A highly lipophilic near-infrared heptamethine cyanine dye and its preparation method and application

Abstract

The present invention belongs to the field of fine chemical technology, and specifically relates to a highly lipophilic near-infrared heptamethine cyanine dye, a preparation method thereof and an application thereof. The present invention aims to provide a highly lipophilic near-infrared heptamethine cyanine dye that can be used for fluorescent imaging of the thoracic duct, and a method for preparing a dye preparation and applying it for intraoperative imaging. Unlike traditional heptamethine cyanine dyes represented by indocyanine green, the dye does not contain water-soluble groups such as sulfonic acid groups, carboxyl groups, and hydroxyl groups, and the lipophilicity of the dye is regulated by the length of the aliphatic hydrocarbon of the dye side chain substituent. Therefore, the dye can be dissolved in lipids represented by olive oil and maintain the performance of high fluorescence brightness. Furthermore, by different substituents R in the dye, high-contrast fluorescent imaging of the thoracic duct can be achieved.

Description

Technical Field

[0001] The invention belongs to the cross field of surgical imaging and fine chemical industry, and specifically relates to a highly lipophilic near-infrared heptamethine cyanine dye and a preparation method thereof. The dye is applied in the field of thoracic duct fluorescence development. Background Art

[0002] The thoracic duct is the longest lymphatic duct in the human body, collecting 3 / 4 of the body's lymph, including the lymph of the left half of the body, the abdomen and pelvis, and both lower limbs. The lower end of the thoracic duct generally starts in front of the 1st to 2nd lumbar vertebrae, which is called the "chyle pool". It passes through the aortic hiatus of the diaphragm and enters the chest cavity and is injected into the left venous angle. Chylothorax is a serious complication after esophageal cancer surgery, which is related to thoracic duct injury during surgery. Thoracic duct injury during surgery is because the liquid in the thoracic duct is transparent and invisible, and the specific trend of the thoracic duct cannot be determined, making it difficult to accurately identify the thoracic duct during surgery. According to the U.S. National Library of Medicine, the incidence of thoracic duct injury during esophagectomy is between 0.2% and 10.5% (https: / / clinicaltrials.gov / study / NCT03292757). After the thoracic duct is damaged, a large amount of lymph fluid will be lost to form "chylothorax", which will cause the body's resistance to decrease, and it will also lead to secondary surgery for thoracic duct ligation.

[0003] Fluorescent surgical navigation is a method that uses fluorescent imaging technology to assist surgical operations. It injects fluorescent dyes into patients so that the target tissue or organ emits near-infrared fluorescence under the irradiation of light of a specific wavelength, thereby intuitively guiding surgeons to perform surgical operations more accurately. At present, thoracic duct imaging mainly relies on traditional imaging techniques such as CT and MRI (Chinese Journal of Clinical Medical Imaging. 2019; 30: 851), which needs to be completed in a specific instrument and cannot provide real-time imaging. Therefore, surgeons cannot use it in the operating room, nor can they develop the thoracic duct in real time to prevent surgical misinjury or accurately locate the site of chyle leakage. At present, there have been reports of attempts to use indocyanine green (ICG) for thoracic duct imaging, such as injecting ICG into the inguinal lymph node area, so that the thoracic duct can be visualized under near-infrared fluorescence, and real-time imaging of the thoracic duct can be achieved (Am. J. Otolaryng., 2020, 41: 102463). However, since ICG imaging is not specific, this method requires extremely precise injection positions, and the differences in injection methods (mucosal injection, ultrasound-guided lymph node injection) and accuracy directly determine the imaging quality.

[0004] Studies have shown that the thoracic duct is a long-chain fatty acid transport channel. Oral administration of heavy cream milk or olive oil before surgery can help identify the thoracic duct during thoracoscopic esophagectomy and reduce the risk of surgical injury to the thoracic duct (Eur. J. Cardiothorac. Surg. 2014, 20: 1035; Ann. Thorac. Surg. 2019, 107: 1540). However, this method provides low contrast and poor repeatability. Therefore, the invention provides a highly lipophilic heptamethine cyanine dye, which is mixed with olive oil or heavy cream milk and taken orally, which can perform fluorescent imaging of the thoracic duct, greatly improve the contrast of the thoracic duct, and effectively reduce the risk of surgical injury to the thoracic duct. Summary of the invention

[0005] Based on the above considerations, the purpose of the present invention is to provide a highly lipophilic heptamethine cyanine dye, the dye molecule contains two straight-chain aliphatic hydrocarbon substituents consisting of 4-12 carbon atoms, which has absorption and emission wavelengths similar to indocyanine green (ICG) in lipids and can be directly used in conjunction with existing fluorescence laparoscope equipment.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0007] A highly lipophilic heptamethine cyanine dye, the general structural formula of the dye molecule is:

[0008]

[0009] In the formula, n1 is any value of 3-11, corresponding to a straight-chain aliphatic hydrocarbon substituent having 4-12 carbon atoms; n2 is any value of 0-8, corresponding to a straight-chain aliphatic hydrocarbon substituent having 1-9 carbon atoms; R is a substituent in the middle of the heptamethine cyanine dye;

[0010] The R group is any one of the following nucleophilic groups:

[0011]

[0012] A method for preparing a highly lipophilic heptamethine cyanine dye, comprising:

[0013] Step 1: Synthesis of 2-chloro-1-formyl-3-hydroxymethylcyclohexene;

[0014] Step 2: Synthesis of 1-hexyl-2,3,3-trimethyl-1H-indole iodide;

[0015] Step 3: 2-chloro-1-formyl-3-hydroxymethylcyclohexene is reacted with 1-hexyl-2,3,3-trimethyl-1H-indole iodide, acetic anhydride and anhydrous sodium acetate to obtain a chlorinated highly lipophilic heptamethine cyanine dye.

[0016] Preferably, the highly lipophilic heptamethine cyanine dye further comprises a chlorinated highly lipophilic heptamethine cyanine dye and a nucleophilic group and N,N-dimethylformamide, prepared by heat treatment and filtration;

[0017] Specifically include:

[0018] S1: Add chlorinated highly lipophilic heptamethine cyanine dye, nucleophilic group and N,N-dimethylformamide into an eggplant-shaped bottle and stir to mix evenly;

[0019] S2: Heat the material in step S1 to 80°C under argon protection and react for 1 hour;

[0020] S3: After the reaction is completed, the reaction solution is diluted with dichloromethane, washed with brine, and the organic solvent is removed by a rotary evaporator to obtain a crude product. The crude product is diluted with dichloromethane and dropped into n-hexane to precipitate a highly lipophilic heptamethine cyanine dye.

[0021] Preferably, the nucleophilic group is any one of the following structural formulas:

[0022]

[0023] The method of the invention prepares the highly lipophilic heptamethine cyanine dye, which is used as a ready-to-use dye-olive oil solution or dye-milk solution.

[0024] Preferably, the preparation method of the ready-to-use dye-olive oil solution is:

[0025] Step 1: dissolving highly lipophilic heptamethine cyanine dye in ethanol;

[0026] Step 2: Add olive oil to the ethanol solution dissolved in step 1 and mix well to obtain a ready-to-use dye-olive oil solution.

[0027] Preferably, the dye-milk solution is prepared by:

[0028] Step 1: dissolving highly lipophilic heptamethine cyanine dye in ethanol to prepare a 20 mg / mL dye ethanol solution;

[0029] Step 2: Add 29 times the volume of milk and 10 times the volume of light cream to the ethanol solution dissolved in step 1, mix well to obtain a 0.5 mg / mL dye-milk solution.

[0030] The ready-to-use dye-olive oil solution or dye-milk solution prepared by the invention is applied to fluorescent development of the thoracic duct.

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] The present invention aims to provide a highly lipophilic near-infrared heptamethine cyanine dye that can be used for fluorescent imaging of the thoracic duct, and the preparation of the dye preparation and the application method of intraoperative imaging. Unlike the traditional heptamethine cyanine dye represented by indocyanine green, the dye does not contain water-soluble groups such as sulfonic acid, carboxyl, and hydroxyl groups, and the water solubility of the dye is greatly reduced. At the same time, the lipophilicity of the dye is regulated by the length of the aliphatic hydrocarbon of the dye side chain substituent. Therefore, this type of dye can be dissolved in lipids represented by olive oil and maintain the performance of high fluorescence brightness. Furthermore, by the difference of the substituent R in the dye, the physiological metabolism, photostability and fluorescence brightness of the dye can be regulated to achieve high-contrast fluorescence imaging of the thoracic duct. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] The accompanying drawings are used to provide further understanding of the present invention and constitute a part of the specification. They are used to explain the present invention together with the embodiments of the present invention and do not constitute a limitation of the present invention.

[0034] In the attached picture:

[0035] Figure 1 Example 1 IR-790 in deuterated chloroform solution H NMR spectrum (5 mg / mL);

[0036] Figure 2 Example 1 IR-790 in deuterated chloroform solution (50 mg / mL) C NMR spectrum;

[0037] Figure 3 Example 2 IR-790 + in deuterated chloroform solution H NMR spectrum (5 mg / mL);

[0038] Figure 4 Example 2 IR-790 + in deuterated chloroform solution carbon nuclear magnetic resonance spectrum (50mg / mL);

[0039] Figure 5 (a) Example 1 UV absorption of IR-790 in dimethyl sulfoxide, chloroform and olive oil solutions (1×10 -6 mol·L -1 ); (b) Fluorescence spectrum of IR-790 in dimethyl sulfoxide, chloroform and olive oil solution (1×10 -6 mol·L -1 );

[0040] Figure 6 .Distribution of ICG and IR790 in a mixture of olive oil and water (concentration 100 μM)

[0041] Figure 7 (a) UV absorption of IR-790+ of Example 2 in dimethyl sulfoxide, chloroform and olive oil solutions (1×10-6 mol·L -1 ); (b) Fluorescence spectra of IR-790+ of Example 2 in dimethyl sulfoxide, chloroform and olive oil solutions (1×10 -6 mol·L -1 );

[0042] Figure 8 .(a) Cytotoxicity of IR-790 on HEK-293T cells; (b) Cytotoxicity of IR-790+ on HEK-293T cells; Dye concentration 0-10 μM;

[0043] Fig. 9 .Fluorescence confocal imaging of IR-790 and IR-790+ in HEK-293T cells, dye concentration 1μM;

[0044] Fig.10 .IR-790 is used for intraoperative thoracic duct fluorescence imaging, with a dye dosage of 0.15 mg / kg.

[0045] Fig.11 The synthetic route of IR-790 is shown in FIG.

[0046] Fig.12 The synthetic route of 2-chloro-1-formyl-3-hydroxymethylcyclohexene is shown in FIG.

[0047] Fig.13 The synthetic route of 1-hexyl-2,3,3-trimethyl-1H-indole iodide is shown;

[0048] Fig.14 A synthetic route for the synthesis of IR-790;

[0049] Fig.15 The synthetic route for the highly lipophilic heptamethine cyanine dye is shown;

[0050] Fig.16 This is the synthetic route of IR790+. DETAILED DESCRIPTION

[0051] The following is combined with Figure 1-16 The preferred embodiments of the present invention are described. It should be understood that the preferred embodiments described herein are only used to illustrate and explain the present invention, and are not used to limit the present invention.

[0052] Terminology explanation:

[0053] Fluorescence imaging: Utilizing the property of fluorescent dyes that release fluorescence of specific wavelengths after absorbing excitation light, the dyes are injected into the target blood vessels or tissues, and equipment such as fluorescence laparoscopes are used to collect bright field and fluorescence signals, and real-time imaging is further output on the screen to achieve targeted imaging of specific tissues or tracing of blood flow during surgery.

[0054] Lipids: Also known as lipids, compounds that are insoluble in water but can be extracted by non-polar organic solvents such as ether, chloroform, and benzene. In the present patent, they mainly refer to triglycerides, phospholipids, and sterols commonly found in mammals.

[0055] Fluorescence brightness: The fluorescence brightness of a dye is determined by its ability to absorb and emit photons. Therefore, the present invention uses the product of the molar absorptivity representing the dye's ability to absorb photons and the fluorescence quantum yield representing its ability to emit photons to represent it.

[0056] A highly lipophilic heptamethine cyanine dye, the general structural formula of the dye molecule is:

[0057]

[0058] In the formula, n1 is any value of 3-11, corresponding to a straight-chain aliphatic hydrocarbon substituent having 4-12 carbon atoms; n2 is any value of 0-8, corresponding to a straight-chain aliphatic hydrocarbon substituent having 1-9 carbon atoms; R is a substituent in the middle of the heptamethine cyanine dye;

[0059] The R group is any one of the following nucleophilic groups:

[0060]

[0061] Embodiment 1:

[0062] like Fig.10 The synthetic route of highly lipophilic heptamethine cyanine dye is shown in FIG.

[0063] Step 1: If Fig.12 , Synthesis of 2-chloro-1-formyl-3-hydroxymethylcyclohexene:

[0064] Step 1.1: In a 50 mL reaction bottle, add phosphorus oxychloride (5.0 g, 32.5 mmol) dropwise into dry N,N-dimethylformamide (5 mL) under ice bath conditions. After the addition is complete, the solution is colorless and transparent and gradually becomes viscous.

[0065] Step 1.2: After the addition is completed, the ice bath is removed and the mixture is reacted at room temperature for 30 min. Then, a mixed solution of cyclohexanone (1.00 g, 10.2 mmol) and dry N,N-dimethylformamide (5 mL) is dripped into the reaction solution drop by drop using a constant pressure dropping funnel. The solution gradually turns yellow and becomes viscous.

[0066] Step 1.3: React in an oil bath at 60°C under argon protection for 3 h. After stopping heating, pour the reaction solution into crushed ice (about 200 mL), and an orange-yellow powder precipitates;

[0067] Step 1.4: The obtained liquid was allowed to stand overnight, and a yellow solid was precipitated on the next day. 1.50 g of a yellow product was obtained by filtration, with a yield of 85.3%. The product was used in the next step without further treatment.

[0068] Step 2: If Fig.13 , Synthesis of 1-hexyl-2,3,3-trimethyl-1H-indole iodide:

[0069] Step 2.1: Add 2,3,3-trimethyl-1H-indole (3.8 g, 23.8 mmol) and iodohexane (10.1 g, 47.6 mmol) into a 100 mL single-necked bottle and stir to dissolve;

[0070] Step 2.2: Then, the mixture was refluxed at 110°C in an oil bath under argon protection for 7 h. The reaction solution changed from yellow to purple-red, and a red solid precipitated.

[0071] Step 2.3: After the reaction is completed, the reaction solution is allowed to stand and cool, and the reaction solution is dissolved with a small amount of dichloromethane and dropped into ether, whereupon a red solid precipitates, which is dried in vacuo to obtain 2.9 g of a red powder, with a yield of 49.7%.

[0072] 1 H-NMR (400MHz, CDCl3, ppm): δ = 7.63-7.55 (m, 4H, Phenyl-H), 7.63-7.55 (t, J = 8.0Hz, 2H, N-CH2-C5H 11 ),3.13(s,3H,-CH3),1.97-1.89(m,2H,-CH2-C4H9),1.67(s,6H,-CH3),1.53-1.4 4(m,2H,-CH2-),1.38-1.30(m,4H,-CH2-),0.91-0.87(t,J=7.2Hz,3H,-CH2-CH3).

[0073] Step 3: Chlorinated highly lipophilic heptamethine cyanine dye (IR-790)

[0074] Step 3.1: In a 200 ml dry single-necked bottle, add 2-chloro-1-formyl-3-hydroxymethylcyclohexene (1.0 g, 5.9 mmol), 1-hexyl-2,3,3-trimethyl-1H-indole iodide (2.9 g, 11.8 mmol), and 20 ml of acetic anhydride, stir to dissolve, and then add anhydrous sodium acetate (1.0 g, 11.8 mmol).

[0075] Step 3.2: The reaction was carried out in an oil bath at 100°C under argon protection for 2 h. As the reaction progressed, the color of the reaction solution gradually turned dark green.

[0076] Step 3.3: After the reaction is completed, acetic anhydride is removed by rotary evaporation to obtain a red solid. The crude product is dissolved with a small amount of dichloromethane, squeezed out with a large amount of n-hexane, and filtered to obtain a green solid. It is recrystallized with 30 ml of ethanol and filtered to obtain 3.0 g of a green metallic luster solid, namely, chlorinated highly lipophilic heptamethine cyanine dye (IR-790), with a yield of 68.9%, recorded as IR790. Figure 1 is the IR790 hydrogen spectrum, Figure 2 This is the carbon spectrum of IR790.

[0077] 1 H NMR (400MHz, CDCl3, ppm): δ = 8.36-8.32 (d, J = 14.0Hz, 2H, Alkene-H), 7.42-7.38 (m, 4H, Phenyl-H), 7.27-7.24 (m, 2H, Pheny l-H),7.17-7.15(d,J=8.0Hz,2H,Phenyl-H),6.26-6.22(d,J=14.4Hz,2H,Alkene-H),4.22-4.18(t,J=7.2Hz,4H,N-CH2-C5H 11 ),2.77-2.74(t,J=6.0Hz,4H,Alkane-H),2.01-1.98(m,4H,Alkane-H),1.89-1.81(m,4H,Alkane-H),1.72(s, 12H,-CH3),1.50-1.43(m,4H,Alkane-H),1.39-1.31(m,9H,Alkane-H),0.91-0.88(t,J=6.8Hz,6H,-CH2-CH3). 13 C-NMR (100MHz, CDCl3, ppm): δ=172.35,150.49,144.29,142.23,141.06,128.82,127.28,125.35 ,122.31,110.85,101.34,49.37,45.06,31.42,28.16,28.13,27.34,26.66,22.44,20.73,13.97.

[0078] The synthetic route of highly lipophilic heptamethine cyanine dye is as follows Fig.15 As shown. The nucleophilic group is The products prepared are:

[0079] 1. Piperazine-substituted heptamethine cyanine dyes

[0080]

[0081] 2. Methylpiperazine-substituted cyanine dyes

[0082]

[0083] 3. Aniline-substituted cyanine dyes

[0084]

[0085] 4. p-Cresol substituted cyanine dyes

[0086]

[0087] 5. p-Methylthiophenol-substituted cyanine dyes

[0088]

[0089] This is Example 2.

[0090] Embodiment 2:

[0091] On the basis of Example 1, taking piperazine as an example, Fig.16 , the preparation method is:

[0092] Step 1: Add chlorinated highly lipophilic heptamethine cyanine dye IR-790 (500 mg, 0.5 mmol), nucleophilic group (piperazine 200 mg, 2.0 mmol)) and N,N-dimethylformamide into a 25 mL eggplant-shaped bottle and stir to mix evenly;

[0093] Step 2: Heat the material in step 1 to 80°C under argon protection and react for 1 hour; TLC tracking DCM:MeOH=10:1 (v / v), the solution turns blue-black after the reaction (TLC is thin layer chromatography, DCM is dichloromethane, MeOH is methanol).

[0094] Step 3: After the reaction is completed, the reaction solution is diluted with dichloromethane, washed with brine, and the organic solvent is removed by a rotary evaporator to obtain a crude product. The crude product is diluted with dichloromethane and dropped into n-hexane under stirring to precipitate a golden red solid with a metallic luster. The highly lipophilic heptamethine cyanine dye (piperazine substituted heptamethine cyanine dye) is filtered off with a yield of 73.7%, which is recorded as IR790+. Figure 3 is the IR790+ hydrogen spectrum of Example 2, Figure 4 This is the IR790+ carbon spectrum of Example 2.

[0095] 1H-NMR (400MHz, CDCl3, ppm): δ = 7.84-7.80 (d, J = 14.0Hz, 2H, Phenyl-H), 7.43-7.42 (d, J = 7.6Hz, 2H, Phenyl-H), 7.36-7.32 (t, J = 7.6, 2H, Phenyl-H) H),7.19-7.16(t,J=7.6Hz,2H,Phenyl-H),7.03-7.01(d,J=16Hz,2H,Alkene-H),5.91-5.88(d,J=13.6Hz,2H,Alkene-H),4.05(s,4H,N-CH2-C5H 11 ),3.98-3.94(t,J=7.2Hz,4H,N-CH2-CH2),3.52(s,4H,CH2-CH2-NH),2.51-2.48(m,4H,CH2-CH2-C4H9),1.88-1.85(m,4H,Alkane-H),1.81(s,12H,-CH3 ),1.78(s,1H,-NH),1.71-1.69(d,J=6Hz,2H,Alkene-H),1.45-1.43(m,4H,Alkane-H),1.37-1.35(m,8H,Alkane-H),1.92-0.89(t,J=6.8Hz,6H,-CH3). 13 C-NMR (100MHz, CDCl3, ppm): δ = 170.78, 170.34, 142.26, 141.77, 141.07, 128.37, 125.04, 124.42, 122.6 1,109.70,97.46,51.11,48.95,45.16,44.03,31.40,29.27,26.93,26.73,25.27,22.47,21.73,13.99.

[0096] test:

[0097] (1) Ultraviolet absorption and fluorescence of IR-790

[0098] The IR-790 prepared in Example 1 was dissolved in analytically pure dimethyl sulfoxide to prepare 1.0×10 -3 2 μL of IR-790 stock solution was added to 2 mL of dimethyl sulfoxide, chloroform and olive oil respectively, mixed evenly and transferred to an optical quartz cuvette (10×10 mm) to test its absorption and fluorescence spectra.

[0099] like Figure 5As shown, the absorption wavelength of IR-790 is about 790nm and the emission wavelength is about 810nm, which is very similar to the absorption (788nm) and emission peak (813nm) of the current clinical fluorescent surgical navigation dye indocyanine green, and can be directly adapted to existing clinical fluorescent laparoscope equipment.

[0100] In addition, if Figure 6 As shown in the figure, IR-790 has a solubility completely opposite to that of ICG because it does not contain a water-soluble sulfonic acid group. Specifically, in a mixture of water and olive oil, IR-790 is almost entirely distributed in the olive oil phase, while ICG is mainly distributed in the water phase, indicating that IR-790 has excellent lipid solubility.

[0101] (2) Ultraviolet absorption, fluorescence, and afterglow luminescence spectra of compound IR-790+

[0102] The IR-790+ prepared in Example 2 was dissolved in analytically pure dimethyl sulfoxide to prepare 1.0×10 -3 2 μL of IR-790+ stock solution was added to 2 mL of dimethyl sulfoxide, chloroform and olive oil respectively, mixed evenly and transferred to an optical quartz cuvette (10×10 mm) to test its absorption and fluorescence spectra.

[0103] like Figure 7 As shown in the figure, its absorption wavelength is significantly blue-shifted compared to IR-790, with the two absorption peaks at approximately 690nm and 778nm respectively, and the emission wavelength is approximately 807nm, which can also be adapted to existing fluorescence cavity mirror equipment. It is worth noting that compared to IR-790 dye, the fluorescence quantum efficiency of IR-790+ dye is significantly improved.

[0104] (3) Evaluation of cytotoxicity of IR-790 and IR-790+ on HEK-293T cells

[0105] Human embryonic kidney 293T cells (HEK-293T cells) were purchased from Shanghai Institute of Cell Biology. DMEM cell culture medium contains 10% fetal bovine serum (FBS, Biological Industry, Kibbutz Beit Haemek, Israel) and 1% double antibody (10,000U mL-1 penicillin and 10mg / ml streptomycin, Solarbio life Science, Beijing, China). The culture conditions are 37°C and a humidified gas atmosphere containing 5% CO2.

[0106] The cells were 1×10 4The cells were seeded at a density of 100 μL / well in a 96-well plate. After the cells were fully attached, 100 μL of culture medium containing different concentrations of IR-790 was added and incubated for 24 h. Subsequently, 10 μL of 5 mg / mL MTT solution was added and cultured for another 4 h. Finally, DMSO was used to dissolve the formazan crystals converted by MTT, and the absorbance at 450 nm was measured using a multifunction microplate reader (Tecan GENios Pro multifunction reader).

[0107] The relative cell viability was calculated by the following formula: Cell viability (%) = (ODtreated / ODcontrol) × 100%. Figure 8 As shown, when the concentration of the two dyes was 10 μM / mL, the cell viability was still higher than 80%, and the dyes showed good biocompatibility.

[0108] (4) Confocal fluorescence imaging of IR-790 and IR-790+ in HEK-293T cells

[0109] HEK-293T cells were cultured at 1×10 5 The density of cells / well was transferred to a glass culture dish containing 1.5 mL of complete culture medium. After the cells were fully attached, a culture medium containing 1 μM IR-790 or IR-790+ was added and incubated for 1 h. After incubation, the cells were washed with PBS (1 mL × 3) and then imaged using a Leica confocal laser microscope Leica TCS SP8 (63× oil objective). Excitation was 688 nm and fluorescence was 700-800 nm. Fig. 9 As shown, obvious red fluorescence was shown in the cytoplasm, indicating that IR-790 and IR-790+ have good cell membrane permeability. In addition, the fluorescence intensity of IR-790+ in the cell is significantly higher than that of IR-790, showing a better fluorescence imaging potential.

[0110] (4) Confocal fluorescence imaging of IR-790 and IR-790+ in HEK-293T cells

[0111] HEK-293T cells were cultured at 1×10 5 The density of cells / well was transferred to a glass culture dish containing 1.5 mL of complete culture medium. After the cells were fully attached, a culture medium containing 1 μM IR-790 or IR-790+ was added and incubated for 1 h. After incubation, the cells were washed with PBS (1 mL × 3) and then imaged using a Leica confocal laser microscope Leica TCS SP8 (63× oil objective). Excitation was 688 nm and fluorescence was 700-800 nm. Fig. 9As shown, obvious red fluorescence was shown in the cytoplasm, indicating that IR-790 and IR-790+ have good cell membrane permeability. In addition, the fluorescence intensity of IR-790+ in the cell is significantly higher than that of IR-790, showing a better fluorescence imaging potential.

[0112] Embodiment 3:

[0113] A dye-olive oil solution was prepared based on IR-790 of Example 1. The preparation method was as follows:

[0114] Step 1: Take 20 mg of IR-790 prepared in Example 1 and dissolve it in 1 mL of ethanol to prepare 20.0 mg / mL -1 Ethanol solution;

[0115] Step 2: Add 39 mL of olive oil to the ethanol solution dissolved in step 1 and mix well to obtain a 0.5 mg / mL dye-olive oil solution.

[0116] Embodiment 4:

[0117] A dye-milk solution was prepared based on IR-790 of Example 1. The preparation method was as follows:

[0118] Step 1: dissolving highly lipophilic heptamethine cyanine dye in ethanol to prepare a 20 mg / mL dye ethanol solution;

[0119] Step 2: Add 29 times the volume of milk and 10 times the volume of light cream to the ethanol solution dissolved in step 1, mix well to obtain a 0.5 mg / mL dye-milk solution.

[0120] Embodiment 5:

[0121] The IR-790 prepared in Example 1 was applied to the fluorescent imaging of the thoracic duct in surgical operations. The specific method is:

[0122] Step 1: 0.5 hours before surgery, add 20 mL of 0.5 mg / mL -1 The ready-to-use IR790 olive oil solution was injected into the submucosal part of the small intestine of pigs or taken orally;

[0123] Step 2: A hole was made in the intercostal space of the experimental pig through the optical cannula channel, and the chest cavity was inflated with 6 mmHg of carbon dioxide inflation pressure.

[0124] like Fig.10 As shown in the figure, through the fluorescent laparoscope, the clear anatomical structure of the thoracic duct can be observed in the fluorescent mode and the fluorescent white light superposition mode, while the boundary between the thoracic duct and the surrounding tissue is not obvious in the pure white light mode, indicating that IR-790 can perform high-contrast fluorescent imaging of the thoracic duct, thereby assisting in guiding thoracic surgery.

[0125] The above shows and describes the basic principles, main features and advantages of the present invention. It should be understood by those skilled in the art that the present invention is not limited to the above embodiments, and the above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, which fall within the scope of the present invention to be protected. The scope of protection of the present invention is defined by the attached claims and their equivalents.

Claims

1. A method for preparing a highly lipophilic heptamethine cyanine dye, characterized in that: include Step 1: Synthesis of 2-chloro-1-formyl-3-hydroxymethylcyclohexene; Step 2: Synthesis of 1-hexyl-2,3,3-trimethyl-1H-indole iodide; Step 3: reacting 2-chloro-1-formyl-3-hydroxymethylcyclohexene with 1-hexyl-2,3,3-trimethyl-1H-indole iodide, acetic anhydride and anhydrous sodium acetate to obtain a chlorinated highly lipophilic heptamethine cyanine dye; Highly lipophilic heptamethine cyanine dyes also include those based on chlorinated highly lipophilic heptamethine cyanine dyes and nucleophilic groups and N,N-dimethylformamide, prepared by heat treatment and filtration methods; Specifically include: S1: Add chlorinated highly lipophilic heptamethine cyanine dye, nucleophilic group and N,N-dimethylformamide into an eggplant-shaped bottle and stir to mix evenly; S2: Heat the material in step S1 to 80°C under argon protection and react for 1 hour; S3: After the reaction is completed, the reaction solution is diluted with dichloromethane, washed with brine, and the organic solvent is removed by rotary evaporation to obtain a crude product, which is diluted with dichloromethane and dropped into n-hexane to precipitate a highly lipophilic heptamethine cyanine dye; Wherein, the nucleophilic group is any one of the following structural formulas:

2. Use of the highly lipophilic heptamethine cyanine dye obtained by the preparation method as claimed in claim 1 as a ready-to-use dye-olive oil solution.

3. Use of the highly lipophilic heptamethine cyanine dye obtained by the preparation method as claimed in claim 1 as a dye-milk solution.

4. The instant dye-olive oil solution as claimed in claim 2 is used for thoracic duct fluorescence imaging.

5. The dye-milk solution as claimed in claim 3 is used for fluorescent imaging of the thoracic duct.

6. The instant dye-olive oil solution according to claim 2, characterized in that: The preparation method is: Step 1: dissolving highly lipophilic heptamethine cyanine dye in ethanol; Step 2: Add olive oil to the ethanol solution dissolved in step 1 and mix well to obtain a ready-to-use dye-olive oil solution.

7. The dye-milk solution according to claim 3, characterized in that: The preparation method is: Step 1: dissolving highly lipophilic heptamethine cyanine dye in ethanol to prepare a 20 mg / mL dye ethanol solution; Step 2: Add 29 times the volume of milk and 10 times the volume of light cream to the ethanol solution dissolved in step 1, mix well to obtain a 0.5 mg / mL dye-milk solution.

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