Zwitterionic fluorescent probe based on click chemical reaction as well as preparation method and application of zwitterionic fluorescent probe

Abstract

The invention belongs to the technical field of novel micromolecular drugs, and particularly relates to a zwitterionic fluorescent probe based on click chemistry reaction as well as a preparation method and application of the zwitterionic fluorescent probe. The chemical structural formula of the zwitterionic fluorescent probe based on the click chemical reaction is as shown in formula (1): # imgabs0 #, the fluorescent probe is symmetrical in structure, the net charge on the surface of the probe is zeroed due to the introduction of tetra-substituted nitrogen cations in the fluorescent probe structure, and the zwitterionic design enables the probe to have lower tissue background signals and good water solubility, so that the probe can be applied to the field of fluorescence detection. The non-specific fluorescence signal is low, and the probe is suitable for tumor diagnosis and treatment. The synthesis method of the probe is simple and efficient, and is very suitable for industrial production.

Description

Technical Field

[0001] The present invention belongs to the technical field of novel small molecule drugs, and specifically relates to a zwitterionized fluorescent probe based on click chemistry reaction, and a preparation method and application thereof. Background Art

[0002] Cancer is the second leading cause of death worldwide and poses a serious threat to human health. Surgical resection is still the main treatment for the vast majority of tumors. Surgical intervention may be required to diagnose and confirm surgical resection of cancerous tissue, locate metastatic lesions, and help plan further treatment. One of the major challenges facing surgeons is to achieve the ideal "negative margin" surgical outcome, that is, the tissue surrounding the resection site is free of cancer cells and the entire tumor mass has been removed.

[0003] Preoperative imaging techniques such as CT, MRI, and PET do not have sufficient sensitivity to detect all tiny lesions. Most patients often only discover small lesions that were not discovered during preoperative diagnosis during laparoscopic surgery. Because malignant tumors are usually highly invasive, it is difficult for doctors to distinguish them by naked eye observation and palpation during surgery. Although intraoperative frozen section analysis can be used to identify tumor boundaries to metastatic lesions, it is limited by sampling errors. Therefore, the development of new real-time tumor imaging technology, improving intraoperative tumor detection rates, and thus achieving complete surgical resection of tumors will hopefully improve the survival rate of cancer patients.

[0004] Intraoperative near-infrared fluorescence imaging is a new technology that has emerged in the past decade. It mainly uses fluorescent molecular probes to achieve targeted labeling of tumors, and then uses near-infrared fluorescence imaging equipment to image the tumor in real time during surgery. Studies have found that the use of fluorescent molecular imaging to guide surgery can provide instant feedback to the surgeon, improve the detection rate of tiny tumor metastases during surgery, and improve the resolution of benign and malignant tissues such as blood vessels and nerves that need to be preserved. Real-time optimization of decisions during surgery not only helps to completely eliminate tumor metastases and reduce the possibility of postoperative recurrence, but also may prevent overtreatment by avoiding the removal of non-tumor tissue. The development of fluorescence imaging technology has promoted the serial clinical application of new tumor molecular imaging technologies. Therefore, the use of fluorescent molecular imaging to guide precision surgery has formed an international expert consensus.

[0005] However, there are various problems with the existing probes. For example, the widely used tumor tracing probe indocyanine green (ICG) in clinical practice cannot be modified to connect targeting groups and can only be used to trace the resection of liver cancer surgery. 5-Aminolevulinic acid (5-ALA) is a small lipophilic zwitterion that is converted into protoporphyrin through the heme synthesis pathway. Its fluorescence characteristics are an absorption peak of 405nm and a red visible light emission peak of 635nm. It is approved for fluorescent tracing of glioblastoma. However, 5-ALA has limited penetration in lesions thicker than 2mm, which affects the imaging effect. Hexylaminolevulinic acid (HAL, ) is a lipophilic ester of the heme precursor 5-aminolevulinic acid. It is instilled into the bladder before surgery and can be metabolized by tumor cells into protoporphyrin to visualize the lesions. However, clinical trial results show that although its cancer detection rate is better than white light cystoscopy, its false positive rate is very high (as high as 43% to 82%).

[0006] LI-COR, an American company, has developed the fluorescent dye IRDye800CW, which is a heptamethine cyanine dye with a hexagonal rigid ring in the middle connected by a phenoxy bridge, and has strong fluorescence intensity and stability. In addition, it contains a carboxyl group, which can be coupled to various tumor-targeting ligands to achieve intraoperative tumor tracing. However, the asymmetric structure of IRDye800CW makes its synthesis difficult and costly. In 2021, the FDA approved the imaging probe Cytalux (formerly known as OTL38) to assist in the intraoperative identification of ovarian cancer. The folate group in the Cytalux structure develops tumor lesions by binding to folate receptors highly expressed in tumor tissues, but its clinical false positive rate is as high as 20.2%. The fluorescent group in Cytalux contains four negatively charged sulfonic acid groups, which can increase the water solubility of the dye, but it also leads to higher nonspecific adsorption, which to a certain extent leads to higher clinical false positives. 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 based on click chemistry reaction and its preparation method and application, so as to overcome the shortcomings of the prior art.

[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 based on click chemistry reaction, wherein the chemical structure of the zwitterionized fluorescent probe based on click chemistry reaction is shown in formula (1):

[0010]

[0011] In formula (1), x, y, z are integers greater than or equal to 1; R is O, S, N or C; Ligand is a small molecule, polypeptide, aptamer or antibody with tumor targeting function.

[0012] 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.

[0013] Preferably, in formula (1), x=3, y=2, z=2, and R is O.

[0014] More preferably, the chemical structure of the zwitterionized click chemistry-based fluorescent probe is as shown in formula (3):

[0015]

[0016] The second aspect of the present invention is to provide a method for preparing the zwitterionized fluorescent probe based on click chemistry reaction according to the first aspect of the present invention, the preparation method comprising the following steps:

[0017] Step 1, mixing 4-hydrazinobenzenesulfonic acid and 3-methyl-2-butanone, heating, and performing reflux reaction, then cooling and filtering, washing and drying the obtained crude product to obtain indolesulfonic acid, dissolving the indolesulfonic acid in methanol, and then dropping it into a propanol solution, stirring, filtering, and evaporating under reduced pressure to obtain 3H-indole-2,3,3-trimethyl-5-sulfonate;

[0018] Step 2, dissolving 3H-indole-2,3,3-trimethyl-5-sulfonate in 1,2-dichlorobenzene, adding compound D, and then heating to reflux reaction, after cooling, discarding the upper solution, washing the obtained residue, filtering, and evaporating under reduced pressure to obtain compound E;

[0019] Step 3, dissolving compound E, compound F (N-[(3-(anilinomethylene)-2-chloro-1-cyclohexen-1-yl)methylene]aniline hydrochloride) and potassium acetate in a mixed solvent, heating to reflux reaction, then adding isopropanol to precipitate to obtain a crude product, and chromatographing and purifying the crude product to obtain compound G;

[0020] Step 4, after dissolving compound H and compound I, heating them for reflux reaction, and then recrystallizing to obtain product J;

[0021] Step 5, dissolving compound J and potassium hydroxide, stirring, then adding compound G, heating to react, and purifying the obtained crude product to obtain compound K;

[0022] Step 6: Dissolve compound K, cuprous iodide and compound L, and stir to obtain a crude product, which is then purified to obtain compound M.

[0023] In step 1,

[0024] Preferably, p-hydrazinobenzenesulfonic acid is dissolved in acetic acid, and then 3-methyl-2-butanone is added, heated to 110-130° C. for reflux reaction for 3-5 hours, then cooled to 20-30° C., and filtered with filter paper to obtain a crude product; and / or,

[0025] Preferably, the molar ratio of hydrazinobenzenesulfonic acid to 3-methyl-2-butanone is 53:(150-170); and / or,

[0026] Preferably, the crude product is washed with ethyl acetate, the solvent is evaporated to obtain indole sulfonic acid, the indole sulfonic acid is dissolved in methanol, and then added dropwise to a propanol solution containing potassium hydroxide, followed by stirring at room temperature for 20 to 30 hours, filtering with filter paper, and evaporating the residue under reduced pressure to obtain 3H-indole-2,3,3-trimethyl-5-sulfonate; and / or,

[0027] Preferably, the mass ratio of indolesulfonic acid to potassium hydroxide in the propanol solution is 5.1:(1.2-1.5).

[0028] In step 2,

[0029] Preferably, under N2 protection, 3H-indole-2,3,3-trimethyl-5-sulfonate is dissolved in 1,2-dichlorobenzene, then compound D is added, the temperature is raised to 100-120°C, and the mixture is refluxed for reaction for 10-13 hours, then cooled to room temperature, the upper layer solution is discarded, isopropanol is added to the obtained residue for washing, the residue is filtered with filter paper, and evaporated to dryness under reduced pressure to obtain compound E; and / or,

[0030] Preferably, the molar ratio of the 3H-indole-2,3,3-trimethyl-5-sulfonate to compound D is (35-45):12.

[0031] In step 3,

[0032] Preferably, under N2 protection, compound E, compound F (N-[(3-(anilinomethylene)-2-chloro-1-cyclohexene-1-yl)methylene]aniline hydrochloride) and potassium acetate are dissolved in an acetic acid-acetic anhydride mixed solvent, the volume ratio of acetic acid:acetic anhydride is (5-8):3, then heated to 100-140°C for reflux reaction for 2-5h, then isopropanol is added for precipitation to obtain a crude product, and finally purified by column chromatography to obtain compound G; and / or,

[0033] Preferably, the molar ratio of compound E, compound F (N-[(3-(anilinomethylene)-2-chloro-1-cyclohexene-1-yl)methylene]aniline hydrochloride) and potassium acetate is (1-1.5):(0.5-0.7):(2-3); and / or,

[0034] In step 4,

[0035] Preferably, compound H (p-dimethylaminoethyl phenol) and compound I (5-iodo-1-pentyne) are dissolved in toluene in a molar ratio of (1-3):3, heated to 110° C. and refluxed for 10-14 hours. After the reaction, product J is obtained by recrystallization using methanol and methyl tert-butyl ether respectively.

[0036] In step 5,

[0037] Preferably, under N2 protection, compound J and potassium hydroxide are dissolved in DMSO (dimethyl sulfoxide) at a molar ratio of 1: (1-3), stirred at room temperature for 20-40 min, then compound G is added, heated to 50-70° C. for reaction for 45-75 min, and the obtained crude product is purified by column chromatography to obtain compound K; and / or,

[0038] Preferably, the molar ratio of compound G to compound J is (0.1-0.3):1; and / or,

[0039] In step 6,

[0040] Preferably, under N2 protection, compound K, cuprous iodide and compound L are dissolved in PBS (phosphate buffered saline) in a molar ratio of (0.4-0.6):(1.5-2.5):0.5, stirred at room temperature for 30-60 min to obtain a crude product, and the crude product is purified by column chromatography to obtain compound M.

[0041] The third aspect of the present invention is to provide an application of the zwitterionized fluorescent probe based on click chemistry reaction described in the first aspect of the present invention in fluorescence imaging and photogenerated imaging.

[0042] The beneficial effects of the present invention are:

[0043] (1) The luminescent group of the zwitterionized click-chemistry-based fluorescent probe of the present invention is a heptamethine cyanine dye structure with indole sulfonate groups at both ends connected by a phenoxy bridge, two polyethylene glycol chains are respectively connected to the indole groups, and the tumor targeting ligand is connected to the luminescent group through an efficient click chemistry reaction. The introduction of tetrasubstituted nitrogen cations in the fluorescent probe structure of the present invention returns the net charge on the probe surface to zero. This zwitterionized design makes the fluorescent probe structure of the present invention symmetrical, has a lower tissue background signal, good water solubility, and low nonspecific fluorescence signal, and has good application prospects in tumor diagnosis and treatment.

[0044] (2) The tumor targeting ligand (Ligand) in the fluorescent probe of the present invention is connected with the luminescent group through a click chemistry reaction. The reaction conditions are mild and the reaction can be completed in PBS buffer with almost no side reactions, thereby simplifying the purification process, with a fast reaction rate and high connection efficiency. The synthesis method of the fluorescent probe is simple and efficient, and is suitable for industrial production and clinical application. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 The absorption spectrum of the zwitterionic fluorescent probe prepared in Example 6 of the present invention in PBS buffer is shown;

[0046] Figure 2 The fluorescence imaging photos of mice at different time points after injection of the zwitterionized fluorescent probe prepared in Example 6 are shown. DETAILED DESCRIPTION

[0047] 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.

[0048] The first aspect of the present invention is to provide a zwitterionized fluorescent probe based on click chemistry reaction, wherein the chemical structure of the zwitterionized fluorescent probe based on click chemistry reaction is shown in formula (1):

[0049]

[0050] In formula (1), x, y, z are integers greater than or equal to 1; R is O, S, N or C; and the ligand is a small molecule, polypeptide, aptamer or antibody with tumor targeting function.

[0051] 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.

[0052] The fluorescent probe of the present invention has a near-infrared absorption and emission heptamethine cyanine dye luminescent group, two polyethylene glycol short chains, and contains a tetrasubstituted nitrogen cation. The introduction of the nitrogen cation returns the net charge on the probe surface to zero, which is beneficial to reducing the nonspecific enrichment of the probe in the body, thereby reducing the tissue background signal.

[0053] Preferably, in formula (1), x=3, y=2, z=2, and R is O.

[0054] In the fluorescent probe of the present invention, two polyethylene glycols are connected to the indole group. The short chain can improve the water solubility of the dye. 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 present invention selects a polyethylene glycol chain with a degree of polymerization of 3, at which time the probe has good water solubility and is easier to synthesize.

[0055] Preferably, the chemical structure of the zwitterionized click chemistry-based fluorescent probe is as shown in formula (2):

[0056]

[0057] More preferably, the chemical structure of the zwitterionized click chemistry-based fluorescent probe is as shown in formula (3):

[0058]

[0059] In addition to being used for fluorescence imaging, the fluorescent probe of 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, which can be used for photodynamic and sonodynamic treatment of tumors respectively.

[0060] The second aspect of the present invention is to provide a method for preparing the zwitterionized fluorescent probe based on click chemistry reaction according to the first aspect of the present invention, the preparation method comprising the following steps:

[0061] Step 1, mixing 4-hydrazinobenzenesulfonic acid and 3-methyl-2-butanone, heating, and performing reflux reaction, then cooling and filtering, washing and drying the obtained crude product to obtain indolesulfonic acid, dissolving the indolesulfonic acid in methanol, and then dropping it into a propanol solution, stirring, filtering, and evaporating under reduced pressure to obtain 3H-indole-2,3,3-trimethyl-5-sulfonate;

[0062] Step 2, dissolving 3H-indole-2,3,3-trimethyl-5-sulfonate in 1,2-dichlorobenzene, adding compound D, and then heating to reflux reaction, after cooling, discarding the upper solution, washing the obtained residue, filtering, and evaporating under reduced pressure to obtain compound E;

[0063] Step 3, dissolving compound E, compound F (N-[(3-(anilinomethylene)-2-chloro-1-cyclohexen-1-yl)methylene]aniline hydrochloride) and potassium acetate in a mixed solvent, heating to reflux reaction, then adding isopropanol to precipitate to obtain a crude product, and chromatographing and purifying the crude product to obtain compound G;

[0064] Step 4, after dissolving compound H and compound I, heating them for reflux reaction, and then recrystallizing to obtain product J;

[0065] Step 5, dissolving compound J and potassium hydroxide, stirring, then adding compound G, heating to react, and purifying the obtained crude product to obtain compound K;

[0066] Step 6: Dissolve compound K, cuprous iodide and compound L, stir to obtain a crude product, and purify to obtain compound M, i.e., a fluorescent probe.

[0067] The above steps are described in detail below.

[0068] In step 1, p-hydrazinobenzenesulfonic acid is dissolved in acetic acid, and then 3-methyl-2-butanone is added, heated to 110-130° C. and refluxed for 3-5 hours, then cooled to 20-30° C., and filtered with filter paper to obtain a crude product.

[0069] Preferably, p-hydrazinobenzenesulfonic acid is dissolved in acetic acid, and then 3-methyl-2-butanone is added, heated to 120° C. and refluxed for 4 h, then cooled to 25° C., and filtered with filter paper to obtain a crude product.

[0070] The molar ratio of hydrazinobenzenesulfonic acid to 3-methyl-2-butanone is 53:(150-170), preferably, the molar ratio of hydrazinobenzenesulfonic acid to 3-methyl-2-butanone is 53:160.

[0071] The crude product was washed with ethyl acetate, and the solvent was evaporated to obtain indole sulfonic acid. The indole sulfonic acid was dissolved in methanol and then added dropwise to a propanol solution containing potassium hydroxide. The mixture was then stirred at room temperature for 20 to 30 hours, filtered with filter paper, and the residue was evaporated under reduced pressure to obtain 3H-indole-2,3,3-trimethyl-5-sulfonate.

[0072] Preferably, the crude product is washed with ethyl acetate, the solvent is evaporated to obtain indole sulfonic acid, the indole sulfonic acid is dissolved in methanol, and then added dropwise to a propanol solution containing potassium hydroxide, 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.

[0073] The mass ratio of the indolesulfonic acid to the potassium hydroxide in the propanol solution is 5.1:(1.2-1.5), preferably, the mass ratio of the indolesulfonic acid to the potassium hydroxide in the propanol solution is 5.1:1.4.

[0074] In step 2, under N2 protection, 3H-indole-2,3,3-trimethyl-5-sulfonate is dissolved in 1,2-dichlorobenzene, and then compound D is added and the temperature is raised to 100-120°C and refluxed for 10-13 hours, followed by cooling to room temperature, discarding the upper solution, adding isopropanol to the obtained residue for washing, filtering with filter paper, and evaporating under reduced pressure to obtain compound E.

[0075] Preferably, under N2 protection, 3H-indole-2,3,3-trimethyl-5-sulfonate is dissolved in 1,2-dichlorobenzene, and then compound D is added, the temperature is raised to 110°C and refluxed for 12 hours, then cooled to room temperature, the upper solution is discarded, isopropanol is added to the obtained residue for washing, filtered with filter paper, and evaporated to dryness under reduced pressure to obtain compound E.

[0076] The molar ratio of the 3H-indole-2,3,3-trimethyl-5-sulfonate to compound D is (35-45):12. Preferably, the molar ratio of the 3H-indole-2,3,3-trimethyl-5-sulfonate to compound D is 40:12.

[0077] D in the present invention is a phenoxy bridge, a phenylsulfide bridge, a phenylnitrogen bridge or is directly connected to a benzene ring. Preferably, it is a phenoxy bridge. When it is a phenoxy bridge, the fluorescence of the dye is the strongest, but the chemical stability is poor, and although the fluorescence of the other three connection modes is weakened, the chemical stability is relatively better. In the present invention, the carbon chain length y on the opposite side of the phenoxy bridge can be any length, and the preferred length is two carbons. z can be any length, and the preferred length is two carbons.

[0078] In step 3, under N2 protection, compound E, compound F (N-[(3-(anilinomethylene)-2-chloro-1-cyclohexen-1-yl)methylene]aniline hydrochloride) and potassium acetate are dissolved in an acetic acid-acetic anhydride mixed solvent, with a volume ratio of acetic acid:acetic anhydride of (5-8):3, and then heated to 100-140°C for reflux reaction for 2-5h, followed by addition of isopropanol to precipitate to obtain a crude product, which is then purified by column chromatography to obtain compound G.

[0079] Preferably, under N2 protection, compound E, compound F (N-[(3-(anilinomethylene)-2-chloro-1-cyclohexene-1-yl)methylene]aniline hydrochloride) and potassium acetate are dissolved in an acetic acid-acetic anhydride mixed solvent, the volume ratio of acetic acid: acetic anhydride = 7:3, and then heated to 120°C for reflux reaction for 3 to 4 hours, followed by addition of isopropanol to precipitate to obtain a crude product, and finally purified by column chromatography to obtain compound G.

[0080] The molar ratio of the compound E, the compound F (N-[(3-(anilinomethylene)-2-chloro-1-cyclohexene-1-yl)methylene]aniline hydrochloride) and potassium acetate is (1-1.5):(0.5-0.7):(2-3).

[0081] Preferably, the molar ratio of compound E, compound F and potassium acetate is 2:1:4.

[0082] In step 4, compound H (p-dimethylaminoethyl phenol) and compound I (5-iodo-1-pentyne) are dissolved in toluene at a molar ratio of (1-3):3, heated to 220° C. and refluxed for reaction for 10-14 hours. After the reaction, product J is obtained by recrystallization using methanol and methyl tert-butyl ether, respectively.

[0083] Preferably, compound H (p-dimethylaminoethyl phenol) and compound I (5-iodo-1-pentyne) are dissolved in toluene at a molar ratio of 2:3, heated to 110° C. and refluxed for 12 hours. After the reaction, product J is obtained by recrystallization using methanol and methyl tert-butyl ether respectively.

[0084] In step 5, under N2 protection, compound J and potassium hydroxide are dissolved in DMSO (dimethyl sulfoxide) at a molar ratio of 1: (1-3), stirred at room temperature for 20-40 min, and then compound G is added, heated to 50-70°C and reacted for 45-75 min, and the crude product is purified by column chromatography to obtain compound K.

[0085] Preferably, under N2 protection, compound J and potassium hydroxide are dissolved in DMSO at a molar ratio of 1:2, stirred at room temperature for 30 min, then compound G is added, heated to 60°C and reacted for 60 min, and the obtained crude product is purified by column chromatography to obtain compound K.

[0086] The molar ratio of the compound G to the compound J is (0.1-0.3):1. Preferably, the molar ratio of the compound G to the compound J is 0.2:1.

[0087] In step 6, under N2 protection, compound K, cuprous iodide and compound L are dissolved in PBS (phosphate buffered saline) at a molar ratio of (0.4-0.6):(1.5-2.5):0.5, and stirred at room temperature for 30-60 min to obtain a crude product, which is purified by column chromatography to obtain compound M.

[0088] Preferably, under N2 protection, compound K, cuprous iodide and compound L are dissolved in PBS (phosphate buffered saline) at a molar ratio of 0.5:1:0.5, stirred at room temperature for 45 min to obtain a crude product, and the crude product is purified by column chromatography to obtain compound M.

[0089] The third aspect of the present invention is to provide an application of the zwitterionized fluorescent probe based on click chemistry reaction described in the first aspect of the present invention in fluorescence imaging and photogenerated imaging.

[0090] Specifically, the zwitterionized click chemistry reaction-based fluorescent probe can be used as a tumor fluorescence imaging or photoacoustic imaging contrast agent in tumor diagnosis.

[0091] The zwitterionized click chemistry-reaction-based fluorescent probe can be used as a photothermal therapy agent, a photodynamic therapy agent or a sonodynamic therapy agent in tumor treatment.

[0092] Example

[0093] 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.

[0094] Example 1

[0095] The preparation process of compound C (3H-indole-2,3,3-trimethyl-5-sulfonate) is shown in the following formula:

[0096]

[0097] 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, then cool to 25°C and filter with filter paper to obtain a solid crude product. Wash the solid crude product with ethyl acetate, evaporate the solvent to obtain pink powder indolesulfonic acid (10 grams, 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).

[0098] Compound C was tested by nuclear magnetic resonance and obtained 1 H NMR (500 MHz, 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).

[0099] Example 2

[0100] The preparation process of compound E is shown in the following formula:

[0101]

[0102] Under N2 protection, compound C (40 mmol) was dissolved in 1,2-dichlorobenzene (15 ml), and then compound D (1-bromo-2-[2-[2-methoxyethoxy]ethoxy]ethane) (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, isopropanol (50 mL) was added to the residue for washing, and then filtered with filter paper, and the solid crude product was evaporated under reduced pressure to obtain pink compound E (1-[2-[2-[2-methoxyethoxy]ethoxy]ethyl]-2,3,3-trimethyl-3H-indole-5-sulfonic acid inner salt).

[0103] Compound E was subjected to NMR detection to obtain 1H NMR(500MHz,Chloroform-d)δ7.98(dd,J=8.1,2.2Hz,1H),7.92(d,J=2.2Hz,1H),7.46(s,1H),4.55(t,J=5.9Hz,2H),3.99(t,J=5 .8Hz,2H),3.69(d,J=1.7Hz,4H),3.63(td,J=4.6,0.8Hz,2H),3.58(td,J=4.6,0.9Hz,2H),3.38(s,3H),3.04(s,3H),1.82(s,5H).

[0104] Example 3

[0105] The preparation process of compound G is shown in the following formula:

[0106]

[0107] Under N2 protection, compound E (1.28 mmol), compound F (0.64 mmol) and potassium acetate 2.56 mmol were dissolved in a mixed solvent (12 mL) of acetic acid-acetic anhydride (volume ratio of acetic acid: acetic anhydride = 7:3), heated to 120°C and refluxed for 3-4 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 was methanol: water, the proportion of methanol was gradually increased from about 5% to 40%).

[0108] Compound G was subjected to NMR detection to obtain 1 H NMR(500MHz,Chloroform-d)δ7.70–7.60(m,2H),6.99–6.93(m,1H),4.69(t,J=5.7Hz,1H),4.03–3.97(m,2H),3.78(t,J=5 .7Hz,1H),3.72–3.60(m,6H),3.58(td,J=4.7,0.9Hz,2H),3.38(s,3H),2.72–2.66(m,2H),1.86(s,3H),1.65–1.57(m,4H).

[0109] Example 4

[0110] The preparation process of compound J is shown in the following formula:

[0111]

[0112] Compound H (p-dimethylaminoethylphenol) (20 mmol) and compound I (5-iodo-1-pentyne) (30 mmol) were dissolved in toluene, heated to 110°C and refluxed for 12 hours. After the reaction, the product J was obtained by recrystallization using methanol and methyl tert-butyl ether (MTBE).

[0113] Compound J was subjected to NMR detection to obtain 1 H NMR(500MHz,Chloroform-d)δ7.08–7.01(m,1H),6.76–6.70(m,1H),3.68–3.61 (m,2H),3.36(s,3H),3.04(tt,J=8.4,0.9Hz,1H),2.87(td,J=7.7,2.5Hz,1H).

[0114] Example 5

[0115] The preparation process of compound K is shown in the following formula:

[0116]

[0117] Under N2 protection, compound J (N-[4-hydroxyphenethyl]-N,N-dimethyl-3-butyn-1-ammonium) (1 mmol) and potassium hydroxide (2 mmol) were dissolved in DMSO (2 mL), stirred at room temperature for 30 min, and then compound G (2-((E)-2-((E)-2-chloro-3-(2-((E)-1-(2-(2-(2-methoxyethoxy)eth oxy)ethyl)-3,3-dimethyl-5-sulfonatoindolin-2-ylidene)ethylidene)cyclohex-1-en-1-yl)vinyl)-1-(2-(2-(2-methoxyethoxy)ethoxy)ethy l)-3,3-dimethyl-3H-indol-1-ium-5-sulfonate) (0.2 mmol) was added, and the mixture was heated to 60°C for 60 min to obtain a crude product. Compound K was purified by column chromatography (reverse phase chromatography, the eluent was acetonitrile:water, the proportion of acetonitrile was gradually increased from about 5% to 80%).

[0118] Compound K was subjected to NMR detection to obtain 1H NMR(500MHz,Chloroform-d)δ7.80–7.72(m,3H),7.68–7.60(m,3H),7.14–7.08(m,3H),7.0 4–6.94(m,5H),4.69(t,J=5.7Hz,3H),4.03–3.97(m,6H),3.78(t,J=5.7Hz,3H),3.72–3.66( m,9H),3.66–3.57(m,20H),3.57(s,1H),3.37(d,J=8.1Hz,18H),3.04(tt,J=8.4,0.9Hz,3H ), 2.87(td,J=7.6,2.5Hz,3H),2.77–2.65(m,6H),1.86(s,9H),1.63(tt,J=7.4,6.3Hz,3H).

[0119] Example 6

[0120] The preparation process of compound M is shown in the following formula:

[0121]

[0122] Under N2 protection, compound K (0.5 mmol), cuprous iodide (2 mmol) and compound L (0.5 mmol) were dissolved in PBS (2 mL) and stirred at room temperature for 45 min to obtain a crude product M. Compound M (i.e., fluorescent probe) was purified by column chromatography (reverse phase chromatography, eluent acetonitrile: water, the proportion of acetonitrile gradually increased from about 5% to 60%).

[0123] Compound M was subjected to NMR detection to obtain 1H NMR(500MHz,Chloroform-d)δ8.95(d,J=8.6Hz,1H),8.08(d,J=2.1Hz,1H),8.03(dd,J=8.1,2 .2Hz,1H),7.94(d,J=8.4Hz,1H),7.82–7.72(m,3H),7.68–7.58(m,4H),7.38(dt,J=15.2,0.9H z,1H),7.31–7.22(m,4H),7.16(t,J=6.3Hz,1H),7.14–7.06(m,3H),7.04–6.98(m,2H),6.97(d ,J=8.0Hz,1H),6.85(d,J=8.7Hz,2H),6.41(dt,J=8.2,1.0Hz,1H),5.62(s,2H),4.81(dt,J=8. 6,7.4Hz,1H),4.69(t,J=5.7Hz,2H),4.48(dt,J=8.6,6.2Hz,1H),4.35(dt,J=8.6,4.7Hz,1H) ,4.23(dt,J=8.8,5.8Hz,1H),4.19–4.05(m,2H),4.03–3.97(m,4H),3.94–3.81(m,4H),3.78(t ,J=5.7Hz,2H),3.72–3.55(m,16H),3.38(s,5H),3.28(s,5H),3.23–3.00(m,6H),2.86–2.72(m ,4H),2.72–2.65(m,2H),2.56(dd,J=17.6,7.3Hz,1H),2.11–1.80(m,9H),1.76–1.45(m,11H).

[0124] The absorption spectrum of compound M in PBS buffer is as follows Figure 1 As shown, in PBS, the characteristic absorption of compound M is around 760 nm, which is consistent with the absorption characteristics of Cy7 cyanine dye compounds.

[0125] Experimental example

[0126] This experimental example studies the in vivo tumor fluorescence imaging performance of the fluorescent probe of the present invention. A mouse breast cancer subcutaneous tumor model was established by injecting 4T1 mouse breast cancer cells into the legs of Balb / c nude mice. After the tumor grew to an appropriate size, the fluorescent probe prepared in Example 6 was injected intravenously (2 nmol of the probe was injected into each mouse), and then the mice were imaged with fluorescence at different time points after the injection. Figure 2 shown.

[0127] from Figure 2It can be seen that a strong fluorescent signal can be detected at the tumor site 2 hours after the fluorescent probe is injected. From 2 hours to 48 hours after the fluorescent probe is injected, the fluorescent signal of the tumor tissue is significantly stronger than that of the surrounding tissue, indicating that the fluorescent probe of the present invention can effectively and specifically develop tumor tissue.

[0128] 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 based on click chemistry reaction, characterized in that: The chemical structure of the zwitterionized click chemistry-based fluorescent probe is shown in formula (1): In formula (1), x, y, z are integers greater than or equal to 1; R is O, S, N or C; Ligand is a small molecule, polypeptide, aptamer or antibody with tumor targeting function.

2. The zwitterionized click chemistry-based fluorescent probe 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.

3. The zwitterionized click chemistry-based fluorescent probe according to claim 1, characterized in that: In formula (1), x=3, y=2, z=2, and R is O.

4. The zwitterionized fluorescent probe based on click chemistry reaction according to any one of claims 1 to 3, characterized in that: The chemical structure of the zwitterionized click chemistry-based fluorescent probe is shown in formula (3):

5. A method for preparing a zwitterionized fluorescent probe based on click chemistry reaction according to any one of claims 1 to 4, characterized in that: The preparation method comprises the following steps: Step 1, mixing 4-hydrazinobenzenesulfonic acid and 3-methyl-2-butanone, heating, and performing reflux reaction, then cooling and filtering, washing and drying the obtained crude product to obtain indolesulfonic acid, dissolving the indolesulfonic acid in methanol, and then dropping it into a propanol solution, stirring, filtering, and evaporating under reduced pressure to obtain 3H-indole-2,3,3-trimethyl-5-sulfonate; Step 2, dissolving 3H-indole-2,3,3-trimethyl-5-sulfonate in 1,2-dichlorobenzene, adding compound D, and then heating to reflux reaction, after cooling, discarding the upper solution, washing the obtained residue, filtering, and evaporating under reduced pressure to obtain compound E; Step 3, dissolving compound E, compound F and potassium acetate in a mixed solvent, heating to reflux reaction, then adding isopropanol to precipitate to obtain a crude product, and chromatographing and purifying the crude product to obtain compound G; Step 4, after dissolving compound H and compound I, heating them for reflux reaction, and then recrystallizing to obtain product J; Step 5, dissolving compound J and potassium hydroxide, stirring, then adding compound G, heating to react, and purifying the obtained crude product to obtain compound K; Step 6: Dissolve compound K, cuprous iodide and compound L, stir to obtain a crude product, and purify to obtain compound M.

6. The preparation method according to claim 5, characterized in that: In step 1, Dissolve p-hydrazinobenzenesulfonic acid in acetic acid, then add 3-methyl-2-butanone, heat to 110-130° C. for reflux reaction for 3-5 hours, then cool to 20-30° C., filter with filter paper to obtain a crude product; and / or, The molar ratio of hydrazinobenzenesulfonic acid to 3-methyl-2-butanone is 53:(150-170); and / or, The crude product is washed with ethyl acetate, and the solvent is evaporated to obtain indole sulfonic acid, which is dissolved in methanol and then added dropwise to a propanol solution containing potassium hydroxide, followed by stirring at room temperature for 20 to 30 hours, filtering with filter paper, and evaporating the residue under reduced pressure to obtain 3H-indole-2,3,3-trimethyl-5-sulfonate; and / or, The mass ratio of the indolesulfonic acid to the potassium hydroxide in the propanol solution is 5.1:(1.2-1.5).

7. The preparation method according to claim 5, characterized in that: In step 2, Under the protection of N2, 3H-indole-2,3,3-trimethyl-5-sulfonate is dissolved in 1,2-dichlorobenzene, and then compound D is added, the temperature is raised to 100-120°C, and the mixture is refluxed for reaction for 10-13 hours, then cooled to room temperature, the upper layer solution is discarded, isopropanol is added to the obtained residue for washing, the residue is filtered with filter paper, and evaporated to dryness under reduced pressure to obtain compound E; and / or, The molar ratio of the 3H-indole-2,3,3-trimethyl-5-sulfonate to compound D is (35-45):

12.

8. The preparation method according to claim 5, characterized in that: In step 3, Under N2 protection, compound E, compound F and potassium acetate are dissolved in a mixed solvent of acetic acid and acetic anhydride, wherein the volume ratio of acetic acid to acetic anhydride is (5-8):3, and then heated to 100-140°C for reflux reaction for 2-5h, and then isopropanol is added for precipitation to obtain a crude product, and finally purified by column chromatography to obtain compound G; and / or, The molar ratio of the compound E, the compound F and potassium acetate is (1-1.5): (0.5-0.7): (2-3); and / or, In step 4, compound H and compound I are dissolved in toluene at a molar ratio of (1-3):3, heated to 110° C. and refluxed for reaction for 10-14 hours. After the reaction, product J is obtained by recrystallization using methanol and methyl tert-butyl ether, respectively.

9. The preparation method according to claim 5, characterized in that: In step 5, Under N2 protection, compound J and potassium hydroxide are dissolved in DMSO (dimethyl sulfoxide) at a molar ratio of 1: (1-3), stirred at room temperature for 20-40 minutes, then compound G is added, heated to 50-70°C for reaction for 45-75 minutes, and the obtained crude product is purified by column chromatography to obtain compound K; and / or, The molar ratio of compound G to compound J is (0.1-0.3):1; and / or, In step 6, under N2 protection, compound K, cuprous iodide and compound L are dissolved in PBS (phosphate buffered saline) at a molar ratio of (0.4-0.6):(1.5-2.5):0.5, and stirred at room temperature for 30-60 min to obtain a crude product, which is purified by column chromatography to obtain compound M.

10. Use of the zwitterionized click chemistry reaction-based fluorescent probe according to any one of claims 1 to 4 in fluorescence imaging and photogenerated imaging.

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