Tris-linker-linked dimerized label precursors and radioactive tracers derived therefrom

Abstract

The present invention relates to a radiotracer-labeled precursor having a structure (I) comprising a first targeting vector (TV1), a second targeting vector (TV2), a labeling group (MG) for complexation or covalent attachment of a radioisotope, a first spacer (S1), a second spacer (S2) and a third spacer (S3).

Description

[Technical field]

[0001] The present invention relates to dimerized labeled precursors and radioactive tracers derived therefrom by conjugation with radioisotopes for the diagnosis and treatment of cancer.

[0002] The label precursor has the following structure: [ka] (wherein TV1 is a first targeting vector, TV2 is a second targeting vector, MG is a chelator or linker for complexation or covalent attachment of a radioisotope, S1 is a first spacer, S2 is a second spacer, S3 is a third spacer, and TL is a tris linker). has.

[0003] The labeled precursors and radiotracers of the present invention are intended for nuclear medicine diagnostics (particularly positron emission tomography (PET) and single photon emission computed tomography (SPECT)) imaging, as well as nuclear medicine / endoscopic therapy of carcinomas and metastases of various cancer types. [Background technology]

[0004] In nuclear medicine diagnosis, tumor cells and metastases are detected using radioisotopes, such as gallium-68 (68Ga), technetium-99m ( 99m Tc), Scandium-44( 44 For the above types of metal radionuclides, complex-forming chelating agents are used.

[0005] Fluorine 18( 18 F), Iodine-123( 123 I), Iodine-131( 131 I), astatine 211( 211 Non-metallic radioisotopes, such as At, are covalently bound and do not require a chelating agent.

[0006] In comparison to diagnostics, nuclear medicine treatment uses higher radiation doses to destroy tumor tissue. For this purpose, for example, lutetium-177 ( 177 Lu), Yttrium 90 ( 90 Y), Iodine-131 ( 131 Beta-negative emitting radioisotopes such as I and actinium-225 ( 225 Alpha-emitting nuclides such as alpha-beta-Cu (Ac) are used. Alpha and beta-negative radiation have short ranges in tissue. This short range allows for localized irradiation of tumors and metastases with minimal radiation dose and damage to surrounding healthy tissue.

[0007] In the last few years, the combination of diagnostics and therapy (known among experts as theranostics) has gained importance. In this context, the same labeled precursor can be used for both diagnostics and therapy. This labeled precursor can be, for example, 68 Ga and 177 For example, Lu is simply labeled with a different radioisotope, and PET diagnosis and radiotherapy can be performed with essentially the same chemical compound. This allows the results of imaging nuclear medicine diagnosis to be translated into nuclear medicine treatment (theranostics) with improved dose regulation.

[0008] Labeling groups, especially chelating groups, change the configuration and chemical properties of the targeting vectors bound to them, generally affecting their affinity for tumor cells. Therefore, labeling precursors must be reevaluated for conjugation with radioisotopes, especially with regard to their biochemical and pharmacological properties in vitro and in vivo. The chemical bond between the labeling group and the targeting vector is crucial for the biological and nuclear medicine efficacy of the corresponding radiotracer.

[0009] Radioisotopes labeled with a radioactive precursor (also called radioactive tracer) are injected intravenously into the blood and then accumulate in tumor cells and metastatic lesions. To minimize the radiation dose in healthy tissues, radioisotopes with short half-lives of several hours to several days are used.

[0010] In summary, the labeled precursors and the radiotracers derived therefrom must meet the following requirements: 1. Rapid and effective complexation or binding of the respective radioisotope; 2. High selectivity for tumor cells and metastases over healthy tissues; 3. In vivo stability, i.e. biochemical stability in serum under physiological conditions; 4. High enrichment in tumors and metastases allows for accurate diagnosis and effective treatment; 5. Low retention and rapid excretion from healthy tissues and blood, minimizing dosage and toxicity to these organs.

[0011] Prostate cancer For men in industrialized countries, prostate cancer is the most common cancer and the third most deadly cancer. The disease is characterized by slow tumor growth, and the 5-year survival rate is nearly 100% for early diagnosis. However, survival rates are significantly reduced if the disease is only discovered after the tumor has metastasized. On the other hand, treating the tumor too early and too aggressively can unnecessarily and significantly impair the patient's quality of life. For example, surgical removal of the prostate can cause incontinence and impotence. A reliable diagnosis and information on the stage of the disease are essential for successful treatment while improving the patient's quality of life. A widespread diagnostic tool, alongside palpation of the prostate by a physician, is the measurement of tumor markers in the patient's blood. The most prominent marker of prostate cancer is the prostate-specific antigen (PSA) concentration in the blood. However, the significance of PSA concentration is controversial, as patients with slightly elevated PSA concentrations often do not have prostate cancer, and 15% of patients with prostate cancer do not show elevated PSA concentrations in the blood. A further target structure for prostate tumor diagnosis is the prostate-specific membrane antigen (PSMA). In contrast to PSA, PSMA is not detectable in blood. PSMA is a membrane-bound glycoprotein with enzymatic activity. Its function is to remove C-terminal glutamic acid from N-acetyl-aspartyl-glutamic acid (NAAG) and folate-(poly)-gamma-glutamic acid. PSMA is almost absent in normal tissues, but is highly overexpressed in prostate cancer cells, and its expression is closely correlated with the stage of the tumor. PSMA expression is also observed in about 40% of lymph node and bone metastases of prostate cancer.

[0012] The strategy for molecular targeting of PSMA is to use antibodies to bind to the protein structure of PSMA. In addition, ligands that correspond to the enzyme-binding pocket of PSMA are used. The central enzyme-binding pocket of PSMA contains two Zn2+ ligands that bind glutamic acid. 2+ions. An aromatic binding pocket is located in front of the central binding pocket. The PSMA protein is capable of inducible fit and expansion to various ligands, including inhibitors and enzymatically cleavable ligands. Thus, PSMA binds folate as well as NAAG, with the pteroate group docking in the aromatic binding pocket. Binding of inhibitors or substrates to the binding pocket of PSMA generally induces uptake into cells (endocytosis).

[0013] PSMA inhibitors are particularly suitable as targeting vectors for imaging radiopharmaceuticals and radiotracers. The radiolabeled inhibitors bind to the central binding pocket of PSMA and are not enzymatically transformed or cleaved, and the inhibitor / targeting vector is not detached from the radiolabel. Facilitated by endocytosis, the radiolabeled inhibitors are taken up by tumor cells and concentrated there.

[0014] Inhibitors with high affinity for PSMA (Scheme 1) generally have a glutamic acid motif and a structure that is not enzymatically cleaved. Highly effective PSMA inhibitors are 2-phosphonomethylglutaric acid or 2-phosphonomethylpentanedicarboxylic acid (2-PMPA), in which the glutamic acid motif is linked to a phosphonate group that is not cleaved by PSMA. In addition, urea-based PSMA inhibitors are also used, for example in clinically relevant radiotracers of PSMA-11 (Scheme 2) and PSMA-617 (Scheme 3).

[0015] In addition to the central binding pocket, it has proven advantageous to address the aromatic binding pocket of PSMA. For example, in the highly active radiotracers of the PSMA-11 type, the L-lysine-urea-L-glutamate binding motif (KuE) is linked to the aromatic HBED chelator (N,N'-bis[2-hydroxy-5-carboxyethyl]benzyl)ethylenediamine-N,N'-diacetate) via a hexyl (hexyl spacer).

[0016] In contrast, conjugation of L-lysine-urea-L-glutamate (KuE) to the non-aromatic DOTA chelator (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetate) establishes reduced affinity and enrichment in tumor tissue. 177 Lu and 225 To use DOTA chelators in radiopharmaceuticals with PSMA affinity to therapeutic radionuclides such as Ac, the spacer needs to be adapted. Controlled substitution of the hexyl spacer with various aromatic structures allowed the synthesis of PSMA-617-labeled precursors and the highly active 1,2-diphenyl-1,2-diphenyl-2,3-diphenyl-1,2-tetrahydrofuran (1,2-diphenyl-2,4-triphenyl-1,2-tetrahydrofuran)-1,2-tetrahydrofuran (1,2-diphenyl-2,4-triphenyl ... 177 The Lu-PSMA-617 radiotracer was found.

[0017] [ka]

[0018] [ka]

[0019] [ka]

[0020] Cancer stroma Malignant epithelial cells are a component of many tumors and tumor types and form the tumor stroma that surrounds the tumor, measuring at least 1–2 mm in size.

[0021] The tumor stroma (tumor microenvironment, TME) is composed of various non-malignant cell types and can account for up to 90% of the tumor mass. The TME plays an important role in tumor progression, i.e., tumor growth and metastasis.

[0022] The most important cellular components of the tumor stroma are the extracellular matrix containing various cytokines, endothelial cells, pericytes, macrophages, immune regulatory cells, and activated fibroblasts. The activated fibroblasts surrounding the tumor are called cancer-associated fibroblasts (CAFs).

[0023] During tumor evolution, CAFs change their morphology and biological functions. These changes are induced by intercellular communication between cancer cells and CAFs. Against this background, CAFs form an environment that promotes cancer cell proliferation. It has been shown that therapies targeting only cancer cells are insufficient. Effective therapies must also include the tumor microenvironment, i.e., CAFs.

[0024] In more than 90% of human epithelial cancers, CAFs overexpress fibroblast activation protein (FAP). Thus, FAP is a promising attack point for nuclear medicine diagnosis and therapy. Like PSMA, FAP inhibitors (FAPI or FAPi) in particular are suitable as targeting vectors for FAP-labeled precursors and radiotracers derived from them. Although the role of FAP in vivo has not yet been fully elucidated, it is known to be an enzyme with specific catalytic activity. It has both dipeptidyl peptidase (DPP) and prolyloligopeptidase (PREP) activities. Therefore, useful inhibitors are those that inhibit the DPP and / or PREP activities of FAP. Of importance is the selectivity of the inhibitor against other similar enzymes such as dipeptidyl peptidases DPPII, DPPIV, DPP8, DPP9, and prolyloligopeptidase (PREP). However, in the case of cancer types in which both FAP and PREP are overexpressed, it is also possible to use inhibitors that do not have high selectivity between PREP and FAP and inhibit both enzymes.

[0025] In 2013, a high affinity and highly selective inhibitor structure was developed and published, based on a modified glycine-proline unit linked to quinoline (Jansen et al. ACS Med. Chem. Lett. 2013, 4, 491-496). The desired compound, (S)-N-(2-(2-cyanopyrrolidin-1-yl)-2-oxoethyl)quinoline-4-carboxamide, is depicted in Scheme 4 (left side). Subsequent structure-activity studies (SAR) found compounds with improved affinity and selectivity, including the difluorinated derivative (S)-N-(2-(2-cyano-4,4'-difluoropyrrolidin-1-yl)-2-oxoethyl)quinoline-4-carboxamide, abbreviated UAMC1110, depicted in Scheme 4 (right side) (JANSEN et al, J. Med. Chem. 2014, 57 (7), 3053-3074).

[0026] [ka]

[0027] UAMC1110 forms the basis of various FAP-labeled precursors and targeting vectors for nuclear medicine radiotracers. Scheme 5 (top) shows the FAPI-04-labeled precursor as an example (LINDNER et al, J. Nucl. Med. 2018, 59 (9), 1415-1422). Scheme 5 (bottom) shows further FAP-labeled precursors, including a DOTA chelator. The DOTA chelator is linked to the quinoline unit of the pharmacological FAPi targeting vector via the 4-aminobutoxy, squaric acid and ethylenediamine groups.

[0028] [ka]

[0029] Bone metastases Farnesyl pyrophosphate synthase (FPPS), an enzyme in the HMG-CoA reductase (mevalonate) pathway, is expressed in bone metastases. Inhibition of FPPS suppresses the production of farnesyl, a molecule important for docking of signaling proteins to the cell membrane. As a result, apoptosis of carcinogenic bone cells is induced. FPPS is inhibited by bisphosphonates such as alendronate, pamidronate, and zoledronate. For example, BPAMD tracers and pamidronate-targeted vectors are routinely used to treat bone metastases.

[0030] A particularly effective tracer for the treatment of bone metastases has proven to be zoledronate (ZOL), a hydroxybisphosphonate with a heteroaromatic imidazole unit. NODAGA- and DOTA-conjugated zoledronic acid chelators (Scheme 6) are currently the most potent radiotherapeutic agents for bone metastases.

[0031] [ka]

[0032] The prior art discloses a number of labelled precursors for cancer diagnosis and theranostics using radioisotopes.

[0033] For example, WO 2015 / 055318 discloses radiotracers for the diagnosis and theranostics of prostate or epithelial cancer, in particular the PSMA-617 labeled precursor shown in Scheme 3.

[0034] Monomeric radiotracers with targeting vectors (TVs) play a central role in nuclear medicine and deserve the name "precision oncology". Recently, dimerized labeling precursors with two targeting vectors have also been investigated. Here, it is hypothesized that radiotracers with two targeting vectors have a higher affinity. The prior art discloses "linear" homodimerized labeling precursors with two identical targeting vectors, each bound to a central chelator, and the first studies in this regard support this hypothesis (Zia, NA et al. Angw. Chem. Int. Ed. 2019, 58, 14991-14994). Summary of the Invention

[0035] In the present invention, homo- and heterodimerized label precursors are provided for the first time, which comprise two identical or two different targeting vectors linked via a label group and a tris linker (TL).The tris linker (TL) used is, for example, an amino acid residue, such as a lysine residue or a glutamic acid residue in particular.

[0036] The Trislinker (TL) of the present invention decouples the chelator and the targeting vector with respect to sterically and electronically induced interactions. The coupling of the Trislinker (TL) and the chelator is designed so as not to impair the conjugation with clinically relevant radioisotopes. For this purpose, it is possible to utilize couplings that have been found to be useful for monomeric labeling precursors. The present invention allows for independent (orthogonal) optimization of radioisotope conjugation, affinity, pharmacokinetics and pharmacodynamics of homo- and heterodimeric radioisotopes. In contrast, known linear homodimerization labeling precursors require complex molecular engineering, which is often accompanied by dysfunction. [Brief description of the drawings]

[0037] [Figure 1] Reaction kinetics of [68Ga]Ga-DOTA.Glu.(FAPi)2 (1M HEPES (pH=4.5 / 5.5), 95℃, n=3) [Diagram 2] [68Ga]Ga-DOTA.Glu.(FAPi)2 (20 nmol, 100 MBq, 30 min, 95 °C, 20-90% CAN (in 20 min): radiochemical purity (RCP) >98%) [Diagram 3] Stability of [68Ga]Ga-DOTA.Glu.(FAPi)2 in HS and PBS at 37°C for 2 hours (~20MBq / mL) [Figure 4] Kinetics of [177Lu]Lu-DOTA.Glu.(FAPi)2 (1M HEPES (pH=5.5), 95°C, n=3 (50MBq) or n=2 (100MBq)) [Diagram 5] [177Lu]Lu-DOTA.Glu.(FAPi)2 (4 nmol, 100 MBq, 60 min, 95 °C, 20-50% ACN (in 20 min): RCP>99%) [Figure 6] Stability of [177Lu]Lu-DOTA.Glu.(FAPi)2 in HS and PBS at 37°C for 2 hours (~20MBq / mL) [Figure 7] Kinetics of [68Ga]Ga-DOTAGA.Glu.(FAPi)2 (1M HEPES (pH=4.5), 95°C, n=4 (100MBq) or n=2 (400MBq) [Figure 8] [68Ga]Ga-DOTAGA.Glu.(FAPi)2 (40nmol, 400MBq, 30 minutes, 95℃, 20~70%CAN (in 20 minutes): RCP>97%) [Figure 9] Stability of [68Ga]Ga-DOTAGA.Glu.(FAPi)2 in HS and PBS at 37°C for 2 hours (~20MBq / mL) [Figure 10] Kinetics of [177Lu]Lu-DOTAGA.Glu.(FAPi)2 (1M HEPES (pH=5.5), 95℃, n=3 (50MBq) or n=1-2 (100MBq)) [Figure 11] [177Lu]Lu-DOTAGA.Glu.(FAPi)2 (4nmol, 100MBq, 60 minutes, 95℃, 20~50%CAN (in 20 minutes): RCP>99%) [Figure 12] Stability of [177Lu]Lu-DOTAGA.Glu.(FAPi)2 in HS and PBS at 37°C for 2 hours (~20MBq / mL) [Figure 13] Kinetics of [225Ac]Ac-DOTAGA.Glu.(FAPi)2 (0.1M sodium ascorbate (pH=7), 95℃, n=3, 1 hour and 1 day exposure) [Figure 14] [225Ac]Ac-DOTAGA.Glu.(FAPi)2 (0.7-0.8MBq / mL) in HS and PBS at 37°C for 20 days each [Figure 15] Reaction kinetics of [68Ga]Ga-DATA5m.Glu.(FAPi)2 (0.5M HEPES (pH=5.5), 95℃, n=4) [Figure 16] [68Ga]Ga-DATA5m.Glu.(FAPi)2 (20 nmol, 50 MBq, 30 min, RT, 10-90% CAN (in 10 min): RCP>98%) [Figure 17] Stability of [68Ga]Ga-DATA5m.Glu.(FAPi)2 in HS and PBS at 37°C for 2 hours (approximately 20MBq / mL) [Specific Description of the Invention]

[0038] The FAP-labeled precursors and radioactive tracers of the present invention further have the following characteristics:

[0039] 1.High binding affinity to FAP, IC 50 Values ​​are in the nanomolar to subnanomolar range.

[0040] 2. Excellent binding specificity to competing PREP proteases and the DPPIV family, in particular DPP4 (a type II integral protein with intracellular and extracellular forms), as well as DPP8 and DPP9 (intracellular proteins) (Hamson et al, Proteomics Clin.). The binding affinities of the compounds of the invention are now in the micromolar range, so that the ratio of binding to the FAP target and to competing proteases is usually greater than 1000. This ratio is expressed as the IC 50 The selectivity index (SI) between the values ​​can be described (see Table 2). This significantly reduces the accumulation of the radiolabeled compounds of the present invention in tissues outside the tumor microenvironment (healthy tissues), ensuring extremely high contrast in molecular imaging.

[0041] 3. High hydrophilicity (low logD value) results in a short residence time in the blood of the compounds of the invention, which ensures extremely high contrast in molecular imaging between the tumor and the surrounding perfused healthy tissue.

[0042] 4. Rapid concentration and long residence time of the compounds of the invention in the tumor microenvironment, which allows to ensure the administration of high doses to the tumor and its environment, even when using relatively long-lived radioisotopes such as lutetium-177 and actinium-225 in intracellular radiotherapy.

[0043] 5. Rapid excretion via the kidneys and bladder results in a short residence time of the compounds of the invention in healthy tissues, which not only ensures extremely high contrast in molecular imaging between the tumor and the surrounding blood-supplied healthy tissues, but also low radiation stress for the patient.

[0044] Furthermore, the concept of the present invention can be easily applied to compounds with two different targeting vectors. For example, it is possible to combine a targeting vector (bisphosphonate) that deals with bone metastasis with a targeting vector (PSMA inhibitor) that deals with prostate cancer. This has the advantage that in prostate cancer patients with bone metastasis, it can deal with bone metastasis more than radiopharmaceuticals with only PSMA targeting vectors. The reason for this is the high heterogeneity of PSMA expression in bone metastasis of patients, which PSMA inhibitor structure can only deal with insufficiently in some situations.

[0045] Only in about 90% of patients suffering from prostate cancer, there is overexpression of PSMA.Therefore, in the context of the present invention, heterodimerized labeled precursors with FAP targeting vector and PSMA targeting vector are also envisioned.Such heterodimerized labeled precursors address both tumor tissues expressing PSMA and stromal cells expressing FAP associated with tumors.Therefore, it is also possible to detect and visualize prostate cancers and metastases that do not overexpress PSMA by PET or SPECT.

[0046] The object of the present invention is to provide labeled precursors and radiotracers for improving the diagnosis and theranostics of cancer diseases, particularly those that provide high selectivity and specificity, effective radioisotope complexation and binding, and rapid absorption and systemic excretion.

[0047] The objective is to have the following structure: [ka] (wherein TV1 is a first targeting vector, TV2 is a second targeting vector, MG is a chelator or linker for complexation or covalent attachment of a radioisotope, S1 is a first spacer, S2 is a second spacer, S3 is a third spacer, and TL is a tris linker). This is achieved by a labeled precursor having the following structure:

[0048] Suitable embodiments of the label precursors of the present invention are characterized by the following features in any combination, to the extent that the features are not mutually exclusive: TV1 and TV2 can independently have the following structures[1]-

[43] : [ka] [ka] [ka] [ka] [ka] [ka] (In the formula, Structures [1]-[8] and

[43] represent peptides, X=H or F Y=H, CH3, CH(CH3)2, C(CH3)3 or (CH2) n CH3, n = 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10); Selected from; · TV1 is the same as TV2 (TV1=TV2); · TV1 and TV2 are different from each other (TV1 ≠ TV2); · TV1 has the structure

[13] ; · TV1 has the structure

[14] ; · TV2 has the structure

[13] ; · TV2 has the structure

[14] ; ·TV1 and TV2 each have the structure

[13] ; ·TV1 and TV2 each have the structure

[14] ; TV1 has one of the structures [9]–

[12] and TV2 has one of the structures

[13] or

[14] ; TV1 has one of the structures [9]–

[12] and TV2 has one of the structures

[40] or

[41] ; TV2 has one of the structures [9]–

[12] and TV1 has one of the structures

[13] or

[14] ; TV2 has one of the structures [9]–

[12] and TV1 has one of the structures

[40] or

[41] ; S1, S2, and S3 are independently selected from the following structures: [ka] (In the formula, A, B, and C each independently represent an amide group, a carboxamide group, a phosphinate group, an alkyl group, a triazole group, a thiourea group, an ethylene group, a maleimide group, an amino acid residue, [ka] (In the formula, s is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; p, q and r are independently selected from the set {0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20}) selected from the group consisting of; S1, S2, and S3 are independently selected from the following structures: [ka] having; S1, S2, and S3 are independently a peptide group having the structure: [ka] is; S1, S2, and S3 are independently a dipeptide group having the structure: [ka] is; S1, S2, and S3 are independently a tripeptide group having the structure: [ka] is; Side chain R 1 , R 2 , R 3 are independently -H, -CH3, -CH(CH3)2, -CH2CH(CH3)2, -CH(CH3)-CH2CH3, -CH2-Phe, -CH2-Phe-OH, -CH2SH, -(CH2)2-S-CH3, -CH2OH, -(CH)(OH)(CH3), -(CH2)4NH2, -(CH2)3NH(C=NH)NH2, -CH2COOH, -(CH2)2COOH, -CH2(C=O)NH2, -(CH2)2(C=O)NH2, [ka] and the peptide spacers S1, S2, S3 are selected from the group consisting of: ·MG, 43 Sc, 44 Sc, 47 Sc, 55 Co, 62 Cu, 64 Cu, 67 Cu, 66 Ga, 67 Ga, 68 Ga, 89 Zr, 86 Y, 90 Y, 89 Zr, 90 Nb, 99m Tc, 111 In, 135 Sm, 140 Pr, 159 Gd, 149 Tb, 160 Tb, 161 Tb, 165 Er, 166 Dy, 166 Ho, 175 Yb, 177 Lu, 186 Re, 188 Re, 211 At, 212 Pb, 213 Bi, 225 Ac and 232It is a chelating agent for the complexation of radioisotopes from the group consisting of Th; MG has the following: H4pypa, EDTA (ethylenediaminetetraacetic acid), EDTMP (diethylenetriaminepenta(methylenephosphonic acid)), DTPA (diethylenetriaminepentaacetate) and its derivatives, NOTA (nona-1,4,7-triaminetriacetate) and its derivatives (e.g., NODAGA (1,4,7-triazacyclononane, 1-glutaric acid, 4,7-acetic acid)), TRAP (triazacyclononanephosphinic acid), NOPO (1,4,7-triazacyclononane-1,4-bis[methylene(hydroxymethyl)phosphinic acid]), phosphinic acid]-7-[methylene(2-carboxyethyl)phosphinic acid]), DOTA (dodeca-1,4,7,10-tetraaminetetraacetate), DOTAGA (2-(1,4,7,10-tetraazacyclododecane-4,7,10)pentanedioic acid) and other DOTA derivatives, TRITA (trideca-1,4,7,10-tetraaminetetraacetate), TETA (tetradeca-1,4,8,11-tetraaminetetraacetate) and its derivatives, PEPA (pentadeca-1,4,7,10,13-pentaaminepentaacetate) ), HEHA (hexadeca-1,4,7,10,13,16-hexamine hexaacetate) and its derivatives, HBED (N,N'-bis(2-hydroxybenzyl)ethylenediamine-N,N'-diacetate) and its derivatives (e.g., HBED-CC (N,N'-bis[2-hydroxy-5-carboxyethyl]benzyl)ethylenediamine-N,N'-diacetate)), DEDPA and its derivatives (e.g., H2dedpa (1,2-[[6-(carboxyl)pyridin-2-yl]methylamine]ethane) and H 4octapa (1,2-[[6-(carboxyl)pyridin-2-yl]methylamine]ethane-N,N'-diacetate)), DFO (deferoxamine) and its derivatives, trishydroxypyridinone (THP) and its derivatives (e.g., H3THP-Ac and H3THP-mal (YM103)), TEAP (tetraazacyclodecane phosphinic acid) and its derivatives, AAZTA (6-amino-6-methylperhydro-1,4-diazepane N,N,N',N'-tetraacetate) and its derivatives (e.g., AAZTA 5(5-[(6-amino)-1,4 diazepane]pentanoic acid-N,N,N',N'-tetraacetate)), DATA 5m (5-[[6-(N-methyl)amino]-1,4-diacetate-1,4-diazepane]-pentanoic acid-N,N',N'-triacetate); Sarcofazine SAR (1-N(4-aminobenzyl)-3,6,10,13,16,19-hexazabicyclo[6.6.6]eicosane-1,8-diamine) and its derivatives (e.g., (NH2)2SAR(1,8-diamino-3,6,10,13,16,19-hexazabicyclo[6.6.6]eicosane)), N4(3-[(2'-aminoethyl)amino]-2-[(2'' N4 derivatives, PnAO (6-(4-isothiocyanatobenzyl)-3,3,9,9-tetramethyl-4,8-diazaundecane-2,10-dione dioxime) and derivatives (e.g., BMS181321 (3,3'-(1,4-butanediyldiamino)-bis(3-methyl-2-butanone) dioxime)), M AG2 (mercaptoacetylglycylglycine) and its derivatives, MAG3 (mercaptoacetylglycylglycine) and its derivatives (e.g., N3S-adipat), MAS3 (mercaptoacetylserylserylserine) and its derivatives, MAMA (N-(2-mercaptoethyl)-2-[(2-mercaptoethyl)amino]acetamide) and its derivatives, EC (ethylenedicysteine) and its derivatives, dmsa (dimercaptosuccinic acid) and its derivatives, DADT (diaminodithiol), DADS (diaminodisulfide), N2S2 chelating agents and their derivatives, aminothiols and their derivatives; salts of the above chelating agents; hydrazine nicotinamide (HYNIC) and hydrazine nicotinamide derivatives. is a chelating agent selected from the group consisting of: The labeling group MG has the following structures

[44] ,

[45] ,

[46] and

[47] : [ka] having a structure selected from the group consisting of: The labeling group MG has the following structures

[48] ,

[49] ,

[50] and

[51] : [ka] having a structure selected from the group consisting of: · MG is DOTA (dodeca-1,4,7,10-tetraaminetetraacetate); ·MG, DATA 5m (1,4-bis(carboxymethyl)-6-[methylcarboxymethylamino]-6-pentanoic acid-1,4-diazepane); · MG is AAZTA (1,4-bis(carboxymethyl)-6-[bis(carboxymethyl)amino]-6-pentanoic acid-1,4-diazepane); ·MG, 18 F, 131 I or 211 is a linker for covalent attachment of At; MG has the following: [ka] Selected from; ·MG, 18 F, 131 I or 211 The following with a leaving group X for substitution by At: [ka] is a linker of the type MG contains a leaving group X selected from the group bromine (Br), chlorine (Cl) or iodine (I), tosyl (Ts), brosylate (Bs), nosylate (Nos), 2-(N-morpholino)ethanesulfonic acid (MES), triflate (Tf) and nonaflate (Non); The Tris linker TL has the following structure

[52] ~

[64] : [ka] Selected from one of; The Tris linker TL has the following structure:

[65] ~

[0116] : [ka] [ka] (selected from one of the following) is selected from one of the following:

[0049] In the peptide or structural formulas [1]-[8], the following terms are used for synthetic amino acids: Aph(Hor) = 4-[2,6-dioxohexahydropyrimidine-4-carbonylamino]-L-phenylalanine Cpa = 4-chlorophenylalanine D-Aph(Cbm) = D-4-aminocarbamoylphenylalanine Pal = 2-, 3- or 4-pyridylalanine

[0050] radioactive isotope 18 F, 131 I or 211 The labeling group MG for the covalent attachment of At includes, in particular, a leaving group X selected from the group consisting of bromine (Br), chlorine (Cl), iodine (I), tosyl (-SO2-CH4-CH3; abbreviated as "Ts"), brosylate (-SO2-CH4-Br; abbreviated as "Bs"), nosylate or nitrobenzenesulfonate (-OSO2-CH4-NO2; abbreviated as "Nos"), 2-(N-morpholino)ethanesulfonic acid (-SO3-(CH2)2-N(CH2)4O; abbreviated as "MES"), triflate or trifluoromethanesulfonyl (-SO2CF3; abbreviated as "Tf") or nonaflate (-OSO2-CF9; abbreviated as "Non").

[0051] The present inventors have surprisingly found that the above-mentioned dimerized labeled precursors or radioactive tracers derived therefrom with two targeting vectors TV1 and TV2 have non-specific enrichment (off-target exposure) and much higher enrichment (target exposure) in tumor tissues at the same systemic dose compared to monomeric radioactive tracers with one targeting vector. This advantageous property is expected to be due to increased docking probability and / or selectivity.

[0052] The targeting vectors TV1 and TV2 used according to the present invention have high binding affinity, in particular to membrane tumor markers such as PSMA (prostate-specific membrane antigen), FAP (fibroblast activation protein) and FPPS (farnesyl pyrophosphate synthase).

[0053] The heterodimerized labeled precursor and radioactive tracer of the present invention can be used to address various tumor tissues and metastatic lesions.This is advantageous for the treatment of bone metastasis induced by prostate cancer.Particularly useful for this purpose is the labeled precursor or radioactive tracer with the first targeting vector TV1 (PSMA targeting vector) for PSMA and the second bone-directed targeting vector TV2 (FPPS targeting vector) for FPPS.

[0054] The labeled precursors and radioactive tracers of the present invention are also suitable for addressing tumor stroma. For example, in the case of triple-negative breast cancer (TNBC), there are no specific receptors on the surface of carcinogenic cells that allow direct addressing. One option here is the "indirect" addressing of tumor stroma. In the case of TNBC, the tumor stroma contains cancer-associated fibroblasts (CAFs) and modified endothelial cells (ECs), which overexpress FAP and PSMA, respectively. Therefore, both homodimerized precursors with PSMAi, FAPi or bisphosphonate vectors, and heterodimerized labeled precursors with a first PSMA-targeting vector and a second FAP-targeting vector are suitable for the diagnosis and treatment of TNBC.

[0055] The situation is similar for PSMA-negative prostate cancer, i.e., prostate cancer that does not overexpress PSMA, which represents approximately 10% of prostate cancer cases. However, PSMA-negative tumors and metastases can be diagnosed and treated by addressing the tumor stroma with the help of FAP-targeting vectors. Thus, heterodimerized labeled precursors with a first PSMA-targeting vector and a second FAP-targeting vector are suitable for the comprehensive diagnosis and treatment of PSMA-positive and PSMA-negative prostate cancer.

[0056] 177 Lu and 225 Theranostic application of radioisotopes such as Ac to the tumor stroma directly damages the tumor microenvironment essential for progression and induces “indirect” radiation damage (radiation-induced bystander effect, RIBE) in adjacent cancer cells.

[0057] The spacers S1, S2, and S3 function as steric spacers and pharmacokinetic modifiers that optimize the biochemical function (binding affinity to the target), the radiochemical function (stable complexation or binding of the radioisotope) of the labeling group, and the blood half-life (hydrophilicity) of the targeting vector. The spacers S1, S2, and S3 preferably contain structural elements that improve affinity to PSMA, such as squaramide or other aromatic units.

[0058] The Tris-linker TL creates the prerequisites for an orthogonal, sterically and pharmacokinetically optimized coupling of the labeling group MG with the two targeting vectors TV1 and TV2, analogous to established monomeric radiopharmaceuticals with only one targeting vector. Thus, the present invention allows the synthesis of effective labeling precursors and radiotracers with high theranostic potency.

[0059] The present invention relates to a method for producing a medicament comprising the steps of: · 43 Sc, 44 Sc, 47 Sc, 55 Co, 62 Cu, 64 Cu,67 Cu, 66 Ga, 67 Ga, 68 Ga, 89 Zr, 86 Y, 90 Y, 89 Zr, 90 Nb, 99m Tc, 111 In, 135 Sm, 140 Pr, 159 Gd, 149 Tb, 160 Tb, 161 Tb, 165 Er, 166 Dy, 166 Ho, 175 Yb, 177 Lu, 186 Re, 188 Re, 211 At, 212 Pb, 213 Bi, 225 Ac and 232 a radioisotope complexed to the label precursor as described above, selected from the group including Th; or · 18 F, 131 I and 211 a radioisotope covalently attached to said label precursor selected from the group including At; The radioactive tracer comprises:

[0060] In a suitable embodiment of the invention, the radioactive tracer is ·NOTA (Nona-1,4,7-triamine triacetate), DATA 5m a labeling group MG selected from the group consisting of (5-[[6-(N-methyl)amino]-1,4-diacetate-1,4-diazepane]pentanoic acid-N,N',N'-triacetate) and NODAGA (1,4,7-triazacyclononane, 1-glutaric acid, 4,7-acetate); and Radioactive aluminum compound complexed to a label precursor [ 18 F] fluoride (i.e. [ 18 F]AlF The label precursor comprises one of the above label precursors having the formula:

[0061] In the case of the labelling group MG in the form of a chelating agent, said chelating agent is 43 Sc, 44 Sc, 47 Sc, 55 Co, 62 Cu, 64 Cu, 67 Cu, 66 Ga, 67 Ga, 68 Ga, 89 Zr, 86 Y, 90 Y, 89 Zr, 90 Nb, 99m Tc, 111 In, 135 Sm, 140 Pr, 159 Gd, 149 Tb, 160 Tb, 161 Tb, 165 Er, 166 Dy, 166 Ho, 175 Yb, 177 Lu, 186 Re, 188 Re, 211 At, 212 Pb, 213 Bi, 225 Ac and 232 The radioisotope serves as a label with a radioisotope selected from Th.

[0062] The invention therefore includes radioactive tracers obtained from said labeled precursors by complexation with a radioisotope, said radioisotope being 43 Sc, 44 Sc, 47 Sc, 55 Co, 62 Cu, 64 Cu, 67 Cu, 66 Ga, 67 Ga, 68 Ga, 89 Zr, 86 Y, 90 Y, 89 Zr, 90 Nb, 99m Tc,111 In, 135 Sm, 140 Pr, 159 Gd, 149 Tb, 160 Tb, 161 Tb, 165 Er, 166 Dy, 166 Ho, 175 Yb, 177 Lu, 186 Re, 188 Re, 211 At, 212 Pb, 213 Bi, 225 Ac and 232 It is selected from the group including Th.

[0063] Chelating Agents The prior art discloses a number of chelating agents for the complexation of radioisotopes. Scheme 7 shows examples of chelating agents that may be used according to the present invention.

[0064] [ka] [ka] [ka] [ka] [ka] [ka] [ka]

[0065] Amide Coupling In the present invention, functional groups such as chelator Chel, targeting vector TV1 and TV2, spacer S1, S2, S3, and tris linker TL are preferably linked by amide coupling reaction. Amide coupling, which forms protein backbone, is the most commonly used reaction in medicinal chemistry. A general example of amide coupling is shown in Scheme 8.

[0066] [ka]

[0067] The amide coupling strategy opens a simple route for the synthesis of new compounds, since carboxylic acid and amine derivatives are virtually unlimitedly available. Those skilled in the art are aware of numerous reagents and protocols for amide coupling. The most commonly used amide coupling strategy is based on the condensation of a carboxylic acid with an amine. For this purpose, the carboxylic acid is generally activated. Before activation, the remaining functional groups are protected. The reaction is carried out in two steps, either by directly transforming the activated carboxylic acid in one reaction medium (single pot) or by isolating the activated "trapped" carboxylic acid and reacting it with the amine.

[0068] The carboxylic acid is then reacted with a coupling reagent to form a reactive intermediate, which can be isolated or reacted directly with an amine. Numerous reagents are available for activating carboxylic acids, including acid halides (chlorides, fluorides), azides, anhydrides, and carbodiimides. In addition, esters such as pentafluorophenyl and hydroxysuccinimide esters can be generated as reactive intermediates. Intermediates formed from acyl chlorides and azides are highly reactive. However, harsh reaction conditions and high reactivity are barriers to their use with sensitive substrates and amino acids. Therefore, amide coupling strategies using carbodiimides such as DCC (dicyclohexylcarbodiimide) and DIC (diisopropylcarbodiimide) open up a wide range of applications. Additives are often used to improve reaction efficiency, especially in solid-phase synthesis. Aminium salts are very efficient peptide coupling reagents, with short reaction times and minimal racemization. With some additives, it is possible to completely avoid racemization, for example HOBt. Aminium reagents are used in equimolar amounts to the carboxylic acid to prevent excessive reaction with the free amines of the peptide. Phosphonium salts react with carboxylic acids and generally require two equivalents of a base, e.g., DIEA. A major advantage of phosphonium salts over iminium reagents is that the phosphonium does not react with the free amino groups of the amine component. This allows the coupling of an equimolar ratio of acid to amine, avoiding intramolecular cyclization of linear peptides and the overuse of expensive amine components.

[0069] For a review of amide coupling strategies and reagents, see: ·Analysis of Past and Present Synthetic Methodologies on Medicinal Chemistry: Where Have All the New Reactions Gone?; DG Brown, J. Bostroem; J. Med. Chem. 2016, 59, 4443-4458; ·Peptide Coupling Reagents, More than a Letter Soup; A. El-Faham, F. Albericio; Chem. Rev. 2011, 111, 6557-6602; ·Rethinking amide bond synthesis; VR Pattabiraman, JW Bode; Nature, Vol. 480 (2011) 22 / 29; Amide bond formation: beyond the myth of coupling reagents; E. Valeur, M. Bradley; Chem. Soc. Rev., 2009, 38, 606-631.

[0070] Many of the chelating agents used according to the invention, such as DOTA and its derivatives, have one or more carboxy or amine groups and can therefore be attached to the spacer S3 in a straightforward manner using one of the amide coupling strategies known in the art.

[0071] In the following the meaning of some terms used in the context of the present invention will be clarified.

[0072] Theranostics : Cancer diagnosis and treatment using nuclear medicine radiotracers and similar targeted vectors.

[0073] Labeling precursors : a compound comprising a first and a second targeting vector, and a chelator or functional group for labeling with a radioisotope.

[0074] Radioactive tracers : A radioisotope-labeled precursor for nuclear medicine diagnostics and theranostics that is used in low concentrations without affecting the patient's metabolism.

[0075] target: A biological target structure, especially a (membrane-bound) receptor, protein, enzyme, or antibody in vivo to which a targeting vector binds.

[0076] Targeting Vectors : A chemical group or radical that functions as a ligand, agonist, antagonist, or inhibitor for a biological target (protein, enzyme, receptor, etc.) and has high binding affinity for that target.

[0077] Trislinker : a structural unit having three functional groups for binding to a first, second and third spacer for a first and second targeting vector and a labeling group.

[0078] Spacer : A structural unit, group or radical that connects the first and second targeting vectors and the labeling group to the Tris linker and functions as a steric and / or pharmacokinetic modulator. EXAMPLES

[0079] The compound (S)-6-(4-aminobutoxy)-N-(2-(2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)-quinoline-4-carboxamide is hereinafter abbreviated as FAPi-NH2. [ka]

[0080] material and method Nuclear magnetic resonance (NMR) spectroscopy: NMR spectra were recorded in deuterated solvents on an Avance II 400 (400 MHz) spectrometer equipped with a 5 mm BBFO sample head (z gradient) from Bruker (Reinstetten, Germany). Chemical shifts δ (ppm) are based on the proton signals of the deuterated solvent relative to the tetramethylsilane standard (=0.00 ppm). Calculated coupling constants were reported in Hertz (Hz). Spin multiplicities were abbreviated as follows: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet or combinations thereof. Spectra were analyzed using MestReNova 14.2.0 software from Mestrelab Research (Santiago de Compostela, Spain).

[0081] ESI-LC / MS: ESI-LC / MS mass spectra were recorded using an Agilent Technologies 1220 Infinity LC coupled with an Agilent Zorbax SB-C18 column (21 × 50 mm, 1.8 μm) and an Agilent Technologies 6130B single quadrupole LC / MS system with a linear gradient of acetonitrile (ACN) / Milli-Q® water (HO) + 0.05% formic acid (HFo) at a flow rate of 0.5 mL / min.

[0082] ESI-HPLC / MS: HPLC-MS measurements were performed using an Agilent Technologies G6545A Q-ToF equipped with electrospray ionization in combination with a 1260 Infinity II HPLC system (Agilent Technologies) (G7111B 1260 quaternary pump, G7129A 1260 vial sampler, G7116A multicolumn thermostat). Separation was performed using an Agilent Poroshell 120 EC-C8 column (2.1 × 100 mm, 2.7 μm) with HO + 2% ACN / ACN + 2% HO + 0.05% HFo at a flow rate of 0.1 mL / min.

[0083] RP-HPLC: Semi-preparative reversed-phase high-pressure liquid chromatography (RP-HPLC) was performed using a Merck Hitachi LaChrom-HPLC (7000 series) equipped with an L-7100 pump, an L-7400 UV detector (λ = 254 nm), a D-7000 interface, and an autosampler. Separation was performed on a Phenomenex Synergi Max-RP C18 column (250 × 10 mm, 4 μm) with a linear gradient of ACN / HO + 0.1% trifluoroacetic acid (TFA) at a flow rate of 5 mL / min.

[0084] radioactive tlc Radio-TLC was evaluated using a Raytest CR-35 Bio Test-Imager and AIDA software.

[0085] Radioactive HPLC Analytical radio-HPLC was performed using the same Merck Hitachi LaChrom-HPLC (7000 series). Separation was performed using a Phenomenex Luna C18 column (250 × 4.6 mm, 5 μm) with a linear gradient of ACN / H2O + 0.1% TFA at a flow rate of 1 mL / min. The radio-HPLC was additionally equipped with an Elysia Raytest Ramona radiodetector. 68 The energy window for Ga measurements is 100-1200 keV. 177 The energy window for Lu measurements is set to 100–250 keV.

[0086] Stability measurements The stability of each labeled compound in human serum (HS) and phosphate buffered saline (PBS) was examined by incubating approximately 10 MBq of labeled solution in 0.5 mL of HS or PBS at 37°C for approximately two half-lives (n=3 in each case) ( 68 Ga: 2 hours, 177 Lu:14 days).

[0087] Measurement of logD (measurement of lipophilicity): The logD value of each labeled compound was measured by diluting the labeled solution 4-fold with approximately 10 MBq each time and diluting it with PBS to 700 μL. 700 μL of 1-octanol was added each time, and the mixture was vigorously shaken for 2 min and then centrifuged for 1 min. The organic and aqueous phases were separated and 400 μL of each were isolated. 3 μL (PBS) and 6 μL (1-octanol) samples were dropped onto a TLC plate. Most of the activity was in the aqueous phase. This was then diluted to 700 μL, extracted twice more with 1-octanol, and dropped onto the TLC plate again. The TLC was exposed for approximately 5 min to determine the integral value of each spot (octanol phase: IO, PBS aqueous phase: IW). The difference in volume between VO = 6 μL and VW = 3 μL was taken into account in the calculation of the logD value using equation (1):

number

[0088] For the evaluation, the values ​​of the second and third extractions of four batches were averaged.

[0089] In vitro assays The rhFAP (fibroblast activation protein), PREP (prolyl endopeptidase), DPP4 (dipeptidyl peptidase IV), DPP8 (dipeptidyl peptidase VIII), and DPP9 (dipeptidyl peptidase IX) enzymes were expressed and subsequently purified before use in in vitro assays.

[0090] I C 50 Measurements were performed on an Infinite 200 instrument (Tecan Group) and evaluated with Magellan software.

[0091] Data were evaluated with GraFit 7 using a nonlinear fit according to the following equation:

number

[0092] Example 1: FAPi-NH 2 [ka]

[0093] Scheme 10 shows the synthesis of FAPi-NH2.

[0094] 4-Bromoethylamine To 4-aminobutanol (5.39 g, 60.47 mmol, 1.00 equiv.) was slowly added 70 mL of 47% hydrobromic acid, and the mixture was heated under reflux for 4 h. A colorless solid (13.521 g, 58.04 mmol, 96%) was obtained, which was used directly in the next synthetic step without further purification. MS (ESI-positive): m / z (%) = 152.0 (100, [M+H] + ), 154.0 (98, [M+H] + ), C4H 10 Calculated for BrN: 151.00[M]. 1 HNMR (400MHz, MeOD): δ [ppm] = 3.51 (t, J=6.4 Hz, 2H), 2.98 (t, J=7.6 Hz, 2H), 2.00-1.78 (m, 4H).

[0095] tert-Butyl (4-bromobutyl)carbamate 4-Bromobutylamine (7.01 g, 30.09 mmol, 1.0 equiv.) was dissolved in dry THF (34 mL) together with di-tert-butyl dicarbonate (Boc2O, 7.34 g, 33.63 mmol, 1.12 equiv.) under argon atmosphere. TEA (4.6 mL, 36.12 mmol, 1.2 equiv.) was then added. MeOH (36 mL) was added to the formed suspension until the solution became clear again, and then it was stirred at RT for 19 h. The aqueous solution was extracted with Et2O (5×80 mL) and the combined organic phases were washed once each with small amounts of NaHCO3 and brine and dried over Na2SO4. The solvent was removed under reduced pressure. Column chromatography (CH / EA 5:1) gave a colorless solid (5.08 g, 20.15 mmol, 66%). MS (ESI-positive): m / z (%) = 196.0 (100, [M-tBu] + ), 198.0 (100, [M-tBu] + ), C9H 18 Calculated for BrNO2: 251.05[M]. 1 HNMR (400MHz, CDCl3): δ [ppm] = 3.36-3.21 (m, 4H), 1.86-1.76 (m, 4H), 1.43 (s, 9H).

[0096] Boc-Gly-Pro-CONH 2 (tert-Butyl (S)-(2-(2-carbamoyl-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)carbamate) Boc-Gly-OH (1.38 g, 7.88 mmol, 1.05 eq.) and HBTU (3.12 g, 8.20 mmol, 1.1 eq.) were dissolved in dry DCM (8 mL) and DMF (8 mL) under argon. DIPEA (1.53 mL, 8.97 mmol, 1.2 eq.) was then added and the mixture was stirred at RT for 1 h. In a further reaction vessel, 4,4-difluoro-L-prolinamide hydrochloride was dissolved in dry DCM (5 mL) and DMF (5 mL) and DIPEA (2.54 mL, 14.90 mmol, 2.0 eq.) was added as well. The precipitated solid was filtered off and the mother liquor was cooled overnight to complete the precipitation. The two precipitates were combined. A colorless solid (1.97 g, 6.41 mmol, 86%) was obtained. MS (ESI-positive): m / z (%) = 207.8 (62, [M-Boc+H] + ), 251.8 (100, [M- t Bu+H] + ), 307.9 (39, [M+H] + ), 329.9 (24, [M+Na] + ), C 12 H 19 Calculated for F2N3O4: 307.13 [M] + . 1 HNMR (400MHz, DMSO-d6): δ [ppm] = 7.40 (s, 1H), 7.16 (s, 1H), 6.87 (dt, J=10.4, 5.8 Hz, 1H), 4.45 (dd, J = 9.0 Hz, 1H), 4.15 - 3.85 (m, 2H), 3.86 - 3.63 (m, 2H), 2.81-2.27 (m, 2H), 1.37 (s, 9H).

[0097] Boc-Gly-Pro-CN (tert-butyl (S)-(2-(2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)carbamate) Boc-Gly-Pro-CONH2 (1.97 g, 6.41 mmol, 1.0 equiv) was dissolved in dry THF (50 mL) under argon and cooled to 0°C. Pyridine (4.1 mL, 51.3 mmol, 8.0 equiv) was added. In a further reaction vessel, TFAA (2.7 mL, 19.2 mmol, 3.0 equiv) was dissolved in dry DCM (35 mL) under argon and slowly added dropwise to the reaction solution. Then, 1 M hydrochloric acid (80 mL) was added and the aqueous solution was extracted with DCM (5 x 80 mL). The combined organic phase was washed once each with small amount of Na2CO3 and brine and dried over Na2SO4. The solvent was removed under reduced pressure and the product was purified by column chromatography (CH / EA=3:2). A colorless solid (1.49 g, 4.81 mmol, 81%) was obtained. MS (ESI-positive): m / z (%) = 190.0 (31, [M-Boc+H] + ), 233.9 (100, [M- t Bu+H] + ), C12 H 17 Calculated for F2N2O3: 289.12 [M] + . 1 HNMR (400MHz, DMSO-d6): δ [ppm] = 5.34 (s, 1H), 4.97 (t, J = 6.5 Hz, 1H), 4.04 - 3.78 (m, 4H), 2.81 - 2.69 (m, 2H), 1.45 (s, 9H).

[0098] Gly-Pro-CN ((S)-4,4-difluoroglycylpyrrolidine-2-carbonitrile) Boc-Gly-Pro-CN (1.15 g, 3.97 mmol, 1.0 equiv.) was dissolved in dry MeCN (2 mL) under argon and TFA (2 mL) was added dropwise slowly. After the mixture was stirred at RT for 5 h, the solvent was removed under reduced pressure and the residue was co-distilled with MeOH (5×25 mL). A yellowish oil was obtained, which was used in the next step without further purification. MS (ESI-positive): m / z (%) = 189.9 (100, [M+H] + ), 231.0 (20, [M+ACN+H] + ), calculated for CHC7H9F2N3O: 189.07 [M] + . 1 HNMR (400MHz, MeOD): δ [ppm] = 8.25 (s, 2H), 5.22 - 5.15 (m, 1H), 4.15 - 3.91 (m, 4H), 3.00 - 2.81 (m, 2H).

[0099] 6-Hydroxyquinoline-4-carboxylic acid hydrobromide 6-Methoxyquinoline-4-carboxylic acid (2.46 g, 12.1 mmol, 1.0 equiv) was dissolved in 47% HBr (28.18 mL, 242.42 mmol, 20 equiv) and heated under reflux for 1 day. After cooling to RT, the hydrobromic acid was partially removed under reduced pressure and the precipitate was filtered and washed first with cold EA (20 mL) and then with a small amount of cold EA / MeOH (90:10). A yellow solid (3.25 g, 12.1 mmol, 100%) was obtained. MS (ESI-positive): m / z (%) = 190.0 (100, [M+H] + ), 191.0 (12, [M+H] + ), C 10 Calculated for H8BrNO3: 189.04[M]. 1 HNMR (400MHz, MeOD): δ [ppm] = 9.04 (d, J=5.6 Hz, 1H), 8.41 (d, J=5.6 Hz, 1H), 8.34 (d, J=2.6 Hz, 1H), 8.19 (d, J=9.3 Hz, 1H), 7.77 (dd, J=9.3, 2.6 Hz, 1H).

[0100] 6-Hydroxyquinoline-4-carboxylic acid methyl ester First, dry MeOH (20 mL) was cooled to 0 °C under argon and SOCl2 (4.43 mL, 61.09 mmol, 5.0 equiv.) was added dropwise. 6-Hydroxyquinoline-4-carboxylic acid hydrobromide was dissolved in dry MeOH (20 mL) and also cooled to 0 °C under argon. Then, SOCl2-MeOH solution was added dropwise. SOCl2 (2.91 g, 24.44 mmol, 2 equiv.) and MeOH (20 mL) were recombined at 0 °C and added dropwise to the reaction mixture. The solution was heated under reflux for another 24 h. The above step was repeated once more and after heating under reflux for another 4 h, the solvent was removed under reduced pressure. A yellow solid was obtained, which was used in the next step without further purification. MS (ESI-positive): m / z (%) = 204.0 (100, [M+H] + ), 205.1 (12, [M+H] + ), C 11 Calculated for H9NO3: 203.06[M]. 1HNMR (400MHz, MeOD): δ [ppm] = 9.02 (d, J=5.5Hz, 1H), 8.38 (d, J=5.5Hz, 1H), 8.24 (d, J=2.6Hz, 1H), 8.17 (d, J=9.3Hz, 1H), 7.75 (dd, J=9.3, 2.6Hz, 1H), 4.09 (s, 3H).

[0101] Boc-Quino-COOMe (6-(4-((tert-butoxycarbonyl)amino)butoxy)quinoline-4-carboxylic acid methyl ester) Under an argon atmosphere, 6-hydroxyquinoline-4-carboxylic acid methyl ester (2.48 g, 12.1 mmol, 1.0 equiv.) and Cs2CO3 (4.37 g, 13.4 mmol, 1.25 equiv.) were suspended in dry DMF (55 mL). The reaction solution was heated to 70°C. Then, tert-butyl (4-bromobutyl)carbamate (3.76 g, 14.91 mmol, 1.22 equiv.) was dissolved in dry DMF (80 mL) and added dropwise to the hot reaction mixture. After confirming the reaction, tert-butyl (4-bromobutyl)carbamate (1.23 g, 4.88 mmol, 0.4 equiv.) was dissolved again in dry DMF (20 mL) and added to the reaction mixture. The mixture was stirred at 70°C overnight. Further (308 mg, 1.22 mmol, 0.1 equiv) was added and after 3 h at 70 °C, the solvent was evaporated under reduced pressure and the residue was dissolved in dilute HBr (150 mL, pH = 2.6). The mixture was extracted with EA (5 x 80 mL) and the combined organic phases were washed with brine and dried over Na2SO4. The solvent was evaporated under reduced pressure and the crude product was obtained via column chromatography (CHCl3 / MeOH, 100:1) as a pale yellow solid (2.68 g, 7.17 mmol, 59%). MS (ESI-positive): m / z (%) = 375.2 (100, [M+H] + ), 376.2 (23, [M+H] + ), C 20 H 26 Calculated for N2O5: 374.18[M]. 1HNMR (400MHz, CDCl3): δ [ppm] = 8.84 (d, J=4.6 Hz, 1H), 8.24 (dd, J=16.7, 2.8 Hz, 1H), 8.11 (d, J=9.2 Hz, 1H), 7.94 (d, J=4.6 Hz, 1H), 7.43 (dd, J=9.2, 2.8 Hz, 1H), 4.74-4.60 (m, 1H), 4.15 (t, J=6.21 Hz, 2H), 4.03 (s, 3H), 3.27-3.16 (m, 2H), 1.95-1.86 (m, 2H), 1.78-1.67 (m, 2H), 1.42 (s, 9H).

[0102] Boc-Quino-COOH (6-(4-((tert-butoxycarbonyl)amino)butoxy)quinoline-4-carboxylic acid) Boc-Quino-COOMe (3.34 g, 8.92 mmol, 1.0 equiv) was dissolved in 1,4-dioxane (40 mL). Then 1M LiOH (17.8 mL, 17.84 mmol, 2.0 equiv) was added and the mixture was stirred at RT for 4 h. After removing the organic solvent under reduced pressure, the pH was set to 3.5 with 1M HCl. The aqueous solution was extracted with EA (8×80 mL), the combined organic phases were dried over Na2SO4 and the solvent was removed under reduced pressure. A pale yellow solid (1.82 g, 5.05 mmol, 57%) was obtained. MS (ESI-positive): m / z (%) = 261.1 (20, [M-Boc+H] + ), 361.2 (100, [M+H] + ), 362.2 (22, [M+H] + ), C 19 H 24 Calculated for N2O5: 360.17[M]. 1HNMR (400MHz, DMSO-d6): δ [ppm] = 8.86 (d, J=4.5Hz, 1H), 8.15 (d, J=2.8Hz, 1H), 8.02 (d, J=9.3Hz, 1H), 7.92 (d, J=4.4 Hz, 1H), 7.49 (dd, 6.87 (t, J=5.8Hz, 1H), 4.10 (t, J = 6.3Hz, 2H), 3.00 (q, J = 6.6Hz, 2H), 1.78 (q, J = 11.8, 6.5Hz, 2H), 1.62-1.51 (m, 2H), 1.37 (s, 9H).

[0103] FAPi-NHBoc (tert-butyl (S)-(4-((4-((2-(2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)carbamoyl)quinolin-6-yl)oxy)butyl)carbamate) Under argon atmosphere, Boc-Quino-COOH (1.64 g, 4.55 mmol, 1.0 eq.) and DIPEA (0.93 mL, 5.46 mmol, 1.2 eq.) were dissolved in dry DMF (16 mL). Then, HOBt (0.68 g, 5.01 mmol, 1.1 eq.) and HBTU (1.90 g, 5.01 mmol, 1.1 eq.) were added and the reaction mixture was stirred at RT for 1 h. Then, Gly-Pro-CN (as dissolved in dry DMF (10 mL) and added thereto together with DIPEA (1.93 mL, 11.38 mmol, 2.5 eq.)) was added and the whole reaction mixture was stirred at RT for another day. Then, the solvent was removed in vacuum and the residue was dissolved in EA. The organic phase was washed with 1 M citric acid, saturated Na2CO3 and brine. The aqueous phase was then extracted with EA (3×100 mL) and the combined organic extracts were dried over Na2SO4. The solvent was removed under reduced pressure and the product was obtained via column chromatography (CHCl3 / MeOH, 100:3) as a colorless solid (1.74 g, 3.27 mmol, 72%). MS (ESI-positive): m / z (%) = 432.0 (33, [M-Boc+H] + ), 476.1 (46, [M- t Bu+H] + ), 532.4 (100, [M+H] + ), C 26 H 31Calculated for F2N5O5: 531.23 [M] + . 1 HNMR (400MHz, MeOD): δ [ppm] = 8.74 (d, J = 4.4 Hz, 1H), 7.96 (d, J = 9.3 Hz, 1H), 7.93 - 7.88 (m, 1H), 7.56 (d, J = 4.4 Hz, 1H), 7.46 (dd, J = 9.3, 2.7 Hz, 1H), 5.15 (dd, J = 9.4, 3.1 Hz, 1H), 4.39 - 3.98 (m, 8H), 3.19 - 3.09 (m, 2H), 3.02 - 2.70 (m, 2H), 1.94 - 1.83 (m, 2H), 1.76 - 1.65 (m, 2H), 1.43 (s, 9H).

[0104] FAPi-NH 2 ((S)-6-(4-aminobutoxy)-N-(2-(2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)quinoline-4-carboxamide FAPi-NHBoc (531.6 mg, 1.0 mmol, 1.0 equiv) was dissolved in dry acetonitrile (10 mL) at 0° C. under argon. It was dissolved in 4 M HCl in 1,4-dioxane (5.0 mL, 5.0 mmol, 5.0 equiv) and allowed to warm slowly to RT. After 3 h, 4 M hydrochloric acid in 1,4-dioxane (2.5 mL, 2.5 mmol, 2.5 equiv) was added once more and after another 4 h at RT, the mixture was diluted with more acetonitrile (30 mL) and concentrated completely in vacuo. A colorless solid (467 mg, 1.0 mmol, 100%) was obtained. MS (ESI-positive): m / z (%) = 216.7 (100, [M+H] 2+ ), 237.2 (27, [M+ACN+H] 2+ ), 432.1 (22, [M+H] + ), C 21 H 23 Calculated for O5F2N5O3: 431.18 [M] + . 1HNMR (400MHz, MeOD): δ [ppm] = 9.10 (d, J = 5.5 Hz, 1H), 8.32 (d, J = 2.7 Hz, 1H), 8.24 (d, J = 9.3 Hz, 1H), 8.08 (d, J = 5.5 Hz, 1H), 7.86 (dd, J = 9.4, 2.6 Hz, 1H), 5.15 (dd, J = 9.4, 3.1 Hz, 1H), 4.48 - 4.33 (m, 4H), 4.32 - 4.07 (m, 2H), 3.06 (t, J=6.5 Hz, 2H), 3.02 - 2.74 (m, 2H), 2.09 - 1.87 (m, 4H).

[0105] Example 2: DOTA.Glu.(FAPi) 2 , DOTAGA.Glu.(FAPi) 2 ,DATA 5m .Glu.(FAPi) 2 Next, the labeled precursors DOTA.Glu.(FAPi)2, DOTAGA.Glu.(FAPi)2 and DATA 5m The synthesis of .Glu.(FAPi)2 is described below. The first synthetic step is the same for all three compounds, and a representative synthesis example is shown in Scheme 11.

[0106] [ka]

[0107] Boc-Glu.(FAPi) 2 (tert-Butyl ((S)-1,5-bis((4-((4-((2-((S)-2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)carbamoyl)quinolin-6-yl)oxy)butyl)amino)-1,5-dioxopentan-2-yl)carbamate) tert-Butoxycarbonyl-L-glutamic acid (Boc-Glu-OH, 40 mg, 162 μmol, 1.0 equiv.), 1-hydroxybenzotriazole (HOBt, 55 mg, 405 μmol, 2.5 equiv.) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC *HCl, 78 mg, 405 μmol, 2.5 equiv.) was dissolved in dry N,N-dimethylformamide (DMF, 4 mL), N,N-diisopropylethylamine (DIPEA, 68.9 μL, 405 μmol, 2.5 equiv.) was added, and the mixture was stirred at room temperature (RT) under an argon atmosphere for 90 min. Then, FAPi-NH2 in DMF (4 mL) was * A solution of TFA (265 mg, 486 μmol, 3 equiv.) and DIPEA (110 μL, 648 μmol, 4 equiv.) was added and stirring was continued at RT overnight. Further HOBt (16 mg, 121 μmol, 0.75 equiv.) and EDC * HCl (23 mg, 121 μmol, 0.75 equiv.) was added and after another 60 min another solution of FAPi-NH2*TFA (88 mg, 162 μmol, 1.0 equiv.) and DIPEA (41.4 μL, 243 μmol, 1.5 equiv.) in DMF (2 mL) was added. Stirring was continued overnight at RT and the solvent was then removed under reduced pressure. After column chromatography (CHCl3 / MeOH (100:10-15)), 127 mg (118 μmol, 73%) of Boc-Glu.(FAPi)2 was obtained as a yellow oil. LC-MS (ESI-positive): m / z (%) = 487.8 (100, [M-Boc+H] 2+ ), 537.8 (73, [M+H] 2+ ), 1074.4 (9, [M+H] + ), 1075.4 (6, [M+H] + ), C 52 H 59 F4N 11 O 10 Calculated for: 1073.44 [M] + .

[0108] Glu.(FAPi) 2 ((S)-2-amino-N 1 ,N 5 -Bis(4-((4-((2-((S)-2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)carbamoyl)quinolin-6-yl)oxy)butyl)pentanediamide To Boc-Glu.(FAPi)2 (127 mg, 118 μmol) was added 50 μL Milli-Q® water, 50 μL triisopropylsilane (TIPS) and 1.9 mL TFA (TFA:TIPS:H2O (95:2.5:2.5)) and the mixture was stirred at RT for 1 h. Then, 5×≈10 mL MeOH was added respectively and the solvent was removed again in vacuo to give a yellow oil, which was used in the next step without further purification. LC-MS (ESI-positive): m / z(%)= 325.6(100, [M-Boc+H] 3+ ), 487.8(28, [M+H] 2+ ),974.3(5,[M+H] + ), C 47 H 51 F4N 11 Calculated for O8: 973.39 [M] + .

[0109] The synthesis of the labeled precursor DOTA.Glu.(FAPi)2 is shown in Scheme 12.

[0110] [ka]

[0111] DOTA(tBu) 3 -NHS(2,2',2''-(10-(2-((2,5-dioxopyrrolidin-1-yl)oxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid tert-butyl ester) DOTA-tris(tert-butyl ester) (129 mg, 224 μmol, 1.0 equiv.) and 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU, 87 mg, 229 μmol, 1.0 equiv.) were dissolved in dry ACN (5 mL). The mixture was stirred at RT under argon for 75 min, after which N-hydroxysuccinimide (NHS, 31 mg, 267 μmol, 1.2 equiv.) was added. Stirring was continued overnight, followed by the addition of HBTU (52.2 mg, 138 μmol, 0.6 equiv.) and, 1 h later, NHS (22 mg, 191 μmol, 0.85 equiv.), and the mixture was stirred for an additional day. After removal of all solvents in vacuo, column chromatography (DCM:MeOH (100:15)) afforded 145 mg (217 μmol, 97%) of DOTA(tBu)3-NHS as a colorless solid. LC-MS (ESI-positive): m / z (%) = 335.7 (100, [M+H] 2+ ), 670.4 (50, [M+H] + ), 671.4 (18, [M+H] + ), C 32 H 55 N5O 10 Calculated for: 669.39 [M] + .

[0112] DOTA(tBu) 3 .Glu.(FAPi) 2 (2,2',2''-(10-(2-(((S)-1,5-bis((4-(4-(2-((S)-2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)carbamoyl)quinolin-6-yl)oxy)butyl)amino)-1,5-dioxypentan-2-yl)amino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid tert-butyl ester) DOTA(tBu) 3- NHS (40 mg, 60 μmol, 1.2 equiv.) was dissolved in dry DMF (2 mL) along with Glu.(FAPi)2 (48.7 mg, 50 μmol, 1.0 equiv.) and DIPEA (200 μL) was added. The mixture was stirred at 40° C. under argon atmosphere for 1 day, after which all solvents were completely removed in vacuo. A yellow oil was obtained and used directly in the next step without further purification. HPLC-MS (ESI-positive): m / z (%) = 382.95 (22, [M+H] 4+ ), 383.20 (19, [M+H] 4+), 491.57 (34, [M-tBu+H] 3+ ), 491.90 (28, [M-tBu+H] 3+ ), 492.24 (13, [M-tBu+H] 3+ ), 510.26 (100, [M+H] 3+ ), 510.59 (90, [M+H] 3+ ), 510.93 (44, [M+H] 3+ ), 511.26 (14, [M+H] 3+ ), 764.88 (42, [M+H] 2+ ), 765.38 (37, [M+H] 2+ ), 765.89 (17, [M+H] 2+ ), 1528.76 (25, [M+H] + ), 1529.76 (22, [M+H] + ), 1530.77 (10, [M+H] + ), calculated for: C 75 H 101 F4N 15 O 15 : 1527.75 [M] + .

[0113] DOTA.Glu.(FAPi) 2 (2,2',2''-(10-(2-(((S)-1,5-bis((4-((4-((2-((S)-2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)carbamoyl)quinolin-6-yl)oxy)butyl)amino)-1,5-dioxopentan-2-yl)amino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid) To the DOTA(tBu)3.Glu.(FAPi)2, 50 μL Milli-Q® water, 50 μL TIPS and 1.9 mL TFA (TFA:TIPS:HO (95:2.5:2.5)) were added and the mixture was stirred at RT for 8 h. Afterwards, about 10 mL (×4) MeOH was added each time and the solvent was removed again in vacuo. The crude product was purified by semi-preparative RP-HPLC (22–23% ACN, t in 16 min). R =14-15 min.) 19.9 mg (14.6 μmol, 29%) of a yellow solid was obtained. LC-MS (ESI-positive): m / z (%) = 340.85 (42, [M+H] 4+ ), 351.00 (57, [M+ACN+H] 4+ ), 361.35 (13, [M+2ACN+H]4+ ), 454.15 (100, [M+H] 3+ ), 468.00 (20, [M+ACN+H] 3+ ), 680.85 (9, [M+H] 2+ ), C 63 H 77 F4N 15 O 15 Calculated for: 1359.57 [M] + .

[0114] [ nat Lu]Lu-DOTA.Glu.(FAPi) 2 DOTA.Glu.(FAPi)2 (2.8 mg, 2.0 μmol, 1.0 equiv.) was dissolved in 500 μL of 1 M HEPES buffer (pH = 5.5), 40 μL of 0.1 M LuCl3 solution (4 μmol, 2.0 equiv.) was added, and the mixture was shaken at 90 °C for 4 h. Subsequent semi-preparative RP-HPLC (20–25% ACN in 20 min, t R = 14-15 min) 0.7 mg (0.46 μmol, 23%) nat Lu]Lu-DOTA.Glu.(FAPi)2 was obtained as a yellow solid. LC-MS (ESI-positive): m / z (%) = 511.55 (100, [M+H] 3+ ), 766.75 (14, [M+H] 2+ ), C 63 H 74 F4LuN 15 O 15 Calculated for: 1531.48 [M] + .

[0115] [ 68 Ga]Ga-DOTA.Glu.(FAPi) 2 Initial charge of 100MBq [ 68A solution of 400 μL of 1 M HEPES buffer (pH = 4.5 or 5.5) and 5–20 nmol DOTA.Glu.(FAPi)2 (5–20 μL of 1 μmol / mL stock solution with Trace-Select HO) was added to [Ga]GaCl3 at 95 °C and the mixture was shaken for 30 min. Labeling was performed at least three times (n = 3) for each molar amount (5, 10, 20 nmol) and analyzed each time by radio-TLC with 0.1 M Na-citrate buffer (pH = 4.0) as mobile phase (see Figure 1). Furthermore, the consistency was checked by comparison with radio-TLC with 1 M AmOAc (pH = 4) / MeOH (1:1) and analytical radio-HPLC (Figure 2). It was possible to achieve a high radiochemical conversion rate of more than 98%. The stability after 2 h in HS and PBS was more than 98% (see Figure 3). The logD value was -2.08 ± 0.07.

[0116] [ 177 Lu]Lu-DOTA.Glu.(FAPi) 2 20-40 μL of 0.04 M HCl with initial charge of 50-100 MBq [ 177 To [Lu]LuCl3, 400 μL of 1 M HEPES buffer (pH = 5.5) and a solution of 2–5 nmol DOTA.Glu.(FAPi)2 (2–5 μL of 1 μmol / mL stock solution with Trace-Select HO) were added at 95 °C, and the mixture was shaken for 60 min. Labeling was performed in duplicate (n = 3 (50 MBq), n = 2 (100 MBq)) and analyzed by developing and evaluating radio-TLC in each case using 0.1 M Na-citrate buffer (pH = 4.0) as mobile phase (see Figure 4). Furthermore, the consistency was examined by comparison with radio-TLC using 1 M AmOAc (pH = 4) / MeOH (1:1) and analytical radio-HPLC (Figure 5). A high radiochemical conversion rate of more than 99% could be achieved. The stability after 14 days was about 99% in HS and 95% in PBS (see Figure 6). The logD value was 1.77±0.10.

[0117] The synthesis of the labeled precursor DOTAGA.Glu.(FAPi)2 is shown in Scheme 12.

[0118] [ka]

[0119] DOTAGA(tBu) 4 .Glu.(FAPi) 2 (2,2',2''-(10-(5-(((S)-1,5-bis((4-((4-(2-(S)-2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)carbamoyl)quinolin-6-yl)oxy)butyl)amino)-1,5-dioxopentan-2-yl)amino)-1-(tert-butoxy)-1,5-dioxopentan-2-yl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid tert-butyl ester) DOTAGA(tBu)4 (60 mg, 85.6 μmol, 1.0 equiv.) and O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU, 36 mg, 94.2 μmol, 1.1 equiv.) were dissolved in dry DMF (2 mL) under argon atmosphere and DIPEA (17.5 μL, 103 μmol, 1.2 equiv.) was added. After 1 h at 30 °C, a solution of Glu.(FAPi)2 (104 mg, 107 μmol, 1.25 equiv.) and DIPEA (43.7 μL, 257 μmol, 3 equiv.) in dry DMF (3 mL) was added. The mixture was stirred overnight at 30 °C, and then HATU (16 mg, 42 μmol, 0.5 equiv.) was added again. After stirring at 30 °C for an additional day, the solvent was removed in vacuo and purified by column chromatography (CHCl3:MeOH:triethylamine (TEA) (100:10 to 15:1)) to give 39 mg (23.5 μmol, 27%) of DOTAGA(tBu)4.Glu.(FAPi)2 as a yellow oil. HPLC-MS (ESI-positive): m / z (%) = 414.97 (13, [M+H] 4+ ), 415.22 (12, [M+H] 4+ ), 552.95 (100, [M+H] 3+ ), 553.29 (97, [M+H] 3+ ), 553.62 (51, [M+H] 3+ ), 553.96 (18, [M+H] 3+ ), 828.93 (82, [M+H] 2+ ), 829.43 (78, [M+H] 2+ ), 829.93 (40, [M+H] 2+ ), 830.43 (15, [M+H] 2+), 1656.85 (87, [M+H] + ), 1657.85 (85, [M+H] + ), 1658.85 (43, [M+H] + ), 1659.86 (15, [M+H] + ), C 82 H 113 F4N 15 O 17 Calculation: 1655.84 [M] + .

[0120] DOTAGA.Glu.(FAPi) 2 (2,2',2''-(10-(4-(((S)-1,5-bis((4-((4-((2-((S)-2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)carbamoyl)quinolin-6-yl)oxy)butyl)amino)-1,5-dioxopentan-2-yl)amino)-1-carboxy-4-oxobutyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid) To DOTAGA(tBu)4.Glu.(FAPi)2, 50 μL Milli-Q® water, 50 μL TIPS and 1.9 mL TFA (TFA:TIPS:H2O (95:2.5:2.5)) were added and the mixture was stirred at RT for 7 h. Afterwards, MeOH was added in portions of about 10 mL each time (×5) and the solvent was again removed in vacuo. The crude product was purified by semi-preparative RP-HPLC (23% ACN isocratic, t R =10-10.5 min) to obtain 16.4 mg (11.5 μmol, 49%) of a yellow solid. LC-MS (ESI-positive): m / z (%) = 358.85 (65, [M+H] 4+ ), 369.05 (24, [M+ACN+H] 4+ ), 478.30 (100, [M+H] 3+ ), 717.30 (6, [M+H] 2+ ), 1432.40 (1, [M+H] + ), 1454.70 (1, [M+Na] + ), C 66 H 81 F4N 15 O 17 Calculated for: 1431.59 [M] + .

[0121] [ nat Lu]Lu-DOTAGA.Glu.(FAPi) 2 DOTAGA.Glu.(FAPi)2 (2.8 mg, 2.0 μmol, 1.0 equiv.) was dissolved in 550 μL of 1 M HEPES buffer (pH = 5.5) and 50 μL of EtOH, 40 μL of 0.1 M LuCl3 solution (4 μmol, 2.0 equiv.) was added, and the mixture was shaken at 90 °C for 4 h. Subsequent semi-preparative RP-HPLC (23% ACN isocratic, t R = 13-14 min) with 0.5 mg (0.31 μmol, 16%) of [ nat Lu]Lu.DOTAGA.Glu.(FAPi)2 was obtained as a yellow solid. LC-MS (ESI-positive): m / z (%) = 535.50 (100, [M+H] 3+ ), 802.95 (36, [M+H] 2+ ), C 66 H 78 F4LuN 15 O 17 Calculation: 1603.50 [M] + .

[0122] [ 68 Ga]Ga-DOTAGA.Glu.(FAPi) 2 Initial charge of 100 or 400 MBq in 0.05 M hydrochloric acid (0.5 or 2 mL) 68 A solution of 0.5 or 2 mL of 1 M HEPES buffer (pH = 4.5) and 10–40 nmol DOTAGA.Glu.(FAPi)2 (10–40 μL of 1 μmol / mL stock solution with Trace-Select HO) was added to [Ga]GaCl3 at 95 °C, and the mixture was shaken for 30 min. Labeling was performed in duplicate (n = 4 (100 MBq), n = 2 (400 MBq)) and the reaction kinetics was examined by radio-TLC with 0.1 M Na3 citrate buffer (pH = 4.0) as the mobile phase (see Figure 7). Furthermore, the consistency was examined by comparison with radio-TLC using 1 M AmOAc (pH = 4) / MeOH (1:1) and analytical radio-HPLC (Figure 8). A high radiochemical conversion rate of more than 97% could be achieved. The stability after 2 h in HS and PBS was more than 95% (see Figure 9). The logD value was -2.48 ± 0.05.

[0123] [ 177 Lu]Lu-DOTAGA.Glu.(FAPi) 2 20-40μL of 0.04M HCl with initial charge of 50-100MBq [ 177 To [Lu]LuCl3, 400 μL of 1 M HEPES buffer (pH = 5.5) and a solution of 1-5 nmol DOTAGA.Glu.(FAPi)2 (1-5 μL of 1 μmol / mL stock solution with Trace-Select H2O) were added at 95 °C, and the mixture was shaken for 60 min. The reaction rate was investigated by developing and evaluating radioactive TLC with 0.1 M NaCitrate buffer (pH = 4.0) as the mobile phase (number of labels: n = 3 (50 MBq), n = 1-2 (100 MBq)) (see Figure 10). In addition, the consistency was examined by comparison with radioactive TLC using 1 M AmOAc (pH = 4) / MeOH (1:1) and analytical radioactive HPLC (Figure 11). A high radiochemical conversion rate of more than 99% could be achieved. The stability after 14 days was more than 99% in HS and PBS (see Figure 12). The logD value was 2.77±0.10.

[0124] [ 225 Ac]Ac-DOTAGA.Glu.(FAPi) 2 Initial charges of 1.6–3.2 MBq in 100 μL of 0.04 M HCl 225To [Ac]AcCl3 was added 1 mL of 0.1 M sodium ascorbate (pH = 7.0) and 30 nmol / MBq of a solution of DOTAGA.Glu.(FAPi)2 (30 μl / MBq of 1 μmol / mL stock solution with Trace-Select H2O) at 95 °C, and the mixture was shaken for 60 min. Labeling was performed in triplicate (n = 3) to study the reaction kinetics. For this purpose, radio-TLC (see Figure 13) with 0.1 M NaCitrate buffer (pH = 4.0) as the mobile phase was developed, exposed and evaluated at different times (1 h and 1 d). A high radiochemical conversion of more than 94.3 ± 2.1% was observed after 15 min (exposure after 1 day). Subsequent purification using a SepPak® Light C18 cartridge finally yielded a product of high radiochemical purity (>98%, determined by radio-TLC and high-resolution gamma spectroscopy using a HPGe detector).

[0125] [ 225 To measure the stability of [Ac]Ac-DOTAGA.Glu.(FAPi)2, 350-400 kBq of labeling solution was added to HS and PBS (n=3 in each case) and incubated at 37 °C for 20 days (see Figure 14).

[0126] The synthesis of the labeled precursor DATA5m.Glu.(FAPi)2 is shown in Scheme 13: [ka]

[0127] DATA 5m (tBu) 3 .Glu.(FAPi) 2 (2,2'-(6-(5-(((S)-1,5-bis((4-((4-)((2-((S)-2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)carbamoyl)quinolin-6-yl)oxy)butyl)amino)-1,5-dioxopentan-2-yl)amino)-5-oxopentyl)-6-((2-(tert-butoxy)-2-oxoethyl)(methyl)amino)-1,4-diazepane-1,4-diyl)diacetic acid tert-butyl ester) DATA 5m(tBu)3 (22.8 mg, 40 μmol, 1.0 equiv.) and HATU (17.5 mg, 46 μmol, 1.15 equiv.) were dissolved in dry DMF (1 mL) and DIPEA (8.5 μL, 50 μmol, 1.25 equiv.) was added. After 1 h at 25 °C under argon atmosphere, a solution of Glu.(FAPi)2 (39 mg, 40 μmol, 1.0 equiv.) and DIPEA (17 μL, 100 μmol, 2.5 equiv.) in dry DMF (2 mL) was added. After removing the solvent under reduced pressure, the product was purified by column chromatography (CHCl3:MeOH:triethylamine (TEA) (100:10 to 15:1)) to give 60 mg (39.2 μmol, 98%) of a yellow oil. LC-MS (ESI-positive): m / z (%) = 510.0 (100, [M+H] 3+ ), 764.5 (24, [M+H] 2+ ), C 76 H 102 F4N 14 O 15 Calculated for: 1526.76 [M] + .

[0128] DATA 5m .Glu.(FAPi) 2 (2,2'-(6-(5-(((S)-1,5-bis((4-((4-)((2-((S)-2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)carbamoyl)quinolin-6-yl)oxy)butyl)amino)-1,5-dioxopentan-2-yl)amino)-5-oxopentyl)-6-((carboxymethyl)(methyl)amino)-1,4-diazepane-1,4-diyl)diacetic acid) DATA 5m To (tBu)3.Glu.(FAPi)2 was added 25 μL Milli-Q® water, 25 μL TIPS and 950 μL TFA (TFA:TIPS:HO (95:2.5:2.5)) and the mixture was stirred at RT for 2.5 h. Afterwards, MeOH was added in approximately 10 mL portions (×3) and the solvent was again removed in vacuo. The crude product was purified by semi-preparative RP-HPLC (23% ACN isocratic, t R =13-13.5 min) to obtain 8.2 mg (6.0 μmol, 15%) of a yellow solid. LC-MS (ESI-positive): m / z (%) = 340.7 (6, [M+H] 4+ ), 454.0 (100, [M+H] 3+ ), 680.4 (48, [M+H] 2+), 706.8 (47, [M+Fe] 2+ ), 707.3 (35, [M+Fe] 2+ ), 1359.5 (6, [M+H] + ), 1360.5 (5, [M+H] + ), C 64 H 78 F4N 14 O 15 Calculated for: 1358.57 [M] + .

[0129] [ 68 Ga]Ga-DATA 5m .Glu.(FAPi) 2 Initial charge of 50MBq [ 68 A solution of 400 μL of 0.5 M HEPES buffer (pH = 5.5) and 10–20 nmol of DOTA.Glu.(FAPi)2 (10–20 μL of 1 μmol / mL stock solution with Trace-Select HO) was added to [Ga]GaCl3 at room temperature, and the mixture was shaken for 30 min. Labeling was performed four times (n = 4) for both molar amounts and analyzed by radio-TLC with 0.1 M NaCitrate buffer (pH = 4.0) as the mobile phase (see Figure 15). Furthermore, the consistency was examined by comparison with radio-TLC using 1 M AmOAc (pH = 4) / MeOH (1:1) and analytical radio-HPLC (Figure 16). A high radiochemical conversion of more than 96% was achieved. The stability after 2 h in HS and PBS was more than 97% (see Figure 17). The logD value was -2.03 ± 0.05.

[0130] Table 1 summarizes the experimentally determined logD values.

[0131] [Table 1]

[0132] In vitro testing FAP: I C 50Measurements were performed using Z-Gly-Pro-7-amino-4-methylcoumarin (AMC) as substrate at a concentration of 50 μM in pH = 8 (0.05 M Tris-HCl buffer, 1 mg / mL bovine serum albumin (BSA), 140 mM NaCl). Eight concentrations of FAP inhibitors were examined, but the concentration of DMSO was always the same. The inhibitors were preincubated for 15 min at 37 °C before adding the Z-Gly-Pro-AMC substrate. The release kinetics of AMC was measured for at least 10 min at an excitation wavelength λex = 380 nm and an emission wavelength λem = 465 nm.

[0133] PREP: I C 50 Measurements were performed using N-Succinyl-Gly-Pro-AMC as substrate at a concentration of 250 μM in pH = 7.4 (0.1 MK phosphate buffer, 1 mM EDTA, 1 mM DTT, 1 mg / mL BSA). Eight concentrations of FAP inhibitors were examined, but the concentration of DMSO was always the same. Before adding the N-Succinyl-Gly-Pro-AMC substrate, the inhibitors were preincubated for 15 min at 37 °C. The release kinetics of AMC was measured for at least 10 min at an excitation wavelength λex = 380 nm and an emission wavelength λem = 465 nm.

[0134] DPP4, DPP8 and DPP9: I C 50 Measurements were performed using Ala-Pro-p-nitroanilide (pNA) as substrate at concentrations of 25 μM (DPP4), 300 μM (DPP8) or 150 μM (DPP9) at pH = 7.4 (0.05 M HEPES-NaOH buffer containing 0.1% Tween-20, 1 mg / mL BSA, 150 mM NaCl). At least eight concentrations of FAP inhibitors were tested, always with the same DMSO concentration. Inhibitors were preincubated for 15 min at 37 °C before adding the Ala-Pro-pNA substrate. pNA release kinetics was measured at a wavelength of λex = 405 nm for at least 10 min.

[0135] Table 2 shows the IC 50The results of the measurements were summarized. The selectivity index (SI) is the IC4 of FAP and each of its competing enzymes (PREP, DPP4, DPP8, and DPP9). 50 The ratio was calculated.

[0136] [Table 2]

[0137] Example 3: DOTA.NPyr.(FAPi) 2 , DOTAGA.NPyr.(FAPi) 2 Next, the synthesis of the labeled precursors DOTA.NPyr.(FAPi)2 and DOTAGA.NPyr.(FAPi)2 will be described. The first synthesis step is the same for both compounds, and a representative synthesis example is shown in Scheme 14.

[0138] [ka]

[0139] Boc-NPyr(OBzl) 2 ((S)-2,2'-((1-(tert-butoxycarbonyl)pyrrolidin-3-yl)azanediyl)diacetic acid benzyl ester) (S)-1-Boc-3-aminopyrrolidine (1.07 g, 5.74 mmol, 1.0 equiv.) and DIPEA (1.5 mL) were initially charged in acetonitrile (6 mL). After 60 min, a solution of benzyl bromoacetate (1.74 g, 7.55 mmol, 1.3 equiv.) in acetonitrile (6 mL) was slowly added dropwise and the mixture was stirred at RT for another 2 h. Subsequent column chromatography (CHCl3:MeOH (30:1) + 1% TEA) afforded Boc-NPyr-OBzl(benzyl-(S)-N-(pyrrolidine-3-tert-butoxycarbamate)glycine (680 mg, 2.03 mmol, 35%) as a by-product along with Boc-NPyr(OBzl)2 (1.31 g, 2.71 mmol, 47%). LC-MS (ESI-positive): m / z (%) = 383.2 (45, [M-Boc+H] + ), 483.2 (100, [M+H] + ), 484.2 (30, [M+H] + ), C27 H 34 Calculated for N2O6: 482.24 [M] + .

[0140] Boc-NPyr ((S)-2,2'-((1-(tert-butoxycarbonyl)pyrrolidin-3-yl)azanediyl)diacetic acid) Boc-NPyr(OBzl)2 (1.21 g, 2.51 mmol, 1.0 equiv.) was added to palladium on activated carbon (10 wt% Pd, 53 mg, 50 μmol, 0.02 equiv.) and dry methanol (8 mL). The mixture was stirred at room temperature under a hydrogen atmosphere for 2 days. The mixture was filtered through Celite, and the methanol was removed under reduced pressure. A colorless solid (643 mg, 2.13 mmol, 85%) was obtained. LC-MS (ESI-positive): m / z (%) = 247.0 (100, [M- t Bu+H] + ), 303.1 (36, [M+H] + ), 605.3 (23, [2M+H] + ), C 13 H 22 Calculated for N2O6: 302.15 [M] + .

[0141] Boc-NPyr.(FAPi) 2 (tert-Butyl (S)-3-(bis(2-((4-((4-((2-((S)-2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)carbamoyl)quinolin-6-yl)oxy)butyl)amino)-2-oxoethyl)amino)pyrrolidine-1-carboxylate) Boc-NPyr (30.2 mg, 100 μmol, 1.0 equiv.), HOBt (36 mg, 266 μmol, 2.7 equiv.) and EDC * HCl (50 mg, 260 μmol, 2.6 equiv.) was dissolved in dry DMF (3 mL) and stirred at 30° C. for 60 min under an argon atmosphere. FAPi-NH2 * A solution of TFA (110 mg, 202 μmol, 2.0 equiv) and DIPEA (51.0 μL, 300 μmol, 3.0 equiv) was added and the mixture was continued to stir at 30° C. for 3.5 h. HOBt (8.5 mg, 63 μmol, 0.63 equiv.) and EDC * HCl (12 mg, 63 μmol, 0.63 equiv.) was added, and after 30 min, FAPi-NH2 in DMF (1 mL) was added. *A solution of TFA (25 mg, 46 μmol, 0.46 equiv.) and DIPEA (17.0 μL, 100 μmol, 1.0 equiv.) in DMF was added. After stirring overnight at 30° C., HOBt (8.5 mg, 63 μmol, 0.63 equiv.), EDC * HCl (12 mg, 63 μmol, 0.63 equiv.), and after another 30 min, FAPi-NH2 in DMF (1 mL) * The addition of TFA (16 mg, 29 μmol, 0.29 equiv.) and DIPEA (17.0 μL, 100 μmol, 1.0 equiv.) was repeated. The mixture was stirred at 30 °C for an additional 5 h and the solvent was removed in vacuo. After column chromatography (CHCl3:MeOH:TEA (100:7.5 to 10:1)), 102 mg (90.3 μmol, 90%) of Boc-NPyr.(FAPi)2 was obtained as a yellow oil. LC-MS (ESI-positive): m / z (%) = 358.6 (86, [M- t Bu+H] 3+ ), 372.2 (58, [M- t Bu+ACN+H] 3+ ), 377.3 (100, [M+H] 3+ ), 390.3 (68, [M+ACN+H] 3+ ), 515.3 (36, [M-Boc+H] 2+ ), 537.5 (8, [M- t Bu+H] 2+ ), 565.5 (84, [M+H] 2+ ), 1129.6 (28, [M+H] + ), 1130.6 (17, [M+H] + ), C 55 H 64 F4N 12 O 10 Calculated for: 1128.48 [M] + .

[0142] NPyr.(FAPi) 2 (6,6'-((((2,2'-(((S)-pyrrolidin-3-yl)azanediyl)bis(acetyl))bis(azanediyl))bis(butane-4,1-diyl))bis(oxy))bis(N-(2-((S)-2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)quinoline-4-carboxamide) To Boc-NPyr.(FAPi)2 (102 mg, 90 μmol) was added 50 μL Milli-Q® water, 50 μL triisopropylsilane (TIPS) and 1.9 mL TFA (TFA:TIPS:H2O (95:2.5:2.5)) and the mixture was stirred at RT for 1 h. Then, about 10 mL (×5) of MeOH was added each time and the solvent was removed again in vacuum to give a yellow oil, which was used in the next step without further purification. LC-MS (ESI-positive): m / z (%) = 344.1 (100, [M+H] 3+ ), 357.6 (45, [M+ACN+H] 3+ ), 515.5 (18, [M+H] 2+ ), 1029.5 (3, [M+H] + ), C 50 H 56 F4N 12 Calculated for O8: 1028.43 [M] + .

[0143] The synthesis of the labeled precursor DOTA.NPyr.(FAPi)2 is shown in Scheme 15.

[0144] [ka]

[0145] DOTA(tBu) 3 .NPyr.(FAPi) 2 (2,2',2''-(10-(2-((S)-3-(bis(2-((4-((4-((2-((S)-2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)carbamoyl)quinolin-6-yl)oxy)butyl)amino)-2-oxoethyl)amino)pyrrolidin-1-yl)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid tert-butyl ester) DOTA(tBu)3-NHS (33.5 mg, 50 μmol, 1.25 equiv.) was dissolved in dry DMF (1 mL) along with NPyr.(FAPi)2 (41.2 mg, 40 μmol, 1.0 equiv.) and DIPEA (50 μL) was added. The mixture was stirred at 40° C. under argon atmosphere for 3 days, after which all solvents were completely removed in vacuo. A yellow oil was obtained and used directly in the next step without further purification. HPLC-MS (ESI-positive): m / z (%) = 396.71 (35, [M+H] 4+ ), 396.96 (33, [M+H]4+ ), 397.21 (15, [M+H] 4+ ), 509.92 (48, [M- t Bu+H] 3+ ), 510.25 (42, [M- t Bu+H] 3+ ), 510.59 (20, [M- t Bu+H] 3+ ), 528.61 (100, [M+H] 3+ ), 528.94 (95, [M+H] 3+ ), 529.27 (50, [M+H] 3+ ), 529.61 (17, [M+H] 3+ ), 792.40 (30, [M+H] 2+ ), 792.91 (28, [M+H] 2+ ), 793.41 (13, [M+H] 2+ ), 1583.80 (18, [M+H] + ), 1584.81 (17, [M+H] + ), 1585.81 (8, [M+H] + ), 1605.79 (8, [M+Na] + ), 1606.79 (8, [M+Na] + ), calculated for: C 78 H 106 F4N 16 O 15 : 1582.80 [M] + .

[0146] DOTA.NPyr.(FAPi) 2 (2,2',2''-(10-(2-((S)-3-(bis(2-((4-((4-((2-((S)-2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)carbamoyl)quinolin-6-yl)oxy)butyl)amino)-2-oxoethyl)amino)pyrrolidin-1-yl)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid) To the DOTA(tBu)3.NPyr.(FAPi)2 was added 50 μL of Milli-Q® water, 50 μL of TIPS and 1.5 mL of TFA (TFA:TIPS:HO (94:3:3)) and the mixture was stirred at RT for 12 h. The crude product was purified by semi-preparative RP-HPLC in 20 min with 21-22% ACN,t R The purification took 18.5-19.5 minutes. 5.6 mg (4.0 μmol, 10%) of a yellow solid was obtained. LC-MS (ESI-positive): m / z (%) = 354.55 (95, [M+H] 4+ ), 364.750 (59, [M+ACN+H] 4+ ), 472.60 (100, [M+H] 3+ ), 708.55 (13, [M+H] 2+ ), 1415.50 (5, [M+H] + ), C 66 H 82 F4N 16 O 15 Calculated for: 1414.61 [M] + .

[0147] The synthesis of the labeled precursor DOTAGA.NPyr.(FAPi)2 is shown in Scheme 16.

[0148] [ka]

[0149] DOTAGA(tBu) 4 .NPyr.(FAPi) 2 (2,2',2''-(10-(5-((S)-3-(bis(2-((4-((4-((2-((S)-2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)carbamoyl)quinolin-6-yl)oxy)butyl)amino)-2-oxoethyl)amino)pyrrolidin-1-yl)-1-(tert-butoxy)-1,5-dioxopentan-2-yl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid tert-butyl ester) DOTAGA(tBu)4 (23.5 mg, 33.5 μmol, 1.0 equiv), NHS (8.0 mg, 70 μmol, 2.0 equiv) and HBTU (26.5 mg, 70 μmol, 2.0 equiv) were dissolved in dry DMF (0.5 mL) and shaken at 30 °C overnight. NHS (4.5 mg, 39.0 μmol, 1.26 equiv) and HBTU (13.5 mg, 35.6 μmol, 1.06 equiv) were added once more. After 4 h, a solution of NPyr.(FAPi)2 (41.2 mg, 40 μmol, 1.0 equiv) and DIPEA (50 μL) in dry DMF (1 mL) was added. The mixture was stirred at 40 °C for 3 days, after which all solvents were completely removed in vacuo. A yellow oil was obtained and used directly in the next step without further purification. HPLC-MS (ESI-positive): m / z (%) = 428.73 (100, [M+H] 4+ ), 428.98 (32, [M+H] 4+), 429.23 (25, [M+H] 4+ ), 571.64 (16, [M+H] 3+ ), 571.97 (10, [M+H] 3+ ), 856.45 (5, [M+H] 2+ ), 856.95 (5, [M+H] 2+ ), 1711.89 (2, [M+H] + ), 1712.89 (2, [M+H] + ), 1733.87 (2, [M+Na] + ), 1734.87 (2, [M+Na] + ), calculated for: C 85 H 118 F4N 16 O 17 : 1710.88 [M] + .

[0150] DOTAGA.NPyr.(FAPi) 2 (2,2',2''-(10-(4-((S)-3-(bis(2-((4-((4-((2-((S)-2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)carbamoyl)quinolin-6-yl)oxy)butyl)amino)-2-oxoethyl)amino)pyrrolidin-1-yl)-1-carboxy-4-oxobutyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid) To the DOTA(tBu)3.NPyr.(FAPi)2 was added 50 μL Milli-Q® water, 50 μL TIPS and 1.9 mL TFA (TFA:TIPS:HO (95:2.5:2.5)) and the mixture was stirred at RT for 8 h. Afterwards, MeOH was added in portions of about 10 mL each time (×4) and the solvent was again removed in vacuo. The crude product was purified by semi-preparative RP-HPLC (21% ACN isocratic, t R =23-24 min) to obtain 3.0 mg (2.0 μmol, 6%) of a yellow solid. LC-MS (ESI-positive): m / z (%) = 372.55 (100, [M+H] 4+ ), 382.90 (38, [M+ACN+H] 4+ ), 496.60 (76, [M+H] 3+ ), 744.40 (5, [M+H] 2+ ), C 69 H 86 F4N 16 O 17 Calculated for: 1486.63 [M] + .

[0151] Example 4: DOTA.PEG2.Glu.(FAPi) 2 , DOTAGA.PEG2.Glu.(FAPi) 2 Next, the synthesis of the labeled precursors DOTA.PEG2.Glu.(FAPi)2 and DOTAGA.PEG2.Glu.(FAPi)2 will be described. The initial synthesis steps are the same for both compounds, and a representative synthesis example is shown in Scheme 17.

[0152] [ka]

[0153] Fmoc-PEG2.Glu(OBzl) 2 ((1-(9H-fluoren-9-yl)-3-oxo-2,7,10-trioxa-4-azatridecan-13-oyl)-L-glutamic acid dibenzyl ester) Fmoc-N-amide-dPEG2 acid (450.0 mg, 1.1 mmol, 1.00 equiv.) and DIPEA (182.0 mg, 240 μL, 1.4 mmol, 1.25 equiv.) were dissolved in dry DMF (9.0 mL), and HBTU (470.3 mg, 1.2 mmol, 1.10 equiv.) and HOBt (167.6 mg, 1.2 mmol, 1.10 equiv.) were added. After 1 h, dibenzyl glutamic acid (460.6 mg, 1.4 mmol, 1.25 equiv.) and DIPEA (320.5 mg, 422 μL, 4.5 mmol, 2.20 equiv.) dissolved in dry DMF (3.0 mL) were added. After the reaction was completed, the solvent was distilled off under reduced pressure, and the yellowish oil was purified by column chromatography (DCM:MeOH (100:2)). Fmoc-PEG2.Glu(OBzl)2 (795.1 mg, 1.1 mmol, 99%) was obtained as a colorless oil. LC-MS (ESI-positive): m / z (%) = 709.4 (100, [M+H] + ), 710.2 (15, [M+H] + ), C 41 H 44 Calculated for N2O9: 708.30 [M] + .

[0154] Fmoc-PEG2.Glu ((1-(9H-fluoren-9-yl)-3-oxo-2,7,10-trioxa-4-azatridecan-13-oyl)-L-glutamic acid) Fmoc-PEG2.Glu(OBzl)2 (196.4 mg, 0.3 mmol, 1.00 equiv.) was dissolved in dry tetrahydrofuran (THF) (2.0 mL) and palladium on activated carbon (10 wt% Pd, 30.0 mg, 0.3 mmol, 1.00 equiv.) was added. The suspension was filtered through Celite, the residue was washed with THF, and the solvent was removed under reduced pressure. Fmoc-PEG2.Glu (122.2 mg, 231.3 μmol, 82%) was obtained as a colorless oil and used in the next step without further manipulation. LC-MS (ESI-positive): m / z (%) = 529.25 (100, [M+H] + ), 530.15 (12, [M+H] + ), C 27 H 32 Calculated for N2O9: 528.21 [M] + .

[0155] Fmoc-PEG2.Glu.(FAPi) 2 ((9H-fluoren-9-yl)methyl((11S)-19-((4-((2-(2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)carbamoyl)quinolin-6-yl)oxy)-11-((4-((4-((2-(2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)carbamoyl)quinolin-6-yl)oxy)butyl)carbamoyl)-9,14-dioxo-3,6-dioxa-10,15-diazanonadecyl)carbamate) Fmoc-PEG2.Glu (32.0 mg, 60.0 μmol, 1.00 equiv.) was dissolved in HOBt (20.4 mg, 150.0 μmol, 2.50 equiv.) and EDC * The mixture was dissolved in dry DMF (1.0 mL) together with HCl (28.8 mg, 150.0 μmol, 2.50 equivalents) and stirred at room temperature under an argon atmosphere. After 1 hour, FAPi * A colorless solution of TFA (65.4 mg, 120.0 μmol, 2.00 equiv.), DIPEA (23.3 mg, 30 μL, 180.0 μmol, 3.00 equiv.) and dry DMF (0.5 mL) was added. After an additional 3 h, HOBt (7.8 mg, 60.0 μmol, 1.00 equiv.) and EDC were added. * HCl (11.4 mg, 60.0 μmol, 1.00 equiv.) was added again. Shortly thereafter, DIPEA (7.8 mg, 10 μL, 60.0 μmol, 1.00 equiv.) and FAPi dissolved in 0.5 mL of dry DMF were added. * An additional 1 / 2 equivalent of HOBt (3.9 mg, 30.0 μmol, 0.50 equiv.) and EDC were added the next day. *HCl (5.7 mg, 30.0 μmol, 0.5 equiv.) was added and the reaction was terminated after an additional 4 h. DMF was removed under reduced pressure and the residue was purified by column chromatography (CHCl3:MeOH (100:10)) to give Fmoc-PEG2.Glu.(FAPi)2 (79.1 mg, 58.4 μmol, 97%) as a pale yellow solid. LC-MS (ESI-positive): m / z (%) = 452.50(31, [M+H] 3+ ), 678.45(100, [M+H] 2+ ), 679.25(13, [M+H] 2+ ), 1355.85 (9, [M+H] + ), C 69 H 74 F4N 12 O 13 Calculated for: 1354.54 [M] + .

[0156] PEG2.Glu.(FAPi) 2 ((2S)-2-(3-(2-(2-aminoethoxy)ethoxy)propanamide)-N 1 ,N 5 -Bis(4-((4-((2-(2-cyano-4,4-difluoropyrrolidin-1)-yl)-2-oxoethyl)carbamoyl)quinolin-6-yl)oxy)butyl)-pentanediamide Fmoc-PEG2.Glu.(FAPi)2 (67.0 mg, 50.0 μmol, 1.00 equiv.) was dissolved in dry DMF (1.0 mL) and 10% piperidine (0.1 mL) was added. The pale yellowish solution was stirred at room temperature for 2 h, after which the solvent was removed under reduced pressure. PEG2.Glu.(FAPi)2 was obtained in quantitative yield and was used without further purification. LC-MS (ESI-positive): m / z (%) = 378.40 (100, [M+H] 3+ ), 567.35 (26, [M+H] 2+ ), 1133.35(3, [M+H] + ), C 54 H 64 F4N 12 O 11 Calculated for: 1132.48 [M] + .

[0157] The synthesis of the labeled precursor DOTA.PEG2.Glu.(FAPi)2 is shown in Scheme 18.

[0158] [ka]

[0159] DOTA(tBu) 3 .PEG2.Glu.(FAPi) 2 (2,2',2''-(10-(2-(((S)-1,5-bis(((4-((4-((2-((S)-2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)carbamoyl)quinolin-6-yl)oxy)butyl)amino)-1,5-dioxopentan-2-yl)amino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid tert-butyl ester) PEG2.Glu.(FAPi)2 (13.4 mg, 20.0 μmol, 1.00 equiv.) was dissolved in DMF (0.4 mL) and 1 vol% DIPEA (10.4 mg, 14 μL, 82.3 μmol) was added, followed by DOTA(tBu)3-NHS (22.7 mg, 20.0 μmol, 1.00 equiv.) also dissolved in DMF (1.0 mL). The mixture was stirred at 35° C. for 3 days, after which the DMF was removed under reduced pressure. The tan oil was further converted without further workup. HPLC-MS (ESI-positive): m / z (%) = 432.70 (55, [M+H] 4+ ), 576.60 (26, [M+H] 3+ ), 864.90 (18, [M+Na] 2+ ), 1687 .84 (1, [M+H] + ), 1709.82 (1, [M+Na] + ), C 82 H 114 F4N 16 O 18 Calculated for: 1686.84 [M] + .

[0160] DOTA.PEG2.Glu.(FAPi) 2 (2,2',2''-(10-(2-(((S)-1,5-bis((4-((4-((2-((S)-2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)carbamoyl)quinolin-6-yl)oxy)butyl)amino)-1,5-dioxopentan-2-yl)amino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid) To the DOTA(tBu)3.PEG2.Glu.(FAPi)2 was added 50 μL of water, 50 μL of TIPS, and 1.5 mL of trifluoroacetic acid (TFA). The brown solution was stirred at room temperature for 5 h and the solvent was removed under reduced pressure. The resulting dark brown oil was purified by semi-preparative RP-HPLC (22-23% ACN in 20 min, tR = 16-17 min) to give DOTA.PEG2.Glu.(FAPi)2 (1.8 mg, 1.2 μmol, 6%) as a yellow solid. LC-MS (ESI-positive): m / z (%) = 380.60 (66, [M+H] 4+ ), 507.30 (100, [M+H] 3+ ), 760.30 (12, [M+H] 2+ ), 1519.55 (4, [M+H] + ), 1541.75 (7, [M+Na] + ), C 70 H 90 F4N 16 O 18 Calculated for: 1518.66 [M] + .

[0161] The synthesis of the labeled precursor DOTAGA.PEG2.Glu.(FAPi)2 is shown in Scheme 19.

[0162] [ka]

[0163] DOTAGA(tBu) 4 .PEG2.Glu.(FAPi) 2 (2,2',2''-(10-((20S)-28-((4-((2-(2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)carbamoyl)quinolin-6-yl)oxy)-20-((4-((4-((2-(2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)carbamoyl)quinolin-6-yl)oxy)butyl)carbamoyl)-2,2-dimethyl-4,8,18,23-tetraoxo-3,12,15-trioxa-9,19,24-triazaoctacosan-5-yl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid tert-butyl ester) DOTAGA(tBu)4 (10.0 mg, 14.3 μmol, 1.00 equiv.) was dissolved in 0.8 mL of dry MeCN with HBTU (10.8 mg, 28.6 μmol, 2.00 equiv.), NHS (3.3 g, 28.6 μmol, 2.00 equiv.) was added, and the colorless solution was stirred under an argon atmosphere. After 6 h, additional HBTU (5.4 mg, 14.3 μmol, 1.00 equiv.) and NHS (1.6 mg, 14.3 μmol, 1.00 equiv.) were added. Glu.(FAPi)2 (8.2 mg, 8.7 μmol, 1.00 equiv.) was dissolved in 0.4 mL of dry MeCN and 1.0 mL of dry DMF, 1 vol% DIPEA (19 mg, 25 μL, 147.0 μmol) was added, and the mixture was added to a red DOTAGA(tBu)4-NHS solution (11.4 mg, 14.3 μmol, 1.65 equiv. in 1.1 mL of MeCN). The reaction was stirred at 40° C. for 24 h, and additional PEG2.Glu.(FAPi)2 (8.2 mg, 8.7 μmol, 1.00 equiv.) was added. After an additional 24 h, the solvent was removed under reduced pressure to give a yellowish oil. HPLC-MS (ESI-positive): m / z (%) = 454.99 (100, [M+H] 4+ ), 606.31 (55, [M+H] 3+ ), 908.97 (34, [M+H] 2+ ), 1815.93 (4, [M+H] + ), 1837.91 (2, [M+Na] + ), C 89 H 126 F4N 16 O 20 Calculation: 1814.93 [M] + .

[0164] DOTAGA.PEG2.Glu.(FAPi) 2 (2,2',2''-(10-((20S)-28-((4-((2-(2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)carbamoyl)quinolin-6-yl)oxy)-20-((4-((4-((2-(2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)carbamoyl)quinolin-6-yl)oxy)butyl)carbamoyl)-2,2-dimethyl-4,8,18,23-tetraoxo-3,12,15-trioxa-9,19,24-triazaoctacosan-5-yl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid) To the DOTAGA(tBu)4.PEG2.Glu.(FAPi)2 was added 50 μL water, 50 μL TIPS, and 1.5 mL trifluoroacetic acid (TFA). The dark brown solution was stirred at room temperature for 6 h and the solvent was removed under reduced pressure. A brown oil was obtained, which was purified by semi-preparative RP-HPLC (22% ACN isocratic, t R =17–18 min) to obtain DOTAGA.PEG2.Glu.(FAPi)2 (2.3 mg, 1.5 μmol, 10%) as a yellow solid. LC-MS (ESI-positive): m / z (%) = 398.70 (93, [M+H] 4+ ), 531.30 (100, [M+H] 3+ ), 796.20 (8, [M+H] 2+), 1591.85 (3, [M+H] + ), C 73 H 94 F4N 16 O 20 Calculated for: 1590.68 [M] + .

[0165] Example 5: DOTA.Glu.Glu.(FAPi) 2 , DOTAGA.Glu.Glu.(FAPi) 2 The synthesis of the labeled precursors DOTA.Glu.Glu.(FAPi)2 and DOTAGA.Glu.Glu.(FAPi)2 is shown in Scheme 20. The first synthetic steps are the same for both compounds.

[0166] [ka]

[0167] Fmoc-Glu(OtBu).Glu(OBzl) 2 ((S)-4-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-(tert-butoxy)-5-oxopentanoyl)-L-glutamic acid dibenzyl ester Fmoc-Glu-OtBu (400.0 mg, 0.94 mmol, 1.00 equiv.) was dissolved in dry DMF (2.0 mL) and DIPEA (151.9 mg, 200 μL, 1.2 mmol, 1.25 equiv.) and HATU (393.2 mg, 1.0 mmol, 1.10 equiv.) were added. The solution was then stirred at 25° C. under an argon atmosphere. After 1 h, dibenzyl glutamic acid (384.7 mg, 1.2 mmol, 1.25 equiv.) and DIPEA (267.3 mg, 352 μL, 2.1 mmol, 2.20 equiv.) dissolved in dry DMF (1.0 mL) were added. The next day, HATU (357.4 mg, 0.9 mmol, 1.00 equiv) and DIPEA (121.5 mg, 156 μL, 0.9 mmol, 1.00 equiv) were added again. After 3 days, 1.00 equiv HATU was added, followed 1 h later by a solution of dibenzylglutamic acid (153.87 mg, 0.5 mmol, 0.50 equiv) and 1.00 equiv DIPEA in 0.5 mL DMF. After an additional day at 25 °C, the solvent was removed under reduced pressure and the product was purified by column chromatography (cyclohexane:ethyl acetate (CH:EA, 3:1)). Fmoc-Glu(OtBu).Glu(OBzl)2 (657.3 mg, 0.89 mmol, 95%) was obtained as a pale yellow solid. LC-MS (ESI-positive): m / z (%) = 679.20 (27, [M- t Bu+H] + ), 680.30 (11, [M- t Bu+H] + ), 735.50 (100, [M+H] + ), 736.15 (15, [M+H] + ), C 43 H 46 Calculated for N2O9: 734.32 [M] + .

[0168] Fmoc-Glu(OtBu).Glu((S)-4-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-(tert-butoxy)-5-oxopentanoyl)-L-glutamic acid) Fmoc-Glu(OtBu).Glu(OBzl)2 (25.0 mg, 34.0 μmol, 1.00 equiv.) was dissolved in 1.0 mL of dry THF and palladium on activated carbon (10 wt% Pd, 7.25 mg, 78.0 μmol, 2.00 equiv.) was added. The suspension was stirred overnight under hydrogen atmosphere and filtered through Celite the next day. The residue was washed with THF and the latter was removed under reduced pressure. Fmoc-Glu(OtBu).Glu (17.8 mg, 32.1 μmol, 94%) was obtained as a colorless solid. LC-MS (ESI-positive): m / z (%) = 499.05 (57, [M- t Bu+H] + ), 500.15 (11, [M- t Bu+H] + ), 555.25 (100, [M+H] + ), 556.15 (21, [M+H] + ), C 29 H 34 Calculated for N2O9: 554.23 [M] + .

[0169] Fmoc-Glu(OtBu).Glu.(FAPi) 2 (N 2 -(((9H-fluoren-9-yl)methoxy)carbonyl)-N 5 -((2S)-1,5-Bis((4-((4-((2-(2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)carbamoyl)quinolin-6-yl)oxy)butyl)-amino)-1,5-dioxopentan-2-yl)-L-glutamic acid tert-butyl ester Fmoc-Glu(OtBu).Glu (33.3 mg, 60.0 μmol, 1.00 equiv.) was dissolved in HOBt (20.4 mg, 15.0 μmol, 2.50 equiv.) and EDC * The mixture was dissolved in dry DMF (2.5 mL) together with HCl (28.8 mg, 15.0 μmol, 2.50 equivalents) and stirred at room temperature for 1 hour under an argon atmosphere. * TFA (65.4 mg, 12.0 μmol, 2.00 equiv.) and DIPEA (23.3 mg, 31 μL, 18.0 μmol, 3.00 equiv.) were added. The next day, an additional equivalent of HOBt (7.8 mg, 60.0 μmol, 1.00 equiv.) and EDC were added. * HCl (11.4 mg, 60.0 μmol, 1.00 equivalent) was added, and after 30 minutes, half an equivalent of FAPi *TFA (16.5 mg, 30.0 μmol, 0.50 equiv.) was dissolved in 1 equiv. of DIPEA (7.8 mg, 10 μL, 60.0 μmol, 1.00 equiv.) and 0.5 mL of DMF was added. After 24 h, HOBt (3.9 mg, 30.0 μmol, 0.50 equiv.) and EDC were added. * HCl (5.7 mg, 30.0 μmol, 0.50 equiv.) was added again and after 1 h, more FAPi*TFA (16.5 mg, 30.0 μmol, 0.50 equiv.) and DIPEA (3.9 mg, 5 μL, 30.0 μmol, 0.50 equiv.) dissolved in DMF (0.5 mL) were added. This step was repeated once more the next day. After stirring the pale yellowish solution for another day, the solvent was removed under reduced pressure. Column chromatography (CHCl3:MeOH (100:10)) afforded Fmoc-Glu(OtBu).Glu.(FAPi)2 (86.7 mg, 62.8 μmol, 79%) as a yellow solid. LC-MS (ESI-positive): m / z (%) = 461.25 (32, [M+H] 3+ ), 691.45 (100, [M+H] 2+ ),692.25 (12, [M+H] 2+ ), 1381.95 (12, [M+H] + ), C 71 H 76 F4N 12 O 13 Calculation: 1380.56 [M] + .

[0170] Glu(OtBu).Glu.(FAPi) 2 (N 5 -((2S)-1,5-bis((4-((4-((2-(2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)carbamoyl)quinolin-6-yl)oxy)butyl)amino)-1,5-dioxopentan-2-yl)-L-glutamic acid tert-butyl ester) Fmoc-Glu(OtBu).Glu.(FAPi)2 (72.2 mg, 52.2 μmol, 1.00 equiv.) was dissolved in dry DMF (1.0 mL), 10% piperidine (0.1 mL) was added, and the mixture was stirred at room temperature for 90 min. The solvent was then removed under reduced pressure to give a yellowish oil, which was used directly in the next step without further purification. LC-MS (ESI-positive): m / z (%) = 387.10 (99, [M+H]3+ ), 580.35 (37, [M+H] 2+ ), 1159.30 (4, [M+H] + ), C 56 H 66 F4N 12 O 11 Calculated for: 1158.49 [M] + .

[0171] The synthesis of the labeled precursor DOTA.Glu.Glu.(FAPi)2 is shown in Scheme 21.

[0172] [ka]

[0173] DOTA(tBu) 3 .Glu(OtBu).Glu.(FAPi) 2 (2,2',2''-(10-(2-(((2S)-5-(((2S)-1,5-bis(((4-((4-((2-(2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)carbamoyl)quinolin-6-yl)oxy)butyl)amino)-1,5-dioxopentan-2-yl)amino)-1-(tert-butoxy))-1,5-dioxopentan-2-yl)amino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid tert-butyl ester) DOTA(tBu)3-NHS (17.5 mg, 26.1 μmol, 1.00 equiv.) was dissolved in 1.0 mL of dry DMF, and Glu(OtBu).Glu.(FAPi)2 (30.3 mg, 26.1 μmol, 1.00 equiv.) dissolved in 0.5 mL of DMF and 1 vol% DIPEA (11.4 mg, 15 μL, 88.2 μmol) were added. The pale yellowish solution was stirred at 40° C. for 24 hours under an argon atmosphere, and the solvent was then removed under reduced pressure. The resulting yellowish oil was then dissolved in 0.5 mL of dry DMF and DIPEA (3.4 mg, 4 μL, 26.1 μmol, 1.00 equiv.) was added. DOTA (17.5 mg, 26.1 μmol, 1.00 equiv.), HATU (14.9 mg, 39.2 μmol, 1.50 equiv.) and DIPEA (6.7 mg, 9 μL, 52.2 μmol, 2.00 equiv.) were added after the mixture had been stirred for 1 h in 0.5 mL of dry DMF. The yellowish solution was stirred for 24 h at 30 °C under argon atmosphere and then another HATU (1.50 equiv.) and DIPEA (2.00 equiv.) were added. After another 6 h at 40 °C, HATU (1.50 equiv.) and DIPEA (2.00 equiv.) were added once more. The next day, the solvent was removed under reduced pressure to give a yellowish oil. HPLC-MS (ESI-positive): m / z (%) = 429.47 (9, [M+H] 4+ ), 571.96 (10, [M+H] 3+ ), 857.43 (3, [M+H] 2+ ), C 84 H 116 F4N 16 O 18 Calculated for: 1712.94 [M] + .

[0174] DOTA.Glu.Glu.(FAPi) 2 (2,2',2''-(10-(2-(((1S)-4-(((2S)-1,5-bis((4-((4-((2-(2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)carbamoyl)quinolin-6-yl)oxy)butyl)amino)-1,5-dioxopentan-2-yl)amino)-1-carboxy-4-oxobutyl)amino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid) To the DOTA(tBu)3.Glu(OtBu).Glu.(FAPi)2 was added 50 μL of water, 50 μL of TIPS, and 1.5 mL of trifluoroacetic acid (TFA). The yellowish solution was stirred at room temperature for 5 h and the solvent was removed under reduced pressure. The crude product was purified by semi-preparative RP-HPLC (22–23% ACN, t in 20 min). R =13–14 min) to give DOTA.Glu.Glu.(FAPi)2 (6.6 mg, 4.4 μmol, 17%) as a yellow solid. LC-MS (ESI-positive): m / z (%) = 373.05 (84, [M+H] 4+ ), 497.15 (100, [M+H] 3+ ), 745.70 (5, [M+H] 2+ ), 1511.35 (1, [M+Na] + ), C 68 H 84 F4N 16 O 18 Calculated for: 1488.61 [M] + .

[0175] The synthesis of the labeled precursor DOTAGA.Glu.Glu.(FAPi)2 is shown in Scheme 22.

[0176] [ka]

[0177] DOTAGA(tBu)4 .Glu(OtBu).Glu.(FAPi) 2 (2,2',2''-(10-((10S,15S)-10-(tert-butoxycarbonyl)-23-((4-((2-(2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)carbamoyl)quinolin-6-yl)oxy)-15-((4-((4-((2-(2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)carbamoyl)quinolin-6-yl)oxy)butyl)-carbamoyl)-2,2-dimethyl-4,8,13,18-tetraoxo-3-oxa-9,14,19-triazatricosane-5-yl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid tert-butyl ester) DOTAGA(tBu)4 (22.4 mg, 32.6 μmol, 1.00 equiv.) was dissolved in dry MeCN (1.0 mL) with HBTU (24.7 mg, 65.3 μmol, 2.00 equiv.) and NHS (7.5 mg, 65.3 μmol, 2.00 equiv.) was added. The colorless solution was stirred under argon for 4 h, and HBTU (12.4 mg, 32.6 μmol, 1.00 equiv.) and NHS (3.8 mg, 32.6 μmol, 1.00 equiv.) dissolved in DMF (0.2 mL) were added. Glu(OtBu).Glu.(FAPi)2 (30.3 mg, 26.1 μmol, 1.00 equiv.) dissolved in DMF (1.0 mL) and 1 vol% DIPEA (19 mg, 25 μL, 147.0 μmol) were then added. The colorless solution was stirred at room temperature overnight and the next day the solvent was evaporated under reduced pressure to give a yellowish oil which was further transformed without further workup. HPLC-MS (ESI-positive): m / z (%) = 461.49 (52, [M+H] 4+ ), 614.99 (100, [M+H] 3+ ), 921.97 (56, [M+H] 2+ ), 1841.94 (35, [M+H] + ), 1863.93 (6, [M+Na] + ), C 91 H 128 F4N 16 O 20 Calculation: 1840.94 [M] + .

[0178] DOTAGA.Glu.Glu.(FAPi) 2 (2,2',2''-(10-(4-(((1S)-4-(((2S)-1,5-bis(((4-((4-((2-(2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)carbamoyl)quinolin-6-yl)oxy)butyl)amino)-1,5-dioxopentan-2-yl)amino)-1-carboxy-4-oxobutyl)amino)-1-carboxy-4-oxobutyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid) To the DOTAGA(tBu)4.Glu(OtBu).Glu.(FAPi)2 was added 50 μL water, 50 μL TIPS, and 1.5 mL trifluoroacetic acid (TFA). The yellowish solution was stirred at room temperature for 6 h and the solvent was removed under reduced pressure. The crude product was purified by semi-preparative RP-HPLC (22% ACN isocratic, t R=14–15 min) to give DOTAGA.Glu.Glu.(FAPi)2 (2.0 mg, 1.3 μmol, 5%) as a yellow solid. LC-MS (ESI-positive): m / z (%) = 391.10 (78, [M+H] 4+ ), 401.15 (19, [M+ACN+H] 4+ ), 521.30 (100, [M+H] 3+ ), 781.75 (6, [M+H] 2+ ), 1561,65 (3, [M+H] + ) C 71 H 88 F4N 16 O 20 Calculated for: 1560.63 [M] + .

[0179] Example 6: Examples of the compounds of the present invention that do not contain spacer units (S1, S2, S3) are shown below.

[0180] [ka] [ka] [ka] [ka] [ka] [ka] [ka]

[0181] Example 7: Examples of the compounds of the present invention having a spacer unit (S3) are shown below.

[0182]

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[0183] Example 8: Examples of compounds of the invention having two spacer units (S1+S2) are shown below.

[0184] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka]

[0185] Example 9: An example of a compound of the present invention having three spacer units (S1+S2+S3) is shown below.

[0186] [ka] [ka] [ka]

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Claims

1. 1. A dimerization label precursor for nuclear medicine diagnostics and theranostics, comprising the following structure: 【Chemistry 1】 where TV1 is a first targeting vector, TV2 is a second targeting vector, MG is a chelator or linker for conjugation or covalent attachment of a radioisotope, S1 is a first spacer, S2 is a second spacer, S3 is a third spacer, and TL is a Tris linker. having TV1 and TV2 are independently selected from the following structures [1] to [43]: 【Chemistry 2-1】 【Chemistry 2-2】 【Chemistry 2-3】 【Chemistry 2-4】 【Chemistry 2-5】 【Chemistry 2-6】 (In the formula, Structures [1]-[8] and [43] represent peptides, X is H or F; Y is H, CH ３ , CH(CH ３ ) ２ , C(CH ３ ) ３ Or (CH ２ ) ｎ CH ３ (n is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10). selected from one of: The tris linker TL has the following structures [52] to [116]: 【Chemistry 3-1】 【Chemistry 3-2】 【Chemistry 3-3】 or The structure: 【Chemistry 4】 having Dimerized labeled precursor.

2. MG is the following: H ４ pypa, EDTA (ethylenediaminetetraacetic acid), EDTMP (diethylenetriaminepenta(methylenephosphonic acid)), DTPA (diethylenetriaminepentaacetate) and its derivatives, NOTA (nona-1,4,7-triaminetriacetate) and its derivatives, TRAP (triazacyclononanephosphinic acid), NOPO (1,4,7-triazacyclononane-1,4-bis[methylene(hydroxymethyl)phosphinic acid]-7-[methylene(2-carboxyethyl)phosphinic acid]), DOTA (dodeca-1,4,7,10-tetraaminetetraacetate), DOTAGA (2-(1,4,7,10-tetraazacyclododecane-4,7,10)pentanedioic acid) and other DOTA derivatives, TRITA (trideca-1,4,7,10-tetraaminetetraacetate) ester), TETA (tetradeca-1,4,8,11-tetraaminetetraacetate) and its derivatives, PEPA (pentadeca-1,4,7,10,13-pentaaminepentaacetate), HEHA (hexadeca-1,4,7,10,13,16-hexaaminehexaacetate) and its derivatives, HBED (N,N'-bis(2-hydroxybenzyl)ethylenediamine-N,N'-diacetate) and its derivatives, DEDPA and its derivatives, DFO (deferoxamine) and its derivatives, trishydroxypyridinone (THP) and its derivatives, TEAP (tetraazacyclodecanephosphinic acid) and its derivatives, AAZTA (6-amino-6-methylperhydro-1,4-diazepane N,N,N',N'-tetraacetate) and its derivatives, DATA ５ｍ (5-[[6-(N-methyl)amino]-1,4-diacetate-1,4-diazepane]-pentanoic acid-N,N',N'-triacetate); Sarcofazine, SAR (1-N(4-aminobenzyl)-3,6,10,13,16,19-hexazabicyclo[6.6.6]eicosane-1,8-diamine) and its derivatives, N4 (3-[(2'-aminoethyl)amino]-2-[(2''-aminoethyl)aminomethyl]propionic acid) and other N ４ derivatives, PnAO (6-(4-isothiocyanatobenzyl)-3,3,9,9-tetramethyl-4,8-diazaundecane-2,10-dionedioxime) and derivatives, MAG2 (mercaptoacetylglycylglycine) and derivatives, MAG3 (mercaptoacetylglycylglycine) and derivatives, MAS3 (mercaptoacetylserylserylserine) and derivatives, MAMA (N-(2-mercaptoethyl)-2-[(2-mercaptoethyl)amino]acetamide) and derivatives, EC (ethylenedicysteine) and derivatives, dmsa (dimercaptosuccinic acid) and derivatives, DADT (diaminodithiol), DADS (diaminodisulfide), N ２ S ２ Chelating agents and their derivatives, aminothiols and their derivatives; salts of said chelating agents; hydrazine nicotinamide (HYNIC) and hydrazine nicotinamide derivatives 2. The method of claim 1, wherein the chelating agent is selected from the group consisting of:

3. MG is DOTA (dodeca-1,4,7,10-tetraaminetetraacetate), DATA ５ｍ 3. The label precursor of claim 2, which is 1,4-bis(carboxymethyl)-6-[methyl-carboxymethylamino]-6-pentanoic acid-1,4-diazepane or AAZTA (1,4-bis(carboxymethyl)-6-[bis(carboxymethyl)amino]-6-pentanoic acid-1,4-diazepane).

4. MG is the following: 【Chemistry 5】 The label precursor of claim 1 , selected from:

5. The spacers S1, S2, and S3 each independently have the following structure: 【Chemistry 6】 (In the formula, A, B, and C each independently represent an amide group, a carboxamide group, a phosphinate group, an alkyl group, a triazole group, a thiourea group, an ethylene group, a maleimide group, an amino acid residue, 【Chemistry 7】 (In the formula, s is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; p, q and r are independently selected from the set {0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20}) The label precursor of claim 1, selected from the group consisting of:

6. The spacers S1, S2, and S3 each independently have the following structure: 【Chemistry 8】 The label precursor of claim 1 , having the formula:

7. The spacers S1, S2, and S3 are independently a peptide, dipeptide, or tripeptide group having the following structure: 【Chemistry 9】 The label precursor of claim 1 , selected from:

8. R １ 、R ２ 、R ３ are, independently, -H, -CH ３ , -CH(CH ３ ), ２ , -CH ２ CH(CH ３ ), ２ , -CH(CH ３ ), -CH ２ CH ３ , -CH ２ , -Phe, -CH ２ , -Phe-OH, -CH ２ SH, -(CH ２ ), ２ , -S-CH ３ , -CH ２ OH, -(CH)(OH)(CH ３ ), -(CH ２ ), ４ NH ２ , -(CH ２ ), ３ , -NH(C=NH)NH ２ , -CH ２ COOH, -(CH ２ ), ２ , -COOH, -CH ２ (C=O)NH ２ , -(CH ２ ), ２ (C=O)NH ２ , 【Chemistry 10】 The label precursor of claim 7, selected from the group consisting of:

9. 2. The labeled precursor of claim 1, wherein TV1 is the same as TV2 (TV1=TV2).

10. The labeled precursor of claim 1 , wherein TV1 and TV2 are different from each other (TV1≠TV2).

11. The label precursor of claim 10, wherein TV1 has one of structures [9] to [12], and TV2 has one of structures [13] or [14].

12. 11. The label precursor of claim 10, wherein TV1 has one of the structures [9] to [12], and TV2 has one of the structures [40] or [41].

13. The structure: 【Chemistry 11】 The label precursor of claim 1 , having the formula:

14. A radioactive tracer for nuclear medicine diagnostics and theranostics, comprising a labeled precursor according to any one of claims 1 to 13, ４４ Sc, ４７ Sc, ５５ Co, ６２ Cu, ６４ Cu, ６７ Cu, ６６ G.A. ６７ G.A. ６８ G.A. ８９ Zr, ８６ Y. ９０ Y. ８９ Zr, ９０ Nb, ９９ｍ Tc, １１１ In, １３５ Sm, １４０ P.R. １５９ Gd, １４９ Tb, １６０ Tb, １６１ Tb, １６５ Er, １６６ Dy, １６６ Ho, １７５ Yb, １７７ Lu, １８６ Re, １８８ Re, ２１１ At, ２１２ Pb, ２１３ Bi, ２２５ A c, ２３２ Th, １８ F. １３１ I or ２１１ and a radioactive isotope selected from the group consisting of At.

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