A dual-targeting multi-modal diagnosis and treatment integrated probe and a preparation method and application thereof

Abstract

The application provides a double-targeting multi-modal diagnosis and treatment integrated probe and a preparation method and application thereof. The probe provided by the application comprises a labeling tracer part, a target point binding ligand and a cyanine fluorescent dye part. After the probe reaches the body, the probe can be quickly combined with a target tissue, can effectively perform double-modal imaging on a target tissue with high expression of a target point and can effectively distinguish the target tissue, and the probe can effectively perform surgical navigation on the target tissue with high expression of the target point, so that the probe is a diagnosis and treatment integrated probe with very promising application in clinical practice.

Description

TECHNICAL FIELD

[0001] The application relates to the technical field of biological probes, in particular to a dual-targeting multi-modal diagnosis and treatment integrated probe and a preparation method and application thereof. BACKGROUND

[0002] Prostate cancer is one of the common malignant tumors, and its incidence ranks the second in the global male malignant tumors. According to the data of the National Cancer Center, prostate cancer has been the highest incidence tumor in the male urinary system in China since 2008, and the incidence in 2014 reached 9.8 / 10 million, ranking the sixth in male malignant tumors; the mortality rate reached 4.22 / 10 million, ranking the ninth in male malignant tumors. In recent years, the incidence of prostate cancer in China has shown an increasing trend year by year, especially in elderly men. The stage composition of prostate cancer patients in China is greatly different from that in western developed countries. Among the newly diagnosed cases in China, only 30% are clinically localized patients, and the rest are patients with locally advanced or extensive metastasis. These patients cannot receive local radical treatment, and the prognosis is poor. Early, comprehensive, non-invasive and dynamic detection of prostate cancer primary and metastatic lesions is of great significance for guiding accurate clinical staging, judging prognosis, selecting treatment options and judging efficacy.

[0003] The most commonly used imaging method for prostate cancer diagnosis at present is MRI, which has the characteristics of high sensitivity and relatively low specificity, high false positive rate, and limited detection of distant metastasis and biochemical recurrence. Recently, prostate specific membrane antigen (PSMA) targeted PET imaging has attracted more and more attention, and it has good application value for prostate diagnosis and treatment. In 2021, the China CSCO guideline first recommended (IA class I level recommendation) PSMAPET / CT for distant staging of prostate cancer. In a meta-analysis, 68 The sensitivity and specificity of Ga-PSMA PET / CT for preoperative regional lymph node metastasis in medium and high risk prostate cancer were 65% and 95% respectively, which had a high level.

[0004] PSMA is a type II transmembrane protein composed of 19 intracellular, 24 transmembrane, and 707 extracellular amino acids. The outer group of PSMA can bind various ligands with different functions, while the inner group contains functional factors that initiate endocytosis. When PSMA ligands or specific antibodies used for imaging bind to the outer group of PSMA, the internalization group initiates endocytosis in the cell. The PSMA ligand or antibody is internalized into the cytoplasm or retained in lysosomes, thus achieving the accumulation of the radioactive imaging agent inside the cell. Unbound ligands, due to their small size, are rapidly cleared by the body, resulting in a high contrast between the tumor and the background. PSMA is significantly highly expressed in prostate cancer cells, 100 to 1000 times higher than in normal cells. Expression is even higher in advanced prostate cancer and castration-resistant cancer cells, and increases with increasing tumor stage and grade. Although PSMA is not specifically expressed in prostate cancer, it can also be observed in the neovascularization of some normal organs, including the kidneys, salivary glands, lacrimal glands, and small intestine, as well as in some tumors such as renal cell carcinoma. However, the expression level of PSMA in these tissues is lower than that in prostate cancer. Based on these biological characteristics, PSMA has become a promising biological target.

[0005] Different PSMA radioligands have slightly different properties. PSMA inhibitors are mainly divided into three categories: phosphorus-based, thiol-based, and urea-based. The most widely used PSMA PET imaging agents are urea-based small molecules, such as PSMA-11. 68 Ga-PSMA-617 68 Ga-PSMA-I&T 18 F-DCFBC, 18 F-DCFPyL and 18 F-PSMA-1007, etc. Multiple studies have shown that PSMA PET imaging has significant advantages compared with other PET imaging or conventional imaging, including higher specificity, sensitivity, and positive predictive value. 68 Ga-labeled PSMA ligands, as the first PSMA PET imaging agents applied to prostate cancer, have shown promising application prospects in various aspects, including tumor localization, local staging, BCR, metastasis detection, and guiding treatment. [2] .

[0006] Of these ligands, PSMA-11 is the most widely used in clinical practice. 68 Ga-PSMA-11 was approved by the FDA in December 2020 for the treatment of suspected metastatic prostate cancer patients scheduled for initial radical therapy and suspected recurrent prostate cancer patients with elevated PSA levels. Another similar product is LOCAMETZ. ® ( 68Gallium radiopharmaceuticals, such as gallium gozetotide, were approved by FDA in March 2022 for use in patients with suspected metastatic prostate cancer who are candidates for initial curative therapy, patients with suspected recurrent prostate cancer with rising PSA levels, and patients who are candidates for salvage therapy 177 Lu-PSMA-617 targeted therapy for patients with metastatic prostate cancer.

[0007] However, such probes are all nuclide diagnosis or diagnosis and treatment type probes. First, such nuclide probes can only provide diagnostic effects and cannot provide guidance in surgery; second, there is a lack of research and development of more diagnosis and treatment type probes, and currently only 177 Lu-PSMA-617 is a type of radiopharmaceutical approved by FDA for the treatment of prostate cancer, so there is a need to develop a more specific treatment type probe; third, there is a lack of development of PSMA targeted multi-modal molecular probes that are integrated with diagnosis and treatment and can be used for intraoperative navigation. Therefore, there is an urgent need to develop a PSMA targeted probe that is specific, has high tumor uptake, has a long retention time, can be used for intraoperative navigation, and is integrated with diagnosis and treatment, in order to achieve early diagnosis, surgery, and treatment for patients with prostate cancer. SUMMARY

[0008] The purpose of the present application is to provide a dual-targeting multi-modal diagnosis and treatment integrated probe and a preparation method and application thereof. The probe of the present application comprises a labeling tracer part, a target binding ligand, and a cyanine fluorescent dye part. After the probe reaches the body, it can quickly bind to the target tissue, can effectively dual-modality image the target tissue with high expression of the target point and effectively distinguish it, and the probe can effectively perform surgical navigation on the target tissue with high expression of the target point, and is a very promising diagnosis and treatment integrated probe in clinical application.

[0009] To achieve the above-mentioned purpose, the present application provides the following technical solutions:

[0010] In a first aspect, the present application provides a dual-targeting multi-modal diagnosis and treatment integrated probe, which comprises the following parts: a labeling tracer part, a target binding ligand, and a cyanine fluorescent dye part, wherein the labeling tracer part is a group that has been labeled with a radioisotope or a group that can chelate a radioisotope, the cyanine fluorescent dye is a double-claw structure group with two end heterocycles and an intermediate bridge chain, the labeling tracer part is connected to the bridge chain of the cyanine fluorescent dye molecular skeleton, and one target binding ligand is connected to each of the two end heterocycles of the cyanine fluorescent dye molecule, forming a double-ligand structure.

[0011] Preferably, the group that has been labeled with a radioisotope is selected from 18 FAlF-labeled RSECA-TFP, 18 F-labeled N-propargyl-N,N-dimethylammonium methyl boron trifluoride, 18 F-labeled OTS group,18 F-labeled quaternary ammonium salt group, 124 / 125 / 131 I-labeled phenol group, 89 any one or more of Zr-labeled DFO group.

[0012] Preferably, the group that can chelate a radioisotope is selected from any one or more of the group consisting of 68 / 69 Ga or 177 any one or more of Lu-labeled DOTA, NOTA, NODA, NODAGA, DOTP, TETA, NOTAM, DO3AP chelating groups.

[0013] Preferably, the cyanine fluorescent dye is a cyanine fluorescent dye with near-infrared fluorescence.

[0014] Preferably, the target binding ligand is selected from any one or more of the group consisting of PSMA targeting ligand for prostate cancer, FAPI ligand targeting fibroblast target FAP for tumor, ligand targeting somatostatin receptor SSTR for neuroendocrine tumor growth, RGD ligand targeting integrin ανβ3 for tumor, ligand targeting GPC-3 for hepatocarcinoma or hepatocellular carcinoma.

[0015] More preferably, the labeling tracer moiety is DOTA, the target binding ligand is PSMA ligand, and the cyanine fluorescent dye is IR-780.

[0016] Further, the probe has the following molecular structure:

[0017] .

[0018] In a second aspect, the present application provides a preparation method of the dual-targeting multi-modal diagnosis and treatment integrated probe as described above, which comprises: firstly reacting and connecting the target binding ligand with the cyanine fluorescent dye on the heterocycle at both ends of the dye molecule, and then reacting and connecting the labeling tracer moiety with the cyanine fluorescent dye on the bridge chain of the dye molecule, or firstly reacting and connecting the labeling tracer moiety with the cyanine fluorescent dye on the bridge chain of the dye molecule, and then reacting and connecting the target binding ligand with the cyanine fluorescent dye on the heterocycle at both ends of the dye molecule, to form the dual-targeting multi-modal diagnosis and treatment integrated probe with dual target binding ligands and simultaneously with radioisotope tracing and fluorescent tracing capabilities.

[0019] Preferably, the reaction is a click chemistry reaction, wherein the alkyne group and the azido group are introduced at the reaction sites of the target-binding ligand and the cyanine fluorescent dye, respectively, and the reaction is continued after the alkyne group and the azido group are introduced at the reaction sites of the labeling tracer moiety and the cyanine fluorescent dye, respectively, after the reaction, or the alkyne group and the azido group are introduced at the reaction sites of the labeling tracer moiety and the cyanine fluorescent dye, respectively, and the reaction is continued after the alkyne group and the azido group are introduced at the reaction sites of the target-binding ligand and the cyanine fluorescent dye, respectively, after the reaction.

[0020] Preferably, the cyanine fluorescent dye used is IR-780, the labeling tracer moiety is DOTA, and the target-binding ligand is PSMA, and the specific reaction is:

[0021] (a) click chemistry reaction of compound IR-780-Alkyne and compound N3-PSMA to introduce a double PSMA targeting group;

[0022] (b) after step (a) is completed, the product is directly subjected to a substitution reaction without purification to introduce an azido group;

[0023] (c) click chemistry reaction of the reaction product of step (b) and compound PG without purification to introduce an alkyne amine short chain to obtain compound IR-780-C-4;

[0024] (d) condensation reaction of compound IR-780-C-4 and DOTA-NHS to introduce a labeling group to obtain a labeling precursor DOTA-IR-780-C-4.

[0025] Further, the method further comprises a step of radioisotope labeling of the obtained labeling precursor.

[0026] In a third aspect, the present application provides use of the dual-targeting multi-modal theranostic probe as described above or prepared by the method as described above in imaging of a target tissue.

[0027] Preferably, the tissue imaging method is PET or SPECT.

[0028] Further, the target tissue is a tumor tissue.

[0029] In a fourth aspect, the present application further provides use of the dual-targeting multi-modal theranostic probe as described above or prepared by the method as described above in guiding fluorescent surgical navigation.

[0030] Compared with existing technologies, the beneficial effects of this invention are as follows: This invention combines receptor-mediated binding, retention effect, and multimodal strategy to design and synthesize a dual-target, long-retention multimodal imaging probe. By binding to the target, the probe can effectively remain in tissues with high target expression, thus avoiding excessively rapid clearance; through radionuclide labeling, the probe can generate a strong radioactive signal at the target tissue, which is acquired by the instrument and corrected by the computer to obtain an image, thereby achieving more accurate and higher signal-to-background ratio imaging detection and analysis of the target tissue; then, through fluorescence imaging, the long retention and high signal-to-background ratio of the probe at the target tissue mediate surgical navigation for lesion resection. In the embodiments, the probe synthesized according to the method of this invention […]. 68 The GaDOTA-IR-780-C-4 probe was used in clinical PET imaging of prostate cancer patients. The results showed that the probe has good specificity and sensitivity, a high signal-to-background ratio, and is beneficial for the diagnosis of prostate cancer. 69 The GaDOTA-IR-780-C-4 probe is used for rapid detection of prostate cancer tumors and biopsies in patients. It measures the PSMA expression and malignancy of prostate cancer tumors, achieving a combination of imaging and detection for the purpose of rapid diagnosis of prostate cancer. Attached Figure Description

[0031] Figure 1 This is a schematic diagram illustrating the principle and mechanism of the dual-target multimodal integrated diagnostic and therapeutic probe of the present invention;

[0032] Figure 2 For probe [ 68 Ga]DOTA-IR-780-C-4 68 HPLC analysis of Ga radiolabeled synthesis and its stability in PBS and serum, and [ 69 UV and fluorescence spectra of Ga]DOTA-IR-780-C-4;

[0033] Figure 3 For probe [ 68 Binding affinity study of Ga]DOTA-IR-780-C-4;

[0034] Figure 4 For probe [ 69 / 68 Specificity study of Ga]DOTA-IR-780-C-4 in 22RV1 and PC-3 cells (cell uptake and fluorescence imaging).

[0035] Figure 5 For probe [ 68 Ga]DOTA-IR-780-C-4 and [ 68 PET imaging and quantitative analysis of Ga]PSMA-617 in different tumor-bearing mice (Lncap, 22RV1, PC-3) showed that the probe [68 Ga]DOTA-IR-780-C-4 exhibits better targeting specificity, higher tumor uptake, and stronger retention, compared to clinically used drugs. 68 Ga]PSMA-617 showed higher tumor uptake and tumor / muscle uptake ratio compared to tumor uptake, indicating good clinical translational value;

[0036] Figure 6 For probe [ 68 Ga]DOTA-IR-780-C-4 and [ 68 Biodistribution of Ga]PSMA-617 in tumor-bearing mice over 3 h;

[0037] Figure 7 for[ 69 Fluorescence imaging and quantitative analysis of Ga]DOTA-IR-780-C-4 in different tumor-bearing mice (Lncap, 22RV1, PC-3) showed that it has good targeting specificity to detect tumors with different PSMA expression, and has the advantage of fluorescent surgical navigation for tumors with high PSMA expression.

[0038] Figure 8 For probe [ 69 Fluorescence imaging of Ga]DOTA-IR-780-C-4 in different tumor-bearing mice (Lncap, 22RV1, PC-3) for 24 h, fluorescence imaging, HE staining and immunohistochemical analysis of tumor sections;

[0039] Figure 9 For injection probes [ 69 The fluorescent surgical navigation map of Lncap tumor-bearing mice after 24 hours of Ga]DOTA-IR-780-C-4 shows that the probe can indeed guide fluorescent surgical navigation and direct the resection of PSMA-highly expressed tumors.

[0040] Figure 10 The fluorescent biodistribution of tumors and various tissues and organs in tumor-bearing mice after surgical navigation;

[0041] Figure 11 For probe [ 69 Fluorescence distribution of Ga]DOTA-IR-780-C-4 in kidney sections;

[0042] Figure 12 For probe [ 177 Lu]DOTA-IR-780-C-4 177 HPLC analysis of Lu radiolabeled synthesis and its stability in PBS and serum;

[0043] Figure 13 For probe [ 177Lu]DOTA-IR-780-C-4 and [ 177 SPECT imaging and quantitative analysis of Lu]PSMA-617 in Lncap tumor-bearing mice showed that the probe[ 177 Lu]DOTA-IR-780-C-4 has better targeting specificity and long retention at the tumor site;

[0044] Figure 14 For the probe[ 177 Lu]DOTA-IR-780-C-4 and [ 177 The biodistribution of Lu]PSMA-617 in Lncap tumor-bearing mice for 84 h, 177 The Lncap tumor uptake of the Lu]DOTA-IR-780-C-4 group is higher, and the clinical drug[ 177 Compared with Lu]PSMA-617, the tumor / muscle, tumor / heart, and tumor / liver uptake ratios are higher, and it has good clinical radiotherapy conversion potential. DETAILED DESCRIPTION

[0045] The present application develops a dual-targeting multi-modal diagnosis and treatment integrated probe. The probe of the present application comprises a marker tracing part, a target binding ligand and a cyanine fluorescent dye part. After the probe reaches the body, it can quickly bind to the target tissue, can effectively dual-modality image the target tissue with high expression of the target point and effectively distinguish, and the probe can effectively perform surgical navigation on the target tissue with high expression of the target point, and is a very promising diagnosis and treatment integrated probe in clinical application.

[0046] Figure 1 The principle mechanism schematic diagram of the dual-targeting multi-modal diagnosis and treatment integrated probe of the present application is shown. The probe of the present application has enhanced binding force due to dual targeting, and the nuclide marker is used for accurate diagnosis, and the near-infrared fluorescent skeleton is beneficial for surgical navigation. Taking prostate cancer tumor tissue and the high expression of PSMA therein as an example of target tissue and target point, the present application provides a dual-PSMA targeting multi-modal molecular probe and a preparation method thereof, so as to realize more accurate and higher signal-to-background ratio PET and SPECT imaging detection and analysis of high PSMA expression prostate cancer, and to perform intraoperative navigation and resect the lesion.

[0047] In the following examples, a dual-PSMA targeting, long retention multi-modal imaging probe[ 68 / 69 Ga / 177 Lu]DOTA-IR-780-C-4. By binding to PSMA, 68 / 69 Ga / 177 Lu]DOTA-IR-780-C-4 can effectively stay at the high PSMA expression tumor site to avoid rapid clearance; by the radionuclide68 Ga and 177 Lu, the probe can produce a strong radioactive signal at the tumor and be acquired by the instrument, the computer corrects the obtained image to realize more accurate and higher signal-to-background ratio imaging detection and analysis of prostate cancer; then through fluorescence imaging, long retention and high signal-to-background ratio of the probe at the tumor mediate surgical navigation resection of lesions. The 68 Ga]DOTA-IR-780-C-4 is used for clinical PET imaging of prostate cancer patients, and it is concluded that the probe has good specificity and sensitivity, and has high signal-to-background ratio, which is beneficial to the diagnosis of prostate cancer. The 69 Ga]DOTA-IR-780-C-4 is used for rapid detection of prostate cancer tumor and section of patients, determination of PSMA expression and malignancy of prostate cancer tumor, realization of imaging and detection combination, and rapid diagnosis of prostate cancer.

[0048] In the illustrative embodiment, the technical solutions adopted by the present application are as follows:

[0049] A dual-PSMA targeting multimodal molecular probe, which is composed of the following parts:

[0050] A short-chain compound PG connecting the fluorescent skeleton and the DOTA labeling group:

[0051] ;

[0052] N3-PSMA, a specific PSMA targeting ligand:

[0053] ;

[0054] has 68 / 69 Ga and 177 Lu chelate labeling function ligand DOTA-NHS:

[0055]

[0056] DOTA-NHS;

[0057] and the cyanine fluorescent dye (IR-780-Alkyne or IR780-SO3 - ) with near-infrared fluorescence:

[0058] .

[0059] Specifically, the probe has the following molecular structure:

[0060] .

[0061] Further, the present application also provides a preparation method of the above-mentioned dual-PSMA multimodal molecular probe, comprising the following steps:

[0062] (a) introducing a PSMA targeting group by carrying out a click chemistry reaction between compound IR-780-Alkyne and compound N3-PSMA;

[0063] (b) after step (a) is completed, introducing an azide (N3) group by directly carrying out a substitution reaction on the product without purification;

[0064] (c) introducing compound IR-780-C-4 by directly carrying out a click chemistry reaction between the reaction product of step (b) and a PG short chain without purification;

[0065] (d) introducing a labeling group DOTA-NHS by carrying out a condensation reaction on compound IR-780-C-4 to obtain a labeling precursor DOTA-IR-780-C-4;

[0066] The reaction formula of the above-mentioned steps is as follows:

[0067] .

[0068] In step (a), the click chemistry reaction is carried out by dissolving compound IR-780-Alkyne, compound N3-PSMA, anhydrous copper sulfate (CuSO4), sodium ascorbate, and tris(3-hydroxypropyltriazolylmethyl)amine (THPTA) in a solvent; the molar ratio of compound IR-780-Alkyne, compound N3-PSMA, sodium ascorbate, anhydrous copper sulfate, and tris(3-hydroxypropyltriazolylmethyl)amine is 1:2.5, 1:1, 1:2.5, 1:1, the reaction temperature is 25°C, and the reaction is carried out under stirring for 30-45 min, preferably 30 min. The solvent includes but is not limited to dimethyl sulfoxide, preferably dimethyl sulfoxide, and a certain amount of H2O is added (V DMSO :V H2O = 10:1).

[0069] In step (b), sodium azide (NaN3) is dissolved in a solvent and added to the reaction product of step (a) to carry out a substitution reaction; the molar ratio of compound IR-780-Alkyne and sodium azide (NaN3) is 1:2, preferably 1:2; the reaction temperature is 25°C, and the reaction is carried out under stirring for 10-15 min, preferably 15 min. The solvent includes but is not limited to double-distilled water, preferably double-distilled water (double-distilled water).

[0070] In step (c), the compound PG is dissolved in a solvent and added to the reaction product of step (b) to react; wherein the molar ratio of the compound IR-7810-Alkyne to the compound PG is 1:1.2, preferably 1:1.2; the reaction temperature is 25°C, preferably room temperature, and the reaction is carried out under stirring for 30-45 min, preferably 40 min, to obtain a reaction solution containing the compound IR-780-C-4, which is purified and freeze-dried to obtain the compound IR-780-C-4; the solvent includes but is not limited to dimethyl sulfoxide, preferably dimethyl sulfoxide.

[0071] In step (d), the compound IR-780-C-4, DOTA-NHS and N,N-diisopropylethylamine are dissolved in a solvent to react; wherein the molar ratio of the compound IR-780-C-4, DOTA-NHS and N,N-diisopropylethylamine is 1:1.2:3, the reaction temperature is 25°C, and the reaction is carried out under stirring for 2-3 h; the reaction solvent includes but is not limited to DMF, preferably DMF. After the reaction is completed, the reaction solution containing the compound is purified and freeze-dried to obtain the labeling precursor DOTA-IR-780-C-4.

[0072] The DOTA-NHS structure is as follows:

[0073]

[0074] Further, the present application also claims the above-mentioned dual-PSMA multi-modal small molecule probe for detecting the binding to PSMA in vitro.

[0075] Further, the present application also claims the above-mentioned dual-PSMA multi-modal small molecule probe for detecting different prostate cancer tumors in PET and SPECT imaging.

[0076] Further, the present application also claims the above-mentioned dual-PSMA multi-modal small molecule probe for detecting prostate cancer tumors in fluorescence surgical navigation.

[0077] Further, the present application also claims the above-mentioned dual-PSMA multi-modal small molecule probe for detecting prostate cancer patients in clinical PET imaging.

[0078] Further, the present application also claims the above-mentioned dual-PSMA multi-modal small molecule probe for rapid detection of prostate cancer tumors.

[0079] The probe DOTA-IR-780-C-4 of the present application contains a compound capable of performing 68 / 69 Ga / 177Lu radiolabeled DOTA group, N3-PSMA group with PSMA targeting and IR-780-Alkyne group (808 nm) with near-infrared fluorescent group. When intravenous injection of [ 68 / 69 Ga / 177 Lu]DOTA-IR-780-C-4, 68 / 69 Ga / 177 Lu]DOTA-IR-780-C-4 can quickly reach the tumor with high expression of PSMA and bind to PSMA. Due to the double PSMA structure of the probe, the probe binds to PSMA strongly, the retention time in the tumor is long, the tumor uptake belongs to the rising and slowly falling trend, the signal-to-background ratio (with muscle tissue, heart and liver, etc.) is continuously enhanced, the probe is metabolized in non-target tissues (such as heart, liver), and the probe is mainly metabolized through the kidney. First, through the PET imaging of [ 68 Ga]DOTA-IR-780-C-4 in prostate cancer tumor-bearing mice, it is preliminarily judged that the probe has high uptake in the tumor, high signal-to-background ratio, and enhanced tumor-to-muscle uptake ratio, which is beneficial to the early diagnosis of PSMA-related cancer. Then, through the fluorescence imaging of [ 69 Ga]DOTA-IR-780-C-4 in PSMA high expression tumor, the tumor uptake is increased, and the signal-to-background ratio is improved, which is beneficial to the fluorescence surgical navigation research of tumor-bearing mice around 24 h to resect the prostate cancer lesion. Further, through the SPECT imaging of [ 177 Lu]DOTA-IR-780-C-4 in PSMA expression tumor-bearing mice, it is obtained that the probe has high uptake in the tumor, long retention time, and high signal-to-background ratio, which indicates that it has the potential for radiotherapy. Then, [ 68 Ga]DOTA-IR-780-C-4 is used for prostate cancer patients for clinical PET imaging, and it is obtained that the probe has good specificity and sensitivity, and the tumor and metastatic lesion uptake are increased within two hours, and the signal-to-background ratio is increased, which is beneficial to the diagnosis of prostate cancer. Finally, [ 69 Ga]DOTA-IR-780-C-4 is used for rapid detection of prostate cancer tumor and section in patients, to determine the PSMA expression and malignancy of prostate cancer tumor, to realize the combination of imaging and detection, and to achieve the purpose of rapid diagnosis of prostate cancer.

[0080] It should be understood that only specific embodiments shown in the above exemplary embodiments are listed for the purpose of illustrating the principles, but under this principle, a variety of labeling groups, dye skeletons and ligand groups can be selected, and the probes composed of these different components should be covered within the protection scope of the present application.

[0081] For synthesizing probe molecules by selecting different labeling groups, dye skeletons and ligand groups, the following general synthesis flow can be referred to.

[0082]

[0083] General synthetic scheme 1

[0084]

[0085] General synthetic scheme 2

[0086] The above general synthetic scheme 1 is the synthetic route of general labeled probe Q1-IR-780-R2-2, taking near-infrared fluorescent dye IR-780-Alkyne as an example of probe skeleton, any alternative binding type targeting ligand (letter R represents the connected targeting ligand) above can be connected through Click reaction, then after connecting N3, again through Click reaction to connect the labeled group, such as through 18 F or 124 / 125 / 131 I labeled connecting group Q1, through the above "one pot three step" Click reaction to obtain the final labeled precursor probe Q1-IR-780-R2-2.

[0087] The above general synthetic scheme 2 is the synthetic route of general labeled probe Q2-IR-780-R2-3, taking near-infrared fluorescent dye IR-780-Alkyne as an example of probe skeleton, any alternative binding type targeting ligand (letter R represents the connected targeting ligand) above can be connected through Click reaction, then after connecting N3, again through Click reaction to connect the connecting short chain of PG, and finally through condensation reaction to connect the chelating group Q2, such as chelating group of metal ions such as 89 Zr, 18 F]AlF3, 68 Ga or 177 Lu, etc. to obtain the final labeled precursor probe Q2-IR-780-R2-3.

[0088] In addition to the replaceability of targeting ligand and labeled group, the person skilled in the art knows that the near-infrared fluorescent skeleton in the above synthetic route can also have multiple alternatives, for example, in addition to IR-780-Alkyne shown in the above examples, IR780-SO3 -ICG, etc. The person skilled in the art knows that the cyanine near-infrared fluorescent dye is a double claw molecule with two end heterocycles and a middle methine conjugated bridge chain. In the present application, a ligand group can be connected to the two end heterocycles, and a labeling group can be connected to the middle bridge chain to form the multimodal probe with a double ligand structure of the present application. Therefore, the dye molecules with such a double claw structure can be used in the present application. The methine conjugated chain can be a linear carbon chain, such as ICG described above, or a ring structure can be introduced into the carbon chain, such as IR-780 described in the present application. The substitution on the methine chain is well known to the person skilled in the art, and therefore will not be described in detail below.

[0089] The technical solutions in the embodiments of the present application will be described clearly and completely below in combination with specific embodiments of the present application and the accompanying drawings. Obviously, the described embodiments are only some of the embodiments of the present application, but not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by the person skilled in the art without creative labor fall within the scope of protection of the present application.

[0090] In the description of the present application, it should be noted that the terms "upper", "lower", "inner", "outer", "front end", "rear end", "two ends", "one end", "the other end" and the like are only for the convenience of describing the present application and simplifying the description, and do not indicate or imply that the devices or elements referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore cannot be understood as a limitation on the present application. In addition, the terms "first" and "second" are only for descriptive purposes and cannot be understood as indicating or implying relative importance.

[0091] In the description of the present application, it should be noted that unless otherwise explicitly specified and limited, the terms "mounting", "provided with", "connection" and the like should be understood broadly, for example, "connection" can be fixed connection, can also be detachable connection, or integral connection; can be direct connection, can also be indirect connection through an intermediate medium, can be internal communication of two elements. For the person skilled in the art, the specific meaning of the above terms in the present application can be understood according to the specific circumstances.

[0092] In the following examples, the experimental methods are described, and if not specifically stated, they are all conventional methods; the reagents and materials are commercially available, unless otherwise specified.

[0093] In the following examples, "%" is used, unless otherwise specified, which is percentage.

[0094] Reagents and instruments: all chemical reagents and solvents were purchased from Bide, Biotized Technology Co., Ltd. (Shanghai, China), Tianhe (Shanghai, China) Chemical Industry Development Co., Ltd. and Sigma-Aldrich. Analytical solvents and reagents were chromatographically pure, and conventional reagents were analytically pure and not further purified.

[0095] 1 H-NMR spectra were obtained using a 400 MHz Bruker Avance III 400 NMR spectrometer. High performance liquid chromatography (HPLC) was performed using a Thermo Scientific Dionex Ultimate 3000 with CH3CN / H2O (1 ‰ CF3COOH) as eluent. Matrix assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analysis was performed using an AB SCIEX 4800 Plus MALDI TOF / TOF™ mass spectrometer. UV-Vis spectra were measured using an Ocean Optics Maya 2000 Pro spectrometer. Fluorescence spectra were measured using a HORIBA Jobin Yvon Fluoromax-4 fluorimeter. Fluorescence images of cells and tissue sections were acquired using an Olympus IX73 fluorescence inverted microscope. In vivo fluorescence images were acquired using an IVIS Lumina XR III system and the Living image software (PerkinElmer) was used to measure the region of interest signal to quantify the fluorescence intensity. Small animal PET imaging was performed using a Siemens PET / CT imager and small animal SPECT imaging was performed using a PMOD software to acquire and process the images.

[0096] Example 1:

[0097] Design, synthesis and characterization of the probe labeling precursor DOTA-IR-780-C-4

[0098] The synthetic route of the intermediate compound cml-2 is as follows:

[0099]

[0100] Reaction conditions: (a) L-glutamic acid di-tert-butyl ester hydrochloride, triethylamine, triphosgene, dichloromethane, -90 °C→25 °C, 16 h, 70%; (b) 10% palladium on carbon, hydrogen, anhydrous methanol, 25 °C, overnight, 68%;

[0101] Synthesis of compound cml-1 : L-glutamic acid di-tert-butyl ester hydrochloride (500 mg, 1.6 mmol), triethylamine (773 μL, 5.5 mmol) were dissolved in super dry dichloromethane (30 mL) and stirred at -90 °C. Then, triphosgene (170.5 mg, 0.58 mmol) was dissolved in super dry dichloromethane (20 mL) and added dropwise to the above reaction mixture at -90 °C. After the addition was completed, the reaction was continued at room temperature for 30 min. Then, (S)-2-amino-6-(((benzyloxy)carbonyl)amino)hexanoic acid tert-butyl ester hydrochloride (378 mg, 1 mmol) and triethylamine (141 μL, 1 mmol) were added to the above reaction mixture and stirred at room temperature for 16 h. After the reaction was completed, cml-1 (yield 70%) was isolated as a transparent colorless oil by column chromatography.

[0102] Synthesis of compound cml-2: Compound cml-1 was dissolved in 10 mL of methanol-free. Then, palladium on carbon was added and the reaction was carried out overnight under hydrogen gas. After the reaction was completed, palladium on carbon was removed by filtration through celite to obtain cml-2 (yield 68%) as a colorless oil.

[0103]

[0104] Reaction conditions: (a) Fmoc-3-(2-naphthyl)-alanine, DIPEA, dichloromethane, room temperature, 3 h; (b) 20% piperidine; (c) azidoacetic acid, HBTU, DIPEA, DMF, room temperature, 3 h; (d) 1% TFA, 45.7%.

[0105] Synthesis of compound cml-6: After washing the solid-phase peptide synthesis tube with 10 mL of dichloromethane twice, the dichloromethane was removed by suction filtration. 250 mg of 2-chlorotrityl chloride resin (loading: 1.1 mmol / g) was weighed into the solid-phase peptide synthesis tube, 10 mL of super-dry dichloromethane was added, the resin was swelled by shaking for 10 min, and the dichloromethane was removed by suction filtration. (2) Fmoc-3-(2-naphthyl)-alanine (131 mg, 0.3 mmol) was added to the solid-phase peptide synthesis tube, 10 mL of super-dry dichloromethane was added, and the pH was adjusted to 8-9 by DIPEA (108 μL, 0.625 mmol) to make the amino acid loaded onto the resin. (3) After the reaction was completed, the reaction solution was removed by suction filtration, 10 mL of dichloromethane was added to wash the resin, and the suction filtration was repeated 3 times. After 10 mL of a capping solution (DCM / MeOH / DIPEA = 17 / 2 / 1) was added, the resin was shaken for 1 h to cap the sites of the resin to which the amino acid was not loaded. Then, 10 mL of DCM was added to wash the resin 3 times, and the suction filtration was performed, and then 10 mL of DMF was added to wash the resin 3 times, and the suction filtration was performed. (4) 10 mL of a 20% piperidine DMF solution was added, shaken for 10 min, and suction filtered, and the operation was repeated 3 times to remove the Fmoc protecting group from the amino acid, and then 10 mL of DMF was added to wash the resin 3 times to remove the piperidine, and the suction filtration was performed. (5) Azidoacetic acid (22.6 μL, 0.3 mmol) and HBTU (113 mg, 0.3 mmol) were weighed into the solid-phase peptide synthesis tube, 10 mL of super-dry DMF was added, and the pH was adjusted to 8-9 by DIPEA (130 μL, 0.75 mmol). After shaking at room temperature for 3 hours, the reaction was completed, the reaction solution was removed by suction filtration, 10 mL of DMF was added to wash the resin, and the suction filtration was repeated 3 times, and then the operation of (4) was repeated to complete the peptide synthesis. 10 mL of a 1% TFA dichloromethane solution was added to the solid-phase peptide synthesis tube, shaken for 10 min, and the filtrate was collected, and the operation was repeated until the color of the resin was wine red and did not fade. The organic solvent was removed from the obtained filtrate by a rotary evaporator under reduced pressure, cold diethyl ether was added, and a yellow oil was precipitated, which was transferred to a 50 mL centrifuge tube, centrifuged at high speed, and the supernatant was removed. The obtained product was placed in a vacuum drying oven and vacuum dried for 1 hour to obtain the peptide cml-6 (yield 45.7%).

[0106]

[0107] Reaction conditions: (a) cml-2, cml-6, HBTU, DIPEA, THF, r.t., 3 h; (b) TFA:DCM:TiPS = 65:33:2, r.t., 2 h, 74%.

[0108] Synthesis of compound N3-PSMA: cml-6 (34 mg, 0.11 mmol), cml-2 (61 mg, 0.13 mmol) and HBTU (50 mg, 0.13 mmol) were dissolved in 5 mL THF, DIPEA (60 μL, 0.33 mmol) was added, the pH was adjusted to 8-9, N2 protection and the reaction was stirred at room temperature for 3 h, then the organic solvent was removed to obtain the crude product cml-7. Next, TFA:DCM:Tips = 65:33:2 (10 mL) was added, the reaction was stirred at room temperature for 2 h, and then the white solid N3-PSMA was precipitated with cold ether.

[0109]

[0110] Synthesis of compound DOTA-IR-780-C-4: First, the near-infrared fluorescent dye IR-780-Alkyne was synthesized according to the reported method (J. Am. Chem. Soc. 2020, 142, 2787-2794.). (a) Compound N3-PSMA (308 mg, 0.51 mmol) and IR-780-Alkyne (100 mg, 0.17 mmol) were dissolved in DMSO, CuSO4(27 mg, 0.17 mmol), sodium ascorbate (67 mg, 0.34 mmol) and THPTA (74 mg, 0.17 mmol) were dissolved in double distilled water and added to the DMSO, and the PSMA group was introduced by stirring at room temperature for 30 min. (b) Subsequently, NaN3(22.1 mg, 0.34 mmol) dissolved in double distilled water was directly added to the reaction solution, and the N3 group was introduced by stirring at room temperature for 12 min. (c) Finally, compound PG (28.6 mg, 0.26 mmol) was dissolved in DMSO, which was added to the reaction solution, and CuSO4(27 mg, 0.17 mmol), sodium ascorbate (67 mg, 0.34 mmol) and THPTA (74 mg, 0.17 mmol) dissolved in double distilled water (200 μL) were added, and the short peptide PG was introduced by continuing to stir at room temperature for 30 min. After the reaction was completed, the residue was purified by preparative HPLC, and the compound IR-780-C-4 was obtained as a dark green solid by lyophilization. Yield: 129 mg (40 %). MS: calcd. for C 99 H 118 N 21 O 19 + [M + ]:1904.8907; found MALDI-MS: m / z 1904.7325.

[0111] Synthesis of compound DOTA-IR-780-C-4: (d) DOTA-NHS (2.6 mg, 0.0057 mmol), compound IR-780-C-4 (10 mg, 0.0053 mmol) and DIPEA (3 μL, 0.0159 mmol) were dissolved in DMF (1 mL) solution, stirring at room temperature for 2-3 hours to introduce the labeling group DOTA. After the reaction was completed, the residue was purified by preparative HPLC, and the compound DOTA-IR-780-C-4 was obtained as a dark green solid by lyophilization. Yield: 10 mg (77%). MS: calcd. for C 116 H 144 N 25 O 28 [M 2 + ]: 2291.0709; found MALDI-MS: m / z 2291.0310.

[0112] Other synthesis examples:

[0113] In addition to DOTA-IR-780-C-4, probe molecules with different targeting ligands, labeling groups and dye backbones were also synthesized, which are summarized as follows in categories:

[0114] 1) The backbone can be connected to different ligands (such as PSMA targeting ligand, FAP targeting ligand and integrin αvβ3 targeting ligand, etc.)

[0115]

[0116] 2) The backbone can be connected to different 18 F or 124 / 125 / 131 I labeling groups (such as 18 F-labeled OTS group, 18 F-labeled quaternary ammonium salt group, 124 / 125 / 131 I-labeled phenol group, etc.)

[0117]

[0118] 3) The backbone can be connected to different 89 Zr, [ 18 F]AlF3, 68 Ga or 177 Lu, etc. metal ion chelating groups (such as DOTA, RESCA-TFP, NOTA and DFO-NCS, etc.)

[0119]

[0120] 4) Can connect to different skeletons, such as IR-780-Alkyne and IR-780-SO3 - wait

[0121]

[0122] The amounts of reactants fed in the above reactions are as follows:

[0123] Fluorescent dye: Targeting ligand: Sodium azide NaN3: CuSO4·5H2O: Sodium ascorbate: THPTA: 18 F or 124 / 125 / 131 I-type labeled groups = 1:2.4-3:2-2.5:1:2-2.5:1:1.2-1.5.

[0124] Fluorescent dye: Targeting ligand: Sodium azide NaN3: CuSO4·5H2O: Sodium ascorbate: THPTA: Propylene amino group: Metal chelating group = 1:2.4-3:2-2.5:1:2-2.5:1:1.2-1.5:1.2-1.5, DIPEA adjusts the alkalinity to pH = 8-9.

[0125] Probe performance testing

[0126] 1. [ 68 Radioactive synthesis and physicochemical properties of Ga]DOTA-IR-780-C-4.

[0127] See Figure 2 In step a, HCl (0.05M) was used to... 68 Ge / 68 Ga generator rinsing yield 68 After Ga, sodium acetate buffer (0.25M) was added to adjust the pH of the reaction solution to 4-4.5, with a hydrochloric acid to sodium acetate volume ratio of 1 mL:0.285 mL. Then, the labeled precursor DOTA-IR-780-C-4 (10 μL, 30 μg) was added and mixed thoroughly. The mixture was reacted at 90℃ for 15 min, followed by HPLC analysis with shaking every 5 minutes. Finally, the C-18 column was activated (with ethanol and water), the probe was attached to the C-18 column, and the column was rinsed with water to remove free precursors. 68 Ga ions were then used to wash the product into a flask with ethanol to obtain the purified product. 68 [Ga]DOTA-IR-780-C-4. The purity of the drug was analyzed by radioactive HPLC before and after purification, showing that the probe had good reactivity, high radiochemical purity, and high yield. Figure 2 (b) The purified samples were reacted with PBS ( Figure 2 c) and serum ( Figure 2(d) After incubation for 1, 2, and 3 hours, the sample purity was analyzed by radioactive HPLC to assess its stability. HPLC results showed that the probe exhibited high stability in both PBS and serum, with a radiochemical purity >97%, indicating good stability. Furthermore, the probe possesses good hydrophilicity, which is beneficial for subsequent biological studies. In addition, cold compounds [ 69 Ga]DOTA-IR-780-C-4 was analyzed by ultraviolet absorption spectroscopy ( Figure 2 (e) and fluorescence spectrum ( Figure 2 As shown in f), the probe also possesses good near-infrared optical properties, which is beneficial for subsequent fluorescent surgical navigation research. Specific results and parameters are shown in Table 1 below.

[0128] Table 1 [ 68 Radiosynthesis and physicochemical properties of Ga]DOTA-IR-780-C-4

[0129]

[0130] 2. Study on the binding affinity of the probe to PSMA

[0131] See Figure 3 In the case of 'a', different concentrations of [ 68 Ga]DOTA-IR-780-C-4 or [ 68 Ga]PSMA-617 and adherent 22RV1 cells (250,000 / well) were incubated at 4℃ for 1 h; and different concentrations of [ 68 Ga]DOTA-IR-780-C-4 or [ 68 [Ga]PSMA-617 and its 1000-fold concentrated inhibitor 2-PMPA were incubated with 22RV1 cells (250,000 cells / well) at 4°C for 1 h. Both experiments were performed in triplicate. After incubation, the supernatant was removed, and the cells were washed twice with PBS. Cells were then cleaved with sodium hydroxide, and their radioactivity dose was measured using a gamma counter. The data were fitted using Graphpad software. 68 Ga]DOTA-IR-780-C-4 and [ 68 Ga]PSMA-617's affinity for PSMA binding to the 22RV1 cell membrane surface. See the detailed results below. Figure 3 In step b, the introduction of dual PSMA targeting significantly enhances the binding affinity between the probe and the target, increasing it by approximately two times. Figure 3 As shown in c in the figure.

[0132] 3. Probe [ 68 / 69 Specificity study of Ga]DOTA-IR-780-C-4 in prostate cancer cells

[0133] (A) will [ 68Ga]DOTA-IR-780-C-4 or [ 68 GaPSMA-617 (5 μCi) and 22RV1, 22RV1+2-PMPA (2-PMPA pre-incubated with 22RV1 cells 0.5 h), and PC-3 cells (250,000 cells / well) were incubated at 37°C for 0.5 h, 1 h, and 2 h, respectively, with three replicates for each group. After incubation, the supernatant was removed, and the cells were washed twice with PBS. Cells were then digested with trypsin, and their radioactivity dose was measured using a gamma counter. Finally, the relative uptake of the probe by the cells was calculated (uptake % = cell radioactivity count / drug-treated radioactivity count * 100%). Results are shown below. Figure 4 As shown in a, [ 68 [Ga]DOTA-IR-780-C-4 exhibits good targeting specificity and can significantly distinguish prostate cancer cells with different PSMA expressions. Furthermore, at 2 hours, 22RV1 cells showed […]. 68 Ga]DOTA-IR-780-C-4's uptake is higher than [ 68 Ga]PSMA-617 2 times, dual PSMA targeting can enhance the uptake of the probe by tumor cells and enhance its specificity.

[0134] (B) 69 [Ga]DOTA-IR-780-C-4 (5 μM) was incubated with 22RV1, 22RV1+2-PMPA (2-PMPA was incubated with 22RV1 cells 0.5 h in advance), and PC-3 cells at 37℃ for 2 h. After removing the supernatant, Hoechst was added and incubation continued for 15 min. After removing the supernatant again, the cells were washed twice with PBS, and serum-free culture medium was added before fluorescence imaging. The results are as follows: Figure 4 As shown in b, the fluorescent probe [ 69 Ga]DOTA-IR-780-C-4 also exhibits good targeting specificity, significantly distinguishing prostate cancer cells with different PSMA expressions, and possesses good near-infrared optical properties that are beneficial for subsequent in vivo fluorescence imaging.

[0135] 4. In vivo PET imaging of tumor-bearing mice with different PSMA expression levels using probes.

[0136] See Figure 5 In step a, 15 male NCG mice aged 5-6 weeks were used to construct tumor-bearing mouse models with different PSMA expression levels by injecting prostate cancer tumor cells into the right axilla. Tumors with a diameter of approximately 0.6-0.8 cm were suitable for imaging studies. [The text then abruptly shifts to a different topic:] ...containing [... 68 Ga]DOTA-IR-780-C-4 or [ 68Ga]PSMA-617 (100 μCi) in saline solution was injected into tumor-bearing mice via the tail vein. Whole-body images were acquired using a PET scanner at 1 h, 2 h, and 3 h post-injection.

[0137] Inject into the tail vein [ 68 Following Ga]DOTA-IR-780-C-4, significant radioactive signals were rapidly detected at Lncap tumor sites overexpressing PSMA, with time-dependent tumor uptake ( Figure 5 b) and the credit-to-back ratio ( Figure 5 c) in the text all showed a continuous increase. Furthermore, this probe exhibited good targeting specificity, distinguishing tumors with different PSMA expression levels (Lncap>22RV1>PC-3). Meanwhile, [ 68 At a time point of 3 h, the uptake of Ga]DOTA-IR-780-C-4 in positive Lncap tumors was significantly higher than that in [[Ga]DOTA-IR-780-C-4]. 68 Ga]PSMA-617 was 2.6 times better (14.27 ± 1.10 % ID / g vs 5.40 ± 1.64 % ID / g), and the signal-to-back ratio was 1.8 times better (22.80 ± 1.23 vs 12.41 ± 1.52).

[0138] After 3 hours of PET imaging, the tumors and major organs of the tumor-bearing mice were removed and their radioactivity levels were measured using a gamma counter. Figure 6 From this, we can see that, compared to other groups, those injected with [ 68 Lncap tumor-bearing mice with Ga]DOTA-IR-780-C-4 showed higher radioactive uptake at the tumor site, significantly higher than those with moderately expressed 22RV1 and low expressed PC-3. Furthermore, [ 68 Ga]DOTA-IR-780-C-4 uptake at the tumor site in Lncap tumor-bearing mice was higher than [ 68 Ga]PSMA-617 2.8 times ( Figure 6 (a) The letter-to-back ratio is 1.5 times higher ( Figure 6 (b) The results are basically consistent with those of PET imaging, demonstrating its excellent targeting specificity, which is beneficial for subsequent clinical diagnostic applications.

[0139] 5. Application of near-infrared fluorescence imaging and fluorescence surgical navigation in tumor-bearing mice with different PSMA expression levels at the in vivo level.

[0140] See Figure 7 In part a, for tumor-bearing mice that have undergone PET imaging, a tail vein injection is performed. 69Ga]DOTA-IR-780-C-4 (50 μM, 200 μL) was subjected to near-infrared fluorescence imaging before injection and at 0.5 h, 1 h, 2 h, 4 h, 6 h, 8 h, 12 h, and 24 h after injection. Figure 7 As observed, this fluorescent probe also exhibits good targeting specificity, distinguishing between tumor-bearing mice with different PSMA expressions. Furthermore, in addition to high fluorescence intensity detected at the tumor site in positive Lncap tumor-bearing mice, the probe demonstrates a prolonged retention effect at the tumor site, maintaining high fluorescence intensity even after 24 hours. Figure 7 (b) and the credit-to-back ratio continues to rise, reaching its highest point at 24 hours. Figure 7 (c) This is beneficial for subsequent research on fluorescent surgical navigation applications. After completing near-infrared fluorescence imaging, the mouse tumor was dissected and removed for near-infrared fluorescence imaging, HE staining, and IHC immunohistochemistry of tissue sections. Figure 8 It can be seen that the probe still has good targeting specificity at the tissue level and high tissue penetration. The results are consistent with HE staining and immunohistochemistry, indicating its high specificity.

[0141] After completing the above fluorescence imaging, the next step is to use the near-infrared fluorescent probe [ 69 Ga]DOTA-IR-780-C-4 applied to surgical navigation. Tail vein injection [ 69 After 24 hours of treatment with Ga]DOTA-IR-780-C-4 (50 μM, 200 μL), fluorescence surgical navigation was performed. Figure 9 In the study, we observed significant near-infrared fluorescence signals only at the tumor site, clearly delineating the tumor margins and facilitating rapid tumor dissection while minimizing damage to normal tissue. After completing the surgical navigation experiment, mice were dissected to examine the fluorescence distribution in tumor and normal tissues. Figure 10 As shown, the probe is mainly distributed in tumors and kidneys, and almost no obvious near-infrared fluorescence can be detected in other normal tissues. Figure 10 (a and b in the text). Because the probe has a relatively long circulation time in the blood, PSMA is expressed to some extent in the renal tubules, leading to tubular reabsorption. Therefore, its metabolism in the kidneys is slightly slower, but this does not affect the probe's performance (e.g., ...). Figure 11 (As shown).

[0142] 6. [ 177 Radiosynthesis and in vitro stability study of Lu]DOTA-IR-780-C-4

[0143] because[ 68 / 69Ga]DOTA-IR-780-C-4 has high uptake and long retention in positive Lncap tumor, which is beneficial for converting to radiotherapy probe to study its radiotherapy potential. Therefore, the diagnostic probe[ 68 Ga]DOTA-IR-780-C-4 is converted to radiotherapy probe[ 177 Lu]DOTA-IR-780-C-4 to study its radiotherapy application potential. See Figure 12 a, first add HCl (0.05M) containing 177 Lu to sodium acetate buffer (0.25M) to adjust the pH of the reaction solution to 4-4.5, wherein the volume ratio of hydrochloric acid to sodium acetate is 1 mL:0.285 mL; then add the labeling precursor DOTA-IR-780-C-4 (10 μL, 30 μg) and mix well, and perform HPLC analysis after 90°C reaction for 15 min, with shaking every 5 min; finally, activate the C-18 column (ethanol and water), hang the probe on the C-18 column, and wash the free 177 Lu ion with water, then elute the product into a bottle with ethanol to obtain the purified product[ 177 Lu]DOTA-IR-780-C-4. The radiochemical purity of the probe before and after purification is analyzed by radio-HPLC, and the results show that the probe has good reactivity, high radiochemical purity and yield (b in Figure 12 c) and serum (d in Figure 12 c) and serum (d in Figure 12 c) and serum (d in

[0144] Table 2 177 Lu]DOTA-IR-780-C-4 radiochemical synthesis and in vitro stability parameters

[0145]

[0146] 7. SPECT imaging of Lncap tumor-bearing mice in vivo by probe

[0147] See Figure 13 a, 6 five to six-week-old male NCG mice are injected with prostate cancer cells through the right axillary to construct a Lncap tumor-bearing mouse model. When the tumor diameter is about 0.6-0.8 cm, it can be used for SPECT imaging study. The 177 Lu]DOTA-IR-780-C-4 or 177Lu]PSMA-617 (300 μCi) in saline solution was injected into tumor-bearing mice via the tail vein. Whole-body images were acquired using SPECT imaging at 24 h, 48 h, and 72 h post-injection.

[0148] like Figure 13 As shown in b and c, injection is performed via the tail vein. 177 Following Lu]DOTA-IR-780-C-4, significant radioactive signals were detected at Lncap tumor sites overexpressing PSMA. 177 At positive Lncap tumors, the uptake of Lu]DOTA-IR-780-C-4 was significantly higher than that of [[ 177 Lu]PSMA-617, and has long retention at the tumor site and high signal-to-back ratio.

[0149] Following SPECT imaging, tumors and major organs of Lncap tumor-bearing mice were removed 84 hours after probe injection, and their radioactivity levels were measured using a gamma counter. The probe showed up to 93.7% ID / g uptake at the tumor site after 84 hours. Furthermore, from... Figure 14 We can monitor that, 177 Lu]DOTA-IR-780-C-4 uptake at the tumor site in Lncap tumor-bearing mice was higher than [ 68 Ga]PSMA-617 21.8 times ( Figure 14 (a) The letter-to-back ratio is 2.8 times higher ( Figure 14 (b) Meanwhile, except for the kidneys, the probe showed higher uptake ratios in tumors compared to the heart, liver, and muscles, indicating […]. 177 Lu]DOTA-IR-780-C-4 has potential for radiotherapy applications.

[0150] Any aspects of this invention not described in detail are well-known to those skilled in the art.

[0151] Finally, it should be noted that the above specific embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to the embodiments, those skilled in the art should understand that modifications and equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications and substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A dual-targeting diagnostic and therapeutic integrated probe, characterized in that, It has the following molecular structure: Where R = H or sulfonic acid group.

2. A method for preparing a dual-targeting, multimodal, integrated diagnostic and therapeutic probe, characterized in that, The method reaction is: (a) The compound IR-780-Alkyne was reacted with the compound N3-PSMA by click chemistry to introduce a dual PSMA targeting group; (b) After step (a) is completed, the product is not purified and directly subjected to a substitution reaction to introduce an azide group; (c) The reaction product of step (b) is not purified and is directly reacted with compound PG by click chemistry to introduce short chains of acetyleneamine to obtain compound IR-780-C-4. (d) Compound IR-780-C-4 was condensed with DOTA-NHS to introduce a labeling group, yielding the labeled precursor DOTA-IR-780-C-4. The resulting labeled precursor was then radiolabeled with an isotope. The reaction equations for the above steps are as follows:

3. The use of the dual-targeting therapeutic probe as described in claim 1 in the preparation of reagents for PSMA-expressing tissue imaging.

4. The use as described in claim 3, wherein the tissue imaging method is PET or SPECT.

5. The use of the dual-targeting therapeutic probe as described in claim 1 in the preparation of reagents for guiding surgical navigation of PSMA-expressing tissues with fluorescence.

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