Near-infrared two-region fluorescent organic molecules, fluorescent targeting molecular probes, and preparation method and application thereof

Abstract

The application discloses a near-infrared two-region fluorophore organic molecule, a fluorescent targeting molecular probe and a preparation method and application thereof. The near-infrared two-region fluorophore organic molecule has the structure shown in the following formula: the fluorescent targeting molecular probe comprises the near-infrared two-region fluorophore organic molecule as a probe main body, and a targeting peptide cRGD coupled and modified on the surface of the near-infrared two-region fluorophore organic molecule. The near-infrared two-region fluorophore organic molecule provided by the application has the advantages of a large molar absorption coefficient, a high quantum yield and a small cytotoxicity, a deep tissue penetration depth, a high spatial resolution and a high sensitivity, and can clearly mark a tumor boundary and a small hidden lesion; the prepared fluorescent targeting molecular probe utilizes the specific recognition ability of the targeting peptide cRGD and tumor cells to transport the organic small molecule to a tumor site, accurately targets tumor cells and enhances the targeting property of the probe to the tumor.

Description

Technical Field

[0001] This invention relates to a near-infrared II cyanine fluorescent organic molecule, particularly to a near-infrared II cyanine fluorescent organic molecule and its preparation method, as well as a near-infrared II fluorescent targeting molecular probe for tumor surgical navigation, its preparation method and application, belonging to the field of targeting molecular probe technology. Background Technology

[0002] The ultimate goal of surgical resection of solid tumors is to achieve complete removal of cancerous tissue while preserving as much healthy tissue as possible. This requires surgeons to accurately identify the tumor (including primary and metastatic tumors) and adjacent healthy tissue (such as nerves, blood vessels, and lymph nodes) during surgery. However, the differences between tumors and healthy tissue are often not directly visible to the naked eye.

[0003] Fluorescence imaging technology boasts a series of advantages, including intuitive visualization, high sensitivity, non-contact operation, and convenient accessibility. Fluorescence-guided surgical treatment can provide a powerful tool for differentiating tumor lesions from normal tissues. Biological organisms possess two optically transparent windows in the near-infrared (NIR) band: the 700-900 nm NIR-I region and the 950-1700 nm NIR-II region. In particular, the NIR-II region, compared to traditional bands, offers deeper tissue penetration (>1.5 cm), lower light scattering, absorption, and autofluorescence interference, and holds promise for providing precise imaging guidance for intraoperative tumor treatment.

[0004] Currently approved fluorescent probes such as indocyanine green (ICG) and methylene blue (MB) have problems such as non-specific tissue uptake and easy fluorescence bleaching, making it difficult to accurately define tumor boundaries and causing some small and hidden metastatic lesions to be missed. Summary of the Invention

[0005] The main objective of this invention is to provide a near-infrared II region anthocyanin-based fluorescent organic molecule and its preparation method, so as to overcome the shortcomings of the prior art.

[0006] Another objective of this invention is to provide a near-infrared II region fluorescent targeting molecular probe and its preparation method.

[0007] Another object of the present invention is to provide the application of the near-infrared II fluorescent targeting molecular probe.

[0008] To achieve the aforementioned objectives, the technical solution adopted by this invention includes:

[0009] This invention provides a near-infrared II region cyanin-based fluorescent organic molecule with the structure shown in formula (I):

[0010]

[0011] This invention also provides a method for preparing near-infrared II region cyanine fluorescent organic molecules, comprising:

[0012] Diethylaminoketo acid was reacted with cyclopentanone in the first reaction to prepare the first compound;

[0013] The second compound was prepared by reacting 1,1,2-trimethyl-1H-benzoindole with 1,4-butyryl lactone in a second reaction.

[0014] The first compound, the second compound, and glutaraldehyde were reacted in a third reaction to prepare near-infrared II region anthocyanin-based fluorescent organic molecules.

[0015] This invention also provides near-infrared II region anthocyanin fluorescent organic molecules prepared by the aforementioned method.

[0016] This invention also provides a near-infrared II fluorescent targeting molecular probe, comprising: a near-infrared II cyanine fluorescent organic molecule as the probe body, and a targeting peptide cRGD, wherein the targeting peptide cRGD is coupled and modified on the surface of the near-infrared II cyanine fluorescent organic molecule.

[0017] Furthermore, the targeting peptide cRGD is a targeting molecule that specifically recognizes the integrin receptor protein αvβ3 on the surface of tumor blood vessels.

[0018] This invention also provides a method for preparing a near-infrared II region fluorescent targeting molecular probe, comprising:

[0019] Near-infrared II region anthocyanin-based fluorescent organic molecules were prepared according to the aforementioned preparation method;

[0020] By modifying the surface of the near-infrared II fluorescent organic cyanide molecules with the coupling targeting peptide cRGD, a near-infrared II fluorescent targeting molecular probe is obtained.

[0021] Furthermore, embodiments of the present invention also provide the application of the aforementioned near-infrared II fluorescent targeting molecular probe in the preparation of products with tumor surgical navigation functions.

[0022] Compared with the prior art, the beneficial effects of the present invention include at least the following:

[0023] 1) The near-infrared II region cyanine fluorescent organic molecules provided by this invention have the advantages of large molar absorptivity, high quantum yield and low cytotoxicity. Compared with clinically approved fluorescent probes indocyanine green (ICG) and methylene blue (MB), the organic small molecule NIR-950 has low biological background signal, deep tissue penetration, high spatial resolution and sensitivity, and can clearly mark tumor boundaries and small occult lesions.

[0024] 2) The near-infrared II fluorescent targeting molecular probe provided by this invention uses cyanide organic small molecules with high quantum yield and targeting peptide cRGD coupled together. By utilizing the ability of the targeting peptide cRGD to specifically recognize tumor cells, the organic small molecules are delivered to the tumor site, accurately targeting tumor cells and enhancing the targeting of the probe to the tumor. Attached Figure Description

[0025] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0026] Figure 1 This is a diagram illustrating the synthesis process of the near-infrared II region cyanine fluorescent organic molecule NIR-950 in Example 1 of this invention;

[0027] Figure 2 The near-infrared II region anthocyanin fluorescent organic molecule NIR-950 in Example 1 of this invention. 1 H-NMR spectrum;

[0028] Figure 3A and Figure 3B The images show the fluorescence emission spectrum and ultraviolet absorption curve of the near-infrared II cyanine fluorescent organic molecule NIR-950 in Example 1 of this invention, respectively.

[0029] Figure 4 This is a diagram illustrating the synthesis process of the targeted molecular probe NIR-950-cRGD in Example 3 of the present invention;

[0030] Figure 5A and Figure 5B The images show the ultraviolet absorption and fluorescence emission spectra of the targeting molecular probe NIR-950-cRGD in Example 3 of this invention.

[0031] Figure 6 This is a diagram illustrating the cell targeting accuracy verification of the targeting molecular probe NIR-950-cRGD in Example 3 of this invention.

[0032] Figure 7 This is a near-infrared imaging image of Hepa1-6 subcutaneous tumor mice in Example 3 of the present invention;

[0033] Figure 8 Fluorescence images of the heart, liver, spleen, lung, kidney, and tumor tissues of the Hepa1-6 subcutaneous tumor model in Example 5 of this invention, obtained by a near-infrared in vivo imaging system after cleaning;

[0034] Figure 9 This is a near-infrared imaging image of a 4T1 orthotopic tumor mouse in Example 6 of the present invention.

[0035] Figure 10 Fluorescence images of the heart, liver, spleen, lung, kidney, and tumor tissues of the 4T1 orthotopic tumor model in Embodiment 6 of the present invention, obtained by a near-infrared in vivo imaging system after cleaning;

[0036] Figures 11A-11P This is a graph showing the hematological and blood biochemical index test results of blood collected from the eyeballs of mice after tail vein injection of the targeted molecular probe NIR-950-cRGD in Example 8 of the present invention. Detailed Implementation

[0037] In order to overcome the shortcomings of the above-mentioned technologies, the inventors of this case, through long-term research and extensive practice, have proposed the technical solution of this invention, which provides a near-infrared II region (hereinafter referred to as NIRII region) cyanin fluorescent organic molecule and a NIR II region fluorescent targeting molecular probe for tumor surgical navigation.

[0038] To facilitate understanding of this application, it will be described in more detail below. However, it should be understood that the invention can be implemented in many different forms and is not limited to the embodiments or examples described herein. Rather, these embodiments or examples are provided to provide a thorough and complete understanding of the disclosure of this invention.

[0039] Specifically, as one aspect of the technical solution of this invention, a near-infrared II cyanin-based fluorescent organic molecule (hereinafter referred to as NIR-950) has the structure shown in formula (I):

[0040]

[0041] As one aspect of the technical solution of this invention, a method for preparing a near-infrared II region cyanin-based fluorescent organic molecule includes:

[0042] Diethylaminoketo acid was reacted with cyclopentanone in the first reaction to prepare the first compound;

[0043] The second compound was prepared by reacting 1,1,2-trimethyl-1H-benzoindole with 1,4-butyryl lactone in a second reaction.

[0044] The first compound, the second compound, and glutaraldehyde were reacted in a third reaction to prepare near-infrared II region anthocyanin-based fluorescent organic molecules.

[0045] In some embodiments, the preparation method specifically includes:

[0046] At 0-5℃, cyclopentanone is added to concentrated sulfuric acid, which acts as a reaction solvent and catalyst. The mixture is stirred for 1-2 hours.

[0047] Diethylaminoketo acid was added at 0-5℃, followed by heating to carry out the first reaction;

[0048] After the first reaction is completed, an acidifying agent is added to form an acidification system. The mixture is then filtered, dried, and the first compound is obtained.

[0049] In some embodiments, the first compound has the structural formula shown in formula (II):

[0050]

[0051] In some more preferred embodiments, the mass-to-volume ratio of the diethylaminoketo acid to cyclopentanone is 5-10 g: 4-10 mL.

[0052] In some more preferred embodiments, the temperature of the first reaction is 80-100°C, and the reaction time is 1-2 hours.

[0053] Furthermore, the acidifying agent may include, but is not limited to, HClO4.

[0054] In some embodiments, the preparation method specifically includes: mixing 1,1,2-trimethyl-1H-benzoindole, 1,4-butyryl lactone with an organic solvent, and then heating to carry out a second reaction to obtain a second compound.

[0055] In some embodiments, the structural formula of the second compound is shown in formula (III):

[0056]

[0057] In some embodiments, the temperature of the second reaction is 100-200°C, and the reaction time is 24-48 hours.

[0058] In some more preferred embodiments, the mass ratio of 1,1,2-trimethyl-1H-benzoindole to 1,4-butyryl lactone is 8-10:5-6.5.

[0059] Furthermore, the organic solvent may include, but is not limited to, acetonitrile.

[0060] In some more preferred embodiments, the preparation method further includes: after the second reaction is completed, rotary evaporation under reduced pressure, recrystallizing the obtained crude product with a first mixed solvent, filtering, and drying to obtain the second compound.

[0061] In some more preferred embodiments, the first mixed solvent comprises a combination of diethyl ether and dichloromethane, wherein the volume ratio of the diethyl ether to dichloromethane is 5-10:1.

[0062] In some embodiments, the preparation method specifically includes: uniformly mixing a first compound, a second compound, glutaraldehyde, and acetic anhydride (as a catalyst), dissolving them in an organic solvent (such as acetonitrile), and carrying out a third reaction under a protective atmosphere to obtain a near-infrared II region cyanine fluorescent organic molecule with the structure shown in formula (I).

[0063] In some embodiments, the mass ratio of the first compound, the second compound, and glutaraldehyde is 500-800:400-600:300-500.

[0064] In some embodiments, the temperature of the third reaction is 120-180°C, and the time of the third reaction is 20-40 min.

[0065] Furthermore, the protective atmosphere includes, but is not limited to, a nitrogen atmosphere.

[0066] In some preferred embodiments, the preparation method further includes: after the third reaction is completed, rotary evaporation under reduced pressure, followed by adding a hexane solution of acetic anhydride and stirring for 1-2 hours to precipitate a solid, filtering, and obtaining a crude product.

[0067] In some preferred embodiments, the preparation method further includes: adding the crude product to a second mixed solvent, refluxing at 120-180°C for 3-6 hours, rotary evaporating under reduced pressure, followed by column chromatography and elution, and then rotary evaporating under reduced pressure to obtain the near-infrared II region anthocyanin fluorescent organic molecule.

[0068] Further, the second mixed solvent comprises a combination of toluene and n-butanol, wherein the volume ratio of toluene to n-butanol is 1-2:1.

[0069] Furthermore, the column chromatography uses 200-300 mesh silica gel.

[0070] In some preferred embodiments, the eluent used for elution comprises a combination of ethyl acetate and methanol, wherein the volume ratio of ethyl acetate to methanol is 3-5:1-2.

[0071] In some preferred embodiments, the pressure used for the reduced pressure rotary evaporation is -0.1 to 0.1 MPa.

[0072] In one of the more specific implementation schemes, the preparation steps of a near-infrared II cyanin-based fluorescent organic molecule, NIR-950, specifically include:

[0073] (1) Add 4.0-10.0 mL of cyclopentanone dropwise to a reaction flask containing 20 mL of concentrated sulfuric acid at 0-5℃, stir for 1-2 hours, continue to add 5-10 g of diethylamino keto acid to the reaction flask at 0-5℃, heat to 80-100℃ and stir for 1-2 hours, add 10 mL of HClO4 aqueous solution dropwise to the reaction flask, filter and dry to obtain the first compound;

[0074] (2) Add 8-10 g of 1,1,2-trimethyl-1H-benzoindole and 5-6.5 g of 1,4-butyryl lactone to a reaction flask, dissolve in 70 mL of acetonitrile, reflux at 100-200 °C for 24-48 hours, then rotary evaporate under reduced pressure. Add the crude product to a mixed solvent V. 乙醚 V DCM =5-10:1 (DCM is dichloromethane), filter and dry to obtain the second compound;

[0075] (3) Add 500-800 mg of the first compound, 400-600 mg of the second compound and 300-500 mg of glutaraldehyde to the reaction flask, add 20 mL of acetonitrile and 10 mL of acetic anhydride to dissolve, heat to 120-180℃ and reflux for 20-40 min under nitrogen atmosphere, then evaporate the solvent under reduced pressure.

[0076] (4) Add 10 mL of acetic anhydride-n-hexane solution to the product obtained in step (3) and stir for 1-2 hours. Filter the mixture, and add the crude product to a mixed solvent V. 甲苯 V 正丁醇 = 1-2:1, reflux at 120-180℃ for 3-6 hours, and rotary evaporate the solvent under reduced pressure;

[0077] (5) Perform column chromatography on the crude product obtained in step (4) (containing approximately 70g of 200-300 mesh silica gel). Elute with 900mL of eluent, wherein the eluent is composed of V EA V MeOH =3-5:1-2 (EA is ethyl acetate, MeOH is methanol) was mixed to obtain the final product. All the eluent was collected, and the solvent was removed by rotary evaporation under reduced pressure (at a pressure of -0.1-0.1 MPa) to finally obtain the organic small molecule NIR-950.

[0078] As another aspect of the technical solution of the present invention, it also relates to near-infrared II region anthocyanin fluorescent organic molecules prepared by the aforementioned preparation method.

[0079] The organic small molecule NIR-950 prepared by this invention has the advantages of large molar absorptivity, high quantum yield and low cytotoxicity; it has low biological background signal, deep tissue penetration, high spatial resolution and sensitivity, and can clearly mark tumor boundaries and tiny occult lesions.

[0080] As another aspect of the technical solution of the present invention, it also relates to a near-infrared II fluorescent targeting molecular probe (which may be referred to as NIR-950-cRGD) for tumor surgical navigation, comprising: the aforementioned near-infrared II cyanine fluorescent organic molecule as the probe body, and a targeting peptide cRGD, wherein the targeting peptide cRGD is coupled and modified on the surface of the near-infrared II cyanine fluorescent organic molecule.

[0081] Furthermore, the fluorescent targeting molecular probe NIR-950-cRGD is composed of NIR-II region cyanin-based fluorescent organic molecular probe NIR-950 coupled with the targeting peptide cRGD.

[0082] Furthermore, the targeting peptide cRGD is a targeting molecule that specifically recognizes the integrin receptor protein αvβ3 on the surface of tumor blood vessels.

[0083] Specifically, the amino acid sequence of the targeting peptide cRGD is cyclo(Arg-Gly-Asp-D-Phe-Lys).

[0084] As another aspect of the technical solution of the present invention, it also relates to a method for preparing a near-infrared II region fluorescent targeting molecular probe, which includes:

[0085] Near-infrared II region anthocyanin fluorescent organic molecules were prepared according to the aforementioned preparation method;

[0086] By modifying the surface of the near-infrared II fluorescent organic cyanide molecules with the coupling targeting peptide cRGD, a near-infrared II fluorescent targeting molecular probe is obtained.

[0087] In some embodiments, the preparation method includes:

[0088]

[0089] Near-infrared II region anthocyanin-type fluorescent organic molecules are added to a dimethyl sulfoxide solution containing N,N′-dicyclohexylcarboimide and stirred at room temperature for 3-6 hours; then a dimethyl sulfoxide solution containing N-hydroxysuccinimide (or ultra-dry acetonitrile, N,N-dimethylformamide) is added and stirred at room temperature for 8-12 hours to form a mixture;

[0090] The targeting peptide cRGD was added to the mixture and stirred at room temperature for 10-12 hours to obtain a near-infrared II fluorescent targeting molecular probe.

[0091] In some preferred embodiments, the mass ratio of the near-infrared II region cyanine fluorescent organic molecule to the target peptide cRGD is 5-10:3-5.

[0092] In some more preferred embodiments, the preparation method further includes: purifying the near-infrared II fluorescent targeting molecular probe.

[0093] In some preferred embodiments, the specific steps for obtaining the targeted molecular probe NIR-950-cRGD by modifying the surface of the probe body NIR-950 with the coupling of the targeting peptide cRGD include the following:

[0094] (1) Add 5-10 mg NIR-950 to a DMSO (dimethyl sulfoxide) solution containing 3.0 mg / mL DCC (N,N′-dicyclohexylcarboimide) and stir at room temperature for 3-5 hours;

[0095] (2) Add a DMSO (dimethyl sulfoxide) solution containing 2.0 mg / mL NHS (N-hydroxysuccinimide) to the above solution and stir at room temperature for 8-12 hours;

[0096] (3) Add 3-5 mg cRGD to a DMSO (dimethyl sulfoxide) solution containing 5-10 mg NIR-950 obtained in step (2) and stir at room temperature for 10-12 hours;

[0097] (4) Filter the solution obtained in step (3) using a 5 kDa filter membrane to remove the byproducts of the reaction;

[0098] (5) After the reaction is complete, the reaction solution can be purified by high performance liquid chromatography (HPLC) to obtain the product;

[0099] (6) The high-performance liquid chromatography (HPLC) purification is specifically performed as follows: methanol (containing 0.05% trifluoroacetic acid) and water (containing 0.05% trifluoroacetic acid) are used as eluents, with the elution gradient being 20% ​​water + 80% methanol for 10 min; 10% water + 90% methanol for 10 min; and 100% methanol for 40 min. The flow rate is 5 mL / min, and the eluent with a retention time of 27 min is collected.

[0100] (7) Collect all the eluent and remove the solvent by rotary evaporation under reduced pressure (at a pressure of 0.1 MPa) to finally obtain the targeted molecular probe NIR-950-cRGD.

[0101] Furthermore, the targeted molecular probe is used to define and remove the surgical boundaries of tumors.

[0102] Accordingly, as another aspect of the technical solution of the present invention, it also relates to the application of the near-infrared II fluorescent targeting molecular probe in the preparation of products with tumor surgical navigation function, wherein the products can specifically target and identify tumor cells.

[0103] Furthermore, the targeted molecular probe in this invention uses cyanide organic small molecules with high quantum yield coupled with the targeting peptide cRGD. By utilizing the ability of the targeting peptide cRGD to specifically recognize tumor cells, the organic small molecules are delivered to the tumor site, accurately targeting tumor cells and enhancing the probe's targeting ability to the tumor.

[0104] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0105] Unless otherwise specified, the various raw materials, reaction equipment, testing equipment and testing methods used in the following embodiments are all known in the art.

[0106] Example 1

[0107] The synthesis process of the near-infrared II region cyanin-based fluorescent organic molecule NIR-950 is as follows: Figure 1 As shown, specifically, the preparation method of the organic molecule NIR-950 includes the following steps:

[0108] 1. Add 4.0 mL of cyclopentanone dropwise to a reaction flask containing 20 mL of concentrated sulfuric acid at 0 °C, stir for 1 hour, continue to add 5 g of diethylamino keto acid to the reaction flask at 0 °C, heat to 80 °C and stir for 1 hour, add 10 mL of HClO4 aqueous solution dropwise to the reaction flask, filter and dry to obtain the first compound (which can be referred to as compound 1), with a yield of 80%.

[0109] 2. Add 10g of 1,1,2-trimethyl-1H-benzoindole and 6.5g of 1,4-butyryl lactone to a reaction flask, dissolve in 70mL of acetonitrile, reflux at 100℃ for 24 hours, then rotary evaporate under reduced pressure. Add the crude product to a mixed solvent V. 乙醚 V DCM = 5:1 (DCM is dichloromethane), filtered and dried to obtain the second compound (which can be simply referred to as compound 2), with a yield of 85%;

[0110] 3. Add 500 mg of compound 1, 400 mg of compound 2, and 300 mg of glutaraldehyde to the reaction flask, add 20 mL of acetonitrile and 10 mL of acetic anhydride to dissolve, heat to 120 °C and reflux under nitrogen atmosphere for 20 min, then evaporate the solvent under reduced pressure.

[0111] 4. Add 10 mL of acetic anhydride-hexane solution to the product obtained in step 3 and stir for 1 hour. A solid precipitates out. Filter the solid and add the crude product to a mixed solvent V. 甲苯 V 正丁醇 = 1:1, reflux at 120℃ for 3 hours, and rotary evaporate the solvent under reduced pressure;

[0112] 5. Perform column chromatography on the crude product obtained in step 4 (containing approximately 70g of 200-300 mesh silica gel). Elute with 900mL of eluent, wherein the eluent is composed of V... EA V MeOH The eluent was obtained by mixing ethyl acetate (EA) and methanol (MeOH) in a 3:1 ratio. All the eluent was collected, and the solvent was removed by rotary evaporation under reduced pressure (-0.1 MPa) to finally obtain the small organic molecule NIR-950. The identification data of the obtained product are as follows: 1 H NMR (400MHz, DMSO-d6) δ = 8.30 (d, 2H), 8.13-8.039 (m, 5H), 7.89 (d, 2H), 7.71 (dt, 2H), 7.59 (m, 4 H), 7.39 (m, 4H), 6.77 (dd, 2H), 6, 64 (s, 1H), 6.55 (d, 2H), 6.44 (t, 1H), 4.36 (s, 2H), 3.43 (d, 1H).

[0113] The 1H NMR spectrum of the synthesized organic small molecule NIR-950 in this embodiment is shown below. Figure 2 As shown, the fluorescence emission spectrum and ultraviolet absorption curve are respectively as follows: Figure 3A and Figure 3B As shown.

[0114] Example 2

[0115] The synthesis process of the near-infrared II region cyanin-based fluorescent organic molecule NIR-950 is as follows: Figure 1 As shown, specifically, the preparation method of the organic molecule NIR-950 includes the following steps:

[0116] 1. At 5°C, 10 mL of cyclopentanone was added dropwise to a reaction flask containing 20 mL of concentrated sulfuric acid and stirred for 2 hours. Then, 10 g of diethylamino keto acid was added dropwise to the reaction flask at 5°C. The mixture was heated to 100°C and stirred for 2 hours. 10 mL of HClO4 aqueous solution was added dropwise to the reaction flask. The mixture was filtered and dried to obtain the first compound (which can be referred to as compound 1) with a yield of 75%.

[0117] 2. Add 8g of 1,1,2-trimethyl-1H-benzoindole and 5g of 1,4-butyryl lactone to a reaction flask, dissolve in 70mL of acetonitrile, reflux at 200℃ for 48 hours, then rotary evaporate under reduced pressure. Add the crude product to a mixed solvent V. 乙醚 V DCM =10:1 (DCM is dichloromethane), filtered and dried to obtain the second compound (which can be simply referred to as compound 2), with a yield of 70%;

[0118] 3. Add 800 mg of compound 1, 600 mg of compound 2, and 500 mg of glutaraldehyde to the reaction flask, add 20 mL of acetonitrile and 10 mL of acetic anhydride to dissolve, heat to 180 °C and reflux under nitrogen atmosphere for 40 min, then evaporate the solvent under reduced pressure.

[0119] 4. Add 10 mL of acetic anhydride-n-hexane solution to the product obtained in step 3 and stir for 2 hours. A solid precipitates out. Filter the mixture, and add the crude product to a mixed solvent V. 甲苯 V 正丁醇 = 2:1, reflux at 180°C for 6 hours, and rotary evaporate the solvent under reduced pressure;

[0120] 5. Perform column chromatography on the crude product obtained in step 4 (containing approximately 70g of 200-300 mesh silica gel). Elute with 900mL of eluent, wherein the eluent is composed of V... EA V MeOH The eluent was obtained by mixing 5:2 (EA is ethyl acetate, MeOH is methanol), collecting all the eluent, and removing the solvent by rotary evaporation under reduced pressure (0.1 MPa) to finally obtain the organic small molecule NIR-950.

[0121] The nuclear magnetic resonance (NMR) spectrum, product identification data, fluorescence emission spectrum, and ultraviolet absorption curve of the synthesized organic small molecule NIR-950 in this embodiment are the same as those in Example 1.

[0122] Example 3

[0123] A fluorescent targeting molecular probe in the NIR II region for tumor surgical navigation, wherein the targeting molecular probe is NIR-950-cRGD, which is composed of probe NIR-950 coupled with targeting peptide cRGD. The probe is an organic small molecule NIR-950, and the targeting peptide cRGD is a targeting molecule that specifically recognizes the integrin receptor protein αvβ3 on the surface of tumor blood vessels.

[0124] The synthesis process of the targeted molecular probe NIR-950-cRGD is as follows: Figure 4 As shown, specifically, the preparation method of the targeted molecular probe NIR-950-cRGD includes the following steps:

[0125] 1. Add 10 mg NIR-950 to a DMSO (dimethyl sulfoxide) solution containing 3.0 mg / mL DCC (N,N′-dicyclohexylcarboimide) and stir at room temperature for 6 hours;

[0126] 2. Add a DMSO (dimethyl sulfoxide) solution containing 2.0 mg / mL NHS (N-hydroxysuccinimide) to the above solution and stir at room temperature for 12 hours;

[0127] 3. Add 5 mg of the targeting peptide cRGD to a DMSO (dimethyl sulfoxide) solution containing 10 mg of NIR-950 obtained in step 1, and stir at room temperature for 12 hours.

[0128] 4. Filter the solution obtained in step 3 through a 5 kDa filter membrane to remove the reaction byproducts;

[0129] 5. After the reaction is complete, the reaction solution can be purified by high performance liquid chromatography (HPLC) to obtain the product;

[0130] 6. The high-performance liquid chromatography (HPLC) purification is specifically performed as follows: methanol (containing 0.05% trifluoroacetic acid) and water (containing 0.05% trifluoroacetic acid) are used as eluents, with the elution gradient being 20% ​​water + 80% methanol for 10 min; 10% water + 90% methanol for 10 min; and 100% methanol for 40 min. The flow rate is 5 mL / min, and the eluent with a retention time of 27 min is collected.

[0131] 7. Collect all the eluent, and remove the solvent by rotary evaporation under reduced pressure (at a pressure of -0.1 MPa) to finally obtain the target molecular probe NIR-950-cRGD.

[0132] The fluorescence emission spectrum and ultraviolet absorption spectrum of the synthesized targeting molecular probe NIR-950-cRGD in this embodiment are shown below. Figure 5A and Figure 5B As shown, the fluorescence spectrum of the targeted molecular probe NIR-950-cRGD is consistent with that of the organic small molecule NIR-950, and the absorption spectrum shows that NIR-950 and cRGD are successfully coupled.

[0133] also, Figure 6 The figure shows the uptake results of the targeting molecular probe NIR-950-cRGD in this embodiment by different cells, i.e., the cell targeting verification diagram. As can be seen from the figure, tumor cells have a higher uptake capacity for the probe NIR-950-cRGD and have tumor targeting.

[0134] Example 4

[0135] The synthesis process of the targeted molecular probe NIR-950-cRGD is as follows: Figure 4 As shown, specifically, the preparation method of the targeted molecular probe NIR-950-cRGD includes the following steps:

[0136] 1. Add 5 mg of NIR-950 to an ultra-dry acetonitrile solution containing 3.0 mg / mL LDC (N,N′-dicyclohexylcarboimide) and stir at room temperature for 3 hours;

[0137] 2. Add an ultra-dry acetonitrile solution containing 2.0 mg / mL NHS (N-hydroxysuccinimide) to the above solution and stir at room temperature for 8 hours;

[0138] 3. Add 3 mg of the targeting peptide cRGD to a DMSO (dimethyl sulfoxide) solution containing 5 mg of NIR-950 obtained in step 1, and stir at room temperature for 10 hours.

[0139] 4. Filter the solution obtained in step 3 through a 5 kDa filter membrane to remove the reaction byproducts;

[0140] 5. After the reaction is complete, the reaction solution can be purified by high performance liquid chromatography (HPLC) to obtain the product;

[0141] 6. The high-performance liquid chromatography (HPLC) purification is specifically performed as follows: methanol (containing 0.05% trifluoroacetic acid) and water (containing 0.05% trifluoroacetic acid) are used as eluents, with the elution gradient being 20% ​​water + 80% methanol for 10 min; 10% water + 90% methanol for 10 min; and 100% methanol for 40 min. The flow rate is 5 mL / min, and the eluent with a retention time of 27 min is collected.

[0142] 7. Collect all the eluent and remove the solvent by rotary evaporation under reduced pressure (0.1 MPa) to finally obtain the target molecular probe NIR-950-cRGD.

[0143] The fluorescence emission spectrum and ultraviolet absorption spectrum of the target molecular probe NIR-950-cRGD synthesized in this embodiment are the same as those in Example 3.

[0144] Example 5

[0145] 1. Experimental Methods

[0146] Establishment of Hepa1-6 subcutaneous tumor model

[0147] A Hepa1-6 subcutaneous tumor model was established using female Balb / C mice (4-6 weeks old). After hair removal on the right hind limb of the mice, 100 μL of Hepa1-6 cell suspension (1 x 10⁻⁶ cells) was injected subcutaneously. 7 (cells). Seven days after inoculation, when the tumor volume reached approximately 100 mm². 3 At that time, live fluorescence imaging experiments were conducted.

[0148] 2. Preparation of injection solution

[0149] Weigh 1 mg of NIR-950-cRGD solid powder, dissolve it in serum, and prepare a 1.0 mg / mL solution.

[0150] 3. In vivo imaging

[0151] Tumor-bearing mice were randomly divided into two groups of three, and administered the drug via tail vein injection at a dose of 7 mg / kg. In vivo imaging of the tumor-bearing mice was performed using a near-infrared in vivo imaging system. Twenty-four hours later, the mice were euthanized by cervical dislocation, and heart, liver, spleen, lung, kidney, and tumor tissues were collected. Fluorescence images were then acquired using the near-infrared in vivo imaging system.

[0152] 4. Experimental Results

[0153] Figure 7 This image shows the near-infrared imaging results of Hepa1-6 subcutaneous tumor mice. Figure 7 As shown in the fluorescence imaging of the Hepa1-6 subcutaneous tumor model, obvious fluorescence signals were captured at the lesion sites in the experimental group, and the imaging boundaries were clear, indicating that the probe was significantly enriched in the Hepa1-6 subcutaneous tumor tissue.

[0154] Figure 8 Fluorescence images of the heart, liver, spleen, lung, kidney, and tumor tissues from the Hepa1-6 subcutaneous tumor model, obtained after washing and using a near-infrared in vivo imaging system. Figure 8 As shown in the comparison diagram of the biodistribution of ex vivo tissues in the Hepa1-6 subcutaneous tumor experimental group, residual fluorescence signals were captured in the liver, strong fluorescence signals were captured in the tumor tissue, and no obvious fluorescence signals were captured in the other tissues and organs. The above results indicate that the probe has high targeting to the tumor site.

[0155] Example 6

[0156] 1. Experimental Methods

[0157] Establishment of 4T1 orthotopic tumor model

[0158] A 4T1 orthotopic breast cancer model was established using female Balb / C mice (4-6 weeks old). 100 μL of 4T1 cell suspension (1 x 10⁻⁶ cells) was injected into the mammary glands of the mice. 7 (cells). Seven days after inoculation, when the tumor volume reached approximately 100 mm². 3 At that time, live fluorescence imaging experiments were conducted.

[0159] 2. Preparation of injection solution

[0160] Weigh 1 mg of NIR-950-cRGD solid powder, dissolve it in serum, and prepare a solution with a concentration of 1.0 mg / mL.

[0161] 3. In vivo imaging

[0162] Balb / C mice with an established 4T1 orthotopic tumor model were randomly divided into two groups of three. The mice were administered the drug via tail vein injection at a dose of 7 mg / kg. In vivo imaging of the tumor-bearing mice was performed using a near-infrared in vivo imaging system. Twenty-four hours later, the mice were euthanized by cervical dislocation, and heart, liver, spleen, lung, kidney, and tumor tissues were collected. Fluorescence images were obtained using the near-infrared in vivo imaging system.

[0163] 4. Experimental Results

[0164] Figure 9 This is a near-infrared imaging image of a 4T1 orthotopic tumor mouse. Figure 9 As shown, the image acquisition results of the experimental group of 4T1 orthotopic tumor mice showed that strong fluorescence signals were captured at the lesion site and the imaging boundary was clear, indicating that the probe was significantly enriched in the tumor tissue.

[0165] Figure 10 Fluorescence images of the heart, liver, spleen, lung, kidney, and tumor tissues from a 4T1 orthotopic tumor model, obtained after washing and using a near-infrared in vivo imaging system. (Example:) Figure 10 As shown in the comparison of the biodistribution of ex vivo tissues in the experimental group of 4T1 orthotopic tumor mice, residual fluorescence signals were captured in the liver, strong fluorescence signals were captured in the tumor tissue, and no obvious fluorescence signals were captured in the other tissues and organs. The above results indicate that the probe has high targeting to the tumor site.

[0166] Example 7

[0167] The targeted molecular probe NIR-950-cRGD prepared in Example 2 can be used for defining and resecting surgical margins in liver cancer. The specific steps are as follows:

[0168] NIR-950-cRGD for intraoperative fluorescent surgical navigation in liver cancer surgery

[0169] 1. A Hepa1-6-luc mouse tumor model was established. After modeling, mice were injected with the targeted molecular probe NIR-950-cRGD, and fluorescence-guided surgery was performed at the optimal imaging time (24 hours). Tumor fluorescence images were acquired using a near-infrared in vivo imaging system, and the tumor was surgically removed under near-infrared fluorescence guidance. The tumor cavity was carefully examined using a fluorescence imaging system, and any tissue with residual signal was re-excised until no obvious signal residue was observed on the wound surface. The wound was then sutured. Postoperatively, the mice were placed in an IVISLumina III small animal in vivo optical imaging system for imaging to verify that the tumor tissue had been completely removed. Histopathological analysis of the removed tumor tissue was then performed.

[0170] 2. In vitro analysis of postoperative tissue specimens: Tumor tissue was fixed in 4% paraformaldehyde at 4°C for 24 hours, followed by routine dehydration, embedding, and sectioning. The tissue sections were then subjected to H&E staining and fluorescence imaging analysis. The degree of co-localization matching between the H&E staining results and the near-infrared fluorescence indicator areas was used to determine whether a negative resection margin had been achieved. An inverted fluorescence microscope was used to image the frozen tissue sections to obtain the signal distribution of the tumor tissue and surrounding normal tissue. The degree of matching between the near-infrared fluorescence signals captured from the tumor tissue and adjacent normal tissue and the pathological diagnosis results was compared to verify the accuracy and sensitivity of near-infrared fluorescence surgical navigation.

[0171] Example 8

[0172] The targeted molecular probe NIR-950-cRGD prepared in Example 2 can be used for defining and removing surgical margins in breast cancer surgery. The specific steps are as follows:

[0173] NIR-950-cRGD for intraoperative fluorescent surgical navigation in breast cancer surgery

[0174] 1. A 4T1-luc mouse tumor model was established. After modeling, mice were injected with the targeted molecular probe NIR-950-cRGD, and fluorescence-guided surgery was performed at the optimal imaging time (24 hours). Tumor fluorescence images were acquired using a near-infrared in vivo imaging system, and the tumor was surgically removed under near-infrared fluorescence guidance. The tumor cavity was carefully examined using a fluorescence imaging system, and any tissue with residual signal was re-excised until no obvious signal residue was observed on the wound surface. The wound was then sutured. Postoperatively, the mice were placed in an IVISLumina III small animal in vivo optical imaging system for imaging to verify that the tumor tissue had been completely removed. Histopathological analysis of the removed tumor tissue was then performed.

[0175] 2. In vitro analysis of postoperative tissue specimens: Tumor tissue was fixed in 4% paraformaldehyde at 4°C for 24 hours, followed by routine dehydration, embedding, and sectioning. The tissue sections were then subjected to H&E staining and fluorescence imaging analysis. The degree of co-localization matching between the H&E staining results and the near-infrared fluorescence indicator areas was used to determine whether a negative resection margin had been achieved. An inverted fluorescence microscope was used to image the frozen tissue sections to obtain the signal distribution of the tumor tissue and surrounding normal tissue. The degree of matching between the near-infrared fluorescence signals captured from the tumor tissue and adjacent normal tissue and the pathological diagnosis results was compared to verify the accuracy and sensitivity of near-infrared fluorescence surgical navigation.

[0176] also, Figures 11A-11PThe above figures show the hematological and blood biochemical indicators of mice after intravenous injection of the targeted molecular probe NIR-950-cRGD. As can be seen from the figures, two groups of normal mice were injected with probe NIR-950-cRGD and PBS via the tail vein, respectively. Peripheral blood serum was collected from the orbital cavity on days 1 and 7. There were no significant differences in the test results of ALT, AST, UREA, CHO, TP, ALB, and GLOB in the serum of the two groups of mice. There were also no significant differences in the test results of WBC, Lymph, MCH, RBC, and HGB in whole blood, indicating that the injection of probe NIR-950-cRGD does not cause significant harm to mice.

[0177] In summary, the above embodiments of the present invention use the targeted molecular probe NIR-950-cRGD to illuminate the tumor and delineate the tumor boundary. Compared with the traditional tumor surgery process where the surgeon subjectively judges the tumor boundary, this solves the problem of finding negative surgical margins that has always troubled clinicians. In addition, the visualization of tumor boundaries guided by NIR II region fluorescence imaging can effectively improve the negative surgical margin rate, which has important clinical significance for improving patients' quality of life and prognosis.

[0178] In addition, the inventors of this case also conducted experiments with other raw materials, process operations, and process conditions described in this specification, referring to the aforementioned embodiments, and obtained relatively ideal results in all cases.

[0179] The above description is merely a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A near-infrared II region anthocyanin-based fluorescent organic molecule, characterized in that, The near-infrared II region cyanin fluorescent organic molecules have the structure shown in formula (Ⅰ): Equation (Ⅰ).

2. The method for preparing near-infrared II region cyanine fluorescent organic molecules as described in claim 1, characterized in that, include: Diethylaminoketo acid was reacted with cyclopentanone in the first reaction to prepare the first compound; The second compound was prepared by reacting 1,1,2-trimethyl-1H-benzoindole with 1,4-butyryl lactone in a second reaction. The first compound, the second compound, and glutaraldehyde were reacted in a third reaction to prepare near-infrared II region anthocyanin-based fluorescent organic molecules.

3. The preparation method according to claim 2, characterized in that, include: Add cyclopentanone to concentrated sulfuric acid at 0-5℃ and stir for 1-2 hours; Diethylaminoketo acid was added at 0-5℃, followed by heating to carry out the first reaction; After the first reaction is completed, an acidifying reagent is added, the mixture is filtered, dried, and the first compound is obtained.

4. The preparation method according to claim 3, characterized in that: The acidifying agent includes HClO4.

5. The preparation method according to claim 2, characterized in that: The mass-to-volume ratio of diethylaminoketo acid to cyclopentanone is 5-10 g: 4-10 mL.

6. The preparation method according to claim 2, characterized in that: The temperature of the first reaction is 80-100℃, and the reaction time is 1-2 hours.

7. The preparation method according to claim 2, characterized in that, The structural formula of the first compound is shown in formula (II): Formula (II).

8. The preparation method according to claim 3, characterized in that, include: 1,1,2-trimethyl-1H-benzoindole, 1,4-butyryl lactone were mixed with an organic solvent, and then heated to carry out a second reaction to obtain a second compound.

9. The preparation method according to claim 3, characterized in that: The mass ratio of 1,1,2-trimethyl-1H-benzoindole to 1,4-butyryl lactone is 8-10:5-6.

5.

10. The preparation method according to claim 3, characterized in that: The temperature of the second reaction is 100-200℃, and the reaction time is 24-48 hours.

11. The preparation method according to claim 8, characterized in that: The organic solvent includes acetonitrile.

12. The preparation method according to claim 8, characterized in that... Also includes: After the second reaction is completed, the crude product is recrystallized under reduced pressure using a first mixed solvent, filtered, and dried to obtain the second compound.

13. The preparation method according to claim 12, characterized in that: The first mixed solvent comprises a combination of diethyl ether and dichloromethane, wherein the volume ratio of the diethyl ether to dichloromethane is 5-10:

1.

14. The preparation method according to claim 3, characterized in that, The structural formula of the second compound is shown in formula (Ⅲ): Formula (III).

15. The preparation method according to claim 3, characterized in that, include: The first compound, the second compound, glutaraldehyde, and acetic anhydride as a catalyst were uniformly mixed and dissolved in an organic solvent. The third reaction was carried out under a protective atmosphere to obtain near-infrared II region cyanine fluorescent organic molecules.

16. The preparation method according to claim 3, characterized in that: The mass ratio of the first compound, the second compound, and glutaraldehyde is 500-800:400-600:300-500.

17. The preparation method according to claim 3, characterized in that: The temperature of the third reaction is 120-180℃, and the time of the third reaction is 20-40 min.

18. The preparation method according to claim 15, characterized in that: The protective atmosphere includes a nitrogen atmosphere.

19. The preparation method according to claim 15, characterized in that... Also includes: After the third reaction is completed, the mixture is rotary evaporated under reduced pressure, and then a hexane solution of acetic anhydride is added and stirred for 1-2 hours to precipitate a solid. The solid is then filtered to obtain the crude product.

20. The preparation method according to claim 19, characterized in that... Also includes: The crude product was added to the second mixed solvent and refluxed at 120-180 °C for 3-6 hours. The mixture was then subjected to rotary evaporation under reduced pressure, followed by column chromatography and elution, and then rotary evaporation under reduced pressure to obtain the near-infrared II region anthocyanin fluorescent organic molecules.

21. The preparation method according to claim 20, characterized in that: The second mixed solvent comprises a combination of toluene and n-butanol, wherein the volume ratio of toluene to n-butanol is 1-2:

1.

22. The preparation method according to claim 20, characterized in that: The column chromatography uses 200-300 mesh silica gel.

23. The preparation method according to claim 20, characterized in that: The eluent used in the elution includes a combination of ethyl acetate and methanol, wherein the volume ratio of ethyl acetate to methanol is 3-5:1-2.

24. The preparation method according to claim 20, characterized in that: The pressure used for the reduced pressure rotary evaporation is -0.1 to 0.1 MPa.

25. A near-infrared II region fluorescent targeting molecular probe, characterized in that, include: The probe consists of the near-infrared II region cyanine fluorescent organic molecule as described in claim 1, and the targeting peptide cRGD, wherein the targeting peptide cRGD is coupled and modified on the surface of the near-infrared II region cyanine fluorescent organic molecule, and the targeting peptide cRGD specifically recognizes the integrin receptor protein α on the surface of tumor blood vessels. v β3 target molecules.

26. The near-infrared II fluorescent targeting molecular probe according to claim 25, characterized in that: The amino acid sequence of the targeting peptide cRGD is cyclo(Arg-Gly-Asp-D-Phe-Lys).

27. A method for preparing a near-infrared II region fluorescent targeting molecular probe, characterized in that, include: Near-infrared II region anthocyanin fluorescent organic molecules are prepared according to the preparation method of any one of claims 2-24; By modifying the surface of the near-infrared II fluorescent organic cyanide molecules with the coupling targeting peptide cRGD, a near-infrared II fluorescent targeting molecular probe is obtained.

28. The preparation method according to claim 27, characterized in that, include: Near-infrared II region anthocyanin-based fluorescent organic molecules were added to a dimethyl sulfoxide solution containing N,N'-dicyclohexylcarboimide and stirred at room temperature for 3-6 hours; then a dimethyl sulfoxide solution containing N-hydroxysuccinimide was added and stirred at room temperature for 8-12 hours to form a mixture. The targeting peptide cRGD was added to the mixture and stirred at room temperature for 10-12 hours to obtain a near-infrared II fluorescent targeting molecular probe.

29. The preparation method according to claim 28, characterized in that: The mass ratio of the near-infrared II region cyanine fluorescent organic molecule to the target peptide cRGD is 5-10:3-5.

30. The preparation method according to claim 28, characterized in that... Also includes: The near-infrared II region fluorescent targeting molecular probe was purified.

31. The application of the near-infrared II fluorescent targeting molecular probe of claim 25 or 26 in the preparation of a product with tumor surgical navigation function, wherein the product is capable of specifically targeting and recognizing tumor cells.

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