< 68 > Ga-labeled targeted VLA-4 molecular probe as well as preparation method and application of < 68 > Ga-labeled targeted VLA-4 molecular probe

Abstract

The invention relates to the technical field of tumor imaging, in particular to a < 68 > Ga-labeled targeted VLA-4 molecular probe and a preparation method and application thereof. The invention relates to a < 68 > Ga-labeled targeted VLA-4 molecular probe and a preparation method and application thereof, the < 68 > Ga-labeled targeted VLA-4 molecular probe not only has the advantage of adopting < 68 > Ga labeling, but also has specific targeting to VLA-4 protein, and is low in background interference, high in specificity and high in sensitivity. Furthermore, malignant melanin tumors or other potential VLA-4 positive tumors can be effectively distinguished from normal tissues with high metabolic activity, and the metastasis potential of black tumors or other potential VLA-4 positive tumors can be accurately evaluated.

Description

Technical Field

[0001] The present invention relates to the field of tumor imaging technology, and in particular to a 68 A Ga-labeled molecular probe targeting VLA-4 and a preparation method and use thereof. Background Art

[0002] Melanoma is a highly malignant skin cancer with an increasing incidence worldwide. Due to its invasiveness and strong metastatic potential, the prognosis of advanced melanoma remains poor, so early diagnosis is crucial to improve patient survival. Although conventional diagnostic methods such as skin biopsy are valuable, it may not adequately assess the full extent of tumor spread and carries the risk of misdiagnosis. Positron emission tomography (PET) as a functional imaging modality offers superior sensitivity by detecting abnormal metabolic activity in tumor cells. PET scanning allows for a comprehensive assessment of primary melanoma lesions and metastatic spread, including lymph node involvement and distant metastasis, thereby facilitating accurate staging and prognosis. In PET imaging, radiolabeled molecular probes are essential for effective imaging. Although targeting glucose metabolism 18 F-FDG is the most widely used PET probe, but it has limited specificity for melanoma due to metabolic similarities between melanoma cells and other highly metabolic cells. Therefore, researchers have focused on developing new PET probes that target specific molecular markers to improve the specificity and accuracy of melanoma imaging.

[0003] VLA-4 (very late antigen-4) is an integrin protein, specifically a heterodimer composed of α4 and β1 subunits. It is mainly expressed on the surface of various immune cells, including lymphocytes and monocytes, as well as certain tumor cells. In the context of tumors, VLA-4 is usually located on the cell membrane and plays a key role in mediating adhesion to the extracellular matrix and endothelial cells. By binding to VCAM-1 on endothelial cells, VLA-4 promotes tumor cell adhesion, migration and metastasis. In particular, VLA-4 plays a key role in melanoma metastasis, and its elevated expression is correlated with the malignancy of metastatic melanoma. Therefore, VLA-4 represents a promising target for molecular imaging and targeted therapy of melanoma. Using a single bead one compound (OBOC) combinatorial chemistry approach, Kit S. Lam's research group at the University of California, Davis, successfully identified the peptidomimetic LLP2A, which has a significant binding affinity to α4β1 integrin, IC 50 As low as 2 picomolar (pM). LLP2A exhibited excellent performance in vitro and, when conjugated to a fluorescent dye, showed high sensitivity and specificity for imaging α4β1-expressing tumors in a xenograft mouse model. These results highlight the great potential of LLP2A as a lead compound for the development of diagnostic and therapeutic agents, especially for α4β1-positive cancers.

[0004] Currently, radiolabeled LLP2A has been extensively studied in preclinical models of various tumors, including melanoma, lymphoma, and multiple myeloma, for diagnostic and therapeutic purposes. Some LLP2A probes show high uptake in α4β1-positive tumors with low background interference. Among them, 64 Cu-labeled LLP2A showed excellent performance and has entered Phase I clinical trials. 64 Cu has a relatively long half-life of about 12.7 hours, which is suitable for delayed imaging and observing slower biological processes. However, its extended half-life and high-energy radiation limit its widespread use. The complexity and high cost of the copper-64 labeling process further pose challenges to clinical application. In contrast, 68 Ga is used 68 Ga / 68 Ge generators, which do not require expensive cyclotron infrastructure, thus facilitating its availability. 68 Ga has a short half-life, which reduces radiation exposure to non-target tissues and improves imaging safety. 68 Related research reports on Ga-labeled LLP2A. Summary of the invention

[0005] Therefore, the technical problem to be solved by the present invention is to provide a 68 Ga-labeled molecular probe targeting VLA-4 and its preparation method and use, the 68 The Ga-labeled molecular probe targeting VLA-4 not only has the 68 The advantages of Ga labeling include specific targeting of VLA-4 protein and low background interference, which can further effectively distinguish malignant melanoma tumors or other potential VLA-4-positive tumors from normal tissues with high metabolic activity, and can accurately evaluate the metastatic potential of melanoma tumors or other potential VLA-4-positive tumors.

[0006] To this end, the present invention provides the following technical solutions:

[0007] The present invention provides a 68 Ga-labeled molecular probe targeting VLA-4, the structural formula is as follows:

[0008]

[0009] In the structural formula, X represents an integer ≥0.

[0010] In some embodiments, X represents an integer ≥ 0 and ≤ 3. In a preferred embodiment, X is 1.

[0011] The embodiment of the present invention discloses a precursor of a molecular probe targeting VLA-4, the structural formula of which is as follows:

[0012]

[0013] In the structural formula, X represents an integer ≥0.

[0014] In some embodiments, X represents an integer ≥ 0 and ≤ 3. In a preferred embodiment, X is 1.

[0015] The present invention provides a 68 The method for preparing a precursor of a Ga-labeled molecular probe targeting VLA-4 comprises the following steps:

[0016] When X in the structural formula represents an integer of =0:

[0017] S1, coupling the compound Fmoc-Lys(Dde)-OH to a solid phase support 2-chlorotrityl chloride resin, and then removing the Fmoc protecting group to obtain compound 1;

[0018] S2, coupling the compound LLP2A(tBu)-OH to the compound 1, and then removing the Dde protecting group to obtain the compound 2; the compound LLP2A(tBu)-OH is a compound in which the carboxyl group on the 2-aminosebacic acid side chain of LLP2A is protected by the tBu group;

[0019] S3, coupling the compound DOTA(OtBu)3 to compound 2, and then cleaving the peptide to remove the protecting group tBu;

[0020] The synthetic route is as follows:

[0021]

[0022] In some embodiments, in step S1, the coupling reaction conditions are 1-2 hours at room temperature. In some embodiments, the reaction time at room temperature can be any one of 1, 1.2, 1.5, 1.8, 2 hours or a range between any two values.

[0023] In some embodiments, in step S2 or step S3, the coupling reaction conditions are 8-12 hours at room temperature. In some embodiments, the reaction time at room temperature can be any one of 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12 hours or a range between any two of the values.

[0024] In some embodiments, the reagents used in the coupling step include a coupling reagent and / or a pH adjuster, the coupling reagent is HOBT and HBTU, and the pH adjuster is DIPEA.

[0025] In step S1, the compound Fmoc-Lys(Dde)-OH and DIPEA are dissolved in an organic solvent at a molar ratio of 1:2-1:3, and then added to a solid support 2-chlorotrityl chloride resin for coupling. In some embodiments, Fmoc-Lys(Dde)-OH and DIPEA are in a molar ratio of 1:2, 1:2.2, 1:2.5, 1:2.8, 1:3, or a range between any two values.

[0026] In some embodiments, in step S2, compound LLP2A(tBu)-OH, DIPEA, HOBT and HBTU are dissolved in an organic solvent at a molar ratio of 1:2:2:2-1:3:3:3, and then added to compound 1 for coupling. In some embodiments, the molar ratio of compound LLP2A(tBu)-OH, DIPEA, HOBT and HBTU can be any one of 1:2:2:2, 1:2.5:2.5:2.5, 1:2:3:2, 1:3:2:3, 1:2:2.5:2, 1:3:2.5:3, 1:3:3:3, or a range between any two of the values.

[0027] In some embodiments, in step S3, the molar ratio of DOTA(OtBu)3, DIPEA, HOBT and HBTU is 1:2:2:2-1:3:3:3 and dissolved in an organic solvent, and then added to compound 2 for coupling. In some embodiments, the molar ratio of DOTA(OtBu)3, DIPEA, HOBT and HBTU can be any one of 1:2:2:2, 1:2.5:2.5:2.5, 1:2:3:2, 1:3:2:3, 1:2:2.5:2, 1:3:2.5:3, 1:3:3:3, or a range between any two of the values.

[0028] When X in the structural formula represents an integer ≥ 1:

[0029] Q1, coupling the compound Fmoc-Lys(Dde)-OH to the solid phase support 2-chlorotrityl chloride resin, and then removing the Fmoc protecting group to obtain compound 1;

[0030] Q2, coupling the compound Fmoc-PEGn-OH to the compound 1, and then removing the Fmoc protecting group to obtain the compound 2; in the compound Fmoc-PEGn-OH, n represents an integer multiple of 2, and n>0;

[0031] Q3, coupling compound LLP2A(tBu)-OH to compound 2, and then removing the Dde protecting group to obtain compound 3; compound LLP2A(tBu)-OH is a compound in which the carboxyl group on the 2-aminosebacic acid side chain of LLP2A is protected by a tBu group;

[0032] Q4, coupling the compound DOTA(OtBu)3 to compound 3, and then cleaving the peptide to remove the protecting group tBu;

[0033] The synthetic route is as follows:

[0034]

[0035] In some embodiments, in steps Q1 and Q2, the coupling reaction conditions are: reaction at room temperature for 1-2 hours. In some embodiments, the reaction time at room temperature can be any one of 1, 1.2, 1.5, 1.8, 2 hours or a range between any two of the values.

[0036] In some embodiments, in steps Q3 and Q4, the coupling reaction conditions are: reaction at room temperature for 8-12 hours. In some embodiments, the reaction time at room temperature can be any one of 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12 hours or a range between any two of the values.

[0037] In some embodiments, the reagents used in the coupling step include a coupling reagent and / or a pH adjuster, the coupling reagent is HOBT and HBTU, and the pH adjuster is DIPEA.

[0038] In some embodiments, in step Q1, the compound Fmoc-Lys(Dde)-OH and DIPEA are dissolved in an organic solvent at a molar ratio of 1:2-1:3, and then added to a solid support 2-chlorotrityl chloride resin for coupling. In some embodiments, Fmoc-Lys(Dde)-OH and DIPEA are in a molar ratio of 1:2, 1:2.2, 1:2.5, 1:2.8, 1:3, or a range between any two values.

[0039] In some embodiments, in step Q2, compounds Fmoc-PEGn-OH, DIPEA, HOBT and HBTU are dissolved in an organic solvent at a molar ratio of 1:2:2:2-1:3:3:3, and then added to compound 1 for coupling; in some embodiments, the molar ratio of compounds Fmoc-PEGn-OH, DIPEA, HOBT and HBTU can be any one of 1:2:2:2, 1:2.5:2.5:2.5, 1:2:3:2, 1:3:2:3, 1:2:2.5:2, 1:3:2.5:3, 1:3:3:3, or a range value between any two values.

[0040] In some embodiments, in step Q3, compounds LLP2A(tBu)-OH, DIPEA, HOBT and HBTU are dissolved in an organic solvent at a molar ratio of 1:2:2:2-1:3:3:3, and then added to compound 2 for coupling; in some embodiments, the molar ratio of compounds LLP2A(tBu)-OH, DIPEA, HOBT and HBTU can be any one of 1:2:2:2, 1:2.5:2.5:2.5, 1:2:3:2, 1:3:2:3, 1:2:2.5:2, 1:3:2.5:3, 1:3:3:3, or a range value between any two values.

[0041] In some embodiments, in step Q4, DOTA(OtBu)3, DIPEA, HOBT and HBTU are dissolved in an organic solvent at a molar ratio of 1:2:2:2-1:3:3:3, and then added to compound 3 for coupling. In some embodiments, the molar ratio of compounds DOTA(OtBu)3, DIPEA, HOBT and HBTU can be any one of 1:2:2:2, 1:2.5:2.5:2.5, 1:2:3:2, 1:3:2:3, 1:2:2.5:2, 1:3:2.5:3, 1:3:3:3, or a range between any two of the values.

[0042] In some embodiments, the compound LLP2A(tBu)-OH is a derivative of LLP2A (CAS: 874148-50-2), which can be obtained by conventional peptide synthesis methods. The first amino acid of LLP2A is cleaved using dichlororesin to expose the carboxyl group, and trifluoroethanol is used for cleavage to protect the side chain carboxyl group of the second amino acid with the tBu group to obtain LLP2A(tBu)-OH.

[0043] In a preferred embodiment, the synthesis method of compound LLP2A(tBu)-OH comprises the following steps:

[0044] (1) coupling the compound Fmoc-Ach-OH to the solid phase support 2-chlorotrityl chloride resin, and then removing the Fmoc protecting group to obtain compound 1;

[0045] (2) coupling Fmoc-Aad(OtBu)-OH to compound 1, and then removing the Fmoc protecting group to obtain compound 2;

[0046] (3) coupling Fmoc-Lys(Dde)-OH to compound 2, and then removing the Fmoc protecting group to obtain compound 3;

[0047] (4), coupling 2-(4-(3-(o-tolyl)ureido)phenyl)acetic acid to compound 3, and then removing the Dde protecting group to obtain compound 4;

[0048] (5) Couple trans-3-(3-pyridyl) acrylic acid to compound 4, and then perform peptide cleavage; the synthetic route is as follows:

[0049]

[0050] In some embodiments, in step (1) to step (5), the coupling reaction conditions are: reaction time at room temperature for 8-12 hours. In some embodiments, the reaction time at room temperature can be any one of 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12 hours or a range between any two of the values.

[0051] In some embodiments, the reagents used in the coupling step include a coupling reagent and / or a pH adjuster, the coupling reagent is HOBT and HBTU, and the pH adjuster is DIPEA.

[0052] In some embodiments, in step (1), Fmoc-Ach-OH and DIPEA are dissolved in an organic solvent at a molar ratio of 1:2-1:3, and then added to a solid support 2-chlorotrityl chloride resin for coupling. In some embodiments, Fmoc-Ach-OH and DIPEA are in a molar ratio of 1:2, 1:2.2, 1:2.5, 1:2.8, 1:3, or a range between any two values.

[0053] In some embodiments, in step (2), Fmoc-Aad(OtBu)-OH, DIPEA, HOBT and HBTU are dissolved in an organic solvent at a molar ratio of 1:2:2:2-1:3:3:3, and then added to compound 1 for coupling. In some embodiments, the molar ratio of the compound Fmoc-Aad(OtBu)-OH, DIPEA, HOBT and HBTU can be any one of 1:2:2:2, 1:2.5:2.5:2.5, 1:2:3:2, 1:3:2:3, 1:2:2.5:2, 1:3:2.5:3, 1:3:3:3, or a range between any two of the values.

[0054] In some embodiments, in step (3), Fmoc-Lys(Dde)-OH, DIPEA, HOBT and HBTU are dissolved in an organic solvent at a molar ratio of 1:2:2:2-1:3:3:3, and then added to compound 2 for coupling. In some embodiments, the molar ratio of the compound Fmoc-Lys(Dde)-OH, DIPEA, HOBT and HBTU can be any one of 1:2:2:2, 1:2.5:2.5:2.5, 1:2:3:2, 1:3:2:3, 1:2:2.5:2, 1:3:2.5:3, 1:3:3:3, or a range between any two of the values.

[0055] In some embodiments, in step (4), 2-(4-(3-(o-tolyl)ureido)phenyl)acetic acid, DIPEA, HOBT and HBTU are dissolved in an organic solvent at a molar ratio of 1:2:2:2-1:3:3:3, and then added to compound 3 for coupling. In some embodiments, the molar ratio of compound 2-(4-(3-(o-tolyl)ureido)phenyl)acetic acid, DIPEA, HOBT and HBTU can be any one of 1:2:2:2, 1:2.5:2.5:2.5, 1:2:3:2, 1:3:2:3, 1:2:2.5:2, 1:3:2.5:3, 1:3:3:3, or a range between any two of the values.

[0056] In some embodiments, in step (5), trans-3-(3-pyridyl)acrylic acid, DIPEA, HOBT and HBTU are dissolved in an organic solvent at a molar ratio of 1:2:2:2-1:3:3:3, and then added to compound 4 for coupling. In some embodiments, the molar ratio of the compound trans-3-(3-pyridyl)acrylic acid, DIPEA, HOBT and HBTU can be any one of 1:2:2:2, 1:2.5:2.5:2.5, 1:2:3:2, 1:3:2:3, 1:2:2.5:2, 1:3:2.5:3, 1:3:3:3, or a range between any two of the values.

[0057] In some embodiments, in the step of removing the Fmoc protecting group, a solution containing 20%-25% piperidine by volume is added to the obtained conjugate to remove the Fmoc protecting group. In some embodiments, the concentration of the piperidine solution can be any one of 20%, 22%, 24%, and 25% by volume, or a range between any two values. In some embodiments, the Fmoc protecting group is removed 1-2 times, each time for 5-10 minutes, and the removal time can be any one of 5, 6, 8, and 10 minutes, or a range between any two values.

[0058] In some embodiments, in the step of removing the Dde group, a solution containing 2%-2.5% hydrazine hydrate by volume is added to the obtained conjugate to remove the Dde group. In some embodiments, the concentration of the hydrazine hydrate solution can be any one of 2%, 2.2%, 2.4%, and 2.5% by volume, or a range between any two values. In some embodiments, the removal of the Dde protecting group is performed 1-2 times, each removal takes 10-20 minutes, and the removal time can be any one of 10, 13, 15, 18, and 20 minutes, or a range between any two values.

[0059] In some embodiments, in the peptide cleavage and tBu group deprotection steps, a DCM solution containing 93%-95% trifluoroacetic acid by volume is used; the deprotection condition is nitrogen gas flow for 40-60 minutes. In some embodiments, the volume percentage of trifluoroacetic acid in the DCM solution of trifluoroacetic acid can be any one of 93, 93.5, 94, 94.5, 95% or a range between any two values. The deprotection condition is nitrogen gas flow for 40, 45, 50, 55, 60 minutes or a range between any two values.

[0060] In some embodiments, in a separate peptide cleavage step, a DCM solution containing 23%-27% trifluoroethanol by volume is used; the peptide cleavage condition is to react at room temperature for 3-6 hours. In some embodiments, a DCM solution containing trifluoroacetic acid of any one of 23%, 24%, 25%, 26%, 27% by volume or a range between any two values ​​is used for peptide cleavage. In some embodiments, the peptide cleavage condition is to react at room temperature for 3, 4, 5, 6 hours or a range between any two values.

[0061] The present invention provides a 68 The preparation method of a Ga-labeled molecular probe targeting VLA-4 comprises:

[0062] The 68 Ga-labeled molecular probe precursor targeting VLA-4 or the one prepared by the preparation method 68 Ga-labeled molecular probe precursor targeting VLA-4 68 Ga marking.

[0063] In some embodiments, the 68 The radioactivity in the Ga mixed solution is 15 to 18 mCi. 68 The radioactivity intensity in the Ga mixed solution can be any one of 15, 16, 17, 18 mCi or a range between any two values.

[0064] In some embodiments, the 68 In the Ga mixed solution, the pH value is 4-5. In some embodiments, the pH value is any one of 4, 4.2, 4.5, 4.8, 5, or a range between any two values.

[0065] In some embodiments, the compound 68 Ga-labeled molecular probe precursor targeting VLA-4 and the like 68 The mass volume ratio of the Ga mixed solution is 20:200-300, μg / μL. In some embodiments, the mass volume ratio can be any one of 20:200, 20:220, 20:240, 20:260, 20:280, 20:300 μg / μL or a range between any two values.

[0066] The present invention provides a 68 Use of Ga-labeled molecular probe targeting VLA-4 in preparing PET tracer targeting VLA-4 or melanoma tumor.

[0067] The present invention provides a PET tracer, comprising the 68Ga-labeled molecular probe targeting VLA-4.

[0068] The technical solution of the present invention has the following advantages:

[0069] 1. The present invention provides a 68 Ga-labeled molecular probe targeting VLA-4, the 68 The Ga-labeled molecular probe targeting VLA-4 not only has the 68 Ga labeling has the advantages of specific targeting of VLA-4 protein and low background interference, which can further effectively distinguish malignant melanoma tumors from normal tissues with high metabolic activity and accurately evaluate the metastatic potential of melanoma tumors. 68 Modification of different sites of LLP2A by Ga and DOTA labeling groups will have different effects on the binding affinity between LLP2A and VLA-4. It has been verified that modification of the Ach site of LLP2A has little effect on the binding affinity between LLP2A and VLA-4. The obtained probe has high specific targeting and low background interference to VLA-4 protein, and can be further used for imaging detection of melanoma tumors with high expression of VLA-4 protein or other potential VLA-4 positive tumors, which can show a higher tumor-background contrast of melanoma.

[0070] Furthermore, in order to improve the imaging clarity, the modified sites Ach and 68 A polyvinyl alcohol (PEG) chain was also introduced between the Ga and DOTA labeling groups.

[0071] Furthermore, the above molecular probe has good stability in vitro and is non-cytotoxic.

[0072] 2. The present invention provides a 68 Ga-labeled molecular probe targeting VLA-4, X represents an integer ≥0 and ≤3, preferably, X is 1; the present invention verifies a series of PEG chains of different lengths synthesized 68 The specific targeting and background interference of Ga-labeled LLP2A PET probe on VLA-4 protein are shown. The results show that it is the best among PEGylated probes. 68 Ga-T2 (PEG-2 linker) and non-PEGylated 68 Compared with Ga-T-CH, the tumor-to-muscle ratio was increased by about three times, showing the most favorable in vivo distribution characteristics, and among the PEGylated probes, 68 Ga-T2 has a high specific targeting ability to VLA-4 protein.

[0073] 3. The present invention provides 68The invention discloses a method for preparing a precursor of a Ga-labeled molecular probe targeting VLA-4, which has the advantages of simple method, mild reaction conditions and easy operation. BRIEF DESCRIPTION OF THE DRAWINGS

[0074] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0075] Figure 1 Mass spectrum of LLP2A-NH2;

[0076] Figure 2 Mass spectrum of LLP2A(tBu)-OH in Example 1;

[0077] Figure 3 Mass spectrum of LLP2A-CH-Lys(DOTA)-OH(T-CH) in Example 2;

[0078] Figure 4 Mass spectrum of LLP2A-AD-Lys(DOTA)-OH(T-AD) in Example 3;

[0079] Figure 5 Mass spectrum of LLP2A-PEG2-Lys(DOTA)-OH(T2) in Example 4;

[0080] Figure 6 Mass spectrum of LLP2A-PEG4-Lys(DOTA)-OH(T4) in Example 5;

[0081] Figure 7 Mass spectrum of LLP2A-PEG6-Lys(DOTA)-OH(T6) in Example 6;

[0082] Figure 8 In Example 7 68 Radio-HPLC profile of Ga-T-CH;

[0083] Fig. 9 In Example 7 68 Radio-HPLC profile of Ga-T-AD;

[0084] Fig.10 In Example 7 68 Radio-HPLC profile of Ga-T2;

[0085] Fig.11 In Example 7 68Radio-HPLC profile of Ga-T4;

[0086] Fig.12 In Example 7 68 Radioactive HPLC profile of Ga-T6;

[0087] Fig.13 In Experimental Example 1 68 Ga-T-CH and 68 PET imaging results of Ga-T-AD; Figure A: B16F10 tumor-bearing mice injected 68 Ga-T-CH and 68 Static PET image of Ga-T-AD after 1 hour; B: Quantitative analysis of A 68 Ga-T-CH and 68 Tumor uptake and tumor / muscle ratio of Ga-T-AD; R1: 68 Schematic diagram of Ga-T-CH structure connection; R2: 68 Schematic diagram of Ga-T-AD structure connection; **P<0.01;

[0088] Fig.14 In Experimental Example 2 68 Ga-T2, 68 Ga-T4 and 68 Stability results of Ga-T6; A: 68 Ga-T2, 68 Ga-T4 and 68 Molecular structure of Ga-T6; B: 68 Radio-HPLC chromatogram of Ga-T2 after incubation in PBS and FBS for 2 hours; C: 68 Radio-HPLC chromatogram of Ga-T4 after incubation in PBS and FBS for 2 hours; D: 68 Radio-HPLC chromatograms of Ga-T6 after 2 h of incubation in PBS and FBS;

[0089] Fig.15 In vitro experimental results of the probe in Experimental Example 3; A: Western blot analysis of VLA-4 expression levels in B16F10 and A375 cells; B: B16F10 and A375 cells at 1 hour and 2 hours 68 Ga-T2 uptake; C: B16F10 and A375 cells at 1 hour and 2 hours 68 Ga-T4 uptake; D: B16F10 and A375 cells at 1 hour and 2 hours 68 Ga-T6 uptake; **P < 0.01, ***P < 0.001;

[0090] Fig.16 In Experimental Example 3 68The cytotoxicity results of Ga-T2 on B16F10 and A375 cells;

[0091] Fig.17 In Experimental Example 3 68 The cytotoxicity results of Ga-T4 on B16F10 and A375 cells;

[0092] Fig.18 In Experimental Example 3 68 The cytotoxicity results of Ga-T6 on B16F10 and A375 cells;

[0093] Fig.19 Probe in Experimental Example 4 68 Ga-T2, 68 Ga-T4 and 68 Ga-T6 PET imaging results; A: B16F10 tumor-bearing mice 1 hour and 2 hours after injection 68 Ga-T2, 68 Ga-T4 and 68 Static PET images of Ga-T6; B: A375 tumor-bearing mice 1 hour and 2 hours after injection 68 Ga-T2, 68 Ga-T4 and 68 Static PET images of Ga-T6; C: Quantified from A in B16F10 tumor-bearing mice after 1 hour 68 Ga-T2, 68 Ga-T4 and 68 Tumor uptake of Ga-T6; D Quantification of Ga-T6 in B16F10 tumor-bearing mice after 1 hour according to A. 68 Ga-T2, 68 Ga-T4 and 68 Tumor to muscle ratio of Ga-T6; E: B16F10 and A375 quantified according to A and B at 1 hour 68 Ga-T2, 68 Ga-T4 and 68 Tumor uptake of Ga-T6; *P < 0.05, **P < 0.01, ***P < 0.001;

[0094] Fig. 20 Probe in Experimental Example 5 68 Ga-T2, 68 Ga-T4 and 68 Biodistribution results of Ga-T6; A: Administration 68 Biodistribution of Ga-T2 1 hour after administration, ***P<0.001; B: 68 Biodistribution of Ga-T4 1 hour after administration, ***P<0.001; C: 68Biodistribution of Ga-T6 1 hour after administration, ***P<0.05; D: Immunofluorescence staining of VLA-4 protein in mouse tissues, magnification: 40 times; E: Quantitative analysis of immunofluorescence intensity from D; F: Administration 68 Ga-T2, 68 Ga-T4 and 68 Histopathological images of major organs 24 hours after Ga-T6, magnification: 20x;

[0095] The data analysis in Experimental Examples 1-5 was performed using Prism 10.2.3 software for statistical significance analysis, using an unpaired t-test. Multiple comparisons were performed using a multiple t-test corrected by the Holm-Sidak method. The p-value format follows the NEJM format, *P<0.05 is considered significant, and **P<0.01 is considered highly significant. DETAILED DESCRIPTION

[0096] The following examples are provided for a better understanding of the present invention, but are not intended to limit the best mode of implementation, nor to limit the content and protection scope of the present invention. Any product identical or similar to the present invention obtained by anyone under the inspiration of the present invention or by combining the features of the present invention with other prior arts shall fall within the protection scope of the present invention.

[0097] If no specific experimental steps or conditions are specified in the examples, the conventional experimental steps or conditions described in the literature in the field can be used. If no manufacturer is specified for the reagents or instruments used, they are all conventional reagent products that can be obtained commercially.

[0098] Reagents and instruments used in the following examples

[0099] The raw materials involved in the synthesis were purchased from Bide Pharmatech (Shanghai), GL Biochem (Shanghai) and AlphaChemical (Zhengzhou), and the solvents were purchased from Sinopharm Chemical Reagents (Shanghai) and Energy Chemical (Anhui). All compounds were reagent grade and did not require further purification. Mass spectrometry data were recorded using a Thermo Fisher Orbitrap Exploris 120 (USA). Two elution buffers [buffer A: deionized water containing 0.1% trifluoroacetic acid (TFA); buffer B: acetonitrile containing 0.1% TFA] were used for precursor purification and radiolabeling analysis. High performance liquid chromatography (HPLC) was performed on an Empower system (Waters) equipped with an e2695 separation module (Waters), a 2988PDA detector (Waters), a flow counting radiation detector (Elysia Raytest, Germany) and an XBridge C18 column (4.6×250 mm, 5 μm, Waters).

[0100] The experimental cells involved in the following examples: B16F10 cells and A375 cells were obtained from Otwo Biotech Inc. (Shenzhen).

[0101] The culture medium involved in the following examples: RPMI 1640 culture medium was purchased from Shanghai Shenggong Bioengineering Co., Ltd., and fetal bovine serum (FBS) was purchased from Zhejiang Tianhang Biotechnology Co., Ltd. Other reagents and materials used in the experiment were purchased from Shanghai Bio-Tech Biotechnology Co., Ltd.

[0102] Experimental animals involved in the following examples: Female BALB / c nude mice were obtained from Changzhou Kevin Experimental Animal Co., Ltd. All mice were raised in the SPF animal facility of Jiangsu Institute of Atomic Medicine, following the ethical guidelines set by the institution's ethics committee. Tumor cell implantation was performed when the mice reached 4-6 weeks of age: 3×10 6 B16F10 or A375 tumor cells were suspended in PBS and injected subcutaneously into the right axilla. Mice were housed under standard conditions for 2-3 weeks, and tumor diameters were measured regularly. When the tumor diameter reached 5-10 mm, the tumor model was considered fully established and subsequent experimental operations could be performed.

[0103] The CAS number of LLP2A (referred to as LLP2A-NH2 in the present invention) in the following example is 874148-50-2. The synthesis method is as follows: Compound LLP2A-NH2 was obtained by standard solid phase peptide synthesis (SPPS) using Rink Amide-MBHA resin and the corresponding Fmoc protected amino acid, and the side chain was appropriately protected. Rink Amide-MBHA resin (4 mmol) was swelled in DMF for 3 hours. The Fmoc protecting group was removed twice (10 minutes and 20 minutes) with 20 v / v% piperidine solution (solvent: DMF). The specific steps are as follows: After washing the resin with DMF (4×100 mL), Fmoc-Ach-OH (8 mmol, CAS: 162648-54-6) was dissolved in a DMF solution containing DIPEA (2.78 mL, 16 mmol), and then added to Rink Amide-MBHA resin (4 mmol). The coupling reaction was carried out overnight at room temperature. The resin was washed with DMF (4×100 mL). A 20 v / v% piperidine in DMF solution was added to the resin to remove the Fmoc protecting group twice (10 minutes for the first time and 20 minutes for the second time). After washing the resin with DMF (4×100 mL), Fmoc-Aad(OtBu)-OH (8 mmol, CAS: 159751-47-0) was dissolved in a DMF solution containing DIPEA (2.78 mL, 16 mmol), HOBT (16 mmol) and HBTU (16 mmol), and then added to the resin. The coupling reaction was carried out at room temperature overnight. The resin was washed with DMF (4×100 mL). A 20 v / v% piperidine in DMF solution was added to the resin to remove the Fmoc protecting group twice (10 minutes for the first time and 20 minutes for the second time). After washing the resin with DMF, Fmoc-Lys(Dde)-OH (8 mmol, CAS: 150629-67-7) was dissolved in a DMF solution containing DIPEA (2.78 mL, 16 mmol), HOBT (2.16 g, 16 mmol) and HBTU (6.08 g, 16 mmol) and then added to the resin for coupling. The coupling reaction was carried out at room temperature overnight. The resin was washed with DMF (4×100 mL). A 20 v / v% piperidine DMF solution was added to the resin to remove the Fmoc protecting group twice (10 minutes for the first time and 20 minutes for the second time). After washing the resin with DMF, 2-(4-(3-(o-tolyl)ureido)phenyl)acetic acid (8 mmol, CAS: 181517-99-7), DIPEA (2.78 mL, 16 mmol), HOBT (16 mmol) and HBTU (16 mmol) were dissolved in DMF and then added to the resin. The reaction was carried out at room temperature overnight. The resin was washed with DMF (4 x 100 mL).The Dde protecting group was removed with 2 v / v% hydrazine hydrate (NH2NH2) in DMF (30 minutes). After washing the resin with DMF (4×100 mL), trans-3-(3-pyridyl)acrylic acid (16 mmol, CAS: 19337-97-4), DIPEA (2.78 mL, 16 mmol), HOBT (16 mmol) and HBTU (16 mmol) were dissolved in DMF and added to the resin. The coupling reaction was carried out at room temperature overnight. The resin was washed with DMF (3×100 mL), IPA (3×100 mL) and HEX (3×100 mL). The resin was then dried under vacuum for 10 minutes, after which a cleavage mixture of 95 v / v% TFA was added: 95 mL TFA + 2.5 mL H2O + 2.5 mL TIS. The cleavage reaction was carried out at room temperature for 1 hour. The liquid was collected and concentrated, and the crude product LLP2A (2.5 g, 3.08 mmol, yield: 77.1%, purity: 96%) was obtained by adding ether for precipitation and centrifugation. The mass spectrum is shown in FIG. Figure 1 As shown, [(M+H) + ] Theoretical calculated value: 811.41; observed value of mass spectrum m / z: 811.91.

[0104] The synthetic route is as follows:

[0105]

[0106] The Chinese name of DMF is N,N-dimethylformamide; the Chinese name of DIPEA is N,N-diisopropylethylamine; the Chinese name of HOBT is 1-hydroxybenzotriazole; the Chinese name of HBTU is benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate; the Chinese name of IPA is isopropanol; the Chinese name of HEX is n-hexane; the Chinese name of TFA is trifluoroacetic acid; the Chinese name of TFE is trifluoroethylene; the Chinese name of TIS is triisopropylsilane; the Chinese name of DCM is dichloromethane; the Chinese name of PMSF is phenylmethylsulfonyl fluoride; in the present invention, the temperature range of room temperature is 10-30°C. Fmoc-Ach-OH, CAS: 162648-54-6; Fmoc-Aad(OtBu)-OH, CAS: 159751-47-0; Fmoc-Lys(Dde)-OH, CAS: 150629-67-7; 2-(4-(3-(o-tolyl)ureido)phenyl)acetic acid, CAS: 181517-99-7; trans-3-(3-pyridyl)acrylic acid, CAS: 19337-97-4; DOTA(OtBu) 3， CAS: 137076-54-1.

[0107] Example 1 Synthesis of LLP2A(tBu)-OH

[0108] This embodiment provides a method for synthesizing LLP2A(tBu)-OH, comprising the following steps:

[0109] Compound LLP2A(tBu)-OH was obtained by standard solid phase peptide synthesis (SPPS) using 2-chlorotrityl chloride resin and the corresponding Fmoc protected amino acids with appropriate side chain protection. Details are as follows:

[0110] (1) Synthesis of Compound 1: 2-Chlorotrityl chloride resin (4 mmol, CAS No.: 934816-82-7) was swelled in DMF for 3 hours. Fmoc-Ach-OH (8 mmol, CAS: 162648-54-6) was dissolved in a DMF solution containing DIPEA (2.78 mL, 16 mmol) and then added to the 2-chlorotrityl chloride resin. The coupling reaction was carried out at room temperature overnight. The resin was washed with DMF (4×100 mL). A 20 v / v% piperidine solution in DMF was added to the resin to remove the Fmoc protecting group twice (the first use volume was 15-20 mL, the reaction was 10 minutes, and the first use volume was 15-20 mL, the reaction was 20 minutes), to obtain Compound 1.

[0111] (2) Synthesis of Compound 2: After washing the compound 1 of step (1) with DMF (4×100 mL), Fmoc-Aad(OtBu)-OH (8 mmol, CAS: 159751-47-0) was dissolved in a DMF solution containing DIPEA (2.78 mL, 16 mmol), HOBT (16 mmol) and HBTU (16 mmol), and then added to the resin. The coupling reaction was carried out at room temperature overnight. The resin was washed with DMF (4×100 mL). A 20 v / v% piperidine DMF solution was added to the resin to remove the Fmoc protecting group twice (the first use volume was 15-20 mL, the reaction was carried out at room temperature for 10 minutes, and the first use volume was 15-20 mL, the reaction was carried out at room temperature for 20 minutes), to obtain Compound 2.

[0112] (3) Synthesis of Compound 3: After washing the resin (Compound 2) obtained in step (2) with DMF, Fmoc-Lys(Dde)-OH (8 mmol) was dissolved in a DMF solution containing DIPEA (2.78 mL, 16 mmol), HOBT (16 mmol) and HBTU (16 mmol), and then added to the resin. The coupling reaction was carried out at room temperature overnight. The resin was washed with DMF (4×100 mL). A 20 v / v% piperidine DMF solution was added to the resin to remove the Fmoc protecting group twice (the first use volume was 15-20 mL, the reaction was carried out at room temperature for 10 minutes, and the first use volume was 15-20 mL, the reaction was carried out at room temperature for 20 minutes), to obtain Compound 3.

[0113] (4) Synthesis of compound 4: After removing Fmoc, 2-(4-(3-(o-tolyl)ureido)phenyl)acetic acid (8 mmol), DIPEA (2.78 mL, 16 mmol), HOBT (16 mmol) and HBTU (16 mmol) were dissolved in DMF and added to compound 3 in step (3). The reaction was carried out at room temperature overnight. The resin was washed with DMF (4 times × 100 mL). 100 mL of a DMF solution containing 2 v / v% hydrazine hydrate (NH2NH2) was added to the resin and reacted at room temperature for 30 minutes. The Dde protecting group was removed in DMF to obtain compound 4.

[0114] (5) Synthesis of LLP2A(tBu)-OH: After washing compound 4 with DMF (4×100 mL), trans-3-(3-pyridyl)acrylic acid (16 mmol), DIPEA (2.78 mL, 16 mmol), HOBT (16 mmol) and HBTU (16 mmol) were dissolved in DMF and then added to the resin (compound 4). The coupling reaction was carried out at room temperature overnight. The resin was washed with DMF (3×100 mL), IPA (3×100 mL) and HEX (3×100 mL). The resin was then dried under vacuum for 10 minutes, after which a cleavage mixture of 25% TFE was added: 30 mL TFE + 90 mL DCM. The cleavage reaction was carried out at room temperature for 4 hours. The liquid was collected and concentrated, and the crude product LLP2A(tBu)-OH (1.9 g, 1.96 mmol, yield: 48.9%, purity: 93%) was obtained by adding ether for precipitation and centrifugation. The HPLC method was as follows: Chromatographic conditions: Mobile phase: a mixture of mobile phase A and mobile phase B, mobile phase A: a sterile aqueous solution containing 0.1 v / v% TFA; mobile phase B: acetonitrile containing 0.1 v / v% TFA; elution program: 0-15 minutes, gradient from 70 v / v% mobile phase A to 40 v / v% mobile phase A; flow rate, 1.0 ml / min; wavelength: 254 nm; column temperature 25°C; chromatographic column: preparative 250×10.0 mm C18 column or analytical 250×4.6 mm C18 column. The mass spectrum is shown in Figure 2 ,[(M+H) + The theoretical calculated value of ] is 868.4603; the observed value of high resolution mass spectrum m / z is 868.4603. The synthetic route is as follows:

[0115]

[0116] Example 2 Synthesis of LLP2A-CH-Lys(DOTA)-OH(T-CH)

[0117] Compound LLP2A-CH-Lys(DOTA)-OH(T-CH) was synthesized by standard solid phase peptide synthesis (SPPS) using 2-chlorotrityl chloride resin and the corresponding Fmoc protected amino acids with appropriate side chain protection. Details are as follows:

[0118] (1) Synthesis of Compound 1: 2-Chlorotrityl chloride resin (0.1 mmol) was swelled in DMF for 3 hours. Fmoc-Lys(Dde)-OH (0.2 mmol) was dissolved in a DMF solution containing DIPEA (70 μL, 0.4 mmol) and then added to the resin. The coupling reaction was carried out at room temperature for 1 hour. The resin was washed with DMF (4×10 mL). A 20 v / v% piperidine solution in DMF was added to the resin to remove the Fmoc protecting group twice (the first use volume was 15-20 mL, the reaction was carried out at room temperature for 10 minutes, and the first use volume was 15-20 mL, the reaction was carried out at room temperature for 20 minutes), to obtain Compound 1.

[0119] (2) Synthesis of Compound 2: After washing the resin (Compound 1) with DMF (4×10 mL), LLP2A(tBu)-OH (0.2 mmol) was dissolved in a DMF solution containing DIPEA (70 μL, 0.4 mmol), HOBT (0.4 mmol) and HBTU (0.4 mmol), and then added to the resin (Compound 1). The reaction was carried out at room temperature overnight. The resin was washed with DMF (4×10 mL). 10 mL of a DMF solution containing 2 v / v% hydrazine hydrate (NH2NH2) was added to the resin, and the reaction was carried out at room temperature for 15 minutes. The Dde protecting group was removed in DMF to obtain Compound 2.

[0120] (3) Synthesis of LLP2A-CH-Lys(DOTA)-OH(T-CH): After washing the resin (compound 2) with DMF (4×10 ml), DOTA(OtBu)3 (0.2 mmol), DIPEA (70 μl, 0.4 mmol), HOBT (0.4 mmol) and HBTU (0.4 mmol) were dissolved in DMF and added to compound 2. The coupling reaction was carried out at room temperature overnight. The resin was washed with DMF (3×10 ml), IPA (3×10 ml) and HEX (3×10 ml). The resin was then dried under vacuum for 10 minutes, after which a cleavage mixture of 95 v / v% TFA was added: 19 ml TFA + 0.5 ml H2O + 0.5 ml TIS. The cleavage reaction was carried out at room temperature under nitrogen atmosphere for 1 hour. The liquid was collected and concentrated. The crude product LLP2A-CH-Lys(DOTA)-OH(T-CH) (70 mg, 0.053 mmol, yield: 53%, purity: 98%) was obtained by adding ether for precipitation and HPLC purification. The HPLC method was the same as the chromatographic conditions in Example 1, except that the elution condition was 0-15 minutes, with a gradient from 80 v / v% mobile phase A to 40 v / v% mobile phase A. The mass spectrum is shown in FIG. Figure 3 As shown, [(M+2H) 2+The theoretical calculated value of ]: 663.84; the observed value of mass spectrum m / z: 664.39. The synthetic route is as follows:

[0121]

[0122] Example 3 Synthesis of LLP2A-AD-Lys(DOTA)-OH(T-AD)

[0123] The compound LLP2A-AD-Lys(DOTA)-OH(T-AD) was synthesized by standard solid phase peptide synthesis (SPPS) using 2-chlorotrityl chloride resin and the corresponding Fmoc protected amino acids with appropriate side chain protection. Details are as follows:

[0124] (1) Synthesis of Compound 1: 2-chlorotrityl chloride resin (0.1 mmol) was swelled in DMF for 3 hours. Fmoc-Lys(Dde)-OH (0.2 mmol) was dissolved in a DMF solution containing DIPEA (70 μL, 0.4 mmol) and then added to the resin. The coupling reaction was carried out at room temperature for 1 hour. The resin was washed with DMF (4×10 mL). A 20 v / v% piperidine solution in DMF was added to the resin to remove the Fmoc protecting group twice (the first use volume was 15-20 mL, the reaction was 10 minutes, and the first use volume was 15-20 mL, the reaction was 20 minutes), and Compound 1 was obtained.

[0125] (2) Synthesis of Compound 2: After washing the resin (Compound 1) with DMF (4×10 mL), LLP2A-NH2 (0.2 mmol) was dissolved in a DMF solution containing DIPEA (70 μL, 0.4 mmol), HOBT (0.4 mmol) and HBTU (0.4 mmol), and then added to Compound 1. The reaction was carried out at room temperature overnight. The resin was washed with DMF (4×10 mL). A DMF solution containing 2 v / v% hydrazine hydrate (NH2NH2) in a volume of 10 mL was added to the resin and reacted at room temperature for 15 minutes. The Dde protecting group was removed in DMF to obtain Compound 2.

[0126] (3) Synthesis of LLP2A-AD-Lys(DOTA)-OH(T-AD): After washing the resin (compound 2) with DMF (4×10 ml), DOTA(OtBu)3 (0.2 mmol), DIPEA (70 μl, 0.4 mmol), HOBT (0.4 mmol) and HBTU (0.4 mmol) were dissolved in DMF and added to compound 2. The coupling reaction was carried out at room temperature overnight. The resin was washed with DMF (3×10 ml), IPA (3×10 ml) and HEX (3×10 ml). The resin was then dried under vacuum for 10 minutes, after which a cleavage mixture of 95% TFA was added: 19 ml TFA + 0.5 ml H2O + 0.5 ml TIS. The cleavage reaction was carried out at room temperature under nitrogen atmosphere for 1 hour. The liquid was collected and concentrated. The crude product LLP2A-AD-Lys(DOTA)-OH(T-AD) (64 mg, 0.048 mmol, yield: 48%, purity: 98%) was obtained by adding ether for precipitation and HPLC purification. The HPLC method was the same as the chromatographic conditions in Example 1, except that the elution condition was 0-15 minutes, with a gradient from 80 v / v% mobile phase A to 40 v / v% mobile phase A. The mass spectrum was as follows: Figure 4 As shown, [(M+2H) 2+ The theoretical calculated value of ]: 663.35; the observed value of mass spectrum m / z: 664.01. The synthetic route is as follows:

[0127]

[0128] Example 4 Synthesis route of LLP2A-PEG2-Lys(DOTA)-OH(T2).

[0129] The compound LLP2A-PEG2-Lys(DOTA)-OH(T2) was synthesized by standard solid phase polysynthesis (SPPS) using 2-chlorotrityl chloride resin and the corresponding Fmoc protected amino acids with appropriate side chain protection. Details are as follows:

[0130] (1) Synthesis of Compound 1: 2-Chlorotrityl chloride resin (0.1 mmol) was swelled in DMF for 3 hours. Fmoc-Lys(Dde)-OH (0.2 mmol) was dissolved in a DMF solution containing DIPEA (70 μL, 0.4 mmol) and then added to the resin. The coupling reaction was carried out at room temperature for 1 hour. The resin was washed with DMF (4×10 mL). A 20 v / v% piperidine solution in DMF was added to the resin to remove the Fmoc protecting group twice (the first use volume was 15-20 mL, the reaction was carried out at room temperature for 10 minutes, and the first use volume was 15-20 mL, the reaction was carried out at room temperature for 20 minutes), to obtain Compound 1.

[0131] (2) Synthesis of Compound 2: After washing the resin (Compound 1) with DMF (4×10 mL), Fmoc-PEG2-OH (0.2 mmol, CAS: 872679-70-4) was dissolved in a DMF solution containing DIPEA (70 μL, 0.4 mmol), HOBT (0.4 mmol) and HBTU (0.4 mmol), and then added to the resin (Compound 1). The coupling reaction was carried out at room temperature for 1 hour. A 20 v / v% piperidine DMF solution was added to the resin to remove the Fmoc protecting group twice (the first use volume was 15-20 mL, the reaction was 10 minutes, and the first use volume was 15-20 mL, the reaction was 20 minutes), and Compound 2 was obtained.

[0132] (3) Synthesis of Compound 3: After washing the resin (Compound 2) with DMF (4×10 ml), LLP2A(tBu)-OH (0.2 mmol) was dissolved in a DMF solution containing DIPEA (70 μl, 0.4 mmol), HOBT (0.4 mmol) and HBTU (0.4 mmol), and then added to the resin. The reaction was carried out at room temperature overnight. The resin was washed with DMF (4×10 ml). A DMF solution containing 2 v / v% of hydrazine hydrate (NH2NH2) in a volume of 10 ml was added to the resin and reacted at room temperature for 15 minutes to obtain Compound 3.

[0133] (4) Synthesis of LLP2A-PEG2-Lys(DOTA)-OH(T2): After washing with DMF (4×10 ml), DOTA(OtBu)3 (0.2 mmol), DIPEA (70 μl, 0.4 mmol), HOBT (0.4 mmol) and HBTU (0.4 mmol) were dissolved in DMF and added to the resin. The coupling reaction was carried out at room temperature overnight. The resin was washed with DMF (3×10 ml), IPA (3×10 ml) and HEX (3×10 ml). The resin was then dried under vacuum for 10 minutes, after which a cleavage mixture of 95% TFA was added: 19 ml TFA + 0.5 ml H2O + 0.5 ml TIS. The cleavage reaction was carried out at room temperature under a nitrogen atmosphere for 1 hour. The liquid was collected and concentrated. The crude product LLP2A-PEG2-Lys(DOTA)-OH(T2) (55 mg, 0.037 mmol, yield: 37%, purity: 98%) was obtained by adding ether for precipitation and HPLC purification. The HPLC method was: 75%-50% aqueous phase for 15 min; the mass spectrum was as follows Figure 5 As shown, [(M+3H) 3+ The theoretical calculated value of ]: 495.9297, [(M+2H) 2+The theoretical calculated value of ]: 743.3849; the observed value of high resolution mass spectrum m / z: 495.9249,743.3843. The synthetic route is as follows:

[0134]

[0135] Example 5 Synthesis of LLP2A-PEG4-Lys(DOTA)-OH(T4)

[0136] The synthesis method of compound LLP2A-PEG4-Lys(DOTA)-OH(T4) is similar to that in Example 4, except that in step (2), Fmoc-PEG2-OH is replaced by Fmoc-PEG4-OH (CAS: 557756-85-1) in equal moles. Finally, crude product LLP2A-PEG4-Lys(DOTA)-OH(T4) (67 mg, 0.043 mmol, yield: 43%, purity: 98%) was obtained by adding ether precipitation and HPLC purification. The HPLC method is the same as that in Example 1, except that 75%-50% mobile phase A was used for 15 min. The mass spectrum is shown in FIG. Figure 6 As shown, [(M+3H) 3+ Theoretical calculated value of ]: 525.2765, [(M+2H) 2+ ] The theoretical calculated value: 787.4111; the observed value of high-resolution mass spectrum m / z: 525.2758,787.4103.

[0137] Example 6 Synthesis of LLP2A-PEG6-Lys(DOTA)-OH(T6)

[0138] The synthesis method of compound LLP2A-PEG6-Lys(DOTA)-OH(T6) is similar to that in Example 4, except that in step (2), Fmoc-PEG2-OH is replaced by Fmoc-PEG6-OH (CAS: 437655-96-4) in equal moles. Finally, crude product LLP2A-PEG6-Lys(DOTA)-OH(T6) (68 mg, 0.041 mmol, yield: 41%, purity: 98%) was obtained by adding ether precipitation and HPLC purification. The HPLC method is the same as that in Example 1, except that 75%-50% mobile phase A15min. The mass spectrum is shown in FIG. Figure 7 As shown, [(M+3H) 3+ Theoretical calculated value: 549.9559, [(M+2H) 2+ ] The theoretical calculated value: 824.4296; the observed value of high-resolution mass spectrum m / z: 549.9559,824.4296.

[0139] Example 7 Preparation of Radioactive Probes

[0140] Prepare a 0.05 M hydrochloric acid solution by diluting 30% Ultrapur hydrochloric acid (Sigma, Germany) with sterile water. Elute with 0.05 M hydrochloric acid using a syringe pump (TYD01-01, Leadfuid) 68 Ga / 68 Ge generator (740 MBq, Isotope Technologies Garching GmbH), to obtain 3 mL 68 GaCl3 solution (15-18 mCi). 200 μl of 68 The pH of GaCl3 was adjusted to 4-5, and then reacted with 20 μg of T-CH, T-AD, T2, T4 or T6 in an oil bath at 95°C for 10 minutes. 68 Ga-T-CH, 68 Ga-T-AD, 68 Ga-T2, 68 Ga-T4 and 68 Radiochemical purity (RCP) of Ga-T6. Radio-HPLC method ( 68 Ga-T-CH and 68 Ga-T-AD): Mobile phase: a mixture of mobile phase A and mobile phase B, mobile phase A: sterile aqueous solution containing 0.1 v / v% TFA; mobile phase B: acetonitrile containing 0.1 v / v% TFA; elution program: 0-15 minutes, gradient from 80 v / v% buffer A (sterile aqueous solution containing 0.1% TFA) to 40 v / v% buffer A; flow rate, 1.0 ml / min; wavelength: 254 nm; column temperature 25°C; chromatographic column: preparative 250×10.0 mm C18 column or analytical 250×4.6 mm C18 column. Radioactive HPLC method ( 68 Ga-T2, 68 Ga-T4 and 68 Ga-T6): Mobile phase: a mixture of mobile phase A and mobile phase B, mobile phase A: a sterile aqueous solution containing 0.1 v / v% TFA; mobile phase B: acetonitrile containing 0.1 v / v% TFA; elution program: 0-15 minutes, a gradient from 80 v / v% buffer A to 40 v / v% buffer A; flow rate, 1.0 ml / min; wavelength: 254 nm; column temperature 25°C; chromatographic column: preparative 250×10.0 mm C18 column or analytical 250×4.6 mm C18 column.

[0141] The results are as follows Figure 8-Figure 12 , it can be concluded that the radiolabeled probe ( 68 Ga-T-CH,68 Ga-T-AD, 68 Ga-T2, 68 Ga-T4 and 68 Ga-T6), with a radiochemical purity (RCP) > 95% and a molar activity between 22-30 MBq / nmol.

[0142] Experimental Example 1 68 Ga-T-CH and 68 PET imaging of Ga-T-AD

[0143] The present study found that the LLP2A molecule contains two key amide condensation sites: the free amino group on aminocyclohexanoic acid (Ach) and the carboxyl group on the side chain of 2-aminodecanedioic acid (Aad). Chemical modifications at these sites may have different effects on the binding affinity of LLP2A to VLA-4 integrin. To this end, two probes were synthesized by attaching Lys-DOTA to these carboxyl or amino sites: 68 Ga-T-CH (Ach modified) and 68 Ga-T-AD (Aad side chain modification). Observed by PET imaging 68 Ga-T-CH and 68 The targeting property of Ga-T-AD was investigated to investigate the effect of different modification sites on the binding affinity of LLP2A and VLA-4 integrin. The method is as follows:

[0144] B16F10 tumor mice were injected i.v. 68 Ga-T-CH or 68 Ga-T-AD (4.3-8.0MBq, 4.2-5.6nmol per mouse, 2 mice per group). Static images were obtained at 1.0 hour and 2.0 hours after injection using an Inveon microPET scanner (Siemens, Germany), with a scan time of 10 minutes. Mice were anesthetized with 0.8L / min of oxygen and 2% halothane during the scan, keeping the heart rate at 30-60BPM. PET data were reconstructed using the OSEM 3D / MAP algorithm and processed by InveonResearch Workplace software (Siemens, Germany). Regions of interest (ROIs) such as tumors and muscles were outlined and calculated using ASIPro VM 6.8.6.9 software. ROI values ​​(tissue uptake rate) are expressed as a percentage of injected dose per gram of tissue (%ID / g).

[0145] The results are as follows Fig.13 As shown, 68 Ga-T-CH and 68The tumor to muscle ratios of Ga-T-AD were similar (4.21±0.21 vs. 4.15±0.31 after 1 hour, P>0.05). 68 The tumor uptake of Ga-T-CH was significantly higher than 68 Ga-T-AD (4.25±0.28 vs. 1.63±0.06% ID / g after 1 hour, P<0.05), indicating a significant difference in targeting efficacy. The above results indicate that the carboxyl group on the Aad side chain plays a dominant role in the interaction between LLP2A and VLA-4, and modification of this site may weaken ligand-receptor binding, thereby reducing targeting specificity. In contrast, the free amino group on Ach has little effect on VLA-4 binding, so modification at this site can maintain a higher affinity. At the same time, it shows that compared 68 Ga-T-AD, 68 Ga-T-CH has more significant advantages as a probe targeting VLA-4 or melanoma VLA-4.

[0146] Experimental Example 2 Probe 68 Ga-T2, 68 Ga-T4 and 68 Stability of Ga-T6

[0147] Based on the relationship between the probe molecular structure and the VLA-4 binding force verified in Experimental Example 1, in order to further improve the binding force between the probe and VLA-4, the present invention introduced polyvinyl alcohol (PEG) linker molecules (PEG-2, PEG-4, PEG-6) of different lengths between the free amino group on Ach and DOTA to ensure the high binding affinity of the probe to VLA-4, and finally obtained the free amino group on Ach modified 68 LLP2A-PEG-derivatized probe of Ga 68 Ga-T2, 68 Ga-T4 and 68 Ga-T6. This experiment evaluates the stability of the three probes obtained as follows:

[0148] Add 50 μL of probe solution 68 Ga-T2 (5-10 MBq, 3-6 nmol), 68 Ga-T4 (5-10 MBq, 3-6 nmol) and 68Ga-T6 (5-10MBq, 3-6nmol) was diluted with 450μl PBS or FBS, and then incubated at 37°C for 2 hours. The probe solution before and after incubation was subjected to radiochemical HPLC analysis. Radiochemical HPLC method: Mobile phase: a mixture of mobile phase A and mobile phase B, mobile phase A: a sterile aqueous solution containing 0.1v / v% TFA; mobile phase B: acetonitrile containing 0.1v / v% TFA; elution program: 0-15 minutes, a gradient from 75v / v% buffer A (sterile aqueous solution containing 0.1% TFA) to 50v / v% buffer A; flow rate, 1.0 ml / min; wavelength: 254nm; column temperature 25°C; chromatographic column: preparative 250×10.0mmC18 column or analytical 250×4.6mmC18 column.

[0149] FBS used: Plasma proteins in FBS samples were precipitated with anhydrous acetonitrile and the supernatant was obtained after centrifugation. PBS used: Concentration 80-85%, pH 7-7.4. Free 68 Ga 3+ It is identified by its characteristic elution peak, appearing at approximately 2.5 minutes.

[0150] The results are as follows Fig.14 As shown, impurity peaks or free 68 Ga 3+ The peak did not increase significantly, and the main product peak remained stable, indicating that the three probes had good stability.

[0151] Experimental Example 3 Probe 68 Ga-T2, 68 Ga-T4 and 68 In vitro cell experiments with Ga-T6

[0152] In order to evaluate the ability of the three probes in Experimental Example 2 to detect the expression level of VLA-4, this experiment selected B16F10 cells with high expression of VLA-4 and A375 cells with low expression of VLA-4 as positive and negative cell models for cell uptake studies. The method is as follows:

[0153] (1) To confirm the differential expression of VLA-4 in B16F10 and A375 cell lines, Western Blot analysis was performed using GAPDH as an internal reference. Specifically:

[0154] B16F10 and A375 cells were washed with PBS and collected by centrifugation. The cell pellet was resuspended in RIPA lysis buffer containing phenylmethylsulfonyl fluoride (PMSF) and incubated on ice for 30 minutes. The lysate was vortexed and centrifuged at 12,000 rpm for 15 minutes to collect the supernatant. The protein concentration was determined using the BCA method. The protein samples were mixed with reducing protein loading buffer and boiled for 10 minutes. After SDS-PAGE separation and transfer to PVDF membrane (0.45 μm), the membrane was blocked with 5% non-fat milk powder in TBST for 30 minutes at room temperature. The membrane was incubated with primary antibodies against VLA-4 and GAPDH overnight at 4°C. After washing, the membrane was incubated with secondary antibody goat anti-rabbit IgG for 30 minutes at room temperature. The signal was visualized using enhanced chemiluminescence (ECL) and analyzed using Image-Pro Plus software.

[0155] The results are as follows Fig.15 In Figure A, the VLA-4 band in B16F10 cells is significantly stronger than that in A375 cells, where the VLA-4 signal is weaker. Quantitative analysis of band intensity showed that the average VLA-4 / GAPDH ratio of B16F10 cells was 0.518±0.041, significantly higher than 0.171±0.015 of A375 cells. This result was consistent with expectations, indicating that the VLA-4 expression level of B16F10 cells was higher than that of A375 cells, and therefore B16F10 cells were used as a model for VLA-4 targeting experiments.

[0156] (2) Cellular uptake of the three probes

[0157] B16F10 and A375 cells were cultured in RPMI 1640 medium supplemented with 20 v / v% FBS and 1 v / v% penicillin-streptomycin (penicillin concentration 100 U / mL, streptomycin concentration 100 μg / mL) at 37°C in an atmosphere of 95% air and 5% CO2 until they reached the logarithmic growth phase. After trypsinization, the cells were diluted to 2×10 5 The concentration of cells / mL was adjusted, and the cell suspension was inoculated into a 24-well plate (500 μL / well). The cells were cultured for 24 hours to allow them to adhere. The culture medium was then removed, and 200 μL of probe diluent (10 μCi / mL, the probe dilution solvent was PBS) was added to each well (n=4). After incubation for 1 hour and 2 hours, respectively, the supernatant was discarded and the cells were washed three times with PBS. After trypsin digestion, the cells were collected in a γ counting tube and measured using a 1470 automatic γ counter wizard (PerkinElmer, USA). The cell uptake percentage was calculated as: (cell CPM value / CPM value of 200 μL probe diluent) × 100%.

[0158] The experimental results are as follows Fig.15In the middle BD, the uptake of all probes in VLA-4-positive B16F10 cells was significantly higher than that in VLA-4-negative A375 cells. Specifically:

[0159] for 68 Ga-T2 probe, after 1 hour incubation, 68 There was no significant difference between the uptake of Ga-T2 in B16F10 cells and in A375 cells. After 2 hours of incubation, 68 The uptake of Ga-T2 in B16F10 cells (0.66±0.06%) was 3.1 times that in A375 cells (0.21±0.02%), with a significant difference of P<0.001.

[0160] for 68 The uptake of Ga-T4 probe in B16F10 cells after 1 and 2 hours of incubation was significantly different from that in A375 cells, and the difference was more significant after 2 hours of incubation. 68 The uptake of Ga-T4 in B16F10 cells (1.58±0.11%) was 2.0 times higher than that in A375 cells (0.79±0.15%).

[0161] for 68 After 1 and 2 hours of incubation, the uptake of Ga-T6 probe in B16F10 cells was significantly different from that in A375 cells. After 2 hours of incubation, the uptake in B16F10 cells (1.05±0.13%) was 1.8 times that in A375 cells (0.59±0.04%).

[0162] The above results show that the three probes preferentially accumulated in VLA-4-positive B16F10 cells, highlighting their strong specificity for VLA-4.

[0163] (3) Cytotoxicity of the three probes

[0164] B16F10 and A375 cell suspensions (1×10 5 Cells / mL) were seeded into 96-well plates, 100 μL per well, and incubated for 24 h to allow cell adhesion, and then probe solution (final concentration as Fig.16 and Fig.17 The volume of the cells was 200 μL. After incubation for 12 hours, 10 μL of cell counting kit-8 (CCK-8) solution (KeyGEN) was added to each well and then incubated for 3 hours to allow for reaction with cell dehydrogenase. The optical density (OD) of the colored product at 450 nm was measured using a microplate reader (BioTek). The cell viability percentage was calculated as: (OD value of samples treated with different concentrations / OD value of 0 μM control sample) × 100%.

[0165] The results are as follows Figure 16-Figure 18 As shown, at a concentration of 100 μM, the cell viability of the three probes in B16F10 and A375 cells remained at around 90%, indicating that the cytotoxicity of the three probes was relatively low, indicating that the three probes had certain in vivo applicability and would not significantly affect the health of the experimental subjects.

[0166] Experimental Example 4 Probe 68 Ga-T2, 68 Ga-T4 and 68 PET imaging of Ga-T6

[0167] This experiment investigated 68 Ga-T2, 68 Ga-T4 and 68 Targeting of Ga-T6 to VLA-4 or melanoma.

[0168] B16F10 and A375 tumor mice were injected i.v. 68 Ga-T2, 68 Ga-T4 or 68 Ga-T6 (4.2-10.7MBq, 3.4-5.0nmol per mouse, one probe corresponding to one tumor mouse in one group, a total of 6 groups, 3 mice in each group). Static images were obtained at 1.0 hour and 2.0 hours after injection using an Inveon microPET scanner (Siemens, Germany), and the scanning time was 10 minutes. Mice were anesthetized with 0.8L / min of oxygen and 2% halothane during the scan, and the heart rate was maintained at 30-60BPM. PET data were reconstructed using the OSEM 3D / MAP algorithm and processed by Inveon Research Workplace software (Siemens, Germany). Regions of interest (ROIs) such as tumors and muscles were outlined and calculated using ASIPro VM 6.8.6.9 software. ROI values ​​(tissue uptake rate) are expressed as a percentage of the injected dose per gram of tissue (%ID / g).

[0169] The results are as follows Fig.19 As shown:

[0170] exist Fig.19 In Figure A, in B16F10 tumor mice, 68 Ga-T2, 68 Ga-T4 and 68The static PET images of Ga-T6 showed high tumor accumulation at 1 and 2 hours after injection, and good contrast between tumor and background. Among the metabolic and excretory organs (low liver and kidney signals and high bladder signals in the figure), the signals of the liver and kidneys were relatively low, while the signals of the bladder were the highest, indicating that the probe may be rarely metabolized by the liver and kidneys, but directly excreted into the bladder in its original form. Among the healthy tissues, only the spleen showed significant uptake, which may be attributed to its VLA-4 positive expression or high macrophage activity due to the negative charge of the probe.

[0171] exist Fig.19 In Figures C and D of the B16F10 tumor mouse model, the 68 Compared with the tumor uptake of Ga-T-CH, 68 Ga-T2, 68 Ga-T4 and 68 The tumor uptake of Ga-T6 decreased, and the tumor signals at 1 hour after injection were 2.69±0.17, 2.22±0.11, and 1.55±0.09%ID / g, respectively. This phenomenon can be attributed to the increased water solubility brought by PEG, which in turn accelerated metabolism. Nevertheless, 68 Ga-T2(11.20±1.08), 68 Ga-T4(8.77±0.32) and 68 The contrast between tumor and muscle of Ga-T6 (6.57±0.69) within 1 hour was higher than that in Experiment 1. 68 Ga-T-CH, indicating that the metabolic rates of the three probes in non-target tissues are faster ( Fig.19 Medium D and Fig.13 (B) The above shows that the PEG-linked molecular structure in the three probes significantly reduces the binding of the probes to nonspecific tissues, thereby enhancing the pharmacokinetic properties and optimizing the imaging performance of the probes.

[0172] In the above Fig.19 Figures A, C, and D show that although the PEGylated probes show similar distribution patterns in the tumor model, there is a significant difference: the length of the PEG chain is not proportional to the binding performance of the probe to nonspecific tissues. The shortest PEG derivative 68 Ga-T2 showed the highest tumor uptake and tumor-to-muscle ratio.

[0173] exist Fig.19 In Figure B, in order to verify the in vivo specificity of the three probes, the three probes were respectively tested in the A375 model expressing low levels of VLA-4. 68 Ga-T2, 68 Ga-T4 and 68Ga-T6 PET imaging. As can be seen in the figure, no significant uptake difference among the three probes was observed in normal tissues; however, Fig.19 In Figure E, the uptake of the three probes in the A375 tumor mouse model was significantly lower than that in the B16F10 tumor mouse model. 68 Ga-T2, 68 Ga-T4 and 68 The uptake amounts of Ga-T6 in 1 hour were 0.72±0.12%ID / g, 0.87±0.03%ID / g and 0.62±0.08%ID / g, respectively, indicating that the three probes have good in vivo specificity.

[0174] Experimental Example 5 Probe 68 Ga-T2, 68 Ga-T4 and 68 Biodistribution of Ga-T6

[0175] This experimental example examines the probe 68 Ga-T2, 68 Ga-T4 and 68 The biodistribution of Ga-T6 is as follows:

[0176] (1) Tissue distribution

[0177] B16F10 and A375 tumor-bearing mice were intravenously injected 68 Ga-T2, 68 Ga-T4 or 68 Ga-T6 (4.2-10.7 MBq, 3.4-5.0 nmol per mouse, one probe corresponding to one tumor mouse in one group, a total of 6 groups, 3 mice in each group). Mice were killed 1 hour after injection, and tumors and other tissues (heart, liver, spleen, lung, kidney, stomach, intestine, muscle, bone, brain and blood) were collected. The mass and radioactivity of each tissue were measured. Tissue uptake rate was standardized as the percentage of injected dose per gram of tissue (%ID / g).

[0178] The experimental results are as follows Fig. 20 As shown in A-C, 68 Ga-T2, 68 Ga-T4 or 68 After Ga-T6, mice with B16F10 tumors were killed 1 hour later. This is consistent with the trend of the PET imaging data of the three probes in Experimental Example 4. 68 The tumor uptake and tumor-to-muscle ratio of Ga-T2 were significantly higher than those of 68 Ga-T4 and 68Ga-T6 (tumor: 3.58±0.28 vs. 2.90±0.16 vs. 1.87±0.22% ID / g, P<0.05 for comparison between any two groups; tumor / muscle: 13.38±0.43 vs. 10.62±0.70 vs. 7.19±1.15, P<0.05 for comparison between any two groups). In addition, 68 The tumor-to-blood ratio of Ga-T2 at 1 hour (8.64±1.12) was significantly better than that of 68 Ga-T4(5.32±0.91) and 68 Ga-T6(4.36±0.59)( 68 Ga-T2 and 68 Ga-T4 comparison and 68 Ga-T2 and 68 Ga-T6 comparison (all P<0.05). 68 Ga-T2, 68 Ga-T4 and 68 The uptake values ​​of Ga-T6 in spleen were 6.04±0.54%ID / g, 3.77±0.32%ID / g, and 2.82±0.41%ID / g, respectively, which were all higher than those in tumors, suggesting that spleen may be the dose-limiting organ for these probes. Bone marrow is also a VLA-4-positive organ, however, the uptake values ​​in bone marrow are relatively low, probably due to rapid clearance from blood: 68 Ga-T2 is 1.44±0.20%ID / g, 68 Ga-T4 is 1.73±0.09%ID / g, 68 Ga-T6 is 0.64±0.07%ID / g, which is 68 Compared with Ga-T4, 68 Ga-T2 and 68 Ga-T6 showed superior distribution characteristics, potentially reducing the potential risk of myelosuppressive toxicity. This was supported by the tumor to bone marrow ratios: 68 Ga-T2 is 2.55±0.47, 68 Ga-T4 is 1.68±0.06, 68 Ga-T6 was 3.08±1.06 (P<0.05). 68 Ga-T2 showed higher tumor uptake and lower background interference. Subsequently, the uptake of the three probes was evaluated in negative models, including A375 xenograft tumors and healthy tissues. Compared with B16F10 tumors, A375 tumors showed significantly reduced uptake. 68 Ga-T2 is 1.41±0.19%ID / g, 68 Ga-T4 is 1.25±0.10%ID / g, 68Ga-T6 was 1.12±0.06%ID / g, while other tissues showed no significant differences. These findings demonstrate the VLA-4 targeting specificity of the three probes.

[0179] Finally, 68 Ga-T2 and existing high-performance probes with similar structural architecture 68 Ga-NODAGA-PEG4-LLP2A was compared to evaluate tumor uptake and target-to-target ratio. 68 Tumor uptake of Ga-T2 was lower than 68 Ga-NODAGA-PEG4-LLP2A (8.7±1.3% ID / g at 1 hour). 68 The tumor-to-muscle ratio (10.3 ± 1.5 at 1 h) and tumor-to-blood ratio (4.4 ± 1.6 at 1 h) of Ga-NODAGA-PEG4-LLP2A were lower than those of 68 Ga-T2. Probe 68 The clearance rate of Ga-T2 in vivo was 68 Ga-NODAGA-PEG4-LLP2A was faster, resulting in a decrease in tumor signal; however, the clearance rate from background tissue was even faster than that from the target, which enhanced the signal contrast, indicating that the probe of the present invention 68 Ga-T2 has stronger signal contrast.

[0180] (2) Hematoxylin-eosin (HE) staining

[0181] Four normal nude mice (BALB / c nude mice, 4-6 weeks old) were intravenously injected with normal saline, 68 Ga-T2, 68 Ga-T4 or 68 Ga-T6 (4.2-10.7MBq, 3.4-5.0nmol per mouse). After 24 hours, mice were killed and major organs (heart, liver, spleen, lung, kidney, stomach, intestine and muscle) were collected. Tissues were fixed in 10% neutral formalin for 24 hours, then paraffin embedded, sliced ​​and stained with hematoxylin-eosin (HE). Full slice scanning data was obtained using a 3DHISTECH midi microscope slice scanner and processed by SlideViewer software to obtain pathological images (magnification: 20X).

[0182] The results are as follows Fig. 20 As shown in Figure F, after injection 68 Ga-T2, 68 Ga-T4 or 68 24 hours after Ga-T6, the morphology of healthy tissues was similar to that of the control group injected with normal saline, and no obvious damage was observed, indicating that these probes have a good safety profile.

[0183] Immunofluorescence staining

[0184] To evaluate the expression of VLA-4 protein in various tissues, immunofluorescence staining was performed on various mouse tissues and B16F10 / A375 tumor specimens. After fixing B16F10 / A375 tumors and normal tissues (heart, liver, spleen, lung, kidney, stomach, intestine, and muscle) with 10% neutral formalin for 24 hours, sections were prepared. The sections were washed with TBSTx solution and blocked with TBSTx containing 5% BSA. Then, the tissue sections were incubated with anti-VLA-4 antibody overnight at 4°C. Cy-3-labeled goat anti-rabbit IgG was incubated with frozen sections for 1 hour, and DAPI was incubated for 2 minutes. Immunofluorescence images were acquired using a fluorescence microscope and analyzed using ImageJ software.

[0185] Ending Fig. 20 In Figure D, the VLA-4 signal in B16F10 tumors is prominent and significantly higher than that in healthy tissues and A375 tumors. Fig. 20 Middle E: Among healthy tissues, the expression level of VLA-4 protein in the spleen is relatively high.

[0186] In summary: Existing studies have demonstrated a strong correlation between VLA-4 expression in melanoma and important clinical parameters such as tumor stage, invasion depth, and metastatic potential. Therefore, imaging of VLA-4 expression becomes an important tool to assess melanoma progression and monitor treatment efficacy, thereby potentially improving patient survival. 18 F-FDG PET imaging is commonly used in clinical practice for the diagnosis and staging of melanoma, but its usefulness is limited by its inability to effectively distinguish malignant tumors from metabolically active normal tissues. This limitation highlights the urgent need for a PET imaging probe that is sensitive and specific for VLA-4 expression in melanoma to accurately assess the metastatic potential of melanoma. LLP2A is a ligand with high affinity for VLA-4. In this context, the present invention combines 68 Ga-DOTA was attached to the Ach or Aad side chain of the molecule LLP2A to develop an innovative probe of LLP2A. In Experimental Example 1, in vivo studies showed that 68 Tumor uptake of Ga-T-CH was significantly higher than 68 Ga-T-AD, while maintaining a similar tumor-to-muscle ratio. Therefore, the present invention concluded that the modification of Ach has little effect on the binding affinity between LLP2A and VLA-4. 68 The Ga-T-CH probe has better targeting in VLA-4 or melanoma VLA-4.

[0187] In order to further improve the binding affinity between the probe and VLA-4 and enhance the imaging clarity, the present invention introduces polyvinyl alcohol (PEG) connection structures (PEG-2, PEG-4, PEG-6) of different lengths between Ach and DOTA to obtain three corresponding probes: 68 Ga-T2, 68 Ga-T4, 68 Ga-T6. In Experimental Example 4, it was verified that this modification significantly improved the melanoma signal clarity of the probe. In the PEGylated probe, 68 Ga-T2 (PEG-2 linker) and non-PEGylated 68 Compared with Ga-T-CH, the tumor-to-muscle ratio increased by about three times, showing the most favorable in vivo distribution characteristics. Although PEGylation of any chain length accelerated clearance from non-target tissues, extending the PEG chain did not significantly change the metabolic rate of the probe in these tissues, especially muscle: at 1 hour, 68 Ga-T2, 68 Ga-T4 and 68 The uptake of Ga-T6 in B16F10 muscle was 0.27±0.04%ID / g, 0.28±0.03%ID / g, and 0.26±0.03%ID / g, respectively, with no statistically significant difference. However, longer PEG chains may weaken the affinity of the probe for tumor-associated proteins, resulting in reduced specific uptake. Therefore, shorter PEG linkage structures may have advantages in optimizing the in vivo distribution of certain ligands. In addition, since bone tissue also expresses VLA-4, it was observed in PET imaging. 68 Significant bone uptake of Ga-T-CH in the limbs was not observed with the PEGylated probes, an important consideration for reducing myelosuppression and associated toxicities.

[0188] 18 F-FDG is well known for its rapid accumulation in high metabolic organs, especially its significant specific uptake in the myocardium, which may interfere with the imaging of chest lesions (such as lung and liver regions). In contrast, in Experimental Example 5, 68 Ga-T2 did not show specific myocardial uptake, and its myocardial signal (0.22±0.03% ID / g) was very close to muscle tissue, allowing melanoma imaging with minimal cardiac background interference. This selective uptake is attributed to 68 Ga-T2 specifically targets VLA-4 protein without recognizing and accumulating in high metabolic organs.

[0189] in conclusion:

[0190] In the present invention, 68 Ga-T-CH and 68Ga-T-AD was compared and analyzed. 68 The binding sites of Ga-DOTA were different, and PEGylated derivatives with different chain lengths were evaluated ( 68 Ga-T2, 68 Ga-T4 and 68 The results showed that all three PEGylated probes were excellent imaging agents; in particular, 68 Ga-T2 stands out for its excellent tumor uptake and enhanced background contrast. These new VLA-4-targeted compounds are able to specifically detect melanoma and may be more effective than conventional VLA-4-targeted compounds in melanoma imaging. 18 F-FDG has significant advantages.

[0191] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. A 68 The Ga-labeled molecular probe targeting VLA-4 is characterized in that: The structural formula is as follows In the structural formula, X represents an integer ≥0.

2. according to claim 1 68 The Ga-labeled molecular probe targeting VLA-4 is characterized in that: X represents an integer ≥0 and ≤3. Optionally, X is 1.

3. A 68 The precursor of the Ga-labeled molecular probe targeting VLA-4 is characterized in that The structural formula is as follows In the structural formula, X represents an integer ≥0.

4. A method as claimed in claim 3 68 A method for preparing a precursor of a Ga-labeled molecular probe targeting VLA-4, characterized in that: The steps include: When X in the structural formula represents an integer of =0: S1, coupling the compound Fmoc-Lys(Dde)-OH to a solid phase support 2-chlorotrityl chloride resin, and then removing the Fmoc protecting group to obtain compound 1; S2, coupling the compound LLP2A(tBu)-OH to the compound 1, and then removing the Dde protecting group to obtain the compound 2; the compound LLP2A(tBu)-OH is a compound in which the carboxyl group on the 2-aminosebacic acid side chain of LLP2A is protected by the tBu group; S3, coupling the compound DOTA(OtBu)3 to compound 2, and then cleaving the peptide to remove the protecting group tBu; The synthetic route is as follows: When X in the structural formula represents an integer ≥ 1: Q1, coupling the compound Fmoc-Lys(Dde)-OH to the solid phase support 2-chlorotrityl chloride resin, and then removing the Fmoc protecting group to obtain compound 1; Q2, coupling the compound Fmoc-PEGn-OH to the compound 1, and then removing the Fmoc protecting group to obtain the compound 2; wherein n in the compound Fmoc-PEGn-OH represents an integer multiple of 2, and n>0; Q3, coupling compound LLP2A(tBu)-OH to compound 2, and then removing the Dde protecting group to obtain compound 3; compound LLP2A(tBu)-OH is a compound in which the carboxyl group on the 2-aminosebacic acid side chain of LLP2A is protected by a tBu group; Q4, coupling the compound DOTA(OtBu)3 to compound 3, and then cleaving the peptide to remove the protecting group tBu; The synthetic route is as follows:

5. According to claim 4 68 A method for preparing a precursor of a Ga-labeled molecular probe targeting VLA-4, characterized in that: The coupling reaction conditions in step S1, step Q1, and step Q2 are: reaction at room temperature for 1-2 hours; And / or, the coupling reaction conditions in step S2, step S3, step Q3, and step Q4 are: reaction time at room temperature for 8-12 hours; And / or, the reagents used in the coupling step include coupling reagents and / or pH adjusters, the coupling reagents are HOBT and HBTU, and the pH adjuster is DIPEA.

6. The method according to claim 4 or 5 68 A method for preparing a precursor of a Ga-labeled molecular probe targeting VLA-4, characterized in that: In step S1, the compound Fmoc-Lys(Dde)-OH and DIPEA are dissolved in an organic solvent at a molar ratio of 1:2-1:3, and then added to a solid support 2-chlorotrityl chloride resin for coupling; and / or, in step S2, compound LLP2A(tBu)-OH, DIPEA, HOBT and HBTU are dissolved in an organic solvent at a molar ratio of 1:2:2:2 to 1:3:3:3, and then added to compound 1 for coupling; and / or, in step S3, DOTA(OtBu)3, DIPEA, HOBT and HBTU are dissolved in an organic solvent at a molar ratio of 1:2:2:2-1:3:3:3, and then added to compound 2 for coupling; And / or, in step Q1, the compound Fmoc-Lys(Dde)-OH and DIPEA are dissolved in an organic solvent at a molar ratio of 1:2-1:3, and then added to a solid support 2-chlorotrityl chloride resin for coupling; and / or, in step Q2, the compounds Fmoc-PEGn-OH, DIPEA, HOBT and HBTU are dissolved in an organic solvent at a molar ratio of 1:2:2:2-1:3:3:3, and then added to compound 1 for coupling; and / or, in step Q3, compound LLP2A(tBu)-OH, DIPEA, HOBT and HBTU are dissolved in an organic solvent at a molar ratio of 1:2:2:2 to 1:3:3:3, and then added to compound 2 for coupling; And / or, in step Q4, DOTA(OtBu)3, DIPEA, HOBT and HBTU are dissolved in an organic solvent at a molar ratio of 1:2:2:2-1:3:3:3, and then added to compound 3 for coupling.

7. The method according to any one of claims 4 to 6. 68 A method for preparing a precursor of a Ga-labeled molecular probe targeting VLA-4, characterized in that: In the step of removing the Fmoc protecting group, a solution containing 20% ​​to 25% piperidine by volume is used to remove the Fmoc protecting group; the removal is performed 1 to 2 times, and each removal time is 5 to 10 minutes; And / or, in the step of removing the Dde protecting group, a solution containing 2% to 2.5% by volume of hydrazine hydrate is used to remove the Dde protecting group; the removal is performed 1 to 2 times, and each removal time is 10 to 20 minutes; And / or, in the peptide cleavage and tBu group deprotection steps, a DCM solution containing 93%-95% trifluoroacetic acid by volume is used; the deprotection condition is nitrogen flow for 40-60 minutes.

8. A method as claimed in claim 1 68 The method for preparing a Ga-labeled molecular probe targeting VLA-4 is characterized in that: The claim 3 68 Ga-labeled molecular probe precursor targeting VLA-4 or prepared by the preparation method according to any one of claims 4 to 7 68 Ga-labeled molecular probe precursor targeting VLA-4 68 Ga label; optionally, the 68 The radioactivity in the Ga mixed solution is 15 to 18 mCi; Optionally, the 68 In the Ga mixed solution, the pH value is 4-5; Optionally, the 68 Ga-labeled molecular probe precursor targeting VLA-4 and the like 68 The mass volume ratio of Ga mixed solution is 20:200-300, μg / μL.

9. A method as claimed in claim 1 68 Use of Ga-labeled molecular probe targeting VLA-4 in preparing PET tracer targeting VLA-4 or melanoma tumor.

10. A PET tracer, characterized in that: Containing the 68 Ga-labeled molecular probe targeting VLA-4.

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