A t cell immunoglobulin and mucin domain-3 targeting compound and uses

Abstract

The application belongs to the field of nuclear medicine and relates to a T cell immunoglobulin and mucin domain-3 targeted compound and application. 68 Ga labeled TIM-3 targeted molecular probes can realize imaging of TIM-3 positive tumors and exhibit good potential as TIM-3 targeted PET imaging agents.

Description

Technical Field

[0001] This invention belongs to the field of nuclear medicine, specifically relating to a T-cell immunoglobulin and mucin domain-3 (TIM-3) targeting compound and its applications. Background Technology

[0002] The tumor immune microenvironment is a complex and dynamic system composed of tumor cells, immune cells, and stromal cells, profoundly regulating anti-tumor immune responses. Immune checkpoint molecules maintain immune homeostasis and prevent autoimmune damage by inhibiting the overactivation of T cells under normal physiological conditions. In the tumor microenvironment, abnormally high expression of immune checkpoint molecules directly inhibits the infiltration and function of effector T cells, inducing them into a state of exhaustion. It also indirectly weakens immune surveillance capabilities by affecting the activity of antigen-presenting cells. Furthermore, these signaling pathways synergistically exacerbate the inhibitory properties of the tumor microenvironment with processes such as tumor metabolic reprogramming. T cell immunoglobulin and mucin domain-3 (TIM-3), as a key immune checkpoint molecule, has received widespread attention in recent years.

[0003] TIM-3 is an important member of the TIM protein family. Its structure includes an extracellular immunoglobulin variable region, a mucin stem region, a transmembrane region, and a unique cytoplasmic tail. This cytoplasmic tail lacks typical inhibitory motifs but possesses conserved tyrosine residues that can bind to intracellular aptamers such as BAT3, mediating non-canonical signal transduction. In adaptive immunity, TIM-3 is a key marker of T cell exhaustion and is often co-expressed with programmed cell death protein 1 (PD-1) in tumors and chronic viral infection environments containing CD8. + T cells synergistically inhibit T cell activation and function, maintaining the immunosuppressive microenvironment. In innate immunity, TIM-3 is widely expressed in dendritic cells, macrophages, and microglia, participating in the regulation of inflammatory responses, phagocytic function, and cellular homeostasis. TIM-3 is an important molecular target in tumors, autoimmune diseases, chronic infections, and neurodegenerative diseases, exerting unique immune functions. Therefore, designing high-affinity probes targeting TIM-3 has a clear theoretical basis and application potential.

[0004] Develop TIM-3-targeting ligands with good target affinity, specificity, pharmacokinetic properties, and stability, and prepare diagnostic radiolabeled ligands (such as...). 68 Ga 18 F or 99m Tc-labeled probes and therapeutic radiolabeled probes (such as Tc-labeled probes and Tc-labeled probes) 177 Therapeutic drugs with Lu markers will provide more efficient tools for lesion detection, efficacy monitoring, and clinical remission of related diseases, and have broad application prospects. Summary of the Invention

[0005] The purpose of this invention is to provide a novel T-cell immunoglobulin and mucin domain-3 targeting compound.

[0006] To achieve the above objectives, the present invention provides a T-cell immunoglobulin and mucin domain-3 targeting compound, wherein the T-cell immunoglobulin and mucin domain-3 targeting compound has the structure shown in Formula I: Formula I In Formula I: R1 is a chemical bond. , ,or ; R2 is .

[0007] Specifically, the T-cell immunoglobulin and mucin domain-3 targeting compound is compound H1, compound H2, compound H3, or compound H4. Compound H1; Compound H2; Compound H3; Compound H4.

[0008] The T-cell immunoglobulin and mucin domain-3 targeting compounds of this invention can be used to prepare nuclear medicine diagnostic probes or immune checkpoint inhibitors.

[0009] Specifically, the present invention provides a T-cell immunoglobulin and mucin domain-3 targeting imaging reagent, wherein the T-cell immunoglobulin and mucin domain-3 targeting imaging reagent is the above-mentioned T-cell immunoglobulin and mucin domain-3 targeting compound labeled with a diagnostic radionuclide.

[0010] The diagnostic radionuclides include, but are not limited to, those used in the diagnosis of radioactive nuclides. 68 Ga、 64 Cu、 18 F, 86 Y、 90 Y、 89 Zr、 111 In、 99m Tc, 11 C 123 I, 125 I and 124 At least one of I.

[0011] The present invention also provides a T-cell immunoglobulin and mucin domain-3 targeted therapeutic agent, wherein the T-cell immunoglobulin and mucin domain-3 targeted therapeutic agent is the above-mentioned T-cell immunoglobulin and mucin domain-3 targeted compound labeled with a therapeutic radionuclide.

[0012] The therapeutic radionuclides include, but are not limited to, those used in treatment. 177 Lu、 125 I, 131 I, 211 At、 111 In、 153 Sm、 186 Re、 188 Re、 67 Cu、 212 Pb, 225 Ac、 213 Bi、 212 Bihe 212 At least one of Pb.

[0013] The present invention provides 68 After injection of Ga-labeled TIM-3 targeting molecular probes (H1, H2, H3, H4) into tumor-bearing mice, significant radioactive concentrations were observed in the subcutaneous tumor areas of all four probes, indicating that they could effectively target the tumors and demonstrate good potential as TIM-3-targeted PET imaging agents. MicroPET / CT imaging results showed that at 60 minutes post-injection, 68 The Ga-H4 probe achieved the highest tumor-to-muscle ratio, and at this time point, its tumor-to-muscle ratio was superior to the other three probes, exhibiting the best targeting performance.

[0014] Other features and advantages of the present invention will be described in detail in the following detailed description section. Attached Figure Description

[0015] The above and other objects, features and advantages of the present invention will become more apparent from the more detailed description of exemplary embodiments of the invention in conjunction with the accompanying drawings.

[0016] Figure 1 The general preparation route for TIM-3 targeting probes H1-H4 is as follows.

[0017] Figure 2 The preparation route for TIM-3 targeting probe H1 is shown.

[0018] Figure 3 This is the mass spectrum of the TIM-3 targeting probe H1.

[0019] Figure 4 The preparation route for the TIM-3 targeting probe H2 is described.

[0020] Figure 5 This is the mass spectrum of the TIM-3 targeting probe H2.

[0021] Figure 6 The preparation route for TIM-3 targeting probe H3 is described.

[0022] Figure 7 This is the mass spectrum of the TIM-3 targeting probe H3.

[0023] Figure 8 The preparation route for the TIM-3 targeting probe H4 is described.

[0024] Figure 9 This is the mass spectrum of the TIM-3 targeting probe H4.

[0025] Figure 10 For injection 68 PET MIP image of tumor-bearing mice 60 min after using the Ga-H1 probe.

[0026] Figure 11 For injection 68 PET MIP image of tumor-bearing mice after 60 min with Ga-H2 probe.

[0027] Figure 12 For injection 68 PET MIP image of tumor-bearing mice after 60 min with Ga-H3 probe.

[0028] Figure 13 For injection 68 PET MIP image of tumor-bearing mice after 30 min with Ga-H4 probe.

[0029] Figure 14 For injection 68 PET MIP image of tumor-bearing mice after 60 min with Ga-H4 probe. Detailed Implementation

[0030] Preferred embodiments of the invention will now be described in more detail. While preferred embodiments of the invention are described below, it should be understood that the invention can be implemented in various forms and should not be limited to the embodiments set forth herein.

[0031] Example 1: Preparation of TIM-3 targeting probes H1, H2, H3 and H4

[0032] The synthesis steps of H1-H4 are as follows: Figure 1 As shown. The coupling of amino acids was carried out according to the standard Fmoc solid-phase synthesis method. Reaction conditions: (a) 20% piperidine N,N - Dimethylformamide (DMF) solution, Fmoc-R1-OH, benzotriazole- N ,N , N’ , N’ -Tetramethylurea hexafluorophosphate (HBTU), 1-hydroxybenzotriazole (HOBt) and N,N - A DMF solution of diisopropylethylamine (DIPEA); or a DMF solution of 20% piperidine, Fmoc-R2-OH, HBTU, HOBt and DIPEA. (b) 1. A DMF solution of 20% piperidine, Fmoc-R2-OH, HBTU, HOBt and DIPEA; 2. Trifluoroacetic acid and dimethyl sulfoxide (DMSO).

[0033] Preparation of H1:

[0034] H1 structural form

[0035] The synthesis steps of H1 are as follows: Figure 2 As shown. The coupling of amino acids was carried out according to the standard Fmoc solid-phase synthesis method. Reaction conditions: (a) 20% piperidine in DMF solution, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid tritert-butyl ester (DOTA) in DMF solution; (b) trifluoroacetic acid, DMSO.

[0036] Specifically, a certain mass of resin (0.04 mmol) was placed in a 10 mL solid-phase synthesis tube, and 3 mL of dichloromethane (DCM) was added for swelling. This process was repeated three times, each time for 5 minutes, followed by washing three times with DMF for 5 minutes each time. The amino protecting group Fmoc was removed using a DMF solution containing 20% ​​piperidine (v / v). Specifically, 3 mL of 20% piperidine DMF solution was added for 10 minutes, followed by washing six times with 2 mL of DMF for 2 minutes each time. Six times the stoichiometric amount of DOTA, activated with six times the stoichiometric amount of HBTU and HOBt in the presence of six times the stoichiometric amount of DIPEA relative to the resin (0.04 mmol), was added to the synthesis tube and reacted under electromagnetic stirring for 1 hour. After the reaction, the tube was washed six times with 2 mL of DMF for 2 minutes each time. The ligand dissociation from the resin and the removal of Tlt were completed by stirring with 4 mL of trifluoroacetic acid / DMSO (9:1, v / v) for 12 hours. The resin was then washed with 2 mL of trifluoroacetic acid, and all filtrates were collected. After removing the trifluoroacetic acid under reduced pressure, the target compound H1 was obtained. The ligand structure was identified by mass spectrometry, as follows: Figure 3 As shown.

[0037] Preparation of H2:

[0038] H2 structural formula

[0039] The steps for synthesizing H2 are as follows: Figure 4 As shown. The coupling of amino acids was carried out according to the standard Fmoc solid-phase synthesis method. Reaction conditions: (a) 20% piperidine DMF solution, Fmoc-L-glycine, HBTU, HOBt and DIPEA DMF solution; (b) 20% piperidine DMF solution, DOTA DMF solution; trifluoroacetic acid, DMSO.

[0040] Specifically, a certain mass of resin (0.04 mmol) was placed in a 10 mL solid-phase synthesis tube, and 3 mL of DCM was added for swelling. This process was repeated three times, each time for 5 minutes, followed by washing three times with DMF, each time for 5 minutes. The amino protecting group Fmoc was removed using a DMF solution containing 20% ​​piperidine (v / v). Specifically, 3 mL of 20% piperidine DMF solution was reacted for 10 minutes, then 10 minutes each time, followed by washing six times with 2 mL of DMF, each time for 2 minutes. Six times the stoichiometric amount of Fmoc-L-glycine, relative to the resin (0.04 mmol), was added to the synthesis tube after activation with six times the stoichiometric amount of HBTU and HOBt in the presence of six times the stoichiometric amount of DIPEA. The reaction was carried out under electromagnetic stirring for 1 hour. The mixture was then washed six times with 2 mL of DMF, each time for 2 minutes. The amino protecting group Fmoc was then removed using 3 mL of DMF solution containing 20% ​​piperidine (v / v), repeated twice, each time for 10 minutes. DOTA, with a stoichiometric concentration of 6 times that of the resin, was activated with HBTU and HOBt in the presence of 6 times that of DIPEA and then added to the synthesis tube. The reaction was carried out under electromagnetic stirring for 1 hour. Afterward, the mixture was washed 6 times with 2 mL DMF for 2 minutes each time. Ligand dissociation from the resin and removal of Trt were completed by stirring with 4 mL trifluoroacetic acid / DMSO (9:1, v / v) for 12 hours. The resin was then washed with 2 mL trifluoroacetic acid, and all filtrates were collected. After removing trifluoroacetic acid under reduced pressure, the target compound H2 was obtained. The ligand structure was identified by mass spectrometry, as shown below. Figure 5 As shown.

[0041] Preparation of H3:

[0042] H3 structural form

[0043] The steps for synthesizing H3 are as follows: Figure 6 As shown. The coupling of amino acids was carried out according to the standard Fmoc solid-phase synthesis method. Reaction conditions: (a) 20% piperidine DMF solution, Fmoc-(4-aminomethyl)benzoic acid, HBTU, HOBt and DIPEA DMF solution; (b) 20% piperidine DMF solution, DOTA DMF solution; trifluoroacetic acid, DMSO.

[0044] Specifically, a certain mass of resin (0.04 mmol) was placed in a 10 mL solid-phase synthesis tube, and 3 mL of DCM was added for swelling. This process was repeated three times, each time for 5 minutes, followed by washing three times with DMF, each time for 5 minutes. The amino protecting group Fmoc was removed using a DMF solution containing 20% ​​piperidine (v / v). Specifically, 3 mL of 20% piperidine DMF solution was reacted for 10 minutes, then 10 minutes each time, followed by washing six times with 2 mL of DMF, each time for 2 minutes. Relative to the resin (0.04 mmol), 6 times the stoichiometric amount of Fmoc-(4-aminomethyl)benzoic acid, activated with 6 times the stoichiometric amount of HBTU and HOBt in the presence of 6 times the stoichiometric amount of DIPEA, was added to the synthesis tube and reacted under electromagnetic stirring for 1 hour. The mixture was then washed six times with 2 mL of DMF, each time for 2 minutes. The amino protecting group Fmoc was then removed using 3 mL of DMF solution containing 20% ​​piperidine (v / v), repeated twice, each time for 10 minutes. DOTA, with a chemical weight of 6 times that of the resin, was activated with HBTU and HOBt in the presence of 6 times that of DIPEA and then added to the synthesis tube. The reaction was carried out under electromagnetic stirring for 1 hour. After the reaction, the mixture was washed 6 times with 2 mL DMF for 2 minutes each time. The dissociation of the ligand from the resin and the removal of Trt were completed by stirring with 4 mL trifluoroacetic acid / DMSO (9:1, v / v) for 12 hours. The resin was then washed with 2 mL trifluoroacetic acid, and all filtrates were collected. After removing the trifluoroacetic acid under reduced pressure, the target compound H3 was obtained. The ligand structure was identified by mass spectrometry, as shown in the figure. Figure 7 As shown.

[0045] Preparation of H4:

[0046] H4 structure

[0047] The steps for synthesizing H4 are as follows: Figure 8 As shown. The coupling of amino acids was carried out according to the standard Fmoc solid-phase synthesis method. Reaction conditions: (a) 20% piperidine DMF solution, Fmoc-11-amino-3,6,9-trioxanedecanoic acid, HBTU, HOBt and DIPEA DMF solution; (b) 20% piperidine DMF solution, DOTA DMF solution; trifluoroacetic acid, DMSO.

[0048] Specifically, a certain mass of resin (0.04 mmol) was placed in a 10 mL solid-phase synthesis tube, and 3 mL of DCM was added for swelling. This process was repeated three times, each time for 5 minutes, followed by washing three times with DMF, each time for 5 minutes. The amino protecting group Fmoc was removed using a DMF solution containing 20% ​​piperidine (v / v). Specifically, 3 mL of 20% piperidine DMF solution was reacted for 10 minutes, then 10 minutes each time, followed by washing six times with 2 mL of DMF, each time for 2 minutes. Relative to the resin (0.04 mmol), 6 times the stoichiometric amount of Fmoc-11-amino-3,6,9-trioxanedecanoic acid, activated with 6 times the stoichiometric amount of HBTU and HOBt in the presence of 6 times the stoichiometric amount of DIPEA, was added to the synthesis tube and reacted under electromagnetic stirring for 1 hour. This was followed by washing six times with 2 mL of DMF, each time for 2 minutes. The amino protecting group Fmoc was then removed using 3 mL of DMF solution containing 20% ​​piperidine (v / v), repeated twice, each time for 10 minutes. DOTA, with a chemical weight of 6 times that of the resin, was activated with 6 times that of HBTU and HOBt in the presence of 6 times that of DIPEA and then added to the synthesis tube. The reaction was carried out under electromagnetic stirring for 1 hour. After the reaction, the mixture was washed 6 times with 2 mL DMF for 2 minutes each time. The dissociation of the ligand from the resin and the removal of Trt were completed by stirring with 4 mL trifluoroacetic acid / DMSO (9:1, v / v) for 12 hours. The resin was then washed with 2 mL trifluoroacetic acid, and all filtrates were collected. After removing the trifluoroacetic acid under reduced pressure, the target compound H4 was obtained. The ligand structure was identified by mass spectrometry, as shown in the figure. Figure 9 As shown.

[0049] Example 2: Labeling and Quality Control

[0050] mark: 68 Ga: Add a certain amount of DMSO to the prepared ligand sample to dissolve it and dilute it to 10 μg / μL. Rinse with 2 mL of fresh solution. 68 Ga 3+ An ionic solution (0.05 mol / L dilute hydrochloric acid solution with a radioactivity of 10⁻¹⁷ mCi) was added to a vial, followed by 10 μL of ligand solution and 260 μL of NaOAc solution (1 mol / L). The solution was shaken well, sealed, and reacted at 90°C for 10 minutes. After cooling to room temperature, the reaction solution was analyzed by high-performance liquid chromatography (HPLC) for quality control.

[0051] Quality control: HPLC determination 68 The radiochemical purity of Ga-labeled molecular probes was determined using an aqueous solution containing 20% ​​acetonitrile (containing 0.1% trifluoroacetic acid) as the mobile phase. All complexes had a radiochemical purity greater than 95% and were used for further study without purification.

[0052] Example 3 Imaging of the labeled product

[0053] Take 100 μL of freshly prepared 68 Ga-labeled molecular probes (200 μCi) were injected via the tail vein into female 7-8 week old mice bearing 4T1 tumors (tumor diameter approximately 1 cm). Sixty minutes later, the mice were anesthetized with isoflurane and subjected to small animal PET / CT (SUPER-NOVA, Ping Sheng Technology, China). The regions of interest were delineated using the normalized maximum uptake value (SUVmax).

[0054] like Figure 10 As shown in Table 1, injection 68 Sixty minutes after the Ga-H1 probe was applied, significant concentrations were observed in the subcutaneous tumor areas of mice. The SUVmax ratio of tumor to muscle was 6.257 ± 1.240, the SUVmax ratio of tumor to liver was 1.003 ± 0.126, and the SUVmax ratio of tumor to kidney was 0.444 ± 0.052.

[0055] like Figure 11 As shown in Table 1, injection 68 After 60 minutes, the Ga-H2 probe showed significant accumulation in the subcutaneous tumor areas of mice. The SUVmax ratio of tumor to muscle was 8.005 ± 0.963, the SUVmax ratio of tumor to liver was 1.611 ± 0.190, and the SUVmax ratio of tumor to kidney was 0.552 ± 0.079. Compared with the other three probes, 68 Ga-H2 showed a higher tumor-to-liver ratio 60 minutes after injection.

[0056] like Figure 12 As shown in Table 1, injection 68 Sixty minutes after the Ga-H3 probe was applied, significant concentrations were observed in the subcutaneous tumor areas of mice. The SUVmax ratio of tumor to muscle was 15.738 ± 3.785, the SUVmax ratio of tumor to liver was 1.225 ± 0.213, and the SUVmax ratio of tumor to kidney was 0.371 ± 0.050.

[0057] like Figure 13 As shown in Table 1, injection 68 Thirty minutes after application of the Ga-H4 probe, significant accumulation was observed in the subcutaneous tumor areas of mice. The SUVmax ratio of tumor to muscle was 14.122 ± 3.550, the SUVmax ratio of tumor to liver was 1.307 ± 0.167, and the SUVmax ratio of tumor to kidney was 0.367 ± 0.098. Figure 14 As shown in Table 1, injection 68After 60 minutes with the Ga-H4 probe, more significant radioactive concentration was observed in the subcutaneous tumor area of ​​mice. The SUVmax ratio of tumor to muscle was 24.510 ± 9.236, the SUVmax ratio of tumor to liver was 1.554 ± 0.252, and the SUVmax ratio of tumor to kidney was 0.482 ± 0.053. Compared with the other three probes, 68 Ga-H4 showed a higher tumor-to-sarcoma ratio 60 min after injection, demonstrating its potential as a TIM-3 targeting molecular probe.

[0058] Table 1. SUVmax values ​​and ratios of the probe in tumors, muscles, liver, and kidneys (mean ± SD, n = 4)

[0059] Table 1 (continued)

[0060] The various embodiments of the present invention have been described above. These descriptions are exemplary and not exhaustive, nor are they limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments.

Claims

1. A T-cell immunoglobulin and mucin domain-3 targeting compound, characterized in that, The compound has the structure shown in Formula I: Equation I In Formula I: R1 is a chemical bond. , ,or ; R2 is .

2. The T-cell immunoglobulin and mucin domain-3 targeting compound according to claim 1, characterized in that, The T-cell immunoglobulin and mucin domain-3 targeting compound is compound H1, compound H2, compound H3, or compound H4. Compound H1; Compound H2; Compound H3; Compound H4.

3. The use of the T-cell immunoglobulin and mucin domain-3 targeting compound as described in claim 1 or 2 in the preparation of nuclear medicine diagnostic probes or immune checkpoint inhibitors.

4. A T-cell immunoglobulin and mucin domain-3 targeted imaging reagent, characterized in that, The T-cell immunoglobulin and mucin domain-3 targeted imaging reagent is a diagnostic radionuclide-labeled T-cell immunoglobulin and mucin domain-3 targeted compound as described in claim 1 or 2.

5. The T-cell immunoglobulin and mucin domain-3 targeted imaging reagent according to claim 4, characterized in that, The diagnostic radionuclide is 68 Ga、 64 Cu、 18 F, 86 Y、 90 Y、 89 Zr、 111 In、 99m Tc, 11 C 123 I, 125 I and 124 At least one of I.

6. A T-cell immunoglobulin and mucin domain-3 targeted therapeutic agent, characterized in that, The T-cell immunoglobulin and mucin domain-3 targeted therapeutic agent is a therapeutically radiolabeled T-cell immunoglobulin and mucin domain-3 targeted compound as described in claim 1 or 2.

7. The T-cell immunoglobulin and mucin domain-3 targeted imaging reagent according to claim 6, characterized in that, The therapeutic radionuclide is 177 Lu、 125 I, 131 I, 211 At、 111 In、 153 Sm、 186 Re、 188 Re、 67 Cu、 212 Pb, 225 Ac、 213 Bi、 212 Bihe 212 At least one of Pb.

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