Nucleotide compounds, methods of making and using the same, radionuclide targeting probes, methods of making and using the same, pharmaceutical compositions

Abstract

The application belongs to the technical field of biological medicine, and particularly relates to a nucleotide compound and a preparation method and application thereof, a nuclide targeting probe and a preparation method and application thereof, and a pharmaceutical composition. The nucleotide compound and the nuclide labeled complex provided by the application have excellent in-vivo biological performance, have high specific uptake in a lesion with high expression of CD73 protein, have a high target / non-target ratio, and have low non-specific background. The characteristics can reduce unnecessary radioactive damage to normal tissues and organs, and are very suitable for use in diagnosis and treatment of tumors. In addition, the nuclide targeting probe can also be combined with other immune checkpoint inhibitors (such as PD-1 / PD-L1 monoclonal antibodies) for combined treatment. The nuclide targeting probe can be used for visualizing and quantitatively analyzing the dynamic expression of CD73 in a tumor microenvironment through a nuclear medicine imaging method, and overcomes the defects of sampling errors and poor accuracy in traditional detection methods.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to nucleotide compounds and their preparation methods and applications, pharmaceutically acceptable salts of nucleotide compounds, radionuclide targeting probes and their preparation methods and applications, pharmaceutically acceptable salts of radionuclide targeting probes, and pharmaceutical compositions. Background Technology

[0002] CD73 (extracellular 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 glucosyl phosphatidylinositol (GPI) via a C-terminal serine residue, forming a non-covalently bound homodimer with variable conformation. 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 various 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 purine pathway. This pathway utilizes the sequential degradation of CD39 (exonucleoside triphosphate diphosphate hydrolase-1) and CD73 to convert extracellular ATP into extracellular adenosine (ADO), involving biological processes including neurotransmission, platelet aggregation, regulation of immune responses, smooth muscle contraction, and control of cell proliferation, differentiation, and apoptosis. In tumor tissues, ADO interacts with specific G protein-coupled receptors (A1R, A2AR, A2BR, and A3R) to remodel different types of cells, including the infiltration of immune cells and tumorigenic angiogenesis, promoting tumorigenesis and metastasis, thus acting as a potent immunosuppressive molecule.

[0003] In recent years, tumor immunotherapy has become a research hotspot in both academia and industry, bringing revolutionary changes to tumor treatment models. While immune checkpoint inhibitors, represented by PD-1 / PD-L1 monoclonal antibodies, have shown promise, low response rates remain a significant challenge for clinical application. The main reasons for this are: individual differences and heterogeneity in biomarker expression; suppression of immune cell infiltration and function in the tumor microenvironment; and the emergence of primary or even adaptive resistance to immunotherapy. Therefore, transforming "cold tumors" that are unresponsive or poorly responsive to immunotherapy into "hot tumors" is a current research focus. In the purine pathway, adenosine, converted from extracellular ATP through the synergistic action of CD39 and CD73, is a key regulator of innate and adaptive immunity in the tumor microenvironment. Increasing research indicates that tumor cells can utilize the CD73-mediated adenosine production mechanism to evade the immune system. The CD73-adenosine pathway is strongly correlated with tumor occurrence and development, and a growing body of research emphasizes the potential value of this target, which is expected to become a new biomarker for personalized cancer treatment.

[0004] CD73 is overexpressed in various 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, and the adenosine produced by this upregulation 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 inhibitory activity, stimulating macrophage M2 polarization, thus providing conditions for tumor formation and metastasis. Furthermore, studies have shown that radiotherapy, chemotherapy, and the use of PD-1 / PD-L1 monoclonal antibodies significantly upregulate CD73 expression, subsequently leading to immunosuppression through the catalytic production of adenosine, resulting in acquired drug resistance. Targeting CD73 can produce good anti-tumor effects, and by inhibiting adenosine production, the inhibitory effect of the tumor microenvironment on immune cells can be alleviated. Therefore, the combination of CD73 inhibitors with immunomodulators (such as CTLA-4 and PD-1 / PD-L1 antibodies) is a highly attractive option. This strategy effectively compensates for the inherent limitations of immunotherapy, transforming "cold tumors" into "hot tumors," thereby producing a "1+1>2" therapeutic effect. Its synergistic effect has become a new hot topic in anti-tumor research. Considering the complexity and heterogeneity of tumor immune targets, image-guided tumor immunotherapy is receiving increasing attention. Following the great success of immunoPET / SPECT targeting immune checkpoints such as PD-1 / PD-L1, how to construct specific probes for the key immune target CD73 and "tailor-make" nuclear medicine imaging protocols around it has become a key focus for researchers.

[0005] Both research into immune mechanisms and clinical needs demonstrate the rationale and necessity of constructing molecular probes targeting CD73. However, to date, research using molecular imaging techniques to obtain in vivo CD73 information is very limited, and there are no reports of using specific radioactive small molecule probes to image tumor CD73 expression and guide or combine with CD73 inhibitor therapy. Therefore, developing high-resolution molecular imaging technology PET / SPECT combined with highly sensitive and specific CD73-targeting probes is of great significance for accelerating research on related immune mechanisms, deepening our understanding of this target, and broadening the application scope of immunotherapy. Summary of the Invention

[0006] In view of this, the object of the present invention is to provide nucleotide compounds, methods for their preparation and applications, pharmaceutically acceptable salts of nucleotide compounds, radionuclide targeting probes, methods for their preparation and applications, pharmaceutically acceptable salts of radionuclide targeting probes, and pharmaceutical compositions. The nucleotide compounds and their pharmaceutically acceptable salts, as well as the radionuclide targeting probes and their pharmaceutically acceptable salts provided by the present invention, all possess CD73 targeting properties.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solutions.

[0008] This invention provides a nucleotide compound having the structure shown in Formula I:

[0009]

[0010] In equation I: L is R1 is n and m are independent integers from 0 to 10; R5 includes -H or -CH3; R2 includes -H or halogen; R4 includes -OH or -F; X includes =N- or =CH-;

[0011] R3 includes -H or any of the following structures:

[0012]

[0013]

[0014] This invention provides a method for preparing the nucleotide compounds described in the above technical solution, comprising the following steps:

[0015] Compound A and compound B were coupled together under Lewis acid catalysis to obtain compound C.

[0016] Compound C and compound D were subjected to a first substitution reaction under alkaline conditions to obtain compound E;

[0017] Compound E was subjected to a deriboprotecting reaction to obtain compound F;

[0018] Compound F was subjected to a second substitution reaction with methylenebis(phosphine dichloride) followed by a deprotection reaction to yield compound G;

[0019] The compound G was subjected to a third substitution reaction with the active compound R3 to obtain the nucleotide compound with the labeled group;

[0020]

[0021] In compounds A to G, R1 to R5, L and X are the same as R1 to R5, L and X in Formula I, A is a -F or -OBz ​​group, and R7 includes a Boc, DDE or Fmoc protecting group.

[0022] The R3 active compound is a compound with an active reactive group corresponding to R3, and can be selected from the following structures:

[0023]

[0024] This invention provides a pharmaceutically acceptable salt of a nucleotide compound, obtained by reacting the nucleotide compound with an acid or base; wherein 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.

[0025] This invention provides a radionuclide targeting probe having the structure shown in Formula II:

[0026]

[0027] In Formula II, R1 to R5, L and X are the same as R1 to R5, L and X in the nucleotide compound of claim 1;

[0028] The R6 is obtained by a coordination reaction between the R3 coordinating group and the labeled nuclide.

[0029] Preferably, 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、 119Sb, 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.

[0030] This invention provides a method for preparing the radionuclide targeting probe described in the above technical solution, the method comprising the following steps:

[0031] A nucleotide compound or its pharmaceutically acceptable salt is coordinated with a labeled nuclide to obtain a nuclide-targeting probe or a pharmaceutically acceptable salt of a nuclide-targeting probe; wherein the nucleotide compound or its pharmaceutically acceptable salt is the nucleotide compound described in the above technical solution or the nucleotide compound or its pharmaceutically acceptable salt prepared by the preparation method described in the above technical solution.

[0032] This invention provides a pharmaceutically acceptable salt of a radionuclide-targeting probe, comprising a trifluoroacetate, hydrochloride, formate, potassium salt, or sodium salt of the radionuclide-targeting probe; wherein the radionuclide-targeting probe is the radionuclide-targeting probe described in the above-described technical solution or a radionuclide-targeting probe prepared by the preparation method described in the above-described technical solution. This invention also provides a pharmaceutically acceptable salt of a radionuclide-targeting probe, obtained by a salt-forming reaction of the radionuclide-targeting probe or a nucleotide compound; wherein the radionuclide-targeting probe is the radionuclide-targeting probe described in the above-described technical solution or a radionuclide-targeting probe prepared by the preparation method described in the above-described technical solution; wherein the nucleotide compound is the nucleotide compound described in the above-described technical solution or a nucleotide compound prepared by the preparation method described in the above-described technical solution.

[0033] This invention provides a pharmaceutical composition comprising an active ingredient and pharmaceutically acceptable excipients; the active ingredient comprises one or more of a nucleotide compound, a radionuclide targeting probe, a pharmaceutically acceptable salt of a nucleotide compound, and a pharmaceutically acceptable salt of a radionuclide targeting probe as described in the above-described technical solutions; the nucleotide compound is a nucleotide compound as described in the above-described technical solutions or a nucleotide compound prepared by the preparation method described in the above-described technical solutions; the radionuclide targeting probe is a radionuclide targeting probe as described in the above-described technical solutions or a radionuclide targeting probe prepared by the preparation method described in the above-described technical solutions.

[0034] This invention provides the use of the nucleotide compounds described in the above-described technical solutions, the nucleotide compounds prepared by the above-described technical solutions, pharmaceutically acceptable salts of the nucleotide compounds described in the above-described technical solutions, the radionuclide targeting probes described in the above-described technical solutions, the radionuclide targeting probes prepared by the above-described technical solutions, the pharmaceutically acceptable salts of the radionuclide targeting probes described in the above-described technical solutions, or the pharmaceutical compositions described in the above-described technical solutions in the preparation of therapeutic or diagnostic drugs for CD73 protein-mediated diseases.

[0035] Preferably, the diseases mediated by the CD73 protein include tumors or immune-related diseases.

[0036] The nucleotide compounds with the structure shown in Formula I or the radionuclide targeting probes shown in Formula II provided by this invention have a coordinating group R3 that exhibits strong labeling ability, short labeling time, and high labeling yield. As a precursor compound, it can chelate with the labeled radionuclide to form a CD73-targeting radionuclide targeting probe with high affinity and high specificity, which is beneficial for the commercial application and clinical promotion of radionuclide targeting probes. This invention introduces a molecular chain L with adjustable chain length between the nucleotide targeting group and the coordinating structure R3, which can adjust the distance between the targeting group and the coordinating structure, reducing the influence of the coordinating structure on the compound's activity. Simultaneously, by connecting with different types of radionuclide chelating groups, this structure can alter the lipid-water distribution properties of the radionuclide targeting probe, thereby obtaining radionuclide targeting probes with different polarities. Furthermore, the small molecule polarity change can improve the pharmacokinetic properties of the radionuclide targeting probe, accelerate the clearance rate of the tracer in non-target tissues, enhance the affinity between the radionuclide targeting probe and the receptor, increase the uptake of the radionuclide targeting probe in lesions with high CD73 protein expression, increase the target / non-target ratio, and thus give it excellent in vivo biological performance. This property can reduce unnecessary radiation damage to normal tissues and organs, making it ideal for the diagnosis and treatment of tumors. Furthermore, this radionuclide marker can also be used in combination with other immune checkpoint inhibitors (such as PD-1 / PD-L1 monoclonal antibodies) for combination therapy.

[0037] The radionuclide markers provided by this invention enable visualized and quantitative analysis of the dynamic expression of CD73 in the tumor microenvironment through nuclear medicine imaging techniques. This overcomes the shortcomings of traditional detection methods, such as sampling errors and poor accuracy, and provides a new research method for exploring the role of CD73 in tumor occurrence and development. Attached Figure Description

[0038] Figure 1 Mass spectrometry chromatogram for the compound DOTA-dPNE;

[0039] Figure 2 The HPLC purity chromatogram for the compound DOTA-dPNE is shown.

[0040] Figure 3 Mass spectrometry chromatogram for the compound NOA-dPNE;

[0041] Figure 4 The HPLC purity chromatogram for the compound NOA-dPNE is shown.

[0042] Figure 5 Mass spectrometry chromatogram for the compound DOTA-dPNH;

[0043] Figure 6 The HPLC purity chromatogram for compound DOTA-dPNH is shown.

[0044] Figure 7 Mass spectrometry chromatogram for the compound NOA-dPNH;

[0045] Figure 8 The HPLC purity identification chromatogram for compound Nota-dPNH is shown.

[0046] Figure 9 Mass spectrometry spectral representation of compound DOTA-dPNPEG;

[0047] Figure 10 The HPLC purity chromatogram for the compound DOTA-dPNPEG is shown.

[0048] Figure 11 Mass spectrometry chromatogram for the compound NOTA-dPNPEG;

[0049] Figure 12 The HPLC purity chromatogram for the compound NOA-dPNPEG is shown.

[0050] Figure 13 This is the mass spectrometry spectrum for identifying the compound HYNIC-dPNPEG.

[0051] Figure 14 The HPLC purity chromatogram for the compound HYNIC-dPNPEG is shown.

[0052] Figure 15 This is the mass spectrometry identification of compound DOTA-dPNPEG2;

[0053] Figure 16 The HPLC purity identification chromatogram for compound DOTA-dPNPEG2 is shown.

[0054] Figure 17 This is the mass spectrometry identification of compound Nota-dPNPEG2;

[0055] Figure 18 The HPLC purity identification chromatogram for compound Nota-dPNPEG2 is shown.

[0056] Figure 19 Mass spectrometry spectral representation of compound DOTA-dPNME;

[0057] Figure 20 The HPLC purity chromatogram for compound DOTA-dPNME is shown.

[0058] Figure 21 This is the mass spectrometry identification of the compound NOTA-dPNME;

[0059] Figure 22 This is the mass spectrometry identification of compound DOTA-dPNPZ;

[0060] Figure 23 The HPLC purity chromatogram for compound DOTA-dPNPZ is shown.

[0061] Figure 24 This is the mass spectrometry identification of compound Nota-dPNPZ;

[0062] Figure 25 For 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);

[0063] Figure 26 For compounds [ 68 Ga]Ga-DOTA-dPNE and [ 18 Stability of F]AlF-NOTA-dPNE in physiological saline and serum;

[0064] Figure 27 For compounds [ 68 Ga]Ga-DOTA-dPNE, [ 68 Ga]Ga-DOTA-dPNH, [ 68 Ga]Ga-DOTA-dPNPEG and [ 68 Ga]Ga-DOTA-dPNPEG2 cellular K d Value measurement results graph;

[0065] Figure 28 For compounds [ 68 Ga]Ga-DOTA-dPNE, [ 68 Ga]Ga-DOTA-dPNH, [68 Ga]Ga-DOTA-dPNPEG and [ 68 Figure 1. Results of in vitro cell uptake and inhibition experiments of Ga]Ga-DOTA-dPNPEG2;

[0066] Figure 29 for[ 68 Ga]Ga-DOTA-dPNE (Figure A) and [ 68 Ga]Ga-DOTA-dPNPEG2 (Figure B) MicroPET images of normal mice at 0.5–2 h;

[0067] Figure 30 for[ 68 Ga]Ga-DOTA-dPNE, [ 68 Ga]Ga-DOTA-dPNH, [ 68 Ga]Ga-DOTA-dPNPEG and [ 68 MicroPET images of Ga]Ga-DOTA-dPNPEG2 in B16F10 model mice from 0.5 to 2 hours;

[0068] Figure 31 for[ 68 Radioactive uptake (A) and target / non-target ratio (B) of Ga-DOTA-dPNPEG in major tissues of B16F10 model mice;

[0069] Figure 32 for[ 68 Ga]Ga-DOTA-dPNE, [ 68 Ga]Ga-DOTA-dPNH, [ 68 Ga]Ga-DOTA-dPNPEG and [ 68 MicroPET images of Ga]Ga-DOTA-dPNPEG2 in MDA-MB-231 model mice from 0.5 to 2 hours;

[0070] Figure 33 for[ 68 Ga]Ga-DOTA-dPNE(A) and [ 68 Radioactive uptake and target / non-target ratio of Ga]Ga-DOTA-dPNPEG2(B) in major tissues of MDA-MB-231 model mice;

[0071] Figure 34 for[ 68 Ga]Ga-DOTA-dPNME and [ 68MicroPET images of Ga]Ga-DOTA-dPNPZ in MDA-MB-231 model mice at 0.5–2 h (A, C), radioactive uptake in major tissues and target / non-target ratio (B, D);

[0072] Figure 35 for[ 18 F]AlF-NOTA-dPNE and [ 18 MicroPET images of F]AlF-NOTA-dPNPEG2 in MDA-MB-231 model mice at 0.5–2 h (A, C), radioactive uptake in major tissues and target / non-target ratio (B, D);

[0073] Figure 36 for[ 68 Distribution of Ga]Ga-DOTA-dPNPEG in tissues and organs of B16F10 tumor mice and target / non-target ratio;

[0074] Figure 37 for[ 68 Distribution of Ga-DOTA-dPNE in tissues and organs of MDA-MB-231 tumor-bearing mice and target / non-target ratio. Detailed Implementation

[0075] This invention provides a nucleotide compound having the structure shown in Formula I:

[0076]

[0077] In this invention, in formula I: L is for n and m are independent integers from 0 to 10, preferably 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; R5 includes -H or -CH3.

[0078] In this invention, R2 comprises -H or a halogen, wherein the halogen preferably comprises fluorine, chlorine or bromine.

[0079] In this invention, R3 includes -H or any of the following structures:

[0080]

[0081] In this invention, R4 includes -OH or -F.

[0082] In this invention, X includes =N- or =CH-.

[0083] In this invention, when R1 is When n is 1 or 5, R3 is R5 is -H, and the nucleotide compound has the structure shown in DOTA-dPNE, NOTA-dPNE, DOTA-dPNH, NOTA-dPNH, DOTAGA-dPNE, DOTA-SCN-dPNE, DOTAGA-dPNH, or DOTA-SCN-dPNH:

[0084]

[0085]

[0086]

[0087] In this invention, when R1 n is 1, 2, or 5, R3 is When R5 is -CH3, the nucleotide compound has the structure shown in DOTA-dPNME, NOTA-dPNME, DOTA-dPNPE, NOTA-dPNPE, DOTA-dPNMH, NOTA-dPNMH, DOTAGA-dPNME, DOTA-SCN-dPNME, DOTAGA-dPNMH, or DOTA-SCN-dPNMH:

[0088]

[0089]

[0090]

[0091] In this invention, when L is R3 is When the nucleotide compound has the structure shown in DOTA-dPNPZ, NOA-dPNPZ, DOTAGA-dPNPZ, or DOTA-SCN-dPNPZ:

[0092]

[0093]

[0094] In this invention, when R1 is m is 2 or 4, R3 is When R5 is -H or -CH3, the nucleotide compound has the structures 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, and DOTA-SCN-dPNMPEG2.

[0095]

[0096]

[0097]

[0098] This invention provides a method for preparing the nucleotide compounds described in the above technical solution, comprising the following steps:

[0099] Compound A and compound B were coupled together under Lewis acid catalysis to obtain compound C.

[0100] Compound C and compound D were subjected to a first substitution reaction under alkaline conditions to obtain compound E; compound E was then subjected to a deribose protecting group reaction to obtain compound F;

[0101] Compound F was subjected to a second substitution reaction with methylenebis(phosphine dichloride), followed by a deprotection reaction to yield compound G;

[0102] The compound G was subjected to a third substitution reaction with the active compound R3 to obtain the nucleotide compound with the labeled group;

[0103]

[0104] In compounds A to G, R1 to R5, L and X are the same as R1 to R5, L and X in Formula I, A is a -F or -OBz ​​group, and R7 includes a Boc, DDE or Fmoc protecting group.

[0105] The R3 active compound is a compound with an active reactive group corresponding to R3, and can be selected from the following structures:

[0106]

[0107] Unless otherwise specified, the materials and equipment used in this invention are all commercially available products in the field.

[0108] This invention involves a coupling reaction between compound A and compound B under Lewis acid catalysis to obtain compound C. Preferably, the coupling reaction involves: mixing compound A and a catalyst under reflux in hexamethyldisilazane (HMDS) to activate the mixture and obtain an activated intermediate; then mixing the activated intermediate, compound B, an organic solvent, and a Lewis acid to perform a coupling reaction to obtain compound C.

[0109] This invention involves mixing compound A, a catalyst, and hexamethyldisilazane (HMDS) for activation to obtain an activated product. In this invention, the catalyst is preferably ammonium sulfate; the molar ratio of compound A to the catalyst is preferably 1:0.01 to 1:1, more preferably 1:0.1. In this invention, the molar ratio 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 this invention, the activation is preferably carried out under reflux conditions, and the activation time is preferably 2 to 6 hours, more preferably 3 hours. After activation, this invention preferably further includes concentrating the obtained activated system to obtain the activated product; this invention does not have specific limitations on the concentration, and concentration to constant weight can be achieved using methods well known to those skilled in the art.

[0110] After obtaining the activated product, the present invention mixes the activated product, compound B, organic solvent, and 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 does not have a special limitation on the amount of organic solvent used, as long as it can dissolve compound A and ensure the smooth progress of the coupling reaction. In the present invention, the mixing is preferably carried out by dissolving the activated product in the organic solvent and then mixing it with compound B, cooling the resulting mixture to 25-0°C (more preferably 0°C), and then adding the Lewis acid dropwise. In this invention, the temperature of the coupling reaction is preferably 0–25°C, more preferably 0°C, and the time of the coupling reaction is preferably 0.5–4 h, more preferably 1 h. In a specific embodiment of this invention, the progress of the coupling reaction is preferably monitored by a thin-layer chromatography silica gel plate. During the coupling reaction, the protected furanose in compound B undergoes a coupling reaction with the hexamethyl group in the activated product. After the coupling reaction, this invention preferably includes post-treatment, which preferably includes: adding a saturated sodium bicarbonate solution to the obtained coupling reaction system to quench the reaction, then extracting with ethyl acetate 3–4 times, combining the organic phases, washing with saturated brine, drying with anhydrous sodium sulfate, filtering, 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.

[0111] After obtaining compound C, the present invention reacts compound C with compound D under alkaline conditions to undergo a first substitution reaction to obtain compound E. In the present invention, the first substitution reaction preferably includes: 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 have a particular limitation on the amount of the high-boiling-point solvent used, as long as it is sufficient to 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, which preferably includes triethylamine (TEA) and / or N,N-diisopropylethylamine (DIPEA); the molar ratio of compound C to organic base is preferably 1:1 to 1:5, more preferably 1:2. In this invention, the temperature of the first substitution reaction is preferably 80–120°C, more preferably 110°C, and the time of the first substitution reaction is preferably 1–12 h, more preferably 2 h. In a specific embodiment of this invention, the progress of the first substitution reaction is preferably monitored by a thin-layer chromatography silica gel plate. After the first substitution reaction, this invention preferably further includes post-treatment, which preferably includes: adding water to the obtained first substitution system to quench the reaction, then extracting with ethyl acetate 3–4 times, combining the organic phases, washing with saturated brine, drying with anhydrous sodium sulfate, filtering, concentrating the obtained filtrate under reduced pressure, and then purifying by column chromatography to obtain compound E; the eluent used for 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.

[0112] After obtaining compound E, the present invention performs a deriboprotecting reaction on compound E to obtain compound F. In the present invention, the deriboprotecting reaction is preferably carried out under alkaline conditions. Specifically, compound E is dissolved in a mixed solvent, and an alkaline aqueous solution is added dropwise to mix and carry out the deriboprotecting reaction. In the present invention, the alkaline in the alkaline aqueous solution preferably includes alkali metal hydroxides, ammonia, etc., more preferably sodium hydroxide. The molar ratio of compound E to the alkaline is preferably 1:3 to 1:10, more preferably 1:4; the concentration of the alkaline aqueous solution is preferably 0.1 to 2, 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 to methanol is preferably 1:1 to 5:1, more preferably 4:1. The present invention does not have a special limitation on the amount of the mixed solvent, as long as it is sufficient to dissolve compound E and ensure the smooth progress of the deriboprotecting reaction. In this invention, the temperature of the deriboprotecting reaction is preferably 0–60°C, more preferably 20°C, and the reaction time is preferably 1–12 h, more preferably 2 h. In a specific embodiment of this invention, the progress of the deriboprotecting reaction is preferably monitored by a thin-layer chromatography silica gel plate. During the deriboprotecting reaction, the ribose protecting group in compound E is removed. After the deriboprotecting reaction, this invention preferably includes a post-treatment, which preferably includes: concentrating the obtained deriboprotecting reaction system, adding water and mixing, then extracting with ethyl acetate 3–4 times, combining the organic phases, washing with saturated brine, drying with anhydrous sodium sulfate, filtering, and concentrating the obtained filtrate under reduced pressure to constant weight to obtain compound F. Compound F is directly used in subsequent reactions without purification.

[0113] After obtaining compound F, the present invention reacts compound F with methylenebis(phosphine dichloride) in a second substitution reaction followed by a deprotecting 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(phosphine dichloride) is added to carry out the second substitution reaction. In the present invention, the molar ratio 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(phosphine dichloride) 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, and the time of the second substitution reaction is preferably 1–12 h, more preferably 3 h. During the second substitution reaction, the ribose-OH in compound F undergoes a substitution reaction with methylenebis(phosphine dichloride). In this invention, 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 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.

[0114] The present invention preferably includes post-treatment after the second substitution reaction. 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 resulting aqueous phase followed by purification using a reversed-phase high-performance chromatography (RP-HPLC) column. The present invention does not have specific limitations on the amount and type of reagents or reaction conditions required for the deprotection reaction, as long as the protecting group (DDE, Boc, or Fmoc) can be removed. In the present invention, the conditions for the RPG purification include: the column is a reversed-phase C18 semi-preparative column; mobile phase A is preferably water + 0.1% (v / v) trifluoroacetic acid (TFA), and mobile phase B is preferably acetonitrile + 0.1% TFA; the elution method is preferably gradient elution, and the gradient elution conditions are preferably 0–30 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.

[0115] After obtaining compound G, the present invention performs a third substitution reaction with the active compound R3 to obtain the nucleotide compound. In the present invention, the third substitution reaction preferably includes: dissolving compound G and the active compound R3 in a high-boiling-point solvent and carrying out 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 includes one or more of N-methylpyrrolidone, dimethyl sulfoxide, and N,N-dimethylformamide. The present invention does not have a special limitation on the amount of the high-boiling-point solvent, as long as it is sufficient 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, which preferably includes 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 this invention, the temperature of the third substitution reaction is preferably 25–50°C, more preferably 25°C, and the time of the third substitution reaction is preferably 1–24 h, more preferably 12 h. After the third substitution reaction, this invention preferably further includes purification of the obtained third substitution system by reversed-phase high-performance chromatography (RP-HPLC). In this invention, the conditions for the RPI purification include: the column is a reversed-phase C18 semi-preparative column; mobile phase A is preferably water + 0.1% trifluoroacetic acid (TFA), and mobile phase B is preferably acetonitrile + 0.1% TFA; the elution method is preferably gradient elution, and the gradient elution conditions are preferably 0–30 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.

[0116] This 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-described technical solution or a nucleotide compound prepared by the preparation method described in the above-described technical solution. In this invention, the pharmaceutically acceptable salt preferably includes trifluoroacetate, phosphate, formate, acetate, potassium salt, or sodium salt. In this invention, the acid preferably includes trifluoroacetic acid, hydrochloric acid, formic acid, or acetic acid; the base preferably includes potassium hydroxide or sodium hydroxide. This invention does not specifically limit the reaction conditions; conditions for salt-forming reactions well known to those skilled in the art can be used.

[0117] This invention provides a radionuclide targeting probe having the structure shown in Formula II:

[0118]

[0119] In this invention, R1 to R5, L and X in Formula II are the same as R1 to R5, L and X in the nucleotide compound of claim 1.

[0120] In this invention, the coordinating group is obtained by coordinating R3 in the nucleotide compound described in the above technical solution with a 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.

[0121] This invention provides a method for preparing the radionuclide targeting probe described in the above technical solution, the method comprising the following steps:

[0122] A nucleoside-based compound is coordinated with a labeled nuclide to obtain a nuclide-targeting probe; the nucleoside-based compound is the nucleoside-based compound described in the above technical solution or the nucleoside-based compound prepared by the preparation method described in the above technical solution. In this invention, the nuclide-targeting probe is preferably prepared by a wet labeling method or a lyophilization labeling method.

[0123] In this invention, the wet labeling method for preparing radionuclide-targeting probes preferably includes the following steps: mixing a solution of a nucleotide compound with a labeled radionuclide solution, performing a coordination reaction, and then diluting to obtain an injection solution of the radionuclide-targeting probe. In this invention, the solvent in the nucleotide compound solution preferably includes one or more of a buffer solution, water, and an organic solvent; the buffer solution preferably includes an acetate solution or an aluminum chloride-acetate solution, and the pH value of the buffer solution is preferably 3-7, more preferably 4-6.5; the concentration of aluminum chloride in the aluminum chloride-acetate solution is preferably 0.2-1 g / L, more preferably 0.4 g / L; the acetate in the 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 nucleotide compound solution is preferably 0.001-1000 mg / mL, more preferably 0.01-1 mg / mL. In this 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 even more preferably 20–200 μg: 0.037–7400 MBq. In this invention, there is no particular limitation on the labeled nuclide solution; any labeled nuclide solution well known to those skilled in the art can be used, specifically as follows: 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 rinsing from a germanium-gallium generator, wherein the Na 99m The TcO4 eluent is preferably obtained by rinsing from a molybdenum technetium generator. In this invention, the temperature of the coordination reaction is preferably 25–100°C, more preferably 80–100°C, and the time of the coordination reaction is preferably 10–60 min, more preferably 20–30 min. When the temperature of the coordination reaction is higher than room temperature, this invention preferably further includes cooling the obtained coordination reaction system to room temperature after the coordination reaction. This invention does not have a specific limitation on the cooling; any cooling method well known to those skilled in the art can be used, such as natural cooling. After dilution, this invention preferably further includes sterile membrane filtration of the obtained diluted system to obtain an injection solution of the radionuclide-targeting probe. In this invention, the dilution preferably uses physiological saline or water for injection. In this invention, the concentration of the injection solution of the radionuclide-targeting probe is preferably 0.037–3700 MBq / mL.

[0124] In this invention, when the labeled nuclide is 18In case F, the coordination reaction is preferably carried out under mixed media in which metal ions are co-co-co-ion, buffer solution and organic solvent are present. The metal ions preferably include aluminum ions, the buffer solution preferably includes aluminum chloride-acetate solution and the organic solvent preferably includes acetonitrile.

[0125] In this invention, when the labeled nuclide is 99m At time Tc, the coordination reaction is preferably carried out in the presence of a reducing agent and a cooperating ligand. The reducing agent preferably includes SnCl2, which is preferably used in the form of a hydrochloric acid solution. Commercially available concentrated hydrochloric acid can be used to dissolve the SnCl2. The amount of SnCl2 used each time for labeling is preferably 0.001–100 mg, more preferably 0.01–0.2 mg. In this invention, the cooperating ligand preferably includes at least one of N-tris(hydroxymethyl)methylglycine (Tricine), sodium triphenylphosphine tris(m-sulfonate) (TPPTS), ethylenediamine-N,N'-diacetic acid (EDDA), ethylenediaminetetraacetic acid (EDTA), and sodium 3-diphenylphosphine benzenesulfonate (TPPMS). The mass ratio of the nucleotide compound to the cooperating ligand is preferably 0.01–0.5:0.1–50, more preferably 0.02–0.1:1–10.

[0126] In this invention, the preferred method for preparing radionuclide-targeted probes using lyophilization labeling includes the following steps: lyophilizing a solution of a nucleotide compound and sealing it to obtain a lyophilized kit; dissolving the compound in a solvent, then adding a labeled radionuclide solution for coordination reaction and diluting to obtain an injection solution of the radionuclide-targeted probe. In this invention, the lyophilization is preferably performed by dispensing the nucleotide compound solution into a lyophilization container before lyophilization. This invention does not impose special limitations on the lyophilization conditions; lyophilization conditions well-known to those skilled in the art can be used. In this invention, the solvent added to the lyophilized kit is preferably the same as the solvent in the nucleotide compound solution. This invention preferably adds at least one of an excipient, an antioxidant, and an acid-base regulator to the lyophilized kit as needed. This invention does not impose special limitations on the excipient, antioxidant, and acid-base regulator; excipients, antioxidants, and acid-base regulators well-known to those skilled in the art can be used. In this invention, other preparation conditions for the radionuclide-targeted probe are preferably the same as those described in the aforementioned wet labeling method, and will not be repeated here.

[0127] In this invention, when the radiochemical purity of the injection solution of the radionuclide targeting probe prepared by the wet labeling method and the lyophilization labeling method is less than 95%, it is preferable to further purify the injection solution of the radionuclide targeting probe. The purification is preferably performed using a Sep-Pak C18 separation column, which is preferably activated and rinsed sequentially with anhydrous ethanol and water before use. In this invention, the eluent used for purification is preferably water and anhydrous ethanol sequentially. The eluent of anhydrous ethanol is collected and the solvent is removed, followed by dilution to obtain a high-purity injection solution of the radionuclide targeting probe. In this invention, the dilution is preferably performed using physiological saline or water for injection. In this invention, the concentration of the high-purity injection solution of the radionuclide targeting probe is preferably 0.037–3700 MBq / mL.

[0128] In this invention, a pharmaceutically acceptable salt of a radionuclide-targeting probe is provided, obtained by a salting reaction of the radionuclide-targeting probe or a radionuclide labeling reaction of a pharmaceutically acceptable salt of a nucleotide compound. The radionuclide-targeting probe is the radionuclide-targeting probe described in the above-described technical solution or a radionuclide-targeting probe prepared by the above-described technical solution. The nucleotide compound is the nucleotide compound described in the above-described technical solution or a nucleotide compound prepared by the above-described technical solution. In this invention, the pharmaceutically acceptable salt preferably includes trifluoroacetate, phosphate, formate, acetate, potassium salt, or sodium salt. This invention does not specifically limit the preparation method of the pharmaceutically acceptable salt of the radionuclide-targeting probe; any pharmaceutically acceptable salt preparation method well known to those skilled in the art can be used.

[0129] This invention provides a pharmaceutical composition comprising an active ingredient and pharmaceutically acceptable excipients. The active ingredient comprises one or more of the following: a nucleotide compound, a radionuclide-targeting probe, a pharmaceutically acceptable salt of the nucleotide compound described in the above-described technical solution, and a pharmaceutically acceptable salt of the radionuclide-targeting probe described in the above-described technical solution. The nucleotide compound is either the nucleotide compound described in the above-described technical solution or a nucleotide compound prepared by the preparation method described in the above-described technical solution. The radionuclide-targeting probe is either the radionuclide-targeting probe described in the above-described technical solution or a radionuclide-targeting probe prepared by the preparation method described in the above-described technical solution. This invention does not specifically limit the pharmaceutically acceptable excipients; any pharmaceutically acceptable excipient well-known to those skilled in the art can be used. In this invention, when the active ingredient includes a radionuclide-targeting probe and / or a pharmaceutically acceptable salt of a radionuclide-targeting probe, the dosage form of the pharmaceutical composition is preferably an injection, preferably administered intravenously. When the active ingredient does not contain a radionuclide-targeting probe or its pharmaceutically acceptable salt, this invention does not specifically limit the dosage form and administration method of the pharmaceutical composition; any dosage form and administration method well-known to those skilled in the art can be used.

[0130] This invention also provides the use of the nucleotide compounds described in the above-described technical solutions, the nucleotide compounds prepared by the above-described technical solutions, pharmaceutically acceptable salts of the nucleotide compounds described in the above-described technical solutions, the radionuclide targeting probes described in the above-described technical solutions, the radionuclide targeting probes prepared by the above-described technical solutions, the pharmaceutically acceptable salts of the radionuclide targeting probes described in the above-described technical solutions, or the pharmaceutical compositions described in the above-described technical solutions in the preparation of therapeutic or diagnostic drugs for CD73 protein-mediated diseases. In this invention, the CD73 protein-mediated diseases include tumors or immune-related diseases. In this invention, the tumors preferably include one or more of breast cancer, ovarian cancer, lung cancer, liver cancer, colorectal cancer, prostate cancer, bone sarcoma, connective tissue sarcoma, renal cell carcinoma, gastric cancer, pancreatic cancer, nasopharyngeal carcinoma, head and neck cancer, neuroendocrine tumors, skin melanoma, and bone metastases. In this invention, the immune-related diseases preferably include one or more of the following: lupus erythematosus, rheumatoid arthritis, hepatitis, autoimmune encephalomyelitis, scleroderma, psoriasis, Sjögren's syndrome, polyarteritis nodosa, asthma, and atherosclerosis. In this invention, the diagnostic methods preferably include single-photon emission computed tomography (SPECT), positron emission tomography (PET), and magnetic resonance imaging (MRI); the treatment methods preferably include radionuclide targeted therapy and / or chemotherapy and / or immunotherapy.

[0131] To further illustrate the present invention, the nucleotide compounds, their preparation methods and applications, radionuclide targeting probes, their preparation methods and applications, and pharmaceutical compositions of the present invention are described in detail below with reference to the accompanying drawings and embodiments. However, these descriptions should not be construed as limiting the scope of protection of the present invention.

[0132] Example 1

[0133] DOTA-dPNE Synthesis

[0134]

[0135] Specifically, the following steps are included:

[0136] 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-chloroperoxybenzoic acid (m-CPBA, 59.91 mmol) was slowly added while controlling the reaction temperature below 20 °C. The reaction was carried out until the starting material was completely consumed. Methyl tert-butyl ether (40 mL) was added to the reaction solution, and the mixture was stirred for 1 h. The precipitated solid was filtered, washed with methyl tert-butyl ether (40 mL), and the filter cake was collected and dried at 45 °C to obtain crude compound 2 (4.9 g, 26.05 mmol).

[0137] 2) Synthesis of compound 3: Compound 2 (26.05 mmol) and a catalytic amount of triethylbenzylammonium chloride (BnEt3N) were synthesized. + Cl - 13.025 mmol) was dissolved in acetonitrile (CH3CN, 50 mL), cooled to 10 °C, and phosphorus oxychloride (POCl3, 52.1 mmol) was added to the reaction solution. The mixture was stirred at room temperature for 2 h until the reactants reacted completely. Water was added to quench the reaction mixture, and the temperature was controlled below 20 °C. The mixture was stirred for 1 h, and the precipitated solid was filtered. The filter cake was washed with saturated sodium bicarbonate solution, collected, and dried at 45 °C to give compound 3 (4.1 g, 21.8 mmol). ESI MS [M+H] + for C6H4Cl2N3, calcd 190.01, found 190.02.

[0138] 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, and acetonitrile (CH3CN, 5 mL) was added to the concentrate to dissolve it. 1-acetyl-2,3,5-tribenzoyloxy-1-β-D-ribofuranosyl (starting material 4, 2.87 mmol) was added. 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. Thin-layer chromatography with silica gel plate was used to monitor the reaction until complete. The reaction was quenched by adding saturated sodium bicarbonate solution (10 mL). Ethyl acetate was added for three extractions. 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 23Cl2N3O7Na, calcd 654.09, found654.26; 1 H 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).

[0139] 4) Synthesis of Compound 7: Compound 5 (0.632 mmol) was dissolved in N-methylpyrrolidone (NMP, 5 mL), and N-Boc-ethylenediamine (starting material 6, 0.759 mmol) and N,N-diisopropylethylamine (DIPEA, 1.26 mmol) were added. The mixture was heated to 110 °C and stirred for 2 h. The reaction was monitored by silica gel plate thin-layer chromatography until complete. The mixture was cooled to room temperature, and the reaction was quenched with water. Ethyl acetate was added for extraction. The organic phases were combined, washed with saturated brine, dried with 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 give compound 7 (pale yellow oil, 390 mg, yield 81%). 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).

[0140] 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). Sodium hydroxide aqueous 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 reactants were completely reacted. 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 give compound 8 (white solid, 180 mg, yield 85.2%, 0.405 mmol). This compound was used directly in the next reaction without purification. ESI MS [M+Na] + for C 18 H 26 ClN5O3Na,calcd 466.16, found 466.28.

[0141] 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, followed by the addition of tetraethylammonium chloride solution (TEAC, pH = 7.4–7.6, 1 mL, 0.5 M). Stirring was continued for 1 h, and 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 reversed-phase high-performance chromatography (HPLC). HPLC purification conditions: reversed-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 flow rate was 3 mL / min. The separated sample was lyophilized to give 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.

[0142] 7) Synthesis of DOTA-dPNE: Compound 10 (9.97 μmol) was dissolved in N,N-dimethylformamide (DMF, 1 mL), and DOTA-activated ester (starting material 11, 24.91 μmol) and N,N-diisopropylethylamine (DIPEA, 49.83 μmol) were added separately. The mixture was stirred at 25 °C for 12 h. After the reaction was complete, the mixture was purified by reversed-phase high-performance chromatography (HPLC). HPLC purification conditions: reversed-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 flow rate was 3 mL / min. The separated sample was lyophilized to obtain DOTA-dPNE (white solid, 3 mg, yield 33.9%, 3.38 μmol); ESI MS [M+H] + forC 30 H 49 ClN9O 16 P2,calcd 888.24,found 888.22. Figure 1 and Figure 2 Mass spectrometry and HPLC chromatograms for the purity identification of compound DOTA-dPNE.

[0143] Example 2

[0144] Preparation of NOTA-dPNE: Prepared according to step 7) of Example 1, except that the DOTA-activated ester was replaced with a NOTA-activated ester, yielding 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. Figure 3 and Figure 4 Mass spectrometry and HPLC chromatograms for the purity identification of compound NOA-dPNE.

[0145] The structural formula of NOTA activated ester is:

[0146] Example 3

[0147] Preparation of DOTA-dPNH: DOTA-dPNH was prepared according to steps 4) to 7) of Example 1, except that N-Boc-ethylenediamine was replaced with N-Boc-1,6-hexanediamine, yielding compound DOTA-dPNH (white solid, 3.2 mg, yield 38%, 3.39 μmol); ESI MS [M+H]+ for C 34 H 57 ClN9O 16 P2,calcd 944.30,found 944.25. Figure 5 and Figure 6 Mass spectrometry and HPLC chromatograms for the purity identification of compound DOTA-dPNH.

[0148] The structural formula of Boc-1,6-hexanediamine is:

[0149] Example 4

[0150] Preparation of NOTA-dPNH: The compound was prepared according to steps 4) to 7) of Example 1, except that N-Boc-ethylenediamine was replaced with N-Boc-1,6-hexanediamine, and the DOTA-activated ester was replaced with a NOTA-activated ester, yielding the 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. Figure 7 and Figure 8 Mass spectrometry and HPLC chromatograms for the purity identification of compound NOA-dPNH.

[0151] Example 5

[0152] Synthesis of DOTA-dPNPEG

[0153]

[0154] Specifically, the following steps are included:

[0155] 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 mixture was heated to 110 °C and stirred for 2 h. The reaction was monitored by silica gel plate for thin-layer chromatography until complete. The mixture was cooled to room temperature, and the reaction was quenched with water. Ethyl acetate was added for extraction. The organic phases were combined, washed with saturated brine, dried with 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, yield 93.6%, 592.2 μmol); ESI MS [M+Na] + for C 43 H 46 ClN5O 11 Na,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).

[0156] 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). Sodium hydroxide aqueous solution (NaOH, 2.37 mL, 1 M) was added dropwise. The mixture was stirred at 20 °C for 2 h, and the reaction was confirmed to be complete by thin-layer chromatography using silica gel plates (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). This compound was used directly in the next reaction without purification; ESIMS[M+Na]+ for C 22 H 34 ClN5O8Na,calcd 554.21,found 554.36.

[0157] 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) (starting material 9, 168.96 μmol) was added. The mixture was stirred at 0 °C for 3 h, followed by the addition of tetraethylammonium chloride solution (TEAC buffer, pH = 7.4–7.6, 2 mL, 0.5 M), and stirring continued for 1 h. Extraction was performed with methyl tert-butyl ether, and the aqueous phase was collected and lyophilized to obtain the crude product. The crude product was purified by reversed-phase high-performance chromatography. The separated sample was lyophilized to obtain compound 15 (white solid, 18 mg, yield 63.7%, 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).

[0158] 4) Synthesis of DOTA-dPNPEG: Compound 15 (6.78 μmol) was dissolved in N,N-dimethylformamide (DMF, 1 mL), and DOTA-activated ester (starting 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 the reaction was complete, the mixture was purified by reversed-phase high-performance chromatography (HPLC). HPLC purification conditions: reversed-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 flow rate was 3 mL / min. The separated sample was lyophilized to obtain DOTA-dPNPEG (white solid, 2.5 mg, yield 37.7%, 2.56 μmol); ESI MS [M+H] + for C 22 H 35 ClN5O8,calcd 976.29,found 976.04. Figure 9 and Figure 10 Mass spectrometry and HPLC chromatograms for the purity identification of compound DOTA-dPNPEG.

[0159] Example 6

[0160] Synthesis of NOTA-dPNPEG

[0161]

[0162] The specific steps include: dissolving compound 15 (5.09 μmol) in N,N-dimethylformamide (DMF, 1 mL), adding the activated ester of Nota (starting material 16, 12.71 μmol) and N,N-diisopropylethylamine (DIPEA, 25.43 μmol), and stirring at 20 °C for 12 h; after the reaction is complete, the sample is purified by reversed-phase high-performance chromatography. HPLC purification conditions: reversed-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 increases from 5% to 95%, and the flow rate is 3 mL / min. The separated sample is lyophilized to obtain Nota-dPNPEG as a white solid (1.5 mg, yield 33.7%, 1.72 μmol); ESI MS [M+H] + forC 30 H 50 ClN8O16P2,calcd 875.24, found 875.19, Figure 11 and Figure 12Mass spectrometry and HPLC chromatograms for the purity identification of compound NOTA-dPNPEG.

[0163] Example 7

[0164] Synthesis of HYNIC-dPNPEG

[0165]

[0166] The specific steps include: dissolving compound 15 (5.09 μmol) in N,N-dimethylformamide (DMF, 1 mL), adding HYNIC activated ester (starting material 17, 12.71 μmol) and N,N-diisopropylethylamine (DIPEA, 25.43 μmol), and stirring at 20 °C for 12 h; after the starting material reaction is complete, the mixture is purified by reversed-phase high-performance chromatography (HPLC). HPLC purification conditions: reversed-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 increases from 5% to 95%, and the flow rate is 3 mL / min. The separated sample is lyophilized to obtain HYNIC-dPNPEG as a yellow solid (1.3 mg, yield 28.6%, 1.46 μmol); ESI MS [M+H] + forC 33 H 45 ClN9O 12 P2,calcd 856.23,found 856.15, Figure 13 and Figure 14 Mass spectrometry and HPLC chromatograms for the purity identification of compound HYNIC-dPNPEG.

[0167] Example 8

[0168] Synthesis of DOTA-dPNPEG2

[0169]

[0170] Specifically, the following steps are included:

[0171] 1) Synthesis of Compound 16: Compound 5 (790.58 μmol) was dissolved in N-methylpyrrolidone (NMP, 5 mL), and 1,1-dimethyl ethyl 16-amino-5,8,11,14-tetraoxa-2-azahexadecanoic acid (starting material 18, 948.69 μmol) and N,N-diisopropylethylamine (DIPEA, 2.37 mmol) were added sequentially. The mixture was heated to 110 °C and stirred for 2 h. The reaction was monitored by silica gel plate for thin-layer chromatography until complete. The mixture was cooled to room temperature, and the reaction was quenched with water. Ethyl acetate was added for extraction. The organic phases were combined, washed with saturated brine, dried with 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 pale yellow oil (0.5 g, yield 67.8%, 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).

[0172] 2) Synthesis of Compound 17

[0173] Compound 16 (536.2 μmol) obtained from reaction 1 was dissolved in a mixed solution of tetrahydrofuran (THF, 5 mL) and methanol (MeOH, 1 mL), and sodium hydroxide aqueous solution (NaOH, 2.14 mL, 1 M) was added dropwise. The mixture was stirred at 20 °C for 2 h, and the reaction was confirmed to be complete by thin-layer chromatography using silica gel plates (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). This compound 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.

[0174] 3) Synthesis of compound 18

[0175] 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, followed by the addition of tetraethylammonium chloride solution (TEAC buffer, pH = 7.4–7.6, 2 mL, 0.5 M), and stirring continued for 1 h. Extraction was then performed with methyl tert-butyl ether, and the aqueous phase was collected and lyophilized to obtain the crude product. The crude product was purified by reversed-phase high-performance chromatography (HPLC). HPLC purification conditions: reversed-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 give compound 18 (white solid, 10 mg, yield 30.5%, 14.75 μmol); ESI MS [M+H] + for C 22 H 39 ClN5O 13 P2,calcd678.16,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).

[0176] 4) Synthesis of DOTA-dPNPEG2

[0177] Compound 18 (8.85 μmol) was dissolved in N,N-dimethylformamide (DMF, 1 mL), and DOTA-activated ester (starting 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 the reaction was complete, the sample was purified by reversed-phase high-performance chromatography (HPLC). HPLC purification conditions: reversed-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 flow rate was 3 mL / min. The separated sample was lyophilized to obtain DOTA-dPNPEG2 as a white solid (5 mg, yield 53.1%, 4.69 μmol); ESI MS [M+H] + for C 38 H 65 ClN9O 20 P2,calcd 976.29,found976.04, Figure 15 and Figure 16 Mass spectrometry and HPLC chromatograms for the purity identification of compound DOTA-dPNPEG2.

[0178] Example 9

[0179] Preparation of NOTA-dPNPEG2: Prepared according to step 4) of Example 8, the only difference from step 7) of Example 1, where the DOTA-activated ester was replaced with a NOTA-activated ester, yielding compound NOTA-dPNPEG2 (white solid, 1.7 mg, yield 39.9%, 1.76 μmol); ESI MS [M+H] + for C 34 H 58 ClN8O18 P2,calcd 963.30,found 963.53. Figure 17 and Figure 18 Mass spectrometry and HPLC chromatograms for the purity identification of compound NOTA-dPNPEG2.

[0180] Example 10

[0181] DOTA-dPNME Synthesis

[0182]

[0183] Specifically, the following steps are included:

[0184] 1) Synthesis of Compound 19: Compound 5 (632.46 μmol) was dissolved in N-methylpyrrolidone (NMP, 5 mL), and N-tert-butoxycarbonyl-1,2-ethylenediamine (starting material 19, 822.2 μmol) and N,N-diisopropylethylamine (DIPEA, 1.26 mmol) were added sequentially. The mixture was heated to 110 °C and stirred for 2 h. The reaction was monitored by silica gel plate for thin-layer chromatography until complete. The mixture was cooled to room temperature, and the reaction was quenched with water. Ethyl acetate was added for extraction. The organic phases were combined, washed with saturated brine, dried with 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 give compound 19 (pale yellow oil, 0.43 g, yield 88.3%, 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).

[0185] 2) Synthesis of Compound 20: Compound 19 (558.27 μmol) obtained from reaction 1 was dissolved in a mixed solution of tetrahydrofuran (THF, 5 mL) and methanol (MeOH, 1 mL). Sodium hydroxide aqueous solution (NaOH, 2.23 mL, 1 M) was added dropwise. The mixture was stirred at 20 °C for 2 h, and the reaction was confirmed to be complete by thin-layer chromatography using silica gel plates (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). This compound 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.

[0186] 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, followed by the addition of tetraethylammonium chloride solution (TEAC buffer, pH = 7.4–7.6, 2 mL, 0.5 M). Stirring was continued for 1 h, and 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 reversed-phase high-performance chromatography (HPLC). HPLC purification conditions: reversed-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) increased 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, yield 26.6%, 29.1 μmol); ESI MS [M+H] + for C 15 H 25 ClN5O9P2,calcd 516.07, found 515.95.

[0187] 4) Synthesis of DOTA-dPNME

[0188] Compound 21 (5 mg, 9.69 μmol) obtained from reaction 3 was dissolved in N,N-dimethylformamide (DMF, 1 mL), and 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. The mixture was stirred at 20 °C for 12 h. After the reaction was complete, the mixture was purified by reversed-phase high-performance chromatography (HPLC). HPLC purification conditions: reversed-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 flow rate was 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] + forC 31 H 51 ClN9O 16 P2,calcd 902.15,found 902.17, Figure 19 and Figure 20 Mass spectrometry and HPLC chromatograms for the purity identification of compound DOTA-dPNME.

[0189] Example 11

[0190] Preparation of NOTA-dPNME: Prepared according to step 4) of Example 10, the only difference from step 7) of Example 1, where the DOTA-activated ester was replaced with a NOTA-activated ester, yielding 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 This is the mass spectrometry identification of the compound NOTA-dPNME.

[0191] Example 12

[0192] DOTA-dPNPZ Synthesis

[0193]

[0194] Specifically, the following steps are included:

[0195] 1) Synthesis of Compound 22: Compound 5 (632.46 μmol) was dissolved in N-methylpyrrolidone (NMP, 5 mL), and N-tert-butoxycarbonyl-1,2-ethylenediamine (starting material 20, 758.95 μmol) and N,N-diisopropylethylamine (DIPEA, 1.26 mmol) were added sequentially. The mixture was heated to 110 °C and stirred for 2 h. The reaction was monitored by silica gel plate for thin-layer chromatography until complete. The mixture was cooled to room temperature, and the reaction was quenched with water. Ethyl acetate was added for extraction. The organic phases were combined, washed with saturated brine, dried with 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, yield 80.9%, 521.11 μmol); ESI MS [M+H] + for C 41 H 41 ClN5O9,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).

[0196] 2) Synthesis of Compound 23: Compound 22 (511.35 μmol) obtained from reaction 1 was dissolved in a mixed solution of tetrahydrofuran (THF, 5 mL) and methanol (MeOH, 1 mL). Sodium hydroxide aqueous solution (NaOH, 2.05 mL, 1 M) was added dropwise. The mixture was stirred at 20 °C for 2 h. Thin-layer chromatography (petroleum ether:ethyl acetate volume ratio = 0:1) was used to confirm the completeness of the reaction. 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). This compound was used directly in the next reaction without purification. ESI MS [M+H] + for C 20 H 29ClN5O6,calcd 470.17,found 470.05.

[0197] 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, followed by the addition of tetraethylammonium chloride solution (TEAC buffer, pH = 7.4–7.6, 2 mL, 0.5 M). Stirring was continued for 1 h, and 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 reversed-phase high-performance chromatography (HPLC). HPLC purification conditions: reversed-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 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] + forC 16 H 25 ClN5O9P2,calcd 528.07, found 527.97.

[0198] 4) Synthesis of DOTA-dPNPZ: Compound 21 (9.97 μmol) was dissolved in N,N-dimethylformamide (DMF, 1 mL), and DOTA-activated ester (starting 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 the reaction was complete, the mixture was purified by reversed-phase high-performance chromatography (HPLC). HPLC purification conditions: reversed-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 flow rate was 3 mL / min. The separated sample was lyophilized to obtain DOTA-dPNPZ (white solid, 2.8 mg, yield 31.6%, 3.15 μmol); ESI MS [M+H] + for C 32 H 51 ClN9O 16 P2,calcd 914.25,found914.17, Figure 22 and Figure 23 Mass spectrometry and HPLC chromatograms for the purity identification of compound DOTA-dPNPZ.

[0199] Example 13

[0200] Preparation of NOTA-dPNPZ: Prepared according to step 4) of Example 12, the only difference from step 7) of Example 1, where the DOTA-activated ester was replaced with a NOTA-activated ester, yielding 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, Figure 24 This is the mass spectrometry spectrum for the compound NOA-dPNPZ.

[0201] Example 14

[0202] 68 The Ga nuclide labeling process is as follows:

[0203] Wet labeling method: 740 MBq 68 GaCl3 hydrochloric acid solution (rinsed from the germanium-gallium generator, 4 mL) was added to a centrifuge tube containing 1 mL of LDOTA-dPNPEG (50 μg) in an acetate-sodium acetate solution (0.5 M). The mixture was incubated at room temperature to 100°C for 20 min, then cooled to room temperature. It 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.

[0204] Lyophilization labeling method: Add 1 mL of buffer solution (acetic acid-sodium acetate solution, 0.5 M) and 740 MBq of... 68 GaCl3 hydrochloric acid solution (rinsed from the germanium-gallium generator) was added to a lyophilized kit containing DOTA-dPNPEG (50 μg), mixed and dissolved, and reacted at room temperature for 20 min. The solution was then 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.

[0205] If the radiochemical purity is below 95%, purification is required. The purification steps are as follows: Take a Sep-Pak C18 separation column and activate and rinse it successively with 10 mL of anhydrous ethanol and 10 mL of water. Dilute the labeled solution with 10 mL of water and load the sample onto the separation column. Rinse the separation column with water to remove unreacted components. 68 Ga 3+ Then rinse with 1 mL of anhydrous ethanol to obtain 68Ga-labeled complexes. Organic solvents were removed by nitrogen blowing, and the resulting solution was diluted with physiological saline and sterile filtered to obtain the radionuclide-targeting probe. 68 Ga]Ga-DOTA-dPNPEG injection (nuclear target probe) 68 The concentration of Ga]Ga-DOTA-dPNPEG was 74 MBq / mL.

[0206] right[ 68 Ga]Ga-DOTA-dPNPEG samples were analyzed and identified by HPLC. The HPLC system was as follows: reversed-phase C18 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%, and the flow rate was 1 mL / min. Radiolabeled target complex [ 68 The retention time of Ga-DOTA-dPNPEG was 10.438 min, and its radiochemical purity was calculated to be greater than 95%. The DOTA-dPNPEG and [ 68 The HPLC spectrum of Ga]Ga-DOTA-dPNPEG is as follows: Figure 25 As shown in Figure A.

[0207] according to[ 68 The method for preparing Ga-DOTA-dPNPEG involves replacing DOTA-dPNPEG with DOTA-dPNE to obtain [ 68 Ga]Ga-DOTA-dPNE.

[0208] according to[ 68 The method for preparing Ga-DOTA-dPNPEG involves replacing DOTA-dPNPEG with DOTA-dPNH to obtain [ 68 Ga]Ga-DOTA-dPNH.

[0209] according to[ 68 The method for preparing Ga-DOTA-dPNPEG involves replacing DOTA-dPNPEG with DOTA-dPNPEG2 to obtain [ 68 Ga]Ga-DOTA-dPNPEG2.

[0210] Example 15

[0211] 18 F nuclide labeling ([ 18 The process of F]AlF-NOTA-dPNE is as follows:

[0212] 740MBq 18 F -The solution was mixed with 50 μL of AlCl3 (20 μg) in a sodium acetate solution (0.5 M, pH = 4.0) and shaken at 100 °C for 10 min. Then, 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 the mixture was shaken at 100 °C for 20 min. A Sep-Pak C18 separation column was activated and rinsed successively with 10 mL of anhydrous ethanol and 10 mL of water. The labeled solution was diluted with 10 mL of water and then loaded onto the separation column. The separation column was rinsed with water to remove unreacted reagents. 18 F - The radionuclide targeting probe was obtained by rinsing with 1 mL of anhydrous ethanol. 18 F]AlF-NOTA-dPNE.

[0213] right[ 18 F]AlF-NOTA-dPNE samples were analyzed and identified by HPLC. The HPLC system was as follows: reversed-phase C18 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%, and the 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%. The relationship between Nota-dPNE and [ 18 The HPLC spectrum of F]AlF-NOTA-dPNE is as follows: Figure 25 As shown in B.

[0214] Example 16

[0215] 177 Lu nuclide labeling ([ 177 The process of Lu-DOTA-dPNPEG is as follows:

[0216] Wet labeling method: 370 MBq 177 LuCl3 solution was added to a centrifuge tube containing 0.2 mL of 0.5 M, pH 4.0 acetate-sodium acetate solution (50 μg) of DOTA-dPNPEG. The mixture was incubated at room temperature to 100°C for 20 min, then cooled to room temperature. It was diluted with physiological saline or water for injection and sterile filtered to obtain the radionuclide-targeted probe injection solution. 177 The concentration of Lu]Lu-DOTA-dPNPEG was 370 MBq / mL.

[0217] Lyophilization labeling method: Mix 0.2 mL of acetate-sodium acetate buffer (0.5 M, pH = 4.0) and 370 MBq of...177 LuCl3 solution was added to a lyophilized kit containing DOTA-dPNPEG (50 μg), mixed and dissolved, and then reacted at room temperature to 100°C for 20 min. After cooling to room temperature, it was diluted with physiological saline or water for injection and 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.

[0218] If the radiochemical purity is below 95%, purification is required. The purification steps are as follows: Take a Sep-Pak C18 separation column and activate and rinse it successively with 10 mL of anhydrous ethanol and 10 mL of water. Dilute the labeled solution with 10 mL of water and load the sample onto the separation column. Rinse the separation column with water to remove unreacted components. 177 Lu 3+ Then rinse with 1 mL of anhydrous ethanol to obtain 177 Lu-labeled complexes. Organic solvents are removed by nitrogen blowing, and the solution is diluted with physiological saline and 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.

[0219] Targeted probes for radionuclides 177 Lu-DOTA-dPNPEG samples were analyzed and identified by HPLC. The HPLC system was as follows: reversed-phase C18 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%, and the flow rate was 1 mL / min. Radiolabeled target complex [ 177 The retention time of Lu-DOTA-dPNPEG was 13.760 min, and its radiochemical purity was calculated to be greater than 97%. DOTA-dPNPEG and [ 177 The HPLC spectrum of Lu-DOTA-dPNPEG is as follows: Figure 25 As shown in C.

[0220] Example 17

[0221] 99m Tc radionuclide labeling: SnCl2 was used as the reducing agent, and N-tris(hydroxymethyl)methylglycine (Tricine) and sodium triphenylphosphine tris(m-sulfonate) (TPPTS) were used as cooperating ligands. 99m The Tc marker.

[0222] Wet labeling method: 20 μL of freshly prepared SnCl2 solution (SnCl2 hydrochloric acid solution, 1 mg / mL) was added to a solution containing HYNIC-dPNPEG (100 μg), 10 mg Tricine, and 10 mg TPPTS, followed immediately by the addition of 740 MBq of freshly rinsed Na+. 99m TcO4 eluent (washed from the molybdenum technetium generator), mixed thoroughly, capped and sealed, reacted at room temperature to 100°C for 30 min, 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.

[0223] Freeze-drying labeling method: 740 MBq of fresh Na 99m TcO4 eluent (eluted from a molybdenum technetium generator) was added to a lyophilized kit containing 100 μg HYNIC-dPNPEG, 10 mg Tricine, and 10 mg TPPTS (containing mannitol and ascorbic acid). After mixing, the kit was capped and sealed. The reaction was carried out at room temperature to 100°C for 30 minutes, then cooled to room temperature. The kit 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.

[0224] If the radiochemical purity is below 95%, purification is required. The purification steps are as follows: Take a Sep-Pak C18 separation column and activate and rinse it successively with 10 mL of anhydrous ethanol and 10 mL of water. Dilute the labeled solution with 10 mL of water and load the sample onto the separation column. Rinse the separation column with water to remove unreacted components. 99m TcO4 - Then rinse with 1 mL of anhydrous ethanol to obtain 99m Tc-labeled complexes were obtained by removing organic solvents with nitrogen blowing, followed by dilution with physiological saline and sterile filtration to yield the radionuclide targeting probe. 99m Tc-HYNIC-dPNPEG injection. The HPLC system was as follows: reversed-phase C18 column; mobile phase A: water + 0.1% trifluoroacetic acid (TFA); mobile phase B: acetonitrile + 0.1% TFA; elution gradient: 0–20 min, with the volume fraction of mobile phase B increasing from 5% to 95%, and a flow rate of 1 mL / min. Radiolabeled target complex. 99m The retention time of Tc-HYNIC-dPNPEG was 11.608 min, and based on this, the radiochemical purity was calculated to be greater than 95%. HYNIC-dPNPEG and99m The HPLC spectrum of Tc-HYNIC-dPNPEG is as follows: Figure 25 As shown in D.

[0225] Test Example 1

[0226] 1. Stability Test

[0227] Stability of physiological saline: The radionuclide targeting probe dissolved in physiological saline ([ 68 Ga]Ga-DOTA-dPNE, [ 18 [F]AlF-NOTA-dPNE) was left at room temperature for different times, and samples were analyzed by HPLC. At the tested time points (0.5h, 1h, and 2h), each probe still maintained a radiochemical purity >95%, indicating that it is stable in the specified solution and does not easily decompose. 68 The HPLC results of the in vitro saline stability determination of Ga]Ga-DOTA-dPNE are as follows: Figure 26 As shown in A, [ 18 The HPLC results of the in vitro saline stability determination of F]AlF-NOTA-dPNE are as follows: Figure 26 As shown in C, it indicates that [ 68 Ga]Ga-DOTA-dPNE and [ 18 F]AlF-NOTA-dPNE maintained high stability (>95%) in physiological saline system up to 2 hours.

[0228] Serum stability: The radionuclide targeting probe ([ 68 Ga]Ga-DOTA-dPNE, [ 18 F]AlF-NOTA-dPNE) was co-incubated with serum at room temperature for different times (0.5h, 1h, and 2h), followed by the addition of acetonitrile to remove proteins. The supernatant was collected by centrifugation and analyzed by HPLC. At the tested time points, each probe maintained a radiochemical purity >95%, indicating its stability and resistance to decomposition in the specified solution. [Compound] 68 The in vitro serum stability HPLC identification results of Ga]Ga-DOTA-dPNE are as follows: Figure 26 As shown in B, [ 18 The in vitro serum stability HPLC identification results of F]AlF-NOTA-dPNE are as follows: Figure 26 As shown in D, it indicates that [ 68 Ga]Ga-DOTA-dPNE and [ 18 F]AlF-NOTA-dPNE maintained high stability (>95%) in the serum system for 2 hours.

[0229] 2. Lipid-water distribution coefficient (LogP) pH＝7.4 ) Measurement

[0230] 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. The tube was vortexed for 3 min, then centrifuged at 12000 rpm for 5 min. 100 μL of liquid was taken from both the aqueous and n-octanol phases and counted for radioactivity using a gamma counter. The experiment was repeated three times, and the average value was taken. The formula for calculating LogP is:

[0231] P = I 有机相 / I 水相

[0232] Among them I 有机相 Represents the radioactivity count measured in the organic phase, I 水相 This represents the radioactivity count measured in the aqueous phase. The lipid-water distribution coefficient of each radiolabeled target probe was calculated. The results are shown in Table 1 below; the measured radionuclide target probes exhibit water solubility.

[0233] Table 1. Lipid-water distribution coefficient of radionuclide targeting probes

[0234] <![CDATA[[ 68 Ga]Ga-DOTA-dPNE]]> -2.74±0.06(n＝6) <![CDATA[[ 68 Ga]Ga-DOTA-dPNH]]> -2.67±0.09(n＝6) <![CDATA[[ 68 Ga]Ga-DOTA-dPNPEG]]> -2.93±0.05(n＝6) <![CDATA[[ 68 Ga]Ga-DOTA-dPNPEG2]]> -2.97±0.05(n＝6)

[0235] As shown in Table 1, the above four 68 Ga-labeled probes are hydrophilic, suggesting that their metabolic pathway primarily involves the kidneys. By linking the chelating group to the parent structure using connecting chains of varying lengths and polarities, the probe's LogP can be appropriately adjusted, resulting in different pharmacokinetic properties.

[0236] Test Example 2

[0237] Cell binding and uptake assays

[0238] 1. Cell binding experiment

[0239] MDA-MB-231 cells cultured in DMEM high-glucose medium were seeded into two 24-well plates (2 × 10⁻⁶ cells / well). 5 Cells per well (10 cells / well) were incubated overnight in a cell culture incubator. After 24 hours, the 24-well plate was removed, the culture medium was aspirated, and 4°C pre-chilled PBS buffer was added along the well walls to wash the wells twice. Different concentrations of radioligand were added to each well of the first plate. In the second plate, diluted inhibitor AB680 was added to each well first, followed by different concentrations of radioligand. After incubation at 37°C for 2 hours, the cells were washed twice with PBS buffer. Finally, 1M sodium hydroxide solution was added, and the cells were incubated at room temperature for 10 minutes to lyse all cells. The cell suspension was collected, and the radioactivity in the cells was counted using a gamma counter. Data processing was performed using Graphpadprism 8.0 to calculate Bmax and K. d(nmol / L) value.

[0240] Figure 27 for[ 68 Ga]Ga-DOTA-dPNE, [ 68 Ga]Ga-DOTA-dPNH, [ 68 Ga]Ga-DOTA-dPNPEG and [ 68 Ga]Ga-DOTA-dPNPEG2 cellular K d Value measurement results graph. (From...) Figure 27 It was learned that in the MDA-MB-231 cell line, which highly expresses CD73 protein, 68 K in Ga-labeled compounds d The value reaching the nanomolar level indicates that it has good affinity.

[0241] 2. In vitro cell uptake and inhibition experiments

[0242] MDA-MB-231 cells, which highly express CD73 protein, were seeded in 24-well plates containing DMEM medium (containing fetal bovine serum and antibiotics). Cell counts were performed using a cell counting chamber (2 × 10⁻⁶ cells / well). 5 After culturing (cells / well) for 24 hours, set up the uptake group and the inhibition group according to the following steps.

[0243] The uptake experiment procedure was as follows: the original culture medium was aspirated, the cells were washed twice with PBS, and the PBS was aspirated; culture medium containing specific radioactive drugs and DMEM culture medium without bovine serum were added, and the cells were incubated at 37°C for 10, 30, 60, and 120 min; after incubation, the radioactive culture medium was aspirated, sodium hydroxide solution was added to each well to lyse the cells, and the cells were incubated for 5 min. The lysed cells were placed in disposable centrifuge tubes and the radioactivity count was measured; the cell uptake percentage (%uptake) was obtained by dividing the count by the total count of radioactive substances added.

[0244] Inhibition group experimental procedure: Aspirate the original culture medium, wash twice with PBS, and aspirate the PBS; add CD73 protein inhibitor AB680 and culture medium containing specific radioactive drugs, and incubate at 37°C for 10, 30, 60, and 120 min; after incubation, aspirate the radioactive culture medium, add sodium hydroxide solution to each well to lyse the cells, incubate for 5 min, and place the lysed cells into disposable centrifuge tubes to measure the radioactivity count; divide this count by the total count of radioactive substances added to each well to obtain the cell uptake percentage (%uptake).

[0245] Figure 28 for[ 68 Ga]Ga-DOTA-dPNE, [ 68 Ga]Ga-DOTA-dPNH, [ 68Ga]Ga-DOTA-dPNPEG and [ 68 Ga]Ga-DOTA-dPNPEG 22 Figure showing the results of in vitro cell uptake and inhibition experiments. (Source: [Insert image here]) Figure 28 It can be seen that, 68 These Ga-labeled radiocomplexes all showed significant intracellular uptake and were significantly inhibited by the CD73 protein inhibitor AB680, indicating that... 68 Ga-labeled compounds have specificity for binding to the CD73 protein.

[0246] Test Example 3

[0247] Micro-PET imaging of normal mice:

[0248] Prepare radiochemically pure samples with a purity greater than 99% according to the examples. 68 Ga nuclide targeting probe ([ 68 Ga]Ga-DOTA-dPNE, [ 68 A solution of Ga[Ga-DOTA-dPNPEG2] was prepared, and 10 MBq was injected into the tail vein of normal female mice. Micro-PET imaging was performed at 0.5 h, 1 h, and 2 h, and regions of interest (ROIs) were delineated on the images. The metabolism of the probe in vivo was calculated. Figure 29 for[ 68 Ga]Ga-DOTA-dPNE(A) and [ 68 Micro-PET imaging of Ga]Ga-DOTA-dPNPEG2(B) in normal mice at 0.5–2 h. Figure 29 It can be seen that, [ 68 Ga]Ga-DOTA-dPNE and [ 68 In normal mice, Ga]Ga-DOTA-dPNPEG2 is mainly metabolized by the kidneys and is also taken up to a certain extent in bones and joints, presumably due to the osteophilicity of (α,β-methylene) diphosphate in its structure.

[0249] Test Example 4

[0250] PET imaging of tumor-bearing mice

[0251] Validation in a B16F10 melanoma model [ 68 Ga]Ga-DOTA-dPNE, [ 68 Ga]Ga-DOTA-dPNH, [ 68 Ga]Ga-DOTA-dPNPEG and [ 68Tumor targeting capability of Ga]Ga-DOTA-dPNPEG2. 7.4 MBq of the radiocomplex was injected via tail vein into mouse models bearing B16F10 tumors. Micro-PET imaging was performed at 0.5 h, 1 h, and 2 h, and regions of interest (ROIs) were delineated on the images.

[0252] Figure 30 for[ 68 Ga]Ga-DOTA-dPNE, [ 68 Ga]Ga-DOTA-dPNH, [ 68 Ga]Ga-DOTA-dPNPEG and [ 68 MicroPET imaging of Ga-DOTA-dPNPEG2 in B16F10 tumor-bearing mice from 0.5 to 2 hours. Figure 30 It can be seen that, compared to [ 68 Ga]Ga-DOTA-dPNE and [ 68 Ga]Ga-DOTA-dPNH,[ 68 Ga]Ga-DOTA-dPNPEG and [ 68 Ga]Ga-DOTA-dPNPEG2 showed more significant uptake at tumor sites, which may be attributed to the hydrophilic modification of the polyethylene glycol linker. Figure 31 for[ 68 [Ga]Ga-DOTA-dPNPEG radioligand uptake (A) and target / non-target ratio (B) in key tissues and organs of mouse B16F10 model mice from 0.5 to 2 hours, from left to right: 0.5 hours, 1 hour, and 2 hours. Figure 31 It can be seen that, [ 68 The ratio of Ga]Ga-DOTA-dPNPEG to tumor tissue reached 2–3 times. These data indicate that the distribution of the radionuclide-targeting probe in the B16F10 model changed due to variations in the length and properties of the linker.

[0253] Validation 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 on tumors. 7.4 MBq of the radiocomplex was injected via the tail vein into mice bearing MDA-MB-231 tumors. Micro-PET imaging was performed at 0.5 h, 1 h, and 2 h, and regions of interest (ROIs) were delineated on the scanned images. Figure 32 for[ 68Ga]Ga-DOTA-dPNE, [ 68 Ga]Ga-DOTA-dPNH, [ 68 Ga]Ga-DOTA-dPNPEG and [ 68 MicroPET imaging of Ga-DOTA-dPNPEG2 in human MDA-MB-231 mouse model for 0.5–2 hours. Figure 32 As shown, the four radionuclide targeting probes showed significant uptake at the tumor site and rapid clearance in normal tissues such as muscle, liver, and kidney, with a high target / non-target ratio.

[0254] Figure 33 for[ 68 Ga]Ga-DOTA-dPNE and [ 68 The uptake of Ga-DOTA-dPNPEG2 in important tissues and organs and the target / non-target ratio of Ga-DOTA-dPNPEG2 in human MDA-MB-231 model mice from 0.5 to 2 h. In A and B, from left to right, the values ​​are 0.5 h, 1 h, and 2 h, respectively. In C and D, from left to right, the values ​​are 10 min, 0.5 h, 1 h, and 2 h, respectively. Figure 33 The diagram in section A shows that... 68 The tumor / sarcoma ratio of Ga-DOTA-dPNE reached approximately 5.8, and non-target sites were rapidly cleared 2 hours after probe injection. A high radioactive signal was observed in the bladder, indicating […]. 68 Ga]Ga-DOTA-dPNE is metabolized in the body through the kidneys and excreted in urine. Figure 33 The B-shaped display [ 68 Ga]Ga-DOTA-dPNPEG2 has high uptake at tumor sites, and compared to [ 68 For Ga]Ga-DOTA-dPNE, [ 68 Ga]Ga-DOTA-dPNPEG2 is metabolized more rapidly in normal organs, with the tumor / sarcoma ratio reaching around 6 within 2 hours.

[0255] Validation in the human breast cancer MDA-MB-231 model [ 68 Ga]Ga-DOTA-dPNME and [ 68 The targeting ability of Ga]Ga-DOTA-dPNPZ on tumors. 7.4 MBq of the radiocomplex was injected via the tail vein into mice bearing MDA-MB-231 tumors. MicroPET imaging was performed at 0.5 h, 1 h, and 2 h, and regions of interest (ROIs) were delineated on the scanned images. Figure 34 A and B are [ 68 [Ga]Ga-DOTA-dPNME uptake of radioligands in key tissues and organs and target / non-target ratios in human MDA-MB-231 mouse model mice from 0.5 to 2 hours.68 Ga-DOTA-dPNME accumulated significantly in the tumor and kidney within 30 minutes; after 2 hours, it showed a high signal at the tumor site and was almost completely metabolized in normal tissue, with a high radioactive signal only observed in the bladder, indicating that... 68 Ga-DOTA-dPNME is primarily metabolized by the kidneys and excreted in urine. Furthermore, in [ 68 AB680 (150 μg) was injected via the tail vein 10 minutes before Ga-DOTA-dPNME injection. Imaging of the inhibition group was performed 1 hour after the radionuclide-targeting probe injection. PET images showed that AB680 completely accumulated the radionuclide-targeting probe at the tumor site, indicating […]. 68 Ga]Ga-DOTA-dPNME has CD73 targeting. Figure 34 The B-value shows that, 68 Ga-DOTA-dPNME exhibits favorable target-to-non-target organ ratios: at the 2-hour time point, the tumor / sarcoma ratio reaches approximately 15, the tumor / heart and tumor / liver ratios both exceed 5, and the tumor / kidney ratio reaches approximately 3. These results show that... 68 Ga]Ga-DOTA-dPNME exhibits good tumor targeting.

[0256] Figure 34 C displays [ 68 Ga-DOTA-dPNPZ accumulated extensively in the tumor area and kidneys within 30 minutes; after 2 hours, it showed a high signal at the tumor site and was almost completely metabolized in normal tissues except the kidneys, with a high radioactive signal only observed in the bladder, indicating that [ 68 Ga]Ga-DOTA-dPNPZ is mainly metabolized by the kidneys and excreted in urine. Figure 34 The D-display shows, [ 68 Ga-DOTA-dPNPZ exhibits favorable target / non-target organ ratios: at the 2-hour time point, the tumor / sarcoma ratio reaches approximately 4, while the tumor / heart and tumor / liver ratios both exceed 3. These results indicate that... 68 Ga]Ga-DOTA-dPNPZ exhibits good tumor targeting properties.

[0257] Also verified in the MDA-MB-231 model [ 18 F]AlF-NOTA-dPNE and [ 18 The targeting ability of F]AlF-NOTA-dPNPEG2 on tumors. Figure 35 for[ 18 F]AlF-NOTA-dPNE and [ 18MicroPET images of F]AlF-NOTA-dPNPEG2 in human MDA-MB-231 model mice from 0.5 to 2 hours, radioactive uptake in major tissues, and target / non-target ratio. Figure 35 From A and B, we know that, 18 [F]AlF-NOTA-dPNE showed significant uptake at tumor sites and rapid clearance in muscle, kidneys, and other areas, with a tumor / tumor ratio of approximately 20. Figure 35 From C and D, we can see that, [ 18 F]AlF-NOTA-dPNPEG2 also showed significant uptake at the tumor site, with a tumor / tumor ratio of 10. In addition, the probe also showed some uptake in the gallbladder and intestines.

[0258] The above results indicate that the radionuclide targeting probe provided by the present invention has significant aggregation in tumor regions with high expression of CD73 protein, and the pharmacokinetic properties of the probe can be altered by changing the length and properties of the linker chain.

[0259] Test Example 5

[0260] Biodistribution experiment in tumor-bearing mice

[0261] Select C57BL / 6 mice (approximately 8 weeks old and weighing about 20g) and inject them with 1×10⁻⁶ mol / L in their right upper limb. 6 B16F10 cells. When the diameter of the subcutaneous tumor was 0.5–1.0 cm, 1.5 MBq was injected via the tail vein of mice. 68 [Ga]Ga-DOTA-dPNPEG was administered to mice at different time points after injection (4 mice per group). Blood, brain, heart, liver, lung, kidney, intestine, spleen, muscle, bone, and tumors of interest were collected. The radioactivity counts were measured after weighing. The results were expressed as the percentage uptake dose per gram of tissue or organ (%ID / g). Figure 36 for[ 68 Distribution of Ga-DOTA-dPNPEG in tissues and organs of mice with B16F10 tumors, corresponding to time points of 0.5h, 1h, and 2h. Figure 36 It was found that 0.5 hours after injection, the tumor uptake of the radionuclide-targeted probe was >3% ID / g, significantly higher than that of muscle tissue. Over time, the retention in blood and non-target organs gradually decreased, while the uptake at the tumor site was maintained, and the ratio of radioactive uptake by the tumor to that by the muscle further increased.

[0262] Female Balb / c mice (approximately 8 weeks old and weighing about 20g) were selected and injected with 1×10⁻⁶ ppm in their right upper limb. 7 MDA-MB-231 cells. When the diameter of the subcutaneous tumor was 0.5-1.0 cm, 1.5 MBq was injected via the tail vein of mice. 68[Ga]Ga-DOTA-dPNE was administered to mice at different time points after injection (10 min, 0.5 h, 1 h, and 2 h, respectively). Four mice were euthanized at each time point. Blood, brain, heart, liver, lung, kidney, intestine, spleen, muscle, bone, and tumors of interest were collected. The radioactivity counts were measured after weighing. The results were expressed as the percentage uptake dose per gram of tissue or organ (%ID / g). Figure 37 for[ 68 Distribution of Ga-DOTA-dPNE in tissues and organs of MDA-MB-231 tumor-bearing mice, corresponding to time points of 10 minutes, 30 minutes, 1 hour, and 2 hours. Figure 37 It was found that 10 minutes after injection, the tumor uptake of the radionuclide-targeted probe was >10% ID / g, significantly higher than that in muscle tissue. The probe showed high uptake in the kidneys, which is consistent with the PET imaging results; the kidney retention decreased significantly at 0.5 h, and over time, the retention in non-target organs, including the kidneys, gradually decreased, while the uptake at the tumor site was maintained.

[0263] In summary, the CD73-mediated radionuclide targeting probes provided by this invention exhibit specific binding to CD73 protein at both the cellular and in vivo levels. Currently, there are no reports of related small-molecule radionuclide targeting probes. Cell uptake and inhibition experiments show that the radionuclide targeting probes provided by this invention can be efficiently taken up by the CD73-highly-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 probes reaches the nanomolar level. In different mouse tumor models, including but not limited to breast cancer and melanoma, the radionuclide targeting probes can accumulate in tumor regions with high CD73 expression, and PET imaging results are good, indicating that this series of radionuclide targeting probes has the potential to become novel diagnostic and therapeutic agents based on the CD73 target.

[0264] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, not all embodiments. People can obtain other embodiments based on the present invention without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A nucleotide compound, characterized in that, It has the structure shown in Equation I: Equation I; In equation I: L is or R1 is or n and m are independent integers from 0 to 10; R5 is selected from -H or -CH3; R2 is selected from -H or halogen; R4 is selected from -OH or -F; X is selected from =N- or =CH-; R3 is selected from -H or any of the following structures: 。 2. The method for preparing the nucleotide compound according to claim 1, characterized in that, Includes the following steps: Compound A and compound B were coupled together under Lewis acid catalysis to obtain compound C. Compound C and compound D were subjected to a first substitution reaction under alkaline conditions to obtain compound E; Compound E was subjected to a deriboprotecting reaction to obtain compound F; Compound F was subjected to a second substitution reaction with methylenebis(phosphine dichloride) followed by a deprotection reaction to yield compound G; The compound G was subjected to a third substitution reaction with the active compound R3 to obtain the nucleotide compound. ； In compounds A to G, R1 to R5, L and X are the same as R1 to R5, L and X in formula I, group 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, selected from the following structures: 。 3. A pharmaceutically acceptable salt of a nucleotide compound, characterized in that, Selected from trifluoroacetate, hydrochloride, formate, potassium or sodium salts of nucleotide compounds; The nucleotide compound is the nucleotide compound of claim 1 or the nucleotide compound prepared by the preparation method of claim 2.

4. A radionuclide targeting probe, characterized in that, It has the structure shown in Formula II: Formula II; In Formula II, R1~R5, L and X are the same as R1~R5, L and X in the nucleotide compound of claim 1; The R6 is obtained by coordinating the R3 coordinating group as described in claim 1 with a labeled nuclide.

5. The radionuclide targeting probe according to claim 4, characterized in that, 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.

6. The method for preparing the radionuclide targeting probe according to claim 4 or 5, characterized in that, The preparation method includes the following steps: A nucleotide compound or its pharmaceutically acceptable salt is coordinated with a labeled nuclide to obtain a nuclide-targeting probe or a pharmaceutically acceptable salt of a nuclide-targeting probe; wherein the nucleotide compound is the nucleotide compound of claim 1, the nucleotide compound prepared by the preparation method of claim 2, or a pharmaceutically acceptable salt of the nucleotide compound of claim 3.

7. A pharmaceutically acceptable salt for a radionuclide targeting probe, characterized in that, It is obtained by a salt-forming reaction of a radionuclide targeting probe or a nucleotide compound; the radionuclide targeting probe is the radionuclide targeting probe according to any one of claims 4 to 5 or the radionuclide targeting probe prepared by the preparation method according to claim 6; the nucleotide compound is the nucleotide compound according to claim 1 or the nucleotide compound prepared by the preparation method according to claim 2.

8. A pharmaceutical composition, characterized in that, It includes an active ingredient and pharmaceutically acceptable excipients; the active ingredient includes one or more of the following: nucleotide compounds, pharmaceutically acceptable salts of nucleotide compounds according to claim 3, radionuclide targeting probes, and pharmaceutically acceptable salts of radionuclide targeting probes according to claim 7; the nucleotide compounds are nucleotide compounds according to claim 1 or nucleotide compounds prepared by the preparation method according to claim 2; the radionuclide targeting probes are radionuclide targeting probes according to any one of claims 4-5 or radionuclide targeting probes prepared by the preparation method according to claim 6.

9. The use of the nucleotide compound of claim 1, the nucleotide compound prepared by the method of claim 2, a pharmaceutically acceptable salt of the nucleotide compound of claim 3, the radionuclide targeting probe of any one of claims 4-5, the radionuclide targeting probe prepared by the method of claim 6, a pharmaceutically acceptable salt of the radionuclide targeting probe of claim 7, or the pharmaceutical composition of claim 8 in the preparation of therapeutic or diagnostic drugs for CD73 protein-mediated diseases.

10. The application according to claim 9, characterized in that, The diseases mediated by the CD73 protein are selected from tumors or immune-related diseases.

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