A multifunctional fluorescent linker based on tumor hypoxic microenvironment response, preparation method and application thereof

Abstract

The application discloses a multifunctional fluorescent linker based on tumor hypoxic microenvironment response, a preparation method and application, and a reaction of cyclohexanone and POCl3 in a DMF solution obtains an intermediate with a dialdehyde group; the intermediate with the dialdehyde group is reacted with 4-(2-methyl-1l4-benzo[cd]indole-1-yl) butane-1-sulfonate to obtain the multifunctional linker based on tumor hypoxic microenvironment response. The probe molecule constructed by using the linker can improve the biological toxicity of a nano delivery drug system in the application aspect of the diagnosis and treatment integration concept, and simultaneously expand the application diversity of the diagnosis and treatment integration concept. The multifunctional fluorescent linker based on tumor hypoxic microenvironment response can be used for constructing a diagnosis and treatment integration drug and verifying the feasibility of the drug in realizing the diagnosis and treatment integration.

Description

Technical Field

[0001] This invention belongs to the field of drug preparation technology, and relates to a multifunctional fluorescent linker based on the response of tumor hypoxic microenvironment, its preparation method and application. Background Technology

[0002] Cancer is one of the most serious diseases threatening human health and life. Therefore, timely, efficient, and precise diagnosis and treatment of cancer is not only related to people's health and quality of life, but also to sustainable economic and social development.

[0003] Therapeutic integration is a novel biomedical technology that organically combines disease diagnosis or monitoring with treatment. Because it integrates diagnostic and therapeutic functions, it offers significant advantages over single diagnostic or treatment methods. Specifically, therapeutic integration in cancer treatment demonstrates great potential in patient stratification and personalized medicine, real-time monitoring of drug therapy processes, and feedback on drug treatment effects.

[0004] Fluorescent labeling technology is widely used in labeled tracking and imaging analysis of organisms due to its high sensitivity and low toxicity. However, traditional fluorescent labeling techniques often involve directly introducing fluorescent groups into target-recognition molecules for tracking and imaging analysis. This inevitably leads to poor targeting, the need for multiple washing processes, and the inability to achieve real-time in vivo tracking and imaging analysis. Therefore, developing a fluorescent tracking and labeling strategy based on fluorescence illumination provides an effective solution for achieving efficient and specific imaging and tracking analysis of organisms. Summary of the Invention

[0005] The purpose of this invention is to provide a multifunctional fluorescent linker based on tumor hypoxia microenvironment response, its preparation method, and its application. This multifunctional fluorescent linker based on tumor hypoxia microenvironment response can be used to chemically modify targeted anti-tumor drug molecules, and can also be used to verify the feasibility of using probes constructed based on tumor hypoxia microenvironment response to achieve "therapeutic integration".

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] A multifunctional fluorescent linker based on the response of tumor hypoxic microenvironment, the structural formula of which is as follows:

[0008]

[0009] A method for preparing a multifunctional fluorescent linker based on the response of tumor hypoxic microenvironment includes the following steps:

[0010] a) Cyclohexanone reacts with POCl3 in DMF solution to give an intermediate with a dialdehyde group;

[0011] b) An intermediate with a dialdehyde group reacts with 4-(2-methyl-114-benzo[cd]indol-1-yl)butane-1-sulfonate in the presence of acetic anhydride to yield sodium 4,4'-(((1E,1'E)-(2-chlorocyclohexene-1,3-diyl)bis(ethylene-2,1-diyl))bis(benzo[cd]indol-1-onthium-2,1-diyl))butane-1-sulfonate;

[0012] c) Sodium 4,4'-(((1E,1'E)-(2-chlorocyclohexene-1,3-diyl)bis(ethylene-2,1-diyl))bis(benzo[cd]indol-1-onthium-2,1-diyl))butane-1-sulfonate reacts with 3-nitrophenol under the action of K2CO3 to yield (E)-4-(2-(2-(6-amino-2,3-dihydro-1H-vinyl)benzo[cd]indol-1-onthium-1-yl)butane-1-sulfonate;

[0013] d)(E)-4-(2-(2-(6-amino-2,3-dihydro-1H-vinyl)benzo[cd]indol-1-onthiol-1-yl)butane-1-sulfonate and phenol were reacted with NaNO2 to obtain a multifunctional linker based on the response to tumor hypoxic microenvironment, with the following structural formula:

[0014]

[0015] Further, the specific process of step a) is as follows: N,N-dimethylformamide and dichloromethane are cooled at 0°C, then a dichloromethane solution of POCl3 is added dropwise, followed by the addition of cyclohexanone and reflux reaction to obtain an intermediate with a dialdehyde group.

[0016] Further, the specific process of step b) is as follows: 4-(2-methyl-1l4-benzo[cd]indol-1-yl)butane-1-sulfonate, the intermediate with a dialdehyde group and sodium acetate are dissolved in acetic anhydride, and then heated and stirred at 70°C for 30 min to obtain sodium 4,4'-(((1E,1'E)-(2-chlorocyclohexene-1,3-diyl)bis(ethylene-2,1-diyl))bis(benzo[cd]indol-1-onthium-2,1-diyl))butane-1-sulfonate.

[0017] Further, the specific process of step c) is as follows: 3-nitrophenol and K2CO3 are dissolved in CH3CN and stirred evenly under N2 atmosphere. Then, a CH3CN solution of sodium 4,4'-(((1E,1'E)-(2-chlorocyclohexene-1,3-diyl)bis(ethylene-2,1-diyl))bis(benzo[cd]indol-1-onthium-2,1-diyl))butane-1-sulfonate is added and stirred for 4 hours to obtain the reactant.

[0018] Under a nitrogen atmosphere, the reactants were added to a hydrochloric acid solution of SnCl2 and then reacted at 70 °C for 12 h to obtain (E)-4-(2-(2-(6-amino-2,3-dihydro-1H-vinyl)benzo[cd]indol-1-onth-1-yl)butane-1-sulfonate.

[0019] Further, the specific process of step d) is as follows: (E)-4-(2-(2-(6-amino-2,3-dihydro-1H-vinyl)benzo[cd]indol-1-onth-1-yl)butane-1-sulfonate is dissolved in concentrated hydrochloric acid solution, and NaNO2 is added at 0°C under N2 atmosphere. After stirring evenly, aminosulfonic acid is added, and after stirring evenly, CH3CN solution containing phenol is added. The reaction is carried out for 1.5 h to obtain a multifunctional fluorescent linker based on the response of tumor hypoxia microenvironment.

[0020] The application of a multifunctional fluorescent linker based on tumor hypoxia microenvironment response as described above in the preparation of therapeutic drugs for the treatment of tumors.

[0021] Furthermore, the antitumor drug, multifunctional fluorescent linker, DCC, and DMAP were dissolved in anhydrous CH2Cl2 and reacted under stirring to obtain a therapeutic drug for treating tumors.

[0022] Furthermore, the ratio of antitumor drug, multifunctional fluorescent linker, DCC, DMAP and CH2Cl2 is 0.22mmol: 0.22mmol: 0.33mmol: 0.07mmol: 20mL.

[0023] Carboxyl-terminated sorafenib (0.10 g, 0.22 mmol, preparation method see literature: Bioconjugchem.2022;33(5):918-928), linker (0.14 g, 0.22 mmol), DCC (0.07 g, 0.33 mmol), and DMAP (0.008 g, 0.07 mmol) were dissolved in anhydrous CH2Cl2 (20 mL). The reaction was stirred overnight at room temperature. The reaction was monitored by TLC until the reaction was complete. The solvent was removed by rotary evaporation, and an appropriate amount of H2O was added.

[0024] The anti-tumor drug is one of the drugs for cervical cancer, breast cancer, human glioma, and melanoma.

[0025] Furthermore, the application of therapeutic drug molecules for tumor treatment in in situ tumor imaging.

[0026] Application of a multifunctional fluorescent linker based on tumor hypoxic microenvironment response as described above in the construction of drug probes.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] This invention synthesizes a multifunctional fluorescent linker based on the response to tumor hypoxia microenvironment using cyclohexanone, 4-(2-methyl-114-benzo[cd]indol-1-yl)butane-1-sulfonate, 4-nitrophenol, and phenol. This linker molecule can chemically modify targeted antitumor drug molecules to construct a "therapeutic" drug. Once in vivo, under the catalysis of the tumor hypoxia microenvironment, fluorescence is activated for labeling and tracking, while the azo bonds release the targeted antitumor drug under hypoxic conditions, thus achieving the goal of therapeutic integration. The preparation method of this multifunctional fluorescent linker based on the response to tumor hypoxia microenvironment is simple, easy to implement, and has a wide range of applications.

[0029] The hypoxia-responsive multifunctional fluorescent linker of this invention can chemically covalently modify targeted antitumor drug molecules to construct "therapeutic" drugs. Then, through stimulation of the tumor microenvironment (liver cancer, lung cancer, breast cancer, cervical cancer, melanoma, glioma), it triggers fluorescence illumination for tumor site fluorescent labeling, simultaneously releasing targeted antitumor drug molecules, thereby achieving tumor labeling and treatment. The probe molecules constructed using this linker can improve the biotoxicity of nanodelivery drug systems in the application of the therapeutic concept, while expanding the application diversity of the therapeutic concept. The tumor hypoxia-responsive multifunctional fluorescent linker of this invention can be used to construct "therapeutic" drugs and verify the feasibility of such drugs in achieving integrated diagnosis and treatment. Attached Figure Description

[0030] Figure 1 A synthetic route diagram for a multifunctional fluorescent linker based on tumor hypoxic microenvironment response provided by the present invention;

[0031] Among them, compound 1 is cyclohexanone, compound 2 is benzo[cd]indol-2(1H)-one, compound 3 is 4-(2-methyl-1l4-benzo[cd]indol-1-yl)butane-1-sulfonate, compound 4 is 4,4'-(((1E,1'E)-(2-chlorocyclohexene-1,3-diyl)bis(ethylene-2,1-diyl))bis(benzo[cd]indol-1-onthium-2,1-diyl))butane-1-sulfonate sodium salt, compound 5 is 4-(2-oxo-1,2-dihydrobenzo[cd]indol-1-onthium-1-yl)butane-1-sulfonate-triisopropylethylamine salt, and compound 6 is 4-nitrobenzene. Reagents and conditions: (a) POCl3, DMF, CH2Cl2; (b) CH3COONa, (CH3CO)2O, 70℃, 1h; (c) 3-Nitrophenol, K2CO3, CH3CN; SnCl2·HCl, CH3OH; (d) NaNO2·HCl, Phenol, CH3CN, CH2Cl2.

[0032] Figure 2 Fluorescence spectra under different hypoxic conditions were constructed for different concentrations of Na2S2O4. The concentrations, from bottom to top along the arrows, are 0, 1.0 mmol / L, 1.5 mmol / L, 2.0 mmol / L, 2.5 mmol / L, 3.0 mmol / L, and 5.0 mmol / L.

[0033] Figure 3 The results of quantitative fluorescence under different hypoxic conditions constructed for different concentrations of Na2S2O4 are shown in the figure.

[0034] Figure 4 Linear fitting plots of fluorescence quantitative PCR results under different hypoxic conditions constructed for different concentrations of Na2S2O4.

[0035] Figure 5 This is a diagram illustrating the fluorescence illumination mechanism.

[0036] Figure 6 This is a cell imaging image.

[0037] Figure 7 This figure shows the effect of inhibiting cell proliferation. Detailed Implementation

[0038] The present invention will now be described in further detail with reference to the accompanying drawings and specific embodiments. These descriptions are intended to explain the invention and not to limit it.

[0039] In addition to exhibiting high reducing and low pH characteristics, the tumor microenvironment often displays severe hypoxia. Moreover, this hypoxic microenvironment tends to improve after tumor treatment. Considering the goal of real-time monitoring of the tumor treatment process, this invention designs a class of bifunctional molecules based on the specific response of the tumor hypoxic microenvironment. These molecules, while killing tumor cells, also achieve real-time in vivo treatment and monitoring of treatment effects based on the relationship between their regulatory effects and the tumor hypoxic microenvironment, as well as the relationship between the hypoxic microenvironment and changes in fluorescence intensity.

[0040] This invention constructs a therapeutic molecule based on small molecules, and uses it to achieve labeling and treatment functions, thereby expanding the means of realizing therapeutic integration in cancer treatment.

[0041] This invention synthesizes a multifunctional fluorescent linker molecule that responds to the hypoxic microenvironment of tumors by using cyclohexanone, 4-(2-methyl-114-benzo[cd]indol-1-yl)butane-1-sulfonate, 4-nitrophenol, and phenol. This linker can be used to chemically modify targeted antitumor drug molecules to construct "therapeutic" drugs.

[0042] The multifunctional fluorescent linker based on tumor hypoxic microenvironment response provided by this invention has the following structure:

[0043]

[0044] The multifunctional fluorescent linker based on tumor hypoxic microenvironment response described in this invention is named as follows:

[0045] 4-(2-((E)-2-(6-((E)-(4-hydroxyphenyl)azo)-2,3-dihydro-1H-oxanthracene-4-yl)vinyl)benzo[cd]indol-1-yl)butane-1-sulfonic acid.

[0046] The following is combined with Figure 1 The synthetic route and specific synthetic examples shown herein illustrate the method for preparing a multifunctional fluorescent linker based on tumor hypoxia microenvironment response for constructing "therapeutic" drugs, provided by the present invention.

[0047] See Figure 1 A synthetic route for a multifunctional fluorescent linker based on tumor hypoxia microenvironment response includes the following steps:

[0048] a) Cyclohexanone reacts with POCl3 in DMF solution to give an intermediate with a dialdehyde group;

[0049] The specific operation of step a) is as follows:

[0050] N,N-dimethylformamide was cooled with dichloromethane at 0°C, followed by the slow addition of a dichloromethane solution of POCl3, and then cyclohexanone was added. The reaction mixture was heated under reflux at 50°C for 3 hours. After cooling, 40 g of ice was added and the mixture was allowed to stand overnight. The intermediate containing a dialdehyde group was obtained by filtration.

[0051] b) An intermediate with a dialdehyde group reacts with 4-(2-methyl-114-benzo[cd]indol-1-yl)butane-1-sulfonate in the presence of acetic anhydride to yield sodium 4,4'-(((1E,1'E)-(2-chlorocyclohexene-1,3-diyl)bis(ethylene-2,1-diyl))bis(benzo[cd]indol-1-onthium-2,1-diyl))butane-1-sulfonate;

[0052] The specific operation of step b) is as follows:

[0053] Sodium 4,4'-(((1E,1'E)-(2-chlorocyclohexene-1,3-diyl)bis(ethylene-2,1-diyl))bis(benzo[cd]indol-1-onthium-2,1-diyl))butane-1-sulfonate, an intermediate with a dialdehyde group, and sodium acetate were dissolved in acetic anhydride. The reaction system was heated and stirred at 70°C for 30 min. The solvent was removed under reduced pressure, and the solution was washed with saturated NaHCO3 to obtain sodium 4,4'-(((1E,1'E)-(2-chlorocyclohexene-1,3-diyl)bis(ethylene-2,1-diyl))bis(benzo[cd]indol-1-onthium-2,1-diyl))butane-1-sulfonate.

[0054] c) Sodium 4,4'-(((1E,1'E)-(2-chlorocyclohexene-1,3-diyl)bis(ethylene-2,1-diyl))bis(benzo[cd]indol-1-onthium-2,1-diyl))butane-1-sulfonate reacts with 3-nitrophenol under the action of K2CO3 to yield (E)-4-(2-(2-(6-amino-2,3-dihydro-1H-vinyl)benzo[cd]indol-1-onthium-1-yl)butane-1-sulfonate;

[0055] The specific operation of step c) is as follows:

[0056] 3-Nitrophenol was dissolved in K₂CO₃ in CH₃CN, and stirred at room temperature for 10 min under N₂ atmosphere. Then, a CH₃CN solution of sodium 4,4'-(((1E,1'E)-(2-chlorocyclohexene-1,3-diyl)bis(ethylene-2,1-diyl))bis(benzo[cd]indole-1-onium-2,1-diyl))butane-1-sulfonate was added to the above reaction system using a syringe. The mixture was stirred at room temperature for 4 h, and the solvent was removed by rotary evaporation. The reaction system was then dissolved in CH₂Cl₂. Next, the mixture was washed with H₂O, and the CH₂Cl₂ phase was collected and evaporated to dryness using a rotary evaporator before being dissolved in methanol. SnCl2 was dissolved in concentrated hydrochloric acid. Under a nitrogen atmosphere, the solution was added and the mixture was reacted overnight at 70°C. After the reaction was complete, Na2CO3 was added to neutralize the reaction system. The precipitate was then removed by filtration, and the filtrate was collected. The filtrate was then washed with saturated NaCl, and the organic phase was collected. Finally, an appropriate amount of Na2SO4 was added and stirred at room temperature for 4 hours. The mixture was concentrated using a rotary evaporator, then stirred with 60-100 mesh silica gel, and purified by column chromatography to obtain (E)-4-(2-(2-(6-amino-2,3-dihydro-1H-vinyl)benzo[cd]indol-1-onthiol-1-yl)butane-1-sulfonate.

[0057] d)(E)-4-(2-(2-(6-amino-2,3-dihydro-1H-vinyl)benzo[cd]indol-1-onth-1-yl)butane-1-sulfonate and phenol were reacted with NaNO2 to obtain a multifunctional linker based on the response of tumor hypoxic microenvironment.

[0058] The specific operation of step d) is as follows:

[0059] (E)-4-(2-(2-(6-amino-2,3-dihydro-1H-vinyl)benzo[cd]indol-1-onthiol-1-yl)butane-1-sulfonate was dissolved in concentrated hydrochloric acid solution. The solution was placed under N2 atmosphere, and NaNO2 was added at 0°C with stirring for 30 min. Then, aminosulfonic acid was added and stirring continued for 10 min. Immediately afterwards, a CH3CN solution containing phenol was added, and the reaction was carried out for another 1.5 h at the same temperature. The reaction mixture was then diluted with water and extracted with CH2Cl2. The organic phase was collected and washed with saturated NaCl. Finally, an appropriate amount of Na2SO4 was added and the mixture was stirred at room temperature for 4 h. The solution was then concentrated using a rotary evaporator, mixed with 60-100 mesh silica gel, and purified by column chromatography to obtain the fluorescent linker based on the response to tumor hypoxia microenvironment.

[0060] The above-mentioned multifunctional fluorescent linkers based on tumor hypoxic microenvironment response are used in the construction of "therapeutic" drugs.

[0061] Example 1

[0062] The preparation process of this multifunctional fluorescent linker based on the response to tumor hypoxia microenvironment is as follows: Figure 1 As shown,

[0063] a) Cyclohexanone reacts with POCl3 in DMF solution to yield an intermediate containing a dialdehyde group, as detailed below:

[0064] N,N-dimethylformamide (4 mL) and dichloromethane (4 mL) were cooled at 0 °C, and then a dichloromethane solution (3 mL) of POCl3 (6.31 g, 0.04 mmol) was slowly added dropwise. Cyclohexanone (1.00 g, 0.01 mmol) was then added, and the reaction system was heated to reflux at 50 °C for 3 h. After the system cooled, 40 g of ice was added and the mixture was allowed to stand overnight. The intermediate containing a dialdehyde group was obtained by filtration.

[0065] b) The intermediate containing the dialdehyde group reacts with 4-(2-methyl-4-benzo[cd]indol-1-yl)butane-1-sulfonate in the presence of acetic anhydride to yield sodium 4,4'-(((1E,1'E)-(2-chlorocyclohexene-1,3-diyl)bis(ethylene-2,1-diyl))bis(benzo[cd]indol-1-onthium-2,1-diyl))butane-1-sulfonate, as detailed below:

[0066] 4-(2-methyl-1l4-benzo[cd]indol-1-yl)butane-1-sulfonate (5.27 g, 17.37 mmol), a dialdehyde intermediate (1.50 g, 8.69 mmol), and sodium acetate (1.43 g, 17.37 mmol) were dissolved in 20 mL of acetic anhydride. The reaction system was heated and stirred at 70 °C for 30 min. The solvent was removed under reduced pressure, and the solution was washed with saturated NaHCO3 to obtain sodium 4,4'-(((1E,1'E)-(2-chlorocyclohexene-1,3-diyl)bis(ethylene-2,1-diyl))bis(benzo[cd]indol-1-onthium-2,1-diyl))butane-1-sulfonate.

[0067] c) Sodium 4,4'-(((1E,1'E)-(2-chlorocyclohexene-1,3-diyl)bis(ethylene-2,1-diyl))bis(benzo[cd]indol-1-onthium-2,1-diyl))butane-1-sulfonate reacts with 3-nitrophenol under the action of K2CO3 to yield (E)-4-(2-(2-(6-amino-2,3-dihydro-1H-vinyl)benzo[cd]indol-1-onthium-1-yl)butane-1-sulfonate, the specific process of which is as follows:

[0068] 3-Nitrophenol (0.25 g, mmol) and K₂CO₃ (0.25 g, mmol) were dissolved in CH₃CN (10 mL). The mixture was stirred at room temperature for 10 min under N₂ atmosphere. Then, a CH₃CN (2 mL) solution of sodium 4,4'-(((1E,1'E)-(2-chlorocyclohexene-1,3-diyl)bis(ethylene-2,1-diyl))bis(benzo[cd]indol-1-onthium-2,1-diyl))butane-1-sulfonate (0.50 g, 0.67 mmol) was added to the above reaction system using a syringe. The mixture was stirred at room temperature for 4 h. The solvent was removed by rotary evaporation, and the reaction system was dissolved in CH₂Cl₂ (10 mL). Next, the mixture was washed with H₂O (30 mL × 3), and the CH₂Cl₂ phase was collected. The phase was then evaporated to dryness using a rotary evaporator and dissolved in methanol (10 mL). SnCl2 (2.56 g, 13.50 mmol) was dissolved in concentrated hydrochloric acid (4 mL). Under a nitrogen atmosphere, the solution was added, and the mixture was reacted overnight at 70 °C. After the reaction was complete, Na2CO3 was added to neutralize the reaction system. The precipitate was then removed by filtration, and the filtrate was collected. The filtrate was washed with saturated NaCl (20 mL × 3) and the organic phase was collected. Finally, an appropriate amount of Na2SO4 was added, and the mixture was stirred at room temperature for 4 h. The solution was then concentrated using a rotary evaporator, mixed with 60-100 mesh silica gel, and purified by column chromatography to obtain (E)-4-(2-(2-(6-amino-2,3-dihydro-1H-vinyl)benzo[cd]indol-1-onthiol-1-yl)butane-1-sulfonate.

[0069] d)(E)-4-(2-(2-(6-amino-2,3-dihydro-1H-vinyl)benzo[cd]indol-1-onthiol-1-yl)butane-1-sulfonate and phenol were reacted with NaNO2 to obtain a multifunctional linker based on the response of the tumor hypoxic microenvironment. The specific process is as follows:

[0070] (E)-4-(2-(2-(6-amino-2,3-dihydro-1H-vinyl)benzo[cd]indol-1-onthiol-1-yl)butane-1-sulfonate (0.10 g, 0.20 mmol) was dissolved in concentrated hydrochloric acid (0.05 mL, CH3CN:CH2Cl2 = 1:1, 10 mL). The solution was placed under a N2 atmosphere, and NaNO2 (0.01 g, 0.14 mmol) was added at 0 °C with stirring for 30 min. Then, aminosulfonic acid (0.04 g, 0.41 mmol) was added and stirring continued for 10 min. Immediately afterwards, a CH3CN solution (2 mL) containing phenol (0.11 g, 1.17 mmol) was added, and the reaction was allowed to proceed further at the same temperature for 1.5 h. The reaction was then purified using water. The sample was diluted and then extracted with CH2Cl2 (20 mL × 3). The collected organic phase was then washed with saturated NaCl (20 mL × 3) and collected. Finally, an appropriate amount of Na2SO4 was added and stirred at room temperature for 4 h. The mixture was then concentrated using a rotary evaporator, mixed with 60-100 mesh silica gel, and purified by column chromatography (dichloromethane:methanol = 10:1, v:v) to obtain a fluorescent linker based on the response of tumor hypoxia microenvironment. The yield was 0.06 g, with a yield of 48.58%, yielding a multifunctional fluorescent linker responsive to tumor hypoxia microenvironment.

[0071] LC-MS (ESI, m / z): 618.30 [M+H] + 616.80 [MH] -- .

[0072] Multifunctional fluorescent linkers containing responses to the tumor hypoxic microenvironment can be used in the preparation of therapeutic drugs.

[0073] The linker of this invention has a wide range of applications, representing a general-purpose, multifunctional fluorescent linker based on the response to tumor hypoxia microenvironment. It can be used to modify small bioactive molecules with antitumor activity, such as drugs for cervical cancer, breast cancer, human glioma, and melanoma. It can also be used to modify monoclonal antibody drug molecules with therapeutic effects, such as the anti-angiogenic monoclonal antibody bevacizumab and the PD1 / PDL1 monoclonal antibody atezolizumab. In this invention, only sorafenib was examined as an application example, demonstrating significant anti-angiogenic activity and a strong killing effect on gliomas.

[0074] This invention utilizes a multifunctional fluorescent linker that responds to the hypoxic microenvironment of tumors to chemically modify the targeted antitumor drug molecule sorafenib, constructing a sorafenib "therapeutic" drug molecule, and using it to perform tracking, localization, imaging analysis, and activity screening of some tumor cells.

[0075] Construction of the sorafenib "therapeutic" drug molecule: Carboxyl-terminated sorafenib (0.10 g, 0.22 mmol, preparation method see literature: Bioconjug chem.2022;33(5):918-928), linker (0.14 g, 0.22 mmol), DCC (0.07 g, 0.33 mmol) and DMAP (0.008 g, 0.07 mmol) were dissolved in anhydrous CH2Cl2 (20 mL). The reaction was stirred overnight at room temperature. The reaction was monitored by TLC until the reaction was complete. The solvent was removed by rotary evaporation. An appropriate amount of H2O was added. Then, dichloromethane (20 mL) was added for extraction. The organic phase was collected and then washed with saturated NaCl (20 mL × 3) and the organic phase was collected. Finally, add an appropriate amount of Na2SO4 and stir at room temperature for 4 hours. Concentrate using a rotary evaporator, then mix with 60-100 mesh silica gel, and purify by column chromatography (petroleum ether: ethyl acetate (v:v) = 1:1) to obtain the sorafenib bifunctional molecule.

[0076] Application of "therapeutic" targeted antitumor drug molecules constructed with multifunctional fluorescent linkers that respond to tumor hypoxia microenvironment in hypoxia response.

[0077] This invention utilizes the Na₂S₂O₄ oxygen-consuming method to simulate the hypoxic microenvironment of tumors. Na₂S₂O₄ is a reagent with strong reducing properties that can remove oxygen without damaging cell membranes. Furthermore, when administered in appropriate doses, it can remove oxygen from the culture medium within 2-10 minutes, creating hypoxia. Therefore, it was chosen to simulate the hypoxic conditions of the tumor microenvironment. Specifically, different concentrations of Na₂S₂O₄ were co-incubated with bifunctional molecules to regulate fluorescence.

[0078] The specific steps are as follows:

[0079] Construction of the Na2S2O4 hypoxia system: 0.0087 g of Na2S2O4 was accurately weighed and dissolved in PBS buffer (pH 7.4, 10 mL) to prepare a 5 mM Na2S2O4 solution. This solution was then diluted to prepare 3 mM, 2.5 mM, 2.0 mM, 1.5 mM, and 1.0 mM Na2S2O4 solutions. Simultaneously, PBS without added Na2S2O4 was used as a blank solution to simulate the normal environment.

[0080] Fluorescence illumination effect evaluation: 3 mL of hypoxic solution was transferred to each system, and 1 mL of sorafenib functional molecule was added to the system. The mixture was stirred at room temperature and then transferred to a 37°C constant temperature incubator for 30 min. After that, the fluorescence spectrum was scanned using a fluorescence spectrophotometer and the fluorescence intensity at the maximum fluorescence wavelength was determined to determine the dose-effect relationship between fluorescence intensity and hypoxic microenvironment.

[0081] The results are as follows Figures 2-5 As shown, the fluorescence intensity increases with increasing Na2S2O4 concentration (intensified hypoxia), exhibiting a certain concentration dependence. Next, since the functionalized sorafenib releases drug molecules after cleavage in a hypoxic microenvironment, the fluorescent fragment is a fluorophore with an amino terminus, which is also a key intermediate in the construction of the fluorescent linker in this invention. Therefore, this invention scanned the fluorescence spectrum of the system after hypoxia treatment and the fluorescence spectrum of the system after hypoxia regulation. The results show that the fluorescence spectrum of the system after hypoxia treatment is consistent with the fluorescence spectrum of the key intermediate, further indicating that the fluorescence illumination is achieved through the breaking of azo bonds.

[0082] In summary, the therapeutic molecule based on tumor hypoxia microenvironment response constructed in this invention can achieve good fluorescence illumination in the infrared region under hypoxic conditions, and the fluorescence intensity gradually increases with the intensification of hypoxia. Furthermore, the mechanism of its fluorescence illumination was verified to be the breaking of azo bonds, which not only elucidates the fluorescence illumination mechanism but also lays a theoretical foundation for selecting it as a controllable release group in this invention.

[0083] Application of "therapeutic" targeted antitumor drug molecules constructed with multifunctional fluorescent linkers that respond to the tumor hypoxic microenvironment in in situ tumor imaging.

[0084] The sorafenib "therapeutic" drug molecule designed and synthesized above was used to image EA.hy926 cells.

[0085] Fluorescence illumination effect evaluation: Fully adherent cells were replaced with a different culture medium and washed with sugar-free Earle's washing buffer. Then, sugar-free Earle's solutions containing different concentrations of Na₂S₂O₄ were added to induce cell hypoxia. Functionalized molecules were then added and incubated with the cells at 37°C for 2 hours at a probe concentration of 4 μM per well. Cells were washed three times with PBS to remove excess probe molecules. After washing, 1 mL of 3.7% paraformaldehyde (925 μL of 4% paraformaldehyde + 75 μL of PBS) was added to the cells at room temperature for 30 minutes. The cells were washed twice with PBS (with gentle stirring for 1-2 minutes) and permeabilized for 10 minutes at room temperature with PBS containing 0.1% Triton X-100. The cells were then washed twice with PBS, gently stirred for 1 minute, blocked for 30 minutes at room temperature with PBS containing 2% BSA (containing 0.05% Tween-20), and washed twice with PBS (containing 0.05% Tween-20). Each treatment lasted 5 minutes, with gentle shaking. Add 5 μL of anti-fluorescence quenching mounting medium to each group, and then seal the slides with nail polish. Finally, perform fluorescence microscopy for examination: cell imaging results are shown below. Figure 6 As shown, the probe concentration is 4 μM, from Figure 6 It can be seen that efficient fluorescence illumination can be achieved under hypoxic conditions for cell labeling, indicating that the constructed probe can achieve the labeling step in the "diagnosis and treatment integration".

[0086] Application of "therapeutic" targeted antitumor drug molecules constructed with multifunctional fluorescent linkers that respond to tumor hypoxia microenvironment in terms of antitumor activity.

[0087] The proliferation inhibition analysis of the linivanil "therapeutic" drug molecule designed and synthesized above was performed on EA.hy926 cells (human umbilical vein cells), HeLa (cervical cancer cells), MDA-MB-231 (breast cancer cells), U87 (human glioma cells), B16 (mouse melanoma cells), and A549 (human non-small cell lung cancer cells).

[0088] Activity was evaluated using the MTT assay.

[0089] 1) Cell seeding plate: 96-well plate seeding plate, with a cell density of 1*10 cells per well. 5 Add 180 μL of cell suspension to each well and incubate overnight at 37°C.

[0090] 2) Cell drug delivery: Six concentration gradients were set up, namely 20 μM, 4 μM, 0.8 μM, 0.16 μM, 0.032 μM and 0.0064 μM. 20 μL was added to each well and incubated at 37 °C for 48 h.

[0091] 3) Cells were given MTT: 22 μM was added to each well and incubated at 37°C for 4 h;

[0092] 4) Cell treatment: Aspirate the liquid from each well, add 150 μL of DMSO, and incubate on a shaker at room temperature for 10 min.

[0093] 6) Absorbance measurement: Place the 96-well plate on a microplate reader and measure its absorbance at 490 nm.

[0094] 7) Inhibition rate calculation: Inhibition rate = (OD of negative wells - OD of donor wells) / (OD of negative wells - OD of empty wells)

[0095] Cell proliferation inhibition results as follows Figure 7 As shown, the constructed probe molecule exhibits anti-cell proliferation activity similar to that of positive drug molecules, indicating that the constructed probe molecule can achieve the treatment step in the concept of "integrated diagnosis and treatment".

Claims

1. The application of a multifunctional fluorescent linker based on tumor hypoxia microenvironment response in the preparation of a therapeutic drug for treating tumors, characterized in that, The aforementioned therapeutic drug for treating tumors is prepared by dissolving carboxyl-terminated sorafenib, a multifunctional fluorescent linker, DCC, and DMAP in anhydrous CH2Cl2 and reacting them under stirring. The ratio of carboxyl-terminated sorafenib, multifunctional fluorescent linker, DCC, DMAP, and CH2Cl2 used was 0.22 mmol: 0.22 mmol: 0.33 mmol: 0.07 mmol: 20 mL. The structural formula of the multifunctional fluorescent linker is as follows: ； The structural formula of the carboxyl-terminated sorafenib is as follows: 。

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