A multifunctional prodrug delivery compound based on in vivo reactive oxygen species response and its preparation method and application

Abstract

The present invention discloses a multifunctional prodrug delivery compound based on in vivo reactive oxygen species response, a preparation method and an application, which are prepared by the following process: methylene blue is reacted in a mixed solution of CH2Cl2 and H2O containing Na2S2O4 and NaHCO3 to obtain reduced methylene blue; the reduced methylene blue reacts with triphosgene in a CH2Cl2 solution of triethylamine to obtain a multifunctional prodrug delivery compound based on in vivo reactive oxygen species response. The preparation method of the multifunctional prodrug delivery compound based on in vivo ROS response of the present invention is simple, easy to implement and has a wide range of applications. The delivery compound can be used to construct "integrated diagnosis and treatment" drugs, and the probe molecules constructed using the compound can improve the biological toxicity of the nano-delivery drug system in the application of the integrated diagnosis and treatment concept, while expanding the application diversity of the integrated diagnosis and treatment concept.

Description

Technical Field

[0001] The present invention belongs to the technical field of drug preparation, and relates to a multifunctional prodrug delivery compound based on in vivo active oxygen response, a preparation method and an application thereof. Background Art

[0002] According to the statistics of global cancer deaths in 2020, the number of cancer deaths in my country is as high as 3 million per year, which seriously affects the health and quality of life of the people. Among them, targeted therapy and photodynamic therapy have achieved certain therapeutic effects in cancer treatment. The combination of the two can improve each other's defects, achieve synergistic drug delivery, and produce more efficient therapeutic effects.

[0003] Photodynamic therapy (PDT) is an emerging treatment method with low toxicity, rapid results, and no drug resistance after repeated use. It has been approved for clinical use in the treatment of tumor diseases. It kills tumors by generating reactive oxygen species under laser irradiation by photosensitizers. However, the current clinical application of PDT still faces certain challenges, the main problem being the lack of sufficient targeting. In addition, although targeted therapy has certain therapeutic efficacy. However, it is easy to develop drug resistance after long-term use, which is caused by the compensatory activation of target proteins. Therefore, in order to exert more efficient anti-tumor therapeutic efficacy, targeted therapy and photodynamic therapy are combined to give play to their respective advantages, thereby producing efficient therapeutic efficacy. In addition, the effectiveness of PDT in generating reactive oxygen species is affected by the tumor microenvironment and the body's own specificity. Therefore, real-time monitoring of PDT efficacy is very necessary to achieve efficient anti-tumor effects. Summary of the invention

[0004] The purpose of the present invention is to provide a multifunctional prodrug delivery compound based on in vivo active oxygen response, a preparation method and an application thereof. The preparation method of the multifunctional prodrug delivery compound is simple, easy to implement and has a high yield, and can be used in the preparation of anti-tumor drugs.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions:

[0006] A multifunctional prodrug delivery compound based on in vivo reactive oxygen species response, the structural formula of the multifunctional prodrug delivery compound is as follows:

[0007]

[0008] A method for preparing a multifunctional prodrug delivery compound based on in vivo reactive oxygen species response comprises the following steps:

[0009] a) reducing methylene blue in a mixed solution of CH2Cl2 and H2O containing Na2S2O4 and NaHCO3 to obtain reduced methylene blue;

[0010] b) The reduced methylene blue reacts with triphosgene in a triethylamine CH2Cl2 solution to obtain a multifunctional prodrug delivery compound based on in vivo reactive oxygen species response, the structural formula of which is as follows:

[0011]

[0012] Furthermore, the specific process of step a) is: dissolving methylene blue in a mixed solution of CH2Cl2 and H2O, then adding a mixed powder of Na2S2O4 and NaHCO3, stirring to change the color from blue to khaki, separating, and obtaining reduced methylene blue.

[0013] Furthermore, the volume ratio of CH2Cl2 and H2O is 1:1; the molar ratio of Na2S2O4 and NaHCO3 is 1:1, the amount ratio of methylene blue to CH2Cl2 is 1.56mmol:10mL, and the amount ratio of Na2S2O4 to CH2Cl2 is 3.13mmol:10mL.

[0014] Furthermore, the specific process of step b) is: adding the reduced methylene blue to a triethylamine solution, cooling to 0°C, adding a CH2Cl2 solution of triphosgene, and stirring to obtain a multifunctional prodrug delivery compound based on in vivo active oxygen response.

[0015] Furthermore, the ratio of reduced methylene blue, triethylamine and triphosgene was 0.94 mmol:1.31 mmol:2.82 mmol, and the ratio of reduced methylene blue to CH2Cl2 was 0.94 mmol:10 mL.

[0016] A use of a multifunctional prodrug delivery compound based on in vivo reactive oxygen species response as described above in the preparation of an integrated diagnosis and treatment drug for treating tumors.

[0017] Furthermore, multifunctional prodrug delivery compounds based on in vivo reactive oxygen species response are used to modify active biological small molecules or monoclonal antibody drug molecules.

[0018] Furthermore, the active biological small molecule is sorafenib or 1-(4-amino-3-(trifluoromethyl)phenyl)-3-(4-(4-oxo-3,4,4a,8a-tetrahydroquinazolin-7-yl)phenyl)urea, and the monoclonal antibody drug molecule is atezolizumab or bevacizumab.

[0019] A multifunctional prodrug delivery compound based on in vivo reactive oxygen species response as described above is used in constructing drug probes and tracing.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] The present invention synthesizes a multifunctional prodrug delivery compound based on in vivo ROS response through methylene blue and triphosgene. The molecule can chemically modify the targeted anti-tumor drug molecule to construct an "integrated diagnosis and treatment" drug. When it enters the body, under the catalysis of reactive oxygen, the fluorescence turns on for labeling and tracing. At the same time, the urea group releases the targeted anti-tumor drug and the photodynamic group (methylene blue) under the action of reactive oxygen. While achieving efficient tumor labeling, it combines targeted therapy and photodynamic therapy, thereby achieving the purpose of integrated diagnosis and treatment. The preparation method of the multifunctional prodrug delivery compound based on in vivo ROS response of the present invention is simple, easy to implement, and has a wide range of applications.

[0022] The multifunctional prodrug delivery compound based on in vivo ROS response in the present invention can chemically covalently modify the targeted anti-tumor drug molecules to construct "integrated diagnosis and treatment" drugs, and then through the stimulation of the tumor (liver cancer, lung cancer, breast cancer, cervical cancer, melanoma, glioma) microenvironment, trigger fluorescence lighting to fluorescently mark the tumor site, and release targeted anti-tumor drug molecules at the same time, thereby achieving tumor labeling and tracing and treatment. The probe molecule constructed using this compound can improve the biological toxicity of the nano-delivery drug system in the application of the concept of integrated diagnosis and treatment, and at the same time expand the application diversity of the concept of integrated diagnosis and treatment. The multifunctional prodrug delivery compound based on in vivo ROS response of the present invention can be used to construct "integrated diagnosis and treatment" drugs and verify the feasibility of the drug in realizing integrated diagnosis and treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 A synthetic route for a multifunctional prodrug delivery compound based on in vivo reactive oxygen species response provided by the present invention;

[0024] Figure 2 A synthetic route for a QDAU5 multifunctional prodrug delivery compound based on in vivo reactive oxygen species response provided by the present invention;

[0025] Figure 3 The fluorescence spectra of a QDAU5 multifunctional prodrug delivery compound based on in vivo reactive oxygen species response provided by the present invention after being treated with H2O2 at different concentrations;

[0026] Figure 4 The fluorescence spectra of a QDAU5 multifunctional prodrug delivery compound based on in vivo reactive oxygen species response provided by the present invention after being treated with HClO at different concentrations;

[0027] Figure 5 A cell imaging labeling diagram of different types of cells by a QDAU5 multifunctional prodrug delivery compound based on in vivo reactive oxygen species response provided by the present invention;

[0028] Figure 6 A quantitative graph showing the inhibitory effect of a QDAU5 multifunctional prodrug delivery compound based on in vivo reactive oxygen species response on cell proliferation of different types of tumor cells provided by the present invention;

[0029] Figure 7 A quantitative graph showing the cell proliferation inhibition effect of a QDAU5 multifunctional prodrug delivery compound based on in vivo reactive oxygen species response provided by the present invention on melanoma cells under photodynamic combined therapy;

[0030] Figure 8 This is a cell imaging diagram of the ROS production at the cellular level under the photodynamic therapy of melanoma cells by a QDAU5 multifunctional prodrug delivery compound based on in vivo reactive oxygen species response provided by the present invention. Among them, (a) is 0μM, (b) is 0.1μM, (c) is 1μM, (d) is 2μM, and (e) is 4μM.

[0031] Wherein, compound 1 is methylene blue, and compound 2 is bis(trichloromethyl) carbonate. Reagents and conditions: Na2S2O4, NaHCO3, TEA, 0℃ to rt, 30min. DETAILED DESCRIPTION

[0032] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments, which are intended to explain the present invention rather than to limit it.

[0033] The present invention constructs an integrated diagnosis and treatment molecule based on small molecules, and utilizes it to achieve labeling and treatment functions, so as to expand the means of realizing integrated diagnosis and treatment in cancer treatment.

[0034] The present invention uses methylene blue and bis(trichloromethyl) carbonate (triphosgene) to synthesize a multifunctional prodrug delivery compound based on in vivo active oxygen response. The compound can be used to chemically modify targeted anti-tumor drug molecules to construct "integrated diagnosis and treatment" drugs.

[0035] The structure of the multifunctional prodrug delivery compound based on in vivo reactive oxygen species response provided by the present invention is as follows:

[0036]

[0037] The multifunctional prodrug delivery compound based on in vivo active oxygen response of the present invention is named: 3,7-bis(dimethylamino)-10H-phenothiazine-10-carbonyl chloride.

[0038] In the present invention, overnight means reacting for 12 hours.

[0039] Combine the following Figure 1The synthetic route and specific synthetic examples shown in the are used to explain in detail the preparation method of the multifunctional prodrug delivery compound with in vivo active oxygen response for constructing "integrated diagnosis and treatment" drugs provided by the present invention.

[0040] See also Figure 1 A method for preparing a multifunctional prodrug delivery compound responsive to active oxygen in vivo, comprising the following steps

[0041] a) Methylene blue (MB) is reduced in a mixed solution of CH2Cl2 and H2O containing Na2S2O4 and NaHCO3 to obtain reduced methylene blue (R-MB);

[0042] The specific operation of step a) is:

[0043] Dissolve methylene blue in a mixed solution of CH2Cl2 and H2O, then add a mixed powder of Na2S2O4 and NaHCO3, stir it for 20 minutes into the aqueous phase to change its color from blue to khaki, separate the organic phase and the aqueous phase, extract with CH2Cl2, combine the organic phases, and dry the organic phase with anhydrous Na2SO4 to obtain the reduced methylene blue.

[0044] b) reacting the reduced methylene blue with triphosgene (BTC) in a triethylamine CH2Cl2 solution to obtain a multifunctional prodrug delivery compound based on in vivo reactive oxygen species response;

[0045] The specific operation of step b) is:

[0046] The reduced methylene blue was added to the triethylamine solution, the reaction system was cooled to 0°C, and then a CH2Cl2 solution of triphosgene was added. The reaction system was stirred at room temperature for 0.5 h to obtain a multifunctional prodrug delivery compound based on the in vivo active oxygen response.

[0047] The above-mentioned multifunctional prodrug delivery compounds containing in vivo reactive oxygen species response are used in the construction of "integrated diagnosis and treatment" drugs.

[0048] Example 1

[0049] The preparation process of the multifunctional prodrug delivery compound containing in vivo reactive oxygen species response is as follows Figure 1 As shown,

[0050] a) Methylene blue (MB) is reduced in a mixed solution of CH2Cl2 and H2O containing Na2S2O4 and NaHCO3 to obtain reduced methylene blue (R-MB):

[0051] Dissolve methylene blue (0.50 g, 1.56 mmol) in a mixed solution of CH2Cl2 (10 mL) and H2O (10 mL), then add a mixed powder of Na2S2O4 (0.54 g, 3.13 mmol) and NaHCO3 (0.26 g, 3.13 mmol), stir it for 20 min into the aqueous phase to change its color from blue to khaki, separate the organic phase and the aqueous phase, extract with CH2Cl2 (30 mL × 3), combine the organic phases, dry the organic phase with anhydrous Na2SO4, and you can get the reduced methylene blue.

[0052] b) The reduced methylene blue reacts with triphosgene (BTC) in a triethylamine CH2Cl2 solution to obtain a multifunctional prodrug delivery compound based on in vivo reactive oxygen species response, and the specific process is as follows:

[0053] The reduced methylene blue (0.3 g, 0.94 mmol) was added to a triethylamine solution (0.08 g, 1.31 mmol), the reaction system was cooled to 0°C, and then a CH2Cl2 (10 mL) solution of triphosgene (0.84 g, 2.82 mmol) was added, and the reaction system was stirred at room temperature for 0.5 h to obtain a multifunctional prodrug delivery compound based on in vivo active oxygen response. The weight was 0.29 g, and the yield was 88.69%.

[0054] LC-MS (ESI, m / z): 348.26 [M+H] + , 346.29[MH] -- .

[0055] Multifunctional prodrug delivery compounds containing in vivo reactive oxygen species responses can be used in the integration of diagnosis and therapy.

[0056] The present invention utilizes a multifunctional prodrug delivery compound that responds to active oxygen in vivo to chemically modify the targeted anti-tumor drug molecule QDAU5 (the full name of QDAU5 is 1-(4-amino-3-(trifluoromethyl)phenyl)-3-(4-(4-oxo-3,4,4a,8a-tetrahydroquinazolin-7-yl)phenyl)urea), constructs the QDAU5 "integrated diagnosis and treatment" drug molecule, and uses it to perform tracing positioning imaging analysis and activity screening of some tumor cells.

[0057] Construction of QDAU5 "integrated diagnosis and treatment" drug molecule (see Figure 2): QDAU5 (0.05 g, 0.11 mmol) at the amino terminal and PyBop (1H-benzotriazol-1-yloxytripyrrolidino hexafluorophosphate, 0.12 g, 0.23 mmol) were dissolved in anhydrous CH2Cl2 (20 mL), triethylamine (0.046 g, 0.46 mmol) was slowly added dropwise at 0°C and stirred at room temperature for 3 min. Malonic acid (0.04 g, 0.34 mmol) was added and reacted at room temperature overnight. The reaction was monitored by TLC until the reaction was completed. The solvent was removed by rotary evaporator, an appropriate amount of H2O was added, and then dichloromethane (20 mL) was added for extraction. The organic phase was collected and then washed with saturated NaCl (20 mL×3). The organic phase was collected. Finally, an appropriate amount of Na2SO4 was added and stirred at room temperature for 4 h, and the mixture was concentrated using a rotary evaporator, and then mixed with 60-100 mesh silica gel, and purified by a chromatographic column (petroleum ether: ethyl acetate (v:v) = 1:1) to obtain the carboxyl-terminal QDAU5 molecule;

[0058] Next, the carboxyl-terminal QDAU5 molecule (0.04 g, 0.07 mmol), EDC·HCl (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 0.01 g, 0.05 mmol), and HOBt (1-hydroxybenzotriazole, 0.008 g, 0.05 mmol) were dissolved in anhydrous CH2Cl2 (20 mL), and triethylamine (0.015 g, 0.15 mmol) was slowly added dropwise at 0°C. After stirring at room temperature for 30 min, tert-butyl (2-(hydroxymethyl)phenyl)carbamate was added and reacted at room temperature overnight. The reaction was monitored by TLC until the reaction was completed. The solvent was removed by rotary evaporator, an appropriate amount of H2O was added, and then dichloromethane (20 mL) was added for extraction. The organic phase was collected and then washed with saturated NaCl (20 mL×3). The organic phase was collected. Finally, an appropriate amount of Na2SO4 was added and stirred at room temperature for 4 h, and the mixture was concentrated using a rotary evaporator, and then mixed with 60-100 mesh silica gel, and purified by a chromatographic column (petroleum ether: ethyl acetate (v:v) = 10:1) to obtain a QDAU5 molecule containing a Boc terminal;

[0059] The QDAU5 molecule containing the Boc terminal (0.01 g, 0.02 mmol) was dissolved in 10 mL of anhydrous CH2Cl2, and 3 mL of trifluoroacetic acid was slowly added at 0°C. After stirring for 1 h, it was stirred at room temperature overnight. TLC was used to monitor the reaction until the reaction was completed, and the solvent was removed by rotary evaporator. An appropriate amount of H2O was added, followed by dichloromethane (20 mL) for extraction, and the organic phase was collected. Then, it was washed with saturated NaCl (20 mL×3) and the organic phase was collected. Finally, an appropriate amount of Na2SO4 was added and stirred at room temperature for 4 h, and concentrated by rotary evaporator. Then, the sample was mixed with 60-100 mesh silica gel, and purified by chromatographic column (petroleum ether: ethyl acetate (v:v) = 10:1) to obtain the QDAU5 molecule with the terminal Boc removed;

[0060] Finally, QDAU5 (0.01 g, 0.02 mmol) with terminal Boc removed and triethylamine (0.005 g, 0.04 mmol) were added to the prodrug delivery system based on in vivo ROS response (0.01 g, 0.04 mmol). After stirring at room temperature overnight, CH2Cl2 (20 mL) was added to the above reaction system, and then an appropriate amount of H2O was added, and the organic phase was collected, and then washed with saturated NaCl (20 mL × 3) and the organic phase was collected. Finally, an appropriate amount of Na2SO4 was added and stirred at room temperature for 4 h, concentrated by a rotary evaporator, and then mixed with 60-100 mesh silica gel, and purified by a chromatographic column (petroleum ether: ethyl acetate (v: v) = 1: 1) to obtain the QDAU5 "diagnosis and treatment integration" drug molecule based on in vivo reactive oxygen response.

[0061] Application of "integrated diagnosis and treatment" drug molecules constructed with multifunctional prodrug delivery compounds based on in vivo reactive oxygen species response in visualizing reactive oxygen species content.

[0062] Here, different concentrations of reactive oxygen molecules H2O2 and HClO were selected to determine the content of reactive oxygen by fluorescence quantitative visualization.

[0063] The specific practical steps are as follows:

[0064] Accompanied by different concentrations of active oxygen molecular systems: H2O2 and HClO were used as active oxygen reagents for investigation, and their concentrations were diluted with pure water to construct systems containing active oxygen with different concentrations. The concentrations were: 0.1μΜ, 0.5μΜ, 1.25μΜ, 2.5μΜ, 5.0μΜ, 10μΜ, and 20μΜ.

[0065] Investigation of fluorescence lighting effect: 3 mL of the test solution was transferred respectively, 1 mL of QDAU5 functional molecule was added to the system, the system was mixed at room temperature, and then moved to a 37°C constant temperature incubator for incubation 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 the fluorescence intensity and the reactive oxygen content.

[0066] The results are as follows Figure 3 , Figure 4 As shown in the figure, with the increase of H2O2 and HClO concentrations, the fluorescence intensity becomes stronger and stronger, showing a certain concentration dependence. It is more sensitive to HClO than H2O2. In general, the integrated diagnosis and treatment molecule based on reactive oxygen response that we constructed can achieve better infrared fluorescence lighting under the condition of reactive oxygen, and the fluorescence intensity gradually increases with the increase of reactive oxygen content.

[0067] Application of "integrated diagnosis and treatment" targeted anti-tumor drug molecules constructed with multifunctional prodrug delivery compounds based on reactive oxygen species response in in situ tumor imaging.

[0068] The QDAU5 "integrated diagnosis and treatment" drug molecule designed and synthesized above was used to image EA.hy926 cells (tumor cells) and normal liver cells (L02).

[0069] Fluorescence lighting effect investigation: Replace the culture medium of the completely adherent cells, add functionalized molecules and incubate the cells, the probe concentration in each well is 4μM, incubate at 37℃ for 2h, wash the cells three times with PBS to remove excess probe molecules. After washing, add 1mL 3.7% paraformaldehyde (925μL 4% paraformaldehyde + 75μL PBS) to the cells at room temperature, fix for 30min, wash twice with PBS (gently stir for 1-2 minutes), and permeabilize with PBS solution containing 0.1% Triton X-100 for 10min at room temperature. Then wash the cells twice with PBS, stir slowly for 1min, block with PBS containing 2% BSA (containing 0.05% Tween-20) for 30min at room temperature, and wash twice with PBS (containing 0.05% Tween-20). Shake gently for 5min each time. Add 5μL of anti-fluorescence quenching sealing agent to each group, and seal with nail polish around. Finally, perform fluorescence microscopy for detection: the cell imaging results are as follows Figure 5 As shown, the probe concentration was 4 μM. Figure 5 It can be seen that the functional molecules can achieve selective and efficient labeling of tumor cells, indicating that the constructed probe can realize the labeling, that is, diagnosis step in "integrated diagnosis and treatment".

[0070] The application of "integrated diagnosis and treatment" targeted anti-tumor drug molecules constructed with multifunctional prodrug delivery compounds based on reactive oxygen species response in anti-tumor activity.

[0071] Since this part plays a role in photodynamic therapy and targeted therapy, it is necessary to investigate and analyze both the photodynamic activity and the targeted therapy effect.

[0072] The above-designed and synthesized Linifanib "integrated diagnosis and treatment" drug molecule was used to conduct proliferation inhibition analysis and evaluation on HT-29 cells (human colon cancer cells), HepG2 (human liver cancer cells), U87 (human glioma cells), and B16-F10 (mouse melanoma cells).

[0073] MTT was used to evaluate its activity:

[0074] 1) Cell seeding plate: 96-well plate, the cell density per well is 1×10 5 cells / mL, 180 μL of cell suspension was added to each well and incubated at 37°C overnight.

[0075] 2) Cell administration: set 6 concentration gradients, namely 20μM, 4μM, 0.8μM, 0.16μM, 0.032μM, 0.0064μM, add 20μL to each well, and incubate at 37℃ for 48h;

[0076] 3) MTT for cells: add 22 μL to each well and incubate at 37°C for 4 h;

[0077] 4) Cell treatment: Aspirate the liquid from each well, add 150 μL DMSO, and incubate on a shaker at room temperature for 10 min.

[0078] 6) Absorbance measurement: Place the 96-well plate on an ELISA reader to measure its absorbance at 490 nm.

[0079] 7) Calculation of inhibition rate: Inhibition rate = (negative well OD - feeding well OD) / (negative well OD - empty well OD)

[0080] The difference between the cell proliferation inhibition experiment under photodynamic therapy and the above operation is that after 12 hours of drug treatment, the cells are irradiated with laser (650nm) for 10 minutes, and then moved to a cell culture incubator for further incubation for 36 hours. The remaining steps are the same as the above steps and will not be repeated.

[0081] Cell proliferation inhibition results Figure 6 As shown in Figure 2, the constructed probe molecule exhibited anti-cell proliferation activity similar to that of the positive drug molecule, and showed a superior therapeutic effect on B16-F10 cells. Figure 7As shown: After laser irradiation, the probe molecule exhibited better anti-cell proliferation inhibition activity. Next, in terms of photodynamic properties, the generation of reactive oxygen species in vivo and in vitro was investigated and analyzed.

[0082] The ROS detection kit was used to investigate the generation of ROS in cells:

[0083] Replace the culture medium of the fully adherent cells, add the functionalized molecules and incubate the cells. The probe concentrations in each well are 4 μM, 2 μM, 1 μM, and 0.1 μM. Incubate at 37°C for 4 h. Dilute DCFH-DA with serum-free culture medium at a ratio of 1:1000 to a final concentration of 10 μmol·L -1 , remove the cell culture medium, add an appropriate volume of diluted DCFH-DA solution, incubate at 37°C for 20 minutes, and then wash the cells three times with serum-free cell culture medium to fully remove the DCFH-DA that has not entered the cells. Finally, fluorescence microscopy was used for detection: The cell imaging results are shown in Figure 8 As shown in (a), (b), (c), (d) and (e), with the increase of concentration, the amount of ROS produced at the cellular level gradually increased, which has a strong anti-tumor effect.

[0084] The above results indicate that the constructed probe molecules can realize the treatment step in the concept of "integrated diagnosis and treatment".

[0085] The multifunctional prodrug delivery compound of the present invention based on the tumor active oxygen response of the methylene blue photosensitizer is linked with the targeted drug molecule to construct a dual-function drug delivery system with both "diagnosis and treatment integration". After entering the tumor targeting site, it generates a small amount of active oxygen through laser irradiation, releases the photodynamic group and the targeting group, thereby lighting up the fluorescence characteristics of the photodynamic group. While achieving efficient and specific marking of the tumor, the photodynamic therapeutic activity of the photodynamic group and the targeted anti-tumor activity of the targeting molecule are exerted, thereby producing a more efficient anti-tumor effect.

[0086] The multifunctional prodrug delivery compound based on the in vivo ROS response of the present invention has a wide range of applications and is a general type. It can be used to modify active biological small molecules with anti-tumor activity, such as sorafenib, QDAU5, etc., and can also be used to modify monoclonal antibody drug molecules with therapeutic effects, such as anti-angiogenic monoclonal antibody drugs bevacizumab, PD1 / PDL1 monoclonal antibody drugs atezolizumab, etc. The multifunctional prodrug delivery compound based on the in vivo ROS response of the present invention is used to chemically modify targeted anti-tumor drug molecules, and can be used to verify the feasibility of realizing efficient "integration of diagnosis and treatment" by constructing a probe based on the in vivo ROS response multifunctional prodrug delivery compound. Fluorescence is activated by the generation of reactive oxygen species, and the strength of the fluorescence signal is used to reflect the efficacy of photodynamic therapy in real time. The combination of the two is expected to achieve real-time monitoring of the efficacy of photodynamic therapy while combining targeted therapy to exert efficient anti-tumor efficacy.

[0087] In the present invention, only QDAU5 was examined in the application example verification, which has a significant anti-angiogenic effect and shows a strong killing effect on melanoma.

Claims

1. A multifunctional prodrug delivery compound based on in vivo reactive oxygen species response for the preparation of an integrated diagnosis and treatment drug for treating tumors, wherein: The structural formula of the multifunctional prodrug delivery compound based on in vivo reactive oxygen species response is: The construction process of the integrated diagnosis and treatment drug is as follows: dissolve the amino-terminal 1-(4-amino-3-(trifluoromethyl)phenyl)-3-(4-(4-oxo-3,4,4a,8a-tetrahydroquinazolin-7-yl)phenyl)urea and 1H-benzotriazol-1-yloxytripyrrolidino hexafluorophosphate in anhydrous CH2Cl2, slowly add triethylamine at 0°C and stir at room temperature for 3 minutes, then add malonic acid and react at room temperature overnight, monitor the reaction by TLC until the reaction is completed, and obtain the carboxyl-terminal QDAU5 molecule. The full name of QDAU5 is 1-(4-amino-3-(trifluoromethyl)phenyl)-3-(4-(4-oxo-3,4,4a,8a-tetrahydroquinazolin-7-yl)phenyl)urea; The carboxyl-terminated QDAU5 molecule, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and HOBt were dissolved in anhydrous CH2Cl2, triethylamine was slowly added dropwise at 0°C and stirred at room temperature for 30 min, and then tert-butyl (2-(hydroxymethyl)phenyl)carbamate was added. The reaction was allowed to proceed at room temperature overnight and monitored by TLC until the reaction was completed to obtain a QDAU5 molecule containing a Boc terminus. The QDAU5 molecule containing the Boc terminal was dissolved in 10 mL of anhydrous CH2Cl2, and 3 mL of trifluoroacetic acid was slowly added dropwise at 0°C. After stirring for 1 h, it was stirred at room temperature overnight. The reaction was monitored by TLC until the reaction was completed to obtain the QDAU5 molecule with the terminal Boc removed. The QDAU5 molecule with the terminal Boc removed and triethylamine were added to the prodrug delivery system based on in vivo ROS response, and after stirring at room temperature overnight, an integrated diagnosis and treatment drug was obtained.

2. Use of the drug according to claim 1 in the preparation of an anti-tumor drug, wherein the anti-tumor drug is a drug for human colon cancer, human liver cancer, human glioma or mouse melanoma.

3. Use of the drug according to claim 1 in visualizing the content of active oxygen, in constructing drug probes and in tracing, which is not for the purpose of diagnosing diseases.

Images