Multi-cyano offset substituted pentamethine cyanine dye as well as synthesis method and application thereof

Abstract

The invention discloses a multi-cyano offset substituted pentamethine cyanine dye and a synthesis method and application thereof, and the dye has the following general formula structure: # imgabs0. According to the multi-cyano offset substituted pentamethine cyanine dye disclosed by the invention, the absorption emission wavelength is increased by introducing an offset substitution structure; the dye has near-infrared first-area imaging and treatment performance, so that the optimal excitation and emission wavelength can be flexibly selected, the dye shows obvious Stokes shift, reduction of self-absorption and scattering effects is facilitated, the imaging definition and depth are improved, the molar extinction coefficient of the prepared dye is up to 190000, and the dye can be applied to near-infrared first-area imaging and treatment of the near-infrared first-area imaging and treatment of the near-infrared first-area imaging and treatment of the near-infrared first-area imaging. According to the present invention, with the application of the dye, the light energy utilization degree of the dye is substantially improved, the long wavelength light with the deeper penetration depth can be strongly absorbed, the light energy capture and conversion can be effectively achieved, the photosensitizer efficiency can be improved, the light dose can be reduced, the phototoxicity can be reduced, the high absolute fluorescence quantum yield can be represented, and the biological imaging effect can be substantially improved; and the imaging quality is higher.

Description

Technical Field

[0001] The present invention relates to the technical field of cyanine dyes, and particularly relates to a polycyano-offset-substituted pentamethine cyanine dye, a synthesis method thereof, and an application thereof. Background Art

[0002] The cyanine dye structure is the most commonly used fluorophore in cell, tissue, and whole organism imaging, and is often used to label biological macromolecules or specific cell structures. In the aspect of optical therapy, the cyanine structure can also be used in the research of photodynamic or photothermal therapy. Utilizing the advantages of multiple modification sites of cyanine dyes, it is convenient to modify and connect functional modules such as targeting groups, antibodies, or biomarkers. In addition, cyanine dyes have good fluorescence characteristics and adjustable energy dissipation pathways, etc., and can achieve the integrated diagnosis and treatment of real-time imaging while meeting better treatment effects.

[0003] As a typical cyanine dye, pentamethine dyes have been widely studied. Such dyes have absorption and emission in the near-infrared band and have been widely used in the fields of fluorescence imaging, staining, etc.

[0004] However, the currently available pentamethine cyanine dyes have short absorption and emission wavelengths, often unable to achieve deep treatment effects, have high cytotoxicity, and the energy dissipation pathways cannot be effectively utilized, which limits the further efficient application of pentamethine cyanine dyes.

[0005] Therefore, it is necessary to develop pentamethine cyanine dyes separately, design and adjust their parent bodies to synthesize dye structures with high performance. Summary of the Invention

[0006] The present invention provides a polycyano-offset-substituted pentamethine cyanine dye, a synthesis method thereof, and an application thereof to solve the above problems.

[0007] To achieve the above object, the technical solution of the present invention is:

[0008] A polycyano-offset-substituted pentamethine cyanine dye, the dye having the following general formula structure:

[0009]

[0010] Wherein,

[0011] R 1 is selected from one of O, S, Se, NH, NCH 3 ;

[0012] R 2 and R 3Each independently selected from one of hydrogen, halogen, methyl, hydroxy, methoxy, phenyl, trifluoromethyl, alkoxy having 1 - 18 carbons, carboxyalkyl having 1 - 18 carbons, alkylsulfonic group having 1 - 18 carbons, ester group having 1 - 18 carbons, amide group having 1 - 18 carbons;

[0013] R 4 Selected from any one of hydrogen, halogen, cyano, methoxy, amino, nitro, hydroxy, carboxy, N,N - dimethylamino, N,N - diethylamino, trifluoromethyl, ester group, amide group, sulfonate or sulfonate salt;

[0014] R 5 Selected from substituents at an indeterminate position on the benzene ring, selected from any one of hydrogen, halogen, carboxyalkyl having 1 - 18 carbons, hydroxyalkyl having 1 - 18 carbons, alkylsulfonic group having 1 - 18 carbons, ester group having 1 - 18 carbons or amide group having 1 - 18 carbons;

[0015] Y is selected from inorganic cations or organic cations.

[0016] 2. The polycyano - meta - substituted pentamethine cyanine dye according to claim 1, wherein R 1 Selected from one of O, NH;

[0017] R 2 and R 3 Each independently selected from one of methyl, phenyl, trifluoromethyl, alkoxy having 1 - 8 carbons;

[0018] R 4 Selected from one of hydrogen, nitro, cyano, amino, trifluoromethyl, methoxy;

[0019] Y is selected from one of Na, K, piperidine, pyridine, triphenylphosphine.

[0020] The synthesis method of the polycyano - meta - substituted pentamethine cyanine dye described above, comprising the following steps:

[0021] (1) In an organic solvent, add the compound of formula S1 and malononitrile, stir, then add an appropriate amount of basic catalyst, and reflux for 1 - 3 h, then cool, filter, wash and dry to obtain the intermediate S3, or,

[0022] Add the compound of formula S2 and 2% NaOH aqueous solution to the malononitrile dimer, then dissolve it in an organic solution, stir at room temperature for 2 h, then cool to 0 °C, filter, wash and dry to obtain the intermediate S3;

[0023] (2) At - 10 - 20 °C, drop phosphorus oxychloride into DMF and mix evenly, then add the compound of formula S4, N 2Stir at 30 - 80 °C for 6 - 24 h under protection, then cool to room temperature, add ice water to quench the reaction, and add inorganic salts to obtain intermediate S5;

[0024] (3) Dissolve intermediate S3 and intermediate S5 in an organic solvent, add a basic catalyst, and reflux under N 2 protection for 4 h. After the reaction is completed, concentrate and purify by silica gel column to obtain the polycyano meta-substituted pentamethine cyanine dye.

[0025]

[0026] Further, the molar ratio of the compound of formula S1 to malononitrile is 1:2.

[0027] Further, the molar ratio of the compound of formula S2 to malononitrile dimer is 1:1.

[0028] Further, in steps (1) and (3), the organic solvent is selected from one of toluene, ethanol, methanol, isopropanol, acetonitrile, and tetrahydrofuran; the base is selected from one of sodium hydroxide, potassium hydroxide, ammonia water, sodium bicarbonate, sodium ethoxide, sodium methoxide, pyridine, piperidine, triethylamine, trimethylamine, and diisopropylethylamine.

[0029] Further, in step (2), the inorganic salt is selected from one of NaCl, CH 3 COONa, CH 3 COONH 4 , NaClO 4 , Na 2 CO 3 one of them.

[0030] The application of the polycyano meta-substituted pentamethine cyanine dye is used in the fields of in vitro or in vivo fluorescence imaging, biomarker labeling, nucleic acid specific analysis, diagnosis and treatment of tumor tissues or cells, phototherapy, and biosensing.

[0031] Further, when applied, its fluorescence imaging emission wavelength is 600 - 900 nm.

[0032] The beneficial effects of the present invention are:

[0033] (1) The multi-cyano meta-substituted pentamethine cyanine dye disclosed in the present invention increases its absorption and emission wavelengths by introducing a meta-substituted structure, enabling the dye to have the performance of near-infrared I imaging and treatment. Thus, the optimal excitation and emission wavelengths can be flexibly selected. The dyes prepared in the examples exhibit an obvious Stokes shift, which helps to reduce self-absorption and scattering effects, improve the clarity and depth of imaging. The molar extinction coefficients of the prepared dyes all exceed 190,000, greatly improving the utilization degree of light energy by such dyes. They can strongly absorb long-wavelength light with a deeper penetration depth, effectively achieve the capture and conversion of light energy, improve the efficiency of photosensitizers, reduce the light dose, and mitigate phototoxicity. In addition, they exhibit a high absolute fluorescence quantum yield, greatly improving the effect of biological imaging and having a higher imaging quality.

[0034] (2) When co-incubating human breast cancer cells with near-infrared fluorescent dyes with different concentrations of the multi-cyano structure in the present invention within two days, the tumor cells still have good survival rates. And when the concentration increases to 20 μmol / L, the survival rate of the cells is still very high. This shows that the meta-multi-cyano structure near-infrared fluorescent dye structure has good biocompatibility and will not produce toxic and side effects on cells within the working range. It can be applied to biological imaging, molecular labeling, tumor treatment, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] 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 description in the embodiments or the prior art. Obviously, the following-described drawings are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0036] Figure 1 is the high-resolution mass spectrometry data of Compound 1;

[0037] Figure 2 is the high-resolution mass spectrometry data of Compound 2;

[0038] Figure 3 is the ultraviolet-visible absorption spectrum of Compound 1 in acetonitrile solvent;

[0039] Figure 4 is the ultraviolet-visible fluorescence spectrum of Compound 1 in acetonitrile solvent;

[0040] Figure 5 is the ultraviolet-visible absorption spectrum of Compound 2 in acetonitrile solvent;

[0041] Figure 6 is the ultraviolet-visible fluorescence spectrum of Compound 2 in acetonitrile solvent;

[0042] Figure 7It is the result of the lysosome-targeted co-localization analysis of Compound 1 on MCF-7 cells;

[0043] Figure 8 It is the result graph of the MTT dark toxicity experiment of Compound 1. Detailed implementation manners

[0044] 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 drawings in the embodiments of the present invention. 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.

[0045] The term "halogen" used herein includes fluorine, chlorine, bromine and iodine.

[0046] The term "alkyl" used in the present invention includes straight-chain alkyl and branched-chain alkyl.

[0047] The term "MTT" used in the present invention refers to a method for detecting cell survival and growth.

[0048] Instruments and equipment used in the examples:

[0049] During the column chromatography process of the present invention, 200-300 mesh and 100-200 mesh column chromatography silica gels purchased from Qingdao Meigao Group Co., Ltd. and 20-40 mesh analytical pure quartz sand purchased from Tianjin Chemical Reagent Factory are used.

[0050] The absorption and emission spectra of the dyes are measured using an Agilent Cary 60 UV-visible spectrophotometer and a Cary Eclipse fluorescence spectrophotometer. The absolute fluorescence quantum yield of the dyes is measured using a C11347 absolute fluorescence quantum yield meter from Hamamatsu Photonics Trading (China) Co., Ltd.

[0051] The cytotoxicity test is measured using a Varioskan LUX Multimode Microplate Reader instrument from Thermofisher, USA.

[0052] Example 1: Preparation of Compound 1

[0053] Structural formula of Compound 1:

[0054]

[0055] (1) Preparation of Compound 1.1:

[0056]

[0057] In 20 mL of ethanol, 3-hydroxy-3-methyl-2-butanone (1.00 g, 9.8 mmol) and malononitrile (1.29 g, 19.6 mmol) were added. After stirring, sodium ethoxide (106 mg, 1.9 mmol) was added, and the mixture was refluxed for 2 h. Then it was cooled to room temperature, filtered with cold ethanol, washed, and dried in vacuo to obtain a yellowish-gray compound 1.1 (1.40 g, 7.0 mmol, Y = 72%);

[0058] (2) Preparation of compound 1.2

[0059]

[0060] At 0 °C, 10 mL of phosphorus oxychloride was added dropwise to 15 mL of DMF, and the mixture was stirred for 1 h. Then it was warmed to room temperature and stirred for another 3 h. Then p-trifluoromethylacetophenone (1 g, 5.31 mmol) was added, and under N 2 protection, it was stirred at 60 °C for 6 h. Then the reaction solution was poured into 150 mL of ice water, and sodium hypochlorite was added with stirring. 4 After the solid was precipitated, it was filtered. The solid was added to 50 mL of saturated sodium hydroxide solution, stirred at 80 °C for 1 h, then cooled to room temperature, 4 M HCl was added dropwise to adjust the pH to acidic, the precipitated solid was collected and washed with cold water to obtain a yellow compound 1.2 (1.25 g, 3.45 mmol, Y = 65%);

[0061] (3) Preparation of compound 1

[0062]

[0063] Compound 1.1 (1 g, 5.02 mmol) and compound 1.2 (826 mg, 2.21 mmol) were dissolved in a methanol solution, 3 drops of piperidine were added to catalyze the reaction, and the mixture was stirred. Under N 2 protection, it was refluxed for 4 h. After the reaction was cooled to room temperature, it was concentrated, and the obtained crude product was purified by silica gel column chromatography to obtain compound 1 (540 mg, 0.90 mmol, Y = 39%). High-resolution mass spectrometry is shown in Figure 1 , 1 1H NMR (600 MHz, DMSO-d 6 ) δ 7.79 (d, J = 8.0 Hz, 2H), 7.59 (d, J = 7.9 Hz, 2H), 7.55–7.49 (m, 1H), 6.82 (d, J = 12.8 Hz, 1H), 6.33 (d, J = 13.9 Hz, 1H), 5.76 (s, 1H), 1.56 (s, 6H), 1.51 (s, 6H).

[0064] Example 2: Preparation of compound 2

[0065] Structural formula of Compound 2:

[0066]

[0067] (1) Preparation of Compound 2.1:

[0068]

[0069] At 0 °C, 10 mL of phosphorus oxychloride was added dropwise to 15 mL of DMF, stirred for 1 h, heated to room temperature and stirred for another 3 h, then acetophenone (1 g, 8.32 mmol) was added, and under N 2 protection, stirred at 60 °C for 6 h, then the reaction solution was poured into 150 mL of ice water, and NaClO was added with stirring 4 . After the solid was precipitated, it was filtered, the solid was added to 50 mL of saturated sodium hydroxide solution, stirred at 80 °C for 1 h, then cooled to room temperature, 4 M HCl was added dropwise to adjust the pH to acidic, the precipitated solid was collected and washed with cold water to obtain yellow Compound 2.1 (1.14 g, 3.88 mmol, Y = 47%);

[0070] (2) Preparation of Compound 2

[0071]

[0072] Compound 1.1 (1 g, 5.02 mmol) prepared in Example 1 and the above Compound 2.1 (446 mg, 2.28 mmol) were dissolved in methanol solution, 3 drops of piperidine were added to catalyze the reaction, stirred, and under N 2 protection, refluxed for 4 h. After the reaction was cooled to room temperature, it was concentrated, and the obtained crude product was purified by silica gel column to obtain Compound 2 (450 mg, 0.84 mmol, Y = 37%). High-resolution mass spectrometry is shown in Figure 2 , 1H NMR (600 MHz, DMSO-d6) δ 7.46 (p, J = 3.6 Hz, 4H), 7.39 (dd, J = 6.6, 3.0 Hz, 2H), 7.07 (t, 2H), 6.81 (t, J = 13.7 Hz, 1H), 6.29 (d, J = 13.9 Hz, 1H), 5.75 (s, 1H), 1.56 (s, 6H), 1.49 (s, 6H).

[0073] Example 3: Preparation of Compound 3

[0074] Structural formula of Compound 3:

[0075]

[0076] (1) Preparation of Compound 3.1:

[0077]

[0078] In 20 mL of ethanol, 2-(trifluoromethyl)buta-1,3-diene (1.00 g, 6.40 mmol) and malononitrile (846 mg, 12.8 mmol) were added. After stirring, sodium ethoxide (1.05 g, 12.86 mmol) was added, and the mixture was refluxed for 2 h. Then it was cooled to room temperature, filtered with cold ethanol, washed, and dried in vacuo to obtain a yellowish-gray compound 3.1 (1.21 g, 4.78 mmol, Y = 74%);

[0079] (2) Preparation of compound 3.2

[0080]

[0081] At 0 °C, 10 mL of phosphorus oxychloride was added dropwise to 15 mL of DMF, and the mixture was stirred for 1 h. Then it was warmed to room temperature and stirred for another 3 h. Then p-nitroacetophenone (1 g, 6.05 mmol) was added, and the mixture was stirred at 60 °C for 6 h. Then the reaction solution was poured into 150 mL of ice water, and NaClO was added with stirring 4 , and after the solid was precipitated, it was filtered. The solid was added to 50 mL of saturated sodium hydroxide solution, stirred at 80 °C for 1 h, then cooled to room temperature, 4 M HCl was added dropwise to adjust the pH to acidic, the precipitated solid was collected and washed with cold water to obtain a yellow compound 3.2 (1.42 g, 4.19 mmol, Y = 69%);

[0082] (3) Preparation of compound 3

[0083]

[0084] Compound 3.1 (1 g, 3.95 mmol) and compound 3.2 (609 mg, 1.80 mmol) were dissolved in a methanol solution, 3 drops of piperidine were added to catalyze the reaction, and the mixture was stirred and refluxed for 4 h. After the reaction was cooled to room temperature, it was concentrated, and the obtained crude product was purified by silica gel column chromatography to obtain compound 3 (350 mg, 0.51 mmol, Y = 28%). 1H NMR (400 MHz, Chloroform-d) δ 8.27–8.20 (m, 2H), 7.52–7.44 (m, 2H), 7.30 (t, J = 1.2 Hz, 1H), 6.78 (dt, J = 8.6, 1.5 Hz, 1H), 6.67 (dd, J = 15.1, 1.6 Hz, 1H), 6.54 (ddd, J = 15.1, 8.6, 1.0 Hz, 1H), 1.46 (d, J = 2.5 Hz, 6H).

[0085] Example 4: Preparation of compound 4

[0086] Structural formula of Compound 4:

[0087]

[0088] (1) Preparation of Compound 4.1:

[0089]

[0090] In 20 mL of ethanol, add 2-(trifluoromethyl)-2-phenyl-but-1,3-diene (1.00 g, 4.58 mmol) and malononitrile (606 mg, 9.17 mmol). After stirring, add sodium ethoxide (752 mg, 9.17 mmol) and reflux for 2 h. Then cool to room temperature, filter with cold ethanol, wash, and dry in vacuo to obtain yellowish-gray Compound 4.1 (946 mg, 3.00 mmol, Y = 65%);

[0091] (2) Preparation of Compound 4.2

[0092]

[0093] At 0 °C, add 10 mL of phosphorus oxychloride dropwise to 15 mL of DMF, stir for 1 h, warm to room temperature and continue stirring for 3 h. Then add p-cyanoacetophenone (1 g, 6.89 mmol), stir at 60 °C for 6 h, and then pour the reaction solution into 150 mL of ice water. Add NaClO with stirring 4 . After precipitation of the solid, filter, add the solid to 50 mL of saturated sodium hydroxide solution, stir at 80 °C for 1 h, then cool to room temperature, add 4 M HCl dropwise to adjust the pH to acidic, collect the precipitated solid and wash with cold water to obtain yellow Compound 4.2 (1.58 g, 4.95 mmol, Y = 72%);

[0094] (3) Preparation of Compound 4

[0095]

[0096] Compound 4.1 (1 g, 3.17 mmol) and compound 4.2 (460 mg, 1.44 mmol) were dissolved in methanol solution. 3 drops of piperidine were added to catalyze the reaction, and the mixture was stirred and refluxed for 4 h. After the reaction was cooled to room temperature, it was concentrated, and the obtained crude product was purified by silica gel column to obtain compound 4 (257 mg, 0.32 mmol, Y = 22%). 1H NMR (400 MHz, Chloroform-d) δ 8.27–8.20 (m, 2H), 7.53–7.39 (m, 12H), 7.36–7.25 (m, 2H), 7.16 (dd, J = 15.1, 1.5 Hz, 1H), 6.80 (dt, J = 8.8, 1.3 Hz, 1H), 6.54 (ddd, J = 14.9, 8.8, 0.9 Hz, 1H).

[0097] Example 5: Preparation of Compound 5

[0098] Structural formula of Compound 5:

[0099]

[0100] (1) Preparation of Compound 5.1:

[0101]

[0102] Diacetyl (1.72 g, 20 mmol) and 2% NaOH aqueous solution (0.6 g) were added to malononitrile dimer (2.64 g, 20 mmol), dissolved in 25 mL of ethanol aqueous solution (20%, v / v), stirred at room temperature for 2 h and then filtered. Then the solution was adjusted to 0 °C and filtered again, washed with cold ethyl acetate, and dried to obtain intermediate 5.1 (2.9 g, 15 mmol, Y = 73%);

[0103] (2) Preparation of Compound 5.2

[0104]

[0105] At 0 °C, 10 mL of phosphorus oxychloride was added dropwise to 15 mL of DMF, stirred for 1 h, heated to room temperature and continued to stir for 3 h. Then p-aminoacetophenone (1 g, 7.40 mmol) was added, and under N 2 protection, stirred at 60 °C for 6 h. Then the reaction solution was poured into 150 mL of ice water, and sodium hypochlorite was added with stirring 4 . After the solid was precipitated, it was filtered. The solid was added to 50 mL of saturated sodium hydroxide solution, stirred at 80 °C for 1 h, then cooled to room temperature, 4M HCl was added dropwise to adjust the pH to acidic, the precipitated solid was collected and washed with cold water to obtain yellow compound 5.2 (1.16 g, 3.74 mmol, Y = 51%);

[0106] (3) Preparation of Compound 5

[0107]

[0108] Dissolve Compound 5.1 (1 g, 5.00 mmol) and Compound 5.2 (702 mg, 2.27 mmol) in methanol solution, add 3 drops of piperidine to catalyze the reaction, stir, and reflux for 4 h under N 2 protection. After cooling the reaction to room temperature, concentrate it, and purify the obtained crude product through a silica gel column to obtain Compound 5 (275 mg, 0.50 mmol, Y = 22%). 1H NMR (400 MHz, Chloroform-d) δ 7.99 (s, 1H), 7.63 (s, 1H), 7.54–7.47 (m, 2H), 6.85–6.78 (m, 2H), 6.67–6.50 (m, 4H), 5.76 (s, 1H), 5.58 (s, 1H), 4.85 (d, J = 5.7 Hz, 1H), 4.71 (d, J = 5.7 Hz, 1H), 1.64 (d, J = 2.8 Hz, 6H).

[0109] Example 6: Preparation of Compound 6

[0110] Structural formula of Compound 6:

[0111]

[0112] (1) Preparation of Compound 6.1:

[0113]

[0114] Add the Compound 5.1 (1.00 g, 20 mmol) prepared in Example 5 to 15 mL of benzyl alcohol solution, and cool it to 0 °C. Slowly add 2 mL of trifluoroacetic acid to the above solution, then stir for 12 h. After raising the temperature to room temperature, continue to stir for 24 h. After the reaction is completed, slowly add the above solution dropwise to methyl tert-butyl ether at 0 °C for recrystallization. Filter, wash, and purify with a silica gel column to obtain Compound 6.1 (950 mg, 32.72 mmol, Y = 65%);

[0115] (2) Preparation of Compound 6.2

[0116]

[0117] Add Compound 6.1 (1.00 g, 3.44 mmol) to 10 mL of acetonitrile solution, and add 0.5 g of solid potassium carbonate, N 2Stir for 1 h, then slowly add methyl iodide (1.46 g, 10.33 mmol) and stir at 50 °C for 24 h. After cooling to room temperature, concentrate, filter, and purify by silica gel column chromatography to obtain the yellow solid compound 6.2 (875 mg, 2.87 mmol, Y = 83%);

[0118] (3) Preparation of compound 6.3

[0119]

[0120] At 0 °C, add 10 mL of phosphorus oxychloride dropwise to 15 mL of DMF, stir for 1 h, warm to room temperature and continue stirring for 3 h, then add p-methylacetophenone (1 g, 7.45 mmol), N 2 Stir at 60 °C for 6 h under protection, then pour the reaction solution into 150 mL of ice water, add NaClO with stirring 4 , filter after precipitation of solid, add the solid to 50 mL of saturated sodium hydroxide solution, stir at 80 °C for 1 h, then cool to room temperature, add 4 M HCl dropwise to adjust the pH to acidic, collect the precipitated solid and wash with cold water to obtain the yellow compound 6.3 (1.46 g, 4.73 mmol, Y = 63%);

[0121] (4) Preparation of compound 6

[0122]

[0123] Dissolve compound 6.2 (1 g, 3.29 mmol) and compound 6.3 (460 mg, 1.49 mmol) in methanol solution, add 3 drops of piperidine to catalyze the reaction, stir, N 2 Reflux for 4 h under protection, cool the reaction to room temperature and concentrate, and purify the obtained crude product by silica gel column chromatography to obtain compound 6 (164 mg, 0.22 mmol, Y = 15%). 1H NMR (400 MHz, Chloroform-d) δ 7.38–7.27 (m, 4H), 7.28 (s, 1H), 6.66–6.49 (m, 1H), 4.68–4.56 (m, 1H), 2.99 (s, 1H), 2.92 (s, 1H), 2.36 (d, J = 0.6 Hz, 1H), 1.46 (d, J = 0.7 Hz, 2H).

[0124] Example 7: Preparation of compound 7

[0125] Structural formula of compound 7:

[0126]

[0127] (1) Preparation of compound 7.1:

[0128]

[0129] Compound 5.1 (1.00 g, 5.00 mmol) prepared in Example 5 was added to 15 mL of n-pentanol solution and cooled to 0°C. 2 mL of trifluoroacetic acid was slowly added dropwise to the solution, followed by stirring for 12 h. After warming to room temperature, stirring was continued for 24 h. After the reaction was completed, the solution was slowly added dropwise to 0°C methyl tert-butyl ether for recrystallization. Filter, wash and purify with a silica gel column to obtain compound 7.1 (461 mg, 1.71 mmol, Y = 34%).

[0130] (2) Preparation of Compound 7.2

[0131]

[0132] At 0°C, 10 mL of phosphorus oxychloride was added dropwise to 15 mL of DMF, stirred for 1 h, heated to room temperature and continued to stir for 3 h, then p-methoxyacetophenone (1 g, 6.66 mmol) and N 2 The mixture was stirred at 60°C for 6 h under protection, and then the reaction solution was poured into 150 mL of ice water, and NaClO4 was added under stirring. After the solid was precipitated, it was filtered, and the solid was added into 50 mL of saturated sodium hydroxide solution, stirred at 80°C for 1 h, and then cooled to room temperature, 4 M HCl was added dropwise, and the pH was adjusted to acidic. The precipitated solid was collected and washed with cold water to obtain yellow compound 7.2 (1.02 g, 3.16 mmol, Y = 47%);

[0133] (3) Preparation of Compound 7

[0134]

[0135] Compound 7.1 (1 g, 3.70 mmol) and compound 7.2 (545 mg, 1.68 mmol) were dissolved in methanol solution, 3 drops of piperidine were added to catalyze the reaction, and the mixture was stirred at N 2The mixture was refluxed for 4 h under protection, the reaction mixture was cooled to room temperature and concentrated, and the crude product was purified by silica gel column to obtain compound 7 (265 mg, 0.376 mmol, Y = 22%). 1H NMR (400 MHz, Chloroform-d) δ7.43–7.35 (m, 1H), 7.05–6.98 (m, 1H), 6.80 (t, J = 1.1 Hz, 0H), 6.66–6.57 (m, 1H), 6.57–6.47 (m, 1H), 3.81 (s, 1H), 3.57–3.37 (m, 2H), 1.70–1.54 (m, 2H), 1.53 (d, J = 2.2 Hz, 3H), 1.46–1.28 (m, 4H), 0.96–0.85 (m, 3H).

[0136] Comparative Example 1: No substitution of methine chain

[0137] The structural formula of the dye of Comparative Example 1 is:

[0138]

[0139] Synthesis of Comparative Example 1 Dye Compound

[0140] Compound 1.1 (1 g, 5.02 mmol) prepared in Example 1 and N-(3-phenylamino-2-propyleneylidene)aniline hydrochloride (590 mg, 2.28 mmol) were dissolved in methanol solution, 3 drops of piperidine were added to catalyze the reaction, and the mixture was stirred at N 2 The mixture was refluxed for 4 h under protection, the reaction mixture was cooled to room temperature and concentrated, and the crude product was purified by silica gel column to obtain the dye compound of comparative example 1 (520 mg, 1.14 mmol, Y = 50%). 1H NMR (400 MHz, Chloroform-d) δ 6.85 (ddt, J = 15.1, 8.2, 0.9 Hz, 1H), 6.69 (dt, J = 8.7, 1.0 Hz, 1H), 6.66-6.54 (m, 2H), 6.20 (ddt, J = 14.8, 8.4, 0.9 Hz, 1H), 1.49 (d, J = 7.5 Hz, 12H).

[0141] Comparative Example 2: Median Substitution

[0142] Comparative Example 2 Dye compound structural formula:

[0143]

[0144] Preparation of Comparative Example Compound 2.1

[0145]

[0146] At 0°C, 12 mL of phosphorus oxychloride was added dropwise to 15 mL of DMF, stirred in an ice bath for 1 h, then heated to 25°C and stirred for 3 h, then p-methoxyacetic acid (1 g, 6.02 mM) was added. 2 The reaction solution was then poured into 200 mL of ice water and NaClO was added under stirring. 4 , until no more solid precipitates, filter to obtain a solid, add the solid to 50 mL of a saturated sodium hydroxide solution, stir at 80°C for 1 h, then cool to room temperature, add HCl dropwise to adjust the pH to 1, collect the precipitated solid, filter and wash with ice water to obtain a yellow powder compound condensing agent 2.1 (0.52 g, 2.92 mM, Y = 48.5%);

[0147] Manufacturing Comparative Example 2

[0148] The compound 3.1 (1 g, 3.95 mmol) prepared in Example 3 and the comparative example compound 2.1 (320 mg, 1.80 mmol) were dissolved in a methanol solution, 3 drops of piperidine were added to catalyze the reaction, stirred and refluxed for 4 h, the reaction was cooled to room temperature and concentrated, and the obtained crude product was purified by silica gel column to obtain the comparative example 2 compound (356 mg, 0.53 mmol, Y = 30%). 1H NMR (400 MHz, Chloroform-d) δ7.57–7.49 (m, 2H), 7.26–7.19 (m, 1H), 7.05–6.98 (m, 2H), 6.58–6.50 (m, 3H), 3.81 (s, 3H), 1.45 (d, J = 12.2 Hz, 5H).

[0149] Tests, results and analysis

[0150] (1) Identification of near-infrared fluorescent dye compounds with polycyano structures

[0151] Compounds 1 and 2 were identified using high-precision, high-sensitivity, high-resolution mass spectrometry. Figure 1 and Figure 2 shown.

[0152] The high-resolution mass spectrometry analysis results for compound 1 showed that the mass spectrum ( Figure 1 ) showed a clear and sharp main peak, the position of which was highly consistent with the theoretically calculated molecular weight of compound 1, and no obvious impurity peaks or fragment peaks were observed, which fully proved the purity of compound 1 and the correctness of its structure, indicating the successful preparation of compound 1. At the same time, the distribution of characteristic peaks in the mass spectrum was consistent with the functional groups and connection methods in the expected structure, further verifying the specified structure of compound 1. Similarly, Figure 2The high-resolution mass spectrometry analysis results of compound 2 are shown. Similar to compound 1, the mass spectrum of compound 2 also shows a main peak, whose position accurately corresponds to the theoretical molecular weight of compound 2. The spectrum is clear and free of interference, reflecting the high purity and structural accuracy of compound 2.

[0153] (2) Test of the photophysical properties of near-infrared fluorescent dye compounds with polycyano structures

[0154] A. Dye preparation method

[0155] This experiment uses a balance with an accuracy of one ten-thousandth to accurately weigh the dye that has been strictly vacuum dried. Subsequently, the weighed dye is dissolved in dimethyl sulfoxide (DMSO) to prepare a dye mother solution with a concentration of 2mmol / L. This mother solution is carefully dispensed into brown sample bottles to prevent light from interfering with its stability, and is stored in a 4°C refrigerator to ensure chemical stability and activity during long-term storage. Before conducting UV-visible absorption and fluorescence spectroscopy tests, the test solution needs to be prepared. The specific steps are: Use a high-precision micropipette to accurately measure 4μL of the dye mother solution, slowly inject it into a quartz cuvette containing 3mL of a predetermined solvent (such as acetonitrile), and gently shake to ensure that the solution is evenly mixed. This operation dilutes the dye concentration to 1.75μmol / L, which is suitable for subsequent spectral analysis.

[0156] B. Broad spectrum conditions and photophysical properties test conditions

[0157] All spectral tests were performed in a strictly controlled constant temperature environment of 25°C to eliminate the potential impact of temperature fluctuations on the experimental results and ensure the repeatability and accuracy of the obtained data. During the photophysical property test, a precisely prepared dye stock solution was used as the test solution. According to the principle of Beer's law, the molar extinction coefficient of each dye was calculated by measuring the absorbance at a specific wavelength and combining the molar extinction coefficient calculation formula; a high-precision absolute fluorescence quantum yield meter (Hamamatsu, model C11347) was used to measure the absolute fluorescence quantum yield of each dye sample under the same conditions to comprehensively evaluate its fluorescence emission efficiency.

[0158] C. Result Analysis

[0159] Figure 3 is the absorption spectrum of compound 1, Figure 4 is the fluorescence spectrum of compound 1, Figure 5 is the absorption spectrum of compound 2, Figure 6 is the fluorescence spectrum of compound 2. Figure 3 and Figure 5 As shown, the test results of the absorption spectra of compound 1 and compound 2 in acetonitrile show that the maximum absorption peaks of compound 1 and compound 2 are 765nm and 762nm respectively.Figure 4 and Figure 6 It was found that the maximum emission peaks of compounds 1 and 2 were 825nm and 810nm, the Stokes shift of compound 1 was 60nm, and the Stokes shift of compound 2 was 48nm. The absorption and emission wavelengths of compounds 1 and 2 were both relatively long, breaking through the upper limit of the absorption and emission wavelengths of common pentamethine cyanine dyes, making them better applicable in the field of near-infrared imaging. In addition, the Stokes shifts of both are relatively large, which can provide a better signal-to-noise ratio in the biological imaging process, and facilitate the provision of higher fluorescence resolution. The above properties enable this type of dye matrix to have better performance in biological imaging and other aspects.

[0160] (3) The photophysical data results of compounds 1-7 are shown in Table 1.

[0161] Table 1 Photophysical properties of compounds 1-7

[0162]

[0163] Table 1 lists in detail the key photophysical properties of compounds 1-7 as near-infrared fluorescent dyes with a polycyano structure, including absorption wavelength, emission wavelength, molar extinction coefficient (ε) and absolute fluorescence quantum yield These data not only reveal the diversity and superiority of this type of dye in spectral characteristics, but also profoundly reflect the remarkable effectiveness of the solution of the present invention in solving the problems of the prior art.

[0164] The absorption wavelengths of compounds 1-7 are all above 700nm, covering the long wavelength region from 729nm to 785nm, while the emission wavelengths are distributed between 777nm and 825nm. The longer absorption and emission wavelengths give the dyes the performance of near-infrared imaging and treatment, allowing for flexible selection of the optimal excitation and emission wavelengths. Among them, compound 1 exhibits a significant Stokes shift, which helps reduce self-absorption and scattering effects and improves the clarity and depth of imaging.

[0165] The molar extinction coefficients of compounds 1-7 all exceeded 190,000, significantly higher than those of traditional cyano dyes. The molar extinction coefficient of compound 1 was as high as 240,841 L / mol. -1 cm -1, the higher molar extinction coefficient greatly improves the utilization of light energy by such dyes, and can strongly absorb long-wavelength light with a deeper penetration depth, effectively realize the capture and conversion of light energy, improve the efficiency of photosensitizers, reduce light doses, and reduce phototoxicity. In addition, compared with the unsubstituted compound of the methine chain in Comparative Example 1 and the substituted compound in Comparative Example 2, compounds 1-7 all exhibit higher absolute fluorescence quantum yields, which can reach up to 0.096 (compound 1). The higher fluorescence quantum yield greatly improves the effect of biological imaging, and combined with the long-wavelength absorption and emission properties of such dyes, compounds 1-7 have higher imaging quality in deep-level imaging.

[0166] (4) Fluorescence labeling of lysosomes in cells by activated compound 1

[0167] After MCF-7 cells were grown in a confocal dish to the logarithmic growth phase, 2 μM compound 1 was added, and after incubation for 10 min, commercial lysosome localization dye was added to co-incubate the cells for 15 min. The cells were observed under a confocal microscope, and the fluorescence channels of compound 1 and commercial dye were superimposed to determine the localization effect of compound 1.

[0168] like Figure 7 As shown, the green channel is a commercial lysosomal localization probe, the red channel is compound 1, and the scale is 20 mm. By observing the superimposed pathway, it is found that its localization and lysosomal effect are excellent. The co-localization effect is quantified, and the Pearson co-localization coefficient is determined to be 0.89. The results show that compound 1 is a dye with good lysosomal localization effect.

[0169] (5) Cytotoxicity test of compound 1

[0170] The toxicity of dye molecules to cells is evaluated by MTT assay. The principle is that succinate dehydrogenase in the mitochondria of living cells can reduce exogenous MTT to water-insoluble blue-purple crystalline formazan and deposit it in cells, while dead cells do not have this function. Dimethyl sulfoxide (DMSO) can dissolve the formazan in cells, and its light absorption value is measured at a wavelength of 570nm using a microplate reader, which can indirectly reflect the number of living cells.

[0171] MCF-7 cells were inoculated in a 96-well plate. After a period of culture, a certain concentration of compound 1 was added to different wells, and the compound concentration was 0-40 μmol / L. After incubating the cells for 24 hours, the cell activity was detected by MTT assay. The experimental data are shown in Figure 2. Figure 8As shown, after culturing MCF-7 cells with different concentrations of compound 1 for 24 hours, the cells still showed a good survival rate. Even when the concentration was increased to 40 μmol / L, the cell survival rate was still very high, indicating that this type of new polycyano near-infrared fluorescent dye has very good biocompatibility and will not cause toxic side effects to cells within the working concentration range. Therefore, it can be used in the biological and medical fields.

[0172] In addition, the phototoxicity of compound 1 was tested. MCF-7 cells were inoculated in a 96-well plate, and after a period of culture, a certain concentration of compound 1 was added to different wells, so that the compound concentration was 0-40 μmol / L. After 2 hours, the cells were irradiated with a 760nm laser, and the cells were incubated for 24 hours before the cell activity was detected by MTT assay. The experimental data are shown in Figure 8 As shown, after culturing MCF-7 cells with different concentrations of compound 1 for 24 hours, the cells showed good phototoxicity, and when the concentration increased to 40 μmol / L, the cell survival rate was only 30%. This shows that this type of new polycyano near-infrared fluorescent dye has a very good cancer killing effect, and can effectively kill cells within the working concentration range, so it can be used in the biological and medical fields.

[0173] In summary, the present invention synthesizes a type of partial-substituted polycyano pentamethine cyanine dye structure by rationally designing the terminal group of the cyanine dye and the intermediate condensing agent structure, and successfully realizes the regulation of the electron supply type and charge separation degree of the dye molecule, and the regulation of the dye aggregation performance. The polycyano partial-substituted pentamethine cyanine dye structure exhibits near-infrared absorption and emission characteristics in the absorption and emission spectrum, has high optical absorption capacity and fluorescence imaging ability, and improves the absorption and utilization of photons; its absolute fluorescence quantum yield reaches a high level, which can significantly enhance the near-infrared deep tissue imaging ability, and provides a solution for the integration of biological diagnosis and treatment; the higher biological safety and photostability ensure that the partial polycyano partial-substituted pentamethine cyanine dye is used in the biological and medical fields to improve the safety performance; the higher photothermal capacity enables it to significantly kill tumor cells at a lower concentration, providing strong support for the application in the fields of cancer treatment.

[0174] 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 aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A polycyano-substituted pentamethine cyanine dye, characterized in that: The dye has the following general structure: in, R1 is selected from one of O, S, Se, NH, and NCH3; R2 and R3 are each independently selected from one of hydrogen, halogen, methyl, hydroxyl, methoxy, phenyl, trifluoromethyl, alkoxy with 1-18 carbon atoms, carboxyalkyl with 1-18 carbon atoms, alkylsulfonic acid with 1-18 carbon atoms, ester with 1-18 carbon atoms, and amide with 1-18 carbon atoms; R4 is selected from any one of hydrogen, halogen, cyano, methoxy, amino, nitro, hydroxyl, carboxyl, N,N-dimethylamino, N,N-diethylamino, trifluoromethyl, ester, amide, sulfonate or sulfonate; R5 is selected from a substituent at an undetermined position of the benzene ring, selected from any one of hydrogen, halogen, a carboxyalkyl group having 1-18 carbon atoms, a hydroxyalkyl group having 1-18 carbon atoms, an alkylsulfonic acid group having 1-18 carbon atoms, an ester group having 1-18 carbon atoms, or an amide group having 1-18 carbon atoms; Y is selected from inorganic cations or organic cations.

2. The polycyano-substituted pentamethine cyanine dye according to claim 1, characterized in that: R1 is selected from one of O and NH; R2 and R3 are each independently selected from methyl, phenyl, trifluoromethyl, and an alkoxy group having 1 to 8 carbon atoms; R4 is selected from one of hydrogen, nitro, cyano, amino, trifluoromethyl, and methoxy; Y is selected from one of Na, K, piperidine, pyridine and triphenylphosphine.

3. A method for synthesizing the polycyano-substituted pentamethine cyanine dye according to claim 1, characterized in that: The following steps are involved: (1) Add the compound of formula S1 and malononitrile to an organic solvent, stir, add an appropriate amount of alkaline catalyst, and reflux for 1-3 hours, then cool, filter, wash and dry to obtain intermediate S3, or The compound of formula S2 and 2% NaOH aqueous solution are added to the malononitrile dimer, which is then dissolved in an organic solvent, stirred at room temperature, cooled, filtered, washed, and dried to obtain an intermediate S3; (2) Add phosphorus oxychloride dropwise to DMF at -10-20°C and mix well, then add the compound of formula S4, stir at 30-80°C under N2 protection for 6-24h, then cool to room temperature, add ice water to quench the reaction, and add an inorganic salt to obtain intermediate S5; (3) The intermediates S3 and S5 are dissolved in an organic solvent, an alkaline catalyst is added, and the reaction is refluxed under N2 protection for 4 hours. After the reaction is completed, the product is concentrated and purified by a silica gel column to obtain a polycyano-substituted pentamethine cyanine dye.

4. The method for synthesizing the polycyano-substituted pentamethine cyanine dye according to claim 3, characterized in that: The molar ratio of the compound of formula S1 to malononitrile is 1:

2.

5. The method for synthesizing the polycyano-substituted pentamethine cyanine dye according to claim 3, characterized in that: The molar ratio of the compound of formula S2 to the malononitrile dimer is 1:

1.

6. The method for synthesizing the polycyano-substituted pentamethine cyanine dye according to claim 3, characterized in that: In step (1) and step (3), the organic solvent is selected from one of toluene, ethanol, methanol, isopropanol, acetonitrile and tetrahydrofuran; and the base is selected from one of sodium hydroxide, potassium hydroxide, ammonia water, sodium bicarbonate, sodium ethoxide, sodium methoxide, pyridine, piperidine, triethylamine, trimethylamine and diisopropylethylamine.

7. The method for synthesizing the polycyano-substituted pentamethine cyanine dye according to claim 3, characterized in that: In step (2), the inorganic salt is selected from one of NaCl, CH3COONa, CH3COONH4, NaClO4, and Na2CO3.

8. An application of the polycyano-substituted pentamethine cyanine dye according to claim 1, characterized in that: It is used in in vitro or in vivo fluorescence imaging, biomarker labeling, nucleic acid-specific analysis, diagnosis and treatment of tumor tissues or cells, phototherapy, and biosensing.

9. Use of the polycyano-substituted pentamethine cyanine dye according to claim 8, wherein the fluorescence imaging emission wavelength is 600-900 nm.

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