A coumarin-like hemicyanine fluorescent dye molecule, preparation method and application

Abstract

The present invention relates to the technical field of organic functional materials, and in particular to a coumarin-like hemicyanine fluorescent dye molecule, a preparation method, and an application thereof. The coumarin-like hemicyanine fluorescent dye molecule uses a tetraphenylethylene moiety as a donor and 1-ethyl-2-methylbenz[c,d]indol-1-ium as an acceptor, has good photostability, a fluorescence emission wavelength reaching the second near-infrared region, good biocompatibility, and excellent photothermal imaging properties, and can be used for cell imaging and small animal in vivo imaging. Under 808nm laser irradiation, the temperature can be increased from 20°C to 60°C, and the dye has an excellent photothermal therapeutic effect, a strong ability to generate reactive oxygen species, and a good photodynamic effect. The dye can achieve synergistic photothermal and photodynamic therapy for tumor cells, and has great potential application prospects in the field of tumor treatment.

Description

Technical Field

[0001] The present invention relates to the technical field of organic functional materials, and in particular to a coumarin-like hemicyanine fluorescent dye molecule, a preparation method and an application thereof. Background Art

[0002] Cancer is one of the leading causes of death and poses a significant threat to human health and life. Currently, the main treatments for cancer include radiotherapy, chemotherapy, and surgical resection. However, these traditional treatments suffer from significant side effects, low efficacy, and high treatment risks. Compared to traditional cancer treatments such as surgery, chemotherapy, and radiotherapy, phototherapy has become an emerging research hotspot due to its highly selective and effective tumor-destroying capabilities, lack of drug resistance, non-invasiveness, and ease of integration with other treatments.

[0003] Tumor phototherapy is generally divided into two types: photothermal therapy (PTT) and photodynamic therapy (PDT). PTT utilizes a photosensitizer to absorb light of a specific wavelength and convert the light energy into heat, thereby killing cancer cells. PDT utilizes photosensitizers in the presence of oxygen to generate reactive oxygen species (ROS), which kill specific tumor cells. With the advancement of medical technology, the limited therapeutic efficacy and persistent side effects of single-modality treatments are no longer sufficient to meet clinical treatment needs. This is because the hypoxic microenvironment surrounding tumors severely affects the production of ROS, thereby severely inhibiting the efficiency of PDT. Furthermore, some tumors, after PTT treatment, may retain heat-resistant residual cancer cells, which may cause tumor recurrence, limiting the use of PTT in cancer treatment. PDT and PTT dual-modality combined therapy (PDT-PTT) can overcome the shortcomings of single-modality treatments and achieve enhanced synergistic therapeutic effects. This is because PTT can increase blood flow, thereby increasing oxygen concentration, thereby enhancing the effectiveness of PDT and promoting the elimination of heat-resistant tumors in PTT. The combination of PTT and PDT can not only cover the entire process of tumor treatment, but also achieve better anti-tumor effects through the synergistic effect of ROS and hyperthermia.

[0004] Traditional photosensitizers include derivatives based on tetrapyrrole, cyanine, and BODIPY backbones, some of which have been approved for clinical use. However, these compounds share a common characteristic: a rigid planar structure. Due to the inherent hydrophobicity of the molecules and strong intermolecular π-π interactions, they can aggregate and quench fluorescence in aqueous environments, resulting in a decrease in the singlet oxygen quantum yield. Therefore, developing a diagnostic and therapeutic agent with near-infrared emission and high singlet oxygen yield is of great significance for improving the effectiveness of phototherapy. Summary of the Invention

[0005] In order to overcome the shortcomings of the above-mentioned technical defects, the present invention provides a coumarin-like hemicyanine fluorescent dye molecule, preparation method and application. By using a tetraphenylethylene moiety as a donor and 1-ethyl-2-methylbenz[c,d]indol-1-ium as an acceptor, a coumarin-like hemicyanine photosensitizer with a D-π-A motif is designed and synthesized, so that the emission wavelength is located in the near-infrared window (650-1000nm) and is conducive to the separation of the HOMO and LUMO of the molecule, thereby obtaining highly efficient singlet oxygen, so as to achieve the purpose of fluorescence imaging of organisms and synergistic photothermal and photodynamic therapy.

[0006] In order to achieve the above object, the technical solution of the present invention is:

[0007] In one aspect, a coumarin-like hemicyanine fluorescent dye molecule is provided, whose structural formula is shown in Formula I to Formula III:

[0008]

[0009] Furthermore, the maximum emission wavelength of the fluorescent dye molecule is 890-920 nm, and the maximum absorption wavelength is 750-890 nm.

[0010] In a second aspect, a method for preparing a coumarin-like hemicyanine fluorescent dye molecule is provided, wherein a compound of formula IV undergoes a condensation reaction with a compound of formula V to obtain a photosensitizer CM-2-1, the structure of which is shown in formula I;

[0011] Or the compound of formula VI reacts with the compound of formula VII to produce photosensitizer CM-2-2, the structure of which is shown in formula II;

[0012] Or the compound of formula VI reacts with the compound of formula VIII to produce photosensitizer CM-2-3, the structure of which is shown in formula III;

[0013] Among them, the structures of each compound are as follows:

[0014]

[0015] Further, under an inert atmosphere, an organic solvent and an activator are added, reacted at 60-70° C. for 30-60 minutes, and separated and purified to obtain the product;

[0016] The organic solvent is selected from one or more of anhydrous ethanol, anhydrous methanol, N,N-dimethylformamide or acetic anhydride;

[0017] The activator is selected from one or more of potassium carbonate, sodium acetate and potassium acetate.

[0018] Furthermore, the synthetic route of the compound of formula IV is as follows:

[0019]

[0020] Furthermore, the synthetic route of the compound of formula VII is as follows:

[0021]

[0022] Furthermore, the synthesis route of the compound of formula VIII is as follows:

[0023]

[0024] In a third aspect, a method is provided for using the above-mentioned coumarin-like hemicyanine fluorescent dye molecule and the coumarin-like hemicyanine fluorescent dye molecule prepared by the above-mentioned method in the preparation of tumor photothermal therapy and / or diagnostic reagents or drugs.

[0025] In a fourth aspect, a method is provided for applying the coumarin-like hemicyanine fluorescent dye molecule and the coumarin-like hemicyanine fluorescent dye molecule prepared by the above method in medical imaging, wherein the medical imaging includes bioluminescence imaging and / or photoacoustic imaging.

[0026] In the fifth aspect, a method is provided for preparing a reagent or drug for combined therapy based on the above-mentioned coumarin-like hemicyanine fluorescent dye molecule or the coumarin-like hemicyanine fluorescent dye molecule prepared by the above-mentioned method, wherein the combined therapy includes a combined therapy of photothermal therapy with photodynamic therapy, chemotherapy and / or radiotherapy.

[0027] In a sixth aspect, a pharmaceutical composition is provided, characterized in that it contains the above-mentioned coumarin-like hemicyanine fluorescent dye molecule, the coumarin-like hemicyanine fluorescent dye molecule prepared by the above-mentioned method, or a pharmaceutically acceptable salt, solvate, enantiomer, diastereomer, or tautomer thereof.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] (1) The coumarin-like hemicyanine fluorescent dye molecules of the present invention have good photostability and excellent photothermal imaging properties;

[0030] (2) The fluorescence emission wavelength of the coumarin-like hemicyanine fluorescent dye molecules of the present invention reaches the near-infrared region II and has good biocompatibility, and can be used for cell imaging and small animal in vivo imaging;

[0031] (3) The coumarin-like hemicyanine fluorescent dye molecules of the present invention can increase from 20°C to 60°C under 808nm laser irradiation and reach a plateau at around 62°C, indicating excellent photothermal therapeutic effects;

[0032] (4) The coumarin-like hemicyanine fluorescent dye molecules of the present invention have enhanced ability to generate active oxygen and have good photodynamic effects, which can achieve synergistic photothermal and photodynamic therapy for tumor cells;

[0033] (5) The molecular synthesis method of the coumarin-like hemicyanine fluorescent dye of the present invention has simple steps and high yield, and is expected to be industrially produced on a large scale at a relatively low cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 The coumarin-like hemicyanine fluorescent dye CM-2-1 of the present invention 1 H NMR spectrum;

[0035] Figure 2 HRMS spectrum of the coumarin-like hemicyanine fluorescent dye CM-2-1 of the present invention;

[0036] Figure 3 The coumarin-like hemicyanine fluorescent dye CM-2-2 of the present invention 1 H-NMR spectrum;

[0037] Figure 4 HRMS spectrum of the coumarin-like hemicyanine fluorescent dye CM-2-2 of the present invention;

[0038] Figure 5 The coumarin-like hemicyanine fluorescent dye CM-2-3 of the present invention 1 H NMR spectrum;

[0039] Figure 6 HRMS spectrum of the coumarin-like hemicyanine fluorescent dye CM-2-3 of the present invention;

[0040] Figure 7 This is the absorption and emission spectrum of the coumarin-like hemicyanine fluorescent dye CM-2-3 of the present invention;

[0041] Figure 8 The photothermal performance test of the coumarin-like hemicyanine fluorescent dyes of different concentrations of the present invention is carried out;

[0042] Figure 9 The photothermal properties of the coumarin-like hemicyanine fluorescent dye irradiated by lasers of different powers of the present invention;

[0043] Figure 10 The photothermal stability of the coumarin-like hemicyanine fluorescent dye of the present invention;

[0044] Figure 11 Infrared thermal imaging of the coumarin-like hemicyanine fluorescent dye of the present invention;

[0045] Figure 12PL spectra of the coumarin hemicyanine fluorescent dye DCFH in PBS after irradiation with white light for different times;

[0046] Figure 13 Photodynamic imaging of the coumarin-like hemicyanine fluorescent dye of the present invention;

[0047] Among them, 13A is the fluorescence imaging image of CM-2-1; 13B is the fluorescence imaging image of CM-2-2; 13C is the fluorescence imaging image of CM-2-3; 13D is the fluorescence emission spectrum of the coumarin-like hemicyanine fluorescent dye. DETAILED DESCRIPTION

[0048] In order to make those skilled in the art better understand the technical scheme of the present invention, the present invention is described in detail below in conjunction with specific embodiments. The experimental methods for which specific conditions are not specified in the following examples are usually based on conventional conditions or the conditions recommended by the manufacturer. The test materials used in the following examples, unless otherwise specified, are purchased from conventional biochemical reagent stores. Unless otherwise specified, percentages and parts are calculated by weight. Unless otherwise defined, all professional and scientific terms used in the text have the same meaning as those familiar to those skilled in the art. In addition, any method and material similar to or equivalent to the described content can be applied to the present invention. The preferred implementation methods and materials described in the text are only for demonstration purposes.

[0049] The endpoints of the ranges and any values disclosed herein are not limited to the precise ranges or values, and these ranges or values should be understood to include values close to these ranges or values. For numerical ranges, the endpoints of each range, the endpoints of each range and individual point values, and the individual point values can be combined with each other to obtain one or more new numerical ranges, which should be considered to be specifically disclosed herein.

[0050] The present invention aims to provide a coumarin-like hemicyanine fluorescent dye molecule with high photothermal stability in the near-infrared region II (NIR-II region), with a tetraphenylethylene moiety as a donor and 1-ethyl-2-methylbenz[c,d]indol-1-ium as an acceptor. It has a high photothermal temperature, can cause thermal damage to the nuclear genetic material of tumor cells, induce acute necrosis, cell apoptosis and microenvironmental destruction of tumors, and has a good ability to generate reactive oxygen species. Therefore, using this compound as a photosensitizer can achieve efficient near-infrared light-guided photodynamic therapy, thereby realizing synergistic photothermal and photodynamic therapy of tumor cells.

[0051] In some specific embodiments, a coumarin-like hemicyanine fluorescent dye molecule is provided, and its structural formula is shown in Formula I:

[0052]

[0053] In another specific embodiment, a method for preparing the above-mentioned coumarin-like hemicyanine fluorescent dye molecule is provided, comprising the following steps:

[0054] S1: Add triphenylethylene bromide, 4-benzoyl borate, tetrakistriphenylphosphine palladium, and potassium carbonate under Ar protection to toluene / water, heat under reflux, cool to room temperature, and then extract with dichloromethane. The organic phase is dried, filtered, and distilled under reduced pressure. The collected phase is passed through a silica gel chromatography column, eluted, separated, and purified to obtain a light yellow solid compound 1.

[0055] S2: The obtained compound 1 and 1-(4-(diethylamino)-2-hydroxyacetophenone were dissolved in acetic acid and HClO4, heated under reflux, cooled to room temperature, and added to water to obtain a pink solid compound 2, whose structural formula is shown in Formula IV:

[0056]

[0057] S3: Compound 2, compound 3 (as shown in formula V), and potassium acetate were added to acetic anhydride. After the reaction was completed by heating under nitrogen protection, the mixture was poured into a saturated solution in an ice bath, stirred vigorously, and filtered to obtain a black solid. The solid was eluted, separated, and purified by silica gel chromatography to obtain photosensitizer CM-2-1.

[0058]

[0059] In some examples of this embodiment, in S1, the specific steps are: triphenylethylene bromide, 4-benzoyl borate, tetrakis(triphenylphosphine)palladium, and potassium carbonate are added with toluene / water under Ar protection, refluxed at 110°C for 24 hours, and cooled to room temperature; then extracted with dichloromethane, dried the organic phase, filtered, distilled under reduced pressure, collected, eluted, separated, and purified by silica gel chromatography to obtain compound 1.

[0060] In some preferred embodiments, the molar ratio of triphenylethylene bromide and 4-benzoyl borate is 1:1; and the eluent is a mixture of petroleum ether and dichloromethane in a volume ratio of 5:1.

[0061] In some examples of this embodiment, in S2, the specific steps are: dissolving the obtained compound 1 and 1-(4-(diethylamino)-2-hydroxyacetophenone in acetic acid and HClO4, heating under reflux at 120°C for 15h, cooling to room temperature, and then adding water to obtain a pink solid compound 2, which can be directly carried out in the next reaction after drying.

[0062] In some preferred embodiments, the molar ratio of compound 1 to 1-(4-(diethylamino)-2-hydroxyacetophenone is 1:1.2.

[0063] In some examples of this embodiment, in S3, the specific steps are: dissolving the obtained compound 2, compound 3, and potassium acetate in acetic anhydride, heating the mixture at 65°C for 30 minutes under nitrogen protection, and after the reaction, pouring the mixture into a saturated NaHCO3 solution in an ice bath, stirring vigorously, and filtering to obtain a black solid, which was eluted, separated, and purified by a silica gel chromatography column to obtain photosensitizer CM-2-1.

[0064] In some preferred embodiments, the molar ratio of Compound 2 to Compound 3 is 1:1, and the eluent is a mixture of dichloromethane and methanol in a volume ratio of 50:1.

[0065] In some specific embodiments, a coumarin hemicyanine fluorescent dye molecule is provided, and its structural formula is shown in Formula II:

[0066]

[0067] In another specific embodiment, a method for preparing the above-mentioned coumarin-like hemicyanine fluorescent dye molecule is provided, comprising the following steps:

[0068] S1: Add triphenylethylene bromide, 4-benzoyl borate, tetrakis(triphenylphosphine)palladium, and potassium carbonate under Ar protection to toluene / water, heat under reflux, cool to room temperature, and extract with dichloromethane. The organic phase is dried, filtered, and evaporated under reduced pressure. The organic phase is collected, eluted, separated, and purified by silica gel chromatography to obtain a light yellow solid compound 1.

[0069] S2: The obtained compound 1 and 1-(4-(diethylamino)-2-hydroxyacetophenone were dissolved in acetic acid and HClO4, heated under reflux, cooled to room temperature, and added to water to obtain a pink solid compound 2, whose structural formula is shown in Formula IV:

[0070]

[0071] S3: Add acetic anhydride to the obtained compound 2, malondialdehyde bisphenylimine monohydrochloride, and potassium acetate. After the heating reaction is completed under nitrogen protection, pour the mixture into a saturated solution in an ice bath, stir vigorously, and filter to obtain a black solid. After elution, separation, and purification through a silica gel chromatography column, a black solid compound 4 is obtained, whose structural formula is shown in Formula VII:

[0072]

[0073] S4. Add acetic anhydride to the obtained compound 4, 1-ethyl-2-methylbenz[c,d]indol-1-ium and potassium acetate. After heating under nitrogen protection, pour the mixture into a saturated solution in an ice bath, stir vigorously, and filter to obtain a black solid. After elution, separation and purification through a silica gel chromatography column, photosensitizer CM-2-2 is obtained.

[0074] In some examples of this embodiment, in S1, the specific steps are: triphenylethylene bromide, 4-benzoyl borate, tetrakis(triphenylphosphine)palladium, and potassium carbonate are added with toluene / water under Ar protection, refluxed at 110°C for 24 hours, and cooled to room temperature; then extracted with dichloromethane, dried the organic phase, filtered, distilled under reduced pressure, collected, eluted, separated, and purified by silica gel chromatography to obtain compound 1.

[0075] In some preferred embodiments, the molar ratio of triphenylethylene bromide and 4-benzoyl borate is 1:1; and the eluent is a mixture of petroleum ether and dichloromethane in a volume ratio of 5:1.

[0076] In some examples of this embodiment, in S2, the specific steps are: dissolving the obtained compound 1 and 1-(4-(diethylamino)-2-hydroxyacetophenone in acetic acid and HClO4, heating under reflux at 120°C for 15h, cooling to room temperature, and then adding water to obtain a pink solid compound 2, which can be directly carried out in the next reaction after drying.

[0077] In some preferred embodiments, the molar ratio of compound 1 to 1-(4-(diethylamino)-2-hydroxyacetophenone is 1:1.2.

[0078] In some examples of this embodiment, in S3, the obtained compound 2, malondialdehyde diphenylimine monohydrochloride, and potassium acetate are dissolved in acetic anhydride, and the mixture is heated at 80°C for 30 minutes under nitrogen protection. After the reaction is completed, the mixture is poured into a saturated NaHCO3 solution in an ice bath, stirred vigorously, and filtered to obtain a black solid. The solid is eluted, separated, and purified by a silica gel chromatography column to obtain a black solid compound 4.

[0079] In some preferred embodiments, the molar ratio of the compound 2 and malondialdehyde bisphenylimine monohydrochloride is 1:1; and the column chromatography eluent is a mixture of dichloromethane and methanol in a volume ratio of 80:1.

[0080] In some examples of this embodiment, in S4, the obtained compound 4, 1-ethyl-2-methylbenz[c,d]indol-1-ium, and potassium acetate are dissolved in acetic anhydride, and the mixture is heated at 65° C. for 30 min under nitrogen protection. After the reaction is completed, the mixture is poured into a saturated NaHCO3 solution in an ice bath, stirred vigorously, and filtered to obtain a black solid, which is then eluted, separated, and purified by a silica gel chromatography column to obtain photosensitizer CM-2-2.

[0081] In some preferred embodiments, the molar ratio of compound 4 and 1-ethyl-2-methylbenz[c,d]indol-1-ium is 1:1; and the column chromatography eluent is a mixture of dichloromethane and methanol in a volume ratio of 50:1.

[0082] In some specific embodiments, a coumarin-like hemicyanine fluorescent dye molecule is provided, and its structural formula is shown in Formula III:

[0083]

[0084] In another specific embodiment, a method for preparing the above-mentioned coumarin-like hemicyanine fluorescent dye molecule is provided, comprising the following steps:

[0085] S1: Add triphenylethylene bromide, 4-benzoyl borate, tetrakis(triphenylphosphine)palladium, and potassium carbonate under Ar protection to toluene / water, heat under reflux, cool to room temperature, and extract with dichloromethane. The organic phase is dried, filtered, and evaporated under reduced pressure. The organic phase is collected, eluted, separated, and purified by silica gel chromatography to obtain a light yellow solid compound 1.

[0086] S2: The obtained compound 1 and 1-(4-(diethylamino)-2-hydroxyacetophenone were dissolved in acetic acid and HClO4, heated under reflux, cooled to room temperature, and added to water to obtain a pink solid compound 2, whose structural formula is shown in Formula IV:

[0087]

[0088] S3: The obtained compound 2, pentadienaldehyde diphenylamine hydrochloride, and potassium acetate were dissolved in acetic anhydride. After the heating reaction was completed under nitrogen protection, the mixture was poured into a saturated solution in an ice bath, stirred vigorously, and filtered to obtain a black solid. The solid was eluted, separated, and purified on a silica gel chromatography column to obtain a black solid compound 5, whose structural formula is shown in Formula VIII:

[0089]

[0090] S4. Add acetic anhydride to the obtained compound 5, 1-ethyl-2-methylbenz[c,d]indol-1-ium, potassium acetate, and the like. After the heating reaction is completed under nitrogen protection, pour the mixture into a saturated solution in an ice bath, stir vigorously, and filter to obtain a black solid. After elution, separation, and purification through a silica gel chromatography column, photosensitizer CM-2-3 is obtained.

[0091] In some examples of this embodiment, in S1, the specific steps are: triphenylethylene bromide, 4-benzoyl borate, tetrakis(triphenylphosphine)palladium, and potassium carbonate are added with toluene / water under Ar protection, refluxed at 110°C for 24 hours, and cooled to room temperature; then extracted with dichloromethane, dried the organic phase, filtered, distilled under reduced pressure, collected, eluted, separated, and purified by silica gel chromatography to obtain compound 1.

[0092] In some preferred embodiments, the molar ratio of triphenylethylene bromide and 4-benzoyl borate is 1:1; and the eluent is a mixture of petroleum ether and dichloromethane in a volume ratio of 5:1.

[0093] In some examples of this embodiment, in S2, the specific steps are: dissolving the obtained compound 1 and 1-(4-(diethylamino)-2-hydroxyacetophenone in acetic acid and HClO4, heating under reflux at 120°C for 15h, cooling to room temperature, and then adding water to obtain a pink solid compound 2, which can be directly carried out in the next reaction after drying.

[0094] In some preferred embodiments, the molar ratio of compound 1 to 1-(4-(diethylamino)-2-hydroxyacetophenone is 1:1.2.

[0095] In some examples of this embodiment, in S3, the obtained compound 2, pentadienaldehyde diphenylamine hydrochloride, and potassium acetate are dissolved in acetic anhydride, and the mixture is heated at 80°C for 30 minutes under nitrogen protection. After the reaction is completed, the mixture is poured into a saturated NaHCO3 solution in an ice bath, stirred vigorously, and filtered to obtain a black solid. The solid is eluted, separated, and purified by a silica gel chromatography column to obtain a black solid compound 5.

[0096] In some preferred embodiments, the molar ratio of the compound 2 and pentadienaldehyde diphenylamine hydrochloride is 1:1; and the column chromatography eluent is a mixture of dichloromethane and methanol in a volume ratio of 50:1.

[0097] In some examples of this embodiment, in S4, the obtained compound 5, 1-ethyl-2-methylbenz[c,d]indol-1-ium, and potassium acetate are dissolved in acetic anhydride, and the mixture is heated at 65° C. for 30 min under nitrogen protection. After the reaction is completed, the mixture is poured into a saturated NaHCO3 solution in an ice bath, stirred vigorously, and filtered to obtain a black solid, which is then eluted, separated, and purified by a silica gel chromatography column to obtain photosensitizer CM-2-3.

[0098] In some preferred embodiments, the molar ratio of the compound 5 and 1-ethyl-2-methylbenz[c,d]indol-1-ium is 1:1; and the column chromatography eluent is a mixture of dichloromethane and methanol in a volume ratio of 50:1.

[0099] The technical solutions of the present invention are further described in detail below in conjunction with specific embodiments and drawings. It should be understood that the following embodiments are only used to explain the present invention and are not used to limit the present invention.

[0100] Example 1 Synthesis of Coumarin-like Hemicyanine Fluorescent Dyes

[0101] (1) Preparation of Compound 1: Triphenylethylene bromide (15 g, 44.74 mmol), 4-benzoyl borate (7.5 g, 45.74 mmol), tetrakis(triphenylphosphine)palladium (100 mg, 0.087 mmol), and potassium carbonate (11.0 g, 79.59 mmol) were added toluene / water (v / v = 4:1) under Ar protection, and the mixture was refluxed at 110°C for 24 h. The mixture was cooled to room temperature. The mixture was extracted with dichloromethane, the organic phase was dried, filtered, and evaporated under reduced pressure. The crude product was purified by silica gel chromatography and eluted with petroleum ether:dichloromethane = 5:1 to obtain Compound 1 as a pale yellow solid in a yield of 88%. 1 H NMR (500MHz, CDCl3) δ7.72 (d, J = 8.3Hz, 2H), 7.14 (dt, J = 4.8, 3.5Hz, 11H), 7.08–7.01 (m, 6H), 2.55 (s, 3H).

[0102] (2) Preparation of Compound 2: 1-(4-(Diethylamino)-2-hydroxyacetophenone) (0.2 g, 1 mmol) and 4-tetraphenylethylene benzoyl (0.4 g, 1.2 mmol) were dissolved in acetic acid (20 mL) and HClO4 (25 mL). The mixture was heated under reflux at 120°C for 15 h. After the reaction was completed, the mixture was cooled to room temperature and added to water to obtain a pink solid Compound 2. The solid was dried and then directly subjected to the next step of the reaction.

[0103] (3) Preparation of compound CM-2-1: Compound 2 (100 mg, 0.184 mmol), compound 3 (78 mg, 0.184 mmol) and potassium acetate (54 mg, 0.54 mmol) were dissolved in 5 mL of acetic anhydride and reacted at 65°C for 30 min under nitrogen protection. After the reaction, the mixture was poured into a saturated NaHCO3 solution in an ice bath, stirred vigorously, and filtered. The crude product was separated and purified by column chromatography and eluted (dichloromethane / methanol = 50:1, v / v) to obtain a black solid compound CM-2-1 (54 mg, 33% yield), which was characterized by mass spectrometry and nuclear magnetic resonance ( Figure 1-2 ). 1 H NMR (500MHz, DMSO) δ8.81(d,J=27.3Hz,2H),8.09(s,3H),7.76(s,1H),7.61(d,J=37.8Hz,2H),7.47–7.37(m,2H ),7.29–6.99(m,20H),6.91(d,J=11.5Hz,1H),6.80(s,1H),4.23(s,2H),3.55(s,4H),1.37(s,3H),1.20(s,6H).

[0104] (4) Preparation of compound 4: Compound 2 (100 mg, 0.182 mmol), malondialdehyde diphenylimine monohydrochloride (46 mg, 0.182 mmol) and potassium acetate (54 mg, 0.546 mmol) were dissolved in 5 mL of acetic anhydride and reacted at 65°C for 30 min under nitrogen protection. After the reaction, the mixture was poured into a saturated NaHCO3 solution in an ice bath, stirred vigorously, and filtered. The crude product was separated and purified by column chromatography and eluted (dichloromethane / methanol = 80:1, v / v) to obtain black solid compound 4 (55 mg, 43% yield). 1 H NMR(500MHz,DMSO)δ8.54–8.48(m,1H),8.40(d,J=13.4Hz,1H),8.24(d,J=9.7Hz,1H), 8.14–8.12(m,2H),7.66(t,J=7.4Hz,2H),7.60(t,J=7.1Hz,1H),7.48(d,J=7.4Hz,2H) ,7.38(d,J=14.7Hz,1H),7.20(ddd,J=19.2,14.7,7.5Hz,15H),7.09–7.01(m,6H),5.5 6 (dd, J = 13.3, 11.2 Hz, 1H), 3.64 (d, J = 7.1 Hz, 4H), 1.97 (s, 3H), 1.20 (t, J = 7.0 Hz, 6H).

[0105] (5) Preparation of compound CM-2-2: Compound 4 (240 mg, 0.34 mmol), 1-ethyl-2-methylbenz[c,d]indol-1-ium (110 mg, 0.34 mmol) and potassium acetate (100 mg, 1.00 mmol) were dissolved in 5 mL of acetic anhydride and reacted at 65°C for 30 min under nitrogen protection. After the reaction, the mixture was poured into a saturated NaHCO3 solution in an ice bath, stirred vigorously, and filtered. The crude product was separated and purified by column chromatography and eluted (dichloromethane / methanol = 50:1, v / v) to obtain a black solid compound CM-2-2 (94 mg, 31% yield), which was characterized by mass spectrometry and nuclear magnetic resonance ( Figure 3-4 ). 1 H NMR (400MHz, DMSO) δ8.50–8.37(m,1H),8.32–8.15(m,1H),8.12(d,J=9.7Hz,1H),8.03(d,J=8.1Hz,2H),7.96(s,1H),7.91(d,J=7.5Hz,2H), 7.79(s,1H),7.57(s,3H),7.16–7.04(m,20H),6.75(s,2H),4.19(s,2H),3.51(d,J=5.7Hz,4H),1.33(t,J=6.7Hz,3H),1.19(d,J=4.9Hz,6H).

[0106] (6) Preparation of Compound 5: Compound 2 (200 mg, 0.364 mmol), pentadienaldehyde diphenylamine hydrochloride (104 mg, 0.364 mmol), and potassium acetate (108 mg, 1.092 mmol) were dissolved in 4 mL of acetic anhydride and reacted at 80°C for 30 min under nitrogen. After the reaction, the mixture was poured into a saturated NaHCO3 solution in an ice bath, stirred vigorously, and filtered. The crude product was separated and purified by column chromatography (dichloromethane / methanol = 50:1, v / v) to obtain Compound 5 as a black solid (105 mg, 39% yield). 1 H NMR (500MHz, DMSO) δ8.16–8.14(m,2H),7.63(t,J=7.5Hz,2H),7.57(d,J=7.3Hz,1H),7.42–7.38(m,3H),7.17(dd,J=17.7,10.0Hz,17 H),7.08(d,J=7.7Hz,3H),7.05–7.01(m,6H),5.25–5.19(m,1H),3.66(d,J=6.6Hz,4H),1.93(d,J=11.8Hz,3H),1.22(t,J=7.0Hz,6H).

[0107] (7) Preparation of compound CM-2-3: Compound 5 (177 mg, 0.24 mmol), 1-ethyl-2-methylbenz[c,d]indol-1-ium (74 mg, 0.24 mmol) and potassium acetate (24 mg, 0.24 mmol) were dissolved in 5 mL of acetic anhydride and reacted at 65°C for 30 min under nitrogen protection. After the reaction, the mixture was poured into a saturated NaHCO3 solution in an ice bath, stirred vigorously, and filtered. The crude product was separated and purified by column chromatography and eluted (dichloromethane / methanol = 50:1, v / v) to obtain a black solid compound CM-2-3 (64 mg, 28% yield), which was characterized by mass spectrometry and nuclear magnetic resonance ( Figure 5-6 ). 1 H NMR (600MHz, DMSO) δ8.27–8.22(m,1H),8.11(d,J=15.8Hz,1H),7.95(d,J=8.2Hz,1H),7.79–7.75(m,1H),7.70(d,J=7.3Hz,1H),7.46(d,J=22.8Hz,2 H),7.17(dd,J=21.9,14.9Hz,20H),7.02(dd,J=13.7,6.4Hz,9H),3.71(d, J=6.9Hz, 2H), 3.38 (d, J=6.2Hz, 4H), 1.18 (d, J=23.9Hz, 6H), 1.08 (s, 3H).

[0108] The synthetic route is as follows:

[0109]

[0110] Example 2 Investigation of the UV Fluorescence Properties of Coumarin-like Hemicyanine Dyes

[0111] Weigh 2 mg of CM-2-1, 2 mg of CM-2-2, and 2 mg of CM-2-3 and dissolve them in 1 mL of dichloromethane. Then, use a pipette to take 0.0172 mL of the solution and dissolve it in 4.9828 mL of dichloromethane to prepare a test solution with a concentration of 10 μM.

[0112] The absorption spectrum of the test solution was measured by a ULC 1503007 UV-visible spectrophotometer, and the emission spectrum of the solution was measured by a F97XP fluorescence spectrophotometer. During the test, the slit of the instrument was adjusted to an appropriate width.

[0113] The photophysical properties of the photosensitizer were investigated using UV-visible absorption and fluorescence emission spectroscopy, such as Figure 7As shown, the photosensitizer has a large π part and high electron delocalization, and exhibits strong absorption in the NIR-II region. The maximum absorption wavelengths of CM-2-1, CM-2-2 and CM-2-3 in dichloromethane are at 759, 858 and 880 nm. With the increase of the conjugated system, the absorption of the photosensitizer also undergoes an obvious red shift. Under the excitation of 808 nm laser, the maximum emission peaks of the photosensitizer appear at 894, 924 and 910 nm.

[0114] Example 3: Photothermal Performance Test of Coumarin-like Hemicyanine Dyes

[0115] Dissolve 1.335 mg of compounds CM-2-1, CM-2-2, and CM-2-3 in 2 mL of DMSO to prepare a 1 mmol / L solution. Then, pipette 125 μL, 250 μL, 375 μL, and 500 μL into 5 mL of PBS solution and dilute to 25, 50, 75, and 100 μM solutions, respectively.

[0116] 100 μL of the photothermal test solution was pipetted into a 1 mL centrifuge tube. Then, an 808 nm laser (1.5 W / cm 2 ) Irradiate the aqueous solution in a centrifuge tube for 10 minutes and record the temperature change. Study the photothermal properties of three photosensitizers at different concentrations and powers.

[0117] like Figure 8 As shown, at a fixed laser power (808 nm, 1.5 W / cm 2 ), with the increase of photosensitizer concentration, the temperature of photosensitizer CM-2-1, CM-2-2, and CM-2-3 solutions also increased. When the photosensitizer concentration increased from 25 μM to 100 μM, the temperature of CM-2-1 increased from 47 to 62 °C, the temperature of CM-2-2 increased from 42 to 60 °C, and the temperature of CM-2-3 increased from 45 to 60 °C within 600 s. The overall trend was that CM-2-1 had the highest temperature, which was 62 °C.

[0118] like Figure 9 As shown, as the power density increases from 0.8W / cm 2 Increased to 1.5W / cm 2 The temperature of photosensitizer CM-2-1 increased from 45°C to 62°C, the temperature of CM-2-2 increased from 37°C to 60°C, and the temperature of CM-2-3 increased from 37°C to 58°C. It can be seen that the power intensity of the photosensitizer and the temperature of the laser are positively correlated.

[0119] like Figure 10As shown, after four photothermal cycles of 808 nm laser irradiation, the temperature of photosensitizer CM-2-1 was stabilized at 56 °C, the temperature of CM-2-2 was stabilized at 56 °C, and the temperature of CM-2-3 was stabilized at 57 °C.

[0120] The coumarin-like hemicyanine fluorescent dyes (CM-2-1, CM-2-2, and CM-2-3) of the present invention exhibit excellent photothermal properties and are potential photothermal agents for effective photothermal anti-tumor therapy. After 10 minutes of continuous laser irradiation at 808 nm, they exhibited excellent photostability with negligible attenuation, and high photothermal stability was observed even after four heating-cooling cycles.

[0121] Example 4 Investigation of Photothermal Imaging of Coumarin-like Hemicyanine Dyes

[0122] like Figure 11 As shown, the photosensitizer CM-2-1, CM-2-2, CM-2-3 solutions (50 μM) were irradiated at a low power density of 1.5 W / cm 2 ) After 10 minutes of continuous irradiation, the temperatures of all three photosensitizers increased significantly. This indicates that the coumarin-like hemicyanine fluorescent dyes (CM-2-1, CM-2-2, and CM-2-3) of the present invention have excellent photothermal imaging properties, and the temperature of CM-2-2 is significantly higher than that of the other two photosensitizers.

[0123] Example 5 Investigation of the Photodynamic Properties of Coumarin-like Hemicyanine Dyes

[0124] In order to evaluate the PDT performance of coumarin-like hemicyanine dyes, DCFH was used as an indicator to evaluate the total ROS-generating capacity of these photosensitizers. DCFH has no fluorescence emission in its natural state, but its green fluorescence at 525 nm can be greatly enhanced in the presence of any type of ROS. Figure 12 As shown in the figure, with the increase of white light irradiation time, the fluorescence intensity of DCFH co-incubated with the photosensitizer continued to increase, while DCFH alone showed negligible fluorescence enhancement; in addition, the ROS generation performance of CM-2-1 was better than that of CM-2-2 and CM-2-3, showing excellent ROS generation ability.

[0125] Example 6 Investigation of Photodynamic Cell Imaging with Coumarin-like Hemicyanine Dyes

[0126] The DCFH-DA probe was used to evaluate the ROS generation ability of three photosensitizers in cells. Figure 13As shown in the figure, only weak green fluorescence was detected in the PBS group and PBS+laser group. Rosup is a positive control reagent that can stimulate cells to produce ROS. However, Hela cells treated with CM-2-1 and CM-2-2 were stimulated by 808nm laser at 1.5W / cm 2 After irradiation with a power of 100 nm for 5 min, strong green fluorescence was emitted, and the fluorescence intensity was comparable, indicating that CM-2-1 and CM-2-2 of the present invention can generate a large amount of ROS in cells.

[0127] Finally, it should be noted that the above description is only a preferred embodiment of the present invention. Under the guidance of the present invention, those skilled in the art can make various similar expressions without violating the purpose and claims of the present invention. Such changes fall within the scope of protection of the present invention.

Claims

1. A coumarin-like hemicyanine fluorescent dye molecule, characterized in that: Its structural formula is shown in Formula I-Formula III:

2. The method for preparing a coumarin-like hemicyanine fluorescent dye molecule according to claim 1, characterized in that: The compound of formula IV reacts with the compound of formula V to produce photosensitizer CM-2-1, the structure of which is shown in formula I; Or the compound of formula VI reacts with the compound of formula VII to produce photosensitizer CM-2-2, the structure of which is shown in formula II; Or the compound of formula VI reacts with the compound of formula VIII to produce photosensitizer CM-2-3, the structure of which is shown in formula III; The condensation reaction conditions are as follows: adding an organic solvent and an activator under an inert atmosphere, reacting at 60-70° C. for 30-60 min, and separating and purifying to obtain the product; The organic solvent is selected from one or more of anhydrous ethanol, anhydrous methanol, N,N-dimethylformamide or acetic anhydride; The activator is selected from one or more of potassium carbonate, sodium acetate, and potassium acetate; Among them, the structures of each compound are as follows:

3. The preparation method according to claim 2, characterized in that The synthetic route of the compound of formula IV is as follows:

4. The preparation method according to claim 2, characterized in that The synthetic route of the compound of formula VII is as follows:

5. The preparation method according to claim 2, characterized in that The synthetic route of the compound of formula VIII is as follows:

6. Use of the coumarin-like hemicyanine fluorescent dye molecule according to claim 1 in the preparation of a drug for photothermal therapy of tumors, wherein: The tumor cells are HeLa cells.

7. Use of the coumarin-like hemicyanine fluorescent dye molecule prepared by the preparation method according to any one of claims 2 to 5 in the preparation of tumor photothermal therapy drugs, wherein: The tumor cells are HeLa cells.

8. Use of the coumarin-like hemicyanine fluorescent dye molecule according to claim 1 in the preparation of a medical imaging preparation, wherein the medical imaging is HeLa cell fluorescence imaging and / or photoacoustic imaging.

9. Use of the coumarin-like hemicyanine fluorescent dye molecule prepared by the preparation method according to any one of claims 2 to 5 in the preparation of a medical imaging preparation, wherein the medical imaging is HeLa cell fluorescence imaging and / or photoacoustic imaging.

10. Use of the coumarin-like hemicyanine fluorescent dye molecule according to claim 1 in the preparation of an agent or drug for combined therapy of HeLa cells, wherein the combined therapy is a combined therapy of photothermal therapy with photodynamic therapy, chemotherapy and / or radiotherapy.

11. Use of the coumarin-like hemicyanine fluorescent dye molecule prepared by the preparation method according to any one of claims 2 to 5 in the preparation of an agent or drug for combined therapy of HeLa cells, wherein the combined therapy is a combined therapy of photothermal therapy with photodynamic therapy, chemotherapy and / or radiotherapy.

12. A pharmaceutical composition, characterized in that Contains the coumarin-like hemicyanine fluorescent dye molecule according to claim 1 or a pharmaceutically acceptable salt thereof.

13. A pharmaceutically acceptable salt of a coumarin-like hemicyanine fluorescent dye molecule obtained by the preparation method according to any one of claims 2 to 5.

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