NIR-II fluorescent small molecule as well as preparation method and application thereof

Abstract

The invention discloses an NIR-II fluorescent small molecule as well as a preparation method and application thereof. The construction (formula I) of the fluorescent small molecule is based on a TQP receptor, and the nano-particles further prepared from the fluorescent small molecule have excellent biocompatibility and light stability, high near-infrared second-region fluorescent brightness, good near-infrared second-region photoacoustic signal and photo-thermal performance, and good application prospects. And near-infrared two-region fluorescence imaging, near-infrared two-region photoacoustic imaging and near-infrared two-region photothermal therapy excited by 1064nm laser can be realized, so that the receptor TQP has an application prospect as a common receptor for constructing a diagnosis and treatment integrated platform excited by 1064nm laser. In addition, the preparation method of the compound and the nano-particles thereof is simple, convenient and easy to implement, good in economical efficiency and beneficial to large-scale production of the pharmaceutical industry.

Description

Technical Field

[0001] The present invention relates to a NIR-II fluorescent small molecule and a preparation method and application thereof, and in particular to a TQP receptor-based DAD-type NIR-II fluorescent small molecule and a preparation method and application thereof. Background Art

[0002] Near-infrared (NIR)-triggered phototherapy utilizes the energy dissipation of different excited states of a material after being excited by near-infrared light. Due to its inherent advantages of being non-invasive, accurate, effective, and well-controllable, it has garnered widespread attention in the treatment of diseases, especially superficial tumors. Currently, phototherapy combines real-time near-infrared fluorescence / photoacoustic imaging (FLI / PAI) with non-invasive photothermal therapy (PTT) / photodynamic therapy (PDT). Compared with FLI and PAI in the first near-infrared window (NIR-I, 700-900nm), FLI and PAI in the second near-infrared window (NIR-II, 1000-1700nm) (NIR-II FLI / PAI) exhibit deeper tissue penetration and better spatiotemporal resolution due to reduced light scattering, self-absorption, and tissue autofluorescence in the NIR-II optical window. This provides higher imaging quality for accurate tumor diagnosis and guidance of surgery or treatment. The same holds true for photothermal performance. Compared to the well-developed NIR-1 photothermal therapy, photothermal therapy using 1064nm excitation in the NIR-2 region offers three key advantages: a higher maximum permissible dose, deeper penetration, and greater safety. Therefore, developing materials for fluorescence imaging and photothermal therapy using NIR-2 laser excitation is crucial.

[0003] In addition, organic small molecules with donor-acceptor-donor (DAD) backbones have attracted extensive attention in NIR-II FLI and PTT due to their superior biocompatibility, negligible toxicity, easy functionalization, and tunable chemical structures. These chemical structures impart specific energy outputs, including: (1) radiative transition of FLI in the form of fluorescence signal; (2) non-radiative transition of PAI / PTT in the form of heat, as well as simultaneously tunable photophysical properties. However, the current DAD-type NIR-II small molecules used for NIR-II FLI and NIR-II PTT mainly rely on the acceptor benzothiadiazole (BBTD). However, its complex synthesis and easy degradation in alkaline and oxidative environments limit the preparation of high-performance NIR-II dyes. Therefore, the synthesis of stable acceptor units for NIR-II FLI and NIR-II PTT is a crucial technical key. Summary of the Invention

[0004] Purpose of the invention: The first purpose of the present invention is to provide a DAD-type NIR-II fluorescent small molecule based on a TQP receptor, the second purpose is to provide a method for preparing the fluorescent small molecule, and the third purpose is to provide an application of the fluorescent small molecule.

[0005] Technical solution: The NIR-II fluorescent small molecule described in the present invention has a structure of formula I:

[0006]

[0007] Among them, the π bridge is Or the π bridge does not exist;

[0008] R is H or Donor D is any of the following structures:

[0009]

[0010] * is the connection site of the structural fragments.

[0011] Preferably, in the structure:

[0012] When the π bridge does not exist, the donor D is * is the connection site of the structural fragment. Preferably, in the structure:

[0013] When the π bridge is When the donor D is any of the following structures:

[0014]

[0015] The definition of R is as described above, and * is the connection site of the structural fragment.

[0016] Further preferably, in the structure:

[0017] When R is When the donor D is * is the connection site of the structural fragments.

[0018] Preferably, the NIR-II fluorescent small molecule of the present invention is selected from any one of the following compounds:

[0019]

[0020] The preparation method of the NIR-II fluorescent small molecule of the present invention is selected from any one of the following methods:

[0021] Methods 1 to 3: React donor D (TPA or F), acceptor A (TQP-Br or TQP-T-Br), tetrakistriphenylphosphine palladium, aqueous potassium carbonate, and anhydrous toluene. After completion of the reaction, post-processing yields the final product, TQP-TPA, TQP-TTPA, or TQP-TF.

[0022] Method 1:

[0023]

[0024] Method 2:

[0025]

[0026] Method 3:

[0027]

[0028] Method 4: The raw materials and iron powder are placed in an acetic acid solution and stirred at high temperature. The reaction is completed and post-processed to obtain the final product M4. This product M4 is then mixed with 9,10-phenanthrenequinone in an acetic acid solution and stirred at high temperature. The reaction is completed and post-processed to obtain the final product M5. Finally, product M5 is placed in anhydrous toluene with the donor D(F'), trisdibenzylideneacetone dipalladium, and tri(o-methylphenyl)phosphine and stirred. The reaction is completed and post-processed to obtain TQP-TC-TF.

[0029]

[0030] The preparation method of TQP-T-Br is as follows:

[0031]

[0032] The two raw materials and the catalyst were reacted in ultra-dry tetrahydrofuran. After completion of the reaction, M1 was obtained through post-processing. M1 and N-bromosuccinimide were then dissolved in a mixture of N,N-dimethylformamide and acetonitrile and stirred at room temperature. A 48% aqueous solution of hydrogen bromide and N-bromosuccinimide were then added. After completion of the reaction, M2 was obtained through post-processing. M2 and iron powder were then stirred in an acetic acid solution. After completion of the reaction, M3 was obtained through post-processing. Finally, M3 was reacted with 9,10-phenanthrenequinone in an acetic acid solution. After completion of the reaction, TQP-T-Br was obtained through post-processing.

[0033] The contrast agent of the present invention is self-assembled by the NIR-II fluorescent small molecule of the present invention and the amphiphilic polymer DSPE-mPEG2000.

[0034] Preferably, the method for preparing the contrast agent comprises the following steps:

[0035] (1) dissolving the near-infrared second region fluorescent molecule of the present invention in tetrahydrofuran;

[0036] (2) Under ultrasonic conditions, the tetrahydrofuran solution was added to an aqueous solution of the amphiphilic polymer DSPE-mPEG2000 for self-assembly;

[0037] (3) dialyzing the self-assembled mixture to obtain the contrast agent.

[0038] The NIR-II fluorescent small molecule of the present invention is used in preparing a reagent for near-infrared second-region fluorescence imaging.

[0039] The NIR-II fluorescent small molecules of the present invention are used in the preparation of photoacoustic imaging reagents.

[0040] The NIR-II fluorescent small molecules described in the present invention are used in the preparation of reagents for photothermal therapy.

[0041] The NIR-II fluorescent small molecule of the present invention is used in preparing drugs for treating vascular diseases and tumors.

[0042] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:

[0043] The present invention designs a series of NIR-II fluorescent small molecules based on TQP receptors through molecular engineering strategies, and further prepares nanoparticles. The nanoparticles have excellent biocompatibility and photostability, high near-infrared zone II fluorescence brightness, good near-infrared zone II photoacoustic signal and photothermal performance, and can realize near-infrared zone II fluorescence imaging, near-infrared zone II photoacoustic imaging, and near-infrared zone II photothermal therapy under 1064nm laser excitation, making the receptor TQP have the application prospect of being a common receptor for constructing an integrated diagnosis and treatment platform under 1064nm laser excitation. In addition, the preparation method of the fluorescent small molecule and its nanoparticles is simple, easy, and economical, which is conducive to the scale-up production of the pharmaceutical industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 This is the mass spectrum of the compound TQP-TPA obtained in Example 1 of the present invention;

[0045] Figure 2 This is the H NMR spectrum (deuterated chloroform) of the compound TQP-TPA obtained in Example 1 of the present invention.

[0046] Figure 3 This is the mass spectrum of the compound TQP-TF obtained in Example 1 of the present invention;

[0047] Figure 4 This is the H NMR spectrum (deuterated chloroform) of the compound TQP-TF obtained in Example 1 of the present invention.

[0048] Figure 5 This is the mass spectrum of the compound TQP-TTPA obtained in Example 1 of the present invention;

[0049] Figure 6 This is the H NMR spectrum (deuterated chloroform) of the compound TQP-TTPA obtained in Example 1 of the present invention;

[0050] Figure 7 This is the mass spectrum of the compound TQP-TC-TF obtained in Example 2 of the present invention;

[0051] Figure 8 This is the H NMR spectrum (deuterated chloroform) of the compound TQP-TC-TF obtained in Example 2 of the present invention;

[0052] Figure 9 Schematic diagram of frontier orbital release and energy levels of the compounds obtained in Examples 1 and 2 of the present invention;

[0053] Figure 10 The standardized ultraviolet absorption spectra of the compounds obtained in Examples 1 and 2 of the present invention;

[0054] Figure 11 is the standardized fluorescence emission spectra of the compounds obtained in Examples 1 and 2 of the present invention;

[0055] Figure 12 The morphology characterization results of the contrast agent obtained in Example 4 of the present invention;

[0056] Figure 13 This is the size characterization result of the contrast agent obtained in Example 4 of the present invention;

[0057] Figure 14 is the absorption spectrum of the contrast agent obtained in Example 4 of the present invention;

[0058] Figure 15 is the fluorescence emission spectrum of the contrast agent obtained in Example 4 of the present invention;

[0059] Figure 16 This is the photothermal heating curve of the contrast agent obtained in Example 4 of the present invention under 1064nm laser;

[0060] Figure 17 This is the in vitro photoacoustic image of the contrast agent obtained in Example 4 of the present invention under 1064nm laser;

[0061] Figure 18 This is the near-infrared second-zone fluorescence imaging of mouse tumors using the contrast agent obtained in Example 4 of the present invention under a 1064 nm laser. DETAILED DESCRIPTION

[0062] The technical solution of the present invention will be further described below in conjunction with embodiments.

[0063] Example 1: Synthesis of small molecules TQP-TPA, TQP-TTPA and TQP-TF in the near-infrared region II

[0064] Donor D (TPA or F, 0.32 mmol), acceptor A (TQP-Br or TQP-T-Br, 0.15 mmol), tetrakistriphenylphosphine palladium (5 mg), aqueous potassium carbonate solution (4 ml), and 20 ml of anhydrous toluene were added to a 50 ml round-bottom flask. After bubbling deoxygenation for 30 minutes, the mixture was stirred at 90°C for 20 hours in the dark. After thin-layer chromatography indicated the reaction was complete, the mixture was cooled to room temperature. The mixture was washed with saturated sodium chloride solution and extracted with dichloromethane three times. The organic phase was collected, dried over anhydrous sodium sulfate, and the solvent was removed in vacuo. The crude product was purified by column chromatography to obtain TQP-TPA (black solid, 80% yield), TQP-TTPA (black solid, 81% yield), and TQP-TF (black solid, 76% yield).

[0065] Structural confirmation: The mass spectrum of the prepared compound TQP-TPA is shown in Figure 1 , NMR hydrogen spectrum see Figure 2 ; The mass spectrum of compound TQP-TF is shown in Figure 3 , NMR hydrogen spectrum see Figure 4 ; The mass spectrum of compound TQP-TTPA is shown in Figure 5 , NMR hydrogen spectrum see Figure 6 .

[0066] Example 2: Synthesis of the Near-Infrared Region II Small Molecule TQP-TC-TF

[0067] The raw material (0.681 mmol), iron powder (10.2 mmol) and acetic acid solution (21.3 ml) were placed in a three-necked flask, connected to a reflux tube, and bubbled for 20-30 min. The mixture was refluxed at 70 °C for 3 h. After the reaction was completed, the mixture was cooled to room temperature and the pH was adjusted to 7 with aqueous sodium hydroxide solution. The mixture was extracted three times. The organic phase was collected and dried over anhydrous sodium sulfate. The solvent was removed in vacuo, and the crude product M4 was directly used for the next step reaction.

[0068] M4 (9,10-phenanthrenequinone, 0.612 mm1) and acetic acid solution (21.3 ml) were placed in a three-necked flask, connected to a reflux tube, and bubbled for 20-30 minutes. The mixture was then refluxed at 70°C in the dark overnight until the color turned into a green insoluble liquid. After the reaction was completed, the mixture was cooled to room temperature, water was added, and the pH was adjusted to 7 with aqueous sodium hydroxide solution. Finally, the mixture was extracted three times, the organic phase was collected and dried over anhydrous sodium sulfate, the solvent was removed in vacuo, and the crude product was purified by column chromatography (n-hexane: petroleum ether = 10:1) to obtain a green solid M5 (TQP-TC-Br, yield 80%).

[0069] Donor D (F', 0.46 mm1), acceptor A (TQP-T-Br, 0.21 mm1), trisdibenzylideneacetone dipalladium (5 mg), tri(o-methylphenyl)phosphine (32 mg) and anhydrous toluene (15 ml) were added to a 50 ml round-bottom flask. After bubbling and deoxygenation for 30 minutes, the mixture was stirred at 110°C for 24 hours in the dark. After thin-layer chromatography showed that the reaction was complete, the mixture was cooled to room temperature. The mixture was washed with saturated sodium chloride aqueous solution and extracted with dichloromethane, which was repeated 3 times. The organic phase was collected and dried over sodium sulfate, and the solvent was removed in vacuo. The crude product was purified by column chromatography to obtain TQP-TC-TF (brown solid, 50% yield).

[0070] Structural confirmation: The mass spectrum of the prepared compound TQP-TC-TF is shown in Figure 7 , NMR hydrogen spectrum see Figure 8 .

[0071] Example 3: Spectroscopic Characterization of Near-Infrared Region II Fluorescent Small Molecules

[0072] The near-infrared second-region fluorescent small molecules (1 mg) obtained in Examples 1 and 2 were dissolved in tetrahydrofuran (2 ml), and then their absorption and emission spectra were measured by ultraviolet absorption spectroscopy and fluorescence emission spectroscopy, respectively.

[0073] like Figure 9 As shown, the wave function diagrams of the highest occupied molecular orbital and the lowest unoccupied molecular orbital simulated by computer.

[0074] From the above results, it can be seen that with the extension of the conjugated skeleton and the improvement of the introduction of strong electron donors, TQP-TPA, TQP-TF, TQP-TTPA and TQP-TC-TF show a gradually decreasing energy gap (ΔE HOMO-LUMO ), which are 1.75, 1.59, 1.38, and 1.28 eV, respectively. This theoretical result shows that a planar electron acceptor, a long conjugated backbone, and a strong electron donor play a crucial role in the design of DAD-type fluorescent small molecules with NIR-II absorption ability.

[0075] like Figure 10 and Figure 11 Shown are the normalized UV absorption and fluorescence emission spectra of four near-infrared region II fluorescent small molecules at the same concentration.

[0076] The above results show that the UV absorption wavelengths of TQP-TPA, TQP-TF, TQP-TTPA, and TQP-TC-TF are located at 676, 793, 843, and 858 nm, respectively. This result is consistent with theoretical calculations. Furthermore, their corresponding emission spectra are 976, 1032, 1103, and 1094 nm, respectively. TQP-TC-TF exhibits excellent absorption at 1064 nm, enabling near-infrared second-zone fluorescence imaging under 1064 nm laser light.

[0077] Example 4: Preparation and characterization of contrast agents

[0078] Dissolve 1 mg of the near-infrared second region molecule TQP-TC-TF in 2 ml of tetrahydrofuran, and then add 10-20 mg of the amphiphilic polymer DSPE-mPEG 2000 Dissolve in 20 ml of water, then add the TQP-TC-TF tetrahydrofuran solution to the DSPE-mPEG 2000 The final water-soluble nanoparticles were obtained by ultrasonic mixing in aqueous solution at room temperature. The excess tetrahydrofuran solution was removed by dialysis and stored in a refrigerator at 4°C.

[0079] like Figure 12 and Figure 13 As shown, the morphology (transmission electron microscopy) and size (dynamic light scattering) of the obtained nanoparticles (0.1 mg / ml) were characterized.

[0080] As can be seen from the above results, transmission electron microscopy results show that the morphology of the contrast agent nanoparticles is solid spherical, with a particle size of approximately 55nm. In addition, dynamic light scattering results show that the hydrodynamic diameter of the contrast agent nanoparticles is approximately 68nm.

[0081] like Figure 14 Shown are the absorption spectra of the obtained nanoparticles (0.1 mg / ml).

[0082] From the above results, it can be seen that the absorption peak of the nanoparticles is 864 nm.

[0083] like Figure 15 As shown, the fluorescence emission spectrum (1064 nm laser excitation) of the obtained nanoparticles (0.1 mg / ml) and its corresponding in vitro near-infrared second-zone fluorescence imaging.

[0084] From the above results, it can be seen that the nanoparticles have two-zone emission, indicating that the nanoparticles can achieve near-infrared two-zone fluorescence imaging under 1064nm laser excitation.

[0085] like Figure 16As shown, the photothermal heating curves of the obtained nanoparticles at different concentrations (under 1064nm laser irradiation).

[0086] The above results show that the nanoparticles' temperature rise curve is positively correlated with their concentration, meaning that their temperature increases with increasing concentration. The maximum temperature change of the nanoparticles at a concentration of 200 μM after 6 minutes of laser irradiation was 36.5°C. This indicates that the nanoparticles possess excellent photothermal properties and can achieve photothermal therapy under 1064 nm laser excitation.

[0087] like Figure 17 As shown, in vitro photoacoustic imaging (under 1064 nm laser irradiation) of the obtained nanoparticles at different concentrations.

[0088] The above results show that the nanoparticles exhibit a significant concentration-dependent NIR II photoacoustic signal, with the signal intensity increasing with increasing concentration. This indicates that the nanoparticles have a good photoacoustic signal and can achieve NIR II photoacoustic imaging under 1064nm laser excitation.

[0089] like Figure 18 As shown, the obtained nanoparticles were prepared into an aqueous solution with a concentration of 2 mg / ml, and 150 μl of the nanoparticle aqueous solution was injected into the tail vein of prostate RM1 subcutaneous C57 black mice. The tumor site of the mice was imaged by near-infrared zone II fluorescence imaging (1064 nm laser) using a near-infrared zone II imager to monitor and observe the accumulation of the nanoparticles in the tumor.

[0090] The above results show that initially, there was almost no signal at the tumor site. Over time, a fluorescent signal began to appear at the tumor site after 4-8 hours, reaching a maximum at 12 hours. Subsequently, the fluorescence signal intensity significantly decreased. Therefore, it can be seen that 12 hours after tail vein injection, the nanoparticles reached maximum accumulation at the tumor site, which is conducive to subsequent near-infrared second-zone photothermal therapy under 1064nm excitation.

[0091] From the above results, it can be seen that the nanoparticles have excellent biocompatibility and photostability, high near-infrared zone II fluorescence brightness, good near-infrared zone II photoacoustic signal and photothermal performance, and can realize near-infrared zone II fluorescence imaging, near-infrared zone II photoacoustic imaging and near-infrared zone II photothermal therapy excited by 1064nm laser.

Claims

1. A NIR-II fluorescent small molecule, characterized in that Having the structure of Formula I: Among them, the π bridge is Or the π bridge does not exist; R is H or Donor D is any of the following structures: * is the connection site of the structural fragments.

2. The NIR-II fluorescent small molecule according to claim 1, characterized in that In the structure: When the π bridge does not exist, the donor D is * is the connection site of the structural fragments.

3. The NIR-II fluorescent small molecule according to claim 1, characterized in that In the structure: When the π bridge is When the donor D is any of the following structures: The definition of R is as described in claim 1, and * is the connection site of the structural fragment.

4. The NIR-II fluorescent small molecule according to claim 3, characterized in that In the structure: When R is When the donor D is * is the connection site of the structural fragments.

5. The NIR-II fluorescent small molecule according to claim 1, characterized in that It is selected from any one of the following compounds:

6. A method for preparing the NIR-II fluorescent small molecule according to claim 1, characterized in that: It is selected from any one of the following methods: Method 1: Method 2: Method 3: Method 4:

7. Use of the NIR-II fluorescent small molecule according to claim 1 in the preparation of a near-infrared second region fluorescence imaging agent.

8. Use of the NIR-II fluorescent small molecule according to claim 1 in the preparation of a photoacoustic imaging agent.

9. Use of the NIR-II fluorescent small molecule according to claim 1 in the preparation of a photothermal therapy agent.

10. Use of the NIR-II fluorescent small molecule according to claim 1 in the preparation of drugs for treating vascular diseases and tumors.

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