LIGANDS OF PROSTATE SPECIFIC MEMBRANE ANTIGEN (PSMA) CONTAINING HETEROAROMATIC LINKER BUILDING BLOCKS

Abstract

The present invention relates to novel compounds that bind to the prostate-specific membrane antigen (PSMA)-binding and their use in the diagnosis and treatment of certain diseases where PSMA is upregulated.

Claims

1. A compound of Formula I ##STR00030## wherein C is represented by any of the structures selected from the group consisting of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), N,N-bis[2-hydroxy-5-(carboxyethyl)benzyl]ethylenediamine-N,N-diacetic acid (HBED-CC), 1,4-bis(carboxymethyl)-6-[bis(carboxymethyl)]amino-6-methylperhydro-1,4-diazepine (AAZTA), 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), 2-(4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-yl)pentanedioic acid (DOTAGA), N.sup.4-[5-[[4-[[5-(acetylhydroxyamino)pentyl]amino]-1,4-dioxobutyl]hydroxyamino]pentyl]-N.sup.1-(5-aminopentyl)-N.sup.1-hydroxybutanediamide (DFO), N.sup.1-[5-(acetylhydroxyamino)pentyl]-N.sup.26 -(5-aminopentyl)-N.sup.26,5,16-trihydroxy-4,12,15,23-tetraoxo-5,11,16,22-tetraazahexacosanediamide (DFO*), or 5-[(2-mercapto-2-methylpropyl)[2-[(2-mercapto-2-methylpropyl)amino]ethyl]amino]pentanoic acid (BAT), L is C1-C5-heteroaryl, optionally substituted with C1-C6 alkyl, X is NH or CH.sub.2, k is 0, 1 or 2, m is an integer from 1 to 7, n is an integer from 0 to 6, p is 0 or 1, R.sub.1 is phenyl, phenyl-NH, benzothiophenyl or naphthyl, wherein phenyl and phenyl-NH is optionally substituted with 1, 2 or 3 groups each independently selected from halo, OH and NO.sub.2, and R.sub.2 is aryl, aryl-C(O), or heteroaryl-C(O), optionally substituted with 1, 2 or 3 groups each independently selected from halo, OH, C1-C6-alkyl, OCH.sub.3 and NO.sub.2, wherein C1-C5-heteroaryl contains 1, 2, 3 or 4 heteroatoms, each independently selected from N, O or S, or a pharmaceutically acceptable salt thereof or a solvate thereof or a solvate of a salt thereof.

2. The compound of Formula I according to claim 1 that is a compound of Formula II ##STR00031## wherein L, X, k, m, n, p, R.sub.1 and R.sub.2 are as defined in claim 1, or a pharmaceutically acceptable salt thereof or a solvate thereof or a solvate of a salt thereof.

3. The compound of Formula I according to claim 1 ##STR00032## wherein C, X, k, m, n, p, R.sub.1 and R.sub.2 are as defined in claim 1, L is selected from the group consisting of isoxazolyl, pyridyl, thiazolyl and thiophenyl, optionally substituted with C1-C6 alkyl, or a pharmaceutically acceptable salt thereof or a solvate thereof or a solvate of a salt thereof.

4. The compound of Formula I according to claim 1 that is a compound of Formula III ##STR00033## wherein X, m, n, R.sub.1 and R.sub.2 are as defined in claim 1, L is selected from the group consisting of isoxazolyl, pyridyl, thiazolyl and thiophenyl, optionally substituted with C1-C6 alkyl, or a pharmaceutically acceptable salt thereof or a solvate thereof or a solvate of a salt thereof.

5. The compound of Formula I according to claim 1 that is a compound of Formula IV ##STR00034## wherein m and R.sub.1 are as defined in claim 1, L is selected from the group consisting of isoxazolyl, pyridyl, thiazolyl and thiophenyl, optionally substituted with C1-C6 alkyl, or a pharmaceutically acceptable salt thereof or a solvate thereof or a solvate of a salt thereof.

6. The compound of Formula I according to claim 1 that is a compound of Formula V ##STR00035## wherein X, m, n, R.sub.1 and R.sub.2 are as defined in claim 1, or a pharmaceutically acceptable salt thereof or a solvate thereof or a solvate of a salt thereof.

7. The compound of Formula I according to claim 1 that is a compound of Formula VI ##STR00036## wherein m and R.sub.1 are as defined in claim 1, or a pharmaceutically acceptable salt thereof or a solvate thereof or a solvate of a salt thereof.

8. The compound according to claim 1, wherein R.sub.1 is benzothiophenyl or naphthyl and m is 1, or a pharmaceutically acceptable salt thereof or a solvate thereof or a solvate of a salt thereof.

9. The compound according to claim 1, that is a compound selected from the list of the following compounds: ##STR00037## ##STR00038## ##STR00039## or a pharmaceutically acceptable salt thereof or a solvate thereof or a solvate of a salt thereof.

10. A method for preparing a radiolabeled compound, comprising radiolabeling a compound according to claim 1 or a pharmaceutically acceptable salt thereof or a solvate thereof or a solvate of a salt thereof.

11. A metal complex comprising a radionuclide and a compound of claim 1 or a pharmaceutically acceptable salt thereof or a solvate thereof or a solvate of a salt thereof.

12. The metal complex according to claim 11, wherein the radionuclide is .sup.111In, .sup.90Y, .sup.68Ga, .sup.177Lu, .sup.99mTc, .sup.64Cu, .sup.67Cu .sup.153Gd, .sup.155Gd, .sup.157Gd, .sup.213Bi, .sup.225Ac, .sup.89Zr, .sup.203Pb, .sup.212Pb.

13. The metal complex according to claim 11, wherein the radionuclide is .sup.177Lu.

14. The metal complex according to claim 11, wherein the radionuclide is .sup.68Ga.

15. The metal complex according to claim 11, wherein the radionuclide is .sup.225Ac.

16. A pharmaceutical composition comprising a compound of claim 1 or a metal complex comprising a radionuclide and said compound, or a pharmaceutically acceptable salt, or a solvate thereof, or a solvate of a salt thereof, and a pharmaceutically acceptable carrier.

17. A metal complex according to claim 11 or a pharmaceutically acceptable salt thereof or a solvate thereof or a solvate of a salt thereof for use in a method of imaging in a patient.

18. A metal complex according to claim 11 or a pharmaceutically acceptable salt thereof or a solvate thereof or a solvate of a salt thereof for use in a method of diagnosing a cancer and/or metastasis thereof, optionally selected from the group of cancers that are positive for expression of PSMA, further optionally prostate cancer.

19. A metal complex according to claim 11 or a pharmaceutically acceptable salt thereof or a solvate thereof or a solvate of a salt thereof for use in a method for treating cancer and/or metastasis thereof, optionally selected from the group of cancers that are positive for expression of PSMA, further optionally prostate cancer.

20. A method of diagnosing cancer and/or metastasis thereof, optionally selected from the group of cancers that are positive for expression of PSMA, further optionally prostate cancer, comprising administering to an individual a metal complex according to claim 11 or a pharmaceutically acceptable salt thereof or a solvate thereof or a solvate of a salt thereof.

21. A method of treating cancer and/or metastasis thereof, optionally selected from the group of cancers that are positive for expression of PSMA, further optionally prostate cancer, comprising administering to an individual a metal complex according to claim 11 or a pharmaceutically acceptable salt thereof or a solvate thereof or a solvate of a salt thereof.

Description

DESCRIPTION OF THE FIGURES

[0256] FIG. 1 shows the general scheme of the synthesis of DOTA conjugated-PSMA ligands of the invention. The signification of the abbreviations used in FIG. 1 is as follows: TFA=Trifluoro acetic acid; CDI=1,1-Carbonyldiimidazole; DCM=Dichloromethane; DMF=N,N-Dimethylformamide; IPA=2-Propanole; NMM=4-Methylmorpholine; coupling reagent=COMU or PyBOP, base=TEA or DIPEA

[0257] FIGS. 2-9 show the results of PET-Imaging for each of the tested compounds at 20-40 minutes (A), 40-60 minutes (B) and 120-140 minutes (C). FIG. 2A-2C: PSMA617. FIG. 3A-3C: ABX271; FIG. 4A-4C: ABX288; FIG. 5A-5C: ABX289; FIG. 6A-6C: ABX338; FIG. 7A-7C: ABX339; FIG. 8A-8C: ABX341; FIG. 9A-9C: ABX346.

EXAMPLES

Example 1: Synthesis of DOTA-Conjugated PSMA Ligands

[0258] The DOTA-conjugated PSMA ligands were synthesized via solid-phase peptide synthesis (FIG. 1). In the first step, 0.3 mmol of Fmoc-Lys(Dde)-OH were immobilized on 2-chloro-trityl resin. After Fmoc-deprotection Di-tert-butyl (1H-imidazole-1-carbonyl)-L-glutamate (intermediate 1) was added and the mixture was reacted for 16 h with gentle agitation. The resin was filtered off and the Dde-protecting group was removed using 1% hydrazine hydrate in DMF according to standard Fmoc-deprotection procedures.

[0259] The subsequent synthesis of the peptidomimetic PSMA binding motif was performed according to standard Fmoc solid phase protocols. The consecutive coupling of each linker part was performed using 2 equivalents of the corresponding Fmoc-protected acid (FMPA1, FMPA2, FMPA3), 2 equivalents of PyBOP, 2 equivalents of HOBt and 3 equivalents of N-ethyl-diisopropylamine in DMF.

[0260] After activation with PyBOP, HOBt and DIPEA, 1.5 eq of tris(t-bu)-DOTA (Chematech) relative to the resin loading were coupled in DMF. The product was cleaved from the resin in a mixture consisting of trifluoroacetic acid, triisopropylsilane, and hydrochloric acid (95:2.5:2.5).

[0261] Alternatively the chelator was conjugated using HBTU activated DOTA-NHS ester (Chematech) or DOTA-TFP ester.

[0262] Analysis of the synthesized molecules was performed using reversed-phase high performance liquid chromatography (RP-HPLC; Ascentis Express C18, 1504.6 mm; Supelco, Germany) with a linear A-B gradient (5% B to 100% B in 10 min) at a flow rate of 1.5 mL/min (analysis). Purification was performed using reversed-phase high performance liquid chromatography (RP-HPLC; Gemini-NX C18, 25050 mm; Phenomenex, Germany) with a linear A-B gradient at a flow rate of 100 mL/min. Solvent A consisted of 0.1% aqueous TFA and solvent B was 0.1% TFA in CH.sub.3CN.

[0263] The HPLC system (Dionex Ultimate 3000; Thermo-Fisher, Germany) was equipped with a UV detector. UV absorbance was measured at 200, 210 and 230 nm. Mass spectrometry was performed with a LC-MS (Dionex 3000, Thermo-Fisher, Germany).

Example 2: Synthesis of Intermediate 1

[0264] Under argon atmosphere 25 g of bis(tert-butyl)-L-glutamate hydrochloride were disssolved in 250 ml of dichloromethane and cooled to 0 C. Then 0.414 g of 4-(Dimethylamino)pyridine and 29.5 ml of triethylamine were added and subsequently, 15 g of 1,1-carbonyldiimidazole were added in small portions. The reaction mixture was stirred overnight. An aqueous solution of NaHCO.sub.3 was added and subsequently extracted with dichloromethane. Solvent was evaporated and the crude product was purified using column chromatography (dichloromethane/methanol, 80:1 (v/v)).

Example 3: Synthesis of Compound ABX 346

[0265] In the first step, 0.3 mmol of Fmoc-Lys(Dde)-OH were immobilized on 2-chloro-tritylresin. After Fmoc-deprotection di-tert-butyl (1H-imidazole-1-carbonyl)-L-glutamate (intermediate 1) was added and the mixture was reacted for 16 h with gentle agitation. The resin was filtered off and the Dde-protecting group was removed using 1% hydrazine hydrate in DMF according to standard Fmoc-deprotection procedures. The consecutive coupling of Fmoc-3-benzothienylalanine (FMPA1), Fmoc-5-(Aminomethyl)-1,2-oxazole-3-carboxylic acid (FMPA2) and Fmoc-Lys(Dde)-OH (FMPA3) linker was performed using 2 equivalents of the corresponding Fmoc-protected acid (FMPA1, FMPA2, FMPA3), 2 equivalents of PyBOP, 2 equivalents of HOBt and 3 equivalents of N-ethyl-diisopropylamine in DMF. After the reaction, the resin was filtered off and the Dde-protecting group was removed using 1% hydrazine hydrate in DMF according to standard Fmoc-deprotection procedures. After Fmoc-deprotection 4-iodobezoic acid was coupled by using 2 equivalents of PyBOP, 2 equivalents of HOBt and 3 equivalents of N-ethyl-diisopropylamine and DMF.

[0266] After activation with PyBOP, HOBt and DIPEA, 1.5 eq of tris(t-bu)-DOTA (Chematech) relative to the resin loading were coupled in DMF. The product was cleaved from the resin in a mixture consisting of trifluoroacetic acid, triisopropylsilane, and hydrochloric acid (95:2.5:2.5).

[0267] Purification was performed using reversed-phase high performance liquid chromatography (RP-HPLC; Gemini-NX C18, 25050 mm; Phenomenex, Germany) with a linear A-B gradient at a flow rate of 100 mL/min. Solvent A consisted of 0.1% aqueous TFA and solvent B was 0.1% TFA in CH.sub.3CN.

##STR00022##

[0268] The compounds in the examples 4-10 have been prepared in a similar way.

Example 4: ABX 341

[0269] ##STR00023##

Example 5: ABX 339

[0270] ##STR00024##

Example 6: ABX 338

[0271] ##STR00025##

Example 7: ABX 289

[0272] ##STR00026##

Example 8: ABX 288

[0273] ##STR00027##

Example 9: ABX 272

[0274] ##STR00028##

Example 10: ABX 271

[0275] ##STR00029##

Example 11: Radiolabeling

Labeling With Ga-68

[0276] Gallium labeling was carried out using 90 L HEPES in water (580 mg/mL; ultrapure-grade, Merck, Darmstadt, Germany) mixed with 40 L of [.sup.68Ga]GaCl.sub.3. The pH of the mixture was adjusted to 3.8 to 4.1 with NaOH (30%; ultrapure-grade, Merck, Darmstadt, Germany). Subsequently 1.0 nmol of a synthesized compound of Example 3 to 10 was added and incubated at 98 C. for 10 to 15 minutes. For the competitive cell assay, the precursor amount was diminished to 0.5 nmol.

Labeling With Lu-177

[0277] Lutetium labelling was carried out using 115 L of sodium acetate buffer (400 mM, pH mixed with 10 to 20 MBq [177Lu]LuCl.sub.3. Subsequently, 1.0 nmol of a synthesized compound of Example 3 to 10 was added and incubated at 98 C. for 20 to 25 minutes.

Quality Control

[0278] The radiochemical purity of the labelled compounds was evaluated by analytical RP-HPLC (Reversed Phase High Performance Liquid Chromatography, Chromolith RP-18e, Merck, Darmstadt, Germany) using a linear gradient from 100% water to 100% acetonitrile in 5 minutes at a flow rate of 4 ml/min. The purity was evaluated by RP-TLC (Reversed Phase Thin Layer Chromatography) using 0.1 M citrate buffer as the mobile phase.

Example 12: Determination of the Competitive Binding Affinity

[0279] Cell binding studies were performed using human PSMA+ LNCaP cells (ATCC CRL-1740) cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (Sera plus, PAN Biotech) and 2 mmol/l stable glutamine (PAN Biotech). Cells were grown at 37 C. in an incubator with humidified air and 5% CO.sub.2. Trypsin/EDTA 0.5%/0.02% in PBS (PAN Biotech) was used to harvest cells.

Internalization

[0280] 110.sup.5 LNCaP cells were seeded in poly-L-lysine coated 24-well-plates for 24 h at 37 C. humidified air with 5% CO.sub.2. After removing the medium, cells were incubated with radiolabelled compounds of Example 11 (30 nM) for 45 minutes at 37 C. and at 4 C. respectively. For determination of specific cell uptake, cells were blocked with PMPA (Tocris) at a concentration of 500 M. The cells were washed three times with 1 ml ice-cold PBS, twice with 500 l glycine-HCl (50mM; pH 2.8) for 5 min at rt to remove the surface-bound radioactivity and with 1 ml ice-cold PBS. Subsequent the cells were lysed with 0.5 ml 0.3M NaOH. The internalized and the surface-bound fractions were measured in a gammacounter.

[0281] The results are shown in table 1.

TABLE-US-00001 TABLE 1 Internalisation rate of PSMA binding compounds compound internalisation rate (%) PSMA-617 31.1 EP2862857-MB17 Example ABX346 46.7 Example ABX341 61.3 Example ABX339 60.6 Example ABX338 40.8 Example ABX289 55.7 Example ABX288 33.2 Example ABX272 41.4 Example ABX271 37.8

[0282] As can be seen from Table 1, the PSMA-binding ligands of the present invention which contain heteroaromatic linker building blocks show significantly higher internalisation rates than compound PSMA-617 known from the prior art.

PET-Imaging

[0283] 510.sup.b 6 LNCAP cells in Opti-MEM 50% Matrigel (Corning) were inoculated subcutaneously into the trunk of male balbc nu/nu mice (Charles River). The tumors were allowed to grow until approximately 1 cm.sup.3 in size.

[0284] For PET studies 60 pmol (3-10 MBq) [68Ga]-compounds were injected via tail vein into anesthetized mice. They were placed into the PET scanner and a dynamic scan over 130 min was performed. The results are shown in FIGS. 2-9. Tumor uptake was observed for all PSMA-binding ligands of the present invention containing heteroaromatic linker building blocks.