A near-infrared II region hemicyanine compound, its preparation method and application

Abstract

This invention provides a near-infrared II hemicyanine compound, its preparation method, and its applications. Based on the distorted intramolecular charge transfer effect, the hemicyanine molecule was modified to obtain three near-infrared II hemicyanine fluorescent molecules: HCY-945, HCY-995, and HCY-1080. These synthesized molecules exhibit near-infrared II fluorescence emission. HCY-995 and HCY-1080 also possess high photothermal conversion efficiency, enabling simultaneous imaging and treatment of in vivo tumors.

Description

Technical Field

[0001] This invention belongs to the field of organic synthesis, and in particular relates to a near-infrared II hemicyanine compound, its preparation method, and its application. Background Technology

[0002] In recent years, phototherapy has attracted much attention because it allows for simultaneous in-situ imaging and treatment upon light initiation, opening up new avenues and providing a novel approach for disease research. Among existing diagnostic and therapeutic methods, fluorescence therapy has unique advantages, including high sensitivity, rapid response, and non-invasiveness. In fluorescence therapy, near-infrared II fluorescence possesses high resolution, high signal-to-noise ratio, and deep tissue penetration capabilities, thus opening new avenues for high-quality imaging of deep tissues. Currently, various materials for near-infrared II fluorescence diagnostic imaging and therapy are being continuously explored and prepared to construct fluorescence diagnostic and therapeutic systems, including quantum dots, rare earth elements, and small organic molecules. Because small organic molecules have well-defined structures, are easily metabolized, and possess great structural versatility, their optical and photothermal properties can be well designed through fine-tuning of their molecular structure, leading to their widespread application.

[0003] Cancer poses a significant threat to human health and life, being one of the leading causes of death. Therefore, early diagnosis and targeted treatment of cancer are crucial. Contrast-assisted medical imaging plays a vital role in cancer diagnosis, with various imaging techniques widely used for early diagnosis due to their convenience, low cost, and real-time imaging capabilities. Among recently developed novel cancer therapies, photothermal therapy has garnered significant attention due to its ease of operation, non-invasiveness, and therapeutic selectivity, and is being applied in ablation therapy research for solid tumors. Traditionally, cancer diagnosis and treatment are two relatively independent processes in clinical practice, requiring separate use of diagnostic contrast agents and therapeutic reagents. The long interval between these two procedures can easily delay optimal treatment, and the cumulative side effects of two injections can increase patient suffering and risks. Therefore, in recent years, phototherapy has attracted considerable attention because it allows for simultaneous in-situ imaging and treatment upon light initiation, providing a new approach to the research and treatment of related diseases.

[0004] Among existing diagnostic and therapeutic methods, fluorescence-photothermal therapy possesses unique advantages due to its high sensitivity, rapid response, and non-invasiveness. Near-infrared II fluorescence, with its high resolution, high signal-to-noise ratio, and deep tissue penetration, provides high-quality images of deep tissues, giving it a unique advantage in in vivo fluorescence diagnosis. Currently, various materials for near-infrared II fluorescence diagnostic imaging and therapy are being continuously explored to construct fluorescence diagnostic and therapeutic systems, including quantum dots, rare earth elements, and small organic molecules. Small organic molecules are widely used because their well-defined structures, easy metabolism, and high structural versatility allow for precise design of their optical and photothermal properties through fine-tuning of molecular structures. However, near-infrared II organic fluorescent molecules with both fluorescence and photothermal properties often face problems such as small Stokes shift, low fluorescence quantum yield (QY), and poor photothermal conversion efficiency (PCE). Since fluorescence generation is a radiative transition of electrons from a singlet excited state to the ground state in a molecule, while photothermal generation is a non-radiative transition, and radiative and non-radiative transitions are a pair of competing photophysical processes that are difficult to balance. Therefore, there are still very few reports on organic near-infrared II molecules with good optical properties and high photothermal conversion efficiency. Twisted intramolecular charge transfer (TICT) is a twisted excited-state electron transfer process, usually consisting of single-bonded donor and acceptor moieties. [3] When a molecule is in an excited state, its structure is distorted, and it then returns to the ground state mainly through nonradiative relaxation, accompanied by a redshift in emission wavelength, an increased Stokes shift, and enhanced photothermal activity. Therefore, TICT (Transient Induction of Light) possesses the properties of redshifting molecular wavelengths, increasing the Stokes shift, and enhancing photothermal activity. Hemicyanine is one of the main members of the cyanine family. Its unique D-π-A structure gives it excellent TICT properties, resulting in a large Stokes shift and good biocompatibility, making it widely used in biomedical imaging. However, the emission wavelengths of existing hemicyanine fluorescent molecules are generally short, making them difficult to apply effectively in vivo. Summary of the Invention

[0005] In view of this, the present invention aims to propose a near-infrared II hemicyanine compound, its preparation method and application, and to adjust its optical and photothermal properties by regulating the TICT (twisted intramolecular charge transfer) properties of the hemicyanine molecule to make it a high-performance near-infrared II phototherapy molecule.

[0006] To achieve the above objectives, the technical solution of the present invention is implemented as follows:

[0007] A hemicyanine compound in the near-infrared II region, the general structural formula of which is shown in formula (I):

[0008]

[0009] X is one of O, S, and Se.

[0010] n is a natural number, preferably 0, 1, 2, 3, or 4.

[0011] The synthetic route for this compound is as follows:

[0012]

[0013] n is a natural number; preferably, n is 0, 1, or 2.

[0014] Compound (Ⅰ) was obtained by coupling a pyridine salt, an aldehyde-containing triphenylamine, and a furan ring.

[0015] By using the Knoevenagel reaction and the Suzuki coupling reaction to couple pyridinium salts, aldehyde-containing triphenylamines, and furan rings, all the target molecules in this work can be synthesized through these two reactions.

[0016] The synthetic route for this compound is as follows:

[0017]

[0018] n is a natural number; preferably, n is 0, 1, or 2.

[0019] Compound (Ⅰ) was obtained by coupling a pyridine salt, an aldehyde-containing triphenylamine, and a furan ring.

[0020] The structural formula of this compound is shown in formula (HCY-945):

[0021]

[0022] The synthesis route is as follows:

[0023]

[0024] A1: Using 1,8-naphthenic acid and 1-iodohexadecane as raw materials, NaH and N,N-dimethylformamide were added, and after the reaction was completed, compound A-1 was obtained;

[0025] A2: Compound A-1 was dissolved in tetrahydrofuran, methyl magnesium chloride was added, and after the reaction was completed, compound A-2 was obtained;

[0026] A3: Compound (HCY-945) was obtained by adding anhydrous ethanol to compound A-2 and 4-diphenylaminobenzaldehyde as raw materials and treating the mixture after the reaction was completed.

[0027] The structural formula of this compound is shown in formula (HCY-995):

[0028]

[0029] The synthesis route is as follows:

[0030]

[0031] B1: Using 4-(diphenylamino)phenylboronic acid pinacol ester and 5-bromofuran-2-carboxaldehyde as raw materials, tetra(triphenylphosphine)palladium, toluene / ethanol solution and sodium carbonate aqueous solution were added. After the reaction was completed, compound A-3 was obtained.

[0032] B2: Using compounds A-3 and A-2 as raw materials, and anhydrous and alcohol-free solvents, the reaction was completed and the compound (HCY-995) was obtained.

[0033] The structural formula of this compound is shown in formula (HCY-1080):

[0034]

[0035] The synthesis route is as follows:

[0036]

[0037] C1: Using 5-bromofuran-2-carboxaldehyde and 2-(tributyltinyl)furan as raw materials, 2-(tributyltinyl)furan and tetra(triphenylphosphine)palladium were added, and after the reaction was completed, compound A-4 was obtained;

[0038] C2: Compound A-4, N-bromosuccinimide and N,N-dimethylformamide are reacted, and after the reaction is completed, compound A-5 is obtained;

[0039] C3: Using compounds A-5, 4-(diphenylamino)phenylboronic acid pinacol ester, and 4-(diphenylamino)phenylboronic acid pinacol ester as raw materials, tetrakis(triphenylphosphine)palladium, N,N-dimethylformamide, and potassium carbonate aqueous solution are added, and after reaction, compound A-6 is obtained;

[0040] C4: Using compounds A-6 and A-2 as raw materials, anhydrous ethanol was added, and after the reaction was completed, compound (HCY-1080) was obtained.

[0041] Nanoparticles of hemicyanine compounds, characterized in that: the compound is prepared as compound nanoparticles,

[0042] The preparation of the compound nanoparticles includes: adding polylactic acid-glycolic acid copolymer and compound (Ⅰ) to chloroform to dissolve them, adding polyvinyl alcohol to mix, sonicating, and stirring overnight.

[0043] Nanoparticles of hemicyanine compounds are used in in vivo vascular imaging and the preparation of antitumor drugs;

[0044] The compound nanoparticles, when combined with light, can be used to inhibit the growth of tumor cells.

[0045] Preferably, the tumor cells include one or more selected from colorectal cancer cells, breast cancer cells, lung cancer cells, gastric cancer cells, liver cancer cells, human placental choriocarcinoma cells, and cervical cancer cells.

[0046] This paper utilizes intramolecular charge transfer effect (TICT) to construct near-infrared II hemicyanine molecules HCY-945, HCY-995, and HCY-1080 for fluorescence imaging and photothermal synergistic therapy of diseases. These molecules share the same electron-withdrawing pyridinium salt and electron-donating triphenylamine groups, linked by varying numbers of furan rings. With increasing furan ring count, the fluorescence quantum yield decreases, the photothermal conversion efficiency increases, and the Stokes shift increases. Compared to HCY-945 and HCY-1080, HCY-995 exhibits a quantum yield of 0.09%, a photothermal conversion efficiency of 54.9%, and a Stokes shift of 232 nm, achieving a good balance between light and heat. This property enables HCY-995 to perform well in high-resolution imaging of blood vessels and photothermal therapy of tumors, realizing photothermal integration.

[0047] In this work, based on the unique D-π-A structure of hemicyanine molecules, we first used a positively charged pyridinium salt and triphenylamine, which has good electron-donating ability, as electron acceptors and donors, respectively. Then, we selected furan as a conjugated π-bridge to better promote the transfer of electrons from triphenylamine to pyridinium salt. Furthermore, since aggregation between small organic molecules can lead to fluorescence quenching, we attached a long alkyl chain to the pyridinium salt as a shielding group to avoid intermolecular aggregation quenching, improve fluorescence brightness, and maintain molecular rotation, which is beneficial for molecular heat generation. This resulted in a near-infrared II hemicyanine molecule with high fluorescence quantum yield, good photothermal conversion efficiency, and large Stokes shift, making it well-suited for the diagnosis and treatment of tumors.

[0048] Compared with existing technologies, the near-infrared II hemicyanine compound, its preparation method, and its application described in this invention have the following advantages:

[0049] This patent modifies hemicyanine molecules based on the distorted intramolecular charge transfer effect to obtain three near-infrared II fluorescent hemicyanine molecules: HCY-945, HCY-995, and HCY-1080. Experimental results show that these synthesized molecules exhibit near-infrared II fluorescence emission. HCY-995 and HCY-1080 also possess high photothermal conversion efficiency, enabling simultaneous imaging and treatment of in vivo tumors. Attached Figure Description

[0050] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings:

[0051] Figure 1 This is a schematic diagram of the ultraviolet absorption spectra of compounds HCY-945, HCY-995, and HCY-1080 in dichloromethane, as described in the embodiments of the present invention.

[0052] Figure 2 This is a schematic diagram of the fluorescence emission spectra of compounds HCY-945, HCY-995, and HCY-1080 in dichloromethane, as described in the embodiments of the present invention.

[0053] Figure 3 The relative quantum yields of HCY-945, HCY-995, and HCY-1080 were determined in CH2Cl2.

[0054] Figure 4 Photothermal images of HCY-945-NPs, HCY-995-NPs, and HCY-1080-NPs under 721nm laser irradiation (1.0Wcm-2);

[0055] Figure 5 The molecular structures of NIR-II fluorophores with different emission wavelengths and HCY-945, HCY-995 and HCY-1080 are reported;

[0056] Figure 6 Photothermal conversion efficiency (PEC) at 721 nm for HCY-945-NPs, HCY-995-NPs, and HCY-1080-NPs;

[0057] Figure 7 The maximum emission wavelengths of HCY-945, HCY-995, and HCY-1080 in different solvents;

[0058] Figure 8 The fluorescence intensity (I / I0) of HCY-945, HCY-995, and HCY-1080 in a tetrahydrofuran (THF) / toluene (PhMe) mixture is shown as a function of the toluene ratio.

[0059] Figure 9 The fluorescence intensity (I / I0) of HCY-945, HCY-995, and HCY-1080 in a mixture of hydrogen furan / acetonitrile (ACN) as a function of the acetonitrile ratio is shown.

[0060] Figure 10 The correlation between solvent polarity parameters and Stock shift for HCY-945, HCY-995, and HCY-1080;

[0061] Figure 11 Theoretical calculations of HOMO and LUMO energy levels for compounds HCY-945, HCY-995, and HCY-1080;

[0062] Figure 12 This is a schematic diagram of the self-assembly and cytotoxicity assays of nanoparticles.

[0063] Figure 13 Cell viability of 4T1 cells after exposure to darkness (B) and near-infrared light (C) (721nm, 1.0W cm-2, 5min) with different concentrations of HCY-995-NPs;

[0064] Figure 14 Fluorescence imaging of 4T1 cells after different treatments with co-staining of calcein AM (green) and propidium iodide (red) (cells were incubated with HCY-995-NPs (0.05mM) and then irradiated with near-infrared light at 721nm, 1.0W cm-2, for 5min, scale bar: 200μm);

[0065] Figure 15 To analyze apoptosis and necrosis of 4T1 cells after different treatments by flow cytometry; after incubating cells with HCY-995-NPs (0.05mM), they were irradiated with near-infrared light (721nm, 1.0W cm-2, 5min).

[0066] Figure 16 Imaging of capillaries filled with ICG(A) and HCY-995-NPs(B) immersed in 1% lipid of different thicknesses under intrinsic 721 nm excitation (0.5 W cm-2, 1000 nm long-pass filter);

[0067] Figure 17 NIR-II fluorescence imaging of blood vessels in the mouse brain (C), hind limbs (D), and abdomen (E);

[0068] Figure 18 The fluorescence intensity distribution (black line) and Gaussian fitting (red line) are shown along the yellow dashed lines (F: SBR = 4.8, G: SBR = 10.1, H: SBR = 10.6);

[0069] Figure 19 This is a diagram illustrating the principle of internal phototherapy.

[0070] Figure 20 In vivo fluorescence imaging of subcutaneous tumor models (0.2 mM, 200 μL, 0.50 W cm⁻², 1000 nm long-pass filter) after intravenous injection of HCY-995-NPs at different time points;

[0071] Figure 21The curves showing the changes in 4T1 tumor temperature in mice before and after HCY-995-NPs and laser irradiation with irradiation time are shown.

[0072] Figure 22 Thermal infrared images of 4T1 tumor-bearing mice after intravenous injection of HCY-995-NPs or saline followed by 1.0W cm-2 (721nm) laser irradiation for 5 min (8 h after injection).

[0073] Figure 23 Photos of tumors taken 15 days after different treatments;

[0074] Figure 24 The average weight of the tumor 15 days after treatment;

[0075] Figure 25 Tumor volume changes during treatment in each group (H&E analysis of tumor tissue in mice at the end of treatment. Scale bar: 100 μm);

[0076] Figure 26 The images are TEM images and dynamic light scattering diagrams (A is a TEM image of HCY-945-NPs, B is a TEM image of HCY-995-NPs, C is a TEM image of HCY-1080-NPs, E is a dynamic light scattering diagram of HCY-945-NPs, F is a dynamic light scattering diagram of HCY-995-NPs, and G is a dynamic light scattering diagram of HCY-1080-NPs).

[0077] Figure 27 The diagram shows the UV absorption of molecular nanoparticle solutions (A is the UV absorption of HCY-945-NPs, B is the UV absorption of HCY-995-NPs, and C is the UV absorption of HCY-1080-NPs).

[0078] Figure 28 The curves are negative natural logarithmic curves of cooling time (t) versus temperature driving force (-lnθ) (A is HCY-945-NPs, B is HCY-995-NPs, C is HCY-1080-NPs);

[0079] Figure 29 This is a schematic diagram illustrating biocompatibility (A is the UV absorption spectrum of HCY-945-NPs, B is the UV absorption spectrum of HCY-995-NPs, and C is the UV absorption spectrum of HCY-1080-NPs). Detailed Implementation

[0080] It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other.

[0081] The present invention will now be described in detail with reference to the accompanying drawings and embodiments.

[0082] Example 1:

[0083] HCY-945 synthesis route map:

[0084]

[0085] A1: 1,8-Naphthyleneamide (0.50 g, 2.95 mmol, 1.0 equiv) was added to a 50 mL three-necked flask, followed by 15 mL of DMF. The mixture was stirred in an ice bath for 10 min, then NaH (0.18 g, 4.40 mmol, 1.5 equiv) was added and the reaction was allowed to proceed for 0.50 h. Hexadecane iodoforme (0.44 g, 3.54 mmol, 1.2 equiv) was then added, and the reaction was carried out at 40 °C for 4 h under an argon atmosphere. After the reaction was complete, the DMF was evaporated to dryness, and the mixture was separated by silica gel column chromatography. The petroleum ether:ethyl acetate ratio was 20:1, yielding 750 mg of a yellow solid compound B-1, with a yield of 64%.

[0086] A2: Add B-1 (0.50 g, 1.3 mmol, 1.0 equiv) and 10 mL of THF to a 50 mL three-necked flask, stir for 10 min in an ice bath, add methylmagnesium chloride (0.7 mL, 2.6 mmol, 2.0 equiv), react in an ice bath for 20 min, heat to 60 °C, and react for 1.5 h. Maintain argon protection throughout the reaction. After cooling to room temperature, add 10 mL of hydrochloric acid solution (2 M), stir for 0.5 h (the solution changes from yellow to green). Add 5 mL of potassium iodide solution (1 M) (the solution changes from green to red). Filter the solid and wash three times with deionized water. 0.55 g of red product B-2 is obtained, yield 83%.

[0087] A3: B-2 (0.21 g, 0.38 mmol, 1.00 equiv.) and 4-diphenylaminobenzaldehyde (0.11 g, 0.40 mmol, 1.00 equiv.) were added to a 50 mL three-necked flask, and 15 mL of anhydrous ethanol was added. The mixture was reacted at 85 °C for 5 h under argon protection. After the reaction was completed, the mixture was cooled to room temperature, the ethanol was evaporated, and the mixture was purified by column chromatography (dichloromethane / methanol = 20 / 1) to obtain 0.12 g of HCY-945 dark green solid, with a yield of 49.5%.

[0088] Example 2

[0089] HCY-995 Synthesis Roadmap:

[0090]

[0091] B1: 5-Bromofuran-2-carboxaldehyde (0.60 g, 3.45 mmol, 1 equiv.), pinacol 4-(diphenylamino)phenylboronic acid (1.11 g, 3.83 mmol, 1.1 equiv.), and tetra(triphenylphosphine)palladium (0.044 g, 0.038 mmol, 0.01 equiv.) were added to a three-necked round-bottom flask. Toluene / ethanol (50 / 5 ml) and 2M sodium carbonate aqueous solution (5 ml) were added via syringe. The reaction was heated at 110 °C for 12 h under argon protection. After cooling to room temperature, the reaction solution was evaporated to dryness. The solution was dissolved in dichloromethane, extracted with water, and the organic phase was collected and dried over anhydrous sodium sulfate. Purification was performed by column chromatography (petroleum ether / ethyl acetate = 10 / 1) to give 0.45 g of yellow solid B-3, yield 38%.

[0092] B2: Add B-2 (0.15 g, 0.29 mmol, 1.00 equiv.) and B-3 (0.15 g, 0.44 mmol, 1.50 equiv.) to a 50 mL three-necked flask, add 15 mL of anhydrous ethanol, and react at 85 °C for 5 h under argon protection. After the reaction is complete, cool to room temperature, evaporate the ethanol, and purify by column chromatography (dichloromethane / methanol = 20 / 1) to obtain 0.16 g of HCY-995 dark green solid, yield 66.1%.

[0093] Example 3:

[0094] Synthetic route of HCY-1080

[0095]

[0096] C1: Under argon protection, 5-bromofuran-2-carboxaldehyde (0.87 g, 5.00 mmol, 1.00 equiv.), 2-(tributyltinyl)furan (2.14 g, 6.00 mmol, 1.20 equiv.), and tetra(triphenylphosphine)palladium (0.15 g, 0.13 mmol, 0.13 equiv.) were added to a 50 mL three-necked flask, along with anhydrous dioxane (15 mL). The reaction was carried out at 80 °C for 24 hours. After the reaction was completed and cooled to room temperature, the residue was dissolved in ethyl acetate, filtered through diatomaceous earth, and the filtrate was collected and evaporated to dryness. The solution was purified by column chromatography (petroleum ether / ethyl acetate = 95 / 5) to give a yellow liquid B-40.60 g, yield 74%.

[0097] C2: Under argon protection, B-4 (0.25 g, 3.00 mmol, 1.00 equiv.) and N-bromosuccinimide (0.65 g, 3.60 mmol, 1.20 equiv.) were added to DMF (7 ml) and stirred in an ice bath for 12 hours, protected from light, and gradually brought to room temperature. After the reaction was complete, the reaction solution was poured into cold water, and the precipitate was collected. The precipitate was washed with water and dried under vacuum to give 0.60 g of purple solid B-5, yield 81%.

[0098] C3: B-5 (0.20 g, 0.83 mmol, 1.10 equiv.), 4-(diphenylamino)phenylboronic acid pinacol ester (0.28 g, 0.75 mmol, 1.00 equiv.), tetra-n-butylammonium bromide (0.048 g, 0.15 mmol, 0.20 equiv.), and tetra(triphenylphosphine)palladium (0.015 g, 0.02 mmol, 0.03 equiv.) were added to a 100 mL three-necked round-bottom flask. N,N-dimethylformamide (30 mL) and 2 M potassium carbonate aqueous solution (5 mL) were added via syringe. The reaction was carried out at 85 °C for 12 h under argon protection. After the reaction was complete and cooled to room temperature, the reaction solution was evaporated to dryness. The solution was dissolved in dichloromethane, extracted with water, and the organic phase was collected and dried over anhydrous Na₂SO₄. Purification was performed using a chromatographic column (petroleum ether / ethyl acetate = 10 / 1) to give 0.24 g of yellow solid B-6, with a yield of 78.6%.

[0099] C4: B-2 (0.29 g, 0.55 mmol, 1.00 equiv.) and B-6 (0.25 g, 0.62 mmol, 1.13 equiv.) were added to a 50 mL three-necked flask, and 15 mL of anhydrous ethanol was added. The mixture was reacted at 85 °C for 5 h under argon protection. After the reaction was completed, the mixture was cooled to room temperature, the ethanol was evaporated, and the mixture was purified by column chromatography (dichloromethane / methanol = 20 / 1) to obtain 0.15 g of HCY-1080 dark green solid, with a yield of 29.6%.

[0100] Example 4:

[0101] Take 25 mg of PLGA (polylactic acid-glycolic acid copolymer) and 2 mg of HCY-995 obtained in Example 2, add 0.5 mL of chloroform to dissolve it, add 2.5 mL of 1% PVA (polyvinyl alcohol) and mix, sonicate, stir overnight to obtain HCY-995 nanoparticles.

[0102] Comparative Example 1:

[0103] Purchase a batch of mice that have already been modeled. The model consists of mice with tumors in their bodies. Treat five mice with saline solution.

[0104] Comparative Example 2

[0105] Using the same mice as in Comparative Example 1, five mice were treated simultaneously with saline and light.

[0106] Example 5

[0107] Five mice were treated with HCY-995 nanoparticles (HCY-995 nanoparticles prepared in Example 4) using the same mice as in Comparative Example 1.

[0108] Example 6

[0109] Using the same mice as Comparative Example 1, five mice were simultaneously treated with HCY-995-NPS (HCY-995 nanoparticles prepared in Example 4) and phototherapy.

[0110] Example 7:

[0111] Take 25 mg of PLGA (polylactic acid-glycolic acid copolymer) and 2 mg of HCY-995 obtained in Example 1, add 0.5 mL of chloroform to dissolve it, add 2.5 mL of 1% PVA (polyvinyl alcohol) and mix, sonicate, and stir overnight to obtain HCY-995-NPs.

[0112] Example 8

[0113] Take 25 mg of PLGA (polylactic acid-glycolic acid copolymer) and 2 mg of HCY-995 obtained in Example 3, add 0.5 mL of chloroform to dissolve it, add 2.5 mL of 1% PVA (polyvinyl alcohol) and mix, sonicate, stir overnight to obtain HCY-1080-NPs.

[0114] It can be seen that HCY-995 nanoparticles have a good therapeutic effect on tumors, and that HCY-995 nanoparticles and phototherapy have a synergistic effect, realizing the integration of photothermal diagnosis and treatment. Furthermore, it has been able to achieve near-infrared II region coverage for hemicyanine molecules.

[0115] Optical performance:

[0116] By coupling a pyridinium salt, an aldehyde-containing triphenylamine, and a furan ring using the Knoevenagel and Suzuki coupling reactions, three hemicyanine molecules, HCY-945, HCY-995, and HCY-1080, were obtained. All the target molecules in this work can be synthesized using these two reactions.

[0117] The products prepared in Examples 1, 2, and 3 were subjected to UV and fluorescence property tests in dichloromethane. The spectra are shown below. Figure 1 , Figure 2As shown, the UV absorption peaks of HCY-945, HCY-995, and HCY-1080 are 645 nm, 763 nm, and 790 nm, respectively, while their fluorescence emission peaks are 945 nm, 995 nm, and 1080 nm, respectively. This indicates that with the increase of furan number, both UV absorption and fluorescence emission peaks redshift. Figure 5 As shown, the structures and wavelengths of existing HCY-945, HCY-995, and HCY-1080 are different. The hemicyanine molecule in this patent has a longer wavelength compared to the hemicyanine molecules that have been reported previously.

[0118] Quantum yield:

[0119] Using IR-26 as a reference, the quantum yields of HCY-945, HCY-995, and HCY-1080 were calculated to be 0.38%, 0.09%, and 0.019%, respectively. Figure 3 As shown, this result indicates that the quantum yield decreases sequentially with increasing furan ring size.

[0120] Photothermal properties:

[0121] Water-soluble nanoparticles of the compounds were obtained by encapsulating HCY-945, HCY-995 and HCY-1080 with PLGA. To characterize the size and morphology of the nanoparticles, we tested them by dynamic light scattering (DLS) and transmission electron microscopy (TEM).

[0122] TEM images show that the compound's nanoparticles have a spherical morphology with an average diameter of 90–110 nm. Furthermore, DLS indicates that the hydrodynamic diameter of the compound's nanoparticles is approximately 200 nm. The relatively small size obtained by TEM measurement is likely due to the shrinkage of the hydration layer in the dried TEM sample. Due to the high permeability and retention effect (EPR) of solid tumors, nanoparticles with diameters of 10–200 nm can aggregate at the tumor site, such as... Figure 26 As shown. Furthermore, we tested the UV absorption of the encapsulated molecular nanoparticle solution, such as... Figure 27 As shown.

[0123] Using a 721nm laser (1.0W / cm) 2 The solution of nanoparticles encapsulating the target molecules was irradiated. The results showed that the HCY-945 nanoparticle solution did not show a significant temperature change after 240 seconds of laser irradiation, maintaining the ambient temperature. However, the temperature of the HCY-995 nanoparticle solution reached its highest value of 51.2℃ after 180 seconds of irradiation, an increase of 23.7℃ (ΔT). The temperature of the HCY-1080 nanoparticle solution reached its highest value of 53.5℃ after 36 seconds of irradiation, an increase of 26℃ (ΔT). Figure 5 , 6As shown. Furthermore, PCE is a very important parameter for evaluating the photothermal properties of molecules. Therefore, at 1.0 W / cm²... 2 The temperature change of the solution containing target molecular nanoparticles was measured under laser irradiation. The laser was turned off when the solution reached its maximum temperature after continuous irradiation. Based on the cooling stages of the solution, a negative natural logarithmic curve of cooling time (t) versus temperature driving force (-lnθ) was obtained, as shown in the figure. Figure 28 As shown, based on the obtained data, the PCE values ​​for HCY-995 and HCY-1080 were calculated to be 54.9% and 73.0%, respectively. This indicates that the photothermal effect of the compounds improves sequentially with the increase of the furan ring. In summary, HCY-945 has a high fluorescence quantum yield but poor photothermal effect; HCY-1080 has better photothermal effect, but its low fluorescence quantum yield is very unfavorable for in vivo fluorescence imaging; while HCY-995 possesses both good optical and photothermal properties.

[0124] This invention utilizes the twisted intramolecular charge transfer (TICT) effect to modulate and balance the optical and photothermal properties of small organic molecules. Hemicyanine, a major member of the cyanine family, possesses excellent TICT properties due to its unique D-π-A structure. Furthermore, its large Stokes shift, good biocompatibility, and low toxicity have led to its widespread application in many research fields. However, its short wavelength limits its suitability for in vivo biological applications. Therefore, we aim to regulate the optical and photothermal properties of hemicyanine molecules by controlling their TICT properties, making them high-performance near-infrared II phototherapy molecules.

[0125] TICT properties:

[0126] Through the above tests on the optical and photothermal properties of the molecule, we concluded that the fluorescence quantum yield increases with the increase of the furan ring, while the photothermal conversion efficiency decreases. This may be because the increase of the furan ring gives the molecule a better TICT effect, which we verified. We used steady-state fluorescence spectroscopy to study the fluorescence spectral properties of the molecule in different polar solvents and discussed in detail the differences in its fluorescence spectra in different solvents: such as... Figure 7As shown, the fluorescence emission wavelengths of HCY-995 and HCY-1080 gradually red-shift with increasing solvent polarity. In stark contrast, the emission wavelength of HCY-945 does not exhibit a red-shift with changing polarity. Furthermore, molecules exhibiting the TICT effect undergo rotation between electron donors and acceptors in polar solvents, resulting in a distorted conformation. Intramolecular charge transfer occurs rapidly in the excited state, and its energy is continuously dissipated through non-radiative processes, leading to a decrease in fluorescence intensity. Therefore, molecules exhibiting the TICT effect experience a decrease in fluorescence intensity with increasing solvent polarity. To address this, we dissolved the molecules in THF and then added a low-polarity toluene solution and a high-polarity acetonitrile solution.

[0127] The results are as follows Figure 8 and Figure 9 As shown, the fluorescence brightness of HCY-995 and HCY-1080 increases with the continuous addition of toluene solution, while it decreases with the continuous addition of acetonitrile. However, the fluorescence brightness of HCY-945 shows no significant change. These results indicate that the wavelength and brightness of HCY-1080 change significantly with polarity, followed by HCY-995, while HCY-945 shows almost no change, suggesting that HCY-1080 exhibits a better TICT effect. To further investigate the TICT characteristics, we used the Lippert-Mataga equation to study the effect of solvent polarity on fluorescence emission:

[0128]

[0129] here and μ represents the maximum absorption and emission wavenumbers, h is Planck's constant, c is the speed of light, and a is the radius of the solvent cage containing the phosphor. G and μ E These are the dipole moments of the phosphor in its electronic ground state and excited state, respectively. The solvent polarity parameter Δf can be calculated using the following formula:

[0130]

[0131] n is the solvent refractive index, and ε is the solvent dielectric constant. From the Stokes displacement... The relationship with Δf yields the ground state and excited state dipole moments Δμ, which can be calculated from the slope of the linear regression. Figure 10 The results show that the slopes of the Stokes shift with respect to solvent orientation polarizability (Δf) of HCY-945, HCY-995 and HCY-1080 are 2148, 2823 and 5047, respectively. The larger the slope, the better the TICT effect. That is, HCY-1080 has a better TICT effect than HCY-995 and HCY-945.

[0132] To further understand the molecular properties, we performed theoretical calculations. Based on the time-dependent density functional theory (TD-DFT) of acetonitrile solvation, we obtained detailed information on the ground state (S0) and excited state (S1) of the molecule at the B3LYP / 6–311G(d,p) level. The calculation results show that with the increase of the furan ring, the conjugated structure of the molecule becomes larger, the HOMO and LUMO become significantly separated, and the intermolecular energy levels decrease. In HCY-945, the HOMO and LUMO are distributed throughout the molecule and overlap. However, in HCY-995 and HCY-1080, the HOMO and LUMO are separated, with the HOMO mainly distributed on the molecular donor and the LUMO mainly distributed on the molecular acceptor. This favors molecular twisting. As expected, compared to HCY-945, the dihedral angle between the donor and acceptor in HCY-995 and HCY-1080 decreases, and the molecular structure becomes twisted, as shown in the figure. Figure 11 As shown.

[0133] Cytotoxicity:

[0134] The phototoxicity of HCY-995-NPs to 4T1 cancer cells was detected by MTT assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide). Figure 12 As shown. The cytotoxicity of the NPs against cancer cells was evaluated under both dark and light conditions. Even with high concentrations of the NPs solution, almost no cytotoxicity was observed under dark conditions, indicating that the NPs have good biocompatibility. Conversely, when using 1.0 W cm⁻¹ solution... -2 After 5 minutes of (721nm) laser irradiation, cell viability decreased significantly with increasing NPs solution concentration. The results indicate that light irradiation can trigger the photothermal effect of HCY-995 cells, and that increased temperature is sufficient to effectively kill cancer cells. Figure 13 As shown. To more intuitively demonstrate the effectiveness of HCY-995 photothermal therapy, we used calcein AM (green) and propidium iodide (red) dyes for live and dead cell staining. Green fluorescence indicates live cells, and red indicates dead cells. As expected, confocal imaging showed that under laser irradiation (1.0W cm⁻¹), live and dead cells were effectively stained. -2 After 5 minutes, 4T1 cells treated with HCY-995-NPs exhibited significant red fluorescence, indicating complete cell death. However, only green fluorescence was observed in the light-only and HCY-995-NPs-only groups, suggesting that strong cytotoxicity is induced only by the coexistence of nanoparticles and laser irradiation. Figure 14 As shown. Next, the cell death pathway induced by HCY-995 at high temperature was further investigated. After irradiation with a 721nm near-infrared laser for 5 minutes, the percentage of necrotic cells increased from 1.07% (blank control) to 71.4%, as shown. Figure 15 As shown, this further confirms the effective necrosis-inducing ability of HCY-995 on cells. These results are consistent with the MTT photocytotoxicity results, indicating that at a power intensity (1.0 W cm⁻¹), HCY-995 effectively induces cell necrosis. -2 Under laser irradiation, the photothermal ability of HCY-995 can cause cell death, which has great potential application prospects in the field of tumor treatment.

[0135] In vivo angiography

[0136] To evaluate the near-infrared II imaging performance of HCY-995, its optical transmission characteristics in simulated biological tissues and its in vivo imaging performance were tested. ICG and HCY-995-NPs-filled capillaries immersed in 1% Intralipid were imaged using a 1000LP filter at 721 nm excitation. The results showed that HCY-995 light was more clearly visible compared to ICG, confirming that HCY-995 has good fluorescence performance. Figure 16 As shown. Subsequently, PLGA-encapsulated HCY-995 molecules were injected into mice via tail vein injection, and clear vascular images were obtained from the blood vessels in the mouse brain, hind limbs, and abdomen. Figure 17 As shown, the SBR values ​​for NIR-II imaging at the aforementioned locations were calculated to be 4.8, 10.1, and 10.3, respectively. Figure 18 As shown in the image, the tiny blood vessels in the abdomen are clearly visible, with a full width at half maximum (FWHM) of only 140 μm. This indicates that the molecule can be used for in vivo tissue imaging.

[0137] Internal phototherapy

[0138] The efficacy of photothermal therapy (PTT) on tumors in tumor-bearing mice was investigated using HCY-995-NPs solution. Figure 19 As shown. To obtain the optimal time point for PTT, fluorescence images of the tumor site were recorded after intravenous injection of HCY-995-NPs via the tail vein. The fluorescence signal at the tumor site increased in a time-dependent manner, reaching a stable intensity 8 hours after injection. Therefore, the nanoparticles exhibit a significant EPR effect, leading to their effective accumulation in tumor tissue. Furthermore, the fluorescence signal helps guide the irradiation time and location during PTT. The fluorescence signal significantly decreased 24 hours after injection, indicating that the nanoparticles can be eliminated from the body after treatment, such as... Figure 20 As shown. We used a safe laser intensity (1.0W cm⁻¹). -2 The irradiation study investigated the tumor-killing effect. In vivo photothermal imaging showed that the temperature of the laser group (saline + laser) remained almost unchanged, such as... Figure 21 , 22 As shown. Therefore, the power is 1.0W cm. -2Continuous irradiation with 721nm laser did not cause tissue overheating, meeting the basic requirements for in vivo phototherapy. Conversely, tumors in the HCY-995-NPs (50μM) + light irradiation group showed a rapid temperature rise of approximately 25°C, indicating that HCY-995 exhibits good photothermal effects in vivo. The PTT effect in each group was assessed by monitoring tumor volume every three days for 15 days post-treatment. Treatment with saline, HCY-995-NPs, and light irradiation alone failed to inhibit tumor growth, with average tumor volume increasing 2-3 times. The results indicate that an intensity of 1.0W cm⁻¹ is sufficient for effective phototherapy. -2 Single laser irradiation (721nm) and HCY-995-NPs alone failed to inhibit tumor growth. However, in the HCY-995-NPs + phototherapy group, tumor suppression and elimination without recurrence were observed. These findings are highly consistent with in vitro phototoxicity results, confirming that HCY-995 exhibits excellent PTT (phototransfer) effects under laser irradiation, such as... Figure 23 , 24 As shown. Histological images of tumor tissue stained with hematoxylin and eosin (H&E) after treatment showed severe necrosis in the HCY-995-NPs+ light-irradiated group, while cancer cells in other groups were unaffected, such as... Figure 25 As shown in the figure. In addition, HCY-995 also demonstrated excellent in vivo physiological safety. The body weight of all mice in both the control and experimental groups was unaffected, indicating that these treatments did not cause systemic toxicity in the mice. Furthermore, the toxicity of HCY-995 was estimated by analyzing the tissues of the major organs (heart, liver, spleen, lungs, and kidneys) of mice euthanized after treatment. No pathological tissue damage was observed in any of these organs in all groups, demonstrating the good biocompatibility of HCY-995. Figure 29 As shown in the figure. These results confirm that HCY-995 has a good PTT effect and does not cause systemic toxicity in vivo.

[0139] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A hemicyanine compound in the near-infrared II region, characterized in that: The structural formula of this compound is shown in formula (HCY-995): (HCY-995).

2. The method for preparing a near-infrared II region hemicyanine compound according to claim 1, characterized in that: The synthesis route is as follows: B1: Using 4-(diphenylamino)phenylboronic acid pinacol ester and 5-bromofuran-2-carboxaldehyde as raw materials, tetrakis(triphenylphosphine)palladium, toluene / ethanol solution and sodium carbonate aqueous solution were added. After the reaction was completed, compound A-3 was obtained. B2: Using compounds A-3 and A-2 as raw materials and anhydrous ethanol as solvent, the reaction was completed and the resulting compound (HCY-995) was obtained.

3. A hemicyanine compound nanoparticle in the near-infrared II region, characterized in that: It was prepared using the following method: Polylactic acid-glycolic acid copolymer and compound (HCY-995) were dissolved in chloroform, polyvinyl alcohol was added and mixed, and the mixture was sonicated and stirred overnight. The structural formula of compound (HCY-995) is shown below: 。 4. The use of the near-infrared II hemicyanine compound of claim 1 and / or the near-infrared II hemicyanine compound nanoparticle of claim 3 in the preparation of a drug that inhibits tumor growth in conjunction with light irradiation.

5. The use according to claim 4, characterized in that: The tumor is selected from one or more of the following: colorectal cancer, breast cancer, lung cancer, stomach cancer, liver cancer, human placental choriocarcinoma, and cervical cancer.

6. The use according to claim 5, characterized in that: The tumor was selected from breast cancer.

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