Radiotracers and precursors for these radiotracers, and use of these radiotracers and precursors as diagnostic agents and therapeutic agents

Abstract

The invention relates to PSMA-targeted radiotracers comprising a linker that is derived from suberic acid and lysine.

Description

Description Radiotracers and precursors for these radiotracers and the use of these radiotracers as diagnostics and therapeutics

[0001] The invention relates to radiotracers and precursors for these radiotracers. Furthermore, it relates to the use of the radiotracers, in particular their use in the diagnosis and treatment of diseases involving the prostate-specific membrane antigen (PSMA).

[0002] In personalized medicine, radiolabeled drugs that bind to disease-specific biological structures for diagnosis and subsequent treatment of that disease are called theranostics. This approach is used to localize and subsequently eliminate metastatic disease.

[0003] Compared to normal prostate epithelial cells, expression of prostate-specific membrane antigen (PSMA) is elevated in human prostate cancers and metastatic malignancies (Wright et al., Urol Oncol 1995, 7, 18-28). This high and specific expression of PSMA on the cell surface is a hallmark of localized and metastatic prostate cancer. Furthermore, PSMA expression has been observed in the neovascularization of several non-prostatic solid malignancies (Chang et al., Clin Cancer Res 1999, 5, 2674).

[0004] Therefore, PSMA is an attractive target for the diagnosis, staging, detection of recurrence, and treatment of prostate cancer (and other diseases in which PSMA is upregulated) using radiolabeled compounds. Such molecules typically comprise a pharmacophoric group for PSMA binding, a linker structure for optimizing pharmacological properties, and a chelating or prosthetic group for the incorporation of a suitable radionuclide. In recent years, mainly 18 F and 68 Ga- labeled radioligands for PET imaging of prostate cancer, including 68 Ga-PSMA-11, 18 F-DCFPyL and 18 F-PSMA-1007, used.

[0005] 18 F is a popular radionuclide for positron emission tomography (PET). It is obtained from [ 18 O]water using a cyclotron and can then be used for the synthesis of18 F-labeled radiopharmaceuticals. The decay results 18 O accompanied by positron (ß+) emission. The high proportion of positron radiation of 97% and the low maximum positron energy of 0.64 MeV are advantageous properties for high image resolution in PET. The comparatively long half-life of 18 F (109.7 min) enables production in centralized facilities and subsequent distribution of the radiopharmaceutical to the applying clinic (Werner et al., Theranostics 2020, 10, 1-16; Preshlock et al., Chem. Rev. 2016, 116, 719; Piron et al., Nuclear Medicine and Biology 2022, 106-107, 29-51).

[0006] 18State-of-the-art F-labeled PSMA ligands can be grouped into two types. The first group consists of ligands that are excreted primarily via the kidneys, which leads to significant accumulation of activity in the bladder and can complicate the detection of metastases near the urinary tract. The second group of ligands is characterized by long retention in the body with concomitant slow and primarily hepatobiliary excretion. This leads to stronger background signals in imaging and may be an explanation for the observed uptake in non-target tissues, such as PSMA-nonspecific uptake in bone (Amfield et al., European Journal of Nuclear Medicine and Molecular Imaging 2021, 48, 4495-4507; Vollnberg et al., European Journal of Nuclear Medicine and Molecular Imaging 2022, 49, 3910-3916; Oh et al., Journal of Nuclear Medicine 2020, 61, 702-709).

[0007] Glu-CO-Lys-Sub-Lys represents a preferred structural motif for binding to PSMA, which has been combined with hydrophobic amino acids such as phenylalanine to synthesize PSMA-binding radiometal complexes for imaging and therapy of prostate cancer (Banerjee et al., J. Med. Chem. 2010, 53, 5333; Banerjee et al., Bioconjug. Chem. 2016, 27, 1447-1455; Weineisen et al., EJNMMI Research 2014, 4, 63; Robu et al., J. Nucl. Med. 2017, 58, 235-242).

[0008] The application of this motif for the development of 18 F-labeled PSMA ligands are currently limited to the radiohybrid ligands rhPSMA-5 and rhPSMA-6, which show only low uptake in LNCaP prostate cancer cells (Wurzer et al., J. Nucl. Med. 2020, 61, 735-742).

[0009] Consequently, there is a need for new radiolabeled PSMA ligands, especially new 18 F-labeled PSMA ligands with optimized pharmacological properties.

[0010] The object of the invention is to eliminate the disadvantages of the prior art. In particular, radiotracers are provided that serve as ligands for the prostate-specific membrane antigen (PSMA) and can be used in the diagnosis and treatment of certain diseases involving PSMA.

[0011] This problem is solved by the features of claims 1, 12, 13, 14, and 15. Practical embodiments of the inventions result from the features of the dependent claims.

[0012] According to the invention, a compound of general formula I is provided (Formula I), where A is an amino acid selected from the group consisting of wherein the amino acids are optionally substituted: k is independently 0, 1, or 2 at each occurrence; m is independently 1, 2, 3, 4, or 5 at each occurrence; n is independently 0, 1, 2, or 3 at each occurrence; p is independently 1, 2, or 3 at each occurrence; q is independently 1, 2, or 3 at each occurrence; X and Y are substituted or unsubstituted amino acids; Z is selected from the group consisting of ZI Z2 Z3 Z4, where v is 0, 1 or 2; Gi N or CR a is; G2 is N or C-Rb; G3 N or CR c is; G4 is N or C-Rd; where 0, 1 or 2 of Gi, G2, G3 and G4 is / are N; Ra, Rb, Rc and Ra, if present, are in each case independently selected from the group consisting of hydrogen, halogen, hydroxyl, carboxyl, Ci-Ce alkyl, Ci-Ce alkoxy, cyano, nitro and Si(tBu)2F, provided that only 1 or 2 of Ra, Rb, Rc and Ra is / are halogen; and R is selected from the group consisting of hydrogen, halogen, hydroxy, Ci-Ce-alkyl and Ci-Ce-alkoxy.

[0013] According to the invention, it can be provided that the halogen in the compounds of general formula I is selected from the group consisting of [ 18 F]Fluorine, [ 123 I]Iodine, [ 124 I]Iodine, [ 125 I]Iodine, [ 126 I]Iodine, [ 128 I]Iodine, [ 130 I]Iodine, [ 131 I]Iodine, [ 209 At]Astatine, [ 210 At]Astatand [ 211 At] astatine. [ 18 F]Fluorine, [ 123 I]Iodine, [ 124 I]Iodine, [ 131 I]Iodine and [ 211 At]astatin is used.

[0014] It can further be provided that the amino acids X and Y in the compounds of the general formula I are independently substituted or unsubstituted glutamic acid, substituted or unsubstituted glutamine, substituted or unsubstituted aspartic acid, substituted or unsubstituted phenylalanine, substituted or unsubstituted histidine, substituted or unsubstituted serine, substituted or unsubstituted tyrosine, substituted or unsubstituted arginine, substituted or unsubstituted asparagine, substituted or unsubstituted Tryptophan, substituted or unsubstituted alanine, and substituted or unsubstituted aminoadipic acid. It may be provided that group X is linked to group Y via a peptide bond.

[0015] In a preferred embodiment of the present invention, the amino acids X and Y are independently glutamic acid, 3-(carboxymethyl)glutamic acid, glutamine, tyrosine, 3-iodotyrosine, arginine, aspartic acid, 2-aminoadipic acid, tryptophan, phenylalanine, 4-methoxyphenylalanine, 4-carboxyphenylalanine, 1-naphthylalanine, 2-naphthylalanine, 3-(anthracen-9-yl)alanine, 3-(quinolin-2-yl)alanine, 3-(quinolin-3-yl)alanine, 3-(isoquinolin-3-yl)alanine, 3-(isoquinolin-7-yl)alanine, 3-(benzothiophen-3-yl)alanine and 3-(Benzothiophen-2-yl)alanine. In a further preferred embodiment, amino acids X and Y are independently glutamic acid, glutamine, tyrosine, 3-iodotyrosine, arginine, aspartic acid, or phenylalanine.

[0016] According to the invention, the group X is an amino acid (q = 1) or a sequence of amino acids (q = 2 or 3). If q is 1, the amino acid is preferably selected from the group consisting of substituted or unsubstituted glutamic acid, substituted or unsubstituted glutamine, substituted or unsubstituted aspartic acid, substituted or unsubstituted phenylalanine, substituted or unsubstituted histidine, substituted or unsubstituted serine, substituted or unsubstituted tyrosine, substituted or unsubstituted arginine, substituted or unsubstituted asparagine, substituted or unsubstituted tryptophan, substituted or unsubstituted alanine, and substituted or unsubstituted aminoadipic acid. It may be provided that group X is bound to group Y via a peptide bond.When q is 2 or 3, preferably each of the amino acids forming the sequence of amino acids is independently selected from the group consisting of substituted or unsubstituted glutamic acid, substituted or unsubstituted glutamine, substituted or unsubstituted aspartic acid, substituted or unsubstituted phenylalanine, substituted or unsubstituted histidine, substituted or unsubstituted serine, substituted or unsubstituted tyrosine, substituted or unsubstituted arginine, substituted or. unsubstituted asparagine, substituted or unsubstituted tryptophan, substituted or unsubstituted alanine, and substituted or unsubstituted aminoadipic acid. Preferably, each of the amino acids in the sequence of amino acids is substituted or unsubstituted glutamic acid. Preferably, p is 1.

[0017] It can be provided that group X is bound to group Y via a first peptide bond and to the unit BM via a second peptide bond. The unit BM is a unit of the general formula where k, m, and n have the meanings given in connection with the general formula I. Thus, the compound of the general formula I can also be represented by the general formula BM-Xp-Yq-Z can be represented.

[0018] If the X group has a sequence of two or three amino acids, the amino acids are linked together by a peptide bond. The BM group is a urea-based pharmacophore group. The X and Y groups form a linker between the BM group on the one hand and the Z group on the other.

[0019] According to the invention, the group Y is an amino acid (q = 1) or a sequence of amino acids (q = 2 or 3). If q is 1, then the amino acid is preferably selected from the group consisting of substituted or unsubstituted glutamic acid, substituted or unsubstituted glutamine, substituted or unsubstituted aspartic acid, substituted or unsubstituted phenylalanine, substituted or unsubstituted histidine, substituted or unsubstituted serine, substituted or unsubstituted tyrosine, substituted or unsubstituted arginine, substituted or unsubstituted asparagine, substituted or unsubstituted trypto- phane, substituted or unsubstituted alanine, and substituted or unsubstituted aminoadipic acid. It may be provided that group X is linked to group Y via a peptide bond. If q is 2 or 3, then preferably each of the amino acids forming the sequence of amino acids is independently selected from the group consisting of substituted or unsubstituted glutamic acid, substituted or unsubstituted glutamine, substituted or unsubstituted aspartic acid, substituted or unsubstituted phenylalanine, substituted or unsubstituted histidine, substituted or unsubstituted serine, substituted or unsubstituted tyrosine, substituted or unsubstituted arginine, substituted or unsubstituted asparagine, substituted or unsubstituted tryptophan, substituted or unsubstituted alanine and substituted or unsubstituted aminoadipic acid.Preferably, each of the amino acids in the sequence of amino acids is substituted or unsubstituted glutamic acid. Preferably, q is 1.

[0020] It may be provided that group Y is bound to group X via a first peptide bond and to unit Z via a second peptide bond. If group X has a sequence of two or three amino acids, the amino acids are bound to each other by a peptide bond.

[0021] The X and Y groups may be the same or different. In one embodiment of the invention, the term "substituted amino acid" refers to an amino acid having a phenyl ring, wherein the phenyl ring has one or two substituents independently selected from the group consisting of halogen and hydroxy. The term "halogen," unless otherwise specified, refers to fluorine, chlorine, bromine, iodine, or astatine. Preferably, the halogen is iodine. In one embodiment, the phenyl ring has two substituents, one of the substituents being iodine and the other substituent being hydroxy.

[0022] It may be provided that X and Y are not both unsubstituted phenylalanine.

[0023] It can be provided that group A in the compounds of general formula I is Al, A2, A3, A6 or A7. It can be provided that group A in the compounds of general formula I is Al or A2. In a more preferred embodiment, A is Al.

[0024] It may further be provided that the group Z in the compounds of general formula I is selected from the group consisting of the following:

[0025] It may also be provided that the group Z in the compounds of the 5 general formula I is selected from the group consisting of the following:

[0026] It may further be provided that the group Z in the compounds of general formula I is selected from the group consisting of the following:

[0027] In a further preferred embodiment, the group Z in the compounds of general formula I is selected from the group consisting of the following:

[0028] In a particularly preferred embodiment, the group Z in the compounds of the general formula i.e. the group ZI, where RH is, Gi C-Ra with Ra = H is, G2 C-Rb with Rb = H is, G3 CR c with Rc = F, G4 = N and v = 0.

[0029] It can further be provided that in the compounds of the general formula I k is 1, m is 3, n is 2, p is 1 or 2 and q is 1 or 2, X and Y are independently glutamic acid, glutamine, tyrosine, 3-iodotyrosine, arginine, aspartic acid, phenylalanine; Z is ZI, where R is H, Gl is C-Ra with Ra = H, G2 is C-Rb with Rb = H, G3 is CR c with R c= F, G4 is N, v is 0 and A is Al. It is preferred that in the compounds of general formula I k is 1, m is 3, n is 2, p is 1 and q is 1, X and Y are independently glutamic acid, glutamine, tyrosine, 3-iodotyrosine, arginine, aspartic acid, phenylalanine; Z is ZI, where R is H, Gi is CR a with Ra = H, G2 C-Rb with Rb = H, G3 CR c with Rc = F, G4 is N, v is 0 and A is Al. It is further preferred that in the compounds of general formula I k is 1, m is 3, n is 2, p is 1 and q is 1, X and Y are glutamic acid; Z is ZI, where R is H, Gi is C-Ra with Ra = H, G2 is C-Rb with Rb = H, G3 CR c with Rc = F, G4 = N, v = 0 and A = Al.

[0030] According to the invention, the following compounds E514, E587, E588, E589, E590, E591, E592 and E593 preferred: E514 Compound E514 is a compound of general formula I, wherein k is 1, m is 3, n is 2, p and q are 1, X and Y are glutamic acid; A is Al and Z is ZI, wherein RH is, Gi C-Ra with Ra = H is, G2 C-Rb with Rb = H is, G3 CR c with Rc = F, G4 = N and v = 0. E587 Compound E587 is a compound of general formula I, wherein k is 1, m is 3, n is 2, p and q are 1, X is tyrosine and Y is glutamic acid; A is Al and Z is ZI, wherein R is H, Gi is C-Ra with R a = H, G2 C-Rb with Rb = H, G3 C-Rc with Rc = F, G4 N is and v is 0. E588 Compound E588 is a compound of general formula I, wherein k is 1, m is 3, n is 2, p and q are 1, X and Y are phenylalanine; A is Al and Z is ZI, wherein R is H, Gi is C-Ra with Ra = H, G2 is C-Rb with Rb = H, G3 is CR c with R c = F, G4 is N and v is 0. E589 Compound E589 is a compound of general formula I, wherein k is 1, m is 3, n is 2, p and q are 1, X is tyrosine and Y is arginine; A is Al and Z is ZI, wherein R is H, Gi is C-Ra with Ra = H, G2 is C-Rb with Rb = H, G3 is CR c with Rc = F, G4 = N and v = 0. E590 Compound E590 is a compound of general formula I, wherein k is 1, m is 3, n is 2, p and q are 1, X and Y are tyrosine; A is Al and Z is ZI, wherein R is H, Gi is C-Ra with R a = H, G2 C-Rb with Rb = H, G3 C-Rc with Rc = F, G4 N and v 0. E591 Compound E591 is a compound of general formula I, wherein k is 1, m is 3, n is 2, p and q are 1, X is iodotyrosine and Y is glutamic acid; A is Al and Z is ZI, wherein R is H, Gi is C-Ra with Ra = H, G2 is C-Rb with Rb = H, G3 is CR c with Rc = F, G4 = N and v = 0. E592 Compound E592 is a compound of general formula I, wherein k is 1, m is 3, n is 2, p and q are 1, X and Y are aspartic acid; A is Al and Z is ZI, wherein R is H, Gi is C-Ra with Ra = H, G2 is C-Rb with Rb = H, G3 is CR c with Rc = F, G4 = N and v = 0. E593 Compound E593 is a compound of general formula I, wherein k is 1, m is 3, n is 2, p and q are 1, X and Y are glutamine; A is Al and Z is ZI, wherein R is H, Gi is C-Ra with R a = H, G2 C-Rb with Rb = H, G3 C-Rc with Rc = F, G4 N and v 0.

[0031] Depending on their structure, the compounds of general formula I can exist in tautomeric or stereoisomeric forms. Therefore, compounds of general formula I include all enantiomers and all diastereomers. The invention therefore also encompasses the tautomers, enantiomers, or diastereomers, and respective mixtures of a compound of general formula I. The stereoisomerically uniform components can be isolated in a known manner from such mixtures of enantiomers and / or diastereomers.

[0032] The compounds of the invention can be used as ligands that bind to the prostate-specific membrane antigen (PSMA). It has been discovered that the compounds can be used as PSMA inhibitors. Therefore, the compounds can be used in the diagnosis and treatment of diseases involving PSMA. More specifically, the compounds can be used in the diagnosis and treatment of certain diseases in which PSMA is upregulated. Therefore, the compounds of the invention can Compounds are used as drugs in prostate cancer diagnosis and therapy.

[0033] The compounds of the invention can bind to the prostate-specific membrane antigen (PSMA). Therefore, they are ligands for the prostate-specific membrane antigen (PSMA). Hereinafter, the compounds of the invention are also referred to as ligands or PSMA ligands. If the compounds of the invention contain a radioactive halogen isotope, they are also referred to as radioligands or PSMA radioligands.

[0034] The compounds of general formula I can be synthesized in various ways using known synthetic routes, for example by applying the Merrifield synthesis (see Robert Bruce Merrifield, Solid phase peptide synthesis, Journal of the American Chemical Society, Volume 85, Item 14, pp. 2149-2154). In a first step, a compound of general formula II (Formula II) where R 11 represents a solid phase, preferably 2-CTC resin, R 12 and R 13orthogonal protecting groups and n has the meanings given in connection with the general formula I. In a preferred embodiment, R 12 Fmoc and R 13 Dde. In another preferred embodiment, R 12 Dde and R 13 Fmoc.

[0035] For example, if R 12 Dde and R 13 Fmoc, the following synthesis route can be used. In this synthesis route, Fmoc deprotection of the compound of general formula II is carried out in a first step, resulting in a first reaction product of general formula III (Formula III). In a second step, an amide coupling is carried out between the first Reaction product of general formula III and a compound of general formula IV (Formula IV), whereby a second reaction product of the general formula V (Formula V). In a third step, an amide coupling is carried out between the second reaction product of general formula V and a compound of general formula VI (Formula VI), whereby a third reaction product of the general formula VII (Formula VII) is obtained. In a fourth step, an Fmoc-Ab cleavage of the compound of general formula VII is carried out, whereby a fourth reaction product of general formula VIII is received.

[0036] In a fifth step, a fifth reaction product of the general Formula IX synthesized, where D corresponds to group A in a compound of general formula I, except that the reactive side groups are protected. For this purpose, a compound of general formula VIII is reacted with one of the compounds D1 to D8 and a compound that provides a carbonyl group in the compound of general formula IX. This compound can be, for example, N,N-carbonyldiimidazole or triphosgene.

[0037] In a sixth step, a Dde-Ab cleavage of the compound of general formula IX is carried out, whereby a sixth reaction product of general formula X (Formula X) is obtained.

[0038] In a seventh step, a seventh reaction product of the general formula XI (Formula XI) synthesized. The unit X p corresponds to the unit X pof general formula I, with the proviso that reactive side chains are protected with protecting groups. For example, -COOH can be protected as -COOtBu. To synthesize a reaction product of general formula XI, an amide coupling is carried out between the sixth reaction product of general formula X and a compound of general formula Fmoc-X-OH when p = 1. When p = 2, a first amide coupling is carried out between the sixth reaction product of general formula X and a first compound of general formula Fmoc-X-OH to obtain an intermediate, and after Fmoc deprotection of the intermediate, a second amide coupling is carried out between the Fmoc-deprotected intermediate and a second compound of general formula Fmoc-X-OH.When p = 3, a first amide coupling is carried out between the sixth reaction product of general formula X and a first compound of general formula Fmoc-X-OH to obtain a first intermediate, and after Fmoc deprotection of the first intermediate, a second amide coupling is carried out between the Fmoc-deprotected first intermediate and a second compound of general formula Fmoc-X-OH to obtain a second intermediate, and after Fmoc deprotection of the second intermediate, a third amide coupling is carried out between the Fmoc-deprotected second intermediate and a third compound of general formula Fmoc-X-OH.

[0039] In a seventh step, a seventh reaction product of the general formula XII (Formula XII) synthesized. The unit Y q corresponds to the unit Y qof general formula I, provided that reactive side chains are protected with protecting groups. For example, -COOH can be protected as -COOtBu. To synthesize a reaction product of general formula XII, Fmoc deprotection of the reaction product of general formula XI is carried out, followed by an amide coupling between the Fmoc-deprotected reaction product of general formula XI and a compound of general formula Fmoc-Y-OH, when q = 1.When q = 2, Fmoc deprotection of the reaction product of general formula XI is carried out and then a first amide coupling is carried out between the Fmoc-deprotected reaction product of general formula XI and a first compound of general formula Fmoc-Y-OH to obtain an intermediate, and after Fmoc deprotection of the intermediate, a second amide coupling is carried out between the Fmoc-deprotected intermediate and a second compound of general formula Fmoc-Y-OH.When q = 3, Fmoc deprotection of the reaction product of general formula XI is carried out and then a first amide coupling is carried out between the Fmoc-deprotected reaction product of general formula XI and a first compound of general formula Fmoc-Y-OH to obtain a first intermediate, and after Fmoc deprotection of the first intermediate, a second amide coupling is carried out between the Fmoc-deprotected first intermediate and a second compound of general formula Fmoc-Y-OH to obtain a second intermediate, and after Fmoc deprotection of the second intermediate, a third amide coupling is carried out between the Fmoc-deprotected second intermediate and a third compound of general formula Fmoc-Y-OH.

[0040] In a next step, an Fmoc deprotection of the reaction product of the general formula XII is carried out and then a coupling between the resulting reaction product and a compound of the general formula Z-AG is carried out, whereby a reaction product of the general formula XIII (Formula XIII) is obtained, where AG is a suitable leaving group. The unit Z corresponds to the unit Z of the general formula I, with the proviso that reactive side chains are protected with protecting groups. For example, -COOH can be protected as -COOtBu. In a next step, the reaction product of the general formula XIII is cleaved from the 2-CTC resin and deprotected by removing all protecting groups, whereby the compound of the general formula I is obtained. In formulas II to XIII, the indices k, m, n, p and q have the meanings given in connection with the general formula I.

[0041] The synthesis of precursors of the compounds of general formula I for subsequent radiosynthesis can be carried out starting from a compound of general formula XII (Formula XII) can be done in different ways.

[0042] In a first possible synthetic route, the Fmoc group of a compound of general formula XII is first cleaved and coupled with a compound of general formula E-AG, where group E corresponds to group Z in the compounds of general formula I, except that the corresponding halogen is replaced by a suitable leaving group for radiolabeling, such as a nitro- or trimethylammonium group, and AG is a suitable leaving group for the coupling reaction. Subsequently, the compound is cleaved from the resin, all protecting groups are removed, and the resulting compound is purified, for example, by reversed-phase high-performance liquid chromatography, and subsequently subjected to radiosynthesis.

[0043] In a second possible synthetic route, the Fmoc group of a compound of general formula XII is first cleaved. The resin is then cleaved, and all protecting groups are removed. This is followed by coupling with a compound of general formula E-AG, where E and AG have the meanings explained in the previous paragraph. The resulting compound is then purified, for example, by reversed-phase high-performance liquid chromatography, and subsequently subjected to radiosynthesis.

[0044] In another possible synthesis route, the Fmoc group of a compound of general formula XII is first cleaved. The resin is then cleaved, all protecting groups are removed, and the resulting compound is purified, for example, by reversed-phase high-performance liquid chromatography. Only then is the coupling carried out with a compound of general formula Z-AG, where Z has the meaning defined in connection with the compounds of general formula I, AG is a suitable leaving group for the coupling reaction, and the halogen is selected from the group consisting of [ 18 F]Fluorine, [ 123 I]Iodine, [ 124 I]Iodine, [ 125 I]Iodine, [ 126 I]Iodine, [ 128 I]Iodine, [ 130 I]Iodine, [ 131 I]Iodine, [ 209 At]Astatine, [ 210 At]Astatine and [ 211 At] Astatine exists.

[0045] This is followed by an explanation of various possibilities of radiosynthesis using the example of a [ 18 F]Fluorine radiosynthesis. To produce a Compound of general formula I, in which Z is ZI, where R is H, Gi is CR a with R a = H, G2 C-Rb with Rb = H, G3 CR c with R c = [ 18 F], G4 is N and v is 0, a precursor can be used which corresponds to the compound of general formula I, except that a leaving group LG is provided instead of the group Rc.

[0046] The radiosynthesis of [ 18 F]Fluorine radioligands according to the invention can be achieved by various methods for nucleophilic aromatic substitution, as known from the literature, which differ only in the nature of the precursor used, in particular the leaving group, which is activated by [ 18F]fluoride is to be replaced. In one embodiment, precursor compounds suitable for a one-step radiosynthesis have been provided with leaving groups that can be selected from a halide (e.g., bromide, iodide), hydroxyl, diazonium, trialkylammonium (e.g., trimethylammonium), sulfonate (e.g., mesylate, tosylate, triflate), stannane, aryliodonium, thiophenium, boronic acid, boronic acid ester, or nitro group. where LG is a leaving group.

[0047] The precursor compounds can also be produced as salts and used in this form for radiosynthesis. These salts of the precursor compounds can be inorganic salts, such as halides (e.g., chloride, iodide), phosphates, and sulfates, or organic salts, such as acetate, formate, benzoate, ascorbate, sulfonates (e.g., triflate, tosylate), or trifluoroacetate (TFA). Precursor compounds particularly suitable for one-step radiosynthesis are where various negatively charged counterions such as trifluoroacetate, acetate, chloride, bromide or iodide are possible, and

[0048] The direct radiolabeling of the precursors is carried out using a fluorine-18 complex in which [ 18 F]fluoride is activated by a phase transfer catalyst, such as basic tetraalkylammonium salts, e.g., tetrabutylammonium phosphate, carbonate or preferably bicarbonate, or aminopolyethers, such as Kryptofix 2.2.2, in the presence of potassium carbonate, lithium carbonate, cesium carbonate or oxalate. In particular, general 18F-activation methods by Kryptofix 2.2.2 or tetrabutylammonium bicarbonate in a polar aprotic solvent selected from acetone, 1,4-dioxane, tetrahydrofuran, N-methylpyrrolidinone, dimethoxyethane, dimethylacetamide, N,N-dimethylformamide and hexamethylphosphoramide, preferably acetonitrile and dimethyl sulfoxide (DMSO), including mixtures thereof.

[0049] In a further embodiment, suitable precursor compounds were prepared by a two-step radiosynthesis according to the following scheme where LG is a leaving group. The coupling was then carried out to a compound of the general formula IN (Formula IN)

[0050] The radiosynthesis of radiohalogenated ligands according to the invention can be carried out by applying generally known methods for radiohalogen labeling as described in the literature (Coenen, HH; Ermert, J. 18F-labelling innovations and their potential for clinical application. Clin Transl Imaging 2018, 6, 169-193; Schubiger, PA; Lehmann, L.; Friebe, M. PET chemistry: The driving force in molecular imaging,' Springer: Berlin, New York, 2007, ISBN 3540495274; Wang, Y.; Lin, Q.; Shi, H.; Cheng, D. Fluor-18: Radiochemistry and Target-Specific PET Molecular Probes Design. Front. Chem. 2022, 10, 884517; Goud, NS; Joshi, RK; Bharath, RD; Kumar, P. Fluorine-18: A Radionuclide with diverse range of radiochemistry and synthesis strategies for target-based PET diagnosis. European Journal of Medicinal Chemistry 2019, 187, 111979). In general, strategies for [ 18F]Fluorine labeling can be divided into electrophilic and nucleophilic substitution, including modular assembly approaches, e.g., using prosthetic groups (Ermert, J. 18 F-Labeled Intermediates for Radiosynthesis by Modular Build-Up Reactions: Newer Developments. BioMed Research International 2014, 2014, 1-15; van der Bom, Dion; Pees, A.; Poot, A.J.; Orru, Romano VA; Windhorst, AD; Vugts, DJ Fluorine-18 labeled building blocks for PET tracer synthesis. Chem. Soc. Rev. 2017, 46, 4709-4773; Kniess, T.; Laube, M.; Brust, P.; Steinbach, J. 2-[18F]Fluorethyl tosylate - a versatile tool for building 18F-based radiotracers for positron emission tomography. Med. Chem. Commun. 2015, 6, 1714-1754) and approaches using umpolung for either the reaction center of the precursor or the radiohalogen-containing reactant (Tredwell, M.; Gouverneur, V. 18F Labeling of Arenes. Angew. Chem. Int. Ed. 2012, 57, 11426-11437). A person skilled in the art is able to develop suitable strategies for radiosynthesis and select suitable methods for this purpose depending on the chemical structure of the halogen ligand.

[0051] The radiosynthesis of 18 F-labelled radioligands according to formula I can be carried out by performing each step of the procedure using general laboratory equipment suitable for radiochemical work, as is the state of the art. Individual steps can also be carried out using automated systems that are commercially available from various manufacturers. Similarly, the entire manufacturing procedure can be carried out using such automated systems, as is widely documented in the literature and is a prerequisite for clinical application (Shamni, O.; Nebeling, B.; Grievink, H.; Mishani, E. Fine-tuning of the automated [ 18 F]PSMA-1007 radiosynthesis. J. Label. Compd. Radiopharm. 2019; I Sachinidis, J.; Poniger, S.; J Tochon-Danguy, H. Automation for Optimized Production of Fluorine-18-Labeled Radiopharmaceuticals. Current Radiopharmaceuticals 2010, 3, 248-253; Barnes, C.; Nair, M.; Aboagye, E.O.; Archibald, S.J.; Allott, L. A practical guide to automating fluorine-18 PET radiochemistry using commercially available cassette-based platforms. React. Chem. Eng. 2022, 7, 2265-2279).

[0052] After the 18 F -labeling step of radiosynthesis of 18Once the F-labeled radioligands of Formula I are complete, purification, isolation, and formulation of these compounds can be performed using semi-preparative HPLC or solid-phase extraction (SPE), or a combination of both. Finally, a pharmaceutically acceptable solvent (e.g., saline) or a suitable buffer (e.g., PBS) is used to formulate the product.

[0053] The synthesis of the compounds of general formula I is described in more detail in the Examples section.

[0054] Surprisingly, imaging studies in a LNCaP xenograft tumor model revealed that compound [ 18F]E514 exhibits advantageous pharmacokinetic properties (low retention in blood, faster and mainly renal excretion) and a more favorable organ distribution (high tumor uptake with simultaneous low to moderate uptake in non-target organs such as kidneys) than those known from the state of the art. The higher tumor-to-muscle, tumor-to-kidney, and tumor-to-blood ratios compared to the state of the art were achieved already 1 hour after injection. In addition, a further increase in the tumor-to-muscle, tumor-to-kidney, and tumor-to-blood ratios was observed up to 2 hours after injection. This significantly higher and faster achieved tumor contrast shows that compound [ 18 F]E514 enables improved visualization of PSMA-positive tumors at earlier acquisition times compared to the state of the art.

[0055] In the following, the invention will be explained in more detail by means of non-limiting examples with reference to the drawings. Fig. 1 the positron emission tomograms of LNCaP tumor-bearing mice at time points 4-6, 50-60 and 105-120 min after injection of the reference compound [ 18 F]PSMA-1007 and the compound according to the invention [ 18 F]E514 in comparison, with both compounds showing the highest activity concentrations in tumors, kidneys, and salivary glands. (B) Bladder, (D) Intestine, (H) Heart, (L) Liver, (M) Muscle, (N) Kidneys, (S) Salivary glands, (T) Tumor; (SUV) standardized uptake value, (BW) Body weight; and Fig. 2 shows the time courses of tissue activity concentrations and tumor contrast during PET of LNCaP tumor-bearing mice with the reference compound [ 18 F]PSMA-1007 and the compound according to the invention [ 18F]E514 in comparison. Quantitative image analysis of positron emission tomograms; (A) Activity concentration in the internal blood volume of the heart; (BC) proportion of excretion or retention of activity in the renal (kidneys + urinary bladder) and hepatobiliary (gallbladder + liver + intestine) organ systems; (DF) Activity concentrations in typical target tissues and demonstration of binding specificity by PSMA blockade with 50 mg / kg 2-PMPA; (GI) Contrast between the activity concentrations in the tumor and selected organs; (SUV) standardized uptake value, mean values ​​(n = 3) ± standard error of the mean. Examples General procedure for the synthesis of compounds E514, E587, E588, E589. E590, E591, E592 and E593

[0056] Compounds E514, E587, E588, E589, E590, E591, E592 and E593 were synthesized by solid phase peptide synthesis (SPPS) on 2-chlorotrityl resin.

[0057] The synthesized molecules were analyzed using reversed-phase high-performance liquid chromatography (RP-HPLC; Ascentis Express CI 8, 150 x 4.6 mm; Supelco, Germany) with a linear AB gradient (5% B to 100% B in 10 min) at a flow rate of 1.5 ml / min (analysis). Purification was performed using reversed-phase high-performance liquid chromatography (RP-HPLC; Gemini-NX CI 8, 250 x 50 mm; Phenomenex, Germany) with a linear AB gradient at a flow rate of 100 ml / min. Solvent A consisted of 0.1% aqueous TFA, and solvent B was 0.1% TFA in ACN.

[0058] The HPLC system (Dionex Ultimate 3000; Thermo-Fisher, Germany) was equipped with a UV detector. UV absorption was measured at 200, 210, and 230 nm. Mass spectrometry was performed using an LC-MS system (Dionex 3000, Thermo-Fisher, Germany). Examples 1 to 6 Synthesis of intermediates

[0059] The following intermediates were prepared for the preparation of compounds E514, E587, E588, E589, E590, E591, E592 and E593. Example 1 Synthesis of intermediate 101: Glu-CO-Im ((Di-tert-butyl-(lH-imidazole-l-carbonylD-L-glutamate))

[0060] H-Glu(OtBu)-OtBu (29.59 g, 1 eq, 100 mmol) was dissolved in 400 mL of DCM. Triethylamine (25.3 g, 2.5 eq, 250 mmol) was added slowly. Carbonyldiimidazole (17.84 g, 1.1 eq, 110 mmol) was added portionwise. The reaction was stirred at RT for 4 h. The reaction solution was washed with water, NaHCl, and brine. The organic phase was dried over Na2SO4 and concentrated in vacuo to give 101 as an oil. Example 2 Synthesis of intermediate 102: N,NN-Trimethyl-5-((2.3.5.6-tetrafluorophenoxy)carbonyl)pyridine-2-aminium chloride © © CI

[0061] 8.0 g (50.78 mmol) of 6-chloronicotinic acid was dissolved in 250 mL of 1,4-dioxane. 8.6 mL (50.78 mmol) of diisopropylcarbodiimide and 8.43 g (50.78 mmol) of 2,3,5,6-tetrafluorophenol were added, and the reaction mixture was stirred overnight. The precipitate was filtered off. The filtrate was concentrated in vacuo and then purified by column chromatography (DCM / petroleum ether, 2:1, v / v). The product-containing fractions were collected, concentrated in vacuo, and dissolved in 150 mL of a 2M solution of trimethylamine in THF. The reaction was stirred for 3 h at RT. The precipitate was filtered off and washed with diethyl ether to afford 102 as a white solid. Example 3 Synthesis of Intermediate 103: 2.5-Dioxopyrrolidin-l-yl 6-nitronicotinate

[0062] To a solution of 0.5 g (2.97 mmol) of 6-nitronicotinic acid in 10 ml of DMF, 1.02 ml (5.94 mmol) of DIPEA and 1.79 g (5.94 mmol) of TSTU (O-[N-succinimidyl)-1,1,3,3-tetramethyluronium tetrafluoroborate) were added. The reaction was carried out at RT for Stirred for 1 h and then diluted with ethyl acetate. The resulting solution was Washed twice with 1 M HCl, twice with saturated NaHCl solution, and 1x with saturated sodium chloride solution. The organic phase was dried over sodium sulfate and concentrated in vacuo to give 103 as a pale yellow solid. Example 4 Synthesis of Intermediate 601: (3S.7S.26R.29S.32S)-32-Amino-29-(2-carboxyethyl)-5.13.20.28.3 l-pentaoxo-4.6.12.21.27.30-hexaazatetratriacontane- L3.7.26.34-pentacarboxylic acid

[0063] Starting from 406, Fmoc deprotection was performed using 20% ​​piperidine in DMF. The resin was washed with DMF (3 x 5 mL), DCM (3 x 5 mL), IPA (3 x 5 mL), and Et2O (3 x 5 mL). Cleavage from the resin and removal of all protecting groups were performed using TFA / TIS / water / DODT (v / v / v; 92.5 / 2.5 / 2.5 / 2.5). The crude product was precipitated from ice-cold diethyl ether and purified by preparative RP-HPLC (gradient: 95% ACN, 5% H2O, and 0.1% TFA: 5–50% in 60 min), yielding 601 as a white solid (isolated yield: 335 mg). RP-HPLC (5 to 100% B in 12 min): tR = 3.28 min. Calculated monoisotopic mass (C36H59N7O17): 861.40; found: m / z = 431.92 [M+2H] 2+ , 862.55 [M+H] + . Example 5

[0064] Starting from 406, Fmoc deprotection was carried out using 20% piperidine in DMF, and then the free amine was treated with 102 and triethylamine in DMF to give 501. The resin was washed with DMF (3 x 5 mL), DCM (3 x 5 mL), IPA (3 x 5 mL), and E^2O (3 x 5 mL). Cleavage from the resin and removal of all protecting groups was performed using TFA / TIS / water / DODT (v / v / v; 92.5 / 2.5 / 2.5 / 2.5). The crude product was precipitated from ice-cold diethyl ether and purified by preparative RP-HPLC (gradient: 95% ACN, 5% H2O and 0.1% TFA: 5 - 50% in 60 min), yielding E529 (TFA salt) as a white to off-white solid (isolated yield: 588 mg). RP-HPLC (5 to 100% B in 12 min): tR = 3.4 min. Calculated monoisotopic mass (C 45 H7oN90i8 + ): 1024.48; found: m / z = 1024.57 [M] + . Example 6 Synthesis of compound E551

[0065] 601 was dissolved in DMF and treated with 103 and triethylamine for 2 hours. The solvent was removed in vacuo, and the residue was purified by preparative RP-HPLC (gradient: 95% ACN, 5% H2O, and 0.1% TFA: 5-30% in 40 min), affording E551 (TFA salt) as a white solid (yield: 52 mg). RP-HPLC (5 to 100% B in 12 min): tR = 3.9 min. Calculated monoisotopic mass (C42H61N9O2O): 1011.40; found: m / z = 1010.39 [MH]'. Example 7 Synthesis of compound E514

[0066] The synthesis of the compound E514 according to the invention E514 is shown in Scheme EX-1. Scheme EX-1 In Scheme EX-1, letter a) indicates the use of 20% piperidine in DMF; letter b) the use of Sub-NHS, DIPEA, and DMF; letter c) the use of TSTU, DIPEA, and DMF; letter d) the use of Fmoc-Lys-OtBu, DIPEA, and DMF; letter e) the use of Glu-CO-Im (101), NMM, and DMF; letter f) the use of 2% N2H4H2O in DMF; letter g) Fmoc-Glu(OtBu)-OH, HATU, HO At, DIPEA, and DMF; letter h) 6-Fluomicotonic acid, PyBOP, HOBt, DIPEA, and DMF; letter i) TFA / TIS / water (vol / vol / vol; 95 / 2.5 / 2.5)

[0067] For the synthesis of compound E514, Fmoc-D-Lys(Dde)-OH was loaded onto 2-CTC resin (in Scheme EX-1, the resin is circled). The synthesis of E514 was carried out on 2-CTC resin. Dde-D-Lys(Fmoc)-OH was loaded onto 2-CTC resin (401), and Fmoc was deprotected with 20% piperidine in DMF. Subsequently, suberic acid mono-NHS ester (2 eq.) and DIPEA (2 eq.) were dissolved in DMF (1 mL) and allowed to react with the resin-bound peptide for 2 h at RT. The free carboxylic acid (402) was then treated with TSTU (2 eq.) and DIPEA (2 eq.) in DMF (10 mL / g resin) at RT for 1 h. After formation of the NHS ester, the resin-bound peptide was treated with Fmoc-Lys-OtBu (2 eq.) and DIPEA (2 eq.) in DMF (2 mL) for 2 h at room temperature to synthesize 403. Fmoc deprotection was achieved using 20% ​​piperidine in DMF. Then, the free amine was treated with Glu-CO-Im (intermediate 101, 3 eq.) and NMM (3 eq.) in DMF for 2 h at room temperature to yield 404.To synthesize 405, Dde deprotection was performed using 2% hydrazine monohydrate in DMF, followed by Fmoc-Glu(OtBu)-OH (2 eq.) activation with HATU (2 eq.), HO At (2 eq.), and DIPEA (5.6 eq.) in DMF and addition to the resin. The reaction mixture was shaken for 2 h at RT. Fmoc deprotection was performed using 20% ​​piperidine in DMF, followed by Fmoc-Glu(OtBu)-OH (2 eq.) activation with HATU (2 eq.), HO At (2 eq.), and DIPEA (5.6 eq.) in DMF and addition to the resin, yielding 406 after 2 h of shaking at RT. To synthesize 407, 6-fluoronicotinic acid (2 eq.) was conjugated to the peptide sequence using PyBOP (2 eq.), HOBt (2 eq.), and DIPEA (2 eq.) in DMF for 2 h at RT. The resin. was washed with DMF (3 x 5 ml), DCM (3 x 5 ml), IPA (3 x 5 ml), and Et2O (3 x 5 ml). Cleavage from the resin and removal of all protecting groups were performed using TFA / TIS / water (v / v / v; 95 / 2.5 / 2.5).

[0068] The crude product was precipitated from ice-cold diethyl ether and purified by preparative RP-HPLC (gradient: 95% ACN, 5% H2O, and 0.1% TFA: 5–50% in 60 min), yielding compound E514 (TFA salt) as a white to off-white solid (isolated yield: 155 mg). RP-HPLC (5–100% B in 12 min): tR = 3.8 min. Calculated monoisotopic mass (C42H61FN8O18): 984.99; found: m / z = 985.22 [M+H] + . Example 8 Synthesis of compound E587 E587

[0069] The synthesis of compound E587 was carried out according to the procedure described in Example 7, except for the use of Fmoc-L-Tyr(OtBu)-OH instead of Fmoc-Glu(OtBu)-OH. The crude product was purified by preparative RP-HPLC to yield E587. Calculated monoisotopic mass (C46H 63 FN8Oi7): 1018.43; found: m / z = 510.21 [M+2H] 2+ , 1019.35 [M+H] + . Example 9 Synthesis of compound E588 E588

[0070] The synthesis of compound E588 was carried out according to the procedure described in Example 7, except for the use of Fmoc-L-Phe-OH instead of Fmoc-Glu(OtBu)-OH. The crude product was purified by preparative RP-HPLC to yield compound E588. Calculated monoisotopic mass (C50H65FN8O14): 1020.46; found: m / z = 1021.28 [M+H] + . Example 10 Synthesis of compound E589

[0071] The synthesis of compound E589 was carried out according to the procedure described in Example 7 except for the use of Fmoc-L-Tyr(OtBu)-OH and Fmoc-L-Arg(Pbf)-OH instead of Fmoc-Glu(OtBu)-OH. The crude product was purified by preparative RP-HPLC to yield E589. Calculated monoisotopic mass (C47H68FN11O15): 1045.49; found: m / z = 523.89 [M+2H] 2+ . Example 11 Synthesis of compound E590

[0072] The synthesis of compound E590 was carried out according to the procedure described in Example 7, except for the use of Fmoc-L-Tyr(OtBu)-OH instead of Fmoc-Glu(OtBu)-OH. The crude product was purified by preparative RP-HPLC to yield E590. Calculated monoisotopic mass (C5oH65FN8Oi6): 1052.45; found: m / z = 527.26 [M+2H] 2+ , 1053.33 [M+H] + . Example 12 Synthesis of compound E591

[0073] The synthesis of compound E591 was carried out according to the procedure described in Example 7, except for the use of Fmoc-L-Tyr(3I)-OH instead of Fmoc-Glu(OtBu)-OH. The crude product was purified by preparative RP-HPLC to yield E591. Calculated monoisotopic mass (C46H66FIN8O17): 1144.33; found: m / z = 573.30 [M+2H] 2+ . Example 13 Synthesis of compound E592

[0074] The synthesis of compound E592 was carried out according to the procedure described in Example 7, except for the use of Fmoc-L-Asp(OtBu)-OH instead of Fmoc-Glu(OtBu)-OH. The crude product was purified by preparative RP-HPLC to yield E592. Calculated monoisotopic mass (C40H57FN8O18): 956.38; found: m / z = 479.37 [M+2H] 2+ , 957.38 [M+H] + . Example 14 Synthesis of compound E593 Compound E593 was synthesized according to the procedure described in Example 7, except for the use of Fmoc-L-Gln(Trt)-OH instead of Fmoc-Glu(OtBu)-OH. The crude product was purified by preparative RP-HPLC to yield E593. Calculated monoisotopic mass (C42H63FN10O16): 982.44; found: m / z = 983.71 [M+H] + . Example 15 Manual radiosynthesis of [18 F]E514

[0075] For manual synthesis, a reactive 18 F-containing phase transfer complex, by loading 18 F-fluoride was applied to a QMA cartridge and eluted with 1.2 ml of TBA bicarbonate (0.075 M), followed by azeotropic drying using a total of 3.7 ml of acetonitrile (ACN) at 105 °C for 17 min. After dissolving the residue in 2.1 ml of DMSO, 1 ml was taken for radiolabeling and 1 mg of E529 was added, followed by heating at 95 °C for 15 min. After cooling, the mixture was diluted with 20 ml of water and loaded onto a Sep-Pak CI 8 Plus short cartridge (Waters, Milford, MA, USA), which was washed with 30 ml of water. The product was eluted with 3 ml of 20% EtOH, to which the same volume of PBS was added, yielding the product for testing and application. Starting from 4.1 GBq 18 F-fluoride for radiolabeling was 760 MBq [ 18F]E514 was obtained within a total synthesis time of ~45 min. The radiochemical purity (RCP) was 89.5% (radio-reverse-phase high-performance liquid chromatography (radio-RP-HPLC) method 2, see below). Example 16 Automated radiosynthesis of [ 18 F]E514

[0076] For the preparation of the compound according to the invention [ 18 F]E514, an automated synthesis was set up on an “RN - plus research self clean” module (Synthra, Hamburg, Germany), which included the following steps: 1. Generation of a reactive 18 F-containing phase-transfer complex; 2. Radiolabeling; 3. Purification by semi-preparative radio-HPLC; 4. Final purification and concentration by SPE; 5. Evaporation of the solvent and formulation. Step 1: 18F-fluoride was concentrated on a QMA cartridge and eluted with 0.75 ml of TBA bicarbonate (0.075 M), followed by azeotropic drying using 1.9 ml of ACN at 98 °C for 12 min. Step 2: Radiolabeling was achieved by adding 0.5 mg of E551 in 0.5 ml of DMSO and heating at 125 °C for 6 min. Step 3: 4 ml of water was added to the reaction mixture, and purification was performed on a ReproSil Gold column (10 pm, 50 x 10 pm; Dr. Maisch HPLC GmbH, Ammerbuch, Germany) using 15% ACN (0.1% TFA) as the mobile phase at a flow rate of 3 ml / min. The product fraction was collected and diluted in 20 ml of water. Step 4: The diluted product fraction was loaded onto a Sep-Pak C18 Plus light cartridge (Waters, Milford, MA, USA), which had been previously conditioned with 1.0 ml of EtOH and 5 ml of water.After evaporation of the solvent by a stream of helium at 40 °C, 250 μl of PBS was added (mixing), obtaining the product for further use.

[0077] Starting from 31 GBq 18 F-fluoride was 2.8 GBq [ 18 F]E514 was obtained within a total synthesis time of ~70 min. The RCP was 99.0% (Radio-RP-HPLC method 2, see below) and the identity of the product was confirmed by Radio-RP-HPLC method 3 and co-injection of unlabeled E514, which was added during the measurement with [ 18 F]E514 co-eluted.

[0078] Quality control of the resulting product was performed by radio-reverse-phase high-performance liquid chromatography (radio-RP-HPLC). A total of four methods (1-4) were used. Table 1 lists the process parameters and conditions of the radio-RP-HPLC methods used for various purposes. In addition, radio-thin-layer chromatography (radio-DSC) was performed on silica gel DSC films (POLYGRAM SIL G UV254, Macherey-Nagel, Düren, Germany) using acetone / ammonium acetate (2 M) 1:1. Table 1 * for all methods: UV detection at 205 nm # I: Poroshell 120 EC-C18 (3.5 pm, 100 x 3 mm; Agilent Technologies Deutschland, Waldbronn, Germany), II: Prodigy 5p C8 (5 pm, 250 x 4.6 mm; Phenomenex, Aschaffenburg, Germany) Example 17 Determination of molar activity

[0079] To determine the molar activity, a sample of the final [ 18 F]E514 product solution was measured with a calibrated activity meter and an aliquot was analyzed by radio-RP-HPLC using method 2. Using a standard calibration of non-radioactive E514 in five different concentrations at 205 nm, the amount of E514 in the product solution was determined and the molar activity (expressed in GBq / pmol at a given time point) was calculated. Example 18 Determination of logD values

[0080] LogD values ​​were determined for various media using the shake-flask method (Andres, A. et al. Setup and validation of shake-flask procedures for the determination of partition coefficients (logD) from low drug amounts. Eur. J. Pharm. Sci. 2015, 76, 181-191). During preparation, n-octanol was saturated with each of the buffer solutions used (Na phosphate, TRIS buffer, PBS). These buffer solutions were also preparatory to saturation with n-octanol. 3-5 μl of the 18 F-labeled radioligand in ethanol (20-25 MBq, samples in quadruplicate) was added and shaken vigorously for 30 min. After centrifugation at 9,000 rpm for 15 min, samples were taken from both separated phases and measured with a gamma counter. LogD values ​​were calculated as the ratio of radioactivity determined in n-octanol and aqueous phase and are presented in Table 2. Table 2 Example 19 Stability studies

[0081] The stability of [ 18 F]E514 was determined in various media using freshly prepared 18 F-labeled radioligand solution (10-20 MBq, 50-150 μl) was added to the respective medium (450-950 μl), rapidly vortexed, and gently shaken at the temperature specified below. Samples were taken at specific time points, diluted, and measured by radio-RP-HPLC. The analysis of plasma samples included protein precipitation with 4 times the volume of an ice-cold mixture of methanol / water (4 / 1, v / v), vigorous shaking (5 min), and centrifugation (14,000 rpm, 10 min) prior to supernatant analysis. Furthermore, the recovery of extracted activity was calculated using a gamma counter after measuring the activity of the supernatant and residue. Table 3: Product stability at room temperature * determined by radio-RP-HPLC: Method 2 for PBS and NaCl, Method 1 for plasma, expressed as mean ± SD (n = 2-6) # for PBS: n = 2, for plasma data: n = 2-3 & the recovery of activity after plasma precipitation is given in parentheses ([]) Example 20 In vitro assays

[0082] Example 20 describes in vitro assays conducted to determine the properties of the compounds of the invention. a) Cell culture

[0083] Displacement and internalization assays were performed using the highly PSMA-expressing human prostate cancer cell line LNCaP (ATCC® CRL-1740) and the PSMA-negative cell line PC3 (ATCC® CRL-1435). Cells were cultured as monolayers at 37°C in a humidified atmosphere with 5% CO2 and 95% air gassing in RPMI medium containing 10% FCS (Merck KGaA, Germany). After washing the confluent cells twice with phosphate-buffered saline (PBS) and detaching them with trypsin / EDTA (0.05% / 0.02%), the cells were mixed in medium and counted (Casy TT, Omni Life Science, Germany). Two days before the assays, LNCaP and PC3 cells were seeded as monolayers, 1 * 10 5 Cells / well in 24-well plates. b) 68 Ga labeling of PSMA-11

[0084] A 68 Ga generator was purchased from iThemba LABS (Republic of South Africa). PSMA-11 (4-8 pg = 4.2-8.4 nmol) was used with 68Ga (100 to 200 MBq) in a mixture of ammonium acetate (2 M) and HCl at pH 4.5. The reaction mixture was incubated for 10 min at 90 °C. Quality control of radiolabeled PSMA-11 was performed using RP-HPLC with a C-18 reversed-phase column (semi-preparative Zorbax 300SB-C18, 9.4 x 250 mm 5 pm; Agilent Technologies, USA). The radiochemical yield was >97% for [ 68 Ga]Ga-PSMA-11 at molar activities between 30 and 60 GBq / pmol. c) Determination of the competitive binding affinity

[0085] For displacement assays, after aspirating the medium from the cells, 100 μl of PBS and 100 μl of various concentrations of the test substance in PBS (final concentrations 10' 12 up to 10' 6 M) was pipetted onto the cell layer. At the same time, 400 μl of medium containing [ 68 Ga]Ga-PSMA-l 1 (final concentration 1 nM) was added. After 1 hour of incubation at 37 °C, the supernatants were aspirated, and the cells were washed twice with cold PBS. The cell layer was lysed with 500 μl of NaOH / SDS (0.1 M / 1%) by shaking for 3 to 5 minutes. After transferring the lysates to volumetric tubes, the activity of the samples was measured in a gamma counter (2480 Automatic Gamma Counter Wizard 2, Perkin Elmer, USA).

[0086] From the displacement assay data, the half-maximal inhibitory concentrations (IC50) were calculated using a non-linear curve fitting program (GraphPad Prism 9). With the Kd value of [ 68 Ga]Ga-PSMA-l 1 on LNCaP (12.4 nM) and the known concentration of [ 68 Ga]Ga-PSMA-l 1 in the displacement assay (1 nM), the inhibition constants (Ki) were determined using the non-linear curve fitting program.

[0087] For the non-radioactive reference compound E514, a Ki of 9.5 ± 1.0 nM was determined. d) Determination of internalization

[0088] For internalization assays, after aspirating the medium from the cells, 100 μl of PBS and in adjacent wells 100 μl of PMPA (final concentration 10' 4 M) for samples for non-specific binding were pipetted onto the cell layer. At the same time, 400 μl of medium with [ 18F]E514 (final concentration 25 nM) was added. After 1 hour of incubation at 37°C and additional samples at 4°C, the supernatants were aspirated, and the cells were washed twice with cold PBS. Surface-bound activity was expelled with 4°C acidic wash buffer (0.2 M glycine, pH 2.8) for 5 min. The acidic wash buffer was transferred from each well into measuring tubes, as was the PBS buffer after a single wash (ligand binding to the cell surface). Cytosolic activity was determined after treatment with cell lysis buffer NaOH / SDS (0.1 M / 1%). Cell surface activity and cytosolic activity were measured separately in a gamma counter.

[0089] To determine the internalized percentage of applied [ 18F]E514 dose per milligram of protein (% AD / mg), the protein content of the cell lysate was determined using a spectrophotometer (NanoDrop, Thermo Fisher Scientific, USA) at an absorbance of 280 nm Table 4 * Mean ± SEM, a Total cell uptake = binding + internalization Example 21 Tumor model and animal testing

[0090] All animal experiments were conducted in accordance with the German Animal Welfare Act and approved by the local ethics committee for animal experiments. A PSMA-positive prostate cancer xenograft model was established by subcutaneous injection of human LNCaP cells into the right shoulder of 8- to 12-week-old male nude mice (Rj :NMRJ-Foxz?7”" / ", Janvier Labs, Le Genest-Saint-Isle, France). Imaging studies were performed when the subcutaneous tumors had reached a diameter of at least 6 mm. General anesthesia of the animals was induced and maintained by inhalation of 10% (v / v) desflurane in 30 / 70% (v / v) oxygen / air. During anesthesia, the animals were continuously warmed to 37°C. Positron emission tomography and image analysis

[0091] Positron emission tomography (PET) was performed using the nanoScan® PET / CT scanner (Mediso Medical Imaging Systems), which is specifically designed for small animals. Each animal received 10 MBq of the reference compound [ 18 F]PSMA- 1007 or the newly developed compound [ 18 F]E514, each administered in phosphate-buffered saline by intravenous injection via a tail vein catheter. The molar activity of both radiotracers was Each injection was 60 MBq / nmol. The specificity of target molecule binding was investigated by blocking the PSMA binding sites with 2-phosphonomethylpentanedicarboxylic acid (2-PMPA) at a dose of 50 mg / kg.

[0092] During PET, the emission of annihilation photons was recorded continuously in coincidence mode of 1:5 for 120 min starting at the time of radiotracer injection. A corresponding CT image was acquired for each PET scan and used for attenuation correction and as an anatomical reference. Events in the energy window of 400–600 keV were extracted from the three-dimensional list-mode images and sorted into 36 time frames with a sequence of 15^10 s, 5 x 30 s, 5 x 60 s, 4 x 300 s, 3 x 600 s, and 4 x 900 s. For all time frames, PET images were reconstructed using the three-dimensional Tera-Tom o™ algorithm with a voxel size of 0.4 mm, applying corrections for random events, scattering, attenuation, and decay.

[0093] All PET images were analyzed with ROVER (ABX, Radeberg, Germany) and displayed as maximum intensity projections with identical scaling of the activity concentration. Three-dimensional regions of interest (ROIs) were defined with fixed thresholds: tumor (39%), muscle (0%), heart (50%), kidneys (15%), liver (39%), salivary glands (39%), and bladder (10%). Activity concentrations in the tissue were determined as standardized uptake values ​​(SUV = [MBq detected activity / ml tissue] / [MBq injected activity / g body weight]) and expressed as SUVmeaw (mean within the ROI). Time-activity curves were generated and analyzed with Prism (GraphPad Software, San Diego, CA, USA). Data points of the time-activity curves were generated by summarizing measured values ​​(means) corresponding to the following mid-frame times: 1, 2, 5, 10, 20, 31, 55, 83, and 113 min. The areas under the curves (AUC) were calculated from 0-120 min.The statistical significance of the mean differences was tested using analysis of variance with Fisher's LSD test. Functional imaging with [ 18 F]E514 in LNCaP tumor-bearing mice

[0094] Fig. 1 shows the positron emission tomograms of LNCaP tumor-bearing mice at time points 4-6, 50-60 and 105-120 min after injection of the reference compound [ 18 F]PSMA-1007 and the compound according to the invention [ 18 F]E514 in comparison. Both compounds show the highest activity concentrations in tumors, kidneys, and salivary glands.

[0095] Fig. 2 shows the time courses of tissue activity concentrations and tumor contrast during PET of LNCaP tumor-bearing mice with the reference compound [ 18 F]PSMA-1007 and the compound according to the invention [ 18 F]E514 in comparison.

[0096] The distribution of [ 18F]E514 is characterized by lower retention in the blood, faster renal excretion and lower retention in the hepatobiliary excretory system compared to [ 18 F]PSMA-1007 (Fig. 1 AC). In addition, [ 18 F]E514 showed lower retention in kidneys and salivary glands compared to [ 18 F]PSMA-1007 (Fig. 2 DE). The enrichment of [ 18 F]E514 in tumor tissue reaches its steady state approximately 55 min after injection and remains almost constant until 120 min, while the activity concentration of [ 18 F]PSMA-1007 increases continuously from 0-120 min (Fig. 2 F). The blockade by 2-PMPA illustrates the PSMA specificity of the accumulation processes in kidneys, salivary glands, and tumors. Despite the lower activity concentration of [ 18 F]E514 in tumor tissue, the positron emission tomograms show a significantly higher tumor contrast after a shorter time compared to [18 F]PSMA-1007, for example, compared to the activity concentrations in blood (>55 min), kidneys (>10 min) and muscles (>55 min) (Fig. 2 G— I).

[0097] Table 5 shows the areas under the time-activity curves (ACU) during PET of LNCaP tumor-bearing mice with [ 18 F]PSMA-1007 and [ 18 F]E514 in comparison. The lower retention in kidneys, salivary glands and tumors, the faster renal excretion as well as the higher tumor-to-blood and Tumor-to-muscle contrast of [ 18 F]E514 is compared to [ 18 F]PSMA-1007 statistically significant. Table 5 (AUC) area under the curve 0-120 min after injection, (SUV) standardized uptake value, mean values ​​(n = 3) ± standard error of the mean; statistical significance of mean differences compared to the reference compound [ 18F ]PSMA-1007: * p < 0.033; ** p < 0.02; *** p < 0.001

[0098] Table 6 shows the tumor-to-background ratios in PET of LNCaP tumor-bearing mice with [ 18 F]PSMA-1007 and [ 18 F]E514 in comparison. The higher tumor-to-blood, tumor-to-kidney, and tumor-to-muscle contrasts of [ 18 F]E514 are compared to [ 18 F]PSMA-1007 statistically significant. Table 6 SUV ratios 105-120 min after injection; (SUV) standardized uptake value; mean values ​​(n = 3) ± standard error of the mean; statistical significance of mean differences compared to the reference compound [ 18 F]PSMA-1007: *p < 0.033; ** p < 0.02; *** p < 0.001 List of abbreviations Ac Acetate ACN Acetonitril AD applied dose AUC area under the curve Boc / c / V-Butoxycarbonyl CT computed tomography 2-CTC 2-chlorotrityl chloride DCM Dichloromethane Dde A-(l-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl) DIPEA AyV-Diisopropylethylamine DMF Dimethylformamide DMSO dimethyl sulfoxide DODT 3,6-Dioxa-1,8-octanedithiol DPBS Dulbecco's Phosphate-Buffered Saline EDTA ethylenediaminetetraacetic acid Et2O diethyl ether FCS fetal bovine serum Fmoc Fluorenylmethoxycarbonyl HATU (l-[Bis(dimethylamino)methylene]-lH-l,2,3-triazolo[4,5-b]pyridinium- 3-oxide hexafluorophosphate HO At l-Hydroxy-7-azabenzotriazole HOBt 1-Hydroxybenzotriazol IPA 2-Propanol LNCaP lymph node carcinoma of the prostate, human prostate cancer cell line logD partition coefficient NaOAc sodium acetate NMM 4-Methylmorpholine NHS A-Hydroxysuccinimid PBS Phosphate-buffered saline PC3 human prostate cancer cell line PET positron emission tomography 2-PMPA 2-(Phosphonomethyl)pentanedicarboxylic acid PSMA prostate-specific membrane antigen PyBOP (benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate RLT radioligand therapy ROI region of interest RP-HPLC reversed-phase high-performance liquid chromatography RPMI growth medium for cell culture RT room temperature SDS sodium dodecyl sulfate SEM Standard error of the mean SPECT single-photon emission computed tomography SPPS solid phase peptide synthesis Sub Suberic acid SUV standardized uptake value, standardized uptake value tBu Zc / V-Butyl TFA trifluoroacetic acid THF Tetrahydrofuran TIS Triisopropylsilane TRIS Tris(hydroxymethyl)aminomethane TSTU O-(A-Succinimidyl)-A,A,A‘,A‘-tetramethyluroniumtetrafluorborat VOI volume of interest, Volumen von Interesse

Claims

Patent claims 1. Compound of general formula I (Formula I) where A is an amino acid selected from the group consisting of , wherein the amino acids are optionally substituted: k is independently 0, 1, or 2 at each occurrence; m is independently 1, 2, 3, 4, or 5 at each occurrence; n is independently 0, 1, 2, or 3 at each occurrence; p is independently 1, 2, or 3 at each occurrence; q is independently 1, 2, or 3 at each occurrence; X and Y are substituted or unsubstituted amino acids; Z is selected from the group consisting of Z3 Z4, where v is 0, 1 or 2; Gi N or CR a is; G2 is N or C-Rb; G3 N or CR c is; G4 is N or C-Rd; where 0, 1 or 2 of Gi, G2, G3 and G4 is / are N; Ra, Rb, Rc and Ra, if present, are in each case independently selected from the group consisting of hydrogen, halogen, hydroxy, carboxyl, Ci-Ce alkyl, Ci-Ce alkoxy, cyano, nitro and Si(tBu)2F, provided that only 1 or 2 of Ra, Rb, Rc and Ra is / are halogen; and R is selected from the group consisting of hydrogen, halogen, hydroxy, Ci-Ce-alkyl and Ci-Ce-alkoxy.

2. A compound of general formula I according to claim 1, wherein the halogen is selected from the group consisting of [ 18 F]Fluorine, [ 123 I]Iodine, [ 124 I]Iodine, [ 125 I]Iodine, [ 126 I]Iodine, [ 128 I]Iodine, [ 130 I]Iodine, [ 131 I]Iodine, [ 209 At]Astatine, [ 210 At]Astatine and [ 211 At]astat exists.

3. A compound of general formula I according to claim 1 or 2, wherein the amino acids X and Y independently of one another are substituted or unsubstituted glutamic acid, substituted or unsubstituted glutamine, substituted or unsubstituted aspartic acid, substituted or unsubstituted phenylalanine, substituted or unsubstituted histidine, substituted or unsubstituted serine, substituted or unsubstituted tyrosine, substituted or unsubstituted arginine, substituted or unsubstituted asparagine, substituted or unsubstituted tryptophan, substituted or unsubstituted alanine and substituted or unsubstituted aminoadipic acid.

4. A compound of general formula I according to any one of the preceding claims, wherein the amino acids X and Y are independently glutamic acid, 3-(carboxymethyl)glutamic acid, glutamine, tyrosine, 3-iodotyrosine, arginine, aspartic acid, 2-aminoadipic acid, tryptophan, phenylalanine, 4-methoxyphenylalanine, 4-carboxyphenylalanine, 1-naphthylalanine, 2-naphthylalanine, 3-(anthracen-9-yl)alanine, 3-(quinolin-2-yl)alanine, 3-(quinolin-3-yl)alanine, 3-(isoquinolin-3-yl)alanine, 3-(isoquinolin-7-yl)alanine, 3-(benzothiophene- 3-yl)alanine and 3-(benzothiophen-2-yl)alanine.

5. A compound of general formula I according to any one of the preceding claims, wherein the amino acids X and Y are independently glutamic acid, glutamine, tyrosine, 3-iodotyrosine, arginine, aspartic acid, phenylalanine.

6. A compound of general formula I according to any one of the preceding claims, wherein X and Y are glutamic acid.

7. A compound of general formula I according to any one of the preceding claims, wherein ZZ is 1, wherein R is H, Gi is C-Ra with R a = H, G2 C-Rb with Rb = H, G3 C-Rc with Rc = F, G4 N and v is 0.

8. A compound of general formula I according to any one of the preceding claims, wherein A is Al.

9. A compound of general formula I according to any one of the preceding claims, wherein k is 1, m is 3, n is 2, p is 1 or 2 and q is 1 or 2, X and Y independently from each other are glutamic acid, glutamine, tyrosine, 3-iodotyrosine, arginine, aspartic acid, phenylalanine; Z is ZI, where R is H, Gi is C-Ra with Ra = H, G2 is C-Rb with Rb = H, G3 is CR cwith Rc = F, G4 is N, v is 0 and A is Al.

10. A compound of general formula I according to any one of the preceding claims, wherein k is 1, m is 3, n is 2, p is 1 and q is 1, X and Y are independently glutamic acid, glutamine, tyrosine, 3-iodotyrosine, arginine, aspartic acid, phenylalanine; Z is ZI, where R is H, Gi is CR a with Ra = H, G2 C-Rb with Rb = H, G3 C-Rc with Rc = F, G4 N, v 0 and A Al.

11. A compound of general formula I according to any one of the preceding claims, wherein the compound of general formula I is selected from the group consisting of 5 E592 and E593 12. A compound according to any one of claims 1 to 11 for use as a medicament.

13. A compound according to any one of claims 1 to 11 for use as a medicament for the diagnosis and / or treatment of diseases involving PSMA.

14. A medicament containing a compound according to any one of claims 1 to 11 or a pharmaceutically acceptable salt thereof.

15. Use of a compound according to any one of claims 1 to 11 for the diagnosis and / or treatment of diseases in which PSMA is involved.

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