CXCR4 targeted dimer molecular probe as well as preparation method and application thereof

Abstract

The invention provides a CXCR4 targeted dimer molecular probe as well as a preparation method and application thereof, and belongs to the technical field of molecular probes. The CXCR4 targeted dimer molecular probe structure shows very high receptor binding activity, can influence migration of cells and release of calcium ions, is used for experimental research of tumor imaging, improves sensitivity and specificity of tumor imaging by targeting different receptors, and is expected to play an important role in future tumor diagnosis and treatment.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular probes, and particularly relates to a CXCR4-targeted dimer molecular probe, a preparation method thereof, and an application thereof. Background Art

[0002] CXCR4 dimer molecular probes have important research value and application potential in the diagnosis and treatment of tumors. CXCR4, that is, C-X-C motif chemokine receptor 4, is a G protein-coupled receptor, which is highly expressed on the surface of a variety of tumor cells and is closely related to the malignancy of tumors and the prognosis of patients. The expression level of CXCR4 is an important biomarker in tumor treatment. Therefore, the development of molecular probes that can target CXCR4 is of great significance for the diagnosis and treatment of tumors.

[0003] In recent years, scientists have made some progress in the research of CXCR4 dimer molecular probes. For example, in a research report published in "PNAS", researchers revealed the dynamic dimerization process of CXCR4 receptors in cancer cells through advanced fluorescence microscopy technology, which provides the possibility for the development of new and efficient cancer drugs targeting CXCR4. In addition, there are also studies developing PET / CT molecular probes targeting CXCR4, and these probes have great application potential in aspects such as tumor diagnosis, case selection for CXCR4-targeted therapy, and efficacy evaluation. Summary of the Invention

[0004] The purpose of the present invention is to provide a CXCR4-targeted dimer molecular probe, a preparation method thereof, and an application thereof, which show high receptor binding activity, can affect cell migration and calcium ion release, are used for experimental research on tumor imaging, and improve the sensitivity and specificity of tumor imaging by targeting different receptors, and are expected to play an important role in future tumor diagnosis and treatment.

[0005] The technical solution of the present invention is realized as follows: The present invention provides a CXCR4-targeted dimer molecular probe having a structure shown in Formula I: Formula I.

[0006] The present invention further protects a preparation method of the above-mentioned CXCR4-targeted dimer molecular probe, including the following steps: (1) React Compd1 and Compd2 to obtain Compd3; the structural formula of Compd1 is as follows: ; the structural formula of Compd2 is as follows: ; the structural formula of Compd3 is as follows: ; (2)Compound 3 and Compound 4 react to produce Compound 5; the structural formula of Compound 4 is as follows: ; the structural formula of Compound 5 is as follows: ; (3)Compound 5 and Compound 6 react to produce a product; the structural formula of Compound 6 is as follows: .

[0007] As a further improvement of the present invention, it includes the following steps: (1)Dissolve Compound 1 in DMF, add EDC.HCl (1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride) and HOBT (3-hydroxy-1,2,3-benzotriazin-4(3H)-one) at 0 - 4 °C, react for 10 min, then add Compound 2 and NMM (N-methylmorpholine), transfer to room temperature and react for 2 h, spin dry the solvent, add 20% piperidine / DMF (dimethylformamide) solution, react at room temperature for 5 - 15 min, concentrate under reduced pressure, and purify by reverse-phase preparative liquid chromatography to obtain Compound 3; (2)Dissolve Compound 4 in DMF, add EDC.HCl and HOBT at 0 - 4 °C, react for 5 - 15 min, then add Compound 3 and NMM and transfer to room temperature to react for 1 - 3 h, spin dry the solvent, add TFA (trifluoroacetic acid) solution, react at room temperature for 2 - 7 min, add ether, precipitate a solid, centrifuge, filter dry, and purify by reverse-phase preparative liquid chromatography to obtain Compound 5; (3)Dissolve Compound 6 in DMF, add HATU (peptide coupling reagent, 2-(7-azabenzotriazol)-N,N,N',N'-tetramethyluronium hexafluorophosphate) and DIPEA (N,N-diisopropylethylamine), stir at room temperature for 2 - 7 min, add Compound 4, react at room temperature for 1 - 3 h, spin off the solvent, add TFA solution, react at room temperature for 1 - 3 h, add ether, precipitate a solid, centrifuge, filter dry, and purify by reverse-phase preparative liquid chromatography to obtain the product.

[0008] The present invention further protects the application of the above CXCR4-targeted dimer molecular probe in the preparation of drugs for the diagnosis and treatment of tumors.

[0009] The present invention further protects a preparation method of a CXCR4-targeted dimer microsphere probe, including the following steps: S1. Preparation of nano-microspheres: Add alkyl orthosilicate to ethanol, add ammonia water, heat and stir to react, centrifuge, wash, and dry to obtain nano-microspheres; S2. Preparation of surface-modified microspheres: Add nano-microspheres to water, add tannic acid and a catalyst, heat and stir to react, centrifuge, wash, and dry to obtain surface-modified microspheres; S3. Preparation of magnetic microspheres: Add the surface-modified microspheres into water, add ferric chloride and ferrous chloride, under the protection of inert gas, dropwise add ammonia water to adjust the pH value, heat and stir for reaction, separate by magnet, wash, and dry to obtain magnetic microspheres; S4. Preparation of modified magnetic microspheres: Add the magnetic microspheres into ethanol, add a composite silane coupling agent, heat and stir for reaction, centrifuge, wash, and dry to obtain modified magnetic microspheres; S5. Preparation of CXCR4-targeted dimer microsphere probe: Add the above-mentioned CXCR4-targeted dimer molecular probe into water, add NHS and EDC, carry out an activation reaction, add the modified magnetic microspheres, stir for reaction, separate by magnet, wash, and dry to obtain the CXCR4-targeted dimer microsphere probe.

[0010] As a further improvement of the present invention, in step S1, the mass ratio of the alkyl orthosilicate, ethanol, and ammonia water is 12 - 15:100:7 - 10, the alkyl orthosilicate is methyl orthosilicate or ethyl orthosilicate, the temperature of the heating and stirring reaction is 40 - 50 °C, and the time is 7 - 10 h; in step S2, the mass ratio of the nano-microspheres, tannic acid, and catalyst is 10:2 - 3:0.2 - 0.5, the catalyst is a Tris-HCl solution with pH = 8.5 - 9.5, the temperature of the heating and stirring reaction is 40 - 50 °C, and the time is 2 - 4 h.

[0011] As a further improvement of the present invention, in step S3, the mass ratio of the surface-modified microspheres, ferric chloride, and ferrous chloride is 10:3.24:1.26, the pH value is adjusted to 9 - 10, the temperature of the heating and stirring reaction is 80 - 90 °C, and the time is 3 - 5 h; in step S4, the composite silane coupling agent is selected from at least two of KH560, KH550, KH692, and KH702. Preferably, the composite silane coupling agent is KH560 and KH550, and the mass ratio is 10:4 - 5; the mass ratio of the magnetic microspheres to the composite silane coupling agent is 10:1 - 2, the temperature of the heating and stirring reaction is 35 - 45 °C, and the time is 2 - 4 h.

[0012] As a further improvement of the present invention, in step S5, the mass ratio of the CXCR4-targeted dimer molecular probe, NHS, EDC, and modified magnetic microspheres is 10:3 - 5:2 - 3:17 - 22, the temperature of the activation reaction is 0 - 4 °C, the time is 30 - 50 min, and the time of the stirring reaction is 10 - 14 h.

[0013] The present invention further protects a CXCR4-targeted dimer microsphere probe prepared by the above preparation method.

[0014] The present invention further protects the use of the above-mentioned CXCR4-targeted dimer microsphere probe in the preparation of drugs for the diagnosis and treatment of tumors.

[0015] The present invention has the following beneficial effects: The structure of the CXCR4-targeted dimer molecular probe of the present invention shows high receptor binding activity and can affect cell migration and calcium ion release. It is used in experimental studies of tumor imaging. By targeting different receptors, the sensitivity and specificity of tumor imaging are improved, and it is expected to play an important role in future tumor diagnosis and treatment.

[0016] The present invention prepared a magnetic nanosphere conjugated with a CXCR4-targeted dimer molecular probe. Using nano-silica as the carrier, after surface modification with tannic acid, the complexation of the microsphere with ferric ions and ferrous ions is improved, enabling in-situ reaction to form magnetic iron tetroxide, endowing the microsphere with magnetic resonance imaging characteristics. However, it is not easy to load the CXCR4-targeted dimer molecular probe on the surface of this magnetic microsphere, and there is a problem of low loading amount of the CXCR4-targeted dimer molecular probe.

[0017] Therefore, the present invention further modifies the surface of the prepared magnetic microsphere with KH560 and KH550, so that its surface is loaded with amino groups and epoxy groups, which can react with the activated CXCR4-targeted dimer molecular probe, greatly improving the loading amount of the CXCR4-targeted dimer molecular probe, thereby improving the detection sensitivity, having almost no cytotoxicity, high safety, good dispersibility in solution, good stability, and being able to protect the CXCR4-targeted dimer molecular probe from being easily degraded. Description of the Drawings

[0018] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0019] Figure 1 It is the mass spectrum of Compd3 in Example 1 of the present invention; Figure 2 It is the mass spectrum of Compd5 in Example 1 of the present invention; Figure 3 It is the mass spectrum of the product in Example 1 of the present invention; Figure 4 It is the 1H NMR spectrum of the product in Example 1 of the present invention Figure 5 It is the infrared spectrum of the product in Example 1 of the present invention. Detailed implementation mode

[0020] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0021] Compd1, 13-(9H-Fluoren-9-yl)-11-oxo-4,7,12-trioxa-10-azatridecanoic acid, Cas: 872679-70-4 Brand: Macklin 200 mg Purity: 98%.

[0022] Compd2, Cyclo [L-arginyl -3-(2-naphthalenyl) -L-alanylglycyl-D-tyrosyl-N2-methyl-D-ornithyl], is synthesized by the following method: Synthesis route: ; Compd4, tert -Butoxycarbonyl-L-glutamic acid, Cas: 2419-94-5 Brand: Aladdin 5 g Purity: 98%; Compd6, 1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid, 1,4,7,10-tetrakis(1,1-dimethylethyl) ester, Cas: 585531-74-4 Brand: Bide 100 mg Purity: 99%.

[0023] Example 1 This example provides a preparation method of a CXCR4-targeted dimer molecular probe, including the following steps: (1) Synthesis of Compd3: Synthesis route: ; Dissolve Compd1 (1 eq) in 10 mL of DMF. Add EDC·HCl (1 eq) and HOOBT (1 eq) at 0 °C and react for 10 min. Then add Compd2 (1 eq) and NMM (3 eq), transfer to room temperature and react for 2 h. Monitor the reaction completion by LC-MS. Rotate to dry the solvent, add 10 mL of 20% piperidine / DMF solution, react at room temperature for 10 min, concentrate under reduced pressure, and purify by reverse-phase preparative liquid chromatography to obtain Compd3 (yield: 30.70%). The mass spectrum is as shown in Figure 1 ; (2)Synthesis of Compd5: Synthetic route: ; Dissolve Compd4 (1 eq) in 10 mL of DMF. Add EDC·HCl (2.5 eq) and HOOBT (2.5 eq) at 0 °C and react for 10 min. Then add Compd3 (2.0 eq) and NMM (6 eq), transfer to room temperature and react for 2 h. Monitor the reaction completion by LC-MS. Rotate to dry the solvent, add 5 mL of TFA solution, react at room temperature for 5 min, add 50 mL of ether, a large amount of solid precipitates, centrifuge, filter, and purify by reverse-phase preparative liquid chromatography to obtain Compd5 (yield: 43.6%). The mass spectrum is as shown in Figure 2 ; (3)Synthesis of the product: Synthetic route: ; Dissolve Compd6 (1 eq) in 10 mL of DMF. Add HATU (1 eq) and DIPEA (5 eq), stir at room temperature for 5 min, add Compd4 (1 eq), and react at room temperature for 2 h. Monitor the reaction completion by LC-MS. Rotate to remove the solvent, add 10 mL of TFA solution, react at room temperature for 2 h, add 100 mL of ether, a large amount of solid precipitates, centrifuge, filter, and purify by reverse-phase preparative liquid chromatography to obtain the product (yield: 18.5%). The mass spectrum is as shown in Figure 3 . The 1H NMR spectrum is as shown in Figure 4, As can be seen from the figure, aromatic ring hydrogens are in the range of 6 - 9 ppm, usually corresponding to the hydrogen atoms on the aromatic ring. Olefinic hydrogens are in the range of 4 - 6 ppm and may appear in the region containing double bonds. Alkyl hydrogens usually appear in the 0 - 4 ppm region, representing the hydrogens on the alkyl chain. Large peak: The most prominent feature in the figure is a high peak located around approximately 5 ppm, indicating a relatively high concentration of hydrogen atoms in this chemical environment. This signal may be related to certain special structures in the molecule (such as olefins, aromatic rings, or other groups). Multiplet: Near 5 ppm, we also observe some complex peaks, which indicates the presence of coupling effects between hydrogen atoms. This is usually due to the interaction between different hydrogen atoms in the molecule (for example, neighboring hydrogen atoms coupling with each other). In multiple signal regions, especially the multiplets in the 5 - 6 ppm range, they represent the intermolecular interactions during dimer formation (such as the hydrogen nucleus coupling between two receptor units). These couplings can generate complex signals in the NMR spectrum, and in particular, the formation of dimers may lead to significant chemical shift offsets and the appearance of multiplets.

[0024] The infrared spectrum of the product is as Figure 5 , Main absorption peaks: 3396.743 cm⁻¹ This absorption peak is in the higher wavenumber region and is usually related to the stretching vibration of hydrogen bonds (such as O - H or N - H). For the CXCR4 dimer, it may represent the presence of hydroxyl or amino groups in the molecule, or the interaction between water molecules and other parts. 2935.823 cm⁻¹ This peak usually appears in the region of C - H stretching vibration, especially related to the C - H bonds of the alkyl chain or aliphatic part. It may be related to the aliphatic part of CXCR4 or the presence of alkyl chains in the ligand. 1651.120 cm⁻¹ This peak is related to the C = O stretching vibration and usually appears in peptide bonds (proteins) or certain ester groups. The CXCR dimer may contain peptide chains, and this peak can be attributed to the peptide bonds of certain amino acid residues in the dimer structure. 1546.031 cm⁻¹ This absorption peak is usually attributed to the N - H bending vibration, especially in proteins and polypeptides. It can also be related to certain groups in aromatic amines or amino acids. This peak may indicate the amino groups of certain amino acids in the CXCR4 dimer. 1396.997 cm⁻¹ This peak is usually related to the C - H bending vibration (especially the bending of methyl and methylene groups) and may be the contribution of some aliphatic parts of the molecule. 1093.454 cm⁻¹ This absorption peak is usually related to the C - O - C stretching vibration or C - O stretching vibration and may indicate the presence of ether groups, ester groups, etc. in the molecule, or be related to sugars or other organic functional groups. This infrared spectrum reveals several important functional groups in the CXCR4 dimer, especially the characteristic absorption peaks related to proteins (peptide bonds, amino acid residues), aliphatics, and other functional groups (such as ether groups).

[0025] Example 2 This embodiment provides a method for preparing a CXCR4-targeted dimer microsphere probe, comprising the following steps: S1. Preparation of nano-microspheres: Add 12 g of methyl orthosilicate to 100 g of ethanol, add 7 g of ammonia water, heat to 40 °C, stir and react for 7 h, centrifuge, wash, and dry to obtain nano-microspheres; S2. Preparation of surface-modified microspheres: Add 10 g of nano-microspheres to 200 mL of water, add 2 g of tannic acid and 0.2 g of catalyst, heat to 40 °C, stir and react for 2 h, centrifuge, wash, and dry to obtain surface-modified microspheres; The catalyst is a Tris-HCl solution with a pH of 8.5; S3. Preparation of magnetic microspheres: Add 10 g of surface-modified microspheres to 150 mL of water, add 3.24 g of ferric chloride and 1.26 g of ferrous chloride, under ammonia protection, dropwise add ammonia water to adjust the pH value to, heat to 80 °C, stir and react for 3 h, separate with a magnet, wash, dry, and calcine at 500 °C for 2 h to obtain magnetic microspheres; S4. Preparation of modified magnetic microspheres: Add 10 g of magnetic microspheres to 200 mL of ethanol, add 1 g of composite silane coupling agent, heat to 35 °C, stir and react for 2 h, centrifuge, wash, and dry to obtain modified magnetic microspheres; The composite silane coupling agent is KH560 and KH550, with a mass ratio of 10:4; S5. Preparation of CXCR4-targeted dimer microsphere probe: Add 10 g of the CXCR4-targeted dimer molecular probe prepared in Example 1 to 500 mL of water, add 3 g of NHS and 2 g of EDC, activate and react at 0 °C for 30 min, add 17 g of modified magnetic microspheres, stir and react for 10 h, separate with a magnet, wash, and dry to obtain a CXCR4-targeted dimer microsphere probe.

[0026] Example 3 This embodiment provides a method for preparing a CXCR4-targeted dimer microsphere probe, comprising the following steps: S1. Preparation of nano-microspheres: Add 15 g of tetraethyl orthosilicate to 100 g of ethanol, add 10 g of ammonia water, heat to 50 °C, stir and react for 10 h, centrifuge, wash, and dry to obtain nano-microspheres; S2. Preparation of surface-modified microspheres: Add 10 g of nano-microspheres to 200 mL of water, add 3 g of tannic acid and 0.5 g of catalyst, heat to 50 °C, stir and react for 4 h, centrifuge, wash, and dry to obtain surface-modified microspheres; The catalyst is a Tris-HCl solution with a pH of 9.5; S3. Preparation of magnetic microspheres: Add 10 g of surface-modified microspheres into 150 mL of water, add 3.24 g of ferric chloride and 1.26 g of ferrous chloride. Under ammonia protection, dropwise add ammonia water to adjust the pH value to 10, heat to 90 °C, stir and react for 5 h, separate by magnet, wash, dry, and calcine at 500 °C for 2 h to obtain magnetic microspheres; S4. Preparation of modified magnetic microspheres: Add 10 g of magnetic microspheres into 200 mL of ethanol, add 2 g of composite silane coupling agent, heat to 45 °C, stir and react for 4 h, centrifuge, wash, dry to obtain modified magnetic microspheres; The composite silane coupling agent is KH560 and KH550, and the mass ratio is 10:5; S5. Preparation of CXCR4-targeted dimer microsphere probe: Add 10 g of the CXCR4-targeted dimer molecular probe prepared in Example 1 into 500 mL of water, add 5 g of NHS and 3 g of EDC, activate and react at 4 °C for 50 min, add 22 g of modified magnetic microspheres, stir and react for 14 h, separate by magnet, wash, dry to obtain CXCR4-targeted dimer microsphere probe.

[0027] Example 4 This example provides a preparation method of a CXCR4-targeted dimer microsphere probe, including the following steps: S1. Preparation of nano-microspheres: Add 13 g of tetraethyl orthosilicate into 100 g of ethanol, add 10 g of ammonia water, heat to 50 °C, stir and react for 10 h, centrifuge, wash, dry to obtain nano-microspheres; S2. Preparation of surface-modified microspheres: Add 10 g of nano-microspheres into 200 mL of water, add 2.5 g of tannic acid and 0.35 g of catalyst, heat to 45 °C, stir and react for 3 h, centrifuge, wash, dry to obtain surface-modified microspheres; The catalyst is Tris-HCl solution with pH = 9; S3. Preparation of magnetic microspheres: Add 10 g of surface-modified microspheres into 150 mL of water, add 3.24 g of ferric chloride and 1.26 g of ferrous chloride. Under ammonia protection, dropwise add ammonia water to adjust the pH value to 9.5, heat to 85 °C, stir and react for 4 h, separate by magnet, wash, dry, and calcine at 500 °C for 2 h to obtain magnetic microspheres; S4. Preparation of modified magnetic microspheres: Add 10 g of magnetic microspheres into 200 mL of ethanol, add 1.5 g of composite silane coupling agent, heat to 40 °C, stir and react for 3 h, centrifuge, wash, dry to obtain modified magnetic microspheres; The composite silane coupling agent is KH560 and KH550, and the mass ratio is 10:4.5; S5. Preparation of CXCR4-targeted dimer microsphere probe: Add 10 g of the CXCR4-targeted dimer molecular probe prepared in Example 1 to 500 mL of water, then add 4 g of NHS and 2.5 g of EDC, and activate the reaction at 2 °C for 40 min. Add 20 g of modified magnetic microspheres, stir and react for 12 h, separate by magnet, wash, and dry to obtain the CXCR4-targeted dimer microsphere probe.

[0028] Example 5 Compared with Example 4, the difference lies in that the composite silane coupling agent is replaced by a single KH560.

[0029] Example 6 Compared with Example 4, the difference lies in that the composite silane coupling agent is replaced by a single KH550.

[0030] Comparative Example 1 Compared with Example 4, the difference lies in that step S2 is not carried out.

[0031] Specifically as follows: S1. Preparation of nano-microspheres: Add 13 g of tetraethyl orthosilicate to 100 g of ethanol, add 10 g of ammonia water, heat to 50 °C, stir and react for 10 h, centrifuge, wash, and dry to obtain nano-microspheres; S2. Preparation of magnetic microspheres: Add 10 g of nano-microspheres to 150 mL of water, add 3.24 g of ferric chloride and 1.26 g of ferrous chloride, under ammonia protection, dropwise add ammonia water to adjust the pH value to 9.5, heat to 85 °C, stir and react for 4 h, separate by magnet, wash, dry, and calcine at 500 °C for 2 h to obtain magnetic microspheres; S3. Preparation of modified magnetic microspheres: Add 10 g of magnetic microspheres to 200 mL of ethanol, add 1.5 g of composite silane coupling agent, heat to 40 °C, stir and react for 3 h, centrifuge, wash, and dry to obtain modified magnetic microspheres; The composite silane coupling agent is KH560 and KH550, and the mass ratio is 10:4.5; S4. Preparation of CXCR4-targeted dimer microsphere probe: Add 10 g of the CXCR4-targeted dimer molecular probe prepared in Example 1 to 500 mL of water, then add 4 g of NHS and 2.5 g of EDC, and activate the reaction at 2 °C for 40 min. Add 20 g of modified magnetic microspheres, stir and react for 12 h, separate by magnet, wash, and dry to obtain the CXCR4-targeted dimer microsphere probe.

[0032] Comparative Example 2 Compared with Example 4, the difference lies in that step S4 is not carried out.

[0033] Specifically as follows: S1. Preparation of nano - microspheres: Add 13 g of tetraethyl orthosilicate to 100 g of ethanol, add 10 g of ammonia water, heat to 50 °C, stir and react for 10 h, centrifuge, wash, and dry to obtain nano - microspheres; S2. Preparation of surface - modified microspheres: Add 10 g of nano - microspheres to 200 mL of water, add 2.5 g of tannic acid and 0.35 g of catalyst, heat to 45 °C, stir and react for 3 h, centrifuge, wash, and dry to obtain surface - modified microspheres; The catalyst is a Tris - HCl solution with pH = 9; S3. Preparation of magnetic microspheres: Add 10 g of surface - modified microspheres to 150 mL of water, add 3.24 g of ferric chloride and 1.26 g of ferrous chloride, under ammonia protection, dropwise add ammonia water to adjust the pH value to 9.5, heat to 85 °C, stir and react for 4 h, separate with a magnet, wash, dry, and calcine at 500 °C for 2 h to obtain magnetic microspheres; S4. Preparation of CXCR4 - targeted dimer microsphere probes: Add 10 g of the CXCR4 - targeted dimer molecular probes prepared in Example 1 to 500 mL of water, add 4 g of NHS and 2.5 g of EDC, activate and react at 2 °C for 40 min, add 20 g of magnetic microspheres, stir and react for 12 h, separate with a magnet, wash, and dry to obtain CXCR4 - targeted dimer microsphere probes.

[0034] Comparative Example 3 Compared with Example 4, the difference is that NHS and EDC were not added for activation reaction in step S5.

[0035] Specifically as follows: S1. Preparation of nano - microspheres: Add 13 g of tetraethyl orthosilicate to 100 g of ethanol, add 10 g of ammonia water, heat to 50 °C, stir and react for 10 h, centrifuge, wash, and dry to obtain nano - microspheres; S2. Preparation of surface - modified microspheres: Add 10 g of nano - microspheres to 200 mL of water, add 2.5 g of tannic acid and 0.35 g of catalyst, heat to 45 °C, stir and react for 3 h, centrifuge, wash, and dry to obtain surface - modified microspheres; The catalyst is a Tris - HCl solution with pH = 9; S3. Preparation of magnetic microspheres: Add 10 g of surface - modified microspheres to 150 mL of water, add 3.24 g of ferric chloride and 1.26 g of ferrous chloride, under ammonia protection, dropwise add ammonia water to adjust the pH value to 9.5, heat to 85 °C, stir and react for 4 h, separate with a magnet, wash, dry, and calcine at 500 °C for 2 h to obtain magnetic microspheres; S4. Preparation of modified magnetic microspheres: Add 10 g of magnetic microspheres into 200 mL of ethanol, add 1.5 g of composite silane coupling agent, heat to 40 °C, stir and react for 3 h, centrifuge, wash, and dry to obtain modified magnetic microspheres; The composite silane coupling agent is KH560 and KH550, and the mass ratio is 10:4.5; S5. Preparation of CXCR4-targeted dimer microsphere probe: Add 10 g of the CXCR4-targeted dimer molecular probe prepared in Example 1 into 500 mL of water, add 20 g of modified magnetic microspheres, stir and react for 12 h, separate with a magnet, wash, and dry to obtain the CXCR4-targeted dimer microsphere probe.

[0036] Test Example 1 Targeted magnetic resonance imaging at the cell line level Take the CXCR4-integrin bispecific targeted molecular magnetic microsphere probe prepared in Examples 2-6 or Comparative Examples 1-3 and prepare a 10 μg / mL solution with PBS solution. The experimental cell lines are: human pancreatic cancer cell line PANC-1; human lung cancer cell line A549.

[0037] Select cells in the logarithmic growth phase, routinely digest the cells according to the subculture method, add culture medium and blow evenly to make a single-cell suspension. Take the cell suspension and adjust the cell concentration to 5 - 9×10 4 cells / mL. Gently add the cell suspension along the wall of the 96-well plate and place it in a CO 2 incubator for culture. When the cells grow to 70 - 80% confluence at the bottom of the wells, proceed to the next experiment.

[0038] Take out the 96-well plate, observe under an inverted microscope, select the wells with good cell growth status and uniform density, aspirate the culture medium, and add PBS solution for washing. Add 200 μL of CXCR4-integrin bispecific targeted molecular magnetic microsphere probe solutions with concentrations of 10 μg / mL to each well, and select two wells for the negative control to add PBS. Incubate in a 4 °C refrigerator for 2 h. Take out, wash the cells, add 200 μL of digestive solution and let stand for 10 min, pipette repeatedly until the cells are almost completely detached, and collect the cell suspension (containing microsphere probe) into numbered EP tubes. Place the above EP tubes on a test tube rack above a cryotube box filled with water so that the cell suspension in the EP tubes is below the water surface.

[0039] On a Signal Excite HD 1.5T superconducting magnetic resonance machine, scan with a knee coil. The scanning sequences include fast spin echo T2WI sequence, fast gradient echo T2* sequence, T2 map sequence and sequence. The scanning parameters are set as follows: 1. FSE T2WI imaging parameters: repetition time 1500 ms, echo time 62.3 ms, slice thickness 2.5 mm, slice gap 0.2 mm, number of excitations 1, matrix 256×192, field of view 18 cm×18 cm.

[0040] 2. FGRE T2* imaging parameters: repetition time 7.3 ms, echo time 2.8 ms, slice thickness 1.0 mm, slice gap 5.0 mm, number of excitations 1, matrix 256×128, field of view 18 cm×18 cm.

[0041] 3. T2 map repetition time 1800 ms, echo time 15.1 ms, slice thickness 2.5 mm, slice gap 0.5 mm, number of excitations 1, matrix 196×160, field of view 18 cm×13.5 cm.

[0042] 4. T2* map repetition time 42.5 ms, echo time 2.1 ms, slice thickness 2.5 mm, slice gap 0.5 mm, number of excitations 2, matrix 256×192, field of view 18 cm×18 cm.

[0043] On the GE SUN ADW 4.2 workstation, the T2map and T2*map sequence images were imported into the software, and the T2 and T2* values of different concentration gradient solutions were measured. According to the specific attenuation of the measured values, the number of echoes was selected. For the T2 value, ten echoes were used, and for the T2* value, three echoes were used. The area of the region of interest for the T2 value and T2* value was 22.0 mm 2 .

[0044] Calculation of the enhancement rate (MR) of the T2 value: MR = (Mo - Ms) / Mo×100%, where Mo is the T2 value of cells and agarose, and Ms is the T2 value after the probe is incubated with cells.

[0045] Calculation of the enhancement rate (MR) of the T2* value: MR* = (Mo* - Ms*) / Mo*×100%, where Mo* is the T2* value of cells and agarose, and Ms* is the T2* value after the probe is incubated with cells. The results are shown in Table 1.

[0046] Table 1 ; As can be seen from the above table, after the CXCR4-targeted dimer microsphere probe prepared in Examples 2-4 of the present invention binds to tumor cells, the enhancement rates of T2 and T2* are high, and the T2 and T2* signal values are high.

[0047] Test Example 2 CCK-8 eukaryotic cell cytotoxicity detection A CXCR4-targeted dimer microsphere probe solution prepared in Examples 2-6 or Comparative Examples 1-3 at a concentration of 50 μg / mL was prepared with PBS solution.

[0048] Digest, centrifuge, and count HaCaT or NIH-3T3 cells, and seed them into a 96-well plate at a density of 2×10 4 / well. Incubate at 37°C until the cell confluence rate reaches 70%-80%. Then, add 200 μL of serum-free DMEM medium (MEM medium for HaCaT cells) to the tube, add 10 μL of the CXCR4-targeted dimer microsphere probe solution, incubate at 37°C for 2 h, add DMEM containing 20% fetal bovine serum, mix well, and transfer to the 96-well plate. Continue to culture for 24 h, add CCK-8 solution, incubate at 37°C for 1.5 h, and measure the D 450 nm value. Use the blank group (deionized water) as the control group and calculate the relative cell viability (%). The results are shown in Table 2.

[0049] Table 2 Group Relative cell viability of HaCaT cells (%) Relative cell viability of NIH-3T3 cells (%) Blank group 100 100 Example 2 111 110 Example 3 112 111 Example 4 114 112 Example 5 107 103 Example 6 106 102 Comparative example 1 105 101 Comparative example 2 98 96 Comparative example 3 102 100 As can be seen from the above table, the CXCR4-targeted dimer microsphere probe prepared in Examples 2-4 of the present invention has almost no cytotoxicity and high safety.

[0050] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A CXCR4-targeted dimer molecular probe, characterized in that: Having the structure shown in Formula I: Formula I.

2. A method for preparing a CXCR4-targeted dimer molecular probe as claimed in claim 1, characterized in that: The following steps are involved: (1) Compd1 and Compd2 react to obtain Compd3; the structural formula of Compd1 is as follows: ; The structural formula of the Compd2 is as follows: ; The structural formula of the Compd3 is as follows: ; (2) Compd3 and Compd4 react to obtain Compd5; the structural formula of Compd4 is as follows: ; The structural formula of the Compd5 is as follows: ; (3) Compd5 and Compd6 react to obtain a product; the structural formula of Compd6 is as follows: .

3. The preparation method according to claim 2, characterized in that: The following steps are involved: (1) Compd1 was dissolved in DMF, EDC.HCl and HOOBT were added at 0-4°C, and the reaction was continued for 10 min. Compd2 and NMM were then added, and the reaction was continued at room temperature for 2 h. The solvent was then dried, and 20% piperidine / DMF solution was added, and the reaction was continued at room temperature for 5-15 min. The product was concentrated under reduced pressure, and purified by reverse phase preparative liquid chromatography to obtain Compd3. (2) Compd4 was dissolved in DMF, EDC.HCl and HOOBT were added at 0-4°C, the reaction was continued for 5-15 min, Compd3 and NMM were added, the reaction was continued at room temperature for 1-3 h, the solvent was dried, TFA solution was added, the reaction was continued at room temperature for 2-7 min, ether was added, solid was precipitated, centrifuged, dried, and purified by reverse phase preparative liquid phase to obtain Compd5; (3) Dissolve Compd6 in DMF, add HATU and DIPEA, stir at room temperature for 2-7 min, add Compd4, react at room temperature for 1-3 h, remove the solvent by vortexing, add TFA solution, react at room temperature for 1-3 h, add ether, precipitate solid, centrifuge, drain, and purify the product by reverse phase preparative liquid phase.

4. Use of the CXCR4-targeted dimer molecular probe as claimed in claim 1 in the preparation of drugs for diagnosis and treatment of tumors.

5. A method for preparing a CXCR4-targeted dimer microsphere probe, characterized in that: The following steps are involved: S1. Preparation of nanospheres: adding alkyl orthosilicate to ethanol, adding ammonia water, heating and stirring to react, centrifuging, washing, and drying to obtain nanospheres; S2. Preparation of surface-modified microspheres: adding nano-microspheres to water, adding tannic acid and a catalyst, heating and stirring to react, centrifuging, washing, and drying to obtain surface-modified microspheres; S3. Preparation of magnetic microspheres: adding the surface-modified microspheres to water, adding ferric chloride and ferrous chloride, adding ammonia water to adjust the pH value under the protection of inert gas, heating and stirring the reaction, separating with a magnet, washing, and drying to obtain magnetic microspheres; S4. Preparation of modified magnetic microspheres: adding magnetic microspheres to ethanol, adding a composite silane coupling agent, heating and stirring the reaction, centrifuging, washing, and drying to obtain modified magnetic microspheres; S5. Preparation of CXCR4-targeted dimer microsphere probe: Add the CXCR4-targeted dimer molecular probe described in claim 1 into water, add NHS and EDC, activate the reaction, add modified magnetic microspheres, stir the reaction, separate with a magnet, wash, and dry to obtain the CXCR4-targeted dimer microsphere probe.

6. The preparation method according to claim 5, characterized in that: The mass ratio of the alkyl orthosilicate, ethanol and ammonia water in step S1 is 12-15:100:7-10, the alkyl orthosilicate is methyl orthosilicate or ethyl orthosilicate, the temperature of the heating and stirring reaction is 40-50°C, and the time is 7-10h; the mass ratio of the nanospheres, tannic acid and catalyst in step S2 is 10:2-3:0.2-0.5, the catalyst is a Tris-HCl solution with a pH of 8.5-9.5, the temperature of the heating and stirring reaction is 40-50°C, and the time is 2-4h.

7. The preparation method according to claim 5, characterized in that: The mass ratio of the surface modified microspheres, ferric chloride and ferrous chloride described in step S3 is 10: 3.24:1.26, the pH value is adjusted to 9-10, the temperature of the heating and stirring reaction is 80-90°C, and the time is 3-5h; the composite silane coupling agent in step S4 is selected from at least two of KH560, KH550, KH692, and KH702; the mass ratio of the magnetic microspheres to the composite silane coupling agent is 10:1-2, the temperature of the heating and stirring reaction is 35-45°C, and the time is 2-4h.

8. The preparation method according to claim 5, characterized in that: In step S5, the mass ratio of the CXCR4-targeted dimer molecular probe, NHS, EDC, and modified magnetic microspheres is 10:3-5:2-3:17-22, the activation reaction temperature is 0-4°C, the time is 30-50 min, and the stirring reaction time is 10-14 h.

9. A CXCR4-targeted dimer microsphere probe prepared by the preparation method according to any one of claims 5 to 8.

10. Use of the CXCR4-targeted dimer microsphere probe as claimed in claim 9 in the preparation of drugs for diagnosis and treatment of tumors.

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