Nucleotide compound, preparation method therefor and use thereof, nuclide targeting probe, preparation method therefor and use thereof, and pharmaceutical composition

Abstract

The present invention relates to the technical field of biomedicine, and particularly relates to a nucleotide compound, a preparation method therefor and a use thereof, a nuclide targeting probe, a preparation method therefor and a use thereof, and a pharmaceutical composition. The nucleotide compound and a nuclide labeled complex provided in the present invention have excellent in-vivo biological properties, high specific uptake in lesions having high expression of CD73 protein, a high target / non-target ratio and a low non-specific background. The characteristics can reduce unnecessary radioactive damage to normal tissues and organs, and are extremely suitable for the diagnosis and treatment of tumors. In addition, the nuclide targeting probe can also be used in combination with other immune checkpoint inhibitors (such as PD-1 / PD-L1 monoclonal antibodies) for combined treatment. In addition, the nuclide targeting probe performs visual quantitative analysis on CD73 dynamic expression in a tumor microenvironment by means of nuclear medicine imaging, thereby overcoming the defects of wrong sampling and poor accuracy of conventional detection methods.

Description

Nucleotide compound and preparation method and application thereof, nuclide targeting probe and preparation method and application thereof, pharmaceutical composition

[0001] This application claims priority to the Chinese patent application filed with the State Intellectual Property Office on September 5, 2023, with application number CN202311133397.7 and invention name “Nucleotide compounds, preparation methods and applications thereof, radionuclide targeted probes, preparation methods and applications thereof, and pharmaceutical compositions”, the entire contents of which are incorporated by reference into this application. Technical Field

[0002] The present invention belongs to the field of biomedicine technology, and specifically relates to nucleotide compounds and preparation methods and applications thereof, pharmaceutically acceptable salts of nucleotide compounds, radionuclide targeting probes and preparation methods and applications thereof, pharmaceutically acceptable salts of radionuclide targeting probes, and pharmaceutical compositions. Background Art

[0003] CD73 (ecto-5′-nucleotidase) is a multifunctional transmembrane protein composed of 532 amino acids encoded by the NT5E gene. It is anchored to the plasma membrane after binding to glucosylphosphatidylinositol (GPI) via a C-terminal serine residue, forming a non-covalently bound homodimer with variable conformations. CD73 exists in two conformations: "open" and "closed." The "closed" conformation exposes the active binding site, allowing binding to the substrate 5'-adenosine monophosphate (AMP), providing a theoretical basis for the rational design of CD73 small molecule inhibitors. CD73 is expressed on the surface of a variety of cells, including dendritic cells (DC cells), regulatory T cells (Treg cells), NK cells, myeloid-derived suppressor cells (MDSC cells), tumor-associated macrophages (TAM cells), and cancer cells, and is an important component of the purinergic pathway. This pathway utilizes the sequential degradation of CD39 (ectonucleoside triphosphate diphosphohydrolase-1) and CD73 to convert extracellular ATP into extracellular adenosine (ADO), and is involved in biological processes including neurotransmission, platelet aggregation, regulation of immune responses, smooth muscle contraction, and control of cell proliferation, differentiation, and apoptosis. In tumor tissue, ADO interacts with specific G protein-coupled receptors (A1R, A2AR, A2BR, and A3R) to reshape different types of cells, including immune cell infiltration and tumorigenic angiogenesis, promoting tumor occurrence and metastasis, and is a powerful immunosuppressive molecule.

[0004] In recent years, tumor immunotherapy has become a research hotspot in both academia and industry, revolutionizing cancer treatment. While immune checkpoint inhibitors, such as PD-1 / PD-L1 monoclonal antibodies, offer promising results, low response rates remain a pressing challenge in clinical application. This is primarily due to individual differences and heterogeneity in marker expression; suppressed immune cell infiltration and function within the tumor microenvironment; and the development of primary or even adaptive resistance to immunotherapy. Therefore, transforming "cold" tumors, which are unresponsive or poorly responsive to immunotherapy, into "hot" tumors is a current research priority. In the purinergic pathway, adenosine, generated by the synergistic conversion of extracellular ATP through CD39 and CD73, is a key regulator of innate and adaptive immunity within the tumor microenvironment. A growing body of research indicates that tumor cells can exploit CD73-mediated adenosine production to evade immune attack. The CD73-adenosine pathway is strongly associated with tumor development and progression, and a growing body of research highlights the potential value of this target as a new biomarker for personalized cancer treatment.

[0005] CD73 is overexpressed in a variety of solid tumors, such as breast cancer, melanoma, and colorectal cancer, directly or indirectly promoting tumor growth, metastasis, angiogenesis, and the development of drug resistance. Due to factors such as tissue hypoxia, inflammation, and cytotoxic stress, the tumor microenvironment upregulates CD73. The adenosine generated by this catalysis can strongly inhibit the immune activity of various immune cells, especially T cells and antigen-presenting cells (APCs). On the other hand, adenosine can promote the proliferation of Treg cells and enhance their suppressive activity, stimulate macrophage M2 polarization, and provide conditions for tumor formation and metastasis. In addition, studies have shown that radiotherapy, chemotherapy, and the use of PD-1 / PD-L1 monoclonal antibodies can significantly upregulate CD73 expression, which in turn produces immunosuppression through catalytically generated adenosine, leading to acquired drug resistance. Targeting CD73 can produce a good anti-tumor effect, and by inhibiting adenosine production, it can alleviate the inhibitory effect of the tumor microenvironment on immune cells. Therefore, the combination of CD73 inhibitors and immune molecule modulators (such as CTLA-4 and PD-1 / PD-L1 antibodies) is an extremely attractive option. This strategy effectively addresses the inherent limitations of immunotherapy, transforming "cold tumors" into "hot tumors," resulting in a "1+1>2" therapeutic effect. Its demonstrated synergistic effect has become a new hotspot in anti-tumor research. Given the complexity and heterogeneity of tumor immune targets, imaging-guided tumor immunotherapy is gaining increasing attention. Following the remarkable success of immunoPET / SPECT targeting immune checkpoints such as PD-1 / PD-L1, researchers are focusing on developing specific probes for CD73, a key immune target, and developing tailored nuclear medicine imaging solutions for it.

[0006] Whether it is research on immune mechanisms or clinical diagnosis and treatment needs, the rationality and necessity of constructing molecular probes around the CD73 target have been demonstrated. However, to date, there are very few studies using molecular imaging technology to obtain in vivo CD73 information, and there are no reports on the use of specific radioactive small molecule probes to image tumor CD73 expression and guide combined CD73 inhibitor treatment. Therefore, the development of high-resolution molecular imaging technology PET / SPECT combined with highly sensitive and specific CD73-targeted probes is of great significance for accelerating related immune mechanism research, deepening people's understanding of this target, and broadening the scope of application of immunotherapy.

[0007] Summary of the Invention

[0008] In view of this, the present invention aims to provide nucleotide compounds, preparation methods and uses thereof, pharmaceutically acceptable salts thereof, radionuclide targeting probes, preparation methods and uses thereof, pharmaceutically acceptable salts thereof, and pharmaceutical compositions. The nucleotide compounds, pharmaceutically acceptable salts thereof, radionuclide targeting probes, pharmaceutically acceptable salts thereof, and pharmaceutical compositions provided herein all have CD73 targeting properties.

[0009] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions.

[0010] The present invention provides a nucleotide compound having the structure shown in Formula I:

[0011] In the formula I: L is R1 is n and m are independently integers of 0 to 10; R5 includes -H or -CH3; R2 includes -H or halogen; R4 includes -OH or -F; X includes =N- or =CH-;

[0012] R3 includes -H or any of the following structures:

[0013] The present invention provides a method for preparing the nucleotide compound described in the above technical solution, comprising the following steps:

[0014] Compound A and compound B are subjected to a coupling reaction under Lewis acid catalysis to obtain compound C;

[0015] Performing a first substitution reaction between the compound C and the compound D under alkaline conditions to obtain a compound E;

[0016] The compound E is subjected to a ribose protecting group removal reaction to obtain a compound F;

[0017] The compound F is subjected to a second substitution reaction with methylenebis(phosphine dichloride) followed by a deprotection reaction to obtain compound G;

[0018] Performing a third substitution reaction between the compound G and the R3 active compound to obtain the nucleotide compound with the labeling group;

[0019] In the compounds A to G, R1 to R5, L and X are the same as R1 to R5, L and X in the formula I, A is a -F or -OBz ​​group, and R7 includes a Boc, DDE or Fmoc protecting group;

[0020] The R3 active compound is a compound with an active reactive group corresponding to R3, and can be selected from the following structures:

[0021] The present invention provides a pharmaceutically acceptable salt of a nucleotide compound, which is obtained by reacting the nucleotide compound with an acid or a base; the nucleotide compound is the nucleotide compound described in the above technical solution or the nucleotide compound prepared by the preparation method described in the above technical solution.

[0022] The present invention provides a nuclide targeting probe having a structure shown in Formula II:

[0023] In the formula II, R1 to R5, L and X are the same as R1 to R5, L and X in the nucleotide compound according to claim 1;

[0024] The R6 is obtained by the coordination reaction between the R3 coordination group and the labeled nuclide.

[0025] Preferably, the labeled nuclides include 18 F. 47 Sc, 64 Cu, 67 Cu, 67 Ga, 68 Ga, 89 Zr, 86 Y. 89 Sr, 90 Y. 99m Tc, 105 Rh, 109 Pd, 111 In, 119 Sb, 149 Tb, 153 Sm, 157 Gd, 161 Tb, 166 Ho, 177 Lu, 186 Re、188 Re、 201 Tl, 203 Pb, 212 Pb, 212 Bi, 213 Bi, 223 Ra, 227 Th and 225 At least one of Ac.

[0026] The present invention provides a method for preparing the nuclide targeting probe described in the above technical solution, the preparation method comprising the following steps:

[0027] A nucleotide compound or a pharmaceutically acceptable salt thereof is subjected to a coordination reaction with a labeled nuclide to obtain a nuclide-targeted probe or a pharmaceutically acceptable salt thereof; the nucleotide compound or the pharmaceutically acceptable salt thereof is the nucleotide compound described in the above technical solution or the nucleotide compound or the pharmaceutically acceptable salt thereof obtained by the preparation method described in the above technical solution;

[0028] The present invention provides a pharmaceutically acceptable salt of a nuclide targeting probe, including a trifluoroacetate, hydrochloride, formate, potassium salt, or sodium salt of the nuclide targeting probe; the nuclide targeting probe is the nuclide targeting probe described in the above technical solution or the nuclide targeting probe prepared by the preparation method described in the above technical solution. The present invention provides a pharmaceutically acceptable salt of a nuclide targeting probe, obtained by a salt-forming reaction of a nuclide targeting probe or a nucleotide compound; the nuclide targeting probe is the nuclide targeting probe described in the above technical solution or the nuclide targeting probe prepared by the preparation method described in the above technical solution; the nucleotide compound is the nucleotide compound described in the above technical solution or the nucleotide compound prepared by the preparation method described in the above technical solution.

[0029] The present invention provides a pharmaceutical composition comprising an active component and a pharmaceutically acceptable excipient; the active component comprises a nucleotide compound, a nuclide targeting probe, a pharmaceutically acceptable salt of the nucleotide compound described in the above technical solution, and one or more of the pharmaceutically acceptable salts of the nuclide targeting probe described in the above technical solution; the nucleotide compound is the nucleotide compound described in the above technical solution or a nucleotide compound prepared by the preparation method described in the above technical solution; the nuclide targeting probe is the nuclide targeting probe described in the above technical solution or a nuclide targeting probe prepared by the preparation method described in the above technical solution.

[0030] The present invention provides the use of the nucleotide compound described in the above technical solution, the nucleotide compound prepared by the preparation method described in the above technical solution, the pharmaceutically acceptable salt of the nucleotide compound described in the above technical solution, the nuclide targeting probe described in the above technical solution, the nuclide targeting probe prepared by the preparation method described in the above technical solution, the pharmaceutically acceptable salt of the nuclide targeting probe described in the above technical solution, or the pharmaceutical composition described in the above technical solution in the preparation of therapeutic drugs or diagnostic drugs for diseases mediated by CD73 protein.

[0031] Preferably, the disease mediated by the CD73 protein includes tumors or immune-related diseases.

[0032] In the nucleotide compound having the structure shown in Formula I or the nuclide targeting probe shown in Formula II provided by the present invention, the coordination group R3 has the characteristics of strong labeling ability, short labeling time, and high labeling yield. As a precursor compound, it can chelate with the labeled nuclide to form a CD73-targeted nuclide targeting probe with high affinity and high specificity, which is conducive to the commercial application and clinical promotion of the nuclide targeting probe. The present invention introduces a molecular chain L with adjustable chain length between the nucleotide targeting group and the coordination structure R3, which can adjust the distance between the targeting group and the coordination structure and reduce the effect of the coordination structure on the activity of the compound. At the same time, the structure changes the lipid-water distribution properties of the nuclide targeting probe by connecting with different types of nuclide chelating groups, thereby obtaining nuclide targeting probes of different polarity types. The change in small molecule polarity can also improve the pharmacokinetic properties of the nuclide targeting probe, accelerate the clearance rate of the tracer in non-target tissues, enhance the affinity between the nuclide targeting probe and the receptor, improve the uptake of the nuclide targeting probe in lesions with high expression of CD73 protein, increase the target / non-target ratio, and make it have excellent in vivo biological performance. This property can reduce unnecessary radiation damage to normal tissues and organs, making it very suitable for the diagnosis and treatment of tumors. In addition, this radionuclide marker can also be used in combination with other immune checkpoint inhibitors (such as PD-1 / PD-L1 monoclonal antibodies) for combined therapy.

[0033] The radionuclide marker provided by the present invention uses nuclear medicine imaging to perform visual quantitative analysis of the dynamic expression of CD73 in the tumor microenvironment, overcoming the shortcomings of traditional detection methods such as sampling errors and poor accuracy, and providing a new research method for exploring the role of CD73 in tumor occurrence and development. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] FIG1 is a mass spectrometric identification diagram of the compound DOTA-dPNE;

[0035] FIG2 is a graph showing the HPLC purity of the compound DOTA-dPNE;

[0036] FIG3 is a mass spectrometric identification diagram of compound NOTA-dPNE;

[0037] FIG4 is a graph showing the HPLC purity of compound NOTA-dPNE;

[0038] FIG5 is a mass spectrometric identification diagram of the compound DOTA-dPNH;

[0039] FIG6 is a graph showing the HPLC purity of the compound DOTA-dPNH;

[0040] FIG7 is a mass spectrometric identification diagram of compound NOTA-dPNH;

[0041] FIG8 is a graph showing the HPLC purity of compound NOTA-dPNH;

[0042] FIG9 is a mass spectrometric identification diagram of the compound DOTA-dPNPEG;

[0043] FIG10 is a graph showing the HPLC purity of the compound DOTA-dPNPEG;

[0044] FIG11 is a mass spectrometric identification diagram of compound NOTA-dPNPEG;

[0045] FIG12 is a graph showing the HPLC purity of compound NOTA-dPNPEG;

[0046] FIG13 is a mass spectrometric identification diagram of the compound HYNIC-dPNPEG;

[0047] FIG14 is a graph showing the HPLC purity of the compound HYNIC-dPNPEG;

[0048] FIG15 is a mass spectrometric identification diagram of the compound DOTA-dPNPEG2;

[0049] FIG16 is a graph showing the HPLC purity of compound DOTA-dPNPEG2;

[0050] FIG17 is a mass spectrometric identification diagram of compound NOTA-dPNPEG2;

[0051] FIG18 is a graph showing the HPLC purity of compound NOTA-dPNPEG2;

[0052] FIG19 is a mass spectrometric identification diagram of compound DOTA-dPNME;

[0053] FIG20 is a graph showing the HPLC purity of compound DOTA-dPNME;

[0054] FIG21 is a mass spectrum identification diagram of compound NOTA-dPNME;

[0055] FIG22 is a mass spectrometric identification diagram of compound DOTA-dPNPZ;

[0056] FIG23 is a graph showing the HPLC purity of compound DOTA-dPNPZ;

[0057] FIG24 is a mass spectrometric identification diagram of compound NOTA-dPNPZ;

[0058] Figure 25 shows the compound DOTA-dPNPEG and [ 68 Ga]Ga-DOTA-dPNPEG(A), NOTA-dPNE and [ 18 F]AlF-NOTA-dPNE(B), DOTA-dPNPEG and [ 177 Lu]Lu-DOTA-dPNPEG(C), HYNIC-dPNPEG and 99m HPLC analysis spectrum of Tc-HYNIC-dPNPEG(D);

[0059] Figure 26 shows the compound [ 68 Ga]Ga-DOTA-dPNE and [ 18 F]AlF-NOTA-dPNE stability in saline and serum;

[0060] Figure 27 shows the compound [ 68 Ga]Ga-DOTA-dPNE, [ 68 Ga]Ga-DOTA-dPNH, [ 68 Ga]Ga-DOTA-dPNPEG and [ 68 Cellular K of [Ga]Ga-DOTA-dPNPEG2 d Value measurement result diagram;

[0061] Figure 28 shows the compound [ 68 Ga]Ga-DOTA-dPNE, [ 68 Ga]Ga-DOTA-dPNH, [ 68 Ga]Ga-DOTA-dPNPEG and [ 68 Figure 3. In vitro cellular uptake and inhibition assay results of Ga]Ga-DOTA-dPNPEG2.

[0062] Figure 29 is [ 68 Ga]Ga-DOTA-dPNE (Figure A) and [ 68 MicroPET imaging of Ga]Ga-DOTA-dPNPEG2 (Figure B) in normal mice at 0.5 to 2 h;

[0063] Figure 30 is [ 68 Ga]Ga-DOTA-dPNE, [68 Ga]Ga-DOTA-dPNH, [ 68 Ga]Ga-DOTA-dPNPEG and [ 68 MicroPET imaging of Ga]Ga-DOTA-dPNPEG2 in B16F10 model mice at 0.5 to 2 h;

[0064] Figure 31 is [ 68 Radioactive uptake of Ga]Ga-DOTA-dPNPEG in major tissues of B16F10 model mice (A) and target / non-target ratio (B);

[0065] Figure 32 is [ 68 Ga]Ga-DOTA-dPNE, [ 68 Ga]Ga-DOTA-dPNH, [ 68 Ga]Ga-DOTA-dPNPEG and [ 68 MicroPET imaging of Ga]Ga-DOTA-dPNPEG2 in MDA-MB-231 model mice at 0.5 to 2 h;

[0066] Figure 33 is [ 68 Ga]Ga-DOTA-dPNE(A) and [ 68 Ga]Ga-DOTA-dPNPEG2 (B) radioactive uptake in major tissues and target / non-target ratio in MDA-MB-231 model mice;

[0067] Figure 34 is [ 68 Ga]Ga-DOTA-dPNME and [ 68 MicroPET images of Ga]Ga-DOTA-dPNPZ in MDA-MB-231 model mice at 0.5 to 2 h (A, C), radioactive uptake in major tissues and target / non-target ratios (B, D);

[0068] Figure 35 is [ 18 F]AlF-NOTA-dPNE and [ 18 MicroPET images of [F]AlF-NOTA-dPNPEG2 in MDA-MB-231 model mice at 0.5 to 2 h (A, C), radioactive uptake in major tissues and target / non-target ratios (B, D);

[0069] Figure 36 is [ 68 Ga]Ga-DOTA-dPNPEG distribution in tissues and organs and target / non-target ratio in B16F10 tumor mice;

[0070] Figure 37 is [ 68The distribution of tissues and organs and target / non-target ratio of Ga]Ga-DOTA-dPNE in MDA-MB-231 tumor mice;

[0071] Figure 38 is [ 177 SPECT imaging of Lu]Lu-DOTA-dPNE in MDA-MB-231 model mice from 1 to 24 h (A) and radioactive uptake in major tissues and target / non-target ratios (B). DETAILED DESCRIPTION

[0072] The present invention provides a nucleotide compound having the structure shown in Formula I:

[0073] In the present invention, in the formula I: L is R1 is n and m are independently integers of 0 to 10, preferably 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; and R5 includes -H or -CH3.

[0074] In the present invention, the R2 includes -H or halogen, and the halogen preferably includes fluorine, chlorine or bromine.

[0075] In the present invention, the R3 includes -H or any one of the following structures:

[0076] In the present invention, the R4 includes -OH or -F.

[0077] In the present invention, the X includes ═N— or ═CH—.

[0078] In the present invention, when the R1 is When n is 1 or 5, R3 is R5 is -H, and the nucleotide compound has the structure shown by DOTA-dPNE, NOTA-dPNE, DOTA-dPNH, NOTA-dPNH, DOTAGA-dPNE, DOTA-SCN-dPNE, DOTAGA-dPNH or DOTA-SCN-dPNH:

[0079] In the present invention, when the R1 is n is 1, 2 or 5, R3 is When R5 is -CH3, the nucleotide compound has the structure shown by DOTA-dPNME, NOTA-dPNME, DOTA-dPNPE, NOTA-dPNPE, DOTA-dPNMH, NOTA-dPNMH, DOTAGA-dPNME, DOTA-SCN-dPNME, DOTAGA-dPNMH or DOTA-SCN-dPNMH:

[0080] In the present invention, when the L is R3 is When the nucleotide compound has the structure shown by DOTA-dPNPZ, NOTA-dPNPZ, DOTAGA-dPNPZ or DOTA-SCN-dPNPZ:

[0081] In the present invention, when the R1 is m is 2 or 4, R3 is When R5 is -H or -CH3, the nucleotide compound has the structure shown in DOTA-dPNPEG, NOTA-dPNPEG, HYNIC-dPNPEG, DOTAGA-dPNPEG, DOTA-SCN-dPNPEG, DOTA-dPNPEG2, NOTA-dPNPEG2, DOTAGA-dPNPEG2, DOTA-SCN-dPNPEG2, DOTA-dPNMPEG, NOTA-dPNMPEG, DOTAGA-dPNMPEG, DOTA-SCN-dPNMPEG, DOTA-dPNMPEG2, NOTA-dPNMPEG2, DOTAGA-dPNMPEG2, DOTA-SCN-dPNMPEG2:

[0082] The present invention provides a method for preparing the nucleotide compound described in the above technical solution, comprising the following steps:

[0083] Compound A and compound B are subjected to a coupling reaction under Lewis acid catalysis to obtain compound C;

[0084] Performing a first substitution reaction between the compound C and the compound D under alkaline conditions to obtain a compound E;

[0085] The compound E is subjected to a ribose protecting group removal reaction to obtain a compound F;

[0086] The compound F is subjected to a second substitution reaction with methylenebis(phosphine dichloride) followed by a deprotection reaction to obtain compound G;

[0087] Performing a third substitution reaction between the compound G and the R3 active compound to obtain the nucleotide compound with the labeling group;

[0088] In the compounds A to G, R1 to R5, L and X are the same as R1 to R5, L and X in the formula I, A is a -F or -OBz ​​group, and R7 includes a Boc, DDE or Fmoc protecting group;

[0089] The R3 active compound is a compound with an active reactive group corresponding to R3, and can be selected from the following structures:

[0090] Unless otherwise specified, the materials and equipment used in the present invention are all commercially available products in the art.

[0091] In the present invention, compound A and compound B are subjected to a coupling reaction under the catalysis of a Lewis acid to obtain compound C. In the present invention, the coupling reaction is preferably carried out as follows: compound A and the catalyst are mixed under heating and reflux in hexamethyldisilazane (HMDS) to activate the mixture to obtain an activated intermediate; and the activated product, compound B, an organic solvent, and a Lewis acid are mixed to carry out a coupling reaction to obtain compound C.

[0092] The present invention mixes compound A with a catalyst and hexamethyldisilazane (HMDS) for activation to obtain an activated product. In the present invention, the catalyst is preferably ammonium sulfate; the molar ratio of the compound A to the catalyst is preferably 1:0.01 to 1:1, more preferably 1:0.1. In the present invention, the ratio of the amount of compound A to the volume of hexamethyldisilazane is preferably 1 mmol:0.5 to 5 mL, more preferably 1 mmol:2.1 mL. In the present invention, the activation is preferably carried out under reflux conditions, and the activation time is preferably 2 to 6 h, more preferably 3 h. After the activation, the present invention preferably further comprises concentrating the obtained activation system to obtain an activated product; the present invention has no special limitation on the concentration, and the concentration method well known to those skilled in the art is used to concentrate to a constant weight.

[0093] After obtaining the activated product, the present invention mixes the activated product, compound B, an organic solvent and a Lewis acid to carry out a coupling reaction to obtain compound C. In the present invention, the molar ratio of compound A to compound B is preferably 1:1 to 1:5, more preferably 1:1.2. In the present invention, the Lewis acid is preferably trimethylsilyl trifluoromethanesulfonate; the molar ratio of compound A to Lewis acid is preferably 1:1 to 1:3, more preferably 1:1.5. In the present invention, the organic solvent preferably includes acetonitrile, N,N-dimethylformamide, N,N-dimethylacetamide and dimethyl sulfoxide, etc. The present invention has no special limitation on the amount of the organic solvent, as long as it can dissolve compound A and ensure that the coupling reaction proceeds smoothly. In the present invention, the mixing is preferably: the temperature of the coupling reaction is preferably such that the activated product is dissolved in the organic solvent and then mixed with compound B, the resulting mixed solution is cooled to 25 to 0°C (more preferably 0°C) and then the Lewis acid is added dropwise. In the present invention, the temperature of the coupling reaction is preferably 0 to 25°C, more preferably 0°C, and the time of the coupling reaction is preferably 0.5 to 4 hours, more preferably 1 hour; in a specific embodiment of the present invention, the progress of the coupling reaction is preferably monitored by thin layer chromatography on a silica gel plate; during the coupling reaction, the protected ribofuranose in compound B undergoes a coupling reaction with the hexamethyl group in the activated product. After the coupling reaction, the present invention preferably further comprises a post-treatment, which preferably comprises: adding a saturated sodium bicarbonate solution to the obtained coupling reaction system to quench the reaction, then extracting with ethyl acetate 3 to 4 times, combining the organic phases, washing with saturated brine, drying with anhydrous sodium sulfate, filtering, and concentrating the obtained filtrate under reduced pressure and then purifying by column chromatography to obtain compound C; the eluent used for the column chromatography purification is preferably a petroleum ether-ethyl acetate solvent, and the volume ratio of petroleum ether to ethyl acetate in the petroleum ether-ethyl acetate solvent is preferably 1:0 to 1:1.

[0094] After obtaining compound C, the present invention conducts a first substitution reaction between compound C and compound D under alkaline conditions to obtain compound E. In the present invention, the first substitution reaction preferably comprises: dissolving compound C and compound D in a high-boiling-point solvent and conducting the first substitution reaction under alkaline conditions. In the present invention, the molar ratio of compound C to compound D is preferably 1:1 to 1:3, more preferably 1:1.2. In the present invention, the high-boiling-point solvent preferably includes one or more of N-methylpyrrolidone (NMP), dimethyl sulfoxide (DMSO), and N,N-dimethylformamide (DMF). The present invention does not specifically limit the amount of the high-boiling-point solvent used, as long as it can dissolve compound C and compound D and ensure the smooth progress of the first substitution reaction. In the present invention, the alkaline conditions are preferably provided by an organic base, preferably triethylamine (TEA) and / or N,N-diisopropylethylamine (DIPEA); the molar ratio of compound C to the organic base is preferably 1:1 to 1:5, more preferably 1:2. In the present invention, the temperature of the first substitution reaction is preferably 80 to 120° C., more preferably 110° C., and the time of the first substitution reaction is preferably 1 to 12 hours, more preferably 2 hours. In a specific embodiment of the present invention, the progress of the first substitution reaction is preferably monitored by thin layer chromatography on a silica gel plate. After the first substitution reaction, the present invention preferably further comprises post-treatment, which preferably comprises: adding water to the obtained first substitution system to quench the reaction, then extracting with ethyl acetate 3 to 4 times, combining the organic phases, washing with saturated brine, drying with anhydrous sodium sulfate, filtering, and concentrating the obtained filtrate under reduced pressure and then purifying by column chromatography to obtain compound E; the eluent used for the column chromatography purification is preferably a petroleum ether-ethyl acetate solvent, and the volume ratio of petroleum ether to ethyl acetate in the petroleum ether-ethyl acetate solvent is preferably 1:0 to 1:1.

[0095] After obtaining compound E, the present invention performs a deribose protecting group reaction on compound E to obtain compound F. In the present invention, the deribose protecting group reaction is preferably carried out under alkaline conditions. Specifically, compound E is dissolved in a mixed solvent, an alkaline aqueous solution is added dropwise to mix, and the deribose protecting group reaction is carried out. In the present invention, the base in the alkaline aqueous solution preferably includes an alkali metal hydroxide, ammonia water, etc., and more preferably includes sodium hydroxide. The molar ratio of compound E to the base is preferably 1:3 to 1:10, more preferably 1:4; the concentration of the alkaline aqueous solution is preferably 0.1 to 2M, more preferably 1M (M represents mol / L). In the present invention, the mixed solvent preferably includes tetrahydrofuran and methanol, and the volume ratio of tetrahydrofuran and methanol is preferably 1:1 to 5:1, more preferably 4:1. The present invention has no special limitation on the amount of the mixed solvent, as long as it can dissolve compound E and ensure that the deribose protecting group is carried out smoothly. In the present invention, the temperature of the deribose protecting group reaction is preferably 0 to 60° C., more preferably 20° C., and the time of the deribose protecting group reaction is preferably 1 to 12 hours, more preferably 2 hours. In a specific embodiment of the present invention, the progress of the deribose protecting group reaction is preferably monitored by thin layer chromatography on silica gel plates. During the deribose protecting group reaction, the protecting group of ribose in compound E is removed. After the deribose protecting group reaction, the present invention preferably further comprises a post-treatment, which preferably comprises: concentrating the resulting deribose protecting group reaction system, adding water to mix, then extracting with ethyl acetate 3 to 4 times, combining the organic phases, washing with saturated brine, drying with anhydrous sodium sulfate, filtering, and concentrating the resulting filtrate under reduced pressure to a constant weight to obtain compound F, which is directly subjected to subsequent reactions without purification.

[0096] After obtaining compound F, the present invention performs a second substitution reaction on compound F and methylenebis(dichlorophosphine) followed by a deprotection reaction to obtain compound G. Specifically, compound F is dissolved in trimethyl phosphate ((MeO)3PO), cooled to 0-25°C (more preferably 0-10°C), and methylenebis(dichlorophosphine) is added and mixed to perform a second substitution reaction. In the present invention, the ratio of the amount of compound F to the volume of trimethyl phosphate is preferably 1 mmol:0.01-0.1 mL, more preferably 1 mmol:0.02 mL. In the present invention, the molar ratio of compound F to methylenebis(dichlorophosphine) is preferably 1:1-5, more preferably 1:3. In the present invention, the temperature of the second substitution reaction is preferably 0-25°C, more preferably 0-10°C, the time of the second substitution reaction is preferably 1-12 h, more preferably 3 h, and during the second substitution reaction, the ribose-OH in compound F undergoes a substitution reaction with methylenebis(dichlorophosphine).

[0097] After the second substitution reaction, the present invention preferably further includes a post-treatment, and the post-treatment preferably includes: subjecting the obtained second substitution reaction system to a deprotection reaction, followed by extraction with methyl tert-butyl ether, and lyophilizing the obtained aqueous phase and then purifying it with a reverse-phase high-performance chromatography column. The present invention has no special restrictions on the amount and type of reagents required for the deprotection reaction, and the reaction conditions, as long as the protecting group (DDE, Boc or Fmoc) can be removed. In a specific embodiment of the present invention, the reagent used in the deprotection reaction is preferably a tetraethylammonium chloride solution, the pH value of the tetraethylammonium chloride solution is preferably 7.4 to 7.6, more preferably 7.5, and the concentration of the tetraethylammonium chloride solution is preferably 0.1 to 1 M, more preferably 0.5 M; the ratio of the amount of the compound F to the volume of the tetraethylammonium chloride solution is preferably 1 mmol: 0.01 to 0.1 mL, more preferably 1 mmol: 0.02 mL. In the present invention, the conditions for purification using a reverse-phase high-performance chromatography column include: the chromatographic column is a reverse-phase C18 semi-preparative column; the mobile phase A is preferably water + 0.1% (volume fraction) trifluoroacetic acid (TFA), and the mobile phase B is preferably acetonitrile + 0.1% TFA; the elution method is preferably gradient elution, and the conditions for the gradient elution are preferably 0 to 30 min: the volume fraction of mobile phase B increases from 5% to 95%, and the flow rate of the mobile phase is preferably 3 mL / min.

[0098] After obtaining compound G, the present invention conducts a third substitution reaction between compound G and the active compound R3 to obtain the nucleotide compound. In the present invention, the third substitution reaction preferably comprises dissolving compound G and the active compound R3 in a high-boiling-point solvent and conducting the third substitution reaction under alkaline conditions. In the present invention, the molar ratio of compound G to the active compound R3 is preferably 1:1 to 1:3, more preferably 1:2.5. In the present invention, the high-boiling-point solvent preferably comprises one or more of N-methylpyrrolidone, dimethyl sulfoxide, and N,N-dimethylformamide. The amount of the high-boiling-point solvent employed is not particularly limited; it only requires sufficient solvent to dissolve compound G and the active compound R3 and ensure the smooth progress of the first substitution reaction. In the present invention, the alkaline conditions are preferably provided by an organic base, preferably triethylamine and / or N,N-diisopropylethylamine. The molar ratio of compound G to the organic base is preferably 1:1 to 1:10, more preferably 1:5. In the present invention, the temperature of the third substitution reaction is preferably 25 to 50°C, more preferably 25°C, and the time of the third substitution reaction is preferably 1 to 24h, more preferably 12h. After the third substitution reaction, the present invention preferably further comprises subjecting the obtained third substitution system to reverse phase high performance chromatography column purification. In the present invention, the conditions for the reverse phase high performance chromatography column purification include: the chromatographic column is a reverse phase C18 semi-preparative column; the mobile phase A is preferably water + 0.1% trifluoroacetic acid (TFA), and the mobile phase B is preferably acetonitrile + 0.1% TFA; the elution method is preferably gradient elution, and the conditions for the gradient elution are preferably 0 to 30min: the volume fraction of mobile phase B increases from 5% to 95%, and the flow rate of the mobile phase is preferably 3mL / min.

[0099] The present invention provides a pharmaceutically acceptable salt of a nucleotide compound, obtained by reacting the nucleotide compound with an acid or a base; the nucleotide compound is the nucleotide compound described in the above technical solution or the nucleotide compound produced by the preparation method described in the above technical solution. In the present invention, the pharmaceutically acceptable salt preferably includes trifluoroacetate, phosphate, formate, acetate, potassium salt, or sodium salt. In the present invention, the acid preferably includes trifluoroacetic acid, hydrochloric acid, formic acid, or acetic acid; the base preferably includes potassium hydroxide or sodium hydroxide. The present invention does not particularly limit the reaction conditions; salt-forming reaction conditions familiar to those skilled in the art can be used.

[0100] The present invention provides a nuclide targeting probe having a structure shown in Formula II:

[0101] In the present invention, R1 to R5, L and X in Formula II are the same as R1 to R5, L and X in the nucleotide compound according to claim 1.

[0102] In the present invention, the coordination group is obtained by the coordination reaction between R3 in the nucleotide compound described in the above technical solution and the labeled nuclide; the labeled nuclide includes 18 F. 47 Sc, 64 Cu, 67 Cu, 67 Ga, 68 Ga, 89 Zr, 86 Y. 89 Sr, 90 Y. 99m Tc, 105 Rh, 109 Pd, 111 In, 119 Sb, 149 Tb, 153 Sm, 157 Gd, 161 Tb, 166 Ho, 177 Lu, 186 Re、 188 Re、 201 Tl, 203 Pb, 212 Pb, 212 Bi, 213 Bi, 223 Ra, 227 Th and 225 At least one of Ac.

[0103] The present invention provides a method for preparing the nuclide targeting probe described in the above technical solution, the preparation method comprising the following steps:

[0104] A nucleotide compound is subjected to a coordination reaction with a labeled nuclide to obtain a nuclide-targeted probe; the nucleotide compound is the nucleotide compound described in the above technical solution or the nucleotide compound prepared by the preparation method described in the above technical solution. In the present invention, the nuclide-targeted probe is preferably prepared by a wet labeling method or a lyophilization labeling method.

[0105] In the present invention, the preparation of a nuclide targeting probe by a wet labeling method preferably includes the following steps: mixing a solution of a nucleotide compound with a labeled nuclide solution, performing a coordination reaction, and then diluting to obtain an injection of the nuclide targeting probe. In the present invention, the solvent in the solution of the nucleotide compound preferably includes one or more of a buffer solution, water, and an organic solvent; the buffer solution preferably includes an acetic acid-acetate solution or an aluminum chloride-acetate solution, and the pH value of the buffer solution is preferably 3 to 7, more preferably 4 to 6.5; the concentration of aluminum chloride in the aluminum chloride-acetate solution is preferably 0.2 to 1 g / L, more preferably 0.4 g / L; the acetate in the acetic acid-acetate solution and the aluminum chloride-acetate solution independently includes one or more of sodium acetate, potassium acetate, and ammonium acetate; the concentration of the solution of the nucleotide compound is preferably 0.001 to 1000 mg / mL, more preferably 0.01 to 1 mg / mL. In the present invention, the ratio of the mass of the nucleotide compound to the activity of the labeled nuclide in the labeled nuclide solution is preferably 20-400 μg: 1 kBq-1000 GBq, more preferably 20-400 μg: 0.037-74000 MBq, and further preferably 20-200 μg: 0.037-7400 MBq. In the present invention, there is no particular limitation on the labeled nuclide solution, and any labeled nuclide solution well known to those skilled in the art can be used, such as 68 GaCl3 hydrochloric acid solution, 18 F - solution, 177 LuCl3 solution or Na 99m TcO4 eluent, the 68 The GaCl3 hydrochloric acid solution is preferably obtained by eluting from a germanium gallium generator. 99m The TcO4 eluent is preferably obtained by eluting from a molybdenum-technetium generator. In the present invention, the temperature of the coordination reaction is preferably 25 to 100°C, more preferably 80 to 100°C, and the time of the coordination reaction is preferably 10 to 60 minutes, more preferably 20 to 30 minutes. When the temperature of the coordination reaction is higher than room temperature, the present invention preferably further comprises cooling the obtained coordination reaction system to room temperature after the coordination reaction. The present invention has no special limitation on the cooling, and a cooling method well known to those skilled in the art can be adopted, such as natural cooling. After the dilution, the present invention preferably further comprises aseptically filtering the obtained dilution system to obtain an injection of a radionuclide targeted probe. In the present invention, the dilution preferably utilizes physiological saline or water for injection. In the present invention, the concentration of the injection of the radionuclide targeted probe is preferably 0.037 to 3700 MBq / mL.

[0106] In the present invention, when the labeling nuclide is 18F, the coordination reaction is preferably carried out under the conditions of a metal ion as a co-ligand ion, a buffer solution and an organic solvent as a mixed medium, the metal ion preferably includes an aluminum ion, the buffer solution preferably includes an aluminum chloride-acetate solution, and the organic solvent preferably includes acetonitrile.

[0107] In the present invention, when the labeling nuclide is 99m When Tc, the coordination reaction is preferably carried out in the presence of a reducing agent and a co-ligand, and the reducing agent preferably includes SnCl2, and the SnCl2 is preferably used in the form of a hydrochloric acid solution of SnCl2. The hydrochloric acid for dissolving SnCl2 can be commercially available concentrated hydrochloric acid. Each time the labeling is performed, the amount of SnCl2 used is preferably 0.001 to 100 mg, more preferably 0.01 to 0.2 mg. In the present invention, the co-ligand preferably includes at least one of N-tris(hydroxymethyl)methylglycine (Tricine), triphenylphosphine tris-sulfonic acid sodium salt (TPPTS), ethylenediamine-N,N'-diacetic acid (EDDA), ethylenediaminetetraacetic acid (EDTA), and 3-diphenylphosphinobenzenesulfonic acid sodium salt (TPPMS). The mass ratio of the nucleotide compound to the co-ligand is preferably 0.01 to 0.5:0.1 to 50, more preferably 0.02 to 0.1:1 to 10.

[0108] In the present invention, the preparation of a radionuclide-targeted probe using a lyophilization labeling method preferably includes the following steps: freeze-drying a solution of a nucleotide compound and then sealing it to obtain a lyophilized kit; adding a solvent to the lyophilized kit to dissolve the solution, then adding a labeled radionuclide solution for coordination reaction and diluting the solution to obtain an injection of the radionuclide-targeted probe. In the present invention, the freeze-drying method preferably involves dispensing the solution of the nucleotide compound into lyophilization containers and then freeze-drying it. The present invention does not specifically limit the freeze-drying conditions; freeze-drying conditions familiar to those skilled in the art can be used. In the present invention, the solvent added to the lyophilization kit is preferably the same as the solvent in the solution of the nucleotide compound. In the present invention, at least one of an excipient, an antioxidant, and an acid-base modifier is preferably added to the lyophilized kit as needed. The excipients, antioxidants, and acid-base modifiers are not specifically limited; excipients, antioxidants, and acid-base modifiers familiar to those skilled in the art can be used. In the present invention, the other preparation conditions for the radionuclide-targeted probe are preferably the same as those for the aforementioned wet labeling method and are not further detailed here.

[0109] In the present invention, when the radiochemical purity of the injection of the radionuclide targeted probe prepared by the wet labeling method and the freeze-drying labeling method is lower than 95%, it is preferred to further include purifying the injection of the radionuclide targeted probe, and the purification is preferably Sep-Pak C18 separation column purification, and the Sep-Pak C18 separation column is preferably activated and eluted with anhydrous ethanol and water in sequence before use. In the present invention, the eluents used for the purification are preferably water and anhydrous ethanol in sequence, and the eluate of anhydrous ethanol is collected and the solvent is removed, and then diluted to obtain an injection of a high-purity radionuclide targeted probe. In the present invention, the dilution is preferably carried out using physiological saline or water for injection. In the present invention, the concentration of the injection of the high-purity radionuclide targeted probe is preferably 0.037 to 3700 MBq / mL.

[0110] In the present invention, a pharmaceutically acceptable salt of a nuclide targeting probe is provided, which is obtained by a salt-forming reaction of the nuclide targeting probe or a nuclide labeling reaction of a pharmaceutically acceptable salt of a nucleotide compound; the nuclide targeting probe is the nuclide targeting probe described in the above technical solution or the nuclide targeting probe prepared by the preparation method described in the above technical solution; the nucleotide compound is the nucleotide compound described in the above technical solution or the nucleotide compound prepared by the preparation method described in the above technical solution. In the present invention, the pharmaceutically acceptable salt preferably includes trifluoroacetate, phosphate, formate, acetate, potassium salt or sodium salt. The present invention does not specifically limit the preparation method of the pharmaceutically acceptable salt of the nuclide targeting probe, and the preparation method of the pharmaceutically acceptable salt well known to those skilled in the art can be used.

[0111] The present invention provides a pharmaceutical composition comprising an active ingredient and a pharmaceutically acceptable excipient; the active ingredient comprises one or more of a nucleotide compound, a nuclide targeting probe, a pharmaceutically acceptable salt of the nucleotide compound described in the above technical solution, and a pharmaceutically acceptable salt of the nuclide targeting probe described in the above technical solution; the nucleotide compound is the nucleotide compound described in the above technical solution or a nucleotide compound produced by the preparation method described in the above technical solution; the nuclide targeting probe is the nuclide targeting probe described in the above technical solution or a nuclide targeting probe produced by the preparation method described in the above technical solution. The present invention does not specifically limit the pharmaceutically acceptable excipients, and pharmaceutically acceptable excipients familiar to those skilled in the art can be used. In the present invention, when the active ingredient comprises a nuclide targeting probe and / or a pharmaceutically acceptable salt of a nuclide targeting probe, the dosage form of the pharmaceutical composition is preferably an injection, preferably administered by intravenous injection; when the active ingredient does not contain a nuclide targeting probe and its pharmaceutically acceptable salt, the present invention does not specifically limit the dosage form and administration method of the pharmaceutical composition, and dosage forms and administration methods familiar to those skilled in the art can be used.

[0112] The present invention also provides the use of the nucleotide compound described in the above technical solution, the nucleotide compound prepared by the preparation method described in the above technical solution, the pharmaceutically acceptable salt of the nucleotide compound described in the above technical solution, the radionuclide targeting probe described in the above technical solution, the radionuclide targeting probe prepared by the preparation method described in the above technical solution, the pharmaceutically acceptable salt of the radionuclide targeting probe described in the above technical solution, or the pharmaceutical composition described in the above technical solution in the preparation of therapeutic drugs or diagnostic drugs for diseases mediated by CD73 protein. In the present invention, the disease mediated by CD73 protein includes tumors or immune-related diseases. In the present invention, the tumor preferably includes one or more of breast cancer, ovarian cancer, lung cancer, liver cancer, colorectal cancer, prostate cancer, skeletal tissue sarcoma, connective tissue sarcoma, renal cell carcinoma, gastric cancer, pancreatic cancer, nasopharyngeal carcinoma, head and neck cancer, neuroendocrine tumor, skin melanoma, and bone metastasis. In the present invention, the immune-related disease preferably includes one or more of lupus erythematosus, rheumatoid arthritis, hepatitis, autoimmune encephalomyelitis, scleroderma, psoriasis, Sjögren's syndrome, polyarteritis nodosa, asthma, and atherosclerosis. In the present invention, the diagnostic method preferably includes single photon emission computed tomography (SPECT), positron emission tomography (PET), and magnetic resonance imaging (MRI); the treatment method preferably includes radionuclide targeted therapy and / or chemotherapy and / or immunotherapy.

[0113] To further illustrate the present invention, the nucleotide compounds of the present invention, their preparation methods and applications, the radionuclide targeting probes, their preparation methods and applications, and the pharmaceutical compositions are described in detail below with reference to the accompanying drawings and examples. However, these should not be construed as limiting the scope of protection of the present invention.

[0114] Example 1

[0115] Synthesis of DOTA-dPNE

[0116] The specific steps include:

[0117] 1) Synthesis of Compound 2: 4-chloro-1H-pyrazolo[4,3-b]pyridine (Compound 1, 26.05 mmol) was dissolved in N-methylpyrrolidone (NMP, 40 mL), and m-chloroperbenzoic acid (m-CPBA, 59.91 mmol) was slowly added while controlling the reaction temperature below 20°C. The reaction was continued until the starting material was completely consumed. Methyl tert-butyl ether (40 mL) was added to the reaction solution and stirring was continued for 1 h. The precipitated solid was filtered and washed with methyl tert-butyl ether (40 mL). The filter cake was collected and dried at 45°C to obtain a crude product of Compound 2 (4.9 g, 26.05 mmol).

[0118] 2) Synthesis of compound 3: Compound 2 (26.05 mmol) and a catalytic amount of triethylbenzylammonium chloride (BnEt3N + Cl - , 13.025 mmol) was dissolved in acetonitrile (CH3CN, 50 mL), cooled to 10°C, phosphorus oxychloride (POCl3, 52.1 mmol) was added to the reaction solution, and stirred at room temperature for 2 h until the reaction of the raw materials was complete; water was added to the reaction solution to quench it, the temperature was controlled below 20°C, and stirred for 1 h. The precipitated solid was filtered, and the filter cake was washed with saturated sodium bicarbonate solution. The filter cake was collected and dried at 45°C to obtain compound 3 (4.1 g, 21.8 mmol); ESI MS [M+H] + for C6H4Cl2N3, calcd 190.01, found 190.02.

[0119] 3) Synthesis of compound 5: Compound 3 (2.39 mmol) and a catalytic amount of ammonium sulfate ((NH4)2SO4, 0.239 mmol) were dissolved in hexamethyldisilazane (HMDS, 5 mL) and stirred under reflux for 3 h; the reaction solution was concentrated, acetonitrile (CH3CN, 5 mL) was added to the concentrate to dissolve it, 1-acetyl-2,3,5-tribenzoyloxy-1-β-D-ribofuranose (raw material 4, 2.87 mmol) was added, and after cooling to 0°C, trimethylsilyl trifluoromethanesulfonate (TMSOTf, 3.59 mmol) was added dropwise to the reaction solution, and stirring was continued for 1 h. The reaction of the starting material was monitored by thin-layer chromatography on silica gel plates until complete, and a saturated sodium bicarbonate solution (10 mL) was added to quench the reaction; ethyl acetate was added and extracted three times, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure; the crude product was purified by column chromatography (petroleum ether:ethyl acetate = 1:0-1:1) to give compound 5 (white solid, 1.2 g, yield 79.5%); ESI MS [M+Na] + for C 32 H 23 Cl2N3O7Na, calcd 654.09, found 654.26; 1H NMR (400MHz, CDCl3-d) δ8.10-8.04(m,3H),8.02-7.94(m,4H),7.55(t,J=7.8Hz,3H),7.39(dd,J=14.6,6.8Hz,6H),7.23(s,1H),6.90(d,J=2 .8Hz,1H),6.43(dd,J=5.4,2.8Hz,1H),6.36(t,J=5.9Hz,1H),4.89-4.83(m,1H),4.77(dd,J=12.0,4.0Hz,1H),4.60(dd,J=12.0,5.0Hz,1H).

[0120] 4) Synthesis of Compound 7: Compound 5 (0.632 mmol) was dissolved in N-methylpyrrolidone (NMP, 5 mL), and N-Boc-ethylenediamine (raw material 6, 0.759 mmol) and N,N-diisopropylethylamine (DIPEA, 1.26 mmol) were added. The temperature was raised to 110°C and stirred for 2 h. The reaction was monitored for completion by thin-layer chromatography on silica gel. The mixture was cooled to room temperature and quenched with water. Ethyl acetate was added for extraction. The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The crude product was purified by column chromatography (petroleum ether:ethyl acetate = 1:0 to 1:1) to obtain compound 7 (pale yellow oil, 390 mg, 81% yield); ESI MS [M+H] + for C 39 H 39 ClN5O9,calcd 758.21, found 758.41; 1 H NMR(400MHz, CDCl3-d)δ8.05(d,J=7.1Hz,2H),7.99(d,J=6.9Hz,2H),7.93(d,J=4.9Hz,2H), 7.92(s,1H),7.58-7.46(m,3H),7.42-7.32(m,6H),6.86(d,J=2.6Hz,1H),6.49(s,1H),6.44- 6.33(m,2H),6.10(s,1H),5.22(q,J=5.5,5.0Hz,1H),4.82(q,J=5.2Hz,1H),4.73(dd,J=11. 9, 4.2Hz, 1H), 4.61 (dd, J = 11.9, 5.3Hz, 1H), 3.45 (s, 2H), 3.36 (t, J = 4.9Hz, 2H), 1.42 (s, 9H).

[0121] 5) Synthesis of Compound 8: Compound 7 (0.476 mmol) was dissolved in a mixed solution of tetrahydrofuran (THF, 4 mL) and methanol (MeOH, 1 mL), and an aqueous sodium hydroxide solution (4 eq., 1.9 mL, 1 M, 1.9 mmol) was added dropwise. The mixture was stirred at 20°C for 2 h until the reaction was complete. The reaction solution was concentrated, water was added, and ethyl acetate was added for extraction. The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain compound 8 (white solid, 180 mg, yield 85.2%, 0.405 mmol), which was used directly in the next step without purification. ESI MS [M+Na] + for C 18 H 26 ClN5O3Na,calcd 466.16, found 466.28.

[0122] 6) Synthesis of Compound 10: Compound 8 (56.32 mmol) was dissolved in trimethyl phosphate ((MeO)3PO, 1 mL), cooled to 0°C, and methylenebis(phosphine dichloride) (starting material 9, 168.96 μmol) was added. The mixture was stirred at 0°C for 3 h. Tetraethylammonium chloride solution (TEAC, pH = 7.4-7.6, 1 mL, 0.5 M) was added and stirred for 1 h. Extraction was performed with methyl tert-butyl ether. The aqueous phase was collected and lyophilized to obtain the crude product. The crude product was purified by reverse-phase high performance chromatography (HPLC) using a reverse-phase C18 semi-preparative column. Mobile phase A: water + 0.1% trifluoroacetic acid (TFA); mobile phase B: acetonitrile + 0.1% TFA. Gradient elution conditions: 0-30 min: the volume fraction of mobile phase B increased from 5% to 95%, and the mobile phase flow rate was 3 mL / min. The separated sample was lyophilized to obtain compound 10 (white solid, 10 mg, yield 35.4%, 19.93 μmol); ESI MS [M+H] + for C 14 H 23 ClN5O9P2,calcd 502.02, found 502.20.

[0123] 7) Synthesis of DOTA-dPNE: Compound 10 (9.97 μmol) was dissolved in N,N-dimethylformamide (DMF, 1 mL), and DOTA activated ester (raw material 11, 24.91 μmol) and N,N-diisopropylethylamine (DIPEA, 49.83 μmol) were added, respectively. The mixture was stirred at 25°C for 12 h. After complete reaction, the mixture was purified by reverse-phase high performance chromatography (HPLC). HPLC purification conditions: reverse-phase C18 semi-preparative column. Mobile phase A: water + 0.1% trifluoroacetic acid (TFA); mobile phase B: acetonitrile + 0.1% TFA. Gradient elution conditions: 0-30 min: mobile phase B volume fraction increased from 5% to 95%, mobile phase flow rate 3 mL / min. The separated sample was lyophilized to obtain DOTA-dPNE (white solid, 3 mg, 33.9% yield, 3.38 μmol); ESI MS [M+H] + for C 30 H 49 ClN9O 16 P 2， Figures 1 and 2 are the mass spectrum and HPLC purity identification diagrams of compound DOTA-dPNE.

[0124] Example 2

[0125] Preparation of NOTA-dPNE: Prepared according to the method of step 7) of Example 1, except that DOTA activated ester was replaced with NOTA activated ester to obtain compound NOTA-dPNE (white solid, 3.5 mg, yield 45%, 4.45 μmol); ESI MS [M+H] + for C 26 H 42 ClN8O 14 P2, calcd 787.19, found 787.16. Figures 3 and 4 are the mass spectrum and HPLC purity identification diagrams of compound NOTA-dPNE.

[0126] The structural formula of NOTA activated ester is

[0127] Example 3

[0128] Preparation of DOTA-dPNH: Prepared according to the method of steps 4) to 7) of Example 1, except that N-Boc-ethylenediamine was replaced with N-Boc-1,6-hexanediamine to obtain compound DOTA-dPNH (white solid, 3.2 mg, 38% yield, 3.39 μmol); ESI MS [M+H] + for C 34H 57 ClN9O 16 P2, calcd 944.30, found 944.25. Figures 5 and 6 are the mass spectrum and HPLC purity identification diagrams of compound DOTA-dPNH.

[0129] The structural formula of Boc-1,6-hexanediamine is

[0130] Example 4

[0131] Preparation of NOTA-dPNH: Prepared according to the method of steps 4) to 7) of Example 1, except that N-Boc-ethylenediamine was replaced with N-Boc-1,6-hexanediamine and DOTA activated ester was replaced with NOTA activated ester to obtain compound NOTA-dPNH (white solid, 2.1 mg, yield 46.4%, 2.49 μmol); ESI MS [M+H] + for C 30 H 50 ClN8O 14 P2, calcd 843.25, found 843.09. Figures 7 and 8 are the mass spectrum and HPLC purity identification diagrams of compound NOTA-dPNH.

[0132] Example 5

[0133] Synthesis of DOTA-dPNPEG

[0134] The specific steps include:

[0135] 1) Synthesis of Compound 13: Compound 5 (632.46 μmol) was dissolved in N-methylpyrrolidone (NMP, 5 mL), and tert-butyl 2-(2-(2-aminoethoxy)ethoxy)ethylcarbamate (starting material 12, 758.95 μmol) and N,N-diisopropylethylamine (DIPEA, 1.9 mmol) were added sequentially. The temperature was raised to 110°C and stirred for 2 h. The reaction was monitored for completion by thin-layer chromatography on silica gel. The mixture was cooled to room temperature and quenched with water. Ethyl acetate was added for extraction. The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The crude product was purified by column chromatography (petroleum ether:ethyl acetate volume ratio = 1:0 to 1:2) to obtain compound 13 (pale yellow oil, 0.5 g, 93.6% yield, 592.2 μmol); ESI MS [M+Na] + for C 43 H 46 ClN5O 11Na,calcd 866.29,found 866.52; 1 H NMR (400MHz, CDCl3-d) δ8.09-8.05(m,2H),8.02-7.97(m,3H),7.95-7.91(m,2H),7.58-7.48(m,3H ),7.41-7.32(m,6H),6.88(d,J=2.9Hz,1H),6.42(dd,J=5.4,3.0Hz,1H),6.35(t,J=5.8Hz,1H),6. 20(s,1H),4.80(q,J=5.2Hz,1H),4.72(dd,J=11.9,4.3Hz,1H),4.61(dd,J=11.9,5.3Hz,1H),3.74 (t,J=4.9Hz,2H),3.64(s,4H),3.56(t,J=5.3Hz,2H),3.54-3.49(m,2H),3.32(s,2H),1.44(s,9H).

[0136] 2) Synthesis of Compound 14: Compound 13 (592.2 μmol) was dissolved in a mixed solution of tetrahydrofuran (THF, 5 mL) and methanol (MeOH, 1 mL), and an aqueous sodium hydroxide solution (NaOH, 2.37 mL, 1 M) was added dropwise. The mixture was stirred at 20°C for 2 h. The reaction was complete by thin-layer chromatography on silica gel (developing system: ethyl acetate). The reaction solution was concentrated, water was added, and ethyl acetate was added for extraction. The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain compound 14 (white solid, 0.3 g, yield 95.2%, 564 μmol), which was used directly in the next step without purification. ESI MS [M+Na] + for C 22 H 34 ClN5O8Na,calcd 554.21, found 554.36.

[0137] 3) Synthesis of Compound 15: Compound 14 (56.32 μmol) was dissolved in trimethyl phosphate ((MeO)3PO, 1 mL), cooled to 0°C, and methylenebis(phosphine dichloride) (raw material 9, 168.96 μmol) was added. The mixture was stirred at 0°C for 3 h. Tetraethylammonium chloride solution (TEAC buffer, pH = 7.4-7.6, 2 mL, 0.5 M) was added and stirred for 1 h. Methyl tert-butyl ether was added for extraction. The aqueous phase was collected and lyophilized to obtain the crude product. The crude product was purified by reverse phase high performance chromatography. The separated sample was lyophilized to obtain compound 15 (white solid, 18 mg, 63.7% yield, 35.87 μmol); ESI MS [M+H]+ for C 22 H 35 ClN5O8,calcd 590.11,found 590.29; 1 H NMR (400MHz, DMSO-d6) δ8.37(s,1H),8.06(s,4H),6.31(s,1H),6.10(d,J=4.2Hz,1H),4.55(t,J=4.7Hz,1H),4.27(t,J=4.7Hz,1H),4.03( s,2H),3.89-3.78(m,1H),3.67(d,J=8.0Hz,1H),3.65-3.62(m,2H),3.60-3.55(m,6H),3.47(s,2H),2.90(s,2H),2.15(t,J=20.0Hz,2H).

[0138] 4) Synthesis of DOTA-dPNPEG: Compound 15 (6.78 μmol) was dissolved in N,N-dimethylformamide (DMF, 1 mL), and DOTA activated ester (raw material 11, 16.95 μmol) and N,N-diisopropylethylamine (DIPEA, 33.91 μmol) were added. The mixture was stirred at 20°C for 12 h. After complete reaction, the product was purified by reverse-phase high performance chromatography (HPLC). HPLC purification conditions: reverse-phase C18 semi-preparative column. Mobile phase A: water + 0.1% trifluoroacetic acid (TFA); mobile phase B: acetonitrile + 0.1% TFA. Gradient elution conditions: 0-30 min: mobile phase B volume fraction increased from 5% to 95%, mobile phase flow rate 3 mL / min. The separated sample was lyophilized to obtain DOTA-dPNPEG (white solid, 2.5 mg, 37.7% yield, 2.56 μmol); ESI MS [M+H] + for C 22 H 35 ClN5O8, calcd 976.29, found 976.04. Figures 9 and 10 are the mass spectrum and HPLC purity identification chart of the compound DOTA-dPNPEG.

[0139] Example 6

[0140] Synthesis of NOTA-dPNPEG

[0141] The specific steps include: dissolving compound 15 (5.09 μmol) in N,N-dimethylformamide (DMF, 1 mL), adding NOTA activated ester (raw material 16, 12.71 μmol) and N,N-diisopropylethylamine (DIPEA, 25.43 μmol), and stirring at 20°C for 12 h. After the raw materials reacted completely, they were purified by reverse-phase high-performance chromatography. HPLC purification conditions: reverse-phase C18 semi-preparative column. Mobile phase A: water + 0.1% trifluoroacetic acid (TFA); mobile phase B: acetonitrile + 0.1% TFA; gradient elution conditions: 0-30 min: the volume fraction of mobile phase B increased from 5% to 95%, and the mobile phase flow rate was 3 mL / min. The separated sample was lyophilized to obtain NOTA-dPNPEG as a white solid (1.5 mg, 33.7% yield, 1.72 μmol); ESI MS [M+H] + for C 30 H 50 ClN8O16P2, calcd 875.24, found 875.19. Figures 11 and 12 are the mass spectrum and HPLC purity identification spectrum of compound NOTA-dPNPEG.

[0142] Example 7

[0143] Synthesis of HYNIC-dPNPEG

[0144] The specific steps involved were as follows: Compound 15 (5.09 μmol) was dissolved in N,N-dimethylformamide (DMF, 1 mL), HYNIC activated ester (raw material 17, 12.71 μmol) and N,N-diisopropylethylamine (DIPEA, 25.43 μmol) were added, and the mixture was stirred at 20°C for 12 h. After the reaction was complete, the mixture was purified by reverse-phase high-performance chromatography (HPLC). HPLC purification conditions were reverse-phase C18 semi-preparative column. Mobile phase A: water + 0.1% trifluoroacetic acid (TFA); mobile phase B: acetonitrile + 0.1% TFA. Gradient elution conditions were as follows: 0-30 min: the volume fraction of mobile phase B increased from 5% to 95%, and the mobile phase flow rate was 3 mL / min. The separated sample was lyophilized to obtain HYNIC-dPNPEG as a yellow solid (1.3 mg, 28.6% yield, 1.46 μmol); ESI MS [M+H] + for C 33 H 45 ClN9O 12 P2, calcd 856.23, found 856.15. Figures 13 and 14 are the mass spectrum and HPLC purity identification spectrum of compound HYNIC-dPNPEG.

[0145] Example 8

[0146] Synthesis of DOTA-dPNPEG2

[0147] The specific steps include:

[0148] 1) Synthesis of Compound 16: Compound 5 (790.58 μmol) was dissolved in N-methylpyrrolidone (NMP, 5 mL), and 1,1-dimethylethyl 16-amino-5,8,11,14-tetraoxa-2-azahexadecanoate (raw material 18, 948.69 μmol) and N,N-diisopropylethylamine (DIPEA, 2.37 mmol) were added sequentially. The temperature was raised to 110°C and stirred for 2 h. Completion of the reaction was monitored by thin-layer chromatography on silica gel. The mixture was cooled to room temperature and quenched with water. Ethyl acetate was added for extraction. The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The crude product was purified by column chromatography (petroleum ether:ethyl acetate volume ratio = 1:0 to 1:2) to obtain compound 16 as a light yellow oil (0.5 g, 67.8% yield, 536.2 μmol); ESI MS [M+H] + for C 47 H 55 ClN5O 13, calcd 932.34,found 932.32; 1 H NMR(400MHz,Chloroform-d)δ8.05(d,J=10.5Hz,3H),7.98(d,J=7.3Hz,2H),7.91(d,J=9.6Hz,2H),7.57-7. 47(m,3H),7.41-7.30(m,6H),6.87(s,1H),6.40(dd,J=5.3,2.9Hz,1H),6.35(t,J=5.7Hz,1H),6.19(s,1H),5 .18(s,1H),4.79(q,J=5.3Hz,1H),4.70(dd,J=11.8,4.4Hz,1H),4.59(dd,J=11.8,5.4Hz,1H),3.75(t,J=5.0 Hz, 2H), 3.66 (s, 8H), 3.61 (d, J = 2.7Hz, 4H), 3.50 (dt, J = 9.5, 5.1Hz, 4H), 3.36 (t, J = 7.1Hz, 2H), 1.40 (s, 9H).

[0149] 2) Synthesis of compound 17

[0150] Compound 16 (536.2 μmol) obtained in reaction 1 was dissolved in a mixed solution of tetrahydrofuran (THF, 5 mL) and methanol (MeOH, 1 mL), and an aqueous sodium hydroxide solution (NaOH, 2.14 mL, 1 M) was added dropwise. The mixture was stirred at 20°C for 2 h. The reaction was complete when detected by thin-layer chromatography on silica gel (developing system: ethyl acetate). The reaction solution was concentrated, water was added, and ethyl acetate was added for extraction. The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain compound 17 (white solid, 0.3 g, yield 94%, 455 μmol), which was used directly in the next reaction without purification. ESI MS [M+H] + for C 26 H 43 ClN5O 10 ,calcd 620.26,found 620.29.

[0151] 3) Synthesis of Compound 18

[0152] Compound 14 (48.38 μmol) was dissolved in trimethyl phosphate ((MeO)3PO, 1 mL), cooled to 0°C, and methylenebis(phosphine dichloride) (starting material 9, 241.90 μmol) was added. The mixture was stirred at 0°C for 3 h. Tetraethylammonium chloride solution (TEAC buffer, pH = 7.4-7.6, 2 mL, 0.5 M) was added and stirred for 1 h. Extraction was performed with methyl tert-butyl ether. The aqueous phase was collected and lyophilized to obtain the crude product. The crude product was purified by reverse-phase high-performance chromatography. HPLC purification conditions: reverse-phase C18 semi-preparative column. Mobile phase A: water + 0.1% trifluoroacetic acid (TFA); mobile phase B: acetonitrile + 0.1% TFA. Gradient elution conditions: 0-30 min: the volume fraction of mobile phase B increased from 5% to 95%, and the mobile phase flow rate was 3 mL / min. The separated sample was lyophilized to obtain compound 18 (white solid, 10 mg, yield 30.5%, 14.75 μmol); ESI MS [M+H] + for C 22 H 39 ClN5O 13 P2,calcd 678.16,found 678.13; 1H NMR (400MHz, DMSO-d6) δ8.28(s,1H),7.87(s,4H),6.31(s,1H),6.09(d,J=4.3Hz,1H),4.54(s,1H),4.28(d,J=4.7Hz,1H),4.01(t,J=4.3Hz,2H),3.8 1(p,J=6.4,4.9Hz,1H),3.62(d,J=5.4Hz,2H),3.53(dd,J=14.4,5.1Hz,8H ), 3.46 (s, 8H), 3.43 (d, J = 5.2Hz, 2H), 2.92 (s, 2H), 2.08 (t, J = 19.8Hz, 2H).

[0153] 4) Synthesis of DOTA-dPNPEG2

[0154] Compound 18 (8.85 μmol) was dissolved in N,N-dimethylformamide (DMF, 1 mL), and DOTA activated ester (raw material 11, 22.13 μmol) and N,N-diisopropylethylamine (DIPEA, 44.25 μmol) were added. The mixture was stirred at 20°C for 12 h. After complete reaction, the mixture was purified by reverse-phase high performance chromatography (HPLC). HPLC purification conditions were: reverse-phase C18 semi-preparative column. Mobile phase A: water + 0.1% trifluoroacetic acid (TFA); mobile phase B: acetonitrile + 0.1% TFA; gradient elution conditions: 0-30 min: mobile phase B volume fraction increased from 5% to 95%, mobile phase flow rate 3 mL / min. The separated sample was lyophilized to obtain DOTA-dPNPEG2 as a white solid (5 mg, 53.1% yield, 4.69 μmol); ESI MS [M+H] + for C 38 H 65 ClN9O 20 P 2, calcd 976.29, found 976.04, Figures 15 and 16 are the mass spectrum and HPLC purity identification chart of compound DOTA-dPNPEG2.

[0155] Example 9

[0156] Preparation of NOTA-dPNPEG2: Prepared according to the method of step 4) of Example 8, the only difference from step 7) of Example 1 is that DOTA activated ester is replaced by NOTA activated ester to obtain compound NOTA-dPNPEG2 (white solid, 1.7 mg, yield 39.9%, 1.76 μmol); ESI MS [M+H] + for C 34 H 58 ClN8O 18P2, calcd 963.30, found 963.53. Figures 17 and 18 are the mass spectrum and HPLC purity identification chart of compound NOTA-dPNPEG2.

[0157] Example 10

[0158] Synthesis of DOTA-dPNME

[0159] The specific steps include:

[0160] 1) Synthesis of Compound 19: Compound 5 (632.46 μmol) was dissolved in N-methylpyrrolidone (NMP, 5 mL), and N-tert-butyloxycarbonyl-1,2-ethylenediamine (raw material 19, 822.2 μmol) and N,N-diisopropylethylamine (DIPEA, 1.26 mmol) were added sequentially. The temperature was raised to 110°C and stirred for 2 h. Completion of the reaction was monitored by thin-layer chromatography on silica gel. The mixture was cooled to room temperature and quenched with water. Ethyl acetate was added for extraction. The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The crude product was purified by column chromatography (petroleum ether:ethyl acetate volume ratio = 1:0 to 1:2) to obtain compound 19 (pale yellow oil, 0.43 g, 88.3% yield, 558.27 μmol); ESI MS [M+H] + for C 40 H 41 ClN5O9,calcd 770.25,found 770.10; 1 H NMR(400MHz,Chloroform-d)δ8.08(dd,J=5.4,3.2Hz,3H),8.02-7.97(m,2H),7.95-7.90(m ,2H),7.53(dd,J=13.2,6.7Hz,3H),7.42-7.31(m,6H),6.91(d,J=3.0Hz,1H),6.42(dd,J=5 .4,3.1Hz,1H),6.36(t,J=5.7Hz,1H),6.20(s,1H),4.80(q,J=5.1Hz,2H),4.73(dd,J=11.9 ,4.1Hz,1H),4.59(dd,J=11.9,5.1Hz,1H),3.37(t,J=7.0Hz,4H),2.84(s,3H),1.40(s,9H).

[0161] 2) Synthesis of Compound 20: Compound 19 (558.27 μmol) obtained in Reaction 1 was dissolved in a mixed solution of tetrahydrofuran (THF, 5 mL) and methanol (MeOH, 1 mL), and an aqueous sodium hydroxide solution (2.23 mL, 1 M) was added dropwise. The mixture was stirred at 20°C for 2 h. The reaction was complete by thin-layer chromatography on silica gel (developing system: ethyl acetate). The reaction solution was concentrated, water was added, and ethyl acetate was added for extraction. The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain compound 20 (white solid, 0.21 g, yield 82.1%, 458.6 μmol), which was used directly in the next reaction without purification; ESI MS [M+Na] + for C 19 H 28 ClN5O6Na, calcd 479.91, found 480.02.

[0162] 3) Synthesis of Compound 21: Compound 20 (109.2 μmol) was dissolved in trimethyl phosphate ((MeO)3PO, 1 mL), cooled to 0°C, and methylenebis(phosphine dichloride) (starting material 9, 436.77 μmol) was added. The mixture was stirred at 0°C for 3 h. Tetraethylammonium chloride solution (TEAC buffer, pH = 7.4-7.6, 2 mL, 0.5 M) was added and stirred for 1 h. Extraction was performed with methyl tert-butyl ether. The aqueous phase was collected and lyophilized to obtain the crude product. The crude product was purified by reverse-phase high-performance chromatography using a reverse-phase C18 semi-preparative column. Mobile phase A: water + 0.1% trifluoroacetic acid (TFA); mobile phase B: acetonitrile + 0.1% TFA; gradient elution conditions: 0-30 min: 5% acetonitrile (0.1% TFA) and 95% water (0.1% TFA) increasing to 95% acetonitrile (0.1% TFA) and 5% water (0.1% TFA), flow rate: 3 mL / min. The separated sample was lyophilized to obtain compound 21 as a white solid (15 mg, 26.6% yield, 29.1 μmol); ESI MS [M+H] + for C 15 H 25 ClN5O9P2,calcd 516.07, found 515.95.

[0163] 4) Synthesis of DOTA-dPNME

[0164] Compound 21 (5 mg, 9.69 μmol) obtained in reaction 3 was dissolved in N,N-dimethylformamide (DMF, 1 mL). DOTA activated ester (starting material 11, 2 eq., 9.72 mg, 19.39 μmol) and N,N-diisopropylethylamine (DIPEA, 5 eq., 6.26 mg, 48.47 μmol) were added and stirred at 20°C for 12 h. After complete reaction, the product was purified by reverse-phase high-performance chromatography (HPLC). HPLC purification conditions used a reverse-phase C18 semi-preparative column. Mobile phase A: water + 0.1% trifluoroacetic acid (TFA); mobile phase B: acetonitrile + 0.1% TFA. Gradient elution conditions: 0-30 min: the volume fraction of mobile phase B increased from 5% to 95%, with a mobile phase flow rate of 3 mL / min. The separated sample was lyophilized to obtain DOTA-dPNME (white solid, 2.5 mg, yield 28.6%, 2.77 μmol); ESI MS [M+H] + for C 31 H 51 ClN9O 16 P 2, calcd 902.15, found 902.17, Figures 19 and 20 are the mass spectrum and HPLC purity identification spectrum of compound DOTA-dPNME.

[0165] Example 11

[0166] Preparation of NOTA-dPNME: Prepared according to the method of step 4) of Example 10, the only difference from step 7) of Example 1 is that DOTA activated ester is replaced by NOTA activated ester to obtain compound NOTA-dPNME (white solid, 1.5 mg, yield 19.32%, 1.87 μmol); ESI MS [M+H] + for C 27 H 44 ClN8O 14 P2, calcd 801.21, found 801.21. Figure 21 is the mass spectrum identification diagram of compound NOTA-dPNME.

[0167] Example 12

[0168] Synthesis of DOTA-dPNPZ

[0169] The specific steps include:

[0170] 1) Synthesis of Compound 22: Compound 5 (632.46 μmol) was dissolved in N-methylpyrrolidone (NMP, 5 mL), and N-tert-butyloxycarbonyl-1,2-ethylenediamine (starting material 20, 758.95 μmol) and N,N-diisopropylethylamine (DIPEA, 1.26 mmol) were added sequentially. The temperature was raised to 110°C and stirred for 2 h. Completion of the reaction was monitored by thin-layer chromatography on silica gel. The mixture was cooled to room temperature and quenched with water. Ethyl acetate was added for extraction. The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The crude product was purified by column chromatography (petroleum ether:ethyl acetate volume ratio = 1:0 to 1:2) to obtain compound 22 (pale yellow oil, 0.4 g, 80.9% yield, 521.11 μmol); ESI MS [M+H] + for C 41 H 41 ClN5O 9, calcd 782.25,found 782.10; 1 H NMR(400MHz,Chloroform-d)δ8.08(d,J=7.0Hz,2H),8.00(d,J=6.9Hz,2H),7.94(d ,J=6.5Hz,3H),7.58-7.50(m,3H),7.42-7.32(m,6H),6.91(d,J=2.9Hz,1H),6.43(d d,J=5.4,3.0Hz,1H),6.36(t,J=5.8Hz,1H),6.33(s,1H),4.81(t,J=5.3Hz,1H),4. 74(dd,J=11.9,4.2Hz,1H), 4.59(dd,J=11.9,5.1Hz,1H), 3.64(s,8H), 1.50(s,9H).

[0171] 2) Synthesis of Compound 23: Compound 22 (511.35 μmol) obtained in Reaction 1 was dissolved in a mixed solution of tetrahydrofuran (THF, 5 mL) and methanol (MeOH, 1 mL), and an aqueous sodium hydroxide solution (2.05 mL, 1 M) was added dropwise. The mixture was stirred at 20°C for 2 h. The reaction was complete when detected by thin-layer chromatography on silica gel (petroleum ether:ethyl acetate volume ratio = 0:1). The reaction solution was concentrated, water was added, and ethyl acetate was added for extraction. The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain compound 23 (white solid, 0.21 g, yield 92.1%, 411.78 μmol), which was used directly in the next step without purification; ESI MS [M+H] + for C 20 H 29ClN5O6,calcd 470.17,found 470.05.

[0172] 3) Synthesis of Compound 24: Compound 23 (50 mg, 106.4 μmol) was dissolved in trimethyl phosphate ((MeO)3PO, 1 mL), cooled to 0°C, and methylenebis(phosphine dichloride) (starting material 9, 532.0 μmol) was added. The mixture was stirred at 0°C for 3 h. Tetraethylammonium chloride solution (TEAC buffer, pH = 7.4-7.6, 2 mL, 0.5 M) was added and stirred for 1 h. Extraction was performed with methyl tert-butyl ether. The aqueous phase was collected and lyophilized to obtain the crude product. The crude product was purified by reverse-phase high-performance chromatography (HPLC) using a reverse-phase C18 semi-preparative column. Mobile phase A: water + 0.1% trifluoroacetic acid (TFA); mobile phase B: acetonitrile + 0.1% TFA. Gradient elution conditions: 0-30 min: the volume fraction of mobile phase B increased from 5% to 95%, and the mobile phase flow rate was 3 mL / min. The separated sample was lyophilized to obtain compound 24 as a white solid (30 mg, yield 53.4%, 56.84 μmol); ESI MS [M+H] + for C 16 H 25 ClN5O9P2,calcd 528.07, found 527.97.

[0173] 4) Synthesis of DOTA-dPNPZ: Compound 21 (9.97 μmol) was dissolved in N,N-dimethylformamide (DMF, 1 mL), and DOTA activated ester (raw material 11, 24.91 μmol) and N,N-diisopropylethylamine (DIPEA, 49.83 μmol) were added. The mixture was stirred at 20°C for 12 h. After complete reaction, the product was purified by reverse-phase high performance chromatography (HPLC). HPLC purification conditions: reverse-phase C18 semi-preparative column. Mobile phase A: water + 0.1% trifluoroacetic acid (TFA); mobile phase B: acetonitrile + 0.1% TFA. Gradient elution conditions: 0-30 min: mobile phase B volume fraction increased from 5% to 95%, mobile phase flow rate 3 mL / min. The separated sample was lyophilized to obtain DOTA-dPNPZ (white solid, 2.8 mg, 31.6% yield, 3.15 μmol); ESI MS [M+H] + for C 32 H 51 ClN9O 16 P 2, calcd 914.25, found 914.17. Figures 22 and 23 are the mass spectrum and HPLC purity identification spectrum of compound DOTA-dPNPZ.

[0174] Example 13

[0175] Preparation of NOTA-dPNPZ: Prepared according to the method of step 4) of Example 12, the only difference from step 7) of Example 1 is that DOTA activated ester is replaced by NOTA activated ester to obtain compound NOTA-dPNPZ (white solid, 2 mg, yield 25.96%, 2.46 μmol); ESI MS [M+H] + for C 28 H 44 ClN8O 14 P2, calcd 813.21, found 813.21. FIG24 is the mass spectrometry identification spectrum of compound NOTA-dPNPZ.

[0176] Example 14

[0177] 68 The Ga nuclide labeling process is as follows:

[0178] Wet labeling method: 740MBq 68 GaCl3 hydrochloric acid solution (eluted from a gallium germanium generator, 4 mL) was added to a centrifuge tube containing 1 mL of acetic acid-sodium acetate solution (0.5 M) of DOTA-dPNPEG (50 μg), and the mixture was allowed to react at room temperature to 100°C for 20 min, then cooled to room temperature, diluted with physiological saline or water for injection, and sterile filtered to obtain a radionuclide targeted probe injection (nuclide targeted probe [ 68 The concentration of Ga]Ga-DOTA-dPNPEG was 74 MBq / mL).

[0179] Lyophilization labeling method: 1 mL of buffered acetic acid-sodium acetate solution (0.5 M) and 740 MBq of 68 GaCl3 hydrochloric acid solution (eluted from the gallium germanium generator) was added to the freeze-dried drug box containing DOTA-dPNPEG (50 μg), mixed and dissolved, and then allowed to react at room temperature for 20 minutes. The solution was diluted with physiological saline or water for injection and sterile filtered to obtain the radionuclide targeted probe injection solution (radionuclide targeted probe [ 68 The concentration of Ga]Ga-DOTA-dPNPEG was 74 MBq / mL).

[0180] If the radiochemical purity is less than 95%, purification is required. The purification steps are as follows: Take a Sep-Pak C18 separation column and activate it by eluting it with 10 mL of anhydrous ethanol and 10 mL of water. Dilute the labeled solution with 10 mL of water and load it onto the separation column. Rinse the separation column with water to remove unreacted 68 Ga 3+ , and then washed with 1 mL of anhydrous ethanol to obtain 68The Ga-labeled complex was purified by nitrogen blowing to remove the organic solvent, and the radionuclide targeting probe was obtained by dilution with physiological saline and sterile filtration. 68 Ga]Ga-DOTA-dPNPEG injection (nuclide targeting probe [ 68 The concentration of Ga]Ga-DOTA-dPNPEG was 74 MBq / mL).

[0181] right[ 68 Ga]Ga-DOTA-dPNPEG was sampled and analyzed by HPLC. The HPLC system was as follows: reverse phase C18 analytical column, mobile phase A: water + 0.1% trifluoroacetic acid (TFA); mobile phase B: acetonitrile + 0.1% TFA, elution gradient: 0-25 min: the volume fraction of mobile phase B increased from 5% to 95%, the mobile phase flow rate was 1 mL / min. Radiolabeled target complex [ 68 The retention time of Ga]Ga-DOTA-dPNPEG was 10.438 min, and the radiochemical purity was calculated to be greater than 95%. DOTA-dPNPEG and [ 68 The HPLC spectrum of Ga]Ga-DOTA-dPNPEG is shown in Figure 25A.

[0182] according to[ 68 The preparation method of Ga]Ga-DOTA-dPNPEG was used to prepare the product, and DOTA-dPNPEG was replaced with DOTA-dPNE to obtain [ 68 Ga]Ga-DOTA-dPNE.

[0183] according to[ 68 The preparation method of Ga]Ga-DOTA-dPNPEG was used to prepare the product, and DOTA-dPNPEG was replaced with DOTA-dPNH to obtain [ 68 Ga]Ga-DOTA-dPNH.

[0184] according to[ 68 The preparation method of Ga]Ga-DOTA-dPNPEG was used to prepare the product, and DOTA-dPNPEG was replaced with DOTA-dPNPEG2 to obtain [ 68 Ga]Ga-DOTA-dPNPEG2.

[0185] Example 15

[0186] 18 F nuclide labeling ([ 18 The process of [F]AlF-NOTA-dPNE is as follows:

[0187] 740MBq 18 F -The solution was mixed with 50 μL of AlCl3 (20 μg) in sodium acetate solution (0.5 M, pH = 4.0) and shaken at 100°C for 10 min. 100 μg of NOTA-dPNE, 100 μL of acetonitrile and 100 μL of sodium acetate (0.5 M, pH = 4.0) were added to the above reaction solution and shaken at 100°C for 20 min. A Sep-Pak C18 separation column was activated and washed with 10 mL of anhydrous ethanol and 10 mL of water. The labeled solution was diluted with 10 mL of water and loaded onto the separation column. The separation column was rinsed with water to remove unreacted 18 F - , and then washed with 1 mL of anhydrous ethanol to obtain the radionuclide targeting probe [ 18 F]AlF-NOTA-dPNE.

[0188] right[ 18 F]AlF-NOTA-dPNE was sampled and analyzed by HPLC. The HPLC system was as follows: reversed-phase C18 analytical column, mobile phase A: water + 0.1% trifluoroacetic acid (TFA); mobile phase B: acetonitrile + 0.1% TFA, elution gradient: 0-25 min: the volume fraction of mobile phase B increased from 5% to 95%, mobile phase flow rate was 1 mL / min. Radiolabeled target complex [ 18 The retention time of F]AlF-NOTA-dPNE was 11.305 min, and its radiochemical purity was calculated to be greater than 95%. 18 The HPLC spectrum of [F]AlF-NOTA-dPNE is shown in Figure 25B.

[0189] Example 16

[0190] 177 Lu nuclide labeling ([ 177 The process of Lu]Lu-DOTA-dPNPEG) is as follows:

[0191] Wet labeling method: 370MBq 177 LuCl3 solution was added to a centrifuge tube containing 0.2 mL of acetic acid-sodium acetate solution (0.5 M, pH = 4.0) containing DOTA-dPNPEG (50 μg), and the mixture was placed at room temperature to 100°C for 20 min, then cooled to room temperature, diluted with physiological saline or water for injection, and sterile filtered to obtain a radionuclide targeted probe injection solution ([ 177 The concentration of Lu]Lu-DOTA-dPNPEG was 370 MBq / mL).

[0192] Lyophilization labeling method: 0.2 mL of acetic acid-sodium acetate solution buffer (0.5 M, pH = 4.0) and 370 MBq of 177LuCl3 solution was added to the freeze-dried drug box containing DOTA-dPNPEG (50 μg), mixed and dissolved, and then placed at room temperature to 100 ° C for 20 min, then cooled to room temperature, diluted with physiological saline or water for injection, and sterile filtered to obtain the radionuclide targeted probe injection (nuclide targeted probe [ 177 The concentration of Lu]Lu-DOTA-dPNPEG was 370 MBq / mL).

[0193] If the radiochemical purity is less than 95%, purification is required. The purification steps are as follows: Take a Sep-Pak C18 separation column and activate it by eluting it with 10 mL of anhydrous ethanol and 10 mL of water. Dilute the labeled solution with 10 mL of water and load it onto the separation column. Rinse the separation column with water to remove unreacted 177 Lu 3+ , and then washed with 1 mL of anhydrous ethanol to obtain 177 The Lu-labeled complex was purified by nitrogen purge to remove the organic solvent, and the solution was diluted with physiological saline and then sterile filtered to obtain the radionuclide targeted probe injection solution (radionuclide targeted probe [ 177 The concentration of Lu]Lu-DOTA-dPNPEG was 370 MBq / mL).

[0194] Radionuclide targeted probes[ 177 Lu]Lu-DOTA-dPNPEG was sampled and analyzed by HPLC. The HPLC system was as follows: reverse phase C18 analytical column, mobile phase A: water + 0.1% trifluoroacetic acid (TFA); mobile phase B: acetonitrile + 0.1% TFA, elution gradient: 0-25 min: the volume fraction of mobile phase B increased from 5% to 95%, the mobile phase flow rate was 1 mL / min. Radiolabeled target complex [ 177 The retention time of Lu]Lu-DOTA-dPNPEG is 13.760min, and its radiochemical purity is calculated to be greater than 97%. 177 The HPLC spectrum of Lu]Lu-DOTA-dPNPEG is shown in Figure 25C.

[0195] according to[ 177 Lu]Lu-DOTA-dPNPEG was prepared by replacing DOTA-dPNPEG with DOTA-dPNE to obtain [ 177 Lu]Lu-DOTA-dPNE.

[0196] Example 17

[0197] 99mTc nuclide labeling: SnCl2 was used as a reducing agent, and N-tris(hydroxymethyl)methylglycine (Tricine) and triphenylphosphine tris(sulfonic acid) sodium salt (TPPTS) were used as co-ligands. 99m Tc labeling.

[0198] Wet labeling method: 20 μL of freshly prepared SnCl2 solution (SnCl2 in hydrochloric acid, 1 mg / mL) was added to a solution containing HYNIC-dPNPEG (100 μg), 10 mg Tricine and 10 mg TPPTS, followed by the addition of 740 MBq of freshly eluted Na 99m The TcO4 eluent (eluted from the molybdenum-technetium generator) was mixed and sealed with a cap. The mixture was reacted at room temperature to 100°C for 30 minutes and then cooled to room temperature. The mixture was diluted with physiological saline or water for injection and sterile filtered to obtain the radionuclide targeting probe ( 99m Tc-HYNIC-dPNPEG) injection ( 99m The concentration of Tc-HYNIC-dPNPEG was 370 MBq / mL).

[0199] Lyophilization labeling method: 740 MBq of fresh Na 99m The TcO4 eluate (eluted from the molybdenum-technetium generator) was added to a freeze-dried drug box containing 100 μg HYNIC-dPNPEG, 10 mg Tricine and 10 mg TPPTS (containing mannitol and ascorbic acid), mixed and sealed, and reacted at room temperature to 100 ° C for 30 minutes, then cooled to room temperature, diluted with physiological saline or water for injection, and sterile filtered to obtain the radionuclide targeting probe ( 99m Tc-HYNIC-dPNPEG) injection ( 99m The concentration of Tc-HYNIC-dPNPEG was 370 MBq / mL).

[0200] If the radiochemical purity is less than 95%, purification is required. The purification steps are as follows: Take a Sep-Pak C18 separation column and activate it by eluting it with 10 mL of anhydrous ethanol and 10 mL of water. Dilute the labeled solution with 10 mL of water and load it onto the separation column. Rinse the separation column with water to remove unreacted 99m TcO4 - , and then washed with 1 mL of anhydrous ethanol to obtain 99m The Tc-labeled complex was purified by nitrogen blowing to remove the organic solvent, and then diluted with physiological saline and sterile filtered to obtain the radionuclide targeting probe ( 99mTc-HYNIC-dPNPEG) injection. The HPLC system is as follows: reversed-phase C18 analytical column, mobile phase A: water + 0.1% trifluoroacetic acid (TFA); mobile phase B: acetonitrile + 0.1% TFA, elution gradient: 0-20 min: mobile phase B volume fraction increases from 5% to 95%, mobile phase flow rate is 1 mL / min. Radiolabeled target complex 99m The retention time of Tc-HYNIC-dPNPEG was 11.608 min, and the radiochemical purity was calculated to be greater than 95%. 99m The HPLC spectrum of Tc-HYNIC-dPNPEG is shown in Figure 25D.

[0201] Test Example 1

[0202] 1. Stability test

[0203] Stability of physiological saline: The radionuclide targeting probe ([ 68 Ga]Ga-DOTA-dPNE, [ 18 F]AlF-NOTA-dPNE) was placed at room temperature for different periods of time, and samples were taken for analysis by HPLC. At the tested time points (0.5h, 1h, and 2h), each probe still maintained a radiochemical purity of >95%, indicating that it is stable in the specified solution and is not easily decomposed. 68 The HPLC identification results of the in vitro physiological saline stability of Ga]Ga-DOTA-dPNE are shown in Figure 26A. 18 The results of HPLC identification of the in vitro physiological saline stability of [F]AlF-NOTA-dPNE are shown in Figure 26C, indicating that [ 68 Ga]Ga-DOTA-dPNE and [ 18 F]AlF-NOTA-dPNE still maintains a high stability (>95%) in physiological saline system for 2 hours.

[0204] Serum stability: The radionuclide targeted probe ([ 68 Ga]Ga-DOTA-dPNE, [ 18 F]AlF-NOTA-dPNE) was incubated with serum at room temperature for various periods of time (0.5 h, 1 h, and 2 h). Acetonitrile was added to remove proteins, and the supernatant was centrifuged and analyzed by HPLC. At all time points tested, each probe maintained a radiochemical purity >95%, indicating its stability in the specified solution and resistance to decomposition. 68 The results of HPLC identification of the in vitro serum stability of [Ga]Ga-DOTA-dPNE are shown in Figure 26B. 18The results of HPLC identification of the in vitro serum stability of [F]AlF-NOTA-dPNE are shown in Figure 26D, indicating that [ 68 Ga]Ga-DOTA-dPNE and [ 18 F]AlF-NOTA-dPNE still maintains a high stability (>95%) in the serum system for 2 hours.

[0205] 2. Lipid-water distribution coefficient (LogP pH＝7.4 ) determination

[0206] 100 μL of the diluted radionuclide targeting probe was added to a centrifuge tube containing 0.9 mL of phosphate buffered saline (PBS, pH = 7.4) and 1 mL of n-octanol. After vortexing for 3 minutes, the tube was centrifuged at 12,000 rpm for 5 minutes. 100 μL of liquid was taken from each of the aqueous and n-octanol phases and the radioactivity was counted using a gamma counter. The experiment was repeated three times and the average value was obtained. The formula for calculating LogP is: P = I 有机相 / I 水相

[0207] Among them I 有机相 represents the radioactive count measured in the organic phase, I 水相 represents the radioactivity count measured in the aqueous phase. The lipid-water distribution coefficient of each radiolabeled targeting probe was ultimately determined through calculation. The results are shown in Table 1 below. The measured radionuclide targeting probes were water-soluble.

[0208] Table 1 Lipid-water distribution coefficient of radionuclide targeting probe

[0209] From Table 1, we know that the above four 68 The Ga-labeled probe is hydrophilic, suggesting that its metabolic pathway primarily involves the kidneys. Connecting the chelating group to the parent structure via linkers of varying lengths and polarities can appropriately adjust the probe's LogP, resulting in varying pharmacokinetic properties.

[0210] Test Example 2

[0211] Cell binding and uptake experiments

[0212] 1. Cell binding assay

[0213] MDA-MB-231 cells cultured in DMEM high glucose medium were plated in two 24-well plates (2×10 5cells / well), and placed in a cell culture incubator overnight. After 24 hours, take out the 24-well plate, aspirate the culture medium, add 4°C pre-cooled PBS buffer along the wall, and wash the well wall twice. Add different concentrations of radioactive ligand to each well of the first well plate, and first add the diluted inhibitor AB680 to each well of the second well plate, and then add different concentrations of radioactive ligand. Incubate at 37°C for 2 hours and then wash twice with PBS buffer. Finally, add 1M sodium hydroxide solution and place at room temperature for 10 minutes to lyse all the cells. Collect the cell suspension, count the radioactivity in the cells with a γ-counter, and use Graphpad prism 8.0 for data processing to calculate Bmax and K d (nmol / L) value.

[0214] Figure 27 is [ 68 Ga]Ga-DOTA-dPNE, [ 68 Ga]Ga-DOTA-dPNH, [ 68 Ga]Ga-DOTA-dPNPEG and [ 68 Cellular K of [Ga]Ga-DOTA-dPNPEG2 d As shown in Figure 27, in the MDA-MB-231 cell line that highly expresses CD73 protein, 68 K of Ga-labeled compounds d The value reached the nanomolar level, indicating that it has good affinity.

[0215] 2. In vitro cell uptake and inhibition experiments

[0216] MDA-MB-231 cells with high expression of CD73 protein were plated in 24-well plates containing DMEM culture medium (containing fetal bovine serum and double antibody) (cell count was performed using a cell counting plate, 2×10 5 After culturing for 24 h, the uptake group and inhibition group were set up according to the following operation.

[0217] Experimental steps for the uptake group: aspirate the original culture medium, wash twice with PBS, and aspirate the PBS; add culture medium containing specific radioactive drugs and DMEM culture medium without bovine serum, and incubate at 37°C for 10, 30, 60, and 120 minutes; after incubation, aspirate the radioactive culture medium, add sodium hydroxide solution to each well to lyse the cells, incubate for 5 minutes, place the lysed cells in a disposable centrifuge tube, and measure the radioactivity count; divide this count by the total count of added radioactivity to obtain the percentage of cell uptake (% uptake).

[0218] Experimental steps for the inhibition group: aspirate the original culture medium, wash twice with PBS, and aspirate the PBS; add the CD73 protein inhibitor AB680 and culture medium containing specific radioactive drugs, and incubate at 37°C for 10, 30, 60, and 120 minutes; after the incubation is completed, aspirate the radioactive culture medium, add sodium hydroxide solution to each well to lyse the cells, incubate for 5 minutes, place the lysed cells in a disposable centrifuge tube, and measure the radioactivity count; divide the count by the total count of radioactivity added to each well to obtain the cell uptake percentage (% uptake).

[0219] Figure 28 is [ 68 Ga]Ga-DOTA-dPNE, [ 68 Ga]Ga-DOTA-dPNH, [ 68 Ga]Ga-DOTA-dPNPEG and [ 68 Ga]Ga-DOTA-dPNPEG 22 The results of in vitro cell uptake and inhibition experiments are shown in Figure 28. 68 These radioactive complexes labeled with Ga were significantly taken up in cells and were significantly inhibited by the CD73 protein inhibitor AB680, indicating that 68 The Ga-labeled compound has specific binding to the CD73 protein.

[0220] Test Example 3

[0221] Micro-PET imaging of normal mice:

[0222] Prepared according to the embodiment with a radiochemical purity greater than 99% 68 Ga nuclide targeted probe ([ 68 Ga]Ga-DOTA-dPNE, [ 68 Ga]Ga-DOTA-dPNPEG2) solution, 10MBq was injected through the tail vein of normal female mice, and Micro-PET imaging was performed at 0.5h, 1h and 2h respectively. The region of interest (ROI) was outlined on the image, and the metabolism of the probe in vivo was obtained by calculation. Figure 29 is [ 68 Ga]Ga-DOTA-dPNE(A) and [ 68 Micro-PET imaging of Ga]Ga-DOTA-dPNPEG2(B) in normal mice for 0.5 to 2 hours. As shown in Figure 29, [ 68 Ga]Ga-DOTA-dPNE and [ 68 Ga]Ga-DOTA-dPNPEG2 is mainly metabolized by the kidneys in normal mice and is taken up to a certain extent in bones and joints, presumably due to the osteophilicity of (α,β-methylene) diphosphate in its structure.

[0223] Test Example 4

[0224] PET imaging of tumor-bearing mice

[0225] Validated in the B16F10 melanoma model [ 68 Ga]Ga-DOTA-dPNE, [ 68 Ga]Ga-DOTA-dPNH, [ 68 Ga]Ga-DOTA-dPNPEG and [ 68 Tumor targeting ability of Ga]Ga-DOTA-dPNPEG2. 7.4 MBq of radioactive complex was injected into B16F10 tumor-bearing mice via the tail vein. Micro-PET imaging was performed at 0.5 h, 1 h, and 2 h, and regions of interest (ROIs) were outlined on the images.

[0226] Figure 30 is [ 68 Ga]Ga-DOTA-dPNE, [ 68 Ga]Ga-DOTA-dPNH, [ 68 Ga]Ga-DOTA-dPNPEG and [ 68 MicroPET imaging of Ga]Ga-DOTA-dPNPEG2 in B16F10 tumor-bearing mice for 0.5 to 2 hours. As shown in Figure 30, compared with [ 68 Ga]Ga-DOTA-dPNE and [ 68 Ga]Ga-DOTA-dPNH, [ 68 Ga]Ga-DOTA-dPNPEG and [ 68 Ga]Ga-DOTA-dPNPEG2 has a more obvious uptake at the tumor site, which may be due to the hydrophilic modification of the polyethylene glycol linker. Figure 31 is [ 68 The radioligand uptake (A) and target / non-target ratio (B) of important tissues and organs of mouse B16F10 model mice of Ga]Ga-DOTA-dPNPEG from 0.5 to 2 hours, respectively, are 0.5 hours, 1 hour and 2 hours. As shown in Figure 31, [ 68 The tumor-to-skin ratio of Ga]Ga-DOTA-dPNPEG reached 2-3 times. The above data show that the distribution of radionuclide targeting probes in the B16F10 model is changed by changing the length and properties of the linker.

[0227] Validated in the human breast cancer MDA-MB-231 model[ 68 Ga]Ga-DOTA-dPNE, [ 68 Ga]Ga-DOTA-dPNH, [ 68 Ga]Ga-DOTA-dPNPEG and [68 The targeting ability of Ga]Ga-DOTA-dPNPEG2 to tumors. 7.4MBq of radioactive complex was injected into mice bearing MDA-MB-231 tumors via the tail vein. Micro-PET imaging was performed at 0.5h, 1h, and 2h, and the region of interest (ROI) was outlined on the scanned image. Figure 32 shows [ 68 Ga]Ga-DOTA-dPNE, [ 68 Ga]Ga-DOTA-dPNH, [ 68 Ga]Ga-DOTA-dPNPEG and [ 68 MicroPET imaging of Ga]Ga-DOTA-dPNPEG2 in human MDA-MB-231 model mice for 0.5 to 2 hours. As shown in Figure 32, the four radionuclide-targeted probes were clearly taken up in the tumor site and rapidly cleared from normal tissues such as muscle, liver, and kidney, resulting in a high target / non-target ratio.

[0228] Figure 33 is [ 68 Ga]Ga-DOTA-dPNE and [ 68 The radioligand uptake and target / non-target ratio of important tissues and organs of human MDA-MB-231 model mice from 0.5 to 2 hours are shown in A and B from left to right as 0.5 hours, 1 hour and 2 hours, and in C and D from left to right as 10 minutes, 0.5 hours, 1 hour and 2 hours. 68 The tumor / muscle ratio of Ga]Ga-DOTA-dPNE reached about 5.8, and non-target sites were rapidly cleared 2 hours after probe injection. There was a higher radioactive signal at the bladder site, indicating that [ 68 Ga]Ga-DOTA-dPNE is metabolized in the kidneys and excreted through urine. Figure 33B shows [ 68 Ga]Ga-DOTA-dPNPEG2 has a high uptake in tumor sites and is significantly higher than that of [ 68 For Ga]Ga-DOTA-dPNE, [ 68 The metabolism of Ga]Ga-DOTA-dPNPEG2 in normal organs was more rapid, and the tumor / muscle ratio reached about 6 within 2 h.

[0229] Validated in the human breast cancer MDA-MB-231 model[ 68 Ga]Ga-DOTA-dPNME and [ 68The targeting ability of Ga]Ga-DOTA-dPNPZ to tumors. 7.4MBq of radioactive complex was injected into mice bearing MDA-MB-231 tumors via the tail vein. MicroPET imaging was performed at 0.5h, 1h, and 2h, and regions of interest (ROIs) were drawn on the scanned images. Figure 34 A and B are [ 68 Ga]Ga-DOTA-dPNME radioligand uptake in important tissues and organs and target / non-target ratios in human MDA-MB-231 model mice at 0.5 to 2 hours. 68 Ga]Ga-DOTA-dPNME accumulated in large quantities in the tumor and kidney within 30 minutes; after 2 hours, a higher signal was observed in the tumor site, and it was basically completely metabolized in normal tissues, with a high radioactive signal only in the bladder site, indicating that [ 68 Ga]Ga-DOTA-dPNME is mainly excreted from the body through urine through renal metabolism. 68 AB680 (150 μg) was injected into the tail vein 10 min before the injection of Ga]Ga-DOTA-dPNME, and the inhibition group was imaged 1 h after the injection of the radionuclide targeting probe. PET images showed that AB680 could completely block the accumulation of the radionuclide targeting probe at the tumor site, indicating that [ 68 Ga]Ga-DOTA-dPNME has CD73 targeting. Figure 34B shows that [ 68 Ga]Ga-DOTA-dPNME has a good ratio of target to non-target organs: at the 2-h time point, the tumor / meat ratio is about 15, the tumor / heart and tumor / liver ratios are both over 5, and the tumor / kidney ratio is about 3. 68 Ga]Ga-DOTA-dPNME has good tumor targeting ability.

[0230] Figure 34C shows [ 68 Ga]Ga-DOTA-dPNPZ accumulated in large quantities in the tumor area and kidney within 30 minutes; after 2 hours, there was a high signal in the tumor area and it was basically metabolized completely in normal tissues except the kidney. Only a high radioactive signal was present in the bladder area, indicating that [ 68 Ga]Ga-DOTA-dPNPZ is mainly metabolized by the kidneys and excreted from the body through urine. Figure 34D shows that [ 68 Ga]Ga-DOTA-dPNPZ has a good target / non-target organ ratio: at the 2-h time point, the tumor / meat ratio is about 4, and the tumor / heart and tumor / liver ratios are both over 3. 68 Ga]Ga-DOTA-dPNPZ has good tumor targeting ability.

[0231] The same results were also verified in the MDA-MB-231 model [18 F]AlF-NOTA-dPNE and [ 18 F]AlF-NOTA-dPNPEG2 targeting ability to tumors. Figure 35 is [ 18 F]AlF-NOTA-dPNE and [ 18 MicroPET images of F]AlF-NOTA-dPNPEG2 in human MDA-MB-231 model mice at 0.5 to 2 hours, radioactive uptake in major tissues, and target / non-target ratios. As shown in Figures 35A and B, [ 18 F]AlF-NOTA-dPNE was significantly taken up in the tumor site and was cleared quickly in the muscle and kidney, among which the tumor / muscle ratio was about 20. As shown in Figure 35C and D, [ 18 F]AlF-NOTA-dPNPEG2 also showed significant uptake at the tumor site, with a tumor / tissue ratio of 10. Furthermore, the probe showed some uptake in the gallbladder and intestine.

[0232] In Figure 38, A is [ 177 SPECT images of Lu]Lu-DOTA-dPNE in the human breast cancer MDA-MB-231 model at 1 hour, 4 hours, and 24 hours. The results show that the radioactive signal is mainly concentrated in the tumor, kidney, bladder, and bones at 1 hour, while the uptake values ​​of other normal organs are low. The radioactive signal can still be observed in the tumor site at 24 hours. Figure 38B is [ 177 Lu]Lu-DOTA-dPNE has a good target / non-target ratio, suggesting that it is suitable for radiotargeted therapy with long-half-life radionuclides and causes less damage to normal tissues.

[0233] The above results indicate that the radionuclide targeting probe provided by the present invention has significant aggregation in tumor areas where CD73 protein is highly expressed, and the pharmacokinetic properties of the probe can be changed by changing the length and properties of the connecting chain.

[0234] Test Example 5

[0235] Biodistribution experiment in tumor-bearing mice

[0236] Eight-week-old C57BL / 6 mice (weighing about 20 g) were selected and injected with 1×10 6 B16F10 cells. When the diameter of the subcutaneous tumor was 0.5-1.0 cm, 1.5 MBq [ 68Ga]Ga-DOTA-dPNPEG, mice were decapitated at different times after injection (4 mice per time point), and the blood, brain, heart, liver, lung, kidney, intestine, spleen, muscle, bone, and tumor organs and tissues of interest were collected and weighed, and the radioactivity counts were measured. The results were expressed as the percentage of uptake dose per gram of tissue or organ (%ID / g). Figure 36 is [ 68 Distribution of [Ga]Ga-DOTA-dPNPEG in tissues and organs of mice with B16F10 tumors at 0.5, 1, and 2 hours. Figure 36 shows that at 0.5 hours post-injection, tumor uptake of the radionuclide-targeted probe was >3% ID / g, significantly higher than that in muscle tissue. Over time, retention in the blood and non-target organs gradually decreased, while uptake in the tumor was maintained, and the ratio of radioactivity uptake in tumor to muscle further increased.

[0237] Female Balb / c mice (weighing about 20 g) aged about 8 weeks were selected and injected with 1×10 7 MDA-MB-231 cells. When the diameter of the subcutaneous tumor was 0.5-1.0 cm, 1.5 MBq [ 68 Ga]Ga-DOTA-dPNE, mice were decapitated at different times after injection (10 min, 0.5 h, 1 h, and 2 h, respectively) (4 mice per time point), and blood, brain, heart, liver, lung, kidney, intestine, spleen, muscle, bone, and tumor and other organs and tissues of interest were collected and weighed, and the radioactivity counts were measured. The results were expressed as the percentage of uptake dose per gram of tissue or organ (%ID / g). Figure 37 is [ 68 Distribution of [Ga]Ga-DOTA-dPNE in tissues and organs of mice bearing MDA-MB-231 tumors at 10 minutes, 30 minutes, 1 hour, and 2 hours. Figure 37 shows that tumor uptake of the radionuclide-targeted probe exceeded 10% ID / g at 10 minutes post-injection, significantly higher than in muscle tissue. Probe uptake was high in the kidneys, consistent with PET imaging results. Renal retention decreased significantly at 0.5 hours. Furthermore, non-target organ uptake, including the kidneys, gradually decreased over time, while tumor uptake remained stable.

[0238] In summary, the CD73-mediated radionuclide targeting probe provided by the present invention exhibits binding specificity to the CD73 protein at the cellular and in vivo levels. There are currently no reports on related small molecule radionuclide targeting probes. Cell uptake and inhibition experiments show that the radionuclide targeting probe provided by the present invention can be efficiently taken up by the CD73 high-expressing cell line MDA-MB-231, and can be significantly inhibited by the CD73 inhibitor AB680; cell affinity experiments show that the CD73 affinity of the radionuclide targeting probe reaches the nanomolar level; in different mouse tumor models, including but not limited to breast cancer, melanoma, etc., the radionuclide targeting probe can be enriched in tumor areas with high CD73 expression, and the PET and SPECT imaging effects are good, indicating that this series of radionuclide targeting probes has the potential to become a new diagnostic and therapeutic reagent based on the CD73 target.

[0239] Although the above embodiments provide a detailed description of the present invention, they are only part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on the embodiments of the present invention without creative work, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. A nucleotide compound, characterized in that: It has the structure shown in Formula I: In the formula I: L is R1 is n and m are independently integers of 0 to 10; R5 includes -H or -CH3; R2 includes -H or halogen; R4 includes -OH or -F; X includes =N- or =CH-; R3 includes -H or any of the following structures:

2. The nucleotide compound according to claim 1, characterized in that In the formula I, when R1 is When n is 1 or 5, R3 is R5 is -H; When R1 is When n is 1, 2 or 5, R3 is R5 is -CH3; When R1 is When m is 2 or 4, R3 is R5 is -H or -CH3; When L is When R3 is 3. The method for preparing the nucleotide compound according to claim 1 or 2, characterized in that: The following steps are involved: Compound A and compound B are subjected to coupling reaction under Lewis acid catalysis to obtain compound C; The compound C and the compound D are subjected to a first substitution reaction under alkaline conditions to obtain a compound E; The compound E is subjected to a ribose protecting group removal reaction to obtain a compound F; The compound F is subjected to a second substitution reaction with methylenebis(dichlorophosphine) followed by a deprotection reaction to obtain a compound G; The compound G is subjected to a third substitution reaction with the R3 active compound to obtain the nucleotide compound with the labeling group; R1-R5, L and X in the compounds A-G are the same as R1-R5, L and X in the formula I, A is a -F or -OBz ​​group, and R7 includes a Boc, DDE or Fmoc protecting group; The R3 active compound is a compound with an active reactive group corresponding to R3, which can be selected from the following structures:

4. The preparation method according to claim 3, characterized in that: The molar ratio of compound A to compound B is 1:1 to 1:5; The Lewis acid is trimethylsilyl trifluoromethanesulfonate; the molar ratio of the compound A to the Lewis acid is 1:1 to 1:3; The coupling reaction is carried out at a temperature of 0 to 25° C. and for a time of 0.5 to 4 hours.

5. The preparation method according to claim 3, characterized in that: The first substitution reaction comprises: dissolving the compound C and the compound D in a high boiling point solvent, and performing a first substitution reaction under alkaline conditions; The molar ratio of compound C to compound D is 1:1 to 1:3; The high boiling point solvent includes one or more of N-methylpyrrolidone, dimethyl sulfoxide and N,N-dimethylformamide; The alkaline condition is provided by an organic base, and the organic base includes triethylamine and / or N,N-diisopropylethylamine; the molar ratio of the compound C to the organic base is 1:1 to 1:5; The temperature of the first substitution reaction is 80-120° C., and the time is 1-12 hours.

6. The preparation method according to claim 3, characterized in that: The deribose protecting group reaction is carried out under alkaline conditions, the temperature of the deribose protecting group reaction is 0 to 60° C., and the time is 1 to 12 hours.

7. The preparation method according to claim 3, characterized in that: The molar ratio of the compound F to methylenebis(phosphine dichloride) is 1:1-5; The temperature of the second substitution reaction is 0 to 25° C., and the time is 1 to 12 hours.

8. The preparation method according to claim 3, characterized in that: The third substitution reaction comprises: dissolving compound G and R3 active compound in a high boiling point solvent, and performing the third substitution reaction under alkaline conditions; The molar ratio of the compound G to the R3 active compound is 1:1 to 1:3; The high boiling point solvent includes one or more of N-methylpyrrolidone, dimethyl sulfoxide and N,N-dimethylformamide; The alkaline condition is provided by an organic base, and the organic base includes triethylamine and / or N,N-diisopropylethylamine; the molar ratio of the compound G to the organic base is 1:1 to 1:10; The temperature of the third substitution reaction is 25 to 50° C., and the time is 1 to 24 hours.

9. A pharmaceutically acceptable salt of a nucleotide compound, characterized in that: Including trifluoroacetate, hydrochloride, formate, potassium salt or sodium salt of nucleotide compounds; The nucleotide compound is the nucleotide compound according to claim 1 or 2 or the nucleotide compound prepared by the preparation method according to any one of claims 3 to 8.

10. A nuclide targeting probe, characterized in that: It has the structure shown in formula II: In the formula II, R1 to R5, L and X are the same as R1 to R5, L and X in the nucleotide compound according to claim 1 or 2; The R6 is obtained by the coordination reaction between the R3 coordination group and the labeled nuclide.

11. The nuclide targeting probe according to claim 10, characterized in that: The labeled nuclides include 18 F. 47 Sc, 64 Cu, 67 Cu, 67 Ga, 68 Ga, 89 Zr, 86 Y. 89 Sr. 90 Y. 99m Tc, 105 Rh, 109 Pd, 111 In, 119 Sb, 149 Tb, 153 Sm, 157 Gd, 161 Tb, 166 Ho, 177 Lu, 186 Re, 188 Re, 201 Tl, 203 Pb, 212 Pb, 212 Bi, 213 Bi, 223 Ra, 227 Th and 225 At least one of Ac.

12. The method for preparing the nuclide targeting probe according to claim 10 or 11, characterized in that: The preparation method comprises the following steps: A nucleotide compound or a pharmaceutically acceptable salt thereof is subjected to a coordination reaction with a labeled nuclide to obtain a nuclide targeting probe or a pharmaceutically acceptable salt of the nuclide targeting probe; the nucleotide compound is the nucleotide compound described in claim 1 or 2, or a nucleotide compound obtained by the preparation method described in any one of claims 3 to 8, or a pharmaceutically acceptable salt of the nucleotide compound described in claim 9.

13. A pharmaceutically acceptable salt of a nuclide targeting probe, characterized in that: It is obtained by subjecting a nuclide targeting probe or a nucleotide compound to a salt-forming reaction; the nuclide targeting probe is the nuclide targeting probe described in any one of claims 10 to 11 or the nuclide targeting probe obtained by the preparation method described in claim 12; the nucleotide compound is the nucleotide compound described in claim 1 or 2 or the nucleotide compound obtained by the preparation method described in any one of claims 3 to 8.

14. A pharmaceutical composition, characterized in that It comprises active components and pharmaceutically acceptable excipients; the active components comprise one or more of a nucleotide compound, a pharmaceutically acceptable salt of the nucleotide compound according to claim 9, a radionuclide targeting probe and a pharmaceutically acceptable salt of the radionuclide targeting probe according to claim 13; the nucleotide compound is the nucleotide compound according to claim 1 or 2 or a nucleotide compound obtained by the preparation method according to any one of claims 3 to 8; the radionuclide targeting probe is the radionuclide targeting probe according to any one of claims 10 to 11 or a radionuclide targeting probe obtained by the preparation method according to claim 12.

15. Use of the nucleotide compound according to claim 1 or 2, the nucleotide compound obtained by the preparation method according to any one of claims 3 to 8, the pharmaceutically acceptable salt of the nucleotide compound according to claim 9, the radionuclide targeting probe according to any one of claims 10 to 11, the radionuclide targeting probe obtained by the preparation method according to claim 12, the pharmaceutically acceptable salt of the radionuclide targeting probe according to claim 13, or the pharmaceutical composition according to claim 14 in the preparation of therapeutic drugs or diagnostic drugs for diseases mediated by CD73 protein.

16. The use according to claim 15, characterized in that The CD73 protein-mediated diseases include tumors or immune-related diseases.

17. The use according to claim 16, characterized in that The tumor includes one or more of breast cancer, ovarian cancer, lung cancer, liver cancer, colorectal cancer, prostate cancer, bone tissue sarcoma, connective tissue sarcoma, renal cell carcinoma, gastric cancer, pancreatic cancer, nasopharyngeal carcinoma, head and neck cancer, neuroendocrine tumor, skin melanoma, and bone metastasis.

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