Labeling precursors and radiotracers for nuclear medicine diagnosis and therapy of prostate cancer-induced bone metastases

Abstract

The invention relates to a labeling precursor for nuclear medicine diagnostics and theranostics that has the structure

##STR00001##

with a first PSMA-specific targeting vector TV1, a second targeting vector TV2 having osteoaffinity, a chelator Chel for complexing a radioisotope, and two or three linkers L1, L2 and L3.

Claims

1. A labeling precursor for complexing radioactive isotopes having the structure ##STR00058## in which a first targeting vector TV1 is selected from the group of PSMA inhibitors comprising ##STR00059## a second targeting vector TV2 is selected from the group of bisphosphonates comprising ##STR00060## a first linker L1 has a structure selected from ##STR00061## in which G is ##STR00062## O1, O2 and O3 are independently selected from the group comprising amide radicals, carboxamide radicals, phosphinate radicals, alkyl radicals, triazole radicals, thiourea radicals, ethylene radicals, maleimide radicals, (CH.sub.2), (CH.sub.2CH.sub.2O), CH.sub.2CH(COOH)NH and (CH.sub.2).sub.qNH with q=1, 2, 3, 4, 5, 6, 7,8,9 or 10; p1, p2 and p3 are independently selected from the set {0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 , 19, 20}; a second linker L2 has a structure selected from ##STR00063## in which R1, R2 and R3 are independently selected from the group comprising amide radicals, carboxamide radicals, phosphinate radicals, alkyl radicals, triazole radicals, thiourea radicals, ethylene radicals, maleimide radicals, furan radicals, azole radicals, oxazole radicals, thiophene radicals, thiazole radicals, azine radicals, thiazine radicals, naphthalene radicals, quinoline radicals, pyrrole radicals, imidazole radicals, pyrazole radicals, tetrazole radicals, thiadiazole radicals, oxadiazole radicals, pyridine radicals, pyrimidine radicals, triazine radicals, tetrazine radicals, thiazine radicals, oxazine radicals, naphthalene radicals, chromene radicals or thiochromene radicals, (CH.sub.2), (CH.sub.2CH.sub.2O), CH.sub.2CH(COOH)NH and (CH.sub.2).sub.qNH with q=1, 2, 3, 4, 5,6, 7, 8,9 or 10; s1, s2 and s3 are independently selected from the set {0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 , 19, 20}; a third linker L3 has a structure selected from ##STR00064## in which T1, T2 and T3 are independently selected from the group comprising amide radicals, carboxamide radicals, phosphinate radicals, alkyl radicals, triazole radicals, thiourea radicals, ethylene radicals, maleimide radicals, (CH.sub.2), (CH.sub.2CH.sub.2O), CH.sub.2CH(COOH)NH and (CH.sub.2).sub.vNH with v=1, 2, 3,4, 5, 6,7,8,9 or 10; u1, u2 and u3 are independently selected from the set {0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 , 19, 20}; QS is a squaric acid radical; ##STR00065## and Chel is a chelator selected from the group comprising H.sub.4pypa, ethylenediaminetetraacetate (EDTA), diethylenetriaminepenta(methylenephosphonic acid) (EDTMP) diethylenetriaminepentaacetate (DTPA) and derivatives thereof, dodeca-1,4,7,10-tetraamine tetraacetate (DOTA), 2-(1,4,7,10-tetraazacyclododecane-4,7,10)pentanedioic acid (DOTAGA) and other derivatives, trideca-1,4,7,10-tetraamine tetraacetate (TRITA), tetradeca-1,4,8,11-tetraamine tetraacetate (TETA) and derivatives thereof, nona-1,4,7-triamine triacetate (NOTA) and derivatives thereof, triazacyclononanephosphinic acid (TRAP), 1,4,7-triazacyclononane-1,4-bis[methylene(hydroxymethyl)phosphinic acid]-7-[methylene(2-carboxyethyl)phosphinic acid] (NOPO), pentadeca-1,4,7,10,13-pentaamine pentaacetate (PEPA), hexadeca-1,4,7,10,13,16-hexaamine hexaacetate (HEHA) and derivatives thereof, hydroxybenzylethylenediamine (HBED) and derivatives thereof, DEDPA and derivatives thereof, deferoxamine (DFO) and derivatives thereof, trishydroxypyridinone (THP) and derivatives thereof, tetraazacyclodecanephosphinic acid (TEAP) and derivatives thereof, 6-amino-6-methylperhydro-1,4-diazepine N,N,N,N-tetraacetate (AAZTA) and derivatives; 1-N-(4-aminobenzyl)-3,6,10,13,16,19-hexaazabicyclo[6.6.6]eicosane-1,8-diamine (SarAr) and salts thereof, 1,8-diamino-3,6,10,13,16,19-hexaazabicyclo[6.6.6]eicosane ((NH.sub.2).sub.2SAR) and salts and derivatives thereof, aminothiols and derivatives thereof.

2. The labeling precursor as claimed in claim 1, wherein the chelator Chel is DOTA, H.sub.4pypa, DATA or DOTAGA.

3. The labeling precursor as claimed in claim 1, wherein the second linker L2 comprises at least one radical selected from ##STR00066##

4. The labeling precursor as claimed in claim 3, wherein the second linker L2 comprises at least one squaric acid radical ##STR00067##

5. The labeling precursor as claimed in claim 3, wherein the second linker L2 comprises at least one radical selected from ##STR00068##

6. The labeling precursor as claimed in claim 1, wherein the second linker L2 comprises at least one imidazole radical ##STR00069##

7. The labeling precursor as claimed in claim 1, wherein two or three of the linkers L1, L2 and L3 are the same.

8. A radiotracer comprising a labeling precursor as claimed in claim 1 and a radioactive isotope selected from the group comprising .sup.44Sc, .sup.47Sc, .sup.55Co, .sup.62Cu, .sup.64Cu, .sup.67Cu, .sup.66Ga, .sup.67Ga, .sup.68Ga, .sup.89Zr, .sup.86Y, .sup.90Y, .sup.89Zr, .sup.90Nb, .sup.99mTc, .sup.111In, .sup.135Sm, .sup.140Pr, .sup.159Gd, .sup.149Tb, .sup.160Tb, .sup.161Tb, .sup.165Er, .sup.166Dy, .sup.166Ho, .sup.175Yb, .sup.177Lu, .sup.186Re, .sup.188Re, .sup.211 At, .sup.212Pb, .sup.213Bi, .sup.225 Ac and .sup.232Th.

9. The radiotracer as claimed in claim 8, wherein the radioactive isotope is .sup.68Ga, .sup.177Lu or .sup.225 Ac.

10. A labeling precursor as claimed in claim 8, wherein the NOTA derivative is 1,4,7-triazacyclononane, 1-glutaric acid,4,7-acetate (NOTAGA), the DEDPA derivative is 1,2-[[6-(carboxylate)pyridin-2-yl]methylamine]ethane (H.sub.2DEDPA), the THP derivative is YM103, and the AAZTA derivatives is (6-pentanoic acid)-6-(amino)methyl-1,4-diazepine triacetate-(DATA).

Description

[0058] The invention is elucidated in detail hereinafter with reference to figures and examples. The figures show:

[0059] FIG. 1 . . . the functional components of a radiotracer;

[0060] FIG. 2 . . . in vivo PET images of Wistar rats;

[0061] FIG. 3 . . . diagrams of the progression of SUV and the PET signal ratio of epiphysis and blood against time; and

[0062] FIG. 4 . . . the result of a docking simulation for the PSMA binding pocket;

[0063] FIG. 5 . . . the labeling kinetics of a labeling precursor;

[0064] FIG. 6 . . . the stability of a radiotracer in physiological medium;

[0065] FIG. 7 . . . binding affinities for hydroxyapatite;

[0066] FIG. 8 . . . ex vivo organ accumulations with and without blocking of PSMA.

EXAMPLE OF AMIDE COUPLING

[0067] In the invention, the chelator Chel, the targeting vectors TV1, TV2, and the linkers L1, L2 are preferably conjugated by an amide coupling reaction. Amide coupling, which forms the backbone of proteins, is the most commonly used reaction in medicinal chemistry. A generic example of an amide coupling is shown in scheme 5.

##STR00020##

[0068] Owing to a virtually unlimited set of readily available carboxylic acid and amine derivatives, amide coupling strategies open up a simple route to the synthesis of novel compounds. Numerous reagents and protocols for amide coupling are known to those skilled in the art. The most common amide coupling strategy is based on the condensation of a carboxylic acid with an amine. The carboxylic acid is generally activated for this purpose. Remaining functional groups are protected prior to activation. The reaction is effected in two steps, either in one reaction medium (single pot) with direct conversion of the activated carboxylic acid or in two steps with isolation of an activated trapped carboxylic acid and reaction with an amine.

[0069] The carboxylate reacts here with a coupling agent to form a reactive intermediate that can be isolated or reacted directly with an amine. Numerous reagents are available for carboxylic acid activation, such as acid halides (chloride, fluoride), azides, anhydrides, or carbodiimides. In addition, esters such as pentafluorophenyl or hydroxysuccinic imido esters can be formed as reactive intermediates. Intermediates derived from acyl chlorides or azides are highly reactive. However, harsh reaction conditions and high reactivity are often a barrier to use for sensitive substrates or amino acids. In contrast, amide coupling strategies that use carbodiimides such as DCC (dicyclohexylcarbodiimide) or DIC (diisopropylcarbodiimide) open up a wide range of applications. Commonly, especially in solid-phase synthesis, additives are used to improve reaction efficiency. Aminium salts are highly efficient peptide coupling reagents with short reaction times and minimal racemization. With some additives, for example HOBt, racemization can even be completely avoided. Aminium reagents are used in equimolar amounts relative to the carboxylic acid in order to prevent excessive reaction with the free amine of the peptide. Phosphonium salts react with carboxylate, which generally requires two equivalents of a base, for example DIEA. A key advantage of phosphonium salts over iminium reagents is that phosphonium does not react with the free amino group of the amine component. This enables couplings in equimolar ratios of acid and amine and helps to avoid intramolecular cyclization of linear peptides and excessive use of costly amine components.

[0070] A comprehensive summary of reaction strategies and reagents for amide coupling can be found in the review articles: [0071] Analysis of Past and Present Synthetic Methodologies on Medicinal Chemistry: Where Have All the New Reactions Gone?; D. G. Brown, J. Bostr?m; J. Med. Chem. 2016, 59 (10), 4443-4458; [0072] Peptide Coupling Reagents, More than a Letter Soup; A. El-Faham, F. Albericio; Chem. Rev. 2011, 111 (11), 6557-6602; [0073] Rethinking amide bond synthesis; V. R. Pattabiraman, J. W. Bode; Nature, Vol. 480 (2011) 471-479; [0074] Amide bond formation: beyond the myth of coupling reagents; E. Valeur, M. Bradley; Chem. Soc. Rev., 2009, 38, 606-631.

[0075] Many of the chelators used in accordance with the invention, such as DOTA in particular, have one or more carboxy or amide groups. Accordingly, these chelators can be readily conjugated to the linkers L1, L2 using any of the amide coupling strategies known in the art. Schemes 6 and 7 show examples of coupling of the linker targeting vector unit L1-TV1 with a chelator Chel; schemes 8-10 show examples of coupling of L2-TV2 with a chelator Chel.

##STR00021##

##STR00022##

##STR00023##

##STR00024##

##STR00025##

Chelator Chel for Radioisotope Labeling

[0076] The chelator Chel is intended for labeling of the labeling precursor of the invention with a radioisotope selected from the group consisting of .sup.44Sc, .sup.47Sc, .sup.55Co, .sup.62Cu, .sup.64Cu, .sup.67Cu, .sup.66Ga, .sup.67Ga, .sup.68Ga, .sup.89Zr, .sup.86Y, .sup.90Y, .sup.89Zr, .sup.90Nb, .sup.90mTc, .sup.111In, .sup.135Sm, .sup.140Pr, .sup.159Gd, .sup.149Tb, .sup.160Tb, .sup.161Tb, .sup.165Er, .sup.166Dy, .sup.166Ho, .sup.175Yb, .sup.177Lu, .sup.186Re, .sup.188Re, .sup.211At, .sup.212Pb, .sup.213Bi, .sup.225Ac and .sup.232Th. A variety of chelating agents for complexing the above radioisotopes are known in the art. Scheme 11 shows examples of chelators used in accordance with the invention.

##STR00026## ##STR00027## ##STR00028## ##STR00029## ##STR00030##

##STR00031##

Radioisotopes

[0077] For nuclear-medical theranostics (diagnosis and therapy), in particular, the radioisotopes .sup.68Ga or .sup.177Lu used. The invention also provides for the use of radioisotopes selected from the group comprising .sup.44Sc, .sup.47Sc, .sup.55Co, .sup.62Cu, .sup.64Cu, .sup.67Cu, .sup.66Ga, .sup.67Ga, .sup.68Ga, .sup.89Zr, .sup.90Nb, .sup.90mTc, .sup.111In, .sup.135Sm, .sup.140Pr, .sup.159Gd, .sup.149Tb, .sup.160Tb, .sup.161Tb, .sup.165Er, .sup.166Dy, .sup.166Ho, .sup.175Yb, .sup.177Lu, .sup.186Re, .sup.188Re, .sup.211At, .sup.212Pb, .sup.213Bi, .sup.225Ac and .sup.232Th.

[0078] Structural formulae of labeling precursors of the invention are listed below:

##STR00032##

##STR00033##

##STR00034##

##STR00035##

##STR00036##

##STR00037##

##STR00038##

##STR00039##

##STR00040##

##STR00041##

Example 1: Squaric Acid as Affinity Promoter for Bisphosphonates

[0079] The inventors have found that, surprisingly, squaric acid as a component of a linker of a bisphosphonate targeting vector increases affinity for hydroxyapatite in bone tissue. This beneficial effect is demonstrated by the adsorption therms of conjugates of the chelator NODAGA with squaric acid pamidronate (NODAGA.QS.Pam) and NODAGA with zoledronate (NODAGA.Zol). For this purpose, the adsorption therms are determined by the method of Langmuir and Freundlich.

[0080] For comparison, schemes 23 and 24 show conjugates of the chelator NODAGA with squaric acid pamidronate (NODAGA.QS.Pam) and NODAGA with zoledronate (NODAGA.Zol), and also the respective coefficients of adsorption K.sub.LF, measured by the method of Langmuir and Freundlich.

##STR00042##

##STR00043##

[0081] The coefficient of adsorption KLF of the conjugate NODAGA.QS.Pam is about three times greater than that of NODAGA.Zol, which contains an imidazole radical instead of a squaric acid group. It is immediately apparent from this that squaric acid significantly increases the affinity of the bisphosphonate group for bone tissue.

[0082] Moreover, in vivo PET (positron emission tomography) studies on young, healthy Wistar rats using the radiotracer [.sup.68Ga]Ga-NODAGA.QS.Pam (cf. FIG. 2) show high accumulation in the epiphyses, which in young animalsanalogously to bone metastasesare characterized by rapid renewal and remodeling of bone tissue.

[0083] Compared to published SUVs (Standardized Uptake Value, https://de.wikipedia.org/wiki/SUV_(Nuklearmedizin)) for the PET radiolabel [.sup.68Ga]Ga-DOTA.Zol with SUV.sub.epiphysis=17.4, it is possible with [.sup.68Ga]NODAGA.QS.Pam to achieve a noticeably increased value of SUV.sub.epiphysis=22.9 (cf. FIG. 3).

[0084] In addition, renal excretion of [.sup.68Ga]NODAGA.QS.Pam (% ID.sub.renal=40+4.60 min p.i.) is faster than for [.sup.68Ga]Ga-DOTA.Zol (% ID.sub.renal=33+17, 60 min p.i.). For [.sup.68Ga]Ga-NODAGA.QS.Pamas shown in FIG. 3this results in a higher epiphysis to blood ratio of 299.1, compared to 30.3 for [.sup.68Ga]Ga-DOTA.Zol, and correspondingly better PET image contrast (or signal-to-noise ratio).

Example 2: Synthesis of the KuE Unit

[0085] Synthesized as targeting vector for PSMA is, for example, the PSMA inhibitor L-lysine-urea-L-glutamate (KuE) using a known method according to Bene?ov? et al. (Linker Modification Strategies To Control the Prostate-Specific Membrane Antigen (PSMA)-Targeting and Pharmacokinetic Properties of DOTA-Conjugated PSMA Inhibitors; J Med Chem, 2016, 59 (5), 1761-1775) (cf. scheme 25). Lysine bound to a solid phase, especially to a polymer resin, and protected with tert-butyloxycarbonyl (tert-butyl) is reacted here with doubly tert-butyl-protected glutamic acid. After activation of the protected glutamic acid by triphosgene and coupling to the lysine bound to the solid phase, L-lysine-urea-L-glutamate (KuE) is eliminated using TFA and at the same time completely deprotected. The product can then be separated from free lysine by semipreparative HPLC with a yield of 71%.

##STR00044##

##STR00045##

[0086] The KuE squaric acid monoester obtained in this way is storable and can be used as a building block for further syntheses.

Example 3: Solid Phase-Based Synthesis of the Kue Unit and of the PSMA-617 Linker

[0087] The glutamate-urea-lysine binding motif KuE is conjugated with an aromatic linker unit by a method developed by Bene?ov? et al. (Linker Modification Strategies To Control the Prostate-Specific Membrane Antigen (PSMA)-Targeting and Pharmacokinetic Properties of DOTA-Conjugated PSMA Inhibitors; J Med Chem, 2016, 59 (5), 1761-1775). The synthesis reported by Bene?ov? et al. was modified slightly (cf. scheme 27).

##STR00046##

##STR00047##

##STR00048##

##STR00049##

Example 4: Synthesis of the Labeling Precursor Pam.QS.DOTAGA.KuE-617

[0088] First, the DOTAGA substructure is synthesized. This was done with a yield of 74%.

##STR00050##

[0089] The synthesis proceeds from the commercially available DO2A(tBu)-GABz, functionalized on the secondary amine with a Boc-protected amino group.

[0090] The benzyl protecting group of the glutaric acid side chain of DOTAGA(COOtBu).sub.3(NHBoc)-GABz (4) is reductively removed to allow coupling to the targeting vector.

[0091] The PSMA-617 linker is then coupled to the chelator (5) via amide coupling.

##STR00051##

[0092] The coupling of the chelator (5) to the KuE-bound linker is described in scheme 29. The protected PSMA-617 derivative (6) obtained by the amide coupling is deprotected using trifluoroacetic acid (TFA) and detached from the solid phase. The overall yield of the two-stage synthesis was 6% after HPLC purification.

[0093] In the last step, the pamidronate-squaric acid unit is synthesized and coupled to compound (7) (scheme 30). Proceeding from B-alanine, pamidronate (8) is first prepared and coupled to squaric diester in aqueous phosphate buffer with a pH of 7. The conjugation of the pamidronate-squaric acid group (9) with DOTAGA.KuE-617 (7) is effected in aqueous phosphate buffer with a pH of 9 (cf. scheme 30). The inventive labeling precursor Pam.QS.DOTAGA.KuE-617 (10) is obtained with a yield of 49% after HPLC purification.

##STR00052## ##STR00053##

Example 5: Synthesis of the Labeling Precursor DOTA.L-Lys(SA.Pam)KuE-617

[0094] The synthesis of the labeling precursor DOTA.L-Lys(SA.Pam)KuE-617 shown in scheme 17 is represented in scheme 31. First, the first targeting vector KuE bound to the solid phase and the aromatic linker conjugated thereto (structure (11) in scheme 31) are synthesized in the same way as shown in scheme 27. Then orthogonally protected lysine is conjugated to the linker as bridging unit X. Fmoc deprotection (structure (12)) is followed by coupling with DOTA-tris(tert-butyl ester), in each case using HATU as reagent for amide formation, to obtain the compound according to structural formula (13). Subsequently, compound (13) is fully deprotected in TFA/DCM and decoupled from the solid phase in order to obtain compound (14). Finally, the second targeting vector (9) consisting of pamidronate and squaric acid is conjugated to compound (14). The second targeting vector is previously synthesized in the same manner as shown in scheme 30.

##STR00054## ##STR00055## ##STR00056##

Example 6: In Vitro Study of the Compounds NH.SUB.2..DOTAGA.KuE-617, NH.SUB.2..DOTAGA.QS.KuE and Pam.SA.DOTAGA.KuE-617

[0095] Using a cell-based assay, the affinity of the KuE targeting vector with a lipophilic linkeranalogously to PSMA-617and with a squaric acid linker was examined using the compounds NH.sub.2.DOTAGA.KuE-617 and NH.sub.2.DOTAGA.QS.KuE (structural formulae (8) and (10) in scheme 32).

##STR00057##

[0096] For the assay, LNCaP cells were pipetted into multiwell plates (Merck Millipore Multiscreen?). To the compounds to be analyzed, in increasing concentrations, was in each case added a defined amount or concentration of the reference compound .sup.68Ga[Ga]PSMA-with known K.sub.dvalue, and the mixture was incubated in the wells with the LNCaP cells for 45 min. After washing several times, the cell-bound activity was determined. The inhibition curves obtained were used to calculate the IC.sub.50 values and K.sub.i values shown in table 1.

TABLE-US-00001 TABLE 1 IC.sub.50 values Compound IC.sub.50 (nM) PSMA-617 15.1 ? 3.8 PSMA-11 26.1 ? 1.2 NH.sub.2.DOTAGA.KuE-617 20.6 ? 3.4 NH.sub.2.DOTAGA.QS.KuE 20.2 ? 3.5 Pam.SA.DOTAGA.KuE-617 49.8 ? 10

[0097] In order to determine non-specific binding, an excess of the PSMA inhibitor 2-PMPA (2-(phosphonomethyl)pentanoic acid) was additionally added to all compounds, and they were subjected to the same LNCaP assay-as described above.

[0098] The affinities of the squaric acid-containing compound NH.sub.2.DOTAGA.QS.KuE (16) and NH.sub.2.DOTAGA. KuE-617 (7) are virtually the same and correspond roughly to those of the established compounds PSMA-617 and PSMA-11.

[0099] FIG. 4 illustrates the interaction of the QS.KuE group with the binding pocket of PSMA.

[0100] The complexity involved in the synthesis of the squaric acid-containing compound NH.sub.2.DOTAGA.QS.KuE (10) is considerably lower compared to the other compounds. The use of squaric acid as a linker between the targeting vector KuE and a chelator additionally opens up a simple means of quantitatively synthesizing more complex labeling precursors with two different targeting vectorsin the present case KuE and a bisphosphonate.

[0101] Furthermore, the PSMA affinity of the final labeling precursor Pam.SA.DOTAGA.KuE-617 (10) was determined. The IC.sub.50 is 49.8?10 nM.

Example 7: Radiochemical Analysis of [.SUP.117.Lu]Lu-DOTA.L-Lys(SA.Pam)KuE-617

[0102] At a temperature of 95? C. , the labeling precursor DOTA.L-Lys(SA.Pam)KuE-617 (see scheme 17 and structural formula (15) in scheme 31) is labeled with .sup.177Lu in 1 ml of an aqueous ammonium acetate buffer solution (1 M, pH 5.5). The radiochemical yield (RCY) as a function of the amount of the labeling precursor present in the ammonium acetate buffer solution (5, and 30 nmol) is shown in FIG. 5. For an amount of the labeling precursor of ?10 nmol, the radiochemical yield (RCY) reaches a value of ?90% after 5 min. In contrast, with an amount of 5 nmol of labeling precursor, the yield after 5 min is only 75% and later reaches a plateau value of 85%. Radiochemical yield and purity are determined by radio thin-layer chromatography (radio-TLC) and radio high-pressure liquid chromatography (radio-HPLC). Radio thin-layer chromatography for the radiotracer [.sup.117Lu]Lu-DOTA.L-Lys(SA.Pam) KuE-617 gives an R.sub.f of 0.0. In contrast, unbound [.sup.177Lu]Lu.sup.3+ in citrate buffer as mobile phase gives an R.sub.f of 0.8 to 1.0. In analytical radio high-pressure liquid chromatography, for the radiotracer, a retention time t.sub.R of 9.8 min is measured.

[0103] FIG. 6 shows measurements of the stability of the radiotracer [.sup.117Lu]Lu-DOTA.L-Lys(SA.Pam)KuE-617 in phosphate-buffered saline (PBS), isotonic saline (NaCl) and human serum (HS). Even after 14 days in PBS and isotonic saline (NaCl), ?98% of the radiotracer [.sup.117Lu]Lu-DOTA.L-Lys(SA.Pam)KuE-617 is present unchanged in complexed form. In human serum (HS), stability is slightly lower at 93% after 9 days, with stability remaining at 93% after 14 days.

[0104] The lipophilicity of [.sup.117Lu]Lu-DOTA.L-Lys(SA.Pam)KuE-617 is determined by determining the partition equilibrium of the compound in a mixture of n-octanol and PBS. Measurements for the logD.sub.7.4 coefficients of [.sup.117Lu]Lu-DOTA.L-Lys(SA.Pam)KuE-617 and [.sup.117Lu]Lu-PSMA-617 are presented in table 2. The results show that [.sup.117Lu]Lu-DOTA.L-Lys(SA.Pam) KuE-617 has virtually the same lipophilicity as PSMA-617.

TABLE-US-00002 TABLE 2 Radiotracer lipophilicity Radiotracer LogD.sub.7,4 (n-octanol/PBS) [.sup.117Lu]Lu-DOTA.L-Lys(SA.Pam)KuE-617 ?2.3 ? 0.12 [.sup.177Lu]Lu-PSMA-617 ?2.2 ? 0.20

Example 8: Affinity for Hydroxyapatite (HAP)

[0105] Calcium-containing crystalline hydroxyapatite is an essential component of mammalian bones and is suitable as a model substrate for the in vitro study of bisphosphonate accumulation in healthy bone tissue and bone metastases. FIG. 7 shows measurements for the accumulation of [.sup.117Lu]Lu-DOTA.L-Lys(SA.Pam)KuE-617, [117Lu]Lu-PSMA-617 and [.sup.117Lu]Lu.sup.3+ on ordinary HAP and on HAP pretreated or blocked with pamidronate, with free [.sup.117Lu]Lu.sup.3+ being known to have a high affinity for HAP and serving as a reference. The proportion of the radiotracer [.sup.117Lu]Lu-DOTA.L-Lys(SA.Pam)KuE-617 bound to HAP is 98.2%, slightly below the value measured for free [.sup.117Lu]Lu.sup.3+ of 99.9%. In contrast, [.sup.117Lu]Lu-PSMA-617 is found to have an HAP-enriched fraction of only 1.2%. In order to determine selectivity, accumulation on HAP previously treated with excess pamidronate was additionally measured. This gives values of 7.3% for [.sup.117Lu]Lu-DOTA.L-Lys(SA.Pam)KuE-617 and 4.9% for free [.sup.117Lu]Lu.sup.3, which demonstrate high selectivity for HAP.

Example 9: In Vitro Affinity for PSMA

[0106] By means of comparative radioligand assays, binding affinity for PSMA is determined for the radiotracer or labeling precursor DOTA.L-Lys(SA.Pam)KuE-617 and reference structures. Corresponding measurements for the inhibition constant K.sub.i are given in table 3. The K.sub.i-value for [.sup.natLu]Lu-DOTA.L-Lys(SA.Pam)KuE-617 corresponds roughly to the value for the labeling precursor DOTA.L-Lys(SA.Pam) KuE-617. It can be seen from this that that complexation with lutetium does not adversely affect binding affinity for PSMA. Compared to DOTA.L-Lys.KuE-617corresponding to structural formula (14) in scheme 31the K.sub.i of [.sup.natLu]Lu-DOTA.L-Lys(SA.Pam)KuE-617 is greater by a factor of about 2. It can be seen from this that the squaric acid-pamidronate group reduces affinity for PSMA.

TABLE-US-00003 TABLE 3 Affinity for PSMA Labeling precursor/compound PSMA K.sub.i [nM] DOTA.L-Lys(SA.Pam)KuE-617 53 ? 4 [.sup.natLu]Lu-DOTA.L-Lys(SA.Pam)KuE-617 42 ? 8 DOTA.L-Lys.KuE-617 20 ? 3 PSMA-617 7 ? 1

Example 10: Ex Vivo Studies

[0107] For the radiotracer [.sup.117Lu]Lu-DOTA.L-Lys(SA.Pam)KuE-617, organ accumulation was examined in Balb/c mice with induced LNCaP tumors. The results are shown in FIG. 8. Enrichment of the radiotracer [.sup.117Lu]Lu-DOTA.L-Lys(SA.Pam)KuE-617 in tumor and femur is comparable, with values of 4.2?0.7% ID/g and 3.4?0.4% ID/g respectively. In contrast to tumors, accumulation in bones cannot be blocked by administering the PSMA inhibitor PMPA (2-phosphonomethylpentanedioic acid). It can be seen from this that that uptake in the bone is not caused by PSMA, since no PSMA is expressed. With a value of 17?2% ID/g, the kidneys show high accumulation of [.sup.117Lu]Lu-DOTA.L-Lys(SA.Pam)KuE-617, which can likewise be greatly reduced by administration of PMPA. In contrast, accumulation in the liver and spleen is not PSMA-specific. The ratio of accumulations in tumors and the blood is exceptionally high with values of 210 and 170 respectively, and suggests minor hematological side effects.

Methods and Materials

General

[0108] All chemicals were sourced from Sigma-Aldrich, Merck, Fluka, AlfaAesar, VWR, AcrosOrganics, TCI, Iris Biotech or Fisher Scientific and used without additional purification. Dry solvents were sourced from Merck and VWR, deuterated solvents for NMR spectra from Deutero. PSMA-617 was purchased from Hycultec. Thin-layer chromatography was performed with Merck silica gel 60 F254-coated aluminum plates. Evaluation was effected by fluorescence absorbance at ?=254 nm and staining with potassium permanganate. The radio TLCs were evaluated with a Raytest CR-35 Bio Test Imager and the AIDA software (Raytest). The .sup.1H and .sup.13C NMR measurements were performed on a Bruker Avance III HD 300 spectrometer (300 MHz, 5 mm BBFO probe with z-gradient and ATM and BACS 60 sample changer), a Bruker Avance II 400 spectrometer (400 MHZ, 5 mm BBFO probe with z-gradient, ATM and SampleXPress 60 sample changer) and a Bruker Avance III 600 spectrometer (600 MHz, 5 mm TCI CryoProbe probe with z-gradient and ATM and SampleXPress Lite 16 sample changer). LC/MS measurements were performed on an Agilent Technologies 1220 Infinity LC system coupled to an Agilent Technologies 6130B Single Quadrupole LC/MS system. Semi-preparative HPLC purification was conducted on a Hitachi LaChrom series 7000 and with the conditions and columns mentioned in each case. For radioactive labeling experiments, [.sup.177Lu]LuCl.sub.3 in 0.04 M HCl, provided by ITM Garching, was used.

Organic Syntheses

Solid-Phase Synthesis of the PSMA Ligand (KuE-617 on Polystyrene Resin)

[0109] The synthesis of the glutamate-urea-lysine binding motif KuE and of the linker of the KuE-617 ligand is in accordance with established solid-phase peptide chemistry by a method proposed by Bene?ov? et al. (Bene?ov?, M.; Sch?fer, M.; Bauder-W?st, U.; Afshar-Oromieh, A.; Kratochwil, C.; Mier, W.; Haberkorn, U.; Kopka, K.; Eder, M. Preclinical Evaluation of a Tailor-Made DOTA-Conjugated PSMA Inhibitor with Optimized Linker Moiety for Imaging and Endoradiotherapy of Prostate Cancer. J. Nucl. Med. 2015, 56 (6), 914-920; Bene?ov?, M.; Bauder-W?st, U.; Sch?fer, M.; Klika, K. D.; Mier, W.; Haberkorn, U.; Kopka, K.; Eder, M. Linker Modification Strategies to Control the Prostate-Specific Membrane Antigen (PSMA)-Targeting and Pharmacokinetic Properties of DOTA-Conjugated PSMA Inhibitors. J.Med.Chem. 2016, 59 (5), 1761-1775) and slightly adapted methods. Bis(tert-butyl)-L-glutamate hydrochloride (4.5 g, 15.21 mmol) and DIPEA (7.98 g, 10.5 ml, 61.74 mmol) are dissolved in dry dichloromethane (200 ml), and cooled to 0? C. Triphosgene (1.56 g, 5.26 mmol) in dichloromethane (30 ml) is added dropwise over a period of 4.5 h. After the addition is complete, the solution is stirred for a further hour. The Fmoc protecting group of Fmoc-L-lysine(Alloc)-Wang resin (1.65 g, 1.5 mmol, 0.9 mmol/g) is removed by stirring it in a piperidine/DMF solution (1:1) for 15 minutes, followed by a wash step with dichloromethane. The deprotected L-lysine (Alloc)-Wang resin is added to the previously prepared solution and stirred at room temperature overnight. The resin is washed with dichloromethane (15 ml) and used without further purification. Tetrakis(triphenylphosphine)palladium (516 mg, 0.45 mmol) and morpholine (3.92 g, 3.92 ml, 45 mmol) are dissolved in dichloromethane (12 ml) and added. The solution is stirred for 24 h in the dark. It is then washed with dichloromethane (15 ml), 1% DIPEA solution in DMF (3?13 ml) and sodium diethyldithiocarbamate trihydrate solution (15 mg/ml) in DMF (9?10.5 ml?5 minutes) to obtain resin-bound and Alloc-deprotected glutamate-urea-lysine conjugate. Fmoc-3-(2-naphthyl)-L-alanine (1.75 g, 4.00 mmol), HATU (1.52 g, 4.00 mmol), HOBt (540 mg, 4 mmol) and DIPEA (780 mg, 1.02 ml, 6.03 mmol) are dissolved in dry DMF (10 ml) and added to the resin. The solution is stirred overnight and then washed with DMF (10 ml) and dichloromethane (10 ml). To remove the Fmoc group, the resin is stirred in a piperidine/DMF solution (1:1, 3?11 ml) for 10 minutes each time and washed with DMF (10 ml) and dichloromethane (10 ml). Fmoc-4-Amc-OH (1.52 g, 4 mmol), HATU (1.52 g, 4 mmol), HOBt (540 mg, 4 mmol) and DIPEA (780 mg, 1.02 ml, 6.03 mmol) are added to the resin in dry DMF (10 ml). The solution is stirred for two days and then washed with DMF (10 ml) and dichloromethane (10 ml). To remove the Fmoc group, the reaction solution is stirred in a piperidine/DMF solution (1:1, 11 ml) for 10 min each time and washed with DMF (10 ml) and dichloromethane (10 ml) to obtain the resin-bound KuE-617 ligand.

Pamidronate Synthesis

[0110] ?-Alanine (1.5 g, 0.017 mol) and phosphoric acid (2.76 g, 0.034 mol) are dissolved in sulfolane (5.5 ml) and cooled to 0? C. Phosphorus trichloride (4.62 g, 2.95 ml, 0.034 mmol) is added dropwise. The solution is stirred at 75? C. for 3 h. Water (15 ml) is added and the mixture is stirred at 100? C. for 12 h. Finally, ethanol (15 ml) is added and, after crystallization at 0? C. for 3 days, the pamidronate product (1.48 g, 0.006 mol, 37%) is obtained as a yellow solid.

[0111] .sup.1H NMR (300 MHZ, D.sub.2O): [?ppm]=3.34 (t, J=7.1 Hz, 2H), 2.31 (tt, J=13,7, 7.1 Hz, 2H).

[0112] .sup.13C NMR (400 MHz, D.sub.2O): [?ppm]=72.58; 36.14; 30.54.

[0113] .sup.13P NMR (121.5 MHZ, D.sub.2O): [?ppm]=17.58 (s, 2P).

[0114] MS (ESI): 236.0 [M+H].sup.+, calculated for C.sub.3H.sub.11NO.sub.7P.sub.2: 235.07 [M].sup.+.

Synthesis of Pamidronate-Ethyl Squarate

[0115] Pamidronate (500 mg, 2.13 mmol) is dissolved in phosphate buffer (0.5 M, pH 7, 5 ml). 3,4-Diethoxycyclobut-3-ene-1,2-dione (diethyl squarate, SADE, 542 mg, 468 ?l, 3.2 mmol) is added and the mixture is stirred at room temperature for 2 days. Ethanol (3 ml) is added for crystallization. The mixture is left in the freezer for 3 days to complete crystallization. The white precipitate is washed with cold ethanol and the pamidronate-ethyl squarate product (0.58 g, 1.62 mol, 76%) is obtained as a white solid.

[0116] .sup.1H NMR (400 MHZ, D2O): [?ppm]=4.79-4.62 (m, 2H), 3.31 (t, J=6.6 Hz, 2H), 2.32-2.15 (m, 2H), 1.42 (dt, J=11.7, 7.2 Hz, 3H).

[0117] .sup.31P NMR (162 MHZ, D2O): [?ppm]=17.92 (s), 2.26 (s).

[0118] MS (ESI): 360.0 [M+H].sup.+, 720.0 2[M+H].sup.+, 763.0 2[M+Na].sup.+, calculated for C.sub.9H.sub.15NO.sub.10P.sub.2: 359.16[M].sup.+.

Fmoc-L-Lys(Boc)-KuE-617 Resin

[0119] Fmoc-L-Lys(Boc)-OH (506 mg, 0.0011 mmol), HATU (415 mg, 0.0011 mg), HOBt (146 mg, 0.0011 mmol) and DIPEA (277 ?l, 211 mg, 0.00162 mmol) are dissolved in acetonitrile (4 ml) and stirred for 30 min. The KuE-617 resin (300 mg, 0.0027 mmol, 0.09 mmol/g) is added and the mixture is stirred at room temperature for 1 day. The resin is mixed with acetonitrile (10 ml) and dichloromethane (10 ml), and kept ready for subsequent synthesis steps.

L-Lys(Boc)-KuE-617 Resin

[0120] The Fmoc-L-Lys(Boc)-KuE-617 resin is stirred in a mixture of DMF and piperidine (1:1, 6 ml) for one hour. The Fmoc-deprotected resin is washed with DMF (10 ml) and dichloromethane (10 ml) and used in the next step without further purification.

DOTA(tBu)3-L-Lys(Boc)-KuE-617 Resin

[0121] DOTA-tris(tert-butyl ester) (310 mg, 0.54 ?mol), HATU (308 mg, 0.00081 mmol), HOBt (110 mg, 0.00081 mmol) and DIPEA (184 ?l, 140 mg, 0.0011 mmol) are dissolved in acetonitrile (4 ml) and stirred for 30 min. L-Lys(Boc)-KuE-617 resin (461 mg, 0.00027 mmol, 0.9 mmol/g) is added and the mixture is stirred at room temperature for one day. The resin is washed with acetonitrile (10 ml) and dichloromethane (10 ml), and used in the next step without further purification.

DOTA-L-Lys-KuE-617

[0122] DOTA(tBu)3-L-Lys(Boc)-KuE-617 resin (536 mg, 0.00027 mmol, 0.9 mmol/g) is stirred in a solution of TFA and dichloromethane (1:1, 4 ml). The TFA/dichloromethane solution is concentrated under reduced pressure, and the product (10.6 mg, 0.0091 mmol, 4%) is obtained as a colorless powder after semipreparative HPLC purification (column: LiChrospher 100 RP18 EC (250?10 mm) 5 m, flow rate: 5 ml/min, H.sub.2O/MeCN+0.1% TFA, 25% MeCN isocratic, tR=10.3 min).

[0123] MS (ESI): 1172.5 [M+2H].sup.+, 585.9 1/2[M+2H].sup.+, 391.0 1/3[M+2H].sup.+, calculated for C.sub.55H.sub.83N.sub.11O.sub.17: 1170.33 [M].sup.+.

DOTA-L-Lys(SA.Pam)-KuE-617

[0124] Compound (14) from scheme 31 (10 mg, 0.0085 mmol) and pamidronate-ethyl squarate (16 mg, 0.043 mmol) are dissolved in phosphate buffer (0.5 M, pH 9, 1 ml) and stirred for 2 days. The DOTA-L-Lys(SA.Pam)-KuE-617 product (10.56 mg, 0.0071 mmol, 84%) is obtained as a colorless powder after semipreparative HPLC purification (column: LiChrospher 100 RP18 EC (250?10 mm) 5 m, flow rate: 5 ml/min, H.sub.2O/MeCN+0.1% TFA, 23% to 28% MeCN in 20 min, tR=8.2 min).

[0125] MS (ESI): 511.3 1/3[M+H+2Na].sup.+, 520.0 [1/3M+2K].sup.+, 781.0 1/2[M+2K].sup.+, calculated for C.sub.62H.sub.92N.sub.12O.sub.26P.sub.2: 1483.42 [M].sup.+.

Radiolabeling of DOTA-L-Lys(SA.Pam)-KuE-617 with lutetium-177

[0126] For radioactive labeling, [.sup.177Lu]LuCl.sub.3 in 0.04 M HCl (ITG, Garching, Germany) is used. Radiolabeling is performed in 1 ml of 1 M ammonium acetate buffer at pH 5.5. Reactions are performed with different amounts of precursor (5, 10 and 30 nmol) and at 95? C. with 40-50 MBq n.c.a. lutetium-177. The reaction was monitored using radio thin-layer chromatography (TLC silica gel 60 F254 from Merck) and citrate buffer (pH 4) as mobile phase and high-pressure liquid chromatography using a HPLC 7000 Hitachi LaChrom analytical instrument (column: Merck Chromolith? RP-18e, 5-95% MeCN (0.1% TFA)/95-5% water (0.1% TFA) in 10 min). Radio thin-layer chromatography samples are measured and evaluated with the TLC Imager CR-35 Bio Test Imager from Elysia-Raytest (Straubenhardt, Germany) with AIDA software.

In Vitro Stability Study

[0127] Stability studies of .sup.177Lu-labeled compounds are performed in human serum (HS, AB human male plasma, USA origin, Sigma-Aldrich) and phosphate-buffered saline (Sigma-Aldrich). 5 MBq of the radioactive compound is incubated in 0.5 ml of the medium for 14 days. Aliquots are taken at different times (1 h, 2 h, 5 h, 1 d, 2 d, 5 d, 7 d, 9 d and 14 d) to determine the radiochemical stability. Each measurement is carried out in triplicate.

Determination of Lipophilicity

[0128] LogD.sub.7.4 of the respective compound is determined via the partition coefficient in n-octanol and PBS. The labeling solution is adjusted to pH 7.4 and 5 MBq is diluted in 700 ?l of n-octanol and 700 ?l of PBS. It is shaken at 1500 rpm for 2 min and then centrifuged. 400 ?l of the n-octanol phase and 400 ?l of the PBS phase were each transferred to a new Eppendorf tube. 3-6 ?l is then pipetted onto a TLC plate and analyzed using a phosphor imager. The logD.sub.7.4 is calculated based on the ratio of the activities of the two phases. The measurement of each phase is also repeated twice more with the sample of higher activity, such that three logD.sub.7.4 values can be obtained and an average can be calculated.

Measurement of Hydroxyapatite Affinity of .SUP.177.Lu-Labeled Compounds

[0129] Hydroxyapatite (20 mg) is incubated in saline (1 ml) for 24 h. 50 ?l of the radiotracer [.sup.177Lu]Lu-DOTA-L-Lys(SA.Pam)-KuE-617 (5 MBq) or [.sup.177Lu]Lu-PSMA-617 (5 MBq) is added. Each suspension is vortexed with a vortex mixer for 20 s and incubated at room temperature for 1 h. Each suspension is then passed through a filter (CHROMAFIL? Xtra PTFE-45/13), and the supernatant is washed with water (500 ?l). The radioactivity of the liquids and HAP-containing supernatants obtained is measured in each case with a curiemeter (Isomed 2010 activimeter, MED Nuclear-Medizintechnik Dresden GmbH). The binding of [.sup.177Lu]Lu-DOTA-L-Lys(SA.Pam)-KuE-617 and [.sup.177Lu]Lu-PSMA-617 is determined as a percentage of the activity absorbed on HAP. As a reference, the HAP binding of free Lu-177 is measured in an analogous manner. Comparative measurements are carried out on blocked hydroxyapatite in an analogous manner. For this purpose, HAP (20 mg) in saline solution (1 ml) is incubated with pamidronate (100 mg) and the respective activities of [.sup.177Lu]Lu-DOTA-L-Lys(SA.Pam)-KuE-617 and free Lu-177 are determined.

In Vitro Study of PSMA Binding Affinity

[0130] Non-active (cold) [natLu]Lu complexes are prepared by shaking a solution containing the labeling precursor DOTA-L-Lys(SA.Pam)-KuE-617 (371 ?l, 1 mg/ml, 250 nmol) with LuCl.sub.3 (129 ?l, 1 mg/ml, 375 nmol, metal to labeling precursor ratio 1.5:1) in 1 M ammonium acetate buffer at 95? C. for 2 hours. Complex formation is monitored by ESI-LC/MS.

[0131] PSMA binding affinity is determined by the competitive radioligand assay described by Bene?ov? et al. (Bene?ov?, M.; Schaefer, M.; Bauder-W?st, U.; Afshar-Oromieh, A.; Kratochwil, C.; Mier, W.; Haberkorn, U.; Kopka, K.; Eder, M. Preclinical Evaluation of a Tailor-Made DOTA-Conjugated PSMA Inhibitor with Optimized Linker Moiety for Imaging and Endoradiotherapy of Prostate Cancer. J. Nucl. Med. 2015, 56 (6), 914-920.). For this purpose, PSMA-positive LNCaP cells from Sigma-Aldrich in RPMI 1640 (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific), 100 ?g/ml streptomycin and 100 units/ml penicillin are cultured at 37? C. in 5% CO.sub.2. The LNCaP cells are incubated with increasing concentrations of solutions containing the labeling precursors in the presence of 0.75 nM [.sup.68Ga]Ga-PSMA-10 for 45 min. Free radioactivity is removed by several washes with ice-cold PBS. The samples obtained are measured in a y counter (2480 WIZARD2 Automatic Gamma Counter, PerkinElmer). The measurement data are evaluated in GraphPad Prism 9 using non-linear regression.

Ex Vivo Studies

[0132] All animal experiments were approved by the ethics committee of the state of Rhineland-Palatinate (according to ? 8 para. 1 Tierschutzgesetz [Animal Protection Act], Landesuntersuchungsamt [State Investigation Office]) and carried out in accordance with the relevant federal laws and institutional guidelines (approval no. 23 177-07/G 21-1-022). 6- to 8-week-old BALB/cAnNRj males (Janvier Labs) were inoculated subcutaneously with 5?10.sup.6 LNCaP cells in 200 ?l 1:1 (v/v) Matrigel/PBS (Corning?). Measurements were conducted after the tumor reached a volume of about 100 cm.sup.3. Before intravenous injection of 0.5 nmol [.sup.177Lu]Lu-DOTA-L-Lys(SA.Pam)-KuE-617, the LNCaP tumor-bearing mice were anesthetized with 2% isoflurane. The specific activity was about 3 MBq/nmol. PSMA selectivity was examined by coinjecting 1.5 mmol of PMPA per mouse. The animals were sacrificed 24 h p.i. The organs were collected and weighed. Radioactivity was measured and calculated as a decay-corrected percentage of injected dose per gram of tissue mass % ID/g.