Patent Description:
Prostate Cancer (PCa) remained over the last decades the most common malignant disease in men with high incidence for poor survival rates. Due to its overexpression in prostate cancer, prostate-specific membrane antigen (PSMA) or glutamate carboxypeptidase II (GCP II) proved its eligibility as excellent target for the development of highly sensitive radiolabelled agents for endoradiotherapy and imaging of PCa. Prostate-specific membrane antigen is an extracellular hydrolase whose catalytic center comprises two zinc(II) ions with a bridging hydroxido ligand. It is highly upregulated in metastatic and hormone-refractory prostate carcinomas, but its physiologic expression has also been reported in kidneys, salivary glands, small intestine, brain and, to a low extent, also in healthy prostate tissue. In the intestine, PSMA facilitates absorption of folate by conversion of pteroylpoly-γ-glutamate to pteroylglutamate (folate). In the brain, it hydrolyses N-acetyl-Laspartyl-L-glutamate (NAAG) to N-acetyl-L-aspartate and glutamate.

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

The general necessary structures of PSMA targeting molecules comprise a binding unit that encompasses a zinc-binding group (such as urea, phosphinate or phosphoramidate) connected to a P1' glutamate moiety, which warrants high affinity and specificity to PSMA and is typically further connected to an effector functionality. The effector part is more flexible and to some extent tolerant towards structural modifications. The entrance tunnel accommodates two other prominent structural features, which are important for ligand binding. The first one is an arginine patch, a positively charged area at the wall of the entrance funnel and the mechanistic explanation for the preference of negatively charged functionalities at the P1 position of PSMA. This appears to be the reason for the preferable incorporation of negative charged residues within the ligand-scaffold. An in-depth analysis about the effect of positive charges on PSMA ligands has been, to our knowledge, so far not conducted. Upon binding, the concerted repositioning of the arginine side chains can lead to the opening of an S1 hydrophobic accessory pocket, the second important structure that has been shown to accommodate an iodo-benzyl group of several urea based inhibitors, thus contributing to their high affinity for PSMA.

Zhang et al. discovered a remote binding site of PSMA, which can be employed for bidentate binding mode (<NPL>)). The so called arene-binding site is a simple structural motif shaped by the side chains of Arg463, Arg511 and Trp541, and is part of the GCPII entrance lid. The engagement of the arene binding site by a distal inhibitor moiety can result in a substantial increase in the inhibitor affinity for PSMA due to avidity effects. PSMA I&T was developed with the intention to interact this way with PSMA, albeit no crystal structure analysis of binding mode is available. A necessary feature according to Zhang et al. is a linker unit (Suberic acid in the case of PSMA I&T) which facilitates an open conformation of the entrance lid of GCPII and thereby enabling the accessibility of the arene-binding site. It was further shown that the structural composition of the linker has a significant impact on the tumor-targeting and biologic activity as well as on imaging contrast and pharmacokinetics, properties which are crucial for both high imaging quality and efficient targeted endoradiotherapy.

Two categories of PSMA targeting inhibitors are currently used in clinical settings. On the one side there are tracers with chelating units for radionuclide complexation such as PSMA I&T or related compounds. On the other side there are small molecules, comprising a targeting unit and effector molecules.

Recently, several groups have focused on the development of novel <NUM>F-labelled urea-based inhibitors for PCa diagnosis. The <NUM>F-labelled urea-based PSMA inhibitor <NUM>F-DCFPyl demonstrated promising results in the detection of primary and metastatic. Based on the structure of PSMA-<NUM>, the <NUM>F-labelled analogue PSMA-<NUM> was recently developed, which showed comparable tumor-to-organ ratios.

An attractive approach for introducing <NUM>F labels is the use of silicon fluoride acceptors (SIFA). Silicon fluoride acceptors are described, for example, in <NPL>). In order to preserve the silicon-fluoride bond, the use of silicon fluoride acceptors introduces the necessity of sterically demanding groups around the silicone atom. This in turn renders silicon fluoride acceptors highly hydrophobic. In terms of binding to the target molecule, in particular to the target molecule which is PSMA, the hydrophobic moiety provided by the silicone fluoride acceptor may be exploited for the purpose of establishing interactions of the radio-diagnostic or -therapeutic compound with the hydrophobic pocket described in <NPL>). Yet, prior to binding, the higher degree of lipophilicity introduced into the molecule poses a severe problem with respect to the development of radiopharmaceuticals with suitable in vivo biodistribution, i.e. low unspecific binding in non-target tissue.

Despite many attempts, the hydrophobicity problem caused by silicon fluoride acceptors has not been satisfactorily solved in the prior art.

To explain further, <NPL>) synthesized different <NUM>F-labelled peptides using the highly effective labelling synthon p-(di-tert-butylfluorosilyl) benzaldehyde ([<NUM>F]SIFA-A), which is one example of a silicon fluoride acceptor. The SIFA technique resulted in an unexpectedly efficient isotopic <NUM>F-<NUM>F exchange and yielded the <NUM>F-synthon in almost quantitative yields in high specific activities between <NUM> and <NUM> GBq/µmol (<NUM>-<NUM><NUM> Ci/mmol) without applying HPLC purification. [<NUM>F]SIFA-benzaldehyde was finally used to label the N-terminal amino-oxy (N-AO) derivatized peptides AO-Tyr3 -octreotate (AO-TATE), cyclo(fK(AO-N)RGD) and N-AO-PEG<NUM>-[D-Tyr-Gln-Trp-Ala-Val-Ala-His-Thi-Nle-NH<NUM>] (AO-BZH3, a bombesin derivative) in high radiochemical yields. Nevertheless, the labelled peptides are highly lipophilic (as can be taken from the HPLC retention times using the conditions described in this paper) and thus are unsuitable for further evaluation in animal models or humans.

In <NPL>), the first SIFA-based Kit-like radio-fluorination of a protein (rat serum albumin, RSA) has been described. As a labelling agent, <NUM>-(di-tert-butyl[<NUM>F]fluorosilyl)benzenethiol (Si[<NUM>F]FA-SH) was produced by simple isotopic exchange in <NUM>-<NUM>% radiochemical yield (RCY) and coupled the product directly to maleimide derivatized serum albumin in an overall RCY of <NUM>% within <NUM>-<NUM>. The technically simple labelling procedure does not require any elaborated purification procedures and is a straightforward example of a successful application of Si-18F chemistry for in vivo imaging with PET. The time-activity curves and µPET images of mice showed that most of the activity was localized in the liver, thus demonstrating that the labelling agent is too lipophilic and directs the in vivo probe to hepatobiliary excretion and extensive hepatic metabolism.

<NPL>) subsequently tried to overcome the major drawback of the SIFA technology, the high lipophilicity of the resulting radiopharmaceuticals, by synthesizing and evaluating new SIFA-octreotate analogues (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, i.e. a carbohydrate and a PEG linker plus a carbohydrate. As a measure of lipophilicity of the conjugates, the log P(ow) was determined and found to be <NUM> for SIFA-Asn(AcNH-β-Glc)-PEG-Tyr<NUM>-octreotate and <NUM> for SIFA-Asn(AcNH-β-Glc)-Tyr<NUM>-octreotate. These results show that the high lipophilicity of the SIFA moiety can only be marginally compensated by applying hydrophilic moieties. A first imaging study demonstrated excessive hepatic clearance /liver uptake and thus has never been transferred into a first human study.

<NPL>) reviews a great plethora of different SIFA species that have been reported in the literature ranging from small prosthetic groups and other compounds of low molecular weight to labelled peptides and most recently affibody molecules. Based on these data the problem of lipophilicity of SIFA-based prosthetric groups has not been solved so far; i.e. a methodology that reduces the overall lipophilicity of a SIFA conjugated peptide to a log D lower than approx -<NUM>,<NUM> has not been described.

In <NPL>) it is described that PEGylated bombesin (PESIN) derivatives as specific GRP receptor ligands and RGD (one-letter codes for arginine-glycine-aspartic acid) peptides as specific αvβ3 binders were synthesized and tagged with a silicon-fluorine-acceptor (SIFA) moiety. To compensate the high lipophilicity of the SIFA moiety various hydrophilic structure modifications were introduced leading to reduced logD values. SIFA-Asn(AcNH-β-Glc)-PESIN, SIFA-Ser(β-Lac)-PESIN, SIFA-Cya-PESIN, SIFA-LysMe3-PESIN, SIFA-γ-carboxy-d-Glu-PESIN, SIFA-Cya2-PESIN, SIFA-LysMe3-γ-carboxy-d-Glu-PESIN, SIFA-(γ-carboxy-d-Glu)<NUM>-PESIN, SIFA-RGD, SIFA-γ-carboxy-d-Glu-RGD, SIFA-(γ-carboxy-d-Glu)<NUM>-RGD, SIFA-LysMe3-γ-carboxy-d-Glu-RGD. All of these peptides - already improved and derivatized with the aim to reduce the lipophilicity - showed a logD value in the range between +<NUM> and -<NUM>.

<CIT> discloses <NUM>F-tagged inhibitors of PSMA.

In view of the above, the technical problem underlying the present invention can be seen in providing radio-diagnostics which contain a silicone fluoride acceptor and which are, at the same time, characterized by favourable in-vivo properties.

As will be become apparent in the following, a proof-of-principle using specific conjugates which bind with high affinity to prostate-specific antigen (PSMA) as target has been established. Accordingly, a further technical problem underlying the present invention can be seen in providing improved diagnostics for the medical indication which is cancer, preferably prostate cancer.

These technical problems are solved by the subject-matter of the appended claims. All described embodiments which fall outside the scope of the appended claims do not represent the invention.

In the first aspect, the present disclosure relates to a ligand-SIFA-chelator conjugate, comprising, within a single molecule: (a) one or more ligands which are capable of binding to PSMA, (b) a silicon-fluoride acceptor (SIFA) moiety which comprises a covalent bond between a silicon and a fluorine atom and which is labeled with <NUM>F, and (c) one or more chelating groups, containing a chelated nonradioactive cation selected from the cations of Sc, Cu, Ga, Y, In, Tb, Ho, Lu, Re, Pb, Bi, Ac, Th or Er.

The ligand-SIFA-chelator conjugate comprises three separate moieties. The three separate moieties are a) one or more ligands which are capable of binding to PSMA, (b) a silicon-fluoride acceptor (SIFA) moiety which comprises a covalent bond between a silicon and a fluorine atom, and (c) one or more chelating groups, containing a chelated nonradioactive cation selected from the cations of Sc, Cu, Ga, Y, In, Tb, Ho, Lu, Re, Pb, Bi, Ac, Th or Er.

For diagnostic imaging, the fluorine atom on the SIFA moiety is <NUM>F. The <NUM>F can be introduced by isotopic exchange with <NUM>F.

Whilst certain ligands which are capable of binding to a disease-relevant target molecule may be cyclic peptides, such cyclic peptides are not chelating groups as envisaged herein, as the problem of the hydrophobic SIFA moiety is not solved in the absence of a further chelating moiety. Thus the compounds require a hydrophilic chelating group in addition to the ligands which are capable of binding to PSMA. The hydrophilic chelating group is required to reduce the hydrophobic nature of the compounds caused by the presence of the SIFA moiety.

The ligand in relation to the first aspect of the disclosure is defined in functional terms. This is the case because the present disclosure does not depend on the specific nature of the ligand in structural terms. Rather, a key aspect is the combination, within a single molecule, of a silicon fluoride acceptor and a chelator or a chelate. These two structural elements, SIFA and the chelator, exhibit a spatial proximity. Preferably, the shortest distance between two atoms of the two elements is less or equal <NUM>Å, more preferably less than <NUM>Å and even more preferably less than <NUM>Å. Alternatively or in addition, it is preferred that not more than <NUM> covalent bonds separate an atom of the SIFA moiety and an atom the chelator, preferably not more than <NUM> chemical bonds and even more preferably not more than <NUM> chemical bonds.

The cation in accordance with item (c) of the first aspect is a non-radioactive cation. It is preferably a non-radioactive metal cation. Examples are given further below.

As a consequence, conjugates fall under the terms of the first aspect which are radioactively labelled at the SIFA moiety and the chelating group may be a complex of a cold (non-radioactive) ion.

The present inventors surprisingly discovered that placement of the silicone fluoride acceptor in the neighbourhood of a hydrophilic chelator such as, but not limited to, DOTAGA or DOTA, shields or compensates efficiently the lipophilicity of the SIFA moiety to an extent which shifts the overall hydrophobicity of compound in a range which renders the compound suitable for in-vivo administration.

A further advantage of the PSMA targeted compounds of the present disclosure is their surprisingly low accumulation in the kidneys of mice when compared to other PSMA targeted radiopharmaceuticals, such as PSMA I&T. Without wishing to be bound by a particular theory, it seems to be the combination of the structural element SIFA with a chelator which provides for the unexpected reduction of accumulation in the kidneys.

In terms of lipophilicity/hydrophilicity, the logP value (sometimes also referred to as logD value) is an art-established measure.

The term "lipophilicity" relates to the strength of being dissolved in, or be absorbed in lipid solutions, or being adsorbed at a lipid-like surface or matrix. It denotes a preference for lipids (literal meaning) or for organic or apolar liquids or for liquids, solutions or surfaces with a small dipole moment as compared to water. The term "hydrophobicity" is used with equivalent meaning herein. The adjectives lipophilic and hydrophobic are used with corresponding meaning to the substantives described above.

The mass flux of a molecule at the interface of two immiscible or substantially immiscible solvents is governed by its lipophilicity. The more lipophilic a molecule is, the more soluble it is in the lipophilic organic phase. The partition coefficient of a molecule that is observed between water and n-octanol has been adopted as the standard measure of lipophilicity. The partition coefficient P of a species A is defined as the ratio P = [A]n-octanol / [A]water. A figure commonly reported is the logP value, which is the logarithm of the partition coefficient. In case a molecule is ionizable, a plurality of distinct microspecies (ionized and not ionized forms of the molecule) will in principle be present in both phases. The quantity describing the overall lipophilicity of an ionizable species is the distribution coefficient D, defined as the ratio D = [sum of the concentrations of all microspecies]n-octanol / [sum of the concentrations of all microspecies] water. Analogous to logP, frequently the logarithm of the distribution coefficient, logD, is reported. Often, a buffer system, such as phosphate buffered saline is used as alternative to water in the above described determination of logP.

If the lipophilic character of a substituent on a first molecule is to be assessed and/or to be determined quantitatively, one may assess a second molecule corresponding to that substituent, wherein said second molecule is obtained, for example, by breaking the bond connecting said substituent to the remainder of the first molecule and connecting (the) free valence(s) obtained thereby to hydrogen(s).

Alternatively, the contribution of the substituent to the logP of a molecule may be determined. The contribution πX X of a substituent X to the logP of a molecule R-X is defined as πX X = logPR-X - logPR-H, wherein R-H is the unsubstituted parent compound.

Values of P and D greater than one as well as logP, logD and πX X values greater than zero indicate lipophilic/hydrophobic character, whereas values of P and D smaller than one as well as logP, logD and πX X values smaller than zero indicate hydrophilic character of the respective molecules or substituents.

The above described parameters characterizing the lipophilicity of the lipophilic group or the entire molecule according to the disclosure can be determined by experimental means and/or predicted by computational methods known in the art (see for example <NPL>)).

In a preferred embodiment, the logP value of the compounds of the disclosure is between -<NUM> and -<NUM>. It is particularly preferred that the logP value is between -<NUM> and -<NUM>.

In a preferred embodiment, a ligand in accordance with the disclosure comprises or consists of a peptide, a peptidomimetic or a substituted urea, substituents including amino acids. It is understood that a ligand which comprises a peptide or peptidomimetic also comprises a non-peptidic and non-peptidomimetic part. In terms of molecular weight, preference is given to molecular weights below <NUM> kDa, below <NUM> kDa or below <NUM> kDa. Accordingly, small proteins are also embraced by the term "ligand". The ligands are preferably high affinity ligands with preferable affinity, expressed as IC<NUM>, being below <NUM>, below <NUM> or below <NUM>.

Especially preferred are those ligands which bind with high affinity to prostate-specific membrane antigen (PSMA).

Preferably, the silicon-fluoride acceptor (SIFA) moiety has the structure represented by formula (I):
<CHM>.

Wherein F is understood to encompass both <NUM>F and <NUM>F, R<NUM> and R<NUM> are independently a linear or branched C3 to C10 alkyl group, preferably R<NUM> and R<NUM> are selected from isopropyl and tert-butyl, and are more preferably R<NUM> and R<NUM> are tert-butyl; R<NUM> is a C1 to C20 hydrocarbon group which may comprise one or more aromatic and one or more aliphatic units and/or up to <NUM> heteroatoms selected from O and S, preferably R<NUM> is a C6 to C10 hydrocarbon group which comprises an aromatic ring and which may comprise one or more aliphatic units; more preferably R<NUM> is a phenyl ring, and most preferably, R<NUM> is a phenyl ring wherein the Si-containing substituent and the bond marked by <IMG> are in a para-position, and wherein the SIFA moiety is attached to the remainder of the conjugate via the bond marked by <IMG>.

More preferably, the silicon-fluoride acceptor (SIFA) moiety has the structure represented by formula (la):
<CHM>.

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

In preferred specific examples, the chelating group is a residue of a chelating agent selected from bis(carboxymethyl)-<NUM>,<NUM>,<NUM>,<NUM>-tetraazabicyclo[<NUM>. <NUM>]hexadecane (CBTE2a), cyclohexyl-<NUM>,<NUM>-diaminetetraacetic acid (CDTA), <NUM>-(<NUM>,<NUM>,<NUM>,<NUM>-tetraazacyclotetradec-<NUM>-yl)-methylbenzoic acid (CPTA), N'-[<NUM>-[acetyl(hydroxy)amino]pentyl]-N-[<NUM>-[[<NUM>-[<NUM>-aminopentyl-(hydroxy)amino]-<NUM>-oxobutanoyl]amino]pentyl]-N-hydroxybutandiamide (DFO), <NUM>,<NUM>-bis(carboxymethyl)-<NUM>,<NUM>,<NUM>,<NUM>-tetraazabicyclo[<NUM>. <NUM>]hexadecan (DO2A) <NUM>,<NUM>,<NUM>,<NUM>-tetracyclododecan-N,N',N",N"'-tetraacetic acid (DOTA), α-(<NUM>-carboxyethyl)-<NUM>,<NUM>,<NUM>,<NUM>-tetraazacyclododecane-<NUM>,<NUM>,<NUM>,<NUM>-tetraacetic acid (DOTAGA), <NUM>,<NUM>,<NUM>,<NUM> tetraazacyclododecane N, N', N", N‴ <NUM>,<NUM>,<NUM>,<NUM>-tetra(methylene) phosphonic acid (DOTMP), N,N'-dipyridoxylethylendiamine-N,N'-diacetate-<NUM>,<NUM>'-bis(phosphat) (DPDP), diethylene triamine N,N',N" penta(methylene) phosphonic acid (DTMP), diethylenetriaminepentaacetic acid (DTPA), ethylenediamine-N,N'-tetraacetic acid (EDTA), ethyleneglykol-O,O-bis(<NUM>-aminoethyl)-N,N,N',N'-tetraacetic acid (EGTA), N,N-bis(hydroxybenzyl)-ethylenediamine-N,N'-diacetic acid (HBED), hydroxyethyldiaminetriacetic acid (HEDTA), <NUM>-(p-nitrobenzyl)-<NUM>,<NUM>,<NUM>,<NUM>-tetraazacyclodecan-<NUM>,<NUM>,<NUM>-triacetate (HP-DOA3), <NUM>-hydrazinyl-N-methylpyridine-<NUM>-carboxamide (HYNIC), tetra <NUM>-hydroxy-N-methyl-<NUM>-pyridinone chelators (<NUM>-((<NUM>-(<NUM>-(bis(<NUM>-(<NUM>-hydroxy-<NUM>-methyl-<NUM>-oxo-<NUM>,<NUM>-dihydropyridine-<NUM>-carboxamido)ethyl)amino)-<NUM>-((bis(<NUM>-(<NUM>-hydroxy-<NUM>-methyl-<NUM>-oxo-<NUM>,<NUM>-dihydropyridine-<NUM>-carboxamido)ethyl)amino)methyl)propyl)phenyl)amino)-<NUM>-oxobutanoic acid), abbreviated as Me-<NUM>,<NUM>-HOPO, <NUM>,<NUM>,<NUM>-triazacyclononan-<NUM>-succinic acid-<NUM>,<NUM>-diacetic acid (NODASA), <NUM>-(<NUM>-carboxy-<NUM>-carboxypropyl)-<NUM>,<NUM>-(carbooxy)-<NUM>,<NUM>,<NUM>-triazacyclononane (NODAGA), <NUM>,<NUM>,<NUM>-triazacyclononanetriacetic acid (NOTA), <NUM>,<NUM>-bis(carboxymethyl)-<NUM>,<NUM>,<NUM>,<NUM>-tetraazabicyclo[<NUM>. <NUM>]hexadecane (TE2A), <NUM>,<NUM>,<NUM>,<NUM>-tetraazacyclododecane-<NUM>,<NUM>,<NUM>,<NUM>-tetraacetic acid (TETA), tris(hydroxypyridinone) (THP), terpyridinbis(methyleneamintetraacetic acid (TMT), <NUM>,<NUM>,<NUM>-triazacyclononane-<NUM>,<NUM>,<NUM>-tris[methylene(<NUM>-carboxyethyl)phosphinic acid] (TRAP), <NUM>,<NUM>,<NUM>,<NUM>-tetraazacyclotridecan-N,N',N",N‴-tetraacetic acid (TRITA), <NUM>-[[<NUM>,<NUM>-bis[[<NUM>-carboxyethyl(hydroxy)phosphoryl]methyl]-<NUM>,<NUM>,<NUM>-triazonan-<NUM>-yl]methyl-hydroxy-phosphoryl]propanoic acid, and triethylenetetraaminehexaacetic acid (TTHA), which residue is provided by covalently binding a carboxyl group contained in the chelating agent to the remainder of the conjugate via an ester or an amide bond.

Particular chelators are shown below:
<CHM>
<CHM>
<CHM>.

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

Metal- or cation-chelating macrocyclic and acyclic compounds are well-known in the art and available from a number of manufacturers. While the chelating moiety in accordance with the present disclosure is not particularly limited, it is understood that numerous moieties can be used in an off-the-shelf manner by a skilled person without further ado.

The chelating group comprises a chelated cation which is non-radioactive selected from the cations of Sc, Cu, Ga, Y, In, Tb, Ho, Lu, Re, Pb, Bi, Ac, Th or Er. The cation may be Ga. The cation may be Lu.

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

More preferably, the ligand has the structure represented by formula (II):
<CHM>
wherein m is an integer of <NUM> to <NUM>, preferably <NUM> to <NUM>, more preferably <NUM>; n is an integer of <NUM> to <NUM>, preferably <NUM> to <NUM>, more preferably <NUM> or <NUM>; R<NUM> is CH<NUM>, NH or O, preferably NH; R<NUM> is CH<NUM>, NH or O, preferably NH; R<NUM> is C or P(OH), preferably C; and wherein the ligand is attached to the remainder of the conjugate via the bond marked by <IMG>.

The ligand can have the structure represented by formula (IIa):
<CHM>
wherein n is an integer of <NUM> to <NUM>; and wherein the ligand is attached to the remainder of the conjugate via the bond marked by
<CHM>.

A number of PSMA binders are known in the art which are all suitable in accordance with the disclosure. The above preferred embodiment is a structural definition of a preferred group of PSMA binders.

It is particularly preferred that the conjugate of the first aspect is a compound of formula (III):
<CHM>
or a pharmaceutically acceptable salt thereof, wherein:.

L<NUM> can be optionally substituted with one or more substituents independently selected from - OH, -OCH<NUM>, -COOH, -COOCH<NUM>, -NH<NUM>, and -NHC(NH)NH<NUM>.

X<NUM> is selected from an amide bond, and ester bond, an ether, an amine, and a linking group of the formula:
<CHM>
wherein the bond marked by <IMG> at the NH group is bound to RB and the other bond marked by <IMG> is bound to SIFA; preferably X<NUM> is an amide bond; RB is a trivalent coupling group.

X<NUM> is selected from an amide bond, an ether bond, a thioether bond, and ester bond, a thioester bond, a urea bridge, an amine bond, a linking group of the formula:
<CHM>
wherein the amide bond marked by <IMG> is formed with the chelating group, and the other bond marked by <IMG> is bound to RB; and a linking group of the formula:
<CHM>
wherein the bond marked by <IMG> at the carbonyl end is formed with the chelating group, and the other bond marked by <IMG> is bound to RB; preferably X<NUM> is an amide bond.

RCH is chelating group containing a chelated nonradioactive cation, preferably a nonradioactive metal cation, wherein preferred embodiments of said chelating group and of the optional chelated cation are as defined above.

The term "oligo" as used in 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 (ether-urea), oligo(thioether-ester), oligo(thioether-thioester), oligo(thioether-urea), oligo(ester-thioester), oligo(ester-urea), and oligo(thioester-urea) is preferably to be understood as referring to a group wherein <NUM> to <NUM>, more preferably wherein <NUM> to <NUM> subunits are linked by the type of bonds specified in the same terms. As will be understood by the skilled reader, where two different types of bonds are indicated in brackets, both types of bonds are contained in the concerned group (e.g. in "oligo (ester-amide)", ester bonds and amide bonds are contained).

It is preferred that L<NUM> comprises a total of <NUM> to <NUM>, more preferably a total of <NUM> to <NUM>, and most preferably a total of <NUM> or <NUM> amide and/or ester bonds, preferably amide bonds, within its backbone.

The term oligoamide therefore describes a moiety having a chain of CH<NUM> or CHR groups interspersed with groups selected from NHCO or CONH. Each occurrence of the R moiety is an optional substituent selected from -OH, -OCH<NUM>, -COOH, -COOCH<NUM>, -NH<NUM>, and -NHC(NH)NH<NUM>.

It is also preferred that -X<NUM>-L<NUM>-X<NUM>- represents one of the following structures (L-<NUM>) and (L-<NUM>):.

-NH-C(O)-R<NUM>-C(O)-NH-R<NUM>-NH-C(O)-     (L-<NUM>).

-C(O)-N H-R<NUM>-NH-C(O)-R<NUM>-C(O)-NH-R<NUM>-NH-C(O)-     (L-<NUM>).

wherein R<NUM> to R<NUM> are independently selected from C2 to C10 alkylene, preferably linear C2 to C10 alkylene, which alkylene groups may each be substituted by one or more substitutents independently selected from -OH, -OCH<NUM>, -COOH, -COOCH<NUM>, -NH<NUM>, and -NHC(NH)NH<NUM>.

Especially preferred is that the total number of carbon atoms in R<NUM> and R<NUM> is <NUM> to <NUM>, more preferably <NUM> to <NUM>, without carbon atoms contained in optional substituents. Especially preferred is that the total number of carbon atoms in R<NUM> to R<NUM>, is <NUM> to <NUM>, more preferably <NUM> to <NUM>, without carbon atoms contained in optional substituents.

It is particularly preferred that -X<NUM>-L<NUM>-X<NUM>- represents one of the following structures (L-<NUM>) and (L-<NUM>):.

-NH-C(O)-R-C(O)-NH-R<NUM>-CH(COOH)-NH-C(O)-     (L-<NUM>).

-C(O)-NH-CH(COOH)-R<NUM>-NH-C(O)-R<NUM>-C(O)-NH-R<NUM>-CH(COOH)-NH-C(O)-     (L-<NUM>).

wherein R<NUM> to R<NUM> are independently selected from C2 to C8 alkylene, preferably linear C2 to C8 alkylene.

Especially preferred is that the total number of carbon atoms in R<NUM> and R<NUM> or R<NUM> to R<NUM> respectively, is <NUM> to <NUM>, more preferably <NUM> to <NUM>, yet more preferably <NUM> or <NUM>.

Preferably, RB has the structure represented by formula (IV):
<CHM>
wherein: A is selected from N, CR<NUM>, wherein R<NUM> is H or C1-C6 alkyl, and a <NUM> to <NUM> membered carbocyclic or heterocyclic group; preferably A is selected from N and CH, and more preferably A is CH; the bond marked by <IMG> at (CH<NUM>)a is formed with X<NUM>, and a is an integer of <NUM> to <NUM>, preferably <NUM> or <NUM>, and most preferably <NUM>; the bond marked by <IMG> at (CH<NUM>)b is formed with X<NUM>, and b is an integer of <NUM> to <NUM>, preferably of <NUM> to <NUM>, and more preferably <NUM> or <NUM>; and the bond marked by <IMG> at (CH<NUM>)c is formed with X<NUM>, and c is an integer of <NUM> to <NUM>, preferably of <NUM> to <NUM>, and more preferably <NUM> or <NUM>.

Even more preferred is a compound of formula (Illa):
<CHM>
or a pharmaceutically acceptable salt thereof, wherein m, n, R<NUM>, R<NUM>, R<NUM>, X<NUM>, L<NUM>, b, c, X<NUM> and RCH are as defined above, including all preferred embodiments thereof.

It is preferred for the compound of formula (Illa) that b+c ≥ <NUM>.

It is also preferred for the compound of formula (Illa) that b+c ≤ <NUM>.

It is more preferred for the compound of formula (Illa) that b is <NUM> and c is <NUM>.

It is also preferred for the compound of formula (III) that -X<NUM>-RCH represents a residue of a chelating agent selected from DOTA and DOTAGA bound with one of its carboxylic groups via an amide bond to the remainder of the conjugate.

In a preferred embodiment of the compound of formula (III), said compound is a compound of formula (IIIb):
<CHM>
or a pharmaceutically acceptable salt thereof, wherein m, n, R<NUM>, R<NUM>, R<NUM>, X<NUM>, L<NUM>, b, c, X<NUM> and RCH are as defined above; and r is <NUM> or <NUM>.

Especially preferred is that -N(H)-RCH represents a residue of a chelating agent selected from DOTA and DOTAGA bound with one of its carboxylic groups via an amide bond to the remainder of the conjugate.

In order to be used in PET imaging, the compounds require a positron emitting atom. The compounds include <NUM>F for medical use. Most preferred compounds of the disclosure are wherein F includes <NUM>F and M<NUM>+ refers to a nonradioactive metal cation.

In the compounds herein, the nonradioactive metal cation M may be chelated to one or more COO- groups. M may be chelated to one or more N atoms. M may be chelated to one or more N atoms or one or more COO- groups. M may be chelated to one or more N atoms and one or more COO- groups. Where the chelated nonradioactive metal cation M is shown, the acid groups to which it is chelated are merely representatively shown as COO-, the equivalent fourth acid may also be partly chelated and hence may not literally be COOH.

<CHM>
and isomers thereof:
<CHM>
<CHM>
<CHM>
<CHM>
<CHM>
<CHM>
<CHM>
<CHM>.

<CHM>
and isomers thereof
<CHM>
<CHM>
<CHM>
<CHM>.

<CHM>
and isomers thereof
<CHM>
<CHM>
<CHM>
<CHM>
<CHM>
<CHM>
<CHM>
<CHM>
<CHM>
<CHM>
<CHM>
<CHM>
<CHM>
<CHM>
<CHM>
<CHM>.

<CHM>
and isomers thereof
<CHM>
<CHM>
<CHM>
<CHM>
<CHM>
<CHM>
<CHM>
<CHM>.

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

In a further aspect, the present disclosure provides a pharmaceutical imaging composition comprising or consisting of one or more conjugates or compounds of the disclosure as disclosed herein above.

In a further aspect, the present disclosure provides a diagnostic composition comprising or consisting of one or more conjugates or compounds of the disclosure as disclosed herein above.

The pharmaceutical composition may further comprise 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/water emulsions, various types of wetting agents, sterile solutions etc. Compositions comprising such carriers can be formulated by well-known conventional methods. These pharmaceutical compositions can be administered to the subject at a suitable dose. Administration of the suitable compositions may be effected by different ways, e.g., by intravenous, intraperitoneal, subcutaneous, intramuscular, topical, intradermal, intranasal or intrabronchial administration. It is particularly preferred that said administration is carried out by injection and/or delivery, e.g., to a site in the pancreas or into a brain artery or directly into brain tissue. The compositions may also be administered directly to the target site, e.g., by biolistic delivery to an external or internal target site, like the pancreas or brain. The dosage regimen will be determined by the attending physician and clinical factors. As is well known in the medical arts, dosages for any one patient depends upon many factors, including the patient's size, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health, and other drugs being administered concurrently. Pharmaceutically active matter may be present in amounts between <NUM>,<NUM> ng and <NUM>/kg body weight per dose; however, doses below or above this exemplary range are envisioned, especially considering the aforementioned factors.

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

Preferred uses in medicine are in nuclear medicine such as nuclear diagnostic imaging, also named nuclear molecular imaging, and/or targeted radiotherapy of diseases associated with an overexpression, preferably of PSMA on the diseased tissue.

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

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

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

The Fmoc-(<NUM>-fluorenylmethoxycarbonyl-) and all other protected amino acid analogs were purchased from Bachem (Bubendorf, Switzerland) or Iris Biotech (Marktredwitz, Germany). The tritylchloride polystyrene (TCP) resin was obtained from PepChem (Tübingen, Germany). Chematech (Dijon, France) delivered the chelators DOTAGA-anhydride, (R)-DOTA-GA(tBu)<NUM> and (S)-DOTA-GA(tBu)<NUM>. All necessary solvents and other organic reagents were purchased from either, Alfa Aesar (Karlsruhe, Germany), Sigma-Aldrich (Munich, Germany) or VWR (Darmstadt, Germany). Solid phase synthesis of the peptides was carried out by manual operation using an Intelli-Mixer syringe shaker (Neolab, Heidelberg, Germany). Analytical and preparative reversed-phase high pressure chromatography (RP-HPLC) were performed using Shimadzu gradient systems (Shimadzu Deutschland GmbH, Neufahrn, Germany), each equipped with a SPD-20A UV/Vis detector (<NUM>, <NUM>). A Nucleosil <NUM> C18 (<NUM> × <NUM>, <NUM> particle size) column ( CS GmbH, Langerwehe, Germany) was used for analytical measurements at a flow rate of <NUM>/min. Both specific gradients and the corresponding retention times tR are cited in the text. Preparative HPLC purification was done with a Multospher <NUM> RP <NUM> (<NUM> × <NUM>, <NUM> particle size) column (CS GmbH, Langerwehe, Germany) at a constant flow rate of <NUM>/min. Analytical and preparative radio RP-HPLC was performed using a Nucleosil <NUM> C18 (<NUM>, <NUM> × <NUM>) column (CS GmbH, Langerwehe, Germany). Eluents for all HPLC operations were water (solvent A) and acetonitrile (solvent B), both containing <NUM>% trifluoroacetic acid. Electrospray ionization-mass spectra for characterization of the substances were acquired on an expressionL CMS mass spectrometer (Advion Ltd. , Harlow, UK). NMR spectra were recorded on Bruker AVHD-<NUM> or AVHD-<NUM> spectrometers at <NUM>. pH values were measured with a SevenEasy pH-meter (Mettler Toledo, Gießen, Germany).

Loading of the tritylchloride polystyrene (TCP) resin with a Fmoc-protected amino acid (AA) was carried out by stirring a solution of the TCP-resin (<NUM> mmol/g) and Fmoc-AA-OH (<NUM> eq. ) in anhydrous DCM with DIPEA (<NUM> eq. ) at room temperature for <NUM>. Remaining tritylchloride was capped by the addition of methanol (<NUM>/g resin) for <NUM>. Subsequently the resin was filtered and washed with DCM (<NUM> × <NUM>/g resin), DMF (<NUM> × <NUM>/g resin), methanol (<NUM>/g resin) and dried in vacuo. Final loading l of Fmoc-AA-OH was determined by the following equation: <MAT>.

For conjugation of a building block to the resin bound peptide, a mixture of TBTU and HOBT is used for pre-activation with DIPEA or <NUM>,<NUM>,<NUM>-trimethylpyridine as a base in DMF (<NUM>/g resin) for <NUM>. The exact stoichiometry and reaction time for each conjugation step is given in the synthesis protocol. After reaction, the resin was washed with DMF (<NUM> × <NUM>/g resin).

The resin-bound Fmoc-peptide was treated with <NUM>% piperidine in DMF (v/v, <NUM>/g resin) for <NUM> and subsequently for <NUM>. Afterwards, the resin was washed thoroughly with DMF (<NUM> × <NUM>/g resin).

The Dde-protected peptide (<NUM> eq. ) was dissolved in a solution of <NUM>% hydrazine monohydrate in DMF (v/v, <NUM>/g resin) and shaken for <NUM> (GP4a). In the case of present Fmoc-groups, Dde-deprotection was performed by adding a solution of imidazole (<NUM>/g resin), hydroxylamine hydrochloride (<NUM>/g reisn) in NMP (<NUM>) and DMF (<NUM>) for <NUM> at room temperature (GP4b). After deprotection the resin was washed with DMF (<NUM> × <NUM>/g resin).

The fully protected resin-bound peptide was dissolved in a mixture of TFA/TIPS/water (v/v/v; <NUM>/<NUM>/<NUM>) and shaken for <NUM>. The solution was filtered off and the resin was treated in the same way for another <NUM>. Both filtrates were combined, stirred for additional <NUM> and concentrated under a stream of nitrogen. After dissolving the residue in a mixture of tert-butanol and water and subsequent lyophilisation the crude peptide was obtained.

For natGa-complexation, the peptide (<NUM> eq. ) was dissolved in a <NUM>:<NUM> (v/v) mixture of tBuOH in H<NUM>O and an aqueous solution of Ga(NO<NUM>)<NUM> (<NUM> eq. ) was added. After heating the resulting mixture for <NUM> at <NUM> the peptide was purified by RP-HPLC.

The tBu-protected Glu-urea-Glu binding motif (EuE) was synthesized according to a previously published procedure (scheme <NUM>) for tBu-protected Glu-urea-Lys (EuK).

A solution of DCM containing <NUM> (<NUM> mmol, <NUM> eq. ) I-di-tert-butyl-L-glutamate·HCl was cooled on ice for <NUM> and afterwards treated with <NUM> TEA (<NUM> mmol, <NUM> eq. ) and <NUM> (<NUM> mmol, <NUM> eq. After additional stirring for <NUM>, <NUM> (<NUM> mmol, <NUM> eq. ) of <NUM>,<NUM>'-carbonyldiimidazole (CDI) dissolved in DCM were slowly added over a period of <NUM>. The reaction mixture was further stirred overnight and enabled to warm to RT. The reaction was stopped using <NUM> saturated NaHCO<NUM> with concomitant washing steps of water (<NUM> ×) and brine (<NUM> ×) and dried over Na<NUM>SO<NUM>. The remaining solvent was removed in vacuo and the crude product (S)-Di-tert-butyl <NUM>-(<NUM>-imidazole-<NUM>-carboxamido)pentanedioate (i) was used without further purification.

<NUM> (<NUM> mmol, <NUM> eq. ) of the crude product (S)-Di-tert-butyl-<NUM>-(<NUM>-imidazole-<NUM>-carboxamido) pentanedioate (i) were dissolved in <NUM>,<NUM>-dichloroethane (DCE) and cooled on ice for <NUM>. To this solution were added <NUM> (<NUM> mmol, <NUM> eq. ) TEA and <NUM> (<NUM> mmol, <NUM> eq. ) H-L-Glu(OBzl)-OtBu·HCl and the solution was stirred overnight at <NUM>. The remaining solvent was evaporated and the crude product purified using silica gel flash-chromatography with an eluent mixture containing ethyl acetate/hexane/TEA (<NUM>:<NUM>:<NUM> ; v/v/v). After removal of the solvent, <NUM>-benzyl-<NUM>-(tert-butyl)-(((S)-<NUM>,<NUM>-di-tert-butoxy-<NUM>,<NUM>-dioxopentan-<NUM>-yl)carbamoyl)-L-glutamate (ii) was obtained as a colorless oil.

To synthesize (tBuO)EuE(OtBu)<NUM>, <NUM> (<NUM> mmol, <NUM> eq. ) of <NUM>-benzyl-<NUM>-(tert-butyl)-(((S)-<NUM>,<NUM>-di-tert-butoxy-<NUM>,<NUM>-dioxopentan-<NUM>-yl)carbamoyl)-L-glutamate (ii) were dissolved in <NUM> EtOH and <NUM> (<NUM> mmol, <NUM> eq. ) palladium on activated charcoal (<NUM>%) were given to this solution. The flask containing the reaction mixture was initially purged with H<NUM> and the solution was stirred over night at room temperature under light H<NUM>-pressure (balloon). The crude product was purified through celite and the solvent evaporated in vacuo. The product (iii) was obtained as a hygroscopic solid (<NUM>%). HPLC
(<NUM>% to <NUM>% B in <NUM>): tR = <NUM>. Calculated monoisotopic mass (C<NUM>H<NUM>N<NUM>O<NUM>): <NUM>; found: m/z = <NUM> [M+H]+, <NUM> [M+Na]+.

SiFA-BA was synthesized according to a previously published procedure (scheme <NUM>). All reactions were carried out in dried reaction vessels under argon using a vacuum gas manifold.

To a stirred solution of <NUM>-bromobenzylalcohol (<NUM>, <NUM> mmol, <NUM> eq. ) in anhydrous DMF (<NUM>) imidazole (<NUM>, <NUM> mmol, <NUM> eq. ) and TBDMSCI (<NUM>, <NUM> mmol, <NUM> eq. ) were added and the resulting mixture was stirred at room temperature for <NUM>. The mixture was then poured into ice-cold H<NUM>O (<NUM>) and extracted with Et<NUM>O (<NUM> × <NUM>). The combined organic fractions were washed with sat. NaHCO<NUM> (<NUM> ×<NUM>) and brine (<NUM>), dried, filtered and concentrated in vacuo to give the crude product which was purified by flash column chromatography (silica, <NUM>% EtOAc/petrol) to give i as a colourless oil (<NUM>, <NUM>%). <NUM>H NMR (<NUM>, CDCl3): δ [ppm] = <NUM> (<NUM>, s, SiMe<NUM>t-Bu), <NUM> (<NUM>, s, SiMe<NUM>tBu), <NUM> (<NUM>, s, CH<NUM>OSi), <NUM> (<NUM>, d), <NUM> (<NUM>, d). HPLC (<NUM> to <NUM>% B in <NUM>): tR = <NUM>.

At -<NUM> under magnetic stirring, a solution of tBuLi in pentane (<NUM>, <NUM> mol/L, <NUM> mmol <NUM> eq. ) was added to a solution of ((<NUM>-bromobenzyl)oxy)(tert-butyl)dimethylsilane (i) (<NUM>, <NUM> mmol, <NUM> eq. ) in dry THF (<NUM>). After the reaction mixture had been stirred for <NUM> at -<NUM>, the suspension obtained was added dropwise over a period of <NUM> to a cooled (-<NUM>) solution of di-tert-butyldifluorosilane (<NUM>, <NUM> mmol, <NUM> eq. ) in dry THF (<NUM>). The reaction mixture was allowed to warm to room temperature over a period of <NUM> and then hydrolyzed with saturated aqueous NaCl solution (<NUM>). The organic layer was separated and the aqueous layer was extracted with diethyl ether (<NUM> × <NUM>). The combined organic layers were dried over magnesium sulfate and filtered. The filtrate was concentrated in vacuo to afford ii as a yellowish oil (<NUM>, <NUM>%). It was used for subsequent reactions without further purification. NMR spectra were in accordance with the data reported in the literature[<NUM>]. HPLC (<NUM> to <NUM>% B in <NUM>): tR = <NUM>.

A catalytic amount of concentrated aqueous HCl (<NUM>) was added to a suspension of ii (<NUM>, <NUM> mmol, <NUM> eq. ) in methanol (<NUM>). The reaction mixture was stirred for <NUM> at room temperature and then the solvent and the volatiles were removed under reduced pressure. The residue was redissolved in diethyl ether (<NUM>) and the solution was washed with saturated aqueous NaHCO<NUM> solution. The aqueous layer was extracted with diethyl ether (<NUM> × <NUM>). The combined organic layers were dried over magnesium sulfate and filtered. The filtrate was concentrated in vacuo to afford iii as a yellowish oil (<NUM>, <NUM>%) that solidified. The product was used without further purification. NMR spectra were in accordance with the data reported in the literature[<NUM>]. HPLC (<NUM> to <NUM>% B in <NUM>): tR = <NUM>.

A solution of the alcohol iii (<NUM>, <NUM> mmol, <NUM> eq. ) in dry dichloromethane (<NUM>) was added dropwise to a stirred ice-cooled suspension of pyridinium chlorochromate (<NUM>, <NUM> mmol, <NUM> eq. ) in dry dichloromethane (<NUM>). After the reaction mixture had been stirred for <NUM> at <NUM> and for <NUM> at room temperature, anhydrous diethyl ether (<NUM>) was added and the supernatant solution was decanted from the black gum-like material. The insoluble material was washed thoroughly with diethyl ether and the combined organic phases were passed through a short pad of silica gel (<NUM> per g crude product) for filtration. The solvents were removed in vacuo to yield aldehyde iv as a yellowish oil (<NUM>, <NUM>%). NMR spectra were in accordance with the data reported in the literature[<NUM>]. HPLC (<NUM> to <NUM>% B in <NUM>): tR = <NUM>.

At room temperature, <NUM> aqueous KMnO<NUM> (<NUM>) was added to a mixture of iv (<NUM>, <NUM> mmol, <NUM> eq. ), tert-butanol (<NUM>), dichloromethane (<NUM>), and <NUM> NaH<NUM>PO<NUM>·H<NUM>O buffer (<NUM>) at pH <NUM>-<NUM>. After the mixture had been stirred for <NUM>, it was cooled to <NUM>, whereupon excess KMnO<NUM> (<NUM>, <NUM> mmol, <NUM> eq. ) was added. The reaction was then quenched by the addition of saturated aqueous Na<NUM>SO<NUM> solution (<NUM>). Upon addition of <NUM> aqueous HCl, all of the MnO<NUM> dissolved. The resulting solution was extracted with diethyl ether (<NUM> × <NUM>). The combined organic layers were washed with saturated aqueous NaHCO<NUM> solution, dried over MgSO<NUM>, filtered, and concentrated under reduced pressure to provide a white solid, which was purified by recrystallization from Et<NUM>O/n-hexane (<NUM>:<NUM>, for <NUM>) to give v (<NUM>, <NUM>%). NMR spectra were in accordance with the data reported in the literature[<NUM>]. HPLC (<NUM> to <NUM>% B in <NUM>): tR = <NUM>.

The first synthetic steps for preparation of the four different isomers of rhPSMA-<NUM> are identical and carried out together, applying the standard Fmoc-SPPS protocol described above, starting from resin bound Fmoc-D-Orn(Dde)-OH. After cleavage of the Fmoc group with <NUM>% piperidine in DMF (GP3), (tBuO)EuE(OtBu)<NUM> (<NUM> eq. ) was conjugated with HOAt (<NUM> eq. ), TBTU (<NUM> eq. ) and DIPEA (<NUM> eq. ) in DMF for <NUM>. After cleavage of the Dde-group with a mixture of <NUM>% hydrazine in DMF (GP4a), a solution of succinic anhydride (<NUM> eq. ) and DIPEA (<NUM> eq. ) in DMF was added and left to react for <NUM>. Conjugation of Fmoc-D-Lys(OtBu)·HCl (<NUM> eq. ) was achieved by adding a mixture of HOAt (<NUM> eq. ), TBTU (<NUM> eq. ) and DIPEA (<NUM> eq. ) in DMF to the resin. After pre-activation for <NUM>, Fmoc-D-Lys(OtBu)·HCl (<NUM> eq. ) dissolved in DMF was added and left to react for <NUM> (GP2). Subsequent cleavage of the Fmoc-group was performed, by adding a mixture of <NUM>% piperidine in DMF (GP3). Finally, the resin was split in order to synthesize rhPSMA-<NUM>-<NUM> (scheme <NUM>).

Fmoc-D-Dap(Dde)-OH (<NUM> eq. ) was pre-activated in a mixture of HOAt (<NUM> eq. ), TBTU (<NUM> eq. ) and <NUM>,<NUM>,<NUM>-trimethylpyridine (<NUM> eq. ) in DMF and added to the resin-bound peptide for <NUM>. Following orthogonal Dde-deprotection was done using imidazole and hydroxylamine hydrochloride dissolved in a mixture of NMP and DMF for <NUM>. SiFA-BA (<NUM> eq. ) was reacted with the free amine of the side chain with HOAt (<NUM> eq. ), TBTU (<NUM> eq. ) and DIPEA (<NUM> eq. ), as activation reagents in DMF for <NUM>. After Fmoc-deprotection with piperidine (GP3), (R)-DOTA-GA(tBu)<NUM> (<NUM> eq. ) was conjugated with HOAT (<NUM> eq. ), TBTU (<NUM> eq. ) and <NUM>,<NUM>,<NUM>-trimethylpyridine (<NUM> eq. ) in DMF for <NUM>. Cleavage from the resin with simultaneous deprotection of acid labile protecting groups was performed in TFA, according to GP5. natGa-complexation of the peptide was carried out, as described in GP6.

Fmoc-L-Dap(Dde)-OH (<NUM> eq. ) was pre-activated in a mixture of HOAt (<NUM> eq. ), TBTU (<NUM> eq. ) and <NUM>,<NUM>,<NUM>-trimethylpyridine (<NUM> eq. ) in DMF for <NUM>. Following orthogonal Dde-deprotection, conjugation of SiFA-BA and Fmoc-cleavage was carried out as described for rhPSMA-<NUM>. (R)-DOTAGA(tBu)<NUM> (<NUM> eq. ) was conjugated with HOAT (<NUM> eq. ), TBTU (<NUM> eq. ) and <NUM>,<NUM>,<NUM>-trimethylpyridine (<NUM> eq. ) in DMF for <NUM>. Cleavage from the resin with simultaneous deprotection of acid labile protecting groups was performed in TFA according to GP5. natGa-complexation of the peptide was carried out, as described in GP6.

Fmoc-D-Dap(Dde)-OH (<NUM> eq. ) was pre-activated in a mixture of HOAt (<NUM> eq. ), TBTU (<NUM> eq. ) and <NUM>,<NUM>,<NUM>-trimethylpyridine (<NUM> eq. ) in DMF for <NUM>. Following orthogonal Dde-deprotection, conjugation of SiFA-BA and Fmoc-cleavage was carried out as described for rhPSMA-<NUM>. (S)-DOTAGA(tBu)<NUM> (<NUM> eq. ) was conjugated with HOAT (<NUM> eq. ), TBTU (<NUM> eq. ) and <NUM>,<NUM>,<NUM>-trimethylpyridine (<NUM> eq. ) in DMF for <NUM>. Cleavage from the resin with simultaneous deprotection of acid labile protecting groups was performed in TFA according to GP5. natGa-complexation of the peptide was carried out, as described in GP6.

Fmoc-L-Dap(Dde)-OH (<NUM> eq. ) was pre-activated in a mixture of HOAt (<NUM> eq. ), TBTU (<NUM> eq. ) and <NUM>,<NUM>,<NUM>-trimethylpyridine (<NUM> eq. ) in DMF for <NUM>. Following orthogonal Dde-deprotection, conjugation of SiFA-BA and Fmoc-cleavage was carried out as described for rhPSMA-<NUM>. (S)-DOTAGA(tBu)<NUM> (<NUM> eq. ) was conjugated with HOAT (<NUM> eq. ), TBTU (<NUM> eq. ) and <NUM>,<NUM>,<NUM>-trimethylpyridine (<NUM> eq. ) in DMF for <NUM>. Cleavage from the resin with simultaneous deprotection of acid labile protecting groups was performed in TFA according to GP5. natGa-complexation of the peptide was carried out, as described in GP6.

For <NUM>F-labeling a previously published procedure <NPL>. was applied, which was slightly modified. Briefly, aqueous <NUM>F- was passed through a SAX cartridge (Sep-Pak Accell Plus QMA Carbonate light), which was preconditioned with <NUM> of water. After drying with <NUM> of air, water was removed, by rinsing the cartridge with <NUM> of anhydrous acetonitrile followed by <NUM> of air. <NUM>F was eluted with <NUM>µmol of [K+⊂<NUM>. <NUM>]OH- dissolved in <NUM>µL of anhydrous acetonitrile. Before labelling, <NUM>µmol of oxalic acid in anhydrous acetonitrile (<NUM>, <NUM>µL) were added. This mixture was used as a whole or aliquot for fluorination of <NUM>-<NUM> nmol of PSMA-SiFA (<NUM> in anhydrous DMSO). The resulting reaction mixture was incubated for <NUM> minutes at room temperature. For purification of the tracer, a Sep-Pak C18 light cartridge, preconditioned with <NUM> EtOH, followed by <NUM> of H<NUM>O was used. The labeling mixture was diluted with <NUM> PBS (pH <NUM>) and passed through the cartridge followed by <NUM> of H<NUM>O. The peptide was eluted with <NUM>µL of a <NUM>:<NUM> mixture (v/v) of EtOH in water. Radiochemical purity of the labelled compound was determined by radio RP-HPLC and radio-TLC (Silica gel <NUM> RP-<NUM> F<NUM>s, mobile phase: <NUM>:<NUM> mixture (v/v) of MeCN in H<NUM>O supplemented with <NUM>% of <NUM> aqueous NaOAc and <NUM>% of TFA).

The reference ligand for in vitro studies ([<NUM>I]I-BA)KuE was prepared according to a previously published procedure<NUM>. Briefly, <NUM> of the stannylated precursor (SnBu<NUM>-BA)(OtBu)KuE(OtBu)<NUM> was dissolved in a solution containing <NUM>µL peracetic acid, <NUM>µL (<NUM> MBq) [<NUM>I]Nal (<NUM> TBq/mmol, <NUM> GBq/mL, <NUM> NaOH, Hartmann Analytic, Braunschweig, Germany), <NUM>µL MeCN and <NUM>µL acetic acid. The reaction solution was incubated for <NUM> at RT, loaded on a cartridge and rinsed with <NUM> water (C18 Sep Pak Plus cartridge, preconditioned with <NUM> MeOH and <NUM> water). After elution with <NUM> of a <NUM>:<NUM> mix (v/v) of EtOH/MeCN, the radioactive solution was evaporated to dryness under a gentle nitrogen stream and treated with <NUM>µL TFA for <NUM> with subsequent evaporation of TFA. The crude product of ([<NUM>I]I-BA)KuE was purified by RP-HPLC (<NUM>% to <NUM>% B in <NUM>): tR = <NUM>.

The PSMA-posivite LNCaP cells were grown in Dublecco modified Eagle medium/Nutrition Mixture F-<NUM> with Glutamax-I (<NUM>:<NUM>) (Invitrigon), supplemented with <NUM>% fetal calf serum and maintained at <NUM> in a humidified <NUM>% CO<NUM> atmosphere. For determination of the PSMA affinity (IC<NUM>), cells were harvested <NUM> ± <NUM> hours before the experiment and seeded in <NUM>-well plates (<NUM> × <NUM><NUM> cells in <NUM>/well). After removal of the culture medium, the cells were treated once with <NUM>µL of HBSS (Hank's balanced salt solution, Biochrom, Berlin, Germany, with addition of <NUM>% bovine serum albumin (BSA)) and left <NUM> on ice for equilibration in <NUM>µL HBSS (<NUM>% BSA). Next, <NUM>µL per well of solutions, containing either HBSS (<NUM>% BSA, control) or the respective ligand in increasing concentration (<NUM>-<NUM> - <NUM>-<NUM> M in HBSS, were added with subsequent addition of <NUM>µL of ([<NUM>I]I-BA)KuE (<NUM>) in HBSS (<NUM>% BSA). All experiments were performed at least three times for each concentration. After <NUM> incubation on ice, the experiment was terminated by removal of the medium and consecutive rinsing with <NUM>µL of HBSS. The media of both steps were combined in one fraction and represent the amount of free radioligand. Afterwards, the cells were lysed with <NUM>µL of <NUM> NaOH and united with the <NUM>µL HBSS of the following wash step. Quantification of bound and free radioligand was accomplished in a γ-counter.

For internalization studies, LNCaP cells were harvested <NUM> ± <NUM> hours before the experiment and seeded in <NUM>-well plates (<NUM> × <NUM><NUM> cells in <NUM>/well). Subsequent to the removal of the culture medium, the cells were washed once with <NUM>µL DMEM-F12 (<NUM>% BSA) and left to equilibrate for at least <NUM> at <NUM> in <NUM>µL DMEM-F12 (<NUM>% BSA). Each well was treated with either <NUM>µL of either DMEM-F12 (<NUM>% BSA) or a <NUM> PMPA solution for blockade. Next, <NUM>µL of the <NUM>Ga/<NUM>F-labeled PSMA inhibitor (<NUM>) was added and the cells incubated at <NUM> for <NUM>. The experiment was terminated by placing the <NUM>-well plate on ice for <NUM> and consecutive removal of the medium. Each well was rinsed with <NUM>µL HBSS and the fractions from these first two steps combined, representing the amount of free radioligand. Removal of surface bound activity was accomplished by incubation of the cells with <NUM>µL of ice-cold PMPA (<NUM> in PBS) solution for <NUM> and rinsed again with another <NUM>µL of ice-cold PBS. The internalized activity was determined by incubation of the cells in <NUM>µL <NUM> NaOH and the combination with the fraction of a subsequent wash step with <NUM>µL <NUM> NaOH. Each experiment (control and bloackde) was performed in triplicate. Free, surface bound and internalized activity was quantified in a γ-counter. All internalization studies were accompanied by reference studies using ([<NUM>I]I-BA)KuE (c = <NUM>), which were performed analogously. Data were corrected for non-specific internalization and normalized to the specific-internalization observed for the radioiodinated reference compound.

Approximately <NUM> MBq of the labeled tracer was dissolved in <NUM> of a <NUM>:<NUM> mixture (by volumes) of phosphate buffered saline (PBS, pH <NUM>) and n-octanol in an Eppendorf tube. After vigorous mixing of the suspension for <NUM> minutes at room temperature, the vial was centrifuged at <NUM> for <NUM> minutes (Biofuge <NUM>, Heraus Sepatech, Osterode, Germany) and <NUM>µL aliquots of both layers were measured in a gamma counter. The experiment was repeated at least six times.

For the determination of HSA binding, a Chiralpak HSA column (<NUM> x <NUM>, <NUM>, H13H-<NUM>) was used at a constant flow rate of <NUM>/min. The mobile phase (A: NH<NUM>OAc, <NUM> in water, pH <NUM> and B: isopropanol) was freshly prepared for each experiment and only used for one day. The column was kept at room temperature and each run was stopped after detection of the signal to reduce the acquisition time. All substances were dissolved in a <NUM>/ml concentration in <NUM>% <NUM>-propanol and <NUM>% <NUM> pH <NUM> ammonium acetate buffer. The chosen reference substances display a range of HSA binding from <NUM>% to <NUM>% since a broad variety of albumin binding regarding the peptides was assumed. All nine reference substances were injected consecutively to establish a non-linear regression with OriginPro <NUM>.

The retention time is shown exemplary for a conducted experiment; tR retention time; Lit. HSA literature value of human serum albumin binding in [%]; Log K HAS logarithmic K of human serum albumin binding.

All animal experiments were conducted in accordance with general animal welfare regulations in Germany and the institutional guidelines for the care and use of animals. To establish tumor xenografts, LNCaP cells (<NUM><NUM> cells / <NUM>µL) were suspended in a <NUM>:<NUM> mixture (v/v) of Dulbecco modified Eagle medium / Nutrition Mixture F-<NUM> with Glutamax-I (<NUM>:<NUM>) and Matrigel (BD Biosciences, Germany), and inoculated subcutaneously onto the right shoulder of <NUM>-<NUM> weeks old CB17-SCID mice (Charles River, Sulzfeld, Germany). Mice were used for when tumors had grown to a diameter of <NUM>-<NUM> (<NUM>-<NUM> weeks after inoculation).

Approximately <NUM>-<NUM> MBq (< <NUM> nmol) of the <NUM>F-labeled PSMA inhibitor was injected into the tail vein of LNCaP tumor-bearing male CB-<NUM> SCID mice and sacrificed after <NUM> post injection (n = <NUM>-<NUM>). Selected organs were removed, weighted and measured in a γ-counter.

Analytical reversed-phase high pressure chromatography (RP-HPLC) were performed using Shimadzu gradient systems (Shimadzu Deutschland GmbH, Neufahrn, Germany), equipped with a SPD-20A UV/Vis detector (<NUM>, <NUM>). A Multospher <NUM> RP18 (<NUM> × <NUM>, <NUM> particle size) column (CS GmbH, Langerwehe, Germany) was used for analytical measurements at a flow rate of <NUM>/min. Eluents for all HPLC operations were water (solvent A) and acetonitrile (solvent B), both containing <NUM>% trifluoroacetic acid. Radioactivity was detected through connection of the outlet of the UV-photometer to a HERM LB <NUM> detector (Berthold Technologies GmbH, Bad Wildbad, Germany). The gradient for all HPLC operations was: <NUM>% B isocratic <NUM>-<NUM>, <NUM> - <NUM> % B <NUM>-<NUM>, <NUM> - <NUM> % B <NUM>-<NUM>.

For radio-thin-layer chromatography, aluminum sheets coated with silica gel <NUM> RP-<NUM> F<NUM>s were used with a mobile phase consisting of a <NUM>:<NUM> mixture (v/v) of MeCN in H<NUM>O supplemented with <NUM>% of <NUM> aqueous NaOAc and <NUM>% of TFA. Analysis was performed using either a Scan-RAM radio-TLC detector (LabLogic Systems Ltd. , Sheffield, United Kingdom) or a CR <NUM> BIO phosphorimager (Duerr Medical GmbH, Bietigheim-Bissingen, Germany).

For in vivo µmetabolism studies, <NUM> - <NUM> MBq (< <NUM> nmol) of the respective <NUM>F-labeled ligand (rhPSMA-<NUM>- <NUM>) was injected into the tail vein of female healthy CB17-SCID mice (n=<NUM>). Mice were left under anesthesia for <NUM> and the urine was collected using a bladder catheter. Urine samples were pooled and centrifuged for <NUM> at <NUM> rpm to remove suspended solids. The supernatant was directly used for radio-HPLC analysis with the above mentioned conditions. In order to demonstrate that isotopic exchange of <NUM>F with peptide-bound <NUM>F is taking place in urine, each compound was incubated for certain time intervals with urine samples of female healthy CB-<NUM>-SCID mice, which where analysed by radio-HPLC and/or radio-TLC. Additionally, this experiment was carried out with the addition of excess Na<NUM>F (<NUM>µmol) and incubation for <NUM> with <NUM>F-labeled rhPSMA-<NUM>.

In order to quantify the relative uptake of each isomer (rhPSMA-<NUM> - <NUM>), a tumor-bearing male CB-<NUM>-SCID mouse was injected with the racemic mixture of rhPSMA-<NUM> (<NUM>-<NUM> MBq, SA = <NUM>-<NUM> GBq/µmol, produced at the Klinikum rechts der Isar in a fully automated procedure). The animal was left under anesthesia for <NUM> and sacrificed. Urine, blood, liver, kidneys and tumor were collected and processed to the hereafter described procedures. The urine sample was centrifuged for <NUM> at <NUM> rpm to yield a clear solution and directly subjected to radio-HPLC analysis. Blood was diluted to <NUM> with H<NUM>O and centrifuged twice at <NUM> for <NUM>. The supernatant was collected and loaded on a Strata X cartridge (<NUM> Polymeric Reversed Phase <NUM>, pre-conditioned with <NUM> MeOH, followed by <NUM> H<NUM>O). After washing with <NUM> H<NUM>O, the cartridge was eluted with a <NUM>:<NUM> mixture (v/v) of MeCN in H<NUM>O, supplemented with <NUM>% TFA. The eluate was diluted with water and analysed by radio-HPLC. Tumour, kidneys and liver were homogenised using either a Potter-Elvehjem tissue grinder (Kontes Glass Co, Vineland, USA) or a MM-<NUM> ball mill (Retsch GmbH, Haan, Germany).

Tumour and kidneys were separately homogenised in the tissue homogeniser with <NUM> of extraction buffer (<NUM>µL <NUM> HEPES pH7. <NUM>, <NUM>µL <NUM> PMPA and <NUM>µL <NUM> NaCl) for <NUM>. The resulting homogenate was collected and centrifuged at <NUM> for <NUM>. Subsequently the supernatant was collected, centrifuged again (<NUM>, <NUM>) and loaded on a Strata X cartridge (<NUM> Polymeric Reversed Phase <NUM>, pre-conditioned with <NUM> MeOH, followed by <NUM> H<NUM>O). After washing with <NUM> H<NUM>O, the cartridge was eluted with a <NUM>:<NUM> mixture (v/v) of MeCN in H<NUM>O, supplemented with <NUM>% TFA. The eluate of each organ was diluted with water and analysed by radio-HPLC.

The organs (tumour, kidney, liver) were separately homogenised in a <NUM> tube together with <NUM> grinding balls (<NUM> diameter) and <NUM> of extraction buffer (<NUM>µL <NUM> HEPES pH7. <NUM>, <NUM>µL <NUM> PMPA and <NUM>µL <NUM> NaCl) for <NUM> at <NUM>. The homogenate was centrifuged at <NUM> for <NUM> and the supernatant was collected. Subsequently, the pellet was suspended in <NUM> of extraction buffer and homogenized again with the ball mill for <NUM> at <NUM>. After centrifugation (<NUM>, <NUM>), both supernatants were combined and loaded on a Strata X cartridge (<NUM> Polymeric Reversed Phase <NUM>, pre-conditioned with <NUM> MeOH, followed by <NUM> H<NUM>O). After washing with <NUM> H<NUM>O, the cartridge was eluted with a <NUM>:<NUM> mixture (v/v) of MeCN in H<NUM>O, supplemented with <NUM>% TFA. The eluate of each organ was diluted with water and analysed by radio-HPLC. In order to demonstrate that the breakthrough during cartridge loading, is not a result of unbound F-<NUM>, the supernatant was also examined by radio-TLC after centrifugation.

Finally the ratios of the individual isomers were determined from the HPLC profiles of the extracted samples and compared to the ratios of the isomers from the quality control of the racemic mixture of rhPSMA-<NUM>. The decay corrected extraction- and cartridge loading-efficiency, as well as the overall extracted activity of the examined samples are given in table <NUM>. The cartridge elution-efficiency was ><NUM>% for all experiments.

The chromatographic peak assignment was carried out by comparison of the UV profiles of.

The following names are used for the different isomers:.

The first set of values (rhPSMA-<NUM> and rhPSMA-<NUM>; <FIG>) were determined by using for the dilution series a solution directly obtained after natGa-complexation of the respective ligand. In the second data set (<FIG>), the complexed ligands were purified by RP-HPLC in order to separate uncomplexed natGa-salts. Since there were no significant differences observed, both series were merged and used for the calculation of the mean values (±SD).

Determianation of the logP values was carried out in phosphate buffered saline (PBS, pH <NUM>) and n-octanol (= logPoct/PBS).

With the aim to quantify the relative changes of each rhPSMA7 isomer in blood, liver, kidney, urine and tumor <NUM> after injection of [<NUM>F]rhPSMA7-rac into a LNCaP tumor bearing mouse, two different homogenization methods (a potter and a ball mill) were used to extract the tracer from kidney, liver and tumor tissue (see Materials and Methods).

Table <NUM> summarizes the observed efficiencies for both homogenization methods and the efficancy of the subsequent solid phase extraction procedure (to separate the tracer from the protein fraction).

Whereas the extraction of activity from the samples using the potter was quite efficient, the use of the ball mill was disappointing. Nevertheless, even with the ball mill ><NUM>% extraction efficiency was reached.

Taking into account the possible species that could be formed by metabolic cleavage of amide bonds of rhPSMA7, only a) species with significantly increase lipophilicity of b) F18-fluoride seem probable. Thus in principle it seem possible that "iL" species depicted in <FIG> are not extracted from tissue sample (aqueous extraction) and thus do not appear in the final analysis. However, it should be noted that such species would appear in vivo in the liver and intestine (hepatobiliary excretion of lipophilic compounds) or should be bound to plasma proteins (resulting in high activity levels for the blood, which on the other hand showed excellent extraction efficiency).

For quantification of each isomer in the racemic mixture and especially for the poorly separated first and second peak (rhPSMA <NUM> and rhPSMA7. <NUM>) a graphical approximation was initially used. This approach was based on the assumption that a) each isomer is eluted from the HPLC column with an identical peak shape and b) the different peak heights can be used as first approximation to calculate by means of linear factors less separated peaks (i.e. rhPSMA <NUM> and rhPSMA7.

Based on these assumptions, the first analysis was performed by using one LNCaP tumor bearing mice coinjected with [<NUM>F][natGa]rhPSMA7-rac. With the aim to validate these experiments by means of three additional experiments and to improve the graphical analyses by a more valid procedure, the Systat softare package 'PeakFit' was used. PeakFit allows for automated nonlinear separation, analysis and quantification of HPLC elution profiles by deconvolution procedures that uses a Gaussian response function with a Fourier deconvolution / filtering algorithm (https://systatsoftware. com/products/peakfit/).

A comparison of the graphical analysis of the first experiments revealed that the graphical analysis overestimated the second peak (rhPSMA7. <NUM>), whereas the first peak was underestimated. Consequently, all data sets were reanalyzed and quantified by means of PeakFit.

It was first examined, whether the deconvolution technique shows similar data for the last two peaks (rhPSMA7. <NUM> and <NUM>) that have a good separation (although they are not baseline separated).

<FIG> and <FIG> summarise the percentage change of each rhPSAM7. n isomer in a given sample with respect to its percentage in the injected solution ([<NUM>F][natGa]rhPSMA7-rac); the results for the individual experiment are shown in <FIG>. The proportion of each isomer was quantified by analysis of the HPLC elution profile by Systat 'PeakFit'. Subsequently, the percentage change of each isomer in a given sample with repect to its percentage in the injected solution was calculated.

The radio-HPLC analyses of the radioactivity extracted from the homogenized (kidney, liver, tumor) or diluted (blood) tissues and subsequently immobilized on and eluted from the solid phase extraction cartridge did show no signs of metabolic instability. Thus, no lipophilic metabolic fragments were observed. It should be noted that F-<NUM>-fluoride cannot be accurately detected by HPLC under the conditions used for sample preparation (see TLC analysis).

Although there is a clear trend towards the D-Dap-derivative rhPSMA7. <NUM> and <NUM>, the overall changes are low (max <NUM>%). It is also important to stress in this context, that <FIG> and <FIG> show "relative changes" without taking the absolute uptake values into account.

Although rhPSMA7. <NUM> has the weakest affinity and internalization of all rhPSMA7 compounds, it shows the largest positive percentage change in blood liver, kidney and tumor.

Although the reason for this result is unclear, one can speculate that homogenization of the tissue samples, even with the ball mill, did not resulted in a quantitative cell disruption. Thus, the rhPSMA7 tracers with the highest internalization (rhPSMA7. <NUM>: <NUM>% ± <NUM>%, rhPSMA7. <NUM>: <NUM> ± <NUM>% and rhPSMA7. <NUM>: <NUM>% ± <NUM>%) might have been extracted in a less efficient manner, whereas rhPSMA7. <NUM> with its low internalization of only <NUM>% ± <NUM>% was efficiently extracted and is consequently overestimated in the HPLC analysis.

In addition, it seems that the rhPSMA compounds <NUM> and <NUM> are somewhat more rapidly excreted (see values for urine). These compounds show generally negative changes in solid tissues and blood, although both compounds exhibit higher affinities and internalization rates when compared with rhPSMA7. Whether this might be caused by metabolic degradation of <NUM> and <NUM> (both are L-Dap derivative) is unclear, since no metabolites, i.e. lyophilic metabolites have been detected. It might however be possible, that such metabolites (see <FIG>), due to their high logP value, are not extractable in aqueous buffer solutions. In this case, they should appear in the liver (see biodistribution) and perhaps in blood samples (high probability for high serum protein binding). Since no elevated activity accumulation has been observed for liver tissue in the course of the biodistribution studies and the activity extraction from blood was highly efficient (see table <NUM>), we assume that no significant degradation for rhPSMA7. <NUM> and <NUM> occurred. This assumption is supported by unsuspicious SUV-values for liver tissue (gall bladder, intestine) in the context of the clinical use of [<NUM>F]rhpsma-rac in humans.

Radio-TLC Analysis was carried out a) on urine samples by directly subjecting a small volume onto a TLC strip, b) by analysis of a small volume of the non-immobilized activity during the SPE process (the 'breakthrough fraction'), and c) by analysis of a small volume of the cartridge eluates.

Since it is very difficult to detect n. <NUM>F-fluoride by means of RP-<NUM> chromatography (due to free Si-OH groups of the matrix that interact with nca fluoride), thin layer chromatography was performed to investigate to quantify F-<NUM>-Fluoride in the extracted solutions.

Since none of the reagents and salts normally used for protein precipitation are tested for cold fluoride and to avoid possible liberation of F-<NUM>-fluoride from the tracer by isotopic exchange, protein precipitation was not implemented in the sample preparation process - although such protein load often result in limited peak separation, peak tailing and activity that sticks at the start line. The solutions obtained after tissue extraction (or blood centrifugation) were directly used for TLC analysis.

Although the activity available for analysis was quite low in all samples, the TLC results reveal that the overall content of F-<NUM>-fluride was below approx. <NUM>% in the tissue investigated, except:.

Whereas the analysis of the urine by TLC is regarded as valid result (see Profile in the <FIG>), the result obtained with the liver sample is caused by extensive tailing of the peak representing the intact tracer (see <FIG>). In addition it can be concluded that the above mentioned max. <NUM>% free fluoride represent an overestimation, since peak tailing, even obtained during the QK and release of [F-<NUM>]rhPSMA7-rac in PBS (<FIG>) for clinical application show a tailing of the product peak. As demonstrated by the phosphoimages, this tailing is observed in almost every TLC analysis and contributes to the integrated area of F-<NUM>-fluoride.

It need to be noted that neither the biodistribution studies, nor the clinical PET scans in humans (status July <NUM>: approx <NUM> scans with [F-<NUM>]rhPSMA7-rac) resulted in any suspicious or identifiable F-<NUM>-acculuation in bone by liberated F-<NUM>-fluoride.

To further investigate the liberation of F-<NUM> fluoride from [F-<NUM>]rhPSMA7-rac (as observed in one urine sample) we investigated the occurrence of F-<NUM>-Fluoride in further urine samples (normal mice) by means of RP-<NUM> HPLC (new RP-<NUM> end-capped column) and TLC analyses.

For this purpose normal mice were used. Urine samples were collected by means of a catheter over a period of <NUM>. The urine was centrifuged and directly subjected to HPLC and TLC.

As shown in <FIG>, left column, free F-<NUM>-fluoride was found in urine samples of all isomers and is also formed when fresh urine is "incubated with [F-<NUM>]rhPSMA7. (right column). Identification of F-<NUM>-fluoride was performed by demonstrating that a) this species is retained on QMA cartridges (data not shown), b) is eluted in the dead volume of RP-<NUM> columns and c) can not be retained or mobilized on RP-<NUM> columns or RP-<NUM> TLC plates, respectively, irrespective of the mobile phases used.

Due to the fact that such high amounts of F-<NUM> fluoride were not detected in the HPLC analyses of blood or organs, such as kidneys, tumor, liver etc., that no elevated activity uptake in bone was observed in the biodistribution studies in mice and no elevated activity uptake in bone was observed during the clinical PET scans with the [F-<NUM>]rhPSMA7-rac compound since [F-<NUM>]rhPSMA7-rac has been established for clinical scanning end of <NUM> at the TUM (status end July, <NUM>: approx. <NUM> PET scans in patients with prostate cancer) we concluded that [F-<NUM>]fluoride might be formed downstream from glomerular filtration of the tracer, resulting in the formation and subsequent excretion of [F-<NUM>]fluoride WITHOUT detectable uptake of F-<NUM>-fluoride in blood, organs or bones.

This assumption is supported by the literature on the toxicology of fluoride that describes relevant amounts of fluoride in KIDNEYS AND URINE. Normal urinary fluoride levels of <NUM>. 3ppm were observed in mice (<NPL>). In another publication, the average fluoride concentration in the urine of normal mice was determined to be <NUM>-<NUM>. 14µg/mL (<NPL>), and Inkielewicz I. found that the fluoride content in the serum of rats is about <NUM>% of the concentration of fluoride in the kidneys (serum: <NUM>µg/mL, kidneys: <NUM>µg/mL) (<NPL>). Taken into account that most of the tracer is specifically taken up into and also physiologically cleared by the kidneys, an elevated fluoride level in the kidney, combined with a body temperature of <NUM>, might result in a continuous elimination of F-<NUM>-fluoride from the rhPSMA-compounds in kidneys.

Consequently, fresh and nonradioactive urine samples collected from normal mice were incubated with [F-<NUM>]rhPSMA7. x for various time periods (see legend to <FIG>). <FIG>, right column, clearly demonstrate that incubation of urine with [F-<NUM>]rhPSMA7. x EX VIVO result in the formation of free [F-<NUM>]fluoride to various degrees, promoted by the different concentrations of cold F-<NUM>-Fluoride in the urine samples and increasing over time.

To further support the hypothesis, <NUM> nmol cold F-<NUM>-fluoride was added to fresh and nonradioactive urine of mice, followed by the addition of [F-<NUM>]rhPSMA7. <NUM> and incubation for <NUM>. According to the hypothesis, the high concentration of [F-<NUM>]fluoride should result in the formation of a significant amount of [F-<NUM>]fluoride. <FIG> shows that under these conditions <NUM> % of the radioactivity is exchanged and forms [F-<NUM>]fluoride within <NUM> (<FIG>).

Since isotopic exchange rates are depending on the concentration of the four relevant species in the equilibrium ([F-<NUM>]Fluoride, [F-<NUM>]fluoride, [F-<NUM>]rhPSMA7. <NUM> and [F-<NUM>]rhPSMA7. <NUM>), it was also investigated, whether the addition of [F-<NUM>]fluoride to fresh and radioactive urine (<NUM>,<NUM>% [F-<NUM>]Fluoride, <NUM>,<NUM>% [F-<NUM>]rhpsma7. <NUM>) followed by the addition of cold [F-<NUM>]rhPSMA7. <NUM> tracer also result in the labeling of the radiopharmaceutical [F-<NUM>]rhPSMA7-<NUM>. Unexpectedly, even a small amount of <NUM> nmol [F-<NUM>]rhPSMA7-<NUM> to the urine above resulted in an increase of [F-<NUM>]rhPSMA7. <NUM> from <NUM>% to <NUM>% (F-<NUM>]Fluoride decreased from <NUM>% to <NUM>%) at room temperature.

The results obtained by isotopic exchange in urine are considered representative for all tracers conjugated with the <NUM>-(di-tert-butyl[(<NUM>)F]fluorosilyl)-benzyl)oxy moiety and thus for all rhPSAM7 isomers.

Please note that in the following 18F-rhPSMA-<NUM> refers to natGa-<NUM>F-rhPSMA7-rac and 18F-rhPSMA-<NUM> to natGa-<NUM>F-rhPSMA7.

Aim was to assess the distribution and excretion of <NUM>F-rhPSMA-<NUM> and <NUM>F-rhPSMA-<NUM> at different time-points up to <NUM> minutes following a single intravenous administration in mice and to perform calculations for internal dosimetry.

<NUM>-<NUM> mice were injected per timepoint with a mean <NUM> ± <NUM> MBq of 18F-rhPSMA-<NUM> and <NUM> ± <NUM> MBq of 18F-rhPSMA-<NUM>, respectively. Mice, severe combined immunodeficiency (SCID) were used for the experiments. All animal experiments were conducted in accordance with general animal welfare regulations in Germany and the institutional guidelines for the care and use of animals.

Mice were sacrificed at the following timepoints:.

Please note that based on initial experiments exhibiting prolonged renal kidney uptake for <NUM>F-rhPSMA-<NUM> a late timepoint (<NUM>) was used for the final experiments.

The following tissues / fluids were harvested:
Urine, blood, heart, lung, spleen, pancreas, liver, stomach (emptied), small intestine (emptied), large intestine (emptied), kidneys, bladder, testis, fat, muscle (partial, femoral), femur, tail and brain. Urine was collected with a pipette in the CO<NUM> gas chamber. In case of missing urination in the chamber the bladder was aspirated with an insulin syringe. Blood was withdrawn instantly after sacrifice with an insulin syringe from the heart. All other tissues and organs were dissected and transferred directly in plastic containers.

The weights of the samples in the plastic containers were measured using an electronic balance. The weights of the empty and pre-labeled plastic containers for the dedicated samples were measured beforehand. The tare weight of the plastic containers was subtracted from the weight of the measurement sample with the plastic container. The thus-calculated weight was designated as the weight of the measurement sample.

The plastic containers containing the measurement samples were placed in specific racks of an automatic gamma counter (PerkinElmer - Wallac, Waltham, USA) for measuring the counting rate over <NUM> seconds (counts per minute = cpm). In addition, a <NUM>% (v/v) standard (n=<NUM>) with a known amount of radioactivity was measured together with the samples to convert the counting rate of the organ samples into activity.

The counting rates of measurement samples were automatically corrected for decay. The radioactivity distribution ratios (unit: percentage of the injected dose (%ID) in the measurement samples were determined using the equation below. The sum of the counting rates from all measurement samples obtained from one mouse was designated as the counting rate for administrated radioactivity.

The radioactivity distribution ratio per unit weight of the measurement sample (unit: %ID/g) excluding urine and feces samples was determined by using the equation below. The weight of the measurement sample was determined by subtraction of the empty measurement container from the container including the sample.

For consistency of statistical calculations for each radiotracer the same number of time-points for <NUM>F-rhPSMA-<NUM> and <NUM>F-rhPSMA-<NUM> was used. Therefore, for <NUM>F-rhPSMA-<NUM> the <NUM> and <NUM> time points were combined creating a <NUM> endpoint.

The time-integral of activity for the accumulation in the significant source organs (AUCs) were generated both with numerical integration and physical decay according to <NPL>.

Kirshner et al. established a method that uses linear scaling of the percent injected dose in the animal by the ratio of the organ weights and total body weights of phantoms in both species.

In brief, to calculate a human dosimetry from the biodistribution in the mice, an extrapolation was necessary to account for the differences between the animals and humans. Normal-organ radiation doses were estimated for the <NUM>-kg Standard Adult anatomic model using time-depending organ activity concentrations (in percent of the injected dose per gram, %ID/g) and total-body activities measured in the biodistribution studies in mice.

Tissue activity concentrations in mice were converted to tissue fractional activities in the <NUM>-kg Standard Adult using the relative fractional organ masses in the Standard Adult and the "standard" <NUM>-gramm mouse. Time-dependent total-body activity was fit to an exponential function and the difference between the injected activity and the total-body activity was assumed to be excreted to the urine because activity concentrations in the liver and Gl tracer were low at all time points studied. Organ residence time was calculated by numerical integration using the trapezoidal rule and the rest-of-body <NUM>F residence times was calculated as the difference between the total-body residence time and the sum of the organ and urine residence times. The bladder contents residence time was estimated using the dynamic voiding model in the OLINDA/EXM <NUM> dosimetry software. Finally, the Standard Adult mean organ dose equivalents (in mSv/MBq) and effective dose (also in mSv/MBq) were then calculated using OLINDA/EXM <NUM>.

Final calculation of radiation absorbed dose and dosimetry from biodistribution in mice: The tissues or organs in which a significant accumulation of radioactivity occurs (i.e., source organ) were kidney, spleen, lung, liver and heart. With respect to activity accumulation and clearance, a rapid clearance from blood and clearance to urine but relatively slow build-up in kidney was found.

The radioactivity distribution ratios were highest in kidneys after administration of both <NUM>F-rhPSMA-<NUM> and <NUM>F-rhPSMA-<NUM> at all examined time points in mice. Moreover, it was high in the spleen and in the bladder for both radiotracers compared to all other assessed tissues, where activity ratios were lower than <NUM>%ID/g.

Since the majority of <NUM>F-rhPSMA-<NUM>/ <NUM>F-rhPSMA-<NUM> activity augment in the kidneys and the excretion via bladder reveal high activities, the main excretion route is defined via kidneys and the urinary system.

Using a <NUM> and <NUM> bladder voiding interval the extrapolated total effective doses were <NUM>. 66E-<NUM> and <NUM>. 22E-<NUM> mSv/MBq for <NUM>F-rhPSMA-<NUM> and <NUM>. 17E-<NUM> and <NUM>. 28E-<NUM> mSv/MBq for <NUM>F-rhPSMA-<NUM>, respectively. An injection of up to <NUM> MBq (<NUM> mCi) for a clinical scan would result in a favorable radiation exposure of less than <NUM> mSv for both agents assuming a <NUM> voiding interval.

Differences worth to mention between both radiotracers are only evident regarding kidney uptake as <NUM>F-rhPSMA-<NUM> tends to accumulate more gradual with longer retention. Yet radiation exposure is comparable between both agents.

The following sections describe biodistribution of 18F-rhPSMA-<NUM> and 18F-rhPSMA-<NUM>. Proof-of-concept evaluation was conducted under compassionate use. The agent was applied in compliance with The German Medicinal Products Act, AMG §<NUM>2b, and in accordance with the responsible regulatory body (Government of Oberbayern).

All subjects were examined on a Biograph mCT scanner (Siemens Medical Solutions, Erlangen, Germany). All PET scans were acquired in 3D-mode with an acquisition time of <NUM>-<NUM> per bed position. Emission data were corrected for randoms, dead time, scatter, and attenuation and were reconstructed iteratively by an ordered-subsets expectation maximization algorithm (four iterations, eight subsets) followed by a postreconstruction smoothing Gaussian filter (<NUM>-mm full width at one-half maximum).

Human biodistribution was estimated by analysing clinical <NUM>F-rhPSMA-<NUM>- and <NUM>F-rhPSMA-<NUM>-PET/CT exams in <NUM> and <NUM> patients, respectively. Mean injected activities were <NUM> (range <NUM>-<NUM>) MBq vs. <NUM> (range <NUM>-<NUM>) MBq and uptake times were <NUM> (range <NUM>-<NUM>) min and vs. <NUM> (range <NUM>-<NUM>) min for <NUM>F-rhPSMA-<NUM> vs. <NUM>F-rhPSMA-<NUM>, respectively.

The mean and maximum standardized uptake values (SUVmean/SUVmax) were determined for background (gluteal muscle), normal organs (salivary glands, blood pool, lung, liver, spleen, pancreas, duodenum, kidney, bladder, bone) and three representative tumor lesions. Tumor uptake was analyzed in <NUM> lesions (<NUM> primary tumors/local recurrences, <NUM> bone, <NUM> lymph node and <NUM> visceral metastases) and <NUM> lesions (<NUM> primary tumors/local recurrences, <NUM> bone, <NUM> lymph node and <NUM> visceral metastases) for <NUM>F-rhPSMA-<NUM> and <NUM>F-rhPSMA-<NUM>, respectively.

For calculation of the SUV, circular regions of interest were drawn around areas with focally increased uptake in transaxial slices and automatically adapted to a three-dimensional volume of interest (VOI) at a <NUM> % isocontour. Organ-background and Tumor-background ratios were calculated.

Human biodistribution of <NUM>F-rhPSMA-<NUM> and <NUM>F-rhPSMA-<NUM> showed the typical pattern known from other PSMA-ligands. Uptake parameters for <NUM>F-rhPSMA-<NUM> and <NUM>F-rhPSMA-<NUM> were very similar with a lower activity retention in the bladder and higher uptake in tumor lesions for <NUM>F-rhPSMA-<NUM>: SUVmean for <NUM>F-rhPSMA-<NUM> vs. <NUM>F-rhPSMA-<NUM> were <NUM> vs. <NUM> (parotid gland), <NUM> vs. <NUM> (submandibular gland), <NUM> vs. <NUM> (blood pool), <NUM> vs. <NUM> (lungs), <NUM> vs. <NUM> (liver), <NUM> vs. <NUM> (spleen), <NUM> vs. <NUM> (kidney), <NUM> vs. <NUM> (pancreas), <NUM> vs. <NUM> (duodenum), <NUM> vs. <NUM> (non-diseased bone) and <NUM> vs. <NUM> (bladder) for <NUM>F-rhPSMA-<NUM> vs. <NUM>F-rhPSMA-<NUM>, respectively. In particular, uptake values of <NUM>F-rhPSMA-<NUM> vs. <NUM>F-rhPSMA-<NUM> were significantly lower for retention in the bladder (<NUM> ± <NUM> vs. <NUM> ± <NUM>, p <<NUM>) and significantly higher for tumor lesions (<NUM> ± <NUM> vs. <NUM> ± <NUM>, p <<NUM>).

Claim 1:
A ligand-SIFA-chelator conjugate, comprising, within a single molecule three separate moieties:
(a) one or more ligands which are capable of binding to PSMA;
(b) a silicon-fluoride acceptor (SIFA) moiety which comprises a covalent bond between a silicon and a <NUM>F fluorine atom;
(c) one or more chelating groups, containing a chelated nonradioactive metal cation; and, wherein the ligand-SIFA-chelator conjugate is a compound of formula
<CHM>
and M<NUM>+ is the chelated nonradioactive cation