D-pi-A type hemicyanine dye, preparation method and application

Abstract

The invention relates to the technical field of organic functional materials, in particular to a D-pi-A type hemicyanine dye and a preparation method and application thereof.The D-pi-A type hemicyanine dye takes a benzopyran derivative part as a donor and a hemicyanine dye part as an acceptor, pi bridges with different conjugation degrees are introduced between the hemicyanine dye and the benzopyran derivative, and the D-pi-A type hemicyanine dye is obtained. The synthesized compound is good in light stability and has good photo-thermal imaging property, the fluorescence emission wavelength reaches the near-infrared second region, the stokes shift can reach 157 nm to the maximum extent, and the compound has good biocompatibility and can be used for cell imaging and small animal living body imaging. And the temperature can be quickly increased to 53 DEG C from 30 DEG C within 2 minutes, and is stable at about 56 DEG C. The material has an excellent photo-thermal treatment effect and has a great potential application prospect in the field of tumor treatment.

Description

Technical Field

[0001] The invention relates to the technical field of organic functional materials, and in particular to a D-π-A type hemicyanine dye, a preparation method and application thereof. Background Art

[0002] Cancer is one of the leading causes of human death and poses a huge threat to human health and life. Rapid diagnosis and targeted treatment of cancer are particularly important as they can significantly improve the cure rate of cancer. In traditional clinical applications, tumor diagnosis and treatment are two largely independent processes that require the use of separate diagnostic contrast agents and therapeutic agents. The extended time between the two medical procedures can easily delay the best treatment opportunity, while the negative effects of two drug injections can cause discomfort and increase patient risks. To address these challenges, the integration of fluorescence imaging and photothermal treatment has emerged as a promising approach that can simultaneously perform in situ imaging and treatment during photoactivation, thereby providing faster and more precise diagnosis with unique advantages such as high sensitivity, rapid response, and non-invasiveness.

[0003] At present, clinical phototherapy is widely used in the treatment of solid tumors such as skin cancer and cancer. In the past few decades, people have been committed to developing functional fluorescent molecules, photosensitizers and photothermal converters that are optically responsive mainly in the short-wavelength region. Such drugs can be used for single-mode or multi-mode combined therapy, but due to the low tissue penetration depth of short-wavelength light, they are only suitable for the treatment and diagnosis of superficial lesions. Therefore, it is necessary to develop a diagnostic and therapeutic agent with a long wavelength that meets the requirements of fluorescence-guided photothermal therapy. Summary of the invention

[0004] In order to overcome the shortcomings of the above-mentioned technical defects, the present invention provides a D-π-A type hemicyanine dye, a preparation method and an application thereof. By using a benzopyran derivative portion as a donor and a hemicyanine dye portion as an acceptor, π bridges with different degrees of conjugation are introduced between the hemicyanine dye and the benzopyran derivative, and a hemicyanine photosensitizer with a D-π-A motif is designed and synthesized to achieve the purpose of fluorescence imaging and photothermal therapy for organisms.

[0005] In order to achieve the above object, the technical solution of the present invention is:

[0006] In one aspect, a D-π-A type hemicyanine dye is provided, and its structural formula is shown in Formula I to Formula III:

[0007]

[0008] Further, the compound of formula V reacts with the compound of formula VI to obtain a hemicyanine photosensitizer CM-1-1, the structure of which is shown in formula I;

[0009] Or the compound of formula IV reacts with the compound of formula VII to obtain the hemicyanine photosensitizer CM-1-2, the structure of which is shown in formula II;

[0010] Or the compound of formula IV reacts with the compound of formula VIII to produce a hemicyanine photosensitizer CM-1-3, the structure of which is shown in formula III;

[0011] The structures of the compounds are as follows:

[0012]

[0013] Furthermore, the condensation reaction conditions are: adding an organic solvent and an activator under an inert atmosphere, reacting at 60-70° C. for 30-60 min, separating and purifying to obtain;

[0014] The organic solvent is selected from one or more of anhydrous ethanol, anhydrous methanol, N,N-dimethylformamide or acetic anhydride;

[0015] The activator is selected from one or more of potassium carbonate, sodium acetate and potassium acetate.

[0016] Furthermore, the synthesis route of the compound of formula V is as follows:

[0017]

[0018] Furthermore, the synthetic route of the compound of formula VI 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 preparing tumor photothermal therapy and / or diagnostic reagents or drugs based on the above-mentioned D-π-A type hemicyanine dye and the D-π-A type hemicyanine dye prepared by the above-mentioned method.

[0025] In a fourth aspect, a method is provided for applying the D-π-A type hemicyanine dye 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 pharmaceutical composition is provided, comprising the third aspect, providing a D-π-A type hemicyanine dye based on the above-mentioned D-π-A type hemicyanine dye, the D-π-A type hemicyanine dye prepared by the above-mentioned method, or a pharmaceutically acceptable salt, solvate, enantiomer, diastereomer, or tautomer thereof.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] (1) The D-π-A type hemicyanine dye of the present invention has good photostability and good photothermal imaging properties;

[0029] (2) The fluorescence emission wavelength of the D-π-A type hemicyanine dye of the present invention reaches the second near-infrared region, the maximum Stokes shift can reach 157nm, and it has good biocompatibility and can be used for cell imaging and small animal in vivo imaging;

[0030] (3) The temperature of the D-π-A type hemicyanine dye of the present invention can be rapidly increased from 30°C to 53°C within 2 minutes and reach a plateau at around 56°C, indicating that it has an excellent photothermal therapeutic effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 The hemicyanine dye CM-1-1 of the present invention 1 H NMR spectrum;

[0032] Figure 2 is the HRMS (ESI) spectrum of the hemicyanine dye of the present invention;

[0033] Figure 3 The hemicyanine dye CM-1-2 of the present invention 1 H-NMR spectrum;

[0034] Figure 4 is the HRMS (ESI) spectrum of the hemicyanine dye of the present invention;

[0035] Figure 5 The hemicyanine dye CM-1-3 of the present invention 1 H NMR spectrum;

[0036] Figure 6 is the HRMS (ESI) spectrum of the hemicyanine dye of the present invention;

[0037] Figure 7 The ultraviolet-visible absorption spectrum and fluorescence emission spectrum of the hemicyanine dye of the present invention in dichloromethane;

[0038] Figure 8 The photothermal performance test of the hemicyanine dyes of different concentrations of the present invention is performed;

[0039] Fig. 9The photothermal properties of the hemicyanine dye irradiated by lasers of different powers of the present invention;

[0040] Fig.10 The light and heat stability of the hemicyanine dyes CM-1-1 (A), CM-1-2 (B), and CM-1-3 (C) of the present invention;

[0041] Fig.11 Photothermal imaging of the photo-hemicyanine dyes CM-1-1, CM-1-2, and CM-1-3 of the present invention;

[0042] Fig.12 In vivo fluorescence imaging of the hemicyanine dye CM-1-3 of the present invention;

[0043] Fig.13 In vivo photothermal imaging of the hemicyanine dye CM-1-3 of the present invention;

[0044] Fig.14 This is a photothermal treatment effect diagram of the hemicyanine dye CM-1-3 in vivo in tumor-bearing mice;

[0045] Among them, 14A is the in vivo imaging test result of mouse Hela cell tumor model; 14B is the in vivo photothermal imaging of semi-cyanine dye CM-1-3; 14C is the photothermal stability of semi-cyanine dye CM-1-3 in mouse Hela cell tumor model; 14D is the photothermal performance test of semi-cyanine dye CM-1-3 and control group; 14E is the in vivo weight change diagram of semi-cyanine dye CM-1-3 and control group after treatment; 14F is the comparison diagram of in vivo tumor after treatment of semi-cyanine dye CM-1-3 and control group;

[0046] Fig.15 This is the H&E staining image of hemicyanine dye CM-1-3. DETAILED DESCRIPTION

[0047] 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.

[0048] 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.

[0049] The present invention aims to provide a D-π-A type hemicyanine dye, by using a benzopyran derivative part as a donor and a hemicyanine dye part as an acceptor, introducing π bridges with different conjugation degrees between the hemicyanine dye and the benzopyran derivative, and designing and synthesizing three hemicyanine dyes with D-π-A motifs CM-1-1, CM-1-2, and CM-1-3. The hemicyanine dyes have good photostability, good near-infrared second-zone active fluorescence imaging, good photothermal imaging properties, and excellent photothermal therapeutic effects.

[0050] In some specific embodiments, a D-π-A type hemicyanine dye is provided, and its structural formula is shown in Formula I:

[0051]

[0052] In another specific embodiment, a preparation method based on the above-mentioned D-π-A type hemicyanine dye is provided, comprising the following steps:

[0053] S1. Add POCl to the mixture of malonic acid and phenol 3 , heated to reflux, cooled to room temperature, poured the supernatant into water, extracted with ethyl acetate, dried the organic layer over anhydrous sodium sulfate, removed the solvent from the crude product under reduced pressure, separated by column chromatography, and placed in a refrigerator to obtain compound 1;

[0054] S2: Add compound 1,3-diethylaminophenol and toluene respectively, reflux the mixture, cool it to room temperature after the reaction, and filter it to obtain compound 2;

[0055] S3: Compound 3 and dilute sulfuric acid were added separately, and the mixture was heated to react for 5 hours. After the reaction was completed, it was cooled to room temperature, neutralized with ammonia water until neutral, and the product was extracted with dichloromethane. The organic layer was dried over anhydrous sodium sulfate, and the solvent was removed by evaporation under reduced pressure. The crude product was separated by column chromatography and placed in a refrigerator to obtain compound 3;

[0056] S4: Add compound 3,4-dimethylaminoacetophenone, acetic acid and perchloric acid, react for 12 hours, cool to room temperature, pour into ice water, and filter to obtain compound 6, whose structural formula is shown in Formula VI:

[0057]

[0058] S5. Dissolve compound 4, N,N-dibutylformamide and triethyl orthoformate in ethanol. 2 The mixture was stirred for 30 min under protection, and then saturated sodium bicarbonate was added, stirred in an ice bath, filtered, and separated by column to obtain compound 5, whose structural formula is shown in Formula V:

[0059]

[0060] S6. Compound 6, compound 5 and potassium acetate were dissolved in acetic anhydride, and the condensation reaction was carried out for 30 min under nitrogen protection. Saturated NaHCO 3 The solution was filtered, separated and purified by column chromatography to obtain the semi-cyanine photosensitizer CM-1-1.

[0061] In some examples of this embodiment, in S1, the specific steps are: adding malonic acid and phenol, slowly adding POCl 3 After the addition was completed, the mixture was heated to 115°C and refluxed, and a tail gas absorption device was used until the HCl gas was completely released and the reaction was stopped. The reaction mixture was cooled to room temperature, and then the supernatant was poured into 150 mL of water and extracted with ethyl acetate. The organic layer was dried over anhydrous sodium sulfate, and the crude product was freed from the solvent under reduced pressure and separated by column chromatography to obtain a colorless transparent oil. After cooling in a refrigerator overnight, a white solid 1 was obtained.

[0062] In some examples of this embodiment, in S2, compound 1 and 3-diethylaminophenol are dissolved in toluene, and the mixture is refluxed at 118° C. for 5 h. After the reaction is completed, the mixture is cooled to room temperature and filtered to obtain a yellow solid 2.

[0063] In some examples of this embodiment, in S3, compound 2 is added with 30% sulfuric acid, and the mixture is reacted at 125° C. for 5 h. After the reaction is completed, the mixture is cooled to room temperature, and the mixture is neutralized with aqueous ammonia until it is neutral. The product is extracted with dichloromethane, and the organic layer is dried with anhydrous sodium sulfate. The solvent is removed by evaporation under reduced pressure, and the crude product is separated by column chromatography to obtain a purple-red oily substance, which is refrigerated overnight to obtain compound 3;

[0064] In some examples of this embodiment, in S4, compound 3 and 4-dimethylaminoacetophenone are dissolved in acetic acid and perchloric acid, and the mixture is reacted at 120° C. for 12 h. After the reaction is completed, the mixture is cooled to room temperature, poured into 150 mL of ice water, and filtered to obtain compound 6;

[0065] In some examples of this embodiment, in S5, at N 2 Under protection, compound 4, N,N-dibutylformamide and triethyl orthoformate were dissolved in ethanol and reacted at 65°C for 30 minutes. The solution turned red and then saturated sodium bicarbonate was added to neutralize it. The mixture was stirred in an ice bath, filtered and separated by column to obtain compound 5.

[0066] In some examples of this embodiment, in S6, compound 6, compound 5 and potassium acetate are dissolved in acetic anhydride, reacted at 65° C. for 30 min under nitrogen protection, and after the reaction is completed, the mixture is poured into a saturated NaHCO 3 The solution was stirred vigorously, filtered, separated and purified by column chromatography to obtain the semi-cyanine photosensitizer CM-1-1.

[0067] In some specific embodiments, a D-π-A type hemicyanine dye is provided, and its structural formula is shown in Formula II:

[0068]

[0069] In another specific embodiment, a preparation method based on the above-mentioned D-π-A type hemicyanine dye is provided, comprising the following steps:

[0070] S1. Add POCl to the mixture of malonic acid and phenol 3 , heated to reflux, cooled to room temperature, poured the supernatant into water, extracted with ethyl acetate, dried the organic layer over anhydrous sodium sulfate, removed the solvent from the crude product under reduced pressure, separated by column chromatography, and placed in a refrigerator to obtain compound 1;

[0071] S2: Add compound 1,3-diethylaminophenol and toluene respectively, reflux the mixture, cool it to room temperature after the reaction, and filter it to obtain compound 2;

[0072] S3: Compound 3 and dilute sulfuric acid were added separately, and the mixture was heated to react for 5 hours. After the reaction was completed, it was cooled to room temperature, neutralized with ammonia water until neutral, and the product was extracted with dichloromethane. The organic layer was dried over anhydrous sodium sulfate, and the solvent was removed by evaporation under reduced pressure. The crude product was separated by column chromatography and placed in a refrigerator to obtain compound 3;

[0073] S4: Add compound 3,4-dimethylaminoacetophenone, acetic acid and perchloric acid, react for 12 hours, cool to room temperature, pour into ice water, and filter to obtain compound 6, whose structural formula is shown in Formula VI:

[0074]

[0075] S5. Compound 6, malondialdehyde diphenylimine monohydrochloride and potassium acetate were dissolved in acetic anhydride, reacted for 30 min under nitrogen protection, and then poured into saturated NaHCO 3 The solution was filtered to obtain compound 7, whose structural formula is shown in Formula VII:

[0076]

[0077] S6. Compound 7, compound 4 and potassium acetate were dissolved in acetic anhydride, reacted for 30 min under nitrogen protection, and then saturated NaHCO 3 The solution was filtered, separated and purified by column chromatography to obtain hemicyanine photosensitizer CM-1-2.

[0078] In some examples of this embodiment, in S1, the specific steps are: adding malonic acid and phenol, slowly adding POCl 3 After the addition was completed, the mixture was heated to 115°C and refluxed, and a tail gas absorption device was used until the HCl gas was completely released and the reaction was stopped. The reaction mixture was cooled to room temperature, and then the supernatant was poured into 150 mL of water and extracted with ethyl acetate. The organic layer was dried over anhydrous sodium sulfate, and the crude product was freed from the solvent under reduced pressure and separated by column chromatography to obtain a colorless transparent oil. After cooling in a refrigerator overnight, a white solid 1 was obtained.

[0079] In some examples of this embodiment, in S2, compound 1 and 3-diethylaminophenol are dissolved in toluene, and the mixture is refluxed at 118° C. for 5 h. After the reaction is completed, the mixture is cooled to room temperature and filtered to obtain a yellow solid 2.

[0080] In some examples of this embodiment, in S3, compound 2 is added with 30% sulfuric acid, and the mixture is reacted at 125° C. for 5 h. After the reaction is completed, the mixture is cooled to room temperature, and the mixture is neutralized with aqueous ammonia until it is neutral. The product is extracted with dichloromethane, and the organic layer is dried with anhydrous sodium sulfate. The solvent is removed by evaporation under reduced pressure, and the crude product is separated by column chromatography to obtain a purple-red oily substance, which is refrigerated overnight to obtain compound 3;

[0081] In some examples of this embodiment, in S4, compound 3 and 4-dimethylaminoacetophenone are dissolved in acetic acid and perchloric acid, and the mixture is reacted at 120° C. for 12 h. After the reaction is completed, the mixture is cooled to room temperature, poured into 150 mL of ice water, and filtered to obtain compound 6;

[0082] In some examples of this embodiment, in S5, compound 6, malondialdehyde diphenylimine monohydrochloride and potassium acetate are dissolved in acetic anhydride, reacted at 80° C. for 30 min under nitrogen protection, and after the reaction is completed, the mixture is poured into saturated NaHCO 3 The solution was stirred vigorously, filtered, and separated and purified by column chromatography to obtain compound 7;

[0083] In some examples of this embodiment, in S6, compound 7, compound 4 and potassium acetate are dissolved in acetic anhydride, reacted at 65° C. for 30 min under nitrogen protection, and after the reaction is completed, the mixture is poured into a saturated NaHCO 3 The solution was stirred vigorously, filtered, separated and purified by column chromatography to obtain the semi-cyanine photosensitizer CM-1-2.

[0084] In some specific embodiments, a D-π-A type hemicyanine dye is provided, and its structural formula is shown in Formula III:

[0085]

[0086] In another specific embodiment, a preparation method based on the above-mentioned D-π-A type hemicyanine dye is provided, comprising the following steps:

[0087] S1. Add POCl to the mixture of malonic acid and phenol 3 , heated to reflux, cooled to room temperature, poured the supernatant into water, extracted with ethyl acetate, dried the organic layer over anhydrous sodium sulfate, removed the solvent from the crude product under reduced pressure, separated by column chromatography, and placed in a refrigerator to obtain compound 1;

[0088] S2: Add compound 1,3-diethylaminophenol and toluene respectively, reflux the mixture, cool it to room temperature after the reaction, and filter it to obtain compound 2;

[0089] S3: Compound 3 and dilute sulfuric acid were added separately, and the mixture was heated to react for 5 hours. After the reaction was completed, it was cooled to room temperature, neutralized with ammonia water until neutral, and the product was extracted with dichloromethane. The organic layer was dried over anhydrous sodium sulfate, and the solvent was removed by evaporation under reduced pressure. The crude product was separated by column chromatography and placed in a refrigerator to obtain compound 3;

[0090] S4: Add compound 3,4-dimethylaminoacetophenone, acetic acid and perchloric acid, react for 12 hours, cool to room temperature, pour into ice water, and filter to obtain compound 6, whose structural formula is shown in Formula VI:

[0091]

[0092] S5. Dissolve compound 6, pentadienaldehyde diphenylamine hydrochloride and potassium acetate in acetic anhydride, react for 30 min under nitrogen protection, and pour into saturated NaHCO 3 The solution was filtered to obtain compound 8, whose structural formula is shown in Formula VIII:

[0093]

[0094] S6. Compound 8, compound 4 and potassium acetate were dissolved in acetic anhydride and reacted at 65°C for 30 min under nitrogen protection. After the reaction was completed, the mixture was poured into saturated NaHCO in an ice bath. 3 The solution was stirred vigorously, filtered, separated and purified by column chromatography to obtain the semi-cyanine photosensitizer CM-1-3.

[0095] In some examples of this embodiment, in S1, the specific steps are: adding malonic acid and phenol, slowly adding POCl 3 After the addition was completed, the mixture was heated to 115°C and refluxed, and a tail gas absorption device was used until the HCl gas was completely released and the reaction was stopped. The reaction mixture was cooled to room temperature, and then the supernatant was poured into 150 mL of water and extracted with ethyl acetate. The organic layer was dried over anhydrous sodium sulfate, and the crude product was freed from the solvent under reduced pressure and separated by column chromatography to obtain a colorless transparent oil. After cooling in a refrigerator overnight, a white solid 1 was obtained.

[0096] In some examples of this embodiment, in S2, compound 1 and 3-diethylaminophenol are dissolved in toluene, and the mixture is refluxed at 118° C. for 5 h. After the reaction is completed, the mixture is cooled to room temperature and filtered to obtain a yellow solid 2.

[0097] In some examples of this embodiment, in S3, compound 2 is added with 30% sulfuric acid, and the mixture is reacted at 125° C. for 5 h. After the reaction is completed, the mixture is cooled to room temperature, and the mixture is neutralized with aqueous ammonia until it is neutral. The product is extracted with dichloromethane, and the organic layer is dried with anhydrous sodium sulfate. The solvent is removed by evaporation under reduced pressure, and the crude product is separated by column chromatography to obtain a purple-red oily substance, which is refrigerated overnight to obtain compound 3;

[0098] In some examples of this embodiment, in S4, compound 3 and 4-dimethylaminoacetophenone are dissolved in acetic acid and perchloric acid, and the mixture is reacted at 120° C. for 12 h. After the reaction is completed, the mixture is cooled to room temperature, poured into 150 mL of ice water, and filtered to obtain compound 6;

[0099] In some examples of this embodiment, in S5, compound 6, pentadienaldehyde diphenylamine hydrochloride and potassium acetate are dissolved in acetic anhydride, reacted at 80° C. for 30 min under nitrogen protection, and after the reaction is completed, the mixture is poured into saturated NaHCO 3 The solution was stirred vigorously, filtered, and separated and purified by column chromatography to obtain compound 8;

[0100] In some examples of this embodiment, in S6, compound 8, compound 4 and potassium acetate are dissolved in acetic anhydride, reacted at 65° C. for 30 min under nitrogen protection, and after the reaction is completed, the mixture is poured into a saturated NaHCO 3 The solution was stirred vigorously, filtered, separated and purified by column chromatography to obtain the semi-cyanine photosensitizer CM-1-3.

[0101] 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.

[0102] Example 1 Synthesis of Hemicyanine Photosensitizer

[0103] (1) Preparation of compound 1: Add malonic acid (11.0 g, 106.0 mmol) and phenol (19.9 g, 212 mmol) into a 500 mL single-necked bottle, and slowly add POCl 3 (11.5mL, 123mmol), after the addition is completed, the mixture is heated to 115°C and refluxed, and a tail gas absorption device is used until the HCl gas is completely released and the reaction is stopped. The reaction mixture is cooled to room temperature, and then the supernatant is poured into 150mL of water, extracted with ethyl acetate, and the organic layer is dried over anhydrous sodium sulfate. The crude product is freed from the solvent under reduced pressure, and separated by column chromatography, and the eluent is: petroleum ether / dichloromethane (v / v=3 / 1) to obtain a colorless transparent oil. After cooling in a refrigerator overnight, a white solid is obtained.

[0104] (2) A 250 mL single-mouth bottle was added with diphenyl malonate (5 g, 19.5 mmol), 3-diethylaminophenol (3.2 g, 19.5 mmol) and 15 mL of toluene, and the mixture was refluxed at 118° C. for 5 h. After the reaction was completed, the mixture was cooled to room temperature and filtered to obtain 2.9 g of a yellow solid with a yield of 64%. The product was directly used for the next step without purification. 1 H NMR (500MHz, MeOD) δ7.68–7.65(m,1H),6.69(dd,J=9.0,2.5Hz,1H),6.48(d,J=2.4Hz,1H),3.47(q,J=7.1Hz,4H),1.22(t,J=7.1Hz,6H).

[0105] (3) Compound 3 (2 g, 8.57 mmol) was added to a 125 mL single-necked bottle, and 60 mL of 30% sulfuric acid was added. The mixture was reacted at 125 °C for 5 h. After the reaction was completed, it was cooled to room temperature and neutralized with aqueous ammonia until it was neutral. The product was extracted with dichloromethane (50 × 3 mL). The organic layer was dried over anhydrous sodium sulfate and the solvent was removed by evaporation under reduced pressure. The crude product was separated by column chromatography to obtain a purple-red oil, which was refrigerated overnight to obtain a red solid 4. 1 H NMR (500 MHz, CDCl 3 )δ12.94(s,1H),7.53(d,J=9.1Hz,1H),6.20(dd,J=9.1,2.5Hz,1H),6.08( d,J=2.5Hz,1H),3.41(q,J=7.1Hz,4H),2.49(s,3H),1.22(t,J=7.1Hz,6H).

[0106] (4) In N 2Under protection, compound 4 (0.323 g, 1 mmol), N,N-dibutylformamide (0.196 g, 1 mmol) and triethyl orthoformate (0.15 g, 1 mmol) were dissolved in 10 mL of ethanol, heated and stirred at 65°C for 30 minutes, and the solution turned red. 50 mL of saturated sodium bicarbonate was added under ice bath conditions, stirred vigorously, filtered, and separated by column to obtain a red solid. 1 H NMR (400 MHz, CDCl 3 )δ9.07(t,J=12.9Hz,1H),8.41(d,J=7.4Hz,1H),8.21(d,J=8.1Hz,1H),7.92(t,J=7.7Hz,1H),7.84(d,J=12.2Hz,1H),7.77(d,J=8.3Hz,1H),7 .64(d,J=1.2Hz,1H),7.62(d,J=1.9Hz,1H),7.43(t,J=7.9Hz,2H),7.27(d,J=2.6Hz,1H),4.24(dd,J=14.2,7.1Hz,2H),1.56(t,J=7.3Hz,3H).

[0107] (5) Compound 3 (250 mg, 1.2 mmol), 4-dimethylaminoacetophenone (195 mg, 1.2 mmol), acetic acid 5 mL and perchloric acid 6 mL were added to a 50 mL single-mouth bottle, and the mixture was reacted at 120°C for 12 h. After the reaction was completed, the mixture was cooled to room temperature and poured into 150 mL of ice water. 84 mg of a black solid with a metallic luster was obtained by filtration, with a yield of 21%. The product was directly used for the next step without purification.

[0108] (6) Compound 6 (50 mg, 0.15 mmol), malondialdehyde bisbenzimidamine monohydrochloride (39 mg, 0.15 mmol) and potassium acetate (44 mg, 0.45 mmol) were dissolved in 2 mL of acetic anhydride and reacted at 80 °C for 30 min under nitrogen protection. After the reaction was completed, the mixture was poured into a saturated NaHCO solution in an ice bath. 3 The solution was stirred vigorously and filtered to obtain a black solid. The product was directly used for the next step without purification. 1H NMR (500MHz, DMSO) δ8.40 (dd, J=28.6, 14.3Hz, 2H), 8.24 (d, J=8.8Hz, 2H), 8.1 5(d,J=8.9Hz,1H),8.01(s,1H),7.71–7.56(m,4H),7.48(d,J=7.3Hz,2H),7.2 6(d,J=15.0Hz,1H),7.10(d,J=9.1Hz,2H),6.91(d,J=9.0Hz,2H),5.57–5.50( m,1H),3.61(d,J=6.9Hz,4H),3.16(s,6H),1.97(s,3H),1.20(t,J=6.9Hz,6H).

[0109] (7) Compound 6 (200 mg, 0.60 mmol), pentadienaldehyde diphenylamine hydrochloride (172 mg, 0.60 mmol) and potassium acetate (176 mg, 1.8 mmol) were dissolved in 4 mL of acetic anhydride and reacted at 80 °C for 30 min under nitrogen protection. After the reaction was completed, the mixture was poured into a saturated NaHCO 3 The solution was stirred vigorously and filtered to obtain a black solid. The product was separated and purified by column chromatography to obtain 67 mg of a black solid with a yield of 21%. 1H NMR (500MHz, DMSO) δ8.23 (d, J = 9.1 Hz, 2H), 8.10 (d, J = 14.6 Hz, 1H), 8.06 (s, 1H), 8.04 (s, 1H), 7.98 (s, 1H), 7.63 (t, J = 7.5 Hz, 2H), 7.58 (d, J = 7.3 Hz, 1H), 7.41 (d, J = 7.4 Hz, 2H), 7.23 (d, J = 14.8 Hz, 1H), 7.17(d,J=11.8Hz,1H),7.10(s,1H),7.06(d,J=11.4Hz,1H),6.88(d,J=9.2Hz,2H),6.50(t,J=10.6 Hz,1H),5.22–5.16(m,1H),3.61(d,J=7.1Hz,4H),3.15(s,6H),1.93(s,3H),1.21(t,J=7.0Hz,6H).

[0110] (8) Compound 6 (300 mg, 0.9 mmol), compound 5 (384 mg, 0.9 mmol) and potassium acetate (264 mg, 2.7 mmol) were added to a 50 mL two-necked bottle, and 5 mL of acetic anhydride was added. The reaction was carried out at 65 °C for 30 min under nitrogen protection. After the reaction was completed, the mixture was poured into a saturated NaHCO solution in an ice bath. 3The solution was stirred vigorously and filtered to obtain CM-1-1. The crude product was separated and purified by column chromatography with a yield of 27%. It was characterized by mass spectrometry and nuclear magnetic resonance ( Figure 1-2 ). 1H NMR (500MHz, DMSO) δ8.93(d,J=8.9Hz,1H),8.84(s,1H),8.29(d,J=8.8Hz,2H),8.18(d,J =9.6Hz,1H),8.13(s,1H),8.08(s,1H),7.89(t,J=7.7Hz,1H),7.64–7.57(m,2H),7.49(d ,J=13.8Hz,1H),7.34(s,1H),7.15(s,1H),7.04(s,1H),6.93(d,J=9.2Hz,2H),6.85(d,J =8.4Hz,1H),4.24(s,2H),3.61(d,J=7.0Hz,4H),3.14(s,6H),1.39(s,3H),1.24(s,6H).

[0111] (9) Compound 7 (200 mg, 0.39 mmol), compound 4 (126 mg, 0.39 mmol) and potassium acetate (114 mg, 1.17 mmol) were added to a 50 mL two-necked bottle, and 5 mL of acetic anhydride was added. The reaction was carried out at 65 °C for 30 min under nitrogen protection. After the reaction was completed, the mixture was poured into a saturated NaHCO solution in an ice bath. 3 The solution was stirred vigorously and filtered to obtain CM-1-2. The crude product was separated and purified by column chromatography with a yield of 20%. It was characterized by mass spectrometry and nuclear magnetic resonance ( Figure 3-4 ). 1 H NMR (400 MHz, CDCl 3 )δ8.28(d,J=6.7Hz,1H),7.94–7.83(m,4H),7.65(t,J=7.6Hz,1H),7.47(t,J=7.4Hz ,2H),7.32(s,1H),7.18(t,J=7.5Hz,1H),7.10(d,J=7.8Hz,1H),6.54(dd,J=28.0,1 4.1Hz,5H),6.38(d,J=6.8Hz,1H),6.19(s,1H),5.85(d,J=12.6Hz,1H),3.52(d,J=6 .9Hz,2H),3.36(d,J=7.0Hz,4H),2.88(s,6H),1.26–1.22(m,3H),1.22–1.17(m,6H).

[0112] (10) Compound 8 (200 mg, 0.38 mmol), compound 4 (122 mg, 0.38 mmol) and potassium acetate (111 mg, 1.14 mmol) were added to a 50 mL two-necked bottle, and 5 mL of acetic anhydride was added. The reaction was carried out at 65 °C for 30 min under nitrogen protection. After the reaction was completed, the mixture was poured into a saturated NaHCO solution in an ice bath. 3 The solution was stirred vigorously and filtered to obtain CM-1-3. The crude product was separated and purified by column chromatography with a yield of 20%. It was characterized by mass spectrometry and nuclear magnetic resonance ( Figure 5-6 ). 1H NMR (400MHz, DMSO) δ8.17 (dd, J=25.4, 8.4Hz, 5H), 7.93 (t, J=18.4Hz, 2H), 7.84 ( s,1H),7.68(s,1H),7.40(dd,J=27.8,11.9Hz,4H),7.12(d,J=8.7Hz,1H),7.02( s,1H),6.89(d,J=8.0Hz,3H),6.75(dd,J=27.3,13.1Hz,2H),6.39(d,J=10.2Hz, 1H), 4.02 (s, 2H), 3.61 (d, J = 5.9Hz, 4H), 3.16 (s, 6H), 1.26 (s, 3H), 1.24 (s, 6H).

[0113] The synthetic route is as follows:

[0114]

[0115] Example 2 Investigation of the UV Fluorescence Properties of Hemicyanine Dyes CM-1-1, CM-1-2, and CM-1-3

[0116] Weigh 2 mg of CM-1-1, 2 mg of CM-1-2 and 2 mg of CM-1-3 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.

[0117] 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.

[0118] The photophysical properties of the photosensitizer were investigated using UV-visible absorption and fluorescence emission spectroscopy, such as Figure 7As shown, the three photosensitizers all show broad absorption bands in dichloromethane, with the maximum absorption peaks at 751nm, 846nm and 863nm, respectively. CM-1-3 has the highest degree of conjugation, so its absorption peak is the farthest. In addition, the maximum emission peaks of CM-1-1, CM-1-2 and CM-1-3 appear at 904, 903 and 1020nm, and the emission peaks all appear in the NIR-II region. It is worth noting that the Stokes shift of the peak of CM-1-3 is the largest, reaching 157nm.

[0119] Example 3 Photothermal performance test of different concentrations of hemicyanine dyes

[0120] 1.335 mg of compound CM-1-1 was dissolved in 2 mL of DMSO to prepare a solution with a concentration of 1 mmol / L. Then 125 μL, 250 μL, 375 μL and 500 μL were respectively transferred to 5 mL of PBS solution to dilute the solution into solutions with concentrations of 25, 50, 75 and 100 μM.

[0121] 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.

[0122] like Figure 8 As shown, at a fixed laser power (808nm, 1.5W / cm 2 ), the photothermal temperature of pure PBS did not change much under the irradiation of 25 μM, while the temperature of photosensitizer CM-1-1, CM-1-2 and CM-1-3 solutions also increased with the increase of photosensitizer concentration. When the photosensitizer concentration increased from 25 μM to 100 μM, the temperature of CM-1-1 increased from 57 to 61°C, the temperature of CM-1-2 increased from 45 to 58°C, and the temperature of CM-1-3 increased from 52 to 62.5°C within 600 s. The overall trend was that the temperature of CM-1-3 was the highest, which was 62.5°C, followed by the temperature of CM-1-1 (61°C) and the temperature of CM-1-2 (58°C). The results showed that the photosensitizer had excellent photothermal performance and CM-1-3 had the best photothermal performance.

[0123] Example 4 Photothermal performance test of hemicyanine dyes irradiated with lasers of different powers

[0124] 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-1-1 increased from 39°C to 61°C, the temperature of CM-1-2 increased from 37°C to 58°C, and the temperature of CM-1-3 increased from 38°C to 62.5°C. This shows that the temperature is positively correlated with the laser power intensity of the photosensitizer.

[0125] Example 5 Investigation of the Photothermal Stability of Hemicyanine Dyes

[0126] Photothermal stability is a key parameter affecting the performance of photothermal preparations, and good stability is also an important indicator for evaluating in vivo imaging performance. Fig.10 As shown in the figure, after four photothermal cycles of 808 nm laser irradiation, the temperature of photosensitizer CM-1-1 was stabilized at 50 °C, the temperature of CM-1-2 was stabilized at 56 °C, and the temperature of CM-1-3 was stabilized at 61 °C. In general, the three photosensitizers showed excellent photostability after 808 nm continuous laser irradiation for 10 min, with negligible attenuation. High photothermal stability was observed even after four heating-cooling cycles.

[0127] Example 6 Investigation of Photothermal Imaging of Hemicyanine Dyes

[0128] like Fig.11 As shown, a photosensitizer solution (50 μM) was placed at a low power density (1.5 W / cm 2 ) After continuous irradiation for 10 min, the temperatures of the three photosensitizers increased significantly, and the temperature of CM-1-3 was significantly higher than that of the other two photosensitizers.

[0129] The above indicates that the hemicyanine dyes CM-1-1, CM-1-2 and CM-1-3 involved in the present invention have good photothermal properties.

[0130] Example 7 Investigation of in vivo fluorescence imaging of hemicyanine dyes

[0131] Since CM-1-1 has strong NIR fluorescence above 800 nm, we explored its ability to perform in vivo fluorescence imaging in the BABL / C mouse model. Fig.12 As shown, the fluorescence signal in the tumor area gradually increases with time, and a significant fluorescence signal can be detected about 2 hours after the tail vein injection of the photosensitizer CM-1-1 (100μM, 200μL), and the tumor outline can be clearly distinguished. Obvious fluorescence signals can still be observed within 6 hours after injection, but the fluorescence intensity decreases a little due to the gradual metabolism of CM-1-1. This shows that CM-1-1 can effectively accumulate in tumors.

[0132] Example 8: Photothermal therapy of tumor-bearing mice using hemicyanine dyes

[0133] Photosensitizer CM-1-3 (100 μM, 200 μL, solvent: PBS) was injected subcutaneously at the thigh of BABL / C nude mice or mice, and the light was irradiated at 808 nm, 1 W / cm 2 The laser was irradiated for 10 minutes and dynamic monitoring was performed. Fig.13 As shown, the tumor temperature increased rapidly from 25°C to 45°C in just 2 minutes and reached a plateau of about 50°C within ten minutes, indicating that the photothermal temperature of the photosensitizer was very stable and ideal.

[0134] Guided by the above results, we further tested the PTT effect of photosensitizer CM-1-3 on tumors in vivo. We used Hela cell tumor-bearing mice as model animals to evaluate the in vivo PTT efficacy of photosensitizer CM-1-3. Fig.14 As shown in (A), a Hela cell tumor model was first established, and then 200 μL of photosensitizer (200 μM) was injected intratumorally for diagnosis and treatment. Tumor-bearing mice were randomly divided into four groups: PBS, PBS+laser, CM-1-3, and CM-1-3+laser. The same volume of PBS was given to the PBS and PBS+laser groups. Fig.14 (B) shows that during laser irradiation, the tumor temperature was monitored in real time by an infrared thermal imager. Fig.14 (C) It can be seen that the tumor temperature of the CM-1-3+laser group increased rapidly from 30°C to 53°C within 2 minutes and then stabilized at around 56°C. In contrast, there was no obvious increase in tumor temperature in the PBS+laser group, indicating that the photothermal effect after laser irradiation was negligible in the absence of photosensitizer CM-1-3.

[0135] During the 18-day monitoring period after laser irradiation, the tumor volume of each group of mice was recorded every two days. Fig.14 As shown in (D), the tumors in the control group, including the PBS group and the PBS+laser group, showed similar growth rates, while the tumor volume in the CM-1-3 group also showed a downward trend, which may be due to the high cytotoxicity, indicating that neither CM-1-3 nor laser irradiation alone has an anti-tumor effect. In contrast, the combined treatment of CM-1-3 and laser irradiation showed a good tumor inhibition effect and eventually eradicated the tumor. In addition, Fig.14 (E) It can be seen that the PBS+laser group, CM-1-3 group, and CM-1-3+laser group showed comparable body weights to the PBS group at each time point, indicating that the systemic toxicity of the photosensitizer was negligible and the laser intensity was well tolerated. Fig.14 (F) It can be seen that the treatment effect of CM-1-3+laser group is obvious, and the tumor volume is significantly smaller than that of the other three groups.

[0136] Example 9 Investigation of biological toxicity of hemicyanine dyes

[0137] To further evaluate the tumor killing effect of CM-1-3 under laser irradiation, the heart, liver, spleen, lung and kidney were collected after treatment for H&E staining ( Fig.15 ), no obvious tissue damage was observed, indicating that CM-1-3 + laser had no toxicity in various organs. The above results show that the photosensitizer CM-1-3 has an excellent PTT effect on mouse tumors.

[0138] 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 D-π-A type hemicyanine dye, characterized in that: Its structural formula is shown in Formula I-Formula III:

2. The method for preparing the D-π-A type hemicyanine dye according to claim 1, characterized in that: The compound of formula V reacts with the compound of formula VI to obtain a hemicyanine photosensitizer CM-1-1, the structure of which is shown in formula I; Or the compound of formula IV reacts with the compound of formula VII to obtain the hemicyanine photosensitizer CM-1-2, the structure of which is shown in formula II; Or the compound of formula IV reacts with the compound of formula VIII to produce a hemicyanine photosensitizer CM-1-3, 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 V is as follows:

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

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

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

8. Use of the D-π-A type hemicyanine dye according to claim 1 or the D-π-A type hemicyanine dye prepared by the method according to any one of claims 2 to 7 in the preparation of tumor photothermal therapy and / or diagnostic reagents or drugs.

9. Use of the D-π-A type hemicyanine dye according to claim 1 or the D-π-A type hemicyanine dye prepared by the method according to any one of claims 2 to 7 in medical imaging, wherein the medical imaging includes bioluminescence imaging and / or photoacoustic imaging.

10. A pharmaceutical composition, characterized in that Containing the D-π-A type hemicyanine dye according to claim 1, the D-π-A type hemicyanine dye prepared by the method according to any one of claims 2 to 7, or a pharmaceutically acceptable salt, solvate, enantiomer, diastereomer, or tautomer thereof.

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