Near-infrared second-region cyanine fluorescent organic molecule, fluorescent targeting molecular probe as well as preparation method and application of fluorescent targeting molecular probe

Abstract

The invention discloses a near-infrared second-region cyanine fluorescent organic molecule, a fluorescent targeting molecular probe as well as a preparation method and application of the near-infrared second-region cyanine fluorescent organic molecule and the fluorescent targeting molecular probe. The near-infrared second-region cyanine fluorescent organic molecule has the following structure: # imgabs0 #, the fluorescent targeting molecular probe comprises the near-infrared second-region cyanine fluorescent organic molecule as a probe main body and a targeting peptide cRGD, and the targeting peptide cRGD is coupled and modified on the surface of the near-infrared second-region cyanine fluorescent organic molecule. The near-infrared two-region cyanine fluorescent organic molecule provided by the invention has the advantages of large molar absorption coefficient, high quantum yield and low cytotoxicity, is deep in tissue penetration depth, high in spatial resolution and sensitivity, and can clearly mark tumor boundaries and tiny hidden focuses; the prepared fluorescent targeting molecular probe utilizes the specific recognition capability of the targeting peptide cRGD and tumor cells to convey small organic molecules to tumor parts, so that the tumor cells are accurately targeted, and the targeting property of the probe to tumors is enhanced.

Description

Technical Field

[0001] The present invention relates to a near-infrared zone II cyanine fluorescent organic molecule, in particular to a near-infrared zone II cyanine fluorescent organic molecule and a preparation method thereof, as well as a near-infrared zone II fluorescent targeted molecular probe for tumor surgery navigation and a preparation method and application thereof, belonging to the technical field of targeted molecular probes. Background Art

[0002] The ultimate goal of surgical resection of solid tumors is to achieve complete resection of cancerous tissue and preserve healthy tissue to the maximum extent possible, which requires doctors to accurately identify tumors (including primary tumors and metastases) and healthy tissues adjacent to the cancer (such as nerves, blood vessels, and lymph nodes) during surgery. However, the differences between tumors and healthy tissues are often not directly identifiable with the naked eye.

[0003] Fluorescence imaging technology has a series of advantages such as intuitive visualization, high sensitivity, non-contact, green and convenient. Surgical treatment based on fluorescence guidance can provide a powerful tool for the identification of tumor lesions and normal tissues. There are two optically transparent windows in the near-infrared (NIR) band in the organism, namely the near-infrared region I (NIR-I) of 700-900nm and the near-infrared region II (NIR-II) of 950-1700nm. In particular, the near-infrared region II has deeper tissue penetration (>1.5 cm) and lower light scattering, absorption and autofluorescence interference compared to traditional bands, and is expected to provide accurate imaging guidance for intraoperative treatment of tumors.

[0004] Currently clinically approved fluorescent probes such as indocyanine green ICG and methylene blue MB have problems such as nonspecific tissue uptake and easy fluorescence photobleaching, making it difficult to accurately define tumor boundaries, and some tiny hidden metastatic lesions are easily missed. Summary of the invention

[0005] The main purpose of the present invention is to provide a near-infrared second-zone cyanine fluorescent organic molecule and a preparation method thereof to overcome the deficiencies in the prior art.

[0006] Another object of the present invention is to provide a near-infrared second-region fluorescent targeting molecular probe and a preparation method thereof.

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

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

[0009] The embodiment of the present invention provides a near-infrared second-zone cyanine fluorescent organic molecule having a structure as shown in formula (I):

[0010]

[0011] The embodiment of the present invention also provides a method for preparing near-infrared second-zone cyanine fluorescent organic molecules, which comprises:

[0012] Allowing diethylamino keto acid and cyclopentanone to undergo a first reaction to obtain a first compound;

[0013] Conducting a second reaction between 1,1,2-trimethyl-1H-benzidindole and 1,4-butanesultone to obtain a second compound;

[0014] The first compound, the second compound and glutaraldehyde are subjected to a third reaction to obtain near-infrared second-zone cyanine fluorescent organic molecules.

[0015] The embodiment of the present invention also provides near-infrared second-zone cyanine fluorescent organic molecules prepared by the above-mentioned preparation method.

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

[0017] Furthermore, the targeting peptide cRGD specifically recognizes the integrin receptor protein αvβ on the surface of tumor blood vessels. 3 targeting molecules.

[0018] The embodiment of the present invention also provides a method for preparing a near-infrared second-region fluorescent targeting molecular probe, which comprises:

[0019] According to the aforementioned preparation method, a near-infrared second-zone cyanine fluorescent organic molecule is prepared;

[0020] The near-infrared zone II cyanine fluorescent organic molecule is surface-modified with a coupling targeting peptide cRGD to obtain a near-infrared zone II fluorescent targeting molecular probe.

[0021] Furthermore, an embodiment of the present invention also provides the use of the aforementioned near-infrared second-zone fluorescent targeting molecular probe in the preparation of a product with the function of tumor surgery navigation.

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

[0023] 1) The near-infrared zone II cyanine fluorescent organic molecules provided by the present invention have the advantages of large molar absorption coefficient, high quantum yield and low cytotoxicity; compared with the 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 depth, high spatial resolution and sensitivity, and can clearly mark tumor boundaries and tiny hidden lesions;

[0024] 2) The near-infrared second-zone fluorescent targeting molecular probe provided by the present invention is coupled with a cyanine organic small molecule with a high quantum yield and a targeting peptide cRGD, and utilizes the ability of the targeting peptide cRGD to specifically recognize tumor cells to deliver the organic small molecule to the tumor site, accurately target tumor cells, and enhance the targeting of the probe to the tumor. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0026] Figure 1 This is a diagram of the synthesis process of the near-infrared second-zone cyanine fluorescent organic molecule NIR-950 in Example 1 of the present invention;

[0027] Figure 2 The near-infrared second zone cyanine fluorescent organic molecule NIR-950 in Example 1 of the present invention 1 H-NMR spectrum;

[0028] Figure 3A and Figure 3B They are respectively the fluorescence emission spectrum curve and the ultraviolet absorption curve graph of the near-infrared second-zone cyanine fluorescent organic molecule NIR-950 in Example 1 of the present invention;

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

[0030] Figure 5A and Figure 5B They are respectively the ultraviolet absorption and fluorescence emission spectra of the targeted molecular probe NIR-950-cRGD in Example 3 of the present invention;

[0031] Figure 6 This is a diagram for verifying the cell targeting of the targeted molecular probe NIR-950-cRGD in Example 3 of the present invention;

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

[0033] Figure 8 This is a fluorescent image of the heart, liver, spleen, lung, kidney, and tumor tissues of the Hepa1-6 subcutaneous tumor model in Example 5 of the present invention, which was obtained by a near-infrared in vivo imaging system after washing;

[0034] Fig. 9 This is a diagram showing the acquisition results of near infrared imaging of 4T1 orthotopic tumor mice in Example 6 of the present invention;

[0035] Fig.10 This is a fluorescent image of the heart, liver, spleen, lung, kidney, and tumor tissues of the 4T1 orthotopic tumor model in Example 6 of the present invention, which is obtained by a near-infrared in vivo imaging system after washing;

[0036] Figure 11A-Figure 11P This is a graph showing the test results of hematological and blood biochemical indicators of blood collected from mouse eyeballs after tail vein injection of the targeted molecular probe NIR-950-cRGD in Example 8 of the present invention. DETAILED DESCRIPTION

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

[0038] For ease of understanding of the present application, the present application will be described in more detail below. However, it should be understood that the present invention can be implemented in many different forms and is not limited to the embodiments or examples described herein. On the contrary, the purpose of providing these embodiments or examples is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

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

[0040]

[0041] As one aspect of the technical solution of the present invention, a method for preparing a near-infrared second-zone cyanine fluorescent organic molecule involves:

[0042] Allowing diethylamino keto acid and cyclopentanone to undergo a first reaction to obtain a first compound;

[0043] Conducting a second reaction between 1,1,2-trimethyl-1H-benzidindole and 1,4-butanesultone to obtain a second compound;

[0044] The first compound, the second compound and glutaraldehyde are subjected to a third reaction to obtain near-infrared second-zone cyanine fluorescent organic molecules.

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

[0046] At 0-5°C, add cyclopentanone into concentrated sulfuric acid, which serves as a reaction solvent and catalyzes the reaction, and stir for 1-2 hours;

[0047] At 0-5°C, continue to add diethylamino keto acid, and then heat to carry out the first reaction;

[0048] After the first reaction is completed, an acidifying agent is added to form an acidifying system, and the mixture is filtered and dried to obtain a first compound.

[0049] In some embodiments, the structural formula of the first compound is as shown in Formula (II):

[0050]

[0051] In some more preferred embodiments, the mass volume ratio of 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 time of the first reaction is 1-2 hours.

[0053] Further, the acidifying agent may include HClO 4 , but it is not limited to this.

[0054] In some embodiments, the preparation method specifically comprises: mixing 1,1,2-trimethyl-1H-benzindole, 1,4-butanesultone and an organic solvent, and then heating the mixture to perform a second reaction to obtain a second compound.

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

[0056]

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

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

[0059] Further, the organic solvent may include acetonitrile, but is not limited thereto.

[0060] In some more preferred embodiments, the preparation method further comprises: after the second reaction is completed, performing rotary evaporation under reduced pressure, recrystallizing the obtained crude product with the 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 the first compound, the second compound, glutaraldehyde, and acetic anhydride (as a catalyst), dissolving them in an organic solvent (such as acetonitrile), heating them under a protective atmosphere to carry out a third reaction, and obtaining a near-infrared second-zone cyanine fluorescent organic molecule with a structure as 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 a nitrogen atmosphere, but is not limited thereto.

[0066] In some more preferred embodiments, the preparation method further comprises: after the third reaction is completed, performing rotary evaporation under reduced pressure, then adding a n-hexane solution of acetic anhydride and stirring for 1-2 hours, precipitating a solid, and filtering to obtain a crude product.

[0067] In some more preferred embodiments, the preparation method further comprises: adding the crude product to a second mixed solvent, reflux at 120-180°C for 3-6 hours, performing reduced pressure rotary evaporation, followed by column chromatography and elution, and then performing reduced pressure rotary evaporation to obtain the near-infrared second zone cyanine fluorescent organic molecule.

[0068] Furthermore, 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 more preferred embodiments, the eluent used for elution includes a combination of ethyl acetate and methanol, wherein the volume ratio of ethyl acetate to methanol is 3-5:1-2.

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

[0072] Among them, in one of some more specific embodiments, the preparation steps of a near-infrared second-zone cyanine fluorescent organic molecule NIR-950 specifically include:

[0073] (1) Add 4.0-10.0 mL of cyclopentanone to a reaction bottle containing 20 mL of concentrated sulfuric acid at 0-5°C, stir for 1-2 hours, continue to add 5-10 g of diethylamino keto acid to the reaction bottle at 0-5°C, heat to 80-100°C and stir for 1-2 hours, then add 10 mL of HClO to the reaction bottle. 4 aqueous solution, filtering and drying to obtain a first compound;

[0074] (2) Add 8-10 g 1,1,2-trimethyl-1H-benzidindole and 5-6.5 g 1,4-butanesultone to a reaction flask, add 70 mL acetonitrile to dissolve, heat to 100-200° C., reflux for 24-48 hours, and evaporate under reduced pressure. Add the crude product to the 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 a reaction bottle, add 20 mL of acetonitrile and 10 mL of acetic anhydride to dissolve, heat to 120-180° C., reflux for 20-40 min under a nitrogen atmosphere, and then evaporate the solvent under reduced pressure;

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

[0077] (5) The crude product obtained in step (4) was subjected to column chromatography (containing about 70 g of 200-300 mesh silica gel). The eluent was 900 mL, and the eluent was V EA :V MeOH =3-5:1-2 (EA is ethyl acetate, MeOH is methanol), all the eluents are collected, and the eluent is decompressed (at a pressure of -0.1-0.1 MPa) to remove the solvent by rotary evaporation, and finally the organic small molecule NIR-950 is obtained.

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

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

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

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

[0082] Furthermore, the targeting peptide cRGD specifically recognizes the integrin receptor protein αvβ on the surface of tumor blood vessels. 3 targeting molecules.

[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 second-region fluorescent targeting molecular probe, which comprises:

[0085] According to the above preparation method, prepare near-infrared second-zone cyanine fluorescent organic molecules;

[0086] The near-infrared zone II cyanine fluorescent organic molecule is surface-modified with a coupling targeting peptide cRGD to obtain a near-infrared zone II fluorescent targeting molecular probe.

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

[0088]

[0089] Adding a near-infrared second-zone cyanine fluorescent organic molecule to a dimethyl sulfoxide solution containing N, N'-dicyclohexylcarboximide, stirring at room temperature for 3-6 hours; then adding a dimethyl sulfoxide solution containing N-hydroxysuccinimide (or ultra-dry acetonitrile, N, N-dimethylformamide), stirring at room temperature for 8-12 hours to form a mixed solution;

[0090] The targeting peptide cRGD is added into the mixed solution and stirred at room temperature for 10-12 hours to obtain a near-infrared second-region fluorescent targeting molecular probe.

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

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

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

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

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

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

[0097] (4) filtering the solution obtained in step (3) using a 5KDa filter membrane to remove the by-products of the reaction;

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

[0099] (6) The high performance liquid chromatography (HPLC) purification is specifically as follows: using methanol (containing 0.05% trifluoroacetic acid) and water (containing 0.05% trifluoroacetic acid) as eluents, the elution gradient is 20% water + 80% methanol, 10 min: 10% water + 90% methanol, 10 min; 100% methanol, 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 eluate, decompress the eluate (at a pressure of 0.1 MPa) and remove the solvent by rotary evaporation to finally obtain the targeted molecular probe NIR-950-cRGD.

[0101] Furthermore, the targeted molecular probe is used for defining surgical boundaries and resection of tumors.

[0102] Correspondingly, as another aspect of the technical solution of the present invention, it also involves the use of the near-infrared second-zone fluorescent targeting molecular probe in the preparation of a product with the function of tumor surgery navigation, and the product can specifically target and identify tumor cells.

[0103] Furthermore, the targeted molecular probe of the present invention is coupled with a cyanine organic small molecule with a high quantum yield and a targeting peptide cRGD, and utilizes the ability of the targeting peptide cRGD to specifically recognize tumor cells to deliver the organic small molecule to the tumor site, accurately target tumor cells, and enhance the targeting of the probe to the tumor.

[0104] In order to make the purpose, technical solution and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

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

[0106] Example 1

[0107] The synthesis process of near-infrared second-zone cyanine fluorescent organic molecule NIR-950 is as follows Figure 1 As shown, specifically, the preparation method of the organic molecule NIR-950 comprises the following steps:

[0108] 1. Add 4.0 mL of cyclopentanone to a reaction bottle 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 bottle at 0°C, heat to 80°C and stir for 1 hour, then add 10 mL of HClO to the reaction bottle. 4 The aqueous solution was filtered and dried to obtain the first compound (referred to as compound 1) with a yield of 80%;

[0109] 2. Add 10g 1,1,2-trimethyl-1H-benzidindole and 6.5g 1,4-butanesultone to the reaction bottle, add 70mL acetonitrile to dissolve, heat to 100℃ and reflux for 24 hours, evaporate under reduced pressure, add mixed solvent V to the obtained crude product 乙醚 :V DCM =5:1 (DCM is dichloromethane), filtered and dried to obtain the second compound (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 a reaction bottle, add 20 mL of acetonitrile and 10 mL of acetic anhydride to dissolve, heat to 120 °C, reflux for 20 min under a nitrogen atmosphere, and evaporate the solvent under reduced pressure;

[0111] 4. Add 10 mL of acetic anhydride in n-hexane to the product obtained in step 3 and stir for 1 hour to precipitate a solid, filter it, and add a mixed solvent V to the obtained crude product. 甲苯 :V 正丁醇 =1:1, reflux at 120°C for 3 hours, and evaporate the solvent under reduced pressure;

[0112] 5. The crude product obtained in step 4 was subjected to column chromatography (containing about 70 g of 200-300 mesh silica gel). Elute with 900 mL of eluent, which is composed of V EA :V MeOH =3:1 (EA is ethyl acetate, MeOH is methanol), collect all the eluents, decompress the eluents (at a pressure of -0.1 MPa) and evaporate the solvent to finally obtain the organic small 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 NMR hydrogen spectrum of the organic small molecule NIR-950 synthesized in this example is as follows Figure 2 The fluorescence emission spectrum curve and the ultraviolet absorption curve are shown in Figure 3A and Figure 3B shown.

[0114] Example 2

[0115] The synthesis process of near-infrared second-zone cyanine fluorescent organic molecule NIR-950 is as follows Figure 1 As shown, specifically, the preparation method of the organic molecule NIR-950 comprises the following steps:

[0116] 1. Add 10 mL of cyclopentanone to a reaction bottle containing 20 mL of concentrated sulfuric acid at 5 °C, stir for 2 hours, continue to add 10 g of diethylamino keto acid to the reaction bottle at 5 °C, heat to 100 °C and stir for 2 hours, then add 10 mL of HClO to the reaction bottle. 4 The aqueous solution was filtered and dried to obtain the first compound (referred to as compound 1) with a yield of 75%;

[0117] 2. Add 8g 1,1,2-trimethyl-1H-benzidindole and 5g 1,4-butanesultone to the reaction bottle, add 70mL acetonitrile to dissolve, heat to 200℃ and reflux for 48 hours, evaporate under reduced pressure, add mixed solvent V to the obtained crude product 乙醚 :V DCM =10:1 (DCM is dichloromethane), filtered and dried to obtain the second compound (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 a reaction bottle, add 20 mL of acetonitrile and 10 mL of acetic anhydride to dissolve, heat to 180 °C, reflux for 40 min under a nitrogen atmosphere, and evaporate the solvent under reduced pressure;

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

[0120] 5. The crude product obtained in step 4 was subjected to column chromatography (containing about 70 g of 200-300 mesh silica gel). Elute with 900 mL of eluent, which is composed of V EA :V MeOH =5:2 (EA is ethyl acetate, MeOH is methanol), all the eluents are collected, and the eluent is subjected to reduced pressure (at a pressure of 0.1 MPa) to rotary evaporate the solvent, and finally the organic small molecule NIR-950 is obtained.

[0121] The H NMR spectrum, product identification data, fluorescence emission spectrum curve and UV absorption curve of the organic small molecule NIR-950 synthesized in this example are the same as those in Example 1.

[0122] Example 3

[0123] A NIR II region fluorescent targeting molecular probe for tumor surgical navigation, wherein the targeting molecular probe is NIR-950-cRGD, which is composed of the probe NIR-950 coupled with the targeting peptide cRGD, wherein the probe is an organic small molecule NIR-950, and the targeting peptide cRGD is a specific receptor protein for integrin αvβ on the surface of tumor blood vessels. 3 targeting molecules.

[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 comprises the following steps:

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

[0126] 2. Add 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 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 with a 5KDa filter membrane to remove the by-products of the reaction;

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

[0130] 6. The high performance liquid chromatography (HPLC) purification is specifically as follows: using methanol (containing 0.05% trifluoroacetic acid) and water (containing 0.05% trifluoroacetic acid) as eluents, the elution gradient is 20% water + 80% methanol, 10 min; 10% water + 90% methanol, 10 min; 100% methanol, 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 eluate, decompress the eluate (at a pressure of -0.1 MPa) and remove the solvent by rotary evaporation, and finally obtain the targeted molecular probe NIR-950-cRGD.

[0132] The fluorescence emission spectrum and ultraviolet absorption spectrum of the targeted molecular probe NIR-950-cRGD synthesized in this example are shown in Figures 1 and 2. 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 is successfully coupled to cRGD.

[0133] also, Figure 6 The results of uptake of the targeted molecular probe NIR-950-cRGD in this example by different cells are shown, namely, a cell targeting verification diagram. As can be seen from the diagram, 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 comprises the following steps:

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

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

[0138] 3. Add 3 mg of 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 with a 5KDa filter membrane to remove the by-products of the reaction;

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

[0141] 6. The high performance liquid chromatography (HPLC) purification is specifically as follows: using methanol (containing 0.05% trifluoroacetic acid) and water (containing 0.05% trifluoroacetic acid) as eluents, the elution gradient is 20% water + 80% methanol, 10 min; 10% water + 90% methanol, 10 min; 100% methanol, 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 eluate, 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.

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

[0144] Example 5

[0145] 1. Experimental methods

[0146] Establishment of Hepa1-6 subcutaneous tumor model

[0147] Female Balb / C mice (4-6 weeks) were used to establish the Hepa1-6 subcutaneous tumor model. After depilation of the right lower limbs of the mice, 100 μL of Hepa1-6 cell suspension (1x10 7 7 days after inoculation, when the tumor volume reached about 100 mm 3 In vivo fluorescence imaging experiments were performed.

[0148] 2. Preparation of injection solution

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

[0150] 3. In vivo imaging

[0151] The tumor-bearing mice were randomly divided into 2 groups, 3 mice in each group, and the drug was administered by tail vein injection at a dose of 7 mg / kg. The tumor-bearing mice were imaged by near-infrared in vivo imaging system. After 24 hours, the mice were killed by cervical dislocation, and the heart, liver, spleen, lung, kidney, and tumor tissues were collected, and fluorescence images were obtained by near-infrared in vivo imaging system.

[0152] 4. Experimental results

[0153] Figure 7 The following is the result of near infrared imaging 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 site of the experimental group, and the imaging boundary was clear, which indicated 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 of the Hepa1-6 subcutaneous tumor model were obtained by a near-infrared in vivo imaging system after washing. Figure 8 As shown in the in vitro tissue biodistribution comparison diagram of the Hepa1-6 subcutaneous tumor experimental group, the liver captured residual fluorescence signals, the tumor tissue captured stronger fluorescence signals, and the other tissues and organs did not capture obvious fluorescence signals. The above results indicate that the probe is highly targeted to the tumor site.

[0155] Example 6

[0156] 1. Experimental methods

[0157] Establishment of 4T1 orthotopic tumor model

[0158] Female Balb / C mice (4-6 weeks old) were used to establish a 4T1 orthotopic breast cancer model. 100 μL of 4T1 cell suspension (1x10 7 7 days after inoculation, when the tumor volume reached about 100 mm 3 In vivo fluorescence imaging experiments were performed.

[0159] 2. Preparation of injection solution

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

[0161] 3. In vivo imaging

[0162] Balb / C mice with established 4T1 orthotopic tumor models were randomly divided into 2 groups, 3 mice in each group, and the drug was injected into the tail vein at a dose of 7 mg / kg. The tumor-bearing mice were imaged in vivo using a near-infrared in vivo imaging system. After 24 hours, the mice were killed by cervical dislocation, and the heart, liver, spleen, lung, kidney, and tumor tissues were collected, and fluorescence images were obtained using a near-infrared in vivo imaging system.

[0163] 4. Experimental results

[0164] Fig. 9 The figure shows the results of near-infrared imaging of 4T1 orthotopic tumor mice. Fig. 9 As shown, the image acquisition results of the experimental group of 4T1 orthotopic tumor mice showed that a strong fluorescent signal was captured at the lesion site, and the imaging boundary was clear, indicating that the probe was significantly enriched in the tumor tissue.

[0165] Fig.10 Fluorescence images of the heart, liver, spleen, lung, kidney, and tumor tissues of the 4T1 orthotopic tumor model were obtained by a near-infrared in vivo imaging system after washing. Fig.10 As shown in the in vitro tissue biodistribution comparison diagram of the experimental group of 4T1 orthotopic tumor mice, the liver was captured with residual fluorescence signals, the tumor tissue was captured with stronger fluorescence signals, and the other tissues and organs were not captured with obvious fluorescence signals. The above results indicate that the probe is highly targeted to the tumor site.

[0166] Example 7

[0167] The targeted molecular probe NIR-950-cRGD prepared in Example 2 can be used for the definition and resection of liver cancer surgical margins, and the specific steps are as follows:

[0168] NIR-950-cRGD for fluorescence surgical navigation during liver cancer surgery

[0169] 1. Construct a Hepa1-6-luc mouse tumor model. After modeling, inject the targeted molecular probe NIR-950-cRGD into the mice, and perform fluorescence-guided surgery at the optimal development time (24 hours). Use a near-infrared in vivo imager to collect tumor fluorescence images, and perform surgery to remove the tumor under the guidance of near-infrared fluorescence. Use a fluorescence imager to carefully examine the tumor cavity, and re-remove the tissue with residual signals until no obvious signal remains on the wound surface, and suture the wound. After surgery, place the mice in the IVISLumina III small animal in vivo optical imaging system for imaging to verify that the tumor tissue has been removed cleanly, and perform histopathological analysis on the removed tumor tissue.

[0170] 2. In vitro analysis of postoperative tissue specimens: Tumor tissue was fixed with 4% paraformaldehyde at 4°C for 24 hours, and then routinely dehydrated, embedded, and sliced. Tissue sections were 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 indication area was used to determine whether a negative resection margin was achieved. An inverted fluorescence microscope was used to image frozen tissue sections to obtain the signal distribution of tumor tissue and normal tissue surrounding the tumor. The degree of matching between the fluorescence signals of tumor tissue and adjacent tissue captured by the near-infrared fluorescence signal 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 the surgical margin and resection of breast cancer. The specific steps are as follows:

[0173] NIR-950-cRGD for fluorescence surgical navigation during breast cancer surgery

[0174] 1. Construct a 4T1-luc mouse tumor model. After modeling, inject the targeted molecular probe NIR-950-cRGD into the mice, and perform fluorescence-guided surgery at the optimal development time (24 hours). Use a near-infrared in vivo imager to collect tumor fluorescence images, and perform surgery to remove the tumor under the guidance of near-infrared fluorescence. Use a fluorescence imager to carefully examine the tumor cavity, and re-remove the tissue with residual signals until no obvious signal remains on the wound surface, and suture the wound. After surgery, place the mice in the IVISLumina III small animal in vivo optical imaging system for imaging to verify that the tumor tissue has been removed cleanly, and perform histopathological analysis on the removed tumor tissue.

[0175] 2. In vitro analysis of postoperative tissue specimens: Tumor tissue was fixed with 4% paraformaldehyde at 4°C for 24 hours, and then routinely dehydrated, embedded, and sliced. Tissue sections were 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 indication area was used to determine whether a negative resection margin was achieved. An inverted fluorescence microscope was used to image frozen tissue sections to obtain the signal distribution of tumor tissue and normal tissue surrounding the tumor. The degree of matching between the fluorescence signals of tumor tissue and adjacent tissue captured by the near-infrared fluorescence signal and the pathological diagnosis results was compared to verify the accuracy and sensitivity of near-infrared fluorescence surgical navigation.

[0176] also, Figure 11A-Figure 11PThese are the identification results of hematological and blood biochemical indicators of mice after tail vein injection of targeted molecular probe NIR-950-cRGD. As can be seen from the above figures, two groups of normal mice were injected with probe NIR-950-cRGD and PBS through the tail vein, respectively, and peripheral blood serum was collected from the mice through the eye sockets on day 1 and day 7, respectively. 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 no significant differences in the test results of WBC, Lymph, MCH, RBC, HGB, etc. in the whole blood, indicating that the injection of probe NIR-950-cRGD will not cause obvious 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 outline the tumor boundary, which solves the problem of finding negative resection margins that has always troubled clinicians compared to the traditional tumor surgery process in which the surgeon subjectively judges the tumor boundary. In addition, the visualized tumor boundary under the guidance of NIR II zone fluorescence imaging can effectively improve the negative resection margin rate, which is of great clinical significance for improving the quality of life of patients and improving their prognosis.

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

[0179] The above is only a preferred specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily thought of by a person skilled in the art within the technical scope disclosed by the present invention should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope of the claims.

Claims

1. A near-infrared second-zone cyanine fluorescent organic molecule, characterized in that: The near-infrared second-zone cyanine fluorescent organic molecule has a structure as shown in formula (I):

2. A method for preparing near-infrared second-zone cyanine fluorescent organic molecules, characterized in that: include: Allowing diethylamino keto acid and cyclopentanone to undergo a first reaction to obtain a first compound; Conducting a second reaction between 1,1,2-trimethyl-1H-benzidindole and 1,4-butanesultone to obtain a second compound; The first compound, the second compound and glutaraldehyde are subjected to a third reaction to obtain near-infrared second-zone cyanine fluorescent organic molecules.

3. The preparation method according to claim 2, characterized in that: include: At 0-5°C, add cyclopentanone to concentrated sulfuric acid and stir for 1-2 hours; At 0-5°C, continue to add diethylamino keto acid, and then heat to carry out the first reaction; After the first reaction is completed, an acidifying agent is added, filtered, and dried to obtain a first compound; Preferably, the acidifying agent comprises HClO4; And / or, the mass volume ratio of the diethylamino keto acid to cyclopentanone is 5-10 g:4-10 mL; And / or, the temperature of the first reaction is 80-100° C., and the time of the first reaction is 1-2 hours; And / or, the structural formula of the first compound is as shown in formula (II):

4. The preparation method according to claim 3, characterized in that: include: Mixing 1,1,2-trimethyl-1H-benzidindole, 1,4-butanesultone and an organic solvent, and then heating to carry out a second reaction to obtain a second compound; And / or, the mass ratio of 1,1,2-trimethyl-1H-benzidindole to 1,4-butanesultone is 8-10:5-6.5; And / or, the temperature of the second reaction is 100-200° C., and the time of the second reaction is 24-48 hours; Preferably, the organic solvent comprises acetonitrile; Preferably, the preparation method further comprises: after the second reaction is completed, performing rotary evaporation under reduced pressure, recrystallizing the obtained crude product with the first mixed solvent, filtering, and drying to obtain the second compound; Particularly preferably, the first mixed solvent comprises a combination of diethyl ether and dichloromethane, wherein the volume ratio of the diethyl ether to the dichloromethane is 5-10:1; And / or, the structural formula of the second compound is as shown in formula (III):

5. The preparation method according to claim 3, characterized in that: include: The first compound, the second compound, glutaraldehyde, and acetic anhydride as a catalyst are uniformly mixed, dissolved in an organic solvent, and heated under a protective atmosphere to perform a third reaction to obtain a near-infrared second-zone cyanine fluorescent organic molecule; And / or, the structural formula of the near-infrared second-zone cyanine fluorescent organic molecule is as shown in formula (I): And / or, the mass ratio of the first compound, the second compound and glutaraldehyde is 500-800:400-600:300-500; And / or, the temperature of the third reaction is 120-180° C., and the time of the third reaction is 20-40 min; Preferably, the protective atmosphere comprises a nitrogen atmosphere; Preferably, the preparation method further comprises: after the third reaction is completed, performing rotary evaporation under reduced pressure, then adding a n-hexane solution of acetic anhydride and stirring for 1-2 hours, precipitating a solid, and filtering to obtain a crude product; Particularly preferably, the preparation method further comprises: adding the crude product to a second mixed solvent, refluxing at 120-180° C. for 3-6 hours, performing vacuum rotary evaporation, then performing column chromatography and elution, and then performing vacuum rotary evaporation to obtain the near-infrared second-zone cyanine fluorescent organic molecule; Particularly preferably, 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; Particularly preferably, the column chromatography uses 200-300 mesh silica gel; Particularly preferably, 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; Particularly preferably, the pressure used in the reduced pressure rotary evaporation is -0.1-0.1 MPa.

6. A near-infrared second-zone cyanine fluorescent organic molecule prepared by the preparation method according to any one of claims 2 to 5.

7. A near-infrared second-region fluorescent targeting molecular probe, characterized in that: include: The near-infrared zone II cyanine fluorescent organic molecule according to claim 1 or 6 as the probe body, and the targeting peptide cRGD, wherein the targeting peptide cRGD is coupled and modified on the surface of the near-infrared zone II cyanine fluorescent organic molecule; Preferably, the targeting peptide cRGD is a peptide that specifically recognizes the integrin receptor protein α on the surface of tumor blood vessels. v B3 targeting molecule; Preferably, the amino acid sequence of the targeting peptide cRGD is cyclo(Arg-Gly-Asp-D-Phe-Lys).

8. A method for preparing a near-infrared second-region fluorescent targeting molecular probe, characterized in that: include: Prepare near-infrared second-zone cyanine fluorescent organic molecules according to the preparation method described in any one of claims 2 to 5; The near-infrared zone II cyanine fluorescent organic molecule is surface-modified with a coupling targeting peptide cRGD to obtain a near-infrared zone II fluorescent targeting molecular probe.

9. The preparation method according to claim 8, characterized in that: include: Adding a near-infrared second-zone cyanine fluorescent organic molecule to a dimethyl sulfoxide solution containing N, N′-dicyclohexylcarboximide, stirring at room temperature for 3-6 hours; then adding a dimethyl sulfoxide solution containing N-hydroxysuccinimide, stirring at room temperature for 8-12 hours to form a mixed solution; Adding the targeting peptide cRGD to the mixed solution, stirring at room temperature for 10-12 hours, to obtain a near-infrared second-region fluorescent targeting molecular probe; Preferably, the mass ratio of the near-infrared second-zone cyanine fluorescent organic molecule to the targeting peptide cRGD is 5-10:3-5; Preferably, the preparation method further comprises: purifying the near-infrared second region fluorescent targeting molecular probe.

10. Use of the near-infrared second-zone fluorescent targeting molecular probe according to claim 7 in the preparation of a product with the function of tumor surgery navigation, wherein the product can specifically target and identify tumor cells.

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