t, J = 6.8 Hz, 2H), 3.64 (t, J = 7.0 Hz, 2H), 2.69 (td, J = 6.9, 2.7 Hz, 2H), 2.44 (s, 3H), 2.22 (m, 5H), 2.08 (t, J = 2.7 Hz, 1H), 1.59 (s, 6H). 13C NMR (151 MHz, CDCl3) δ 151.3, 151.3, 149.9, 149.9, 131.5, 130.5, 129.1, 117.4, 81.0, 77.2, 77.0, 76.8, 69.9, 69.7, 61.5, 44.9, 39.9, 32.2, 20.2, 16.2, 15.9. HRMS (ESI) m / z calcd for C 17 H 23 O3[M - H] - : 275.1653, found: 275.1659. Example 2: Experiment on Loading Trigger Group, Drug and Targeting Group in a Three-Branched Loading System The synthetic route is as Figure 5 shown: The specific preparation method includes: Step 1: Introduction of a silyl ether protecting group at the 4'-position. Dissolve compound I (1.5 g, 5.4 mmol) in 20.0 mL of DCM, transfer it to an ice bath at 0 °C, and successively add TBSCl (1.6 g, 10.9 mmol) and triethylamine (2.3 mL, 16.3 mmol). Stir at room temperature for 24 h. Monitor the reaction by TLC until completion, concentrate the solvent under reduced pressure, and separate by column chromatography (pe:ea = 2:1) to obtain 1.7 g of product A1 with a yield of 81.2%. 1 1H NMR (600 MHz, CDCl3) δ 6.44 (s, 1H), 6.03 (s, 1H), 3.81 (t, J J = 6.9 Hz, 2H), 3.64 (t, J J = 6.8 Hz, 2H), 2.69 (td, J J = 6.9, 2.7 Hz, 2H), 2.42 (s, 3H), 2.21 (s, 3H), 2.14 (t, J J = 6.8 Hz, 2H), 2.07 (t, J J = 2.7 Hz, 1H), 1.59 (s, 6H), 0.90 (s, 9H), 0.05 (s, 6H). 13 13C NMR (151 MHz, CDCl3) δ 151.7, 149.8, 131.1, 131.1, 128.8, 118.0, 81.0, 77.2, 77.0, 76.8, 69.9, 69.7, 61.9, 44.7, 39.8, 32.4, 26.0, 25.9, 20.2, 20.1, 18.3, 16.2, 15.9, -5.4. HRMS (ESI) m / z calcd for C 23 H 37 O3Si [M-H] - : 389.2517, found: 389.2520. Step 2: Connect the trigger group to the 1-position phenolic hydroxyl group. Compound A1 (1.6 g, 4.1 mmol), bis(p-nitrophenyl) carbonate A2 (2.5 g, 8.2 mmol), and DIPEA (2.9 mL, 16.4 mmol) were successively dissolved in 20.0 mL of THF, heated to 45 °C, and stirred overnight. The reaction was monitored by TLC until completion, and the solvent was removed under reduced pressure. The residue was separated by column chromatography (pe:ea = 10:1) to obtain a white solid. The above solid (2.0 g, 3.6 mmol), compound A3 (847.6 mg, 4.3 mmol), and DMAP (527.6 mg, 4.3 mmol) were successively added to 20.0 mL of DCM and stirred at room temperature for 24 h. After monitoring the reaction by TLC until completion, the solvent was removed under reduced pressure. The residue was separated by column chromatography (pe:ea = 5:1) to obtain 1.7 g of product A4 with a yield of 75.6%. 1 H NMR (600 MHz,CDCl3) δ 6.70 (s, 1H), 4.51 (t, J = 6.6 Hz, 2H), 4.36 (t, J = 6.5 Hz, 2H),3.84 (t, J = 6.8 Hz, 2H), 3.52 (dd, J = 8.1, 6.9 Hz, 2H), 3.04 (t, J = 6.6Hz, 2H), 2.98 (t, J = 6.5 Hz, 2H), 2.71 (td, J = 6.8, 2.7 Hz, 2H), 2.49 (s,3H), 2.27 (s, 3H), 2.12–2.05 (m, 6H), 1.52 (s, 6H), 0.87 (s, 9H). 13 C NMR (151MHz, CDCl3) δ 170.7, 154.3, 154.1, 145.9, 136.2, 132.1, 129.6, 123.7, 80.7,77.3, 77.1, 76.8, 69.9, 69.6, 66.0, 62.2, 60.7, 45.7, 39.5, 37.3, 36.9, 31.7,25.9, 20.8, 20.2, 18.2, 16.3, 16.0, -5.3. HRMS (ESI) m / z calcd for C 30 H 49O7S2Si[M+H] + : 613.2684, found: 613.2689. In the third step, the 4'-position protecting group was removed.

[0024] Compound A4 (1.5 g, 2.5 mmol) was dissolved in 15.0 mL of THF and placed in an ice bath at 0 °C. A solution of pyridine hydrofluoride (70%) (3.8 mL, 146.8 mmol) was added dropwise, and the mixture was stirred in the ice bath for 3 h. After the reaction was completed as monitored by TLC (PE:EA = 2:1), saturated sodium bicarbonate solution was added to quench the reaction, and then ethyl acetate was added for liquid separation to obtain the organic layer. The organic layer was washed with saturated brine, then dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and separated by column chromatography (pe:ea = 2:1) to obtain 1.1 g of product A5 with a yield of 90.3%. 1 1H NMR (600 MHz, CDCl3) δ 6.68 (s, 1H), 4.50 (t, J J = 6.7 Hz, 2H),4.34 (t, J J = 6.6 Hz, 2H), 3.82 (t, J J = 6.9 Hz, 2H), 3.54 (t, J J = 7.3 Hz, 2H),3.02 (t, J J = 6.6 Hz, 2H), 2.96 (t, J J = 6.6 Hz, 2H), 2.68 (t, J J = 6.6 Hz, 2H),2.47 (s, 3H), 2.24 (s, 3H), 2.10–2.05 (m, 6H), 1.51 (s, 6H). 13 13C NMR (151 MHz,CDCl3) δ 170.9, 170.8, 154.4, 154.3, 145.9, 135.9, 132.2, 129.9, 123.8, 80.7,77.3, 77.1, 76.8, 69.9, 69.6, 66.1, 62.3, 60.4, 45.7, 39.6, 37.3, 37.0, 31.9,20.9, 20.2, 16.4, 16.0. HRMS (ESI) m / z calcd for C 24 H 35 O7S2[M+H] + : 499.1819, found: 499.1814. In the fourth step, after oxidizing the exposed 4′-alcohol hydroxyl group to a carboxylic acid, a drug or a probe is linked thereto. Compound A5 (1.0 g, 2.0 mmol) and pyridinium dichromate (PDC) (7.5 g, 20.1 mmol) were added to a round-bottom flask, and 20.0 mL of DMF was added until dissolved. The mixture was stirred at room temperature for 24 h. After monitoring the reaction by TLC (PE:EA = 1:1) until completion, water and ethyl acetate were added for liquid separation to obtain the organic layer. The organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and separated by column chromatography (pe:ea = 1:1) to obtain 462.6 mg of product A6. Compound A6 (400.0 mg, 780.3 µmol) and docetaxel A7 (756.5 mg, 936.3 µmol) were placed in a round-bottom flask, and 10.0 mL of DCM was added and stirred until dissolved. The reaction flask was placed in an ice-water bath, and then EDCI (179.5 mg, 936.3 µmol) and DMAP (114.4 mg, 936.3 µmol) were successively added to the solution, and the mixture was stirred at room temperature for 6 h. After monitoring the reaction by TLC (DCM:MeOH = 15:1) until completion, saturated ammonium chloride solution was added to quench the reaction, and liquid separation was performed to obtain the organic layer. The organic layer was washed with saturated brine and dried over anhydrous sodium sulfate. After concentration under reduced pressure, column chromatography separation (DCM:MeOH = 15:1) was carried out to obtain 711.4 mg of product A8 with a yield of 70.1%. 1 H NMR (400 MHz, CDCl3) δ 8.13 (d, J J =7.1 Hz, 2H), 7.63 (t, J J = 7.3 Hz, 1H), 7.53 (t, J J = 7.6 Hz, 2H), 7.37 (t, J J =7.3 Hz, 2H), 7.32 (s, 1H), 7.18 (d, J J = 7.0 Hz, 2H), 6.72 (s, 1H), 6.23 (s,1H), 5.71 (d, J J = 7.0 Hz, 1H), 5.51–5.15 (m, 4H), 4.98 (dd, J J = 9.7, 2.3 Hz,1H), 4.52 (t, J J = 6.6 Hz, 2H), 4.37 (t, J= 6.5 Hz, 2H), 4.34–4.25 (m, 2H), 4.24–4.19 (m, 2H), 3.97–3.88 (m, 2H), 3.82 (t, J = 6.9 Hz, 2H), 3.08–2.87 (m, 6H), 2.70 (td, J = 6.9, 2.7 Hz, 2H), 2.66–2.54 (m, 1H), 2.48–2.35 (m, 6H), 2.27 (s, 3H), 2.13–2.08 (m, 4H), 2.02–1.96 (m, 2H), 1.93 (s, 3H), 1.89–1.85 (m, 1H), 1.77 (s, 3H), 1.54 (s, 3H), 1.50 (s, 3H), 1.36 (s, 9H), 1.25 (s, 3H), 1.15 (s, 3H). 13 C NMR (151 MHz, CDCl3) δ 211.6, 171.0, 170.7, 169.7, 168.0, 167.8, 167.1, 155.2, 154.3, 153.9, 145.6, 139.3, 136.0, 135.4, 135.1, 133.6, 131.8, 130.2, 130.0, 129.2, 128.7, 128.7, 127.9, 126.2, 126.1, 123.6, 84.2, 80.9, 80.8, 80.2, 78.9, 77.2, 77.0, 76.8, 76.5, 75.0, 74.5, 74.1, 71.8, 71.7, 70.0, 69.6, 66.4, 66.2, 62.3, 61.4, 57.5, 47.1, 46.4, 43.1, 39.3, 37.2, 36.9, 36.9, 35.5, 31.2, 31.1, 28.2, 26.3, 22.6, 20.9, 20.9, 20.2, 18.3, 16.2, 16.2, 14.2, 9.9. HRMS (ESI) m / z calcd for C 67 H 84 NO 21 S2[M+H] + :1302.4972,found:1302.4981. Step 5: Introduce the targeting group through the alkyne group at the end of the ether bond at the 4″ position.

[0025] Compound A9 (200 mg) and compound A8 (228.2 mg, 175.2 μmol) were dissolved in 5.0 mL of THF. An aqueous solution of copper sulfate (14.0 mg, 87.6 μmol) and sodium ascorbate (17.4 mg, 87.6 μmol) was added to the solution in sequence. The reaction was monitored by TLC (DCM:MeOH = 15:1) until completion, concentrated under reduced pressure, and purified by reverse-phase column chromatography to obtain 49.8 mg of product A10 with a yield of 15.2%. 1 H NMR (600 MHz, CDCl3) δ 8.07 (d, J J = 7.4 Hz, 2H), 7.73 (s,1H), 7.59 (t, J J = 7.4 Hz, 1H), 7.49 (t, J J = 7.5 Hz, 2H), 7.34 (t, J J = 7.5 Hz,2H), 7.26–7.17 (m, 3H), 6.67 (s, 1H), 6.37 (s, 2H), 6.11 (s, 1H), 5.63 (s,1H), 5.49 (s, 1H), 5.41–5.10 (m, 3H), 4.93 (d, J J = 8.2 Hz, 1H), 4.61–4.43 (m,4H), 4.43–4.08 (m, 7H), 4.01–3.78 (m, 5H), 3.70 (s, 2H), 3.59 (s, 12H), 3.18(s, 3H), 3.00 (t, J J = 6.6 Hz, 2H), 2.98–2.80 (m, 4H), 2.57–2.39 (m, 4H), 2.32(s, 6H), 2.15–2.09 (m, 3H), 2.06 (s, 3H), 2.00 (s, 4H), 1.79 (s, 3H), 1.71(s, 4H), 1.48 (s, 7H), 1.33 (s, 10H), 1.26 (s, 3H), 1.17 (s, 3H), 1.10 (s,3H). 1313C NMR (151 MHz, CDCl3) δ 211.3, 172.8, 171.0, 170.8, 169.6, 168.2, 167.0, 158.8, 155.3, 154.4, 153.9, 145.5, 144.4, 139.1, 137.5, 135.6, 134.8, 133.6, 132.0, 130.2, 129.9, 129.4, 128.8, 128.6, 128.1, 126.5, 123.7, 123.4, 84.5, 81.0, 80.2, 78.8, 77.3, 77.1, 76.8, 76.5, 75.1, 74.4, 74.3, 71.7, 71.3, 70.6, 70.3, 70.2, 70.0, 69.4, 67.2, 66.1, 62.3, 57.7, 54.1, 53.4, 52.8, 50.3, 47.0, 46.4, 43.1, 39.4, 37.2, 36.9, 36.6, 35.3, 31.3, 31.2, 29.8, 29.7, 28.2, 26.7, 26.4, 22.6, 20.9, 16.1, 16.0, 14.1, 10.1. HRMS (ESI) m / z calcd for C 90 H 123 N7O 33 S2Na [M+Na] + :1916.7495,found:1916.7559. Example 3: Verification experiment on the action of the targeting group after the three-branched loading system is loaded with the triggering group, drug and targeting group Taking the three-branched loading system I in the present invention as an example, after it is loaded with the triggering group, drug and targeting group (Compound A10), it can bind to a specific disease target protein through the targeting group to achieve precise delivery of the molecule. Among them, in this example, human prostate cancer cell line 22Rv1 is taken as an example.

[0026] In prostate cancer, prostate-specific membrane antigen (PSMA) is highly expressed. The targeting group in this example (mainly the Lys-Urea-Glu structure at the end plays a role) can bind to PSMA highly.

[0027] Test the proliferation inhibitory activity of Compound A10 against 22Rv1 with high expression of PSMA. The method is as follows: Prepare a cell suspension and count using a cell counting plate. 8×10 3Cells were seeded in a 96-well plate at a density of (cells / well), with a volume of 100 μL of culture medium per well. The experiment set up a blank control group (containing only culture medium), a negative control group (cells + culture medium without drugs), and a drug treatment group (containing gradient concentrations of the drug to be tested), with 3 replicates in each group. To reduce errors caused by evaporation from the edge wells, 100 μL of PBS buffer was added to the peripheral wells, and then transferred to an incubator for 24 h.

[0028] After 24 h of cell adhesion, the old culture medium in each well was aspirated, and 100 μL of fresh culture medium containing different concentrations of A10 (5000 nM, 500 nM, 50 nM, 5 nM, 0.5 nM, 0.05 nM, 0.005 nM, 0.0005 nM) was added respectively, and incubated in an incubator for 72 h. The old culture medium was aspirated, 100 μL of culture medium solution containing 10% CCK-8 was added, incubated in the dark for 1 - 4 h, and the absorbance (OD value) of each well was measured at a wavelength of 450 nm using a microplate reader.

[0029] Cell viability (%) = (OD of control group - OD of blank group) / (OD of experimental group - OD of blank group) × 100%; A dose - effect curve was plotted using GraphPad Prism 9.0 software, and the half - inhibitory concentration (IC 50 ) was calculated by non - linear regression fitting.

[0030] As measured, the proliferation inhibitory activity IC 50 of compound A10 against 22Rv1 cells was 10.3 ± 0.6 nM, while the IC 50 of docetaxel (the drug prototype loaded in this example) under the same conditions was 21.8 ± 0.8 nM. This indicates that after loading docetaxel into the multi - branched loading system of the present invention, its anti - tumor activity is significantly improved, suggesting that it may be caused by the targeting of the loading system.

[0031] To further verify its targeting, a PSMA high - affinity blocker 2 - PMPA can be added separately during the test. In this case, PSMA is preferentially blocked by 2 - PMPA. When the proliferation inhibitory activity of compound A10 against 22Rv1 cells was tested again, it was found that its IC 50 >5000 nM. This fully demonstrates that the anti - tumor activity of compound A10 is caused by its binding to PSMA.

[0032] This example proves that after loading the trigger group, drug, and targeting group into the three - branched loading system of the present invention, it indeed acts on the disease target through the targeting group.

[0033] Example 4: Drug release experiment triggered by the trigger group after loading the trigger group, drug, and targeting group into the three - branched loading system Glutathione (GSH) was prepared into PBS solutions (pH = 7.4) containing 20% acetonitrile at 10 mM and 0.1 mM. 2 mg of A10 was added. At 37 °C, reaction solutions at time points of 0 min, 20 min, 1 h, 2 h, 4 h, and 8 h were taken and monitored by HPLC. The mobile phase was acetonitrile:water = 55:45, the flow rate was 1.0 mL / min, the detection wavelength was 230 nm, the column temperature was 25 °C, and the injection volume was 10 μL. Thus, the release of docetaxel was monitored.

[0034] High concentrations of glutathione (GSH) in tumor cells can specifically reduce disulfide bonds (-S-S-), thereby releasing active drug molecules. To simulate the drug release behavior of macromolecular materials in vivo in vitro, we selected glutathione as the trigger molecule for disulfide bonds and monitored the change in docetaxel release over time under the action of GSH on macromolecular materials at 37 °C. Under the condition of 10 mM GSH, compound A10 could release more than 70% of docetaxel in 8 h, while under the condition of 0.1 mM GSH, the released docetaxel was less than 20%. This result indicates that compound A10 can rapidly release docetaxel after entering tumor cells, playing a role in killing tumor cells, while in an environment with low-concentration GSH, the drug release is slower and basically remains stable.

[0035] This example proves that after the three-branch loading system of the present invention is loaded with a trigger group, a drug, and a targeting group, under specific circumstances, the drug can be released through the trigger group.

[0036] Combining Example 3 and Example 4, it can be proved that after the three-branch loading system of the present invention is loaded with a trigger group, a drug, and a targeting group, it binds to a specific target through the targeting group, and then the drug is released through the trigger group in the microenvironment here, so as to achieve the purpose of targeted drug delivery and precise treatment.

[0037] The above-described examples are only descriptions of the preferred embodiments of the present invention and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. A three-branch load system, characterized in that Its structural formula is shown in Formula (I):

2. A synthesis method of the three-branch load system according to claim 1, characterized in that, The synthetic reaction route is as follows:

3. The synthesis method of the three-branch load system according to claim 2, wherein The synthetic method of the three-branch loading system includes the following steps: In the first step, 2,6-dimethyl-1,4-benzenediol reacts with methyl 3,3-dimethylacrylate under the catalysis of methanesulfonic acid to generate Compound III; In the second step, Compound III reacts with 3-alkyn-1-ol under the action of diethyl azodicarboxylate and triphenylphosphine to generate Compound II; In the third step, the lactone ring on Compound II undergoes a reduction ring-opening reaction under the action of a reducing agent to generate Compound I.

4. Use of a three-branch loading system according to claim 1 in the preparation of a targeted drug or a targeted probe.

5. Use of the three-branch load system according to claim 4 in the preparation of a targeted drug or a targeted probe, characterized in that, The targeted drug includes drugs for treating tumors, inflammation or neurodegenerative diseases; the targeted probe includes probes for diagnosing tumors, inflammation or neurodegenerative diseases.

6. Use of the three-branch load system according to claim 4 in the preparation of a targeted drug or a targeted probe, characterized in that, A trigger group, a drug or a probe, and a targeting group are connected to Compound I.

7. Use of the three-branch load system according to claim 6 in the preparation of a targeted drug or a targeted probe, characterized in that, Compound I has 3 group connection sites, including: the phenolic hydroxyl site at the 1-position, the alcoholic hydroxyl site at the 4'-position, and the alkynyl site at the end of the ether bond at the 4''-position; Among them, the phenolic hydroxyl site at the 1-position is used to connect the trigger group, the hydroxyl group at the alcoholic hydroxyl site at the 4'-position is oxidized to a carboxyl group and then used to connect the drug or the probe, and the alkynyl site at the end of the ether bond at the 4''-position is used to connect the targeting group.

8. Use of the three-branch load system according to claim 4 in the preparation of a targeted drug or a targeted probe, characterized in that, After the targeted drug or the targeted probe reaches the target position of the organism or cell, its action mode includes: The targeting group on Compound I binds to the target protein. Under the microenvironment near the target, the phenolic hydroxyl group at the 1-position is released through the trigger group, and then the intramolecular spontaneous cyclization of Compound I is triggered, releasing the drug or the probe for targeted treatment or targeted diagnosis of the disease.

9. A method for preparing a targeted drug or a targeted probe using the three-branch loading system according to claim 1: In the first step, a silyl ether protecting group of an alcoholic hydroxyl group is first introduced at the 4'-position of Compound I; In the second step, a trigger group is connected to the phenolic hydroxyl group at the 1-position of Compound I; In the third step, the silyl ether protecting group at the 4'-position of Compound I is removed; In the fourth step, the alcoholic hydroxyl group at the 4'-position of Compound I is oxidized to a carboxyl group, and a drug or a probe is connected to the carboxyl group at the 4'-position; In the fifth step, a targeting group is connected to the alkynyl group at the end of the ether bond at the 4''-position of Compound I.

10. The method for preparing a targeted drug or a targeted probe using the three-branch load system according to claim 9, wherein, The trigger group includes a group that can be cleaved under the in vivo microenvironment.

Images