A zwitterionic fluorescent probe containing a dimeric targeting ligand, and a preparation method and application thereof

Abstract

The application belongs to the technical field of new small-molecule drugs, and particularly relates to a zwitterionic fluorescent probe containing a dimeric targeting ligand as well as a preparation method and application thereof.The chemical structural formula of the zwitterionic fluorescent probe containing the dimeric targeting ligand is shown as formula (1): The fluorescent probe can be used for near-infrared fluorescence imaging of tumor targeting, and the two identical tumor targeting ligands contained in the structure have better targeting function compared to a single targeting ligand.The introduction of basic amino acids in the fluorescent probe makes the charge on the surface of the probe zero, and the zwitterionic design makes the probe have a lower tissue background signal.The probe structure is completely symmetrical, the synthesis method is simpler, the water solubility is good, the non-specific fluorescence signal is low, and the probe is suitable for industrialized production and clinical application.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of new small-molecule drugs, and particularly relates to a zwitterionic fluorescent probe containing a dimeric targeting ligand, and a preparation method and application thereof. BACKGROUND

[0002] Minimally invasive surgery (MIS) gradually replaces traditional open surgery with the advantages of less trauma, fewer complications, lower risk of infection, shorter hospitalization and recovery time, and so on. The proportion of minimally invasive surgery is increasing, and minimally invasive surgery has become one of the trends in the development of surgery in recent years.

[0003] However, current minimally invasive surgery still mainly relies on the observation of the lesion site by the doctor, experience judgment, and auxiliary case section for resection, which leads to the inability to clearly distinguish the tumor boundary and the micro lesion, so as to completely resect the tumor tissue, and the high risk of recurrence. There is also a possibility of damaging normal tissue and causing loss of body function due to excessive resection caused by unclear boundaries. The use of fluorescent molecular probes to label tumors for specific fluorescence imaging of tumors can provide timely feedback to the surgeon, improve the detection rate of micro metastases during surgery, and optimize real-time decision-making during surgery. The application of fluorescent molecular imaging technology in clinical practice brings new opportunities for precision surgery. With the rapid development of minimally invasive surgery, fluorescent navigation surgery has become the mainstream, and the market for fluorescent endoscopic diagnostic equipment has expanded rapidly. However, the development of targeted fluorescent contrast agents has become the main bottleneck restricting the development of fluorescent endoscopy.

[0004] The commonly used tumor tracer near-infrared dye in clinical practice is indocyanine green (ICG), which is used to trace liver cancer resection due to the difference in metabolic rate between liver cancer tissue and normal liver tissue. However, ICG is difficult to use for fluorescence tracing of most tumors other than liver cancer. Therefore, the development of high specificity and high sensitivity fluorescent molecular probes has important clinical significance.

[0005] Due to the huge market prospect of fluorescent contrast agents, foreign medical device giants have begun to layout. In November 2021, the world's first fluorescent molecular probe Cytalux was approved by FDA for listing in the United States, which is currently the only tumor-targeting fluorescent contrast agent approved 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, which is difficult for latecomers to break through. Taking the fluorescent dye necessary for the synthesis of fluorescent contrast agents as an example, the American LI-COR company developed the fluorescent dye IRDye800CW and provided it to the world's fluorescent contrast agent companies as the sole supplier. 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 effect, its synthesis is difficult and the cost is high.

[0006] However, the existing probes all have various problems. Taking IRDye800CW as an example, due to its completely asymmetric structure, the structures connected to the sulfonic acid indole on both sides are completely different, so two different intermediates need to be synthesized, the synthesis route will be longer, and the yield of the asymmetric dye in synthesis will be much lower than that of the symmetric dye. In addition, the hydrophilic structure in the structure of IRDye800CW is a negatively charged sulfonic acid group, so the overall charge of the probe is negative, and the dye with a negative structure will cause more non-specific tissue signals. SUMMARY

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

[0008] To achieve the foregoing purposes of the application, the technical solutions adopted by the present application include:

[0009] The first aspect of the present application provides a zwitterionic fluorescent probe containing a dimeric targeting ligand, and the chemical structural formula of the zwitterionic fluorescent probe containing a dimeric targeting ligand is shown as formula (1):

[0010] Formula (1);

[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, a polypeptide, an aptamer, or an antibody with tumor targeting function.

[0012] Preferably, R is selected from one of N-methyl lysine, 2,5-diaminopentanoic acid, 2,4-diaminobutanoic acid, 2,3-diaminopropionic acid, 2-amino-4-guanidinobutanoic acid, 2-amino-3-guanidinopropionic acid.

[0013] Preferably, Ligand is selected from one of folate targeting folate receptor-alpha (FRa), RGD peptide targeting integrin, glutamic acid urea and derivatives targeting PSMA, rk polypeptide targeting Her2, nucleic acid aptamer NOX-A12 targeting chemokine CXCL12, trastuzumab, bevacizumab, PD-L1 mAb.

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

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

[0016] Formula (3).

[0017] The second aspect of the present application is to provide a preparation method of the zwitterionic fluorescent probe containing dimeric targeting ligand according to the first aspect of the present application, which comprises the following steps:

[0018] Step 1, dissolve p-hydrazinobenzenesulfonic acid, then add 3-methyl-2-butanone, and then perform a reflux reaction under heating. After the reaction is completed, cool, filter, wash, and dry to obtain indole sulfonic acid. Dissolve the indole sulfonic acid, and then drop into a propanol solution. After post-treatment, 3H-indole-2,3,3-trimethyl-5-sulfonate is obtained.

[0019] Step 2, dissolve 3H-indole-2,3,3-trimethyl-5-sulfonate, then add 6-bromohexanoic acid, and then perform a reflux reaction under heating. After the reaction is completed, cool, wash, filter, and evaporate to dryness under reduced pressure to obtain compound E.

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

[0021] Step 4, under N2 protection, dissolve compound G and DSC in CH3CN, then add pyridine, triethylamine, or DIPEA, and then perform a stirring reaction. Then add compound J, continue to perform a stirring reaction, and then perform concentration under reduced pressure, washing, and purification to obtain compound K, i.e., the fluorescent probe.

[0022] In step 1,

[0023] Preferably, hydrazinobenzenesulfonic acid is dissolved in acetic acid solution, then 3-methyl-2-butanone is added, the molar ratio of hydrazinobenzenesulfonic acid and 3-methyl-2-butanone is 50-55:160, then heated to 110-130°C to reflux for 3-5 hours, cooled to 20-30°C, filtered with filter paper, the obtained solid crude product is washed with ethyl acetate, and indole sulfonic acid is obtained after the solvent is evaporated; and / or,

[0024] Preferably, indole sulfonic acid is dissolved in methanol solution, then added dropwise into a propyl alcohol solution containing potassium hydroxide, the mass ratio of potassium hydroxide and indole sulfonic acid in the propyl alcohol solution is (1-1.5):5.1, then stirred at room temperature for 20-25 hours, filtered with filter paper, and the residue is evaporated 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, then 6-bromohexanoic acid is added and heated to 100-120°C to reflux 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, cooled to room temperature, the upper solution is discarded, isopropyl alcohol is added to the residue for washing, then filtered with filter paper, and compound E is obtained by evaporating under reduced pressure; and / or,

[0027] In step 3,

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

[0029] In step 4,

[0030] Preferably, compound G and DSC (di(N-succinimidyl) carbonate) are dissolved in CH3CN under N2 protection, the molar ratio is 1:(1.2-1.7), then pyridine, triethylamine or DIPEA (N,N-diisopropyl ethylamine) is added, stirred at room temperature for 5-8 hours, then compound J is added, and stirred at room temperature for 6-10 hours, then concentrated under reduced pressure, washed with dichloromethane and ethyl acetate in sequence, and finally compound K, i.e. the fluorescent probe, is obtained by column chromatography purification; 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 application provides a use of the zwitterionic fluorescent probe containing a dimeric targeting ligand according to the first aspect of the present application, which can be used as a tumor fluorescence imaging contrast agent, a photoacoustic imaging contrast agent, a photothermal therapy agent, a photodynamic therapy agent or an sonodynamic therapy agent.

[0033] The present application has the following beneficial effects:

[0034] (1) The luminescent group of the fluorescent probe according to the present application is a heptamethine cyanine dye structure with sulfonic acid indole 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 according to the present application can be used for tumor-targeted near-infrared fluorescence imaging, and contains two identical tumor targeting ligands in its structure, which greatly improves the affinity of the single targeting ligand to the receptor, and has better targeting function and higher targeting efficiency.

[0036] The fluorescent probe is made to have a whole of zero charge on the surface of the probe by introducing a positively charged basic amino acid, and this zwitterionic design makes the probe have a lower tissue background signal.

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

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

[0039] Figure 1 An absorption spectrum diagram of the zwitterionic fluorescent probe prepared in Example 4 of the present application in water is shown;

[0040] Figure 2 A fluorescence imaging photo of a mouse at different time points after injection of the fluorescent probe prepared in Example 4 is shown. DETAILED DESCRIPTION

[0041] The present application will be described in detail below, and the features and advantages of the present application will become clearer and more explicit with these descriptions.

[0042] The first aspect of the present application provides a zwitterionic fluorescent probe containing a dimeric targeting ligand, the chemical structure of which is shown as formula (1):

[0043] Formula (1).

[0044] In formula (1), x and y are integers greater than or equal to 1; Z is a halide 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, a polypeptide, an aptamer or an antibody with tumor targeting function.

[0045] Preferably, R is a non-natural basic amino acid, and R is selected from one of N-methyl lysine, 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, glutamic acid 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 and y = 4. The length of the alkane chain on both sides in the present application can be any length, and the preferred length is 5 carbons. The length of the polyethylene glycol chain between the tumor targeting ligand and the basic amino acid in the present application can be any length, and the 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, but the longer the degree of polymerization, the higher the synthesis cost. The preferred length is 4, and the connection mode of the polyethylene glycol chain and the ligand can be through amide reaction, thiol-maleimide reaction, esterification reaction, click chemistry reaction, etc.

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

[0049] Formula (2).

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

[0051] Formula (3).

[0052] The fluorescent probe can be used for fluorescence imaging, and can also be used for photoacoustic imaging. In addition, the fluorescent probe can generate heat under excitation of near-infrared light in a specific wave band, and thus can be used for photothermal treatment of tumors. Meanwhile, the probe can generate singlet oxygen under irradiation of laser or ultrasound in a specific wave band, and thus can be used for photodynamic and sonodynamic treatment of tumors.

[0053] The second aspect of the present application provides a preparation method of the zwitterionic fluorescent probe containing a dimeric targeting ligand according to the first aspect of the present application, and the preparation method comprises the following steps:

[0054] Step 1: dissolve p-hydrazinobenzenesulfonic acid, then add 3-methyl-2-butanone, and then perform a reflux reaction under heating; after the reaction is completed, perform cooling, filtration, washing, and drying to obtain indole sulfonic acid; then dissolve the indole sulfonic acid, and drop it into a propanol solution; after post-treatment, 3H-indole-2,3,3-trimethyl-5-sulfonate is obtained;

[0055] Step 2: dissolve 3H-indole-2,3,3-trimethyl-5-sulfonate, then add 6-bromohexanoic acid, and then perform a reflux reaction under heating; after the reaction is completed, perform cooling, washing, filtration, and drying under reduced pressure to obtain compound E;

[0056] Step 3: under N2 protection, dissolve compound E (1-[5-carboxyhexyl]-2,3,3-trimethyl-3H-indole-5-sulfonic acid inner salt), compound F (N-[[1E,3E,5E]-5-[phenylimino]pent-1,3-dien-1-yl]aniline hydrochloride), and potassium acetate in a mixed solvent of acetic acid-acetic anhydride, then perform a reflux reaction under heating, and then perform precipitation and purification to obtain compound G (E)-1-(5-carboxypentyl)-2-((2E,4E,6E)-7-(1-(5-carboxypentyl)-3,3-dimethyl-5-sulfonato-3H-indol-1-ium-2-yl)hepta-2,4,6-trien-1-ylidene)-3,3-dimethylindolin-5-sulfonic acid inner salt;

[0057] Step 4: under N2 protection, dissolve compound G and DSC in CH3CN, then add pyridine, triethylamine, or DIPEA, and perform stirring reaction; then add compound J, continue to perform stirring reaction, then perform concentration under reduced pressure, washing, and purification to obtain compound K, i.e., the fluorescent probe.

[0058] The above steps are specifically described as follows.

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

[0060] Preferably, p-hydrazinobenzenesulfonic acid is dissolved in acetic acid solution, then 3-methyl-2-butanone is added, the molar ratio of p-hydrazinobenzenesulfonic acid and 3-methyl-2-butanone is 53:160, then heated to 120°C to reflux for 4 hours, cooled to 25°C, filtered with filter paper, the obtained solid crude product is washed with ethyl acetate, and the solvent is evaporated to obtain indole sulfonic acid.

[0061] In step 3, indole sulfonic acid is dissolved in methanol solution, then added dropwise to a propyl alcohol solution containing potassium hydroxide, the mass ratio of potassium hydroxide and indole sulfonic acid in the propyl alcohol solution is (1-1.5):5.1, then stirred at room temperature for 20-25 hours, filtered with filter paper, and the residue is evaporated under reduced pressure to obtain 3H-indole-2,3,3-trimethyl-5-sulfonate.

[0062] Preferably, indole sulfonic acid is dissolved in methanol solution, then added dropwise to a propyl alcohol solution containing potassium hydroxide, the mass ratio of potassium hydroxide and indole sulfonic acid in the propyl alcohol solution is 1.4:5.1, then stirred at room temperature for 24 hours, filtered with filter paper, and the residue is evaporated 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, then 6-bromohexanoic acid is added and heated to 100-120°C to reflux 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, cooled to room temperature, the upper solution is discarded, isopropyl alcohol is added to the residue for washing, 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, then 6-bromohexanoic acid is added and heated to 110°C to reflux 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, cooled to room temperature, the upper solution is discarded, isopropyl alcohol is added to the residue for washing, 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 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℃ to reflux for 2-5h, followed by adding isopropyl alcohol to precipitate the 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 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℃ to reflux for 3-4h, followed by adding isopropyl alcohol to precipitate the crude product, and finally purified by column chromatography to obtain compound G.

[0067] In step 4, under N2 protection, compound G and DSC (di(N-succinimidyl) carbonate) are dissolved in CH3CN in a molar ratio of 1:(1.2-1.7), followed by adding pyridine, triethylamine or DIPEA (N,N-diisopropyl ethylamine), stirring at room temperature for 5-8h, followed by adding compound J, continuing to stir at room temperature for 6-10h, then concentrated under reduced pressure, washed with dichloromethane and ethyl acetate in sequence, and finally purified by column chromatography to obtain compound K, i.e. the fluorescent probe.

[0068] Preferably, under N2 protection, compound G and DSC (di(N-succinimidyl) carbonate) are dissolved in CH3CN in a molar ratio of 1:1.5, followed by adding pyridine, triethylamine or DIPEA (N,N-diisopropyl ethylamine), stirring at room temperature for 6h, followed by adding compound J, continuing to stir at room temperature for 8h, then concentrated under reduced pressure, washed with dichloromethane and ethyl acetate in sequence, and finally purified by column chromatography to obtain compound K, i.e. 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 application provides an application of the zwitterionic fluorescent probe containing a dimeric targeting ligand according to the first aspect of the present application, which 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 according to the present application can be used in tumor diagnosis and treatment.

[0072] The fluorescent probe can be used as a tumor fluorescent imaging or photoacoustic imaging contrast agent in tumor diagnosis. It can be used as a photothermal therapeutic agent, a photodynamic therapeutic agent or an acoustic power therapeutic agent in tumor treatment.

[0073] Embodiments

[0074] The present application is further described by the following specific examples, which are only intended to illustrate the present application and not used to limit the scope of the present application. The raw materials used in the embodiments of the present application are all commercially available.

[0075] Example 1

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

[0077]

[0078] Dissolve p-hydrazinylbenzenesulfonic 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 reflux for 4 hours, cool to 25°C, then filter with filter paper. Wash the obtained solid crude product with ethyl acetate, evaporate the solvent to obtain pink powder indole sulfonic acid (10 grams, 79%). Dissolve the obtained indole sulfonic acid (5.1 g) in methanol (35 mL) solution, then add dropwise to a solution containing potassium hydroxide (1.4 g) in propanol (35 mL), stir at room temperature for 24 hours, filter with filter paper, evaporate the residue under reduced pressure to obtain compound C (3H-indole-2,3,3-trimethyl-5-sulfonate).

[0079] NMR detection of compound C gives 1 H NMR (500 MHz, Chloroform-d) δ 7.94 (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-sulfonate) is as shown in the following formula:

[0082]

[0083] Compound C (11 g, 39.7 mmol) was dissolved in 1,2-dichlorobenzene (15 ml) under N2protection, then 6-bromohexanoic acid (Compound D) (2.3 g, 12.0 mmol) was added and heated to 110°C to reflux for 12 hours. After the reaction was completed, it was cooled to room temperature, the upper solution was discarded, the residue was washed with isopropyl alcohol (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 nuclear magnetic detection to obtain 1 H NMR (500 MHz, Chloroform-d) δ 8.11-8.04 (m, 2H), 7.60-7.55 (m, 1H), 4.36 (t, J = 6.8 Hz, 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 as shown in the following formula:

[0087]

[0088] 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]pent-1,3-dien-1-yl]aniline hydrochloride) (181 mg, 0.64 mmol) and potassium acetate (251 mg, 2.56 mmol) were dissolved in a mixed solvent of acetic acid-acetic anhydride (acetic acid: acetic anhydride = 7:3) (12 mL) under N2protection, heated to 120°C to reflux for 3-4 hours, then a large amount of isopropyl alcohol was added to precipitate the crude product. Purification was performed by column chromatography (reverse phase chromatography, eluent: methanol: water, the proportion of methanol was gradually increased from about 5% to 20%) to obtain compound G.

[0089] Compound G was subjected to nuclear magnetic detection to obtain 1H NMR (500 MHz, Chloroform-d) δ 8.13 (dd, J = 8.1, 1.8 Hz, 2H), 8.08 (d, J = 1.9 Hz, 2H), 7.77 (d, J = 2.2 Hz, 2H), 7.67 - 7.59 (m, 4H), 7.41 - 7.35 (m, 2H), 7.06 (d, J = 8.0 Hz, 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, 0.9 Hz, 2H), 4.43 (t, J = 7.0 Hz, 4H), 3.78 (t, J = 5.5 Hz, 4H), 2.27 (dt, J = 11.0, 9.0 Hz, 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] Compound G (1 mmol) and 1.5 mmol of DSC (di(N-succinimidyl) carbonate) were dissolved in CH3CN under N2protection, 3 mmol of pyridine was added, and the reaction was stirred at room temperature for 6 hours to obtain the crude product compound I (mixture). 1.5 mmol of compound J was added to the above mixture, and the stirring was continued at room temperature for 8 hours. After concentration under reduced pressure, washing with dichloromethane and ethyl acetate in turn, and purification by column chromatography (reverse phase chromatography, eluent acetonitrile: water, acetonitrile ratio gradually increased from about 5% to 30%), compound K, the fluorescent probe, was obtained.

[0094] Compound K was detected by nuclear magnetic resonance to obtain 1H NMR (500 MHz, 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, 1H), 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, 2H), 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), 1.84 - 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 shown in Figure 1 In PBS, the characteristic absorption of the fluorescent probe is around 766 nm, which is consistent with the absorption characteristics of Cy7 cyanine dye compounds.

[0096] Experimental Example

[0097] The experiment example studies the performance of the fluorescent probe prepared by the embodiment 4 of the present application in the tumor fluorescence imaging of mice. The subcutaneous tumor model of mouse breast cancer is established by injecting 4T1 mouse breast cancer cells into the right hind leg of Balb / c nude mice. After the tumor grows to a suitable size, the fluorescent probe is injected intravenously (2 nmol of the fluorescent probe is injected into each mouse), and then the mice are subjected to fluorescence imaging at different time points after the injection of the fluorescent probe. As shown in Figure 2

[0098] As can be seen from Figure 2 2h~48h after the injection of the probe, strong fluorescence signals are detected at the tumor site, and the fluorescence signals of the tumor tissue are significantly stronger than those of the surrounding tissues, indicating that the fluorescent probe of the present application can effectively specifically develop the tumor tissue.

[0099] The present application is described in detail above in combination with the specific embodiments and exemplary examples, but these descriptions cannot be understood as limitations of the present application. Those skilled in the art understand that the technical solutions and embodiments of the present application can be variously replaced, modified or improved without departing from the spirit and scope of the present application, and these all fall within the scope of the present application. The protection scope of the present application is subject to the appended claims.​

Claims

1. A zwitterionic fluorescent probe containing a dimerizing targeting ligand, characterized in that, The chemical structure of the zwitterionic fluorescent probe containing a dimer targeting ligand is shown in formula (3): Equation (3).

2. A method for preparing the zwitterionic fluorescent probe containing a dimerizing targeting ligand as described in claim 1, characterized in that, The preparation method includes the following steps: Step 1: Dissolve p-hydrazinebenzenesulfonic acid, add 3-methyl-2-butanone, and then heat to reflux. After the reaction is completed, cool, filter, wash and dry to obtain indolesulfonic acid. Dissolve indolesulfonic acid and add it dropwise to propanol solution. After post-treatment, 3H-indole-2,3,3-trimethyl-5-sulfonate is obtained. Step 2: Dissolve 3H-indole-2,3,3-trimethyl-5-sulfonate, add 6-bromohexanoic acid, and then heat to reflux. After the reaction is complete, cool, wash, filter, and evaporate under reduced pressure to obtain compound E. The chemical structure of compound E is shown in formula (4): Equation (4); Step 3: Under N2 protection, compound E, compound F, and potassium acetate are dissolved in a mixed solvent of acetic acid and acetic anhydride, and then heated under reflux. After precipitation and purification, compound G is obtained. Compound F is N-[[1E,3E,5E]-5-[phenylimino]pent-1,3-dien-1-yl]aniline hydrochloride, and compound G is [formula missing]. The chemical formula of compound F is shown in formula (5), and the chemical formula of compound G is shown in formula (6). Equation (5), Equation (6); Step 4: Under N2 protection, compound G and DSC are dissolved in CH3CN, then pyridine, triethylamine or DIPEA are added and the mixture is stirred. Compound J is then added and the mixture is stirred again. The mixture is then concentrated under reduced pressure, washed and purified to obtain compound K, which is the fluorescent probe. The chemical structure of compound J is shown in formula (7). Equation (7).

3. The preparation method according to claim 2, characterized in that, In step 1, Hydrazinesulfonic acid was dissolved in acetic acid solution, and then 3-methyl-2-butanone was added. The molar ratio of hydrazinesulfonic acid to 3-methyl-2-butanone was 50-55:

160. The mixture was then heated to 110-130°C and refluxed for 3-5 hours. After cooling to 20-30°C, the mixture was filtered through filter paper. The resulting crude solid product was washed with ethyl acetate, and the solvent was evaporated to obtain indolesulfonic acid. Indolesulfonic acid was dissolved in a methanol solution and then added dropwise to a propanol solution containing potassium hydroxide. The mass ratio of potassium hydroxide to indolesulfonic acid in the propanol solution was (1-1.5):5.

1. The mixture was then stirred at room temperature for 20-25 hours, filtered through filter paper, and the residue was evaporated under reduced pressure to obtain 3H-indole-2,3,3-trimethyl-5-sulfonate.

4. The preparation method according to claim 2, characterized in that, In step 2, 3H-indole-2,3,3-trimethyl-5-sulfonate was dissolved in 1,2-dichlorobenzene, and then 6-bromohexanoic acid was added. The mixture was heated to 100-120°C and refluxed for 10-15 hours. The molar ratio of 3H-indole-2,3,3-trimethyl-5-sulfonate to 6-bromohexanoic acid was (35-45):

12. After the reaction was completed, the mixture was cooled to room temperature, the supernatant was discarded, and the residue was washed with isopropanol. The residue was then filtered through filter paper and evaporated under reduced pressure to obtain compound E. In step 3, under N2 protection, compounds E, F, and potassium acetate were dissolved in a mixed solvent of acetic acid and acetic anhydride in a molar ratio of (1-1.5):(0.6-0.7):(2-3), wherein the volume ratio of acetic acid to acetic anhydride was (6-8):

3. The mixture was then heated to 110-130°C and refluxed for 2-5 hours. Isopropanol was then added to precipitate the crude product, which was finally purified by column chromatography to obtain compound G.

5. The preparation method according to claim 2, characterized in that, In step 4, Under N2 protection, compound G and DSC (di(N-succinimide) carbonate) were dissolved in CH3CN at a molar ratio of 1:(1.2-1.7). Then, pyridine, triethylamine, or DIPEA (N,N-diisopropylethylamine) were added, and the mixture was stirred at room temperature for 5-8 hours. Subsequently, compound J was added, and the mixture was stirred at room temperature for another 6-10 hours. The mixture was then concentrated under reduced pressure, washed successively with dichloromethane and ethyl acetate, and purified by column chromatography to obtain compound K, which is the fluorescent probe. The molar ratio of pyridine, triethylamine or DIPEA to compound G is (2-4):1.

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