PSMA LIGANDS FOR IMAGING AND ENDORADIOTHERAPY

Abstract

The present invention relates to compounds which bind and/or inhibit prostate-specific membrane antigen (PSMA) comprising at least one group electron dense substituent (EDS), and at least one moiety which is amenable to radiolabeling; and therapeutic and diagnostic uses thereof.

Claims

1. A compound of formula (I), or a pharmaceutically acceptable salt thereof, ##STR00085## wherein: m is an integer of 2 to 6; n is an integer of 2 to 6; R.sup.1L is CH.sub.2, NH or O; R.sup.2L is C or P(OH); R.sup.3L is CH.sub.2, NH or O; X.sup.1 is selected from an amide bond, an ether bond, a thioether bond, an ester bond, a thioester bond, a urea bridge, and an amine bond; L.sup.1 is a divalent linking group with a structure selected from an oligoamide, an oligoether, an oligothioether, an oligoester, an oligothioester, an oligourea, an oligo(ether-amide), an oligo(thioether-amide), an oligo(ester-amide), an oligo(thioester-amide), oligo(urea-amide), an oligo(ether-thioether), an oligo(ether-ester), an oligo(ether-thioester), an oligo(ether-urea), an oligo(thioether-ester), an oligo(thioether-thioester), an oligo(thioether-urea), an oligo(ester-thioester), an oligo(ester-urea), and an oligo(thioester-urea), which linking group may carry a group EDS; X.sup.2 is selected from an amide bond, an ether bond, a thioether bond, an ester bond, a thioester bond, a urea bridge, and an amine bond; R.sup.2 is an optionally substituted aryl group or an optionally substituted aralkyl group, which aryl group or aralkyl group may be substituted on its aromatic ring with one or more substituents selected from halogen and —OH; R.sup.3 is an optionally substituted aryl group or an optionally substituted aralkyl group, which aryl group or aralkyl group may be substituted on its aromatic ring with one or more substituents selected from halogen and —OH; r is 0 or 1; p is 0 or 1; q is 0 or 1; R.sup.4 is selected from an optionally substituted aryl group and a group EDS, which aryl group may be substituted on its aromatic ring with one or more substituents selected from halogen, —OH and —NH.sub.2; X.sup.3 is selected from an amide bond, an ether bond, a thioether bond, an ester bond, a thioester bond, a urea bridge, an amine bond, and a group of the formula ##STR00086## wherein the marked bond at the carbonyl group attaches X.sup.3 to R.sup.M and the other marked bond attaches X.sup.3 to the remainder of the compound of formula (I); R.sup.M is a marker group which comprises a chelating group optionally containing a chelated non-radioactive or radioactive cation; and wherein the group EDS is contained at least once in the compound of formula (I) and has a structure selected from (E-1A), (E-1B), (E-2A) and (E-2B): ##STR00087## wherein marks the bond which attaches the group EDS, to the remainder of the compound of formula (I); s is 1, 2 or 3, preferably 1 or 2, and more preferably 1; t is 1, 2 or 3, preferably 1 or 2, and more preferably 2; R.sup.5A is, independently for each occurrence for s>1, an electron withdrawing substituent, which is preferably selected from —NO.sub.2 and —COOH, and which is more preferably —COOH, and wherein the bond between R.sup.5A and the phenyl ring indicates that the s groups R.sup.5A replace s hydrogen atoms at any position on the phenyl ring; R.sup.5B is, independently for each occurrence for s>1, a substituent carrying an electron lone pair at the atom directly attached to the phenyl ring shown in formula (E-1B), which substituent is preferably selected from —OH and —NH.sub.2, and which is more preferably —NH.sub.2, and wherein the bond between R.sup.5B and the phenyl ring indicates that the s groups R.sup.5B replace s hydrogen atoms at any position on the phenyl ring; R.sup.6A is, independently for each occurrence for t>1, an electron withdrawing substituent, which is preferably selected from —NO.sub.2 and —COOH, and which is more preferably —COOH, and wherein the bond between R.sup.6A and the phenyl ring indicates that the t groups R.sup.6A replace t hydrogen atoms at any position on the phenyl ring; and R.sup.6B is, independently for each occurrence for t>1, a substituent carrying an electron lone pair at the atom directly attached to the phenyl ring shown in formula (E-1B), which substituent is preferably selected from —OH and —NH.sub.2, and which is more preferably —OH, and wherein the bond between R.sup.6B and the phenyl ring indicates that the t groups R.sup.6B replace t hydrogen atoms at any position on the phenyl ring.

2. The compound or salt of claim 1, wherein m is 2, n is 2 or 4, R.sup.1L is NH, R.sup.2L is C, and R.sup.3L is NH.

3. The compound or salt of claim 1, wherein L.sup.1 is a divalent linking group with a structure selected from an oligoamide which comprises a total of 1 to 5, more preferably a total of 1 to 3, and most preferably a total of 1 or 2 amide bonds within its backbone, and an oligo(ester-amide) which comprises a total of 2 to 5, more preferably a total of 2 to 3, and most preferably a total of 2 amide and ester bonds within its backbone, which linking group may carry a group EDS.

4. The compound or salt of claim 2, wherein the moiety —X.sup.2-L.sup.1-X.sup.1— in formula (I) has a structure selected from:
*—C(O)—NH—R.sup.7—NH—C(O)—R.sup.8—C(O)—NH—  (L-1),
*—C(O)—NH—R.sup.9A—NH—C(O)—R.sup.10A—C(O)—NH—R.sup.11A—NH—C(O)—  (L-2A), and
*—C(O)—NH—R.sup.9B—C(O)—NH—R.sup.10B—C(O)—NH—R.sup.11B—NH—C(O)—  (L-2B); wherein the amide bond marked with * is attached to the carbon atom carrying R.sup.2 in formula (I), and wherein R.sup.7, R.sup.8, R.sup.9A, R.sup.9B, R.sup.11A and R.sup.11B are independently selected from optionally substituted C2 to C10 alkanediyl, which alkanediyl groups may each be substituted by one or more substituents independently selected from —OH, —OCH.sub.3, —COOH, —COOCH.sub.3, —NH.sub.2, —NHC(NH)NH.sub.2, and a group EDS, and R.sup.10A and R.sup.10B are selected from optionally substituted C2 to C10 alkanediyl, and optionally substituted C6 to C10 arenediyl, which alkanediyl and arenediyl group may each be substituted by one or more substituents independently selected from —OH, —OCH.sub.3, —COOH, —COOCH.sub.3, —NH.sub.2, —NHC(NH)NH.sub.2, and a group EDS.

5. The compound or salt of claim 4, wherein the moiety —X.sup.2-L.sup.1-X.sup.1— has a structure selected from:
*—C(O)—NH—CH(COOH)—R.sup.12—NH—C(O)—R.sup.13—C(O)—NH—  (L-3),
*—C(O)—NH—CH(COOH)—R.sup.14—NH—C(O)—R.sup.15—C(O)—NH—R.sup.16—CH(COOH)—NH—C(O)—   (L-4), and
*—C(O)—NH—CH(COOH)—R.sup.17—C(O)—NH—R.sup.18—C(O)—NH—R.sup.19—CH(COOH)—NH—C(O)—   (L-5); wherein the bond marked with * is attached to the carbon atom carrying R.sup.2 in formula (I), R.sup.12 and R.sup.14 are independently selected from linear C2 to C6 alkanediyl, R.sup.13 is a linear C2 to C10 alkanediyl, R.sup.15 and R.sup.16 are independently selected from linear C2 to C6 alkanediyl, and wherein each of R.sup.13 and R.sup.15 may carry one group EDS as a substituent, R.sup.17 is a linear C2 to C6 alkanediyl, R.sup.18 is a phenylene group, and R.sup.19 is a linear C2 to C6 alkanediyl.

6. The compound or salt of claim 1, wherein R.sup.2 is an optionally substituted aralkyl group selected from optionally substituted —CH.sub.2-phenyl and optionally substituted —CH.sub.2-naphthyl, wherein the phenyl and the naphthyl group are optionally substituted with a substituent selected from halogen, preferably I, and —OH.

7. The compound or salt of claim 1, wherein R.sup.3 is an optionally substituted aralkyl group selected from optionally substituted —CH.sub.2-phenyl and optionally substituted —CH.sub.2-naphthyl, wherein the phenyl and the naphthyl group are optionally substituted with a substituent selected from halogen and —OH.

8. The compound or salt of claim 1, wherein r is 1, and wherein R.sup.4 is selected from optionally substituted phenyl, optionally substituted naphthyl, and a group EDS, which phenyl group and naphthyl group are optionally substituted with a substituent selected from halogen, —OH and —NH.sub.2.

9. The compound or salt of claim 1, wherein X.sup.3 is an amide bond or a group of the formula ##STR00088## wherein the marked bond at the carbonyl group attaches X.sup.3 to R.sup.M and the other marked bond attaches X.sup.3 to the remainder of the molecule.

10. The compound or salt of claim 1, wherein R.sup.M is a chelating group optionally containing a chelated non-radioactive or radioactive cation.

11. The compound or salt of claim 1, wherein the compound of formula (I) either contains one group EDS which is carried by the linking group L.sup.1, or contains two groups EDS, one being represented by R.sup.4 and one being carried by L.sup.1.

12. The compound or salt of claim 1, which contains a group EDS which has the formula (E-2A): ##STR00089## wherein marks the bond which attaches the group EDS to the remainder of the compound of formula (I); and t is 1 or 2, and R.sup.6A is selected from —NO.sub.2 and —COOH.

13. (canceled)

14. A pharmaceutical or diagnostic composition comprising or consisting of one or more compounds or salts in accordance with claim 1.

15. (canceled)

16. A method for imaging or treating prostate-specific membrane antigen (PSMA)-associated cancer in a subject, comprising administering to the subject an effective amount of the composition of claim 14.

17. The method of claim 16, wherein the chelated cation is .sup.177Lu.

18. (canceled)

19. (canceled)

20. The method of claim 16, wherein the PSMA-associated cancer is glioma, lung cancer, or prostate cancer.

21. (canceled)

22. A method for treating prostate cancer in a subject, comprising administering to the subject an effective amount of a composition of claim 14.

Description

[0257] The figures show:

[0258] FIG. 1: Web chart of characteristics for [.sup.nat/177Lu]PSMA I&T and [.sup.nat/177Lu]PSMA-62 and [.sup.nat/177Lu]PSMA-66.

[0259] FIG. 2: Externalization kinetics of selected .sup.177Lu-labeled PSMA inhibitors from LNCaP cells. 1.25*10.sup.5 cells/well were incubated 1 h with the respective radioligand (c=1.0 nm) at 37° C. in DMEM-solution (5% BSA). Then, the supernatant was removed and once washed with DMEM-solution (5% BSA, 37° C.). Afterwards, either A) only DMEM-solution (5% BSA) or B) blockade DMEM-solution (5% BSA, 10 μm 2-PMPA) were added for replacement. The total cellular internalized activity at t=0 min was corrected for non-specific binding (10 μm 2-PMPA) and normalized to 100%. All data are expressed as mean±SD (n=3).

[0260] FIG. 3: Biodistribution (in % ID/g) of 2.5 to 3.0 MBq (0.15 to 0.25 nmol) of [.sup.177Lu]PSMA-66 and [.sup.177Lu]PSMA I&T in LNCaP-tumor bearing CB-17 SCID mice (n=4, respectively).

[0261] FIG. 4: Maximum intensity projection (MIP) of a pPET scan in LNCaP-tumor bearing CB-17 SCID mice after injection of approx. 10.3 MBq (0.19 nmol tracer) of [.sup.68Ga]PSMA-36 (dynamic scan, summed up frames 1 to 1.5 h p.i.) (top left). TACs (logarithmic plot) in % ID/mL of [.sup.68Ga]PSMA-36 derived from dynamic PET data (90 min acquisition time, OSEM 3D reconstruction) in a LNCaP-tumor bearing CB-17 SCID mouse of blood pool (heart), kidney, tumor, muscle, lacrimal- and salivary gland.

[0262] FIG. 5: Maximum intensity projection (MIP) of μPET scans in LNCaP-tumor bearing CB-17 SCID mice after injection of approx. 11 and 13 MBq (0.15 to 0.25 nmol tracer) of the .sup.68Ga-labeled PSMA inhibitor PSMA-62 and PSMA-66, respectively (dynamic scan, summed up frames 1 to 1.5 h p.i.) (top left). TACs (logarithmic plot) in % ID/mL of the respective .sup.68Ga-labeled PSMA inhibitor derived from dynamic PET data (90 min acquisition time, OSEM 3D reconstruction) in LNCaP-tumor bearing CB-17 SCID mice of blood pool (heart), kidney, tumor and muscle for both .sup.68Ga-labeled tracer.

[0263] FIG. 6: Biodistribution (in % ID/g) of 2.5 to 6.0 MBq (0.15 to 0.25 nmol) of [.sup.177Lu]PSMA-62, [.sup.177Lu]PSMA-66, [.sup.177Lu]PSMA-71 and [.sup.177Lu]PSMA I&T in LNCaP-tumor bearing CB-17 SCID mice (n=4, respectively).

[0264] The examples illustrate the invention.

EXAMPLE 1: MATERIALS AND METHODS

1. General Information

[0265] The Fmoc-(9-fluorenylmethoxycarbonyl-) and all other protected amino acid analogs were purchased from Bachem (Bubendorf, Switzerland) or Iris Biotech (Marktredwitz, Germany). The 2-chlorotrityl chloride (2-CTC) resin was obtained from PepChem (Tübingen, Germany). Chematech (Dijon, France) delivered the chelator DOTAGA-anhydride. PSMA-DKFZ-617 was purchased from ABX advanced chemical compounds (Radeberg, Germany). 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 reversed-phase high performance liquid chromatography (RP-HPLC) was performed on a Nucleosil 100 C18 column (5 μm, 125×4.0 mm, CS GmbH, Langerwehe, Germany) using a Shimadzu gradient RP-HPLC System (Shimadzu Deutschland GmbH, Neufahrn, Germany). Analysis of the peptides was performed by applying different gradients of 0.1% (v/v) trifluoroacetic acid (TFA) in H.sub.2O (solvent A) and 0.1% TFA together with (v/v) in acetonitrile (MeCN) (solvent B) with a constant flow of 1 mL/min (specific gradients are cited in the text). The Shimadzu SPD 20 A prominence UV/VIS detector (Shimadzu Deutschland GmbH) was used at A=220 nm and 254 nm. HSA binding was determined using a Chiralpak HSA (5 μm, 50×3 mm) analytical column connected to a Chiralpak HSA (5 μm, 10×3 mm) guard cartridge (Daicel Chemical Industries) purchased from Chiral Technologies Europe (Illkirch, France). Non-linear regression for the HSA binding was performed using OriginPro 2016G (Northampron, USA). Retention times t.sub.R as well as the capacity factors K′ are cited in the text. Preparative RP-HPLC of the peptides was achieved on a Shimadzu RP-HPLC system using a Multospher 100 RP 18-5 column (250×20 mm, CS GmbH) with a constant flow of 5 mL/min. Analytical and preparative Radio RP-HPLC of the radioiodinated reference ligand was performed using a Nucleosil 100 C18 column (5 μm, 125×4.0 mm). Radioactivity was detected through connection of the outlet of the UV-photometer to a NaI(Tl) well-type scintillation counter from EG&G Ortec (Munich, Germany). The .sup.68Ga- and .sup.177Lu-labeled compounds were analyzed as published previously [1, 2]. Electrospray ionization mass spectrometry (ESI-MS) spectra were acquired on an expression.sup.L CMS mass spectrometer (Advion Ltd., Harlow, UK) and on a Varian 500-MS IT mass spectrometer (Agilent Technologies, Santa Clara, USA). For the Bradford-Assay a V-630 UV-Vis spectrophotometer from JASCO Germany GmbH (Gross-Umstadt, Germany) was used and centrifugation of the S9-fractions was performed in an Avanti JXN-26 centrifuge from Beckman Coulter GmbH (Krefeld, Germany). The centrifugation of the radioactive S9-metabolite assays was performed using a Heraeus PICO 17 centrifuge from Thermo Fisher Scientific Messtechnik GmbH (Munich, Germany). NMR Data were obtained applying 300 K using an AV 300 (300 MHz) or an AV 400 (400 MHz) from Bruker (Billerica, USA). The incubation of the S9-fractions for ex vivo metabolite analysis was performed in a Biometra UNO Thermoblock (Biometra, Göttingen, Deutschland).

2. Synthesis Protocols (SP)

[0266] SP-1: 2-CTC-resin loading: 2-CTC-resin (1.6 mmol/g) is loaded with Fmoc-AA-OH (1.5 eq.) in anhydrous dichloromethane (DCM) with N,N-Diisopropylethylamine (DIPEA) (4.5 eq.) at room temperature (RT) for 2 h. The remaining tritylchloride is capped by addition of 2 mL/g methanol (MeOH) for 15 min. After that, the resin is filtered and thoroughly washed with DCM (2.sup.x), with dimethylformamide (DMF) (2.sup.x) and MeOH (2.sup.x), respectively and stored under vacuum overnight. The loading is determined using the weight differences:

[00001]$\frac{m_{\mathrm{total}}-m_{\mathrm{net}\mathrm{weight}})\times 1000}{\left(M_{\mathrm{As}}-M_{\mathrm{HCl}}\right)\times m_{\mathrm{weight}\mathrm{of}\mathrm{resin}}}=\mathrm{mmol}/g$$\mathrm{Formula}1.\mathrm{Determination}\mathrm{of}\mathrm{resin}-\mathrm{loading}:m_{\mathrm{total}}:\mathrm{mass}\mathrm{of}\mathrm{loaded}\mathrm{resin}\left(\mathrm{Fmoc}-\mathrm{AA}-\mathrm{OH}\mathrm{and}\mathrm{HCI}\right);M_{\mathrm{As}}:\mathrm{molar}\mathrm{mass}\mathrm{of}\mathrm{amino}\mathrm{acid};m_{\mathrm{net}}\mathrm{weight}:\mathrm{mass}\mathrm{of}\mathrm{used}\mathrm{resin};M_{\mathrm{HCl}}:\mathrm{molar}\mathrm{mass}\mathrm{of}\mathrm{hydrochloric}\mathrm{acid}$

[0267] SP-2: Peptide synthesis via TBTU/HOBt coupling: A solution of Fmoc-AA-OH (2.0 eq.), N,N,N′,N′-Tetramethyl-O-(benzotriazol-1-yl)uronium tetrafluoroborate (TBTU) (2.0 eq.), N-Hydroxybenzotriazole (HOBt) (2.0 eq.), DIPEA (4.5 eq.) in DMF (8 ml/g resin) was added to the resin-bound free amine peptide and shaken for 2 h at RT and washed with DMF (6.sup.x). The coupling with secondary or aromatic amines was performed employing a different protocol. Fmoc-AA-OH (3.0 eq.) was dissolved in DMF (8 mL/g resin) together with 1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluoro-phosphate (HATU) (3.0 eq.), 1-Hydroxy-7-azabenzotriazol (HOAt) (3.0 eq.) and DIPEA (6.0 eq.) and stirred for 15 min. The pre-activated solution was added to the resin bound peptide and shaken for 2 h at RT. After completion of the reaction, the resin was washed with DMF (6.sup.x). In general, all peptidic scaffolds were synthesized as previously described (Weineisen, M.; Schottelius, M.; Simecek, J.; Eiber, M.; Schwaiger, M.; Wester, H. Development and first in human evaluation of PSMA I&T—A ligand for diagnostic imaging and endoradiotherapy of prostate cancer. Journal of Nuclear Medicine 2014, 55, 1083-1083; Weineisen, M.; Simecek, J.; Schottelius, M.; Schwaiger, M.; Wester, H.-J. Synthesis and preclinical evaluation of DOTAGA-conjugated PSMA ligands for functional imaging and endoradiotherapy of prostate cancer. EJNMMI research 2014, 4, 1).

[0268] SP-3: On-resin Fmoc-deprotection: The resin-bound Fmoc-protected peptide was treated with 20% piperidine in DMF (v/v) for 5 min and a second time for 15 min. Afterwards, the resin was washed thoroughly with DMF (8.sup.x).

[0269] SP-4: On-resin Dde-deprotection: The N-(1-(4,4-dimethyl-2,6-dioxocyclohexylidene)-ethyl) (Dde) protected peptide (1.0 eq.) was dissolved in a solution of 2.0% hydrazine monohydrate (N.sub.2H.sub.4.H.sub.2O) in DMF (v/v). After 15 min, the deprotected peptide, if bound to resin, was washed with DMF (6.sup.x) or precipitated in diethyl ether (Et.sub.2O) to give the crude product. If Fmoc- and Dde-protecting groups were present and only Dde-deprotection was necessary, the resin-loaded peptide was treated with a solution containing NH.sub.2OH.HCl (630 mg), imidazole (460 mg), DCM (0.5 mL), DMF (0.5 mL) and N-methyl-2-pyrrolidone (NMP) (2.5 mL) for 3 h at RT. Afterwards, the resin-loaded peptide was washed with DMF (6.sup.x).

[0270] SP-5: On-resin Alloc/Allyl-deprotection: The Alloc/Allyl-protecting group was removed from the resin-bound peptide using a solution of DCM (6.0 mL) containing triisopropylsilane (TIPS) (50.0 eq.) and (triphenyl)palladium(0) (Pd(PPh.sub.3).sub.4) (0.3 eq.). The resin was treated with this solution for 1.5 h at RT. Finally, the resin was washed with DCM (3.sup.x) to remove the Pd(PPh.sub.3).sub.4.

[0271] SP-6: tBu/Boc deprotection: Removal of the tert-butyl (tBu)/tert-butyloxycarbonyl (Boc)-protecting groups was carried out by dissolving the crude product in TFA (approx. 500 μL) and stirring for 40 min at RT. Afterwards, the TFA was almost completely removed using nitrogen stream. After precipitation in Et.sub.2O, the crude product was centrifuged and the supernatant removed. The dried pellet was further used for the following synthesis-steps.

[0272] SP-7.1: A) Peptide cleavage from the resin with preservation of side-chain protecting groups: The fully protected, resin-bound peptide was dissolved in a mixture of DCM/trifluoroethanol (TFE)/acetic acid (AcOH) (6/3/1; v/v/v) and shaken for 30 min. The solution was filtered off and the resin was dissolved in another cleavage solution for another 30 min. The fractions were combined and the solvent was concentrated under reduced pressure. The filtrate was redissolved in toluene and concentrated under reduced pressure to remove the AcOH. Precipitation in water or Et.sub.2O resulted in the crude, side chain protected peptide.

[0273] SP-7.2: B) Peptide cleavage from the resin with concurrent deprotection of all acid labile protecting groups: The fully protected, resin-bound peptide was dissolved in a mixture of TFA/TIPS/water (95/2.5/2.5; v/v/v) and shaken for 30 min. The solution was filtered off and the resin was treated in the same way for another 30 min. Afterwards, the fractions were combined and the solvent was concentrated under a constant flow of nitrogen. The crude peptide was precipitated in Et.sub.2O and left to dry overnight.

[0274] SP-8: Deacetylation of carbohydrate-moieties: Deacetylation was accomplished by dissolving the PSMA inhibitor in MeOH containing KCN (0.5 eq.) (Herzig, J.; Nudelman, A.; Gottlieb, H. E.; Fischer, B. Studies in sugar chemistry. 2. A simple method for O-deacylation of polyacylated sugars. The Journal of Organic Chemistry 1986, 51, 727-730) with concomitant stirring overnight at RT. The final product was purified by RP-HPLC.

[0275] SP-9: Preparation of non-radioactive metal-complexed PSMA inhibitors:

[0276] SP-9.1: .sup.natGa-compounds: For the preparation of the .sup.natGa.sup.III-complexes, a 2.0 mm aqueous (aq.) solution of the PSMA inhibitor (50 μL) and a 2.0 mm aq. solution of Ga(NO.sub.3).sub.3 (50 μL) were mixed and heated at 40° C. for 30 min. The chelate formation was assessed using RP-HPLC and ESI-MS. The resulting 1.0 mm solution was diluted and used for in vitro IC.sub.50 determination and HSA binding.

[0277] SP-9.2: .sup.natLu-compounds: The corresponding .sup.natLu.sup.III-complexes were prepared from a 2.0 mm aqueous solution of the PSMA inhibitor with a 2.5 molar excess of LuCl.sub.3 (20 mm aq. solution) and heated to 95° C. for 30 min. After cooling, the .sup.natLu.sup.III-chelate formation was confirmed using RP-HPLC and ESI-MS. The resulting 1.0 mm aqueous solutions of the respective .sup.natLu-complexes were then diluted and used in the in vitro IC.sub.50 studies without further processing.

3. Building Blocks for PSMA-36 and the EuE Based PSMA Inhibitors

Di-tert-butyl(((S)-6-amino-1-(tert-butoxy)-1-oxohexan-2-yl)carbamoyl)-L-glutamate

[0278] ##STR00055##

[0279] ((OtBu)KuE(OtBu).sub.2) (1): The synthesis of the tert-butyl-protected Lys-urea-Glu binding motif (EuK) was synthesized as previously described by solution phase synthesis [3]. In short, a solution of DCM containing L-di-tert-butyl-glutamate-HCl (2.0 g, 7.71 mmol, 1.0 eq.) was cooled on ice for 30 min and afterwards treated with trimethylamine (TEA) (2.69 mL, 19.28 mmol, 2.5 eq.) and 4-(dimethylamino)pyridine (DMAP) (3.3 mg, 0.3 mmol, 0.04 eq.). After additional stirring for 5.0 min, 1,1′-carbonyldiimidazole (CDI) (1.38 g, 8.84 mmol, 1.1 eq.) was dissolved in DCM and slowly added over a period of 30 min. The reaction mixture was further stirred overnight and enabled to warm to RT. The reaction was stopped using saturated (sat.) NaHCO.sub.3 solution (8 mL) with concomitant washing steps of water (2.sup.x) and brine (2.sup.x) and dried over sat. Na.sub.2SO.sub.4 solution. The remaining solvent was removed in vacuo and the crude product (S)-Di-tert-butyl 2-(1H-imidazole-1-carboxamido)pentanedioate used without further purification. RP-HPLC (10 to 90% B in 15 min): t.sub.R=12.2 min; K′=5.8. Calculated monoisotopic mass (C.sub.17H.sub.27N.sub.3O.sub.5): 353.4; found: m/z=376.1 [M+Na].sup.+. The crude product (S)-Di-tert-butyl 2-(1H-imidazole-1-carboxamido)pentanedioate (2.72 g, 7.71 mmol, 1.0 eq.) was dissolved in 1,2-dichloroethane (DCE) and cooled on ice for 30 min. To this solution was added TEA (2.15 mL, 15.42 mmol, 2.0 eq.) and H-Lys(Cbz)-OtBu.HCl (2.87 g, 7.71 mmol, 1.0 eq.) and the solution stirred overnight at 40° C. The remaining solvent was evaporated and the crude product purified using silica gel flash-chromatography with an eluent mixture containing ethyl acetate (EtOAc)/hexane/TEA (500/500/0.8; v/v/v). After removal of the solvent, (9R,13S)-tri-tert-butyl-3,11-dioxo-1-phenyl-2-oxa-4,10, 12-triazapentadecane-9,13,15-tricarboxylate was obtained as colorless oil. RP-HPLC (40 to 100% B in 15 min): t.sub.R=14.5 min; K′=6.25. Calculated monoisotopic mass (C.sub.32H.sub.51N.sub.3O.sub.9)=621.8; found: m/z=622.3 [M+H].sup.+. To synthesize (OtBu)KuE(OtBu).sub.2 (1), (9R,13S)-tri-tert-butyl-3,11-dioxo-1-phenyl-2-oxa-4,10, 12-triazapentadecane-9,13,15-tricarboxylate (3.4 g, 5.47 mmol, 1.0 eq.) was dissolved in ethanol (EtOH) (75 mL) and palladium on activated charcoal (0.34 g, 0.57 mmol, 0.1 eq.) (10%) was given to this solution. The reaction mixture containing flask was initially purged with hydrogen stream and the solution allowed to stir overnight at RT under light hydrogen-pressure (balloon). The crude product was purified through celite and the solvent evaporated in vacuo. The desired product 1 was obtained as a waxy solid (1.9 g, 3.89 mmol, 71.6% yield). RP-HPLC (10 to 90% B in 15 min): t.sub.R=12.6 min; K′=6.4. Calculated monoisotopic mass (C.sub.24H.sub.45N.sub.3O.sub.7)=487.6; found: m/z=488.3 [M+H].sup.+, 510.3 [M+Na].sup.+.

(S)-5-(tert-butoxy)-4-(3-((S)-1,5-di-tert-butoxy-1,5-dioxopentan-2-yl)ureido)-5-oxopentanoic acid ((OtBu)EuE(OtBu).SUB.2.) (2)

[0280] ##STR00056##

[0281] The synthesis of the tert-butyl-protected Glu-urea-Glu binding motif (EuE) was similarly synthesized as described for 1 [3] using H-L-Glu(OBzl)-OtBu.HCl instead of H-L-Lys(Cbz)-OtBu.HCl. The desired product was obtained as waxy and strongly hygroscopic solid (4.10 g, 8.39 mmol, 84% yield). RP-HPLC (10 to 90% B in 15 min): t.sub.R=11.3 min; K′=7.69. Calculated monoisotopic mass (C.sub.23H.sub.49N.sub.2O.sub.9)=488.3; found: m/z=489.4 [M+H].sup.+, 516.4 [M+Na].sup.+.

[0282] (S)-NHFmoc-Asu(OtBu)-OBzl (5): To a solution of (S)-Fmoc-Asu(OtBu)-OH (50 mg, 107.0 μmol, 1.0 eq.) in DMF was added HOAt (21.8 mg, 0.16 mmol, 1.5 eq.), HATU (61.0 mg, 161.0 μmol, 1.5 eq.) and DIPEA (73.2 μL, 0.48 mmol, 4.5 eq.). After 15 min of stirring at RT, benzyl alcohol (22.2 μL, 0.32 mmol, 3.0 eq.) was further added and the solution stirred overnight. Finally, the solvent was removed in vacuo. Completion of reaction of 5 was analyzed by RP-HPLC (10 to 90% B in 15 min): t.sub.R=17.1 min K′=7.55. Calculated monoisotopic mass for 5 (C.sub.34H.sub.39NO.sub.6)=557.28; found: m/z=580.7 [M+Na].sup.+.

[0283] (S)-NHFmoc-Asu-OBzl (6): tBu deprotection of the crude product 5 was performed with a stirring mixture (v/v) of TFA (95%) and DCM (5%) at RT for 45 min. After evaporation of the solvent, the crude product 6 was purified using preparative RP-HPLC (60 to 80% B in 15 min): t.sub.R=9.3 min; K′=8.9. Calculated monoisotopic mass for 6 (C.sub.30H.sub.31NO.sub.6)=501.22; found m/z=524.5 [M+Na].sup.+.

[0284] OBzl-(S)-Fmoc-Asu[(OtBu)KuE(OtBu).sub.2] (7): To a solution of 6 (51.8 mg, 10.3 μmol, 1.0 eq.) in DMF was added HOBt (20.9 mg, 0.15 mmol, 1.5 eq.), TBTU (36.3 mg, 15.5 μmol, 1.5 eq.) and DIPEA (79.4 μL, 59.7 mg, 0.46 mmol, 4.5 eq.). After 15 min stirring, 1 (75.6 mg, 15.5 μmol, 1.5 eq.) was added and further stirred for 20 h at RT. The crude product 7 was purified using preparative RP-HPLC (70 to 80% B in 15 min): t.sub.R=8.9 min; K′=1.97. Calculated monoisotopic mass for 7 (C.sub.54H.sub.74N.sub.4O.sub.12)=970.53; found: m/z=971.8 [M+H].sup.+.

[0285] (S)-Fmoc-Asu[(OtBu)KuE(OtBu).sub.2] (8): For benzyl alcohol (Bzl) deprotection, of 7 (57.2 mg, 65.0 μmol, 1.0 eq.) was dissolved in EtOH (2.0 mL) and palladium on activated charcoal (10%) (5.72 mg, 9.0 μmol, 0.1 eq.) was added. The flask was purged beforehand with hydrogen stream and the solution stirred under light hydrogen-pressure (balloon). After 70 min stirring, the crude product was filtered through celite, the EtOH evaporated in vacuo and the product purified using preparative RP-HPLC (70 to 70.5% B in 15 min): t.sub.R=6.5 min; K′=0.54. Calculated monoisotopic mass for 5 (C.sub.47H.sub.68N.sub.4O.sub.12)=880.48; found: m/z=881.8 [M+H].sup.+.

[0286] OPfp-(S)-Fmoc-Asu[(OtBu)KuE(OtBu).sub.2] (9):

##STR00057##

[0287] To a solution of 8 (13.6 mg, 15.4 μmol, 1.0 eq.) in dry DMF was added DIC (4.77 μL, 1.94 mg, 30.8 μmol, 2.0 eq.) and PfpOH (5.67 mg, 30.8 μmol, 2.0 eq.). After 5 min stirring, pyridine (2.49 μL, 31.0 μmol, 2.0 eq.) was added and the solution was allowed to stir overnight at RT. Completion of reaction of 9 was analyzed by RP-HPLC (10 to 90% B in 15 min): t.sub.R=17.2 min; K′=7.6. Calculated monoisotopic mass for 9 (C.sub.53H.sub.67F.sub.5N.sub.4O.sub.12)=1046.47; found: m/z=1069.8 [M+Na].sup.+.

[0288] NHS-2,4-dinitrobenzoate (NHS-DNBA) (27):

##STR00058##

[0289] To a solution of 2,4-dinitrobenzoic acid (DNBA) (10.0 mg, 47.1 μmol, 1.0 eq.) in dry THF was given N, N′-dicyclohexylcarbodiimide (DCC) (9.7 mg, 47.1 μmol, 1.0 eq.) and N-hydroxysuccinimide (NHS) (10.8 mg, 94.3 μmol, 2.0 eq.) and the reaction mixture was allowed to stir overnight. The crude product was purified using RP-HPLC. RP-HPLC (10 to 90% B in 15 min): t.sub.R=10.21 min K′=4.1. Calculated monoisotopic mass (C.sub.11H.sub.7N.sub.3O.sub.8)=309.02; found: not detectable in ESI-MS

[0290] DOTAGA-3-iodo-D-Tyr-D-Phe-D-Lys-OH (DOTAGA-y(3-I)fk) (30):

##STR00059##

[0291] The synthesis of 30 was accomplished via solid phase strategy as previously described [2, 3]. In short: The initial starting point was the 2-CTC resin loading according to SP-1 of Fmoc-D-Lys(Boc)-OH. After conjugation of lysine, Fmoc was deprotected according to SP-3 and Fmoc-D-phenylalanine coupled applying SP-2. The same procedure was used to couple Fmoc-D-Tyr(3-1)-OH. After completion of reaction, the Fmoc protecting group was cleaved according to SP-3 and the resin bound peptide condensed with the chelator using DOTAGA-anhydride (2.0 eq.) and DIPEA (2.0 eq.) in DMF. The reaction was allowed to stir for 48 h at RT. Finally, the crude product was cleaved from the resin according to SP-7.2 and precipitated in Et.sub.2O and centrifuged. The supernatant was removed and 30 purified using RP-HPLC. RP-HPLC (10 to 90% B in 15 min): t.sub.R=6.2 min K′=2.1. Calculated monoisotopic mass (C.sub.43H.sub.61IN.sub.8O.sub.14)=1,040.34; found: m/z=1,040.5 [M+H].sup.+, m/z=521.3 [M+2H].sup.2+, m/z=1,063.4=[M+Na].sup.+.

[0292] DOTAGA-y(3-I)fk(L-Asu[KuE]) (PSMA-8):

##STR00060##

[0293] To a solution of DMF containing 30 (5.0 mg, 4.8 μmol, 1.0 eq.), 9 (7.5 mg, 7.2 μmol, 1.5 eq.) and DIPEA (3.3 μL, 21.6 μmol, 4.0 eq.) were added. The reaction solution was allowed to stir overnight at RT. After completion of reaction, the solvent was removed in vacuo and the crude product treated with a mixture of piperidine in DMF (20/80; v/v) for 15 min to achieve Fmoc-deprotection. The solvent was reduced to approx. 300 μL via evaporation in vacuo, precipitated in Et.sub.2O and centrifuged. With the resulting pellet was processed according to SP-6 for tBu-removal. The final product was purified via RP-HPLC (10 to 90% B in 15 min): t.sub.R=6.09 min K′=2.05. Calculated monoisotopic mass (C.sub.63H.sub.93IN.sub.12O.sub.23)=1,512.55; found: m/z=1, 513.9 [M+H].sup.+, 757.8 [M+2H].sup.2+.

[0294] DOTAGA-y(3-I)fk(L-Asu[KuE]-2,4-DNBA) (PSMA-36):

##STR00061##

[0295] The synthesis of PSMA-36 was achieved by dissolving PSMA-8 (3.0 mg, 3.3 μmol, 1.0 eq.) in DMF and addition of 27 (4.1 mg, 13.2 μmol, 4.0 eq.) and DIPEA (2.3 μL, 13.2 μmol, 4.0 eq.). The solution was stirred for 10 h at RT and the final product purified by RP-HPLC (10 to 50% B in 15 min): t.sub.R=12.12 min K′=5.06. Calculated monoisotopic mass (C.sub.70H.sub.95IN.sub.14O.sub.28)=1,706.55; found: m/z=1,707.8 [M+H].sup.+, 854.7 [M+2H].sup.2+.

[0296] [.sup.natLu]DOTAGA-y(3-I)fk(L-Asu[KuE]-2,4-DNBA) ([.sup.natLu]PSMA-36): RP-HPLC (10 to 60% B in 15 min): t.sub.R=9.81 min K′=3.91. Calculated monoisotopic mass (C.sub.70H.sub.92IN.sub.14O.sub.28Lu)=1,878.47; found: m/z=1,879.9 [M+H].sup.+.

##STR00062##

[0297] Schematic illustration of the synthesis of PSMA-36. (a) HOAt, HATU, DIPEA, benzyl-alcohol, [DMF]; (b) 95% TFA, 5% DCM; (c) 1, HOBt, TBTU, DIPEA, [DMF]; (d) Pd/C (10%), H.sub.2, [EtOH]; (e) DIC, PFP, pyridine, [DMF]; (f) 30, DIPEA, [DMF]; (g) 20% piperidine in DMF, [DMF]; (h) TFA; (i) 27, DIPEA [DMF]

4. Synthesis of EuE-Based PSMA Inhibitors PSMA-52 and PSMA-53

[0298] DOTAGA-F(4-NO.sub.2)-y-2-naI-k(Suc-N.sup.5-orn-C.sup.4-EuE) (PSMA-52):

##STR00063##

[0299] The initial resin loading with Fmoc-D-Orn(NHDde)-OH was performed as described in SP-1. After Fmoc-deprotection according to SP-3, 2 (1.5 eq.) was coupled to D-Orn(NHDde) according to SP-2. In the next step, the Dde-protecting group was cleaved according to SP-4 and the free amino group treated with succinic anhydride (4.0 eq.) and DIPEA (1.5 eq.) dissolved in DMF. The reaction mixture was allowed to react overnight at RT. Next, Fmoc-D-Lys-OtBu.HCl (1.5 eq.) was coupled according to SP-2 and Fmoc-deprotected as described in SP-3. The following conjugations with the Fmoc-protected amino acids Fmoc-D-2-NaI—OH, Fmoc-D-Tyr(OtBu)-OH and Fmoc-L-Phe(4-NO.sub.2)—OH were conducted as described in SP-2. The N-terminal Fmoc-deprotected amino acid was conjugated with the chelator in the final step using DOTAGA-anhydride (2.0 eq.) and DIPEA (2.0 eq.). The reaction was allowed to stir for 48 h at RT. After completion of reaction with DOTAGA-anhydride, the peptide was cleaved from the resin according to SP-7.2, the crude product precipitated in Et.sub.2O, centrifuged and the supernatant removed. The final product was purified via RP-HPLC. RP-HPLC (10 to 60% B in 15 min): t.sub.R=9.71 min K′=3.86. Calculated monoisotopic mass (C.sub.76H.sub.100N.sub.14O.sub.29)=1,672.68; found: m/z=1,673.0 [M+H].sup.+.

[0300] [.sup.natLu]DOTAGA-F(4-NO.sub.2)-y-2-naI-k(Suc-N.sup.5-orn-C.sup.4-EuE) ([.sup.natLu]PSMA-52): RP-HPLC (10 to 60% B in 15 min): t.sub.R=9.4 min K′=3.7. Calculated monoisotopic mass (C.sub.76H.sub.97N.sub.14O.sub.29Lu)=1,844.6; found: m/z=1,846.0 [M+H].sup.+.

[0301] 2,4-DNBA-Dap(DOTAGA)-y-2-naI-k(Suc-N.sup.5-orn-C.sup.4-EuE) (PSMA-53):

##STR00064##

[0302] The initial resin loading with Fmoc-D-Orn(NHDde)-OH was performed as described in SP-1. After Fmoc-deprotection according to SP-3, 2 (1.5 eq.) was coupled to D-Orn(NHDde) according to SP-2. In the next step, the Dde-protecting group was cleaved according to SP-4 and the free amino group treated with succinic anhydride (4.0 eq.) and DIPEA (1.5 eq.) dissolved in DMF. The reaction mixture was allowed to react overnight at RT. Next, Fmoc-D-Lys-OtBu.HCl (1.5 eq.) was coupled according to SP-2 and Fmoc-deprotected as described in SP-3. The following conjugations with the Fmoc-protected amino acids Fmoc-D-2-NaI—OH, Fmoc-D-Tyr(OtBu)-OH and of Fmoc-L-Dap(NHDde)-OH were conducted as described in SP-2. After coupling of Fmoc-L-Dap(NHDde)-OH, Fmoc-deprotection was achieved as described in SP-3. Next, the free amino group was conjugated to 2,4-dinitrobenzoic acid (2,4-DNBA) using 2,4-DNBA (2.0 eq.), HOBt (2.0 eq.), TBTU (2.0 eq.) and DIPEA (4.0 eq.) in DMF. After completion of reaction, Dde-deprotection was achieved using SP-5. The N-terminal free amino acid L-Dap was conjugated with the chelator in the final step using DOTAGA-anhydride (2.0 eq.) and DIPEA (2.0 eq.). The reaction was allowed to stir for 48 h at RT. After completion of reaction with DOTAGA-anhydride, the peptide was cleaved from the resin according to SP-7.2, the crude product precipitated in Et.sub.2O, centrifuged and the supernatant removed. The final product was purified via RP-HPLC.

[0303] RP-HPLC (10 to 60% B in 15 min): t.sub.R=11.71 min K′=4.86. Calculated monoisotopic mass (C.sub.77H.sub.100N.sub.16O.sub.32)=1,760.67; found: m/z=1,762.1 [M+H].sup.+.

[0304] [.sup.natLu]2,4-DNBA-Dap(DOTAGA)-y-2-naI-k(Suc-N.sup.5-orn-C.sup.4-EuE) ([.sup.natLu]PSMA-53): RP-HPLC (10 to 60% B in 15 min): t.sub.R=8.3 min K′=3.15. Calculated monoisotopic mass (C.sub.77H.sub.97N.sub.16O.sub.32Lu)=1,932.59; found: m/z=1,933.7 [M+H].sup.+.

##STR00065##

[0305] Schematic illustration of the general synthesis procedure of EuE-based PSMA inhibitors PSMA-52 and PSMA-53 exemplified by PSMA-52. (a) 20% piperidine in DMF, 2, HOBt, TBTU, DIPEA [DMF]; (b) succinic anhydride, DIPEA [DMF]; (c) Fmoc-D/L-Lys-OAII.HCl, HOBt, TBTU, DIPEA [DMF]; (d) 20% piperidine in DMF, Fmoc-D-2-NaI—OH, HOBt, TBTU, DIPEA [DMF]; (e) 20% piperidine in DMF, Fmoc-D-Tyr(OtBu)-OH, HOBt, TBTU, DIPEA [DMF]; (f) 20% piperidine in DMF, Fmoc-D-Phe(4-NH.sub.2)—OH, HOBt, TBTU, DIPEA [DMF]; (g) DOTAGA-anhydride, DIPEA [DMF]; (h) TFA;

5. Synthesis of PSMA-61 and PSMA-62

[0306] DOTAGA-F(4-NH.sub.2)y-2-naI-k(d[N.sup.5-orn-C.sup.4-EuE]-2,4-DNBA) (PSMA-61):

##STR00066##

[0307] The initial resin loading with Fmoc-D-Orn(NHDde)-OH was performed as described in SP-1. After Fmoc-deprotection according to SP-3, 2 (1.5 eq.) was coupled to D-Orn(NHDde) according to SP-2. In the next step, the Dde-protecting group was cleaved according to SP-4 and the free amino group treated with Fmoc-D-Asp-OAII.HCl (1.5 eq.) according to SP-2. The amino group of Fmoc-D-Asp-OAII.HCl was deprotected according to SP-3 and conjugated to 2,4-DNBA using 2,4-DNBA (1.5 eq.), HOBt (2.0 eq.), TBTU (2.0 eq.) and DIPEA (4.0 eq.) in DMF. After completion of reaction, Allyl-deprotection was achieved according to SP-5. The next steps included the repetitive conjugation with Fmoc-D-Lys-OtBu.HCl (1.5 eq.), Fmoc-D-2-NaI—OH, Fmoc-D-Tyr(OtBu)-OH and Fmoc-L-Phe(4-NHBoc)-OH according to SP-2. The N-terminal Fmoc-deprotected amino acid L-Phe(4-NHBoc)-OH was conjugated with the chelator in the final step using DOTAGA-anhydride (2.0 eq.) and DIPEA (2.0 eq.). The reaction was allowed to stir for 48 h at RT. After completion of reaction with DOTAGA-anhydride, the peptide was cleaved from the resin according to SP-7.2, the crude product precipitated in Et.sub.2O, centrifuged and the supernatant removed. The final product was purified via RP-HPLC.

[0308] RP-HPLC (10 to 90% B in 15 min): t.sub.R=6.40 mi K′=2.2. Calculated monoisotopic mass (C.sub.83H.sub.105N.sub.17O.sub.32)=1,851.71; found: m/z=1,852.5 [M+H].sup.+, 926.7 [M+2H].sup.2+.

[0309] [.sup.natLu]DOTAGA-F(4-NH.sub.2)y-2-naI-k(d[N.sup.5-orn-C4-EuE]-2,4-DNBA) ([.sup.natLu]PSMA-61): RP-HPLC (10 to 90% B in 15 min): t.sub.R=8.22 min K′=3.11. Calculated monoisotopic mass (C.sub.83H.sub.102N.sub.17O.sub.32Lu)=2,023.63; found: m/z=1,013.1 [M+2H].sup.2+.

[0310] DOTAGA-F(4-NH.sub.2)y-2-naI-k(d[N.sup.5-orn-C.sup.4-EuE]-TMA) (PSMA-62):

##STR00067##

[0311] The initial resin loading with Fmoc-D-Orn(NHDde)-OH was performed as described in SP-1. After Fmoc-deprotection according to SP-3, 2 (1.5 eq.) was coupled to D-Orn(NHDde) according to SP-2. In the next step, the Dde-protecting group was cleaved according to SP-4 and the free amino group treated with Fmoc-D-Asp-OAII.HCl (1.5 eq.) according to SP-2. The amino group of Fmoc-D-Asp-OAII.HCl was Fmoc-deprotected according to SP-3 and protected with Dde-OH (2.0 eq.) and DIPEA (4.0 eq.) in DMF at RT. The reaction was allowed to stir overnight. Afterwards, Allyl-deprotection of D-Asp was achieved applying SP-5. The next steps included the repetitive conjugation with Fmoc-D-Lys-OtBu.HCl (1.5 eq.), Fmoc-D-2-NaI—OH, Fmoc-D-Tyr(OtBu)-OH and Fmoc-L-Phe(4-NHBoc)-OH according to SP-2. In order to conjugate TMA to D-Asp, selective Dde-deprotection was achieved applying SP-4 to afford the free amino group. TMA was coupled using TMA (2.0 eq.), HOBt (1.5 eq.), TBTU (1.5 eq.) and DIPEA (10 eq.) in DMF. The reaction was allowed to stir for 8 h at RT. After conjugation of TMA, Fmoc-deprotection of Fmoc-L-Phe(4-NHBoc)-OH was achieved using SP-3. The N-terminal Fmoc-deprotected amino acid L-Phe(4-NHBoc)-OH was conjugated with the chelator in the final step using DOTAGA-anhydride (2.0 eq.) and DIPEA (2.0 eq.). The reaction was allowed to stir for 48 h at RT. After completion of reaction with DOTAGA-anhydride, the peptide was cleaved from the resin according to SP-7.2, the crude product precipitated in Et.sub.2O, centrifuged and the supernatant removed. The final product was purified via RP-HPLC. RP-HPLC (10 to 70% B in 15 min): t.sub.R=7.48 min K′=2.74. Calculated monoisotopic mass (C.sub.85H.sub.107N.sub.15O.sub.32)=1,849.72; found: m/z=1,850.5 [M+H]*, 925.7 [M+2H].sup.2+.

[0312] [.sup.natLu]DOTAGA-F(4-NH.sub.2)y-2-naI-k(d[N.sup.5-orn-C.sup.4-EuE]-TMA) ([.sup.natLu]PSMA-62): RP-HPLC (10 to 70% B in 15 min): t.sub.R=7.27 min K′=2.64. Calculated monoisotopic mass (C.sub.85H.sub.104N.sub.15O.sub.32Lu)=2,021.64; found: m/z=1,012.3 [M+2H].sup.2+.

6. Synthesis of PSMA-65, PSMA-66 and PSMA-71

[0313] 2,4-DNBA-Dap(DOTAGA)y-2-naI-e(Abz-N.sup.5-orn-C.sup.4-EuE) (PSMA-65):

##STR00068##

[0314] The initial resin loading with Fmoc-D-Orn(NHDde)-OH was performed as described in SP-1. After Fmoc-deprotection according to SP-3, 2 (1.5 eq.) was coupled to D-Orn(NHDde) according to SP-2. In the next step, the Dde-protecting group was cleaved according to SP-4 and the free amino group treated with Fmoc-4-Abz-OH (1.5 eq.), HOAt (1.5 eq.), HATU (1.5 eq.) and DIPEA (4.0 eq.) in DMF. The reaction was allowed to stir overnight at RT. In the next step, the Abz-residue was Fmoc-deprotected according to SP-3. The next steps included the repetitive conjugation with Fmoc-D-Glu-OtBu, Fmoc-D-2-NaI—OH, Fmoc-D-Tyr(OtBu)-OH and Fmoc-L-Dap(Dde)-OH according to SP-2. After Fmoc-deprotection of Fmoc-L-Dap(Dde)-OH according to SP-3, 2,4-DNBA was coupled using 2,4-DNBA (1.5 eq.), HOBt (2.0 eq.), TBTU (2.0 eq.) and DIPEA (4.0 eq.) in DMF. After completion of reaction, the L-Dap(Dde)-residue was Dde-deprotected according to SP-4 and conjugated to the chelator using DOTAGA-anhydride (2.0 eq.) and DIPEA (2.0 eq.). The reaction was allowed to stir for 48 h at RT. After completion of reaction with DOTAGA-anhydride, the peptide was cleaved from the resin according to SP-7.2, the crude product precipitated in Et.sub.2O, centrifuged and the supernatant removed. The final product was purified via RP-HPLC. RP-HPLC (10 to 60% B in 15 min): t.sub.R=10.2 min K′=4.1. Calculated monoisotopic mass (C.sub.79H.sub.96N.sub.16O.sub.32)=1,780.64; found: m/z=1,781.3 [M+H].sup.+.

[0315] [.sup.natLu]2,4-DNBA-Dap(DOTAGA)y-2-naI-e(Abz-N.sup.5-orn-C.sup.4-EuE) ([.sup.natLu]PSMA-65): RP-HPLC (10 to 60% B in 15 min): t.sub.R=9.8 min K′=3.9. Calculated monoisotopic mass (C.sub.79H.sub.93N.sub.16O.sub.32Lu)=1,952.56; found: m/z=1.954.0 [M+H].sup.+.

[0316] DOTAGA-Dap(TMA)y-2-naI-k(d[N.sup.5-orn-C.sup.4-EuE]-TMA) (PSMA-66):

##STR00069##

[0317] The initial resin loading with Fmoc-D-Orn(NHDde)-OH was performed as described in SP-1. After Fmoc-deprotection according to SP-3, 2 (1.5 eq.) was coupled to D-Orn(NHDde) according to SP-2. In the next step, the Dde-protecting group was cleaved according to SP-4 and the free amino group treated with Fmoc-D-Asp-OAII.HCl (1.5 eq.) according to SP-2. The amino group of Fmoc-D-Asp-OAII.HCl was Fmoc-deprotected according to SP-3 and protected with 2.0 eq. Dde-OH and 4.0 eq. DIPEA in DMF. The reaction was allowed to stir overnight. Afterwards, Allyl-deprotection of D-Asp was achieved applying SP-5. The next steps included the repetitive conjugation with Fmoc-D-Lys-OtBu.HCl (1.5 eq.), Fmoc-D-2-NaI—OH, Fmoc-D-Tyr(OtBu)-OH and Fmoc-L-Dap(Dde)-OH according to SP-2. In order to conjugate TMA to D-Asp and L-Dap, selective Dde-deprotection was achieved applying SP-4 to afford the free amino groups. TMA was coupled using TMA (4.0 eq.), HOBt (3.0 eq.), TBTU (3.0 eq.) and DIPEA (20 eq.) in DMF. The reaction was allowed to stir for 8 h at RT. After conjugation of TMA, Fmoc-deprotection of Fmoc-L-Dap(TMA)-OH was achieved using SP-3. The N-terminal Fmoc-deprotected amino acid L-Dap was conjugated with the chelator in the final step using DOTAGA-anhydride (2.0 eq.) and DIPEA (2.0 eq.). The reaction was allowed to stir for 48 h at RT. After completion of reaction with DOTAGA-anhydride, the peptide was cleaved from the resin according to SP-7.2, the crude product precipitated in Et.sub.2O, centrifuged and the supernatant removed. The final product was purified via RP-HPLC. RP-HPLC (10 to 70% B in 15 min): t.sub.R=7.48 min K′=2.74. Calculated monoisotopic mass (C.sub.88H.sub.107N.sub.15O.sub.37)=1,965.70; found: m/z=1,966.4 [M+H].sup.+, 984.1 [M+2H].sup.2+.

[0318] [.sup.natLu]DOTAGA-Dap(TMA)y-2-naI-k(d[N.sup.5-orn-C.sup.4-EuE]-TMA) (PSMA-66) RP-HPLC (10 to 70% B in 15 min): t.sub.R=7.46 mi K′=2.73. Calculated monoisotopic mass (C.sub.88H.sub.108N.sub.15O.sub.37Lu)=2,137.62; found: m/z=1,070.4 [M+2H].sup.2+.

[0319] DOTAGA-2-NaI-y-2-naI-k(d[N.sup.5-orn-C.sup.4-EuE]-TMA) (PSMA-71):

##STR00070##

[0320] The initial resin loading with Fmoc-D-Orn(NHDde)-OH was performed as described in SP-1. After Fmoc-deprotection according to SP-3, 2 (1.5 eq.) was coupled to D-Orn(NHDde) according to SP-2. In the next step, the Dde-protecting group was cleaved according to SP-4 and the free amino group treated with Fmoc-D-Asp-OAII.HCl (1.5 eq.) according to SP-2. The amino group of Fmoc-D-Asp-OAII.HCl was Fmoc-deprotected according to SP-3 and protected with Dde-OH (2.0 eq.) and DIPEA (4.0 eq.) in DMF at RT. The reaction was allowed to stir overnight. Afterwards, Allyl-deprotection of D-Asp was achieved applying SP-5. The next steps included the repetitive conjugation with Fmoc-D-Lys-OtBu.HCl (1.5 eq.), Fmoc-D-2-NaI—OH, Fmoc-D-Tyr(OtBu)-OH and Fmoc-L-2-NaI—OH according to SP-2. In order to conjugate TMA to D-Asp, selective Dde-deprotection was achieved applying SP-4 to afford the free amino group. TMA was coupled using TMA (2.0 eq.), HOBt (1.5 eq.), TBTU (1.5 eq.) and DIPEA (10 eq.) in DMF. The reaction was allowed to stir for 8 h at RT. After conjugation of TMA, Fmoc-deprotection of Fmoc-L-2-NaI—OH was achieved using SP-3. The N-terminal Fmoc-deprotected amino acid L-2-NaI—OH was conjugated with the chelator in the final step using DOTAGA-anhydride (2.0 eq.) and DIPEA (2.0 eq.). The reaction was allowed to stir for 48 h at RT. After completion of reaction with DOTAGA-anhydride, the peptide was cleaved from the resin according to SP-7.2, the crude product precipitated in Et.sub.2O, centrifuged and the supernatant removed. The final product was purified via RP-HPLC. RP-HPLC (10 to 80% B in 15 min): t.sub.R=7.57 min K′=2.79. Calculated monoisotopic mass (C.sub.89H.sub.108N.sub.14O.sub.32)=1,884.73; found: m/z=1,886.1 [M+H].sup.+, 943.5 [M+2H].sup.2+.

[0321] [.sup.natLu]DOTAGA-2-NaI-y-2-naI-k(d[N.sup.5-orn-C.sup.4-EuE]-TMA) ([.sup.natLu]PSMA-67): RP-HPLC (10 to 90% B in 15 min): t.sub.R=7.81 min K′=2.91. Calculated monoisotopic mass (C.sub.89H.sub.105N.sub.14O.sub.32Lu)=2,056.64; found: m/z=1,029.7 [M+2H].sup.2+.

7. Radiolabeling

[0322] .sup.68Ga-labeling: The .sup.68Ge/.sup.68Ga generator was eluted with aq. HCl (1.0 M), from which a fraction of 1.25 mL, containing approximately 80% of the activity (600 to 800 MBq), was transferred into a reaction vial (ALLTECH, 5 mL). The vial was beforehand loaded with the respective compound (5.0 nmol) and an aq. 2-(4-(2-hydroxyethyl)-1-piperazinyl)-ethanesulfonic acid (HEPES) solution (950 μL, 2.7 M). The reaction vial was heated for 5 min at 95° C. with subsequent fixation of the radiolabeled compound on a preconditioned SPE cartridge (C8 light, SepPak). After purging the cartridge with water (10 mL) in advance, the elution of the radiolabeled PSMA inhibitor from the cartridge was achieved with a mixture of EtOH and water (1/1; v/v), phosphate buffered saline (PBS) (1.0 mL) and again water (1.0 mL). At the end of radiolabeling, the EtOH was evaporated in vacuo and the tracer used without any further purification. Radiochemical purity was controlled using Radio-TLC (1.0 M sodium citrate buffer and 0.06 M NH.sub.4OAc/MeOH buffer (1/1; v/v)).

[0323] .sup.177Lu-labeling: The .sup.77Lu-labeled compounds were prepared as previously described [5] with minor modifications and used without further purification. In short, to NH.sub.4OAc-buffer (10 μL, 1.0 M, pH=5.9) was added the respective tracer (0.75 to 1.0 nmol, 7.5 to 10 μL), .sup.177LuCl.sub.3 (10 to 40 MBq; A.sub.S>3000 GBq/mg, 740 MBq/mL, 0.04 M HCl, ITG, Garching, Germany) and finally filled with trace-pure water (up to 100 μL) (Merck, Darmstadt, Germany). The reaction mixture was heated for 40 min at 95° C. and the radiochemical purity was determined using radio-TLC.

[0324] .sup.125I-labeling: Briefly, the stannylated precursor (SnBu.sub.3-BA)(OtBu)KuE(OtBu).sub.2 (PSMA-45) (approx. 0.1 mg) was dissolved in a solution containing peracetic acid (20 μL), [.sup.125]NaI (5.0 μL, approx. 21.0 MBq) (74 TBq/mmol, 3.1 GBq/mL, 40 mM NaOH, Hartmann Analytic, Braunschweig, Germany), MeCN (20 μL) and AcOH (10 μL). The reaction solution was incubated for 10 min at RT, loaded on a cartridge (C18 Sep Pak Plus, preconditioned with 10 mL MeCOH and 10 mL water) and rinsed with water (10 mL). After elution with a 1/1 mix (v/v) of EtOH and MeCN (2.0 mL), the solution was evaporated to dryness under a gentle nitrogen stream and treated with TFA (200 μL) for 30 min with subsequent evaporation of TFA. The crude product of ([.sup.125I]I-BA)KuE was purified by radio-RP-HPLC (20 to 40% B in 20 min): t.sub.R=13.0 min; K′=6.2.

8. Determination of HSA Binding

[0325] HSA binding experiments were performed as previously described [6]. The mobile phase consisted of a binary gradient system with a constant total flow rate of 0.5 mL/min. Mobile phase A was a 50 mm pH 6.9 NH.sub.4OAc-solution, mobile phase B was 2-Propanol (RP-HPLC grade, VWR, Germany). The gradient of mobile phase A was 100% from 0 to 3 min and from 3 min to the end of each run mobile phase B was set 20%. At each experimental day, the column was calibrated with nine reference substances to confirm the performance and to establish the non-linear regression. PSMA inhibitors were dissolved in a 0.5 mg/mL concentration in a mixture of 2-Propanol and NH.sub.4OAc-buffer (50 mm pH 6.9) (1/1; v/v). For each run, 10 μL of the solution containing the inhibitor was injected into the RP-HPLC system and the retention time measured. The literature HSA binding [%] was obtained from Valko et. al. or Yamazaki et al. [6, 7]. Non-linear regression was established with OriginPro 2016G.

9. Determination of Lipophilicity

[0326] Lipophilicity: The radiolabeled PSMA inhibitor (0.5 to 1.0 MBq) dissolved in PBS (500 μL, pH=7.4), was added to n-octanol (500 μL) in a reaction vial (1.5 mL), which was rigorously vortexed for 3 min (n=6). For quantitative phase separation, the mixture was centrifuged at 6,000 g for 5 min (Biofuge 15, Heraus Sepatech, Osterode, Germany). The activity from samples of each phase (100 μL) were measured in a γ-counter to obtain the log P.sub.(o/w) value.

10. Cell Experiments

[0327] Cell culture: PSMA-positive LNCAP cells (300265; Cell Lines Service GmbH) were cultivated in Dulbecco modified Eagle medium/Nutrition Mixture F-12 (1/1) (DMEM-F12, Biochrom) supplemented with fetal calf serum (FCS) (10%, Biochrom) and kept at 37° C. in a humidified CO.sub.2 atmosphere (5%). One day (24 h±2 h) prior to all experiments with LNCaP cells, the cultivated cells were harvested using a mixture of trypsin/ethylendiaminetetraacetate (0.05%/0.02%) and PBS and centrifuged. After centrifugation, the supernatant was disposed and the cell pellet resuspended in culture medium. Afterwards, cells were counted with a hemocytometer (Neubauer) and seeded in 24-well plates. IC.sub.50 values were determined transferring 150,000 cells/mL per well into 24-well plates, whereas internalization rates were obtained by transferring 125,000 cells/mL per well into 24-well PLL-coated plates.

11. Affinity (IC.SUB.50.)

[0328] After removal of the culture medium, the cells were treated once with HBSS (500 μL, Hank's balanced salt solution, Biochrom, Berlin, Germany, with addition of 1% BSA) and left 15 min on ice for equilibration in HBSS (200 μL, 1% BSA). Next, solutions (25 μL per well) containing either HBSS (1% BSA, control) or the respective ligand in increasing concentration (10.sup.−10 to 10.sup.−4 m in HBSS (1% BSA)) were added with subsequent addition of ([.sup.125I]I-BA)KuE (25 μL, 2.0 nm) in HBSS (1% BSA). All experiments were performed at least three times for each concentration. After 60 min incubation on ice, the experiment was terminated by removal of the medium and consecutive rinsing with HBSS (200 μL). The media of both steps were combined in one fraction and represent the amount of free radioligand. Afterwards, the cells were lysed with NaOH (250 μL, 1.0 m) and united with the HBSS (200 μL) of the following washing step. Quantification of bound and free radioligand was accomplished in a γ-counter.

12. Internalization

[0329] Subsequent to the removal of the culture medium, the cells were washed once with DMEM-F12-solution (500 μL, 5% BSA) and left to equilibrate for at least 15 min at 37° C. in DMEM-F12-solution (200 μL, 5% BSA). Afterwards, each well was treated with either DMEM-F12-solution (25 μL, 5% BSA) or 2-PMPA-solution (25 μL, 100 μm) for blockade. Next, the respective .sup.68Ga- or .sup.177Lu-labeled PSMA inhibitor (25 μL; 2.0 nm and 10 nm, respectively) was added and the cells incubated at 37° C. for 5, 15, 30 and 60 min, respectively. The experiment was terminated by placing the 24-well plate on ice for 3 min and the consecutive removal of the medium. Each well was rinsed with HBSS (250 μL) 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 ice-cold 2-PMPA-solution (250 μL, 10 μm in PBS) for 5 min and subsequent rinsing with ice-cold PBS (250 μL). The internalized activity was determined through incubation of the cells in NaOH (250 μL, 1.0 m) and the combination with the fraction of the subsequent washing step with again NaOH (250 μL, 1.0 m). Each experiment (control and blockade) was performed in triplicate for each time point. Free, surface bound and internalized activity was quantified in a γ-counter.

13. Externalization

[0330] Externalization kinetics of the radiolabeled PSMA inhibitors were determined using LNCaP cells, which were similarly prepared as described for the internalization assay. After an initial cell-washing step with DMEM-F12-solution (5% BSA), the cells were left to recondition for at least 15 min at 37° C. Subsequently, the LNCaP cells were incubated with the respective radiolabeled peptide (25 μL, 10.0 nm) at 37° C. for 60 min in a total volume of 250 μL in each well. After 60 min, the supernatant with the unbound free fraction was removed and measured in a γ-counter for the calculation of total added radioactivity. An acid wash step was avoided to warrant enzyme integrity during the following externalization and recycling study. To determine the recycling rate, fresh DMEM-F12-solution (250 μL, 5% BSA) was given to the cells to allow re-internalization. In contrast, re-internalization was inhibited by addition of DMEM-F12-solution containing 2-PMPA (225 μL DMEM-F12 (5% BSA) and 25 μL of 100 μm 2-PMPA-solution (PBS)). The cells were then incubated for 0, 20, 40 and 60 min at 37° C. Consequently, the supernatant was removed and the cells were washed with ice-cold HBSS (250 μL). The combination of the supernatant and the volume of the concomitant washing step with HBSS (200 μL) account for externalized radioligand at the investigated time point. Further, the cells were then washed with ice-cold 2-PMPA HBSS solution (250 μL, 10 μm) twice, combined and thus represented the fraction of membrane-bound radioligand. The determination of the internalized fraction was achieved by lysis as described for the internalization assay with NaOH (250 μL, 1.0 m). The activities of free, externalized, membrane-bound and internalized radioligand were quantified in a γ-counter.

14. Animal Experiments

[0331] All animal experiments were carried out in accordance with the general animal welfare regulations in Germany (Deutsches Tierschutzgesetz, approval #55.2-1-54-2532-71-13). For the tumor model, LNCaP cells (approx. 10.sup.7 cells) were suspended in serum-free DMEM-F12 medium and Matrigel (1/1; v/v) (BD Biosciences, Germany) and inoculated onto the right shoulder of male, 6 to 8 weeks old CB-17 SCID mice (Charles River Laboratories, Sulzfeld, Germany). Animals were used after the tumor size reached 4 to 8 mm in diameter for experiments.

15. PET

[0332] Imaging experiments were conducted using a Siemens Inveon small animal PET and the data analyzed by the associated Inveon Research Workplace software. Mice were anaesthetized with isoflurane and approx. 4.0 to 17 MBq of the .sup.68Ga-labeled compounds were injected via tail vein (approx. 150 to 300 μL). Dynamic imaging was carried out after on-bed injection for 90 min. The static blockade image was obtained after 1 h p.i. with 15 min acquisition time. PSMA-blockade was achieved by coinjection of 8 mg/kg of 2-PMPA-solution (PBS). All images were reconstructed using an OSEM3D algorithm without scanner and attenuation correction.

16. Biodistribution

[0333] Approximately 4.0 to 12.0 MBq (approx. 150 to 300 μL) of the respective .sup.68Ga- or .sup.177Lu-labeled PSMA inhibitors were injected into the tail vein of LNCaP tumor-bearing male CB-17 SCID mice, which were sacrificed after a specific timeframe (n=4, respectively). Selected organs were removed, weighted and measured in a γ-counter.

17. References in Example 1

[0334] 1. Šimeček, J., et al., A Monoreactive Bifunctional Triazacyclononane Phosphinate Chelator with High Selectivity for Gallium-68. Chem Med Chem, 2012. 7(8): p. 1375-1378. [0335] 2. Weineisen, M., et al., Development and first in human evaluation of PSMA I&T—A ligand for diagnostic imaging and endoradiotherapy of prostate cancer. Journal of Nuclear Medicine, 2014. 55(supplement 1): p. 1083-1083. [0336] 3. Weineisen, M., et al., Synthesis and preclinical evaluation of DOTAGA-conjugated PSMA ligands for functional imaging and endoradiotherapy of prostate cancer. EJNMMI research, 2014. 4(1): p. 1. [0337] 4. Weineisen, M., et al., 68Ga- and 177Lu-Labeled PSMA I&T: Optimization of a PSMA-Targeted Theranostic Concept and First Proof-of-Concept Human Studies. Journal of Nuclear Medicine, 2015. 56(8): p. 1169-1176. [0338] 5. Sosabowski, J. K. and S. J. Mather, Conjugation of DOTA-like chelating agents to peptides and radiolabeling with trivalent metallic isotopes. Nat. Protocols, 2006. 1(2): p. 972-976. [0339] 6. Valko, K., et al., Fast gradient HPLC method to determine compounds binding to human serum albumin. Relationships with octanol/water and immobilized artificial membrane lipophilicity. Journal of pharmaceutical sciences, 2003. 92(11): p. 2236-2248. [0340] 7. Yamazaki, K. and M. Kanaoka, Computational prediction of the plasma protein-binding percent of diverse pharmaceutical compounds. Journal of pharmaceutical sciences, 2004. 93(6): p. 1480-1494.

EXAMPLE 2: RESULTS

1. Effect of the Introduction of 2,4-Dinitrobenzoic Acid into the Linker Area of PSMA I&T

[0341] DOTAGA-y(3-I)fk(Sub-KuE) (PSMA I&T):

##STR00071##

[0342] DOTAGA-y(3-I)fk(L-Asu[KuE]-2,4-DNBA) (PSMA-36):

##STR00072##

TABLE-US-00003 TABLE 3 IC.sub.50 Internalization HSA PSMA Inhibitor Configuration [nM] [%] logP [%] [.sup.nat/177Lu]PSMA I&T —  7.9 ± 2.4* 75.5 ± 1.6* −4.12 ± 0.11* 78.6 [.sup.nat/177Lu]PSMA-36 2,4-DNBA- 5.3 ± 1.0 189.8 ± 37.5  n.d. 82.5 .fwdarw. Slightly higher affinity and increase of internalization by 251%.

2. The Binding Motif was Changed from EuK to EuE and the Peptide Spacer from -y(3-I)fk- to -y-2-naI-k-

[0343] DOTAGA-y(3-I)fk(Sub-KuE) (PSMA I&T):

##STR00073##

[0344] DOTAGA-y-2-naI-k(Suc-N.sup.5-orn-C.sup.4-EuE) (PSMA-46):

##STR00074##

TABLE-US-00004 TABLE 4 IC.sub.50 Internalization HSA PSMA inhibitor Configuration [nM] [%] logP [%] [.sup.nat/177Lu]PSMA I&T -y(3-I)fk- ∥ -KuE 7.9 ± 2.4  75.5 ± 1.6 −4.12 ± 0.11 78.6 [.sup.nat/177Lu]PSMA-46 -y-2-nal-k- ∥ -EuE 3.2 ± 1.1 216.2 ± 9.2 −4.21 ± 0.08 57.7 .fwdarw. Compared to the reference PSMA I&T, the improved reference compound PSMA-46 showed higher internalization and exhibited improved affinity. Thus, based on the structure PSMA-46, electron deficient aromatic residues were introduced in a further development step.

3. 4-Nitrophenylalanine and 2,4-DNBA were Introduced into the Peptide Spacer of PSMA-46

[0345] DOTAGA-y-2-naI-k(Suc-N.sup.5-orn-C.sup.4-EuE) (PSMA-46):

##STR00075##

[0346] DOTAGA-F(4-NO.sub.2)-y-2-naI-k(Suc-N.sup.5-orn-C.sup.4-EuE) (PSMA-52):

##STR00076##

[0347] 2,4-DNBA-Dap(DOTAGA)-y-2-naI-k(Suc-N.sup.5-orn-C.sup.4-EuE) (PSMA-53):

##STR00077##

TABLE-US-00005 TABLE 5 IC.sub.50 Internalization HSA PSMA inhibitor Configuration [nM] [%] logP [%] [.sup.nat/177Lu]PSMA-46 -y-2-nal-k- ∥ -EuE 3.2 ± 1.1 216.2 ± 9.2 −4.21 ± 0.08 57.7 [.sup.nat/177Lu]PSMA-52 -F(4-NO.sub.2)y-2-nal-k- 3.4 ± 0.2 229.9 ± 8.0 −4.11 ± 0.07 95.4 [.sup.nat/177Lu]PSMA-53 2,4 DNBA-Dap-y-2- 3.2 ± 0.5  293.6 ± 10.0 −4.08 ± 0.04 95.9 nal-k- .fwdarw. While the affinity remained similar, the introduction of 4-nitrophenylalanine increased slightly the internalization, however, by introduction of a further nitro group through 2,4-DNBA, a significant increase of internalization was possible. .fwdarw. Two electron withdrawing groups are preferred in order to increase the internalization.

4. Introduction of 4-amino-phenylalanine

[0348] DOTAGA-F(4-NH.sub.2)y-2-naI-k(Suc-N.sup.5-orn-C4-EuE) (PSMA-49):

##STR00078##

[0349] DOTAGA-F(4-NH.sub.2)y-2-naI-k(d[N.sup.5-orn-C.sup.4-EuE]-2,4-DNBA) (PSMA-61):

##STR00079##

[0350] DOTAGA-F(4-NH.sub.2)y-2-naI-k(d[N.sup.5-orn-C.sup.4-EuE]-TMA) (PSMA-62):

##STR00080##

TABLE-US-00006 TABLE 6 IC.sub.50 Internalization HSA PSMA inhibitor Configuration [nM] [%] logP [%] [.sup.nat/177Lu]PSMA I&T -k(Sub-ε-KuE) 7.9 ± 2.4  75.5 ± 1.6 −4.12 ± 0.11 78.6 [.sup.nat/177Lu]PSMA-49 -k(Suc-δ-orn(γ-EuE)) 2.5 ± 0.6 245.0 ± 4.2 −4.01 ± 0.11 74.2 [.sup.nat/177Lu]PSMA-61 -k((2,4-DNBA)-d-δ- 4.5 ± 0.4  359.5 ± 22.6 −4.07 ± 0.05 63.3 orn(γ-EuE)) [.sup.nat/177Lu]PSMA-62 -k((TMA)-d-δ-orn(γ- 4.0 ± 0.2 343.9 ± 6.0 −4.12 ± 0.05 >91.0 EuE)) .fwdarw. Both modifications, 2,4-DNBA and Trimesic acid, were able to further increase the internalization.

5. Introduction of an Electron Deficient Group in the Peptide Spacer

[0351] DOTAGA-F(4-NH.sub.2)y-2-naI-e(Abz-N.sup.5-orn-C.sup.4-EuE) (PSMA-60):

##STR00081##

[0352] 2,4-DNBA-Dap(DOTAGA)y-2-naI-e(Abz-N.sup.5-orn-C.sup.4-EuE) (PSMA-65):

##STR00082##

TABLE-US-00007 TABLE 7 IC.sub.50 Internalization HSA PSMA inhibitor Configuration [nM] [%] logP [%] [.sup.nat/177Lu]PSMA-60 -e(Abz-δ-orn(γ-EuE)) 6.6 ± 1.5 267.4 ± 7.9  −3.85 ± 0.13 98.5 [.sup.nat/177Lu]PSMA-65 2,4-DNBA-Dap 3.5 ± 0.3 340.2 ± 18.9 −4.15 ± 0.08 98.7 (DOTAGA)-y-2-nal- e(Abz-[HO-δ-orn-[γ- EuE]])-OH .fwdarw. The electron deficient aromatic modification 2,4-DNBA was able to increase the internalization.

6. Trimesic Acid was Incorporated into the Linker and Peptide Spacer of the PSMA Inhibitors

[0353] DOTAGA-F(4-NH.sub.2)y-2-naI-k(d[N.sup.5-orn-C.sup.4-EuE]-TMA) (PSMA-62):

##STR00083##

[0354] DOTAGA-Dap(TMA)y-2-naI-k(d[N.sup.5-orn-C.sup.4-EuE]-TMA) (PSMA-66):

##STR00084##

TABLE-US-00008 TABLE 8 IC.sub.50 Internalization HSA PSMA inhibitor Configuration [nM] [%] logP [%] [.sup.nat/177Lu]PSMA I&T -k(Sub-ε-KuE)  7.9 ± 2.4*  75.5 ± 1.6*  −4.12 ± 0.11* 78.6 [.sup.nat/177Lu]PSMA-62 -k((TMA)-d-δ-orn(γ- 4.0 ± 0.2 343.9 ± 6.0 −4.12 ± 0.05 >91.0 EuE)) [.sup.nat/177Lu]PSMA-66 DOTAGA- 3.8 ± 0.3 297.8 ± 2.0 −4.25 ± 0.14 64.4 Dap(TMA)y-2-nal- k(d[N5-orn-C4- EuE]-TMA) .fwdarw. The exchange of 4-amino-phenylalanine to Dap(TMA) resulted in similar affinity but a slightly reduced internalization capacity. Since both ligands seemed highly promising, both tracer were evaluated in further experiments. .fwdarw. The conclusion from these experiments is that an electron deficient aromatic residue is transferable and able to increase the internalization while maintaining a high affinity.

7. The Influence of Internalization on the Cell Retention In Vitro was Evaluated for the Compounds [.SUP.177.Lu]PSMA-62 and [.SUP.177.Lu]PSMA-66 in Comparison to [.SUP.177.Lu]PSMA I&T and [.SUP.177.Lu]PSMA-617

[0355] [.sup.177Lu]PSMA-66 demonstrated the highest intracellular activity in the tumor cells after 1 h followed by [.sup.177Lu]PSMA-62, although the internalization of [.sup.177Lu]PSMA-62 was found to be higher than for [.sup.177Lu]PSMA-66 (343.9% vs. 297.8%; respectively). Interestingly, even when re-internalization was blocked with 100 μM 2-PMPA-solution, the intracellular clearance [.sup.177Lu]PSMA-66 was lower than for all other investigated compounds. The difference compared to reference [.sup.117Lu]PSMA I&T was more than twofold, if re-internalization was blocked.

[0356] [.sup.177Lu]PSMA-66 has nine free carboxylic groups, which equal nine negative charges in vivo (pH=7.4). The extensively charged character of this compound could be a possible explanation for the protracted intracellular retention due to electrostatic repulsive effects from the negatively charged cell membranes.

8. In Vivo Experiments: Biodistribution

[0357]

TABLE-US-00009 TABLE 9 Biodistribution data of [.sup.177Lu]PSMA-49, [.sup.177Lu]PSMA-62 and [.sup.177Lu]PSMA-66 (in % ID/g) in LNCaP-tumor xenograft bearing CB-17 SCID mice at 1 h p.i. (n = 4, respectively). Between 3.5 MBq and 5.5 MBq of the respective .sup.177Lu-labeled radioligand were injected (0.15 to 0.25 nmol tracer). [.sup.177Lu]PSMA I&T [.sup.177Lu]PSMA-49 [.sup.177Lu]PSMA-62 [.sup.177Lu]PSMA-66 1 h p.i. 1 h p.i. 1 h p.i. 1 h p.i. blood 0.37 ± 0.10 0.46 ± 0.06 0.52 ± 0.03 0.50 ± 0.03 heart 0.58 ± 0.15 0.57 ± 0.11 0.58 ± 0.06 0.49 ± 0.06 lung 1.32 ± 0.45 1.47 ± 0.25 0.87 ± 0.11 0.62 ± 0.12 liver 0.37 ± 0.10 0.99 ± 0.19 0.37 ± 0.04 0.32 ± 0.02 spleen 13.8 ± 4.59 20.53 ± 6.79  4.62 ± 1.81 1.93 ± 0.04 pancreas 1.30 ± 1.39 0.56 ± 0.07 0.18 ± 0.05 0.15 ± 0.04 stomach 0.29 ± 0.06 0.35 ± 0.10 0.51 ± 0.2  0.28 ± 0.04 intestine 0.59 ± 0.50 0.30 ± 0.08 0.49 ± 0.25 0.21 ± 0.03 kidney 128.90 ± 10.74  162.96 ± 23.20  106.45 ± 17.18  117.47 ± 6.86  adrenal gland 6.25 ± 2.59 5.66 ± 1.66 1.92 ± 0.80 0.78 ± 0.07 muscle 0.18 ± 0.07 0.17 ± 0.02 0.12 ± 0.03 0.19 ± 0.07 bone 0.14 ± 0.04 0.29 ± 0.14 0.30 ± 0.08 0.37 ± 0.13 brain 0.03 ± 0.01 0.03 ± 0.01 0.03 ± 0.01 0.02 ± 0.01 tumor 4.69 ± 0.95 8.21 ± 0.23 8.00 ± 0.78 10.00 ± 0.44  tumor/blood 12.7 17.8 15.4 20.0 tumor/kidney  0.04  0.05  0.08  0.09 tumor/muscle 26.1 48.3 66.7 52.6

[0358] Compared to the tumor uptake of [.sup.177Lu]PSMA I&T after 1 h p.i. (4.69±0.95%), a significant increase in tumor activity was achieved through the improvement of internalization and affinity. As already observed for [.sup.177Lu]PSMA-16, [.sup.177Lu]PSMA-40 and [.sup.177Lu]PSMA-41, the extension of the peptide spacer with 4-amino-D-phenylalanine led to high kidney uptake and confirmed that this modification increases renal accumulation.

[0359] The introduction of trimesic acid into the linker of [.sup.177Lu]PSMA-62 led to a reduction of renal uptake (106.45±17.18% vs. 162.96±23.20%, respectively) and slightly lower tumor uptake compared to the reference. Since the internalization for [.sup.177Lu]PSMA-62 was higher in direct comparison to [.sup.177Lu]PSMA-49, the lower tumor uptake was unexpected. It is unclear to what extent internalization contributes to tumor uptake and if it is less important than affinity. The direct comparison of [.sup.177Lu]PSMA-49 and [.sup.177Lu]PSMA-62 indicates that affinity is more crucial since [.sup.177Lu]PSMA-49 was more affine towards PSMA (2.5±0.6 nM vs. 4.0±0.2 nM, respectively).

TABLE-US-00010 TABLE 10 Biodistribution data of [.sup.177Lu]PSMA I&T, [.sup.177Lu]PSMA-62 and [.sup.177Lu]PSMA- 66 and [.sup.177Lu]PSMA-71 (in % ID/g) in LNCaP-tumor xenograft bearing CB-17 SCID mice after 24 h p.i. (n = 4, respectively). Between 3.5 MBq and 5.5 MBq of the respective .sup.177Lu-labeled radioligand were injected (0.15 to 0.25 nmol tracer). [.sup.177Lu]PSMA I&T [.sup.177Lu]PSMA-62 [.sup.177Lu]PSMA-66 [.sup.177Lu]PSMA-71 24 h p.i. 24 h p.i. 24 h p.i. * 24 h p.i. blood 0.012 ± 0.01  0.004 ± 0.001 0.003 ± 0.001 0.008 ± 0.001 heart 0.05 ± 0.03 0.02 ± 0.01  0.03 ± 0.007 0.07 ± 0.01 lung 0.16 ± 0.03 0.03 ± 0.02  0.03 ± 0.007 0.05 ± 0.01 liver 0.05 ± 0.01 0.17 ± 0.07 0.14 ± 0.02 0.38 ± 0.14 spleen 1.94 ± 1.01 0.09 ± 0.06 0.09 ± 0.02 0.27 ± 0.15 pancreas 0.05 ± 0.02  0.01 ± 0.002  0.02 ± 0.007 0.15 ± 0.15 stomach 0.05 ± 0.02 0.03 ± 0.01 0.07 ± 0.03 0.20 ± 0.10 intestine 0.12 ± 0.06 0.03 ± 0.02 0.11 ± 0.07 0.27 ± 015  kidney 34.66 ± 17.20 5.26 ± 1.58 20.92 ± 2.51  32.36 ± 2.49  adrenal g. 1.06 ± 0.24 0.04 ± 0.02 0.09 ± 0.09 0.32 ± 0.15 muscle 0.01 ± 0.01 0.007 ± 0.001  0.02 ± 0.006  0.01 ± 0.001 bone 0.01 ± 0.01  0.04 ± 0.006 0.02 ± 0.01 0.04 ± 0.02 brain 0.02 ± 0.01  0.01 ± 0.006  0.01 ± 0.001  0.01 ± 0.002 tumor 4.06 ± 1.12 7.70 ± 1.35 5.73 ± 1.39 14.29 ± 0.89  t/blood 406 1925.0 1910.0 1786.3 t/kidney 0.1 1.5 0.3 0.4 t/muscle 406 1100.0 286.5 1429.0 t/liver 81.2 45.3 40.9 37.6

[0360] The results in Table 10 show distinct differences between the evaluated tracer [.sup.177Lu]PSMA-62, [.sup.177Lu]PSMA-66 and [.sup.177Lu]PSMA-71. Regarding renal clearance, it was visible that for all ligands a decrease in renal uptake compared to 1 h p.i. (Table 9) was observed. While [.sup.177Lu]PSMA I&T demonstrated after 24 h p.i. the highest renal uptake, [.sup.177Lu]PSMA-62 showed the lowest, which is in concordance to the observed renal clearance in the PET-study. The tumor uptake of [.sup.177Lu]PSMA-61 after 24 h p.i. remained almost stable over 23 h (8.00±0.75 vs. 7.70±1.35% ID/g, 1 h p.i. and 24 h p.i., respectively). Although [.sup.177Lu]PSMA-62 and [.sup.177Lu]PSMA-66 demonstrated similar in vitro parameter regarding internalization and affinity, the tumor uptake of [.sup.177Lu]PSMA-66 decreased to a greater extent from 1 h p.i. to 24 h p.i. (10.00±0.44 vs. 5.73±1.39% ID/g, 1 h p.i. and 24 h p.i., respectively) compared to [.sup.177Lu]PSMA-62. The stronger tumor retention together with the more beneficial tumor to liver and tumor to muscle ratios render [.sup.177Lu]PSMA-62 superior compared to [.sup.177Lu]PSMA-66. The highest tumor uptake was observed for PSMA-71, which also exhibited the highest HSA-binding value. While the kidney uptake 24 h p.i. was similar to [.sup.177Lu]PSMA I&T, the tumor uptake was more than threefold higher for [.sup.177Lu]PSMA-71 (4.06±1.12 vs. 14.29±0.89% ID/g, [.sup.177Lu]PSMA I&T and [.sup.177Lu]PSMA-71, respectively).

[0361] In this respect, [.sup.177Lu]PSMA-71 may be considered as a particularly valuable tracer for endoradiotherapeutic application and is a candidate for clinical application.

9. In Vivo Experiments: PET-Imaging

[0362] Effect of 2,4-Dinitrobenzoic Linker Substitution on EuK-Based Inhibitors

[0363] The EuK-based inhibitor PSMA-36 was evaluated in a small animal PET scan to examine the influence of the 2,4-dinitrobenzoic acid in the linker on the in vivo distribution.

[0364] The logarithmic TACs plot shows specific kidney and tumor uptake of [.sup.68Ga]PSMA-36. Linear decrease of the blood pool activity and in the muscle region imply low unspecific binding and fast excretion. Accumulation in the tumor remained steady over the observed period. Although [.sup.177Lu]PSMA-36 exhibited a more than threefold higher internalization rate than [.sup.177Lu]PSMA I&T, tumor uptake was only moderate with 3.5% ID/mL after 85 min p.i. The most significant difference compared to [.sup.68Ga]PSMA I&T was the high and steady uptake in the lacrimal and salivary gland, displaying approx. 2% ID/mL in both regions. Since the only structural difference to the reference [.sup.68Ga]PSMA I&T is the introduction of 2,4-dinitrobenzoic acid, the linker modification must be the reason for this enhanced uptake. However, further studies are necessary to confirm this effect.

[0365] It is also interesting, that the clearance in these regions was slower compared to the blood pool and muscle, which implies that a distinct retaining mechanism is involved. It was reported that PSMA participates in angiogenesis during ocular neovascularization in mice and might therefore explain the uptake of [.sup.68Ga]PSMA-36 [1]. Tracer accumulation in the salivary glands is a common problem during clinical therapeutic approaches with .sup.177Lu-labeled PSMA inhibitors [2]. Drug uptake into the salivary glands depends on intra- or extracellular pathways and most commonly on simple diffusion among the phospholipid bilayer of the acinar cells. Saliva drug concentrations are reflected predominantly by the free, non-ionized fraction in the blood plasma regarding passive diffusion [3-5]. In this respect, it seems highly unlikely that passive diffusion is responsible for the salivary gland uptake. Other mechanisms have to be involved since EuK-based PSMA inhibitors are highly charged in vivo and thus exhibit high polarity. Further, passive diffusion would be visualized during PET scans in every region as high background activity, which does not occur for most PSMA ligands since the rapid clearance removes the tracer from the blood pool.

[0366] Effect of Trimesic Acid on EuE-Based Inhibitors

[0367] Substitution of the PSMA ligands with electron deficient aromatic systems resulted in enhanced internalization rates of [.sup.177Lu]PSMA-62 and [.sup.177Lu]PSMA-66 (343.9% and 297.8%, respectively). Both ligands were therefore evaluated and compared among each other in PET studies.

[0368] Both tracer exhibited excellent tracer kinetics regarding kidney, muscle and blood pool uptake. Specific uptake in the kidneys was slightly higher for [.sup.68Ga]PSMA-66 compared to [.sup.68Ga]PSMA-62 (45.3% ID/mL vs. 34.8% ID/mL, respectively). The higher renal accumulation in the PET scan of [.sup.68Ga]PSMA-66 compared to [.sup.68Ga]PSMA-62 nicely correlated with the biodistribution experiments. TACs for muscle and blood pool activity showed linear uptake and ongoing clearance from these compartments.

10. References in Example 2

[0369] 1. Grant, C. L., et al., Prostate specific membrane antigen (PSMA) regulates angiogenesis independently of VEGF during ocular neovascularization. PloS one, 2012. 7(7): p. e41285. [0370] 2. Kulkarni, H. R., et al., PSMA-Based Radioligand Therapy for Metastatic Castration-Resistant Prostate Cancer: The Bad Berka Experience Since 2013. Journal of Nuclear Medicine, 2016. 57(Supplement 3): p. 97S-104S. [0371] 3. Haeckel, R., Factors influencing the saliva/plasma ratio of drugs. Annals of the New York Academy of Sciences, 1993. 694(1): p. 128-142. [0372] 4. Jusko, W. J. and R. L. Milsap, Pharmacokinetic Principles of Drug Distribution in Salivaa. Annals of the New York Academy of Sciences, 1993. 694(1): p. 36-47. [0373] 5. Aps, J. K. and L. C. Martens, Review: the physiology of saliva and transfer of drugs into saliva. Forensic science international, 2005. 150(2): p. 119-131. [0374] 6. Young, J. D., et al., 68Ga-THP-PSMA: a PET imaging agent for prostate cancer offering rapid, room temperature, one-step kit-based radiolabeling. Journal of Nuclear Medicine, 2017: p. jnumed. 117.191882. [0375] 7. Wüstemann, T., et al., Design of Internalizing PSMA-specific Glu-ureido-based Radiotherapeuticals. Theranostics, 2016. 6(8): p. 1085. [0376] 8. Hao, G., et al., A multivalent approach of imaging probe design to overcome an endogenous anion binding competition for noninvasive assessment of prostate specific membrane antigen. Molecular pharmaceutics, 2013. 10(8): p. 2975-2985. [0377] 9. Soret, M., S. L. Bacharach, and I. Buvat, Partial-volume effect in PET tumor imaging. Journal of Nuclear Medicine, 2007. 48(6): p. 932-945. [0378] 10. Bao, Q., et al., Performance evaluation of the inveon dedicated PET preclinical tomograph based on the NEMA NU-4 standards. Journal of Nuclear Medicine, 2009. 50(3): p. 401-408.