Near-infrared two-region fluorescent dye molecule diphenylamine acridine and preparation method and application thereof

Abstract

The application belongs to the technical field of fluorescent small molecule dyes, and particularly relates to a near-infrared two-region fluorescent dye molecule with a large stoke shift as well as a preparation method and application thereof. The near-infrared two-region fluorescent dye molecule is diphenylamine acridine; the molecule has a large molar extinction coefficient, absorption and emission wavelength, a large stoke shift, good light stability and chemical stability, and is easy to synthesize and modify. The fluorescent dye molecule can be used as a multifunctional near-infrared two-region fluorescent imaging reagent, and is used for biomedical imaging of deep tissue of a living body, and has excellent medical imaging effect.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of fluorescent dye molecules, and particularly relates to a near-infrared second window fluorescent dye molecule diphenylamine acridine with a large stokes shift, a preparation method thereof and application thereof. BACKGROUND

[0002] In recent years, in vivo fluorescence imaging in the near-infrared second window (1000-1700 nm) has been rapidly developed in the biomedical field due to its strong deep tissue penetration ability and high spatiotemporal resolution. Among them, near-infrared second window small molecule fluorescent dyes play an important role in biological imaging research. At present, the strategies for developing near-infrared second window small molecule imaging reagents mainly include extending the π-π conjugated system, donor and acceptor modification, and constructing fluorophore J-aggregates. Based on the existing construction strategies, the existing near-infrared second window fluorophore scaffolds mainly include polymethine cyanine dye (D-π-A), donor-acceptor-donor (D-A-D) type fluorophore, aza-bodipy (Aza-BODIPY) dye and xanthene fluorescent dye have been successfully constructed and applied to in vivo biological imaging. However, most of these dyes are easily oxidized and decomposed by the rich active oxygen substances in the biological system, and most of them exhibit small stokes shift. It is well known that fluorophores with large stokes shift help to reduce self-quenching and avoid interference caused by excitation light, thereby improving the signal-to-noise ratio of biological imaging. Although the existing D-A-D type fluorophore exhibits a large stokes shift, it has the disadvantages of low fluorescence brightness, large conjugated system and difficult preparation. In the near-infrared second window, the construction strategy of the dye still faces deficiencies. Therefore, there is an urgent need to develop new strategies for constructing near-infrared second window fluorophores with specific properties. SUMMARY

[0003] The purpose of the present application is to provide a near-infrared second window fluorophore with a large stokes shift, high fluorescence brightness, a small conjugated system and easy preparation, and a preparation method and application thereof.

[0004] The present application first constructs a near-infrared second window fluorescent dye molecule with a large stokes shift based on the planarization intramolecular charge transfer mechanism, specifically diphenylamine acridine, denoted as DPA, and the compound structure general formula I is as follows:

[0005]

[0006] Among them, R1, R2, R3 and R4 groups are independently selected from hydrogen (-H), methyl (-CH3), methoxy (-OCH3), hydroxyl (-OH), tert-butyl (-C(CH3)3), fluorine (-F) and 2-(2-ethyl sulfonic acid) amide-1-ethoxy

[0007] The near-infrared two-region fluorescent dye molecule provided by the present application has a large molar extinction coefficient, long absorption and emission wavelength, large stoke shift, good light stability and chemical stability, and is easy to synthesize and modify. In dimethyl sulfoxide (DMSO) solvent, the maximum absorption peak is located at about 800 nm, the maximum emission peak is located at about 1000 nm, and the stoke shift is greater than 200 nm.

[0008] Further, the near-infrared two-region fluorescent dye molecule of the present application, preferably R1 and R2 are hydrogen, and R3 and R4 are 2-(2-ethyl sulfonic acid) amide-1-ethoxy:

[0009]

[0010] The structure is shown in formula II: the water-soluble near-infrared two-region fluorescent dye molecule is DPA-2SO3H;

[0011]

[0012] The DPA-2SO3H has strong absorption and emission in DMSO and water, the absorption peak is located at about 800 nm, the maximum emission peak is located at about 1000 nm, and the stoke shift is greater than 200 nm.

[0013] The present application also provides a preparation method of the above-mentioned near-infrared two-region fluorescent dye molecule with large stoke shift, and the synthesis route is as follows:

[0014]

[0015] The specific steps are as follows:

[0016] (1) Synthesis of intermediate 1:

[0017] First, compound 2,7-dibromo-9,9-dimethyl-9,10-dihydroacridine and di-tert-butyl dicarbonate are dissolved in tetrahydrofuran solution, a catalytic amount of 4-dimethylaminopyridine is added, and the reaction is carried out at 60-80℃ for 12-24 hours; after cooling and concentration, the intermediate product 1 is separated and purified by silica gel column chromatography;

[0018] (2) Synthesis of intermediate 2:

[0019] First, compound 1 and substituted diphenylamine are added to a reaction bottle, an inorganic base is added, a palladium catalyst and a ligand reagent are added, the reaction bottle is evacuated and filled with argon in turn, and the operation is repeated three times; then anhydrous dioxane or toluene is added by syringe, and the reaction mixture is reacted at 60-100°C for 20-24 hours; after the reaction is completed, the organic solvent is removed under reduced pressure, and the reaction mixture is extracted and washed with dichloromethane and water three times, the organic phase is combined and dried with anhydrous sodium sulfate to remove water, and finally purified by column chromatography to obtain intermediates 2a-2f; when the substituent groups R1, R2, R3, R4 are all hydrogen, intermediate 2a is obtained; when the substituent groups R1 and R4 are methyl, and R2 and R3 are hydrogen, intermediate 2b is obtained; when the substituent groups R1, R2, R3, R4 are all methoxy, intermediate 2c is obtained; when the substituent groups R1, R2, R3, R4 are all hydroxy, intermediate 2d is obtained; when the substituent groups R1, R2, R3, R4 are all tert-butyl, intermediate 2e is obtained; when the substituent groups R1 and R4 are fluoro, and R2 and R3 are hydrogen, intermediate 2f is obtained;

[0020] (3) Synthesis of near-infrared two-region fluorescent dye molecule DPA:

[0021] The intermediate compound 2 is dissolved in a mixed solvent of dichloromethane and trifluoroacetic acid, stirred at room temperature for 3-6 hours, and after the reaction is completed, extracted and washed with dichloromethane and water three times, the organic phase is dried with anhydrous sodium sulfate, and then purified by column chromatography to obtain the target near-infrared two-region dye molecule DPA; when the substituent groups R1, R2, R3, R4 are all hydrogen, intermediate DPA-1 is obtained; when the substituent groups R1 and R4 are methyl, and R2 and R3 are hydrogen, intermediate DPA-2 is obtained; when the substituent groups R1, R2, R3, R4 are all methoxy, intermediate DPA-3 is obtained; when the substituent groups R1, R2, R3, R4 are all hydroxy, intermediate DPA-4 is obtained; when the substituent groups R1, R2, R3, R4 are all tert-butyl, intermediate DPA-5 is obtained; when the substituent groups R1 and R4 are fluoro, and R2 and R3 are hydrogen, intermediate DPA-6 is obtained.

[0022] Further, in step (2):

[0023] The inorganic base is selected from one of sodium tert-butoxide, potassium tert-butoxide, sodium carbonate, potassium carbonate, cesium carbonate and potassium phosphate;

[0024] The palladium catalyst is selected from one of palladium acetate, bis(dibenzylideneacetone)palladium, tris(dibenzylideneacetone)dipalladium, diphenylphosphin ferrocene dichloropalladium, tetrakis(triphenylphosphine)palladium and dichlorobis(triphenylphosphine)palladium;

[0025] The reagent is one of 2-dicyclohexylphospho-2',4',6'-triisopropyl biphenyl, 1,1'-binaphthalene-2,2'-diphenylphosphine, 4,5-bis-diphenylphosphine-9,9-dimethyl xanthene and 2-dicyclohexylphosphine-2',6'-dimethoxy-1,1'-diphenyl.

[0026] The application also provides the application of the above-mentioned near-infrared two-region fluorescent dye molecule (diphenylamine acridine) with large Stokes shift in biomedical imaging of deep tissue of organisms.

[0027] For example, the water-soluble near-infrared two-region dye DPA-2SO3H can be used for gastrointestinal fluorescence imaging of mice, specifically, by gavage of the water-soluble near-infrared two-region fluorescent dye DPA-2SO3H aqueous solution to mice, irradiating the mouse abdomen with an 808 nm external laser, and collecting 1100-1500 nm fluorescence using 850 nm and 1100 nm long-pass filters, high-resolution, high-signal-to-noise ratio and high-definition imaging of the mouse gastrointestinal tract can be achieved as shown in Figure 6 .

[0028] In addition, the effects of anesthetics on the intestinal peristalsis of mice are studied according to the different fluorescence signals, by gavage of the water-soluble near-infrared two-region fluorescent dye DPA-2SO3H aqueous solution to mice, then anesthetizing the mice with anesthetics, irradiating the mouse abdomen with an 808 nm external laser, and collecting 1100-1500 nm fluorescence using 850 nm and 1100 nm long-pass filters, it can be used for research related to the process of mouse gastrointestinal peristalsis as shown in Figure 7 . BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1 Normalized absorption spectrum of near-infrared two-region dye DPAs in DMSO solution (corresponding to Example 1).

[0030] Figure 2 Normalized fluorescence emission spectrum of near-infrared two-region dye DPAs in DMSO solution (corresponding to Example 1).

[0031] Figure 3 Normalized absorption and fluorescence emission spectrum of near-infrared two-region dye DPA-3 in DMSO solution (corresponding to Example 1).

[0032] Figure 4 Normalized absorption and emission spectrum of near-infrared two-region dye DPA-2SO3H in DMSO solution (corresponding to Example 2).

[0033] Figure 5 Normalized absorption and emission spectrum of near-infrared two-region dye DPA-2SO3H in aqueous solution (corresponding to Example 2).

[0034] Figure 6 In vivo near-infrared two-region fluorescence imaging map of near-infrared two-region dye DPA-2SO3H for non-invasive gastrointestinal tract imaging of mice.

[0035] Figure 7 In vivo near-infrared two-region fluorescence imaging map of near-infrared two-region dye DPA-2SO3H for imaging of gastrointestinal tract of anesthetized mice for studying real-time detection of intestinal peristalsis. DETAILED DESCRIPTION

[0036] In order to make the objectives, technical solutions and advantages of the present application clearer, the present application is described below with the following specific examples, but the present application is not limited to these examples. The following description is only a better embodiment of the present application, which is used to explain the present application, and cannot be understood as a limitation on the scope of the present application. It should be noted that any modification, substitution or improvement made within the spirit and principles of the present application should be included in the protection scope of the present application.

[0037] Example 1:

[0038] The preparation route of the near-infrared two-region fluorescent dye DPAs is as follows:

[0039]

[0040] wherein the R group is selected from methyl (-CH3), methoxy (-OCH3), tert-butyl (-C(CH3)3), fluorine (-F), sulfonate (-SO3H). The specific steps are as follows:

[0041] (1) Synthesis of intermediate 1:

[0042] First, compound 2,7-dibromo-9,9-dimethyl-9,10-dihydroacridine (0.583 g, 1.6 mmol) and di-tert-butyl dicarbonate (0.768 g, 3.52 mmol) were dissolved in a tetrahydrofuran solution, 4-dimethylaminopyridine (30 mg, 0.24 mmol) was added, and the reaction was carried out at 80°C for 12 hours; after cooling and concentration, the intermediate product 1 was separated and purified by silica gel column chromatography; the yield was 0.707 g, 95%. 1 H NMR (400 MHz, Chloroform-d) δ 7.49 (d, J = 2.5 Hz, 3H), 7.46 (s, 1H), 7.37 (d, J = 2.2 Hz, 1H), 7.35 (d, J = 2.1 Hz, 1H), 1.52 (s, 6H), 1.51 (s, 9H). 13C NMR (101 MHz, Chloroform-d) δ 152.20, 142.95, 137.37, 129.19, 127.26, 126.53, 119.13, 82.76, 38.10, 28.47. HRMS (ESI) m / z:

[0043] [M-C(CH3)3] + Calcd:C 16 H 14 Br2NO2 + , 411.9371; Found 411.9373.

[0044] (2) Synthesis of Intermediate 2:

[0045] Compound 1 (232 mg, 0.5 mmol, 1 eq) and substituted diphenylamine (1.5 mmol, 3 eq) were added to the reaction flask, sodium tert-butoxide (1.5 mmol, 3 eq) was added, tris(dibenzylideneacetone)dipalladium (46 mg, 0.05 mmol, 10% mmol) and 2-biscyclohexylphosphino-2',4',6'-triisopropylbiphenyl (47.6 mg, 0.1 mmol, 20% mmol) were added. The reaction flask was evacuated and filled with argon repeatedly three times, then anhydrous dioxane was added by syringe. The mixture was reacted at 100 °C for 24 hours. After the reaction was completed, the organic solvent was removed under reduced pressure, and the reaction mixture was washed with dichloromethane and water three times, the combined organic phase was dried with anhydrous sodium sulfate, and finally purified by column chromatography to obtain intermediates 2a-2f.

[0046] 2a (82%), 1 H NMR (400 MHz, Chloroform-d) δ 7.48 (d, J = 8.6 Hz, 2H), 7.23 (dd, J = 8.5, 7.3 Hz, 8H), 7.11 - 7.05 (m, 10H), 7.02 - 6.92 (m, 6H), 1.54 (s, 9H), 1.34 (s, 6H). 13 C NMR (101 MHz, Chloroform-d) δ 153.09, 148.15, 145.17, 142.35, 133.89, 129.48, 126.39, 124.17, 122.81, 122.29, 119.36, 81.98, 38.17, 28.68. HRMS (ESI) m / z: [M+H] + Calcd:C 44 H 42 N3O2, 644.3272; Found 644.3270.

[0047] 2b (83%) 1 H NMR (400 MHz, Chloroform-d) δ 7.45 (d, J = 8.6 Hz, 2H), 7.20 (t, J = 7.7 Hz, 4H), 7.09 - 7.02 (m, 10H), 6.99 (d, J = 8.1 Hz, 4H), 6.97 - 6.88 (m, 4H), 2.31 (s, 6H), 1.53 (s, 9H). 13 C NMR (101 MHz, Chloroform-d) δ 153.15, 148.41, 145.57, 145.35, 142.29, 133.59, 132.77, 130.19, 129.38, 126.30, 124.95, 123.57, 122.31, 121.77, 118.84, 81.87, 38.18, 28.69, 21.19. HRMS (ESI) m / z: [M + H] + Calcd: C 73.62, H 5.62, N 8.62; Found C 73.64, H 5.63, N 8.63. 46 H 46 N3O2, 672.3585; Found 672.3586.

[0048] 2c (67%) 1 H NMR (400 MHz, Chloroform-d) δ 7.38 (d, J = 8.9 Hz, 2H), 7.06 - 6.99 (m, 8H), 6.94 (d, J = 2.6 Hz, 2H), 6.83 - 6.76 (m, 10H), 3.79 (s, 12H), 1.52 (s, 9H), 1.30 (s, 6H). 13 C NMR (101 MHz, Chloroform-d) δ 155.65, 153.23, 146.05, 142.10, 141.58, 132.29, 126.25, 126.00, 119.20, 116.26, 114.79, 81.63, 55.73, 38.11, 28.65. HRMS (ESI) m / z: [M + H] + Calcd: C 73.62, H 5.62, N 8.62; Found C 73.64, H 5.63, N 8.63. 48 H 50 N3O6, 764.3694; Found 764.3698.

[0049] 2e (65%) 1H NMR (400 MHz, Chloroform-d) δ 7.45 (d, J = 8.7 Hz, 2H), 7.24 - 7.19 (m, 8H), 7.10 (d, J = 2.5 Hz, 2H), 7.04 - 6.97 (m, 8H), 6.91 (dd, J = 8.7, 2.4 Hz, 2H), 1.53 (s, 9H), 1.35 (s, 6H), 1.30 (s, 36H). 13 C NMR (101 MHz, Chloroform-d) δ 153.18, 145.58, 145.35, 145.33, 142.28, 133.57, 126.25, 126.19, 123.62, 121.87, 118.89, 81.72, 38.19, 34.54, 31.82, 28.72, 27.25. HRMS (ESI) m / z: [M+H] + Calcd: C 60 H 74 N3O2, 868.5776; Found 868.5799.

[0050] 2f (68%) 1 H NMR (400 MHz, Chloroform-d) δ 7.47 (d, J = 8.7 Hz, 2H), 7.24 - 7.19 (m, 4H), 7.09 - 7.00 (m, 10H), 7.00 - 6.88 (m, 8H), 1.53 (s, 9H), 1.33 (s, 6H). 13 C NMR (101 MHz, Chloroform-d) δ 160.30, 157.89, 153.06, 148.22, 145.25, 144.19, 144.17, 142.36, 133.76, 129.50, 126.60, 126.52, 126.42, 123.42, 122.59, 121.68, 118.75, 116.43, 116.21, 82.01, 38.16, 28.64. HRMS (ESI) m / z: [M+H] + Calcd: C 44 H 40 F2N3O2, 702.2903; Found 702.2918.

[0051] (3) Synthesis of near-infrared dye molecule DPA:

[0052] The intermediate compound 2 was dissolved in a mixed solvent of dichloromethane and trifluoroacetic acid, and reacted at room temperature for 3 hours. After the reaction was completed, the reaction solution was extracted with dichloromethane and washed with water three times. The organic phase was dried over anhydrous sodium sulfate and concentrated, and then purified by column chromatography to obtain the target near-infrared two-region dye molecule DPA.

[0053] DPA-1 (39%), 1 H NMR (400 MHz, Chloroform-d) δ 7.69 (d, J = 9.3 Hz, 2H), 7.54 (t, J = 7.6 Hz,

[0054] 8H), 7.45 (t, J = 7.5 Hz, 4H), 7.36 (dd, J = 7.6, 1.6 Hz, 8H), 7.07 (d, J = 2.5 Hz, 2H), 7.01 (d, J = 9.0 Hz, 2H), 1.37 (s, 6H). HRMS (ESI) m / z: [M+H] + Calcd: C 39 H 33 N3 + , 543.2669; Found 543.2649.

[0055] DPA-2 (42%), 1 H NMR (400 MHz, Chloroform-d) δ 7.67 (d, J = 9.2 Hz, 2H), 7.52 (t, J = 7.6

[0056] Hz, 4H), 7.44 (t, J = 7.3 Hz, 2H), 7.32 (d, J = 7.9 Hz, 8H), 7.20 (d, J = 8.1 Hz, 4H), 7.06 (s, 2H), 6.99 (d, J = 8.9 Hz, 2H), 2.43 (s, 6H), 1.36 (s, 6H). HRMS (ESI) m / z: [M+H] + Calcd: C 41 H 36 N3 + , 570.2904; Found 570.2920.

[0057] DPA-3 (45%), 1 H NMR (400 MHz, Chloroform-d) δ 7.61 (d, J = 9.3 Hz, 2H), 7.23 (dd, J = 8.7,

[0058] 1H NMR (400 MHz, DMSO-d6) δ 8.02 (d, J = 8.2 Hz, 4H), 7.62 (d, J = 8.2 Hz, 4H), 7.06 - 6.99 (m, 10H), 6.99 - 6.93 (m, 2H), 3.88 (d, J = 1.1 Hz, 12H), 1.37 (s, 6H). HRMS (ESI) m / z: [M+H] + Calcd: C 73.49; H 4.85; N 21.66. 43 Found: C 73.48; H 4.86; N 21.66. 41 N3O4 + , 662.3013; Found 662.2995.

[0059] DPA-4 (37%), 1 H NMR (400 MHz, Chloroform-d) δ 7.65 (d, J = 9.2 Hz, 2H), 7.50 (d, J = 8.3

[0060] 1.29 (s, 6H). HRMS (ESI) m / z: [M+H] + Calcd: C 73.49; H 4.85; N 21.66. 39 Found: C 73.48; H 4.86; N 21.66. 33 N3O4 + , 606.2387; Found 606.2386.

[0061] DPA-5 (47%), 1 H NMR (400 MHz, Chloroform-d) δ 7.65 (d, J = 9.2 Hz, 2H), 7.50 (d, J = 8.3

[0062] Hz, 8H), 7.23 (d, J = 8.4 Hz, 8H), 7.06 (d, J = 2.5 Hz, 2H), 7.00 (t, J = 7.5 Hz, 2H), 1.38 (s, 6H), 1.36 (s, 36H). HRMS (ESI) m / z: [M+H] + Calcd: C 73.49; H 4.85; N 21.66. 55 Found: C 73.48; H 4.86; N 21.66. 65 N3 + , 767.5173; Found 767.5159.

[0063] DPA-6 (37%), 1 H NMR (400 MHz, Chloroform-d) δ 7.81 - 7.30 (m, 16H), 7.24 (t, J = 8.3 Hz,

[0064] 4H), 7.11 - 6.88 (m, 4H), 1.38 (s, 6H). HRMS (ESI) m / z: [M+H] + Calcd: C 73.49; H 4.85; N 21.66. 39 Found: C 73.48; H 4.86; N 21.66. 31 F2N3+ 579.2481 ; Found 579.2456.

[0065] Example 2:

[0066] The preparation of water-soluble near-infrared two-region fluorescent dye DPA-2SO3H, the structural formula of the compound is as follows:

[0067]

[0068] The specific synthesis route is as follows:

[0069]

[0070] The specific synthesis steps are as follows:

[0071] (1) Synthesis of intermediate 1:

[0072] In a three-necked round-bottom flask equipped with a magnetic stirrer, 4-hydroxydiphenylamine (1.85 g, 10 mmol), tert-butyl bromoacetate (2.34 g, 12 mmol) and potassium carbonate (4.14, 30 mmol) were added in sequence, acetonitrile was added as the reaction solvent, the mixture was refluxed under N2 protection for 3 hours. The solvent was removed under reduced pressure, extracted with dichloromethane and water three times, and finally purified by column chromatography to obtain white solid intermediate compound 1; yield 2.680 g, 89.6%. 1 H NMR (400 MHz, Chloroform-d) δ 7.22 (t, J = 7.7 Hz, 2H), 7.05 (d, J = 8.3 Hz, 2H), 6.93 (d, J = 8.0 Hz, 2H), 6.85 (d, J = 8.5 Hz, 3H), 4.49 (s, 2H), 1.50 - 1.49 (m, 9H). 13 C NMR (101 MHz, CDC13) δ 168.57, 153.56, 145.00, 136.99, 129.57, 121.68, 120.04, 116.28, 115.85, 82.54, 66.53, 28.34. HRMS (ESI) m / z: [M+H]+Calcd: C 18 H 22 NO3, 300.1600; Found 300.1609.

[0073] (2) Synthesis of intermediate 2:

[0074] First, intermediate compound 1 (232 mg, 0.5 mmol, 1 eq), compound 3 (450 mg, 1.5 mmol, 3 eq), CsCO3(1.5 mmol, 3 eq), Pd2(OAc)2(12 mg, 0.05 mmol, 10% mmol) and BINAP (62 mg, 0.1 mmol, 20% mmol) were sequentially added into a dry reaction bottle, the reaction bottle was evacuated with an oil pump and then flushed with argon, the operation was repeated three times, and then anhydrous toluene was added as the reaction solvent by a syringe, and the reaction was refluxed at 100 °C for 20-24 hours. After the reaction was completed, the solvent was removed by rotary evaporation under reduced pressure, and the reaction mixture was washed with dichloromethane and water three times, the organic phase was combined and dried with anhydrous sodium sulfate. Finally, further separation and purification by column chromatography (eluent: PE / EtOAc = 50:10-10:1) to obtain intermediate 2; 1 H NMR (400 MHz, Chloroform-d) δ 7.44 (d, J = 8.7 Hz, 2H), 7.22 - 7.16 (m, 4H), 7.03 (td, J = 10.4, 9.8, 5.0 Hz, 10H), 6.95 - 6.86 (m, 4H), 6.84 - 6.78 (m, 4H), 4.50 (s, 4H), 1.53 (s, 9H), 1.49 (s, 18H), 1.32 (s, 6H). 13 C NMR (101 MHz, Chloroform-d) δ 168.38, 154.40, 153.07, 148.39, 145.30, 142.19, 141.84, 133.34, 129.27, 126.92, 126.16, 122.82, 121.91, 121.24, 118.32, 115.72, 82.58, 81.82, 66.30, 38.07, 28.58, 28.31. HRMS (ESI) m / z: [M] + Calcd: C 56 H 61 N3O8, 903.4459; Found 903.4540, [M-Boc] + 803.3991.

[0075] (3) Synthesis of water-soluble near-infrared two-region dye DPA-2SO3H

[0076] In a three-necked round bottom flask equipped with a magnetic stirrer, intermediate compound 2 (360 mg, 0.4 mmol) was dissolved in ethanol, then potassium hydroxide (67 mg, 1.2 mol) dissolved in water was added to the reaction system, then the reaction was refluxed at 60 °C for 3 hours. After the reaction was completed, the organic solvent was removed under reduced pressure, and the residue was added to diethyl ether to obtain a precipitate intermediate, which was dried and directly used for the next reaction. Then, TSTU (1.5 eq), DIPEA (3 eq) were added in turn, anhydrous DMF was used as the reaction solvent, then the reaction was stirred at room temperature under N2protection. When the mass spectrum tracking monitoring showed complete conversion to the activated NHS ester product, 2-aminoethanesulfonic acid (20 eq) was added, and the reaction was continued for 24 hours. Finally, the solvent was removed by rotary evaporation under reduced pressure, and a mixture of CH2Cl2and trifluoroacetic acid was added, and the reaction was stirred at room temperature for 3 hours. After the reaction was completed, it was separated and purified by reverse phase column (filling material C18) to obtain the target product DPA-2SO3H. 1 H NMR (400 MHz, DMSO-d6) δ 8.35 (d, J = 5.5 Hz, 2H), 7.77 (d, J = 9.2 Hz, 2H), 7.58 (t, J = 7.7 Hz, 4H), 7.46 (dd, J = 19.5, 7.9 Hz, 10H), 7.13 (d, J = 8.4 Hz, 4H), 7.05 - 6.98 (m, 2H), 6.89 (d, J = 8.7 Hz, 2H), 4.54 (s, 4H), 3.36 (s, 4H), 2.58 (t, J = 6.7 Hz, 4H), 1.30 (s, 6H). HRMS (ESI) m / z: [M] + Calcd: C 47 H 46 N5O 10 S2 + , 904.2686; Found 904.2661.

[0077] Example 3:

[0078] The water-soluble near-infrared two-region fluorescent dye DPA-2SO3H was used for near-infrared two-region fluorescent bioimaging of mouse gastrointestinal tract. The specific steps are as follows:

[0079] By intragastrically injecting 200 μL of DPA-2SO3H solution with a concentration of 100 μM into the mouse, and irradiating the mouse abdomen with an 808 nm external laser, the power density of the laser was 50 mW / cm2. 2 The fluorescence of 1000-1500 nm was collected using 850 nm and 1100 nm long-pass filters, and the fluorescence signals of different tumors were used to monitor the mouse's stomach, small intestine and other organs (as shown in FIG. 6). Figure 5The NIR-II fluorescence imaging of the mouse gastrointestinal tract was non-invasively imaged by using the NIR-II dye DPA-2SO3H (see FIG. 1). The NIR-II fluorescence imaging of the mouse gastrointestinal tract was non-invasively imaged by using the NIR-II dye DPA-2SO3H (see FIG. 1). Figure 6 The NIR-II fluorescence imaging of the mouse gastrointestinal tract was non-invasively imaged by using the NIR-II dye DPA-2SO3H (see FIG. 1). The NIR-II fluorescence imaging of the mouse gastrointestinal tract was non-invasively imaged by using the NIR-II dye DPA-2SO3H (see FIG. 1). Figure 5 The NIR-II fluorescence imaging of the mouse gastrointestinal tract was non-invasively imaged by using the NIR-II dye DPA-2SO3H (see FIG. 1). The NIR-II fluorescence imaging of the mouse gastrointestinal tract was non-invasively imaged by using the NIR-II dye DPA-2SO3H (see FIG. 1).

[0080] Example 4:

[0081] The NIR-II fluorescence imaging of the mouse gastrointestinal tract was non-invasively imaged by using the NIR-II dye DPA-2SO3H (see FIG. 1). The NIR-II fluorescence imaging of the mouse gastrointestinal tract was non-invasively imaged by using the NIR-II dye DPA-2SO3H (see FIG. 1).

[0082] The NIR-II fluorescence imaging of the mouse gastrointestinal tract was non-invasively imaged by using the NIR-II dye DPA-2SO3H (see FIG. 1). The NIR-II fluorescence imaging of the mouse gastrointestinal tract was non-invasively imaged by using the NIR-II dye DPA-2SO3H (see FIG. 1). 2 The NIR-II fluorescence imaging of the mouse gastrointestinal tract was non-invasively imaged by using the NIR-II dye DPA-2SO3H (see FIG. 1). The NIR-II fluorescence imaging of the mouse gastrointestinal tract was non-invasively imaged by using the NIR-II dye DPA-2SO3H (see FIG. 1). Figure 7 The NIR-II fluorescence imaging of the mouse gastrointestinal tract was non-invasively imaged by using the NIR-II dye DPA-2SO3H (see FIG. 1). The NIR-II fluorescence imaging of the mouse gastrointestinal tract was non-invasively imaged by using the NIR-II dye DPA-2SO3H (see FIG. 1).

Claims

1. A near-infrared II fluorescent dye molecule, characterized in that, Its structure is shown in Formula II: The water-soluble near-infrared II fluorescent dye molecule is denoted as DPA-2SO3H; 。 2. A method for preparing a near-infrared II fluorescent dye molecule as described in claim 1, characterized in that, Its synthetic route is as follows: 。 3. The application of the near-infrared II fluorescent dye molecule as described in claim 1 in medical imaging of deep tissues of organisms, wherein the application is not for the purpose of disease diagnosis or treatment.

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