Multifunctional probe based on protein dynamic binding and preparation and application thereof

Abstract

The invention provides a series of cyanine or cyanine-like multifunctional probe molecules which are derivatives with a near-infrared cyanine dye molecular skeleton, and the skeleton is modified with any one or a combination of more than two of a protein affinity agent structure, a targeting group or a functional group; the near-infrared cyanine dye molecule is a near-infrared indole cyanine dye molecule or a near-infrared naphthopyrrole cyanine or flavonoid dye molecule; the structure of the protein affinity agent is from a 4 (p-iodophenyl) butyric acid derivative, Evans blue or a derivative thereof; the targeting group is a structure containing any one pharmaceutically acceptable targeting peptide; the functional group comes from any nuclide chelating agent or a functional drug molecule with a modification site. The multifunctional probe molecule provided by the invention can realize clear near-infrared first-region and second-region lymphatic imaging and angiography, and has good biological safety and adjustable circulation metabolism time at the same time. The invention also provides a method for preparing the multifunctional probe molecule and application of the multifunctional probe molecule in multi-mode diagnosis and treatment.

Description

Technical Field

[0001] The present invention relates to the technical field of fluorescent probes, and particularly to a multifunctional fluorescent probe having a dynamic binding ability with proteins, its preparation method, and its application in multimodal diagnosis and treatment. Background Art

[0002] With the continuous progress in the medical field, the rise of multimodal fluorescent probe technology has provided innovative solutions for clinical treatment.

[0003] As a non-invasive, highly sensitive imaging and treatment means, fluorescent probe technology has gradually become one of the key technologies in clinical medicine. Its advantages are mainly reflected in high-resolution imaging, real-time monitoring, low toxicity, and no radiation exposure. Such technologies help to more clearly observe tissue structures and abnormal lesions in clinical diagnosis and treatment, contribute to early diagnosis and treatment, and provide timely information for clinical decision-making. At the same time, fluorescent probes usually have low toxicity, reducing side effects on patients, making it a relatively safe imaging and treatment method. These advantages make fluorescent probe technology a core component of the multimodal platform, providing comprehensive information support for clinical practice. Among many fluorescent probes, near-infrared (Near Infrared, NIR, 700-1700 nm) fluorescence imaging technology has unique advantages in multimodal imaging. In this light region, the absorption and scattering of biological tissues are relatively low, and this technology can achieve multi-scale tracer imaging from individual organelles to the whole organism, which enables NIR probes to achieve deeper penetration. This makes it possible to obtain high-resolution imaging of deep tissues and makes it an ideal choice for fluorescence imaging. By integrating NIR probes into the multimodal platform, we can obtain biological information more comprehensively and accurately during the imaging process, providing a more reliable diagnostic basis for doctors.

[0004] Near-infrared fluorescent probes can not only be used for imaging but also have the potential for photodynamic therapy. This treatment method activates the fluorescent probe to release reactive oxygen species, triggering apoptosis or death of tumor cells, thus achieving precise treatment of cancer. Photodynamic therapy is a highly promising cancer treatment method. Its core lies in delivering photosensitizers directly to the tumor site, and then generating highly cytotoxic reactive oxygen species, such as singlet oxygen, under light irradiation. These reactive oxygen species can oxidize key macromolecules in tumor cells, ultimately leading to the death of tumor cells. Compared with traditional treatment methods, photodynamic therapy exhibits a series of significant advantages, including low invasiveness, repeatability, and no cumulative toxicity. Its low invasiveness makes photodynamic therapy a relatively mild treatment method, reducing the discomfort of patients and also helping to protect surrounding normal tissues. The repeatability of this treatment method allows patients to receive multiple rounds of treatment, thus better coping with the complexity of cancer. Compared with the possible cumulative toxicity caused by some traditional treatments, photodynamic therapy does not leave accumulated harmful substances in the body, further reducing the risk during the treatment process. In practical applications, photodynamic therapy has shown obvious effectiveness in the treatment of various cancers, such as bladder cancer, lung cancer, esophageal cancer, head and neck cancer, and skin cancer, etc. This indicates its wide applicability in various cancer types. At the same time, such local treatment methods can be non-invasive, maximizing the protection of surrounding normal tissues, reducing the side effects of treatment, and the pain and recovery time of patients. Materials that can be used for near-infrared imaging are roughly divided into two categories: inorganic materials and organic materials. Due to the good biocompatibility, easy metabolism, and less residue of near-infrared organic fluorescent materials, they have received extensive attention from researchers in recent years. Based on different mechanisms and luminescent structures, a variety of organic fluorescent materials have been developed. Among them, cyanine dyes stand out because of their adjustable emission wavelength, low synthesis difficulty, and ultra-bright luminescence performance. However, the regular conjugated structure makes most cyanine dyes have poor water solubility and are prone to aggregation-caused quenching (ACQ) in water, significantly affecting the biocompatibility of these dyes and the imaging quality when used for in vivo monitoring.

[0005] Currently, most researchers overcome this problem through the following two methods. The most common method is to encapsulate hydrophobic dyes in phospholipid polyethylene glycol (mPEG-DSPE) or other water-soluble polymers to form hydrophilic nanoparticles; or directly connect one or more hydrophilic polyethylene glycol (PEG) chains to the hydrophobic dyes, so that the modified amphiphilic dyes can self-assemble into nanoparticles in buffer solutions or biological fluid environments. However, this strategy can only retain partial brightness of the dyes. Meanwhile, with the in-depth research, the sensitization problem of PEG has been revealed, which makes its biosafety highly questionable. Another very effective improvement strategy is to load or encapsulate dyes with proteins to form stable complexes, thereby alleviating the inherent fluorescence quenching phenomenon of the dyes. Among them, albumin has the characteristics of being rich in blood and easily binding to poorly soluble exogenous substances, and has been widely used as a drug delivery carrier. Previous patent content has fully reported that many cyanine dyes can rapidly bind to endogenous albumin, and the formed stable complexes can greatly improve the dye brightness and alleviate the photobleaching problem of the dyes. Although this method greatly improves the biocompatibility of the dyes and prolongs the circulation time of the probes in vivo, due to the widespread distribution of albumin in organisms, this irreversible covalent binding between the dyes and albumin inevitably leads to the long-term retention and systemic non-specific distribution of dye molecules in vivo, which severely limits its application in many clinical scenarios that require long-term monitoring. In addition, the fixed circulation half-life of albumin in the organism determines that the dyes have a fixed retention time in vivo, which makes it impossible for us to freely regulate the circulation half-life and metabolism time of the dyes as needed. Therefore, developing a series of cyanine dyes with adjustable circulation time and high brightness is the key to solving the above problems.

[0006] Meanwhile, by further integrating functional structures such as targeting structures, specific drug molecules, and metal chelator structures, the multimodal platform can simultaneously possess the high-precision quantitative imaging of PET imaging and the composite imaging of fluorescence imaging, further improving the imaging quality. By integrating means such as photodynamic / photothermal therapy and drug combination therapy of fluorescent probes in the multimodal platform, targeted and highly efficient treatment of tumors can be achieved while achieving accurate diagnosis, providing a more comprehensive and personalized treatment plan for patients. Summary of the Invention

[0007] The present invention aims to provide a series of synthetic and modification routes and applications of novel cyanine / cyanine-like multifunctional probe molecules. The multifunctional probe molecules mentioned in the present invention can achieve dynamic binding between the dye and albumin through different molecular modification means, thereby regulating the lymph node grading lighting time window and blood circulation time of the dye; at the same time, after further modifying the dye with a targeting group, it exhibits a highly specific tumor targeting effect; at the same time, the multifunctional probe molecule has photothermal and photodynamic therapy effects, and can further modify functional groups such as radionuclide chelators to realize the construction of an optical / PEG multimodal diagnosis and treatment integrated probe platform.

[0008] To achieve the above object, the present invention provides the following technical solutions:

[0009] A series of cyanine or cyanine-like multifunctional probe molecules, which are derivatives with a near-infrared cyanine dye molecule backbone, and any one or a combination of two or more of a protein affinity structure, a targeting group or a functional group is modified on the backbone; the near-infrared cyanine dye molecule can be a near-infrared indole cyanine dye molecule or a near-infrared naphthopyrrole cyanine or flavonoid dye molecule; the protein affinity structure can be derived from 4-(p-iodophenyl)butyric acid derivative, Evans blue or its derivative; the targeting group can be a structure containing any pharmaceutically acceptable targeting peptide; the functional group can be derived from any radionuclide chelator or a functional drug molecule with a modification site.

[0010] In the solution of the present invention, the protein affinity structure is a structure that has an affinity for multiple proteins; further, the protein is preferably albumin; more preferably, the albumin is any one of bovine serum albumin and human serum albumin. In a preferred embodiment of the present invention, the multifunctional probe molecule is a derivative with a near-infrared cyanine dye molecule backbone, and any one or a combination of two or more of a protein affinity structure, a targeting group or a functional group is modified on the backbone; the near-infrared cyanine dye molecule can be a near-infrared indole cyanine dye molecule or a near-infrared naphthopyrrole cyanine or flavonoid dye molecule; the protein affinity structure is derived from 4-(p-iodophenyl)butyric acid derivative; the targeting group can be a structure containing any pharmaceutically acceptable targeting peptide; the functional group can be any radionuclide chelating group.

[0011] In a preferred embodiment of the present invention, the multifunctional probe molecule is a derivative having a near-infrared cyanine dye molecule skeleton, and any one of a protein affinity agent structure, a targeting group or a functional group is modified on the skeleton; the near-infrared cyanine dye molecule may be a near-infrared indole-based cyanine dye molecule or a near-infrared naphthopyrrole-based cyanine or flavonoid dye molecule; the protein affinity agent structure is derived from 4-(p-iodophenyl)butyric acid derivative; the targeting group may be a structure containing any pharmaceutically acceptable targeting peptide; the functional group may be any radionuclide chelating group.

[0012] In a preferred embodiment of the present invention, the multifunctional probe molecule is a derivative having a near-infrared cyanine dye molecule skeleton, and any two of a protein affinity agent structure, a targeting group or a functional group are simultaneously modified on the skeleton; the near-infrared cyanine dye molecule may be a near-infrared indole-based cyanine dye molecule or a near-infrared naphthopyrrole-based cyanine or flavonoid dye molecule; the protein affinity agent structure is derived from 4-(p-iodophenyl)butyric acid derivative; the targeting group may be a structure containing any pharmaceutically acceptable targeting peptide; the functional group may be any radionuclide chelating group.

[0013] In a preferred embodiment of the present invention, the multifunctional probe molecule is a derivative having a near-infrared cyanine dye molecule skeleton, and a protein affinity agent structure, a targeting group and a functional group are simultaneously modified on the skeleton; the near-infrared cyanine dye molecule may be a near-infrared indole-based cyanine dye molecule or a near-infrared naphthopyrrole-based cyanine or flavonoid dye molecule; the protein affinity agent structure is derived from 4-(p-iodophenyl)butyric acid derivative; the targeting group may be a structure containing any pharmaceutically acceptable targeting peptide; the functional group may be any radionuclide chelating group.

[0014] In a preferred embodiment of the present invention, the multifunctional probe molecule is a derivative having a near-infrared cyanine dye molecule skeleton, and a protein affinity agent structure and a targeting group are simultaneously modified on the skeleton; the near-infrared cyanine dye molecule is a near-infrared indole-based cyanine dye molecule or a near-infrared naphthopyrrole-based cyanine or flavonoid dye molecule; the protein affinity agent structure is derived from 4-(p-iodophenyl)butyric acid derivative; the targeting group may be a structure containing any pharmaceutically acceptable targeting peptide.

[0015] In a preferred embodiment of the present invention, the multifunctional probe molecule is a derivative having a near-infrared cyanine dye molecule backbone, and a protein affinity agent structure and a functional group are simultaneously modified on the backbone; the near-infrared cyanine dye molecule is a near-infrared indole cyanine dye molecule or a near-infrared naphthopyrrole cyanine or flavonoid dye molecule; the protein affinity agent structure is derived from 4-(p-iodophenyl)butyric acid derivative; the functional group can be any one of radionuclide chelating groups.

[0016] In a preferred embodiment of the present invention, the targeting group is a structure containing a targeting peptide having a targeting effect on any one of the following targets: PSMA, integrin, SSTR, EGFR, HER2, VEGFR, PD-1, PD-L1, CD20, CDK4 / 6, BRAF, ALK, RET, CD33, CDK, FLT3, KIT, ROS1, MET, PARP, CD19, GRPR, CC2KR, CXCR4, uPAR, etc.; preferably, it contains a structure of any one of the following targeting peptides: RGD, PSMA, TATE or NGR.

[0017] Further, in addition to the targeting peptide structure, the targeting group may further contain a linker structure (Linker).

[0018] In a preferred embodiment of the present invention, the radionuclide chelating group is derived from any one of the following radionuclide chelating agents: NOTA, DOTA, CB-TE2A, HBED, PSMA-11, FACBC, FDG, FES, FDOPA, FMISO, etc.; preferably, it is derived from NOTA or DOTA.

[0019] In a preferred embodiment of the present invention, the functional drug molecule having a modification site is selected from: Ibuprofen, Lisinopril, Metformin, Omeprazole, Clopidogrel, Metoprolol, Levothyroxine, Fluoxetine, Warfarin, Imatinib Mesylate, Trastuzumab, Cisplatin, Tamoxifen, Paclitaxel, Dasatinib, Lapatinib, Bortezomib, Sorafenib, Trametinib, Pembrolizumab, Atezolizumab, Ibrutinib, Everolimus, Vemurafenib, etc.

[0020] The preferred multifunctional probe molecule of the present invention has the following structures shown in Formula I, II or III:

[0021]

[0022] Among them, the X group can be a halogen or any one of the following structures:

[0023]

[0024]

[0025]

[0026] R 1 has one of the following structures:

[0027]

[0028] where n is an integer from 0 to 20, preferably an integer from 0 to 10, more preferably 1 to 5, and still more preferably an integer from 2 to 5; R 2 has one of the following structures:

[0029]

[0030] where n is an integer from 0 to 20, preferably an integer from 0 to 10, more preferably 1 to 5, and still more preferably an integer from 2 to 5;

[0031] and when X is a halogen, R1 and R2 are not both -OH at the same time.

[0032] A further preferred multifunctional probe molecule of the present invention has a structure as shown in formula I, where X is a halogen, Ac, IP-S, RGD or PSMA; and when X is a halogen, R1 and R2 are not both -OH at the same time.

[0033] A further preferred multifunctional probe molecule of the present invention has a structure as shown in formula I, where X is a halogen, Ac, IP-S, RGD or PSMA, and R1 is -OH, IP or NOTA; and when X is a halogen, R1 and R2 are not both -OH at the same time.

[0034] A further preferred multifunctional probe molecule of the present invention has a structure as shown in formula I, where X is a halogen, Ac, IP-S, RGD or PSMA, and R1 is -OH, IP or NOTA; R2 is -OH, IP or Evans blue; and when X is a halogen, R1 and R2 are not both -OH at the same time.

[0035] A further preferred multifunctional probe molecule of the present invention has a structure as shown in formula I, where X is a halogen, Ac, IP-S, or RGD, and R1 is -OH or IP; R2 is -OH or IP; and when X is a halogen, R1 and R2 are not both -OH at the same time.

[0036] The multifunctional probe molecule described in the present invention can be prepared through a general modification route of near-infrared cyanine dyes. The modification route includes modifying near-infrared cyanine dyes with protein affinity agents represented by 4-(p-iodophenyl)butyric acid derivatives, modifying near-infrared cyanine dyes with mercapto-containing amino acids, mercapto-containing functional peptide segments or other drug molecules with modification sites, and / or modifying near-infrared cyanine dyes with functional groups such as NOTA, DOTA, CB-TE2A, HBED, PSMA-11, FACBC, FDG, FES, FDOPA or FMISO.

[0037] As a protein non-covalent binding site (Albumin binding moiety, ABM), the above 4-(p-iodophenyl)butyric acid derivative can dynamically carry albumin, realizing the tunability of the circulation time of dye molecules for in vivo bioimaging.

[0038] When the near-infrared cyanine dye is a protein-binding dye such as IR-808, IR-780, IR-783, IR-820, etc., shielding its halogen sites with the above-mentioned functional peptide segment or other drug molecules with modification sites can effectively prevent the binding of dye molecules to albumin. When the molecule retains halogen sites, the molecule can covalently bind to albumin, but the binding ability is affected by other substitution structures on the molecule; when the halogen sites of the molecule are replaced by any mercapto-containing amino acid or mercapto-containing functional polypeptide or its derivative, the molecule does not covalently bind to albumin at all, and its interaction with the protein is completely determined by the protein affinity structure. The dynamic binding ability between the molecule and the protein is affected by the number of protein affinity agents and the strength of the interaction with the protein. At this time, the multifunctional probe molecule has the non-covalent dynamic binding ability of albumin. This method hardly loses the brightness and wavelength of the dye, and can achieve high-resolution and residue-free vascular imaging. The modified dye molecule can accumulate in a specific target environment to achieve specific imaging of tumors.

[0039] Further modifying the above dye molecule with the above-mentioned functional group can simultaneously achieve NIR fluorescence imaging and imaging modalities such as PET and SPECT, and further improve the imaging effect in a combined evaluation manner to better achieve guiding the localization and removal of tumors during surgery.

[0040] The present invention also provides a preparation method for the above series of near-multifunctional probe molecules.

[0041] When the multifunctional probe molecule is modified with a protein affinity agent represented by 4-(p-iodophenyl)butyric acid derivative on its skeleton, the preparation method specifically includes the following steps:

[0042] 1) Synthesize 4-(p-iodophenyl)butyric acid derivative, and the synthesis route is as follows:

[0043]

[0044] The steps are as follows:

[0045] a) Under the conditions that 2-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) is used as a polypeptide condensation reagent, an organic base provides a basic environment, and N,N-dimethylformamide (DMF), dichloromethane (DCM), or tetrahydrofuran is used as a solvent, 4-(4-iodophenyl)butyric acid (I) and tert-butyl (2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethyl)carbamate (II) undergo a condensation reaction to form an intermediate (III).

[0046] b) In a DCM solvent, trifluoroacetic acid is added dropwise as a Boc-deprotecting reagent to the intermediate (III) for deprotection to form a 4-(4-iodophenyl)butyric acid derivative (IV).

[0047] 2) Modify the cyanine dye with the 4-(4-iodophenyl)butyric acid derivative. Taking IR-808 as an example of the cyanine dye, the synthesis route is as follows:

[0048]

[0049] The specific steps are as follows:

[0050] c) Optionally, HATU and DIPEA or 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and 4-dimethylaminopyridine (DMAP) are used as amide-forming reaction condensation reagents, and IR-808 and the compound (IV) obtained in step b) are reacted in an organic solvent. By adjusting the feeding ratio of IR-808 and the compound (IV), the carboxyl hydroxyl group on either one or both sides of IR-808 is condensed with the amino group of the compound IV, and finally a series of multifunctional probe molecules are obtained, which are summarized by the general formula (V), where R 1 and R 2 are the same or different and each independently takes -OH or IP.

[0051] In some specific embodiments of the present invention, the organic base used in step a) is diethylamine (DEA), triethylamine (TEA), or N,N-diisopropylethylamine (DIPEA). First, HATU is mixed and stirred with the 4-(4-iodophenyl)butyric acid molecule containing a carboxyl group to activate the carboxyl group; then the organic base and the tert-butyl (2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethyl)carbamate molecule containing an amino group are dissolved in an organic solvent and added dropwise into the reaction system; the reaction is carried out at room temperature.

[0052] In some specific embodiments of the present invention, after the reaction in step b) is completed, the system is extracted with saturated sodium bicarbonate solution and then washed with saturated brine until neutral.

[0053] In some specific embodiments of the present invention, both steps a) and b) are purified by column chromatography.

[0054] In some specific embodiments of the present invention, when the feed ratio of compound (IV) to IR-808 in step c) is 1:1, a single-sided substitution product IR-808-IP is preferably obtained, i.e., R 1 =-OH, R 2 =IP; when the feed ratio of compound (IV) to IR-808 is 2:1, a double-sided substitution product IR-808-IP2 is preferably obtained, i.e., R 1 =R 2 =IP.

[0055] In some specific embodiments of the present invention, when HATU and DIPEA are used as amidation condensation reagents in step c), HATU should be first mixed and stirred with the IR-808 molecule containing a carboxyl group to activate the carboxyl group; then DIPEA and the compound (IV) molecule containing an amino group are dissolved in an organic solvent and added dropwise into the reaction system.

[0056] In some specific embodiments of the present invention, EDC and DMAP can also be selected as amidation condensation reagents in step c). At this time, the temperature of the reaction system should be 0 °C, and after stirring for 30 minutes, the temperature is restored to room temperature, and the reaction time is 4-24 hours.

[0057] The preparation method of the present invention further includes modifying an existing near-infrared indocyanine dye or the multifunctional probe molecule obtained in step c) with a thiol-containing compound. The thiol-containing compounds include: IP-SH, thiol-containing amino acids, and thiol-containing polypeptides; further, the thiol-containing polypeptides can be thiol-containing targeting peptides or other functional polypeptides. Taking the modification of the multifunctional probe molecule obtained in step c) as an example, the synthesis route of this step is as follows:

[0058]

[0059] The specific steps include:

[0060] Using the multifunctional probe molecule (V) obtained in step c) as a raw material, reacting it with a thiol-containing amino acid or a thiol-containing polypeptide in an organic solvent under the catalysis of an organic base, and replacing the halogen on the cyclohexene structure of the raw material with the thiol-containing compound to obtain a series of multifunctional probe molecules with a protein affinity structure modified on the skeleton, or multifunctional probe molecules with both a protein affinity structure and a functional group modified on the skeleton, which are summarized by the general formula (VII), where R1 and R 2 are the same or different and each independently takes -OH or IP, and the S atom on the cyclohexene structure is connected with a protein affinity agent IP, a functional amino acid structure or a functional polypeptide structure.

[0061] In some specific embodiments of the present invention, the organic base used in step d) can be DEA or TEA or DIPEA; the reaction temperature is between 30 and 70 °C; the reaction time is 1 to 24 hours; the organic solvent is any one of DMSO, DMF and methanol; the reaction crude product is purified by column chromatography.

[0062] In some specific embodiments of the present invention, the mercapto-containing amino acid or mercapto-containing polypeptide in step d) can be selected from cysteine, acetylated cysteine, c(RGDfC), PMSA-SH, TATE-SH, etc.

[0063] To solve the above technical problems, the present invention also provides an evaluation of the metabolic behavior of the synthesized multifunctional probe molecule, and provides the application of the multifunctional probe molecule in constructing a lymph node imaging time window in the first and second near-infrared regions and the application of high-resolution near-infrared first and second region vascular imaging with adjustable imaging time.

[0064] To solve the above technical problems, the present invention also provides an evaluation of the near-infrared first and second region tumor targeting imaging effects of the multifunctional probe molecule before and after modifying the targeting peptide.

[0065] Further, the first and second near-infrared regions are near-infrared light bands with wavelengths above 800 nanometers.

[0066] The present invention relates to a multi-modal diagnosis and treatment multifunctional fluorescence probe platform with adjustable pharmacokinetics based on albumin dynamic binding. This platform integrates a variety of advanced technologies, including near-infrared probes, PET technology, photodynamic therapy of fluorescence probes, and the regulation mechanism of albumin interaction, bringing more accurate and personalized solutions for clinical treatment.

[0067] Compared with the prior art, the beneficial effects of the present invention are as follows: A series of multifunctional probe molecules provided by the present invention can achieve clear near-infrared first and second region lymphatic imaging and vascular imaging, and at the same time have good biosafety and adjustable circulation and metabolism time; the synthetic route proposed by the present invention is concise and can be extended to other dyes with similar structures and properties; the modification method proposed by the present invention effectively regulates the interaction between the dye molecule and the protein, realizes the transformation between the covalent interaction and the non-covalent interaction between the dye molecule and the protein, and can be used for the regulation of in vivo imaging time; the method for shielding the halogen sites of the protein-binding dye proposed by the present invention effectively improves the long-term in vivo retention caused by the binding of the dye to the protein and the systemic non-specific fluorescence signal, and realizes a high-resolution and residue-free vascular imaging effect superior to traditional commercial dyes; the present invention further shows precise tumor localization and treatment through the modification of targeting peptides and radionuclide chelators. And further realizes a personalized treatment effect of highly efficient diagnosis and treatment integration through the combination of drugs and photodynamic therapy.

[0068] By introducing a multimodal fluorescence probe platform, combining near-infrared probes, PET technology, photodynamic therapy of fluorescence probes, and the regulation mechanism of albumin interaction, this technology platform has broad application prospects in the field of tumor diagnosis and treatment. At the same time, this technology also involves drug delivery and pharmacokinetic regulation, providing new ideas for improving the drug treatment effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0069] Figure 1 Schematic structural diagram of the multimodal fluorescence probe platform described in Example 2 of the present invention.

[0070] Figure 2 For the compound of formula (IV) in Example 1 of the present invention 1 1H-NMR spectrum.

[0071] Figure 3 For the compound IR-808(Ac) in Example 1 of the present invention 1 1H-NMR spectrum.

[0072] Figure 4 For the compound IR-808-IP in Example 1 of the present invention 1 1H-NMR spectrum.

[0073] Figure 5 For the compound IR-808(Ac)-IP in Example 1 of the present invention 1 1H-NMR spectrum.

[0074] Figure 6 For the compound IR-808-IP2 in Example 1 of the present invention 1 1H-NMR spectrum.

[0075] Figure 7 For the compound IR-808(Ac)-IP2 in Example 1 of the present invention 1 1H-NMR spectrum.

[0076] Figure 8 For the structural characterization of the 1080-COOH backbone structure in Example 1 of the present invention

[0077] Figure 9 For the 1 1H-NMR spectrum of the IR-820 backbone structure in Example 1 of the present invention

[0078] Figure 10 For the 1 1H-NMR spectrum of the compound IR-820-IP structure in Example 1 of the present invention

[0079] Figure 11 For the 1 1H-NMR spectrum of the compound IR-820-IP2 structure in Example 1 of the present invention

[0080] Figure 12 Absorption spectra of the cyanine dyes involved in Experimental Example 1 of the present invention in DMSO, PBS, and BSA solutions; wherein, the concentration of BSA is 10 μM; the dye concentrations in DMSO, PBS, and BSA solutions are 10 μM.

[0081] Figure 13 Fluorescence spectra of the cyanine dyes involved in Experimental Example 1 of the present invention in DMSO, PBS, and BSA solutions; wherein, the concentration of BSA is 10 μM; the dye concentrations in DMSO, PBS, and BSA solutions are 10 μM; excited by an 808-nm laser.

[0082] Figure 14 For the reaction solution obtained by mixing BSA solution and dye solution with the same molar concentration and incubating at 37 °C for 2 hours in Experimental Example 1 of the present invention, and evaluating the binding of the dye and BSA by SDS-PAGE gel electrophoresis.

[0083] Figure 15 For the fluorescence brightness of the reaction solution obtained by mixing BSA solution and dye solution with the same molar concentration and incubating at 37 - 70 °C for 2 hours or immediately after mixing in Experimental Example 1 of the present invention, and evaluating the binding of the dye and BSA by SDS-PAGE gel electrophoresis.

[0084] Figure 16 For the evaluation diagram of the in vivo metabolism of the cyanine dyes involved in Experimental Example 2 of the present invention in mice.

[0085] Figure 17It is the evaluation diagram of the near-infrared imaging time window of different dyes on mouse lymph nodes under anesthesia in Experimental Example 2 of the present invention.

[0086] Figure 18 It is the evaluation diagram of the near-infrared imaging time window of different dyes on mouse lymph nodes in the free activity state in Experimental Example 2 of the present invention.

[0087] Figure 19 It is the evaluation diagram of the near-infrared angiography effect and imaging time of different dyes on mice in Experimental Example 2 of the present invention.

[0088] Figure 20A and Figure 20B It is the evaluation diagram of the in vivo imaging effect of different dyes on the mouse tumor-bearing model in Experimental Example 3 of the present invention.

[0089] Figure 21A and Figure 21B It is the evaluation diagram of the ex vivo imaging effect of different dyes on the mouse tumor-bearing model in Experimental Example 3 of the present invention.

[0090] Figure 22 It is the 1 1H-NMR spectrum of the compound IR-808-IP-PEG-Boc structure in Example 2 of the present invention.

[0091] Figure 23A and Figure 23B It reflects the analysis result of the accumulation of dye molecules' fluorescence signals in tumors after intravenous injection of 808-IP-DOTA-PSMA into tumor-bearing mice in Experimental Example 4 of the present invention.

[0092] Figure 24A and Figure 24B It reflects the uptake and fluorescence signals of the material by the tumors and infiltrating tissues of tumor-bearing mice after intravenous injection of 808-IP-DOTA-PSMA for 24 hours in Experimental Example 4 of the present invention.

[0093] Figure 25 It reflects the radionuclide labeling result of 808-IP-DOTA-PSMA in Experimental Example 5 of the present invention.

[0094] Figure 26 It reflects the color of the material after radionuclide labeling in Experimental Example 5 of the present invention.

[0095] Figure 27 It reflects the change of the labeling rate of 808-IP-DOTA-PSMA over time in Experimental Example 5 of the present invention. Detailed implementation mode

[0096] The present invention is not limited to the specific methods, protocols, and reagents described herein, as they can vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention, which is defined solely by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0097] Preferably, the terms used herein are defined as in "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)", edited by Leuenberger, H.G.W, Nagel, B. and Klbl, H.b (1995), Helvetica Chimica Acta, CH-4010 Basel, Switzerland.

[0098] Unless the context otherwise requires, throughout the specification and the following claims, the word "comprising" and variations thereof such as "comprises" and "comprising" will be understood to imply the inclusion of the stated integer or step, or group of integers or steps, but not the exclusion of any other integer or step, or group of integers or steps. In the following paragraphs, aspects of the present invention will be defined in more detail. Each aspect so defined may be combined with any other one or more aspects, unless there is an express contrary indication. In particular, any optional, preferred, or advantageous feature may be combined with any other optional, preferred, or advantageous feature or features.

[0099] Throughout this specification, several documents are cited. Each document cited herein (including all patents, patent applications, scientific publications, manufacturer's instructions, operating guides, etc.), whether supra or infra, is hereby incorporated by reference in its entirety. Nothing in this application shall be construed as an admission that the present invention is not entitled to antedate such disclosure by virtue of prior invention. Certain of the documents cited herein are identified as "incorporated by reference". In the event of a conflict between the definitions or teachings of the incorporated reference and the text of this specification, the text of this specification shall govern.

[0100] To make the objectives, technical solutions, and advantages of the present invention more clearly understood, the present invention will be further described in detail below with reference to the embodiments and the accompanying drawings. The illustrative embodiments and descriptions thereof of the present invention are only for the purpose of explaining the present invention and shall not be construed as limiting the present invention.

[0101] The multifunctional probe molecule of the present invention has a structure shown in Formula I, II, or III as follows:

[0102]

[0103] Among them, the X group can be a halogen or any one of the following structures and their thio derivatives:

[0104]

[0105]

[0106]

[0107] R 1 The structure of is any one of the following

[0108]

[0109] R 2 The structure of is any one of the following

[0110]

[0111] In the above structures of R1 and R2, n is an integer from 0 to 20, preferably an integer from 0 to 10, more preferably from 1 to 5, and still more preferably an integer from 2 to 5; and when X is a halogen, R1 and R2 are not both -OH at the same time.

[0112] In the present invention, the multifunctional probe molecule having the structure shown in Formula I can be obtained by modifying a protein affinity agent, a targeting group, and / or a functional group based on a cyanine dye molecule having a similar skeleton to Formula I, such as IR-780, IR-808, IR-783, or IR-775;

[0113]

[0114] Specifically, it can be any compound in Table 1 below:

[0115] Table 1. List of Structures of Multifunctional Probe Molecules Shown in Formula I

[0116]

[0117]

[0118]

[0119]

[0120]

[0121]

[0122]

[0123]

[0124]

[0125]

[0126]

[0127]

[0128]

[0129] In the present invention, the multifunctional probe molecule with the structure shown in Formula II can be obtained by modifying a cyanine dye molecule with a skeleton similar to Formula II, such as an IR-820 modified structure, with a protein affinity agent, a targeting group, and / or a functional group;

[0130]

[0131] Specifically, it can be any compound in Table 2 below:

[0132] Table 2. List of Structures of Multifunctional Probe Molecules Shown in Formula II

[0133]

[0134]

[0135]

[0136]

[0137]

[0138]

[0139]

[0140]

[0141]

[0142]

[0143]

[0144]

[0145]

[0146] In the present invention, the multifunctional probe molecule with the structure shown in Formula III can be obtained by modifying a cyanine or flavonoid dye molecule such as FD-1080, Et-1080 or CO-1080, which has a similar skeleton to Formula III, with a protein affinity agent, a targeting group and / or a functional group;

[0147]

[0148] Specifically, it can be any compound in Table 3 below:

[0149] Table 3. List of Structures of Multifunctional Probe Molecules Shown in Formula III

[0150]

[0151]

[0152]

[0153]

[0154]

[0155]

[0156]

[0157]

[0158]

[0159]

[0160]

[0161]

[0162]

[0163] Furthermore, based on the above modification basis, the multifunctional probe molecule described in the present invention can be named in the following manner: When IR-808 is used as the modification basis, the naming rule is as follows: X=-Cl, R 1 =IP and R 2 =-OH, and this molecule is IR-808-IP; X=-Cl, R 1 =IP and R 2 =IP, and this molecule is IR-808-IP2; X=Ac, R 1 =-OH and R 2 =-OH, and this molecule is IR-808(Ac); X=Ac, R1 =IP and R 2 =-OH, the molecule is IR-808(Ac)-IP; X = Ac, R 1 =IP and R 2 =IP, the molecule is IR-808(Ac)-IP2; X = IP-S-, R 1 =IP and R 2 =IP, the molecule is IR-808-IP3; X = RGD, R 1 =-OH and R 2 =-OH, the molecule is IR-808 (RGD); X = RGD, R 1 =IP and R 2 =-OH, the molecule is IR-808(RGD)-IP; X = RGD, R 1 =IP and R 2 =IP, the molecule is IR-808(RGD)-IP2; X = PSMA, R 1 =-OH and R 2 =-OH, the molecule is IR-808 (PMSA); X = PSMA, R 1 =IP and R 2 =-OH, the molecule is IR-808(PMSA)-IP; X = PSMA, R 1 =IP and R 2 =IP, the molecule is IR-808(PMSA)-IP2. Other combinations can be deduced by analogy.

[0164] In some specific embodiments of the present invention, in the structure of the product (VII) of step d), R 1 =-OH, R 2 =IP, it is a unilateral substitution product, such as IR-808(Ac)-IP / IR-808(RGD)-IP / IR-808(PSMA)-IP; R 1 =R 2 =IP is a bilaterally substituted product, such as IR-808(Ac)-IP2 / IR-808(RGD)-IP2 / IR-808(PSMA)-IP2; R 1 =R 2 =-OH, it is IR-808(Ac) / IR-808(RGD) / IR-808(PSMA). Other combinations are similar;

[0165]

[0166] The present invention is further explained below in conjunction with embodiments and experimental examples.

[0167] Example 1: Preparation of dynamic protein-binding multifunctional probes with structures of Formula I, Formula II, and Formula III respectively

[0168] (1) Preparation method of the multifunctional probe with dynamic protein-binding of Formula I series with IR-808 as the core

[0169] In this method, by modifying the protein affinity structure, targeting group or functional group on the skeleton of the near-infrared cyanine dye molecule IR-808, a series of the above-mentioned dynamic protein-binding multifunctional probes described in Table 1 can be obtained. The said IR-808 can be purchased through commercial channels or synthesized according to existing methods.

[0170] When modifying a protein affinity agent containing a mercapto group, the synthesis route is as follows:[[]]

[0171]

[0172]

[0173] The specific synthesis steps are as follows:[[]]

[0174] (1) Select a 50 mL two-necked flask, dissolve 4-(p-iodophenyl)butyric acid (I) (500 mg, 1 eq) and N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)uronium hexafluorophosphate (HATU) (655 mg, 1 eq) in dichloromethane (10 mL), and stir at room temperature for 30 minutes. Dissolve tert-butyl (2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethyl)carbamate (II) (479.5 μL, 1 eq) and N,N-diisopropylethylamine (DIPEA) (354.5 μL, 1.24 eq) in dichloromethane, and slowly drop this mixed solution into the reaction system. After reacting overnight, extract with dichloromethane and saturated brine, and distill under reduced pressure to obtain a crude product of a pale yellow viscous liquid. Purify it with a silica gel chromatography column (ethyl acetate: n-hexane = 1:3) to obtain a pale yellow viscous liquid, which is compound (III).

[0175] (2) Select a 50 mL two-necked flask, dissolve compound (III) (400 mg) in dichloromethane (10 mL), add trifluoroacetic acid (4.5 mL) to the constant pressure dropping funnel, and slowly drop it into the reaction system at 0 °C while controlling the dropping rate. After the dropping is completed, restore the reaction system to room temperature. After reacting overnight, wash the reaction system with saturated sodium bicarbonate solution multiple times to remove the excess acid in the system, and then wash it three times with saturated brine. The collected organic phase is added with anhydrous magnesium sulfate for drying, filtered to remove the desiccant, and the filtrate is collected and the solvent is removed by distillation under reduced pressure to obtain a pale yellow viscous liquid, which is compound (IV). The 1 1H-NMR spectrum for characterizing its structure can be seen in Figure 2。

[0176] (3) A 25 mL round-bottom flask was selected, and compound (IV) (300 mg, 0.83 eq) and N,N,N’,N’-tetramethyl-O-(7-azabenzotriazol-1-yl)uronium hexafluorophosphate (HATU) (296 mg, 1 eq) were dissolved in dichloromethane (2 mL), and stirred at room temperature for 30 minutes. Mercaptopropionic acid (83 mg, 1 eq) and N,N-diisopropylethylamine (DIPEA) (125 mg, 1.24 eq) were dissolved in dichloromethane (2 mL), and this mixed solution was slowly added dropwise to the reaction system. After reacting overnight, it was extracted with dichloromethane and saturated brine, and the solvent was removed by distillation under reduced pressure to obtain a pale yellow viscous liquid crude product, which was purified by silica gel column chromatography (ethyl acetate: n-hexane = 1:3) to obtain a white solid, namely compound (IP-SH).

[0177] (4) A 5 mL round-bottom flask was selected, and IR-808 (20 mg, 1 eq) and N,N,N’,N’-tetramethyl-O-(7-azabenzotriazol-1-yl)uronium hexafluorophosphate (HATU) (10 mg, 1 eq) were dissolved in dichloromethane (0.2 mL), and stirred at room temperature for 30 minutes. The compound (IV) (20 mg, 1 eq) prepared in (2) and N,N-diisopropylethylamine (DIPEA) (5.3 μL, 1.24 eq) were dissolved in dichloromethane (0.2 mL), and this mixed solution was slowly added dropwise to the reaction system. After reacting overnight, it was extracted with dichloromethane and saturated brine, the solvent was removed by distillation under reduced pressure, and it was purified by silica gel column chromatography (methanol: dichloromethane = 1:5) to obtain a green solid, namely compound (V), where R1 is OH and R2 is IP in its structure, and it can be named IR-808-IP.

[0178] (5) A 10 mL round-bottom flask was selected, and compound (V) (20 mg, 1 eq) was dissolved in 2 mL of dimethyl sulfoxide, and then the mercapto compound (IP-SH) prepared in (3) above (3 eq) was added. After complete dissolution, DIPEA (13 μL, 3 eq) was added. After reacting at room temperature for two hours, it was extracted with dichloromethane and saturated brine, the organic phase was collected and dried with anhydrous magnesium sulfate, the solvent was removed by distillation under reduced pressure, and the crude product was purified by silica gel column chromatography (methanol: dichloromethane = 1:5) to obtain a green solid, namely compound (VI), where X is IP, R1 is OH, and R2 is IP in its structure, and it can be named IR-808-IP-X.

[0179] Referring to the above synthesis steps, by adjusting the feeding ratio of IR-808 and compound (IV) in step (4) above, and / or using an appropriate amount of other mercapto-containing compounds to replace compound (IP-SH) used in step (5), other multifunctional probe molecules conforming to general formula I of the present invention in Table 1 can be obtained. The other mercapto-containing compounds may be mercapto-containing amino acids or mercapto-containing functional polypeptides or their derivatives (such as mercapto-containing cysteine, PSMA, integrin, SSTR, EGFR, HER2, VEGFR, PD-1, PD-L1, CD20, CDK4 / 6, BRAF, ALK, RET, CD33, CDK, FLT3, KIT, ROS1, MET, PARP, CD19, GRPR, CC2KR, CXCR4 or uPAR, etc.).

[0180] In addition, referring to synthesis step (5) above, directly reacting a mercapto-containing amino acid or a mercapto-containing functional polypeptide or its derivative with IR-808 to replace the -Cl in the structure of IR-808, a variety of multifunctional probe molecules after No. 230 in Table 1 can be obtained, such as IR-808(RGD), IR-808(PSMA), IR-808(Ac), IR-808(TATE), IR-808(NGR), and so on.

[0181] According to the above method, the present embodiment can synthesize at least the following multifunctional probe molecules based on the backbone of formula I: IR-808-IP, IR-808-IP2, IR-808-IP3, IR-808(Ac), IR-808(Ac)-IP, IR-808(Ac)-IP2, IR-808(RGD), IR-808(RGD)-IP, IR-808(RGD)-IP2, IR-808(PSMA), IR-808(PSMA)-IP, IR-808(PSMA)-IP2, and so on. Among them, the 1 H-NMR spectrum of IR-808(Ac) is shown in Figure 3 , the 1 H-NMR spectrum of IR-808-IP is shown in Figure 4 , the 1 H-NMR spectrum of IR-808(Ac)-IP is shown in Figure 5 , the 1 H-NMR spectrum of IR-808-IP2 is shown in Figure 6 , the 1 H-NMR spectrum of IR-808(Ac)-IP2 is shown in Figure 7 .

[0182] (II) Preparation method of multifunctional probe for dynamic protein binding of formula II series with IR-820 as the core

[0183] In this method, by modifying a protein affinity agent structure, a targeting group or a functional group on the skeleton of the near-infrared cyanine dye molecule IR-820, a series of multifunctional probes for dynamic protein binding described in Table 2 above can be obtained. The shown IR-820 can be purchased through commercial channels or synthesized according to existing methods, and its structure is characterized by the Figure 9 of 1 1H-NMR spectrum.

[0184] Based on the skeleton of the near-infrared cyanine dye molecule including IR-820, it is prepared with reference to the synthesis steps described in (I). For example, by replacing IR-808 with IR-820 in step (4) of (I) above, and adjusting the feeding ratio of the IR-820 molecule and compound (IV), and / or using an appropriate amount of other mercapto-containing compounds to replace compound (IP-SH) in step (5), other multifunctional probe molecules conforming to general formula II of the present invention in Table 2 such as IR-820-IP and IR-820-IP2 can be obtained. The other mercapto-containing compounds may be mercapto-containing amino acids or mercapto-containing functional polypeptides or their derivatives.

[0185] The structures of the obtained IR-820-IP and IR-820-IP2 are respectively characterized by the Figure 10 and Figure 11 of 1 1H-NMR spectrum.

[0186] (III) Preparation method of multifunctional probes for dynamic protein binding of formula III series with CO-1080 as the core

[0187] In this method, by modifying a protein affinity agent structure, a targeting group or a functional group on the skeleton of the near-infrared cyanine dye molecule 1080-COOH, a series of multifunctional probes for dynamic protein binding described in Table 3 above can be obtained. The 1080-COOH can be synthesized according to existing methods. For example, the method described in Example 3 of the specification in the patent document CN116283726A can be used to synthesize 1080-COOH, and the synthesis route is as follows:

[0188]

[0189] The molecular structure of 1080-COOH synthesized according to this method is characterized by Figure 8 for characterization.

[0190] Based on the skeleton of near-infrared cyanine dye molecules including 1080-COOH, it is prepared with reference to the synthesis steps described in (I). For example, by replacing IR-808 with 1080-COOH in step (4) of the above (I), and adjusting the feeding ratio of 1080-COOH molecules and compound (IV), and / or using an appropriate amount of other mercapto-containing compounds to replace compound (IP-SH) in step (5), multifunctional probe molecules conforming to general formula III of the present invention in Table 3 such as 1080-COOH-IP and 1080-COOH-IP2 can be obtained. The other mercapto-containing compounds may be mercapto-containing amino acids or mercapto-containing functional polypeptides or their derivatives.

[0191] Experimental Example 1:

[0192] It was experimentally verified that all the multifunctional probe molecules synthesized in the first part of Example 1 have excellent near-infrared luminescence ability and exhibit different albumin-binding abilities.

[0193] (1) As Figure 12 and Figure 13 shown, all the multifunctional probe molecules synthesized in the first part of Example 1 of the present invention have absorption and emission peaks in the first near-infrared region in organic phases represented by dimethyl sulfoxide (the fluorescence spectra were measured under laser excitation at a wavelength of 808 nm), and all have absorption and emission wavelengths similar to those of the traditional commercial cyanine dye IR-808, and have good near-infrared luminescence ability. In particular, it should be noted that all the molecules designed and synthesized in the first part of Example 1 have an emission tail extended to more than 1100 nm, enabling them to have good second near-infrared imaging ability and allowing imaging at more than 1500 nm. Among them, the fluorescence spectra of all the multifunctional probe molecules retaining -Cl based on IR-808 in bovine serum albumin (BSA) solution are different from those in phosphate buffer solution (PBS), which is caused by the covalent binding of these dyes to albumin to varying degrees. The absorption and emission behaviors of all the multifunctional probe molecules modified to replace -Cl based on IR-808 are similar in BSA and PBS.

[0194] (2) As Figure 14As shown, both IR-808 and IR-808-IP exhibited bright fluorescent bands at the corresponding position of BSA, indicating the formation of covalent bonds between the dye molecules and BSA. However, IR-808-IP2 did not show obvious fluorescent signals at the molecular weight of BSA, suggesting that as the number of IPs linked to the side chain increased, the binding ability between the dye molecules and BSA decreased significantly. All the multifunctional probe molecules modified on the basis of IR-808 by substituting -Cl did not exhibit bright fluorescent bands at the corresponding position of BSA, indicating that stable covalent bonds were not formed between the dye molecules and BSA.

[0195] (3) Further, equimolar concentrations of BSA and IR-808, IR-808-IP, and IR-808-IP2 were mixed and incubated at different incubation temperatures from 37 to 60 °C for 2 hours. As Figure 15 shown, when IR-808 was mixed with BSA and heated between 37 and 60 °C, the brightness increased significantly. The decrease in brightness when heated to 70 °C was due to the destruction of the protein structure caused by overheating; the phenomenon of IR-808-IP was basically similar to that of IR-808. For the mixture system of IR-808-IP2 and BSA, significant fluorescence enhancement was only observed when heated to 50 - 60 °C, indicating that IR-808-IP2 still had the ability to covalently bind to BSA.

[0196] Experimental Example 2:

[0197] It was experimentally verified that the different binding abilities of the multifunctional probe molecules synthesized in the first part of Example 1 to proteins significantly affected the metabolic behavior of the molecules and the imaging time of lymph nodes and blood vessels.

[0198] (1) As Figure 16 shown, the change in the distribution of the fluorescent signals of different dyes in mice over time after tail vein injection. Due to the rapid binding of IR-808 and IR-808-IP to proteins at room temperature, the blood vessels throughout the mouse body were clearly visible after tail vein injection of the dyes; and as the observation time extended, the fluorescent signals were dispersed throughout the body with albumin, resulting in severe skin absorption. The binding behavior between IR-808-IP2 and albumin was not active, so no vascular fluorescent signals could be observed. All the multifunctional probe molecules modified on the basis of IR-808 by substituting -Cl (including IR-808(Ac), IR-808(Ac)-IP, IR-808(Ac)-IP2, and IR-808-IP3) had lower skin absorption, and the metabolic rate of the dyes decreased as the number of modified IPs in the molecule increased.

[0199] (2) As Figure 17As shown, after isoflurane anesthesia, IR-808 and IR-808-IP were respectively injected into the mouse footpads. The popliteal lymph nodes of the mouse were illuminated 2 minutes after the injection of IR-808, and the sacral lymph nodes of the mouse were illuminated 7 minutes later; the popliteal lymph nodes of the mouse were illuminated 7 minutes after the injection of IR-808-IP, and the sacral lymph nodes of the mouse were illuminated 25 minutes later. By regulating the interactions between proteins and molecules, a lymph node imaging time window with adjustable illumination intervals was constructed. As Figure 18 shown, IR-808, IR-808-IP, IR-808-IP2 and IR-808-IP3 were injected into the footpads of normal mice. The mice were allowed to move freely at other times except when anesthetized during observation. Several dyes showed different lymph node imaging behaviors: the lymph nodes were illuminated by IR-808 for less than 24 hours; the illumination time of the lymph nodes by IR-808-IP was extended, but the signal of the leg skin also increased over time; IR-808-IP2 and IR-808-IP3 had slower illumination speeds and longer illumination times, and the popliteal lymph nodes and popliteal lymph nodes of the mouse could still be accurately traced at 24 hours.

[0200] The present invention Figures 17 - 18 The imaging of the leg lymph nodes in mice was carried out in the second near-infrared imaging window greater than 1100 nm and excited by an 808 nm laser.

[0201] (3) As Figure 19 shown, IR-808(Ac), IR-808(Ac)-IP and IR-808(Ac)-IP2 were injected into the tail vein of mice for NIR-II region imaging of the leg blood vessels of mice. Different dyes maintained a similar blood vessel-skin signal-to-background ratio, and the change in the number of modified IPs significantly changed the imaging time of the blood vessels: IR-808(Ac) could clearly image the leg blood vessels of mice for 5 minutes; IR-808(Ac)-IP could clearly image the leg blood vessels of mice for 1 hour; IR-808(Ac)-IP2 could clearly image the leg blood vessels of mice for 2 hours.

[0202] The present invention Figure 19 The imaging of the leg blood vessels in mice was carried out in the second near-infrared imaging window greater than 1300 nm and excited by an 808 nm laser.

[0203] Experimental Example 3:

[0204] It was experimentally verified that the multifunctional probe molecules obtained by modifying the dye IR-808 with thiol-containing targeting peptides such as RGD and PSMA in the first part of Example 1 and replacing its -Cl had higher tumor targeting than IR-808 and achieved clear tumor localization.

[0205] (1) After intravenous injection of IR-808, IR-808(RGD), IR-808(RGD)-IP, and IR-808(RGD)-IP2 into tumor-bearing mice, the accumulation of the fluorescence signal of the dye molecules in the tumor was analyzed, and the analysis results are as shown in Figure 20A and Figure 20B . It can be seen from Figure 20A that IR-808(RGD), IR-808(RGD)-IP, and IR-808(RGD)-IP2 have lower skin fluorescence signals than IR-808; it can be seen from Figure 20B that IR-808(RGD) reaches the maximum tumor-to-skin signal-to-noise ratio 3 hours after intravenous injection; IR-808(RGD)-IP reaches the maximum tumor-to-skin signal-to-noise ratio 24 hours after intravenous injection; IR-808(RGD)-IP2 reaches the maximum tumor-to-skin signal-to-noise ratio 48 hours after intravenous injection; among them, IR-808(RGD)-IP has the largest SBR, and compared with the dye molecules without targeting peptide modification (including IR-808-IP and IR-808-IP2), the above dye molecules modified with targeting peptides all showed better tumor targeting (see Figure 20A ).

[0206] (2) 24 hours after intravenous injection of the dye molecules IR-808, IR-808(RGD), IR-808(RGD)-IP, and IR-808(RGD)-IP2 into tumor-bearing mice, ex vivo imaging of tumor, muscle, and skin tissues was performed. The imaging results are as shown in Figure 21A and Figure 21B . After intravenous injection of IR-808, the ex vivo tissues of the mice showed obvious fluorescence signals and had a low tumor-to-skin signal-to-noise ratio; after intravenous injection of IR-808(RGD), IR-808(RGD)-IP, and IR-808(RGD)-IP2, they all showed relatively high tumor-to-skin signal-to-noise ratios and had good tumor localization effects.

[0207] Example 2: Construction of a multimodal diagnosis and treatment integrated probe platform

[0208] Based on the IR-808-IP structure synthesized in the first part of Example 1, targeting molecules (such as RGD, PSMA, etc.), protein affinity agents, and functional groups - radionuclide chelating groups (such as DOTA ring, NOTA ring, etc.) are simultaneously modified on the same molecule to construct a multimodal fluorescence probe platform with a structural form as shown in Figure 1 .

[0209] Taking the simultaneous modification of IP, RGD, and DOTA on the basis of IR-808-IP as an example, the synthesis route is as follows.

[0210]

[0211] The specific synthesis steps are as follows:

[0212] (1) HATU (13 mg, 0.1 eq) and IR-808-IP prepared in Example 1 (40 mg, 1 eq) were dissolved in 5 mL of DCM and stirred for 30 minutes under a nitrogen atmosphere. Then, a solution of compound (II) (tert-butyl (2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethyl)carbamate) (19 mg, 0.2 eq) and DIPEA (5 mg, 0.15 eq) in DCM (5 mL) was slowly added dropwise to the reaction system, and the mixture was stirred overnight at room temperature. After the reaction was completed, the reaction system was quenched with 0.1 M aqueous hydrochloric acid solution, extracted three times with DCM and 0.1 M aqueous hydrochloric acid solution, and then extracted three times with saturated brine. The organic phase was collected, dried over anhydrous magnesium sulfate, filtered, and the organic phase was collected and concentrated under reduced pressure to obtain a dark green product (VII) (45.1 mg). This structure was named IR-808-IP-PEG-Boc, and its 1 1H-NMR spectrum is shown in Figure 22 .

[0213] (2) 30 mg of compound (VII) was dissolved in 2 mL of dichloromethane. After the reaction system was cooled to 0 °C, 1 mL of trifluoroacetic acid was slowly added dropwise. After reacting for two hours, the mixture was washed three times with saturated sodium bicarbonate solution and three times with saturated salt solution. The organic phase was collected, dehydrated with anhydrous sodium sulfate, filtered, and the organic phase was collected and concentrated under reduced pressure to obtain an oily green product (VIII). Since this step is a continuous reaction, the reaction process can be a continuous reaction or can be purified independently. The structural characterization of this step was carried out using a liquid chromatography-mass spectrometry instrument, and the calculated value of the nuclear mass ratio was [M] + = 1303.6046, and the measured value [M] 2+ = 652.3061.

[0214] (3) Compound (VIII) (13.9 mg, 1 eq) was dissolved in 2 mL of dimethyl sulfoxide, and then DOTA-NHS and DIPEA were added. The mixture was stirred and reacted at 30 °C for 4 hours. Without further purification, cRGD (10.6 mg, 2 eq) was directly added and reacted for 4 hours. The crude product was purified using a preparative C18 high-performance liquid chromatography column to obtain compound (IX), and this structure was named IR-808-IP-DOTA-RGD. Its specific structure is as follows:

[0215]

[0216] Since this step is a continuous reaction, its reaction process can be a continuous reaction or can be independently purified. The structural characterization of 808-IP-DOTA was carried out using a liquid chromatography-mass spectrometry instrument, and the calculated value of its mass-to-charge ratio was [M] + = 1689.7847, and the measured value was [M] 3+ = 563.9364. The structural characterization of 808-IP-DOTA-RGD was carried out using a liquid chromatography-mass spectrometry instrument, and the calculated value of its mass-to-charge ratio was [M] + = 2233.0385, and the measured value was [M] 4+ = 558.2591; [M] 3+ = 744.3464. Maldi-Tof was used to identify [M+H] + = 2234.3.

[0217] (3) We can also dissolve compound (VIII) (13.9 mg, 1 eq) in 2 mL of dimethyl sulfoxide, then add DOTA-NHS and DIPEA, stir and react at 30 °C for 4 hours, without further purification, and directly add PSMA-SH (14.5 mg, 2 eq) and react for 4 hours. The crude product was purified using a preparative C18 high-performance liquid chromatography column to obtain compound (X), and the structure was named IR-808-IP-DOTA-PSMA. Its specific structure is as follows:

[0218]

[0219] Since this step is a continuous reaction, its reaction process can be a continuous reaction or can be independently purified. The structural characterization of 808-IP-DOTA was carried out using a liquid chromatography-mass spectrometry instrument, [M] + = 1689.7847, and the measured value was [M] 3+ = 563.9364.

[0220] The structural characterization of 808-IP-DOTA-PSMA was carried out using a liquid chromatography-mass spectrometry instrument, and the calculated value of its mass-to-charge ratio was [M] + = 2384.1160, and the measured value was [M] 3+ = 794.7052. Maldi-Tof was used to identify [M+H] + = 2385.1.

[0221] Referring to the above synthesis steps, by using an appropriate amount of other mercapto - containing amino acids or mercapto - containing functional polypeptides or their derivatives to replace cRGD - SH in step (3), and / or using an appropriate amount of other radionuclide chelating agents to replace DOTA - NHS in step (3), other multifunctional probe molecules of the present invention can be obtained. The other mercapto - containing amino acids or mercapto - containing functional polypeptides or their derivatives include: mercapto - containing cysteine, PSMA, integrin, SSTR, EGFR, HER2, VEGFR, PD - 1, PD - L1, CD20, CDK4 / 6, BRAF, ALK, RET, CD33, CDK, FLT3, KIT, ROS1, MET, PARP, CD19, GRPR, CC2KR, CXCR4, uPAR, etc. The other radionuclide chelating agents include: DOTA, CB - TE2A, HBED, PSMA - 11, FACBC, FDG, FES, FDOPA or FMISO, etc.

[0222] Experimental Example 4

[0223] It was experimentally verified that the RGD, PSMA and other targeted bimodal probes constructed in Example 2 can achieve clear tumor localization.

[0224] (1) Analyze the accumulation of the fluorescence signal of the dye molecule in tumors after intravenous injection of 808 - IP - DOTA - PSMA into tumor - bearing mice via the tail vein. From Figure 23A and Figure 23B , it can be seen that 808 - IP - DOTA - PSMA has good tumor targeting, and high - definition near - infrared imaging of the tumors in the subcutaneous tumor model mice constructed by PIP - PC3 was achieved (see Figure 23A ). Its tumor uptake reached the maximum 24 hours after intravenous injection, and showed low uptake signals in the liver and skin (see Figure 23B ).

[0225] (2) As shown in Figure 24A and Figure 24B , 24 hours after intravenous injection of 808 - IP - DOTA - PSMA into tumor - bearing mice, the tumors and infiltrating tissues of the tumor - bearing mice showed good material uptake and obvious fluorescence signals, and the R^2 simulated by the Gaussian signal could reach 0.9077 (see Figure 24B ), with good tumor imaging effect and can well guide surgical resection.

[0226] Experimental Example 5

[0227] It was experimentally verified that the RGD, PSMA and other targeted bimodal probes constructed in Example 2 can efficiently label radionuclides for imaging diagnosis or treatment.

[0228] (1) As Figure 25 shown, 808-IP-DOTA-PSMA can be labeled with 68 Ga at a relatively high temperature (90 - 130 °C) under acidic conditions, and the labeling rate can reach 99% without further purification. The UV channel data of HPLC shows that the substance is not damaged or degraded after high-temperature labeling. The labeled material, as Figure 26 shown, still maintains the intact light green color of the material.

[0229] (1) As Figure 27 shown, 808-IP-DOTA-PSMA can be labeled with 177 Lu at room temperature with a longer reaction time under acidic conditions, and the labeling rate can reach 99% without further purification. The iTLC data shows that the labeling rate increases continuously within 4 hours over time.

Claims

1. A series of cyanine or cyanine-like multifunctional probe molecules, which are derivatives having a near-infrared cyanine dye molecular skeleton, wherein the skeleton is modified with any one or a combination of two or more of a protein affinity agent structure, a targeting group or a functional group; the near-infrared cyanine dye molecule can be a near-infrared indole cyanine dye molecule or a near-infrared naphthopyrrole cyanine or flavonoid dye molecule; the protein affinity agent structure can be derived from 4 (p-iodophenyl) butyric acid derivatives, Evans blue or its derivatives; the targeting group can be a structure containing any pharmaceutically acceptable targeting peptide; the functional group can be derived from any radionuclide chelator or a functional drug molecule with a modification site.

2. The multifunctional probe molecule according to claim 1, characterized in that: The multifunctional probe molecule is a derivative having a near-infrared cyanine dye molecular skeleton, and the skeleton is modified with any one or a combination of two or more of a protein affinity agent structure, a targeting group or a functional group; the near-infrared cyanine dye molecule can be a near-infrared indole cyanine dye molecule or a near-infrared naphthopyrrole cyanine or flavonoid dye molecule; the protein affinity agent structure is derived from a 4 (p-iodophenyl) butyric acid derivative; the targeting group can be a structure containing any one pharmaceutically acceptable targeting peptide; and the functional group can be any one nuclide chelating group.

3. The multifunctional probe molecule according to claim 1, characterized in that: The multifunctional probe molecule is a derivative having a near-infrared cyanine dye molecular skeleton, and the skeleton is modified with any one of a protein affinity agent structure, a targeting group or a functional group; the near-infrared cyanine dye molecule can be a near-infrared indole cyanine dye molecule or a near-infrared naphthopyrrole cyanine or flavonoid dye molecule; the protein affinity agent structure is derived from a 4 (p-iodophenyl) butyric acid derivative; the targeting group can be a structure containing any one of a pharmaceutically acceptable targeting peptide; and the functional group is any one of a nuclide chelating group.

4. The multifunctional probe molecule according to claim 1, characterized in that: The multifunctional probe molecule is a derivative having a near-infrared cyanine dye molecular skeleton, and the skeleton is simultaneously modified with any two of a protein affinity agent structure, a targeting group or a functional group; the near-infrared cyanine dye molecule can be a near-infrared indole cyanine dye molecule or a near-infrared naphthopyrrole cyanine or flavonoid dye molecule; the protein affinity agent structure is derived from a 4 (p-iodophenyl) butyric acid derivative; the targeting group can be a structure containing any pharmaceutically acceptable targeting peptide; and the functional group can be any nuclide chelating group.

5. The multifunctional probe molecule according to claim 1, characterized in that: The multifunctional probe molecule is a derivative having a near-infrared cyanine dye molecular skeleton, and the skeleton is simultaneously modified with a protein affinity agent structure, a targeting group and a functional group; the near-infrared cyanine dye molecule can be a near-infrared indole cyanine dye molecule or a near-infrared naphthopyrrole cyanine or flavonoid dye molecule; the protein affinity agent structure is derived from a 4 (p-iodophenyl) butyric acid derivative; the targeting group can be a structure containing any pharmaceutically acceptable targeting peptide; and the functional group can be any nuclide chelating group.

6. The multifunctional probe molecule according to claim 1, characterized in that: The multifunctional probe molecule is a derivative having a near-infrared cyanine dye molecular skeleton, and the skeleton is modified with a protein affinity agent structure and a targeting group at the same time; the near-infrared cyanine dye molecule is a near-infrared indole cyanine dye molecule or a near-infrared naphthopyrrole cyanine or flavonoid dye molecule; the protein affinity agent structure is derived from a 4 (p-iodophenyl) butyric acid derivative; and the targeting group can be a structure containing any pharmaceutically acceptable targeting peptide.

7. The multifunctional probe molecule according to claim 1, characterized in that: The multifunctional probe molecule is a derivative having a near-infrared cyanine dye molecular skeleton, and the skeleton is modified with a protein affinity agent structure and a functional group at the same time; the near-infrared cyanine dye molecule is a near-infrared indole cyanine dye molecule or a near-infrared naphthopyrrole cyanine or flavonoid dye molecule; the protein affinity agent structure comes from a 4 (p-iodophenyl) butyric acid derivative; and the functional group can be any nuclide chelating group.

8. The multifunctional probe molecule according to any one of claims 1 to 7, characterized in that: The protein affinity agent structure is a structure having affinity with albumin; further, the albumin is any one of bovine serum albumin and human serum albumin.

9. The multifunctional probe molecule according to any one of claims 1 to 8, characterized in that: The targeting group is a structure comprising a targeting peptide having a targeting effect on any of the following targets: PSMA, integrin, SSTR, EGFR, HER2, VEGFR, PD-1, PD-L1, CD20, CDK4 / 6, BRAF, ALK, RET, CD33, CDK, FLT3, KIT, ROS1, MET, PARP, CD19, GRPR, CC2KR, CXCR4 or uPAR; preferably, a structure comprising any of the following targeting peptides: RGD, PSMA, TATE or NGR.

10. The multifunctional probe molecule according to any one of claims 1 to 9, characterized in that: The targeting group may include a linker structure in addition to the targeting peptide structure.

11. The multifunctional probe molecule according to any one of claims 1 to 10, characterized in that: The nuclide chelating group is derived from any one of the following nuclide chelating agents: NOTA, DOTA, CB-TE2A, HBED, PSMA-11, FACBC, FDG, FES, FDOPA or FMISO; preferably from NOTA or DOTA.

12. The multifunctional probe molecule according to any one of claims 1 to 11, characterized in that: The functional drug molecule with modification sites is selected from: Ibuprofen, Lisinopril, Metformin, Omeprazole, Clopidogrel, Metoprolol, Levothyroxine, Fluoxetine, Warfarin, Imatinib Mesylate, Trastuzumab, Cisplatin, Tamoxifen, Paclitaxel, Dasatinib, Lapatinib, Bortezomib, Sorafenib, Trametinib, Pembrolizumab, Atezolizumab, Ibrutinib, Everolimus or Vemurafenib.

13. The multifunctional probe molecule according to claim 1, characterized in that: Its structure is shown in the following formula I, II or III: Wherein, the X group can be a halogen or any of the following structures and their thio derivatives: The structure of R1 is any of the following: wherein n is an integer of 0-20, preferably an integer of 0-10, more preferably an integer of 1-5, and further preferably an integer of 2-5; the structure of R2 is any one of the following: wherein n is an integer of 0-20, preferably an integer of 0-10, more preferably an integer of 1-5, and further preferably an integer of 2-5; When X is halogen, R1 and R2 are not -OH at the same time.

14. A multimodal integrated diagnosis and treatment probe platform composed of the multifunctional probe molecules described in any one of claims 1 to 13.

15. A method for preparing the multifunctional probe molecule according to any one of claims 1 to 13, characterized in that: The steps include: Step S1: 4-(p-iodophenyl)butyric acid or other protein affinity agent and N,N,N',N'-tetramethyl-O-(7-azabenzotriazole-1-yl)urea hexafluorophosphate are mixed and dissolved in an organic solvent in equal molar amounts, stirred at room temperature for 30 minutes, and then tert-butyl (2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethyl)carbamate and N,N-diisopropylethylamine are added to react at room temperature and pressure; Step S2: deprotecting the intermediate product obtained in step S1 using trifluoroacetic acid; Step S3: reacting the intermediate product obtained in step S2 with IR-808 or other cyanine dyes with similar structures, the reaction steps refer to S1; Step S4: reacting the intermediate obtained in S3 with a polyethylene glycol chain terminated with amino groups at both ends, with the reaction steps referring to S1, and deprotecting the intermediate obtained with trifluoroacetic acid; Step S5: reacting the intermediate obtained in S4 with a functional group such as a DOTA ring; Step S6: The intermediate obtained in step S5 is further reacted with a thiol-containing amino acid or other thiol-containing polypeptide under the catalysis of an organic base to prepare an integrated tumor diagnosis and treatment probe platform with multimodality.

16. The method according to claim 15, characterized in that Steps S1, S2, S3 and S4 should be carried out in an organic solvent, the organic solvent is any one of dichloromethane, dimethylformamide and tetrahydrofuran, the reaction temperature is 0°C or room temperature, and the reaction time is 4-12 hours; step S6 should be carried out in an organic solvent, the organic solvent is any one of dimethylformamide, dimethyl sulfoxide, methanol and ethanol, the reaction temperature is 30-70°C, and the reaction time is 1-24 hours; after the reaction of steps S1 and S3 is completed, the excess alkali should be washed with dilute hydrochloric acid, saturated sodium bicarbonate solution and saturated saline in sequence; after the reaction of step S2 is combined, the excess trifluoroacetic acid is neutralized with saturated sodium bicarbonate solution; the products obtained by the reactions of steps S3 and S4 need to be purified by silica gel chromatography, and the solvent used for silica gel chromatography purification is a mixture of ethyl acetate, dichloromethane, methanol or acetone.

17. The multifunctional probe molecule described in any one of claims 1 to 13 is prepared for near-infrared zone 1 and 2 biological in vivo imaging, cell imaging and fluorescence surgical navigation.

18. The use according to claim 18, characterized in that The near infrared regions 1 and 2 are near infrared light bands with wavelengths above 700 nanometers.

19. The use according to claim 17, characterized in that: The near-infrared zone one and zone two biological in vivo imaging is near-infrared zone one and zone two lymph node in vivo imaging and realizes the construction of a lymph node graded imaging time window of 5 minutes to 3 hours.

20. The use according to claim 17, characterized in that: The near infrared one and two zone biological in vivo imaging is near infrared one and two zone high-resolution blood vessel development and can achieve imaging time regulation from 5 minutes to 2 hours.

21. Use of the multifunctional probe molecule according to any one of claims 1 to 13 in tumor-targeted multimodal imaging and therapy.

22. Use of the multifunctional probe molecule described in any one of claims 1 to 13 in targeted imaging and surgical navigation tumor resection of various cancers.

23. Use of the multifunctional probe molecule according to any one of claims 1 to 13 in the screening and resection of tumor-infiltrating lymph nodes.

24. Use of the multifunctional probe molecules described in claims 1-13 in photothermal therapy and photodynamic therapy.

25. The multifunctional probe molecules described in claims 1-13 are chelated or substituted with radionuclides such as Tc-99m, Lu-177, Cu-64, F-18, I-131, I-123, Ga-67, In-111, Tl-201, Cs-137, Y-90, etc., for targeted PET imaging or radiotherapy.

Images