WO2025146433A1

WO2025146433A1 - Precursor and theranostic radiotracer with prolonged tumor retention

Info

Publication number: WO2025146433A1
Application number: PCT/EP2024/088680
Authority: WO
Inventors: Alexi Nikolai ZOUNEK
Original assignee: Radiovaxx GmbH
Current assignee: Radiovaxx GmbH
Priority date: 2024-01-05
Filing date: 2024-12-31
Publication date: 2025-07-10
Anticipated expiration: 2026-07-05

Abstract

A precursor compound for a therapeutic, FAP-targeted radioligand comprises an FAP ligand that contains a covalent warhead.

Description

Precursor and Theranostic Radiotracer with Prolonged Tumor Retention

The present invention pertains to a precursor compound and a radiotracer or radioligand for cancer diagnosis and treatment.

The precursor compound of the invention and the thereon based radioligand can comprise one, two or three ligands PL, PL', PL" that bind covalently to fibroblast activation protein (FAP) and either a chelator Ch for complexation of a radioisotope or a leaving group LR for substitution with a radioisotope.

The precursor compound of the invention can have a structure selected from the group comprising

wherein

- PL, PL', PL" independently of one another are ligands that bind covalently to fibroblast activation protein (FAP),

- Ch is a chelator for complexation of a radioisotope,

- LR is a leaving group for substitution with a radioisotope, - PM is a pharmacokinetic modulator group,

- Bl, B2, B3, SI, S2 independently of one another are absent or bivalent spacers,

- TL, TL' independently of one another are trivalent linkers, and

- TL" is a tetravalent linker. Each ligand PL, PL', PL" independently of one another can comprise a covalent warhead CW selected from the group comprising

- X = -H or -CH₃ , Y¹ = -H or -F , Y² = -H or -F ,

- V is absent or a radical selected from the group comprising

- W1 is =0 or =NH ,

- W2 is =0 or =NH ,

- W1 is =NH and W2 is =0 if V is absent or -CH2- ,

- BT is a benzotriazole radical selected from the group comprising

- ArNASA is a /V-methylJV-arylmethanesulfonamide radical selected from the group comprising

wherein - Ml is =0 or =NH₂ ,

- M2 is =O or =NH₂ ,

- M3 is -CH₃ , -OH , -NH₂ or alkyl ,

- Z¹ is absent or selected from the group comprising -F, -Cl, -Br and — N0₂ ,

- Z² is absent or selected from the group comprising -F, -Cl, -Br and — N0₂ ,

- Z³ is absent or selected from the group comprising -F, -Cl, -Br and — N0₂ , and

- ML is a malolactone radical selected from the group comprising

wherein Al is a radical of a linear or branched alkyl.

Each ligand PL, PL', PL" independently of one another can also comprise a conjugate of the covalent warhead CW with a radical Z, wherein the conjugate has a structure selected from the group comprising

wherein

Z is a radical selected from the group comprising

and a terminal carbonyl (-CO-) of Z is bound to the terminal amine (-NH-) of the covalent warhead CW.

In an expedient embodiment of the invention the precursor compound has the structure PL— SI— Ch, wherein PL is a ligand that binds covalently to FAP, SI is a bivalent spacer and Ch is a chelator for complexation of a radioisotope.

The invention employs ligands that bind covalently to fibroblast activation protein (FAP) with high specificity. In order to mitigate off-target side effects the ligands are configured to have low to moderate reactivity with non-catalytic nucleophilic (Nu) amino acids, such as serine. Accordingly, the ligands of the invention can be designated as "slow" or "latent". The reactivity of the covalent ligand of the invention can be precisely adjusted through selection of proper substituents. For example, the reactivity of covalent SuFEx warheads can be significantly reduced by fluorosulfonate or sulfonimidoyl fluoride moieties of type

This affords covalent warheads CW that are stable under physiological conditions and covalently bind to nucleophilic amino acid sidechains in the FAP catalytic cleft, such as FAP Ser⁶²⁴. The beneath scheme illustrates the covalent bond formation for amino acids lysine (Lys) and aspartic acid (Asp) with covalent warheads CW based on benzotriazole, /V-methyl-/V- arylmethanesulfonamide and malolactone, respectively. benzotriazole

N-m ethyl- /V-aryl- methane-sulfonamide

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 ¹⁸F, ⁶⁸Ga, ¹⁷⁷Lu 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 ¹⁷⁷Lu or ²²⁵Ac 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 ¹⁷⁷Lu-DOTATATE Therapy: An In Silica 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_on/k_off of kinetic rate constants k_on and k_Off for ligation with and dissociation from FAP, respectively. For small molecules, such as the radiotracers or radioligands of the invention and the prior art, k_on approaches the diffusion limit of 10⁸ M ^-1-s ^-1. For irreversibly binding radiotracers k_Off equals zero and the affinity (KD = k_on/k_off) becomes infinite. Therefore, for the radioligands of the invention, affinity or the dissociation constant KD = k_On/k_Off is not adequate as an indicator for therapeutic potency. Moreover, chemical modification of peripheral structural groups of the radioligands of the invention - apart from the characteristic FAP-ligand motifs, such as exemplified beneath - does not measurably affect their tumor retention and radiological efficacy.

Examples (a), (b), (c), (d), (e), (f) of characteristic FAP-ligand motifs comprising one of amino acids glycine or alanine, one of proline derivates 4,4-difluoropyrrolidin-2-yl-sulfurofluoridate or pyrrolidin-2-yl-sulfurofluoridate and an optional quinoline group.

Example 36 of this application further illustrates the profound impact that an irreversible radioligand has on radiological efficacy.

Examples 37 and 38 pertain to the electronic properties of SuFEx warheads and FAP ligands and provide guidance for the adaptation of their reactivity.

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 ¹⁸F and ⁶⁸Ga or ¹⁷⁷Lu. 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 ⁶⁴Ga and ¹⁷⁷Lu 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 ¹⁷⁷Lu or ²²⁵Ac 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 ⁶⁸Ga and ¹⁷⁷Lu 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 precursors of the invention. 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. Tarrago, 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.

Methods for synthesis and conjugation of benzotriazole, /V-methyl-/V-arylmethanesulfon- amide, strain-release alkylating malolactone, and derivatives thereof are described in:

- X. Xin, Y. Zhang, M. Gaetani, S.L. Lundstrbm, R.A. Zubarev, Y. Zhou, D.P. Corkery, Y.-W. Wu; Ultrafast and selective labeling of endogenous proteins using affinity-based benzotriazole chemistry; Chem. Sci., 2022, 13, 7240; https://doi.org/10.1039/dlsc05974b;

- M. Kawano, S. Murakawa, K. Higashiguchi, K. Matsuda, T. Tamura, I. Hamachi; Lysine-Reactive N -Acyl -N -aryl Sulfonamide Warheads: Improved Reaction Properties and Application in the Covalent Inhibition of an Ibrutinib-Resistant BTK Mutant; J. Am. Chem. Soc. 2023, 145, 26202-26212; https://doi.org/10.1021/jacs.3c08740;

- Q. Zheng, Z. Zhang, K.Z.Guiley, K.M. Shokat; Strain-release alkylation of Aspl2 enables mutant selective targeting of K-Ras-G12D; Nat Chem Biol. 2024 Sep; 20(9):1114-1122; doi: 10.1038/s41589-024-01565-w; https://www.nature.com/articles/s41589-024- 01565-w.

The disclosure of the above cited articles by Xin et al., Kawano et al., and Zheng et al., particularly the therewith associated supporting information in its entirety is 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 radioligands warrants further improvement. Accordingly, the present invention has the object to provide a precursor for FAP-targeted radioligands that yield higher tumor uptake, prolonged tumor retention time and increased ratio of tumor-absorbed to whole-body-absorbed radiation dose.

In particular, the invention is directed to radioligands which covalently bind to FAP with high specifity and little or no off -target loss.

The above objects are achieved by a precursor compound that comprises one, two or three ligands PL, PL', PL" that bind covalently to fibroblast activation protein (FAP), and either a chelator Ch for complexation of a radioisotope or a leaving group LR for substitution with a radioisotope. The precursor compound of the invention can have a structure selected from the group comprising

wherein

- PL, PL', PL" independently of one another are ligands that bind covalently to fibroblast activation protein (FAP), - Ch is a chelator for complexation of a radioisotope,

- LR is a leaving group for substitution with a radioisotope,

- PM is a pharmacokinetic modulator group,

- TL, TL' independently of one another are trivalent linkers, and - TL" is a tetravalent linker.

Each ligand PL, PL', PL" independently of one another can comprise a covalent warhead CW selected from the group comprising

wherein - X = -H or -CH₃ , Y¹ = -H or -F , Y² = -H or -F ,

- V is absent or a radical selected from the group comprising

- W1 is =0 or =NH ,

- W2 is =0 or =NH ,

- W1 is =NH and W2 is =0 if V is absent or -CH2- ,

- BT is a benzotriazole radical selected from the group comprising

wherein - Ml is =0 or =NH₂ ,

- M2 is =O or =NH₂ ,

- M3 is -CH₃ , -OH , -NH₂ or alkyl ,

- ML is a malolactone radical selected from the group comprising

wherein Al is a radical of a linear or branched alkyl.

Expedient embodiments of the precursor compound of the invention 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:

- ligands PL, PL', PL" independently of one another comprise a conjugate of a covalent warhead CW with a radical Z, wherein the conjugate has a structure selected from the group comprising

- Z is a radical selected from the group comprising

wherein the terminal carbonyl (-CO-) of Z is bound to the terminal amine (-NH-) of the covalent warhead CW;

- ligands PL, PL', PL" independently of one another comprise a covalent warhead CW selected from the group comprising

- W1 is =0 or =NH , and

- W2 is =0 or =NH ; ligands PL, PL', PL" independently of one another comprise a covalent warhead CW selected from the group comprising

wherein

- X = -H or -CH₃ , Y^ -H or -F , Y² = -H or -F ,

- W1 is =0 or =NH ,

- W2 is =0 or =NH , and - BT is a benzotriazole radical selected from the group comprising

- ligands PL, PL', PL" independently of one another comprise a covalent warhead selected from the group comprising

- X = -H or -CH₃ , Y^ -H or -F , Y² = -H or -F ,

- W1 is =0 or =NH ,

- W2 is =0 or =NH,and - ArNASA is a /V-methylJV-arylmethanesulfonamide radical selected from the group comprising

Ml is =Oor=NH₂, - M2 is =0 or =NH₂ ,

- M3 is -CH₃ , -OH , -NH₂ or alkyl ,

- Z¹ is absent or selected from the group comprising -F, -Cl, -Br and — N0₂ , - Z² is absent or selected from the group comprising -F, -Cl, -Br and — N0₂ , and

- Z³ is absent or selected from the group comprising -F, -Cl, -Br and -NO2 ;

wherein - X = -H or -CH₃ , Y^ -H or -F , Y² = -H or -F ,

- W1 is =0 or =NH ,

- W2 is =0 or =NH , and

- ML is a malolactone radical selected from the group comprising

wherein Al is a radical of a linear or branched alkyl;

- ligands PL, PL', PL" independently of one another comprise a radical selected from the group comprising

- the precursor compound has a structure selected from the group comprising

PL— S1— Ch PL— S1— LR

PL— S2— TL— S1— Ch PL— S2— TL— S1— LR and

PM PM

- ArNASA is a /V-methyl-/V-arylmethanesulfonamide radical selected from the group comprising

wherein

- Z¹ is absent or selected from the group comprising -F, -Cl, -Br and -NO2 ,

- Z² is absent or selected from the group comprising -F, -Cl, -Br and -NO2 , and

- Z³ is absent or selected from the group comprising -F, -Cl, -Br and -NO2 ; - X is -H ;

- Y¹ is -F and Y² is -F ;

- X is -CH₃ ;

- Y¹ is -H and Y² is -H ;

- Bl, B2, B3, SI, S2 independently of one another are bivalent alkyl spacers; - Bl, B2, B3, SI, S2 independently of one another are bivalent heteroalkyl spacers;

- Bl, B2, B3, SI, S2 independently of one another are bivalent heteroalkyl spacers with one, two, three, four, five, six, seven, eight, nine or ten substituents selected independently of one another from the group comprising

- Bl, B2, B3, SI, S2 independently of one another are bivalent heteroalkyl spacers with one, two, three, four or five substituents selected independently of one another from the group comprising

- bivalent spacers Bl, B2, B3, SI, S2 independently of one another comprise one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen or twenty methylene groups (-CH2-);

- bivalent spacers Bl, B2, B3, SI, S2 independently of one another comprise one, two, three, four, five, six, seven, eight, nine or ten methylene groups (-CH2-);

- bivalent spacers Bl, B2, B3, SI, S2 independently of one another comprise one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty-two, twenty-three, twenty-four, twenty-five, twenty-six, twenty-seven, twenty-eight, twenty-nine, thirty, thirty-one, thirty-two, thirty-three, thirty-four, thirty-five, thirty-six, thirty-seven, thirtyeight, thirty-nine or forty linearly conjugated radicals selected independently of one another from the group comprising -CH2- , — C(=O)— , -CHf-CHs)- , -CH(-CH2COOH)- , -CH=CH- , -NH- , -Nf-CHs)- , -O- , -CH2CH2O- and radicals of C4-C10 aryl or heteroaryl, substituted C4-C10 aryl or heteroaryl, C4-C10 heteroaryl, substituted C4-C10 heteroaryl, alanine, glycine, phenylalanine, arginine, histidine, proline, asparagine, isoleucine, serine, aspartic acid, leucine, threonine, cysteine, lysine, tryptophan, glutamine, methionine, tyrosine, glutamic acid, ornithine, valine, alcohols, aminoalkylcarboxylic acids;

- bivalent spacers Bl, B2, B3, SI, S2 independently of one another comprise a radical selected from the group comprising

wherein u = 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 and R' and R" are selected independently of one another from the group comprising hydrogen, alkyl, substitued alkyl, cycloalkyl, substituted cycloalkyl, heterocycloalkyl, substituted heterocycloalkyl, aryl, substituted aryl, heteroaryl and substituted heteroaryl;

- bivalent spacers Bl, B2, B3, SI, S2 independently of one another comprise a radical having the structure

wherein

- each P' 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 Q^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;

- each P^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(CH₃)-, -O-,

-S-, -C(O)- and -C(CH₃)- ;

- each Q^l 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(CH₃)-, -O-, -S-, -C(O)- and -C(CH₃)- ; - T is absent or a radical selected from the group comprising

- bivalent spacers Bl, B2, B3, SI, S2 independently of one another comprise a radical having the structure -[CH₂]_P- with p = 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20;

- bivalent spacers Bl, B2, B3, SI, S2 independently of one another comprise a radical having the structure -(NH)-[CH₂]_P- with p = 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20;

- bivalent spacers Bl, B2, B3, SI, S2 independently of one another comprise a radical having the structure -(NH)-[CH₂]_P-(NH)- with p = 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20;

- bivalent spacers Bl, B2, B3, SI, S2 independently of one another comprise a radical having the structure -[CH₂CH₂O]_P- with p = 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20;

- bivalent spacers Bl, B2, B3, SI, S2 independently of one another comprise a radical having the structure -(N H)-[ CH₂CH₂O]_P- with p = 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20;

- bivalent spacers Bl, B2, B3, SI, S2 independently of one another comprise a radical having the structure -(N H)-[ CH₂CH₂O]_P-(NH)- with p = 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20;

- bivalent spacers Bl, B2, B3, SI, S2 independently of one another comprise a radical of ethylene diamine having the structure

- bivalent spacers Bl, B2, B3, SI, S2 independently of one another comprise a radical 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, He, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, Vai, Pyl, Sec, GABA or y-Aminobutyric acid, Homoserine, DOPA or 3,4-Dihydroxyphenylalanine, Citrulline, P-Alanine and Thyroxine;

- bivalent spacers Bl, B2, B3, SI, S2 independently of one another comprise a naphthol radical having the structure

wherein I = 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20;

- bivalent spacers Bl, B2, B3, SI, S2 independently of one another comprise a phenylalanine radical;

- bivalent spacers Bl, B2, B3, SI, S2 independently of one another comprise a radical of N,N-dimethylarginine; - bivalent spacers Bl, B2, B3, SI, S2 independently of one another comprise a radical having the structure

- the pharmacokinetic modulator group PM comprises one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen or twenty conjugated radicals selected independently of one another from the group comprising -CH2- , — C(=O)— , -CHf-CHs)- , -CH(-CH2COOH)- , -CH=CH- , -NH- , -Nf-CHs)- , - O- , -CH2CH2O- and radicals of C4-C10 aryl or heteroaryl, substituted C4-C10 aryl or heteroaryl, C4-C10 heteroaryl, substituted C4-C10 heteroaryl, alanine, glycine, phenylalanine, arginine, histidine, proline, asparagine, isoleucine, serine, aspartic acid, leucine, threonine, cysteine, lysine, tryptophan, glutamine, methionine, tyrosine, glutamic acid, ornithine, valine, alcohols, aminoalkylcarboxylic acids; trivalent linkers TL, TL' independently of one another are radicals selected from the group comprising radicals of an alkyl, substituted alkyl, heteroalkyl, substituted heteroalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alcohol, aminoalkylcarboxylic acid, dipeptide and tripeptide; trivalent linkers TL, TL' independently of one another comprise a radical selected from the group comprising

the tetravalent linker TL" is a radical selected from the group comprising radicals of an alkyl, substituted alkyl, heteroalkyl, substituted heteroalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alcohol, aminoalkylcarboxylic acid, dipeptide, tripeptide, and tetrapetide;

- the tetravalent linker TL" comprises a radical selected from the group comprising

- Ch comprises a radical selected from the group comprising

wherein Fl is -OH or -NH₂, F2 is -OH or -NH₂, F3 is -OH or -NH₂, F4 is -OH or -NH₂ ;

- Ch comprises a structure selected from the group comprising structures (I), (II), (III), (IV), (V) and (VI) with

- Ch comprises a structure selected from the group comprising structures (VII), (VIII), (IX) and (X) with

- Ch comprises a structure selected from the group comprising structures (XI), (XII), (XIII) and (XIV) with

(XIII) (XIV)

- Ch is a radical of DOTAM (l,4,7,10-Tetrakis(carbamoylmethyl)-l,4,7,10-tetraazacyclo- dodecane), DOTAM-mono-acid (l,4,7,10-Tetraazacyclododecane-l,4,7-tri(carbamoyl- methyl)-10-acetic acid) or DOTAM-bis-acid (l,4,7,10-Tetraazacyclododecane-l,7- bis(acetate)-4,10-bis(acetamide) );

- Ch comprises a radical selected from the group comprising

wherein Fl is -OH or -NH₂, F2 is -OH or -NH₂, F3 is -OH or -NH₂, F4 is -OH or -NH₂, and at least one of Fl, F2 and F3 is -NH₂ or at least one of Fl, F2, F3 and F4 is -NH₂ ;

- the precursor compound comprises a chelator Ch having the structure

wherein D¹ is H, CH3 or NH2 ; - the precursor compound comprises a chelator radical selected from the group comprising

- Ch is a chelator selected from the group comprising H4pypa, 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)-phosphinic acid]-7-[meth- ylene-(2-carboxyethyl)-phosphinic acid]), DOTP^H (1,4,7,10-tetraazacyclododecane- l,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 H2dedpa (l,2-[[6-(Carboxyl)pyridine-2-yl]methylamine]ethane) and l-Uoctapa (l,2-[[6-(Carboxyl)pyridine-2-yl]methylamine]ethane-N,N'-diacetate), DFO (Deferoxamine) and derivatives thereof, Trishydroxypyridinone (THP) and derivatives thereof, such as HsTHP-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-^hSAR (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 N4-derivates, PnAO (6-(4-lsothiocyanatobenzyl)-3,3,9,9,-tetramethyl-4,8-diaza-undecane-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;

- the leaving group LR for substitution with a radioisotope is selected from the group comprising dinitrogen, dialkyl ether, perfluoroalkylsulfonates, triflate, iodide, tosylates, mesylates, sulfonates, bromide, hydrogen, alcohols, chloride, nitrate, phosphate, inorganic esters, thioether, amines, ammonia, fluoride, carboxylate, phenoxides, hydroxide, alkoxides, amides, hydride, arenide, alkanide and sulfur fluorides;

wherein W1 is =NH and W2 is =0 if V is absent or -CH2- ;

wherein V is =CH- or =N- , W1 is =0 or =NH , and W2 is =0 or =NH ;

- ligands PL, PL', PL" independently of one another comprise a radical having the structure

The invention has the further object to provide a radioligand for cancer diagnosis and treatment. This object is achieved through a radioligand comprised of any the above described precursor compounds including a chelator Ch and a therewith complexed radioisotope or radioactive compound selected from the group comprising ⁴⁴Sc, ⁴⁷Sc, ⁵⁵Co, ⁶²Cu, ⁶⁴Cu, ⁶⁷Cu, ⁶⁶Ga, ⁶⁷Ga, ⁶⁸Ga, ⁸⁹Zr, ⁸⁶Y, ⁹⁰Y, ⁹⁰Nb, ^mln, ¹³⁵Sm, ¹⁴⁰Pr, ¹⁵⁹Gd, ¹⁴⁹Tb, ¹⁶⁰Tb, ¹⁶¹Tb, ¹⁶⁵Er, ¹⁶⁶Dy, ¹⁶⁶Ho, ¹⁷⁵Yb, ¹⁷⁷Lu, ²¹²Pb, ²¹³Bi, ²²⁵Ac and ¹⁸FAI, or through a radioligand comprised of any of the above described precursor compounds wherein a leaving group LR is substituted with ¹⁸F, ¹³¹l or ²¹¹At.

Expedient embodiments of the radioligand of the invention 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 ⁶⁸Ga;

- the radioisotope is ¹⁷⁷Lu;

- the radioisotope is ²²⁵Ac;

- the radioisotope is ²¹²Pb;

- the radioactive compound is ¹⁸FAI (aluminum fluoride);

- the radioligand 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 ⁴⁴Sc, ⁴⁷Sc, ⁵⁵Co, ⁶²Cu, ⁶⁴Cu, ⁶⁷Cu, ⁶⁶Ga, ⁶⁷Ga, ⁶⁸Ga, ⁸⁹Zr, ⁸⁶Y, ⁹⁰Y, ⁹⁰Nb, ^mln, 1³⁵Sm, ¹⁴⁰Pr, ¹⁵⁹Gd, ¹⁴⁹Tb, ¹⁶⁰Tb, ¹⁶¹Tb, ¹⁶⁵Er, ¹⁶⁶Dy, ¹⁶⁶Ho, ¹⁷⁵Yb, ¹⁷⁷Lu, ²¹²Pb, ²¹³Bi and ²²⁵Ac;

- the radioligand 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 ⁶⁸Ga, ¹⁷⁷Lu, ²¹²Pb and ²²⁵Ac;

- the radioligand comprises a chelator having the structure (XI), (XII), (XIII) or (XIV) and therewith complexed radioactive compound ¹⁸FAI (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 (Ser⁶²⁴) 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 fluorine atom of the SuFEx FAP ligand of the present invention acts as leaving group when situated adajacent to FAP's catalytic amino acid Ser⁶²⁴, such that upon deprotonation of the Ser⁶²⁴ hydroxy group a permanent covalent bond is formed between the FAP ligand of the invention and Ser⁶²⁴.

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 NH2-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 FAP ligands of the invention is illustrated beneath in Example 1. Auxiliary methods for covalent bond formation between the linker L and a chelator or the FAP ligand of the present invnetion 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.

In the present invention

- the term "radical" refers to a monovalent, bivalent, trivalent or multivalent atom, molecule, residue, chemical group, chemical unit, chemical structure or chemical moiety that is covalently coupled to or covalently conjugated with one, two, three or more radicals of atoms, molecules, chemical groups, chemical units, chemical structures or chemical moieties of the same or different types;

- the terms "sulfur fluoride exchange group", "SuFEx warhead" and "sulfur fluoride radical" are used interchangeably and refer to a chemical unit comprising a radical of type

- radicals can be conjugated by one, two, three or more single covalent bonds, each with two shared electrons, or by one, two, three or more double covalent bonds, each with four shared electrons;

- radicals of atoms, molecules, chemical groups or chemical units are specified by structural formulas or by letters, digits, brackets, hyphens and equal signs;

- unless otherwise indicated, the symbols C, F, H, N and S have their usual meaning according to standard chemical notation and refer to a carbon, fluorine, hydrogen, nitrogen or sulfur atom or radical;

- radicals can also be denoted by symbols and hyphens, for example -NH- or -NFh for amine radicals, -CH2- for methylen radicals, and -CO- , — C(O)— , -C=O- , -O=C- , — C(=O)— or — O(=C)— for carbonyl radicals, wherein a terminal hyphen corresponds to one shared electron of a single covalent bond or one "half" of a single covalent bond, and a terminal equal sign corresponds to two shared electrons of a double covalent bond or one "half" of a double covalent bond;

- the term "benzotriazole" is also used for derivatives of benzotriazole;

- the term "/V-methyl-ZV-arylmethanesulfonamide" is also used for derivatives of /V-methyl- /V-arylmethanesulfonamide;

- the terms "radioligand" and "radiotracer" have the same meaning and are used interchangeably.

EXAMPLES

Schemes la-lc illustrate the synthesis of FAP ligands. Benzyl 2-hydroxypyrrolidine-l-carboxy- late (CAS No. 69261-54-7) or alternatively tert-Butyl 2-hydroxypyrrolidine-l-carboxylate (CAS No. 84766-91-6) and N-(4-acetamidophenyl)-N-fluorosulfonyl-sulfamoyl fluoride (CAS No. 2172794-56-6 ) are commercially available. Similar applies regarding commercial alternatives for the quinoline group.

6-Methoxyquinoline-4-carboxylic acid tert-Butyl (4-bromoquinolin-6-yl)carbamate

CAS No. 86-68-0 CAS No. 1260784-05-1

Scheme la: Synthesis of (S)-l-(aminoacetyl)pyrrolidin-2-yl-sulfurofluoridate (a) THF, DBU (l,8-diazabicyclo[5.4.0]undec-7-ene), 10 min, 20 °C, H2O quench, EtOAc extraction, 12%;

(b) HBr, AcOH, MeCN, Dowex-CI, 90%.

Scheme lb: Synthesis of 6-methoxyquinoline-4-carboxylic acid (a) PBr₂, DMF, 3 h, 63%;

(b) Zn(CN)₂, Pd/C, Zn⁺⁺(COO-)₂, dppf, 110°C, 3 h, 72%; (c) NaOH, H₂O/EtOH, reflux, 94%.

Scheme lc: Conjugation of (S)-l-(aminoacetyl)pyrrolidin-2-yl-sulfurofluoridate 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; Focile one- pot synthesis of sulfonyl fluorides from sulfonates or sulfonic acids; RSC Adv., 2019, 9, 13863; doi: 10.1039/c9ra02531f).

Example 3: Amide bond formation

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

Scheme 2: 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. Bostrbm; 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 3 follows:

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

R₁ = aryl , R₂, R₃ = alkyl or aryl

Scheme 3: I ron(l I l)-catalyzed etherification of two different alcohols

Secondary alcohol (0.5 mmol), primary alcohol (0.5 mmol), Fe(OTf)s (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(NOs)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. 1161415-28-6 CAS No. 438553-50-3

Scheme 4: Chelator building blocks Example 6: DATA^5m Prochelator Synthesis

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

Scheme 5: Synthesis of 3‘Bu-protected DATA^5m prochelator (i) Amberlyst-21, EtOH; (ii) CH₂O, EtOH; (iii) CH₃COOH, Pd(OH)₂/C, H₂, EtOH; (iv) BrCH₂COO‘Bu, K₂CO₃, MeCN; (v) CH₃I, K₂CO₃, DCM : MeCN; (vi) LiOH, THF : H₂O Examples 6-25: Precursor Compounds of the Invention

Schemes 6-25 depict exemplary embodiments 6-25 of precursor compounds according to the invention.

Scheme 6: Exemplary precursor compound 6

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 13: Exemplary precursor compound 13

Scheme 14: Exemplary precursor compound 14

Scheme 15: Exemplary precursor compound 15

Scheme 16: Exemplary precursor compound 16

Scheme 17: Exemplary precursor compound 17

Scheme 19: Exemplary precursor compound 19

Scheme 20: Exemplary precursor compound 20

Scheme 21: Exemplary precursor compound 21

Scheme 22: Exemplary precursor compound 22

Scheme 23: Exemplary precursor compound 23

Scheme 24: Exemplary precursor compound 24

Scheme 25: Exemplary precursor compound 25

Scheme 26: Exemplary precursor compound 26

Scheme 27: Exemplary precursor compound 27

Scheme 28: Exemplary precursor compound 28

Scheme 29: Exemplary precursor compound 29

Scheme 30: Exemplary precursor compound 30

Scheme 31: Exemplary precursor compound 31

Scheme 34: Exemplary precursor compound 34

Example 35: 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 precursor compound of Scheme 7; (b) the prior art precursor compound FAPI-46 (CAS No. 2374782-04-2; GSRS UNII 59QC5DY68A); and as reference (c).

Scheme 35: FAPI-46

About 160 pL of 2 pM solutions of the precursor compound of Scheme 7 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 (X_ex = 380 nm, Xem = 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 precursor compound of Scheme 7 binds irreversibly to FAP.

Example 36: Irreversible Radioligand Efficacy

For PSMA-617 (Pluvicto®) Begum et al. (N.J. Begum, G. Glatting, H.-J. Wester, M. Eiber, AJ. Beer, P. Kletting; The effect of ligand amount, affinity and internalization on PSMA-targeted imaging and therapy: A simulation study using a PBPK model; Nature Scientific Reports (2019) 9:20041; https://doi.org/10.1038/s41598-019-56603-8) cite a dissociation constant (affinity) of KD = k_off/k_on = 0.06 nM . For small molecules, such as PSMA-617, the association rate k_on ~ 10⁷ L-mol ^-1-s ¹ is mainly determined by diffusion. From k_Off = KD-k_on results a value of k_Off = 0.036 min ¹ , which is 497 times faster than the ¹⁷⁷Lu decay constant T = In2 / 6.647 d = 7.241-10’⁵ min ¹ . Accordingly, an irreversible radioligand with k_Off = 0 is 497 times more effective than PSMA-617. Known FAP radioligands have a dissociation constant KD (affinity) of KD > 89 pM and dissociate about 1.5 times faster from their target receptor compared to PSMA-617 (A. Simkova, T. Ormsby, N. Sidej, L. Postova Slavetinska, J. Brynda, J. Beranova, P. Sacha, P. Majer, J. Konvalinka; Structure-activity relationship and biochemical evaluation of novel fibroblast activation protein and prolyl endopeptidase inhibitors with a-ketoamide warheads; European Journal of Medicinal Chemistry 224 (2021) 113717; https://doi.Org/10.1016/j.ejmech.2021.113717). Therefore, an irreversible FAP radioligand is 737 times more effective than the most affine prior art FAP radioligand. Fig. 1 illustrates the relation between radioligand dissociation from FAP and the radiocative decay of ¹⁷⁷Lu. Example 37: Partial Charges of Sulfur Fluoride Exchange (SuFEx) Warheads and FAP Ligands

Table 1 lists calculated partial charges for various SuFEx warheads and FAP ligands. The calculations were performed using the algorithm provided by Racek et al. (T. Racek, O. Schindler, D. Tousek, V. Horsky, K. Berka, J. Koca, R. Svobodova; Atomic Charge Calculator II: web-based tool for the calculation of partial atomic charges; Nucleic Acids Research, Volume 48, Issue Wl, 02 July 2020, Pages W591-W596; https://doi.org/10.1093/nar/gkaa367)

Scheme 37 illustrates the substituent designation in Table 1.

Scheme 37: Substituent designation in Tables 1 and 2; V = absent, CH₂, O, NH or CO ; Wl = O or NH ; W2 = O or NH ; Y¹ = H or F ; Y² = H or F

Table 1: Partial charges of SuFEx warheads and FAP ligands

Example : Hammett Constants of Sulfur Fluoride Exchange (SuFEx) Warheads and FAP Ligands

Table 2 lists calculated Hammett constants for various SuFEx warheads and FAP ligands relative to fluorine. The calculations were performed using the algorithm provided by Ertl (P.

Ertl; A Web Tool for Calculating Substituent Descriptors Compatible with Hammett Sigma Constants**; Chemistry-Methods 2022, 2, e202200041; https://doi.org/10.1002/cmtd. 202200041). Scheme 28 illustrates the substituent designation in Table 2. The SMILES strings contain [R] as fluorine reference. Table 2: Hammett constants of SuFEx warheads and FAP ligands

Claims

1. A precursor compound for a radioligand having a structure selected from the group comprising

PL— S1 — Ch PL— S1 — LR

PL— B1 PL— Bl

TL'— S1 — Ch TL'— S1 — LR / /

PL'— B2 PL'— B2

wherein - Ch is a chelator for complexation of a radioisotope,

- LR is a leaving group for substitution with a radioisotope,

- PM is a pharmacokinetic modulator group,

- TL, TL' independently of one another are trivalent linkers, - TL" is a tetravalent linker, - PL, PL', PL" are ligands that bind covalently to fibroblast activation protein (FAP) and independently of one another comprise a covalent warhead CW selected from the group comprising

wherein - X = -H or -CH₃ , Y¹ = -H or -F , Y² = -H or -F ,

- V is absent or a radical selected from the group comprising

- W1 is =0 or =NH ,

- W2 is =0 or =NH ,

- W1 is =NH and W2 is =0 if V is absent or -CH2- ,

- BT is a benzotriazole radical selected from the group comprising

- ArNASA is a /V-methy-N-arylmethanesulfonamide radical selected from the group comprising

wherein - Ml is =0 or =NH₂ ,

- M2 is =O or =NH₂ ,

- M3 is -CH₃ , -OH , -NH₂ or alkyl ,

- ML is a malolactone radical selected from the group comprising

wherein Al is a radical of a linear or branched alkyl.

2. The precursor compound of claim 1, characterized in that PL, PL', PL" independently of one another comprise a radical Z selected from the group comprising

and a terminal carbonyl (-CO-) of Z is bound to a terminal amine (-NH-) of the covalent warhead CW.

3. The precursor compound of claim 1 or 2, characterized in that PL, PL', PL" independently of one another comprise a radical selected from the group comprising

4. The precursor compound of claim 1, 2 or 3, characterized in that it has the structure

PL— SI— Ch or PL-S1-LR

5. The precursor compound of any one of claims 1 to 4, characterized in that it

6. The precursor compound of any one of claims 1 to 5, characterized in that it

7. The precursor compound of any one of claims 1 to 6, characterized in that the bivalent spacers Bl, B2, B3, SI, S2 independently of one another comprise one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty-two, twenty-three, twenty- four, twenty-five, twenty-six, twenty-seven, twenty-eight, twenty-nine, thirty, thirty-one, thirty-two, thirty-three, thirty-four, thirty-five, thirty-six, thirty-seven, thirty-eight, thirty- nine or forty linearly conjugated radicals selected independently of one another from the group comprising -CH₂- , -C(=O)- , -CH(-CH₃)- , -CH(-CH₂COOH)- , -CH=CH- , -NH- , -Nj-CHs)- , - O- , -CH₂CH₂O- and radicals of C4-Cw aryl or heteroaryl, substituted C4- Cw aryl or heteroaryl, C4-C10 heteroaryl, substituted C4-C10 heteroaryl, alanine, glycine, phenylalanine, arginine, histidine, proline, asparagine, isoleucine, serine, aspartic acid, leucine, threonine, cysteine, lysine, tryptophan, glutamine, methionine, tyrosine, glutamic acid, ornithine, valine, alcohols, aminoalkylcarboxylic acids.

8. The precursor compound of any one of claims 1 to 6, characterized in that the bivalent spacers Bl, B2, B3, SI, S2 independently of one another comprise a radical having the structure

wherein - each P' is present for 1 ≤ i ≤ k and absent for i > k with k = 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

-S-, -C(O)- and -C(CH₃)- ;

9. The precursor compound of any one of claims 1 to 8, characterized in that the trivalent linker TL and TL' independently of one another are radicals selected from the group comprising radicals of an alkyl, substituted alkyl, heteroalkyl, substituted heteroalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alcohol, aminoalkylcarboxylic acid, dipeptide and tripeptide, or

TL and TL' independently of one another comprise a radical selected from the group comprising

10. The precursor compound of any one of claims 1 to 9, characterized in that it comprises a chelator Ch selected from the group comprising

wherein Fl is -OH or -NH₂, F2 is -OH or -NH₂, F3 is -OH or -NH₂, F4 is -OH or -NH₂ .

11. The precursor compound of any one of claims 1 to 9, characterized in that it comprises a chelator Ch selected from the group comprising

wherein D¹ is H, CH3 or NH2 .

12. The precursor compound of any one of claims 1 to 9, characterized in that it comprises a chelator Ch selected from the group comprising H4pypa, 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)-phosphinic acid]-7- [methylene-(2-carboxyethyl)-phosphinic acid]), DOTP^H (1,4,7,10-tetraazacyclododecane- l,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 H2dedpa (l,2-[[6-(Carboxyl)pyridine-2-yl]methylamine]ethane) and H4octapa (l,2-[[6-(Carboxyl)pyridine-2-yl]methylamine]ethane-N,N'-diacetate), DFO (Deferoxamine) and derivatives thereof, Trishydroxypyridinone (THP) and derivatives thereof, such as HsTHP-Ac and HsTHP-mal (YM103), TEAP (Tetraazycyclodecane- phosphinic acid) and derivatives thereof, Sarcophagin SAR (l-N-(4-aminobenzyl)- 3,6,10,13,16,19-hexaazabicyclo[6.6.6]-eicosan-l,8-diamine) and derivatives thereof, such as (Nf^hSAR (l,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 N4-derivates, PnAO (6-(4-lsothiocyanatobenzyl)-3,3,9,9,-tetramethyl-4,8-diaza- undecane-2,10-dione-dioxime) and derivatives thereof, such as BMS181321 (3,3'-( 1,4- 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.

13. A radioligand comprising the precursor compound of any one of claims 1 to 12 and a therewith complexed radioisotope or radioactive compound selected from the group comprising ⁴⁴Sc, ⁴⁷Sc, ⁵⁵Co, ⁶²Cu, ⁶⁴Cu, ⁶⁷Cu, ⁶⁶Ga, ⁶⁷Ga, ⁶⁸Ga, ⁸⁹Zr, ⁸⁶Y, ⁹⁰Y, ⁹⁰Nb, ^mln, 1³⁵Sm, ¹⁴⁰Pr, ¹⁵⁹Gd, ¹⁴⁹Tb, ¹⁶⁰Tb, ¹⁶¹Tb, ¹⁶⁵Er, ¹⁶⁶Dy, ¹⁶⁶Ho, ¹⁷⁵Yb, ¹⁷⁷Lu, ²¹²Pb, ²¹³Bi, ²²⁵Ac and ¹⁸FAI.

14. A radioligand comprising the precursor compound of any one of claims 1 to 9 wherein the leaving group LR is substituted with a radioisotope selected from the group comprising ¹⁸F, ¹³¹l and ²¹¹At.