The present invention pertains to a precursor compound and a radiotracer for cancer diagnosis and treatment. The inventive precursor comprises a chelator Ch for complexation of a radioisotope and one, two, three or four targeting vectors TV ¹ , TV ² , TV ³ , TV ⁴ , wherein - each of targeting vectors TV ¹ , TV ² , TV ³ , TV ⁴ independently of one another has a structure selected from the group of structures according to the formula with X - -H or -CH3 ; Y = -H or -F ; m - 0, 1, 2, 3 or 4 ; where

Z is selected from the group comprising structures (1), (2), (3), (4), (5), (6), (7), (8), (9),

Many cancer tumors comprise a tumor micro environment or stroma that surrounds cancer cells (carcinogenic cells). The tumor stroma includes various non-malignant cell types and accounts for up to 90% of the total tumor mass. It plays an important role in the supply of cancer cells as well as in tumor progression and metastasis. Major components of the tumor stroma are the extracellular matrix (ECM), endothelial cells, pericytes, macrophages, immune regulatory cells and activated fibroblasts, commonly referred to as cancer-associated fibroblasts (CAFs). During tumor progression, CAFs change their morphology and biological function. These changes are induced by intercellular communication between cancer cells and CAFs. CAFs create an environment that promotes cancer cell growth. It has been shown that therapies which merely target cancer cells are inadequate. Effective therapies must also address the tumor microenvironment and in particular CAFs. In more than 90% of all human epithelial tumors CAFs overexpress fibroblast activation protein (FAP). Contrary thereto, FAP expression in healthy tissue is practically negligible. Hence, FAP constitutes a promising cellular receptor for targeted drug delivery and theranostic radiopharmaceuticals or radiotracers. In particular, FAP-targeted cancer drugs are equipped with a suitable cytotoxin or radioistope such as ¹⁸ F, ⁶⁸ Ga, ¹⁷⁷ Lu or ²²⁵ Ac.

The entire content of all prior art documents cited in this patent application is incorporated by reference. In particular, the chemical synthesis methods described in the cited prior art documents are used directly or in an analogous, suitably adapted manner to prepare the precursor compounds of the present invention.

The role of FAP in vivo is not fully understood, however, it is known to be a serine protease with unique enzymatic activity. It exhibits both dipeptidyl peptidase (DPP) and prolyl oligopeptidase (PREP) activity. Hence, for CAF targeting, substrates and inhibitors of DPP, PREP and FAP come into consideration as homing ligands. A suitable FAP ligand must possess high selectivity over related enzymes, such as dipeptidyl peptidases DPPII, DPPIV, DPP8, DPP9 and homologous prolyl oligopeptidases (PREP) that are ubiquitous in healthy tissue.

In order to target CAFs a drug or radiotracer is equipped with a ligating moiety or ligand having high binding affinity for FAP. Depending on their interaction FAP-ligands are classified as inhibitors or substrates. Inhibitor ligands bind at the FAP enzymatic cleft for prolonged time periods whereas substrate ligands are efficiently cleaved and subsequently released. The binding, cleavage and dissociation kinetics depend on various factors such as FAP and ligand concentration as well as reaction rate constants. According to Jimenez-Franco et al., the tumor release rate - or conversely the tumor retention - of a therapeutic radiotracer comprising a radioisotope, such as ¹⁷⁷ Lu or ²²⁵ Ac with half-life (ti/2) of 6.7 and 9.9 days, determines its therapeutic efficacy (cf. L.D. Jimenez-Franco, G. Glatting, V. Prasad, W.A. Weber, AJ. Beer, P. Kletting; Effect of Tumor Perfusion and Receptor Density on Tumor Control Probability in ¹⁷⁷ Lu-DOTATATE Therapy: An In Silico Analysis for Standard and Optimized Treatment; Journal of Nuclear Medicine January 2021, 62 (1) 92-98; DOI: https://doi.org/10.2967/jnumed.120.245068). The longer the radiotracer remains in the tumor tissue, the greater its therapeutic effectiveness.

Accordingly, the prior art endeavors to improve the therapeutic efficacy of FAP-targeted radiotracers by endowing them with prolonged "tumor retention" or greater "avidity" and "affinity". Tumor retention and avidity are assessed in vivo or ex vivo via radiological measurement of the signal uptake value (SUV) or biodistribution (i.e. the residual radioactivity in excised tissue). Contrary thereto, affinity is quantified in vitro by enzymatic assay methods and expressed as ratio k _O n/k _O ff of kinetic rate constants k _on and k _O ff for ligation with and dissociation from FAP, respectively. For small molecules, such as the inventive and various prior art radiotracers, k _on approaches the diffusion limit of 10 ⁸ M ^s ¹ . For irreversibly binding radiotracers, such as the inventive radiotracers, k _O ff equals zero and the affinity (k _on /koff) becomes infinite. Therefore, for the inventive radiotracers affinity loses its importance as an indicator of therapeutic potency. Moreover, chemical modification of peripheral structural groups of the inventive radiotracers - apart from the characteristic FAP-ligand motifs, such as exemplified beneath - does not measurably affect their tumor retention and therapeutic efficacy.

Examples (a) and (b) of characteristic FAP-ligand motifs comprising the amino acid glycine, the proline derivate 4,4-difluoro-2-[(fluorosulfonyl)methyl]pyrrolidine and an auxiliary quinoline group, respectively.

Small molecule ligands with high affinity and selectivity for FAP are known since 2014 (cf. K. Jansen, L. Heirbaut, R. Verkerk, J.D. Cheng, J. Joossens, P. Cos, L. Maes, A.-M. Lambeir, I. De Meester, K. Augustyns, P. Van der Veken; Extended Structure-Activity Relationship and Pharmacokinetic Investigation of (4-Quinolinoyl)glycyl-2-cyanopyrrolidine Inhibitors of Fibroblast Activation Protein (FAP); J. Med. Chem. 2014 Apr 10; 57(7): 3053-74, DOI 10.1021/jm500031w; A. De Decker, G. Vliegen, D. Van Rompaey, A. Peeraer, A. Bracke, L. Verckist, K. Jansen, R. Geiss-Friedlander, K. Augustyns, H. De Winter, I. De Meester, A.-M. Lambeir, P. Van der Veken, Novel Small Molecule-Derived, Highly Selective Substrates for Fibroblast Activation Protein (FAP), ACS Med. Chem. Lett. 2019, 10, 8, 1173-1179). These ligands comprise a modified glycine-proline unit and therewith coupled quinoline group.

Theranostic radiopharmaceuticals or radiotracers consist of a precursor compound and a therewith conjugated or complexed radioisotope such as ¹⁸ F and ⁶⁸ Ga or ¹⁷⁷ Lu. The precursor compound comprises a ligand for a relevant cellular receptor such as somatostatin receptor 2 (SSR2), prostate specific membrane antigen (PSMA) or FAP.

For labeling with radioisotopes such as ⁶⁴ Ga and ¹⁷⁷ Lu the precursor compound also includes a chelator moiety such as l,4,7,10-tetraazacyclododecane-l,4,7,10-tetraacetic acid (DOTA) or 6-amino-l,4-diazepine-triacetic acid (DATA).

For cancer radioendotherapy FAP-targeted radiotracers comprising a highly ionizing beta- or a-emitter such as ¹⁷⁷ Lu or ²²⁵ Ac with half-life (ti/2) of 6.7 and 9.9 days, respectively, constitute promising treatment modalities.

Various radiotracers comprising one or more PSMA or FAP inhibitor-homing-ligands conjugated with a chelator such as DOTA or DATA for complexation of radioistopes such as ⁶⁸ Ga and ¹⁷⁷ Lu are known in the prior art.

Banerjee et al. propose multivalent radiotracer compounds comprising a DOTA chelator and two or more therewith conjugated PSMA inhibitor ligands (cf. S.R. Banerjee, M. Pullambhatla, H. Shallal, A. Lisok, R.C. Mease, M.G. Pomper; A Modular Strategy to Prepare Multivalent Inhibitors of Prostate-Specific Membrane Antigen (PSMA); Oncotarget 2011; 2: 1244 - 1253; doi: 10.18632/oncotarget.415).

WO 2019/083990 A2 discloses a compound of formula B-L-A, wherein B is a targeting moiety for FAP-a, B is a radiolabeled functional group suitable for PET imaging or radiotherapy and L is a linker having bi-functionalization adapted to form a chemical bond with B and A.

WO 2019/154886 Al pertains to radiotracers comprising FAP-ligands, such as FAPI-46 (CAS No. 2374782-04-2).

WO 2021/016392 Al and WO 2022/258637 Al are directed to multivalent FAP-targeted imaging and treatment agents for cancers and other fibrotic diseases.

WO 2019/083990 A2 (pages 48-51), WO 2019/154886 Al (pages 61-75), WO 2021/016392 Al (pages 63-71) and WO 2022/258637 Al (pages 37-49) describe synthesis methods, which in conjunction with Examples 1 and 2 of the present application enable the skilled person to prepare the inventive precursors. Accordingly, the disclosure of WO 2019/083990 A2 (pages 48-51), WO 2019/154886 Al (pages 61-75), WO 2021/016392 Al (pages 63-71) and WO 2022/258637 Al (pages 37-49) is incorporated by reference. Various researchers, e.g. Narayanan et al., report the use of sulfonyl fluorides in pharmaceutical compounds (cf. A. Narayanan, L.H. Jones; Sulfonyl fluorides as privileged warheads in chemical biology; Chem. Sci., 2015, 6, 2650; doi: 10.1039/c5sc00408j).

Guardiola et al. describe ligand compounds for highly selective inhibition of prolyl oligopeptidase (cf. S. Guardiola, R. Prades, L. Mendieta, AJ. Brouwer, J. Streefkerk, L. Nevola, T. Tarragd, R.M.J. Liskamp, E. Giralt; Targeted Covalent Inhibition of Prolyl Oligopeptidase (POP): Discovery of Sulfonylfluoride Peptidomimetics; Cell Chemical Biology 25, 1031-1037, August 16, 2018; https://doi.Org/10.1016/j.chembiol.2018.04.013; in particular pages e2-e3 and Supplementary Information, Figure S1A).

Further methods for synthesis of sulfonyl fluoride comprising compounds are described in:

- Y. Jiang, N.S. Alharbi, B. Sun, H.-L. Qin; Facile one-pot synthesis of sulfonyl fluorides from sulfonates or sulfonic acids; RSC Adv., 2019, 9, 13863; doi: 10.1039/c9ra02531f; Supporting Information, pages S3-S11;

- R. Xu, T. Xu, M. Yang, T. Cao, S. Liao; A rapid access to aliphatic sulfonyl fluorides; Nature

Communications (2019) 10:3752; https://doi.org/10.1038/s41467-019-11805-6;

Supplementary Information, pages 2-41;

- T. Zhong, J.-T. Yi, Z.-D. Chen, Q.-C. Zhuang, Y.-Z. Li, G. Lu, J. Weng; Photoredox-catalyzed aminofluorosulfonylation of unactivated olefins; Chem. Sci., 2021, 12, 9359; doi: 10.1039/dlsc02503a; Supplementary Material, pages S2-S47;

- G. Laudadio, A. de A. Bartolomeu, L.M.H.M. Verwijlen, Y. Cao, K.T. de Oliveira, T. Noel; Sulfonyl Fluoride Synthesis through Electrochemical Oxidative Coupling of Thiols and Potassium Fluoride; J. Am. Chem. Soc. 2019, 141, 11832-11836; doi: 10.1021/jacs.9b06126; Supporting Information, pages S3-S31;

- S.N. Carneiro, S.R. Khasnavis, J. Lee, T.W. Butler, J.D. Majmudar, C.W. am Ende; N.D. Ball; Sulfur(VI) fluorides as tools in biomolecular and medicinal chemistry; Org. Biomol. Chem., 2023, 21, 1356; DOI: 10.1039/d2ob01891h; pages 1356-1360; all of which - including the synthesis method of Guardiola et al. - are incorporated by reference in the present patent application.

Tumor uptake, tumor retention time and the ratio of tumor-absorbed to whole-body- absorbed radiation dose of known FAP-targeted radiotracers warrants further improvement.

Accordingly, the present invention has the object to provide a precursor for FAP-targeted radiotracers that yield higher tumor uptake, prolonged tumor retention time and increased ratio of tumor-absorbed to whole-body-absorbed radiation dose. This object is achieved by a precursor compound comprising a chelator Ch for complexation of a radioisotope and one, two, three or four targeting vectors TV ¹ , TV ² , TV ³ , TV ⁴ , wherein each of targeting vectors TV ¹ , TV ² , TV ³ , TV ⁴ independently of one another has a structure selected from the group of structures according to the formula with X = -H or -CH3 ; Y = -H or -F ; m = 0, 1, 2, 3 or 4 ; where

Z is selected from the group comprising structures (1), (2), (3), (4), (5), (6), (7), (8), (9),

Expedient embodiments of the inventive precursor compound are characterized by one of the following features or a combination of two or more of the following features insofar the combined features are not mutually exclusive or contradictory and according to which: the chelator Ch has a structure selected from the group comprising structures (I), (II), (III), (IV), (V) and (VI) with the chelator Ch has a structure selected from the group comprising structures (VII), (VIII), (IX) and (X) with

(IX) (X) the chelator Ch has a structure selected from the group comprising structures (XI), (XII), (XIII) and (XIV) with

(XI) (XII)

(XIII) (XIV) the chelator Ch is selected from the group comprising H ₄ pypa, EDTA (Ethylenediamine tetraacetate), EDTMP (Ethylenediaminetetra(methylenephosphonic acid)), DTPA (Diethylenetriamine pentaacetate) and derivatives thereof, NOTA (1,4,7-triazacyclo- nonane-l,4,7-triacetic acid) and derivatives thereof, such as NODAGA (1,4,7-triazacyclo- nonane,l-glutaric acid-4, 7-acetic acid), TRAP (Triazacyclononane-phosphinic acid), NOPO (l,4,7-triazacyclononane-l,4-bis[methylene-(hydroxymethyl)-p hosphinic acid]-7-[meth- ylene-(2-carboxyethyl)-phosphinic acid]), DOTP ^H (1,4,7, 10-tetraazacyclododecane- 1,4,7, 10-tetrakis[methylenephosphinic acid]) and derivatives thereof, such as DOTPI (l,4,7,10-tetraazacyclododecane-l,4,7,10-tetrakis[methylene( 2-carboxyethylphosphinic acid)]) and DOTPI(azid)4, TRITA (Trideca-l,4,7,10-tetraamine-tetraacetate), TETA (Tetradeca-l,4,8,ll-tetraamine-tetraacetate) and derivatives thereof, PEPA (Pentadeca- 1,4,7,10,13-pentaamine pentaacetate), HEHA (Hexadeca-l,4,7,10,13,16-hexaamine- hexaacetate) and derivatives thereof, HBED (N,N'-Bis-(2-hydroxybenzyl)ethylene- diamine-N,N'-diacetate) and derivatives thereof such as HBED-CC (N,N'-Bis-[2-hydroxy-5- carboxyethyl)benzyl)ethylene-diamine-N,N'-diacetate), DEDPA and derivatives thereof, such as Hzdedpa (l,2-[[6-(Carboxyl)pyridine-2-yl]methylamine]ethane) and H ₄ octapa (l,2-[[6-(Carboxyl)pyridine-2-yl]methylamine]ethane-N,N'-dia cetate), DFO (Deferoxamine) and derivatives thereof, Trishydroxypyridinone (THP) and derivatives thereof, such as H3THP-AC and HsTHP-mal (YM103), TEAP (Tetraazycyclodecane-phosphinic acid) and derivatives thereof, Sarcophagin SAR (l-N-(4-aminobenzyl)-3,6,10,13,16,19-hexaaza- bicyclo[6.6.6]-eicosan-l,8-diamine) and derivatives thereof, such as (NI-hhSAR (1,8- diamino-3,6,10,13,16,19-hexaazabicyclo [6.6.6]icosane), N4 (3-[(2'-Aminoethyl)amino]- 2-[(2"-aminoethyl) aminomethyl] propionic acid) and other N ₄ -derivates, PnAO (6-(4-lsothiocyanatobenzyl)-3,3,9,9,-tetramethyl-4,8-diaza-u ndecane-2,10-dione- dioxime) and derivatives thereof, such as BMS181321 (3,3'-(l,4-Butanediyldiamino)- bis(3-methyl-2-butanone)dioxime), MAG2 (Mercaptoacetyl-glycyl-glycine) and derivatives thereof, MAG3 (Mercaptoacetyl-glycyl-glycyl-glycine) and derivatives thereof, such as NsS-adipate, MAS3 (Mercaptoacetyl-seryl-seryl-serine) and derivatives thereof, MAMA (N-(2-Mercaptoethyl)-2-[(2-mercaptoethyl)amino]acetamide) and derivatives thereof, EC (Ethylene dicysteine) and derivatives thereof, dmsa (Dimercaptosuccinic acid) and derivatives thereof, DADT (Diamine dithiol), DADS (Diamine disulfide), N2S2-chelators and derivatives thereof, Aminothiol and derivatives thereof; salts of the preceding chelators; HYNIC (Hydrazinonicotinamide) and derivatives thereof; one, two, three or all of targeting vectors -TV ¹ , -TV ² , -TV ³ , -TV ⁴ independently of each other have the structure one, two, three or all of targeting vectors -TV ¹ , -TV ² , -TV ³ , -TV ⁴ independently of each other have the structure one, two, three or all of targeting vectors -TV ¹ , -TV ² , -TV ³ , -TV ⁴ independently of each other have the structure one, two, three or all of targeting vectors -TV ¹ , -TV ² , -TV ³ , -TV ⁴ independently of each other have the structure one, two, three or all of targeting vectors -TV ¹ , -TV ² , -TV ³ , -TV ⁴ independently of each other have the structure - one, two, three or all of targeting vectors -TV ¹ , -TV ² , -TV ³ , -TV ⁴ independently of each other have the structure one, two, three or all of targeting vectors -TV ¹ , -TV ² , -TV ³ , -TV ⁴ independently of each other have the structure one, two, three or all of targeting vectors -TV ¹ , -TV ² , -TV ³ , -TV ⁴ independently of each other have the structure one, two, three or all of targeting vectors -TV ¹ , -TV ² , -TV ³ , -TV ⁴ independently of each other have the structure - one, two, three or all of targeting vectors -TV ¹ , -TV ² , -TV ³ , -TV ⁴ independently of each other have the structure one, two, three or all of targeting vectors -TV ¹ , -TV ² , -TV ³ , -TV ⁴ independently of each other have the structure one, two, three or all of targeting vectors -TV ¹ , -TV ² , -TV ³ , -TV ⁴ independently of each other have the structure one, two, three or all of targeting vectors -TV ¹ , -TV ² , -TV ³ , -TV ⁴ independently of each other have the structure - one, two, three or all of targeting vectors -TV ¹ , -TV ² , -TV ³ , -TV ⁴ independently of each other have the structure

one, two, three or all of targeting vectors -TV ¹ , -TV ² , -TV ³ , -TV ⁴ independently of each other have the structure one, two, three or all of targeting vectors -TV ¹ , -TV ² , -TV ³ , -TV ⁴ independently of each other have the structure - one, two, three or all of targeting vectors -TV ¹ , -TV ² , -TV ³ , -TV ⁴ independently of each other have the structure one, two, three or all of targeting vectors -TV ¹ , -TV ² , -TV ³ , -TV ⁴ independently of each other have the structure one, two, three or all of targeting vectors -TV ¹ , -TV ² , -TV ³ , -TV ⁴ independently of each other have the structure - one, two, three or all of targeting vectors -TV ¹ , -TV ² , -TV ³ , -TV ⁴ independently of each other have the structure one, two, three or all of targeting vectors -TV ¹ , -TV ² , -TV ³ , -TV ⁴ independently of each other have the structure one, two, three or all of targeting vectors -TV ¹ , -TV ² , -TV ³ , -TV ⁴ independently of each other have the structure - one, two, three or all of targeting vectors -TV ¹ , -TV ² , -TV ³ , -TV ⁴ independently of each other have the structure one, two, three or all of targeting vectors -TV ¹ , -TV ² , -TV ³ , -TV ⁴ independently of each other have the structure one, two, three or all of targeting vectors -TV ¹ , -TV ² , -TV ³ , -TV ⁴ independently of each other have the structure - one, two, three or all of targeting vectors -TV ¹ , -TV ² , -TV ³ , -TV ⁴ independently of each other have the structure one, two, three or all of targeting vectors -TV ¹ , -TV ² , -TV ³ , -TV ⁴ independently of each other have the structure one, two, three or all of targeting vectors -TV ¹ , -TV ² , -TV ³ , -TV ⁴ independently of each other have the structure - one, two, three or all of targeting vectors -TV ¹ , -TV ² , -TV ³ , -TV ⁴ independently of each other have the structure one, two, three or all of targeting vectors -TV ¹ , -TV ² , -TV ³ , -TV ⁴ independently of each other have the structure one, two, three or all of targeting vectors -TV ¹ , -TV ² , -TV ³ , -TV ⁴ independently of each other have the structure - one, two, three or all of targeting vectors -TV ¹ , -TV ² , -TV ³ , -TV ⁴ independently of each other have the structure one, two, three or all of targeting vectors -TV ¹ , -TV ² , -TV ³ , -TV ⁴ independently of each other have the structure one, two, three or all of targeting vectors -TV ¹ , -TV ² , -TV ³ , -TV ⁴ independently of each other have the structure - one, two, three or all of targeting vectors -TV ¹ , -TV ² , -TV ³ , -TV ⁴ independently of each other have the structure one, two, three or all of targeting vectors -TV ¹ , -TV ² , -TV ³ , -TV ⁴ independently of each other have the structure one, two, three or all of targeting vectors -TV ¹ , -TV ² , -TV ³ , -TV ⁴ independently of each other have the structure - one, two, three or all of targeting vectors -TV ¹ , -TV ² , -TV ³ , -TV ⁴ independently of each other have the structure one, two, three or all of targeting vectors -TV ¹ , -TV ² , -TV ³ , -TV ⁴ independently of each other have the structure one, two, three or all of targeting vectors -TV ¹ , -TV ² , -TV ³ , -TV ⁴ independently of each other have the structure - targeting vectors TV ¹ and TV ² are identical; targeting vectors TV ¹ , TV ² and TV ³ are identical; targeting vectors TV ¹ , TV ² , TV ³ and TV ⁴ are identical; the precursor compound has a structure selected from the group comprising structures

(A), (B), (C), (D), (E), (F) and (G) with

(A) (B) (C) (D) wherein each of L, L ¹ , L ² , L ³ , L ⁴ independently of one another is absent or a bifunctional covalent linker moiety and M is a trifunctional, tetrafunctional or pentafunctional covalent linker moiety;

- the trifunctional, tetrafunctional or pentafunctional covalent linker moiety M is a residue of a peptide comprising 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids independently selected from the group comprising Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, Vai, Pyl, Sec, GABA or y-Aminobutyric acid, Homoserine, DOPA or 3,4-Dihydroxyphenylalanine, Citrulline, 0-Alanine and Thyroxine; each of linkers -L ¹ - , - L ² - , -L ³ - , -L ⁴ - independently of one another comprises or consists of the structure each of linkers -L ¹ - , - L ² - , -L ³ - , -L ⁴ - independently of one another comprises or consists of the structure - each of moieties -L^TV ¹ , -L ² -TV ² , -L ³ -TV ³ , -L ⁴ -TV ⁴ independently of one another comprises or consists of the structure each of moieties -L ¹ -TV ¹ _; -L ² -TV ² , -L ³ -TV ³ , -L ⁴ -TV ⁴ independently of one another comprises or consists of the structure each of moieties -l^-TV ¹ , -L ² -TV ² , -L ³ -TV ³ , -L ⁴ -TV ⁴ independently of one another comprises or consists of the structure each of moieties -IJ-TV ¹ , -L ² -TV ² , -L ³ -TV ³ , -L ⁴ -TV ⁴ independently of one another comprises or consists of the structure each of moieties -iJ-TV ¹ , -L ² -TV ² , -L ³ -TV ³ , -L ⁴ -TV ⁴ independently of one another comprises or consists of the structure each of moieties -iJ-TV ¹ , -L ² -TV ² , -L ³ -TV ³ , -L ⁴ -TV ⁴ independently of one another comprises or consists of the structure each of moieties -l^-TV ¹ , -L ² -TV ² , -L ³ -TV ³ , -L ⁴ -TV ⁴ independently of one another comprises or consists of the structure each of moieties -iJ-TV ¹ , -L ² -TV ² , -L ³ -TV ³ , — L ⁴ — TV ⁴ independently of one another comprises or consists of the structure each of moieties -l^-TV ¹ , -L ² -TV ² , -L ³ -TV ³ , -L ⁴ -TV ⁴ independently of one another comprises or consists of the structure each of moieties -iJ-TV ¹ , -L ² -TV ² , -L ³ -TV ³ , -L ⁴ -TV ⁴ independently of one another comprises or consists of the structure each of linkers -L-, -L ¹ - -L ² -, -L ³ -, -L ⁴ - independently of one another comprises or consists of a structure of type -[C Jp- with p = 1, 2, ... , 19 or 20; - each of linkers -L-, -L ¹ - -L ² -, -L ³ -, -L ⁴ - independently of one another comprises or consists of a structure of type -(NH)-[CH2] _P - with p = 1, 2, ... , 19 or 20; each of linkers -L-, -L ¹ - -L ² - -L ³ -, -L ⁴ - independently of one another comprises or consists of a structure of type -(NH)-[CH2] _P -(NH)- with p = 1, 2, ... , 19 or 20; each of linkers -L-, -L ¹ - -L ² - -L ³ -, -L ⁴ - independently of one another comprises or consists of a structure of type -[CH2CH2O] _P - with p = 1, 2, ... , 19 or 20; each of linkers -L-, -L ¹ - -L ² -, -L ³ -, -L ⁴ - independently of one another comprises or consists of a structure of type — (NH)— [ CF C OJp- with p = 1, 2, ... , 19 or 20; each of linkers -L-, -L ¹ - -L ² -, -L ³ -, -L ⁴ - independently of one another comprises or consists of a structure of type — (NH)— [ CH2CH2O] _P -(NH)- with p = 1, 2, ... , 19 or 20; - each of linkers -L-, -L ¹ - -L ² - -L ³ -, -L ⁴ - independently of one another comprises or consists of a residue of ethylene diamine each of linkers -L-, -L ¹ - -L ² -, -L ³ -, -L ⁴ - independently of one another comprises or consists of a residue having the structure - each of linkers -L-, -L ¹ -, -L ² -, -L ³ -, -L ⁴ - independently of one another comprises or consists of a residue of a peptide comprising 1, 2, 3, 4, 5, 6, 7 , 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids independently selected from the group comprising Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, Vai, Pyl, Sec, GABA or y-Aminobutyric acid, Homoserine, DOPA or 3,4-Dihydroxyphenylalanine, Citrulline, 0-Alanine and Thyroxine;

- each of linkers -L-, -L ¹ -, -L ² -, -L ³ -, -L ⁴ - independently of one another comprises a phenylalanine residue;

- each of linkers -L-, -L ¹ -, -L ² -, -L ³ -, -L ⁴ - independently of one another comprises the residue

- each of linkers -L-, -L ¹ -, -L ² -, -L ³ -, -L ⁴ - independently of one another comprises an N,N-dimethylarginine residue;

- each of linkers -L-, -L ¹ -, -L ² -, -L ³ -, -L ⁴ - independently of one another comprises the residue

each of linkers -L- -L ¹ -, -L ² -, -L ³ -, -L ⁴ - independently of one another comprises or consists of the residue each of linkers -L- -L ¹ -, -L ² -, -L ³ -, -L ⁴ - independently of one another comprises or consists of the residue each of linkers -L- -L ¹ -, -L ² - -L ³ -, -L ⁴ - independently of one another comprises or consists of a residue having structure wherein each Q' is present for 1 < i < k and absent for i > k with k = 1, 2, 3, 4, 5, 6, 7 , 8, 9 or 10; each R ^j is present for 1 < j < h and absent for j > h with h = 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

T is absent or present; each Q ^s with s = 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, if present, independently of one another is selected from the group comprising -CH2-, -CH2CH2O-, -N(H)- , -N(CH ₃ )-, -O-, -S-, -C(O)- and -C(CH ₃ )- ; each R‘ with t = 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, if present, independently of one another is selected from the group comprising -CH2-, -CH2CH2O-, — N(H)— , -N(CH ₃ )-, -O-, -S-, -C(O)- and -C(CH ₃ )- ;

T, if present, is selected from the group comprising

The invention has the further object to provide a radiotracer for cancer diagnosis and treatment.

This object is achieved through a radiotracer comprised of the above described precursor compound and a therewith complexed radioisotope or radioactive compound selected from the group comprising ¹³⁵ Sm, ¹⁴⁰ Pr, ¹⁵⁹ Gd, ¹⁴⁹

Expedient embodiments of the inventive radiotracer are characterized by one of the following features or a combination of the following features insofar the combined features are not mutually exclusive or contradictory and according to which: the radioisotope is ⁶⁸ Ga;

- the radioisotope is ¹⁷⁷ Lu;

- the radioisotope is ²²⁵ Ac;

- the radioactive compound is ^1S FAI (aluminum fluoride);

- the radiotracer comprises a chelator having the structure (I), (II), (III), (IV), (V), (VI), (VII), (VIII), (IX) or (X) and a therewith complexed radioisotope selected from the group comprising ⁴⁴ Sc, ⁴⁷ Sc, ⁵⁵ Co, ⁶² Cu, ⁶⁴ Cu, ⁶⁷ Cu, ⁶⁶ Ga, ⁶⁷ Ga, ⁶⁸ Ga, ⁸⁹ Zr, ⁸⁶ Y, ⁹⁰ Y, ⁹⁰ Nb, ^m ln, ¹³⁵ Sm, 1 ⁴⁰ Pr, ¹⁵⁹ Gd, ¹⁴⁹ Tb, ¹⁶⁰ Tb, ¹⁶¹ Tb, ¹⁶⁵ Er, ¹⁶⁶ Dy, ¹⁶⁶ Ho, ¹⁷⁵ Yb, ¹⁷⁷ Lu, ²¹³ Bi and ²²⁵ Ac;

- the radiotracer comprises a chelator having the structure (I), (II), (III), (IV), (V), (VI), (VII), (VIII), (IX) or (X) and a therewith complexed radioisotope selected from the group comprising ⁶⁸ Ga, ¹⁷⁷ Lu and ²²⁵ Ac; the radiotracer comprises a chelator having the structure (XI), (XII), (XIII) or (XIV) and therewith complexed radioactive compound ¹⁸ FAI (aluminum fluoride). Sulfonyl fluoride electrophiles have found significant utility as reactive probes in chemical biology and molecular pharmacology. As warheads they possess the right balance of biocompatibility (including aqueous stability) and protein reactivity. Their functionality is privileged in this regard as they are known to modify not only reactive serines (resulting in their common use as protease inhibitors), but also context-specific threonine, lysine, tyrosine, cysteine and histidine residues (cf. A. Narayanan, L.H. Jones; Sulfonyl fluorides as privileged warheads in chemical biology; Chem. Sci., 2015, 6, 2650).

Known FAP inhibitors feature a C-terminal reactive functionality, such as carbonitrile which covalently bind to the hydroxyl group of the catalytic serine (Ser ⁶²⁴ ) of FAP. However, these molecules form a transient covalent bond with FAP that is hydrolyzed after a short time. Hence, FAP regains its enzymatic activity.

Contrary thereto, the sulfonly fluoride group of the inventive FAP ligand acts as leaving group when situated adajacent to FAP's catalytic amino acid Ser ⁶²⁴ , such that upon deprotonation of the Ser ⁶²⁴ hydroxy group a permanent covalent bond is formed between the inventive FAP ligand and Ser ⁶²⁴ .

Numerous chelators for complexation of radioisotopes, in particular chelators based on the DOTA- and DATA-scaffold, are readily available from commercial vendors (e.g. https://www.macrocyclics.com/). Many of the commercially available chelators comprise a terminal OH- or NHz-group for facile coupling with a linker (cf. Example 5).

Likewise, a large variety of heterobifunctional linkers are commercially available either as ready-made compound, crosslinking kit or service (e.g. from https://www.carbolution.de/, https://bezwadabiomedical.com/, https://broadpharm.com, https://p3bio.com/amino- acids/fmoc-amino-acids/, https://www.thermofisher.com, https://www.profacgen.com). Some vendors offer comprehensive libraries of Fmoc- and tBu-protected amino acids. The Crosslinking Technical Handbook, ThermoFisher® Scientific (2022; https://assets.thermo- fisher.com/TFS-Assets/BID/Handbooks/bioconjugation-technical -handbook.pdf) describes numerous linker chemistries and bioconjugation strategies.

The synthesis of inventive FAP ligands is illustrated beneath in Example 1. Auxiliary methods for covalent bond formation between linkers L, L ¹ , L ² , L ³ , L ⁴ and a chelator or the inventive FAP ligand are presented in Examples 2 and 3.

If required, methoxy groups may be dealkylated using known protocols such as described in S.A. Weissman, D. Zewge; Recent advances in ether dealkylation; Tetrahedron 61 (2005) 7833- 7863; and A. Boto, D. Hernandez, R. Hernandez, E. Suarez; Selective Cleavage of Methoxy Protecting Groups in Carbohydrates; J. Org. Chem. 2006, 71, 1938-1948. EXAMPLES

Example 1: Synthesis of FAP ligand

Schemes la-lc illustrate the synthesis of FAP ligands.

Scheme la: Synthesis of (S)-4,4-difluoro-l-(aminoacetyl)pyrrolidine-2-[(sulfonyl fluoride)methyl] (a) TEMPO (0.02 eq), l,3,5-Trichloro-l,3,5-triazinane-2,4,6-trione, DCM, 1 h, 0°C, 74%; (b) DAST, DCM, 16 h, 61%; (c) KOH, MeOH, 16 h, 86%; (d) 1. LiAl H ₄ , 2. H ₃ O ⁺ , 72%;

(e) 1. MsCI, Et ₃ N, CH ₂ CO ₃ , 2. AcSH, Cs ₂ CO ₃ , DMF, 68%; (f) H ₂ O ₂ , AcOH, AcONa, 97%;

(g) Et ₃ .3HF, CH ₂ CI ₂ , 76%; (h) HBr, AcOH, MeCN, Dowex-CI, 90%; (i) HATU, Gly-OH, DIPEA, DCM, 3 h, 77%.

Scheme lb: Synthesis of 6-methoxyquinoline-4-carboxylic acid (a) PBr ₃ , DMF, 3 h, 63%;

(b) Zn(CN) ₂ , Pd/C, Zn ⁺⁺ (COO-) ₂ , dppf, 110°C, 3 h, 72%; (c) NaOH, H ₂ O/EtOH, reflux, 94%.

Scheme 1c: Conjugation of (S)-4,4-difluoro-l-(aminoacetyl)pyrrolidine-2-[(sulfonyl fluoridejmethyl] with 6-methoxyquinoline-4-carboxylic acid.

Example 2: Alternative strategy for synthesis of FAP ligands with sulfonyl fluoride group

Jiang et al. describe a one-pot synthesis of sufonyl fluorides that can be employed as an alternative to the method of Example 1 (cf. Y. Jiang, N.S. Alharbi, B. Sun, H.-L. Qin; Facile one- pot synthesis of sulfonyl fluorides from sulfonates or sulfonic acids; RSC Adv., 2019, 9, 13863; doi: 10.1039/c9ra02531f).

Commercially available tert-butyl 4,4-difluoro-2-(hydroxymethyl)pyrrolidine-l-carboxylate (CAS No. 215918-21-1) is reacted with triphenylphosphine dihalide ( (CeHs)3PX2 , X - Cl, Br, I) displace the hydroxy group with a halide (SN2 reaction), which is then converted to sulfonate (Strecker sulfite alkylation) using alkali sulfite in the presence of an iodine catalyst (Scheme 2a). The obtained sufonate is reacted with cyanuric chloride (2,4,6-trichloro-l,3,5- triazine, tetrabutylammonium bromide, acetonitrile, potassium bifluoride, acetone) to obtain 4,4-difluoro-2-[(sulfonyl fluoridejmethyl] pyrrolidine-l-carboxylate (Scheme 2b), the amine of which is subsequently deprotected (Scheme 2c).

Scheme 2a: Conversion of tert-butyl 4,4-difluoro-2-(hydroxymethyl)pyrrolidine-l- carboxylate to corresponding halide and sulfonate (side products are not shown).

Scheme 2b: One-pot conversion of sulfonate to 4,4-difluoro-2- [(sulfonyl fluoride)methyl]pyrrolidine-l-carboxylate (side products are not shown).

Scheme 2c: Deprotection to 4,4-difluoro-2-[(sulfonyl fluoride)methyl]pyrrolidine (side products are not shown).

4,4-dif luoro-2-[(sulfonylf luoride) methyl]pyrrolidine may be conjugated with quinolines, such as commercially available 6-methoxyquinoline-4-carboxylic acid or tert-butyl (4-bromo- quinolin-6-yl)carbamate (cf. Scheme 2d) using reactions analogous to Scheme la(i), lc and la(i), lb, lc, respectively.

6-Methoxyquinoline-4-carboxylic acid tert-Butyl (4-bromoquinolin-6-yl)carbamate CAS No. 86-68-0 CAS No. 1260784-05-1

Scheme 2d: Commercially available quinolines Example 3: Amide bond formation

A generic example of an amide coupling reaction is shown in scheme 2.

Scheme 3: Amide coupling Owing to a virtually unlimited set of readily available carboxylic acid and amine derivatives, amide coupling strategies open up a simple route for the synthesis of novel compounds. The person skilled in the art is aware of numerous reagents and protocols for amide coupling. The most commonly used amide coupling strategy is based on the condensation of a carboxylic acid with an amine. For this purpose, the carboxylic acid is generally activated. Prior to the activation, remaining functional groups are protected. The reaction is carried out in two steps, either in one reaction medium (single pot) with direct conversion of the activated carboxylic acid, or in two steps with isolation of activated "trapped" carboxylic acid and reaction with an amine.

The carboxylic reacts here with a coupling agent to form a reactive intermediate which can be reacted in isolated form or directly with an amine. Numerous reagents are available for carboxylic acid activation, such as acid halide (chloride, fluoride), azides, anhydrides or carbodiimides. In addition, reactive intermediates formed may be esters such as pentafluorophenyl or hydroxysuccinimido esters. Intermediates formed from acyl chlorides or azides are highly reactive. However, harsh reaction conditions and high reactivity are frequently a barrier to use for sensitive substrates or amino acids. By contrast, amide coupling strategies that utilize carbodiimides such as DCC (dicyclohexylcarbodiimide) or DIC (diisopropylcarbodiimide) open up a broad spectrum of application. Frequently, especially in the case of solid-phase synthesis, additives are used to improve reaction efficiency. Aminium salts are highly efficient peptide coupling reagents having short reaction times and minimal racemization. With some additives, for example HOBt, it is impossible to completely prevent racemization. Aminium reagents are used in an equimolar amount with the carboxylic acid in order to prevent excess reaction with the free amine of the peptide. Phosphonium salts react with carboxylate, which generally requires two equivalents of a base, for example DIEA. A significant advantage of phosphonium salts over iminium reagents is that phosphonium does not react with the free amino group of the amine component. This enables couplings in a molar ratio of acid and amine and helps to prevent the intramolecular cyclization of linear peptides and excessive use of costly amine components.

An extensive summary of reaction strategies and reagents for amide couplings can be found in the following review articles:

- Analysis of Past and Present Synthetic Methodologies on Medicinal Chemistry: Where Have All the New Reactions Gone?,- D. G. Brown, J. Bostrom; J. Med. Chem. 2016, 59, 4443-4458;

- Peptide Coupling Reagents, More than a Letter Soup; A. El-Faham, F. Albericio; Chem. Rev. 2011, 111, 6557-6602;

- Rethinking amide bond synthesis; V. R. Pattabiraman, J. W. Bode; Nature, Vol. 480 (2011) 22/29;

- Amide bond formation: beyond the myth of coupling reagents; E. Valeur, M. Bradley; Chem.

Soc. Rev., 2009, 38, 606-631. Example 4: Ether bond formation between alcohols

The synthetic strategy outlined beneath in Scheme 4 follows:

P.K. Sahoo, S.S. Gawali, C. Gunanathan; Iron-Catalyzed Selective Etherification and Transetherification Reactions Using Alcohols; ACS Omega 2018, 3, 124-136.

R ₁ = aryl , R ₂ , R ₃ = alkyl or aryl Scheme 4: I ron(l I l)-cata lyzed etherification of two different alcohols

Secondary alcohol (0.5 mmol), primary alcohol (0.5 mmol), Fe(0Tf)3 (0.025 mmol, 5 mol %) and NH4CI (0.025 mmol, 5 mol %) in DCM (2 mL) are heated at 45 °C for 1 to 24 h.

Fe(NO3)3 ■ 9 H2O (0.025 mmol, 5 mol %) is used as catalyst. Reaction is carried out at 70 °C.

Product is isolated via column chromatographic purification with typical yield between 40 and 93 %.

Example 5: Chelator building blocks

Numerous chelators for complexation of radioisotopes, in particular chelators based on the DOTA- and DATA-scaffold, are readily available from commercial vendors (e.g. https://www.macrocyclics.com/; https://www.macrocyclics.com/wp-content/uploads/2022/ 07/2022-Product-Catalog.pdf; https://www.chematech-mdt.com/wp-content/uploads/ 2020/09/Brochure_Chematech-2020-web.pdf). Many of the commercially available chelators comprise a terminal OH- or NH2-group for facile coupling with a linker.

CAS No. 137076-54-1 CAS No. 306776-79-4

CAS No. 1161415-28-6 CAS No. 438553-50-3

Scheme 5: Chelator building blocks

Example 6: DATA ^5m Prochelator Synthesis

Scheme 6 illustrates the synthesis of the DATA ⁵ " ¹ prochelator (cf. J. Seemann, B. Waldron, D. Parker, F. Roesch; DATATOC: a novel conjugate for kit-type ⁶⁸ Ga labelling of TOC at ambient temperature; EJNMMI Radiopharmacy and Chemistry (2016) 1:4, DOI 10.1186/s41181-016- 0007-3).

Schema 6: Synthesis of 3 ^l Bu-protected DATA ^5m prochelator (i) Amberlyst-21, EtOH; (ii) CH ₂ O, EtOH; (iii) CH3COOH, Pd(OH) ₂ /C, H ₂ , EtOH; (iv) BrCH ₂ COO ^l Bu, K ₂ CO ₃ , MeCN; (v) CH ₃ I, K ₂ CO ₃ , DCM : MeCN; (vi) LiOH, THF : H ₂ O Examples 7-14: Inventive Precursor Compounds

Schemes 7-15 depict exemplary embodiments 7-15 of inventive precursor compounds.

Scheme 7: Exemplary precursor compound 7

Scheme 8: Exemplary precursor compound 8

Scheme 9: Exemplary precursor compound 9

Scheme 10: Exemplary precursor compound 10

Scheme 11: Exemplary precursor compound 11

Scheme 12: Exemplary precursor compound 12

Scheme 14: Exemplary precursor compound 14

Scheme 15: Exemplary precursor compound 15

Example 16: Fluorescence Binding Assay

Biotinylated human fibroblast activation protein (AcroBiosystems Inc., Human FAP Protein, His, Avitag™, product no. FAP-H82Q6) is immobilized on streptavidin precoated 96-well plates (AcroBiosystems Inc., SP-11, polystyrene, clear, 100 pL streptavidin tetramer) with about 0.5 pg (2,9 pM) FAP per well.

On each plate 1/3 of wells (i.e. 32 wells) each are assigned to (a) the inventive precursor compound of Scheme 8; (b) the prior art precursor compound FAPI-46 (CAS No. 2374782-04-2; GSRS UN II 59QC5DY68A); and as reference (c).

Scheme 16: FAPI-46

About 160 pLof 2 pM solutions of the inventive precursor compound of Scheme 8 and FAPI-46 are incubated for lh at 37.5 °C in each of 32 wells (a) and (b), respectively. Subsequently, the precursor solutions are removed, each of 32 wells (a), (b) rinsed three times with physiological saline and incubated with about 160 pL of human serum (Merck, H4522) for periods of 96 h and 168 h at 37.5 °C while being stirred at 50 rpm using a plate shaker with 3 mm orbital travel diameter. Each 24 h wells (a) and (b) are emptied and refilled with fresh human serum. After 96 h and 168 h incubation period the human serum is removed and wells (a), (b) and (c) incubated with about 160 pL of 1 mM solution of fluorogenic FAP-substrate Z-Gly-Pro-AMC (MedChemExpress, Cat. No. HY-D1670, CAS No. 68542-93-8). The fluorescence of cleavage product 7-amino-4-methylcoumarin (A _ex = 380 nm, A _e m = 465 nrn) in each well is measured with a plate reader (Thermo Scientific™ Fluoroskan™ FL) and averaged over each 32 wells (a),

(b) and (c).

The averaged fluorescence signal from wells (a) is negligible compared to that of wells (b) and

(c) - less than 1% and l%o, respectively- which demonstrates that the inventive precursor compound of Scheme 8 binds irreversibly to FAP.