Near-infrared two-region aggregation-induced emission type photosensitizer with photo-thermal generation capability as well as preparation method and application of near-infrared two-region aggregation-induced emission type photosensitizer

Abstract

The invention provides a near-infrared two-region aggregation-induced emission type photosensitizer with photo-thermal generation capacity as well as a preparation method and application thereof, and belongs to the field of biochemical materials. The molecular structure of the organic fluorescent compound provided by the invention has electron donor-electron acceptor interaction, and the introduction of an R group in the acceptor part is beneficial to promoting D-A interaction, promoting absorption wavelength to generate red shift, increasing the penetration depth to biological tissues and providing a fluorescence imaging result with higher resolution; besides, a donor part of the organic fluorescent compound provided by the invention is julolidine coupled with thienyl ethylene and 4, 4 '-bis (N, N-dimethylamino) benzophenone coupled with conjugated diene, and the 4, 4'-bis (N, N-dimethylamino) benzophenone has a highly twisted space structure, so that the occurrence of a non-radiative transition process is facilitated, and the organic fluorescent compound has a good application prospect. Therefore, the improvement of the photo-thermal performance is promoted.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biochemical materials, in particular to a near-infrared region II aggregation-induced emission luminophore with photothermal generation capability and a preparation method and application thereof. BACKGROUND

[0002] Cancer is a disease that seriously endangers human life and health. Traditional cancer treatment methods mainly include surgical resection, radiotherapy, chemotherapy, etc. Although these methods have been widely used in clinical practice, there are still problems such as invasive operation, drug resistance, easy recurrence, and high toxicity and side effects, which further limit their development. In recent years, with the rapid development of nanotechnology and the biomedical field, new optical diagnosis and treatment technology integrating cancer diagnosis and treatment has received widespread attention.

[0003] New optical diagnosis and treatment technology mainly uses optical imaging technology such as fluorescence imaging and photoacoustic imaging for disease diagnosis and detection, and uses optical means such as photodynamic and photothermal therapy for disease treatment. Among them, photothermal therapy uses a specific wavelength of laser to irradiate a photothermal agent, so that it absorbs light energy and converts it into heat energy, thereby locally heating the tumor tissue to kill cancer cells or inhibit tumor growth. Compared with traditional treatment methods, photothermal therapy has the advantages of minimally invasive, strong selectivity, small toxicity and side effects, and can reduce damage to normal tissues through targeted therapy, thus showing broad prospects in the field of tumor treatment.

[0004] The performance of the optical diagnosis and treatment agent has a great influence on the diagnosis and treatment effect. Traditional photothermal materials often face problems such as low light absorption efficiency and insufficient light-heat conversion efficiency, which to some extent limits their application. Therefore, it is of great significance to improve the light absorption and light-heat conversion efficiency of molecules in the near-infrared region II. SUMMARY

[0005] The present application provides a near-infrared region II aggregation-induced emission luminophore with photothermal generation capability and a preparation method and application thereof. The organic fluorescent compound of the present application has high light absorption and light-heat conversion efficiency in the near-infrared region II.

[0006] The present application provides an organic fluorescent compound having a structural formula as shown in formula I:

[0007]

[0008] The R1 is The R2 is

[0009] Preferably, the structural formula as shown in any one of formula I-a to I-f:

[0010]

[0011] The application further provides a preparation method of the organic fluorescent compound.

[0012] (1) mixing a compound with a structure shown in formula II, a compound with a structure shown in formula III and acetic anhydride to perform a first Bredt reaction to obtain a compound with a structure shown in formula IV;

[0013]

[0014] The R is

[0015] (2) mixing the compound with a structure shown in formula IV, a compound with a structure shown in formula V, piperidine and an organic solvent to perform a second Bredt reaction to obtain the organic fluorescent compound.

[0016] The compound with a structure shown in formula V is a compound with a structure shown in formula V-a or a compound with a structure shown in formula V-b.

[0017]

[0018] Preferably, the molar ratio of the compound with a structure shown in formula II to the compound with a structure shown in formula III is 1:(1-3).

[0019] The use amount ratio of the compound with a structure shown in formula II to acetic anhydride is 1 mmol:(1-4) mL.

[0020] Preferably, the temperature of the first Bredt reaction is 100-130 DEG C, and the time is 12-24 h.

[0021] The first Bredt reaction is performed in a protective gas.

[0022] Preferably, the molar ratio of the compound with a structure shown in formula IV to the compound with a structure shown in formula V to piperidine is 1:(2-3):(0.83-6).

[0023] The use amount ratio of the compound with a structure shown in formula IV to the organic solvent is 1 mmol:(4-10) mL, and the organic solvent includes trichloromethane.

[0024] Preferably, the temperature of the second Bredt reaction is 20-40 DEG C, and the time is 12-24 h.

[0025] The second Bredt reaction is performed in a protective gas.

[0026] The application further provides application of the organic fluorescent compound in preparation of an anti-tumor drug, an anti-tumor diagnostic reagent or high-resolution blood vessel imaging for non-disease diagnosis and treatment purposes.

[0027] The application further provides an organic fluorescent compound water-dispersed nanoparticle, characterized in that the nanoparticle comprises the organic fluorescent compound or the organic fluorescent compound prepared by the preparation method and a coating agent coated on the surface of the organic fluorescent compound.

[0028] Preferably, the coating agent comprises one or more of methoxy polyethylene glycol amine, distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000, phosphatidyl ethanolamine-polyethylene glycol 2000-maleimide, distearoyl phosphatidyl ethanolamine-polyethylene glycol-folic acid, distearoyl phosphatidyl ethanolamine-polyethylene glycol-thiol, distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000-carboxylic acid, distearoyl phosphatidyl ethanolamine-polyethylene glycol 5000-azide, distearoyl ethanolamine-polyethylene glycol 2000-biotin, 1-palmitoyl-2-oleoyl ethanolamine, 1-stearoyl-2-oleoyl lecithin, dipalmitoyl phosphatidyl ethanolamine-polyethylene glycol 2000 and poloxamer F127.

[0029] The organic fluorescent compound provided by the application has a molecular structure with electron donor (from the structural formula shown in formula V)-electron acceptor (from the structural formula shown in formula IV) interaction (D-A). The introduction of the R1 group in the acceptor part facilitates the D-A interaction, promotes red shift of the absorption wavelength, increases the penetration depth of the biological tissue, and provides a fluorescent imaging result with higher resolution. In addition, the donor part of the organic fluorescent compound provided by the application is thienyl ethylene-coupled julolidine and conjugated diene-coupled 4,4'-di(N,N-dimethylamino) benzophenone. The 4,4'-di(N,N-dimethylamino) benzophenone has a highly twisted spatial structure, which is more conducive to the occurrence of non-radiative transition process, thereby promoting the improvement of the photothermal performance. The results of the examples show that the organic fluorescent compound with the structure shown in formula I-a, formula I-b, formula I-c, formula I-d, formula I-e or formula I-f has near-infrared two-zone emission, which is beneficial to penetrate deep biological tissues and improve the imaging resolution. After being coated into corresponding nanoparticles, the organic fluorescent compound still maintains good light absorption and emission properties in the solution state, has a large Stokes shift, can reduce the background noise interference in fluorescent imaging, and realizes high-resolution imaging of leg blood vessels for non-diagnostic purposes and non-treatment purposes. Meanwhile, the photodiagnosis and treatment agent provided by the application also has high-efficiency generation of active oxygen and excellent photothermal generation capacity, and can generate active oxygen and heat under light activation to efficiently perform photodynamic and photothermal synergistic killing on cancer cells.

[0030] The application further provides a preparation method of the near-infrared two-region organic fluorescent compound with light-heat generation capability, and the preparation method is simple, and the separation and purification process is easy to operate, Suitable for industrial production. BRIEF DESCRIPTION OF DRAWINGS

[0031] Figure 1 Absorption and emission spectra of BPN-M, BPN-P, BPN-BP, BPTQ-M, BPTQ-P and BPQ-BP in DMSO solution;

[0032] Figure 2 Particle size diagrams of BPN-M NPs, BPN-P NPs, BPN-BP NPs, BPTQ-M NPs, BPTQ-P NPs and BPTQ-BP NPs;

[0033] Figure 3 Absorption and emission spectra of BPN-M NPs, BPN-P NPs, BPN-BP NPs, BPTQ-M NPs, BPTQ-P NPs and BPTQ-BP NPs in aqueous solution;

[0034] Figure 4 Real-time fluorescence intensity ratio at 520 nm to initial fluorescence intensity of BPN-M NPs, BPN-P NPs, BPN-BP NPs, BPTQ-M NPs, BPTQ-P NPs and BPTQ-BP NPs mixed with an active oxygen trapping agent DCFH in a PBS buffer solution under 660 nm laser irradiation (light power: 0.4 W / cm 2 ) with the change of irradiation time;

[0035] Figure 5 Photo-thermal performance of BPN-M NPs, BPN-P NPs, BPN-BP NPs, BPTQ-M NPs, BPTQ-P NPs and BPTQ-BP NPs (molar concentration: 15 μM) under 660 nm laser irradiation and calculation of photo-thermal conversion efficiency (light power: 0.4 W / cm 2 );

[0036] Figure 6 Temperature rise of BPN-M NPs, BPN-P NPs and BPN-BP NPs in 5 cycles of 660 nm laser opening / closing (molar concentration: 15 μM, light power: 0.4 W / cm 2 );

[0037] Figure 7 Dark toxicity and photo-toxicity of BPN-P NPs of different concentrations on mouse breast cancer cells (4T1 cells);

[0038] Figure 8 Fig. 3 is a near-infrared fluorescence imaging diagram of BPN-P NPs on the leg blood vessels of a normal mouse under different filter conditions, and a resolution analysis diagram under different filter conditions;

[0039] Figure 9 Fig. 4 is a synthetic route diagram for preparing the compound of the structural formula shown in 3, 5 and 7;

[0040] Figure 10 Fig. 5 is a synthetic route diagram for preparing the compound of the structural formula shown in formula I in the embodiment. DETAILED DESCRIPTION

[0041] The present application provides an organic fluorescent compound, which has a structural formula shown in formula I:

[0042]

[0043] The R1 is

[0044] The R2 is

[0045] In the present application, the organic fluorescent compound preferably has a structural formula shown in any one of formula I-a to I-f:

[0046] The present application further provides a preparation method of the organic fluorescent compound in the above technical solution, which comprises the following steps:

[0047] (1) mixing a compound having a structure shown in formula II, a compound having a structure shown in formula III and acetic anhydride to perform a first brain text reaction, to obtain a compound having a structure shown in formula IV;

[0048]

[0049] The R is

[0050] (2) mixing the compound having a structure shown in formula IV, a compound having a structure shown in formula V, piperidine and an organic solvent to perform a second brain text reaction, to obtain the organic fluorescent compound;

[0051] The compound having a structure shown in formula V is a compound having a structure shown in formula V-a or a compound having a structure shown in formula V-b;

[0052]

[0053] The present application mixes a compound having a structure shown in formula II, a compound having a structure shown in formula III and acetic anhydride to perform a first brain text reaction, to obtain a compound having a structure shown in formula IV.

[0054] In the present application, the molar ratio of the compound having the structure shown in formula II to the compound having the structure shown in formula III is preferably 1:3; the usage ratio of the compound having the structure shown in formula II to acetic anhydride is preferably 1 mmol:(1-4) mL, and in specific embodiments of the present application, the usage ratio of the compound having the structure shown in formula II to acetic anhydride can be 1 mmol:2 mL or 1 mmol:3 mL.

[0055] In the present application, the temperature of the first Brain's reaction is preferably 100-130°C, and the time is preferably 12-24 h, and in specific embodiments of the present application, the temperature of the first Brain's reaction can be 110°C or 120°C, and the time can be 15 h, 18 h, 20 h or 22 h.

[0056] The first Brain's reaction is preferably carried out in a protective gas.

[0057] After the first Brain's reaction, the present application preferably carries out post-treatment on the obtained product; the post-treatment preferably includes cooling the product to 25°C under ultrasonic state, and then adding saturated sodium carbonate aqueous solution and acetic anhydride, until no more bubbles are generated, then adding dichloromethane and water to the above mixture for extraction, collecting the organic phase for reduced pressure concentration, and then purifying the obtained crude product by silica gel chromatography, and recrystallizing with a mixed solvent of dichloromethane and n-hexane to obtain the final product; the volume ratio of dichloromethane to n-hexane in the mixed solvent is preferably 1:10.

[0058] After obtaining the compound having the structure shown in formula IV, the present application carries out a second Brain's reaction on the compound having the structure shown in formula IV, the compound having the structure shown in formula V, piperidine and an organic solvent, to obtain the organic fluorescent compound.

[0059] In the present application, the molar ratio of the compound having the structure shown in formula IV to the compound having the structure shown in formula V to piperidine is preferably 1:(2-3):(0.83-6), and in specific embodiments of the present application, the molar ratio of the compound having the structure shown in formula IV to the compound having the structure shown in formula V to piperidine can be 1:2.5:4.

[0060] The usage ratio of the compound having the structure shown in formula IV to the organic solvent is preferably 1 mmol:4-10 mL; and the organic solvent preferably includes trichloromethane.

[0061] In the present application, the temperature of the second Brain's reaction is preferably 20-40°C, and the time is preferably 12-24 h, and in specific embodiments of the present application, the temperature of the second Brain's reaction can be 25°C, 30°C or 35°C, and the time can be 15 h, 18 h, 20 h or 22 h.

[0062] The second brain text reaction is preferably carried out in a protective gas.

[0063] After the second brain text reaction, the application preferably carries out post-treatment on the obtained product; the post-treatment preferably includes concentrating the obtained product under reduced pressure, then recrystallizing it using methanol and dichloromethane, dissolving the obtained crude product in dichloromethane, further purifying it through silica gel chromatography, and recrystallizing it using a mixed solvent of dichloromethane and n-hexane to obtain the final product; the volume ratio of dichloromethane to n-hexane in the mixed solvent is preferably 1:10.

[0064] The application also provides the use of the organic fluorescent compound in the preparation of an antitumor drug, the preparation of an antitumor diagnostic reagent, or high-resolution blood vessel imaging for non-disease diagnosis and treatment purposes.

[0065] The application also provides an organic fluorescent compound water-dispersible nanoparticle, characterized in that it comprises the organic fluorescent compound or the organic fluorescent compound prepared by the preparation method described in the above technical solution and a coating agent coated on the surface of the organic fluorescent compound.

[0066] In the application, the coating agent preferably comprises one or more of methoxy polyethylene glycol amine, distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000, phosphatidyl ethanolamine-polyethylene glycol 2000-maleimide, distearoyl phosphatidyl ethanolamine-polyethylene glycol-folic acid, distearoyl phosphatidyl ethanolamine-polyethylene glycol-thiol, distearoyl phosphatidyl ethanolamine-polyethylene glycol-biotin, 1-palmitoyl-2-oleoyl ethanolamine, 1-stearoyl-2-oleoyl lecithin, dipalmitoyl phosphatidyl ethanolamine-polyethylene glycol 2000, and poloxamer F127. Fatty acyl ethanamide-polyethylene glycol 2000- Carboxylic acid, distearoyl phosphatidyl ethanolamine-polyethylene glycol 5000-azido, In the application, the coating agent preferably comprises one or more of methoxy polyethylene glycol amine, distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000, phosphatidyl ethanolamine-polyethylene glycol 2000-maleimide, distearoyl phosphatidyl ethanolamine-polyethylene glycol-folic acid, distearoyl phosphatidyl ethanolamine-polyethylene glycol-thiol, distearoyl phosphatidyl ethanolamine-polyethylene glycol-biotin, 1-palmitoyl-2-oleoyl ethanolamine, 1-stearoyl-2-oleoyl lecithin, dipalmitoyl phosphatidyl ethanolamine-polyethylene glycol 2000, and poloxamer F127.

[0067] In the application, the preparation method of the organic fluorescent compound water-dispersible nanoparticle comprises the following steps:

[0068] Mixing the organic fluorescent compound, the coating agent, and the polar organic solvent to obtain a mixed solution;

[0069] Mixing the mixed solution with deionized water, performing ultrasonic co-precipitation, and obtaining the organic fluorescent compound water-dispersible nanoparticle.

[0070] In the application, the mass ratio of the organic fluorescent compound to the coating agent is preferably 1:5; the mass ratio of the organic fluorescent compound to the polar organic solvent is preferably 3 mg:1 mL; and the polar organic solvent preferably comprises tetrahydrofuran.

[0071] In the present application, the power of the ultrasound during the ultrasonic co-precipitation is preferably 150 W, and the time is preferably 10 min.

[0072] After the ultrasonic co-precipitation, the product obtained is preferably subjected to post-treatment; the post-treatment preferably comprises transferring the product obtained into a dialysis bag for dialysis, and then concentrating, filtering and drying the dialysis product obtained.

[0073] In the present application, the molecular weight cut-off of the dialysis bag used for the dialysis is preferably 3600, and the present application does not have special limitations on the dialysis, which can be sufficiently cleaned by dialysis with polar organic solvents.

[0074] The near-infrared two-region aggregation-induced emission type photosensitizer with light-heat generation capability provided by the present application, the preparation method and the application thereof will be described in detail below with reference to the examples, but they should not be understood as limitations on the protection scope of the present application.

[0075] Example 1

[0076] Figure 9 The synthesis route chart for preparing the compounds of the structural formula shown in 3, 5 and 7 is as follows:

[0077] Preparation of the compound of the structure shown in 3:

[0078] Compound 1 (3 mmol) and 2 (9 mmol) were dissolved in 3 mL of acetic anhydride, and stirred at 110°C under N2for 12 h. After the completion of the reaction was monitored by TLC, the reaction liquid was cooled to room temperature, and saturated sodium carbonate aqueous solution was added to neutralize the acetic anhydride until no more bubbles were generated under ultrasonic state. Then dichloromethane and water were added to extract the mixture, and the organic phase was collected and concentrated under reduced pressure. The obtained crude product was purified by silica gel chromatography (pure dichloromethane), and recrystallized with dichloromethane / n-hexane to obtain the final product.

[0079]

[0080] The calculated yield of the obtained red solid was 37%:

[0081] The obtained red solid was characterized, and the specific data are as follows:

[0082] 1H NMR (600 MHz, Chloroform-d) δ 6.66 (s, 1H), 6.27 (s, 1H), 5.19 (s, 1H), 2.41 (s, 3H), 2.30 (s, 3H), 1.55 (s, 6H).

[0083] Preparation of the compound of the structure shown in 5:

[0084] Instead of using the compound of formula 1 to prepare the compound of formula 5, the compound of formula 4 is used to prepare the compound of formula 7:

[0085]

[0086] The obtained red-orange solid has a calculated yield of 42%:

[0087] The obtained red-orange solid is characterized, and the specific data are as follows:

[0088] 1H NMR (600 MHz, Chloroform-d) δ 7.64 (d, J = 8.2 Hz, 2H), 7.58 (d, J = 7.6 Hz, 2H), 7.46 (t, J = 7.5 Hz, 2H), 7.40-7.37 (m, 3H), 6.48 (s, 1H), 6.20 (s, 1H), 5.30 (s, 1H), 2.28 (s, 3H), 2.25 (s, 3H), 2.00 (s, 3H).

[0089] According to the above nuclear magnetic characterization data, the compound 7 (i.e., formula IV) is prepared.

[0090] Preparation of the compound of formula 7

[0091] Instead of using the compound of formula 4 to prepare the compound of formula 7, the compound of formula 6 is used to prepare the compound of formula 7:

[0092]

[0093] The obtained red-orange solid has a calculated yield of 42%:

[0094] The obtained red-orange solid is characterized, and the specific data are as follows:

[0095] 1H NMR (600 MHz, Chloroform-d) δ 7.64 (d, J = 8.2 Hz, 2H), 7.58 (d, J = 7.6 Hz, 2H), 7.46 (t, J = 7.5 Hz, 2H), 7.40-7.37 (m, 3H), 6.48 (s, 1H), 6.20 (s, 1H), 5.30 (s, 1H), 2.28 (s, 3H), 2.25 (s, 3H), 2.00 (s, 3H).

[0096] According to the above nuclear magnetic characterization data, the compound 7 (i.e., formula IV) is prepared.

[0097] Preparation of the compound of formula I-a, R is methyl:

[0098] Compound 3 (1 mmol), compound 8 (2.5 mmol) and piperidine (340 mg, 4 mmol) were dissolved in 10 mL of chloroform and reacted at 25°C for 12 h under nitrogen. After the reaction was completed, the reaction solution was concentrated under reduced pressure and recrystallized using a small amount of methanol and dichloromethane. The obtained crude product was dissolved in dichloromethane and further purified by silica gel chromatography (volume ratio: dichloromethane:methanol = 400:1), and recrystallized using a mixed solvent of dichloromethane and n-hexane (volume ratio of 1:10) to obtain a compound having a structure represented by Formula I.

[0099]

[0100] The obtained black-green solid had a calculated yield of 8.8%:

[0101] The obtained black-green solid was characterized, and the specific data are as follows:

[0102] 1H NMR (600 MHz, TCE-d2) δ 7.40-7.36 (m, 2H), 7.25 (d, J = 8.5 Hz, 4H), 7.10 (d, J = 8.2 Hz, 4H), 6.68 (d, J = 8.2 Hz, 4H), 6.65 (d, J = 12.0 Hz, 2H), 6.62 (d, J = 8.5 Hz, 4H), 6.59 (s, 2H), 6.29 (d, J = 15.0 Hz, 2H), 5.19 (s, 1H), 2.98 (d, J = 8.7 Hz, 24H), 1.50 (s, 6H).13C NMR (151 MHz, TCE-d2) δ 179.24, 169.02, 161.57, 153.08, 150.89, 150.61, 139.26, 132.40, 130.33, 129.49, 129.18, 128.35, 126.46, 122.01, 119.26, 115.70, 114.99, 114.74, 111.65, 111.61, 99.03, 95.85, 47.26, 40.47, 40.28, 26.96. HRMS (MALDI-TOF): m / z: [M+Na]+calcd for C56H55N7NaO2: 880.4315; found: 880.4312.

[0103] According to the above nuclear magnetic characterization data, the compound I-a (denoted as BPN-M) was prepared in this embodiment.

[0104] The obtained black-green solid had a calculated yield of 8.8%:

[0105] Example 2

[0106] The only difference from Example 1 is that compound 3 is replaced by compound 5.

[0107] The obtained black-green solid was characterized, and the specific data are as follows:

[0108] 1 H NMR (600 MHz, TCE-d2) δ 7.29-7.24 (m, 4H), 7.21 (d, J = 8.8 Hz, 4H), 7.17 (d, J = 7.4 Hz, 3H), 7.06 (d, J = 8.6 Hz, 4H), 6.67 (d, J = 8.5 Hz, 4H), 6.61 (d, J = 8.8 Hz, 4H), 6.57 (d, J = 11.4 Hz, 2H), 6.25 (s, 2H), 6.05 (d, J = 15.0 Hz, 2H), 5.42 (s, 1H), 2.96 (s, 24H), 1.89 (s, 3H).13C NMR (151 MHz, TCE-d2) δ 161.60, 153.23, 150.95, 150.70, 139.27, 137.96, 132.43, 130.35, 129.34, 129.17, 128.35, 126.39, 125.88, 125.44, 121.83, 118.92, 115.48, 114.90, 114.33, 111.61, 111.57, 47.27, 40.45, 40.26, 29.77, 21.61. HRMS (MALDI-TOF): m / z: [M+Na]+calcd for C61H57N7NaO2: 942.4471; found: 942.4467.

[0109] According to the above nuclear magnetic characterization data, compound I-b (denoted as BPN-P) is prepared in this embodiment.

[0110] Example 3

[0111] The only difference from Example 1 is that compound 3 is replaced by compound 7.

[0112] The obtained black-green solid has a calculated yield of 11%:

[0113] The obtained black-green solid was characterized, and the specific data are as follows:

[0114] 1H NMR (600 MHz, DMSO-d6) δ 7.62 (s, 4H), 7.46 (t, J = 6.78 Hz, 3H), 7.39 (t, J = 7.2 Hz, 2H), 7.20 (d, J = 8.4 Hz, 4H), 7.00 (d, J = 7.8 Hz, 4H), 6.77-6.71 (m, 12H), 6.38 (s, 2H), 5.33 (s, 1H), 2.98 (s, 12H), 2.92 (s, 12H), 2.01 (s, 3H).13C NMR (151 MHz, TCE-d2) δ 161.70, 153.29, 150.97, 150.75, 139.77, 139.28, 138.00, 132.53, 130.40, 129.33, 129.17, 128.35, 127.95, 127.16, 126.41, 125.44, 121.80, 118.88,115.55,114.93,114.21,111.61, 111.54,47.22,40.45,40.27,29.77,21.61. HRMS (MALDI-TOF): m / z: [M+Na]+calcd for C67H61N7NaO2: 1018.4784; found: 1018.4785.

[0115] According to the above nuclear magnetic characterization data, the compound I-c (denoted as BPN-BP) was prepared in this embodiment.

[0116] Example 4

[0117] The only difference from Example 1 is that compound 8 is replaced by compound 9.

[0118]

[0119] The obtained black-green solid has a calculated yield of 9%:

[0120] The obtained black-green solid was characterized, and the specific data are as follows:

[0121] 1H NMR (600 MHz, TCE-d2) δ 7.56 (d, J = 15.4 Hz, 2H), 7.25 (d, J = 3.6 Hz, 2H), 7.08 (d, J = 3.6 Hz, 2H), 7.05 (s, 4H), 6.60 (s, 2H), 6.44 (d, J = 15.3 Hz, 2H), 5.23 (s, 1H), 3.18 (t, J = 5.9 Hz, 8H), 2.73 (t, J = 5.9 Hz, 8H), 1.95 - 1.94 (m, 8H), 1.54 (s, 6H).13C NMR (151 MHz, TCE-d2) δ 179.19, 170.52, 159.52, 150.72, 150.60, 143.76, 136.68, 133.74, 131.18, 124.90, 121.53, 121.47, 119.97, 115.31, 115.01, 114.46, 112.67, 99.71, 96.52, 83.91, 49.91, 49.11, 41.09, 27.77, 26.82, 21.75. HRMS (MALDI-TOF): m / z: [M+H]+calcd for C52H46N5O2S2: 836.3093; found: 836.3098.

[0122] According to the above nuclear magnetic characterization data, the compound I-d (denoted as BPTQ-M) was prepared in this embodiment.

[0123] Example 5

[0124] The only difference between Example 2 and Example 5 is that compound 8 is replaced by compound 9.

[0125] The obtained black-green solid has a calculated yield of 10%:

[0126] The obtained black-green solid was characterized, and the specific data are as follows:

[0127] 1H NMR (600 MHz, DMSO-d6) δ 7.85 (d, J = 15.6 Hz, 2H), 7.52-7.51 (m, 5H), 7.48 (d, J = 3.8 Hz, 2H), 7.32 (d, J = 3.8 Hz, 2H), 7.12 (s, 4H), 6.82 (s, 2H), 6.58 (d, J = 15.9 Hz, 2H), 5.66 (s, 1H), 3.20 (t, J = 5.46 Hz, 8H), 2.73 (t, J = 6.1 Hz, 8H), 2.07 (s, 3H), 1.91-1.88 (m, 8H).13C NMR (151 MHz, TCE-d2) δ 169.97, 159.55, 150.78, 143.79, 136.62, 133.75, 131.20, 130.06, 129.47, 125.88, 124.91, 121.57, 121.47, 119.91, 115.14, 114.84, 114.34, 114.13, 101.76, 97.75, 67.96, 49.90, 49.23, 27.77, 25.71, 21.60. HRMS (MALDI-TOF): m / z: [M+H]+calcd for C57H48N5O2S2: 898.3250; found: 898.3239.

[0128] According to the above nuclear magnetic characterization data, the compound I-e (denoted as BPTQ-P) was prepared in this embodiment.

[0129] Example 6

[0130] The only difference between Example 3 and this example is that compound 8 is replaced by compound 9.

[0131] The obtained black-green solid has a calculated yield of 12%:

[0132] The obtained black-green solid was characterized, and the specific data are as follows:

[0133] 1H NMR (600 MHz, DMSO-d6) δ 7.86 (d, J = 15.4 Hz, 2H), 7.81 (d, J = 7.9 Hz, 2H), 7.71 (d, J = 7.6 Hz, 2H), 7.61 (d, J = 5.8 Hz, 2H), 7.48 - 7.46 (m, 4H), 7.39 (t, J = 7.3 Hz, 1H), 7.32 (d, J = 3.8 Hz, 2H), 7.12 (s, 4H), 6.84 (s, 2H), 6.57 (d, J = 15.4 Hz, 2H), 5.71 (s, 1H), 3.20 (t, J = 5.4 Hz, 8H), 2.72 (t, J = 6.1 Hz, 8H), 2.10 (s, 3H), 1.91 - 1.87 (m, 8H).13C NMR (151 MHz, TCE-d2) δ 159.58, 150.80, 143.80, 139.75, 136.60, 133.71, 128.07, 127.23, 126.40, 124.91, 121.56, 121.48, 119.92, 115.17, 114.82, 114.30, 49.90, 49.37, 27.76, 21.72, 14.30. HRMS (MALDI-TOF): m / z: [M]+calcd for C63H51N5O2S2: 973.3484; found: 973.3487.

[0134] According to the above nuclear magnetic characterization data, the compound I-f (denoted as BPTQ-BP) is prepared in this embodiment.

[0135] Figure 10 The synthetic route chart of the compound of the formula I prepared in the embodiment is shown in the following.

[0136] Comparative Example 1

[0137] The only difference between the preparation of I-a in Example 1 is that sodium acetate is used instead of piperidine, and ethanol is used instead of chloroform.

[0138] The organic fluorescent compound represented by I-a is not obtained.

[0139] Comparative Example 2

[0140] The only difference between the preparation of I-a in Example 1 is that sodium hydroxide is used instead of piperidine, and DMF is used instead of chloroform.

[0141] The organic fluorescent compound represented by I-a is not obtained.

[0142] Application Example

[0143] In order to make BPN-M, BPN-P, BPN-BP, BPTQ-M, BPTQ-P and BPTQ-BP be applied to biological environment water system, the embodiment takes BPN-M, BPN-P, BPN-BP, BPTQ-M, BPTQ-P and BPTQ-BP as organic compounds, takes amphiphilic copolymer DSPE-PEG2000 as coating matrix, and adopts ultrasonic method to prepare nanoparticle water dispersion, which are respectively recorded as BPN-MNPs, BPN-PNPs, BPN-BP NPs, BPTQ-MNPs, BPTQ-PNPs and BPTQ-BPNPs. The preparation steps are as follows:

[0144] 3mg of BPN-M, BPN-P, BPN-BP, BPTQ-M, BPTQ-P and BPTQ-BP are respectively dissolved in 1mL of tetrahydrofuran together with 15mg of distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000 (DSPE-mPEG2000) and mixed uniformly. Under the working condition of cell crusher (150W), the above mixture is injected into 10mL of double-distilled water at one time, and worked for 10min. Subsequently, the solution is transferred into a dialysis bag (with a molecular weight cut-off of 3600), and continuously dialyzed with deionized water for 2-3 days until tetrahydrofuran is completely dialyzed clean. The obtained near-infrared two-region organic fluorescent compound nanoparticles can be used for next step test after concentration, filtration (0.45μm filter membrane), and concentration calibration (concentration is 700μM).

[0145] Performance test

[0146] (1) The ultraviolet absorption spectrum and fluorescence emission spectrum test of BPN-M, BPN-P, BPN-BP, BPTQ-M, BPTQ-P and BPTQ-BP in DMSO solution, the specific steps are as follows:

[0147] BPN-M, BPN-P, BPN-BP, BPTQ-M, BPTQ-P and BPTQ-BP are respectively configured into 1mM DMSO stock solution. The corresponding stock solution is transferred into DMSO solution, configured into 10μM standard test solution, placed in ultraviolet spectrophotometer to collect 300nm-1100nm waveband ultraviolet absorption spectrum, placed in fluorescence spectrometer, and excited by external 660nm laser to collect 700nm-1600nm waveband fluorescence emission spectrum, and the results are shown in Figure 1 .

[0148] from Figure 1It can be seen that the maximum absorption peaks of BPN-M, BPN-P and BPN-BP are located at 680 nm, 686 nm and 689 nm, respectively, while the maximum absorption peaks of BPTQ-M, BPTQ-P and BPTQ-BP are located at 675 nm, 685 nm and 689 nm, respectively; the maximum emission peaks of BPN-M, BPN-P and BPN-BP with the same donor unit are all located near 1000 nm, while the maximum emission peaks of BPTQ-M, BPTQ-P and BPTQ-BP can be red-shifted to 1100 nm, and the fluorescence emission wavelength can reach the near-infrared two region. Although the longer emission wavelength of BPTQ series helps to reduce the background interference of imaging and improve the resolution of imaging, the more planar donor structure of BPTQ series molecules is prone to π-π stacking, and the weak fluorescence emission further limits the practical application. Among them, the Stokes shifts of BPN-M, BPN-P and BPN-BP are 320 nm, 314 nm and 311 nm, respectively.

[0149] (2) The particle size of BPN-MNPs, BPN-PNPs, BPN-BPNPs, BPTQ-MNPs, BPTQ-PNPs and BPTQ-BPNPs was tested, and the specific steps were as follows:

[0150] 10 μL of nanoparticles were added to 3 mL of deionized water, and then the particle size was determined by dynamic light scattering instrument, and the results are shown in Figure 2 .

[0151] Figure 2 The particle size of BPN-MNPs, BPN-PNPs, BPN-BPNPs, BPTQ-MNPs, BPTQ-PNPs and BPTQ-BPNPs was tested, and the specific steps were as follows: BPTQ-PNPs and BPTQ- Particle size distribution of BPNPs, Figure 2 BPN-MNPs, BPN-PNPs, The corresponding transmission electron microscopy images of BPN-BP NPs.

[0152] It can be seen from Figure 2 that the sizes of BPN-MNPs, BPN-PNPs, BPN-BPNPs, BPTQ-MNPs, BPTQ-PNPs and BPTQ-BPNPs are 59 nm, 55 nm, 60 nm, 198 nm, 71 nm and 72 nm, respectively. It can be further seen from the transmission electron microscopy images in the insert that BPN-MNPs, BPN-PNPs and BPN-BPNPs all present uniform spherical morphology.

[0153] (3) The ultraviolet absorption spectrum and fluorescence emission spectrum of BPN-MNPs, BPN-PNPs, BPN-BPNPs, BPTQ-MNPs, BPTQ-PNPs and BPTQ-BPNPs in aqueous solution were tested, and the specific steps were as follows:

[0154] BPN-M NPs, BPN-PNPs, BPN-BPNPs, BPTQ-M NPs, BPTQ-PNPs, and BPTQ-BP NPs were transferred to deionized water to prepare a 10 μM standard test solution. The solution was then used in a UV spectrophotometer to collect UV absorption spectra in the 300 nm–1000 nm band. Finally, the solution was used in a fluorescence spectrometer with an external 660 nm laser for excitation, and fluorescence emission spectra in the 700 nm–1500 nm band were collected. The results are as follows: Figure 3 As shown.

[0155] Figure 3 The absorption and emission spectra of BPN-MNPs, BPN-PNPs, BPN-BPNPs, BPTQ-MNPs, BPTQ-PNPs, and BPTQ-BPNPs in aqueous solution are shown.

[0156] Depend on Figure 3 It is known that, in the nanoparticle state, the absorption peaks of BPN-M NPs, BPN-PNPs, and BPN-BPNPs are 661 nm, 669 nm, and 673 nm, respectively. The maximum emission spectra of all three NPs are located near 960 nm, with Stokes shifts of 299 nm, 291 nm, and 287 nm. Among the BPTQ series molecules, the optimal absorption peaks of BPTQ-M NPs, BPTQ-P NPs, and BPTQ-BPNPs are 673 nm, 674 nm, and 675 nm, respectively, with the maximum emission peak located near 1100 nm, still exhibiting a relatively weak fluorescence emission signal. After being prepared into corresponding nanoparticles, the six nanoparticles still maintained good light absorption and emission properties in the solution state.

[0157] (4) The total reactive oxygen species (ROS) generation capacity of BPN-MNPs, BPN-PNPs, BPN-BPNPs, BPTQ-MNPs, BPTQ-PNPs, and BPTQ-BPNPs was tested, and the specific steps are as follows:

[0158] The total reactive oxygen species (ROS) generation capacity was assessed using 2',7'-dichlorodihydrofluorescein (DCFH) fluorescent indicator. DCFH (50 μL, 40 μM) was mixed with BPN-MNPs, BPN-PNPs, BPN-BPNPs, BPTQ-MNPs, BPTQ-PNPs, and BPTQ-BPNPs nanoparticles (1 μM) in PBS buffer (pH 7.4) and then placed under a 660 nm laser (0.4 W / cm²). 2 Irradiation was performed under the following conditions: fluorescence spectrometry was used (490nm excitation) to record the changes in fluorescence spectra at regular intervals. The group irradiated with only DCFH was used as the blank control. White control group.

[0159] Figure 4 The change of the absorbance value of BPN-MNPs, BPN-PNPs, BPN-BPNPs, BPTQ-MNPs, BPTQ-PNPs and BPTQ-BPNPs mixed with active oxygen trapping agent DCFH in PBS at 520 nm with light irradiation time.

[0160] From Figure 4 it can be seen that in the presence of BPN-MNPs, BPN-PNPs, BPN-BPNPs, BPTQ-MNPs, BPTQ-PNPs and BPTQ-BPNPs, the fluorescence intensity of DCFH rapidly increases with the increase of 660 nm laser irradiation time, while the fluorescence signal of the group with only DCFH is negligible, indicating that BPN-MNPs, BPN-PNPs, BPN-BPNPs, BPTQ-MNPs, BPTQ-PNPs and BPTQ-BPNPs all have high active oxygen production capacity, which is expected to be applied in efficient photodynamic therapy.

[0161] (5) The photothermal properties of BPN-MNPs, BPN-PNPs, BPN-BPNPs, BPTQ-MNPs, BPTQ-PNPs and BPTQ-BPNPs under 660 nm laser irradiation and the calculation of their photothermal conversion efficiency, the specific steps are as follows:

[0162] The aqueous solution (200 μL, 15 μM) of BPN-MNPs, BPN-PNPs, BPN-BPNPs, BPTQ-MNPs, BPTQ-PNPs and BPTQ-BPNPs was irradiated under 660 nm laser (0.4 W / cm 2 ) for 6 min, and the laser was turned off at this time point to cool the sample to room temperature (about 10 min), and the temperature change in this process was monitored using an infrared thermal imager. The results are shown in Figure 5 .

[0163] Figure 5 The photothermal properties of BPN-MNPs, BPN-PNPs, BPN-BPNPs, BPTQ-MNPs, BPTQ-PNPs and BPTQ-BPNPs under 660 nm laser irradiation (0.4 W / cm 2 ) and the calculation of their photothermal conversion efficiency.

[0164] From Figure 5The photothermal conversion efficiencies of BPN-MNPs, BPN-PNPs, BPN-BPNPs, BPTQ-MNPs, BPTQ-PNPs, and BPTQ-BPNPs were 46%, 63%, 60%, 40%, 40%, and 37%, respectively. These six nanoparticles exhibit controllable photothermal generation and high photothermal conversion efficiency. Among them, the BPN series molecules with higher donor twisting exhibit superior photothermal properties and hold promise for application in highly efficient photothermal therapy.

[0165] (6) The photothermal stability test of BPN-MNPs, BPN-PNPs and BPN-BPNPs is conducted using the following steps:

[0166] Use 0.4W / cm 2 A 15 μM aqueous solution of BPN-MNPs, BPN-PNPs, and BPN-BPNPs was irradiated with a 660 nm laser for 6 min, followed by a 6 min cooling period. This cycle was repeated five times, and the temperature change during this process was monitored. The results are as follows: Figure 6 As shown,

[0167] Figure 6 For BPN-M NPs, BPN-PNPs, and BPN-BPNPs, the activation was achieved through five 660nm laser pulses. Ramp up in the closed cycle Case (molar concentration: 15 μM, light power: 0.4 W / cm 2 ).

[0168] Depend on Figure 6 It can be seen that at 0.4 W / cm 2 Under 660nm laser irradiation, the temperatures of the three types of NPs did not show significant changes after five heating and cooling cycles, indicating that they have good photothermal stability.

[0169] (7) Cytotoxicity test of BPN-PNPs, the specific steps are as follows:

[0170] 4T1 cells in the logarithmic growth phase were evenly seeded into 96-well plates (approximately 5000 cells / well) and incubated at 37°C with 5% CO2. 2 Pre-incubate in an incubator for 24 hours. Then, replace the medium in the 96-well plate with fresh medium containing different concentrations of BPN-PNPs, and continue incubation for another 20 hours. Next, place it under 660nm laser irradiation (0.4W / cm²). 2)5 min, and cells without light were reserved as dark control group. After 4 h incubation, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT solution, 10 μL, 5 mg / mL) was added for another 4 h incubation. Then, the medium containing MTT was removed, and 100 μL of DMSO was added to each well to dissolve the formazan. Finally, the absorbance at 490 nm was measured by a microplate reader. Cells incubated with medium only were used as control. The results were expressed as the percentage of viable cells of BPN-PNPs treated cells relative to control cells. The relative cell viability was calculated according to the following equation 1: Cell viability (%) = (ODsample- ODbackground) / (ODcontrol- ODbackground) x 100% (equation 1). The results are shown in Figure 7 .

[0171] Figure 7 The dark toxicity and phototoxicity of different concentrations of BPN-PNPs on 4T1 cells.

[0172] As can be seen from Figure 7 , under the condition of no light, even if the concentration of BPN-PNPs is as high as 10 μM, the survival rate of 4T1 cells can still remain above 90%, indicating that BPN-PNPs have good biological safety and negligible toxicity in the dark. After irradiation with 0.4 W / cm2 660 nm laser for 5 min, BPN-PNPs showed obvious concentration-dependent cytotoxicity, and the survival rate of 4T1 cells was as low as 25% or less at a concentration of 10 μM, showing high efficiency of light-activated killing of tumor cells.

[0173] (8) Test of the high-resolution NIR-II fluorescence imaging ability of BPN-PNPs on the blood vessels of the legs of mice, with the specific steps as follows:

[0174] 100 μL of BPN-PNPs with a concentration of 700 μmol / L were injected into healthy mice through the tail vein, and then the mice were subjected to in vivo fluorescence imaging using a near-infrared two-zone small animal in vivo imaging instrument. The excitation light source was a 660 nm laser, and the fluorescence images were collected by adjusting different wavelength filters (LP1100, LP1000, and LP900). The results are shown in Figure 8 . The imaging resolution under different filter conditions was analyzed by imaging software.

[0175] Figure 8 The near-infrared two-zone fluorescence imaging of BPN-PNPs on the blood vessels of the legs of normal mice under different filter conditions, and the resolution analysis chart under different filter conditions.

[0176] It can be seen from Figure 8 that as the long pass filter filters longer wavelengths (900 nm, 1000 nm and1100nm), the background interference of the collected images gradually decreased, the signal-to-noise ratio gradually increased, thereby realizing higher imaging resolution. Since the emission intensity of the BPN-PNPs was weak at a wavelength of 1000nm or above, the LP900 filter showed a lower full width at half maximum (FWHM = 610 μm). These results indicate that the BPN-PNPs are expected to be applied to high-resolution near-infrared two-region fluorescence imaging.

[0177] It can be seen from the above examples that the preparation method of the near-infrared two-region organic fluorescent compound with light-heat generation capacity provided by the present application is simple, the separation and purification operation is simple, the near-infrared two-region fluorescent emission, large stokes shift, excellent active oxygen generation capacity and light-heat generation capacity are achieved; under light activation, the cancer cells can be efficiently killed by photodynamic and photothermal synergy, and at the same time, high-resolution vascular imaging can be realized, and the present application is expected to be used for photodynamic and photothermal synergy treatment guided by near-infrared fluorescence imaging.

[0178] The above only describes the preferred embodiments of the present application, and it should be noted that for those skilled in the art, without departing from the principles of the present application, a number of improvements and refinements can be made, and these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. An organic fluorescent compound, characterized in that, It has the structural formula shown in Equation I: R1 is R2 is 2. The organic fluorescent compound according to claim 1, characterized in that, It has the structure shown by any one of the terms Ia to If:

3. The method for preparing the organic fluorescent compound according to claim 1 or 2, characterized in that, Includes the following steps: (1) A compound having the structure shown in Formula II and a compound having the structure shown in Formula III were mixed with acetic anhydride and subjected to a first Nevon reaction to obtain a compound having the structure shown in Formula IV. The R is (2) A second brain-Wenger reaction was carried out by mixing a compound having the structure shown in Formula IV, a compound having the structure shown in Formula V, piperidine, and an organic solvent to obtain the organic fluorescent compound; The compound having the structure shown in formula V is a compound having the structure shown in formula Va or a compound having the structure shown in formula Vb.

4. The preparation method according to claim 3, characterized in that, The molar ratio of the compound having the structure shown in Formula II to the compound having the structure shown in Formula III is 1:(1-3); The ratio of the compound having the structure shown in Formula II to acetic anhydride is 1 mmol: (1-4) mL.

5. The preparation method according to claim 3, characterized in that, The temperature of the first brain von Willebrand reaction is 100–130°C, and the time is 12–24 hours; The first brain Wenger reaction was carried out in a protective gas.

6. The preparation method according to claim 3, characterized in that, The molar ratio of the compound having the structure shown in Formula IV, the compound having the structure shown in Formula V, and piperidine is 1:(2-3):(0.83-6); The ratio of the compound with the structure shown in Formula IV to the organic solvent is 1 mmol: (4-10) mL; the organic solvent includes chloroform.

7. The preparation method according to claim 3, characterized in that, The temperature for the second brain von Willebrand reaction is 20–40°C, and the time is 12–24 hours. The second brain ventricle reaction is carried out in a protective gas atmosphere.

8. The use of the organic fluorescent compound of claim 1 or 2 or the organic fluorescent compound prepared by the preparation method of any one of claims 3 to 7 in the preparation of antitumor drugs, the preparation of antitumor diagnostic reagents or high-resolution vascular imaging for non-disease diagnosis and treatment purposes.

9. An organic fluorescent compound water-dispersible nanoparticle, characterized in that, It includes the organic fluorescent compound as described in claim 1 or 2, or the organic fluorescent compound prepared by the preparation method described in any one of claims 3 to 7, and a coating agent coating the surface of the organic fluorescent compound.

10. The organic fluorescent compound water-dispersible nanoparticles according to claim 9, characterized in that, The coating agent includes one or more of the following: methoxy polyethylene glycol amine, distearyl phosphatidylethanolamine-polyethylene glycol 2000, phosphatidylethanolamine-polyethylene glycol 2000-maleimide, distearyl phosphatidylethanolamine-polyethylene glycol-folic acid, distearyl phosphatidylethanolamine-polyethylene glycol-mercapto, distearyl phosphatidylacetamide-polyethylene glycol 2000-carboxylic acid, distearyl phosphatidylethanolamine-polyethylene glycol 5000-azide, distearyl phosphatidylethanolamine-polyethylene glycol 2000-biotin, 1-palmitoyl-2-oleoylethanolamine, 1-stearoyl-2-oleoyl lecithin, distearyl phosphatidylethanolamine-polyethylene glycol 2000, and poloxamer F127.

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