EA046402B1 - DUAL-MODE RADIOACTIVE LABEL AND RADIOTHERAPEUTIC DRUG - Google Patents

Description

The present invention relates to a ligand-chelator conjugate (SIFA - silicofluoride acceptor) containing in one molecule: (a) one or more ligands that are capable of binding to a target molecule corresponding to the disease, (b) a silicofluoride acceptor (SIFA) moiety that contains a covalent bond between silicon and a fluorine atom and which may be labeled with ¹⁸ F by isotopic exchange of ¹⁹ F with ¹⁸ F or which is labeled with ¹⁸ F, and (c) one or more chelating group, optionally containing a chelated non-radioactive or radioactive cation.

A number of documents are cited herein, including patent applications and manufacturer's manuals. The disclosure of these documents, although not considered material to the patentability of the present invention, is incorporated herein by reference in its entirety. More specifically, all referenced documents are incorporated by reference to the same extent as if each individual document were specifically and individually incorporated by reference.

Prostate cancer

Prostate cancer (PCa) has remained the most common malignant disease with low survival rates in men over the past decades. Due to overexpression in prostate cancer (Silver et al., Clinical Cancer Research 3, 81-85 (1997)), prostate-specific membrane antigen (PSMA) or glutamate carboxypeptidase II (GCP II) have proven to be excellent targets for the development of highly sensitive radiolabeled agents for endoradiotherapy and imaging of prostate cancer (Afshar-Oromieh et al., European journal of nuclear medicine and molecular imaging 42, 197-209 (2015), Benesova et al., Journal of Nuclear Medicine 56, 914-920 (2015), Robu et al ., Journal of Nuclear Medicine, jnumed. 116.178939(2016), Weineisen et al., Journal of Nuclear Medicine 55, 1083-1083 (2014), Rowe et al., Prostate cancer and prostatic diseases (2016), Maurer et al. , Nature Reviews Urology (2016)). Prostate-specific membrane antigen is an extracellular hydrolase whose catalytic center contains two zinc(II) ions bridged by a hydroxy ligand. It is highly elevated in metastatic and hormone-refractory prostate cancer, but its physiological expression is also noted in the kidney, salivary glands, small intestine, brain and, to a lesser extent, also in healthy prostate tissue. In the intestine, PSMA promotes folate absorption by converting pteroylpoly-γ-glutamate to pteroylglutamate (folate). In the brain, it hydrolyzes N-acetyl-laspartyl-G-glulamate (NAAG) to N-acetyl-Gaspartate and glutamate.

Prostate-specific membrane antigen (PSMA)

Prostate-specific membrane antigen (PSMA) is a type II transmembrane glycoprotein that is highly overexpressed in prostate cancer epithelial cells. Despite its name, PSMA is also expressed to varying degrees in neovascularization of a wide range of nonprostate cancers. The most common nonprostate cancers that demonstrate PSMA expression include breast, lung, colorectal, and renal cell cancers.

The general required structures of molecules that target PSMA contain a binding unit that includes a zinc-binding group (such as urea (Zhou et al., Nature Reviews Drug Discovery 4, 1015-1026 (2005)), phosphinate or phosphoramidate), associated with the P1'-glutamate fragment, which guarantees high affinity and specificity for PSMA and, as a rule, is additionally associated with an effector functional group (Machulkin et al., Journal of drug targeting, 1-15 (2016)). The effector part is more flexible and somewhat resistant to structural changes. The entry tunnel houses two other important structural features that are important for ligand binding. The first is an arginine patch, a positively charged site on the entrance tunnel wall, and a mechanistic explanation for the preference for negatively charged functional groups at the P1 position of PSMA. This appears to be the reason for the preferential inclusion of negatively charged residues in the ligand framework. To the best of the inventors' knowledge, an in-depth analysis of the effect of positive charges on PSMA ligands has not yet been carried out. Upon binding, the concerted movement of arginine side chains can result in the opening of the hydrophobic accessory pocket S1, a second important structure that has been shown to accommodate the iodobenzyl group of more urea-based inhibitors, thereby contributing to their high affinity for PSMA (Barinka et al. , Journal of medicinal chemistry 51, 7737-7743 (2008).

Zhang et al. discovered a remote PSMA binding site that can be applied to the bidentate binding mode (Zhang et al., Journal of the American Chemical Society 132, 12711-12716 (2010)). The so-called arene binding site is a simple structural motif formed by the side chains of Arg463, Arg511 and Trp541 and is part of the GCPII entry cap. Engagement of the arene binding site by a distal inhibitory moiety can result in a significant increase in the affinity of the inhibitor for PSMA due to avidity effects. PSMA I&T was designed specifically to interact with PSMA in this manner, although crystal structure analysis of the binding mode is not available. The required characteristic, according to Zhang et al., is a linker unit (suberic acid in the case of PSMA I&T) that promotes open

- 1 046402 conformation of the GCPII entry cap and thus ensures accessibility of the arene binding site. It has further been shown that the structural composition of the linker has a significant impact on tumor targeting and biological activity, as well as image contrast and pharmacokinetics (Liu et al., Bioorganic & medicinal chemistry letters 21, 7013-7016 (2011)) , properties that are critical for both high image quality and effective targeted endoradiotherapy.

There are currently two categories of inhibitors in clinical use that target PSMA. On the one hand, there are radioactive tags with chelating units for complexation with radionuclides such as PSMA I&T or related compounds (Kiess et al., The quarterly journal of nuclear medicine and molecular imaging 59, 241 (2015)). On the other hand, there are small molecules that include a targeting element and effector molecules.

The most commonly used agents for selective imaging of PSMA are PSMA HBED-CC (Eder et al., Bioconjugate chemistry 23, 688-697 (2012)), PSMA-617 (Benesova et al., Journal of Nuclear Medicine 56, 914-920 (2015)) and PSmA I&T (Weineisen et al., Journal of Nuclear Medicine 55, 1083-1083 (2014)), which are predominantly labeled with ⁶⁸ Ga (88.9% β+, Εβ+, max = 1.89 MeV, t1/ ₂ = 68 min). Among these, ⁶⁸ Ga-PSMA-HBED-CC (also known as ⁶⁸ Ga-PSMA-11) is still considered the gold standard for PET imaging (positron emission tomography) of PCa.

Marking ¹⁸ F

Recently, several groups have focused on developing new urea-based ¹⁸ F-labeled inhibitors for PCa diagnosis. Unlike the radiometal ⁶⁸ Ga, which can be obtained from commercially distributed radionuclide generators ⁶⁸ Ge/6 ⁸ Ga ( ⁶⁸ Ge, t1/2 = 270.8 d), the radioisotope ¹⁸ G-fluoride (96.7% β+, Εβ+ , max = 634 keV) requires a cyclotron to obtain it. Despite this limitation, ¹⁸ F has, due to its longer half-life (t1/2 = 109.8 min) and lower positron energy, significant advantages in terms of routine processing and image quality. In addition, there is the possibility of large-scale production in a cyclotron, which would be beneficial for higher patient throughput and lower production costs. The ^-labeled urea-based PSMA inhibitor ¹⁸ F-DCFPyl has shown promising results in the detection of primary and metastatic PCa (Rowe et al., Molecular Imaging and Biology, 1-9 (2016)) and is superior to ⁶⁸ Ga-PSMA-HBED-CC in a comparative study (Dietlein et al., Molecular Imaging and Biology 17, 575-584 (2015)). Based on the structure of PSMA-617, a β-labeled analogue of PSMA-1007 was recently developed, which showed comparable tumor-to-organ ratios (Cardinale et al., Journal of nuclear medicine: official publication, Society of Nuclear Medicine 58, 425-431 (2017) , Giesel et al., European journal of nuclear medicine and molecular imaging 43, 1929-1930 (2016)). A comparative study with ⁶⁸ Ga-PSMA-HBED-CC found similar diagnostic accuracy of both radiotracers and reduced urinary clearance ^{of 18} FPSMA-1007, which allowed for improved assessment of the prostate gland (Giesel et al., European journal of nuclear medicine and molecular imaging 44, 678 -688 (2017)).

An attractive approach for introducing ^18F tags is the use of silicofluoride acceptors (SIFAs). Silicofluoride acceptors are described, for example, in Lindner et al., Bioconjugate Chemistry 25, 738-749 (2014). To maintain the silicon-fluoride bond, the use of silicofluoride acceptors necessitates the introduction of sterically demanding groups around the silicon atom. This, in turn, makes silicofluoride acceptors highly hydrophobic. With regard to binding to a target molecule, in particular to a target molecule that is PSMA, the hydrophobic moiety provided by the silicofluoride acceptor can be used to establish interactions of a radiodiagnostic or therapeutic compound with the hydrophobic pocket, as described in Zhang et al., Journal of American Chemical Society 132, 12711-12716 (2010). However, prior to binding, the higher degree of lipophilicity introduced into the molecule poses a major challenge to the development of radiopharmaceuticals with suitable in vivo biodistribution, i.e., low nonspecific binding to non-target tissue.

Inability to solve the problem of hydrophobicity

Despite numerous attempts, the problem of hydrophobicity caused by silicofluoride acceptors has not been satisfactorily solved in the prior art.

In more detail, Schirrmacher E. et al (Bioconjugate Chem. 2007, 18, 2085-2089) synthesized various ¹⁸ G-labeled peptides using the highly efficient labeled p-(di-tertbutylfluorosilyl)benzaldehyde ([ ^18F ]SIFA-A) synthon. which is one example of a silicofluoride acceptor. The SIFA method resulted in an unexpectedly efficient ^19F - ^18F isotopic exchange and yielded ^{the 18G} synthon in near quantitative yield at high specific activities between 225 and 680 GBq/µmol (608,118,378 Ci/mmol) without the use of HPLC (high performance liquid chromatography) purification ). [ ¹⁸ F]SIFA-benzaldehyde was finally used to label the N-terminal aminooxy (N-AO) derivatized peptides AO-Tyr3-octreotate (AO-TATE), cyclo(fK(AO-N)RGD) and N-AO-PEG ₂ -[D-Tyr-Gln

- 2 046402

Trp-Ala-Val-Ala-His-Thi-Nle-NH ₂ ](AO-BZH3, bombesin derivative) with high radiochemical yields. However, the labeled peptides are highly lipophilic (as can be inferred from HPLC retention times using the conditions described therein) and are thus not suitable for further evaluation in animal or human models.

In Wangler S. et al (Bioconjugate Chem., 2009, 20 (2), pp. 317-321), the first SIFA Kit-based radiofluorination of a protein (rat serum albumin, RSA) was described. As a labeling agent, 4-(di-tert-butyl[%]fluorosilyl)benzenethiol (Si[ ^18F ]FA-SH) was prepared by simple isotope exchange with a radiation chemical yield (RCY) of 40 to 60% and bound the product directly with maleimide-derivatized serum albumin with a total RCV of 12% for 20 to 30 minutes. The technically simple labeling method does not require any complex purification procedures and is a direct example of the successful application of Si- ^18F chemistry for in vivo PET imaging. Time-activity relationships and μPET images in mice showed that most of the activity was localized to the liver, suggesting that the labeling agent is too lipophilic and directs the probe in vivo to clearance by the hepatobiliary system and extensive metabolism in the liver.

Wangler S. et al (see Bioconjug Chem. 2010 Dec 15, 21 (12): 2289-96) subsequently tried to overcome the main drawback of the SIFA technique, the high lipophilicity of the resulting radiopharmaceuticals, by synthesizing and evaluating new analogues of SIFA-octreotate (SIFA- Tyr3-octreotate, SIFA-Asn(AcNH-β-Glc)-Tyr3-octreotate and SIFA-Asn(AcNH-β-Glc)-PEG-Tyr3-octreotate). In these compounds, hydrophilic linkers and pharmacokinetic modifiers were introduced between the peptide and the SIFA moiety, that is, the carbohydrate and the PEG plus carbohydrate linker. As a measure of the lipophilicity of the conjugates, log P(ow) was determined to be 0.96 for SIFA-Asn(AcNH-e-Glc)-PEG-Tyr3octreotate and 1.23 for SIFA-Asn(AcNH-e-Glc)-Tyr3 -octreotate. These results indicate that the high lipophilicity of the SIFA moiety can only be slightly compensated for by the use of hydrophilic moieties. The first imaging study showed excessive hepatic clearance/absorption efficiency in the liver and therefore no further first study in humans was performed.

Bernard-Gauthier et al (Biomed Res Int. 2014, 2014:454503) review the large number of different types of SIFA reported in the literature, from small prosthetic groups and other low molecular weight compounds to labeled peptides and more recently described affibody molecules. Based on these data, the problem of lipophilicity of SIFA-based prosthetic groups has not been addressed, that is, a method that would reduce the overall lipophilicity of a SIFA-conjugated peptide to a log D below approximately minus 2.0 has not been described.

Lindner S. et al (Bioconjug Chem. 2014 Apr 16, 25 (4):738-49) describe pegylated bombesin (PESIN) derivatives as specific ligands for the GRP receptor and RGD peptides (one-letter codes for arginine-glycine-aspartic acids) as specific ανβ3 binders were synthesized and labeled with a silicofluoride acceptor moiety (SIFA). To compensate for the high lipophilicity of the SIFA fragment, various modifications to the hydrophilic structure were introduced, resulting in lower logD values. SIFA-Asn(AcNH-e-Glc)-PESIN, SIFASer(e-Lac)-PESIN, SIFA-Cya-PESIN, SIFA-LysMe3-PESIN, SIFA-γ-carboxy-d-Glu-PESIN, SIFA-Cya2PESIN, SIFA-LysMe3-γ-carboxy-d-Glu-PESIN, SIFA-(γ-carboxy-d-Glu)2-PESIN, SIFA-RGD, SIFA-γcarboxy-d-Glu-RGD, SIFA-(γ-carboxy- d-Glu)2-RGD, SIFA-LysMe3-γ-carboxy-d-Glu-RGD. All of these peptides, already improved and derivatized to reduce lipophilicity, showed logD values ranging from plus 2 to minus 1.22.

Niedermoser S. et al (J Nucl Med. 2015 Jul, 56 (7): 1100-5) compared the newly developed ¹⁸ F-SIFA- and ¹⁸ F-SIFA1in- (SIFA - silicofluoride acceptor) modified TATE derivatives with the current clinical gold standard ⁶⁸ Ga-DOTATATE for high-quality imaging of tumors harboring the somatostatin receptor. For this purpose, ¹⁸ F-SIFA-TATE and two rather complex analogues, ¹⁸ F-SIFA-Glc-PEG1-TATE, ¹⁸ F-SIFA1in-Glc-Asp2-PEG1-TATE, were developed. None of the agents showed logD less than minus 1.5.

In view of the above, the technical problem underlying the present invention can be defined as providing radiodiagnostic and radiotherapeutic agents that contain a silicofluoride scavenger and at the same time are characterized by favorable properties in vivo.

As will become clear below, the present invention discloses testing and proof of principle for the use of specific conjugates that bind with high affinity to prostate specific antigen (PSMA) as a target. Accordingly, another technical object underlying the present invention is to provide improved radiotherapeutic and radiodiagnostic agents for the medical indications of cancer, preferably prostate cancer.

The solution to these technical problems is provided by the claimed subject matter of the present invention. Accordingly, in a first aspect, the present invention relates to a ligand-SIFA-chelator conjugate,

- 3 046402 containing in one molecule: (a) one or more ligands that can bind to the target molecule corresponding to the disease, (b) a silicon fluoride acceptor moiety (SIFA) which contains a covalent bond between silicon and a fluorine atom and which can be labeled ¹⁸ F by isotopic exchange ^{of 19} F for ¹⁸ F or which is labeled with ¹⁸ F, and (c) one or more chelating group, optionally containing a chelated non-radioactive or radioactive cation.

The ligand-SIFΆ-chelator conjugate contains three separate moieties. The three separate moieties are: a) one or more ligands that are capable of binding to a disease target molecule, (b) a silicon fluoride acceptor (SIFA) that contains a covalent bond between silicon and a fluorine atom, and (c) one or more a chelating group optionally containing a chelated non-radioactive or radioactive cation

The fluorine atom in the SIFA fragment may be ¹⁹ F or ¹⁸ F.

Although certain ligands that can bind to a disease-related target molecule may be cyclic peptides, such cyclic peptides are not chelating groups as contemplated herein because the function of the hydrophobic moiety of SIFA is not accomplished in the absence of an additional chelating moiety. Thus, the compounds of the present invention require a hydrophilic chelating group in addition to ligands that are capable of binding to the target molecule relevant to the disease. A hydrophilic chelating group is required to reduce the hydrophobic nature of the compounds caused by the presence of the SIFA moiety.

The ligand according to the first aspect of the invention is defined functionally. This is the case because the present invention is not structurally dependent on the specific nature of the ligand. Rather, the key aspect of the invention is the combination, within a single molecule, of a silicofluoride acceptor and a chelator or chelate. These two structural elements, SIFA and the chelator, exhibit spatial proximity. Preferably, the shortest distance between two atoms of two elements is less than or equal to 25 A, more preferably less than 20 A, and even more preferably less than 15 A. Alternatively or in addition, it is preferred that no more than 25 covalent bonds separate a SIFA fragment atom and a chelator atom , preferably no more than 20 chemical bonds and even more preferably no more than 15 chemical bonds.

The cation according to paragraph (c) of the first aspect is a radioactive or non-radioactive cation. It is preferably a radioactive or non-radioactive metal cation, and more preferably a radioactive metal cation. Examples are given below.

As a consequence, conjugates that are radiolabeled on both the SIFA moiety and the chelating group, molecules that are radiolabeled on only one of the two sides, and molecules that are not radiolabeled at all are included in the scope of the first aspect. In the latter case, the chelating group may be either a cold (non-radioactive) ion complex or may not contain any ion.

We have surprisingly discovered that placing a silicofluoride acceptor near a hydrophilic chelator such as, but not limited to, DOTAGA ((1-(1,3carboxypropyl)4,7,10(carboxymethyl)-1,4,7,10-tetraazacyclododecane ) or DOTA (tetraxetane), screens or effectively compensates for the lipophilicity of the SIFA moiety to an extent that shifts the overall hydrophobicity of the radiotherapeutic or radiodiagnostic compound to a range that makes the compound suitable for in vivo administration.

In addition, the combination of chelator use and isotope exchange at SIFA with ¹⁸ G-fluoride also results in coupled diagnostic radiolabels that can be used as [ ¹⁸ F][ ^nat Ion]labels in centers with a local cyclotron or centers that receive ¹⁸ Ffluoride by shipment from cyclotron centers, while in centers that do not have access to ¹⁸ Ffluoride but have access to radioisotope generators such as the Ge-68/Ga-68 generator, appropriate versions can be used, for example |'Г|| ⁶⁸ Tags.

Importantly, in both cases a chemically identical radiopharmaceutical is administered and therefore no differences in in vivo behavior are expected. While, at this time, due to chemical differences, clinical data for an ¹⁸ F-labeled compound provided on a patient population at one site cannot be directly compared with clinical data for the ⁶⁸ Ga analogue reported on another cohort at a different site, radiopharmaceutical and/or the radiodiagnostic tool of the present invention can be compared directly and thus such data can be linked (eg, data from a European center working with the F-18 and another center in India working with the Ga-68).

In addition, with appropriate selection, the chelate can also be used for labeling with a therapeutic isotope such as the beta-emitting isotopes Lu-177, Y-90 or the alpha-emitting isotope Ac-225, allowing the concept of coupled radiopharmaceuticals to be extended to connect diagnostic agents (| ¹⁸ E||'Gi|tags) and therapeutic radiopharmaceuticals ([ ^nat F][ ¹⁷⁷ Lu].

Another advantage of the compounds, especially the PSMA targeting compounds, of the present invention is their surprisingly low accumulation in the kidneys

- 4,046,402 mice compared to other PSMA-targeting radiopharmaceuticals such as PSMA I&T. Without being limited to any particular theory, it is believed that the reason is the combination of the SIFA building block with a chelator, which provides a surprising reduction in accumulation in the kidneys.

From a lipophilicity/hydrophilicity perspective, the logP value (sometimes also called the logD value) is a generally accepted value in the art.

The term lipophilicity refers to the strength of dissolution or absorption in lipid solutions or adsorption onto a lipid-like surface or matrix. It denotes a preference for lipids (literal meaning) or for organic or non-polar liquids, or for liquids, solutions or surfaces with a small dipole moment compared to water. The term hydrophobicity is used here with an equivalent meaning. The adjectives lipophilic and hydrophobic are used in the meaning corresponding to the nouns described above.

The mass velocity of a molecule at the interface between two immiscible or practically immiscible solvents is determined by its lipophilicity. The more lipophilic a molecule, the more soluble it is in the lipophilic organic phase. The partition coefficient of the molecule, which is observed between water and n-octanol, has been adopted as a standard measure of lipophilicity. The distribution coefficient P of species A was determined as the ratio P = [A] _n-octanol / [A] _water . A typical value is the logP value, which is the logarithm of the distribution coefficient. If the molecule is ionizable, there will in principle be many different microspecies (ionized and non-ionized forms of the molecule) present in both phases. The value characterizing the overall lipophilicity of the ionizable species is the partition coefficient D, defined as the ratio D = [sum of concentrations of all microspecies] _n-octanol / [sum of concentrations of all microspecies] _water . Similar to logP, logD, the logarithm of the partition factor, is often used. Often a buffer system, such as phosphate-buffered saline, is used as an alternative to water in the above-described logP determination.

If the lipophilic nature of a substituent in a first molecule is to be assessed and/or quantified, a second molecule corresponding to that substituent can be assessed, wherein said second molecule is obtained, for example, by breaking the bond connecting said substituent to the rest of the first molecule and attaching the thus obtained free valence(s) to hydrogen(s).

Alternatively, the contribution of the substituent to the logP of the molecule can be determined. The contribution of the p _{X x} substituent X to the logP of the RX molecule is determined as p _{X x} = logP _RX -logP _RH , where RH is the unsubstituted parent compound.

P and D values greater than one and logP, logD and n _{X x} values greater than zero indicate lipophilic/hydrophobic nature, while P and D values less than one and logP, logD and n _{X x} values less than zero indicate the hydrophilic nature of the respective molecules or substituents.

The above-described parameters characterizing the lipophilicity of the lipophilic group or the entire molecule of the present invention can be determined experimentally and/or predicted using computational methods known in the art (see, for example, Sangster, Octanol-water Partition Coefficients: fundamentals and physical chemistry, John Wiley & Sons, Chichester. (1997)).

In a preferred embodiment, the logP value for the compounds of the present invention is from minus 5 to minus 1.5. It is particularly preferred that the logP value be between minus 3.5 and minus 2.0.

In a preferred embodiment, the ligand of the present invention contains or consists of a peptide, peptidomimetic or substituted urea, substituents including amino acids. It is understood that a ligand that contains a peptide or peptidomimetic also contains a non-peptide and a non-peptidomimetic portion. In terms of molecular weight, preference is given to molecular weights below 15 kDa, below 10 kDa or below 5 kDa. Accordingly, small proteins are included in the definition of the term ligand. Target molecules are not particularly limited and include enzymes, receptors, epitopes, transporters, cell surface molecules and extracellular matrix proteins. Targets that are relevant to the disease are preferred. Particularly preferred are targets that are causally related to the disease or that are highly overexpressed in the disease and/or the inhibition of which can produce a benefit in a patient suffering from the disease. The ligands are preferably high affinity ligands, with a preferred affinity, expressed as _IC50 , being less than 50 nM, less than 20 nM or less than 5 nM.

Particularly preferred are those ligands that bind with high affinity to disease-related target molecules or disease-related biomolecules, including, but not limited to, somatostatin receptors, bombesin receptors, chemokine receptors, integrin receptors, cholecystokinin receptors, melanocortin, receptors

- 5 046402 vasoactive intestinal peptide, neurotensin receptors, neuropeptide Y receptors, neurokinin receptors, glucacon-like peptide 1 receptors, Her2 receptors, PD-L1, PD-1, gonadotropin-releasing hormone receptors and prostate-specific membrane antigen (PSMA).

The term disease-related refers preferably to causal involvement in a disease.

Preferably, the silicofluoride acceptor moiety (SIFA) has the structure represented by formula (I)

where R ^1S and R ^2S independently represent a linear or branched C3-C10 alkyl group, preferably R ^1S and R ^2S are selected from isopropyl and tert-butyl and more preferably R ^1S and R ^2S are tert-butyl, R ^3S is a C1- _C20 hydrocarbon group which may contain one or more aromatic and one or more aliphatic units and/or up to 3 heteroatoms selected from O and S, preferably ^R3S is a _C6 - _C10 hydrocarbon group which contains an aromatic ring, and which may contain one or more aliphatic units, more preferably R ^3S is a phenyl ring, and most preferably R ^3S is a phenyl ring, wherein the Si-containing substituent and the bond designated *"*** are in para position, and where the SIFA moiety is attached to the rest of the conjugate through a bond designated '/wwvw.

More preferably, the silicofluoride acceptor moiety (SIFA) has the structure represented by formula (Ia)

where t-Bu denotes a tert-butyl group.

A preferred chelating group contains at least one of the following (i), (ii) or (iii).

(i) A macrocyclic ring structure with 8 to 20 ring atoms, of which 2 or more, more preferably 3 or more, are selected from oxygen atoms or nitrogen atoms. Preferably, the 6 or fewer ring atoms are selected from oxygen atoms or nitrogen atoms. It is particularly preferred that 3 or 4 ring atoms are nitrogen atoms or oxygen atoms. Among oxygen and nitrogen atoms, preference is given to nitrogen atoms. In combination with the macrocyclic ring structure, a preferred chelating group may contain 2 or more, for example 2 to 6, preferably 2 to 4, carboxyl groups and/or hydroxyl groups. Among carboxyl groups and hydroxyl groups, preference is given to carboxyl groups.

(ii) An open-chain acyclic chelating structure with 8 to 20 backbone atoms, of which 2 or more, more preferably 3 or more, are heteroatoms selected from oxygen atoms or nitrogen atoms. Preferably, the 6 or fewer backbone atoms are selected from oxygen atoms or nitrogen atoms. Among oxygen and nitrogen atoms, preference is given to nitrogen atoms. More preferably, the open chain chelating structure is a structure that contains a combination of 2 or more, more preferably 3 or more heteroatoms selected from oxygen atoms or nitrogen atoms, and 2 or more, for example 2 to 6, preferably 2 to 4 carboxyl groups and/or hydroxyl groups. Among carboxyl groups and hydroxyl groups, preference is given to carboxyl groups.

(iii) Branched chelating structure containing a quaternary carbon atom. Preferably, the quaternary carbon atom is replaced with 3 identical chelating groups in addition to the SIFA/ligand moiety. Substituted chelating groups may contain an amide. Substituted chelating groups may contain an aromatic group. Substituted chelating groups may contain hydroxypyridinone.

In preferred specific examples, the chelating group is a chelating agent residue selected from bis(carboxymethyl)-1,4,8,11-tetraazabicyclo[6.6.2]hexadecane

- 6 046402 (CBTE2a), cyclohexyl-1,2-diaminetetraacetic acid (CDTA), 4-(1,4,8,11-tetraazacyclotetradec-1yl)-methylbenzoic acid (CPTA), K'-[5-[acetyl( hydroxy)amino]pentyl]-K-[5-[[4-[5-aminopentyl(hydroxy)amino]-4-oxobutanoyl]amino]pentyl]-K-hydroxybutanediamide (DFO), 4,11-bis(carboxymethyl) -1,4,8,11-tetraazabicyclo[6.6.2]hexadecane (DO2A), 1,4,7,10-tetracyclododecane-K,K',KK'tetraacetic acid (DOTA), a-(2-carboxyethyl) -1,4,7,10-tetraazacyclododecane-1,4,7,10tetraacetic acid (DOTAGA), 1,4,7,10-tetraazacyclododecane-H№,№',№-1,4,7,10-tetra (methylene)phosphonic acid (DOTMP), K,K'-dipyridoxylethylenediamine-K,K'-diacetate-5,5'-bis(phosphate) (DPDP), diethylenetriamine-M,Ni,Ni'-penta(methylene)phosphonic acid (DTMP), diethylenetriaminepentaacetic acid (DTPA), ethylenediamine-K,K'-tetraacetic acid (EDTA), ethylene glycol-O,Obis(2-aminoethyl)-K,K,K',K'-tetraacetic acid (EGTA) , K,K-bis(hydroxybenzyl)-ethylenediamineK,K'-diacetic acid (HBED), hydroxyethyldiaminetriacetic acid (HEDTA), 1-(pnitrobenzyl)-1,4,7,10-tetraazacyclodecane-4,7,10-triacetate (HP-DOA3), 6-hydrazinyl-K-methylpyridine-3-carboxamide (HYNIC), tetra-3-hydroxy-M-methyl-2-pyridinone chelators (4-((4-(3-(bis(2( 3-hydroxy-1-methyl-2-oxo-1,2-dihydropyridine-4-carboxamido)ethyl)amino)-2-((bis(2-(3-hydroxy-1methyl-2-oxo-1,2- dihydropyridine-4-carboxamido)ethyl)amino)methyl)propyl)phenyl)amino)-4-oxobutanoic acid), abbreviated as Me-3,2-HOPO, 1,4,7-triazacyclononane-1-succinic acid-4,7 diacetic acid (NODASA), 1-(1-carboxy-3-carboxypropyl)-4,7-(carboxy)-1,4,7-triazacyclononane (NODAGA), 1,4,7-triazacyclonone triacetic acid (NOTA), 4, 11bis(carboxymethyl)-1,4,8,11-tetraazabicyclo[6.6.2]hexadecane (TE2A), 1,4,8,11-tetraazacyclododecane1,4,8,11-tetraacetic acid (TETA), tris(hydroxypyridinone) (THP), terpyridinebis(methyleneaminetetraacetic acid (TMT), 1,4,7-triazacyclononane-1,4,7-tris[methylene(2carboxyethyl)phosphinic acid] (TRAP), 1,4,7,10-tetraazacyclotridecane-H No,No',No''-tetraacetic acid (TRITA), 3-[[4,7-bis[[2-carboxyethyl(hydroxy)phosphoryl]methyl]-1,4,7-triazonan-1-yl]methylhydroxy -phosphoryl]propanoic acid and triethylenetetraamine-hexacetic acid (TTHA), where the residue is obtained by covalently linking the carboxyl group contained in the chelating agent to the rest of the conjugate via an ester or amide bond.

Specific chelators are listed below:

But

TNR

HBED Me-3,2-HOPO.

Among the above examples of chelating agents, particular preference is given to a chelating agent selected from TRAP, DOTA and DOTAGA.

Macrocyclic and acyclic metal chelating compounds or cations are well known in the art and are available from a number of manufacturers. Although the chelating moiety of the present invention is not particularly limited, it is understood that numerous moieties can be used by one skilled in the art without modification.

The chelating group may contain a chelated cation, which may be radioactive or non-radioactive, preferably a chelated metal cation, which may be radioactive or non-radioactive. More preferred is a chelated isotope of the radioactive metal.

Preferred examples of cations that can be chelated by a chelating group are the cations ⁴³ Sc, ⁴⁴ Sc, ^{47 Sc, 51 Cr, 52 mMn} , ⁵⁸ Co, ⁵² Fe, ⁵⁶ Ni, ⁵⁷ Ni, ⁶² Cu, ⁶⁴ Cu, ⁶⁷ Cu, ⁶⁶ Ga, ⁶⁷ Ga, ⁶⁸ Ga, ⁸⁹ Zr, ⁹⁰ Y, ⁸⁹ Y, <Tc, ⁹⁹ mTc, ⁹⁷ Ru, ¹⁰⁵ Rh, ¹⁰⁹ Pd, ¹¹¹ Ag, ¹¹⁰ mIn, ¹¹¹ In, ¹¹³ mIn, ¹¹⁴ mln , ¹¹⁷ mSn, ¹²¹ Sn, ¹²⁷ Te, ¹⁴² Pr, ¹⁴³ Pr, ¹⁴⁹ Pm, ¹⁵¹ Pm, ¹⁴⁹ Tb, ¹⁵² Tb, ¹⁵⁵ Tb, ¹⁶¹ Tb, ¹⁵³ Sm, ¹⁵⁷ Gd, ¹⁶¹ Tb, ¹⁶⁶ Ho, ¹⁶⁵ Dy, ¹⁶⁹ Er, ¹⁶⁹ Yb, ¹⁷⁵ Yb, ¹⁷² Tm, ¹⁷⁷ Lu, ¹⁸⁶ Re, ¹⁸⁸ Re, ¹⁹¹ Pt, ¹⁹⁷ Hg, ¹⁹⁸ Au, ¹⁹⁹ Au, ²¹² Pb, ²⁰³ Pb, ²¹¹ At, ²¹² Bi, ²¹³ Bi, ²²³ Ra, ²²⁵ Ac, ²²⁷ Th, a cationic molecule containing ¹⁸ F or a cation such as ¹⁸ F-[AlF] ² +, more preferably the cations ⁴⁴ Sc, ⁴⁷ Sc, ⁶⁴ Cu, ⁶⁷ Cu, ⁶⁸ Ga, ⁹⁰ Y, ¹¹¹ In, ¹⁶¹ Tb, ¹⁶⁶ Ho, ¹⁷⁷ Lu, ¹⁸⁸ Re, ²¹² Pb, ²¹² Bi, ²¹³ Bi, ²²⁵ Ac and ²²⁷ Th.

Using PSMA binding agents as an example, the present inventors put the above-disclosed invention into practice. This is the subject of preferred aspects and embodiments disclosed below. However, the present inventors have surprisingly discovered that compensation for the lipophilicity of the SIFA moiety is, to an unexpected extent, not limited to molecules containing PSMA binding agents.

Accordingly, the ligand is preferably capable of binding to prostate-specific membrane antigen (PSMA).

More preferably, the ligand has the structure represented by formula (II)

NOOS R^ .R ³ L SDS ₂ )р—i

Y Г γ ' * _ig _/< ^CH 2)t O COOH nUUL (P), where m is an integer from 2 to 6, preferably from 2 to 4, more preferably 2, n is an integer from 2 to 6 , preferably from 2 to 4, more preferably from 2 to 3, R ^1l represents CH2, NH or O, preferably NH, R ^3L represents CH2, NH or O, preferably NH, R ^2L represents C or P(OH) , preferably C, and where the ligand is attached to the rest of the conjugate through a bond designated llllllllll.

The ligand may have the structure represented by formula (IIa)

HOOS ΝγΝγ,(ΟΗ ₂ ) _η ^ ⁰ coon coon _(Pa) where n is an integer from 2 to 6, and where the ligand is attached to the rest of the conjugate through a bond designated as ^lllllllll ''

Several PSMA binding agents are known in the art, all of which are suitable in accordance with the present invention. The above preferred embodiment is the structural definition of a preferred group of PSMA binding agents.

Particularly preferably, the conjugate according to the first aspect of the present invention is a compound of formula (III)

SIFA

1z

X

NOOS 2L-R ^3L SDS ₂ )p—x ¹ —l ¹ —x—r —x—r ^ch γ RV *n I (CH ₂ ) _m o COOH

HOOC(III) or a pharmaceutically acceptable salt thereof, where

SIFA is a silicofluoride acceptor fragment (SIFA) containing a covalent bond between silicon and a fluorine atom and which can be labeled with ¹⁸ F by isotopic exchange of ¹⁹ F with ¹⁸ F or which is labeled with ¹⁸ F, preferably the SIFA is a SIFA fragment of formula (I) and more preferably formula (Ia) as defined above;

m is an integer from 2 to 6, preferably 2 or 3, more preferably 2;

n is an integer from 2 to 6, preferably 2 or 3, more preferably 2 or 4;

R ^1L is CH ₂ , NH or O, preferably NH;

R ^3L is CH ₂ , NH or O, preferably NH;

R ^2L represents C or P(OH), preferably C;

X ¹ is selected from an amide bond, an ether bond, a thioether bond, an ester bond, a thioester bond, a urea bridge and an amine bond, preferably an amide bond;

X ² is selected from an amide bond, an ether bond, a thioether bond, an ester bond, a thioester bond, a urea bridge and an amine bond, preferably an amide bond;

L ¹ represents a divalent linking group, the structure of which is selected from oligoamide, oligoether, oligothioether, oligoester, oligothioester, oligourea, oligo(ether-amide), oligo(thioether-amide), oligo(esteramide), oligo(thioester-amide), oligo(urea-amide), oligo(ether-thioether), oligo(ether-ester), oligo(ether-thioester), oligo(ether-urea), oligo(thioether-ester), oligo(thioether-thioester),

- 8 046402 oligo(thioether-urea), oligo(ester-thioether), oligo(esterurea) and oligo(thioester-urea), preferably the structure is selected from oligoamide and oligo(ester-amide).

L ¹ may be optionally substituted with one or more substituents independently selected from -OH, -OCH3, -COOH, -COOCH3, -NH2 and -NHC(NH)NH2.

X ³ is selected from an amide bond and an ester bond, an ether bond, an amine bond, and a linking group of the formula

wherein the bond designated as ' ^llllllll/ ' in the NH group is linked to ^RB and the other link designated as mlink is linked to SIFA, preferably X ³ is an amide linkage, ^RB is a trivalent linking group, X ⁴ is selected of amide bond, ether bond, thioether bond and ester bond, thioester bond, urea bridge, amine bond, linking group of the formula:

where the amide bond, designated as ***, is formed with a chelating group, and the other link, designated as ****, is connected with R ^B , and the linking group of the formula

where a bond designated as , at the carbonyl end, is formed with a chelating group, and another bond, designated as *»*«*, is linked to ^RB , preferably X ⁴ is an amide linkage,

R ^CH is a chelating group optionally containing a chelated radioactive or non-radioactive cation, preferably a radioactive or non-radioactive metal cation, wherein preferred embodiments of said chelating group and the optional chelated cation are as defined above.

The term oligo, in such expressions as, oligoamide, oligoether, oligothioether, oligoester, oligothioester, oligourea, oligo(ether-amide), oligo(thioether-amide), oligo(ester-amide), oligo( thioether-amide), oligo(urea-amide), oligo(ether-thioether), oligo(ether-ester), oligo(ether-thioester), oligo(ether-urea), oligo( ester thioether), oligo(thioether-thioester), oligo(thioether-urea), oligo(ester-thioester), oligo(ester-urea) and oligo(thioether-urea) are preferably to be understood as referring to a group in which from 2 to 20, more preferably from 2 to 10 subunits are connected by the types of bonds specified in the same conditions. As one skilled in the art will appreciate, when two different types of bonds are indicated in parentheses, both types of bonds are contained in the corresponding group (eg, ester and amide bonds are contained in oligo(ester-amide).

Preferably, L ¹ contains a total of 1 to 5, more preferably 1 to 3, and most preferably a total of 1 or 2 amide and/or ester bonds, preferably amide bonds, in its backbone.

Thus, the term oligoamide describes a group having a chain of CH2 or CHR groups alternating with groups selected from NHCO or CONH. In each case, moiety R is an optional substituent selected from -OH, -OCH ₃ , -COOH, -COOCH ₃ , -NH ₂ and -NHC(NH)NH2.

It is also preferable that -X ¹ -L ¹ -X ² - be one of the following structures (L-1) and (L-2):

-NH-C(O)-R ⁶ C(O)-NH-R ⁷ -NH-C(O)- (L-1),

-C(O)-NH-R ⁸ -NH-C(O)-R ⁹ -C(O)-NH-R ¹⁰ -NH-C(O)- (L-2) where R ⁶ -R ¹⁰ regardless selected from C ₂ -C ₁₀ -alkylene, preferably linear C ₂ -C ₁₀ -alkylene, each alkylene group of which may be substituted by one or more substituents independently selected from -OH, -OCH3, -COOH, -COOCH3, -NH2 and -NHC(NH)NH2.

It is particularly preferred that the total number of carbon atoms in R ⁶ and R ⁷ is from 4 to 20, more preferably from 4 to 16, without carbon atoms in optional substituents. It is particularly preferred that the total number of carbon atoms in R ⁸ -R ¹⁰ is from 6 to 20, more

- 9 046402 preferably from 6 to 16, without carbon atoms in optional substituents.

It is particularly preferred that -X ¹ -L ¹ -X ² - be one of the following structures (L-3) and (L-4):

-NH-C(O)-R ⁿ -C(O)-NH-R ¹² -CH(COOH)-NH-C(O)- (L-3),

-C(O)-NH-CH(COOH)-R ¹³ -NH-C(O)-R ¹⁴ -C(O)-NH-R ¹⁵ -CH(COOH)-NH-C(O)- (L -4) where R11-R ¹⁵ are independently selected from ^-^-alkylene, preferably linear ^-C^alkylene.

It is particularly preferred that the total number of carbon atoms in R ¹¹ and R ¹² or in R ¹³ R ¹⁵ , respectively, is from 8 to 18, more preferably from 8 to 12, even more preferably from 9 to 10.

Preferably R ^B has the structure represented by formula (IV) (CH ₂ ) _b

--(CH ₂ ) _a --A—(CH ₂ )^U

16 ^(IV) 'where A is selected from N, CR ¹⁶ , where R ¹⁶ represents H or ^-^-alkyl and a 5-7 membered carbocyclic or heterocyclic group, preferably A is selected from N and CH and more preferably A represents CH, the bond designated as ***** at (CH2)a is formed by X ² and a is an integer from 0 to 4, preferably 0 or 1 and most preferably 0, bond designated as ' ^/wvww ' at (CH2) _b is formed by X ³ and b is an integer from 0 to 4, preferably 0 to 2 and more preferably 0 or 1, and the bond designated ***** at (CH ₂ ) _c is formed by X ⁴ , and c is an integer from 0 to 4, preferably from 0 to 2, and more preferably 0 or 1.

Even more preferred as a conjugate of the present invention is a compound of formula (IIIa:

or a pharmaceutically acceptable salt thereof, wherein m, n, R ^1L , R ^2L , R ^3L , X ¹ , L ¹ , b, c, X ⁴ and R ^CH are as defined above, including all preferred embodiments thereof.

For a compound of formula (IIIa), preferably b + c is greater than or equal to 1.

For the compound of formula (IIIa), b + c is also preferably less than or equal to 3.

For a compound of formula (IIIa), more preferably b is 1 and c is 0.

For the compound of formula (III), it is also preferable that -X ⁴ -R ^CH is a chelating agent moiety selected from DOTA and DOTAGA linked to one of its carboxyl groups via an amide bond to the rest of the conjugate.

In a preferred embodiment of a compound of formula (III), said compound is a compound of formula (IIIb) noos noos

R ^CH (nib) or a pharmaceutically acceptable salt thereof, wherein m, n, R ^1L , R ^2L , R ^3L , X ¹ , L ¹ , b, c, X ⁴ and R ^CH are as defined above, and r is equal to 0 or 1.

It is particularly preferred that -N(H)-R ^CH is a chelating agent moiety selected from DOTA and DOTAGA linked to one of its carboxyl groups via an amide bond to the rest of the conjugate.

The most preferred compounds of the present invention are

- 10 046402 and its isomers and its isomers

PSMA-SIFAl (5)

He

He

- 11 046402 and its isomers

- 12 046402

- 13 046402

- 14 046402

- 15 046402 and its isomers

- 16 046402

PSMA-SIFA5 (9) and its isomers

- 19 046402

- 20 046402 and its isomers

- 22 046402

- 23 046402 and its isomers

PSMA-SIFA 11

- 24 046402

- 25 046402

Preferred labeling schemes for these most preferred compounds are as defined herein above.

In a further aspect, the present invention relates to a pharmaceutical composition containing or consisting of one or more conjugates or compounds of the present invention, as disclosed hereinabove.

- 26 046402

In a further aspect, the present invention relates to a diagnostic composition containing or consisting of one or more conjugates or compounds of the present invention, as disclosed hereinabove.

In a further aspect, the present invention relates to a therapeutic composition containing or consisting of one or more conjugates or compounds of the present invention, as disclosed hereinabove.

The pharmaceutical composition may further contain pharmaceutically acceptable carriers, excipients and/or diluents. Examples of suitable pharmaceutical carriers, excipients and/or diluents are well known in the art and include phosphate-buffered saline solutions, water, emulsions such as oil-in-water emulsions, various types of wetting agents, sterile solutions, etc. Compositions containing such carriers can be prepared by well known conventional methods. Such pharmaceutical compositions can be administered to a subject at a suitable dose. Administration of suitable compositions can be accomplished by various routes, for example, intravenous, intraperitoneal, subcutaneous, intramuscular, topical, intradermal, intranasal or intrabronchial administration. It is particularly preferred that said administration be carried out by injection and/or delivery, for example, to a site in the pancreas or to a cerebral artery or directly to brain tissue. The compositions can also be administered directly to the target site, for example, by biolistic delivery to an external or internal target site, such as the pancreas or brain. The dosage regimen will be determined by the attending physician and clinical factors. As is well known in the medical field, dosages for any one patient depend on many factors, including the patient's size, body surface area, age, the specific compound to be administered, sex, time and route of administration, general health, and other drugs being administered. the same time. The pharmaceutically active agent may be present in amounts from 0.1 ng to 10 mg/kg body weight per dose, but dosages lower or higher than this exemplary range are contemplated, especially taking into account the above factors.

In a further aspect, the present invention provides one or more compounds of the invention, as described above herein, for use in medicine.

Preferred medical applications are nuclear medicine, such as nuclear diagnostic imaging, also called nuclear molecular imaging, and/or targeted radiotherapy for diseases associated with overexpression, preferably PSMA, on affected tissue.

In a further aspect, the present invention provides a compound of the present invention, as defined herein above, for use in a method for diagnosing and/or staging cancer, preferably prostate cancer. Prostate cancer is not the only cancer characterized by PSMA expression. Non-prostate cancers that exhibit PSMA expression include breast, lung, colorectal, and renal cell cancers. Thus, any compound described herein having a PSMA binding moiety can be used for the diagnosis, imaging, or treatment of cancer characterized by PSMA expression.

Preferred indications are detection or staging of cancers such as, but not limited to, high-grade gliomas, lung cancer and especially prostate cancer and metastatic prostate cancer, detection of metastatic disease in patients with intermediate to high risk primary prostate cancer and detection of metastatic sites, even at low serum PSA values, in patients with biochemically recurrent prostate cancer. Another preferred indication is visualization and imaging of neoangiogenesis.

In terms of medical indications for therapy, especially radiotherapy, cancer is the preferred indication. Prostate cancer is a particularly preferred indication.

In a further aspect, the present invention provides a conjugate or compound of the present invention, as defined herein above, for use in a method for diagnosing and/or staging cancer, preferably prostate cancer.

The present invention further relates to the following points.

1. Ligand-SIFA-chelator conjugate containing in one molecule:

(a) one or more ligands that are capable of binding to a disease-relevant target molecule, (b) a silicofluoride acceptor moiety (SIFA) that contains a covalent bond between silicon and a fluorine atom and that can be labeled with ¹⁸ F by isotope exchange with ¹⁹ F at ¹⁸ F or which is labeled with ¹⁸ F and (c) one or more chelating group, optionally containing a chelated non-radioactive or radioactive cation.

2. The conjugate according to claim 1, where the silicofluoride acceptor fragment (SIFA) has a structure represented by

- 27 046402 defined by formula (I) _r <v ^r2S

R ^3S wherein R ^1S and R ^2S independently represent a linear or branched C3-C ₁₀ alkyl group, preferably R ^1S and R ^2S are selected from isopropyl and tert-butyl and more preferably R ^1S and R ^2S are tert-butyl;

R ^3S represents a C1-C20 hydrocarbon group, which may contain one or more aromatic and one or more aliphatic units and/or up to 3 heteroatoms selected from O and S, preferably R ^3S represents a C6-C ₁₀ hydrocarbon group, which contains an aromatic ring, and which may contain one or more aliphatic units, more preferably R ^3S is a phenyl ring, and most preferably, R ^3S is a phenyl ring, wherein the Si-containing substituent and the bond designated ' ^{ΛΛΛΛΛΛΛΖ} ' are in the para position and where the SIFA moiety is attached to the rest of the conjugate through a bond designated άλλαλλλλ.

3. The conjugate according to claim 2, wherein the silicofluoride acceptor moiety (SIFA) has the structure represented by formula (Ia)

where t-Bu denotes a tert-butyl group.

4. Conjugate according to any one of claims 1-3, where the chelating group contains at least one of:

(i) a macrocyclic ring structure with 8 to 20 ring atoms, of which 2 or more, more preferably 3 or more, are selected from oxygen atoms or nitrogen atoms; and (ii) an open-chain acyclic chelating structure with main chain atoms in an amount of 8 to 20, of which 2 or more, more preferably 3 or more, are heteroatoms selected from oxygen atoms or nitrogen atoms.

5. The conjugate according to any one of claims 1 to 3, where the chelating group is the remainder of a chelating agent selected from bis(carboxymethyl)-1,4,8,11-tetraazabicyclo[6.6.2]hexadecane (CBTE2a), cyclohexyl-1 ,2-diamintetraacetic acid (CDTA), 4-(1,4,8,11-tetraazacyclotetradec-1yl)-methylbenzoic acid (CPTA), N-[5-[acetyl(hydroxy)amino]pentyl]-M-[5 -[[4-[5-Aminopentyl(hydroxy)amino]-4-oxobutanoyl]amino]pentyl]-M-hydroxybutanediamide (DFO), 4,11-bis(carboxymethyl)-1,4,8,11-tetraazabicyclo[ 6.6.2]hexadecane (DO2A), 1,4,7,1 O-tetracyclododecane-N,N',N'',N'tetraacetic acid (DOTA), N-dipyridoxylethylenediamine-M, N-diacetate-5, 5'-bis(phosphate) (DPDP), diethylenetriaminepentaacetic acid (DTPA), ethylenediamine-MA-tetraacetic acid (EDTA), ethylene glycol-O,O-bis(2-aminoethyl)-NN,N-tetraacetic acid (EGTA) , N,Nbis(hydroxybenzyl)-ethylenediamine-M,N-diacetic acid (HBED), hydroxyethyldiaminetriacetic acid (HEDTA), 1-(p-nitrobenzyl)-1,4,7,10-tetraazacyclodecane-4,7,10- triacetate (HP-DOA3), 6hydrazinyl-M-methylpyridin-3-carboxamide (HYNIC), 1,4,7-triazacyclononane-1-succinic acid-4,7-diacetic acid (NODASA), 1-(1-carboxy-3- carboxypropyl)-4,7-(carboxymethyl)-1,4,7triazacyclononane (NODAGA), 1,4,7-triazacyclononetriacetic acid (NOTA), 4,11bis(carboxymethyl)-1,4,8,11-tetraazabicyclo[6.6 .2]hexadecane (TE2A), 1,4,8,11-tetraazacyclododecane1,4,8,11-tetraacetic acid (TETA), terpyridine-bis(methyleneaminetetraacetic acid (TMT), 1,4,7,10-tetraazacyclotridecane- M,Ni,Ni',Ni-tetraacetic acid (TRITA) and triethylene tetraamine hexacetic acid (TTHA), where the residue is obtained by covalently linking the carboxyl group contained in the chelating agent to the rest of the conjugate via an ester or amide bond, preferably via an amide bond.

6. The conjugate according to claim 5, where the chelating agent is selected from DOTA and DOTAGA.

7. Conjugate according to any one of claims 1 to 6, where the chelating group contains a chelated cation, preferably a chelated radioactive cation selected from ⁴⁴ Sc, ⁴⁷ Sc, ⁵¹ Cr, ^52m Mn, ⁵⁸ Co, ⁵² Fe, ⁵⁶ Ni, ⁵⁷ Ni, ⁶² Cu, ⁶⁴ Cu, ⁶⁷ Cu, ⁶⁶ Ga, ⁶⁸ Ga, ⁶⁷ Ga, ⁸⁹ Zr, ⁹⁰ Y, ⁸⁹ Y, <Tc, ^99m Tc, ⁹⁷ Ru, ¹⁰⁵ Rh, ¹⁰⁹ Pd, ¹¹¹ Ag, ^110m In, ¹¹¹ In , ^113m In, ^114m In, ^117m Sn, ¹²¹ Sn, ¹²⁷ Te, ¹⁴² Pr, ¹⁴³ Pr, ¹⁴⁹ Pm, ¹⁵¹ Pm, ¹⁴⁹ Tb, ¹⁵³ Sm, ¹⁵⁷ Gd, ¹⁶¹ Tb, ¹⁶⁶ Ho, ¹⁶⁵ Dy, ¹⁶⁹ Er, ¹⁶⁹ Yb, ¹⁷⁵ Yb, ¹⁷² Tm, ¹⁷⁷ Lu, ¹⁸⁶ Re, ¹⁸⁸ Re, ¹⁹¹ Pt, ¹⁹⁷ Hg, ¹⁹⁸ Au, ¹⁹⁹ Au, ²¹² Pb, ²⁰³ Pb, ²¹¹ At., ²¹² Bi, ²¹³ Bi, ²²³ Ra, ²²⁵ Ac , ²²⁷ Th, a cationic molecule containing ¹⁸ F or a cation such as ¹⁸ F-[AlF] ² +, more preferably the cations ⁴⁴ Sc, ⁴⁷ Sc, ⁶⁴ Cu, ⁶⁷ Cu, ⁶⁸ Ga, ⁹⁰ Y, ¹¹¹ In, ¹⁶¹ Tb , ¹⁶⁶ Ho, ¹⁷⁷ Lu, ¹⁸⁸ Re, ²¹² Pb, ²¹² Bi, ²¹³ Bi, ²²⁵ Ac and ²²⁷ Th or cati

- 28 046402 ion molecule containing ¹⁸ F.

8. A conjugate according to any one of claims 1 to 8, wherein the ligand can bind to PSMA.

9. Conjugate according to any one of claims 1 to 8, where the ligand has the structure represented by formula (II) noos.

noos ^1L 3L

II (CH ₂ ) _m o , _H \ (CH ₂ ) _P --coon (II), where m is an integer from 2 to 6, preferably from 2 to 4, more preferably 2;

n is an integer from 2 to 6, preferably from 2 to 4, more preferably 2 or 3;

R ^1L is CH ₂ , NH or O, preferably NH;

R ^3L is CH ₂ , NH or O, preferably NH;

R ^2L is C or P(OH), preferably C and where the ligand is attached to the rest of the conjugate through a bond designated as

10. Conjugate according to any one of claims 1 to 9, which is a compound of formula (III)

SIFA

₁₃

X ³

NOOS^ ,R ¹ J: _2L ,R ³ ^ X ¹ L ¹ —x ² —R ^B —X—R ^CH

Ίί h I hooc- ^(CH2 ^° ^C00H noos _(IIIX or a pharmaceutically acceptable salt thereof, where SIFA is a silicofluoride acceptor moiety (SIFA) which contains a covalent bond between silicon and a fluorine atom and which can be labeled with ¹⁸ F by isotope exchange ¹⁹ F by ¹⁸ F or which is labeled with ¹⁸ F, preferably the SIFA is a SIFA fragment according to claim 2;

m is an integer from 2 to 6, preferably from 2 to 4, more preferably 2;

n is an integer from 2 to 6, preferably from 2 to 4, more preferably 2 or 3;

R ^1L is CH ₂ , NH or O, preferably NH;

R ^3L is CH ₂ , NH or O, preferably NH;

R ^2L represents C or P(OH), preferably C;

X ¹ is selected from an amide bond, an ether bond, a thioether bond, an ester bond, a thioester bond, a urea bridge and an amine bond, preferably an amide bond;

X ² is selected from an amide bond, an ether bond, a thioether bond, an ester bond, a thioester bond, a urea bridge and an amine bond, preferably an amide bond;

L ¹ represents a divalent linking group, the structure of which is selected from oligoamide, oligoether, oligothioether, oligoester, oligothioester, oligourea, oligo(ether-amide), oligo(thioether-amide), oligo(ester-amide ), oligo(thioester-amide), oligo(urea-amide), oligo(ether-thioether), oligo(ether-ester), oligo(ether-thioester), oligo(etherurea), oligo(thioether-ester), oligo(thioether-thioester), oligo(thioether-urea), oligo(ester-thioether), oligo(ester-urea) and oligo(thioester-urea) , preferably the structure is selected from oligoamide and oligo(ester-amide);

X ³ is selected from an amide linkage, an ester linkage, an ether linkage, an amine linkage and a linking group of the formula

wherein the bond designated as ***** in the NH group is linked to ^RB and the other bond designated as ***** is linked to SIFA, preferably X is an amide linkage;

R ^B represents a trivalent linking group;

X ⁴ is selected from an amide bond, an ether bond, a thioether bond, an ester bond, a thioester bond, a urea bridge, an amine bond, and a linking group of the formula

where the amide bond, denoted by "^, is formed with the chelating group, and the other bond,

- 29 046402 designated as ^^^. linked to ^RB , preferably X ⁴ is an amide bond;

R ^CH represents a chelating group, optionally containing a chelating radioactive or non-radioactive cation, preferably a radioactive or non-radioactive metal cation, preferably a chelating group according to claim 4, more preferably a chelating group according to claim 5, and most preferably a chelating group according to claim 6.

11. The conjugate according to claim 10, wherein L ¹ contains a total of 1 to 5, more preferably a total of 1 to 3, and most preferably a total of 1 or 2 amide and/or ester bonds, preferably amide bonds, in the backbone.

12. The conjugate according to claim 10, where -X ¹ -L ¹ -X ² - represents one of the following structures (L-1) and (L-2): -NH-C(O)-R ⁶ -C( O)-NH-R ⁷ -NH-C(O)- (L-1)

-C(O)-NH-R ⁸ -NH-C(O)-R ⁹ -C(O)-NH-R ¹⁰ -NH-C(O)- (L-2), where R6-R ¹⁰ independently selected from C ₂ -C ₁₀ -alkylene, preferably linear C ₂ -C ₁₀ -alkylene, each alkylene group may be substituted by one or more substituents independently selected from -OH, -OCH ₃ , -COOH, -COOCH ₃ , - NH ₂ and -NHC(NH)NH ₂ .

13. The conjugate according to claim 12, wherein the total number of carbon atoms in ^R6 and ^R7 or in R8- ^R10 , respectively, is from 8 to 20, more preferably from 8 to 14, without carbon atoms in optional substituents.

14. The conjugate according to claim 10, where -X ¹ -L ¹ -X ² - is one of the following structures (L-3) and (L-4):

-NH-C(O)-R ⁿ -C(O)-NH-R ¹² -CH(COOH)-NH-C(O)- (L-3),

-C(O)-NH-CH(COOH)-R ¹³ -NH-C(O)-R ¹⁴ -C(O)-NH-R ¹⁵ -CH(COOH)-NH-C(O)- (L -4), where R ¹¹ -R ¹⁵ are independently selected from C ₂ -C ₈ alkylene, preferably linear C ₂ -C ₈ alkylene.

15. The conjugate according to claim 14, where the total number of carbon atoms in R ¹¹ and R ¹² or in R ¹³ -R ¹⁵ , respectively, is from 8 to 18, more preferably from 8 to 12, even more preferably from 9 to 10.

16. Conjugate according to any one of claims 10-15, where R ^B has the structure represented by formula (IV)

where A is selected from N, CR ¹⁶ , where R ¹⁶ represents H or C1-C ₆ -alkyl and a 5-7 membered carbocyclic or heterocyclic group, preferably A is selected from N and CH and more preferably A represents CH;

the bond designated mvww at (CH ₂ ) _a is formed by X ² , and a is an integer from 0 to 4, preferably 0 or 1, and most preferably 0;

the bond designated as llllllll/>at (CC^ is formed by X ³ , and b is an integer from 0 to 4, preferably 0 to 2, and more preferably 0 or 1, and the bond designated as llllllllll at (C11 ₂ ) _c is formed by X ⁴ , and c is an integer from 0 to 4, preferably from 0 to 2, and more preferably 0 or 1.

17. Conjugate according to any one of claims 10-16, which is a compound of formula (IIIa)

or a pharmaceutically acceptable salt thereof, wherein m, n, R ^1L , R ^2L , R ^3L , X ¹ , L ¹ , b, c, X ⁴ and Rdl are as defined in claims 10-16.

18. Conjugate according to claim 17, where b + c is greater than or equal to 1.

19. Conjugate according to claim 17 or claim 18, where b + c is less than or equal to 3.

20. Conjugate according to any one of claims 17-19, where b is equal to 1 and c is equal to 0.

21. The conjugate according to any one of claims 10 to 20, wherein -X^R^ represents the remainder of a chelating agent selected from DOTA and DOTAGA, linked to one of its carboxyl groups through an amide bond to the rest of the conjugate.

22. Conjugate according to any one of claims 10-20, which is a compound of formula (IIIb)

- 30 046402

or a pharmaceutically acceptable salt thereof, wherein m, n, R ^1l , R ^2L , R ^3L , X ¹ , L ¹ , b, c, X ⁴ and R ^CH are as defined in claims 10-20, and r is equal to 0 or 1.

23. The conjugate of claim 22, wherein -N(H) ^-RCH is a chelating agent moiety selected from DOTA and DOTAGA linked to one of its carboxyl groups via an amide bond to the rest of the conjugate.

Brief description of graphic materials

Fig. 1. Illustrative correlation of binding determination of nine reference substances to human serum albumin (OriginPro 2016G).

Fig. 2. Illustrative PET image (maximum intensity projection, dorsal frame) ^{of 68 Ganat} F-5 in SCID mice bearing the LNCaP tumor (1 hour after injection, exposure time 15 minutes) and quantitative assessment of the ROI of selected organs. Data are expressed as mean ± standard deviation (n = 4). % IV/ml represents the % of dose administered per ml. The position of the tumor is indicated by the arrow.

Fig. 3. Illustrative PET image (maximum intensity projection, dorsal frame) ^{of 68 Ganat} F-6 in SCID mice bearing the LNCaP tumor (1 h after injection, exposure time 15 min) and quantification of the ROI of selected organs. Data are expressed as mean ± standard deviation (n = 4). % IV/ml represents the % of dose administered per ml. The position of the tumor is indicated by the arrow.

Fig. 4. Illustrative PET images (maximum intensity projection, dorsal frame) ^{of 68 Ganat} F-7 and ^{68 Ganat} F-7 co-injected with PMPA (8 mg/kg) in SCID mice bearing LNCaP tumor (1 h after injection, exposure time 15 min) and quantification of the ROI of selected organs on PET scans without blocking. Data are expressed as mean ± standard deviation (n = 2). % IV/ml represents the % of dose administered per ml. The positions of the tumor are indicated by arrows.

Fig. 5. Activity versus time curves (log plot) in % ID/ml obtained from dynamic PET data (exposure time 90 min, OSEM spatial reconstruction) in the blood pool (heart), muscle, kidney, liver and LNCaP ⁶⁸ Ga xenograft tumor ^nat F-7 in SCID mice carrying the LNCaP tumor.

Fig. 6. Illustrative PET images (maximum intensity projection, dorsal frame) ^{of 18} F-7 (with free chelate) in SCID mice bearing LNCaP tumor (1 h after injection, exposure time 15 min) and quantification of ROI of selected organs at PET scanning without blocking. Data are expressed as mean ± standard deviation (n = 3). % IV/ml represents the % of dose administered per ml. The positions of the tumor are indicated by arrows.

Fig. 7. Activity versus time curves (log plot) in % ID/ml obtained from dynamic PET data (exposure time 90 min, OSEM spatial reconstruction) in the blood pool (heart), muscle, kidney, liver and LNCaP ¹⁸ F xenograft tumor -7 (with free chelate) in SCID mice bearing the LNCaP tumor.

Fig. Fig. 8. Biodistribution (% WD/g) ^{of 68 Ganat} F-7 (gray columns) and ¹⁸ F-7 (with free chelate) (white columns) 1 hour after administration in SCID mice bearing the LNCaP tumor. Data are expressed as mean ± standard deviation (n = 3).

Fig. 9. Illustrative PET images (maximum intensity projection, dorsal frame) ^{of 68 Ganat} F-8 and ^{68 Ganat} F-8 co-injected with PMPA (8 mg/kg) in SCID mice bearing LNCaP tumor (1 h after injection, exposure time 15 min) and quantification of the ROI of selected organs on PET scans without blocking. Data are expressed as mean ± standard deviation (n = 2). % IV/ml represents the % of dose administered per ml. The positions of the tumor are indicated by arrows.

Fig. 10. Activity versus time curves (log plot) in % ID/ml obtained from dynamic PET data (exposure time 90 min, OSEM spatial reconstruction) in a blood pool (heart), muscle, kidney, liver and LNCaP ⁶⁸ Ga xenograft tumor ^nat F-8 in SCID mice carrying the LNCaP tumor.

Fig. 11. Illustrative PET images (maximum intensity projection, dorsal

- 31 046402 frame) ¹⁸ F-8 (free chelate) and ¹⁸ F-8 (free chelate) co-injected with PMPA (8 mg/kg) in SCID mice bearing LNCaP tumor (1 hour after injection, exposure time 15 min) and quantitative assessment of the ROI of selected organs in PET scanning without blocking. Data are expressed as mean ± standard deviation (n = 3). % IV/ml represents the % of dose administered per ml. The positions of the tumor are indicated by arrows. Note the different scaling (0-10 on the left, 0-20 on the right)

Fig. 12. Activity versus time curves (log plot) in % ID/ml obtained from dynamic PET data (exposure time 90 min, OSEM spatial reconstruction) in a blood pool (heart), muscle, kidney, liver and LNCaP ¹⁸ F xenograft tumor -8 in SCID mice carrying the LNCaP tumor.

Fig. 13. Biodistribution (% WD/g) ^{of 68 Ganat} F-8 (free chelate), (gray bars) and ¹⁸ F-8 (white bars) 1 hour after administration in SCID mice bearing the LNCaP tumor. Data are expressed as mean ± standard deviation (n = 3).

Fig. 14. Illustrative PET images (maximum intensity projection, dorsal frame) ^{of 68 Ganat} F-9 (free chelate) and ^{68 Ganat} F-9 (free chelate) co-injected with PMPA (8 mg/kg) in strain mice SCID bearing LNCaP tumor (1 h post-injection, 15 min exposure time) and quantification of the ROI of selected organs on unblocked PET scans. Data are expressed as mean ± standard deviation (n = 3). % IV/ml represents the % of dose administered per ml. The positions of the tumor are indicated by arrows.

Fig. 15. Activity versus time curves (log plot) in % ID/ml obtained from dynamic PET data (90 min exposure time, OSEM spatial reconstruction) in a blood pool (heart), muscle, kidney, liver and LNCaP ⁶⁸ Ga tumor xenograft - ^nat F-9 in SCID mice carrying the LNCaP tumor.

Fig. 16. Illustrative PET images (maximum intensity projection, dorsal frame) ^{of 18} F-9 (free chelate) and ¹⁸ F-9 (free chelate) co-injected with PMPA (8 mg/kg) in SCID mice bearing LNCaP tumor (1 hour after injection, exposure time 15 minutes) and quantification of the ROI of selected organs in PET scanning without blocking. Data are expressed as mean ± standard deviation (n = 4). % IV/ml represents the % of dose administered per ml. The positions of the tumor are indicated by arrows.

Fig. 17. Activity versus time curves (log plot) in % ID/ml obtained from dynamic PET data (exposure time 90 min, OSEM spatial reconstruction) in a blood pool (heart), muscle, kidney, liver and LNCaP ¹⁸ F xenograft tumor -9 (free chelate) in SCID mice bearing the LNCaP tumor.

Fig. 18. Biodistribution (% WD/g) ^{of 68 Ganat} F-9 (gray columns) and ¹⁸ F-9 (free chelate) (white columns) 1 hour after administration in SCID mice bearing the LNCaP tumor. Data are expressed as mean ± standard deviation (n = 3).

Fig. 19. Illustrative PET images (maximum intensity projection, dorsal frame) ^{of 18} F- ^nat Ga-7 (free chelate) and ¹⁸ F- ^nat Ga-7 (free chelate) co-injected with PMPA (8 mg/kg) in mice SCID lines bearing the LNCaP tumor (1 h after injection, exposure time 15 min) and quantification of the ROI of selected organs on unblocked PET scans. Data are expressed as mean ± standard deviation (n = 3). % IV/ml represents the % of dose administered per ml. The positions of the tumor are indicated by arrows.

Fig. 20. Biodistribution (% WD/g) ^{of 18} F- ^nat Ga-7 (free chelate) (white bars) compared to the structurally identical compound ^{68 Ganat} F-7 (free chelate) (gray bars) 1 h after administration in SCID mice carrying the LNCaP tumor. Data are expressed as mean ± standard deviation (n = 3).

Fig. 21. The image on the left shows the maximum intensity projection (MIR) from a PET scan of a subject with normal biodistribution (no tumor lesions detected). Images were acquired 76 min after administration of 272 MBq PSMA-SIFA3 (7) labeled at ¹⁸ F. FIG. 21 on the right shows a maximum intensity projection (MIP) from a PET scan of a subject with moderately advanced disease demonstrating multiple tumor lesions with a high lesion-to-background ratio. Images were obtained 102 min after injection of 312 MBq PSMA-SIFA3 (7) labeled with ¹⁸ F.

Fig. 22. Graphic representation of table. 6.

Fig. 23. Graphic representation of table. 7.

Fig. 24. Graphic representation of table. 8.

Fig. 25. Graphic representation of table. 9.

Fig. 26. Shows: MIR (A) and transaxial images (B-D) of a 70-year-old patient with biochemical recurrence 1.5 years after radical prostate removal (Gleason 8, pT2c, pN1). There is a single typical prostate cancer lesion measuring 5 mm in diameter on the right side of the pelvis with high uptake of PSMA-SIFA3 (7) labeled F. Malignant in nature by

- 32 046402 lesions were confirmed by histopathology.

Fig. 27. A set of images of an 80-year-old patient with advanced castration-resistant prostate cancer (PSA 66.4 ng/ml). Images show high uptake of ^18F -labeled PSMASIFA3 (7) in different classes of prostate cancer lesions (local tumor, lymph node metastases, bone metastases, liver metastases). The lesions shown are within 2 mm (arrows indicate representative, not all tumor lesions).

Fig. 28. Proof of concept experimental proof of action of a ^68Ga , SiFA-substituted chelator-based PET label is shown.

Fig. 29. Illustrative PET images (maximum intensity projection, dorsal frame) ^{of 18} F- ^nat Lu-rh-7 in SCID mice bearing the LNCaP tumor (1 h after injection, exposure time 15 min) and quantification of ROI of selected organs during scanning PET without blocking. Data are expressed as mean ± standard deviation (n = 3). % IV/ml represents the % of dose administered per ml. The positions of the tumor are indicated by arrows.

Fig. 30. Activity versus time curves (log plot) in % ID/ml obtained from dynamic PET data (exposure time 90 min, OSEM spatial reconstruction) in a blood pool (heart), muscle, kidney, liver and LNCaP ¹⁸ F xenograft tumor ^nat Lu-rh-7 in SCID mice carrying the LNCaP tumor.

Fig. Fig. 31. Biodistribution (% ID/g) ^{of 177 Lunat} F-7, ^{177 Lunat} F-8 and ^{177 Lunat} F-10 24 hours after administration in SCID mice bearing the LNCaP tumor. Data are expressed as mean ± standard deviation (n = 4).

Fig. Fig. 32. Biodistribution (% ID/g) ^{of 177 Lunat} F-10 1 and 24 hours after administration in SCID mice bearing the LNCaP tumor. Data are expressed as mean ± standard deviation (n = 4).

Fig. 33. Comparative biodistribution (in % ID/g) of established and new rhPSMA ligands 24 hours after administration in SCID mice bearing the LNCaP tumor. Data are expressed as mean ± standard deviation (n = 4–5).

Fig. Fig. 34. Biodistribution (% WD/g) ^{of 177 Lunat} F-10 and ^{68 Ganat} F-10 1 hour after administration in SCID mice bearing the LNCaP tumor. Data are expressed as mean ± standard deviation (n = 4).

The invention is further illustrated by examples.

Example 1. Materials and methods

General.

Fmoc-(9-fluorenylmethoxycarbonyl-) and all other protected amino acid analogs were purchased from Bachem (Bubendorf, Switzerland) or Iris Biotech (Marktredwitz, Germany). Trityl chloride polystyrene resin (TCP) was obtained from PepChem (Tübingen, Germany). The chelators DOTAGA anhydride and NOTA were obtained from Chematech (Dijon, France). The TRAP chelator (1,4,7-triazacyclononane1,4,7-tris[methylene(2-carboxyethyl)phosphinic acid]) was prepared as previously described (Notni et al., Chemistry (Weinheim an der Bergstrasse, Germany) 16), 7174-85 (2010)). The preparation of the silicofluoride SIFA-benzoic acid acceptor is carried out in accordance with a previously published method (Iovkova et al., Chemistry (Weinheim an der Bergstrasse, Germany) 15, 2140-7 (2009)). All required solvents and other organic reagents were purchased from Alfa Aesar (Karlsruhe, Germany), Sigma-Aldrich (Munich, Germany), or VWR (Darmstadt, Germany). Solid-phase synthesis of peptides was carried out manually using an Intelli-Mixer syringe shaker (Neolab, Heidelberg, Germany). Analytical and preparative reverse-phase high-performance liquid chromatography (RP-HPLC) was performed using Shimadzu gradient systems (Shimadzu Deutschland GmbH, Neufarn, Germany), each equipped with an SPD-20A UV/Vis detector (220 nm, 254 nm). A Nucleosil 100 C18 column (125 x 4.6 mm, particle size 5 μm) (CS GmbH, Langerwee, Germany) was used for analytical measurements at a flow rate of 1 mL/min. Both the specific gradients and the corresponding retention times t _R are given in the text. Preparative HPLC purification was carried out on a Multospher 100 RP 18 column (250x10 mm, particle size 5 μm) (CS GmbH, Langerwee, Germany) at a constant flow rate of 5 ml/min. Analytical and preparative RP-HPLC with radiometric detection was performed using a Nucleosil 100 C18 column (5 μm, 125x4.0 mm) (CS GmbH, Langerwee, Germany). The eluents for all HPLC runs were water (solvent A) and acetonitrile (solvent B), both containing 0.1% trifluoroacetic acid. Electrospray ionization mass spectra for substance characterization were obtained on an Expression ¹ ' CMS mass spectrometer (Advion Ltd., Harlow, UK). Radioactivity is detected by coupling the UV photometer output to a NaI(Tl) scintillation detector from EG&G Ortec (Munich, Germany). Gel permeation chromatography (GPC) was performed on Sephadex GP-10 (100 g, bed size approximately 30x3 cm) with water as eluent, separating the eluate into 20 ml fractions. NMR spectra were recorded on Bruker AVHD-300 or AVHD-400 spectrometers at 300 K. pH values were measured using a SevenEasy pH meter (Mettler Toledo, Giessen, Germany).

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Reception protocols

1) Solid-phase synthesis of peptides using the Fmoc strategy.

Loading TCP resin.

Loading of trityl chloride polystyrene resin (TCP) with Fmoc-protected amino acid (AA) is accomplished by mixing a solution of TCP resin (1.95 mmol/g) and Fmoc-AA-OH (1.5 eq.) in anhydrous DCM (Dichloromethane) with DIPEA (Diisopropylethylamine) (4.5 eq.) At room temperature for 2 hours. The remaining trityl chloride is capped by adding methanol (2 ml/g resin) over 15 minutes. The resin was then filtered and washed with DCM (2x5 ml/g resin), DFMA (2x5 ml/g resin), methanol (5 ml/g resin) and dried in vacuum. The final loading of 1 Fmoc-AA-OH is determined by the following equation:

?η?ηοί1 _ (?η ₂ -?η ₁ )χ1000 9 -I (Mw-Mhci) ^t 2 m ₂ is the mass of loaded resin [g];

m1 is the mass of unloaded resin [g];

MW represents the molecular weight of AA [g/mol];

MHCl is the molecular weight of HCl [g/mol].

Formation of peptide on resin

The corresponding side chain-protected Fmoc-AA-OH (1.5 eq.) is dissolved in DMF (8 ml/g resin) and pre-activated by adding TBTU (2-(N-Benzotriazol-1-yl)-1,1,3 ,3tetramethylaminium tetrafluoroborate 2-(1H-Benzotriazole-1-yl)-1,1,3,3-tetramethylaminium tetrafluoroborate) (1.5 eq.), HOBt (Hydroxybenzotriazole) (1.5 eq.) and DIPEA (4, 5). e.). Pre-activation for SIFA-BA is carried out similarly. For azido-substituted amino acids (2.0 eq.), use HATU (1[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium hexafluorophosphate 3-oxide) (3.0 eq. ), HOAt (1-hydroxy-7-azabenzotriazole) (3.0 eq.) and DIPEA (6.0 eq.). After activation for 15 minutes, the solution is added to the free amino peptide TCP-AA-NH2 bound to the resin and shaken for 2 hours at room temperature. The resin is then washed with DMF (6x5 ml/g resin) and after deprotection of the Fmoc, the next amino acid is coupled in a similar manner.

Removing Fmoc protection on resin

The resin-bound Fmoc peptide was treated with 20% piperidine in DMF (v/v, 8 ml/g resin) for 5 min and then for 15 min. Then the resin is thoroughly washed with DMF (8x5 ml/g resin).

Removal of Dde (2-acetylmedone) protection on resin

Dde-protected peptide (1.0 eq.) was dissolved in a solution of 2% hydrazine monohydrate in DMF (v/v, 5 ml/g resin) and shaken for 15 min. In the case of the presence of Fmoc groups, deprotection of Dde is carried out by adding a solution of imidazole (0.46 g), hydroxylamine hydrochloride (0.63 g) in n-methyl-2-pyrrolidone (2.5 ml) and DMF (0. 5 ml) for 3 hours at room temperature. After deprotection, the resin was washed with DMF (6x5 ml/g resin).

Removing Alloc (allyloxycarbonyl) protection on resin

The allyloxy protecting group was removed by adding triisopropylsilane (TIPS) (50.0 eq.) and tetrakis(triphenylphosphine)palladium(0) (Pd(PPh ₃ ) ₄ ) (0.3 eq.) dissolved in DCM (6 ml). After 1.5 hours at room temperature, the resin is washed with DCM (6x5 ml/g resin).

Removing tBu/Boc protection

Removal of tBu/tBoc protecting groups is accomplished by dissolving the crude product in TFA and stirring for 40 min at room temperature. After TFA is removed under a stream of nitrogen, the residue is dissolved in a mixture of tert-butanol and water. After lyophilization, the crude peptide is obtained.

Cleavage of peptide from resin

a) Preservation of labile acid protecting groups: the resin-bound peptide is dissolved in a mixture of DCM/TFE/AcOH (TFE - tetrafluoroethylene, AcOH - acetic acid) (v/v, 6/3/1.8 ml/g resin) and shake for 30 minutes. The solution containing the fully protected peptide is filtered and the resin is treated with another portion of the digestion solution for 30 minutes. Both fractions are combined and acetic acid is removed under reduced pressure by sequential addition of toluene and water. After lyophilization of the remaining water, the crude, fully protected peptide is obtained.

b) Deprotection of all labile acid protecting groups: the fully protected resin bound peptide is dissolved in a mixture of TFA/TIPS/water (TFA - trifluoroacetic acid, TIPS - triisopropylsilyl) (v/v/v 95/2.5/ 2.5) and shake for 30 minutes. The solution is filtered and the resin is treated in the same way for another 30 minutes. Both filtrates are combined and concentrated under a stream of nitrogen. After dissolving the residue in a mixture of tert-butanol and water, and subsequent lyophilization, the crude peptide is obtained.

2) Synthesis of Lys-urea-Glu binding motifs ((tBuO)KuE(OtBu) ₂ ) (1)

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Synthesis of the tBu-protected Lys-MO4eeuHa-Glu (EuK) binding motif was carried out as previously described using solution phase synthesis (Weineisen et al., EJNMMI study 4, 63 (2014)). The product is obtained as a waxy solid (91.5%). HPLC (10 to 90% B in 15 min): t _R is 12.6 min. Calculated monoisotopic mass (C ₂₄ H ₄₅ N ₃ O ₇ ): 487.6, found: m/z is 488.3 [M+H]+, 510.3 [M+Na]+.

Glu-urea-Glu ((tBuO)EuE(OtBu) ₂ ) (2)

The tBu-protected Glu-urea-Glu (EuE) binding motif was prepared according to a previously published method (Weineisen et al., EJNMMI study 4, 63 (2014)). The product is obtained as a hygroscopic solid (84%). HPLC (10 to 90% B in 15 min): t _R is 11.3 min. Calculated monoisotopic mass (C ₂₃ H ₄₉ N ₂ O ₉ ): 488.3, found: m/z is 489.4 [M+H]+, 516.4 [M+Na]+.

PfpO-Sub-(tBuO)KuE(OtBu) ₂ (Pfp - pentafluorophenyl, Sub - suberic acid) (3) θ H n?

®- _Ό ^Λ Τ ^Ν γ ^Ν - ^Λ οιΒυ

G ° Ί ® ^and hLo %o

HN

OF.

Conjugation of EuK to the suberic acid spacer was performed as previously described (Weineisen et al., EJNMMI study 4, 63 (2014)). The product is obtained as a colorless oil (72%). HPLC (10 to 90% B in 15 min): t _R is 15.5 min. Calculated monoisotopic mass (C ₃₈ H ₅₆ F ₅ N ₃ O ₁₀ ): 809.4, found: m/z is 810.6 [M+H]+, 832.4 [M+Na]+.

Conjugation of the EuK subunit of motif (3) to the peptide

N-terminal protected peptide (1.0 eq) was added to a solution of 3 (1.2 eq) in DMF and TEA (8 eq) was added. After stirring the solution for 2 hours at room temperature, the DMF is removed in vacuo. To cleave tBu esters, TFA is added and the solution is stirred for 45 minutes at room temperature. After TFA is removed under a nitrogen stream, the crude product is purified by RP-HPLC.

3) Obtaining propargyl-TRAP (4) he no-no

Preparation of propargyl-TRAP was carried out as described previously (Reich et al., Chemical Communications (Cambridge, England) 53, 2586-2589 (2017)). Final purification was accomplished by preparative HPLC to provide 60.2 mg (82.4 μmol, 46%) of propargyl-TRAP as a colorless solid. HPLC (2 to 40% B after 20 min): t _R is 6.0 min. Calculated monoisotopic mass (C ₂₁ H ₃₉ N ₄ O11P ₃ ): 616.5, found: m/z is 617.5 [M+H]+.

1H-NMR (300 MHz, D ₂ O, 300 K) δ = 1.96-2.08 (m, 6H, C(O)-CH ₂ ), 2.46-2.55 (m, 2H, P ^B -CH2-C), 2.592.70 (m, 4H, P ^a -CH2-C), 3.36 (d, 2H, ² JPH=6 Hz, P ^B -CH2-N), 3.41 (d , 4H, ² JPH=6 Hz, PA-CH2-N), 3.47-3.48 (m, 12H, ring-CH2), 3.95 (d, 2H, CH2-C=CH, ⁴ Jhh= 3 Hz) ppm*, 13C{1H}-NMR (101 MHz, D2O, 300 K) δ = 24.61 (d, 1JPP=95 Hz, PB-CC), 25.07 (d, 1J _PP =94 Hz, RA-S-S), 26.71 (d, ² JPP=4 Hz, RV-S-S), 27.82 (d, ² JPP=3 Hz, RA-S-S), 28 .89 (C-C^C), 51.29/51.41/51.50 (three different rings-C), 53.66 (d, 1J _PP =91 Hz, NC-PA), 53.66 ( d, 1J _PP =90 Hz, NC-PB), 71.79 (С-С^С), 79.65 (С-С^С), 174.46 (d, ³ JPP=14 Hz, N(H )-C=O ^B ), 177.24 (d, ³ JPP=13 Hz, C=O ^A ) ppm*, 31Р{1Н}-NMR (162 MHz, D ₂ O, 300 K) δ = 37.99 (Р ^А ), 38.68 (Р ^В ) ppm*. *: subscripts A and B indicate the P and O atoms belonging to the uncolored side arm A and colored B, respectively.

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Binding of propargyl-TRAP (4) to peptide

A previously developed method was used to conjugate azide-functionalized peptides to propargyl-TRAP via copper(I)-catalyzed alkynazide cycloaddition (Reich et al., Chemical communications (Cambridge, England) 53, 2586-2589 (2017)). Briefly, propargyl-TRAP (1.0 eq.) was dissolved in water (40 mM solution) and combined with a solution of peptide (1.1 eq.) in a 1:1 (v/v) mixture of tBuOH (2-methylpropanol- 2) and water. Then add a solution of sodium ascorbate (0.5 M, 50 eq.) in water. To initiate the reaction, aqueous Cu(OAc)2(copper acetate) _-H2O (0.05 M, 1.2 eq) was added, resulting in a brown precipitate that dissolved after stirring in a clear green solution. To demetalate TRAP, an aqueous solution of 1,4,7-triazacyclononane1,4,7-triacetic acid (NOTA, 8 mM, 12 eq.) was added and the pH was adjusted to 2.2 with 1 M aq. HCl. After either 1 hour at 60°C or 48 hours at room temperature, the mixture is directly purified using preparative HPLC.

4) DOTAGA conjugation.

The condensation of peptides and the corresponding DOTAGA anhydride chelator has been described in several publications and is summarized as follows: N-terminal exposed peptide (1.0 eq) was dissolved together with DOTAGA anhydride (1.5 eq) and DIPEA (10.0 eq) .) in dry DMF. After stirring the reaction mixture overnight, the DMF was removed in vacuo to give the crude product.

5) Preparation of PSMA-SIFA inhibitors based on EuK PSMA-SIFA1 (5)

PSMA-SIFA1 was prepared according to standard Fmoc solid-phase peptide synthesis (SPPS) on trityl chloride polystyrene resin (TCP) using the above methods. Briefly, resin-bound Fmoc-D-Lys(Boc) (^)-6-[(tert-butoxycarbonyl)amino]-2-[[(9H-fluoren-9yl)methoxy]carbonyl]amino]hexanoic acid) is stripped protection with Fmoc with 20% piperidine in DFMA and Fmoc-D-Dap(Dde)-OH ((2R)-3-{[1-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl]amino}- 2-({[(9H-fluoren-9-yl)methoxy]carbonyl}amino)propanoic acid) (2.0 eq.) was conjugated using HOBt (2.0 eq.), TBTU (2.0). eq.) and DIPEA (6.0 eq.) in DMF. After orthogonal deprotection with Dde (2-Acetyldimedone) imidazole and hydroxylamine hydrochloride in a mixture of n-methyl-2-pyrrolidone and DFMA, SIFA-BA (1.5 eq.) was conjugated in a similar manner. Subsequent deprotection of Fmoc and gentle cleavage from the resin using TFE and AcOH in DCM resulted in the formation of a Boc-protected peptide backbone. Condensation of DOTAGA anhydride (1.5 eq) was carried out by adding DIPEA (10 eq) to DMF. After deprotecting Boc in TFA, add the fragment PfpO-Sub-(tBuO)KuE(OtBu) ₂ (1.2 eq.) in a mixture of TEA (8 eq.) and DMF. Final cleavage of tBu esters by TFA and RP-HPLC purification yielded PSMA-SIFA1 (70%) as a colorless solid. HPLC (10 to 90% B in 15 min): t _R is 9.1 min. Calculated monoisotopic mass (C63H102FN11O22Si): 1411.7, found: m/z is 1412.3 [M+H]+, 706.8 [M+2H]2 ⁺ .

Scheme 1. Preparation of PSMA-SIFA1: a) 20% piperidine (DMF), b) Fmoc-D-Dap(Dde)-OH, HOBt, TBTU, DIPEA (DFMA), c) imidazole, hydroxylamine hydrochloride, (n-methyl -2-pyrrolidone, DPMA), d) SIFA-BA, HOBt, TBTU, DIPEA (DFMA), e) TFE, AcOH (DCM), f) DOTAGA anhydride, DIPEA (DMF), g) TFA, h) PfpO -Sub-(tBuO)KuE(OtBu)2, TEA (DMF).

PSMA-SIFA2 (6)

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Preparation of PSMA-SIFA2 is carried out using the general methods and procedures mentioned above. Briefly, resin-bound Fmoc-D-Lys(Boc)-OH is deprotected from Fmoc with 20% piperidine in DMF and conjugated to N3-L-Dap(Fmoc)-OH (A)-2-azido-3- [(9fluorenylmethyloxycarbonyl)amino]propanoic acid) (2.0 eq) with HATU (3.0 eq), HOAt (3.0 eq) and DIPEA (6.0 eq) in DMF. After cleavage of the Fmoc group, SIFA-BA (1.5 eq) was added with HOBt (1.5 eq), TBTU (1.5 eq) and DIPEA (4.5 eq) in DMF. Subsequent cleavage from the resin with TFA produced a fully deprotected peptide backbone. To conjugate the EuK fragment, PfpO-Sub(tBuO)KuE(OtBu) ₂ (1.2 eq.) was added to a mixture of TEA (8 eq.) and DFMA. Cleavage of tBu-esters is carried out by adding TFA. In the final step, the purified peptide (1.1 eq.) is reacted with propargyl-TRAP (1.0 eq.) in a copper(I)-catalyzed alkynazide cycloaddition as above. After purification by RP-HPLC, PSMA-SIFA2 (4%) was obtained as a colorless solid. HPLC (10 to 90% B in 15 min): t _R is 8.5 min. Calculated monoisotopic mass (C ₆₅ H ₁ 0 ₉ FN ₁ 3O24P ₃ Si): 1595.7, found: m/z is 1596.5 [M+H]+, 799.1 [M+2H] ²⁺ .

Scheme 2. Preparation of PSMA-SIFA2: a) 20% piperidine (DMF), b) N ₃ -L-Dap(Fmoc)-OH, HATU, HOAt, DIPEA (DFMA), c) SIFA-BA, HOBt, TBTU, DIPEA (DFMA), d) TFA, e) PfpO-Sub-(tBuO)KuE(OtBu) ₂ , TEA (DMF), f) propargyl-TRAP, Cu(OAc) ₂ -H ₂ O, sodium ascorbate (tBuOH, _H2O ).

6) Preparation of PSMA-SIFA inhibitors based on EuE.

PSMA-SIFA3 (7):

PSMA-SIFA3 was prepared using the standard Fmoc-SPPS protocol described above. Briefly, resin-bound Fmoc-D-Orn(Dde)-OH (D-Orn D-ornithine) was deprotected Fmoc with 20% piperidine in DFMA and (tBuO)EuE(OtBu) ₂ (2.0 eq.) conjugated with HOBt (2.0 eq.), TBTU (2.0 eq.) and DIPEA (6.0 eq.) in DMF. After cleavage of the Dde group with a mixture of 2% hydrazine in DMF, a solution of succinic anhydride (7.0 eq.) and DIPEA (7.0 eq.) in DMF is added. Conjugation of Fmoc-D-Lys-OAll-HCl (1.5 eq) was achieved by adding a mixture of HOBt (1.5 eq), TBTU (1.5 eq) and DIPEA (4.5 eq) in DMF. After cleavage of the Fmoc group with 20% piperidine in DMF, the free amine is conjugated with Fmoc-D-Dap(Dde)-OH (2.0 eq.) after preliminary activation of the amino acid in a mixture of HOBt (2.0 eq.), TBTU (2 .0 eq.) and DIPEA (6.0 eq.) in DFMA. Subsequent orthogonal deprotection of Dde is carried out using imidazole and hydroxylamine hydrochloride dissolved in a mixture of n-methyl-2-pyrrolidone and DFMA. SIFA-BA (1.5 eq) was reacted with the free side chain amine with HOBt (1.5 eq), TBTU (1.5 eq) and DIPEA (4.5 eq) as activating reagents in DMF. Al

- 37 046402 the lyloxy protecting group is removed by adding TIPS (50.0 eq.) and Pd(PPh ₃ ) ₄ (0.3 eq.) dissolved in DCM. After deprotection of Fmoc with piperidine, the peptide is cleaved from the resin while retaining the labile acid protecting groups using a mixture of TFE and AcOH in DCM. Final condensation of DOTAGA anhydride (1.5 eq) was achieved with piperidine (10 eq) in DMF. After cleavage of the tBu esters of the EuE moiety with TFA, the crude peptide was purified by RP-HPLC to yield PSMA-SIFA3 (24%) as a colorless solid. HPLC (10 to 70% B in 15 min): t _R is 10.4 min. Calculated monoisotopic mass (C ₆₃ H ₉₉ FN ₁₂ O ₂₅ Si): 1470.7, found: m/z is 1471.8 [M+H]+, 736.7 [M+2H] ²⁺ .

Scheme 3. Preparation of PSMA-SIFA3: a) 20% piperidine (DMF), b) (tBuO)EuE(OtBu) ₂ , HOBt, TBTU, DIPEA, (DFMA), c) 2% hydrazine (DMF), d) succinic anhydride, DIPEA (DMF), f) Fmoc-D-LysOAll-HCl (All - allyl), HOBt, TBTU, DIPEA (DFMA), f) Fmoc-D-Dap(Dde)-OH, HOBt, TBTU, DIPEA ( DPMA), g) imidazole, hydroxylamine hydrochloride, (n-methyl-2-pyrrolidone, DPMA), h) SIFA-BA, HOBt, TBTU, DIPEA (DFMA), i) TIPS, Pd(PPh3)4 (DCM), j) TFE, AcOH (DCM), k) DOTAGA anhydride, DIPEA (DMF), l) TFA.

PSMA-SIFA4 (8):

PSMA-SIFA4 is prepared by SPPS as one-step compound 7, after conjugation with Fmoc-D-Dap(Dde)-OH, the Fmoc protecting group is first cleaved with 20% piperidine in DMF, and SIFA-BA is reacted with the free N-terminus of the peptide . The remaining Dde group is cleaved after removal of the allyloxy protecting group using imidazole and hydroxylamine hydrochloride dissolved in a mixture of n-methyl-2-pyrrolidone and DFMA. The subsequent reaction steps are identical to the preparation of PSMA-SIFA3. After purification by RP-HPLC, PSMA-SIFA4 (11%) was obtained as a colorless solid. HPLC (10 to 70% B in 15 min): t _R is 10.4 min. Calculated monoisotopic mass (C ₆₃ H ₉₉ FN ₁₂ O ₂₅ Si): 1470.7, found: m/z is 1471.7 [M+H]+, 736.8 [M+2H] ²⁺ .

- 38 046402

Scheme 4: Preparation of PSMA-SIFA4: a) 20% piperidine (DMF), b) Fmoc-D-Dap(Dde)-OH, HOBt, TBTU, DIPEA (DFMA), c) SIFA-BA, HOBt, TBTU, DIPEA (DPMA), d) TIPS, Pd(PPh3)4 (DCM), e) imidazole, hydroxylamine hydrochloride (n-methyl-2-pyrrolidone, DFMA), f) TFE, AcOH (DCM), g) DOTAGA anhydride, DIPEA ( DMF), h) TFA.

PSMA-SIFA5 (9):

are obtained similarly to ligands 7 and 8. The difference was the use of N ₃ -L-Dap(Fmoc)-OH instead of Fmoc-D-Dap(Dde)-OH, which was necessary for the final click reaction with propargyl-TRAP. Azide-substituted amino acid (2.0 eq.) was conjugated with HATU (3.0 eq.), HOAt (3.0 eq.) and DIPEA (6.0 eq.) in DMF. After deprotecting Fmoc from its side chain with 20% piperidine, SIFA-BA (1.5 eq.) was reacted as above. Prior to conjugation of the chelator moiety, all remaining reaction steps are identical to peptides 7 and 8. After cleavage from the resin with TFA while deprotecting all labile acid protecting groups, the purified E&E azido conjugate (1.1 eq.) was reacted with propargyl-TRAP ( 1.0 eq) in a copper(I)-catalyzed alkinazide cycloaddition. Purification by RP-HPLC gave PSMA-SIFA5 (9%) as a colorless solid. HPLC (10 to 90% B in 15 min): t _R is 8.7 min. Calculated monoisotopic mass (C ₆₅ H ₁₀₆ FN ₁₄ O ₂₇ P ₃ Si): 1654.6, found: m/z is 1655.6 [M+H]+, 828.4 [M+2H] ²⁺ .

Scheme 5. Preparation of PSMA-SIFA5: a) 20% piperidine (DMF), b) N ₃ -L-Dap(Fmoc)-OH, HOBt, TBTU, DIPEA (DFMA), c) SIFA-BA, HOBt, TBTU, DIPEA (DFMA), d) TIPS, Pd(PPh3)4 (DCM), e) TFA, f) propargyl-TRAP, Cu(OAc) ₂ -H ₂ O, sodium ascorbate (tBuOH, H ₂ O).

Complexation experiments

Preparation of ^nat Ga-TRAP complexes: 500 μl of a 2 mM PSMA precursor stock solution in DMSO is combined with 750 μl of a 2 mM Ga(NO ₃ ) ₃ solution in water. Complexation occurred instantly at room temperature. The completeness of the reaction is monitored using RP-HPLC and subsequent mass spectrometry.

^nat Ga-PSMA-SIFA2: HPLC (10 to 90% B after 15 min): t _R is 9.5 min. Calculated monoisotopic mass (C ₆₅ H ₁₀₆ FGaN ₁₃ O ₂₄ P ₃ Si): 1661.6 found: m/z is 1663.9 [M+H] ⁺ , 832.6 [M+2H] ²⁺ .

^nat Ga-PSMA-SIFA5: HPLC (10 to 90% B after 15 min): t _R is 9.0 min. Calculated monoisotopic mass (C ₆₅ H ₁₀₃ FGaN ₁₄ O ₂₇ P ₃ Si): 1720.5 found: m/z is 1720.8 [M+H] ⁺ , 861.1

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[M+2H]2+.

Preparation of 'Ga-DOTAGA complexes: 500 µL of 2 mM PSMA precursor stock solution in DMSO is combined with 1500 µL of 2 mM Ga(NO3)3 solution in water. The reaction mixture is heated for 30 minutes at 60°C. The reaction result is monitored using RP-HPLC and subsequent mass spectrometry. For ¹⁸ E radiolabeling of [ ^nat Ga]PSMA-SIFA ligands, the complex was purified by RP-HPLC prior to the labeling reaction.

^nat Ga-PSMA-SIFA1: HPLC (10 to 90% B after 15 min): t _R is 9.1 min. Calculated monoisotopic mass (C ₆₃ H ₉₉ FGaN _n O ₂₂ Si): 1477.6 found: m/z is 1479.5 [M+H]+, 740.2 [M+2H]2+.

^nat Ga-PSMA-SIFA3: HPLC (10 to 70% B after 15 min): t _R is 10.4 min. Calculated monoisotopic mass (C ₆₃ H ₉₆ FGaN ₁₂ O ₂₅ Si): 1536.6 found: m/z is 1539.4 [M+H]+, 770.3 [M+2H] ²⁺ .

^nat Ga-PSMA-SIFA4: HPLC (10 to 70% B after 15 min): HPLC (10 to 70% B after 15 min): t _R is 10.4 min. Calculated monoisotopic mass (C ₆₃ H ₉₆ FGaN ₁₂ O ₂₅ Si): 1536.6 found: m/z is 1539.1 [M+H]+, 770.5 [M+2H] ²⁺ .

Complexation of ^nat Lu: the corresponding ^nat Lu ^nI complexes are prepared from a 2 mM aqueous solution of PSMA inhibitor with a 2.5 molar excess of LuCl3 (20 mM solution), heated to 95°C for 30 min. After cooling, the formation of the ''^^chelate was confirmed using HPLC and MS. The resulting 1 mM aqueous solutions of the respective ^nat Lu complexes were then diluted and used in in vitro IC50 studies without further processing.

Radiolabeling ⁶⁸ Ga-labeling: ⁶⁸ Ga-labeling is carried out using an automated system (GallElut ⁺ from Scintomics, Germany) as described previously (Notni et al., EJNMMI study, 28 (2012)). Briefly, the ⁶⁸ Ge/6 ⁸ Ga generator with SnO ₂ matrix (IThemba LABS) is eluted with 1.0 M aqueous HCl, from which a fraction (1.25 ml) representing approximately 80% activity (500-700 MBq) is transferred to the reaction bottle (ALLTECH, 5 ml). Before elution, the reactor is charged with 2-5 nmol of the appropriate chelator conjugate in an aqueous 2.7 M HEPES solution (DOTAGA conjugates: 900 μl, TRAP conjugates: 400 μl). After elution, the vial is heated for 5 minutes at 95°C. Purification is carried out by passing the reaction mixture through a solid phase extraction cartridge (C 8 light, SepPak), which is purged with water (10 ml) and the product is eluted with 50% aqueous ethanol (2 ml), phosphate buffered saline (PBS, 1 ml) and again with water (1 ml). After removal of ethanol in vacuo, the purity of radiolabeled compounds is determined by radio-TLC (ITLC-SG chromatography paper, mobile phase: 0.1 M trisodium citrate and a 1:1 (v/v) mixture of 1 M ammonium acetate and methanol) .

¹⁸ P-labeling: To carry out ¹⁸ P-labeling, a previously published method is used (Wangler et al., Nat Protoc 7, 1946-55 (2012)), which was slightly modified. Briefly, aqueous ^- is passed through a SAX cartridge (Sep-Pak Accell Plus QMA Carbonate light), which is pretreated with 10 ml of water. After drying with 10 ml air, the water is removed by washing the cartridge with 10 ml anhydrous acetonitrile and then 20 ml air. ¹⁸ F is eluted with 100 µmol [K+c2.2.2]OH ^- dissolved in 500 µl anhydrous acetonitrile. Before labeling, add 25 µmol oxalic acid in anhydrous acetonitrile (1 M, 25 µl). This mixture is used as a whole or an aliquot to fluoridate 10-25 µM PSMA-SIFA (1 M in anhydrous DMSO). The resulting reaction mixture was incubated for 5 minutes at room temperature. To purify the labeled substance, use a light Sep-Pak C18 cartridge, pre-treated with 10 ml of EtOH (ethyl alcohol), and then 10 ml of H ₂ O. The mixture for introducing the radioactive label is diluted with 9 ml of 0.1 M HEPES buffer (pH 3) and pass through the cartridge and then 10 ml of H ₂ O. The peptide is eluted with 500 μl of a mixture of 4:1 (v/v) EtOH in water. The radiochemical purity of the labeled compound is determined by RP-HPLC with radiometric detection and radio-TLC (silica gel 60 RP-18 F254s, mobile phase: a mixture of 3:2 (v/v) MeCN in H ₂ O with the addition of 10% 2M NaOAc (acetate sodium) solution and 1% TFA).

^|2 5[-labeling: reference ligand for in vitro studies ([ ¹²⁵ I]I-BA)KuE is prepared according to a previously published method (Weineisen et al., EJNMMI research, 4, 63 (2014)). Briefly, 0.1 mg of stannylated precursor (SnBu3-BA)(OtBu)KuE(OtBu)2 is dissolved in a solution containing 20 μL peracetic acid, 5.0 μL (21 MBq) [ ¹²⁵ I]NaI (74 TBq/mmol, 3.1 GBq/ml, 40 mM NaOH, Hartmann Analytic, Braunschweig, Germany), 20 μl MeCN (Acetonitrile) and 10 μl acetic acid. The reaction solution is incubated for 10 minutes at room temperature, loaded into a cartridge and washed with 10 ml of water (C18 Sep Pak Plus cartridge, pretreated with 10 ml of MeOH (Methanol) and 10 ml of water). After eluting with 2.0 mL of 1:1 (v/v) EtOH/MeCN, the radioactive solution was evaporated to dryness under a gentle stream of nitrogen and treated with 200 μL of TFA for 30 min, followed by evaporation of the TFA. The crude product ([ ¹²⁵ I]I-BA)KuE is purified by RP-HPLC (20 to 40% B for 20 min): t _R is 13.0 min.

^^labeling: to a reaction mixture volume of 10 μl of 1.0 M NH ₄ OAc buffer (ammonium acetate), pH 5.9, add the labeled substance from 0.75 to 1.0 nmol (from 7.5 to 10 μl) , 10 to 40 MBq ¹⁷⁷ LuCl3 (specific activity (AS) > 3000 GBq/mg, 740 MBq/ml, 0.04 M HCl, ITG, Garching, Germany) and finally fill to 100 µl with label-free water (Merck, Darmstadt, Germany). Reactionary

- 40 046402 the mixture is heated for 30 min at 95°C and the radiochemical purity is determined using radio-TLC (silica gel 60 RP-18 F _254S , a mixture of 3:2 (v/v) MeCN in H2O with the addition of 10% 2 M solution of NaOAc and 1% TFA, Rf is 0.5).

In vitro experiments Determination of IC ₅₀

PSMA-positive LNCaP cells were grown in Dulbecco's modified Eagle's medium/F-12 with Glutamax-I (1:1) (Invitrigon) supplemented with 10% fetal bovine serum and maintained at 37°C in a 5% humidified atmosphere. _CO2 . To determine the affinity of PSMA (IC ₅₀ ), cells are collected 24 ± 2 hours before the experiment and seeded in 24-well plates (1.5x105 cells in 1 ml/well). After removal of the culture medium, cells are treated once with 500 μl HBSS (Hank's balanced salt solution, Biochrom, Berlin, Germany, supplemented with 1% bovine serum albumin (BSA)) and left for 15 min on ice to equilibrate in 200 μl HBSS (1% BSA). Then add 25 µl per well of solutions containing either HBSS (1% BSA, control) or the appropriate ligand in increasing concentrations (from 10 ^-10 to 10 ^-4 M in HBSS, followed by the addition of 25 µl ([ ¹²⁵ I]I- BA)KuE (2.0 nM) in HBSS (1% BSA). All experiments were performed at least three times for each concentration. After a 60-minute incubation on ice, the experiment was stopped by removing the medium and then washing with 200 μl HBSS. Both media stages are combined into one fraction and the amount of free radioligand is represented.The cells are then lysed with 250 μl of 1 M NaOH and combined with 200 μl of HBSS in the next washing step. Quantification of bound and free radioligand is carried out in a γ-counter.

Internalization

For internalization studies, LNCaP cells were harvested 24 ± 2 h before the experiment and seeded in 24-well plates (1.25 x 105 cells in 1 ml/well). After removal of the culture medium, cells are washed once with 500 μl DMEM-F12 (5% BSA) and allowed to equilibrate for at least 15 min at 37°C in 200 μl DMEM-F12 (5% BSA). Each well is treated with either 25 μl of either DMEM-F12 (5% BSA) or 100 μM PMPA block solution. Then 25 μl of PSMA inhibitor labeled with ⁶ 8Ga/18F (5.0 nM) was added and the cells were incubated at 37°C for 60 min. The experiment is stopped by placing the 24-well plate on ice for 3 min and then removing the medium. Each well is washed with 250 μl of HBSS and fractions from these first two steps are pooled to represent the amount of free radioligand. Removal of surface-bound activity is accomplished by incubating the cells with 250 μl of ice-cold PMPA solution (10 μM in PBS) for 5 min and washing again with another 250 μl of ice-cold PBS. Internalized activity was determined by incubating cells in 250 μl 1 M NaOH and combining with the subsequent wash step fraction 250 μl 1.0 M NaOH. Each experiment (control and blockade) is carried out in triplicate. Free, surface and internalized activities are quantified in a γ-counter. All internalization studies were accompanied by control studies using ([ ¹²⁵ I]I-BA)KuE (c is 0.2 nM), which were performed in a similar manner. Data were adjusted for nonspecific internalization and normalized for specific internalization observed for the radioiodinated reference compound.

Octanol-water partition coefficient

Approximately 1 MBq of tracer is dissolved in 1 ml of a 1:1 (v/v) mixture of phosphate-buffered saline (PBS, pH 7.4) and n-octanol in an Eppendorf tube. After vigorously stirring the suspension for 3 min at room temperature, the vial is centrifuged at 15,000 g for 3 min (Biofuge 15, Heraus Sepatech, Osterode, Germany) and 100 μl aliquots of both layers are measured in a gamma counter. The experiment is repeated at least six times.

HSA binding

To determine HSA binding, a Chiralpak HSA column (50x3 mm, 5 μm, H13H-2433) was used at a constant flow rate of 0.5 ml/min. The mobile phase (A: NH ₄ OAc, 50 mM in water, pH 7 and B: isopropanol) was freshly prepared for each experiment and was used for one day only. The column is stored at room temperature and each run is stopped after signal detection to reduce data acquisition time. All substances are dissolved at a concentration of 0.5 mg/ml in 50% 2-propanol and 50% 50 mM ammonium acetate buffer with a pH value of 6.9. The selected reference substances show a range of HSA binding from 13 to 99%, as a wide variety of albumin binding to peptides was expected. All nine reference substances (see Table 1) are introduced sequentially to establish a non-linear regression with OriginPro 2016G, see Fig. 1.

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Table 1. Reference substances (Yamazaki et al., Journal of pharmaceutical sciences 93, 1480-94 (2004)) used to calibrate the HSA column

Reference tR Logt _R Lit. HSA % Log To HSA p-benzyl alcohol 2.40 0.38 13.15 minus 0.82 Aniline 2.72 0.43 14.06 minus 0.79 Phenol 3.28 0.52 20.69 minus 0.59 Benzoic acid 4.08 0.61 34.27 minus 0.29 Carbamazepine 4.15 0.62 75.00 0.46 p-nitrophenol 5.62 0.75 77.65 0.52 Estradiol 8.15 0.91 94.81 1.19 Probenecid 8.84 0.95 95.00 1.20 Glibenclamide 29.18 1.47 99.00 1.69

The retention time is shown as an example for the experiment performed, t _R retention time, Lit. HSA literature value of human serum albumin binding in [%], Log K HAS log K of human serum albumin binding.

In vivo experiments

All experiments on animals are carried out in accordance with the general rules for keeping animals in Germany and the rules adopted in the institution for the care and use of animals. To establish tumor xenografts, LNCaP cells (10 ⁷ cells/200 μl) are suspended in a 1:1 (v/v) mixture of Dulbecco's modified Eagle's medium/F-12 nutrient mixture with Glutamax-I (1:1) and Matrigel (BD Biosciences, Germany) and inoculated subcutaneously into the right shoulder of 6-8 week old CB17-SCID mice (Charles River, Sulzfeld, Germany). Mice are used for cases where tumors have grown to a diameter of 5-8 mm (3-4 weeks after inoculation).

μPET image

Imaging studies are carried out using a Siemens Inveon small animal PET system. Data were reconstructed as single frames using the 3D ordered maximum expected subset (OSEM3D) algorithm, followed by data analysis (ROI-based quantification) using Inveon Research Workplace software. For PET studies, mice are anesthetized with isoflurane and 0.15 to 0.25 nmol (2-20 MBq) of ⁶⁸ Ga or ¹⁸ F labeled substance is injected into the tail vein. Dynamic imaging was performed after injection on the bedside for 90 min. Static images were recorded one hour after injection with an acquisition time of 15 min. For blockade, 8 mg/kg PMPA is administered immediately before the tracer injection.

Biodistribution

Approximately 2 to 20 MBq (0.2 nmol) of PSMA inhibitors labeled with ⁶⁸ Ga or ¹⁸ F were injected into the tail vein of male SCID CB-17 mice bearing the LNCaP tumor and sacrificed 1 hour after injection (n = 3) . Selected organs were removed, weighed and measured in a γ-counter.

In experiments on humans.

Human proof-of-concept testing was conducted as part of a compassionate use trial. The agent is used in accordance with the German Medicines Law, AMG §13 2b, and in accordance with the responsible regulatory authority (Government of Oberbayern).

All objects are examined on a Biograph mCT scanner (Siemens Medical Solutions, Erlangen, Germany) or a Biograph mMR scanner (Siemens Medical Solutions, Erlangen, Germany). All PET scans were acquired in spatial mode with an acquisition time of 2–4 min per position. Outlier data were corrected for randomness, dead time, scatter, and attenuation and iteratively reconstructed using an ordered subset expectation maximization algorithm (four iterations, eight subsets) followed by a post-reconstruction Gaussian smoothing filter (5 mm full width at half maximum). Images in 53 prostate cancer patients were obtained after administration of an average of 324 (range, 236 to 424) MBq of ¹⁸ F-labeled PSQ-SIFA3 (7) at an average of 84 (range, 42 to 166) min after injection. 47 subjects were imaged on a PET/CT (Positron Emission Tomography/Computed Tomography) scanner, 6 subjects on a PET/MR (Positron Emission Tomography/Magnetic Resonance Imaging) scanner. In 33 subjects, furosemide was used at the time of tag injection; in 20 patients, furosemide was not administered.

The average and maximum standardized accumulation values (SUVavg/SUVmax) of the parotid glands, submandibular glands, lungs, mediastinal blood pool, liver, spleen, head of the pancreas, duodenum, kidneys, bladder and unaffected bone are analyzed. To calculate SUV, circular ROIs are drawn around areas of focal increased uptake in transaxial slices and are automatically adapted to the spatial volume of interest (VOI) at 50% isocontour. Lesions that are visually seen as evidence of recurrence or metastasis of prostate cancer are counted.

- 42 046402 glands. One or two lesions of the same type (local tumor, lymph node metastases, bone metastases, internal organ metastases) are analyzed on a patient using SUVmax and SUVmedia as described above. The gluteal muscle was used as a background.

Example 2. Results

Overview of the resulting PSMA-SIFA ligands

PSMA-SIFA1 (5)

He

PSMA-SIFA 10

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Lipophilicity

The determined octanol/water partition coefficients (log D) for compounds labeled with ⁶⁸ Ga or ¹⁸ F are presented in Table. 2. Within the EuK-based inhibitors labeled with ⁶⁸ Ga, the TRAP-functionalized compound (5) was found to be more hydrophilic than 6, where DOTAGA was used as a chelator. This result was also shown for EuE-based agents labeled with ⁶⁸ Ga, where the TRAP derivative (9) showed the highest lipophilicity. All compounds labeled with ¹⁸ F showed lower hydrophilicity compared to substances labeled with ⁶⁸ Ga.

Table 2. Log D values of the resulting radiolabeled PSMA-SIFA ligands (n is 6)

Ligand logD ^{68 Ganat} FL logD ¹⁸ fl logD ^{177 Lunat} FL 5 minus 2.75 ± 0.07 Not detected 6 minus 3.00 ± 0.09 Not detected 7 minus 3.18 ± 0.05 minus 1.98 ± 0.04 minus 4.20 ± 0.09 8 minus 2.59 ± 0.04 minus 2.25 ± 0.07 minus 3.75 ± 0.06 9 minus 3.26 ± 0.06 minus 2.23 ± 0.07 10 minus 3.59 ± 0.05 eleven minus 3.62 ± 0.06

PSMA Affinity Determination

The resulting compounds bearing the EuE-binding motif (7, 8, 9) showed higher affinity for PSMA compared to EuK-based agents (5, 6). TRAP chelator compounds (6 and 9) showed slightly reduced affinity compared to their DOTAGA counterparts (5 and 7, respectively). No significant differences in affinity for PSMA were observed between ^nat Ga complexing agents and the corresponding non-complexing compound (Table 3).

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Table 3.

Binding affinity (IC50 in nM) of PSMA-SIFA ligands to PSMA. Affinity was determined using LNCaP cells (150,000 cells/well) and ([ ¹²⁵ I]I-BA)KuE (c is 0.2 nM) as radioligand (1 h, 4°C, HBSS + 1% BSA). Data are expressed as mean ± standard deviation (n is 3 from 3 different experiments).

Standards: (I-BA)KuE DCFPyL PSMA-1007 IC ₅₀ = 7.l ± 2.4 hM IC ₅₀ = 12.3 ± 1.2 hM IC ₅₀ = 4.2 ± 0.5 hM Ligand 1C ₅₀ [nM] ^Ga-^FL IC ₅₀ [hM] ^mt FL IC ₅₀ [hM] ^Lu-^FL 5 7.3 ± 0.2 6.4 ± 0.2 6 10.8 ±2.5 8.5 ± 1.7 7 3.0 ±0.7 3.5 ±0.2 3.9 ±0.5 8 3.8 ±0.7 2.5 ± 0.2 3.0 ±0.2 9 4.5 ± 0.3 4.3 ± 0.2 10 2.8 ± 0.7 AND 4.8 ± 0.7

Internalization

In analogy with binding affinity, EuE-based compounds (7, 8, 9) showed significantly higher internalization values compared to peptides bearing the EuK-binding motif (5, 6). Regarding chelator effects, TRAP showed a positive effect on internalization (5 and 9 versus 6 and 7, respectively), although binding affinity was higher for analogs

DOTAGA (Table 3). All ^18F -labeled compounds showed higher internalization values compared to the corresponding ^68Ga -labeled compounds (Table 4).

Table 4.

Total internalized activity (c is 0.5 nM) over 1 hour as % of reference ligand ([ ¹²⁵ I]I-BA)KuE (c is 0.2 nM) determined on LNCaP cells (37°C, DMEM F12 + 5% BSA, 125,000 cells/well). Data are corrected for nonspecific binding (10 μmol PMPA) and expressed as mean ± standard deviation (n is 3).

Standards: [%] DCFPyL PSMA-1007 118 ± 5 118 ± 4 Ligand Internalization [%] ^{68 Ganat} FL Internalization [%] ¹⁸ fl Internalization [%] ¹⁷⁷ Lu- ^m 'FL 5 33.0 ±2.1 Not detected 6 43.0 ±3.4 Not detected 7 126.0 ± 13.1 164.8 ±4.5 185.8 ±4.5 8 98.2 ± 12.4 130.3 ±5.5 165.9 ±8.5 9 176.9 ± 11.7 211.6 ± 5.3 10 184.1 ± 16 AND 134.5 ± 18.5

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Human serum albumin binding

Table 5. HSA binding of the resulting PSMA-SIFA ligands determined on the column

see fig. 2.

2. [ ⁶⁸ Ga][ ^nat F]PSMA-SIFA2 ( ⁶⁸ Ga- ^nat F-6) see Fig. 3.

3. PSMA-SIFA3 (7)

a) static image of ⁶⁸ Ga-H)T see fig. 4;

b) dynamic image of ⁶⁸ Ga-H)T see fig. 5;

c) static image ¹⁸ Fn)T see FIG. 6;

d) dynamic image ^l8 Fl Г)Т see fig. 7;

e) biodistribution studies, see Fig. 8.

4. PSMA-SIFA4 (8)

a) static image of ⁶⁸ Ga-H)T see Fig. 9;

b) dynamic image of ⁶⁸ Ga-H)T see fig. 10;

c) static image ¹⁸ Fl)T see Fig. eleven;

d) dynamic image ¹⁸ FH)T see fig. 12;

e) biodistribution studies, see Fig. 13.

5. PSMA-SIFA5 (9)

a) static image of ⁶⁸ Ga-H)T see Fig. 14;

b) dynamic image of ⁶⁸ Ga-H)T see fig. 15;

c) static image ¹⁸ Fl)T see Fig. 16;

d) static image ¹⁸ Fl)T see fig. 17;

e) biodistribution studies, see Fig. 18.

6. Experimental proof of concept ^nat Ga-PSMA-SIFA3 ( ^nat Ga-7)

a) static image ¹⁸ Fl)T see Fig. 19;

b) biodistribution studies, see FIG. 20.

7. Image 1 Small animal DEG using luteinium-rhPSMA ligands

a) static l)T image: ¹⁸ F- ^nat Lu-rh-7 see fig. 29;

b) dynamic l)T image: ¹⁸ F- ^nat Lu-rh7 see fig. thirty;

c) studies of the biodistribution of ^{177 Lunat} F-7, ^{177 Lunat} F-8 and ^{177 Lunat} F-10 after 24 hours

- 46 046402 see fig. 31;

d) biodistribution of ^{177 Lunat} F-10 after 1 and 24 hours, see Fig. 32;

e) Comparative biodistribution of established and new rhPSMA ligands after 24 hours, see FIG. 33;

f) comparative biodistribution of ^{177 Lunat} F-rhPSMA-10 and ^{68 Ganat} F-rhPSMA-10 over 1 hour, see FIG. 34.

Biodistribution and uptake of PSMA-SIFA3 (7) in human tumor lesions

No side effects or clinically detectable pharmacological effects were noted.

Fig. 21 shows a maximum intensity projection (MIR) from a PET scan of a subject with normal biodistribution (no tumor lesions detected). Images were taken 76 min after administration of 272 MBq PSMA-SIFA3 (7) labeled at ¹⁸ F. FIG. 21 on the right shows a maximum intensity projection (MIP) from a PET scan of a subject with moderately advanced disease demonstrating multiple tumor lesions with a high lesion-to-background ratio. Images were acquired 102 min after injection of 312 MBq PSMA-SIFA3 (7) labeled with ¹⁸ F.

Absorbance parameters reflect background PSMA expression for different tissue types. Significant uptake of the radioactive tracer was detected only for the salivary glands, kidneys, liver, spleen and duodenum. Absorbance in the background tissue was low. Uptake in tumor lesions was significantly higher than in tissue with low PSMA expression.

Table 6.

Average SUVmax (left) and SUVav (right) in different tissues (tissues/organs: n is 53, tumor lesions: n is 72) with standard error. See fig. 22 for graphical representation.

Average SUVMaKC minimum maximum Average SUVcpcg minimum maximum Background ι,θ 0.6 1.9 0.6 0.4 1.2 Blood pool 2.4 1.6 3.9 2.0 1.1 17.0 Parotid gland 23.5 8.2 42.3 16.8 5.5 32.7 Submandibular gland 26.7 10.1 43.8 19.6 7.0 29.7 Lungs 1.0 0.5 3.1 0.7 o, s 2.0 Liver 9.5 4.5 25.2 7.0 3.2 17.7 Spleen 11.8 4.7 21 9.1 3.4 17.1 Pancreas gland 3.9 1.8 9.2 2.7 1.3 7.4 duodenum intestine 14.2 2.8 32.7 10.5 1.9 23.9 Bone 1.7 0.8 3.1 1.1 0.6 2.1 Bud 44.3 19.1 75.2 32.1 13.2 54.7 Bladder 8.3 0.5 112.0 6.1 o, s 85.7 Tumor lesions 26.6 4.0 95.4 19.2 2.7 71.7

Due to the low ratio of background SUV activity to background of organs and tumor lesions, it is favorable for clinical imaging. Tumor lesions are displayed in high contrast compared to the background.

- 47 046402

Table 7.

Average ratio of SUVMaKC (left) and SUVcpeg (right) with standard error in different tissues (tissues/organs: n is 53, tumor lesions: n is 72) with standard error. See fig. 23 for graphical representation._________________________________________

SUVmbkc to background ratio SUVcpeg to background ratio Average minimum maximum Average minimum maximum Blood pool 2.5 1.3 4.8 3.1 1.5 21.3 Parotid gland 24.3 8.2 45.3 27.5 9.2 54.5 Submandibular gland 27.8 10.1 54.7 32.5 I,7 61.8 Lungs 1.1 0.4 z, z 1.1 0.4 4.0 Liver 10.1 2.9 42.0 11.7 3.7 44.3 Spleen 12.3 4.7 35.0 14.9 5.7 39.5 Pancreas 4.0 1.5 from 4.3 1.9 10.8 Duodenum 14.8 2.8 31.3 17.3 3.2 35.3 Bone 1.7 0.9 2.9 1.8 1.0 3.2 Bud 46.7 16.9 98.7 53.8 19.8 109.3 Bladder 8.3 0.6 112.0 9.8 0.5 142.8 Tumor lesions 28.6 5.0 83.4 31.9 5.4 83.2

Tumor lesion uptake and contrast to background were relatively similar between different tumor types (local tumor [n is 24], lymph node metastasis [n is 23], bone metastasis [n is 21], visceral metastasis [n is 4]).

Table 8.

Mean SUVmax, SUVav, ratio of SUVmax to background, and ratio of SUVmax to background for different tumor types with standard error. See fig. 24 for graphical representation.

Local tumor SUVMaKC Attitude SUVmhkc SUVcpeg Attitude SUVcpeg Average 26.9 29.6 19.3 32.4 minimum 4 5 2.7 5.4 maximum 75.1 83.4 19.3 83.2 Metastases in lymph nodes SUVMaKC Attitude SUVmbkc SUVcpeg Attitude SUVcpeg Average 22.2 23.7 16.6 27.3 minimum 8.1 5.8 6.4 7.1 maximum 67.5 63.6 44.6 70.7 Metastases in bones SUVmbkc Attitude SUVmbkc SUVcpeg Attitude SUVcpeg Average 31.2 32.8 22.2 36.6 minimum 7.9 7.9 5.3 8.8 maximum 95.4 73.4 71.7 80 Metastases in internal organs SUVMaKC Attitude SUVmbkc SUVcpeg Attitude SUVcpeg Average 26.1 28.9 17.4 30.2 minimum 20.4 18.5 15.5 22.1 maximum 32.5 40.6 19.1 38.2

Retention of PSMA-SIFA3 (7) tag in the human bladder

Retention of tracer in the urinary excretory system is a common drawback of PSMA ligand imaging. PSF-SIFA3 (7), labeled with ¹⁸ F, as a potential prototype compound, PET agent labeled with ⁶⁸ Ga, SiFA substituted, chelator-based, is excreted through the urinary excretory system, but to a much lesser extent than most other PET agents. In addition, on his

- 48 046402 bladder retention can be significantly affected by the use of furosemide during injection of the label. T-test 8 showed a statistically significant lower label latency with furosemide (p is 0.018 for both SUVmax and SUVmax).

Table 9.

Mean SUVmax (left) and SUVav (right) of label latency in the bladder in subjects with and without furosemide administration with standard error. See fig. 25, for graphical representation.

ZiUmax SUVcpeg Average minimum maximum Average minimum maximum With furosemide 4.8 1.6 32 3.4 1.1 26.6 Without furosemide 13.9 0.5 112 10.5 0.3 85.7

Clinical results for detection of PSMA-SIFA3 (7) tumor lesions and histopathological confirmation

Subjects are imaged by primary stage (n is 6) and recurrent disease (n is 47). Lesions consistent with prostate cancer were found in 39 patients. 72 lesions were analyzed. 21 of 72 lesions had no correlation on morphologic imaging. 14 of the 72 lesions measured were equal to or less than 5 mm in size on the morphological image. Both demonstrate the high clinical utility of ^18F -labeled PSMA-SIFA3 (7) for identifying lesions that are otherwise occult on morphological imaging. The uptake parameters of 35 uncorrelated or small lesions showed favorable uptake parameters on morphologic imaging.

Table 10. Average SUVmax, SUVav, ratio of SUVmax to background and ratio of SUVmax to background in prostate cancer tumors with standard error

SUVmhkc Attitude ZiUmax SUVcpcg Attitude SUVcpcg Average 16.7 18.3 13.3 22.9 minimum 9.2 5.8 6.4 7.1 maximum 25 43.8 28.3 56.6

Example visualizations show favorable characteristics. Both small subcentimeter lesions and diffuse metastatic disease involving various tissue types are indicated.

In fig. Figure 26 shows: MIR (A) and transaxial images (BD) of a 70-year-old patient with biochemical recurrence 1.5 years after radical removal of the prostate gland (Gleason 8, pT2c, pN1). There is a single typical prostate cancer lesion measuring 5 mm in diameter on the right side of the pelvis with high uptake of PSMA-SIFA3 (7) labeled with ¹⁸ F. The malignant nature of the lesion was confirmed by histopathology.

In fig. 27 shows a set of images of an 80-year-old patient with advanced castration-resistant prostate cancer (PSA 66.4 ng/ml).

Images show high uptake of ^18F -labeled PSMA-SIFA3 (7) in different classes of prostate cancer lesions (local tumor, lymph node metastases, bone metastases, liver metastases). The lesions shown are within 2 mm (arrows indicate representative, not all tumor lesions).

Clinical application of ⁶⁸ Ga labeled PSMA-SIFA3 (7)

As a proof-of-concept PET study of a ⁶⁸ Ga-labeled, SiFA-substituted, chelator-based PET tracer, one subject with biochemical relapse following radical prostate ablation (PSA 0.44 ng/mL, pT2c, pNO, Gleason 7b) underwent PET/MR 66 min after injection of 144 MBq PSMA-SIFA3 (7) labeled with ⁶⁸ Ga. Uptake typical of recurrent prostate cancer was demonstrated in a 2 mm lymph node.

In fig. 28 shows a proof-of-concept proof of concept for the action of a ⁶⁸ Ga-labeled SiFA-substituted chelator-based ⁶⁸ Ga-labeled PET tag.

Human PSMA-SIFA3(7) studies

1. Distribution and absorption in tumor lesions.

(a) Maximum intensity PET projection of a subject with normal biodistribution.

See fig. 21.

(b) Average standardized absorbance values in different tissues.

See fig. 22.

(c) Average ratio of standardized absorbance values in different tissues.

See fig. 23.

(d) Mean standardized uptake values across tumor types.

See fig. 24.

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Claims (3)

2. Retention of the mark in the bladder. (a) Mean standardized tracer retention uptake values in the bladder. See fig. 25. 3. Clinical results for detection of tumor lesions and histopathological confirmation. See fig. 26 and 27. 4. Clinical application of ⁶⁸ Ga labeled PSMA-SIFA3 (7). See fig. 28. CLAIM 1. Connection selected from He - 50 046402 and pharmaceutically acceptable salts and individual isomers thereof, optionally containing a chelated non-radioactive or radioactive cation, and wherein the fluorine atom is optionally ¹⁸ F. 2. The connection according to claim 1, which is or a pharmaceutically acceptable salt or single isomer thereof, optionally containing a chelated non-radioactive or radioactive cation, and wherein the fluorine atom is optionally ¹⁸ F. 3. The connection according to claim 1, which is -