A targeted near-infrared fluorescent compound and its preparation method and application

Abstract

The present invention provides a targeted near-infrared fluorescent compound and a preparation method and application thereof, and relates to the technical field of near-infrared fluorescent molecules. The targeted near-infrared fluorescent compound has a structure as shown in the following formula I. The S0456 near-infrared small molecule modified on a COX-2 inhibitor by an organic total synthesis method of the present invention obtains a targeted near-infrared fluorescent compound as shown in formula I. The targeted near-infrared fluorescent compound has a good active targeting effect when identifying COX-2 overexpressing liver tumors; and the targeted near-infrared fluorescent compound retains the water solubility of the dye and the specificity for tumor cells, and has the advantages of good water solubility and high fluorescence quantum yield.

Description

Technical Field

[0001] The invention relates to the technical field of near-infrared fluorescent molecules, and in particular to a targeted near-infrared fluorescent compound and a preparation method and application thereof. Background Art

[0002] Cancer remains the leading cause of death worldwide. Cancer-related deaths can be reduced by 30% in cancer patients who are diagnosed and treated early, highlighting the importance of developing new strategies for early diagnosis. Therefore, significant efforts have been made over the past decade to develop imaging technologies for early diagnosis of cancer. A variety of methods have been used for cancer imaging, including single-photon emission computed tomography (SPECT) and positron emission tomography (PET), computed tomography (CT), radionuclide imaging using single photons and positrons, magnetic resonance imaging (MRI), ultrasonography (US), and optical imaging. However, conventional examination techniques do not provide sufficient contrast to sensitively and reliably identify early neoplastic disease.

[0003] To address this problem, fluorescence imaging methods have attracted widespread attention due to their high selectivity and sensitivity, relatively low cost, and non-invasiveness. Fluorescent probes have been used for early diagnosis by detecting enzymes that are highly expressed in different types of cancer. Clinical results have shown that COX-2 is an enzyme overexpressed in tumors of all stages, while it is not expressed or expressed weakly in normal tissues. Therefore, detecting and imaging COX-2 with specific fluorescent probes can provide valuable information, thereby achieving early diagnosis of cancer. Therefore, COX-2 fluorescent probes have become a promising tool for detecting early cancer. In recent years, many COX-2 fluorescent probes have been developed and have been shown to determine the expression levels of COX-2 in cells and early tumor tissues. Traditionally, most fluorescent probes are synthesized by coupling macromolecular fluorophores to target drugs through appropriate flexible linkers.

[0004] In view of this, the present invention is proposed. Summary of the invention

[0005] The purpose of the present invention is to provide a targeted near-infrared fluorescent compound and its preparation method and application. The targeted near-infrared fluorescent compound of the present invention is a near-infrared fluorescent molecule IMCS obtained by connecting a near-infrared fluorescent molecule S0456 to a COX2 inhibitor, which can be specifically targeted for identifying COX-2 overexpressing tumors and has the advantages of good water solubility and high fluorescence quantum yield.

[0006] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are particularly adopted:

[0007] In a first aspect, the present invention provides a targeted near-infrared fluorescent compound, wherein the targeted near-infrared fluorescent compound has a structure as shown in the following formula I:

[0008]

[0009] Formula 1.

[0010] In the present invention, a novel near-infrared fluorescent probe indomethacin-S0456 probe (the targeted near-infrared fluorescent compound shown in Formula 1, hereinafter referred to as IMCS) with the ability to target COX-2 was designed and synthesized.

[0011] In a second aspect, the present invention provides a method for preparing the targeted near-infrared fluorescent compound as described in the first aspect, the preparation method comprising the following steps:

[0012] (1) Compound S0456 reacts with p-hydroxyphenylpropionate to obtain compound A;

[0013] The structural formula of the compound A is shown below:

[0014] ;

[0015] (2) After indomethacin and N-Boc-ethylenediamine are reacted, deprotection treatment is performed to obtain compound B;

[0016] The structural formula of the compound B is shown below:

[0017] ;

[0018] (3) Compound A reacts with compound B to obtain a targeted near-infrared fluorescent compound as shown in formula I.

[0019] Preferably, in step (1), the molar ratio of the compound S0456 to p-hydroxyphenylpropionate is 1:(2-4).

[0020] Preferably, in step (1), the salt of p-hydroxyphenylpropionate is selected from sodium p-hydroxyphenylpropionate.

[0021] Preferably, in step (1), the reaction is carried out in a solvent selected from dimethyl sulfoxide.

[0022] Preferably, in step (1), the reaction is carried out under a protective gas atmosphere, and the protective gas is selected from nitrogen.

[0023] Preferably, in step (1), the reaction temperature is 60-70° C., and the reaction time is 5-7 h.

[0024] Preferably, in step (1), after the reaction is completed, a post-treatment step is also included:

[0025] The reaction solution obtained after the reaction in step (1) is added dropwise to a mixture of ethyl acetate and ethanol, and the mixture is filtered and dried to obtain the compound A.

[0026] Preferably, in the mixed solution, the volume ratio of ethyl acetate to ethanol is 1:(0.5-2).

[0027] Preferably, in step (2), the molar ratio of indomethacin to N-Boc-ethylenediamine is 1:(1-1.5).

[0028] Preferably, in step (2), the reaction is carried out in the presence of a condensing agent, and the condensing agent is selected from dipyrrolidino (N-succinimidyloxy) carbonium hexafluorophosphate.

[0029] Preferably, in step (2), the molar ratio of indomethacin to dipyrrolidino(N-succinimidyloxy)carbonium hexafluorophosphate is 1:(1.2-2).

[0030] Preferably, in step (2), the reaction is carried out in the presence of a base, and the base is selected from N,N-diisopropylethylamine.

[0031] Preferably, in step (2), the molar ratio of indomethacin to base is 1:(0.5-5).

[0032] Preferably, in step (2), the reaction is carried out in a solvent selected from dichloromethane.

[0033] Preferably, in step (2), the reaction temperature is 30-40° C., and the reaction time is 1-3 h.

[0034] Preferably, in step (2), the deprotection treatment comprises the following steps:

[0035] Trifluoroacetic acid is added to the reaction solution obtained after the reaction of indomethacin and N-Boc-ethylenediamine, and the mixture is stirred and concentrated to obtain the compound B.

[0036] Preferably, the molar ratio of N-Boc-ethylenediamine to trifluoroacetic acid is 1:(1-5).

[0037] Preferably, the stirring temperature is 20-40° C., and the stirring time is 1-3 h.

[0038] Preferably, in step (3), the molar ratio of compound A to compound B is 1:(0.5-2).

[0039] Preferably, in step (3), the reaction is carried out in the presence of a condensing agent, and the condensing agent is selected from dipyrrolidino (N-succinimidyloxy) carbonium hexafluorophosphate.

[0040] Preferably, in step (3), the molar ratio of compound A to dipyrrolidino(N-succinimidyloxy)carbonium hexafluorophosphate is 1:(1.2-2).

[0041] Preferably, in step (3), the molar ratio of compound A to base is 1:(0.5-5).

[0042] Preferably, in step (3), the reaction is carried out in a solvent, and the solvent is selected from dimethyl sulfoxide.

[0043] Preferably, in step (3), the reaction temperature is 30-40° C., and the reaction time is 18-30 h.

[0044] Preferably, in step (3), after the reaction is completed, a post-treatment step is also included:

[0045] The reaction solution obtained after the reaction in step (3) is added dropwise to an acetone solution, and the mixture is filtered and dried to obtain a targeted near-infrared fluorescent compound represented by formula I.

[0046] In a third aspect, the present invention provides a use of the targeted near-infrared fluorescent compound or its racemate, stereoisomer, or pharmaceutically acceptable salt as described in the first aspect in the preparation of a tumor imaging agent;

[0047] Wherein, the tumor includes any one of liver cancer tumor, pancreatic cancer tumor, colon cancer tumor, gastric cancer tumor, breast cancer tumor or head and neck cancer tumor.

[0048] In a fourth aspect, the present invention provides a use of the targeted near-infrared fluorescent compound or its racemate, stereoisomer, or pharmaceutically acceptable salt as described in the first aspect in the preparation of a reagent for identifying COX-2 overexpressing tumors.

[0049] Compared with the prior art, the present invention has the following beneficial effects:

[0050] (1) The present invention synthesizes IMCS by organic total synthesis. When indomethacin is combined with the fluorescent dye S0456, it has a higher selectivity for COX-2. It can be quickly cleared in normal tissues, thereby quickly achieving a tumor-to-background signal ratio (TBR>1.5) that meets clinical needs, and has a good active targeting effect in identifying COX-2 overexpressing tumors.

[0051] (2) The targeted near-infrared fluorescent compound IMCS of the present invention has the advantages of water solubility of the dye and high fluorescence quantum yield, etc. It has great development potential in the fields of near-infrared fluorescence navigation tumor surgery and medical cell labeling. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0053] Figure 1 It is an IMCS fluorescent molecule 1 HNMR spectrum.

[0054] Figure 2 This is the IMCS fluorescence molecular mass spectrum.

[0055] Figure 3 This is the IMCS fluorescent molecular liquid chromatography diagram.

[0056] Figure 4 This is the fluorescence performance diagram of IIMCS fluorescent molecules.

[0057] Figure 5 It is the specific targeting effect of different doses of IMCS on liver tumors.

[0058] Figure 6 This is a trend chart of TBR changes at different IMCS doses.

[0059] Figure 7 This is the distribution diagram of the fluorescence signal of IMCS in various organs. DETAILED DESCRIPTION

[0060] Unless otherwise defined herein, scientific and technical terms used in conjunction with the present invention shall have the meanings commonly understood by those of ordinary skill in the art. The meaning and scope of the terms should be clear, however, in the case of any potential ambiguity, the definitions provided herein take precedence over any dictionary or external definitions. In this application, unless otherwise stated, the use of "or" means "and / or". In addition, the use of the term "including" and other forms is non-limiting.

[0061] It should be noted that specific details are described in the following description to facilitate a full understanding of the present invention. However, the present invention can be implemented in a variety of other ways different from those described herein, and those skilled in the art can make similar generalizations without violating the connotation of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0062] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0063] In a first aspect, the present invention provides a targeted near-infrared fluorescent compound, wherein the targeted near-infrared fluorescent compound has a structure as shown in the following formula I:

[0064]

[0065] Formula 1.

[0066] In the present invention, a novel near-infrared fluorescent probe indomethacin-S0456 probe (IMCS) with the ability to target COX-2 is designed and synthesized. With indomethacin as the recognition group, it is connected to S0456 through ethylenediamine to construct a targeted COX-2 fluorescent probe (targeted near-infrared fluorescent compound shown in Formula 1, hereinafter referred to as IMCS) with excitation (775 nm) and emission (797 nm), which has higher selectivity for COX-2. The obtained IMCS can be quickly cleared in normal tissues, thereby quickly achieving a tumor-to-background signal ratio (TBR>1.5) that meets clinical needs, and has a good active targeting effect in identifying COX-2 overexpressing tumors, further expanding the application value of the drug for surgical navigation and tumor resection. And IMCS also has the advantages of water solubility of the dye and high fluorescence quantum yield.

[0067] Among them, indometacin is a nonsteroidal anti-inflammatory drug (NSAID) that plays a tumor-selective diagnostic and therapeutic role by targeting the biosynthesis of COX-derived prostaglandins (PG). It becomes more selective when combined with fluorescent dyes. Indometacin combined with fluorescent tracers shows significant targeting ability to COX2-overexpressing cancer cells but not normal cells. It has certain research value and can be used for the development of specific targeted fluorescent probes.

[0068] In a second aspect, the present invention provides a method for preparing the targeted near-infrared fluorescent compound as described in the first aspect, the preparation method comprising the following steps:

[0069] (1) Compound S0456 reacts with p-hydroxyphenylpropionate to obtain compound A;

[0070] The structural formula of the compound A is shown below:

[0071] ;

[0072] (2) After indomethacin and N-Boc-ethylenediamine are reacted, deprotection treatment is performed to obtain compound B;

[0073] The structural formula of the compound B is shown below:

[0074] ;

[0075] (3) Compound A reacts with compound B to obtain a targeted near-infrared fluorescent compound as shown in formula I.

[0076] In an optional embodiment, the method for preparing the targeted near-infrared fluorescent compound comprises the following steps:

[0077] (1) Compound S0456 reacts with p-hydroxyphenylpropionate to obtain compound A; wherein the reaction formula is as follows:

[0078] ;

[0079] (2) After indomethacin and N-Boc-ethylenediamine are reacted, a deprotection treatment is performed to obtain compound B; the structural formula of compound B is shown below:

[0080] ;

[0081] (3) Compound A reacts with compound B to obtain a targeted near-infrared fluorescent compound represented by formula I; wherein the reaction formula is as follows:

[0082] .

[0083] In the present invention, a targeted near-infrared fluorescent compound shown in Formula I of S0456 near-infrared small molecule modified with COX-2 inhibitor indomethacin was synthesized by organic total synthesis method, which has significant targeting ability in identifying COX2 overexpressing cancer cells rather than normal cells. The targeted near-infrared fluorescent compound shown in Formula I retains the water solubility and specificity of the dye to tumor cells, and as a tracer, has the advantages of good water solubility and high fluorescence quantum yield, and has great development potential in the fields of fluorescence navigation tumor surgery and medical cell labeling.

[0084] In an optional embodiment, in step (1), the molar ratio of the compound S0456 to p-hydroxyphenylpropionate is 1:(2-4), for example, it can be 1:2, 1:2.2, 1:2.4, 1:2.6, 1:2.8, 1:3, 1:3.2, 1:3.4, 1:3.6, 1:3.8, 1:4, etc.

[0085] In an optional embodiment, in step (1), the salt of p-hydroxyphenylpropionate is selected from sodium p-hydroxyphenylpropionate.

[0086] In an optional embodiment, in step (1), the reaction is carried out in a solvent.

[0087] In an optional embodiment, in step (1), the solvent is selected from dimethyl sulfoxide.

[0088] In an optional embodiment, in step (1), the reaction is carried out under a protective gas atmosphere.

[0089] In an optional embodiment, in step (1), the protective gas is selected from nitrogen.

[0090] In an optional embodiment, in step (1), the reaction temperature is 60-70°C, for example, it can be 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C, 70°C, etc.

[0091] In an optional embodiment, in step (1), the reaction time is 5 to 7 h, for example, 5 h, 5.2 h, 5.4 h, 5.6 h, 5.8 h, 6 h, 6.2 h, 6.4 h, 6.6 h, 6.8 h, 7 h, etc.

[0092] In an optional embodiment, in step (1), after the reaction is completed, a post-treatment step is further included:

[0093] The reaction solution obtained after the reaction in step (1) is added dropwise to a mixture of ethyl acetate and ethanol, and the mixture is filtered and dried to obtain the compound A.

[0094] In an optional embodiment, in the mixed solution, the volume ratio of ethyl acetate to ethanol is 1:(0.5~2), for example, it can be 1:0.5, 1:0.6, 1:0.7, 1:0.8, 1:0.85, 1:0.9, 1:0.95, 1:1, 1:1.05, 1:1.1, 1:1.15, 1:1.2, 1:1.4, 1:1.6, 1:1.8, 1:2, etc.

[0095] In an optional embodiment, in step (2), the molar ratio of indomethacin to N-Boc-ethylenediamine is 1:(1-1.5), for example, it can be 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, etc.

[0096] In an optional embodiment, in step (2), the reaction is carried out in the presence of a condensing agent, and the condensing agent is selected from dipyrrolidino (N-succinimidyloxy) carbonium hexafluorophosphate.

[0097] In an optional embodiment, in step (2), the molar ratio of indomethacin to dipyrrolidino (N-succinimidyloxy) carbonium hexafluorophosphate is 1:(1.2~2), for example, it can be 1:1, 1:1.2, 1:1.25, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2, etc.

[0098] In an optional embodiment, in step (2), the reaction is carried out in the presence of a base, and the base is selected from N,N-diisopropylethylamine.

[0099] In an optional embodiment, in step (2), the molar ratio of indomethacin to base is 1:(0.5~5), for example, it can be 1:0.5, 1:0.6, 1:0.7, 1:0.8, 1:0.9, 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5, 1:5, etc.

[0100] In an optional embodiment, in step (2), the reaction is carried out in a solvent.

[0101] In an optional embodiment, in step (2), the solvent is selected from dichloromethane.

[0102] In an optional embodiment, in step (2), the reaction temperature is 30-40°C, for example, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, etc.

[0103] In an optional embodiment, in step (2), the reaction time is 1 to 3 h, for example, 1 h, 1.2 h, 1.4 h, 1.6 h, 1.8 h, 2 h, 2.2 h, 2.4 h, 2.6 h, 2.8 h, 3 h, etc.

[0104] In an optional embodiment, in step (2), the deprotection treatment comprises the following steps:

[0105] Trifluoroacetic acid is added to the reaction solution obtained after the reaction of indomethacin and N-Boc-ethylenediamine, and the mixture is stirred and concentrated to obtain the compound B.

[0106] In an optional embodiment, in step (2), the molar ratio of N-Boc-ethylenediamine to trifluoroacetic acid is 1:(1-5), for example, it can be 1:1, 1:1.5, 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5, 1:5, etc.

[0107] In an optional embodiment, in step (2), the stirring temperature is 20-40°C, for example, it can be 20°C, 22°C, 24°C, 26°C, 28°C, 30°C, 32°C, 34°C, 36°C, 38°C, 40°C, etc.

[0108] In an optional embodiment, in step (2), the stirring time is 1 to 3 h, for example, 1 h, 1.2 h, 1.4 h, 1.6 h, 1.8 h, 2 h, 2.2 h, 2.4 h, 2.6 h, 2.8 h, 3 h, etc.

[0109] In an optional embodiment, in step (3), the molar ratio of compound A to compound B is 1:(0.5~2), for example, it can be 1:0.5, 1:0.6, 1:0.7, 1:0.8, 1:0.85, 1:0.9, 1:0.95, 1:1, 1:1.05, 1:1.1, 1:1.15, 1:1.2, 1:1.4, 1:1.6, 1:1.8, 1:2, etc.

[0110] In an optional embodiment, in step (3), the reaction is carried out in the presence of a condensation agent.

[0111] In an optional embodiment, in step (3), the condensing agent is selected from dipyrrolidino (N-succinimidyloxy) carbonium hexafluorophosphate.

[0112] In an optional embodiment, in step (3), the molar ratio of compound A to dipyrrolidino (N-succinimidyloxy) carbonium hexafluorophosphate is 1:(1.2-2), for example, it can be 1:1, 1:1.2, 1:1.25, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2, etc.

[0113] In an optional embodiment, in step (3), the reaction is carried out in the presence of a base.

[0114] In an optional embodiment, in step (3), the base is selected from N,N-diisopropylethylamine.

[0115] In an optional embodiment, in step (3), the molar ratio of compound A to base is 1:(0.5~5), for example, it can be 1:0.5, 1:0.6, 1:0.7, 1:0.8, 1:0.9, 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5, 1:5, etc.

[0116] In an optional embodiment, in step (3), the reaction is carried out in a solvent, and the solvent is selected from dimethyl sulfoxide.

[0117] In an optional embodiment, in step (3), the reaction temperature is 30-40°C, for example, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, etc.

[0118] In an optional embodiment, in step (3), the reaction time is 18 to 30 h, for example, 18 h, 19 h, 20 h, 21 h, 22 h, 23 h, 24 h, 25 h, 26 h, 27 h, 28 h, 29 h, 30 h, etc.

[0119] In an optional embodiment, in step (3), after the reaction is completed, a post-treatment step is also included:

[0120] The reaction solution obtained after the reaction in step (3) is added dropwise to an acetone solution, and the mixture is filtered and dried to obtain a targeted near-infrared fluorescent compound represented by formula I.

[0121] In a third aspect, the present invention provides a use of the targeted near-infrared fluorescent compound or its racemate, stereoisomer, or pharmaceutically acceptable salt as described in the first aspect in the preparation of a tumor imaging agent;

[0122] Wherein, the tumor includes any one of liver cancer tumor, pancreatic cancer tumor, colon cancer tumor, gastric cancer tumor, breast cancer tumor or head and neck cancer tumor.

[0123] In a fourth aspect, the present invention provides a use of the targeted near-infrared fluorescent compound or its racemate, stereoisomer, or pharmaceutically acceptable salt as described in the first aspect in the preparation of a reagent for identifying COX-2 overexpressing tumors.

[0124] In an optional embodiment, the application is specifically: using the targeted near-infrared fluorescent compound or its racemate, stereoisomer, pharmaceutically acceptable salt as described in the first aspect to establish a tumor imaging model, and using intraoperative fluorescence navigation equipment to diagnose the tumor effect.

[0125] In an optional embodiment, the present invention provides a liver cancer tumor development model and a method for establishing the same, which specifically comprises the following steps: inoculating a human liver cancer cell line with high COX-2 expression into the back of a mouse, and injecting the targeted near-infrared fluorescent compound (IMCS) of the present invention via tail vein injection.

[0126] It should be noted that after the above-mentioned liver tumor imaging model is established, the intraoperative fluorescent navigation device is used to diagnose the liver tumor effect of small molecules; the liver tumor can be removed during surgery, and the pathological section can be used to determine whether IMCS can specifically target the liver tumor.

[0127]

Term Explanation

[0128] As mentioned in the present invention, the term "pharmaceutically acceptable salt" means that the salt is not only physiologically acceptable to the subject, but also refers to a synthetic substance with pharmaceutical use value, such as a salt formed as an intermediate when performing chiral separation. Although the salt of this intermediate cannot be directly administered to the subject, the salt can play a role in obtaining the final product of the present invention.

[0129] As mentioned in the present invention, the pharmaceutically acceptable salt of the compound of formula I is a salt formed with a base or an alkali metal. The acid that forms a pharmaceutically acceptable salt with the compound of formula I includes an inorganic acid and an organic acid. More specifically, the alkali metal that forms a pharmaceutically acceptable salt with the compound of formula I includes but is not limited to lithium, sodium, potassium, magnesium, calcium, aluminum, zinc, etc.; the base that forms a pharmaceutically acceptable salt with the compound of formula I includes but is not limited to choline, diethanolamine, morpholine, etc.

[0130] The present invention is further described below by way of examples. Unless otherwise specified, the materials in the examples are prepared according to existing methods or directly purchased from the market.

[0131] Example 1

[0132] This embodiment provides a targeted near-infrared fluorescent compound IMCS having a structure as shown in the following formula 1:

[0133]

[0134] Formula 1.

[0135] The synthesis route of the targeted near-infrared fluorescent compound IMCS is as follows:

[0136]

[0137]

[0138] The preparation method of the targeted near-infrared fluorescent compound IMCS specifically comprises the following steps:

[0139] (a) Preparation of Compound A:

[0140] S0456 (888 mg, 1 mmol) was dissolved in 10 mL DMSO and stirred until completely dissolved. Sodium p-hydroxyphenylpropionate (424 mg, 2 mmol) was added to the flask. The mixture was reacted at 65 °C for 6 h under nitrogen protection. The reaction solution was added dropwise to a mixed solution of 200 mL ethyl acetate and ethanol (V 乙酸乙酯 :V 乙醇 =1:1), a dark green precipitate was produced, which was filtered and dried in a vacuum to obtain 1.05 g of solid (i.e., compound A), with a yield of 95%.

[0141] (b) Preparation of compound B:

[0142] Indomethacin (0.357 g, 1 mmol) was placed in a round-bottom flask and dissolved in 20 mL of dichloromethane. Dipyrrolidino (N-succinimidyloxy) carbonium hexafluorophosphate (483 mg, 1.176 mmol), N-Boc-ethylenediamine (320 mg, 2 mmol), and DIEA (151 mg, 1.176 mmol) were added in sequence. The mixture was stirred at 37 ° C for 2 h. After the reaction was completed, 2 mL of trifluoroacetic acid was added to the reaction system, and the mixture was stirred at 37 ° C for 2 h. The solution was spin-dried to obtain compound B, and 0.3 g (i.e., compound B) was obtained.

[0143] (c) Preparation of compound IMCS:

[0144] Compound A (1.01 g, 1 mmol) was placed in a round-bottom flask, dissolved in 20 mL DMSO, and dipyrrolidino (N-succinimidyloxy) carbonium hexafluorophosphate (483 mg, 1.176 mmol), DIEA (258 mg, 2 mmol), and compound B (0.8 g, 2 mmol) were added in sequence. The mixture was stirred at 37 ° C for 24 h and precipitated in acetone solution. The precipitate was filtered and dried in vacuo. The product was purified by preparative liquid chromatography to obtain the targeted near-infrared fluorescent compound IMCS shown in Formula 1.

[0145] The structure was characterized by H-NMR, HPLC and MS, and the results of structure determination are as follows:

[0146] like Figure 1 As shown, 1HNMR (400MHz, DMSO-d6) δ: δ 8.06, 7.82, 7.78, 7.71, 7.69,7.63, 7.63, 7.62, 7.61, 7.61, 7.59, 7.59, 7.33, 7.31, 7.20, 7.18, 7.11, 7.10,7.05, 7.03, 6.94, 6.92, 6.69, 6.68, 6.67, 6.66, 6.23, 6.20, 4.11, 3.83, 3.73,3.49, 3.03, 2.72, 2.71, 2.69, 2.67, 2.20, 1.93, 1.76, 1.73, 1.25.

[0147] like Figure 2 As shown, LCMS (ESI): m / z: Chemical Formula: C 68 H 76 ClN5O 17 S4, [M-3H]found 465.15, [M-2H]found 698.65.

[0148] like Figure 3 As shown, the purity of the targeted near-infrared fluorescent compound IMCS is 96.66%.

[0149] like Figure 4 As shown, the targeted near-infrared fluorescent compound IMCS has a maximum excitation wavelength Ex max = 775 nm and a maximum fluorescence emission wavelength Em max = 797 nm, indicating that the molecule is a near-infrared fluorescent probe.

[0150] Application Example 1

[0151] This application example provides an application of IMCS near-infrared fluorescent molecules in the specific identification of liver cancer.

[0152] HepG2 cell line was used to construct a liver cancer model: HepG2, a human liver cancer cell line with high COX-2 expression, was inoculated on the back of Balb / c nude mice, 10 nmol of IMCS molecules were injected into the tail vein, and intraoperative fluorescence navigation equipment was used to diagnose liver cancer effects of small molecules. The liver cancer was removed during the operation, and pathological sections were used to determine whether IMCS could specifically target liver tumors.

[0153] The test results are as follows Figure 5 , Figure 6 and Figure 7 As shown:

[0154] like Figure 5~Figure 6As shown in the figure, 1 h, 2 h, 4 h, 8 h, 12 h, 24 h, 48 h, and 72 h after injection of 1, 5, 10, 50, and 100 nmol IMCS, the back of the mouse showed high fluorescence signals only in the tumor area, while IMCS in the surrounding normal tissues was gradually metabolized, and the fluorescence intensity decayed faster than the tumor. Not only did it reach the clinical imaging requirement TBR after 1 h, but it also had a wider imaging window. This shows that IMCS has good specificity in identifying liver tumors with overexpression of COX2.

[0155] like Figure 7 As shown in the figure, the fluorescence signal distribution of the isolated organs over a period of 24 hours, at each time point, it can be seen that the fluorescence intensity of the tumor tissue is significantly higher than that of other tissues, indicating that IMCS can accumulate specifically in tumors.

[0156] In summary, the present invention aims to provide a near-infrared fluorescent probe and its preparation method and application, which can actively target COX2, can be quickly cleared in normal tissues, and stay in the tumor site for a long time, so as to achieve the effect of in vivo diagnosis. It has a good active targeting effect in identifying liver cancer. This near-infrared fluorescent dye retains the water solubility of the dye and the specificity for tumor cells. The tracer has the advantages of good water solubility and high fluorescence quantum yield, and has great development potential in the fields of near-infrared fluorescence navigation tumor surgery and medical cell labeling.

[0157] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A targeted near-infrared fluorescent compound, characterized in that: The targeted near-infrared fluorescent compound has a structure as shown in the following formula I: Formula 1.

2. A method for preparing a targeted near-infrared fluorescent compound according to claim 1, characterized in that: The preparation method comprises the following steps: (1) Compound S0456 reacts with p-hydroxyphenylpropionate to obtain compound A; wherein the reaction formula is as follows: ； (2) in the presence of a condensing agent and a base, indomethacin and N-Boc-ethylenediamine are reacted, and then deprotected to obtain compound B; Wherein, the condensing agent is selected from dipyrrolidino (N-succinimidyloxy) carbonium hexafluorophosphate; the base is selected from N, N-diisopropylethylamine; The deprotection treatment comprises the following steps: adding trifluoroacetic acid to the reaction solution obtained after the reaction of indomethacin and N-Boc-ethylenediamine, stirring, and concentrating to obtain the compound B; The structural formula of the compound B is shown below: ； (3) Compound A reacts with compound B in the presence of a condensing agent and a base to obtain a targeted near-infrared fluorescent compound as shown in Formula I; Wherein, the condensing agent is selected from dipyrrolidino (N-succinimidyloxy) carbonium hexafluorophosphate; and the base is selected from N, N-diisopropylethylamine.

3. The method for preparing the targeted near-infrared fluorescent compound according to claim 2, characterized in that: In step (1), the molar ratio of the compound S0456 to p-hydroxyphenylpropionate is 1:(2-4); In step (1), the p-hydroxyphenylpropionate is selected from sodium p-hydroxyphenylpropionate; In step (1), the reaction is carried out in a solvent selected from dimethyl sulfoxide; In step (1), the reaction is carried out under a protective gas atmosphere, and the protective gas is selected from nitrogen; In step (1), the reaction temperature is 60-70° C., and the reaction time is 5-7 h.

4. The method for preparing the targeted near-infrared fluorescent compound according to claim 2 or 3, characterized in that: In step (1), after the reaction is completed, a post-treatment step is also included: The reaction solution obtained after the reaction in step (1) is added dropwise to a mixture of ethyl acetate and ethanol, and the mixture is filtered and dried to obtain the compound A; In the mixed solution, the volume ratio of ethyl acetate to ethanol is 1:(0.5-2).

5. The method for preparing the targeted near-infrared fluorescent compound according to claim 2, characterized in that: In step (2), the molar ratio of indomethacin to N-Boc-ethylenediamine is 1:(1-1.5); In step (2), the molar ratio of indomethacin to dipyrrolidino(N-succinimidyloxy)carbonium hexafluorophosphate is 1:(1.2-2); In step (2), the molar ratio of indomethacin to base is 1:(0.5-5); In step (2), the reaction is carried out in a solvent selected from dichloromethane; In step (2), the reaction temperature is 30-40° C., and the reaction time is 1-3 h.

6. The method for preparing the targeted near-infrared fluorescent compound according to claim 2, characterized in that: In step (2), the molar ratio of N-Boc-ethylenediamine to trifluoroacetic acid is 1:(1-5); The stirring temperature is 20-40° C., and the stirring time is 1-3 h.

7. The method for preparing the targeted near-infrared fluorescent compound according to claim 2, characterized in that: In step (3), the molar ratio of compound A to compound B is 1:(0.5-2); In step (3), the molar ratio of compound A to dipyrrolidino (N-succinimidyloxy) carbonium hexafluorophosphate is 1:(1.2-2); In step (3), the molar ratio of compound A to base is 1:(0.5-5); In step (3), the reaction is carried out in a solvent selected from dimethyl sulfoxide; In step (3), the reaction temperature is 30-40° C., and the reaction time is 18-30 h.

8. The method for preparing a targeted near-infrared fluorescent compound according to claim 2 or 7, characterized in that: In step (3), after the reaction is completed, a post-treatment step is also included: The reaction solution obtained after the reaction in step (3) is added dropwise to an acetone solution, and the mixture is filtered and dried to obtain a targeted near-infrared fluorescent compound represented by formula I.

9. Use of the targeted near-infrared fluorescent compound or a pharmaceutically acceptable salt thereof as claimed in claim 1 in preparing a reagent for tumor imaging; in, The tumor includes any one of a liver cancer tumor, a pancreatic cancer tumor, a colon cancer tumor, a stomach cancer tumor, a breast cancer tumor or a head and neck cancer tumor.

10. Use of the targeted near-infrared fluorescent compound or a pharmaceutically acceptable salt thereof as claimed in claim 1 in the preparation of a reagent for identifying a COX-2 overexpressing tumor.

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