A metasubstituted heptamethine cyanine dye and its synthesis method and application

Abstract

The application discloses a kind of offsite aromatic ketone substituted heptamethine cyanine dyes and its synthesis method and application.The offsite aromatic ketone substituted heptamethine cyanine dye is substituted by heptamethine cyanine dye methine chain, changes the electronic cloud arrangement of cyanine dye, so that the absorption and emission wavelength of dye all have blue shift, has good intersystem crossing ability, can quickly pass through cell membrane, can be concentrated in cell mitochondria, can detect physiological process using the change of cell mitochondria, and can produce a large amount of active oxygen after illumination, cause cell death, realize the purpose of photodynamic therapy.The application can be applied to the field of photocatalysis, as near-infrared region luminescent material, biological marker probe, photodynamic therapy, water photolysis hydrogen production and the field of biological sensing.

Description

Technical Field

[0001] This invention relates to the field of fine chemical technology, specifically to the field of fluorescent materials technology, and in particular to a heptamethine cyanine dye substituted with a dissimilar aromatic ketone, its synthesis method, and its application. Background Technology

[0002] Photodynamic therapy, as a novel non-invasive treatment method with short treatment cycles and no drug resistance, has been applied in the clinical treatment of certain diseases. Utilizing the fluorescence properties and intersystem crossing capabilities of organic dyes, fluorescence imaging and precise photodynamic therapy of diseased tissues can be achieved, thus realizing the integration of diagnosis and treatment. Therefore, researchers have developed many organic dyes as triplet photosensitizers for disease diagnosis and treatment. Currently, the most common are BODIPY dyes and ruthenium complexes, but these dyes generally have short absorption and emission wavelengths and lack targeting effects on organelles, making it difficult to achieve accurate diagnosis and treatment of deep-seated diseases in the body. Therefore, designing and developing dye molecules with near-infrared absorption and emission properties and high-efficiency intersystem crossing capabilities has become a pressing challenge.

[0003] Heptamethrin dyes, as typical cyanine dyes, have been extensively studied. These dyes exhibit absorption and emission in the near-infrared band, possessing very high molar extinction coefficients and fluorescence quantum yields, and have been widely used in fluorescence imaging and staining. However, because existing heptamethrin cyanine dyes typically release a large amount of energy through non-radiative inactivation via thermal and fluorescence emission upon photoexcitation, they cannot efficiently generate reactive oxygen species (ROS) through intersystem crossing, thus hindering their use in photodynamic therapy. Current photosensitization modifications of heptamethrin cyanine dyes mainly rely on the "heavy atom effect." However, like many other fluorophores, the heavy atom effect often leads to decreased dye solubility and increased cytotoxicity, hindering their further biological applications.

[0004] Therefore, it is urgent to rationally design the heptamethrin cyanine dye matrix while ensuring the near-infrared absorption and emission characteristics of heptamethrin dye and overcoming the low intersystem crossing efficiency and poor photodynamic therapy effect. Summary of the Invention

[0005] This invention addresses the aforementioned problems by researching and designing a heptamethine cyanine dye with a partial aromatic ketone substitution, along with its synthesis method and applications. The technical means employed in this invention are as follows:

[0006] A heptamethine cyanine dye substituted with a paraaromatic ketone has the following general formula:

[0007]

[0008] R1 and R2 are each independently selected from any one of hydrogen, alkyl having 1-18 carbons, carboxylalkyl having 1-18 carbons, aryl, arylcarboxylic acid, alkylsulfonate having 1-18 carbons, arylsulfonate, alkyl sulfonate or aryl sulfonate.

[0009] R3 and R4 are substituents at indeterminate positions on the benzene ring, each independently selected from any one of hydrogen, halogen, methoxy, amino, nitro, hydroxyl, carboxyl, carboxylalkyl having 1-18 carbons, alkylsulfonic acid having 1-18 carbons, ester having 1-18 carbons, amide having 1-18 carbons, N,N-dimethylamino, N,N-diethylamino, trifluoromethyl, sulfonic acid, or sulfonate.

[0010] R5, R6 and R7 are each independently selected from any one of hydrogen, alkyl groups having 1 to 8 carbons, halogens, methoxy groups, trifluoromethyl groups, ester groups or amide groups;

[0011] X is selected from any one of oxygen, sulfur, selenium, or gem-dimethyl;

[0012] Y is selected from inorganic negative ions or organic negative ions.

[0013] Furthermore, R1 and R2 are each independently selected from any one of hydrogen, alkyl groups having 1-18 carbons, alkyl sulfonates having 1-18 carbons, aryl groups, and aryl carboxylic acid groups;

[0014] R3 and R4 are each independently selected from any one of hydrogen, halogen, carboxyl, alkyl sulfonic acid group having 1-18 carbons, ester group having 1-18 carbons or amide group having 1-8 carbons;

[0015] R5, R6 and R7 are each independently selected from one of hydrogen, an alkyl group having 1 to 8 carbons, a halogen, an ester group having 1 to 8 carbons or an amide group having 1 to 8 carbons;

[0016] Y is selected from halide ions, ClO4 - BF4 - CH3COO - CF3COO - or OTs - Any one of them.

[0017] Furthermore, R5, R6, and R7 are each independently selected from hydrogen, methyl, or isopropyl; Y is selected from halide ions, ClO4, etc. - CH3COO - Any one of them.

[0018] Furthermore, R1 and R2 are methyl groups, R3 and R4 are hydrogen groups, X is gem-dimethyl, and Y is an iodide ion.

[0019] A method for synthesizing a heptamethine cyanine dye substituted with a para-aromatic ketone, used to synthesize the heptamethine cyanine dye substituted with a para-aromatic ketone as described in this invention, includes the following steps:

[0020] S1: Synthesis of quaternary ammonium salts J-1 and J-2:

[0021]

[0022] Indole Y-1 modified with R3 substituent was mixed with halo-N alkylating agent Y-2 with R1 substituent at a molar ratio of 1:(1-10), and a first organic solvent was added. The mixture was reacted at 40-120℃ to obtain quaternary ammonium salt J-1.

[0023] Indole Y-3 modified with R4 substituent was mixed with halo-N-alkylating agent Y-4 with R2 substituent at a molar ratio of 1:(1-10), a second organic solvent was added, and the mixture was reacted at 40-120℃ to obtain quaternary ammonium salt J-2.

[0024] S2: Synthesis of Zincke salt:

[0025]

[0026] Compound P-2 and 2,4-dinitrohalobenzene were mixed evenly, a third organic solvent was added, and the mixture was reacted at 40-120℃ to obtain compound P-3, wherein Z is a halogen.

[0027] S3: Synthesis of the target product:

[0028]

[0029] Compound P-3 obtained in step S2 and 4-bromoaniline were mixed evenly at a molar ratio of 1:1.2. Methanol was added, and the mixture was stirred at room temperature for more than 0.5 h. Then, quaternary ammonium salts J-1 and J-2 obtained in step S1 and sodium acetate were added, and the mixture was stirred at room temperature for 5-60 min. The solvent was removed to obtain the target product.

[0030] Furthermore, the method for synthesizing the ketone-substituted pyridine in step S2 is as follows:

[0031]

[0032] Under N2 protection, R5, R6, and R7 substituted bromobenzenes were dissolved in anhydrous tetrahydrofuran and cooled to -78°C. Then, [the following was added]... nBuLi, and heated to room temperature, continued stirring, added a tetrahydrofuran solution of 3-pyridinecarboxaldehyde at -78°C to the above reaction mixture, continued stirring to react, after the reaction was completed, allowed to stand and heated to room temperature, quenched the reaction with saturated NH4Cl solution, removed the solvent, and obtained reaction intermediate P-1;

[0033] Intermediate P-1 was mixed with pyridine chlorochromate at a molar ratio of 1:(4-6), dichloromethane was added, and the mixture was reacted at room temperature for 3-6 hours. The solvent was then removed to obtain compound P-2.

[0034] Further, in the synthesis of ketone-substituted pyridine in step S2, the molar ratio of 3-pyridinecarboxaldehyde and R-substituted bromobenzene is 1:(1-1.5); in step S2, the molar ratio of compound P-2 and 2,4-dinitrohalobenzene is 1:(1-5); in step S3, the molar ratio of compound P-3 to the total amount of quaternary ammonium salt J-1 and quaternary ammonium salt J-2 is 1:(2-3), and the molar ratio of compound P-3 to sodium acetate is 1:6.

[0035] The heptamethine cyanide dye substituted with a terroir ketone described in this invention can be applied in the fields of photocatalysis, as a near-infrared luminescent material, a biolabeled probe, photodynamic therapy, photocatalytic water splitting for hydrogen production, and biosensing. Specifically, it can be used as a near-infrared luminescent material, or in the preparation of photocatalytic agents, biolabeled probes, photodynamic therapy agents, photocatalytic water splitting agents, or biosensor devices.

[0036] The aforementioned ketone-substituted heptamethine cyanine dyes can serve as triplet photosensitizers, initiating chemical reactions in photocatalysis, generating reactive oxygen species in photodynamic therapy, and producing phosphorescent signals in phosphorescent materials. The light signals of these ketone-substituted heptamethine cyanine dye molecules are particularly prominent in near-infrared imaging systems, with the maximum emission wavelengths of this series of dyes all exceeding 710 nm, making them suitable as near-infrared luminescent materials. These ketone-substituted heptamethine cyanine dye molecules exhibit excellent aqueous solubility, enabling them to rapidly and efficiently cross cell membranes, enter cells, and accumulate in subcellular organelles. Through low-density near-infrared light irradiation, they efficiently generate reactive oxygen species, disrupting intracellular homeostasis and killing cancer cells with high efficiency at nanomolar levels, thus finding applications in photodynamic therapy.

[0037] Compared with the prior art, the present invention has the following beneficial effects:

[0038] First, the heptamethine cyanide dye substituted with a teryl ketone described in this invention alters the electron cloud arrangement of the cyanide dye by substituting the methyl ketone chain, resulting in a blue shift in both the absorption and emission wavelengths of the dye. Compared to ordinary heptamethine cyanide dyes and meso-substituted heptamethine cyanide dyes, the heptamethine cyanide dye substituted with a teryl ketone described in this invention exhibits a certain blue shift in both the maximum absorption and emission wavelengths.

[0039] Second, the heptamethine cyanine dyes with partial substitution of aromatic ketones described in this invention have excellent intersystem crossing ability: the singlet oxygen quantum yield of compounds 1-4 described in this invention is 3.8%-8.1% in vitro, while the yield of the comparative compounds with partial substitution of aromatic ketones in vitro is less than 1%.

[0040] Third, the heptamethrin dye substituted with teratophore described in this invention can rapidly cross the cell membrane and accumulate in the mitochondria of cells. Changes in the mitochondria can be used to detect physiological processes. Furthermore, it can generate a large amount of reactive oxygen species after light exposure, causing cell death and achieving the purpose of photodynamic therapy. Attached Figure Description

[0041] Figure 1 The normalized UV-Vis absorption spectra of compounds 1-4 and Comparative Example 1 in dichloromethane as disclosed in the embodiments of this invention;

[0042] Figure 2 The normalized fluorescence emission spectra of compounds 1-4 and Comparative Example 1 in dichloromethane are shown in the embodiments of this invention.

[0043] Figure 3 These are the spectra of DPBF degradation in dichloromethane for compounds 1-4 and Comparative Example 1 disclosed in the embodiments of the present invention;

[0044] Figure 4 These are single-photon microscope images of organelle colocalization of compound 4 disclosed in the embodiments of this invention.

[0045] Figure 5 This is an MTT assay diagram of compound 4 disclosed in an embodiment of the present invention. Detailed Implementation

[0046] Unless otherwise stated, the terms used herein have the following meanings.

[0047] The term "halogen" as used in this article includes fluorine, chlorine, bromine, and iodine.

[0048] The term "alkyl" as used in this application includes straight-chain alkyl and branched-chain alkyl.

[0049] Y is used in this article -This refers to negative ions, which can be any suitable negative ion, including inorganic and organic negative ions. Examples include, but are not limited to, halide ions and ClO4. - PF6 - BF4 - CH2COO - CF3C0O - or OTs - .

[0050] The instruments and equipment used in the embodiments are as follows:

[0051] In the column chromatography process of this invention, 200-300 mesh and 500-600 mesh silica gel for column chromatography purchased from Qingdao Meigao Group Co., Ltd., and 20-40 mesh analytical grade quartz sand purchased from Tianda Chemical Reagent Factory are used.

[0052] The absorption and emission spectra of the dyes were measured using an Agilent Cary 60 UV-Vis spectrophotometer and a Cary Eclipse fluorescence spectrophotometer.

[0053] The dye organelle localization experiment was performed using an FV1000 single-photon confocal microscope from Olympus Corporation, Japan.

[0054] Cytotoxicity assays were performed using the Varioskan LUX Multimode Microplate Reader instrument from Thermofisher, Inc.

[0055] Example 1

[0056] The following is a method for synthesizing a class of heptamethrin cyanides substituted with terroir:

[0057] Synthesize using R1 as methyl, R3 as hydrogen, and Y as I - Quaternary ammonium salt J-1:

[0058]

[0059] Iodoethane (3.10 g, 20.0 mmol) was added to 2,3,3-trimethyl-3H-indole (1.60 g, 10.0 mmol) dissolved in 50 mL of acetonitrile at room temperature. The mixture was stirred and heated to reflux under nitrogen protection for 12 h, after which the reaction was stopped. The mixture was cooled to room temperature. Most of the acetonitrile was removed, and the mixture was recrystallized from ethyl acetate to give compound 1.1 (2.76 g, 87%) as a pink powder.

[0060] In this embodiment, R1 and R2 are both methyl groups, R3 and R4 are both hydrogen groups, and quaternary ammonium salt J-1 is the same as quaternary ammonium salt J-2.

[0061] Synthesis of 3-benzoyl-substituted Zincke salts:

[0062]

[0063] 3-Benzoylpyridine was purchased directly from Anaiji Chemical. 3-Benzoylpyridine (1.00 g, 5.5 mmol) and 2,4-dinitrobromobenzene (1.48 g, 6.0 mmol) were added to a 50 mL sealed quartz tube, and 4 mL of acetonitrile was added to dissolve them. The reaction mixture was then heated to 82 °C with stirring and reacted for 20 h. After the reaction was complete, the mixture was cooled to room temperature, and the reaction solution was slowly added dropwise to diethyl ether and stirred. Intermediate 1.2 was a yellow powder (1.63 g, 69.4%).

[0064] Synthesis of the target product:

[0065]

[0066] The Zincke salt 1.2 (200 mg, 0.46 mmol) and 4-bromoaniline (96 mg, 0.56 mmol) were dissolved in 2 mL of methanol, and the reaction mixture was stirred at room temperature for 30 min. Then, indole salt 1.1 (440 mg, 1.39 mmol) and sodium acetate (230 mg, 2.79 mmol) were added, and the reaction mixture was stirred again at room temperature for 16 h. The solvent was then removed by evaporation under reduced pressure, and the product was separated by silica gel column chromatography with a dichloromethane:methanol ratio of 30:1, v / v to obtain green solid compound 1 (75 mg, 24.1%).

[0067] 1 H NMR (400MHz, DMSO-d6) δ8.16(t,J=13.0Hz,1H),8.02(d,J=14.1Hz,1H),7.81(dd,J=24.5,10.8Hz,2H),7.73–7.47(m,7H),7.44–7.23(m,3H ),7.18(t,J=7.4Hz,1H),6.77(d,J=14.2Hz,1H),5.94(s,1H),4.32–4.23(m,2H),3.93(s,2H),1.70(s,6H),1.51(s,6H),1.35–1.18(m,6H).

[0068] ESI-MS(C 13 H 18 N + )m / z:[M–I] + Calculated value: 188.14, Measured value: 188.21.

[0069] Example 2

[0070] The following is a method for synthesizing a class of heptamethrin cyanides substituted with terroir:

[0071] Synthesis of quaternary ammonium salt J-1 and quaternary ammonium salt J-2: Same as in Example 1;

[0072] Synthesis of pyridines substituted with methyl aromatic ketones at R5, R6, and R7:

[0073]

[0074] 1,3,5-Trimethylbromobenzene (10.0 g, 50.0 mmol) was added to a 250 mL three-necked flask, the air was exchanged three times with dry N2, 100 mL of anhydrous tetrahydrofuran was added, and the mixture was cooled to -78 °C. Then, it was slowly added through a syringe. n BuLi (50 mL, 126 mmol) was slowly heated to room temperature under N2 protection and stirred for 1 hour. Next, 3-pyridinecarboxaldehyde (5.9 g, 55.3 mmol) was dissolved in 10 mL of tetrahydrofuran and slowly added to the above reaction mixture at -78 °C, and stirred for another 1 hour. After the reaction was completed, the mixture was allowed to stand and then heated to room temperature, followed by quenching with 30 mL of saturated NH4Cl solution. The tetrahydrofuran was then removed by vacuum rotary evaporation. The mixture was extracted with DCM (2 × 30 mL), and the combined organic phases were dried over anhydrous sodium sulfate, filtered, and the solvent was evaporated. The resulting product was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 50 / 1, v / v). The product was a pale yellow solid (7.1 g, 62%).

[0075] The above intermediate alcohol (2.0 g, 8.8 mmol) and pyridinium chlorochromate (7.6 g, 35.2 mmol) were added to a 100 mL single-necked flask, and 50 mL of dichloromethane was added to dissolve them. The reaction solution was stirred at room temperature for 4 h. The reaction solution was filtered, and the solvent was removed by rotary evaporation under reduced pressure. The product was then purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate = 20 / 1, v / v). Product 2.1 was a white solid (0.6 g, 30%).

[0076] Synthetic Zincke salt:

[0077]

[0078] Intermediate pyridine (1.0 g, 4.4 mmol) and 2,4-dinitrobromobenzene (1.3 g, 5.3 mmol) were added to a 50 mL sealed quartz tube, and 4 mL of acetonitrile was added to dissolve them. The reaction mixture was then heated to 82 °C with stirring and reacted for 20 hours. After the reaction was completed, the mixture was cooled to room temperature, and the reaction solution was slowly added dropwise to diethyl ether to form a slurry. The slurry was filtered and dried. Intermediate 2.2 was a yellow powder solid (1.6 g, 76%).

[0079] Synthesis of the target product:

[0080]

[0081] Intermediate 2.2 (200 mg, 0.89 mmol) was dissolved in 2 mL of methanol with 4-bromoaniline (87 mg, 0.51 mmol), and the reaction mixture was stirred at room temperature for 30 min. Then, indole quaternary ammonium salt 1.1 (400 mg, 1.27 mmol) and sodium acetate (208 mg, 2.54 mmol) were added, and the reaction mixture was stirred again at room temperature for 0.5 h. The solvent was then removed by vacuum evaporation, and the product was separated by silica gel column chromatography with a dichloromethane:methanol ratio of 40:1, v / v to obtain green solid compound 2 (92 mg, 30.6%).

[0082] 1 H NMR (400MHz, DMSO-d6) δ8.32(dd,J=14.7,11.2Hz,1H),8.14–8.03(m,1H),7.98(d,J=14.3Hz,1H),7.77(dd,J =20.4,7.7Hz,2H),7.66(d,J=14.2Hz,1H),7.59–7.46(m,3H),7.40–7.33(m,1H),7.30(d,J=7.8Hz,1H),7.18 –7.13(m,1H),7.09(d,J=14.6Hz,1H),6.99(s,2H),6.41(d,J=14.2Hz,1H),4.45(q,J=7.1Hz,2H),4.14(d,J= 7.3Hz,2H),2.33(s,3H),2.10(s,6H),1.77(s,6H),1.39(t,J=7.2Hz,3H),1.28(d,J=7.0Hz,3H),1.12(s,6H)

[0083] ESI-MS:C 41 H 47 N2O + [M–I - Calculated value: 583.37; Detected value: 583.28.

[0084] Example 3

[0085] The following is a method for synthesizing a class of heptamethrin cyanides substituted with terroir:

[0086] Synthesis of quaternary ammonium salt J-1 and quaternary ammonium salt J-2: Same as in Example 1;

[0087] Synthesis of pyridines substituted with aromatic ketones at R5, R6, and R7:

[0088]

[0089] 2-Isopropylbromobenzene (10.0 g, 50 mmol) was added to a 250 mL three-necked flask, the air was exchanged three times with dry N2, 100 mL of anhydrous tetrahydrofuran was added, and the mixture was cooled to -78 °C. Then, it was slowly added through a syringe. n BuLi (50 mL, 126 mmol) was slowly heated to room temperature under N2 protection and stirred for 1 hour. Next, 3-pyridinecarboxaldehyde (5.9 g, 55.3 mmol) was dissolved in 10 mL of tetrahydrofuran and slowly added to the above reaction mixture at -78 °C, and stirred for another 1 hour. After the reaction was completed, the mixture was allowed to stand and then heated to room temperature, followed by quenching with 30 mL of saturated NH4Cl solution. The tetrahydrofuran was then removed by vacuum rotary evaporation. The mixture was extracted with DCM (2 × 30 mL), and the combined organic phases were dried over anhydrous sodium sulfate, filtered, and the solvent was evaporated. The mixture was then purified by silica gel column chromatography (eluent: dichloromethane / methanol = 50 / 1, v / v). The intermediate product was a pale yellow solid (7.3 g, 64%).

[0090] The above intermediate alcohol (2.0 g, 8.8 mmol) and pyridinium chlorochromate (7.6 g, 35.2 mmol) were added to a 100 mL single-necked flask, and 50 mL of dichloromethane was added to dissolve them. The reaction solution was stirred at room temperature for 4 h. The reaction solution was filtered, and the solvent was removed by rotary evaporation under reduced pressure. The product was then purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate = 20 / 1, v / v). Product 3.1 was a white solid (0.5 g, 25%).

[0091] Synthetic Zincke salt:

[0092]

[0093] The above-mentioned pyridine intermediate (1.0 g, 4.4 mmol) and 2,4-dinitrobromobenzene (1.3 g, 5.3 mmol) were added to a 50 mL quartz sealed tube, and 4 mL of acetonitrile was added to dissolve them. The reaction mixture was then refluxed with stirring for 20 h. After the reaction was completed, the mixture was cooled to room temperature, and the reaction solution was slowly added dropwise to diethyl ether to form a slurry. The slurry was filtered and dried. Intermediate 3.2 was a yellow powder solid (1.2 g, 57%).

[0094] Synthesis of the target product:

[0095]

[0096] The Zincke salt 3.2 (200 mg, 0.89 mmol) and 4-bromoaniline (87 mg, 0.51 mmol) were dissolved in 2 mL of methanol, and the reaction mixture was stirred at room temperature for 30 min. Then, indole quaternary ammonium salt 1.1 (400 mg, 1.27 mmol) and sodium acetate (208 mg, 2.54 mmol) were added, and the reaction mixture was stirred again at room temperature for 16 h. The solvent was then removed by evaporation under reduced pressure, and the product was separated by silica gel column chromatography (eluent: dichloromethane / methanol = 40 / 1, v / v) to give green solid compound 3 (92 mg, 30.6%).

[0097] 1 H NMR (400MHz, DMSO-d6) δ8.36–8.30(m,1H),8.01(d,J=4.1Hz,3H),7.80(d,J=7.1Hz,1H),7.74(d, J=8.0Hz,1H),7.66(d,J=14.2Hz,1H),7.59–7.54(m,1H),7.50(d,J=5.0Hz,2H),7.48–7.44(m,1H) ,7.39–7.26(m,4H),7.20–7.05(m,3H),4.45(d,J=7.2Hz,2H),4.14(d,J=7.1Hz,2H),2.93(p,J=7. 1Hz,1H),1.77(s,6H),1.39(t,J=7.2Hz,3H),1.24(s,3H),1.18(s,3H),1.17(s,3H),1.14(s,6H).

[0098] ESI-MS:C 41 H 47 N2O + [M–I - Calculated value: 583.37; Detected value: 583.28.

[0099] Example 4

[0100] The following is a method for synthesizing a class of heptamethrin cyanides substituted with terroir:

[0101] Synthesis of quaternary ammonium salt J-1 and quaternary ammonium salt J-2: Same as in Example 1;

[0102] Synthesis of pyridines substituted with isopropyl aromatic ketones at R4, R5, and R6:

[0103]

[0104] 1,3,5-Triisopropylbromobenzene (10.0 g, 35 mmol) was added to a 250 mL three-necked flask, the air was exchanged three times with dry N2, 100 mL of anhydrous tetrahydrofuran was added, and the mixture was cooled to -78 °C. Then, it was slowly added through a syringe. n BuLi (35 mL, 88 mmol) was slowly heated to room temperature under N2 protection and stirred for 1 h. Next, 3-pyridinecarboxaldehyde (5.29 g, 49.4 mmol) was dissolved in 10 mL of tetrahydrofuran and slowly added to the above reaction mixture at -78 °C, and stirred for another 1 h. After the reaction was completed, the mixture was allowed to stand and then heated to room temperature, followed by quenching with 30 mL of saturated NH4Cl solution. The tetrahydrofuran was then removed by vacuum rotary evaporation. The mixture was extracted with DCM (2 × 30 mL), and the combined organic phases were dried over anhydrous sodium sulfate, filtered, and the solvent was evaporated. The resulting product was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 50 / 1, v / v). The product was a pale yellow solid (5.2 g, 47%).

[0105] The above intermediate alcohol (2.0 g, 6.4 mmol) and pyridinium chlorochromate (5.5 g, 25.7 mmol) were added to a 100 mL single-necked flask, and 35 mL of dichloromethane was added to dissolve them. The reaction solution was stirred at room temperature for 4 h. The reaction solution was filtered, and the solvent was removed by rotary evaporation under reduced pressure. The product was then purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate = 20 / 1, v / v). Product 4.1 was a white solid (0.5 g, 25%).

[0106] Synthetic Zincke salt

[0107]

[0108] The intermediate pyridine (1.0 g, 3.2 mmol) and 2,4-dinitrobromobenzene (1.0 g, 3.9 mmol) were added to a 50 mL sealed quartz tube, and 4 mL of acetonitrile was added to dissolve them. The reaction mixture was then refluxed with stirring for 20 h. After the reaction was completed, the mixture was cooled to room temperature, and the reaction solution was slowly added dropwise to diethyl ether to form a slurry. The slurry was filtered and dried. Product 4.2 was a yellow powder (0.8 g, 45%).

[0109] Synthesis of the target product:

[0110]

[0111] The Zincke salt 4.2 (200 mg, 0.89 mmol) and 4-bromoaniline (87 mg, 0.51 mmol) were dissolved in 2 mL of methanol, and the reaction mixture was stirred at room temperature for 30 min. Then, indole quaternary ammonium salt (400 mg, 1.27 mmol) and sodium acetate (208 mg, 2.54 mmol) were added, and the reaction mixture was stirred again at room temperature for 16 h. The solvent was then removed by vacuum evaporation, and the product was separated by silica gel column chromatography with a dichloromethane:methanol ratio of 40:1, v / v to obtain green solid compound 4 (92 mg, 30.6%).

[0112] 1 H NMR (500MHz, DMSO-d6) δ8.37–8.28(m,1H),8.19(t,J=13.0Hz,1H),7.98(d,J=14.4Hz,1H),7.80(d,J=7.4Hz,1H), 7.75(d,J=8.1Hz,1H),7.62–7.53(m,2H),7.50(t,J=7.5Hz,1H),7.42(d,J=7.5Hz,1H),7.35(t,J=7.6Hz,1H),7.2 9(d,J=7.9Hz,1H),7.14(d,J=15.0Hz,4H),6.36(d,J=14.2Hz,1H),4.46(d,J=7.9Hz,2H),4.14(s,2H),2.96(q,J= 6.9Hz,1H),2.71(q,J=6.8Hz,2H),1.77(s,6H),1.29(d,J=6.9Hz,6H),1.24(s,6H),1.14(dd,J=13.8,6.8Hz,12H).

[0113] ESI-MS:C 41 H 47 N2O + [M–I - Calculated value: 667.4622; Detected value: 667.38.

[0114] Comparative Example 1

[0115] The synthetic method of a class of aromatic ketone-substituted heptamethine is as follows:

[0116] Synthesis of quaternary ammonium salt J-1 and quaternary ammonium salt J-2: Same as in Example 1;

[0117] Synthesis of 4-benzoyl-substituted Zincke salts:

[0118]

[0119] 4-Benzoylpyridine was purchased directly from Anaiji Chemical. 4-Benzoylpyridine (1.0 g, 5.5 mmol) and 2,4-dinitrobromobenzene (1.5 g, 6 mmol) were added to a 50 mL sealed quartz tube, and 4 mL of acetonitrile was added to dissolve them. The reaction mixture was then refluxed with stirring for 20 h. After the reaction was complete, the mixture was cooled to room temperature, and the reaction solution was slowly added dropwise to diethyl ether and stirred. The product was a yellow powdery solid, 5.1 g (1.79 g, 76.2%).

[0120] Synthesis of the target product:

[0121]

[0122] The Zincke salt 5.1 (200 mg, 0.46 mmol) and 4-bromoaniline (96 mg, 0.56 mmol) were dissolved in 2 mL of methanol, and the reaction mixture was stirred at room temperature for 30 min. Then, indole salt (440 mg, 1.39 mmol) and sodium acetate (230 mg, 2.79 mmol) were added, and the reaction mixture was stirred again at room temperature for 16 h. The solvent was then removed by vacuum evaporation, and the mixture was separated by silica gel column chromatography (dichloromethane:methanol = 30:1, v / v) to obtain a green solid, Comparative Example 1 (90 mg, 28.9%).

[0123] 1 H NMR (400MHz, DMSO-d6) δ8.05(d,J=7.7Hz,2H),7.78(t,J=7.4Hz,1H),7.67(t,J=7.6Hz,2H),7.50(d,J=7.4Hz,2H),7.43–7.28(m,6H),7.2 3(ddd,J=8.1,5.1,3.3Hz,2H),6.70(d,J=13.5Hz,2H),6.49(d,J=13.4Hz,2H),4.12(q,J=7.1Hz,4H),1.27(d,J=7.1Hz,6H),1.24(s,12H).

[0124] ESI-MS:C 38 H 41 N2O + [M–I - Calculated value: 541.32; Detected value: 541.25.

[0125] Performance testing

[0126] Compounds 1-4 obtained in the above examples and Comparative Example 1, and the dye of Comparative Example 1 after vacuum drying were accurately weighed using a 0.01 g balance. A 5 mmol / L DMSO dye stock solution was prepared in a brown sample bottle and stored in a refrigerator at 4°C for later use as a dye stock solution.

[0127] 1. Determination of UV-Vis absorption and fluorescence spectra of compounds 1-4 and Comparative Example 1.

[0128] Test method: When testing the UV-Vis absorption and fluorescence spectra, 1.2 μL of the dye stock solution was measured using a micropipette and dissolved in a quartz cuvette containing 3 mL of the test solvent. The mixture was thoroughly mixed to obtain a dye concentration of 2.0 μmol / L, which was used for the absorption and fluorescence emission spectra. All tests were performed at 25℃.

[0129] Figure 1 The normalized UV-Vis absorption spectra of compounds 1-4 and Comparative Example 1 in dichloromethane are given by [source missing]. Figure 1 It can be seen that, compared with Comparative Example 1, which has a meso-substituted aromatic ketone, the absorption wavelength of Example 1, which has a partial aromatic ketone substitution, exhibits a blue shift. Furthermore, after increasing the steric hindrance on the aromatic ketone benzene ring of the partial-substituted heptamethrin, the absorption wavelengths of Examples 2-4 show a further blue shift compared to Example 1. This indicates that meso-substituted aromatic ketones have little effect on the ground-state properties of the dye, while partial-substituted dyes affect the electron cloud density of the compound in the ground state, thereby affecting the ground-state properties of the dye.

[0130] Figure 2 The normalized fluorescence emission spectra of compounds 1-4 and Comparative Example 1 in dichloromethane are given by [the data source is missing]. Figure 2 It can be seen that, compared with Comparative Example 1 which has a meso-substituted aromatic ketone, the emission wavelengths of Examples 1-4 which have a tert-substituted aromatic ketone exhibit a blue shift. This indicates that meso-substituted dyes do not significantly affect the excited-state properties of the dyes, while tert-substituted dyes affect the electron cloud density of the excited-state of the compound, thereby affecting the fluorescence properties of the excited-state of the dyes.

[0131] 2. Tests on the singlet oxygen generation capacity of compounds 1-4, Comparative Example 1, and the commercially available photosensitizer ICG in dichloromethane.

[0132] Test method: 3 mL of dichloromethane solution was added to a cuvette, followed by a certain amount of DPBF to achieve an absorbance of approximately 1.00 at 415 nm. Then, the dye stock solutions of compounds 1-4 from Examples and Comparative Example 1 were added to adjust the absorbance of the dye at 660 nm to approximately 0.4. The cuvette was illuminated under a 660 nm LED lamp, and the UV absorption of the solution was measured every 30 seconds. The DPBF degradation spectrum of ICG was then measured using the same method. The singlet oxygen quantum yield of ICG in dichloromethane was used as a reference to calculate the relative singlet oxygen quantum yields of compounds 1-4 and Comparative Example 1. Test results are as follows Figure 3 As shown in Table 1, the calculation results are as follows.

[0133] Table 1

[0134]

[0135] Analysis of the data from Examples 1-4, Comparative Example 1, and Table 1 shows that the relative singlet oxygen quantum yield of the partially substituted compounds 1-4 is 5.4-11.6 times that of ICG, while the relative singlet oxygen quantum yield of the meso-substituted Comparative Example 1 is not significantly different from that of ICG. This indicates that the partially substituted compounds can significantly alter the excited-state properties of heptamethrin dye, promoting more electrons to undergo intersystem crossings to reach the triplet excited state, thereby transferring energy with oxygen and generating a large amount of singlet oxygen. This proves that the partially substituted compounds are good photosensitizer dyes.

[0136] 3. Dye co-localization experiment with organelles

[0137] 4T1 cells were selected as the test cell line. After incubating them in confocal culture dishes for 24 hours, the culture medium was discarded, and the cells were washed three times with PBS buffer. DMEM medium containing 2 μmol / L of compound 4 was added, followed by the addition of 2 μmol / L of commercially available dyes for different organelles: mitochondrial MitoTrackerGreen, lysosomal localization probe LysoTrackerGreen, and nuclear localization probe Hoechst33342. After incubation in a cell culture incubator for 30 minutes, the cells were imaged under a confocal microscope. The results are shown in the figure. Figure 4 .

[0138] Confocal parameters: 60× oil immersion lens; excitation wavelength of compound 4 channel was 660 nm, and acquisition and emission wavelengths were 690-750 nm; excitation wavelength of mitochondrial MitoTrackerGreen and lysosomal localization probe LysoTrackerGreen channels was 488 nm, and acquisition and emission wavelengths were 500-550 nm; excitation wavelength of nuclear localization probe Hoechst33342 channel was 405 nm, and acquisition and emission wavelengths were 440-480 nm.

[0139] from Figure 4 As shown in the colocalization experiment results, compound 4 can be clearly localized to the mitochondrial region of cells. Its colocalization coefficient with the commercial nuclear localization probe Hoechst33342 is as high as 0.871, which proves that the heptamethrin dye substituted with teratophore can be localized to the mitochondrial region of cells. It can be used to image the mitochondria of cells, detect physiological processes, and generate reactive oxygen species under light to destroy the mitochondrial membrane potential of cells, successfully killing cells and playing an integrated diagnostic and therapeutic role.

[0140] 4. The cytotoxicity of dye molecules to cells was assessed using the MTT assay.

[0141] The testing principle is as follows: Succinate dehydrogenase in the mitochondria of living cells can reduce exogenous MTT to water-insoluble blue-purple formazan crystals, which are then deposited in the cells. Dead cells do not have this function. Dimethyl sulfoxide can dissolve the formazan in the cells, and its absorbance value is measured at a wavelength of 490 nm using an ELISA reader, which can indirectly reflect the number of living cells.

[0142] Test method: Four T1 cells were seeded into two 96-well plates. After a period of culture, a certain concentration of compound 4 was added to different wells to make the concentration of compound 4 0-2 μmol / L. After incubation for 2 hours, one of the 96-well plates was placed under a 660 nm LED lamp for 20 minutes at an optical density of 30 mW / cm². 2 Cells were then incubated in two 96-well plates for another 24 hours, and cell viability was assessed using an MTT assay. The results are shown below. Figure 5 .

[0143] Experimental data such as Figure 5 As shown, without light exposure, the cell survival rate after incubation with 2 μmol / L compound 4 remained above 80%, demonstrating that compound 4 exhibits very low dark toxicity within this concentration range. However, under light exposure, the cell survival rate after incubation with 2 μmol / L compound 4 was below 20%, demonstrating that compound 4 possesses high phototoxicity. This indicates that compound 4, as a photosensitizer, can generate a large amount of reactive oxygen species to kill cells under light exposure, while having almost no effect on cell viability under no-light conditions, thus enabling targeted cell killing.

[0144] Combined with Examples 1-4 and Comparative Example 1, and in conjunction with Table 1 and Figure 3 It can be seen that the heptamethrin dyes substituted with phenoxy ketones have a superior ability to generate singlet oxygen compared with those substituted with meso-substituted dyes. This indicates that phenoxy substitution can effectively promote intersystem crossing in dyes; combined with Figure 4-5 It is known that heptamethrin dyes substituted with teratophores can accumulate well in the mitochondria of cells, exhibit low cytotoxicity in the absence of light, and can generate reactive oxygen species to damage cell mitochondria after light exposure, thereby killing tumor cells.

[0145] In conclusion, heptamethine cyanide dyes substituted with terroaryl ketones exhibit superior photodynamic therapy capabilities compared to dyes substituted with mesoaryl ketones.

[0146] Compared with existing technologies, the dye provided by this invention has the following inventive aspects: a new dye structure has been successfully developed and its photophysical properties have been studied; both absorption and emission wavelengths are located in the near-infrared region, resulting in strong tissue penetration; it has high intersystem crossing efficiency; it can generate a large amount of reactive oxygen species in cell mitochondria for photodynamic therapy; and it has low biological dark toxicity, thus enabling it to be better used in practical applications.

[0147] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A heptamethine cyanine dye substituted with a pseudo-aromatic ketone, characterized in that, It has the following general formula: R1 and R2 are each independently selected from hydrogen and any alkyl group having 1 to 18 carbons; R3 and R4 are substituents at uncertain positions on the benzene ring, each independently selected from hydrogen or halogen. R5, R6 and R7 are each independently selected from any one of hydrogen, alkyl groups having 1 to 8 carbons, and halogens; X is selected from any one of oxygen, sulfur, selenium, or gem-dimethyl; Y is selected from inorganic negative ions or organic negative ions.

2. The heptamethine cyanide dye substituted with a para-aromatic ketone according to claim 1, characterized in that, Y is selected from halide ions, ClO4 - BF4 - CH3COO - CF3COO - or OTs - Any one of them.

3. The heptamethine cyanide dye substituted with a para-aromatic ketone according to claim 2, characterized in that, R5, R6, and R7 are each independently selected from hydrogen, methyl, or isopropyl; Y is selected from halide ions, ClO4, etc. - CH3COO - Any one of them.

4. The heptamethine cyanide dye substituted with a para-aromatic ketone according to claim 3, characterized in that, R1 and R2 are methyl groups, R3 and R4 are hydrogen groups, X is gem-dimethyl, and Y is an iodide ion.

5. A method for synthesizing a heptamethine cyanine dye substituted with a terroirone, characterized in that, The method for synthesizing the heptamethine cyanide dye substituted with a para-aromatic ketone according to any one of claims 1 to 4 comprises the following steps: S1: Synthesis of quaternary ammonium salts J-1 and J-2: Indole Y-1 modified with R3 substituent was mixed with halo-N alkylating agent Y-2 with R1 substituent at a molar ratio of 1:(1-10), and a first organic solvent was added. The mixture was reacted at 40-120℃ to obtain quaternary ammonium salt J-1. Indole Y-3 modified with R4 substituent was mixed with halo-N-alkylating agent Y-4 with R2 substituent at a molar ratio of 1:(1-10), a second organic solvent was added, and the mixture was reacted at 40-120℃ to obtain quaternary ammonium salt J-2. S2: Synthesis of Zincke salt: Compound P-2 and 2,4-dinitrohalobenzene were mixed evenly, a third organic solvent was added, and the mixture was reacted at 40-120℃ to obtain compound P-3, wherein Z is a halogen. S3: Synthesis of the target product: Compound P-3 obtained in step S2 and 4-bromoaniline were mixed evenly at a molar ratio of 1:1.

2. Methanol was added, and the mixture was stirred at room temperature for more than 0.5 h. Then, quaternary ammonium salts J-1 and J-2 obtained in step S1 and sodium acetate were added, and the mixture was stirred at room temperature for 5-60 min. The solvent was removed to obtain the target product.

6. The method for synthesizing heptamethrin dye substituted with a para-aromatic ketone according to claim 5, characterized in that, The method for synthesizing the ketone-substituted pyridine described in step S2 is as follows: Under N2 protection, R5, R6, and R7 substituted bromobenzenes were dissolved in anhydrous tetrahydrofuran and cooled to -78°C. Then, [the following was added]... n BuLi, and heated to room temperature, continued stirring, added a tetrahydrofuran solution of 3-pyridinecarboxaldehyde at -78°C to the above reaction mixture, continued stirring to react, after the reaction was completed, allowed to stand and heated to room temperature, quenched the reaction with saturated NH4Cl solution, removed the solvent, and obtained reaction intermediate P-1; Intermediate P-1 was mixed with pyridine chlorochromate at a molar ratio of 1:(4-6), dichloromethane was added, and the mixture was reacted at room temperature for 3-6 hours. The solvent was then removed to obtain compound P-2.

7. The method for synthesizing heptamethrin dye substituted with a para-aromatic ketone according to claim 6, characterized in that, In the synthesis of ketone-substituted pyridine in step S2, the molar ratio of 3-pyridinecarboxaldehyde to R-substituted bromobenzene is 1:(1-1.5); in step S2, the molar ratio of compound P-2 to 2,4-dinitrohalobenzene is 1:(1-5); in step S3, the molar ratio of compound P-3 to the total amount of quaternary ammonium salt J-1 and quaternary ammonium salt J-2 is 1:(2-3), and the molar ratio of compound P-3 to sodium acetate is 1:

6.

8. The application of a heptamethine cyanide dye substituted with a terroirone as described in any one of claims 1 to 4 as a near-infrared luminescent material, or its application in the preparation of photocatalytic agents, biolabeled probes, photodynamic therapy agents, photolyzed water agents, or biosensor devices.

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