A fibroblast activation protein FAP and integrin α v β3 dual-targeting compound and its preparation method and application

Abstract

The present invention relates to a method for targeting FAP and integrin α v The dual targeting compound of β3 has a structure as shown in the following formula (I) or formula (II), wherein R1, R2, R3 and R4 are the same or different and are independently selected from H or F; A is a v β3 specific binding ligand structure; Z1 is Z, Q, V and U are the same or different connecting structures, independently selected from NH-, or based on -(CH2) n The targeted compound of the present invention can synergistically target FAP targets and integrin α in tumors. v β3 target, which can increase the number and utilization efficiency of effective receptors in tumors. The present invention also provides radionuclide labeled compounds based on the targeting compounds, and their preparation methods and methods for diagnosing or treating tumors with fibroblast activation protein (FAP) and / or integrin α v Application in diseases characterized by β3 overexpression.

Description

Technical Field

[0001] The present invention relates to the field of nuclear medicine and molecular imaging, and in particular to a compound, a pharmaceutical composition comprising or consisting of the compound, a kit comprising or consisting of the compound or pharmaceutical composition, and the use of the compound or pharmaceutical composition in diagnosing or treating diseases caused by fibroblast activation protein (FAP) and / or integrin α v Use in diseases characterized by overexpression of β3. Background Art

[0002] Fibroblast activation protein (FAP) is a membrane serine peptidase expressed on the surface of activated fibroblasts in the tumor stroma and plays an important role in the occurrence and development of tumors. Previous studies have shown that FAP is generally not expressed in normal human tissues, but is selectively and highly expressed on the surface of stromal fibroblasts in more than 90% of epithelial malignancies, including breast cancer, ovarian cancer, lung cancer, colorectal cancer, gastric cancer, and pancreatic cancer. Given its widespread expression and important role in tumors, FAP has become an important target for tumor imaging and treatment. Radionuclide-labeled fibroblast activation protein inhibitors (FAPI), represented by quinolinic acid derivatives, have made important progress in the field of precise tumor imaging. For example, PET / CT imaging agents such as FAPI-02 and FAPI-04 have achieved specific imaging of more than 30 different types of tumors.

[0003] Integrin α v β3 (integrin α v β3) is a heterodimeric receptor located on the cell surface. It is rarely expressed in normal vascular endothelial and epithelial cells, but is highly expressed on the cell surface of various solid tumors such as lung cancer, osteosarcoma, neuroblastoma, breast cancer, prostate cancer, bladder cancer, glioblastoma and invasive melanoma. It is also highly expressed on the membrane of new blood vessels in all tumor tissues, suggesting that integrin α v β3 plays a key role in tumor growth, invasion and metastasis. Peptides containing the arginine-glycine-aspartic acid (RGD) sequence can bind to integrin α v β3 specific binding. Various radionuclide-labeled RGD peptides have been successfully used in imaging studies of various tumor-bearing animal models. 18 F-Galacto-RGD has become the first non-invasive integrin α to enter clinical trials v β3-targeted tumor imaging agents have been successfully used in PET diagnosis of tumor patients and have shown good biological distribution and specific target recognition in clinical trials of glioblastoma.

[0004] Considering the heterogeneity of tumors, it is necessary to develop a new approach targeting FAP and integrin α in order to further improve the efficiency of tumor diagnosis and treatment. v Drugs that can target both β3 targets. Summary of the Invention

[0005] In view of the above background, the primary purpose of the present invention is to develop a new compound structure that can synergistically target FAP targets and integrin α in tumors. v The β3 target can increase the number and utilization efficiency of effective receptors in tumors, thereby improving tumor uptake efficiency and positive tumor detection efficiency and / or treatment efficiency.

[0006] Another object of the present invention is to provide a method for preparing the novel compound, so as to synthesize the novel compound that can synergistically target the FAP target and integrin α in tumors through a convenient and readily available synthetic route. v Compounds that target β3.

[0007] Another object of the present invention is to provide the compound for diagnosis or treatment of fibroblast activation protein (FAP) and / or integrin α v Application in diseases characterized by β3 overexpression.

[0008] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0009] In the first aspect, the present invention provides a method for targeting FAP and integrin α v β3 targeting compounds, whose structures contain both FAP and integrin α v The specific binding ligand structure of β3 is recorded as FAPI-RGD structure in the present invention. The compound structure is shown in the following formula (I) or formula (II):

[0010]

[0011] in:

[0012] R1, R2, R3 and R4 are the same or different and are independently selected from H or F;

[0013] Z, Q, V and U are the same or different linking structures, independently selected from -NH-, Or based on -(CH2) n -Substitution structure; Z1 is When Z, Q, V and U are based on -(CH2) n-replacement structure, wherein n is an integer from 0 to 30, wherein each -CH2- is individually replaced with or without -O-, -NH-, -(CO)-, -NH-(CO)-, -CH(NH2)- or -(CO)-NH-, provided that no two adjacent -CH2- groups are replaced.

[0014] A is associated with integrin α v The ligand structure that specifically binds to β3 is selected from:

[0015] or

[0016] R5 in the formula (III) is selected from H or OH;

[0017] R5 and R6 in the formula (IV) are the same or different and are independently selected from H or OH; M and P are based on -(CH2) n -substituted structure, wherein n is an integer from 0 to 30, wherein each -CH2- is individually replaced with or without -O-, -NH-, -(CO)-, -NH-(CO)-, -CH(NH2)- or -(CO)-NH-, provided that no two adjacent -CH2- groups are replaced; G is selected from or

[0018] In a preferred embodiment of the present invention, Z in the formula (I) or (II) is -NH-CH2-(CH2-O-CH2)2-CH2-(CO)-, -NH-CH2-(CH2-O-CH2)3-CH2-(CO)-, -NH-CH2-(CH2-O-CH2)4-CH2-(CO)-, or -(CH2)0-; Q is V is -NH-CH2-(CH2-O-CH2)2-CH2-(CO)-, -NH-CH2-(CH2-O-CH2)3-CH2-(CO)-, -NH-CH2-(CH2-O-CH2)4-CH2-(CO)- or -(CH2)O-; U is -NH- or Z1 in the formula (II) is

[0019] Based on the targeting compound of the present invention, the present invention further provides a compound that can be labeled with a radionuclide, which is composed of an amino group in any structure of Z, Q or V in formula (I) or formula (II) connected to a radionuclide chelating group, and its general formula is shown in the following formula (V) or (VI):

[0020]

[0021] Wherein, W is a fragment with a nuclide chelating group, which is derived from any one of 1,4,7,10-tetraazacyclododecane-N,N',N,N'-tetraacetic acid (DOTA), ethylenediaminetetraacetic acid (EDTA), 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), triethylenetetramine (TETA), iminodiacetic acid, diethylenetriamine-N,N,N',N',N"-pentaacetic acid (DTPA), bis-(carboxymethylimidazole)glycine or 6-hydrazinopyridine-3-carboxylic acid (HYNIC), or any of the following structures:

[0022]

[0023] Where D is based on -(CH2) p -substituted structure, wherein p is an integer from 0 to 30, wherein each -CH2- is individually replaced with or without -O-, -NH-, -(CO)-, -NH-(CO)-, -CH(NH2)- or -(CO)-NH-, provided that no two adjacent -CH2- groups are replaced.

[0024] Furthermore, the present invention also provides a radionuclide-labeled targeting compound, which is a compound in which the W group in the compound represented by formula (V) or formula (VI) is chelated with a radionuclide.

[0025] In the solution of the present invention, the radioactive nuclide can be selected from isotopes that emit α rays, isotopes that emit β rays, isotopes that emit γ rays, isotopes that emit Auger electrons, or isotopes that emit X rays, for example 18 F. 51 Cr, 67 Ga, 68 Ga, 111 In, 99m Tc, 186 Re、 188 Re、 139 La, 140 La, 175 Yb, 153 Sm, 166 Ho, 86 Y. 90 Y. 149 Pm, 165 Dy, 169 Second, 177 Lu, 47 Sc, 142 Pr, 159 Gd, 212 Bi, 213 Bi,72 As、 72 Se, 97 Such as 109 Pd, 105 Rh, 101m Rh, 119 Sb, 128 Ba, 123 I. 124 I. 131 I. 197 Hg, 211 At 151 Eu, 153 Eu, 169 Eu, 201 Tl, 203 Pb, 212 Pb, 64 Cu, 67 Cu, 198 Au, 225 Ac, 227 Th, 89 Zr or 199 Any one of Ag; more preferably the radioactive nucleus is 18 F. 64 Cu, 68 Ga, 89 Zr, 90 Y. 111 In, 99m Tc, 177 Lu, 188 Re or 225 Ac.

[0026] The present invention also provides the targeting compound, the compound that can be labeled with a radionuclide, or the pharmaceutically acceptable tautomer, racemate, hydrate, solvate or salt of the targeting compound labeled with a radionuclide.

[0027] In a second aspect, the present invention provides a method for preparing a partially targeted compound represented by formula (V) and a radionuclide-labeled compound thereof, comprising:

[0028] The carboxyl group of 6-hydroxyquinoline-4-carboxylic acid first undergoes an amide condensation reaction with the amino group of glycine tert-butyl ester. A Boc-protected piperazine group is then attached to the hydroxyl position of the amide condensation product via an alkyl chain. The Boc and tert-butyl protecting groups are removed under acidic conditions, and a Boc protecting group is introduced into the piperazine ring. This is followed by an amide condensation reaction with (S)-pyrrolidine-2-carbonitrile hydrochloride or (S)-4,4-difluoropyrrolidine-2-carbonitrile hydrochloride. After removal of the Boc protecting group, the product undergoes a condensation reaction with N-Boc-3-[2-(2-aminoethoxy)ethoxy]propionic acid. The Boc protecting group is then removed, and the product reacts sequentially with propionic acid maleimide, protected cysteine, or protected glutamic acid or lysine. Finally, RGD (c(RGDyK), c(RGDfK), or c(RGDyK) / c(RGDfK) with a short PEG chain) is introduced via an activated ester reaction to yield a dual-targeting compound.

[0029] ② reacting the dual-targeting compound obtained in ① with a radionuclide chelator, wherein the radionuclide chelator is selected from any one of hydroxysuccinimide-tetraazacyclododecane-N,N',N,N'-tetraacetic acid (DOTA-NHS), NOTA-succinimide ester (NOTA-NHS), succinimide active ester of iminodiacetic acid, succinimide active ester of diethylenetriamine-N,N,N',N',N"-pentaacetic acid (DTPA-NHS), bis-(carboxymethylimidazole)glycine or succinimide active ester of 6-hydrazinopyridine-3-carboxylic acid (HYNIC-NHS), to obtain a portion of the compound represented by formula (V) that can be labeled with a radionuclide;

[0030] ③ The radionuclide-labeled compound obtained in ② is reacted with a compound containing a radionuclide according to an existing wet labeling method or a freeze-drying labeling method to prepare a radionuclide-labeled targeting compound of the present invention.

[0031] In a third aspect, the present invention provides a pharmaceutical composition, which comprises the targeting compound described in the first aspect of the present invention, the compound that can be labeled with a radionuclide, the radionuclide-labeled compound, or any pharmaceutically acceptable tautomers, racemates, hydrates, solvates or salts thereof; or is composed of the targeting compound described in the first aspect of the present invention, the compound that can be labeled with a radionuclide, the radionuclide-labeled compound, or any pharmaceutically acceptable tautomers, racemates, hydrates, solvates or salts thereof and any pharmaceutically acceptable carriers and / or excipients.

[0032] In a fourth aspect, the present invention provides a method for preparing a targeted compound, a compound that can be labeled with a radionuclide, a compound labeled with a radionuclide, or a pharmaceutical composition according to the first aspect for use in the diagnosis or treatment of a fibroblast activation protein (FAP) and / or integrin α-positive disease in an animal or human individual. v Application of drugs in diseases characterized by β3 overexpression.

[0033] In the application of the present invention, the fibroblast activation protein (FAP) and / or integrin α v Diseases characterized by overexpression of β3 include, but are not limited to, cancer, chronic inflammation, atherosclerosis, fibrosis, tissue remodeling, and scarring; preferably, the cancer is further selected from breast cancer, pancreatic cancer, small intestine cancer, colon cancer, rectal cancer, lung cancer, head and neck cancer, ovarian cancer, hepatocellular carcinoma, esophageal cancer, hypopharyngeal cancer, nasopharyngeal cancer, laryngeal cancer, myeloma cells, bladder cancer, bile duct cell carcinoma, clear cell renal carcinoma, neuroendocrine tumors, carcinogenic osteomalacia, sarcoma, CUP (cancer of unknown primary), thymic cancer, glioma, glioma, astrocytoma, cervical cancer, or prostate cancer.

[0034] In a fifth aspect, the present invention also provides a kit comprising or consisting of a targeted compound represented by formula (I) or (II) of the present invention, a compound represented by formula (V) or (VI), a radionuclide-labeled targeted compound of the present invention, or a pharmaceutical composition of the present invention, and instructions for diagnosing a disease.

[0035] The FAPI-RGD compound structure provided by the present invention can synergistically target FAP targets and integrin α in tumors v β3 target, which can increase the number and utilization efficiency of effective receptors in tumors. The radiolabeled compounds provided based on this structure are expected to be used in the diagnosis or treatment of fibroblast activation protein (FAP) and / or integrin α v Diseases characterized by overexpression of β3. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 This is the mass spectrum of compound 2 in Example 1 of the present invention.

[0037] Figure 2 This is the H NMR spectrum of compound 2 in Example 1 of the present invention.

[0038] Figure 3 This is the NMR carbon spectrum of compound 2 in Example 1 of the present invention.

[0039] Figure 4 This is the mass spectrum of compound 3 in Example 1 of the present invention.

[0040] Figure 5 This is the H NMR spectrum of compound 3 in Example 1 of the present invention.

[0041] Figure 6 This is the mass spectrum of compound 4 in Example 1 of the present invention.

[0042] Figure 7 This is the H NMR spectrum of compound 4 in Example 1 of the present invention.

[0043] Figure 8 This is the C NMR spectrum of compound 4 in Example 1 of the present invention.

[0044] Figure 9 This is the mass spectrum of compound 7 in Example 1 of the present invention.

[0045] Figure 10 This is the H NMR spectrum of compound 7 in Example 1 of the present invention.

[0046] Figure 11 This is the C NMR spectrum of compound 7 in Example 1 of the present invention.

[0047] Figure 12 This is the mass spectrum of compound 9 in Example 1 of the present invention.

[0048] Figure 13 This is the mass spectrum of compound 10 in Example 1 of the present invention.

[0049] Figure 14 This is the mass spectrum of compound 11 in Example 1 of the present invention.

[0050] Figure 15 This is the mass spectrum of compound V-1 in Example 1 of the present invention.

[0051] Figure 16 This is the mass spectrum of the intermediate M in Example 2 of the present invention.

[0052] Figure 17 This is the mass spectrum of intermediate O in Example 2 of the present invention.

[0053] Figure 18 This is the mass spectrum of intermediate B in Example 2 of the present invention.

[0054] Figure 19 This is the mass spectrum of intermediate C in Example 2 of the present invention.

[0055] Figure 20 This is the mass spectrum of intermediate D in Example 2 of the present invention.

[0056] Figure 21 This is the mass spectrum of intermediate E in Example 2 of the present invention.

[0057] Figure 22 This is the mass spectrum of intermediate F in Example 2 of the present invention.

[0058] Figure 23 This is the mass spectrum of intermediate G in Example 2 of the present invention.

[0059] Figure 24 This is the mass spectrum of intermediate H in Example 2 of the present invention.

[0060] Figure 25 This is the mass spectrum of the intermediate I in Example 2 of the present invention.

[0061] Figure 26 This is the mass spectrum of intermediate J in Example 2 of the present invention.

[0062] Figure 27 This is the mass spectrum of intermediate Q in Example 2 of the present invention.

[0063] Figure 28 This is the mass spectrum of compound V-14 in Example 2 of the present invention.

[0064] Figure 29 This is the mass spectrum of intermediate K in Example 3 of the present invention.

[0065] Figure 30 This is the mass spectrum of compound V-23 in Example 3 of the present invention.

[0066] Figure 31 This is the mass spectrum of intermediate B1 in Example 4 of the present invention.

[0067] Figure 32 This is the mass spectrum of intermediate D1 in Example 4 of the present invention.

[0068] Figure 33 This is the mass spectrum of intermediate G1 in Example 4 of the present invention.

[0069] Figure 34 This is the mass spectrum of intermediate H1 in Example 4 of the present invention.

[0070] Figure 35 This is the mass spectrum of the intermediate I1 in Example 4 of the present invention.

[0071] Figure 36 This is the mass spectrum of intermediate J1 in Example 4 of the present invention.

[0072] Figure 37 This is the mass spectrum of the intermediate V-25 in Example 4 of the present invention.

[0073] Figure 38 This is the mass spectrum of intermediate H3 in Example 6 of the present invention.

[0074] Figure 39 This is the mass spectrum of the intermediate I2 in Example 6 of the present invention.

[0075] Figure 40 This is the mass spectrum of intermediate O1 in Example 6 of the present invention.

[0076] Figure 41 This is the mass spectrum of the intermediate P1 in Example 6 of the present invention.

[0077] Figure 42 This is the mass spectrum of compound V-30 in Example 6 of the present invention.

[0078] Figure 43 This is the mass spectrum of the intermediate N2 in Example 7 of the present invention.

[0079] Figure 44 This is the mass spectrum of intermediate F3 in Example 7 of the present invention.

[0080] Figure 45 This is the mass spectrum of compound V-35 in Example 7 of the present invention.

[0081] Figure 46 is the compound V-1 in Example 1 of the present invention 68 HPLC quality control results of Ga-labeled FAPI-RGD (Formula V-1) complex.

[0082] Figure 47 For the present invention 68 MicroPET imaging results of Ga-labeled FAPI-RGD (Formula V-1) complex in HepG2-FAP tumor-bearing mice.

[0083] Figure 48 For the present invention 68 MicroPET imaging results of Ga-labeled FAPI-RGD (Formula V-1) complex and FAPI-02 co-injected in HepG2-FAP tumor-bearing mice.

[0084] Figure 49 For the present invention 68 MicroPET imaging results of Ga-labeled FAPI-RGD (Formula V-1) complex and RGD co-injected in HepG2-FAP tumor-bearing mice.

[0085] Figure 50 For the present invention 68Statistical graph of the uptake results of the Ga-labeled FAPI-RGD (Formula V-1) complex and C(RGDfK) or FAPI-02 in tumors and important organs 30 minutes after co-injection (the horizontal axis in the figure represents different organs, and the bar graphs from left to right in each organ correspond to 68 Uptake of Ga-labeled FAPI-RGD complex and co-injection of FAPI-02 to block FAP protein 68 Uptake of Ga-FAPI-RGD and after co-injection of RGD to block integrin 68 uptake of Ga-FAPI-RGD).

[0086] Figure 51 For the present invention 68 Figure 3 shows the stability test results of Ga-labeled FAPI-RGD (Formula V-25) complex in normal saline.

[0087] Figure 52 For the present invention 68 Figure 3 shows the results of cellular uptake and cell binding experiments of Ga-labeled FAPI-RGD (Formula V-25) complex.

[0088] Figure 53 For the present invention 68 Ga-labeled FAPI-RGD (Formula V-25) complex and monomer 68 Ga-FAPI-02 and 68 MicroPET imaging results of Ga-C(RGDfK) in HT1080-FAP tumor-bearing mice.

[0089] Figure 54 For the present invention 68 MicroPET imaging results and statistical graphs of tumor and vital organ uptake results 30 minutes after co-injection of Ga-labeled FAPI-RGD (Formula V-25) complex with C(RGDfK) or / and FAPI-02.

[0090] Figure 55 For the present invention 68 Ga-labeled FAPI-RGD complex, 18F-FDG and 68 PET / CT imaging results of Ga-FAPI46 in patients with pancreatic cancer, non-small cell lung cancer, small cell lung cancer and nasopharyngeal carcinoma 3 hours after intravenous injection.

[0091] Figure 56 This is the mass spectrum of intermediate G2 in Example 5 of the present invention.

[0092] Figure 57 This is the mass spectrum of the intermediate N1 in Example 5 of the present invention.

[0093] Figure 58 This is the mass spectrum of the intermediate P in Example 5 of the present invention.

[0094] Figure 59 This is the mass spectrum of the intermediate V-26 in Example 5 of the present invention. DETAILED DESCRIPTION

[0095] The technical solution of the present invention is further illustrated and described below through specific implementation methods in conjunction with the accompanying drawings.

[0096] Example 1: Preparation of FAPI-RGD linker (V-1)

[0097] Synthesis of compound 2:

[0098] In a 100 mL flask, compound 1 (6-hydroxyquinoline-4-carboxylic acid, 1.89 g, 10.0 mmol), glycine tert-butyl ester (1.89 g, 10.0 mmol), HATU (3.8 g, 10.0 mmol) and N, N-diisopropylethylamine (2.6 g, 20.0 mmol) were added to 30 mL of N, N-dimethylformamide. The reaction mixture was stirred overnight and the solvent was removed by distillation under reduced pressure to obtain a crude product. The product was purified by silica gel column (dichloromethane / methanol = 30:1) to obtain a white solid compound 2 with a yield of 87%. Figure 1 is the mass spectrum of compound 2, Figure 2 is the H NMR spectrum of compound 2, Figure 3 is the C NMR spectrum of compound 2.

[0099] Synthesis of compound 3:

[0100] In a 100 mL flask, compound 2 (1.51 g, 5.0 mmol), 1-bromo-3-chloropropane (1.55 g, 10.0 mmol), and potassium carbonate (1.38 g, 10.0 mmol) were added to 50 mL of N,N-dimethylformamide. The system was heated to 60 degrees Celsius and stirred overnight. The solvent was removed by vacuum distillation to obtain a crude product. The product was purified by silica gel column (dichloromethane / methanol = 50:1) to obtain a white solid compound 3 with a yield of 63%. Figure 4 is the mass spectrum of compound 3, Figure 5 is the H NMR spectrum of compound 3.

[0101] Synthesis of compound 4:

[0102] In a 100 mL flask, compound 3 (0.76 g, 2.0 mmol), 1-tert-butyloxycarbonylpiperazine (0.55 g, 3.0 mmol), and potassium iodide (0.49 g, 3.0 mmol) were added to 30 mL of acetonitrile. The system was heated to 60 degrees Celsius and stirred overnight at 60 degrees Celsius. The solvent was removed by distillation under reduced pressure to obtain a crude product. Purification by silica gel column (dichloromethane / methanol = 30:1) gave compound 4 as a white solid with a yield of 58%. MS (ESI) m / z calculated for [C 28 H 40 N4O6]:528.29; found:529.10[M+H] + . Figure 6 is the mass spectrum of compound 4, Figure 7 The H NMR spectrum of compound 4 is shown. Figure 8 is the C NMR spectrum of compound 4.

[0103] Synthesis of compound 5:

[0104] In an ice bath, compound 4 (0.52 g, 1.0 mmol) was dissolved in 10 mL of a mixed solution of dichloromethane and trifluoroacetic acid (volume ratio 9:1), and the system was heated to room temperature for 2 h. After the reaction, the solvent was distilled off under reduced pressure and the product was dissolved in 10 mL of N,N-dimethylformamide to obtain compound 5 for later use.

[0105] Synthesis of compound 6:

[0106] To compound 5 in N,N-dimethylformamide, di-tert-butyl dicarbonate (0.22 g, 1.0 mmol) and N,N-diisopropylethylamine (0.39 g, 3.0 mmol) were added, respectively. The mixture was stirred at room temperature overnight, and the solvent was removed by distillation under reduced pressure to obtain a crude product. The product was purified on a silica gel column (dichloromethane / methanol = 10:1) to obtain compound 6 as a white solid in a 72% yield.

[0107] Synthesis of compound 7:

[0108] In a 100 mL flask, compound 6 (0.47 g, 1.0 mmol), (S)-pyrrolidine-2-carbonitrile hydrochloride (0.13 g, 1.0 mmol), HATU (0.38 g, 1.0 mmol), and N,N-diisopropylethylamine (0.26 g, 2.0 mmol) were added sequentially to 10 mL of N,N-dimethylformamide. The reaction mixture was stirred at room temperature until the reaction was complete, and the solvent was removed by distillation under reduced pressure to obtain a crude product. The product was purified by silica gel column chromatography (dichloromethane / methanol = 50:1) to obtain compound 7 as a white solid in an 85% yield. Figure 9 is the mass spectrum of compound 7, Figure 10 is the H NMR spectrum of compound 7, Figure 11 is the C NMR spectrum of compound 7.

[0109] Synthesis of compound 8:

[0110] In a 100 mL flask, compound 7 (0.55 g, 1.0 mmol) and p-toluenesulfonic acid monohydrate (0.27 g, 1.5 mmol) were added to 10 mL of acetonitrile. The reaction system was heated to 60°C and stirred until the reaction was complete. The solvent was then distilled off under reduced pressure to obtain a crude product of compound 8.

[0111] Synthesis of compound 9:

[0112] To the reaction flask containing compound 8, N-Boc-3-[2-(2-aminoethoxy)ethoxy]propionic acid (0.27 g, 1.0 mmol), HATU (0.38 g, 1.0 mmol), N,N-diisopropylethylamine (0.26 g, 2.0 mmol), and 10 mL of N,N-dimethylformamide were added. The reaction mixture was stirred overnight, and the solvent was removed by distillation under reduced pressure to obtain a crude product. The product was purified by silica gel column chromatography (dichloromethane / methanol = 50:1) to obtain compound 9 as a white solid in a 64% yield. Figure 12 is the mass spectrum of compound 9.

[0113] Synthesis of compound 10:

[0114] In a 100 mL flask, compound 9 (0.66 g, 1.0 mmol) and p-toluenesulfonic acid monohydrate (0.27 g, 1.5 mmol) were added to 10 mL of acetonitrile. The reaction system was heated to 60 degrees Celsius and stirred until the reaction was completed. The solvent was removed by distillation under reduced pressure to obtain a crude product. The crude product was dissolved in 10 mL of N, N-dimethylformamide, HATU (0.38 g, 1.0 mmol) and N, N-diisopropylethylamine (0.26 g, 2.0 mmol) and propionic acid maleimide (0.17 g, 1.0 mmol) were added, and the reaction was continued for 3 h. The solvent was removed by distillation under reduced pressure to obtain a crude product. The white solid compound 10 was purified by silica gel column (dichloromethane / methanol = 50:1) with a yield of 59%. MS (ESI) m / z calculated for [C 35 H 51 N7O8]:697.38; found:698.43[M+H] + . Figure 13 is the mass spectrum of compound 10.

[0115] Synthesis of compound 11:

[0116] In a 25 mL flask, compound 10 (0.072 g, 0.1 mmol), Boc-protected cysteine ​​(0.022 g, 0.1 mmol), and 10 mL of N,N-dimethylformamide were placed and stirred at room temperature for 3 h. After monitoring the reaction, 0.12 mmol of DCC and NHS were added to the system, and the system was stirred for 12 h. c(RGDyK) (0.06 g, 0.1 mmol) and N,N-diisopropylethylamine (0.065 g, 0.05 mmol) were added and the reaction was continued for 12 h. The solvent was removed by distillation under reduced pressure to obtain a crude product. The crude product was purified by reverse phase column chromatography and freeze-dried to obtain pure compound 11 with a two-step yield of 43%. Figure 14 is the mass spectrum of compound 11.

[0117] Synthesis of compound V-1:

[0118] Compound 11 (0.146 g, 0.1 mmol) was added to a 25 mL flask, and tert-butyl ester and Boc protection were removed using thioanisole: 1,2-ethanedithiol: anisole: TFA (5:3:2:90) at room temperature. After the reaction, TFA was removed by argon flow, and then dissolved with 10 mL of N, N-dimethylformamide, and DOTA-NHS (0.05 g, 0.1 mmol) and N, N-diisopropylethylamine (0.04 g, 0.3 mmol) were added in sequence. The reaction system was stirred at room temperature, and the reaction was monitored by HPLC until the reaction was complete. The solvent was then distilled off under reduced pressure to obtain a crude product. The crude product was subjected to reverse phase column chromatography and freeze-dried to obtain pure compound V-1 with a yield of 53%. Figure 15 is the mass spectrum of compound V-1.

[0119] The synthetic route of the above steps is as follows:

[0120]

[0121] Example 2: Preparation of FAPI-RGD linker (Compound V-14)

[0122] Preparation of Intermediate M: Dissolve SM (6-hydroxyquinoline-4-carboxylic acid) in 100 mL of methanol, add 1 mL of concentrated sulfuric acid, and allow to react overnight in a 90°C bath. Monitor the reaction by TLC. Drain the methanol and add the resulting mixture dropwise to 40 mL of saturated NaHCO₃. Stir and crystallize for 1 hour after addition. Filter and dry to obtain Intermediate M in a 59% yield. The theoretical molecular weight is 203.0582, the measured molecular weight is 203.06767, and the mass spectrometry results are consistent with the target compound. Figure 16 is the mass spectrum of intermediate M.

[0123] Preparation of intermediate N: Intermediate M was dissolved in 30 mL of DMF, and potassium carbonate (2.00 g, 14.5 mmol) and 1-bromo-3-chloropropane (2.19 g, 13.9 mmol) were added in sequence. The reaction was allowed to proceed overnight in an external bath at 25°C and monitored by TLC. After the reaction was completed, 70 mL of purified water was added and the mixture was extracted twice with 70 mL of DCM. The organic phases were combined, dried, and then reduced to dryness to obtain the crude intermediate N with a crude yield of 83.8%.

[0124] Preparation of Intermediate O: Intermediate N,1-Boc-piperazine (1.67 g, 6.8 mmol) and KI (1.11 g, 6.7 mmol) were dissolved sequentially in 10 mL of DMF and reacted at 100°C. After completion of the reaction, monitored by TLC, 60 mL of purified water was added and the mixture was extracted twice with 30 mL of DCM. The combined organic phases were washed twice with 30 mL of purified water, dried over anhydrous sodium sulfate, and then column purified. The crude yield was 86%. The theoretical molecular weight was 429.2264, the measured molecular weight was 429.24041, and the mass spectrometry results were consistent with the target compound. Figure 17 This is the mass spectrum of intermediate O.

[0125] Preparation of Intermediate B: Dissolve Intermediate O in 10 mL of methanol, add a 1V / LiOH aqueous solution, and react in an external bath at 25°C. After completion of the reaction, monitor by TLC. Drain the methanol, add 2 mL of water, and slowly add 1N HCl dropwise. Adjust the pH to 6-7. Crystallize for 1 hour, filter, and dry to obtain Intermediate B in an 85% yield. The theoretical molecular weight is 415.2107, the measured molecular weight is 415.21775, and the mass spectrometry results are consistent with the target compound. Figure 18 is the mass spectrum of intermediate B.

[0126] Preparation of Intermediate C: Intermediate B, (S)-4,4-difluoro-1-glycylpyrrolidine-2-carbonitrile hydrochloride (1.11 g, 2.7 mmol), HATU (1.06 g, 2.8 mmol), and DIPEA (1.1 g, 8.1 mmol) were dissolved sequentially in 10 mL of DMF and reacted in an external bath at 25°C. After completion of the reaction, monitored by TLC, 30 mL of purified water was added to the system, followed by extraction twice with 3 mL of DCM. The combined organic phases were dried over anhydrous sodium sulfate, reduced to dryness, and column purified to obtain Intermediate C in a 73.4% yield. The theoretical molecular weight was 586.2715, the measured molecular weight was 586.28448, and the mass spectrometry results were consistent with the target compound. Figure 19 is the mass spectrum of intermediate C.

[0127] Preparation of Intermediate D: Dissolve Intermediate C in 10 mL of acetonitrile, add p-toluenesulfonic acid monohydrate (1.54 g, 8.1 mmol), and allow to react in a 65°C external bath. Monitor the reaction by TLC to obtain crude Intermediate D. The theoretical molecular weight is 486.2191, the measured molecular weight is 486.22858, and the mass spectrometry results are consistent with the desired product. Figure 20 is the mass spectrum of intermediate D.

[0128] Preparation of Intermediate E: Intermediate D was dissolved in 10 mL of DMF, and DIPEA (2.71 g, 21.1 mmol) and t-Boc-N-amido-PEG2-NHS-ester (1.22 g, 6.3 mmol) were added sequentially. The reaction was incubated at 25°C under HPLC monitoring. After completion of the reaction, 30 mL of purified water was added to the system, and the mixture was extracted twice with 30 mL of DCM. The combined organic phases were dried, concentrated, and then column purified to obtain Intermediate E in a two-step yield of 64.7%. The theoretical molecular weight was 745.3611, and the measured molecular weight was 745.37466. The mass spectrometry results were consistent with those of the target compound. Figure 21 is the mass spectrum of intermediate E.

[0129] Preparation of Intermediate F: Intermediate E was dissolved in 10 mL of acetonitrile, and p-toluenesulfonic acid monohydrate (2.42 g, 12.7 mmol) was added. The reaction was incubated at 65°C under HPLC monitoring. After completion of the reaction, the system was dried to obtain crude Intermediate F. The theoretical molecular weight was 645.3086, the measured molecular weight was 645.31807, and the mass spectrometry results were consistent with those of the desired product. Figure 22 This is the mass spectrum of intermediate F.

[0130] Preparation of Intermediate G: Intermediate F and DIPEA (1.28 g, 10.1 mmol) were dissolved sequentially in 5 mL of DMF to obtain system ①. HATU (0.84 g, 2.2 mmol) and Fmoc-Glu(OtBu)OH (0.61 g, 2.2 mmol) were dissolved sequentially in 5 mL of DMF to obtain system ②. System ② was stirred at 25°C for 1 hour and then added to system ①. The reaction was allowed to proceed at 25°C and monitored by TLC. After completion of the reaction, 20 mL of purified water was added to the system, followed by two extractions with 20 mL of DCM. The combined organic phases were washed once with saturated sodium chloride, concentrated, and purified by column chromatography to obtain Intermediate G in a two-step yield of 63.6%. The theoretical molecular weight was 1052.4819, the measured molecular weight was 1052.49330, and the mass spectrometry results were consistent with those of the target compound. Figure 23 This is the mass spectrum of intermediate G.

[0131] Preparation of Intermediate H: Intermediate G was dissolved in 10 mL of acetonitrile, and p-toluenesulfonic acid monohydrate (2.87 g, 15.1 mmol) was added. The reaction was carried out at 65°C and monitored by HPLC. After completion of the reaction, the system was dried and column purified to obtain Intermediate H. The theoretical molecular weight was 996.4193, the measured molecular weight was 996.42947, and the mass spectrometry results were consistent with the target compound. Figure 24 This is the mass spectrum of intermediate H.

[0132] Preparation of Intermediate I: Intermediate H and DIPEA (1.62 g, 10.1 mmol) were dissolved in 10 mL of DMF. HATU (1.37 g, 3.6 mmol) and NHS (1.28 g, 5.85 mmol) were added sequentially and stirred at 30°C to obtain System ①. RGDfK (2.17 g, 3.6 mmol) was dissolved in 10 mL of DMSO to obtain System ②. After the reaction of System 1 was completed, System ② was added to System 1 in three batches, with 15-min intervals between each batch. After complete addition, the mixture was reacted in an external bath at 30°C and monitored by HPLC. After completion of the reaction, the mixture was dried and sent for further preparation to obtain Intermediate I in a 34.2% yield. The theoretical molecular weight was 1581.7216, the measured molecular weight was 1581.7372, and the mass spectrometry results were consistent with those of the target compound. Figure 25 is the mass spectrum of intermediate I.

[0133] Preparation of Intermediate J: Dissolve Intermediate I in 30 mL of DMF, add 2 mL of piperidine, and react at 25°C under HPLC monitoring. After the reaction, add 200 mL of MTBE to crystallize the mixture. Allow to stand, aspirate the supernatant, and reduce the remaining mixture to dryness to obtain crude Intermediate J, which was used directly in the next step. The theoretical molecular weight was 1359.6536, the measured molecular weight was 1359.66432, and the mass spectrometry results were consistent with the target compound. Figure 26 is the mass spectrum of intermediate J.

[0134] Preparation of Intermediate Q: Intermediate J was dissolved in 20 mL of DMF, and DIPEA (0.81 g, 5.0 mmol) and DOTA-TRIS-TBU-NHS Ester (0.50 g, 1.0 mmol) were added sequentially. The reaction was monitored by HPLC. After completion, the system was concentrated and sent for preparative purification to obtain Intermediate Q in a two-step yield of 15.7%. The theoretical molecular weight was 1914.0215, and the measured molecular weight was 1914.03418. Mass spectrometry results were consistent with the desired product. Figure 27 is the mass spectrum of intermediate Q.

[0135] Preparation of Compound V-14: Intermediate Q was dissolved in 10 mL of TFA and reacted at 25°C in an external bath under HPLC monitoring. After completion of the reaction, 100 mL of MTBE was added to the system for crystallization. The reaction was allowed to stand, and the supernatant was aspirated. The remaining system was concentrated to dryness and then spun with MTBE until no significant TFA residue remained. The system was then sent for preparative purification to obtain Compound V-14. The theoretical molecular weight was 1745.8337, the measured molecular weight was 1745.84714, and the mass spectrometry results were consistent with the target compound. Figure 28 is the mass spectrum of compound V-14.

[0136] The synthetic route of the above steps is as follows:

[0137]

[0138]

[0139] Example 3: Preparation of FAPI-RGD linker (Compound V-23)

[0140] Preparation of Intermediate K: Intermediate J prepared according to the method of Example 2 was dissolved in 30 mL of DMF, and DIPEA (0.97 g, 7.5 mmol) and 2 eq of NOTA-Bis-TBU-NHS Ester (calculated based on Intermediate J) were added. The reaction was incubated at 25°C in an external bath and monitored by HPLC. After completion of the reaction, the system was dried and sent for further preparation to obtain Intermediate K in a two-step yield of 25.1%. The theoretical molecular weight was 1756.9112, the measured molecular weight was 1756.92282, and the mass spectrometry results were consistent with those of the target compound. Figure 29 This is the mass spectrum of intermediate K.

[0141] Preparation of V-23: Intermediate K was dissolved in 30 mL of TFA and reacted at 25°C in an external bath under HPLC monitoring. After completion of the reaction, 200 mL of MTBE was added to the system for crystallization. The reaction was allowed to stand, and the supernatant was aspirated. The remaining system was concentrated to dryness and then stripped with MTBE until no significant TFA residue remained. Compound V-23 was obtained after purification in a 14.2% yield. The theoretical molecular weight was 1644.7860, the measured molecular weight was 1644.8104, and the mass spectrometry results were consistent with the target compound. Figure 30 This is the mass spectrum of V-23.

[0142] The synthetic route of the above steps is as follows:

[0143]

[0144]

[0145] Example 4: Preparation of FAPI-RGD linker (Compound V-25)

[0146] Preparation of intermediate B1: Compound 7 (2.50 g, 4.5 mmol), p-toluenesulfonic acid monohydrate (2.58 g, 13.6 mmol), and 25 mL of acetonitrile were added to a reaction flask and reacted at 65°C for 1 h. The SM1 reaction was complete (methanol: dichloromethane = 5:1) after TLC monitoring. The mixture was evaporated to dryness under reduced pressure at 40°C. 14 mL of DMF and DIPEA (3.05 g, 23.6 mmol) were added and stirred at 25°C. Reaction number (1), i.e., piperazine deprotection of compound 7, was performed to obtain intermediate 8. N-tert-butyloxycarbonyl-diethylene glycol-carboxylic acid (1.62 g, 4.8 mmol), HATU (2.60 g, 6.8 mmol), and 10 mL of DMF were added to another reaction flask and reacted at 25°C for 30 min. Reaction number (2), reaction (2) was added to reaction (1) and reacted for 1 h. The mixture was evaporated to dryness under reduced pressure at 40°C, and 50 mL of purified water was added. The mixture was extracted twice with 50 mL of DCM each time. The combined DCM was dried over anhydrous sodium sulfate, filtered, and evaporated to dryness to obtain a crude product. Purification by column chromatography afforded 1.68 g of the desired product. The theoretical molecular weight was 709.3799, the measured molecular weight was 709.38801, and the mass spectrometry results were consistent with those of the desired product. Figure 31 is the mass spectrum of intermediate B1.

[0147] Preparation of Intermediate D1: Add Intermediate B1, p-toluenesulfonic acid monohydrate (1.61 g, 8.5 mmol), and 20 mL of acetonitrile to a reaction flask, react at 65°C for 1 h, and evaporate to dryness under reduced pressure at 40°C. Add 20 mL of DMF and DIPEA (1.83 g, 14.2 mmol), stir at 25°C, and perform reaction number (1). Intermediate B1 is deprotected to obtain Intermediate C1. Add Fmoc-O-tert-butyl-L-glutamic acid (1.43 g, 3.4 mmol), HATU (1.29 g, 3.4 mmol), and 20 mL of DMF to another reaction flask, react at 25°C for 30 min, and perform reaction number (2). Add the reaction (2) stream to reaction (1) and react for 1 h. Evaporate to dryness under reduced pressure at 40°C to obtain a crude product, which is purified by column chromatography to obtain 1.19 g of the target compound. The theoretical molecular weight is 1016.5008, the measured molecular weight is 1016.51094, and the mass spectrometry results are consistent with the target compound. Figure 32 is the mass spectrum of intermediate D1.

[0148] Preparation of Intermediate G1: c(RGDfK) (1.00 g, 1.7 mmol), t-Boc-N-amido-PEG2-NHS ester (0.74 g, 1.9 mmol), DIPEA (0.44 g, 3.4 mmol), and 20 mL of DMF were added to a reaction flask and reacted at 30°C for 20 h. Evaporate to dryness under reduced pressure at 40°C, add 10 mL of methanol, and dropwise add 60 mL of MTBE to precipitate a solid, affording Intermediate F1. This solid was filtered and dried under vacuum at 40°C for 2 h. The solid Intermediate F1 was added to a reaction flask, along with 30 mL of TFA and 1.5 mL of purified water. The reaction was continued at 30°C for 1 h. The temperature was then lowered to 0-5°C, and 200 mL of MTBE was added dropwise. Stir at 0-5°C for 30 min, filter, rinse with MTBE, and dry under vacuum at 40°C to obtain the product. The theoretical molecular weight is 762.4024, the measured molecular weight is 762.40768, and the mass spectrometry results are consistent with the target compound. Figure 33 This is the mass spectrum of intermediate G1.

[0149] Preparation of Intermediate H1: Intermediate D1, p-toluenesulfonic acid monohydrate (0.34 g, 1.8 mmol), and 20 mL of acetonitrile were added to a reaction flask, reacted at 65°C for 4 h, and evaporated to dryness under reduced pressure at 40°C. Then, 20 mL of DMF, DIPEA (0.36 g, 2.8 mmol), DCC (0.14 g, 0.7 mmol), and NHS (0.08 g, 0.7 mmol) were added and reacted at 35°C for 15-20 h to afford Intermediate E1. The temperature was lowered to 25°C, and Intermediate G1 was added. The reaction was continued for 1 h, and the crude product was evaporated to dryness under reduced pressure at 40°C. Liquid phase preparation was performed to obtain 66.5 mg of the desired compound. The theoretical molecular weight was 1704.8300, and the measured molecular weight was 1704.84518. Mass spectrometry results were consistent with the desired compound. Figure 34 This is the mass spectrum of intermediate H1.

[0150] Preparation of Intermediate I1: Intermediate H1, 0.5 mL of piperidine, and 2 mL of DMF were added to a reaction flask and reacted at 25°C for 1 hour. 10 mL of ethyl acetate was added dropwise to crystallize the mixture, stirred for 30 minutes, filtered, and the solid dried under vacuum at 40°C for 2 hours to yield 50.8 mg of the product. The theoretical molecular weight was 1482.7619, the measured molecular weight was 1482.7759, and the mass spectrometry results were consistent with the desired product. Figure 35 is the mass spectrum of intermediate I1.

[0151] Preparation of Intermediate J1: Intermediate I1, NOTA-Bis-TBU-NHS Ester, DIPEA (0.010 g, 0.08 mmol), and 2 mL of DMF were added to a reaction flask. The reaction was allowed to proceed at 25°C for 1 h. The mixture was then evaporated to dryness under reduced pressure at 40°C. 2 mL of ethyl acetate and 2 mL of MTBE were added for crystallization. The mixture was stirred for 20 min, filtered, and the solid was dried under vacuum at 40°C to yield 43.2 mg of the product. The theoretical molecular weight was 1880.0196, the measured molecular weight was 1880.0369, and the mass spectrometry results were consistent with those of the desired compound. Figure 36 is the mass spectrum of intermediate J1.

[0152] Preparation of compound V-25: Intermediate J1 and 2 mL of trifluoroacetic acid were added to a reaction flask, reacted at 25°C for 1 h, and evaporated to dryness under reduced pressure at 40°C to obtain a crude product. The crude product was purified by preparative liquid phase purification and freeze-dried to obtain the product with a theoretical molecular weight of 1767.8944 and an actual molecular weight of 1767.91036. The mass spectrometry results were consistent with those of the target compound. Figure 37 This is the mass spectrum of compound V-25.

[0153] The synthetic route of the above steps is as follows:

[0154]

[0155]

[0156]

[0157] Example 5: Preparation of FAPI-RGD linker (Compound V-26)

[0158] Preparation of Intermediate B1: Starting material 7 (5.50 g, 10 mmol) and p-toluenesulfonic acid (5.71 g, 30 mmol) were weighed and added to a reaction flask. 10 mL of acetonitrile was added and the mixture was stirred and heated to 65°C for 1 h. The protecting group on the piperazine ring was removed to obtain Intermediate 8. TLC (developing solvent: dichloromethane:methanol = 5:1) confirmed the reaction was complete, and the mixture was evaporated to dryness under reduced pressure at 40°C. 4 mL of DMF and DIPEA (9.05 g, 70 mmol) were added and dissolved with stirring. t-Boc-N-amido-PEG2-NHS ester (Intermediate C2, 5.62 g, 15 mmol) was added and the mixture was allowed to react at 25°C for 3 h. TLC (developing solvent: dichloromethane:methanol = 5:1) confirmed the reaction was complete, and the mixture was evaporated to dryness under reduced pressure at 40°C. Add 5 mL of purified water and extract twice with 5 mL of DCM each time. Combine the organic phases, dry over anhydrous sodium sulfate (1 m / m) for 30 min, filter, and evaporate the mother liquor to dryness under reduced pressure at 40°C to obtain the crude product. Purify by column chromatography to obtain intermediate B1, which is used directly in the next step.

[0159] Preparation of Intermediate G2: Intermediate B1 and p-toluenesulfonic acid (5.14 g, 27 mmol) were weighed and added to a reaction flask. 10 mL of acetonitrile was added, and the mixture was stirred and heated to 65°C for 1 hour. The protecting group was removed to obtain Intermediate C1. TLC analysis (developing solvent: dichloromethane:methanol = 10:1) indicated the reaction was complete, and the mixture was evaporated to dryness under reduced pressure at 40°C. DIPEA (5.82 g, 45 mmol), 10 mL of DMF, and 3-maleimidopropionic acid N-hydroxysuccinimide ester (2.88 g, 10.8 mmol) were added, and the mixture was allowed to react at room temperature for 1 hour. TLC analysis (developing solvent: dichloromethane:methanol = 10:1) indicated the reaction was complete, and the mixture was evaporated to dryness under reduced pressure at 40°C. Column chromatography was performed to obtain Intermediate G2. The target compound had a theoretical molecular weight of 760.35443, and the molecular weight was 760.37090 as determined by LC / MS / MS. The mass spectrometry results were consistent with the target compound. Figure 52 This is the mass spectrum of intermediate G2.

[0160] Preparation of Intermediate N1: Intermediate G2 and Boc-cysteine ​​(1.77 g, 8 mmol) were weighed and added to a reaction flask. 10 mL of DMF was added and the mixture was allowed to react at 25°C for 2 h to yield Intermediate H2. TLC analysis (developing solvent: dichloromethane:methanol = 5:1) confirmed the reaction was complete. DCC (1.98 g, 9.6 mmol) and NHS (1.86 g, 9.6 mmol) were added and the mixture was allowed to react at 35°C for 2 h. TLC analysis (developing solvent: dichloromethane:methanol = 5:1) confirmed the reaction was complete. Cyclopeptide Cyclo (Arg-Gly-Asp-DPhe-Lys) (4.83 g, 8 mmol) and DIPEA (3.10 g, 24 mmoleq) were added and the mixture was allowed to react at 25°C for 1 h. TLC analysis (developing solvent: dichloromethane:methanol = 5:1) confirmed the reaction was complete. The crude product was evaporated to dryness under reduced pressure at 40°C and purified by preparative liquid chromatography to yield Intermediate N1. The theoretical molecular weight of the target compound is 1566.72893, and the molecular weight shown by liquid chromatography-mass spectrometry is 1566.74480. The mass spectrometry results are consistent with the target compound. Figure 53 This is the mass spectrum of intermediate N1.

[0161] Preparation of Intermediate P: Intermediate N1 was weighed and added to a reaction flask. 2 mL of methyl phenyl sulfide, 2 mL of 1,2-ethanedithiol, and 20 mL of trifluoroacetic acid were added under nitrogen and allowed to react at room temperature for 1 hour. 20 mL of methyl tert-butyl ether was added, and a solid precipitated. This solid was filtered and dried under vacuum at 40°C for 1 hour to obtain Intermediate P. The target compound had a theoretical molecular weight of 1466.67650, and its molecular weight was 1466.69746 as measured by liquid chromatography-mass spectrometry. The mass spectrometry results were consistent with the target compound. Figure 54 is the mass spectrum of intermediate P.

[0162] Preparation of Product Compound V-26: Intermediate P and NOTA-Bis-TBU-NHS Ester were weighed and added to a reaction flask. 40 mL of DMF was added and the mixture was allowed to react at room temperature for 1 hour. 4 mL of DIPEA was added and the mixture was allowed to react at room temperature for 3 hours. Evaporation under reduced pressure at 40°C yielded Intermediate S. 30 mL of trifluoroacetic acid was added and the mixture was stirred at room temperature for 1 hour. Evaporation under reduced pressure at 40°C yielded the crude product, which was purified by preparative liquid chromatography to yield Product Compound V-26. The theoretical molecular weight of the target compound was 1751.80898, and the LC / MS results indicated a molecular weight of 1751.83088, which was consistent with the mass spectrometry results. Figure 55 This is the mass spectrum of the intermediate compound V-26.

[0163] The above-mentioned synthetic route is as follows:

[0164]

[0165]

[0166] Example 6: Preparation of FAPI-RGD linker (Compound V-30)

[0167] Preparation of intermediate Cmpd3: Fmoc-PEG4-CH2CH2COOH (1.46 g, 3.0 mmol) was dissolved in DMF, and DCC (0.68 g, 3.3 mmol) and HOSu (0.38 g, 3.3 mmol) were added. The reaction was carried out at room temperature for 6 hours, filtered, and TEA (0.90 g, 9.0 mmol) was added to the filtrate. Cyclo (RGDfK) (2.23 g, 3.6 mmol) was added and the reaction was carried out at room temperature for 3 hours. The reaction solution was spin-dried and redissolved in 25% DEA / THF. The reaction was carried out at room temperature for 4 hours. The solution was concentrated to a small amount and added to 10 volumes of diethyl ether. A large amount of solid precipitated and was filtered to obtain the crude product Cyclo (RGDfK) -PEG4. The fine product Cyclo (RGDfK) -PEG4 was obtained after purification by reverse phase preparative liquid chromatography. The eluent was (solution A: 0.1% TFA in H2O; solution B: acetonitrile).

[0168] Synthesis of intermediate (RGDfK)2-PEG4-Glu: Boc-Glu-OH (0.4 g, 2.0 mmol) was dissolved in DMF, and then DCC (0.45 g, 2.2 mmol) and HOSu (0.25 g, 2.2 mmol) were added. The mixture was reacted at room temperature for 6 hours, filtered, and TEA (0.60 g, 6.0 mmol) was added to the filtrate. Cyclo (RGDfK)-PEG4 (2.61 g, 2.4 mmol) was added and the mixture was reacted at room temperature for 3 hours. The reaction solution was spin-dried and then dissolved in TFA. The mixture was reacted at room temperature for 10 minutes and added to 10 volumes of ether. A large amount of solid precipitated and was filtered to obtain the crude product 2 (RGDfK)-PEG4-Glu. The refined product (RGDfK)2-PEG4-Glu was obtained after purification by reverse phase preparative liquid chromatography. The eluent was (liquid A: 0.1% TFA in H2O; liquid B: acetonitrile), and then the pH of the fine product (RGDfK)2-PEG4-Glu was adjusted to neutral with TEA, and then the reverse phase was used to prepare the liquid phase, and the finished product (RGDfK)2-PEG4-Glu was lyophilized.

[0169] Preparation of Intermediate H3: Intermediate 8 (0.45 g, 1 mmol), prepared according to the method of Example 1, Fmoc-O-tert-butyl-L-glutamic acid (0.42 g, 1 mmol), HATU (0.38 g, 1 mmol), and DIPEA (0.58 g, 4.5 mmol) were dissolved sequentially in 10 mL of DMF. The reaction was allowed to proceed at 25°C in an external bath and monitored by HPLC. After completion of the reaction, 20 mL of purified water was added to the reaction system, and the mixture was extracted twice with 20 mL of DCM. The combined organic phases were dried over anhydrous sodium sulfate, concentrated, and column purified to obtain Intermediate H3 in a two-step crude yield of 97%. The theoretical molecular weight was 735.3956, the measured molecular weight was 735.40744, and the mass spectrometry results were consistent with those of the target compound. Figure 38 This is the mass spectrum of intermediate H3.

[0170] Preparation of Intermediate I2: Dissolve Intermediate H3 in 20 mL of acetonitrile, add p-toluenesulfonic acid monohydrate (0.65 g, 3.4 mmol), and react at 70°C in an external bath under HPLC monitoring. After completion of the reaction, remove the acetonitrile and use it directly in the next step. The theoretical molecular weight is 579.2805, and the measured molecular weight is 579.28563. Mass spectrometry results are consistent with the target compound. Figure 39 This is the mass spectrum of intermediate I2.

[0171] Preparation of Intermediate O1: Intermediate I2 and DIPEA (0.64 g, 5.0 mmol) were dissolved in 10 mL of DMF, followed by the addition of DOTA-TRIS-TBU-NHS Ester (1.7 g, 2.5 mmol). The reaction was monitored by HPLC. After the solvent was evaporated, the remaining system was purified and prepared to obtain Intermediate O1 in a two-step yield of 27.15%. The theoretical molecular weight was 1133.6485, and the measured molecular weight was 1133.65551. Mass spectrometry results were consistent with the desired product. Figure 40 This is the mass spectrum of intermediate O1.

[0172] Preparation of Intermediate P1: Intermediate O1 was dissolved in 5 mL of DMF, and HATU (0.076 g, 0.2 mmol) was added. The mixture was stirred at room temperature for 1 h to obtain system ①. DIPEA (0.090 g, 0.7 mmol) and (RGDfK)2-PEG4-Glu (0.24 g, 0.13 mmol) were dissolved in 5 mL of DMSO to obtain system ②. System ① was added to system ②, and the mixture was stirred at 28°C and monitored by HPLC. After completion of the reaction, the DMF was reduced to dryness, and 100 mL of MTBE was added for crystallization. The reaction was allowed to stand, and the supernatant was decanted. The remaining oil was sent for preparative purification to obtain Intermediate P1 in a yield of 17.09%. The theoretical molecular weight was 2927.5797, the measured molecular weight was 2927.60652, and the mass spectrometry results were consistent with the target compound. Figure 41 is the mass spectrum of intermediate P1.

[0173] Preparation of V-30: Intermediate P1 was dissolved in 5 mL of TFA and reacted at 25°C in an external bath under HPLC monitoring. After completion of the reaction, 25 mL of MTBE was added to the system for crystallization. The mixture was allowed to stand, and the supernatant was aspirated. The remaining system was stripped with MTBE until no significant TFA residue was present. The product was then sent for preparative purification to obtain V-30 in a yield of 32.13%. The theoretical molecular weight was 2759.3919, the measured molecular weight was 2759.40972, and the mass spectrometry results were consistent with the target compound. Figure 42 This is the mass spectrum of V-30.

[0174] The above-mentioned synthetic route is as follows:

[0175]

[0176]

[0177] Example 7: Preparation of FAPI-RGD linker (Compound V-35)

[0178] Preparation of Intermediate N2: Intermediate I2, prepared according to the method of Example 6, and DIPEA (3.90 g, 30 mmol) were dissolved in 10 mL of DMF. NOTA-Bis-TBU-NHS Ester (7.65 g, 15 mmol) was then added to the reaction mixture. The reaction was monitored by HPLC. After the reaction, the DMF was evaporated to dryness, and the remaining mixture was purified to obtain Intermediate N2 in a two-step yield of 22.88%. The theoretical molecular weight was 976.5382, and the measured molecular weight was 976.56026. The mass spectrometry results were consistent with those of the desired compound. Figure 43 This is the mass spectrum of intermediate N2.

[0179] Preparation of Intermediate F3: Intermediate N2 was dissolved in 10 mL of DMF, and HATU (0.46 g, 1.2 mmol) was added. The reaction was incubated at 30°C for 1 h to obtain system ①. c(RGDfK)2-PEG4-Glu (1.54 g, 0.8 mmol) and DIPEA (0.62 g, 4.8 mmol) were dissolved in 5 mL of DMF and 5 mL of DMSO to obtain system ②. System ① was added to system ②, stirred at 30°C in a 4-well bath, and monitored by HPLC. After completion of the reaction, the solvent was evaporated to dryness, and the remaining system was purified to obtain Intermediate F3 in a yield of 15.34%. The theoretical molecular weight was 2770.4894, the measured molecular weight was 2770.49229, and the mass spectrometry results were consistent with the target compound. Figure 44 is the mass spectrum of intermediate F3.

[0180] Preparation of V-35: Intermediate F3 was dissolved in 20 mL of TFA and reacted in an external bath at 25°C under HPLC monitoring. After completion of the reaction, 50 mL of MTBE was added to the reaction mixture for crystallization. The mixture was allowed to stand and the supernatant was decanted. The remaining mixture was then stripped with MTBE until no significant TFA residue remained. The mixture was then purified to obtain V-35 in a 2.89% yield. The theoretical molecular weight was 2658.3442, the measured molecular weight was 2658.36508, and the mass spectrometry results were consistent with those of the target compound. Figure 45 This is the mass spectrum of V-35.

[0181] The above-mentioned synthetic route is as follows:

[0182]

[0183] Example 8-48

[0184] The structures of the compounds of Examples 8-48 are shown in Formulas (V-2) to (V-13), (V-15) to (V-22), (V-24), (V-27) to (V-29), (V-31) to (V-34), (V-36) to (V-40), and (VI-1) to (VI-8), respectively. Their preparation methods can refer to the above examples. For example, based on the above examples, some raw materials are replaced, including replacing c(RGDfK) with c(RGDyK), replacing c(RGDyK) with c(RGDfK), replacing (S)-difluoropyrrolidine-2-carbonitrile hydrochloride with (S)-4,4-difluoropyrrolidine-2-carbonitrile hydrochloride, etc., to obtain the following corresponding structures:

[0185]

[0186]

[0187]

[0188]

[0189]

[0190]

[0191]

[0192]

[0193]

[0194]

[0195]

[0196]

[0197]

[0198] Examples 49-86:

[0199] With reference to the preparation methods of Examples 1-48, the following FAPI-RGD compounds of formula (V) in Table 1 or formula (VI) in Table 2 were prepared:

[0200]

[0201] Table 1

[0202]

[0203]

[0204]

[0205]

[0206] Table 2

[0207]

[0208]

[0209]

[0210]

[0211] Example 99. Preparation of Radioactive Ga-68 Labeled FAPI-RGD (Formula V-1) Complex:

[0212] Wet method: about 18.5 to 1850 megabecquerels (MBq) 68 GaCl3 hydrochloric acid solution (eluted from the gallium germanium generator) was added to a centrifuge tube containing 0.5 mL of acetic acid-acetate solution (1.0 g / L) of compound V-1 prepared in Example 1 and allowed to react at 37°C for 20 minutes. A C18 separation column was first slowly rinsed with 10 mL of anhydrous ethanol and then with 10 mL of water. The labeled solution was diluted with 10 mL of water and loaded onto the separation column. Unlabeled 68 Ga ions were then washed with 0.3 mL of 10 mM HCl in ethanol to obtain 68 Ga-labeled FAPI-RGD (V-1) complex. The eluent was diluted with physiological saline and sterile filtered to obtain 68 Injection of Ga-labeled FAPI-RGD (V-1) complex.

[0213] Lyophilization: About 18.5 to 1850 megabecquerels (MBq) 68 Add GaCl3 hydrochloric acid solution (eluted from the gallium germanium generator) to the freeze-dried drug box containing compound V-1, mix well and react at 37°C for 20 minutes. Take a C18 separation column and slowly elute it with 10mL of anhydrous ethanol first, then with 10mL of water. Dilute the labeled solution with 10mL of water and load it onto the separation column. First, remove the unlabeled 68 The Ga ions were then washed with 0.3 mL of 10 mM HCl in ethanol to obtain a complex eluent. The eluent was diluted with physiological saline and sterile filtered to obtain 68 Injection of Ga-labeled FAPI-RGD (V-1) complex.

[0214] Experimental example. Analysis and application effect

[0215] 1. 68 HPLC Analysis and Identification of Ga-labeled FAPI-RGD(V-1) Complex

[0216] The HPLC system was as follows: a SHIMADZULC-20A C18 column (YMC, 3 μm, 4.6 × 150 mm) was used for analysis. Detection wavelength was 254 nm, flow rate was 1 mL / min, and elution gradient was as follows: 0-3 min: 10% acetonitrile and 90% water (50 mM ammonium acetate) maintained constant; 3-16 min: increased to 90% acetonitrile and 10% water (50 mM ammonium acetate); 16-18 min: maintained at 90% acetonitrile and 10% water (50 mM ammonium acetate); 18-20 min: decreased to 10% acetonitrile and 90% water (50 mM ammonium acetate); 20-22 min: maintained at 10% acetonitrile and 90% water (50 mM ammonium acetate). 68 The HPLC quality control results of Ga-labeled FAPI-RGD (V-1) complex are shown in the figure. Figure 46 .

[0217] 2. 68 MicroPET imaging of Ga-labeled FAPI-RGD(V-1) complex in tumor-bearing mice

[0218] Prepared according to the method of Example 99 68 In HepG2-FAP tumor-bearing mice, 7.4 MBq of Ga-FAPI-RGD (V-1) was injected into the tail vein. 68 Ga-FAPI-RGD (V-1) was then anesthetized with isoflurane and MicroPET imaging was performed at 0 to 120 minutes after administration. Figure 47 . Figure 47 Intravenous injection is shown 68 Representative coronal MicroPET images of HepG2-FAP tumor-bearing mice (n=3) at different times after Ga-FAPI-RGD (V-1). Tumors were clearly visible at the time points of imaging (30 min and 2 h). 68 The ability of Ga-FAPI-RGD (V-1) to specifically bind to integrin and FAP in vivo was confirmed by blocking experiments. 68 Ga-FAPI-RGD (V-1) was co-injected with RGD or FAPI-02 into HepG2-FAP tumor-bearing mice. The MicroPET imaging results and organ uptake results are shown in the figure. Figures 48-50 As shown. Figures 48-49 As can be seen in the figure, co-injection of RGD or FAPI-02 can reduce the tumor68 Uptake of Ga-FAPI-RGD(V-1). Figure 50 As can be seen in the figure, the uptake values ​​of major organs and tumors (%ID / g) were obtained 0.5 hours after injection, and the uptake of tumors was 68 Ga-FAPI-RGD(V-1) was partially inhibited by RGD or FAPI-02, as confirmed by blocking experiments 68 Ga-FAPI-RGD(V-1) can achieve tumor-specific targeting in vivo by binding to integrins and FAP proteins.

[0219] 3. 68 Stability Analysis of Ga-labeled Complex of Formula (V-25) FAPI-RGD

[0220] With reference to the method of Example 99, prepare 68 Ga-labeled complex of formula (V-25) FAPI-RGD. Pipette 20 μL 68 A solution of Ga-FAPI-RGD (3.7 MBq activity / 20 μL) was added to a centrifuge tube containing 100 μL of saline or PBS (pH = 7.4) and incubated at 37°C for 0.5 h, 1 h, and 4 h. 20 μL of the incubation solution was taken and filtered through a 0.22 μm needle filter membrane and analyzed for radiochemical purity by HPLC. The test results are as follows: Figure 51 As shown, 68 After incubation in physiological saline, Ga-FAPI-RGD (V-25) showed no obvious decomposition and the radiochemical purity was greater than 99%, indicating that the Ga-FAPI-RGD prepared by the present invention is 68 Ga-FAPI-RGD (V-25) has excellent stability.

[0221] 4. 68 Cellular Assay Analysis of the Complex of Ga-labeled (V-25) FAPI-RGD

[0222] In HT1080-FAP tumor cells 68 The cell uptake experiment of Ga-FAPI-RGD (V-25) showed the following results: Figure 52 As shown in Part A, 68 Ga-FAPI-RGD(V-25) has a rapid cellular uptake, reaching a maximum at 30 minutes of incubation and maintaining a similar uptake level for up to 2 hours. In addition, blocking experiments confirmed that 68 The cellular uptake of Ga-FAPI-RGD(V-25) was partially inhibited by C(RGDfK) or FAPI-02 and completely blocked by FAPI-RGD (see Figure 52Cell binding experiments were performed on HT1080-FAP and U87MG tumor cells, and the test results were shown in Figure 2. Figure 52 As shown in B and C, in the HT1080-FAP cell experiment, the 68 Ga-FAPI-RGD(V-25) and 68 Ga-FAPI-02 IC 50 The two were 11.17nM and 4.14nM respectively. In the HT1080-FAP cell experiment, the 68 Ga-FAPI-RGD(V-25) and 68 Ga-C(RGDfK) IC 50 The two groups were 18.93nM and 11.49nM respectively. The experimental results showed that FAPI-RGD, compared with the corresponding monomer, had a strong affinity for the corresponding receptor FAP protein and integrin α v β3 has similar affinity.

[0223] 5. 68 MicroPET imaging of Ga-labeled (V-25) FAPI-RGD complex in tumor-bearing mice

[0224] In HT1080-FAP tumor-bearing mice, 7.4 MBq of 68 Ga-FAPI-RGD(V-25), 68 Ga-FAPI-02 and 68 Ga-C(RGDfK), and then under isoflurane anesthesia, 68 The Ga-FAPI-RGD (V-25) group underwent MicroPET imaging from 0 to 240 minutes after administration, and the other groups underwent MicroPET imaging from 0 to 120 minutes after administration. Figure 53 . Figure 53 Figures A, C, and E show the maximum intensity projection images of MicroPET of HT1080-FAP tumor-bearing mice (n=3) at different times after intravenous injection of the above three groups of mice. Figures B, D, and F show the uptake of various organs or tissues (blood, liver, kidney, tumor, and muscle) of the above three groups of mice at different time points after injection. The three uptake amounts in each group correspond from left to right to 0.5 h, 1 h, and 2 h after injection, respectively. Figure 53 shows that at the time point of image acquisition, the tumor was clearly visible, and 68 The tumor uptake of Ga-FAPI-RGD(V-25) is higher than that of the monomer 68 Ga-FAPI-02 and 68 Tumor uptake of Ga-C(RGDfK). 68Ga-FAPI-RGD(V-25) specifically binds to integrin α in vivo v The performance of β3 and FAP was confirmed by blocking experiments. 68 Ga-FAPI-RGD (V-25) and C (RGDfK) or FAPI-02 were co-injected into HT1080-FAP tumor-bearing mice. The MicroPET imaging results and organ uptake results are shown in Figure 2. Figure 54 shown. Figure 54 The four images in A correspond to the individual injections from left to right. 68 Ga-FAPI-RGD(V-25), 68 Ga-FAPI-RGD (V-25) and C (RGDfK) were co-injected. 68 Ga-FAPI-RGD (V-25) and FAPI-02 were co-injected, 68 Images obtained by co-injection of Ga-FAPI-RGD (V-25) with C (RGDfK) and FAPI-02; B and C respectively reflect the effects of the four groups of injections on the various organs or tissues (blood, liver, kidney, tumor and muscle) of mice. 68 Uptake and target / non-target ratio of Ga-FAPI-RGD (V-25). The four columns in each organ or tissue in B and C correspond to the four injection methods in A from left to right. Figure 54 As can be seen in 68 Co-injection of Ga-FAPI-RGD (V-25) with RGD or FAPI-02 can reduce the tumor 68 The uptake of Ga-FAPI-RGD(V-25) 68 The co-injection of Ga-FAPI-RGD (V-25) with RGD + FAPI-02 further reduced the tumor 68 Uptake of Ga-FAPI-RGD(V-25) confirmed by blocking experiments 68 Ga-FAPI-RGD(V-25) can achieve tumor-specific targeting in vivo by binding to integrins and FAP proteins.

[0225] 6. 68 PET / CT imaging of Ga-labeled (V-25) FAPI-RGD complex in tumor patients

[0226] Approved by the Clinical Research Ethics Committee of the First Affiliated Hospital of Xiamen University 68 All subjects signed written informed consent for the clinical trial of Ga-FAPI-RGD (V-25), including one pancreatic cancer patient, one non-small cell lung cancer patient, one small cell lung cancer patient, and one nasopharyngeal carcinoma patient. 68The dose of Ga-FAPI-RGD (V-25) was 1.8-2.2 MBq [0.05-0.06 mCi] / kg. Three hours after intravenous injection, data were acquired using a hybrid PET / CT scanner (Discovery MI, GE Healthcare, Milwaukee, WI, USA). The imaging results are shown in Figure 2. Figure 55 The maximum standardized uptake value (SUV) was automatically calculated using a region of interest (ROI) drawn on the transaxial image. max ). Dual-targeted 68 SUV of Ga-FAPI-RGD(V-25) in different types of tumors max Both were higher than those of FAP protein single targeting 68 Ga-FAPI-46, SUV max The increase was about 30-50%, proving that the dual-target design can increase the number and utilization efficiency of effective receptors in tumors and thus improve tumor uptake.

[0227] In summary, the present invention has developed a FAPI-RGD dual-targeting structure for FAP targets and integrin α v β3 targets have high affinity and can synergistically target FAP targets and integrin α in tumors v β3 targets, showing excellent metabolic kinetics, high tumor uptake and tumor retention time, and are expected to be used in the diagnosis or treatment of fibroblast activation protein (FAP) and / or integrin α v Diseases characterized by overexpression of β3.

[0228] Although the present invention has been described in detail above using general explanations, specific embodiments, and experiments, it will be apparent to those skilled in the art that modifications and improvements may be made based on the present invention. Therefore, such modifications and improvements, which do not depart from the spirit of the present invention, are intended to be within the scope of protection claimed herein.

Claims

1. A radionuclide-labeled dual-targeting compound, wherein: The structure of the dual-targeting compound is shown in Formula V-1 or V-25: The radionuclide is 68 Ga.

2. A pharmaceutical composition, characterized in that: A dual-targeting compound labeled with radionuclides according to claim 1 or a pharmaceutically acceptable hydrate, solvate or salt thereof.

3. A pharmaceutical composition, characterized in that: The invention is composed of any pharmaceutically acceptable carrier and / or excipient and the radionuclide-labeled dual-targeting compound according to claim 1 or a pharmaceutically acceptable hydrate, solvate or salt thereof.

4. The dual-targeting compound labeled with radionuclide according to claim 1, or the pharmaceutical composition according to any one of claims 2 to 3, in the preparation of a fibroblast activation protein (FAP) and / or integrin α-antigen for use in animals or human subjects. v Application of an imaging agent for imaging tumors characterized by overexpression of β3; the tumor is breast cancer, pancreatic cancer, small intestine cancer, colon cancer, rectal cancer, lung cancer, head and neck cancer, ovarian cancer, hepatocellular carcinoma, esophageal cancer, hypopharyngeal cancer, nasopharyngeal cancer, laryngeal cancer, myeloma, bladder cancer, bile duct cell carcinoma, clear cell renal carcinoma, neuroendocrine tumor, sarcoma, CUP, thymic cancer, glioma, cervical cancer or prostate cancer.

5. The use according to claim 4, characterized in that The glioma is a neuroglioma or an astrocytoma.

6. A kit comprising or consisting of: ① the radiolabeled dual-targeting compound according to claim 1 or the pharmaceutical composition according to any one of claims 2 to 3; and ② instructions for tumor imaging.

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