Coumarin-like hemicyanine fluorescent dye molecule, preparation method and application

Abstract

The invention relates to the technical field of organic functional materials, in particular to a coumarin-like hemicyanine fluorescent dye molecule as well as a preparation method and application thereof.The coumarin-like hemicyanine fluorescent dye molecule takes a tetraphenylethylene part as a donor and 1-ethyl-2-methylbenzo [c, d] indole-1-onium as an acceptor, and is good in light stability, high in fluorescence intensity and high in fluorescence sensitivity. The fluorescence emission wavelength reaches the near-infrared second region, and the fluorescent probe has good biocompatibility and good photo-thermal imaging property and can be used for cell imaging and small animal living body imaging; the temperature can be increased to 60 DEG C from 20 DEG C under 808 nm laser irradiation, the photothermal therapy effect is excellent, the active oxygen generation capacity is high, the photodynamic effect is good, the synergistic photothermal and photodynamic therapy on tumor cells can be achieved, and the potential application prospect in the field of tumor therapy is achieved.

Description

Technical Field

[0001] The 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 main causes of death in humans and poses a huge threat to human health and life. At present, the main treatments for cancer include radiotherapy, chemotherapy and surgical resection. These traditional treatments have disadvantages such as large toxic side effects, low treatment efficiency and high treatment risks. Compared with traditional cancer treatments such as surgery, chemotherapy and radiotherapy, phototherapy has become an emerging research hotspot due to its high selectivity and efficient tumor destruction ability, no drug resistance, non-invasiveness and easy combination with other treatments.

[0003] Tumor phototherapy is generally divided into two types, photothermal therapy (PTT) and photodynamic therapy (PDT). PTT uses photosensitizers to absorb light of a specific wavelength and convert light energy into heat energy, thereby killing cancer cells. PDT uses photosensitizers to produce reactive oxygen species (ROS) in the presence of oxygen to kill specific tumor cells. With the development of medical technology, the limited therapeutic effect of a single mode and the side effects that have always existed can no longer meet the needs of the clinical treatment field. This is because the hypoxic microenvironment around the tumor seriously affects the efficiency of ROS generation, thereby severely inhibiting the efficiency of PDT. In addition, after some tumors are treated with PTT, the residual cancer cells are heat-resistant, which may cause tumor recurrence, making PTT limited in the treatment of tumor diseases. PDT and PTT dual-mode combined therapy (PDT-PTT) can overcome the shortcomings of single-mode treatment and achieve enhanced synergistic treatment effects. The reason is that PTT can increase the oxygen concentration by increasing the blood flow rate, thereby enhancing the PDT effect, while reversely 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 with tetrapyrrole, cyanine and BODIPY as skeletons, some of which have been approved for clinical use. However, these compounds have a common feature, that is, they all have a rigid planar structure. Due to the hydrophobicity of the molecules themselves and the strong π-π interaction between molecules, they will cause aggregation fluorescence quenching effect in an aqueous environment, resulting in a decrease in the singlet oxygen quantum yield. Therefore, the development of a diagnostic and therapeutic agent with near-infrared emission and high singlet oxygen yield is of great significance to improve the effect 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 hemicyanine fluorescent dye molecule, a preparation method and an application thereof. A coumarin hemicyanine photosensitizer with a D-π-A motif is designed and synthesized by using a tetraphenylethylene part as a donor and 1-ethyl-2-methylbenz[c,d]indol-1-ium as an acceptor, so that the emission wavelength is located in the near-infrared window (650-1000nm), and is beneficial 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] On the one hand, a coumarin-like hemicyanine fluorescent dye molecule is provided, and its 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 obtain 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] The structures of the compounds 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 min, separated and purified to obtain;

[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 or 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 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 medical imaging, wherein the medical imaging includes bioluminescence imaging and / or photoacoustic imaging.

[0026] In a 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 obtained by the above-mentioned method, wherein the combined therapy includes a combined therapy of photothermal therapy and 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 obtained 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 good 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 be raised from 20°C to 60°C under 808nm laser irradiation and reach a plateau at around 62°C, indicating that they have 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 method for synthesizing the coumarin-like hemicyanine fluorescent dye molecule 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 is the 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 is the 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 is the HRMS spectrum of the coumarin-like hemicyanine fluorescent dye CM-2-3 of the present invention;

[0040] Figure 7 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] Fig. 9 The photothermal properties of the coumarin-like hemicyanine fluorescent dye irradiated by lasers of different powers of the present invention;

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

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

[0045] Fig.12The PL spectra of DCFH of the coumarin hemicyanine fluorescent dye of the present invention in PBS after being irradiated with white light for different time periods;

[0046] Fig.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 indicated in the following examples are usually carried out under normal conditions or according to 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 equal to the recorded content can all be applied to the present invention. The preferred implementation methods and materials described in the text are only for demonstration purposes.

[0049] The endpoints and any values ​​of the ranges disclosed in this article 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 endpoint values ​​of each range, the endpoint values ​​of each range and the 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 as specifically disclosed in this article.

[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 part as a donor and 1-ethyl-2-methylbenz[c,d]indol-1-ium as an acceptor, having a high photothermal temperature, being able to cause thermal damage to the nuclear genetic material of tumor cells, inducing acute necrosis, apoptosis and microenvironmental destruction of the tumor, and having a good ability to generate reactive oxygen species, so that the compound can be used as a photosensitizer to achieve efficient photodynamic therapy guided by near-infrared light, thereby achieving 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, tetrakis(triphenylphosphine)palladium and potassium carbonate under Ar protection to toluene / water and heat under reflux, cool to room temperature, extract with dichloromethane, dry the organic phase, filter, distill under reduced pressure, collect, pass through a silica gel chromatography column, elute, separate and purify to obtain a light yellow solid compound 1.

[0055] S2: The obtained compound 1 and 1-(4-(diethylamino)-2-hydroxyacetophenone are dissolved in acetic acid and HClO4, heated to 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: Add acetic anhydride to the obtained compound 2, compound 3 (as shown in formula V) and potassium acetate, and 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-2-1 is obtained.

[0058]

[0059] In some examples of this implementation mode, in S1, the specific steps are: triphenylethylene bromide, 4-benzoyl borate, tetrakis(triphenylphosphine)palladium, potassium carbonate, under Ar protection, then add toluene / water, reflux at 110°C for 24 hours, and cool to room temperature; then extract with dichloromethane, dry the organic phase, filter, distill under reduced pressure, collect, pass through a silica gel chromatography column, elute, separate, and purify 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 implementation mode, 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 subjected to the next step of 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 embodiments of this implementation mode, in S3, the specific steps are: dissolving the obtained compound 2, compound 3, and potassium acetate in acetic anhydride, heating the reaction 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, filtering to obtain a black solid, and eluting, separating, and purifying through 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, extract with dichloromethane, dry the organic phase, filter, distill under reduced pressure, collect, pass through a silica gel chromatography column, elute, separate, and purify to obtain a light yellow solid compound 1;

[0069] S2: The obtained compound 1 and 1-(4-(diethylamino)-2-hydroxyacetophenone are dissolved in acetic acid and HClO4, heated to 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 diphenylimine 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, and its structural formula is shown in Formula VII:

[0072]

[0073] S4. Add acetic anhydride to the obtained compound 4, 1-ethyl-2-methylbenzo[c,d]indol-1-ium and potassium acetate. After heating reaction 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 implementation mode, in S1, the specific steps are: triphenylethylene bromide, 4-benzoyl borate, tetrakis(triphenylphosphine)palladium, potassium carbonate, under Ar protection, then add toluene / water, reflux at 110°C for 24 hours, and cool to room temperature; then extract with dichloromethane, dry the organic phase, filter, distill under reduced pressure, collect, pass through a silica gel chromatography column, elute, separate, and purify 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 implementation mode, 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 subjected to the next step of 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 heated to 80°C for 30 minutes under nitrogen protection. After the reaction, the mixture is poured into a saturated NaHCO3 solution in an ice bath, stirred vigorously, and filtered to obtain a black solid. The black solid is eluted, separated, and purified through 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 diphenylimide 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-methylbenzo[c,d]indol-1-ium and potassium acetate are dissolved in acetic anhydride, and heated at 65°C for 30 min under nitrogen protection. After the reaction, 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 through a silica gel chromatography column to obtain photosensitizer CM-2-2.

[0081] In some preferred embodiments, the molar ratio of the 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, extract with dichloromethane, dry the organic phase, filter, distill under reduced pressure, collect, pass through a silica gel chromatography column, elute, separate, and purify to obtain a light yellow solid compound 1;

[0086] S2: The obtained compound 1 and 1-(4-(diethylamino)-2-hydroxyacetophenone are dissolved in acetic acid and HClO4, heated to 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 black solid was eluted, separated and purified through 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-methylbenzo[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 implementation mode, in S1, the specific steps are: triphenylethylene bromide, 4-benzoyl borate, tetrakis(triphenylphosphine)palladium, potassium carbonate, under Ar protection, then add toluene / water, reflux at 110°C for 24 hours, and cool to room temperature; then extract with dichloromethane, dry the organic phase, filter, distill under reduced pressure, collect, pass through a silica gel chromatography column, elute, separate, and purify 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 implementation mode, 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 subjected to the next step of 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 heated to 80°C for 30 minutes under nitrogen protection. After the reaction, 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 through 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 pentadienal 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-methylbenzo[c,d]indol-1-ium and potassium acetate are dissolved in acetic anhydride, and heated at 65°C for 30 minutes under nitrogen protection. After the reaction, 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 through 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 solution of the present invention is 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 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), potassium carbonate (11.0 g, 79.59 mmol), toluene / water (v / v=4:1) were added under Ar gas protection, refluxed at 110°C for 24 h, and cooled to room temperature. The mixture was extracted with dichloromethane, the organic phase was dried, filtered, and distilled under reduced pressure to collect the obtained crude product. The obtained crude product was eluted with petroleum ether: dichloromethane=5:1 to obtain a light yellow solid compound 1 with 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 to 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 directly used for 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 diphenylimide 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 a 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-methylbenzo[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 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 5 (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-methylbenzo[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 respectively 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 tested by ULC 1503007 UV-visible spectrophotometer, and the emission spectrum of the solution was tested by F97XP fluorescence spectrophotometer. During the test, the slit of the instrument was adjusted to a suitable 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 solution with a concentration of 1 mmol / L. Then transfer 125 μL, 250 μL, 375 μL and 500 μL to 5 mL of PBS solution to dilute the solutions to concentrations of 25, 50, 75 and 100 μM.

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

[0117] like Figure 8 As shown, at a fixed laser power (808nm, 1.5W / 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, within 600 s, 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. The overall trend was that CM-2-1 had the highest temperature, which was 62 °C.

[0118] like Fig. 9 As shown, as the power density increases from 0.8W / cm 2 Increase 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 Fig.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] It can be seen that the coumarin-like hemicyanine fluorescent dye (CM-2-1, CM-2-2, CM-2-3) of the present invention has excellent photothermal properties and is a potential photothermal agent that can be used for effective photothermal anti-tumor therapy. After continuous laser irradiation at 808 nm for 10 min, it exhibits excellent photostability with negligible attenuation, and high photothermal stability is observed even after four heating-cooling cycles.

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

[0122] like Fig.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 continuous irradiation for 10 minutes, the temperatures of the three photosensitizers all increased significantly. It can be seen that the coumarin-like hemicyanine fluorescent dyes (CM-2-1, CM-2-2, and CM-2-3) of the present invention have good 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 assess 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. Fig.12 As shown, with the increase of white light irradiation time, the fluorescence intensity of DCFH co-incubated with 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 of Coumarin-like Hemicyanine Dyes

[0126] The DCFH-DA probe was used to evaluate the ROS-generating ability of the three photosensitizers in cells. Fig.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 500 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 a variety of similar expressions without violating the purpose and claims of the present invention, and such changes all fall within the protection scope 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 the 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 obtain 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 obtain 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 structures of the compounds are as follows:

3. The method according to claim 2, characterized in that The condensation reaction conditions are: adding an organic solvent and an activator under an inert atmosphere, reacting at 60-70° C. for 30-60 minutes, 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.

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

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

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

7. Use of the coumarin-like hemicyanine fluorescent dye molecule according to claim 1 or the coumarin-like hemicyanine fluorescent dye molecule prepared by the method according to any one of claims 2 to 6 in the preparation of tumor photothermal therapy and / or diagnostic reagents or drugs.

8. Use of the coumarin-like hemicyanine fluorescent dye molecule according to claim 1 or the coumarin-like hemicyanine fluorescent dye molecule prepared by the method according to any one of claims 2 to 6 in medical imaging, wherein the medical imaging includes bioluminescence imaging and / or photoacoustic imaging.

9. Use of the coumarin-like hemicyanine fluorescent dye molecule according to claim 1 or the coumarin-like hemicyanine fluorescent dye molecule obtained by the method according to any one of claims 2 to 6 in the preparation of a reagent or drug for combined therapy, wherein the combined therapy includes a combined therapy of photothermal therapy and photodynamic therapy, chemotherapy and / or radiotherapy.

10. A pharmaceutical composition, characterized in that A coumarin-like hemicyanine fluorescent dye molecule comprising the coumarin-like hemicyanine fluorescent dye molecule according to claim 1, a coumarin-like hemicyanine fluorescent dye molecule prepared by the method according to any one of claims 2 to 6, or a pharmaceutically acceptable salt, solvate, enantiomer, diastereomer, or tautomer thereof.

Images