Side chain modified pentamethine cyanine dye as well as synthesis method and application thereof

Abstract

The invention relates to a side chain modified pentamethine cyanine dye as well as a synthesis method and application thereof. The dye has a general formula CY5-R, has strong absorption and emission characteristics at 650nm or above, and is suitable for red light excitation, mitochondrial localization and I-type photodynamic therapy. Experiments show that the co-localization coefficient of the dye and mitochondria reaches 0.860, the dye shows excellent deep tissue penetrating power and low dark toxicity, IC50 is 0.15 mu M after illumination, superoxide anion free radicals can be efficiently generated, cancer cells can be remarkably killed, and diagnosis and treatment integration is achieved. The synthesis method comprises the steps of quaternary ammonium salt synthesis, condensation reaction, side chain modification and the like, and has the characteristics of cheap raw materials, high yield and simplicity and convenience in operation. The dye has wide application in the fields of photocatalysis, photodynamic therapy, biomarkers, deep tissue imaging and the like, and is particularly suitable for fluorescence imaging and photodynamic therapy of deep tumors.

Description

Technical Field

[0001] The present invention relates to the technical field of the synthesis of red fluorescent dyes, and particularly to a class of pentamethine cyanine dyes with side-chain modification, a synthesis method thereof, and applications thereof. Background Art

[0002] As a new non-invasive, short-cycle, and non-drug-resistant treatment method, photodynamic therapy has been applied to the clinical treatment of certain diseases. By utilizing the fluorescence characteristics and electron transfer properties of organic dyes, fluorescence imaging and precise photodynamic therapy can be performed on diseased tissues, thereby achieving the purpose of integrated diagnosis and treatment of diseases. Therefore, researchers have developed many organic dyes as photosensitizers for the diagnosis and treatment of diseases. Currently, the most common ones are BODIPY dyes and ruthenium complexes, etc. However, these dyes generally have short absorption and emission wavelengths and do not have a targeting effect on cell organelles, making it difficult to achieve precise diagnosis and treatment of deep-seated diseases in the body. Due to the severe hypoxic microenvironment in solid tumors restricting the use of photosensitizers, in dealing with the difficult problem of hypoxic tumor treatment, type I photodynamic therapy that generates superoxide anion radicals or hydroxyl radicals as the main reactive oxygen species shows better advantages and application potential. However, most of the existing clinically used photosensitizers are of the type II-dominated photosensitization mode that generates singlet oxygen as the main reactive oxygen species, and type I photosensitizers are relatively lacking.

[0003] As a classic cyanine dye system, the photophysical properties and application research of pentamethine cyanine dyes have been relatively in-depth. This series of compounds exhibit characteristic absorption and emission spectra in the red light region (600 - 750 nm), and have both a high molar extinction coefficient (ε > 10^5 L·mol-1·cm-1) and an excellent fluorescence quantum yield (Φ f ≈0.2 - 0.5), and have been successfully applied in the fields of biological fluorescence labeling and tissue staining. However, after being excited, this type of dye mainly releases energy through non-radiative transition and fluorescence emission pathways, resulting in a low intersystem crossing efficiency and making it difficult to achieve the photosensitized electron transfer process required for effective photodynamic therapy. The existing modification strategies are mainly based on the heavy atom effect (such as iodine / bromine substitution), but this approach has significant limitations: ① The introduction of heavy atoms leads to a decrease in molecular polarity (the LogP value increases by 2 - 3 units), significantly affecting its water solubility (<0.1 mg / mL); ② The cytotoxicity is significantly enhanced (the IC 50 value decreases by about 2 orders of magnitude); ③ The introduction of complex substituents extends the synthesis route (an average increase of 3 - 5 reaction steps), and the isomer separation efficiency decreases (HPLC purity <85%). The above defects seriously restrict its biomedical transformation potential.

[0004] Therefore, aiming at the inherent defect that the singlet oxygen generation efficiency of current type II photosensitizers drops sharply in the tumor hypoxic microenvironment, it is urgent to develop new pentamethine type I photosensitizers through molecular engineering strategies. On the basis of maintaining their inherent red light (650 - 700 nm) absorption and emission characteristics, a photosensitive system with a controllable electron transfer path is constructed, enabling it to generate long-lived reactive oxygen species such as superoxide anion radicals and hydroxyl radicals through the electron transfer mechanism, thereby breaking through the limitations of the tumor microenvironment on traditional photodynamic therapy. Summary of the Invention

[0005] Aiming at the problem that existing pentamethine cyanine dyes in the prior art cannot undergo efficient electron transfer to generate superoxide anion radicals, the first object of the present invention is to provide a synthesis method and application of a class of pentamethine cyanine dyes with side chain modification. The pentamethine cyanine dyes have red light excitation, mitochondrial localization, and good type I photodynamic effects.

[0006] The second object of the present invention is to provide a synthesis method of a class of pentamethine cyanine dyes with side chain modification. The preparation method has the advantages of simple synthesis method, cheap raw materials, high yield, and high generality.

[0007] The third object of the present invention is to provide an application of a class of pentamethine cyanine dyes with side chain modification. The pentamethine cyanine dyes with side chain modification can be used as dyes to initiate chemical reactions in the field of photocatalysis and generate reactive oxygen species in the field of photodynamic therapy.

[0008] To achieve the above first object, the present invention provides the following technical solution: A class of pentamethine cyanine dyes with side chain modification, including the following general formula CY5-R:

[0009]

[0010] Wherein, R1 and R2 are each independently selected from any one of hydrogen, alkyl with 1 - 6 carbons, carboxyalkyl with 1 - 6 carbons, arylethyl, 2-(thiophen-2-yl)ethyl, 2-(5-(methylthio)thiophen-2-yl)ethyl, 2-(bithiophen-2-yl)ethyl, 2-(thieno[3,2-b]thiophen-2-yl)ethyl, 2-([2,2':5',2'-terthiophen]-5-yl)ethyl and its derivatives. Preferably, R1 and R2 are selected from any one of arylethyl, 2-(thiophen-2-yl)ethyl, 2-(5-(methylthio)thiophen-2-yl)ethyl, 2-(bithiophen-2-yl)ethyl, 2-(thieno[3,2-b]thiophen-2-yl)ethyl, 2-([2,2':5',2'-terthiophen]-5-yl)ethyl and its derivatives;

[0011] R3 and R4 are substituents at undefined positions on the benzene ring, each independently selected from any one of hydrogen, halogen, methoxy, amino, nitro, hydroxy, and carboxyl. Preferably, the substitution positions of R3 and R4 on the benzene ring are preferably the 5-position substitution, and each is independently selected from any one of hydrogen, halogen, methoxy, and nitro.

[0012] Y− is selected from inorganic anions or organic anions: halide ions, ClO4 - , BF4 - , PF6 - , CH3COO−, CF3COO - or OTs - Any one of them. Preferably, Y− is selected from halide ions, ClO4 - , PF6 - Any one of them.

[0013] Furthermore, the emission wavelength of the side-chain modified pentamethine cyanine dye is above 650 nm. The emission wavelengths used in Examples 1-5 are 650-720 nm.

[0014] By adopting the above technical solution: Since the maximum emission wavelength of the dye is above 650 nm, it can be used as a luminescent material in the red light region. The maximum emission wavelength is in the range of 650-720 nm, which is suitable for deep tissue imaging; both the absorption and emission wavelengths are in the red light region; it has the ability of mitochondrial targeting and localization, and the co-localization coefficient with the mitochondrial probe is as high as 0.860.

[0015] To achieve the above second object, the present invention provides the following technical solution: A class of side-chain modified pentamethine cyanine dyes, including:

[0016]

[0017] (1) Synthesis of quaternary ammonium salts H-1 and H-2:

[0018] Mix the benzindole J-1 modified with the R3 substituent with 6-bromohexanoic acid evenly, then add the first organic solvent, and react at 40-120 °C for 4-12 h to obtain the quaternary ammonium salt H-1;

[0019] Mix the benzindole J-2 modified with the R4 substituent with 6-bromohexanoic acid evenly, then add the second organic solvent, and react at 40-120 °C for 4-12 h to obtain the quaternary ammonium salt H-2;

[0020] (2) Synthesis of compound H-3:

[0021] Mix the quaternary ammonium salts H-1, H-2 and the condensing agent J-3 evenly, then add the third organic solvent and inorganic base, and react at 50-150 °C for 4-12 h, remove the solvent to obtain the target product.

[0022] (3) Synthesis of Compound H-4:

[0023] Mix Compound H-3 evenly with the corresponding alcohol with R1 substituent, then add the fourth organic solvent and organic base I, add catalyst I, react at 25 - 50 °C for 4 - 12 h, remove the solvent, and perform column chromatography to obtain the target product.

[0024] (4) Synthesis of Compound H-5:

[0025] Mix Compound H-4 evenly with the corresponding alcohol with R2 substituent, then add the fifth organic solvent and organic base II, add catalyst II, react at 25 - 50 °C for 4 - 12 h, remove the solvent, and perform column chromatography to obtain the target product.

[0026] For the technical solution described above, further preferably, in the above step (1), the first organic solvent or the second organic solvent is independently selected from at least one of ethanol, acetonitrile, toluene, N,N-dimethylformamide, o-dichlorobenzene, and sulfolane;

[0027] For the technical solution described above, further preferably, in the above step (1), the molar ratio of the benzindole, 6-bromohexanoic acid, and the first or second organic solvent is 1:1 - 8:8 - 20, and the further preferred ratio is 1:1 - 3:8 - 12; the most preferred molar ratio is 1:2:10;

[0028] For the technical solution described above, further preferably, in the above step (1), the reaction temperature is 60 - 100 °C, and the reaction time is 6 - 10 h.

[0029] For the technical solution described above, further preferably, in the above step (2), the third organic solvent is selected from at least one of ethanol, acetic anhydride, n-butanol, isopropanol, and acetic acid, and the inorganic base is selected from one of potassium carbonate, sodium carbonate, sodium acetate, sodium bicarbonate, cesium carbonate, potassium hydroxide, sodium hydroxide, and sodium tert-butoxide;

[0030] For the technical solution described above, further preferably, in step (2), the molar ratio of the quaternary ammonium salts H-1, H-2, condensing agent J-3, the third organic solvent, and the inorganic base is 1 - 4:1 - 4:1:8 - 20:1 - 5, and the further preferred ratio is 1 - 2:1 - 2:1:8 - 15:1 - 3; the most preferred molar ratio is 1:1:1:10:2;

[0031] For the technical solution described above, further preferably, in step (2), the reaction temperature is 60 - 100 °C, and the reaction time is 6 - 10 h.

[0032] For the technical solutions described above, more preferably, in the above step (3), the fourth organic solvent is selected from at least one of dichloromethane, N,N-dimethylformamide, 1,2-dichloroethane, acetonitrile, and toluene; the organic base I is selected from one of triethylamine, pyridine, and N,N-diisopropylethylamine; the catalyst I is selected from one of DCC, EDC-HCl, HATU, HBTU, HCTU, TNTU, and TSTU;

[0033] For the technical solutions described above, even more preferably, in the above step (3), the molar ratio of compound H-3, the corresponding alcohol with R1 substituent, catalyst I, organic base I, and the fourth organic solvent is 1:1-4:1-4:1-5:8-20;

[0034] For the technical solutions described above, even more preferably, in the above step (3), the molar ratio is 1:1-2:2-3:4-5:8-12; the most preferred molar ratio is 1:1:2.5:3:10;

[0035] For the technical solutions described above, even more preferably, in the above step (3), the reaction temperature is 25-35 °C and the reaction time is 6-10 h.

[0036] For the technical solutions described above, more preferably, in the above step (4), the fifth organic solvent is selected from at least one of dichloromethane, N,N-dimethylformamide, 1,2-dichloroethane, acetonitrile, and toluene; the organic base II is selected from one of triethylamine, pyridine, and N,N-diisopropylethylamine; the catalyst II is selected from one of DCC, EDC-HCl, HATU, HBTU, HCTU, TNTU, and TSTU;

[0037] For the technical solutions described above, even more preferably, in the above step (4), the molar ratio of compound H-4, the corresponding alcohol with R2 substituent, catalyst II, organic base II, and the fifth organic solvent is 1:1-4:1-4:1-5:8-20, and the even more preferred molar ratio is 1:1-2:1-3:2-3:8-12; the most preferred molar ratio is 1:1.5:1.5:3:10;

[0038] For the technical solutions described above, even more preferably, in the above step (4), the reaction temperature is 25-35 °C and the reaction time is 6-10 h;

[0039] To achieve the above-mentioned third objective, the present invention is characterized in that the side-chain modified pentamethine cyanine dye has excellent red light excitation characteristics, efficient electron transfer ability and low dark toxicity, showing great potential in the fields of biomedicine and energy. It can be applied to the field of photocatalysis, as a luminescent material in the red light region, a biological labeling probe, a preparation for photodynamic therapy, photocatalytic water splitting for hydrogen production, and the field of biosensing.

[0040] Furthermore, the side-chain modified pentamethine cyanine can act as a photosensitizer to initiate chemical reactions in the field of photocatalysis, generate reactive oxygen species in the field of photodynamics, and produce phosphorescence signals in the field of phosphorescent materials.

[0041] Furthermore, the optical signal of the side-chain modified pentamethine cyanine is particularly prominent in the red light system. The maximum emission wavelength of this series of dyes is above 680 nm, having excellent tissue penetration and deep imaging ability. It can be used as a luminescent material in the red light region, especially suitable for fluorescence imaging and photodynamic therapy of deep tissues.

[0042] Furthermore, the side-chain modified pentamethine cyanine molecule can quickly and efficiently penetrate the cell membrane, enter the cell and accumulate in subcellular organelles. By irradiating with low-light-density red light, it can efficiently generate reactive oxygen species, disrupt the intracellular homeostasis balance, and kill cancer cells with high efficiency at a concentration as low as the nanomolar level. It can be applied to the field of photodynamic therapy, and at the same time has low dark toxicity and high selectivity, enabling integrated diagnosis and treatment.

[0043] Compared with the prior art, the present invention has the following beneficial effects:

[0044] First, for the side-chain modified pentamethine cyanine molecule of the present invention, by modifying the side chain of the pentamethine cyanine molecule, it has good molar extinction coefficient and fluorescence quantum yield in organic solvents.

[0045] Second, the side-chain modified pentamethine cyanine dye of the present invention has good electron transfer ability. Compared with the comparative example in vitro, it can generate more superoxide anion radicals. It is suitable for photodynamic therapy. Through the DHR123 fluorescence probe test, the fluorescence intensity of the dye of the present invention is significantly enhanced after illumination, indicating that it can efficiently generate superoxide anion radicals. Compared with Comparative Example 1, the generation amount of superoxide anion radicals in Examples 1-5 is significantly increased, and with the increase of the electron cloud density of the side-chain modification group, the radical generation amount gradually increases. This efficient electron transfer ability makes it show excellent reactive oxygen species generation effect in photodynamic therapy.

[0046] Third, the pentamethine cyanine dye with side-chain modification according to the present invention can rapidly penetrate cell membranes and accumulate in cell mitochondria. It can detect physiological processes by utilizing changes in cell mitochondria, and can achieve targeted imaging and treatment of cell organelles. Through confocal microscopy imaging, the co-localization coefficient of the dye of the present invention with the mitochondrial green fluorescence probe is as high as 0.860, indicating that it can be localized in cell mitochondria. Example 5 has low dark toxicity to 4T1 cells under dark conditions, and the cell survival rate is higher than 90%; while after irradiation with 660 nm light for 20 min, its phototoxicity shows a dose-dependent change, and the half inhibitory concentration (IC 50 ) is 0.15 μM, which is significantly better than Comparative Example 1. This low dark toxicity and high selectivity enable it to achieve the integration of diagnosis and treatment.

[0047] Fourth, the pentamethine cyanine dye with side-chain modification according to the present invention has low cytotoxicity to cells under dark conditions, generates a large amount of superoxide anion radicals after irradiation, causing cell death, and achieving the purpose of photodynamic therapy. Through MTT experiments, the dye of the present invention has low cytotoxicity to cells under dark conditions and can efficiently kill cancer cells after irradiation, and the IC 50 is 0.15 μM. This highly efficient photodynamic therapy effect makes it have important application value in tumor treatment.

[0048] Fifth, the pentamethine cyanine dye with side-chain modification according to the present invention has strong absorption and emission characteristics above 650 nm, can penetrate deep tissues, and is suitable for fluorescence imaging and photodynamic therapy of deep tumors. Through ultraviolet-visible absorption spectroscopy and fluorescence emission spectroscopy tests, the dye of the present invention shows strong absorption and emission characteristics in the range of 650 - 720 nm. This red light excitation characteristic gives it excellent tissue penetration ability, and is particularly suitable for fluorescence imaging and photodynamic therapy of deep tumors.

[0049] Sixth, the synthesis method of the present invention includes steps such as quaternary ammonium salt synthesis, condensation reaction, and side-chain modification, and has the characteristics of cheap raw materials, high yield, and simple operation. In the synthesis steps of Examples 1 - 5, the yields of each intermediate and the target product are all higher than 45%, and the highest can reach 90.25%. This highly efficient synthesis method provides technical guarantee for its large-scale production and application. Description of the Drawings

[0050] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0051] Figure 1UV-Vis absorption spectra of Examples 1-5 and Comparative Example 1 disclosed in the present invention in dichloromethane;

[0052] Figure 2 Fluorescence emission spectra of Examples 1-5 and Comparative Example 1 disclosed in the present invention in dichloromethane;

[0053] Figure 3 Line graph of the production effect of superoxide anion radicals of Examples 1-5 and Comparative Example 1 disclosed in the present invention in water;

[0054] Figure 4 Confocal single-photon microscope images of cell organelles of Example 5 disclosed in the present invention;

[0055] Figure 5 MTT experiment graph of Example 5 disclosed in the present invention. Detailed implementation manners

[0056] To make the objectives, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying Figures 1-5 , and the technical solutions in the embodiments of the present invention will be described clearly and completely. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0057] Unless otherwise specified, the terms used herein have the following meanings.

[0058] The term "halogen" used herein includes fluorine, chlorine, bromine and iodine.

[0059] The term "alkyl" used in this application includes straight-chain alkyl and branched-chain alkyl.

[0060] Y used herein - represents a negative ion, which can be any suitable negative ion, including inorganic negative ions and organic negative ions, and examples include but are not limited to halogen ions, ClO4 - , PF6 - , BF4 - , CH3COO - , CF3COO - or OTs - .

[0061] Instruments and equipment used in the examples:

[0062] In the column chromatography process of the present invention, 200-300 mesh and 500-600 mesh column chromatography silica gel purchased from Qingdao Meigao Group Co., Ltd. and 20-40 mesh analytical pure quartz sand purchased from Tianjin Chemical Reagent Factory were used.

[0063] The absorption and emission spectra of the dyes were measured using an Agilent Cary 60 UV-Vis spectrophotometer and a Cary Eclipse fluorescence spectrophotometer.

[0064] The dye organelle localization experiment was measured using an Olympus FV1000 single-photon confocal microscope.

[0065] The cytotoxicity test was measured using a Thermofisher Varioskan LUX Multimode Microplate Reader. Specific Examples

[0067]

[0068] The synthesis method of the side-chain modified pentamethine cyanine molecule is as follows:

[0069] Example 1:

[0070] Synthesis of Intermediate 1-1

[0071]

[0072] 2,3,3-Trimethylindole (5.00 g, 31.40 mmol) and 6-bromohexanoic acid (12.25 g, 62.80 mmol) were added to a reaction flask, 50 mL of acetonitrile was added, and the temperature was raised to 85 °C, followed by heating and stirring for 12 h. After the reaction was completed, the temperature was restored to room temperature. 20 mL of dichloromethane was added to the reaction flask and stirred until clear. The mixture was added dropwise to 500 mL of ether, and the solid was collected by filtration. The solid was dried to obtain 8.92 g, with a yield of 80.18%. No purification was required, and it could be used for the next reaction.

[0073] Synthesis of Intermediate 1-2

[0074]

[0075] Intermediate 1-1 (5.00 g, 14.11 mmol), condensing agent malondialdehyde diphenylamine hydrochloride (1.28 g, 4.70 mmol), and sodium acetate (1.93 g, 23.52 mmol) were added to 20 mL of acetic anhydride, and the temperature was raised to 50 °C, followed by heating and stirring for 12 h. After the reaction was completed, the temperature was restored to room temperature. The reaction solution was added dropwise to 500 mL of ether, and the blue solid was collected by filtration. The solid was dried to obtain 2.33 g, with a yield of 74.63%. No purification was required, and it could be used for the next reaction.

[0076] Synthesis of Intermediate 1-3

[0077]

[0078] Intermediate 1-2 (2.00 g, 3.01 mmol), DIPEA (1.36 g, 10.55 mmol), and HATU (2.85 g, 7.52 mmol) were successively added to 20 mL of dichloromethane, and the mixture was stirred at room temperature for 30 min. Methanol (96.56 mg, 3.01 mmol) was added dropwise to the reaction flask, and the mixture was stirred overnight at room temperature. After the reaction was completed, the reaction solution was concentrated and purified by silica gel column chromatography to obtain 2.02 g of a blue solid with a yield of 90.25%.

[0079] Synthesis Example 1

[0080]

[0081] Intermediate 1-3 (100.00 mg, 134.63 μmol), DIPEA (60.90 mg, 471.20 μmol), and HATU (61.43 mg, 161.55 μmol) were successively added to 1 mL of dichloromethane, and the mixture was stirred at room temperature for 30 min. 2-Thiopheneethanol (20.71 mg, 161.55 μmol) was added dropwise to the reaction flask, and the mixture was stirred overnight at room temperature. After the reaction was completed, the reaction solution was concentrated and purified by silica gel column chromatography to obtain 76.00 mg of a blue solid with a yield of 60.18%. 1 H NMR (400 MHz, DMSO-d6) δ 8.33 (t, J = 13.0 Hz, 2H), 7.62 (d, J = 7.4 Hz, 2H), 7.40 (d, J = 5.7 Hz, 4H), 7.32 (dd, J = 5.1, 1.3 Hz, 1H), 7.29–7.19 (m, 2H), 6.97–6.87 (m, 2H), 6.58 (t, J = 12.2 Hz, 1H), 6.29 (d, J = 13.6 Hz, 2H), 4.18 (t, J = 6.5 Hz, 2H), 4.07 (d, J = 8.5 Hz, 4H), 3.55 (s, 3H), 3.07 (t, J = 6.5 Hz, 2H), 2.29 (t, J = 7.3 Hz, 4H), 1.68 (s, 16H), 1.60–1.53 (m, 4H), 1.37 (s, 4H). +ES-HRMS: C44H55N2O4S+ calculated value 707.3877, detected value 707.3885. -ES-HRMS: F6P- calculated value 144.9647, detected value 144.9649.

[0082] Example 2

[0083] Synthesis Example 2

[0084]

[0085] Intermediate 1-3 (100.00 mg, 134.63 μmol), DIPEA (60.90 mg, 471.20 μmol), and HATU (61.43 mg, 161.55 μmol) were successively added to 1 mL of dichloromethane, and the mixture was stirred at room temperature for 30 min. 2-(5-(Methylthio)thiophen-2-yl)ethanol (23.56 mg, 135.17 μmol) was added dropwise to the reaction flask, and the mixture was stirred at room temperature overnight. After the reaction was completed, the reaction solution was concentrated and purified by silica gel column chromatography to obtain 56.00 mg of a blue solid with a yield of 47.62%. 1 H NMR (400 MHz, DMSO-d6) δ 8.33 (t, J = 13.0 Hz, 2H), 7.62 (d, J = 7.5 Hz, 2H), 7.40 (d, J = 5.6 Hz, 4H), 7.25 (t, J = 6.7 Hz, 2H), 6.95 (d, J = 3.5 Hz, 1H), 6.79 (d, J = 3.5 Hz, 1H), 6.58 (t, J = 12.3 Hz, 1H), 6.29 (d, J = 13.7 Hz, 2H), 4.17 (t, J = 6.4 Hz, 2H), 4.07 (d, J = 8.1 Hz, 4H), 3.55 (s, 3H), 3.02 (t, J = 6.4 Hz, 2H), 2.41 (s, 3H), 2.29 (dt, J = 7.5, 4.1 Hz, 4H), 1.68 (s, 16H), 1.57 (q, J = 7.5 Hz, 4H), 1.37 (s, 4H). +ES-HRMS: C45H57N2O4S2 + calculated value 753.3754, detected value 753.3749. -ES-HRMS: F6P - calculated value 144.9647, detected value 144.9645.

[0086] Example 3

[0087] Synthesis Example 3

[0088]

[0089] Intermediate 1-3 (100.00 mg, 134.63 μmol), DIPEA (60.90 mg, 471.20 μmol), and HATU (61.43 mg, 161.55 μmol) were successively added to 1 mL of dichloromethane, and the mixture was stirred at room temperature for 30 min. 2-(Thieno[3,2-b]thiophen-2-yl)ethanol (29.77 mg, 161.55 μmol) was added dropwise to the reaction flask, and the mixture was stirred at room temperature overnight. After the reaction was completed, the reaction solution was concentrated and purified by silica gel column chromatography to obtain 39.00 mg of a blue solid with a yield of 31.87%. 11H NMR (400 MHz, DMSO-d6) δ 8.33 (t, J = 13.1 Hz, 2H), 7.58 (dd, J = 27.1, 6.3 Hz, 3H), 7.39 (dd, J = 6.9, 4.6 Hz, 4H), 7.33 (d, J = 5.3 Hz, 1H), 7.28–7.18 (m, 3H), 6.57 (s, 1H), 6.28 (d, J = 13.9 Hz, 2H), 4.24 (t, J = 6.4 Hz, 2H), 4.14–3.97 (m, 4H), 3.55 (d, J = 2.2 Hz, 3H), 3.15 (t, J = 6.4 Hz, 2H), 2.30 (q, J = 7.3 Hz, 4H), 1.68 (d, J = 3.3 Hz, 16H), 1.56 (q, J = 7.5 Hz, 4H), 1.36 (s, 4H). +ES-HRMS: C46H55N2O4S2 + calculated value 763.3598, found value 763.3595. -ES-HRMS: F6P - calculated value 144.9647, found value 144.9646.

[0090] Example 4

[0091] Synthesis Example 4

[0092]

[0093] Intermediate 1-3 (100.00 mg, 134.63 μmol), DIPEA (60.90 mg, 471.20 μmol), and HATU (61.43 mg, 161.55 μmol) were successively added to 1 mL of dichloromethane, and the mixture was stirred at room temperature for 30 min. Then, 2-([2,2'-bithiophen]-5-yl)ethanol (33.98 mg, 161.55 μmol) was added dropwise to the reaction flask, and the mixture was stirred at room temperature overnight. After the reaction was completed, the reaction solution was concentrated and purified by silica gel column chromatography to obtain 45.00 mg of a blue solid with a yield of 35.75%. 1HNMR (400 MHz, DMSO-d6) δ 8.33 (t, J = 13.0 Hz, 2H), 7.62 (d, J = 7.4 Hz, 2H), 7.40 (dq, J = 14.4, 7.6, 6.3 Hz, 5H), 7.25 (d, J = 7.2 Hz, 2H), 7.19 (d, J = 3.7 Hz, 1H), 7.09 (d, J = 3.5 Hz, 1H), 7.04–6.97 (m, 1H), 6.85 (d, J = 3.6 Hz, 1H), 6.57 (t, J = 12.3 Hz, 1H), 6.28 (d, J = 13.7 Hz, 2H), 4.21 (t, J = 6.3 Hz, 2H), 4.07 (s, 4H), 3.55 (s, 3H), 3.06 (t, J = 6.4 Hz, 2H), 2.30 (dt, J = 10.7, 7.2 Hz, 4H), 1.67 (s, 16H), 1.58 (q, J = 7.3 Hz, 4H), 1.38 (q, J = 7.9 Hz, 4H). +ES-HRMS: C48H57N2O4S2 + calculated value 789.3754, detected value 789.3748. -ES-HRMS: F6P - calculated value 144.9647, detected value 144.9646.

[0094] Example 5

[0095] Synthesis Example 5

[0096]

[0097] Intermediate 1-3 (100.00 mg, 134.63 μmol), DIPEA (60.90 mg, 471.20 μmol), and HATU (61.43 mg, 161.55 μmol) were successively added to 1 mL of dichloromethane, and the mixture was stirred at room temperature for 30 min. Then, 2-([2,2':5',2'-terthiophen]-5-yl)ethanol (47.24 mg, 161.55 μmol) was added dropwise to the reaction flask, and the mixture was stirred at room temperature overnight. After the reaction was completed, the reaction solution was concentrated and purified by silica gel column chromatography to obtain 52.00 mg of a blue solid with a yield of 37.97%. 11H NMR (400 MHz, DMSO-d6) δ 8.32 (t, J = 13.2 Hz, 2H), 7.61 (dd, J = 7.5, 2.8 Hz, 2H), 7.50 (d, J = 5.1 Hz, 1H), 7.39 (t, J = 6.6 Hz, 4H), 7.25 (dd, J = 9.2, 4.2 Hz, 3H), 7.19 (d, J = 3.8 Hz, 1H), 7.16–7.11 (m, 2H), 7.08 (dd, J = 5.2, 3.6 Hz, 1H), 6.88 (d, J = 3.7 Hz, 1H), 6.56 (t, J = 12.2 Hz, 1H), 6.27 (d, J = 13.7 Hz, 2H), 4.22 (t, J = 6.4 Hz, 2H), 4.07 (s, 4H), 3.55 (d, J = 3.5 Hz, 3H), 3.08 (t, J = 6.4 Hz, 2H), 2.29 (dd, J = 15.4, 7.7 Hz, 4H), 1.67 (t, J = 4.1 Hz, 16H), 1.61–1.53 (m, 4H), 1.37 (s, 4H). +ES-HRMS: C52H59N2O4S3+ calculated 871.3631, found 871.3628. -ES-HRMS: F6P- calculated 144.9647, found 144.9642.

[0098] Comparative Example 1

[0099] Synthesis of Comparative Example 1

[0100]

[0101] Intermediate 1-3 (100.00 mg, 134.63 μmol), DIPEA (60.90 mg, 471.20 μmol), and HATU (61.43 mg, 161.55 μmol) were successively added to 1 mL of dichloromethane, and the mixture was stirred at room temperature for 30 min. Then, 2 mL of methanol was added dropwise to the reaction flask, and the mixture was stirred overnight at room temperature. After the reaction was completed, the reaction solution was concentrated and purified by silica gel column chromatography to obtain 87.00 mg of a blue solid with a yield of 85.39%. 1HNMR(400 MHz, DMSO-d6) δ 8.33 (t, J = 13.0 Hz, 2H), 7.62 (d, J = 7.5 Hz, 2H), 7.40 (d, J = 2.6 Hz, 4H), 7.26 (d, J = 7.8 Hz, 2H), 6.58 (t, J = 12.4 Hz, 1H), 6.30 (d, J = 13.8 Hz, 2H), 4.08 (d, J = 8.1 Hz, 4H), 3.56 (s, 6H), 2.30 (t, J = 7.3 Hz, 4H), 1.68 (s, 16H), 1.57 (q, J = 7.4 Hz, 4H), 1.38 (d, J = 7.1 Hz, 4H). +ES-HRMS: C39H51N2O4+ calculated value 611.3843, detected value 611.3849. -ES-HRMS: F6P- calculated value 144.9647, detected value 144.9641.

[0102] Performance detection test

[0103] After vacuum drying the above-mentioned examples and Comparative Example 1, accurately weigh them with a ten-thousandth balance, prepare a 3 mmol / L DMSO dye mother liquor in a brown sample bottle, and store it in a refrigerator at 4 °C for standby as the dye mother liquor.

[0104] 1. Measure the ultraviolet-visible absorption spectra and fluorescence spectra of Examples 1-5 and Comparative Example 1.

[0105] Test method: When measuring the ultraviolet-visible absorption spectrum and fluorescence spectrum, use a micropipette to measure 4 μL of the dye mother liquor, and dissolve it in a quartz cuvette containing 3 mL of the solvent to be measured, mix well, and obtain a dye concentration of 4.0 μmol / L for the measurement of the absorption spectrum and fluorescence emission spectrum. All tests are completed at 25 °C.

[0106] Figure 1 is the ultraviolet-visible absorption spectrum of Examples 1-5 and Comparative Example 1 in water. From Figure 1 it can be seen that compared with Comparative Example 1, the ultraviolet absorption curves of Examples 1-5 are all broad peaks in the range of 500-700 nm, indicating that assemblies or aggregations are formed by Examples 1-5 in water, which also shows that the above-mentioned examples can form donor-acceptor complexes in water.

[0107] Figure 2 is the normalized fluorescence emission spectrum of Examples 1-5 and Comparative Example 1 in water. From Figure 2 it can be seen that compared with Comparative Example 1, the fluorescence peak positions of Examples 1-5 remain unchanged, but the fluorescence intensity is quenched to varying degrees, and the degree of fluorescence quenching increases with the increase of the donor electron cloud density.

[0108] 2. Test on the ability of generating superoxide anion radicals in water for Examples 1-5 and Comparative Example 1

[0109] Test method: Add 3 mL of aqueous solution into a cuvette, add 1 μL of DMSO solution of DHR123 (3 mmol / L), and then add the dye mother liquors of Examples 1-5 and Comparative Example 1 respectively. Place the cuvette under an LED lamp at 660 nm for illumination, and test the fluorescence spectrum of the solution every 2 s. Excitation wavelength: 495 nm, collection wavelength: 495 - 500 nm. DHR123 itself has no fluorescence, but it will generate strong fluorescence at 525 nm after being oxidized by superoxide anion radicals in aqueous solution. The quantitative detection of superoxide anion radicals can be achieved through the change of fluorescence intensity at this wavelength. The test results are as Figure 3 shown. It can be analyzed that compared with Comparative Example 1, the generation of superoxide anion radicals in Examples 1-5 has increased significantly, and with the enhancement of the electron cloud density of the side-chain modified donor, the generation of superoxide anion radicals also gradually increases. Since the generation of superoxide anion radicals is positively correlated with the electron transfer between the donor and acceptor, enhancing the electron cloud density of the side-chain modification group can enhance the electron transfer between the donor and acceptor and generate more superoxide anion radicals.

[0110] 3. Organelle co-localization experiment of the dye

[0111] Select 4T1 cells as the test cell line. After incubating them in a confocal dish for 24 h, pour out the culture medium, wash three times with PBS buffer solution, add DMEM medium containing 3 μmol / L of Example 5, and then add 2 μmol / L of commercial dyes for different organelles: mitochondrial green fluorescence probe, lysosomal green fluorescence probe, endoplasmic reticulum blue fluorescence probe, and nuclear blue fluorescence probe Hoechst33342. After incubating in a cell culture incubator for 30 min, place it under a confocal microscope for imaging. The detection results are shown in Figure 4 .

[0112] Confocal parameters: 60× oil immersion objective lens. The excitation wavelength of Example 5 is 640 nm, and the collected emission wavelength is 690 - 740 nm; the excitation wavelengths of the mitochondrial green fluorescence probe and the lysosomal green fluorescence probe are both 488 nm, and the collected emission wavelength is 500 - 550 nm; the excitation wavelength of the endoplasmic reticulum blue fluorescence probe and the nuclear blue fluorescence probe Hoechst33342 is 405 nm, and the collected emission wavelength is 440 - 480 nm.

[0113] From Figure 4From the co-localization experiment results graph, it can be seen that in Example 5, it can be clearly localized to the mitochondrial part of the cell. Its co-localization coefficient with the commercial mitochondrial green fluorescent probe is as high as 0.860, proving that the pentamethine cyanine dye with side-chain modification substitution can be localized to the mitochondrial part of the cell, can image the mitochondria of the cell, detect physiological processes, and can also generate reactive oxygen species under light irradiation, successfully killing cells, playing the role of integrated diagnosis and treatment.

[0114] 4. The cytotoxicity of the dye molecule to cells was evaluated by MTT assay

[0115] The test principle is as follows: Succinate dehydrogenase in the mitochondria of living cells can reduce exogenous MTT to water-insoluble blue-violet crystalline formazan and deposit it in the cells, while dead cells do not have this function. Dimethyl sulfoxide can dissolve the formazan in the cells, and its light absorption value is measured at a wavelength of 490 nm with an enzyme-linked immunosorbent assay, which can indirectly reflect the number of living cells.

[0116] Test method: 4T1 cells were seeded in two 96-well plates respectively. After culturing for a period of time, a certain concentration of Example 5 was added to different wells, so that the concentration of Compound 5 was 0 - 30 μmol / L. After incubating for 1.5 h, one of the 96-well plates was placed under an LED lamp at 660 nm for 20 min, and the light density was 25 mW / cm 2 , and then the two 96-well plates were incubated with cells for another 12 h, and the cell viability was detected by MTT experiment. The detection results are shown in Figure 5 .

[0117] The experimental data is as Figure 5 shown. Example 5 showed relatively low dark toxicity to 4T1 cells, with low toxicity within the range of 10 μM. Under normoxia, after 20 min of light irradiation at 660 nm, its phototoxicity to 4T1 cells showed a dose-dependent change. The half-maximal inhibitory concentration (IC 50 ) was 0.15 μM. When the photosensitizer concentration was 1.0 μM, it showed the most obvious killing effect on cancer cells, and the cell survival rate was close to 20%. It shows that Example 5 as a photosensitizer can generate a large amount of reactive oxygen species to kill cells under light irradiation, and has almost no effect on cell viability without light irradiation, and can specifically complete cell killing.

[0118] Combining Examples 1 - 5 and Comparative Example 1 and combining Figure 3 , it can be seen that the pentamethine cyanine dye with side-chain modification has more excellent superoxide anion radical generation ability than the control dye, which indicates that the electron-donating group with side-chain substitution can well carry out electron transfer with the dye; combining Figures 4-5It can be seen that the pentamethine cyanine dye modified with side chains can be well enriched in the mitochondria in cells, has low cytotoxicity in the absence of light, and can generate reactive oxygen species after light irradiation to damage cell mitochondria, thereby killing tumor cells.

[0119] In summary, the pentamethine cyanine dye modified with side chains has excellent photodynamic therapy ability.

[0120] Compared with the prior art, the dye provided by the present invention has creativity in the following aspects: successfully developed a new dye structure and studied its photophysical properties; both the absorption and emission wavelengths are in the red light region, with strong tissue penetration; has high-efficiency electron transfer ability; can generate a large amount of reactive oxygen species in cell mitochondria for photodynamic therapy; has low biological dark toxicity, so it can be better used in practical applications.

[0121] Finally, it should be noted that: the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that: they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements on some or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A side chain modified pentamethine cyanine dye, characterized in that: The dye has the following general formula CY5-R: wherein R1 and R2 are each independently selected from any one of hydrogen, an alkyl group having 1 to 6 carbon atoms, a carboxyalkyl group having 1 to 6 carbon atoms, an arylethyl group, a 2-(thiophen-2-yl)ethyl group, a 2-(5-(methylthio)thiophen-2-yl)ethyl group, a 2-(bithiophene-2-yl)ethyl group, a 2-(thieno[3,2-b]thiophen-2-yl)ethyl group, a 2-([2,2':5',2'-terthiophene]-5-yl)ethyl group, and derivatives thereof; R3 and R4 are substituents at undetermined positions of the benzene ring, each independently selected from any one of hydrogen, halogen, methoxy, amino, nitro, hydroxyl, and carboxyl; Y - Selected from halogen ions, ClO4 - 、BF4 - PF6 - 、CH3COO - CF3COO - or OTs - Any one of .

2. The dye according to claim 1, characterized in that: The emission wavelength of the dye is above 650 nm.

3. The method for preparing a side-chain modified pentamethine cyanine dye according to claim 1, characterized in that: The steps include: ⑴Synthesis of quaternary ammonium salts H-1 and H-2: The benzindole J-1 modified with the R3 substituent is uniformly mixed with 6-bromohexanoic acid, and then a first organic solvent is added to fully react at 40-120° C. to obtain a quaternary ammonium salt H-1; The benzindole J-2 modified with the R4 substituent is mixed evenly with 6-bromohexanoic acid, and then a second organic solvent is added to fully react at 40-120° C. to obtain a quaternary ammonium salt H-2; (2) Synthesis of compound H-3: The quaternary ammonium salts H-1, H-2 and the condensing agent J-1 are uniformly mixed, and then a third organic solvent and an inorganic base are added, and the mixture is fully reacted at 50-150° C., and the solvent is removed to obtain a compound H-3; (3) Synthesis of compound H-4: The compound H-3 is mixed evenly with the corresponding alcohol having an R1 substituent, and then a fourth organic solvent and an organic base I are added, and a catalyst I is added, and the mixture is fully reacted at 25-50° C., and the solvent is removed, and column chromatography is performed to obtain a compound H-4; (4) Synthesis of compound H-5: The compound H-4 is mixed evenly with the corresponding alcohol having an R2 substituent, and then the fifth organic solvent and organic base II are added, and the catalyst II is added, and the mixture is fully reacted at 25-50° C., and the solvent is removed and column chromatography is performed to obtain the target product.

4. The method according to claim 3, characterized in that: In step (1), the molar ratio of the benzindole, 6-bromohexanoic acid, and the first organic solvent or the second organic solvent is 1:1-8:8-20.

5. The method according to claim 3, characterized in that: In step (2), the molar ratio of the quaternary ammonium salt H-1, H-2, the condensing agent J-1, the third organic solvent, and the inorganic base is 1-4:1-4:1:8-20:1-5.

6. The method according to claim 3, characterized in that: In step (3), the molar ratio of compound H-3, the corresponding alcohol having an R1 substituent, catalyst I, organic base I, and the fourth organic solvent is 1:1-4:1-4:1-5:8-20.

7. The method according to claim 3, characterized in that: In step (4), the molar ratio of compound H-4, the corresponding alcohol having an R2 substituent, catalyst II, organic base II, and the fifth organic solvent is 1:1-4:1-4:1-5:8-20.

8. The method according to claim 3, characterized in that: In step (1), the first organic solvent or the second organic solvent is independently selected from at least one of ethanol, acetonitrile, toluene, N,N-dimethylformamide, o-dichlorobenzene, and sulfolane; In the above step (2), the third organic solvent is selected from at least one of ethanol, acetic anhydride, n-butanol, isopropanol, and acetic acid, and the inorganic base is selected from one of potassium carbonate, sodium carbonate, sodium acetate, sodium bicarbonate, cesium carbonate, potassium hydroxide, sodium hydroxide, and sodium tert-butoxide. In the above step (3), the fourth organic solvent is selected from at least one of dichloromethane, N,N-dimethylformamide, 1,2-dichloroethane, acetonitrile, and toluene, the organic base I is selected from one of triethylamine, pyridine, and N,N-diisopropylethylamine, and the catalyst I is selected from one of DCC, EDC-HCl, HATU, HBTU, HCTU, TNTU, and TSTU; In the above step (4), the fifth organic solvent is selected from at least one of dichloromethane, N,N-dimethylformamide, 1,2-dichloroethane, acetonitrile, and toluene, the organic base II is selected from one of triethylamine, pyridine, and N,N-diisopropylethylamine, and the catalyst II is selected from one of DCC, EDC-HCl, HATU, HBTU, HCTU, TNTU, and TSTU.

9. The method according to claim 3, characterized in that: In step (1), the reaction temperature is 60-100°C, and the reaction time is 6-10h; in step (2), the reaction temperature is 60-100°C, and the reaction time is 6-10h; in step (3), the reaction temperature is 25-35°C, and the reaction time is 6-10h; in step (4), the reaction temperature is 25-35°C, and the reaction time is 6-10h.

10. Application of the side-chain modified pentamethine cyanine dye as claimed in claim 1 in the fields of photocatalysis, red light luminescent materials, biomarker probes, preparations for photodynamic therapy, photolysis of water to produce hydrogen, and biosensing.

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