Zwitterionic cyanine dyes, methods of making and using the same

Abstract

The present application relates to a kind of zwitterionic cyanine dyes and its preparation method and application.The present application introduces sulfonic acid group in the specific position of existing cyanine dye, which not only allows the cyanine dye to maintain good hydrophilicity, but also makes the charge distribution of cyanine dye molecule more balanced, and the protein binding rate is lower, thereby improving its metabolic rate as a kidney clearance probe, and then ureter imaging can be realized.At the same time, since the kidney metabolic rate of the zwitterionic cyanine dye of the present application is fast, the metabolism in vivo is completed in about 6 hours, so the biological safety is higher.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and more specifically, to a zwitterionic anthocyanin dye, its preparation method, and its application. Background Technology

[0002] The urinary system is a vital metabolic and excretory pathway in the human body, primarily composed of the kidneys, ureters, bladder, and urethra. During excretion, abnormal metabolic waste, foreign microorganisms, and medications can easily cause infections and damage to the kidneys, ureters, and bladder, leading to a range of urinary system diseases. In recent years, the widespread use of diagnostic tools such as ultrasound, computed tomography (CT), and magnetic resonance imaging (MRI) has enabled imaging of the urinary system's tissue structures and early detection of small tumors. However, while achieving early diagnosis, this also presents challenges for clinicians. Furthermore, surgeons rely heavily on visual information during operations; increasing tissue contrast and achieving precise intraoperative manipulation remains another critical issue that needs to be addressed.

[0003] Near-infrared fluorescence imaging (NIIR) is a novel imaging method that has been widely applied in sentinel lymph node visualization, tumor localization, and angiography. By injecting near-infrared fluorescent dye intravenously or locally, it accumulates at a specific site. Under excitation light with a wavelength of 700–900 nm, the near-infrared fluorescent dye emits fluorescence that is invisible to the naked eye, longer than the incident light wavelength. This fluorescence can be captured by the near-infrared fluorescence imaging device. After passing through a filter and entering the camera, the light signal is converted into an electronic signal through data processing, and the signal intensity is ultimately displayed on the screen as grayscale values. Biological tissues absorb light quanta weakly in this wavelength range. Compared to traditional imaging methods, near-infrared fluorescence imaging can achieve real-time intraoperative imaging, providing surgeons with intuitive visual guidance. Furthermore, because tissues have low absorption and scattering of light within this wavelength range, it has strong tissue penetration (1–2 cm) and poses no hazards such as ionizing radiation.

[0004] Hydrophilic fluorescent substances can be excreted through the urinary system and used in organ imaging to determine the presence of lesions. However, unlike the kidneys and bladder, the ureter is difficult to capture in near-infrared fluorescence imaging of the urinary system because it primarily functions as a transport organ and lacks storage capabilities. Currently, it is believed that to achieve in vivo ureter imaging, the probe must possess not only good hydrophilicity but also a high renal metabolic rate. A high renal metabolic rate allows for sufficient accumulation of fluorescent signals in the ureter, thus enabling imaging.

[0005] Furthermore, if the probe remains in the body for a long time and cannot be metabolized and eliminated in a timely manner, it can cause certain toxicity to the circulatory system. Therefore, if the probe is metabolized by the kidneys quickly, i.e., its retention time in the body is short, its biosafety can be further improved.

[0006] Patent CN117186151A discloses a near-infrared zwitterionic cyanine dye, indicating that it enables real-time monitoring of urinary system lesions and visual identification of lesion areas through fluorescence imaging. This is of great significance for diagnosing kidney damage and ureteral obstruction. Furthermore, real-time fluorescence imaging can provide more precise guidance for ureteral surgery. However, experiments on its renal clearance efficiency show that it takes approximately 8 hours to be essentially metabolized and excreted (i.e., it takes approximately 8 hours to reach the maximum urine recovery rate in the renal clearance efficiency test). This metabolic rate is still relatively slow, making it difficult to sufficiently enrich the signal in the ureter for ureteral imaging. Moreover, further shortening its metabolic time could improve its biocompatibility. Summary of the Invention

[0007] The primary objective of this invention is to overcome the problems of the need for further improvement in the renal metabolic rate of current renal clearance fluorescent probes and the difficulty of achieving ureteral imaging with existing renal clearance fluorescent probes, by providing a zwitterionic cyanine dye. This invention introduces sulfonic acid groups at specific positions in existing cyanine dyes. This not only allows the cyanine dye to maintain good hydrophilicity but also makes the charge distribution of the cyanine dye molecule more balanced and the protein binding rate lower, thereby improving its metabolic rate as a renal clearance probe and enabling ureteral imaging. Furthermore, because the zwitterionic cyanine dye of this invention has a rapid renal metabolic rate, completing metabolism in vivo in approximately 6 hours, its biosafety is higher.

[0008] A further object of the present invention is to provide a method for preparing the above-mentioned zwitterionic anthocyanin dyes.

[0009] Another object of the present invention is to provide the application of the above-mentioned zwitterionic cyanine dyes or their pharmaceutically acceptable salts, solvates, enantiomers, diastereomers, and tautomers in the preparation of fluorescent probes.

[0010] Another object of the present invention is to provide a fluorescent probe containing the above-mentioned zwitterionic cyanine dye and / or its pharmaceutically acceptable salts, solvates, enantiomers, diastereomers, and tautomers.

[0011] The above-mentioned objective of this invention is achieved through the following technical solution: This invention provides a zwitterionic anthocyanin dye having the structure shown in any of formulas (I) to (IV): , Where Y is F, Cl, Br or I; X is NH, SH or O; n is an integer from 0 to 30; n1 is an integer from 2 to 30.

[0012] This invention is based on existing cyanine dyes Introducing sulfonic acid groups at specific positions not only allows the cyanine dye to maintain good hydrophilicity but also makes the charge distribution of the cyanine dye molecule more balanced and the protein binding rate lower, thereby increasing its metabolic rate when used as a renal clearance probe, thus enabling ureteral imaging. Furthermore, because the zwitterionic cyanine dye of this invention has a rapid renal metabolic rate, completing its metabolism in vivo in about 6 hours, it exhibits higher biosafety.

[0013] Furthermore, the zwitterionic cyanine dye of the present invention is a single molecule, label-free, amphoteric and electrically neutral, with high specificity, high sensitivity, good biocompatibility and good optical stability. Moreover, it can be rapidly metabolized by the kidneys without being non-specifically taken up by other tissues or organs. With its high specificity, it can play an important role in future medical optical examinations and has excellent application prospects.

[0014] Furthermore, this invention can introduce carboxyl groups (Formula (II) or (III)) or PEG-carboxyl groups (Formula (IV)) at specific positions of the six-membered ring of the cyanine dye core structure. The carboxyl groups can undergo condensation reactions with amino acids, thereby providing more modifiable sites for subsequent targeted modification of fluorescent probes. By introducing different functional groups (e.g., oligopeptide or polypeptide sequences), the probes can have higher specificity and targeting, which can then be further used for cancer tracking and detection. PEG chains can not only improve the solubility, stability, and biocompatibility of drugs, but also enhance drug targeting through the binding of targeting ligands. In addition, functionally modified PEG chains can be further used to construct nanoscale drug delivery systems.

[0015] Preferably, n is an integer from 1 to 30.

[0016] More preferably, n is an integer from 1 to 15.

[0017] More preferably, n is an integer from 1 to 3.

[0018] Preferably, n1 is an integer from 2 to 20.

[0019] Preferably, the zwitterionic anthocyanin dye has any of the following structures: , , , , or .

[0020] A method for preparing the above-mentioned zwitterionic anthocyanin dye includes the following steps: When zwitterionic anthocyanin dyes have the structure of formula (Ⅰ): S1. Compound (14) and compound (15) undergo a condensation reaction to obtain the zwitterionic anthocyanin dye; ; When zwitterionic anthocyanin dyes have the structure of formula (II): S1. Compound (14) and compound (15) undergo a condensation reaction to obtain the compound of formula (I); S2. The compound of formula (I) undergoes a condensation reaction with compound (2) to obtain the zwitterionic anthocyanin dye; ; When zwitterionic anthocyanin dyes have the structure of formula (Ⅲ): S1. Compound (14) and compound (15) undergo a condensation reaction to obtain the compound of formula (I); S3. The compound of formula (I) undergoes a condensation reaction with compound (5) to obtain the zwitterionic anthocyanin dye; ; When zwitterionic anthocyanin dyes have the structure of formula (Ⅳ): S1. Compound (14) and compound (15) undergo a condensation reaction to obtain the compound of formula (I); S4. The compound described in formula (Ⅰ) and compound (6) undergo a condensation reaction under anaerobic conditions to obtain the zwitterionic anthocyanin dye; .

[0021] The inventors of this invention have discovered that a condensation reaction needs to be carried out under anaerobic conditions in order to successfully introduce PEG flexible segments onto the rigid six-membered ring of the cyanine dye core structure.

[0022] Preferably, in step S1, the reaction temperature of the condensation reaction is 80~100℃ and the time is 12~24 h.

[0023] More preferably, in step S1, the solvent for the condensation reaction is methanol, and the catalyst is acetic anhydride and sodium acetate.

[0024] Preferably, in steps S2 to S4, the reaction temperature of the condensation reaction is 65 to 85°C, and the reaction time is 6 to 12 hours.

[0025] Preferably, in steps S2 to S4, the condensation reaction is carried out in the presence of a solvent, wherein the solvent is at least one of water or N,N-dimethylformamide.

[0026] Preferably, in steps S2 to S4, the condensation reaction is carried out in the presence of an activator, which is selected from one or more of potassium carbonate, cesium carbonate, sodium acetate, potassium acetate, or triethylamine.

[0027] Preferably, compound (14) is obtained by a substitution reaction between compound (12) and compound (13); .

[0028] The inventors of this invention discovered that, in the process of preparing the zwitterionic cyanine dye of this invention, due to the effects of steric hindrance and charge distribution, it is necessary to modify the end of compound (12) with iodine atoms in order to successfully convert the zwitterionic cyanine dye into a zwitterionic cyanine dye. Introduced to middle.

[0029] More preferably, the substitution reaction is carried out in the presence of sodium iodide at a temperature of 110-130°C, and the solvent is one or both of o-dichlorobenzene and toluene.

[0030] More preferably, the compound (12) is obtained by a substitution reaction between the compound (11) and sodium iodide.

[0031] More preferably, the solvent for the substitution reaction is acetonitrile, the reaction temperature is 60~70℃, and the reaction time is 8~12 h.

[0032] More preferably, the compound (11) is obtained by a condensation reaction of compound (9) and compound (10).

[0033] More preferably, the condensation reaction is carried out in the presence of sodium hydroxide at a temperature of 70-90°C.

[0034] The structures of the compounds involved are as follows:

[0035] Preferably, compound (13) is obtained by a condensation reaction of compound (16) and compound (17).

[0036] More preferably, the condensation reaction is carried out in the presence of acetic acid at a temperature of 110-130°C.

[0037] The structures of the compounds involved are as follows:

[0038] This invention also protects the use of the above-mentioned zwitterionic cyanine dyes or their pharmaceutically acceptable salts, solvates, enantiomers, diastereomers, and tautomers in the preparation of fluorescent probes.

[0039] Preferably, the fluorescent probe is a fluorescent probe for organ imaging, wherein the organ is the kidney, ureter, or bladder.

[0040] Preferably, the pharmaceutically acceptable salt is a hydrochloride, hydrobromide, nitrate, methyl nitrate, sulfate, hydrogen sulfate, aminosulfate, phosphate, acetate, glycolate, phenylacetate, propionate, butyrate, isobutyrate, valerate, maleate, hydroxymaleate, acrylate, fumarate, malate, tartrate, citrate, salicylate, para-aminosalicylic acid, glycolate, lactate, heptanoate, phthalate, oxalate, succinate, benzoate, or o-acetyl At least one of the following: oxybenzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, mandelate, tannic acid salt, formate, stearate, ascorbate, palmitate, oleate, pyruvate, dihydroxynaphthyl salt, malonate, laurate, glutamate, propionate, lauryl sulfate, methanesulfonate, ethanesulfonate, 2-hydroxyethanesulfonate, benzenesulfonate, p-aminobenzenesulfonate, p-toluenesulfonate (toluenesulfonate), or naphthalene-2-sulfonate.

[0041] This invention also protects a fluorescent probe containing the above-mentioned zwitterionic cyanine dye and / or its pharmaceutically acceptable salts, solvates, enantiomers, diastereomers, and tautomers.

[0042] Compared with the prior art, the beneficial effects of the present invention are: This invention introduces sulfonic acid groups at specific positions in existing cyanine dyes. This not only maintains the cyanine dye's good hydrophilicity but also results in a more balanced charge distribution and lower protein binding rate, thereby increasing its metabolic rate as a renal clearance probe and enabling ureteral imaging. Furthermore, because the zwitterionic cyanine dye of this invention exhibits rapid renal metabolism, completing its metabolism in vivo in approximately 6 hours, it demonstrates higher biosafety.

[0043] Furthermore, the zwitterionic cyanine dye of the present invention is a single molecule, label-free, amphoteric and electrically neutral, with high specificity, high sensitivity, good biocompatibility and good optical stability. Moreover, it can be rapidly metabolized by the kidneys without being non-specifically taken up by other tissues or organs. Its high specificity allows for more accurate early diagnosis of diseases, intraoperative navigation therapy, and evaluation of tissue and organ function. It can play an important role in future medical optical examinations and has excellent application prospects. Attached Figure Description

[0044] Figure 1 The UV absorption and fluorescence emission spectra of the zwitterionic anthocyanin dyes in Example 2 are shown.

[0045] Figure 2 The UV absorption and fluorescence emission spectra of the zwitterionic anthocyanin dye in Example 3 are shown.

[0046] Figure 3 This is a statistical chart showing the recovery rate of zwitterionic cyanine dye in urine within 24 hours after injection, as described in Example 2. Figure 3 In the graph, A represents the change in urine recovery rate over time. Figure 3 Figure B in the graph represents the results of three replicate experiments on urine recovery rate.

[0047] Figure 4 This is a statistical chart showing the recovery rate of zwitterionic cyanine dye in urine within 24 hours after injection, as described in Example 3. Figure 4 In the graph, A represents the change in urine recovery rate over time. Figure 4 Figure B in the graph represents the results of three replicate experiments on urine recovery rate.

[0048] Figure 5 The image shows organ imaging of the fluorescent probe prepared by zwitterionic cyanine dye in normal mice in Example 2; where He represents heart, Li represents liver, Sp represents spleen, Lu represents lung, Ki represents kidney, Bl represents bladder, Sk represents skin, Mu represents muscle, and In represents small intestine.

[0049] Figure 6 This is a quantitative organ imaging diagram of the fluorescent probe prepared by zwitterionic cyanine dye in normal mice in Example 2; where He represents heart, Li represents liver, Sp represents spleen, Lu represents lung, Ki represents kidney, Bl represents bladder, Sk represents skin, Mu represents muscle, and In represents small intestine.

[0050] Figure 7The image shows organ imaging of the fluorescent probe prepared by zwitterionic cyanine dye in normal mice in Example 3; where He represents heart, Li represents liver, Sp represents spleen, Lu represents lung, Ki represents kidney, Bl represents bladder, Sk represents skin, Mu represents muscle, and In represents small intestine.

[0051] Figure 8 This is a quantitative organ imaging diagram of the fluorescent probe prepared by zwitterionic cyanine dye in Example 3 in normal mice; where He represents heart, Li represents liver, Sp represents spleen, Lu represents lung, Ki represents kidney, Bl represents bladder, Sk represents skin, Mu represents muscle, and In represents small intestine.

[0052] Figure 9 In Figure A, the image shows the ureteral imaging result of zwitterionic anthocyanin dye in Example 2. Figure 9 B in the image represents the ureter imaging result of zwitterionic anthocyanin dye in Example 3. Detailed Implementation

[0053] To more clearly and completely describe the technical solution of the present invention, the present invention is further described in detail below through specific embodiments. It should be understood that the specific embodiments described herein are only for explaining the present invention and are not intended to limit the present invention. Various changes can be made within the scope of the claims of the present invention. Unless otherwise specified, the reagents and materials used in the following embodiments are all commercially available.

[0054] Example 1 This embodiment provides a zwitterionic anthocyanin dye, the synthetic route of which is as follows:

[0055] Specifically, the steps include the following: 1) In a 100 mL double-necked round-bottom flask, under an argon atmosphere, compound 9 (CAS: 5407-04-5, 10.3 g, 65 mmol), compound 10 (CAS: 1120-71-4, 8 mL, 97.5 mmol), solvent (1,2-dichloroethane, 6 mL), and sodium hydroxide (2.6 g, 65 mmol) were added sequentially. The mixture was heated to 70 °C and stirred overnight. After the reaction was complete, the supernatant was discarded, and a white viscous liquid was collected to obtain compound 11 (9.5 g, 60%). LRMS (ESI) m / z: [MH]- Calcd for C8H18ClNO3S2 43.07; Found 243.75.

[0056] 2) In a 100 mL double-necked round-bottom flask, under an argon atmosphere, compound 11 (15 g, 65 mmol), sodium iodide (19.5 g, 130 mmol), and acetonitrile (20 mL) were added sequentially. The mixture was heated to 65 °C and stirred for 12 h. After the reaction was complete, the mixture was evaporated to dryness, recrystallized from ethyl acetate, and a red viscous solid was collected to give compound 12. LRMS (ESI) m / z: [MH]- Calcd for C8H18INO3S 355.01, Found 355.20.

[0057] 3) In a 100 mL double-necked round-bottom flask, under an argon atmosphere, add acetic acid (30 mL), sodium acetate (3.2 g), phenylhydrazine-4-sulfonic acid (3.76 g, 20 mmol), and methyl isopropyl ketone (3.2 mL, 30 mmol). Heat to 110 °C and stir overnight. After the reaction is complete, no further treatment is needed; directly load the sample onto a column for chromatography, eluting the target product with dichloromethane:methanol at a volume ratio of 15:1. Collect the yellow liquid, evaporate to dryness, and then dry to obtain a pink solid compound 13 (3.8 g, 80%). 1H NMR (400 MHz, DMSO-d6): δ 7.63 (s, 1H), 7.55 (d, J = 7.9 Hz, 1H), 7.34 (d, J = 7.9 Hz, 1H), 2.21(s, 3H), 1.25 (s, 6H). LRMS (ESI) m / z: [M+H]+ Calcd for C11H14NO3S 240.07;Found 240.19.. 4) Take a 100 mL round-bottom flask and add compound 12 (2 g, 6 mmol), compound 13 (2 g, 8 mmol), sodium iodide (900 mg, 6 mmol), and o-dichlorobenzene (50 mmol) in sequence. Heat in an oil bath to 110°C and react for 48 h. After the reaction is complete, recrystallize with ethanol and collect the pink solid to obtain compound 14 (1.2 g, yield 46%). 1H NMR (500 MHz, Methanol-d4): δ 8.23–8.09 (m, 2H), 8.06 (s, 1H), 4.84–4.61 (m, 2H), 3.88–3.76 (m, 2H), 3.72–3.55 (m, 4H), 3.46–3.37 Found 445.22.

[0058] 5) Take a 10 mL round-bottom flask and add compound 14 (120 mg, 0.27 mmol), compound 15 (18.6 mg, 0.11 mmol), anhydrous ethanol (2 mL), and sodium acetate (54 mg, 0.65 mmol) sequentially. Heat in an oil bath to 80°C and react for 12 h. After the reaction is complete, perform high-performance liquid chromatography (HPLC) separation and analysis to obtain the green compound 1, which is the zwitterionic anthocyanin dye (166 mg, 60%). LRMS (ESI) m / z: [MH]- Calcd for C46H68ClN4O12S43+ 1031.34 ; Found 1028.07.

[0059] Example 2 This embodiment provides a zwitterionic anthocyanin dye, the synthetic route of which is as follows:

[0060] Specifically, the steps include the following: A 10 mL round-bottom flask was filled with compound 1 (102.8 mg, 0.1 mmol), compound 2 (CAS: 2393-17-1, 83 mg, 0.5 mmol), water (2 mL), and sodium bicarbonate (25 mg, 0.3 mmol). The mixture was heated in an oil bath to 80°C and reacted for 6 h. After the reaction was complete, high-performance liquid chromatography (HPLC) analysis was performed to obtain blue compound 3, which was the zwitterionic anthocyanin dye (53 mg, 50%). LRMS (ESI) m / z: [MH]- Calcd for C55H78N5O14S43+1160.44 ; Found 1156.07.

[0061] Example 3 This embodiment provides a zwitterionic anthocyanin dye, and the synthetic route is as follows:

[0062] Specifically, the steps include the following: A 10 mL round-bottom flask was filled with compound 1 (102.8 mg, 0.1 mmol), compound 6 (CAS: 791028-27-8, 83 mg, 0.5 mmol), water (2 mL), and sodium bicarbonate (25 mg, 0.3 mmol). An inert gas was then introduced to replace the air, and the mixture was heated in an oil bath to 80°C for 6 h under an inert gas atmosphere. After the reaction was complete, high-performance liquid chromatography (HPLC) analysis revealed the blue compound 4, which was identified as the zwitterionic cyanine dye (53 mg, 50%). LRMS (ESI) m / z: [MH]- Calcd for C53H82N5O16S43+1172.46 ; Found 1168.03.

[0063] Performance testing 1. Spectral testing The zwitterionic cyanine dyes from Examples 2 and 3 were used to prepare 0.05 mg / mL PBS solutions of constant-brightness fluorescent probes. Absorption spectra of each sample from 400 to 900 nm were measured using a UV spectrometer, and fluorescence spectra were measured using a fluorescence spectrometer. The excitation wavelengths used in Examples 2 and 3 were 700 nm and 600 nm, respectively. The results are as follows: Figure 1 and 2 As shown. Figure 1 The UV absorption and fluorescence emission spectra of the zwitterionic anthocyanin dyes in Example 2 are shown. Figure 2 The UV absorption and fluorescence emission spectra of the zwitterionic anthocyanin dyes in Example 3 are shown. Figure 1It is known that the absorption wavelength of ultraviolet absorption spectrum is between 650 and 750 nm, and the emission wavelength of fluorescence emission spectrum is between 700 and 800 nm. From... Figure 2 It is known that the absorption wavelength of ultraviolet absorption spectrum is between 550 and 650 nm, and the emission wavelength of fluorescence emission spectrum is between 700 and 800 nm.

[0064] 2. Renal clearance efficiency test The zwitterionic cyanine dyes from Examples 2 and 3 were used to prepare PBS solutions containing 2 mg / mL of a stable fluorescent probe. Nine Balb / c mice were randomly divided into three groups (A, B, and C), with three mice in each group. 150 μL of the probe solution was injected via the tail vein. After injection, the mice were placed individually in clean metabolic cages, and urine was collected and its volume recorded over 24 hours. A standard curve of probe concentration versus UV absorption intensity was prepared using UV absorption spectroscopy, and the probe recovery rate in mouse urine was calculated. The results are shown below. Figure 3 and 4 As shown.

[0065] Figure 3 This is a statistical chart showing the recovery rate of zwitterionic cyanine dye in urine within 24 hours after injection, as described in Example 2. Figure 3 In the graph, A represents the change in urine recovery rate over time. Figure 3 Figure B in the graph represents the results of three replicate experiments on urine recovery rate. Figure 4 This is a statistical chart showing the recovery rate of zwitterionic cyanine dye in urine within 24 hours after injection, as described in Example 3. Figure 4 In the graph, A represents the change in urine recovery rate over time. Figure 4 Figure B in the graph represents the results of three replicate experiments on urine recovery rate. From... Figure 3 As can be seen from A, the zwitterionic anthocyanin dye in Example 2 was basically metabolized and excreted from the body 5-6 hours after tail vein injection, and the urine recovery rate reached its maximum value. Figure 4 As can be seen from A, the zwitterionic cyanine dye in Example 3 was essentially metabolized and excreted from the body approximately 5 hours after tail vein injection, with the urine recovery rate reaching its maximum value, indicating that the zwitterionic cyanine dye of the present invention has a rapid renal metabolic rate. From Figure 3 B and Figure 4 As shown in B, the urine recovery rate of the probe is relatively high, averaging about 97%, indicating that the zwitterionic anthocyanin dye provided by this invention is mainly metabolized by the kidneys.

[0066] 3. Study on probe tissue distribution The zwitterionic cyanine dyes from Examples 2 and 3 were used to prepare PBS solutions of 2 mg / mL constant-brightness fluorescent probes. Fifteen healthy Balb / c mice were randomly divided into five groups (1h group, 2h group, 3h group, 24h group, and control group), and 150 μL of the probe solution was injected via the tail vein. Rats were sacrificed at 1h, 2h, 3h, and 24h after injection, and major organs (heart (He), liver (Li), spleen (Sp), lung (Lu), kidney (Ki), bladder (Bl), small intestine (In), skin (Sk), and muscle (Mu)) were collected and subjected to fluorescence imaging. The results are as follows: Figures 5-8 As shown.

[0067] Figure 5 and Figure 7 The images show organ imaging of fluorescent probes prepared with zwitterionic cyanine dyes in normal mice, as shown in Examples 2 and 3, respectively; where He represents heart, Li represents liver, Sp represents spleen, Lu represents lung, Ki represents kidney, Bl represents bladder, Sk represents skin, Mu represents muscle, and In represents small intestine. Figure 6 and Figure 8 The images show quantitative organ imaging of fluorescent probes prepared with zwitterionic cyanine dyes in normal mice, as shown in Examples 2 and 3, respectively; where He represents heart, Li represents liver, Sp represents spleen, Lu represents lung, Ki represents kidney, Bl represents bladder, Sk represents skin, Mu represents muscle, and In represents small intestine. Figures 5-8 It is evident that the fluorescent probe prepared from the zwitterionic cyanine dye provided by this invention is mainly metabolized by the urinary system, and rapidly accumulates in the kidneys and / or bladder after probe injection. No obvious fluorescent signals were observed in other organs, indicating that the non-specific uptake of the probe by other tissues and organs is extremely low, and it can be used for the detection of urinary system diseases.

[0068] 4. Applications of ureteral imaging The zwitterionic cyanine dyes from Examples 2 and 3 were used to prepare PBS solutions of 2 mg / mL of constant-brightness fluorescent probes. These solutions were injected into mice via the tail vein. After 30 minutes, laparotomy and in vivo imaging of the small animals were performed. The results are as follows: Figure 9 As shown. Among them, Figure 9 In Figure A, the image shows the ureteral imaging result of zwitterionic anthocyanin dye in Example 2. Figure 9 Image B in the image represents the ureteral imaging result of zwitterionic anthocyanin dye in Example 3. Figure 9 As can be seen, the kidneys, ureters, and bladder of the urinary system are all fully displayed, indicating that the zwitterionic cyanine dye provided by this invention not only has the ability to image the kidneys and bladder, but also the ability to image the ureters, thus it can be applied to the diagnosis and treatment of ureter-related diseases.

[0069] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. Those skilled in the art can make other variations or modifications based on the above description. It is neither necessary nor possible to exhaustively describe all embodiments here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A zwitterionic anthocyanin dye, characterized in that, It has a structure shown in any of Equations (I), (II), or (IV): Where Y is F, Cl, Br or I; X is NH, SH or O; n is an integer from 1 to 3; n1 is an integer from 2 to 30.

2. The zwitterionic anthocyanin dye according to claim 1, characterized in that, The zwitterionic anthocyanin dye has any of the following structures: 、 、 、 , or .

3. A method for preparing the zwitterionic anthocyanin dye according to claim 1 or 2, characterized in that, Includes the following steps: When zwitterionic anthocyanin dyes have the structure of formula (Ⅰ): S1. Compound (14) and compound (15) undergo a condensation reaction to obtain the zwitterionic anthocyanin dye; When zwitterionic anthocyanin dyes have the structure of formula (II): S1. Compound (14) and compound (15) undergo a condensation reaction to obtain the compound of formula (I); S2. The compound of formula (I) undergoes a condensation reaction with compound (2) to obtain the zwitterionic anthocyanin dye; When zwitterionic anthocyanin dyes have the structure of formula (Ⅳ): S1. Compound (14) and compound (15) undergo a condensation reaction to obtain the compound of formula (I); S4. The compound described in formula (Ⅰ) and compound (6) undergo a condensation reaction under anaerobic conditions to obtain the zwitterionic anthocyanin dye; The specific compounds involved are as follows: 。 4. The preparation method according to claim 3, characterized in that, The compound (14) is obtained by a substitution reaction between the compound (12) and the compound (13); The specific compounds involved are as follows: 。 5. The preparation method according to claim 3, characterized in that, In step S1, the condensation reaction is carried out at a temperature of 80-100°C for 12-24 hours.

6. The preparation method according to claim 3, characterized in that, In steps S2 to S4, the condensation reaction is carried out at a temperature of 65 to 85°C for 6 to 12 hours.

7. The use of the zwitterionic cyanine dye of claim 1 or 2, or its pharmaceutically acceptable salts, enantiomers, diastereomers, or tautomers, in the preparation of fluorescent probes for non-disease diagnostic purposes.

8. The application according to claim 7, characterized in that, The fluorescent probe is a fluorescent probe used for organ imaging, wherein the organ is the kidney, ureter, or bladder.

9. A fluorescent probe, characterized in that, Contains zwitterionic anthocyanin dyes as described in claim 1 or 2 and / or their pharmaceutically acceptable salts, enantiomers, diastereomers, and tautomers.

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