LABELED INHIBITORS OF PROSTATE SPECIFIC MEMBRANE ANTIGEN (PSMA), THEIR USE AS IMAGING AGENTS AND PHARMACEUTICAL AGENTS FOR THE TREATMENT OF PSMA-EXPRESSING CANCERS

Abstract

The present invention relates to a compound of formula (1)

##STR00001##

or a pharmaceutically acceptable salt or solvate thereof, wherein Y.sup.3 is O or S, wherein s, t, u and w are, independently of each other, 0 or 1, wherein i is an integer of from 1 to 3, wherein j is an integer of from 3 to 5, and wherein Z.sup.1, Z.sup.2 and Z.sup.3 are independently of each other, selected from the group consisting of CO.sub.2H, SO.sub.2H, SO.sub.3H, OSO.sub.3H, and OPO.sub.3H.sub.2, R.sup.1 is CH.sub.3 or H, preferably H, X is selected from the group consisting of alkylaryl, aryl, alkylheteroaryl and heteroaryl, Y.sup.1 and Y.sup.2 are independently, of each other, selected from the group consisting of aryl, alkylaryl, cycloalkyl, heterocycloalkyl, heteroaryl and alkylheteroaryl, and wherein A is a chelator residue having a structure selected from the group consisting of (Ia), (Ib) and (Ic)

##STR00002##

wherein R.sup.2, R.sup.3, R.sup.4 and R.sup.5 are, independently of each other, selected from the group consisting of H, CH.sub.2COOH and CH.sub.2C(O)NH.sub.2 or wherein R.sup.2 and R.sup.4 form a (CH.sub.2).sub.n bridge with n being an integer of from 1 to 3, wherein n is preferably 2, and wherein r, v and q, are independently of each other, 0 or 1, with the proviso that in case u and w are 0, q and v are 0, and (A) wherein u and w are 1, or (B) wherein u is 0 and w is 1, and wherein A is selected from (Ia) or (Ib), or (C) wherein A is not

##STR00003##

Further, the present invention relates to a complex comprising (a) a radionuclide, and (b) the compound, as described above or below, or a salt, solvate, metabolite or prodrug thereof.

Further, the present invention relates to a pharmaceutical composition comprising a compound, as described above or below, or a complex, as described above or below. Further, the present invention relates to a compound, as described above or below, or a complex, as described above or below, or a pharmaceutical composition, as described above or below, for use in treating, ameliorating or preventing PSMA-expressing cancer and/or metastases thereof, in particular prostate cancer and/or metastases thereof. Further, the present invention relates to a compound, as described above or below, or a complex, as described above or below, or a pharmaceutical composition, as described above or below, for use in diagnostics. In addition, the present invention relates to a compound, as described above or below, or a complex, as described above or below, or a pharmaceutical composition, as described above or below, for use in the diagnosis of cancer, in particular of PSMA-expressing cancer and/or metastases thereof, in particular prostate cancer and/or metastases thereof.

Claims

1-16. (canceled)

17. A compound of formula (1): ##STR00075## or a pharmaceutically acceptable salt or solvate thereof, wherein: Y.sup.3 is O or S; s, t, u and w are, independently of each other, 0 or 1; i is an integer of from 1 to 3; j is an integer of from 3 to 5; Z.sup.1, Z.sup.2 and Z.sup.3 are independently of each other, selected from the group consisting of CO.sub.2H, SO.sub.2H, SO.sub.3H, OSO.sub.3H, and OPO.sub.3H.sub.2; R.sup.1 is CH.sub.3 or H, preferably H; X is ##STR00076## Y.sup.1 and Y.sup.2 are independently, of each other, selected from the group consisting of optionally substituted aryl, optionally substituted alkylaryl (-alkyl-aryl-), optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted heteroaryl and optionally substituted alkylheteroaryl (-alkyl-heteroaryl); and A is a chelator residue having the structure: ##STR00077##

18. The compound of claim 17, wherein Y.sup.1 is ##STR00078##

19. The compound of claim 17, wherein: i is 2; j is 4; and the compound has preferably the structure (1a) ##STR00079##

20. The compound of claim 17, wherein all amino acid residues present in the compound have L-configuration.

21. The compound of claim 17, wherein: u and w are 1 and Y.sup.3 is S; and Y.sup.2 is ##STR00080## and wherein R.sup.6, R.sup.7, R.sup.8 and R.sup.9 are, independently of each other H or alkyl, preferably H.

22. The compound of claim 17, wherein A is ##STR00081##

23. The compound of claim 19, wherein: u and w are 1; and Y.sup.3 is S; and Y.sup.2 is ##STR00082## R.sup.6, R.sup.7, R.sup.8 and R.sup.9 are, independently of each other H or alkyl, preferably H; and A is ##STR00083##

24. The compound of claim 17 having a structure selected from the group consisting of: ##STR00084##

25. The compound of claim 17 having a structure selected from the group consisting of ##STR00085##

26. The compound of claim 17, wherein u and w are 0.

27. The compound of claim 17, wherein A is: ##STR00086##

28. The compound of claim 19, wherein u and w are 0 and wherein A is: ##STR00087##

29. The compound of claim 17, having the structure: ##STR00088##

30. The compound of claim 17, having the structure: ##STR00089##

31. A Complex comprising: (a) a radionuclide of Pb; and (b) the compound of claim 17 or a salt thereof.

32. The complex of claim 31, wherein, the radionuclide is selected from the group consisting of .sup.203Pb, .sup.212Pb, .sup.211Pb, .sup.213Pb, .sup.214Pb, .sup.209Pb, .sup.198Pb, and .sup.197Pb.

33. The complex of claim 31, wherein the radionuclide is .sup.203Pb or .sup.212Pb.

34. A pharmaceutical composition comprising the complex of claim 31.

35. A method for treating, ameliorating or preventing PSMA-expressing cancer and/or metastases thereof, in particular prostate cancer and/or metastases thereof, comprising administering to a subject in need the complex of claim 31.

36. The method of claim 35, wherein the radionuclide is .sup.212Pb.

37. A method for diagnosing PSMA-expressing cancer and/or metastases thereof, in particular prostate cancer and/or metastases thereof, comprising using a diagnostic method comprising the complex of claim 31.

38. The method of claim 37, wherein the diagnostic method is tumor imaging using positron emission tomography (PET) or single photon emission computed tomography (SPECT), and wherein the radionuclide is preferably .sup.203Pb.

39. A compound or a pharmaceutically acceptable salt or solvate thereof, the compound having the following formula: ##STR00090## or the following structure ##STR00091##

40. A complex comprising: (a) a radionuclide selected from .sup.68Ga and .sup.177Lu, and (b) the compound of claim 39 or a salt thereof.

41. A pharmaceutical composition comprising the complex of claim 40.

42. A method for treating, ameliorating or preventing PSMA-expressing cancer and/or metastases thereof, in particular prostate cancer and/or metastases thereof, comprising administering to a subject in need the complex of claim 40.

43. A method for diagnosing PSMA-expressing cancer and/or metastases thereof, in particular prostate cancer and/or metastases thereof, comprising using a diagnostic method comprising the complex of claim 40.

44. The method of claim 43, wherein the diagnostic method is tumor imaging using positron emission tomography (PET) or single photon emission computed tomography (SPECT).

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0193] FIG. 1: Table 1A: Overview over preferred compounds. It is to be understood that if a stereocenter in the respective shown structure is not specified, this shall mean that all respective stereoisomers shall be encompassed, in isolated form as well in form of a mixture of the respective stereoisomers, Table 1B: Overview over very preferred compounds.

[0194] FIG. 2: Reaction scheme for the synthesis of the PSMA-chelator conjugates (a) triphosgene, DIPEA, CH.sub.2Cl.sub.2, 0 C.; (b) H-Lys(Alloc)-2CT-resin, CH.sub.2Cl.sub.2 (c) Pd[P(C.sub.6H.sub.5).sub.3].sub.4, morpholine, CH.sub.2Cl.sub.2; (d) Fmoc-2-Nal-OH, HBTU, DIPEA, DMF; (e) 20% piperidine, DMF; (f) trans-4-(Fmoc-aminomethyl)-cyclohexanecarboxylic acid, HBTU, DIPEA, DMF; (g) 20% piperidine, DMF; (h) chelator, HBTU (if necessary), DIPEA, DMF; (i) 95% TFA, 2.5% H.sub.2O, 2.5% TIPS.

[0195] FIG. 3: PSMA inhibition potencies and specific internalization values of the novel PSMA ligands labeled with the nuclide specified in the table. Competitive cell binding was performed using the PSMA-positive C4-2 cell line. The new ligands showed good inhibition potencies with K.sub.i in the low nanomolar range and partially higher internalization values (specific lysate) than PSMA-617.

[0196] FIGS. 4A-B: A.PET scan and time-activity curves of C4-2 tumour bearing mice showing biodistribution of .sup.64Cu-PSMA-617, .sup.64Cu-CA003 and .sup.64Cu-CA023. .sup.64Cu-PSMA-617 shows high accumulation in the tumor and kidneys but also in the liver. A. Visualization of biodistribution of .sup.64Cu-PSMA-617, .sup.64Cu-CA003 and .sup.64Cu-CA023 at different time points (0-20 min, 40-60 min, 2 h, 48 h). B. Time-activity curve of dynamic PET scan (0-60 min) of C4-2 tumour bearing mice showing biodistribution of .sup.64Cu-PSMA-617 and .sup.64Cu-CA003. Standard-uptake-values (SUV) for tumor and liver show higher uptake of .sup.64Cu-PSMA-617 in the liver than in the tumor, contrary to .sup.64Cu-CA003.

[0197] FIG. 5: Maximum standard-uptake values (mSUV) of .sup.64Cu-PSMA-617, .sup.64Cu-CA003 and .sup.64Cu-CA023 in PET images of C4-2 tumour bearing mice at different time points (0-48 h). .sup.64Cu-CA003 shows high accumulation in the tumor and kidneys, .sup.64Cu-CA023 shows high accumulation in the tumor and fast clearance from the kidneys.

[0198] FIG. 6A: Accumulation of .sup.64Cu-CA003 in dissected organs of interest in C4-2 tumour bearing mice confirms tumor specificity and fast clearance from the kidneys.

[0199] FIG. 6B: Accumulation of .sup.64Cu-CA023 in dissected organs of interest in C4-2 tumour bearing mice confirms tumor specificity and fast clearance from the kidneys

[0200] FIG. 6C: Organ distribution of 0.025 nmol of .sup.64Cu-CA003 at the time points: 10 min, 1 h, 4 h, 24 h and 72 h after injection. Values are expressed as % ID/g of tissue standard deviation; n=3 for all tissues.

[0201] FIG. 6D: In a blockade experiment (B), the radiotracer .sup.64Cu-CA003 (0.030 nmol) was injected at the same time as 2 mg of PSMA-617 per kilogram of body weight. Values are expressed as % ID/g of tissue standard deviation; n=3 for all tissues.

[0202] FIG. 7: .sup.64Cu-CA003 (200 MBq, 0.5 nmol) PET/CT maximum intensity projections of a patient at 2 h (A) and 20 h post injection (B). The red arrows point to selected right shoulder soft-tissue infiltration from scapula origin, lung, bone and lymph-node metastases increased in contrast over time. The hepatobiliary clearance causes hot-spots inside the intestine; cross-sectional slices (C) are mandatory to avoid false-positive readings.

[0203] FIG. 8: Planar scintigraphic imaging of different .sup.203Pb-labeled compounds A) at 1 h after tail vein injection and (B) time course of the distribution of .sup.203Pb-CA012 in BALB/c nu/nu C4-2 tumor bearing mice. The radiolabelled derivatives of CA009 and CA012 show high uptake of the tracer in the tumor tissue. The kinetics of the uptake determined for .sup.203Pb-CA012 reveals a long retention of the radiotracer in the tumor tissue. The selectivity of uptake of .sup.203Pb-CA012 is enhanced when compared to of .sup.203Pb-CA009. This is the result of the rapid clearance of the non-target organs.

[0204] FIG. 9: Organ distribution of .sup.203Pb-PSMA-CA012 in tumor bearing mice 0.025 nmol of .sup.203Pb-PSMA-CA012. This quantification confirms the results of the imaging experiment. The high ratio of the tumor to kidney uptake is achieved due to the high excretion valued observed for the kidneys.

[0205] FIG. 10: Geometric mean images of .sup.203Pb-CA012 planar scans over time (A) in comparison to a treatment scan with .sup.177Lu-PSMA-617 (B); both acquired with a Medium Energy collimator.

[0206] FIG. 11: Safety Dosimetry estimate of diagnostic .sup.203Pb-CA012 (left column) and therapeutic .sup.212Pb-CA012 (right column) based on the male-adult phantom in OLINDA (ULI=upper large intestine, LLI=lower large intestine)

[0207] FIG. 12: Dosimetry of .sup.212Pb-CA012 (TCMC-PSMA-617) for salivary glands, randomly chosen tumor lesions (sphere model) and the presumably dose-limiting organs in comparison .sup.213Bi-PSMA-617 and .sup.225Ac-PSMA-617.

[0208] FIG. 13A: Maximum intensity projection of a .sup.68Ga-PSMA-CA028 PET scan of a patient with multiple lymph node prostate cancer metastases at 1 h and 3 h post injection. Cross-sectional slices demonstrate lymph-node metastases (indicated by red arrows) axillar and hilar (C); also delineable on the correlated CT which serves as a standard of reference.

[0209] FIG. 13B. Maximum intensity projections of PSMA-PET performed 1 h (A) and 3 h (B) post injection of 295 MBq/20 nmol .sup.68Ga-CA030. Arrows point to the position of the cross-sectional slices demonstrating bone-metastases in multiple regions of the axial skeleton (C-E). In CT (F), no typical osteoblastic reactions allowed tumor-delineation by morphological information alone.

[0210] FIG. 14: Organ distribution expressed as % ID/g of tissue SD (n=3) of .sup.68Ga-PSMA-CA028 at 1 h, 2 h and 4 h after injection.

[0211] FIG. 15: Comparison of the whole-body small-animal PET imaging of selected .sup.68Ga-PSMA ligands 2 h after injection (A) and time course of .sup.68Ga-CA028 (B) and the time course of .sup.68Ga-CA030 (C), in BALB/c nu/nu mice bearing a C4-2 tumor xenograft.

[0212] FIG. 16: Serum stability of .sup.177Lu-CA028, .sup.177Lu-CA029, .sup.177Lu-CA030 in comparison with .sup.177Lu-PSMA-617 at 37 C. over 72 h (meanSD, n=3) as determined by radio-ITLC.

[0213] FIG. 17: Serum stability of .sup.64Cu-CA003, .sup.64Cu-CA005 and .sup.64Cu-PSMA-617 at 37 C. over 72 h (meanSD, n=4) as determined by radio-ITLC.

[0214] FIG. 18: Serum stability of .sup.64Cu-CA003, .sup.64Cu-CA005 and .sup.64Cu-PSMA-617 at 37 C. over 72 h (meanSD, n=4) as determined by activity measurement.

[0215] FIG. 19: In vivo metabolite analysis of .sup.64Cu-CA003 in a BALB/c nude mouse (no tumor) at 10 min p.i. Radio-HPLC chromatograms of extracts from the kidney, the blood and the liver show that the activity elutes at the retention time of the intact tracer. This proves the integrity of the copper complex within the main distribution period.

[0216] FIG. 20: Radio-HPLC chromatograms of extracts of .sup.64Cu-CA003 in liver in comparison with of .sup.64Cu-chloride in the liver in a BALB/c nude mouse (no tumor) at 10 min p.i.

[0217] FIG. 21A: Whole-body small-animal PET scans as maximum-intensity projections of BALB/c nu/nu mice bearing C4-2 tumor xenografts. PET imaging of .sup.64Cu-PSMA-617 (10 MBq, 0.2 nmol), .sup.64Cu-PSMA-CA003 (10 MBq, 0.2 nmol),

[0218] FIG. 21B: .sup.64Cu-PSMA-CA003 (5 MBq, 0.030 nmol) co-injected with an excess amount of non-labeled PSMA-617 (2 mg per kilogram of body weight) and .sup.64Cu-chloride (10 MBq). The color bar gives a link between the SUV and the color scale of the PET image with 0=minimum and 4=maximum.

[0219] FIG. 22: Comparison of the whole body small animal PET scans as maximum-intensity projections of BALB/c nu/nu mice bearing C4-2 tumor xenografts. PET imaging of the four newly PSMA ligands radiolabeled with .sup.68Ga (20 MBq; 0.2 nmol) 2 h post injection (A), the time course of .sup.68Ga-CA028 (B) and the time course of .sup.68Ga-CA030 (C). The color bar gives a link between the SUV and the color scale of the PET image with 0=minimum and 4E0=maximum.

[0220] FIG. 23: Blood time-activity curve for .sup.68Ga PSMA-CA027 (0.6 nmol, 5 MBq) and .sup.68Ga PSMA-CA028 (0.6 nmol, 6 MBq), including bi-exponential curve fit.

[0221] FIG. 24. In vivo metabolite analysis of .sup.177Lu-CA028 in comparison with .sup.177Lu-PSMA-617 (10 MBq, 0.2 nmol in approximately 100 l of 0.9% saline) in a BALB/c nude mouse (tumor) at 1 h p.i. Radio-HPLC chromatograms of extracts from the kidney, the blood, the liver and tumor show that the activity elutes at the retention time of the intact tracer. This proves the integrity of the complex within the main distribution period.

[0222] FIG. 25. Organ distribution of 0.05 nmol of .sup.68Ga-CA028 expressed as % ID/g of tissue SD (n=3) at 20 min, 1 h, 2 h and 4 h after injection.

[0223] FIG. 26. Radio-HPLC chromatograms of the novel compounds labeled with .sup.64Cu.

[0224] FIG. 27: Time-activity curves of the novel PSMA ligands labeled with .sup.68Ga. (A) Time activity curves for kidney and (B) time activity curves for the tumor up to 1 h post injection. Data are mean standardized uptake values (SUV mean).

[0225] FIG. 28. (A) PET image of 9 MBq (0.30 nmol) .sup.64Cu-CA003 10 min post injection in a female Swiss mouse. The maximum intensity projection (MIP) illustrates circulation in the blood and renal uptake. (B) PET image of a female Swiss mouse at 10 min p.i. of 10 MBq .sup.64Cu 10 min post injection. The maximum intensity projection (MIP) illustrates a strong uptake in the liver and the kidneys.

DETAILED DESCRIPTION

[0226] The following examples shall merely illustrate the invention. Whatsoever, they shall not be construed as limiting the scope of the invention.

EXAMPLES

Materials and Methods

[0227] Solvents and chemicals were purchased from Merck (Darmstadt, Germany) and Sigma-Aldrich (Munich, Germany) and used without further purification. The in vitro experiments were conducted in triplicate and at least three independent sets of data were obtained for each experiment performed. The PET imaging of the prostate cancer patient was consented by the University Hospital Heidelberg following the German laws in vigor and granted the Helsinki Declaration (permit S321/2012).

Synthesis of the Chelator Moieties

[0228] The chelator moieties were synthesized in high yields and characterized by LC-MS. The synthesis of the chelator 4-[(1,4,8,11-tetraazacyclotetradec-1-yl)-methyl]benzoic acid, a bifunctional macrocyclic cyclam analogue, was described by Studer and Kaden (Studer M, and Kadan, T. A. One-step synthesis of mono-N-substituted azamacrocycles with a carboxylic group in the side-chain and their complexes with Cu.sup.2+ and Ni.sup.2+. Helvetica. 1986; 69:2081-2086), while 4-carboxymethyl-11-(1,3-dicarboxypropyl)-1,4,8,11-tetraazabicyclo[6.6.2]hexadecane-pentanedioic acid, a cross-bridged chelator, was reported by Boswell et al. (Boswell C A, Regino C A, Baidoo K E, et al. Synthesis of a cross-bridged cyclam derivative for peptide conjugation and .sup.64Cu radiolabeling. Bioconjug Chem. 2008; 19:1476-1484).

I. General Procedure: Synthesis of Novel PSMA Ligands

[0229] The PSMA-binding motif was prepared by solid-phase synthesis on a 2-chlorotrityl resin (2CT-resin), as previously described by Eder et al. (Eder M, Schfer M, Bauder-Wst U, et al. .sup.68Ga-complex lipophilicity and the targeting property of a urea-based PSMA inhibitor for PET imaging. Bioconjug Chem. 2012; 23:688-697) and Benesovi et al. (Benesova M, Schfer M, Bauder-Wst U, et al. 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; 56:914-920) see FIG. 2. For this purpose Fmoc-Lys(Alloc)-OH was immobilized on an equimolar amount of 2-chlorotrityl resin. Afterwards, the isocyanate (2) of the glutamyl moiety by was generated using triphosgene. The -allyloxycarbonyl-protected lysine immobilized on 2-chloro-tritylresin was added and reacted for 16 h with careful agitation resulting in compound 3. The resin was filtered off and the allyloxycarbonyl-protecting group was cleaved to obtain (4). In order to obtain compounds CA001 and CA027, the respective chelator was coupled to this intermediate. Subsequently, the PSMA coupled to the chelator was cleaved from the resin. Alternatively, the coupling of Fmoc-2-naphthylalanine was proceeded to obtain (5). In order to obtain compounds CA002, CA005, CA008 and CA011 the respective chelator was coupled to this intermediate. Subsequently, the PSMA coupled to the chelator was cleaved from the resin. Alternatively, trans-4-(Fmoc-aminomethyl) cyclohexanecarboxylic acid was coupled to obtain (6), the compound to which the respective chelator was coupled to obtain compounds CA003, CA006, CA009, CA012, CA022, CA023, CA024, CA025, CA026, CA028, CA029, and CA030. Subsequently, the PSMA coupled to the chelator was cleaved from the resin. The structures were confirmed by HPLC and MS-LC. The substances were isolated by preparative HPLC using water-acetonitrile gradients containing trifluoroacetic acid. For this, the compounds were purified using a gradient of 20-50% of acetonitrile in water over 15 min. The purified compounds were analyzed by analytical HPLC (0-100%) acetonitrile in water containing trifluoroacetic acid over 5 min, Monolith RP HPLC column 1003 mm and LC/MS. The product fractions were pooled and lyophilized.

II. Ligands for Imaging and Therapy with Copper Isotopes

Specification for (CA001)

[0230] The product was obtained by incubating the resin (compound 4) with 1.5 equivalents of CTPA-NHS-ester (4-[(1,4,8,11-tetraazacyclotetradec-1-yl)-methyl]benzoic acid) and 10 equivalents of DIPEA in 500 l of DMF. The compound was purified and the final product was analyzed by HPLC as described above (see section I.). HPLC-retention time: 1.68 min; ESI-MS (m/z): [M+H].sup.+ (calculated C.sub.30H.sub.50N.sub.7O.sub.8): 636.37 (636.36)

##STR00063##

[0231] Chemical structure of the chelator CTPA-NHS-ester, the compound used in the synthesis of CA001, CA002 and CA003.

Specification for CA002

[0232] The product was obtained by incubating the resin (compound 5) with 1.5 equivalents of CTPA-NHS-ester (4-[(1,4,8,11-tetraazacyclotetradec-1-yl)-methyl]benzoic acid) and 10 equivalents of diisopropylamine (DIPEA) in 500 l of DMF. The compound was purified and the final product was analyzed by HPLC as described above (see section I.). HPLC-retention time: 2.39 min; ESI-MS (m/z): [M+H]+(calculated C.sub.43H.sub.61N.sub.8O.sub.9): 833.42 (833.45)

Specification for CA003

[0233] The product was obtained by incubating the resin (compound 6) with 1.5 equivalents of CTPA-NHS-ester (4-[(1,4,8,11-tetraazacyclotetradec-1-yl)-methyl]benzoic acid) and 10 equivalents of DIPEA in 500 l of dimethylformamide (DMF). The compound was purified and the final product was analyzed by HPLC as described above (see section I.). HPLC-retention time: 2.50 min; ESI-MS (m/z): [M+H]+(calculated C.sub.51H.sub.74N.sub.9O.sub.10): 972.52 (972.55)

Specification for CA005

[0234] The product was obtained by incubating the resin (compound 5) with 1.5 equivalents of Cross bridged-TE2A chelator, 0.98n.sub.chelator HBTU and 10 equivalents of DIPEA in 500 l of DMF. The compound was purified and the final product was analyzed by HPLC as described above (see section I.). HPLC-retention time: 2.38 min; ESI-MS (m/z): [M+H].sup.+ (calculated C.sub.44H.sub.64N.sub.8O.sub.13): 913.45 (913.47)

##STR00064##

[0235] Chemical structure of the chelator chelator 8-carboxymethyl-cross bridged-TE2A, the compound used in the synthesis of CA005, and CA006.

Specification for CA006

[0236] The product was obtained by incubating the resin (compound 6) with 1.5 equivalents of Cross bridged-TE2A chelator, 0.98n.sub.chelator HBTU and 10 equivalents of DIPEA in 500 l of DMF. The compound was purified and the final product was analyzed by HPLC as described above (see section I.). HPLC-retention time: 2.55 min; ESI-MS (m/z): [M+H]+(calculated C.sub.52H.sub.78N.sub.9O.sub.14): 1052.62 (1052.56)

Specification for CA022

[0237] The product was obtained by incubating the resin (compound 6) with 1.5 equivalents of cross bridged-CTPA chelator and 10 equivalents of DIPEA in 500 l of DMF. The compound was purified and the final product was analyzed by HPLC as described above (see section I.). HPLC-retention time: 2.72 min; ESI-MS (m/z): [M+H]+(calculated C.sub.53H.sub.76N.sub.9O.sub.10): 998.56 (998.57)

##STR00065##

[0238] Chemical structure of the chelator Cross-bridged-CTPA, the compound used in the synthesis of CA022

Specification CA023

[0239] The product was obtained by incubating the resin (compound 6) with 1.5 equivalents of 8-carboxymethyl-CTPA chelator and 10 equivalents of DIPEA in 500 l of DMF. The compound was purified and the final product was analyzed by HPLC as described above (see section I.). HPLC-retention time: 2.54 min; ESI-MS (m/z): [M+H]+(calculated C.sub.53H.sub.76N.sub.9O.sub.12): 1030.55 (1030.56)

##STR00066##

[0240] Chemical structure of the chelator 8-carboxymethyl-CTPA, the compound used in the synthesis of CA023.

Specification for CA024

[0241] The product was obtained by incubating the resin (compound 6) with 1.5 equivalents of 8-carboxymethyl-cross bridged-CTPA chelator and 10 equivalents of DIPEA in 500 l of DMF. The compound was purified and the final product was analyzed by HPLC as described above (see section I.). HPLC-retention time: 2.60 min; ESI-MS (m/z): [M+H]+(calculated C.sub.55H.sub.78N.sub.9O.sub.12): 1056.56 (1056.57)

##STR00067##

[0242] Chemical structure of the chelator 8-carboxymethyl-cross bridged-CTPA, the compound used in the synthesis of CA024.

Specification for CA025

[0243] The product was obtained by incubating the resin (compound 6) with 1.5 equivalents of 8,11-bis(carboxymethyl)-CTPA chelator [CPTA=4-[(1,4,8,11-tetraazacyclotetradec-1-yl)methyl]benzoic acid] and 10 equivalents of DIPEA in 500 l of DMF. The compound was purified and the final product was analyzed by HPLC as described above (see section I.). HPLC-retention time: 2.60 min; ESI-MS (m/z): [M+H]+(calculated C.sub.55H.sub.78N.sub.9O.sub.14): 1088.55 (1088.56)

##STR00068##

[0244] Chemical structure of the chelator 8,11-bis(carboxymethyl)-CTPA, the compound used in the synthesis of CA025.

Specification for CA026

[0245] The product was obtained by incubating the resin (compound 6) with 1.5 equivalents of 8,11-bis(carboxymethyl)-CTPA chelator and 10 equivalents of DIPEA in 500 l of DMF. The compound was purified and the final product was analyzed by HPLC as described above (see section I.). HPLC-retention time: 2.53 min; ESI-MS (m/z): [M+H]+(calculated C.sub.57H.sub.80N.sub.9O.sub.16): 1146.56 (1146.57)

##STR00069##

[0246] Chemical structure of the chelator 4, 8,11-tris(carboxymethyl)-CTPA, the compound used in the synthesis of CA026.

III. PSMA Ligands for Alpha Therapy with Lead-Isotopes (.sup.203Pb/.sup.212Pb)

Specification for CA007

[0247] The product was obtained by incubating the resin (compound 5) with 1.5 equivalents of p-SCN-Bn-TCMC chelator [TCMC=1,4,7,10-tetraaza-1,4,7,10-tetra(2-carbamoylmethyl)cyclododecane] and 10 equivalents of DIPEA in 500 l of DMF. The compound was purified and the final product was analyzed by HPLC as described above (see section I.). HPLC-retention time: 2.41 min; ESI-MS (m/z): [M+H]+(calculated C.sub.49H.sub.70N.sub.13O.sub.12S): 1064.49 (1064.50)

##STR00070##

[0248] Chemical structure of the chelator p-SCN-Bn-TCMC, the compound used in the synthesis of CA007, and CA008, CA009.

Specification for CA009

[0249] The product was obtained by incubating the resin (compound 6) with 1.5 equivalents of p-SCN-Bn-TCMC chelator and 10 equivalents of DIPEA in 500 l of DMF. The compound was purified and the final product was analyzed by HPLC as described above (see section I.). ESI-MS (m/z): [M+H]+(calculated C.sub.57H.sub.83N.sub.14O.sub.13S): 1203.59 (1203.60)

Specification for CA011

[0250] The product was obtained by incubating the resin (compound 5) with 1.5 equivalents of 2-(4,7,10-tris(2-amino-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-1-yl)acetic acid, the monocarboxylate derivative of the chelator 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetamide (DO3AM), 0.98n.sub.chelator HBTU and 10 equivalents of DIPEA in 500 l of DMF. The compound was purified and the final product was analyzed by HPLC as described above (see section I.). HPLC-retention time: 2.07 min; ESI-MS (m/z): [M+H]+(calculated C.sub.41H.sub.62N.sub.11O.sub.12): 900.45 (900.46)

##STR00071##

Chemical Structure of the Chelator

[0251] 2-(4,7,10-tris(2-amino-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-1-yl)acetic acid, the monocarboxylate derivative of the chelator 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetamide (DO3AM), the compound used in the synthesis of CA010, CA011 and CA012.

Specification for CA012

[0252] The product was obtained by incubating the resin (compound 6) with 1.5 equivalents of DO3AM chelator, 0.98n.sub.chelator HBTU and 10 equivalents of DIPEA in 500 l of DMF. The compound was purified and the final product was analyzed by HPLC as described above (see section I.). HPLC-retention time: 2.21 min; [M+H]+(calculated C.sub.49H.sub.75N.sub.12O.sub.13): 1039.54 (1039.56)

IV. Chelator Spacer Moieties Enhancing the Pharmacokinetic Properties of PSMA-617

Specification for CA027

[0253] The product was obtained by incubating the resin (compound 4) with 1.5 equivalents of p-NHS-Bn-DOTA chelator and 10 equivalents of DIPEA in 500 l of DMF. The compound was purified and the final product was analyzed by HPLC as described above (see section I.). HPLC-retention time: 1.47 min; [M+H]+(calculated C.sub.34H.sub.52N.sub.7O.sub.14): 782.33 (782.36)

##STR00072##

Chemical Structure of p-NHS Ester-Bn-DOTA, the Chelator Used for the Synthesis of CA027 and CA028

Specification for CA028

[0254] The product was obtained by incubating the resin (compound 6) with 1.5 equivalents of p-NHS-Bn-DOTA chelator and 10 equivalents of DIPEA in 500 l of DMF. The compound was purified and the final product was analyzed by HPLC as described above (see section I.). HPLC-retention time: 2.36 min; [M+H]+(calculated C.sub.55H.sub.76N.sub.9O.sub.16): 1119.53 (1118.54)

Specification for CA029

[0255] The product was obtained by incubating the resin (compound 6) with 1.5 equivalents of p-SCN-Bn-DOTA chelator and 10 equivalents of DIPEA in 500 l of DMF. The compound was purified and the final product was analyzed by HPLC as described above (see section I.). HPLC-retention time: 2.49 min; [M+H]+(calculated C.sub.57H.sub.79N.sub.10O.sub.17S): 1207.52

##STR00073##

[0256] Chemical structure of the chelator p-SCN-Bn-DOTA, the compound used in the synthesis of CA029.

Specification for CA030

[0257] The product was obtained by incubating the resin (compound 6) with 1.5 equivalents of p-NCS-benzyl-DOTA-GA chelator and 10 equivalents of DIPEA in 500 l of DMF. The compound was purified and the final product was analyzed by HPLC as described above (see section I.). HPLC-retention time: 2.50 min; [M+H]+(calculated C.sub.60H.sub.84N.sub.11O.sub.18S): 1278.56

##STR00074##

[0258] Chemical structure of the chelator p-NCS-benzyl-DOTA-GA, the compound used in the synthesis of CA030.

V Synthesis of Radiolabeled Complexes:

V.1 Radiochemical Synthesis of the .SUP.64.Cu-PSMA-Derivatives

[0259] The conjugates (1 mM in water, 5 l, 5 nmol) were added to a mixture of 400 l sodium acetate buffer (0.4 M in water, pH 5.0), 10 l ascorbic acid (20% in water) and 282 l [.sup.64Cu]CuCl.sub.2 in 0.1 M HCl (200 MBq). The mixture was heated at 95 C. for 5 min. The labeling was controlled by radio-HPLC (0-100% MeCN in 5 min, Monolith column), with a flow rate of 2 mL/min and retention time of 2.3 min.

[0260] The labeling led to radiolabeling yields >98% within 10 min (as illustrated by the radiochromatograms in FIG. 26). The specific activity of e.g. .sup.64Cu-PSMA-CA003 was approximately 40 MBq/nmol. The same protocol was used for the labeling with .sup.67Cu.

[0261] V.2 Radiochemical Synthesis of the .sup.203/212Pb-PSMA-Ligands

[0262] Eighty nmol of the conjugates (1 mM in water, 80 l, 80 nmol) were added to 400 l sodium acetate buffer (0.4 M in water, pH 5.0), 10 l ascorbic acid (20% in water) and 140 l .sup.203Pb-chloride solution in 0.04 M HCl, with specific activity approx. 102.6 TBq/g (Lantheus Medical Imaging, USA). The mixture was then heated at 95 C. for 5 min. The labeling was controlled by radio-HPLC.

V.3 Radiochemical Synthesis of the .SUP.68.Ga-PSMA-CA028 (CA027, CA029, CA030)

[0263] .sup.68Ga was eluted from a .sup.68Ge/Ga generator (iThemba LABS, South Africa). The conjugate (1 mM in DMSO, 20 l, 20 nmol) was added to a mixture of 320 l sodium acetate buffer (0.4 M in water, pH 4-5), 10 l ascorbic acid (20% in water) and 400 MBq .sup.68Ga in 0.6 M HCl. The mixture was heated at 95 C. for 5 min. The labeling was controlled by radio-HPLC (0-100% MeCN in 5 min, Monolith column), with a flow rate of 2 mL/min and retention time of 2.4 min.

TABLE-US-00001 Analytic Data of selected Novel Ligands Molecular weight [.sup.68Ga-Ligand]-HPLC m/z* Compound (g/mol) retention time (min) experimental CA027 781.35 1.55 782.33 CA028 1117.53 2.37 1118.53 CA029 1206.52 2.60 1207.52 CA030 1277.56 2.61 1278.56 *Mass spectrometry of non-labeled ligands detected as [M + H].sup.+

V.4 Radiochemical Synthesis of the .SUP.17.Lu-PSMA-CA028 (CA027, CA029, CA030)

[0264] For .sup.177Lu labeling, approx. 20 MBq was mixed with 200 l of 0.4 M sodium acetate buffer containing Chelex (pH=5). 2 l of a 1 mM solution of the compound in 10% DMSO in water, 2 l of a saturated solution of ascorbic acid and 40 l of the solution [.sup.177Lu]LuCl.sup.3 were mixed and heated to 95 C. for 10 min. The labelling was checked by radio-HPLC (0-100% ACN in water within 5 min, Monolith column).

VI. Preclinical Evaluation

[0265] In vitro and in vivo experiments were performed using the PSMA-positive C4-2 cell line, a subline of the LNCaP (lymph node carcinoma of the prostate) cell line (CRL-3314; American Type Culture Collection). C4-2 cells were cultivated in RPMI 1640 (PAN Biotech) medium supplemented with 10% fetal calf serum and stable glutamine (PAN Biotech). Cells were grown at 37 C. and incubated with humidified air equilibrated with 5% CO.sub.2.

VI.1 In Vitro

VI.1.1 Competitive Binding Assay and Internalization Ratio

[0266] A MultiScreen.sub.HTS-DV filter plate was incubated at room temperature with 100 l PBS containing 1% BSA per well for 30 min. After removal of the PBS/BSA solution 110.sup.5 C4-2 cells were added Opti-MEM to each well. The inhibitory potency of the synthesized compounds was determined using 0.75 nM of .sup.68Ga-labeled PSMA-HBED-CC dimer (.sup.68Ga-PSMA-10) (Schfer M, Bauder-Wst U, Leotta K, et al. A dimerized urea-based inhibitor of the prostate-specific membrane antigen for .sup.68Ga-PET imaging of prostate cancer. EJNMMI research. 2012; 2:23-23.) as a standard. All non-labeled compounds were dissolved in Opti-MEM at a volume of 300 l with the following concentrations: 0, 0.5, 1, 2.5, 5, 10, 25, 50, 100, 500, 1000 and 5000 nM. Subsequently, 3 l of the radiolabeled compound was added. 50 l of this mixture was taken to obtain a 0.75 nM concentration of the radiolabeled ligand. After 45 min incubation at 37 C., the cells were washed twice with PBS on a multiscreen vacuum manifold (Millipore, Billerica, MA) and the cell-bound radioactivity was measured with a gamma counter (Packard Cobra II, GMI, Minnesota, USA). The inhibitory potency was determined using .sup.68Ga-labeled PSMA-HBED-CC dimer (i.e. PSMA-11) as reference. The Ki was calculated using a nonlinear regression algorithm (Graph Pad Prism 5.01 software). The experiments were performed in quadruplicate.

[0267] For determination of the specific internalization ratio, 24-well plates were incubated for 20 min with 0.1% poly-L-lysine in PBS at room temperature and washed once with PBS. In the next step, 1 ml RPMI medium containing 110.sup.1 C4-2 cells was added to each well and incubated overnight. The conditions during the experiment for each compound were: incubation at 37 C. or 4 C. with or without receptor blocking via 2-(phosphonomethyl)pentanedioic acid (2-PMPA; Axxora) at a final concentration of 500 PM. Afterwards, the cells were incubated with 250 l of a 30 nM solution of the labeled compounds. The plates were either incubated for 45 min in a water bath at 37 C. or on ice at 4 C. Subsequently, the cells were washed 3 times with 1 mL of ice-cold PBS and incubated with 0.5 ml glycine (50 mM in HCl pH 2.8) for 5 min. After an additional washing step with 1 mL of ice-cold PBS, the cells were lysed with 0.5 ml of 0.3 M NaOH, collected and the radioactivity was measured with a gamma-counter for 1 min. The specific cellular uptake was determined as percentage of initially added radioactivity bound to 10.sup.6 cells (% IA/10.sup.6 cells) by subtraction of the respective uptake under blocking conditions. All experiments were performed in triplicate.

[0268] The results are given in FIG. 3. The K.sub.i determination showed nanomolar binding affinities of the synthesized ligands to PSMA.

[0269] For the .sup.203Pb labeled compounds, compounds CA009 and CA012 revealed the highest affinity to inhibit PSMA. Moreover, the .sup.203Pb-labeled compounds CA009 and CA012 showed high rates of specific internalization in the PSMA positive cell line. The compounds .sup.203Pb-CA009 and .sup.203Pb-CA012 showed an internalization ratio up to 28.362.23 and for 7.331.26 injected activity/10.sup.6 C4-2 cells (n=3).

[0270] As shown in the table in FIG. 3, for the Cu-labeled ligands, e.g, CA003 revealed a particularly high affinity to PSMA, followed by CA006, CA002 and CA026. Moreover, the .sup.64Cu-labeled compounds showed specific binding to C4-2 cells. 34.632.77% of .sup.4Cu-CA003, 18.634.46% of .sup.64Cu-CA005 and 38.76.69% of .sup.64Cu-CA022 were internalized (n=3). 10.sup.6 C4-2 cells were used for these experiments.

[0271] Further, the results of the K.sub.i determination for e.g. the Ga-labeled ligands revealed e.g., nanomolar binding affinities of the synthesized ligands to PSMA. As shown in FIG. 3, among all new compounds CA030 showed particularly advantageous properties.

VI.1.2 Serum Stability

VI.1.2.1 Serum Stability of .SUP.203.Pb Labeled Compounds

[0272] The stability of the radiolabeled compounds was determined by incubation in 300 l of human serum at 37 C. after 1 h, 2 h, 3 h, 6 h, 24 h, 48 h and 72 hours. The serum was precipitated by addition of 2 parts acetonitrile. Subsequently, the samples were vortexed and centrifuged for 5 min at 13,000 rpm (2 times) and the supernatant was analyzed by radio-HPLC (0-100% MeCN in 5 min, Monolith column).

[0273] All compounds showed a high stability in human serum for at least 48 h. CA011 and CA012 showed a particularly advantageous stability and were stable for at least 72 h.

VI.1.2.2 Serum Stability of 6.sup.8Ga, .sup.77Lu and .sup.64Cu Labeled Compounds

[0274] After radiolabeling of the compounds the serum stability was determined by iTLC and HPLC analysis. 50 L (20 MBq) of the labeled ligands was added to 200 L of human serum (H4522; Sigma-Aldrich, Germany) and incubated at 37 C. for different time-points (0, 2 h, 24 h, 48 h and 72 h). 0.55 cm strips of iTLC-SG-glass microfiber chromatography paper (Folsom, California USA) were used. 0.5 L of the radiolabeled compound in serum was applied to each strip at 1 cm from the bottom (origin) and the solvent (sodium citrate buffer (0.5 M, pH=5.0) in case of .sup.177Lu ligands, (1% Na-EDTA, pH=4) in case of Cu labeled ligands) front was allowed to rise to 5 cm from the bottom. Finally, each strip was cut in 8 pieces; each piece was measured in a gamma-counter. For HPLC analysis an equal volume of ACN was added to the samples to precipitate the serum proteins. Subsequently, the samples were centrifuged for 10 min at 13,000 rpm and the pellet and supernatant were separated and the relative activity was measured. The results are expressed as percent. In addition, an aliquot of the supernatant was analyzed by radio-HPLC (0-100% ACN in 5 min, Monolith column), with a flow rate of 2 mL/min.

[0275] The results of the stability testings of the compounds radiolabeled with .sup.68Ga and .sup.177Lu is shown in FIG. 16) As indicated by HPLC and radio TLC, the .sup.68Ga-labeled compounds did not show degradation after incubation for 2 h in human serum. .sup.177Lu-CA028 showed 40% of free .sup.177Lu after incubation for 24 h whereas CA029, CA030 and the reference compound PSMA-617 revealed no free activity at this time point (FIG. 16).

[0276] The results of the stability testings of .sup.64Cu-labeled compounds is shown in FIG. 17 and FIG. 18. Until 2 h incubation, ITLC showed that all compounds dissociate only to a degree of less than 20.6%. After 24 h incubation only 64% of .sup.64Cu-CA003 and 31% of .sup.64Cu-CA006 were found to be dissociated. In contrast .sup.64Cu-PSMA-617 showed 133% of free .sup.64Cu activity. Long term stability examination (72 h) showed that .sup.4Cu-CA006 (84%) possesses stability comparable to .sup.64Cu-CA003 (113%). At this point in time 186% of .sup.64Cu-PSMA-617 was found to be dissociated. After incubation for 2 h, activity measurement in the pellet (FIG. 18) revealed that 19.05.2% of .sup.64Cu-CA003, 127.2% of .sup.64Cu-CA006 and 407.5% of .sup.64Cu-PSMA-617 was precipitated with the protein fraction. The percentage of the activity in the pellet increased over time. The highest amount of activity was measured in the pellet of .sup.64Cu-PSMA-617 followed by .sup.64Cu-CA003, and less activity was found to be precipitated for .sup.64Cu-CA006

VI.2 In Vivo Experiments

[0277] The in vivo experiments were carried out in accordance with the laws of the German Federal Republic. For PET imaging and biodistribution studies, male nude mice (Balb/c nu/nu mice) (19-23 g) were obtained from Charles River at 4-5 weeks of age and kept under specific-pathogen-free condition for 1 week prior to the study. The mice were housed with a 12-hour/12-hour light/dark cycle and had free access to water and food. The mice were anesthetized with 2% sevoflurane and inoculated subcutaneously on the right trunk with 510.sup.7 C4-2 cells in 50% Matrigel in Opti-MEM I (1) medium. Organ distribution studies were performed, when the size of the tumor was approximately 1 cm.sup.3.

VI.2.1 Lead-Labeled Compounds:

VI.2.1 a Scintigraphic Imaging and Biodistribution

[0278] For small-animal imaging the mice were anesthetized with 2% sevoflurane. 0.1 nmol (1.0 MBq) of the .sup.203Pb-ligand was injected into the tail vein. Serial planar scans were performed using a Gamma Imager SCT (Biospace Lab, Paris, France) with a parallel collimator (35 mm/1.8 mm/0.2 mm) after 10 min, 1 h, 4 h, 24 h. and 72 h. Based on the imaging results, compound CA012 was chosen for biodistribution studies. Experiments were performed in triplicate.

[0279] The results are given in FIGS. 8 and 9.

VI.2.2 .SUP.64.Cu-Labeled Compounds:

VI.2.2 a) Stability in the Blood and In Vivo Fate of .sup.64Cu-Chloride and .sup.64Cu-CA003

[0280] The stability of .sup.64Cu-labeled CA003 in vivo was determined by ITLC and HPLC. Male BALB/c nude mice without tumor (n=3) were injected via the tail vein with .sup.64Cu-CA003 (3.6 MBq; 0.26 nmol, dissolved in a total volume of approximately 100 l of 0.9% saline) and 800 l blood was harvested 10 min post injection. The blood sample was centrifuged for 10 min at 13,000 rpm. Subsequently, the pellet and supernatant were separated and the relative activity was determined. ITLC was performed in order to assess the stability of the radiolabeled compound in the blood as described above. Furthermore, an aliquot of the supernatant was analyzed by radio-HPLC (0-100% ACN in 5 min, Monolith column), with a flow rate of 2 mL/min after addition an equal volume of ACN and removal of the proteins by centrifugation

[0281] The metabolization in vivo was studied by radio-HPLC analysis. Female Swiss mice (n=3) without tumor were injected via the tail vein with .sup.64Cu-chloride (10 MBq in approximately 100 L of 0.9% saline) or .sup.64Cu-CA003 (9 MBq, 0.30 nmol in approximately 100 L of 0.9% saline). PET imaging was performed 10 min post injection and subsequently blood, the liver and the kidneys were harvested. The tissues were rinsed with precooled saline, blotted dry and treated with 2 mL of 0.1 M NH.sub.4OAc/EtOH (35:65). The tissues were homogenized using an Ultra-Turrax T8 (IKA Labortechnik, Germany). The samples were centrifuged for 10 min at 13,000 rpm (4 C.). Subsequently, the pellet and supernatant were separated and the relative activity was measured. The results are expressed as percent. Additionally, an aliquot of supernatant was prepared for HPLC measurement by precipitation of the proteins with ACN as described above. The sample was analyzed by radio-HPLC (0-100% ACN in 5 min, Monolith column), with a flow rate of 2 mL/min. Fractions were collected every ten seconds over the whole course of the chromatography and the relative activity of the samples was measured in a gamma-counter to reconstruct a chromatogram.

[0282] ITLC results of the blood stability showed that .sup.64Cu-CA003 undergo 3% of .sup.64Cu dissociation or 972.3% of the intact tracer (see FIG. 19). Radio HPLC chromatograms likewise confirms the integrity of the copper complex (see FIG. 20). In vivo fate of .sup.64Cu-chloride or .sup.64Cu-CA003 studies was conducted and PET imaging of .sup.64Cu-chloride and .sup.64Cu-CA003 showed different pharmacokinetics (Figure S21). Maximum intensity projection PET imaging of .sup.64Cu-chloride indicated, lower blood circulation (1.3), high liver (2.7) and kidney uptake (3.4). Whereas .sup.64Cu-CA003 demonstrated longer blood circulation (2.3), higher kidney (5.7) uptake and lower liver accumulation (1.0) than .sup.64Cu-chloride (FIG. 28). The integrity of the .sup.64Cu-CA003 was proven by radio HPLC chromatograms of tissue extracts of kidney, blood and liver (FIG. 19). The chromatogram for .sup.64Cu-CA003 showed different retention time of the tracer than free copper, .sup.64Cu-chloride (FIG. 20).

VI.2.2 b) Organ Distribution Experiments (.SUP.64.Cu Ligands) and Small Animal PET

[0283] Based on the results of the PET imaging, CA003, and CA023, were chosen for a biodistribution analysis using the C4-2 tumor bearing mice. Experiments were performed in triplicate. 0.025 nmol of the .sup.64Cu-labeled compound (1 MBq per mouse in approximately 100 L of 0.9% saline) was administered by tail vein injection. At the time points: 10 min, 1 h, 4 h, 24 h and 72 h the organs were dissected and weighed and the activity was measured using a -counter (Packard Cobra Auto-gamma). The percentage of the injected dose per gram (% ID/g) was calculated. (See FIG. 6A, 6B)

[0284] In addition to this, the experiment with simultaneous administration of PSMA-617 to block PSMA binding at 1 h (n=3) is represented (FIGS. 6C and 6D).

[0285] For small-animal PET imaging with various .sup.64Cu-labeled PSMA ligands, 0.2 nmol, 10 MBq of approximately 100 L in 0.9% saline the radiolabeled compound were injected into a C4-2 tumor bearing mouse. The dynamic PET was recorded in a small animal PET scanner (Siemens Inveon D-PET, Malvern, PA USA). The SUV values were obtained from conventional (non-dynamic) PET images. The formula for the SUV was:

[00001]$\mathrm{SUV}=\frac{\mathrm{activity}\mathrm{in}\mathrm{ROI}\left(\frac{\mathrm{Bq}}{\mathrm{ml}}\right)\mathrm{animal}\mathrm{weight}\left(g\right)}{\mathrm{injected}\mathrm{dose}\left(\mathrm{Bq}\right)}$

[0286] The volumes-of-interest (VOIs) were obtained by manual delineation of the appropriate whole tissue (heart, kidneys, bladder, tumorwith an approximate volume of 100-500 l)or parts of the tissue liver and muscle. The images were reconstructed based on the procedure: OSEM 3D/SP MAP with 16 subsets, 2 iterations and an image x-y size: 256, image z size: 161. The data were not modified with a post processing filter. The software used to analyze images and TACs was Inveon Acquisition Workplace (IAW) from Siemens IRW 4.1. Dynamic PET scans were performed 0-60 min post injection, and images were reconstructed in three time frames of 20 min (0-20 min, 20-40 min and 40-60 min) for visual display. For some compounds that showed long retention later time points (2 h, 4 h, 20 h, 45/48 h) were included as shown in FIG. 2/FIG. 3 and Table S1+S2. After 1 h a static PET scan was generated. In order to compare the different radiotracers, the mean SUVs were plotted over time.

[0287] The results obtained for the biodistribution of the PSMA ligand .sup.64Cu-CA003 (n=3) are shown in FIGS. 6A-6D. 10 min after injection 11.334.11% ID/g tumor uptake is observed. After 4 h the amount of the tracer accumulation (32.3410.6% ID/g) in the tumor is much higher than in the kidneys (13.333.36% ID/g). Time activity curves generated from the dynamic PET imaging, showed a tumor-to-muscle ratio of 10.5 and 3.0 for tumor-to-blood at 1 h post injection, see following table VI.2.2 b_1:

TABLE-US-00002 TABLE VI.2.2 b_1 Mean standardized uptake values (mSUV) derived from the time-activity curves from small-animal PET of .sup.64Cu-CA003 in a BALB/c nu/nu mouse bearing a C4-2 tumor xenograft meanSUV Heart Liver Kidneys Bladder Muscle Tumor T.sub.1 = 1 h 0.25 0.19 4.0 5.3 0.07 0.76 T.sub.2 = 2 h 0.04 0.19 1.3 23 0.01 1.2 T.sub.3 = 4 h 0.04 0.12 0.70 2.8 0.01 1.0 T.sub.4 = 20 h 0.02 0.08 0.15 0.44 0.01 0.92 T.sub.5 = 45 h 0.01 0.06 0.08 0.11 0.00 0.66

[0288] These curves demonstrated a rapid renal uptake. The organ distribution study showed that the high kidney uptake at 1 h (67.0420.89% ID/g) was largely cleared (7.488.51% ID/g) within 24 h. In contrast the high tumor uptake value (30.8312.61% ID/g at 1 h p.i.) remained almost constant (19.996.43% ID/g at 24 h p.i.). PET imaging confirmed the strong accumulation of the radiotracer in the tumor (FIG. 4). At 1 h post injection, the amount of the radioactivity in the background organs such as kidneys decreased whereas the tumor-to-background ratio increased. 24 h after injection the PET scans demonstrated a very high tumor uptake confirming the enrichment in the tumor.

[0289] The specificity of the binding to PSMA was proven with a blockade experiment: co-injection of non labelled PSMA-617 [2 mg/kg] led to a strong decrease of the accumulation of .sup.64Cu-CA003 in C4-2 tumors (30.8312.61% ID/g to 2.350.38% ID/g) and in the kidneys (67.0420.89% ID/g to 3.470.48% ID/g) at 1 h post injection. PET imaging of .sup.64Cu-CA003 with excess of non-labeled PSMA (FIG. 6C/6D) clearly confirmed the biodistribution results.

[0290] Time activity curves generated from the dynamic PET imaging, showed a tumor-to-muscle ratio of 10.5 and 3.0 for tumor-to-blood at 1 h post injection (Table S1). These curves demonstrated a rapid renal uptake. The organ distribution study (FIG. 6A) showed that the high kidney uptake at 1 h (67.0420.89% ID/g) was largely cleared (7.488.51% ID/g) within 24 h. In contrast the high tumor uptake value (30.8312.61% ID/g at 1 h p.i.) remained almost constant (19.996.43% ID/g at 24 h p.i.). PET imaging confirmed the strong accumulation of the radiotracer in the tumor (FIG. 4). At 1 h post injection, the amount of the radioactivity in the background organ such as kidneys decreased whereas the tumor-to-background ratio increased. 24 h after injection the PET scans demonstrated a very high tumor uptake confirming the enrichment in the tumor. The high tumor accumulation was retained at long time points i.e. at 45 h post injection (FIG. 4).

IV.2.2 c) Comparison of .sup.64Cu-PSMA-CA003 with .sup.64Cu-PSMA-617 and .sup.64Cu-chloride In Vivo

[0291] In order to prove the in vivo stability of the copper complexes of PSMA-CA003, .sup.64Cu-PSMA-CA003 was compared to .sup.64Cu-PSMA-617 as well as to .sup.64Cu-chloride (FIG. 20). The compounds were studied in a small animal PET study in a C4-2 tumor xenograft. The results are shown in FIG. 3. The time activity curves obtained from dynamic PET for .sup.4Cu-CA003 showed a high tumor-to-liver ratio (4.0) at 1 h after injection, whereas for .sup.64Cu-PSMA-617 the tumor-to-liver ratio was 0.37 (FIG. 20/21 and Table S1/S2).

TABLE-US-00003 TABLE S2 Mean standardized uptake values (mSUV) derived from the time-activity curves from small-animal PET of .sup.64Cu-PSMA-6I7 in a BALB/c nu/nu mouse bearing a C4-2 tumor xenograft meanSUV Heart Liver Kidneys Bladder Muscle Tumor T.sub.1 = 1 h 0.30 1.8 1.7 9.6 0.20 0.67 T.sub.2 = 2 h 0.25 1.6 0.84 3.0 0.10 0.5 T.sub.3 = 4 h 0.21 2.0 0.64 0.19 0.09 0.86 T.sub.4 = 20 h 0.25 1.5 0.44 0.12 0.07 0.64 T.sub.5 = 45 h 0.21 1.2 0.34 0.10 0.06 0.46

[0292] To prove that the species which is taken up into the tumor is actually .sub.64Cu-CA003 and not free .sub.64Cu, PET imaging of .sub.64Cu-chloride performed in C4-2 tumor bearing mice (FIG. 20/21) was followed by homogenization, extraction and subsequent HPLC analysis of the respective tissue. The pharmacokinetic observed for .sub.64Cu-chloride is different to that of 64Cu-CA003. PET imaging of .sub.64Cu-chloride at maximum-intensity projections reveal an increasing tumor uptake up to 2 h after injection. In contrast to .sub.64Cu-CA003, 64Cu-chloride showed a very high liver accumulation (FIG. 20/21). The tumor-to-liver ratio at 2 h for .sub.64Cu-chloride was 0.38, whereas the tumor-to-liver ratio of .sub.64Cu-CA003 was 6.3.

[0293] 1 h post injection the time activity curves and the mean SUV body weight values generated from the dynamic PET imaging of .sup.68Ga-CA028 demonstrated a tumor-to-kidney ratio of 0.78. At 2 h, this ratio was increased to 3.0 (FIG. 15, and Table S1.1), while the ligands .sup.68Ga-CA030 and .sup.68Ga-CA029 showed lower tumor-to-kidneys ratio of 0.52 and 0.33, respectively (FIGS. 3, 25 and 23).

TABLE-US-00004 TABLE S1.1 Mean standardized uptake values (mSUV) derived from the time- activity curves from small-animal PET of .sup.68Ga-PSMA-CA028 in a BALB/c nu/nu mouse bearing a C4-2 tumor xenograft. mSUV Heart Liver Kidneys Bladder Tumor Muscle T.sub.1 = 1 h 0.36 0.22 0.39 6.50 0.81 0.13 T.sub.2 = 2 h 0.13 0.09 0.29 2.5 0.78 0.05

[0294] The time-activity curves revealed a fast clearance of the tracer.sup.68Ga-CA028 showed a high tumor accumulation and high kidney values. In contrast, for .sup.68Ga-CA027 a faster clearance by the kidney at a tumor accumulation was found (FIG. 22). Even though .sup.68Ga-CA030 demonstrated higher kidney uptake values than .sup.68Ga-CA028, it showed the highest tumor uptake among all compounds (FIG. 15C). The small animal PET images demonstrated a very fast kidney clearance and low tumor accumulation of .sup.68Ga-CA027. Distinctly, the radiotracers .sup.68Ga-CA028, .sup.68Ga-CA029 and .sup.68Ga-CA030 showed high tumor accumulation (FIG. 27B). .sup.68Ga-CA029 showed the highest kidney uptake, followed by .sup.68Ga-CA030 (FIG. 27A).

VII. In-Human Studies in a First Patient

VII.1 PET with .sup.64Cu-PSMA-CA003

[0295] The PET imaging of the prostate cancer patient shown in FIG. 7 was consented by the University Hospital Heidelberg following the German laws in vigor and granted the Helsinki Declaration (permit S321/2012).

[0296] The first-in-human study was performed with 200 MBq of .sup.64Cu-PSMA-CA003. The first PET imaging is presented in FIG. 7 and shows imaging of a patient with high level of the prostate-specific membrane antigen. It is clearly evident, that the patient suffered from PCa in metastasized stage notably through multiple lymph node metastases, on the right shoulder the main tumor is localized.

[0297] With the new copper ligands a significant improvement of the pharmacokinetics and the tumor targeting for copper isotopes was observed. Despite the high labeling yields of PSMA-617 with .sup.64Cu in vitro (>99%), a poor in vivo stability with occurrence high liver uptake was observed (Cui C, Hanyu M, Hatori A, et al. Synthesis and evaluation of [(64)Cu]PSMA-617 targeted for prostate-specific membrane antigen in prostate cancer. Am J Nucl Med Mol Imaging. 2017; 7:40-52).

[0298] The novel .sup.64Cu-labeled PSMA ligands are promising agents to target PSMA and visualize PSMA positive tumor lesions as shown in the preclinical evaluation by small-animal PET studies, organ distribution and the first-in-human application.

[0299] The imaging in a patient with the .sup.64Cu-PSMA-CA003 ligand demonstrated its successful translation in clinical studies.

VII.2 Experiments with .sup.203Pb-CA012

[0300] Two patients, with castration-resistant metastasized prostate cancer underwent planar whole-body scans (GE Hawkeye Millennium, 1 crystal, ME-collimator, 279 keV peak+/10%, 8 cm/min) at 0.4 h, 4 h, 18 h, 28 h and 42 h post-injection of 258 and 310 MBq .sup.203Pb-CA012, respectively. Images were loaded into the QDOSE dosimetry software suite (ABX-CRO, Dresden) and coregistered. Kidneys, liver, spleen, urinary bladder, salivary glands (both left and right of parotid and submandibular glands) and several tumor lesions as well as a total-body region-of-interest (ROI) were segmented using organ-dependent percentage of maximum thresholds (15-65%) in the most suitable time-point and propagated to all other time-points performing an additional organ-based automatic rigid co-registration step. These ROIs were used to determine time-activity-curves (TAC) for each organ, tumors and total body. The first time-point (before voiding) of the uncorrected geometric mean images was used to calibrate the ROI-counts to injected activity (MBq). Red marrow TAC was calculated from venous blood (6 samples/patient) using established model assumptions [Shen, S., Meredith, R. F., Duan, J., Macey, D. J., Khazaeli, M. B., Robert, F., LoBuglio, A. F.: Improved Prediction of Myelotoxicity Using a Patient-Specific Imaging Dose Estimate for Non-Marrow-Targeting 90Y-Antibody Therapy. J Nucl Med, 43: 1245-1253, 2002. Sgouros, G. Bone Marrow Dosimetry for Radioimmunotherapy: Theoretical Considerations. J Nucl Med, 34: 689-694, 1993.].

[0301] All TACs derived from the .sup.203Pb data were re-calculated using the replacement nuclide function of QDOSE, which automatically corrects all time-points for the physical decay of the source isotope, leaving only its biological clearance; then the physical decay of the replacement radionuclide is applied.

[0302] Bi-exponential curve fitting was applied to all organ TACs (with exception of few tumors and glands, which had to be fitted mono-exponentially). The cumulated activity was integrated assuming a linear increase from 0 to first measured time-point, numerically from first to last measured time-point using trapezoidal approximations and from last measured time-point to infinity using the fit-function. The remainder body was calculated by subtracting all source organs from the total body. The residence times of kidneys, liver, spleen, urinary bladder content, red marrow and remainder body were exported for dose calculation in OLINDA 1.1 [Stabin, M. G., Sparks, R. B., Crowe, E. OLINDA/EXM: the second-generation personal computer software for internal dose assessment in nuclear medicine. J Nucl Med, 46(6):1023-1027, 2005] using the organ masses of the male-adult-phantom.

[0303] The potential therapeutic nuclide .sup.212Pb decays further to .sup.212Bi, .sup.212Po and .sup.208T1. Assuming that the daughter nuclides remain at the site of decay of the parent nuclide and transient equilibrium between .sup.212Pb and its daughters, the same residence time as for 212Pb was applied to the daughter nuclides. OLINDA calculations were performed for all nuclides individually. Respective decay steps were summed up, using weighting factors of 64.07% for .sup.212Po and 35.93% for .sup.208T1 according to their branching ratios. According to the suggestions from the Committee on Medical Internal Radiation Dose and the US Department of Energy [Sgouros G, Roeske J C, McDevitt M R, et al. MIRD pamphlet no. 22 (abridged): radiobiology and dosimetry of alpha-particle emitters for targeted radionuclide therapy. J Nucl Med. 2010; 51:311-28. Feinendegen L E, McClure J J. Meeting report: alpha-emitters for medical therapy-workshop of the United States Department of Energy, Denver, Colorado, may 30-31, 1996. Radiat Res. 1997; 148:195-201.], physical absorbed doses were translated into equivalent doses using weighting factors of 5 for alpha and 1 for beta and photon radiation; Thus, reflecting the relative biological efficacy in regard to deterministic radiation effectsconsidered the leading factor in therapeutic settings. Tumor and salivary gland volumes were measured individually based on CT-segmentation and their absorbed doses were approximated by using a power function interpolating the spherical model estimates [Stabin, M. G., Konijnenberg, M. Re-evaluation of Absorbed Fractions for Photons and Electrons in Small Spheres. J Nucl Med, 41: 149-160, 2000.].

[0304] .sup.203Pb Imaging Data of Patients

[0305] With an injected activity of 258-310 MBq, the 279 keV gamma rays emitted from .sup.213Pb with an 80% abundance probability were found to be sufficient to obtain clear planar scans (FIG. 10). These scans show the tracer labeled with the lead isotope specifically enriches in the target tissue. Consequently the method provides a successful approach to use isotopes of lead for the intended applications.

[0306] (The Organ distribution of .sup.203Pb-PSMA-CA012 in tumor bearing mice is shown in the Table in FIG. 11)

VII.3 Dosimetry Estimates .SUP.203.Pb-CA012

Dosimetry Estimates

[0307] The dosimetry estimate for diagnostic .sup.203Pb-CA012 is presented in the left column of the table in FIG. 11. All organ absorbed doses are dominated by photons (primary emission at 279 keV), low probability emissions in sum contribute <10% in all organs, respectively. A typical clinical examination with 250-300 MBq, e.g. for individual dosimetry prediction, translates into a radiation burden of 6.0-7.5 mSv.

[0308] During decay from .sup.212Pb to stable .sup.208Pb, regardless whether by the Polonium or Thallium branch, two beta- and one alpha-particle are emitted per atom. The safety dosimetry estimate for therapeutic .sup.212Pb-CA012, taking into account the complete succeeding decay chain, is presented on the right column of FIG. 11. Assuming an RBE=5 for alpha and RBE=1 for beta and gamma radiation the equivalent doses for therapeutic .sup.212Pb-CA012 consist of 96.4% alpha, 2.2% beta and 1.4% gamma contribution. Remarkably: the initial beta decay of .sup.212Pb, which is directly traced by .sup.203Pb, contributes less than 1% to the total equivalent dose; 99% of the equivalent dose arises from the succeeding daughter nuclides.

[0309] Amongst the OLINDA organs, kidneys and red-marrow might be dose limiting, together with the salivary glands which were assessed using the spherical model. The therapeutic range of a radiopharmaceutical is defined by the ratio between tumor dose and dose-limiting organs. The most relevant dosimetry information are summarized and compared to other PSMA-targeting alpha therapies in FIG. 12.

VII.4 Human PET-scan-.sup.68Ga-CA028 and .sup.68Ga-CA030

[0310] Method for CA028 radiation dosimetry was performed as previously described in Afshar-Oromieh et al., 2015 (Afshar-Oromieh A, Hetzheim H, Kratochwil C, et al. The Theranostic PSMA Ligand PSMA-617 in the Diagnosis of Prostate Cancer by PET/CT: Biodistribution in Humans, Radiation Dosimetry, and First Evaluation of Tumor Lesions. J Nucl Med. 2015; 56:1697-1705.)

[0311] Images were obtained with .sup.68Ga-CA028 and .sup.68Ga-CA030, respectively, which were applied via intravenous (either 339 MBq/20 nmol .sup.68Ga-CA028 or 295 MBq/20 nmol .sup.68Ga-CA030 per patient). The diagnostic examination of .sup.68Ga-CA028 was conducted at the point of time after 1 h and 3 h injection. (See FIG. 14)

[0312] Diagnostic PSMA-PET/CT examinations were performed 1 h and 3 h after antecubital injection of 339 MBq/20 nmol .sup.68Ga-CA028 or 295 MBq/20 nmol .sup.68Ga-CA030 per patient, respectively. The method for assessing the biodistribution was performed as previously described by Afshar-Oromieh et al. (Afshar-Oromieh A, Hetzheim H, Kratochwil C, et al. The Theranostic PSMA Ligand PSMA-617 in the Diagnosis of Prostate Cancer by PET/CT: Biodistribution in Humans, Radiation Dosimetry, and First Evaluation of Tumor Lesions. J Nucl Med. 2015; 56:1697-1705). The activity distributions of the source organs were determined with the clinical standard software Syngo (Siemens), which was used to define the VOIs in the PET images. This reference, Afshar-Oromieh et al, was also used as a standard of reference for .sup.68Ga-PSMA-617

[0313] In order to prove the clinical applicability of .sup.68Ga-CA028 and .sup.68Ga-CA030, PET/CT imaging in first patients was performed. The resulting images are illustrated in FIG. 13A and FIG. 13B. The imaging with .sup.68Ga-CA028 and .sup.68Ga-CA030 confirmed the results obtained in vitro and in the animal models. The PET scans and the SUVs were acquired with standard scanner settings, calibrated for pure positron emitters (Wadas T J, Pandya D N, Solingapuram Sai K K, Mintz A. Molecular targeted alpha-particle therapy for oncologic applications. AJR Am J Roentgenol. 2014; 203:253-260). The SUV-values obtained at 1 h versus 3 h post injection are presented in the following table. As reflected by these values a high and stable accumulation in the tumor is achieved.

TABLE-US-00005 TABLE Safety dosimetry of diagnostic SUVmean values of .sup.68Ga-CA028, .sup.68Ga-PSMA-617 (values from Afshar-Oromieh et al., 2015 (P)) and .sup.68Ga-CA030 and based on an adult male phantom in OLINDA. .sup.68Ga-CA028 .sup.68Ga-PSMA-617 .sup.68Ga-CA030 Tissue 1 h 3 h 1 h 3 h 1 h 3 h Lacrimal gland 7.4 7.6 4.9 5.9 7.1 9.0 Nasal mucosa 2.9 3.5 2.9 3.4 3.3 3.8 Parotid gland 11.0 8.0 10.4 13.1 6.1 6.7 Submandibular gland 14.4 8.9 10 12.4 8.7 11.3 Sublingual gland 3.8 2.8 4.6 4.0 4.1 4.9 Blood pool, mediastinal 3.2 2.9 2.5 2.4 5.6 4.5 Liver 4.3 1.8 3.3 2.7 5.8 5.9 Spleen 5.1 2.5 4.3 3.5 7.2 6.1 Prox. Small intestine 8.4 10.4 4.7 5.5 4.1 5.0 Colon 4.9 4.2 3.5 4.0 4.9 4.7 Kidneys 13.8 10.9 15.6 17.0 13.4 16.5 Gluteal muscle 0.7 0.4 0.7 0.7 0.8 0.7 Bone met 4.5 4.1 9.4 6.3 27.2 37.7 Lymph node 13.5 10.7 7.1 13.5 Bone met 6.9 5.7 30.9 32.6 Bone met 4.6 4.4 31.0 33.1 Lymph node 17.0 25.4 Lymph node 5.0 4.1 Lung metastases 5.7 4.7