Cancer-associated protein-targeted strong or covalently binding precursor compounds and radiotracers

Abstract

A precursor compound and a radioligand for cancer diagnosis and treatment comprises a high-affinity ligand PL for a cancer- associated protein conjugated to a covalent warhead CW for strong or covalent binding to an amino acid sidechain of the cancer-associated protein and a chelator Ch for complexation of a radioisotope or a leaving group LR for substitution with a radioisotope, wherein the covalent warhead CW is configured for quasi-covalent ionic binding using a sulfonic acid group or for covalent binding using a sulfur fluoride exchange (SuFEx) group, a benzotriazole or N-methyl-N- arylmethanesulfonamide leaving group or a malolactone group for strain-release alkylation.

Description

[0001] CANCER-ASSOCIATED PROTEIN-TARGETED STRONG OR COVALENTLY BINDING PRECURSOR COMPOUNDS AND RADIOTRACERS

[0002] The present invention pertains to a precursor compound and a radiotracer or radioligand for cancer diagnosis and treatment. The precursor compound of the invention comprises a high- affinity ligand PL for a cancer-associated protein conjugated to a covalent warhead CW for strong or covalent binding to an amino acid sidechain of the cancer-associated protein, and a chelator Ch for complexation of a radioisotope or a leaving group LR for substitution with a radioisotope. Prior to ligation to the cancer-associated protein, the covalent warhead CW is practically inert against hydrolysis under physiological conditions.

[0003] The ligand PL is configured to bind with high affinity to a cancer-associated protein selected from the group comprising fibroblast activation protein (FAP), prostate specific membrane antigen (PSMA), somatostatin receptor SSTR1, somatostatin receptor SSTR2, somatostatin receptor SSTR3, somatostatin receptor SSTR4, somatostatin receptor SSTR5, chemokine receptor CXCR4, nectin cell adhesion molecule 4 (nectin4), fibroblast growth factor receptor 1 (FGFR1 or CD331), fibroblast growth factor receptor 2 (FGFR2 or CD332), fibroblast growth factor receptor 3 (FGFR3 or CD333), fibroblast growth factor receptor 4 (FGFR4 or CD334), fibroblast growth factor receptor-like 1 (FGFR6) and integrin receptors.

[0004] The covalent warhead CW is configured for

[0005] - quasi-covalent ionic binding using a sulfonic acid group, or

[0006] - covalent binding using

[0007] - a sulfur fluoride exchange (SuFEx) group with fluorine (F“) leaving group,

[0008] - electrophiles with benzotriazole or / V-methyl- / V-arylmethanesulfonamide leaving group, or

[0009] - for strain-release alkylation using a malocatone group.

[0010] The covalent warhead of the invnetion can comprise one, two, three or more radicals selected from the group comprising radicals of derivatives and analogues of sulfonic acid, SuFEx groups, benzotriazole, / V-methyl- / V-arylmethanesulfonamide, and malolactone.

[0011] The present invention is partly inspired by the concept of targeted covalent inhibitors (TCI; https: / / en.wikipedia.org / wiki / Targeted_covalent_inhibitors). Prominent examples of TCI pharmaceuticals are aspirin, penicillin, afatinib, rociletinib, omeprazole, clopidogrel and ibrutinib. TCIs are rationally designed inhibitors that bind and then bond to their target proteins. They possess a bond-forming functional group of low chemical reactivity that, following binding to the target protein, is positioned to react rapidly with a proximate nucleophilic residue at the target site to form a covalent bond. Alternatively, the low reactivity functional group can be configured as ionic moiety, in particular sulfonic acid.

[0012] Transient inhibitor ligation combined with strong ionic or covalent binding leads to the formation of a long-lived or irreversible protein-TCI complex. The probability of forming a stable protein-TCI complex increases with the affinity of the inhibitor ligand PL for the target protein.

[0013] The precursor or radioligand of the invention can comprise a high-affinity inhibitor ligand for fibroblast activation protein (FAP) or prostate-specific membrane antigen (PSMA). FAP inhibitor ligands can be based on UAMC1110, i.e. A / -[2-[(2S)-2-cyano-4,4-difluoropyrrolidin- l-yl]-2-oxoethyl]quinoline-4-carboxamide, which has a dissociation constant KD of about 3 nM, or ketoamide derivatives thereof with KD = 0.089 nM. PSMA inhibitor ligands can be based on the lysine-urea-glutamate (KuE) motif and have a dissociation constant KD of about 0.06 nM. Respective inhibitors are described in WO 2013 / 107820 Al, WO 2021 / 197519 Al and WO 2018 / 108287 Al.

[0014] The precursor compound of the present invention can have the structure

[0015] wherein

[0016] - R is a radical of a substituted or unsubstituted alkyl, a substituted or unsubstituted Ce aryl, or a substituted or unsubstituted a Ikyl-a ryl, or a radical having the structure - Y1is H or F;

[0017] - Y2is H or F;

[0018] - G is -NH-C(=0)-(Ce-Cio)-aryl- or -NH-C(=0)-(Ce-Cio)-heteroaryl- with 1, 2 or 3 nitrogen substituents;

[0019] - S2 is absent or a bivalent spacer; - Ch is a chelator for a radioisotope;

[0020] - S3 is absent or a bivalent spacer;

[0021] - TL is absent or a trivalent linker; with the proviso (a), (b) or (c) that

[0022] (a) XI is -S1-O=C-LG , -S1-U-V-(W1=S=W2)-F or -S1-U-S(=O)2OH and X2, S3, TL, X3 are absent, or

[0023] (b) X2 is -S1-O=C-LG , -S1-U-V-(W1=S=W2)-F or -S1-U-S(=O)2OH and XI is absent, -H or -CH3 , and S3, TL, X3 are absent, or (c) X3 is -S1-O=C-LG , -S1-U-V-(W1=S=W2)-F or -S1-U-S(=O)2OH and XI is absent, -H or -CH3 , and X2 is absent, wherein

[0024] - SI is absent or a bivalent spacer, - LG is a benzotriazole or / V-methyl- / V-arylmethanesulfonamide leaving group, or LG is strain-release alkylating malolactone,

[0025] - U is absent, a substituted or unsubstituted alkyl or heteroalkyl, or a substituted or unsubstituted C4 to C10 aryl or heteroaryl,

[0026] - V is absent, -CH2- , =CH- , -O- , -NH- or -C(=O)- , - W1 is =0 or =NH , and

[0027] W2 is =0 or =NH .

[0028] In expedient embodiments of the invention, the precursor compound or radioligand comprises a radical having the structure

[0029] In expedient embodiments of the invention the leaving group LG is a benzotriazole or / V-methyl- / V-arylmethanesulfonamide radical selected from the group comprising

[0030]

[0031] - Z1is absent or selected from the group comprising -F, -Cl, -Br and -NO2 ;

[0032] - Z2is absent or selected from the group comprising -F, -Cl, -Br and -NO2 ;

[0033] - Z3is absent or selected from the group comprising -F, -Cl, -Br and -NO2 .

[0034] In expedient embodiments of the invention the covalent warhead CW comprises a malolactone radical selected from the group comprising wherein Al is a radical of a linear or branched alkyl.

[0035] Upon ligation of the precursor compound or thereon based radioligand of the invention with a cancer-associated protein the covalent warhead CW is located proximal to an accessible sidechain of an amino acid, such as aspartic acid (Asp, D), cysteine (Cys, C), glutamic acid (Glu, E), asparagine (Asn, N), lysine (Lys, K), histidine (His, H), serine (Ser, S), threonine (Thr, T) or tyrosine (Tyr, Y) which promotes nucleophilic attack. Depending on the chemical configuration of the covalent warhead CW, nucleophilic attack leads to abstraction of a benzotriazole, / V-methyl- / V-arylmethanesulfonamide or fluorine leaving group and subsequent formation of a covalent bond or strain-release alkylation with malolactone. Thereby the precursor compound or radioligand of the invention is irreversibly attached to the cancer-associated protein. This allows for prolonged retention in tumor tissue with increased expression of the cancer-associated protein.

[0036] 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 / V-met y - / V-ary - methane-sulfonamide

[0037] The reactivity of the covalent warhead CW of the invention can be precisely adjusted through selection of proper substituents. For example, the reactivity of covalent SuFEx warheads can be susbtantially 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 vicinity of the ligation site of the targeted cancer-associated protein, such as FAP or PSMA. Covalent binding occurs due to the so-called proximity effect and can be understood in terms of increased collision probability between the covalent warhead and nucleophilic amino acid sidechains during transient or reversible ligation of the precursor or radioligand of the invention to the targeted cancer- associated protein. The increased reaction probability can also be explained by the locally elevated reactand concentration.

[0038] 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 radioligands. In particular, FAP-targeted cancer drugs are equipped with a suitable cytotoxin or radioistope such as18F,68Ga,177Lu or225Ac.

[0039] 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 pharmaceutical compounds of the present invention.

[0040] 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 or POP) 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.

[0041] In order to target CAFs a drug or radioligand 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, particularly konfor ligation and kOff for dissociation. According to Jimenez-Franco et al., the tumor release rate - or conversely the tumor retention - of a therapeutic radioligand comprising a radioisotope, such as177Lu or225Ac 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 in177Lu-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 radioligand remains in the tumor tissue, the greater its therapeutic effectiveness.

[0042] Accordingly, the prior art endeavors to improve the therapeutic efficacy of radioligands 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 via radio dosimetry of excised tissue. Contrary thereto, affinity is quantified in vitro by enzymatic assay methods and expressed as ratio on / off of kinetic rate constants konand kOff for ligation with and dissociation from FAP, respectively. For small molecules, such as prior art radioligands and those of the present invention, konapproaches the diffusion limit of 108M^-s1. For irreversibly binding radioligands, such as the radioligands of the invention, kOff equals zero and affinity ( kOn / kOff) becomes infinite. Therefore, for the radioligand of the invention affinity is not a meaningful measure for tumor retention and therapeutic potency.

[0043] Examples of FAP and PSMA inhibitor ligands according to the invention are shown beneath

[0044] Examples (a), (b) of FAP ligands with X = H or CH3 , Y1= H or F , Y2= H or F, and examples (c), (d) of PSMA ligands.

[0045] Small molecule inhibitors and substrates 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.

[0046] Theranostic radiopharmaceuticals or radioligands are comprised of a precursor compound and a therewith conjugated or complexed radioisotope such as18F and68Ga or177Lu. The precursor compound comprises a ligand for a relevant cellular receptor such as somatostatin receptor 2 (SSR2), prostate specific membrane antigen (PSMA) or fibroblast activation protein (FAP).

[0047] For labeling with radioisotopes such as64Ga and177Lu the precursor compound can also include a chelator Ch, such as l,4,7,10-tetraazacyclododecane-l,4,7,10-tetraacetic acid (DOTA) or 6-amino-l,4-diazepine-triacetic acid (DATA).

[0048] For cancer radioendotherapy or radioligand therapy (RLT), radioligands comprising a highly ionizing |3- or a-emitter such as177Lu or225Ac with half-life (ti / 2) of 6.7 and 9.9 days, respectively, constitute promising treatment modalities.

[0049] Prostate cancer is the most common cancer and the third deadliest cancer among men in developed countries. Tumor growth progresses slowly in this disease and the 5-year survival rate when diagnosed early is almost 100%. However, if the disease is only discovered after the tumor has metastasized, the survival rate drops significantly. Taking action against a prostate tumor too early and too aggressively can significantly impact the patient's quality of life. For example, surgical removal of the prostate can lead to incontinence and impotence. A reliable diagnosis and information about the stage of the disease are essential for successful treatment and a high quality of life for the patient. A widely used diagnostic method, in addition to medical palpation of the prostate, is the determination of tumor markers in the patient's blood. The most noticeable marker for prostate cancer is the serum level of prostate-specific antigen (PSA). However, the significance of the PSA serum level is controversial because patients with slightly elevated values often do not have prostate cancer, but 15% of patients with prostate cancer do not have an elevated PSA blood concentration. Another target structure for the diagnosis of prostate tumors is the prostatespecific membrane antigen (PSMA). Unlike PSA, PSMA cannot be detected in the blood. It is a membrane-bound glycoprotein with enzymatic activity. It cleaves C-terminal glutamate from N-acetyl-aspartyl-glutamate (NAAG) and folic acid (poly)-y-glutamate. PSMA is rare in normal tissue but is highly overexpressed in prostate cancer cells, with a close correlation of expression with tumor stage. Lymph node metastases and bone metastases from prostate cancer also show PSMA-expression to an extent of 40%. Various radioligands comprising one or more PSMA or FAP inhibitor-homing-ligands conjugated with a chelator such as DOTA or DATA for complexation of radioisotopes such as68Ga and177Lu are known in the prior art.

[0050] Banerjee et al. propose multivalent radioligand compounds comprising a DOTA chelator and two or more therewith conjugated PSMA inhibitor ligands (cf. S.R. Banerjee, M.

[0051] Pullambhatla, H. Shallal, A. Lisok, R.C. Mease, M.G. Pom per; A Modular Strategy to Prepare Multivalent Inhibitors of Prostate-Specific Membrane Antigen (PSMA); Oncotarget 2011; 2: 1244 - 1253; doi: 10.18632 / oncotarget.415).

[0052] Known FAP inhibitors feature a C-terminal reactive functionality, such as carbonitrile which covalently binds 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, commonly quantified via the dissociation rate kOff- Accordingly, known FAP inhibitors detach from their target receptor FAP and are subsequently washed out from tumor tissue.

[0053] 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.

[0054] WO 2019 / 154886 Al pertains to radioligands comprising FAP-ligands, such as FAPI-46 (CAS No. 2374782-04-2).

[0055] 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.

[0056] 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.

[0057] Methods for synthesis and conjugation of benzotriazole and / V-methyl- / V-arylmethane- sulfonamide derivates, strain-release alkylating malolactone groups, and sulfur(VI) fluorides, i.e. SuFEx warheads are described in:

[0058] - 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;

[0059] - M. Kawano, S. Murakawa, K. Higashiguchi, K. Matsuda, T. Tamura, I. Hamachi;

[0060] 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 AsplZ 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;

[0061] - 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.

[0062] The disclosure of the above cited articles by Xin et al., Kawano et al., Zheng et al., and Carneiro et al., particularly the therewith associated supporting information in its entirety is incorporated by reference in the present patent application.

[0063] Example 33 of this application illustrates the effect that a strongly or irreversibly binding radioligand has on radiological efficacy.

[0064] Examples 34 and 35 pertain to the electronic properties of SuFEx warheads and FAP ligands and provide guidance for the adaptation of their reactivity. The polarity of the S-F bond (i.e. the difference between the partial charges of the sulfur and fluorine atoms) is indicative for the bond strength. Apart from electronic effects the reactivity of a SuFEx warhead or FAP ligand is strongly influenced by steric effects. Amine (NH) substituents tend to lower the polarity of the S-F bond. Notwithstanding, SuFEx warheads or FAP ligands with amine substituents can exhibit substantially lower reactivity compared to methanesulfonyl fluoride or sulfonyl fluoride groups, which likely is attributable to their chirality.

[0065] Known drugs for chemotherapy and radioligand therapy (RLT) warrant further improvement of their therapeutic index, in particular the ratio of tumor to whole-body dose.

[0066] For PSMA-targeting and to a lesser extent for FAP-targeting radioligands, renal retention of radioactive metabolites increases the risk of nephrotoxicity. Various structural modifications can mitigate renal retention. Renal retention can be significantly reduced through modification of the Glu-Urea binding motif of established PSMA ligands, as indicated beneath

[0067] Renal retention can furthermore be reduced by incorporating a renal enzyme cleavable peptide into the spacer S2, such as -Gly-Lys-, -Gly-Tyr-, -Gly-Phe-Lys-, -Met-Val-Lys-, -His-Glu-, -His-Glu-His-Glu- and -His-Glu-His-Glu-His-Glu- . Release of radioactive metabolites from renal vesicles can be enhanced through adaptation of the lipophilicity of the -S2-Ch group, such that it comprises one of the beneath depicted radicals

[0068] The present invention has the object to provide precursor compounds and thereon based radioligands that enable efficient and selective delivery of radioactive payloads to tumor tissue. For radioactive payloads high tumor uptake, prolonged tumor retention and increased ratio of tumor-absorbed to whole-body-absorbed dose are sought.

[0069] This object is achieved through a precursor compound for a radioligand comprising a ligand PL, a covalent warhead CW and either a chelator Ch for complexation of a radioisotope or a leaving group LR for substitution with a radioisotope, wherein

[0070] - the ligand PL is configured to bind with high affinity to a cancer-associated protein selected from the group comprising fibroblast activation protein (FAP), prostate specific membrane antigen (PSMA), somatostatin receptor SSTR1, somatostatin receptor SSTR2, somatostatin receptor SSTR3, somatostatin receptor SSTR4, somatostatin receptor SSTR5, chemokine receptor CXCR4, nectin cell adhesion molecule 4 (nectin4), fibroblast growth factor receptor 1 (FGFR1 or CD331), fibroblast growth factor receptor 2 (FGFR2 or CD332), fibroblast growth factor receptor 3 (FGFR3 or CD333), fibroblast growth factor receptor 4 (FGFR4 or CD334), fibroblast growth factor receptor-like 1 (FGFR6) and integrin receptors,

[0071] - the covalent warhead CW comprises

[0072] - a sulfur fluoride radical having the structure -U-V-(W1=S=W2)-F, wherein

[0073] - U is absent, a substituted or unsubstituted alkyl or heteroalkyl, or a substituted or unsubstituted C4 to C10 aryl or heteroaryl,

[0074] - V is absent, -CH2- , =CH- , -O- , -NH- or -C(=O)- , - W1 is =0 or =NH , and

[0075] - W2 is =0 or =NH,

[0076] - a sulfonic acid radical having the structure -U-S(=0)20H, wherein U is absent, a substituted or unsubstituted alkyl or heteroalkyl, or a substituted or unsubstituted C4 to C10 aryl or heteroaryl,

[0077] - a benzotriazole radical selected from the group comprising

[0078] - a / V-methyl- / V-arylmethanesulfonamide radical selected from the group comprising wherein

[0079] - Ml is =0 or =NH2, - M2 is =0 or =NH2,

[0080] - M3 is -CH3, -OH , -NH2or alkyl ,

[0081] - Z1is absent or selected from the group comprising -F, -Cl, -Br and — N02,

[0082] - Z2is absent or selected from the group comprising -F, -Cl, -Br and — N02,

[0083] - Z3is absent or selected from the group comprising -F, -Cl, -Br and — N02, a malolactone radical selected from the group comprising wherein Al is a radical of a linear or branched alkyl.

[0084] The object of the invention is also achieved through a precursor compound having a structure selected from the group comprising

[0085]

[0086] R is a substituted or unsubstituted alkyl, a substituted or unsubstituted Ce aryl, or a substituted or unsubstituted a Ikyl-a ryl,

[0087] Y1is H or F, - Y2is H or F,

[0088] G is -NH-C(=0)-(Ce-Cio)-aryl- or -NH-C(=0)-(Ce-Cio)-heteroaryl- with 1, 2 or 3 nitrogen substituents,

[0089] S2 is absent or a bivalent spacer,

[0090] Ch is a chelator for a radioisotope, - S3 is absent or a bivalent spacer,

[0091] TL is absent or a trivalent linker, with the proviso (a), (b) or (c) that

[0092] (a) XI is -S1-C(=O)-LG , -S1-U-V-(W1=S=W2)-F or -S1-U-S(=O)2OH and X2, S3,

[0093] TL, X3 are absent, or

[0094] (b) X2 is -S1-C(=O)-LG , -S1-U-V-(W1=S=W2)-F or -S1-U-S(=O)2OH and XI is absent, -H or -CH3 and S3, TL, X3 are absent, or (c) X3 is -S1-C(=O)-LG , -S1-U-V-(W1=S=W2)-F or -S1-U-S(=O)2OH and XI is absent, -H or -CH3 and X2 is absent, wherein

[0095] - SI is absent or a bivalent spacer,

[0096] - LG is a benzotriazole or / V-methyl V-arylmethanesulfonamide leaving group, or LG is strain-release alkylating malolactone,

[0097] - U is absent, a substituted or unsubstituted alkyl or heteroalkyl, or a substituted or unsubstituted C4 to C10 aryl or heteroaryl,

[0098] - V is absent, -CH2- , =CH- , -O- , -NH- or -C(=O)- ,

[0099] - W1 is =0 or =NH , and

[0100] - W2 is =0 or =NH .

[0101] The precursor compound of the invention can have a structure selected from the group comprising

[0102] / PL^ . / Ch , / PL _ / LR

[0103] X1 S2 ' X1 S2

[0104] X3 X3

[0105] PL. / -TL Cha ndPL. , / TL / LR

[0106] S3 S2 S3 S2 wherein

[0107] - PL is a ligand for a cancer-associated protein,

[0108] - S3 is absent or a bivalent spacer,

[0109] - TL is a trivalent linker,

[0110] - S2 is absent or a bivalent spacer,

[0111] - Ch is a chelator for a radioisotope,

[0112] - LR is a leaving group for substitution with a radioisotope,

[0113] - XI is -S1-C(=O)-LG , -S1-U-V-(W1=S=W2)-F or -S1-U-S(=O)2OH ,

[0114] - X3 is -S1-C(=O)-LG , -S1-U-V-(W1=S=W2)-F or -S1-U-S(=O)2OH , with the proviso that

[0115] - SI is absent or a bivalent spacer,

[0116] - LG is a benzotriazole or / V-methyl- / V-arylmethanesulfonamide leaving group, or LG is strain-release alkylating malolactone,

[0117] - U is absent, a substituted or unsubstituted alkyl or heteroalkyl, or a substituted or unsubstituted C4 to C10 aryl or heteroaryl, - V is absent, -CH2- , =CH- , -O- , -NH- or -C(=O)- ,

[0118] - W1 is =0 or =NH , and

[0119] - W2 is =0 or =NH .

[0120] 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:

[0121] - the precursor compound has the structure wherein TL is a trivalent linker, SI is absent or a bivalent spacer, S2 is absent or a bivalent spacer, and S3 is absent or a bivalent spacer; - G is a radical selected from the group comprising

[0122] - R is a radical selected from the group comprising

[0123]

[0124] - LG is a benzotriazole or / V-methyl- / V-arylmethanesulfonamide radical selected from the group comprising

[0125]

[0126] - Z1is absent or selected from the group comprising -F, -Cl, -Br and -NO2 ;

[0127] - Z2is absent or selected from the group comprising -F, -Cl, -Br and -NO2 ;

[0128] - Z3is absent or selected from the group comprising -F, -Cl, -Br and -NO2 ; - LG a / V-methyl V-arylmethanesulfonamide radical selected from the group comprising wherein

[0129] - Ml is =0 or =NH ;

[0130] - M2 is =0 or =NH ; - M3 is -CH3 , -OH , -NH2or alkyl ,

[0131] - Z1is absent or selected from the group comprising -F, -Cl, -Br and -N02;

[0132] - Z2is absent or selected from the group comprising -F, -Cl, -Br and -N02; and

[0133] - Z3is absent or selected from the group comprising -F, -Cl, -Br and -N02;

[0134] - LG is a strain-release alkylating malolactone radical selected from the group comprising wherein Al is a radical of a linear or branched alkyl;

[0135] - the covalent warhead CW comprises a benzotriazole radical selected from the group comprising

[0136] - the covalent warhead CW comprises a / V-methyl V-arylmethanesulfonamide radical selected from the group comprising wherein

[0137] - Ml is =0 or =NH , - M2 is =0 or =NH ,

[0138] - M3 is -CH3, -OH , -NH2or alkyl ,

[0139] - Z1is absent or selected from the group comprising -F, -Cl, -Br and -N02,

[0140] - Z2is absent or selected from the group comprising -F, -Cl, -Br and -NO2, and

[0141] - Z3is absent or selected from the group comprising -F, -Cl, -Br and -N02, - the covalent warhead CW comprises a malolactone radical selected from the group comprising

[0142] wherein Al is a radical of a linear or branched alkyl;

[0143] - the covalent warhead CW comprises a sulfonic acid radical selected from the group comprising

[0144] - the ligand PL comprises a trivalent or tetravalent linker 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 tetrapeptide;

[0145] - the ligand PL comprises a trivalent linker radical selected from the group comprising

[0146]

[0147] - the ligand PL comprises one, two or three radicals having a structure selected from the group comprising

[0148] wherein X = H or CH3, Y1= H or F and Y2= H or F ; - the ligand PL comprises one, two or three radicals having the structure wherein optionally a hydrogen radical, respectively a proton of a hydroxy (-OH), amine (-NH2, -NH-) or methylene (-CH2-) group is abstracted for coupling with a covalent warhead CW or a bivalent spacer SI and a thereto conjugated covalent warhead CW; - the ligand PL comprises one, two or three radicals having the structure wherein optionally a hydrogen radical, respectively a proton of a hydroxy (—OH), amine (-NH2, -NH-) or methylene (-CH2-) group is abstracted for coupling with a covalent warhead CW or a bivalent spacer SI and a thereto conjugated covalent warhead CW; - the ligand PL comprises one, two or three radicals having the structure wherein optionally a hydrogen radical, respectively a proton of a hydroxy (-OH), amine (-NH2, -NH-) or methylene (-CH2-) group is abstracted for coupling with a covalent warhead CW or a bivalent spacer SI and a thereto conjugated covalent warhead CW; - the ligand PL comprises one, two or three radicals having the structure wherein optionally a hydrogen radical, respectively a proton of a hydroxy (-OH), amine (-NH2, -NH-) or methylene (-CH2-) group is abstracted for coupling with a covalent warhead CW or a bivalent spacer SI and a thereto conjugated covalent warhead CW;

[0149] - the ligand PL comprises one, two or three radicals having the structure wherein optionally a hydrogen radical, respectively a proton of a hydroxy (—OH), amine (-NH2, -NH-) or methylene (-CH2-) group is abstracted for coupling with a covalent warhead CW or a bivalent spacer SI and a thereto conjugated covalent warhead CW, and Z4is a radical selected from the group comprising - the ligand PL comprises one, two or three radicals having the structure wherein

[0150] - W = -H or -CH3;

[0151] - four of V1, V2, V3, V4, V5are terminal radicals selected independently from one another from the group comprising -H, -CH2COOH, -(CH2)3NH2, -(CH2)4NH2, -(CH2)3(NH)C(=NH)NH2, -CH2(C6-CIO aryl) and -CH2(Ce-Cio substituted aryl); and

[0152] - one of V1, V2, V3, V4, V5is a bivalent connector radical selected from the group comprising -(CH2)3NH- -(CH2)4NH- -(CH2)3(NH)C(=NH)NH- -CH2(C6-CIO aryl)- and -CH2(C6-CIO substituted aryl)- ; the ligand PL comprises one, two or three radicals having the structure wherein optionally a hydrogen radical, respectively a proton of a hydroxy (-OH), amine (-NH2, -NH-) or methylene (-CH2-) group is abstracted for coupling with a covalent warhead CW or a bivalent spacer SI and a thereto conjugated covalent warhead CW; - the ligand PL comprises one, two or three radicals having the structure wherein optionally a hydrogen radical, respectively a proton of a hydroxy (-OH), amine (-NH2, -NH-) or methylene (-CH2-) group is abstracted for coupling with a covalent warhead CW or a bivalent spacer SI and a thereto conjugated covalent warhead CW; wherein optionally a hydrogen radical, respectively a proton of a hydroxy (-OH), amine (-NH2, -NH-) or methylene (-CH2-) group is abstracted for coupling with a covalent warhead CW or a bivalent spacer SI and a thereto conjugated covalent warhead CW; wherein optionally a hydrogen radical, respectively a proton of a hydroxy (—OH), amine (-NH2, -NH-) or methylene (-CH2-) group is abstracted for coupling with a covalent warhead CW or a bivalent spacer SI and a thereto conjugated covalent warhead CW;

[0153] - the ligand PL comprises one, two or three radicals of a compound having a structure selected from the group comprising

[0154] wherein one or two hydrogen radicals, respectively protons of hydroxy (-OH), amine (-NH-) or methylene (-CH2-) groups are abstracted for coupling within the precursor compound, and optionally for coupling with a covalent warhead CW or a bivalent spacer SI and a thereto conjugated covalent warhead CW; - the ligand PL comprises one, two or three radicals of a compound having a structure selected from the group comprising

[0155] wherein one or two hydrogen radicals, respectively protons of hydroxy (-OH), amine (-NH-) or methylene (-CH2-) groups are abstracted for coupling within the precursor compound, and optionally for coupling with a covalent warhead CW or a bivalent spacer SI and a thereto conjugated covalent warhead CW; - the precursor compound comprises a radical having a structure selected from the group comprising wherein

[0156] - X = H or CH3, Y1= H or F and Y2= H or F , - SI is a bivalent spacer, and

[0157] - the covalent warhead CW comprises

[0158] - a sulfur fluoride radical having the structure -U-V-(W1=S=W2)-F, wherein

[0159] - U is absent, a substituted or unsubstituted alkyl or heteroalkyl, or a substituted or unsubstituted C4 to C10 aryl or heteroaryl, - V is absent, -CH2- , =CH- , -O- , -NH- or -C(=O)- ,

[0160] - W1 is =0 or =NH , and

[0161] - W2 is =0 or =NH, - a sulfonic acid radical having the structure -U-S(=O)2OH, wherein U is absent, a substituted or unsubstituted alkyl or heteroalkyl, or a substituted or unsubstituted C4 to C10 aryl or heteroaryl,

[0162] - a benzotriazole radical selected from the group comprising - a / V-methyl- / V-arylmethanesulfonamide radical selected from the group comprising

[0163] - Ml is =0 or =NH2,

[0164] - M2 is =O or =NH2,

[0165] - M3 is -CH3, -OH , -NH2or alkyl ,

[0166] - Z1is absent or selected from the group comprising -F, -Cl, -Br and — N02,

[0167] - Z2is absent or selected from the group comprising -F, -Cl, -Br and — N02,

[0168] - Z3is absent or selected from the group comprising -F, -Cl, -Br and — N02, or a malolactone radical selected from the group comprising

[0169] wherein Al is a radical of a linear or branched alkyl.

[0170] - the precursor compound comprises a radical having a structure selected from the group comprising - the precursor compound comprises a radical having a structure selected from the group comprising

[0171] wherein X = H or CH3, Y1= H or F and Y2= H or F ;

[0172] SI is a bivalent alkyl spacer;

[0173] SI is a bivalent heteroalkyl spacer;

[0174] SI is a bivalent heteroalkyl spacer with one, two, three, four, five, six, seven, eight, nine or ten substituents selected independently of one another from the group comprising

[0175] - SI is a bivalent heteroalkyl spacer with one, two, three, four or five substituents selected independently of one another from the group comprising

[0176] - the bivalent spacer SI comprises one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen or twenty methylene groups (-CH2-);

[0177] - the bivalent spacer SI comprises one, two, three, four, five, six, seven, eight, nine or ten methylene groups (-CH2-);

[0178] - the bivalent spacer SI comprises 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- , -N(-CH3)- , -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;

[0179] - the bivalent spacer SI comprises a radical selected from the group comprising

[0180] 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; - the bivalent spacer SI comprises a radical having the structure wherein

[0181] - 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;

[0182] - each QJis present for 1 < j < h and absent for j > h with h = 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0183] - each Pswith 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-,

[0184] -S-, -C(O)- and -C(CH3)- ;

[0185] - each Qlwith 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

[0186] LG and SI are coupled via a -C(=O)-NH- radical;

[0187] LG and SI are coupled via a -C(=O)-O- radical; - the conjugate group LG-S1 comprises a terminal methylene (-CH2-), amine (-NH-), oxy (— O— ) or carbonyl (— C(=0)— ) coupling radical;

[0188] - the conjugate group LG-S1 comprises a terminal -CH2- coupling radical;

[0189] - the conjugate group LG-S1 comprises a terminal -NH- coupling radical;

[0190] - the conjugate group LG-S1 comprises a terminal -O- coupling radical;

[0191] - the conjugate group LG-S1 comprises a terminal -0=C- coupling radical;

[0192] - the moiety -V-(W1=S=W2)-F has a structure selected from the group comprising

[0193] - the moiety -U-V-(W1=S=W2)-F has the structure

[0194] , wherein

[0195] - V is absent, -CH2- , =CH- , -O- , -NH- or -C(=0)- ,

[0196] - W1 is =NH or =0 ,

[0197] - W2 is =NH or =0 ,

[0198] - A is a C4-C10 aryl, C4-C10 substitued aryl, C4-C10 heteroaryl or substitued C4-C10 heteroaryl,

[0199] - R1is absent, H, NH2, NH, OH, O, linear or branched alkyl, or linear or branched substituted alkyl,

[0200] - R2is absent, H, NH2, NH, OH, O, linear or branched alkyl, or linear or branched substituted alkyl,

[0201] - R3is absent, H, NH2, NH, OH, O, linear or branched alkyl, or linear or branched substituted alkyl, and

[0202] - R4is absent, H, NH2, NH, OH, O, linear or branched alkyl, or linear or branched substituted alkyl; - the radical -U-V-(W1=S=W2)-F has a structure selected from the group comprising

[0203] - the radical -U-V-(W1=S=W2)-F has a structure selected from the group comprising - the radical -U-V-(W1=S=W2)-F has a structure selected from the group comprising

[0204] - the radical -U-V-(W1=S=W2)-F has a structure selected from the group comprising

[0205] - the radical -U-V-(W1=S=W2)-F has a structure selected from the group comprising

[0206] - the radical -U-V-(W1=S=W2)-F has a structure selected from the group comprising

[0207]

[0208] - the precursor compound has a structure selected from the group comprising

[0209] - the precursor compound has a structure selected from the group comprising

[0210] - the precursor compound has a structure selected from the group comprising - the precursor compound has a structure selected from the group comprising

[0211] - the group G comprises a radical selected from the group comprising

[0212] - the precursor compound comprises a radical selected from the group comprising

[0213] - the precursor compound comprises a radical selected from the group comprising

[0214] - the precursor compound comprises a radical selected from the group comprising

[0215] - the precursor compound comprises a radical selected from the group comprising

[0216] - the group G comprises a radical selected from the group comprising

[0217]

[0218] - the precursor compound comprises a radical selected from the group comprising

[0219]

[0220] - the precursor compound comprises a radical selected from the group comprising - the precursor compound comprises a radical selected from the group comprising

[0221] - the precursor compound comprises a radical selected from the group comprising

[0222]

[0223] - the precursor compound comprises a radical selected from the group comprising

[0224]

[0225] - the precursor compound comprises a radical selected from the group comprising

[0226]

[0227] - the precursor compound comprises a radical selected from the group comprising

[0228]

[0229] - the precursor compound comprises a radical selected from the group comprising

[0230]

[0231] - the precursor compound comprises a radical selected from the group comprising

[0232] - the precursor compound comprises a radical selected from the group comprising

[0233] - the precursor compound comprises a radical having the structure

[0234] - the precursor compound comprises a radical having the structure

[0235] - the precursor compound comprises a radical having the structure - the precursor compound comprises a radical having the structure

[0236] - the precursor compound comprises a radical having the structure

[0237] - the precursor compound comprises a radical having the structure

[0238] - the precursor compound comprises a radical having the structure - the precursor compound comprises a radical having the structure

[0239] - the precursor compound comprises a radical having the structure

[0240] - the precursor compound comprises a radical having the structure - the precursor compound comprises a radical having the structure

[0241] - the precursor compound comprises a radical having the structure

[0242] - the precursor compound comprises a radical having the structure

[0243] - S2 is a bivalent alkyl spacer; - S2 is a bivalent heteroalkyl spacer;

[0244] - S2 is a bivalent heteroalkyl spacer with one, two, three, four, five, six, seven, eight, nine or ten substituents selected independently of one another from the group comprising

[0245] S2 is a bivalent heteroalkyl spacer with one, two, three, four or five substituents selected independently of one another from the group comprising the bivalent spacer S2 comprises one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen or twenty methylene groups (-CH2-); the bivalent spacer S2 comprises one, two, three, four, five, six, seven, eight, nine or ten methylene groups (-CH2-); the bivalent spacer S2 comprises a naphthol group having the structure

[0246] - the bivalent spacer S2 comprises a group having the structure wherein the terminal amine group (-NH-) is coupled to G or TL; the bivalent spacer S2 comprises a group having the structure wherein the terminal amine group (-NH-) is coupled to G or TL; the bivalent spacer S2 comprises a group having the structure wherein the terminal amine group (-NH-) is coupled to G or TL and I = 1, 2, 3, 4, 5, 6, 7,

[0247] 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20; the bivalent spacer S2 comprises a radical of type — [CH2]P— with p = 1, 2, 3, 4, 5, 6, 7, 8,

[0248] 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20; the bivalent spacer S2 comprises a radical of type — ( N H)— [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; the bivalent spacer S2 comprises a radical of type -(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; the bivalent spacer S2 comprises a radical of type -[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; the bivalent spacer S2 comprises a radical of type — ( 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; the bivalent spacer S2 comprises a radical of type — ( 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; the bivalent spacer S2 comprises a radical of ethylene diamine the bivalent spacer S2 comprises a radical having the structure

[0249] - the bivalent spacer S2 comprises 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;

[0250] - the bivalent spacer S2 comprises a phenylalanine radical;

[0251] - the bivalent spacer S2 comprises a radical having the structure

[0252] - the bivalent spacer S2 comprises a N,N-dimethylarginine radical; - the bivalent spacer S2 comprises a radical having the structure the bivalent spacer S2 comprises a radical having the structure the bivalent spacer S2 comprises a radical having the structure the bivalent spacer S2 comprises a radical having the structure the bivalent spacer S2 comprises a radical having the structure wherein

[0253] - 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;

[0254] - each QJis present for 1 < j < h and absent for j > h with h = 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0255] - each Pswith 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 Qlwith 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)- ;

[0256] - T is absent or a radical selected from the group comprising - the bivalent spacer S2 comprises a peptide radical selected from the group comprising

[0257] -Gly-Lys-, -Gly-Tyr-, -Gly-Phe-Lys-, -Met-Val-Lys-, -His-Glu-, -His-Glu-His-Glu- and -His-Glu-His-Glu-His-Glu-;

[0258] - the bivalent spacer S2 comprises a peptide radical selected from the group comprising

[0259]

[0260] - the bivalent spacer S2 comprises 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- , -N(-CH3)- , -O- , -CH2CH2O- and radicals of C4-C10 aryl or heteroaryl, substituted C4-C10 aryl or 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;

[0261] - the bivalent spacer S2 comprises 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. - the moiety -S2-Ch comprises a radical selected from the group comprising

[0262] - the moiety -S2-Ch comprises a radical selected from the group comprising - the trivalent 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 and tripeptide; the trivalent linker TL comprises a radical or is a radical selected from the group comprising

[0263] - S3 is a bivalent alkyl spacer;

[0264] - S3 is a bivalent heteroalkyl spacer;

[0265] - S3 is a bivalent heteroalkyl spacer with one, two, three, four, five, six, seven, eight, nine or ten substituents selected independently of one another from the group comprising - S3 is a bivalent heteroalkyl spacer with one, two, three, four or five substituents selected independently of one another from the group comprising

[0266] - the bivalent spacer S3 comprises one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen or twenty methylene groups (-CH2-);

[0267] - the bivalent spacer S3 comprises one, two, three, four, five, six, seven, eight, nine or ten methylene groups (-CH2-);

[0268] - the bivalent spacer S3 comprises comprises a radical of type — [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;

[0269] - the bivalent spacer S3 comprises comprises a radical of type — (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;

[0270] - the bivalent spacer S3 comprises comprises a radical of type -(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;

[0271] - the bivalent spacer S3 comprises comprises a radical of type -[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;

[0272] - the bivalent spacer S3 comprises comprises a radical of type — (NH)— [ 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;

[0273] - the bivalent spacer S3 comprises comprises a radical of type — ( 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;

[0274] - the bivalent spacer S3 comprises 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 selected independently of one another 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;

[0275] - the bivalent spacer S3 comprises a phenylalanine radical;

[0276] - the bivalent spacer S3 comprises a naphthol radical having the structure the bivalent spacer S3 comprises a radical having the structure wherein the terminal amine group (-NH-) is covalently coupled to the ligand PL; the bivalent spacer S3 comprises a radical having the structure wherein the terminal amine group (-NH-) is covalently coupled to the ligand PL; the bivalent spacer S3 comprises a group having the structure wherein the terminal amine group (-NH-) is coupled to the ligand PL and I = 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20; the bivalent spacer S3 comprises a radical having the structure the bivalent spacer S3 comprises a N,N-dimethylarginine radical; - the bivalent spacer S3 comprises a radical having the structure

[0277] - the bivalent spacer S3 comprises a radical having the structure

[0278] - the bivalent spacer S3 comprises a radical having the structure - the bivalent spacer S3 comprises a radical having the structure

[0279] - the bivalent spacer S3 comprises a radical having the structure

[0280] - the bivalent spacer S3 comprises 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;

[0281] - each QJis present for 1 < j < h and absent for j > h with h = 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0282] - each Pswith 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 Qlwith 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)- ;

[0283] - T is absent or a radical selected from the group comprising

[0284] - the bivalent spacer S3 comprises a radical of ethylene diamine

[0285] - the bivalent spacer S3 comprises a radical having the structure

[0286] - the bivalent spacer S3 comprises 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- , -N(-CH3)- , -O- , -CH2CH2O- and radicals of C4-C10 aryl or heteroaryl, substituted C4-C10 aryl or 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; - the bivalent spacer S3 comprises 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. - 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;

[0287] - Ch comprises a structure selected from the group comprising structures (I), (II), (III), (IV), (V) and (VI) with

[0288]

[0289] - 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

[0290] - 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) );

[0291] - 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 -NH2or at least one of Fl, F2, F3 and F4 is -NH2;

[0292] - the precursor compound comprises a chelator Ch having the structure wherein D1is H, CH3 or NH2 ; - the precursor compound comprises a chelator radical selected from the group comprising

[0293] - 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]), DOTPH(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;

[0294] - 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.

[0295] The invention has the further object to provide a radioligand for cancer diagnosis and treatment.

[0296] 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 comprising44Sc,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 and18FAI, or through a radioligand comprised of any of the above described precursor compounds wherein a leaving group LR is substituted with18F,131l or211At.

[0297] 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:

[0298] - the radioisotope is68Ga;

[0299] - the radioisotope is177Lu;

[0300] - the radioisotope is225Ac;

[0301] - the radioisotope is212Pb;

[0302] - the radioactive compound is18FAI (aluminum fluoride);

[0303] - 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 comprising44Sc,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 and225Ac;

[0304] - 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 comprising68Ga,177Lu,212Pb and225Ac; the radioligand comprises a chelator having the structure (XI), (XII), (XIII) or (XIV) and therewith complexed radioactive compound18FAI (aluminum fluoride). The radioligand compound according to the present invention comprises a high-affinity ligand PL conjugated to a covalent warhead CW comprising a benzotriazole, / V-methyl- / V- arylmethanesulfonamide or fluorine leaving group or malolactone as strain-release alkylator, and a chelator Ch with a complexed radioisotope (e.g.177Lu3+). When ligated to a cancer- associated protein, such as FAP, the covalent warhead CW is positioned proximal to a nucleophilic amino acid sidechain, e.g. -(CFhhNH- of FAP Lys120. The nucleophilic amino acid sidechain readily reacts with the covalent warhead CW and forms a covalent bond. Thus the radioactive payload of the radioligand is irreversibly attached to the cancer-associated protein, as illustrated beneath.

[0305]

[0306] Cleavage of benzotriazole leaving group LG via nucleophilic attack by the E-amino group of a FAP lysine sidechain and covalent coupling to the remaining carbonyl radical. The dotted arrow indicates reversible binding of the FAP ligand motif to FAP. The catalytic triad of FAP comprises amino acids Ser624, Asp702and His734. Amino acid Lys120is located adjacent thereto at a distance of 18.9 A from Ser624. The distances Ser624-Asp702and Ser624-His734are 9.7 A and 6.7 A, respectively. The corresponding Ser624-Asp702- His734-Lys120dihedral angle is 156.6° (https: / / www.rcsb.org / structure / lz68). Fig. 1 shows a perspective view of the FAP catalytic triad and Lys120.

[0307] Beneath Schemes Al and Bl depict exemplary scaffolds comprising a FAP ligand motif and a benzotriazole leaving group. The corresponding molecule conformations are shown in Fig. 2A and 2B. The molecule conformations have been determined using a 3d-optimizer provided as part of opensource or commercial software packages, such as https: / / pymol.Org / 2 / , https: / / molview.org / and https: / / www.acdlabs.com / products / chem- sketch / , which employ computational methods based on molecular force fields.

[0308] Scheme Al Scheme Bl

[0309] Exemplary compounds of Scheme Al and Bl with FAP ligand motif and benzotriazole leaving group. The corresponding molecular conformations depicted in Figures 2A and 2B are computed using a chemistry software 3d-optimizer. The distances between the carbonitrile N-atom and the carbonyl 0-atom coupled to the benzotriazole leaving group are 7.3 A for compound Al and 19.4 A for compound Bl.

[0310] As demonstrated by the above examples Al and Bl, the compound of the present invention can be readily adapted by changing the length of spacer SI between the FAP ligand motif and a benzotriazole or / V-methyl- / V-arylmethanesulfonamide leaving group LG or SuFEx group with structure -U-V-(W1=S=W2)-F in order to address the FAP catalytic triad and simultaneously enable cleavage of LG or fluorine by and covalent bonding to a nucleophilic amino acid sidechain, such as that of FAP Lys120.

[0311] Table 1 lists examples of FAP amino acid residues that are located on the surface of the FAP cavity and can be addressed by the strong binding or covalent warheads of the present invention.

[0312] Table 1: Examples of FAP amino acid residues for strong or covalent binding; *protonated partial charges are determined based on https: / / alphacharges.ncbr.muni.cz / and https: / / www.uniprot.org / uniprotkb / Q12884 / entry; score corresponds to accessible surface area divided by the square of the distance to Ser624and multiplied by 100

[0313] Fig. 4a is a perspective view of the surface of PSMA (https: / / www.rcsb.org / 3d-view / 4NGN / !) with the ligation site (i.e. enzymatic pocket or cleft) in the vicinity of the amino acid residue

[0314] Glu424. The entry funnel to the ligation site comprises amino acid residues Asp316, Lys617, Pro504, Glu542, which can be used as reference points for dimensional assessment, as depicted in Fig. 4b.

[0315] When a PSMA targeted compound of the present invention binds to PSMA, a major portion thereof, respectively the compound backbone occupies the entry funnel to the PSMA ligation site. As illustrated by Fig. 4b, during transient or reversible ligation, the average distance between the compound backbone and the PSMA surface is about 5 A to 10 A which affords proximity mediated strong ionic or covalent binding of the sulfonic acid group, benzotriazole, / V-methyl- / V-arylmethanesulfonamide, malolactone or SuFEx warhead. The probability of covalent binding largely depends on the reactivity of the instant warhead and the accessible surface, protonated charge and deprotonation of nucleophilic amino residues that reside on the PSMA entry funnel surface. Table 2 lists amino acid residues that are located on the surface of the PSMA entry funnel and can be addressed by the strong binding or covalent warheads of the present invention.

[0316] Table 2: Examples of PSMA amino acid residues for strong or covalent binding; *protonated partial charges are determined based on https: / / alphacharges.ncbr.muni.cz / and https: / / www. uniprot.org / uniprotkb / Q04609 / entry

[0317] As demonstrated by Tables 1 and 2, the ligation cavity or pocket of FAP and PSMA contain numerous nucleophilic amino acid residues that are suitable for strong ionic or covalent binding with a ligand that comprises an auxiliary sulfonic acid group, a leaving group based on benzotriazole or / V-methyl- / V-arylmethanesulfonamide, strain-release alkylating malolactone or a sulfur fluoride exchange (SuFEx) group.

[0318] The precursor compounds expressly disclosed in this patent application can be readily altered and optimized using industrial drug design techniques, such as in silica virtual screening (also designated as molecular docking simulation) followed by in vitro high- throughput screening (https: / / en.wikipedia.org / wiki / Virtual_screening, https: / / en.wikipedia.org / wiki / Docking_ (molecular), https: / / en.wikipedia.org / wiki / High- throughput_screening). High-throughput screening enables analysis of large compound classes with several hundred thousand members in less than 24 h in order to identify effective precursor compounds. Therefore, the present patent application enables improvement of expressly disclosed embodiments by industry-established methods without departing from the spirit of the invention.

[0319] Suitable strategies and methods for mechanistic lead optimization are described in the articles cited below, particularly by Li et al. and Borsari et al.

[0320] - Q. Li; Application of Fragment-Based Drug Discovery to Versatile Targets; Front. Mol. Biosci. 7:180; https: / / www.frontiersin.org / articles / 10.3389 / fmolb.2020.00180 / full ;

[0321] - C. Borsari, E. Keles, J. A. McPhail, A. Schaefer, R. Sriramaratnam, W. Goch, T. Schaefer, M. De Pascale, W. Bal, M. Gstaiger, J.E. Burke, M.P. Wymann; Covalent Proximity Scanning of a Distal Cysteine to Target PI3Ka; J. Am. Chem. Soc. 2022, 144, 6326-6342; https: / / pubs.acs.org / doi / 10.1021 / jacs.lcl3568 ;

[0322] - B. Yu, L. Cao, S. Li, P.C. Klauser, L. Wang; The proximity-enabled sulfur fluoride exchange reaction in the protein context; Chem. Sci., 2023, 14, 7913; https: / / pubs.rsc.org / en / content / articlelanding / 2023 / sc / d3sc01921g ;

[0323] - P.C. Klauser, S. Chopra, L. Cao, K. Naidu Bobba, B. Yu, Y. Seo, E. Chan, R.R. Flavell, MJ. Evans, L. Wang; Covalent Proteins as Targeted Radionuclide Therapies Enhance Antitumor Effects; ACS Central Science 2023 9 (6), 1241-1251; https: / / pubs.acs.org / doi / 10.1021 / acscentsci.3c00288 ;

[0324] - X.-Y. Cui, Z. Li, Z. Kong, Y. Liu, H. Meng, Z. Wen, C. Wang, J. Chen, M. Xu, Y. Li, J. Gao, W. Zhu, Zhixin Hao, Li Huo, S. Liu, Z. Yang, Z. Liu; Covalent targeted radioligands potentiate radionuclide therapy; https: / / doi.org / 10.1038 / s41586-024-07461-6 . Lead optimization comprises the steps of

[0325] - selecting a preferred binding pair of a covalent warhead with a nucleophilic amino acid residue of FAP or PSMA; and

[0326] - adjusting the spacer SI that links the covalent warhead to the conjugate comprising the chelator and the reversible FAP or PSMA ligand.

[0327] SuFEx compound libraries are readily available from vendors, such as Enamine Ltd. (https: / / enamine.net / compound-collections / covalent-compounds / sulfonyl-fluorides). The reaction kinetics of covalent warheads with nucleophilic amino acid residues of PSMA and FAP can be assayed in a highly efficient and rapid "shot-gun" manner by incubating a large variety of covalent warheads with a cancer-associated protein, such as PSMA or FAP. The incubation time can be increased in preset increments in order to rank binding pairs according to their reaction rate. The covalent adducts formed in each assay are determined by mass spectrometry, preferably by electrospray ionization time-of-flight mass spectrometry.

[0328] A suitable spacer SI is readily determined by the approach described by Borsari et al. This is mainly a matter of adjusting the length of the spacer SI in order to allow the covalent warhead to engage the selected amino acid residue of the targeted cancer-associated protein. According to Borsari et al. the reaction rate for covalent binding is described by the equation wherein kchemdesignates the intrinsic warhead reactivity and a is an experimentally determined compensation factor for nonintrinsic warhead reactivity due to steric effects and

[0329] 4 Q the warhead's chemical environment. Ktand 1^ = - 7T GT represent the dissociation constant for reversible ligation and a reaction volume with equivalent radius d, respectively, wherein the radius d corresponds to a minimal distance between the covalent warhead and the amino residue. The above equation by Borsari et al. provides a practical guideline for effectively extrapolating and adjusting the length of the spacer SI.

[0330] Numerous commercial providers of GMP (Good Manufacturing Practice) and CRO (Clinical Research Organization) services have advanced assay and screening capabilities that enable highly efficient and rapid lead optimization as well as in vitro and in vivo studies.

[0331] 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 7).

[0332] 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. thermofisher. com / TFS-Assets / BID / Handbooks / bioconjugation-technical-handbook.pdf) describes numerous linker chemistries and bioconjugation strategies.

[0333] The synthesis of a FAP ligand according to the present invention is illustrated beneath in Example 1.

[0334] Auxiliary methods for covalent bond formation between groups G and S2, G and S3, S3 and TL, TL and S2, S2 and Ch are presented in Examples 2 and 3.

[0335] 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.

[0336] In the present invention

[0337] - 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;

[0338] - 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

[0339] - 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;

[0340] - 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;

[0341] - 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 " / V-methyl- V-arylmethanesulfonamide" is also used for derivatives of / V-methyl-

[0342] / V-arylmethanesulfonamide;

[0343] - the terms "radioligand" and "radiotracer" have the same meaning and are used interchangeably.

[0344] EXAMPLES Example 1: Synthesis of FAP ligand

[0345] Schemes la-lc illustrate the synthesis of FAP ligands.

[0346] 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. LiAIH4, 2. H3O+, 72%;

[0347] (e) 1. MsCI, Et3N, CH2CO3, 2. AcSH, Cs2CO3, DMF, 68%; (f) H2O2, AcOH, AcONa, 97%;

[0348] (g) Et3.3HF, CH2CI2, 76%; (h) HBr, AcOH, MeCN, Dowex-CI, 90%; (i) HATU, Gly-OH, DIPEA, DCM, 3 h, 77%.

[0349] Scheme lb: Synthesis of 6-methoxyquinoline-4-carboxylic acid (a) PBrs, DMF, 3 h, 63%;

[0350] (b) Zn(CN)2, Pd / C, Zn++(COO-)2, dppf, 110°C, 3 h, 72%; (c) NaOH, H2O / EtOH, reflux, 94%.

[0351] Scheme lc: Conjugation of (S)-4,4-difluoro-l-(aminoacetyl)pyrrolidine-2-[(sulfonyl fluoride)methyl] with 6-methoxyquinoline-4-carboxylic acid.

[0352] Alternative strategy for synthesis of FAP ligands with sulfonyl fluoride group

[0353] Jiang et al. describe a one-pot synthesis of sufonyl fluorides that can be employed as an alternative to the method of Schemes la, lb and lc (cf. Y. Jiang, N.S. Alharbi, B. Sun, H.-L. Qin; Focile one-pot synthesis of sulfonyl fluorides from sulfonotes or sulfonic ocids; RSC Adv., 2019, 9, 13863; doi: 10.1039 / c9ra02531f).

[0354] Commercially available tert-butyl 4,4-difluoro-2-(hydroxymethyl)pyrrolidine-l-carboxylate (CAS No. 215918-21-1) is reacted with triphenylphosphine dihalide ( (C6Hs)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 Id). 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 fluoride)methyl] pyrrolidine-l-carboxylate (Scheme le), the amine of which is subsequently deprotected (Scheme If).

[0355] Scheme Id: Conversion of tert-butyl 4,4-difluoro-2-(hydroxymethyl)pyrrolidine-l- carboxylate to corresponding halide and sulfonate (side products are not shown).

[0356] Scheme le: One-pot conversion of sulfonate to 4,4-difluoro-2-

[0357] [(sulfonyl fluoride)methyl]pyrrolidine-l-carboxylate (side products are not shown).

[0358] Scheme If: Deprotection to 4,4-difluoro-2-[(sulfonyl fluoride)methyl]pyrrolidine (side products are not shown).

[0359] 4,4-difluoro-2-[(sulfonylfluoride) 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 lg) using reactions analogous to Scheme la(i), lc and la(i), lb, lc, respectively.

[0360] 6-Methoxyquinoline-4-carboxylic acid tert-Butyl (4-bromoquinolin-6-yl)carbamate CAS No. 86-68-0 CAS No. 1260784-05-1

[0361] Scheme lg: Commercially available quinolines

[0362] Example 2: Ether bond formation between different alcohols

[0363] The synthetic strategy outlined beneath in Scheme 2 follows:

[0364] P.K. Sahoo, S.S. Gawali, C. Gunanathan; Iron-Catalyzed Selective Etherification and Transetherification Reactions Using Alcohols; ACS Omega 2018, 3, 124-136.

[0365] R1= aryl , R2, R3= alkyl or aryl Scheme 2: I ron( 111 )-cata lyzed etherification of two different alcohols

[0366] 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.

[0367] Fe(N0s)3 • 9 H2O (0.025 mmol, 5 mol %) is used as catalyst. Reaction is carried out at 70 °C.

[0368] Product is isolated via column chromatographic purification with typical yield between 40 and 93 %.

[0369] Example 3: Synthesis ofsulfonimidoyl fluoride

[0370] Sulfonimidoyl fluoride compounds are prepared according the synthesis methods described by Wu et al. (X. Wu, W. Zhang, G. Sun, X. Zou, X. Sang, Y. He, B. Gao; Turning sulfonyl and sulfonimidoyl fluoride electrophiles into sulfur(VI) radicals for alkene ligation; Nature Communications (2023) 14:5168; https: / / doi.org / 10.1038 / s41467-023-40615-0) as depicted beneath in Scheme 3 in a generic manner.

[0371] Scheme 3: General procedure for synthesis of sulfonimidoyl fluoride

[0372] Example 4: Conjugation of alcohol and amine through N-0 bond formation

[0373] The reaction strategy outlined beneath in Scheme 4a-4e bears on:

[0374] J. Hill, A. A. Hettikankanamalage, D. Crich; Diversity-Oriented Synthesis of N,N,O-Tri- substituted Hydroxylamines from Alcohols and Amines by N-0 Bond Formation; J. Am. Chem. Soc. 2020, 142, 14820-14825.

[0375] Scheme 4a: Synthesis of 2-hydroperoxytetrahydro-2H-pyran

[0376] H2SO4 (18.4 M, 0.05 mL, 0.92 mmol, 0.01 eq) is added to a stirred solution of H2O2 (50% v / v) (3.8 mL, 58.8 mmol, 2 eq.) at 0 °C. The solution is stirred for 10 min, after which, 3,4-dihydro- pyran (2.68 mL, 29.4 mmol, 1 eq.) is added dropwise at 0 °C and the solution is stirred for

[0377] 1 h. Following, the reaction mixture is diluted with Et20 (15 mL) and quenched by addition of saturated NH4CI (30 mL) solution. The resulting biphasic mixture is transferred to a separatory funnel and the layers separated. The aqueous layer is extracted with ethyl acetate (5 x 40 mL) and the organic layers are combined, dried over Na2SO4, filtered, and concentrated in vacuo. The obtained residue is purified by flash column chromatography on silica (eluent: 5:95 EtOAc:Hexanes) to obtain the compound 2-hydroperoxytetrahydro-2H- pyran as a colorless oil (2.04 g, 17.3 mmol, 59%).

[0378] Scheme 4b: Synthesis of 2-hydroperoxy-2-methyltetrahydro-2H-pyran (MTHP)

[0379] CHsMgCI 3.0 M solution in THF (20 mmol, 6.67 mL, 1.0 eq) is added dropwise over 10 minutes in to a solution of 6-valerolactone (20 mmol, 1.86 mL, 1.0 equiv.) in 40 mL of anhydrous THF at -40 °C under an argon atmosphere. The reaction is stirred for 1 h at -40 °C. After consumption of starting material as indicated by TLC and MS, the reaction mixture is brought to -20 °C and quenched with a saturated solution of NH4CI (40 mL) followed by diluting with DI water (20 mL) at room temperature. The resulting biphasic mixture is separated and the aqueous layer extracted with EtOAc (5 x 40 mL). The organic layers are combined and dried over Na2SO4, filtered, and concentrated in vacuo. The crude reaction mixture is used subsequently without further purification.

[0380] Sulfuric acid (18.4 M, 109 pL, 2.0 mmol, 0.10 eq.) is added to a stirred solution of 2-methyl- tetrahydro-2H-pyran-2-ol (2.3 g, 20 mmol, 1 eq.) obtained in the previous step in 100 mL of DCM at 0 °C. Aqueous hydrogen peroxide solution (50% w / w) (6.8 mL, 100 mmol, 5.00 eq.) is added dropwise over 5 min and stirred another 10 min at 0 °C. The reaction is brought to room temperature and stirred for 2 h. The reaction is quenched with a saturated NH4CI solution (40 mL) and the resulting biphasic mixture is separated and the aqueous layer extracted with EtOAc (5 x 40 mL). The combined organic layers are dried over Na2SO4, filtered and concentrated in vacuo. The obtained residue is purified by flash column chromatography on silica (eluent: 0:100 DCM - 8:92 Et2O:DCM) to obtain 2-hydroperoxy-2 methyltetrahydro-2H-pyran as a clear, colorless oil (1.72 g, 13.0 mmol, 65% total yield).

[0381] Scheme 4c: Synthesis of THP and MTHP monoperoxy acetal of simple alcohol

[0382] Anhydrous DCM (0.17 - 0.60 M), alcohol (1.0 eq.) and base (1.5 eq.) are added to an oven dried flask under argon atmosphere at 0 °C. The solution is stirred for 10 min, after which, Tf20 (1.2 - 1.5 eq.) is added dropwise. The solution is stirred for 30 to 60 min at 0 °C. Following, HCI (10%, 10 mL) is added and the layers are separated. The organic layer is washed with saturated NaHCCh (1 x 10 mL). The aqueous layer is extracted with EtOAc (3 x 5 mL) and the organic layers are combined and washed with saturated NaCI solution (1 x 10 mL), dried over MgSO4, filtered, and concentrated in vacuo. The triflate is extracted via flash silica column chromatography (eluent: EtOAc: Hexanes) and used in the following step.

[0383] Lithium tert-butoxide or potassium tert-butoxide (1.2 - 1.5 eq.) is added in a single portion under argon atmosphere (balloon) to a stirred solution of THP or MTHP (1.0 - 2.0 eq.) in anhydrous THF (0.2 - 0.5 M). The solution is stirred for 10 min at 0 °C, after which, a portion of the triflate (1.0 - 2.0 eq.) obtained in the previous step is added dropwise via syringe. The solution is stirred for 1 h at 0 °C, after which, the mixture is allowed to reach room temperature and stirred for an additional 1-24 h. The reaction mixture is quenched with NaHCCh (20 mL) and diluted with EtOAc (10 mL). The layers are separated and the aqueous layer extracted with EtOAc (3 x 5 mL). The combined organic layers are dried over MgSO4 / Na2SO4, filtered, and concentrated in vacuo. Flash column chromatography on silica (eluent: EtOAc:Hexanes) yields the monoperoxy acetal.

[0384] Scheme 4d: Synthesis of MTHP monoperoxy acetal of complex alcohol

[0385] Anhydrous DCM (0.13 - 0.50 M), alcohol (1.0 eq.) and pyridine (2.0 eq.) are added to an oven dried flask under argon atmosphere at 0 °C. The solution is stirred for 10 min, thereafter, Tf20 (1.2 - 1.5 eq.) is added dropwise. The solution is stirred for 30 to 60 min at 0 °C and subsequently diluted with a few drops of MeOH and 10% HCI (1 x 10 mL). The layers are separated and the organic layer washed with saturated NaHCOs (1 x 10 mL). The aqueous layer is extracted with EtOAc (3 x 5 mL) and the organic layers are combined and washed with saturated NaCI solution (1 x 10 mL), dried over MgSO4, filtered, and concentrated in vacuo. The triflate is extracted via flash silica column chromatography (eluent: EtOAc:Hexanes) and used in the following step.

[0386] NaH (60% dispersion in mineral oil, 1.2 - 1.5 eq.) is added in a single portion under argon atmosphere to a stirred solution of MTHP (1.0 eq.) in anhydrous DMF. The solution is stirred for 10 min at 0 °C, thereafter, the triflate (1.3 eq.) obtained in the previous step is added dropwise via syringe. The solution is stirred for 1 h. The mixture is allowed to settle at room temperature and stirred for an additional 1-16 h. The reaction mixture is then diluted with EtOAc (10 mL) and quenched with saturated NaHCOs (10 mL). The layers are separated and the aqueous layer extracted with EtOAc (3 x 5 mL). The organic layers are dried over Na2$O4 / MgSO4, filtered, and concentrated in vacuo. The monoperoxy acetal is extracted via flash column chromatography on silica (eluent: EtOAc:Hexanes).

[0387] Scheme 4e: Synthesis of N,N,O-trisubstituted hydroxylamine with N-0 bond

[0388] Amine (0.25 - 11.0 mmol, 2.5 eq.) and 0.25 - 11.0 mL anhydrous THF are added to an oven / flame dried flask at 0 °C under an argon atmosphere. To this solution EtMgBr (3M in diethyl ether) (0.2 - 8.7 mmol, 2.0 eq.) is added dropwise and the reaction mixture is stirred for 10 - 30 min at 0 °C (magnesium amide formation produces substantial amount of gas, hence, at larger scale caution is mandated). The magnesium amide is subsequently transferred via syringe to a stirred solution of THP or MTHP monoperoxyacetal (0.10 - 4.36 mmol, 1.0 eq.) stirred in additional 0.25 - 11.0 mL anhydrous THF (0.2 M total) under argon atmosphere at 0 °C. The solution is stirred until the starting material is consumed as indicated by TLC and MS, after which, the mixture is quenched by addition of ice water and the layers are separated. The aqueous layer is extracted with EtOAc and the combined organics are dried over MgSC / IX^SC , filtered, and concentrated in vacuo. Flash column chromatography on silica or neutral alumina (eluent: EtOAc:Hexanes or DCM:EtOAc) yield the N,N,O-trisubstituted hydroxylamines.

[0389] A generic example of an amide coupling reaction is shown in scheme 5.

[0390] Scheme 5: Amide coupling

[0391] 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.

[0392] 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.

[0393] An extensive summary of reaction strategies and reagents for amide couplings can be found in the following review articles:

[0394] - 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;

[0395] - Peptide Coupling Reagents, More than a Letter Soup; A. El-Faham, F. Albericio; Chem. Rev. 2011, 111, 6557-6602;

[0396] - Rethinking amide bond synthesis; V. R. Pattabiraman, J. W. Bode; Nature, Vol. 480 (2011) 22 / 29;

[0397] - Amide bond formation: beyond the myth of coupling reagents; E. Valeur, M. Bradley; Chem. Soc. Rev., 2009, 38, 606-631.

[0398] 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.

[0399] CAS No. 1161415-28-6 CAS No. 438553-50-3

[0400] Scheme 6: Chelator building blocks no

[0401] Example 7: DATA5mProchelator Synthesis

[0402] Scheme 7 illustrates the synthesis of the DATA5mprochelator (cf. J. Seemann, B. Waldron, D. Parker, F. Roesch; DATATOC: a novel conjugate for kit-type68Ga labelling of TOC at ambient temperature; EJNMMI Radiopharmacy and Chemistry (2016) 1:4, DOI 10.1186 / s41181-016- 0007-3).

[0403] Scheme 7: Synthesis of 3‘Bu-protected DATA5mprochelator (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 8-30: Radioligand precursor compounds

[0404] Schemes 8-30 depict exemplary embodiments 8-30 of precursor compounds of the present invention that include a FAP or PSMA ligand conjugated to various covalent warheads based on sulfonic acid, electrophiles with benzotriazole or / V-methyl- / V-arylmethanesulfonamide leaving group, strain-release alkylating malolactone, or sulfur fluoride exchange (SuFEx), and a DOTA chelator for radioisotope complexation.

[0405] Scheme 8: Exemplary precursor compound 8

[0406]

[0407] Scheme 9: Exemplary precursor compound 9

[0408] Scheme 10: Exemplary precursor compound 10

[0409]

[0410] Scheme 11: Exemplary precursor compound 11

[0411] Scheme 12: Exemplary precursor compound 12

[0412]

[0413] Scheme 13: Exemplary precursor compound 13

[0414] Scheme 14: Exemplary precursor compound 14

[0415]

[0416] Scheme 15: Exemplary precursor compound 15

[0417] Scheme 16: Exemplary precursor compound 16

[0418]

[0419] Scheme 17: Exemplary precursor compound 17

[0420] Scheme 18: Exemplary precursor compound 18

[0421]

[0422] Scheme 19: Exemplary precursor compound 19

[0423] Scheme 20: Exemplary precursor compound 20

[0424]

[0425] Scheme 21: Exemplary precursor compound 21

[0426] Scheme 22: Exemplary precursor compound 22

[0427]

[0428] Scheme 23: Exemplary precursor compound 23

[0429] Scheme 24: Exemplary precursor compound 24

[0430]

[0431] Scheme 24: Exemplary precursor compound 25

[0432] Scheme 26: Exemplary precursor compound 26

[0433]

[0434] Scheme 27: Exemplary precursor compound 27

[0435] Scheme 28: Exemplary precursor compound 28

[0436]

[0437] Scheme 29: Exemplary precursor compound 29

[0438] Scheme 30: Exemplary precursor compound 30

[0439] Scheme 31: Exemplary precursor compound 31

[0440] Example 33: Radioligand Clearance from Tumor Tissue

[0441] For therapy radioligands with rapid systemic excretion and, conversely, high uptake and long retention in tumor tissue are sought. For tumor uptake and retention the affinity of the radioligand to its target receptor plays a crucial role. Affinity is quantified by its reciprocal, the dissociation constant KD, preferably in units of nM (IO-9mol-L-1). For the clinically approved radioligand [177Lu]-PSMA-617, Wen et al. (X. Wen, P. Xu, X. Zeng, J. Liu, C. Du, X. Zeng, X. Cheng, X. Wang, Y. Liang, T. Zhao, H. Yang, H. Li, L. Meng, J. Fang, H. Liu, Z. Zhou, J. Zhang, X. Zhang, Z. Guo, X. Chen; Development of [177Lu]Lu-LNC1003 for radioligand therapy of prostate cancer with a moderate level of PS MA expression; Eur. J. Nucl. Med. Mol. Imaging 50, 2846-2860 (2023); https: / / doi.org / 10.1007 / s00259-023-06229-w) report values of KD= 4.7 nM and koff= 3.44 x 10-4s-1. koffdenotes the equilibrium dissociation rate, which in the case of [177Lu]-PSMA-617 corresponds to a binding half-life of

[0442] I n (2) / 3.44 x 104s = 33.6 min. The latter is 285 times smaller than the 9751.0 min (6.65 d) half-life of the therapeutic radioisotope177Lu. The large difference between the binding halflife of radioligands and therapeutic radioisotopes used for them is remarkable and deserves explanation. Due to 90 % cellular internalization [177Lu]-PSMA-617 has a retention half-life in tumor tissue in the range from 68,0 h to 74,5 h which exceeds its binding half-life by a factor of about 127. However, for radioligands that bind to FAP-positive CAFs, the internalization ratio appears to be considerably lower. Accordingly, the retention time in tumor tissue is largely limited by radioligand dissociation from extracellular FAP.

[0443] Without cellular internalization, retention in tumor tissue depends on radioligand pharmacokinetics and KDor affinity to the target receptor R. The effective rate constant Kefor radioligand clearance from tumor tissue is linked to the dissociation constant KDand the intratumoral receptor concentration [ / ?]tvia the following relationship: wherein KPKdenotes the physiological clearance rate in the absence of the target receptor R. According to this equation, Ketends towards KPKfor large dissociation constant KD» [ / ?]ti.e. low affinity. The physiological clearance rate KPKin tumor tissue largely depends on intercellular diffusion and convection. Convection can be accelerated due to fenestrated vasculature or shunts between arterial and venous capillaries, as commonly found in tumor tissue. In contrast to normal tissue, lymphatic clearance in tumor tissue is severely impaired. Regardless of the different physiology of tumor and normal tissue, the latter can serve as a useful approximation for KPK. Alternatively, KPKcan be experimentally determined using multilayered cell cultures or multicellular spheroids.

[0444] As can be gathered from the above equation, the effective clearance rate Kecan be lowered by reducing either KPKor KD. Modification of radioligand chemistry affects both KPKand KD. Therefore, it may be difficult to distinguish or decouple the effect of reduced KDfrom the effect of altered KPKin tumor tissue and pharmacokinetics in healthy tissue. This interdependence must be heeded especially if clearance from other tissues such as kidney, liver or bone marrow is slow. Example 34: Partial Charges of Sulfur Fluoride Exchange (SuFEx) Warheads and FAP Ligands

[0445] Table 3 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 33 illustrates the substituent designation in Table 3.

[0446] Scheme 33: Substituent designation in Table 3; V = absent, CH2, O, NH or CO ; W1 = O or NH ; W2 = O or NH ; Y1= H or F ; Y2= H or F

[0447] Table 3: Partial charges of SuFEx warheads and FAP ligands

[0448] Example 35: Hammett Constants of Sulfur Fluoride Exchange (SuFEx) Warheads and FAP Ligands

[0449] Table 4 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.

[0450] 202200041). Scheme 31 illustrates the substituent designation in Table 4. The SMILES strings contain [R] as a fluorine reference or proxy. Table 4: Hammett constants of SuFEx warheads and FAP ligands

Claims

Claims1. A precursor compound for a radioligand comprising a ligand PL, a covalent warhead CW and either a chelator Ch for complexation of a radioisotope or a leaving group LR for substitution with a radioisotope, wherein- the ligand PL is configured to bind with high affinity to a cancer-associated protein selected from the group comprising fibroblast activation protein (FAP), prostate specific membrane antigen (PSMA), somatostatin receptor SSTR1, somatostatin receptor SSTR2, somatostatin receptor SSTR3, somatostatin receptor SSTR4, somatostatin receptor SSTR5, chemokine receptor CXCR4, nectin cell adhesion molecule 4 (nectin4), fibroblast growth factor receptor 1 (FGFR1 or CD331), fibroblast growth factor receptor 2 (FGFR2 or CD332), fibroblast growth factor receptor 3 (FGFR3 or CD333), fibroblast growth factor receptor 4 (FGFR4 or CD334), fibroblast growth factor receptor-like 1 (FGFR6) and integrin receptors,- the covalent warhead CW comprises- a sulfur fluoride radical having the structure -U-V-(W1=S=W2)-F, wherein- U is absent, a substituted or unsubstituted alkyl or heteroalkyl, or a substituted or unsubstituted C4 to C10 aryl or heteroaryl,- V is absent, -CH2- , =CH- , -O- , -NH- or -C(=O)- ,- W1 is =0 or =NH , and- W2 is =0 or =NH,- a sulfonic acid radical having the structure -U-S(=0)20H, wherein U is absent, a substituted or unsubstituted alkyl or heteroalkyl, or a substituted or unsubstituted C4 to C10 aryl or heteroaryl,- a benzotriazole radical selected from the group comprising- a / V-methyl V-arylmethanesulfonamide radical selected from the group comprisingwherein- Ml is =0 or =NH2,- M2 is =O or =NH2,- M3 is -CH3, -OH , -NH2or alkyl ,- Z1is absent or selected from the group comprising -F, -Cl, -Br and — N02,- Z2is absent or selected from the group comprising -F, -Cl, -Br and — N02,- Z3is absent or selected from the group comprising -F, -Cl, -Br and — N02, or- a malolactone radical selected from the group comprisingwherein Al is a radical of a linear or branched alkyl.

2. The precursor compound of claim 1, characterized in that it has the structurewherein TL is a trivalent linker, SI is absent or a bivalent spacer, S2 is absent or a bivalent spacer, and S3 is absent or a bivalent spacer.

3. The precursor compound of claim 1 or 2, characterized in that the covalent warhead CW comprises a / V-methyl- / V-arylmethanesulfonamide radical having a structure selected from the group comprisingwherein- Z1is absent or selected from the group comprising -F, -Cl, -Br and -NO2 ,- Z2is absent or selected from the group comprising -F, -Cl, -Br and -NO2 , and- Z3is absent or selected from the group comprising -F, -Cl, -Br and -NO2 .

4. The precursor compound of claim 1, 2 or 3, characterized in that the ligand PL comprises one, two or three radicals having a structure selected from the group comprisingwherein X = H or CH3, Y1= H or F and Y2= H or F .

5. The precursor compound of claim 1, 2 or 3, characterized in that the ligand PL comprises one, two or three radicals having a structure selected from the group comprisingwherein optionally a hydrogen radical, respectively a proton of a hydroxy (-OH), amine (-NH2, -NH-) or methylene (-CH2-) group is abstracted for coupling with a covalent warhead CW or a bivalent spacer SI and a thereto conjugated covalent warhead CW, and Z4is a radical selected from the group comprising6. The precursor compound of claim 1, 2 or 3, characterized in that the ligand PL comprises one, two or three radicals having the structurewherein- W = -H or -CH3;- four of V1, V2, V3, V4, V5are terminal radicals selected independently of one another from the group comprising -H, -CH2COOH, -(CH2)3NH2, -(CH2)4NH2, -(CH2)3(NH)C(=NH)NH2, -CH2(C6-CIO aryl) and -CH2(Ce-Cio substituted aryl); and - one of V1, V2, V3, V4, V5is a bivalent connector radical selected from the group comprising -(CH2)3NH- -(CH2)4NH- -(CH2)3(NH)C(=NH)NH- -CH2(C6-CIO aryl)- and -CH2(C6-CIO substituted aryl)-.

7. The precursor compound of claim 1, 2 or 3, characterized in that the ligand PL comprises one, two or three radicals having a structure selected from the group comprisingwherein optionally a hydrogen radical, respectively a proton of a hydroxy (-OH), amine (-NH2, -NH-) or methylene (-CH2-) group is abstracted for coupling with a covalent warhead CW or a bivalent spacer SI and a thereto conjugated covalent warhead CW.

8. The precursor compound of claim 1, 2 or 3, characterized in that the ligand PL comprises one, two or three radicals of a compound having a structure selected from the group comprisingwherein one or two hydrogen radicals, respectively protons of hydroxy (-OH), amine (-NH2, -NH-) or methylene (-CH2-) groups are abstracted for coupling within the precursor compound, and optionally for coupling with a covalent warhead CW or a bivalent spacer SI and a thereto conjugated covalent warhead CW.

9. The precursor compound of claim 1, 2 or 3, characterized in that the ligand PL comprises one, two or three radicals of a compound having a structure selected from the group comprisingwherein one or two hydrogen radicals, respectively protons of hydroxy (-OH), amine (-NH2, -NH-) or methylene (-CH2-) groups are abstracted for coupling within the precursor compound, and optionally for coupling with a covalent warhead CW or a bivalent spacer SI and a thereto conjugated covalent warhead CW.

10. The precursor compound of claim 1, 2 or 3, characterized in that it has a structure selected from the group comprisingwherein- R is a substituted or unsubstituted alkyl, a substituted or unsubstituted Ce aryl, or a substituted or unsubstituted a Ikyl-a ryl;- Y1is H or F; - Y2is H or F;- G is -NH-C(=0)-(Ce-Cio)-aryl- or -NH-C(=0)-(Ce-Cio)-heteroaryl- with 1, 2 or 3 nitrogen substituents;- S2 is absent or a bivalent spacer;- Ch is a chelator for a radioisotope;- S3 is absent or a bivalent spacer;- TL is absent or a trivalent linker; with the proviso (a), (b) or (c) that(a) XI is -S1-C(=O)-LG , -S1-U-V-(W1=S=W2)-F or -S1-U-S(=O)2OH and X2, S3, TL, X3 are absent, or(b) X2 is -S1-C(=O)-LG , -S1-U-V-(W1=S=W2)-F or -S1-U-S(=O)2OH and XI is absent, -H or -CH3 and S3, TL, X3 are absent, or(c) X3 is -S1-C(=O)-LG , -S1-U-V-(W1=S=W2)-F or -S1-U-S(=O)2OH and XI is absent, -H or -CH3 and X2 is absent, wherein- SI is absent or a bivalent spacer,- LG is a benzotriazole or / V-methyl V-arylmethanesulfonamide leaving group, or LG is strain-release alkylating malolactone,- U is absent, a substituted or unsubstituted alkyl or heteroalkyl, or a substituted or unsubstituted C4 to C10 aryl or heteroaryl,- V is absent, -CH2- , =CH- , -O- , -NH- or -C(=O)- ,- W1 is =0 or =NH , and- W2 is =0 or =NH .

11. The precursor compound of claim 10, characterized in that G is a radical selected from the group comprising12. The precursor compound of any one of claims 1 to 11, characterized in that the covalent warhead CW is conjugated to the ligand PL directly or through a bifunctional spacer SI.

13. The precursor compound of any one of claims 1 to 12, characterized in that any of bivalent spacers SI, S2, S3 independently of one another comprises 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- , -N(-CH3)- , -O- , -CH2CH2O- and radicals of C4-C10 aryl or heteroaryl, substituted C4-C10 aryl or 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.

14. The precursor compound of any one of claims 1 to 12, characterized in that any of bivalent spacers SI, S2, S3 independently of one another comprises a radical selected from the group comprisingwherein 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.

15. The precursor compound of any one of claims 1 to 12, characterized in that any of bivalent spacers SI, S2, S3 independently of one another comprises a radical having the structurewherein- 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 QJis present for 1 < j < h and absent for j > h with h = 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; - each Pswith 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 Qlwith 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 comprising16. The precursor compound of any one of claims 1 to 15, characterized in that the chelator Ch comprises a radical selected from the group comprisingwherein Fl is -OH or -NH2, F2 is -OH or -NH2, F3 is -OH or -NH2, F4 is -OH or -NH2.

17. The precursor compound of any one of claims 1 to 15, characterized in that the chelator Ch is selected from the group comprising18. The precursor compound of any one of claims 1 to 15, characterized in that the chelator Ch has the structurewherein D1is H, CH3 or NH2 .

19. The precursor compound of any one of claims 1 to 15, characterized in that the chelator Ch is selected from the group comprising20. The precursor compound of any one of claims 1 to 15, characterized in that the chelator Ch is 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]), DOTPH(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 l-hdedpa (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 (NI-^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'-(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.

21. A radioligand comprising the precursor compound of any one of claims 1 to 20 and a therewith complexed radioisotope or radioactive compound selected from the group comprising44Sc,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 and18FAI.

22. A radioligand comprising the precursor compound of any one of claims 1 to 15 wherein the leaving group LR is substituted with a radioisotope selected from the group comprising18F,131l and211At.

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