Thrombolytic small molecule compound, nano particle, and preparation method and application of thrombolytic small molecule compound and nano particle

Abstract

The invention provides a thrombolytic small molecule compound, nanoparticles and a preparation method and application thereof, and belongs to the technical field of chemical synthesis and biomedicine. The molecular formula of the thrombolytic micromolecule is shown as a formula 1 or a formula 2. The invention provides a preparation method of a thrombolytic small molecule compound, and further provides nano particles formed by biologically encapsulating the small molecule compound into an amphiphilic polymer distearoyl phosphatidyl ethanolamine-polyethylene glycol. The nanoparticles have excellent bioluminescence performance, and can specifically recognize the position of a suppository block. The prepared nanoparticles have a photodynamic and photo-thermal combined thrombolysis effect.

Description

Technical Field

[0001] The present invention belongs to the field of chemical synthesis and biomedical technology, and specifically relates to thrombolytic small molecule compounds, nanoparticles, and preparation methods and applications thereof. Background Art

[0002] Thrombosis refers to a blood clot formed in a blood vessel due to a disorder in the hemostatic regulatory mechanism. It can obstruct blood flow and lead to serious or even fatal cardiovascular diseases such as myocardial infarction, ischemic stroke, and pulmonary embolism. Acute embolism can obstruct the blood supply to tissues and can be fatal in severe cases. The main treatment method is thrombolysis to restore blood supply to tissues. Thrombolytic drugs such as recombinant tissue plasminogen activator (rt-PA) have been widely used. However, its administration can lead to a severe decrease in coagulation parameters, resulting in a high risk of complications such as intracranial hemorrhage. As an alternative, the protein drug urokinase (Uk) can be used as a safer thrombolytic drug, but it has a short half-life, poor targeting, and requires repeated administration in a short period of time, which increases the risk of side effects. Therefore, the rational design of efficient and safe new strategies has always been a top priority for the treatment of thrombotic diseases.

[0003] Non-drug thrombolytic strategies such as photothermal therapy (PTT), photodynamic therapy (PDT), and mechanical thrombolysis have attracted increasing attention due to their good efficacy and relative non-invasiveness. PTT is a non-invasive thrombus treatment method that uses near-infrared (NIR) light to convert light energy into heat energy through the Landau damping effect, thus providing a safe and non-invasive thrombolytic method. 1O2 generated by photodynamic therapy can destroy the fibrin skeleton of the thrombus and prevent secondary embolism caused by photothermal therapy.

[0004] Sustained luminescence, including afterglow luminescence and chemiluminescence, is a unique luminescence process that can release photons continuously over time (i.e., afterglow luminescence) or trigger luminescence (i.e., chemiluminescence) by utilizing chemical reactions, such as chemical reactions with reactive oxygen and nitrogen species (RONS), after the excitation light stops. As a real-time, light-free optical imaging method, sustained luminescence imaging can avoid the interference of autofluorescence noise, has a higher signal-to-background ratio (SBR) and specificity than traditional fluorescence, and is more promising in in vivo biological applications. Porphyrin derivatives can be used to generate luminescent materials after the excitation light stops or reacts with peroxynitrite (ONOO - ) have an inherent persistent luminescence in the near-infrared region after interaction, which can report the location of thrombus in real time and be used for in vivo thrombus diagnosis. Summary of the invention

[0005] The present invention provides a thrombolytic small molecule compound, nanoparticles, and a preparation method and application thereof. The small molecule compound of the present invention has the property of chemiluminescence and simultaneously achieves the synergistic therapeutic effect of photothermal and photodynamic therapy.

[0006] The present invention first provides a thrombolytic small molecule compound, the structural formula of which is shown in Formula 1 or Formula 2:

[0007]

[0008] The present invention also provides a method for preparing a thrombolytic small molecule compound, comprising the following steps:

[0009] Step 1: dissolving 2-(2,6-dioxo-piperidin-3-yl)-4-fluoro-isoindole-1,3-dione or 2-(2,6-dioxo-piperidin-3-yl)-5-fluoro-isoindole-1,3-dione and 1-tert-butyloxycarbonylpiperazine in a solvent, adding N,N-diisopropylethylamine (DIEA) and reflux reaction to obtain 4-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)piperazine-1-carboxylic acid tert-butyl ester or 4-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)piperazine-1-carboxylic acid tert-butyl ester;

[0010] Step 2: reacting tert-butyl 4-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)piperazine-1-carboxylate or tert-butyl 4-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)piperazine-1-carboxylate in step 1 with trifluoroacetic acid in a solvent to obtain 2-(2,6-dioxopiperidin-3-yl)-4-(piperazine-1-yl)isoindole-1,3-dione or 2-(2,6-dioxopiperidin-3-yl)-5-(piperazine-1-yl)isoindole-1,3-dione;

[0011] Step 3: Mix and stir the 2-(2,6-dioxopiperidin-3-yl)-4-(piperazine-1-yl)isoindole-1,3-dione or 2-(2,6-dioxopiperidin-3-yl)-5-(piperazine-1-yl)isoindole-1,3-dione obtained in step 2 with pyropheophorbide a in a solvent, add N,N,N′,N′-tetramethyl-O-(7-azabenzotriazole-1-yl)urea hexafluorophosphate and N,N-diisopropylethylamine to react to obtain the small molecule compound ppa-o shown in formula 1 or the small molecule compound ppa-m shown in formula 2.

[0012] Preferably, the reaction temperature of step 1 is 80-90° C. and the reaction time is 4-5 h.

[0013] Preferably, the molar ratio of 2-(2,6-dioxo-piperidin-3-yl)-4-fluoro-isoindole-1,3-dione or 2-(2,6-dioxo-piperidin-3-yl)-5-fluoro-isoindole-1,3-dione:1-tert-butyloxycarbonylpiperazine:DIEA in step 1 is (1.2-1.5):1:6.

[0014] Preferably, the molar ratio of tert-butyl 4-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)piperazine-1-carboxylate or tert-butyl 4-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)piperazine-1-carboxylate to trifluoroacetic acid in step 2 is 1:45.

[0015] Preferably, the reaction temperature in step 2 is room temperature, and the reaction time is 0.5h-1h.

[0016] Preferably, in the step three, the molar ratio of 2-(2,6-dioxopiperidin-3-yl)-4-(piperazine-1-yl)isoindole-1,3-dione or 2-(2,6-dioxopiperidin-3-yl)-5-(piperazine-1-yl)isoindole-1,3-dione:pyropheophorbide a:N,N,N′,N′-tetramethyl-O-(7-azabenzotriazole-1-yl)urea hexafluorophosphate:N,N-diisopropylethylamine is (4-5):1:2.5:4.

[0017] Preferably, the reaction temperature in step 3 is room temperature and the stirring time is 4h-6h.

[0018] The present invention also provides a nanoparticle prepared from the thrombolytic small molecule compound shown in Formula 1 or Formula 2.

[0019] The present invention also provides the use of the nanoparticles in preparing medicines for treating thrombotic diseases.

[0020] Beneficial effects of the present invention

[0021] The present invention provides a thrombolytic small molecule compound PPA-O, PPA-M and its preparation method and application. The compound of the present invention can react chemically with excessive ROS at the thrombus inflammation site without the need for an external excitation light source to produce bioluminescence, and has a high penetration of 12 mm in the chicken breast coverage test, successfully avoiding the excitation wavelength band required by the compound itself and the problem of limited light penetration depth. And the test results show that the compound of the present invention has good photodynamic photothermal ability, which indicates that the material can have a good therapeutic effect on thrombolysis. The carefully designed nanoassembly has multiple advantages such as simple preparation, strong drug co-loading capacity, long body circulation, targeted accumulation of thrombus, photothermal enhancement of thrombus penetration and thrombolysis, and has a high therapeutic effect. As expected, the thrombolytic small molecule compound of the present invention not only shows bright chemiluminescent signals in embolic blood vessels, but also has a photothermal / photodynamic synergistic thrombolytic effect in vivo. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1The particle size potential diagram of ppa-o NPs and ppa-m NPs prepared in the present invention;

[0023] Figure 2 The ultraviolet absorption spectra and fluorescence emission spectra of ppa-o NPs and ppa-m NPs prepared in the present invention in aqueous solution;

[0024] Figure 3 The UV absorption spectra of ppa-o NPs, ppa-m NPs and DPBF prepared in the present invention in aqueous solution without illumination;

[0025] Figure 4 The UV absorption spectra of ppa-o NPs, ppa-m NPs and DPBF prepared in the present invention under single illumination in aqueous solution;

[0026] Figure 5 The UV absorption spectra of ppa-o NPs, ppa-m NPs and DPBF prepared in the present invention under illumination in aqueous solution;

[0027] Figure 6 This is a graph of ROS generation in aqueous solution of ppa-o NPs and ppa-m NPs prepared in the present invention;

[0028] Figure 7 The photothermal data of ppa-o NPs and ppa-m NPs prepared in the present invention in aqueous solution;

[0029] Figure 8 This is a thermal imaging data diagram of ppa-o NPs and ppa-m NPs prepared in the present invention in aqueous solution;

[0030] Fig. 9 The chemiluminescent properties of ppa-o NPs and ppa-m NPs prepared in the present invention are shown in FIG.

[0031] Fig.10 This is a test diagram of the antioxidant capacity of ppa-o NPs and ppa-m NPs prepared in the present invention;

[0032] Fig.11 This is a hemolysis test diagram of ppa-o NPs and ppa-m NPs prepared in the present invention;

[0033] Fig.12 The thrombolytic data of ppa-o NPs and ppa-m NPs prepared in the present invention under different conditions are shown;

[0034] Fig.13 This is an in vivo photothermal imaging test image of the ppa-m NPs prepared in Example 2 of the present invention;

[0035] Fig.14 This is a diagram showing the in vivo thrombolytic effect of ppa-m NPs prepared in Example 2 of the present invention;

[0036] Fig.15 This is a graph showing the in vivo hemodynamic changes of ppa-m NPs prepared in Example 2 of the present invention;

[0037] Fig.16 This is a line graph of the in vivo hemodynamic changes of ppa-m NPs prepared in Example 2 of the present invention;

[0038] Fig.17 4-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)piperazine-1-carboxylic acid tert-butyl ester prepared in Example 1 of the present invention 1 H NMR spectrum.

[0039] Fig.18 The ppa-o prepared in Example 1 of the present invention 1 H NMR spectrum.

[0040] Fig.19 The ppa-o prepared in Example 1 of the present invention 13 C NMR spectrum.

[0041] Fig. 20 This is the mass spectrum of ppa-o prepared in Example 1 of the present invention.

[0042] Fig.21 4-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)piperazine-1-carboxylic acid tert-butyl ester prepared in Example 2 of the present invention 1 H NMR spectrum.

[0043] Fig. 22 The ppa-m prepared in Example 2 of the present invention 1 H NMR spectrum.

[0044] Fig.23 The ppa-m prepared in Example 2 of the present invention 13 C NMR spectrum.

[0045] Fig.24 This is the mass spectrum of ppa-m prepared in Example 2 of the present invention. DETAILED DESCRIPTION

[0046] The present invention first provides a thrombolytic small molecule compound ppa-o, the structural formula of which is shown in Formula 1:

[0047]

[0048] The present invention also provides a method for preparing the thrombolytic small molecule compound PPA-O shown in Formula 1, comprising:

[0049] Step 1: Dissolve 2-(2,6-dioxo-piperidin-3-yl)-4-fluoro-isoindole-1,3-dione and 1-tert-butyloxycarbonylpiperazine in a solvent, preferably DMSO, add N,N-diisopropylethylamine DIEA for reaction, preferably heat under reflux at 80°C for 4-5h, stop the reaction and cool to room temperature, extract with appropriate amount of ethyl acetate and saturated sodium chloride, combine the organic phases, add anhydrous sodium sulfate, The mixture was dried, the solvent in the system was removed by a rotary evaporator, and the mixture was purified by column chromatography to obtain a black solid 4-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)piperazine-1-carboxylic acid tert-butyl ester; the molar ratio of the 2-(2,6-dioxopiperidin-3-yl)-4-fluoro-isoindole-1,3-dione:1-tert-butyloxycarbonylpiperazine:DIEA is preferably (1.2-1.5):1:6;

[0050] Step 2: reacting the tert-butyl 4-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)piperazine-1-carboxylate and trifluoroacetic acid in a solvent, wherein the solvent is preferably dichloromethane, the reaction temperature is preferably room temperature, and the reaction time is preferably 0.5-1h. When the reaction is completed, extracting with ethyl acetate and saturated sodium chloride, combining the organic phases, adding anhydrous sodium sulfate for drying, removing the solvent in the system by a rotary evaporator, and purifying by column chromatography to obtain a black solid 2-(2,6-dioxopiperidin-3-yl)-4-(piperazine-1-yl)isoindole-1,3-dione, wherein the molar ratio of tert-butyl 4-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)piperazine-1-carboxylate to trifluoroacetic acid is preferably 1:45;

[0051] Step 3: The 2-(2,6-dioxopiperidin-3-yl)-4-(piperazine-1-yl)isoindole-1,3-dione obtained in step 2 is mixed with pyropheophorbide a in DMF, and N,N,N′,N′-tetramethyl-O-(7-azabenzotriazole-1-yl)urea hexafluorophosphate (HATU) and N,N-diisopropylethylamine (DIEA) are added, preferably reacted at room temperature for 4-6 hours, extracted with ethyl acetate and saturated sodium chloride, the organic phases are combined, anhydrous sodium sulfate is added for drying, the solvent in the system is removed by a rotary evaporator, and purified by column chromatography to obtain a black solid small molecule compound ppa-o, wherein the molar ratio of the 2-(2,6-dioxopiperidin-3-yl)-4-(piperazine-1-yl)isoindole-1,3-dione: pyropheophorbide a: HATU: DIEA is preferably (4-5): 1: 2.5: 4.

[0052] The specific reaction process is as follows:

[0053]

[0054] The present invention also provides a thrombolytic small molecule compound ppa-m, the molecular formula of which is C 50 H 50 N 8 O 6 , the relative molecular mass is 858.39 g / mol, and its structural formula is shown in Formula 2:

[0055]

[0056] The present invention also provides a method for preparing the thrombolytic small molecule compound ppa-m of formula 2, comprising the following steps:

[0057] Step 1: Dissolve 2-(2,6-dioxo-piperidin-3-yl)-5-fluoro-isoindole-1,3-dione and 1-tert-butyloxycarbonylpiperazine in a solvent, preferably DMSO, add N,N-diisopropylethylamine (DIEA) for reaction, preferably heat under reflux at 80°C for 4-5h, stop the reaction and cool to room temperature, extract with appropriate amount of ethyl acetate and saturated sodium chloride, combine the organic phases, and add anhydrous sodium sulfate The mixture was dried, the solvent in the system was removed by a rotary evaporator, and the mixture was purified by column chromatography to obtain a black solid 4-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)piperazine-1-carboxylic acid tert-butyl ester; the molar ratio of the 2-(2,6-dioxo-piperidin-3-yl)-5-fluoro-isoindole-1,3-dione:1-tert-butyloxycarbonylpiperazine:DIEA is preferably (1.2-1.5):1:6;

[0058] Step 2: reacting the tert-butyl 4-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)piperazine-1-carboxylate and trifluoroacetic acid in a solvent, wherein the solvent is preferably dichloromethane, the reaction temperature is preferably room temperature, and the reaction time is preferably 0.5-1h. When the reaction is completed, extracting with ethyl acetate and saturated sodium chloride, combining the organic phases, adding anhydrous sodium sulfate for drying, removing the solvent in the system by a rotary evaporator, and purifying by column chromatography to obtain a black solid 2-(2,6-dioxopiperidin-3-yl)-5-(piperazine-1-yl)isoindole-1,3-dione, wherein the molar ratio of tert-butyl 4-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)piperazine-1-carboxylate to trifluoroacetic acid is preferably 1:45;

[0059] Step 3: Mix the 2-(2,6-dioxopiperidin-3-yl)-5-(piperazine-1-yl)isoindole-1,3-dione obtained in step 2 with pyropheophorbide a in a solvent, preferably DMF, add N,N,N′,N′-tetramethyl-O-(7-azabenzotriazole-1-yl)urea hexafluorophosphate (HATU) and N,N-diisopropylethylamine (DIEA), preferably react at room temperature for 4-6 hours, extract with ethyl acetate and saturated sodium chloride, combine the organic phases, add anhydrous sodium sulfate for drying, remove the solvent in the system by rotary evaporator, and purify by column chromatography to obtain a black solid small molecule compound ppa-m, wherein the molar ratio of 2-(2,6-dioxopiperidin-3-yl)-5-(piperazine-1-yl)isoindole-1,3-dione: pyropheophorbide a: HATU: DIEA is preferably ((4-5):1:2.5:4

[0060] The specific reaction process is as follows:

[0061]

[0062] The present invention also provides a nanoparticle prepared from the thrombolytic small molecule compound shown in Formula 1 or Formula 2.

[0063] The method for preparing the nanoparticles of the present invention preferably comprises:

[0064] Water is added to the reaction containers respectively, ppa-o or ppa-m and distearoyl phosphatidylethanolamine-polyethylene glycol are dissolved in tetrahydrofuran respectively, and then added dropwise into the stirred water, ultrasonically applied (150W) for 10 minutes, stirred and volatilized overnight at room temperature, and then the RGD targeting peptide is added into the water and stirred for 16 hours. After the stirring is completed, the nanoparticles are dialyzed with a 1000 molecular weight dialysis membrane and filtered with a filter (0.45 / 0.22 μm) to obtain nanoparticles ppa-o NPs or ppa-m NPs with uniform particle size and stability, respectively. The mass ratio of ppa-o or ppa-m: distearoyl phosphatidylethanolamine-polyethylene glycol: RGD targeting peptide is preferably 1:2:1.5.

[0065] The present invention also provides the use of the nanoparticles in preparing medicines for treating thrombotic diseases.

[0066] The present invention is further described by way of the following examples, which do not limit the present invention in any way. Without departing from the technical solution of the present invention, any modification or change made to the present invention that is easily implemented by a person of ordinary skill in the art will fall within the scope of the claims of the present invention.

[0067] Example 1

[0068] Add 10 ml of dry DMSO to a 50 ml round-bottom flask containing 0.2 g of 2-(2,6-dioxo-piperidin-3-yl)-4-fluoro-isoindole-1,3-dione and 0.16 g of 1-tert-butyloxycarbonylpiperazine, and add 0.6 ml of DIEA while stirring. React at 80 ° C for 4 hours. After the reaction is cooled to room temperature, water and ethyl acetate are added to extract, the organic phases are combined, anhydrous sodium sulfate is added to dry, the solvent in the system is removed by rotary evaporator, and column chromatography is used for purification. The solid obtained is dried to obtain tert-butyl 4-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)piperazine-1-carboxylate.

[0069] To a 50 mL round-bottom flask, 0.2 g of tert-butyl 4-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)piperazine-1-carboxylate was added, 10 ml of dichloromethane was added, and 1.5 ml of trifluoroacetic acid was added under an ice bath. The mixture was reacted at room temperature for 1 h. When the reaction was completed, ethyl acetate and saturated sodium chloride were used for extraction. The organic phases were combined and dried by adding anhydrous sodium sulfate. The solvent in the system was removed by a rotary evaporator, and the mixture was purified by column chromatography to obtain a black solid, which was 2-(2,6-dioxopiperidin-3-yl)-4-(piperazine-1-yl)isoindole-1,3-dione.

[0070] Add 0.05g of 2-(2,6-dioxopiperidin-3-yl)-4-(piperazine-1-yl)isoindole-1,3-dione and 0.2g of pyropheophorbide a to a 50mL flask, add 15ml of DMF, add 0.03g of HATU, 0.02ml of DIEA and stir at room temperature for 4 hours. After the reaction is completed, add saturated sodium chloride and ethyl acetate for extraction, combine the organic phases, remove the solvent in the system by rotary evaporator, and purify by column chromatography to obtain black solid ppa-o. The yield is 81.1%, and the molecular formula is C 50 H 50 N 8 O 6 , the molecular mass is 858.39 g / mol.

[0071] The NMR hydrogen spectrum and mass spectrum of ppa-o prepared in Example 1 are as follows Figure 17-20 shown.

[0072] Example 2

[0073] To a 50ml round-bottom flask containing 0.2g of 2-(2,6-dioxo-piperidin-3-yl)-5-fluoro-isoindole-1,3-dione and 0.16g of 1-tert-butyloxycarbonylpiperazine, add 10ml of dry DMSO, and add 0.6ml of DIEA while stirring. React at 80°C for 4h. After the reaction is cooled to room temperature, water and ethyl acetate are added to extract, the organic phases are combined, anhydrous sodium sulfate is added to dry, the solvent in the system is removed by rotary evaporator, and column chromatography is used for purification. The resulting solid is tert-butyl 4-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)piperazine-1-carboxylate. The NMR spectrum is shown in Fig.21 shown.

[0074] To a 50 mL round-bottom flask, add 0.2 g of tert-butyl 4-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)piperazine-1-carboxylate, add 10 ml of dichloromethane, add 1.5 ml of trifluoroacetic acid under an ice bath, and react at room temperature for 1 h. When the reaction is completed, extract with ethyl acetate and saturated sodium chloride, combine the organic phases, add anhydrous sodium sulfate to dry, remove the solvent in the system by a rotary evaporator, and purify by column chromatography to obtain a black solid, which is 2-(2,6-dioxopiperidin-3-yl)-5-(piperazine-1-yl)isoindole-1,3-dione.

[0075] Add 0.05g of 2-(2,6-dioxopiperidin-3-yl)-5-(piperazine-1-yl)isoindole-1,3-dione and 0.2g of pyropheophorbide a to a 50mL flask, add 15ml of DMF, add 0.03g of HATU, 0.02ml of DIEA and stir at room temperature for 4 hours. After the reaction is completed, add saturated sodium chloride and ethyl acetate for extraction, combine the organic phases, remove the solvent in the system by rotary evaporator, and purify by column chromatography to obtain black solid ppa-m. The yield is 81.1%, and the molecular formula is C 50 H 50 N 8 O 6 , the molecular mass is 858.39 g / mol.

[0076] The NMR hydrogen spectrum and mass spectrum of PPA-M prepared in Example 2 are as follows: Figure 22-24 shown.

[0077] The thrombolytic small molecule compounds ppa-o and ppa-m prepared in Examples 1 and 2 were characterized for their performance as follows:

[0078] 1. Preparation and properties of PPA-O and PPA-M nanoparticles:

[0079] The preparation method of ppa-o and ppa-m in the present invention is to prepare water-soluble nanoparticles in a simple and convenient manner. The specific operation is as follows: 10 ml of water is added to a 20 ml vial, ppa-o or ppa-m (0.0010 g) and distearoyl phosphatidylethanolamine-polyethylene glycol (0.0020 g) are respectively dissolved in 1 ml of tetrahydrofuran, and added dropwise to 10 ml of stirred water, ultrasonic (150 W) for 10 minutes, stirred and volatilized overnight at room temperature, and then RGD targeting peptide (0.0015 g) is added to the water and stirred for 16 hours. After the stirring is completed, the nanoparticles are dialyzed with a 1000 molecular weight dialysis membrane and filtered with a filter (0.45 / 0.22 μm) to obtain nanoparticles ppa-o NPs and ppa-m NPs with uniform particle size and stability.

[0080] The particle size and potential of ppa-o NPs and ppa-m NPs in the present invention are as follows: Figure 1 , Figure 1 is the particle size of ppa-oNPs and ppa-m NPs of the present invention ( Figure 1 a, 1b) and potential diagram ( Figure 1 c), as can be seen from the figure, the particle sizes of ppa-oNPs and ppa-m NPs in aqueous solution are 98.74nm and 98.7nm respectively, and the measured potentials of ppa-o NPs and ppa-m NPs are -7.77mV and -8.22mV respectively. The uniform particle size of ppa-m NPs indicates that it has good biocompatibility, and the potential is negative, which can have a good circulation effect in the body.

[0081] 2. Photophysical properties of ppa-o NPs and ppa-m NPs:

[0082] Figure 2 is the UV absorption of ppa-o NPs and ppa-m NPs in DMSO solution of the present invention ( Figure 2 a) and fluorescence emission ( Figure 2 b), as can be seen from the figure, ppa-m NPs show bright red light emission in DMSO solution, and its emission peak is 680nm.

[0083] 3. Singlet oxygen production capacity of PPA-O NPs and PPA-M NPs:

[0084] In order to verify the photodynamic therapeutic effect of ppa-o NPs and ppa-m NPs, DPBF was used as an indicator and evaluated by measuring the absorbance of DPBF at 415 nm under light irradiation. Figure 3In vitro singlet oxygen generation experiment of ppa-o NPs and ppa-m NPs of the present invention, under non-illumination conditions, in the presence of DPBF and PSs: ppa-o NPs ( Figure 3 a), ppa-m NPs ( Figure 3 b), no obvious absorbance change was found at the characteristic absorption peak of DPBF at 415nm, indicating that singlet oxygen cannot be produced under non-light irradiation. Figure 4 Laser irradiation of ppa-o NPs alone ( Figure 4 a), ppa-m NPs( Figure 4 b), DPBF( Figure 4 c) No obvious absorbance changes were found, indicating that singlet oxygen could not be produced under laser irradiation of ps and DPBF alone.

[0085] Figure 5 Under illumination, DPBF and PSs:ppa-o NPs ( Figure 5 a), ppa-m NPs( Figure 5 b), the characteristic absorption peak of DPBF at 415nm will decrease continuously, which also proves that PSs can effectively produce 1 O 2 .

[0086] Figure 6 a is the time-dependent generation of ppa-o NPs and ppa-m NPs 1 O 2 The formation kinetic curve of ppa-oNPs and ppa-m NPs. 0 The curve can also better illustrate its good 1 O 2 Generate capacity, such as Figure 6 As shown in b 1 O 2 The generation also conforms to first-order linear dynamics.

[0087] 4. Photothermal properties of ppa-o NPs and ppa-m NPs:

[0088] Figure 7 The photothermal properties of ppa-m NPs were measured. Compared with the control group ppa-o NPs and water, the experimental group ppa-m NPs had better photothermal generation ability ( Figure 7 a), and the concentration dependence of ppa-m NPs at different concentrations in the experimental group ( Figure 7 b) Under different powers, the stronger the power, the higher the heat generation and temperature rise ( Figure 7 c), after five cycles of heating and cooling, it can be seen that the drug has good photothermal cycling ability ( Figure 7 d)

[0089] Figure 8 Thermal images of A) water, B) ppa-o NPs, and C) ppa-m NPs corresponding to photothermal data. Under illumination conditions, the solution temperature increases with the extension of illumination time.

[0090] 5. Chemiluminescence experiment of ppa-m NPs:

[0091] Fig. 9 The relevant data characterization of the chemiluminescence of ppa-m NPs of the present invention. First, when responding to different types of ROS, ONOO - The luminescence intensity of ONOO - The best selectivity ( Fig. 9 a) ONOO in the inflammatory site of mice - As a marker substance, it can respond to PPA-M NPs to produce chemiluminescence, illuminate the inflammation site, and diagnose the occurrence and treatment of inflammation. At different illumination times, the test results showed that the drug luminescence intensity was the highest when the illumination was 240s ( Fig. 9 b), this time is selected as the illumination time for in vivo drug imaging. The dotted line graph of chemiluminescence intensity at different wavelengths shows that the drug has the highest intensity of bioluminescence emission at 680nm ( Fig. 9 c). Then, at different ratios of ONOO - The results of coexistence measurements with drugs showed a linear correlation ( Fig. 9 d). Using chicken breasts of different thicknesses to cover the drug, the chemiluminescence can penetrate up to 12 mm. The high penetration depth is conducive to imaging testing of drugs at deep inflammatory sites ( Fig. 9 e). It has significant advantages over photosensitizers that rely on short-wavelength excitation light.

[0092] 6. Antioxidant performance experiment of ppa-o NPs and ppa-m NPs:

[0093] Fig.10 The test kit is used to test the antioxidant capacity of ppa-o NPs and ppa-m NPs of the present invention. When the drug concentration reaches 300 μg / mL, the scavenging rate of DPPH can reach 70% ( Fig.10 a). Incubation of the drug with hydrogen peroxide revealed a clearance rate of 80% ( Fig.10 b). It can be seen from the figure that ppa-m NPs have better antioxidant and anti-inflammatory capabilities and can have a good therapeutic effect on inflammation in the body.

[0094] 7. Hemolysis experiment of ppa-m NPs:

[0095] Fig.11For the ppa-m NPs hemolysis test of the present invention, different concentrations of ppa-m NPs were incubated with red blood cells in hot water at 37°C for 2 h, the PBS group was the negative control group, and the water group was the positive control group (the upper row was ppa-m NPs, and the lower row was ppa-mNPs+RBC). The measured drug hemolysis rate was still lower than 5% at 200 μg / mL, showing good biosafety.

[0096] 8. In vitro thrombolytic experiments of ppa-o NPs and ppa-m NPs:

[0097] Fig.12 In vitro thrombolysis test of ppa-o NPs and ppa-m NPs of the present invention, the ppa-o NPs and ppa-m NPs groups were simultaneously activated with PDT / PTT dual-mode thrombolysis under laser irradiation, and the thrombolysis rate was higher than that of the control group, reaching 71% ( Fig.12 a, 12b). Fig.12 As shown in c, the amount of hemoglobin and fibrin released into the supernatant during the clot dissolution process showed a similar trend to the dissolution efficiency. The experimental results showed that ppa-m NPs have good in vitro thrombolytic ability.

[0098] 9. In vivo photothermal imaging experiment of ppa-m NPs:

[0099] Fig.13 This is a photothermal imaging test of ppa-m NPs in vivo. The temperature change of the thrombus site under the light stimulation of 689nm laser was monitored by infrared thermal imaging. The results showed that the temperature of the thrombus site gradually increased with the extension of light exposure time.

[0100] 10. In vivo thrombolytic experiment of ppa-m NPs:

[0101] Fig.14 Figure 2 shows the in vivo thrombolytic effect of PPA-M NPs. After successful thrombus formation, the drug was injected through the tail vein and laser (689nm 0.8W cm -2 ) irradiated the thrombus site, and the results showed that it had a good thrombolytic effect.

[0102] 11. In vivo thrombolytic hemodynamics experiment of ppa-m NPs:

[0103] Fig.15 The following is a graph showing the changes in hemodynamics of PPA-M NPs in vivo, where the blue graph is a color graph of hemodynamic changes and the gray graph is the corresponding black and white graph. After successful thrombus formation, the blood flow at the thrombus site was the lowest as detected by the laser speckle imaging device. As the laser irradiation time increases, the thrombus dissolves and the blood flow gradually recovers. Fig.16As shown, the thrombolysis rate of the ppa-m NPs group can reach 80%, indicating that the drug has a good thrombolytic effect.

Claims

1. A thrombolytic small molecule compound, characterized in that: Its structural formula is shown in Formula 1 or Formula 2:

2. The method for preparing a thrombolytic small molecule compound according to claim 1, characterized in that: The following steps are involved: Step 1: dissolving 2-(2,6-dioxo-piperidin-3-yl)-4-fluoro-isoindole-1,3-dione or 2-(2,6-dioxo-piperidin-3-yl)-5-fluoro-isoindole-1,3-dione and 1-tert-butyloxycarbonylpiperazine in a solvent, adding N,N-diisopropylethylamine DIEA and reflux reaction to obtain 4-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)piperazine-1-carboxylic acid tert-butyl ester or 4-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)piperazine-1-carboxylic acid tert-butyl ester; Step 2: reacting tert-butyl 4-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)piperazine-1-carboxylate or tert-butyl 4-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)piperazine-1-carboxylate in step 1 with trifluoroacetic acid in a solvent to obtain 2-(2,6-dioxopiperidin-3-yl)-4-(piperazine-1-yl)isoindole-1,3-dione or 2-(2,6-dioxopiperidin-3-yl)-5-(piperazine-1-yl)isoindole-1,3-dione; Step 3: Mix and stir the 2-(2,6-dioxopiperidin-3-yl)-4-(piperazine-1-yl)isoindole-1,3-dione or 2-(2,6-dioxopiperidin-3-yl)-5-(piperazine-1-yl)isoindole-1,3-dione obtained in step 2 with pyropheophorbide a in a solvent, add N,N,N′,N′-tetramethyl-O-(7-azabenzotriazole-1-yl)urea hexafluorophosphate and N,N-diisopropylethylamine to react to obtain the small molecule compound ppa-o shown in formula 1 or the small molecule compound ppa-m shown in formula 2.

3. The method for preparing a thrombolytic small molecule compound according to claim 2, characterized in that: The reaction temperature of step 1 is 80-90°C and the reaction time is 4-5h.

4. The method for preparing a thrombolytic small molecule compound according to claim 2, characterized in that: The molar ratio of 2-(2,6-dioxo-piperidin-3-yl)-4-fluoro-isoindole-1,3-dione or 2-(2,6-dioxo-piperidin-3-yl)-5-fluoro-isoindole-1,3-dione:1-tert-butyloxycarbonylpiperazine:DIEA in step 1 is (1.2-1.5):1:

6.

5. The method for preparing a thrombolytic small molecule compound according to claim 2, characterized in that: The molar ratio of tert-butyl 4-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)piperazine-1-carboxylate or tert-butyl 4-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)piperazine-1-carboxylate to trifluoroacetic acid in step 2 is 1:

45.

6. The method for preparing a thrombolytic small molecule compound according to claim 2, characterized in that: The reaction temperature of step 2 is room temperature, and the reaction time is 0.5h-1h.

7. The method for preparing a thrombolytic small molecule compound according to claim 2, characterized in that: In the step 3, the molar ratio of 2-(2,6-dioxopiperidin-3-yl)-4-(piperazine-1-yl)isoindole-1,3-dione or 2-(2,6-dioxopiperidin-3-yl)-5-(piperazine-1-yl)isoindole-1,3-dione:pyropheophorbide a:N,N,N′,N′-tetramethyl-O-(7-azabenzotriazole-1-yl)urea hexafluorophosphate:N,N-diisopropylethylamine is (4-5):1:2.5:

4.

8. The method for preparing a thrombolytic small molecule compound according to claim 2, characterized in that: The reaction temperature of step 3 is room temperature, and the stirring time is 4h-6h.

9. A nanoparticle, characterized in that: It is prepared from the thrombolytic small molecule compound represented by formula 1 or formula 2 as claimed in claim 1.

10. Use of the nanoparticles according to claim 9 in the preparation of drugs for treating thrombotic diseases.

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