A D-π-A type semi-cyanine dye, preparation method and application

Abstract

The present invention relates to the technical field of organic functional materials, and particularly relates to a D-π-A type semi-cyanine dye, a preparation method and an application thereof. The D-π-A type semi-cyanine dye uses a benzopyran derivative part as a donor and a semi-cyanine dye part as an acceptor, and a π-bridge with different conjugation degrees is introduced between the semi-cyanine dye and the benzopyran derivative. The synthesized compound has good photostability, good photothermal imaging properties, the fluorescence emission wavelength reaches the second near-infrared region, the Stokes shift can reach up to 157 nm at most, and has good biocompatibility, and can be used for cell imaging and small animal in vivo imaging; and can rapidly rise from 30 °C to 53 °C within 2 minutes and reach a steady state at about 56 °C, and has excellent photothermal therapy effect, 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 specifically relates to a D-π-A type semi-cyanine dye, a preparation method and an application thereof. Background Art

[0002] Cancer is one of the main causes of human death and poses a great threat to human health and life. The rapid diagnosis and targeted treatment of cancer are particularly important because 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 reagents. The extended time between the two medical procedures easily delays the optimal treatment opportunity, and the negative effects of two drug injections can cause discomfort and increase the risk to the patient. To address these challenges, the integration of fluorescence imaging and photothermal therapy has emerged as a promising approach that can simultaneously perform in-situ imaging and treatment during the photoactivation process, thus providing faster and more accurate diagnosis with unique advantages such as high sensitivity, rapid response, and non-invasiveness.

[0003] Currently, clinical phototherapy is widely used in the treatment of solid tumors such as skin cancer and cancer. In the past few decades, efforts have been made to develop functional fluorescent molecules, photosensitizers, and photothermal conversion agents that mainly have optical responsiveness in the short wavelength region. Such drugs can be used for single-mode or multimode combination 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 very necessary to develop a long-wavelength diagnostic and therapeutic reagent that meets fluorescence-guided photothermal therapy. Summary of the Invention

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

[0005] To achieve the above object, the technical solution of the present invention is as follows:

[0006] On the one hand, a D-π-A type semi-cyanine dye is provided, and its structural formula is shown in Formula I - Formula III:

[0007]

[0008] Furthermore, when the compound of Formula V reacts with the compound of Formula VI through a condensation reaction, the semi-cyanine photosensitizer CM-1-1 is obtained, and its structure is shown in Formula I;

[0009] The condensation reaction of the compound of formula IV with the compound of formula VII gives the hemicyanine photosensitizer CM-1-2, the structure of which is shown in formula II;

[0010] The condensation reaction of the compound of formula IV with the compound of formula VIII gives the hemicyanine photosensitizer CM-1-3, the structure of which is shown in formula III;

[0011] Among them, the structures of the compounds are as follows:

[0012]

[0013] Furthermore, the conditions for the condensation reaction are as follows: under an inert atmosphere, an organic solvent and an activator are added, and the reaction is carried out at 60 - 70 °C for 30 - 60 min, followed by separation and purification;

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

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

[0016] Furthermore, the synthetic 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 synthetic route of the compound of formula VIII is as follows:

[0023]

[0024] In the third aspect, there is provided an application of the above-mentioned D-π-A type hemicyanine dye and the D-π-A type hemicyanine dye prepared by the above method in the preparation of tumor photothermal therapy and / or diagnostic reagents or drugs.

[0025] In the fourth aspect, there is provided an application of the above-mentioned D-π-A type hemicyanine dye and the D-π-A type hemicyanine dye prepared by the above method in medical imaging, and the medical imaging includes biofluorescence imaging and / or photoacoustic imaging.

[0026] Fifth aspect, there is provided a pharmaceutical composition containing the D-π-A type hemicyanine dye described in the third aspect, the D-π-A type hemicyanine dye prepared by the above method, or a pharmaceutically acceptable salt, solvate, enantiomer, diastereomer, or tautomer thereof.

[0027] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0028] (1) The D-π-A type hemicyanine dye of the present invention has good photo-stability and excellent 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 Stokes shift can reach up to 157 nm at most, and it has good biocompatibility and can be used for cell imaging and small animal in vivo imaging;

[0030] (3) The D-π-A type hemicyanine dye of the present invention can rapidly increase from 30 °C to 53 °C within 2 min and reach a plateau at about 56 °C, indicating excellent photothermal therapy effect. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

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

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

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

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

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

[0038] Figure 8 Photothermal performance test of the hemicyanine dye with different concentrations of the present invention;

[0039] Figure 9Photothermal properties of hemicyanine dyes irradiated by lasers with different powers of the present invention;

[0040] Figure 10 Photothermal stability of hemicyanine dyes CM-1-1(A), CM-1-2(B), and CM-1-3(C) of the present invention;

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

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

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

[0044] Figure 14 Photothermal therapy effect diagram of hemicyanine dye CM-1-3 in tumor-bearing mice of the present invention;

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

[0046] Figure 15 H&E staining diagram of hemicyanine dye CM-1-3. Detailed implementation manners

[0047] To enable those skilled in the art to better understand the technical solutions of the present invention, the present invention will be described in detail below in conjunction with specific implementation manners. The experimental methods without specific conditions noted in the following examples are usually carried out according to conventional conditions or according to the conditions recommended by the manufacturer. The test materials used in the following examples are all obtained from regular biochemical reagent stores without special instructions. Unless otherwise stated, percentages and parts are calculated by weight. Unless otherwise defined, all professional and scientific terms used herein have the same meaning as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to the described content can be applied to the present invention. The preferred implementation methods and materials described herein are for illustrative purposes only.

[0048] The endpoints and any values within the ranges disclosed herein are not limited to the exact ranges or values. 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, between the endpoint values of each range and individual point values, and between individual point values can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be considered to be specifically disclosed herein.

[0049] The present invention aims to provide a D-π-A type hemicyanine dye. By using a benzopyran derivative part as the donor and a hemicyanine dye part as the acceptor, π-bridges with different conjugation degrees are introduced between the hemicyanine dye and the benzopyran derivative, and three D-π-A motif hemicyanine dyes CM-1-1, CM-1-2, and CM-1-3 are designed and synthesized. They have good photostability, good near-infrared second-region active fluorescence imaging properties, good photothermal imaging properties, and excellent photothermal therapy effects.

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

[0051]

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

[0053] S1. Drop POCl 3 into the mixture of malonic acid and phenol, heat under reflux, cool to room temperature, pour the supernatant into water, extract with ethyl acetate, dry the organic layer with anhydrous sodium sulfate, remove the solvent under reduced pressure for the crude product, separate by column chromatography, and place in the refrigerator to obtain Compound 1;

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

[0055] S3: Add Compound 3 and dilute sulfuric acid respectively, heat the mixture to react for 5 h, cool to room temperature after the reaction ends, neutralize the reaction to neutrality with ammonia water, extract the product with dichloromethane, dry the organic layer with anhydrous sodium sulfate, remove the solvent by evaporation under reduced pressure, separate the crude product by column chromatography, and place in the refrigerator to obtain Compound 3;

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

[0057]

[0058] S5. Dissolve compound 4, N,N-dibutylformamide and triethyl orthoformate in ethanol. Under N 2 protection, add and stir for 30 min, then add saturated sodium bicarbonate, stir in an ice bath, filter, and separate by column chromatography to obtain compound 5, whose structural formula is as shown in Formula V:

[0059]

[0060] S6. Dissolve compound 6, compound 5 and potassium acetate in acetic anhydride. Under nitrogen protection, carry out a condensation reaction for 30 min, pour it into a saturated NaHCO 3 solution, filter, and purify by column chromatography to obtain the hemicyanine photosensitizer CM-1-1.

[0061] In some embodiments of this embodiment, in S1, the specific steps are as follows: Add malonic acid and phenol, slowly dropwise add POCl 3 to the mixture at 0 °C. After the addition is completed, heat the mixture to reflux at 115 °C, use a tail gas absorption device, and stop the reaction until all HCl gas is released. Cool the reaction mixture to room temperature, then pour the upper clear liquid into 150 mL of water, extract with ethyl acetate, dry the organic layer with anhydrous sodium sulfate, remove the solvent under reduced pressure for the crude product, separate by column chromatography, and obtain a colorless transparent oil. After cooling in the refrigerator overnight, a white solid 1 is obtained.

[0062] In some embodiments of this embodiment, in S2, dissolve compound 1 and 3-diethylaminophenol in toluene, reflux the mixture at 118 °C for 5 h, cool to room temperature after the reaction is completed, and filter to obtain a yellow solid 2.

[0063] In some embodiments of this embodiment, in S3, add compound 2 to 30% sulfuric acid, react the mixture at 125 °C for 5 h. After the reaction is completed, cool to room temperature, neutralize the reaction to neutrality with ammonia water, extract the product with dichloromethane, dry the organic layer with anhydrous sodium sulfate, remove the solvent by evaporation under reduced pressure, separate the crude product by column chromatography, obtain a purple-red oil, and refrigerate it in the refrigerator overnight to obtain compound 3;

[0064] In some embodiments of this embodiment, in S4, dissolve compound 3 and 4-dimethylaminoacetophenone in acetic acid and perchloric acid, react the mixture at 120 °C for 12 h. After the reaction is completed, cool to room temperature, pour it into 150 mL of ice water, and filter to obtain compound 6;

[0065] In some embodiments of this embodiment, in S5, under N 2 protection, dissolve compound 4, N,N-dibutylformamide and triethyl orthoformate in ethanol, react at 65 °C for 30 minutes, the solution turns red, then add saturated sodium bicarbonate for neutralization, stir in an ice bath, filter, and separate by column chromatography to obtain compound 5;

[0066] In some embodiments of this embodiment, in S6, compound 6, compound 5 and potassium acetate are dissolved in acetic anhydride, and the reaction is carried out at 65 °C for 30 min under nitrogen protection. After the reaction is completed, the mixture is poured into saturated NaHCO 3 solution in an ice bath, stirred vigorously, filtered, and purified by column chromatography to obtain the hemicyanine 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 D-π-A type hemicyanine dye is provided, including the following steps:

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

[0071] S2: Compound 1, 3-diethylaminophenol and toluene are added respectively, and the mixture is refluxed. After the reaction is completed, it is cooled to room temperature and filtered to obtain compound 2;

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

[0073] S4: Compound 3,4-dimethylaminobenzeneacetone, acetic acid and perchloric acid are added, reacted for 12 h, cooled to room temperature, poured into ice water, and filtered to obtain compound 6, whose structural formula is shown in Formula VI:

[0074]

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

[0076]

[0077] S6. Dissolve compound 7, compound 4 and potassium acetate in acetic anhydride. Under nitrogen protection, react for 30 min, then pour into saturated NaHCO 3 solution, filter, and purify by column chromatography to obtain the hemicyanine photosensitizer CM-1-2.

[0078] In some embodiments of this embodiment, in S1, the specific steps are as follows: Add malonic acid and phenol, and slowly drop POCl 3 to the mixture at 0 °C. After the dropping is completed, heat the mixture to reflux at 115 °C and use a tail gas absorption device until the HCl gas is completely released, then stop the reaction. Cool the reaction mixture to room temperature, then pour the supernatant into 150 mL of water, extract with ethyl acetate, dry the organic layer with anhydrous sodium sulfate, remove the solvent from the crude product under reduced pressure, separate by column chromatography to obtain a colorless transparent oil. After cooling in the refrigerator overnight, a white solid 1 is obtained.

[0079] In some embodiments of this embodiment, in S2, dissolve compound 1 and 3-diethylaminophenol in toluene, reflux the mixture at 118 °C for 5 h, after the reaction is completed, cool to room temperature, filter to obtain yellow solid 2.

[0080] In some embodiments of this embodiment, in S3, add compound 2 to 30% sulfuric acid, react the mixture at 125 °C for 5 h. After the reaction is completed, cool to room temperature, neutralize the reaction to neutral with ammonia water, extract the product with dichloromethane, dry the organic layer with anhydrous sodium sulfate, remove the solvent by evaporation under reduced pressure, separate the crude product by column chromatography to obtain a purplish-red oil, and refrigerate in the refrigerator overnight to obtain compound 3;

[0081] In some embodiments of this embodiment, in S4, dissolve compound 3 and 4-dimethylaminoacetophenone in acetic acid and perchloric acid, react the mixture at 120 °C for 12 h. After the reaction is completed, cool to room temperature, pour into 150 mL of ice water, and filter to obtain compound 6;

[0082] In some embodiments of this embodiment, in S5, dissolve compound 6, malondialdehyde bis(phenylimine) monohydrochloride and potassium acetate in acetic anhydride. Under nitrogen protection, react at 80 °C for 30 min. After the reaction is completed, pour the mixture into saturated NaHCO in an ice bath 3 solution, stir vigorously, filter, and purify by column chromatography to obtain compound 7;

[0083] In some embodiments of this embodiment, in S6, dissolve compound 7, compound 4 and potassium acetate in acetic anhydride. Under nitrogen protection, react at 65 °C for 30 min. After the reaction is completed, pour the mixture into saturated NaHCO in an ice bath 3 solution, stir vigorously, filter, and purify by column chromatography to obtain the hemicyanine photosensitizer CM-1-2.

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

[0085]

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

[0087] S1. Drop POCl 3 into a mixture of malonic acid and phenol, heat under reflux, cool to room temperature, pour the upper clear liquid into water, extract with ethyl acetate, dry the organic layer with anhydrous sodium sulfate, remove the solvent under reduced pressure for the crude product, separate by column chromatography, and place in the refrigerator to obtain Compound 1;

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

[0089] S3: Add Compound 3 and dilute sulfuric acid respectively, heat the mixture to react for 5 h, cool to room temperature after the reaction ends, neutralize the reaction to neutrality with ammonia water, extract the product with dichloromethane, dry the organic layer with anhydrous sodium sulfate, remove the solvent by evaporation under reduced pressure, separate the crude product by column chromatography, and place in the refrigerator to obtain Compound 3;

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

[0091]

[0092] S5. Dissolve Compound 6, pentadienal dianiline hydrochloride and potassium acetate in acetic anhydride, react for 30 min under nitrogen protection, pour into saturated NaHCO 3 solution, and filter to obtain Compound 8, whose structural formula is as shown in Formula VIII:

[0093]

[0094] S6. Dissolve Compound 8, Compound 4 and potassium acetate in acetic anhydride, react at 65 °C for 30 min under nitrogen protection. After the reaction ends, pour the mixture into saturated NaHCO 3 solution in an ice bath, stir vigorously, filter, and purify by column chromatography to obtain the semi-cyanine photosensitizer CM-1-3.

[0095] In some embodiments of this embodiment, in S1, the specific steps are as follows: Add malonic acid and phenol, and slowly drop POCl into the mixture at 0 °C. 3 After the dropping is completed, heat the mixture to reflux at 115 °C, use a tail gas absorption device, and stop the reaction until all HCl gas is released. Cool the reaction mixture to room temperature, then pour the supernatant into 150 mL of water, extract with ethyl acetate, dry the organic layer with anhydrous sodium sulfate, remove the solvent under reduced pressure for the crude product, and separate by column chromatography to obtain a colorless transparent oil. After cooling in the refrigerator overnight, a white solid 1 is obtained.

[0096] In some embodiments of this embodiment, in S2, dissolve compound 1 and 3 - diethylaminophenol in toluene, reflux the mixture at 118 °C for 5 h, cool to room temperature after the reaction ends, and filter to obtain yellow solid 2.

[0097] In some embodiments of this embodiment, in S3, add compound 2 to 30% sulfuric acid, react the mixture at 125 °C for 5 h, after the reaction ends, cool to room temperature, neutralize the reaction to neutrality with ammonia water, extract the product with dichloromethane, dry the organic layer with anhydrous sodium sulfate, remove the solvent by evaporation under reduced pressure, separate the crude product by column chromatography to obtain a purplish - red oil, and place it in the refrigerator overnight to obtain compound 3;

[0098] In some embodiments of this embodiment, in S4, dissolve compound 3 and 4 - dimethylaminoacetophenone in acetic acid and perchloric acid, react the mixture at 120 °C for 12 h, after the reaction ends, cool to room temperature, pour it into 150 mL of ice water, and filter to obtain compound 6;

[0099] In some embodiments of this embodiment, in S5, dissolve compound 6, pentadienal - bis - aniline hydrochloride and potassium acetate in acetic anhydride, react at 80 °C for 30 min under nitrogen protection, after the reaction ends, pour the mixture into a saturated NaHCO 3 solution in an ice bath, stir vigorously, filter, and purify by column chromatography to obtain compound 8;

[0100] In some embodiments of this embodiment, in S6, dissolve compound 8, compound 4 and potassium acetate in acetic anhydride, react at 65 °C for 30 min under nitrogen protection, after the reaction ends, pour the mixture into a saturated NaHCO 3 solution in an ice bath, stir vigorously, filter, and purify by column chromatography to obtain the hemicyanine photosensitizer CM - 1 - 3.

[0101] The technical solutions of the present invention will be 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] Synthesis of Hemicyanine Photosensitizer in Example 1

[0103] (1) Preparation of Compound 1: Take a 500 mL single-necked flask and add malonic acid (11.0 g, 106.0 mmol) and phenol (19.9 g, 212 mmol). Slowly add POCl 3 (11.5 mL, 123 mmol) dropwise to the mixture at 0 °C. After the addition is complete, heat the mixture to reflux at 115 °C. Use a tail gas absorption device and stop the reaction until all HCl gas is released. Cool the reaction mixture to room temperature, then pour the upper clear liquid into 150 mL of water, extract with ethyl acetate, dry the organic layer with anhydrous sodium sulfate, remove the solvent under reduced pressure from the crude product, and separate by column chromatography. The eluent is: petroleum ether / dichloromethane (v / v = 3 / 1) to obtain a colorless transparent oil. After cooling in the refrigerator overnight, a white solid is obtained.

[0104] (2) Take a 250 mL single-necked flask and add diphenyl malonate (5 g, 19.5 mmol), 3-diethylaminophenol (3.2 g, 19.5 mmol) and 15 mL of toluene. Reflux the mixture at 118 °C for 5 h. After the reaction is completed, cool to room temperature and filter to obtain 2.9 g of a yellow solid with a yield of 64%. The product does not need to be purified and is directly used for the next reaction. 1 H NMR (500 MHz, MeOD) δ 7.68–7.65 (m, 1H), 6.69 (dd, J = 9.0, 2.5 Hz, 1H), 6.48 (d, J = 2.4 Hz, 1H), 3.47 (q, J = 7.1 Hz, 4H), 1.22 (t, J = 7.1 Hz, 6H).

[0105] (3) Add Compound 3 (2 g, 8.57 mmol) to a 125 mL single-necked flask and add 60 mL of 30% sulfuric acid. React the mixture at 125 °C for 5 h. After the reaction is completed, cool to room temperature, neutralize the reaction to neutral with ammonia water, extract the product with dichloromethane (50 × 3 mL), dry the organic layer with anhydrous sodium sulfate, remove the solvent by evaporation under reduced pressure, and separate the crude product by column chromatography to obtain a purplish-red oil. After refrigerating in the refrigerator overnight, a red solid 4 is obtained. 1 H NMR (500 MHz, CDCl 3 ) δ 12.94 (s, 1H), 7.53 (d, J = 9.1 Hz, 1H), 6.20 (dd, J = 9.1, 2.5 Hz, 1H), 6.08 (d, J = 2.5 Hz, 1H), 3.41 (q, J = 7.1 Hz, 4H), 2.49 (s, 3H), 1.22 (t, J = 7.1 Hz, 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 chromatography to obtain a red solid. 1 H NMR (400 MHz, CDCl 3 ) δ 9.07 (t, J = 12.9 Hz, 1H), 8.41 (d, J = 7.4 Hz, 1H), 8.21 (d, J = 8.1 Hz, 1H), 7.92 (t, J = 7.7 Hz, 1H), 7.84 (d, J = 12.2 Hz, 1H), 7.77 (d, J = 8.3 Hz, 1H), 7.64 (d, J = 1.2 Hz, 1H), 7.62 (d, J = 1.9 Hz, 1H), 7.43 (t, J = 7.9 Hz, 2H), 7.27 (d, J = 2.6 Hz, 1H), 4.24 (dd, J = 14.2, 7.1 Hz, 2H), 1.56 (t, J = 7.3 Hz, 3H).

[0107] (5) A 50 mL single-necked flask was charged with compound 3 (250 mg, 1.2 mmol), 4-dimethylaminobenzophenone (195 mg, 1.2 mmol), 5 mL of acetic acid and 6 mL of perchloric acid. The mixture was reacted at 120 °C for 12 h. After the reaction was completed, it was cooled to room temperature and poured into 150 mL of ice water. Filtration gave 84 mg of a black solid with a metallic luster, and the yield was 21%. The product was used directly in the next step without purification.

[0108] (6) Compound 6 (50 mg, 0.15 mmol), malondialdehyde bis(phenylimine) monohydrochloride (39 mg, 0.15 mmol) and potassium acetate (44 mg, 0.45 mmol) were dissolved in 2 mL of acetic anhydride. Under nitrogen protection, the reaction was carried out at 80 °C for 30 min. After the reaction was completed, the mixture was poured into a saturated NaHCO 3 solution in an ice bath, stirred vigorously, and filtered to obtain a black solid. The product was used directly in the next step without purification. 11H NMR (500 MHz, DMSO) δ 8.40 (dd, J = 28.6, 14.3 Hz, 2H), 8.24 (d, J = 8.8 Hz, 2H), 8.15 (d, J = 8.9 Hz, 1H), 8.01 (s, 1H), 7.71–7.56 (m, 4H), 7.48 (d, J = 7.3 Hz, 2H), 7.26 (d, J = 15.0 Hz, 1H), 7.10 (d, J = 9.1 Hz, 2H), 6.91 (d, J = 9.0 Hz, 2H), 5.57–5.50 (m, 1H), 3.61 (d, J = 6.9 Hz, 4H), 3.16 (s, 6H), 1.97 (s, 3H), 1.20 (t, J = 6.9 Hz, 6H).

[0109] (7) Compound 6 (200 mg, 0.60 mmol), pentadienal diphenylamine hydrochloride (172 mg, 0.60 mmol) and potassium acetate (176 mg, 1.8 mmol) were dissolved in 4 mL of acetic anhydride. Under nitrogen protection, the reaction was carried out at 80 °C for 30 min. After the reaction, the mixture was poured into a saturated NaHCO 3 solution in an ice bath, 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 (500 MHz, DMSO) δ 8.23 (d, J = 9.1 Hz, 2H), 8.10 (d, J = 14.6 Hz, 1H), 8.06 (s, 1H), 8.04 (s, 1H), 8.00 (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 = 11.8 Hz, 1H), 7.10 (s, 1H), 7.06 (d, J = 11.4 Hz, 1H), 6.88 (d, J = 9.2 Hz, 2H), 6.50 (t, J = 10.6 Hz, 1H), 5.22–5.16 (m, 1H), 3.61 (d, J = 7.1 Hz, 4H), 3.15 (s, 6H), 1.93 (s, 3H), 1.21 (t, J = 7.0 Hz, 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 flask, 5 mL of acetic anhydride was added, and the reaction was carried out at 65 °C for 30 min under nitrogen protection. After the reaction, the mixture was poured into a saturated NaHCO 3In a solution, stir vigorously, filter to obtain CM-1-1. The crude product is separated and purified by column chromatography with a yield of 27%. Perform mass spectrometry and NMR characterization on it ( Figure 1-2 ). 1H NMR (500 MHz, DMSO) δ 8.93 (d, J = 8.9 Hz, 1H), 8.84 (s, 1H), 8.29 (d, J = 8.8 Hz, 2H), 8.18 (d, J = 9.6 Hz, 1H), 8.13 (s, 1H), 8.08 (s, 1H), 7.89 (t, J = 7.7 Hz, 1H), 7.64–7.57 (m, 2H), 7.49 (d, J = 13.8 Hz, 1H), 7.34 (s, 1H), 7.15 (s, 1H), 7.04 (s, 1H), 6.93 (d, J = 9.2 Hz, 2H), 6.85 (d, J = 8.4 Hz, 1H), 4.24 (s, 2H), 3.61 (d, J = 7.0 Hz, 4H), 3.14 (s, 6H), 1.39 (s, 3H), 1.24 (s, 6H).

[0111] (9) Add compound 7 (200 mg, 0.39 mmol), compound 4 (126 mg, 0.39 mmol) and potassium acetate (114 mg, 1.17 mmol) to a 50 mL two-necked flask, add 5 mL of acetic anhydride, and react at 65 °C for 30 min under nitrogen protection. After the reaction is completed, pour the mixture into saturated NaHCO in an ice bath 3 solution, stir vigorously, filter to obtain CM-1-2. The crude product is separated and purified by column chromatography with a yield of 20%. Perform mass spectrometry and NMR characterization on it ( Figure 3-4 ). 1 1H NMR (400 MHz, CDCl 3 ) δ 8.28 (d, J = 6.7 Hz, 1H), 7.94–7.83 (m, 4H), 7.65 (t, J = 7.6 Hz, 1H), 7.47 (t, J = 7.4 Hz, 2H), 7.32 (s, 1H), 7.18 (t, J = 7.5 Hz, 1H), 7.10 (d, J = 7.8 Hz, 1H), 6.54 (dd, J = 28.0, 14.1 Hz, 5H), 6.38 (d, J = 6.8 Hz, 1H), 6.19 (s, 1H), 5.85 (d, J = 12.6 Hz, 1H), 3.52 (d, J = 6.9 Hz, 2H), 3.36 (d, J = 7.0 Hz, 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 flask. 5 mL of acetic anhydride was added. Under nitrogen protection, the reaction was carried out at 65 °C for 30 min. After the reaction was completed, the mixture was poured into a saturated NaHCO 3 solution in an ice bath, 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 (400 MHz, DMSO) δ 8.17 (dd, J = 25.4, 8.4 Hz, 5H), 7.93 (t, J = 18.4 Hz, 2H), 7.84 (s, 1H), 7.68 (s, 1H), 7.40 (dd, J = 27.8, 11.9 Hz, 4H), 7.12 (d, J = 8.7 Hz, 1H), 7.02 (s, 1H), 6.89 (d, J = 8.0 Hz, 3H), 6.75 (dd, J = 27.3, 13.1 Hz, 2H), 6.39 (d, J = 10.2 Hz, 1H), 4.02 (s, 2H), 3.61 (d, J = 5.9 Hz, 4H), 3.16 (s, 6H), 1.26 (s, 3H), 1.24 (s, 6H).

[0113] The synthetic route is as follows:

[0114]

[0115] Example 2 investigated the ultraviolet 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 and dissolve them in 1 mL of dichloromethane respectively. 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 spectra of the test solutions were measured by a ULC 1503007 ultraviolet-visible spectrophotometer, and the emission spectra of the solutions were measured by an F97XP fluorescence spectrophotometer. During the measurement, the slit of the instrument was adjusted to an appropriate width.

[0118] The photophysical properties of the photosensitizer were investigated using ultraviolet-visible absorption spectra and fluorescence emission spectra, such as Figure 7As shown, all three photosensitizers showed broad absorption bands in dichloromethane, with the maximum absorption peaks located at 751 nm, 846 nm, and 863 nm 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 appeared at 904, 903, and 1020 nm, and the emission peaks all appeared in the NIR-II region. It is worth noting that CM-1-3 has the largest Stokes shift peak, reaching 157 nm.

[0119] Example 3 examines the photothermal performance test of different concentrations of hemicyanine dyes

[0120] Weigh 1.335 mg of compound CM-1-1 and dissolve it 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 respectively into 5 mL of PBS solution and dilute them to 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. Subsequently, use an 808 nm laser (1.5 W / cm 2 ) to irradiate the aqueous solution in the centrifuge tube for 10 min and record its temperature change. Study the photothermal performance of the three photosensitizers at different concentrations and different powers.

[0122] As Figure 8 shown, under the irradiation of a fixed laser power (808 nm, 1.5 W / cm 2 ), the photothermal temperature change of pure PBS is not significant. However, as the concentration of the photosensitizer increases, the temperatures of the photosensitizer CM-1-1, CM-1-2, and CM-1-3 solutions also increase. When the concentration of the photosensitizer increases from 25 μM to 100 μM, within 600 s, the temperature of CM-1-1 increases from 57 to 61 °C, the temperature of CM-1-2 increases from 45 to 58 °C, and the temperature of CM-1-3 increases from 52 to 62.5 °C. The overall trend is that the temperature of CM-1-3 is the highest at 62.5 °C, followed by the temperature of CM-1-1 (61 °C) and CM-1-2 (58 °C). The results show that the photosensitizer has excellent photothermal performance and CM-1-3 has the best photothermal performance.

[0123] Example 4 examines the photothermal performance test of laser irradiation with different powers of hemicyanine dyes

[0124] As Figure 9 shown, as the power density increases from 0.8 W / cm 2 to 1.5 W / 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. It shows that the temperature is positively correlated with the laser power intensity of the irradiated photosensitizer.

[0125] Example 5 examines the photothermal stability of hemicyanine dyes

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

[0127] Example 6 examines the photothermal imaging of hemicyanine dyes

[0128] As Figure 11 shown, after continuously irradiating the photosensitizer solution (50 μM) at a low power density of 808 nm (1.5 W / cm 2 ) for 10 min, the temperatures of the three photosensitizers all increased significantly, and the temperature of CM-1-3 was significantly higher than that of the other two photosensitizers.

[0129] The above shows 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 examines the in vivo fluorescence imaging of hemicyanine dyes

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

[0132] Example 8 examines the in vivo photothermal therapy of tumor-bearing mice with hemicyanine dyes

[0133] Subcutaneously inject the photosensitizer CM-1-3 (100 μM, 200 μL, with PBS as the solvent) into the subcutaneous tissue at the root of the thigh of BABL / C nude mice or mice. Irradiate with a 1W / cm 2 laser at 808 nm for 10 min and conduct dynamic monitoring. The results are as Figure 13 shown. The tumor temperature rapidly increased from 25°C to 45°C within only 2 minutes and reached a plateau of approximately 50°C within ten minutes, indicating that the photothermal temperature of the photosensitizer is very stable and ideal.

[0134] Guided by the above results, we further tested the PTT effect of the photosensitizer CM-1-3 on tumors in vivo. The in vivo PTT efficacy of the photosensitizer CM-1-3 was evaluated by using Hela cell tumor-bearing mice as model animals. As Figure 14 (A) shows, first, a Hela cell tumor model was established, and then 200 μL of the photosensitizer (200 μM) was injected into the tumor for diagnosis and treatment. The 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. As Figure 14 (B) shows, during the laser irradiation, the tumor temperature was monitored in real time by an infrared thermal imager. As can be seen from Figure 14 (C), the tumor temperature of the CM-1-3+laser group rapidly increased from 30°C to 53°C within 2 minutes and then reached a plateau at around 56°C. In contrast, the tumor temperature of the PBS+laser group did not show a significant increase, indicating that the photothermal effect can be ignored in the absence of the photosensitizer CM-1-3 after laser irradiation.

[0135] During the 18-day monitoring period after laser irradiation, the tumor volume of each group of mice was recorded every two days. As Figure 14 (D) shows, the tumors in the control groups, including the PBS group and the PBS+laser group, showed similar growth rates, while the tumor volume of the CM-1-3 group also showed a downward trend, probably due to high cytotoxicity, indicating that neither CM-1-3 nor laser irradiation alone has antitumor effects. In contrast, the combined treatment of CM-1-3 and laser irradiation showed good tumor suppression effects and ultimately eradicated the tumors. In addition, as can be seen from Figure 14 (E), the PBS+laser group, the CM-1-3 group, and the 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 is negligible and the laser intensity is well tolerated. The mice were sacrificed 18 days after photothermal therapy, and the tumor masses were removed. As can be seen from Figure 14 (F), the treatment effect of the CM-1-3+laser group was obvious, and the tumor volume was significantly smaller than that of the other three groups.

[0136] Example 9: Investigation of the biological toxicity of semi-cyanine dyes

[0137] To further evaluate the tumor-killing effect of CM-1-3 under laser irradiation, the heart, liver, spleen, lungs, and kidneys were collected after treatment and subjected to H&E staining ( Figure 15 ). No obvious tissue damage was observed, indicating that CM-1-3 + laser is non-toxic in various organs. The above results indicate that the photosensitizer CM-1-3 has excellent PTT effects on mouse tumors.

[0138] Finally, it should be noted that the above description is only the preferred embodiment of the present invention. Those skilled in the art can make various similar representations under the inspiration of the present invention without departing from the purpose and claims of the present invention. Such transformations 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 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; The structures of the compounds are as follows:

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

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

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

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

7. Use of the D-π-A type hemicyanine dye according to claim 1 in the preparation of drugs for photothermal therapy of tumors, wherein: The tumor cells are Hela cells.

8. Use of the D-π-A type hemicyanine dye prepared by the preparation method according to any one of claims 2 to 6 in the preparation of drugs for photothermal therapy of tumors, wherein: The tumor cells are Hela cells.

9. Use of the D-π-A type hemicyanine dye according to claim 1 in the preparation of a medical imaging preparation, wherein the medical imaging comprises bioluminescence imaging and / or photoacoustic imaging.

10. Use of the D-π-A type hemicyanine dye prepared by the preparation method according to any one of claims 2 to 6 in the preparation of medical imaging preparations, wherein the medical imaging includes bioluminescence imaging and / or photoacoustic imaging.

11. A pharmaceutical composition, characterized in that Contains the D-π-A type hemicyanine dye according to claim 1 or a pharmaceutically acceptable salt thereof.

12. A pharmaceutical composition, characterized in that Contains a D-π-A type hemicyanine dye or a pharmaceutically acceptable salt thereof prepared by the preparation method according to any one of claims 2 to 6.

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