An inhibitory radioactive probe targeting fibroblast activation protein and its preparation method and application

Abstract

The present invention relates to the field of radiopharmaceutical chemistry, and more particularly to a radioactive probe that targets fibroblast activation protein (FAP), a preparation method, and applications thereof. The structural formula of the compound targeting FAP provided by the present invention is: #imgabs0#. This compound possesses excellent radionuclide labeling properties. The resulting radioactive probe, a FAP inhibitor, can simultaneously target tumor-associated fibroblast activation protein (FAP) and bone metabolism-related targets, thereby improving diagnostic accuracy and specificity, increasing retention time and effectiveness, and enhancing treatment outcomes. This allows for more comprehensive diagnosis and evaluation of tumors and bone metastases, and is particularly suitable for patients with both primary tumors and bone metastases.

Description

Technical Field

[0001] The present invention relates to the technical field of radiopharmaceutical chemistry, and in particular to an inhibitor-type radioactive probe targeting fibroblast activation protein, and a preparation method and application thereof. Background Art

[0002] Patients with malignant tumors often have bone metastases, which can lead to bone pain, pathological fractures, spinal cord and nerve compression, and calcium and phosphate balance disorders, among other bone-related events (SREs). The incidence of SREs in lung cancer patients with bone metastases is as high as 53.4%. Without active treatment, the median survival time of these patients is only 10 months. Studies have shown that phosphate compounds can bind to the bone surface through chemical adsorption. As bone metabolism increases due to tumors, infections, etc., the bone surface area increases, and phosphate deposition on the bone surface increases. Bone imaging agents with phosphate as the targeting group have been widely used in clinical bone scans, such as [ 99m Tc]Tc-MDP, etc.

[0003] Malignant tumors encompass not only tumor cells but also the tumor microenvironment (TME). Fibroblast Activating Protein (FAP) is abundantly expressed only on the surface of activated fibroblasts within the TME and is absent from benign tumors or normal adult tissues. FAP is overexpressed in the TME of over 90% of cancers, including lung cancer, breast cancer, myeloma, gastric cancer, and brain tumors, and is rapidly and effectively internalized. FAP-targeted inhibitor radiopharmaceuticals show great promise in tumor imaging and treatment and are currently a hot topic of research.

[0004] FAP, also known as prolyl endopeptidase or serine protease, is a 97kDa type II transmembrane protein containing 760 amino acids. FAP is active only when it exists as a 170kDa dimer (FAP-FAP). FAP has two enzymatic activities: dipeptidyl peptidase activity and endopeptidase activity. Since 2014, researchers have conducted in-depth studies on the structure-activity relationship of FAP proteins and have vigorously explored FAP small molecule inhibitors (FAPI) targeting UAMC-1110: [ 68 Ga]Ga-FAPI-02, [ 68 Ga]Ga-FAPI-04, [ 68 Ga]Ga-FAPI-21, [ 68 Ga]Ga-FAPI-46 and [ 68 However, existing FAPIs have disadvantages such as insufficient retention time in tumors and low uptake in bone metastases.

[0005] Therefore, the existing technology still needs to be improved and developed. Summary of the Invention

[0006] In view of the above-mentioned deficiencies in the prior art, the purpose of the present invention is to provide an inhibitor-type radioactive probe targeting fibroblast activation protein, and its preparation method and application, aiming to solve the problems of insufficient retention time of existing FAPI in tumors and low uptake in bone metastases.

[0007] The technical solutions of the present invention are as follows:

[0008] The first aspect of the present invention provides a compound targeting fibroblast activation protein, wherein the structural formula is:

[0009]

[0010] The second aspect of the present invention provides a method for preparing a compound targeting fibroblast activation protein, wherein the synthesis route of the compound targeting fibroblast activation protein is:

[0011] The method for preparing the compound targeting fibroblast activation protein comprises the steps of:

[0012] Compound 1 is reacted with trifluoroacetic acid at a first temperature for a first predetermined time, and then reacted with DOTA-tri-tert-butyl succinimide ester at a second temperature for a second predetermined time to obtain Compound 2;

[0013] reacting the compound 2 with trifluoroacetic acid at a third temperature for a third predetermined time to obtain compound 3;

[0014] The compound 3 is reacted with trimethylsilyl bromide at a fourth temperature for a fourth predetermined time, and then reacted with trifluoroacetic acid at a fifth temperature for a fifth predetermined time to obtain the compound DFP targeting fibroblast activation protein.

[0015] The third aspect of the present invention provides a use of a compound targeting fibroblast activation protein in the preparation of an inhibitory radioactive probe targeting fibroblast activation protein.

[0016] In a fourth aspect of the present invention, there is provided an inhibitory radioactive probe targeting fibroblast activation protein, wherein the probe comprises a compound targeting fibroblast activation protein labeled with a radionuclide M, wherein the radionuclide M is 68 Ga, 177 Lu, 64 Cu or 89 Zr, the structural formula of the probe is:

[0017]

[0018] A fifth aspect of the present invention provides a method for preparing an inhibitory radioactive probe targeting fibroblast activation protein, wherein the preparation method comprises:

[0019] The compound targeting fibroblast activation protein according to claim 1 is dissolved in sodium acetate buffer, and then [ 177 Lu]LuCl3 solution, [ 68 Ga]GaCl3 solution, [ 64 Cu]CuCl2 solution or

[0020] [ 89 Zr]ZrCl3 solution, mixed and reacted to obtain an inhibitor-type radioactive probe targeting fibroblast activation protein.

[0021] A sixth aspect of the present invention provides a compound targeting fibroblast activation protein, or a radioactive probe that is an inhibitor of fibroblast activation protein, for use in preparing a preparation for tumor imaging or tumor treatment.

[0022] Beneficial effects: The compounds targeting fibroblast activation protein provided by the present invention have excellent radionuclide labeling properties. The inhibitor-type radioactive probes targeting fibroblast activation protein formed after radionuclide labeling can simultaneously target tumor-related fibroblast activation protein (FAP) and bone metabolism-related targets, which can improve the accuracy and specificity of diagnosis, increase retention time and retention effect, improve diagnosis and treatment effects, and achieve more comprehensive diagnosis and treatment evaluation of tumors and bone metastases. It is particularly suitable for patients with both primary tumors and bone metastases. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 is the mass spectrum of compound 3 prepared in Example 1.

[0024] Figure 2 This is the high-resolution mass spectrum of the compound DFP prepared in Example 1.

[0025] Figure 3 Prepared in Example 2 68 Radioactive HPLC spectrum of the Ga-DFP labeling reaction solution.

[0026] Figure 4 Prepared in Example 3 177 Radioactive HPLC spectrum of Lu-DFP labeling reaction solution.

[0027] Figure 5 For the experimental group in Example 4 68 Ga-DFP and control group68 Cellular uptake curves of Ga-FAPI-04 in U87MG cells with high FAP expression at 30 min, 60 min, and 120 min.

[0028] Figure 6 For the experimental group in Example 5 177 Lu-DFP and control group 177 Cellular uptake curves of Lu-FAPI-04 in U87MG cells with high FAP expression at 1h, 4h, and 24h.

[0029] Figure 7 This is a graph showing the binding affinity between DFP in the experimental group and DOTA-FAPI-04 and FAP in the control group in Example 6.

[0030] Figure 8 For the experimental group in Example 7 68 Ga-DFP and control group 68 SUV of the target area of ​​bone tumor in A549-FAP bone tumor mouse model after 1 hour of PET / CT with Ga-FAPI-04 max curve chart.

[0031] Figure 9 For the experimental group in Example 7 68 Ga-DFP and control group 68 Typical images of Ga-FAPI-04 uptake in A549-FAP bone tumor mouse model after 1 hour of PET / CT.

[0032] Figure 10 For the experimental group in Example 8 68 Ga-DFP and control group 68 Biodistribution results of Ga-FAPI-04 in the A549-FAP bone tumor mouse model.

[0033] Figure 11 For the experimental group in Example 9 177 Lu-DFP and control group 177 Biodistribution results of Lu-FAPI-04 in the A549-FAP bone tumor mouse model. DETAILED DESCRIPTION

[0034] The present invention provides a radioactive probe that targets fibroblast activation protein as an inhibitor, as well as its preparation method and application. To clarify the objectives, technical solutions, and effects of the present invention, the present invention is described in further detail below. It should be understood that the specific embodiments described herein are intended only to illustrate the present invention and are not intended to limit the present invention.

[0035] According to one embodiment of the present invention, a compound targeting fibroblast activation protein is provided, wherein the structural formula is:

[0036]

[0037] The compound targeting fibroblast activation protein provided in the embodiments of the present invention has a FAPI and phosphate structure and has excellent radionuclide labeling properties. The molecular probe formed after radionuclide labeling can simultaneously target tumor-related fibroblast activation protein (FAP) and bone metabolism-related targets, which can increase the retention time and uptake of bone metastases, thereby improving the diagnosis and treatment effect.

[0038] According to one embodiment of the present invention, a method for preparing a compound targeting fibroblast activation protein is provided, wherein the synthesis route of the compound targeting fibroblast activation protein is:

[0039]

[0040] The method for preparing the compound targeting fibroblast activation protein comprises the steps of:

[0041] S1, reacting compound 1 with trifluoroacetic acid at a first temperature for a first predetermined time, and then reacting with DOTA-tri-tert-butyl succinimide ester at a second temperature for a second predetermined time to obtain compound 2;

[0042] S2, reacting the compound 2 with trifluoroacetic acid at a third temperature for a third predetermined time to obtain compound 3;

[0043] S3. Reacting the compound 3 with trimethylsilyl bromide at a fourth temperature for a fourth predetermined time, and then reacting with trifluoroacetic acid at a fifth temperature for a fifth predetermined time to obtain the compound DFP targeting fibroblast activation protein.

[0044] In step S1, in one embodiment, the first temperature is 25°C-30°C, and the first predetermined time is 5h-8h.

[0045] In one embodiment, the second temperature is 25° C.-30° C., and the second predetermined time is 6 hours-9 hours.

[0046] In one embodiment, the step of reacting compound 1 with trifluoroacetic acid at a first temperature for a first predetermined time, and then reacting with DOTA-tri-tert-butyl succinimide ester at a second temperature for a second predetermined time to obtain compound 2 specifically includes:

[0047] Compound 1 was dissolved in dichloromethane, trifluoroacetic acid was added, and the mixture was reacted at room temperature for 6 h to obtain a deprotected product. The deprotected product, DOTA-tri-tert-butyl ester-succinimide ester, and N,N-diisopropylethylamine were then dissolved in N,N-dimethylformamide and the mixture was reacted at room temperature for 6 h to obtain compound 6.

[0048] In step S2, in one embodiment, the third temperature is 25°C-30°C, and the third predetermined time is 4h-6h.

[0049] In one embodiment, the step of reacting the compound 2 with trifluoroacetic acid at a third temperature for a third predetermined time to obtain the compound 3 specifically comprises:

[0050] Compound 2 was dissolved in trifluoroacetic acid and reacted at room temperature for 5 h to obtain compound 3.

[0051] In step S3, in one embodiment, the fourth temperature is 25°C-30°C, and the fourth predetermined time is 36h-40h.

[0052] In one embodiment, the fifth temperature is 25° C.-30° C., and the fifth predetermined time is 20 h-24 h.

[0053] In one embodiment, the step of reacting the compound 3 with trimethylsilyl bromide at a fourth temperature for a fourth predetermined time, and then reacting with trifluoroacetic acid at a fifth temperature for a fifth predetermined time to obtain the compound DFP targeting fibroblast activation protein specifically includes:

[0054] Compound 3 was dissolved in anhydrous acetonitrile, trimethylsilyl bromide was slowly added, and the mixture was reacted at room temperature for 36 hours, and then reacted with trifluoroacetic acid at room temperature for 24 hours to obtain the compound DFP targeting fibroblast activation protein.

[0055] According to one embodiment of the present invention, the synthetic route of compound 1 is:

[0056]

[0057] In this embodiment, the preparation method of compound A2 by reacting compound A1 includes dissolving compound A1, Trityl resin and N,N-diisopropylethylamine in DCM, and brewing the mixture with nitrogen at 25° C. for 48 hours to obtain compound A2.

[0058] In this embodiment, the preparation method of compound A3 by reacting compound A2 includes: adding piperidine / dimethylformamide solution to compound A2, bubbling the mixture with nitrogen for 20 minutes, and repeating the operation four times to obtain compound A3.

[0059] In this embodiment, the preparation method of compound A4 by reacting compound A3 includes: dissolving compound A3, succinic acid, N,N-diisopropylethylamine, 1-hydroxybenzotriazole and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride in N,N-dimethylformamide, and reacting at room temperature overnight to obtain compound A4.

[0060] In this embodiment, the preparation method of compound A6 by reacting compound A4 and compound A5 includes: dissolving compound A3, compound A4, N,N-diisopropylethylamine, 1-hydroxybenzotriazole and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride in N,N-dimethylformamide, and reacting at room temperature overnight to obtain compound A6.

[0061] In this embodiment, the preparation method of compound A7 by reacting compound A6 comprises: dissolving compound A6 in dichloromethane / trifluoroacetic acid solution, bubbling the mixture with nitrogen for 20 minutes and filtering, and repeating the operation 5 times to obtain compound A7.

[0062] In this embodiment, the preparation method of compound 1 by reacting compound A7 includes: dissolving compound A7, (aminomethylene) tetraethyl bisphosphate, N,N-diisopropylethylamine and O-(7-azabenzotriazole-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate in N,N-dimethylformamide, and reacting at room temperature overnight to obtain compound 1.

[0063] According to one embodiment of the present invention, there is provided a use of a compound targeting fibroblast activation protein in preparing an inhibitory radioactive probe targeting fibroblast activation protein.

[0064] According to one embodiment of the present invention, a radioactive probe of the inhibitor type targeting fibroblast activation protein is provided, wherein the probe comprises a compound targeting fibroblast activation protein labeled with a radionuclide M, wherein the radionuclide M is 68 Ga, 177 Lu, 64 Cu or 89 Zr, the structural formula of the probe is:

[0065]

[0066] According to a preferred embodiment of the present invention, the radionuclide M is 68 Ga or 177 Lu.

[0067] In this embodiment, when the radionuclide M is 68 When Ga, the structural formula of the probe is Name 68 Ga-DOTA-FAPI-BPs, abbreviated as 68 Ga-DFP.

[0068] In this embodiment, when the radionuclide M is 177 When Lu, the structural formula of the probe is Name 177 Lu-DOTA-FAPI-BPs, abbreviated as 177 Lu-DFP.

[0069] The embodiments of the present invention provide a novel inhibitor-type radioactive probe targeting fibroblast activation protein, specifically a type of integrated diagnostic and therapeutic molecular probe having FAPI and phosphate structures. The probe has two separate targeting structures: the FAPI group can target FAP expressed by tumor cells, and the bisphosphonate group can target the bone surface. By binding to its corresponding target through two separate targeting moieties, the total number of effective binding sites can be increased and the dissociation rate can be reduced, providing a better chance of binding to its target. It has a better retention effect than the existing FAPI, improves the uptake of bone metastases, and has important clinical value and application prospects.

[0070] The inhibitor-type radioactive probe targeting fibroblast activation protein provided in the embodiments of the present invention has the following advantages:

[0071] On the one hand, the probe structure is innovative. The heterodimer structure combines FAPI with bisphosphonates to form a bifunctional or multifunctional targeted probe. This structure can simultaneously target tumor-associated fibroblast activation protein (FAP) and targets related to bone metabolism, which can improve diagnostic accuracy and specificity, increase retention time and retention effect, enhance diagnosis and treatment effectiveness, and achieve more comprehensive diagnosis and treatment evaluation of tumors and bone metastases. It is particularly suitable for patients with both primary tumors and bone metastases.

[0072] Furthermore, the probe possesses the properties of bisphosphonates, which bind with high affinity to hydroxyapatite on the bone surface, inhibiting osteoclast activity and reducing bone resorption. This property allows the bisphosphonate-binding heterodimer probe to have enhanced targeting and retention time in bone tissue, enabling more precise localization of lesions and improving therapeutic efficacy in the diagnosis and treatment of bone metastases.

[0073] According to one embodiment of the present invention, a method for preparing an inhibitory radioactive probe targeting fibroblast activation protein is provided, wherein the preparation method comprises:

[0074] The compound targeting fibroblast activation protein was dissolved in sodium acetate buffer and then added [ 177 Lu]LuCl3 solution, [ 68 Ga]GaCl3 solution, [ 64 Cu]CuCl2 solution or [ 89 Zr]ZrCl3 solution, mixed and reacted to obtain an inhibitor-type radioactive probe targeting fibroblast activation protein.

[0075] According to one embodiment of the present invention, when the radionuclide M is 68 When Ga, the preparation method of the inhibitor radioactive probe targeting fibroblast activation protein specifically includes:

[0076] The compound targeting fibroblast activation protein was dissolved in 1N sodium acetate buffer at pH 4, and then [ 68 The mixture was stirred for 10 minutes at 95 °C and cooled to room temperature to obtain an inhibitory radioactive probe targeting fibroblast activation protein.

[0077] According to one embodiment of the present invention, when the radionuclide M is 177 When Lu, the preparation method of the inhibitor radioactive probe targeting fibroblast activation protein specifically includes:

[0078] The compound targeting fibroblast activation protein was dissolved in 3N sodium acetate buffer at pH 5.2, and then [ 177 The mixture was mixed and reacted at 95°C for 30 minutes, and then cooled to room temperature to obtain an inhibitory radioactive probe targeting fibroblast activation protein.

[0079] According to one embodiment of the present invention, the method for preparing the inhibitory radioactive probe targeting fibroblast activation protein further comprises:

[0080] After obtaining the inhibitory radioactive probe targeting fibroblast activation protein, its labeling rate was measured using high performance liquid chromatography with a radioactivity detector.

[0081] According to a preferred embodiment of the present invention, the high performance liquid chromatography with a radioactivity detector comprises:

[0082] The first mobile phase is an acetic acid solution with 0.1% trifluoroacetic acid, the second mobile phase is an aqueous solution with 0.1% trifluoroacetic acid, and the gradient elution conditions are: 100%-0% of the second mobile phase from 0 to 2 minutes, 100%-0% of the second mobile phase from 2 to 4 minutes, 100% of the first mobile phase from 4 to 9 minutes, 0%-100% of the second mobile phase from 9 to 10 minutes, and 100% of the second mobile phase from 10 to 15 minutes; the flow rate of the mobile phase is 1 mL / min.

[0083] According to one embodiment of the present invention, there is provided a use of a compound targeting fibroblast activation protein, or an inhibitor-type radioactive probe targeting fibroblast activation protein, in the preparation of a preparation for tumor imaging or tumor treatment.

[0084] The present invention will be further described below with reference to specific examples.

[0085] Example 1

[0086] This embodiment provides a compound DFP targeting fibroblast activation protein and a preparation method thereof, wherein the structural formula of DFP is:

[0087] The preparation method comprises the following steps:

[0088] (1) Synthesis of Compound A2

[0089]

[0090] To a solution of Trityl resin (0.4 g, 7 mmol) in DCM (20 mL) were added compound A1 (4 g, 9 mmol) and N,N-diisopropylethylamine (DIPEA, 3 g, 23 mmol), and the mixture was bubbled with nitrogen at 25° C. for 48 h. The resin was then washed with dichloromethane (50 mL×5), methanol (50 mL×5), and dimethylformamide (20 mL×5), and the crude product compound A2 in the resin was used directly in the next step without further purification.

[0091] (2) Synthesis of Compound A3

[0092]

[0093] To the resin obtained in the previous step, piperidine / dimethylformamide solution (v / v = 1:5, 100 mL) was added to remove the Fmoc group. The mixture was sparged with nitrogen for 20 min, and the Fmoc group removal procedure was repeated four times. The resin was then washed with dimethylformamide (50 mL x 5), and the crude product, Compound A3 (1.5 g, 6 mmol), was used directly in the next step without further purification.

[0094] (3) Synthesis of Compound A4

[0095]

[0096] Under argon protection and an ice-water bath, compound A3 (1.5 g, 6 mmol), succinic acid (0.8 g, 7 mmol), N,N-diisopropylethylamine (DIPEA, 2.5 g, 21 mmol), 1-hydroxybenzotriazole (HOBt, 1.35 g, 10 mmol), and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC, 1.5 g, 10 mmol) were dissolved in N,N-dimethylformamide (DMF, 30 mL) in a 50 mL round-bottom flask. After reacting overnight at room temperature, saturated sodium chloride solution (10 mL) was added in an ice-water bath. The reaction solution was extracted with ethyl acetate (30 mL) and water (15 mL x 2). The organic phases were combined, dried over anhydrous magnesium sulfate, and filtered. The filtrate was evaporated under reduced pressure to remove the solvent, and compound A4 was obtained without further purification.

[0097] (4) Synthesis of Compound A6

[0098]

[0099] Under argon protection and an ice-water bath, compound A4 (1 g, 1.6 mmol), compound A5 (0.78 g, 1.6 mmol), N,N-diisopropylethylamine (DIPEA, 0.5 g, 4 mmol), 1-hydroxybenzotriazole (HOBt, 0.32 g, 2.4 mmol), and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC, 0.37 g, 4.2 mmol) were dissolved in N,N-dimethylformamide (DMF, 15 mL) in a 50 mL round-bottom flask. After reacting overnight at room temperature, saturated sodium chloride solution (10 mL) was added in an ice-water bath. The reaction solution was extracted with ethyl acetate (30 mL) and water (15 mL x 2). The organic phases were combined, dried over anhydrous magnesium sulfate, and filtered. The filtrate was evaporated under reduced pressure to remove the solvent, yielding compound A6 (1.8 g, 100% yield) without further purification.

[0100] (5) Synthesis of Compound A7

[0101]

[0102] Compound A6 was dissolved in dichloromethane / trifluoroacetic acid solution (v / v=100:1, 600 mL) for cleavage, and the mixture was bubbled with nitrogen for 20 min and filtered. The operation was repeated 5 times. After cleavage and filtration, the filtrate was adjusted to pH=7 with NaHCO3 solution and extracted between dichloromethane (100 mL×3) and water (100 mL×3) to obtain an organic phase. The combined organic phases were then washed with citric acid (100 mL×3) and the mixture was finally concentrated under high vacuum to obtain compound A7 (0.4 g, 0.5 mmol) as a white solid. MS calcd.for C 39 H 52 F2N8O9, [M+H]+815.4, found 815.2.

[0103] (6) Synthesis of Compound 1

[0104]

[0105] Under argon protection and an ice-water bath, compound A7 (0.4 g, 0.5 mmol), tetraethyl (aminomethylene)bisphosphate (0.45 g, 1.5 mmol), N,N-diisopropylethylamine (DIPEA, 0.9 g, 3 mmol), and O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU, 1.1 g, 3 mmol) were dissolved in N,N-dimethylformamide (DMF, 15 mL) in a 50 mL round-bottom flask and reacted overnight at room temperature. Saturated sodium chloride solution (10 mL) was then added under an ice-water bath, and the reaction solution was extracted with ethyl acetate (30 mL) and water (15 mL x 2). The organic phases were combined, dried over anhydrous magnesium sulfate, and filtered. The filtrate was evaporated under reduced pressure to remove the solvent, and the residue was purified by flash chromatography using the mobile phase of dichloromethane / methanol / aqueous ammonia = 95 / 5 / 1 (v / v / v) to obtain compound 1 (0.4 g, 72% yield) as a colorless oil. 48 H 73 F2N9O 14 P2, [M+H]+1100.5, found 1100.4.

[0106] (7) Synthesis of Compound 2

[0107]

[0108] Under argon protection and an ice-water bath, compound 1 (0.4 g, 0.36 mmol) was dissolved in dichloromethane in a 50 mL round-bottom flask. 1 mL of trifluoroacetic acid was added and the mixture was allowed to react at room temperature for 6 h. The solvent was then removed by rotary evaporation under reduced pressure to obtain the deprotected product, which was then carried out without further purification. The deprotected product, DOTA-tris(tBu)ester NHS ester (0.24 g, 0.36 mmol) and N,N-diisopropylethylamine (DIPEA, 0.36 g, 1.2 mmol), were dissolved in N,N-dimethylformamide (DMF, 15 mL) and allowed to react at room temperature for 6 h. Saturated sodium chloride solution (10 mL) was then added under an ice-water bath, and the reaction solution was extracted with ethyl acetate (30 mL) and water (15 mL x 2). The organic phases were combined, dried over anhydrous magnesium sulfate, and filtered. The filtrate was evaporated under reduced pressure to remove the solvent, and the residue was purified by flash chromatography using the mobile phase of dichloromethane / methanol / aqueous ammonia = 90 / 9 / 1 (v / v / v) to obtain compound 2 (0.31 g, 55% yield) as a colorless oil. 71 H 115 F2N 13 O 19 P2, [M+H]+1554.8, found 1554.7.

[0109] (8) Synthesis of Compound 3

[0110]

[0111] In a 10 mL round-bottom flask, compound 2 (0.31 g, 0.2 mmol) was dissolved in 1 mL trifluoroacetic acid (TFA) and reacted at room temperature for 5 h. The reaction solution was evaporated under reduced pressure to remove the solvent, and the residue was dissolved in 1 mL DMSO solution and separated using semi-HPLC to obtain compound 3 (0.17 g, yield 61%) as a white solid. 59 H 91 F2N 13 O 19 P2, [M+H]+1386.6, found 1385.9. Figure 1 is the mass spectrum of the prepared compound 3.

[0112] (9) Synthesis of compound DFP

[0113]

[0114] Under argon protection and ice-water bath conditions, compound 3 (0.3 g, 0.2 mmol) was dissolved in 5 mL of anhydrous acetonitrile in a 50 mL round-bottom flask, and trimethylsilyl bromide (TMSBr, 91 mg, 0.6 mmol) was slowly added. After reacting at room temperature for 36 h, 20 mL of methanol was added, and the reaction solution was evaporated to remove the solvent under reduced pressure. The residue was then reacted with 1 mL of trifluoroacetic acid at room temperature for 24 h. The reaction solution was evaporated to remove the solvent under reduced pressure and separated using semi-HPLC to obtain the compound DFP targeting fibroblast activation protein (0.19 g, yield 75%) as a white oil. HRMS calcd. for C 51 H 75 F2N 13 O 19 P2, [M+H]+1274.4824, found 1274.4841. Figure 2 This is the high-resolution mass spectrum of the prepared compound DFP.

[0115] Example 2

[0116] This embodiment provides an inhibitory radioactive probe targeting fibroblast activation protein 68 Ga-DFP and its preparation method, wherein 68 The structural formula of Ga-DFP is:

[0117] The preparation method comprises the following steps:

[0118] 30 micrograms of DFP prepared in Example 1 was dissolved in 900 μL of 1N sodium acetate buffer solution with pH=4 to obtain a precursor sodium acetate buffer solution. The germanium gallium generator was rinsed with 0.6N hydrochloric acid solution. 68 1.5 ml of Ga]GaCl3 was added to the precursor sodium acetate buffer, mixed evenly, reacted at 95°C for 10 minutes, and cooled to room temperature. The labeling rate was determined using high performance liquid chromatography (radio-HPLC) with a radioactivity detector, and the radiochemical yield was >95%. 68 Ga-DFP. Figure 3 It is prepared 68 Radioactive HPLC spectrum of the Ga-DFP labeling reaction solution.

[0119] In the above-mentioned radio-HPLC determination, the first mobile phase was an acetic acid solution with 0.1% trifluoroacetic acid, the second mobile phase was an aqueous solution with 0.1% trifluoroacetic acid, and the gradient elution conditions were: 100%-0% of the second mobile phase from 0 to 2 minutes, 100%-0% of the second mobile phase from 2 to 4 minutes, 100% of the first mobile phase from 4 to 9 minutes, 0%-100% of the second mobile phase from 9 to 10 minutes, and 100% of the second mobile phase from 10 to 15 minutes; the flow rate of the mobile phase was 1 mL / min.

[0120] Example 3

[0121] This embodiment provides an inhibitory radioactive probe targeting fibroblast activation protein 177 Lu-DFP and its preparation method, wherein 177 The structural formula of Lu-DFP is:

[0122] The preparation method comprises the following steps:

[0123] 50 μg of DFP prepared in Example 1 was dissolved in 30 μL of 3N sodium acetate buffer solution with pH=5.2 to obtain precursor sodium acetate buffer solution, and then 200 μL of [ 177 Lu]LuCl3 solution, uniformly mixed, reacted at 95 ° C for 30 minutes, and cooled to room temperature. The labeling rate was determined using high performance liquid chromatography (radio-HPLC) with a radioactive detector, and the radiochemical yield was >95%. 177 Lu-DFP. Figure 4 It is prepared 177 Radioactive HPLC spectrum of Lu-DFP labeling reaction solution.

[0124] In the above-mentioned radio-HPLC determination, the first mobile phase was an acetic acid solution with 0.1% trifluoroacetic acid, the second mobile phase was an aqueous solution with 0.1% trifluoroacetic acid, and the gradient elution conditions were: 100%-0% of the second mobile phase from 0 to 2 minutes, 100%-0% of the second mobile phase from 2 to 4 minutes, 100% of the first mobile phase from 4 to 9 minutes, 0%-100% of the second mobile phase from 9 to 10 minutes, and 100% of the second mobile phase from 10 to 15 minutes; the flow rate of the mobile phase was 1 mL / min.

[0125] Example 4

[0126] This example explores 68 The cellular uptake experiment of Ga-DFP in U87MG cells is as follows:

[0127] (1) Experimental group: U87MG cells in the logarithmic growth phase were taken and cultured at a concentration of approximately 2×105 Cells / well were seeded in a 12-well plate, and the labeled 68 The Ga-DFP solution was diluted with MEM solution, and about 0.5 uCi of 68 Mix Ga-DFP with 1 mL of MEM solution.

[0128] Control group: The operation is the same as that of the experimental group, except that 68 Ga-DFP is replaced by 68 Ga-FAPI-04, where 68 The structural formula of Ga-FAPI-04 is:

[0129]

[0130] (2) The experimental and control groups were placed in a CO2 incubator (37°C) and incubated for 30 min, 60 min, and 120 min, and then rinsed with PBS buffer.

[0131] (3) The cells in the experimental and control groups were lysed with NaOH solution for 5 min, the lysates were collected, and the radioactivity counts were measured.

[0132] Figure 5 The experimental group 68 Ga-DFP and control group 68 The cell uptake curve of Ga-FAPI-04 in U87MG cells with high FAP expression at 30min, 60min and 120min. 68 Ga-DFP was rapidly taken up by U87MG cells and showed an increasing trend, reaching 2.418±0.35%ID / million cells at 60 min and 3.635±1.33%ID / million cells at 120 min, both higher than 68 Ga-FAPI-04, which shows that the present invention 68 Ga-DFP has a relatively high uptake in U87MG cells.

[0133] Example 5

[0134] This example explores 177 The cellular uptake experiment of Lu-DFP in U87MG cells is as follows:

[0135] (1) Experimental group: U87MG cells in the logarithmic growth phase were taken and cultured at a concentration of approximately 2×10 5 Cells / well were seeded in a 12-well plate, and the labeled 177 Lu-DFP solution was diluted with MEM solution, and about 0.5 uCi of 177Lu-DFP was mixed with 1 mL of MEM solution.

[0136] Control group: The operation is the same as that of the experimental group, except that 177 Lu-DFP is replaced by 177 Lu-FAPI-04, which 177 The structural formula of Lu-FAPI-04 is:

[0137]

[0138] (2) The experimental and control groups were placed in a CO2 incubator (37°C) and incubated for 1 h, 4 h, and 24 h, and then rinsed with PBS buffer.

[0139] (3) The cells in the experimental and control groups were lysed with NaOH solution for 5 min, the lysates were collected, and the radioactivity counts were measured.

[0140] Figure 6 The experimental group 177 Lu-DFP and control group 177 Cell uptake curves of Lu-FAPI-04 in U87MG cells with high FAP expression at 1h, 4h and 24h. 177 Lu-DFP was rapidly absorbed in U87MG cells and showed an increasing trend, reaching 2.036±0.03% ID / million cells at 4 h and 8.976±1.39% ID / million cells at 24 h, both higher than 177 Lu-FAPI-04, indicating 177 Lu-DFP has a relatively high uptake in U87MG cells.

[0141] Example 6

[0142] This example explores 177 The competitive binding experiment of Lu-DFP is as follows:

[0143] (1) Experimental group: A549-FAP cells in the logarithmic growth phase were cultured at 1×10 5 Cells / well were seeded in 24-well plates, and different gradients (10 -5 , 10 -6 , 10 -7 , 10 -8 , 10 -9 , 10 -10 , 10 -11 , 10 -12 ) after dilution 177 Lu-FAPI-04+DFP (DFP is 177Lu-DFP probe precursor, not labeled with radioactive elements, can be used as 177 Lu-FAPI-04 inhibitor), 0.5 mL of DMEM solution (0.5 uCi) was added to each well.

[0144] Control group: The operation is the same as that of the experimental group, except that 177 Lu-FAPI-04+DFP is replaced by 177 Lu-FAPI-04+DOTA-FAPI-04 (DOTA-FAPI-04 is 177 Lu-FAPI-04 probe precursor, not labeled with radioactive elements, can be used as 177 inhibitor of Lu-FAPI-04).

[0145] (2) The experimental and control groups were placed in a CO2 incubator (37°C) and incubated for 60 min. The solution was removed and the cells were rinsed with PBS. The cells were then lysed with NaOH solution for 5 min. The lysates were collected and the IC50 values ​​were measured.

[0146] Figure 7 The binding affinity curves of DFP in the experimental group and DOTA-FAPI-04 in the control group to FAP are shown in the figure. 50 The value was 16.76 ± 3.3 nmol, and the IC of DOTA-FAPI-04 50 The value was 6.09 ± 3.5 nmol, both of which were in the nanomolar level, indicating that DFP had a similar FAP binding affinity as DOTA-FAPI-04.

[0147] Example 7

[0148] This example explores 68 The micro PET / CT imaging experiment of Ga-DFP in bone tumor model mice is as follows:

[0149] (1) Establishment of osteoma animal model: A549-FAP cells expressing positive FAP receptors in the logarithmic growth phase were digested and prepared into PBS cell suspension. The cell suspension (about 1×10 6 Cells were injected into the left tibia of 4-5 week old male SCID mice and observed for 30-60 days.

[0150] (2) Experimental group: The labeling prepared in Example 2 was completed. 68 After diluting the Ga-DFP solution with water, take about 200uCi of 68 Ga-DFP solution was injected into mice via the tail vein, and dynamic imaging was performed by micro PET / CT for 1 h.

[0151] Control group: The operation is the same as that of the experimental group, except that 68 Ga-DFP is replaced by 68 Ga-FAPI-04.

[0152] Figure 8 The experimental group 68 Ga-DFP and control group 68 SUV of the target area of ​​bone tumor in A549-FAP bone tumor mouse model after 1 hour of PET / CT with Ga-FAPI-04 max curve chart. Figure 9 The experimental group 68 Ga-DFP and control group 68 Typical images of PET / CT bone tumor uptake of Ga-FAPI-04 in A549-FAP bone tumor mouse model for 1 hour. 68 Ga-DFP has a high specific uptake in bone tumors, and the uptake is relatively stable within 1 hour. 68 Ga-FAPI-04 uptake showed a significant downward trend, and at 60 min 68 Ga-DFP uptake was higher than 68 Ga-FAPI-04.

[0153] Example 8

[0154] This example explores 68 The biodistribution experiment of Ga-DFP in osteoma model mice is as follows:

[0155] (1) Establishment of osteoma animal model: A549-FAP cells expressing positive FAP receptors in the logarithmic growth phase were digested and prepared into PBS cell suspension. The cell suspension (about 1×10 6 Cells were injected into the left tibia of 4-5 week old male SCID mice and cultured and observed for 30-60 days.

[0156] (2) Experimental group: The labeling prepared in Example 2 was completed. 68 The Ga-DFP solution was diluted with water and SCID osteoma model mice (4 mice) were injected with 30uCi of 68 Ga-DFP, 68 After Ga-DFP was metabolized in the body for 2 h, the mice were killed by cervical dislocation, and the tissues of interest were taken to measure the radioactivity count.

[0157] Control group: The operation is the same as that of the experimental group, except that 68 Ga-DFP is replaced by 68 Ga-FAPI-04.

[0158] Figure 10 The experimental group68 Ga-DFP and control group 68 The biodistribution results of Ga-FAPI-04 in A549-FAP bone tumor mouse model. 68 The uptake of Ga-DFP in normal organs of osteoma model mice was low, but its uptake in osteoma (tibia) was high, reaching 4.338±0.28%ID / g at 2 hours.

[0159] Example 9

[0160] This example explores 177 The biodistribution experiment of Lu-DFP in osteoma model mice is as follows:

[0161] (1) Establishment of osteoma animal model: A549-FAP cells expressing positive FAP receptors in the logarithmic growth phase were digested and prepared into PBS cell suspension. The cell suspension (about 1×10 6 Cells were injected into the left tibia of 4-5 week old male SCID mice and cultured and observed for 30-60 days.

[0162] (2) Experimental group: The labeling prepared in Example 3 was completed. 177 Lu-DFP solution was diluted with water, and SCID osteoma model mice (4 mice) were injected with 30uCi of Lu-DFP via the tail vein. 177 Lu-DFP, 177 After 24 h of Lu-DFP metabolism in the body, the mice were killed by cervical dislocation, and the tissues of interest were taken to measure the radioactivity count.

[0163] Control group: The operation is the same as that of the experimental group, except that 177 Lu-DFP is replaced by 177 Lu-FAPI-04.

[0164] Figure 11 The experimental group 177 Lu-DFP and control group 177 The biodistribution results of Lu-FAPI-04 in A549-FAP bone tumor mouse model showed that 177 The uptake of Lu-DFP in normal organs of osteoma model mice was low, but it was higher in osteoma (tibia), showing a continuous increasing trend. The uptake value in 24 hours was 17.13±1.15% ID / g.

[0165] In summary, the compound targeting fibroblast activation protein of the present invention has excellent radionuclide labeling properties, is rapidly and efficiently prepared by radioactivity, and the resulting molecular probe has high stability. The molecular probe comprises a FAPI group, which has good FAP affinity, and further comprises a bisphosphonate group, which can bind with high affinity to hydroxyapatite on the bone surface. Therefore, the inhibitor radioactive probe targeting fibroblast activation protein (FAP) of the present invention is a bifunctional targeting probe, which not only has good affinity for tumors expressing fibroblast activation protein, high tumor uptake value and long tumor retention time, but also has higher targeting and retention time in bone tissue, thereby improving the diagnosis and treatment effect. The inhibitor radioactive probe targeting fibroblast activation protein (FAP) of the present invention can be used in tumor imaging or tumor treatment.

[0166] It should be understood that the application of the present invention is not limited to the above examples. For those skilled in the art, improvements or changes can be made based on the above description. All these improvements and changes should fall within the scope of protection of the claims attached to the present invention.

Claims

1. A compound targeting fibroblast activation protein, characterized in that The structural formula is:

2. A method for preparing a compound targeting fibroblast activation protein according to claim 1, characterized in that: The synthetic route of the compound targeting fibroblast activation protein is: The preparation method of the compound targeting fibroblast activation protein comprises the steps of: Compound 1 is reacted with trifluoroacetic acid at a first temperature for a first predetermined time, and then reacted with DOTA-tri-tert-butyl succinimide ester at a second temperature for a second predetermined time to obtain Compound 2; reacting the compound 2 with trifluoroacetic acid at a third temperature for a third predetermined time to obtain compound 3; The compound 3 is reacted with trimethylsilyl bromide at a fourth temperature for a fourth predetermined time, and then reacted with trifluoroacetic acid at a fifth temperature for a fifth predetermined time to obtain the compound DFP targeting fibroblast activation protein.

3. The method for preparing a compound targeting fibroblast activation protein according to claim 2, characterized in that: The first temperature is 25°C-30°C, and the first predetermined time is 5 hours-8 hours; the second temperature is 25°C-30°C, and the second predetermined time is 6 hours-9 hours; the third temperature is 25°C-30°C, and the third predetermined time is 4 hours-6 hours.

4. The method for preparing a compound targeting fibroblast activation protein according to claim 2, characterized in that: The fourth temperature is 25° C.-30° C., and the fourth predetermined time is 36 hours-40 hours; the fifth temperature is 25° C.-30° C., and the fifth predetermined time is 20 hours-24 hours.

5. Use of the compound targeting fibroblast activation protein according to claim 1 in preparing inhibitory radioactive probes targeting fibroblast activation protein.

6. An inhibitory radioactive probe targeting fibroblast activation protein, characterized in that: Contains the compound according to claim 1 labeled with a radionuclide M, wherein the radionuclide M is 68 Ga, 177 Lu, 64 Cu or 89 Zr, the structural formula of the probe is:

7. A method for preparing an inhibitory radioactive probe targeting fibroblast activation protein according to claim 6, characterized in that: The preparation method comprises: The compound targeting fibroblast activation protein according to claim 1 is dissolved in sodium acetate buffer, and then [ 177 Lu]LuCl3 solution, [ 68 Ga]GaCl3 solution, [ 64 Cu]CuCl2 solution or [ 89 Zr]ZrCl3 solution, mixed and reacted to obtain an inhibitor-type radioactive probe targeting fibroblast activation protein.

8. The method for preparing the inhibitory radioactive probe targeting fibroblast activation protein according to claim 7, characterized in that: When the radionuclide M is 68 When Ga, the preparation method specifically includes: The compound targeting fibroblast activation protein according to claim 1 is dissolved in 1N sodium acetate buffer at pH = 4, and then [ 68 The mixture was stirred for 10 minutes at 95 °C and cooled to room temperature to obtain an inhibitory radioactive probe targeting fibroblast activation protein.

9. The method for preparing the inhibitory radioactive probe targeting fibroblast activation protein according to claim 7, characterized in that: When the radionuclide M is 177 When Lu, the preparation method specifically comprises: The compound targeting fibroblast activation protein according to claim 1 is dissolved in 3N sodium acetate buffer at pH 5.2, and then [ 177 The mixture was mixed and reacted at 95°C for 30 minutes, and then cooled to room temperature to obtain an inhibitory radioactive probe targeting fibroblast activation protein.

10. Use of the compound targeting fibroblast activation protein according to claim 1, or the inhibitor-type radioactive probe targeting fibroblast activation protein according to claim 6, in the preparation of a preparation for tumor imaging or tumor treatment.

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