Pentamethine cyanine dyes with side chain modification, synthesis method and application thereof

Abstract

The present application relates to a kind of side chain modified pentamethine cyanine dye and its synthesis method and application.The dye has general formula CY5-R, the dye has strong absorption and emission characteristics above 650nm, suitable for red light excitation, mitochondrial localization and type I photodynamic therapy.Experiments show that the dye and mitochondrial co-localization coefficient reaches 0.860, exhibit excellent deep tissue penetration ability and low dark toxicity, after illumination IC 50 0.15 μM, can efficiently generate superoxide anion free radical, significantly kill cancer cells, realize diagnosis and treatment integration.The synthesis method of the present application includes quaternary ammonium salt synthesis, condensation reaction and side chain modification and the like, with the characteristics of cheap raw material, high yield, simple operation.The dye has wide application in photocatalysis, photodynamic therapy, biomarker and deep tissue imaging and the like, especially suitable for deep tumor fluorescence imaging and photodynamic therapy.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of red fluorescent dye synthesis, and in particular to a kind of side chain modified pentamethine cyanine dye, its synthesis method and application. BACKGROUND

[0002] As a new treatment method, photodynamic therapy is non-invasive, short cycle and does not produce drug resistance, which has been applied to the clinical treatment of some diseases. By using the fluorescence characteristics and electron transfer properties of organic dyes, fluorescence imaging and precise photodynamic therapy can be performed on diseased tissues, so as to achieve the purpose of diagnosis and treatment integration of diseases. Therefore, researchers have developed many organic dyes as photosensitizers for diagnosis and treatment of diseases. Currently, the most common ones are BODIPY dyes and ruthenium complexes, but these dyes generally have short absorption and emission wavelengths, and do not have organelle targeting effect, making it difficult to achieve accurate diagnosis and treatment of deep diseases in the body. Due to the severe hypoxic microenvironment in solid tumors, the use of photosensitizers is limited. In response to the problem of treating hypoxic tumors, type I photodynamic therapy, which generates superoxide anion radicals or hydroxyl radicals as the main active oxygen, shows better advantages and application potential. However, most of the existing clinical photosensitizers are type II photosensitization mode dominated by generating singlet oxygen as the main active oxygen, and type I photosensitizers are relatively scarce.

[0003] As a classic cyanine dye system, pentamethine cyanine dye has been deeply studied in terms of photophysical properties and application. This series of compounds exhibits characteristic absorption and emission spectra in the red light region (600-750 nm), with high molar extinction coefficient (ε>10^5 L·mol-1·cm-1) and excellent fluorescence quantum yield (Φ f ≈0.2-0.5), which has been successfully applied in the field of biological fluorescence labeling and tissue staining. However, after excitation, the dye mainly releases energy through non-radiative transition and fluorescence emission pathway, resulting in low intersystem crossing efficiency, which makes it difficult to achieve the photosensitization electron transfer process required for effective photodynamic therapy. The existing modification strategy is mainly based on 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 (LogP value increases by 2-3 units), which significantly affects its water solubility (<0.1 mg / mL); ② the cytotoxicity is significantly enhanced (IC 50 value decreases by about 2 orders of magnitude); ③ the introduction of complex substituents prolongs the synthesis route (an average of 3-5 additional reactions), and the isomer separation efficiency decreases (HPLC purity <85%). The above defects seriously restrict the biomedical conversion potential of the dye.

[0004] Therefore, in view of the inherent defect that the efficiency of singlet oxygen generation of the current type II photosensitizer is suddenly reduced in the hypoxic microenvironment of tumors, it is urgent to develop a new pentamethine type I photosensitizer through molecular engineering strategy. On the basis of maintaining its inherent red light (650-700 nm) absorption and emission characteristics, a photosensitizer system with controllable electron transfer path is constructed, so that it can generate long-lived reactive oxygen species such as superoxide anion radicals and hydroxyl radicals through electron transfer mechanism, thereby breaking through the limitation of tumor microenvironment on the traditional photodynamic effect. SUMMARY

[0005] In view of the problem that the existing pentamethine cyanine dye cannot generate superoxide anion radicals through efficient electron transfer in the prior art, a first object of the present application is to provide a synthesis method and application of a side chain modified pentamethine cyanine dye, wherein the pentamethine cyanine dye has red light excitation, mitochondrial localization and good type I photodynamic effect.

[0006] A second object of the present application is to provide a synthesis method of a side chain modified pentamethine cyanine dye, wherein the preparation method has the advantages of simple synthesis method, low cost of raw materials, high yield and high universality.

[0007] A third object of the present application is to provide an application of a side chain modified pentamethine cyanine dye, wherein the side chain modified pentamethine cyanine dye can be used as a dye to initiate chemical reactions in the field of photocatalysis and generate reactive oxygen in the field of photodynamic.

[0008] To achieve the above-mentioned first object, the present application provides the following technical scheme: a side chain modified pentamethine cyanine dye comprising the following general formula CY5-R:

[0009]

[0010] wherein R1 and R2 are each independently selected from any one of hydrogen, an alkyl group with 1-6 carbons, a carboxyalkyl group with 1-6 carbons, an arylethyl group, a 2-(thiophene-2-yl)ethyl group, a 2-(5-(methylthio)thiophene-2-yl)ethyl group, a 2-(bithiophene-2-yl)ethyl group, a 2-(thieno[3,2-b]thiophene-2-yl)ethyl group, a 2-([2,2':5',2'-terthiophene]-5-yl)ethyl group and derivatives thereof, and preferably R1 and R2 are selected from any one of an arylethyl group, a 2-(thiophene-2-yl)ethyl group, a 2-(5-(methylthio)thiophene-2-yl)ethyl group, a 2-(bithiophene-2-yl)ethyl group, a 2-(thieno[3,2-b]thiophene-2-yl)ethyl group, a 2-([2,2':5',2'-terthiophene]-5-yl)ethyl group and derivatives thereof;

[0011] R3 and R4 are substituents at uncertain positions of the benzene ring, and each is independently selected from any one of hydrogen, halogen, methoxy, amino, nitro, hydroxyl, and carboxyl; preferably, R3 and R4 are at the 5th substituting position of the benzene ring, and each is independently selected from any one of hydrogen, halogen, methoxy, and nitro.

[0012] Y- is selected from any one of inorganic negative ions or organic negative ions: halogen ions, CIO4 - , BF4 - , PF6 - , CH3COO-, CF3COO - , or OTs - ; preferably, Y- is selected from any one of halogen ions, CIO4 - , and PF6 - .

[0013] Further, the side chain modified pentamethine cyanine dye has an emission wavelength of 650 nm or more. The emission wavelength used in Examples 1-5 is 650-720 nm.

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

[0015] To achieve the second object, the application provides the following technical scheme: a kind of side chain modified pentamethine cyanine dye, comprising:

[0016]

[0017] (1) synthesis of quaternary ammonium salt H-1 and H-2:

[0018] The benzindole J-1 with R3 substituent modification is uniformly mixed with 6-bromohexanoic acid, then a first organic solvent is added, and the reaction is carried out at 40-120℃ for 4-12h to obtain quaternary ammonium salt H-1;

[0019] The benzindole J-2 with R4 substituent modification is uniformly mixed with 6-bromohexanoic acid, then a second organic solvent is added, and the reaction is carried out at 40-120℃ for 4-12h to obtain quaternary ammonium salt H-2;

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

[0021] The quaternary ammonium salt H-1 and H-2 and the condensing agent J-3 are uniformly mixed, then a third organic solvent and an inorganic base are added, and the reaction is carried out at 50-150℃ for 4-12h, and the solvent is removed to obtain the target product.

[0022] 5. Synthesis of compound H-5:

[0023] Mixing compound H-3 with the corresponding alcohol with R1substituent uniformly, then adding the fourth organic solvent and organic base I, adding catalyst I, reacting at 25-50℃ for 4-12h, removing the solvent, column chromatography to obtain the target product.

[0024] 5. Synthesis of compound H-5:

[0025] Mixing compound H-4 with the corresponding alcohol with R2substituent uniformly, then adding the fifth organic solvent and organic base II, adding catalyst II, reacting at 25-50℃ for 4-12h, removing the solvent, column chromatography to obtain the target product.

[0026] For the technical solutions 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 solutions described above, further preferably, in the above step 1, the molar ratio of the benzindole, 6-bromohexanoic acid, the first or second organic solvent is 1:1-8:8-20, and more preferably the ratio is 1:1-3:8-12; and the most preferred molar ratio is 1:2:10.

[0028] For the technical solutions described above, further preferably, in the above step 1, the reaction temperature is 60-100℃, and the reaction time is 6-10h.

[0029] For the technical solutions 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, isopropyl alcohol, 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 solutions described above, further preferably, in step 2, the molar ratio of the quaternary ammonium salt H-1, H-2, the condensing agent J-3, the third organic solvent, and the inorganic base is 1-4:1-4:1:8-20:1-5, and more preferably the ratio is 1-2:1-2:1:8-15:1-3; and the most preferred molar ratio is 1:1:1:10:2.

[0031] For the technical solutions described above, further preferably, in step 2, the reaction temperature is 60-100℃, and the reaction time is 6-10h.

[0032] For the technical solutions described above, further preferably, in the step ⑥, the fourth organic solvent is selected from at least one of dichloromethane, N,N-dimethylformamide, 1,2-dichloroethane, acetonitrile, toluene, the organic base I is selected from one of triethylamine, pyridine, N,N-diisopropyl ethylamine, and the catalyst I is selected from one of DCC, EDC-HCl, HATU, HBTU, HCTU, TNTU, and TSTU;

[0033] For the technical solutions described above, further preferably, in the step ⑥, the molar ratio of the compound H-3, the corresponding alcohol with the R1 substituent, the catalyst I, the 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, further preferably, in the step ⑥, the molar ratio is 1:1-2:2-3:4-5:8-12; and the most preferred molar ratio is 1:1:2.5:3:10;

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

[0036] For the technical solutions described above, further preferably, in the step ⑷, the fifth organic solvent is selected from at least one of dichloromethane, N,N-dimethylformamide, 1,2-dichloroethane, acetonitrile, toluene, the organic base II is selected from one of triethylamine, pyridine, N,N-diisopropyl ethylamine, and the catalyst II is selected from one of DCC, EDC-HCl, HATU, HBTU, HCTU, TNTU, and TSTU;

[0037] For the technical solutions described above, further preferably, in the step ⑷, the molar ratio of the compound H-4, the corresponding alcohol with the R2 substituent, the catalyst II, the organic base II, and the fifth organic solvent is 1:1-4:1-4:1-5:8-20, and the further preferred molar ratio is 1:1-2:1-3:2-3:8-12; and the most preferred molar ratio is 1:1.5:1.5:3:10;

[0038] For the technical solutions described above, further preferably, in the step ⑷, the reaction temperature is 25-35°C, and the reaction time is 6-10h.

[0039] To achieve the above-mentioned third object, the side chain modified pentamethine cyanine dye has excellent red light excitation characteristics, high efficient electron transfer ability and low dark toxicity, which shows great potential in the fields of biomedicine and energy.

[0040] Further, the side chain modified pentamethine cyanine can be used as a photosensitizer to initiate chemical reactions in the field of photocatalysis, generate reactive oxygen species in the field of photodynamic therapy, and produce phosphorescent signals in the field of phosphorescent materials.

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

[0042] Further, the side chain modified pentamethine cyanine molecule can quickly and efficiently pass through the cell membrane, enter the cell and enrich in the subcellular organelles, and generate reactive oxygen species by low light density red light irradiation, destroy the homeostatic balance in the cell, and kill cancer cells at a high efficiency at a low level of nanomolar, which can be applied in the field of photodynamic therapy, and has low dark toxicity and high selectivity, and can realize the integration of diagnosis and treatment.

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

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

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

[0046] Third, the side chain modified pentamethine cyanine dye described in the present application can quickly pass through the cell membrane, is enriched in the cell mitochondria, can detect physiological processes by using changes in the cell mitochondria, and can achieve targeted imaging and treatment of organelles. Through confocal microscope imaging, the colocalization coefficient of the dye of the present application and the mitochondrial green fluorescent probe is as high as 0.860, indicating that it can be located in the cell mitochondria. The dark toxicity of Example 5 to 4T1 cells under lightless conditions is low, and the cell survival rate is higher than 90%; while after 660 nm light irradiation for 20 min, its phototoxicity shows a dose-dependent change, and the half inhibitory rate (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 diagnosis and treatment integration.

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

[0048] Fifth, the side chain modified pentamethine cyanine dye described in the present application has strong absorption and emission properties above 650 nm, can penetrate deep tissues, and is suitable for fluorescence imaging and photodynamic therapy of deep tumors. Through ultraviolet-visible absorption spectrum and fluorescence emission spectrum tests, the dye of the present application shows strong absorption and emission properties in the range of 650-720 nm. This red light excitation property enables it to have 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 application includes steps of 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 yield of each intermediate and target product is higher than 45%, and the highest can reach 90.25%. This efficient synthesis method provides technical support for large-scale production and application of the dye. BRIEF DESCRIPTION OF DRAWINGS

[0050] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed to be used in the embodiment or prior art description will be briefly introduced below. Obviously, the drawings in the following description are some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without creative labor.

[0051] Figure 1UV-Vis absorption spectra of the present disclosed embodiments 1-5, comparative example 1 in dichloromethane;

[0052] Figure 2 Fluorescence emission spectra of the present disclosed embodiments 1-5, comparative example 1 in dichloromethane;

[0053] Figure 3 SOD generation effect of the present disclosed embodiments 1-5, comparative example 1 in water;

[0054] Figure 4 Cell organelle co-localization single-photon microscopy picture of the present disclosed embodiment 5;

[0055] Figure 5 MTT experiment picture of the present disclosed embodiment 5. DETAILED DESCRIPTION

[0056] In order to make the objects, technical solutions and advantages of the embodiments of the present application clearer, the following will combine the accompanying drawings of the embodiments of the present application to clearly and completely describe the technical solutions of the embodiments of the present application. Obviously, the described embodiments are some but not all of the embodiments of the present application. Based on the embodiments of the present application, all other embodiments obtained by those of ordinary skill in the art without creative work fall within the protection scope of the present application. Figures 1-5 The technical solutions of the embodiments of the present application are clearly and completely described. Obviously, the described embodiments are some but not all of the embodiments of the present application. Based on the embodiments of the present application, all other embodiments obtained by those of ordinary skill in the art without creative work fall within the protection scope of the present application.

[0057] Unless otherwise defined, the terms used in the present application have the following meanings.

[0058] The term "halogen" used in the present application includes fluorine, chlorine, bromine and iodine.

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

[0060] Y - represents a negative ion, which can be any suitable negative ion, including inorganic negative ions and organic negative ions, which can be exemplified but not limited to halogen ions, CIO4 - , PF6 - , BF4 - , CH3COO - , CF3COO - or OTs - .

[0061] Instruments and equipment used in the embodiments:

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

[0063] The dye absorption and emission spectra were measured with an Agilent Cary 60 UV-Vis spectrophotometer and a Cary Eclipse fluorescence spectrophotometer.

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

[0065] The cytotoxicity test was measured with a Varioskan LUX Multimode Microplate Reader instrument from Thermofisher. DETAILED EMBODIMENTS

[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] Put 2,3,3-trimethylindole (5.00 g, 31.40 mmol), 6-bromohexanoic acid (12.25 g, 62.80 mmol) into a reaction bottle, add 50 mL of acetonitrile, warm up to 85°C, and stir for 12 h. After the reaction is completed, return to room temperature, add 20 mL of dichloromethane to the reaction bottle, and stir until the solution is clear. Add the mixture dropwise to 500 mL of ether, and filter to obtain a solid. Dry the solid to obtain 8.92 g, with a yield of 80.18%, which can be used in the next reaction without purification.

[0073] Synthesis of intermediate 1-2

[0074]

[0075] Put 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) into 20 mL of acetic anhydride, warm up to 50°C, and stir for 12 h. After the reaction is completed, return to room temperature. Add the reaction solution dropwise to 500 mL of ether, and filter to obtain a blue solid. Dry the solid to obtain 2.33 g, with a yield of 74.63%, which can be used in the next reaction without purification.

[0076] Synthesis of intermediate 1-3

[0077]

[0078] Into intermediate 1-2 (2.00 g, 3.01 mmol), DIPEA (1.36 g, 10.55 mmol), HATU (2.85 g, 7.52 mmol) in 20 mL of dichloromethane, was added methanol (96.56 mg, 3.01 mmol) dropwise, and stirred at room temperature for 30 min. The reaction bottle 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 2.02 g of a blue solid, with a yield of 90.25%.

[0079] Synthesis Example 1

[0080]

[0081] Into intermediate 1-3 (100.00 mg, 134.63 μmol), DIPEA (60.90 mg, 471.20 μmol), HATU (61.43 mg, 161.55 μmol) in 1 mL of dichloromethane, was added 2-thiophene ethanol (20.71 mg, 161.55 μmol) dropwise, and stirred at room temperature for 30 min. The reaction bottle 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 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: calcd for C44H55N2O4S+707.3877, found 707.3885. -ES-HRMS: calcd for F6P-144.9647, found 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), HATU (61.43 mg, 161.55 μmol) in 1 mL of dichloromethane were added successively, stirred at room temperature for 30 min, 2-(5-(methylthio)thiophen-2-yl)ethanol (23.56 mg, 135.17 μmol) was added dropwise into the reaction flask, stirred at room temperature overnight. After the reaction was completed, the reaction solution was concentrated, purified by silica gel column chromatography to give blue solid 56.00 mg in 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 + calc'd], 753.3754, found, 753.3749. -ES-HRMS: [F6P- calc'd], 144.9647, found, 144.9645.

[0086] Example 3

[0087] Synthesis of Example 3

[0088]

[0089] Intermediate 1-3 (100.00 mg, 134.63 μmol), DIPEA (60.90 mg, 471.20 μmol), HATU (61.43 mg, 161.55 μmol) in 1 mL of dichloromethane were added successively, 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 into the reaction flask, stirred at room temperature overnight. After the reaction was completed, the reaction solution was concentrated, purified by silica gel column chromatography to give blue solid 39.00 mg in yield of 31.87%. 1H 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: calcd for C46H55N2O4S2+ 763.3598, found 763.3595. -ES-HRMS: calcd for F6P- 144.9647, found 144.9646.

[0090] Example 4

[0091] Synthesis of Example 4

[0092]

[0093] To a solution of Intermediate 1-3 (100.00 mg, 134.63 μmol), DIPEA (60.90 mg, 471.20 μmol), HATU (61.43 mg, 161.55 μmol) in 1 mL of dichloromethane, stirred at room temperature for 30 min, then 2-([2,2'-bithiophen]-5-yl)ethanol (33.98 mg, 161.55 μmol) was added dropwise into the reaction vial, stirred at room temperature overnight. After the reaction was completed, the reaction solution was concentrated and purified by silica gel column chromatography to give blue solid 45.00 mg in 35.75% yield. 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: calcd for C48H57N2O4S2+789.3754, found 789.3748. -ES-HRMS: calcd for F6P- 144.9647, found 144.9646.

[0094] Example 5

[0095] Synthesis of Example 5

[0096]

[0097] To a solution of Intermediate 1-3 (100.00 mg, 134.63 μmol), DIPEA (60.90 mg, 471.20 μmol), HATU (61.43 mg, 161.55 μmol) in 1 mL of dichloromethane, was added 2-([2,2':5',2'-terthienyl]-5-yl)ethanol (47.24 mg, 161.55 μmol) dropwise. The reaction mixture was stirred at room temperature for 30 min, then stirred at room temperature overnight. After the reaction was completed, the reaction mixture was concentrated and purified by silica gel column chromatography to give blue solid 52.00 mg in 37.97% yield. 1H 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: calcd for C52H59N2O4S3+ 871.3631, found 871.3628. -ES-HRMS: calcd for F6P- 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), HATU (61.43 mg, 161.55 μmol) in 1 mL of dichloromethane were added successively, stirred at room temperature for 30 min, 2 mL of methanol was added dropwise into the reaction bottle, stirred at room temperature overnight. After the reaction was completed, the reaction solution was concentrated, purified by silica gel column chromatography to give 87.00 mg of blue solid, with a yield of 85.39%. 1H 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 = 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: calcd for C39H51N2O4+ 611.3843, found 611.3849. -ES-HRMS: calcd for F6P- 144.9647, found 144.9641.

[0102] Performance test

[0103] The above examples and Comparative Example 1 were vacuum dried and accurately weighed using a millionth scale, and 3 mmol / L of a DMSO dye stock solution was prepared in a brown sample bottle and stored in a 4°C refrigerator as a dye stock solution for standby use.

[0104] 1. UV-Vis absorption spectrum and fluorescence spectrum of Examples 1-5 and Comparative Example 1 were determined.

[0105] Test method: When testing the UV-Vis absorption spectrum and fluorescence spectrum, 4 μL of the dye stock solution was taken using a micropipette gun and dissolved in a quartz cuvette containing 3 mL of the solvent to be tested, mixed uniformly to obtain a dye concentration of 4.0 μmol / L for absorption spectrum and fluorescence emission spectrum testing. All tests were completed at 25°C.

[0106] Figure 1 The UV-Vis absorption spectrum of Examples 1-5 and Comparative Example 1 in water was determined by Figure 1 It can be seen that, compared with Comparative Example 1, the UV absorption curves of Examples 1-5 in the range of 500-700 nm are all wide peaks, indicating that Examples 1-5 form assemblies or aggregates in water, which also shows that the above examples can all form donor-acceptor complexes in water.

[0107] Figure 2 The normalized fluorescence emission spectrum of Examples 1-5 and Comparative Example 1 in water was determined by Figure 2 It can be seen that, compared with Comparative Example 1, the fluorescence peak position of Examples 1-5 remains unchanged, but the fluorescence intensity appears different degrees of quenching, and the fluorescence quenching degree increases with the increase of the electron cloud density of the donor.

[0108] 2. Superoxide anion radical production ability test of Examples 1-5 and Comparative Example 1 in water

[0109] Test method: 3 mL of aqueous solution was added to a cuvette, 1 μΐ of DHR123 (3 mmol / L) DMSO solution was added, and then dye stock solution of Examples 1-5 and Comparative Example 1 was added, respectively. The cuvette was irradiated under a 660 nm LED lamp, and the fluorescence spectrum of the solution was tested every 2 s, the excitation wavelength was 495 nm, and the collection wavelength was 495-500 nm. DHR123 itself has no fluorescence, but when it is oxidized by superoxide anion radicals in an aqueous solution, it produces strong fluorescence at 525 nm, and the change in fluorescence intensity at this wavelength can be used to quantitatively detect superoxide anion radicals. The test results are shown in Figure 3 As can be seen from the analysis, compared with Comparative Example 1, the production of superoxide anion radicals of Examples 1-5 is obviously increased, and as the electron cloud density of the side chain modified donor increases, the production of superoxide anion radicals also gradually increases. Since the production of superoxide anion radicals is positively correlated with the electron transfer between the donor and the acceptor, increasing the electron cloud density of the side chain modified group can enhance the electron transfer between the donor and the acceptor, and produce more superoxide anion radicals.

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

[0111] 4T1 cells were selected as the test cell line, which were incubated in a confocal culture dish for 24 h, the culture medium was poured out, and the cells were washed with PBS buffer solution for three times, then 3 μmol / L of Example 5 was added to the DMEM culture medium, and then 2 μmol / L of different organelle commercial dyes were added, namely mitochondrial green fluorescent probe, lysosome green fluorescent probe, endoplasmic reticulum blue fluorescent probe and nuclear blue fluorescent probe Hoechst 33342, and then the cells were incubated in a cell incubator for 30 min and then imaged under a confocal microscope. The detection results are shown in Figure 4 .

[0112] Confocal parameters: 60x oil lens, the excitation wavelength of Example 5 was 640 nm, and the collection emission wavelength was 690-740 nm; the excitation wavelength of the mitochondrial green fluorescent probe and the lysosome green fluorescent probe was 488 nm, and the collection emission wavelength was 500-550 nm; the excitation wavelength of the endoplasmic reticulum blue fluorescent probe and the nuclear blue fluorescent probe Hoechst 33342 was 405 nm, and the collection emission wavelength was 440-480 nm.

[0113] From Figure 4From the co-localization experiment results, it can be seen that Example 5 can be obviously located in the mitochondria of cells, and the co-localization coefficient with the commercial mitochondrial green fluorescent probe is as high as 0.860, which proves that the side chain modified pentamethine cyanine dye can be located in the mitochondria of cells, can image the mitochondria of cells, detect physiological processes, and also produce reactive oxygen under light, successfully killing cells, and play a role of diagnosis and treatment integration.

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

[0115] The test principle is that succinate dehydrogenase in the mitochondria of living cells can reduce exogenous MTT to water-insoluble blue-violet crystalline formazan and deposit in the cells, while dead cells do not have this function. Dimethyl sulfoxide can dissolve formazan in cells, and its light absorption value is measured at 490 nm wavelength by an enzyme-labeled instrument, which can indirectly reflect the number of living cells.

[0116] Test method: 4T1 cells were inoculated in two 96-well plates, respectively, and after a period of culture, a certain concentration of Example 5 was added to different wells, so that the concentration of the dye 5 was 0-30 μmol / L, and after 1.5 h of incubation, one of the 96-well plates was placed under a 660 nm LED lamp for irradiation for 20 min, and the light density was 25 mW / cm 2 Then, the two 96-well plates were further incubated for 12 h, and the cell activity was detected by MTT experiment, and the detection results are shown in Figure 5 .

[0117] The experimental data are shown in Figure 5 , Example 5 shows a small dark toxicity to 4T1 cells, and has low toxicity in the range of 10 μM. Under normal oxygen, 660 nm light irradiation for 20 min, the phototoxicity of Example 5 to 4T1 cells shows a dose-dependent change, and the half inhibitory rate (IC 50 ) is 0.15 μM. When the photosensitizer concentration is 1.0 μM, it shows the most obvious killing effect on cancer cells, and the cell survival rate is close to 20%. It is proved that Example 5 as a photosensitizer can generate a large amount of reactive oxygen under light to kill cells, and has almost no effect on cell activity without light, and can complete the directional killing of cells.

[0118] In combination with Examples 1-5 and Comparative Example 1 and in combination with Figure 3 , it can be seen that the side chain modified pentamethine cyanine dye has a more excellent superoxide anion radical generation capacity than the control dye, which shows that the side chain substituted electron-donating group can well transfer electrons with the dye; in combination with Figures 4-5It can be seen that the side chain modified pentamethine cyanine dye can be well enriched in mitochondria in cells, has low cytotoxicity in the absence of light, and can produce reactive oxygen to destroy cell mitochondria after light, thereby killing tumor cells.

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

[0120] Compared with the prior art, the dye provided by the present application has the following aspects: a new dye structure is successfully developed and its photophysical properties are studied; the absorption and emission wavelengths are both in the red light region, and the tissue penetration is strong; it has high efficient electron transfer ability; a large amount of reactive oxygen can be generated in cell mitochondria for photodynamic therapy; and it has low biological dark toxicity, so that 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 application, and not to limit them; although the present application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that: it can still modify the technical solutions recorded in the foregoing embodiments, or make equivalent replacement for part 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 application.

Claims

1. A class of side chain modified pentamethine cyanine dyes characterized in that, The dye has the following general formula CY5-R: wherein R1 is selected from any one of alkyl of 1-6 carbons; R2 is selected from 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; R3 and R4 are both hydrogen; Y - selected from the group consisting of halogen ions, CIO4 - , BF4 - , PF6 - , CH3COO - , CF3COO - or OTs - .

2. The dye of claim 1, wherein: The dye has an emission wavelength of 650 nm or above.

3. A process for the preparation of a side chain modified pentamethine cyanine dye of the class of claim 1, characterized in that, The method comprises the following steps: (1) synthesis of quaternary ammonium salt H-1, H-2: mixing the benzindole J-1 with R3 substituent modification and 6-bromohexanoic acid uniformly, then adding a first organic solvent, and reacting sufficiently at 40-120℃ to obtain quaternary ammonium salt H-1; mixing the benzindole J-2 with R4 substituent modification and 6-bromohexanoic acid uniformly, then adding a second organic solvent, and reacting sufficiently at 40-120℃ to obtain quaternary ammonium salt H-2; (2) synthesis of compound H-3: mixing quaternary ammonium salt H-1, H-2 and condensing agent J-3 uniformly, then adding a third organic solvent and inorganic base, and reacting sufficiently at 50-150℃, and removing the solvent to obtain compound H-3; (3) synthesis of compound H-4: mixing compound H-3 and the corresponding alcohol with R1 substituent uniformly, then adding a fourth organic solvent and organic base I, adding catalyst I, and reacting sufficiently at 25-50℃, removing the solvent, and column chromatography to obtain compound H-4; catalyst I is selected from one of DCC, EDC-HCl, HATU, HBTU, HCTU, TNTU, TSTU; (4) synthesis of compound H-5: mixing compound H-4 and the corresponding alcohol with R2 substituent uniformly, then adding a fifth organic solvent and organic base II, adding catalyst II, and reacting sufficiently at 25-50℃, removing the solvent, and column chromatography to obtain the target product; catalyst II is selected from one of DCC, EDC-HCl, HATU, HBTU, HCTU, TNTU, TSTU.

4. The method of claim 3, wherein: In step (1), the molar ratio of the indole, 6-bromohexanoic acid, the first organic solvent or the second organic solvent is 1:1-8:8-20.

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

6. The method of claim 3, wherein: In step (3), the molar ratio of compound H-2, the corresponding alcohol with R1 substituent, catalyst I, organic base I, the fourth organic solvent is 1:1-4:1-4:1-5:8-20.

7. The method of claim 3, wherein: In step (4), the molar ratio of compound H-3, the corresponding alcohol with R2 substituent, catalyst II, organic base II, the fifth organic solvent is 1:1-4:1-4:1-5:8-20.

8. The method of claim 3, wherein: 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, sulfolane; In the step 2 above, the third organic solvent is selected from at least one of ethanol, acetic anhydride, n-butanol, isopropyl alcohol, 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 step 4 above, the fourth organic solvent is selected from at least one of dichloromethane, N,N-dimethylformamide, 1,2-dichloroethane, acetonitrile, and toluene, and the organic base I is selected from one of triethylamine, pyridine, and N,N-diisopropylethylamine. In the step 5 above, the fifth organic solvent is selected from at least one of dichloromethane, N,N-dimethylformamide, 1,2-dichloroethane, acetonitrile, and toluene, and the organic base II is selected from one of triethylamine, pyridine, and N,N-diisopropylethylamine.

9. The method of claim 3, wherein: In the step 1 above, the reaction temperature is 60-100℃, and the reaction time is 6-10h; in the step 2 above, the reaction temperature is 60-100℃, and the reaction time is 6-10h; in the step 3 above, the reaction temperature is 25-35℃, and the reaction time is 6-10h; and in the step 4 above, the reaction temperature is 25-35℃, and the reaction time is 6-10h.

10. Use of the side chain modified pentamethine cyanine dye according to claim 1 in the fields of photocatalysis, red light emitting material, biological marker probe preparation, preparation for photodynamic therapy for non-disease diagnosis and treatment purposes, preparation for hydrogen production by water photolysis, and preparation for biosensing.

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