Zwitterionic fluorescent probe containing dimer targeting ligand as well as preparation method and application of zwitterionic fluorescent probe

Abstract

The invention belongs to the technical field of novel small-molecule drugs, and particularly relates to a zwitterionic fluorescent probe containing a dimer targeting ligand and a preparation method and application thereof. The chemical structural formula of the zwitterionic fluorescent probe containing the dimer targeting ligand is as shown in formula (1): # imgabs0 #, the fluorescent probe can be used for near-infrared fluorescence imaging of tumor targeting, and the structure of the fluorescent probe contains two same tumor targeting ligands, so that the zwitterionic fluorescent probe has a better targeting function compared with a single targeting ligand. The introduction of the alkaline amino acid in the fluorescent probe enables the charge on the surface of the probe to return to zero, and the zwitterionic design enables the probe to have a lower tissue background signal. The probe disclosed by the invention is completely symmetrical in structure, simpler in synthesis method, good in water solubility, low in non-specific fluorescence signal and suitable for industrial production and clinical application.

Description

Technical Field

[0001] The present invention belongs to the technical field of novel small molecule drugs, and in particular relates to a zwitterionized fluorescent probe containing a dimerized targeting ligand, and a preparation method and application thereof. Background Art

[0002] Minimally invasive surgery (MIS) has gradually replaced traditional open surgery due to its many advantages, such as less trauma, fewer complications, lower risk of infection, shorter hospitalization and recovery time, etc. The proportion of minimally invasive surgery has continued to increase, and minimally invasive surgery has become one of the trends in the development of surgery in recent years.

[0003] However, minimally invasive surgery currently relies mainly on the doctor's observation and empirical judgment of the lesion site, supplemented by case slices for resection, which makes it impossible to clearly identify the tumor boundaries and tiny lesions, and even to completely remove the tumor tissue, resulting in a high risk of recurrence. It is also possible that due to unclear boundaries, excessive resection may damage normal tissue and cause loss of function of the pathogenic body. Using fluorescent molecular probes to mark tumors for specific fluorescent imaging of tumors can provide timely feedback to the surgeon, improve the detection rate of micro-metastases during surgery, and optimize decisions in real time during surgery. The application of fluorescent molecular imaging technology in clinical practice has brought new opportunities for precision surgery. With the rapid development of minimally invasive surgery, fluorescent navigation surgery has become mainstream, and the market for fluorescent endoscopic diagnostic equipment has expanded rapidly, but the research and development of matching targeted fluorescent contrast agents has become the main bottleneck restricting the development of fluorescent endoscopes.

[0004] At present, the commonly used tumor tracing near-infrared dye in clinical practice is indocyanine green (ICG). Due to the difference in the metabolic rate of ICG in liver cancer tissue and normal liver tissue, it is used to trace the surgical resection of liver cancer. However, ICG is difficult to use for fluorescent tracing of most tumors other than liver cancer. Therefore, the development of highly specific and sensitive fluorescent molecular probes has important clinical significance.

[0005] Due to the huge market prospects of fluorescent contrast agents, foreign medical device giants have begun to make plans. In November 2021, the world's first fluorescent molecular probe Cytalux was approved by the FDA for listing in the United States. This is also the only approved tumor-targeted fluorescent contrast agent in the world. Compared with foreign countries, China started late in the field of fluorescent contrast agents, and foreign companies and research institutions have applied for a large number of patents in this field, building a strict intellectual property barrier that is difficult for latecomers to break through. Taking the fluorescent dyes necessary for the synthesis of fluorescent contrast agents as an example, the American LI-COR company has developed the fluorescent dye IRDye800CW and provided it as an exclusive supplier to fluorescent contrast agent companies around the world. According to statistics, there are currently about 30 clinical trials in the world based on LI-COR's IRDye800CW, including several domestic companies. Although IRDye800CW has excellent imaging effects, its synthesis is difficult and costly.

[0006] However, existing probes all have various problems. Take IRDye800CW as an example. Due to its completely asymmetric structure, the structures connected to the indole sulfonate on both sides are completely different. Therefore, it is necessary to synthesize two different intermediates, which takes a longer synthesis route. In addition, the yield of dyes with asymmetric structures is much lower than that of dyes with symmetric structures. In addition, the hydrophilic structure in the IRDye800CW structure is a negatively charged sulfonic acid group, which makes the overall charge of the probe negative. Negatively charged dyes will lead to more nonspecific tissue signals. Summary of the invention

[0007] Based on the above technical background, the main purpose of the present invention is to provide a zwitterionized fluorescent probe containing a dimeric targeting ligand and its preparation method and application, which can overcome the problems of complex synthesis routes, high tissue background signals, instability in vivo, and low targeting efficiency of existing fluorescent probes.

[0008] In order to achieve the above-mentioned invention object, the technical solution adopted by the present invention includes:

[0009] The first aspect of the present invention is to provide a zwitterionized fluorescent probe containing a dimeric targeting ligand, wherein the chemical structure of the zwitterionized fluorescent probe containing a dimeric targeting ligand is shown in formula (1):

[0010]

[0011] In formula (1), x and y are integers greater than or equal to 1; Z is a halogen ion; R is lysine, arginine, histidine or a non-natural basic amino acid with a positively charged side chain under physiological conditions; Ligand is a small molecule, polypeptide, aptamer or antibody with tumor targeting function.

[0012] Preferably, R is selected from one of N-methyllysine, 2,5-diaminopentanoic acid, 2,4-diaminobutyric acid, 2,3-diaminopropionic acid, 2-amino-4-guanidinobutyric acid, and 2-amino-3-guanidinopropionic acid.

[0013] Preferably, Ligand is selected from one of folic acid targeting folate receptor-α (FRα), RGD polypeptide targeting integrin, glutamate urea and derivatives targeting PSMA, rk polypeptide targeting Her2, nucleic acid aptamer NOX-A12 targeting chemokine CXCL12, trastuzumab, bevacizumab, and PD-L1 monoclonal antibody.

[0014] Preferably, in formula (1), x=5, y=4.

[0015] More preferably, the chemical structure of the zwitterionized fluorescent probe containing a dimeric targeting ligand is as shown in formula (3):

[0016]

[0017] The second aspect of the present invention is to provide a method for preparing the zwitterionized fluorescent probe containing a dimeric targeting ligand according to the first aspect of the present invention, the preparation method comprising the following steps:

[0018] Step 1, dissolving p-hydrazinobenzenesulfonic acid, adding 3-methyl-2-butanone, and then heating to perform reflux reaction. After the reaction is completed, cooling, filtering, washing, and drying are performed to obtain indolesulfonic acid. After the indolesulfonic acid is dissolved, it is added dropwise to a propanol solution, and after post-treatment, 3H-indole-2,3,3-trimethyl-5-sulfonate is obtained;

[0019] Step 2, after dissolving 3H-indole-2,3,3-trimethyl-5-sulfonate, adding 6-bromohexanoic acid, and then heating to perform reflux reaction, cooling, washing, filtering, and evaporating under reduced pressure after the reaction is completed to obtain compound E;

[0020] Step 3, under N2 protection, dissolving compound E, compound F and potassium acetate in a mixed solvent of acetic acid-acetic anhydride, and then heating to reflux reaction, followed by precipitation and purification to obtain compound G;

[0021] Step 4: Under N2 protection, compound G and DSC are dissolved in CH3CN, and then pyridine, triethylamine or DIPEA are added and stirred for reaction. Compound J is then added and the reaction is continued by stirring, and then the mixture is concentrated under reduced pressure, washed and purified to obtain compound K, i.e., the fluorescent probe.

[0022] In step 1,

[0023] Preferably, p-hydrazinobenzenesulfonic acid is dissolved in an acetic acid solution, and then 3-methyl-2-butanone is added, the molar ratio of hydrazinobenzenesulfonic acid to 3-methyl-2-butanone is 50-55:160, followed by heating to 110-130° C. and reflux reaction for 3-5 hours, cooling to 20-30° C., filtering with filter paper, washing the obtained solid crude product with ethyl acetate, and evaporating the solvent to obtain indolesulfonic acid; and / or,

[0024] Preferably, indolesulfonic acid is dissolved in a methanol solution and then added dropwise to a propanol solution containing potassium hydroxide, wherein the mass ratio of potassium hydroxide to indolesulfonic acid in the propanol solution is (1-1.5):5.1, followed by stirring at room temperature for 20-25 hours, filtering with filter paper, and evaporating the residue under reduced pressure to obtain 3H-indole-2,3,3-trimethyl-5-sulfonate.

[0025] In step 2,

[0026] Preferably, 3H-indole-2,3,3-trimethyl-5-sulfonate is dissolved in 1,2-dichlorobenzene, and then 6-bromohexanoic acid is added, heated to 100-120° C. and refluxed for 10-15 hours, and the molar ratio of 3H-indole-2,3,3-trimethyl-5-sulfonate to 6-bromohexanoic acid is (35-45):12. After the reaction is completed, it is cooled to room temperature, the upper solution is discarded, isopropanol is added to the residue for washing, and then filtered with filter paper, and evaporated to dryness under reduced pressure to obtain compound E; and / or,

[0027] In step 3,

[0028] Preferably, under N2 protection, compound E, compound F and potassium acetate are dissolved in an acetic acid-acetic anhydride mixed solvent in a molar ratio of (1-1.5): (0.6-0.7): (2-3), wherein the volume ratio of acetic acid to acetic anhydride is (6-8): 3, and then heated to 110-130°C for reflux reaction for 2-5h, and then isopropanol is added to precipitate to obtain a crude product, and finally purified by column chromatography to obtain compound G.

[0029] In step 4,

[0030] Preferably, under N2 protection, compound G and DSC (bis(N-succinimidyl) carbonate) are dissolved in CH3CN at a molar ratio of 1: (1.2-1.7), and then pyridine, triethylamine or DIPEA (N,N-diisopropylethylamine) are added, and the reaction is stirred at room temperature for 5-8 hours, and then compound J is added, and the reaction is continued to be stirred at room temperature for 6-10 hours, and then concentrated under reduced pressure, washed with dichloromethane and ethyl acetate in turn, and purified by column chromatography to obtain compound K, i.e., the fluorescent probe; and / or,

[0031] Preferably, the molar ratio of pyridine, triethylamine or DIPEA to compound G is (2-4):1.

[0032] The third aspect of the present invention is to provide an application of the zwitterionized fluorescent probe containing a dimeric targeting ligand as described in the first aspect of the present invention, wherein the fluorescent probe can be used as a tumor fluorescence imaging contrast agent, a photoacoustic imaging contrast agent, a photothermal therapy agent, a photodynamic therapy agent or a sonodynamic therapy agent.

[0033] The beneficial effects of the present invention are:

[0034] (1) The luminescent group of the fluorescent probe described in the present invention is a heptamethine cyanine dye structure with indole sulfonate groups at both ends, and the tumor targeting ligand is connected to the indole groups at both ends through a polyethylene glycol chain, a basic amino acid and a saturated alkane single chain.

[0035] The probe of the present invention can be used for tumor-targeted near-infrared fluorescence imaging. Its structure contains two identical tumor-targeting ligands, and its affinity with the receptor is greatly improved compared with a single targeting ligand, thereby having better targeting function and higher targeting efficiency.

[0036] The fluorescent probe introduces positively charged basic amino acids to return the charge on the probe surface to zero. This zwitterionization design enables the probe to have a lower tissue background signal.

[0037] (2) The fluorescent probe of the present invention has a completely symmetrical structure, a simple synthesis method, good water solubility, low nonspecific fluorescence signal, is more stable in vivo, has high targeting efficiency, and is suitable for industrial production and clinical application.

[0038] (3) The fluorescent probe of the present invention can be used as a tumor fluorescent imaging contrast agent, a photoacoustic imaging contrast agent, a photothermal therapy agent, a photodynamic therapy agent or a sonodynamic therapy agent. The fluorescent probe has a good application prospect in tumor diagnosis and treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 The absorption spectrum of the zwitterionized fluorescent probe prepared in Example 4 of the present invention in water is shown;

[0040] Figure 2 The fluorescent imaging photos of mice at different time points after injection of the fluorescent probe prepared in Example 4 are shown. DETAILED DESCRIPTION

[0041] The present invention will be described in detail below, and the characteristics and advantages of the present invention will become clearer and more distinct with the description.

[0042] The first aspect of the present invention is to provide a zwitterionized fluorescent probe containing a dimeric targeting ligand, wherein the chemical structure of the zwitterionized fluorescent probe containing a dimeric targeting ligand is shown in formula (1):

[0043]

[0044] In formula (1), x and y are integers greater than or equal to 1; Z is a halogen ion; R is lysine, arginine, histidine or a non-natural basic amino acid with a positively charged side chain under physiological conditions; and the ligand is a small molecule, polypeptide, aptamer or antibody with tumor targeting function.

[0045] Preferably, R is a non-natural basic amino acid, and R is selected from one of N-methyllysine, 2,5-diaminopentanoic acid, 2,4-diaminobutyric acid, 2,3-diaminopropionic acid, 2-amino-4-guanidinobutyric acid, and 2-amino-3-guanidinopropionic acid.

[0046] Preferably, the ligand is selected from one of folic acid targeting folate receptor-α (FRα), RGD polypeptide targeting integrin, glutamate urea and derivatives targeting PSMA, rk polypeptide targeting Her2, nucleic acid aptamer NOX-A12 targeting chemokine CXCL12, trastuzumab, bevacizumab, and PD-L1 monoclonal antibody.

[0047] More preferably, in formula (1), x=5, y=4. The length of the alkane chains on both sides in the present invention can be any length, and the preferred length is 5 carbons. The length y of the polyethylene glycol chain between the tumor targeting ligand (Ligand) and the basic amino acid in the present invention can be any length. These two polyethylene glycol chains can improve the water solubility of the probe. Generally, the longer the degree of polymerization of the polyethylene glycol chain, the better the water solubility. However, the longer the degree of polymerization, the higher the synthesis cost. The preferred length is 4. The polyethylene glycol chain and the ligand can be connected by amide reaction, thiol-maleimide reaction, esterification reaction, click chemistry reaction and other methods.

[0048] Preferably, the chemical structure of the zwitterionized fluorescent probe containing a dimeric targeting ligand is as shown in formula (2):

[0049]

[0050] More preferably, the chemical structure of the zwitterionized fluorescent probe containing a dimeric targeting ligand is as shown in formula (3):

[0051]

[0052] In addition to being used for fluorescence imaging, the fluorescent probe described in the present invention can also be used for photoacoustic imaging. In addition, the fluorescent probe can generate heat under the excitation of near-infrared light in a specific band, so it can also be used for photothermal treatment of tumors. At the same time, the probe will generate singlet oxygen under the irradiation of laser or ultrasound in a specific band, so it can be used for photodynamic and sonodynamic treatment of tumors, respectively.

[0053] The second aspect of the present invention is to provide a method for preparing the zwitterionized fluorescent probe containing a dimeric targeting ligand according to the first aspect of the present invention, the preparation method comprising the following steps:

[0054] Step 1, dissolving p-hydrazinobenzenesulfonic acid, adding 3-methyl-2-butanone, and then heating to perform reflux reaction. After the reaction is completed, cooling, filtering, washing, and drying are performed to obtain indolesulfonic acid. After the indolesulfonic acid is dissolved, it is added dropwise to a propanol solution, and after post-treatment, 3H-indole-2,3,3-trimethyl-5-sulfonate is obtained;

[0055] Step 2, after dissolving 3H-indole-2,3,3-trimethyl-5-sulfonate, adding 6-bromohexanoic acid, and then heating to perform reflux reaction, cooling, washing, filtering, and evaporating under reduced pressure after the reaction is completed to obtain compound E;

[0056] Step 3, under N2 protection, compound E (1-[5-carboxyhexyl]-2,3,3-trimethyl-3H-indole-5-sulfonic acid inner salt), compound F (N-[[1E,3E,5E]-5-[phenylimino]penta-1,3-dien-1-yl]aniline hydrochloride) and potassium acetate are dissolved in an acetic acid-acetic anhydride mixed solvent, and then heated to reflux reaction, followed by precipitation and purification to obtain compound G (E)-1-(5-carboxypentyl)-2-((2E,4E,6E)-7-(1-(5-carboxypentyl)-3,3-dimethyl-5-sulfonic acid 3H-indol-1-ium-2-yl)-hept-2,4,6-triene-1-ylidene) 3,3-dimethylindole-5-sulfonic acid inner salt;

[0057] Step 4: Under N2 protection, compound G and DSC are dissolved in CH3CN, and then pyridine, triethylamine or DIPEA are added and stirred for reaction. Compound J is then added and the reaction is continued by stirring, and then the mixture is concentrated under reduced pressure, washed and purified to obtain compound K, i.e., the fluorescent probe.

[0058] The above steps are described in detail below.

[0059] In step 1, p-hydrazinobenzenesulfonic acid is dissolved in an acetic acid solution, and then 3-methyl-2-butanone is added, the molar ratio of hydrazinobenzenesulfonic acid to 3-methyl-2-butanone is 50-55:160, and then heated to 110-130° C. for reflux reaction for 3-5 hours, cooled to 20-30° C., filtered with filter paper, and the obtained solid crude product is washed with ethyl acetate, and the solvent is evaporated to obtain indolesulfonic acid.

[0060] Preferably, p-hydrazinobenzenesulfonic acid is dissolved in an acetic acid solution, and then 3-methyl-2-butanone is added, the molar ratio of hydrazinobenzenesulfonic acid to 3-methyl-2-butanone is 53:160, followed by heating to 120° C. for reflux reaction for 4 hours, cooling to 25° C., filtering with filter paper, washing the obtained solid crude product with ethyl acetate, and evaporating the solvent to obtain indolesulfonic acid.

[0061] Indolesulfonic acid is dissolved in a methanol solution, and then added dropwise into a propanol solution containing potassium hydroxide, wherein the mass ratio of potassium hydroxide to indolesulfonic acid in the propanol solution is (1-1.5):5.1, followed by stirring at room temperature for 20-25 hours, filtering with filter paper, and evaporating the residue under reduced pressure to obtain 3H-indole-2,3,3-trimethyl-5-sulfonate.

[0062] Preferably, indolesulfonic acid is dissolved in a methanol solution and then added dropwise to a propanol solution containing potassium hydroxide, wherein the mass ratio of potassium hydroxide to indolesulfonic acid in the propanol solution is 1.4:5.1, followed by stirring at room temperature for 24 hours, filtering with filter paper, and evaporating the residue under reduced pressure to obtain 3H-indole-2,3,3-trimethyl-5-sulfonate.

[0063] In step 2, 3H-indole-2,3,3-trimethyl-5-sulfonate is dissolved in 1,2-dichlorobenzene, and then 6-bromohexanoic acid is added and heated to 100-120° C. and refluxed for 10-15 hours. The molar ratio of 3H-indole-2,3,3-trimethyl-5-sulfonate and 6-bromohexanoic acid is (35-45):12. After the reaction is completed, it is cooled to room temperature, the upper solution is discarded, isopropanol is added to the residue for washing, and then filtered with filter paper, and evaporated under reduced pressure to obtain compound E.

[0064] Preferably, 3H-indole-2,3,3-trimethyl-5-sulfonate is dissolved in 1,2-dichlorobenzene, and then 6-bromohexanoic acid is added and heated to 110°C for reflux reaction for 12 hours. The molar ratio of 3H-indole-2,3,3-trimethyl-5-sulfonate and 6-bromohexanoic acid is 39.7:12. After the reaction is completed, it is cooled to room temperature, the upper solution is discarded, isopropanol is added to the residue for washing, and then filtered with filter paper, and evaporated under reduced pressure to obtain compound E.

[0065] In step 3, under N2 protection, compound E, compound F and potassium acetate are dissolved in an acetic acid-acetic anhydride mixed solvent in a molar ratio of (1-1.5): (0.6-0.7): (2-3), wherein the volume ratio of acetic acid to acetic anhydride is (6-8): 3, and then heated to 110-130°C for reflux reaction for 2-5h, and then isopropanol is added to precipitate to obtain a crude product, and finally purified by column chromatography to obtain compound G.

[0066] Preferably, under N2 protection, compound E, compound F and potassium acetate are dissolved in an acetic acid-acetic anhydride mixed solvent in a molar ratio of 1.28:0.64:2.56, wherein the volume ratio of acetic acid to acetic anhydride is 7:3, and then heated to 120°C for reflux reaction for 3 to 4 hours, and then isopropanol is added to precipitate to obtain a crude product, and finally purified by column chromatography to obtain compound G.

[0067] In step 4, under N2 protection, compound G and DSC (bis(N-succinimidyl) carbonate) are dissolved in CH3CN at a molar ratio of 1:(1.2-1.7), and then pyridine, triethylamine or DIPEA (N,N-diisopropylethylamine) are added, and the reaction is stirred at room temperature for 5-8 hours. Subsequently, compound J is added, and the reaction is continued to stir at room temperature for 6-10 hours, and then concentrated under reduced pressure, washed with dichloromethane and ethyl acetate in turn, and purified by column chromatography to obtain compound K, i.e., the fluorescent probe.

[0068] Preferably, under N2 protection, compound G and DSC (bis(N-succinimidyl) carbonate) are dissolved in CH3CN at a molar ratio of 1:1.5, and then pyridine, triethylamine or DIPEA (N,N-diisopropylethylamine) are added, and the reaction is stirred at room temperature for 6 hours, and then compound J is added, and the reaction is continued to be stirred at room temperature for 8 hours, and then concentrated under reduced pressure, washed with dichloromethane and ethyl acetate in turn, and purified by column chromatography to obtain compound K, which is the fluorescent probe.

[0069] The molar ratio of pyridine, triethylamine or DIPEA to compound G is (2-4):1. Preferably, the molar ratio of pyridine, triethylamine or DIPEA to compound G is 3:1.

[0070] The third aspect of the present invention is to provide an application of the zwitterionized fluorescent probe containing a dimeric targeting ligand as described in the first aspect of the present invention, wherein the fluorescent probe can be used as a tumor fluorescence imaging contrast agent, a photoacoustic imaging contrast agent, a photothermal therapy agent, a photodynamic therapy agent or a sonodynamic therapy agent.

[0071] The fluorescent probe of the present invention can be used in tumor diagnosis and treatment.

[0072] The fluorescent probe can be used as a tumor fluorescence imaging or photoacoustic imaging contrast agent in tumor diagnosis, and can be used as a photothermal therapy agent, photodynamic therapy agent or sonodynamic therapy agent in tumor treatment.

[0073] Example

[0074] The present invention is further described below by specific examples, which are limited to illustrating the present invention but not to limiting the scope of the present invention. The raw materials used in the examples of the present invention were all purchased.

[0075] Example 1

[0076] The preparation process of compound C (3H-indole-2,3,3-trimethyl-5-sulfonate) is shown in the following formula:

[0077]

[0078] Dissolve p-hydrazinobenzenesulfonic acid (10.0 g, 53.0 mmol) in acetic acid (30 mL) solution, then add 3-methyl-2-butanone (17 mL, 160.0 mmol) and heat to 120 ° C for reflux reaction for 4 hours, cool to 25 ° C and filter with filter paper. Wash the obtained solid crude product with ethyl acetate, evaporate the solvent to obtain pink powder indolesulfonic acid (10 g, 79%). Dissolve the above-obtained indolesulfonic acid (5.1 g) in methanol (35 mL) solution, then add dropwise to a propanol (35 mL) solution containing potassium hydroxide (1.4 g), stir at room temperature for 24 hours, filter with filter paper, and evaporate the residue under reduced pressure to obtain compound C (3H-indole-2,3,3-trimethyl-5-sulfonate).

[0079] Compound C was tested by nuclear magnetic resonance and obtained 1 H NMR (500MHz, Chloroform-d) δ7.94 (d, J = 2.2 Hz, 1H), 7.76 (dd, J = 8.7, 2.3 Hz, 1H), 7.59 (d, J = 8.6 Hz, 1H), 1.29 (s, 5H).

[0080] Example 2

[0081] The preparation process of compound E (3H-indole, 1-(4-carboxybutyl)-2,3,3-trimethyl-5-sulfonic acid inner salt) is shown in the following formula:

[0082]

[0083] Under N2 protection, compound C (11 g, 39.7 mmol) was dissolved in 1,2-dichlorobenzene (15 ml), and then 6-bromohexanoic acid (compound D) (2.3 g, 12.0 mmol) was added and heated to 110 ° C for reflux reaction for 12 hours. After the reaction was completed, it was cooled to room temperature, the upper solution was discarded, and the residue was washed with isopropanol (50 mL), then filtered with filter paper, and the solid crude product was evaporated under reduced pressure to obtain pink compound E.

[0084] Compound E was subjected to NMR detection to obtain 1 H NMR(500MHz,Chloroform-d)δ8.11–8.04(m,2H),7.60–7.55(m,1H),4.36(t,J=6. 8Hz,2H),2.93(s,3H),2.32–2.25(m,2H),1.98–1.80(m,2H),1.68–1.59(m,10H).

[0085] Example 3

[0086] The preparation process of compound G is shown in the following formula:

[0087]

[0088] Under N2 protection, compound E (1-[5-carboxyhexyl]-2,3,3-trimethyl-3H-indole-5-sulfonic acid inner salt) (500 mg, 1.28 mmol), compound F (N-[[1E,3E,5E]-5-[phenylimino]penta-1,3-diene-1-yl]aniline hydrochloride) (181 mg, 0.64 mmol) and potassium acetate (251 mg, 2.56 mmol) were dissolved in acetic acid-acetic anhydride (acetic acid: acetic anhydride = 7:3) mixed solvent (12 mL), heated to 120 ° C and refluxed for 3 to 4 hours, and then a large amount of isopropanol was added to precipitate to obtain a crude product. Compound G was purified by column chromatography (reverse chromatography, eluent is methanol: water, the proportion of methanol is gradually increased from about 5% to 20%).

[0089] Compound G was subjected to NMR detection to obtain 1H NMR(500MHz,Chloroform-d)δ8.13(dd,J=8.1,1.8Hz,2H),8.08(d,J=1.9Hz,2H),7.77(d,J=2.2Hz,2H),7.67– 7.59(m,4H),7.41–7.35(m,2H),7.06(d,J=8.0Hz,2H),7.00–6.92(m,2H),6.70–6.58(m,4H),6.56–6.48(m,2H) ,6.39–6.30(m,2H),5.97(dt,J=7.7,0.9Hz,2H),4.43(t,J=7.0Hz,4H),3.78(t,J=5.5Hz,4H),2.27(dt,J=11.0 ,9.0Hz,8H), 1.98–1.80(m,4H), 1.80–1.71(m,15H), 1.68–1.59(m,21H), 1.59–1.53(m,3H), 1.46–1.37(m,4H).

[0090] Example 4

[0091] The preparation process of compound K is shown in the following formula:

[0092]

[0093] Under N2 protection, compound G (1 mmol) and 1.5 mmol DSC (bis(N-succinimidyl) carbonate) were dissolved in CH3CN, 3 mmol pyridine was added, and the mixture was stirred at room temperature for 6 hours to obtain a crude product compound I (mixture). 1.5 mmol compound J was added to the above mixture, and stirring was continued for 8 hours at room temperature. After concentration under reduced pressure, washing with dichloromethane and ethyl acetate in turn, column chromatography was used for purification (reverse chromatography, eluent was acetonitrile: water, and the proportion of acetonitrile was gradually increased from about 5% to 30%) to obtain compound K, i.e., a fluorescent probe.

[0094] Compound K was subjected to NMR detection to obtain 1H NMR(500MHz,Chloroform-d)δ=8.01–7.94(m,1H),7.85(d,J=8.4,1H),7.72(d,J=8.4,1H),7. 61(t,J=3.6,2H),7.51(dd,J=8.7,1.9,2H),7.30–7.17(m,6H),7.14(t,J=5.1,1H),7.03(s,1 H),6.94(s,1H),6.70(t,J=4.9,1H),6.66–6.61(m,1H),6.23(t,J=6.2,1H),5.90(d,J=6.6,2 H),5.84(d,J=6.6,2H),4.76(dt,J=8.6,7.3,1H),4.52–4.39(m,2H),4.30(ddt,J=8.6,7.3,5 .7,2H),4.25–4.19(m,1H),3.91(dd,J=16.9,6.2,1H),3.86–3.70(m,2H),3.70–3.59(m,12H) ,3.59–3.51(m,4H),3.44–3.30(m,2H),3.16–3.04(m,7H),2.90(ddt,J=14.0,6.1,1.0,1H),2 .72–2.58(m,2H),2.47(t,J=6.0,2H),2.19(tdd,J=8.5,3.4,1.5,2H),1.98–1.84(m,1H),1.8 4–1.76(m,2H),1.76–1.51(m,17H),1.51–1.44(m,3H),1.44–1.38(m,1H),1.38–1.31(m,2H).

[0095] The absorption spectrum of the fluorescent probe in PBS is as follows: Figure 1 In PBS, the characteristic absorption of the fluorescent probe is around 766nm, which is consistent with the absorption characteristics of Cy7 cyanine dye compounds.

[0096] Experimental example

[0097] This experimental example studies the performance of the fluorescent probe prepared in Example 4 of the present invention in tumor fluorescence imaging in mice. A mouse breast cancer subcutaneous tumor model was established by injecting 4T1 mouse breast cancer cells into the right hind leg of Balb / c nude mice. After the tumor grew to an appropriate size, the fluorescent probe was injected intravenously (2 nmol of fluorescent probe was injected into each mouse), and then the mice were imaged with fluorescence at different time points after the injection of the fluorescent probe. Figure 2 shown.

[0098] from Figure 2It can be seen that a strong fluorescence signal was detected at the tumor site 2h to 48h after the probe injection, and the fluorescence signal of the tumor tissue was significantly stronger than that of the surrounding tissue, indicating that the fluorescent probe of the present invention can effectively and specifically develop tumor tissue.

[0099] The present invention has been described in detail above in conjunction with specific implementations and exemplary examples, but these descriptions cannot be understood as limiting the present invention. Those skilled in the art understand that, without departing from the spirit and scope of the present invention, a variety of equivalent substitutions, modifications or improvements may be made to the technical solution of the present invention and its implementation methods, all of which fall within the scope of the present invention. The scope of protection of the present invention shall be subject to the attached claims.

Claims

1. A zwitterionized fluorescent probe containing a dimeric targeting ligand, characterized in that: The chemical structure of the zwitterionized fluorescent probe containing a dimeric targeting ligand is shown in formula (1): In formula (1), x and y are integers greater than or equal to 1; Z is a halogen ion; R is lysine, arginine, histidine or a non-natural basic amino acid with a positively charged side chain under physiological conditions; Ligand is a small molecule, polypeptide, aptamer or antibody with tumor targeting function.

2. The zwitterionized fluorescent probe containing a dimeric targeting ligand according to claim 1, characterized in that: R is selected from one of N-methyllysine, 2,5-diaminopentanoic acid, 2,4-diaminobutyric acid, 2,3-diaminopropionic acid, 2-amino-4-guanidinobutyric acid, and 2-amino-3-guanidinopropionic acid.

3. The zwitterionized fluorescent probe containing a dimeric targeting ligand according to claim 1, characterized in that: Ligand is selected from one of folic acid targeting folate receptor-α (FRα), RGD peptide targeting integrin, glutamate urea and derivatives targeting PSMA, rk peptide targeting Her2, nucleic acid aptamer NOX-A12 targeting chemokine CXCL12, trastuzumab, bevacizumab, and PD-L1 monoclonal antibody.

4. The zwitterionized fluorescent probe containing a dimeric targeting ligand according to claim 1, characterized in that: In formula (1), x=5, y=4.

5. The zwitterionized fluorescent probe containing a dimeric targeting ligand according to any one of claims 1 to 4, characterized in that: The chemical structure of the zwitterionized fluorescent probe containing a dimeric targeting ligand is shown in formula (3):

6. A method for preparing a zwitterionized fluorescent probe containing a dimeric targeting ligand according to any one of claims 1 to 5, characterized in that: The preparation method comprises the following steps: Step 1, dissolving p-hydrazinobenzenesulfonic acid, adding 3-methyl-2-butanone, and then heating to perform reflux reaction. After the reaction is completed, cooling, filtering, washing, and drying are performed to obtain indolesulfonic acid. After the indolesulfonic acid is dissolved, it is added dropwise to a propanol solution, and after post-treatment, 3H-indole-2,3,3-trimethyl-5-sulfonate is obtained; Step 2, after dissolving 3H-indole-2,3,3-trimethyl-5-sulfonate, adding 6-bromohexanoic acid, and then heating to perform reflux reaction, cooling, washing, filtering, and evaporating under reduced pressure after the reaction is completed to obtain compound E; Step 3, under N2 protection, dissolving compound E, compound F and potassium acetate in a mixed solvent of acetic acid-acetic anhydride, and then heating to reflux reaction, followed by precipitation and purification to obtain compound G; Step 4: Under N2 protection, compound G and DSC are dissolved in CH3CN, and then pyridine, triethylamine or DIPEA are added and stirred for reaction. Compound J is then added and the reaction is continued by stirring, and then the mixture is concentrated under reduced pressure, washed and purified to obtain compound K, i.e., the fluorescent probe.

7. The preparation method according to claim 6, characterized in that: In step 1, Dissolving p-hydrazinobenzenesulfonic acid in an acetic acid solution, then adding 3-methyl-2-butanone, the molar ratio of hydrazinobenzenesulfonic acid to 3-methyl-2-butanone being 50-55:160, then heating to 110-130° C. for reflux reaction for 3-5 hours, cooling to 20-30° C., filtering with filter paper, washing the obtained solid crude product with ethyl acetate, and evaporating the solvent to obtain indolesulfonic acid; and / or, Indolesulfonic acid is dissolved in a methanol solution, and then added dropwise into a propanol solution containing potassium hydroxide, wherein the mass ratio of potassium hydroxide to indolesulfonic acid in the propanol solution is (1-1.5):5.1, followed by stirring at room temperature for 20-25 hours, filtering with filter paper, and evaporating the residue under reduced pressure to obtain 3H-indole-2,3,3-trimethyl-5-sulfonate.

8. The preparation method according to claim 6, characterized in that: In step 2, Dissolve 3H-indole-2,3,3-trimethyl-5-sulfonate in 1,2-dichlorobenzene, then add 6-bromohexanoic acid, heat to 100-120° C. and reflux for reaction for 10-15 hours, wherein the molar ratio of 3H-indole-2,3,3-trimethyl-5-sulfonate to 6-bromohexanoic acid is (35-45):

12. After the reaction is completed, cool to room temperature, discard the upper solution, add isopropanol to the residue for washing, filter with filter paper, and evaporate to dryness under reduced pressure to obtain compound E; and / or, In step 3, under N2 protection, compound E, compound F and potassium acetate are dissolved in an acetic acid-acetic anhydride mixed solvent in a molar ratio of (1-1.5): (0.6-0.7): (2-3), wherein the volume ratio of acetic acid to acetic anhydride is (6-8): 3, and then heated to 110-130°C for reflux reaction for 2-5h, and then isopropanol is added to precipitate to obtain a crude product, and finally purified by column chromatography to obtain compound G.

9. The preparation method according to claim 6, characterized in that: In step 4, Under N2 protection, compound G and DSC (bis(N-succinimidyl) carbonate) are dissolved in CH3CN at a molar ratio of 1: (1.2-1.7), and then pyridine, triethylamine or DIPEA (N,N-diisopropylethylamine) are added, and the reaction is stirred at room temperature for 5-8 hours, and then compound J is added, and the reaction is continued at room temperature for 6-10 hours, and then concentrated under reduced pressure, washed with dichloromethane and ethyl acetate in turn, and purified by column chromatography to obtain compound K, i.e., the fluorescent probe; and / or, The molar ratio of pyridine, triethylamine or DIPEA to compound G is (2-4):

1.

10. Use of a zwitterionic fluorescent probe containing a dimeric targeting ligand as claimed in any one of claims 1 to 5, wherein the fluorescent probe can be used as a tumor fluorescence imaging contrast agent, a photoacoustic imaging contrast agent, a photothermal therapy agent, a photodynamic therapy agent or a sonodynamic therapy agent.

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