Preparation and application of targeted near-infrared fluorescent probes based on cyanine dyes

Abstract

The application discloses preparation and application of a targeting near-infrared fluorescent probe based on a cyanine dye, and relates to the field of fluorescent probes. In view of the problems of indocyanine green, a targeting cyanine structure skeleton is synthesized, the quantum efficiency of the targeting cyanine structure skeleton in a PBS solution for biological imaging is three times that of conventional indocyanine green dye, and in addition, biotin is a vitamin necessary for the growth of all cell division, especially cancer cells, and the application selects biotin as a final model verification, and good targeting effect is achieved in breast cancer tumor-bearing mice.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of fluorescent probes, and particularly relates to preparation and application of a targeting near-infrared fluorescent probe based on a cyanine dye. BACKGROUND

[0002] Fluorescence navigation precise tumor resection has been applied in various clinical operations. At present, the most widely used is the near-infrared region one dye indocyanine green (ICG), but it has no targeting type for tumors, although it has been reported that its signal is 2.5 times stronger in the tumor site than in the surrounding normal tissue, but the differentiation degree is not high. Introducing a targeting group into the near-infrared dye has become a trend of current development. There are two commonly used cyanine skeletons that can introduce targeting groups at present, one is a seven-methine cyanine structure with asymmetric end groups, one end is a sulfonic acid group to give the dye water solubility and metabolic capacity in vivo imaging, and the other end is a reaction site that can access the target, such as carboxyl, alkyne, azide, etc.; the other is to introduce a rigid ring with Cl, Br and other halogens in the middle position of the seven-methine cyanine, and then access the subsequent target by coupling reaction with transition metals or substitution reaction with N, O, S and other strong nucleophilic groups. But the first skeleton has a very low yield due to its asymmetric structure, for example, the cyanine molecule synthesized by Draney, D.R et al. resulted in a final reaction yield of only 30% due to the formation of symmetric cyanine structures IR-783 and MHI148 in the reaction. The second cyanine skeleton requires specific metal-catalyzed coupling reactions or strong nucleophilic groups to access the target, which has requirements for the structure of the target, and needs to modify the coupling reaction structure of the target or have strong nucleophilic N, O, S and other functional groups with certain limitations. Therefore, it is urgent to develop new materials with a single reaction site for connecting a targeting unit while maintaining the universality of target access. In addition, although ICG has been reported to use its emission spectrum tail peak in the near-infrared region two for clinical fluorescence surgery navigation, since its overall emission spectrum is still in the near-infrared region one, it is still of clinical significance to improve its overall brightness. In order to emit fluorescence in the near-infrared region, the fluorophore usually has a large conjugated skeleton to stimulate low band gap, which spontaneously induces the interaction between the conjugated skeleton and other molecules to be enhanced, resulting in low quantum yield, especially in water system. Therefore, the present application introduces a length of alkyl chain into the cyanine skeleton to effectively improve the overall brightness of the entire cyanine fluorophore in the near-infrared region. SUMMARY

[0003] The present application aims to provide a preparation and application of a near-infrared fluorescence probe based on a cyanine dye, the synthesis raw material of the preparation method is easy to obtain, the synthesis operation is simple, and the preparation is convenient; in addition, a reaction site of N3 is introduced, so that a subsequent targeting group is conveniently connected; and a length of alkyl chains is introduced into the intermediate carbon chain skeleton, so that the fluorescence quantum yield of the probe is greatly improved.

[0004] The technical scheme adopted by the present application to achieve the above-mentioned purpose is:

[0005] A cyanine fluorescent dye, namely ICG-AlkylN3, has a structure as shown in formula I:

[0006] I;

[0007] In the formula, R is selected from an alkyl group.

[0008] The present application generates a necessary intermediate of a transition state generated according to Zincke reaction (a reaction of N-(2,4-dinitrophenyl) pyridine salt with aniline or an aliphatic amine to perform amine exchange to generate a corresponding N-aryl or N-alkyl pyridine salt) and performs nucleophilic reaction with p-bromoaniline to generate a heptamethine cyanine, then performs condensation with a quaternary ammonium heterocycle, to generate a cyanine skeleton with a single reaction site connected with a targeting unit in a one-pot method in series, and a benzindole with a sulfonic acid group is selected as the heterocycle structure at both ends according to the actual needs of in-vivo imaging, and a targeting reaction site azide and a length of alkyl chains are introduced at the 3rd position of the cyanine carbon chain, to finally design the cyanine structure ICG-AlkylN3 with targeting ability and higher brightness.

[0009] The present application further discloses a preparation method of the cyanine fluorescent dye, comprising:

[0010] Step one, hydrolysis of 1-chloro-2,4-dinitrobenzene to prepare 2,4-dinitrophenol;

[0011] Step two, substitution reaction of 2,4-dinitrophenol and p-toluenesulfonyl chloride to prepare 2,4-dinitrobenzene p-methylsulfonic acid toluene;

[0012] Step three, amidation reaction of 3-aminopyridine and bromoalkyl acid, and substitution reaction of sodium azide, to prepare a pyridine derivative;

[0013] Step four, preparation of a pyridine salt by using 2,4-dinitrobenzene p-methylsulfonic acid toluene and the pyridine derivative;

[0014] Step five, preparation of ICG-AlkylN3 by using the pyridine salt and an amine compound.

[0015] Specifically, the preparation method of the cyanine fluorescent dye comprises the following steps:

[0016] Step 1, 1-chloro-2,4-dinitrobenzene is added to a 1.3-1.8 M aqueous NaOH solution, and reacted at 95-105°C for 1.5-2 h. Excess NaOH is neutralized by adding acetic acid, followed by saturated brine and ethyl acetate (EA) for extraction. Finally, anhydrous sodium sulfate is used for drying, and rotary evaporation is performed to obtain 2,4-dinitrophenol;

[0017] Step 2, 2,4-dinitrophenol is dissolved in dichloromethane (1 g:20-35 mL), and p-toluenesulfonyl chloride and triethylamine are added sequentially. The reaction is carried out at room temperature for 15-20 h. Water is added, and dichloromethane is used for extraction. The organic phase is washed with saturated brine, dried with anhydrous sodium sulfate, and the organic solvent is evaporated under reduced pressure. Methanol is used for washing to obtain 2,4-dinitrobenzene p-toluenesulfonic acid methyl.

[0018] Step 3, bromoalkyl acid is dissolved in anhydrous dry dichloromethane (1 g:6-10 mL), and a catalytic equivalent of N,N-dimethylformamide is added. Then oxalyl chloride is slowly added. After stirring at room temperature for 25-50 min, the residue is dissolved in anhydrous N,N-dimethylformamide (1 g:4-6 mL), and triethylamine is added. Then 3-aminopyridine is dissolved in anhydrous dry N,N-dimethylformamide (1 g:2.5-3 mL), and then slowly added to the above reaction system. The reaction mixture is stirred at room temperature for 1.5-2.5 h. Sodium azide (NaN3) is added, and stirred overnight under nitrogen. The reaction is quenched with water, extracted with dichloromethane, and the organic layer is washed with water. Anhydrous sodium sulfate is used for drying, and the organic solvent is evaporated under reduced pressure. Column chromatography is performed to obtain the pyridine derivative.

[0019] Step 4, 2,4-dinitrobenzene p-toluenesulfonic acid methyl and the pyridine derivative are dissolved in toluene (1 g:2-2.8 mL), and refluxed for 15-20 h. After cooling to room temperature, filtration is performed, and toluene and diethyl ether (Et2O) are used for washing. Drying is performed to obtain the pyridine salt.

[0020] Step 5, the pyridine salt and amine compound are dissolved in methanol, and stirred at room temperature for 25-35 min. Then 1,1,2-trimethyl-3-(4-sulfonic acid propyl)-1H-benzo[E]indolium inner salt and sodium acetate are added. The reaction mixture is stirred at room temperature for another 15-20 h. After completion, diethyl ether is added, and placed in a -15°C to -18°C freezer. After freezing treatment for 20-30 h, filtration is performed, and washed with water and diethyl ether. Drying is performed, and column chromatography is performed to obtain the product ICG-AlkylN3.

[0021] Preferably, in step one, the mass-volume ratio of 1-chloro-2,4-dinitrobenzene to NaOH aqueous solution is 1g:3-5mL; the volume ratio of saturated brine to ethyl acetate is 1:1.5-2.5.

[0022] Preferably, in step two, the molar ratio of 2,4-dinitrophenol to p-toluenesulfonyl chloride is 1:1-1.3; the molar ratio of 2,4-dinitrophenol to triethylamine is 1:2.3-3.

[0023] Preferably, in step three, the molar ratio of bromoalkyl acid to oxalyl chloride is 1:1.3-1.8; the molar ratio of bromoalkyl acid to triethylamine is 1:0.8-1.2; the molar ratio of bromoalkyl acid to 3-aminopyridine is 1:0.8-1.2; the molar ratio of bromoalkyl acid to sodium azide is 1:1.7-2.5.

[0024] Preferably, in step four, the molar ratio of 2,4-dinitrophenyl p-tolyl methyl sulfonic acid to pyridine derivative is 1:0.6-1.

[0025] Preferably, in step five, the molar ratio of pyridine salt to amine compound is 1:1.1-1.4; the mass-volume ratio of pyridine salt to methanol is 1g:45-55mL; the molar ratio of pyridine salt to 1,1,2-trimethyl-3-(4-sulfopropyl)-1H-benzo[E]indolium inner salt is 1:2.5-4; the molar ratio of pyridine salt to sodium acetate is 1:5-6.5.

[0026] Preferably, the amine compound is selected from one of aniline or aliphatic amine.

[0027] The application also discloses a use of the cyanine fluorescent dye prepared by the preparation method in preparation of a targeted near-infrared fluorescent probe.

[0028] The application aims at two problems of indocyanine green, one is insufficient brightness in the near-infrared two area, and the other is lack of access sites of a targeted group. In combination with a reported synthesis method and actual in-vivo imaging needs, a targeted cyanine structure skeleton is designed and synthesized, quantum efficiency of which in PBS solution suitable for biological imaging is three times of that of conventional indocyanine green dye. In addition, biotin is a vitamin necessary for all cell division, especially cancer cell growth. Studies have shown that biotin receptors are overexpressed in various tumor cells such as ovarian cancer, lung cancer and breast cancer. In the application, biotin is selected as a final model verification, and plays a good targeting role in breast cancer tumor-bearing mice.

[0029] The application further discloses a preparation method of a targeted near-infrared fluorescent probe, comprising the following steps:

[0030] The target-type near-infrared fluorescent probe is prepared by subjecting 5-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanoic acid and tert-butyl 2-(2-(2-aminoethoxy)ethoxy)ethylcarbamate to an amidation reaction, then to a hydrolysis reaction under acidic conditions, followed by an amidation reaction with diphenylcyclooctyne-carboxylic acid, and then an addition reaction with the above cyanine fluorescent dye.

[0031] Specifically, the preparation method of the target-type near-infrared fluorescent probe includes the following steps:

[0032] The 5-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanoic acid and tert-butyl 2-(2-(2-aminoethoxy)ethoxy)ethylcarbamate are dissolved in N,N-dimethylformamide, stirred at room temperature for 15-20 h, extracted with dichloromethane, the organic layer is washed with deionized water, dried with anhydrous sodium sulfate, evaporated under reduced pressure, and then subjected to column chromatography to obtain product 6. Then, the product 6 is added to a dichloromethane solution of 8-12 wt% trifluoroacetic acid and reacted for 5-8 h, washed with saturated sodium bicarbonate (NaHCO3) solution for 2-3 times, washed with deionized water for 2-3 times, dried with anhydrous sodium sulfate, evaporated under reduced pressure, and then subjected to column chromatography to obtain a crude product. Diphenylcyclooctyne-carboxylic acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide are added to the crude product, and then dissolved in anhydrous N,N-dimethylformamide (DMF). After reacting at room temperature for 13-20 h, the reaction mixture is extracted with dichloromethane, the organic layer is washed with water, dried with anhydrous sodium sulfate, and evaporated under reduced pressure to obtain product 7. Then, the product 7 is dissolved in dichloromethane, and then the cyanine fluorescent dye is added and reacted for 20-30 h. After column chromatography, the target-type near-infrared fluorescent probe is obtained.

[0033] Preferably, the molar ratio of the above-mentioned 5-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanoic acid to tert-butyl 2-(2-(2-aminoethoxy)ethoxy)ethylcarbamate is 1:0.8-1.3; the mass-volume ratio of tert-butyl 2-(2-(2-aminoethoxy)ethoxy)ethylcarbamate to N,N-dimethylformamide is 1g:45-55mL; the mass-volume ratio of product 6 to a dichloromethane solution of trifluoroacetic acid is 1g:90-110mL; the mass ratio of the crude product to diphenylcyclooctyne-carboxylic acid is 1:0.8-1; the molar ratio of diphenylcyclooctyne-carboxylic acid to 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride is 1:0.9-1.3; the molar ratio of diphenylcyclooctyne-carboxylic acid to N-hydroxysuccinimide is 1:0.9-1.3; and the molar ratio of product 7 to a cyanine fluorescent dye is 1:0.8-1.3.

[0034] A fluorescent imaging reagent, comprising the targeting near-infrared fluorescent probe prepared by the preparation method.

[0035] The beneficial effects of the present application include:

[0036] The present application obtains the preparation and application of a cyanine dye-based targeting near-infrared fluorescent probe, the present application designs and synthesizes a targeting cyanine structure skeleton in view of the problems of indocyanine green, the quantum efficiency of the present application is three times that of conventional indocyanine green dye in PBS solution for biological imaging, in addition, biotin is a vitamin necessary for all cell division, especially cancer cell growth, the present application selects biotin as the last model verification, and plays a good targeting role in breast cancer tumor-bearing mice.

[0037] Therefore, the present application provides the preparation and application of a cyanine dye-based targeting near-infrared fluorescent probe, the synthetic raw materials of the preparation method are easy to obtain, the synthesis operation is simple, and the production is convenient; in addition, the reaction site of N3 is introduced, which is convenient for subsequent introduction of a targeting group; and a length of alkyl chain is introduced into the intermediate carbon chain skeleton, so that the fluorescence quantum yield of the probe is greatly improved. BRIEF DESCRIPTION OF DRAWINGS

[0038] Figure 1 The hydrogen spectrum test result of 2,4-dinitrophenol prepared for Example 1;

[0039] Figure 2 The carbon spectrum test result of 2,4-dinitrophenol prepared for Example 1;

[0040] Figure 3 The hydrogen spectrum test result of 2,4-dinitrophenol prepared for Example 1;

[0041] Figure 4 Test results of the carbon spectrum of 2,4-dinitrobenzene p-toluenesulfonic acid prepared for Example 1;

[0042] Figure 5 Test results of the hydrogen spectrum of the pyridine derivative prepared for Example 1;

[0043] Figure 6 Test results of the carbon spectrum of the pyridine derivative prepared for Example 1;

[0044] Figure 7 Test results of the hydrogen spectrum of the pyridine salt prepared for Example 1;

[0045] Figure 8 Test results of the carbon spectrum of the pyridine salt prepared for Example 1;

[0046] Figure 9 Test results of the hydrogen spectrum of ICG-AlkylN3 prepared for Example 1;

[0047] Figure 10 Test results of the carbon spectrum of ICG-AlkylN3 prepared for Example 1;

[0048] Figure 11 Test results of the hydrogen spectrum of Product 6 prepared for Example 1;

[0049] Figure 12 Test results of the carbon spectrum of Product 6 prepared for Example 1;

[0050] Figure 13 Test results of the hydrogen spectrum of Product 7 prepared for Example 1;

[0051] Figure 14 Test results of the carbon spectrum of Product 7 prepared for Example 1;

[0052] Figure 15 Test results of the hydrogen spectrum of Product 8 prepared for Example 1;

[0053] Figure 16 Test results of the carbon spectrum of Product 8 prepared for Example 1;

[0054] Figure 17 Test results of the fluorescence spectrum;

[0055] Figure 18 Test results of the absorption spectrum;

[0056] Figure 19 Test results of the quantum efficiency;

[0057] Figure 20 Test results of the molar absorption coefficient;

[0058] Figure 21 The test results of the luminance of the near-infrared two-zone imaging system;

[0059] Figure 22 The test results of the tumor imaging effect of the fluorescent probe in a tumor-bearing mouse model. DETAILED DESCRIPTION

[0060] In order to make the objects, technical solutions and advantages of the present application clearer and more apparent, the technical solutions of the present application are further described in detail below with reference to specific embodiments:

[0061] Embodiment 1:

[0062] A preparation method of a fluorescent dye of a cyanine type, and a synthetic route is as follows:

[0063] .

[0064] A preparation method of a fluorescent dye of a cyanine type, and specific steps include:

[0065] Step one, 14.81 mmol of 1-chloro-2,4-dinitrobenzene is added to 10 mL of a 1.5 M NaOH aqueous solution, and after 2 h of reaction at 100 ℃, 3 mL of acetic acid is added to neutralize the excess NaOH, followed by extraction with saturated brine and EA (volume ratio of saturated brine to EA is 1:2), and finally dried with anhydrous sodium sulfate, and rotary evaporation to obtain product 1, i.e. 2,4-dinitrophenol, with a yield of 95%;

[0066] 1 H NMR (400 MHz, Chloroform-d) δ 9.01 (d, J = 2.8 Hz, 1H), 8.40 (dd, J =9.2, 2.8 Hz, 1H), 7.28 (d, J = 9.2 Hz, 1H). 13 C NMR (101 MHz, Chloroform-d) δ158.04, 130.63, 120.88, 120.24.

[0067] Step two, 1.95 mmol of 2,4-dinitrophenol is dissolved in 10 mL of dichloromethane, 2.15 mmol of p-toluenesulfonyl chloride and 4.88 mmol of triethylamine are added in sequence, and stirred at room temperature for 16 h, 50 mL of water is added, extracted with dichloromethane twice, 50 mL each time, the organic phase is washed with 50 mL of saturated brine, dried with anhydrous sodium sulfate, the organic solvent is evaporated under reduced pressure, and then washed with 100 mL of methanol to obtain product 2, i.e. 2,4-dinitrobenzene p-toluenesulfonic acid methyl, with a yield of 86%.

[0068] 1 H NMR (400 MHz, Chloroform-d) δ 8.77 (d, J = 2.7 Hz, 1H), 8.49 (dd, J =9.0, 2.7 Hz, 1H), 7.81 (d, J = 8.4 Hz, 2H), 7.74 (d, J = 9.0 Hz, 1H), 7.40 (d, J =8.4 Hz, 2H), 2.50 (s, 3H). 13 C NMR (101 MHz, Chloroform-d) δ 146.20, 144.93, 144.29, 141.50, 129.89, 129.32, 127.71, 127.58, 125.34, 120.60, 20.87.

[0069] Step three, 19.6 mmol of 6-bromopropionic acid was dissolved in 30 mL of dry and anhydrous dichloromethane, a catalytic equivalent (3 drops) of N,N- dimethylformamide was added, followed by the slow addition of 29.4 mmol of oxalyl chloride, after stirring at room temperature for 30 min, the volatiles were evaporated under reduced pressure, the residue was dissolved in 20 mL of anhydrous N,N- dimethylformamide, 19.6 mmol of triethylamine was added, then 19.6 mmol of 3- aminopyridine was dissolved in 5 mL of anhydrous and dry N,N-dimethylformamide, then added dropwise to the above reaction system, the reaction mixture was stirred at room temperature for 2 h, 39.3 mmol of NaN3 was added and stirred under N2 overnight, the reaction was quenched with 50 mL of water, extracted with dichloromethane 5 times, 40 mL each time, the organic layer was washed with water 2 times, 50 mL each time, dried over anhydrous sodium sulfate, the organic solvent was evaporated under reduced pressure, the crude product obtained was column chromatographed to obtain the product 3, i.e. the pyridine derivative, with a yield of 60%;

[0070] 1 H NMR (400 MHz, Chloroform-d) δ 8.91-8.59 (m, 2H), 8.23-8.22 (m, 1H), 7.45-7.41 (m, 1H), 3.49 (t, J = 6.6 Hz, 1H), 3.23 (t, J = 6.6 Hz, 1H), 2.49-2.43 (m, 2H), 1.80-1.18 (m, 2H). 13C NMR (101 MHz, Chloroform-d) δ 170.88, 143.80, 139.90, 134.24, 126.39, 122.86, 43.82, 36.11, 31.20, 25.42, 23.62.

[0071] Step four, dissolve a mixture of 0.618 mmol 2,4-dinitrobenzene p-toluenesulfonic acid methyl and 0.52 mmol of the corresponding product 3 pyridine derivative in 5 mL of toluene, reflux the reaction mixture for 16 h, cool to room temperature, filter the resulting precipitate, wash with toluene and Et20, 2 times each 5 mL, dry to obtain product 4, pyridine salt, yield 70%;

[0072] 1 H NMR (400 MHz, Chloroform-d) δ 9.80-9.71 (m, 1H), 9.28-9.15 (m, 1H), 9.05-8.98 (m, 1H), 8.71-8.55 (m, 2H), 8.40-8.23 (m, 1H), 8.18-8.03 (m, 1H), 7.55 (d, J = 7.8 Hz, 2H), 7.14 (d, J = 7.8 Hz, 2H), 3.50-3.47 (m, 1H), 3.22 (t, J = 6.8 Hz, 1H), 2.36 (s, 3H), 1.76-1.52 (m, 4H), 1.46-1.19 (m, 4H). 13 C NMR (101 MHz, Chloroform-d) δ 170.88, 143.80, 139.90, 134.24, 126.39, 122.86, 43.82, 36.11, 31.20, 25.42, 23.62.

[0073] Step five, dissolve 0.18 mmol of the corresponding pyridine salt and 0.22 mmol of 4-bromoaniline in 5 mL of methanol, stir at room temperature for 30 min, then add 0.54 mmol of 1,1,2-trimethyl-3-(4-sulfopropyl)-1H-benzo[E]indolium inner salt (purchased from Shanghai Bide Pharmaceutical Technology Co., Ltd.) and 1.08 mmol of sodium acetate, stir the reaction mixture at room temperature for another 16 h, after the reaction is completed, add 20 mL of Et2O, put the mixture into a-16 °C refrigeration room for refrigeration treatment for 24 h, filter the obtained precipitate, wash with water twice, each time 10 mL, wash with Et2O twice, each time 10 mL, dry, the obtained crude product is subjected to column chromatography to obtain product 5, that is, ICG-AlkylN3, with a yield of 50%.

[0074] 1 H NMR (400 MHz, Methanol-d4) δ 8.20-8.18 (m, 1H), 8.11-8.06 (m, 3H),8.02-8.00 (m, 2H), 7.89-7.80 (m, 4H), 7.69-7.65 (m, 1H), 7.61-7.55 (m, 2H),7.53-7.46 (m, 3H), 7.38-7.28 (m, 2H),4.35-4.27 (m, 4H), 3.58-3.47 (m, 2H),3.21-3.20 (m, 2H), 2.98-2.88 (m, 6H), 2.57-2.53 (m, 2H), 2.37-2.30 (m, 2H),2.21-2.14 (m, 4H), 1.84 (s, 6H), 1.82 (s, 6H). 13C NMR (101 MHz, Methanol-d4) δ 196.68, 174.04, 139.68, 138.45, 137.27, 133.82, 132.18, 131.87, 131.10, 130.52, 130.34, 129.67, 128.27, 128.12, 127.96, 127.71, 127.38, 127.28, 124.88, 124.45, 122.98, 122.06, 112.54, 56.95, 55.93, 51.10, 48.49, 46.74, 44.75, 42.93, 42.51, 35.61, 32.12, 30.56, 29.36, 28.37, 26.48, 26.38, 26.26, 26.16, 25.29, 25.02, 23.46, 23.19, 22.89, 22.35, 20.99, 19.43, 17.01.

[0075] A preparation method of a targeted near-infrared fluorescent probe, and a synthetic route is as follows:

[0076] .

[0077] A preparation method of a targeted near-infrared fluorescent probe, and a synthetic route is as follows:

[0078] Dissolve 0.278 mmol of 5-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanoic acid (purchased from Shanghai Bide Pharmaceutical Technology Co., Ltd.) and 2-(2-(2-aminoethoxy)ethoxy)ethyl carbamic acid tert-butyl ester 0.278 mmol in 5 mL of DMF, stir at room temperature for 16 h, extract 5 times with dichloromethane, 40 mL each time, wash the organic layer with deionized water twice, 50 mL each time, dry over anhydrous sodium sulfate, evaporate the organic solvent under reduced pressure, and the obtained crude product is subjected to column chromatography to obtain product 6, yield 80%;

[0079] 1 H NMR (400 MHz, Chloroform-d) δ 4.48 (dd, J = 8.0, 4.5 Hz, 1H), 4.29(dd, J = 8.0, 4.5 Hz, 1H), 3.59-3.51 (m, 8H), 3.41 (q, J = 5.2 Hz, 2H), 3.29 (q, J= 5.2 Hz, 2H), 3.12 (td, J = 7.2, 4.8 Hz, 1H), 2.87 (dd, J = 12.8, 4.8 Hz, 1H),2.72 (d, J = 12.8 Hz, 1H), 2.21 (t, J = 7.2 Hz, 2H), 1.75-1.58 (m, 4H), 1.42 (s,9H),1.24-1.23 (m, 2H). 13 C NMR (101 MHz, Chloroform-d) δ 172.51, 163.29, 155.05, 78.39, 69.02, 60.79, 59.24, 54.72, 39.50, 39.30, 38.11, 34.96, 30.90, 30.49, 29.10, 28.67, 27.28, 27.09, 24.65, 21.67, 13.12.

[0080] The 0.21 mmol product 6 was dissolved in 10 mL of dichloromethane solution with 10 wt% trifluoroacetic acid and reacted for 6 h, 50 mL of saturated NaHC03solution was added and washed twice, 50 mL of deionized water was added and washed twice, dried with anhydrous sodium sulfate, evaporated the organic solvent under reduced pressure, the crude product was directly used in the next step, 0.24 mmol of diphenylcyclooctyne-carboxylic acid, 0.262 mmol of 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and 0.262 mmol of N-hydroxysuccinimide were added to 0.22 mmol of the crude product, dissolved in 10 mL of anhydrous DMF, reacted at room temperature for 16 h, then extracted with dichloromethane for 3 times, 40 mL each time, the organic layer was washed with water for 2 times, 20 mL each time, dried with anhydrous sodium sulfate, evaporated the organic solvent under reduced pressure, the obtained crude product was column chromatographed to obtain product 7, with a yield of 75%;

[0081] 1H NMR (400 MHz, CDC13) δ 7.53-7.47 (m, 2H), 7.36-7.31 (m, 3H), 7.26-7.18 (m, 2H), 7.13-7.11 (m, 1H), 4.99 (d, J = 14.0 Hz, 1H), 4.50 (s, 1H), 4.36 (dd, J = 7.8, 4.4 Hz, 1H), 4.17 (dd, J = 7.8, 4.4 Hz, 1H), 3.55 (d, J = 14.0 Hz, 1H), 3.42-3.40 (m, 2H), 3.36-3.30 (m, 2H), 3.25-3.20 (m, 2H), 3.15-3.12 (m, 2H), 3.09-3.04 (m, 1H), 2.81-2.77 (m, 1H), 2.63-2.56 (m, 2H), 2.29-2.22 (m, 1H), 2.10-2.02 (m, 3H), 1.88-1.81 (m, 1H), 1.65-1.41 (m, 4H), 1.34-1.16 (m, 5H). 13 C NMR (101 MHz, CDC13) δ 175.38, 171.29, 150.01, 146.69, 131.23, 128.10, 127.56, 127.37, 127.31, 126.84, 126.18, 124.56, 123.34, 121.99, 121.64, 114.00, 113.05, 106.46, 54.63, 32.81, 30.90, 30.48, 29.28, 29.10, 28.68, 28.64, 28.47, 28.42, 28.34, 28.30, 28.14, 28.04, 26.19, 21.67, 13.11.

[0082] The 0.033 mmol product 5 ICG-AlkylN3 and 0.033 mmol product 7 were dissolved in 10 mL dichloromethane and reacted for 24 h. The resulting crude product was column chromatographed to obtain product 8, ICG-AlkylBiotin, in 65% yield.

[0083] 1H NMR (400 MHz, Methanol-d4) δ 8.22-7.94 (m, 4H), 7.91-7.78 (m, 6H),7.68-7.10 (m, 12H), 4.59-4.45 (m,10H), 4.40-4.23 (m, 5H),3.58 (t, J = 6.4Hz, 2H), 3.44-3.35 (m, 2H) 3.23-3.14 (m, 6H),2.95-2.89 (m, 4H), 2.62-2.49(m, 2H), 2.25-2.03 (m, 6H), 1.90-1.72 (m, 18H), 1.61-1.45 (m, 4H), 1.33-1.18(m, 6H). 13 C NMR (101 MHz, Methanol-d4) δ 174.13, 142.93, 140.20, 139.67,139.42, 138.76, 135.38, 134.79, 132.19, 131.88, 131.29, 131.20, 130.54,130.36, 129.74, 129.70, 128.13, 127.97, 127.39, 127.26, 124.89, 124.46,124.14, 123.84, 123.45, 122.06, 121.93, 113.34, 110.96, 110.61, 69.84, 69.17,68.11, 61.93, 60.19, 55.61, 51.25, 50.69, 50.67, 44.76, 42.93, 42.52, 39.69,38.88, 35.60, 35.35, 34.10, 33.51, 32.11, 31.67, 30.44, 29.51, 29.38, 29.19,29.07, 28.81, 28.72, 28.35, 28.08, 26.47, 26.27, 26.16, 25.46, 25.03, 23.15,22.86, 22.35, 13.08。

[0084] Example 2:

[0085] The present application also discloses a cyanine fluorescent dye, wherein R in the structure of Formula I is selected from .

[0086] A preparation method of a cyanine fluorescent dye is different from that of Example 1 in that 2-bromothiazole-5-carboxylic acid is used instead of 6-bromopropionic acid, and a structural formula of the cyanine fluorescent dye is as follows:

[0087] .

[0088] 1 H NMR (400 MHz, Methanol-d4) δ 8.65-8.61 (s, 1H), 8.21-8.15 (m, 1H),8.10-8.04 (m, 3H), 8.00-7.87 (m, 2H), 7.81-7.75 (m, 4H), 7.65-7.62 (m, 1H),7.60-7.56 (m, 2H), 7.51-7.45 (m, 3H), 7.35-7.26 (m, 2H), 4.34-4.25 (m, 4H),3.41-3.20 (m, 2H), 2.95-2.87 (m, 6H), 2.56-2.51 (m, 2H), 2.38-2.32 (m, 2H),2.23-2.15 (m, 2H), 1.86 (s, 2H), 1.80-1.83 (s, 4H)。

[0089] A preparation method of a target near-infrared fluorescent probe is different from that of Example 1 in that the cyanine fluorescent dye is the cyanine fluorescent dye prepared in the present example.

[0090] 1 H NMR (400 MHz, Methanol-d4) δ 8.66-8.63 (s, 1H), 8.23-7.92 (m, 4H),7.90-7.81 (m, 6H), 7.69-7.07 (m, 12H), 4.62-4.48 (m,10H), 4.40-4.21 (m, 5H),3.56 (t, J = 6.4 Hz, 2H), 3.33-3.12 (m, 6H), 2.97-2.85 (m, 4H), 2.64-2.48(m, 2H), 2.26-2.01 (m, 4H), 1.93-1.70 (m, 12H), 1.60-1.43 (m, 4H), 1.31-1.16(m, 6H)。

[0091] Test Example:

[0092] Preparation of probe solution: The synthesized probe sample, commercial ICG probe, and commercial IR-820 probe were dissolved in dimethyl sulfoxide (DMSO) to prepare a 5 x 10⁻⁶ solution. -3 The stock solution of M was prepared by dissolving 4 μL of the stock solution in PBS buffer, deionized water, DMSO, and methanol (MeOH) solvent to obtain final volumes of 2 mL each, with a probe concentration of 1 x 10⁻⁶. -5 The test solution of M.

[0093] 1. Fluorescence spectroscopy test

[0094] The fluorescence excitation wavelength was 808nm, the emission wavelength range was 820nm-1300nm, the emission slit width was 1.5nm, the fluorescence testing instrument used was an Edinburgh FLS1000 fluorescence spectrophotometer, the excitation source was an external 808nm semiconductor laser (Changchun New Industrial Optoelectronics Technology Co., Ltd.), and a quartz cuvette (1cm) was used for emission measurement.

[0095] The fluorescent probes prepared in Examples 1 and 2 were subjected to the above tests, and the results are as follows: Figure 17 As shown, by Figure 17 It can be seen that the fluorescent probe prepared in Example 1 has higher emission intensity in PBS buffer solution and water, commonly used in biological systems, while the intensity is relatively low in organic solvents DMSO and MeOH. This demonstrates that introducing alkyl chains with targeting sites into the carbon chain can effectively prevent dye interactions in aqueous solutions. The fluorescent probe prepared in Example 2 also has high emission intensity in PBS buffer solution and water, while the intensity is relatively low in organic solvents DMSO and MeOH, indicating that the fluorescent probe prepared in Example 2 can also effectively prevent dye interactions in aqueous solutions.

[0096] 2. Absorption Spectrum Testing

[0097] The instrument used for UV-Vis absorption spectroscopy was a PerkinElmer LAMBDA1050+, and a quartz cuvette (1 cm) was used for absorbance measurement.

[0098] The fluorescent probe prepared in Example 1 was subjected to the above tests, and the results are as follows: Figure 18 As shown in the figure, the fluorescent probe backbone synthesized in this invention has higher absorbance in commonly used biological systems such as PBS buffer solution and water, while the absorbance in organic solvents such as DMSO and MeOH is relatively low. This proves that introducing alkyl chains with targeting sites into the carbon chain can effectively prevent the interaction of dyes in aqueous solutions and improve their fluorescence quantum yield.

[0099] 3. Quantum efficiency testing

[0100] The relative strategy was used to measure the quantum yield, using standard dye IR-26 as reference to measure. The calculation formula is:

[0101]

[0102] is the quantum efficiency of the probe sample in PBS solution, is the quantum efficiency of IR-26 in 1,2-dichloroethane is 0.5%, is the slope of the integrated intensity area of the fluorescence spectrum of the probe sample solution of different concentrations in the region of 820~1300nm relative to its absorbance at 808nm, is the slope of the integrated intensity area of the fluorescence spectrum of the IR-26 probe solution of different concentrations in the region of 820~1300nm relative to its absorbance at 808nm, and is the refractive index of the respective solvent (PBS: 1.333, 1,2-dichloroethane: 1.4448), and the probe solution of different concentrations is obtained by diluting the mother liquor with PBS buffer.

[0103] The above test was performed on the fluorescent probe prepared in Example 1, and the results are shown in Figure 19 As can be seen from the figure, the quantum yield of ICG-AlkylN3 is 7.44%, while that of ICG is only 2.33%; it is shown that the quantum yield of the fluorescent probe prepared by the present application is superior to that of the commercial probe.

[0104] 4. Test of molar absorption coefficient

[0105] According to Lambert-Beer law, the absorbance of the solution and the molar concentration were linearly fitted, and the molar extinction coefficient was calculated as follows:

[0106] A=ε×c×l

[0107] In the formula, A is the absorbance at the maximum absorption wavelength or 808nm, ε is the molar extinction coefficient related to the inherent properties of the compound, l is the light path absorption thickness, and c is the solution concentration. The mother liquor of ICG-AlkylN3 and ICG was diluted with PBS buffer to obtain solutions of different concentrations, and the absorbance was measured using solutions of different concentrations.

[0108] The above test was performed on the fluorescent probe prepared in Example 1, and the results are shown in Figure 20 As can be seen from the figure, the molar absorption coefficient of ICG-AlkylN3 at 808nm is 4.97×10 4 , and the molar absorption coefficient of ICG is 5.94×10 4; the molar absorption coefficient of ICG-AlkylN3 at the maximum absorption wavelength is 1.64*10 5 , and the molar absorption coefficient of ICG is 9.29*10 4 ; it is shown that the fluorescent probe prepared in the application has a lower molar absorption coefficient.

[0109] 5. Test of brightness of fluorescent probe sample under near-infrared two-zone imaging system

[0110] VanGogh IGS1000 type NIR-II imaging system was used to perform near-infrared fluorescence imaging under 808nm irradiation (laser output power density = 0.2W·cm -2 , flux = 40mW·cm -2 ), and the concentration of the three fluorescent probes was 50uM.

[0111] The fluorescent probe prepared in Example 1 was tested, and the results are shown in Figure 21 From the figure, it can be seen that, whether under the condition of the 900nm long wavelength filter or under the condition of the 1000nm long wavelength filter, the brightness of the probe synthesized in the application is greatly improved compared with the commercial ICG and IR-820.

[0112] 6. Test of tumor imaging effect of fluorescent probe in tumor-bearing mouse model

[0113] The mouse model used a 4T1 cell breast cancer model, and the probe sample with a volume of 200uL and a concentration of 50uM was injected through the tail vein.

[0114] The fluorescent probe prepared in Example 1 was tested, and the results are shown in Figure 22 From the figure, it can be seen that ICG-AlkylBiotin with the target biotin has a good tumor targeting effect, can be retained and enriched in the tumor site for a long time, and the brightness decreases significantly at 48h, while ICG and ICG-AlkylN3 without the target have obvious brightness decrease at 4h and 8h, respectively.

[0115] The conventional techniques in the above examples are prior art known to those skilled in the art, and therefore will not be described in detail here.

[0116] The above is only a specific implementation of the application, but the protection scope of the application is not limited thereto, and any person skilled in the art can easily think of changes or replacements within the technical range disclosed in the application, which should be covered within the protection scope of the application. Therefore, the protection scope of the application should be subject to the protection scope of the claims.

Claims

1. A cyanin-based fluorescent dye, namely ICG-AlkylN3, has the structure shown in Formula I: Ⅰ。 2. The method for preparing the cyanine fluorescent dye according to claim 1, comprising: Step 1: Hydrolyze 1-chloro-2,4-dinitrobenzene to prepare 2,4-dinitrophenol; Step 2: 2,4-Dinitrophenol undergoes a substitution reaction with p-toluenesulfonyl chloride to prepare 2,4-dinitrophenyl p-methylsulfonic acid toluene; Step 3: 3-Aminopyridine undergoes an amidation reaction with a bromoalkyl acid, followed by a substitution reaction with sodium azide to prepare pyridine derivatives; Step 4: Prepare pyridine salts using 2,4-dinitrobenzene-toluene-2,4-methanesulfonic acid and pyridine derivatives; Step 5: ICG-AlkylN3 was prepared using pyridine salt, 1,1,2-trimethyl-3-(4-sulfopropyl)-1H-benzo[E]indole inner salt and amine compound, wherein the amine compound was 4-bromoaniline; The synthetic route for cyanine fluorescent dyes is shown below: 。 3. The method for preparing cyanine fluorescent dyes according to claim 2, characterized in that: In step four, the molar ratio of 2,4-dinitrobenzene toluene p-methanesulfonate to the pyridine derivative is 1:0.6-1.

4. The method for preparing cyanine fluorescent dyes according to claim 2, characterized in that: In step five, the molar ratio of pyridine salt to amine compound is 1:1.1-1.

4.

5. A method for preparing a targeted near-infrared fluorescent probe, comprising: Amidation reaction is performed using 5-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)valeric acid and 2-(2-(2-aminoethoxy)ethoxy)ethylcarbamate tert-butyl ester, followed by hydrolysis under acidic conditions, and then amidation reaction with diphenylcyclooctyne-carboxylic acid. Finally, an addition reaction is performed with the cyanine-based fluorescent dye described in claim 1 to obtain a targeted near-infrared fluorescent probe. The structure of diphenylcyclooctyne-carboxylic acid is as follows: ; The synthetic route for the targeted near-infrared fluorescent probe is shown below: 。 6. The method for preparing a targeted near-infrared fluorescent probe according to claim 5, characterized in that: The molar ratio of 5-((3aS,4S,6aR)-2-oxohexahydro-1H-thiopheno[3,4-d]imidazol-4-yl)valeric acid to 2-(2-(2-aminoethoxy)ethoxy)ethylcarbamate tert-butyl ester is 1:0.8-1.

3.

7. The use of the cyanine fluorescent dye prepared by the method according to any one of claims 2-4 in the preparation of targeted near-infrared fluorescent probes.

8. A fluorescence imaging reagent comprising a targeted near-infrared fluorescent probe prepared by the preparation method according to any one of claims 5-6.

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