FAPI theranostic compounds assembled with tetrazine ligation

Abstract

The present invention relates to fibroblast activation protein (FAP) - based ligands assembled through tetrazine ligation and their use in radiotherapy and imaging. The FAP inhibitor (FAPi) ligand compounds provided herein comprises a FAP targeting moiety, a linker, an IsoF-TCO and a radiolabeled tetrazine. The FAPi targeting compounds are possessing advantageous pharmacokinetic properties and enable quick and efficient modifications of the radiopharmaceuticals allowing a fast renal excretion and reduced liver accumulation.

Description

[0001] FAPi THERANOSTIC COMPOUNDS ASSEMBLED WITH TETRAZINE LIGATION

[0002] Field of the Invention

[0003] The present invention relates to fibroblast activation protein (FAP) - based ligands assembled through tetrazine ligation and their use in radiotherapy and imaging.

[0004] Background

[0005] Fibroblast activation protein alpha (FAP) is a S9b family serine protease and an integral type- II trans-membrane glycoprotein. FAP is mainly expressed on activated fibroblasts such as cancer-associated fibroblasts (CAFs), which are a major part of the tumor microenvironment (TME). The TME plays an essential role in tumor genesis, tumor growth, metastasis and angiogenesis and can account for up to 90% of the total tumor mass. Each epithelial tumor of a certain size (ca. > 1 mm) is surrounded by an extensive TME. More than 90% of these epithelial tumors strongly express FAP including breast, lung, colorectal, prostate, ovarian among other cancers as well as sarcomas. In contrast, FAP is not expressed by resting fibroblasts in adult, healthy tissue. The selective expression makes FAP an interesting pantumor target in medicinal use, as FAP-based ligands can be used in the diagnosis and / or treatment of a wide range of cancers, including breast, lung, colorectal, prostate, ovarian, pancreatic, and hepatocellular cancers, as well as sarcomas and non-small cell lung cancer (NSCLC).

[0006] Beyond cancer, FAP expression is significant in several non-cancerous conditions such as fibrosis, arthritis, atherosclerosis, and inflammatory diseases like spondyloarthritis. This broad range of expression underscores FAP’s potential as a target for diagnostic and therapeutic applications in various pathological conditions.

[0007] In 2018, the high-affinity and selective FAP inhibitor (FAPi) UAMC-1110 was used as a targeting vector in the first small-molecule radiotracers successfully visualizing various epithelial tumors via positron emission tomography (PET). Within a few years, molecular imaging with these68Ga-labeled radiotracers such as FAPI-04, FAPI-46, DOTA.SA.FAPi and OncoFAP showed highly valuable diagnostic results outperforming or at least being on par with18F-FDG. Other diagnostic tracers using different radionuclides such as18F-FAPI-42 / 74 and99mTc-FAPI-34 among others have also been developed and have shown diagnostic value.

[0008] For diagnostic purposes, the amount of radioactivity administered can vary based on the specific radiotracer and imaging modality used. Typically, for PET imaging with radiolabeled FAP inhibitors such as 68Ga-FAPI tracers, a dose in the range of 100-200 MBq is commonly used to achieve high-quality images while minimizing radiation exposure to patients. For SPECT imaging using 99mTc-FAPI tracers, the administered dose might range from 300 to 740 MBq, depending on the specific clinical protocol and the patient's condition. The dosage can vary significantly depending on factors such as the type of radionuclide used, the sensitivity of the imaging equipment, and the specific clinical scenario. For instance, wholebody PET scans, which are highly sensitive, may require lower doses of radiotracer to achieve effective imaging results.

[0009] However, translation into effective therapeutic radiopharmaceuticals for radioligand therapy (RLT) has proven to be challenging. The main problem is the short tumor residence time of the above-mentioned FAPi monomers (one targeting vector) resulting in a low radiation tumordose when labeled with therapeutic radiometals such as177Lu,90Y or225Ac. Substantial progress regarding the tumor-dose could be achieved with FAPi dimers177Lu- DOTAGA.(SA.FAPi)2and177Lu-DOTAGA.GIu.(FAPi)2 containing two FAPi targeting vectors. They can be administered safely, and the therapeutic efficacy has been investigated mostly in thyroid cancer, with a response rate of >50% in 15 heavily pre-treated RR-DTC patients and an overall survival after a 3-year follow-up of >50%. Other groups adapted the dimer approach and found prolonged tumor retention and improved therapeutic efficacy. Simultaneously, FAP-targeting peptides FAP-2286 and 3BP-3940 labeled with various therapeutic radiometals were developed and performed promisingly in preclinical xenograft models, but the translation into clinical studies could not confirm the therapeutic efficacy (Zboralski et al., Eur. J. Nucl. Med. Mol. Imaging, 49, 3651-3667, 2022; Baum et al., J. Nucl. Med., 63, 415-423, 2022; Baum et al., J. Nucl. Med., 63, 2269, 2022).

[0010] Radiohalogens have also been tested with FAP-directed therapy. For instance, radioiodine, especially1311, since it is available in large quantities and relatively cheap as it formed as a fission product in nuclear reactors would be beneficial to apply since the potentially high demand for FAP therapeutics can be met with1311 as the therapeutic radionuclide. The only published radioiodine-FAP compound131l-FAPI-04 thus far showed suboptimal properties in preclinical models including short tumor retention time and high doses to liver and Gl tract due to hepatobiliary excretion (Ma et al., Mol. Pharm., 18, 4179-4187, 2021).

[0011] Astatine-211 (211At) could also be relevant in FAP-directed therapy as a valid alternative to225Ac based on its short half-life of 7.2 hours and its emission that does produce any long-lived alpha-emitting daughters. Both of these properties can significantly reduce cytotoxicity to the patients compared to225Ac-based FAPi derivatives (Albertsson et al., Front Med (Lausanne), 9, 1076210, 2022). Radiometals, such as Gallium-68, Copper-64, and Zirconium-89, are typically introduced into molecules through chelation. This process involves using chelator groups that form coordinate bonds with the metal ions, ensuring stable attachment. The chelation reaction usually involves mixing the radiometal ions with a precursor molecule containing the chelator group. This mixture is then heated to facilitate the chelation process. Common chelators include DOTA (1 ,4,7,10-tetraazacyclododecane-1 ,4,7,10-tetraacetic acid) and NOTA (1 ,4,7- triazacyclononane-1 ,4,7-triacetic acid), which form strong, stable complexes with radiometals. This method ensures that the radiometal remains securely bound to the molecule, maintaining the integrity and functionality of the radiopharmaceutical. For example, Gallium-68 is often incorporated into molecules using the DOTA chelator, resulting in compounds like Ga-68 DOTATATE, which targets somatostatin receptors in neuroendocrine tumors.

[0012] Radiohalogens, such as lodine-123, Fluorine-18, and Astatine-211 , are incorporated into molecules through covalent bonding, typically via nucleophilic substitution reactions. This process involves the replacement of a leaving group in the molecule with the radiohalogen. For instance, in the case of iodine, an electrophilic substitution reaction can be employed, where radioactive iodide is oxidized to a positively charged iodine species that then replaces a leaving group, usually a stannyl group, in an aromatic substitution reaction. This reaction is conducted at room temperature and often yields high efficiency. Another method for introducing iodine into molecules is isotopic exchange, where a non-radioactive iodine atom in the molecule is replaced by a radioactive one. This process usually requires elevated temperatures and acidic conditions, often catalyzed by copper. Astatine-211 , being a halogen, can also be attached to aryl rings forming astatoaryl moieties. However, due to its unique chemistry, astatine cannot be stably coupled to tyrosine residues of proteins like iodine can. Instead, it forms bonds with sulfhydryl groups of cysteine, necessitating the synthesis of dedicated precursors with suitable leaving groups like trialkylstannyl. The labeling reaction for astatine often uses oxidation agents such as chloramine-T or N-chlorosuccinimide, which can degrade biomolecules used as targeting vectors.

[0013] Overall, the reactions to incorporate chelators usually needs a purification process and heating, which can degrade temperature-sensitive vectors. In contrast, radiohalogens form covalent bonds with targeting vectors, often under harsh and lengthy conditions incompatible with peptides and other sensitive biomolecules. To overcome these challenges, synthon- based methods have been developed, allowing radiolabeling under mild conditions without exposing the vector to harsh environments. Tetrazine ligation exemplifies this approach and the dual-functional tetrazine compounds facilitate these radiolabeling processes, providing a versatile platform for creating advanced radiopharmaceuticals. However, common synthesis and radiolabeling procedures are based on standard methodologies that are not always easy to implement in radiopharmacies, hospitals and clinics. Click chemistry has shown great potential in the synthesis of radiopharmaceuticals for imaging and therapy. In particular, the inverse electron demand Diels- Alder cycloaddition reaction (I EDDA) between tetrazines and dienophiles has shown the fastest reaction kinetics in click chemistry, and it has been utilized in radiosynthesis of various labeled radiopharmaceuticals. The main limitation of this reaction was the formation of multiple isomers after the click. This issue has been recently solved by the synthesis of a new type of isomer-free frans-cyclooctenes (IsoF-TCOs), that combined with an oxidation step result in the formation of a single isomeric product (PCT / EP2023 / 055930).

[0014] The FAP targeting moiety of the present invention is based on a dimeric FAPi that has already been successfully applied for therapy in the clinic. High affinity, selectivity as well as good and prolonged tumor accumulation are reported for this structure. As shown herein, we have found that by synthesizing a compound targeting FAPi, wherein the compound comprises a linker, an IsoF-TCO and a radiolabeled tetrazine, FAPi targeting compounds possessing selected pharmacokinetic properties are obtained. The present compounds can be labeled either with a radiohalogen or a radiometal. The modularity of this approach enables quick and efficient modifications of the radiopharmaceuticals allowing the fast optimization of some properties such as renal excretion and liver accumulation.

[0015] Moreover, the radiolabeled compounds provided herein showed a surprisingly higher tumor accumulation compared to [111ln]ln-DOTAGA.GIu.(FAPi)2 (Martin et al., Cancers, 15(6), 1889, 2023).

[0016] Summary of the invention:

[0017] In a first aspect, the present invention relates to a FAP inhibitor ligand of formula (I): wherein:

[0018] Ri is H or F

[0019] -(CH2)nNH-, -CH2(OCH2CH2)nNH-, -(CH2)nNHAm-, -CH2(OCH2CH2)nNHAm-,

[0020] CH2C(COOH)NHAm-, -CH2CH2C(COOH)NHAm- wherein n and m are integers independently selected from 0-20, and A is an amino acid independently selected from the group consisting of: -COCH2NH-, -COCH(CH3)NH-, -COCH(CH2SH)NH-, -COCH(CH2COOH)NH-, - COCH(CH2CH2COOH)NH-, -COCH(CH2C6H5)NH-, -COCH(CH2C3H3N2)NH-, - COCH(CH(CH3)2)NH-, -COCH((CHCH3)CH2CH3)NH-, -COCH((CH2)4NH2)NH-, - COCH(CH2CH(CH3)2)NH-, -COCH(CH2CH2SCH3)NH-, -CO(CHCH2CH2N)-, - COCH(CH2CONH2)NH-, -COCH(CH2CH2CONH2)NH-, -COCH((CH2)3NH- C(NH)NH2)NH-, -COCH(CH2OH)NH-, -COCH(CH2CH2OH)NH-, COCH(CH(OH)CH3)NH-, -COCH(CH2SeH)NH-, -COCH(CH2C8H6N)NH-, - COCH(CH2C6H4OH)NH-, -COCH(CH2CH2SeCH3)NH-;

[0021] Xi is a pyridazine selected from the group consisting of: wherein L2is -(CH2)nCO-, -(CH2CH2O)n(CH2)mCO or -C(CH3)2(CH2)CO-, wherein n and m are integers independently selected from 1-10, and

[0022] R2is selected from -H, -Me, and R3, wherein the curly sign indicates the link to the tetrazine; and where R4 is selected from: -H, -Y1, -OCH2CH2Y1, -OCH2CH2 CH2Y1, -SCH2CH2Y1, -NHCH2CH2Y1, - OCH2C6H4YI, -OCH2CH2C6H4Y1, -NHCOCH2CH2Y1, -CONHCH2CH2Y1, -NHSO2CH2CH2Y1, -

[0023] SO2NHCH2CH2Y1, wherein Y1 is selected from the group constisting of:

[0024] 1H,2H,3H,11C,12C,13C,14C,18F,19F,76Br,77Br,79Br,80Br,80mBr,81Br,123l 124| 125| 126| 127| 131|210At,211At, and wherein L3 is selected from: -H, -(CH2)nNH-, -O(CH2)nNH-, -S(CH2)nNH-, - SO2NH(CH2)nNH-, -NHSO2(CH2)nNH-, -CONH(CH2)nNH-, -NHCO(CH2)nNH-, - (OCH2CH2)n(CH2)mNH-, -(CH2)n(OCH2CH2)mNH-, -O(CH2)n(OCH2CH2)mNH-, - S(CH2)n(OCH2CH2)mNH-, -SO2NH(CH2)n(OCH2CH2)mNH-, - NHSO2(CH2)n(OCH2CH2)mNH-, -CONH(CH2)n(OCH2CH2)mNH-, - NHCO(CH2)n(OCH2CH2)mNH-, -CH2(OCH2CH2)(CH2)mNH-, - OCH2(OCH2CH2)n(CH2)mNH-, -SCH2(OCH2CH2)n(CH2)mNH-, - SO2NHCH2(OCH2CH2)n(CH2)mNH-, -NHSO2CH2(OCH2CH2)n(CH2)mNH-, - CONHCH2(OCH2CH2)n(CH2)mNH-, -NHCOCH2CH2(OCH2CH2)n(CH2)mNH-, - CH2CH2(OCH2CH2)(CH2)mNH-, -OCH2CH2(OCH2CH2)n(CH2)mNH-, - SCH2CH2(OCH2CH2)n(CH2)mNH-, -SO2NHCH2CH2(OCH2CH2)n(CH2)mNHCO-, - NHSO2CH2CH2(OCH2CH2)n(CH2)mNHCO-, -CONHCH2CH2(OCH2CH2)n(CH2)mNHCO- , -NHCOCH2CH2(OCH2CH2)n(CH2)mNHCO-, where n and m are independently selected from the group consisting of 0-25; and wherein Z is a chelator selected from: -H, 1 ,4,7, 10-tetraazacyclododecane- / V, / V', / \ / ', / \ / "-tetraacetic acid (DOTA), 2-(4,7, 10-tris(2-(tert-butoxy)-2-oxoethyl)-1 ,4,7, 10- tetraazacyclododecan-1-yl)acetic acid (fBu-DOTA), / V, / V'-bis(2-hydroxy-5- (carboxyethyl)benzyl)ethylenediamine / V, / V'-diacetic acid (HBED-CC), 14,7- triazacyclononane-1 ,4,7-triacetic acid (NOTA), 2,2',2"-(2-(4-((l2- azaneyl)methanethioamido)benzyl)-1 ,4,7-triazonane-1 ,4, 7-triyl)triacetic acid (benzyl- NOTA), 2-(4.7-bis(carboxymethyl)-1 ,4,7-triazonan-1-yl)pentanedioic acid (NODAGA), 2-(4,7, 10-tris(carboxymethyl)-1 ,4,7, 10-tetraazacyclododecan-1- yl)pentanedioic acid (DOTAGA), 14,7-triazacyclononane phosphinic acid (TRAP), 14,7-triazacyclononane- 1-methyl(2-carboxyethyl)phosphinic acid-4, 7-bis(methyl(2-hydroxymethyl)phosphinic acid (NOPO), 3,6,9, 15-tetraazabicyclo9.3.1.pentadeca-1 (15), 11 , 13-triene-3,6,9- triacetic acid (PCTA), / \ / '-(5-acetyl (hydroxy)aminopentyl- / V-(5-(4-(5- aminopentyl)(hydroxy)amino-4-oxobutanoyl)amino)pentyl- / \ / -hydroxysuccinamide (DFO), diethylenetriaminepentaacetic acid (DTPA), frans-cyclohexyl- diethylenetriaminepentaacetic acid (CHX-DTPA), 1-oxa-4,7, 10-triazacyclododecane- 4,7, 10-triacetic acid (OXO-Do3A), p-isothiocyanatobenzyl-DTPA (SCN-BZ-DTPA), 1- (p-isothiocyanatobenzyl)-3-methyl-DTPA (1 B3M), 2-(p-isothiocyanatobenzyl)-4- methyl-DTPA (1 M3B), and 1-(2)-methyl-4-isocyanatobenzyl-DTPA (MX-DTPA), and wherein Z is optionally labeled with a metal selected from: and wherein Rs is selected from a -H, a halogen, -OH, -OMe, -OEt, -OPr, -NHCOH, -NHCOMe, NHCOEt, -NHCOPr, -CONH2, -CONHMe, -CONHEt, -CONHPr, NHSOH, -NHSO2Me, NHSO2Et, -NHSO2Pr, -SO2NH2, -SO2NHMe, -SO2NHEt, - SO2NHPr, -NH2, -NHMe, -NHEt, -NHPr; and pharmaceutically acceptable salts thereof.

[0025] In a second aspect, the present invention relates to a pharmaceutical formulation comprising a FAP inhibitor lignad of formula I.

[0026] In a third aspect, the present invention relates to FAP inhibitor ligand of formula I: wherein:

[0027] -(CH2)nNH-, -CH2(OCH2CH2)nNH-, -(CH2)nNHAm-, -CH2(OCH2CH2)nNHAm-, -

[0028] CH2C(COOH)NHAm-, -CH2CH2C(COOH)NHAm- wherein n and m are integers independently selected from 0-20, and A is an amino acid independently selected from the group consisting of: -COCH2NH-, -COCH(CH3)NH-, -COCH(CH2SH)NH-, -COCH(CH2COOH)NH-, - COCH(CH2CH2COOH)NH-, -COCH(CH2C6H5)NH-, -COCH(CH2C3H3N2)NH-, COCH(CH(CH3)2)NH-, -COCH((CHCH3)CH2CH3)NH-, -COCH((CH2)4NH2)NH-, - COCH(CH2CH(CH3)2)NH-, -COCH(CH2CH2SCH3)NH-, -CO(CHCH2CH2N)-, COCH(CH2CONH2)NH-, -COCH(CH2CH2CONH2)NH-, -COCH((CH2)3NH-C(NH)NH2)NH-, -COCH(CH2OH)NH-, -COCH(CH2CH2OH)NH-, -COCH(CH(OH)CH3)NH-,

[0029] COCH(CH2SeH)NH-, -COCH(CH2C8H6N)NH-, -COCH(CH2C6H4OH)NH-;

[0030] COCH(CH2CH2SeCH3)NH-,

[0031] Xi is a pyridazine selected from the group consisting of: wherein L2is -(CH2)nCO-, -(CH2CH2O)n(CH2)mCO or -C(CH3)2(CH2)CO-, wherein n and m are integers independently selected from 1-10,

[0032] R2is selected from -H, -Me, and R3, , wherein the curly sign indicates the link to the tetrazine; and where

[0033] R4is selected from: -H, -Y1, -OCH2CH2YI, -OCH2CH2CH2YI, -SCH2CH2YI, - NHCH2CH2YI, -OCH2C6H4YI, -OCH2CH2C6H4YI, -NHCOCH2CH2YI, CONHCH2CH2YI, -NHSO2CH2CH2YI, -SO2NHCH2CH2YI, wherein Y1 is selected from the group constisting of; and wherein L3 is selected from: -H, -(CH2)nNH-, -O(CH2)nNH-, -S(CH2)nNH-, -

[0034] SO2NH(CH2)nNH-, -NHSO2(CH2)nNH-, -CONH(CH2)nNH-, -NHCO(CH2)nNH-, -

[0035] (OCH2CH2)n(CH2)mNH-, -(CH2)n(OCH2CH2)mNH-, -O(CH2)n(OCH2CH2)mNH-, -

[0036] S(CH2)n(OCH2CH2)mNH-, -SO2NH(CH2)n(OCH2CH2)mNH-,

[0037] NHSO2(CH2)n(OCH2CH2)mNH-, -CONH(CH2)n(OCH2CH2)mNH-,

[0038] NHCO(CH2)n(OCH2CH2)mNH-, -CH2(OCH2CH2)(CH2)mNH-,

[0039] OCH2(OCH2CH2)n(CH2)mNH-, -SCH2(OCH2CH2)n(CH2)mNH-,

[0040] SO2NHCH2(OCH2CH2)n(CH2)mNH-, -NHSO2CH2(OCH2CH2)n(CH2)mNH-,

[0041] CONHCH2(OCH2CH2)n(CH2)mNH-, -NHCOCH2CH2(OCH2CH2)n(CH2)mNH-,

[0042] CH2CH2(OCH2CH2)(CH2)mNH-, -OCH2CH2(OCH2CH2)n(CH2)mNH-,

[0043] SCH2CH2(OCH2CH2)n(CH2)mNH-, -SO2NHCH2CH2(OCH2CH2)n(CH2)mNHCO-,

[0044] NHSO2CH2CH2(OCH2CH2)n(CH2)mNHCO-, -CONHCH2CH2(OCH2CH2)n(CH2)mNHCO-

[0045] , or -NHCOCH2CH2(OCH2CH2)n(CH2)mNHCO-, where n and m are independently selected from the group consisting of 0-25; and wherein Z is a chelator selected from: -H, 1 ,4,7, 10-tetraazacyclododecane- N,N',N',N "-tetraacetic acid (DOTA), 2-(4,7, 10-tris(2-(tert-butoxy)-2-oxoethyl)-1 ,4,7, 10- tetraazacyclododecan-1-yl)acetic acid (fBu-DOTA), / V, / V'-bis(2-hydroxy-5- (carboxyethyl)benzyl)ethylenediamine N,N -diacetic acid (HBED-CC), 14,7- triazacyclononane-1 ,4,7-triacetic acid (NOTA), 2,2',2"-(2-(4-((l2- azaneyl)methanethioamido)benzyl)-1 ,4,7-triazonane-1 ,4, 7-triyl)triacetic acid (benzyl- NOTA), 2-(4.7-bis(carboxymethyl)-1 ,4,7-triazonan-1-yl)pentanedioic acid (NODAGA), 2-(4,7, 10-tris(carboxymethyl)-1 ,4,7, 10-tetraazacyclododecan-1- yl)pentanedioic acid (DOTAGA), 14,7-triazacyclononane phosphinic acid (TRAP), 14,7-triazacyclononane- 1-methyl(2-carboxyethyl)phosphinic acid-4, 7-bis(methyl(2-hydroxymethyl)phosphinic acid (NOPO), 3,6,9, 15-tetraazabicyclo9.3.1.pentadeca-1 (15), 11 , 13-triene-3,6,9- triacetic acid (PCTA), / \ / '-(5-acetyl (hydroxy)aminopentyl- / V-(5-(4-(5- aminopentyl)(hydroxy)amino-4-oxobutanoyl)amino)pentyl- / \ / -hydroxysuccinamide (DFO), diethylenetriaminepentaacetic acid (DTPA), frans-cyclohexyl- diethylenetriaminepentaacetic acid (CHX-DTPA), 1-oxa-4,7, 10-triazacyclododecane- 4,7, 10-triacetic acid (OXO-Do3A), p-isothiocyanatobenzyl-DTPA (SCN-BZ-DTPA), 1- (p-isothiocyanatobenzyl)-3-methyl-DTPA (1 B3M), 2-(p-isothiocyanatobenzyl)-4- methyl-DTPA (1 M3B), and 1-(2)-methyl-4-isocyanatobenzyl-DTPA (MX-DTPA); and wherein Z is optionally labeled with a metal selected from:

[0046] 43Sc,44Sc,45Sc,47Sc,45Ti,46Ti,47Ti,48Ti,49Ti,50Ti,51Cr,52Cr,53Cr,54Cr,54Fe,56Fe,57Fe, 58Fe,55Co,58mCo,59Co,60Cu,61Cu,63Cu,64Cu,65Cu,67Cu,67Ga,68Ga,69Ga,71Ga, 72As,75As,84Sr,86Sr,87Sr,88Sr,89Sr,86Y,89Y,90Y,89Zr,90Zr,91Zr,92Zr,94Zr,94Tc,99mTc, 103Rh103mRh193mPt195mpt192Pt194Pf 195p| 196p| 198p| 111 In 113|p] 114m|n 115m|n 119gp 121Sb,123Sb,135La,138La,139La,149Tb,154Gd,155Gd,156Gd,157Gd,158Gd,160Gd152Tb, 159Tb,156Dy,158Dy,160Dy,161Dy,162Dy,163Dy,164Dy,165Dy ,161Tb,162Er,164Er,165Er,166Er, 167Er,168Er,170Er,165Ho,166Ho,175Lu,177Lu,185Re,186Re,188Re,201TI,203TI,205TI,203Pb, 206Pb,207Pb,208Pb,212Pb,209Bi,212Bi,213Bi,223Ra,225Ac,227Th,232Th; and wherein Rs is selected from a -H, a halogen, -OH, -OMe, -OEt, -OPr, -NHCOH, -NHCOMe, NHCOEt, -NHCOPr, -CONH2, -CONHMe, -CONHEt, -CONHPr, NHSOH, -NHSO2Me, NHSO2Et, -NHSO2Pr, -SO2NH2, -SO2NHMe, -SO2NHEt, - SO2NHPr, -NH2, -NHMe, -NHEt, -NHPr; and wherein said FAP inhibitor ligand of formula (I) comprises at least one radionuclide; and pharmaceutically acceptable salts thereof, for use as a medicament in therapy, imaging, diagnostics, or theranostics.

[0047] In a fourth aspect, the present invention relates to FAP inhibitor ligand of formula I: wherein:

[0048] Ri is H or F

[0049] -(CH2)nNH-, -CH2(OCH2CH2)nNH-, -(CH2)nNHAm-, -CH2(OCH2CH2)nNHAm-, - CH2C(COOH)NHAm-, -CH2CH2C(COOH)NHAm- wherein n and m are integers independently selected from 0-20, and A is an amino acid independently selected from the group consisting of: -COCH2NH-, -COCH(CH3)NH-, -COCH(CH2SH)NH-, -COCH(CH2COOH)NH-, - COCH(CH2CH2COOH)NH-, -COCH(CH2C6H5)NH-, -COCH(CH2C3H3N2)NH-, - COCH(CH(CH3)2)NH-, -COCH((CHCH3)CH2CH3)NH-, -COCH((CH2)4NH2)NH-, - COCH(CH2CH(CH3)2)NH-, -COCH(CH2CH2SCH3)NH-, -CO(CHCH2CH2N)-, - COCH(CH2CONH2)NH-, -COCH(CH2CH2CONH2)NH-, -COCH((CH2)3NH- C(NH)NH2)NH-, -COCH(CH2OH)NH-, -COCH(CH2CH2OH)NH-, COCH(CH(OH)CH3)NH-, -COCH(CH2SeH)NH-, -COCH(CH2C8H6N)NH-, COCH(CH2C6H4OH)NH-; -COCH(CH2CH2SeCH3)NH-,

[0050] Xi is a pyridazine selected from the group consisting of: wherein L2is -(CH2)nCO-, -(CH2CH2O)n(CH2)mCO or -C(CH3)2(CH2)CO-, wherein n and m are integers independently selected from 1-10,

[0051] R2is independently selected from -H, -Me, and R3, , wherein the curly sign indicates the link to the tetrazine; and where R4is selected from: -H, -Y1, -OCH2CH2YI, -OCH2CH2CH2YI, -SCH2CH2YI, - NHCH2CH2YI, -OCH2C6H4YI, -OCH2CH2C6H4YI, -NHCOCH2CH2YI, CONHCH2CH2YI, -NHSO2CH2CH2YI, -SO2NHCH2CH2YI, wherein Y1 is selected from the group constisting of;

[0052] 1H,2H,3H,11

[0053] 126| 127| 1311 and wherein L3 is selected from: -H, -(CH2)nNH-, -O(CH2)nNH-, -S(CH2)nNH-, - SO2NH(CH2)nNH-, -NHSO2(CH2)nNH-, -CONH(CH2)nNH-, -NHCO(CH2)nNH-, - (OCH2CH2)n(CH2)mNH-, -(CH2)n(OCH2CH2)mNH-, -O(CH2)n(OCH2CH2)mNH-, - S(CH2)n(OCH2CH2)mNH-, -SO2NH(CH2)n(OCH2CH2)mNH-, - NHSO2(CH2)n(OCH2CH2)mNH-, -CONH(CH2)n(OCH2CH2)mNH-, - NHCO(CH2)n(OCH2CH2)mNH-, -CH2(OCH2CH2)(CH2)mNH-, - OCH2(OCH2CH2)n(CH2)mNH-, -SCH2(OCH2CH2)n(CH2)mNH-, - SO2NHCH2(OCH2CH2)n(CH2)mNH-, -NHSO2CH2(OCH2CH2)n(CH2)mNH-, - CONHCH2(OCH2CH2)n(CH2)mNH-, -NHCOCH2CH2(OCH2CH2)n(CH2)mNH-, - CH2CH2(OCH2CH2)(CH2)mNH-, -OCH2CH2(OCH2CH2)n(CH2)mNH-, - SCH2CH2(OCH2CH2)n(CH2)mNH-, -SO2NHCH2CH2(OCH2CH2)n(CH2)mNHCO-, - NHSO2CH2CH2(OCH2CH2)n(CH2)mNHCO-, -CONHCH2CH2(OCH2CH2)n(CH2)mNHCO- , -NHCOCH2CH2(OCH2CH2)n(CH2)mNHCO-, where n and m are independently selected from the group consisting of 0-25; and wherein Z is a chelator selected from: -H, 1 ,4,7, 10-tetraazacyclododecane- N,N',N',N "-tetraacetic acid (DOTA), 2-(4,7, 10-tris(2-(tert-butoxy)-2-oxoethyl)-1 ,4,7, 10- tetraazacyclododecan-1-yl)acetic acid (fBu-DOTA), / V, / V'-bis(2-hydroxy-5- (carboxyethyl)benzyl)ethylenediamine N,N -diacetic acid (HBED-CC), 14,7- triazacyclononane-1 ,4,7-triacetic acid (NOTA), 2,2',2"-(2-(4-((l2- azaneyl)methanethioamido)benzyl)-1 ,4,7-triazonane-1 ,4, 7-triyl)triacetic acid (benzyl- NOTA), 2-(4.7-bis(carboxymethyl)-1 ,4,7-triazonan-1-yl)pentanedioic acid (NODAGA), 2-(4,7, 10-tris(carboxymethyl)-1 ,4,7, 10-tetraazacyclododecan-1- yl)pentanedioic acid (DOTAGA), 14,7-triazacyclononane phosphinic acid (TRAP), 14,7-triazacyclononane- 1-methyl(2-carboxyethyl)phosphinic acid-4, 7-bis(methyl(2-hydroxymethyl)phosphinic acid (NOPO), 3,6,9, 15-tetraazabicyclo9.3.1.pentadeca-1 (15), 11 , 13-triene-3,6,9- triacetic acid (PCTA), / \ / '-(5-acetyl (hydroxy)aminopentyl- / V-(5-(4-(5- aminopentyl)(hydroxy)amino-4-oxobutanoyl)amino)pentyl- / \ / -hydroxysuccinamide (DFO), diethylenetriaminepentaacetic acid (DTPA), frans-cyclohexyl- diethylenetriaminepentaacetic acid (CHX-DTPA), 1-oxa-4,7, 10-triazacyclododecane- 4,7, 10-triacetic acid (OXO-Do3A), p-isothiocyanatobenzyl-DTPA (SCN-BZ-DTPA), 1- (p-isothiocyanatobenzyl)-3-methyl-DTPA (1 B3M), 2-(p-isothiocyanatobenzyl)-4- methyl-DTPA (1 M3B), and 1-(2)-methyl-4-isocyanatobenzyl-DTPA (MX-DTPA); and wherein Z is optionally labeled with a metal selected from:

[0054] 43Sc,44Sc,45Sc,47Sc,45Ti,46Ti,47Ti,48Ti,49Ti,50Ti,51Cr,52Cr,53Cr,54Cr,54Fe,56Fe,57Fe, 58Fe,55Co,58mCo,59Co,60Cu,61Cu,63Cu,64Cu,65Cu,67Cu,67Ga,68Ga,69Ga,71Ga, 72As,75As,84Sr,86Sr,87Sr,88Sr,89Sr,86Y,89Y,90Y,89Zr,90Zr,91Zr,92Zr,94Zr,94Tc,99mTc, 103Rh103mRh193mPt195mpt192Pt194Pf 195p| 196p| 198p| 111 In 113|p] 114m|n 115m|n 119gp and wherein Rs is selected from a -H, a halogen, -OH, -OMe, -OEt, -OPr, -NHCOH, -NHCOMe, NHCOEt, -NHCOPr, -CONH2, -CONHMe, -CONHEt, -CONHPr, NHSOH, -NHSO2Me, NHSO2Et, -NHSO2Pr, -SO2NH2, -SO2NHMe, -SO2NHEt, - SO2NHPr, -NH2, -NHMe, -NHEt, -NHPr; and wherein said FAP inhibitor ligand of formula (I) comprises at least one radionuclide; and pharmaceutically acceptable salts thereof for use in the treatment and / or in the diagnosis of FAP expressing diseases including cancer, such as breast cancer, lung cancer, colorectal cancer, prostate cancer, ovarian cancer, pancreatic cancer, hepatocellular cancers, sarcomas and non-small cell lung cancer; and fibrosis, arthritis, atherosclerosis, and inflammatory diseases like spondyloarthritis.

[0055] In a fifth aspect, the present invention relates to FAP inhibitor precursor to formula (I) of formula (II): wherein:

[0056] Ri is H or F

[0057] Li is selected from:

[0058] -(CH2)nNH-, -CH2(OCH2CH2)nNH-, -(CH2)nNHAm-, -CH2(OCH2CH2)nNHAm-,

[0059] CH2C(COOH)NHAm-, -CH2CH2C(COOH)NHAm- wherein n and m are integers independently selected from 0-20, and A is an amino acid independently selected from the group consisting of;

[0060] -COCH2NH-, -COCH(CH3)NH-, -COCH(CH2SH)NH-, -COCH(CH2COOH)NH-, - COCH(CH2CH2COOH)NH-, -COCH(CH2C6H5)NH-, -COCH(CH2C3H3N2)NH-, - COCH(CH(CH3)2)NH-, -COCH((CHCH3)CH2CH3)NH-, -COCH((CH2)4NH2)NH-, - COCH(CH2CH(CH3)2)NH-, -COCH(CH2CH2SCH3)NH-, -CO(CHCH2CH2N)-, - COCH(CH2CONH2)NH-, -COCH(CH2CH2CONH2)NH-, -COCH((CH2)3NH- C(NH)NH2)NH-, -COCH(CH2OH)NH-, -COCH(CH2CH2OH)NH-, COCH(CH(OH)CH3)NH-, -COCH(CH2SeH)NH-, -COCH(CH2C8H6N)NH-, - COCH(CH2C6H4OH)NH-; -COCH(CH2CH2SeCH3)NH-,

[0061] X2is a cyclooctene selected from the group consisting of: wherein L2is -(CH2)nCO-, -(CH2CH2O)n(CH2)mCO or -C(CH3)2(CH2)CO-, wherein n and m are integers independently selected from 1-10, Brief description of the drawings

[0062] Figure 1 : Scheme showing the synthesis of compound 12.

[0063] Figure 2A and 2B: Scheme showing the synthesis of compound 16.

[0064] Figure 3: Scheme showing the synthesis of compound 17.

[0065] Figure 4A and 4B: Scheme showing the synthesis of compound LVIII.

[0066] Figure 5: Scheme showing the synthesis of compound 20.

[0067] Figure 6A and 6B: Scheme showing the synthesis of compound 22.

[0068] Figure 7: Scheme showing the synthesis of compound 23.

[0069] Figure 8: Scheme showing the synthesis of compound I.

[0070] Figure 9A: Scheme showing the synthesis of compound [68Ga]24.

[0071] Figure 9B: Scheme showing the synthesis of compound [111ln]25.

[0072] Figure 10: Scheme showing the synthesis of compound [68Ga]19.

[0073] Figure 11 : Scheme showing the synthesis of compound XXXIV.

[0074] Figure 12: Scheme showing the synthesis of compound XXXV.

[0075] Figure 13: Scheme showing the synthesis of compound [68Ga]25.

[0076] Figure 14: Scheme showing the synthesis of compound [111ln]16.

[0077] Figure 15: Scheme showing the synthesis of compound XXXVII.

[0078] Figure 16: Graphs showing A: Time-activity curves (%ID / mL tissue over time, h) and B: tumor- to-muscle ratios over time (h) for111ln-labeled FAPi derivatives.

[0079] Figure 17: Bar diagram showing the effect of FAP blockade (%ID / g) on the uptake of111Inlabeled FAPi derivatives.

[0080] Figure 18: Table showing biodistribution (%ID / g) of XXXVII based on ex vivo gamma counting results.

[0081] Figure 19: Table showing biodistribution (%l D / g) of [111ln]25 based on ex vivo gamma counting results.

[0082] Figure 20: Table showing biodistribution (%l D / g) of [111ln]16 based on ex vivo gamma counting results.

[0083] Figure 21 : Scheme showing the synthesis of compound LVIa.

[0084] Figure 22: Scheme showing the synthesis of compound [18F]-LVI.

[0085] Figure 23: Graph showing tumor accumulation for [18F]-LVI in in NMRI nude mice with LI87MG xenografts.

[0086] Figure 24: Scheme showing the synthesis of compound [123l][natln]24.

[0087] Figure 25: Scheme showing the synthesis of compound [131l][natln]24.

[0088] Figure 26: Ex vivo biodistribution of compound [131l][natln]24 in healthy rats. Detailed description of the invention

[0089] The FAP inhibitor (FAPi) ligand compounds of the present invention comprises a FAP targeting moiety, a linker, an IsoF-TCO and a radiolabeled tetrazine. The FAPi targeting compounds are possessing advantageous pharmacokinetic properties and enable quick and efficient modifications of the radiopharmaceuticals allowing the fast renal excretion and reduced liver accumulation.

[0090] The present FAP inhibitor ligand compounds were developed by labeling tetrazines synthon and ligate this synthon to a precursor moiety containing an IsoF-TCO.

[0091] The radiolabeled compounds provided herein show a surprisingly higher tumor accumulation compared to [111ln]ln-DOTAGA.GIu.(FAPi) ([111ln]25).

[0092] The present compounds can be labeled with either a radiohalogen or a radiometal providing improved flexible use of these FAP inhibitor ligands compared to ligands that are designed to for labeling with a radiohalogen only or with a radiometal only.

[0093] The structure of the FAP inhibitor ligands herein is of formula (I):

[0094] -(CH2)nNH-, -CH2(OCH2CH2)nNH-, -(CH2)nNHAm-, -CH2(OCH2CH2)nNHAm-, -

[0095] CH2C(COOH)NHAm-, -CH2CH2C(COOH)NHAm- wherein n and m are integers independently selected from 0-20, and A is an amino acid independently selected from the group consisting of: -COCH2NH-, -COCH(CH3)NH-, -COCH(CH2SH)NH-, -COCH(CH2COOH)NH-, - COCH(CH2CH2COOH)NH-, -COCH(CH2C6H5)NH-, -COCH(CH2C3H3N2)NH-, - COCH(CH(CH3)2)NH-, -COCH((CHCH3)CH2CH3)NH-, -COCH((CH2)4NH2)NH-, - COCH(CH2CH(CH3)2)NH-, -COCH(CH2CH2SCH3)NH-, -CO(CHCH2CH2N)-, - COCH(CH2CONH2)NH-, -COCH(CH2CH2CONH2)NH-, -COCH((CH2)3NH- C(NH)NH2)NH-, -COCH(CH2OH)NH-, -COCH(CH2CH2OH)NH-, COCH(CH(OH)CH3)NH-, -COCH(CH2SeH)NH-, -COCH(CH2C8H6N)NH-, -

[0096] COCH(CH2C6H4OH)NH-, -COCH(CH2CH2SeCH3)NH-;

[0097] Xi is a pyridazine selected from the group consisting of: wherein L2is -(CH2)nCO-, -(CH2CH2O)n(CH2)mCO or -C(CH3)2(CH2)CO-, wherein n and m are integers independently selected from 1-10, and

[0098] R2is selected from -H, -Me, and R3, wherein the curly sign indicates the link to the tetrazine; and where R4is selected from: -H, -Yi, -OCH2CH2YI, -OCH2CH2CH2YI, -SCH2CH2YI, -NHCH2CH2YI, - OCH2C6H4YI, -OCH2CH2C6H4YI, -NHCOCH2CH2YI, -CONHCH2CH2YI, -NHSO2CH2CH2YI, -

[0099] SO2NHCH2CH2YI, wherein Yi is selected from the group constisting of: and wherein L3 is selected from: -H, -(CH2)nNH-, -O(CH2)nNH-, -S(CH2)nNH-, - SO2NH(CH2)nNH-, -NHSO2(CH2)nNH-, -CONH(CH2)nNH-, -NHCO(CH2)nNH-, - (OCH2CH2)n(CH2)mNH-, -(CH2)n(OCH2CH2)mNH-, -O(CH2)n(OCH2CH2)mNH-, - S(CH2)n(OCH2CH2)mNH-, -SO2NH(CH2)n(OCH2CH2)mNH-,

[0100] NHSO2(CH2)n(OCH2CH2)mNH-, -CONH(CH2)n(OCH2CH2)mNH-,

[0101] NHCO(CH2)n(OCH2CH2)mNH-, -CH2(OCH2CH2)(CH2)mNH-,

[0102] OCH2(OCH2CH2)n(CH2)mNH-, -SCH2(OCH2CH2)n(CH2)mNH-,

[0103] SO2NHCH2(OCH2CH2)n(CH2)mNH-, -NHSO2CH2(OCH2CH2)n(CH2)mNH-, CONHCH2(OCH2CH2)n(CH2)mNH-, -NHCOCH2CH2(OCH2CH2)n(CH2)mNH-, CH2CH2(OCH2CH2)(CH2)mNH-, -OCH2CH2(OCH2CH2)n(CH2)mNH-,

[0104] SCH2CH2(OCH2CH2)n(CH2)mNH-, -SO2NHCH2CH2(OCH2CH2)n(CH2)mNHCO-, NHSO2CH2CH2(OCH2CH2)n(CH2)mNHCO-, -CONHCH2CH2(OCH2CH2)n(CH2)mNHCO- , -NHCOCH2CH2(OCH2CH2)n(CH2)mNHCO-, where n and m are independently selected from the group consisting of 0-25; and wherein Z is a chelator selected from: -H, 1 ,4,7, 10-tetraazacyclododecane- N,N',N',N "-tetraacetic acid (DOTA), 2-(4,7, 10-tris(2-(tert-butoxy)-2-oxoethyl)-1 ,4,7, 10- tetraazacyclododecan-1-yl)acetic acid (fBu-DOTA), / V, / V'-bis(2-hydroxy-5- (carboxyethyl)benzyl)ethylenediamine / V, / V'-diacetic acid (HBED-CC), 14,7- triazacyclononane-1 ,4,7-triacetic acid (NOTA), 2,2',2"-(2-(4-((l2- azaneyl)methanethioamido)benzyl)-1 ,4,7-triazonane-1 ,4, 7-triyl)triacetic acid (benzyl- NOTA), 2-(4.7-bis(carboxymethyl)-1 ,4,7-triazonan-1-yl)pentanedioic acid (NODAGA), 2-(4,7, 10-tris(carboxymethyl)-1 ,4,7, 10-tetraazacyclododecan-1- yl)pentanedioic acid (DOTAGA), 14,7-triazacyclononane phosphinic acid (TRAP), 14,7-triazacyclononane- 1-methyl(2-carboxyethyl)phosphinic acid-4, 7-bis(methyl(2-hydroxymethyl)phosphinic acid (NOPO), 3,6,9, 15-tetraazabicyclo9.3.1.pentadeca-1 (15), 11 , 13-triene-3,6,9- triacetic acid (PCTA), / \ / '-(5-acetyl (hydroxy)aminopentyl- / V-(5-(4-(5- aminopentyl)(hydroxy)amino-4-oxobutanoyl)amino)pentyl- / \ / -hydroxysuccinamide (DFO), diethylenetriaminepentaacetic acid (DTPA), frans-cyclohexyl- diethylenetriaminepentaacetic acid (CHX-DTPA), 1-oxa-4,7, 10-triazacyclododecane- 4, 7, 10- triacetic acid (OXO-Do3A), p-isothiocyanatobenzyl-DTPA (SCN-BZ-DTPA), 1- (p-isothiocyanatobenzyl)-3-methyl-DTPA (1 B3M), 2-(p-isothiocyanatobenzyl)-4- methyl-DTPA (1 M3B), and 1-(2)-methyl-4-isocyanatobenzyl-DTPA (MX-DTPA), and wherein Z is optionally labeled with a metal selected from:

[0105] 43Sc,44Sc,45Sc,47Sc,45Ti,46Ti,47Ti,48Ti,49Ti,50Ti,51Cr,52Cr,53Cr,54Cr,54Fe,56Fe,57Fe,58Fe,55Co,58mCo,59Co,60Cu,61Cu,63Cu,64Cu,65Cu,67Cu,67Ga,68Ga,69Ga,71Ga, and wherein R5is selected from a -H, a halogen, -OH, -OMe, -OEt, -OPr, -NHCOH, -NHCOMe, NHCOEt, -NHCOPr, -CONH2, -CONHMe, -CONHEt, -CONHPr, NHSOH, -NHSO2Me, NHSO2Et, -NHSO2Pr, -SO2NH2, -SO2NHMe, -SO2NHEt, - SO2NHPr, -NH2, -NHMe, -NHEt, -NHPr; and pharmaceutically acceptable salts thereof.

[0106] The FAP inhibitor targeting region of the FAP inhibitor ligand of formula (I) is based on the UAMC1110molecule which has shown to effectively bind to FAP with high specificity in cancer-associated fibroblasts.

[0107] Apart from the FAP inhibitor targeting region, the compounds of formula (I) comprises the entities K, Li and Xi.

[0108] K is an triazole, which allows for ease of connection of the targeting region to the linker region.

[0109] Li is a linker linking Xi to the FAPi targeting site via K. Li is selected from: -(CH2)nNH-, - CH2(OCH2CH2)nNH-, -(CH2)nNHAm-, -CH2(OCH2CH2)nNHAm-, -CH2C(COOH)NHAm-, - CH2CH2C(COOH)NHAm-. wherein n and m are integers independently selected from 0-20, and A is an amino acid. Thus, the linker may comprise no amino acids or one or more amino acids up to 20 amino acids.

[0110] In a preferred embodiment, the FAP inhibitor ligand of formula (I) comprises a linker Li that includes one or more amino acids, wherein A represents a polar natural amino acid or its D- enantiomer. The inclusion of polar amino acids in the linker significantly enhances the solubility and stability of the inhibitor in aqueous environments, which is critical for effective biological activity and delivery. Polar amino acids, such as serine, threonine, asparagine, glutamine, or their D-enantiomers, possess side chains that can engage in hydrogen bonding and other interactions with water molecules. This improves the overall hydrophilicity of the linker, thereby facilitating better dispersion and bioavailability of the FAP inhibitor in biological fluids.

[0111] Additionally, polar amino acids can influence the overall conformation and flexibility of the linker, allowing for optimal positioning of the inhibitor's active site relative to its target. This can enhance the binding affinity and specificity of the inhibitor, leading to improved inhibition of FAP activity. Furthermore, the use of D-enantiomers of these amino acids may confer resistance to proteolytic degradation, increasing the stability and half-life of the inhibitor in vivo. This stability is particularly advantageous in therapeutic applications, as it can lead to sustained inhibition of FAP activity, thereby enhancing the therapeutic efficacy of the compound.

[0112] Xi is a pyridazine comprising one or two R3 groups: wherein R4 optionally comprises Y1 which is a halogen such as a radiohalogen. The group also comprises Z which is a chelator. Chelators are suitable for binding metals including radiometals.

[0113] The halogen Y1 in R4is selected from1H,2H,3H,11C,12C,13C,14C,18F,19F,76Br,77Br,79Br, 80Br, 80mBn81Bn123| 124| 125| 126| 127| 131| 210^, 211At The fo||Owing halogens from this selection are radionuclides:3H,14C,18F,76Br,77Br,80Br,80mBr,123l,124l,125l,126l,131l,210At, and211At. FAP inhibitor ligands of formula (I) comprising a radionuclide halogen, also referred to as radiohalogens, can be used for imaging for example in relation to diagnostics, and in therapy.18F,76Br,123l,124l,125l,131l, are used in PET or SPECT imaging whereas76Br,77Br,126l,210At, and211At are used in therapy.3H,14C,80Br,80mBr, are primarily used for scientific purposes such as tracers or proof-of-concept tools.

[0114] The following halogens from the Y1 selection are stable nuclides:1H,2H,11C,12C,13C,19F,79Br,81Br, and127l. FAP inhibitor ligands of formula (I) comprising a stable halogen can be used for scientific purposes or when the compound comprises another radionuclide for example at position Z, the chelator, which can optionally be labelled with a radiometal.

[0115] The chelator Z may be unlabled, or labled with a metal selected from:43Sc,44Sc,45Sc,47Sc,

[0116] 45Ti,46Ti,47Ti,48Ti,49Ti,50Ti,51Cr,52Cr,53Cr,54Cr,54Fe,56Fe,57Fe,58Fe,55Co,58mCo,59Co,

[0117] 60Cu,61Cu,63Cu,64Cu,65Cu,67Cu,67Ga,68Ga,69Ga,71Ga,72As,75As,84Sr,86Sr,87Sr,88Sr,89Sr,

[0118] 86y 89y soy 892|-9°Zr91Zr92Zr94Zr94Tc99mTc103Rh103mRh193mpt195mpt192pt194pt sp^ 201TI,203TI,205TI,203Pb,206Pb,207Pb,208Pb,212Pb,209Bi,212Bi,213Bi,223Ra,225Ac,227Th,232Th. This group comprises stable metals as well as radionuclides, also referred to as radiometals. 213Bi,223Ra,225Ac,227Th,232Th are primarily used in therapy and / or for scientific purposes.

[0119] The stable metal can be selected when use is for scientific purposes or when the FAP inhibitor ligand of formula I comprises a radiohalogen.

[0120] A FAP inhibitor ligand according to formula I is accordingly applicable for several different purposes depending on the choice of halogen, optionally the choice of metal and moreover whether the halogen and / or metal is a radionuclide.

[0121] This flexibility of the structure is of particular relevance in theranostic which combines diagnostic imaging and targeted therapy, a powerful concept in personalized medicine aiding patient selection, dose-finding, and therapy response monitoring. A theranostic pair consists of two radionuclides interchangeable without altering the radiopharmaceutical's pharmacokinetics, enabling applications in both diagnostic imaging and radionuclide therapy.

[0122] When the application of the FAP inhibitor ligand of formula (I) is based on the features of a radiohalogen, the chelator is preferably unlabeled or labeled with a stable metal. When the application of the FAP inhibitor ligand of formula (I) is based on the features of a radiometal, R4 is preferably H. Thus, when the FAP inhibitor ligand of formula (I) to be applied in imaging, therapy, diagnostic or theranostics, it is preferred that the compound comprises moiety capable of being labelled with a radionuclide.

[0123] In a preferred embodiment, the FAP inhibitor ligand of formula (I) is a compound wherein R4is selected from Yi, -OCH2CH2YI, CH2YI, -SCH2CH2YI, -NHCH2CH2YI, -OCH2C6H4YI, - OCH2CH2C6H4YI, -NHCOCH2CH2YI, -CONHCH2CH2YI, -NHSO2CH2CH2YI, SO2NHCH2CH2YI,

[0124] In another preferred embodiment the FAP inhibitor ligand of formula (I) is a compound wherein R4is selected from Yi, -OCH2CH2YI, CH2YI, -SCH2CH2YI, -NHCH2CH2YI, -OCH2C6H4YI, - OCH2CH2C6H4YI, -NHCOCH2CH2YI, -CONHCH2CH2YI, -NHSO2CH2CH2YI, SO2NHCH2CH2YI, and wherein Yi is a halogen selected from3H,11C,18F,123l,131l,210At and211At.

[0125] These radionuclides are particularly relevant as they offer distinct advantages due to their diverse nuclear properties, making them highly suitable for both diagnostic imaging and therapeutic applications.

[0126] For diagnostic imaging,11C and18F are positron-emitting radionuclides commonly used in positron emission tomography (PET).11C, with a short half-life of approximately 20 minutes, is ideal for labeling small molecules in metabolic studies, allowing real-time imaging of physiological processes.18F, on the other hand, has a longer half-life of about 110 minutes, providing greater flexibility in synthesis and broader distribution. Its high positron emission efficiency results in excellent image resolution, making it a popular choice for PET imaging.

[0127] 123l and1311 serve both diagnostic and therapeutic purposes.123l, with a half-life of approximately 13 hours and gamma emissions, is well-suited for single-photon emission computed tomography (SPECT), enabling high-quality imaging with minimal patient radiation exposure.1311, with a longer half-life of about 8 days, emits both beta and gamma radiation, making it effective for imaging and therapy, especially in the treatment of thyroid diseases.

[0128] For therapeutic applications,210At and211At are alpha-emitting radionuclides known for their high linear energy transfer (LET). This characteristic leads to substantial cytotoxic effects confined to targeted cells, minimizing damage to surrounding healthy tissue.211At, in particular, with its half-life of approximately 7.2 hours, offers a practical balance between therapeutic efficacy and patient safety, making it suitable for targeted cancer therapies.

[0129] 3H, with its low-energy beta emissions and long half-life of about 12.3 years, is primarily used in biological and biochemical research. It is beneficial in tracing and analyzing biological pathways and processes, due to its stability and minimal radiological hazard.

[0130] The selection of these radionuclides enhances the versatility and functionality of the FAP compounds. They provide a broad range of options for radiolabeling, accommodating both short-lived and long-lived radionuclides.

[0131] In a more preferred embodiment, the FAP inhibitor ligand of formula (I) is a compound wherein R4is selected from Yi, -OCH2CH2YI, CH2YI, -SCH2CH2YI, -NHCH2CH2YI, -OCH2C6H4YI, - OCH2CH2C6H4YI, -NHCOCH2CH2YI, -CONHCH2CH2YI, -NHSO2CH2CH2YI, SO2NHCH2CH2YI, and wherein Yi is a radionuclide selected from131l or211At.

[0132] For all embodiments wherein the FAP inhibitor ligand of formula (I) comprises a radiohalogen, that is when Yi is selected from:1H,2H,11C,12C,13C,19F,79Br,81Br,127l, the chelator Z is unlabeled or Z should be labelled with a stable metal selected from:45Sc,46Ti,47Ti,48Ti,49Ti,50Ti,52Cr,53Cr,54Cr,54Fe,56Fe,57Fe,58Fe,59Co,63Cu,65Cu,69Ga,71Ga,75As,84Sr,86Sr,87Sr,88Sr,89Y,90Zr,91Zr,92Zr,94Zr,103Rh,192Pt,194Pt,195Pt,196Pt,121Sb,123Sb,139La,154Gd,155Gd,156Gd,157Gd,158Gd,160Gd,156Dy,158Dy,160Dy,162Dy,163Dy,164Dy,165Dy,162Er,164Er,165Er,167Er,168Er,170Er,165Ho,175Lu,185Re,203TI,205TI,206Pb,207Pb,208Pb, and209Bi.

[0133] In a preferred embodiment, the FAP inhibitor ligand of formula (I) is selected from the group consisting of I-XXVII I and LVIa:

[0134]

[0135]

[0136]

[0137]

[0138]

[0139]

[0140]

[0141] 

[0142]

[0143] LVIa wherein Re is -H or -CH3.

[0144] In other embodiments, the FAP inhibitor ligand of formula (I) comprises a metal. In a preferred embodiment, the FAP inhibitor ligand of formula (I) comprises a radiometal.

[0145] Thus, in a preferred embodiment, the FAP inhibitor ligand of formula (I) is a compound the one or two chelator(s) Z is labeled with a radiometal selected from:43Sc,44Sc,47Sc,45Ti,51Cr,55Co,58mCo,60Cu,61Cu,64Cu,67Cu,67Ga,68Ga,72As,89Sr,86Y,90Y,89Zr,96Zr,94Tc,99mTc,103mRh, i93mp| i95mpf i98p| 1111 q ii3|q ii4m|p) ii5m|p) n9gp135La138La149Tb152Tb159Tb161Tb161Dy166Er,166Ho,177Lu,185Re,201TI,203Pb,212Pb,209Bi,212Bi,213Bi,223Ra,225Ac,227Th,232Th.

[0146] In a more preferred embodiment, the FAP inhibitor ligand of formula (I) is a compound wherein the chelator Z is labeled with a radiometal selected from64Cu,67Cu,68Ga,90Y,89Zr,99mTc,111In,177Lu,203Pb,212Pb,225Ac.

[0147] These radiometals are of particular interest because of their superior nuclear properties, making them highly effective for a range of diagnostic and therapeutic applications.

[0148] For diagnostic imaging,68Ga and89Zr are particularly valuable.68Ga, a positron emitter with a half-life of approximately 68 minutes, is used in positron emission tomography (PET) imaging. Its availability from a generator system makes it convenient for use in clinical settings, providing high-resolution images for the diagnosis of various conditions.89Zr, with a longer half-life of around 78.4 hours, is suitable for immuno-PET imaging, enabling the tracking of labeled antibodies over several days to study disease progression.

[0149] 99mTc is a cornerstone in diagnostic nuclear medicine due to its ideal physical properties, including a half-life of approximately 6 hours and gamma emissions that are optimal for imaging. It is widely used in single-photon emission computed tomography (SPECT) for a variety of diagnostic procedures due to its versatility and excellent imaging characteristics.

[0150] For therapeutic applications,90Y and177Lu are beta-emitting radionuclides commonly used in targeted radionuclide therapy.90Y has a half-life of approximately 64 hours and is known for its high-energy beta particles, making it effective in treating larger tumors.177Lu, with a halflife of about 6.7 days, offers both beta and gamma emissions, allowing for therapeutic treatment and post-therapy imaging. Its relatively long half-life allows for extended radiation delivery, which is beneficial in treating smaller or residual tumor tissues.

[0151] 64Cu and67Cu provide both diagnostic and therapeutic capabilities.64Cu, with a half-life of about 12.7 hours, emits positrons for PET imaging and beta particles for therapy, making it a dual-purpose radionuclide.67Cu, with a half-life of approximately 61.9 hours, emits beta particles and gamma rays, providing both therapeutic and diagnostic capabilities.

[0152] 111ln is used for diagnostic imaging and radiotherapy, with a half-life of around 2.8 days and gamma emissions that are suitable for SPECT imaging. It is particularly useful in radiolabeling antibodies and peptides, providing valuable information on disease localization and progression.

[0153] The alpha-emitting radionuclides212Pb and225Ac offer potent therapeutic effects due to their high linear energy transfer (LET).212Pb, with a half-life of about 10.6 hours, decays to212Bi, providing alpha and beta emissions that are highly effective for targeted therapy.225Ac, with a longer half-life of approximately 10 days, emits multiple alpha particles, making it exceptionally potent for targeted cancer therapy, especially in cases where conventional therapies may fail.

[0154] 203Pb serves as a diagnostic counterpart to the therapeutic212Pb, allowing for paired imaging and therapy applications. Its half-life of around 51.9 hours and gamma emissions are suitable for imaging, complementing the therapeutic effects of212Pb.

[0155] Thus, these radionuclides maximizes the versatility and applicability of the FAP compounds.

[0156] For all embodiments wherein the FAP inhibitor ligand of formula (I) comprises a radiometal, no radiohalogen should be included in the compound. Accordingly, when the FAP inhibitor ligand of formula (I) is a compound wherein the chelator Z is labeled with a radiometal selected from:43Sc,44Sc,47Sc,45Ti,51Cr,55Co,58mCo,60Cu,61Cu,64Cu,67Cu,67Ga,68Ga,72As,89Sr, 86y soy89Zr94Tc99mTc103mRh193mpt195mpt i98p| ni|q113|n114m|n115m|n119Sb135La138La149Tb,152Tb,159Tb,161Tb,161Dy,166Er,166Ho,177Lu,185Re,201TI,203Pb,212Pb,209Bi,212Bi,213Bi,223Ra,225Ac,227Th,232Th; R4is selected from -H, -Yi, -OCH2CH2YI, -OCH2CH2CH2YI, - SCH2CH2YI, -NHCH2CH2YI, -OCH2C6H4YI, -OCH2CH2C6H4YI, -NHCOCH2CH2YI, - CONHCH2CH2YI, -NHSO2CH2CH2YI, -SO2NHCH2CH2YI, wherein Yi is selected from the group constisting of:1H,2H,11C,12C,13C,19F,79Br,81Br,127l.

[0157] In a preferred embodiment, the FAP inhibitor ligand of formula (I) is selected from the group consisting of:

[0158]

[0159]

[0160]

[0161]

[0162] [68Ga]19

[0163]

[0164] wherein Re is -H or -CH3. In a second aspect, the present invention relates to a pharmaceutical formulation comprising FAP inhibitor ligand of formula (I): wherein:

[0165] Ri is H or F

[0166] -(CH2)nNH-, -CH2(OCH2CH2)nNH-, -(CH2)nNHAm-, -CH2(OCH2CH2)nNHAm-,

[0167] CH2C(COOH)NHAm-, -CH2CH2C(COOH)NHAm- wherein n and m are integers independently selected from 0-20, and A is an amino acid independently selected from the group consisting of: -COCH2NH-, -COCH(CH3)NH-, -COCH(CH2SH)NH-, -COCH(CH2COOH)NH-, - COCH(CH2CH2COOH)NH-, -COCH(CH2C6H5)NH-, -COCH(CH2C3H3N2)NH-, - COCH(CH(CH3)2)NH-, -COCH((CHCH3)CH2CH3)NH-, -COCH((CH2)4NH2)NH-, - COCH(CH2CH(CH3)2)NH-, -COCH(CH2CH2SCH3)NH-, -CO(CHCH2CH2N)-, - COCH(CH2CONH2)NH-, -COCH(CH2CH2CONH2)NH-, -COCH((CH2)3NH- C(NH)NH2)NH-, -COCH(CH2OH)NH-, -COCH(CH2CH2OH)NH-, COCH(CH(OH)CH3)NH-, -COCH(CH2SeH)NH-, -COCH(CH2C8H6N)NH-, - COCH(CH2C6H4OH)NH-, -COCH(CH2CH2SeCH3)NH-;

[0168] Xi is a pyridazine selected from the group consisting of: wherein L2is -(CH2)nCO-, -(CH2CH2O)n(CH2)mCO or -C(CH3)2(CH2)CO-, wherein n and m are integers independently selected from 1-10, and

[0169] R2is selected from -H, -Me, and R3, R3 is , wherein the curly sign indicates the link to the tetrazine; and where R4is selected from: -H, -Y1, -OCH2CH2Y1, -OCH2CH2 CH2Y1, -SCH2CH2Y1, -NHCH2CH2Y1, -

[0170] OCH2C6H4YI, -OCH2CH2C6H4Y1, -NHCOCH2CH2Y1, -CONHCH2CH2Y1, -NHSO2CH2CH2Y1, -

[0171] SO2NHCH2CH2Y1, wherein Y1 is selected from the group constisting of:

[0172] 1H,2H,3H,11C,12C,13C,14C,18F,19F,76Br,77Br,79Br,80Br,80mBr,81Br,123l 124| 125| 126| 127| 131|210At,211At, and wherein L3 is selected from: -H, -(CH2)nNH-, -O(CH2)nNH-, -S(CH2)nNH-, - SO2NH(CH2)nNH-, -NHSO2(CH2)nNH-, -CONH(CH2)nNH-, -NHCO(CH2)nNH-, - (OCH2CH2)n(CH2)mNH-, -(CH2)n(OCH2CH2)mNH-, -O(CH2)n(OCH2CH2)mNH-, - S(CH2)n(OCH2CH2)mNH-, -SO2NH(CH2)n(OCH2CH2)mNH-, - NHSO2(CH2)n(OCH2CH2)mNH-, -CONH(CH2)n(OCH2CH2)mNH-, - NHCO(CH2)n(OCH2CH2)mNH-, -CH2(OCH2CH2)(CH2)mNH-, - OCH2(OCH2CH2)n(CH2)mNH-, -SCH2(OCH2CH2)n(CH2)mNH-, - SO2NHCH2(OCH2CH2)n(CH2)mNH-, -NHSO2CH2(OCH2CH2)n(CH2)mNH-, - CONHCH2(OCH2CH2)n(CH2)mNH-, -NHCOCH2CH2(OCH2CH2)n(CH2)mNH-, - CH2CH2(OCH2CH2)(CH2)mNH-, -OCH2CH2(OCH2CH2)n(CH2)mNH-, - SCH2CH2(OCH2CH2)n(CH2)mNH-, -SO2NHCH2CH2(OCH2CH2)n(CH2)mNHCO-, - NHSO2CH2CH2(OCH2CH2)n(CH2)mNHCO-, -CONHCH2CH2(OCH2CH2)n(CH2)mNHCO- , -NHCOCH2CH2(OCH2CH2)n(CH2)mNHCO-, where n and m are independently selected from the group consisting of 0-25; and wherein Z is a chelator selected from: -H, 1 ,4,7, 10-tetraazacyclododecane- / V, / V', / \ / ', / \ / "-tetraacetic acid (DOTA), 2-(4,7, 10-tris(2-(tert-butoxy)-2-oxoethyl)-1 ,4,7, 10- tetraazacyclododecan-1-yl)acetic acid (fBu-DOTA), / V, / V'-bis(2-hydroxy-5- (carboxyethyl)benzyl)ethylenediamine / V, / V'-diacetic acid (HBED-CC), 14,7- triazacyclononane-1 ,4,7-triacetic acid (NOTA), 2,2',2"-(2-(4-((l2- azaneyl)methanethioamido)benzyl)-1 ,4,7-triazonane-1 ,4, 7-triyl)triacetic acid (benzyl- NOTA), 2-(4.7-bis(carboxymethyl)-1 ,4,7-triazonan-1-yl)pentanedioic acid (NODAGA), 2-(4,7, 10-tris(carboxymethyl)-1 ,4,7, 10-tetraazacyclododecan-1- yl)pentanedioic acid (DOTAGA), 14,7-triazacyclononane phosphinic acid (TRAP), 14,7-triazacyclononane- 1-methyl(2-carboxyethyl)phosphinic acid-4, 7-bis(methyl(2-hydroxymethyl)phosphinic acid (NOPO), 3,6,9, 15-tetraazabicyclo9.3.1.pentadeca-1 (15), 11 , 13-triene-3,6,9- triacetic acid (PCTA), / \ / '-(5-acetyl (hydroxy)aminopentyl- / V-(5-(4-(5- aminopentyl)(hydroxy)amino-4-oxobutanoyl)amino)pentyl- / \ / -hydroxysuccinamide (DFO), diethylenetriaminepentaacetic acid (DTPA), frans-cyclohexyl- diethylenetriaminepentaacetic acid (CHX-DTPA), 1-oxa-4,7, 10-triazacyclododecane- 4, 7, 10- triacetic acid (OXO-Do3A), p-isothiocyanatobenzyl-DTPA (SCN-BZ-DTPA), 1- (p-isothiocyanatobenzyl)-3-methyl-DTPA (1 B3M), 2-(p-isothiocyanatobenzyl)-4- methyl-DTPA (1 M3B), and 1-(2)-methyl-4-isocyanatobenzyl-DTPA (MX-DTPA), and wherein Z is optionally labeled with a metal selected from: and wherein Rs is selected from a -H, a halogen, -OH, -OMe, -OEt, -OPr, -NHCOH, -NHCOMe, NHCOEt, -NHCOPr, -CONH2, -CONHMe, -CONHEt, -CONHPr, NHSOH, -NHSO2Me, NHSO2Et, -NHSO2Pr, -SO2NH2, -SO2NHMe, -SO2NHEt, - SO2NHPr, -NH2, -NHMe, -NHEt, -NHPr; or pharmaceutically acceptable salts thereof.

[0173] The formulation is preferably an intravenous formulation and could be formulated in accordance with other radiopharmaceutical formulations in the art. Such formulations may typically comprise buffering agents, stabilizers, chelating agents, solvents, isotonicity adjusting agents.

[0174] The FAP inhibitor ligand of formula (I) are particularly useful in therapy, imaging such as diagnosis and theranostics when the compound comprises at least one radionuclide, such as a radiohalogen or a radiometal.

[0175] In a third aspect, the present invention accordingly relates to FAP inhibitor ligand of formula (I):

[0176] -(CH2)nNH-, -CH2(OCH2CH2)nNH-, -(CH2)nNHAm-, -CH2(OCH2CH2)nNHAm-,

[0177] CH2C(COOH)NHAm-, -CH2CH2C(COOH)NHAm-, wherein n and m are integers independently selected from 0-20, and A is an amino acid independently selected from the group consisting of: -COCH2NH-, -COCH(CH3)NH-, -COCH(CH2SH)NH-, -COCH(CH2COOH)NH-, - COCH(CH2CH2COOH)NH-, -COCH(CH2C6H5)NH-, -COCH(CH2C3H3N2)NH-, - COCH(CH(CH3)2)NH-, -COCH((CHCH3)CH2CH3)NH-, -COCH((CH2)4NH2)NH-, - COCH(CH2CH(CH3)2)NH-, -COCH(CH2CH2SCH3)NH-, -CO(CHCH2CH2N)-, - COCH(CH2CONH2)NH-, -COCH(CH2CH2CONH2)NH-, -COCH((CH2)3NH-

[0178] C(NH)NH2)NH-, -COCH(CH2OH)NH-, -COCH(CH2CH2OH)NH-, COCH(CH(OH)CH3)NH-, -COCH(CH2SeH)NH-, -COCH(CH2C8H6N)NH-, COCH(CH2C6H4OH)NH-; -COCH(CH2CH2SeCH3)NH-,

[0179] Xi is a pyridazine selected from the group consisting of:

[0180] wherein L2is -(CH2)nCO-, -(CH2CH2O)n(CH2)mCO or -C(CH3)2(CH2)CO-, wherein n and m are integers independently selected from 1-10, and R2is selected from -H, -Me, and R3, , wherein the curly sign indicates the link to the tetrazine; and where

[0181] R4is selected from: -H, -Y1, -OCH2CH2YI, -OCH2CH2CH2YI, -SCH2CH2YI, - NHCH2CH2YI, -OCH2C6H4YI, -OCH2CH2C6H4YI, -NHCOCH2CH2YI, CONHCH2CH2YI, -NHSO2CH2CH2YI, -SO2NHCH2CH2YI, wherein Y1 is selected from the group constisting of;

[0182] 1H,2H,3H,11126| 127| 1311 and wherein L3 is selected from: -H, -(CH2)nNH-, -O(CH2)nNH-, -S(CH2)nNH SO2NH(CH2)nNH-, -NHSO2(CH2)nNH-, -CONH(CH2)nNH-, -NHCO(CH2)nNH-, (OCH2CH2)n(CH2)mNH-, -(CH2)n(OCH2CH2)mNH-, -O(CH2)n(OCH2CH2)mNH-,

[0183] S(CH2)n(OCH2CH2)mNH-, -SO2NH(CH2)n(OCH2CH2)mNH-,

[0184] NHSO2(CH2)n(OCH2CH2)mNH-, -CONH(CH2)n(OCH2CH2)mNH-,

[0185] NHCO(CH2)n(OCH2CH2)mNH-, -CH2(OCH2CH2)(CH2)mNH-,

[0186] OCH2(OCH2CH2)n(CH2)mNH-, -SCH2(OCH2CH2)n(CH2)mNH-,

[0187] SO2NHCH2(OCH2CH2)n(CH2)mNH-, -NHSO2CH2(OCH2CH2)n(CH2)mNH-,

[0188] CONHCH2(OCH2CH2)n(CH2)mNH-, -NHCOCH2CH2(OCH2CH2)n(CH2)mNH-, CH2CH2(OCH2CH2)(CH2)mNH-, -OCH2CH2(OCH2CH2)n(CH2)mNH-,

[0189] SCH2CH2(OCH2CH2)n(CH2)mNH-, -SO2NHCH2CH2(OCH2CH2)n(CH2)mNHCO-, NHSO2CH2CH2(OCH2CH2)n(CH2)mNHCO-, -CONHCH2CH2(OCH2CH2)n(CH2)mNHCO- , -NHCOCH2CH2(OCH2CH2)n(CH2)mNHCO-, where n and m are independently selected from the group consisting of 0-25; and wherein Z is a chelator selected from: -H, 1 ,4,7, 10-tetraazacyclododecane- N,N',N',N "-tetraacetic acid (DOTA), 2-(4,7, 10-tris(2-(tert-butoxy)-2-oxoethyl)-1 ,4,7, 10- tetraazacyclododecan-1-yl)acetic acid (fBu-DOTA), / V, / V'-bis(2-hydroxy-5- (carboxyethyl)benzyl)ethylenediamine N,N -diacetic acid (HBED-CC), 14,7- triazacyclononane-1 ,4,7-triacetic acid (NOTA), 2,2',2"-(2-(4-((l2- azaneyl)methanethioamido)benzyl)-1 ,4,7-triazonane-1 ,4, 7-triyl)triacetic acid (benzyl- NOTA), 2-(4.7-bis(carboxymethyl)-1 ,4,7-triazonan-1-yl)pentanedioic acid (NODAGA), 2-(4,7, 10-tris(carboxymethyl)-1 ,4,7, 10-tetraazacyclododecan-1- yl)pentanedioic acid (DOTAGA), 14,7-triazacyclononane phosphinic acid (TRAP), 14,7-triazacyclononane- 1-methyl(2-carboxyethyl)phosphinic acid-4, 7-bis(methyl(2-hydroxymethyl)phosphinic acid (NOPO), 3,6,9, 15-tetraazabicyclo9.3.1.pentadeca-1 (15), 11 , 13-triene-3,6,9- triacetic acid (PCTA), / \ / '-(5-acetyl (hydroxy)aminopentyl- / V-(5-(4-(5- aminopentyl)(hydroxy)amino-4-oxobutanoyl)amino)pentyl- / \ / -hydroxysuccinamide (DFO), diethylenetriaminepentaacetic acid (DTPA), frans-cyclohexyl- diethylenetriaminepentaacetic acid (CHX-DTPA), 1-oxa-4,7, 10-triazacyclododecane- 4,7, 10-triacetic acid (OXO-Do3A), p-isothiocyanatobenzyl-DTPA (SCN-BZ-DTPA), 1- (p-isothiocyanatobenzyl)-3-methyl-DTPA (1 B3M), 2-(p-isothiocyanatobenzyl)-4- methyl-DTPA (1 M3B), and 1-(2)-methyl-4-isocyanatobenzyl-DTPA (MX-DTPA); and wherein Z is optionally labeled with a metal selected from:

[0190] 43Sc,44Sc,45Sc,47Sc,45Ti,46Ti,47Ti,48Ti,49Ti,50Ti,51Cr,52Cr,53Cr,54Cr,54Fe,56Fe,57Fe,58Fe,55Co,58mCo,59Co,60Cu,61Cu,63Cu,64Cu,65Cu,67Cu,67Ga,68Ga,69Ga,71Ga,72As,75As,84Sr,86Sr,87Sr,88Sr,89Sr,86Y,89Y,90Y,89Zr,90Zr,91Zr,92Zr,94Zr,96Zr,94Tc,99mTc,103Rh103mRh193mPt195mpt192Pt194Pf 195p| 196p| 198p| 111 In 113|p] 114m|n 115m|n 119gp121Sb,123Sb,135La,138La,139La,149Tb,154Gd,155Gd,156Gd,157Gd,158Gd,160Gd152Tb,159Tb,156Dy,158Dy,160Dy,161Dy,162Dy,163Dy,164Dy,165Dy ,161Tb,162Er,164Er,165Er,166Er,167Er,168Er,170Er,165Ho,166Ho,175Lu,177Lu,185Re,186Re,188Re,201TI,203TI,205TI,203Pb,206Pb,207Pb,208Pb,212Pb,209Bi,212Bi,213Bi,223Ra,225Ac,227Th,232Th; and wherein R5is selected from a -H, a halogen, -OH, -OMe, -OEt, -OPr, -NHCOH, -NHCOMe, NHCOEt, -NHCOPr, -CONH2, -CONHMe, -CONHEt, -CONHPr, NHSOH, -NHSO2Me, NHSO2Et, -NHSO2Pr, -SO2NH2, -SO2NHMe, -SO2NHEt, - SO2NHPr, -NH2, -NHMe, -NHEt, -NHPr; and wherein said FAP inhibitor ligand of formula (I) comprises at least one radionuclide; and pharmaceutically acceptable salts thereof, for use as a medicament in therapy, imaging, diagnostics, or theranostics.

[0191] Together with the benefits of the FAP inhibitor ligand of formula (I) in relation to the advantageous pharmacokinetic properties and high tumor accumulation, the fact that the FAP inhibitor ligand of formula (I) is targeting fibroblast activation protein (FAP), which is overexpressed in various cancer-associated fibroblasts (CAFs) and other diseases such as Fibrotic diseases, arthritis, wound healing and inflammatory diseases makes the FAP inhibitor ligand of formula (I) makes it a highly relevant candidate for use in the treatment of and / or diagnosis of such diseases, particularly treatment and / or in the diagnosis of FAP expressing diseases including cancer, such as breast cancer, lung cancer, colorectal cancer, prostate cancer, ovarian cancer, pancreatic cancer, hepatocellular cancers, sarcomas and non-small cell lung cancer; and fibrosis, arthritis, atherosclerosis, and inflammatory diseases like spondyloarthritis.

[0192] Thus, in a fourth aspect, the present invention relates to FAP inhibitor ligand of formula I:

[0193] (I) wherein:

[0194] -(CH2)nNH-, -CH2(OCH2CH2)nNH-, -(CH2)nNHAm-, -CH2(OCH2CH2)nNHAm-, -

[0195] CH2C(COOH)NHAm-, -CH2CH2C(COOH)NHAm- wherein n and m are integers independently selected from 0-20, and A is an amino acid independently selected from the group consisting of: -COCH2NH-, -COCH(CH3)NH-, -COCH(CH2SH)NH-, -COCH(CH2COOH)NH-, - COCH(CH2CH2COOH)NH-, -COCH(CH2C6H5)NH-, -COCH(CH2C3H3N2)NH-, COCH(CH(CH3)2)NH-, -COCH((CHCH3)CH2CH3)NH-, -COCH((CH2)4NH2)NH-, COCH(CH2CH(CH3)2)NH-, -COCH(CH2CH2SCH3)NH-, -CO(CHCH2CH2N)-, COCH(CH2CONH2)NH-, -COCH(CH2CH2CONH2)NH-, -COCH((CH2)3NH-C(NH)NH2)NH-, -COCH(CH2OH)NH-, -COCH(CH2CH2OH)NH-, -COCH(CH(OH)CH3)NH-,

[0196] COCH(CH2SeH)NH-, -COCH(CH2C8H6N)NH-, -COCH(CH2C6H4OH)NH-;

[0197] COCH(CH2CH2SeCH3)NH-,

[0198] Xi is a pyridazine selected from the group consisting of: wherein L2is -(CH2)nCO-, -(CH2CH2O)n(CH2)mCO or -C(CH3)2(CH2)CO-, wherein n and m are integers independently selected from 1-10, and R2is selected from -H, -Me, and R3, , wherein the curly sign indicates the link to the tetrazine; and where R4is selected from: -H, -Y1, -OCH2CH2YI, -OCH2CH2CH2YI, -SCH2CH2YI, - NHCH2CH2YI, -OCH2C6H4YI, -OCH2CH2C6H4YI, -NHCOCH2CH2YI, CONHCH2CH2YI, -NHSO2CH2CH2YI, or -SO2NHCH2CH2Yi, wherein Y1 is selected from the group constisting of;1H,2H,3H,11C,12C,13C,14C,18F,19F,76Br,77Br,79Br,80Br,80mBr,81Br,123l,124l,125l, 126| 127| 1311 210^|211At and wherein L3 is selected from: -H, -(CH2)nNH-, -O(CH2)nNH-, -S(CH2)nNH-, -

[0199] SO2NH(CH2)nNH-, -NHSO2(CH2)nNH-, -CONH(CH2)nNH-, -NHCO(CH2)nNH-, - (OCH2CH2)n(CH2)mNH-, -(CH2)n(OCH2CH2)mNH-, -O(CH2)n(OCH2CH2)mNH-, S(CH2)n(OCH2CH2)mNH-, -SO2NH(CH2)n(OCH2CH2)mNH-, -NHSO2(CH2)n(OCH2CH2)mNH- -CONH(CH2)n(OCH2CH2)mNH-, -NHCO(CH2)n(OCH2CH2)mNH-, CH2(OCH2CH2)(CH2)mNH-, -OCH2(OCH2CH2)n(CH2)mNH-, -SCH2(OCH2CH2)n(CH2)mNH-, -SO2NHCH2(OCH2CH2)n(CH2)mNH-, -NHSO2CH2(OCH2CH2)n(CH2)mNH-,

[0200] CONHCH2(OCH2CH2)n(CH2)mNH-, -NHCOCH2CH2(OCH2CH2)n(CH2)mNH-, CH2CH2(OCH2CH2)(CH2)mNH-, -OCH2CH2(OCH2CH2)n(CH2)mNH-,

[0201] SCH2CH2(OCH2CH2)n(CH2)mNH-, -SO2NHCH2CH2(OCH2CH2)n(CH2)mNHCO-, NHSO2CH2CH2(OCH2CH2)n(CH2)mNHCO-, -CONHCH2CH2(OCH2CH2)n(CH2)mNHCO-, - NHCOCH2CH2(OCH2CH2)n(CH2)mNHCO-, where n and m are independently selected from the group consisting of 0-25; and wherein Z is a chelator selected from: -H, 1 ,4,7,10-tetraazacyclododecane- / V, / \ / ', / \ / ', / \ / "- tetraacetic acid (DOTA), 2-(4,7,10-tris(2-(ferf-butoxy)-2-oxoethyl)-1 ,4,7,10- tetraazacyclododecan-1-yl)acetic acid (fBu-DOTA), / V, / V'-bis(2-hydroxy-5- (carboxyethyl)benzyl)ethylenediamine / V, / V -diacetic acid (HBED-CC), 14,7- triazacyclononane-1 ,4,7-triacetic acid (NOTA), 2,2',2"-(2-(4-((l2- azaneyl)methanethioamido)benzyl)-1,4,7-triazonane-1 ,4,7-triyl)triacetic acid (benzyl-NOTA), 2-(4.7-bis(carboxymethyl)-1 ,4,7-triazonan-1-yl)pentanedioic acid (NODAGA), 2-(4,7,10- tris(carboxymethyl)-1 ,4,7,10-tetraazacyclododecan-1- yl)pentanedioic acid (DOTAGA), 14,7- triazacyclononane phosphinic acid (TRAP), 14,7-triazacyclononane-1-methyl(2- carboxyethyl)phosphinic acid-4, 7-bis(methyl(2-hydroxymethyl)phosphinic acid (NOPO), 3,6,9,15-tetraazabicyclo9.3.1.pentadeca-1 (15), 11 , 13-triene-3,6,9- triacetic acid (PCTA), / V'- (5-acetyl (hydroxy)aminopentyl- / V-(5-(4-(5- aminopentyl)(hydroxy)amino-4- oxobutanoyl)amino)pentyl- / \ / -hydroxysuccinamide (DFO), diethylenetriaminepentaacetic acid (DTPA), frans-cyclohexyl-diethylenetriaminepentaacetic acid (CHX-DTPA), 1-oxa-4,7,10- triazacyclododecane-4,7,10-triacetic acid (OXO-Do3A), p-isothiocyanatobenzyl-DTPA (SCN- BZ-DTPA), 1-(p-isothiocyanatobenzyl)-3-methyl-DTPA (1 B3M), 2-(p-isothiocyanatobenzyl)-4- methyl-DTPA (1M3B), and 1-(2)-methyl-4-isocyanatobenzyl-DTPA (MX-DTPA), and wherein Z is optionally labeled with a metal selected from: and wherein R5is selected from a -H, a halogen, -OH, -OMe, -OEt, -OPr, -NHCOH, -NHCOMe, NHCOEt, -NHCOPr, -CONH2, -CONHMe, -CONHEt, -CONHPr, NHSOH, -NHSO2Me, NHSO2Et, -NHSO2Pr, -SO2NH2, -SO2NHMe, -SO2NHEt, - SO2NHPr, -NH2, -NHMe, -NHEt, -NHPr; and wherein said FAP inhibitor ligand of formula (I) comprises at least one radionuclide; and pharmaceutically acceptable salts thereof for use in the treatment and / or in the diagnosis of FAP expressing diseases including cancer, such as breast cancer, lung cancer, colorectal cancer, prostate cancer, ovarian cancer, pancreatic cancer, hepatocellular cancers, sarcomas and non-small cell lung cancer; and fibrosis, arthritis, atherosclerosis, and inflammatory diseases like spondyloarthritis.

[0202] In a fifth aspect, the present invention relates to a FAP inhibitor precursor to formula (I) of formula (II):

[0203] Li is selected from: -(CH2)nNH-, -CH2(OCH2CH2)nNH-, -(CH2)nNHAm-, -CH2(OCH2CH2)nNHAm-, - CH2C(COOH)NHAm-, -CH2CH2C(COOH)NHAm- wherein n and m are integers independently selected from 0-20, and A is an amino acid independently selected from the group consisting of;

[0204] -COCH2NH-, -COCH(CH3)NH-, -COCH(CH2SH)NH-, -COCH(CH2COOH)NH-, - COCH(CH2CH2COOH)NH-, -COCH(CH2C6H5)NH-, -COCH(CH2C3H3N2)NH-, - COCH(CH(CH3)2)NH-, -COCH((CHCH3)CH2CH3)NH-, -COCH((CH2)4NH2)NH-, - COCH(CH2CH(CH3)2)NH-, -COCH(CH2CH2SCH3)NH-, -CO(CHCH2CH2N)-, - COCH(CH2CONH2)NH-, -COCH(CH2CH2CONH2)NH-, -COCH((CH2)3NH- C(NH)NH2)NH-, -COCH(CH2OH)NH-, -COCH(CH2CH2OH)NH-, COCH(CH(OH)CH3)NH-, -COCH(CH2SeH)NH-, -COCH(CH2C8H6N)NH-, - COCH(CH2C6H4OH)NH-; -COCH(CH2CH2SeCH3)NH-,

[0205] X2is a cyclooctene selected from the group consisting of: wherein L2is -(CH2)nCO-, -(CH2CH2O)n(CH2)mCO or -C(CH3)2(CH2)CO-, wherein n and m are integers independently selected from 1-10.

[0206] These precursors have been found to ligate with a tetrazine in an inverse-electron demand Diels-Alder reaction, to provide the provide the desired pyridazines in a mild, rapid, chemoselective fashion.

[0207] A precursor is generally a compound that is transformed into another compound during a chemical reaction, thereby preceding that compound in the synthetic pathway. In the context of the present invention, a precursor of formula (II) precedes its final corresponding compound of formula (I) in the synthetic pathway. Where compounds according to formula (I) comprises a pyridazine (Xi), the precursors according to formula (II) is identical with the final compound according to formula (I) except it comprises instead a cyclooctene (X2) selected from the group consisting of: wherein L2is -(CH2)nCO-, -(CH2CH2O)n(CH2)mCO or -C(CH3)2(CH2)CO-, wherein n and m are integers independently selected from 1-10.

[0208] In a preferred embodiment, the FAP inhibitor precursors to formula (I) of formula (II) is selected from the group consisting of LVI-LXIX:

[0209]

[0210]

[0211] wherein Re is -H or -CH3. Examples:

[0212] Example 1 : Synthesis of general reagents (Compound 1)

[0213] The compound was synthesized as previously described in the literature to afford 0.36 g (37%) of the desired compound as a red oil (Garcia-Vazquez et al., Pharmaceuticals, 15, 2022). Rf= 0.33 (Heptane / EtOAc 80 / 20);1H NMR (400 MHz, CDCI3) 6 10.07 (s, 1 H), 8.53 (d, J = 8.9 Hz, 2H), 7.06 (d, J = 8.9 Hz, 2H), 4.89 - 4.78 (m, 1 H), 4.74 - 4.62 (m, 1 H), 4.43 - 4.29 (m, 1 H), 4.27 - 4.16 (m, 1 H);13C NMR (101 MHz, CDCI3) 6 166.06, 162.56, 157.40, 130.26, 124.56, 115.38, 81.63 (d, J = 171.5 Hz), 67.29 (d, J = 20.6 Hz).

[0214] The compound was synthesized as described in the literature 0.12 g (65%) of the desired product as a red solid (Garcia- azquez et al., Pharmaceuticals, 15, 2022). Rf = 0.41 (Heptane / EtOAc 50 / 50);1H NMR (400 MHz, DMSO) 5 10.52 (s, 1 H), 8.97 - 8.31 (m, 5H), 8.33 - 7.87 (m, 2H), 7.11 (d, J = 8.9 Hz, 2H), 4.97 - 4.50 (m, 2H), 4.48 - 4.06 (m, 2H);13C NMR (101 MHz, DMSO) 5 165.57, 161.88, 158.28, 154.85, 147.71 , 130.25, 127.38, 125.26, 123.79, 116.05, 75.01 , 67.33.

[0215] Synthesis of 2,2',2"-(10-(2-((3-lodo-5-(1,2,4,5-tetrazin-3-yl)benzyl)amino)-2-oxoethyl)- 1,4, 7, 10-tetraazacyclo dodecane-1 ,4, 7-triyl)triacetic acid (Compound 9)

[0216]

[0217] To a solution of compound 8 (0.05 g, 0.14 mmol) and DIPEA (0.19 mL, 1.14 mmol) in DMF (1 mL) was added DOTA-NHS HPF6 / TFA salt (0.12 g, 0.15 mmol). The reaction was stirred at rt for 12 hours and directly purified by preparative HPLC to give 0.065 g (65%) of the desired compound as a red solid.1H NMR (600 MHz, D2O) 5 10.35 (s, 1 H), 8.60 (s, 1 H), 8.24 (s, 1 H), 7.92 (s, 1 H), 4.44 (s, 2H), 3.71 (br s, 6H), 3.24 (br s, 16H).

[0218] Scheme 1 :

[0219] Reagents and conditions-, i) NBS, AIBN, MeCN, reflux 12 h; ii) NF H, THF, 50 °C, 5 h; iii)

[0220] Boc O, Et N, DCM, rt, 12 h; iv) CH Cl , S , NH NH H O, EtOH, 50 °C, 24 h; v) HCI, dioxane, 2 3 2 2 8 2 2 2 rt, 2 h.

[0221] Synthesis of 2, 5-dioxopyrrolidin- 1-yl (E)-2-( 1, 3-dioxo- 1, 3, 6, 7, 10, 11-hexahydro-2H- (Compound 10)

[0222] The compound was synthesized as previously reported to give 0.056 g (72%) of the desired compound as a yellow solid (PCT / EP2023 / 055930). Rf = 0.41 (n-Heptane / EtOAc 50 / 50);1H NMR (400 MHz, CDCI3) 6 8.56 - 8.47 (m, 2H), 6.10 (td, J = 12.1 , 5.9 Hz, 1 H), 5.25 - 5.07 (m, 1 H), 4.87 (s, 2H), 3.40 - 3.30 (m, 2H), 3.30 - 3.21 (m, 1 H), 3.15 - 3.04 (m, 1 H), 2.92 - 2.68 (m, 6H), 2.45 (q, J = 11.9 Hz, 1 H), 2.33 (dt, J = 12.1 , 6.1 Hz, 1 H);13C NMR (101 MHz, CDCI3) 5 168.16, 165.58, 163.15, 161.67, 161.31 , 143.40, 143.24, 136.54, 132.67, 130.16, 130.00, 126.26, 126.07, 46.74, 41.81 , 37.05, 34.19, 28.26, 25.55.

[0223] Example 2: Synthesis of Compound 12 (Figure 1)

[0224] Figure 1 is a scheme showing the synthesis of compound 13. The following reagents and conditions were used in the steps i - iv: i) Boc-Glu-OH; HOBT, EDCI HCI, DMF, rt, 12 h; ii) HCI, dioxane, rt, 6 h; iii) 10, N-methyl morpholine, DMF, rt, 12 h.; iv) Tz-1 , TFA, H2O, ACN, rt, 24 h. Synthesis of Compound 11 (Figure 1)

[0225] The compound was obtained as previously by Martin et al., Cancers, 15, 2023. The spectral data were in agreement what was previously reported.1H NMR (400 MHz, MeOD) 5 8.72 (t, J = 4.5 Hz, 2H), 8.00 - 7.86 (m, 4H), 7.55 (dd, J = 4.5, 2.6 Hz, 2H), 7.43 (dd, J = 9.3, 2.7 Hz, 2H), 5.17 (dt, J = 9.5, 2.8 Hz, 2H), 4.40 - 3.90 (m, 15H), 3.30 - 3.18 (m, 4H), 3.03 - 2.75 (m, 4H), 2.26 (t, J = 7.5 Hz, 2H), 1.96 - 1.80 (m, 4H), 1.80 - 1.65 (m, 4H); MS (ESI+): m / z (%) = 325.5 (100, [M+3H]3+), 487.8 (35, [M+2H]2+), 974.3 (10, [M+H]+), calculated fo^Hs^NnOs:

[0226] 973.3 [M],

[0227] Synthesis of Compound LVI (Figure 1)

[0228] To a solution of 11 (0.033 g, 0.033 mmol) and 10 (0.014 g, 0.033 mmol) in dry DMF (2 mL) under argon, was added 4-methylmorpholine (0.009 mL, 0.083 mmol). The reaction was stirred at room temperature for 12 h. The mixture was then diluted with 10 mL of 1%TFA in H2O and purified by preparative HPLC. The collected fractions were freeze-dried to give 0.03 g (69%) of the desired compound as a white solid. MS (ESI+): m / z (%) = 432.0 (30, [M+3H]3+),

[0229] 647.3 (100, [M+2H]2+), 1293.4 (20, [M+H]+), calculated for C65H64F4N14O11: 1292.4 [MJ.

[0230] Synthesis of Compound 12 (Figure 1)

[0231] To a solution of LVI (0.003 g, 0.0023 mmol) in ACN (2 mL) and 1% TFA in H2O (2 mL) was added Tz 1 (0.001 g, 0.0026 mmol) in 1%TFA in H2O (1 mL). The reaction was stirred at room temperature for 12 h. The mixture was directly purified by preparative HPLC. The collected fractions were freeze-dried to give 0.002 g (62%) of the desired compound as a white solid. MS (ESI+): m / z (%) = 495.4 (100, [M+3H]3+), 742.4 (60, [M+2H]2+), 1483.4 (30, [M+H]+), calculated for C76H72F5I NI50I2: 1482.4[M],

[0232] Example 3: Synthesis of Compound 13 (Scheme 2)

[0233] Scheme 2 shows the synthesis of compound 13. The grey dot in the intermediate structures symbolizes a resin. The following reagents and conditions were used in the steps i - iii: i) a) DIPEA, CH2CI2, rt, 2 h, b) 50% piperidine in DMF, rt, 20 min.; ii) Fmoc-tBu-D-Ser-OH, HATU, DIPEA, DMF, rt, 2 h, b) 50% piperidine in DMF, rt, 20 min.; iii) TFA, TIPS, H2O, rt, 2 h.

[0234] Chlorotrityl resin (1.28 mmol, 1.2 g) was stirred in a round bottom flask in CH2CI2 (20 mL) for 2 hours. Subsequently, Fmoc-propargyl-glycine-OH (1.53 mmol, 0.51 g) and DIPEA (6.14 mmol, 1.08 mL) were added, and the reaction stirred for 12 hours at room temperature. The resin was then transferred to a reactor and washed twice with CH2CI2 (10 mL). The resin was capped by reacting two times for 20 minutes with 5 mL of a mixture of CH2CI2, MeOH, DIPEA (17:2:1). The Fmoc-protecting group was then cleaved with a mixture of DMF and piperidine (1 :1). The product reacted with 2 equiv. of Fmoc-D-Ser(tBu)-OH activated with 1.95 equivalent of HATU and 2 equivalent of DI PEA. Deprotection of the Fmoc group with a mixture of DMF and piperidine (1 :1) gave the unprotected immobilized peptide. The D-Ser coupling was repeated four more times to afford the final protected peptide. The product was cleaved from the resin and deprotected with trifluoroacetic acid (TFA), triisopropylsilane (TIPS) and water (95:2.5:2.5). Preparative HPLC afforded 0.14 g of the desired peptide. MS (ESI+): m / z (%) = 547.3 (100, [M-H]-), calculated for C20H30N6O12: 546.2 [MJ.

[0235] Example 4: Synthesis of Compound 16 (Figure 2A and 2B)

[0236] Figure 2A and 2B is a scheme showing the synthesis of compound 16. The following reagents and conditions were used in the steps i - iii: i) DI PEA, DMF, rt, 5 h; ii) 13, CuSCL H2O, sodium ascorbate, BPDS, DMF, H2O, rt, 12 h; iii) 10, N-methyl morpholine, DMF, rt, 12 h, iv) 9, ACN, H2O, TFA, rt, 5 h.

[0237] To a solution of 11 (0.049 mmol, 0.05 g) in DMF (2 mL) was added DIPEA (0.12 mmol, 0.021 mL) and 2-azidoacetic NHS ester (0.054 mmol, 0.011 g). The resulting mixture was stirred for 4 hours. The volatiles were then removed under reduced pressure and the residue purified by reversed phase chromatography to give 0.02 g (43%) of the desired product as a white solid. Rf =0.15 (CH2CI2 / MeOH, 90 / 10);1H NMR (600 MHz, MeOD) 5 8.90 - 8.84 (m, 2H), 8.06 (t, J = 2.3 Hz, 2H), 8.03 (d, J = 9.3 Hz, 2H), 7.78 (dd, J = 5.0, 2.1 Hz, 2H), 7.61 (dt, J = 9.3, 3.0 Hz, 2H), 5.16 (dt, J = 9.4, 2.6 Hz, 2H), 4.40 - 4.20 (m, 12H), 4.19 - 4.09 (m, 2H), 3.91 (s, 2H), 3.26 (t, J = 7.0 Hz, 2H), 2.99 - 2.75 (m, 4H), 2.25 (t, J = 7.4 Hz, 2H), 2.08 (dtd, J = 13.0, 7.7, 5.1 Hz, 1 H), 1.90 (pd, J = 7.8, 2.9 Hz, 6H), 1.74 (qd, J = 12.5, 7.1 Hz, 5H);13C NMR (151 MHz, MeOD) 5 174.73, 173.35, 170.15, 169.43, 169.38, 169.34, 161.37, 161.12, 160.61 , 160.58, 146.83, 146.72, 145.43, 145.37, 129.22, 128.34, 127.65, 127.55, 127.32, 127.25, 125.92, 120.66, 118.30, 118.28, 105.74, 69.95, 69.91 , 57.48 (dt, J = 43.0, 21.5 Hz), 54.41 , 53.17, 52.95, 52.78, 52.74, 45.97, 45.94, 42.97, 42.96, 40.17, 40.15, 33.10, 29.22, 27.43, 27.34, 26.98, 26.87, 17.28 (dt, J= 38.4, 19.1 Hz); MS (ESI+): m / z (%) = 538.3 (100, [M+2H]2+), 1057.4 (30, [M+H]+), calculated for C49H52F4N14O9: 1056.4 [MJ.

[0238] Synthesis of Compound 15 (Figure 2 A)

[0239] An aqueous solution of CuSOrSFW (100 mg / mL; 0.0089 mmol) was mixed with an aqueous solution of sodium ascorbate (300 mg / mL, 0.036 mmol), when the color of the mixture turned yellow a solution of BPDS (25 mg / mL, 0.0089 mmol) in water was added. The resulting blue / green mixture was added to a solution of the peptide (0.032 g, 0.048 mmol) in DMF (0.5 mL). Afterward, 14 (0.047 g, 0.044 mmol) dissolved in DMF (1 mL) was stirred at room temperature for 4 h. Full click was observed. The compound was diluted in water (12 mL) and purified by reversed flash HPLC to give 0.027 g of the compound as a white solid. MS (ESI+): m / z (%) = 402.5 (40, [M+4H]4+), 536.3 (100, [M+3H]3+), 803.7 (80, [M+4H]4+), calculated for C69H85F4N20O21: 1605.6 [MJ. Synthesis of Compound LIX (Figure 2A)

[0240] To a solution of 15 (0.03 g, 0.013 mmol) and 10 (0.007 g, 0.015 mmol) in dry DMF (2 mL) under argon, was added 4-methylmorpholine (0.012 mL, 0.11 mmol). The reaction was stirred at room temperature for 12 h. The mixture was then diluted with 10 mL of 1%TFA in H2O and purified by preparative HPLC. The collected fractions were freeze-dried to give 0.007 g (24%) of the desired compound as a white solid. MS (ESI+): m / z (%) = 642.4 (60, [M+2H]2+), 962.9 (100, [M+2H]2+), 1925.6 (100, [M+H]+), calculated for C87H97F4N23O24: 1923.7 [MJ.

[0241] Synthesis of Compound 16 (Figure 2B)

[0242] To a solution of LIX (0.005 g, 0.0026 mmol) in ACN (2 mL) and 1% TFA in H2O (2 mL) was added 9 (0.002 g, 0.0027 mmol) in 1%TFA in H2O (1 mL). The reaction was stirred at room temperature for 12 h. The mixture was directly purified by preparative HPLC. The collected fractions were freeze-dried to give 0.004 g (59%) of the desired compound as a white solid. MS (ESI+): m / z (%) = 649.4 (100, [M+4H]4+), 865.4 (70, [M+3H]3+), 1297.9 (40, [M+2H]2+), calculated for C112H129F.I N30O31: 2592.8 [MJ.

[0243] Example 5: Synthesis of Compound 17

[0244] (Figure 3)

[0245] Figure 3 is a scheme showing the synthesis of compound 17. The grey dot in the intermediate structures symbolizes a resin. The following reagents and conditions were used in the steps i - iv: : i) a) DI PEA, CH2CI2, rt, 2 h, b) 50% piperidine in DMF, rt, 20 min.; ii) Fmoc-D-Arg(Pbf)- OH, HATU, DIPEA, DMF, rt, 2 h, b) 50% piperidine in DMF, rt, 20 min.; iii) Fmoc-D-Glu-OH, HATU, DIPEA, DMF, rt, 2 h, b) 50% piperidine in DMF, rt, 20 min.; iv) TFA, TIPS, H2O, rt, 2 h.

[0246] Chlorotrityl resin (1.28 mmol, 1.2 g) was stirred in a round bottom flask in CH2CI2 (20 mL) for 2 hours. Subsequently, Fmoc-propargyl-glycine-OH (1.53 mmol, 0.51 g) and DIPEA (6.14 mmol, 1.08 mL) were added, and the reaction stirred for 12 hours at room temperature. The resin was then transferred to a reactor and washed twice with CH2CI2 (10 mL). The resin was capped by reacting two times for 20 minutes with 5 mL of a mixture of CH2CI2, MeOH, DIPEA (17:2:1). The Fmoc-protecting group was then cleaved with a mixture of DMF and piperidine (1 :1). The product reacted with 4 equiv. of Fmoc-D-Arg(Pbf)-OH activated with 3.92 equiv. of HATLI and 4 equiv. of DIPEA. Deprotection of the Fmoc group with a mixture of DMF and piperidine (1 :1) gave the desired product. Subsequently, 4 equiv. of Fmoc-D-Glu-OH were activated with 3.92 equiv. of HATLI and 4 equiv. of DIPEA in DMF. The solution was added to the resin-immobilized peptide and shaken for 1 hour. The Fmoc-protecting group was cleaved with a mixture of DMF and piperidine. The product reacted with 4 equiv. of Fmoc-D-Arg(Pbf)- OH activated with 3.92 equiv. of HATLI and 4 equiv. of DI PEA. Deprotection of the Fmoc group with a mixture of DMF and piperidine (1 :1) gave deprotected immobilized resin. The D-Glu and D-Arg coupling was repeated one more time to afford the final protected peptide. The product was cleaved from the resin and deprotected with trifluoroacetic acid (TFA), triisopropylsilane (TIPS) and water (95:2.5:2.5). Preparative HPLC afforded 0.15 g of the desired peptide. MS (ESI+): m / z (%) = 280.9 (100, [M+3H]3+), 420.8 (90, [M+2H]2+), 840.6 (30, [M+H]+), calculated for C33H57N15O11: 839.4 [MJ.

[0247] Example 6: Synthesis of Compound 22,

[0248] (Figure 4A and 4B)

[0249] Figure 4A and 4B is a scheme showing the synthesis of compound 22. The following reagents and conditions were used in the steps i - iii: i) 19, CuSO4 H2O, sodium ascorbate, BPDS, DMF, H2O, rt, 12 h; ii) 10, N-methyl morpholine, DMF, rt, 12 h, iii) 9, ACN, H2O, TFA, rt, 5 h.

[0250] Synthesis of Compound 18 (Figure 4 A)

[0251] An aqueous solution of CUSO4 5H2O (100 mg / mL; 0.01 mL; 0.0038 mmol) was mixed with an aqueous solution of sodium ascorbate (300 mg / mL, 0.01 mL; 0.015 mmol), when the color of the mixture turned yellow a solution of BPDS (25 mg / mL, 0.01 mL, 0.0038 mmol) in water was added. The resulting blue / green mixture was added to a solution of 17 (0.024 g, 0.021 mmol) in DMF (0.5 mL). Afterward, 14 (0.02 g, 0.019 mmol) dissolved in DMF (1 mL) was stirred at room temperature for 4 h. Full click was observed. The compound was diluted in water (12 mL) and purified by reversed flash HPLC to give 0.028 g of the compound as a white solid. MS (ESI+): m / z (%) = 380.3 (80, [M+5H]5+), 475.2 (100, [M+4H]4+), 633.2 (90, [M+3H]3+), 949.0 (50, [M+2H]2+), 1896.8 (5, [M+H]+), calculated for C82H109F4N29O20: 1895.9 [MJ. Synthesis of Compound LVIII (Figure 4B)

[0252] To a solution of LVIII (0.03 g, 0.013 mmol) and 10 (0.07 g, 0.015 mmol) in dry DMF (2 mL) under argon, was added 4-methylmorpholine (0.012 mL, 0.11 mmol). The reaction was stirred at room temperature for 12 h. The mixture was then diluted with 10 mL of 1%TFA in H2O and purified by preparative HPLC. The collected fractions were freeze-dried to give 0.007 g (21%) of the desired compound as a white solid. MS (ESI+): m / z (%) = 444.2 (100, [M+5H]5+), 555.2 (30, [M+4H]4+), 739.8 (30, [M+3H]3+), 1108.8 (20, [M+2H]2+), calculated for C100H122F4N32O23: 2214.9 [MJ.

[0253] Synthesis of Compound 19 (Figure 4B)

[0254] To a solution of 11 (0.006 g, 0.0023 mmol) in ACN (2 mL) and 1% TFA in H2O (2 mL) was added 9 (0.002 g, 0.0026 mmol) in 1%TFA in H2O (1 mL). The reaction was stirred at room temperature for 12 h. The mixture was directly purified by preparative HPLC. The collected fractions were freeze-dried to give 0.005 g (71 %) of the desired compound as a white solid. MS (ESI+): m / z (%) = 482.0 (100, [M+6H]6+), 578.4 (40, [M+5H]5+), 722.7 (30, [M+4H]4+), 962.8 (50, [M+3H]3+), calculated for C125H154F4I N39O30: 2884.1 [M],

[0255] Example 7: Synthesis of Compound 0

[0256] (Figure 5)

[0257] Figure 5 is a scheme showing the synthesis of compound 20. The grey dot in the intermediate structures symbolizes a resin. The following reagents and conditions were used in the steps

[0258] 1 - iii: i) a) DIPEA, CH2CI2, rt, 2 h, b) 50% piperidine in DMF, rt, 20 min.; ii) Fmoc-tBu-D-Ser- OH, HATU, DIPEA, DMF, rt, 2 h, b) 50% piperidine in DMF, rt, 20 min.; iii) TFA, TIPS, H2O, rt,

[0259] 2 h.

[0260] Chlorotrityl resin (1.28 mmol, 1.2 g) was stirred in a round bottom flask in CH2CI2 (20 mL) for 2 hours. Subsequently, Fmoc-propargyl-glycine-OH (1.53 mmol, 0.51 g) and DIPEA (6.14 mmol, 1.08 mL) were added, and the reaction stirred for 12 hours at room temperature. The resin was then transferred to a reactor and washed twice with CH2CI2 (10 mL). The resin was capped by reacting two times for 20 minutes with 5 mL of a mixture of CH2CI2, MeOH, DIPEA (17:2:1). The Fmoc-protecting group was then cleaved with a mixture of DMF and piperidine (1 :1). The product reacted with 2 equiv. of Fmoc-D-Ser(tBu)-OH activated with 1.95 equivalent of HATLI and 2 equivalent of DI PEA. Deprotection of the Fmoc group with a mixture of DMF and piperidine (1 :1) gave the unprotected immobilized peptide. The D-Ser coupling was repeated six more times to afford the final protected peptide. The product was cleaved from the resin and deprotected with trifluoroacetic acid (TFA), triisopropylsilane (TIPS) and water (95:2.5:2.5). Preparative HPLC afforded 0.12 g of the desired peptide. MS (ESI+): m / z (%) = 721.2 (100, [M-H]-), calculated for C20H30N6O12: 722.3 [MJ.

[0261] Example 8: Synthesis of Compound 26

[0262] (Figure 6A and 6B)

[0263] Figure 6A and 6B is a scheme showing the synthesis of compound 26. The following reagents and conditions were used in the steps i - iii: i) 20, CuSC>4 H2O, sodium ascorbate, BPDS, DMF, H2O, rt, 12 h; ii) 10, N-methyl morpholine, DMF, rt, 12 h, iii) 9, ACN, H2O, TFA, rt, 5 h.

[0264] Synthesis of Compound 21 (Figure 6A)

[0265] An aqueous solution of CUSO4 5H2O (100 mg / mL; 0.01 mL; 0.0066 mmol) was mixed with an aqueous solution of sodium ascorbate (300 mg / mL, 0.03 mL; 0.053 mmol), when the color of the mixture turned yellow a solution of BPDS (25 mg / mL, 0.3 mL, 0.0133 mmol) in water was added. The resulting blue / green mixture was added to a solution of 20 (0.061 g, 0.073 mmol) in DMF (0.5 mL). Afterward, 14 (0.07 g, 0.066 mmol) dissolved in DMF (1 mL) was stirred at room temperature for 4 h. Full click was observed. The compound was diluted in water (12 mL) and purified by reversed flash HPLC to give 0.015 g of the compound as a white solid. MS (ESI+): m / z (%) = 594.1 (100, [M+3H]3+), 890.5 (60, [M+2H]2+), 1779.7 (5, [M+H]+), calculated for C75H94F4N22O25: 1778.7 [M],

[0266] Synthesis of Compound LX (Figure 6B)

[0267] To a solution of 21 (0.02 g, 0.011 mmol) and 10 (0.06 g, 0.013 mmol) in dry DMF (2 mL) under argon, was added 4-methylmorpholine (0.006 mL, 0.053 mmol). The reaction was stirred at room temperature for 12 h. The mixture was then diluted with 10 mL of 1%TFA in H2O and purified by preparative HPLC. The collected fractions were freeze-dried to give 0.005 g (22%) of the desired compound as a white solid. MS (ESI+): m / z (%) = 700.7 (60, [M+3H]3+), 1050.2 (100, [M+2H]2+), calculated for C93H107F4N25O28: 2097.8 [MJ.

[0268] Synthesis of Compound 22 (Figure 6B)

[0269] To a solution of LX (0.003 g, 0.0014 mmol) in ACN (2 mL) and 1% TFA in H2O (2 mL) was added 9 (0.001 g, 0.0014 mmol) in 1%TFA in H2O (1 mL). The reaction was stirred at room temperature for 12 h. The mixture was directly purified by preparative HPLC. The collected fractions were freeze-dried to give 0.001 g (30%) of the desired compound as a white solid. MS (ESI+): m / z (%) = 554.8 (45, [M+5H]5+), 693.2 (20, [M+4H]4+), 923.7 (100, [M+3H]3+), calculated for C118H139F4I N32O35: 2776.9[M],

[0270] Example 9: Synthesis of Compound 23

[0271] (Figure 7)

[0272] Figure 7 is a scheme showing the synthesis of compound 23. The following reagents and conditions were used in step i: i) H-DOTAGA-ITz, ACN, H2O, TFA, rt, 5 h.

[0273] Synthesis of Compound 23 (Figure 7)

[0274] To a solution of LX (0.003 g, 0.0014 mmol) in ACN (2 mL) and 1% TFA in H2O (2 mL) was added 9 (0.001 g, 0.0014 mmol) in 1%TFA in H2O (1 mL). The reaction was stirred at room temperature for 12 h. The mixture was directly purified by preparative HPLC. The collected fractions were freeze-dried to give 0.002 g (59%) of the desired compound as a white solid. MS (ESI+): m / z (%) = 569.3 (20, [M+5H]5+), 711.2 (40, [M+4H]4+), 947.5 (100, [M+3H]3+), calculated for Ci2iH143F4I N32O37: 2838.9[M],

[0275] Example 10: Radiolabeling of [18F]1 before IEDDA conjugation

[0276] Figure 8 shows the18F-radiolabeling of [18F]1 , IEDDA conjugation and oxidation steps (step i- ii).

[0277] The radiolabeling was performed according to published procedure (Garcia-Vazquez et al., Pharmaceuticals, 15, 2022). The aqueous [18F]fluoride solution received from the cyclotron was passed through a Sep-Pak Light QMA cartridge preconditioned with 5 mL 0.5 M K3PO4. [18F]Fluoride was eluted from the QMA cartridge into a 4 mL v-shaped vial using Bu4NOTf solution (20 mM in MeOH, 1 mL). The eluate was dried at 100°C for 5 min under nitrogen or helium flow. After MeOH had evaporated, acetonitrile (0.5 mL) was added to the same vial and evaporated under the same conditions to remove traces of water. Two additions of acetonitrile were performed. Nosyl precursor 2 (1.5 mg) was dissolved in anhydrous acetonitrile (0.3 mL), diluted with tBuOH (0.7 mL) and added to the dried [18F]fluoride residue. After reacting for 5 min at 100 °C, the reaction was cooled to 80 °C with ambient air flow, diluted with water (2 mL) and purified by semipreparative HPLC: Discovery HS F5 5 pm, 250 mm x 10 mm column, isocratic elution with 50% acetonitrile in 20 mM citrate buffer pH 6.1 , elution speed 5 mL / min. The product peak (retention time 9.0 min) was collected, diluted with water (50-100 mL) and passed through a Sep-pak Plus C18 Short solid phase extraction cartridge (Waters, USA) that was preconditioned by flushing it with EtOH / water mixture (1 / 1 v / v, 10 mL). The Sep-pak cartridge was then flushed with extra water (5 mL), blown with nitrogen, eluted with 1-2 mL of organic solvent (ACN or EtOH) and diluted with water to achieve the necessary concentration of the organic solvent for the next step. [18F]1 was confirmed in a radiochemical yield of 16±10% (n=4) and >95% radiochemical purity by analytical radio-HPLC (tret= 6.7 min): Luna C18(2) 5pm column 150x4.6 mm; mobile phase H2O / ACN + 0.1% TFA (30-65% ACN in 10 min); flow 1.5 mL / min.

[0278] Example 11 : IEDDA conjugation of TCO functionalized FAPi derivatives with [18F]1

[0279] Click and oxidation step:

[0280] To the solution of [18F]1 in acetonitrile (1 mL, ca. 1.1 GBq) 100 pg LVI (50 pL 2 mg / mL in DMSO) was added. Full click consumption / conversion was confirmed via radio-HPLC after 20 min. 10 pL TFA (1% v / v) was added to the solution and left standing at room temperature for 30 min. Figure 8 shows the full synthesis starting from the nosyl precursor 2.

[0281] Purification and formulation:

[0282] Crude oxidized I was diluted with water (2.7 mL) and purified by semipreparative HPLC: Luna C18 5p 250x10 mm column; mobile phase H2O / ACN +0.1 % TFA (30-55% ACN in 11.7 min); flow 4 mL / min. The product peak was collected (tret= 620-645 s), diluted with water (20 mL) and passed through Sep-Pak C18 Plus cartridge (Waters; preconditioning: 10 mL EtOH / water mixture (1 / 1 v / v). The Sep-Pak cartridge was washed with water (2 mL) and eluted with 0.7 mL of ethanol.

[0283] Ethanolic solution of purified I (324 MBq, RCP = 100%) was diluted with 9 mL 20 mM citrate buffer (pH 6) and used for injections. The final product was confirmed via analytical radio- HPLC (tret = 4.0 min): Luna C18(2) 5pm column 150x4.6 mm; mobile phase H2O / ACN + 0.1% TFA (30-65% ACN in 10 min); flow 1.5 mL / min.

[0284] Example 12: Radiolabeling of [68Ga]Ga-DOTAGA.GIu.(FAPi)2 (Compound [68Ga]24, Figure 9A)

[0285] Figure 9A is a scheme showing the synthesis of compound [68Ga]24.

[0286] Precursor 24 was synthesized according to published procedure (Martin et al., Cancers, 15(6), 1889, 2023). Precursor 24 (8 pg, 4 pL of 2 mg / mL stock solution in DMSO) and 80 pL 1 M NH4OAC (pH = 5.5) was added to a solution of [68Ga]GaCI3(500 pL in 0.1 M HCI, 210 MBq) and was heated / shaken at 60 °C for 5 min (RCC > 90%). After dilution with 5 mL H2O it was rinsed through Strata-X cartridge from Phenomenex, washed with 0.3 mL H2O and eluted with 0.5 mL EtOH. After evaporating EtOH (40 °C, 5 min), the product (97 MBq, d.c. RCY = 61% after 30 min synthesis time) was redissolved in 20|JL EtOH and 2 mL 0.1 M sterile phosphate buffer (PB, pH=7.0). The final product was obtained with a RCP of 90.5% determined via analytical radio-HPLC (tret = 5.3 min): Aeris Peptide XB-C183.6p 150x4.6 mm column; mobile phase H2O / ACN + 0.1% TFA (5-75% ACN in 8 min); flow 1.5 mL / min. The compound was confirmed via radio-iTLCs (mobile phases: 0.2 M citrate buffer (pH=4.0) and 0.25 M NH4OAc (pH=4.0) / MeOH(1 :1)).

[0287] Example 13: Radiolabeling of [111ln]ln-DOTAGA.GIu.(FAPi)2 (Compound [111ln]25, (Figure 9B)

[0288] Figure 9B is a scheme showing the synthesis of compound [111ln]25.

[0289] Precursor 25 was synthesized according to published procedure (Martin et al., Cancers, 15(6), 1889, 2023). Precursor 25 (5 pg, 5 pL 1 mg / mL stock solution in H2O) and 25 pL 1 M NH4OAc (pH = 5.5) was added to a solution of [111 ln]lnCI3(100 pL, ca. 100 MBq) and was shaken at 90 °C for 15 min (RCC > 95%). This was followed by SPE with Sep-Pak tC2 Plus Light cartridge (preconditioning: 2 mL EtOH and 5 mL H2O; cartridge rinsed with reaction solution, washed with 1 mL H2O and eluted with 1 mL EtOH / H2O (1 :1) to yield the final product with RCY = 95% and RCP = 95.2%. This was determined via analytical radio-HPLC (tret = 5.8 min): Luna C18(2) 5pm 150x4.6 mm column; mobile phase H2O / ACN + 0.1% TFA (10-50% ACN in 8 min); flow 1.5 mL / min. Also confirmed via radio-iTLCs (mobile phases: 0.2 M citrate buffer (pH=4.0) and 1 M NH4OAc (pH=4.0) / MeOH(1 :1)).

[0290] Example 14: Radiolabeling of compound [68Ga]19 (Figure 10)

[0291] Figure 10 is a scheme showing the radiolabeling of compound 19 ([68Ga]19).

[0292] Compound 19 (20 pg, 10 pL of 1 mg / mL stock solution in H2O / DMSO (9:1)) and 125 pL 1 M AmOAc (pH = 5.5) was added to a solution of [68Ga]GaCh (850 pL in 0.1 M HCI, 290 MBq) and was heated / shaken at 75 °C for 5 min (RCC = ca. 93%). This was followed by solid-phase extraction (SPE) with Sep-Pak tC2 Plus Light cartridge from Waters (preconditioning: 2 mL EtOH and 5 mL H2O; cartridge rinsed with reaction solution, washed with 1 mL H2O and eluted with 1 mL EtOH / H2O (1 :1) and further diluted with 4 mL 0.1 M sterile phosphate buffer (PB, pH=7.0) to yield the final product with d.c. RCY = 82% (195 MBq after 20 min synthesis time) and RCP = 99.5% (determined via analytical radio-HPLC: Luna C185p column (150x4.6 mm); mobile phase H2O / ACN+0.1% TFA (10-50% ACN in 8 min); elution flow 1.5 mL / min) and confirmed via radio-iTLCs (mobile phases: 0.2 M citrate buffer (pH=4.0) and 0.25 M NH4OAc (pH=4.0) / MeOH(1 :1)). The radiotracer was injected within 30 min after the synthesis was finished. The RCP of the final formulation after 120 min was determined to be 94.5% via radio-HPLC using the same conditions.

[0293] Example 15: Radiolabeling of compound XXXIV (Figure 11)

[0294] Figure 11 is a scheme showing the radiolabeling of compound 16 (XXXIV).

[0295] Compound 16 (20 pg, 10 pL of 2 mg / mL stock solution in DMSO) and 100 pL 1 M AmOAc (pH = 5.5) was added to a solution of [68Ga]GaCh (750pL in 0.1 M HCI, 215 MBq) and was heated / shaken at 75 °C for 5 min (RCC = 92.4%). This was followed by SPE with Sep-Pak tC2 Plus Light cartridge (preconditioning: 2 mL EtOH and 5 mL H2O; cartridge rinsed with reaction solution, washed with 1 mL H2O and eluted with 0.5 mL EtOH / H2O (1 :1) and further diluted with 5 mL 0.1 M sterile PB (pH=7.0) to yield the final product with d.c. RCY = 69% (112 MBq after 30 min synthesis time) and RCP = 99.3% (determined via radio-HPLC: Luna C18 5p column (150x4.6 mm); mobile phase H2O / ACN+0.1% TFA (10-50% ACN in 8 min); elution flow 1.5 mL / min) and confirmed via radio-iTLCs (mobile phases: 0.2 M citrate buffer (pH=4.0) and 1 M NH4OAc(pH=4.0) / MeOH(1 :1)).

[0296] The radiotracer was injected within 30 min after the synthesis was finished. The RCP of the final formulation after 60 min was determined to be 89.2% via radio-HPLC using the same conditions.

[0297] Example 16: Radiolabeling of compound XXXV (Figure 12)

[0298] Figure 12 is a scheme showing the radiolabeling of compound 22 (XXXV).

[0299] Compound 22 (20 pg, 10 pL of 2 mg / mL stock solution in DMSO) and 125 pL 1 M AmOAc (pH = 5.5) was added to a solution of [68Ga]GaCh (950pL in 0.1 M HCI, 310 MBq) and was heated / shaken at 60 °C for 10 min (RCC = 91.5%). This was followed by SPE with Sep-Pak tC2 Plus Light cartridge (preconditioning: 2 mL EtOH and 5 mL H2O; cartridge rinsed with reaction solution, washed with 1 mL H2O and eluted with 0.5 mL EtOH / H2O (1 :1) and further diluted with 5 mL 0.1 M sterile phosphate buffer (pH=7.0) + 10 mg / mL ascorbic acid to yield the final product with d.c. RCY = 67% (155 MBq after 30 min synthesis time) and RCP = 98.7% (determined via radio-HPLC: Luna C18 5p column (150x4.6 mm); mobile phase H2O / ACN+0.1 % TFA (10-50% ACN in 8 min); elution flow 1.5 mL / min) and confirmed via radio- iTLCs (mobile phases: 0.2 M citrate buffer (pH=4.0) and 1 M NH4OAc(pH=4.0) / MeOH(1 :1)).

[0300] The radiotracer was injected within 30 min after the synthesis was finished. The RCP of the final formulation after 120 min was determined to be 93.6% via radio-HPLC using the same conditions. Example 17: Radiolabeling of compound [68Ga]23 (Figure 13)

[0301] Figure 13 is a scheme showing the radiolabeling of compound 23 ([68Ga]23).

[0302] Compound 23 (20 pg, 10 pL of 2 mg / mL stock solution in DMSO) and 125 pL 1 M AmOAc (pH = 5.5) was added to a solution of [68Ga]GaCl3 (1000pL in 0.1M HCI, 290 MBq) and was heated / shaken at 60 °C for 10 min (RCC = 91.0%). This was followed by SPE with Sep-Pak tC2 Plus Light cartridge (preconditioning: 2 mL EtOH and 5 mL H2O; cartridge rinsed with reaction solution, washed with 1 mL H2O and eluted with 0.6 mL EtOH / H2O (1 :1) and further diluted with 5.4 mL 0.1 M sterile phosphate buffer (pH=7.0) + 10 mg / mL ascorbic acid to yield the final product with d.c. RCY = 68% (149 MBq after 25 min synthesis time) and RCP = 96.1% (determined via radio-HPLC: Luna C18 5p column (150x4.6 mm); mobile phase H2O / ACN+0.1 % TFA (10-50% ACN in 8 min); elution flow 1.5 mL / min) and confirmed via radio- iTLCs (mobile phases: 0.2 M citrate buffer (pH=4.0) and 1 M NH4OAc(pH=3.5) / MeOH(1 :1)).

[0303] The radiotracer was injected within 30 min after the synthesis was finished. The RCP of the final formulation after 120 min was determined to be 94.7% via radio-HPLC using the same conditions.

[0304] Example 18: Radiolabeling of compound [111ln]16 (Figure 14)

[0305] Figure 14 is a scheme showing the radiolabeling of compound 16 ([111ln]16).

[0306] Compound 16 (206 pg, 103 pL 2 mg / mL stock solution in DMSO) and 500 pL 1 M NH4OAc (pH = 4.0) was added to a solution of [111ln]lnCh (2.0 mL, 1.382 GBq) and was shaken at 75 °C for 15 min. More precursor 16 (88 pg, 44 pL 2 mg / mL stock solution in DMSO) was added and heated for another 5 min at 75 °C.

[0307] After purification via SPE with Sep-Pak tC2 Plus Light cartridge (preconditioning: 2 mL EtOH and 5 mL H2O; cartridge rinsed with reaction solution, washed with 1 mL H2O and eluted with 1 mL EtOH / H2O (1 :1). The product was obtained with RCY = 80% and RCP = 95.6%, determined via radio-HPLC, (tret = 4.2 min; RCP = 95.6%, ca. 371 MBq / mL in EtOH / H2O (1 :1)) using Luna C18(2) 5pm 150x4.6 mm column; mobile phase H2O / ACN + 0.1% TFA (10-50% ACN in 9 min); flow 1.5 mL / min. The product was confirmed via radio-TLCs (mobile phases: 0.2 M citrate buffer (pH=4.0) and 1 M NH4OAc(pH=4.0) / MeOH(1 :1)). The radiotracer was reformulated in 3 mL saline (371 MBq / mL).

[0308] Example 19: Radiolabeling of compound XXXVII (Figure 15)

[0309] Figure 15 is a scheme showing the synthesis of compound 22 (XXXVII).

[0310] Compound 22 (190 pg, 95 pL 2 mg / mL stock solution in DMSO) and 465 pL 1 M NH4OAc (pH = 4.0) was added to a solution of [111ln]lnCh (1.86 mL, 1.267 GBq) and was shaken at 60 °C for 30 min (RCC = 91.7%). This was followed by SPE with Sep-Pak tC2 Plus Light cartridge (preconditioning: 2 mL EtOH and 5 mL H2O; cartridge rinsed with reaction solution, washed with 1 mL H2O and eluted with 1 mL EtOH / H2O (1:1). The final product was obtained with RCY = 51% and RCP = 98.4%, determined via radio-HPLC, (tret= 4.1 min; RCP = 98.4%, ca. 258 MBq / mL in EtOH / H2O (1 :1)) using Luna C18(2) 5 m 150x4.6 mm column; mobile phase H2O / ACN + 0.1% TFA (10-50% ACN in 9 min); flow 1.5 mL / min. The product confirmed via radio-TLCs (mobile phases: 0.2 M citrate buffer (pH=4.0) and 1 M NH4OAc(pH=4.0) / MeOH(1 :1)). The radiotracer was reformulated in 2.5 mL saline (258 MBq / mL).

[0311] Examples 20: In vivo SPET / CT imaging and biodistribution of [111ln]ln-labeled FAPi derivatives in tumor bearing mice (u87-mg)

[0312] General information

[0313] The biodistribution of radiolabeled FAPi derivatives was investigated in vivo in CD1 nude mice with subcutaneous U87MG tumor xenografts at 7 timepoints after intravenous injection of the radioligands - 1 h, 4 h, 8 h, 24 h, 48 h and 72 h. Three radiolabeled FAPi derivatives were evaluated: [111ln]16, [111ln]25 and XXXVII. In addition, blocking of FAP-specific tumor uptake by unlabeled ligand UAMC1110 was investigated at 1 h post-injection for all three radiolabeled derivatives.

[0314] All experiments were approved by the Ethics Committee of the University of Antwerp, Belgium (file number 2022-63). The use of laboratory rodents was carried out in strict accordance with all mandatory guidelines (EU directives, including the Revised Directive 2010 / 63 / EU on the Protection of Animals used for Scientific Purposes that came into force on 01 / 01 / 2013, and the declaration of Helsinki in its latest version).

[0315] Tumor inoculation and follow up

[0316] Six- to eight week- old female CD1 nude mice (Charles River) were subcutaneously inoculated in the right hind leg with 6-7 x 106U87MG cells in 100pL sterile saline. The tumors were allowed to grow until they reached the size of approximately 150 mm3(9-15 days postinoculation), whereupon the imaging and biodistribution studies were performed.

[0317] Experimental groups and time points

[0318] The mice were split into three cohorts, according to the number of radiolabeled FAPi derivatives tested. Each cohort was further split into groups of 3 animals: 6 groups to investigate baseline biodistribution + 1 group to investigate the effect of FAP blocking with UAMC1 110. For [1111 n] 16, only 5 groups were used for baseline biodistribution. Radiolabeled FAPi derivatives were injected into the lateral tail vein in 0.2 mL sterile saline. In the FAP blocking groups, the mice received 2 mg / kg (50-55 pg per animal) of UAMC1110 intravenously in sterile saline 1 h before the radioligand injection.

[0319] Baseline biodistribution was investigated at 1 h, 4 h, 8 h, 24 h, 48 h and 72 h after radioligand injection. The 8 h timepoint was not investigated for 5S[111 ln]8. The effect of FAP blocking was investigated at 1 h post-injection for all three radioligands.

[0320] Tumor sizes for cohorts and injected activities per experimental group are presented in Table 1.

[0321] Table 1.

[0322] SPECT / CT imaging and ex vivo dissection

[0323] For all timepoints longer than 1 hour post-injection, shortly before the designated timepoint was reached, the mice were anesthetized, placed into the SPECT camera (Vector, Milabs) and imaged for 30 min (8 bed positions, 6 frames of 5 min). For 1 hour timepoint SPECT acquisition (8 bed positions, 12 frames of 5 min) was started immediately after tracer injection.

[0324] Total body SPECT images were obtained, and a 4 min CT scan was performed at the end of all each SPECT acquisition. At the end of the SPECT / CT scan, blood was collected through cardiac puncture, mice were euthanized by cervical dislocation and dissected for ex vivo gamma counting.

[0325] SPECT / CT image processing

[0326] The calibration factor from image gray scale value to activity concentration (kBq / cc) was calculated from a uniform phantom scan with known activity. Raw images from the scanner, reconstructed with OSEM iterative reconstruction (16 subsets, 10 iterations), were then calibrated for activity concentration. Activities in each SPECT frame were decay-corrected relative to the time of injection using the injection time of every animal and start time of every scan frame. Finally, for all scans, all frames were concatenated, also adjusting the time duration of every frame.

[0327] SPECT images were analyzed in PMOD software (PMOD Technologies, Zurich, Switzerland). Radioligand uptake was assessed for the tumor, located in the right leg, and muscle in the leg opposite to the tumor. Ellipsoid volumes of interest were drawn over the tumor and muscle, on the organs of interest and adjusted to the size of the organ. Tumor delineation was performed by drawing an iso contour defined by the 50% of the max-min activity range within the volume of interest encompassing the tumor. Uptake was calculated as average % injected dose per mL tissue. For each timepoint, reported tumor uptake values are time-averaged over all frames in the respective acquisition.

[0328] Ex vivo gamma counting

[0329] Samples of blood, solid organs, tissues and tumors harvested from mice were weighed, and the radioactivity in the samples was measured using an automatic y-counter. The uptake levels of the tracers in the organs, tissues and tumors were expressed as percentage of the injected dose per gram (%l D / g) .

[0330] Results

[0331] Uptake of111ln-labeled FAPi derivatives in various tissues and organs of mice determined by ex vivo gamma counting is shown in Tables XA-XC. Biodistribution and excretion profiles of [111ln]XXXVII and [111ln]16, ligands assembled via TCO-tetrazine ligation, are similar to that of the reference ligand [111ln]25 ([111ln]ln-DOTAGA.GIu.(FAPi)2). All three radioligands are primarily excreted via kidneys. Compared to the reference ligand [111ln]ln- DOTAGA.GIu.(FAPi)2, [111ln]XXXVII and [111ln]16 have considerably higher liver uptake at 1 h post-injection, but at later timepoints liver uptake decreases to same level as the reference ligand. Figure Y shows tumor uptake of111ln-labeled FAPi derivatives. [111ln]XXXVII and [111ln]16 showed slightly lower peak uptake in the tumor compared to [111ln]ln-DOTAGA.GIu.(FAPi)2, but similar long-term retention (Fig. YA) and higher tumor-to-muscle ratios at timepoints of 24 h post-injection and later.

[0332] Figure 16 shows the effect of FAP blockade on tumor uptake of111In-labeled FAPi derivatives. Administration of UAMC1110, a selective FAP ligand, led to the decrease in tumor uptake by 24% for [111ln]ln-DOTAGA.GIu.(FAPi)2, by 32% for [111ln]16 and by 23% for [111ln]XXXVII . This indicates that tumor uptake of all three radioligands is dues to specific binding to FAP in the tumor.

[0333] Figure 16 shows time-activity curves (A) and tumor-to-muscle ratios (B) for111ln-labeled FAPi derivatives determined for LI87MG tumor xenografts by SPECT imaging.

[0334] Figure 17 shows the effect of FAP blockade (2 mg / kg UAMC1110) on the uptake of111ln- labeled FAPi derivatives.

[0335] UAMC1110 was administered 1 h before radioligand injection. Tumor uptake is shown at 1 h after radioligand injection.

[0336] Figure 18 is a table showing the biodistribution (%ID / g) of [111ln]XXXVII based on ex vivo gamma counting results

[0337] All data are presented as mean±SD, n=3 for all experimental groups

[0338] Figure 19 is a table showing the biodistribution (%l D / g) of [111ln]25 based on ex vivo gamma counting results

[0339] All data are presented as mean±SD, n=3 for all experimental groups

[0340] Figure 20 is a table showing the biodistribution (%l D / g) of [111ln]16 based on ex vivo gamma counting results

[0341] All data are presented as mean±SD, n=3 for all experimental groups.

[0342] Example 21 : Synthesis of Compound LVI

[0343] (Figure 21)

[0344] Figure 21 is a scheme showing the synthesis of compound LVIa. The following reagents and conditions were used in step i: i) ACN, H2O, TFA, rt, 5 h.

[0345] Synthesis of Compound LVIa (Figure 22)

[0346] To a solution of LX (0.004 g, 0.0019 mmol) in ACN (2 mL) and 1% TFA in H2O (2 mL) was added 1 (0.001 g, 0.0038 mmol) in ACN (1 mL). The reaction was stirred at room temperature for 12 h. The mixture was directly purified by preparative HPLC. The collected fractions were freeze-dried to give 0.002 g (46%) of the desired compound as a white solid. MS (ESI+): m / z (%) = 569.3 (20, [M+5H]5+), 763.7 (10, [M+3H]3+), calculated for C103H114F5 N27O29: 2287.8[M],

[0347] Example 22: IEDDA conjugation of LX derivative with [18F]1

[0348] Click and oxidation step:

[0349] To a solution of 3-(4-(2-[18F]fluoroethoxy)phenyl)-1 ,2,4,5-tetrazine [18F]1 in acetonitrile (1 mL), 40 - 200 pug LX (20 - 100 pL 2 mg / mL in DMSO) was added. Full consumption of [18F] 1 was confirmed by radio-HPLC after 5 min.

[0350] After the click step, 10 pL TFA or 15 pL 9.5 M HCI (suprapure) was added to the click mixture. The mixture was left standing at room temperature for 30 min.

[0351] Crude oxidized [18F]LVI was diluted with water (3 mL) and purified by semi-preparative HPLC (Luna C18(2) 5 pm column, 250x10 mm; mobile phase A: water + 0.1% TFA, B: acetonitrile + 0.1 % TFA; Gradient: 0 min - 20% acetonitrile, 11.7 min - 55% acetonitrile, 16.7 min - 55% acetonitrile, 30 min - 80% acetonitrile; elution flow 4 mL / min). The product peak (tret = 10.5 - 11.5 min) was collected, diluted with water (50 - 100 mL) and passed through a Sep-Pak tC2 Plus Light solid phase extraction cartridge (Waters, USA, preconditioned with 2.5 mL EtOH and 5 mL water). The Sep-Pak cartridge was then flushed with extra water (5 mL), and dried with nitrogen stream before eluting the product (RCY = 50 - 66% (from [18F] 1 ), RCP > 95%) with 1 mL of ethanol.

[0352] Formulation for experiments in mice: Purified [18F]LVI was eluted from the Sep-pak cartridge with 0.6 mL of gentisic acid solution (5 mg / mL) in ethanol. Ethanolic solution was evaporated to dryness while heating to 40°C under nitrogen flow and redissolved in citrate buffer (20 mM, pH = 6) containing 5% ethanol. If necessary, extra gentisic acid was added (as concentrated stock solution in ethanol) to bring its concentration to 1 mg / mL in the formulated solution (pH = 5).

[0353] EXAMPLE 23: In vivo Positron Emission Tomography (PET) imaging in tumor-bearing mice (U87- MG) of [18F] LVI

[0354] The biodistribution of radiolabeled [18F]LVI was investigated in vivo in NMRI nu / nu mice with subcutaneous U87MG tumor xenografts at 2 timepoints after intravenous injection of the radioligand - 1 h, 4.5 h,

[0355] All animal procedures were approved by the Danish Animal Experimentation Council (2021- 15-0201-01041) and conducted in compliance with EU Directive 2010 / 63 / EU. Female BALB nu / nu mice or NMRI nu / nu mice were housed at the University of Copenhagen under standard conditions (12:12 light / dark cycle, food and water ad libitum). Mice were subcutaneously inoculated in the right flank with the respective tumor cells. After a 7-day acclimatization period and sufficient tumor growth, mice were included in PET imaging experiments.

[0356] PET procedure

[0357] For the PET scans, mice were injected with 4 MBq of [18F]LVI (200 pmol, 100-150 pL formulated solution) via the tail vein. Injected activities were corrected for residual waste.

[0358] PET / CT scans were acquired at applicable timepoints post-injection using an Inveon scanner (Siemens Medical Solutions, USA). Mice were anesthetized with 3.5-4.0% sevoflurane in 30% oxygen-enriched air and scanned in groups of four with body temperature maintained using a water-heated bed. PET scans were performed with a 3.438 ns timing window and a 350- 650 keV energy window.

[0359] Image reconstruction and data analysis

[0360] PET images were reconstructed using OSEM3D / SP-MAP with scatter and attenuation correction in the Inveon Acquisition Workplace software. PET imaging data were then exported in DICOM format and analyzed in PMOD software (PMOD Technologies, Zurich, Switzerland). Maximum intensity projection (MIP) images were prepared using summed PET data. The lower end of the image color map corresponded to zero activity concentration, while the higher end was set to either a certain percentile of the hottest voxel value or to a defined standardized uptake value (SUV). For the extraction of time-activity curves from bright organs such as tumors, kidneys, urinary bladder, or liver, volumes of interest (VOIs) were drawn using the isocontouring tool. Uptake values were expressed as %l D / g or SUV.

[0361] Results

[0362] Figure 23 shows tumor accumulation at 1 and 4.5 hours post injection.

[0363] Example 24 Synthesis of Compound 24

[0364] Synthesis of 22,2',2"-(10-(2-((3-(1,2,4,5-tetrazin-3-yl)-5-(trimethylgermyl)benzyl)amino)-2- oxoethyl)-1,4, 7, 10-tetraazacyclododecane-1,4, 7-triyl)triacetic acid chelated with Indium (Compound [natln]24)

[0365] A solution of tetrazine 24 (0.01 g, 0.014 mmol) and indium chloride (0.064 g, 0.29 mmol) in in ammonium acetate buffer (5 ml) (0.1 M, pH 4) was heated at 70 °C for 15 minutes. The crude was directly purified by preparative HPLC to give 10 mg (86%) of the selected compound as a pink powder. [M+H]+= 804.1.

[0366] Example 25: Radiolabeling of [123l][natln]24 before IEDDA conjugation

[0367] [123l]Nal (3.5 GBq) in sodium hydroxide (250 pL) was evaporated to dryness at 60 °C. Precursor [natl n]24 (600 pg, 60 pL 10 mg / mL stock in MeCN + 0.1% TFA) and the oxidant CAT (100 pg, 10 pL 10 mg / mL stock in MeOH) was evaporated to dryness in a separate vial and redissolved in TFA (100 pL). 25 pL TFA was added to the dried 1-123. The precursor-oxidant solution in TFA was added to 1-123 shortly after that (max. 1 min). The closed vial (with septum) was shaken shortly and then heated at 60 °C for 10 - 12 min. The heating was stopped, a carbon filter was connected to the septum and the reaction was quenched with MeCN / 10 mM Na2S2C>3 (1 :1 , 100 pL). The reaction vial was purged with moderate air / N2 flow for ca. 10 - 15 seconds and subsequently diluted with MilliQ-H2O (300 pL). The solution was injected onto a semiPrep-HPLC (Knauer, Luna C18(2) 5pm, 250x10mm column (20-25% in 16min + 100% 4min, 20% 5min, H2O / MeCN + 0.1 % TFA, 3 mL / min). The product [123l][natln]24 was collected manually (tret = 11.8 - 12.8 min, A = 2.0 GBq, RCY = 60.4%, ACY = 56.7%). The HPLC fraction was diluted with MilliQ-H2O (30 mL) and purified with a Sep-Pak C18 Plus Short cartridge (preconditioned with 5 mL EtOH and 10 mL water). The cartridge was rinsed with 3 mL H2O and the product was eluted quantitively using a) MeCN + 0.1% TFA (0.8 mL) or b) EtOH (1.0 mL) into a vial already containing 10 mg gentisic acid (GenOH, 200 pL 50 mg / mL stock in MeCN or 100 pL 200 mg / mL stock in EtOH). Figure 24 shows the radiolabeling of [natln]24

[0368] Example 26: IEDDA conjugation of LX derivative with [123l][natln]24

[0369] Click and oxidation step: Purified [123l][natln]24 (186 MBq, RCP > 99%) in acetonitrile + 0.1 % TFA (ca. 1.5 mL) and 10 mg / mL gentisic acid (GenOH) was clicked to LX (21 pg, 10 nmol, 10 pL 1 mM stock in DMSO). After 15 min, 9.5 M HCI (30 pL, 2 vol%) was added and left standing at r.t. for 35 min. Complete click and oxidation was confirmed via analytical radio-HPLC. Figure 24 shows the radiolabeling of LX.

[0370] Formulation:

[0371] The crude oxidized product was diluted with MilliQ-H2O (12 mL) and purified with a Sep-Pak tC2 Plus Light cartridge (preconditioned with 2.5 mL EtOH and 5 mL water). The crude product was trapped on the cartridge using counter-flow. Subsequently, the cartridge was rinsed with 3 mL water (counterflow) and the product (138 MBq, RCY = 79% (from [131l]l-natln-DOTA-(3-l- H-Tz)), RCP = 92%) was eluted in normal flow direction with 0.1 M citrate (pH=6) / EtOH (1 :1 , 0.6 mL). 0.1 M citrate containing 10 mg / mL sodium gentisate (NaOGen, 2.5 mL) was added.

[0372] Example 27: Radiolabeling of [131l][natln]24 before IEDDA conjugation

[0373] [131l]Nal in carbonate buffer (max. 50 pL, 1.8 GBq) was evaporated to dryness at 60 °C. Precursor [natln]24 (300 pg, 30 pL 10 mg / mL stock in MeCN + 0.1% TFA) and the oxidant NCS (25 pg, 2.5 pL 10 mg / mL stock in MeCN) was evaporated to dryness in a separate vial and redissolved in TFA (75 pL). 25 pL TFA was added to the dried 1-131. The precursoroxidant solution in TFA was added to 1-131 shortly after that (max. 1 min). The closed vial (with septum) was shaken shortly and then heated at 60 °C for 5 - 6 min. The heating was stopped, a carbon filter was connected to the septum and the reaction was quenched with MeCN / 10 mM Na2S2O3 (1 :1 , 100 pL). The reaction vial was purged with moderate air / N2flow for a couple of seconds and subsequently diluted with M illiQ- H2O (300 pL). The solution was injected onto a semiPrep-HPLC (Knauer, Luna C18(2) 5pm, 250x10mm column (20-25% in 16min + 100% 4min, 20% 5min, H2O / MeCN + 0.1% TFA, 3 mL / min). The product fraction 20 was collected manually (tret= 11.5 - 12.5 min, A = 1.1 GBq, RCY = 60.4%). The HPLC fraction was diluted with MilliQ-H2O (20 mL) and purified with a C18 Light SepPak cartridge (preconditioned with 2.5 mL EtOH and 5 mL water). The cartridge was rinsed with 3 mL H2O and the product was eluted using MeCN + 0.1% TFA (0.6 mL) into a vial already containing 7.5 mg gentisic acid (GenOH, 150 pL 50 mg / mL stock in MeCN). Figure 25 shows the radiolabeling of [natln]24

[0374] Example 28 IEDDA conjugation of LX derivative with [131l][natln]24 Click and oxidation step

[0375] Purified [131l][natln]24 (420 MBq, RCP = 100%) in acetonitrile + 0.1% TFA (ca. 3 mL) and 5 mg / mL gentisic acid (GenOH) was clicked to LX (80 pg, 20 pL 4 mg / mL stock in DMSO). After 10 min, 9.5 M HCI (75 pL, 2.5 vol%) was added and left standing at r.t. for 30 min. Complete click and oxidation was confirmed via analytical radio-HPLC.

[0376] Formulation:

[0377] The crude oxidized product was diluted with MilliQ-H2O (13 mL) and purified with a Sep-Pak C18 Plus Light cartridge (preconditioned with 2.5 mL EtOH and 5 mL water). The crude product was trapped on the cartridge using counter-flow. Subsequently, the cartridge was rinsed with 3 mL water (counterflow) and the product (321 MBq, RCP = 100%) was eluted in normal flow direction with EtOH (1.0 mL). Sodium gentisate (NaOGen, 10 mg) was added and EtOH is evaporated at 40 - 50 °C until dryness. The product can be kept at -20 °C for storage (for at least 1 day). 0.1 M citrate or phosphate buffer (pH = 6) + 10% EtOH (1 mL) was added to achieve a 10 mg / mL concentration of sodium gentisate.

[0378] Examples 29: Ex vivo biodistribution of [131l][natln]24 in healthy rats

[0379] Long-Evans female rats weighing 220 - 250 g (Charles River, Calco, Italy) were anesthetized using 3% isoflurane in oxygen flow and cannulated in a lateral tail vein with BD Neoflon 24G vein catheter.

[0380] In order to study the influence of injected carrier dose on the biodistribution of [131l][natln]24 , two tracer batches with different molar activities were prepared: a batch with low molar activity (0.7 GBq / pmol) for the high injected dose (high ID) group and a batch with high molar activity (8 GBq / pmol) for the low injected dose (low ID) group.

[0381] [131l][natln]24 dissolved in 0.4 mL citrate buffer (0.1 M, pH6) with 10% ethanol and 10 mg / mg sodium gentisate was injected through the tail vein catheter. For each investigated timepoint and injected dose, 3 rats were injected. Injected activity was ~2 MBq, corresponding to 0.8 nmol / kg for the low ID group and 9.3 nmol / kg for the high ID group.

[0382] Once the designated timepoint was reached, blood was collected through cardiac puncture and the rats were euthanized by decapitation. The organs, tissues and tumors were harvested, weighed and the radioactivity in the samples was measured using an automatic y-counter (Hidex, Finland). The uptake levels of the tracers in the organs, tissues and tumors were expressed as percentage of the injected dose per gram (%ID / g).

[0383] Results

[0384] Figure 26 shows the biodistribution of [131l][natln]24 in selected organs at 1 and 4 hours at low and high injected dose.

Claims

1. CLAIMS1. FAP inhibitor ligand of formula (I):-(CH2)nNH-, -CH2(OCH2CH2)nNH-, -(CH2)nNHAm-, -CH2(OCH2CH2)nNHAm-, -CH2C(COOH)NHAm-, -CH2CH2C(COOH)NHAm- wherein n and m are integers independently selected from 0-20, and A is an amino acid independently selected from the group consisting of:-COCH2NH-, -COCH(CH3)NH-, -COCH(CH2SH)NH-,COCH(CH2COOH)NH-, -COCH(CH2CH2COOH)NH-,COCH(CH2C6H5)NH-, -COCH(CH2C3H3N2)NH-, -COCH(CH(CH3)2)NH-COCH((CHCH3)CH2CH3)NH-, -COCH((CH2)4NH2)NH-,COCH(CH2CH(CH3)2)NH-, -COCH(CH2CH2SCH3)NH-,CO(CHCH2CH2N)-, -COCH(CH2CONH2)NH-,COCH(CH2CH2CONH2)NH-, -COCH((CH2)3NH-C(NH)NH2)NH-, -COCH(CH2OH)NH-, -COCH(CH2CH2OH)NH-,COCH(CH(OH)CH3)NH-, -COCH(CH2SeH)NH-,COCH(CH2C8H6N)NH-, -COCH(CH2C6H4OH)NH-,COCH(CH2CH2SeCH3)NH-;Xi is a pyridazine selected from the group consisting of:and wherein L3is selected from: -H, -(CH2)nNH-, -O(CH2)nNH-, -S(CH2)nNH , -SO2NH(CH2)nNH-, -NHSO2(CH2)nNH-, -CONH(CH2)nNH-, -NHCO(CH2)nNH , -(OCH2CH2)n(CH2)mNH-, -(CH2)n(OCH2CH2)mNH-, -O(CH2)n(OCH2CH2)mNH-S(CH2)n(OCH2CH2)mNH-, -SO2NH(CH2)n(OCH2CH2)mNH-, NHSO2(CH2)n(OCH2CH2)mNH-, -CONH(CH2)n(OCH2CH2)mNH-, NHCO(CH2)n(OCH2CH2)mNH-, -CH2(OCH2CH2)(CH2)mNH-, OCH2(OCH2CH2)n(CH2)mNH-, -SCH2(OCH2CH2)n(CH2)mNH-, SO2NHCH2(OCH2CH2)n(CH2)mNH-, -NHSO2CH2(OCH2CH2)n(CH2)mNH-, -CONHCH2(OCH2CH2)n(CH2)mNH-, -NHCOCH2CH2(OCH2CH2)n(CH2)mNH-, - CH2CH2(OCH2CH2)(CH2)mNH-, -OCH2CH2(OCH2CH2)n(CH2)mNH-, SCH2CH2(OCH2CH2)n(CH2)mNH-, -SO2NHCH2CH2(OCH2CH2)n(CH2)mNHCO-, -NHSO2CH2CH2(OCH2CH2)n(CH2)mNHCO-, CONHCH2CH2(OCH2CH2)n(CH2)mNHCO-, NHCOCH2CH2(OCH2CH2)n(CH2)mNHCO-, where n and m are independently selected from the group consisting of 0-25; and wherein Z is a chelator selected from: -H, 1 ,4,7, 10- tetraazacyclododecane- / \ / , / \ / ', / \ / ', / \ / "-tetraacetic acid (DOTA), 2-(4,7, 10-tris(2- (tert-butoxy)-2-oxoethyl)-1 ,4,7, 10-tetraazacyclododecan-1-yl)acetic acid (fBu- DOTA), / V, / V'-bis(2-hydroxy-5-(carboxyethyl)benzyl)ethylenediamine N,N'- diacetic acid (HBED-CC), 14,7-triazacyclononane-1 ,4,7-triacetic acid (NOTA), 2,2',2"-(2-(4-((l2-azaneyl)methanethioamido)benzyl)-1 ,4,7-triazonane-1 ,4,7- triyl)triacetic acid (benzyl-NOTA), 2-(4.7-bis(carboxymethyl)-1 ,4,7-triazonan- 1-yl)pentanedioic acid (NODAGA), 2-(4,7, 10-tris(carboxymethyl)-1 ,4,7,10- tetraazacyclododecan-1- yl)pentanedioic acid (DOTAGA), 14,7- triazacyclononane phosphinic acid (TRAP), 14,7-triazacyclononane-1- methyl(2-carboxyethyl)phosphinic acid-4, 7-bis(methyl(2- hydroxymethyl)phosphinic acid (NOPO), 3,6,9,15- tetraazabicyclo9.3.

1. pentadeca- 1 (15), 1 1 , 13-triene-3,6,9- triacetic acid(PCTA), / \ / '-(5-acetyl (hydroxy)aminopentyl- / V-(5-(4-(5- aminopentyl)(hydroxy)amino-4-oxobutanoyl)amino)pentyl- / \ / - hydroxysuccinamide (DFO), diethylenetriaminepentaacetic acid (DTPA), frans-cyclohexyl-diethylenetriaminepentaacetic acid (CHX-DTPA), 1-oxa- 4,7, 10-triazacyclododecane-4,7, 10-triacetic acid (OXO-Do3A), p- isothiocyanatobenzyl-DTPA (SCN-BZ-DTPA), 1-(p-isothiocyanatobenzyl)-3- methyl-DTPA (1 B3M), 2-(p-isothiocyanatobenzyl)-4-methyl-DTPA (1 M3B), and 1-(2)-methyl-4-isocyanatobenzyl-DTPA (MX-DTPA), and wherein Z is optionally labeled with a metal selected from:196Pt,198Pt,111ln,113ln,114mln,115mln,119Sb,121Sb,123Sb,135La,138La,139La, 149Tb,152Tb,159Tb,161Tb,154Gd,155Gd,156Gd,157Gd,158Gd,160Gd,156Dy,158Dy, leoQy 161 Dy i620y i63Dy164Dy165Dy162Er164Er165Er166Er167Er168Er 170Er,165Ho,166Ho,175Lu,177Lu,185Re,186Re,188Re,201TI,203TI,205TI,203Pb, 206Pb,207Pb,208Pb,212Pb,209Bi,212Bi,213Bi,223Ra,225Ac,227Th,232Th; and wherein Rs is selected from a -H, a halogen, -OH, -OMe, -OEt, -OPr, - NHCOH, -NHCOMe, NHCOEt, -NHCOPr, -CONH2, -CONHMe, -CONHEt, -CONHPr, NHSOH, -NHSO2Me, NHSO2Et, -NHSO2Pr, -SO2NH2, - SO2NHMe, -SO2NHEt, -SO2NHPr, -NH2, -NHMe, -NHEt, -NHPr; and pharmaceutically acceptable salts thereof.

2. FAP inhibitor ligand according to claim 1 , wherein A is a polar natural amino acid or its D-enantiomer.

3. FAP inhibitor ligand according to claim 1 or 2, wherein Yi is a radionuclide selected from3H,11C,18F,123l,131l,210At and211At.

4. FAP inhibitor according to claim 3, wherein Yi is1311 or211At.

5. FAP inhibitor ligand according to claims 1 or 2, wherein the chelator Z is labeled with a metal selected from64Cu,67Cu,68Ga,90Y,89Zr,99mTc,111In,177Lu,203Pb, 212Pb,225Ac.

6. FAP inhibitor ligand according to any of claims 1 - 3 selected from the group consisting of compounds l-XXVIll and LVIa:LVIa wherein R6is -H or -CH37. FAP inhibitor ligand according to claim 1 , 2 or 5 selected from the group consisting of:03 kLZLSSLizZL[68Ga]19wherein Re is -H or -CH3 8. FAP inhibitor ligand according to claim 1 , wherein said FAP inhibitor ligand of formula (I) comprises at least one radionuclide; for use as a medicament in therapy, imaging, diagnostics, or theranostics.

9. FAP inhibitor ligand according to claim any of the previous claim, wherein said FAP inhibitor ligand of formula (I) comprises at least one radionuclide, for use in the treatment and / or in the diagnosis of FAP expressing diseases including cancer, such as breast cancer, lung cancer, colorectal cancer, prostate cancer, ovarian cancer, pancreatic cancer, hepatocellular cancers, sarcomas and non-smallcell lung cancer; and fibrosis, arthritis, atherosclerosis, and inflammatory diseases like spondyloarthritis.

10. A pharmaceutic formulation comprising a FAP inhibitor ligand according to any of the previous claims.

11. FAP inhibitor precursor to formula (I) of formula (II):-(CH2)nNH-, -CH2(OCH2CH2)nNH-, -(CH2)nNHAm-, -CH2(OCH2CH2)nNHAm-, -CH2C(COOH)NHAm-, -CH2CH2C(COOH)NHAm- wherein n and m are integers independently selected from 0-20, and A is an amino acid independently selected from the group consisting of;-COCH2NH-, -COCH(CH3)NH-, -COCH(CH2SH)NH-,COCH(CH2COOH)NH-, -COCH(CH2CH2COOH)NH-,COCH(CH2C6H5)NH-, -COCH(CH2C3H3N2)NH-, -COCH(CH(CH3)2)NH-COCH((CHCH3)CH2CH3)NH-, -COCH((CH2)4NH2)NH-,COCH(CH2CH(CH3)2)NH-, -COCH(CH2CH2SCH3)NH-,CO(CHCH2CH2N)-, -COCH(CH2CONH2)NH-,COCH(CH2CH2CONH2)NH-, -COCH((CH2)3NH-C(NH)NH2)NH-, -COCH(CH2OH)NH-, -COCH(CH2CH2OH)NH-,COCH(CH(OH)CH3)NH-, -COCH(CH2SeH)NH-,COCH(CH2C8H6N)NH-, -COCH(CH2C6H4OH)NH-;COCH(CH2CH2SeCH3)NH-,X2is a cyclooctene selected from the group consisting of:wherein L2is -(CH2)nCO-, -(CH2CH2O)n(CH2)mCO or -C(CH3)2(CH2)CO-, wherein n and m are integers independently selected from 1-10,12. FAP inhibitor precursor according to claim 11 selected from the group consisting of compounds LVI-LXIX:wherein Re is -H or -CH3.

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